Foodborne Disease Handbook, Volume 3: Plant Toxicants 2nd Edition

Foodborne Disease Handbook Second Edition, Revised and Expanded Volume 3: Plant Toxicants

edited by

Y. H. Hui Science Technology System West Sacramento, California

R. A. Smith University of Kentucky Lexington, Kentucky

David G. Spoerke, Jr. Bristlecone Enterprises Denver, Colorado

MARCEL

MARCEL DEKKER, INC. D E K K E R

NEWYORK BASEL

ISBN: 0-8247-0343-X This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 2 12-696-9000; fax:2 12-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 41-61-261-8896 World Wide Web http://www.dekker.conl The publisher offers discountson this book when ordered in bulk quantities. Formore information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright 0 2001 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, by or any infornlation storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 1 0 9 8 7 6 5 4 3 3 1 PRINTED IN THE UNITED STATES OF AMERICA

Introduction to the Handbook

The Foodbome Disease Handbook, Second Edition,Revised am?Expanded, could not be appearing at a more auspicious time. Never before has the campaign for food safety been pursued so intensely on so many fronts in virtually every country around the world. This new edition reflects at least one of the many aspects of that intense and multifaceted campaign: namely, that research on food safety has been very productive in the years since the first edition appeared. The Handbook is now presented in four volumes instead of the three of the 1994 edition. The four volumes are composed of 86 chapters, a 22% increase over the 67 chapters of the first edition. Much of the information in the first edition has been carried forward to this new edition because that information is still as reliable and pertinent as it was in 1994. This integration of the older data with the latest research findings gives the reader a secure scientific foundation on which to base important decisions affecting the public's health. We are not so naive as to think that only scientific facts influence decisions affecting food safety. Political and economic factors and compelling national interests may carry greater weight in the minds of decision-makers than the scientific findings offered in this new edition. However, if persons in the higher levels of national governments and international agencies, such as the Codex Alimentarius Commission, the World Trade Organization, the World Health Organization, and the Food and Agriculture Organization, who must bear the burden of decision-making need and are willing to entertain scientific findings, then the infomation in these four volumes will serve them well indeed. During the last decade of the previous century, we witnessed an unprecedentedly intense and varied program of research on food safety, as we have already noted. There are compelling forces driving these research efforts. The traditional food-associated pathogens, parasites, and toxins of forty years ago still continue to cause problems today, and newer or less well-known species and strains present extraordinary challenges to human health. These newer threats may be serious even for the immunocompetent, but for the inlmunocompromised they can be devastating. The relative numbers of the immunocompromised in the world population are increasing daily. We include here not just those affected by the human immunodeficiency virus (HIV), but also the elderly; the very young; the recipients of radiation treatments, chemotherapy, and immunosuppressive drugs: iii

iv

to

Introduction

the Handbook

patients undergoing major invasive diagnostic or surgical procedures: and sufferers of debilitating diseases such as diabetes. To this daunting list of challenges must be added numerous instances of microbial resistance to antibiotics. Moreover, it is not yet clear how the great HACCP experiment will play out on the worldwide stage of food safety. Altruism and profit motivation have always made strange bedfellows in the food industry. It remains to be seen whether HACCP will succeed in wedding these two disparate motives into a unifying force for the benefit of all concerned-producers, manufacturers, retailers, and consumers. That HACCP shows great promise is thoroughly discussed in Volume 2, with an emphasis on sanitation in a public eating place. All the foregoing factors lend a sense of urgency to the task of rapidly identifying toxins, species, and strains of pathogens and parasites as etiologic agents, and of determining their roles in the epidemiology and epizootiology of disease outbreaks, which are described in detail throughout the Foodborne Disense Hmdbook. It is very fortunate for the consumer that there exists in the food industry a dedicated cadre of scientific specialists who scrutinize all aspects of food production and bring their expertise to bear on the potential hazards they know best. A good sampling of the kinds of work they do iscontained in these four new volumes of the Handbook. And the benefits of their research are obvious to the scientific specialist who wants to learn even more about food hazards, to the scientific generalist who is curious about everything and who will be delighted to find a good source of accurate, up-to-date information, and to consumers who care about what they eat. We are confident that these four volumes will provide competent, trustworthy, and timely information to inquiring readers, no matter what roles they may play in the global campaign to achieve food safety.

Y. H. Hui J. Richard Gorham Dcrvid Kitts K. D. Murre11 Wai-Kit Nip Merle D. Pierson Sved A. Suttar R. A. Smith David G. Spoerke, Jr. Peggv S. Stanjield

Preface

The world ofnature offers many pleasant attractions. Concurrent with theincreased crowding ofurban areas inmuchof the developed world, there is a growing tendency for stressed-out city dwellers to seek peace in the wilderness, the more or less easily accessible natural areas, both terrestrial and aquatic. Much of the fauna and flora of these natural areas are quite innocuous-for the most part, only specialists are aware of exceptions. And even some of the specialists might be unaware of hazards originating outside their own sphere of expertise. Among consumers, mushroom hunters and fishermen are probably the best informed about potential hazards in their favored haunts. However, without access to specialized equipment and laboratory protocols, even the most competent specialist may be quite as unable to detect a hazard in food as the most naive consumer. While poisonous mushrooms figure prominently in this volume of the Foodborne Diseuse Handbook, other dangerous botanicals are by no means neglected. By “dangerous,” we refer to a very broad range of effects on human and animal health. The poisonous plants, their toxins, and the symptoms they cause are all discussed in detail, but more than that, the reader will find current and helpful information on methods of chemical analysis and recommendations for the medical management of poisoning episodes. Mushrooms are enormously popular around the world as a food item. Fortunately for the average consumer, grocery stores and restaurants get their mushrooms from commercial growers. Such mushrooms have no inherent toxic properties and thus are considered safe to eat and, in fact, are safe to eat. However, even with commercially produced mushrooms, the potential for microbial and insecticidal contamination should not be ignored. In the category of organisms known as fungi, mushrooms and toadstools are relatively large and easy to recognize for what they are. There are other fungi, however, that most of us will never see and that many consumers do not even know exist. Yet they, or the toxins they elaborate, may be just as dangerous as the much more obvious poisonous mushrooms. These are the fungi that produce mycotoxins (e.g., aflatoxin). For example, edible plant foods may contain natural poisons. We have heard about molds in peanut, which are a form of fungi-and contain aflatoxin. Poisons in cotton seed, cabbage, and

V

vi

Preface

potatoes are usually either removed during processing or destroyed during cooking. Plant toxins are described in great detail-detection, identification, effects on human and animal health, epidemiology-in this volume.

Y. H. Hui R. A. Snzith David G. Spoerke, JK

Contents

htroduction to the Handbook Preface Corn-ibutors Colttents of Other Volumes

...

111

V

i.x xi

I. Poison Centers 1. U.S. Poison Centers for Exposures to Plant and Mushroom Toxins David G. Spoerke, Jr.

1

11. Selected Plant Toxicants 2. Toxicology of Naturally Occurring Chemicals in Food Ross C. Beier and Herbert N. Nigg

37

3. Poisonous Higher Plants Doreell Grace Lnng and R. A. Smith

187

4. Alkaloids R. A. Smith

247

5. Antinutritional Factors Related to Proteins and Amino Acids Irvin E. Liener

257

6. Glycosides Walter Majak and Michael H. Benn

299

vii

Contents

viii

7. Analytical Methodology for Plant Toxicants Alister David Muir

351

8. Medical Management and Plant Poisoning Robert H. Poppenga

413

9. Plant Toxicants and Livestock: Prevention and Management Michael H. RnIphs

44 1

111. Fungal Toxicants 10. Aspergillus ZoJin Kozakiewicz

47 1

11. Claviceps and Related Fungi Gretchen A. Kuldau and Charles W. Bacon

503

12. Fusarium Walter F. 0. Marasas

535

13. Penicillium John I. Pitt

581

14. Foodborne Disease and Mycotoxin Epidemiology S a m Hale Herwy and F. Xavier Bosch

593

15. Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Koshinshy, Adrienne Woytowich, and George G. Khnchntourians

627

16. Analytical Methodology for Mycotoxins James K. Porter

653

17. Mycotoxin Analysis: Immunological Techniques Fun S. Chu

683

18. Mushroom Biology: General Identification Features David G. Spoerke, Jr.

715

19. Identification of Mushroom Poisoning (Mycetismus), Epidemiology, and Medical Management David G. Spoerke, Jr.

20.

Index

Fungi in Folk Medicine and Society David G. Spoerke, Jr.

739

78 1

803

Contributors

Charles W. Bacon Toxicology and Mycotoxin Research Unit, Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia Ross C. Beier Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas Michael H. Benn Chemistry Department, University of Calgary, Calgary, Alberta, Canada F. Xavier Bosch lona, Spain

Epidemiology Unit, Institute of Oncology, Llobregat Hospital, Barce-

Fun S. Chu Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin Sara Hale Henry Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C. George G. Khachatourians Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Heather A. Koshinsky Investigen, Alameda, California ZofiaKozakiewicz Biotechnology and Utilization of Biodiversity, CAB1 Bioscience, Egham, Surrey, England Gretchen A. Kuldau Department of Plant Pathology, Pennsylvania State University, University Park, Pennsylvania Doreen Grace Lang ington, Kentucky

Department of Veterinary Science, University of Kentucky, Lex-

Irvin E. Liener Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota WalterMajak Range Research Unit, Agriculture and Agri-Food Canada, Kamloops, British Columbia, Canada ix

Contributors

X

Walter F. 0. Marasas Programme on Mycotoxins and Experimental Carcinogenesis, Medical Research Council, Tygerberg, South Africa Alister David Muir Crop Utilization, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada Herbert N. Nigg

University of Florida, Lake Alfred, Florida

John I. Pitt Food Science Australia, North Ryde, New South Wales, Australia Robert H. Poppenga New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania James K. Porter Toxicology and Mycotoxin Research Unit, R. B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia Michael H. Ralphs Poisonous Plant Research Lab, Agriculture Research Service, U.S. Department of Agriculture, Logan, Utah R.A.Smith Kentucky

Department of Veterinary Science, University of Kentucky, Lexington,

David G. Spoerke, Jr.

Bristlecone Enterprises, Denver, Colorado

Adrienne Woytowich Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Contents of Other Volumes

VOLUME 1: BACTERIAL PATHOGENS

I. Poison Centers 1.

The Role of U.S. Poison Centers in Bacterial Exposures David G. Spoerke, Jr.

11. Bacterial Pathogens 2. Bacterial Biota (Flora) in Foods James M. Jay 3. Aeromonns hydrophila Carlos Abeyta, Jr., Samuel A. Palumbo, and Gerard N. Stelma, Jr.

4. Update: Food Poisoning and Other Diseases Induced by Bacillus cereus Kathleerl T. Rnjkowski and James L. Smith 5.

Brucella Shirley M. Hallirzg and Edward J. Young

6. Campylobucter jejuni Don A. Franco and Charles E. Williams 7.

Clostridium botulirzum John W. Austift and Karerl L. Dodds

8.

Clostridium yel@irzgerzs Dorothy M. Wrigley xi

Contents of Other Volumes

xii

9. Escherichia coli Marguerite A. Neill, Phillip I. Tarr, David N. Taylor, and Marcia Wolf 10. Listeria nzonocytogenes Catherine W. Donnelly 11. Bacteriology of Salmonella Robin C. Arzderson and Richard L. Ziprirl

12. Salmonellosis in Animals David J. Nisbet and Richard L. Ziprin

13. Human Salmonellosis: General Medical Aspects Richard L. Ziprin and Michael H . Hume 14. Shigella Anthony T. Mnurelli crnd Keith A. Lnmpel

15. Stuphylococcus aureus Scott E. Martin, Eric R. Myers, and John J. Inndolo 16. Vibrio cholerae Charles A, Kaysner and June H. Wetherington 17. Vibrio yarahaernolyticus Tm-jyi Chai and John L. Pace

18. Vibrio vulnificus Anders Dalsgaard, Lise H@i, Debi Lirlkous, and James D. Oliver

19. Yersinia Scott A. Minnich, Michael J. Smith, Steven D. Weagant, and Peter Feng 111. Disease Surveillance, Investigation, and Indicator Organisms 20.

Surveillance of Foodborne Disease Ewen C. D. Todd

21. Investigating Foodborne Disease Dale L. Morse, Guthrie S. Birkhead, crnd Jack J. Guzewich

22. Indicator Organisms in Foods James M. Jay Index

Contents of Other Volumes

VOLUME 2: VIRUSES, PARASITES, PATHOGENS, AND HACCP I. Poison Centers 1. The Role of Poison Centers in the United States David G. Spoerke, Jr.

11. Viruses 2. Hepatitis A and E Viruses Theresa L. Cromeam, Michael 0. Favorov, Ornana V. Nainan, and Harold S. Margolis 3. Norwalk Virus and the Small Round Viruses Causing Foodborne Gastroenteritis Hazel Appleton

4. Rotavirus Syed A. Sattar, V. Susan Springthorpe, and Jason A. Tetro 5. Other Foodborne Viruses Syed A. Sattar and Jason A. Tetro

6. Detection of Human Enteric Viruses in Foods Lee-Ann Jaykus 7. Medical Management of Foodborne Viral Gastroenteritis and Hepatitis Suzanne M. Mntsui and Ramsey C. Chemg

8. Epidemiology of Foodborne Viral Infections Thomas M. Liithi 9. Environmental Considerations in Preventing the Foodborne Spread of Hepatitis A Syed A. Sattnr and Sabah Bidawid

111. Parasites 10. Taeniasis and Cysticercosis ZbignieMt S. Pawlowski and K. D. Murre11

11. Meatborne Helminth Infections: Trichinellosis William C. Campbell 12. Fish- and Invertebrate-Borne Helminths John H. Cross 13. Waterborne and Foodborne Protozoa Ronald Fayer

Contents of Other Volumes

xiv

14. Medical Management Paul Prociv

15. Immunodiagnosis of Infections with Cestodes Bruno Gottsteirr 16. Immunodiagnosis: Nematodes H. Ray Ganlble 17. Diagnosis of Toxoplasmosis Alan M. Johnson and J. P. Dubey 18. Seafood Parasites: Prevention, Inspection, and HACCP Arm M. A d a m and Debra D. DeVlieger

IV. HACCP and the Foodservice Industries 19. Foodservice Operations: HACCP Principles 0. Peter Snyder, Jr. 20. Foodservice Operations: HACCP Control Programs 0. Peter Srlyder, Jr. Irzdex

VOLUME 4: SEAFOOD AND ENVIRONMENTAL TOXINS I. Poison Centers 1. Seafood and Environmental Toxicant Exposures: The Role of Poison Centers Dmid G. Spoerke, Jr.

11. Seafood Toxins 2. Fish Toxins BrmP W. Hulstend

3. Other Poisonous Marine Animals Bruce W. Hdstend 4.

Shellfish Chemical Poisoning Ljwdolz E. Llewellyn

5. Pathogens Transmitted by Seafood Russell P. Herwig

Contents of Other Volumes

6. Laboratory Methodology for Shellfish Toxins David Kitts 7. Ciguatera Fish Poisoning Yoshitsugi Hokcrrna and Joanrle S. M. Yoshih-awa-Ebesu 8. Tetrodotoxin Joanne S. M. Yoshikawa-Ebesu, Yoshitsugi Hokanln, and Tarnno Noguchi

9. Epidemiology of Seafood Poisoning Lora E. Flemit1g, Dolores Kat:, Judv A. Bean, and Roberta Hammond 10. The Medical Management of Seafood Poisoning Donna Glad Blvthe, Eileerl Hack, Giavnnni Wnshington, and Lorn E. Fleming 11. The U.S. National Shellfish Sanitation Program Rebecca A. Reid m d Timothv D. Durance 12. HACCP, Seafood, and the U.S. Food and Drug Administration Kim R. Young, Miguel Rodrigues Kanznt, arrd George Perly Hoskin 111. Environmental Toxins

13. Toxicology and Risk Assessment Donuld J. Ecobichorr 14. Nutritional Toxicology David Kitts 15. Food Additives Laszlo P. Somogyi

16. Analysis of Aquatic Contaminants Joe W. Kiceniuk 17. Agricultural Chemicals Debra L. Browning and Carl K. Winter 18. Radioactivity in Food and Water Hank Kocol

19. Food Irradiation Hank Kocol 20. Drug Residues in Foods of Animal Origin Austin R. Long and Jose E. Roybnl

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Contents of Other Volumes

21. Migratory Chemicals from Food Containers and Preparation Utensils Yvonne V. Yuan 22. Food and Hard Foreign Objects: A Review J. Richard Gorhnm

23. Food, Filth, and Disease: A Review J. Richard Gorhnrn 24. Food Filth and Analytical Methodology: A Synopsis J. Richard Gorham

1 U S . Poison Centers for Exposures to Plant and Mushroom Toxins David G. Spoerke, Jr. Bristlecorte Enterprises, Denver, Colorado

Epidemiology I. A. B. C. D. E. F. G. H. 1. J. K.

1

AAPCC 2 Staffing poison a center 4 Types of calls received 5 How calls are handled 6 References used 7 How poisoncentersaremonitoredforquality Professionalandpubliceducationprograms Related toxicology organizations 8 International affiliations 10 Toxicology and poisoncenterWeb sites North American mycological association

11. PoisonInformationCenters

intheUnited

States

7

8

11 11 12

111. National and InternationalMycologicalAssociations/Clubs/ Organizations 23 References 36

1.

EPIDEMIOLOGY

Epidemiological studies aid treatment facilities in determining risk factors, determining who may become exposed, and establishing the probable outcomes of various treatments. A few organizations have attempted to gather such information and organize it into yearly reports. The American Associations of Poison Control Centers (AAPCC), North American Mycological Association (NAMA), and some federal agencies all work toward obtaining epidemiological information, while the AAPCC has an active role in assisting with the treatment of exposures. Epidemiological studies assist government and industry in determining package safety, effective treatment measures, conditions of exposure, and frequency of exposure. In 1987 there were 7023 cases of mushroom poisoning reported to the AAPCC. In 1988, that figure increased to 7,834 (1). These numbers were approxif

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mately 0.6% of the total cases called to poison centers. The NAMA mushroom poisoning case registry was provided with 156 reports (4.6% of reported mushroom cases), and, in 1988, 116 cases (3.4%) were registered (1). Studies on mushroom poisonings provide information on the type of people most commonly involved in exposures. Are these patients children experimenting in the backyard, hikers, or mycophiles looking for dinner? Studies can also tell us which species are most commonly involved and what species were being sought. What types of symptoms are seen first, onset of symptoms, and any sequelae may also be determined and compared to accepted norms.

A.

AAPCC

1. What Are Poison Centers and the AAPCC? The group in the United States most concerned on a daily basis with poisonings due to household agents, industrial agents, and biologics (including plants and mushrooms) is the AAPCC. This is a national resource that provides information concerning all aspects of poisoning and often refers patients to treatment centers. This group of loosely affiliated centers is often supported by both government and industrial sources. Poison centers were started in the late 1950s, the first thought to be in the Chicago area. The idea caught on quickly and at the peak of the movement there were hundreds of centers throughout the United States. Unfortunately, there were little or no standards to define what might be called a poison center, the type of staff, hours of operation, or information resources. One center may have had a dedicated staff of doctors, pharmacists, and nurses trained specifically in handling poison cases: the next center may just have had a book on toxicology in the emergency room or hospital library. In 1993, the Health and Safety Code (Sec. 777.002) specified that a poison center provide a 24-hr service for public and health care professionals and meet requirements established by the AAPCC. This action helped the AAPCC to standardize activities and staffing of the various poison centers. The federal government does not fund poison centers, even though for every dollar spent on poison centers there is a savings of $2-9 in unnecessary expenses (2, 3). The federal agency responsible for the Poison Prevention Packaging Act is the U.S. Consumer Product Safety Commission (CPSC). The National Clearinghouse for Poison Control Centers initially collected data on poisonings and information on commercial product ingredients and biologic toxic agents. For several years the National Clearinghouse provided product and treatment information to the poison centers that handled the day-to-day management of the centers. At first most poison centers were funded by the hospital in which they were located. As the centers grew in size and number of calls being handled, both city and state governments took on the responsibility of contributing funds. In recent years, the local governments have found it very difficult to fund such operations and centers have had to look to private industry for additional funding. Government funding may take several forms, either as a line-item on a state’s budget, as a direct grant, or as moneys distributed on a per call basis. Some states with fewer residents may contract with a neighboring state to provide services to its residents. Some states are so populous that more than one center is funded by the state. Industrial funding also varies, sometimes as a grant, sometimes as

Poison Centers for Plant Toxin Exposure

3

Table 1 AAPCC MushroomExposures ~

~

~~~

~

Year

# of exposures

% accidental

% of total AAPCC calls

1989 1990

9388 9570

95 95

5.9 5.7

payment for handling the company’s poison or drug information-related calls, sometimes as payment for collection of data regarding exposure to the company’s product. Every year the AAPCC reports a summary of plant and mushroom exposures. As an example, data on mushroom exposures from 1989 and 1990 are listed in Tables 1 and 2. The totals do not equal loo%, as not everyone who was exposed to a mushroom went to anemergency department, and not all calls concerning mushroom exposures were due to poisonings. As can be seen by these statistics, there are a large number of exposures, but very few serious outcomes due to mushroom exposures. The same type of information is available for plant exposures. Each plant and mushroom has its own code number in the POISINDEX‘ reference system, which is entered by the poison center specialist taking the call. Thus, if the plant or mushroom is known at the time of exposure and the right code is entered, the database will describe ages, sexes, signs and symptoms, treatment, and outcomes for any particular plant or mushroom.

2. RegionalCenters The number of listed centers has dropped significantly since its peak of 600 plus. Many centers have been combined into regional centers. These regional poison centers provide poison information and telephone management and consultation, collect pertinent data, and deliver professional and public education. Cooperation between regional poison centersand poison treatment facilities is crucial. The regional poison information center should work with various hospitals to determine the capabilities of the treatment facilities of the region and to identify and have a working relationship with analytical toxicology laboratories, emergency departments, critical care wards, medical transportation systems, and extracorporeal elimination methods availability. This should be done for both adults and children. A “region” is usually determined by state authorities in conjunction with local health agencies and health care providers. Documentation of these state designations must be in writing unless a state chooses (in writing) not to designate any poison center or accepts a designation by other political or health jurisdictions. Regional poison infomation centers should serve a population base of greater than one million people and must receive at least 10,000 human exposure calls per year. The number of certified regional centers in the United States is now under SO. Certification as a regional center requires the following (4): Table 2 Outcomes of AAPCC Mushroom Exposures-% Outcomes Sx minor No or

Tx EDYear 1989 1990

24.4% 24.8%

78.5% 76.1%

of Total Calls: ED Visits and

Death Mod Sx Major Sx 2.5% 2.0%

0.16% 0.10%

3 cases 1 case

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1. Maintenance of a 24 hr/day, 365 dayslyear service. 2. Service to both health care professionals and the public. 3. Availability of at least one specialist in poison information in the center at all times. 4. A medical director or qualified designee, on call by telephone, at all times. 5. Service readily accessible by telephone from all areas within the region. 6. Comprehensive poison information resources and comprehensive toxicology information covering both general and specific aspects of acute and chronic poisoning. 7. A list of on-call poison center specialty consultants. 8. Written operational guidelines, which provide a consistent approach to evaluation, follow-up, and management of toxic exposures. These guidelines must be approved in writing by the medical director of the program. 9. A staff of certified professionals manning the phones (at least one of the persons on the phone has to be a pharmacist or nurse with 2000 hr and 2000 cases of supervised experience). 10. A 24-hr/day physician (board certified) consultation service. 11. An ongoing quality assurance program. 12. Other criteria, as determined by the AAPCC, may be established with membership approval. 13. The regional poison information center must be an institutional member in good standing of the AAPCC. Many hospital emergency rooms still maintain a toxicology reference such as the POISINDEX system to handle routine exposure cases, but rely on regional poison centers to handle most of the calls in their area.

B. Staffing a PoisonCenter The staffing of a poison center varies considerably from center to center. The three professional groups most often involved are physicians, nurses, and pharmacists. Who answers the phones is somewhat dependent on the local labor pool, moneys available, and the types of calls being received. Other groups called on to serve in a center (with appropriate supervision) include students in medically related fields, toxicologists, and biologists. Persons responsible for answering the phones are either certified by the AAPCC or are in the process of obtaining the certification. Passage of an extensive examination in toxicology is required for initial certification, with periodic recertification required. Regardless of who takes the initial call, there is a medical director and other physician backup available. These physicians have specialized training or experience in toxicology, and are able to provide in-depth consultations for health care professionals calling a center. 1. Medical Director A poison center medical director should be board certified in medical toxicology, internal medicine, pediatrics, family medicine, or emergency medicine. The medical director should be able to demonstrate ongoing interest and expertise in toxicology as evidenced

PoisonPlant Centers for

Toxin Exposure

5

by publications, research, and meeting attendance. The medical director must have a medical staff appointment at a comprehensive poison treatment facility and must be involved in the management of poisoned patients.

2. ManagingDirector The managing director must be a registered nurse, pharmacist, or physician, or hold a degree in a health science discipline. The individual should be certified by the American Board of Medical Toxicology (for physicians) or by the American Board of Applied Toxicology (for nonphysicians). They must be able to demonstrate ongoing interest and expertise in toxicology. 3. Specialists in PoisonInformation These individuals must be registered nurses, pharmacists, or physicians, or be currently certified by the AAPCC as a specialist in poison information. Specialists in poison information must complete a training program approved by the medical director and must be certified by the AAPCC as a specialist in poison information within two examination administrations of their initial eligibility. Specialists not currently certified by the Associations must spend an annual average of no less than 16 hr/week in poison center related activities. Specialists currently certified by the AAPCC must spend an annual average of no less than 8 hr/week. Other poison information providers must have sufficient background to understand and interpret standard poison information resources and to transmit that information understandably to both health professionals and the public. 4. Consultants In addition to physicians specializing in toxicology, most centers also have lists of experts in many other fields. Poison center specialty consultants should be qualified by training or experience to provide sophisticated toxicology or patient care information in their area(s) of expertise. In regard to botanic exposures, the names and phone numbers of persons in a botanic garden, various nurseries, gardening clubs, or mushroom clubs are often available, with experts willing to donate their expertise in identification and handling cases within their specialty.

C. Types of Calls Received All types of calls are received by poison centers, most of which are handled immediately with a few others referred to more appropriate agencies. Which calls are referred depends on the center, its expertise, and the appropriateness of a referral. Below are types of calls that generally fall into each group. There is considerable variation between poison centers, and if there is doubt, call the poison center and they will tell you if your case is more appropriately referred. Poison centers do best on calls regarding acute exposures. Complicated calls regarding exposure to several agents over a long period that produce nonspecific symptoms are often referred to another medical specialist, to the toxicologist associated with the center, or to an appropriate government agency. The poison center will often follow up on these cases to track outcome and type of service given. Types of Calls Usually Accepted Drug identification. Actual acute exposure to a drug or chemical.

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Actual acute exposure to a biologic agent (plants, mushrooms, various animals). Information regarding the toxic potential of an agent. Possible food poisoning. Drug information calls. Types of Calls Often Referred Questions regarding treatment of a medical condition (not poisoning). General psychiatric questions, with no drugs or chemicals involved. Proper disposal of household agents such as batteries, bleach, insecticides. Use of insecticides (e.g., which insecticide to use, how to use it) unless related to a health issue-for example, a person allergic to pyrethrins wanting to know which product does not contain pyrethrins). Drug information calls. 1. Data Collection AAPCC has certain rules about data collection. Records of all calls/cases handled by the center must be kept in a form that is acceptable as a medical record. The regional poison information center must submit all its human exposure data to the American Association of Poison Control Centers' National Data Collection System. The regional poison information center must tabulate its experience for regional program evaluation on at least an annual basis. In 1983 the AAPCC formed the AAPCC Toxic Exposure Surveillance System (TESS) from the former National Data Collection System. Currently TESS contains nearly 16.2 million human poison exposure cases. Sixty-five poison centers, representing 181.3 million people, participate in the data collection. The information has various uses to both governmental agencies and industry, providing data for product reformulations, repackaging, recall, bans, injury potential, and epidemiology. The summation of each year's surveillance is published in the Americcrn Jozmal of Emergency Medicine each year in late summer or fall (1 1, 12).

D. HowCallsAreHandled Most poison centers receive requests for information via the telephone. Calls come from both health care professionals and consumers. Only a few requests are received by mail or in person; these are often medicolegal or complex cases. Most centers can be reached by a toll-free phone number in the areas they serve, as well as a local number. Busy centers will have a single number that will ring on several lines. Calls are often direct referrals from the 91 1 system. In most cases, poison center specialists are unable to determine the exact plant or mushroom species, so it is difficult to give plant/mushroom-specific information. Often there is an attempt to at least identify the genus involved so as to estimate toxicity. In cases where few, if any, symptoms occur, and the more seriously toxic biologics can be ruled out, there is often minimal additional effort put forth to determine the plant/mushroom species. The patient is followed by telephone to assure no signs or symptoms develop. When symptoms are present, experienced plant/mushroom identifiers are often utilized to make identification as precise as possible withthe available botanic material. Poison information specialists listen to the caller, recording the history of the case on a standardized form developed by the AAPCC. Basic information such as the agent

Poison Centers for Plant Toxin Exposure

7

involved, the amount of agent, time of ingestion, symptoms, previous treatment, and current condition are recorded, as well as patient information such as sex, age, phone number, who is with the patient, relevant medical history, and sometimes patient address. All information is considered a part of a confidential medical record. The case is evaluated (using various references) as: 1. information only, no patient involved 2. harmless andnot requiring follow-up 3. slightly toxic, no treatment necessary but a follow-up call is given 4. potentially toxic, treattnent given at home and follow-up given until case resolution 5. potentially toxic, treatment may or may not be given at home, but it is necessary for the patient to be referred to a medical facility 6. emergency-an ambulance and/or paramedics are dispatched to the scene Cases are usually followed until symptoms have resolved. In cases where the patient is referred to a health care facility, the received agency is notified that the patient is in transit. The history is relayed, toxic potential discussed, and suggestions for treatment given. E. References Used References used also vary from center to center, but virtually all U.S. centers use a toxicology system called POISINDEX (5), which contains lists of products, their ingredients, and suggestions for treatment. The system is compiled using medical literature and medical specialists from throughout the world. Biologic products such as plants, insects, mushrooms, animal bites, and so forth are handled similarly. An entry for an individual plant might contain a description, toxic substance present, potential toxic amounts, and most dangerous plant part. The physician or poison information specialist is then referred to a treatment protocol that may be used for a general class of agents. An example would be: Pl?ilodend~-onexposures are referred to a protocol on oxalate-containing plants. This system is available on microfiche, a CD ROM, over a network, or on a mainframe. It is updated every 3 months. Not every plant and mushroom is on the system, but a great tnany are listed by both their scientific and common names. Various texts are also used. Among those mushroom sources stated as helpful in a survey of poison centers, Miller (6), Kingsbury (7), Rumack and Salzman (8), POISINDEX (5), Lincoff and Mitchel (9), and Stamets (10) are frequently mentioned. It is very difficult to identify plants and mushrooms using a description given over the phone, so often the assistance of garden club or mushroom club members, local greenhouses, botanic gardens, and various specialists is requested. Some poison centers have more experience with certain types of poisonings than do others, and these centers are often consulted during a more complex case. A recent trend has been for various manufacturers not to provide product information to all centers via POISINDEX, but to contract with one poison center to provide for poison information services for the whole country. Product information is given to only that center and cases throughout the country can only be handled by that one center.

F. How Poison Centers Are Monitored for Quality Most poison centers have a system of peer review. One person takes a call, another reviews it. Periodic spot review is done by supervisory and physician staff. General competence

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is assured by certification and recertification via examination of physicians and poison informition specialists. Most regional centers have journal clubs where challenging cases are discussed.

G. ProfessionalandPublicEducationPrograms The regional poison information center is required to provide information on the management of poisoning to the health professionals throughout the region who care for poisoned patients. Public education programs, aimed at educating both children and adults about poisoning dangers and other necessary concepts related to poison control, should be provided. In the past, several centers provided stickers or logos such as Officer Ugh, Safety Sadie, and Mr. Yuck. These stickers could be placed on or near potentially toxic substances. While the intent was to identify potentially toxic substances that the children should keep away from, the practice has been much curtailed on the new assumption that in some cases the stickers actually attracted the children to the products. In the spring of every year there is a poison prevention week. National attention is focused on the problem of potentially toxic exposures. During this week many centers run special programs for the public. This may include lectures on prevention, potentially toxic agents in the home, potentially toxic biologic agents, or general first aid methods. Although this week is an important time for poison centers, public and professional education is a year-round commitment. Physicians often have medical toxicology rounds, journal clubs, and lectures by specialty consultants. Health fairs, school programs, and various women’s clubs are used to educate the public. The extent of these activities is often determined by the amount of funding from government, private organizations, and public donations.

H. RelatedProfessionalToxicologyOrganizations AACT American Association of Clinical Toxicologists Address: c/o Medical Toxicology Consultants; Four Columbia Drive; Suite 8 10; Tampa, FL 33606 AAPCC American Association of Poison Control Centers Address: 3201 New Mexico Avenue NW; Washington, DC 20016 Phone: 202-362-7217 FAX: 202-362-8377 ABAT American Board of Applied Toxicology Address: Truman Medical Center, West; 2301 Holmes St.; Kansas City, MO 64108 Phone: 8 16-556-3112 FAX: 816-881-6282 ABEM American Board of Emergency Medicine Address: 300 Coolidge Road; East Lansing, MI 48823 Phone: 5 17-332-4800 FAX: 517-332-2234 ACEP American College of Emergency Physicians (Toxicology Section) Address: P.O. Box 619911; Dallas, TX 75261-9911

Poison Centers forPlant Toxin Exposure

9

Phone: 800-798-1822 FAX: 214-580-2816 ACGIH American Conference of Governmental and Industrial Hygienists Address: Kemper Woods Center; Cincinnati, OH 45240 Phone: 5 13-742-2020 FAX: 513-742-3355 ACMT American College of Medical Toxicology (formerly ABMT) Address: 777 E. Park Drive; P.O. Box 8820; Harrisburg, PA 17105-8820. Phone: 717-558-7846 FAX: 7 17-558-7841 e-mail: [email protected] (Linda L. Koval) ACOEM American College of Occupational and Environmental Medicine Address: 55 West Seegers Road; Arlington Heights, IL 60005 Phone: 708-228-6850 FAX: 708-228- 1856 ACS Association of Clinical Scientists Address: Dept. of Laboratory Medicine; University of Connecticut Medical School; 263 Farmington Ave.; Farmington, CT 06030-2225 Phone: 203-679-2328 FAX: 203-679-2328 ACT American College of Toxicology Address: 9650 Rockville Pike; Bethesda, MD 20814 Phone: 30 1-571- 1840 FAX: 301-571-1852 AOEC Association of Occupational and Environmental Clinics Address: 1010 Vermont Ave., N W , #513; Washington, DC 20005 Phone: 202-347-4976 FAX: 202-347-4950 e-mail: [email protected] ASCEPT Australian Society of Clinical and Experimental Pharmacologists and Toxicologists Address: 145 Macquarie St.; Sydney N.S.W. 2000, Australia Phone: 6 1-2-256-5456 FAX: 6 1-2-252-3310 BTS British Toxicology Society Address: MJ Tucker, Zeneca Pharmaceuticals; 22B 11 Mareside; Alderley Park, Macclesfield; Cheshire, SKlO 4TG; United Kingdom Phone: 0428 65 5041 CAPCC Canadian Association of Poison Control Centers Address: Hopital Sainte-Justine; 3 175 Cote Sainte-Catherine; Montreal, Quebec H3TlC5 Phone: 5 14-345-4675 FAX: 5 14-345-4822 CSVVA (CEVAP) Center for the Study of Venoms and Venomous Animals Address: UNESP; Alameda Santos; N 647; CEP 01419-901; Sao Paulo, SP, Brazil Phone: 55 01 1 252 0233 FAX: 55 01 1 252 0200

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EAPCCT European Association of Poison Control Centers Address: J. Vale; National Poisons Information Centre; P.O. Box 81898 d; N-0034 Oslo, Norway Phone: 47-260-8460 HPS Hungarian Pharmacological Society Address: Central Research Institute for Chemistry; Hungarian Academy of Sciences; H-1525 Budapest; P.O. Box 17; Pusztaszeri ut 59-67 Phone: 36-1-135-21 12 ISOMT International Society of Occupational Medicine and Toxicology Address: USC School of Medicine; 222 Oceanview Ave., Suite 100; Los Angeles, CA 90057 Phone: 213-365-4000 JSTS Japanese Society of Toxicological Sciences Address: Gakkai Center Building; 4-16, Yayoi 2-chome; Bunkyo-ku; Tokyo 113, Japan Phone: 3-38 12-3093 FAX: 3-38 12-3552 SOT Society of Toxicology Address: 1101 14th Street, Suite 1100; Washington, DC 20005-5601 Phone: 202-37 1- 1393 FAX: 202-371-1090 e-mail: [email protected] SOTC Society of Toxicology of Canada Address: P.O. Box 517; Beaconsfield, Quebec, H9W 5V1, Canada Phone: 5 14-428-2676 FAX: 5 14-482-8648 SSPT Swiss Society of Pharmacology and Toxicology Address: Peter Donatsch; Sandoz Pharma AG; Toxicologtie 88 1/130; CH4132 Muttenz, Switzerland Phone: 4 1-61-469-537 1 FAX: 4 1-61-469-6565 STP Society of Toxicologic Pathologists Address: 875 Kings Highway, Suite 200; Woodbury, NJ 08096-3172 Phone: 609-845-7220 FAX: 609-853-04 11 WFCT World Federation of Associations of Clinical Toxicology Centers and Poison Control Centers Address: Centre Anti-Poisons; Hopital Edonard Herriot; 5 pl d’Arsonva1; 69003 Lyon, France Phone: 33 72 54 80 22 FAX: 33 72 34 55 67 I. InternationalAffiliations AAPCC members attend various world conferences to learn of toxicology problems and new methods used by these agencies. An especially close relationship has formed between the American and Canadian Poison Center (CAPPC) associations. Once a year the AAPCC and CAPPC hold a joint scientific meeting and invite speakers and other toxicology spe-

Poison Centers for Plant Toxin Exposure

11

cialist from throughout the world to attend. Some international affiliated organizations are listed with the North American groups above.

J. Toxicology and Poison Center Web Sites Association of Occupational and Environmental Clinics This group is dedicated to higher standards of patient-centered, multidisciplinary care emphasizing prevention and total health through information sharing, quality service, and collaborative research. Address: [email protected]~~.edu Directory of Mycologists List of U.S. and Latin American mycologists who may be available for consultation. Address: http://www.keil.ukans.edu/-fungi Directory for MycorrhizalandEdibleFungi Address: http:// www.mykopat. slu.se/mycorrhiza/edible/home.pht1nl FingerLakesRegionalPoisonCenter Address: [email protected] Latin American Mycologists A site for identifying some Latin American mycologists. Address: http://bragg.ivic.velvic/ALM/directorio/direct/html Medical/Clinical/Occupational Toxicology Professional Groups A list of primarily U.S. professional groups interested in toxicology. There is a description of each group, its address, phone numbers, and contact names. Keyword: poison centers, toxicology. Address: http://www.pitt.edu/-martint/pages/motoxorg.htm Poison Net A mailing list dedicated to sharing information, problem solving, and networking in the areas of poisoning, poison control centers, hazardous materials, and related topics. The list is intended for health care professionals, not the lay public. The moderators do not encourage responses to individual poisoning cases from the public: Key word(s): poisoning, poison control centers K. NorthAmericanMycologicalAssociation In 1984 Ken Cochran started the Mushroom Poisoning Case Registry for the North American Mycological Association. This was kept at the University of Michigan until 1988, then was transferred toKen Lampe at the American Medical Association in Chicago. Since his death in 1990, John Trestrail TI1 has kept the records at the Blodgett Regional Poison Center in Grand Rapids, Michigan (1). Reports to the registry are made on a standard form, which is available free of charge or may be photocopied. The reporting is voluntary, and most often comes from physicians,

Table 3 NAMA MushroomExposures ~

~~

# of exposures

110

% nonhuman

# of genera

% unk. genera

6

26

15.5

Patient agea Under 6

6 13 to 12

10% ~~~

1%

to 17 0%

18 to 49

50 to 69

Over 70

37%

13%

6%

~~

These reports represent a different age distribution than seen in the AAPCC cases, where 80.5% are under 6 years of age.

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poison centers, and mushroom club representatives. Because reporting is voluntary, it is also irregular. The number of cases recorded by NAMA is smaller than that reported by AAPCC, and they are not necessarily the same cases. Intepretation of these data and comparison to the AAPCC data is difficult, if not impossible. Other shortcomings of this database are that no species identifications are questioned, nor are any symptoms eliminated or evaluated based on the type of mushroom exposure. It is possible that symptoms reported could be due to coincidental illness, infection, or environmental conditions. Still, it is an attempt at gathering information on adverse reactions involving mushrooms. Table 3 is based on reports from 21 U.S. states or Canadian providences, but two ,states (Colorado and Oregon) represented 43% of the cases. This is thought to be due to more diligent reporting, rather than actual higher incidence of poisonings. These cases represent exposures reported in 1989 and 1990.

II. POISON INFORMATION CENTERS IN THE UNITED STATES The Poison Control Center telephone numbers and addresses listed below are thought to be accurate as of the date of publication. Poison Control Center telephone numbers or addresses may change. The address and phone number of the Poison Control Center nearest you should be frequently checked. If the number listed does not reach the poison center, contact the nearest emergency service, such as 9 11 or local hospital emergency rooms. The author disclaims any liability resulting from or relating to any inaccuracies or changes in the phone numbers provided below.

ALABAMA

ALASKA

Birmingham Regional Poison Control Center* Children’s Hospital of Alabama 1600 Seventh Avenue, South Birmingham, AL 35233-171 1 (800) 292-6678 (AL only) (205) 933-4050

Anchorage Anchorage Poison Center Providence Hospital P.O. Box 196604 3200 Providence Drive Anchorage, AK 99519-6604 (800) 478-3193 (AK only)

Tuscnloosa Alabama Poison Control System, Inc. 408 A Paul Bryant Drive, East Tuscaloosa, AL 35401 (800) 462-0800 (AL only) (205) 345-0600

Fairbanks Fairbanks Poison Center Fairbanks Memorial Hospital 1650 Cowles St. Fairbanks, AK 99701 (907) 456-7 182

* Indicates a Regional Center designated by the American Association of Poison Control Centers.

13

Poison Centers for Plant Toxin Exposure

ARIZONA Phoenix Samaritan Regional Poison Center* Good Samaritan Medical Center 1130 East McDowell Road, Suite A-5 Phoenix, AZ 85006 (602) 253-3334 Tucsorl Arizona Poison and Drug Information Center" Arizona Health Sciences Center, Room 1156 1501 N. Campbell Ave Tucson, A2 85724 (800) 362-0101 ( A Z only) (602) 626-6016

ARKANSAS Little Rock Arkansas Poison and Drug Information Center University of Arkansas College of Pharmacy 4301 West Markham, Slot 522 Little Rock, AR 77205 (800) 482-8948 (AR only) (501) 661-6161

CALIFORNIA Fresno Fresno Regional Poison Control Center* Fresno Community Hospital & Medical Center 2823 Fresno Street Fresno, CA 93721 (800) 346-5922 (CA only) (209) 445- 1222

Los Artgeles Los Angeles County University of Southern California Regional Poison Center* 1200 North State, Room 1107 Los Angeles, CA 90033

(800) 825-2722 (213) 222-2312 Orange University of California Irvine Medical Center Regional Poison Center" 101 The City Drive, South Route 78 Orange, CA 92668-3298 (800) 544-4404 (CA only) (714) 634-5988 Richmond Chevron Emergency Information Center 15299 San Pablo Avenue P.O. Box 4054 Richmond, CA 94804-0054 (800) 457-2202 (510) 233-3737 or 3738 Sacramento Regional Poison Control Center": University of California at Davis Medical Center 2315 Stockton Boulevard Rm HSF-124 Sacrmento, CA 95817 (800) 342-3293 (northern CA only) (916) 734-3692 Sun Diego San Diego Regional Poison Center* University of California at San Diego Medical Center 225 West Dickinson Street San Diego, CA 92013-8925 (800) 876-4766 (CA only) (619) 543-6000 Sun Francisco San Francisco Bay Area Poison Center* San Francisco General Hospital 1001 Potrero Avenue Rm 1E86 San Francisco, CA 94122 (800) 523-2222 (4 15) 476-6600 San Jose Regional Poison Center Santa Clara Valley Medical Center 751 South Bascom Avenue San Jose, CA 95 128 (800) 662-9886, 9887 (CA only) (408) 299-5112, 5113, 5114

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COLORADO DerzverRocky Mountain Poison Center* 8802 Ninth Avenue Denver, CO 80220-6800 (800) 332-3073 (CO only) (303) 629-1123

CONNECTICUT Farnzington Connecticut Poison Control Center University of Connecticut Health Center 263 Farmington Avenue Farmington, CT 06030 (800) 343-2722 (CT only) (203) 679-3456

DELAWARE Wilmington Poison Information Center Medical Center of Delaware Wilmington Hospital 501 West 14th Street Wilmington, DE 19899 (302) 655-3389

DISTRICT OF COLUMBIA Wmhington National Capital Poison Center* Georgetown University Hospital 3800 Reservoir Road, North West Washington, DC 20007 (202) 625-3333

Tallahassee Tallahassee Memorial Regional Medical Center 1300 MiccosukEr Road Tallahassee, FL 32308 (904) 681-541 1 Tampa Tampa Poison Information Center* Tampa General Hospital Davis Islands P.O. Box 1289 Tampa, FL 33601 (800) 282-3171 (FL only) (813) 253-4444

GEORGIA Atlanta Georgia Regional Poison Control Center* Grady Memorial Hospital 80 Butler Street South East Box 26066 Atlanta, GA 30335-3801 (800) 282-5846 (GA only) (404) 6 16-9000

Macoil Regional Poison Control Center Medical Center of Central Georgia 777 Hemlock Street Macon, GA 31208 (912) 744-1146. 1100 or 1427 Sul~aililah Savannah Regional Poison Control Center Memorial Medical Center Inc. 4700 Waters Avenue Savannah, GA 31403 (912) 355-5228 or 356-5228

HAWAII FLORIDA Jacksonville Florida Poison Information Center University Medical Center 655 West Eighth Street Jacksonville, FL 32209 (904) 549-4465 or 764-7667

Honolulu Kapiolani Women's and Children's Medical Center 1319 Punahou Street Honolulu, HI 96826 (800) 362-3585, 3586 (HI only) (808) 941-4411

Poison Centers for Plant Toxin Exposure

IDAHO Boise Idaho Poison Center St. Alphonsus Regional Medical Center 1055 North Curtis Road Boise, ID 83706 (800) 632-8000 (ID only) (208) 378-2707

ILLINOIS Chicago Chicago and NE Tllinois Regional Poison Control Center Rush Presbyterian-St. Luke’s Medical Center 1653 West Congress Parkway Chicago, IL 60612 (800) 942-5969 (Northeast IL only) (312) 942-5969 Normal Bromenn Hospital Poison Center Virginia at Franklin Normal, IL 61761 (309) 454-6666

SprinRfield Central and Southern Illinois Poison Resource Center St. John’s Hospital 800 East Carpenter Street Springfield, IL 62769 (800) 252-2022 (IL only) (217) 753-3330 Urbancl National Animal Poison Control Center University of Illinois Department of Veterinary Biosciences 2001 South Lincoln Avenue, 1220 VMBSB Urbana, IL 61801 (800) 548-2423 (Subscribers only) (217) 333-2053

INDIANA Indianapolis Indiana Poison Center* Methodist Hospital

1701 North Senate Boulevard Indianapolis, IN 46202-1367 (800) 382-9097 (317) 929-2323

IOWA Des Moirles Variety Club Drug and Poison Information Center Iowa Methodist Medical Center 1200 Pleasant Street Des Moines, IA 50309 (800) 362-2327 ( 515 ) 24 1-6254

Iowa City University of Iowa Hospitals and Clinics 200 Hawkins Drive Iowa City, IA 52246 (800) 272-6477 or (800) 362-2327 (TA only) (319) 356-2922

Sioux C i v St. Luke’s Poison Center St. Luke’s Regional Medical Center 2720 Stone Park Boulevard Sioux City, IA 51 104 (800) 352-2222 (TA, NE, SD) (7 12) 277-2222

KANSAS Kunsas City Mid America Poison Center Kansas University Medical Center 39th and Rainbow Boulevard Room B-400 Kansas City, KS 66160-7231 (800) 332-6633 (KS only) (9 13) 588-6633 Topeka Stormont Vail Regional Medical Center Emergency Department 1500 West 10th Topeka, KS 66604 (9 13) 354-6100

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Wichita Wesley Medical Center 550 North Hillside Avenue Wichita, KS 67214 (3 16)688-2222

Portland, ME 04102 (800) 442-6305 (ME only) (207) 87 1-2950

MARYLAND KENTUCKY St. Thomas Northern Kentucky Poison Information Center St. Luke Hospital 85 North Grand Avenue Ft. Thomas, KY 41075 (513) 872-5111

Louisville Kentucky Poison Control Center of Kosair Children’s Hospital 3 15 East Broadway P.O. Box 35070 Louisville, KY 40232 (800) 722-5725 (KY only) (502) 589-8222

LOUISIANA Houma Terrebonne General Medical Center Drug and Poison Information Center 936 East Main Street Houma, LA 70360 (504) 873-4069 Monroe Louisiana Drug and Poison Information Center Northeast Louisiana University School of Pharmacy, Sugar Hall Monroe. LA 7 1209-6430 (800) 256-9822 (LA only) (318) 362-5393

MAINE Portland Maine Poison Control Center Maine Medical Center 22 Bramhall Street

Baltimore Maryland Poison Center* University of Maryland School of Pharmacy 20 North Pine Street Baltimore, MD 21201 (800) 492-2414 (MD only) (410) 528-7701

MASSACHUSETTS Bostorl Massachusetts Poison Control System* The Children’s Hospital 300 Longwood Avenue Boston, MA 02115 (800) 682-9211 (MA only) (617) 232-2120 or 735-6607

MICHIGAN Adriarl Bixby Hospital Poison Center Emma L. Bixby Hospital 818 Riverside Avenue Adrian, MI 49221 (517) 263-2412 Detroit Poison Control Center Children’s Hospital of Michigan 3901 Beaubien Boulevard Detroit, MI 48201 Outside metropolitan Detroit; (800) 4626642 (MI only) (3 13) 745-57 1 1 Grand Rapids Blodgett Regional Poison Center 1840 Wealthy Street, South East Grand Rapids, MI 49506 Within MI: (800) 632-2727

Poison Centers for Plant Toxin Exposure Kalamazoo Bronson Poison Information Center 252 East Love11 Street Kalamazoo, MI 49007 (800) 442-4112 616 (MI only) (6 16)34 1-6409

MINNESOTA Minneapolis Hennepin Regional Poison Center” 701 Park Avenue South Minneapolis, MN 55415 (612) 347-3144 (612) 347-3141 (Petline) St. Paul Minnesotal Regional Poison Center* St. Paul-Ramsey Medical Center 640 Jackson Street St. Paul, MN 55101 (800) 222- 1222 (MN only) (612) 221-21 13

17 1465 South Grand Boulevard St. Louis, MO 63104 (800) 392-91 11 (MO only) (800) 366-8888 (MO, West IL) (314) 772-5200

MONTANA Denver Rocky Mountain Poison and Drug Center 645 Bannock St. Denver, CO 80204 (800) 525-5042 (MT only)

NEBRASKA Onmlza The Poison Center* Children’s Memorial Hospital 8301 Dodge Street Omaha, NE 681 14 (800) 955-9119 (WY, NE) (402) 390-5400, 5555

MISSISSIPPI Jackson University of Mississippi Medical Center 2500 North State Street Jackson, MS 39216 (601) 354-7660 Hattiesburg Forrest General Hospital 400 S. 28th Avenue Hattiesburg, MS 39402 (601) 288-4235

MISSOURI Kansas City Poison Control Center Children’s Mercy Hospital 2401 Gillham Road Kansas City, MO 64108-9898 (816) 234-3000 or 234-3430 St. Louis Regional Poison Center* Cardinal Glennon Children’s Hospital

NEVADA

L m Vegas Humana Hospital-Sunrise* 3 186Maryland Parkway Las Vegas, NV 89109 (800) 446-6179 (NV only) Reno Washoe Medical Center 77 Pringle Way Reno, NV 89520 (702) 328-4144

NEW HAMPSHIRE Lebanon New Hampshire Poison Center Dartmouth-Hitchcock Medical Center 1 Medical Center Drive Lebanon, NH 03756 (800) 562-8236 (NH only) (603) 650-5000

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NEW JERSEY Newark New Jersey Poison Information and Education Systems* 201 Lyons Avenue Newark, NJ 07 112 (800) 962-1253 (NJ only) (201) 923-0764 Phillipsburg Warren Hospital Poison Control Center 185 Rosberg Street Phillipsburg, NJ 08865 (800) 962-1253 (908) 859-6768

NEW MEXICO Albuquerque New Mexico Poison and Drug Information Center” University of New Mexico Albuquerque, NM 87 131 (800) 432-6866 (NM only) (505) 843-255 1

NEW YORK BtrfSalo Western New York Poison Control Center Children’s Hospital of Buffalo 219 Bryant Street Buffalo, NY 14222 (800) 888-7655 (NY only) (716) 878-7654 Mineola Long Island Regional Poison Control Center* Winthrop University Hospital 259 First Street Mineola, NY 11501 (516) 542-2323, 2324, 2325 New York Cily New York City Poison Control Center* 455 First Avenue, Room 123 New York, NY 10016 (212) 340-4494 (2 12) 764-7667

Nyack Hudson Valley Regional Poison Center Nyack Hospital 160 North Midland Avenue Nyack. NY 10920 (800) 336-6997 (NY only) (914) 353-1000 Rochester Finger Lakes Regional Poison Control Center University of Rochester Medical Center 601 Elmwood Avenue Rochester, NY 14642 (800) 333-0543, (NY only) (716) 275-5151 Syracuse Central New York Poison Control Center SUNY Health Science Center 750 E Adams Street Syracuse, NY 13210 (800) 252-5655 (3 15) 476-4766

NORTH CAROLINA Asheville Western North Carolina Poison Control Center Memorial Mission Hospital 509 Biltmore Avenue Asheville, NC 28801 (800) 542-4225 (NC only) (704) 255-4490 or 258-9907 Charlotte Carolinas Poison Center Carolinas Medical Center 100 Blythe Boulevard Charlotte, NC 28232-2861 (800) 848-6946 (704) 355-4000 Durham Duke Regional Poison Control Center P.O. Box 3007 Durham, NC 27710 (800) 672-1697 (NC only) (919) 684-8111 Greensboro Triad Poison Center Moses H. Cone Memorial Hospital

19

Poison Centers for Plant Toxin Exposure 1200 North Elm Street Greensboro, NC 27401-1020 (800) 953-400 I (NC only) (919) 574-8105 Hickory Catawba Memorial Hospital Poison Control Center 810 Fairgrove Church Road, South East Hickory, NC 28602 (704) 322-6649

NORTH DAKOTA Fnrgo North Dakota Poison Center St. Luke’s Hospital 720 North 4th Street Fargo, ND 58122 (800) 732-2200 (ND only) (701 ) 234-5575

OHIO Akron Akron Regional Poison Center 281 Locust Street Akron, OH 44308 (800) 362-9922 (OH only) (216) 379-8562 Canto11 Stark County Poison Control Center Timken Mercy Medical Center 1320 Timken Mercy Drive, North West Canton, OH 44667 (800) 722-8662 (OH only) (216) 489-1304 Cincinnati South West Ohio Regional Poison Control System and Cincinnati Drug and Poison Infornlation Center* University of Cincinnati College of Medicine 231 Bethesda Avenue ML #144 Cincinnati, OH 45267-0144 (800) 872-51 11 (Southwest OH only) (513) 558-5111

Cleveland Greater Cleveland Poison Control Center 2074 Abington Road Cleveland, OH 44106 (216) 231-4455

Columbus Central Ohio Poison Center* 700 Children’s Drive Columbus, OH 43205 (800) 682-7625 (OH only) (6 14)228- 1323 Dayton West Ohio Regional Poison And Drug Information Center Children’s Medical Center One Children’s Plaza Dayton, OH 45404- 18 15 (800) 762-0727 (OH only) (5 13) 222-2227

Lorain County Poison Control Center Lorain Community Hospital 3700 Kolbe Road Lorain, OH 44053 (800) 821-8972 (OH only) (216) 282-2220 Snndusky

Firelands Community Hospital Poison Information Center 1101 Decatur Street Sandusky, OH 44870 (4 19) 626-7423

Toledo Poison Information Center of Northwest Ohio Medical College of Ohio Hospital 3000 Arlington Avenue Toledo, OH 49614 (800) 589-3897 (OH only) (419) 381-3897 YouilgstoM’n Mahoning Valley Poison Center St. Elizabeth Hospital Medical Center 1044 Belmont Avenue Youngstown, OH 44501 (800) 426-2348 (OH only) (216) 746-2222

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Zclnesville Bethesda Poison Control Center Bethesda Hospital 2951 Maple Ave Zanesville, OH 43701 (800) 686-4221 (OH only) (6 14) 454-422 1

OKLAHOMA Oklahoma Cit?, Oklahoma Poison Control Center Children's Memorial Hospital 940 Northeast 13th Street Oklahoma City, OK 73104 (800) 522-4611 (OK only) (405) 27 1-5454

OREGON Portland Oregon Poison Center Oregon Health Sciences University 3181 South West Sam Jackson Park Road Portland, OR 97201 (800) 452-7165 (OR only) (503) 494-8968

PENNSYLVANIA Hershey Central Pennsylvania Poison Center* Milton Hershey Medical Center Pennsylvania State University P.O. Box 850 Hershey, PA 17033 (800) 521-61 10 (717) 531-6111 Lancclster Poison Control Center St. Joseph Hospital and Health Care Center 250 College Avenue Lancaster, PA 17604 (717) 299-4546 Philadelphia Philadelphia Poison Control Center* One Children's Center

34th and Civic Center Boulevard Philadelphia, PA 19104 (215) 386-2100 Pittsburgh Pittsburgh Poison Center" One Children's Place 3705 Fifth Avenue at DeSoto Street Pittsburgh, PA 15213 (412) 68 1-6669 Williclnlsport The Williamsport Hospital Poison Control Center 777 Rural Avenue Williamsport, PA 17701 (717) 321-2000

RHODE ISLAND Providence Rhode Island Poison Center" 593 Eddy Street Providence, RI 02903 (401) 444-5727

SOUTH CAROLINA Charlotte Carolinas Poison Center Carolinas Medical Center 1000 Blythe Boulevard Charlotte, NC 28232-2861 (800) 848-6946 Col~rmbia Palmetto Poison Center University of South Carolina College of Pharmacy Columbia, SC 29208 (800) 922-1 117 (SC only) (803) 765-7359

SOUTH DAKOTA Aberdeen Poison Control Center St. Luke's Midland Regional Medical Center 305 S. State Street Aberdeen, SD 57401

21

Poison Centers for Plant Toxin Exposure (800) 592-1889 (SD, MN, ND, WY) (605) 622-5678 Rapid City Rapid City Regional Poison Control Center 835 Fairmont Boulevard P.O. Box 6000 Rapid City, SD 57709 (605) 341-3333 Sioux Falls McKennan Poison Center McKennan Hospital 800 East 21st Street P.O. Box 5045 Sioux Falls, SD 571 17-5045 (800) 952-0123 (SD only) (800) 843-0505 (IA, MN, NE) (605) 336-3894

TENNESSEE Kno.uville Knoxville Poison Control Center University of Tennessee Memorial Research Center and Hospital 1924 Alcoa Highway Knoxville, TN 37920 (6 15) 544-9400 Memphis Southern Poison Center, Inc. Lebanheur Children’s Medical Center 848 Adams Avenue Memphis, TN 38103-2821 (901) 528-6048 Nushville Middle Tennessee Regional Poison Center, Inc . 501 Oxford House 1161 21st Avenue South B-101VUII Nashville, TN 37232-4632 (800) 288-9999 (TN only) (615) 322-6435

TEXAS

Medical Center Hospital 504 Medical Center Blvd. Conroe, TX 77304 (409) 539-7700 Drrllas North Central Texas Poison Center* Parkland Memorial Hospital 5201 Harry Hines Boulevard P.O. Box 35926 Dallas, TX 75235 (800) 441-0040 (TX only) (214) 590-5000 El Paso El Paso Poison Control Center Thomas General Hospital 4815 Alameda Avenue El Paso, TX 79905 (915) 533-1244 Galveston Texas State Poison Control Center University of Texas Medical Branch 8th and Mechanic Street Galveston, TX 77550-2780 (800) 392-8548 (TX only) (713) 654-1701 (Houston) (409) 765-1420 (Galveston) Lubbock Methodist Hospital Poison Control 3615 19th Street Lubbock, TX 79413 (806) 793-4366

UTAH Salt Like City Utah Poison Control Center* 410 Chipeta Way, Suite 230 Salt Lake City, UT 84108 (800) 456-7707 (UT only) (801) 581-2151

VERMONT

Conroe Burlington Vermont Poison Center Montgomery County Poison Information Center Hospital CenterMedical of Vermont

Spoerke

22 11 1 Colchester Avenue Burlington, VT 05401 (802) 658-3456

Parkersburg St. Joseph’s Hospital Center 19th Street and Murdoch Avenue Parkersburg, WV 26 101 (304) 424-4222

VIRGINIA Clzarlotteslille Blue Ridge Poison Center* University of Virginia Health Science Center Box 67 Charlottesville, VA 22901 (800) 45 1-1428 (VA only) (804) 924-5543

Richmond Virginia Poison Center Virginia Commonwealth University MCV Station Box 522 Richmond, VA 23298-0522 (800) 5526337 (VA only) (804) 786-9123

WASHINGTON Seattle Washington Poison Center 155 NE 100th Street, Suite #400 Seattle, WA 98105-8012 Within WA: (800) 732-6985 (206) 526-2121

WEST VIRGINIA Charleston West Virginia Poison Center* 31 10 MacCorkal Avenue S.E. Charleston, WV 25304 (304) 348-4211 (800) 642-3625 (WV only)

WISCONSIN Madison Regional Poison Control Center University of Wisconsin Hospital 600 Highland Avenue Madison, Wi 53792 (608) 262-3702 Milwaukee Poison Center of Eastern Wisconsin Children’s Hospital of Wisconsin 9000 West Wisconsin Avenue P.O. Box 1997 Milwaukee, WI 53201 (4 14) 266-2222

WYOMING Onraha. Nebraska The Poison Center c/o Mid-Plains Poison Center* Children’s Memorial Hospital 8301 Dodge Street Omaha, NE 68 114 (402) 390-5555 (800) 955-9119 (NE, ID, IA, KS, MO, SD)

Poison Centers for Plant Toxin Exposure

111.

23

NATIONAL AND INTERNATIONALMYCOLOGICAL ASSOCIATIONS/CLUBS/ORGANlZATlONS

ARGENTINA Asociacion Argentina de Micologia 1141 Parque San Francisco CP 5010 Cordoba, Argentina Instituto de Botanica C. Spegazzini Facultad de Ciencias Naturales y Museo de la Plata Calle 53 No 477 1900 La Plata, Buenos Aires, Argentina Universidad de Buenos Aires Laboratorio de Micologia Depart de Ciencias Biologicas Facultad de Ciencias Exactas y Naturales I1 Pabellon. 4 piso Ciudad Universitaria (Nunez) 1428 Buenos Aires, Argentina

Australian National Reference Laboratory in Medical Mycology The Royal North Shore Hospital of Sydney St. Leonards New South Wales 2065, Australia Plant Pathology Branch Herbarium New South Wales New South Wales Agriculture Biological and Chemical Research Institute Private Mailbag No. 10 Rydalmere New South Wales 2116, Australia Women’s and Children’s Hospital Mycology Laboratory Mycology Unit North Adelaide 5006, Australia

AUSTRIA ARMENIA Botanical Institute of the Academy of Sciences of Armenia 375063 Yerevan 63 Armenia

AUSTRALIA Australian Federation for Medical and Veterinary Mycology Mycology Laboratory Royal North Shore Hospital St Leonards New South Wales, 2065 Australia Australian Mushroom Growers Association PTY PO Box 265 Windsor, New South Wales, NSW 2756 Australia Australian Mycological Society Inc. Australian Biological Resources Study GPO Box 636 Canberra 2601, Australia

Osterreichische Mykologische Gesellschaft Institut fur Botanik Universitat Wien Rennweg 14 A-1300 Wein, Austria Verein fur Pilzkunde Tirol AchenseestrarSe 21 A 6200 Jenbach Tirol. Austria

BELGIUM Antwerpse Mykologische Kring Alfons Schneiderlaans 126 2100 Deurne Antwerpen, Belgium Institute of Hygiene and Epidemiology Mycology Section Rue Juliette Wytstnan 14 B-1050 Bruxelles, Belgium Mycotheque de L’Universite Catholique de Louvain Place Croix du Sud 3

Spoerke te6

B B-1348 Louvain-la-Neuve, Belgium National Garden of Belgium Domein van Bouchout B- 1860 Meise, Belgium

BRAZIL Instituto de Botanica Secao de Micologia i Liquenologia Caixa Postal 4.005 Sao Paulo, SP 01061-970 Brazil Unversidade Federal de Pernambuco Departmento de Micologia Centro de Ciencias Biologicas Cidade Universitaria 50670-420 Recife Pernambuco, Brazil

BRITAIN British Lichen Society Department of Botany The Natural History Museum Cromwell Road London, SW7 5BD, United Kingdom British Mycological Society PO Box 30 Stourbridge. West Midlands DY9 9PZ, United Kingdom International Mycological Institute Bakeham Lane Egham, Surrey TW209TY, United Kingdom

CANADA Alberta Department of Botany University of Toronto Toronto, Ontario, Canada M5S 1Al Edmonton Mycological Club 6003 109 B Ave Edmonton, Alberta, Canada T6A 1S7 University of Alberta Microfungus Collection and Herbarium Devonian Botanic Garden Edmonton, Alberta, Canada TG6 2E1 British CoIzmbin Department of Botany and Biology University of British Columbia Vancouver, BC, Canada V6T 2B1

Vancouver Mycological Society 403 Third Street New Westminster, BC V3L 2S1 Quebec Chibougamau Mycological Club 804 5e Rue Chibougamau, PQ, Canada G8P 1V4 Les Cercle des Mycologues de Montreal 4101 Rue Sherbrooke, Est. 125 Montreal, PQ, Canada HlX2B2 Le Cercle des Mycologues de Quebec Pavillon Comtois Universitaire de Lava1 Ste-Foy, PQ, Canada G1K 7P4 Les Cercle des Mycologues de Rimouski University of Quebec, Rimouski Rinlouski. PQ, Canada Les Cercle des Mycologues de Saguenay 438 Rue Perrault Chicoutimi, PQ, Canada G7J3Y9

Mycology Reference Laboratory Public Health Laboratory Myrtle Road Kingsdown, Bristol, BS2 8EL United Kingdom

Manitoba Department of Botany University of Manitoba Winnipeg, Manitoba, Canada

Royal Botanic Gardens The Herbarium Kew, Richmond Surrey, TW9 3AE, United Kingdom

Nova Scotia Acadia University Biology Department Wolfville, Nova Scotia, Canada BOP 1 x 0

25

Poison Centers for Plant Toxin Exposure Atlantic Regional Laboratory National Research Council 1411 Oxford Street Halifax, Nova Scotia Canada B3H3Z1 Ontario Canadian National Mycological Herbarium Centre for Land and Biological Resources Research William Saunders Building Agriculture Canada Ottawa, Ontario KIAOC6

Cultivated Mushroom Report University of Toronto Mississauga, Ontario Mycological Society of America Department of Botany University of Toronto, Erindale Campus Mississauga, Ontario, Canada L5LlC6 Mycological Society of Toronto 2 Deepwood Crescent North York, Ontario, Canada M3ClN8 Royal Ontario Museum Cryptogamic Herbarium c/o Department of Botany University of Toronto 25 Willcocks Street Toronto, Canada, M5S 3B2

CHINA (PEOPLE’S REPUBLIC) Academia Sinica Institute of Microbiology Zhong Guan Cun Hai Dian Beijing 100080 People’s Republic of China World Society for Mushroom Biology and Mushroom Products c/o Department of Biology The Chinese University of Hong Kong Shatin, New Territories, Hong Kong Mycological Society of the Republic of China Mycology Laboratory Department of Botany

National Taiwan University Roosevelt Road Section 4, No. 1 Taipei, Taiwan 10764. Republic of China Veterans General Hospital-Taipei Mycosis Research Laboratory Room 533, Medical Research Building c/o Departments of Pathology and Dermatology Shih-pai, Taipei City 11217 Taipei, Taiwan, Republic of China

COSTA RlCA University of Costa Rica School of Biology Escuela de Biologia Facultad de Ciencias Universidad de Costa Rica Cuidad Universitaria ‘Rodrigo Facio’ 2050 San Pedro de M. de Oca San Jose, Costa Rica

CUBA Asociacion Latinoamericana de Micologia Jardin Botanic0 Nacional Carretera del Rocio km 3.5 Calabazar, Boyeros CP 19230 Ciudad La Habana, Cuba

CZECH REPUBLIC Culture Collection of Basidiomycetes Laboratory of Biochemistry of WoodRotting Fungi Institute of Microbiology Academy of Sciences of the Czech Republic, Videnska 1083 142 20 Praha 4 Krc, Czech Republic Czech National Collection of Type Cultures National Institute of Public Health Srobarova 48 100 42 Praha 10 Czech Republic

Spoerke

26 Czech Scientific Society for Mycology PO Box 106 CZ-111 21 Praha 1 Czech Republic Itest Brozikova 451 Hradec Kralove 50012 Czech Republic

DENMARK Foreningen Til Svampekundskabens Fremme PO Box 102 DK-2860 Soborg, Denmark

ESTONIA Estonian Academy of Sciences Institute of Zoology and Botany/Tartu University Laboratory of Mycology 21 Vanemuise Street 202400 Tartu, Estonia Estonian Naturalists' Society Mycology Section Kompanii Street 3 EE2400 Tartu, Estonia

FINLAND Finnish Mycological Society Societas Mycologica Fennica Unioninkatu 44 SF-00170 Helsinki, Finland

FRANCE Association d'Ecologie et de Mycologie Laboratorie de Systematique et d'Ecologie Vegetale U.E.R. Pharmacie Rue Laguesse 59045 Little Cedex, France

Association Francaise de Lichenologie Laboratoire de Cryptogamie Universite Pierre et Marie Curie 7 quai Saint-Bernard 75230 Paris Cedex 05. France Museum National D'Historire Naturelle Laboratoire de Cryptogamie 12, rue Buffon 75005 Paris, France Observatoire Mycologique Neronde 7 1250 Mazille, France Societe Francaise de Mycologie Medicale Institut Pasteur 25 rue du Docteur Roux 75724 Paris, Cedex, France Societe Mycologique de France 18, rue de 1'Ermitage 75010 Paris, France

GERMANY Arbeitskreis Mykologie Deutsche Phytomedizinische Gesellschaft Technische Universitat Munchen Lehrstuhl fur Phytopathologie 85350 Freising-Weihenstephan, Gemany Bayerische Landesanstalt Fur Weinbau und Gartenbau Residenzplatz 3 D-97070 Wurzburg, Germany Botanischer Garten und Botanisches Museum Berlin-Dahlem Konigin-Luise Strass 6-8 D-14191 Berlin. Gernlany Deutsche Gesellschafr Fur Mykologie E.V. Rathausstrasse 16 D-78594 Gunnigen, Germany Deutschsprachige Mykologische Gesellschaft Mykologische Laboratorium Univ. Hautklinik Martinistrasse 52 D-2000 Hamburg 20, Germany

Poison Centers for Plant Toxin Exposure Gesellschaft Fur Natur und Umwelt Fachhaus Mykologie Abteilung Natur and Umwelt Postfach 34 1030 Berlin, Germany Institut fur Pflanzenschutz im Forst Biologische Bundesanstalt fur Land-und Forstwirtschaft Messeweg 11/ 12 38104 Braunschweig, Germany International Society for Human and Animal Mycology Brandelweg 24 D-793 12 Emmendingen, Germany International Society for Mushroom Science Institut fur Bodenbiologie Bundesfurschungsanstalt fur Landwirtschaft D-3300 Braunschweig Bundesalle 50, West Germany

GREECE University of Athens Culture Collections of Fungi Department of Biology Section of Ecology and Systematics Panepistinliopolis GR- 157 84 Athens, Greece

GUAM College of Agriculture and Life Sciences V 0 6 Station, Mangilao, Guam 96913 734-292 1x376

27

HUNGARY Hungarian Mycological Society Department of Botany University of Horticulture and Food Industry H- 118 Budapest Menesi ut 44, Hungary

ICELAND Akureyri Museum of Natural History PO Box 180 IS-602 Akureyri, Iceland

INDIA Banaras Hindu University Department of Mycology and Plant Pathology Herbarium Faculty of Agriculture Varanasi-221005, India Indian Mushroom Grower’s Association Indian Research Laboratory College of Agriculture Solon, Himachal Pradesh India International Journal of Research and Development National Centre for Mushroom Research and Training Chambaghat, Solan 173213 (HP) India Mycological Society of India Centre for Advanced Study in Botany University of Madras Madras 600 025. India

HONG KONG Mushroonl Journal of the Tropics The International Mushroom Society for the Tropics c/o Department of Botany Chinese University of Hong Kong Shatin, New Territories, Hong Kong

ITALY Associazione Micologica Ecologica Romana Piazza C. Finocchario Aprile 3 1-00081 Roma, Italy

Spoerke

28 La Rivista del Fungicoltore Modern0 40016 South Giorgio di Plano (BO) Postale Grupo IIII70 Bologna, Italy Universita Degli Studi Di Palermo Dipartimento di Scienze Botaniche Via Archirafi 38 1-90 123 Palmer0 Sicily, Italy

MEXICO College of Postgraduates Laboratory of Edible Mushroom Production Colegio de Postgraduados Apartado Postal 701 Puebla 72001 Puebla, Mexico Sociedad Mexicana de Micologia Apartado Postal 2-378 Mexico D.F. Mexico CP 02860, Mexico

JAPAN Japanese Association for Mycotoxicology Science University of Tokyo 12 Ichigaya Funagawara-Machi Shinjuku-Ku, Tokyo 162 Japan Mycological Society of Japan c/o Business Center for Academic Societies. Japan 4-16, Yayoi 2-chonle Bunkyo-ku, Tokyo 113, Japan Tottori Mycological Institute The Japan Kinoko Research Centre Foundation Kokoge 211, Tottori 689-11, Japan

KlRGHlZlSTAN National Academy of Sciences of Grghizistan Biological Institute Herbarium XXII Partesda Street 265 720071 Frunze, Kirghizistan

KOREA Korean Society of Mycology Department of Agrobiology College of Agriculture Dongguk University Seoul 100-715 Republic of Korea

NEPAL Department of Forests Forest Research and Infornlation Centre Babar Mahal PO Box 106 Kathmandu, Nepal

NETHERLANDS Centraalbureau Voor Schimmelcultures Oosterstraat 1 Post Office Box 273 3740 AG Baarn. Netherlands Centre for Soil Ecology: Biological Station Kampsweg 27 9418 PD Wijster, Netherlands International Association for Plant Taxonomy Nomenclature Cornnlittee for Fungi Centraalbureau voor Schimmelcultures PO Box 213 3740 AG Baarn, Netherlands Netherlands Mycological Society Nederlandse Mycologische Vereniging Biological Station Center for Soil Ecology Kampsweg 27 9418 PD Wijster, Netherlands Onderzoekinstituut RijksherbariuIdHortus Botanicus Department of Mycology PO Box 9514 2300 RA Leiden, Netherlands

29

Poison Centers for Plant Toxin Exposure

ZEALAND

NEW Victoria University Mycology Group School of Biological Sciences PO Box 600 Wellington, New Zealand

NORWAY Fungiflora AIS PO Box 95 Blindern, N-03 14 Research All-Russia Norway Oslo. Mycological Society of Fredrikstad Fredrikstad Soppforening PO Box 167 N-1601 Fredrikstad, Norway Norsk Soppforening PO Box 282 N- 1301 Sandvika, Norway

PHILIPPINES University of the Philippines at Los Banos Mycological Herbarium UPLB Museum of Natural History College, Laguna, Philippines

POLAND Polish Botanical Society Mycological Section Polskie Towarzystwo Botaniczne Aleje Ujazdowslue 4 00-478 Warszawa, Poland

All-Russia Plant Protection Institute Unit of Microbiological Plant Protection Podbelskogo Shosse 3 St. Petersburg-Pushkin 8 189620, Russia All-Russia Plant Protection Institute Jaczewski’s Mycology & Plant Phytopathology Laboratory Podbelskogo Shosse 3 St. Petersburg-Pushkin 8 189620, Russia Institute for Agricultural Microbiology Culture Collection of Microorganisms Podbelskogo Shosse 3 St. Petersburg-Pushkin 8 189620, Russia Komarov Botanical Institute Culture Collection of Basidiomycetes Russian Academy of Sciences Prof. Popov Street 2 Saint Petersburg, 197376 Russia Kornarov Botanical Institute Mycological and Lichenological Herbaria Russian Academy of Sciences Prof. Popov Street 2 Saint Petersburg, 197376 Russia Russian Botanical Society Conmission for the Investigation and Application of Mushrooms Komarov Botanical Institute Russian Academy of Sciences Prof. Popov Street 2 St. Petersburg 197376, Russia

SCOTLAND ROMANIA Societatea Micologica din Romania Aleea M. Sadoveanu Nr. 3 R-6600-Iasi-6, Romania

Botanical Society of Scotland Royal Botanic Garden Endinburgh, EH3 5LR, Scotland, United Kingdom

Spoerke

30 Royal Botanic Garden Edinburgh Inverleith Row Edinburgh. EH3 5LR, Scotland, United Kingdom

SINGAPORE National University of Singapore Botany Department Lower Kent Ridge Road Singapore 0511 Republic of Singapore

SOUTH AFRICA

,

National Collection of Fungi of the Republic of South Africa Mycology Unit Plant Protection Research Institute Private X134 Bag Pretoria 0001, Republic of South Africa South African Society for Plant Pathology Fruit and Fruit Technology Research Institute Private Bag X5013 Stellenbosch 7600 Republic of South Africa University of Pretoria Department of Botany Pretoria 0002. Republic of South Africa

SPAIN Asociacion Espanola de Espe.cialistas en Micolotia Servei de Microbiologia Clinica Hospital de Mar Passeig Maritim 25-29 08003 Barcelona, Spain Societat Catalana de Micologia Catedra de Botanica Facultat de Farmacia Universitat de Barcelona

Avenida Diagonal 643 08038 Barcelona Catalunya, Spain Universidad de Alcala de Henares Departamento de Biologie Vegetal (Seccion Mycologia) Facultad de Ciencias-28871 Madrid, Spain

SWEDEN Goteborg Mycology Club Goteborgs Svampklubb Halltorpsgatan 14 S-461 41 Trollhattan, Sweden Swedish Mycological Society Sveriges Mykologiska Forening Swedish Museum of Natural History PO Box 50 007 S-10405 Stockholm, Sweden University of Uppsala Botanical Museum Villavagen 6 S-752 36 Uppsala, Sweden

SWITZERLAND Swiss Mycological Society Societe Mycologique Suisse Institute de Botanique Universite de Nsuchatel Chantemerle 22, CH-2000 Neuchatel, Switzerland International Society for Human and Animal Mycology Gellerstrasse 11A CH-4052 Basel, Switzerland

THAILAND Chuylalongkorn University: Mushroom Research Unit Department of Botany Bangkok, 10330 Thailand

Poison Centers for Plant Toxin Exposure UNITED STATES National North American Mycological Society 4245 Redinger Rd Portsmouth, OH 45662

United States Federation for Culture Collections Roche Molecular Systems 1145 Atlantic Ave Alameda, CA US National Fungus Collections Systematic Botany and Mycology Laboratory USDA-Agricultural Research Service BOllA Room 304 10300 Baltimore Ave Beltsville, MD 20705-2350 Wadsworth Center for Laboratories and Research Laboratories for Mycology New York State Department of Health The Governor Nelson A. Rockefeller Empire State Plaza PO Box 509 Albany. NY 12201-0509 Alaska Alaska Mycological Society Box 2526 Homer, AK 99603 Glacier Bay Mycological Society PO Box 65 Gustavus, AK 99826-0065 Arkamus Arkansas Mycological Society 55 15 S Main St Pine Bluff, AR 71601-7452 Ccllifom icr Fungus Federation of Santa Cruz 1305 East Cliff Dr (Museum) Santa Cruz, CA 95062

Humboldt Bay Mycological Society PO Box 4419 Arcata, CA 95521-1419

31 Los Angeles Mycological Society Biology Department 5151 State University Dr Los Angeles, CA 90032 Mendocino County Mycological Society PO Box 87 Philo, CA 95466-0087 Mount Shasta Mycological Society 623 Pony Trail Mount Shasta, CA 96067 Mycological Society of San Francisco PO Box 11321 San Francisco, CA 94101-7321 Colorado Colorado Mycological Society PO Box 9621 Denver, CO 80209-0621 Denver Botanic Gardens Herbarium of Fungi 900 York St Denver, CO 80206 Pikes Peak Mycological Society PO Box 1961 Colorado Springs, CO80901- 196 1 Connecticut Connecticut Agricultural Experimental Station 123 Huntington St Box 1106 New Haven, CT 06504 Connecticut Valley Mycological Society 21 Johnson St Maugatuck, CT 06770 Nutmeg Mycological Society PO Box 530 Groton, CT 06340-0530 Georgia Centers for Disease Control Infectious Disease Section Atlanta, GA 30333 Southeastern Forest Experiment Station Forest Sciences Laboratory 320 Green St Athens, GA 30602-2044

Spoerke

32 Idaho Northern Idaho Mycological Association 5936 North Mount Carrol St Coeur d’Alene, ID 83814-9609 Southern Idaho Mycological Association PO Box 843 Boise, ID 83701 Illinois Agricultural Research Service Culture Collection Northern Regional Research Center 1815 North University St Peoria. IL 61604 Illinois Mycological Society 1183 Scott Ave Winnetka, IL 60093 International Mycological Association National Center for Agricultural Utilization Research 1815 North University St Peoria, IL 61604 Ioula Prairie States Mushroom Club 3 10 Central Dr Pella, IA 502 19- 190 1 Kansas Botany Department Department of Biology Pittsburgh State University Pittsburgh, KS 66762 Kaw Valley Mycological Society 601 Mississippi St Lawrence, KS 66044-2349 Department of Botany University of Kansas Lawrence. KS 66045 Kentrrch?! School of Biological Science University of Kentucky Lexington, KY 40506

Louisiana Gulf States Mycological Society 21 1 Lake Tahoe Dr Slidell. LA 70461-8536

Mavland American Type Culture Collection Mycology and Botany Department 12301 Parklawn Dr Rockville, MD 20852- 1776 Lower East Shore Mushroom Club RR 1, Box 94B Princess Anne, MD 21853-9711 Mycological Association of Washington 9408 Byeforde Rd Kensington, MD 20895-3606 Mycology Lab, USDA, ARS, NE Region Agr Research Center Beltsville, MD 20705 National Fungus Collections Plant Industry Station Beltsville, MD 20705 Massachusetts Berkshire Mycological Society Pleasant Valley Sanctuary Lenox, MA 02140 Boston Mycological Club 100 Memorial Dr Cambridge, MA 02142-1314 Farlow Reference Library and Herbarium of Cryptogamic Botany Harvard University 20 Divinity Ave Cambridge, MA 021 38 Michigan Blodgett Memorial Medical Center 1840 Wealthy. SE Grand Rapids, MI 49506 Department of Biology Central Michigan University Mt Pleasant, MI 48859 Michigan Mushroom Hunters Club 4255 19th St Wyandotte, MI 48192 239 Plant Biology Laboratory Michigan State University East Lansing, MI 48823

33

Poison Centers for Plant Toxin Exposure University Herbarium University of Michigan Ann Arbor, MI 48109 West Michigan Mycological Society 923 E Ludington Ave Ludington, MI 3943 1-2437 Minnesota Minnesota Mycological Society 7637 E River Rd Fridley, MN 55432-3058

304 Plant Pathology Building University of Minnesota St Paul, MN 55101 Shiitake News Forest Resource Center Rt. 2, Box 156A Lanesboro, MN 55949

Missouri Missouri Mycological Society Rural Route 3, Box 190 Concordia, MO 64020-9505 Nebraska American Bryological and Lichenological Society, Inc. Department of Biology University of Nebraska at Omaha Omaha. Nebraska 68 182-0072 New Hantpslzise Monadnock Mushroomers Unlimited PO Box 6296 Keene, NH 0343 1-6296

New Hampshire Mycological Society 84 Cannongate I11 Nashua, NH 03063-1948 New Jerse?) New Jersey Mycological Association 20 Lorraine Terr Boonton, NJ 07005

Maittake Inc. (Medicinal Mushrooms) PO Box 7634 6 Aster Ct Paramus, NJ 07653 New Mexico New Mexico Mycological Society 1511 Marble Ave N W Albuquerque, NM 87 104- 1347

New York Central New York Mycological Society 343 Randolph St Syracuse, NY 13205-2357

College of Forestry Syracuse University Syracuse, NY 13210 COMA RR 3, Box 137B Pound Ridge, NY 10576-9803 Come11 University Plant Pathology Herbarium Plant Science Building Cornel1 University Ithaca, NY 14853 Long Island Mycological Club PO Box 180081 Brooklyn, NY 11318 Mid-Hudson Mycological Association 43 South St Highland, NY 12528-9803 Mid-York Mycological Society 2995 Mohawk St Sauquoit, NY 13456 Mycologia Official Publication of the Mycological Society of America The New York Botanical Garden Bronx, NY 10458 Mycological Research Cambridge University Press North American Branch 40 West 20th St New York. NY 10011-4211 Mycotaxon PO Box 264 Ithaca, NY 14850 New York Mycological Society 140 W 13th St New York, NY 1001 1-7802 Rochester Area Mycological Society 71 1 Corwin Rd Rochester, NY 14610-2124 New York Botanical Gardens Bronx, NY 10458

Spoerke

34 North Cnrolinu Asheville Mushroom Club Nature Center, Gashes Center Road Asheville, NC 28805 Blue Ridge Mushroom Club PO Box 2032 North Wilkesboro, NY 28659-2032 Botany Library University of North Carolina 301 Coker, CB #3280 Chapel Hill, NC Cape Fear Mycological Society 10 Scots Hill Road Wilmington, NC 28405 Triangle Area Mushroom Club PO Box 61061 Durham, NC 3,7705 Ohio Ohio Mushroom Society 288 E North Ave East Palestine, OH 44413-2369 Oregon Department of Botany Oregon State University Corvallis, OR 97331 Eclectic Institute (Medicinal Mushrooms) 14385 S.E. Lusted Rd Sandy, OR 97055 Florence Mushroom Club Siltcoos Station Westlake, OR 97493 Lincoln County Mycological Society 207 Hudson Loop Toledo. OR 9739 1-9608 Mount Mazarna Mushroom Association 417 Garfield St Medford, OR 9750 1-4028

North American Truffling Society PO Box 296 Corvallis. OR 97339-0296 Oregon Coast Mycological Society PO Box 1590 Florence, OR 97439 Oregon Mycological Society 2781 S W Shenvood Dr Portland, OR 97201-2250 Willamette Valley Mushroom Society 2610 East Nob Hill Street SE Salem, OR 97302-4429 Pemsyhmia Dept of Biological Sciences Mellon Institute Carneige-Mellon University Pittsburgh, PA 15213

Mushroom News American Mushroom Institute 907 East Baltimore Pike Kennett Square, PA 193587

Rltocle Islmcl Mycological Society of America The Department of Botany University of Rhode Island Kingston. RI 02881 Temessee Department of Botany University of Tennessee Knoxville, TN 37916 Texus Association of Allergists for Mycological Investigations 444 Hermann Professional Building Houston, TX 77030

The Mushroom Grower's Newsletter c/o The Mushroom Company 464 Fulton St Klamath Falls, OR 97601

Medical Mycological Society of the Americas Department of Pathology University of Texas Health Service Center at San Antonio 7703 Floyd Curl Dr San Antonio, TX 78284-7750

M L ~ S ~ W OThe O ~ IJLo m w l Box 3156 Moscow, ID 83843

Texas Mycological Society 7445 Dillon Houston, TX 77061-2721

Poison Centers for Plant Toxin Exposure Utah Biology Dept, UMC53 Utah State University Logan, UT 84322 Vernlorlt Montshire Mycological Club RD No 1, Box 336 Windsor. VT 05089 Virginicc Department of Biology Virginia Polytechnic Institute and State University Blackburg, VA 24061 Wclshirlgton Fungi Perfecti P.O. Box 7634 Olympia, WA 98507 Kitsap Peninsula Mycological Society P.O. Box 265 Bremerton. WA 983 10-0054 Northwest Mushroomers Association 831 Mason St Bellingham, WA 98225 Olympic Mountain Mycological Society P.O. Box 270 Forks, WA 9833 1-0720 Pacific Northwest Key Council 124 Panorama Dr Chehalis, WA 98532-8628 Puget Sound Mycological Society University of Washington Urban Hort. GF- 15 Seattle, WA 98195-0001 Snohomish County Mycological Society P.O. Box 2822 Everett. WA South Sound Mushroom Club 6439 32nd Ave. NW Olympia, WA 98203-0822

35 Spokane Mushroom Club P.O. Box 2791 Spokane, WA 99220-2791 Tacoma Mushroom Society P.O. Box 99577 Tacoma. WA 98499-0577 Tri-Cities Mycological Society Rural Route 1, Box 5250 Richland, WA 99352 Twin Harbors Mushroom Club Route 2, Box 193 Hoquiam. WA 98550 Wenatchee Valley Mushroom Society 287 North Iowa Ave East Wenatchee, WA 98802-5205 Wisconsin Center for Forest Mycology Research Forest Products Laboratory USDA Forest Service 1 Gifford Pinchot Dr Madison. WI 53705 Section for Botany Milwaukee Public Museum 800 W Wells St Milwaukee. WI 53233 Northwestern Wisconsin Mycological Society Rural Route 03 Box 17 Frederic, WI 54837 Parkside Mycological Club 5219 85th St Kenosha, WI 53 142-4358 Wisconsin Mycological Society Room 614, MPM, 800 W Wells Milwaukee, WI 53233 Wyoming University of Wyoming Wilhelnl G. Solheim Mycological Herbarium Laramie, WY 82071

36

Spoerke

REFERENCES by certified regional poison 1. JH Trestrail, JLF Lampe. Mushroom toxicology resources utilized centers in the United States. Clin Toxicol 28:169-176, 1990. 2. CPSC. CPSC Chairman Ann Brown suggests information technology study to support work of poison centers. News Release #94-047, Tuesday, March 15, 1994. of regional poison con3. DL Harrison, JR Draugalis, MK Slack, PC Langly. Cost effectiveness trol centers. Arch Intern Med 156:2601-2608, 1996. 4. TG Martin. Summarizationof the American Associationof Poison Control Centers Certification Criteria for Regional Poison Information Centers. Internet Address: motoxorg.htm. 5. POISINDEXB Information System. Micronledex Inc. Englewood, CO, 1998. 6. OK Miller Jr. Mushrooms of North America. New York: EP Dutton, 1982. 7. JM Kingsbury. Poisonous Plants of the United States and Canada. Englewood Cliffs, NJ: Prentice-Hall,1964. 8. BH Rumack, E Salzman, eds. Mushroom Poisonings: Diagnosis and Treatment. West Palm Beach. FL: CRC Press, 1978. and Hallucinogenic Mushroom Poisoning. Dallas TX: Van 9. G Lincoff, DH Mitchel. Toxic Nostrand Reinhold, 1977. 10. P Stamets. Growing Gourmet and Medicinal Mushrooms. Berkeley, CA: Ten Speed Press, 1993. 11. TL Litovitz, BF Schmitz, KM Bailey. 1989 annual report of the American Association of Poison Centers National Data Collection System. Am J Emerg Med 8:394-442, 1990. 12. TL Litovitz, KM Bailey, BF Schmitz. KC Holm, W Klein-Schwartz. 1990 annual report of the American Association of Poison Centers National Data Collection System. Am J Emerg Med 9:461-509, 1991.

2 ToxicologJ of Naturally Occurring Chemicals in Food I

Ross C. Beier U.S. Department of Agriculture, College Station, Tesm

Herbert N. Nigg Univers-sihof Florida, Lake Aljjred, Florida

I. Introduction 39 A. Milk sickness 39 Phytoalexins B. 42 11. CyanogenicFoods46 A. General perspective 46 Cassava B. (Manihot) 48 111. Citrus 50

A. General perspective Limes B. 53

50

IV. Crucifers (Cruciferae,Brassica) A. Goitrogens 53 Carcinogenicity B. modulation V. Fruits and Vegetables(flavonoids) Dietary A. flavonoids 61 B. Biological effects

53

55 59

of flavonoids 64

67 VI. Herbs A. Asian medicinal herbs 68 B. Onion and garlic C. Yarrow 71 Herbal D. teas 71 E. Bay leaf 71

70

Mention of a trade name. proprietary product,or specific equipment doesnot constitute a guarantee or warranty by the U.S. Department of Agriculture and doesnot imply its approval to the exclusion of other products that may be suitable. This book chapter was prepared by a U.S. government employee as part of his official duties and legally cannot be copyrighted.

37

Beier and Njgg

38 F. G. H. I.

Bishop's weed seed 74 Rosemary and sage 74 Abortifacients 77 Psychoactive substances 77

VII. Mushrooms

77

A. Agaricus bisportls B. Gyromitra esculentn VIII. Mycotoxins A. B. C. D. E.

77 80

81

A Global perspective of food safety Food safety and public health hazard Ergot alkaloids in grain foods 86 Ergot alkaloids in cattle 88 Fumonisins 88

82 83

IX. Nightshades (Solanaceae) 91

A. B. C. D. E. F. G.

White potatoes 91 Cholinesterase inhibition 93 Glycoalkaloid content 93 Teratogens 97 Eggplant 98 Green peppers 99 Tomatoes 99

X. Nitrate-Rich Foods A. B. C. D. E.

100

Reduction of nitrate 100 Nitrosation of amines 100 Quantification of N-nitroso compounds Dietary nitrate intake 102 Nitrate levels in plants 104

101

XI. Parsleys (Uuzbellifeme) 105 A. Biological activities of linear furanocoumarins B. Celery 107 C. Parsley 110 D. Parsnips 112 E. Figs 112 XII. Oxalate-Rich Foods

105

113

A. General perspective 113 B. Mineral balance 114 C. Absorption 114 XIII.

Sweet Potatoes (Zpolnoen Baruras) 118

A. Proposed lung toxins 118 B. Average concentration ofthe lung toxin ipomeamarone C. Activation of the lung toxins 121 D. Sweet potato connection to high rates of asthma 122 XIV. Tannin-Rich Foods 122 A. General perspective B. Effects of tannins xv.

Conclusion

131

Acknowledgment References

137

137

122 123

120

Naturally Occurring Toxic Chemicals in Foods

1.

39

INTRODUCTION

The purpose of exploring the potential naturally occurring toxic hazards of food plants is not to suggest an irrational avoidance of these common foods. However, it is important to identify, define, and investigate the natural toxicants in our foods, provide some perspective on these chemicals, and show clearly that their toxicology is unknown in most cases. Many natural toxicants have functions similar to synthetic pesticides or other biohazardous chemicals. Humans apply synthetic pesticides to food and ornamental plants to prevent insect, fungal, and other pest damage. However, plants produce natural toxicants to protect themselves from pathogens and pests. The natural pesticide concentration in our foods may be as much as 10,000 times higher than that of synthetic pesticide residues (1). Because of the protection they provide to plants, these natural chemicals are prime candidates to be bred into plants by plant breeders and producers (2). The main consideration of the Committee on Food Protection, National Research Council, when reviewing natural toxicants in foods, was “the hope that it may contribute to a more informed, realistic, and sensible attitude on the part of the public toward the food supply” ( 3 ) . Natural toxic components in foods receive little study today, as was the case in 1966 (4). Most people routinely accept that plants eaten in their “pristine” state not only are absolutely safe for one’s health but are better than plants “manipulated” (e.g., pesticide treated or fertilized with manufactured nutrients) by humans. Many people believe that if thefood is natural, it naturally is good for you; however, consider these cases. The plant family Solanaceae includes species that are highly poisonous and also are used for some common medicinal drugs. For example, Solanurn nigl-urn L. and Atropa belltdonna L. are extracted for their bioactive drugs, including atropine, scopolamine, and hyoscyamine. Tobacco also is related to these plants, as are such common food plants as eggplants, garden peppers, tomatoes, and white potatoes. Livestock have died after ingesting potato vines, green potatoes, or tomato vines. Human poisoning episodes and fatalities also have been reported (5-10). There are a number of foods in the human food chain through which exposure to natural toxicants may occur. Some of these are meat, milk, eggs, fish, grains, fruits, herbs, vegetables, and liquids (beer, water, wine, etc.). The first example discussed here of the occurrence of a naturally occurring toxicant appearing in our food chain involves milk. This chapter then discusses notable examples of natural toxins in various food plants and contamination by mycotoxins.

A.

Milk Sickness

The disease in humans referred to as milk sickrzess, was first noted in North Carolina by the time of the American Revolution and today it still remains the classic example of tnilk poisoning. In animals, the disease is called frerrlbles, which is based on the signs of muscle trembling of poisoned animals. White snakeroot (Erqmtoriurn rugosum Houtt) is the etiological agent responsible for milk sickness in humans and trembles in animals, but over a century went by before the plant was connected with the disease. Milk sickness in humans was caused by the use of milk or milk products from animals consuming this plant. Trembles in animals was caused by directly ingesting the plant or, in young animals, by utilizing milk from poisoned mothers. A thorough description of the plant, habitat, historical aspects, and isolation of components are presented by Beier and Norman (1 1). White snakeroot may be found in damp open areas of the woods,

40

Beier and Nigg

shaded areas, along rivers, and in steep canyons. Figure 1 indicates the distribution of white snakeroot throughout the United States (12-18). The first written description of the milk sickness disease was in 1809 by Dr. Thomas Barbee (19). The first published name for the disease, sick stomach, was coined by an anonymous author in 1811. Nancy Hanks Lincoln was among those who died in 1818 during an epidemic at Pigeon Creek, Illinois, of the disease called milk sickness when her son, Abraham Lincoln, was 7 years old (20). Nancy’s great aunt and great uncle, as well as two neighbors, died within a few weeks of each other during the same epidemic. The disease progresses slowly in humans and is characterized by restlessness with vague pains, vomiting, loss of appetite, constipation, acetone breath, severe acidosis, coma, and death. Recovery from an attack is slow and may never be complete (21). The literature on white snakeroot is very vast as it has been written about since 1809. Unfortunately, information is so diverse and inconsistent that it is very difficult to follow the true story surrounding white snakeroot poisoning. It is interesting to note that a recent article by Molyneux and James incorrectly gives Drake credit for establishing the causal relationship for the disease (22). In fact, Drake’s outstanding reputation in the scientific community prompted the acceptance of his incorrect theory that poison ivy was the plant responsible for milk sickness, and stopped research into the real cause of the disease (19). White snakeroot poisoning is still a problem in horses and goats (1 1). In a single episode during the spring of 1985, 53 angora goats died of white snakeroot poisoning in central Texas, and the total loss at that ranch during the 1985 season was 85 goats (23). It was observed by ranchers and diagnostic laboratory personnel that the goats in the

Figure 1 Theshadedareaindicatesthedistribution States.

of white snakeroot throughout the United

Naturally Occurring Toxic Chemicals in Foods

41

central Texas area that had survived white snakeroot poisoning then apparently would leave the plant alone (J. C. Reagor, personal communications, 1987). The question of palatability of white snakeroot to the goat arises (24). The toxin in white snakeroot apparently is effective enough to prevent goats from eating the plant. Since goats are very sensitive to the white snakeroot toxin, they probably are using one of the mechanisms of learning in diet selection (25). The mechanism may be trial and error, since the toxin so quickly is devastating to the goat (J. C. Reagor, personal communications, 1987). The suspected causative agent in white snakeroot poisoning was tremetone (26,27). Three main ketones were isolated from white snakeroot: dehydrotremetone, tremetone, and hydroxytremetone (Fig. 2). However, synthetic tremetone was not toxic in animal tests (28), and again there was a lull in research on the cause of milk sickness. The availability of a good bioassay was the main stumbling block in determining the toxic components of white snakeroot. Many bioassays were evaluated for possible success in showing toxic activity to components from white snakeroot; a microsomal activation assay was selected (29). The assay allowed the isolation and identification of tremetone as the primary activatable toxic component in white snakeroot (Fig. 2) (30). Tremetone readily converts to dehydrotremetone in the plant and cell-free homogenates and also decomposes to dehydrotremetone in extracts. Dehydrotremetone is not toxic with or without microsomal activation. This efficient conversion and spontaneous decomposition of tremetone to dehydrotremetone explains why white snakeroot plant material and extracts have varied toxic activities (30). It also explains why Bowen et al. did not observe toxic activity with synthetic tremetone (28). Crude extracts of rayless goldenrod (Zsoconzn wrightii, jimmyweed) cause trembles and milk sickness in the southwestern United States

Tremetone

Hydroxytremetone

Oehydrotremetone Figure 2 Three main ketones isolated from white snakeroot. (From Ref. 26.)

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42

(31) and are positive in the microsomal activation assay, which may be due to tremetone (30). Other phytotoxins are commonly found in milk and meat. These are reviewed in Ref. 32. B. Phytoalexins Bell has reviewed ways in which plants may express resistance to pathogens (33). The production of phytoalexins (toxic chemicals) is a major mechanism of plant defense. There have been many definitions for the termphytonlexin; certainly, none seem to cover the complexity of biosynthesis or the range of biological activities of these compounds. A working definition of phytoalexins is “low molecular weight, antimicrobial compounds that are both synthesized by and accumulated in plants after exposure to microorganisms” (34, p. 734). Phytoalexins exhibit toxicity across much of the biological spectrum, and their activity is not confined just to microorganisms (35). Various chemical groups that comprise some of these phytoalexins are discussed in a number of reviews (36-38). These chemical groups include the coumarins, glycoalkaloids, isocoumarins, isoflavonoids, linear furanocoumarins, stilbenes, and terpenoids. An abbreviated list of phytoalexins found in some common food plants is presented in Table 1. Induction of phytoalexin synthesis can result from a plant’s exposure to many kinds of stimuli, for instance, bacterial or viral infection (39,40), exposure to cell-wall fragments Table 1 Phytoalexins in Some Food Plants

Plant Alfalfa Pea Soybean Bean Broadbean Grapes Cotton Peanut Celery Parsley Parsnip Rice Castor bean Potato Pepper Sweet potato Carrot Tomato Lima bean Tobacco Eggplant

Phytoalexins Vesitol, sativan, medicarpin Pisatin, cinnamylphenols, 2’-methoxychalcone Glyceollin Phaseolin Wyerone Viniferin, resveratrol Gossypol, cadalenes, lacinilenes, hemigossypol Resveratrol Furanocoumarins Furanocoumarins Furanocoumarins Momilactones, oryzalexins Casbene Rishitin, hydroxylubimin, phytuberin, a-solanine, a-chaconine, lubimin, solavetivone, phytuberol Capsidiol lpomeamarone 6-Methyoxymelleint falcarinol a-Tomatine, rishitin, falcarindiol, falcarinol 5-Deoxykievitol, 8,2’-dihydroxygenistein Rishltin, lubimin, phytuberin, phytuberol, solavetivone, capsidiol, glutinosone Lubimin

in Chemicals Toxic Occurring Naturally

Foods

43

(41-43), cold, ultraviolet (UV) light, heavy-metal salts (44), antibiotics, fungicides (38), herbicides (45), and at feeding sites of nematodes (46-48). Acidic fog can stimulate the phytoalexin response in celery (49). A single stimulus like the herbicide acifluorfen can increase the production of phytoalexins and stress metabolites in crops as diverse as bean, broad bean, celery, cotton, pea, pinto bean, soybean, and spinach (45). Since plants have the ability to increase the levels of phytoalexins in response to external stitnuli, it is important to know the foods in which an abundance of such natural chemicals tnay bea potential problem for humans. It also is important to understand how the chemical content of our foods can be altered unfavorably by various treatments during production, handling, processing, shipping, and marketing. In a hypothetical society that does not use synthetic pesticides, an environmental health scientist would be well advised to take a close look at the food consumed by the inhabitants of that society. Items for consideration include (50): 1. The number and types of naturally occurring compounds present in foods 2. The immense nutnber of chemically uncharacterized compounds present in foods 3. The unknown toxic effects of these chemicals 4. The level and frequency of human consumption of the compounds in foods These four reasons for scientific investigation of the food consumed in a hypothetical society do not differ from the real society in which we live. Thus, the chemical and toxicological study of food deserves serious attention. To help focus on naturally occurring pesticides as potential human toxicants, a Thanksgiving dinner menu developed by the American Council on Science and Health is presented in Table 2, and the potential toxicants are listed (5 1). The health effects of these toxicants include blood pressure elevation from tyramine (in wine) and antithyroid activity from glucosinolates (in broccoli). The dinner also includes a variety of such mutagens as eugenol (from cranberry sauce) and rodent carcinogens like the hydrazines (from mushrooms). In the 1978 review “Phytoalexins and Human Health,’’ phytoalexins of the garden pea (with pisatin), green bean (with phaseolin), and carrot (with chlorogenic acid and myristicin) (Fig. 3) were discussed (52). Carrot also contains the acetylenes carotatoxin (fruns-1,10-heptadecadiene-5,7-diyn-3-01)10 pg/g (53); falcarinol, 18.2 pg/g; falcarindiol, 41.6 pg/g; acetylfalcarindiol; and falcarinolone. Falcarindiol has antifungal activity and is possibly a phytoalexin (54). Carotatoxin is neurotoxic to mice with an LDso of about 100 pg/g and is toxic to Daphnin rnugna Straus (53). A number of food toxicants also are described in the books Food Toxicology, by J. M. Concon (55, 56). Theobromine (Fig. 4) in tea and cocoa powder (2%) may be a potential health hazard. Theobromine can produce testicular atrophy and spermatogenic cell abnormalities in rats (1). Achronic toxicity and Carcinogenicity study of cocoa powder in rats resulted in no carcinogenicity and only limited involvement of the heart, kidneys, and testes of rats at the highest levels of cocoa powder administered (57). Alfalfa sprouts fed to monkeys cause a severe syndrome sitnilar to lupus erythematosus (58). Crude cottonseed oil contains gossypol (Fig. 4) even when it is obtained from glandless cotton (59); this compound causes pathological changes in rat and human testes, and reversible sterility in males at an oral dose of about 10 tng/day (1). Gossypol is very toxic to swine and causes hydrothorax, hydropericardium, edema of the lungs, and hepatic necrosis (60). Hydrogenated oils used in margarine have a cis-trans isomerization of lipids that may

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44

Table 2 Thanksgiving Dinner Menu includescomposition Chemical

Course Appetizer Cream of mushroom soup Fresh vegetable tray Carrots Radishes Cherry tomatoes Celery Entree Roast turkey Bread stuffing with onions, celery, black pepper, mushrooms Cranberry sauce Choice of vegetable Lima beans Broccoli spears Baked potato Sweet potato Rolls with butter

Dessert Pumpkin pie with cinnamon and nutmeg Apple pie with cinnamon Beverages Coffee Tea Red wine Water Assorted nuts

Hydrazines Carotatoxin, myristicin, isoflavones, nitrate Glucosinolates, nitrate Hydrogen peroxide, nitrate, quercetin glycoside, tomatine Nitrate, psoralens Heterocyclic amines, malonaldehyde BenzoIaIpyrene, di- and trisulfides, ethyl carbamate, furan derivatives, hydrazines, psoralens, safrole Eugenol, furan derivatives Cyanogenic glycosides Allyl isothiocyanate, glucosinolates, goitrin,nitrate Amylase inhibitors, arsenic, chaconine, isoflavones, nitrate, oxalic acid, solanine Cyanogenic glycosides, furan derivatives, nitrate Amylase inhibitors, benzo[e]pyrene, ethyl carbamate, furan derivatives, diacetyl Myristicin, nitrate, safrole Acetaldehyde, isoflavones, phlorizin, quercetin glycosides, safrole Benzo(a]pyrene, caffeine, chlorogenic acid, hydrogen peroxide, methylglyoxal, tannins BenzoIaJpyrene, caffeine, quercetin glycosides, tannins Alcohol, ethyl carbamate, methylglyoxal, tannins, tyramine Nitrate Aflatoxins

Source: From Ref. 5 1.

play a role in cancer and aging. These are a few examples of the many naturally occurring potential toxicants in foods. As scientists, we have more than the simple responsibility of advancing the production rates and postharvest quality of our foods; we also are responsible for the wholesomeness and the subtle toxicological effects that foods may have on humans. It has been

Naturally Occurring Toxic Chemicals in Foods

45

HO

Phaseolin

Pisatin

OH

e

/

CH =CH H oHOo c a H OH

kH2CH =CH2

Myristicin

Chlorogenic acid Figure 3 Phytoalexinsfromvariousvegetables.

Theobromine 0

0

Gossypol Figure 4 Theobromine from tea and cocoa powder and gossypol from cotton-seed.

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46

estimated that as much as 35% of all cancer might be related to diet (61). Cancer is only one ailment that may be related to the presence of natural toxicants in our diet. There are many more subtle problems that potentially may be diet related, including the ubiquitous condition arthritis.

II. CYANOGENIC FOODS A.

GeneralPerspective

Cyanide wastes are a 5-6-billion-gallon problem in the United States (62) from industrial and food- and feed-production effluents (63). Cyanide poisoning is not uncommon (64, 65). Cyanide as hydrogen cyanide (HCN) is readily absorbed through skin, mucous membranes, and lungs (66). The American Conference of Governmental Industrial Hygienists threshold limit value (TLV) for exposure to cyanides is 10 mg/m3 and the TLV for hydrogen cyanide should not exceed 10 parts per million (ppm) or 11 mg/m3 (67). Skin is listed as a major route of exposure (67). Methacrylonitrile. with TLVs of 1 ppm or 2.7 mg/m3, is widely used in industry, and it is metabolized to hydrogen cyanide in mammals (67, 68). Hydrogen cyanide can also be a component in smoke and may lead to poisonings during fires (69-73). Cyanide poisoning from fire products may be confounded with or overshadowed by carbon monoxide poisoning (74-76); in rats, carbon monoxide and cyanide potentiate one another (77). The diagnosis of cyanide poisoning in fire victims is not unequivocal due to delays in analysis and possible postmortem cyanide production in blood (78) and direct cyanide diffusion (79). Newer, more sensitive analytical methods and more rapid postmortem analysis may clear up these unanswered questions (80). Many plants produce compounds that contain a cyano group and when eaten or crushed produce HCN, a process termed cymogenesis (81). For an excellent review of the biochemistry of selected cyanogenic glycosides, see Ref. 82. For reviews of cyanogenic compound occurrence, isolation, and characterization, see Refs. 83 and 84. Cyanide production requires p-glucosidases and other enzymes or acid conditions (81, 85-88). The pH of the human stomach (about pH 2) may not be low enough for this hydrolysis (81). Cyanogenic compounds usually are more toxic orally than by injection because the gut contains microorganisms with the necessary p-glucosidases to produce free HCN. p-Glucosidases also occur in various animal organs (89). Cyanogenic plants may be aproblem for livestock (90-92), but some range animals like sheep may adapt or tolerate consumption of these plant toxins (90,93,94). Mule deer consuming Saskatoon serviceberry (Arz-zelanchiernhifolia) went offtheir feed, lost muscle control, and died. Saskatoon serviceberry contains prunasin (95j. Adelzia volkensii, a cyanogenic perennial plant in Kenya, is used to kill hyenas and as a poison to commit suicide (96). In controlled experiments, Jackson administered 1.2, 0.7, and 0.4 mg/kg potassium cyanide orally to young swine (97). The following behavior changes were noted: fighting, decreased dominance behavior, vocalization, investigation of new environments, aggressive feeding, rooting, water overturning, increased distractibility from eating, anesthesia recovery, limping, limb stiffness, vomiting, and shivering. Poisonous plants present hazards to humans (98), but it is unexpected that food would present an acute or a chronic hazard. Around 1900 and also during World War I, lima beans imported into Europe from the tropics caused serious incidences of cyanide poisoning (99, 100). Lima beans implicated in fatal cases contained 200-300 mg/100 g or 2000-3000 yg/g of potential HCN. Lima beans that are acceptable for consumption

Naturally Occurring Toxic Chemicals in Foods

47

contain 100-200 pg/g of cyanogenic glycosides (100). Cyanide exposure has been linked to retrobulbar neuritis in pernicious anemia, tobacco amblyopia, subacute combined degeneration of the optic nerve in vitamin B I Zdeficiency, Leber’s hereditary optic nerve atrophy, and dominantly inherited optic nerve atrophy (101). These diseases apparently are linked to cyanide in tobacco smoke and to metabolic disorders in cyanide metabolism (101, 102). The lethal adult dose of liquid HCN is 50 mg; for cyanide salts it is 200-300 mg. The calculated lethal dosage in children is 1.2-5.0 mg/kg (pg/g) (71). The seeds of peaches, plums, cherries, bitter almonds, pears, apples, crab apples, apricots, pears, and cassavas all are cyanogenic. Canned unstoned peaches, apricots, plums, and morellos contained below 1.0 yg/g (ppm) of HCN in the pulp and syrup (1 03). Bamboo shoots may contain as much as 650 pg/g HCN potential ( 104). Sorghum, cassavas, peas, beans, and grams are cyanogenic (Table 3). Flax seed may contain up to 35,200 pg/g HCN potential.

Table 3 Cyanide Potential of Selected Foods (mg/kg)

Food

Pulp

Peach, fresh Peach, canned Plum, fresh Plum, canned Apricot, fresh Apricot, canned Morello, fresh Lima bean(Phaseolus

6.8 (amygdalin, av1.8 (prunasin) 103 erage of 4 varieties)

-

2.6

-

3.1 (averageof 8 varieties) 65

lunatus)

Fatalitiesa Normal (United States) Sorghum Cassava Linseed meal Black eyed pea (Vlgna

Reference No.

Seed

21 00-3120 140-1 67 2500 1130 530

0.12 9.8 1.5

103 103 103 103

0

103

29.5

103

0.5

-

-

99,100 99 99 99

21

99 99

sinensis)

Garden pea(Plsum satlvum) Kidney bean(Phaseolus vulgaris) Bengal gram(Cicer

23

99

20

99

0

99

arletinum) Red gram(Calanus calan)

5

99

29,000 2000 6000

85 85 85

Almond Bitter seed Young leaves Apricot, seed %lplicated in human fatalities.

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Cyanogenic glycosides in foodstuffs also may be acute poisons, sometimes resulting in death. Children poisoned from eating apricot seeds have been reported from Turkey (l05), where the apricot is a popular fruit and the seeds are processed and detoxified to produce tempe (106). Townsend and Boni reported a fatal apricot-ingestion case (107). A milk shake that included dried apricot kernels purchased at a health food store was the culprit. Humbert et al. (108) and Braico et al. (109) reported a child fatality from ingestion of one to five laetrile (amygdalin) tablets (Fig. 5). Sadoff et al. reported a laetrile death case in which a cancer patient taking the drug intravenously (IV) was unable to inject the drug and swallowed approximately 10 g instead (1 10). This 17-year-old girl died 24 hr later. Rubino and Davidoff reported a nonfatal poisoning case of a 49-year-old woman with nodular lymphoma who ate 20-40 apricot pits for lunch ( 1 1). Later analysis revealed that these pits contained 4090 pg/g of cyanide potential. Brian reported one death (a 7year-old girl) and one very ill 6-year-old girl after consumption of lima beans in New Guinea (1 12). A less-critical case of poisoning from cassava consumption also was reported ( 1 12). Stavric and Klassen studied the ability of fecal flora of the mouse, rat, hamster, guinea pig, monkey, and humans to hydrolyze amygdalin to HCN and benzaldehyde (1 13). Humans appeared to be the most-sensitive species because humans averaged 29% hydrolysis of the added amygdalin. One individual hydrolyzed 45%. Adults (35-55 years old) and children (3-6 years old) were about equal in this ability. Values for the other species were mouse (0.7%), guinea pig (2.2%), monkey (2.7%), rat (3.4%), and hamster (1 3%). Treated rats recovered quite quickly, resulting from a smaller rate of hydrolysis than humans. The oral LD25 forthe rat is 6.5 mg HCN/kg compared to the lethal oral dosage for humans of 0.5-1 .O mg HCN/kg (1 13).

B. Cassava (Manihof) Cassava (~nrzihot esculenta Crantz) is the major calorie source of an estimated 300 million people (1 14) and is the fourth most important food energy source in the tropics (1 15). It is native to tropical America and was used as food at least 4000 years ago (116). Cassava grows only in the tropics (it is cold sensitive), is bulky, high in energy and low in protein provision, and deteriorates rapidly after harvest (1 16, 117). It was projected to be used as food by 600 million people by the year 2000 (1 17). Cassava contains linamarin (Fig. 6) as the main cyanogenic glycoside, with smaller amounts of lotaustralin plus the enzyme linamarase, which liberates HCN from both com-

(R)-Amygdalin Figure 5 The cyanogenicdrug laetrile (amygdalin).

Naturally Occurring Toxic Chemicals in Foods

49

Linamarin Figure 6 Linamarin, the main cyanogenic glycoside in cassava (Mclnilzotesculerzta Crantz).

pounds (1 18). Cassava cultivars are classed as bitter or sweet. In general, bitter cultivars contain higher cyanogenic glycosides (1 18), but there is overlap between these classes and no correlation between taste and cyanogenic glycoside content has been made (1 18120). The cassava plant contains cyanogenic glycosides in all structures and content varies with cultivar, plant part, and growing conditions (1 18, 121, 122). Considerable research has been conducted on processing methods and their effect on cyanogenic glycoside content; for reviews, see Coursey (1 18) and Cooke and Coursey (1 16). Additional reviews include those on drying, wilting, chopping, and chemical treatments (124); fermentation (124, 125); flour source and fermentation (126); such traditional methods as blanching, crushing, and boiling (127, 128); cooking time and water temperature (129); extended processing and taste (fufu) (130); storage and water content (13 1); wheat/cassava flour bread (132); cassava variety floudwheat bread (133); and food analysis for cyanogenic glycosides (134). High-cassava diets are common in West Africa and an adult who consumes 750 g/ day may beexposed to 35 mg HCN, about one-half the lethal dose (135). The consumption of cassava is associated with goiter in iodine-deficient populations (136, 137). Cassava diets have been associated with goiter perhaps through thiocyanate (138, 139). Equivalent antithyroid activity in rats is produced by 10 g of cassava tubers and 1-2 mg SCN- (140). In Nigeria, a cassava diet also has been associated with ataxic neuropathy, myelopathy, bilateral optic atrophy, bilateral perceptive deafness, polyneuropathy (141), and death (1 42). Motor neuron disease, Parkinson's disease, cerebellar degeneration, psychosis, and dementia may accompany the disease and 35% of the patients displayed stomatoglossitis. The disease is linked to low sulfur intake, cassava cultivation, frequency of cassava meals, and plasma thiocyanate levels (141, 143, 144). In Zaire, symmetrical spastic paraparesis epidemics are associated with low sulfur amino acid intake and consumption of inadequately processed cassava (145). The disease, termed konzo, also has appeared in Mozambique (146), the United Republic of Tanzania (145, 147), and Liberia (148). Consumption of short-soaked cassava (one-day soaking) leads to the disease, whereas unaffected persons consumed cassava that had been soaked for 3 days during processing (149). Ankle clonus in children has been correlated with cassava intake and cyanide exposure in Mozambique (150). The consumption of food prepared from the cyanogenic cycad nut has been suggested as the cause of amyotrophic lateral sclerosis, Parkinson's disease, and dementia on Guam (15 1). However, cycads contain cycasins, which are carcinogens (152), mutagens, and neurotoxins. Metabolism of these compounds does not lead to HCN. For a review, see Ref. 153. Whole blood cyanide may be elevated in subjects, particularly those with sickle cell anemia, consuming cyanogenic plants (cassava) (154). Cyanide from smoking and from

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7-Hydroxy-6-methoxycoumarin Figure 7 Scopoletin (7-hydroxy-6-methoxycoumarin) in cassava may be the cause of optic atrophy and nerve deafness.

cassava consumption is postulated to cause calcific pancreatitis (155). Cruciferous plants contain l-cyano-2-hydroxy-3-butene,which, when administered by gavage to rats, resulted in modest elevation of urinary thiocyanate, but appeared to be a pancreatic toxin (156). Rhodanase, which converts cyanide to thiocyanate, is found in mammalian liver, lung, kidney, and brain (157). It is postulated that areas of the brain lacking rhodanase are targets for cyanide (157). Humans adapt to cyanide intake with increased conversion of cyanide to thiocyanate and production of antibodies (thiocyanate bound to a protein as an immunogen) (158). It is postulated that increased methionine and cysteine mobilization to provide sulfur for thiocyanate production and vitamin BI3utilization may operate as well (158). It has been suggested that eating cassava may be prophylactic for sickle cell anemia, bowel cancer, and schistosomiasis (159). A recent study suggests that optic nerve atrophy, nerve deafness, and endemic ataxia associated with cassava consumption may be due to scopoletin (7-hydroxy-6-methoxycoumarin) (Fig. 7), which also is found in cassava (160).

111.

CITRUS

A.

General Perspective

The total number of coumarins reported from natural sources was over 600 in 1977 (161), and that number increased by the year 1982 (162). Coumarins are naturally occurring chemicals that are distributed throughout the citrus species. Linear furanocoumarins are potent photosensitizing toxins (see Sec. XI) that also act as phytoalexins in citrus (163). Bergamot oil has been obtained from the peel of Citrus bergnmin for centuries and is used for its fragrant properties in perfumes. The use of a long-wavelength [320-400 nanometers (nm)] (UVA) sunscreen is more efficient for decreasing the phototoxic properties of bergamot oil than is a short wavelength (290-320 nm) (UVB) sunscreen (164). However, UVA and UVB sunscreens at the low concentrations found in perfumes cannot suppress the phototoxicity of bergamot oil on human skin. Citrus oils are pressed from the peel and used for flavoring candies, soft drinks, and baked goods. The coumarin content of cold-pressed lime oil is about 7% by weight, and that of orange oil is less than 0.5% (165). Solids recovered by column chromatography of cold-pressed citrus peel oils are shown in Table 4. These solids reflect the coumarin content of the citrus oils, except for bitter orange oil, which consists primarily of flavonoids. The coumarins and linear furanocoumarins present in citrus peel are nonvolatile and are not found in distillates, but, the best-quality oils are pressed directly from the peel without distillation.

Naturally Occurring Toxic Chemicals in Foods

51

Table 4 SolidsRecoveredfrom Cold-Pressed Citrus Peel Oils by Column Chromatography on Silicic Acid

Citrus oil

Weight Oo/

Lime, Mexican Grapefruit Bergamot Lemon Bitter orange

6.67 1.37 0.56

0.47 0.23

So~rrce:From Ref. 165.

In citrus juice, the main aromatic component is d-limonene (Fig. 8); it is present as 70-95% of the total volatile substances (166). Table 5 shows the d-limonene content of various orange juices and a grapefruit juice obtained by different techniques. These data led to the understanding that the retention behavior of citrus volatiles during the freezedrying process is based on physicochemical and biological characteristics of the food (166). The flavor of d-limonene was very dependent upon other nonvolatile, interfering constituents including acids, pectins, and sugars (167). There have been large improvements in the quantification of limonin, a compound that causes bitterness in juice processed from early-harvested citrus (168). More than 100 volatile flavor compounds (at very low concentrations) are found in citrus products. For a review, see Ref. 169. The quantitative analysis of the volatile constituents of lemon peel oil resulted in 51 constituents that accounted for approximately 99.7% of the total volatiles in both Sicilian and California commercial lemon peel oils (170). The three main components by weight of Sicilian lemon peel oil and California lemon peel oil, respectively, are limonene

FH3

&Limonene

O w o c H 3 6CH3

Lirnettin Figure 8 Components of citrus.

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Table 5 Concentrations of d-Limonene in Citrus Juices Obtained by Different Processes

Fruit Orange

Variety

Type"

squeezed Navel (2)b Fresh Orange Valencia Fresh squeezed Commercial Brands Brand A (2)b Orange Commercial pasteurized, single strength Brand B (3)' Orange Commercial frozen, reconstituted Grapefruit Brand Commercial reconstituted frozen,

53.8' 41.4 109.Ob 161.Oc 176.0

'The type of process used to produce the citrus juice. hNumber of observations of d-limonene content obtained from similarly processed citrus juice used to calculate the mean. cMean value. Source: From Ref. 166.

(70.43% and 70.53%), p-sinene (1 1.07%and 11.92%), and y-terpinene (1 0.03% and 8.89%). 7-Geranoxycoumarin is present in grapefruit, and isopimpinellin (Fig. 9) is present in lime oil, but neither occurs in lemon oil (165). Using such differences, reliable methods have been developed for detecting cross contamination of these citrus oils (171). With few exceptions, the compounds found in the citrus family are derived from psoralen (Fig. 9) and coumarin (165). The linear furanocoumarins found in citrus are psoralen (172), bergaptol, bergapten (Fig. 9), and bergamottin in grapefruit: phellopterin, 8-geranoxypsoralen, and bergamottin in lemon; and bergaptol and bergapten in orange (173). For an extensive review of the chemical constituents in the family Rutaceae, to which the commercial citrus varieties belong, see Gray and Waterman (161) and Stanley and Jurd (165). The biogenesis, structural diversity, and distribution of simple furano- and pyranocoumar-

bCH3

Psoralen

Bergapten

bCH3

Xanthotoxin

lsopimpinellin

Figure 9 Major linear furanocounmins found in food plants.

Naturally Occurring Toxic Chemicals in Foods

53

Table 6 Furanocoumarins inLimes

Concentrations (pglg fresh weight) ~~~

~

limes Persian Rind

Key limes

Compound

Psoralen

4/5*

Xanthotoxin Bergapten lsopimpinellin Limettin

7 = ND~, ND one fruit = 0.1 ND 0.1 i 0.1 5.9 f 5.1 20.9 i 34.1a 1.1 f 0.9 128.7 f 32.9 22.0 f 31.4 2.9 f 2.5 53.7 A 14.1 1.7 f 1.3 291.1 f 85.4 310.1 f 136.3

3.9

ND

ND 0.4 f 0.6 1.7 f 2.0 2.8 + 2.1

"can ? standard deviation, N = 8: N = 13. bND = Not detected. Sorrrce: From Ref. 181.

ins in the family Rutaceae are described by Gray and Waterman (161). Herniarin and 7ethoxycoumarin were the most active coumarins against yeasts, molds, and bacteria (165).

B. Limes Sams reported 11 cases of dermatitis from lime oil, of which three were documented cases of dermatitis caused by limeade preparation (174). Limes contain the photosensitizing compounds psoralen, bergapten, and xanthotoxin (Fig. 9). Limes also contain isopimpinellin and limettin (161). However, isopimpinellin is not a photosensitizer (175, 176). Limettin is 1/200 as photoactive as bergapten on rabbit skin (177). Contact dermatitis and photodermatitis have been described in children handling limes (178, 179) (Table 6) and in children making limeade from limes (180, 181). The culprit compound appeared to be bergapten (181), but limettin was present in lime rind at about 300 ppm. Interaction assays between photoactive compounds should be conducted.

IV. CRUCIFERS (CRUC/F€RA€,BRASSICA) Cruciferous vegetables (Cruciferae, Brassica) contain natural compounds that exhibit a variety of biological activities. In ancient times, these crops were cultivated primarily for medicinal purposes (182). The first adverse biological activity investigated was their goitrogenic activity (6, 183). Plants that contain natural goitrogens and belong to this group of vegetables are listed in Table 7.

A.

Goitrogens

As early as 1928, laboratory animals fed cabbage were induced to develop goiters (184, 185). Also, lambs from ewes that were fed plants containing goitrogenic components died. Experiments with rats and guinea pigs in 1964 showed that cabbage has a marked goitrogenic capacity (186). The first goitrogen isolated from cabbage was thiocyanate, but the concentration found in cabbage could not explain the total observed effects (187).

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Table 7 Plants Containing Goitrogenic Compounds nameCommonname

Latin Beta vulgaris var. ciela Brassica canlorapa Brassica hirta Brassica napus Brassica nigra Brassica oleracea var. acephala var . bottytis var. capitata var. gemmifera var. napobrassica Brasslca pekinensis Brassica rapa Glycine max Linum usitatissimum Juglans regia' Arachis hypogaea"

Chard Kohlrabi White mustard seed Rapeseed or meal Black mustard seed Kate Broccoli Cabbage Brussels sprouts Rutabaga Chinese cabbage Turnip root Soybean Flax Walnut Peanut

'See Ref. 183. Source: From Ref. 6.

The goitrogenicity of cabbage and other cruciferous plants can be explained by the combined action of thiocyanate, goitrin, and allyl isothiocyanate (188). These compounds are hydrolyzed enzymatically from various glucosinolates (1 89). Therange of glucosinolate concentrations found in various cruciferous vegetables is listed in Table 8 (182, 190193). Brussels sprouts have the highest observed levels of glucosinolates, 1430 to 1760 pg/g of fresh sprouts. The types and quantities of glucosinolates have been determined in 22 different varieties and various head sizes of cabbage (193). The total glucosinolate concentration was 663 pg/g of fresh cabbage. The highest total glucosinolate concentraTable 8 Glucosinolate Content of Cruciferous Vegetables Concentration (pg/g) no.

ReferenceRange

Mean Vegetable Broccoli 1590 sprouts Brussels Cauliflower Red cabbage

740

2000 480 770

320

White cabbage (kraut) 61

0 890

White cabbage (market)

530 650

450-1 480 1430-1 760 600-3900 270-830 470-1 240 160-460 430-760 670-1 020 260- 060 1 300-1 070

190 190 191 190 192 190 192 193 192 193

Naturally Occurring Toxic Chemicals in Foods

55

tions found in four cabbage varieties, Red Hollander, Savoy Perfected Drumhead, Wisconsin Hollander,and Stonehead, were 1203, 1288, 1014, and 1065 pg/g of fresh cabbage, respectively (193). Goitrogenic compounds also are found in rapeseed. Rapeseed meals are used in animal feed because of the lower glucosinolate content in newer varieties (194). The enzymatic degradation product of progoitrin, 5-vinyl- 1,3-oxazolidine-2-thione (5-VOT), is goitrogenic, produces antinutritional effects, and inhibits synthesis of thyroid hormones, which causes metabolic disturbances (182). Therefore, its use in animals or indirect consumption by humans presents a potential danger (194).

B. CarcinogenicityModulation 1. Protection from Cancer Rabbits fed cabbage leaves in 1931 survived a lethal dose of uranium (195). This led to the epidemiological conclusion in the 1970s and 1980s that cruciferous vegetables can provide protection from cancer. Indoles in vegetables of the Brassica genus were known to inhibit carcinogenesis in experimental animals. Three 3-substituted indoles [indole-3carbinol (I3C), 3-indolylacetonitrile, and 3,3’-diindolylmethane] (Fig. 10) are inhibitors of induced cancer (196). These three indoles are produced by enzymatic hydrolysis of indolylmethylglucosinolate (glucobrassicin) by the pH-dependent plant enzyme myrosinase following disruption of plant material (197). Nearly 80 naturally occurring glucosinolates have been described. Isothiocyanates or nitriles formed from the glucosinolates are dependent on the type of plant material and the treatment of the material prior to and during hydrolysis (1 98). Seven isothiocyanates were tested for mutagenicity on S. pphimwiuwz TAlOO and all tested positive, with allyl isothiocyanate having the highest potency. Allyl isothiocyanate glucoside (sinigrin) showed an equivalent mutagenicity potency to allyl isothiocyanate itself. Thiocyanates were found to be nonmutagenic, while isocyanates showed mutagenicity on S. ~yphimzlr - i u n z TAlOO strain even without activation (199). Sedation, ataxia, loss of righting reflex, and sleep were induced in rats by 3-indolylacetonitrile and I3C. Phenylpropyl isothiocyanate and allyl isothiocyanate were not teratogenic to rat fetuses, but they did cause embryonal death and decreased fetal weight (200). Animals fed diets high in cruciferous vegetables and then exposed to various carcinogens expressed lower tumor yields and increased survival rates (201-203 j. Arylalkyl isothiocyanates have been determined to be inhibitors of lung tumorigenesis induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) in rats and mice (204-206). It now is known that manydrugs and other chemicals induce metabolizing enzymes. Animal feeding studies have demonstrated induction of mixed-function oxidases (MFOs) in rats fed Brussels sprouts or cabbage (207) and cauliflower (208); the indoles present in these vegetables were shown to cause induction of metabolizing enzytnes (197, 209). Especially noted is induction of the intestinal aryl hydrocarbon hydroxylase (Ah) system (207, 210). The Ah receptor is involved in the induction of cytochrome P-450 -I A1 and A2, and other enzymes that participate in xenobiotic metabolism (311). 13C isolated from Brassica olerzrcea var. gemmifercl cv. Jade Cross was a significant inducer of hepatic and intestinal MFOs (197). 13C was shown to enhance the activities of rat intestinal Ah and ethoxycoumarin 0-deethylase, which are capable of metabolizing benzo[n]pyrene and other xenobiotics. In addition, both hepatic and intestinal glutathione S-transferase and

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56 NOS03K

QLT?T

glucose

I

H

lndolylmethylglucosinolate

>?-

I

H

3-lndolylacetonitrile

.+

sulfate, & sulfur

glucose,

& sulfate

lndolylrnethyl isothiocyanate

Indole-3-carbinol 3,3’-Diindolylmethane

Figure 10 Enzymatic hydrolysis of indolylmethylglucosinolate. (Adapted from Ref. 197.)

microsomal epoxide hydrolase of the small intestine were induced by I3C. Hepatic cytochrome P-450 was increased by Brussels sprouts and 13C (212). A diet containing Brussels sprouts and cabbage increases the apparent metabolic clearance rate of antipyrine, phenacetin (213), and acetaminophen, while also enhancing its glucuronide conjugation in humans (214). I3C administered orally to humans increased estradiol 2-hydroxylation (215). The effect of 13C on estradiol 2-hydroxylation is similar to that caused by smoking. Epidemiological studies indicate that colon cancer risk is higher in individuals who ate fewer cruciferous vegetables (216) and consumed more fat (2 17-222). Epidemiological studies of people who had gastric cancer also suggested that those who ate cruciferous vegetables were protected (223-225). Other epidemiological studies indicated a lower incidence of breast cancer (226,227) and prostate cancer (227,228) in vegetable-consuming populations. Evidence from an epidemiological case/control study of diet and cancer

Naturally Occurring Toxic Chemicals in Foods

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also suggested that consumption of cruciferous vegetables was associated with a decreased incidence of cancer (229). Recently, approximately 200 studies that examined the relationship between fruit and vegetable intake and cancer of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, bladder, pancreas, and ovary were reviewed (230). A protective effect of fruit and vegetable consumption was found in 82% of the studies. For most types of cancer, persons with low fruit and vegetable intake experienced approximately twice the risk of cancer compared with those people with high intake (230). The public has been advised by various studies to include more cruciferous vegetables, such as cabbage, broccoli, Brussels sprouts, kohlrabi, and cauliflower, in their diets (230-234). Today, some physicians are making the same recommendation. 2. Promotion of Cancer Unfortunately, the early feeding experiments generally used only one type of protocol; cruciferous vegetables or 13C was given prior to or during administration of a carcinogen. When this protocol was changed so that a carcinogen was given before consumption of cruciferous vegetables or I3C, higher cancer rates were obtained in laboratory animals treated with either cruciferous vegetables or I3C. When 13C was given to trout before aflatoxin B I, the trout were protected against liver carcinogenesis (235). However, when aflatoxin B I was given before I3C, hepatocarcinogenesis was strongly promoted (236). This promoter effect also was observed with 1,2-dimethylhydrazine enhancement of colon cancer in rats (237). 13C was mostresponsible for tumor morbidity and appears to promote tumorigenesis by inducing Ah receptor activity. Colon tumor incidence increased when 1,2-dimethylhydrazine was administered to mice fed a diet containing cabbage (238). The level of cabbage used in these studies was comparable with human intake. Feeding cabbage to hamsters elevated the incidence of gallbladder adenocarcinoma, plus feeding high-fat diets elevated pancreatic ductular carcinoma in hamsters administered N-nitroso-bis-(2-oxopropyl)amine (BOP). Skin papilloma, initiated by 7,12-dimethylbenz[n]anthracene(DMBA) and promoted by 12-0-tetra-decanoylphorbol-13-acetate (TPA), increased when mice were fed diets containing 10% dried cabbage (239). Possible biochemical mechanisms behind the promotional effects of 13C are under study. I3C, given intraperitoneally, does not induce hepatic ethoxyresorufin 0-deethylase (EROD) activity. Given orally, it does induce EROD (196). Acid treatment of I3C, under conditions similar to stomach acid conditions, produced a reaction mixture that induced EROD by either the intraperitoneal or oral route. Chromatographic separation of the acid reaction mixture suggested that at least four 13C condensation products will induce EROD (240). The mechanism by which 13C and its analogs induce cytochrome P-448-dependent monooxygenases is mediated via condensation products generated in the acidic conditions of the stomach (196). Glucobrassicin (241) and neoglucobrassicin (242) are proposed as the sources of 3indolylacetonitrile, I3C, and other simple indolic compounds. The glucobrassicin content of fresh Brussels sprouts varies from 220 to 11 10pg/g (191). The pH-dependent enzymatic hydrolysis of these indole glucosinolates by myrosinase produces 13C and a series of simple indoles (197, 243) (Fig. 10). I3C, 3-indolylacetonitrile, and the well-known MFO inducer, 3,3’-diindolylmethane, from cabbage and cauliflower were all demonstrated to induce the Ah receptor (197). Since compounds such as indolo[3.2-b]carbazole (ICZ) (Fig. 11) can be generated from 3,3’-diindolylmethane in the presence of acid, air, and

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LT

CT

H

ICZ Figure 11 Condensation products of indole-3-carbinol in acidic conditions similarto those in the stomach (LT = 2-[indole-3-ylmethyl]-3,3’-diindolylmethane: CT = 5,6,11,12.17,18-hexadrocyclonona[ 1.2-b:4.5-t7’:7.8-b’]triindole;ICZ = indolo[3.2-b]carbazole).

light (244), it was postulated that ICZ may be present (196), ICZ was also the most active of all the indoles studied for inducing the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor (245). The three most prevalent UV-absorbing compounds in the reaction mixture of acidtreated 13C are 2-(indol-3-ylmethyl)-3,3’-diindolylmethane(LT), 5,6,11,12,17,18-hexahydrocyclonona[ 1.2-b:4.5-b’:7.8-b’’]triindole (CT) (Fig. 1l), and 3,3’-diindolylmethane (Fig. 10) (246). Molar yields of the three were in the range of 2-6% of the original amount of I3C. The presence of LT and 3,3’-diindolylmethane was consistent with previous findings (247). Upon further analysis, ICZ was shown to bepresent in theacid condensation reaction mixture (248). ICZ is produced from 13C in yields on the order of 0.01% in vitro and, after oral intubation, in vivo. The binding affinity of IC2 is a factor of 3.7 X 10” lower than that ofTCDD. The most potent Ah receptor agonist identified in the acid condensation reaction mixture is ICZ. ICZ and related condensation products appear responsible for the inducing effects of dietary I3C. Due to the higher yields of the weaker-binding oligomers, ICZ appears of roughly equal importance to other oligomers in the inducing activity of the mixture. TCDD has well-known and established activities as both an anti-initiator and as a promotor of carcinogenesis. Similar effects are observed for the cancer-modulating activity of I3C. 13C or TCDD given before a carcinogen protects against carcinogenesis; when either is given after a carcinogen, it strongly promotes carcinogenesis.

Naturally Occurring Toxic Chemicals in Foods

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The Ah receptor bindings of ICZ and TCDD are similar in all respects. A 100-g portion of Brussels sprouts could provide a dose of 0.256-1.28 pg ICZ. This dose is considerably in excess of the maximum acceptable daily human dose for TCDD, which is 400 fg/70-kg person, established by the U.S. Environmental Protection Agency (EPA). However, there may be a number of factors that could lower the relative hazard or benefit as well as the half-life of ICZ compared to TCDD. Bjeldanes et al. concluded that it appears unlikely that normal levels of ICZ in the diet are a significant hazard compared with the benefit of the micronutrients in Brassica vegetables (248). 3. Induction of Protective Enzymes Zhang et al. isolated a single isothiocyanate, sulforaphane (Fig. 12),from SAGA broccoli that was the major inducer of phase I1 protective enzymes (249). The assay used resulted in the determination of quinone reductase in Hepa IC lc7 murine hepatoma cells. However, Zhang et al. did not include references that describe 13C as themajor component of cruciferous vegetables responsible for the observed biological activities of cruciferous vegetables in animals (see Sec. 1V.B.l and IV.B.2) (249). Since similar effects are observed for both cruciferous vegetables and I3C, an interesting question is, What role does sulforaphane actually play in protection against carcinogens with respect to 13C in cruciferous vegetables? Cruciferous vegetables, I3C, or TCDD given before a carcinogen protects against carcinogenesis, but when any one of the three is given after a carcinogen, each promotes carcinogenesis.

V.

FRUITS AND VEGETABLES(FLAVONOIDS)

The amount of flavonoid literature is enormous. We refer the reader to Refs. 250-255 for a general view of flavonoid chemistry, biochemistry, and distribution. A mass spectral analysis study of 79 flavonoids provided data on 26 compounds that were not found in the National Institute of Standards and Technology database (256). McClure reviewed the function and physiology of flavonoids (257). Plant flavonoid content may be influenced by light, water, temperature, sugars, mineral nutrition, mechanical damage, pathogens, plant growth regulators, and various other chemicals (257-260). Microorganism-resistant cultivars may contain higher flavonoids than susceptible cultivars (261), and flavonoids may vary during a plant’s life cycle (262). Flavonoids may be localized in plant tissues and cells and secreted in various exudates. They may function as antioxidants, enzyme inhibitors, pigments for light absorbance, visual attractants for pollinators, light screens, promoters or inhibitors of plant growth, plant growth regulators, legume Rhizobimz root nodule gene inducers (263, 264), phytoalexins, analgesic agents (265), and plant morphogenic and sex-determination agents (257).

and

60

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Nigg

Kiihnau (266), Hackett (267), and Adzet and Camarasa (268) reviewed flavonoid metabolism. Less-hydroxylated or -methoxylated flavonoids are more active biologically and are more resistant to intestinal microflora metabolism. An exception is the biflavan, 3,3’,4’,5,7-pentahydroxyflavonoid (cyanidin) (see the flavonoid general structure in Fig. 13), which is resistant to gut microflora. The flavonoids are metabolized to phenolic acids or lactones in the gut, are absorbed by the gut as aglycones, may be excreted unchanged in bile, and may be metabolized by the liver. They may be oxidized, reduced, methylated, and conjugated. Although most of these experiments have been in small animals, human experiments suggest similar processes (267). Overall, the absorption, metabolism, and excretion of flavonoids in any animal, particularly humans, have been poorly studied. No study has accounted for the entire dose. In radiolabeled studies, the form of the radiolabel recovered, in most cases, was not determined. Radiochemical purity of starting materials was not determined. Elimination halflives are helpful but are not necessarily related to dose. There are no mass-balance studies.

6

4

Flavonoid general structure

Q

OH

0

Flavonols

0

Flavones

AH

Q

Leucoanthocyanins

Flavanones

fJ

OH

Q 0O

H

Flavanonols

0

0 OH

Catechins

Anthocyanins

Figure 13 Flavonoid general structures.

Naturally Occurring Toxic Chemicals Foods in

61

The pharmacokinetics of absorption, metabolism, and excretion of flavonoids is critical for estimating benefits and risks.

A.

Dietary Flavonoids

Flavonoids usually are conjugated to a sugar and are widespread in the plant kingdom (253, 269). The occurrence of the flavonoid aglycone is less pronounced compared with the carbohydrate moiety of the conjugates and appears to be associated with secretory structures and lipophilic plant products (see Ref. 270 for a review). Pierpoint lists several difficulties in estimating flavonoid intake (271-273). Many compounds are involved. Flavonoid content varies by season, plant cultivar, plant maturity, and plant condition. National diets vary, so an estimate for the United States may not apply to other countries and cultures. Kuhnau published an estimate of the total U.S. flavonoid daily intake per person of 1020 mg/day (winter) and 1070 mg/day (summer) (266). Of these totals, 41% (winter) and 39% (summer) are from cocoa, cola, coffee, beer, and wine. Fruit juices contribute an additional 26% (winter) and (29% (summer), followed by spices at 16% (winter) and 15% (summer). These three food groups contribute 83% of the total daily U.S. intake per person of flavonoids in winter and summer. Pierpoint discussed culture differences for flavonoid intake (272, 273). In the United Kingdom, for instance, the average tea consumption of 4.7 cups daily per person would provide about 900 mg flavonoids, whereas the total estimated U.S. daily consumption per person from all sources is about 1000 mg (266). Dependent on diet, total flavonoid content in some cultures may be 2000-3000 mg/day (273). Many vegetables contain flavonoid compounds: bell pepper, broad bean, broccoli, cabbage (regular and red), chive, endive, garlic, green potato, horseradish, kale, kohlrabi, leek, lettuce, onion, and radish (274-277). Most of these vegetables contained less than 100 pg/g of either quercetin and/or kaernpferol (Fig. 14). However, the colored, outer skin of onion contained up to 65,000 pg/g of quercetin (276), broad bean pods 1340 pg/g of quercetin (276), and green endive 9400 pg/g of quercetin (275). Shallots contained more than 800 pg/g of total flavonoids; 20 cultivars of yellow and red onions contained 60-1000 pg/g, but flavonoids were not detected in white onions (278). Quercetin and kaempferol have been quantified in the following fruit: apples, apricot, bilberry (wild and cultivated), blackberry, black current, highbush blueberry, cherry, cranberry, currant (red and white), elderberry, gooseberry, grape juice, pear, peach, plum, prune (dry plum), apple juice, and quince (276, 279-281). These mostly contained less than 75 pg/g except for apples (about 100 pg/g) (276), bilberry (about 125 pg/g) (276), cranberry (about 175 pg/g) (279), elderberry (about 150 pg/g) (276),prunes (22,000 pg/ g) (282), and quince (about 390 pg/g) (276). Other flavones found in fruit include catechin, in the apple, apricot, cherry, peach, and plum (283);naringin, in various citrus including juice (284); and 5,7-dihydroxychromone, eriodictyol, and luteolin, in peanut hulls (285). Flavonoid content of varieties of the same fruit differ as well as flavonoid content of plant' parts at different stages of maturity. Light conditions affect flavonoid content (276). For example, covering cauliflower curds reduced floret flavonoids 20-40% and improved the quality of the cauliflower (286). Plant disease status affects flavonoid content (287, 288). Processing, shipping, and handling also may affect final flavonoid levels. Some human flavonoid consumption undoubtedly comes from food contamination. Commercial grain may be contaminated with toxic weed seeds, sicklepod (Cassia obtusifolicr), jimson weed (Datum stranzoniunl), velvetleaf (Abzltilo~theophrasti), and morning

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62

Kaempferol

QH

PH

OH OH

OH

Myricetin Figure 14 Mutagenicflavonols from fruits andvegetables.

glory (Ipomoea species) (289). Sicklepod seeds contain anthraquinone derivatives, P-sitosterol, and flavonoids, UV-quenching compounds, and fluorescent blue compounds of unknown structure (289). Jimson weed contains tropane alkaloids; velvet bean contains delphinidin, quercetin, catechin, myricetin (Fig. 14), (- )-epicatechin, and cyanidin (291). Morning glory seeds contain chlorogenic acid (Fig. 3) (289). Range plants also may contain flavonoids. Gutierre,-in microceyhnln A. Gray (broomweed, perennial snakeweed, broom snakeweed, stinkweed, turpentine weed) consumption by cattle in the American Southwest is associated with abortion, placental retention, hemorrhage, and nonsurviving weak offspring (292-294). Over 20 oxygenated flavonol methyl ethers were isolated from the Gutierrezicr species (294). Soybeans contain daidzein (aglycone of daidzein), genistein (aglycone of genistin) (Fig. 15) (295), and 3-@malonyl and 3-acetyl-isoflavones (296). These compounds are bitter or astringent (296). Daidzein and genistein increase due to P-glucosidases in soybean during the processing of soy milk. On a dry soybean basis, these compounds increased

Naturally Occurring Toxic Chemicals in Foods

63

OH

Diethylstilbestrol

Equol

HO

OH

OH

Daidtein

Genistein

OH

Formononetin

Coumestrol

Figure 15 Structural formulas of equol, diethylstilbestrol, diadzein. genistein, formononetin, and coumestrol.

from about 50 pg/g to 350 pg/g at pH 6.0 (close to the pH of soy milk) and 20°C in the soak step of processing (295). In the soybean plant, daidzein, genistein, and coumestrol (Fig. 15) increased in the roots over a 12-day period after transplanting and inoculation with Br.ndyrlzizobiunzjclporzicum. Nitrogen application decreased the isoflavone (Fig. 16) concentration in roots (297, 298). Conjugates of daidzein and genistein are selectively

Figure 16 Isoflavonoidgeneralstructure.

64

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excreted into root and seed exudates at a level of 1-10 yM. Their levels in the seed may be more than 1000 pg/g seed tissue and it is postulated they may act as signal molecules in chemoattraction of nodule-forming bacteria (299). Subterranean clover (Trifolium subterrcrneum L.) is a forage legume widely grown in Mediterranean climates (300). Identified isoflavones in subterranean clover include biochanin A, formononetin, genistein, and daidzein (Fig. 15). Of four cultivars, only one showed an isoflavone content difference based on harvest date. Total isoflavones were almost unchanged from the comparison of fresh to frozen samples, but dried samples showed a 30-50% decrease in isoflavone content (300). Production of isoflavones was induced in the bean (Phaseolus vzrlgnris L.) by ozone, SO:, and several herbicides (301). 6. BiologicalEffects of Flavonoids There are perhaps more good health claims for flavonoids than there are adverse effects reported because humans perceive the healthy aspects of citrus, pome fruits, and vegetables. The U.S. surgeon general and the National Academy of Sciences have strongly recommended consuming more fruits and vegetables. The Committee on Diet, Nutrition, and Cancer, National Research Council, emphasized the importance of including fruits (especially citrus fruits), vegetables (especially carotene-rich and cruciferous vegetables), and whole-grain cereal products in the daily diet (232, p. 15). Kuhnau (266), Singleton (302), and Attaway (303) reviewed the nutritional effects of flavonoids. Flavonoids are considered “semiessential’ ‘ food components and not harmful. Flavonoids are antioxidants (304). Quercetin, myricetin (Fig. 14), quercetagetin, and gossypetin are the best antioxidants; catechin has some activity. Hesperidin is inactive. Daidzin, genistin, malonyl-daidzin, and malonyl-genistin were antioxidants (262, 305). Morin, quercetin, myricetin, and fisetin were the most active inhibitors of induced lipid peroxidation in the presence of ferrous ions (306). Diosmetin, apigenin, hesperetin, naringenin, and 4’,5,7-trihydroxyflavone(all lack the 3-OH group) (Fig. 13) were least active. With ascorbic acid as the inducer, 3-hydroxy-flavone, fisetin, taxifolin, (+)-catechin (Fig. 13), quercetin, myricetin, and morin were the most effective inhibitors of lipid peroxidation (306). These all have a 3-OH group. Isovitexin, a glycosyl flavonoid from rice hull, is a better antioxidant than BHA (butylated hydroxyanisole) and a-tocopherol (307). Isovitexin is absent in rice seeds that deteriorate or rot. Only long-lived rice seed hulls contain isovitexin. Epicatechin 3-0-gallate and other procyanidins from grape seeds were found to be potent, oxygen-free, radical scavengers. The best scavenger was procyanidin B23’0-gallate (308). Some flavonoids can chelate metal ions, particularly copper (266, 309). Copper flavonoid complexes inhibit hyaluronidases, stabilize structural proteins, and strengthen fragile membranes (266). Flavonoids hydroxylated in the 5, 3’,4’ or 5, 3’,4’,5’ positions (Fig. 13), may be competitive antagonists to catecholamines like epinephrine and norepinephrine and extend the effect of these compounds. Flavonoids also extend the action of vitamin C and, at one time, were referred to as vitamin P (310). The term vitamin P was replaced by bioflavonoid. Vitamin P was a mixture of eriodictyol and hesperidin. The interaction between vitamin C and flavonoids is considered to be an antioxidant effect (for reviews, see Refs. 31 1 and 312). Flavonoids reportedly affect glucose uptake by lymphocytes and fibroblasts, and for their immune functions act as smooth muscle antispasmodics, and anti-inflammatory agents. They inhibit platelet aggregation, act as antiviral agents, are synergistic with other

Naturally Occurring Toxic Chemicals in Foods

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antiviral agents, and are bacteriostatic. Flavonoids also are potent multifunction oxidase inducers (thus increasing metabolism of xenobiotics), correct abnormal capillary permeability and fragility after X-radiation and a variety of diseases, and act as anticancer and antimutagenic agents. Quercetin (Fig. 14) is the principal flavonoid tested for these effects (266, 303, 309, 310, 312). Quercetin at 25,000 pg/g in laboratory chow or 70 pg/g in water prevented diabetic cataracts in the degu (Octodon degus), a rodent native to the Andes of South America, possibly through inhibition of lens aldose reductase (313, 314). Flavonoids inhibit a variety of enzymes in vitro that are required for normal physiological function (3 12, 3 15-3 17). Some of these enzymes are involved in axonal transport, basophil/mast cell secretion, cell locomotion/chemotaxus, DNA synthesis, endo-/exocytosis, insulin secretion, intestinal chloride ion secretion, membrane phosphorylation, microtubular dissociation, mitogenesis, neurotransmitter release, platelet function, and smooth muscle contraction (312, 317). Flavonoids may interfere with thyroid function (T,-T, balance) (3 18). A particularly fascinating biological activity of quercetin is its ability to inhibit heatshock protein by interacting with heat-shock factor. This activity is of practical consequence in reducing the heat tolerance of tumors during hyperthermic therapy (3 19). Although many foods have notbeen shown to be mutagenic in microorganism assays, red wine, grape juice, instant coffee, strawberries, raspberries, peaches, raw onions, raisins, and grapes displayed potent mutagenic activity and the primary natural mutagen appeared to be quercetin (Fig. 14) (320-323). In 1977, Bjeldanes and Chang reported that quercetin, a large fraction of the total flavonoids in the daily diet (324), was mutagenic without cytochrome P-450 activation, but was more mutagenic with activation (325). In further studies, quercetin and myricetin (Fig. 14) were mutagenic without activation; kaempferol (Fig. 14) was mutagenic only with activation (326, 327). Quercetin was mutagenic to mouse cell lines (328). Flavonoid content correlated with the mutagenicity of nutritional supplements and tobacco (snuff) containing rutin (326). Of the 70 naturally occurring and synthetic flavonoids tested, 33 were positive in the Ames assay (329). All active flavonoids have maximum active response toward S. ~ p h i m u r i u nstrain ~ TA98 and a highly significant response toward strain TAlOO (309, 329). The flavonoid structural features essential for mutagenic activity in S. typhin~uri~m strains TAlOO and TA98 were determined, and only the flavonols (3-hydroxyflavones) (Fig. 13) appeared to be mutagenic (327). Structural requirements for mutagenic activity appear to be: (1) a free 3-hydroxyl group; (2) a 2,3 double bond; and (3) a 4-keto group (Figs. 13, 14). However, there are flavonoids without the 3-OH group that are mutagenic toward the TAlOO strain, but not strain TA98 (309). Quercetin is the most-active flavonoid mutagen with strain TA98. Oxygen, tyrosinase, and alkaline pH irreversibly inactivate the mutagenicity of quercetin with strain TA98 (330). Quercetin also was positive in the SOS chromotest, apparently without activation (33 1). Norwogonin and sexangularetin are more mutagenic than quercetin with strain TA100. These flavonoid mutagens require activation with the S9 or cytosol cell fraction, and have hydroxy- or methoxy-substitution at positions 5,7, and 8 of the A ring (see Fig. 13). The B ring and the 2, 3 positions apparently are not involved in this second class of flavonoid mutagens (309j. Flavonoids may be responsible for the mutagenicity of weed seeds that contaminate grain (289). The mutagenicity of quercetin probably prompted its inclusion in the National

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Toxicology Program’s testing regimen. Quercetin was mutagenic in Snlrnonellcr assays and cytogenic in Chinese hamster ovary cells (332). Quercetin subsequently was shown to exhibit some carcinogenicity in male mice because of increased renal tubular cell adenomas when fed at 40,000 ppm (333, 334). Cancer studies on flavonoids have been reviewed (309, 335, 336). Quercetin (Fig. 14) was positive for intestinal tumors, bile-duct tumors, bladder tumors, hepatomas, and liver preneoplastic foci in three rat dietary studies. In 10 other rat/mouse/hamster dietary studies, there was no increase in carcinomas (309, 336). Rutin, kaempferol (Fig. 14), tiliroside, and catechin (Fig. 13) were negative (309, 329). In chromosomal aberration tests, quercetin has been both positive and negative (309, 329, 337). Flavan-3-ols, (+)catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin-3-O-gallate, and procyanidin B-1 and C-1 break double-stranded DNA in the presence of cupric ions (338). Quercetin at 0.1% in the diet reduced the life span of “shorter-living” male mice (339). Flavone acetic acid, a synthetic compound, is used as an anticancer drug (340, 341), though not as a single agent (342). A wide variety of flavones have been synthesized to obtain cytotoxic agents (343). Various flavonoids are antimutagenic agents (344-346). Quercetin reduced mortality and the cytotoxic effects of the T-2 mycotoxin in mice (347). Various flavonoids inhibited the mutagenicity of aflatoxin B 1 toward S. ?yphimurilrm strains TAlOO and TA98 (348). Some flavonoids have been found to be antitumorigenic, antimutagenic, and anticarcinogenic (266, 278, 316, 349-353). Flavonoids appear to provide a cancer protective effect at the cellular and molecular level (278, 351), and also by reducing the bioavailability of carcinogenic compounds (351, 353). It is important to note that mutagenicity testing is usually with a single compound whereas “anti” effects usually involve use of a known carcinogen plus a flavonoid. Canada et al. challenged isolated guinea pig enterocytes with kaempferol, quercetin, and myricetin (Fig. 14) (354). All three compounds produced cellular damage at 450 pM. Quercetin and myricetin appeared to be more toxic than kaempferol. These authors suggested that flavonoids might exacerbate or cause inflammatory bowel diseases (354). Kumar et al. administered kaempferol orally at 250 mg/kg body weight/day for 60 days to male rats (355). Spermatids were reduced by 73.7%. Mature and immature Leydig cells decreased by 39.2% and 46.6%, respectively. Testicular cholesterol increased and androgen-dependent sialic acid and protein declined in the testes, epididymides, and sex accessory glands (355). Shore and Lyttle studied the inhibition of rat uterine peroxidase, an enzyme that increases in the uterus in response to estrogen (356). Diethylstilbesterol, genistein (isoflavone) (Fig. 15), and zeralenone and zearalenol (Fig. 17) were competitive inhibitors; coumesterol (isoflavone) (Fig. 15) was a noncompetitive inhibitor of rat uterine peroxi-

Zearalenol

Zearalenone Figure 17 Natural estrogens that inhibit rat uterine peroxidase.

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67

dase. Coumesterol was 2-6 times as inhibitory to peroxidase as diethylstilbestrol (Fig. 15), whereas genistein was 25 times less active than diethylstilbestrol (356). Flavonoids have been linked with abortion in cattle in theAmerican Southwest from consumption of the genus Gutierrezin (294). The isoflavones (Fig. 16) of subterranean clover, formononetin, biochanin A, genistein, and daidzein (Fig. 15) are associated with an infertility syndrome in sheep (357-360). Estrous ewes fed oats or subterranean clover averaged 17,160 and 350 sperm, respectively, per fallopian tube 24 hr after mating. The percentages of motile sperm recovered from the cervix and ova with sperm attached to the zona pellucida were lower in ewes fed clover (361). The isoflavone infertility problem has prompted immunization attempts against genistein and equol (Fig. 15) with temporary success (362). Equol has been identified in the urine of pregnant mares (363), goats (364), cows (365), hens (366, 367), sheep (368, 369), and men, women, and rats (370). The metabolism of formononetin (Fig. 15) and biochanin A in bovine rumen fluid has been studied (371). Analytical methods for isoflavones in soy protein (372) and in human urine (373) are available. Equol (Fig. 15) and other phytoestrogens possess weak estrogenic activity (374377). Estrogenic responses to isoflavones vary by species and, for bioassays using mice, by strain (378). Human volunteers excreted large quantities of equol, a phytoestrogen, after consuming 40 g of textured soya for 5 days. Fecal flora also were incubated with textured soya and intestinal microbes produced equol. Equol excretion exceeded 6 mg/ day in one subject after a 40-g soya meal. Estrone-glucuronide, the principal urinary estrogen in the follicular phase of women, is excreted at 2-27 pg/day (379). Urinary excretion of equol in humans not consuming soy products is about 80 pg/day (379). This study is important for several reasons. It showed conversion in humans to a biologically active flavonoid from a dietary source. Equol crossed into the bloodstream in quantity and was excreted as a glucuronide. Phytoestrogens apparently cotnpete for the same estrone binding sites on a-fetoprotein in rats and in humans (380). Phytoestrogens have a wide range of biological activities, from anticancer to antiestrogen effects (380). Flavones and isoflavones (Fig. 16), including genistein (Fig. 15), biochanin A, prunetin, kaemferol, and quercetin (Fig. 14), inhibit tyrosine protein kinase, which is necessary for retrovirus carcinogenicity (381). Genistein induces mouse erythroleukemia cells synergistically with mitomycin C (382). Immunological data on genistein suggest it is a powerful immunosuppressant (383). Flavonoids also affect the immune system and inflammatory cell functions. These effects are reviewed in Ref. 384. Flavonoids also reduced benzo[a]pyrene DNA adduct formation in vitro and also in vivo after animals were fed flavonoids for 2 weeks (385). Perhaps other flavonoids in the diet are converted to biologically active molecules that have yet to be isolated. Increased dietary fat has been linked to prostate cancer and increased plasma levels of male hormones (386). Low-fat diets can influence plasma levels and excretion of estrogens and influence the incidence of breast cancer (387-390). We suggest that a better understanding of the changes in hormone levels caused by bioactive flavonoids and flavonoid metabolites in the vegetarian diet would be a good starting point in searching for ‘‘anticancer nutrients.’’

VI.

HERBS

The use of herbs and herbal preparations has been reviewed with a historical perspective (391). Many people believe that plant remedies are naturally superior to synthetic drugs

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and that when herbal preparations are used they cannot be harmful to human beings. In 1977, the Consumer Response Corporation reported that a survey showed that the most convincing sales claim to put on a food or beverage label is that it is “natural”; 42% of the consumers surveyed believed that natural products have no adverse effects and are more healthful and safer (392). In thepast two decades, natural foods and herbal medicines have gained substantial popularity in the United States (393). In 1978, the sale of herbs and other related commodities in health food stores alone amounted to $1.1 billion (39 l), and in 1990 was estimated to be approximately $2 billion (394). There is no doubt that some plants do contain biologically active compounds that are medicinally useful. More than 20% of the commercially prepared drugs originate from plants, but these plants contain many active ingredients that also can provoke adverse reactions. Many people, including physicians, are not aware of the dangerous side effects and sometimes fatal adverse reactions that may occur when using these plants (395).

A.

AsianMedicinal Herbs

Throughout history, infectious diseases have been treated with herbal medications, and scientists at present continue to evaluate and identify their active principles. Various biologically active plants and active ingredients in medicinal plants from Aztec-derived Mexican folk medicines to Chinese herbal preparations have been reviewed (396). Of the traditional Chinese medicinal herbs, 178 were investigated for an anti-Bacteroides fragilis substance. B. fingilis is found predominantly in fecal material and produces butaric acid. The bacterium is often obtained from soft-tissue infections. Only one of these herbs, rhubarb root (Rheunz oflcinnle), was found to possess anti-B. fragilis activity. The active substance subsequently was isolated and shown to be 1,8-dihydroxyanthraquinone (397). Also evaluated against 10 microbial pathogens were 18 herbs. Of these, 11 preparations were active against at least one pathogen, six were active against at least three pathogens, and two were active against five pathogens (398). Chinese medicinal herbs, first used more than 2000 years ago, are still used today to treat heart problems (399). When Chinese and Western medicines were combined in the treatment of coronary heart disease in China, a decrease in the mortality rate from 20-30% down to 10-15% was observed. Extracts of 11 out of 27 Chinese medicinal herbs were active against the human imnunodeficiency virus (HIV), and Chinese medicinal herbs appear to be a rich source of drugs for the treatment of HIV (400). Immunomodulatory activity was documented in fractions of Astragalus membrmnceus. Fractions from this Chinese medicinal herb fully corrected an in vitro T-cell function deficiency in cancer patients (401). When 10 Korean medicinal herbs were evaluated for mutagenicity by the Ames test, false-negative reactions were obtained. The substances that inhibited the production of positive reactions were removed through solvent fractionation, which improved the reliability of the results (402). Morimoto et al., while examining 104 medicinal herbs, also found that medicinal herbs contained cytotoxic materials that can limit the applicability of the Ames test (403). Of Pakistani medicinal herbs or mixtures used in treating children, 10 were tested with the Ames test. Extracts of Peshwar (mixture of unknown herbs), Saussuren lappa, Swertia chiraitn, and Skimmia lulrredn were mutagenic. The addition of liver microsomal enzymes increased the activities of two extracts. 1. Adulterated Herbal Preparations There is no doubt that herbal medicines contain effective drugs for specific illnesses. Unfortunately, the herbal medications usually are not prescribed by individuals with the

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scientific knowledge of their contents, and the herbs may contain many biologically active components. At times, herbal preparations are laced with drugs to improve their effectiveness (395). The manufacturers of some Chinese herbal medicines for the treatment of arthritis and back pain have adulterated these herbal products with aminopyrine and phenylbutazone (probably to promote the commercial potential of their products). Both compounds are well-known causes of agranulocytosis and have caused many fatalities. Aminopyrine was removed from over-the-counter sale in the United States in 1938 (404). In addition to adulteration by humans, herbal drugs also may contain mycotoxins and mycotoxin-producing fungi (405).

2. DiverseBiological Activities Because of the increased popularity of natural foods, health foods, and herbal products, the public needs to be aware of and concerned about the potential dangers associated with extensive use of these herbal products (406). The biological activities found in a single herb are truly diverse. A given herb usually will contain many components having quite varied, and often opposite, biological activities. At least 25 psychoactive substances have been identified in herbal preparations, and a number of intoxications have resulted from their use. Plants used in herbal preparations that contain psychoactive substances include broom. California poppy, catnip, cinnamon, hops, hydrangea, juniper, kola nut, nutmeg, periwinkle, thorn apple, and wild lettuce (407). Natural pesticides and other biologically active materials in health foods and herbal products constitute a pharmacopoeia of uncontrolled substances in our nation's health food stores.

3. PyrrolizidineAlkaloids Consumption of herbal medicines that contain pyrrolizidine alkaloids may contribute to the high incidence of chronic liver diseases, including cancer, in Asia and Africa (408). In one study, three of 50 medicinal plant species from Sri Lanka contained pyrrolizidine alkaloids (409); these were Crotcdnrin ver'rucosnL., Holarrhenn antidysenterica (L.) Br., and Cassia nuriculata L. In another study of 75 medicinal plants from Sri Lanka, only Crotnlaria juncea L. contained pyrrolizidine alkaloids. Of the other plant species not containing pyrrolizidine alkaloids, three produced hepatic lesions in rats, and two produced marked renal lesions (4 10). Pyrrolizidine alkaloids can cause cirrhosis of the liver and occur in at least eight plant families (41 1). They are genotoxic and mutagenic (412). Pneumotoxicities may result from pyrrolizidine alkaloids (413). These compounds also crosslink DNA (414). A single intravenous (IV) 3.5 mg/kg body weight dose of monocrotaline pyrrole produced delayed pulmonary microvascular leak, interstitial inflammation, and pulmonary hypertension after 14 days in rats (415). A single oral dose of 120 mg/kg body weight of monocrotaline produced a variety of biochemical and pathological liver changes in rats (416). The guinea pig appears to be metabolically resistant to pyrrolizidine alkaloids (417) and male rats are more resistant than females (418). Humans exhibit wide variations in their ability to metabolize these compounds (419). Pyrrolizidine alkaloids contain a basic moiety consisting of one nitrogen at the bridgehead of two five-membered rings (Fig. 18a). Cordell has reviewed the chemistry of these highly biologically active alkaloids (41 1). Indian herbal teas caused hepatic venoocclusive disease in people who had consumed them (420). The herbs, identified as Heliotropium lnsiocnrpum Fisch. and Mey., contained pyrrolizidine alkaloids. The total alkaloid content of this herbal mixture was 0.47%, dry weight. The major compounds identified were heliotrine and lasiocarpine (Fig. 18b), and the minor constituents were europine and

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Heliotrine

Lasiocarpine

Figure 18 Pyrrolizidine alkaloid chemical structures: (a), basic structural moiety containing one nitrogen at the bridgehead of two five-membered rings; (b), major compounds identified are heliotrine and lasiocarpine.

heleurine (421). The LDsoof heliotrine is 300 mg/kg body weight, and of lasiocarpine is 72 mg/kg body weight in rats (420). Comfrey (SymphytLlm species) is an herb used as a green vegetable, beverage, or remedy. The leaves and roots of a species from Japan (Synzphytunl offkinale)were hepatocarcinogenic in rats; this species contains at least eight pyrrolizidine alkaloids. Large differences in alkaloid concentrations occur between young and old comfrey leaves. The large, mature leaves have the lowest concentrations (422). The alkaloid contents of dried leaves are 0.003-0.2%, and those of dried roots are 0.2-0.4%. The amount of alkaloid consumed in a cup of comfrey root tea is 12-36 mg. A gelatinous residue forms during the process of making the tea; if it is eaten, as much as 26 mg of alkaloids could be consumed. Reliable data on effects of comfrey on humans are scarce, but available data indicate that the use of comfrey root tea could have serious health consequences (423). Comfrey also may be contaminated with other plants, for instance, deadly nightshade (424). It has been recommended, in no uncertain terms, that comfrey be removed from public consumption (425).

B. OnionandGarlic Historically, people have taken onion and garlic juices as a remedy for a long list of ailments. Both onion and garlic juices can prevent the rise of serum cholesterol after a fatty meal (426). Garlic inhibits lipid synthesis; reverses cholesterol-induced atherosclerosis in rabbits; decreases serum cholesterol, triglycerides, low-density lipids (LDLs), and verylow-density lipids (VLDL); increases high-density lipid (HDL) levels (391); and onion and garlic oils have anticancer activity (427). However, in high doses, wild garlic can cause gastroenteritis, diarrhea, rash, and leukocytosis. Long-term ingestion of wild garlic or onion also will block iodine uptake by the thyroid (395).

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Epidemiological studies have demonstrated an inverse correlation between the dietary intake of garlic and onion and stomach cancer risk (428, 429). The antineoplastic effect of these plant oils may be due to the presence of an allyl group containing organosulfur compound (430-432). Eight organosulfides were tested for their inhibitory effects on benzo[a]pyrene-induced neoplasia of the forestomach and lung in the mouse (431). Of the organosulfides tested, diallyl sulfide (DAS), allyl methyl trisulphide, allyl methyl disulfide (AMD), and daillyl trisulfide inhibited benzo[cr]pyrene-induced forestomach tumors, and DAS and AMD also inhibited pulmonary adenoma (43 1). Studies were undertaken to elucidate the mechanism of the antineoplastic effect of DAS (427). It was observed that DAS exerts an antineoplastic effect by modulating glutathione (GSH) dependent detoxification enzymes. Stomach GSH peroxidase activity was increased in a dose-dependent manner. The GSH peroxidase activity also was elevated in the lungs of female CD-1 mice treated with DAS, but it was not dose dependent (427).

C. Yarrow Herbs and natural herbal preparations, in most cases, do not have just a single active component; rather, they have an elaborate array of biologically active components. For example, over 40 indigenous naturally occurring chemical components of the herb yarrow (Adzillen rnillefoliurn) and their biological activities are listed in Table 9 (433, 434). Yarrow is reportedly a hemostatic herb, but it also contains coumarins, which are anticoagulants (433). Thus, varied biological activities can be manifested from crude plant preparations when the proportions of indigenous chemicals change as a result of variable growing conditions or crop treatments during and after the growing period. D. HerbalTeas 1. Chamomile Chamomile tea is a herbal drink commonly sold in supermarkets; people may have allergic or anaphylactic reactions to it. Allergens from chamomile flower heads cross-react with ragweed, chrysanthemums, or other species of the family Compositae (406). The MexicanAmerican population in southern Colorado commonly treat childhood illnesses with the tea. However, chamomile is low insodium, and its continued use without other food intake can cause water intoxication with subsequent hyponatremic seizures (435). Excessive use of chamomile also can cause diarrhea (395). 2. Sassafras Root Herbal teas are used by many people for medicinal purposes as well as for enjoyment. The carcinogenicity of safrole, the natural flavor in root beer, was discovered in 19601961. This discovery led to its banning as a food additive (436). The essential oil of sassafras is about 75% safrole (437). Sassafras root bark continues to be freely available in health food stores despite evidence indicating its carcinogenicity, and despite legal restrictions prohibiting the use of safrole in foods. Safrole is hepatocarcinogenic in rats and mice and is a major constituent of the oil of sassafras root bark (438).

E. Bay Leaf At least one herb, bay leaf, exhibits some biological effects because of its physical size and shape. Bay leafcan become physically stuck in the pharyngeal pouch (439), becoming

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Table 9 Natural Chemicals in the Herb Yarrow (Achillea millefolium) and Their Biological Activities

Chemical Achilleine Achilletin Apigenin Atulene Betaine Borneol Bornyl acetate 6-Cadinene Caffeic acid Camphene Camphor

Caryophyllene Chamazulene

Concentration (P94.I dry weight)

0-1 40 255 210 8

Humulene lsoartemisia ketone Limonene Luteolin

Insectifuge Antitumor, choleretic, hepatotropic

1779

(LDLoa 990 mg ipr in rats) analgesic, anesthetic, antiseptic, antipruritic, carminative, deliriant, emetic, rubefacient, stimulant

159

959

Essential oil Eugenol

Hemostat Hemocoagulant Antihistaminic, antispasmodic Antiinflammatory, antipyretic Antimyoatrophic, emmenagogue

602

Choline

Copaene Coumarins Cuminaldehyde p-Cymene

Activity

Anodyne, antiinflammatory, antiseptic, antispasmodic (LDm400 mg iprin rats) lipotropic, hypotensive (LDm2480,mg orallyin rats) antibronchitic, antilaryngitic, antipharyngitic, antirhinitic, expectorant, insectifuge

59 11 369

Anticoagulant (LDS0 1390 mg orally in rats) (LDm4750 mg orallyin rats) fungitoxic, insectifuge

1000-1 4,000 (LDm3000 mg orallyin mice) analgesic, anesthetic, antiseptic, fungicide, larvicide

22 860

171 Antiinflammatory, antispasmodic, antitussive

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Table 9 Continued

Concentration (&I dry weight)

Activity Chemical

(LDW3180 mg orallyin rats) analgesic, anesthetic, counterirritant, antipruritic

Menthol Myrcene a-Pinene

22 941

&Pinene Quercetin

713

Rutin

Sabinene Salicylic acid

Ailelochemic, beetle-attractant,expectorant Expectorant, insectifuge (LDm 161 mg/kg orallyIn rats) antiinflammatory, antispasmodic (LOW950 mg/kg ivnin mice) antiatherogenic, antiedemic, antiinflammatory, antithrombogenic, hypotensive, spasmolytic, vasopressor

1235

0-Sitosterol

Stachydrine Tannins a-Terpinene 7-Terpinene Terpinen-4-ol

131 371 431

Terpinolene Thujone abortifacient

48

Tricyclene Triganelline

27

(LDW891 mg/kg body weight orally in rats) analgesic, antipyretic, antirheumatic Antihypercholesterolemic, antiprostatitic, antiprostatadenomic, antitumor, aphrodisiac, estrogenic Cardiotonic Antidiarrhetic, bactericide, viricide Insectifuge

Antiallergenic, antiasthmatic, antiseptic, antitussive, bactericide, expectorant, fungicide, insectifuge (LDLo 120 mg/kg body weight ipr in rats) (LDLo 5000 mglkg body subcutaneously in rats) hypoglycemic

-~~

~~~

"DLo, lowest dose proven lethal in experimental animals. Source: From Refs. 433 and 434.

lodged in the mucous membrane, and blocking the esophagus. It also can rupture Meckel's diverticulum, resulting in severe rectal pain (440). Panzer suggests that bay leaf complications may be a more important source of morbidity than the present literature suggests. Perioric dermatitis can be caused by bay leaf, marjoram, and cinnamon; its cause and pathogenesis are not known (441).

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Table 10 Compounds in Bishop's Weed (Amnzi mujus L.) Seed

Compound Alloimperatorin Ammirin' Bergapten Graveoloneb Heraclenin Herniarin' lsoimperatorin' lsopimpinellin' Marmesin Marmesinin Oxypeucedanin Oxypeucedanin hydrate Pabulenol Saxalin Umbelliferonea Umbelliferone-(3'-hydroxymethyl-1 t.-buten-l '-yl)-ether' Umbelliferone-(3'-methyl-buta-lt.3-dien-1 '-yl)-ether' Xanthotoxin Total psoralens

1 400-31 00 100-8000

< 100 2300 400-3300 100-1 450 3000

c 100


c 100

2300-1 0,000 15,000-20,000

'See Ref. 444. 'See Ref. 435. cSee Ref. 346. Source: From Ref. 443.

F. Bishop's WeedSeed Bishop's weed (Amrrzi majus L.) is an annual plant that is used for the cut-flower trade and for medicinal purposes (442). The chemical contents of the herb's seed are shown in Table 10 (443-446). Bishop's weed has been used to treat skin depigmentation in the Middle East for centuries, and this weed also induces phototoxic responses in livestock and poultry (446). The compounds in Bishop's weed can cause cataract formation in both humans and animals (447). See Sec. XI for a discussion of the biological properties and mode of action of the furanocoumarins found in Bishop's weeds and other similar plants.

G.

RosemaryandSage

Rosemary and sage are commonly used herbs that contain bioactive components. Extracts from both plants are toxic to yeasts such as C c ~ d i d ncdbicam, causing cell-wall destruction and impairment of metabolism. Eucalyptol appears to be the fungicidal constituent in these herbs (448). Compounds found in rosemary (Rosrnnrir?nsofJicinnlis) are listed in Table 11 (434). There also are known allergic symptoms from ingesting sage (Salvia ojjicinalis). Small amounts (like those used in dressings) can cause various symptoms, including severe headache, nausea, and vomiting, that may be reduced by taking caffeine soon after the onset of symptoms. Compounds found in sage are listed in Table 12 (434).

Naturally Occurring Toxic Chemicals in Foods Table 11 Compounds in Rosemary (Rosnrarirms oficirralis)

Activity Compound

Concentration (ccg/g dry weight)

Borneol

120-470

Camphor

539-291 0

Carvacrol Carvone 1,8-Cineole

852-5 1 20

Essential oil

4000-1 9,000

Epirosmanol GIycoIIc acid lsorosmanol Linalool

40-1 20

a-Pinene

1030-3226

Rosmarinic acid Rosmanol Safrole

32-95

Terpinen-4-01

10-520

Thymol

Ursolic acid

39,000

(LDLoa 2000 mg/kg body weight orally in rabbits) Carminative, deliriant, analgesic, anesthetic, antiseptic, antipruritic, rubefacient, stimulant (LDso810 mg/kg body weight orally in rats) anthelmintic, antiseptic, fungicide, tracheorelaxant (LDso1640 mg/kg body weight orally in rats) carminative, CNS stimulant, insecticide (LDso 2480 mg/kg body weight orally in rats) antibronchitic, antilaryngitic, antipharyngitic, antirhinitic, expectorant, insectifuge Has pesticidal and medicinal properties Antioxidant Diuretic Antioxidant (LDw 2790 mg/kg body weight orally in rats) anticonvulsant, antiseptic Allelochemic, beetle attractant, expectorant Antioxidant (LDs0 1950 mg/kg body weight orally in rats) anesthetic, antiseptic (LD= 4300 mg/kg body weight orally in rats) antiallergenic, antiasthmatic, antiseptic, antitussive, bactericide, expectorant, fungicide, insectifuge (LDS 980 rng/kg body weight orally in rats) bactericide, fungicide, larvicide, tracheorelaxant, vermicide Antitumor (EDso= 50 mg/kg body weight), diuretic

“LDLo, lowest dose proven lethal i n experimental animals. Ref. 334.

Sot4i-ce: From

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Table 12 Compounds in Sage (Salvirr oflcinalis)

Concentration (P9b dty weight)

Compound

-324

81

Borneol

140-2636

Bornyl acetate Camphor

57-656 28-1 410

Carnosic acid 1,&Cineole

550-541 0

p-Cymene Essential oil Labiatic acid Linalool

30-856

7000-10,000 191

1

-3500

a-Pinene acid Rosmarinic Salvin Terpinen-4-01 29-1

Thymol

21,000

acid

Ursolic

2000 018

Activity (LDLoa2000 mg/kg body weight orally in rabbits) Insectifuge (LDLo 900 mglkg body weight IPR in rats) analgesic, anesthetic, antiseptic, antipruritic, carminative, deliriant, emetic, rubefacient, stimulant Antioxidant (LDm 2480mglkg body weight orally in rats) antibronchitic, antilaryngitic, antipharyngitic, antirhinitic, expectorant, insectifuge (LDm 4750 mg/kg body weight orally in mice) fungicide, insectifuge Has pesticidal and medicinal properties Antioxidant (LDm2790 mglkg body weight orally in rats) anticonvulsant, antiseptic Allelochernic, beetle attractant,expectorant Antioxidant Bacterlclde (LD, 4300 mglkg body weight orally in rats) antiallergenic, antiasthmatic, antiseptic, antitussive, bactericide, expectorant, fungicide, insectifuge (LD, 980 mg/kg body weight orally in rats) bactericide, fungicide, larviclde, tracheorelaxant, vermicide Antitumor (EDm = 50 mglkg), diuretic

"DLo, lowest dose proven lethal in experimental animals. Source: From Ref. 434.

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77

Abortifacients H.

Sage, along with rue (Ruta gruveolens L.), apiol, cohosh, and pennyroyal oil are used by women in the United States as herbal abortifacients. The reasoning for turning to herbal substances in part is thought to be a movement away from conventional medical services. The following are three reasons for not using herbal abortifacients (449): (1) there is a chance of toxic reactions with increased dosages of herbal preparations; (2) herbal preparations, if unsuccessful, could have a teratogenic effect on the developing child; and (3) by delaying an abortion, through failure of an herbal remedy, a women could significantly increase her risks for a later, induced abortion. Individuals of Spanish and Mexican descent in New Mexico have used a number of plants other than those mentioned above for emmenagogues and abortifacients (450). Some of these plants are cotton root bark (Gossypium species), inmortal (Asclepias capric o r m Woodson), poleo chino [Hedeoma obloqifolin (Gray) Heller], and wormseed (Ctzenopodiurn arnbrosioides L.). When used as an abortifacient, cotton root bark seems to exhibit the lowest toxicity. Two to four ounces of fresh root bark is boiled in one quart of water for one-half hour. The entire decoction is drunk the first thing in the morning, and the abortion occurs 2-6 days later, with about a 30% success rate (450). The attention of the medical profession was attracted to cotton root bark in 1840 because it was a popular abortifacient among Negro slaves. It was then dispensed as a pharmaceutical drug having a weak stimulating effect upon the uterus, as well as a vasoconstrictor action (451). 1.

PsychoactiveSubstances

A 400-mg dose of myristicin (Fig. 3), a psychoactive substance, administered to humans will produce cerebral excitations, and larger doses may produce hallucinations (452). Myristicin is found in carrots at 0.6 pg/g (54), which means it would require about 1468 lb of carrots to produce hallucinations. Nutmeg contains approximately 2.5% myristicin (453). A dose of 16-20 g of nutmeg would be expected to cause a narcotic or hallucinogenic effect. See Ref. 454 for historical aspects of nutmeg, its psychoactive components, and metabolic studies.

VII.

MUSHROOMS

About 50 volatile compounds have been identified from each of eight species of edible fresh mushrooms, including Cantlzarellus, Cibnrius, Gyromitra esculenta, Boletus edulis, Lnctm-iustrivialis, Lmtarius torminosus,Lactarius rrgus, and Agaricusbisporus. The main volatile compound in most fresh wild mushrooms is 1-octen-3-01.In the commonly cultivated Agaricus bisporus, benzyl alcohol is the most abundant and 1-octen-3-01 is the second most abundant volatile (455). Two edible mushroom species contain hydrazine analogs: Agaricus bisporus contains p-N-[y-~-( +)-glutamyl]-4-hydroxyn~ethylphenylhydrazine (agaritine, GHPH, Fig. 19) and Gvromitra esculenta (wild false morel) contains acetaldehyde N-methyl-N-formylhydrazone (gyromitrin, acetaldehyde MFM, Fig. 20). Gyromitrin readily yields various hydrazines that are toxic and hepatocarcinogenic (456).

A.

Agaricus bisporus

Agaricus bisporus is the main mushroom of commerce in the United States (457), and the hydrazine analogs isolated from it are shown in Table 13 (458-462). Fresh A. bisporus

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CHZOH

I

Q

NH3+

0

HN-NH -!-CH2CH2CL

-COO'

Agaritine

CH20H

I

+

0 II L-Glutamic acid

4-Hydroxymethylphenyt hydrazine

4-Methylphenyl hydrazine Figure 19 Hydrolysisproducts hydrazine).

of agaritine (p-N-[y-L-( +)-glutamyl]-4-hydroxymethylphenyl-

contains as much as 700 pg/g of agaritine, and fresh frozen mushrooms contain as much as 300 pg/g. Cooking or canning with water destroys the agaritine, but cooking in olive oil at 300°C for 7 min left 300 v g / g of agaritine (461). Agaritine does not induce tumors in mice (463, 464), but forms 4-hydroxymethylphenylhydrazine as a breakdown product, which can be transformed into 4-methylphenylhydrazine (4-MPH) in vitro (460,465) (Fig. 19). Soft-tissue tumors developed at injection sites after 4-MPH was administered to mice (466). 4-MPH was the first diazonium compound discovered to be a rodent carcinogen (467). The N'-acetyl derivative of 4-hydroxymethylphenylhydrazine is an enzymatic product of agaritine. When it was given to Swiss albino mice, the lung tumor incidence rose

Naturally Occurring Toxic Chemicals in Foods

79

I

Gyromitrin H

HpN-N, 0CH3

f;=O

+

CH3-CHO

k N-Methyl-N-formyl hydrazine

N-Methylhydrazine Figure 20 Chemical structure of gyromitrin and its hydrolysis products produced in vitro and in vivo. (From Ref. 479.)

Table 13 Hydrazine Compounds Isolated from Fresh Agaricus bisporzls Mushrooms

Concentration (pglg fresh weight)

Compounds ~

p-Hydrazinobenzoic acid (HB)" M-[yL-(+)-GIutamylJ-4-carboxyphenylhydrazine (GCPH)b /@-[y-L-( )-Glutarnyl]-4-(hydroxymethyl) phenylhydrazine (agaritine, GHPH)" 4-(Hydroxymethyl)benzenediazoniumiond

+

~~

5 e e Ref. 458. %ee Ref. 459. 'See Refs. 460 and 461 dSee Ref. 362.

10.7 42.0

400-700 0.6

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25% in females and 26% in males, and the incidence of blood vessel tumors rose 24% in females and 25% in males (468). Subcutaneous (s.c.) injections of 4-MPH into Swiss mice induced a 36% incidence of lung tumors in females and a 44% incidence in males, whereas intragastric treatments caused a 40% incidence of lung tumors in females (468). Agaritine also breaks down to the 4-(hydroxymethy1)-benzenediazonium ion (HMBD) (465, 469). Subcutaneous injections of 4-(hydroxymethyljbenzet~ediazoniun1tetrafluoroborate were administered to Swiss mice and induced tumors in the subcutis and skin in a concentration-dependent manner (470). A high and low dose of the sulfate form of HMBD was given (s.c.) to Swiss mice. Tumors of the subcutis and skin developed in 32% and 14% of the females and 40% and 4% of the males, respectively (471). A single intragastric instillation of 4-(hydroxymethylj-benzenediazoniumtetrafluoroborate caused glandular stomach tumors in an incidence of 30% and 32% in female and male Swiss albino mice, respectively (469). B.

Gyromitra esculenta

Gyror~itruescuZer?tn (false morel) is highly poisonous fresh, but edible when cooked by boiling (472). Toxicity is caused by the volatile indigenous hydrazone, gyromitrin (473). Concentrations of hydrazone analogs isolated from Gyrorzlitrn esczdelztn are shown in Table 14. During cooking, care also must be taken because highly toxic vapors are volatilized from the mushrooms (472). Boiling for 10 min is required to reduce hydrazone concentrations below 1 pg/g. A minimum of 3 L of water is recommended per kilogram of mushrooms. False morels can be used as food after cooking or drying (474). The LDSo’sof hydrazine and hydrolysis products of gyromitrin in various animals are presented in Table 15 (475-477) (Fig. 20). Hydrazine and N-methylhydrazine are toxic to Escherichin coli and N-methyl-N-formylhydrazine (MFH) is not, but MFH is more toxic than hydrazine to mice (476). Rabbits are more susceptible than rats or mice to gyromitrin. and dogs are more susceptible than rats or mice to N-methylhydrazine or hydrazine. The route of administration (intraperitoneal, intravenous, or oral) essentially

Table 14 Compounds and Concentrations in False Morel Compounds

N-methyl-N-formylhydrazine(MFH) (dry weight)a Methylhydrazine” N-methyl-N-formylhydrazones (MFHO) Acetaldehyde (gyromitrin) Propanol Butanol 3-Methylbutanol Pentanal Hexanal Octanal trans-2-Octenal cis-2-Octenal “See Ref. 365. Source: From Ref. 473.

Concentration fresh weight)

(&

500.0 14.0 49.9 1.o 0.6 2.2 0.8 1,4 0.2 0.6

0.3

Naturally Occurring Toxic Chemicals in Foods

81

Table 15 LDS,,of Hydrazine and the Hydrolysis Products of Gyromitrin in Various Animals ~~

~~

~

~

~~

~~

LDmconcentration(pglg body weight) N-Methyl-NAnimal Gyromitrin formylhydrazine N-Methylhydrazine Hydrazine

-

-

Miceb

344

159

12 124

Rabbit" Rata

70 320

-

-

Doga

-

33

25

(low toxicity)

-

55

'See Ref. 475. bSee Ref. 476. 'See Ref. 477.

has no influence on the toxicity of these compounds (475). The difference between the no-effect amount and a lethal dosage of N-methylhydrazine is small in apes (477), but there is a large variation in individual human tolerances, as indicated by case histories (472). Calculated lethal doses for children and human adults, based on reports of false morel intoxication, are 10-30 pg/g and 20-50 pg/g body weight, respectively (474,478). N-Methylhydrazine caused a decisive increase of lung tumors in Swiss mice, whereas it caused malignant histiocytomas and tumors of the cecum in hamsters (468). Gyromitrin can cause serious liver injury. When given by intragastric administration to Swiss mice, it also caused tumors of the lungs, preputial glands, forestomach, and clitoral glands (468). Gyromitrin forms MFH and N-methylhydrazine upon hydrolysis (479); Swiss nice treated with MFH produced tumors of the liver, lungs, gallbladder, and bile duct. MFH-treated hamsters developed liver cell tumors, malignant histiocytomas, and tumors of the gallbladder and bile ducts (468). Both gyromitrin and N-methylhydrazine are mutagenic and bactericidal. As much as 20-30% of the hydrazones in the false morel decompose during cooking to N-methylhydrazine, which is present in the steam (480). However, the hepatotoxic and carcinogenic MFH is more mutagenic after metabolic transformation (481) into the highly reactive Nnitroso-N-methylformamide(NMFA) by liver microsomal monooxygenase (479). NMFA is unstable and can spontaneously decompose to molecular nitrogen, formic acid, and a methyl cation alkylating agent (482). Cytochrome P-450 is required for the biological activity of gyromitrin or other hydrazines that result in the destruction of cytochrome P450 during their activation. Hydrazine exposures that decrease liver cytochrome P-450 levels may cause subsequent xenobiotic exposures to be toxic. Pretreatment with the cytochrome P-450 inhibitor, SKF-525A, abolishes the loss of cytochrome P-450 by N-methylformylhydrazine. Braun et al. suggest that the methyl cation formation from nitrosamide could be responsible for the hepatocarcinogenicity of MFH (479), but hydrazine carcinogens also may act by producing oxygen radicals (1).

VIII. MYCOTOXINS The term ~~~ycotoxi~z is derived from the Greek word mvkes, meaning fungus, and the Latin word toxicunz, meaning poison (483). Mycotoxins are toxic compounds produced by fungi

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on grains and other foods and can be health hazards to both animals and humans. Mycotoxicosis is the toxicity syndrome resulting from the intake of mycotoxin-contaminated foods. Fungal diseases and mycotoxicoses are discussed in detail in Volume 3, Part C of the Foodhome Disease Handbook. The following is a presentation of the overall global perspective of mycotoxins in foods and an overview on the most recently discovered hazardous mycotoxins, fumonisins B1 through B4 (Fig. 21), in foods. A.

A Global Perspective of Food Safety

The outbreak of turkey X disease in England during 1960 was caused by aflatoxin, a mycotoxin. This led to the realization that low levels of mold metabolites in feeds and foods could cause disease in animals and humans (484). Despite impressions that mycotoxins are recent causes of diseases, they were recorded globally as early as 5000 years ago in China (485). Schoental discussed how mycotoxins might have played an important part in the biblical account of the 10 plagues of Egypt (486). During the 16th century, ergot poisoning caused what is recognized as the first reported mycotoxicosis of man (487). Ergot and ergotism are reviewed in Ref. 488. An epidemic apparently resulting from ergotism was reported in 430 B.C. in Sparta (489) and also in France in 1951 (488). As late as 1962, mycotoxicosis was described by Hayes as the “neglected disease” (490). Widespread public interest in mycotoxins was aroused by the discovery that “yellow rain” in Southeast Asia and Afghanistan contained mycotoxins. By 1982, over 100 mycotoxins were described (491j, and by 1988, over 200 mycotoxins were structurally characterized (492). Mycotoxins are nonantigenic, small molecules that usually have a molecular weight below 500 (493). Molds that produce mycotoxins are quite ubiquitous. Problems in the United States resulting from mycotoxins are very similar to those of the rest of the world as similar mycotoxins occur worldwide. Since molds know no borders, the same fungal species that produce aflatoxin in India or Africa will produce aflatoxin inthe United

Furnonisin Al: R1= COCH3, R2 = OH, R3 = OH Furnonisin A2: R1 = COCH3,R2 = OH, R3 = H Furnonisin B1: Rl = HI R2 = OH, R3 = OH Fumonisin B2: R, = H, R2 = OH, R3 = H Fumonisin 63: R1= H, R2 = HI R3 = OH Furnonisin Bq: R, = H, R2 = H, R3 = H Figure 21 Fumonisins, cancer-promoting mycotoxins from Fzrsnr-iunt nzoni1iforr)te.

Naturally Occurring Toxic Chemicals in Foods

83

States. The major mycotoxins, ranked in declining order of their relative worldwide importance based on an opinion poll of researchers in 30 countries, are aflatoxins, ochratoxin A, trichothecenes, zearalenone, deoxynivalenol, citrinin, sterigmatocystin, patulin, cyclopiazonic acid, nivalenol, stachybotrys toxin, diplodia toxin, ergot, and phomopsin (494). Cyclopiazonic acid is toxic to animals and mutagenic to S. typhimurium strain TA98 in the Ames test. Cyclopiazonic acid is produced by several species of Aspergillus and Perzic i l l i m and has been found in co-occurrence with aflatoxins in corn and peanuts in Georgia (495) using an HPLC (high-performance liquid chromatography) (496). We are now obligated to add the fumonisins to the list of major mycotoxins of worldwide importance. Based on projected relative importance, the fumonisins must be listed close to aflatoxin. Schlatter describes various mycotoxicoses resulting from contaminated foods and thoroughly examines these events (497). Humans are exposed to mycotoxins through several different avenues, including (1) cereal crops, (2) meat products, (3) milk and eggs, (4) peanuts (490), (5) occupational exposure (498), and (6) fruit products and juice (499, 500). Many mycotoxins were first studied as potential antibiotics in the 1930s and 194Os, although they almost invariably were too toxic to higher life forms to be of value. In addition to acute toxicities, aflatoxin B I , sterigmatocystin, and penicillic acid are also potential carcinogens (484). In fact, aflatoxin B is one of the most potent, if not the most potent, carcinogen found in nature (484, 501j. and is carcinogenic in rats, trout, and rhesus monkeys. sterigmatocystin is a liver carcinogen in rats and mice. The major aflatoxin-producing fungi are Asper.giZZusJ m u s and A. parasiticus, and the major aflatoxins are aflatoxin BI, B2, G I , and G2 (502). The discovery of naturally occurring carcinogens in such conmon human foodstuffs as corn, milk, and peanuts was so surprising that, for a short time, the animal toxicoses were nearly forgotten (493). Corn and peanuts are contaminated far more easily by mycotoxin-producing fungi compared to other foodstuffs. Fu-Sun and Kong-Nien suggested that corn is more important in China because corn is a staple food (501), but others believe that, on a worldwide scale, peanuts are consumed in the highest quantity. With few exceptions, the percentages of corn in Chinese staple foods correlated well with the observed mortality rates from liver cancer (501). However, as in Sri Lanka, the consumption of herbal medicines containing hepatotoxic substances also could be of importance in chronic liver disease. Indeed, if aflatoxin is the most significant cause of liver disease in China, consumption of herbs may contribute synergistically to the development of liver disease (409). Commodities that have mycotoxin-contamination problems are apple juice, barley, cheese, corn, cottonseed, milk, peanuts, rice, sorghum, tree nuts, and wheat. The United Nations (UN) Food and Agriculture Organization (FAO) estimated that 25% of theworld’s Food crops are contaminated by mycotoxins. This includes 10-50% of the grain crops in Africa and the Far East (503). Conditions that lead to mycotoxin contamination of feeds and foods are reviewed in Ref. 504. Extensive surveys have demonstrated a measurable incidence of aflatoxin residues in U.S. grain mills and of ochratoxin residues in Danish pork (493). B. Food Safety andPublic HealthHazard The significant involvement of mycotoxins in human health was recognized by Wilson (505j. Human illnesses caused by mycotoxins may be a larger public health problem than anyone realizes because a long period elapses before an illness is recognized unless large

84

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amounts of mycotoxins are consumed, resulting in acute symptomology (494). Mycotoxins also are relatively stable to cooking and processing (506); therefore, food-preparation procedures cannot be expected to remove mycotoxins safely. They may be taken up from the soil and are long-lived in plants (507). A small intake of mycotoxins for long periods can be detrimental; thus, endemic nephropathy and primary liver-cell carcinoma may take decades to become evident. Endemic nephropathy or chronic kidney disease took at least 15-20 years to present manifestations in people who moved into a region in Romania that was known for the disease. The age group of individuals primarily affected by the disease in that region was 30-50 years. In Bulgaria, it was noticed that none of the children aged 4-14 years were affected by endemic nephropathy (508). Because of the long-term effects, a direct relationship of human health to mycotoxins as the etiological agents of disease syndromes in humans is difficult to show. A similar case exists in understanding the role that naturally occurring pesticides play in human diseases. Do long-term effects occur from natural pesticides? How do they manifest themselves? The liver and kidneys are important in the metabolism of chemical neurotransmitters that control brain function. In southern Georgia in the United States, consumption of foods that are potential sources of high levels of aflatoxins was significantly related to mental retardation of the local children (509). There also is a reported problem with mental retardation in a region of East Africa known to have high concentrations of aflatoxins in the food supply (5 10). Aflatoxins have been suggested as being involved in Reye’s syndrome (509). A review of this disease and its possible link with aflatoxin was explored (51 1j. Although the data linking aflatoxin to Reye’s syndrome are preliminary, there is evidence in favor of a role for aflatoxin in some cases. Reye’s syndrome occurs in epidemic proportions in children in northeast Thailand and is characterized by vomiting, hypoglycemia, convulsions, and coma followed by death (512). Autopsy reports of victims in Thailand have shown aflatoxin B I in human tissue specimens from 22 of 23 cases (5 13). Results showed that aflatoxin B I appears to be linked to Reye’s syndrome and supports the suggestion that aflatoxin B may be involved in its etiology (490). Denning concluded that the data linking aflatoxin exposure to Reye’s syndrome was preliminary (5 11). 1. EstrogenicAgents In Canada during 1980, there were epidemic proportions of fungi causing mycotoxin contamination of human foods and animal feeds. The predominant mycotoxin was identified as 4-deoxynivalenol (vomitoxin or DON, Fig. 23) (514). Livestock in northern Bel,’olum fed moldy feeds containing mycotoxins such as the estrogenic zearalenone and zearalenol (Fig. 17) may produce milk and milk products that contain these estrogenic substances (515). Estrogenic agents can increase the plasma levels of cholesterol and triglycerides in females, and an association between oral-estrogen use and myocardial infarction and stroke has been described (516). Patients treated for prostate cancer with the estrogen diethylstilbestrol suffered increased mortality rates from cardiovascular disease (517). Excessive amounts of estrogens, regardless of their structure, are known to affect conception, induce thromboembolic disease, and cause liver hepatomas (5 18j. However, there is a conflict in the understanding of risks and benefits of estrogen replacement in postmenopausal women. One study indicated that the survival rate of postmenopausal women with estrogen replacement was increased (519). Others have reported increased mortality from myocardial infarction and cerebrovascular accidents in women using estrogen replacement (520, 521).

Naturally Occurring Toxic Chemicals in Foods

85

Because barley used for beer production can contain Fusarium species, Schoental suggests that it also may be appropriate not only to monitor milk for these estrogenic substances but also to monitor beer since many Fusarium species will produce estrogenic substances (515). Zearalenol, though less active than other estrogens, affects the same target organs as do some commonly known estrogens (522). Livestock fodder containing zearalenone (Fig. 17) can produce estrogenism, including abortions, sterility, and vulvovaginitis in pigs and cattle (523). The ability of theFz~sariunrspecies to produce estrogenic metabolites may explain the variations in symptomology observed in mycotoxicosis. Zearalenone also is carcinogenic to mice. A common fungal contaminant of barley is Aspergillus terreus (524), which produces the hypocholesteroletnic agent mevinolin (Fig. 22) as a metabolic product (525). Thus, products prepared from barley might contain this compound. 2. LiverCancer Epidemiological studies indicate that environmental contaminants such as mycotoxins, independently or in combination with hepatitis B virus, may be important factors in primary hepatocellular carcinoma (526). Human liver cancer is concentrated most heavily in areas in which climate and food-storage methods favor growth of Aspergillus Jlavus (487). About one-quarter of a million people die from hepatocellular cancer each year; this disease is one of the major malignant diseases worldwide. Symptoms of liver cirrhosis also are evident in about 80% of the cases. In populations at low risk of hepatocellular cancer, alcoholic cirrhosis is a more-important disease (527). There would appear to be the possibility that some alcoholic cirrhosis cases maybe due inpart to aflatoxins in alcoholic beverages. There is a growing belief that high rates of liver cancer, like those observed in adult males in some countries of tropical Africa, may have multiple causes: food contaminants, herbal teas, and environmental chemicals, among others (528). Epidemiological studies have revealed a positive correlation between esophageal cancer in China and foodstuffs contaminated with fungi. F. morriliforme is reported to be one of the most predominant fungi associated with foodstuffs in the high-cancer area of the Chinese county of Linxian (529-531). Feed contaminated with F. mo~2iZiformeis acutely toxic to pigs (532, 533), horses, sheep, baboons (534), ducklings (534-536), chickens (535, 537-539), turkey poults (535). rats (534,540-546), and monkeys (547). Since primates fed diets containing corn inoculated with F. monilifornze strain MRC 826 developed hepatitis (547), it then follows that F. monilifonne may play a role in the etiology of human liver disease. Afla-

Figure 22 Mevinolin,a hypocholesterolenlicagentproduced barley.

by Aspergillus terreeus found on

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toxins may play a role in alcoholic cirrhosis of the liver in beer drinkers (548); however, F. moniZ(fonnemycotoxins may play an even larger role in that same disease.

3. Leukemia A link has been suggested between mycotoxins and leukemia in humans. Houses in which there have been more than one case of leukemia in Cracow, Poland, are damper than other houses, and molds isolated from such houses produce carcinogens (549). Similar observations have been made in Texas (550). 4. Teratogenic Effects The carcinogenicity potential of mycotoxins has been reviewed thoroughly in Ref. 490. However, a report by Schoental indicates that the role of mycotoxins in human abnormalities has yet to beinvestigated appropriately (522). Some mycotoxins are also potent teratogens; the first indication of such effects were shown for aflatoxin in 1964. Hamsters are the most susceptible test animal, and malformations develop in the head region when aflatoxin is adtninistered on the eighth day of gestation. A number of mycotoxins can induce teratogenic effects when administered to pregnant experimental animals (Table 16). Rubratoxin B is also teratogenic when administered during organogenesis (55 1). Ochratoxin A (Fig. 23) is the only known mycotoxin that can induce fetal abnormalities in a large number of animal species, including chick embryos, hatnsters, mice, and rats. It is also nephrotoxic. Zearalenone (Fig. 17) is teratogenic in pregnant rats and may be teratogenic in humans since other estrogenic agents are known to be teratogenic in both rats and humans (515, 5 18, 552, 553).

C. Ergot Alkaloids inGrain Foods Ergot alkaloids, found in grain foods, have extremely diverse biological activities. The dramatic effect that ergot has on pregnancy has been known for over 2000 years and was used first by physicians for uterine stimulation almost 400 years ago (554). After 1582, ergot became widely used in France, Germany, and the United States to produce uterine contractions, but its dangers finally were recognized. The number of stillborn children had increased greatly after the introduction of ergot and, in 1824, it was recommended

Table 16 Mycotoxin Induction of FetalAbnormalitiesinAnimals Dosage (mglkg-body weight) Animal

Mycotoxin

Hamster, rat Rat Hamster Mouse Chick embryo Chick embryo Mouse Rat

Aflatoxin 6, Ochratoxin A

Patulin T-2 toxin Zearalenone ~~~

~

~~

~

"The dosage units are in pg/egg. Source: From Ref. 522.

1-1.5 1-5 7-20 5 0.5-5' 2-68'

1-1.5 5-1 0

Naturally Occurring Toxic Chemicals in Foods

COOH

0

87

OH

Ochratoxin A

Deoxynivalenol (Vomitoxin) Figure 23 Ochratoxin A anddeoxynivalenol (vomitoxin), worldwide importantmycotoxins.

that the drug only be used in the control of postpartum hemorrhage. See Ref. 554 for historical aspects surrounding ergotism and the medicinal use of ergot. There are three main actions of ergot alkaloids: (1) peripheral, (2) neurohormonal, and (3) adrenergic blockage. The most important peripheral effect is smooth-muscle contraction, typified by vasoconstriction, and uterotonic effects (555). Ergot neurohormonal effects are observed in serotonin and adrenaline antagonism. Adrenergic blocking agents prevent the stimulation of sympathetic nerves by antagonizing the effects of other drugs like epinephrine. Ergonovine is a powerful uterine contractant with low vasoconstrictor action and is used for treatment of postpartum hemorrhage. Ergonovine and ergotamine are examples of adrenergic blocking agents (555). Ergot is produced by the fungus Clcwiceys yurpzwen and is known to be more of a problem on rye than other grains, but it is found on other grains used for food stuffs, including wheat and triticale flour (556). C. pryureu is the cause of gangrenous ergotism in livestock and humans (557). Cereal grain contaminated by ergot alkaloids from the fungus C. purpurea is a wellknown problem (488). Over a 6 year period, a survey was conducted for six pharmacologically active ergot alkaloids in rye flour, wheat flour, and bradbran cereal in Canada (556). Ergonovine, ergosine, ergotamine, ergocornine, a-ergokryptine, and ergocristine were the six alkaloids analyzed. Rye flour was the most Contaminated, with 118 alkaloid-positive satnples out of 128 samples, ranging from 70 to 414 ng total alkaloids/%of flour. One of the samples contained 3972 ng total alkaloiddg flour. The alkaloid content of the wheat flour was much less than therye flour, with 68 alkaloid-positive samples out of 93 samples, with a range of 15-68 ng total alkaloiddg flour. Bradbran cereal had alkaloid concentrations similar to those found in wheat flour, with 29 alkaloid-positive samples out of 35 samples, with a range of 12-69 ng total alkaloiddg cereal. These investigations show that it would be quite possible to obtain an exposure to the very biologically active ergot alkaloids from cereal grain flours in modern society.

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D. ErgotAlkaloids In Cattle Tall fescue (Festuca nrurzdirzncea Schreb.) is a perennial grass grown on approximately 14 tnillion hectares in the United States from Florida to Canada (558). The fungus associated with tall fescue toxicosis in cattle is Acrenlorliurn coermpphinlurn (558, 559), and it is related to the fungus, Clnviceps purpz4rea, which is the cause of gangrenous ergotism in livestock and humans (557). The causative agent(s) for fescue toxicosis have not been demonstrated, but current evidence implicates the ergopeptide alkaloids (558). The presence of ergot alkaloids in animals grazing on fescue has not been investigated. Cattle fed sufficient sclerotia of Clnviceps to develop signs of ergotism did not have detectable alkaloids in the milk or body tissues (560). However. some of the sensitive methods used today would detect lower levels than could be observed earlier. The longterm ingestion of the A. coerzophicrlztrn alkaloids from contaminated meat, milk, or other dairy products could pose a public health concern (558).

E. Fumonisins Fz4scrrizm moniliforrne Sheldon is a phytopathogenic fungus that occurs worldwide on a variety ofplant hosts, including wheat, barley, soybeans,rice, and oats (538) and produces fumonisins. It is one of the mostimportant ear rot pathogens of maize (Zecr mays L.) (561,562). 1. Correlation with Human EsophagealCancer Marasas et al. observed that two known Fuscrrizmz mycotoxins, deoxynivalenol (Fig. 23) and zearalenone (Fig. 17), were at high levels in naturally contaminated corn kernels in the Republic of Transkei, Union of South Africa (563). The main staple food of that republic is corn. Transkei has a high incidence of esophageal and liver cancer (564). It was suggested that Fusarium mycotoxins tnay play a role in the development of tumors of the digestive tract (553, 565-567). It was concluded that food contaminated with F. rnoniliforme was positively associated with the esophageal cancer risk in Transkei (568, 569). A positive correlation was found between the incidence of F. rnonilifornze in homegrown maize and human esophageal cancer (542). 2.

EquineLeukoencephalomalacia

In 1971, several species of fungi obtained from an outbreak of equine leukoencephalomalacia (ELEM) were isolated and cultured on autoclaved corn and fed to donkeys. The corn infected with F. moniltforrne Sheldon produced clinical signs and lesions characteristic of ELEM (570, 571).F. moniliform was shown to produce ELEM under other experimental conditions (534, 572, 573) and was consistently isolated from feed corn in all episodes of ELEM investigated (574). However, commercial pelleted and nonpelleted horse rations also have been implicated as a source of F. morziliforrne (575). ELEM is a fatal neurotoxic syndrome of horses and other equines (534) and often is characterized pathologically by liquefaction necrosis of the white matter of one or both cerebral hemispheres (575). ELEM is a disease that has been reported in the United States since the early 1900s and is known in Egypt, Africa, China, Japan, and European countries (574, 576-578). Outbreaks of ELEM that end in acute deaths are characterized by various neurological signs, including ataxia, head pressing, circling, and blindness of 1-2 days’ duration (579). The levels of fumonisins B and B2(Fig. 21) in feed (579) were determined to be 1.3-27.0 pg/g and 0.1-12.6 pg/g, respectively (580). An HPLC method was used toanalyze feed samples collected between mid- 1985 and 1990 from outbreaks of confirmed and suspected mycotoxicoses in the state of Parani, Brazil

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(581). Fumonisins B1and Br,were detected in 20 and 18 of 21 feed samples, respectively, at concentrations of 0.2-38.5 yg/g fumonisin B and 0.1-12.0 yg/g fumonisin B2.This was the firstreport on the natural occurrence of fumonisins in animal feeds from Brazil (58 1). Additional lesions associated with ELEM include perivascular hemorrhage and neuronophagia (574). Corn obtained from an outbreak of ELEM was fed to male Fisher 344 rats, and all rats had multiple hepatic nodules (546). In another study, corn samples associated with ELEM were fed to male Sprague-Dawley rats in a short-term bioassay and caused hepatotoxicity and renal toxicity (545). 3. Esophageal and LiverCancer An isolate of F. morzilifonne strain MRC 826 was obtained from corn collected in Transkei, Union of South Africa, where the esophageal cancer rate is high (563). MRC 826 was found to be highly toxic and to produce ELEM (533); it also was mutagenic (582). The isolate was shown to be hepatocarcinogenic to rats (541, 542). The main mutagen was isolated from strain MRC 826 and was shown to be fusarin C (Fig. 24) (583). Although fusarin C is a potent mutagen, it failed to exhibit carcinogenicity (584). which makes it an unlikely candidate to be involved in the carcinogenic effects of the fungus. Culture material from MRC 826 was fed to a variety of animals and caused ELEM in horses, pulmonary edema in pigs, acute nephrosis and hepatosis in sheep, and cirrhosis, intraventricular cardiac thrombosis, and nephrosis in rats (534). A lifelong feeding experiment of MRC 826 in rats demonstrated that the culture material not only was highly hepatotoxic, but it also was hepatocarcinogenic at low dietary concentrations (542). At dietary concentrations as low as 2%, the culture material caused hepatocellular carcinoma in 80% and ductular carcinoma of the liver in 63% of the surviving rats. Many of the rats also had basal cell hyperplasia of the esophageal epithelia (542). MRC 826 caused significant enhancement of nitrosamine-induced esophageal carcinoma in rats (544). Male white leghorn chickens fed diets containing varying levels of MRC 826 culture material had decreased bursae weights and were immunosuppressed in both primary and secondary immune responses (538). Culture material from this same strain was highly toxic to vervet monkeys (Cel-opithecus pygerytl~us)and caused acute, subacute, and chronic toxic hepatitis (547). A mammalian cell line of MDCK dog kidney epithelial cells was sensitive to fumonisins B and B1 [ICso(dose at which 50% of the tests respond)-2.5 and 2 pglml, respectively] (585). MDCK cells exhibited morphological changes within 24 hr of treatment. Fumonisin B2inhibited renal epithelial cell (LLC-PK) proliferation, possibly by inhibiting sphingolipid biosynthesis, resulting in cell death after a 24-hr lag period (586).

Figure 24 Fusarin C is the main mutagen isolated from F~rsariu~n rnonilifornte strain MRC 526.

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4. SphingolipidBiosynthesis Early biochemical findings show that fumonisins B and B (Fig. 21) are the first naturally occurring inhibitors of sphingosine and sphinganine N-acyltransferase in rat primary hepatocytes (587). The fumonisins bear considerable structural similarity to the backbone structure of sphingolipids. Fumonisins inhibit the conversion of sphinganine to N-acylsphinganines. The disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases associated with fumonisin consumption (587). Since altered levels of sphinganine and sphingosine were observed before other biochemical changes or visual signs from ponies with fumonisin exposure, monitoring of these sphingolipids may be used as early markers of exposure to fumonisins (588). It was determined in serum of pigs with exposure to fumonisin-containing feeds that the serum sphinganineto-sphingosine ratio was an early biomarker of fumonisin exposure (589).

5. CancerPromoter Utilizing a short-term cancer initiation-promotion bioassay, the cancer-promoting activity in cultures of F. n.rorziZ(foi-rnestrain MRC 826 was isolated (540) and chemically characterized (590). The cancer-promoting effect of fumonisin B l in rats was associated with a toxic effect, and the principal pathological change was an insidious and progressive toxic hepatitis similar to that of the culture material of F. n.ronilifoi-v~e strain MRC 826 (540). Four fumonisins were isolated and characterized, fumonisins A and A?and fumonisins B I and B? (Fig. 21). Fumonisins Aland A? are acetyl amides. Since Plattner et al. were unable to observe A, and A2 in extracts, it was thought that they may be artifacts as a result of the NH2 group on fumonisins B I and B2 reacting with acetic acid from the chromatography solvent system (591). However, fumonisins A , and A? were isolated by Cawood et al. and their findings confirmed that there was no indication that either fumonisin B or fumonisin B2 was converted into these compounds (592). This work confirms the earlier work by Bezuidenhout et al. showing that fumonisins A , and A? are metabolites produced by F. mordifor7ne (590). 6. Biological Activities of Pure Fumonisin B, The first experimental evidence that fumonisin B I isolated from F. mo~ilifomecaused ELEM was presented by Marasas et al. (577). A horse was injected intravenously 7 times over a period of 9 days and on Day 10 was euthanized. The principal lesions were severe edema of the brain and early, bilaterally symmetrical, focal necrosis in the medulla oblongata (577). Fumonisins B I and B2 were identified first by Norred et al. (543) and Voss et al. (545) in naturally contaminated corn screenings that caused field cases of ELEM and were hepatotoxic to rats. Funlonisin B I also was identified in moldy homegrown corn collected from an area of Transkei, Union of South Africa, which has a high incidence of esophageal and liver cancer in humans (593). Outbreaks of porcine pulmonary edema (PPE) syndrome have been noted to overlap at the same places and times that ELEM outbreaks have occurred. PPE has now been reproduced by IV injection of fumonisin B I in swine (532). 7. Fumonisin Analysis Methods and Levels in Feeds Fumonisins B I and B2 levels were evaluated in feeds associated with 13 horses that died during an outbreak of ELEM (579). Levels ranged from 37 to 122 pg/g fumonisin B I and from 2 to 23 pg/g fumonisin B2. Feeds associated with 44 cases of ELEM and 42 cases of PPE were analyzed for fumonisin B I (594). The feeds associated with ELEM contained fumonisin B I levels ranging from less than 1 pg/g up to 126 pglg, with 75%

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of the cases on feeds containing levels above 10 pg/g (594, 595). The feeds associated with the PPE had fumonisin B I levels ranging from less than 1 pg/g up to 330 pg/g, with 71% of the cases on feeds above 10 pg/g (594). Recently, the third and fourth members of the fumonisin mycotoxin family, fumonisins B3 and B4 (Fig. 21), were characterized (591, 604). Fumonisin B? has one less hydroxyl group than fumonisin B I, and fumonisin B4 has one less hydroxyl group than fumonisin BL.Methodology for the quantitative purification of fumonisins B B’, and B from corn cultures of F. rnordifonne strain MRC 826 is described by Cawood et al. (592). Three separation methods are described for fumonisin B I but were not fully developed analytical procedures for the mycotoxin (593). The methods consisted of thin-layer chromatography (TLC), reverse-phase HPLC, and capillary gas Chromatography (GC) analysis of various derivatives of fumonisin B Another study also used TLC, HPLC, and GC/MS (mass spectrometry) to determine levels of fumonisins B I and B2 in feed samples (579). The methods used were similar to those of Sydenharn et al. (593). The levels of fumonisins B I and B2 (596-598) and fumonisins B B2, and B3 (58 1) were determined by HPLC with fluorescence detection (597). An analytical method was presented by Plattrier etal. for the detection of fumonisins by hydrolysis and silylation followed by GUMS quantification (599). The hydrolysis removes the ester moieties at C14 and C1S leaving a C1 to C20 backbone typical for each fumonisin. A comparative analytical study utilizing a fluorescanline procedure with four laboratories resulted in acceptable agreement, with fumonisin concentrations ranging from LC to 1800 pg/g (600). Korfmacher et al. made a comparison of MS characterization methods of fumonisin B using thermospray, fast atom bombardment (FAB), and electrospray (601). The first reported determination of fumonisin B in plasma and urine was conducted by reverse-phase HPLC with fluorescence detection (602). Monoclonal antibodies have been produced to fumonisin B but they also cross-react with fumonisins B 2and B1 (603). Primarily, isolate MRC 826 has been used in studies by most researchers until now. Most isolates of F. monilifome examined by Plattner et al. predominantly produce fumonisin B (591), typically about 70% of the total fumonisins detected. However, F. srtbglutirzms appears to make only low, if any, amounts of the fumonisins.

IX. NIGHTSHADES (SOLANAC€A€)

A.

White Potatoes

The white potato, a member of the nightshade family, originally came from the highlands of Peru and was brought to this country by the Irish in 1719. Scottish highlanders ate large amounts of potatoes during the famine of 1782 and noticed a high incidence of dropsy (accumulation of fluid in the joints) (605).Livestock also have died after ingesting potato vines, sprouts, peels, or green and cull potatoes. Severe human illness (7, 606) and fatalities have been caused by eating greened potatoes (5, 6, 8). Forsyth stated, “Fatality in human beings and innumerable instances of severe illness have been reported from all over the world, after people have eaten greened or sprouted potatoes” (607). Consumers have the notion that most of the potato’s nutrients are in the peel and are unaware that toxic compounds also are in thepeel (608). Potatoes contain antinutritional and toxic compounds, including inhibitors of trypsin and chymotrypsin (609). Phytoalexins (natural pesticides) like hydroxylubimin and rhisitin (610). lubimin and phytuberin (38), and constitutive antibiotics like caffeic acid, chlorogenic acid (Fig. 3),

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Solanidine Figure 25 Solanidine, potentialteratogen that is storedin the body for prolonged periods of time. (From Ref. 61 1.)

scopolin, and solanidine (611) (Fig. 25) (612) are present in potato. Two major constitutive glycosteroid alkaloids, a-solanine and a-chaconins (Fig. 26), are fungitoxic and are synthesized at cut (wound) surfaces (613, 614). The synthesis of a-solanine and a-chaconine also is stimulated in tubers by mechanical injury and aging (614-616). Table 17 lists various glycoalkaloids found in the potato (617, 618). Table 17 Potato Tuber Glycoalkaloids

Compound

Formula

a-Solanine @-Solanine YSolanine a-Chaconine P-Chaconine 7-Chaconine Solanidine Demissine Commersonine Demissidine S&SoIanidan-3a-ol Leptidine' Leptine I Leptine Ila 0(23)-Acetylleptinidine LeptinineI LeptinineII Leptinidine a-Solamarine @-Solamarine Tomatidenol 'See Ref. 8. Source: From Refs. 617 and 618. See Ref. 618 for a listing of hydroxy and acetoxy derivativesof solanines and chaconines in wild potato.

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Compounds found in the stressed potato have been reviewed by Maga (618) and Stoessl et al. (619). These include hydroxycinnamic acid, esculin, umbelliferone, rishitinol, anhydro-P-rotunol, solavetivone, vetispiranes, and desacetylphytuberin. Scopoletin (7-hydroxy-6-methoxy coumarin) is produced in virus-infected potatoes (620,621). Glycosides of tomatidenol are also induced in specific varieties by slicing (622). Any general disturbance of plant metabolism may trigger phytoalexin production in the potato (619). B. CholinesteraseInhibition a-Solanine (Fig. 26) is a weak-to-moderate inhibitor of both specific and nonspecific cholinesterases (623), and a-chaconine (Fig. 36) is a potent inhibitor of the cholinesterase isoenzymes (624). When extracts of potato were used to inhibit human plasma cholinesterase, extracts from the peel were 10-40 times more active than those from the innermost flesh, showing the accumulation of alkaloids in the peel (625, 626). The potato glycoalkaloids, leptine I (Fig. 27)and demissidine (Table 17), were among the most-inhibitory natural products to humanplasma cholinesterase (627). Cholinesterase inhibition was used to categorize 24 potato varieties (628). No carcinogenicity data exist for the cholinesterase inhibitors found in the potato (629). C.GlycoalkaloidContent

Potato breeders, in general, have attempted to keep the a-solanine (Fig. 26) content of commercial potatoes below 200 pg/g fresh weight (630). The U.S. Department of Agricul-

Glu,

Gal -

Rh;

a-Solanine

Rh&, Glu

Rh;

a-Chaconine

Figure 26 The major glycoakaloids in the potato. a-solanine and a-chaconine.

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Rha, Glu Rh;

Leptine I Figure 27 The potato glycoalkaloid, leptine I, is one of the most inhibitory natural products to human cholinesterase. (From Ref. 627.)

ture (USDA) potato-breeding program has an accepted guideline of 200 pg/g tuber for the total glycoalkaloid (TGA) content of parents and offspring of potential potato varieties. Although this is not a mandated or regulated TGA limit, it is accepted as being appropriate for insuring that growers and the public have high-quality potatoes (Sinden, personal communication, January 1989; 63 1j. It must be noted that the TGA content may be 12-30% greater than the sum of the a-chaconine and a-solanine content (632). The a-solanine content of 32 varieties of potatoes grown in Wisconsin ranged from 20 to 130 yg/g of tuber (633). These glycoalkaloids usually are present at about 75 yg/g of potato or 15,000 pg per 200-g serving of potatoes (617). In comparison, malathion, a synthetic insecticide and cholinesterase inhibitor, is present in our total daily diet at about 17 pg (629). Under certain weather conditions, potato tubers may synthesize considerable amounts of a-solanine. Exposure to light in the field or in the marketplace can increase the a-solanine content to dangerous levels (634). The glycoalkaloid content also can increase during potato growth as well as after harvest (631). The amounts of a-solanine and a-chaconine increased with light or wound induction (635). Several outbreaks of illnesses have been traced to the use of potatoes with a-solanine contents ranging from 100 to 400 yg/g (636). Acute illnesses induced by glycoalkaloid poisoning probably are more prevalent than is indicated by the few recorded medical cases (606). The TGAcontent of potato tissues and related products is given in Table 18. Bruising can increase the TGA content dramatically at various temperatures (617), significantly enhance the phenolics, and decrease ascorbic acid levels. In fresh Katahdin potatoes, the greatest increase of phenols from about 210 pg/g to 1720 yg/g occurred at 5OC, and an increase in TGA content from about 50 yg/g to 220 yg/g occurred at 20°C (638). At 10 days after wounding, Juliver variety tubers held in the dark had a-chaconine and a-solanine concentrations of 685.4 pg/g and 499.6 pg/g, respectively; unwounded control tubers contained 37 1.7 pg/g of a-chaconine and 27 1.6 pg/g of a-solanine. Final concentrations of glycoalkaloids in wounded potatoes usually were higher in the light. Potato chips produced from unwounded tubers had a-chaconine and a-solanine concentrations of 107.1 pg/g and 69.2 pg/g, respectively, and those frotn wounded tubers had 245.7 pg/g and 197.6 pg/g, respectively (639). Potatoes that contain more than 200 yg/g TGA per tuber are considered unfit for consumption (606, 630). Average TGA contents of potato tubers

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Table 18 TotalGlycoalkaloids in Potato Tuber Tissues and Products

Totalglycoalkaloids (pglg fresh weight)

Potato tissue

300-600 150-300 300-500

Skin, 20/0-3% of tuber Peel, 100/0-15% of tuber Peel and eye, %-in(3-mm) disk

12-50

Flesh

75 1500-2200 250-800 17-40 11-54 67

Whole tuber Peels from bitter tubers Bitter whole tubers Whole tubers" Chips" Frozen croquettes" ~

~

~

~-

3See Ref. 637. Source: From Ref. 618.

from Lenape, Kennebec, Russet Burbank, Katahdin, Irish Cobbler, and RedPontiac potato varieties were 293, 97, 79, 79, 62, and 43 yg/g potato, respectively (631). Consumption of potato peels in restaurants has increased (608). At least one company is producing a potato chip made from potato peels. Fried potato peels are a source of large quantities of glycoalkaloids. In one study, quantities of a-solanine plus a-chaconine in fried peels ranged from 1390 to 1450 yg/g (640). These quantities are more than seven times the recommended upper safety limit of 200 pg/g of potato for potato glycoalkaloids. In another study, cooked peels had glycoalkaloid levels 2-8 times that of the upper safety limit (641). Table 19 lists a-solanine and a-chaconine concentrations in the baked-fried peels of commercial potato varieties.

Table 19 CombinedAlpha-SolanineandAlphaChaconine Concentrations in Baked-Fried Peels of Cornmercial Potato Varieties

a-solanine

+ a-chaconine

Variety Russet Burbank Kennebec Katahdin Superior Allagash russet Round white Green Mountain Round white Bel Rus Russet Source: From Ref. 641.

558.6-1 256.8 1364.0-1 557.4 537.0-1 041.4 289.2-495.4 368.1-41 4.0 252.4-314.1 341.4-41 1.O 215.6-230.2 113.0-1 87.0 41.7-1 36.8

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a-Solanine and a-chaconine contents of potatoes are not affected by baking, boiling, or microwaving and are only slightly reduced by frying (640). During cooking, glycoalkaloids move into the cortex region, whereas phenols migrate from the peel into both the cortex and internal tissues. When potatoes were peeled before cooking, their phenolic contents were decreased but the TGA contents in the internal tissues were unchanged (608j. Symptoms of TGA poisoning can be easily mistaken for gastroenteritis. Common symptoms are nausea, diarrhea, vomiting, stomach cramps, headaches, and dizziness (606). Potato glycoalkaloids have a saponin-like activity and can lyse cells (642). Syrian hamsters receiving dried potato sprouts or alkaloid extracts had severe gastric and intestinal mucosal necrosis. Lesions observed in the intestinal epithelium of hamsters are similar to those caused by alkaloids. These lesions are consistent with the gastroenteritis observed in humans and animals suspected of being intoxicated by potatoes (643). Bitterness, indicative of high TGA content in potato tubers, can be detected readily by chewing a small piece of the raw peel. TGA levels higher than about 100 pg/g of tuber cause a slowly developing, hotly burning, persistent irritation of the sides of the tongue and back of the mouth. Potatoes that contain more than 200 p g l g give an immediate burning sensation (606). Potato varieties have been investigated for toxic glycoalkaloids that could be utilized against the Colorado potato beetle (CPB), a devastating potato pest (644). The potato glycoalkaloid leptine I (Fig. 27j, was a better pest deterrent than a-solanine or a-chaconine (Fig. 26) (645). Its biological activity may be due to its cholinesterase-inhibiting effects. Leptine I is the most inhibitory to human plasma cholinesterase among the natural products tested (627). Kuhn and Low did not find leptines in tubers of potato varieties that contained high concentrations in foliar tissues (646). Leptines were not observed in tubers of three SoZnrwTl clones with high leptine foliar concentrations (645). But leptinines, which are obtained from leptines by the loss of an acetyl group, were found in tubers in concentrations ranging from 20 pg/g to 390 pg/g fresh tuber. Leptinines are weak deterrents of pest feeding. Also, beetles resistant to dichloro-diphenyl-trichloro-ethane(DDT) are less sensitive to leptines or leptinines than normal beetles (647). New potato cultivars produced by cell fbsion, a genetic-engineering technique, contained high foliar leptine concentrations but contained no measurable atnounts in their tubers (648). These varieties have good resistance against CPBs without increasing the tuber leptine content. High TGA concentrations in foliar tissues can be a significant factor in the defense of the potato plant against pests (649). Breeding can produce pest-resistant potato varieties without elevating the glycoalkaloid levels in their tubers (Sinden, personal communication, January 1989). However, plant-breeder efforts to produce more pest-resistant potatoes can result in a potato with high levels of glycoalkaloids. High a-solanine content (650) or other glycoalkaloids appear to be a varietal characteristic. High levels of steroid glycoalkaloids in the potato cultivar Lenape caused its quick withdrawal from the marketplace because of acute toxicity to humans (617). This is exactly the same situation as recently observed with celery, in which human toxicity problems resulted from increased levels of linear furanocoumarins inadvertently bred into celery to obtain better pest resistance (65 1, 652). The second edition of the book A Diet to Stop Arthritis, the Nightshades nrzd Ill Hedth contains over 200 case histories of people who removed nightshades (glycoalkaloids) from their diets and reportedly recovered to varying degrees, from just feeling better to becoming ambulatory, from arthritis symptoms (653). Removing nightshades from the diet may not be as easy as diet changes. Alfalfa, cantaloupes, cotton, sorghum, peanuts,

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Chemicals in Foods

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rice, watermelon, and wheat commonly are contaminated with silverleaf nightshade, Solnnum elcreagnifolium (654).

D. Teratogens Many common commercial food products contain potatoes; there is even a potato ice cream. We serve potatoes in many ways in our home, and they also are used in baby foods. Potato glycoalkaloids, possible teratogens, are contaminants in almost all potato starches commercially available (637). It is difficult for an adult or child to avoid large exposures to potatoes or potato products. Because of this fact, it is important to breed potato varieties to obtain low levels of glycoalkaloids in their tubers. The glycoalkaloids in potato may be possible teratogens (1). Renwick hypothesized that potato avoidance by pregnant women would reduce the incidence of human anencephaly and congenital spina bifida (ASB) by 95% in certain geographical areas (655). Severity of potato late-blight (which causes an increase in glycoalkaloid content of potatoes) correlates with the incidence of ASB in humans. Ireland has highly suitable weather for the blight fungus and also has the world’s highest incidence for ASB (656). Similar correlations between late-blight and ASBexist in potato-growing regions around the world. Areas that have changed to potato varieties (having more blight resistance) with greater glycoalkaloid content also have undergone as much as a doubling in the frequency of anencephaly (655). Late-blight or other fungi causing elevated natural plant chemicals in the potato that might result in toxicity problems are not new phenomena. Many plant poisoning syndromes are known to occur as a result of fungal invasion with subsequent production of natural toxic chemicals. One of these, the increase of phototoxic linear furanocoumarins in celery infected with Sclet-otinia sclelvfiorunr (657-659), causes photodermatitis in celery workers and handlers and is due to an increase of plant phytoalexins (linear furanocoumarins) as a result of the fungal attack (44). Another is the salivary syndrome caused by slaframine and characterized by excessive salivation. Legume forages parasitized by the fungus Rhizoctonia legrrrzzirzicola, when consumed by small animals and cattle, result in salivary episodes ranging from 6 hr to 3 days (660). Neurological diseases of cattle that are characterized by sustained tremors can be produced by at least six tremorgenic mycotoxins. These mycotoxins are produced by several species of fungi belonging to the genera Asper-gillus and Penicillium (661). A problem of both animals and humans is the nervous ergotism syndrome. This problem arises from alkaloids produced by fungi on rye, wheat, or barley. These alkaloids stimulate smooth muscle (662). Toxic problems that are caused directly by a fungus or caused by a plant in response to f h g a l invasion are well known. A requirement for onset of toxic episodes is to have the appropriate weather conditions for fungal growth. Initial reports cast doubt on the epidemiological relationships that formed the basis of Renwick’s hypothesis concerning ASB (663-669). Keeler also was unable to cause teratogenic effects in animals during 1973 to 1975 (670). Then, both pure a-solanine and glycoalkaloid extracts from blighted potatoes were shown to produce teratogenic effects in chick embryos (671, 672),and teratogenic effects also were produced in NAW/pr mice by a-chaconine but not by a-solanine (673). Neural-tube defects were caused in Syrian hamsters by a-chaconine and, to a lesser extent, by a-solanine (674). Keeler et al. showed that the new sprouts of all potato varieties tested (Kennebec, Nampa, Norchip, Pioneer, Russet, Sebago, and Targhee) were teratogenic in Simonsen

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hamsters (675). Thetype of deformities of offspring were mainly cranial blebs and exencephalies, with some microphthalmies and spina bifidas. The tubers and peels of Kennebec potatoes were not teratogenic. Sprouts from the British potato cultivar, Anan Pilot, also were teratogenic and caused cranial bleb, encephalocele, exencephaly, and spina bifida (676). Most of the teratogenic activity was traced to a-chaconine and, at higher doses, a solanine. Women who had not passed the reproductive age were discouraged from eating desprouted potatoes (677). Nevin and Merrett emphasized that some mothers on potatoavoidance diets during pregnancy still gave birth to ASB children and claimed that this result refuted Renwick’s hypothesis (668). Only limited information is known of the effects of solanidine (Fig. 25), the alkaloid aglycone of a-solanine and a-chaconine (Fig. 26). Zitnak reasoned that solanidine may be themost active participant in toxic episodes and suggested that the physiological effects of solanidine needs to be better understood (678). Kinetic and retention studies of solanidine in humans and verification of this alkaloid in postmortem liver samples by mass spectrometry suggested that solanidine is stored in the body for prolonged periods of time (611). Claringbold et al. concluded that, in times of metabolic stress (pregnancy, starvation, or illness), stored solanidine might be mobilized (611). The evidence for human teratogenic risk from consumption of potatoes is still only circumstantial; however, in a review of the toxicity and teratogenicity of Solanaceae glycoalkaloids, Morris and Lee suggested that green or damaged potato tubers especially should be avoided by women likely to become pregnant (8). The concentrations of a-chaconine and a-solanine in potato plants, potato berries, and potato sprouts can vary by a factor of about 500. The ratio of a-chaconine to a solanine varied from 1.2: 1 to 2.7: 1 (632). These findings may have implications in food safety and plant breeding since a-chaconine is more embryotoxic than a-solanine. It may be preferable to use potato varieties with a low a-chaconine-to-a-solanineratio (632).

E.

Eggplant

The spirosolane alkaloid, solasodine (Fig. 28). occurs in eggplant as well as other food plants (679). Solasodine produced teratogenic effects in hamsters during the primitive streakheural plate stage. The deformities were cranial bleb, exencephaly, and spina bifida (680,681). Solasodine was not teratogenic in rats (682). However, Russian workers found

Solasodine Figure 28 Solasodine. a teratogen found in eggplant.

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Capsidiol Figure 29 Capsidiol,aphytoalexin.

solasodine to be embryotoxic and teratogenic in rats at 5-10 mg/specimen/day and also that a-solanine (Fig. 26) had no effect at 5 mg/specimen/day (683).

F. GreenPeppers Green peppers contain the sesquiterpene phytoalexin capsidiol (Fig. 29). About half of the extractable material from pepper is capsidiol(684). Phenolic compounds also accumulate in peppers after fungal or viral infection or chilling injury (619).

Tomatoes G.

The tomato produces a-tomatine (Fig. 30) as its primary toxic agent; this is a steroidal glycoalkaloid and it is distributed throughout the plant (617). The a-tomatine content is responsible for making tomato plants unpalatable to livestock. Consumption of green tomato plants by pigs resulted in illness or death (~607). Cultures of several organisms are inhibited by the antibiotic properties of a-tomatine (683, and it is toxic to a wide range of living organisms (617). The aglycone of a-tomatine is tomatidine. In mice, intravenous injections of tomatidine were slightly more toxic than those of theparent glycoside (686). There are about 360 pg/g of a-tomatine in red tomato fruit, 450 pg/g in yellow fruit, and 870 pg/g in green fruit (617). The substances atomatine and tomatidine have not been tested in other toxicity or cancer bioassays.

XYl,

-

Glu Gat -

/

Glu

a-Tomatine Figure 30 The steroidalglycoalkaloid

a-tomatine.

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X.

NITRATE-RICH FOODS

Worldwide estimates in the early 1980s showed that the stomach was the most colnnlon cancer site, with 670,000 new cases each year (687). In 1930, stomach cancer was the leading cancer in the United States: however, in 1990, itwasthe 10th and 12th most common cancer site in men and women, respectively. Overall results from epidemiological studies conducted as early as 1932 through 1990 do not show a consistent pattern of factors associated with stomach cancer (688). However, nitrate exposure has been positively correlated with the incidence of stomach cancer in 12 countries (689). Studies also have shown either no correlation or even an inverse correlation between nitrate exposure and the incidence of stomach cancer (690). It is estimated that 80% of our nitrate intake is from vegetables and less than 20% comes from cured meats. The excretion of nitrate by humans follows first-order kinetics with an elimination half-life of about 5 hr (691). Nitrate levels in body fluids (serum, saliva, urine) peaked within 1-3 hr after ingestion in food or water. However, from a toxicological point of view, the most important feature of nitrate metabolism is its reduction to nitrite (692).

A.

Reduction of Nitrate

Reduction of nitrate to nitrite can be effected by mammalian nitrate reductase, by the orogastrointestinal microflora, and also by many normal microbial inhabitants of the alimentary tract (692). Nitrite can be further reduced to ammonia; however, the reduction of nitrate to nitrite by the gut flora in vitro occurs more rapidly than the fllrther reduction of nitrite to ammonia, so nitrite tends to accumulate (693). Nitrates also can react with secondary and tertiary amines to form carcinogenic and mutagenic N-nitroso compounds (NOC) (694). Potentially carcinogenic compounds, such as N-nitrosamines may be generated from nitrate in whole saliva or in the saliva-gastric juice mixture after swallowing.

6. Nitrosation of Amines NOC can be synthesized from reaction with nitrite and some nitrogen-containing compounds. This formation can take place in thehuman stomach, and thenitrite can be derived from reduction of ingested or endogenous nitrate (695, 696). The nitrosation of various secondary amines is favored by high salivary (or gastric) concentrations of thiocyanate (a normal constituent of human saliva) andlow pH (696). Thiocyanate is a powerful catalyst of nitrosation reactions of nitrite and amines (697). In the currently accepted model of gastric carcinogenesis (698), it is proposed that there is a long phase of chronic gastric disease preceding endogenous formation of NOC from nitrate. The endogenous formation of carcinogenic NOC is an intermediate step in the development of gastric carcinoma. Endogenous NOC synthesis also can occur when bacterial enzymes catalyze nitrosation from nitrate or nitrite (699). Many other factors may be involved besides nitrate ingestion during the precursor stage and nitrosation. For example, vegetables not only may be a source of nitrate, but they also may be a source of ascorbic acid, which is an inhibitor of endogenous nitrosation (699,700). Physiological factors such as the in vivo reduction of nitrate or nitrite and gastric juice pH affect the rate and extent of endogenous nitrosation (701). The determination of nitrate or nitrite in body fluids is insufficient to assess the extent of nitrosation in humans in vivo (699).

Naturally Occurring Toxic Chemicals in Foods

C.

101

Quantification of %NitrosoCompounds

A sensitive method to quantify human exposure to endogenous NOC has been devised (700). The method is based on the excretion of N-nitrosoproline (NPRO) and other Nnitrosamino acids in the urine. These are measured as an index of endogenous nitrosation following ingestion of precursors. The measurement of urinary NPRO can provide a direct measurement of NPRO synthesis (700). In most cases, higher exposures to endogenous NOC were found in subjects living in high-risk areas for stomach cancers in northern Japan and inPoland, in subjects with esophageal cancer in the People’s Republic of China, in subjects with different habits of betel quid chewing and tobacco use, in European patients with urinary bladder infections, and in subjects infested with liver flukes in Thailand (700). It was suggested that the reduction of exposure to NOC would be a good preventive measure in human cancer. With the measurement of intragastric formation of NOC by monitoring the urinary excretion of NPRO, it was shown that consumption of cooked vegetables increased endogenous nitrosation of proline, while consumption of raw vegetables had only a marginal effect on the nitrosation of proline (702). The result may reflect the destruction of ascorbic acid in the cooked vegetables and the loss of its protective affect. It has been shown that ascorbic acid efficiently lowers the body burden of NOC (700), and ascorbic acid also significantly decreased the urinary NPRO levels (695). Ingestion of fruits and vegetables has been shown to reduce the risk of stomach cancer and the fact that both are sources of nitrosation inhibitors fits quite well (703). Tyramine (Fig. 31) was among three compounds isolated from Japanese soy sauce that were nitrosatable mutagen precursors (704, 705). Tyramine formed 3-diazotyramine (Fig. 3 1) upon direct exposure to nitrite. Indole-3-acetonitrile (3-indolyacetonitrile) (Fig. 10) was one of three indoles isolated from Chinese cabbage that were nitrosatable mutagen precursors (705). Again, direct exposure of 3-indolylacetonitrile to nitrite resulted in form-

Q

Tyramine

CH2CH2NH2

@+ NcFj

0

3-Diazotyramine

m

CEN

I

NO

I-Nitroso-3-indolylacetonitrile

Figure 31 The nitrosationproducts of tyramineand3-indolylacetonitroleare3-diazotyramine respectively. and l-nitroso-3-indolylacetonitrile,

Beier and Nigg

102

ing 1-nitroso-3-indolylacetonitrile(Fig. 31). The initial rate of nitrosation of indole compounds is much faster than that of methylurea or thioproline, and they can be formed in the human stomach. Other nitrosatable mutagen precursors were isolated from soy sauce and Chinese cabbage (706). Goitrin (5-vinyl-2-oxazolidinethione), a naturally occurring compound found in cruciferous vegetables and rape, reacts readily with nitrite to form N-nitroso-5vinyl-2-oxazolidineone under stomach conditions (707,708). Alkylating activity was also found in sauerkraut after incubation with nitrite under quasi-gastric conditions (709). Bartsch et al. shows the structure of compounds that are tobacco-specific N-nitrosamines that have been identified in urine from human subjects (700j. N-Nitrosodimethylamine (NDMA), NOC, and nitrate levels were determined in 170 retail samples of beer (710). Levels of NDMA ranged from below 0.1 pg/kg to 1.2 pg/kg, levels of NOC were detected in 42% of the samples in concentrations up to 569 pg/kg, while nitrate levels ranged from less than 0.2 pg/g to 143 pg/g with a mean value of 16.8 pg/g.

D. DietaryNitrateIntake Estimates of the dietary nitrate intake are listed in Table 20 for various people around the world (690, 701, 71 1-7 14). Singapore has one of the highest incidences of gastric cancer in the world at 38 per 100,000 persons per year (715), compared to 23.3 for the United Kingdom and 13.5 for the United States in the early 1980s. The Chinese population of Singapore was 79%of the total and had anestimated intake of 250 mg nitrate/day, whereas the other 21% is made up of Malays and Indians who had an estimated intake of 113 mg nitrate/day (714). Male gastric cancer incidence rates per 100,000 population of 44.8 for the Chinese, 10.3 for the Malays, and 19.4 for the Indians appear to be directly associated with nitrate intake.

Table 20 Estimated Dietary Intake of Nitrate ~~

~

Nitrate intake People adults'

21 250

Dutch Great Britain (aged 15-74 years)b Havana (aged 18-23 years)c 63.7-1 years)d 16-60 Italian (aged 105-1 58-1 analysisSalivary Northern Japane High-risk area (aged 24-66 years) 95-1 Low-risk area (aged 21-60 years) 88-1 Singapore' population Chinese 113 Indians Malays and ,'See Ref. 71 1. 'See Ref. 712. 'See Ref. 713. "See Ref. 701. 'See Ref. 690. 'See Ref. 713.

(mg/day) 52 95

08.8

50 07

30 77 5

Naturally Occurring Toxic Chemicals in Foods

103

In Japan, nitrate levels were higher in subjects of the low-risk stomach cancer area than in thehigh-risk area. The nitrate levels correlated well with theamounts of vegetables consumed. Even though the subjects in the high-risk area had lower nitrate levels, their potential for endogenous nitrosation was shown to be higher (690). The evaluation of nitrate in Italian populations resulted from residents in four regions of Italy with long-standing, contrasting gastric cancer mortality rates. No association was found with nitrate intake and the occurrence of gastric cancer. However, nitrate could not be ruled out as playing a part in the process (701). The concern in relation to nitrate levels is in the exposure to biologically effective doses of NOC. However, there is no straightforward relationship between nitrate exposure

Table 21 NitrateContent of Vegetables

Vegetable Asparagus Beans, snap Beans, lima Beans, dry Beets Broccoli Cabbage Cabbagea Carrots Cauliflower Celery CeleqP Corn Cress' Cucumbers Eggplants Leeksb Lettuce Lettucea Melons Onions Peas Pickles Potatoes Pumpkin Sauerkraut Spinach Sweet peppers Sweet potatoes Tomatoes Turnipsb 'See Ref. 717. %ee Ref. 718. Satrce: From Ref. 716.

Concentration (pg/g fresh weight) 21 253 54

13 2760 703 635 <500-7000 (dry weight) 119 547 2340 3000-25,000(dry weight) 45 2000-20,000 (dry weight)

24 302 471 850

e 1000-19,000 (dry weight) 433 134

20 59 119 413 191 1860 125

53 62

447

eier

and Nigg

104

and the production of NOC and risk of cancer. It has not been established whether a reduction in nitrate exposure might have a beneficial effect on health (712). E. NitrateLevels in Plants The nitrate content of 10 different fruits was obtained and showed that apples had the lowest nitrate levels (7 pg/g) and banana had the highest levels (130 pg/g) (714). A list of vegetables and their average nitrate levels are shown in Table 21 (716-7 18). The nitrate content of celery, lettuce, and cress frequently was found to exceed the legal limits in force in Switzerland and Holland, which are in the range of 4000-5000 pg/g dry weight (717). Nitrate levels in 46 selected species of wild edible herbs and vegetables common in Jordan varied from 29 pg/g in leaves of tetragonolbus to 6743 pg/g in star fenugreek (719). Potatoes contribute approximately 14% of the per capita ingestion of nitrate in the United States. The average nitrate level in potatoes is 119 pg/g fresh weight. The highest average levels of nitrate found in vegetables are in beets, celery, and spinach with 2760, 2340, and 1860 pg of nitrate per g of fresh weight, respectively (716). In a study of Katahdin and Kennebec potatoes, the application of the auxin (a naturally occurring hormone), indoleacetic acid (IAA), significantly decreased the nitrate-nitrogen content of both varieties (720). Tubers have been found with nitrate-nitrogen levels over 1200 pg/g. Improper irrigation along with high nitrogen fertilizer rates can dramatically increase nitratenitrogen levels in tubers (721, 722). High rates of nitrogen fertilization can increase the nitrate content of plants (723). Several studies in experimental field and on-farm trials have shown increased levels of nitrates in vegetables grown according to conventional farming (7 18, 724-728). The nitrate content of leeks and turnips grown under different fertilizer regimes is shown in Table 22 (718). Both leeks and turnips show significantly higher levels of nitrates when raised using mineral NPK and blood meal fertilizers. The data presented here continue to reinforce the concept that nitrate accumulation heavily depends on the kind of fertilizer

Table 22 Nitrate Content of Leeks and Turnips Grown under Different Fertilizer Regimes

Vegetable Fertilization regime

Nitrates (pg/g fresh weight)

Leeks Control Mineral NPK Manure compost Woodchip compost Bloodmeal

471 1718

Control Mineral NPK Manure compost Woodchip compost Bloodmeal

447 1477 810 193 1646

1035 992

1565

Turnips

Source: From Ref. 718.

Naturally Occurring Toxic Chemicals in Foods

105

used. Manure-based composts maintain low nitrate levels in vegetables. However, if fertilizers contain magnesium, the nitrate content of food plants can be reduced considerably (723). High levels of nitrate and nitrite in both human food and animal feed are harmful because of methemoglobinemic and carcinogenic effects like those observed in rats (729). In infants, nitrates can cause methemoglobinemia (723). Sinios reported cases of infant deaths caused by the high nitrate content of spinach (730). Some vegetables show a steady decrease in nitrate levels and others show an initial rise when cooked in boiling water. Cooking in boiling water releases nitrate from vegetables into the boiling water. However, with only a 5-min cooking time, celery, spring greens, and turnips were measured to have a higher nitrate content than the uncooked vegetables (731). Cabbage and leeks significantly release their nitrate after only 5 min of cooking. A recent review recommended the intake of raw vegetables and fresh fruit as inhibitory for the onset of stomach cancer. The publicizing of reservations about a high intake of salt or particularly processed meat andfishwas apparently clearly justified (688).

XI.

PARSLEYS (UMBELLIFERAE)

A.

BiologicalActivities of LinearFuranocoumarins

Umbelliferous plants have caused contact dermatitis for centuries (732,733). Linear furanocoumarins are potent photosensitizing toxins (446) that also act as phytoalexins in celery (44, 734), parsley (735, 736), parsnips (737), and fig leaves (163). Contact dermatitis and photodermatitis have been described in individuals who handle figs (163), in parsley pickers and cutters (738, 739), and in celery handlers, field workers, and processors (740, 741). Celery dermatitis of the fingers, hands, and forearms is known to be caused by photosensitizing linear furanocoumarins (658). It initially was thought that only diseased celery, especially celery infected with Sclerotinin sclerotiorurn, contained these coinpounds and caused the photosensitization in humans (658,659). However, grocery workers have experienced phytophotodermatitis caused by apparently healthy celery; these incidents were documented by workers at the Centers for Disease Control (CDC) (651) and the National Institute for Occupational Safety and Health (NIOSH) (652). Other studies have shown the presence of large concentrations of linear furanocoumarins on the leaf surfaces of rutaceous plants (742). Linear furanocoumarins (psoralen, bergapten, and xanthotoxin) were determined on the surface and inside the seeds of Rutaceae and the fruits of various Umbelliferae and Leguminosae (Table 23) (743). From analysis of 10 species, the lowest levels of linear furanocoumarins found on the surface were less than 1 pg/g fresh weight and the highest were 40 pg/g fresh weight. The levels found on the inside of seeds or fruits ranged from 1.25 to 3200 pg/g fresh weight. A general review of the chemistry, historical drug use, and photosensitization of furanocoumarins in humans and animals is available (744). 1. DNA and RNA Adducts Biological activities of linear furanocoumarins are expressed in a broad spectrum of living things, from fungi to humans. This diverse activity is due to intercalation of linear furanocoumarins into D N A (745-749) and RNA (750). where they form covalent bonds in the presence of long-wave UV light, resulting in both mono- and diadducts (i.e., cross-links).

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Table 23 Combined Total Concentration of Psoralen, Bergapten, and Xanthotoxin On and Inside Fruits or Seeds

Concentration (pg1g fresh weight) Species

On surfaces

Inside

Umbelliferae Angelica archangelica Heracleum lanatum Pastlnaca sativa Pimplnella anlsum Foeniculum vulgare Apium graveolens Daucus carota

Leguminosas Psoralea bituminosa Rutaceae Ewodia danielli Phellodendron

14 20 40

1.3 1.25 15

0.85 40

6.4 0.7

2550 1250 3200 4.1 4.5 59 1.25 1350

230

Source: From Ref. 743.

Excellent discussions of the photochemistry of psoralens and their DNA and RNA adducts are presented by Hearst (751) and Woo et al. (752).

2. Medicinal Use Because linear furanocounlarins are potent photoactive compounds, they have been used clinically to treat skin depigmentation (vitiligo, a type of leukoderma) (749,753), psoriasis (754, 7.53, and prurigo nodularis lesions (756). An oral or topical dose of psoralen followed by exposure to long-wavelength (320-400 nm) UVA is known as PUVA (psoralen + UVA). PUVA still appears to be the best available medical treatment for vitiligo (757), and sequential combined treatment of UVB irradiation and topical PUVA appears to be the best for the treatment of generalized prurigo nodularis and prevention of new lesions (756). The medicinal use of these compounds has caused concern (758). PUVA caused mice to develop papillomas, keratoacanthomas, and squamous cell carcinomas (759). Stern et al. suggested, following a study of PUVA patients, that PUVA treatment poses an increased carcinogenic risk (760). An animal study indicated that PUVA therapy may initiate tumors (761). PUVA therapy unequivocally can cause cataract formation in both animals and humans (447). An 8-year follow-up of patients failed to show an increase of tumors from PUVA treatments (762) unless the patients had been exposed previously to known carcinogens (763). However, a 12.3-year study of 892 men that received PUVA treatment for psoriasis resulted in notation of the incidence of invasive squamous-cell carcinoma on the genitalia that was 286 times that of the general population (764). Stern et al. concluded that “the strongly dose-dependent increase in the risk of genital tumors associated with exposure to PUVA and ultraviolet B radiation that we observed makes it

Naturally Occurring Toxic Chemicals in Foods

707

prudent for men to use genital protection whenever they are exposed to PUVA or other forms of ultraviolet radiation for therapeutic, recreational, or cosmetic reasons” (764, p. 1093). The advent of tanning booths can be a definite health hazard to those individuals taking psoralen treatments or those exposed to celery juice or other produce containing a high level of linear furanocoumarins. A 45-year-old woman died from burns she received in a tanning booth while she was taking psoralen (765). Produce workers exposed to celery juice were badly burned when theyvisited a tanning parlor after work (C.G. Toby Mathias, NIOSH, personal communication, 1986). A 65-year-old woman developed a severe generalized phototoxic reaction following a visit to a suntan parlor resulting in erythema, edema, and blistering, which was restricted to UV-exposed skin. The woman had eaten a celery root, weighting approximately 450 g that was cooked the evening before, 1 hr prior to visiting the suntan parlor. She also drank the juice in which the celery was cooked. The total amount of psoralens in the consumed root was approximately 45 mg, an equivalent dosage to that used in PUVA therapy (766). Even with the knowledge of photosensitization with the use of linear furanocoumarins, a patent application was made for 5-methoxypsoralen (bergapten) (Fig. 9) and related linear furanocoumarins to be used for suppressing or preventing jet lag (767).

B. Celery Celery contains at least four linear furanocoumarins: psoralen, bergapten. xanthotoxin, and isopimpinellin (Fig. 9) (768). Isopimpinellin is not a photosensitizer (175, 176) and therefore has not been quantified in many studies. Scheel et al. described another linear furanocoumarin, 4,5’,8-trimethylpsoralen (TMP) as being the causal agent for photosensitized skin reactions in farm workers (658). A number of researchers have stated that they were not able to observe TMP in fresh or diseased celery samples (44, 659, 768-771). A report (772) describing the effect of virus infection on the linear furanocoumarins in celery demonstrated the presence of small amounts of TMP and angelicin, and trace amounts of sphondin and peucedanin also were observed. The small amount of TMP observed in celery would be additive to the photosensitizing skin reactions of the other more prominent furanocoumarins. Linear furanocoumarins are phytoalexins in celery (44). Their phytoalexin behavior results in increased levels in celery as a response to general elicitors, including copper sulfate, Hg” and Cu2+ions, UV light, cold, fungicides, herbicides, polyamines, and fungal cell-wall fragments. Some pesticides do not induce these compounds in celery (773). It is quite understandable that acidic fog at pH levels similar to that experienced in commercial celery production near major population centers in California would stimulate the phytoalexin response of the linear furanocoumarins in celery (49). A single 4-hr acidic fog resembling air pollution near major population centers stimulated the production of psoralen, bergapten, xanthotoxin, and isopimpinellin in celery foliage within 24 hr. The linear furanocoumarin content of celery tubers coated with polysaccharide gels over longtime storage was conducted (774). Based on the toxicity levels of linear furanocoumarins in celery, probably all treatments by Rober et al. for long-term storage would not be appropriate (774). The coating of celery tubers with kappa-carrageenan resulted in the lowest production of linear furanocoumarins. Coatings of Ca-alginate and pectin both resulted in very high levels of linear furanocoumarins after 6 months. This could have

108

Beier and Nigg

happened if both the Ca-alginate and pectin functioned as elicitors. It is well known that both Cu” and Hg’+ salts and cell-wall fragments elicit linear furanocoumarin production. The pectin coating resulted in over 40 yg/g total linear furanocoumarins in the edible portion after six months (774). 1. Photophytodermatitis inFood Handlers Concentrations of photoactive linear furanocoumarins in some common celery cultivars (768) are compared in Table 24 with those of the celery cultivar implicated by a CDC study in 1984 (651) and a NIOSH study in 1986 (652) in photophytodermatitis of grocery store workers. The average quantity of linear furanocoumarins in the implicated cultivar was 14 times higher than that in other cultivars; the concentration of the tnost photoactive linear furanocoumarin, psoralen, was 19 times higher in the implicated cultivar. In both studies, the same celery cultivar was determined to be the causative agent of the photophytodermatitis. Linear furanocoumarin concentrations of about 8-10 pg/g fresh weight of the implicated cultivar were responsible for the grocery workers’ photophytodermatitis. These workers could be expected to have multiple exposures to trimmed celery, and concentrations as low as 12.5 1-16” are known to cause contact dermatitis (770). It is interesting to note that the amount of linear furanocoumarins observed in diseased celery was about 25 times that in healthy celery (769). In a number of studies designed to demonstrate phytoalexin production by celery, CuS04 treatments elevated the levels of linear furanocoumarins from 23 to 104 times that observed in the controls (775). Photophytodermatitis in grocery store workers was a direct result of plant breeding to obtain a more pest resistant variety of celery. All celery varieties contain photoactive linear furanocoumarins. and humans have coexisted well with them. However, when chemical concentrations were increased 10- to 15-fold in this new more pest-resistant variety, the toxicity threshold was exceeded and human illness resulted. Thus, the often related theory, “humans have evolved with the common food plants, and are not susceptible to any of nature’s chemicals that are found in those plants,” is not always true. The FDA was concerned that new plant varieties may contain larger amounts of natural toxicants (776). This concern now has become reality in both celery and the potato (see Sec. IX). A celery-breeding project evaluated Apium species for resistance to a key insect pest of celery, Lirionlyzn trifolii (Burgess) (777). L. trifolii is a leaf miner in celery and reduces yield and marketability. The celery cultivar Apium llodiflorurn had the lowest levels of linear furanocoumarins and also the best resistance against L. trifolii. No significant correlation could be observed between the foliar content of linear furanocoumarins and L. trifolii adult production (778). These results suggest that it might be possible to obtain resistance to other insect pests of celery without raising the indigenous levels of linear furanocoumarins. 2. NewBreedingStrategy A new program strategy was suggested for breeders of plants containing known toxic compounds (779). It is called the integrated breeding and environmental chemicals (IBEC) strategy. During the plant-breeding process, the breeder evaluates levels of the toxic compound(s) that are produced by the variety in question. In celery, the toxic compounds currently recognized would be psoralen, bergapten, and xanthotoxin. The variety with the lowest levels of these compounds and the best disease resistance is selected and used in the marketplace.

' I

Table 24 Concentrations of Linear Furanocoutiiarins in Celery Cultivars

Tall Utah 5270-R8 Florida21928 FloridaImplicated brand, lWb Implicated brand, 1986'

grown

Psoralen

5-MOP

California

0.15 i 0.06 <0.03 0.07 f 0.03 2.50 f 1.90 1.70 0.20

0.14 f 0.04 0.04 f 0.03 0.35 f 0.05 0.82 f 0.w 0.70 f 0.10

Florida Michigan

-

C

-

C

g.

Mean concentration =L: SD (pugfresh weight)

State where

Celery cultivar

0

*

&MOP 0.61 0.04 0.47 6.35 5.50

f 0.14 f 0.06 f 0.05 f 2-79

* 0.50

Total 0.90 f 0.24 0.11 f 0.09 0.89 0.13 9.67 f 4.98 7.90 0.80

*

SD, standard deviation; 5-MOP. S-methoxypsoralen: 8-MOP. 8-niethoxypsoralen. 'See Ref. 768. hSee Ref. 651. 'The state where grown. grower. and cultivar were undisclosed by the Centers for Disease Control (1984) and NOSH (1986). "Values were unreported in Ref. 651. 5 e e Ref. 652.

5' 3

0 0

8

Beier and Nigg

110

C. Parsley

Concentrations of linear furanocoumarins found in parsley by Chaudhary et al. (780) and by Beier et al. (445) are compared in Table 25. There is considerable difference between the two data sets of Tables 24 and 25 in the description of the quantities of linear furanocoumarins found. Linear furanocoumarins are more concentrated in parsley (Table 25) than in celery (Table 24). Parsley also contains the linear furanocoumarins, isoimperatorin, oxypeucedanin, oxypeucedanin hydrate, and graveolone (Fig. 32) (445, 736, 780), in addition to those found in celery (Fig. 9). The linear furanocoumarin oxypeucedanin is weakly mutagenic in the dark with the Ames test (781) and it also causes photodermatitis. In Table 25, the brand of commercial parsley flakes marked “high” contained over 300 pg/g dry weight of the phototoxic linear furanocoumarins psoralen, bergapten (5methoxypsoralen, 5-MOP), and xanthotoxin (8-methoxypsoralen, 8-MOP). Double-curled parsley had 112 pg/g of total active linear furanocoumarins per fresh weight, which is about 175-fold more than that found in celery. A constituent of parsley leaf oil, myristicin (Fig. 3), was shown to be a potential cancer chemopreventive agent (752). Myristicin showed high activity in its ability to induce the detoxifying enzyme system glutathione S-transferase in mouse tissue. Carrot and nutmeg also contain myristicin. However, myristicin is converted to an amphetamine in the liver and has been a chemical of abuse for many years (see Sec. VI). Myristicin is a methylenedioxyphenyl inhibitor of the mixed-function oxidase (MFO) (783) detoxification enzymes that metabolize many xenobiotics (784) and is a phytosynergist of xanthotoxin. A phytosyrzergist isa plant compound that is present at concentrations producing no toxic effect but that has a synergistic effect on coocculring toxins. Myristicin has a synergistic effect on the toxicity of xanthotoxin to Heliothis Zen, and it increased the phototoxicity of xanthotoxin in the presence of UV light (785).

lsoimperatorin

Oxypeucedanin

Oxypeucedanin hydrate Figure 32 Three linear furanocoumarins and graveolone in parsley.

Table 25 Concentrations of Linear Furanocoumai-ins in Parsley

-i

0

5.

Mean concentration * SD (pg/g)" Leaves of cutled parslef

Psoralen +MOP &MOP TW Imperatorin lsopimpinellin Oxypeucedanin

2.5 f 10.2 f 3.6 f 16.3 f 0.5 f 1.8 f 102.9 f

0.6 1.7 0.8 3.1 0.1 0.4 14.0

Double curled

Parsley flakes

Parsleyflakes

parsley"

(high)"

(l0W)c

parsley flakesb 1.6 6.6 2.3 10.5 25 3.1 88.6

1.6 1.8 0.6 4.0 f 0.8 f 0.1 17.9

f f f f

0

32.7 f 6.2 63.9 f 13.4 15.4 f 5.1 112.0 f 24.7

*

SD. standard deviation. aValues are given for fresh weight except for parsley flakes, which were dry. 'See Ref. 780. 'See Ref. 445.

*

104.7 4.2 146.7 f 19.4 53.0 f 5.3 304.6 f 28.9

32.3 f 56.7 f 5.3 f 94.3 f

1.8 6.5 2.9 11.2

5'

and

112

Beier

Nigg

D. Parsnips Mixed crystals of linear furanocoumarins were detected on surfaces of parsnip roots by scanning electron microscopy (SEM) and concentrations of the compounds were determined (786) (Table 26). The concentrations of linear furanocoumarins also have been determined in raw, boiled, and microwaved parsnip root (787). Ivie et al. pointed out that consumption of moderate quantities of parsnip root could result in ingestion of appreciable amounts of linear furanocoumarins (787). This study also demonstrated that these chemicals are stable to cooking. The natural synergist myristicin also is found in the edible parts of parsnip root (788).

E.

Figs

Fig (Ficzrs cmica) phytophotodermatitis occurs in some individuals who handle figs and in 10% of all the individuals who handle figs in Turkey. The skin reaction results from contact with the milky sap (latex) of the plant followed by exposure to the sun. The major photoactive compounds isolated from fig leaf are psoralen and bergapten (Fig. 9). Higher levels of these photoactive compound are produced in the spring and summer and are responsible for the increased incidence of fig dermatitis during these times. The peak psoralen concentration measured in fig leaf is 1650 pg/g fresh weight and in fig leaf sap this concentration is 2090 pg/ml. The peak bergapten concentration measured in fig leaf is 480 pg/g fresh weight and in fig leaf sap the concentration is 620 pg/ml. Photoactive furanocoumarins were not detected in any part of the fruits (163). Fig fruits have been investigated for linear furanocoumarins, and these photoactive compounds were not detected at a 0.03 pglg detection limit (7893. Figs also may be contaminated with the mycotoxin aflatoxin. Aflatoxin-contaminated dried figs were observed at a rate of about 1 in 100 fruits and contained aflatoxins B and G I at less than 0.2 ng/g to over 1000 ng/g; most contaminated figs could be removed by observing their fluorescence (790). A method of assessing quantities of phototoxic linear furanocoumarins may beapplicable for monitoring skin patches or skin surfaces. A portable fiberoptics lunlinoscope

Table 26 Concentrations of Linear FuranocoumarinsinParsnipRoot

Mean concentration f SD (pg/g fresh weight) ~~

Psoralen Wholea 7.1 f 7.3 1.4 Peela 9.4 f 2.9 Whole, diseaseda 537.0 f 220.0 Rawb 10.5 f 0.5 Boiledb 11.8 f 4.1 1.8 Microwavedb 10.7 f 3.3 0.6

5-MOP f 0.7

22.5

f

154.4 4.4

~

48.0

*

62.4 6.5

f

30.0

f 8.6 186.3 f 38.2

90.8 f 15.8 1109.0 f 185.0 1736.8 3.2 0.3 26.1 f 39.8 1.8

*

f:20.8 0.4 f

0.5

f 44.7 2.4 27.9 f 41.9 1.6

SD. standard deviation: 5-MOP, 5-methoxypsoralen; %MOP, 8-methoxypsoralen. “See Ref. 786. hSee Ref. 787.

~~

Total

8-MOP

f

* *

420.8 2.6 4.6 2.7

Naturally Occurring Toxic Chemicals in Foods

113

developed for monitoring skin contamination in various settings (791, 792) has been applied to the measurement of linear furanocoumarins (psoralens) in vegetable products (793j.

XII.

OXALATE-RICH FOODS

The consumption of oxalate-rich food plants may lead to calcium deficiency as well as poor absorption of iron, magnesium, and copper. Acute toxic effects from oxalate include muscle cramps, cardiovascular collapse, and renal insufficiency. The spinach leaf may contain a mean concentration of 68,500 pg/g fresh weight (see Ref. 815). The lethal dose of oxalic acid for humans varies from 2 to 30 g (794). The data suggest that it would require approximately 1.2. pounds of spinach to produce lethal toxicity from oxalate.

A.

GeneralPerspective

Oxalic acid is caustic and corrosive to skin and the mucous membranes and ingestion can cause severe gastroenteritis. Renal damage from calcium oxalate, and convulsions, coma, or death from cardiovascular collapse may occur. The rat oral LD50is 9.5 ml/kg body weight of a 5% solution of the dihydrate or about 475 mg/kg (see Tox. Appl. Ph. 42:417 (1977) in Ref. 437). Oxalic acid is used as an analytical reagent in printing and dyeing calico and for bleaching straw hats and leather. It also is used for removing paint and varnish, rust and ink stains, cleaning wood, and in blue-ink, dyes, metal polish, and for purifying methanol. The ceramic, pigment, metal, paper, photography, engraving, and rubber industries use oxalic acid (437). American Conference of Governmental Industrial Hygienists (ACGIH) lists a time-weighted average for human exposure of 1 mg/m’ (67j. Oxalic acid causes renal damage from the deposition of crystals after ethylene glycol ingestion. Ethylene glycol ingestion causes about 50 deaths per year (795). Ascorbic acid also is metabolized to oxalic acid (and also to diketogulonate) in humans (796). Calcium oxalate (dihydrate and monohydrate) crystals occur in the urine of nomlal persons, but are more frequent and larger in calcium oxalate “stone formers’’ (797-799). The concentration of oxalate in the urine and its total excretion is higher in stone-forming patients (800). Calcium oxalate crystals were found in most urine samples below pH 6.5, whereas calcium phosphate crystals were found in urine above pH 6.5 (801). Primary agglomeration appears to be one mechanism for oxalate stone formation (802) and specific substances favoring stone formation have been theorized (803j. Patients with primary hyperoxaluria remain asymptomatic until renal failure is far advanced. Systemic oxalosis may display dysrhythmias, digital gangrene, and peripheral neuropathy due to deposition of calcium oxalate in the myocardium, small muscular arteries, and small peripheral nerves (804). Peritoneal dialysis and hemodialysis have been used to reduce blood oxalate levels in patients with end-stage renal disease, but dialysis is considered a temporary solution to overproduction of oxalic acid (804, 805j. Calcium oxalate stone formation is linked to intrinsic overproduction, vitamin D1 administration to dialysis patients (805), pH changes (801, 806), contraceptive hormones (806), citrate, pyrophosphate, and heparin (807), and to food intake (808). In a study of 11,114 kidney stones, 67% contained calcium oxalate. About 32% of all stones were pure calcium oxalate (808). The remaining stones were either pure or

and

I14

Beier

Nigg

mixed phosphate (19%), uric acid (14%), and cystine (0.8%) (808). One risk factor in secondary hyperoxaluria is high exogenous oxalate intake: in a survey of 10,130 subjects in the Federal Republic of Germany, overconsumption of chocolate, rhubarb, and spinach was identified as a risk factor for stone formation (see Ref. 808 for a review). Oxalate absorption may be increased in patients with an ileal resection of greater than 30 cm (809). B.

MineralBalance

Because oxalic acid can bind minerals, the effect of oxalate-rich foods on mineral balance has been studied. Mean balances of calcium, magnesium. and zinc were negative on a high-fiber diet with or without spinach, but were more negative with spinach (810). The administration of magnesium reduces urinary oxalic acid excretion from spinach, though whether this observation is due to reduced intestinal absorption or increased oxalic acid metabolism is unknown (81 1). Calcium, magnesium, iron, zinc, and tnanganese weekly balances were not affected by 100 g of spinach per day. Copper balances were lowered, but were apparently a result of lower intake (812). Calcium from milk is more available than calcium from spinach (813, and calcium oxalate did not interfere with calcium uptake from milk (814). Oxalic acid appeared to enhance iron utilization by rats (815) and did not interfere with zinc absorption by rats (816). In therat, calcium in spinach is not absorbed as well as calcium from milk (8 17). Oxalic acid enhanced iron absorption in the rat (818).

C. Absorption In early studies, only 2-5% of dietary oxalate was absorbed from the intestine (819, 820), but up to 12% may be absorbed after fasting (821), and 6.6% is absorbed on a routine basis (822). Only 2.4% plus or minus 0.7% of I4C-oxalic acid added to spinach and rhubarb meals was excreted in urine, indicating low absorption (823). However, by measuring urinary oxalate from persons on low, high, and unrestricted oxalate diets, Finch et al. concluded that up to two-thirds of urinary oxalate is contributed by the diet, withan absorption range of 1.3% (16.9-19.1 mmol oxalate intake, rhubarb) to 22% (0.5-0.6 mmol oxalate intake, tea) of dietary oxalate (824). Human oxalate absorption was 0.7% from beet fiber, 4.5% from spinach, and6.2% from oxalate solutions (825). Bioavailability (absorption) of oxalate in humans was 0.08% (tea, brewed), 0.03% (tea with milk), 5.8% (turnip greens), 0.00001% (okra), 3.8% (peanuts), and 2.8% (almonds) (826). Table 27 presents the oxalate content of selected foods (794, 815-8 18, 820, 826842). The listed food with the highest oxalate content is rhubarb, with one sample of 216,000 pg/g. Spinach has a consistently high oxalate content and the oxalate contents of peanuts, almonds, okra, and brewed tea range from 1000 to 2800 pg/g. Beets have a high content, about 7000 pg/g. Oxalate consumption may produce an irritant effect from crystals (common in many plants and fungi) (843-845), crystal punctures (846), and local tissue necrosis with crystal aspiration (847). Oxalate also may be conjugated to other compounds and consequently not be analyzed with normal methods (848). In addition to foods, oxalate is a common constituent of many wild plants, including range plants (794, 849). Since the diurnal rhythm of oxalate excretion is abolished with fasting (850, 851) and significantly reduced on a low-oxalate diet (824), the contribution of diet to oxalate levels can be assessed by urine testing before and after fasting. Such testing should con-

Naturally Occurring Toxic Chemicals in Foods Table 27 ~

115

Oxalate Content of Selected Foods and Plants

~-

Food/plant Almond Almond, dry roasted Alternanthera sessiiis (Racaba) leaf (vegetable) (dry weight) Amaranth Apple Apple Apple juice Apples, raw Arachis hvpOgae8 (peanut) Raw Roasted Asparagus Atrlplex hortensis (garden orach) Banana Banana, raw Beet root, boiled Beets Beta vulgaris (sugar beet, table beet, chard, etc.) Beet Beet root Cabbage, boiled Cabbage, boiled Came///asinensis (tea) Brewed Brewed Carambola (16 varieties) Carrots Carrots, boiled Cashew nuts Cauliflower, boiled Celery, raw Celery stem, raw Chives Chives, raw Chocolate, unsweetened Chocolate, plain Coca-Cola Cocoa

Oxalate content Reference Wg)

no.

2610' 1310 68,000

827 826 828

5980' 15"

827 820 829 830 830

300' Trace 300 2250' 1870 180 12,000-1 6,000" Trace 7a 1210 1270

829 830 829 831 830 820 820 829

600" 6750' 6-2 1 ND~

833

460-1 260 50-2600 800-7300' 600a 227 2310"

820 834 832 829 820 027 820 830 820 829 820

175

200' 175" 1850' 1I'

3250 1170 Trace 9080

830 830 830

829

830 830 829

Beier and Nigg

116

Table 27

Continued

Food/plant Cocoa, powder Cocoyam (taro)

Oxalate content (d9)

Reference no.

6230

820

2200 7000 443-842 31 1-464 330 12,680 56 40' 10' 13,520' 2350 1 80' 50-320 120 ND ND 370' 2170' 7320*

794 794 835 835 830 836 820 829 830 836 827 829 837 830 830 820 829 827 827 829 833 830 836 820 826 829 829 830 830 830 820 826 829 829 830 820 836

Colocasia antiquorum

Main tuber, peeled Mature leaf (dry weight) Colocasia esculenfaL. Xanthosoma sagittifolium

Coffee, instant Coriandrum sativum(cotlander), leaf Cornflakes Cucumber Cucumber, raw Curry leaves Drumstick leaves Eggplant Fermented fish paste (India) Fruit salad, canned Grapefruit juice Grapefruit, raw Green beans Horse gram Kilkeerai Lettuce Lettuce Lettuce Lettuce Oatmeal, porridge Okra, cooked Okra Onions, green Orange, raw Ovaltine Parsley, raw Parsley, raw Peanuts, dry roasted Peas Pecans Pepsi-Cola Pineapple, canned Piper befle (betel leaf)

110'

200' 30' 3660' 10 1320 1460a

760' 40' 350 1000' 1660' 1160 60' 2020' Trace ND 1 3,500'

Naturally Occurring Toxic Chemicals in Foods Table 27

117

Continued

Foodlplant

no.

Plums, stewed Potatoes, boiled Radish Raspberries, raw Rheum species (rhubarb) Rhubarb Rhubarb, stewed Rhubarb, stewed Petiole Petiole (10 varieties) Petiole (78 varieties), dry weight Petiole, field cultivated Petiole, forced Rice pudding Sesame seed Soybean crackers Spinach Spinach Spinach Spinach Tuftegard, dry weight Variety unknown (dry weight) Variety unknown (raw) Variety unknown (raw, powdered) (boiled, powdered) Winter Bloomsdale (dryweight) Winter Bloomsdale (dry weight) Spinach, boiled Spinach, boiled Spinach, boiled Spinach, raw Spinacia oleracea L. (spinach leaf) Strawberries Sweet potato Tea, brewed(10 gm1500 mi water) Tea with milk (10 g/250ml water,250 mi milk) Tetragonia tetragonioides(New Zealand spinach), leaf Tomato Tomatoes, raw

Oxalate content Reference (cc9/9) 340 ND 6760' 220'

820 830 836 820

2750' 2600-6200 8600 18,000-21 6,000' 3300-6600' 33,500-95,000 61,000-85,000 50,000-94,000 ND 15,000 2070 1 3,700a 10,1 40' 10,500'

829 820 830 794 830 839 840 840 820 827 829 832 836 829

68,000-74,000 140,000 4800 147,400 137,900 70,000 78,000-81,000 4580-7800 7500 1 625-444 1 3444-6571 4400-6500 470a 1410a 2800 2804

815 81 8 825 81 7 81 7 816 81 5 820 830 834 834 841 829 829 826 826

1 17,000

842

130' 2oa

829 830

Beier and Nigg

118

Table 27

Continued

Food/plant

Oxalate content Reference (d9)

no.

~

Turnip greens, cooked Turnip greens Vegetables, canned Wheat germ White potato

60 530' 500 2690a 560'

826 829 830

829 829

'Fresh weight. hND. not detected.

sider ascorbic acid intake (one end-point of ascorbic acid metabolism in humans is oxalic acid), and also xylitol intake, which has been linked to oxalate crystal formation in humans (852). A variety of gas chromatographic techniques is available for oxalic acid analysis (834,853-855), although without careful analysis spurious results tnay be obtained (856).

XIII. SWEETPOTATOES (IPOMOEA BATATAS) Sweet potatoes are used widely as human food. A large proportion (about 35%) of the total production is lost by spoilage (857). Sweet potatoes produce numerous natural pesticides or phytoalexins. The first phytoalexin isolated from infected sweet potatoes was the furanosesquiterpene, ipomeamarone (Fig. 33) (858), which is hepatotoxic to mice with an i.p. LD5(,of 230 mg/kg body weight, as well as being toxic to fungi, bacteria, and yeasts (859). Other chemicals (furanoterpenes and eudesmanes) isolated from fungal-infected sweet potatoes are listed in Table 28 (860-867). Sweet potatoes stressed by HgClz respond by producing the phytoalexins or toxic chemicals shown in Table 29 (868-870). Mold-damaged sweet potatoes can have catastrophic effects in cattle: in one case, 69 of 275 exposed cattle died of lung edema (871). Phytoalexins apparently are responsible for the toxicity of sweet potatoes to animals (872). A scheme for the biogenesis of sweet potato stress metabolites was presented by Schneider et al. (862). A.

Proposed Lung Toxins

Four closely related 174-dioxygenated-1-(3-furyl)pentanes (ipomeanine, 4-ipomeanol, 1ipomeanol, and 1,4-ipomeadiol) (Fig. 34) have been suggested by Boyd et al. as being responsible for the observed lung edema caused by sweet potato (864). The LDSovalues of these chemicals, and ipomeamarone, in mice are presented in Table 30 (859, 864, 873). Each of the lung toxins produces identical reactions whether they are given orally, intraperitoneally, or by intravenous injection (865). At LDSnconcentrations, 1-ipomeanol and 1,4-ipomeadiol are toxic also to the kidneys. Tubular nephrosis and accumulation of intratubular debris occur in mice (864). High concentrations of some toxic furanoterpenoids can accumulate in infected sweet potatoes (Table 31). Intraperitoneal injections of 200-250 yg/g body weight of the stress metabolite 7-hydroxymyoporone (Fig. 35) are toxic to the liver as well as the lungs

ht)

Naturally Occurring Toxic Chemicals in Foods

119

Table 28 Chemicals Isolated from Sweet PotatoesInfected with Cercrtocystis jktbriata and Other Pathogens

Concentration Reference (P9/9) no.

Chemical

860

20 7-Hydroxymyoporone 861 125 7-Hydroxycostal 861 1120 7-Hydroxycostol 9-Hydroxyfarnesoic acid 6-Oxodendrolasinolide Ipomeatetrahydrofuran (Z)-1,6-Dioxoisodendrolasin (€)-I ,6-Dioxoisodendrolasin 10-Hydroxyipomeabisfuran lpomeamaronolide 4-Hydroxymyoporonol 4-Hydroxymyoporonol ketal lpomeararone 864,865 6724 4-lpomeanol 1-Ipomeanole lporneanine' 1 ,4-ipomeadiola Chlorogenic acidb Caffeic acidb lsochlorogenic acidb Pseudochlorogenic acidb Dehydroipomeamarone" lpomeamaronol'

862 862 862 862 862 862 862 862 862

863 864 864 864 866 866 866 866 867 867 867 867

4-HydroxydehydromyoporoneC

4-Hydroxymyop~rone~ 'Sweet potatoes were infected with F u s a r i m solmi. bSweet potatoes were infected with Cernmstonlella jifirdwiatcr. 'Sweet potatoes were infected.

Table 29 PhytoalexinsIsolated from Sweet Potatoes Stressed with HgC1: ~~

Concentration (pg/g fresh Reference no.

Chemical ~

~~~

Myoporone 6-Hydroxymyoporol 1-Hydroxymyoporol 6-Oxodendrolasin 6-Hydroxydendrolasin 9-Oxofarnesol 9-Hydroxyfarnesol 6-Dihydro-7-hydroxymyoporone

39.0 109.0 53.0

8.5 1.2 0.7

12.6 26.0

868 868 868 869 869 869 869 870

120

Beier and Nigg

Table 30 LD5, Values for Chemicals from Sweet Potatoes in Mice Concentration(pg1g)

Reference No.

Intraperitoneal intravenous OralChemical ~~

~~~

~

-

~

lpomeamarone 4-lpomeanol 4-lpomeanol l-lpomeanol lpomeanine 1,4-lpomeadiol

230 30

-

21 24'

073

864 49 25 67

34 14 68

864 864

064 36

-

79 26 104

859

'The LD5(,was obtained in the rat.

of mice. This compound is equivalent in hepatotoxicity to ipomeamarone (Fig. 33) (860). 6-Myoporol (shown in Fig. 35) is two to three times more hepatotoxic than ipomeamarone (868).

B. Average Concentration of the Lung Toxin lpomeamarone Blemished, damaged, or infected sweet potatoes from U.S. (874) and U.K. (875) food stores have been analyzed for ipomeamarone. An average concentration for the sweet potatoes in U.S. stores was 1832 pg/g, while a maximum of 900 pg/g was found in those from the United Kingdom. Samples that apparently were free from damage contained a total of 68-328 pg/g ipomeamarone and also were positive for the lung-edema toxin ipomeanine (Fig. 34) (875). Lung-edema toxins occur in blemished and diseased areas of sweet potatoes (876), and toxic sweet potatoes may show only slightly darkened areas without significant color changes (877). The actual levels of toxins are not predictable by

Table 31 MaximumConcentrations in Sweet Potatoes of Chemicals Produced by Infection with Different Sweet Potato Pathogens

Concentration (dg)

Chemical ~

46gb

~~~

lpomeamarone 4-lpomeanol 1,4-lpomeadiol furanoterpenoids Total 106,429'

23,346' 236b

~

~~

~~

'Maximum concentration from sweet potatoes moculated with Sclerotium rnlfsil. hMaxlmurn concentration from sweet potatoes inoculated with Frrsnrbm solmi. L"aximun1 concentration from sweet potatoes inoculated wlth Dirrpnrthe brrtrrtm. Sowce: From Ref. 863.

121

Naturally Occurring Toxic Chemicals in Foods

lpornearnatone Figure 33 Iporneamarone, the first hepatotoxic phytoalexin isolated

from sweet potatoes.

visual examination (875), and normal baking or boiling does not affect thetoxin levels (877).

C. Activation of the Lung Toxins The lung-edema toxin 4-ipomeanol (Fig. 34) is oxidatively metabolized by lung Clara cells in hamsters, mice, and rats (878). Boyd suggested that the xenobiotic-metabolizing Clara cells, containing the cytochrome P-450-dependent MFO system, may be a prime site for bronchogenic cancers (878, 879). The toxicity of 4-ipomeanol to rats apparently is due to a reactive metabolite. The cytochrome P-450 inhibitors pyrazole, piperonyl butoxide, and cobaltous chloride decreased the pulmonary alkylation and the toxicity although SKF-525A had no effect. Radiolabeled 4-ipomea1101 administered by intraperitoneal injection bound more to lung tissue than to any other site. Liver tissue only bound 20% of the4-ipomeanol and was second in binding potential to lung tissue (873). Repeated intraperitoneal injections of 4-ipomeanol in rats resulted in extensive Clara cell necrosis ( . S O ) and in mice caused interstitial edema (881). When mice with viral pneumonia were given 4-ipomeanol, the severity of pneumonia increased with increasing doses of the lung toxin (882).

4-lpomeanol lpomeanine

1-1pomeanol

1,4-lpomeadiol

Figure 34 Closely related lung toxins isolated from sweet potatoes.

Beier and Nigg

122

7-Hydroxymyoporone

OH j

C

CHa

H

3

0

6-Myoporol Figure 35 Hepatotoxic compounds isolated from sweetpotatoes.

D. Sweet Potato Connection to High Rates of Asthma In studies of chronic lung disease in New Guinea populations, Woolcock et al. pointed out that men and women suffer from chronic respiratory disease of unknown etiology (883). They suggest that the etiology may be an environmental factor. It was suggested that humans may be affected adversely by sweet potato consumption (884). Wilson reports that natives from the highlands of NewGuinea raise and consume large quantities of sweet potatoes as a major dietary item (884). Dawson and Mitchell state that the Moari and Pacific Island children have a higher mortality rate from asthma than European children (885). They also state that the cause of the high rates of asthma and asthma-related deaths is not known, and continued research in New Zealand and other countries is needed to identify the causative agent(s). Although the death rate among young people (aged 5-34 years) in New Zealand has decreased from over 4 per 100,000in 1980, it is still persistently high as shown in the death rate of 1988, which was 2.6 per 100,000 (886). It should be noted that other furanoterpenes that may have delayed pneumonotoxicity also may be found in plants (887). Since it is known that 4-ipomeanol (Fig. 34), found in sweet potatoes, is metabolized by lung Clara cells and results in toxicity (878), and also that mice with viral pneumonia given 4-ipomeanol had an increase in the severity of the pneumonia (883) there is the possibility that the lung toxins found in sweet potatoes may exacerbate the severity of asthma in humans.

XIV.

A.

TANNIN-RICH FOODS GeneralPerspective

Tannins are polyphenolic molecules widely distributed in plants and not found in prokaryotes, protists, fungi, or animals (888, 889). Compounds of this type were usedto tan leather, which provided the name tumin. By definition, tannins are biologically active; that is, tannins bind proteins, a process that is pH dependent (890). Most of the phenolic

Naturally Occurring Toxic Chemicals in Foods

123

groups in tannins are free. Tannins have a molecular weight of 500 or more and generally are soluble in water. Condensed tannins are oligomers and polynms of flavonoid units and, because the units are linked by carbon-carbon bonds, are not subject to hydrolysis. Hydrolyzable tannins are linked by carboxylic ester links, are hydrolyzed by acid, base, and esterase enzymes, and are classed as gallotannins (hydroxyl groups esterified by gallic acid) and ellagitannins (hydroxyl groups esterified by hexahydroxydiphenic acid) (888, 889, 891). The chemistry of tannin-protein complexes is reviewed in Ref. 892. A variety of methods for analyzing tannins are available (893-903). Procedures used to quantify tannins and appropriate standards have been reviewed by Hagerman and Butler (897) and Okuda et al. (902). An understanding of the specificity of the assay is essential before interpreting tannin analytical and toxicological data. Many of the biological effects of tannins have been reviewed (888, 895, 902, 904) for protein, metal ion, carbohydrate, alkaloid and phospholipid binding, antifeedant activity for herbivores, nutritional effects (food consumption, food utilization, and growth mechanisms), and physiological adaptations to tannins. However, tannins and tannin complexes may be an important component of the “taste” of some products (e.g., wine) (905). B. Effects of Tannins Tannins generally are considered to be astringent; that is, they lead to a puckering sensation of the mouth (906), although this sensation may be mild with some tannins (907). Astringency is also a drying sensation of the mouth that develops with time (908). Theperception of astringency often is confounded with bitterness as most compounds producing astringency also produce a bitter taste (908). Astringency of any particular foodstuff may be modified by the amount and type of available compound (907, 909), intake of sweetening agents (908). and the amount of polymerization, monomers being more bitter and polymers being more astringent (9 10j. Tannins are the apparent active ingredient in some natural drugs: geraniin from Gemnium thur~bergii,an antidiarrhetic; geraniin and mallotusinic acid from Mallotus juporzicus for gastric ulcers; granatin A and B, punicalagin, and punicalin from Pzmica g l a r m t l m (pomegranate), anthelmintics; and terchebin, chebulinic acid, chebulagic acid, and punicalagih from Tet-mirzaliacl?ebuln,astringent drugs (907). Rhubarb contains a very high tannin level and is used as a purgative, whereas tannins normally are used as antidiarrhetics (907). Rhubarb’s purgative action may be due to its high oxylate content (437, 820, 829, 830). Ethnophamacological studies usually include assays for tannins (91 1, 912). Tannic acid extracted from plants is used infood processing, burn treatment, barium enemas, for clarifying beer and wine, and inconfectionaries and chewing gum (913). Because of their analytical complexity and variety, the level of tannins in foods is represented best on a relative basis, that is, whether it is high or low. Even when the same analytical method is used on different foods, the results may be only relatively dependable unless the basic chemistry is understood. The polyphenolics of sorghum, including tannins, are reviewed in Ref. 895. Butler makes the important point that “sorghum cultivars rich in ‘tannin’ including low-molecular-weight flavonoids are nutritionally inferior to tannin-free cultivars” (895, p. 100).Sorghum tannins negatively influence the digestion of fiber (914). Bird-resistant sorghum cultivars may be tannin rich or tannin free, but, in general, high-tannin cultivars are bird resistant (895). The postulated antinutritional effects of sorghum tannins (protein complex-

124

Beier and Nigg

ing and erosion of the intestinal mucosa) have not been proven (895). Rats undergo adaptation to tannin-rich diets by hypertrophy of the parotid salivary glands and production of proline-rich proteins that are very efficient tannin binders. Mice display a similar adaptation, but hamsters do not. Hamsters are more vulnerable to tannins than rats and mice. In the human, proline-rich proteins make up 75% of salivary proteins (895) and these proteins contain carbohydrates that affect tannin binding (915,916). It has been suggested that tamidprotein interactions may be very specific in vivo. Other detoxification mechanisms for sorghum tannins, including food processing, are reviewed in Ref. 895. Betel nut (Areca catechu L.) contained from 135-489 absorbance units of total phenols (tannins) compared to 3.6 for tannin-free sorghum and 39.9 for high-tannin sorghum (917). Using a tannic acid equivalents (TAE) assay, black teas were the most astringent, with a TAE of 42.5 (cut black tea), whereas green tea from China had a TAE of 11.7, almost 4 times lower (906). Tea grown under shade is claimed to be less astringent (918). The tannin content of selected foods is presented in Table 32 (849, 894, 919-940). The comparison of foods for tannin content is frustrating, to say the least. Preparation and analytical methods differ. Different standards such as tannic acid, catechin, or tannin extracted from a different source may be used. Data presentation may be in mg/100 g, mg/g, % equivalents, mg/L, or pg/mg and either dry or fresh weight. If a reader is not careful, the impression might be that a foodstuff is low in tannin when the opposite is true. We suggest that all tannin data be reported as pg/g (ppm), afairly standard practice in analytical chemistry. Data for tannin content reported as yg/berry or mg/stalk, and so on, purposely have been left out of this review because these data are useless beyond a specific point in a specific study. A variety of presumed beneficial effects of tannins are listed in Table 33 (920, 941971). These should be viewed with caution. Anticarcinogenic and antiplaque activities have been reported for foods but, in one case in the rat (97 1j, the effect was correlated with fluoride content and not with tannin content. A hot-water extract of green tea given orally to the rat 24 hr prior to injection suppressed aflatoxin B I chromosomal aberrations. Green tea extract given 2 hr before or after the aflatoxin B I dose did not suppress chromosomal aberrations (968). Low concentrations of tea tannin suppressed sister chromatid exchanges and chromosome aberrations in cultured mammalian cell and in mice whereas, at 20 pg/g, a comutagenic effect was observed (969). Antimutagenic activities of tannins also may be confounded with activities of other compounds such as ascorbic acid (vitamin C) (966). Green tea hot-water extracts inhibited platelet aggregation supposedly through the action of epigallocatechin gallate, which has the same in vitro potency as aspirin (965). Many health claims have been made for tea. Tea has been claimed to help digestion, the nervous system, blood vessels, cardiovascular function, blood pressure, and also to increase energy (972). Tea is supposed to have a vitamin P effect (see Sect. V.B) that provides antioxidants and radiation protection, delays arteriosclerosis, is antimicrobial, is effective against dysentery, is therapeutic for chronic hepatitis, and has been termed ‘‘an elixir of life” (972). Tannin from cotton bract promoted platelet aggregation and may contribute to the pulmonary symptonx of byssinosis (973). Tannin may affect niacin utilization (974) and negatively affects protein utilization (922, 929, 975). Tannins in tea appear to be responsible for reduced iron absorption in humans (976), apparently through formation of iron complexes (977). In fact, immobilized tannin has been proposed as an absorbent for protein and iron (978). Tea and coffee (and presumably their tannins) decrease thiamine utilization

Naturally Occurring Toxic Chemicals in Foods

125

Table 32 Tannin Content of Selected Foods

Food

Assay

Alfalfa leaf concentrate Almond H20extract (1/10) Apple juice Armagnac Banana H20extract (1/10) Barley Barley Bean, red, common (Phaseolusvulgaris) Dry bean(Phaseolus vulgaris) Dry bean (Phaseolus vu/garis) Beer

Content (PSlS)

Reference no.

3600-1 0,340 Folin-Ciocalteau chlorogenic acid standard Folin-Ciocalteau gall nut tannic acid standard Folin-Ciocalteau gall 100 nut tannic acid standard Acid hydrolysis, HPLC 13-1 27 240-nm castalagin equivalent Folin-Ciocalteau gall 0 nut tannic acid standard 4100 Butanol/HCI 1100 Folin-Denis, tannic 8600 acid equivalent Vanillin-HCI catechin 0-3060 equivalent Vanillin-HCI catechin 0-9800 equivalent Iron-phenanthroline, 74-1 01 tannic acid equiva-

-

919 920

920 921

920

922 923 924 925 926 927

lent Black cherryjuice

300

Brewer's grain Carob pods (food additive) Cassava

1400 192,000

Cinnamon

38,700

Cocoa Cocoa, 30 g/lOO ml

142,400

Coffee

13,500

2000 4100

Folin-Ciocalteau gall nut tannic acid standard ButanoVHCI Anthocyanidin standard Vanillin-HCI catechin equivalent FAS" reagent, tannic acid equivalent

-

Folin-Ciocalteau gall nut tannic acid standard FASa reagent, gallic acid equivalents

920

923 928 929 894 922 920 894

Beier and Nigg

126

Table 32 Continued

Food Coffee, green

Content no. Spectrophotometric, 6800

Coffee, ground Coffee powder

6600 84,100 5600

Coffee pulp Coffee, roasted

1600-1 4,800 1700

Coffee, 1 tablespoon/

Re Assay

1800 400

200 ml Cognac

14-71

Common bean Cottonseed Cranberry juice

89,100 16,000 1300

930

tannic acid equivalents S S A ~precipitation

-

FAS' reagent, tannic 894 acid equivalent Acid hydrolysis, HPLC Spectrophotometric, 930 tannic acid equivalent B S A precipitation ~ Folin-Ciocalteau gall nut tannicacid standard Acid hydrolysis, HPLC 921 240-nm castalagin equivalent

-

ButanollHCl Folin-Ciocalteau gall nut tannicacid standard

930 922

931

930 920

932 923 920

Crop seed Adansonia digltata Azadiracta indica Bixa orellana Blighia sapida Carapa procera Cedrella ordorata Coula edulis Dioscoreophyllum cumminsii Eucalyphus deglupta Hyphaene thebaica Lophiza data Monodora termifolia Rouvolia vomitoria Spondias mombin

9800 6000 5100 7100 8900 4600 1100 1600

Protein precipitation Protein precipitation Protein precipitation Protein precipitation Protein precipitation Protein precipitation Protein precipitation Protein precipitation

849 849 849 849 849 849 849 849

3300 11,100 11,500 1700 3400 5900

Protein precipitation Protein precipitation Protein precipitation Protein precipitation Protein precipitation Protein precipitation

849 849 849 849 849 849

Naturally Occurring Toxic Chemicals in Foods

127

Table 32 Continued

no.

Food

Assay

Terminalia catappa Treculia africana Vitex doniana Xylopia aethiopica

Forage legumes Forage tree leaves Grape juice Green tea leaves Lentil Dehulled seed

Content (Pglg) 4000 Protein precipitation 3600 Protein precipitation 6700 Protein precipitation 6600 Protein precipitation 1500-1 87,000 ButanoVHCI 0-450,000 Folin-Ciocalteau gall 1700 nut tannic acid standard 195,500

849 849 849 849 923 933 920

Vanillin-HCI, catechin equivalent Vanillin-HCI, catechin equivalent Vanillin-HCI, catechin equivalent FAS" reagent, tannic acid equivalent FASareagent, tannic acid equivalent Vanillin-HCI catechin equivalent

934

0-1 0

Seed coat

0-5720

Whole seed

0-5300

Oats

100

Oregano

20,500

Peach Boone County (low quality) Loring (yellow)

21,800 9310

Belle of Georgia (white) Peanut H20extract (1/10)

4410

Peanuts

NDC

Prune juice

500

Rapeseed Rapeseed meal

Reference

100

5900 682-772

Vanillin-HCL catechin equivalent Vanillin-HCI catechin equivalent Folin-Ciocalteau gall nut tannicacid standard FAS" reagent, tannic acid equivalent Folin-Ciocalteau gall nut tannic acid standard Butanol/HCI Vanillin-HCI, catechin equivalent

922

934 934 894 894 935 935 935 920 894 920 923 936

Beier and Nigg

128

Table 32 Continued

Food

Content no. (dg)

Rum

31-37

Ref Assay Acid hydrolysis, HPLC 240-nmcastalagin equivalent Catechin equivalent

921

'

Sorghum grain Sorghum leaf, 47 varieties Sorghum, red Sorghum Low tannin

690-1 970 0

FAS' reagent, tannic acid equivalent

094

1400

ButanollHCl, Lotus pedunculatus standard

923

FAS" reagent, tannic acid equivalent

894

11,8OO 200

Sorghum, dehulled Soybean Soybean meal Spinach

82,400 1000 1700 150 0

Tea

12,970-

Tea, black

39,600 52,500

Tea, green

26,600

Tea, Indian

3700

Tea leaves, black Tea leaves, black

2400 300,000 105,000

Tea leaves, green

84,000

Tea (Lipton)

937 938

5500

High tannin Sorghum, white

Swiss cheeseH20extract (NO)

-

200

-

Butanol/HCI

-

FAS' reagent, tannic acid equivalent Folin-Ciocalteau gall nut tannic acid standard

FAS' reagent, gallic acid equivalent FAS" reagent, gallic acid equivalent Spectrophotometric, tannic acid equivalent BSAbprecipitation

-

FAS" reagent, tannic acid equivalent FAS" reagent, tannic acid equivalent Folin-Ciocalteau gall nut tannic acid standard

932 923 922 894 920

894 094

930 930 922 094 894 920

Naturally Occurring Toxic Chemicals in Foods

129

Table 32 Continued

Food ~~~

Assay

Content (CLE)/g)

Reference

no.

~

Tea (Salada)

400

Tomato (total phenols), 3 varieties Tomatoes

23,900

Wine, red

87,000

NDc

Wine, red (5 month)

566

Wine, white

ND

Whiskey

7-30

Folin-Ciocalteau gall nut tannic acid standard Folin-Ciocalteau, gallic acid standard FAS’ reagent, tannic acid equivalent FASareagent, gallic acid equivalents Cinchonine precipitation gallic acid standard FAS’ reagent, gallic acid equivalent hydrolysis, Acid HPLC 240-nm castalagin equivalent

920

939 a94

894 940 894 921

”FAS, Fenic Ammonium Sulfate. ’BSA, Bovine Serum Albumin. ‘ND, not detected.

in humans (979). Gallotannic acid at 20 m g h l formed precipitates in vitro with amitriptyline, chlorpromazine, haloperidol, imipramine, loxapine, prochlorperazine, thioridazine, and trifluoperazine. Most of these also were precipitated by tea and four were precipitated by coffee (980). Tea accelerated benzo[n]pyrene-induced skin cancer in mice (98 1), and other tannin infusions produced cancer in rats when injected S.C.(982). Betel nut water extracts and betel nut tannins increased sister chromatid exchanges in mouse bone marrow cells after intraperitoneal (i.p.1 injection (983). Miang leaf (fermented wild tea leaves) and betel nut extracts inhibited DNA synthesis in cultured lymphocytes and tumor cells (984). Betel nut carcinogens are reviewed in Ref. 336. Betel quid in combination with tobacco is carcinogenic to humans (985). In a very insightful publication, Morton reviews the links between esophageal cancer and tannins in “bush tea,” tea without milk (the Netherlands), tea-rice gruel, dark sorghum (China, Rwanda), cider and calvados (France), liquor, and khat (986; also see Refs. 987 and 988). Tannins in tea are listed as one of the carcinogens in food (989) and in tea, red wine, and mate as a factor influencing cancer mortality patterns (990). It is noteworthy that removal of phenolics, including tannins, with the fining agent polyvinylpolypyrrolidone reduces red wine mutagenicity by 63-95% (99 1). One study of red kidney bean combined lectin and tannic acid and found that together they did not inhibit a-amylase, though both inhibit it individually (992). Hagernlan and Robbins suggested that tannins, in addition to forming insoluble protein complexes, also may form soluble complexes with metabolic effects that are not known (993).

Beier

and Njgg

130 Table 33 Presumed Beneficial Biological Activities of Tannins

Reference Tannin no. source Assay

Activity

Glucosyltransferase inhibition Bacterial adherAntiplaque Chewing sticks ence, in vitro No effect on mouse Ellagic acid (tannin Anticancer skin tumor formaconstituent) tion S. typhimurium, Antimutagenic Ellagic acid TA100 Antimutagenic Ellagic acid S. typhimurium, TA100 Antioxidant Oil oxidation Onion skin Osbeckia chinensis Antioxidant Thiocyanate and tannins TBA Pistacia lentiscus" Hypotensive Rat, i.p. Anti-herpes 1 and 2 Green monkey kidPlants ney cells CV-1; human adenocarcinoma A549 HIV-RT, H9 lymphoPlants (9 tannins) Anti-AIDS cYte Rhubarb Anti-hepatitis B HBV in cellline 2.2.15 (HBV) Rhubarb Rat, in vivo Antiuremic toxin Tannic acid stanAntimutagenic S. typhimurium dard (benzo(8)pyrene) TA100 Tannic acid stanAnticancer HeLa and Raji lymdard phoma cells Tannic acid stanAntitumor Mouse epidermis, In dard vivo Tannins (57) Antimelanoma (cyto- Human cell line toxic) Tea leaf infusiona Antiviral Bonnet monkeykidney cells,in vitro Tea tannin Reduction in intesti- Rat and rabbit intestines, in vitro nal movement Teaa Anti-Clostriudium Growth inhibition species Tea, green extract,' Antiatherosclerotic Mouse, in vivo 11 tannins Isolated Beverages, food

Antiplaque

920

941 942

943 944 945 946 947 948

949 950 951 952

953 954 955 956

957 958

959

Naturally Occurring Toxic Chemicals in Foods

131

Table 33 Continued

Content (P9M no.

Food

Anti-lung tumor Antiinflammatory, antitumor, dermal Antitumor, antiTea, greena cancer Tea, green, tannins Decreased cholesterol Radioprotective Tea, green Platelet aggregation Tea, green inhibition Antimutagenic Tea, green Tea, black Tea, green Tea, green

Reference Assay Mouse, in vivo Mouse, in vivo

960 961

In vitro, in vivo

962

PO, rat

963

Mouse, in vivo Rabbit platelets,in

964 965

vitro

E. coli, reverse mutation

966

Tea, green Tea, black

Antimutagenic

Sister chromatidexchange, chromosomal aberrations

967

Tea, green

Antimutagenic

Aflatoxin B,, rat bone marrow

968

Tea, black

No effect

Tea, green

Antitumor, skin

Tea, green Tea

Anticytotoxic Anticarcinogenic

968 Benzo(a)pyrene, mouse, in vivo Mouse hepatocytes Rat, in vivo

969 970 971

AIDS, acquired immunodeficiency syndrome; i.p., Intraperitoneal. ”Extract activity presumed to be from tannins.

XV.

CONCLUSION

One tenet of toxicology is a principle that originated with Paracelsus (994). If you want to explain each poison correctly: what is there that is not poison’? All things are poison and nothing (is) without poison. Solely the dose determines that a thing is not poison.-Paracelsus, 1492-1541; The Third Defense

This often misquoted principle generally is understood to mean that a dose-response relationship exists for all chemicals (994). For Paracelsus, this principle referred to “things,’ ’ including food and drink, as the first example appearing in the next sentence is “every food substance and every drink is poisonous when ingested above its dose” (994, p. 210). Many studies referenced here tested individual compounds in their experimental design. The dose-response relationship is a current concept. Testing procedures for chronic effects like cancer and birth defects nornlally evaluate single compounds.

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Occasionally, two-compound studies are used to test for potentiation, synergism, and antagonism. There are a few compounds in food that scientific studies have shown to have no redeeming value to humans. The potato alkaloids, psoralens in citrus and the parsleys, 4ipomeanol in the sweet potato, N-methylhydrazine in mushrooms, and cyanogenic compounds in cassava would seem to provide nothing for humans but toxic responses, acute and chronic. Of particular concern are the mycotoxins. These insidious contaminants could and should be eliminated from foods without any deleterious effect on humans based on the present data. Some of the studies we cited in this chapter used both a toxicant and an antitoxicant. Two-compound studies of this type are valuable for suggesting the potential for a dietary chemical to prevent a toxic effect. Other studies used an extract or the food itself. The positive results of studies with natural products (anticancer, antitumor, antimutagenic, etc., tendencies) tend to show up in the news media. This practice also seems common especially for preliminary medical studies. The public perception thatnatural ingredients, herbs, spices, and particularly all natural foods are valuable therapeutically is thus collectively ingrained in our intuitive knowledge and we subsequently believe that “everyone knows that it (substitute any natural food here) is healthful and therapeutic.” The key word is thempeutic. Paracelsus was defending his therapeutic use of mercury compounds for treating syphilis when he formulated his dose/poison principle. Is therapeutic use desirable when one consumes a product for a lifetime? What is the difference between a vitatnin or essential element and a therapeutic dose of a food chemical? The vitatnin or essential element participates directly in the biochemistry of the organism whereas the food chemical participates by affecting an organism’s biochemical and physiological processes. Does therapeutic use count in a risk/benefit analysis’?Is a small amount of a bad compound good if the small amount is therapeutic? Is a chronic dose better because we can tolerate that dose? Paracelsus was skirting the fine line between curing a patient and killing a disease organism with an acute dose. Quoting from Paracelsus, “And while a thing may be a poison, it may not cause poisoning” (994, p. 210). This statement of threshold values and no-effect levels is useful for many basic toxicological studies. Do these principles precisely apply to a small therapeutic dose of a toxic compound that may have a long-term negative effect but produces a beneficial effect in the short term? Perhaps they do, but we do not have the data on foods to answer this question definitely. What is a large dose anyway? It is given that the measurement of a dose in grams or moles is an absolute. One thousand yg/g (pptn) obviously is greater than 1 yg/g (ppm). If we compare the level of toxic (the definition of toxic depends on the study) compounds in food, synthetic pesticides and herbicides might be consumed at 60 pg/day and many natural compounds are consumed at 4.8-6 million pg/day. These numbers result in a natural pesticide potential of 80,000-100,000 times that of synthetic chemicals in the diet. This argument is used to emphasize the importance of food toxicants and to emphasize how little we know about their occurrence, levels, mode of action, biological effects, and, ultimately, risk. It is probably the only argument that has an impact on the general public. A “large” dose may not be a toxic dose. But for many natural compounds our toxicological knowledge is either nonexistent or very limited. Central to this discussion is the concept of hormesis. Homesis is defined as “the stimulating effect of subinhibitory concentrations of any toxic substance on any organism” (995). References 996-1000 are several excellent articles on hormesis. For our

Chemicals in Toxic Occurring Naturally

Foods

133

purposes, if a good effect (hormesis) occurs with a naturally occurring toxic food chemical, is there an advantage to chronically consuming the chemical? Drug dealers add strychnine to illegal drugs to improve the drugs’ performance. The desired effect is obtained, but is the consumption of a small amount of strychnine or any poison on a daily basis beneficial or healthy? The answer that respects scientific uncertainty is that we do not know. We are exposed to a plethora of natural food chemicals as mixtures. A small percentage of these chemicals have known toxic effects when tested alone. But most of the naturally occurring foodstuff chemicals have never been evaluated for toxicity or benefit of any type. For those that have been tested, the testing procedures were limited in scope, design, and dose range. Some of this testing is for “anti” or antagonistic responses. Yet, the same “good” compound may be protective at a low dose and toxic or carcinogenic at a high dose, perhaps to another animal or perhaps to the same animal. We cannot evaluate either the beneficial or the toxic effects on one end-point and ignore all other end-points, yet this is the common practice. We now live long enough to conduct epidemiological studies. These studies give us clues as to which mixtures of foods provide for lack of disease or pathological conditions. It is proper that we study these differences, but we rush to judgment on anindividual food component when a mixture of natural chemicals may be thecorrect answer. Although epidemiological studies provide clues, they do not provide absolute scientific answers. Epidemiological studies seldotn consider evolution. Could a stable diet for a society select for humans who survived longer on that diet? There is every reason to suspect that natural selection operates for diet just as for disease and environment. Is it fair to epidemiologically compare one society with a stable historical diet with another society that has dietary habits that are different and are historically recent? An example might be the comparison of epidemiological data of Japan, having an old established diet, to data from the United States, which on the evolutionary time scale has a historically recent diet that is changing constantly. Such a study might discover that anold dietary toxin that has selected survivors in one society is a new and potent dietary toxin for another society. For naturally occurring mixtures of chemicals, recommending the reduction of exposure or an increase in exposure will be very complex. For example, crucifers (broccoli, Brussels sprouts, cabbage, cauliflower) contain naturally occurring goitrogenic components resulting from the combined action of allyl isothiocyanate, goitrin, and thiocyanate. Although crucifers may provide some protection from cancer when taken prior to a carcinogen, crucifers act as promoters of carcinogenesis in animals when taken after a carcinogen. The acid-condensed mixture of 13C (an enzyme-released component of crucifers) binds to the TCDD receptor and causes responses similar to those of TCDD. Flavonoids may act as “vitamins,” but other compounds in this class may cause abortion. Herbs contain many biologically active components, with more than 20% of the commercially prepared human drugs coming from these plants. Onion and garlic juices can help to prevent the rise of serum cholesterol and protect against stomach cancer. Many herbs used in treatments have natural constituents that act oppositely from their intended use. Some herbs, like bishop’s weed seed, contain carcinogens, and others, may contain pyrrolizidine alkaloids, which are toxic to the liver. Two major potato compounds are a-solanine and a-chaconine. These are human plasma cholinesterase inhibitors and teratogens in animals. Because of toxicity to humans, the potato variety Lenape was withdrawn from the market. Celery, citrus, parsley, and parsnips contain the linear furanocoumarin phytoalexins psoralen, bergapten, and xantho-

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toxin, which can cause photosensitization and also are photomutagenic and photocarcinogenic. CDC and NIOSH studies definitively linked one new celery variety with outbreaks of photophytodermatitis in grocery store workers. At least one sweet potato compound, 4-ipomeanol, can cause extensive lung Clara cell necrosis and increase the severity of pneumonia in mice. Some sweet potato phytoalexins are hepatotoxic and nephrotoxic to mice. Might these lung toxins also exacerbate asthma conditions? A commonly consumed mushroom, Agaricus bispor-us, contains benzyl alcohol as its most abundant volatile compound. A. bisporus and Gyr-omitr-cresculenta both contain hydrazine analogs, and several hydrazines in mushrooms are rodent carcinogens. As much as 20-30% of the hydrazones in G. esculentc~decompose during cooking to N-methylhydrazine, causing the steam to be toxic. N-Methylhydrazine is metabolized to a reactive species that decomposes to produce a methyl cation alkylating agent. Mycotoxins are found worldwide in corn, cottonseed, fruits, grains, grain sorghums, and nuts (especially peanuts). They also occur in apple juice, bread, peanut butter, and other products made from contaminated starting materials. Mycotoxins have many biological activities that result in toxicity, mutagenicity, or carcinogenicity. Aflatoxins are linked to leukemia, liver cancer, mental retardation, and Reye’s syndrome. Aflatoxins are hepatotoxic to fish, mammals, and poultry. They attack the immune system. Fumonisin B causes equine leukoencephalomalacia, is toxic to a wide range of animals, including monkeys, is hepatotoxic to rats, causes porcine pulmonary edema syndrome, is toxic to cultured mammalian cells, and has been identified in moldy homegrown corn collected from southern Africa in anarea with a high human incidence of esophageal and liver cancer. Zearalenone and zearalenol are estrogenic agents and are barley grain contaminants. These agents could occur in beer or other barley-containing products. Zearalenone has been shown to be a teratogen. Most plants probably contain high concentrations of toxic chemicals and/or produce phytoalexins in response to disease, foreign chemicals, or injury. The herbicide acifluorfen increases the production of phytoalexins and stress metabolites in crops as diverse as bean, broad bean, celery, cotton, pea, pinto bean, soybean, and spinach. A foreign chemical may be as common as acidic air pollution. Celery was shown to increase its levels of phototoxic psoralens in a phytoalexin response to acidic fog at pH levels similar to that experienced by commercial celery production near major population centers. Plants also absorb and translocate toxic chemicals from the soil. Endive and lettuce absorb and translocate selenium and mutagens from fly ash. Other food plants known to actively translocate chemicals include cantaloupes, carrots, celery, coffee, green beans, herbal plants used for teas, mushrooms, peppers, radishes, rhubarb, and spinach (1001). If we understood the intake of natural chemicals (i.e., the dose), we could begin to estimate risks. An article by Ames and Gold speaks directly to the alar issue and asks, ‘‘What risks might we incur by banning alar?’’ ( 1002). They point out that alar plays a role in reducing pesticide use in some apples. It helps produce firmer apples and results in fewer apples falling to the ground, which hence is a potential for less pesticide use in the following year and also possibly less mycotoxin (patulin) production contaminating the apples. Life is a tradeoff of risks. Hopefully, we will be able to make informed choices among risks. There appears to be little direct evidence that normal exposure to natural chemicals (with certain exceptions) poses significant risks, but there has been no risk assessment study of naturally occurring toxicants (except aflatoxin) in foods. About half

Naturally Occurring Toxic Chemicals in Foods

135

(29 of 57) of the few plant toxins that have been tested in animal cancer bioassays are rodent Carcinogens; these chemicals are present in more than 50 of the most common foods (1003). Ames et al. concluded that both natural and synthetic chemicals are equally likely to be carcinogenic in animal cancer tests (1003). About 50% of both natural and synthetic chemicals tested in animal cancer tests at the maximum tolerated dose (MTD) are carcinogens. In studies conducted at the MTD, 30-50% of both natural and synthetic chemicals are also mutagens, teratogens, and clastogens. These data undermine the current regulatory efforts to protect public health by monitoring only for synthetic chemicals based on these tests (1004). Animal cancer tests are carried out at the MTD of a particular chemical, a near-toxic dose that can cause chronic mitogenesis (1005). Mitogenesis increases mutagenesis (1005, 1006). Chronic mitogenesis appears to be an important factor for many, if not most, of the known causes of human cancer (1005, 1006). Ames and Gold point out that “the idea that mitogenesis increases mutagenesis helps to explain promotion and other aspects of carcinogenesis” (1006, p. 970). Monro (1007) has challenged the traditional dosing regimen in carcinogenicity testing with rodents and biological criteria for exposure determinations have been proposed (1008, 1009). Humans have developed numerous defense systems that are active against natural as well as synthetic toxicants (1002). Animal defenses are mostly of a general type. Because the number of natural chemicals that might have toxic effects is large, general defenses not only offer protection from natural toxicants but also may offer protection from synthetic toxicants as well (1004). Concentrations well above normal dietary levels are required to produce biological effects caused by most natural toxicants in foods. A few natural chemicals cause adverse biological effects at normal consumption levels. However, based on the overall epidemiological studies concerning foods and the risk of cancer, fruits and vegetables are, and continue to be, recommended to decrease the risk of cancer (230). Human concerns about synthetic pesticide residues in foods have prompted aggressive U.S. EPA regulation of pesticides in the environment and in foods. The U.S. Food and Drug Administration (FDA) found, in a “total diet” study of eight population groups from 1982 to 1984, that none of the pesticide residue levels measured approached the tolerances set by the U.S. EPA (1010). FDA’s monitoring for pesticides in 14,492 samples from October 1, 1986 to September 30, 1987 showed that exposure of the U.S. population to pesticide residues is consistently below established limits. Less than 1% of the samples contained pesticide residues that exceeded regulatory limits (101 1). During 1987, the California Department of Food and Agriculture (CDFA) analyzed 7010 samples from marketplaces; only 1.5% of these samples had detectable levels of illegal pesticides, and only 0.3% of the samples had pesticide residues in excess of tolerance limits for specific foods (1012). FDA’s pesticide residue monitoring for October 1990 through September 1991 continued to show that the levels of pesticide residues in the U.S. food supply generally are below established safety limits (1013). The total diet study measured the intake of pesticide residues and, for fiscal year 1991 (FY91), these were well below established standards. These findings were consistent with those from earlier years. The analytical methods used in the total diet study were modified to pennit measurement at levels 5-10 times lower than those in regulatory monitoring. The frequency of occurrence as well as the amounts of the four pesticides most frequently found has decreased over the years. These four pesticides are DDE, DDT, TDE, and dieldrin as monitored from 1964 through the pesticide residue monitoring of 1991 (1013).

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There are many misconceptions concerning the relationship among synthetic chemicals, environmental pollution, human cancer, and natural toxicants. Ames and Gold point out eight of these leading misconceptions and cover the scientific findings that undermine each (1014). Ames et al. concluded “that at the low doses of most human exposures the comparative hazards of synthetic pesticide residues are insignificant” (1003, p. 7777). Phytoalexins and other naturally occurring pesticides produced by plants represent 99.99% of all the toxic chemicals we consume (1002, 1003). The Committee on Food Protection suggested that plant breeders attempting to develop higher-yielding or diseaseresistant crop varieties must be alerted to the possible production of undesirable components (3). That suspicion now has become reality in at least two known plant species, celery and potatoes. In an effort to obtain an understanding with a broad perspective of natural and synthetic chemicals, a comparison was made of the possible hazards from 80 daily exposures to rodent carcinogens (1015). Gold et al. used an index that related human exposure to carcinogenic potency in rodents (HERP, Human Exposure dose/Rodent Potency dose) (1015). The results confirm that synthetic pesticide residues or environmental pollutants rank low compared to the naturally occurring carcinogens that are in typical portions of common foods. Although the findings by Gold et al. do not show that these natural carcinogens play a part in human cancer (1015), they do cast doubt on the relative importance of low-dose exposures to synthetic chemicals. If Doll and Peto’s estimate that as much as 35% of all cancer might be related to diet is correct (61), then it follows that chemicals other than synthetic pesticides may be responsible for cancer and other disease syndromes in humans. Dietary unbalances, like deficiencies of antioxidants and vitamins or excesses of alcohol and salt, also may play an important role in modulating diet-related cancer. Because this is an enormous risk to humans, extensive, basic research on food-related diseases should receive the highest priority. A new area of high interest and deserved questioning is the genetic engineering of new food plant varieties. We are going to make mistakes with genetic engineering. All new and unexplored technologies make mistakes. Genetic engineering is no exception. We do not understand the levels and toxicology of chemicals in our present foods. Without this scientific base, we cannot know what we have produced or might produce with genetic engineering. The concern should not be centered on genetic engineering per se but rather on the results, which might be the production of varieties with larger quantities of toxicants. Older, more-established methods of breeding may arrive at essentially the same result, only more slowly. Increasing food plant toxicants could prove to be devastating and only hinder the progress of genetic engineering. We need to generate a scientific base and a better understanding of the levels and toxicology of the natural chemicals in today’s foods. There are no guidelines or regulations regarding naturally occurring toxicants in food. It isimmaterial whether a new variety of our food is bred traditionally or by geneticengineering techniques because the variety that has a production advantage will be produced and marketed. Do we know what we have placed in the marketplace? We do not. New food varieties traditionally are placed on the market with no toxicological testing. We have always experimented with foods on ourselves. A basis for chemical change(s) in our food supply should be established now. We have the tools and scientific talent to obtain the knowledge for that base. A new strategy was suggested for plant breeders (779): integrated breeding and environmental chemicals (IBEC). During the plant-breeding process, the breeder evaluates

Naturally Occurring Toxic Chemicals Foods in

137

levels of a toxic compound(s) that is produced by the variety in question. The variety with the lowest level(s) of the compound(s) and best disease resistance ultimately would be selected and marketed. This method was used with success while breeding for leaf miner resistance in Apium accessions. Using IBEC would bea very important step forward in breeding programs for crops like celery, sweet potatoes, nightshade family crops, and other crops that we know contain natural toxicants. Rodricks states that ‘‘we remain abysmally ignorant of the chemical and toxicological properties of most of the chemicals to which humans are exposed throughout their lifetimes” (50, p. 2592). In our opinion, we need to identify human health hazards due to naturally occurring chemicals in food. Scientific progress in this new frontier will require hard thinking and solid facts.

ACKNOWLEDGMENT

Special thanks to the following individuals for their stimulating discussions or helpful critiques: D. E. Corrier, J. A. Duke, L. S. Gold, N.I. Mondy, R. J. Nachman, L. D. Rowe, S. L. Sinden, and C. H. VanEtten.

REFERENCES 1. Ames, B. N. (1983). Dietary carcinogens and anticarcinogens: Oxygen radicals and degenerative diseases, Sciewce, 221: 1256-1263. 2. Barz, W.. Bless, W., Borger-Papendorf, G., Gunia, W., Mackenbrock, U., Meier, D., Otto. Ch., and Super, E. (1 990). Phytoalexins as part of induced defence reactions in plants: Their elicitation. function and metabolism. Bioactil-e Compozlndsfrom Plants, Ciba Foundation Symposium 154. Wiley. Chichester, pp. 140-156. 3. Committee on Food Protection, National Research Council (1973). Toxicants Occzrrring Naturally in Foods, National Academy of Science, Washington, D.C. 3. Committee on Food Protection, National Research Council (1966). To.yicants Occzrrring Natura//y in Foods, National Academy of Science, Washington, D.C. 5. Hansen. A. A. (1925). Two fatal cases of potato poisoning, Science, 61:340-341. 6. Kingsbury, J. M. (1964). Poisonous Plants of the United States and Carzucia. Prentice-Hall, Englewood Cliffs, New Jersey, p. 287. 7. McMillan, M.. and Thompson, J. C. (1979). An outbreak of suspected solanine poisoning in schoolboys: Examination of criteria of solanine poisoning, Q.J. Med., 48227-243. 8. Morris, S. C., and Lee, T. H. (198.1). The total toxicity and teratogenicity of Solanaceae glycoalkaloids, particularly those of the potato (Solanum tzrberoszcnz):A review, Food Technol. Aztst.. 36:118-124. 9. Willimott, S. G. (1933). An investigation of solanine poisoning, Analyst, 58:431-439. 10. Wilson, G. S. (1959). A small outbreak of solanine poisoning, Morz. Bull. Min. Health Publ. Health Lab. Sen?., 18:207-210. 11. Beier, R. C., and Nornlan, J. 0. (1990). The toxic factor in white snakeroot: Identity, analysis, and prevention, Vet. Hzmzan Toxicol., 32(Supplement): 81-88. 12. Committee Report (1981). Weeds ofthe North Central States, North Central Regional Research Bulletin No. 772, Publication No. 281, College of Agriculture. University of Illinois at Urbana-Champaign, p. 208. 13. Hardin, J. W. (1973). Stock-Poisoning Plants of North Carolina. North Carolina Agricul-

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14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30.

31. 32. 33.

34. 35. 36.

Beier and Nigg tural Experiment Station, North Carolina State University, Raleigh, Technical Bulletin No. 414 (Revised), p. 121. Harvill, A. M., Jr.. Bradley, T. R., Stevens, C. E., Wieboldt, T. F., Ware, D. M. E., and Ogle, D. W. (1986). Atlas of the Virginia Flora, 2d Edition, Virginia Botanical Association, Farmville, Virginia. Jones, S. B., Jr., and Coile, N.C. (1988). Distribution of the vascular flora in Georgia, Department of Botany, University of Georgia, Athens, p. 97. Ownbey, G. B.. and Morley, T. (1991). Vascrdar Plants of Mirmesota: A Checklist and Atlas, University of Minnesota Press, Minneapolis. Sperry, 0. E., Dollahite, J. W., Hoffman, G. O., and Camp, B. J. (1964). Texas plants poisonous to livestock, Texas Agric. Exp. Stat. Bzdl., B-1028:59. Stotts. R. (1984). White snakeroot toxicityin dairy cattle, Vet. Med. S??zallAnim. Clirz., 79: 118-120. Niederhofer, R. E. (1985). The milk sickness, J. Am. Med. Assoc., 2542123-2125. Christensen, W. I. (1965). Milk sickness: A review of the literature, Econ. Bot., 19:293300. Couch. J. F. (1927). The toxic constituent of richweed or white snakeroot (Ez4patoriun1 urticaefolium). J. Agric. Res., 35:547-576. Molyneux, R. J., and James, L. F. (1990). Pyrrolizidine alkaloids in milk: Thresholds of intoxication, Vet. Hz4m. Tosicol., 32(Supplement):94-103. Reagor, J. C. (1985). TVMDL Accession No. C85 137234, Texas Veterinary Medical Diagnostic Laboratory, Texas A&M University System, College Station, Texas. Molyneux, R. J., and Ralphs, M. H. (1992). Plant toxins and palatability to herbivores,J. Range Mamge., 45:13-18. Provenza, F. D., Pfister, J. A., and Cheney. C. D. (1992). Mechanisms of learning in diet selection with reference to phytotoxicosis in herbivores, J. Range Manage.. 45:36-45. Bonner, W. A., and DeGraw, J.I., Jr. (1962). Ketones from “white snakeroot”Enyntorilc~n zcrticaefoliwn, Tetrahedron, 18:1295- 1309. Bonner, W. A., DeGraw, I., J. Jr., Bowen, D.M., and Shah, V. R. (1961). Toxic constituents of white snakeroot, Tetrahedron Lett., 417-420. Bowen, D. M., DeGraw, J. I., Jr., Shah, V. R., and Bonner, W. A. (1963). The synthesis and pharmacological action of tremetone, J. Med. C h n . , 6:315-319. Beier, R. C., Norman, J. 0..Irvin, T. R., and Witzel, D. A. (1987). Microsomal activation of constituents of white snakeroot (Ezlpatoriu~~z rugosunz Houtt) to form toxic products. A m J. Vet. Res., 48:583-585. Beier, R. C., Norman,J. O., Reagor, J. C., Rees, M. S.. and Mundy, B. P. (1993). Isolation of the major component in white snakerootthat is toxic after microsomal activation: Possible explanation of sporadic toxicity of white snakeroot plants and extracts, Natural Tosim, 1: 286-293. Snively. W. D.,Jr.. and Furbee, L. (1966). Discovererof the cause of milk sickness, J. Am. Med. Assoc., 196: 1055-1060. Cheeke, P. R. (1990). Perspectives on human health concerns arising from natural toxicants in feeds and poisonous plants, Vet. Hum. Toxicol.. 3271-75. Bell, A. A. (1974). Biochemical basesof resistance of plants to pathogens. In Proceedirgs of the SlmnzerInstitute on Biological Control of Plant Imects and Diseases (F. G. Maxwell and F. A. Harris. eds.), University of Mississippi Press, Jackson, pp. 403-362. Paxton, J. (1980). A new working definition of the term “phytoalexin.” Plant Dis., 64: 734. Smith, D. A. (1982). Toxicity of phytoalexins. In P1zytoale.xilz.s (J. A. Bailey and J. W. Mansfield. eds.), John Wiley and Sons. New York, p. 218. Bailey, J. A., and Mansfield, J. W.. eds. (1982). Pltytoalesirts. John Wiley and Sons, New York.

Naturally Occurring Toxic Chemicals in Foods

139

37. Bell, A. A. (1 981). Biochemical mechanisms of disease resistance, A m . Rell. Plant Pltysiol., 32~21-81. 38. Grisebach, H., and Ebel, J. (1978). Phytoalexins, chemical defense substances of higher plants? Artgew. Chem. Int. Ed. Engl., 17:635-647. 39. Lord, K. M., Epton, H. A. S., and Frost, R. R. (1988). Virus infection and furanocoumarins in celely, Plant Pathol., 37:385-389. 40. Ryan, C. A. (1973). Proteolytic enzymes and their inhibitors in plants, Ann. Rell. Plar~t Physiol., 24:173-196. 41. Tietjen, K. G., and Matern, U. (1 984). Induction and suppression of phytoalexin biosynthesis in cultured cells of safflower. Carthanzus ti~rctoriusL.. by metabolites of Altermria carthmli Chowdhury, Arch. Bioclzertt. Biophys., 329: 136-144. 42. Ryan. C. A. (1988). Oligosaccharides as recognition signals for the expression of defensive genes in plants, Biochemist]-\,,27:8879-8883. 43. Ryan, C. A., Bishop. P. D., Graham, J. S., Broadway, R. M., and Duffey, S. S. (1986). Plant and fungal cell wall fragments activate expression of proteinase inhibitor genes for plant defense, J. Chenz. Ecol., 121025-1036. 44. Beier. R. C., and Oertli, E. H. (1983). Psoralen and other linear furocoumarins as phytoalexins in celery, Phytocherrlistl?, 22:2595-2597. 45. Komives, T., and Casida, J. E. (1983). Acifluorfen increases the leaf content of phytoalexins and stress metabolites in several crops, J. Agric. Food Clzem., 31 :751-755. 46. Kaplan, D. T., Keen, N. T., and Thomason, I. J. (1980). Association of glyceollin with the inconlpatible response of soybean roots to Meloidogye incognita, Pllysiol. Plant Pathol., 161309-318. 47. Rich, J. R., Keen, N. T., and Thomason, I. J. (1977). Association of coumestans with the hypersensitivity of lima bean roots to Pratylencltus scribneri, Physiol. Plant Pathol., 10:105-116. 48. Veech, J. A. (1979). Histochemical localization and nematoxicity of terpenoid aldehydes in cotton. J. Nenratol., 11240-246. 49. Dercks, W., Trurnble, J., and Winter, C. (1990). Impact of atmospheric pollution on linear furanocoumarin content in celery, J. Clzenl. Ecol., 16:443-454. 50. Rodricks, J. V. (1978). Food hazard of natural origin, Fed. Proc., 37:2587-2593. 51. American Council on Science and Health (1987). Mother Nature ai7d Her Chemicals Join Us for ThnnX-sgia,ingDinner, American Council on Science and Health, New York. 52. Surak, J. G. (1978). Phytoalexins and human health-A review, Proc. Fla. State Hortic. SOC.,91 :256-258. 53. Crosby, D. G., and Aharonson. N. (1967). The structure of carotatoxin, a natural toxicant from carrot, Tetrahedron Lett., 23:465-472. 54. Yates. S. G., and England, R. E. (1 982). Isolation and analysis of carrot constituents: Myristicin, falcarinol, and falcarindiol, J. Agric. Food Chem., 30:317-320. 55. Concon, J. M. (1988). Food Toxicology: Principles and Concepts, Part A, Marcel Dekker, New York. 56. Concon, J. M. (1988). Food Toxicology: Contarninants and Additives, Part B. Marcel Dekker, New York. 57. Tarka, S. M., Jr., Morrissey. R. B., Apgar, J. L., Hostetler, K. A.. and Shively, C. A. (1991). Chronic toxicity/carcinogenicity studies of cocoa powder in rats, Food Chem. Toxicol., 29: 7-19. 58. Malinow, M. R., Bardana, E. J., Jr., Pirofsky, B., Craig, S., and McLaughlin, P. (1982). Systemic lupus erythematosus-like syndrome in monkeys fed alfalfa sprouts: Role of a nonprotein amino acid, Science, 216:415-417. 59. Fisher, G.S., Frank, A. W., and Cherry, J. P. (1988). Total gossypol content of glandless cottonseed, J. Agric. Food Chem., 36:42-44. 60. Smith, H. A., Jones, T. C., and Hunt, R. D. (1 972). Veterirtary Pathology, 4th ed.. Lea and Febiger, Philadelphia. pp. 873-875.

140

Beier and Nigg

61. Doll, R., and Peto, R. (1981). The causes of cancer: Quantitative estimates of avoidable risks of cancer in the United States today, J. Nut. Cancer Znst., 66:1191-1308. 62. Bhakta, D., Shukla, S. S., Chandrasekharalah, M. S., and Margrave, J. L. (1992). A novel photocatalytic method for detoxification of cyanide wastes, Erniron. Sci. Technol., 36:625626. 63. Basheer, S., Kut. 0 . M., Prenosil, J. E., and Bourne, J. R. (1992). Kinetics of enzymatic degradation of cyanide, Biotechnol. Bioeng., 39:629-634. 64. Baselt, R. C., and Cravey, R. H. (1980). Forensic toxicology. In: Toxicology. The Basic Scierzce of Poisons (J. Doull, C. D. Klasson. and M. 0. Amdur, eds.), Macmillan, New York, pp. 667-682. 65. Gonazles. J., and Sabatini, S. (1989). Cyanide poisoning: Pathophysiology and current approaches to therapy, Zrzt. J. Art$ Organs, 12:347-355. 66. Rumack, B. H., and Peterson, R. G. (.1980). Clinical toxicology. In Tosicology. The Basic Scierzce of Poisons (J. Doull, C. D. Klassen, and M. 0. Amdur, eds.), Macmillan, New York, pp. 677-698. 67. American Conference of Governtnental Industrial Hygienists (ACGIH) (1991). 1991-1992 Tlweshold Limit Valuesfor Chenzical Substances am! Physical Agents and Biological Exposure Indices. Author, Cincinnati. 68. Farooqui, M. Y. H.. Diaz. R. G., and Deleon. J. H. (1992). Methacrylonitrile: In vivo metabolism to cyanide in rats, mice, and gerbils, Drug Metab. Dispos.. 20:156-160. 69. Autian. J. (1980). Plastics. In Toxicology. The Basic Science of Poisons (J. Doull, C. D. Klasson, and M. 0. Amdur, eds.), Macmillan, New York, pp. 531-556. 70. Baud. F. J., Barriot, P.. Toffis, V., Riou, B., Vicaut, E., Lecarpentier, Y., Bourdon, R., Astier, A., and Bismuth, C. (1991). Elevated blood cyanide concentrations in victims of smoke inhalation, N. Engl. J. Med., 325:1761-1766. 71. Ellenhorn. M. J., and Barceloux, D. G. (1988). Medical Toxicolog?,, Elsevier, New York, p. 829-835. 72. Kulig, K. (1991). Cyanide antidotes and fire toxicology, N. Engl. J. Med., 325:1801-1803. 73. Yoshida, M., Adachi, J., Watabiki, T., Tatsuno, Y., and Ishida, N. (1991). A study on house fire victims: Age, carboxyhemoglobin. hydrogen cyanide and hemolysis, Forensic Sci. Znt., 52: 13-20. 74. Levin, B. C., Rechani. P. R., Guman. J. L., Landron. F., Clark, H. M.. Yoklavich. M. F., Rodriguez, J. R., Droz, L., de Cabrera, F. M.. and Kaye, S. (1990). Analysis of carboxyhemoglobin and cyanide in blood from victims of the Dupont plaza hotel fire in Puerto Rico, J. Forensic Sci., 35:151-168. 75. Lundquist. P., Rammer, L.. Sorbo, B. (1989). The role of hydrogen cyanide and carbon monoxide in fire casualties: A prospective study, Forerzsic Sci. Int., 43:9-14. 76. Okae, M., Yamamoto, K., Yamanloto, Y., and Fukui, Y. (1989). Cyanide, carboxyhemoglobin and blood acid-base state in animals exposed to combustion products of various combinations of acrylic fiber and gauze, Forensic Sci. Zrzt., 42:33-41. 77. Moore, S. J.. Ho, I. K., and Hume, A. S. (1991). Severe hypoxia produced by concomitant intoxication with sublethal doses of carbon monoxide and cyanide. Toxicol. Appl. PharmaC O ~ . , 109:412-420. 78. Lokan, R. J.. James, R. A., and Dymock, R. B. (1987). Apparent post-mortem production of high levels of cyanide in blood, J. Forensic Sci. SOC.,27:253-259. 79. Karhunen, P. J., Lukkari. I., and Vuori, E. (1991). High cyanide level in a homicide victim burned after death: Evidence of post-mortem diffusion, Forensic Sci. Int., 49:179183. 80. Shiono, H.. Maseda, C., Akane, A., and Matsubara, K. (1991). Rapid and sensitive quantitation of cyanide in blood and its application to fire victims, Am. J. Forensic Med. Pathol., 1250-53. 81. Conn. E. E. (1978). Cyanogenesis. the production of hydrogen cyanide. by plants. In Efsects

Naturally Occurring Toxic Chemicals in Foods

82. 83. 83. 85.

86.

87.

88.

89.

90.

91. 92.

93.

94.

95. 96.

97.

98.

99.

141

of Poisorzous Plarzts or2 Livestock (R. F. Keeler, K. R. Van Kampen, and L. F. James, eds.), Academic Press, New York, pp. 301 -3 10. Conn, E. E. (1991). The metabolism of a natural product: Lessons learned from cyanogenic glycosides, Plarrta Med., 57:s 1-S9. Seigler, D. S. (1975). Isolation and characterization of naturally occurring cyanogenic compounds, Phytocltenzistty, 14:9-29. Seigler, D. S. (1977). The naturally occurring cyanogenic glycosides, Prog. Phytoclzem., 4183-120. Conn, E. E. (1 979). Cyanogenic glycosides. In I~lternatiorzalReview of Biochemistg, Biochenzist~,of N~4tritionZA, Volume 27 (A. Neuberger and T. H. Jukes, eds.), University Park Press, Baltimore, Maryland, pp. 21-43. Hosel, W. (1981). Theenzymatic hydrolysis of cyanogenic glucosides. In Cyanide in Biology (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, eds.), Academic Press. New York, pp. 217-232. Nahrstedt, A., Erb, N.. Zinsmeister, H.-D. (1981). Methods of liberating and estimating hydroxyanic acid from cyanogenic plant material. In Cyanide in Biology (B. Vennesland, E. E. Conn. C. J. Knowles. J. Westley, and F. Wissing, eds.), Academic Press. New York, pp. 46 1-37 1. Rimington, C. (1935). The occurrence of cyanogenetic glucosides in South African species of Acacia. TI. Determination of the chemical constitution of Acaciapetalin and its isolation Accrcicr stolorrfera Burch., Orderstepoort J. Vet. Res., 5:445. Veibel, S. (1950). j3-Glucosidase. In The Enzynzes, Chemistry and Mechnrzisnz of Action, Volume I, Part 1 (J. B. Summer and Myrback, eds.), Academic Press, New York. pp. 583619. Hill, D. C. (1973).Chronic cyanide toxicity in domestic animals.In Chronic Cassalla Toxicity: Proceedings of an Interdisciplinay Workshop (B.Nesteland R. MacIntyre. eds.). MonographIDRC-OlOe,InternationalDevelopmentResearchCenter. Ottawa,Canada, pp. 105-1 11. Majak, W. (1992). Metabolism and absorption of toxic glycosides by ruminants, J. Range Marlage., 45:67-7 1. VanEtten, C. H., and Tookey, H. L. (1978). Glucosinolates in cruciferous plants. In Effects of Poisonom Plcrnts on Lillestock (R. F. Keeler, K. R. Van Kampen, and L. F. James, eds.), Academic Press, New York, pp. 507-520. Duncan, A. J., and Milne, J. A. (1992). Effect of long-term intra-ruminal infusion of the glucosinolatemetabolite allyl cyanide on thevoluntaryfoodintakeandmetabolismof lambs, J. Sci. Food Agric., 58:9-14. Duncan, A. J., and Milne, J. A. (1992). Rumen microbial degradation of allyl cyanide as a possible explanation for the tolerance of sheep to brassica-derived glucosinolates, J. Sci. Food Agric., 58:15-19. Majak. W., Bose, R. J., and Quinton, D. A. (1978). Prunasin, the cyanogenic glycoside in Amelandrier ahifolia, Phytochewisti?!, 17:803. Gondwe, A. T. D., Seigler, D. S., andDunn, J. E. (1978). Two cyanogenic glucosides, tetraphyllin B andepi-tetraphyllin B,from Aderzia volkemii, Phytochetrzistl?. 1727 1274. Jackson, L. C. (1988). Behavioral effects of chronic sublethal dietary cyanide in an animal model: Implications for humans consuming cassava (Manillot esculenta), Hmz. Biol., 60: 597-614. Oehme, F. W. (1978). The hazard of plant toxicities to the human population. 111EfSects of Poisonous Plants on Lilvestock (R. F. Keeler, K. R. Van Kampen, and L. F. James, eds.), Academic Press New York, pp. 67-80. Montgomery, R. D. (1964). Observations on the cyanide content and toxicity of tropical pulses, West btclian Mecl. J., 13:1- 11.

142

Beier and Nigg

100. Montgomery, R. D. (1980).Cyanogens. In Toxic Co~zstitzterztsof Plarlt Foodstzrfls, 2d Edition (I. E. Liener, ed.), Academic Press, New York. pp. 143-160. 101. Wilson, J. (1973). Cyanide and human disease. In Clzrorlic Cassava Toxiciv: Proceedings ofcrn blterdisciplina~? Workshop (B. Nestel and R. MacIntyre. eds.), Monograph IDRCOlOe, International Development Research Center, Ottawa, Canada, pp. 121-125. 102. Wilson, J. ( 1983). Cyanide in human disease: A review of clinical and laboratory evidence, Fund. Appl. Toxicol., 3:397-399. 103. Voldrich, M., and Kyzlink, V. (1992). Cyanogenesis in canned stone fruits, J. Food Sci., 57:161-162,189. 104. Ferreira, V. L. P., Yotsuyanagi, K., Teixeria Neto, R. O., Vitali, A. A., and Carvalho, C. R. L. (1992). Elimination of cyanogenic compounds of bamboo shoots (De~~dr-ocrrlantz4s giganteus Munro) by different processes, Rev. ESP. Cierzc. Tecrlol. Alimellt., 32175-184 (in Portuguese). 105. Sayre. J. W.,and Kaymakcalan, S. (1964).Hazards tohealth, Cyanide poisoning from apricot seeds among children in central Turkey, New Engl. J. Med., 270: 1113-1 115. 106. Tuncel, G., Nout. M. J. R.. Brimer. L., and Goktan, D. (1990). Toxicological, nutritional and microbiological evaluation of tempe fermentation with Rhizopus oligosporzrs of bitter and sweet apricot seeds, Int. J. Food Microbiol., 11:337-344. 107. Townsend, W. A., andBoni, B.(1 975). Cyanide poisoning from ingestion of apricot kernels, MMWR, (December 13):427. 108. Humbert, J. R., Trees, J. H., and Braico. K. T. (1977). Fatal cyanidepoisoning: Accidental ingestion of amygdalin, J. Am. Med. Assoc., 2383482. 109. Braico, K. T., Humbert, J. R., Terplan, K. L., and Lehotay, J. M. (1979). Laetrile intoxication, report of a fatal case, N. Engl. J. Med.. 300238-230. 110. Sadoff, L., Fuchs, K., and Hollander, J. (1978). Rapid death associated with laetrile ingestion, J. Am. Med. Assoc., 239:1532. 111. Rubino, M. J., and Davidoff, F. (1979). Cyanidepoisoning from apricot seeds, J. Am. Med. Assoc., 241:359. 112. Brian, M. J. (1990). Case reports, cyanide poisoning in children in Goroka, Papua Nebt? Gtrirrea Med. J., 33:151-153. 113. Stavric. B., and Klassen, R. (1980). Enzymatic hydrolysis of amygdalin by fecal samples and some foods. In Natural Toxins (D. Eaker and T. Wadstrom, eds.), Pergamon Press, New York, pp. 655-660. 113. Nestel. B., and MacIntyre. R. (1973). Foreword. In Chromk Cassrrva Toxicio, Proceedings of nrz I~rterdiscipli~zary Workshop(B. Nestel and R. MacIntyre, eds.), Monograph IDRCOlOe, International Development Research Center, Ottawa, Canada, pp. 5-7. 115. Cock, J. H. (1982). Cassava: A basic energy source in the tropics, Science. 218:755-762. 116. Cooke, R. D., and Coursey, D. G. (1 981). Cassava: A major cyanide-containing food crop. In Cyarzide i n Biology (B. Vennesland, E. E. Conn, C.J. Knowles, J. Westley, and F. Wissing, eds.), Academic Press, New York, pp. 93-114. 117 Nestel, B. (1973). Current utilization and future potential for cassava. In Chronic Casscrva Toxicity: Proceedingsof an hrterdisciplirrary Workshop,(B. Nestel and R. MacIntyre,eds.), Monograph IDRC-01 Oe, InternationalDevelopment ResearchCenter, Ottawa,Canada, pp.11-26. 118. Coursey, D. G. (1973).Cassava as food: Toxicityandtechnology.In Clrronic Cassnva Toxicity: Proceedings of an Zrzterdisciplinaql Workshop,(B. Nestel and R. MacIntyre, eds.), Monograph IDRC-01 Oe, InternationalDevelopment Research Center, Ottawa. Canada, pp. 27-36. 119. Pereira, J. F., Seigler, D. S.. and Splittstoesser, W. E. (1981). Cyanogenesis in sweet and bitter cultivars of cassava, HortScierrce, 16:776-777. 120. Singh, S. K., and Nair, T. V. R. (1968). Studies on the variability of cyanogenic glucoside content in cassava tubers, Im!. J. Agric. Sci.. 38:958-963.

Naturally Occurring Toxic Chemicals in Foods

143

131. Butler. G. W., Reay, P. F., and Tapper, B. A. (1973). Physiological and genetic aspects of cyanogenesis in cassava and other plants. In Chronic Ccrsscrvcr Toxicity: Proceedilzgs of CUI Iizterdisciplirm? Workshop, (B. Nestel and R. MacIntyre, eds.), Monograph IDRC-O10e, Tnternational Development Research Center, Ottawa, Canada, pp. 65-7 1. 122. deBruijn, G. H. (1973). The cyanogenic character of cassava (ManihOt esculentcr). In Chronic Cassava ToxiciQ: Proceedings of arz Irlterdiscipli~zanlWorkslzop, (€3. Nestel and R. MacIntyre, eds.), Monograph IDRC-OlOe, International Development Research Center, Ottawa, Canada, pp. 43-48. 123. Padmaja. G.(1989). Evaluation of techniques to reduce assayable tannin and cyanide in cassava leaves, J. Agric. Food Chem., 37:712-716. 124. Regez, P. F.. Rigamonti, C., and Geiges, 0. (1989). Evaluation de l’activite amylolytique et de la formation de biomasse de quelques champignons isoles de la pulpe de manioc et du pain de manioc (cikwangue) au Zaire (Amylolytic activity and formation of biomass of some fungi isolated from the manioc pulp and bread (chikwangue) of Zaire), Microbial. Aliitlerlts-NLrtl-., 7: 17-24. 125. Thambirajah, J. J. (1989). Safety evaluation of cassava fermented with microfungi, Troy. Agric. (Trinidad), 66:326-328. 126. Ukhun, M. E., and Nkwocha, F. 0. (1989). The hydrocyanic acid (HCN) content of gcrrri flour made from cassava (Marzihot spp.) and the influence of length of fermentation and location of source, Food Clzem., 33: 107-1 13. Umoh, I. B.(1982).Detoxificationof cassava leaves bysimple 127. Maduagwu,E.N.,and traditional methods, Tosicol. Lett., 10245-248. 128. O’Brien, G. M., Mbome. L., Taylor, A. J., and Poulter, N. H. (1992). Variations incyanogen content of cassava during village processing in Cameroon, Food Chem., 44: 131-1 36. 129. Essers. A. J. A. (1989). Reducing toxicity by simple changes in the traditional preparation of cassava leaves-A small trial in Mozambique, Trop. Sci.. 29:269-272. 130. Akingbala, J. O., Oguntimein, G. B.,and Abass, A.B. (1991).Effect of processing methods on quality and acceptability of fufu from lowcyanide cassava.J. Sci. Food Agric., 57: 151154. 131. Ukhun, M. E., and Dibie, E. N. (1989). Cyanide content of cassava mash and gari flour and influence of water activity (a),.)during storage, Bull. Erwirorz.Corztanz. Tosicol., 42: 548-552. 132. Ogunshua, A. 0.(1989). Total cyanidelevels in breadmade from wheatlcassava composite flours, Int. J. Food Sci. Technol., 24:361-365. 133. Almazan, A. M. (1990). Effect of cassava flour variety and concentration of bread loaf quality, Cereal Chem., 67:97-99. 134. Hirata, K., Ohnishi, K., and Nishijima, M. (1990). Studies on food analysis by using an enzyme. 11. Application to processing foods on the determination of cyanogenic glycoside by using a Conway’s microdiffusional apparatus. Eisei Kagaku, 36:344-348. feeds and foodstuffs. In Cyanide in Biology 135. Nartey. F. (1981). Cyanogenesis in tropical (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley. and F. Wissing, eds.), Academic Press, New York, pp. 115-132. 136. Cliff, J., Lundquist, P., Rosling, H., Sorbo, B., and Wide, L. (1986). Thyroid function in a cassava-eating population affected byepidemic spastic paraparesis, Acta Erzdocrirzol., 1 13: 523-528. 137. Ermans, A. M., Delange, F., van der Velden, M., and Kinthaert, J. (1972). Possible role of cyanide and thiocyanate in the etiology of endemic cretinism, A h . Exp. Med. Biol., 30: 455-486. 138. Delange, F., van des Velden, M., and Ermans, A. M. (1973). Evidence of an antithyroid action of cassava in man and in animals. In Chronic Cassalla Toxicity: Proceedirzgs of an Interdiscipliizan Workshop, (B. Nestel and R. MacTntyre, eds.), Monograph IDRC-OlOe, Tnternational Development Research Center, Ottawa, Canada, pp. 147-1 51.

144

Beier and Nigg

139. Ekpechi, 0. L. (1973). Endemicgoitre and high cassava diets in eastern Nigeria. InChronic Cassava ToxiciQ: Proceedings of ail Interdisciplinary Workshop, (B. Nestel and R. MacIntyre, eds.), Monograph IDRC-OlOe, Tnternational Development Research Center, Ottawa, Canada, pp. 139-145. 140. Ermans. A. M., van der Velden, M.. Kinthaert, J.. and Delange, F. (1973). Mechanism of the goitrogenic action of cassava.Tn Chronic Crrsscnw TosiciQ: Proceedings of an Illterclisciplinay Wor-kshop(B. Nestel and R. MacIntyre, eds.). Monograph IDRC-OlOe, International Development Research Center, Ottawa, Canada, pp. 153-157. cassava diets in West 141. Osuntokun, B. 0. (1973). Ataxic neuropathy associated with high Africa. Tn Chronic CassavcrTo.xici@: An Irzterdisciplincrn Workshop, (B. Nesteland R. MacIntyre, eds.). MonographIDRC-OlOe, International Development Research Center, Ottawa, Canada, pp. 127-138. 142. Aregheore. E. M., and Agunbiade. 0. 0. (1991). The toxic effects of cassava (Manihot esczrlentrr Grantz) diets on humans: A review. Vet. H L ~Toxicol., . 33274-275. 133. Cliff. J., Lundquist, P., Martensson, J., Rosling. H., and Sorbo, B. (1985). Association of high cyanide and low sulphur intakeincassava-inducedspasticparaparesis, Lancet, 2: 121 1-1213. 144. Osuntokun, B. 0. (1981). Cassava diet, chronic cyanideintoxication and neuropathy in the Nigerian Africans. World Rev. Nuti-. Diet., 36: 141-1 73. 145. Tylleskar, T., Banea, M.. Bikangi, N., Fresco, L., Persson, L. A., and Rosling, H. (199 1). Epidemiological evidence from Zaire for dietary a etiology ofkonzo, an upper motor neuron disease. Bull. WHO. 69581-589. 146. Casadei. E., Cliff, J.. and Neves, J. (1990). Surveillanceof urinary thiocyanate concentration after epidemic spastic paraparesis in Mozambique, J. Ti-op. Med. Hyg., 93257-261. 147. Howlett, W. P.. Brubaker, G. R.. Mlingi. N.. and Rosling, H. ( 1990). Konzo, an epidemic upper motor neuron disease studied in Tanzania, Brain, 113223-235. 148. NJoh, J. ( 1990). Tluplcai ataxic neuropathy in Liberians, T ~ o p .Geogr. Med., 49%94. 149. Tylleskar, T., Banea.M., Bikangi. N., Cooke, R. D., Poulter. N. H.. and Rosling, H. (1992). Cassava cyanogens and konzo. an upper motoneuron disease found in Africa. January 25, 1992, Lancet, 3393208-21 1. 150. Cliff, J., Essers, S., and Rosling. H. (1986). Ankle clonus correlating with cyanide intake from cassava in rural children from Mozambique, J. Trop. Pediatr., 32:186-189. 151. Anon. (1972). Food and cyanide, Br. Med. J., 2481-482. 152. International Agency for Research on Cancer Working Group (1975). Cycasin. In IARC Monogrrrphs on the Elwlurrtiorl of the Carcinogenic Risk of Chernicals to m r n : Sonte Naturcrlly Occurring Substances, Vol. 10, WHO IARC. Lyon, France. pp. 121- 130. 153. Woo, Y.-T., Lai, D. Y.. Arcos, J. C., and Argus, M. F. (1988). Cycasin and related compounds. In Chernical Induction of Cancel; Structural Bases and Biological Meclmlisms (Y.-T. Woo, D. Y. Lai, J. C. Arcos, and M. F. Argus, eds.), Academic Press, New York, pp. 179-21 1. 154. Uwakwe, A. A., Monanu. M. O., and Anosike, E. 0. (1991). Whole blood cyanide levels of mainly dietary origin in a human population sample in Port Harcourt, Nigeria, Plant Foods Hum. Nutr., 4 1:117- 124. 155. Pitchumoni,C. S.. Jain, N. K.. Lowenfels. A. B..andDiMagno. E. P. (1988). Chronic cyanide poisoning: Unifying concept for alcoholic and tropical pancreatitis, Pancreas, 3: 220-322. 156. Wallig. M. A., Gould, D. H., and Fettman, M. J. (1988). Selective pancreatotoxicity in the rat inducedbythenaturallyoccurringplant nitrile l-cyano-2-hydroxy-3-butene,Food Cl1enl. Tosicol.. 26:137-147. 157. Sylvester, D. M., and Sander, C. ( 1990). Immunohistochemical localization of rhodanese, Histochenz. J., 23: 197-200.

Chemicals in Toxic Occurring Naturally

Foods

145

158. Jackson, L. C. (1988). Possible adaptation to serum thiocyanate overload associated with chronic sublethal dietary cyanide ingestion, H m l . Biol., 60:615-622. 159. Oke, 0. L. (1 977). The prophylactic action of cassava. In Proceedings of the Fourth Symposiwn of the Intenzationnl Socieq for Tropical Root Crops (J. McIntyre, and R. Graham, eds.), International Development Research Center, Ottawa, Canada. pp. 232-236. 160. Obidoa, O., and Obasi, S. C. (1991). Coumarin compounds in cassava diets: 2 health implications of scopoletin in gari, Plalzt Foods Hum. Nut., 41:283-289. 161. Gray, A. I., and Waterman, P. G. (1978). Coumarins in the Rutaceae, Phytochernistn, 17: 845-864. 162. Murray, R. D. H.. Mindez, J., and Brown, S. A. (1982). The Natural Counlarins: O c c ~ ~ r rence. Chemistry arld Bioclzemistq, John Wiley and Sons, New York. 163. Zaynoun, S. T., Aftimos, B. G., Ali, L. A., Tenekjian, K. K., Khalidi, U., and Kurban, A. K. (1984). Ficus caricn: Isolation and quantification of the photoactive components, Contact Dennutitis, 11:21-25. 164. Dubertret, L., Serraf-Tircazes, D., Jeanmougin, M., Morlikre, P., Averbeck, D., and Young, A. R. (1990). Phototoxic properties of perfumes containing bergamot oil on human skin: photoprotective effect of UVA and UVB sunscreens, J. Photochenz. Photobiol., B: Biol., 7:25 1-259. 165. Stanley, W. L., and Jurd. L. (1971). Citrus coumarins, J. Agric. Food Clzem., 19:11061110. 166. Massaldi, H. A., and King, C. J. (1974). Retention of d-limonene during freeze drying of orange juice, J. Food Sci., 39:445-448. 167. Ahmed, E. M.. Dennison, R. A., Dougherty, R. H., and Shaw, P. E.(1978). Effect of nonvolatile orange juice components, acid, sugar, and pectin on the flavor threshold of d-limonene in water, J. Agric. Food Chenl., 26:192-194. 168. Widmer, W. W.(1991). Improvements in quantitation of limonin in citrus juice by reversedphase high-performance liquid chromatography, J. Agric. Food Clzenl., 39: 1472- 1476. 169. Kealey, K. S., and Kinsella, J. E. (1979). Orange juice quality with an emphasis on flavor components, CRC Crit. Rely. Food Sci. Nutr., 11: 1-40. 170. Chamblee, T. S.. Clark, B. C., Jr., Brewster, G. B., Radford, T., and Iacobucci, G. A. (1991). Quantitative analysis of the volatile constituents of lemon peel oil. Effects of silica gel chromatography on the composition of its hydrocarbon and oxygenated fractions, J. Agric. Food Chenz., 39:162-169. 171. Vannier, S. H., and Stanley, W. L. (1958). Fluorometric determination of 7-geranoxycoumarin in lemon oil: Analysis of mixtures of grapefruit oil in lemon oil, J. Assoc. OffAgric. Chenl., 41:432-435. 172. Tatum, J. H., and Berry, R. E. (1979). coumarins and psoralens in grapefruit peel oil. Phytochemistry, 18:500-502. 173. Fisher, J. F., and Trama. L. A. (1979). High-performance liquid chromatographic determination of some coumarins and psoralens found in citrus peel oils, J. Agric. Food Clzem., 27: 1334-1337. 174. Sams, W. M. (1941). Photodynamic action of lime oil (Citrus crurarztifolia),Arch. Dermatol. Syplzilol., 44:571-587. 175. Ashwood-Smith, M. J., Poulton, G. A., Ceska, O., Liu, M., and Furniss, E. (1983). An ultrasensitive bioassay for the detection of furocoumarins and other photosensitizing molecules, Photochem. Plzotobiol.. 38: 113-1 18. 176. Hudson, J. B., Miki. N., and Towers, G. H. N. (1987). Isopimpinellin is not phototoxic to viruses and cells. Plantcr Med., 53:306-307. 177. Naganuma, M., Hirose. S., Nakayama, Y., Nakajima, K., and Somey, T. (1985). A study of the phototoxicity of lemon oil, Arch. Dermutol. Res., 278:31-36. 178. Gross, T. P., Ratner, L., DeRodriguez. O., Farrell, K. P., and Israel, E. (1987). An outbreak of phototoxic dermatitis due to limes, Am. J. Epidenliol., 125:509-514.

146

Beier and Nigg

of phototoxicderma179. Maryland Department of Health and Mental Hygiene (1984). Outbreak titis from limes, MMWR, 34:462-464. 180. Coffman, K., Boyce, W. T., and Hansen, R. C. (1985). Phytophotodermatitis simulating child abuse. Am. J. Dis. Child., 139:239-240. 181. Nigg, H. N., Nordby, H. E., Beier,R. C., Dillman. A., Macias, C.,and Hansen, R.C. (1993). Phototoxic coumarins in limes, Food Cheln. Toxicol., 31:33 1-335. 182. Fenwick. G. R.. Heaney, R. K., and Mullin, W. J. (1983). Glucosinolates and their breakdown products in food and food plants, Crit. Rev. Food Sci. Nzltr., 18:123-201. 183. Matovinovic. J. (1983). Endemic goiterand cretinism at the dawn of the third millennium, Ann. Rely. Nutr., 3:341-412. 184. Chesney, A. M., Clawson, T. A., and Webster, B. (1928). Endemic goiterinrabbits. I. Incidence and characteristics, Bull. Jolzns Hopkirzs Hosy., 43961-277. 185. Webster, B., Marine. D., and Cipra, A. (1931). The occurrence of seasonal variations in the goiter of rabbits produced by feeding cabbage, J. Exp. Men., 53:81-91. 186. Langer, P., Stolc, V., and Kutka, M. (1964). Naturally occurring goitrogens and thyroid function, Publ. House of the Slovak Acad. of Sci.. Bratislava. Czechoslovakia, pp. 201208. 187. Langer, P., and Stolc, V. (1964). Studien uber Beziehungen zwischen Rhodanbildung und kropfbildender Eigenschaft von Nahrungsmitteln, V. Vergleich der Wirkungvon Weisskohl und Rhodanid auf die Rattenschilddriise, Hoppe-Seyler's 2. Physiol. Chew., 335216-220. doses of some naturally occurring 188. Langer, P. (1966). Antithyroid action in rats of small compounds, Endocrinology, 79: 11 17-1122. 189. McDanell, R., McLean. A. E. M.. Hanley, A. B., Heaney. R. K., and Fenwick, G.R. (1988). Chemical and biological properties of indole glucosinolates (glucobrassicins): A review, Food Clzenz. Tosicol., 26:59-70. 190. Mullin, W. J., and Sahasrabudhe,M. R. (1977). Glucosinolate content of cruciferous vegetable crops, Can. J. Plant Sci., 57:1227-1230. 191. Heaney, R. K., and Fenwick. G. R. (1980). Glucosinolates inBrassica vegetables: Analysis of 22 varieties of Brussels sprout (Brassica oleracea L. var. gernnzifera),J. Sci. Food Agric., 31 ~785-793. 192. VanEtten, C. H., Daxenbichler, M. E., Tookey, H. L., Kwolek, W. F., Williams, P. H., and Yoder, 0. C. (1980). Glucosinolates: Potential toxicants in cabbage cultivars, J. Ant. SOC. Hortic. Sci., 105:710-714. 193. VanEtten, C. H., Daxenbichler, M. E., Williams, P. H., and Kwolek, W. F. (1976). Glucosinolates and derived products in cruciferous vegetables: Analysis of the edible part from twenty-two varieties of cabbage, J. Agric. Food Clzem., 24:452-455. 194. Quinsac, A., Ribaillier, D.,Rollin. P., and Dreux, M. (1992). Analysisof 5-vinyl1.3-oxazolidine-2-thione by liquid chromatography, J. Assoc. Of Anal. Chem., 75529536. 195. Eisner. G. (1931). Ueber die lebensrettende Wirkung von Pflanzenteilen und daraus isolierten Saften bei der todlich verlaufenden, subakuten Uranvergiftung, Biochenz. Z., 232218-228. 196. Bradfield, C. A., and Bjeldanes, L. F. (1987). Structure-activityrelationshipsofdietary indoles: A proposed mechanism of action as modifiers of xenobioticmetabolism, J. To.vico1. Environ. Health, 21:311-323. 197. Loub, W. D., Wattenberg, L. W., and Davis, D. W. (1975).Aryl hydrocarbon hydroxylase induction in rat tissues by naturally occurring indoles of cruciferous plants, J. Nat. Cancer Illst., 54:985-988. 198. VanEtten, C. H., and Tookey, H. L. (1983).Glucosinolates. In CRC Handbook of Naturally Occurring Food Toxicants (M. Rechcigl, Jr., ed.), CRC Press, Boca Raton, Florida, pp. 1530. 199. Yamaguchi. T. ( 1980). Mutagenicity of isothiocyanates. isocyanates and thioureas on Salrnorlellat;vylzinzuriunz, Agric. Biol. Chern., 44:3017-3018.

Chemicals in Toxic Occurring Naturally

Foods

147

200. Nishie, K.. and Daxenbichler, M. E. (1980). Toxicology of glucosinolates, related compounds (nitriles, R-goitrin, isothiocyanates) and vitamin U found in cruciferae. Food Cosmet. Toxicol., 18:159-172. 201. Boyd. J. N., Babish, J. G., and Stoewsand, G. S. (1982). Modification by beet and cabbage diets of aflatoxin B ,-induced rat plasma a-foetoprotein elevation, hepatic tumorigenesis, and mutagenicity of urine, Food Chern. Toxicol., 20:47-52. 203. Stoewsand, G. S., Babish, J. B., and Wimberly, H. C. (1978). Inhibition of hepatic toxicities from polybrominated biphenyls and aflatoxin B in rats fed cauliflower, J. Environ. Pntlzol. Toxicol., 2:399-406. 203. Wattenberg. L. W. (1983). Inhibition of neoplasia by minor dietary constituents, Cancer Res., 43:2448~-2453s. 204. Chung, F.-L., Morse. M. A., Eklind, K. I.. and Lewis, J. (1992). Quantitation of human uptake of the anticarcinogen phenethyl isothiocyanate after a watercress meal, Cancer Epidemiology, Bionz.Prell.. 1:383-388. 205. Morse, M. A., Wang, C.-X., Stoner, G. D., Mandal, S., Conran, P. B.. Amin, S. G., Hecht, S. S., and Chung, F.-L. (1989). Inhibition of 4-(.methylnitrosamino)-l-(3-pyridyl)1-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate, Cancer Res., 49:549-553. 206. Morse, M. A., Eklind, K. I., Hecht, S. S., Jordan, K. G., Choi, C.-I., Desai, D. H.. Amin, S. G., and Chung, F.-L. (1991). Structure-activity relationships for inhibition of 4-(methylnitrosamin0)- 143-pyridyl)- 1-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice, Cancer Res., 5 1: 1846- 1850. 207. Wattenberg, L. W. (1971). Studies of polycyclic hydrocarbon hydroxylases of the intestine possibly related to cancer: Effect of diet on benzpyrene hydroxylase activity, Cancer, 28: 99-102. 208. Babish, J. G., and Stoewsand, G. S. (1975). Hepatic microsomal enzyme induction in rats fed varietal cauliflower leaves, J. Nutr., 105:1592-1 599. 209. Pantuck. E. J., Hsiao, K.-C., Loub, W. D., Wattenberg, L. W., Kuntzman, R., and Conney, A. H. (1976). Stimulatory effect of vegetables on intestinal drug metabolism in the rat, J. Pllarmacol. Exp. Ther., 198:278-283. 210. Babish, J. G., and Stoewsand, G. S. (1978). Effect of dietary indole-3-carbinol on the induction of the mixed-function oxidases of rat tissue, Food Cosmet. Toxicol., 16:151-155. 21 1. Reyes, H., Reisz-Porszasz, S., and Hankinson, 0. (1992). Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor, Science, 356:1193-1195. 212. Bradfield, C. A.. and Bjeldanes, L. F. (1984). Effect of dietary indole-3-carbinol on intestinal and hepatic monooxygenase. glutathione S-transferase and epoxide hydrolase activities in the rat, Food Chem. Toxicol., 22977-982. 213. Pantuck, E. J.. Pantuck, C. B.. Garland, W. A., Min, B. H., Wattenberg, L. W., Anderson, K. E., Kappas, A., and Conney, A. H. (1979). Stimulatory effect of Brussels sprouts and cabbage on human drug metabolism, Clin. Plzarnzacol. Ther.. 1:88-95. 214. Pantuck, E. J., Pantuck, C. B., Anderson, K. E., Wattenberg, L. W., Conney, A. H., and Kappas, A. (1984). Effect of Brussels sprouts and cabbage on drug conjugation, Clirt. P l m mrcol. Tlzer., 2: 161- 169. 215. Michnovicz, J. J., and Bradlow, H. L. (1990). Induction of estradiol metabolism by dietary 13C in humans, J. Nat. Cancer Inst., 82947-949. 216. Graham, S., Dayal, H., Swanson, M., Mittelman, A., and Wilkinson, G. (1978). Diet in the epidemiology of cancer of the colon and rectum, J. Nnt. Cancer Inst., 61:709-714. 21 7. Newmark, H. L., Lipkin, M., and Maheshwari, N. (1990). Colonic hyperplasia and hyperproliferation induced by a nutritional stress diet with four components of western-style diet, J. Nut. Cancer Inst., 82:491-496. 21 8. Weisburger, J. H. (1 988). Colorectal cancer: Etiologic factors and their mode of action as

148

219.

220.

221.

222.

223. 224.

225.

226.

227.

22.8. 229. 230. 231. 232. 233. 234. 235.

236.

237.

238.

Beier and Nigg an effective approach to prevention. In Diseases of the Colon, Rectum, and Anal Canal (J. B. Kirsner and R. G. Shorter, eds.), Williams and Wilkins, Baltimore, pp. 371-394. Weisburger, J. H. (1988). Comparison of nutrition as customary in the western world, the orient, and northern populations (Eskimos) in relation to specific disease risks, Arct. Med. Res.. 47:llO-120. Weisburger, J. H. (1989). Dietary prevention of colorectal cancer. In Colorectal Cancer: From Pathogenesis to Prevention? (H. K. Seitz. U. A. Simanowski, and N. A. Wright, eds.), Springer-Verlag, New York, pp. 361-374. Weisberger, J. H. (1991). Carcinogens in our food and cancer prevention. In Nutritional a d Toxicological Consequerzces of Food Processing (M. Friedman, ed.). Plenum Press, New York, pp. 137-351. Weisburger, J. H. (1992). Mechanisms of macronutrient carcinogenesis. In Macronutrients, Investigating Their RoZe in Cancer (M. S. Micozzi and T. E. Moon, eds.), Marcel Dekker, New York, pp. 3-31. Graham, S., Schotz, W.. and Martino. P. (1972). Alimentary factors in the epidemiology of gastric cancer, Cancer, 30:927-938. Hirayama, T. (1977). Changing patterns of cancer in Japan with special reference to the decrease in stomach cancer mortality. In Origins of Hzmtalz Cancer. Book A. Incidence of Carzcer in Hurrzcrns Volunle 4 (H. H. Hiatt, J. D. Watson. and J. A. Winsten. eds.), Cold Spring Harbor Laboratory Press, New York, pp. 55-75. Weisburger, J. H., and Horn, C. L. (1991). The causes of cancer. In American Cancer Society Textbook of Clinical Oncolog!! (A. I. Holleb, D. J. Fink, and G. P. Murphy, eds.), American Cancer Society, Atlanta, pp. 80-98. Armstrong, B., and Doll, R. (1975).Environmental factors and cancer incidence and mortality in different countries, with special reference to dietary practices, Int. J. Cancer. 15:617631. Phillips, R. L. ( 1975). Role of life-style and dietary habits in risk of cancer among SeventhDay Adventists, Cancer Res.. 353513-3522. Correa, P. (1981). Epidetniological correlations between diet and cancer frequency, Ccnzcer Res., 41 3685-3690. Graham, S. (1983). Results of case-control studies of diet and cancer in Buffalo, New York, Cancer Res., 43:2409~--2413s. Block, G., Patterson, B., and Subar. A. (1992). Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence, Nzrtr. Cancer, 18:1-29. American Cancer Society (1984). Nutrition and cancer: Cause and prevention, Ca-A Cancer, J. Clin., 34:121-126. Assembly of Life Sciences Committee on Diet, Nutrition, and Cancer (1982). Diet, Nutrition. and Cancer, National Academy Press, Washington, D.C. Weisburger, J. H. (1990). Progress against cancer: We can win the war, Carlcer J., 3:120. Weisburger, J. H. (1991). Nutritional approach to cancer prevention with emphasis on vitamins. antioxidants, and carotenoids, Am. J. Clin. N m . , 53:226S-237S. Bailey, G.. Goeger, D., Hendricks, J.. Nixon, J.. and Pawlowski, N. (1985). Indole-3-carbino1 promotion and inhibition of aflatoxin B I carcinogenesis in rainbow trout, Abstract No. 456, Proc. 67th Ann. Mtg. Am. Assoc. Cancer Res., 26: 115. Bailey, G. S., Hendricks, J. D., Shelton. D. W., Nixon, J. E., and Pawlowski, N. E. (1987). Enhancement of carcinogenesis by the natural anticarcinogen indole-3-carbinol, J. Nat. Cancer Irzst., 78:931-934. Pence, B. C., Buddingh, F., and Yang, S. P. (1986). Multiple dietary factors in the enhancement of dimethylhydrazine carcinogenesis: Main effect of indole-3-carbinol, J. Not. Ccrncer Illst., 77:269-276. Temple, N. J., and El-Khatib, S. M. (1987). Cabbage and vitamin E: Their effect on colon tumor formation in mice, Cancer Lett., 35:71-77.

Naturally Occurring Toxic Chemicals in Foods

149

239. Birt. D. F., Pelling, J. C., Pour, P. M., Tibbels, M. G., Schweickert, L., and Bresnick, E. (1987). Enhanced pancreatic and skin tumorigenesis in cabbage-fed hamsters and mice, Carcinogenesis. 8:913-917. 240. Bradfield, C. A., and Bjeldanes, L. F. (1987). High-performance liquid chromatographic analysis of anticarcinogenic indoles in Brassica oleracea, J. Agric. Food Cltent., 35:46-49. 241. Gmelin, R.. and Virtanen, A. I. (1961). Glucobrassicin. the precursor of the thiocyanate ion, 3-indolylacetonitrile, and ascorbigen, in Brassica oleracea [and related] species, Ann. Acad. Sci. Ferlnicae, Ser. A, ZI. 107:l-25 (in German). 242. Gmelin, R., and Virtanen, A. I. (1962). Neoglucobrassicin, ein zweiter SCN"Precursor vom Indoltyp in Brassica-Arten. Acta Chent. Scand., 16:1378-1384 (in German). 243. Virtanen, A. I. (1965). Studies on organic sulphur compounds and other labile substances in plants, Pllytochemistiy, 4207-228. 244. Bergman, J. (1970). Condensation of indole and formaldehyde in the presence of air and sensitizers: A facile synthesis of indolo[3.2-b]carbazole, Tetrahedron, 263353-3355. 245. Gillner, M., Bergman, J., Cambillau, C.. Fernstrom, B., and Gustafsson. J. (1985). Interactions of indoles with specific binding sites for 2,3,7,8-tetrachlorodibenzo-y-dioxin in rat liver, Mol. Phamacol., 28:357-363. 346. Bradfield, C. A., and Bjeldanes, L. F. (1991). Modification of carcinogen metabolism by indolyic autolysis products of Brassica oleracaa. In Nutritional and Toxicological Consequences of Food Processirtg (M. Friedman, ed.), Plenum Press, New York, pp. 153-163. 247. Amat-Guerri, F., Martinez-Utrilla, R., and Pascual. C. (1984). Condensation of 3-hydroxymethylindoles with 3-substituted indoles. Formation of 2,3'-1nethylene-diindole derivatives, J. Cherit. Res. (Synop.), 160-161. 248. Bjeldanes, L. F., Kim, J.-Y., Grose, K. R., Bartholomew, J. C., and Bradfield, C. A. (1991). Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro andin vivo: Comparisons with 2,3.7,8-tetrachlorodibenzo-p-dioxin, Proc. Not. Acacl. Sci., 88:9543-9547. 249. Zhang, Y., Talalay. P.. Cho, C.-G., and Posner, G. H. (1992). A major inducer of anticarcinogenic protective enzymes from broccoli: Isolation and elucidation of structure, Proc. Nat. Acad. Sci., 89:2399-2403. 250. Cody, V., Middleton, E., Jr.. and Harborne, J. B., eds. (1986). Plarzt Flavonoids in Biologv artd Medicine, Biocheriticul. Pl~ar~rracological, and Structure-Activiv Relutionshiys, Alan R. Liss, New York. 251. Cody, V., Middleton, E., Jr., Harborne, J. B., and Beretz, A., eds. (1988). Plmt Flavonoids in Biology artd Medicine II, Biochemical, Cellular, and Medicinal Properties, Alan R. Liss, New York. 252. Das. N. P.,ed. (1990). Flavonoids in Biology and Medicine III, Current Issues in Flmorzoids Research, National University of Singapore. 253. Harborne, J. B., ed. (1988). The Flavolloids, Advances in Research Since 1980, Chapman and Hall, New York. 254. Harborne, J. B., Mabry, T. J., and Mabry, H., eds. (1975). The Flavonoids, Part 1,Academic Press, New York. 255. Harborne, J. B.,Mabry, T. J., and Mabry, H., eds. (1975). The Flavonoids, Pcrrt 2, Academic Press, New York. 256. Hedin, P. A., and Phillips, V. A. (1992). Electron impact mass spectral analysis of flavonoids, J. Agric. Food Clzem.. 40:607-611. 257. McClure, J. W. (1975). Physiology and function of flavonoids. In The Flavonoids Purt 2 (J. B. Harborne, T. J. Mabry, and H. Mabry, eds.), Academic Press, New York, pp. 9701055. 258. Berhow, M. A., and Vandercook, C. E. (1992). The reduction of naringin content of grapefruit by applications of gibberellic acid, Plant Growth Regul.. 11 :75-80. 259. de Riera, M. V. Q., Noriega, M. T., Gonzalez, J. A., and Seeligmann, P. (1988). Influencia

150

260.

261.

262.

263.

264. 265. 266. 267.

268.

269.

270. 271.

272.

273.

274.

275.

276.

Beier and Nigg de la luz solar la sintesis de flavonoides en la germinacion de Arachis hypogaea, Lilloa, 37:33-35. DownunI, K. R.. Swain, L. A., and Faleiro, L. J. (1991). Influence of light on plant allelochemicals: A synergistic defense in higher plants, Arch. Insect Bioclzern. Plzysiol., 17201211. Kogan, M., and Fischer, D. C. (1991). Inducible defenses in soybean against herbivorous insects. In Phytocltenlical Inclzlctiorz by Herbivores (D. W. Tallamy and M. J. Raupp. eds.), John Wiley and Sons, New York, pp. 347-378. Fleury. Y., and Magnolato, D. (1990). Changes in isoflavone content during maturation of soybean (Glycine 111a.x.Mer). In Flavonoids in Biology & Medicine III: Current Issues in Flallorroid Resecrrch (N. P. Das, ed.). National University of Singapore, pp. 19-26. Hungria. M., Joseph, C. M., and Phillips, D. A. (1991). Anthocyanidins and flavonols, major nod gene inducers from seeds of a black-seeded common bean (Phaseolus vulgaris L.), Plant Physiol., 97:751-758. Hungria, M., Joseph, C. M., and Phillips, D. A. (1 991). Rhizobium izod gene inducers exuded naturally from roots of common bean (Pl~aesol~u Idgaris L.), Plant Plzysiol., 97:759-764. Picq, M., Cheav. S. L., and Prigent, A. F. (1991). Effect of two flavonoid compounds on central nervous system. Analgesic activity, Life Sci., 49:1979-1988. Kuhnau. J. (1976). The flavonoids. A class of semi-essential food components: Their role in human nutrition, World Rei!. Nutr. Diet.. 24: 117-191. Hackett, A. M. (1986). The metabolism of flavonoid compounds in mammals. In Plant Flavonoids in Biologv crnd Medicine, Biochernictd, Pharn1acologica1,and Structure-Activie Relationships (V. Cody, E. Middleton, Jr.. and J. B. Harborne, eds.), Alan R. Liss, New York, pp. 177-194. Adzet, T., and Camarasa, J. (1986). Pharmacokinetics of polyphenolic compounds. In Plaizt Flrrvonoids in Biology and Medicine: Biochemical, Pharmacological, arld Stlucture-Acrillify Relltionships (~V.Cody, E. Middleton, Jr. and J. B. Harborne. eds.), Alan R. Liss, New York, pp. 25-47. Harborne, J. B. (1986). Nature, distribution and function of plant flavonoids. In Plant Flavonoids in Biologyand Medicine, Biochemical,Plznmzacological, and Stiuctzlre-Acti\yih Relatiowships (V. Cody, E. Middleton, Jr., and J. B. Harborne, eds.), Alan R. Liss. New York, pp. 15-24. Wollenweber, E., and Dietz, V. H. (1981). Occurrence and distribution of free flavonoid aglycones in plants, Pl1ytochemisttv, 20:869-932. Pierpoint, W. S. (1985). Phenolics in food and feedstuffs: The pleasures and perils of vegetarianism. In The Bioclzenzistry of Plant Pherzolics (C.F. Van Sumere and P. J. Lea, eds.), Annual Proceedings of the Phytochemical Society of Europe, Volume 29, Clarendon Press, Oxford, England, pp. 427-45 1. Pierpoint, W. S. (1986). Flavonoids in the human diet. In Plant Flm9onoids iiz Biology and Medicine, Biochemical, Phamacological, crnd Strtlctrcre-Activih Relationships (V. Cody, E. Middleton. Jr., and J. B. Harborne, eds.), Alan R. Liss. New York, pp. 125-140. Pierpoint, W. S. (1990). Flavonoids in human food and animal feedstuffs. In Flavo~zoids in Biology and Medicine III. Cztrrerzt Issues in Flavonoid Research (N. P. Das, ed.), National University of Singapore, pp. 497-514. Bilyk, A., and Sapers, G. M. (1985). Distribution of quercetin and Kaempferol in lettuce, kale. chive, garlic chive, leek, horseradish, red radish, and red cabbage tissues, J. Agric. Food Chem., 33226-228. Goupy, P. M., Varoquaux, P. J. A., Nicolas, J. J.. and Macheix, J. J. (1990). Identification and localization of hydroxycinnamoyl and flavonol derivatives from endive (Cichorium endivia L. cv. gearzte maraichere) leaves, J. Agric. Food Chern., 3821 16-2121. Herrmann, K. (1976). Flavonols and flavones in food plants: A review, J. Food Technol., 111433448.

Naturally Occurring Toxic Chemicals in Foods

151

277. Tomis-Barberin, F. A., Garcia-Grau, M. M., and Tomis-Lorente,F.(1991). Flavonoid concentration changes in maturing broad bean pods, J. Agric. Food Chem., 39255-258. 278. Leighton, T., Ginther. C., Fluss, L., Harter, W. K., Cansado, J.. and Notario, V. (1992). Molecular characterization of quercetin and quercetin glycosides in A l l i ~ uvegetables: ~ Their effects on malignant cell transformation. In Phenolic Compounds in Food arzcl Their Effects on Health 11 Anti-oxidants & Cancer Prelleiztioiz (M.-T. Huang, C.-T. Ho, and C. Y. Lee, eds.). ACS Symposium Series No. 507, American Chemical Society, Washington, D.C., pp. 220-238. 279. Bilyk, A., and Sapers, G. M. (1986). Varietal differences in the quercetin, kaempferol, and myricetin contents of highbush blueberry, cranberry, and thornless blackberry fruits, J. Agric. Food Chenz., 34:585-588. 280. Spanos, G. A., and Wrolstad, R. E. (1990). Influence of processing and storage on the phenolic composition of Thompson seedless grape juice, J. Agric. Food Chenz., 38:15651571. 281. Spanos. G. A., Wrolstad, R. E., and Heatherbell, D. A. (1990). Influence of processing and storage on the phenolic composition of apple juice, J. Agric. Food Chem., 38:1572-1579. 282. Raynal, J., Moutounet, M., and Souquet, J.-M. (1989). Intervention of phenolic compounds in plum technology. 1. Changes during drying, J. Agric. Food Clzewz., 37:1046-1050. 283. Mosel, H.-D., and Herrmann, K. (1974). The phenolics of fruits. 111. The contents of catechins and hydroxycinnamic acids in pome and stone fruits, 2. Lebens~n.-Urzters.-Forsclt.. 154~6-11. 284. Yusof. S., Ghazali, H. M., and King, G. S. (1990). Naringin content in local citrus fruits, Food Clzenz.. 37:113-121. 285. Daigle, D. J., Conkerton. E. J., Sanders, T. H., and Mixon, A. C. (1988). Peanut hull flavonoids: Their relationship with peanut maturity, J. Agric. Food Chew., 36: 1179-1 181. 286. Thomas, D. S., Turner. D. W.. and Tan, S. C. (1992). Covering cauliflower curds during growth reduces floret flavonoids and improves quality, N.Z.J. Crop Hortic. Sci., 20:147151. 287. Omidiji, O., and Ehimidu. J. (1990). Changes in the content of antibacterial isorhamnetin 3-glucoside and quercetin 3’ -glucoside following inoculation of onion (Allium cepct L. cv. Red Creole) with Pse~rdor~zortas cepacia, Physiol. Mol. Plalzt Putllol., 37:28 1-292. 288. Recourt, K.. van Tunen, A. J.. Mur, L. A., van Brussel, A. A. N., Lugtenberg, B. J. J., and Kijne, J. W. (1992). Activation of flavonoid biosynthesis in roots of Vicin scttilw subsp. nigra plants by inoculation with Rhizobizrrlz Iegwninosarum biovar liciae, Plant Mol. Biol.. 19:411-420. 259. Friedman, M., and Henika, P. R. (1991). Mutagenicity of toxic weed seeds in the Ames test: Jimson weed (Dat1u-astrclntoniunt),velvetleaf (Abutilon theoplzrmti), morning glory (Ipouzoecl spp.), and sicklepod (Ccwicr obtusifolicr), J. Agric. Food Clzent., 39:394-501. 290. Crawford, L.. McDonald, G. M.. and Friedman. M. (1990). Conlposition of sicklepod (Cassicr obtlrsifolia) toxic weed seeds, J. Agric. Food Chem., 38:2169-2175. 291. Paszkowski, W. L., and Kremer, R. J. (1988). Biological activity and tentative identification of flavonoid components in velvetleaf (Abutilon tlzeoplzrctsti Medik.) seed coats, J. Cltenz. Eco~.,14: 1573-1552. 292. Dollahite. J. W., and Anthony, W. V. (1957). Poisoning of cattle with Gutierrezirr microcephala. a perennial broomweed, J. Am. Vet. Med. Assoc.. 130:525-530. 293. Mathews. F. P. (1936). The toxicity of broomweed (Gutierrecia microcephuln)for sheep. cattle and goats, J. Ant. Vet. Med. Assoc., 88:55-61. 294. Roitman, J. N., and James, L. F. (1985). Chemistry of toxic range plants. Highly oxygenated flavonol methyl ethers from Gutierrezin wicrocepheh, Phytoclzentist~y,24:835-848. 295. Matsuura, M., Obata, A., and Fukushima, D. (1989). Objectionable flavor of soy milk developed during the soaking of soybeans and its control, J. Food Sci., 54:602-605. 296. Kudou, S., Fleury, Y.. Welti. D., Magnolato, D., Uchida. T., Kitamura, K., and Okubo, K.

152

297. 298.

299. 300.

301.

302.

303. 304.

305.

306.

307

308.

309. 3 10.

311. 3 12.

313. 3 14.

Beier and Nigg (1991). Malonyl isoflavone glycosides in soybean seeds (Glvcine mcrx Merrill), Agric. Biol. Chew., 552227-2233. Cho, M.-J.. andHarper, J. E. (1991). Effect of inoculation and nitrogen on isoflavonoid concentration in wild-type and nodulation-mutant soybean roots, Plant Physiol., 95:435-442. Cho, M.-J.. and Harper, J. E. (1991). Effect of localized nitrate application on isoflavonoid concentration and nodulation in split-root systems of wild-type and nodulation-mutant soybean plants. Plant Plrysiol., 95: 1106-1 112. Graham, T. L. (1991). Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates, Plunt Physiol., 95:594-603. Smith, G. R., Randel, R. D., and Bradshaw, C. (1986). Influence of harvest date, cultivar, and sample storage method on concentration of isoflavones in subterranean clover, Crop Sci., 26:1013-1016. Rubin. B., Penner, D.. and Saettler. A. W. (1983). Induction of isoflavonoid production in Phaseolus \wlgcrris L. leaves by ozone, sulfur dioxide and herbicide stress, Enviro-on.Toxicol. Chem., 2295-306. Singleton, V. L. (1981). Naturally occurring food toxicants: Phenolic substances of plant origin common in foods. In Aclvances in Food Reseur-ch, Volume 27 (C. 0. Chichester, E. M. Mark, and G. F. Stewart, eds.), Academic Press, New York. pp. 149-242. Attaway, J. A. (1992). Medical benefits of juice flavonoids, Proceedings of the X I hternatiom1 Congress of Fruit Juices. Sao Taulo, Brazil. Cotelle, N.. Bernier, J. L., Hhichart, J. P.. Catteau, J. P., Gaydou, E., and Wallet, J. C. (1992). Scavenger and antioxidant properties of ten synthetic flavones. Free Radiccrl Biol. & Med., 13211-219. Fleury, Y., Welti, D. H., Philippossian, G., and Magnolato. D. (1991). Soybean (Malonyl) isoflavones: Characterization and antioxidant properties. In Pherlolic Corlrpourrds in Food a d Their Effects O H Health II Antioxidants and Ccrncer Prevention (M.-T. Huang, C.-T. Ho. and C. Y. Lee, eds.), ACS Symposium Series No. 507, American Chemical Society, Washington, D.C., pp. 98-113. Das, N. P., and Ratty, A. K. (1986). Effects of flavonoids on induced nonenzymic lipid peroxidation. In Plant Flavonoids irz Biology and Medicine: Biochemical Pharnmcologiccrl crnd Structure-ActiviQ Relatiomhips (V. Cody, E. Middleton, and J. B. Harborne, eds.), Alan R. Liss, New York. pp. 243-247. Ramarathnam, N., Osawa, T.. Namiki, M., and Kawakishi. S. (1989). Chemical studies on novel rice hull antioxidants. 2. Identification of isovitexin, a C-glycosyl flavonoid, J. Agr-ic. Food Chem., 37:316-319. da Silva, J. M. R., Darmon,N., Fernandez. Y.. and Mitjavila, S. (1991). Oxygen free radical scavenger capacity in aqueous models of different procyanidins from grape seeds, J. Agric. Food Clrem., 39:1549-1552. MacGregor. J. T. (1984). Genetic and carcinogenic effects of plant flavonoids: An overview, -4dv. Exp. Med. Biol., 177:497-526. Robbins, R. C. (1980). Medical and nutritional aspects of citrus bioflavonoids. In Citrus Nutrition a d Qnalih (S. Nagy and J. A. Attaway. eds.), Am. Chem. SOC.Symp. Ser. 143, American Chemical Society, Washington, D.C., pp. 34-59. Huet, R. (1982). Constituants des agrumes i effet pharmacodynamique: Les citroflavonoi'des. Fruits, 37267-27 1. Middleton, E. (1988). Plant flavonoid effects on mammalian cell systems. In Herbs, Spices crtrd Medicinal Plants, Volume 3 (L. E. Craker and J. E. Simon. eds.), Oryx Press, Phoenix. Arizona, pp. 103-144. Varma, S. D.. Mikuni, I., and Kinoshita, J. H. (1975). Flavonoids as inhibitors of lens aldose reductase, Science, 188:1215-1216. Varma, S. D., Mizuno, A., and Kinoshita, J. H. (1977). Diabetic cataracts and flavonoids, Science, 195205-206.

Naturally Occurring Toxic Chemicals in Foods 315.

316. 317.

318.

319.

320. 321. 322.

323.

324.

325. 326. 327. 338.

329.

330.

331. 332.

333.

153

Nikaido, T., Ohmoto, T.,Kinoshita, T., Sankawa,U., Monache, F. D., Botta, B., Tomimori, T., Miyaichi, Y., Shirataki, Y., Yokoe, I.. and Komatsu, M. (1989). Inhibition of adenosine 3'.5'-cyclic monophosphate phosphodiesterase by flavonoids, 111, Clzenz. Pharnz. Bull.. 37: 1392- 1395. Stavric. B. (1983). Mutagenic food flavonoids, symposium report, Fed. Proc., 33:24542458. Stavric.B..andMatula.T. I. (1988). Biologicalsignificanceofflavonoids in foods: A Bulletin de Liaisolz No 14 du Grollpe review of some recentissuesandproblems.In Polvplterlols, Nrrrbonne,Frcmce, Compte-Rendudes Jounze'e Intenzatiorzeles d'Etude et de I'Assemblhe Gkne'role 1988, INRA Publications, Versailles, France, pp. 95-1 04. Schroder-van der Elst, J. P., van der Heide, D., and Kohrle. J. (1991). In vivo effects of flavonoid EMD 21388 on thyroidhormone secretion and metabolism inrats. Am. J. Plpsiol.. 26 1 (E~zdocrinol.' Metab.24):E227-E232. Hosokawa. N., Hirayoshi, K.. Kudo, H., Takechi, H., Aoike, A., Kawai, K., and Nagata. K. (1992). Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. Mol. Cell. Biol., 12:3490-3498. Stavric, B.. Stoltz. D. R., and Klassen, R. (1983). Toxicants in foods with special emphasis on mutagens in beverages, fruits and vegetables, Toxico?z,3(Supplement):409-412. Stavric, B., Klassen, R., and Stolz, D. R. (1983). Natural toxins in foods: Mutagens,Period. Biol., 86:165-172. Stoltz, D.R.. Stavric, B., Klassen,R..and Muise, T. (1982). The health significance of mutagens in foods.In Cewcinogerls and Mutagens in the Ertvironment. Volur~eI, Food Products (H. F. Stich, ed.). CRC Press, Boca Raton, Florida, pp. 185-197. Stoltz. D. R., Stavric, B., Stapley, R., Klassen, R., Bendall, R., and Krewski, D. (1984). Mutagenicity screening of foods. 11. Results with fruits and vegetables, Enviroolt. Mzrtcrgen.. 6:343-354. Brown, J. P., and Dietrich, P. S. (1978). Promutagenic plant phenolics in man's diet: Do mutagenic flavonols and anthraquinones play a role in environmental mutagenesis/carcinogenesis? Abstract Cb-5, 9th Anizual Meetiizg of EmVronrlzentrtl Mutagerz Society. Bjeldanes, L. F., and Chang, G. W. (1977). Mutagenic activity of quercetin and related compounds. Science, 197577-578. Hardigree. A. A., and Epler, J. L. (1978). Comparative mutagenesis of plant flavonoids in microbial systems, Mutat. Res., 58:23 1-239. MacGregor. J. T.. and Jurd, L. (1 978). Mutagenicity of plant flavonoids: Structural requirements for mutagenic activity in Salmonella ~pltinturiunl,Mutat. Res., 54:297-309. Meltz, M. L., and MacGregor, J. T. (1981). Activity of the plant flavonol quercetin in the mouse lymphoma L5 178Y TK' mutation, DNA single-strand break and Balb/c 3T3 chemical transformation assays, Mutat. Res., 88:317-324. Woo, Y.-T., Lai, D. Y., Arcos, J. C., and Argus, M.F. (1988). Plant flavonoids. In Cltenziccrl Incl~~tiort of Ccrncer, Stnrcturd Bases arzd Biological Mechnnisnzs (Y.-T. Woo, D. Y. Lai, J. C. Arcos, and M. F. Argus, eds.), Academic Press, New York, pp. 383-396. Friedman. M., and Smith, G. A. (1984). Factors which facilitate inactivation of quercetin mutagenicity. In Advarzces in Experimental Medicine and Biology: Nutritional m t l Toxicologicd Aspects sf Food Sufefy, Volume 77 (M. Friedman, ed.), Plenum Press, New York, pp. 527-534. Rueff, J.. Laires, A., Gaspar, J.. Borba, H., and Rodrigues, A. (1992). Oxygen species and the genotoxicity of quercetin, Mutcrt. Res., 265:75-8 1. National Toxicology Program (1989). Fiscal Year 1989 Airma1 Plan, U.S. Department of Health and Human Services, NTP-89-167, June 1989, Public Health Service, Research Triangle Park, North Carolina. Dunnick, J. K., and Hailey, J. R. (1992). Toxicity and carcinogenicity studies of quercetin, a natural component of foods, Funcl. Appl. Toxicol., 19:423-431.

154

Beier and Nigg

1). NTP Technical Report on the Toxicology and Carci334. National Toxicology Program (199 nogenesis Studies of Quercetirt (CAS No. 117-39-5) in F343/N Rats (Feed Studies) NTP TR 409, NIH Publication No. 91-3140, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, North Carolina. 335. Lai, D. Y., and Woo, Y.-T. (1987). Naturally occurring carcinogens: An overview, J. Envirow Sci. Health, C5:121-173. 336. Woo, Y.-T., Lai, D. Y., Arcos, J. C., and Argus, M. F. (1988). Miscellaneousplant carcinogens. In Chemical htduction of Cancer, Structlrral Bases und Biological Mechanism (Y.T. Woo, D.Y. Lai,J.C. Arcos, and M. F.Argus,eds.),Academic Press, NewYork, pp. 357-435. 337. Hayashi, M., Kishi, M., Sofuni, T.. and Ishidate, M., Jr. (1988). Micronucleustests in mice on 39 food additivesand eight miscellaneous chemicals,Food Chern. Tosicol., 26:487-500. 338. Shirahata. S., Murakami, H., Nishiyama, K., Yamada, K.. Nonaka, G., Nishioka, I., and Omura, H. (1989). DNA breakageflavan-3-01s by andprocyanidins inthe presence of cupric ion, J. Agt-ic. Food Chem., 37:299-303. 339. Jones, E., and Hughes, R. E. (1982). Quercetin, flavonoids and the life-span of mice, E.%perirtt. Gerontol., 17213-217. 340. Futami, H., Eader, L. A., Komschlies, K. L., Bull, R., Gruys, M. E., Ortaldo, J. R., Young. H. A.. and Wiltrout, R. H. (1991). Flavone acetic acid directly induces expression of cytokine genes in mouse splenic leukocytes but not in humanperipheral blood leukocytes, Cancer Res., 51:6596-6602. 341. Pratesi, G., Manzotti,C.. Tortoreto. M., Audisio. R. A., and Zunino, F. (1991). Differential efficacy of flavone acetic acid against liver versus lung metastases in a human tumour xenograft, Br. J. Cancer, 63:71-74. 342 Ching, L.-M., McKeage, M. J., Joseph, W. R., Kestell, P., Zwi, L. J., and Baguley, B. C. (1991).Haematologicaleffectsinmice oftheantitumour agentsxanthenone-4-acetic acid,5,6-methyl-xanthenone-4-aceticacid andflavoneaceticacid, Cuncer Clzentother. Phorittacol.. 28:414-419. 333. Cushman, M.. and Nagarathnam, D. (1991). Cytotoxicities of some flavonoid analogues, J. Nut. Prod. (Lloyclia). 54:1656-1660. 344. Huang. M.-T., Wood. A. W.. Newmark, H. L., Sayer, J. M., Yagi, H., Jerina, D. M., and Conney, A. H. (1983). Inhibition of the mutagenicity of bay-region diol-eposides of polycyclic aromatic hydrocarbons by phenolic plant flavonoids, Carcinogenesis, 4: 1631-1637. 345. Stich. H. F., and Rosin, M. P. (1984). Naturally occun-ing phenolics as antimutagenic and anticarcinogenic agents, AcZtv. Exp. Med. Biol., 177: 1-29. 346. Wall. M. E., Wani, M. C.. Manikumar. G., Abraham, P., Taylor, H., Hughes, T. J., Warner, J., and McGivney,R.(1988). Plant antimutagenic agents, 2. Flavonoids, J. Nut. Prod. (Lloydiu), 5 1:1084- 1091. 347. Markham. R. J. F., Erhardt. N. P., Dininno, V. L., Penman, D.. and Bhatti, A. R. (1987). Flavonoids protect against T-2 mycotoxins both in vitro and in vivo. J. Gen. Microbiol., 13311589-1592. 348. Francis. A. R., Shetty, T. K., and Bhattacharya, R. K. (1989). Modifying role of dietary factors on the mutagenicity of aflatoxin B In vitro effect of plant flavonoids, Mutat. Res., 222393-401. 349. Chang, R. L., Huang, M.-T., Wood, A. W., Wong, C.-Q., Newmark, H. L., Yagi, H., Sayer, J. M., Jerina, D. M., and Conney, A. H. (1985). Effect of ellagic acid and hydroxylated flavonoids onthetumorigenicityof benzo[u]pyrene and (t-)-7,8,8mdihydroxy-9a, loaepoxy-7.8.9. IO-tetrahydrobenzo[r]pyreneon mouse skin and in the newborn mouse, Cllrcinogertesis, 6:1127-1133. 350. Deschner, E. E., Ruperto, J., Wong, G., and Newmark, H. L. (1991). Quercetin and rutin as inhibitorsof azoxymethanol-induced colonicneoplasia, Curcirzogerzesis.12:1193-1 196.

Naturally Occurring Toxic Chemicals in Foods 351.

352.

353.

354. 355.

356.

357. 358.

359. 360. 361.

362.

363. 364. 365. 366. 367.

368. 369.

155

Stavric, B., and Matula, T. I. (1992). Flavonoids in foods: Their significance for nutrition and health. In Lipid-Soluble Antioxidants: Biochenzistn and Clirlical Applications (A. S. H. Ong and L. Packer, eds.), Birkhalsuser, Basel, pp. 274-294. Stavric. B., Matula, T. I., Klassen, R.. and Downie, R. H. (1990). Inhibitory effect of flavonoids against mutagenic and carcinogenic xenobiotics in foods. In Flm,o~zoid,sin Biology and Medicine 111: Current I s s ~ e sin Flavonoids Research, (N. P. Das, ed.),National University of Singapore, pp. 515-529. Stavric, B., Matula, T. I., Klassen, R., Downie, R. H., and Wood, R. J. (1992). Effect of flavonoids on mutagenicity and bioavailability of xenobiotics in foods. In Phenolic Cowpomds irl Food and Their EfSect on Health 11 Antioxidcum and Cancer Preljention (M. T. Huang, C.-T. Ho., and C. Y. Lee, eds.), ACS Symposium Series No. 507, American Chemical Society, Washington. D. c., pp. 239-249. Canada, A. T., Watkins, W. D., and Nguyeen. T. D. (1989). The toxicity of flavonoids to guinea pig enterocytes, Tosicol. Appl. Plzumzacol., 99:357-361. Kumar, P.,Dixit, V. P., and Khanna, P. (1989). Antifertility studies of kaenlpferol: Isolation and identification from tissue culture of some medicinally important plant species, Plcmtes Mbdicinales et Phytotlze‘rapie, 23: 193-201. Shore, L.S., and Lyttle, C. R. (1986).Interactions of isoflavones, zearalenone and DES with rat uterine peroxidase enzyme. In Plmt Flal~orzoirlsin Biology and Medicine, Biochenzical, Pharlmrological, and Structure-Activity Relationships. (V. Cody, E. Middleton, Jr., and J. B. Harborne, eds.), Alan R. Liss, New York, pp. 253-256. Bennetts. H. W., Underwood, E. J., and Shier, F. L. (1946). A specific breeding problem of sheep on subtemanean clover pastures in western Australia, Aust. Vet. J.,322-12. Cox, R. I. (1978). Plant estrogens affecting livestock in Australia. In EfSects of Poisonozls Plants on Livestock (R. F. Keeler. K. R. Van Kampen, and L. F. James, eds.). Academic Press, New York, pp. 451-464. Morley, F. H. W., Axelsen, A., and Bennett, D. (1964). Effects of grazing red(Trijolilan clover prateme L.) during thejoining season onewe fertility, Proc. Aust. SOC.Anim Prod., 5:58-61. Moule, G. R.. Braden, A. W. H., and Lamond, D. R. (1963). Thesignificance of oestrogens in pasture plants in relation to animal production, Anim. Breed. Alxtr., 31:139-157. Lightfoot. R. J., Croker,K. P., and Neil, H. G. (1967). Failure of sperm transport in relation to ewe infertility following prolonged grazing on oestrogenic pastures, Aust. J. Agric. Res., 18:755-765. Cox, R. I. ( 1985). Immunophysiological control of phyto-oestrogen toxicity. In Plant Tosicology (A. A. Seawright. M. P. Hegarty, L. F. James, R. F. Keeler, and J. Hrdlicka. eds.), Dominion Press-Hedges & Bell, Melbourne. pp. 98-108. Marrian, G. F., and Haslewood, G. A. D. (1932). Equol, a new inactive phenol isolated from the ketohydroxyoestrin fraction of mares’ urine. Biochenz. J., 26:1227-1232. Klyne, W., and Wright, A. A. (1957). Steroids and other lipids of pregnant goat’s urine. Bioche~n.J.. 66:92-101. Klyne, W., and Wright, A. A. (1959). Steroids and other lipids of pregnant cow’s urine, J. Endocrinol., 18:32-45. Common, R. H.. and Ainsworth, L. (1961 ). Identification of equolin the urine of thedomestic fowl. Biocl1im. Biophys. Acta, 53:403-404. MacRae, H. F., Dale, D. G., and Common, R. H. ( 1960). Formation in vivo or 16-epiestriol and 16-ketoestradiol-17P from estriol by the laying hen and occurrence of equol in hen’s urine and feces. Can. J. Bioclzenz. Physiol., 38:523-532. Braden, A. W. H., Hart, N. K., and Lamberton, J. A. (1967). The oestrogenic activity and metabolism of certain isoflavones in sheep. Aust. J. Agric. Res., 18:335-348. Shutt, D. A., and Braden. A. W. H. (1968). The significance of equol in relation to the oestrogenic responses in sheep ingesting clover with a high formononetin content, Aust. J. Agric. Res., 19:545-553.

156

Beier and Nigg

370. Axelson, M.. Kirk. D. N., Farrant, R. D., Cooley, G., Lawson, A. M.. and Setchell, K. D. R. (1982). The identification of the weak oestrogen equol [7-hydroxy-3-(4’-hydroxyphenyl)chroman] in human urine, Biochern. J., 201353-357. 371. Dickinson, J. M., Smith,G. R., Randel, R. D.. and Pemberton. I. J. (1988). In vitro metabolism of formononetin and biochanin A in bovine rumen fluid, J. Arzim. Sci., 66:1969-1973. 372. Barbuch, R. J., and Coutant, J. E., Welsh, M. B., and Setchell, K. D. R. (1989). The use of thermospray liquid chronlatography/tandem mass spectrometry for the class identification and structural verification of phytoestrogens in soy protein preparations, Biorrred. Erzviron. Mass Spectrom., 18:973-977. 373. Adlercreutz, H., Fotsis, T., Bannwart, C., Wahala, K., Brunow, G., and Hase, T. ( 1991). Isotope dilution gas chromatographic-mass spectrometric method for the determination of lignans and isoflavonoids in human urine, including identification of genistein. Clirr. Clzint. Act(!, 1991263-278. 374. Kitts, D. D. (1987). Studies on the estrogenic activity of a coffee extract, J. Toxicol. Erniron. Hedth, 20137-49. 375. Newsome, F. E., and Kitts, W. D. (1980). Action of phyto-estrogens coumestrol and genistein on cytosolic and nuclear oestradiol-17P receptors in immature rat uterus. Arzim Reprod. Sci., 3933-245. 376. Shutt, D. A., and Cox, R. I. (1972). Steroid and phyto-oestrogen binding to sheep uterine receptors in vitro. J. Ertdocr-irtol.,52299-3 10. 377. Tang, B. Y., and Adams, N. R. (1980). Effect of equol on oestrogen receptors and on synthesis of DNA and protein in the immature rat uterus, J. Erzdocrirtol., 85:291-297. 378. Farmakalidis, E., and Murphy. P. A. (1984). Oestrogenic response of the CD-1 mouse to the soya-bean isoflavones genistein. genistin and daidzin, Food Chem. Tosicol., 22237-239. 379. Setchell, K. D. R., Borriello, S. P., Hulme, P.. Kirk, D. N., and Axelson, M. (1984). Nonsteroidal estrogens of dietary origin: Possible roles in hormone-dependent disease. Am. J. Clirl. Nutr., 40:569-578. 380. Garreau, B., Vallette, G., Adlercreutz, H., Wihali, K.. Makela, T., Benassayag, C.. and Nunez, E. A. (1991). Phytoestrogens: New ligands for rat and human a-fetoprotein, Bioclzim. Biopkys. Acta, 1094:339-345. 381. Ogawara, H.. Akiyama. T., and Watanabe. S.-I. (1989).Inhibition of tyrosine protein kinase activity by synthetic isoflavones and flavones, J. Arrtibiot. (Tokyo), 42340-343. 382. Watanabe, T., Shiraishi, T., Sasaki, H., and Oishi, M. (1989).Inhibitors for protein-tyrosine kinases, ST638 and genistein. induce differentiation of mouse erythroleukemia cells in a synergistic manner, E . YCell ~ Res., 183:335-342. 383. Atluru. S., and Atluru, D. (1991). Evidence that genistein, a protein-tyrosine kinase inhibitor, inhibits CD2smonoclonal-antibody-stimulated human T cellproliferation, Transplantatiorz (Baltimore), 51:448-450. 384. Middleton, E., Jr., and Kandaswami, C. (1992). Effects of flavonoids on immune and inflammatory cell functions, Biochem. Phnn~acol.,43: 1167-1 179. 385. Le Bon. A.-M., Siess, M.-H., and Suschetet, M. ( 1992). Inhibition of microsome-mediated binding of benzo[a]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats, Cizem.-Biol. Interact., 83:65-7 1. 386. Howie, B. J., and Shultz, T. D. (1985). Dietary and hormonal interrelationships among vegetarian Seventh-Day Adventists and nonvegetarian men, Am. J. Clirz. Nutr., 42: 127134. 387. Goldin, B. R.. and Gorbach, S. L. (1988). Effect of diet on the plasma levels, metabolism, and excretion of estrogens, Am. J. Clin. Nutr., 48:787-790. 388. Goldin, B. R., Adlercreutz, H., Gorbach, S. L., Woods, M. N., Dwyer, J. T., Conlon. T.. Bohn, E., and Gershoff, S. N. (1986). The relationship between estrogen levels and diets of Caucasian American and Oriental immigrant women, Ant. J. Clirz. Nutr., 41:945-953. 389. Hagerty, M. A., Howie. B. J., Tan. S.. and Shultz, T. D. (1988). Effect of low- and high-

Naturally Occurring Toxic Chemicals in Foods

390.

391. 392. 393. 394. 395. 396. 397. 398. 399. 400. 401.

402.

403.

404.

405. 406. 307.

408.

309.

410.

31 1.

157

fat intakes on the hormonal milieu of premenopausal women, Anz. J. Clin. Nutr., 47:653659. Shultz, T. D., Wilcox, R. B., Spuehler, J.M., and Howie, B. J. ( 1987). Dietary and hormonal interrelationships in premenopausal women: Evidence for a relationship between dietary nutrients and plasma prolactin levels, Am. J. Clin. Nutr., 46:905-911. Dubick, M. A. (1986). Historical, perspectives on the use of herbal preparations to promote health, J. N w . , 116:1348-1353. Stephenson, M. (1978). The confusing world of health food, FDA Consum.. 1218-22. herbal tea, J. Am. Hogan, R. P., I11 (1983). Hemorrhagic diathesis caused by drinking an Med. Assoc., 2492679-2680. American Spice Trade Association (1992). Estimate of total herb and related commodities in 1990, American Spice Trade Association, New York. Saxe, T. G. (1987). Toxicity of medicinal herbal preparations, Am. Farrz. Phys. (U.S.), 35: 135-142. Steiner. R. P. (1986).Folk Medicine: The Art and the Science, American Chemical Society, Washington, D.C. Cyong,J.-C.,Matsumoto, T.. Arakawa, K., Kiyohara, H.,Yamada,H., and Otsuka, Y. (1987). Anti-Bacteroidesfi.agilissubstance from rhubarb,J. Etluloplzarmacol., 19:279-283. Franzblau. S. G., and Cross, C. (1986). Comparative in vitro antimicrobial activity of Chinese medicinal herbs. J. Etllnopharnmcol., 15279-288. Keji, C. (1981). Certain progress in the treatment of coronary heart disease with traditional medicinal plants in China, Am. J. Cliw Med., 9:193-196. Chang, R. S., and Yeung, H. W. (1988). Inhibition of growth of human immunodeficiency virus in vitro by crude extracts of Chinese medicinal herbs. Antiviral Res., 9:163-176. Chu, D.-T.. Wong, W. L., and Mavlight, G.M. (1988). Imnlunotherapy withChinese medicinal herbs. I. Immune restoration of local xenogeneic graft-versus-host reaction in cancer patients by fractionated Astragalus membranaceus in vitro. J. Clirz. h r n m o l . , 35: 119-123. Lee, H. K., Kim, Y. K., Kim. Y.-H., and Roh, J. K. (1987). Effect of bacterial growthinhibiting ingredients on the Ames mutagenicity of medicinal herbs. Mutat. Res., 19299104. Morimoto. I., Watanabe, F., Osawa, T.. and Okitsu. T. (1982). Mutagenicity screening of crude drugs with Bacilllrs slrbtilis rec-assay and Salrrzowellalmicrosome reversion assay, Mutrrt. Res., 97:81-102. Ries. C. A., and Sahud, M. A. (1975). Agranulocytosis caused by Chinese herbal medicines: Dangers of medications containing aminopyrine and phenylbutazone, J. Am. Mecl. Assoc., 231:352-355. Roy, A. K., and Chourasia. H. K. (1990). Mycoflora. mycotoxin producibility and mycotoxins in traditional herbal drugs from India, J. Gen. Appl. Microbiol.. 36295-302. Casterline, C. L. (1980). Allergy to Chamomile tea. J. Ant. Med. Assoc.. 344:330-33 1. Siegel, R. K. (1976). Herbal intoxication: Psychoactive effects from herbal cigarettes, tea, and capsules. J. Aril. Med. Assoc., 236:473-476. Woo, Y.-T., Lai, D. Y., Arcos, J. C., and Argus. M. F. (1988). Plant Alkaloids. In Clzerrlical Inductiorz of Cancer, Strzrctw-crl Bases am1 Biological Meclzrrrtisnls (Y.-T. Woo, D. Y. Lai. J. C. Arcos, and M. F. Argus. eds.), Academic Press. New York, pp. 212-266. Arseculeratne, S. N., Gunatilaka, A.A. L., and Panabokke, R. G. (1981). Studieson medicinal plants of Sri Lanka: Occurrence of pyr-rolizidine alkaloids and hepatotoxic properties in some traditional medicinal herbs, J. Etlz~lo~?ltnrirlrrcol.. 4: 159-177. Arseculeratne, S. N., Gunatilaka, A. A. L., and Panabokke,R. G. (1985). Studieson medicinal plants of Sri Lanka,Part 14: Toxicity of some traditional medicinalherbs, J. Etlozophcrri?lacol.. 13~323-335. Cordell, G. A. (1981). Pyrrolizidine alkaloids. In Irztroduction to Alkrrloids: A Biogenetic Approach. John Wiley and Sons, New York, pp. 118-137.

158

Beier and Nigg

312. Frei, H., Luthy, J.. Brauchli, J., Zweifel, U., Wurgler, F. E., and Schlatter, C. (1992). Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila 1nelunogaster, Chem-Biol. Interact., 83: 1-22. 413. Schoental, R. (1968).Toxicology and carcinogenic action of pyrrolizidine alkaloids. Carzcer Res., 28:2237-2246. 314. Hincks, J. R., Kim, H.-Y., Segall, H. J., Molyneux, R. J., Stermitz, F. R., and Coulombe, R. A., Jr. (1991). DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: Structure-activity relationships, Tosicol. Appl. Phamtacol., 111:90-98. 415. Reindel. J. F., Ganey. P. E., Wagne. J. C., Slocombe. R. F., and Roth, R. A. (1990). Development of morphologic, hemodynamic, and biochemical changes in lungs of rats given monocrotaline pyrrole, Toxicol. App., Pharnzacol., 106:179-200. 41 6. Dwivedi, Y., Rastogi, R., Sharma, S. K., Mehrotra, R., Garg, N. K., and Dhawan. B. N. (1 991). Picroliv protects against monocrotaline-induced hepatic damage in rats. Phamcrcol. Res., 23:399-307. 417. Miranda, C. L., Chung, W., Reed. R. E., Zhao, X., Henderson, M. C., Wang, J.-L., Williams, D. E., and Buhler, D. R. (1991). Flavin-containing monooxygenase: A major detoxifying enzyme for the pyrrolizidine alkaloid senecionine in guinea pig tissues, Biochenz. Biophys. Res.Comntun., 178:546-552. 41 8. Williams, D. E., Reed, R. L., Kedzierski, B., Dannan, G. A., Guengerich, F. P., and Buhler. D. R. (1989). Bioactivation and detoxication of the pyrrolizidine alkaloid senecionine by cytochrome P-450 enzymes in rat liver, Drug Metub. Dispos., 17:387-392. 419. Miranda, C. L.. Reed, R. L., Guengerich, F. P., and Buhler, D. R. (1991). Role of cytochrome P450IIIA.1. in the metabolism of the pyrrolizidine alkaloid senecionine in human liver, Carcinogenesis, 1 2 5 15-519. 4’20. McLean, E. K. (1970). The toxic actions of pyrrolizidine (senecio) alkaloids, Plmmacol. Rely., 22429-483. 321. Culvenor, C. C. J., Edgar, J. A., Smith, L. W., Kumana, C. R.. and Lin, H. J. ( 1986). Heliotropium lasiocrrlpurit Fisch. and Mey. identified as cause of veno-occlusive disease due to a herbal tea. Lancet, 1:978. 423,. Mattocks, A. R. (1980). Toxic pyrrolizidine alkaloids in comfrey, Lancet, 21136-1 137. 423. Roitman, J. N. (1981). Comfrey and liver damage, Lancet, 1:943. 424. Huxtable, R. J., and Awang.D. V. C. (1990).Pyrrolizidine poisoning,Am. J.Men., 89:547-548. 425. Ridker, P. M., and McDermott, W. V. (1990). The reply to pyrrolizidine poisoning, An!. J. Men.. 89:547-548. 4’26. Bordia. A., Bansal, H. C.. Arora, S. K., and Singh, S. V. (1975). Effect of the essential oils of garlic and onion on alimentary hyperlipemia, Atherosclerosis, 21:15-19. 427. Gudi. V. A., and Singh, S. V. (1991). Effect of diallyl sulfide. a naturally occurring anticarcinogen. on glutathione-dependent detoxification enzymes of female CD-1 mouse tissues, Biochent. Phasmrrcol., 42: 1261- 1265. 438. You, W. C.. Blot, W. J., Chang. Y. S., Ershow, A. G., Yang, Z. T., An, Q., Henderson, B. E., Fraumeni. J. F., Jr., and Wang, T. G. (1989). Allium vegetables and reduced risk of stomach cancer, J. Ncrt. Cancer Inst., 8 1:162- 163. 429. You, W. C., Blot, W. J.. Chang, Y. S., Ershow, A. G.. Yang. Z. T., An, Q., Henderson, B.. Xu, G. W., Fraumeni, J. F., Jr., and Wang. T. G. (1988). Diet and high risk of stomach cancer in Shandong, China, Cancer Res., 48:3518-3523. 330. Sparnins, V. L., Mott. A. W., Barany, G., and Wattenberg, L. W. (1986). Effects of allyl methyl trisulfide on glutathione S-transferase activity and BP-induced neoplasia in the mouse, Nuts. Cancel-.. 8:211-215. 431. Sparnins, V. L., Barany, G., and Wattenberg, L. W. (1988). Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione Stransferase activity in the mouse. Ccrrcinogerzesis,9: 131-134.

Naturally Occurring Toxic Chemicals in Foods 432.

433. 434. 335. 436.

437. 438. 439. 440. 441. 442. 443. 444.

445. 446. 447. 448.

449. 350. 351. 452.

453. 454.

455.

159

Summiyoshi, H., and Wargovich. M. J. (1990). Chemoprevention of 1,2-dimethylhydrazine-induced colon cancer in mice by naturally occurring organosulfur compounds, Carzcer Res., 505084-5087. Duke, J. A. (1987). Father nature’s farmacy: Yarrow (Achillea nrillefoliurtr,) HelBalCrarn, 12~6-7. Duke, J. A. (1987). Father nature’s farmacy: The mint family, HerbalGram, 14:6. Lipsitz, D. J. (1984). Herbal teas and water intoxication in a young child, J. Fum. Pract.. 18~933-937. Woo, Y.-T., Lai, D. Y.. Acros, J. C., and Argus, M. F. (1988). Safrole, estragole. and related compounds. In Chenriccrl Induction of Cancer, S t r ~ ~ t u rBases a l and Biological Mechanisms (Y.-T. Woo, D. Y. Lai, J. C. Arcos, and M. F. Argus, eds.) Academic Press. New York, pp. 267-3 19. Budavari, S., O’Neil. M. J., and Smith, A., eds. (1989). The Merck Index, 11th Edition, Merck and Co., Rahway, New Jersey, p. 1322. Segelman, A. B., Segelman, F. P., Karliner, J., and Sofia, D. (1976). Sassafras and herb tea, J. Am. Med. Assoc., 236:477. Johns, A. N. (1980). Beware the bay leaf, BY. Med. J., 281:1682. Panzer. P. E. (1983). The dangers of cooking with bay leaves. J. Am. Med. Assoc.. 250: 164-165. Farkas. J. (.1981).Perioral dermatitis from majoram, bay leaf and cinnamon, Colztcrct Dermatitis, 7: 121. Duke, J. A. (1988). Bishop’s weed (Avrnli nrajcrs L., Apiaceae), Notes on economic plants, Ecofz. Bot., 42~442-445. Duke, J. A. (1988). Father nature’s farnlacy: The carrot family, HerbalCram, 15:ll. Hamerski. D., Beier, R. C., Kneusel, R. E., Matem. U., and Himmelspach, K. ( 1990). Accumulation of coumarins in elicitor-treated cell suspension cultures of Ammi majus, Phytochemist?, 29: l 137-1 142. Beier, R. C., Ivie, G. W., and Oertli, E. H. (1994). Linear furocournarins and graveolone from the common herb parsley, Phytochentistry, 36:869-872. Ivie, G. W. (1978). Linear furocoumarins (psoralens) from the seed of Texas Amwi nrnjus L. (Bishop’s weed), J. Agric. Food Chenr., 26:1394-1403. Lerman, S. (1986). Photosensitizing drugs and their possible role in enhancing ocular toxicity, Ophthalmology. 93:304-318. Steinmetz. M.-D., Monlin-Traffort, J.. and Rbgli, P. (1987). Transmission and scanning electronmicroscopy study of the action of sage and rosemary essential oils and eucalyptol on Ccrnclida albicans, Mycoses, 3 1:40-5 1. Gold, J., and Cates. W., Jr. (1980). Herbal abortifacients.J. Aln. Med. Assoc., 243: 1365-1366. Conway, G. A., and Slocumb, J. C. (1979). Plants used as abortifacients and emmenagogues by Spanish New Mexicans, J. Et~aropllariilncol.,1231-261. Osol, A.. and Farrar, G. E. (1947). The Dispematonl of the Utlitecl Stcrtes of America, Volume 24. J. B. Lippincott, Philadelphia, pp. 334-335. Truitt, E. B., Jr., Callaway, E., Braude, M. C., and Krantz, J. C., Jr., (1961). Thepharmacology of myristicin, a contribution to the psychopharmacology of nutmeg, J. Neurupsychicttn. 2:205-3,10. Archer, A. W. (1988). Determination of safrole and myristicin in nutmeg and mace by highperformance liquid chromatography, J. Chrom, 338: 117-1 21. Beier. R. C. (1994). Role of metabolism and the toxic response of some naturally occurring chemicals in food. In Progress in Pesticide Biochemistlv crrrd Toxicology, Vol. 8: The Mammdiarr Mercrbolisrir of Agruchenricals (D. H. Hutson and G. D. Paulson, eds.). John Wiley and Sons, Ltd., Sussex, England, pp. 309-332. Pyysalo. H. (1976). Identification of volatile compounds in seven edible fresh mushrooms, Acta Chem. Scancl. B, 30235-244.

160

Beier and Nigg

456. Toth, B. (1979). Hepatocarcinogenesis by hydrazine mycotoxins of edible mushrooms, J. Toxicol. Emliron. Health, 5: 193-202. 457. Levenberg, B. (1961). Structure and enzymatic cleavage of agaritine, a phenylhydrazide of L-glutamic acid isolated from Agaricaceae, J. Arm Chem. SOC.,83:503-504. 458. Chauhan, Y.. Nagel, D., Issenberg, P., and Toth, B. (1984). Identification of p-hydrazinobenzoic acid in the commercial mushroom Agaricus bisponls, J. Agric. Food Clzen~.,33: 1067-1069. 459. Chauhan, Y., Nagel, D., Gross, M., Cerny, R.. and Toth, B. (1985). Isolation of N ’ - [ ~ L (+)-glutamyl]-4-carboxyphenylhydrazine in the cultivated mushroom Agaricus bispor~s. J. Agric. Food Chenz., 33:817-820. 460. Kelly, R. B., Daniels. E. G., and Hinman, J. W. (1962). Agaritine: Isolation, degradation, and synthesis, J. Org. Clzem., 27:3229-323 1. 461. Ross, A. E., Nagel. D. L., and Toth, B. (1982). Occurrence, stability and decomposition of E N - [ YL( + )-glutamyl]-4-hydroxyn~ethylphenylhydrazine(agaritine) from the mushroom Agaricus bisporus, Food Chenz. Toxicol., 20:903-907. 462. Ross, A. E., Nagel, D. L., and Toth. B. (1982). Evidence for the occurrence and formation of diazonium ions in the Agaricus bisporlw mushroom and its extracts, J. Agric. Food Clzern., 30:521-535. 463. Toth, B., and Sornson, H. (1984). Lack of carcinogenicity of agaritine by subcutaneous administration in nlice, Mycopatlzologin. 85:75-79. 464. Toth, B., Raha, C. R., Wallcave, L., and Nagel. D. (1981). Attempted tumor induction with agaritine in mice, Anticarzcer Res., 1:255-258. 465. Gigliotti, H.. and Levenberg, B. (1964). Studies on the y-glutamyltransferase of Agariczls bisporus, J. Biol. Chem., 2392274-2284. 466. Toth, B.. and Nagel, D. (1981). Studies of the tumorigenic potential of4-substituted phenylhydrazines by the subcutaneous route, J. Tos-icol. Emirorl. Heultlz, 8:l-9. 367. Toth, B., Nagel, D.. and Ross. A. (1980). Occurrence and the carcinogenic action of 4(hydroxymethy1)benzene diazonium ion (4-HMBD), Proc. Anz. Assoc. Cancer Res.. 21 :73. 468. Toth. B. (1983). Carcinogens in edible mushrooms. In Carcinogens and Mutagens in the E~~vironn~erzt: Natzudy Occurring Compounds, Volume 3 (H. F. Stich, ed.), CRC Press, Boca Raton. Florida, pp. 99-108. 469. Toth, B., Nagel, D., and Ross, A. (1982). Gastric tumorigenesis by a single dose of 4(hydroxymethyl)benzenediazonium ion of Agaricus bisporlas. Br. J. Cancer, 46:417-422. 470. Toth, B., Patil, K., Jae, H.-S. (1981). Carcinogenesis of 4-(hydroxymethy1)benzenediazonium ion (tetrafluoroborate) of Agaricus bisporus, Cancer Res., 422444-2449. 471. Toth, B. ( 1987). Cancer induction by the sulfate form of 3-(hydroxymethyl)benzenediazonium ion of Agaricus bisporus, 111 Vivo, 1:39-42. 372. Franke, S., Freimuth, U.. and List, P. H. (1967). Ueber die Giftigkeit der Friihjarhrslorchel. Gyromitra (Helvella) esculerzta Fr.. Arch. To.\-icol., 32293-332. 473. Pyysalo, H., and Niskanen, A. ( 1977). On theoccurrence of N-methyl-N-formylhydrazonesin fresh and processed false morel. Gyronlitra esculerzta, J. Agric. Food Chem.. 25:644-647. 474. Schmidlin-Miszaros, Von J. (1975). Sind die getrockneten Lorcheln Gyrontitrcr (Helvella) esclrlenta, ungiftig? Scl~weic.2. Pilzh-.. 53: 106-1 11. 475. Witkin. L. B. (1956). Acute toxicity of hydrazine and some of its methylated derivatives, Am. Med. Assoc. Arch. I d . Health, 13:34-36. 476. Wright, A. V., Niskanen, A.. Pyysalo, H., and Korpela. H. (1978). The toxicity of some N-methyl-N-formylhydrazones from Gyrornitra esczdenta and related compounds in mouse and microbial tests, Toxicol. Appl. Pharmcrcol., 45:429-434. 477. Mikinen, S. M., Kreula, M., and Kauppi, M. (1977). Acute oral toxicity of ethylidene gyromitrin in rabbits, rats and chickens, Food Cosnzet. Toxicol., 15575-578. 478. Schmidlin-Misziros, J. (1973). Gyromitrin in Trockenlorcheln (Gyromitra esczdenta sicc.). Mitt. Geb. Lebells~?zittellfnters.Hyg.. 65:453-465.

Naturally Occurring Toxic Chemicals in Foods 479. 480. 481. 482.

483. 484. 485.

486. 487.

488. 489. 490. 491. 492.

493. 494. 495.

496. 497. 498. 499. 500. 501. 502. 503. 504.

161

Braun, R., Greeff, U., and Netter, K. J. (1980). Indications for nitrosamide formation from the mushroom poison gyromitrin by rat liver microsomes, Xenobioticn. 10:557-564. Pyysalo, H., Niskanen, A., and von Wright, A. (1979). Formation of toxic methylhydrazine during cooking of false morels, Gyromitra esculenta, J. Food Sa$, 1:295-299. Von Der Hude, W., and Braun, R. (1983). On the mutagenicity of metabolites derived from the mushroom poison gyronitrin, Toxicology, 26: 155-160. Huisgen, R., and Reinilinger, H. (1956). Nitroso-acyl-amine und diazoester. X. Die isomerisierung der nitroso-acyl-allcylarnine zu diazo-estern und ihre kinetik, Justzts Liebigs Ann. Chern., 599: 161- 182. Goldblatt, L. A. (~1972).Implications of mycotoxins, Clin. Tosicol., 5:453-464. Bullerman, L. B. (.1979). Significance of mycotoxins to food safety and human health, J. Food Prot., 42:65-85. Hesseltine, C. W. (1979). Introduction, definition, and history of mycotoxins of importance to animal production. In Intemctiom of Mycotoxins in Aniinnl Production, National Academy of Science, Washington, D.C., pp. 3-18. Schoental, R. (1984). Mycotoxins and the Bible, Perspect. Biol. Med., 28:117-120. Rutledge, A. E. (1977). The public health significance of mycotoxins, A m . Nlctr. Aliment., 31:1019-1030. Lorenz. K. (1979). Ergot on cereal grains, CRC Crit. Rev. Food Sci. Nutr., 11:311-354. Linsell, C. A. (1977). The mycotoxins and human health hazards, A m . Nutr. Aliwelzt., 31: 997- 1004. Hayes, A. W. (1980). Mycotoxins: A review of biological effects and their role in human diseases, Clin. Toxicol.. 17:45-83. Pollock, G. A. (1982). Mycotoxins in animal feeds, Mod. Vet. Prac.. 4:285-287. Kubena, L. F., Harvey, R. B., Phillips, T. D.. and Heidelbaugh, N. D. (1987). Novel approach to the preventive management of aflatoxicosis in poultry. In Proceedings of the Ninety-First Annual Meeting Uizited States Arzimd Health Association, United States Animal Health Association. Lewis Printing Company, Richmond, VA, pp. 302-304. Hamilton, P. B. (1982). Mycotoxins and farm animals. Ref.. Vet., 39: 17-45. Hesseltine, C. W. (1985). Global significance of mycotoxins. In Mycotoxins and P1zycoto.xins (P. S. Steyn and R. Vleggaar, eds.). pp. 1-18. Urano, T.. Trucksess. M. W., Beaver, R. W., Wilson. D. M., Dorner, J. W., and Dowell, F. E. (1992). Co-occurrence of cyclopiazonic acid and aflatoxins in corn and peanuts, J. Assoc. OffAnal. Chern., 75:838-841. Urano, T., Trucksess, M. W., Matusik, J., and Dorner, J. W. (1992). Liquid chromatographic detemination of cyclopiazonic acidin corn and peanuts,J. Assoc. OffAnd. Chem.. 75:319-322. Schlatter, C. (1988). The importance of mycotoxins in foods, Bibl. Nutr. Dieta, 31:55-65. in Sorenson, W. G. (1990). Mycotoxins as potential occupational hazards, De~~elop~nents Industrial Microbiology. 31:205-211. (Reprinted in J. Ind. Microbiol., Supp. No. 5.) Fritz, W., and Engst, R. (1981). Survey of selected mycotoxins in food. J. Emiron. Sci. Health, B 16:193-1210. Stoloff, L. (1982).Mycotoxins as potential environmental carcinogens. In Ccrrcinoge~tsam1 Mutagens in the Enlirortment, Volume 1, CRC Press, Boca Raton, Florida, pp. 97-120. Fu-Sun, Y., and Kong-Nien, S. (1986). Epidemiology and early diagnosis of primary liver cancer in China, Adv. Cancer Res., 47:297-329. World Health Organization (1987). Evaluation of certain food additives and contaminants, World Health Organization, Technical Report Series, 759: 1-53. Mannon, J., and Johnson. E. (1985). Fungi down on the farm, New Sci., 105:12-16. Hesseltine, C. W. (1976). Conditions leading to mycotoxin contamination of foods and feeds. In Mycotoxins and Other Fungal Related Food Problerm, Volume 149 (J. V. Rodricks, ed.), ACS Advances in Chemistry Series, American Chemical Society, Washington, D.C., pp. 1-22

.

162

Beier and Nigg

505. Wilson, B. J. (1978). Hazards of mycotoxins to public health, J. Food Prot., 41:375-384. 506. Rutledge, A. E. (1976). The environmental-public health significance of mycotoxins, Proceedings of the1st International Symnposium 011 Feed Composition,Animal Nmient Requirements, and Computerizatiorl of Data, Utah State University. Logan. Utah, pp. 345-350. 507. Rao, G. J., Govindaraju, G., Sivasithanlparam, N., and Shanmugasundaram, E. R. B. (1982). Uptake, translocation and persistence of mycotoxins in rice seedlings, Plant Soil, 66: 121-123. 508. Akkmeteli, M. A. (1977). Epidemiological features of the mycotoxicoses, Ann. Nutr. Aliment., 3 1:957-976. 509. Caster, W. O., Burton, T. A., Irvin, T. R.. and Tanner. M. A. (1986). Dietary aflatoxins, intelligence and school performance in Southern Georgia, Int. J. Vitanz.Nutr. Res., 56291295. 5 10. Brown, J. (1981). Care of the mentally handicapped, East Afi: Med. I., 58:469-471. 511. Denning, D. W. (1987). Aflatoxin and human disease, Adverse Drug React. AcutePoisoning Rely.. 4: 175-209. 512. Bourgeois, C. H., Shank, R. C., Grossman. R. A., Johnsen, D. 0.. Wooding, W. L., and Chandavimol, P. (1971). Acute aflatoxin B toxicity in the macaque and its similarities to Reye’s syndrome, Lab. Irlrlest.. 24206-216. 513. Shank, R. C., Bourgeois, C. H., Keschamras. N., and Chandavimol. P. (1971). Aflatoxins in autopsy specimens from Thai children with an acute disease of unknown aetiology, Food Costnet. To.vicol., 9501-507. 5 14. Greenhalgh, R., Blackwell, B. A., Pare, J. R. J., Miller, J. D., Levandier, D., Meier, R.M., Taylor, A., and ApSimon, J. W. (1985). Isolation and characterization by mass spectrometry and NMR spectroscopy of secondary metabolites of some Fusuriunz species. In Mycotoxins and Phycotoxins (P. S. Steyn and R. Vleggaar, eds.), Elsevier Science Publishers B. V., Amsterdam, pp. 22-25. 515. Schoental, R. (1977). Health hazards due to oestrogenic mycotoxins in certain foodstuffs, bzt. J. Emirow Stud., 1 1:149- 150. 516. Wallace, R. B., Hoover, J., Sandler, D.. Rifkind, B. M.. and Tyroler, H. A. (1977). Altered plasma-lipids associated with oral contraceptive or oestrogen consumption: The lipid research clinic program. Lancet, 2: 11-14. 517. Blackard, C. E., Doe, R. P., Mellinger, G. T.. and Byar, D. P. (1970).Incidence of cardiovascular disease and death in patients receiving diethylstilbestrol for carcinoma of the prostate, Cancel-, 26249-256. 518. Schoental, R. (1976). Hazards of oral contraceptives to future generations. h t . J. Ewiron. Stud.. 9:8 1. 5 19. Sullivan, J. M.. Zwaag. R. V.. Hughes, J. P., Maddock, V., Kroetz. F. W., Ramanathan, K. B., and Mirvis, D. M. (1990). Estrogen replacement and coronary artery disease: Effect on survival in postmenopausal women, Arch. Irzt. Med., 150:2557-2562. 520. Barrett-Connor, E., and Bush, T. L. (1989). Estrogen replacement and coronary heart disease, Curdiovas. Cliu., 19:159-172. 521. Ernster, V. L., Bush, T. L., Huggins, G. R., Hulka, B. S.. Kelsey, J. L., and Schottenfeld, D. (1988). Benefits and risks of menopausal estrogen and/or progestin hormone use, Pr-ev. Med., 17901-223. 522. Schoental, R. (1981). Mycotoxins and fetal abnormalities, Int. J. Emiron. Sttrd., 17:25-29. 523. Mirocha. C. J., Pathre, S. V., and Christensen, C. M. (1977). Zearlenone. In Mycotoxins i n H~mzana d Arzirtzrrl Health (J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, eds.), Pathotox Publishers, Park Forest South, Illinois, pp. 345-364. 524. Tuite, J.(1977). Mycology of mycotoxic fungi, The genus Aspergillus. In Mycotoxic F ~ m g i , Mycoto.~ins,and M~coto,~icoses, Volume 1 (T.D. Wyllie and L. G. Morehouse, eds.), Marcel DekEcer, New York, pp. 21-39. 525. Vederas, J. C. (1985). Biosynthetic studies on mycotoxins using multiple stable isotope

Naturally Occurring Toxic Chemicals Foods in

526. 527. 528. 529.

530. 531. 532.

533.

534.

535. 536. 537. 538.

539. 540.

541. 542.

543.

544.

545.

163

labelling and NMR spectroscopy. In Mycotoxins and Phycotoxins. (P. S. Steyn and R. Vleggaar, eds.), Elsevier Science Publishers B. V.. Amsterdam, pp. 97-108. Harris, C. C., and Tsung-Tang, S. (1986). Interactive effects of chemical carcinogens and hepatocellular carcinoma, Cancer Sun?., 5:765-780. Kew, M. C. (1986). The development of hepatocellular cancer in humans. Cancer Szm)., 51719-739. Bababunxni. E. A. (1978). Toxins and carcinogens in the environment: An observation in the tropics, J, Toxicol. Erzviron. Health, 4:691-699. Li, M., Lu, S., Ji, C., Wang, Y., Wang. M., Cheng, S., and Tian, G. (1980). Experimental studies on the carcinogenicity of fungus contaminated food from Linxian county. In Genetic and Emironmental Factors in Experirtzental and Hznnnn Culrcer (H. V. Gelboin, ed.), Jpn. Sci. SOC.Press, Tokyo, Japan. pp. 139-148. Lin, P., and Tang, W. (1980). Zur epidemiologie und atiologie des oesophaguscarcinoms in China, J. Cancer Res. Clin. Oncol., 96: 121-130. Yang, C. S. (1980). Research on esophageal cancer in China: A review, Cancer Res., 40: 2633-2644. Harrison, L. R.. Colvin, B. M., Greene, J. T., Newman, L. E., and Cole, J. R. ( 1990). Pulmonary edema and hydrothorax in swine produced by fumonisin B ], a toxic metabolite of Fr4sarizm monilijornze, J. Vet. Diagiz. h e s t . , 2210-212. Kriek, N.P. J., Marasas, W. F. 0..and Thiel, P. G. (1981). Hetapo- and cardiotoxicity of Fzwwizrriz verticillioides (F. ~tzoniliforme)isolates from southern African maize. Food Cosmet. Toxicol., 19:437-456. Kriek. N. P. J., Kellerman, T. S., and Marasas, W. F. 0. (1981). A comparative study of the toxicity of F~rsariurt~ Ilerticillioides (= F. moniliforn?e)to horses, primates, pigs, sheep, and rats, Onderstepoort J. Vet. Res.. 48: 129-13 1. Engelhardt, J. A., Carlton, W. W., and Tuite, J. F. (1989).Toxicity of Fusariunz rtzonil(forrne var Subglrctinans for chicks, ducklings and turkey poults, Avian Dis., 33:357-360. Jeschke. N., Nelson, P. E., and Marasas, W. F. 0. (1987). Toxicity to ducklings of Fzrsurizrm nzoniliforme isolated from corn intended for use in poultry feed, Poult. Sci.,66:1619-1623. Fritz, J. C., Mislivec, P. B., Pla, G. W., Harrison, B. N., Weeks, C. E., and Dantzman, J. G. (1973). Toxicogenicity of moldy feed for young chickens, P014lt. Sci., 521523-1530. Maruanovic, D. R., Holt, P., Norred, W. P., Bacon. C. W., Voss, K. A., Stancel, P. C., and Ragland. W. L. (1991). Immunosuppressive effects of Fz4sarizm rtzoniliformecorn cultures in chickens, Poult. Sci., 70:1895-1901. Sharby, T. F., Templeton, G. E., Beasley, J. N., andStephenson, E. L. (1973). Toxicity resulting from feeding experimentally molded corn to broiler chicks, Poult. Sci., 5 2 1007-1014. Gelderblom, W. C. A., Jaskiewicz, K., Marasas, W. F. O., Thiel, P. G., Horak, R. M., Vleggaar, R., and Kriek, N.P. J. (1988). Fumonisins-Novel mycotoxins with cancerpromoting activity produced by Fusrtriutn monilijorine, Appl. Emiron. Microbiol., 54: 1806-1811. Jaskiewicz, K., van Rensburg.S. J., Marasas, W. F. O., and Gelderblom.W. C. A. (1987). Carcinogenicity of FLrsariunz monilifontze culture materialin rats, J. Nat. Carlcer Instit., 78:321-325. Marasas, W. F. O., Kriek, N. P. J.. Fincham, J. E., and van Rensburg, S. J. (1984). Primary liver cancer and oesophageal basal cell hyperplasia in rats caused by Fusm-iunz monilifomte. Irzt. J. Cancer, 34:383-387. Norred, W. P.. Plattner, R. D., Voss. K. A., Bacon, C. W., and Porter, J. K. ( 1989). Natural occurrence of fumonisins in corn associated with equine leukoencephalomalacia (ELEMI, Toxicologist, 9:258, Abstract No. 1084. van Rensburg, S. J., Marasas, W. F. O., Gelderblom, W. C. A., Thiel, P. G., and Rabie, C. J. (.1982). Mycotoxins and oesophageal cancer, Proc. Vth lilt. IUPAC Synp. Mycotoxins and Plzycoto.~ins,Vienna, pp. 265-268. Voss, K. A., Norred, W. P.. Plattner, R. D., and Bacon, C. W. (1989). Hepatotoxicity and

164

546.

547. 548. 549. 550. 551. 552. 553. 554. 555. 556. 557. 558. 559. 560. 561. 562.

563.

564. 565. 566.

567.

Beier and Nigg renal toxicity in rats of corn samples associated with field cases of equine leukoencephalomalacia, Food Clzem. Tosicol., 27:89-96. Wilson, T. M., Nelson, P. E., and Knepp, C. R. (1985). Hepatic neoplastic nodules, adenofibrosis. and cholangiocarcinomas in male Fisher 344 rats fed corn naturally contaminated with F~rsariummoniliforme, Carcilzogenesis, 6: 1155-1 160. Jaskiewicz, K., Marasas, W. F. O., and Taljaard, J. J. F. (1987). Hepatitis in vervet monkeys caused by FLrsariunz nzonilifome, J. Conzp. Potlzol., 97:281-291. Schoental, R. (1980). Disorders and tumors associated with alcoholic drinks and Mycotoxins. Nutr. Cancel-. 288-92. Aleksandrowicz, J., and Smyk. B. (1973). Theassociation of neoplastic diseases and mycotoxins in the environment, Tes. Rep. Biol. Med., 3 1:715-726. Wray, B. B., and O'Steen, K. G. (1975). Mycotoxin-producing fungi fromhouse associated with leukemia, Ar-clz. E1zviroIz. Health, 30:571-573. Abramovici, A. (1977). Mycotoxins and abnormal fetal development, ContribzltioizsMicr-obiol. I n ~ ~ n u ~ 3:s o l .1-94. , Henderson, B. E., Benton. B., Jing. J., Yu, M. C., and Pike, M. C. (1979). Risk factors for cancer in the testis in young men, Znt. J. Cancer, 23:598-602. Schoental, R. (1977). The role of nicotinamide and of certain other modifying factors in diethylnitrosmine carcinogenesis, Concer, 40: 1833- 1840. Gilman, A. G.. Goodman, L. S., and Gilman. A. (1980). The Phrr~~~~zcrcologiccrl Bcrsis of Therapeutics, 6th Edition, Macmillan, New York. Cordell, G. A. (198 1). Ergot alkaloids. In Irztrod~rctionto Alkaloids: ABiogerzetic Approach, John Wiley and Sons. New York. pp. 622-654. Scott, P. M., Lonlbaert, G. A., Pellaers, P., Bacler, S., and Lappi, J. (1992). Ergot alkaloids in grain foods sold in Canada, J. Assoc. OffAnal. Clter~.,75:773-779. Groger, D. (1972). Ergot. In Microbial Toxiru, Volume 8 (S. Daidis, A. Ciegler, and S. J. Ajl, eds.), Academic Press, New York, pp. 321-373. Thompson, F. N.. and Porter, J. K. (1990). Tall fescue toxicosis in cattle: Could there be a public health problem here'? Vet. Hzrmnn Tosicol., 32. (Suppl.):51-57. Bacon, C. W., Porter, J. K., Robbins, J. D.. and Luttrell. E. S. (1977). Epichloe Qphina from toxic tall fescue grasses, Appl. Emiron. Microbiol.. 34:576-581. Cunningham. I. J.. McIntosh. I. C., and Swan. J. B. (1944). The nontoxicity of milk and meat from ergotized cattle, N. Z. J. Sci. Technol., 26:125-136. Booth. C. (1971). The Gerzlrs Fusarium, Commonwealth Mycological Institute, Kew, Surrey, England. Marasas, W. F. 0..Nelson, P. E., and Toussoun, T. A. (1984). Toxigenic Fusarium Species Identih and Mycotoxicology, Pennsylvania State University Press. University Park, Pittsburgh, p. 216. Marasas. W. F. 0.. van Rensburg, S. F., and Mirocha, S. J. (1979). Incidence of Fusarium species and the mycotoxins. deoxynivalenol and zearalenone, in corn produced in esophageal cancer areas in Transkei. J. Agr-ic. Food Chew., 27: 1108-1 112. Rose. E. F., and Fellingham, S. A. (1981). Cancer patterns in Transkei, South Africa, Afr: J. Sci., 77:555-561. Schoental. R., and Joffe, A. Z. (1974). Lesions induced in rodents by extracts from cultures of Fuscrriunz pone and F. sporotrichioides, J. Pcrthol.. 112:37-42. Schoental, R., Joffe, A. Z., and Yagen, B. (1976).Chronic lesions in rats treated with crude and F. sporotrichioides. The role of mouldy food in the incidence extracts of F~rsari~nz poae of oesophageal, mammary and certain other abnormalities and tumours in livestock and man, Br. J. Cancer. 34:310. Schoental, R., Joffe, A. Z., and Yagen, B. (1978). The induction of tumours of the digestive tract and of certain other organs i n rats given T-2 toxin: A secondary metabolite of Fl~so~.imz sporotrichioides, BI: J. Cmzcer, 38: 171.

Naturally Occurring Toxic Chemicals in Foods

165

568. Marasas, W. F. 0. (1982). Mycotoxicological investigations on corn produced in oesophageal cancer areas in Transkei. In Cancer of the Oesophagus, Volume 1 (C. J. Pfeifer, ed.), CRC Press, Boca Raton, Florida, pp. 29-40. 569. Marasas, W. F. O., Wehner. F. C.. van Rensburg, S. J., and van Schalkwyk, D. J. (1981). Mycoflora of corn produced in human oesophageal cancer areas in Transkei, southern Africa, Phytopathology, 7 1:792-796. 570. Wilson, B. J., and Maronpot, R. R. (1971). Causative fungal agent of leucoencephalonlalacia in equine animals, Vet. Rec., 88:484-485. 571. Wilson, B. J., Maronpot, R. R., and Hildebrandt, P. K. (1973). Equine leukoencephalomalacia, J. Am. Vet. Med. Assoc., 163:1293-1295. 572. Kellerman, T. S., Marasas, W. F. O., Pienaar, J. G., and Naude, T. W. ( 1 9 7 3 Mycotoxicosis of equidae caused by Fusariunz monilifornle Sheldon. A preliminary communication, Orzderstepoort J. Vet. Res., 39905-208. 573. Pienaar, J. G., Kellerman. T. S., and Marasas, W. F. 0. (1981). Field outbreaks of leukoencephalomalacia in horses consuming maize infected by Fusarizmz Iferticillioides (F. moniliforrne) in South Africa, J. South Afr. Vet. Assoc., 5221-24. 574. Buck, W. B., Haliburton, J. C., Thilsted, J. P., Lock. T. F., and Vesonder, R. F. (1979). Equine leukoencephalomalacia: Comparative pathology of naturally occurring and experimental cases, Proc. Ann. Meet. Am. Assoc. Vet. Lab. Diagn., 22239-258. 575. Wilson, T. M., Nelson, P.E., Ryan, T. B., Rouse. C. D., Pittman, C. W.. Neal, T. P., Porterfield, M. L., and Saunders, G. K. (1985). Linking leukoencephalomalacia to commercial horse rations, Equine Prac., 80:63-69. 576. Haliburton, J. C., and Buck, W. B. (1986). Equine leukoencephalomalacia, Curr. Top. Vet. Med. Aninl. Sci., 33175-79. 577. Marasas, W. F. O., Kellernlan, T. S., Gelderblom, W. C. A., Coetzer, J. A. W., Thiel, P. G., and van der Lugt. J. J. (1988).Leucoencephalomalacia in a horse induced by fumonisin B l isolated from Fzuarium nlonil(forme, Onderstepoort J. Vet. Res., 55:197-203. 578. McCue, P. M. (1989). Equine leukoencephalomalacia, Compendium, 11:646-65 1. 579. Wilson, T. M., Ross, F. P., Rice, L. G.. Osweiler, G. D., Nelson, H. A., Owens, D. L., Plattner, R. D., Reggiardo, C., Noon, T. H., and Pickrell, J. W. (1990). Fumonisin B I levels associated with an epizootic of equine leukoencephalomalacia. J. Vet. Diagn. Znl-est., 2: 213-216. 580. Thiel. P. G., Shephard, G. S., Sydenham, E. W., Marasas, W. F. O., Nelson. P. E.. and Wilson, T. M. ( I 991). Levels of fumonisins B I and B2 in feed associated with confirmed cases of equine leukoencephalomalacia. J. Agric. Food Cheul., 39: 109-1 11. 581. Sydenham, E. W., Marasas, W. F. O., Shephard. G. S., Thiel, P. G., and Hirooka, E. Y. (1992). Fumonisin concentrations in Brazilian feeds associated with field outbreaks of confirmed and suspected animal mycotoxicoses, J. Agric. Food Chenl.. 40:994-997. 582. Gelderblom, W. C. A.. Thiel, P. G., van der Merwe, K. J., Marasas, W. F. O., and Spies, H. S. C. (1983). A mutagen produced by Fusarium nloniliforme, Toxicon, 21:467-373. 583. Gelderblom, W. C. A., Marasas, W. F. O., Steyn, P. S., Thiel, P. G., van der Merwe, K. J., van Rooyen, P. H.. Vleggaar, R., and Wessels, P. L. (1984). Structure elucidation of fusarin C, a mutagen produced by Fzlsariun~nloniliforme, J. Chern. SOC.,Chem. Comnt.. 122-124. 584. Gelderblom, W. C. A., Thiel, P. G., Jaskiewicz, K., and Marasas, W. F. 0. (1986). Investigations on the carcinogenicity of fusarin C-A mutagenic metabolite of Fusarizan ntoniliforme, Carcinogenesis, 7: 1899-1901. 585. Shier, W. T., Abbas, H. K., and Mirocha, C. J. (1991). Toxicity of the mycotoxins fumonisins B and B, and Alternaria alterr~ataf. sp. Zycopersici toxin (AAL) in cultured mammalian cells, Mycopnthologia, 116:97-104. 586. Yoo. H.-S., Norred. W. P., Wang, E., Mel-rill, A. H., Jr., and Riley, R. T. ( 1 992). Fumonisin inhibition of de novo sphingolipid biosynthesis and cytotoxicity are correlated in LLC-PK cells, Tosicol. Appl. Pllarnlacol., 114:9-15.

,

166

Beier and Nigg

587. Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991). Inhibition of sphingolipid biosynthesis by fumonisins: Implications for diseases associated with Fusarium ntoniliforwe, J. Biol. Chem., 266: 14486-14490. 588. Wang, E., Ross, P. F., Wilson, T. M., Riley, R. T., and Merrill, A. H., Jr. (1992). Increases in serum sphingosine and sphinganine and decreases in complex sphingolipids in ponies given feed containing fumonisins,mycotoxins produced by Fusarium ntoizilifornze, J. Nutr., 122:1706-1716. 589. Riley, R. T., An, N.-H., Showker, J. L.. Yoo, H.-S., Norred, W. P.. Chamberlain, W. J., Wang, E.. Merrill,A.H., Jr., Motelin, G., Beasley, V.R.,andHaschek,W. M. (1993). Alteration of tissue and serum sphinganine to sphingosine ratio: An early biomarker of exposure to fumonisin-containing feeds in pigs, Toxicol. Appl. Pltannucol., 118: 105-1 12. 590. Bezuidenhout, S. C.. Gelderblom, W. C. A., Gorst-Allman, C. P., Horak, R. M.. Marasas. W. F. O., Spiteller, G., and Vleggaar, R. (1988). Structure elucidation of the fumonisins. mycotoxins from Fusurium moniliforme, J. Chem. SOC.,Chenl. Coitlm., 733-745. 591. Plattner, R. D., Weisleder, D., Shackelford, D. D., Peterson, R., and Powell, R. G. ( 1992). A new fumonisin from solid cultures ofF~~snrium monilifornze, Mycoparhologiu, 117:23-28. 592. Cawood, M. E., Gelderblom. W. C.A., Vleggaar, R.,Behrend, Y., Thiel, P. G., andMarasas, W. F. 0. (1991 ). Isolation of the fumonisin mycotoxins: A quantitative approach. J. Agric. Food Chent., 39:1958-1962. 593. Sydenham, E. W.,Gelderblom. W. C. A., Thiel, P. G., andMarasas, W. F. 0. (1990). Evidence for the natural occurrence of fumonisin B ,, a mycotoxin produced by Ftumizm nzoniliforme, in corn. J. Agric. Food Chenz., 38:285-290. 593. Ross, P. F., Rice, L. G.. Plattner, R. D.. Osweiler, G. D.. Wilson, T. M.. Owens. D. L., Nelson, H. A.,and Richard, J. L. (1991). Concentrationsof fumonisin BI in feeds associated with animal health problems, Mycopathologiu, 114: 129-135. 595. Ross, P. F., Rice, L. G., Reagor. J. C., Osweiler. G. D., Wilson, T. M., Nelson. H. A., Owens, D. L., Plattner, R. D.. Harlin, K. A., Richard, J. L., Colvin, B. M., and Banton, M. I. (1991). Fumonisin B I concentrations in feeds from confirmed 45 equine leukoencephalomalacia cases, J. Vet. Diagil. Imv.. 3238-241. 596. Scott. P. M., and Lawrence, G.A. (1992). Liquidchromatographic determinationof fumonisins with 4-fuora-7-nitrobenzofurazan, J. Assoc. Of Anal. Chew., 75:829-834. 597. Shephard, G.S., Sydenham, E. W., Thiel,P. G., and Gelderblom,W. C. A. (1990). Quantitative determination of fumonisins B I and B2 by high-performance liquid chromatography with fluorescence detection, J. Liquid Chroonzatogr.. 132077-2087. 598. Stack, M. E., and Eppley, R. M. (1992). Liquid chromatographic determination of fumonisins B , and B7 in corn and corn products, J. Assoc. 08 A n d . CIzenl., 75:834-837. 599. Plattner,R. D.,Norred, W.P.,Bacon,C. W.. Voss, K. A.,Peterson,R., Shackelford, D. D., and Weisleder. D. (1990). A method of detection of fumonisins in corn samples associated with field cases of equine leukoenceyhalomalacia, Mycologia, 82:698-702. 600. Plattner, R. D., Ross, P. F., Reagor, J., Stedelin, J., and Rice, L. G. (1991). Analysis of corn and cultured corn for fumonisin B I by HPLC and G U M S by four laboratories, J. Vet. Dicqn. Iml., 3:357-358. 601. Korfmacher, W. A., Chiarelli, M. P., Lay, J. O., Jr., Bloom, J., Holcomb,M.. and McManus, K. T. (1991). Characterization of the mycotoxinfumonisin B,: Comparison of thermospray, fast-atom bombardment and electrospray mass spectrometry. Rcipid Contrtt. Mass Spec.. 5 : 463-468. 602. Shephard. G. S., Thiel, P. G., and Sydenham, E. W. (1992). Determination of fumonisin B in plasma and urine by high-performance liquid chromatography, J. Chronzutogr., 574: 299-304. 603. Azcona-Olivera, J. I.. Abouzied, M. M., Plattner, R. D., and Pestka, J. J. (1992). Production of monoclonal antibodies to the mycotoxins fumonisins B I. B2, and B i. J. Agric. Food Chew., 4053 1-534.

Naturally Occurring Toxic Chemicals in Foods

167

604. Gelderblonl, W. C. A., Marasas, W. F. O., Vleggaar, R., Thiel, P. G., and Cawood, M. E. (1992). Fumonisins: Isolation, chemical characterization and biological effects, Mycopthologiu, 117:1 1 16. 605. Salaman, R. N.(1985). The Histon crnd Sociul Influeme of the Potuto, Cambridge University Press, Cambridge, England. 606. Wood, F. A.. and Young. D. A. (1974). TGA in Potatoes, Publication No. 1533, Canadian Department of Agriculture. 607. Forsyth, A. A. (1954). Brirish Poisonous Plranfs,Bulletin No. 161, Her Majesty’s Stationery Office, London, pp. 1-1 16. 608. Mondy, N.I., and Gosselin, B. ( 1 988). Effect of peeling on total phenols, total glycoalkaloids, discoloration and flavor of cooked potatoes, J. Food Sci., 53:756-759. 609. Friedman, M. (1992). Composition and safety evaluation of potato berries, potato and tomato seeds, potatoes, and potato alkaloids. In ACS Syntposium Series No. 484, Food Sufety Assessrnent (J. W. Finley, S. F. Robinson, and D. J. Armstrong, eds.), American Chemical Society, Washington, D.C., pp. 429-462. 610. Sato. K., Ishiguri, Y., Doke, N., Tomiyama, K.. Yagihashi, F., Murai. A., Katsui, N., and Masamune, T. (1978).Biosynthesis of the sesquiterpenoid phytoalexin rishitin from acetate via oxylubimin in potato, Phytoclzenristty, 17:1901- 1902. 61 1. Claringbold, W. D. B., Few, J. D., and Renwick, J. H. (1982). Kinetics and retention of solanidine in man, Xenobioticu. 12293-302. 612. Kud, J. (1972). Phytoalexins, A m . Rev. Phytopnthol., 10:207-232. 613. Allen, E. H., and Kud, J. (1968). a-Solanine and a-chaconine as fungitoxic compounds in extracts of Irish potato tubers, Phyoputhology, 58:776-781. 614. Locci, R., and KuC, J. (1967). Steroid alkaloids as conlpounds produced by potato tubers under stress, PhJvopathology, 57: 1272-1273. 615. McKee, R. K. (1955). Host-parasite relationships in the dry-rot disease of potatoes, Ann. Appl. Biol., 43: 147- 148. 616. Sinden, S. L. (1972). Effect of light and mechanical injury on the glycoalkaloid content of greening resistant potato tubers, Am. Potuto J., 49:368. 617. Jadhav, S. J., Sharma, R. P., and Salunkhe, D. K. (1981). Naturally occurring toxic alkaloids in foods, Crit. Rely. Toxicol.. 921-104. 618. Maga, J. A. (1980). Potato glycoalkaloids, CRC Crit. Rev. Food Sci. Nzrtr., 12:371-405. 619. Stoessl, A., Stothers, J. B., and Ward, E. W. B. (1976). Sesquiterpenoid stress compounds of the Solanaceae, Plrytochemist~y,15:855-872. 620. Andreae, W. A. (1948). The isolation of a blue fluorescent compound scopoletin, from green mountain potato tubers, infected with leaf roll virus, Cun. J. Res. Sect.. 26:3134. 621. Andreae, S. R., and Andreae. W. A. (1949). The metabolism of scopoletin by healthy and virus infected potato tubers, Can. J. Res., 27:15-22. 622. Shih, M. J., and Kud, J. (1974). a- and PSolamarine in Kennebec Solunzrm tuheroswn leaves and aged tuber slices, Phytochemistly, 13:997-1000. 623. Patil, B. C., Sharma, R. P., Salunkhe. D. K., and Salunkhe, K. (1973). Evaluation of solanine toxicity, Food Comet. Toxicol., 10:395-398. 624. Alozie, S. O., Sharma, R. P., and Salunkhe, D. K. (1979). Inhibition of rat cholinesterase isoenzymes in vitro and in vivo by the potato alkaloid, a-chaconine, J. Food Bioclzem., 2: 259-276. 625. Orgell, W. H.. Vaidya, K. A., and Dahm, P. A. (1958). Cholinesterase inhibition in vitro by extracts of potato, Proc. Zowu Acud. Sci., 65:160-162. 626. Orgell. W. H., Vaidya, K. A., and Dahm, P. A. (1958). Inhibition of human plasma cholinesterase in vitro by extracts of solanaceous plants, Science, 128:1136-1137. 627. Orgell, W. H. (1963). Inhibition of human plasma cholinesterase in vitro by alkaloids, glycosides, and other natural substances, Lloydiu, 26:36-43.

168

Beier and Nigg

628. Orgell, W. H.. and Hibbs, E. T. (1963). Cholinesterase inhibition in vitro by potato foliage extracts, Am. Potato J.,40:403-405. 629. Ames, B. N.. Magaw, R., and Gold, L. S. (1987). Ranking possible carcinogenic hazards, Science, 23627 1-280. 630. Smith, 0. (1977). Chemical composition of the potato. In Potatoes: Production. Storing, Processing, 2d Edition, AVI Publishing, Westport, Connecticut, pp. 59-109. 631. Sinden, S. L., and Webb, R. E. (1972). Effect of variety and location on the glycoalkaloid content of potatoes, Am. Potato J., 49334-338. 632. Friedman, M., and Dao, L. (1992). Distribution of glycoalkaloids in potato plants and commercial potato products, J. Agric. Food Clzenz., 40:419-423. 633. Wolf. M. J., and Duggar, B. M. (1946). Estimation and physiological role of solanine in the potato, J. Agric. Res., 73:l-32. 634. Griebel, C. (1924). Solanine content of potatoes of the 1932 crop, 2. Unters. Nahxngsrmd Genusswittel sowie der Gebraucltsgegaizstaencle, 47:436-438. 635. Wu, M. T., and Salunkhe, D. K. (1978). Difference between light- and wound-induced biosynthesis of a-solanine and a-chaconine in potato tubers, Biol. Plant. (Prague),20:149151. 636. Alfa, J., and Heyl. E. (1923). Potatoes of the 1922 harvest with an unusually high solanine content, Z. Untersuchung der Nul~rmgs--lmcl Gerzzlssmittel sowie der Gebrauchsgeganstnende, 46:306-309. 637. Saito, K., Horie, M., Hoshino, Y., and Nose, N. (1990). High-performance liquid chromatographic determination of glycoalkaloids in potato products, J. Chronzatogr., 508: 141147. 638. Mondy, N. I., Leja, M., and Gosselin, B. (1987). Changes in total phenolic, total glycoalkaloid, and ascorbic acid content of potatoes as a result of bruising, J. Food Sci., 52631633. Effect of wound-damages on the glyco-alkaloid con639. Ahmed, S. S., and Muller, K. (~1978). tent in potato tubers and chips, Lebensn1.-Wiss. & Teclznol.. 11:144-146. 640. Bushway, R. J., and Ponnampalam, R.(1981). 0-Chaconine and a-solanine content of potato products and their stability during several modes of cooking, J. Agr-ic. Food Cl1enl., 291814-817. 641. Bushway, R. J., Bureau, J. L., and McGann. D. F. (1983). Alpha-chaconine and alphasolanine content of potato peels and potato peel products, J. Food Sci., 48:84-86. 642. Konig, H. (1953). Untersuchungen uber solaninwirkung bei rind und schaf in1 zusamnlenhang mit kartoffelkraut-fiitterung, Schweiz. Arch. Tierlzeilkd., 95:97- 120. 643. Baker, D., Keeler, R., and Gaffield, W. (1987). Lesions of potato sprout and extracted potato sprout alkaloid toxicity in Syrian hamsters, Clin. Toxicol., 25:199-208. 644. Sinden, S. L., Sanford, L. L., and Osman, S. F. (1980). Glycoalkaloids and resistance to the Colorado potato beetle in Solmzmr clzacoerzse Bitter, Am. Potuto J., 5731-343. 645. Sinden, S. L., Sanford, L. L., and Deahl, K. L. (1986). Segregation of leptine glycoalkaloids in Solarzum chncoerzse Bitter, J. Agr-ir. Food Chew., 34:372-377. 646. Kuhn. R., and Low, I. (1961). Zur konstitution der leptine, Clzem. Bere.. 94:1088-1095 (in German). 647. Sturckow, B., and Low, I. (1961). Die wirkung einiger solanrrrta-alkaloidglykoside auf den kurtoffelkafer. Leptirzotarso decemlinenta say, Entomol. Exp. Appl., 4: 133- 142 (in German). 648. Berberich, S. (1988). Potato plants make their own insect repellent, Agric. Res., 36:15. 649. Tingey. W. M., Mackenzie, J. D., and Gregory, P. (1978). Total foliar glycoalkaloids and resistance of wild potato species to Empoascufabue (Harris), Am. Potuto J.,55577-585. 650. Lepper, W. (1949). Solanine content of 58 varieties of potatoes, 2. Lebensm-Urzters. Foysclt., 89~264-273. 65 I Berkley, S. F., Hightower, A. W., Beier, R. C., Fleming, D. W., Brokopp, C. D., Tvie.

Naturally Occurring Toxic Chemicals in Foods

652.

653. 654. 655.

656. 657. 658.

659.

660.

661.

662.

663.

664.

665. 666. 667. 668.

669. 670. 671. 672.

169

G. W., and Broome, C. V. (1986). Dermatitisingroceryworkersassociatedwithhigh natural concentrations of furanocoumarins in celery, Ann. Intern. Mer]., 105:351-355. Seligman, P. J., Mathias, C. G. T., O’Malley, M. A..Beier.R. C., Fehrs, L. J., Serrill. W. S.. and Halperin, W. E. (1987). Phytophotodermatitis from celery among grocery store workers. Arch. Dermatol., 123: 1478-1482. Childers, N. F. (1981). A Diet to Stop Arthritis, the Nightsltades rrnd Ill Health, Somerset Press, Somerville, New Jersey. Boyd, J. W., Murray, D. S., and Tyrl, R. J. (1984). Silverleaf nightshade, Solanum elrreagnifoliunr. origin, distribution, and relation to man, Econ. Bot., 38:210-217. Renwick, J. H. (1972). Hypothesis: Anencephaly and spina bifida are usually preventable by avoidance of a specific but unidentified substance present in certain potato tubers, Br. J. Prev. SOC. Med., 26:67-88. Penrose, L. S. (1957). Genetics of anencephaly, J. Ment. Dejic. Res.. 1:4-15. Floss, H. G., Guenther, H., and Hadwiger, L. A. (1969). Biosynthesis of furanocoumarins in diseased celery, Phytochemistq, 8:585-588. Scheel, L. D.,Perone, V. B., Larkin, R. L., andKupel,R. E. (1963). The isolationand characterization of two phototoxic furanocoumarins (psoralens) from diseased celery. Biocltenlistt?, 2: 1127- 1 133. Wu. C. M., Koehler, P. E., and Ayres. J. C. (1972). Isolation and identification of xanthotoxin (8-methoxypsoralen) and bergapten (5-methoxypsoralen) from celery infected with Sclerotinrr sclerotiorum, Appl. Microbiol.. 23:852-856. Smalley, E. B. (1978). Salivary syndrome in cattle. In Mycotoxic Fungi, Mycotoxins, and Mycotoxicoses (T. D. Wyllie and L.G.Morehouse, eds.). Marcel Dekker, NewYork, pp.121-141. Buck, W. B.. and Cysewski, S. J. (1986). Tremorgenictoxins. In Current Veterinap Tltera p : Food Anirttal Practice, Volume 2 (J. L. Howard, ed.), W. B. Saunders, Philadelphia, pp. 378-379. Buck, W. B., and Cysewski. S. J. (1986). Nervous form of ergotism. In Current Veterinav Therapy: Food Animal Practice, Volume 2 (J. L. Howard, ed.), W. B. Saunders, Philadelphia. p. 369. Elwood, J. H.,and MacKenzie, G. (1973). Associations between the incidence of neurological malformations and potato blight outbreaks over 50 years in Ireland, Nclture. 243:476477. Emanuel, I., and Sever, L. E. (1973). Questions concerningthe possible association of potatoes and neural-tube defects, and an alternative hypothesis relating to maternal growth and development, Teratology, 8:325-332. C. (1973).Potato blight andneural-tube defects, Lancet, 2:507Field.B.,andKerr, 508. Kinlen. L., and Hewitt, A. (1973). Potato blight and anencephalus in Scotland, Br. J. Prel,. SOC.Med., 27:208-213. McMahon, B., Yen, S., and Rothman. K. J. (1973). Potato blight and neural-tube defects, Lancet, 1598-599. Nevin, N. C., and Merrett, J. D. (1975). Potato avoidance during pregnancy in women with a previous infant with either anencephaly and/or spina bifida, Br. J. Prev. SOC.Med.. 29: 111-115. Spiers, P. S., Pietrzyk, J. J., Piper, J. M., and Glebatis, D. M. (1974). Human potato consumption and neural-tube malformation, Temtology, 10:125-128. Keeler, R. F. (1986). Teratologyof steroidal alkaloids. In Alkaloids: Chentical and Biological Perspectives (S. W. Pelletier, ed.), John Wiley and Sons, New York, pp. 389-425. Jelinek. R., Kyzlink, V., and Blattny, C., Jr. (1976). An evaluation of the embryo-toxic effects of blighted potatoes on chicken embryos, Teratology, 14:335-342. Mun, A. M., Barden, E. S., Wilson. J. M., and Hogan, J. M. (1975). Teratogenic effects

170

673. 674. 675. 676.

677. 678. 679. 680.

681.

682. 683.

684.

685. 686. 687. 688. 689. 690.

691.

692. 693. 694.

Beier and Nigg in early chick embryos of solanine and glycoalkaloids from potatoes infected with lateblight Phytophtlzorn infestans, Teratology, 11:73-78. Pierro. L. J.. Haines, J. S., and Osman, S. F. (1977). Teratogenicity and toxicity of purified a-chaconine and a-solanine, Teratology, 15:31A. Renwick, J. H. (1982). Vitamin supplementation and neural tube defects, Lancet. 1:738. Keeler, R. F., Young, S., Brown, D., Stallknecht, G. F., and Douglas, D. (1978). Congenital deformities produced in hamsters by potato sprouts, Teratology, 17:327-334. Renwick, J. H., Claringbold, W. D. B., Earthy, M. E., Few, J. D., and McLean, A. C. S. (1 984). Neural-tube defects produced in Syrian hamsters by potato glycoalkaloids. TeratolOgy, 30:371-381. Renwick, J. H. (1982). Food and malformation, Practitioner, 226:1947-1953. Zitnak, A. (1961). The occurrence and distribution of free alkaloid solanidine in netted gem potatoes, Can. J. Bioclzenz. Plzysiol., 39:1257-1265. Schrieber, K. (1968). Steroid Alkaloids: The Solanum group. In The Alkaloids. Volume 10 (R. H. F. Manske, ed.), Academic Press, New York, pp. 1-192. Keeler, R. F., Brown, D., Douglas, D. R., Stallknecht. G. F., and Young, S. (1976). Teratogenicity of the solanum alkaloid solasodine and of “Kennebec” potato sprouts in hamsters. Bull. Emiron. Contctm.To.vicol., 15:522-524. Keeler, R. F., Young, S.. and Brown, D. (1976). Spina bifida, exencephaly, and cranial bleb produced in hamsters by the solanum alkaloid solasodine. Res. Comnl. Chew. Pathol. Pharrnucol., 13:723-730. Keeler, R. F. (1973). Comparison of the teratogenicity in rats of certain potato-type alkaloids and the veratrum teratogen cyclopamine, Lancet, 1:1187- 1188. Seifulla, K. I., and Ryzhova, K. E. (1972). Effect of hydrocortisone, solasodine, and solanine on the growth and development of fetuses of pregnant rats, Mater. Vses Konf Zssled Lek Rust Perspekt Ikh Zspol’z Proizvod Lek Prep, 1970:160-162. (Abstracted in Chem. Abstr., 83:22383t, 1975.) Stoessl, A., Unwin, C. H., and Ward, E. W. B. (1973,).Postinfectional inhibitors from plants. I. Capsidiol. an antifungal compound from Capsicun?frutescens, Phytopathol. Z., 74: 141152. Irving, G. W., Fontaine, T. D., and Doolittle, S. P. (1946). Partial antibiotic spectrum of tomatin. an antibiotic agent from the tomato plant, J. Bncteriol., 52601-607. Wilson, R. H., Poley, G. W., and DeEds, F. (1961). Some pharmacologic and toxicologic properties of tomatine and its derivatives, Toxicol. Appl. Plzurmacol.. 3:39-48. Parkin, D. M., Laiira, E., Muir, C. S. (1988). Estimates of the worldwide frequency of sixteen major cancers in 1980, Int. J. Cancer., 41:184-197. Boeing, H. (199 1). Epidemiological research in stomach cancer: Progress over the last ten years, J. Cancer Res. Clin. Oncol, 117:133-143. Hartman, P. E. (1983). Review: Putative mutagens and carcinogens in foods. 1. Nitrate/ nitrite ingestion and gastric cancer mortality, Emiron. Mutagen., 5:lll-121. Kamiyama. S., Ohshima, H., Shimada, A., Saito, N., Bourgade, M.-C., Ziegler, P., and Barsch, H. (1987). Urinary excretion of N-nitrosamino acids and nitrate by inhabitants in high- and low-risk areas for stomach cancer in northern Japan, ZARC Sci. Publ., 84:497502. Green, L. C., Ruiz de Luzuriaga, K., Wagner, D. A., Rand, W., Istfan, N., Young, V. R., and Tannenbaum, S. R. (1981). Nitrate biosynthesis in man, Proc. Nut. Acad. Sci., 78: 7764-7768. Walker, R. (1990). Nitrates, nitrites and N-nitrosocompounds: A review of the occurrence in food and diet and the toxicological implications, Food Addit. Contam., 7:717-768. Wise, A., Mallett, A. K., and Rowland, I. R. (1982). Dietary fibre. bacterial metabolism and toxicity of nitrate in the rat, Xenobiotica, 12:lll-118. Walters, C. L., Carr, F. P. A., Dyke, C. S., Saxby, M. J., Smith, P. L. R., and Walker, R.

Naturally Occurring Toxic Chemicals in Foods

695. 696. 697. 698. 699. 700.

701.

702.

703. 704.

705.

706. 707.

708. 709.

7 10.

71 1.

712. 713.

171

(1979). Nitrite sources and nitrosamine formation in vitro and in vivo, Food Cosnzet. TOXicol.. 17:473-479. Knight, T. M., Forman, D., Ohshima, H., and Bartsch, H. (1991). Endogenous nitrosation of L-proline by dietary-derived nitrate, Nzrtr. Cancer, 15:195-203. Tenovuo, J. (1986). The biochemistry of nitrates, nitrites, nitrosamines and other potential carcinogens in human saliva, J. Oral Pnthol., 15:303-307. Boyland, E., and Walker, S. A. (1973). Effect of thiocyanate on nitrosation of amines, Notwe, 248:601-602. Correa, P. (1988). A human model of gastric carcinogenesis, Curzcer Res., 48:3554-3560. Bartsch, H. (1991). N-nitroso compounds and human cancer: Where do we stand'? ZARC Sci. Publ., 105:l-10. Bartsch. H.. Ohshima, H., Pignatelli, B., and Calmels, S. (1989). Human exposure to endogenous N-nitroso compounds: Quantitative estimates in subjects at high risk for cancer of the oral cavity, oesophagus, stomach and urinary bladder, Cancer S z r m . , 8:335-362. Knight, T. M., Forman. D., Pirastu, R., Comba, P., Iannarilli, R., Cocco, P. L., Angotzi, G., Ninu, E., and Schierano, S. (1990). Nitrate and nitrite exposure in Italian populations with different gastric cancer rates, Znt. J. Epidemiol., 19:5 10-515. Mflller, H., Landt, J., Pedersen, E., Jensen, P., Autrup, H., and Jensen, 0.M. (19911. Urinary excretion of N-nitrosoproline in relation to consumption of raw and cooked vegetables in Danish rural population, ZARC Sci. Pub., 105:168-171. Bartsch, H.. Ohshima, H.. and Pignatelli, B. (1988). Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention, Mutat. Res., 202307-324. Wakabayashi, K., Ochiai, M., Saito, H.. Tsuda, M., Suwa, Y., Nagao, M., and Sugimura, T. ( 1983). Presence of 1-methyl-1,2.3,4-tetrahydro-~carboline-3-carboxylic acid, a precursor of a mutagenic nitroso compound, in soy sauce, Proc. Nut. Acrrd. Sci. (USA), 8029122916. Wakabayashi, K., Nagao. M., Ochiai, M., Fujita, M.. Tahira, T., Nakayasu, M.. Ohgaki, H., Takayama. S., and Sugimura, T. (1987). Recently identified nitrite-reactive compounds in food: Occurrence and biological properties of the nitrosated products, ZARC Sci. Pub., 84~287-291. Wakabayashi, K. (1990). Identification of food mutagens, Prog. Clin. Biol.Res., 340E: 107-1 16. Luthy, J., Carden, B., Bachmann, M., Friederich, U., and Schlatter, C. (1984). Nitrosatable compounds in foods: Identification and mutagenicity of the reaction product of goitrin and nitrite, Mitt. Geb. Lebensnlittelzuzters. Hyg., 75:lOl-109. Luthy, J., Carden, B., Friederich, U., and Bachmann, M. (1984). Goitrin-A nitrosatable constituent of plant foodstuffs, Evperientia, 40:452-453. Groenen. P. J., and Busink, E. (1988). Alkylating activity in food products-Especially sauerkraut and sour fermented dairy products-After incubation with nitrite under quasigastric conditions, Food Chem. Toxicol., 26215-225. Massey, R., Dennis, M. J., Pointer, M., and Key, P. E. (1990). An investigation of the levels of N-nitrosodimethylamine, apparent total N-nitroso compounds and nitrate in beer. Food Addit. Contam.. 7:605-615. Ellen, G., Egmond, E., Van Loon, J. W., Sahertian, E. T., and Tolsma, K. (1990). Dietary intakes of some essential and non-essential trace elements, nitrate, nitrite, and N-nitrosamines, by Dutch adults: Estimated via a 24-hour duplicate portion study, Food Addit. Contam., 7:207-221. Knight, T. M.. Forman, D., Al-Dabbagh, S. A., and Doll, R. (1987). Estimation of dietary intake of nitrate and nitrite in Great Britain, Food Chenl. Tosicol., 25:277-285. RochC, M. 0. G., Beltrin, G., Hernindez, A. M., and Wettig, K. (1988). Estimation of the daily intake of nitrates and nitrites which may be consumed by two groups of population 18-23 years old in the city of Havana, Nrlhrung. 32:5 17-5 18.

172

Beier and Nigg

714. Dutt, M. C.. Lim, H. Y., and Chew, R. K. H. (1987). Nitrate consumption and the incidence of gastric cancer in Singapore, Food Clzenz. Toxicol., 25:515-520. 715. Shanmugaratnam, K., Lee. H. P., and Day, N. E. (1983). Cancer incidence in Singapore 1968-1977. IARC Sci. Put)., 47:20. 716. White, J. W., Jr. (1975). Relative significance of dietary sources of nitrate and nitrite, J. Agric. Food Chem., 23:886-891. 717. Walters, A. H., Fletcher, J. R., and Law, S. J. (1986). Nitrate in vegetables: Estimation by HPLC, Nutr. Hecrlth, 4:141-149. 718. Termine, E.. Lairon, D., Taupier-Letage, B., Gautier, S., LaFont, R., and LaFont, H. (1987). Yield and content in nitrates, minerals and ascorbic acid of leeks and turnips grown under mineral or organic nitrogen fertilizations, Plcrnt Foods Hum. Nutr., 37:321-332. 719. Takruri, H. R., and Humeid. M. A. (1988). Nitrate levels in edible wild herbs and vegetables common to Jordan. Nutr. Healtlz, 6:89-98. 720. Ponnampalam, R.. and Mondy, N. I. (1986). Effect of foliar application of indoleacetic acid on the total glycoalkaloids and nitrate nitrogen content of potatoes. J. Agric. Food Clzent., 341686-688. 721. Augustin, J., McDole, R. E., and Painter, G. C. (1977). Influence of fertilizer, irrigation, and storage treatments on nitrate-N content of potato tubers, Anz. Potato J., 54:125-136. 723. Carter, J. N., and Bosma, S. M. (1974). Effect of fertilizer and irrigation on nitrate-nitrogen and total nitrogen in potato tubers. Agron. J., 66:263-266. 723. Kiss, A. S. (1990). Nitrate content of plants reduced by magnesium fertilizers, Mngnesizlrtz Res., 3:263-265. 724. Biedermann, R., Lew. D., and Vogelsanger, W. (1 981). Nitrate in der nahrung-eine stand76:S.149-S.156 (in German). ortbestimmung, Deutsche Let)ensnzittel-Run~sc~z~z~. 725. Temperli, A. T. (1982). EinflussBzweier anbauweisen auf den nitratgehalt von kopfsalat, Sclzweiz. Landu-irtsch. Forsclz., 21:s. 167-S. 196 (in German). 726. Vogtmann, H. (1988). From healthy soil to healthy food: An analysis of the quality of food produced under contrasting agricultural systems, Nzctr. Health. 621-35. 727. Vogtmann. H., and Biedermann, R. (1985). The nitrate story-No end in sight, Nutr. Health, 3:217-239. 728. Vogtmann, H., Temperli, A. T., Kiinsch, U., Eichenberger, M., and Ott, P. (1984).Accunmlation of nitrates in leafy vegetables grown under contrasting agricultural systems, Biol. Agric. & Hortic., 351-68. 729. Kiss, A. S., and Pozsir. B. I. (1975). Stimulative effect on protein synthesis of magnesium applied by foliage spray, Acta Agron. A c ~ LSci. ~ . Hung., 24:61-65. 730. Sinios, A. (1964). Methemoglobinemia von nitrat gehalte in spinat, Miinch.Med. Wochensclzr., 106:1180-1182 (in German). 731. Fletcher. J. R., Law.. S. J., and Walters, A. H. (1987). Effect of cooking on the nitrate levels in vegetables, Nutr. Health, 5:61-63. 732. Camm, E.. Buck, H. W. L., and Mitchell, J. C. (1976). Phytophotodermatitis from Heracleuvr rnategnxianum, Co~zracrDermatitis. 2:68-72. 733. Pathak, M. A. (1 986). Phytophotodermatitis, Clin. Dernratol., 4:102-121. 734. Beier. R. C., Ivie, G. W., and Oertli, E. H. (1983). Psoralens as phytoalexins in food plants of the family Umbelliferae: Significance in relation to storage and processing. In Xerzobioticsin Foods a d Feeds (J. W. Finley and D. E. Schwass, eds.), ACS Symposium Series 234, American Chemical Society. Washington. D.C., pp. 295-310. 735. Knogge, W., Kombrink. W., Schmelzer, E., and Hahlbrock, K. (1987). Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants, Planta, 171~279-287. 736. Tietjen, K. G., Hunkler, D., and Matern, U. (1983). Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 1. Identification of induced products as coumarin derivatives. Ezwop. J. Bioclzem.. 131 :401-407.

Naturally Occurring Toxic Chemicals in Foods

I 73

737. Johnson, C., Brannon, D. R.. and Ku6, J. (1973). Xanthotoxin: A phytoalexin of Pastinaca sativa root, Phytochenristq, 122961-2962. 738. Smith, D. M. (1 985). Occupational photodermatitis from parsley, Prcrctitioner, 229:673675. 739. Stransky, L., and Tsankov. N. (1980). Contact dermatitis from parsley (Petroselirzzm),Cont m t Derntutitis, 6:233-234. 740. Birmingham, D. J., Key. M. M., Tubich, G. E.,and Perone. V. B. (1961). Phototoxic bullae among celery harvesters, Arch. Dermatol., 83:73-85. 741. Legrain, P, MM., and Barthe, R. (1926). Dermite professionnelle des mains et des avantbras chez unramasseur de cileris, Bull. SOC.Fr. Dermatol. Syphiligr., 33:662-664(in French). 742. Zobel, A. M., and Brown, S. A. (1989). Histological localization of furanocoumarins in Ruta graveolens, Can. J. Bot., 67:915-921. 743. Zobel, A. M., and Brown, S. A. (1991). Psoralens on the surface of seeds of Rutaceae and fruits of Umbelliferae and Leguminosae, Can. J. Bot., 69:485-488. 744. Ivie, G. W. (1978).Toxicological significance of plant furocoumarins. InEfsects of Poisonous Plartts on Livestock (R. F. Keeler, K. R. Van Kampen, and L. F. James, eds.), Academic Press, New York, pp. 475-485. 745. Belogurw, A. A., and Zavilgelsky, G. B. (1981). Mutagenic effect of furocoumarin monoadducts and cross-links on bacteriophage lambda, Mutat. Res., 84:ll-1 5. 746. Cassier, C., and Moustacchi, E. (1981). Mutagenesis induced by mono- and bi-functional alkylating agents in yeast mutants sensitive to photo-addition of furocoumarins (pso). Mutat. Res., 84:37-47. 747. Grekin, D. A., and Epstein, J. H. (1981). Psoralens, UVA (PUVA) and photocarcinogenesis, Photochent. Photobiol.. 33:957-960. 748. Parsons, B. J. (1980). Psoralen photochemistry, Pllotochenr. Photobiol., 32813-821. 749. Scott, B. R., Pathak. M. A., and Mohn, G. R. (1976). Molecular and genetic basis of furocoumarin reactions, Mutat. Res., 39:29-74. 750. Talib, S., and Banerjee, A. K. (1982). Covalent attachment of psoralen to a single site on vesicular stomatitis virus genome RNA blocks expression of viral genes. Virologv, 118: 430-438. 751. Hearst, J. E. (1989). Photochemistry of the psoralens. Chem. Res. Toxicol., 2:69-75. 752. Woo, Y.-T.. Lai. D. Y.. Arcos, J. C.. and Argus, M. F. (1988). Linear and angular furocoumarins: psoralens, angelicins, and related compounds. In ClzenticcrlIrtclzrction of Cancer, Strmtwal Bases and Biologicctl Mechanisms (Y.-T. Woo, D. Y. Lai, J. C. Arcos,and M. F. Argus, eds.), Academic Press, New York, pp. 320-356. 753. Pathak, M. A., Daniels, F., and Fitzpatrick, T. B. (1962). Thepresently known distribution of furocoumarins (psoralens) in plants, J. hzvest. Dermatol., 39:225-240. 754. McEvoy, M. T., and Stern. R. S. (1987). Psoralens and related compounds in the treatment of psoriasis, Plrarrwcol. Tlter., 34:75-97. 755. Van Scott. E. J. (1976). Therapy of psoriasis 1975, J. Am. Med. Assoc., 235:197-198. 756. Hann, S. IS.. Cho, M. Y., and Park, Y.-K. (1990). UV treatment of generalized prurigo nodularis, h t . J. Dermatol., 19:436-437. 757. Plott, R. T.. and Wagner. R. F. (1990). Modern treatment approaches to vitiligo, CUTIS, 45~311-316. 758. National Institute of Environmental Health Sciences (1982). Psorulens, National Toxicology Program, Technical Bulletin No. 6, p. 8. 759. Hannuksela, M., Stenback, F., and Lahti, A. (1986). The carcinogenicproperties of topical PUVA, Arch. Dennatol. Res., 278:347-351. 760. Stern, R. S., Thibodeau, L. A., Kleinerman. R. A., Parrish, J. A., Fitzpatrick, T. B.. and 22. participating investigators (1979). Risk of cutaneous carcinoma inpatients treated with oral methoxalen photochemotherapy for psoriasis, New Engl. J. Med., 300:809-813.

174

Beier and Njgg

761. Young, A. R. (1986). Aspectsof psoralen phototumorigenesis withemphasis on the possible role of tumour promotion, Biocltinzie. 68:885-889. 762. Henseler,T., Christophers, E., Honigsmann,H.,Wolff, K.,and 19other investigators (1987). Skin tumors in the European PUVA study, J. Am. Accld. Dernzntol., 16:108-116. 763. Tanew, A., Honigsmann, H., Ortel, B., Zussner, C., and Wolff, K. (1986). Nonmelanoma skin tumors in long-term photochemotherapy treatment of psoriasis. J. Am. Acad. Dennutol.. 15:960-965. 764. Stern, R. S., Abel, E., Wintroub, B., Epstein, J. H., Tschen. J., Wolf, J., Nigra, T. P., Voorhees, J., Anderson, T. F., Armstrong, R., Harber. L., Muller, S., Taylor, J. R., Frost, P., Horwitz, S., Urbach, F., Arndt. K. A., Baughman, R. D., Braverman, I. M., Murray, J., Petrozzi, J., Gonzalez, E., Parrish, J. A., Fitzpatrick, T. B.. Lange, R., and Bleich. H. L. (1990). Genital tumors among men with psoriasis exposed to psoralens and ultraviolet A radiation (PUVA) and ultraviolet B radiation, New Erzgl. J. Med., 3221093-1097. 765. Woman dies after tanning, Bryan-College Stcrtion Eagle, May 28, 1989. 766. Ljunggren, B. (1990). Severephototoxic burn following celery ingestion. Arch. Derutatol., 12611334-1336. 767 Forlot, P.(1 990). 5-Methoxypsoralen arzd Other Fzrrocoumarins as Jet Lag Suppressants, European Patent Application 352198. 768. Beier, R. C., Ivie, G. W., Oertli, E. H., and Holt. D. L. (1983). HPLC analysis of linear furanocoumarins (psoralens) in healthy celery (Apium graveolens), Food Chenz. Toxicol., 21:163-165. 769. Ashwood-Smith, M. J., Ceska, O., and Chaudhary, S. K. ( 1 985). Mechanism of photosensitivity reactions to diseased celery. Br. Med. J., 290:1249. 770. Austad, J., and Kavli, G. (1983). Phototoxic dermatitis caused by celery infected by Sclerotinia sclerotiorm, Contact Dermatitis, 9:448-45 1 . 771. Chaudhary, S. K., Ceska, O., Warrington, P. J., and Ashwood-Smith, M. J. (1985). Increased furocoumarin content of celery during storage, J. Agric. Food Clzem., 33: 1153-1 157. 772. Karasawa, D., Shibata, H., Horiuchi, N., Andou, Y., and Simada, M. (1990). Photoactive furocoumarins in diseased celery (Apium gmveolence),Agric. Biol. Chem., 54:2141-2142. 773. Trumble, J. T., Millar, J. G., Ott, D. E., and Carson, W. C. (1992). Seasonal patterns and pesticidal effects on the phototoxic linear furanocoumarins in celery,Apizan grm~eolensL.. J. Agric. Food Chem., 40:1501-1506. 774. Rober, M., Pydde, E., and Knorr, D. ( 199 1). Storage time dependent accumulationof furanocoumarins in polysaccharide gel coated celery tubers. Lebensm. - Wiss. & Techrlol., 24:466-468. 775. Beier and Oerlti, studies conducted during 1982, 79 and 96 hours after a 30minute CuSO, treatment celery petiols contained 21.1 pg/g and 26 pg/g linear furanocoumarins and controls contained 0.92 pg/g and 0.25 pg/g, respectively. This results in a 22.9 and 104 fold increase, respectively, in linear furocoumarin content after CuSO, treatment. 776. Hanson, C. H., ed. (1974). The EfSect of FDA Regzrlatiorts (GRAS)O H Plrrnt Breeding arzd Am., Madison, Wisconsin. Processing, CSSA SpecialPublication Number 5, Crop Sci. SOC. 777. Trumble, J. T.. and Quiros. C. F. (1988). Antixenotic and antibiotic resistance in Apizrm species to Lirionry:a trifolii (Burgess) (Diptera: Agromyzidae), J. Econ. E~ton~ol.. 8 1:602607. 778. Trumble, J. T., Dercks. W., Quiros, C. F., and Beier, R. C. (1990). Host plant resistance and linear furanocoumarin content of Apizrrzt accessions, J. Ecorz. Erztontol., 83:519-525. 779. Beier, R. C., and Nigg, H. N. (1992). Natural toxicants in foods. In Pltytochemicnl Resozrrces for Medicine and Agriczlltw-e (H. N. Nigg and D.Seigler, eds.), Plenum, New York. pp. 247-367. 780. Chaudhary, S. K., Ceska, O., Titu, C., Warrington, P. J., Ashwood-Smith, M. J., and Poulton, G. A. (1 986). Oxypeucedanin. a major furocoumarin inparsley, Petroseli~tun~ crispunt, Plnnta Med., 52:462-464.

Naturally Occurring Toxic Chemicals Foods in

175

781. Ivie. G. W.. MacGregor, J. T., and Hammock, B.D. (1980).Mutagenicity of psoralen epoxides, Mutat. Res., 79:73-77. 782. Zheng, G.-Q., Kenney, P. M., and Lam, L. K. T. (1992). Myristicin: A potential cancer chemopreventive agent from parsley leaf oil, J. Agric. Food Cltent., 40: 107-1 10. 783. Hodgson, E., and Philpot. R.M. (1974).Interaction of ~nethylenedioxyphenyl(1 ,3-benzodioxole) compounds with enzymes and their effect on mammals, Drug Metnb. Rev., 3:23 1-301. 784. Hodgson. E. (1 985). Microsomal mono-oxygenases. In Contprelzensive Insect Plzysiology, Biochentistn crnd Phrrnacology (G. A. Kerkut and L. I. Gilbert, eds.), Pergamon Press, New York, pp. 225-321. 785. Neal, J. J. (1989). Myristicin, safrole, and fagaramide as phytosynergists of xanthotoxin, J. Chem. Ecol.. 15:309-3 15. M. (1986). 786. Ceska, O., Chaudhary, S., Warrington, P., Poulton. G.. and Ashwood-Smith, Naturally-occurring crystals of photocarcinogenic furocoumarins on the surface of parsnip roots sold as food, Experientia, 42: 1302-1304. 787. Ivie, G.W., Holt, D. L., and h e y , M. C. (1981).Natural toxicants in human foods: Psoralens in raw and cooked parsnip root, Science, 213:909-910. oc788. Lichtenstein, E. P., and Casida, J. E. (1963). Myristicin, an insecticide and synergist curring naturally in the edible parts of parsnip, J. Agric. Food Chew., 11:410-415. 768, and at 789. Beier, R. C. (1984). Fig fruits were extracted and analyzed according to Ref. a 0.03 yg/g detection limit no furanocoumarins were detected. 790. Steiner, W. E., Rieker, R. H., and Battaglia, R. (1988). Aflatoxin contamination in dried figs: Distribution and association with fluorescence, J. Agric. Food Chenz., 36:88-9 1. 791. Vo-Dinh, T.( 1987). Evaluation of an improved fiberopticsluminescence skin monitor with background correction, Ana.Incl. Hyg. Assoc. J . , 48:594-598. 792. Vo-Dinh, T.. and Gammage, R. B.(1981). The lightpipe luminoscope for monitoring occupational skin contamination, Arw htd. Hyg. Assoc. J.. 42: 112- 120. 793. Vo-Dinh,T.,White, D. A.,O’Malley, M. A., Seligman.P. J., andBeier, R. C. (1988). Fluorescence detection of phototoxic psoralens in vegetable products, J. Agric. Food Clzern., 36:333-337. 794. Libert, B., and Franceschi. V. R. (1987). Oxalate in crop plants, J. Agric. Food Chem., 35: 926-938. 795. Stryer, L. (1988). Biochemistry, W. H. Freeman, New York. 796. White, A., Handler, P., and Smith, E. L. (1964). Primiples of Bioclzewistty, 4th Edition, McGraw-Hill, New York. 797. Achilles, W. (1991).Microphotometric quantification of crystal growthin gels for the study of calcium oxalate urolithiasis, Scamring Microsc., 5: 1001 -1017. 798. Hallson. P. C., and Rose, G. A. (1976). Crystalluria in normal subjects and in stone formers with and without thiazide and cellulose phosphate treatment, Br. J. Urol., 48515-524. 799. Robertson, W. G., Peacock, M., and Nordin, B. E. C. (1969). Calcium crystalluria in recurrent renal-stone formers, Lancet, 2 2 1 -24. 800. Berg, W., Brundig, P., Bothor. C., Schneider, H.-J. (1982). Biological rhythmicity and crystallization-Urineprofilesandse-studieson calcium oxalate stone genesis, Znt. Urol. Nephrol., 14:363-372. 801. Ahlstrand, C., Tiselius, H.-G., and Larsson, L. (1984). Studies on crystalluria in calcium oxalate stone formers, Urol. Res., 12103-106. 802. Grases. F.. Millan, A., and Sohnel, 0. (1992). Role of agglomeration in calcium oxalate monohydrate urolith development, Nephron, 61: 145-150. 803. Iwata, H., Iio, S., Nishio, S., and Takeuchi, M. (1992). Architecture of mixed calcium oxalate dihydrate and monohydrate stones, Scanning Microsc., 6:231-238. 804. Watts, R. W. E., Veall, N., and Purkiss, P. (1984). Oxalate dynamics and removal rates during haemodialysis and peritoneal dialysis in patients with primary hyperoxaluria and severe renal failure, Clin. Sci., 66:591-597.

I 76

Beier and Nigg

805. Oren, A., Husdan, H., Cheng, P.-T., Khanna, R., Pierratos, A., Digenis, G., and Oreopoulos, D. G. (1984). Calcium oxalate kidney stones in patients on continuous ambulatory peritoneal dialysis, Kidney Int., 25:534-538. 806. Tawashi. R., Cousineau, M., and Denis, G. (1983). Calcium oxalate crystal growth in normal urine: Role of contraceptive hormones. Ut-01. Res., 12:7-9. 807. Martin, X., Smith, L. H., and Wemess, P. G. (1984). Calcium oxalate dihydrate formation in urine, Kidney Int., 25:938-952. 808. Vahlensieck, E. W., Bach. D.. Hesse, A.. and Strenge, A. (1982). Epidemiology,pathogenesis and diagnosis of calcium oxalate urolithiasis, h t . U r d . Neyhrol., 14:333-347. 809. Piepmeyer. J. (1974). Planning diets controlled in oxalic acid content, J. Am. Diet Assoc., 65:438-340. consuming 8 10. Kelsay, J. L., and Prather, E. S. (1983). Mineral balances of human subjects spinach in a low-fiber diet and in a diet containing fruits and vegetables. Am. J. Clin. Nzrtr., 38: 12-1 9. 811. Berg. W., Bothor, C., Pirlich. W., and Janitzky, V. (1986). Influence of magnesium on the absorption and excretion of calcium and oxalate ions, Eur. Urol.. 12:274-282. 812. Kelsay, J. L., Prather, E. S., Clark, W. M.. and Canary. J. J. (1988). Mineral balances of men fed a diet containing fiber in fruits and vegetables and oxalic acid in spinach for six weeks. J. N~rtr.,118:l 197-1204. 81 3. Heaney. R. P.. Weaver, C. M.. and Recker, R. R. (1988). Calcium absorbability from spinach, Am. J. Clin. Nrm., 47:707-709. 8 14. Heaney, R. P., and Weaver, C. M. (1989). Oxalate: Effect on calcium absorbability, Am. J. Clin. Nutt-.. 50:830-832. 8 15. Van Campen, D. R.. and Welch, R. M. (1980). Availability to rats of iron from spinach: Effects of oxalic acid, J. Nzm., 110:1618-1621. 8 16. Welch, R. M., House, W. A., and Van Campen, D. (1977). Effects of oxalic acid on availability of zinc from spinach leaves and zinc sulfate to rats. J. Nutr., 107:929-933. 817. Kikunaga, S., Arimori, M.. and Takahashi, M. (1988). The bioavailability of calcium in spinach andcalcium-oxalatetocalcium-deficient rats, J. Nutr. Sci. Vitrrminol., 33:195207. 818. Gordon. D. T., and Chao, L. S. (1984).Relationshipof components i n wheatbranand spinach to iron bioavailability in the anemic rat, J. Nutr., 114:526-535. 819. Archer, H. E., Dormer, A. E., Sconven. E. F., and Watts, R. W. E. (1957). Studies on the urinary excretion of oxalate by normal subjects, Clin. Sci. (Lo~zdon),16:305-411. 820. Zarembski, P. M., and Hodgkinson. A. (1962). The oxalic acid content of English diets. BI.. J. Nl.{tr.- 161627-634. 821. Earnest, D. L.. Johnson, G., Williams, H. E., and Admirand. W. H. ( 1974). Hyperoxaluria in patients withileal resection: An abnormality in dietary oxalate absorption.Gastroenterology, 6611114-1122. 822. Chadwick, V. S., Modha. K., and Dowling, R. H. ( 1973). Mechanism for hyperoxaluria in patients with ileal dysfunction. N. E@ J. Men., 289: 172-1 76. 823. Prenen, J. A. C., Boer, P., and Dorhout Mees. E. J. (1984). Absorption kinetics of oxalate from oxalate-rich food in man, A m J. cliiz. Nrctl-., 40:1007-1010. 824. Finch, A. M.. Kasidas, G. P., and Rose. G. A. (1981). Urinecomposition in normal subjects after oral ingestion of oxalate-rich foods, Clin. Sci., 60:411-318. 825. Hanson, C. F., Frankos, V. H., and Thompson. W. 0. (1989). Bioavailability of oxalic acid from spinach, sugarbeet fibre and a solution of sodium oxalate consumed by female volunteers, Food Clwnr. Toxicol., 27: 181-1 84. 826. Brinkley, L. J., Gregory. J.. and Pak, C.Y. C. (1990). Afurther study ofoxalate bioavailability in foods, J. Urol., 143:94-96. 827. Meena. B. A.. Ulnapathy, K. P., Pankaja, N., and Prakash, J. (1987). Soluble and insoluble oxalates in selected foods, J. Food Sci. Tuclmol., 24:43-44.

Naturally Occurring Toxic Chemicals in Foods

177

828. Hoover, A.A.,and Karunairatnam, M. C. (1945) Oxalate content of some leafy green vegetables and its relation to oxaluria and calcium utilization. Biochem. J.. 39237-238. 829. Andrews, J. C., and Viser, E. T. (1951). The oxalic acid content of some common foods, Food Res., 16:306-312. 830. Kasidas. G. P., and Rose, G. A. (1980). Oxalate content of some common foods: Determination by an enzymatic method, J. H L I I Nzltr., ~. 34:255-266. 831. Halen. B. ( 1985). Vatskekror?latogt,crJiskBestumning av Osalsyra i Bludgronsaker. Statens Livsmedelsverks Rapport 1985:2, Statens Livsnledelsverk, Uppsala. 832. Wilson, C. W., 111. Shaw. P. E., and Knight, R. J.. Jr. (1982). Analysis of oxalic acid in carambola (Averrlloa carambola L.) and spinach by high-performance liquid chromatography, J. Agric. Food Chem, 30:1106-1108. 833. Srivastava, S. K., and bishnan, P. S. (1962). Oxalate content of plant tissues, J. Sci. Ind. Res., 18:146-148. 834. Ohkawa, H. ( I 985). Food composition, gas chromatographic determination of oxalic acid in foods, J. Assoc. Of A n d . Cllenz.. 68: 108- 111. 835. Onayemi. O., and Nwigwe, N.C. (1987). Effect of processing on the oxalate content of cocoyam, Lebensm-Wiss. Technol., 20293-295. 836. Anantha Samy, T. S., Kamat, V. N., and Pandya, H. G. (1960). Oxalate content of leafy vegetables, Clrrr. Sci. (Bangalore), 29: 133. 837. Vishwanath, W., and Sarojnalini, C. (1989). Changes in the total oxalate conteiit in the fermentation of fish paste hentak. J. Food Sci., 54:754-755. 838. Allison, R. M. ( I 966). Soluble oxalates, ascorbic and other constituents of rhubarb varieties, J. Sci. Food Agric., 17554-557. 839. Libert, B.. and Creed, C.(1985). Oxalate content of seventy-eight rhubarb cultivars and its relation to some other characters, J. Hort. Sci., 60257-261. 840. Kmiecek, W. (1974). Ocena przydatnoschi pieciu odnlian rabarbam do pedzenia, Acto Agt-m. Silvestria Ser. Ag’nr., 14:3-56. 841. Sengbusch, R. V., Sucker. I., and Handke, S. (1965). Untersuchungen iiar den gehalt an oxalsaure in spinat (Spinnciu oleraceu) als guundlage fur die zuchterische bearbeitung dieses merkmals, Zzrechter, 35:90-98 (in German). 842. Kohman, E. F. (1939). Oxalic acid in foods and its behaviour and fate in the diet. J. Nutr.. 18:233. 843. Awott, H. J.. and Webb, M. A. (1983). The structure and formation of calcium oxalate crystal deposits on the hyphae of a wood rot fungus, Scuming Electron Microsc., 4: 17471758. 844. Rao, K. S., and Dave, Y. S. (1984). Occurrence of crystals in vascular cambium, Protoplnsnza, 1 1 9 219-32 1. 845. Wagner, B. L. (1983). Genesis of the vacuolar apparatus responsible for druse formation in Capsiczm a m u z m L. (Solanaceae) anthers, Sccrnlring Electron Microsc., 2:905-912. 846. Perera, C. O., Hallett, I. C., Nguyen, T. T., and Charles, J. C. (1990). Calcium oxalate crystals: The irritant factor in kiwifruit, J. Food Sci., 55:1066-1 080. 847. Weiner, E. S. (1979). Lccalized endotracheal oxalosis probably secondary to aspiration of rhubarb, Arch. h t . Med.. 139:602. 848. Fairbairn, J. W. (1980). Oxalated. sulphated and primary glycosides, Pharmacology, 20: 83-87. 849. Balogun, A. M., and Fetuga, B. L. (1988). Tannin. phytin and oxalate contents of some wild under-utilized crop-seeds in Nigeria. Food Cherrt., 30:37-43. 850. Zarembski, P. M.. and Hodgkinson, A. (1969). Some factors influencing the urinary excretion of oxalic acid in man. C h . Clzinl. Acta, 25:l-10. 851. Hargreaves, T. B., Sali, A., McKay, C., and Sullivan, M. (1977). Diurnal variation in urinaq oxalate, Br. J. Urol., 49597-600. 852. Barngrover. D. A., and Dills, W. L., Jr. (1983). The involvement of liver fructokjnase in

I 78

853. 853. 855. 856. 857.

858. 859. 860. 861. 862.

863.

864.

865. 866. 867. 868. 869.

870. 871.

872.

873.

and

Beier

Nigg

the metabolism of D-xylulose and xylitol in isolated rat hepatocytes, J. Nutr., 113:522530. Moye, H.A.. Malagodi, M. H.. Clarke, D.H.. and Miles, C.J. ( 1981). A rapid gas chromatographic procedure for the analysis of oxalate ion in urine, Clin. Chinz. Acta, 114: 173-185. Gottstein, H. D., Zook. M. N., and K L I ~J., A. (1989). Detection and quantitation of oxalic acid by capillary gas chromatography, J. Chronzntogr.. 38 1:55-61. Fatoki, 0. S. (1991). Ambient temperature acid extraction method for the determination of oxalic acid contents of vegetables, J. Agric. Food Chem.. 39293-295. Cole, F. E., Gladden, K. M.. Bennett, D. J..and Erwin, D. T. (1984). Humanplastnaoxalate concentration re-examined, Clin. Chinz. Acta., 139: 137-1 43. Steinbauer. C. E., and Cushman, L. J. (1971). Sweet Potato Cultwe and Diseuses, Agriculture Handbook No. 388, Agricultural Research Service, U.S. Department of Agriculture, Washington, D.C. Takeuchi, T. (1949). Bitter substance, produced in black-rotted sweet potatoes. Sci. Irzsect Colztl: (Kyoto), 12:26-29. (Clzenl. Abs., 43:8453, 1949.) Taira, T., and Fukagawa. Y. (1958). Bitter substance separated from alcohol distillation of sweet potato mash,Nippon Nhgei Kugaku Kaishi, 32513-514. (Clzenz.Abs., 53:4645c, 1959.) Burka, L. T., Bowen. R. M., and Wilson, B. J. (1974). 7-Hydroxymyoporone, a new toxic furanosesquiterpene from mold-damaged sweet potatoes, J. Org. Chenz., 39:3241-3244. Schneider, J. A., and Nakanishi, K. (1983). A new class of sweet potato phytoalexins, J. Chern. SOC..Chenl. Cortanm.. 353-355. Schneider, J. A., Lee, J..Naya.Y..Nakanishi, K., Oba, K., andUritani, I. (1984). The fate of the phytoalexin ipotneamarone: Furanoterpenes and butenolides from Cemtocystis jintbriata-infected sweet potatoes, Phytochemistn. 23:759-764. Clark. C. A., Lawrence, A., and Martin, F. A. (1981). Accumulation of furanoterpenoids in sweet potato tissue following inoculation with different pathogens, Phytopcrthology, 71: 708-7 1I. Boyd, M.R., Burka, L.T., Harris, T. M., and Wilson, B.J. (1973). Lung-toxicfuranoterpenoids produced by sweet potatoes (lponroerrbutatas) following microbial infection, Biochim. Biophys. Actu. 337:184-195. Wilson, B. J.. Boyd, M. R., Harris, T. M., and Yang, D. T. C. (1971). A lung oedema factor from mouldy sweet potatoes (Ipomoerr butatcrs), Nutwe, 23 1:52-53. Uritani, I., and Miyano. M. (1955). Derivativesof caffeic acid in sweet potato attacked by black rot, Nrrtlwe, 175:812. Inoue, H., and Uritani, I. (1979). Biosynthetic correlation of various phytoalexins in sweet potato root tissue infected by Cemtocystis jiwzbricrta. Plant Cell Physiol., 20: 1307-1314. Burka, L. T., and Iles. J. ( 1979). Myoporone and related keto alcohols from stressed sweet potatoes, Phytochen~istr~, 18:873-874. Burka. L. T.,Felice. L. J.. and Jackson. S. W. (1981). 6-Oxodendrolasin, 6-hydroxydendrolasin, 9-oxofarnesol and 9-hydroxyfarnesol. stress metabolites of the sweet potato, Phytochemistry, 20:647-652. Burka, L. T.( 1 978). 1-(3’-Furyl)-6,7-dihydroxy-4,S-dirnethylnonan-l-one, a stress metabolite from sweet potatoes, Ipornoeu bututus, Phytochemistry, 17:317-3 18. Peckham, J. C., Mitchell, F. E., Jones, 0. H., and Doupnik, B.. Jr. (1979). Atypical interstitial pneumonia in cattle fed moldy sweet potatoes, J. An]. Vet. Med. Assoc.. 160: 169172. In Wilson, B. J., and Burka. L. T. (1983). Sweet potato toxins and related toxic furans. Handbook of Natllral Toxins: Plant and F m g a l Toxins, Volume 1 (R.F. Keeler and A. T. Tu, eds.), Marcel Dekker, New York. pp. 3-42. Boyd, M. R., and Burka, L. T. (1978). In vivo studies on the relationship between target organ alkylation and the pulmonary toxicity of a chemically reactive metabolite of 4-ipomeanol, J. Pltantlacol. E . Y ~Ther., 207:687-697.

Naturally Occurring Toxic Chemicals in Foods

179

874. Boyd, M. R., and Wilson, B. J. (1971). Preparative and analytical gas chromatography of ipomeamarone, a toxicmetaboliteof sweet potatoes (Ipomoea batatas), J. Agric. Food Chern., 19:547-550. 875. Coxon, D. T., Curtis, R. F., and Howard. B. (1975). Ipomeamarone. a toxic furanoterpenoid in sweet potatoes (Iponzoea batatas) in the United Kingdom, Food Cosmet. Toxicol., 13: 87-90. 876. Catalono, E. A., Hasling, V. C., Pons, W. A.. Jr.. and Schuller, W. H. (1979). Analysis of sweet potato products for lung edema toxins, Hort-Science, 14:124-125. 877. Wilson, B. J., Yang, D. T. C., and Boyd, M. R. (1970). Toxicity of mould-damaged sweet potatoes (Ipomoea batatas), Nature, 227:531-522. 878. Boyd, M. R. (1977). Evidence for the Clara cell as a site of cytochrome P450-dependent mixed-function oxidase activity in lung, Nature, 269:7 13-7 15. 879. Boyd, M. R. (1976). Role of metabolic activation in the pathogenesis ofchemically induced pulmonary disease: Mechanism of action of the lung-toxic furan, 4-ipomeanol, Environ. Health Perspect., 16:127-I 38. 880. Doster. A. R., Farrell, R. L., and Wilson, B.J. (1983). An ultrastructural study of bronchiolar lesions in ratsinduced by 4-ipomeanol, aproduct from mold-damaged sweet potatoes,Anz. J. Pathol., 1 1 1:56-61. 881. Durham, S. K., Boyd, M. R.,and Castleman, W. L. (1985). Pulmonary endothelial and bronchiolar epithelial lesions induced by 4-ipomeanol in mice, Anz. J. Patlzol., 118:66-75. 882. Durham, S. K., Babish, J. G., and Castleman, W. L. (1987). 4-Ipomeanol-induced effects on sendai viral pneumonia in mice, Am. J. Pathol., 126:364-375. 883. Woolcock, A. J.. Blackburn, C.R. B.. Freeman,M. H.,Zylstra, W., and Spring, S. R. (1970). Studies of chronic (nontuberculous) lung disease in New Guinea populations: The nature of the disease, 41n. Re)?.Respir. Dis., 102:575-590. 884. Wilson, B. J. (1973). Toxicity of mold-damaged sweetpotatoes, Nutr. Rev., 31:73-78. 885. Dawson, K. P., and Mitchell, E. A. (1990). Asthma in New Zealand children, J. Astlzma, 271291-297. 886. Sears, M. R. (1991). Growing number of asthma cases, deaths, baffles physicians, Can. Med. Assoc. J., 145:379-380. 887. Hrdlicka, J., Dinsdale,D., and Seawright, A.A. (1990). A comparison ofpathological changes in the mouse lung after dosing with the 3-substituted furans, myomontanone and 3-(N-ethylcarbamoyloxymethyl)furan,Int. J. Exp. Patlzol., 7 1:441-453. 888. Hagerman, A.E., and Butler, L. G. (1991). Tanninsand lignins. In Herbivores: Their Interactions with Secondan Plant Metabolites, 2E Volwne I. The Chemical Participants, Academic Press, New York, pp. 355-388. 889. Swain, T. (1979). Tannins and lignins. In Herbivores, Their Interaction with Seconclaq Plant Metabolites (G. A. Rosenthal and D. H. Janzen, eds.), Academic Press, New York, pp. 657-682. 890. Oh, H.-I., and Hoff, J. E. (1987). pH dependenceof complex formation between condensed tanins and proteins, J. Food Sci., 52: 1267-1273. 891. Pierpoint, W. S. (1 983). Reactions of phenolic compounds with proteins, and their relevance to the production of leaf protein. InLeafprotein Corzcentrates (L. Telek and H. D. Graham, eds.), AVT Publishing Company, Westport, Connecticut, pp. 235-267. 892. Spencer, C.M., Cai, Y., Martin, R., Gaffney, S. H., Goulding, P. N., Magnolato, D., Lilley, T. H., and Haslam, E.(1988). Polyphenol complexation-Some thoughts and observations. Phytochenlistry. 27:2397-2409, Review Article Number 35. 893. Amory, A. M., and Schubert, C. L. (1987). A method to determine tannin concentration by the measurement and quantification of protein-Tannin interactions. Oecologia (HeidelB.), 73:420-424. 894. Brune, M., Hallberg, L., and Skinberg, A.-B. (1991). Determination of iron-binding phenolic groups in foods, J. Food Sci., 56: 128-13 l .

180

Beier and Nigg

895. Butler, L. G. (1989). Sorghum Polyphenols. In To.ticants of Plant Origin, VolzrnzeIV Phenolics (P. R. Cheeke, ed.), CRC Press, Boca Raton, Florida, pp. 95-121. 896. Hagerman. A. E. (1938). Extraction of tannin from fresh and preserved leaves. J. Chenz. Eco~.,14:453-461. 897. Hagerman, A. E., and Butler. L. G. (1 989). Choosing appropriate methods and standards for assaying tannin, J. Cherrz. Ecol., 15:1795-1810. 898. Hatano, T., Yoshida, T., and Okuda. T. (1988). Chromatography of tannins, 111. Multiple peaks in high-performance liquid chromatography of some hydrolyzable tannins, J. CItroittcrtogr., 4351285-289. 899. Ittah. Y. (1991). BSA-bound persimmon tannin interactions with other proteins, J. Agric. Food Chem., 39:2119-2122. 900. Makkar, H. P. S. (1989). Protein precipitation methods for quantitation of tannins: A review, J. Agric. Food Chenl., 37: 1197-1202. 901. MakEtar, H. P. S., Dawra, R. K., and Singh, B. (1988). Determination of both tannin and protein in a tannin-protein complex, J. Agric. Food Chenl., 36:523-525. 902. Okuda, T., Yoshida, T.. and Hatano, T. (1989). New methods of analyzing tannins, J. Ncrt. Prod. (Lloydin), 52: 1-3 1. 903. Scalbert, A., Monties, B., and Janin, G. ( 1989). Tannins in wood: Comparison of different estimation methods, J. Agric. Food Chew., 37: 1324-1 329. 904. Liener. 1. E. (1980). Miscellaneous toxic factors. In Toxic Comtitzre~tsofP1crnt FoodstlrfSs, 2d Edition (I. E. Liener, ed.). Academic Press, New York, pp. 430-467. 905. Montedoro, G. F.. and Bertuccioli, M. (1988). Organoleptic importance of certain tannic fractions of aged red wines. In Frontiers of Flcrvor (G. Charalambous, ed.). Elsevier Science Publishers B. V., Amsterdam, pp. 687-696. 906. Rider, P. J., Marderosian. A. D.. and Porter, J. R. (1992). Evaluation of total tannins and relative astringency in teas. In Phellolic Compotlncls i n Food c r d Their Effects 011 Hecrlth I Analysis, Occurrence, & Cltemistn (C.-T. Ho, C. Y. Lee, and M.-T. Huang. eds.), ACS Symposium Series No. 506, American Chemical Society. Washington, D.C., pp. 93-99. 907. Okuda, T.. Yoshida, T.. Mori. K., and Hatano. T. (1981). Tannins of medicinal plants and drugs, Heterocyles, 15:1323-1335. 908. Lyman, B. J., and Green, B. G. ( 1990). Oral astringency: Effects of repeated exposure and interactions with sweeteners, Chem. Senses, 15:151-164. 909. Yonemori, K., and Matsushima, J. (1985). Property of development of the tannin cells in non-asgringent type fruits of Japanese persimmon (Diospyros h k i ) and its relationship to natural deastringency. J. Jpn. Soc. Hortic. Sci., 54201-208 (in Japanese). 910. Robichaud, J. L., and Noble, A. C. ( 1 990). Astringency and bitterness of selected phenolics in wine. J. Sci. Food Agric., 53:343-353. 911. Chandler. R. F., and Hooper. S. N.(1982). Herbal remedies of the Maritime Indians: A preliminary screening. Part 111. J. Ethnoyhni-n~acol., 6:275-285. 912. Tariq. M., Mossa, J. S., Al-Yahya, M. A., Al-Meshal, I. A., and Al-Badr. A. A. (1 987). Phytochemical and biological screening of Saudi medicinal plants. Part 10, A study on Saudi plants of family compositae, Int. J. Crude Drug Res., 25:17-25. 913. Anon. (1969). Tannic acid again! Food Cosnlet. Tosicol.. 7:364-365. 9 14. Reed, J. D. (1987). Phenolics, fiber. and fiber digestibility in bird resistant and non bird resistant sorghum grain, J. Agric. Food Chertl., 35:461-464. 915. Asquith, T. N.(1986). Evidence for functional heterogeneity both between and within four sources of condensed tannin, Dissertation Abstracts Itlternntioncrl,46:3821. 916. Asquith, T. N., Uhlig, J.. Mehansho. H., Putman, L., Carlson, D. M., and Butler. L. (1987). Binding of condensed tannins to salivary proline-rich glycoproteins: The role of carbohydrate, J. Agric. Food Chem., 35:331-334. 917. Butler, L. G., and Pushpamma, P. (1991). Tannins in betel nut and its products consumed in India: Comparison with high-tannin sorghum, J. Agric. Food Chew., 39:322-326.

Naturally Occurring Toxic Chemicals in Foods

181

918. Mahanta, P. K., and Baruah, S. (1992). Changes in pigments and phenolics and their relationship with black tea quality. J. Sci. Food Agric., 59:21-26, 919. Hernindez, T., Hernandez, A., and Martinez. C. (1991).Polyphenols in alfalfa leaf concentrates, J. Agric. Food Clwm., 39:1120-1122. 920. Kashket. S.. Paolino, V. J., Lewis, D. A., and van Houte, J. (1985). In-vitro inhibition of glucosyltransferase from the dental plaque bacterium Stwptococczrs vzutnns by common beverages and food extracts, Arch. Ora1 Biol., 30:821-826. 921. Puech, J.-L., Rabier, P., Bories-Azeau, J., Sarni, F., and Moutounet, M. ( 1990). Alcoholic beverages. Determination of ellagitannins in extracts of oak wood andin distilled beverages matured in oak barrels, J. Assoc. OffAnal. Chet?1.,73:498-501. 922. Eggum, B. 0.. Pedersen, B., and Jacobsen, I. (1983). The influence of dietary tea, coffee and cocoa on protein and energy utilization of soya-bean meal and barley in rats, BI: J. Nutr., 50:197-205. 933. Terrill, T. H., Rowan, A. M., Douglas, G. B., and Barry, T. N. (1992). Determination of extractable and bound condensed tanninconcentrations in forage plants, protein concentrate meals and cereal grains, J. Sci. Food Agric., 58321-329. 924. Bressani, R., Elias, L. G., Wolzak. A., Hagerman, A. E., and Butler, L. G. (1983). Tannin in common beans: Methods of analysis and effects on protein quality. J. Food Sci., 48: 1000- 1003. 925. Deshpande, S. S., and Cheryan, M. (1987). Determination of phenolic compounds of dry beans using vanillin, redox and precipitation assays, J. Food Sci., 52332-334. 926. Wassimi, N.N.,Hosfield, G. L., Uebersax, M. A. (1988).Combining ability of tannin content and protein characteristics of raw and cooked dry beans, Crop Sci., 28:452-458. 927. Lau, 0.-W.. Luk, S.-F., andHuang.H.-L. (1989).Spectrophotometric determinationof tannins in tea and beer samples with iron(111) and 1,lO-phenanthrolineas reagents, Aml?w, 114~631-633. 928. Saura-Calixto, F. (1988). Effect of condensed tannins in the analysis of dietary fiber in carob pods. J. Food Sci., 53:1769-1771. 929. Osuntogun, B. A., Adewusi, S. R. A., Telek, L., and Oke, 0. L. (1987). Theeffect of tannin content on the nutritive value of some leaf protein concentrates, H w ~ a nNzrtr.: Food Sci. N z ~ ~41 T . ,F:41-46. 930. Savolainen, H. (1992). Tannin content of tea and coffee. J. Appl. To.t-ico1.. 12: 191-192. 931. Clifford. M. N..and Ramirez-Martinez, J. R. (1991). Tanninsin wet-processed coffee beans and coffee pulp, Food Chenz., 40: 191-200. 933. Mugula, J. K. (1992). The nutritive quality of sorghum-com~nonbeantempe, Plunt Foods H ~ U TNUtr., I. 42:247-256. 933. Kumar. R.. and Vaithiyanathan, S. (1990). Occurrence, nutritional significance and effect on animal productivity of tannins in tree leaves, Anim Feed Sci. Teclznol., 30:21-38. 934. Vaillancourt, R., Slinkard, A. E., and Reichert. R. D. (1986). The inheritanceof condensed tannin concentration in lentil, C a n J. Plmt Sci., 66241-246. 935. Senter, S. D., and Callahan, A. (1990). Variability in the quantities of condensed tannins andothermajorphenolsinpeach fruit duringmaturation, J. Food Sci., 55:1585-1587. 1602. 936. Shahidi, F., and Naczk. M.(1989). Effect of processing on the content of condensed tannins in rapeseed meals. J. Food Sci., 53:1082-1083. 937. Onlolayo. 0. O., Wedzicha, B. L.. and Muller, H. G. (1988). On the interaction between tannins and aflatoxin B I in sorghum, Food Chenl., 28:239-241. 938. Watterson, J. J., and Butler, L. G. (1983). Occurrence of an unusual leucoanthocyanidin and absence of proanthocyanidins in sorghum leaves, J. Agric. Food Chenz., 31:41-45. 939. Senter, S. D., Horvat, R. J., and Forbus, W. R., Jr. (1988). Quantitative variation of total phenols in fresh market tomatoes at three stages of maturity, J. Food Sci., 53:639-640. 940. Huang. P.-J. D., Cash, J. N., and Santerre, C. R. (1988). Influence of stems, petioles and

182

941. 942.

943.

944.

945. 946. 947. 948.

949.

950.

951. 952.

953. 954.

955.

956. 957. 958.

Beier and Nigg leaves on the phenolic content of concord and aurora blanc juice and wine, J. Food Sci., 53:173-175. Wolinsky, L. E., and Sote, E. 0. (1984). Isolation of plaque-inhibiting substances from “Nigerian chewing sticks,” Curies Res., 15216-225. Smart, R. C., Huang, M.-T., Chang, R. L., Sayer, J. M., Jerina, D. M.. Wood, A. W., and Conney, A. H. (1986). Effect of ellagic acid and 3-0-decylellagic acid on the formation of benzo[a]pyrene-derived DNA adducts in vivo and on the tumorigenicity of 3-methylcholanthrene in mice, Carcinogenesis, 7: 1669-1 675. Smart, R. C., Huang, M.-T., Chang, R. L., Sayer, J. M., Jerina, D. M., and Conney, A. H. (1986). Disposition of the naturally occurring antimutagenic plant phenol, ellagic acid, and its synthetic derivatives, 3-0-decylellagic acid and 3,3’-di-O-methylellagic acid in mice, Cnrcirzogenesis, 7: 1663- 1667. Wood. A. W., Huang, M.-T.. Chang, R. L., Newmark. H. L., Lehr. R. E., Yagi, H., Sayer, J. M., Jerina, D. M., and Conney. A. H. (1982). Inhibition of the mutagenicity of bay-region diol expoxides of polycyclic aromatic hydrocarbons by naturally occurring plant phenols: Exceptional activity of ellagic acid, Proc. Nat. Acucl. Sci., 795513-5517. Akaranta, O., and Odozi, T. 0. (1986). Antioxidant properties of red onion skin (AIliLm cepa) tannin extract, Agric, Wastes, 18:299-303. Su, J.-D., Osawa, T.. Kawakishi, S., and Namiki, M. (1988). Tannin antioxidants from Osbeckia chinensis. Plzytoclzenzistry, 27:1315-1319. Villar, A.. Sanz, M. J., and Paya. M. (1987). Hypotensive effect of Pistacia leiztisczls L., Int. J. Crude Drug Res.. 25:l-3. Fukuchi, K.. Sakagami, H., Okuda, T.. Hatano, T., Tanuma, S., Kitajima, K., Tnoue, Y., Inoue, S., Ichikawa, S.. Nonoyama. M., and Konno, K. (1989). Inhibition of herpes simplex virus infection by tannins and related compounds, Antiviral Res.. 11:285-298. Nonaka, G.-I., Nishioka, I., Nishizawa, M., Yamagishi, T., Kashiwada. Y., Dutschman, G. E., Bodner, A. J., Kilkuskie, R. E., Cheng, Y.-C., and Lee, K.-H. ( 1990). Anti-AIDS AGENTS, 3: Inhibitory effects of tannins on HIV reverse transcriptase and HTV replication in H9 lymphocyte cells, J. Nnt. Prod. (Lloydia). 53587-595. Kashiwada, Y., Shi, H., Nonaka, G., Nishioka, I., Newbold, J. E., and Lee. K. (1991). Inhibitory effects of the phenolic constituents of rhubarb on cell cultures producing hepatitis B virus, Irzt. J. Orient. Mecl., 16:135-143. Yokozawa, T., Fujioka, K., Oura, H.. Nonaka, G.-I., and Nishioka, I. (1991). Effects of rhubarb tannins on uremic toxins, Nephron. 58:155-160. Huang, M.-T., Chang, R. L., Wood, A. W., Newmark, H. L., Sayer, J. M., Yagi, H.. Jerina, D. M., and Conney, A. H. (1985). Inhibition of the mutagenicity of bay-region diol-epoxides of polycylic aromatic hydrocarbons by tannic acid, hydroxylated anthraquinones and hydroxylated cinnamic acid derivatives, Carcinogenesis, 6:237-242. Ramanathan, R., Tan, C. H., and Das, N. P. (1992). Cytotoxic effect of plant polyphenols and fat-soluble vitamins on malignant human cultured cells, Cmcer Lett., 62217-224. Gali, H. U., Perchellet, E. M.. and Perchellet, J.-P. (1991). Inhibition of tumor promoterinduced ornithine decarboxylase activity by tannic acid and other polyphenols in mouse epidermis in vivo, Cancer Res., 51:2820-2825. Kashiwada. Y., Nonaka, G.-I.. Nishioka, I., Chang, J.-J., and Lee, K.-H. (1992). Antitumor agents, 129. Tannins and related compounds as selective cytotoxic agents, J. Nat. Prod., 5511033-1043. John. T. J., and Mukundan, P. (1979). Virus inhibition by tea, caffeine and tannic acid, Indim J. Med. Res., 69:542-545. Zhang, Y. (1983). Effect of tea tannin on intestinal movement in vitro, J. Tradit. Chin. Med., 3271-272 (in Chinese). Ahn, Y.-J., Kawamura, T., Kim, M., Yamanloto, T., and Mitsuoka, T. (1991). Tea polyphenols: Selective growth inhibitors of ClostridiLm spp., Agric. Biol. Clrem., 55: 1425-1426.

Naturally Occurring Toxic Chemicals in Foods

183

959. Yamaguchi, Y., Hayashi, M., Yamazoe, H., and Kunitomo. M. (1991). Preventive effects of green tea extracton lipid abnormalities inserum, liverand aorta of mice fed a atherogenic diet, Folio Plmrrnacol. J a p . , 97:329-337 (in Japanese). 960. Xu, Y.. Ho, C.-T., Amin. S. G., Han, C., and Chung, F.-L. (1992). Inhibition of tobaccospecific nitrosamine-induced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants, Cancer Res., 523875-3879. 961. Huang. M.-T., Ho, C.-T.. Wang, Z. Y., Ferraro. T., Finnegan-Olive, T., Lou. Y.-R., Mitchell, J. M., Laskin, J. D., Newmark, H., Yang, C. S., and Conney. A. H. (1992). Inhibitory effect of topical application of a green tea polyphenol fraction on tumor initiation and promotion in mouse skin. Carcinogenesis, 13:947-954. 963. Worthy. W. (1991). Fruits, vegetables, green tea may cut cancer risk,Cllem. E q . N., (September16):27-29. 963. Muramatsu, K., Fukuyo, M.. and Hara, Y. (1986). Effect of green tea catechins on plasma cholesterol level in cholesterol-fed rats, J. Nzrrr. Sci. Vitam., 32613-622. 964. Uchida, S., Ozaki, M., Suzuki, K., and Shikita, M. (1991). Radioprotective effects of (-)epigallocatechin 3-0-gallate (green-tea tannin) in mice, Life Sci., 50:147-152. 96s. Sagesaka-Mitane, Y., Miwa, M., and Okada, S. (1990). Platelet aggregation inhibitors in hot water extract of green tea. Chenz. P h a m . BdI. (ToLyo). 38:790-793. 966. Jain. A. K., Shimoi. K., Nakamura, Y., Kada, T., Hara, Y.. and Tomita, I. (1989). Crude tea extracts decreasethemutagenicactivityof N-methyl-N’-nitro-N-nitrosoguanidine in vitro and intragastric tract of rats, Mutat. Res., 21O:l-8. 967. Imanishi, H., Sasaki, Y. F., Ohta, T.. Watanabe. M., Kato. T., and Shirasu, Y. (1991). Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cellsand mice, Mutat. Res., 259:79-87. 968. Ito, Y.. Ohnishi, S., and Fujie, K. (1989). Chromosome aberrations induced by aflatoxin B I in rat bone marrow cells in vivo and their suppression by green tea, Mutat. Res., 222: 253-261. 969. Khan, W. A., Wang, Z. Y.. Athar, M., Bickers, D. R., and Mukhtar, H. (1988). Inhibition of the skin tumorigenicity of (~)-7~,8a-dihydroxy-9a,lO~-epoxy-7,8,9,lO-tetr~ydrobenzo[a]pyrene by tannic acid, green tea polyphenols and quercetin in Sencar mice, Comer Lett.. 42:7-12. 970. Ruch, R. J., Cheng,S.-J., and Klaunig, J. E. (1989).Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea, Carcinogerzesis, 10: 1003-1008. 971. Rosen, S., Elvin-Lewis, M., Beck, F. M., and Beck, E. X. (1984). Anticariogenic effects of tea in rats, J. Derrtal Res., 63:658-660. 972. Bokuchava, M. A., and Skobeleva, N. I. (1980). The biochemistry and technology of tea manufacture, CRC Crit. Rely. Food Sci. Nutr., 12:303-370. 973. Rohrbach, M. S., Rolstad, R. A., Tracy. P. B., and Russell, J. A. (1984). Platelet 5-hydroxytryptamine release and aggregation promoted by cotton bracts tannin, J. Lab. Clin. Men., 103:152-160. 974. Wang, R. S. (1989). Niacin bioavailability as affected by tannin from tea and sorgum, Diss. Abs. hlt.. 49:3 117-b. 97s. Sahle, M., Coleou, J., and Haas, C. (.1992). Carob pod (Ceraronin siliqzca) meal in geese diets, BY. Poult. Sci., 33:531-531. 976. Disler, P. B., Lynch, S. R.. Charlton, R.W., Torrance, J. D., Bothwell, T. H., Walker, R. B.. and Mayet, F. (1975). The effect of tea on iron absorption, Gut, 16:193-300. 977. Disler, P. B., Lynch, S. R., Torrance, J. D., Sayers, M. H.. Bothwell, T. H., and Charlton, R, W. (1975). The mechanism of the inhibition of iron absorption by tea, South A@. J. Med. Sci., 40:109-1 16. 978. Chibata, I., Tosa, T., Mori,T., Watanabe, T., and Sakata, N. (1986). Immobilized tanninA novel adsorbent for protein and metal ion, Enzyme Microb. Teclznol., 8:130-136.

184

Beier and Nigg

979. Hilker, D. M.. and Somogyi, J. C. (1982). Antithiamins of plant origin: Their chemical nature and mode of action, A m . N. Y. Acad Sci., 378:137-135. 980. Lasswell, W. L., Jr., Weber. S. S.,and Wilkins, J. M. (1984). In vitro interaction of neuroleptics and tricyclic antidepressants with coffee, tea, and gallotannic acid, J. Plznrrrz. Sci., 73: 1056-1058. 98 1. Bogoviski, P., Day, N., Chvedoff. M., and Lafaverges. F. (1977). Accelerating action of tea on mouse skin carcinogenesis. Cancer Lett., 3:9-13. 982. Kapadia, G. J., Paul, B. D., Chung, E. B.. Ghosh, B., and Pradhan, S. N.(1976). Carcinogenicity of Camellia sinensis (tea) and some tannin-containing folk medicinal herbs administered subcutaneously in rats, J. Nat Cancer Inst., 57207-209. 983. Panigrahi, G. B., and Rao, A. R. (1986). Study of the genotoxicity of the total aqueous extract of betel nut and its tannin. Carcinogenesis, 7:37-39. 984. Yang, J. A.. Huber. S. A., and Lucas. Z. J. (1979). Inhibition of DNA synthesis in cultured lymphocytes and tumor cells by extracts of betel nut, tobacco, and miang leaf, plant substances associated with cancer of the ororespiratory epithelium, Cancel- Res., 39:48024809. 985. IARC ad-hoc Working Group (1987). Betel quid with tobacco (group 1) and betel quid without tobacco (group 3). In: IARC Monographs on the E~duationsof Carcinogenic Risks to H m a n s . Ollerall Evaluations of Carcinogenicity: An Upclating of IARC Monogr-aplzs, Volumes 1 to 42. Supplement 7. WHO International Agency for Research on Cancer, Lyon, France. pp. 128-130. 986. Morton, J. F. (1987). Tannin and oesophageal cancer, Lancet, (August 8, 1957):327-328. 987. Morton. J. F. (1979). Tea with milk, Scieizce, 204:909. 988. Wheeler. S. R. (1979). Tea and tannins, Science. 204:7-8. 989. Hilker, D. M. (1979). Carcinogens occurring naturally in food. Nutr. Cancel-, 2217-223. 990. Groves, F. D., Zavala, D. E., and Correa, P. (1987). Variation in international cancer mortality: Factor and cluster analysis, Irzt. J. E~idenziol.,16501-508. 991. Fluss, L.. Nguyen, T.. Ginther. C., and Leighton, T. (1990). Reduction in the direct-acting mutagenic activity of red wine by treatment with polyvinyl-polypyrrolidone. J. Wine Res., 1:35-43. 993. Fish, B. C.. and Thompson, L. U. (1991). Lectin-tannin interactions and their influence on pancreatic amylase activity and starch digestibility, J. Agl-ic. Food Chem., 39:727-731. 993. Hagerman. A. E., and Robbins, C. T. (1987). Implications of soluble tannin-protein complexes for tannin analysis and plant defense mechanisms, J. Clzenz. Ecol., 13:12431259. 994. Deichmann, W. B., Henschler. D., Holmstedt, B.. and Keil. G. (1986). What is there that is not poison? A study of the Third Defense by Paracelsus. Arch. Tosicol., 58207-213. 995. Dot-land's Illustrated Medical Dictionar?. 27th Edition, S.V."hormesis." W. B. Saunders Co, Harcourt Brace Jovanovich, Inc.. Philadelphia. 996. Calabrese, E. J.. McCarthy. M. E., and Kenyon. E. (1987). The occurrence of chemically induced honnesis, Health Phys., 52:53 1-541. 997. Davis, J. M., and Svendsgaard, D. J. (1990). U-shaped dose-response curves: Their occurrence and implications for risk assessment, J. Toxicol. Enjiron. Health, 30:71-83. 998. Laughlin, R. B.. Jr.. Ng. J.. and Guard, H. E. (1981). Hormesis: A response to low environmental concentrations of petroleum hydrocarbons. Science, 2 1 1:705-707. 999. Sagan, L. A. (1987). What is hormesis and why haven't we heard about it before? Health Phys., 52:521-525. 1000. Stebbing, A. R. D. (1982). Hormesis-The stimulation of growth by low levels of inhibitors, Sci. Total Em~iron.,2213-234. 1001. Shane, B. S.. Littrnan, C. B., Essick. L. A.. Gutenmann, W. H., Doss. G. J., and Lisk. D. J. (1988). Uptake of selenium and mutagens by vegetables grown in fly ash containing greenhouse media, J. Ayric. Food Chent.. 36:328-333.

Naturally Occurring Toxic Chemicals in Foods

185

1002. Ames, B. N., and Gold, L. S. (1989). Pesticides, risk, and applesauce, Science. 244:755757. 1003. Ames, B. N., Profet, M., and Gold, L. S . (1990). Dietary pesticides (99.99% all natural), Proc. Nrrt.Acncl. Sci., 87:7777-7781. 1004. Ames, B. N., Profet, M., and Gold, L. S. (1990). Nature’s chenlicals and synthetic chemicals: Comparative toxicology, Proc. Nnt. Acad. Sci.. 87:7782-7786. 1005. Ames. B. N., and Gold. L. S. (1990). Chemical carcinogenesis: Too many rodent carcinogens. Pt-oc. Nut. Acacl. Sci., 87:7772-7776. 1006. Ames, B. N., and Gold, L. S. ( 1990). Too many rodent carcinogens: Mitogenesis increases mutagenesis, Science, 239:970-97 1. 1007. Monro, A. (1992). What is anappropriate measure of exposurewhentesting drugs for carcinogenicity in rodents? Tosicol. Appl. P/zarmrcol.,1 12:17 1 181. 1008. Hildebrandt, A. G. (1987). Overdosetoxicity studies versus threshold: Elements of biology must be incorporated into risk assessment, Arch. Toxicol., 60:217-223. 1009. Wagner, B. M. ( 1992). Returning biology to carcinogenicity testing,Toxicol. Appl.PhcrrmCOI., 112:169-170. of 1010. Gunderson, E. L. (1988). FDA total diet study, April 1982-April 1984, dietary intakes pesticides, selected elements, and other chemicals, J. Assoc. 08 Anal. Chem., 71:12001209. (1988). Residues in foods--1987. J. 1011. Foodand Drug AdnlinistrationPesticideProgram ASSOC.08 Allal. C h ~ ~ ~71: ~ 156A-174A. r., 1012. 1987 Pesticide Residue Armunl Reports. California Department of Food and Agriculture, 1988, p. 1-32. 1013. Yess, N. J., Gunderson, E. L., and Roy, R. R. (1993). U.S. Food and Drug Administration monitoring of pesticide residues in infant foods and adult foods eaten by infantdchildren, J. Assoc. OffAnul. Chem., 76:392-507. 1014. Ames. B. N., and Gold, L. S. (1993). Environmental pollution and cancer: Some misconceptions. In Phantom Risk (P. W. Huber and K. R. Foster, eds.), MIT Press,Cambridge. Massachusetts, pp. 153-181. 1015. Gold. L. S., Slone, T. H., Stern, B. R., Manley, N. B., and Ames, B. N. (1992). Rodent carcinogens: Setting priorities, Science, 258:261-265.

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Poisonous Higher Plants Doreen Grace Lang and R. A. Smith U n i ~ ~ e r so i fh KejztucL?, Le.~ir.rgton,Kerzttrch?

1S9

I. Introduction

IT. Plant Families Containing Particularly Troubleso~neSpecies 189

Aceraceae

Amaranthaceae

189

190

Anacardiaceae

191

Apocynaceae Araceae

192.

194

Asclepiadaceae Asteraceae

194

Berberidaceae

196

Boraginaceae

197

Brassicaceae

198

Buxaceae

189

199

Carnpanulaceae

199

Cannabinaceae

199

Caryophyllaceae 200 Chenopodiaceae 200 Corynocarpaceae 201 Crassulaceae 202 Cucurbitaceae 202

203

Dennstaedtiaceae

Dichapetalaceae 203 Dioscoreaceae 204 Ephedraceae 204 Equisetaceae 204 Ericaceae 205 Erythroxylaceae Euphorbiaceae Fagaceae

206

207

209 187

Lang and Smith

Fumariaceae 209 Gramineae 2 10 Hippocastanaceae

212

Hypericaceae 2 12 Iridaceae 2 13 Juglandaceae 2 13 Juncaginaceae 2 13 Lamiaceae 213 Lauraceae 2 14 Leguminosae(orFabaceae)

215

Liliaceae 220 Loganiaceae 222 Loranthaceae 323 Lycopodiaceae

223

Malvaceae 223 Meliaceae

224

Myoporaceae 224 Oleaceae 224 Palmae

334

Papaveraceae

225

Pinaceae 226 Polygonaceae 226 Ranunculaceae 227 Rhamnaceae 229 Rosaceae 229 Rubiaceae Rutaceae

230 230

Santalaceae23

1

Sapindaceae23

1

Saxifragaceae 232 Scrophulariaceae 232 Solanceae 232 Taxaceae 235 Thymelaeaceae 235 Umbelliferae

236

Urticaceae 237 Verbenaceae 237 Zygophyllaceae 238 Appendix: Alphabetical List of All Species Discussed i n This Chapter 238 References

24 1

Poisonous Higher Plants

1.

189

INTRODUCTION

In the first edition one of us (RAS) pointed out the remarkable association between plant families as determined by taxonomic botany and the toxins that they contained as organized by chemists. At that time I pointed out that a veritable plethora of plant toxins was found in the Leguminosae: since then the botanists themselves have renamed the family the Fabaceae and have now recognized three subfamilies: the Mimosoideae, the Papilonoideae, and the Caesalpinoideae. Where possible we have given the subfamily for toxic members of the Fabaceae. On reading the present chapter, I find myself wondering about the plethora of toxins in two more families, the Ranunculaceae and the Solanaceae. Only time will tell if these two will come to be divided into subfamilies. The authors from whom we have taken most guidance are Cronquist and Gleason (1991), Kingsbury (1964) and Heywood (1993). Cronquist was, and Heywood is, primarily a botanist with little interest in phytotoxicology. Most readers will be familiar with Kingsbury (a botanist first and a phytotoxicologist second), who considered only poisonous plants of the United States and Canada. Cronquist deals at great length with all of the vascular plants of the northeastern United States and adjacent Canada. Heywood, on the other hand, who discusses the flowering plants ofthe world, is short on detail, but manages to cover the flowering flora globally. The previous grouping of plants into divisions has been dropped in favor of a system based on families alone, arranged in alphabetical order. This will, hopefully, make the book more useful to poison control centers and some other readers.

II. PLANTFAMILIESCONTAININGPARTICULARLY TROUBLESOME SPECIES Aceraceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Acet-.

A. rr~brzmL. (red maple) Throughout temperate North America, Asia, and Europe. Wilted leaves. An unknown oxidant. Hemolytic anemia, methemoglobinemia, icterus, in horses, abortion, and in some cases death. (Stair et aI., 1993; Weber et al., 1997). Horses, cattle, and zebra.

Amaranthaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin:

Ar?m-crnfhrls. A. retroJe.ws (red-root pigweed). Ubiquitous weed. Whole plant. In earlier works, nitrate was cited, but the situation is more complicated than that (Cheeke and Shull, 1985). Oxalates (Murphy, 1996).

190

Symptoms:

Animals affected: Treatment:

Lang and Smith

For nitrates symptoms include perirenal edema, labored breathing, abortions, methemoglobinemia, trembling, and death. For oxalates acute: salivation, weakness, bradycardia, labored breathing, convulsions, and death (Murphy, 1996). For oxalates chronic: anorexia, weight loss, depression, diarrhea, and death (Murphy, 1996). Cattle and pigs. Remove animals from the source of the plant. Treatment for oxalates: give fluids and “dicalcium phosphate [and] sodium chloride; 1:3 [ratio] to form calcium oxalate in the gastrointestinal tract thus preventing oxalate absorption” (Murphy, 1996). Treatment for nitrates; “a 1% solution of methylene blue is generally given at a dose of 4-15 m g k g at 4-6 hour intervals as needed” (Murphy, 1996).

Anacardiaceae Genus: Species: Distribution and habitat:

Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Notes:

Toxicoderdron. T. diversilobmr (T. & G.) (poison oak), T. rnclicnrrs (L.) (poison ivy), T. vernix (L.) (poison sumac). T. clilJersilobais found in western North America. T. mdicnns grows throughout North America (with the exception of the West coast), Central America, Bermuda, the Bahamas, Japan, Taiwan, and in central and western China. T. wrnix is found in eastern North America, in bogs and wetlands (Kingbury, 1964). Whole plant. 3-Pentadecylcatechol and urushiol, oleoresins. Dermatitis upon contact with the plant, chewing leaves can cause inflammation of the oral mucosa. The smoke of burning poison ivy may contain particulates containing oleoresin and may cause lung irritation. ‘‘Hyperpigmentation is a common complication of poison ivy dermatitis in black skin but is rare in white skin“ (Fisher, 1996). Humans and primates. Sewcarpus. S. nrritcardirm. India. Sap. 3-Pentadecylcatechol and urushiol, oleoresins. Dhobi mark dermatitis (Fisher, 1996). Humans. S e n ~ e c a r p sa~~crcnrdium “is used to make laundry marks in India. . . . Once marked, the clothing may cause dermatitis indefinitely because washing or boiling does not destroy the black mark nor does it render the ink inert” (Fisher, 1996).

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191

Apocynaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Ackocnnthern. A. oblongifolia (wintersweet). Africa in damp locations. All, but young growing parts are especially toxic. Ouabain and ackocantherin, which are cardiac glycosides. Rapid heart rate, arrhythmia, bradycardia, and ventricular fibrillation in the terminal stages. When a sublethal dose has been consumed, there is evidence of labored breathing, urination, and defecation developing into diarrhea. Extracts of this plant have been used as arrow poisons (Cheeke, 1989b).

Genus: Species: Distribution and habitat: Poisonous portions: Toxin:

Nerium. N. oleander L. (common oleander). Now global, but native to the eastern Mediterranean. All, but especially leaves and twigs. Oleandrin, oleandroside and neroside, which are cardiac glycosides ‘‘(oleandrigenin is the aglycone of oleandrinj and neriine (Galey et al., 1996);’ folinerin, digitoxigenin. Sudden death usually occurs with oleander poisoning. Rapid heart rate, arrhythmia, bradycardia, and ventricular fibrillation in the terminal stages. “Excitement, intermittent convulsions, dyspnea, and coma may precede death, whichmay occur within 2 or as late as 12-36 hours after the onset of signs” (Galey et al., 1996). When a sublethal dose has been consumed, there is evidence of labored breathing, urination, defecation developing into diarrhea, lethargy, nausea, vomiting, increased salivation, colic, headache, numbness of the tongue, slurred speech, altered mental abilities, and visual disturbances. “The visual disturbances associated with oleander toxicity are rather peculiar, with victims sometimes reporting seeing yellow and green colors intermixed with geometric patterns surrounding objects in the visual field’‘ (Langford and Boor, 1996). “Contact dermititis may result from skin contact to the sap of freshly cut leaves of oleanders” (Langford and Boor, 1996). Humans, geese, cattle, llama, horses, rhea, big horn sheep, dogs, goats, buffalo, and bear. Removal of unabsorbed toxins. Supportive measures designed at maintaining hemodynamic stability. Administration of cholestyramine. Fab fragments from specific antibodies directed against digoxin bind oleander-derived glycosides in both humans and canines. (Langford and Boor, 1996). Plant clippings are the most frequent source of oleander poisoning in cattle in California (Galey et al., 1996). “Oleander is an extremely toxic plant; as little as 0.005% of an animal’s body weight in dry oleander leaves may be lethal (e.g., 1020 leaves for an adult horse)’’ (Kingsbury, 1964). “There are

Symptoms:

Animals affected: Treatment:

Notes:

192

Lang and Smith

numerous reports from around the world of accidental animal poisonings from eating parts of oleander plants or ingestion of water contaminated with oleander toxins. . . . most of the accidental oleander ingestions . . . are the result of animals consuming oleander contaminated feed, rather than being intoxicated through foraging on fresh plant material'' (Langford and Boor, 1996). In one case a woman prepared tea from oleander leaves, which she mistook for eucalyptus (Haynes et al., 1985). Genus: Species:

Animals affected: Treatment: Notes:

Thevetin. T. yeruvima Schum. (= T. riel-eifolicr Juss.) (yellow oleander, be-still tree). All.but especially the seeds and leaves. Cardiac glycosides seem to be in higher concentrations when plant is flowering (Langford and Boor, 1996). Oleandroside, thevetin A, thevetin B, thevetoxin, which are cardiac glycosides. Neriifolin, peruviside, nlvoside. Rapid heart rate, arrhythmia, bradycardia, and ventricular fibrillation in the terminal stages. When a sublethal dose has been consumed, there is evidence of labored breathing, urination, and defecation developing into diarrhea. The yellow oleander is considered to be the most dangerous plant on Oahu, Hawaii (Kingsbury, 1964). Thesymptoms are the same as for N . olennder. Humans, birds. The treatment is the same as for N. olenrlder. Same as above.

Genus: Species: Poisonous portion: Toxin: Symptoms:

Urechitis. U. s1rberectlls. Whole plant. Urechitin and urechitoxin, which are cardiac glycosides. The same as for Ackocmthem.

Poisonous portions:

Toxin: Symptoms:

Araceae Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms:

Animals affected:

Ar-isrremn. A. Atrorubem (jack-in-the-pulpit. indian turnip). Northern Hemisphere, mostly in the Old World. Whole plant. Oxalates. Intense burning sensation and irritation of the mucous membranes (Muenscher, 1951) evidenced by salivation, head shaking, pawing at the mouth, dyspnea, vomiting, diarrhea, and dehydration. This can lead to electrolyte imbalance and shock. (Murphy, 1996). Cats, dogs.

Poisonous Higher Plants

Treatment:

Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

793

Apomorphine (an emetic) and charcoal. Supportive treatment involves rinsing mouth, antihistamines if dyspneic, give fluids if dehydrated, and glucocorticoids. “Oral calcium (milk) may help precipitate any soluble oxalates inthe gastrointestinal tract thus preventing their absorption and preventing renal oxalosis” (‘Murphy, 1996). “Ingestion of ornamental and/or household plants by pets is primarily influenced by non-seasonal factors, i.e., age. boredom, or environmental changes, therefore, if the plant is available in or around the house it may be ingested at any time of year” (Murphy, 1996). Cakdium. Various (angel’s wing). Popular houseplant. Whole plant. Calcium oxalate crystals. “Ingestion causes immediate intense pain, local irritation to mucous membranes, excessive salivation, swollen tongue and pharynx, diarrhea, and dyspnea” (Aiello and Mays, 1998). Dogs, cats. “Emetic (apomorphine), charcoal. Oral calcium (milk) may help precipitate any soluble oxalates inthe gastrointestinal tract thus preventing their absorption and preventing renal oxalosis. [Supportive treatment] rinse mouth; antihistamines (if dyspneic), fluids (if dehydrated), glucocorticoids‘’ (Murphy, 1996).

Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms:

Colocasia. C. mtiquorml (elephant’s ear). Widely dispersed i n tropical re,’~ l 0 l l S . Whole plant. Oxalates. Intense burning sensation and irritation of the mucous membranes (Muenscher, 1951).

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Diefleel1bucl1in. D. seyuine. Global, common omamental indoor plant. Whole plant. Oxalates. Intense burning sensation and irritation of the mucous membranes (Muenscher, 1951) evidenced by salivation, head shaking, pawing at the mouth, dyspnea, vomiting, diarrhea, and dehydration. This can lead to electrolyte imbalance and shock (Murphy, 1996). Humans, dogs. and cats. Apomorphine (an emetic) and charcoal. Supportive treatment involves rinsing mouth, antihistamines if dyspneic, give fluids

Animals affected: Treatment:

Lang and Smith

194

Notes:

Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms:

if dehydrated, and glucocorticoids. “Oral calcium (milk) may help precipitate any soluble oxalates inthe gastrointestinal tract thus preventing their absorption and preventing renal oxalosis” (Murphy, 1996). “Ingestion of ornamental and/or household plants by pets is primarily influenced by non-seasonal factors, i.e., age, boredom, or environmental changes, therefore, if the plant is available in or around the house it may be ingested at any time of year’ ’ (Murphy, 1996). Philoderdr-on.

P. coi’d~ltldm. Global, common ornamental indoor plant. Whole plant. Oxalates. Intense burning sensation and irritation of the mucous membranes (Muenscher, 1951).

Asclepiadaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment: Notes:

Asclepias. A. erioccrrpcr, A. lrbl-{fonuis (milkweeds). Global. All. Eriocarpin and labriformin (cardiac glycosides). Cardenolides inhibit the functioning of the heart muscle, resulting in depression, weakness, ataxia, tetanic seizures, staggering, collapse, labored respiration, and death. Stock losses have been reported in Australia, South Africa, and North America (Clarke and Clarke, 19673. Horses, cattle, dogs, cats, sheep, goats, and chickens. “Treatment is rarely possible. Charcoal, mineral oil, [and] saline cathartics” (Murphy, 1996) should be used. ‘‘Calciurn containing solutions are CONTRAINDICATED since milkweeds are associated with increase intranlyocardial calcium concentration” (Murphy, 1996).

Asteraceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Eupntoriun~. E. rugoswn Houtt. Eastern Canada to Saskatchewan, south to eastern Texas to Georgia (Kingsbury, 1964). Whole plant, either fresh or less toxic when dried or in hay. Tremetol. Muscle tremors; ataxia; heavy sweating; reluctance to move; sluggish behavior; stiff gait; myoglobinuria; listlessness: at-

Poisonous Higher Plants

Animals affected: Treatment:

Notes:

195

tempting to eat, but drinking little: constipation with passage of small amounts of compact feces; dark urine; slobbering; anorexia; greenish discharge from nostrils; labored or accelerated respiration; (in horses) if made to exercise, will abruptly stand still with feet wide apart as though to prop itself up and begin to tremble; prostration and death. 1-10% of an animal‘s body weight of the green plant may be lethal to cattle, sheep, and horses, whether ingested at one time or in successive small doses. Signs occur less than 2 days to 3 weeks after ingestion. Death occurs 1 day to 3 weeks after first signs of symptoms appear (for horses, generally within 1-3 days after symptoms appear) (Kingsbury, 1964: Olsen et al., 1984) Horses, sheep, cattle, hogs, chickens, and humans. For horses, cattle, and sheep: removal from pasturage containing the plant: milking lactating animals; and treatment to counter ketosis, relieve constipation, and promote elimination of toxins (Kingsbury, 1964). “Since the days of the American Revolution, an afebrile disease affecting human beings, characterized by weakness, nausea, and prostration, has occassionally reached epidemic proportions in certain areas of the United States, locally and sporadically causing loss of human life second to no other disease. . . . In time, it became associated inthe minds of the early settlers with the ingestion of milk from cattle that were themselves ill, and was designated ‘milksickness.’ (Kingsbury, 1964) Tremetol in milk, cheese, butter, and possibly meat from affected animals caused a 50% mortality rate in affected areas and it is “stated that this disease was responsible for the death of Abraham Lincoln’s mother.” (Kingsbury, 1964). ”

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment: Notes:

Senecio. Numerous worldwide; S. jncoben (stinking Willie, tansy ragwort). The genus has global distribution. Whole plant. Retrorsine, ruwenine, and rusorine (Naude et al., 1996), which are pyrrolizidine alkaloids (PAS), are hepatotoxins. Acute poison symptoms are depression, weakness, ruminal stasis, constipation, and icterus. Chronic symptoms are rough hair coat, head pressing, emaciation, depression, stupor, aimless wandering, locomotor disturbance, and frenzy (Murphy, 1996; Naude et al., 1996). Horses, cattle, sheep, goats, humans, and pigs. Remove animals from access to the plant. Most human fatalities are from the consumption of commercially available ‘‘health food” teas. Pigs are very susceptible to senecio alkaloids (Hooper, 1978).

196

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Treatment:

Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Lang and Smith

Verbesim. V. erzcelioides (crownbeard, wild sunflower). Native to North America, naturalized in Australia, found in Argentina; grows in modified soils in spring and summer. Whole plant. Nitrite, nitrate, and galegine (3-methyl-2-butenylguanine). Dullness and anorexia, “blood-tinged fluid flowing from nostrils’, (Lopez et al., 1996), death from respiratory arrest. Cattle, sheep, and pigs. There is no tun-ent treatment regimen for this plant intoxication. The only action listed is to remove the animals to other fields. Animals generally avoid the plant unless no other forage is available. “. . . is considerably toxic, as determined by the minimum lethal dose, and as such ranks among the most toxic plants in this Argentinian region” (Lopez et al., 1996). XLUI th i m . X. strzmlnrirrnl (cocklebur). Global in areas under water in spring and that dry out in summer. Seedlings, also seeds (not normally consumed due to outer spiny covering, burs) (Kingsbury, 1964). Carboxyatractyloside (a glycoside). Hunched posture, nausea, vomition, prostration, dyspnea, anorexia, weak heartbeat, muscle weakness. ‘ ‘Opisthotonus, spasmodic running motions, or convulsions are sometimes displayed by severely poisoned, prostrated animals” (Kingsbury, 1964) coma, and death. Cattle, horses, sheep, pigs, and chickens. Supportive treatment: “oral administration of fatty substances may slow absorption (e.g., milk in monogastrics)” (Murphy, 1996). Remove the animal from the source of the plant.

Berberidaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Cnrrlopllylll4m. C. thcrlictroicles (blue cohosh). Eastern North America. Berries and roots. N-Methyl-cytisine. N-Methyl-cytisine is hallucinogenic. A cytotoxin is also present (Lampe and McCann, 1985).

Genus: Species:

Podopllylllrnl. P. peltufrrrn L. [mayapple, mandrake (North American usage 0nlY)lNorth America in wet meadows and woods. Root and young shoots.

Distribution and habitat: Poisonous portion:

Poisonous Higher Plants

Toxin: Symptoms:

Animals affected:

197

Podophyllin, a cytotoxin. The plant itself is not palatable and most cases of toxicity arise from the ingestion of “medicinal” preparations. Victims undergo a severe gastroenteritis and display vomition (Kingsbury, 1964; McIntosh, 1928). Humans, cattle, pigs, and sheep.

Boraginaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Genus: Species: Distribution and Habitat: Poisonous portion: Toxin: Symptoms:

Aminckin. A. irztemedicr (tarweed, fiddleneck). Western North America. All. Pynolizidine alkaloids (PAS), including lycopsamine, intermedine, echiumine, and sincamidine. PAS from this family destroy the liver; symptoms are those of hepatotoxicity. Cattle deaths have been reported. Cattle. Eclliunz. E. yln~ztngineurnand others (Paterson’s curse, Paterson being the individual responsible for its introduction to Australia). Now global, native to countries surrounding the Mediterranean. All. PAS, including echimidine and echiumine. PAS destroy the liver: symptoms are those of hepatotoxicity. Sheep have been poisoned in feeding trials (Seaman et al., 1989). Sheep. Heliotr.opium. H. nrrzylexiccrde (blue heliotrope) and others. Global, native to Europe. All. PAS, including lasiocarpine, heliotrine, europine, and heleurine. PAS destroy the liver; symptoms are those of hepatotoxicity. The deaths of cattle have been reported (Ketterer et al., 1987). Cattle.

Syn1ylzytum. S. oficirlule (comfrey). Eurasia. The root is the most dangerous part. PAS, including symphytine, lasiocarpine, and heliotrine. PAS destroy the liver; symptoms are those of hepatotoxicity. Sales of comfrey are tobe restricted in Australia (Abbott, 1988).

198

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Lang and Smith

Tricodesmn. T. incanurn and others. Tropical Africa and Asia. All. PAS, including tricodesmine and incanine. PAS destroy the liver; symptoms are those of hepatotoxicity. Contamination of grain with Tricodesmn incnrzurn led to bread that caused an epidemic that killed 44 people (Cheeke, 1989a). The symptoms were not those normally seen, but the cooking process may have altered the toxins. Death was from respiratory paralysis. Humans.

Brassicaceae Genus: Species:

Distribution and habitat: Poisonous portion: Toxin:

Symptoms:

Animals affected: Treatment:

Notes: Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms: Animals affected: Treatment:

Brassica. Numerous, including mustards, charlock, kale, rape, brussels sprouts, cabbage, cauliflower, broccoli, kohlrabi, rutabaga, and turnip. Global, mostly cultivated plants. Whole plant. Mustards contain isothiocyanates; many of the other species contain ~-5-vinyl-2-thiooxazolidone, a goitrogenic substance, or nitrates. Constipation, icterus of mucous membranes, in some cases blindness, photosensitivity, hemoglobinuria, reddish-brown urine, and anemia (Kingsbury, 1964). Thiocyanates and isothiocyanates cause severe erosion of the gastrointestinal (GI) tract (Kernaleguen et al., 1989). Thiooxazolidones inhibit thyroid function and nitrates cause liver and kidney lesions. Cattle, horses, sheep, goats, dogs, cats, chickens, and pigs. Removal of animal from access to the plant. Supportive treatment involves demulcents. In cases of photosensitivity, move animals to shelter and give antibiotics (penicillin, orally) and steroids topically or systemically (Murphy, 1996). It is mostly found "as a contaminant in grain (oats, wheat, etc.)" (Murphy, 1996). Thlnsyi. T. nr-velzse (fanweed). Eurasia and North America, especially in disturbed areas. All, especially the seeds. Sinigrin and the enzyme myrosin [which forms Allylisothiocyanate (AITC) in the rurnen]. Severe erosion of the GI tract (Smith RA et al., 1987a). Cattle. Removal of animal from source of fanweed. Formation of allylisothiocyante can be halted by lowering the ph to 5 by orally

Poisonous Higher Plants

199

administering a gallon of vinegar shortly after consumption. The AITC already formed can be countered by administering an antheltnintic such as piperazine (Smith and Crowe, 1987a). Buxaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected:

Buxzrs. B. sernper-virerzs (common box). Native to Eurasia; global ornamental in other than temperate climates. All. Buxene, an alkaloid. Severe gastroenteritis and death. One-and-one-half pounds of leaves have been shown to be lethal to horses (Volker, 1950). Horses.

Campanulaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: symptoms:

Lobelia. L. berlcmdieri, L. ccrrdinnlis, L. itflcltu, L. siplziliticu (Indian tobacco). North and Central America; habitat is species dependent. Leaves especially. Lobeline and other pyridine alkaloids with nicotine-like structures. Vomition, sweating, paralysis, lowered temperature, rapid and feeble pulse, collapse, coma, and death (Dollahite and Allen, 1962).

Cannabinaceae

Genus: Species: Distribution and habitat:

Poisonous portion: Toxin: Symptoms:

Ccmnnbis. C. sntivn (hemp, marijuana). Native to Central Asia, now global, especially in discreet, remote, and well-concealed locations. Often grown as an indoor plant under artificial light. Leaves, stems, and buds. Cannabinoids. Animal fatalities have occurred in areas where dense spreads of C.sativa have been grown, but most exposures, both animal and human, result from consumption of medicinal preparations from the plant. In animals, ingestion of such preparations leads to lowered body temperature and deep coma (Smith, 1988). Ataxia, hyperesthesia, depression, dyspnea, muscle trembling, salivation, sweating, and in some cases death (Murphy, 1996). Recovery is normal when supporting treatment is maintained for along enough period. Exposure to smoke from this plant leads to hallucinations in humans.

200

Animals affected: Treatment:

Lang and Smith

Ferrets, humans, dogs. and cats. Remove animal from the source of the plant. Give oral tannic acid, apomorphine, charcoal, mineral oil or saline, and diazepam for hyperesthesia (Aiello and Mays, 1998; Murphy. 1996).

Caryophyllaceae

Genus: Species: Distribution and habitat:

Poisonous portion: Toxin: Synlptotns: Animals affected: Treattnent: Notes:

Agrostelrznlci. A. githngo (L.) Scop (corn cockle). Native to Europe, and is found as a weed in grain fields and waste places in the United States (Cronquist and Gleason, 199I). Seeds, calyx tubes (which still contain numerous seeds, can be found in unscreened hay) (Smith et al., 1997). and roots. Gypsogenin (also known as githagenin or albasapogenin) is a sapogenin (Kingsbury, 1964). Symptoms of severe gastroenteritis followed by death. Cattle, pigs, horses, and chickens. Detnulcents used for supportive treatment. (Murphy, 1996) Retnove animals from the source of the plant. “Primary hazard lies in the feeding of wheat screenings” (Murphy, 1996).

Chenopodiaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected:

Betn . B. wlgaris L. (beet, turnip). “Temperate and subtropical, principally in saline habitats” (Heywood, 1993). Leaves. Nitrates and oxalates. Hypocalcemia, salivation, weakness, bradycardia, dyspnea, convulsions, anorexia, depression, weight loss, diarrhea, and death (Murphy, 1996). Cattle, sheep. Remove animals from access to the plant. Chenoyodiurrl. C. albzm L. (lamb’s-quartersj. ‘‘Temperate and subtropical, principally in saline habitats” (Heywood, 1993). Whole plant. Nitrates. “Dyspnea, sudden death, abortion storms, methemoglobinemia” (Murphy, 1996). Cattle.

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Poisonous

201

Treatment:

Remove animals from the source of the plant. ‘‘A I % solution of methylene blue is generally given at a dose of 4- 15 mg/ kg at 4-6 hour intervals as needed’’ (Murphy, 1996). “Loss of animals ‘due to eating these species has not been reported, but their presence in toxic hay may have been a factor contributing to the toxicity of the hay” (Kingsbury, 1964).

Notes:

Genus: Species: Distribution and habitat:

Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Halogetost. H. glomeratr~~ (Bieb.) C. A. Mey (halogeton, barilla) Russia near Caspian Sea, accidentally brought to United States and is now found throughout the desert regions of the Western United States (Kingsbury, 1964). Green leaves and fruiting structures (Kingsbury, 1964). Oxalates. Acute: salivation, weakness, bradycardia, labored breathing, convulsions, and death (Murphy, 1996). Chronic: anorexia, weight loss, depression, diarrhea, and death (Murphy, 1996). Sheep and cattle. “Good alfalfa hay has a marked protective affect.” (Kingsbury, 1964). Give fluids and “dicalcium phosphate [and] sodium chloride: 1 : 3 [ratio] to form calcium oxalate in the gastrointestinal tract thus preventing oxalate ab~orption’~ (Murphy, 1996). Remove animals from the source of the plant. “Halogeton is normally distasteful to sheep and cattle. Under certain circumstances sheep may consume quantities of it even when other palatable forage is available“ (Kingsbury, 1964). Kochia. K. scoycwio (L.) Schrad. (Mexican fireweed). Temperate and subtropical (Heywood, 1993). Whole plant. Nitrates, oxalates, and a photosensitizer. Photosensitization; “poisoning in cattle has been shown to produce polioencephalomalacia, blindness, gastrointestinal disorders, cirrhotic liver, and rumen impaction” (James et al., 1992). Cattle and sheep.

Corynocarpaceae Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms:

Col~~nocurplrs. C. InelYgatus. New Zealand. Whole plant. Karakin, a nitropropanol glycoside. Acute symptoms include incoordination, cyanosis, weakness, collapse, and death. Chronic symptoms are weight loss, a poor hair coat, poor exercise tolerance, and eventual death.

Lang and Smith

Crassulaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Notes:

Coiyledon. C. orbiculutu. South Africa, grown as an ornamental. All. Bufadienolides. Death. “The animals that survive a large single dose, or repeated small doses, lag behind the flock, assume a characteristic pose (with the feet together and the back arched), lie down frequently and develop protracted paresis/paralysis, often lasting for weeks” (Kellerman et al., 1996). Sheep, cattle, and humans. “Chronic cardiac glycoside poisoning or krimpsiekte . . . is a paretic condition of small stock (and more rarely of cattle) brought about by members of the family Crassulaceae, which contain cumulative bufadienolides. Krimpsiekte is the only plant poisoning reputed to cause secondary poisoning; humans and animals that eat the meat of krimpsiekte carcases may themselves become affected” (Kellerman et al., 1996).

Tylecodon. T.wullichii.T.velltricosus, T. grnndflor-is. South Africa, where it grows on hills and ridges. All. Bufadienolides. Death. “The anitnals that survive a large single dose, or repeated small doses, lag behind the flock, assume a characteristic pose (with the feet together and the back arched), lie down frequently and develop protracted paresis/paralysis, often lasting for weeks” (Kellerman et al., 1996). Sheep, cattle, and humans. “Chronic cardiac glycoside poisoning or krimpsiekte . . . is a paretic condition of small stock (and more rarely of cattle) brought about by members of the family Crassulaceae, which contain cumulative bufadienolides. Krimpsiekte is the only plant poisoning reputed to cause secondary poisoning; humans and animals that eat the meat of krimpsiekte carcases may themselves become affected” (Kellerman et al., 1996).

Cucurbitaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected:

Momonhka. M. churantin (balsam pear). New Zealand, Australia, India, and Africa. Whole plant. Momordin, a lectin (hemagglutinin). Diarrhea and emesis; human poisonings have been recorded in Africa, Australia, and India (Kingsbury, 1964). Humans.

Poisonous Higher Plants

203

Dennstaedtiaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Pteridiurn. P. nq~rilirzzrn?(bracken fern). Worldwide. Leaves and rhizomes. “Ptaquiloside, a nor~esquiterpene’~ (Alonso-Amelot et al., 1996). Symptoms of thiamine deficiency in monogastric animals and bone marrow depletion in ruminants. In horses, anorexia, incoordination, a crouching stance with arched neck and feet placed wide apart, tachycardia, convulsions, clonic spasms, opisthotonus, and death. In pigs, anorexia, weight loss, recumbency, dyspnea, and heart failure. In cattle, weakness, pale mucous metnbranes with petechiae, bloody feces, bleeding from body orifices, and death (Aiello and Mays, 1988). “Its potential for causing tumors and other ailments in farm animals is well established. . . . It is certainly likely, in our view, that ptaquiloside in milk is responsible for the connection between bracken infestation and the incidence of gastric cancer in populations of farmers inhabiting cattle-range areas in Costa Rica and in other countries where bracken growth is dense (Andean South America, Central America and southern Mexico” (Alonso-Amelot et al., 1996). “Bright blindness is a progressive retinal atrophy . . . hyperreflectivity of the tapetum in affected animals” (Aiello and Mays, 1998). Cattle, humans, horses, pigs, mice, rats, rabbits, guinea pigs, quial, and Egyptian toads. Remove the animals from access to the plant. “Injection of a thiamine solution at 5 mg/kg body wt is suggested, given initially IV every 3 hr. then IM for several days. Oral supplementation may be required for additional 1-2 wk, although SC injection of 100-200 mg daily for 6 days has been successful in some cases” (Aiello and Mays, 1998). Cattle will eat Pteridium nquilinizm when no other forage is available. Ptaquiloside is detectable in the milk after 38 hr and will not disappear until 86 hr after the last ingestion (AlonsoAmelot et al., 1996).

Dichapetalaceae

Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms:

Dichpetdum. D. cymoszrm (gifblaar “poison leaf”). South Africa. Whole plant. Monofluoroacetate ion. Death by cardiac arrest on exertion or after drinking water (Naude et al., 1996); the fluoroacetate ion leads to a break-

204

Animals affected: Treatment:

Notes:

Lang and Smith down of the carboxylic acid (Krebs) cycle and an accumulation of citric acid in the blood and liver. Vomition is an invariable and often early symptom, and excitability and distress are obvious; on postmortem analysis, the blood appears to have the color of India ink. Fluoroacetate has been used widely to control problem animals in Alberta. Cattle and goats. There is no antidote. Animals rarely survive, the ones that do are rested and are withheld from water for 36 hr (Naude et al., 1996). Cattle eat the plant in early spring when itis green and no other forage is available.

Dioscoreaceae

Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms: Animals affected:

Dioscore-ecr. Numerous (yams). Grown as a food crop in West Africa, Southeast Asia, Oceania, and the Caribbean. Whole plant. Dioscorine (low levels in cultivars, high levels in wild yams). General paralysis of the central nervous system, with death in 3-7 days (Jadhav et al., 1981). Humans.

Ephedraceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected:

Ephedra. E. viridis. Temperate Northern Hemisphere. Whole plant. Ephedrine. Ephedrine is an adrenomimetic (Clarke, 1969). Sheep and cattle.

Equisetaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Equisetum. E. nnlerzse L. (horsetail). North America and Europe. Whole plant. Unknown. In horses, weakness; ataxia; difficulty in turning; exercising produces trembling and muscle exhaustion; prostration with frequent attempts to stand; tnuscle rigidity; constipation;

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205

rapid, weak pulse; cold extremities; corneal opacity; opisthotonus: coma; and death. In cattle, hyperexcitability, poor condition, muscle weakness, diarrhea, and lowered milk production. Tn sheep, weakness, staggering, and trembling (Kingsbury, 1964). Horses, cattle, sheep, goats, and rats. Remove animals from access to the plant.

Ericaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxi n : Symptoms: Animals affected: Treatment: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Treatment: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Treatment:

Knlrnin. K. Zntifolicr (mountain laurel) and other Kchnin species. North America. Leaves, twigs, and honey made from the plant. Grayanotoxins. Salivation, tear formation, vomition, convulsions, slow pulse, low blood pressure (BP), paralysis, and death. Cattle. Retnove animals from access to the plant. Leduln. L. gkeuzdulosrlm (Western labrador tea). Western North America. Leaves. Grayanotoxins. Salivation, tear formation, vomition, convulsions, slow pulse, low BP, paralysis, and death. Remove animals from access to the plant. Leucothoe. L. dmisicle (black laurel). Pacific Coast of North America. Leaves. Grayanotoxins. Salivation, tear formation, vomition, convulsions, slow pulse, low BP, paralysis, and death. Remove animals from access to the plant.

Treatment:

Ly onin . L. ligustritzcr (maleben-y). North America and Asia. Whole plant, Grayanotoxins. Salivation, tear formation, vomition, convulsions, slow pulse, low BP, paralysis, and death. Remove animals from access to the plant.

Genus: Species: Distribution and habitat:

Meuciesin. M. fewrlgirzen (mock azalea). Western cordillera of North America.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

206

Poisonous portion: Toxin: Symptoms: Treatment: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Treatment: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Treatment: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Lang and Smith

Leaves, twigs, and honey made from the plant. Grayanotoxins. Salivation, tear formation, vomition, convulsions, slow pulse, low BP, paralysis, and death. Remove animals from access to the plant. Pernepcr. Several. South America, New Zealand, and Tasmania. Whole plant. Grayanotoxins. Salivation, tear formation, vomition, convulsions, slow pulse, low BP, paralysis, and death. Remove animals from access to the plant. Pieris. P. japojzicn (Japanese pieris). Native to Japan, used globally as an ornamental plant. Leaves. Grayanotoxins. Salivation, tear formation, vomition, convulsions, slow pulse, low BP, paralysis, and death. Remove animals from access to the plant. Rhododendron. R. nzcuinwn (rhododendron) and other Rhododendron species. Now global as an ornamental. Native to the northern temperate zone. All, but especially the pollen. Grayanotoxins. Most human sickness results from the consumption of honey made by bees from these plants (Biberoglu et al., 1988). The symptoms are those caused by grayanotoxin and are the same for other Ericaceae. Humans.

Erythroxylaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Elythroxylum. E. coca (coca). South America, introduced to Oceania. Leaves. Ecgonine bases. Problems normally result fron the use of medicinal preparations of these plants, and sensory aberrations, vomiting, and muscular spasms occur, followed by irregular respiration, convulsions, coma, and death from circulatory failure. Humans.

Poisonous Higher Plants

207

Euphorbiaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Genus: Species: Distribution and habitat:

Aleuritis. A. fordii Hemsl. (tung-oil tree) and related species. Tropical and temperate areas, originating from China. Seeds, leaves. Saponin and a phytotoxin. In cattle, symptoms appearing after 3-7 days include: hemorrhagic diarrhea, anorexia, listlessness, emaciation, and death after 1-3 weeks. (Kingsbury, 1964) In humans, symptoms appearing after 1/2 hr include: nausea, abdominal cramps, severe vomiting, diarrhea, weakness, exhaustion, dehydration, shock, cyanosis, respiratory depression, and lowered reflexes (Kingsbury, 1964). Humans, cattle, and horses. “The nuts are attractive to human beings, both in appearance and taste. The open kernel may be mistaken for that of the Brazil nut. More than 40 cases of human poisoning have been reported in medical literature” (Kingsbury, 1964). Euphorbia. E. margi~zatc~ Pursh (snow-on-the-mountain), E. pulcherri~na Willd, (poinsettia) and other related species (spurges). Temperate and tropical re,’olons. Whole plant. Unknown. E. nznrginatn “produces scours and emaciation in cattle’’ (Kingsbury, 1964). E. yulcherrimu symptoms include vomiting, delirium, and death (Kingsbury, 1964). “Severe irritation and blistering of the mouth and gastrointestinal tract with diarrhea and/or hemorrhage’‘ (Murphy, 1996). Irritation and blistering of skin on contact with the plant. Cattle, humans, dogs, and cats. “Emetic (apomorphine), adsorbant (charcoal), cathartic (mineral oil or saline.) Supportive [treatment use a] dilution with milk and egg” (Murphy, 1996). For skin contact wash with isopropyl alcohol, then wash with soap and water. “Euphorbia nzurginnta . . . has been thought responsible for evil tasting, poisonous honey. . . . None of the spurges are relished by livestock, but the tender young growth of some may be eaten more or less readily when other forage is not available. Toxicity is not lost upon drying, and at least in some instances hay seems more palatable to livestock than the fresh plant” (Kingsbury, 1964). Hura , H. creyitans (sandbox tree). Tropical America, grown as an ornamental in such warm parts of the world as the southern United States.

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208

Poisonous portion: Toxin: Symptoms:

Seeds. Unknown, a phytotoxin. Severe emesis and purging.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Jcrtrophu. J. rnult(fidcr (physic nut). Peninsular Florida. Fruits. A phytotoxin destroyed on roasting. Severe emesis and purging.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin:

Mmihot. M. escuenta (cassava, tapioca plant). Global in warm regions, cultivated as a food plant. Root. The root is capable of forming toxic levels of hydrocyanic acid (Espinoza et al., 1992). Those of cyanide poisoning. By and large, the plant is eaten only after processing by the food industry to produce tapioca, from which all toxin has been removed. The plant can be cooked elaborately to destroy the toxin. When in the tropics, the senior author treats the root as a potato and makes French fries from it; these fries are delicious and he has never experienced signs of toxicosis after eating them. Humans.

Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Memuicrlis. M. crrzrzuc~(annual mercury), M. peren~zis(dog‘s mercury). Northern Hemisphere. Whole plant. “Annual mercury is most toxic and in the flowering and seed bearing stages’’ (Welchman et al., 1995). A saponin. Death “preceded by recutnbency with tachycardia and pale greyish brown coloration of the mucous membranes and haematuria. . . . repeated ingestion of small quantities of annual mercury can have a cumulative effect” (Welchman et al., 1995). Sheep and cattle. Ricinus. R. c o r n ~ ~ ~(castor z i s bean). Native to Africa, now widespread in the tropics and semitropics as a crop plant and source of castor oil. The seed or bean. Ricin. Injection of chemically isolated ricin produces agglutination of defibrinated blood and red blood cells in vitro. Ricin is far less toxic by ingestion, and the beans, in many cases at least, seem to be nontoxic. A report of a suicide attempt by ingestion of a handful of diced beans was reported (Rauber and Heard,

Poisonous Higher Plants

Animals affected:

209

1985): the patient developed no symptoms of poisoning. The dangers of the bean may well have been exaggerated by Dame Agatha Christie et al. The plant is included here only because of its reputation. Humans.

Fagaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Syn1ptoms:

Animals affected: Treatment:

Notes:

Quercus. All oaks should be considered potentially toxic (Kingsbury, 1964). Northern temperate regions and mountainous tropics (excluding tropical and southern Africa) (Zomlefer, 1994). Most toxic partis the acorn, though buds and immature leaves may also be toxic. Tannins and gallotannins. (Though more research is needed, it is purported to be tannic acid.) “Symptoms appear in a week or more and rapidly become acute’. (Kingsbury, 1964). Anorexia; rumen stasis; constipation or small amounts of hard, brownish-black pelleted feces (if the animal lives long enough) will change over time to diarrhea containing blood and mucus; rough coat and drymuzzle; there may be a brownish discharge from the nostrils; abdominal pain sometimes accompanied by swelling; excessive thirst; and frequent urination; thin, rapid pulse; and death (Kingsbury, 1964). Cattle, sheep, goats, horses, and pigs. 10% “calcium hydroxide in a pelleted ration prevents tannin absorption. . . . Supportive [treatment], provide supplemental feed, fluidtherapy for emaciated animals” (Murphy, 1996) Remove animals from the source of the plant. “Oak poisoning is a severe economic problem on the ranges of the Southwest where annual losses greater than $10 million have been estimated, and occurs sporadically elsewhere” (Kingsbury, 1964).

Fumariaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Corydalis. C. aurea Willd (golden corydalis) and other Colydalis species. Northern Hemisphere. Whole plant. Isoquinoline alkaloids, including capaurine and tetrahydropalmatine (Smith and Lewis, 1990). Stiffening of muscles, convulsions, bleating and bawling, death have been recorded in sheep following ingestion of C. cnsenna Gray (Kingsbury, 1964). Cattle and sheep.

210

Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptotns:

Animals affected: Treatment: Notes:

Lang and Smith

Dicelztm. D. cucullnr-ia (L.) Beonh. (Dutchman’s breeches), D. eximin (Kerr-Gawl) Torr. (bleeding heart), and others. Northern Hemisphere. Whole plant. Tsoquinoline alkaloids, predominately protopine. Stiffening of muscles, trembling, hyperexcitability, salivation, recumbency, convulsions, bleating and bawling (in sheep), forceful ejection of ingesta (in cattle), and in some cases, death (Kingsbury, 1964; Murphy, 1996). Cattle, sheep, dogs, cats, goats, horses, and chickens. Remove animals from access to the plant (Murphy, 1996). “Poisoning appears confined to cattle on early spring woodland pasture. . . . Cattle find plants distasteful, but will consume them when other feed is scarce”(Kingsbury, 1964).

Gramineae Genus: Species: Distribution and habitat: Poisonous portion: Toxin:

Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Avenrr. A. sativa L. (cultivated oats). Global, native to Eurasia. The hay. Nitrate ion and especially nitrite ion formed by bacteria on hay stored in a less than ideal manner (Smith and Suleiman, 1991b). Death in cattle, which are highly susceptible to nitrite ion. A pronounced conversion of hemoglobin to methemoglobin is noted and the blood is a chocolate-brown color. Cattle.

Cyodon. C. dactylon (L.) Pers. (Bermuda grass). Worldwide. Seedhead and upper stem. Cyanogenic glycosides (Murphy, 1996). Appearing a few days to a week, involve ataxia, photosensitization, reddish-brown urine, muscle twitching, inability to stand, in some cases prostration and death (Kingsbury, 1964: Murphy, 1996). Cattle. Remove animals from areas containing the plant. Festuca. F. arurzdirla~zceae L. (tall fescuej. Southern Hemisphere, introduced to North America. Whole plant. Perloline and other alkaloids. Lameness in hind feet, gangrene in limbs, weight loss, arched back, rough coat, hypersalivation, hyperthermia, lowered re-

Poisonous Higher Plants

Animals affected: Treatment: Notes:

Genus: Species: Distribution and habitat: Toxin: Symptoms: Animals affected: Treatment: Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Genus: Species:

21 1

productivity, (in sheep convulsions), incoordination, (in horses thickened placentas and weak foals), increased pulse and respiration, and coma. (Aiello and Mays, 1998; Cheeke and Shull, 1985; Kingsbury, 1964). Horses, cattle, and sheep. Remove animals from affected feed source. Frequent grazing or topping of pastures prone to ergot infestation during the summer months will help prevent flower-head production, which helps to control the disease (Aiello and Mays, 1998). Pmicurn. P. colorntunl, P. dichotoml~orum,P. schinzii. South Africa, Australia, New Zealand, North and South America. Unknown. Photosensitivity, depression, icterus, and poor performance. Sheep, horses, and goats. Remove animal from source of the plant. “As in the case of T. terrestris, Parziczm grass periodically becomes toxic under certain specific conditions, for instance when wilted during hot, dry spells following summer rains” (Kelleman et al., 1996). Sorgl?um. S. halepeme (L.) Pers. (Johnson grass, Sudan grass). Global, native to Asia and Africa. Whole plant. Dhurrin, a cyanogenetic glycoside, also nitrates. Rapid death from the formation of cyanide, other symptoms rarely seen include: excitement, muscle tremors, salivation, defecation and urination, and clonic convulsions. Nitrate poisoning symptoms; dyspnea, abortion, methemoglobinemia, and death (Murphy, 1996). Cattle, sheep, and horses. Remove animals from the source of the plant. For cyanide poisoning, “sodium nitrite at 10-20 mg/kg with 500 mg/kg sodium thiosulfate as needed” (Murphy, 1996). For nitrate toxicity causing methemoglobinemia “a 1% solution of methylene blue is generally given at a dose of 4-15. mg/kg at 46 hour intervals as needed’‘ (Murphy, 1996). “The frequency of cattle poisoning compared with that of other animals may be explained by conditions in the rumen which promote greater and more rapid enzymatic breakdown of the cyanogenetic glycoside as compared with the digestive process in other animals” (Kingsbury, 1964). Zen. 2. mays (maize, Indian corn, sweet corn, corn on the cob).

212

Distribution and habitat: Poisonous portion:

Toxin: Symptoms:

Animals affected: Treatment:

Lang and Smith

Global, native to the Americas, but cultivated varieties now are grown worldwide. The stalk, especially before tasseling.[The cob may become infected with cob-rot fungus, Diplodicr nlnydis (Kellerman et a1., 1996).] Nitrate ion. (An unknown neurotoxin is implicated in cob-rot fungus.) Dyspnea, abortion, methemoglobinemia, and death. (The symptoms of cob-rot fungus are: “ataxia (stiff-legged high stepping gaitj, paresis and paralysis . . . . animals may die of hunger or thirst if neglected” (Kellerman et al., 1996). Horses, cattle, and sheep. Remove animals from the source of the plant. “(Cob-rot fungus): There is now specific treatment. New cases can appear up to 10 d after withdrawal from toxic lands and the prognosis is usually good if stock are removed as soon as the first signs appear” (Kellerman et al., 1996). For nitrate toxicity causing methemoglobinemia “a 1% solution of methylene blue is generally given at a dose of 4-15 m g k g at 4-6 hour intervals as needed’‘ (Murphy, 1996).

Hippocastanaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Notes:

Aesculus. A. &bra Willd. (Ohio buckeye) and A. Jzippocc~sta~~~m (horse chestnut). Northern Hemisphere. Nuts, but buds and leaves also are dangerous (Casteel and Wagstaff, 1992). Aesculin, a saponin. Gastroenteritis in humans, nervous signs in cattle, in which rumen microorganisms produce the aglycone that then is absorbed. Humans and cattle. Extracts from several species of A e s c h s were used “by North American Indians to stupefy fish”(Heywood, 1993).

Hypericaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Hypericum. H. peifor*cltuwzL. (Saint-John’s-Wort). Native to Europe, now a global weed. Glandular dots in the foliage and petals. Hypericin. Hypericin is a primary photosynthesizer. The skin is covered by white hair patches that slough off and great sores and open wounds develop (Rogers, 1914).

Poisonous Higher Plants

Animals affected: Treatment:

213

Sheep, cattle, horses, goats, rabbits, and rats. Remove animals from the source of the plant. “Shelter from the sun if possible. [Supportive treatment consists ofl topical demulcents (calamine), antibiotics, and steroids may be applied. Steroids and antibiotics (penicillin) may be given systemically” (Murphy, 1996).

lridaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Treatment:

Mornen. M. polystachya (blue tulp), M. bipmtita. Eastern and southern Africa. All. Bufadienolides. Difficulty breathing, asphyxia, tachycardia, arrythmia, bloat, partial paralysis of the hind quarters (Kelleman TS et al., 1996). “. . . activated charcoal administered at a dose of 2 g/kg. Care should be taken not to stress intoxicated animals during treatment as this could lead to fibrillation or heart block” (Kellerman et al., 1996).

Juglandaceae

Genus: Species: Distribution and habitat:

Poisonous portion: Toxin: Symptotns: Animals affected:

Juglcrns. J. nigra L. (black walnut). Primarily in temperate to subtropical of the Northern Hemisphere (Heywood, 1993). Commonly found in rich woods (Wofford, 1989). Bark, leaves. Unknown. Laminitis, mild depression, limb edema (Galey et al., 1991). Horses.

Juncaginaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Triglochin. T. maritima, T. palmtrus(arrow grasses). North America in or near brackish water or bogs. Whole plant. Triglochinin, a cyanogenetic substance. Livestock deaths with signs of cyanide poisoning.

Lamiaceae

Genus: Species: Distribution and habitat:

Hedeoma. H. pulegioides (L.) (pennyroyal). Fields and open woods (Wofford, 1989).

Lang and Smith

214

Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin:

Symptoms: Animals affected: Treatment: Notes:

Leaves. Pulegone (a nlonoterpenej, which also is broken down to menthofuran, a toxic metabolite. Symptoms are dose dependent, “. . . 15 mL of pennyroyal oil or more ingested by an adult has been fatal’‘ (Anderson et al., 1996). In small amounts can cause depression, dizziness, and weakness. In larger doses causes abdominal cramps, seizures within 3 hr of ingestion, and coma (Anderson et al., 1996). Humans and rats. Prompt aggressive cleansing of the stomach with gastric lavage and activated charcoal. . . . N-acetylcysteine [should] be administered as soon as possible after all pennyroyal overdoses that are in the toxic range ( > l o mL of pennyroyal oil). Care is supportive, with close attention to mental status, respiratory status, and hepatic and renal function” (Anderson et al., 1996). ‘‘Its wide commercial availability and reputation as an abortifacient continue to make pennyroyal a serious public health concern’’ (Anderson et al., 1996). < b

Teucriunz T. cllnmaeclq~s,T. yoliwn. Eurasia. Whole plant. “The chemical composition of germander comprises furancontaining neoclerodane diterpenoids, saponins, glycosides and flavonoids”(Larrey, 1997). A hepatotoxin. Hepatitis, cirrhosis, and in one case in France death occured from long-term ingestion of tea made with germander. Humans. Discontinue use of plant. “Germander was given a marketing agreement as a traditional herbal medicine in 1986 in France as an adjuvant to weight control. Germander has been rapidly incriminated in more than 30 cases of liver injury in France, mostly in middle-aged women. Germander has been withdrawn from the market of herbal medicines in France and its free sale has been forbidden. However, it is still used in some other countries and new cases have been recently observed in Canada’’ (Larrey, 1997).

Lauraceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Sassc$ws.

S. cdbidum (sassafras). Originated in the United States, now global in temperate regions. Leaves, root. Safrole (hepatotoxin) (Larrey, 1997). Hepatotoxic affects (Larrey, 1997).

Poisonous Higher Plants

215

Leguminosae (or Fabaceae)

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Abrus. A. precatorius (precatory bean, jequirity bean). Tropics. Seeds; a pretty seed that is made into necklaces and the like. Abrin, a phytotoxin. Purified abrin is avery dangerous toxin when injected into the bloodstream. Ingested seeds are less dangerous, but one can kill an adult human (Davis 1978.) Humans.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Acacia (Mimosaceae). A. berlandieri (Guanjillo). Southern North America. Leaves and fruits. N-Methyl-beta-phenylethylamine. Ataxia develops after months of consumption (Boughton and Hardy, 1940).

Genus: Species: Distribution and habitat: Poisonous portion: Toxin:

Astrl~galus. Numerous species are troublesome. Widespread throughout North America. Whole plant. Astragalus species are poisonous in three distinct ways. Certain Astragalus species (e.g., A. rucemosus) are selenium accumulators and if eaten, these quickly cause death by classical selenium poisoning when they occur on soils rich in that element (Baker et al., 1989). A second type of toxin is found in a group of Astrugalus species (e.g., A. nrgillophilr4s) in which an indolizidine alkaloid, swainsonone, is found; this causes “locoism” and anorexia after a few weeks of grazing on the plant. In locoism, a locomotor ataxia develops and becomes increasingly severe, eventually leading to death (Cheeke, 1989a). The third group of poisonous Astragalus species, typifiedby A. miser (titnber milk vetch), contains miserotoxin, a glucoside of 3-nitropropanol. Some similar species contain glucosides of 3-nitropropionic acid, but only one or the other toxin is found in any one species . A. canacler~siscontains the propionic acid derivative. Rumen microorganisms release 3nitropropanol from miserotoxin; this then is metabolized to nitrite ion and possibly other toxic components (Cheeke and Shull, 1985). Extensive methemoglobin formation is noted in dead cattle. Varies, as discussed above. Cattle, sheep, and horses. Remove animals from access to the plant (Murphy 1996). “Horses which have been ‘locoed’ should not be considered fit for riding ever again” (Murphy 1996).

Symptoms: Animals affected: Treatment: Notes:

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Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat:

Crotolcrrin. C. spectabilis Roth. (rattlebox, crotalaria, pulmonary hypertension plant). Southeastern United States. Seeds, leaves, and stems. Monocrotaline and related Pyrrolizidine alkaloids (PAS). A veno-occlusive disease develops in people who eat Crotolcrria-contaminated grain (Tandon et al., 1976). The victims exhibit distended bellies and prominent veins. In chickens, acute poisoning symptoms include: a dark, scaly, congested comb; depression; ruffled feathers; greenish-yellow diarrhea: and death. The carcass smells of crushed C r o t ~ h r i aleaves. In chickens, chronic signs include: a pale comb and anemia (Kingsbury, 1964). In cattle, loss of appetite, poor condition, nervousness and excitability, excessive straining during defecation, bloody feces, prostration, and death (Kingsbury, 1964). Humans, horses, chickens, cattle, pigs, sheep, goats, mules, and dogs. Cvtisus (Papilionaceae). C. scoparius (Scotch broom).

Temperate Europe and western Asia, now introduced into North America. Legume pod and leaves. Quinolizidine alkaloids, especially sparteine and isosparteine. Weak pulse, intestinal paralysis, weakness, and fall of blood pressure. Gcrstrolobilrrn. G. grcrncliJlorur71 (heart leaf poison bush). Australia. Whole plant. Fluoroacetate ion. Death: the fluoroacetate ion leads to a breakdown of the carboxylic acid (Krebs) cycle and an accumulation of citric acid in the blood and liver. Vomition isan invariable and often early symptom and excitability and distress are obvious; on postmortem examination, the blood appears to have the color of India ink. Fluoroacetate has been used widely to control problem animals in Alberta. The deaths of numerous sheep in western Queensland were reported for this plant (Campbell and Kingston, 1992). Sheep. GymnocledLm. G. clioiccr (L.) K. Koch. (Kentucky coffee tree). Grows in moist woods from New York south to Virginia and westto South Dakota and Oklahoma. Occasionally planted elsewhere (Kingsbury, 1964).

Poisonous Higher Plants

Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin:

Symptoms: Animals affected: Genus: Species: Distribution and habitat:

217

Fruit pulp, leaves, young shoots. Unknown. Gastrointestinal imitation, nervousness, and death in livestock caused by eating the newly fallen seed pods in early spring, by eating pruned foliage, and by eating young shoots. A human death occurred due to mistaking the Kentucky coffee tree pods for honey locust and eating the fruit pulp (Kingsbury 1964). Humans, sheep, cattle, and horses. Laburnurn. L. m~agyoicZesMedic. (goldenchain). Native to southern Europe, now a widespread ornamental plant in warmer climates. Whole plant. The quinolizidine alkaloid, cytisine. Cytisine gives rise to actions similar to nicotine, which include excitement, incoordination, convulsions, coma, and death (Kingsbury, 1964). Horses.

LclthyrLls. L. scrtivus (chick-pea). Arid and poor soil areas in India. Seeds and flour made from the seeds. Beta-N-oxalyl-L-alpha-beta-diaminopropionic acid. Paralysis of the legs after damage to nerves in the spinal cord (Cheeke and Shull, 1985). Humans and horses. Leuccrerza (Mimosaceae). L. glauca L. (lead tree), L. lezrcocephc~lc~ (leuceana). North America and Oceania. All, but especially the seeds. L. leucocephaln contains mimosine, a nonprotein amino acid. In ruminants, mimosine is converted to 3-hydroxy-4( lH)-pyridone (DHP), which is toxic to cattle unable to degrade it further (Pratchett et al., 1991). Mimosine leads to poor weight gain in cattle, poor general condition, and loss of hair. Cattle, horses, mules, donkeys, swine, and rabbits. Lupinus. L. sericerrs (silky lupine) and many other Lupinus species. The genus has global distribution as ornamentals and is native to North America and Europe.

218

Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment: Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Lang and Smith

All, but especially the seeds (including edible lupines). Quinolizidine alkaloids, especially lupinine and sparteine. Breathing becomes heavy and labored, the animal becomes comatose, sometimes after convulsions. Death occurs as a result of respiratory paralysis (Kingsbury, 1964). Sheep, cattle, horses, goats, swine, and deer. Remove animals from access to the plant. ‘‘Hay containing plants with seeds may be toxic. . . . Causes more losses of sheep than any other plant in the NW US” (Murphy 1996). Cattle consuming the plant during gestation can have congenital defects in their offspring, which has been termed “crooked calf disease’’ (Keeler 1989). Oxytropis. 0. sei-icen (white locoweed). Western North America. Whole plant (green and dry). Indolizidine alkaloids swainsonine and swainsonine N-oxide. The same as for indolizidine and alkaloidal Astrngnlrrs species; however, symptoms appear to vary with altitude (Panter et al., 1988). Cattle, sheep, and horses. Physostigma. P. ~ ? e ~ ~ e ~ ~(Calabar o s z r m bean). Native to the Gulf of Guinea (West Africa). Seed (bean). Physostigmine. Physosotimine is an anticholinesterase. Symptoms start within 2 hr of ingestion of the bean. The skin becomes ashen gray. A child has been killed by the ingestion of six beans (Clarke, 1969). Humans.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Robinia. R. ysez.doncncia L. (black locust). Eastern and central United States and southern Canada. Bark. Robin, a phytotoxin. Colic, diarrhea, irregular pulse, hyperexcitability, paralysis, and death.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected:

Senna. S. a ~ g ~ s t i f o l (senna). in The Americas. Leaf and fruit. Sennosides (laxative alkaloids) (Larrey, 1997). Hepatitis. Humans.

Poisonous Higher Plants

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

219

Sesbarl iu. S. vesicuri(l (coffee bean). Southeastern regions of North America. All, but especially the seeds. Possibly sesbanine, a cytotoxic alkaloid. Hemorrhagic diarrhea. Sheep are killed by ingestion of seeds in excess of 0.05% of the animal’s weight (Boughton and Hardy, 1939). Sheep.

Sophorn. S. securzdiforn (mescal bean). Central America and the very southern United States; on limestone. All, but especially the seeds. Quinolizidine alkaloids, including N-acetylcytisine. Breathing becomes heavy and labored, and the animal becomes comatose, sometimes after convulsions. Death occurs as a result of respiratory paralysis. Feeding experiments with sheep, goats, and cattle have demonstrated the toxicity (Boughton and Hardy, 1935). Sheep, goats, and cattle. Thernzopsis. T. rnorztunu (false lupine). Dry plains of central North America. Seeds. Quinolizidine alkaloids, including cytisine, anagyrine (a teratogen), thermopsine, and N-methylcystisine. Breathing becomes heavy and labored, andthe animal becomes comatose, sometimes after convulsions. Death occurs as a result of respiratory paralysis. Cattle have been killed by this plant (Keeler et al., 1986). Cattle.

Animals affected:

Vicin. V. Fuba L. (broad bean, fava bean). Native to the Mediterranean: cultivated globally. Bean, inhalation of the pollen is also dangerous. Aglycones of vicine and convicine (glycosides). Only individuals with a genetic deficiency of a red blood cell enzyme, glucose-6-phosphate dehydrogenase, are susceptible and acute hemolytic anemia occurs. Death of children is a common occurrence after eating V. fuba (Cheeke and Shull, 1985). Humans.

Genus: Species: Distribution and habitat:

Wisteria. W. jloribrrrzdu Willd. (Japanese wisteria). Temperate Northern Hemisphere.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

220

Poisonous portion: Toxin: Symptoms:

Animals affected:

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Seeds and pods. Unknown. Gastroenteritis, vomition, abdominal pain, and diarrhea. Children have been poisoned by this plant (Jacobziner and Raybin, 1960). Humans.

Liliaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Treatment: Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Chlorophytunl. Various (spider plant). Popular houseplant. Leaves and plantlets. Unknown. “Vomiting, salivation, retching, and transcient anorexia . . . within hours of ingestion” (Aiello and Mays, 1998). Cats. Treat symptomatically. “Pet animals (especially cats) reach these plants either by climbing or when plantlets fall from mature stems.’ (Aiello and Mays, 1998). Colchicum. C. nutuwmale L. (autumn crocus, meadow saffron). Ornamental plant now grown worldwide, native to Europe. Whole plant. Colchicine, an alkaloid useful in the treatment of gout, and demecolcine. Thirst, difficulty swallowing, abdominal pain, profuse vomiting and diarrhea, shock (Aiello and Mays, 1998), death of humans and animals from exhaustion or respiratory paralysis; alopecia and severe bone-marrow depression arise from sublethal doses. Animals graze the plant, humans sometimes chew on the plant, but most human fatalities arise from the drug. Seven cows died in France after consumption of the plant (Chareyre et al., 1989). Two sheep in a flock died in Albania and the milk of the flock contained colchicine (Panariti, 1996). Cattle, sheep, and humans. ‘‘Gastric lavage; supportive care for dehydration and electrolyte losses (fluid therapy). . . . Analgesics and atropine recommended for abdominal pain and diarrhea“ (Aiello and Mays, 1998). “Colchicine is well known for its beneficial effects in the treatment of gout and liver cirrhosis, but cases of intoxications following its accidental or premeditated ingestion are well described both in hutnans and animals” (Panariti, 1996).

Poisonous Higher Plants

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptollls:

Animals affected:

221

Comwllcrria. C. majnlis L. (lily-of-the-valley).

North America and Europe. Whole plant. Cardiac glycosides (convallarin, convallamarin, convallotoxinj and saponins. Vomiting, trembling, abdominal pain, diarrhea, irregular heartbeat, hyperkalemia, petechial hemorrhages, and death (Aiello and Mays, 1998). Cattle, dogs, and cats. “Aimed at gut decontamination (gastric lavage) and at correcting bradycardia (atropine), conduction defects (phenytoin), and electrolyte imbalance such as hyperkalemia (IV electrolytes). Electrocardiographic and serum potassium monitoring as necessary” (Aiello and Mays, 1998). Charcoal, mineral oil, assist in respiration (Murphy, 1996). “Calcium containing solutions are corlt~~~indicntecl.’ ’ (Murphy, 1996). Gloriosa. G. srdperba (glory lily). Native to tropical Africa, now global as a houseplant. Tubers. Colchicine. Gastrointestinal irritation, nervous symptoms, and death (Kingsbury, 1964). Humans. Ncrrtheci~m. N. ossifr-ngrm (bog asphodel). European bogs. Flower stems are the most toxic portion, leaves. Unknown . Death. The toxins cause liver and kidney damage, the former causes photosensitization, depression, reduced appetite, and dehydration (Flaoyen et al., 1997). Cattle. Urginea and the closely related Scilla. U. mrritinza (red squill), U. sanguinea. Native to countries surrounding the Mediterranean, grown worldwide as a source of rodenticide. The bulb. Proscillaridin, a cardiac glycoside. An ideal rat poison. Rats lack the vomiting reflex that tends to protect humans and livestock from ingestion of the distasteful plant itself. Sheep have been killed by Urgiflea yhysocles (Ne1 et al., 1987). Rats and sheep.

Lang and Smith

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Loganiaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Verutrum. V. cal~fornicum, V.v i d e (false hellebore). North America and Europe in low, moist areas. Roots, but the whole plant is toxic. Glycoalkaloids and ester alkaloids. Hypotension in humans. The alkaloids of V. cnliforrlicurn contain teratogenic compounds that cause deformed lambs when the ewe eats the plant at the appropriate (or perhaps inappropriate) stage of gestation (Keeler, 1978). Humans, sheep, and cattle. Zvgnderlus (also Zigndews). 2. grcl~ninem(death camas) and other Zygndenrrs species. Rangelands in western North America. Bulbs, but the whole plant is toxic. Zygacine and similar steroid alkaloids and glycoalkaloids. Salivation, hyperexcitability, open mouth breathing, loose green stool, nausea, vomition, muscular weakness and ataxia, trembling, rapid heart rate, prostration, and death (Collett et al., 1996). Animals (Smith and Lewis, 1991a) and humans both eat the plant, the latter often mistaking it for a wild onion. Humans, cattle, and sheep. “Early treatment (in cattle) of Zlrpdelzrrs sp. alkaloid toxicosis with 2 mg atropine and 8 mg picrotoxin in 5 In1 water given subcutaneously for 50 kg/bw may be effective” (Collett et al., 1996). ‘‘The distinguishing feature at the vegetative stage is that wild onion bulbs have a characteristic onion smell and death camas sp. bulbs do not” (Collett et al., 1996).

Gelsemirun. G. senzyenirens (jessaminej. North America and Eurasia. Whole plant. Gelsemine and related alkaloids. Muscular weakness, convulsions, sweating, and death from respiratory failure. Children have been poisoned by licking nectar from the flowers (Morton, 1958). Humans. stlyclzllos. S. nux-vowica. India. Seed. Strychnine and related alkaloids. Convulsive seizures leading to death due to medullary paralysis. (Until recently, strychnine was used by the staff of Alberta Agriculture to control problem animals.)

Poisonous Higher Plants

223

Loranthaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Viscum. V. album (European mistletoe). Europe. Berries. Viscotoxin A, a polypeptide. Incoordination, dilated pupils, polyuria, hypersensitivity, and death.

Lycopodiaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Notes:

Lycopodium. L. serratum. Eurasia. All. Levotetrahydropalmatine (structurally similar to hepatotoxic pyrrolizidine alkaloids) (Larrey, 1997). Hepatitis. Humans. Lycopodiunz is one of several herbs used in Jin Bu Huan, a Chinese herbal remedy (Larrey, 1997).

Malvaceae

Gknus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Notes:

Gossypium. G. burbademe L. (sea-island cotton), G. hirsutunz L. (upland cotton), and several other varieties. Worldwide. Seeds. Gossypol. In cattle andpigs: “dyspnea with gasping or ‘thumping’ breathing and occasionally the development of froth or bloody froth at the mouth. . . . poisoned animals often develop symtoms while gaining weight and while in apparently good condition. Emaciation and weakness appear with the other symptoms even though the appetite maybe normal until shortly before death. Convulsions may accompany death, and cyanosis may be noted immediately before death” (Kingsbury, 1964). In poultry: decreased growth and a lowered hatching rate, discolored yolks and whites, or abnormal white consistency (Kingsbury, 1964). Cattle, pigs, poultry, dogs, sheep, rabbits, rats, and guinea p1gs. Milling procedures can extract gossypol from cottonseeds. Cottonseed meal is an important source of protein for livestock. To benefit from the nutrition available in cottonseed and ensure it is harmless, the levels of gossypol should not exceed 0.01 in the diet (Kingsbury, 1964).

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224

Meliaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Azndir-ncllzn. A. irdicct. “Indomalaysia and the Pacific” (Heywood, 1993). Oil extracts from the seeds (Larrey, 1997). Azadirachtin. Causes fatty degeneration of the microvasculature (Larrey, 1997).

Myoporaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

M?10~OT141?1.

M. l c r m m (ngaio). New Zealand. Branches. Ngaione, a sesquiterpene ketone. Abdominal pain, constipation, and death. Photosensitization is common. Hemouhagic inflammation of the abomasum and intestines is noted at postmortem. M. Zaetzm is one of the most dangerous native plants in New Zealand (Clarke and Clarke, 1967).

Oleaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected:

Ligust~~m. L. w1geu-e (privet). Native to the temperate Northern Hemisphere and Australia. Berries and leaves. Unknown. Horses are reputed to have been poisoned by eating this plant (Clarke and Clarke, 1967). Human fatalities are very rare, but symptoms do occur (Dreisbach, 1963). Horses.

Palmae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

A r-eccl. A. catechu (betelnut). India. Seeds. Arecoline. Vomition, diarrhea, difficulty breathing, impaired vision, convulsions, and death. Arecoline is used as a veterinary anthelmintic andhas resulted in the loss ofmany cats and dogs (Clarke, 1969). Cats and dogs.

Poisonous Higher Plants

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Treatment: Notes:

225

Metroxylon. 28 species of sago palm. Tropical, some subtropical and temperate (Heywood, 1993). Whole plant. Cycasin (Glycoside). Abdominal pain; vomiting; diarrhea; in some cases, rear limb paralysis; coma and death (Murphy, 1996). Dogs and cats. Remove animal from the source of the plant. The plant is a popular houseplant and ornamental. Intoxication generally occurs with young, curious, or bored animals (Murphy, 1996).

Papaveraceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Argemone. A. mexicmcr (Mexican poppy). Mexico and the West Indies. Seeds (poisonings have occurred when the seeds contaminate grain) (‘Watt and Breyer-Brandwijk, 1962). Isoquinoline alkaloids, including protopine and berberine. Vomition, diarrhea, edema of the feet and legs, and intense pain all over the body.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Chelidoniurn. C. n7njzrs (greater celandine). Global ornamental plant, native to the Old World. Sap. Isoquinolir~ealkaloids, including protopine and berberine. Gastroenteritis; death has been reported (Volker, 1950).

Genus: Species: Distribution and habitat:

Papaver. P. somniferzun (opium poppy). Native to Southeast Asia, now global, especially in warm, discrete, remote, and well-concealed locations. Whole plant. Morphine and related alkaloids (narcotic analgesics). Euphoria, followed by somnolence and death. Two dogs were suspected of having a nonfatal toxicosis after chewing on the plant (Odendaal, 1986).

Poisonous portion: Toxin: Symptoms:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Sangllirlaricr. S. carzcrdensis (blood root).

North America. All portions, especially the rhizome. Sanguinarine. Nausea, vomition, diarrhea, fainting, and coma. Death is not common.

226

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Pinaceae

Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms: Animals affected:

Pinus. P. ponderosa (Western yellow pine). Western North America. Needles and buds. Labdane resin acids (Panter et al., 1998). Cattle browsing on the needles are predisposed to abortion (Panter et al., 1992). Cattle.

Polygonaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Fagopyr~r~. F. sngittcrtr4m Gilib. (buckwheat). Northern temperature hemisphere. Seeds, stems, and leaves. Fagopyrin (photosensitizer). Photosensitization of nonpigmented or white areas of skin. ‘‘In acute cases, nervous symptoms are observed. Erythema is followed by subdermal edematous swelling and eventual necrosis. Nervous symptoms include cerebral excitement, running about, grunting, squealing or bellowing, jumping, convulsions, and prostration’ ’ (Kingsbury, 1964). Cattle, horses, sheep, goats, pigs, chickens, dogs, and cats. Remove animals from the source of the plant. “Shelter from the sun if possible. [Supportive treatment of] topical demulcents (calamine), antibiotics, and steroids may be applied. Steroids and antibiotics (penicillin’)may be given systemically” (Murphy, 1996).

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Rheum. R. rhuponticurn (rhubarb). Native to Siberia, now grown globally as a food crop. The leaves and the upper stems in late summer. Anthraquinone glycosides, oxalates. Nausea, vomiting, diarrhea, abdominal pain, hemorrhage, and death (Cooper and Johnson, 1984).

Genus: Species:

Ru1ne.r. R. crispus L. (curly dock), R. ucetoselln L. (sheep sorrel), R. acetosct L. (sour dock). Mainly temperate Northern Hemisphere, with a few tropical and subtropical species (Heywood, 1993). Whole plant. Oxalates. Acute: salivation, weakness, bradycardia, labored breathing, convulsions, and death (Murphy, 1996). Chronic: anorexia, ataxia, weight loss, depression, diarrhea, prostration, and death (Kingsbury, 1964; Murphy, 1996).

Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Poisonous Higher Plants

Animals affected: Treatment:

227

Sheep. Give fluids and “dicalcium phosphate [and] sodium chloride; 1 :3 [ratio] to form calcium oxalate in the gastrointestinal tract thus preventing oxalate absorption” (Murphy, 1996). Remove animals from the source of the plant.

Ranunculaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Aconitum. A. napellus (wolfsbane). Europe and North America, now a global ornamental. All parts, especially the root. Aconitine and other alkaloids. Ventricular fibrillation and respiratory paralysis, followed by death. Aconitine is well absorbed from the gastrointestinal (GI) tract and is a potent and quick-acting poison. A. rzupellus is considered to beone of the mostdangerous plants in England (Clarke and Clarke, 1967j.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Acfuecr. A. rubm (baneberry). Northern temperate zone woodlands. Berries. Ranunculin. Blistering of lips, mouth, and GI tract, salivation, colic, and diarrhea (Murphy, 1996). Severe gastroenteritis and death of children has been reported (Kingsbury, 1964). Humans and cattle. Milk and egg white as a supportive treatment; the protein may help to dilute the toxin (Murphy, 1996). Remove animals from the source of the plant. “Most poisoning occurs in spring and early summer. Fairly large amounts of the fresh plant must be consumed by livestock for toxicosis. The toxin is not active in the hay” (Murphy, 1996). Animals will not consume the plant unless no other forage is available.

Animals affected: Treatment:

Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected:

Adonis. A. venzulis (pheasant’s-eye). Native to Eurasia, now a global ornamental. Whole plant. Adonidin (a series of phenanthrene glycosides). Adonidin has cardiac activity. Gastroenteritis also is noted. Horses (cases in Australia and Hungary) (Kingsbury, 1964).

Genus: Species: Distribution and habitat: Poisonous portion:

Ccdthn.

C. pnlustris (marsh marigold, cowslip). Wetlands in Europe and North America. Whole plant.

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228

Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Protoanemonin. Tingling and burning of the mouth and skin. Vomition. diarrhea, fall of blood pressure, convulsions, and death (Long, 1917). Cattle. Milk and egg white as a supportive treatment; the protein may help to dilute the toxin (Murphy, 1996) Remove animals from the source of the plant. “Most poisoning occurs in spring and early summer. Fairly large amounts of the fresh plant must be consumed by livestock for toxicosis. The toxin is not active in the hay” (Murphy, 1996). Animals willnot consume theplantunlessno other forage is available.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Clematis. C. virgirliancr (virgin‘s bower). Global as an ornamental, native to the Northern Hemisphere. Whole plant. Ranunculin. Severe gastroenteritis and death (Hurst, 1942).

Genus: Species:

Delphir~i~m. D.bicolor, D. ~zrltt~rllianran (low larkspur), D. brottnii (tall 1arkspur). Temperate Northern Hemisphere. Whole plant, especially the flowers andpods (Majak and Engelsjord, 1988). Methyllycaconitine (MLA), an alkaloid that is a neuromuscular blocker (Nation et al., 1982). MLA has a curare-like action of postsynaptic blockage of nicotinic cholinergic receptors. Signs include nausea, abdominal pain, weakness, rapid pulse and respiration, and involuntary muscle twitching. Death occurs from respiratory paralysis. Sheep, though cattle are most affected. Rumenotomy and complete emptying of the rumen when practicable. Intravenous injection of physostigmine (0.08 mg/kg) as an antidote in the case of collapse. Repeated administration of physostigmine may be required at intervals over several hours until clinical signs have abated when large amounts of larkspur has been ingested resulting in continuous absorption from the GI tract (Nation et al., 1982). “Larkspur(s) are one of the major causes of poisoning of range cattle of (Western) North America” (Majak and Engelsjord, 1988).

Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Genus: Species: Distribution and habitat: Poisonous portion:

Hellebor-us. H. rliger (Christmas rose). Europe. Whole plant.

Plants

Poisonous Higher

Toxin: Symptoms: Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

229

Hellebrin, helleborus, and helleborin (cardiac glycosides). Depression, tachycardia, and death. H. foetidm poisoned cattle in Britain (Holliman and Milton, 1990). Cattle.

Ramwxlus. R. repem (creeping buttercup). Global, especially in wet locations. Whole plant. Ranunculin. Severe gastrointestinal imitation, convulsions, and death in extreme cases. Abortions in a dairy herd in Chile were attributed to consumption of R. repens (Morales, 1989). Cattle and horses. Milk and egg white as a supportive treatment; the protein may help to dilute the toxin (Murphy, 1996).Remove animals from the source of the plant. “Most poisoning occurs in spring and early summer. Fairly large amounts of the fresh plant must be consumed by livestock for toxicosis. The toxin is not active in the hay” (Murphy, 1996). Animals will not consume theplantunlessno other forage is available.

Rhamnaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Karwimkia. K. lzutrzbolticmn (Coyotillo). Central America, extending into the southwest United States. Whole plant. Anthracenones, phenolic compounds (Dreyer et al., 1975). Symptoms do not appear for several weeks after exposure. Symptoms involve a progressive bilateral ascending paralytic neuropathy, terminating in respiratory paralysis.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Rhcmmas. R. catlzurticn (buckthorn). Temperate Northern Hemisphere. Leaves and twigs. Hydroxymethylanthraquinones (phenolic compounds). The principal symptom is diarrhea (Clarke and Clarke, 1967).

Rosaceae

Genus: Species: Distribution and habitat: Poisonous portion:

PYLUl U S .

P. virgirzinrzn L. (Chokecherry), P. serotinu Ehrh. (wild black cherry), P. pensyhaniccr L. (pin cherry). Temperate Northern Hemisphere. Fruits, seeds, and leaves.

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230

Toxin: Symptoms:

Animals affected: Treatment:

Prunasin, a cyanogenic glycoside. Generally rapid death from hydrogen cyanide poisoning. Symptoms sometimes include excitement, muscle tremors, dyspnea, salivation, defecation and urination, and convulsions before death (Murphy, 1996). Cattle, sheep, pigs, and humans. Remove animals from the source of the plant. “Sodium nitrite at10-20 mg/kg with 500 mg/kg sodium thiosulfate as needed” (Murphy, 1996).

Rubiaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Cephaellrs. C. ipecncumhu. South America. The root. Emetine, cephaeline, and related alkaloids. Emetine is a severe emetic. Continued consumption leads to a cumulative action. Emetine is a protoplasmic poison acting on all tissues, leading to degenerative changes in all organs. Palicourea.

P. nzal-cgl-mlii (rat weed). South America. Whole plant. Fluoroacetate ion (Oliveria, 1963). Fluoroacetate inhibits the Krebs cycle. Death by cardiac arrest on exertion or after drinking water (Naude et al., 1996); the fluoroacetate ion leads to a breakdown of the carboxylic acid (Krebs) cycle and an accumulation of citric acid in the blood and liver. Vomition is an invariable and often early symptom, and excitability and distress are obvious; on postmortem analysis, the blood appears to have the color of India ink.

Rutaceae

Genus: Species: Distribution and habitat:

Toxin: Symptoms:

Animals affected:

Zuntho.x-ylm1. 2. cla~)n-lzer.culis (Southern prickly ash). ‘‘West Virginia to Florida and westward in the southern United States to Louisiana, Texas, Oklahoma, and Arkansas’’ (Bowen et al., 1996). Magnoflorine and laurifoline (neuromuscular blockers), neoherculin. “blindness, high stepping gait, inability to drink water, difficulty in swallowing, and considerable struggling in therecumbent state’‘ (Bowen et al., 1996). Cattle and sheep.

,

Plants

Poisonous Higher

Treatment:

Notes:

231

“In the absence of direct studies on clinical treatment of Southern prickly ash toxicosis, use of calcium and neostigmine treatment and positive-pressure artificial respiration with oxygen should be considered’‘ (Bowen et al., 1996). “Chewing the bark of 2. clava-herdis and certain other Zant h o q h m spp has been reported to cause a persistent burning, paralyzing sensation on the lips and tongue and may be the basis for referring to this tree as the ‘toothache tree.’ (Bowen et al., 1996). ”

Santalaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Treatment:

Thesium. T. linearum. Global. All. Bufadienolides (Anderson et al., 1987). Difficulty breathing, asphyxia, tachycardia, arrythmia, bloat, and partialparalysis of the hindquarters (Kellerman et al.,1996). Sheep. “activated charcoal administered at a dose of 2 g/kg. Care should be taken not to stress intoxicated animals during treatment as this could lead to fibrillation or heart block” (Kellerman et al., 1996).

Sapindaceae

Genus: Species: Distribution and habitat:

Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Blighia. B. sapida (ackee, ankye, ishin) (McTague and Forney, 1994). Native to West Africa, introduced to the Caribbean by Captain William Bligh of Bomty fame. Now can be found in Central America and southern Florida. Unripe fruit. Hypoglycin A and B, nonprotein amino acids. Consumption of the unripe fruit leads to violent vomiting (vomiting sickness); headache, nausea, a weak feeling with numbness and tingling over entire body (McTague and Forney, 1994), convulsions, coma, and death follow in a few hours. A severe hypoglycemia is noted. Incidences of hypoglycin A levels at various stages of the ripening process have been published (Brown et al., 1992). Humans. Lavage with normal saline and give activated charcoal with sorbitol, correction for hypokalemia, closely monitor and maintain normal serum glucose levels (McTague and Forney, 1994). Even though ackee is illegal in the United States, it is sold in Canada where several cases of ackee poisoning have been

232

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reported. There is one case of a 37-year-old woman poisoned by canned ackee that was purchased in Canada. “Unripe ackee retains its toxicity even if cooked” (McTague and Forney, 1994). Saxif ragaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Syrnptotns:

Hydr-angea. H. rmcropkyllcr (hydrangea). Temperate to subtropical Northern Hemisphere. Leaves and roots. Hydrangin, a cyanogenic glycoside. Although hydrangea contains cyanogenic glycosides, the symptoms normally encountered are those of dermatitis (Bruce, 1920).

Scrophulariaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Notes:

Digitalis. D. put-purea (foxglove). Europe, now global. Whole plant. Digitoxin, gitoxin, and gitalin (cardiac glycosides). Gastric distress, bloody feces, drowsiness, lack of appetite, frequent attempts to urinate. In humans; nausea, diarrhea, abdominal pain, drowsiness, tremors, convulsions, and death (Kingsbury, 1964). Cattle, humans, pigs, horses, and turkeys. “Toxicity of digitalis is not lost upon drying or boiling, and hay containing the plant has proven lethal’’ (Kingsbury, 1964).

Solanaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Genus: Species: Distribution and habitat: Poisonous portion: Toxin:

Atropcr. A. belladonrzn (deadly nightshade). Native to Europe, cultivated globally. Whole plant. Atropine. These are similar to, but milder than, those described below for Datum and Hyoscy~mus. Humans, cats, dogs, horses, cattle, sheep, and pigs. Bwlfelsin. B. yarzciJEot-a var. floribunda (yesterday-today-and-tomorrow). Temperate and tropical. Leaves, bark, and roots. Alkaloids.

Plants

Poisonous Higher

Symptoms: Treatment:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment: Notes:

233

“On ingestion, animals show ataxia, tremors, depression, and sometimes coma (deep sedation)” (Aiello and Mays, 1998). “In severely depressed animals, stimulants (respiratory and cardiac) along with supportive therapy recommended’‘ (Aiello and Mays, 1998). Daturct. D. strmzorzirrm (jimsonweed, thornapple, angel‘s trumpet, Jamestown weed). Global. Whole plant, especially the seeds. Atropine, hyoscyamine, hyoscine, and scopolamine (tropane alkaloids). These were well discussed by Virgil and include disturbance of vision, fixed or sluggish dilated pupils, dry mucous membranes, flushed skin, headache, fatigue, delirium, thirst, difficulty swallowing and speaking, tachycardia, convulsions, coma, and death. A report details toxicosis from wine made from D.sucrveolens (moonflower) (Smith et al., 1991). Humans, horses, sheep, pigs, birds, and cattle. Activated charcoal, gastric lavage, and physostigmine in severe cases (Perrotta et al., 1995). The highest incidence of poisoning occurs in teenagers who drink jimsonweed tea. “Although all parts of the plant are toxic, the highest concentrations of anticholinergic occur in the seeds (equivalent to 0.1 mg of atropine per seed). The estimated lethal doses of atropine and scopolamine in adults are 2 1 0 mg and >2-4 mg, respectively” (Perrotta et al., 1995).

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Hyoscymus. H. rziger (henbane). Europe, but now global. Whole plant. Atropine, hyoscyamine, and hyoscine (tropane alkaloids). These were well discussed by Virgil and include disturbance of vision, fixed or sluggish dilated pupils, dry mucous nlenlbranes, flushed skin, headache, fatigue, delirium, thirst, difficulty swallowing and speaking, tachycardia, convulsions, coma, and death.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Lycopersicorz. L. escrrlentu~n(tomato). Grown globally as a popular garden plant. Vines and unripe fruit. Alpha-tomatine, a glycoalkaloid. Depression, lethargy, ataxia, diarrhea, and death.

Genus: Species: Distribution and habitat:

Mandragora. M. oficirzarum (the dreaded mandrake). Europe.

234

Poisonous portion: Toxin: Symptoms: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Lang and Smith

Whole plant. Hyoscyamine, scopolamine, and mandragorine (tropane alkaloids). Depression, lethargy, ataxia, diamhea, and death. Nicotiana. N. tabacrun (tobacco). North America, now grown globally as an important cash crop. The leaves. Nicotine, as well as anabasine and anatabine (pyridine alkaloids). Tobacco plants are teratogenic (Keeler, 1988). Acute effects arise from the nerves and consist of excitement, tachypnea, salivation, vomiting, diarrhea, fasciculations, depression,shaking, twitching, weakness, prostration, and death. Cattle, dogs, horses, sheep, cats, and birds. Activated charcoal, an emetic, a carthartic, diazapam, and atropine (Murphy, 1996).

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Phvsdis. P. yeruviarzn (ground cherry). Global. Whole plant. Solanine, a glycoalkaloid. Depression, lethargy, ataxia, diarrhea, and death.

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Solar1dr.a. Solandm species (trumpet flowers). Semitropical. Whole plant. Solanaceous alkaloids. Depression, lethargy, ataxia, diarrhea, and death.

Genus: Species:

Solanum. S. sarrachoides (hairy nightshade), S. dulccrrnaln L. (European bittersweet nightshade), S. elreagnifoliurn Cav. (silverleaf nightshade), S. tuberosunz L. (potato). The nightshades have global distribution. ‘‘Toxicity of a given species can vary widely due to environment, part of the plant. or degree of maturity. Green berries are more toxic than red or black berries which are more toxic than leaves which are more toxic than stems or roots. Mature plants may have more toxin than immature plant,” (Murphy, 1996). Solanaceous alkaloids. Depression, salivation, anorexia, nausea, abdominal pain, vomiting, mydriasis, lethargy, ataxia, constipation or diarrhea, in some cases bloody diarrhea, trembling, weakness, prostration, unconsciousness, and death. A report details the death

Distribution and habitat: Poisonous portion:

Toxin: Symptoms:

her

Poisonous

Animals affected: Treatment:

Notes:

235

of ruffled lemurs (Drew and Fowler, 1991). S. tuberosurn (potato) is also a problem. The vines are toxic (solanine), as are green tubers (Jadhav et al., 1981). Sublethal doses of S. dulcnmnm and S. elaengnifoliu~nin pregnant animals can cause congenital malformations (Keeler et al., 1990). Lemurs, cats, dogs, horses, cattle, sheep, pigs, and birds. “Emetic (apomorphine), charcoal, cathartic (mineral oil or saline). Supportive [treatment] demulcents (milk), fluids (if dehydrated)” (Murphy, 1996). “Cattle grazing stubble containing the nightshade have resulted in deaths. The animals prefer the green weed to stubble” (Murphy, 1996).

Taxaceae

Genus: Species: Distribution and habitat: Poisonous portions: Toxin: Symptoms: Animals affected: Treatment: Notes:

Taxus. T. baccnta (English yew), T. cuspidntn (Japanese yew). Northern portion of Northern Hemisphere. Bark, needles, and drupes. Several alkaloids, of which taxine A and taxine B are the most important. Death, incoordination, nervousness, difficulty breathing, bradycardia, diarrhea, and convulsions (Parkinson, 1996). Humans, cattle, horses, cats, dogs, and sheep. None. T. baccata is widely recognized as the most poisonous tree or shrub in the United Kingdom, where it has been cultivated in churchyards for centuries for the express purpose of discouraging sacrilegious grazing by farm animals on holy ground. Numerous reports of the poisoning of livestock and humans continue to occur worldwide. Children are attracted to the large, bright-red, fleshy drupes and, when a small number of whole drupes are eaten (seed included), death occurs. In cattle, death occurs so rapidly that the animals are found in close proximity to the tree and often have undigested twigs and leaves in their rumens (Ogden, 1988).

Thymelaeaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected:

Dcphne. D. mezerurn (daphne). Temperate Northern Hemisphere. All, but especially the berries and bark. Daphnetoxin and mezerein (diterpene alcohols). Vomition, blood, diarrhea, stupor, weakness, convulsions, and death. The berries are very attractive to children and very dangerous. Pigs and a horse have died after ingestion of the berries (Cooper and Johnson, 1984). Pigs and horses.

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Umbelliferae Genus: Species:

Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Notes:

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Treatment: Notes: Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Ciclrtn. C. douglcrsii (water hemlock, beaver poison, children’s bane, death-of-man, poison parsnip, false parsley (Sweeney et al., 1992). The genus is found in North America. Very similar plants in Eurasia are placed in the genus Oemrrlthe. All, but especially the root. Cicutoxin, a polyunsaturated diol. Death. Water hemlock is the most dangerous plant in western Canada. The onset of death is very rapid and the plant material often is found in the esophageal groove of cattle (Smith and Lewis, 1987b). Symptoms include: vomiting, tremors, convulsions, cyanosis, tachycardia, dilated pupils, profuse salivation, delirium, nausea, abdominal pain, dizziness, perspiration, and respiratory distress (Sweeney et al., 1992). Humans, cattle, and horses. Gastric lavage and administration of activated charcoal “No antidotes exist, and treatment is supportive” (Sweeney et al., 1992). A case where two brothers mistook water hemlock for wild ginseng, two bites of the root killed one man. “Ingestion of a 2-3 cm portion of the root can be fatal in adults, and the use of toy whistles made from the water hemlock stem has been associated with deaths in children. Poisonings typically result from ingestions; however, cicutoxin also maybe absorbed through the skin” (Sweeney et al., 1992). Conizrm. C. rz~aczdntllnl(poison hemlock). Native to Europe, now global. Whole plant. coniine and related piperidine alkaloids. Salivation, excitement, gastroenteritis; the patient becomes recumbent and death occurs from respiratory paralysis. Humans, cattle, horses, and pigs. “Treatment is rarely possible” (Murphy, 1996). Remove animals from access to the plant. Socrates was executed by “suicide,” being forced to drink a decoction made from this plant.

Oenm~tl~e. 0. crocntn (water dropwort). Europe. All, especially the root. Oenanthotoxin. Oenanthotoxin is similar to cicutoxin. Death. The onset of death is very rapid. Symptoms include: vomiting, tremors,

Poisonous Higher Plants

Animals affected:

237

convulsions, cyanosis, tachycardia, dilated pupils, profuse salivation, delirium, nausea, abdominal pain, dizziness, perspiration, and respiratory distress (Sweeney et al., 1992). Cattle.

Urticaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Urtica. U. dioicn (common stinging nettle). Tropical and temperate regions. Whole plant. Acetylcholine, histamine. Edema, erythema on areas of skin exposed to the plant. “Animals rarely ingest the plant, but chewing on it will produce salivation and edema of the mucous membranes’’ (Murphy, 1996). Dogs, cats, horses, and ducks. Remove animals from the source of the plant. Supportive treatment involves the use of antihistatnines and glucocorticoids (Murphy, 1996).

Verbenaceae

Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms:

Durnnta D. repens (golden dewdrop). Global, in warm areas of the world. Fruits. Saponins. Alleged to have caused death.

Genus: Species: Distribution and habitat:

Lmtana. L. carnnra (lantana). Native to tropical America and Africa, now widely grown as a garden plant. Leaves and berries. Lantadene A (pentacyclic terpene). Hemorrhagic gastroenteritis, staggering, progressive weakness, bloody diarrhea, in some cases conjunctivitis, reddening of the muzzle, icterus, dyspnea, hepatogenus photosensitization in mild cases, but mild cases are rare and death normally occurs before signs of photosensitization are evident (Fourie et al., 1987; Kingsbury, 1964). Cattle, sheep, humans, goats, horses, dogs, and cats. “Poisoned animals should be removed from infested paddocks, kept in the shade, dosed with activated charcoal and treated symptomatically” (Kellerman et al., 1996). Steroids and antibiotics (penicillin, orally) given topically or systemically (Murphy, 19963.

Poisonous portion: Toxin: Symptoms:

Animals affected: Treatment:

Lang and Smith

238

Zygophyllaceae Genus: Species: Distribution and habitat: Poisonous portion: Toxin: Symptoms: Animals affected: Treatment: I

Notes:

Tribhs. T. terrestris. Global. Fruits. A steroidal saponin. “Photosensitivity and icterus” (Kellerman et al., 1996). Sheep and goats. “Affected sheep must be kept in the shade and, where practical, treated symptomatically” (Kellerman et al., 1996). “Hepatogenous photosensitivity (geeldikkop) results from failure of the liver to excrete phylloerythrin (a photodynamic porphyrin) produced by the degradation of chlorophyll in the gut of herbivorous animals. In geeldikkop, phylloerythrin is believed to accumulate in the blood as a result of the occlusion of bile ducts by crystalloid tnaterial composed of the Ca” glucuronides of epismilagenin and episarsasapogenin” (Kellerman et al., 1996).

APPENDIX Alphabetical List of All Species Discussed in This Chapter Toxin(s)

Group

Genus

in genus

Abrus Acacia Acer Ackocarzthern Acortiturlt Actaea Adorlis Aesculm Agrostemma Aleuritis Amaranthus Amsirtckin Areca Argemorze Arisaema Asclepias Astragalus Atropa Avena A:adirachza Beta Blighia Brassica

Leguminosae Leguminosae (Mimosaceae) Aceraceae Apocynaceae Ranunculaceae Ranunculaceae Ranunculaceae Hippocastanaceae Caryophyllaceae Euphorbiaceae Amaranthaceae Boraginaceae Palmae Papaveraceae Araceae Asclepiadaceae Leguminosae (Fabaceae) Solanaceae Gramineae Meliaceae Chenopodiaceae Sapindaceae Brassicaceae

Brmfelsia

Solanaceae

Phytotoxin it-Methyl-beta-phenylethylamine An unknown oxidant Cardiac glycosides Alkaloids (other) Ranunculin Phenanthrene glycosides Saponin Sapogenin Saponin and a phytotoxin Nitrate ion, oxalates Pyrrolizidine alkaloids (PAS) Arecoline Isoquinoline alkaloids Oxalates Cardiac glycosides 3-Nitropropanolics, swainsonine, selenium Atropine Nitrate ion Azadirachtin Nitrates and oxalates Amino acids Isothiocyanates, nitrates. L-5-vinyl-2-thiooxazolidone and a goitrogenic compound Alkaloids

Poisonous Higher Plants

239

APPENDIX Continued Group

Genus

genus Buxaceae Araceae Ranunculaceae Cannabinaceae Berberidaceae Rubiaceae Papaveraceae Chenopodiaceae Liliaceae Unlbelliferaceae Ranunculaceae Liliaceae Areceae Unlbelliferaceae Liliaceae Fumariaceae Corynocarpaceae Crassulaceae Leguminosae (Fabaceae) Gramineae Leguminosae (Papilionaceae) Thymelaeaceae Solanaceae Ranunculaceae Fumariaceae Dichapetalaceae Araceae Scrophulariaceae Dioscoreaceae Verbenaceae Boraginaceae Ephedraceae Equisetaceae Erythroxylaceae Asteraceae Euphorbiaceae Polygonaceae Gramineae Leguminosae Loganiaceae Liliaceae Malvaceae Legurninosae Chenopodiaceae Lamiaceae Boraginaceae Ranunculaceae Euphorbiaceae

in

Alkaloid (other) Calcium oxalate crystals Protoanemonin Cannabinoids Cytotoxin, hallucinogen Alkaloids (other) Isoquinoline alkaloids Nitrates Unknown Polyunsaturated diol Ranunculin Alkaloid (other) Oxalates Piperidine alkaloids Cardiac glycosides Tsoquinoline alkaloids Nitropropanolic glycoside Bufadienolides Pyrrolizidine Alkaloids Cyanogenic glycosides Quinolizidine alkaloids Diterpene alcohols Tropane alkaloids Methyllycaconitine (MLA) Isoquinoline alkaloids Fluoroacetate ion Oxalates Cardiac glycosides Dioscorine (an alkaloid) Saponins Pyrrolizidine alkaloid (PAS) Andrenomimetic Unknown Ecgonine bases Tremetol Unknown Fagopyrin (photosensitizer) Alkaloids (other) Fluroacetate ion Alkaloids (other) Alkaloids (other) Gossypol Unknown Oxalates Pulegone Pyrrolizidine alkaloids Cardiac glycosides Phytotoxin

Lang and Smith

240

APPENDIX Continued Toxin(s)

Group

Genus

genus Saxifragaceae Solanaceae Hypericaceae Euphorbiaceae Juglandaceae Ericaceae Rhamnaceae Chenopodiaceae Leguminosae Verbenaceae Leguminosae (Fabaceae) Ericaceae Leguminosae (Mimosaceae) Ericaceae Oleaceae Campanulaceae Leguminosae (Fabaceae) Solanaceae Lycopodiaceae Ericaceae Solanaceae Euphorbiaceae Ericaceae Euphorbiaceae Palmae Cucurbitaceae Iridaceae Myoporaceae Liliaceae Apocynaceae Solanaceae Umbelliferae Leguminosae (Fabaceae) Rubiaceae Graminaceae Papaveraceae Ericaceae Araceae Solanaceae Leguminosae Ericaceae Pinaceae Berberidaceae Rosaceae Dennstaeditaceae Fagaceae Ranunculaceae Rhatnnaceae Polygonaceae

Cyanide formers Tropane alkaloids Hypericin Phytotoxin Unknown Grayanotoxins Phenolics Nitrates, oxalates, and a photosensitizer Quinolizidine alkaloid Pentacyclic terpene Amino acid Grayanotoxins Amino acid Grayanotoxins Unknown Pyridine alkaloids Quinolizidine alkaloids Glycoalkaloid Levotetrahydropalmi tine Grayanotoxins Tropane alkaloids Cyanide formers Grayanotoxins Saponin Cycasin (glycoside) Lectin Bufadienolides Sesquiterpene Unknown Cardiac glycosides Pyridine alkaloids Polyunsaturated diol Indolizidine alkaloids Fluoroacetate ion Unknown Alkaloids (narcotic analgesics) Grayanotoxins Oxalates Glycoalkaloid Anticholinesterase Grayanotoxins Labdane resin acids Cytotoxin Cyanide formers Ptaquiloside Tannins and gallotannins Ranunculin Phenolics Oxalates, anthraquinone glycosides

Poisonous Higher Plants

APPENDIX Group

241

Continued

Genus

genus

Rhododendron Ricinus Robinia Rwtex Sanguinclria Scrssqfras Sernecnrpus

Ericaceae Euphorbiaceae Leguminosae (Fabaceae) Polygonaceae Papaveraceae Lauraceae Anacardiaceae

Senecio Senna Sesbania Solnndrn Solanml Sophora Sorghum Strychzos Synlphytum Tasus Te~rcritm

Asteraceae Legunlinosae Leguminosae (Fabaceae) Solanaceae Solanaceae Leguminosae (Fabaceae) Grarninaceae Loganaceae Boraginaceae Taxaceae Larniaceae

Therwoysis Thesium Theveticl Thlaspi Toxicodendron

Leguminosae (Fabaceae) Santalaceae Apocyanaceae Brassicaceae Anacardiaceae

Tribulus Tricodesma Triglochin Tylecodon Urechitis Urginen Urtica Veratrum Verbesincr Vicia Viscurn Wisteria Xunthiunj Zantho.qdum Zea Zygndenus

Zygophyllaceae Boraginaceae Juncaginaceae Crassulaceae Apocynaceae Liliaceae Urticaceae Liliaceae Asteraceae Legunlinaceae (Fabaceae) Loranthaceae Leguminosae (Fabaceae) Asteraceae Rutaceae Graminaceae Liliaceae

in

Grayanotoxins Ricin Phytotoxin Oxalates Sanguinarine Hepatotoxin 3-Pentadecylcatechol and urushiol, oleoresins Pyrrolizidine alkaloids (PAS) Sennosides (laxative alkaloids) Cytotoxic alkaloid Solanaceous alkaloids Solanaceous alkaloids Quinolizidine alkaloids Cyanogenic glycoside, nitrates Alkaloids Pyrrolizidine alkaloids Alkaloids Neoclerodane deterpenoids, saponins. glycosides, flavonoids, hepatotoxins Quinolizidine alkaloids Bufadenolides Cardiac glycosides Sinigrin. myrosin 3-Pentadecylcatechol and urushiol, oleoresins Steroidal saponin Pyrrolizidine alkaloids Cyanide formers Bufadienolides Cardiac glycosides Cardiac glycoside Acetylcholine, histamine Glycoalkaloids, ester alkaloids Nitrates, galegine Glycosides Polypeptide Unknown Glycoside Neuromuscular blockers, neoherculin Nitrate ion Steroid alkaloids, glycoalkaloids

REFERENCES 1. Abbott, P. J. (1988), Comfrey: assessing the low-dose health risk. Med. J. Allst. 149(11/12): 678-682.

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5.

6.

7.

8. 9. 10. 11.

12.

13. 14. 15. 16. 17. 18. 19.

20. 21. 33.

23. 23.

25.

Lang and Smith Aiello, S. E., A. Mays (eds.) (1998)The Merck Veterimr? Mama/. Eighth Edition. Merck & Co., Whitehouse Station. N.J. Alonso-Amelot. M. E., U. Castillo, B. L. Smith, D. R. Lauren (1996), Bracken ptaquiloside i n milk. Nature 382(6592):587. Anderson, I. B., W. H. Mullen, J. E. Meeker, S. C. Khojasteh-Bakht. S. Oishi, S. D. Nelson. and P. D. Blanc (19961, Pennyroyal toxicity: measurement of toxic metabolite levels in two cases and review of the literature. Ann. Znt. Mecl. l X ( 8 ) : 726-734. Anderson, L. A., J. P. Joubert. R. A.Schultz, T.S. Kellerman, B. J. Pienaar (1 987), Experimental evidence that the active principle of the poisonous plant Tllesizlrn linecrtumL.f. (Santalaceae) is a bufadienolide. Oizclerstepoort J. Vet. Res. 54(4):635-650. Baker, D. C., L. F. James. W. L. Hartley. K. E. Panter. H. F. Maynard, and J. Pfister (1989). Toxicosis in pigs fed seleniunl-accumulating astragalus plant species or sodium selenate. A m J. Vet. Res. 50(8):1396-1 399. Biberoglu, S., K. Biberoglu, andB.Kornsuoglu(1988).Madhoney. J. An?. Med. Assoc. 259(13):1943. Boughton, I. B., and W. T. Hardy (1935). Mescalbean (Sophora securzdiflor-cr)poisonous for livestock. Texas Agr. Expt. Stcr.. Bull. 519. Boughton, I. B.. and W. T. Hardy (1939). Toxicity of the coffee bean (Sesbania Ivsicaria) for sheep. J. A m Vet. Med. Assoc. 95239. Boughton, I. B., and W. T. Hardy (1940), Feeding trials of suspected plants. Texas Agr. Exp. Stn. Ann. Rep. 53236. Bowen. J. M., R. J. Cole, D. Bedell,and D. Schabdach (1996). Neuromusculx effects of toxins isolated from Southern prickly ash (Zcrntho.~ylumclnlw-herculis) bark. A. J. V. R. 57(8): 1239- 1244. Brown, M., R. P. Bates, C. McGowan, and J. A. Cornel1 (1992), Influence of fruit maturity on the hypoglycin-A level in ackee (Blighia sapidel). J. Food Safe9 12(2):167-177. Bruce, E. A. (1920), Hydrangea poisoning. J. Am. Vet. Med. Assoc. 56:72. Campbell. G. W. J., and G. C. Kingston (1992). Heart leaf poison bush (Gastrolobiunz grmzdijlorwr) toxicity in a Western Queensland sheep flock. Vet. H m . Toxicol. 34(3):265-266. Casteel, S. W.. and D. J. Wagstaff (1992), Aesczhs glnbra intoxication in cattle. Vet. H m . Toxicol. 34( 1):55. Chareyre, S., D. Meram, C. Pulce, and J. Descotes (1989), Acute poisoning by autumnal crocus. Vet. H m . Toxicol. 31(3):261-262. Cheeke, P. R., and L. R. Shull ( 1985), Ne~tr41-d Toxicants in Feeds nrzd Poisonorls Plants. Avi Publishing, Westport. Connecticut, pp. 154-161. Cheeke. P. R. (ed.) (1989a). Tosicarlts of Plant Origin, Vol. 1. Alkaloids. CRC Press, Boca Raton, Fla. p. 64. Cheeke, P. R. (ed.) (1989b), Toxicernts of Plant Origirl, Vol. 2, Glycosides. CRC Press. Boca Raton. Fla.. p. 80. Clarke. E. G. C.. and M. L. Clarke (1967), Garrler’s Veterinary Toxicology. Tundall and Cassell, London, p. 3 10. Clarke, E. G. C. (1969), Zsolertioiz crnd Zclentification of Drugs. Pharmaceutical Press. London, p. 348. Collett. S., D. Grotelueschen. R. A. Smith, R. Wilson (1996), Deathsof 23 adult cows attributed to intoxication by the alkaloids of Z\lgadenus \wwnoslls (meadow death c‘amas). Agriprcrctice 17(7):5-9. Cooper, M. S.. and A. W. Johnson (198;C),Poisonous Plants i n Britain, Ministry of Agriculture Food Report Bonk 161. Her Majesty’s Stationery Office, London. Cronquist, A., and H. A. Gleason (1991), M a n l d of Vascular Plmts of northeaster^ United Srnres and Adjacent Ccrncrdcr. New York Botanical Garden, Bronx, NY. Davis. J. H. (1 978L Abrus preccrtorius (rosary pea): the most conunon lethal plant poison. J. Flcr. Med Amoc. 65: 1 89- 191 .

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26. Dollahite. J. W., and T. J. Allen (1962), South West Vet. 15: 126. 27. Dreisbach, R. H. (1963), Handbook of Poisoniizg: Diaglzosis cmd Treatnzelrt, 4th Ed. Lange Medical Publications, Los Altos, Calif., p.367. 28. Drew, M. L., and M. E. Fowler (1991),Poisoning of black and white ruffled lemurs (Varecia variegcrta variegata) by hairy nightshade (Sola~ziumsarrachoides). J. Zoo Wildl$e Men. 233): 494-496. 29. Dreyer, D. L., I. Arai, C. D. Bachman, W. R. Anderson, Jr., R. G. Smith. and G. D. Daves. Jr. (1975), Toxins causing non inflammatory paralytic neuronopathy. Isolation and structure elucidation. J. Am. Clzenz. SOC. 97:4985-4990. 30. Espinoza, 0. B., M. Perez, and M. S. Ramirez (1992), Bitter cassava poisoning in eight children: a case report. Vet. Hum. Toxicol. 34(1):65. 31. Fisher, A.A. (1996), Poison ivy/oak/sumac.Part 11: Specificfeatures. CUTIS 58(1):2224. 32. Flaoyen, A., B. Bratberg. A. Froslie. H. Gronstol, W. Langseth, P. G. Mantle. and A. Von Krogh (1997), Further studies on the presence, qualities and effects of the toxic principles from Nurthecitrrtz oss$ragm plants. Vet. Res. Corn. 2 1:137- 148. 33. Fourie, N., J. J. Van der Lugt, S. J. Newsholme, and P. W. Ne1 (1987), Acute Lantana carnai-c! toxicity in cattle. J. South. Afi. Vet. Assoc. 58(4):173-178. 34. Galey F. D., H. E. Whiteley, T. E. Goetz, A. R. Knuenstler. C. A. Davis, and V. R. Beasley (1 991 ), Black walnut (Jl4glansnigra) toxicosis: a model for equinelaminitis. J. Corny. Patlzol. 104(3):313-326. 35. Galey, F. D.. D. M. Holstege, K. H. Plumlee, E. Tor, B. Johnson, and M. L. Anderson, P.C. Blanchard, F. Brown (1996), Diagnosis ofoleanderpoisoninginlivestock. J. Vet. Diagn. Iwest. 8(3):358-364. 36. Haynes. B. E., H. A. Bessen, and W. D. Wightman (1985), Oleander tea: herbal draught of death. Ann. Enzet-g. Med. 14:350-353. 37. Heywood, V. H. (ed.) (1993), Flowering Plants of the World. Oxford University Press, New York. 38. Holliman, A., and D. Milton(1990), Hellebor14sfoetidtrs poisoning of cattle. Vet. Rec. 127( 13): 339-340. reference 39. Hooper, P. T. (1978). Pyrrolizidine alkaloid poisoning-pathology with particular to differences in animal and plant species. In Efsects of Poisonous P1crrrt.s O H Livestock. R. F. Keeler, K. R. Van Kampen, and L. F. James (Eds.). Academic Press, New York. 40. Hurst, E. (1942), The Poisonous Plants of New South Wales, N. S. W. Poison Plants Committee, Sydney. 41. Jacobziner, H., and H. W. Raybin (1960), Plant and insecticide poisonings. New Yoi-k State J. Med. 60:3 139-3 142. 42. Jadhav, S. J., R. P. Shalma, and D. K. Salunkhe (1981), Naturally occurring toxic alkaloids in foods. In Critical review:^ in To.xicology. L. Golberg (Ed.). CRC Press, Boca Raton, Fla., p. 95. 43. James, L. F., K. E. Panter, and R. J. Molyneux (1992). Selenium poisoning in livestock. In Poisorzozls Plarzts,Proceedings of the Third hternational Synzposiunt. L. F. James, R. F. Keeler, E. M. Bailey, Jr., R. R. Cheeke, and M. P. Hegarty (Eds.). Iowa State University Press, Ames. p. 153-158. 44. Keeler, R. F. (1978), Alkaloid teratogens from Lzlyirztrs, Co~izcnz,Veratrzrttz and related genera. In Efsects of Poisoilolrs P l u m orz Lillestock. R. F. Keeler, K. R. Van Kampen, andL. F. James (Eds.). Academic Press, New York, pp. 397-408. 45. Keeler, R. F.. A. E. Johnson, and R. L. Chase (1986), Toxicity of Thernzopsis rtzontarta in cattle. Cornell Vet., 76: 115-127. 46. Keeler, R. F. (1988), Livestock models of human birth defects, reviewed in relation to poisonous plants. J. Atzinz. Sci. 66:2414-2427. 47. Keeler. R. F. (1989), Quinolizidine alkaloids in range and grain lupins. In Toxicants of Plant

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50. 51. 52. 53. 54. 55. 56. 57.

58. 59. 60. 61. 62. 63. 64. 65.

66.

67.

68. 69. 70. 71.

Lang and Smith Origin.Vol I. Alkaloids. P. R. Cheeke(Ed.). CRC Press,BocaRaton,Florida,pp.133167. Keeler, R. F., D. C. Baker, and W. Gaffield (1990), Spirosolane-containing Solcrmm species and induction of congenital craniofacial malformations. Toxicon 28(8):873-884. Kellerman, T. S., T. W. Naude, and N. Fourie (1996), The Distribution, diagnosis, and estimated economic impactof plant poisonings and mycotoxicoses in South Africa. Onderstepoort J. Vet. Res. 63(2):65-90. Kernaleguen, A., R. A. Smith, and C. W. Yong (1989). Acute mustard seed toxicosis in beef cattle. Con. Vet. J. 30(6):524. Ketterer. P. J., P. E. Glover, and L. W. Smith (1987), Blue heliotrope(Heliotropium nmplexicnule) poisoning in cattle. Aust. Vet. J. 64(4):115-1 17. Kingsbury, J. M. (1964), Poisonous Plants of the United Stcrtes and Cnnadr. Prentice-Hall, Englewood Cliffs, N.J. Lampe, K. F., and M. A. McCann (1985). AMA Handbook of Poisonorrs and Injurious Plants. Chicago Review Press, Chicago. Langford, S. D. and P. J. Boor(1996), Oleander toxicity:an examination of human and animal toxic exposures. Toxicology 109:1-1 3. Larrey, D. (1997), Hepatotoxicity of herbal remedies. J. Hepatol. 26 (Suppl. 1):47-51. Long, H. C. (1917), Plants Poisonous to Livestock. Cambridge University Press. England. Lopez, T. A., C.M. Campero, R. Chayer,B. Cosentino, and M. Caracino (1996), Experimental toxicity of Verbesina encelioides in sheep and isolation of galegine. Vet. H m . Tosicol. 38(6): 417-419. Majak, W., M. Engelsjord (1988). Levelsof a neurotoxic alkaloidin a speciesof low larkspur. J. Range Mmage. 41(3):224-226. McIntosh, R. A. (1928), May apple poisoning in a cow. Ont. Vet. Coll. Rep. 29: 18-20. McTague, J. A., R. Forney, Jr. (1994), Jamaican vomiting sickness in Toledo, Ohio. Ann. Emerg. Med. 23(5): 1116-1 1 18. Morales, H. (1989), Abortions in a dairy herd in the VI11 region of Chile attributed to the consumption of creeping buttercup (Ramrzculus repem). Arch.Med. Vet. 21 (3):163-166. Morton, J. F. (1958), Ornamental plantswith poisonous properties. Proc. FZorida Stole. Hort. SOC. 7 1:372. Muenscher. W. C. (1951),Poisonous Plants qftlze United States.2d Edition. Macmillan,New York. Murphy, M. (1996),A Field Guide to Common Anirlzal Poisons. Iowa State University Press, Ames. Nation, P. N., M. H. Bean, S. H. Roth, and J. L. Wilkens (1982), Clinical signs and studies of the site of action of larkspur alkaloid, methyllycaconitine, administered parenterally to calves. Can. Vet. J. 23:264-266. Naude, T. W., T. S. Kellerman, and J. A. W. Coetzer (1996). Plant poisonings and mycotoxicoses as constraints in livestock production in East Africa: the Southern African experience. J. S. Afr. Vet. Assoc. 67(1):8-11. Nel, P. W.. R. A. Schultz, P. Jordaan, L. A. P. Anderson,T. S. Kellerman. and C. Reid (1987), Cardiac glycoside poisoning in sheep caused by Ul-girzecrphjlsodes and the isolated physodine A. Onderstepport. J. Vet. Res. 54(4):641-644. Odendaal, J. S. J. (1986), Suspected opiumpoppy poisoning in two young dogs. J. South. Afr. Vet. Assoc. 57(2): 113-1 14. Ogden, L. (1988). Tcrws (yews)-a highly toxic plant. Vet. Hum. Toxicol. 30(6):563-564. Oliveria, M. M. (1963), Chromatographic isolationof monofluoroacetic acid from Palicouren nzarcgravii. Expel-ierztia 19:586. Olsen, C.T., W. C. Keller,D. F. Gerken, and S. M. Reed (19841, Suspected tremetol poisoning in horses. J. Am. Vet. Med. Assoc. 185(9):1001-1003.

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72. Panariti. E. (1996). Meadow saffron (Colchiczrm autumnale) intoxication in a nomadic Albanian sheep flock. Vet. Hum. Toxicol. 38(3):227-228. 73. Panter, K. E., L. F. James, D. Nielson. R. J. Molyneux, M. H. Ralphs, and J. D. Olsen (1988), The relationship of Osytropis sericea (green and dry) and Astragalus lentigirzosus with high mountain disease in cattle. Vet. Hum. Toxicol. 30(4):318-323. 74. Panter, K. E., L. F. James, and R. J. Molyneux (1992), Ponderosa pine needle-induced parturition in cattle. J. Anim. Sci. 70(5):1604-1608. 75. Panter, K. E., D. R. Gardner, L. F. James, B. L. Stegelmeier, J. A. Pfester. R. J. Molyneux, M. H. Ralphs, and J. N. Roitman (1998), Pine needle (Pinus ponderosa)and broom snakeweed (Gzttierrezia spp.) abortion in livestock. In Toxic Plmts amiOther Natural Toxicants. T. Garland and A. C. Barr (Eds.) CAB International, Wallingford, UK. pp. 307-31 1. 76. Parkinson. N. (1996), Yew poisoning in horses. Can. Vet. J . 37(11):687. 77. Perrotta, D. M., L. N. Nickey, M. Raid, T. Caraccio, H. C. Mofenson, C. Waters, D. Morse, A. M. Osorio, S. Hoshiko, and G. W. Rutherford 111. (1995), Jimson weed poisoning-Texas, New York, and California, 1994. MMWR U.S. Department of Health and Human Services. 44(3):41-44. 78. Pratchett, D., R. J. Jones. and F. X. Syrch (19911, Use of DHP-degrading rumen bacteria to overcome toxicity in cattle grazing irrigated Leucaena pasture. Trop. Grasslands 25:3. 79. Rauber. A., and J. Heard (1985), Castor bean toxicity re-examined: a new perspective. Vet. Hum. Toxicol. 27(6):498-502. 80. Rogers, T. B. (19141, On the action of St. Johnswort as a sensitizing agent for non-pigmented skin. Am. Vet. Rev. 46:145. 81. Seaman, J. T., W. S. Turvey, S. J. Ottaway, R. J. Dixon. and A. R. Gilmour (1989). Investigations into the toxicity of Echizcm plurztagirzeunz in sheep. 1. Field grazing experiments. Aust. Vet. J. 66(9):279-285. 82. Smith, E. A., C. E. Meloan, J. A. Pickwell, and F. W. Oehme (1991), Scopolamine poisoning from homemade “moon flower” wine. J. Anal. TOX.15(July/Aug):216-219. 83. Smith, R. A., and S. P. Crowe (1987a), Fanweed toxicosis in cattle: case history, suggested treatment, and fanweed detoxification. Vet. Hum. Toxicol. 29(2): 155- 156. 84. Smith, R. A., and D. Lewis (1987b), Cicutn toxicosis in cattle: case history and simplified analytical method. Vet. Hum. Toxicol. 29(3):240-241. 85. Smith. R. A. (1988), Coma in a ferret after ingestion of cannabis. Vet. Hum. Toxicol. 30(5): 486. 86. Smith, R. A., and D. Lewis (1990). Apparent Coqdalis aurea intoxication of cattle. Vet. Hum. Toxicol. 32(1):63-64. 87. Smith, R. A., and D. Lewis (1991a), Death camas poisoning in cattle. Vet. Hum. Toxicol. 33(6):615-616. 88. Smith, R. A., and A. Suleiman (1991b), Nitrite intoxication from large round bales. Vet. H u m To.ricol.33(4):349-350. 89. Smith, R. A., R. E. Miller, and D. G. Lang (1997), Presumptive intoxication of cattle by corn cockle, Agrostenzma g i t h g o (L). Scop. Vet. Hunt. To.xicol. 39(4):250. 90. Stair E. L., W. C. Edwards, G. E. Burrows, K. Torbeck (1993), Suspected red maple (Acer rubrum) toxicosis with abortion in two Percheron mares. Vet. Hum. Toxicol. 35(3):229-230. 91. Sweeney, K., K. F. Gensheimer, J. Knowlton-Field, and R. A. Snuth (1992), Water hemlock poisoning-Maine, 1992. JAMA 27 1 (19):1475. 92. Tandon, B. N., H. D. Tandon, R. K. Tandon, M. Marendranathan, and Y. K. Joshi (1976), Epidemic of veno-occlusive disease in central India. Lancet (August):271. 93. Volker, R. (1950), Eugen Frohner’s Lehrbuch der Toxicologie, 6th Ed. Ferdinand Enke Verlag. Stuggart. 94. Watt, J. M., and M. G. Breyer-Brandwijk (19621, The Medicirlal arrdPoisorlozrs Plants of Southern and Eastern Africa. E. and S. Livingstone, Edinburgh, UK.

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95. Weber. M., and R. E. Miller (1997) Presumptive red maple (Acer nrbrarm) toxicosis in Grevy’s zebra (Equus grevyi). J. Zoo Wild. Mecl. 28(1):105-108. 96. Welchman, D. de B., J. C. Gibbens, N. Giles, D. W. T. Piercey, and P. H. Skinner (1995), Suspected annual mercury (Mercurialis an~zua)poisoning of lambs grazing fallow arable land. Vet. Rec. 137(1023):592-593. 97. Wofford. B. E. (1989). Guide to the Vascrhr Plants of the Blue Ridge. University of Georgia Press, Athens, Ga. 98. Zomlefer, W. B. (1994), Guide to Flowering P l a ~ Families. t University of North Carolina Press, Chapel Hill. N. C.

4 Alkaloids

I.

Introduction

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11. Plant Families Containing Alkaloids that Cause Illness or Death in Humans A. B. C. D.

E. F. G. H.

Solanaceae 248 Liliaceae 249 Boraginaceae 249 Papaveraceae 250 Umbelliferae 250 Taxaceae 250 Ranunculaceae 25 1 Fabaceae 25 1

111. Plant Families Containing Alkaloids that Cause Illness or Death in Livestock A. B. C. D. E. F. G.

H.

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Fabaceae 25 1 Asteraceae 252 Umbelliferae 252 Boraginaceae 252 Solanaceae 252 Liliaceae 252 Taxaceae 253 Cyanophyceae 253 Ranunculaceae 253 Canlpanulaceae 253 Convolvulaceae 253

I. J. K. References 254

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1.

INTRODUCTION

In discussing the alkaloids we need to define what an alkaloid is and what it is not. In the first edition of this book Sinden and Deahl wrote, “Alkaloids are nitrogen-containing compounds of plant origin that generally have complex molecular structures and manifest significant physiological activity in animals.” They then went on to discuss phalloidin, a polypeptide from Arnanita yhalloides, the death cup mushroom. Kingsbury (1964) listed several poisonous principles and defined alkaloids as “products of chemical analysis of plants which are not true bases (alkalis) but share certain chemical similarities with them. They are basic in reaction and form salts with acids. Gerzerully irzsoluble in water [my emphasis], but extractable in organic solvents.’’ In this list Kingsbury distinguished polypeptides from alkaloids and treated them separately; he also separated amines from alkaloids. Most amines would besomewhat soluble in water and would therefore fail to comply with his definition of an alkaloid thus justifying a separate category for them. Kingsbury specifically mentions P-phenethylamine (a primary amine) and N-methyl-P-phenethylanline (a secondary amine) as examples of amines; both are somewhat soluble in water. Another group of authors insists that for a compound to be considered an alkaloid at least one nitrogen must be in a ring; hence this group does not consider taxine from Trrsus spp. to be an alkaloid. Presumably an alkali-like material obtained from a plant would have to have its molecular structure determined before this group would know if it was an alkaloid or not. Clearly taxine (which exhibits very significant physiological activity and complies with Kingsbury’s definition of an alkaloid) is an alkaloid, even though it is an amine with the nitrogen located in a side chain. The nitrogen in taxine is a tertiary amine. Zwitterions such as muscazone and quaternary ammonium compounds such as muscarine, both from the mushroom Anlnnitu muscar‘ia, are not alkali-like and are soluble in water and cannot be considered alkaloids. In this chapter the definition of alkaloids given by Kingsbury will be used, with the one caveat that tertiary amines will be considered alkaloids rather than amines. Of necessity nitrogen atoms in ring structures must be at least secondary. This chapter will be divided into the following sections: plant families containing alkaloids that cause illness or death in humans, and plant families containing alkaloids that cause illness or death in livestock. II. PLANT FAMILIES CONTAINING ALKALOIDS THAT CAUSE ILLNESS OR DEATH IN HUMANS Although many plant families have a reputation for being very dangerous to humans, publications in the literature of the last 20 years involve only a handful. The absence of scientific or case reports should not be taken as evidence that these are the only examples to have happened; many case histories are never reported. Many causes of a failure to publish can be given, hospital staff too busy or a sense of d i j i vu being chief among them. I hope, however, that the iceberg reasonably closely resembles the tip that we do see. Only species from eight families have been commonly implicated in the last 20 years. These families are treated in order of decreasing importance. A.

Solanaceae

In a prophetic publication in 1977 on jimsonseed poisoning, Levy listed no fewer that 27 individuals aged 13-21 who were intoxicated with the seeds of Datura strumorzizm

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(jimsonweed). Two years later Shervette et al. reported on 29 patients hospitalized because of intentional jimsonweed ingestion. All had mydriasis, hallucinations, and were disoriented. In 1982 Urich et al. reported on a fatality following ingestion of this plant. Within a few years jimsonweed poisoning was reported in Saudi Arabia where drug abuse is unheard of and the ingestions in three cases had therefore to be accidental (Taha and Mahdi, 1984). Henbane (Hyoscyamus sp.) was reported as a problem in children in Turkey where a common children’s game involves the ingestion of various plants. Henbane is common in Turkey and in a 2-year period 76 children were intoxicated, with two fatalities (Tugrul, 1985). In 1991 four cases of intoxication were reported following the smoking of the plant (Guharoy and Barajas, 1991). The saga continues with a report of 11 very recent intoxications of young individuals after ingestion of jimsonweed pods and seeds (Tiongson and Salen, 1998). A similar situation exists for angel’s trumpet (Datura sauveolerzs): the ingestion of teas brewed from the plant causes intoxication (Hall et al. 1977). Nonadolescents prefer to drink wine made from this plant (Smith et al., 1991). Recently there was a report from Venezuela of multiple poisonings, on different occasions, with atropine-tainted honey (Ramirez et al., 1999). The honey was believed to come from wasps that had gathered nectar from Datura inoxia. Nicotiana species, particularly Nicotiana glnuca, are also troublesome. Manoguerra and Freeman (1982) report neuromuscular blockade and respiratory failure in an elderly man who ingested the plant. Five years later a fatality involving a young adult man found in a field was reported (Castorena et al., 1987). Potatoes, Solarzu-tuberosunz varieties, continue to be problematical. Plant breeders have developed cultivars such as Lenape that have caused poisoning from seemingly normal potatoes. Improperly stored potatoes are still hazardous, however, as was shown a generation ago when 78 schoolboys became ill after eating old potatoes (McMillan and Thompson, 1979).

B. Liliaceae The glory lily (Gloriosasuperbn),containing colchicine, is another plant cited in therecent literature. Attempted suicide with attendant symptoms in the patient has been reported (Mendis, 1989). Symptoms included gastroenteritis, acute renal failure, cardiotoxicity, and hematological abnormalities. Nagaratnam et al. (1973) reported a fatality caused by the same plant. Colchicine, the alkaloid from G. superba, has been used for centuries in the treatment of gout, but it is known that alopecia and bone marrow depression occur in patients who use this drug. Another problem plant in this family is the false hellebore (Veratrum v i d e j. Six cases of poisoning by the root of this plant were reported from New England (Jaffe et al., 1990). They reported bradycardia and hypotension and recommended atropine for therapy, but stressed that pressors may also be needed to maintain blood pressure. Twenty years ago three cases of Zigaderzus sp. poisoning were reported (Spoerke and Spoerke, 1979). These were nonfatal cases and both heart rate and blood pressure were altered in the victims. The treatment recommended was emesis or lavage, activated charcoal, and a saline cathartic.

C.

Boraginaceae

Herbal medicines can cause veno-occlusive disease. The herb HeZiotropiurn eichwnldi was reported to cause liver disease in India (Datta et al., 1978). Despite this early warning,

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use of the Boraginaceae as herbal teas or medicines continues. For example, comfrey (Spzylzynl sp.) continues to be used in herbal medicine despite numerous problems. Veno-occlusive disease was diagnosed in a 49-year-old woman who used ground comfrey root (Ridker et al. 1985). The death of a 23-year-old vegetarian man was reported from New Zealand some months after he began to consume comfrey leaves (Yeong et al. 1990j. In Boston, a review of 29 patients operated upon for intractable ascites showed that 18 had Budd-Chiari syndrome, which is associated with the consumption of pyrrolizidine alkaloids (McDermott and Ridker, 1990). Tragically the disease is not limited to adults and continues to be a problem. An 18-month-old boy in Austria was recently treated for veno-occlusive disease after being given herbal tea for 15 months by his parents. They had gathered wild herbs themselves and had mistaken Aderzosvles allinriae for coltsfoot (Tussilogofrrfnm) (Sperl et al., 1995).

D. Papaveraceae Until 1986 Danes could grow opium poppies (Pcqmer somnifemm), as decorative plants in their gardens. They may still be grown in Denmark for the production of poppy seeds. I myself have gathered several specimens of this plant at Sandy Beach, Alberta, 54”N, 114”W. and found them to contain substantial amounts of morphine and codeine. The conventional wisdom that they have to be grown near the equator to produce narcotics is manifestly false. Given that Danes may still grow the plant, some of them naturally abuse it. Between 1982 and 1985 seven fatalities occurred after subjects drank “opium tea” (Steentoft et al., 1988).

E. Umbelliferae A preschool child became seriously ill after consuming poison hemlock (Co~ziu~n nmulntzm) (Frank et al., 1995). This plant has been known to be poisonous since the time of Socrates. The plant contains a number of alkaloids, which can vary from hour to hour; the alkaloids found in this case, were coniine and y-coniceine. An Italian group reported the clinical findings in 17 nonfatal cases involving coniine from C. r~znc~rlatzrn~ (Rizzi et al., 1991). All 17 patients developed rhabdomyolysis and five developed acute renal failure. Interestingly, ingestion of wildfowl that have consumed hemlock buds can also cause rhabdomyolysis and acute renal failure (Scatizzi et al., 1993)

F. Taxaceae The yew tree is oneofthemost poisonous plants inthe world. In medieval England yews (Tcr-xus bnccntlcl) were invariably planted in churchyards with the express purpose of discouraging sacrilegious grazing by livestock. Thousands of animal deaths occur each year and some 400 head of cattle die each year in the bluegrass area of Kentucky alone. Yews have also been implicated in human poisonings. Sinn and Porterfield (199 1) report the death of a young college student in Nebraska. A recent paper from Dusseldorf, Germany reported the death of a 19-year-old man who had suicidally ingested yew leaves; a major component found on analysis of stomach content was 3,5-dimethoxyphenol, the aglycone of taxicatine (Musshoff et al., 1993).

Alkaloids

G.

251

Ranunculaceae

A case of fatal ingestion of torikabuto, mistaken for an edible plant, was recently reported from Japan (Yoshioka et al., 1996). After 2 h a 61-year-old man developed symptoms of nausea, dialrhea, and general discomfort. Despite aggressive care he died 6 days later. Aconitine and the related alkaloids mesaconitine, jesaconitine, and hypaconitine were all clearly detected in antemortem urine.

H. Fabaceae Lupi~zussp. belong to this family, which includes many wholesome and nutritious species. In recent years there has a been a move to market “edible” lupines that have been developed by the plant breeders. These edible species require elaborate detoxification. Cases have been reported of illness following failure to follow the arcane detoxification steps (Smith, 1987), and also from consumption of the “debittering” water used to remove the toxins from the “edible” product (Luque Marquez et al., 1991). Historically mass poisonings from this family have been reported from the less developed regions of the world, but based on recent literature, these seem to be a thing of the past.

111.

PLANTFAMILIESCONTAININGALKALOIDSTHAT CAUSE ILLNESS OR DEATH IN LIVESTOCK

Livestock poisoning differs from human poisoning in a number of ways. The most obvious of these is that animals are often deliberately poisoned either by plants or by decoctions from them in the name of science. A considerable body of literature can be found in these cases of deliberate poisoning. I intend, by and large, to discuss only natural accidental exposures to actual plant material. I have included only a handful of deliberate poisonings limited to those cases where there was, in my opinion, an overwhelming and compelling need to test a hypothesis or a strong suspicion of teratogenicity, intended to support a theory enabling science to predict teratogenic potential from molecular structure alone. If such knowledge can be used to avert another thalidomide tragedy, then it is justifiable. Livestock are also more prolific grazers than humans and get into more things: however, the same families of plants as with humans are involved, with a few examples of other families involved occasionally. These families involved in livestock poisoning will also be dealt with in decreasing order of importance, although the families are nearly the same, the order differs considerably. A.

Fabaceae

Crooked calf disease (a congenital deformity) is of widespread occurrence i n western North America. It is known to be caused by ingestion, during certain times of gestation, of various Lupirzus sp. Early work suggested that those Lzy~inussp. rich in anagyrine were to blame, but some species, such as Lqirurs ~forrz~osus, containing little anagyrine were still highly teratogenic. Later work showed that ammodendrine, a major alkaloid in L. ~ O ~ ~ I ~ O was S U Salso , teratogenic (Keeler and Panter, 1989). Recently the presence of large amounts of ammodendrine in Lupinus nrbustus was also reported to cause cleft palate and front leg abnormalities in calves (Panter et al., 1998). Crotcdal-inj w l c e ~is another

Smith

252

dangerous member of this family and contains pyrrolizidine alkaloids. In Brazil 20 horses died 30 days after being fed a diet of 40% pulverized seeds. The findings at necropsy included diffuse fibrosing alveolitis in the lungs and congested liver (Nobre et al., 1994).

B. Asteraceae Senecio sp.. once the scourge of livestock, are less important today. Although the problem is diminishing, partly as a result of education, certain species continue to be problematical. The death of a foal, small and jaundiced from birth, was recently reported from Australia. The pasture was heavily infested with Serzecio mndagnscnrierzsis (Small et al., 1993). The loss of yaks in Bhutan in the Himalayas was attributed to the consumption of this plant (Winter et al., 1994). The deaths of 226 cattle were reported from central Queensland following exposure to Senecio lnutus (Noble et al., 1994). A mean of 8% of cattle died in affected groups (range 2-58%). Sickness and death occurred several months after exposure. Senecio poisonings have also been reported recently from Albania (Smith and Panariti, 1995). C.

Umbelliferae

Rabbits were poisoned in Oklahoma after children fed them Conizrm mzczrlntzm in the belief that it was a garden vegetable (Short and Edwards, 1989). A situation of greater economic consequences arose in California when cattle in two herds were fed hay containing C. ~ncrculntzrrn(Galey et al., 1992). The clinical signs included increased salivation and lacrimation, depression, respiratory distress, ataxia, and death. Coniine and y-coniceine were found in the hay and the urine of the affected animals. Unlike the living plant where the alkaloids vary from hour to hour (Kingsbury, 1964), the alkaloids appear to be fixed by cutting.

D. Boraginaceae Many plants can contaminate hay and this is an all too common route for exposure of livestock to poisonous plants. Six calves out of a group of nine being fed hay contaminated with CynogZossur~zoflcinnle died, probably because of exposure to pyrrolizidine alkaloids (Baker et al., 1989). Horses are also susceptible to the alkaloids of this plant and the death of 10 animals had been reported earlier (Knight et al., 1984).

E. Solanaceae Hay is not the only animal feed to be contaminated with weed seeds. A sunflower-based feed supplement grossly contaminated with the seeds of Datum sp. resulted in the death of a horse following gastric rupture (Schulman and Bolton, 1998). F. Liliaceae The death camas (Zigrrclenzrs sp., also spelled Zygmenus) is common in western North America. A number of livestock deaths have been attributed to this plant. Recently 23 cattle deaths in the Nebraska panhandle were reported (Collett et al., 1996). Symptoms included prostration, muscle tremors, hyperexcitability, open mouth breathing, excessive

Alkaloids

253

salivation, rapid heart rate, borborygmus, and loose green stool. Reports of cattle losses have also been reported in Alberta (Smith and Lewis, 1991). Many sheep have been lost in Idaho to this plant; in one case the loss of 250 sheep in a 2-day period was reported (Panter et al., 1987) The meadow saffron (Colchium autz4n~znale)was recently reported as having poisoned sheep in Albania (Panariti, 1996).

G. Taxaceae The numerous cases of Tmus sp. poisoning are undoubtedly underreported as a result of dijii vu. Occasional reports still surface, however, when the deaths of many animals occur in a matter of hours (Panter et al., 1993). Following exposure animals became sick within 2 h. Gross hyperemia in the abomasum and small intestine was noted.

H. Cyanophyceae A great deal of literature exists on the toxicity of, and analytical methods for the detection of, anatoxin-A, but reports of livestock poisoning are sporadic, although dogs are often poisoned. Anatoxin-A is very rapidly toxic and cattle die within a matter of minutes of consuming water covered with a thick bloom of the blue-green algae Annbaelza.fEos-aqune (Smith and Lewis, 1987). Conventional wisdom was that among freshwater algae only those inthe genera Annbaerza, Aphanizomenon, and Microcystis were dangerous (Kingsbury, 1964). This must be reconsidered following the report of dog deaths from anatoxin seemingly produced by a bloom of Oscillutorin sp. (Edwards et al., 1992).

1.

Ranunculaceae

The Ranunculaceae are a diverse family with many toxin types in its various genera. A few genera contain diterpene alkaloids and the most problematic genus is Delyhi~ziur~z. The early literature abounds with reports of multiple cattle losses following ingestion of select species in this genus. I have investigated more than a dozen such cases, but a sense of d6jL vu has prevented me from making case reports on any of them. Most of the toxic species contain methyllycaconitine or very similar alkaloids. Many larkspurs are innocuous, but those that are not contain this small related group of alkaloids. An examination of a tall larkspur (Delphir1iurn barbeyi) revealed this to be true for this plant also (Pfister et al., 1994).

J. Campanulaceae Each year in Tamaulipas (northeastern Mexico) in late winter and early spring, grazing sheep and cattle develop toxicoses empirically associated with consumption of a plant known locally as moradilla. Recent work suggested that moradilla was Lobelia ber-larzdieri, a plant well known in Texas (Lopez et al., 1994). The alkaloids were not affected by making silage, drying in hay, or freezing.

K. Convolvulaceae Pastures heavily infested with field bindweed (Comolvulis amemis)have caused illness and death in horses in localized areas of Colorado (Todd et al., 1995). Several alkaloids

254

Smith

were found in the plants upon analysis and included the tropane alkaloids tropine, pseudotropine, and tropinone, and the pyrrolidine alkaloids cuscohygrine and hygrine. At post morteln intestinal fibrosis and vascular sclerosis of the small intestine were identified.

REFERENCES 1. Baker DC, Smart RA, Ralphs M, Molyneux RJ. (1989).Hound‘s-tongue (Cyg~~oglosswrz oj’icinrile) poisoning in a calf. J Am Vet Med Assoc 194(7):929-930. 2 . Castorena J, Garde J, Banhardt FE, Shaw RF. (1987). A fatal poisoning from Nicotiar~a glrruccr. J Tosicol Clirr Toxic01 25(5):429-435. . 3. Collett S. Grotelueschen D, Smith RA, Wilson R. ( 1996). Deaths of 23 adult cows attributed to intoxication by the alkaloids of Zlyaderzzrs )wzerzous~4s(meadow death camas). Agri-pattics 17(7):5-9. 3. Datta DV. Khuroo MS, Mattocks AR, Aikat BK, Chhuttani PN. (1978). Herbal medicines and veno-occlusive disease in India. Postgrad Med J 54(634):511-515. 5. Edwards C, Beattje KA. Scrimgeour CM, Codd GA. (1992). Identification of anatoxin-A in benthic cyanobacteria (blue-green algae)and in associated dog poisonings at Loch Insh, Scotland. To.vicon 30(10):1165-1175. 6. Frank BS, Michelson WB. Panter KE, Gardner DR. ( 1995). Ingestion of poison hemlock (Conirrrlr rmculcrtum). West J Med 163(6):573-574. 7. Galey FD, Holstege DM, Fisher EG. ( 1992). Toxicosis in dairy cattle exposed to poison hemlock ( C o n i w ~nzncz~lrrtzo~z) in hay: isolation of Corrium alkaloids in plants. hay and urine. J Vet Diag Zmyest 4 1):60-64. 8. Guharoy SR, Barajas M. (1991). Atropine intoxication from the ingestion and smoking of jimson weed (Datzlm stinnzoniwlz. Vet Hrtrn Toxicol 33(6):588-589. 9. Hall RC. Popkin MK. McHenry LE. (1977). Angel’s trumpet psychosis: a central nervous system anticholinergic syndrome. Am J Psychiati?? 134(3):312-313. 10. Jaffe AM. Gephardt D, Courtemanche L. (1990). Poisoning due to ingestion of Vemtmm v i d e (false hellebore). J Emerg Med S(2):161-167. 11. Keeler RF. Panter KE. (1989). Piperidine alkaloid composition and relation to crooked calf disease-inducing potential of Lupimrs .formosrts. Temtology 40(5):423-432. 12. Kingsbury JM. ( 1964). Poisonotts Plants of the United States rrrd Canado. Prentice-Hall, Englewood Cliffs, NJ. 13. Knight AP, Kimberling CV. Stermitz FR, Roby MR. ( 1984). Cygrroglossrtm ofJicirzale (hound’s t o n g u e k a case of pyrrolizidine alkaloid poisoning in horses. J Am Vet Med Assoc 155(6): 637-650. 14. Levy R. (1977). Jimson “loco” weed abuse in adolescents. JACEP (6)2:59-61. 15. Lopez R, Martinez-Burnes J. Vargas G, Loredo J, Medellin J. Rosiles R. (1994). Taxonomical, clinical and pathological findings in moradilla (lobeline-like) poisoning in sheep. Vct Hzcm Toxic01 36(3):195-198. 16. Luque Marquez R, Gutierrez-Ravew M. Infante Miranda F. (1991 ). Acute poisoning by lupine seed debittering water. Vet H w z To.-\-icol33(3):265-267. 17. Manoguerra AS. Freeman D. ( 1 982). Acute poisoning from ingestion of Nocoticrrza glcrucn. J Toxicol Cliil To.vico1 (19)8:861-863. 18. McDernlott WV, Ridker PM. ( 1990). The Budd-Chiari syndrome and hepatic veno-occlusive ~ 125(3):525-527. disease. Recognition and treatment. A I T Sz41-g 19. McMillan M, Thompson J. (1979). An outbreak of suspected solanine poisoning in schoolboys: examinations of the criteria of solanine poisoning. Q J Med 48(190):227-243. 20. Mendis S. (1989). Colchicine cardiotoxicity following ingestion of Gloriosa srrperbn tubers. Postgrad Men J 65(768):752-755.

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21. Musshoff F. Jacob B, Fowinkel C, Daldrup T. (1993). Suicidal yew leaf ingestion-phloroglucindirnethylether (3,5-dimethoxyphenol) as a marker for poisoning from Taxus bcrccata. Z n t J Legal Men 106(1j:45-50. 22. Nagaratnam N, De Silva DP, De Silva N. (1973). Colchicine poisoning following ingestion of Gloriosa szrperba tubers. Tr0l-1Georg Med 25( 1):15-17. 23. Noble JW. Crossley J, Hill BD, Pierce RJ. McKenzie RA, Debritz M, Morely AA. ( I 994). Pyrrolizidinealkaloidosisof cattle associatedwith Senecio lazctus. Azrst Vet J 71(7):196200. 24. Nobre D, Dagli ML, Haraguchi M. (19943. Ct-otnlcrriaj m c e a intoxication in horses. Vet Hzam To.yicol 36(5):445-448. 25. Panariti E. (1996).Meadow saffron (Colchilml rrutum~zcrle) intoxication in a nomadic Albanian sheep flock. Vet Huwz Toxicol 38(3):227-228. 26. Panter K E , Ralphs MH. Smart RA,Duelke B. ( 1987). Death camas poisoning in sheep: a case report. Vet Hum Toxicol 29(1):45-48. 27. Panter KE, Gardener DR, MolyneuxRJ. (1998). Teratogenic and fetotoxic effectsof two piperidine alkaloid-containing lupines (L. formosz4s and L. arbustlas) in cows. J Ncrt Tosins 7(2):131-140. 28. Panter KE. Molyneux RJ, Smart RA, Mitchell L, Hansen S. (1993). English yew poisoning in 43 cattle. J Am Vet Med Assoc 202(9):1476- 1477. 29. Pfister JA, Panter KE, Manners GD. (1994). Effective dose in cattle of toxic alkaloids from tall larkspur (Delplzirzium berbeyij. Vet Hum Toxicol 36( 1): 10- 11. 30. Ramirez M. Rivera E, Ereu C. ( 1999). Fifteen cases of atropine poisoning after honey ingestion. Vet HwIl Toxicol 31(1):19-20. 31. Ridker PM. OhkumaS, McDermott WV, Trey C. Huxtable RJ.(1985). Hepatic veno-occlusive disease with the consumption of pyrrolizidine-containing dietary supplements. Gastroentet-ology 88(4):1050-1054. 32. Rizzi D, Basile C, Di Maggio A. Sebastio A. Introna F Jr., Rizzi R. Scatizzi A, De Marco S. Smialek J. E. (1991). Clinical spectrum of accidental poisoning: neurotoxic manifestations. rhabdonlyolysis and acute tubular necrosis. N e p h o l Dial Transplant 6(12):939-943. 33. Scatizzi A, Di Maggio A, Rizzi D, Sebastio AM, Basile C. (1993). Acute renal failure due to tubular necrosis caused by wildfowl-mediated hemlock poisoning. Re11 Fail 15(1):93-96. 34. Schulman ML, Bolton LA. (1998). Dcrtula seed intoxication in two horses. J S Afi- Vet Assoc 69( 1):27-29. 35. Shervette RE 111. Schydlower M, Lanlpe R. M., Fe‘arnow RG. (1979). Jimson “loco” weed abuse in adolescents. Pediatrics 63(4):520-523. 36. Short SB, Edwards WC. (1989). Accidental Corlizrrlt mmlatw11 poisoning in the rabbit. Vet H z m To.xicol 31( 1):54-57. 37. Sinn LE, Porterfield JF. (1991). Fatal taxine poisoning from yew leaf ingestion. J Forensic Sci 36(2):599-601. 38. Small AC, Kelly WR, Seawright AA, Mattocks AR,Jukes R. (1993).Pyrrolizidine alkaloidosis in a two month old foal. Zeittralb Veterirzarmecl40(3):213-218. 39. Smith EA, Meloan CE, Pickell JA, Oehrne FW. (1991). Scopolamine poisoning from homemade n noon flower” wine. J AHNIToxicol 15(4):216-219. for anatoxin-A, theunstabletoxic 40. Smith RA. Lewis D. (1987). A rapidanalysisofwater alkaloid from Anabnerw jlos-crquae. Vet Hlrm Tosicol 29(2): 153-153. 41. Smith RA. (1987). Potential edible lupinepoisonings in humans. Vet Hzrrn Tosicof 29(6):444445. 42. Smith RA. Lewis DL. (1991). Death camas poisoning in cattle. Vet H w n Toxicol 33(6j:615616. 43. Smith RA, Panariti E. (1995). Intoxication of Albanian cattle after ingestion of Serwcio subalpi~ms.Vet Hum To.xkd 37(5):478-479. 44. Sperl W, Stuppner H. Gassner I, Judmaier W. Dietz 0, Vogel W. (1995). Reversible hepatic

256

45. 46. 47. 48. 49. 50. 51. 52.

53. 54. 55.

Smith veno-occlusive disease in an infant after consumption of pyrrolizidine-containing herbal tea. Eur J Pediatr 154(2):112-116. Spoerke DG, Spoerke SE. (1979). Three cases of Zigadenus (death camas) poisoning. Vet Hun? Tosicol 21(5):346-347. Steentoft A, Kaa E, Worm K, (1988). Fatal intoxications in Denmark following intake of morphine from opium poppies. 2 Reclztsrlzecls lOl(3):197-204. Taha SA, Mahdi AH. (1984). Datura intoxication in Riydah. Trans R SOCTrop Med Hyg 78(1):134-135. Tiongson J, Salen P. (1998). Mass ingestion of jinlson weed by eleven teenagers. Del Med J 70( 11):471-476. Todd FG, Stermitz FR, Schultheis P, Knight AP, Traub-Dargatz J. (1995). Tropane alkaloids and toxicity of Convohwl~tsonlensis. Phytocltemistry 39(3):301-303. Tugrul L. (1985). Abuse of henbane by children in Turkey. Bull Narc 37(2-3):75-78. Urich RW, Bowerman DL, Levinski JA, Pflug J. (1982). Datztm straamoiliurn, a fatal poisoning. J Forensic Sci 27(4):948-954. Van Ingen G, Visser R, Peltenburg H, Van Der Ark AM, Voortman M. (1992). Suddenunexpected death due to Taxus poisoning. A report of five cases. with review of the literature. Forensic Sci Znt 56( 1):81-87. Winter H, Seawright AA, Noltie H. J., Mattocks A. R., Jukes. (1994). Pyrrolizidine alkaloids poisoning of yaks: identification of the plants involved. Vet Rec 134(6). Yeong ML, Swinburn B, Kennedy M, Nicholson G. (1990). Hepatic veno-occlusive disease associated with comfrey ingestion. J Gustroenterol Hepcrtol 5(2):211-214. Yoshioka N,Gonmori K, Tagashira A, Boonhooi 0,Hayashi M, Saito Y, Mizugaki M. (1996). A case of aconitine poisoning with analysis of aconitine alkaloids by GC/SIM. Forensic Sci Znt 81(2-3):117-123.

Antinutritional Factors Related to Proteins and Amino Acids lrvin E. Liener Uttisersiq of Minnesofn, Sf. Paul, Mimesofa

I. Protease Inhibitors A. B. C. D. E. F.

258

Introduction 258 Biochemical properties 258 Nutritional significance 259 Detoxification 264 Physiological role in plants 267 Analytical techniques 267

11. Amylase Inhibitors

268

A. Nutritional significance B. Role in plant 268 111. Lectins

268

269

A. Introduction 269 B. Physicochemical properties 269 C. Nutritional significance 272 D. Mode of action 272 E. Role in plants 274 F. Analytical techniques 276 IV. Toxic Amino Acids 276 A. B. C. D. E. F. G.

H. I. J. K.

Neurotoxins 276 Hypoglycin 278 Mimosine 279 Djenkolic acid 280 Dihydroxyphenylalanine 280 Selenoamino acids 28 1 Indospicine 282 Canavanine 282 Linatine 282 Lysinoalanine 283 Biogenic Anlines 284

References

286

257

Liener

258

1. A.

PROTEASEINHIBITORS Introduction

Among the many factors that have been implicated as having an adverse effect on the nutritional value of plant proteins is a class of proteins that have the ability to inhibit the proteolytic activity of proteases of diverse origin. Because of the important role that soybeans play as a dietary ingredient for animals as well as humans, the protease inhibitor found in this legume has received the most attention since it was first reported by Read and Haas in 1938 (1). The protein fraction responsible for the inhibition of trypsin was partially purified by Bowman (2) and Ham and Sandstedt (3) in 1944 and subsequently isolated in crystalline form by Kunitz (4) 1 year later. The existence of a heat-labile inhibitor of trypsin seemed to offer a reasonable explanation for the observation made many years before by Osborne and Mendel that heat treatment improved the nutritive value of soybean protein (5). The realization that protease inhibitors might be of nutritional significance in soybeans stimulated an intensive search for similar factors in other legumes that provide an important source of protein in the diet of many segments of the world's population. Table 1 provides a partial list of the many plants known to contain protease inhibitors as well as their specificity with respect to the proteases they inhibit. B.

BiochemicalProperties

The protease inhibitors that have been isolated from soybeans and other legumes fall into two main categories: those that have a molecular weight of 20,000-25,000 with relatively few disulfide bonds and possessing a specifity that is directed primarily toward trypsin, and those that have a molecular weight of only 6000-10,000 with a high proportion of cystine residues and are capable of inhibiting chymotrypsin as well as trypsin at independent binding sites. The most thoroughly characterized examples of these two classes of inhibitors are the so-called Kunitz and Bowman-Birk inhibitors isolated from the soybean. The complete amino acid sequence of the Kunitz inhibitor is shown in Fig. 1. It consists of 181 amino acid residues with the reactive site (site directly involved in its interaction with trypsin) being located at residues Arg 63 and Ile 64. This molecule combines with trypsin in a stoichiometric fashion; i.e., one molecule of theinhibitor inactivates one tnolecule of trypsin. The complex that forms is analogous to an enzyme-substrate complex that, unlike the usual enzyme-substrate complex, which readily dissociates, is tightly bound with a Ki of 10"" M (6). X-ray crystallography has given a closer insight into the detailed nature of the enzyme-inhibitor complex (Fig. 2); the molecular forces involved in this interaction have been reviewed by Laskowski and Kato (7). Five closely related Bowman-Birk type of inhibitors have been isolated and characterized from soybeans; they are referred to as PI-I through PI-V (8). The amino acid sequence of one of these (PI-I) is shown in Fig. 3. A unique feature of this molecule is that it has two independent binding sites: a trypsin-reactive site !Lys 18-Ser 17) and a chymotrypsin-reactive site (Leu 44-Ser 45) (9). In contrast to the Kunitz inhibitor, the Bowman-Birk inhibitors are very rich in disulfide bonds, possessing seven disulfide bonds. This feature is responsible for the verytight, compact, three-dimensional structure revealed by x-ray crystallography (10a,b) and by nuclear magnetic resonance spectroscopy (1Oc). The sequences of amino acids surrounding these two reactive sites are remarkably similar to each other, and a high degree of homology has been found between the Bowman-Birk

Antinutritional Factors Related to Proteins

259

Table 1 Distribution of ProteaseInhibitors Present in Legumes ~~~

Botanical name

Voandzeicr subterrailca

Proteases inhibited

Common name Peanut, groundnut Pigeon pea, red gram Jack bean, sword bean Partridge pea Chickpea. Bengal gram, garbanzo Butterfly pea Cluster bean Horse gram Hyacinth bean. field bean, Hakubenzu bean Double bean Soybean Sweet pea Chickling vetch Lentil Lupine Florida velvet bean Moth bean Adzuki bean Mung bean, green gram Scarlet runner bean Lima bean, butter bean B1ack gram Navy bean, kidney bean, pinto bean, French bean, white bean, wax bean, haricot bean, garden bean Field bean, garden pea Winged bean, Gao bean Velvet bean Broad bean, field bean, faba bean Cowpea. black-eyed pea, Southern pea, serido pea Bambara bean

T. C, PI, K T T. C, S T T, c T, C, S T, C, S T T, C, Th T T, C T T, c T T T T T. C T, endopeptidase T, c T. C T, c, s T, C, E. S

T T T T. C, Th, Pr, Pa T. C

T

C , chymotrypsin; E, elastase: K, kallikrein; Pa, papain; P1, plasmin; Pr, pronase: S, subtilisin; T, trypsin; Th. thrombin. Source: From Ref. 6.

inhibitor and a number of other low-molecular-weight inhibitors that have been isolated from other legumes (Table 2).

C. NutritionalSignificance 1. Biological Effects Not long after soybeans were introduced into the United States, primarily as a source of oil, Osborne and Mendel ( 5 ) made the significant observation that soybeans had to be heat-treated to support the growth of rats. With the isolation of of a trypsin inhibitor from

260

< m

9 5

2 ?

G

m

5

H

H

v“ xv2 x

2 ?

vw

>-

1

U

U

Liener

Antinutritional Factors Related to Proteins

261

Figure 1 Amino acid sequence of the Kunitz soybean trypsin inhibitor. (From T. Koide and T. Ikenaka, Studies on soybean trypsin inhibitors. 3. Amino acid sequence of the carboxyl region and the complete amino acid sequence of soybean trypsin inhibitor (Kunitz), Eur. J. Biochent., 32417, 1973.)

raw soybeans by Kunitz (4), it was generally assumed the the beneficial effect of heat treatment could be attributed to the destruction of this factor, which interfered with the digestion of protein in the intestinal tract. Purified fractions from the soybean, which were rich in antitryptic activity, were in fact capable of inhibiting the growth of rats (1 l), chicks (12), and mice (13), an effect that was generally accompanied by a depression in the 1

25

Figure 2 Folding of the polypeptide backbone chain of the Kunitz inhibitor is shown on the left. Amino acid residues in intimate contact with trypsin shown in black. Shown on the right is a model of the Kunitz inhibitor-trypsin complex. The part representing trypsin is less heavily shaded. (From R. M. Sweet, H. T. Wright,J. Janin, C.H. Clothis, and D. M. Bloy, Crystal structure of the complex of porcine trypsin and soybean trypsin inhibitor (Kunitz) at 2.6 A Biochemistty, 13:4212; 1974.)

Liener

262 I

30

70

Figure 3 Amino acid sequence of the Bowman-Birk inhibitor. The disulfide bonds and reactive sites involved in its interaction with trypsin (Lys 16-Ser 17) and chymotrypsin (Leu 44-Ser 45) are shown in black. (From S. Odani and T. Ikenaka, Studies on the soybean trypsin inhibitors. VII. Disulfide bridges in soybean Bowman-Birk proteinaseinhibitor, J. B i o c h e ~(Tokyo), ~. 74:697, 1973.)

digestibility of the protein in the diet. Furthermore, the feeding to rats of a raw soybean extract from which the trypsin inhibitors had been removed by affinity chromatography produced improved growth performance compared to controls from which the trypsin inhibitors had not been removed (14). Despite these observations, it remained unclear why preparations of trypsin inhibitor were capable of inhibiting growth even when incorporated into diets containing predigested protein or free amino acids (15). Such experiments obviously rule out an inhibition of proteolysis as the sole factor responsible for growth inhibition and thus served to focus attention on some alternative mode of action of the trypsin inhibitors. Perhaps the most significant observation that has ultimately led to a better understanding of the modeof action of the soybean inhibitors was the finding that raw soybeans or trypsin inhibitor itself could cause hypertrophy and hyperplasia of the pancreas, an effect acconlpanied by an increase in the secretory activity of the pancreas ( 16-1 8). This has led to the suggestion that the growth depression caused by trypsin inhibitors might be the consequence of an endogenous loss of amino acids being secrected by a hyperactive pancreas (19, 20). Since pancreatic enzymes such as trypsin and chytnotrypsin are particularly rich in sulfllr-containing amino acids, pancreatic hypertrophy and/or hyperplasia diverts these amino acids from the synthesis of body tissue protein to the synthesis of these enzymes. This loss in sulfur-containing amino acids exacerbates an already critical situation with respect to soybean protein, which is inherently deficient in these amino acids. Related to the cellular proliferation of pancreatic tissue evoked by trypsin inhibitors is the observation that raw soy flourpotentiates the carcinogenic effect of azaserine (21a,b) and di(2-hydroxypropyl) nitrosamine (23) on the pancreas of the rat. Even more significant is the fact the the long-term feeding (60 or more weeks) of raw soy flour alone causes the appearance of adenomatous nodules on the pancreas (33). This carcinogenic effect was positively correlated with the level of trypsin inhibitor in the diet when various levels of raw soy flour were incorporated into the diet (24). Similar studies with the mouse (25)

Antinutritional Factors Related to Proteins

263

and hamster (26), however, revealed no evidence of pancreatic lesions with the long-term feeding of raw soy flour. 2. Mode of Action Many studies have been conducted in an attempt to elucidate the mechanism whereby trypsin inhibitors induce pancreatic hypertrophy. Green and Lyman were the first topostulate that pancreatic secretion is controlled by negative feedback inhibition, which depends on the level of trypsin present at any given time in the small intestine (37). When the level of this enzyme falls below a certain critical threshold, the pancreas responds in a compensatory fashion by producing more enzyme. Suppression of this negative-feedback mechanism occurs if the trypsin becomes complexed with the inhibitor. It is believed that the mediating agent between the trypsin and the pancreas is the hormone cholecystokinin (CCK), which is released from the intestinal mucosa when the level of trypsin becomes depleted. This is supported by the observation that elevated levels of plasma CCK accompanied the feeding of raw soy flour or concentrates of the trypsin inhibitor to rats (28a,b), and chicks (2%). These relationships are shown in Fig. 4. Negative feedback control has been found to be operative in most species of animals with the exception of the dog (29). In the case of the pig and calf. however, the existence of the negative feedback mechanism is not accompanied by pancreatic hypertrophy. Evidence that such a mechanism occurs in humans comes from experiments involving the infusion of the Bowman-Birk inhibitor into the duodenum, which evoked an increase in the level of trypsin, chymotrypsin, and elastase into the small intestine (30). The feeding of a meal containing raw soy flour (31a) or the intraduodenal administration of the purified soybean inhibitors (31b) to human subjects also led to an increase in plasma CCK. Until quite recently it has not been clear how trypsin manages to suppress the secretion of CCK, or, conversely, how its inactivation by trypsin inhibitor causes an increase in CCK production. A trypsin-sensitive peptide (“monitor peptide”) with 61 amino acids has been isolated from rat pancreatic juice, which acts as a signal for the release of CCK from the intestinal mucosa (32). This peptide is inactivated by trypsin, thus causing a suppression of CCK release, but when trypsinis complexed with the inhibitor, this peptide

Trypsinogen 4 Ipanereas) Dietary

CCK

\ /

Imucosa’

(intestine)

Trypsin-TI

Proteolysis

Figure 4 Mode of action of soybean trypsin inhibitors onpancreas (CCK = cholecystokinin). (From R. L. Anderson, J. J. Rackis, and W. H. Tallent. Biologically active substances in soy products. In SO?^ Proteirz a d Hurtran Nutritiorr. H. L. Wilcke. D. T. Hopkins, and D. H. Waggle, eds., Academic Press, New York, 1979, p. 209.)

Liener

264 TI

DIET

TRYSIN L T R Y p s I N TI

+FECES

GI TRACT

BLOOD

Figure 5 Role of ‘‘monitor peptide’’ in negative feedback control. (TI = trypsin inhibitor; CCK = cholecystokinin). (Based on studies from Ref. 33.)

is free to signal the release of CCK, which in turn causes pancreas hypertrophy and/or hyperplasia with a concomitant increase in the secretion of enzymes. These relationships are shown in Fig. 5. 3. Nutritional Significance in Humans Since trypsin inhibitors are present in a wide variety of foods commonly consumed by humans, including not only legumes but also cereal grains, tubers, fruits, vegetables, nuts, and eggs (33, 34), the question may be raised as to whether the presence of trypsin inhibitors poses any risk to human health when the above-mentioned foods are consumed in the diet. In a documented instance, inadequately processed soy protein used as an extender for tuna fish caused an outbreak of gastrointestinal illness (35). Although this in itself does not prove that trypsin inhibitors were responsible for this adverse reaction, it is interesting to note that there is at least one case report ofan allergy to the Kunitz trypsin inhibitor (36). D. Detoxification 1. Effect of HeatTreatment Because of its economic importance, the soybean has received the most attention with respect to the effect of heat treatment on trypsin inhibitor activity and the consequences of such treatment on the nutritional quality of the protein. In general, the extent to which the trypsin inhibitory activity is destroyed by heating is a function of the temperature, duration of heating, particle size, and moisture conditions-variables that are carefully controlled in the commercial processing of soybeans to insure a product will have maximum nutritional value. An example of the relationship that exists between the destruction of the trypsin inhibitor by heat treatment and the concomitant improvement in the nutritional quality of the protein using a rat assay is illustrated in Fig. 6. Most commercially available soybean products intended for human consumption (e.g., tofu, soy milk, soy protein isolates and concentrates, and textured meat analogs)

Antinutritional Factors Related to Proteins

265

M

E

\

=> c

-

0 2 4 6 10 Minutes at 100°C

20

Figure 6 Effect of heat treatment on the trypsin inhibitory activity (TI) and nutritive value of soybean meal, as measuredby the protein efficiencyratio (PER). (FromJ. J. Rackis, Biologicaland physiological factors in soybeans, J. Ant. Oil Cher~.Soc., 51:161A, 1974.)

have received sufficient heat treatment so that they contain less than 10% of the trypsin inhibitor activity originally present in the beans or raw soy flour from which they were derived (37). This level of activity is believed to be well below the threshold level necessary to cause pancreatic hypertrophy in rats (38, 39aj. Although live steam treatment of soybeans (a process referred to as “toasting”) is the most commonly used method for inactivating the trypsin inhibitor (39b), other modes ofheat treatment or processing have proved equally effective. These include boiling soybeans in water (40), dry roasting (41), dielectric heating (42), microwave irradiation (43a,b), extrusion cooking (44), gamma irradiation (45j, infrared radiation (46a), and radio frequency energy (46b). The direct infusion of steam into soy milk also inactivates the trypsin inhibitor (47aj. Rouhana et al. have studied the kinetics of the inactivation of trypsin inhibitors during high-temperature, short-time processing of soy milk (47bj. Because of the compact structure of the Bowman-Birk inhibitor and its stability toward heat in its isolated form (48), it has been generally assumed that the residual trypsin inhbibitor activity found in processed soybean products is due to the Bowman-Birk inhibitor. However, using analytical techniques that differentiate between the Kunitz and Bowman-Birk inhibitors, it has been found that the Bowman-Birk inhibitor is more readily destroyed than the Kunitz inhibitor in its natural milieu (e.g., soy flour) (49a,b). The reason for this anomolous situation is not clear, but may be the result of the interaction of the many disulfide groups of the Bowman-Birk inhibitor with other cysteine-rich components comprising the soybean matrix, reactions that may be accelerated by heat.

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The popular use of legumes other than soybeans as a staple food item in many parts of the world has prompted numerous studies on the effect of different forms of heat treatment on the inactivation of the trypsin inhibitors present in these legumes. Those that have received the most attention in this regard include the navy and kidney beans (Phnseolus w l g m i s ) , broad beans (Vicin.fc/bn),peanuts (Arachis lrypognen),lima beans (PhnseoIus lurrntus),cowpeas ( V i g m sinensis or mgmiculntn), winged bean (Psoplzocnrpus tetrngo11olobr1s), moth bean (Vigna nconitifolium), chick-pea (Cicer ctrietirzum), and pigeon pea (Ccrja~uscnjnrzj. In general, the effect of heat treatment on mostother legumes follows the same general pattern as that observed with the soybean (see Ref. 6). 2. Germination Although the germination of soybeans has been reported by some investigators to result in an improvement in the nutritive value of the protein (50, 5 I), the relationship this bears to changes in trypsin inhibitor activity is obscure (52). Changes in net trypsin inhibitor activity are relatively slight (53-55), although the relative distribution of the Kunitz and Bowman-Birk inhibitors becomes altered. The content of the Bowman-Birk inhibitor in the coyledons of soybean gradually disappeared, and by the 13th day, only the Kunitz inhibitor could be detected (56). In this case new forms of the inhibitor were observed that presumably arose as the result of limited proteolysis during germination (57, 58). Several reviews (59,60a,b) have docutnented the changes that take place during the germination of many different legume seeds. No consistent pattern could be discerned; some legumes showed little or no change, while others displayed a decrease in trypsin inhibitor activity. In general, however, there appears to be little or no correlation between changes in trypsin inhibitor activity and the nutritive value of the germinated seed. 3. TraditionalModes of Preparation Tofu is comprised mainly of protein that has been precipitated from a hot water extract of soybeans with calcium-magnesium salts. Soy milk is simply a hot water extract of whole soybeans that may have undergone clarification. Since the preparation of both tofu and soy milk involves the cooking or steaming of soybeans prior to extraction with water, such products are generally quite low in trypsin inhibitor activity (61). Fermented preparations of soybeans and other legumes such as tempeh, miso, and natto are virtually devoid of trypsin inhibitor activity since the beans are subjected to boiling prior to fermentation (62). Fermented legumes have been shown to have superior nutritive value compared to their unfermented counterparts (63-65), but since both types of preparations were derived from heated beans, this improvement probably has little to do with the trypsin inhibitor. In only a few instances has an attempt been made to determine the effect of fermentation alone in the absence of heat. Varying degrees in the reduction of trypsin inhibitory activity by natural fermentation in the absence of heat treatment have been reported for chick-peas (66. 67) and cowpeas (67). In the absence of further studies it can only be assumed that such decreases in trypsin inhibitory activity were due to proteolytic attack by enzymes secreted by the microorganisms involved in the fermentation. 4. GeneticStudies The screening of 56 genotypes of grain-type soybean and 17 vegetable-type soybean collections has revealed over a 10-fold variation in trypsin inhibitor activity (68). Kakade et al. had also noticed a wide variation in the trypsin inhibitor activity of over 100 different varieties and strains of soybeans, but no correlation was obtained between trypsin inhibitor content and the nutritional value of the protein as measured in rats (69). In a continuing

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267

search for soybean genotypes that might be devoid of trypsin inhibitors, 2944 accessions of soybean germ plasm were screened; only two accessions lacked the Kunitz inhibitor but still retained about 50% trypsin inhibitor activity (70, 71), presumably due to the presence of the Bowman-Birk inhibitor (72). Although feeding studies with rats, chicks, and swine have indicated some improvement in the nutritional value of the protein, the strain lacking the Kunitz inhibitor was still inferior to that of heat-processed soybean meal (44, 73-75). E. Physiological Role in Plants The physiological role of the protease inhibitors has sometimes been presumed to be one that regulates protein catabolism during germination or the degradation of storage protein during seed maturation. Attractive as these hypotheses may seem, there is very little experimental evidence for their support. For example, the protease inhibitors isolated from specific plants are in fact incapable of inhibiting the endogenous proteases of the same plant (76,77). Reference was made above to the fact that a diminution in protease inhibitor activity is not consistently observed during germination. It is now generally accepted that theprotease inhibitors play a key role in the defense mechanism that plants have evolved against insects (78-80) and microbial pathogens (8 1 ). Studies of the effect of protease inhibitors artificially introduced into defined diets have been shown to be detrimental to the growth and developtnent of insects from a wide variety of genera (82-85a,b,c.d). The mechanism for protection against insects appears to be very similar to what has been observed in animals, namely, the ability of these inhibitors to interfere with the digestive enzymes in the gut of these insects (86-88a,b). The genes from a number of protease inhibitors from different plants, including soybeans (89-92), potatoes (93, 94). tomatoes (95), rice (96), and barley (97), have been cloned, and concerted efforts are being made to transfer these genes into other plants to confer resistance to insect attack. Two examples illustrating the successful application of this approach may be cited. The cowpea (Vignn ungzliculcrtn) trypsin inhibitor, which is a variant of the Bowman-Birk inhibitor, has been introduced into Agr-obncter-iumturnifnsciem and expressed in the tobacco plant (98). These genetically altered tobacco plants had enhanced resistance to the tobacco budworm (98). The introduction of the gene of potato inhibitor 11, an inhibitor of trypsin and chymotrypsin, into tobacco plants using the cauliflower mosaic virus as the vector severely retarded the growth of the larvae of the tobacco hornworm (99a). The development of transgenic plants with protease inhibitor activity offers exciting possibilities in agriculture (99b). For example, advantage could be taken of the differences in the specificity of various protease inhibitors to provide protection against a wide variety of insects depending on the type of digestive enzymes that are present in their gut. It might also be possible to alter the specificity of a given protease inhibitor by site-directed mutagenesis of the amino acids comprising its reactive site. In the final analysis a combination of several different inhibitors may be required to achieve complete protection against insect predation.

F. AnalyticalTechniques Rackis et al. have critically evaluated themost commonly employed techniques for assaying for protease inhibitor activity, which generally involves a measurement of the

268

Liener

degree to which the activity of a pure sample of a given protease, usually bovine trypsin or chymotrypsin, on casein or a synthetic substrate is inhibited by the test sample (100). The limitation to this type of assay is the fact that it does not serve to distinguish among the different molecular species of inhibitors that might have the same specificity toward a given protease. Furthermore, this type of assay fails to reveal to what extent nonprotein components such as phytates and tannins might contribute to the observed protease inhibitor activity. These problems can be circumvented by using monoclonal antibodies that distinguish between the Kunitz and Bowman-Birk inhibitors in soybeans (49, 101- 103), or by a procedure that involves the specific absorption of trypsin inhibitors by affinity chromatography on immobilized trypsin (104, 105).

II. AMYLASE INHIBITORS

A.

NutritionalSignificance

Protein inhibitors of alpha-amylase are widely distributed throughout the plant kingdom and have been purified from wheat, barley, rye, corn, millet, kidney bean, colocasia, and yam. Many of these have been extensively studied with respect to their structure, physicochemical properties, and mechanism of action (see Ref. 106 for details). It is questionable, however, whether these amylase inhibitors should really be considered true antinutritional factors since biological studies have not been very conclusive regarding their adverse nutritional effects. For example the alpha-amylase inhibitor isolated from the kidney bean (Phaseolus vulgaris) did not affect the growth rate of rats, nor did it affect the availability of energy from dietary starch (107). Although the growth of rats (108) and chickens (109) was not retarded by the feeding of alpha-amylase inhibitor from wheat, there was evidence of pancreatic hypertrophy indicative of degenerative changes in cellular morphology. In summary, therefore, it does not appear that the amylase inhibitors from legumes and cereals are particularly deleterious to animals, and perhaps humans, other than a possible stimulatory effect on pancreatic function similar to what has beenobserved with the trypsin inhibitors. It would be desirable, therefore, that foods possessing high levels of amylase inhibitors, such as beans and cereals, be thoroughly processed by heat treatment to insure their inactivation. At one time a number of alpha-amylase inhibitor preparations from kidney beans (so-called starch blockers) were introduced in the market. These were supposedly capable of inhibiting the digestion of starch in the intestinal tract to produce a reduction in caloric intake and consequent weight loss. Clinical studies, however, did not support these claims (1 10-1 12). Moreover, the amylase inhibitor represented only a minor constituent of these commercial formulations, which also contained significant levels of trypsin inhibitor and lectins (113,114). A subsequent study using a more highly purified preparation of amylase inhibitor from white beans showed that such a preparation was in fact capable of inactivating amylase in the human intestinal lumen (1 15). It remains to be proven, however, whether this effect will actually cause weight loss. B.

RoleinPlant

Since most amylase inhibitors of plant origin are only active against animal alpha-amylase, it does not appear likely that they regulate carbohydrate metabolism in the plant. A much more likely role for the these amylase inhibitors is that they protect seeds against insect

Antinutritional Related Factors

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269

predators. A considerable body of evidence has demonstrated that most of the plant amylase inhibitors are strongly active against amylases from insect species known to attack these same plants (106, 116). The addition of atnylase inhibitors from wheat (1 17) or kidney beans (1 18) to synthetic diets fed to the cowpea weevil (Cullosobruchusmncz~lntzrs) adversely affected the development and increased the mortality of this pest. Earlier reports on the toxic effect of a bean lectin on the development of the cowpea weevil (1 19, 120) now have been shown to bedue to theamylase inhibitor content of such lectin preparations (121). The successful transfer of the bean amylase inhibitor gene to the tobacco plant (122) presents the possibility that genetic engineering involving the transfer of the gene for the amylase inhibitor to other plants may be employed to enhance the resistance of such plants to insects toward which they would be normally be susceptible.

111.

LECTINS

A.

Introduction

Paralleling the distribution of protease inhibitors in plants is a class of proteins referred to as “lectins.” This term was first introduced by Boyd and Shapleigh (123, who pointed out that this class of proteins exhibited a high degree of specificity toward human blood cells of various blood group types. It was this high degree of specificity that led them to coin the the word “lectin” from the Latin word legere, meaning to pick or choose, to emphasize the specificity that these proteins exhibit toward blood groups. One obvious manifestation of this property is their ability to agglutinate the red blood cells from various species of animals due to the interaction of multiple binding sites of the lectins with specific glycoconjugate receptors on the cell metnbrane. In fact, the term “phytohemagglutinin” sometimes is used in referring to lectins of plant origin. Over the ensuing years, it has become increasingly apparent that the lectins exhibit a wide variety of other interesting biological effects that enable them to play a key role as mediators of cell recognition in living systems as well as providing powerful tools for the study of carbohydrates and their derivatives, both in solution and on cell surfaces (1 24). This brief overview will give primary consideration to their physicochemical properties, nutritional significance, mode of action, and role in the plant. More specific details on the many varied aspects of lectins may be found in Ref. 125, a book devoted to this subject. B. PhysicochemicalProperties A cursory examination of the physicochemical properties of some representative lectins shown in Table 3 suggests that, although there is wide diversity in their properties (126), certain broad generalizations can be made. The most important common feature is that most lectins are comprised of either two or four subunits, each of which contains a specific sugar-binding site. It is this feature of multivalency that accounts for the ability of lectins to agglutinate cells or to precipitate glycoproteins or large polysaccharide polymers. For example, concanavalin A, the lectin from the jack bean, is a tetramer comprised of four identical subunits, each of which has a molecular mass of 26,000 daltons (Fig. 7). A rather more complex situation is exemplified by the lectins of the kidney bean (Phaseolzrs vzdgnris). In this bean there is a family of five lectins (isolectins), each of which is a tetramer of four subunits designated as L or E (Fig. 8). These two different subunits confer leukoagglutinating activity (L) or erythroagglutinating activity (E) to the parent tetramer.

Liener

270

Table 3 Physiochemical Properties and Sugar Specificity of Plant Lectins’

Plant source

Molecular SubSugar units specificity weight“

Abr-2~~ precatorizts (jequirity bean)

a-D-Gal

Arachis hypogene (peanut) Bnndlern sirnplicifolin Baztlrinia yzarprwea crlbn Ccu1a~dlaensifortm’s (jack bean)

a-D-Gal a-D-Gal a-D-GalNAc a-D-Man. a-D-Glc a-D-GalNAc

Dolichos t)$orzrs (horse gram) Glycine mclx (soybean) Lens culinnris‘ (lentil) Plrnseolus coccinelrs (scarlet runner bean) Phaseolzts lrt~zntzrs~ (lima bean) Plznseolzrs vrtlgaris (black bean) Phaseolzrs ~ . d g a r i(red s kidney bean) Pllcrseolus vulgaris (wax bean Plznseolzrs vulgaris (navy bean) Pisunt sativzor? (garden pea) Psophocnrpus tetragotlolobus (winged bean) Ricimts corwmnis (castor bean) Vicin Jnba (field bean)

D-Gal, a-D-GalNAc a-D-Man. a-D-Glc GlcNAc D-GalNAc 4 D-GalNAc D-GalNAc D-GalNAc a-D-Man, a-D-Glc a-L-Fuc D-GalNAc D-Gal D-Man. D-GIcNAc

Carbohydrate content 65,000” 134,000 110.000 114.000 195,000 55,000 1 10,000 1 13,000 109,000 122,000

2 4

2 4 4 4 4

3.8

52.000

2

2

120,000 134,000 237,000 128,000 120,000 10.4 120,000 128.000 5 3,000

4 2 4

4

120,000 4.8 5 8,000 60,000h 120,000 50.000

4

4

0

4

4 4 4 4

11 0

6

5.7 4.1

0.3 9.4

2

4

Glc, glucose; GlcNAc. N-acetylglucosamine; Gal. galactose; GalNAc, N-acetylgalactosamine; Fuc. fucose; Man, mannose. If more than one value is glven for the molecular weight, there is evidence for the existence of multiple forms of the lectins (isolectins). Nonhemagglutinating toxins. ‘ Also known as L e w esculerzta. Also known as Pllaseolus linlerlsis. Source: From Ref. 126 wherein more examples may be found.

Thus, the isolectins referred to as L4and E4would have exclusively either leukoagglutinating or hemagglutinating activity, respectively, whereas the other three isolectins (EjLI, E2L,, and EL,) would display both activities depending on the relative proportion of these two subunits. Not shown in Table 3 is the fact that metal ions (of calcium, manganese, or magnesium) are required for the agglutinating activity by most lectins. Note also the fact that most lectins are glycoproteins. Concerted efforts are being made to determine the primary, secondary, tertiary, and quaternary structure of the various lectins, and such studies have

Antinutritional Factors Related to Proteins

271

Figure 7 Schematic :epresentation of the tetramer of concanavalin A. Each subunit is approximately 42 X 40 X 39 A. Manganese and calcium sites are indicated by Mn and Ca, respectively. The saccharide-binding site near the metals is indicated by S and the hydrophobic-binding site in the cavity by I. (From J. W. Becker, G. N. Reeke. B. A. Cunningham, and G. M. Edelman, New Evidence on the location of the saccharide-binding site of concanavalin A. Reprinted by permission from Nature, vol. 259, pp.4406-4409; copyright 1976 Macmillan Magazines Limited.)

revealed a high degree of homology among the lectins of diverse origin (127). In addition to concanavalin A, the three-dimensional structure based on x-ray crystallography has been reported for the wheat germ agglutinin (128), pea (129), peanut (130), soybean (131a,b), and lentil (13 IC) lectins. Despite the vast amount of information we now have regarding the structural features of many lectins (1 36, 1321, the reason for differences in their sugar specificity remains elusive. An x-ray crystallographic study of the lectin from

Figure 8 Schematic representationofthetetramericstructureofthefiveisolectins inthe red kidney bean,where L and E are thesubunits responsible for leukoagglutinating and hemagglutinating activities, respectively. (From J. B. Miller, R. Hsu, R. Heinrikson,and S. Yachnin, Extensive homology between the subunits of the phytohemagglutinin mitogenic proteins derived from Pllaseolus vulgaris, Proc. Natl. Acad. Sci. USA. 72:1388, 1975.)

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272

Erytlzrirzn coraZloderzdron (133) suggests that extensive differences in the topography of the binding pockets of lectins most likely account for differences in sugar specificity.

C.

NutritionalSignificance

The extreme toxicity of the castor bean had been known for a long time, but it remained for Stillmark to demonstrate its hemagglutinating property, in 1888 (134). Twenty years later Landsteiner and Raubitschek showed that even the seeds of many edible species of legumes contained substances capable of agglutinating the red blood cells of various animals in a very specific fashion (135). Following the pioneering work of Jaffi and coworkers, subsequent research by Liener and Pusztai firmly established the fact that the lectins of the common bean (Phaseolus vulgaris) are mainly responsible for the toxic effects resulting from the consumption of the raw bean (see Ref. 136 for Liener's review of this). Other legumes from which lectins have been isolated and shown to be toxic upon oral ingestion include the jack bean (Ccrrzavalln ensiforrnis), horse gram (Dolichos b$or.us), hyacinth bean (Dolichos lnblab), Adzuki bean (Phaseolus ~wlgaris),lima bean (Phaseolus lrmatus), and winged bean (Psophocmpus tetragonolobus). The fact that lectins are so widely distributed in food items commonly consumed in the human diet (137) raises the important question of whether thay pose any significant risk to human health. Although the lectins of most food items are inactivated by the heat involved in processing or household cooking (137), lectin activity has nevertheless been detected in such food items as dry cereals and peanuts (137), dry roasted beans (138), and processed wheat germ (139). The literature includes a number of reports of human intoxication in which lectins appear to have been the causative agents. For example, in 1948 a severe outbreak of gastroenteritis occurred among the population of West Berlin because of the consumption of partially cooked beans that had been air-lifted into the city during its blockade (140). A mixture of beans and maize, prepared by mothers in Tanzania as a porridge for infant food, was found to still possess lectin activity because of insufficient cooking (141). Outbreaks of intoxication have been reported in England because of the consumption of raw or partially cooked beans (142-144). Most of these cases have involved individuals who had eaten raw beans as part of a salad or as an ingredient in dishes such as chili con carne prepared in a slow cooker. In the latter case the conditions of heating were such that the lectin activity was not completely destroyed even though the beans were considered to be acceptable in terms of texture and palatability (145-148). Prompted by these reports, warning labels may now be found on most labels of dry beans sold in retail food stores in England (Fig. 9).

D. Mode of Action Jaffi and co-workers were the first to propose that the toxicity of bean lectins could be attributed to their ability to bind to specific sites on the surface of cells lining the intestinal tract (149). Subsequent studies by other investigators have fully confirmed the fact that bean lectins bind to the intestinal mucosa (150-154). This is illustrated in Fig. 10, which shows that the lectin ingested by rats in the form of raw kidney beans binds to the brush border region of the small intestines and may in part be endocytosed by cells underlying the brush border membrane. As shown in Fig. 11, the binding of the kidney bean lectin

Antinutritional Factors Related to Proteins

273

IMPORTANT These beans must be boiled for at leastten minutes before eating. Do not eat partially cooked. Figure 9 Warning label that has been placed on packets of dry beans sold in the retail market in England. (From Ref. 136.)

is accompanied by severe disruption of the brush border and abnormal development of the microvilli. One of the major consequences of lectin-induced damage to the intestinal mucosa is a serious impairment in the absorption of nutrients across the intestinal barrier. This phenomenon was first demonstrated by Jaffi and Comejo (155) and later confirmed by Donatucci et al. (156) involving the use of radioactive glucose and the technique of vascular intestinal perfusion. This interference with the absorption of nutrients is nonspecific since it can also be demonstrated with amino acids (1 57, 158), lipids (159), and vitamins (1 60). Superimposed upon this impairment in intestinal absorption is the finding that enterokinase (161) and many of the brush border hydrolases are inhibited by lectins (158, 162a), a factor that would also contribute to an interference with nutrient availability and the ability of lectins to cause an increase in the weight and number of cells of the small intestine as well as the pancreas (162b, 163). As in the case of the trypsin inhibitors, the stimulation of pancreatic growth occurs as a result of the release of CCK from the small intestines (163). An alternative explanation for the toxic effects of dietary lectins is suggested by the observation that germ-free animals are better able to tolerate raw beans in their diet compared to conventional animals (164, 165). This observation may be related to the fact that rats fed raw beans exhibit an overgrowth or colonization of coliform bacteria in the small intestine (154, 159). It has been suggested that, as a result of the altered permeability of the intestinal mucosa, these bacteria, or the endotoxins they produce, gain entrance into the bloodstream causing toxic systemic effects (166). The exact mechanism whereby lectins induce colonization of the small intestine is not known. A possible explanation may be that lectins, because of their polyvalency, bind to receptor sites on the brush border as well as to the bacterial coat and thus “glue” bacteria to the luminal surface of the small intestines.

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Liener

Figure 10 Immunofluorescencemicrograph of part of a transverse section through the duodenum of a rat fed a diet containing raw kidney beans. Incubation with rabbit antilectin immunoglobulins and fluorescein isothiocyanate-conjugated antirabbit IgG, showing immunofluorescence in brush border region and within apical cytoplasm of mature enterocytes (arrows). L, intestinal lumen. Bar: 50 pm. (From Ref. 15 1 .)

Lectins and antibodies to lectins can be detected immunochemically in the blood of rats and pigs fed raw kidney beans (167, 168). This would indicate that lectins themselves, either intact or partially digested, may be absorbed and enter the circulatory system. In addition to a direct toxic effect on certain target organs, other systemic effects that may ensue include an increase in protein and fat catabolism, a depletion of muscle glycogen, and an elevation in blood insulin levels (169).

E. Role in Plants Among the various hypotheses that have been proposed for the role of lectins in plants, at least two have attracted the most attention: that they act as mediators of the symbiotic relationship between N-fixing microorganisms, primarily of the genus Rhizobiurn, and leguminous plants, and that they are part of a defense mechanism against insects and microbial pathogens. The association between legumes and N-fixing bacter is highly specific. For example, the rhizobia that infect soybeans cannot nodulate garden peas or white clover, and

AnthUtrkion8l Factors Related to Proteins

275

Figure 11 Electron micrographs of sections through apical regions from rats fed diets containing (A) 5% raw kidney beans and 5% casein compared with (B) 10% casein. (From A. Pusztai, E. M. W. Clarke, T. P. King, and J. C. Stewart, Nutritional evaluation of kidney beans (Phaseolus vulguns): chemical composition, lectin content, and nutritional value of selected cultivars, J. Sci. Food Agr., 305343, 1979.)

vice versa. That lectins are responsible for this specific interaction is based on the finding that the lectin from a particular legume such as the soybean binds in a sugar-specific manner to the the corresponding rhizobial species but not to bacteria that are symbiotic to other legumes. However, a number of exceptions to this general pattern have been reported, so the lectin recognition hypothesis continues to be a subject of controversy (1 70). Nevertheless, it is possible to alter the lectin-binding specificity by the transfer of genes essential for nodulation between plants. For example, the pea lectin gene has been introduced into white clover roots using Agrobacterium rhizogenes as a vector. The clover roots that resulted could then be nodulated by a Rhizobium usually specific for the pea (171). The transfer of the genes coding for the lectin of a N-fixing plant to nonlegumes remains an exciting challenge of obvious agricultural importance. Various lines of evidence suggest that lectins may be involved in the defense of plants against insects, bacteria, fungi, and viruses (170, 172). For example, the lectin from Phaseolus vulgaris was found to have a lethal effect on the larvae of the bruchid beetle (1 19), presumably due to the binding of the lectin to epithelial cells lining the midgut of this insect (173). As pointed out above, however, it is believed that the amylase inhibitor is responsible for the resistance of the common bean to the cowpea weevil (121). A protein isolated from the seeds of a a wild variant of Phaseolus vulgaris, referred to as arcelin, was found to be toxic to the bruchid beetle, an important bean pest (174). It appears that lectins, amylase inhibitors, and arcelin, because of their similarity in 3-D structure, act to a greater or lesser extent as insecticidal proteins (175a). Deletions of one (arcelin) or two (alpha-amylaseinhibitor) loops located in the region of the sugar-binding site of the lectin molecule are believed to be responsible for the different activities exhibited by these molecules. The pea lectin has been expressed in the potato (175b) and in the tobacco plant (176). In the latter case the transgenic tobacco plant was found to have increased resistance to the tobacco bud worm; an additive protective effect was obtained with the introduction

Liener

276

of the cowpea trypsin inhibitor (176). In addition to their role as a defensive measure against insects, the lectins from various plants have also been shown to inhibit the growth of phytopathogenic bacteria and fungi (177- 179). All of these studies suggest that plant lectins offer considerable promise for the genetic engineering of disease-resistant plants. Since this strategy would raise the specter of increasing the toxicity of such plants, careful attention would have to be paid to their elimination by suitable processing techniques. Although the lectins, as well as the protease inhibitors, can usually be inactivated by proper heat treatment, the application of industrial scale technological methods, such as air classification, extraction, and texturization in the absence of heat, may notbe fully effective as a means of detoxification. The best insurance against this possibility is careful monitoring of all newly introduced transgenic plants for the presence of antinutritional factors. F. AnalyticalTechniques Lectin activity is most commonly determined by measuring the degree to which erythrocytes from the blood of a given animal are agglutinated, the cells sometimes being sensitized by treatment with trypsin or some other protease. The simplest assay is one involving serial dilution in which the end-point is determined by visual inspection of the clumped cells. Although this method is rapid and simple, it gives only semiquantitative results. A spectrophotometric method has been proposed to increase the precision of such an assay (180). The most serious limitation of an assay that depends on the agglutination of erythrocytes is the fact that one must choose the blood of an animal for which the lectin is specific. Improper selection of red blood cells may result in very low sensitivity or even negative results. Furthermore, there is no assurance that agglutinating activity per se bears any relevance to the in vivo effects of the lectin. Assuming that the toxicity of lectins to a given animal species depends on their ability to bind to the intestinal mucosa, the most relevant technique would be one that measures the degree to which a certain lectin binds to the epithelial cells of the target animal. Such a method has been proposed that involves an ELISA-type assay in which one measures the binding of an enzyme-linked lectin to preparations of the brush border membrane of the animal under study (181).

IV. TOXIC AMINOACIDS A.

Neurotoxins

Lathyrism, as it is known to occur in humans, is a paralytic disease associated with the consumption of Lathyus sativus (more commonly known as chickling or grass pea) or related species such as L. cZynwrzutn and L. cicera. Although this disease has been recognized since ancient times, today the disease is restricted to India, Bangladesh, and Ethiopia. It surfaces during periods of famine resulting from droughts when field crops become blighted and, as an alternative crop, this particular crop is cultivated. As recently as 1975, over 100,000 cases of lathyrism in men between the ages of 15 and 45 years were reported in India (182). The disease is characterized by a nervous paralysis of the lower limbs that forces the victim to walk with short, jerky steps; in extreme cases death may result (183). Spencer and Schaumberg have described an outbreak of lathyrism that occurred during World War I1 among Roumanian Jews confined to a forced-labor camp in the

Antinutritional Factors Related to Proteins

277

Ukraine (1 84). For 4 months their daily ration consisted of 400 g of L. sntivus peas cooked in saltwater plus 200 g of bread. The neurological symptoms of this disease persist even today in those survivors who now live in Israel. Attempts to identify the causative agent of human lathyrism have been complicated by the fact that the sweet pea (L. odoratus) produces another form of lathyrism (osteolathyrism), which is characterized in rats by skeletal deformities (185). This is in contrast to what is observed with rats who thrive quite well when they are fed L. sntivus and do not display the nervous disorder associated with the consumption of this species in humans. Historically, the osteolathyrogen of the sweet pea was the first to be isolated and was identified as 3-aminopropionitrile (I) (1 86). See Fig. 12 for structures and distribution of the various lathyrogenic neurotoxins. Several groups of workers in India (187-189) have succeeded in isolating a compound from L. sativus that may be the causative factor of human lathyrism. This com-

Structure and Name

Occurrence

Lathyrus odoratus L. pusillus

L. hlrsutu 0

NH2

II

1

HOOC-C-NH- CH2- CH-COOH

3-N-oxalyl-2,3-diarnino-

L. sativus L. cicera L. dymenum

propionic acid NH2 H,N

- CHz-

1

CHF CH-COOH

2,4-diaminobutyric acid

L. latifolius L. sylvestris

NH2

I

Vicia sativa

NsC-CH*-CH-COOH

3-cyanaoatanineb N"2

I

CH3-NH- CH2- CH-

COOH

Cycas circinalis

3-N-methylaminoalanine

Figure 12 Structures anddistributionoflathyrogenicfactors. gen; 'possible cause of amyolateral sclerosis in man.

aOsteolathyrogen;bneurolathyro-

278

Liener

pound, identified as 3-N-oxalyl-2,3-diaminopropionicacid (11), produced severe neurotoxic symtoms when injected into rats, chicks, and monkeys. This compound, as well as 2,4-diaminobutyric acid (111), has also been isolated from other Lcrthyr-us species, and has been shown to produce neurotoxic effects when administered by injection into several different species of animals. However, attempts to reproduce neurolathyrism in animals by the oral administration of these neurotoxic amino acids have generally proved unsuccessful (1 90, 191a); thus the true causative agent of human lathyrism has not beenunequivocally established. A neurotoxic amino acid, 3-cyanoalanine (IV) and its gamma-glutamyl derivative, has been isolated from Vicia sativa by Ressler et al. and was found to exhibit neurotoxic effects after oral or intrapertoneal administration to rats and chickens (191b,c). This toxin, however, can be readily eliminated by steeping and cooking provided the broth is discarded prior to consumption (19 Id). A high incidence of amyotrophic lateral sclerosis (ALS) is known to occur among the residents of certain islands in the Western Pacific such as Guam and Rota. One of the traditional foods consumed by the natives in these islands is the seeds of the false sago palm (Cycns circinalis), which has led to the search for the toxic agent(s) in this plant that might be responsible for this neurological disorder. An unusual nonprotein amino acid, 3-N-methylamino-~-alanine (V), was first isolated from the cycad plant by Vega and Bell (192). It was subsequently shown by Spencer et al. that the repeated oral administration of this compound to macaques produced behavioral dysfunction and neuropathological changes that resembled the prominent features of ALS noted in Guam (193). However, since 3-N-methylamino-~-alanine is only one of several potential neurotoxins present in the cycad seed, it is premature to assign a casual role to any single factor pending further research (194).

B. Hypoglycin Consumption of the fruit of the plant Blighia sapida (known in Jamaica as ackee and in Nigeria as isin) has been linked to a disease of undernourished people, especially in Jamaica, known as vomiting sickness [see review by Kean (195)l. This plant was named after Captain Bligh, who introduced it into the West Indies after he survived the mutiny on the Bomly. The characteristic symptom of violent vomiting that accompanies consumption ofthe unripe fruit is followed by convulsions, coma, and even death in some instances. Hypoglycemia is the principal clinical sign with sugar levels as low as 20 mg/100 ml compared to a normal value of 100 mg/100 ml. The causative principle has been identified as beta-(methylenecyclopropy1)alanine and is referred to as hypoglycin A (Fig. 13). It may also occur as conjugate as the gamma-glutamyl dipeptide. Hypoglycin follows essentially the same pattern of metabolism as branched-chain amino acids. Some of these intermediates are shown in Fig. 13. It is first deaminated to beta-methylenecyclopropyl pyruvate, which then undergoes oxidative deamination to betamethylenecyclopropyl acetyl-coA. Formation of the latter interferes with the transfer of long chain fatty acyl-CoA to carnitine, thus blocking the process of beta-oxidation. This results in an impairment in gluconeogenesis, which is accompanied by depletion of stored glycogen and hypoglycemia. Because of its structural similarity to leucine, hypoglycin may also interfere with the metabolism of this amino acid. This results in an accumulation of isovaleric acid and alpha-methylbutyric acid, amino acids that could act as depressants

Antinutritional Factors Related to Proteins

279

\'

C%=CH-CH-CH,-CH-COOH CH2

p-( Methylenecyclopropyl)a lanine ( hypoglycin A ) 0

II

CH~=CH-FH-CH~-C-S-COA \

CH2

p-( Methy1enecyclopropyl)acetyl-CoA

p -( Methylenecyclopropyl) pyruvate Figure 13 Structure of hypoglycin A and some of the intermediates involved in its metabolism.

of the central nervous system (196). This could explain the syndrome of vomiting that accompanies the ingestion of akee. Hypoglycin has also been reported to produce teratogenic effects in rat and chick embryos (197).

C. Mimosine

The National Academy of Sciences has published a monograph that points out the potential value of the legume Leuccrem leucocephnln(called kcdo hnole in Hawaii) as a forage crop for livestock and human feeding (198). One of the principal factors limiting the use of this plant, however, is the fact that an unusual amino acid, minosine (Fig. 14), comprises 3-5% of the dry weight of the protein. This amino acid is believed to be responsible for

Mirnosine

3,4-Dihydroxypyridine

Figure 14 Structure of mimosineand its goitrogenicmetabolite, 3,4dihydroxypyridine.

Liener

280

the poor growth performance of cattle when Lezlcaetza makes up more than one-half of the diet. This adverse effect on growth has been attributed to the underproduction of thyroxine, presumably due to the fact that the rumen bacteria convert mimosine to 3,4dihydroxypyridine (Fig. 14), which acts as a goitrogenic agent (199a-c). In Hawaii, nminants consume greater amounts of the Leucaerza than ruminants in Australia before toxicity is manifested, presumably due to a difference in the microfloro of the rumen. Jones and Megarrity have, in fact, isolated bacteria capable of detoxifying mimosine from goats in Hawaii and inoculating Australian ruminants with these organisms, thus giving them the ability to consume greater amounts of the legume (199d). In nonruminants, such as the horse, pig, and rabbit, the goitrogenic effect is not very marked. These animals nevertheless do very poorly on diets containing Leuccrena, one of the characteristic features being a loss of hair (200, 201). It has been suggested, in fact, that mimosine might be used as a defleecing agent in sheep (203,).Certain segments of the human population, particularly in Indonesia, are known to consume portions of the Leucaenn in their diet, and a loss of hair has frequently been observed among those individuals who have eaten the leaves, pods, and, and seeds in the form of a soup (203). Although the goitrogenic effect of mimosine seems to be well established, the precise mechanism of toxicity in other animal species remains obscure. It can act as an inhibitor of pyridoxal-containing transaminases (204), tyrosine decarboxylase (205), and both cystathione synthetase and cystathionase (206). The latter effect may have particular relevance, since an inhibition of the conversion of methionine to cysteine, a major component of hair protein, could account for the hair loss that is so characteristic of mimosine toxicity. Mimosine may exert a more direct efftect on hair growth, since it has been reported that Leucaena extracts destroyed the matrix of the hair follicles of mice (207). Matsumoto et al. reported that the mimosine content of the seeds and leaves of Leucaerza could be decreased by storing the plant in temperatures in excess of 70°C in the presence of moisture (208). Yoshida showed that the addition of ferrous sulfate to the diet of rats fed Leucaerza leaf meal reduced mimosine toxicity, presumably due to a decrease in the absorption of this amino acid from the gastrointestinal tract (209). D.

Djenkolicacid

In certain parts of Sumatra, and particularly in Java, the djenkol bean is a popular item of consumption (203). The bean is actually the seed of the leguminous tree Pithecolobiurn lobntzmz and resembles the horse chestnut in size and color. Consumption of this seed sometimes leads to kidney failure, which is accompanied by the appearance of blood and white needle-like clusters in the urine. The latter substance has been identified as as a sulfur-containing amino acid known as djenkolic acid (Fig. 15), which comprises 1 4 % of the seed (210). Despite its structural resemblance to cystine, it cannot replace cystine in the diet of rats, although it can apparently be metabolized by the animal body (21 1). However, because of its relative insolubility, much of the djenkolic acid escapes metabolic degradation and tends to crystallize out in the urine.

E. Dihydroxyphenylalanine The amino acid 3,4-dihydroxyphenylalanine(dopa) (Fig. 16) is present in fairly high concentrations in the fava bean (Vicin fnba) (212-215), the velvet bean (Stizolobium deerirlginmun) (216), and wheat and oats (217). Since the consumption of the fava bean is fre-

Antinutritional Factors Related to Proteins

281

S-CH2-CH-COOH I I CH2 NH2 I S- CH2-CH- COOH I NH2

Djenkolic acid

Cystine

Figure 15 Structure of djenkolic acid, an amino acidpresentin structure of the amino acid cystine is shown for comparison.

Pitlzecolobium lobatum. The

quently associated with a disease in humans known as favism, the question has been raised as to whether dopa might not play a causative role in the etiology of this disease (218). Persons genetically deficient in the enzyme glucose-6-phosphate dehydrogenase appear to be particularly susceptible to this disease, and one of the characteristic clinical features of this disease is believed to be due to a marked lowering of the glutathione content of the erythrocytes (219). In view of these facts, it may be pertinent to note that the in vitro addition of dopa to the red blood cells from individuals deficient in glucose-6-phosphate dehydrogenase produced a significant lowering of the glutathione content of such cells (219). Because of its high content of dopa, it has beeen suggested that V. fnbn might have therapeutic value in the treatment of Parkinson's disease (220).

F. SelenoaminoAcids Selenium poisoning in livestock due to the consumption of selenium-accumulating plants of the genus Astragalus has been well documented (221). The selenium of such plants is present in the protein in the form of selenomethionine, selenocysteine, and selenocystine residues (Fig. 17). Digestion of this protein in the digestive tract results in the liberation and absorption of these amino acids, and, because of their structural similarity to their natural sulfur analogs, they compete for the synthesis of animal protein. Defective proteins thus formed could account for the loss of hair and sloughing of hoofs, which are characteristic features of selenium poisoning in livestock. Chronic selenosis in human beings, presumably caused by eating corn grown in seleniferous soil, has been reported in Colombia, South America (222). In certain parts

HO

Figure 16 Structure of 3,4-dihydroxyphenylalanine(dopa),an amino acid present in the faba bean (Viciafnbn). the velvet bean (Stizolobium derringinnzm~),and wheat and oats.

Liener

282 CH3Se- CH2-CH-COOH I NH2

CH3-Se-CH2-CH2-CH-COOH I NH2

Selenomethionine

Methylselenocysteine

y 2

y 2

S,e-CHZCH-COOH Sb-CH,-CH-COOH I

0

CHz-CHzCH-COOH CH~-CH~ I CHNH2

Selenocystine

Selenocystathionine

Figure 17 Structure of selenoamino acids.

of Venezuela, the ingestion of nuts from a tree known as coco de rnorzo (Lecythis ollcu-ia) produces a toxic syndrome in humans characterized by abdominal distress, nausea, vomiting, dialrhea, and loss of scalp and body hair. Using an assay system involving the measurement of the cytotoxicity activity against mouse fibroblasts, the factor responsible for this toxic effect was identified as selenocystathionine (223) (Fig. 17).

G. lndospicine Zrzdigofem spicata, or creeping indigo, is a tropical legume with potential value as a forage and soil improvement crop. It is known to contain a toxic amino acid, indospicine, which is a structural analog of arginine (224) (Fig. 18). This compound was shown to cause cirrhosis and other pathological changes of the liver when fed to rats, an effect that was attributed to its role as an antagonist of arginine (225). It has also been reported that indospicine may produce cleft palate in the fetuses of rats given a single oral dose of this compound on the 13th day of gestation (226). H. Canavanine Canavanine, like indospicine, is an analog of arginine (Fig. 18), andit occurs in high concentrations (up to 5 % ) in the seeds of the jack bean, Celrrctvrrlia er?s(fomis,and in a number of other legumes in lesser amounts. Its role as a protective agent against insects has been discussed in detail by Rosenthal (227a,b). Alfalfa sprouts contain about 1.570 of their dry weight of canavanine. A severe lupus erythematosus-like syndrome is produced in monkeys fed alfalfa sprouts, an effect that has been attributed to its canavanine content (228). 1.

Linatine

Flax or linseed (Lirlurn usitc~tissirnu~~~) meal is considered a poor source of protein for chicks, but considerable improvement may be effected by extracting the meal with water and autoclaving for 30 min or by supplementation with pyridoxine (229, 230). Klosterman

Antinutritional Factors Related to Proteins

283

Figure 18 Structures of indospicine and canavanine, antimetabolites of arginine found in creeping indigo (Irdigqfern spicotn) and jack beam (Camlwlicr erzs$orruis), respectively.

and co-workers reported the isolation of a pyridoxine antagonist from flaxseed, which was identified as 1-amino-D-proline thatoccurs naturally in combination, via a peptide linkage, with glutamic acid (231) (Fig. 19). This peptide was given the name linatine. 1-AminoD-proline was actually four times as toxic as linatine when injected into chicks, and its toxicity could effectively be counteracted by the simultaneous injection of pyridoxine. Sasaoka et al. carried out similar studies with rats and noted that 1-amino-D-prolinecaused marked changes in amino acid metabolism (332). These effects are most likely related to its effect on enzyme systems that require pyridoxal phosphate as a cofactor (233). J.

Lysinoalanine

Alkaline extraction of soybeans, which is frequenly used in the preparation of soy protein isolates, isknownto reduce the nutritive value of the protein, attributable, at least in

Linatine

I - Aminoo - proline

Glutamic acid

Figure 19 Structure of 1-amino-D-proline, the antipyridoxine factor of linseed (Limon zisitatissin z z r m ) and its natural precursor. linatine.

Liener

284

part, to the destruction of cystine (234). One of the decomposition products of cystine is dehydroalanine (may also be derived from the decomposition of serine), which can interact withthe epsilon-amino group of lysine to form lysinoalanine (Fig. 20). Alkali-treated soybeans produced kidney lesions in rats, an effect that could be reproduced by the administration of lysinoalanine (235, 236). Sternberg et al. have shown lysinoalanine to be widely distributed in cooked foods, commercial food preparations, and food ingredients, many of which had never been subjected to alkaline treatment (237). Many of these foods had levels of lysinoalanine considerably higher than those found in commercial samples of soy protein isolate. The widespread distribution of lysinoalanine among commonly cooked foods indicates that this is not a novel or serious problem because humans have long been exposed to proteins containing lysinoalanine with apparent impunity. This conclusion is strengthened by a recent report that the feeding to preterm babies of a heat-processed infant milk formula containing high levels of lysinoalanine had no effect on their renal function (238). K. BiogenicAmines A wide variety of common foods such as cheese, meat and fish products, wine, beer, and other fermented foods contain appreciable levels of amines, which are referred to collectively as biogenic or pressor amines (239). Some of the most common of these amines are shown in Fig. 21. These amines have aliphatic, aromatic, or heterocyclic structures and are usually generated by the microbial decarboxylation of free amino acids. High levels of of some biogenic amines may cause toxic effects with a wide variety of symptoms, including migraine, headache, gastric and intestinal ulcers, and pseudoallergic responses (240). Additive effects of the consumption of a number of different foods such as wine, cheese, and meat or fish products over a short period may result in biogenic

Lysine

Dehydroalanine

Lysinoalanine

CH29 S-S-CH,

I - CH I

t

-CH t

Cystine

0

- CH denotes alpha carbon of a peptide linkage (-NH-CH-C I

I

I

I

n

-)

as it occurs in the intact protein.

Figure 20 Formation of lysinoalanine from cystine (and/or serine) and lysine.

285

Antinutritional Factors Related to Proteins

H O n - C H 2 - CH2- NH2 W

ne

Hlstamine

w

'4

CH2- CH2- NH,

-CH2- CH2- NH,

H

Serotonin

Q-Phenylethyleneamine

CH2- CH2- NH2

Q-J

NH2- (CH2)5- NH,

H Cadaverine

Tryptamine

H2 N-(CH2)4- NH2

H,N-C-NH-(CH,)4-NH, NH

Putrescine

Agmatine

NH2-(CH2),-NH-(CH2),-NH-(CH,)3-NH2

Spermine

NH,-(CH2)3-NH-(CH2)4-NH2

Spermidine Figure 21 Structures ofbiogenicaminescommonly

found infoods.

Liener

286

amine intoxication, whereas consumption of these individual foods may not cause any problem. Of particular concern is the observation that individuals receiving monoamine oxidase inhibitors in the treatment of psychiatric depression may undergo severe hypertensive crisis upon the ingestion of foods rich in biogenic amines (241, 242). Since monoamine oxidase, an enzyme produced by the liver, detoxifies amines by deamination, an inhibition of this enzyme exacerbates the toxic effects of biogenic amines.

REFERENCES 1.

3,. 3.

4. 5. 6. 7.

8.

9.

1Oa. 1Ob.

1oc.

11. 12.

13. 14. 15.

16.

Read, J. W.and Haas, W. (1938). Studieson the baking quality of flour as affected by certain enzyme actions. V. Further studies concerning potassium bromateand enzyme activity. Cereal Chem., 15:59. Bowman, D. E.( 1944). Fractions derived from soybeans and navy beans which retard digestion of casein. Proc. SOC.Exp. Biol. Med., 57:139. Ham, W. E. and Sandstedt, R. M. (1944). A proteolytic inhibitor substance in the extract from unheated soybean meal. J. Biol. Cltenz., 154:505. Kunitz, M. (1945). Crystallization of a trypsin inhibitor from soybeans. Science, 101:688. Osborne, T. B. and Mendel, L. B. (1917). The use of soybean as food. J. Biol. Client., 32: 369. Liener, I. E. and Kakade, M. L. (1980). Protease Inhibitors. In Toxic Corlstitzlents of Plant Foodstztffs (I. E. Liener, ed.). Academic Press, New York, p. 7. Laskowski, M. and Kato, I. (1980). Protein inhibitors of proteinases. Annu. Re,?. Biochew., 49:593. Hwang, D. L.-R., Lin, K.-T. D., Yang, W.-K., and Foard. D. E. (1977). Purification, partial characterization, and immunological relationship of multiple low molecular weight protease inhibitors of soybean. Bioclzim. Bioplzys. Acta, 495:369. Chen,P.,Rose,J., Love.R.,Wei, C. H.,and Wang, B. C.(1992). Reactive sites ofan anticarcinogenic Bowman-Birk inhibitor are similar to other trypsin inhibitors. J. Biol. Clzent., 267: 1990. Werner, M. H. and Wemmer, D. E. ( 1992). Three-dimensional structure of soybean trypsin/ chymotrypsin Bowman-Birk inhibitor in solution. Biochenristp, 3 1:999. Prakash, B., Murthy. M. R. N., Ssreerama, Y. N., Sarma. P. R. R.. and Rao, D. R. (1994). Crystallization and preliminary x-ray diffraction studies on a trypsin chymotrypsin doubleheaded inhibitor from horse gram. J. Mol. Biol., 235:364. Voss, R. H.. Ermler, U.. Essen, L.-O., Wenzel, G., Kim, Y.-M.. and Flecker, P. (1996). Crytal structure of the bifunctional soybean Bowman-Birk inhibitor at 0.28 nm resolution. Structural peculiarities in the folded protein conformation. Eur. J. Biochent., 3,43:122. Klose. A. A.. Hill. B., and Fevold, H. L. (1946). Presence of a growth inhibiting substance in raw soybean. Proc. SOC.Exp. Biol. Med., 63: 10. Ham, W. E., Sandstedt, R.M.. and Mussehl, F. E. (1945). Theproteolytic inhibiting substance in the extract from unheated soybean meal and its effect upon growth in chicks. J. Biol. Chern.. 161:635. Westfall, R. J. and Hauge, S. M. (1948). The nutritive quality and trypsin inhibitor content of soybean flour heated at various temperatures. J. Nzttr.. 35:373. Kakade, M. L., Hoffa. D. E., and Liener. I. E. (1973). Contribution of trypsin inhibitors to the deleterious effects of unheated soybeans fed to rats. J. Nutr., 103:1772. Liener. I. E., Deuel. H. J., Jr., and Fevold, H. L. (1949). The effect of supplemental methionine on the nutritive value of diets containing concentrates of the soybean trypsin inhibitor. J. Nutr., 39:335. Chernick, S. S.. Lepkovsky, S., and Chaikoff, I. L. (1976). A dietary factor regulating the

Antinutritional Factors Related to Proteins

17.

18. 19. 20.

21a.

21b.

22.

23.

24.

25.

26. 27.

28a.

28b.

28c. 29.

30.

3 la.

287

enzyme content of the pancreas: changes inducedin size and proteolytic activity of thechick Anz. J. Plzysiol., 155:33. pancreas by the ingestion of raw soybean meal. Yanatori, Y. and Fujita, T. (1976). Hypertrophy and hyperplasia in the endocrine and exocrine pancreas of ratsfed soybean trypsin inhibitoror repeatedly injected with pancreozymen. Arch Histol. Jprl., 39:67. Nitsan, Z. and Liener, I. E. (1976). Enzyme activities in the pancreas, digestive tract. and feces of rats fed raw or heated soyflour. J. Nutr. 106:300. Lyman, R. L. and Lepkovsky, S. (1957). The effect of raw soybean meal and trypsin inhibitor diets on pancreatic enzyme secretion in the rat. J. Nzm. 62269. Booth. A. N., Robbins, D. J., Ribelin, W. E., and DeEds, F. (1960). Effect of raw soybean meal and anuno acids on pancreatic hypertrophy in rats. Proc. SOC.Exp. Biol. Med., 104: 681. Morgan. R. G. H., Levison. D. A., Hopwood, D., Saunders, J. H. B., and Wormsley, K. G. (1977). Potentiation of the action of azaserine on the rat pancreas by raw soy bean flour Cancer Lett., 3~87. Ellwood, K.C., Roebuck, B. D.. and Hathcock. J. N. (1994). Marginal zinc status does not exacerbate pancreatic carcinogenesis associated with dietary soybean trypsin inhibitor concentrate in rats. J. Nutr., 124:894. Levison, D.A., Morgan, R. G. H., Brimacombe, J. S., Hopwood. D., Coghill, G., and Wormsley, K. G. (1979).Carcinogenic effects of di(2-hydroxypropyl) nitrosamine (DHPN) inmale Wistar rats: promotion of pancreaticcancer by a raw soya flourdiet. S c a d J. Gastl-oenterol., 14:223. McGuiness, E. E., Morgan, R. G. H.,Levison, D. A., Frape, D. L., Hopwood, D., and Wormsley, K. G. (1980). Theeffects of long-term feeding of soya flour on the rat pancreas. Scwzd. J. Gastroellterol.. 15:497. Liener, I. E., Nitsan, Z., Srisangnam, C., Rackis, J. J., and Gumbmann. M. R. (1985). The USDA trypsin inhibitor study. 11. Time related biochemical changes in the pancreas of rats. Qual. Plant. Plant Foods H u m Nutr., 35:243. Hasdai. A. and Liener, I. E. (1986). The failure of long-term feeding of raw soyflour to induce carcinogenic changes in the pancreas of micein the presence or absence of azaserine. Nutt-. Cancel-, 8:85. Hasdai, A. and Liener, 1. E. (1985). The effectsofsoyflourandN-nitroso-(2-oxypropyl) amine on the pancreas of the hamster. Drug Nzrtrierzt Interact., 3:173. Green, G. M. and Lyman, R. L. (1 972). Feedbackregulation of pancreatic enzyme secretion as a mechanism for trypsin-induced hypersecretion in the rat. Proc. SOC.Exp. Biol. Med., 130:6. Roebuck, B. D.. Kaplita, P. V., Edwards, B. R.. and Praissman, M. (1986). Effects of dietary fats and soybean protein on azaserine-induced pancreatic carcinogenesis and plasma cholecystokinin in the rat. Cnrzcer Res., 47:1333. Ozawa, S. and Hata. Y. (1991). Development of pancreatic exocrine functionincluding negative feedback regulation using oral trypsin inhibitor in rats. Niljpon Shokrzkibyo Gakkai 20sshi, 88:2804. Furuse, M., Yang. S. I., Muramatsu, T.. and Okumura, J. (1990). Enhanced release of cholecystokinin by soya-bean trypsin inhibitor in chickens. S c m d J. Gastroenterol.. 25:1242. Schneeman, B. 0. and Gallaher. D. (1986). Pancreatic response to dietary trypsin inhibitor: variations among species. In Nutritinal arld Toxicological Sign$cance of Enzyme Ivrkit5itor.s in Foods (M. Friedman. ed.). Plenum Press, New York, p. 185. Liener, I. E., Goodale, R. L., Deshmukh, A., Satterberg, T. L., Ward. G., DiPietro, C. M., Bankey, P. E., and Borner, J. W. (1988). Effect of a trypsin inhibitor from soybeans (BowGastroenterology. 94:419. man-Birk) on the secretory activity of the human pancreas. Calam, J., Bojarski, J. C., and Springer, C . J. (1987). Raw soya bean flourincreases cholecystokinin release in man. Br. J. Nutr.. 58:175.

288

Liener

3 lb. Roseland, J. E., Holm, H., Jenssen, T., Jacobson,M. B., and Hanssen, L. E. (1995).Inhibitorstimulated non-parallel pancreatic secretion in man: hormonal and neutral regulation. Scand. J. Gastroenterol., 30:72. 32. Iwai, K., Fushiki, T., and Fukuoka, S.-I. (1988). Pancreatic enzyme secretion mediated by novel peptide: monitor peptide hypothesis. Pancreas, 3:720. 33. Doell, B.H., Ebden, C. J.,and Smith, C.A. (1981). Trypsin inhibitor activity of conventional foods which are part of the British diet and some soya products. Plairf Foods Hmz. Nut r... 31:139. 34. Billings, P. C., Longnecker, M. P., Kearny, M., and Taylor, P. R. (1990). Protease inhibitor content of human dietary samples. Nutr. Cancer, 14:85. 35. Gunn, R. A., Taylor. P. R., and Gangarosa, E. J. (1980). Gastrointestinal illness associated with consumption of a soy protein extender. J. Food Protect., 43525. 36. Moroz, L. A. and Young,W. H. (1980). Kunitz soybean trypsin inhibitor. A specific allergen in food anaphylaxis. N. Engl. J. Med., 302:1126. 37. DiPietro. C. M. and Liener, I. E. (1989). Soybean protease inhibitors in foods. J. Food Sci., 54:606. 38. Rackis, J. J., McGhee. J. E., and Booth, A. N. (1975). Threshold levels of soybean trypsin inhibitors by rat bioassay. Cered Chern., 5285. 39a. Churella, H. R., Yao, B. C.,and Thomson, W. A. B. (1976). Soybean trypsin inhibitor activity of soy infant formulas and its nutritional significance for the rat. J. Agr. Food Chent., 24:393. 39b.Albrecht, W. J., Mustakas, G. C., andMcGhee, J. E. (1966).Ratstudies on atmospheric steaming and immersion cooking of soybeans. Cereal Clzem.. 43:400. 40. Collins, J. L. and Beaty, B. F. (1980). Heat inactivation of trypsin inhibitor in fresh green beans and physiological responses of rats fed the beans. J. Food Sci., 45542. 41. Jansen, G. R.,Harper, J. M.,and O’Deen, L. A.(1978).Nutritionalevaluation of full-fat soy flour produced by dry heat roasting. J. Food Sci., 43:1350. 42. Borchers, R., Manage, L. D., Nelson, S. O., and Stetson, L. E. (1972). Rapid improvement in nutritional quality of soybeans by dielectric heating. J. Food Sci., 37:331. 43a. Hafez, Y. S., Singh, G.. McLellan, M. E., and Monroe-Lord, L. (1983). Effect of microwave heating on nutritional quality of soybeans. Nutr. Rep. Int, 28:413. 43b. Rajko, R., Szabo. G., Vidal-Valverde, C., and Kovacs, E. (1997). Designed experiments for reducing antinutritive agents in soybeans by microwave energy. J. Agr. Food Clzern., 45: 3365. 44.Zhang, Y.. Parsons, C. M.,Weingartner,K. E., and Wijeratne, W. B. (1993). Effects of extrusion and expelling on the nutritional quality of conventional and Kunitz trypsin inhibitor-free soybean. Poultl? Sci., 72:2299. 45. Hafez, Y. S., Mohamed, A. I., Singh. G., and Hewedy. F. M. (1985). Effect of gamma irradiation on proteins and fatty acids of soybean. J. Food Sci., 50:1271. 46a. Kouzeh-Kamani, M., van Zuilichem, D. J., Roozen, J. P., Pilnik, W., van Delden, J. R., and Stolp, W. (1983). Infrared processing of soybeans. Qucrl. Plant. Plant Foods Hrtm. Nutr., 33:139. 46b. Ponne, C. T., Meijer, M. M. T., and Bartels, P. V. (1994). Effect of radio frequency energy on the activity of Bowman-Birk trypsin/chymotrypsin inhibitor. J. Agr. Food Chelrr.. 43: 2583. 47a. Johnson, L. A.. Deyoe, C. W., Hoover. W. J., and Schwenke, J. R. (1980). Inactivation of trypsin inhibitors in aqueous soybean extracts by direct steam infusion. Cered Chenz., 57: 376. 47b. Rouhana. A., Adler-Nissen, J., Cogan, U., and Frokaer, H. (1 996). Heat inactivation kinetics of trypsin inhibitors during high temperature short time processing of soymilk. J. Food Scj., 61 :265. 48.Birk,Y. (1961). Purificationandpropertiesof a highlyactiveinhibitorand chymotrypsin from soybeans. Biochinr. Bioyhys. Acta, 54:378.

Antinutritional Related Factors

to Proteins

289

49a. DiPietro, C. M. and Liener, I. E. (1989). Heat inactivation of the Kunitz and Bowman-Birk soybean protease inhibitors. J. Agr. Food Chertl, 37:39. 49b. Brandon, D. L., Bates, A. H., and Friedman, M. (1991). ELISA analysis of soybean trypsin inhibitors in processed foods. In Nutritional and To.xicologiccd Consequences of Food Processing (M. Friedman, ed.), Plenum Press, New York, p. 321. 50. Everson, G., Steenbock, H., Cederquist, D. C.. and Parsons, H. T. (1944). The effect of germination, the stage of maturity, and variety upon the nutritive value of soybean protein. J. Nutr.. 27:225. 51. Mattingly, J. P. and Bird, H. R. (1945). Effect of heating, under various conditions, and of sprouting on the nutritive value of soybean oil meals and soybeans. Poultly Sci., 24:344. 52. Liener. I. E. (1989). Control of antinutritive and toxic factors in oil seeds and legumes. In Food Uses of Whole Oil m c l Protein Seeds (E. W. Lusas, D. R. Erikson, and W.-K. Nip, eds.), American Oil Chemists Society, Champaign, IL, p. 344. 53. Desikachar, H. S. R. and De. S. S. (1950). Tryptic inhibitor and the availability of cystine and methionine in raw and germinated soya beans. Biochim. Biophys. Acta, 5:285. 54. Collins, J. L. and Sanders, G. G. (1976). Changes in trypsin inhibitory activity in some soybean varieties during maturation and germination. J. Food Sci, 41:168. 55. Bau, H. M. and Debry, G. (1979). Germinated soybean products: chemical and nutritional evaluation. J. Am. Oil. Chem. SOC..56:160. 56. Tan-Wilson, A. L., Rightmire, B. R., and Wilson, K. A. (1982). Different rates of metabolism of soybean proteinase inhibitors during germination. Plant Physiol., 70:493. 57. Harte. P. M., Tan-Wilson, A. L., and Wilson, K. A. (1986). Proteolysis of Kunitz soybean trypsin inhibitor during germination. Phytocltemistry. 2523. 58. Wilson, K. A., Papastoisis, G., Harte, P. M., and Tan-Wilson, A. L. (1988). Survey of proteolytic activities of the Kunitz tlypsin inhibitor and glycinin in germinating soybeans (Glycine mas) Plant Physiol., 88:355. 59. Gupta, Y. P. (1987).Anti-nutritional and toxic factors in food legumes: a review. Plant Foods H m . Nutr., 37201. 60a. Savelkoul, F. H. M. G., van der Poel. A. F. B., and Tamminga, S. (1992). The presence and inactivation of trypsin inhibitors, tannins, lectins, and amylase inhibitors in legume seeds during germination. A review. Plant Foods Hum. Nutr., 42:71. 60b. Bau, H. M., Villaume, C., Nicolas, J. P., and Mejcan, L. (1997). Effect of germination on chemical composition, biochemical constituents and antinutritional factors of soya bean (Glycilre n r m ) . J. Sci. Food Agr., 73:l. 61. Hackler, L. R., van Buren, J. P., Steinkrause, K. H., el-Rawi, I.. and Hand, D. B. ( 1965). Effect of heat treatment on nutritive value of soymilk protein fed to weanling rats. J. Food Sci., 30:723. 62. Chah, C. C., Carlson. C. W., Semeniuk, G., Palmer, I. S., and Hesseltine, C. W. (1975). Growth-promoting effects of fermented soybeans for broilers. Poultry Sci.. 54:600. 63. Reddy, N. R., Pierson, M. D., and Salunkhe, D. K. (1986). Legume-Based Fermeltted Foods. CRC Press, Boca Raton, FL. 64. Zamora, A. F. and Fields, M. L. (1979). Sensory evaluation and nutritive value of soups and chips made from fermented and unfermented beans. J. Food Sci., 44:930. 65. Zamora. R. G. and Veum, T. L. (1 979). Whole soybeans fermented with Aspergilhrs orycae and Rhizopus oligosporus for growing pigs. J. Anim. Sci, 48:63. 66. Rajalakshmi, R. and Vanaja, K. (1967). Chemical and biological evaluation of the effects of fermentation on the nutritive value of foods prepared from rice and grams. Br. J. Nutr., 21:467. 67. Zamora, A. F. and Fields, M. L. (1979). Nutritive quality of fermented cow peas (Vigrza sinensis) and chick peas (Cicer arietinunt). J. Food Sci, 44:234. 68. Mohamed, A. I. and Rangappa, M. (1992). Screening soybean (grain and vegetable) genotypes for nutrients and antinutritional factors. Plant Foods H u m Nutr.. 42:87.

290

Liener

69.

Kakade, M. L., Sirnons, N. R.. Liener. I. E., and Lambert. J. W. (1972). Biochemical and nutritional assessment of different varities of soybeans. J. Agr. Food Cl~er~z., 20:87. Orf, J. H. and Hymowitz, T. (1979). Inheritanceof the absenceof the Kunitz trypsininhibitor of soybeans. Crop Sci., 19:107. Hymowitz, T. (1983). Variation in and genetics of certain antinutritional and biologically active components of soybean seed. Cibn Fourzdution Syrtzp., 97:49. Liener, I. E. and Tomlinson, S. (1981). Heat inactivation of soybean line lacking the Kunitz trypsin inhibitor. J. Food Sci., 46:1354. Yen, J. T., Jensen, A. H., Hymowitz, T., and Baker, D. H. (1973). Utilization of different varieties of raw soybeans by male and female chicks. Poultry Sci., 521875. Yen, J. T., Hymowitz, T., and Jensen,A. H. (1974). Effects of soybeans of different inhibitor activities on performance of growing swine. J. Anim. Sci., 38:304. Friedman, M., Brandon, D. L., Bates. A. H., and Hymowitz, T. (1991). Comparison of commercial soybean cultivars and an isoline lacking the Kunitz trypsin inhibitor: composition, nutritional value and effects of heating. J. Agr. Food Clzern., 39:327. Birk, Y. (1968). Chemistryandnutritionalsignificanceof protease inhibitors from plant sources. A m . N. Y. Acacl. Sci., 146:388. Hobday, S. M., Thurman. D. A., and Barber, D. J. (1973). Proteolytic and trypsin inhibitory activities in extracts of germinating Pisum sati\w?t seeds. Phytoclzemisti?, 12: 1041. Lipke, H., Fraenkel, G. S., and Liener. I. E. (1954). Effect of soybean inhibitors on growth of Tribolium cortfuswz. J. Ayr. Food Chem., 2410. Birk, Y. and Applebaum, S. W. (1960). Effect of soybean trypsin inhibitors on the development and midgut proteolytic activity of Triboliunt crrstarrzmt larvae. Erzzvvmologia, 2 2 3 18. Ryan, C. A. (1978). Proteinase inhibitors in plantleaves: a biochemical model for pestinduced natural plant protection. Trends Biochem SOC.,5 :148. Mosolov. V. V. Loginova, M. D., Fedurkina, N. V., and Benken, I. I. (1976). Biological significance of proteinase inhibitors in plants. Plant Sci. Lett., 7:77. Shukle, R. H. and Murdock. L. L. (1983). Lipoxygenase, trypsin inhibitor, and lectin from soybeans: effects on larval growth of Mmduca sextrr (Leoiclptet-a:sphingidae). Erniroiz. Errtomol., 12787. Gatehouse, A.M.R.and Boulter, D. (1983). Assessmentoftheantimetabolic effects of trypsin inhibitor from cowpeaand other legumeson development of the bruchid beetle(Callobrzrclrus mnc~tlcrt~rs). J. Sci. Food Agr., 34:345. Gatehouse, A. M. R., Gatehouse, J. A.. Doble, P.. Kilminster. A. M., and Boulter, D. (1979). Biochemical basis of insect resistance in Vigrla za~guiculcrta.,J. Sci. Food Agr., 30:948. Wolfson, J. L. and Murdock, L. L. (1987). Suppression of Colorado potato beetle growth and development by digestive proreinase inhibitors. Etrtonzol. Evp. Appl., 43235. Oppert. B., Morgan, T. D., Culbertson. C.. and Kramer, K. J. (1993). Dietary mixtures of cysteine and serine proteinase inhibitors exhibitsynergistic toxicity toward the red flour beetle (Tribolizrm casterzemz). Conzp. Biochem. Physiol., 105C:379. Zhu, K. Y., Huessing, J. E., Shade, R. E.. and Murdock, L. L. (1993). Cowpeatrypsin inhibitor and resistence to cowpea weevil (Coleoptin bl?lC17id).Emiron. E17tOT?70/.,23:987. Health, R. L., McDonald, G.. Christeller, J. T., Lee,M., Bateman, K., West, J., Vanheeswijck, R., andAnderson, M. A. (1997). Protease inhibitors from Nicottiam alata enhance plant resistance to insect pest. J. Imect Plzyiol.. 43:833. Eckelkamp, C., Ehmann, M.B., and Shopfer, P. (1993). Wound-inducedsystemic accumulation of a transcript coding for a Bowman-Birktype trypsin inhibitor-related protein in maize (Zea m a y s L.) seedlings. FEBS Lett., 323:73. Liang, C..Brookhart. G., Feng, G. H.. Reeck. G. R., and Kramer. K. J. (1991). Inhibition of digestive proteinases of stored grain coleoptera by oryzacystatin. a cysteine proteinase inhibitor from rice seed. FEBS Lett.. 278:139. Broadway. R. M. and Duffy, S. S. (1986). Plant proteinase inhibitors: mechanism of action

70. 71. 72. 73. 74. 75.

76. 77. 78. 79. 80.

81. 82.

83.

83. 85a.

85b.

85c.

85d.

85e.

86.

87.

Antinutritional Factors Related to Proteins

291

and effect on growth and digestive physiology larval of Heliothis ,-ea and Spudoptera e.yiqun.

J. Insect Physiol., 32827. 88a. Laroque, A. M. and Houseman, J. G. (1990). Effect of ingested soybean, ovomucoid, and corn proteaseinhibitors on digestive processes of theEuropean cornborer. Ostrinia nubitalis (Lepidoptera, Pyralidae). J. Insect Physiol., 36:69 1. 88b. Burgess, E. P. J., Main, C. A., Stevens, P. S., Christeller, J. T., Gatehouse, A. M. R., and Laing, W. A. (1994). Effects of protease inhibitor concentration and combinations on the survival, growth. and gut enzyme. J. Imect Physiol., 40:803. 89. Hoffman, L.M., Sengupta-Gopalan, C.,and Paaren, H.E.,(1984). Structure of soybean KunitztrypsininhibitormRNAdetermined fromcDNA byusingoligodeoxynucleotide primer. Plarlt Mol. Biol., 3:111. 90. Hammond, R. W., Foard, D. E., and Larkins, B. A. (1984). Molecular cloning and analysis of a gene coding for the Bowman-Birk protease inhibitor in soybeans. J. Biol. Chenz., 259: 9883. 91. Joudrier, P. E., Foard, D. E., Floenor, L. A., and Larkins, B. (1987). Isolation and sequence of cDNAencoding thesoybeanproteinaseinhibitorPI-IVand C-11. Plant Mol.Biol., 10:35. 92. Flecker, P. (1987). Chemical synthesis, molecular cloning and expression of gene coding for a Bowman-Birk type proteinase inhibitor. Ew. J. Biochern., 166: 151. 93. Cleveland, T. E., Thornburg, R. W., and Ryan, C. A. (1987). Molecular characterization of a wound-inducible inhibitor I gene frompotato and the processing of its mRNA and protein. Plant Mol. Biol.. 8:199. 94. Sanchex-Serrano. J. J.. Keil, M.. O’Connor, A., Schell, J., and Willmitzer, L. (1987) Woundinduced expression of a potato proteinase inhibitor I1 gene in transgenic plants. EMBO J. 6: 303. 95. Wingate, V. P. M. and Ryan, C. A. (1991). Uniquely regulated proteinase inhibitor-I family gene is wound inducible in leaves and developmentally regulated in fruit. Plant Physiol., 97:496. 96. Abe, K., Emori, Y.,Kondo, H., Suzuki, K., andArai, S. (1987). Molecular cloningof a cysteine proteinase inhibitor of rice (oryzastatir). J. Biol. Clzem., 262: 16793. 97. Williamson, M. S., Forde, J.,Buxton, B., and Kreis,M. (1987) Nucleotide sequence of barley chymotrypsin inhibitor-2 (CI-2)and its expression in normal and high-lysine barley. E m . J. Biochem., 165:99. 98. Hilder, V. A., Gatehouse. A. M. R., Sheerman, S. E.. Barker, R. F., and Boulter, D. (1987). A novel mechanism of insect resistance engineered into tobacco. Nature, 330:160. 99a. Johnson, R.,Narvaez, J., An, G.. and Ryan, C. A. (1989). Expression of proteinase inhibitors I and I1 in transgenic tobacco plants: effects on naturaldefense against Mnnduca sextu larvae. Proc. Ncrtl. Accrd. Sci. USA, 86:9871. 99b. Boulter, D. ( 1993) Insect pest control by copying nature using genetically engineered crops. Phytochernistgl, 34: 1453. 100. Rackis, J. J.. Wolf, W. J., and Baker, E. C. (1986). Proteaseinhibitors in plant foods: content and inactivation. InNutritional and To.uicologica1SigniJicance of Enzyme Inhibitorsin Foods (M. Friedman., ed.), Plenum Press, New York. p. 299. 101. Brandon, D. L., Haque, S., and Friedman, M. (1987). Interaction of monoclonal antibodies with soybean trypsin inhibitors. J. Agr. Food Chenr., 35:195. 102. Brandon, D. L., Bates, A. H., and Friedman, M. (1988). Enzyme-linked immunoassay of soybean Kunitz trypsin inhibitor using monoclonal antibodies. J. Food Sci.. 53:102. 103. Brandon, D. L.. Bates, A. H., and Friedman. M. (1989). Monoclonal antibody-based enzyme immunoassay of the Bowman-Birk inhibitor of soybeans. J. Agr. Food Chern., 37: 1192. in 104. Roozen. J. P. and Groot, J. (1987). Analysis of low levels of trypsin inhibitor activity food. Lebertsnz.- Wiss. u . -Teclznol., 20:395. Roozen. J. P. and Groot. J. (1991). Analvsis of trvtxin inhibitors and lectins in white kidney

292

106.

107. 108. 109.

110.

111. 112. 113. 114. 115.

116.

117

118.

119. 120.

121.

122. 123. 124. 125. 126.

Liener beans (Pl1aseolus~w/gccris,var. processor) in a combined method. J. Assoc. OffA d . Chem., 74940. Buonocore, V. and Silano, V. (1986). Biochemical, nutritional, and toxicological aspects of alpha-amylase inhibitors from plant foods, In Nutritiortal and Toxicological Significance of Enzyme Inhibitors in Foods (M. Friedman, ed.), Plenum Press, New York, p. 483. Saviano, D. A., Powers, J. R., Costello, M. J., Whitaker. J. R., and Clifford, A. J. (1977). Effect of an alpha-amylase inhibitor on growth rate of weanling rats. Nratr. Rep. Int., 15:443. Granum, P. E. and Eskeland, B. (1981). Nutritional significance of alpha amylase inhibitors from wheat. Nzrtr. Rep. Int, 23:155. Macri, A., Parlamenti, R., Silano, V., and Valfre, F. (1977). Adaptation of the domestic chicken, Gallus do~nestic~~s, to continuous feeding of albuman amylase inhibitors from wheat flour as gastro-resistant microgranules. Poultry Sci.. 56:434. Bo-Linn, G. W., Santa Ana, C. A., Morawski, S. G., and Fordtran, J. S. (1982). Starch blockers-their effect on caloric absorption froma high-starch meal. N. Erzgl. J. Med., 307: 1413. Carlson, G. L., Li, B. U. K., Bass, P., and Olsen. W. A. (1983). A bean alpha-amylase inhibitor formulation (starch blocker) is ineffective in man. Science, 219393. Garrow, J. S., Scott, P. F., Heels, S., Nair, K. S., and Halliday, D. (1983). A study of “starch blockers” in man using ‘jC-enriched starch as a tracer. Hum. Nutr. Clirz. Nzm., 37301. Granum, P. E., Holm, H., Wilcox, E., and Whitaker, J. R. (1983). Inhibitory properties of two commercially available starch blocker preparations. Nrctr. Rep. bzt., 28: 1233. Liener, I. E., Donatucci, D. A., and Tarcza, J. (1984). Starch blockers: a potential source of trypsin inhibitors and lectins. An?. J. Clin. Nutr., 39:196. Layer, P., Carlson, G. L., and DiMagno, E. P. (1985). Partially purified white bean amylase inhibitor reduces starch digestion in vitro and inactivates intraduodenal amylase in humans. Gastroenterology, 88: 1895. Garcia-Olmedo, F., Salcedo, G., Sanchez-Monge, R., Gomez, L., Royo, J., and Carbonero, P. (1987). Plant proteinaceous inhibitors of proteinases and alpha-amylases. Oxford Sur-v. Plant Mol. Cell Biol., 4:275. Gatehouse, A. M. R., Fenton, K. A., Jepson, I., and Pavey, D. J. (1986). Theeffects of alphaamylase inhibitors on insect storage pests: .inhibition of alpha-amylase in vitro and effects on development in vivo. J. Sci. Food. Agr., 37:727. Ishimoto, M. and Kitamura, K. (1989). Growth inhibitory effects of an alpha-amylase inhibitor from the kidney bean (Plzuseolrrs vztlgrrrisL.) on three species of bruchids (Coleoptera: Brmhidae). Appl. Eiztonzol. Zool.. 24:28 1. Janzen, D. H., Juster, H. B.. and Liener. I. E. (1976). Insecticidal action of a phytohemagglutinin in black beans on a bruchid beetle. Science. 192:795. Gatehouse, A. M. R., Dewey, F. M., Dove, J., Fenton, K. A., and Pusztai, A. (1984). Effect of seed lectins from Phaseolus ~~u/gnris on the development of larvae of Crrllobrrrchusnzaculatus: mechanism of toxicity. J. Sci. Food. Agr., 35:373. Huessing, J. E., Shade, R. E., Chrispeels, M. J., and Murdock, L. L. (1991) Alpha-amylase inhibitor, not phytohemagglutinin, explains resistance of common bean seeds to cowpea weevil. Plant Plzysiol., 96:993. Altabella, T. and Chrispeels, M. J. (1990). Tobacco plants with bean alpha-ai-gene express an inhibitor of insect alpha-amylase in their seeds. Plant Phpiol., 93:805. Boyd, W. C. and Shapleigh, E. (1954). Specific precipitating activity of plant agglutinins (lectins). Science, 119:419. Sharon. N. and Lis. H. (1989). Lectins as cell recognition molecules. Science, 246:227. Liener, I. E., Sharon. N. and Goldstein, I. J. (eds.) (1986). The Lectins. Properties, F~rnctions, and Applications in Biology and Medicine, Academic Press, New York. Goldstein, I. J. and Poretz, R. D. (1986). Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins. In The Lectins. Properties. Fz4nctiorzs, and Appli-

Proteins Antinutritional to Related Factors

127.

128. 129. 130.

131a. 131b.

131c. 132. 133. 134.

135. 136.

137. 138. 139. 140. 141. 142. 143. 144. 145.

146.

293

cntiom in Biology and Medicine (I. E. Liener, N. Sharon, and I. J. Goldstein, eds.), Academic Press, New York, p. 35. Strosberg, A. D., Buffard, D.. Lauwereys, and Foriers, A. (1986). Legume lectins: a large family of homologous proteins, In The Lectins, Properties, Functions, arzd Applications in Biology and Medicine (I. E. Liener. N. Sharon, and I. J. Goldstein, eds.), Academic Press, New York, p. 25 1. Wright, C. S. (1977). The crystal structure of wheat germ agglutinin at 2.2 resolution. J. Mol. Biol., 111:439. Meehan, E. J., Jr., McDuffie, J., Einspahr, H., Bugg. C. E., and Suddath, F. L. (1982). The crystal structure of pea lectin at 6 Ao resolution. J. Bid. Chem., 257:13278. Salunke, D. M., Swamy, M. J.. Khan, M. I., Mande, S. C., Surolla. A., and Vijayan, M. (1985). Arrangement of subunits in peanut lectin. Rotation function and chemical crosslinking studies. J. Biol. Chem., 260: 13576. Swamy. M. J. and Surolia, A. (1988). The quatemary structure of soybean agglutinin: chemical cross-linking studies. Indian J. Biochem. Biophys., 25:566. Desenm A.. Gupta, D., Sabesan, S., Brewer, C. F., and Sacchettini, J. C. X-ray crystal structure of the soybean agglutinin crosslinked with a biantennary analog of the blood group I carbohydrate antigen. Biochemistry, 34:4933. Loris, R., (1992). Two crystal forms of the lentil lectin diffract to high resolution. J. Mol. Biol., 223:579. Sharon, N., Lectin-carbohydrate complexes of plants and animals-an atomic view. Trends Biochem. Sci., 18221. Shaaran, B., Lis, H., and Sharon, N. (1991). Structure of a legume lectin with an ordered N-linked carbohydrate in complex with lactose. Science, 254:862. Stillmark (1888). Uber Ricin; Ein Giftiges Ferment aus den Samen von Ricinus corlzrnunis und eigen andererz Euyhoribinceen. Dissertation, University of Dorpat (now Tartu, Estonia). Landsteiner, K. and Raubitschek, H. (1908). Beobachtungen uber Hamolyse und Hemagglutination. Zent. Bakteriol. Pamsititenk.. 45:660. Liener. I. E. (1986). Nutritional significance of lectins in the diet, In The Lectins. Properties, Fzlrzctions, and Applications 1i7 Biology and Medicine (I. E. Liener, N. Sharon, and 1. J. Goldstein. eds), Academic Press, New York, p. 527. Nachbar, M. S. and Oppenheim, J. 0. (1980). Lectins in the US diet: a survey of lectins in commonly consumed foods and a review of the lierature. Am. J. Clin. Nzltr., 332338. DeMuelenaere. H. J. H. (1964). Effect of heat treatment on the hemagglutinating activity of legumes. Nature, 201: 1029. Brady, P. G., Vannier, A. M., and Banwell, J. G. (1978). Identification of the dietary lectin, wheat germ agglutinin, in human intestinal contents. Gastroenterology, 75236. Griebel, C. (1950). Erkrankunungen durch Bohnenflochen (Phaseolzas\wlgaris L.) und Plattererbsen (Lathyrus tigitanus L.). 2. Lebertsm. Unterszd?,Forsch., 90: 191. Korte, R. (1972). Heat resistance of phytohemagglutinins in weaning food containing beans (Phaseolus vulgaris). Ecol. Food Nratr., 1303. Noah, N. D., Bender, A. E., Reaidi, G. B., and Gilbert, R. J. (1980). Food poisoning from raw red kidney beans. Br. Med. J., 281:236. Bender, A. E., and Reaidi, G. B. (1982). Toxicity of kidney beans (Phaseohls Iwlgaris) with particular reference to lectins. J. Plant Foods, 4:15. Bender, A. E. (1983). Hemagglutinins (lectins) in beans. Food Chew., 11:309. Grant, G., More, L. J., McKenzie, N. H.. and Pusztai, A. (1982). The efect of heating on the hemagglutinating activity and nutritional properties of bean (Phaseolus vulgaris) seeds. J. Sci. Food Agr., 33:1324. Thompson, L. U., Rea, R. L., and Jenkins, D. J. A. (1983). Effect of heat processing on hemagglutinin activity in red kidney beans. J. Food Sci., 48:235.

A

294 147. 148.

149.

150. 151. 152.

153.

154.

155. 156.

157. 158.

159.

160. 161. 162a.

162b.

163.

164.

165.

Liener Coffey, D. G., Uebersax, M. A.. Hosfield, G. L., and Brunner, J. R. (1985). Evaluation of hemagglutinating activity of low-temperature cooked kidney beans. J. Food Sci., 50:78. Lowgren, M. and Liener, I. E. (1986). The effect of slow cooking on the trypsin inhibitor and hemagglutinating activities and in vitro digestibility of brown beans (Phaseolzrsvulgcrris, var. Stella) and kidney beans (Pltcrseolrls twlgaris, var. Montcalm). Qrral. Plant. Plant Foods H1mt. Nratr., 36: 147. Jaffk. W. G., Planchert, A., Paez-Pumar, J. I., Torrealba, R.. and Franceschi, D. N. (1955). Nuevos esterdios sobre un factor toxico de las caraotas crudas (Phaseolus Idgaris). Arch. Venez. Nutr., 6: 195. Etzler, M. E. (1979). Lectins as probes in studies of intestinal glycoproteins and glycolipids. Am. J. Clin. Nrrtr., 32:133. King, T. P., Pusztai, A., and Clarke. E. M. (1980). Immunocytochemical localization of ingested kidney bean (Phaseolzrs ~wlgaris)lectins in rat gut. Histochenr. J., 12901. King. T. P.. Pusztai, A., and Clarke. E. M. W. (1982). Kidney bean (Phaseohrs ~~u1grrr-i~) lectin-induced lesions in rat small intestine. 3. Ultrastructure studies. J. Conrp. Pcrthol., 92: 357. King, T. P., Begbie, R., and Cadenhead. A. (1983). Nutritional toxicity of raw kidney beans in pigs. Immunocytochemical and cytopathological studies on the gut and the pancreas. J. Sci. Food Agr.. 34: 1404. Banwell, J. G., Howard, R.. Cooper, D., and Costerton, J. W. (1985). Intestinal microbial flora after feeding phytohemagglutinin lectins (Plzaseolzrs Idgaris) to rats. Appl. Environ. Microbiol., 50:68. Jaffk, W. G. and Comejo, G. (1961 ). The action of a toxic protein from the black bean (Phrrseolus Iwlgaris) on the intestinal absorption in the rat. Acta Cient. Vene:., 1259. Donatucci, D. A., Liener, I. E.. and Gross, C. J. (1987). Binding of navy bean (Phaseolus \mlgrrris) lectin to the intestinal cells of the rat and its effect on the absorption of glucose. J. Nutr., 1172154. Kawatra, B. L. and Bhatia. I. S. (1979). Isolation and characterization of glycoprotein from field pea (Pisrcnr aevertse L.). Biochem. Physiol. Pjnn:., 174283. Dhaunsi, G. S., Garg. U. C., Sidhu. G. S., and Bhatnagar, R. (1985). Enzymatic and transport studies on the rat intestinal brush border membrane vesicles bound to pea and lentil lectins. IRCS Med. Sci.. 13:469. Banwell, J. G., Boldt, D. R., Meyers, J., Weber. F. L.. Jr.. Miller, B., and Howard. R., (1983). Phytohemagglutinin derived from red kidney bean (Plmeolzrs tdgnris):a cause for intestinal malabsorption with bacterial overgrowth in the rat. Gastroenterology. 84506. Dobbins, J. W., Laurenson. J. P., Gorelick, F. S., and Banwell. J. G. (1983). Effect of phytohemagglutinin (PHA) on ion transport in rabbit ileum. Gastroenterology. 83:1138. Rouanet. J.-M., Besancon, P., and F Lafont, J. (1983). Effect of lectins from leguminous seeds on rat duodenal enterokinase activity. Esyerientia, 39: 1356. Lasheras. B., Cenamzabeitia, M. N., Fontan, J., Lluch, M.. and Larralde, J. (1980). Effect of raw legume diets on disaccharidase activity in the small intestine of chicks. Rei,. Espmr. Fisiol., 36:331. Grant, G., Watt, W. B., Stewart, J. C.. and Pusztai. A., Effects of dietary soybean (Glycine mas) lectin and trypsin inhibitors upon the pancreas of rats. Med. Sci. Res., 15:1197. Jordinson, M., Deprez. P. H., Playford, R. J., Heal. S., Freeman, T. C., Alison. M., and Calam, J. Soybean lectin stimilates pancreatic exocrine secretion via CCK-A receptors in rats. A ~ IJ.. Physiol., 33:G653. Hewitt, D., Coates, M. E., Kakade. M. L., and Liener, I. E. (1973). A comparison of fractions from navy bean (Phaseolus vrdgnris) in diets fed germ-free and conventional chicks. Br. J. Nutr., 29:423. Rattray, E. A. S.. Palmer, R., and Pusztai, A. (1974). Toxicity of kidney beans (PhnseoZzls vrrlgcrris) to conventional and gnotobiotic rats. J. Sci. Food Agr., 25:1035.

Antinutritional Factors Related to Proteins 166.

167.

168. 169.

170.

171.

172. 173.

174. 175a.

175b. 176.

177. 178. 179. 180. 181.

182. 183. 184. 185.

295

Jayne-Williams. D. J. (1973). The influence of dietary jack beans (Canavalin ensifornzis) and of concanavalin A onthe growth of conventional and gnotobiotic Japanese quail (Coturnix cotzrrnixjcrponica). Nature New Biol.. 2431150. Begbie, R., and King, T. P. (1985). The interaction of dietary lectin with porcine small intestine and production of lectin-specific antibodies. In Lectins. Biology. Bioclrenzistn, Clinical Biochernistt?, Vol. 4, (T. C. Bog-Hansen and Breborowicz, J., eds.), Walter de Gruyter, Berlin, p. 15. Grant, G., Greer, F., McKenzie, N. H., and Pusztai, A. (1985). Nutritional response of mature rats to kidney bean (Phaseolus ~?ulgc~i-is) lectins. J. Sci. Food Agr., 36:409. Pusztai, A. (1989). Effects on gut structure, function, and the metabolism of dietary lectins. The nutritional toxicity of the kidney bean lectin. In Advances in Lectirz Research (H. Franz, ed.), VEB Verlag Volk und Gesundheit, Berlin, p. 74. Etzler, M. E. (1986). Distribution and function of plant lectins. In The Lectins. Properties, Functions and Applications in Biology nncl Medicine (I. E. Liener, N. Sharon, and I. J. Goldstein, eds.), Academic Press, New York, p. 371. Diaz, C. L., Melchers, L. S., Hooykaas. P. J. J., Lugtenberg, B. J. J., and Kijne, J. W. (1989). Root lectin as determinant of hosy-plant specificity in the Rhi,-obium-legumesymbiosis. Nature, 3381579. Chrispeels, M. J. and Raikhel. N. V. (1991). Lectins, lectin genes, and their role in plant defense. Plant Cell, 3: 1. Gatehouse, A. M. R., Dewey, G. M., Dove. J., Fenton, K. A., and Pusztai, A. (1984). Effect of seed lectins from Plzaseolzrs vztlgclris on the development of larvae of Callobruclzus ntaczilatus; mechanism of toxicity. J. Sci. Food Sci., 35:373. Osborne, T. C.,Alexander, D. C., Su,S. S. M., Cardona, C., and Bliss, F. A. (1988). Insecticidal activity and lectin homology of arcelin seed protein. Science, 340207. Rouge. P., Barre, A., Causse, H., Chatelain, C., and Porthe, G. (1993). Arcelin and amylase inhibitor from the seeds of common bean (PhcrseolusIdgaris L.) are truncated lectins. Biochent. System. Eco., 21:695. Edwards, G. A., Hepher, A., Clerk, S. P., and Boulter, D. (1991). Pea lectin is correctly processed, stable, and active in leaves of transgenic potato plants. Plarzt Mol. Biol. 17:89. Boulter. D., Edwards, G. A., Gatehouse, A. M. R., and Hilder, V. A. (1990). Additive protective effects of different plant-derived insect resistant genes in transgenic plants. Crop Protect., 9:35 1. Pistole, T. G. (1981). Interaction of bacteria and fungi with lectins and lectin-like substances. Anrlu. Rev. Microbiol., 35185. Callow, J. A. (1977). Recognition, resistance, and the role of plant lectins in host-parasite interactions. Adv. Bot. Res., 4: 1. Sequeira, L. (1978). Lectins and their role in host-pathogen specificity. Annu. Rev. Phytopnthol., 16:453. Liener, I. E. (1955). A photometric method for measuring the hemagglutinating activity of soyin and crude soybean extracts. Arch. Biochent. Biophys., 54:223. Hendlicks, H. G. C. J. M., Koninkx, J. F. J. G., Draaijer, M., van Dijk, J. E., Raaijmakers, J. A. M., and Mouwen, J. M. V. M. (1987). Quantitative determination of the lectin-binding capacity of small intestinal brush-border membrane. An enzyme-linked sorbent assay (ELLSA). Biochirlz. Biophys. Acta, 905:371. Natarajan, K. R. (1976). India’s poison peas. Chernistty, 49:12. Attal. H. C., Kulkami, S. W., Chowbey, B. S.. Palkar, N. D., and Deotale, P. G. (1978). A field study of lathyrism-some clinical aspects. Indian J. Med. Res., 67:608. Spencer, P. S. and Schaumberg, H. H. (1983). Lathyrism: a neurotoxic disease. Neurobehav. Toxicol. Teratol.,5:625. Lewis, H. B., Fajans, R. S., Esterer. M. B., Shen, C., and Oliphant, M. (1948). The nutritive value of some legume. Lathyrism in the rat. J. Nzrtr., 36:537.

296

Liener

186. Bachhuber. T. E., Lalich, J. J., Angevine, D. M., Schilling, E.D., and Strong, F. M. (1955). Lathyrus factor activity of beta-aminopropionitrile and related compounds. Proc. SOC.E.xp Biol. Med., 89:294. 187. Adiga, P. G., Padmanaban, G., Rao, S. L. N., and Sarma, P. S. (1962). The isolation of a toxic principle from Latlrynrs sativus. J. Sci. Indust. Res. India, 21284. 188. Murti, V. V. S., Seshadri, T. R., and Venkitasubramanian, T. A., (1964). Neurotoxic compounds of the seeds of Lathyus sativus. Plzytochenzistr?, 3:73. 189. Rao. S. L. N., Adiga, P. R., and Sarma, P. S. (1964). The isolation and characterizationof beta-N-oxalyl-alpha, beta-diaminopropionic acid: a neurotoxin from the seeds of Lathyrus sativus. Biochenzistt?, 3:432. 190. Mehta, T., Zarghami, N.S., Parker, A. J., Cusick, P.K., and Haskell, B.E. (1979). Neurotoxicity of orally or intraperitoneally administeredN-3-oxalyl-3,,3-diaminopropionic acid in the mouse. Toxicol. Appl. Pharnzacol., 48: 1. 191a. Mehta, T., Parker, A. J., Cusick, P. K., Zarghami, N. S., and Haskell, B. E. (1983). The Lathvrzls sativus neurotoxin: resistance of the squirrel monkey to prolongedand high doses. Toxicol. Appl. Pharmacal., 69:480. 191b. Ressler, C.. Nigam, S. N., Giza, Y.-H., and Nelson, J. (1963). Isolation and identification from common vetch of y-L-glutamyl-P-cyano-L-alanine, a bound form of the neurotoxin P-cyano-L-alanine.J. Am. Cltenz. SOC.,85:3311. 191c. Ressler, C., Nigam, S. N., and Giza, Y.-H. (1969). Toxic principle in vetch. Isolation and identification of y-L-glutamyl-L-P-cyanoalanine from common vetch seeds. Distribution in some legumes. J. Ant. Clzenf. SOC.,91:2758. 191d. Ressler, C., Tatake, J. G., Kaizer, E., and Putnam, D. H. (1997). Nerotoxins in vetch foods: stability to cooking and removal of y-glutamyl-P-cyanoalanine and cyanoalalnine and acute toxicity from common vetch (Vicia sativa L.) legumes. J. Agr. Food Clzem., 45:189. 192. Vega, A. and Bell, E. A. (1967). Alpha-amino-beta-methylaminopropionic acid, new a amino acid from seeds of Cycas circinalis. Phytochemistry, 6:759. 193. Spencer, P. S.. Nunn, P. B., Hugon,J., Ludolph, A. C.. Ross,S. M., Roy, D, N.,and Robertson, R. C. (1987). Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant neurotoxin. Science, 237517. 194. Spencer, P. S.. Allen, R. G.. Kisby, G. E., and Ludolph, A. C. (1990). Excitotoxic disorders. Science, 248: 144. 195. Kean, E. A. (1989). Hypoglycin. In Toxicants of Plant Origin, Vol. I11 (P.R. Cheeke, ed.), CRC Press, Boca Raton, FL., p. 229. 196. Tanka, K., Isselbacher, K. J., and Shih, V.(1 972). Isovalericand alpha-methylbutyric acidemia induced by hypoglycin A: mechanism of Jamaican vomiting sickness. Science, 175: 69. 197. Persaud, T. V. N. (1968). Teratogenic effects of hypoglycin A. Nature, 217:471. 198. Vietmeyer, N., Cotton, B., and Ruskin, F. R. (1977). Leucaena, Pronrising Forage and Tree Crop for the Tropics, National Academy of Sciences, Washington, DC. 199a. Hegarty. M. P.. Court, R. D.. Christie, G. S., and Lee, C. P. (1976). Milnosine in Lemaena leucoplzala is metabolized to a goltrogen in ruminants. Aust. Vet. J., 52490. 199b. Hallengrein, B., Hegarty, M. P. Forsgren, A., Ericson, L. E., and Melander, A. (1987). 3,3Dihydroxypyridine: a potential antithyroid drug. Acta Emlocrinol., 114:305. 199c. Lalitha, K., Vargheese, C. M.,and Balasubramanian, N (1993). Spectrophotometric determination of mimosine and 3-hydroxyy-4-( 1H)-pyridine, the toxic principles of Leucaena leucocephala. Anal. Chem., 21357. 199d. Jones, R. J.and Megarrity, R. G. (1986). Successful transfer of DHP-degrading bacteria from Hawaiian goats to Australian ruminants to overcome the toxicity of Leucaenn. ALrst. Vet. Res. J., 63259. 200. Owen, L. N. (1958). Hair loss and other toxic effects of LetlcaentlJlazrca (“jumbey”). Vet. Rec., 70:454.

Antinutritional Factors Related to Proteins 201. 202. 203.

204.

205. 206. 207. 208.

209. 210. 21 1.

212. 213. 214.

215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225.

297

Hegarty, M. P., Schinklel. P. G., and Court, R. D. (1964). Reactionof sheep to the consumption of Leucaena glauca Benth. and to its toxic principle. Aust. J. Agr. Res., 15:153. Reis, P. J., Tunks, D. A., and Hegarty, M. P. (1975). Fate of mimosine administered orally to sheep and its effectiveness as a defleecing agent. Aust. J. Biol. Sci., 28:495. Van Veen, A. G. (1966). Toxic properties of some unusual foods. In Toxicants Occurring Naturally in Foods. Publ.1354,Nat.Acad. Sci. Natl.Res.Council,Washington,DC, p. 176. Lin, K.-C., and Tung, T.-C. (1966). Biochemical studyof nlimosine. 111. Comparative study of the activities of metal-containing enzymes in B,-deficient and mimosine-fed rats. J. Forrnosan Med. Assoc., 65:119. Crounse, R. G., Maxwell, J. D., and Blank, H., (1962). Inhibition of growth of hair by mimosine. Nature, 194:694. Hylin, J. W. (1969). Toxic peptides and anlino acids in foods. J. Agr. Food Chem., 17:492. Montagna, W. and Yun, J. S . (1963). The effects of the seeds of Laecaena glauca on the hair follicles of the mouse. J. Inlvest. Dermztol., 40:325. Matsumoto, H., Smith,E. G., and Sherman, G. D. (1951). The effect of elevated temperatures on the mimosine content and toxicity of kao haole (Leucaena glutlca). Arch Biochem. Bioyhys., 33201. Yoshida, R. K. (1944). A chemical and physiological study of the nature and properties of the toxic principle in Leucaenn glauca (kao haole). Ph.D. thesis, University of Minnesota. Van Veen, A. G. and Latuasan, H. E. (1949). The state of djenkolic acid in the plant.Chron. Natur., 105288. Van Veen, A. G. and Hyman, A. J. (1933). On the toxic component of the djenkol bean. Geneeskd. Tydschr. Ned. Indie, 73:991. Torquati. T. (1913). Presence of a nitrogenous substance in the seeds of Vicia faha. Arch. Farm. Sper., 15:213. Guggenheim, M. (1913). Dihydroxyphenylalanine, a new amino acid from Vicia faba. Z. Physiol. Clzem., 88276. Nagasawa, T., Takagl, H., Kawakami, K., Sukuki, T., and Sahashi, Y. (1951). Studies on the browning productsof broad bean, Vicia fabu L. 111. Isolation of dopa-0-beta-D-glucoside and enzymic mechanism for the color change of broad bean. Agr. Biol. Chem., 25:441. Andrews, H. V. and Pridham, J. B. (1965). Structure of a dopa glucoside from Viciclfabn. Nature, 205:1213. Miller, E. R. (1920).Dihydroxyphenylalanine,aconstituent of thevelvetbean. J. Biol. Client., 44:481. Hoeldtke. R., Baliga, B. S . , Issenberg, P., and Wurtman, R. J. (1972). Dihydroxyphenylalanine in rat food containing wheat and oats. Science, 175:761. Kosower, N. S. and Kosower, E. M. ((1967). Does 3,4-dihydroxyphenylalanineplay a part in favism? Nature, 215:285. Davies, J. P.H. and Gower,D.B.(1964).Conditionsaffectingtheglucose-6-phosphate dehydrogenase activity and glutathione stability of human erythrocytes. Nature, 203:310. Natelson, B. H. (1969). Beans, a source of L-dopa. Lancet, 3:640. Cheeke, P. R. and Shull, L. R. (1985 ). Nnt~4ral Toxicantsin Feeds and Poisonous Plants. Avi Publishing Co.. Westport, CT, p. 265. Rosenfield, I. and Beath, 0. (1964). Seleniurn. Academic Press, New York. Aronow. L and Kerdel-Vegas, F. (1 965). Seleno-cystathionine, a pharmacologically active factor in the seeds of Lecythis ollaria. Nature, 205: 1185. Hegarty, M. P. and Pound, A. W. (1968). Indospicine, a new hepatoxic amino acid from Indigo spicnta. Nutwe, 2 17:354. Christie, G. S., Wilson, M., and Hegarty, M. P. (1975). Effects on the liver in the rat of ingestion of Indigofera spicata, a legume containingan inhibitor of arginine metabolism. J. Pnthol., 117195.

298

Liener

Pearn, J. H. (1967). Studies of a site-specific cleft palate teratogen-the toxic extract from Imligofera spiccltcr Forssk. Br. J. Exp. Patlrol., 48:620. 227 a. Rosenthal. G. A. (1991). Nonprotein amino acids as protective allochemicals. In Herbivores. Their Interaction nitlt Secondar??Plant Metabolites (G. A. Rosenthal and M. R. Berenbaum. eds.), Academic Press, New York, p. 10. 227b. Rosenthal, G. A., Dahlman, D. L., Crooks, P. A., Phukey, S. N., and Trifonov, L. S. (1 995). Insecticidal properties of some derivatives of L-canavanine. J. Agr. Food Cherlz., 43:2728. 228. Malinow, M. R., Bardana, E. J., Jr., Pirofsky, B., Craig, S., and McLaughlin, P. (1982). Systemic lupus erythematosus-like syndrome in monkeys fed alfalfa sprouts: role of a nonprotein amino acid. Science, 216:415. 229. Kratzer, F. H. (1947). Effect of duration of water treatment on the nutritive value of linseed meal. Pozrltn, Sci., 26:90. 230. Kratzer, F. H., Williams, D. E., Marshall, B., and Davis, P. N. (1954). Some properties of the chick growth inhibitor in linseed oil meal. J. Nutr.., 52:555. 231. Klosterman. H. J., Lamoureux, G. L., and Parsons, J. L. (1967). Isolation, characterization, and synthesis of linatine. A vitamin B6 antagonist from flaxseed (Limulz usitatissirnunt). Biochernistr?,, 6: 170. 232. Sasaoka, K., Ogawa, T., Moritiki. K., and Kimoto, M. (1976). Antivitamin B, effect of 1anlinoproline in rats. Biochirn. Biophys. Acta, 428:396. 233. Klosterman, H. J. (1981 1. Vitamin B6 antagonists in natural products. In Antinzltrients and Natural Toxicants inFoods (R. L. Ory, ed.), Food and Nutrition Press, Westport, CT. p. 295. 234. DeGgroot, A. P. and Slump. P. (1969). Effect of severe alkali treatment of proteins on amino acid composition and nutritive value. J. Nzrtr., 98:45. 235. Van Beck. L., Feron, V. J., and de Groot, A. P. (1974). Nutritional effects of alkali-treated soy protein in rats. J. Nzrtr., 194:1630. 236. Woodard, J. C . , Short, D. D., Alvarez, M. R., and Reyniers, J. (1975). Biologic effects of N-epsilon-(m-2-anlino-2-carboxyethyl)-L-lysine, lysinoalanine. In Protein Qualit?,of Foocls a d Feeds (M. Friedman, ed.), Marcel Dekker, New York. p. 595. 237. Sternberg, M., Kim, C. Y., and Schwende, F. J. (1975). Lysinoalanine: presence in foods and food ingredients. Science, 190:992. 238. Langhendries, J. P., Hurrell. R. F., Furniss, D. E., Hischenhuber, C., Finot. P. A., Bernard, A., Battisti, O., Bertrand, J. M., and Senterre, J. (1992). Maillard reaction products and lysinoalanine: urinary excretion and the effects on kidney function of preterm infants fed heatprocessed milk formula. J. Pediatr. Gastroenterol. Nutr., 14:62. 239. Stratton, J. E., Hutkins, R. W., and Taylor, S. L. (1991). Biogenic amines in cheese and other fermented foods. A review. J. Food Prof., 54:460. 240. Brink, B., Dammink, C., Joosten, H. M. L. J., and Huis in’t Veld (1990). Occurrence and formation of biologically active amines in foods. Int. J. Food Microbiol., 11:73. 241. Blackwell. B., Marley, E., Price, J., and Taylor, D. (1967). Hypertensive interaction between monoamine oxidase inhibitors and foodstuffs. Br. J. Psychiaty, 113:349. 242. Taylor. S. A., Shulman, K. I., Walker, S. E., Moss, J., and Gardner, D. (1994). Hypertensive episode associated with phenylzine and tap beer. A reanalysis of the role of pressor amines in beer. J. Clin. Ps?~clzopl~ar?nacol., 14:5. 226.

6 Glycosides Walter Majak Agriculture and Agri-Food Canrrda, Krmtloops, British Colwnbin. Canada

Michael H. Benn University of Calgar?. Culgay, Alberta. Canada

I.

Background

300

Hemiacetals, acetals, and esters 300 B. Hydrolysis of the glycosidic bond 301 C. Inhibitors of P-glucosidase 303

A.

11. HistoricalAspects.Distribution,and

Chemistry 304

A. Aliphatic nitrocompounds 304 B. Cyanogenic glycosides 305 308 C. Cardiac glycosjdes 309 D. Saponins 3 E. Glucosinolates 12. F. Diterpenoid glycosides 3 13 G. Brackenand other sesquiterpeneglycosides glycosides 3 15 H. Calcinogenic 3 I. Phenolic glycosides 16 3 18 J. Ranunculin K. Glycosidesofmethylazoxymethanol19 3 and convicine 3 19 L. Vicine

314

111. Mode of Action,Clinical Signs of Poisoning, and Treatment of Livestock 320 Neurotoxic A. glycosides (nitrocompounds) 320 Cardiovascular B. glycosides 321 C. Glycosidesinducinggastrointestinal or hepatic effects323 D. Glucosinolates 325 E. Glycosides affecting reproduction (isoflavones) 327 F. Glycosides inducing specific disorders 328 IV.AspectsofHumanPoisoning

330

A. The potential for transfer of toxic glycosides or aglycones to milk or meat products 330 B. Review ofincidentsof acute and chronicpoisoning and potentialrisks 332 References

334

299

Majak and Benn

300

1.

BACKGROUND

A.

Hemiacetals,Acetals, and Esters

Glycosides, which are classified chemically as acetals, are conjugated alcohols. By definition, hemiacetals are formed when alcohols add to aldehydes or when the aldehyde function of an open-chain form of an hydroxy aldehyde (e.g., D-glucose) reacts with an internal hydroxyl group to form a cyclic hemiacetal (Table 1). Isomerization at C,, of glucose results in the formation of the a,P-anomers. Further catalysis or biocatalysis pemits the addition of a second alcohol to form the glycoside. Discussion of the biogenesis of these glycosides is beyond the scope of this chapter but, in general, glycosides normally are fonned through the action of glycosylating enzymes on nucleotide-activated sugars and aglycones (1). The aglycone, or the nonsugar portion of the glycoside, should be distinguished from the nonsugar portion of the closely related sugar esters, which are derived from organic acids (see Table 1). Additional esterification or glycosylation also can occur at the hydroxyl groups situated on nonanomeric carbons. However, the addition of an alcohol aglycone to these hydroxyl groups (etherification) is uncommon, ifnot extremely rare. Glycosides occur most commonly as 0-Pa-glucosides but both anomers of glucose are found among esterified conjugates. In C-glycosides, aglycones are linked to sugars through carbon-carbon bonding without an oxygen bridge. Thioglycosides utilize a sulfur bridge. An N-glycoside was recently isolated from a fungus (2). A diverse array of toxic glycosides occurs in higher plants, the most abundant of these glycosides being the glucosinolates (thioglycosides), the cyanogenic glycosides, the aliphatic nitrotoxins, the saponins, and the cardiac glycosides. Glycoalkaloids, discussed in another chapter, are also glycosides and the aglycone is a nitrogen-containing sterol (steroid alcohol). Glycosides are secondary plant products and currently there is sufficient Table 1 Hemiacetal,Acetal, and EsterFormation Products

Reactants

name

RCHO, R'OH

(Hemiacetal) a-D-Glucose

CHO"(CH0H)J"CH:OH P-D-Glucose

ROH

m

w OH

mw-R P-D-Glucoside (acetal)

ON

RCOOH

P-D-Glucose Ho OH

Glycosides

301

evidence to assume that they are not waste products or end-products of metabolism that are put into storage. Many secondary products, including glycosides, are recognized as active metabolites or allelochemicals that interact with other plants, other microorganisms, insects, and animals (3, 4). The roles of these compounds include the attraction of pollinators or seed dispersers and the repulsion or inhibition of herbivores and microorganisms, including pathogens (5,6). Secondary plant metabolism also responds to water deficits and nutrient deficiencies and the concentrations of toxic glycosides in plants can be affected significantly by physiological stress (7). Herbicide treatments to eradicate poisonous plants also can alter toxic glycoside concentrations (8). Other factors that may affect glycoside levels include stage of plant development, translocation and accumulation of glycosides in specific plant tissues, geographic location, and the seasonal effects of climate and soil. The metabolism of the glycosides is considered in Sect. I11 but only in terms of their mode of action. Metabolism in relation to detoxification or excretion is beyond the scope of this review but the reader is directed to pertinent references on the subject. B. Hydrolysis of the Glycosidic Bond

The significance of the glycosidic bond in the toxicity and biological activity of cyanogenic glycosides is well established. In essence, the bond renders the glycosides innocuous through stabilization of the aglycone. Enzymatic hydrolysis of the glycosidic bond yields unstable aglycones with the resultant, spontaneous production of hydrogen cyanide (HCN), the lethal metabolite (9). Glycoside hydrolases such as P-D-glucosidase (EC 3.2.1.21) are distributed widely in higher plants and microorganisms (1,9, 10) and poisoning in mammals is exacerbated if such sources of the plant enzyme as sweet almonds are ingested with a cyanogen (1 1, 12). However, it now is accepted widely that, in mammals, microbial enzymes of the digestive tract are mainly responsible for the hydrolysis of cyanogenic glycosides; the reader is referred to Ref. 13, a thorough review on the subject by Scheline. Hydrolysis of these glycosides by the acid contents of the stomach is unlikely because the glycosides are stable to acid under physiological conditions (14). Also, a 2hr incubation of the nitropropyl glycoside miserotoxin in sheep abomasal (stomach) fluid did not produce detectable levels of the aglycone (15). Cardenolides, however, are susceptible to acid hydrolysis in the stomach (16). In ruminants, such as cattle and sheep, an important feature of the digestive tract is the occurrence of microbial fermentation prior to gastrointestinal activity. The fermentation occurs in the rumen, which has a capacity of up to 100 L in cattle. The rumen accommodates an array of anaerobic microorganisms, including bacteria ( 109/ml),protozoa ( lo5/ ml), and fungi (103-5/ml)(17). When pure cultures of rumen bacteria were incubated with cyanogenic glycosides, P-glucosidase activity was detected in about 50% of the strains that were examined (18, 19). In addition, P-glucosidase, also known as cellobiase, is distributed widely among species of rumen ciliate protozoa and anaerobic fungi in which the enzyme participates in cellulose digestion at the level of cellobiose hydrolysis (17). The bovine ruminal enzyme has a broad pH optimum in the normal pH range (5.57.5) of rumen fluid but significant decreases in activity occur at the extremes of pH 5 and 8 (20). The activity of the enzyme also is affected by feed quality and feed composition. The microbial enzyme shows substrate specificity (18, 19), indicating that cyanogenic glycosides may be differentially toxic owing to the nature of their aglycones. In short, the fermentative conditions of the ruminant forestomach provide a rich source of hy-

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drolytic enzymes for the release of cyanide from cyanogenic glycosides, but prolonged fasting (more than 24 hr) can result in a depletion of microbial activity and depressed rates of hydrolysis (9). In monogastric mammals, the upper regions of the digestive tract contain a-glucosidases for starch digestion but they largely are devoid of P-glucosidase activity. Accordingly, P-D-glucosides, for the most part, are not hydrolyzed in the small intestine but they can be absorbed from the intestine and excreted intact in the urine, hence their decreased toxicity to rats and humans compared to cattle. For example, the lethal dose for the cyanogenic glycoside prunasin was 56 mg/kgbody weight in cattle (21) but the LD5,, for prunasin was 560 mg/kg in rats (22). The hindgut, especially the large intestine, of monogastric mammals has been compared to the rumen (23). The cecal and fecal anaerobic bacteria of monogastric animals contain a number of hydrolytic and reductive enzymes that are similar to those that occur in the rumen (24-27). Consequently, P-D-glycosides that are not absorbed from the small intestine (28) or those that are excreted from the bile duct (29) are subject to hydrolysis by microflora of the hindgut. In monogastric mammals, the toxicity of cyanogenic glycosides probably is determined by the proportion of the dose that reaches the hindgut. The in vitro microsomal degradation of cyanogenic glycosides by nonhydrolytic pathways also was reported but hepatic catabolites were not identified (30). Hydrolysis of the glycosidic bond also is essential for the toxic expression of such aliphatic nitrocompounds as miserotoxin. In ruminants, the aglycone 3-nitropropanol (NPOH) is released rapidly from miserotoxin by ruminal microbes (20) but in rats, the glycoside is relatively innocuous and it can be absorbed and excreted intact (31, 32). The toxicity of miserotoxin also is contingent upon the hepatic biotransformation of the aglycone NPOH to 3-nitropropionic acid (31). In contrast, glucose esters of nitropropionic acid are substrates for esterases that are abundant in mammalian tissues as well as in ruminal microbes. The resultant rapid hydrolysis of these conjugates results in the release of the nitroacid in the forestomach of ruminants and in thesmall intestine of monogastric mammals through the action of intestinal esterases and possibly lipases. Hydrolysis in monogastric animals also could occur after absorption of the intact esters from the small intestine. Glucosinolates differ from other glycosides in that they are S-P-D-glucosides and as such they are not substrates for 0-P-D-glucosidases. However, glucosinolate-containing plants all contain myrosinase (thioglucoside glucohydrolase, EC 3.2.3. l), the hydrolytic enzyme that releases the thiol aglycone (33). The aglycone is unstable at physiological pH, and a nonenzymatic Lossen rearrangement with the elimination of bisulfate results in the formation of isothiocyanates. Three major groups of isothiocyanates can be formed that are characteristic of three classes of glucosinolates. By far the most common breakdown products are the alkyl or alkenyl isothiocyanates. Less abundant are the unstable hydroxyisothiocyanates and the indole isothiocyanates, which yield epithionitriles and nitriles (33). Rumen fluids from cattle on four different diets (34) and sheep on three different diets (15) were screened for myrosinase activity. It was concluded that both bovine and ovine rumen fluid are devoid of thioglucosidase activity (20, 34). Isothiocyanates could be generated from glucosinolates in rumen fluid by the addition of exogenous myrosinase. They also could be generated when ground plant material containing active myrosinase was incubated with rumen fluid. Thus, the hydrolysis of glucosinolates in rumen fluid is contingent upon the presence of the endogenous plant enzyme (20, 34). Although various studies have indicated the degradation of glucosinolates in the digestive tract of monogastric mammals, breakdown products, for the most part, have not

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been identified and the presence of mammalian myrosinase has yet to be unequivocally demonstrated. Earlier reports, summarized by Scheline (13), indicated the presence of thioglucosidase activity in intestinal bacteria but the accuracy of these studies has been questioned recently (33). Glucosinolates can be excreted intact, especially in germfree rats ( 3 5 ) ,and it is known that germfree animals are protected from the goitrogenic effects of glucosinolate breakdown products (33). Hydrolysis of the glycosidic bond is not a prerequisite for the biological activity of such polycyclic glycosides as glycoalkaloids, saponins, and cardenolides. In fact, removal of the monosaccharide or oligosaccharide units resulted in reduced activity with saponins (36, 37) and cardenolides (38) and loss of activity with glycoalkaloids (39). The sugar portion of a glycoside may affect the rate of absorption of the glycoside into cells or cell membranes. For example, such monosaccharide glycosides as salicin, arbutin, and y-nitrophenyl-P-D-glucopyranoside were absorbed readily by human erythrocytes, but the membranes were largely impermeable to such disaccharides as glycyrrhizin and y-nitrophenyl-P-D-lactopyranoside(40). In addition, the aglycones were absorbed at a much more rapid rate than monosaccharide glycosides (40).

C. Inhibitors of P-Glucosidase Considerable effort has been devoted to the isolation and synthesis of glucohydrolase inhibitors in thelast two decades. This has been prompted in part by the potential treatment of such metabolic disorders as diabetes and obesity through the use of enzymatic inhibitors of a-glucosidase and sucrase. These compounds can delay the intestinal enzymatic release of glucose and fructose from starch and sucrose, the two major dietary carbohydrates (41). Some of the inhibitors of a-glucosidases are also inhibitors of P-glucosidase and it has been suggested that they could protect ruminants from toxic glycosides by decreasing the rate of hydrolysis and delaying the release of the aglycone in the rumen (31 ). To our knowledge, invivo studies have not been conducted on theefficacyof P-glucosidase inhibitors as potential inactivators of hydrolytic enzymes that release toxic aglycones in the digestive tract of mammals. Listed in Table 2 are examples of a number of naturally occurring inhibitors of Pglucosidase as well as synthetic ones that showed enzyme inhibition in vitro. Many of these compounds are structural analogs of glucose that bind tightly to the active site of

Table 2 Naturaland Synthetic Inhibitors of P-Glucosidases ~~

source

Plant

G H I

Inhibitor

5-Amino-5-deoxy-~-glucopyranose(nojirimycin) 2,5-Dihydroxy-tnethyI-3,4-dihydroxy pyrrolidine 1,5-Dideoxy-l,5-imino-~-glucitol (deoxynojirirnycin) 1,6,7,8-Tetrahydroxy-indoliziditle (castanospermine) Glucono-l:5-1actone (delta gluconolactone) 1,2-Dideoxy- l,?-epimino-myoinositol (conduritol aziridine) L-Histidine P-naphthylamide R-N-Acetylhistamine Amidine derivative of D-glucose

Ref. Str-eptomyces species Derris ellipticcr Loldlocarplrs sericem Crrstrrnospenmm nustr-nle Synthetic Synthetic

42 43 43,44 44,45 35

Synthetic Synthetic Synthetic

47 47 48

46

Benn 304

and

Majak

the enzyme. Inhibitor C was 10 times more potent than Inhibitor E when assayed with apricot enzyme (emulsin) or fungal enzyme (43). Inhibitor B was 10 times more effective than Inhibitor C when assayed with almond enmlsin (43). In addition to 0-P-glucosidase inhibition (45), castanospermine (D) also inhibited S-P-glucosidase, myrosinase (44). Inhibitor F inactivated a bacterial 0-glucosidase as well as a yeast a-glucosidase (46). The imidazoles (G and H j and the amidine derivative (I) were potent inhibitors of almond emulsin (47, 48). Naturally occurring inhibitors of P-glucosidase have also been implicated in neurological disorders in livestock. Prominent among these are the polyhydroxylated nortropane alkaloids (calysteginesj and castanosperrnine (Table 2), which are powerful inhibitors of mammalian a-glucosidases and galactosidases (49-52). The calystegines also occur as glycosides and as such they represent a small but new group of toxic glycosides (49). Finally, some naturally occurring polyphenols (e.g., tannins) also have the capacity to precipitate and inactivate p-glucosidase (53. 54). Condensed tannins had an inhibitory effect on endoglucanase activity and cellulose digestion by rumen bacteria and fungi (55, 56) and one would also expect inhibition of cellobiase (P-glucosidase) activity, which is part of the microbial cellulolytic system (17). Natural products such as alkaloids and phenolic acids that affect the growth and cellulolytic activity of bacteria (17) may also inhibit the activity of the enzyme indirectly. II. HISTORICALASPECTS,DISTRIBUTION, AND CHEMISTRY A.

AliphaticNitrocompounds

Relatively few natural products contain the nitro group but, of those that do, two important sets correspond to sugar conjugates of NPOH and 3-nitropropanoic acid (NPA). Although the latter are not glycosides, they often resemble them structurally, and their mammalian toxicities are similar; it is for these reasons that they receive attention here. Both types of compound were discovered as a result of searches for the substances responsible for the poisonous properties of particular plants. The Maori used the fruit of a New Zealand tree, Colyocaryus Zrevigcrtzss, known to them as karaka, for food, but only after a carefully prescribed processing, without which there could be fatal poisoning (57). In 1872, Skey reported the isolation from untreated karaka fruit of a crystalIine toxin, which he called karakin (58). Although not recognized as such at the time, this was the first characterization of a natural product containing the nitro function, for when the structure of karakin finally was elucidated (59), it proved to be 1,2,6-tris-O-(3-nitropropanoyl )-P-D-glucopyranose(1) (bold numbers refer to accotnpanying structures). Subsequent investigations of the fruit and foliage of C. Zaevigntzls have shown that X-cwcrkirz is accompanied by a number of other NPA esters of glucose (60, 61).

OR

Glycosides

305

Similarly, an investigation of Astmgallrs miservar. oblongifolius followed the observation that it was responsible for losses of stock on the rangelands of the western United States, and resulted in the isolation and identification of the principal poison, miserotoxin (2), 3-nitropropyl-~-~-g~ucopyranoside (62,63). Other NPOH glycosides may be present, for studies of A. rrliser var. ser-otinus from the interior of British Columbia have shown that, besides 2, extracts contained disaccharidic analogs, that is, 3 (64), 4 (65), and 5 (66).

Aliphatic nitrocompounds such as miserotoxin and karakin usually are detected and estimated either by coupling their aci-form salts with a benzenediazonium ion (a JappKlingemann reaction) to give a product suitable for photometric analysis (67) or by measuring the release of nitrite ion when they are heated with alkali, using the Gries-llosvay reagent or some similar procedure (68). Enzytnatic or nonenzymatic hydrolysis of karakin and its congeners releases NPA and D-glucose, while miserotoxin similarly yields NPOH. The toxicological consequences of these hydrolyses are discussed in Sect. 111 and IV, Apart from their occurrence in the Corynocarpaceae, NPA esters of glucose have been found in the Leguminosae (Astragalus, Coronillcl, hdigofem, and Lotus) and also in the Malphigiaceae and Violaceae (69). Miserotoxin occurs in numerous species of Astrclgcllus, but not with NPA esters as originally thought (69, 70). All of these matters are covered in Ref. 31. B. CyanogenicGlycosides Numerous organisms release HCN when damaged or stressed, a phenomenon known as cyanogenesis. In plants, this is associated with the disruption of cells, often by crushing, followed by autolysis of the contents. The HCN-storage compounds are often, although not always, glycosides and much less toxic than HCN. A classic example is amygdalin (6), a bitter constituent of almond kernels that seems to have been recognized since antiquity, although it was not until 1923, 120 years after its chemical characterization, that its

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structure finally was established. These and other historical aspects are covered in some of the reviews of cyanogenic glycosides (71-77).

y

It has become customary to subclassify the cyanogenic glycosides according to the nature of the aglycone, and a favored system does this by biosynthetic origin (73j. Linamarin (7),lotaustralin (Sj, and proacacipetalin (9j are derived, respectively, from the aliphatic a-amino acids (S)-valine, (S)-isoleucine, and (S)-leucine. Such others as (R)-prunasin (1Sj and (S)-dhurrin (11) come from the aromatic amino acids (S)-phenylalanine and (X)-tyrosine, respectively. The biosynthetic pathways have been mapped in some detail, and involve oxidative processing of the amino acids with intermediate nitrocompounds and oximes, a scheme also followed during the early stages of the S-glycosidic glucosinolate biogenesis (78).

CN

0-Glc

I

Gic-0-C-H

I

Less obviously, the aglycone of triglochinin (12)also is derived from (3)-phenylalanine, apparently by way of (S)-mandelonitrile (13), which is first converted to a 3,4-dihydroxy system and then subjected to oxidative ring scission (79). Gynocardin (14) (80) exemplifies another set of cyanogenic glycosides in which the aglycone is a cyanocyclopentene (74). Their biosynthetic origin remains enigmatic.

CN HO-C-H

I

307

Glycosides

The release of HCN from all these glycosides is brought about by hydrolysis. The cyanohydrin aglycones liberated are in equilibrium with the corresponding carbonyl compounds and HCN (15- 3 16 17). Both the hydrolytic cleavage and final equilibrium can be enzyme or acid catalyzed. Care must be taken when isolating and storing the glycosides since alkaline conditions may induce epimerization at C-2 of the aglycone (6 S 18) (8 1). Aqueous bases usually result in the preferential hydrolysis of the nitrile rather than the glycosidic linkage (6 3 19) (82). However, the y-hydroxyphenyl compound (ll), dhurrin. readily cleaves to HCN and p-hydroxybenzaldehyde under even mildly basic conditions (83).

*

1

1

y

0

(18)

Cassava, which contains 7 and 8, is an important food in the tropics. An International Workshop on this crop included papers dealing with the removal or destruction of the cyanogenic glycosides (84, 85). Methods for the detection and estimation of cyanogenic glycosides, which largely rely on measuring the HCN released following enzymatic and nonenzymatic hydrolyses, have been reviewed, as has their isolation, characterization, and spectrometric identification (72, 75, 86). Their distribution in the plant kingdom also has been reviewed (73-75, 87). The Leguminosae, Rosaceae, and Proteaceae have been particularly rich sources of the aliphatic and aromatic a-amino acid-derived compounds, although they also are found in a number of other families. Triglochinin is associated with the Juncaginaceae, but also occurs elsewhere. The gynocardin type appears to be confined more narrowly to the Flacourtiaceae, Tumeraceae, and Passifloraceae (87-89). The classification of some glycosides as pseudocyanogenic refers to those compounds that do not contain cyano groups but that can be decomposed, usually under rather drastic conditions, to yield HCN. Examples are such nitro esters as karakin and glycosides of methylazoxymethanol (MAM) (see Sec. 11. K). Also, there are some poisonous glycosides that contain the cyano functionality but that do not release HCN readily, at least under hydrolytic conditions. These have been called noncyanogenic. The jojoba (Sirnmondsia clzinerzsis) glycoside simmondsin (20) (90) and its congeners fall into this class (91 ).

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C.

CardiacGlycosides

Cardiac glycosides are characterized by a steroidal aglycone, the genin, which is one of two structural types: cardenolides, in which a conjugated butenolide group is attached to C-17, as exemplified by digitalin (21); or bufadienolides, in which the C-17 substituent is a conjugated pentadienolide as in scilliroside (22) (92-94). These steroidal systems invariably are glycosylated via a C-3 hydroxyl group. The glycosyl unitis usually an oligosaccharide that often is branched and containing deoxy- and monomethyl ether monosaccharides that otherwise seldom are encountered. Labriformin, isolated from Asclepias species, illustrates a rather unusual type of cardenolide in which there is a second, ketaltype linkage between the genin and the glycosyl unit (95).

G k-

I

OH Both cardenolides and bufadienolides are plant products, although bufadienolides commonly are associated with the poison glands of toads (Bufo species), in which they occur as conjugates of suberoylarginine. A recent comprehensive review is available on the distribution of bufadienolides in plants and animals (96). Since steroids are degraded triterpenes, the cardiac glycosides are a subset of triterpenoidal glycosides (see Sec. 11. D). However, because of their exceptional cardiac activity, they often are considered separately from the other saponin type of triterpenes, as is done here. As a consequence of their powerful effect on the heart, preparations containing cardiac glycosides have a long history as medicinals and poisons. Probably tnost famous in the Western world is the story of the introduction, in 1795, by William Withering of digitalis, an extract of Digitalis purpurea (foxglove) glycosides for the treatment of heart disease (97), but records of the similar usage of Scilla ~nnritinm(squill, sea onion) preparations, which also owe their activity to their content of cardiac glycosides, date back to the ancient Egyptian, Greek, and Roman civilizations (92-94). Cardenolides can be detected by a variety of colorimetric tests and spray reagents, of which the Legal (alkaline sodium nitroprusside), Raymond (alkaline 1,3-dinitrobenzenej, and Kedde (alkaline 3,5-dinitrobenzoic acid) reagents are used most commonly (92,93,98). Although equivalent tests for bufadienolides remain to beestablished, these compounds can be detected and estimated by their UV absorption. The presence of 2-deoxy sugars in the glycosides also can be revealed colorimetrically by the Keller-Kiliani test (93).

Glycosides

309

There is an extensive chemistry to the cardiac glycosides, most of which is outside the scope of this review and has been covered elsewhere (92, 93). We confine ourselves to noting that, although the hydrolytic scission of the glycosides can be carried out with aqueous acid, this should be done with caution as such artifacts as dehydration products of the genin may be formed (93). In this regard, enzymatic cleavage is safer (93). With the advent of such improved separation procedures as modern countercurrent techniques for highly polar substances (99), it is possible to isolate pure cardiac glycosides from complex mixtures. These then can be subjected to such spectrometric analyses as 'H and I3Cnuclear magnetic resonance (NMR) (loo), mass spectrometry (MS), and others and their structures determined without the need to split them into their constituent genin and sugars as was done in the past. This is illustrated in Refs. 101 and 102. In terms of botanical distribution, cardiac glycosides are conmon particularly in the Asclepiadaceae, Apocynaceae, Scrophulariaceae, and Liliaceae, but also are found elsewhere (94).

Saponins D. Saponins are glycosidic (usually oligosaccharidic) conjugates of triterpenes. The name derives from their soap-like property of causing the formation of stable foams when their dilute solutions in water are shaken. Also noteworthy is their ability to induce the hernolysis of red blood cells, even at high dilution (93, 103-105). The saponins are divided into subclasses according to the structure of the aglycone, which is known as the sapogenin (or genin). The triterpenoid saponins are characterized by a sapogenin that is an intact triterpene hydroxylated at C-3, with that being the site of attachment of the glycosyl unit. Further subclassification can be made on the basis of the skeleton of thetriterpenoid. Thus, narcissiflorine (23) is an example of a triterpenoid glycoside withan oleanane skeleton (106), while in pavophylline (24) the sapogenin has a lupane skeleton (107). For other examples, consult the catalogs of saponins (105, 108112). Steroidal saponins are characterized by a sapogenin that is steroidal (a degraded triterpene), similarly hydroxylated at C-3 with this being the attachment point for the glycosylation. Tigonin (25) exemplifies this group of saponins.

OH

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4 0

HOH$

OH

Hay--" Xyl-0

0

G IC - 3 G a f

The so-called glycoalkaloids are the glycosides of nitrogenous (alkaloidal) steroids that are found in various members of the Solanaceae, Asclepiadaceae, and Liliaceae, most famously in the cultivated potato (Solanunz tuberosr~mL.) and exemplified by solanine (26j The term “glycoalkaloid” actually encompasses other compounds, such as the 3-pD-glucopyranoside of calystegine B, found in Nicmrdra physolodes (1 13) (a discovery that suggests that other members of the Solanaceae and Convolvulaceae may contain glycosidic conjugates of polyhydroxy-nor-tropane alkaloidsj. Alkaloidal toxins are discussed in another chapter.

Glycosides

31 1

As thus defined, the steroidal saponins would encompass the cardiac and calcinogenic glycosides. However, as noted above, because of their respective exceptional effects on the heart and calcium salt deposition, those particular subsets of glycosides customarily receive separate treatment, and we follow that approach here. The abilities of saponins to cause foaming and to hemolyze red blood cells form the bases of tests for their presence in plant extracts (93, 103). There are also some useful, but nonspecific, color reactions that can be used to visualize saponins on thin-layer chromatography (TLC) plates (98). The isolation of pure saponins and their structure identification were formerly tasks of considerable difficulty (93). However, as with the cardiac glycosides, the advent of high-performance liquid chromatography (HPLC) and improved countercurrent fractionation procedures (99) largely have solved the isolation problems. Modern two-dimensional (3D) NMR techniques often permit the structure of the intact saponin to be established without the need to follow the old procedures, which involved cleaving the glycoside (often after methylation) enzymatically or nonenzytnatically and then identifying the constituent sapogenin and monosaccharide fragments (93, 114). This is illustrated by the work discussed in Ref. 115. As a consequence, the number of known saponins is increasing rapidly. A recent monograph (105) covers all of the above matters, as well as reviewing the occurrence and distribution of triterpenoid and steroidal saponins (including the glycoalkaloids) and their pharmacological and biological properties. Thirteen commercially important saponin-containing preparations (saoparilla, licorice, aescine, ivy leaves, tea, senega root, primula root, asiaticoside, Bupleuri root, Gspsoyhiln, quillaia bark, ginseng, and Elelltherococcus root) receive particular attention. The glycoalkaloids of the cultivated potato have also recently been reviewed (1 16), as has the toxic hazard associated with the consumption of green potato tops ( 117). Historically, Medicrrgo sntilw (alfalfa) and Dioscor-ea species (yams) have been recognized as rich in saponins. However, as the catalogs of known saponins and their plant sources reveal (105, 108-1 13), saponins are distributed very widely throughout the plant kingdom, to the point that they appear to be ubiquitous constituents of plants (a finding that suggests they may have some as yet not understood physiological role).

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E. Glucosinolates The glucosinolates are a set of anions corresponding to salts of substituted glucosinolic acid (27). Usually, they are isolated with potassium as the countercation, although one historically famous case (28) involves a choline derivative. Originally, these compounds were described collectively as mustard oil glucosides since they were recognized to be precursors of organic isothiocyanates (mustard oils), and individuals were named arbitrarily, often by prefixing gluco to an indication of their botanical source. Thus, the substance now known as potassium (3-indolyl) methylglucosinolate (29) formerly was called glucobrassicin. Sinalbin (28) and sinigrin (30), the archetypical members of this group of natural products, isolated from white (Sirzqis nlbcr) and black ( S i m p i s nigrcr) mustard, now are described, respectively, as sinapinium 4-hydroxybenzylglucosinolate and potassium allylglucosinolate.

'OCH:,

/

(28)

S-GLU

An excellent review of the early work on the glucosinolates is available (118); this subsequently was augmented by another that reviewed all glucosinolates known in 1967 (119). These seminal reviews have been supplemented by other good ones (33, 120, 121). A remarkable chemistry is associated with the catabolism of the glucosinolates. Hydrolytic fission by the enzyme myrosinase, thioglucoside glucohydrolase (EC 3.2.3. I), liberates the aglucone (31), the fate of which is pH dependent. At low pH, it gives the corresponding nitrile (32) sulfate, and elemental sulfur, while at neutral or slightly higher pH a rearrangement accompanies the loss of sulfate and leads to isothiocyanates (33). However, while all glucosinolates appear to be able to follow these two pathways, a few are known to be capable of other modes of decomposition. Allyl, benzyl, and 4-(methylthio)butylglucosinolate can be catabolized to give the corresponding thiocyanates (34) and allyl and butenylglucosinolates similarly can be directed enzymatically to give epithioni-

Glycosides

313

triles (e.g., 30 + 35). Isothiocyanates do not appear to be intermediates in these transformations, but they are reactive and can give rise to other products, some of which are biologically active. The goitrogenic principle goitrin (36) arises from the intramolecular cyclization of (S)-2-hydroxybut-3-enyl isothiocyanate (122). The chemistry of these various catabolic processes has been reviewed but remains imperfectly understood (33, 120, 12 1). On the other hand, the biosynthesis of the glucosinolates has been explored in depth (78).

R ‘SH

Because of the biological consequences of their catabolism, a good deal of attention has been paid to the detection and both qualitative and quantitative analysis of glucosinolates (33, 121, 123). Their presence may be inferred by the release of glucose and isothiocyanate (33) following treatment of an extract with myrosinase; the detection of the isothiocyanates that accompanies this release, either as such (by gas chromatography [GC] or by GC/MS)or following their conversion to thioureas, provides an alternative or supplementary indication of their presence. Once a reasonably clean glucosinolate preparation has been obtained, a favored analytical procedure involves treating it with a sulfatase and then derivatizing and analyzing the desulfoglucosinolate (33, 121). The distribution of glucosinolates in the plant kingdom has been scrutinized (1 19, 124). They are invariably constituents of members of the order Capparales, that is, the families Brassicaceae (Cruciferae), Capparidaceae, Moringaceae, Resedaceae, and Tovariaceae, but they also are found in some other genera.

F. DiterpenoidGlycosides The poisonous and medicinal properties of root extracts of the thistle Atl-act?,lis gzmnz$era (Ctrl-lirzagz.mm$el-a) native to the Mediterranean basin were recorded more than two millennia ago. A toxic constituent, atractylic acid, subsequently renamed atractyloside (37), was isolated in 1868, and in 1964 another was isolated, first called gummiferin and later carboxyatractyloside (38). It was not until 1967 that the structure of the first was established, and shortly thereafter that of the other followed. These historical studies, as well as the chemistry and toxicology of 37 and 38, have been reviewed (125, 126). Recently, both 37 and 38 were found to occur in a camel-poisoning member of the Asteraceae (127). The 13-hydroxy derivative of carboxyatractylogenin has been encountered in some toxic aminoglycosides isolated from Australian Wedelia asperrirna (128, 129).

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R =

COOH

(38)

An enzyme-linked immunosorbent assay (ELISA) procedure for determining atracyloside has been described (130), but usually it is detected by TLC or HPLC procedures (13 1). As expected for a gem-dicarboxylic acid, carboxyatractyloside decarboxylates fairly readily to give atractyloside. Thus far, these atractyligenin conjugates have been associated particularly with genera of the Asteraceae (Atmcglis, Cnllilepsis, Wedelin,and Xmthizm), but they also occur in coffee (13 1, 132), in which they were found following the detection of an atractyligenin-like metabolite in the urine of coffee drinkers (133). G.

Bracken and Other Sesquiterpene Glycosides

The bracken glycosides provide a good example of the difficulties that can arise when dealing with an unstable glycoside. Poisoning of cattle by bracken (species of the fern Pter-idiunz)had been suspected for many years, but the firstreport of lesions in experirnental animals maintained on a diet that included bracken did not appear until 1965, when rats were found to develop cancers (134). Despite intensive efforts to identify the carcinogenic agents, another 18 years elapsed before the isolation and identification of the principal toxin as the norrilludane sesquiterpenoid glycoside ptaquiloside (39) was announced (135, 136). This glycoside is unstable and is degraded rapidly under acid or alkaline conditions, with the formation of nonglycosidic indanones (40, 41) (135-137).

0-Glc

Glycosides

315

The generation of these indanones forms the basis of a method for detecting ptaquiloside and its congeners (138). This approach was used to screen the Pteridaceae and guided investigations that resulted in the isolation and identification of ptaquiloside and several analogs (hypolosides A-C (139) and dennstoside-D (140)) from other ferns. An HPLC procedure for detection and quantification of ptaquiloside and pterosin B in plant extracts also has been described, and the procedure revealed 39 to be present in rock fern (CheiZcuztJzes sieberi) (141). Agood synopsis of the properties of these toxic bracken glycosides is available (142), and the toxicity of bracken fern has been reviewed recently (143). Other toxic sesquiterpene glucosides have been isolated from Bnccherris cordjfolin, a plant' responsible for livestock poisoning in southern Brazil and Argentim. These were found to be the P-D-glucopyranoside conjugates of the macrocyclic trichothecenes verrucarin A (42), roridins A and E, and miotoxin A and F (144). Trichothecenes are fungal toxins responsible for mycotoxicoses when contaminated foodstuffs are eaten; however, in the case of the Bcrcchal-is it appears that the plant synthesizes the glucosides de novo, possibly after having acquired the genes from a fungus (144, 145). H

OH

1

OH

H. CalcinogenicGlycosides The history of the discovery of the calcinogenic glycosides illustrates the problem of identifying highly polar toxic substances when they are present in the plant only in small amounts. Pathological calcinogenesis, cedcinosis, refers to the deposition of calcium salts in soft tissues, for example, in arteries. A causative link between the ingestion of So1anur)l glartcopl~yllzn~~ (S. mnlaco,x$orzj and the incidence of a calcinotic disease of livestock in Argentina and Brazil was suspected for some time, as it was for Cestrznncliumunz in Florida and Tl-isetum.fEcrvescerzsin the European alps (146-148). However, it took several years of work involving bioassay-directed fractionation of aqueous extracts of S. glnucoll l u ~leaves before minute amounts of a relatively pure biologically active fraction were obtained. From its polarity, it was suspected that this material was glycosidic, and when it was treated with P-glucosidases or a mixture of glycosidases from the sea worm Charonin lcrnzpns, it released a compound identified as calcitriol (la,25-dihydroxy vitamin D,j (43) (149, 150). This identification was carriedout with a few microgratns of substance and relied heavily on GUMS and UV measurements. Subsequently, vitaminD3 (44) and its 25-hydroxy and la,24,2S-trihydroxy derivatives also were found to be present in glucosidase-treated

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extracts (15 1,152). The identity of the glycosyl moiety of these conjugates remains uncertain, although evidence has been presented to show that some are 2a-fructofuranosyl-terminated oligomers of 1p-+2 linked-D-glucopyranosyl units ( 153). These oligosaccharides (45) are thought to be attached to the 3-hydroxyl of the vitamin D3system.

1

R = OH 1

R=H

R‘

2

R = OH

R

2

= H

(43) (44)

’

CH20H

PI

CHBH

The calcinogenic glycosides of C. d i w ~ z u nand T.flm*escemalso proved to be conjugates of these vitamin Di derivatives, although, on the basis of the relative biological activities of synthetic glucosides of 43, the T. flmescens toxin is thought to be the 25-0p-D-glucoside of calcitriol (148, 154). Not many chemical reactions of these glycosides have been reported, although, as expected for the seco-cholestatrienyl system, they are unstable toward oxidizing agents or acids (146). Various analytical procedures have been described for measuring calcinogenic activity in plant extracts (146, 155), and the vitamin DXconjugates have been detected by radioimmunoassay (156 ). In terms of botanical distribution, vitamin D3 and its hydroxylated derivatives have been detected in a number of plants besides those associated with calcinogenesis, including some grasses and palm and corn extracts (146). It is suspected that the precursor is 7dehydrocholesterol, which is photolyzed to yield 44 with enzymatic hydroxylations giving the other derivatives. However, the occurrence of the calcinogenic glycosides has not been explored to a comparable extent. Reference 146 provides a review of these glycosides. 1.

PhenolicGlycosides

A very large number of phenolic substances have been isolated from plants, in which it appears that they, or their precursors, often are stored as glycosidic conjugates, the pheno-

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lic glycosides. The aglycones exhibit considerable structural diversity, with a particularly important group, known as phenylpropanoids, containing a C6-C3 structural unit. Preeminent among these are the flavonoids, which have been the subject of a major treatise (157) and supplementary reviews (158. 159). Although there is evidence that the phenolic glycosides of plants provide a defense against herbivorous insects ( 160, 161), only a few are regarded as dangerously toxic to mammals; in most cases, the toxic agents have been identified as the phenolic aglycones. Classic examples include the isoflavonoid daidzein (46), found in Trifolium subterranerrnr, and the coumestan coumestrol (47), which occurs in lucerne, Medicngo sativa;both of these toxic agents induce abortions in sheep grazing on these plants (163). The glycosidic conjugates daidzin (48) and coumestrin (49) co-occur with the aglycones (163, 164) and, in view of the ease with which phenolic glycosides undergo enzymatic or nonenzymatic hydrolysis, represent a reservoir of the toxins. Likewise, the phenylpropanoid lignan podophyllotoxin (50), a cytotoxic substance. co-occurs with the glucoside (51) (165, 166). Coumarins often arise from the spontaneous cyclization of the aglycones released from glycosidic precursors, for example, 52+53+54 (167). In a similar vein, the nonphenylpropanoid stypandrol(55), which causes blindness and death of sheep feeding on Stypandra imbricata (168), likely arises by oxidative coupling of 56, which is stored in the plant asa glycoside (169).

\

0

\

OH

OH

R=H

(46)

R = H

(47)

Fi = Glc

(48)

R = GIC

(49)

I

n

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OR

(55) A discussion of the chemistry of the phenolic glycosides would be lengthy. Instead, we refer the reader to the standard texts, which provide good summaries of their detection, isolation, and identification (157-159,170-173) as well as toxicological properties (174).

J.

Ranunculin

The recognition that chewing the fresh leaves or blossoms of buttercups released a vesicant substance must be prehistoric. However, although the identity of this toxin has been established as the unsaturated y-lactone protoanemonin (57) (175), the storage form of this unstable compound has been in doubt. The circumstances surrounding its formation suggest that it is liberated enzymatically from some glycosidic conjugate, and Hill and van Heyningen identified this as the glycoside ranunculin (58) (176). They showed that treatment of 57 with aqueous weak base (sodium acetate) gave protoanemonin. But Tschesche et al. (177) have produced evidence that indicates that ranunculin is an artifact. Three other glycosides were identified but also ruled out as genuine precursors of 57 (177). Subsequently, Tschesche et al. hypothesized that the P-D-glucoside of 5-hydroxylevulinic acid might be the elusive precursor, perhaps forming ranunculin en route to 58 (178). However, recent work (179) indicates ranunculin to be a genuine natural product and provides an explanation for the observations of Tschesche et al. (177).

= ( : y o

(57)

The various analytical procedures, which include HPLC analysis, that have been described to detect “ranunculin” in plant tissues are based on measuring the protoanemonin released upon autolysis (180). Protoanemonin is a very reactive substance that

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319

should readily undergo Michael-type addition reactions with nucleophilic groups in protein side chains, leading to their alkylation. In the absence of such reactions, it readily dimerizes to anemonin (181). An HPLC method for the direct measurement of intact ranunulin has been described ( 179). Protoanemonin has been obtained from numerous species of Rai~unculusand Arzenzone, and also has been obtained from Helleborus and Clernntis, which are also members of the Ranunculaceae (182, 183).

K. Glycosides of Methylazoxymethanol Polynesians prepared a flour from the nuts of cycads but, as with the Maori and karaka, also knew that it wasimportant to follow a protocol to remove a toxic constituent. Failure to do so resulted in the poisoning of members of Captain Cook’s crew of the voyage of 1768- 1771 (184). Studies of the toxins of Cycas and Macro,-crnlia (Zamia)species resulted in their identification as glycosides of methylazoxymethanol, MAM (59). The P-D-glucoside, cycasin (60) (1 85), and the P-D-primveroside, macrozamin (61) ( 186), have received most attention, although a number of other oligosaccharidic derivatives have been characterized ( 187).

HJc\ - ,N=N,

t

CH20 -R

0

R = GIC

The revelation that MAM was mutagenic and a potent carcinogen stimulated studies of its chemistry that showed it to bea biological methylating agent (188). These glycosides appear to be confined to the Cycadales (189).

L. VicineandConvicine Favism is a potentially fatal disorder brought about by eating faba beans (Vicia fnba). The disease is associated with populations of countries surrounding the Mediterranean Sea, and now is known to involve an inherited inborn error of metabolism. The beans contain the toxic pyrimidine glycosides vicine (62) and convicine (63), substances that were isolated first by Ritthausen more than a century ago from vetch (V. sativa) seeds but, as is so often the case with glycosidic toxins, the poisonous effects of vicine and convicine appear to be due to the release of their aglycones, divicine (64) and isouramil (65).

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0

0

References 190 and 191 provide excellent coverage of the detection, isolation, and quantification of convicine and divicine in plants, as well as the chemistry and metabolic effects of these glycosides and their aglucones. Within the Leguminosae, convicine and divicine appear to be present in various species of Vicia, but absent from a number of other legumes (192).

111.

MODE OF ACTION, CLINICAL SIGNS OF POISONING, AND TREATMENT OF LIVESTOCK

A.

NeurotoxicGlycosides(Nitrocompounds)

It has become well established that 3-nitropropionic acid (NPA) is the lethal metabolite formed in the biotransformation of nitropropyl glycosides or nitropropanoyl glucose esters of NPA (3 1, 193). The esters yield NPA directly upon hydrolysis, but for the bioactivation of the glycosides, which yield 3-nitropropanol (NPOH). a second metabolic step is required after the hydrolytic release of the aglycone. This second step probably occurs in the liver, in which the oxidation of NPOH to NPA is catalyzed by alcohol dehydrogenase (194). The in vivo conversion of the nitroalcohol to the nitroacid has been demonstrated in sheep, cattle, and rats (194-196). 3-Nitropropionic acid is a potent inhibitor of Krebs (tricarboxylic acid) cycle enzymes essential to respiration. Specifically, it inhibits succinate dehydrogenase irreversibly and fumarase competitively (197-199). In plants and microorganisms it also inhibits isocitrate lyase (2OOj. In 1982, Gould and Gustine suggested that neuroexcitotoxic mechanisms involving glutamate or aspartate may participate in the pathogenesis of NPA toxicity (201). The central nervous system lesions caused by subcutaneous injections of NPA or NPOH were similar in rats and mice (202) andthe basis of intoxication appeared to be histotoxic hypoxia resulting from marked inhibition of succinate dehydrogenase (203). Brain lesions resulting from NPA intoxication were correlated to physiological changes in the circulatory system of rats (204). Methemoglobin levels were elevated moderately during nitroacid intoxications (201, 205), but the nonfunctioning hemoglobin was not the cause of the neurological disorders nor was it present in sufficient concentration to be fatal by itself. The in vitro conversion of NPOH to acrolein can be shown (206). but the evidence from animal studies does not support this pathway of metabolism in vivo (193). More recently, interest in NPAtoxicity has been rekindled due to outbreaks of moldy sugarcane poisoning in humans in China. The causative agent was identified as NPA

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321

occuming as a mycotoxin (207). The epidemiology and the etiology of the syndrome have been extensively reviewed (208-210). Typically, there is development of acute encephalopathy in children leading to necrosis of the basal ganglia often clinically manifested as dystonia. The syndrome induced by NPA in rats has been studied as a model for neurodegenerative disorders, specifically Huntington’s disease (21 1-21 3). Chronic administration of NPA produces selective lesions in the basal ganglia that replicate many of the neurochemical and histological features of the disease. Neuronal degeneration induced by NPA could be linked to an impairment of energy metabolism due to succinate dehydrogenase inhibition and reduced ATP synthesis (214, 215). The clinical syndromes of nitrotoxin poisoning in livestock and laboratory animals have been reviewed thoroughly in Ref. 3 1. The major clinical signs in the acute syndrome include incoordination, distress, labored breathing, cyanosis, muscular weakness, and collapse, with death occurring from a few hours to a day after the ingestion of the toxin. In chronic poisoning, the animals lose weight and develop respiratory distress, a poor hair coat, hind limb paresis progressing to paralysis, and a nasal discharge. Early signs of nitropropanol poisoning, prominent among which were frothy salivation, stupefaction (205), and diarrhea, have been documented in cattle under field conditions. Recent field observations (216) do not support the concept of livestock addiction to NPOH-containing plants, which has long been suspected. No specific antidote is available for the treatment of livestock poisoned with aliphatic nitrotoxins but dietary protein supplements can enhance the activity of rumen bacteria capable of NPOH detoxification (216, 217). At least two mechanisms are known for NPA and NPOH detoxification (218). Thiamine, given intramuscularly, has been recommended for the treatment of affected sheep and cattle but it was ineffective in treating acute or chronic nitrotoxin poisoning in rats (219). Inhibitors of alcohol dehydrogenase also were ineffective in rats but rats were protected if they were pretreated with ethanol or 4-methylpyrazole (194). Routine hematological and serum biochemical parameters are not useful in the diagnosis of NPOH ingestion or intoxication in cattle (220).

B. CardiovascularGlycosides 1. Cyanogens The aglycones resulting from the hydrolysis of cyanogenic glycosides are unstable cyanohydrins (a-hydroxynitriles). At physiological pH, they undergo a rapid dissociation to yield HCN and either an aldehyde (e.g., benzaldehyde in the case of the cyanogenic glycoside prunasin) or a ketone (e.g., acetone in the case of linamarin). The dissociation is a pH-dependent reaction with higher rates of HCN formation occurring at a pH greater than 6 and slower rates at pH 5-6 (9). Thus, in the diurnal pH cycle of rumen fluid, it was predicted that cattle would be least susceptible to cyanogenic glycosides during feeding and digestion when the pH of rumen fluid is depressed owing to the production of volatile fatty acids and carbon dioxide. They would be most susceptible after a 24-hr fast when the pH of rumen fluid is elevated in response to a slower rate of microbial fermentation and a depletion of volatile fatty acids (9, 20). HCN is the lethal metabolite in the hydrolysis of cyanogenic glycosides but there could be potential side effects from the other noncarbohydrate metabolites (e.g., benzaldehyde). It is interesting that benzaldehyde has the smell of almonds, a fragrance that is detected readily, but the reputed bitter almond smell of HCN can only be detected by 4060% of the population (221).

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HCN is extremely toxic because it blocks aerobic cellular respiration, preventing the utilization of oxygen and causing a decrease in energy production. The toxic effect of HCN is attributed to inhibition of cytochrome oxidase, the terminal oxidase in the mitochondrial respiratory chain, but other enzymes and metabolic processes also may be affected directly or indirectly by HCN (222). For example, HCN impairs normal sulfur metabolism since it can deplete the sulfane (neutral sulfur) pool rapidly (223, 224). Physiologically, HCN poisoning results in histotoxic anoxia with the initial manifestation of hyperpnea, followed by dyspnea, and then convulsive seizures (222). Clinical signs of poisoning with cyanogenic glycoside develop at a much slower rate than they do for sodium cyanide; this has been attributed to the slower release of HCN from the glycoside. The delayed release of HCN permits a greater degree of HCN detoxification and, in contrast to free HCN, twice as much glycoside cyanide is required for a lethal dose in ruminants (21, 225). Clinical signs of subacute and acute poisoning in cattle resulting from prunasin administration have been described; these include tachycardia, hyperpnea, recumbency, increased pinkness of the mucous membranes, and tonic convulsive contractions (21). The classic nitrite-thiosulfate treatment for HCN intoxication is still the preferred therapeutic method, especially if it is supplemented with oxygen (222). Nitrite generates methemoglobin, which is an efficient scavenger of HCN, but newer studies suggest that the unique vasodilator properties of nitrite may be more important in the antagonism of HCN by nitrite (322). Similarly, it has long been accepted that thiosulfate contributes sulfur in the detoxification of HCN to thiocyanate through the enzymatic action of rhodanese (EC 2.8.1.1). However, studies indicate that the critical role in mammalian HCN detoxification probably is effected by serum albumin sulfane carriers that bindHCN tightly (223). Rhodanese mainly is involved in mitochondrial sulfur metabolism (223, 224).

2. Cardenolides It generally is accepted that the Na+K+-adenosinetriphosphatase (NaSK+-ATPase)in cardiac muscle is the major pharmacological receptor of cardiac glycosides (38, 94). This enzyme, which hydrolyses ATP to ADP, requires the presence of both Na+ and K' for maximum activity. NafKf-ATPase is responsible for cellular uptake of K+ and extrusion of Na', the metabolic pump of cell membranes that generates the action potential for the electrical flow of ions along axons and myocardial fibers. Historically, the most familiar inhibitors of this system have been the cardenolides ouabain and digitoxin, therapeutic at medicinal doses for the treatment of congestive heart failure in humans but toxic to domestic herbivores when consumed at the natural concentrations in plants. The inhibitors affect the intracellular electrolyte concentrations, resulting in more forceful contractions of the myocardium. Life-threatening arrhythmias may develop due to changes in the excitability of cardiac muscle (94). The acute toxicities of ouabain and a number of Ascleyins cardenolides were compared in mice. The LDjO values were similar for the glycosides in spite of large differences in chemical structure (226). Inhibition of lamb cardiac Na+K+-ATPaseby the same group of cardenolides, including digitoxin, also was compared and differences among glycosides were even smaller (226). The ventricular effects of digitoxin, digoxin (12-hydroxy digitoxin), and lanatoside C (glycosyl digoxin) were compared to their respective aglycones in human patients with congestive heart conditions. It was concluded that the glycosides and their aglycones, given intravenously, have the same kind of action in depressing the ventricular rate. Unlike the glycosides, the onset of the effect was almost immediate with

Glycosides

323

the aglycones, but the duration of the effect was much longer with the glycosides than with the aglycones (227). In human patients, the variability in the cardioactivity of digoxin was attributed partly to differences in intragastric pH, which influenced the partial hydrolysis of the glycoside. The resultant aglycone and monoglycoside were conjugated rapidly but digoxin and the diglycoside were not metabolized significantlyin vitro (228). The susceptibility of cardenolides to acid hydrolysis in the stomach has long been recognized (16). Seiber et al. reviewed the LDSnof several cardenolides and their aglycones given intravenously to cats (38); it was concluded that, in general, cats are more sensitive to the glycosides than to the aglycones. The authors also stated that all cardenolides may be regarded as highly toxic and that sensitivity may vary considerably with route of administration. Studies on the mammalian absorption and excretion of cardiac glycosides have been reviewed extensively by Scheline (13). Nonpolar glycosides, such as digitoxin, are absorbed readily from the gastrointestinal tract, a property directly attributed to the low degree of hydroxylation of the steroid ring, which reduces ionization in gastrointestinal fluids (13). Subacute-to-acute signs of poisoning in cattle and sheep include restlessness, dyspnea, ruminal atony, cyanosis, frequent urination and defecation, tachycardia, arrhythmia, and ventricular fibrillation (94). The closely related bufadienolides produced similar signs of poisoning in cattle (229). Unexpected mortalities and unthriftiness in farmed red deer were attributed to foxglove poisoning and digitoxin was detected in the tissues of poisoned animals (230). Various treatments for cardiac glycoside poisoning in humans and livestock have been reviewed (94); these include the use of activated charcoal, potassium chloride, atropine, digoxin-specific antibodies, P-adrenergic blocking agents, procainamide, and phenytoin. Barbecue charcoal, however, was ineffective in the treatment of bufadienolide poisoning in cattle, presumably due to its low capacity for adsorption (231).

C. GlycosidesInducingGastrointestinal or Hepatic Effects 1. Saponins In spite of the vast array and wide distribution of saponins in higher plants, only a small number of species contain saponins that are toxic to mammals (104, 105, 232, 233). This has been attributed to the low oral toxicity of saponins arising from the negligible degree of absorption of these glycosides from the gastrointestinal tract (103, 104). Recently, however, there appears to be a growing list of saponin-containing forage species that are implicated in hepatogenous photosensitization of livestock, with sapogenins being detected as birefringent crystals in obstructed bile ducts (234, 235). Saponins are well known for their detergent properties, which are responsible for their important pharmacological action, the ability to disrupt and lyse cell membranes. This enhances their toxicity when given intravenously (236) and is responsible for their much greater toxicity to fish and fungi (103, 104). The toxic effect of saponins in mammals usually is initiated by the saponin interacting with intestinal mucosal membranes, causing permeability changes or loss of membrane-bound enzymes (103, 104). The toxic mode of action of saponins on cell membranes has been summarized as follows (103, 237). First, the hydrophobic aglycone portion of the saponin penetrates the inner phospholipid bilayer of the membrane, in which it specifically interacts with such lipid components as cholesterol. Next, the presence of the saponin aglycone (sapogenin)

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enriches the cholesterol complexes of localized membrane areas causing phase separation and producing conducting channels and leakage. Third, the disruption increases cell permeability, resulting in the efflux of K+, which predominates in intracellular fluid, and the influx of water and Na+, which predominates in extracellular fluid. Then, lysis occurs, with the leakage of macromolecules. Lysis of the mucosal cell membranes results in intestinal lesions and severe gastroenteritis (238). Under these conditions, saponins may be absorbed from the gastrointestinal tract and produce such systemic effects as liver damage, respiratory failure, violent convulsions, and coma (236). It has long been recognized that saponins are also antinutritional factors in livestock feeds such as alfalfa and the topic has been comprehensively reviewed by Cheeke (239). The adverse effects of sapogenins such as medicagenic acid have been reversed by the inclusion of dietary cholesterol, presumably because, in the gut, saponins form insoluble complexes with cholesterol or they perturb cholesterol-containing micelles (103). It is not known whether cholesterol supplements could alleviate acute saponin poisoning, but the protective effect of plasma cholesterol was recognized when the glycosides were given intravenously (236). Other components of plasma also have the capacity to depress the hemolytic activity of saponins (240). Conversely, saponin-containing foods have been shown to be effective in lowering plasma cholesterol levels in humans (103). The site of action appears to be the intestine, in which saponins can prevent the reabsorption of cholesterol after it is excreted in the bile. Cattle poisoned by saponin-containing seeds have been treated with calcium magnesium borogluconate given intravenously (233). 2. Cycasin Two distinct field diseases have been recognized in livestock poisoning by cycads and zamias (241, 242). One is a neurotoxic syndrome characterized by ataxia and permanent weakness of the hindquarters. To our knowledge, the neurotoxic agents involved in this syndrome have not been identified. A rare amino acid was implicated (243) but others have excluded this agent (244). The second syndrome is a hepatic and gastrointestinal disease attributed to cycasin, methylazoxymethanol-P-D-glucoside, and its disaccharide derivatives. Acute cycasin toxicity in sheep and cattle is characterized by hepatitis, gastroenteritis, hemorrhages, and liver cirrhosis. The histopathological changes have been described by Hooper (241). In chronic poisoning, animals lose appetite and develop mild liver cirrhosis and nephrosis. The labile aglycone methylazoxymethanol (MAM) is the lethal metabolite that is also a potent carcinogen in laboratory animals (242, 245). In normal rats, intestinal bacteria differed in their capacity to hydrolyze cycasin: this could explain the biological variation in the toxicity and carcinogenicity of cycasin (246). However, cycasin was nontoxic in germfree rats and the glycoside was excreted intact (246). It has been reported that the toxicity of the aglycone may be attributed, in part, to the oxidation product methylazoxynlethanal, formed by liver alcohol dehydrogenase. The putative unstable aldehyde was found to rapidly release highly reactive carbocations that interacted with cellular macromolecules (247). Others indicate that the methanediazonium cation would be generated from MAM (248). There is no known therapeutic treatment for cycasin poisoning.

3. Ranunculin The lactone protoanemonin, the unstable product derived from the glycoside ranunculin, contains a highly reactive exomethylene group. The irritant oil is responsible for the vesi-

Glycosides

325

cant properties attributed to members of the Ranunculaceae or the buttercup family (179, 180, 183). The oil is derived by autolysis during the maceration of plant tissue, producing an intense burning sensation when chewed or causing erythema, dermatitis, and blistering when applied to the skin (243). Ranunculin is a substrate for P-glucosidase and the hydrolysis products are glucose and the aglycone (S)-5-hydroxy-2-penten-4-olide (15,249), also known as (S)-(-)-5-hydroxymethyl-2(5H)-furanone (Aldrich MSDS no. 34,686-1). To our knowledge the toxicological properties of the aglycone have not been thoroughly investigated. The aglycone is stable and it is not an intermediate in the formation of protoanemonin. The latter is formed through an elimination reaction during autolysis of plant material or when the glycoside is subjected to heat during steam distillation. The aglycone is generated in vitro when the purified glycoside is incubated in rumen fluid. Protoanemonin is generated in vitro only when fresh or freeze-dried plant material is incubated (15). Thus, as with myrosinase, this is another example of the requirement for an endogenous plant enzyme to effect the release of a toxic metabolite. In summary, protoanemonin formation occurs in vivo due to the rapidity of an endogenous elimination reaction that well exceeds the velocity of any hydrolysis reaction. Ingestion of the plant material can cause gastric distress, including irritation of the digestive tract, abdominal pain, and diarrhea. When Cemtocephalus testiculatus was given by gavage to sheep, clinical signs of poisoning were similar to those seen under field conditions; they included weakness, depression, tachycardia, dyspnea, anorexia, diarrhea, and sometimes fever (250). Characteristic lesions included edema of the peritoneal surface of the reticulorumen, hemorrhage in the left ventricle, and congestion of heart, kidneys, liver, and lungs. The irritant effects of protoanemonin were recognized, but the direct effect of this agent on the cardiovascular system was questioned (250). In contrast, clinical signs of poisoning were not detected when R. ncris was fed to sheep and cattle for prolonged periods in Canada (251). However, the authors neglected to test for the toxin either qualitatively or quantitatively. Recent studies in Italy indicate that R. acris may contain high potential levels of protoanemonin (180), in agreement with earlier reports from England (252j. Very little therapeutic information is available on treatment of protoanemonin poisoning. Dermatitis may be treated by standard nonspecific procedures using compresses, corticosteroids, and antibiotics (243).

D. Glucosinolates 1. Goitrogens It is well established that some glucosinolate breakdown products are goitrogenic agents that cause hyperplasia and hypertrophy of the thyroid gland (33, 253, 254) with resultant muscular weakness and hypotonia. Two types of goitrogens are derived from glucosinolate glycosides. The cyclic thiouracils, such as goitrin (5-vinyl- 1,3,-0xazolidine-2-thione), are derived from the hydrolysis of glycosinolates containing a P-hydroxyl substituent. The thiocyanate ion (-SCN), a much less potent goitrogen, is derived from the breakdown of alkyl isothiocyanates or indole isothiocyanates (33, 253). The two types of goitrogens act on the thyroid gland in different ways. Thiocyanate inhibits the uptake of inorganic iodide by the thyroid gland, apparently in a competitive way since the inhibition can be reversed with iodide supplements (253). After the iodide

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is trapped in the gland, it is oxidized to iodine and the amino acid tyrosine is iodinated to form the hormonal precursors of thyroxine (TJ or triiodothyronine (T3). Goitrin and other thiouracil analogs interfere with tyrosine iodination and the coupling reactions that synthesize T4and T, and their effects cannot be reversed by iodide supplementation. Antithyroid drugs are available for the treatment of hyperthyroidism; these include the potent thiocarbamides methimazole and propylthiouracil and such other antithyroid drugs as the sulfonamides, amphenone, and chlorpromazine (253). It is abundantly clear that myrosinase catalyzes the hydrolysis of glucosinolates in higher plants, but the pathway of glucosinolate catabolism in mammals is unclear. A number of studies have concluded that progoitrin (2-hydroxy-3-butenyl glucosinolate) is hydrolyzed to goitrin by intestinal microflora, but the hydrolysis products have not been identified definitively (35, 255-257). A much higher excretion of progoitrin was demonstrated in germfree rats than in normal rats with commensal microflora (35). The assumption was made that the glycoside was “split” in normal rats to goitrin, but the detection or identification of goitrin in tissues or excreta was not reported (35). Sinigrin (2-propenyl glucosinolate) was incubated with 58 strains of intestinal bacteria, but only four strains were able to metabolize the glycoside (257). Theassumption was made that the disappearance of sinigrin from the cultures indicated sinigrin hydrolysis, but the hydrolysis product allylisothiocyanate (AITC) was not identified. A strain of bacteria, Pal-acolobuctnrn~ aerogenoides, that previously had metabolized progoitrin (256) also was incapable of sinigrin metabolism (257). In earlier studies, the putative conversion of progoitrin to goitrin by intestinal bacteria usually was monitored by UV detection, but the identity of the hydrolysis product was not confirmed by different criteria (255,256). Corroborative evidence on goitrin formation was obtained in one study with ovine rumen fluid but rates of hydrolysis of progoitrin (258) were much slower than those obtained for other glycosides in rumen fluid (20). Also, a large portion (50-60%) of theprogoitrin disappeared by unknown pathways (258). The presence of microbial myrosinase in ovine or bovine lumen fluid was not confirmed in our laboratory (15, 20, 34). The best evidence for a myrosinase enzyme of microbial origin comes from a recent screening of Lactobctcillus spp. in which a strain of L. ngilis showed a capacity for sinigrin degradation as evidenced by critical identification of endproducts (259). 2. Mustard Oils and Nitriles The possibility has been considered that plant enzymes could affect the rates of glycoside hydrolysis in rumen fluid (15, 34). Indeed, sinigrin hydrolysis in bovine rumen fluid was effected only in the presence of myrosinase of plant origin (20, 34); this well may explain the mechanism of mustard (Bmssicn species) seed poisoning in cattle. In acute cases, the ingested seeds usually are distributed abundantly throughout the digestive system (260, 261). Initially, the seed coats protect the plant enzyme from digestive enzymes, but as the seeds swell and split such vesicant mustard oils as AITC are released by autolysis. Characteristic lesions in the gastrointestinal tract included profuse edema of the forestomachs and abomasum (260) and mucosal necrosis and hemorrhage of the cecum and colon (261). In addition to thefamiliar isothiocyanates of the Cruciferae, thiocyanates and nitriles also are produced during the autolysis of glucosinolates (262). Allylthiocyanate is formed during stinkweed (Thlnspi atlwzse) autolysis (34), and the irritant oil may cause severe gastric distress (263). Nitriles such as 1-cyano-2-hydroxybutene, 1-cyano-3,4-epithiobu-

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tane, and l-cyano-2-hydroxy-3,4-epithiobutane can be generated during the autolysis of Bmssicn species and other crucifers (264, 265). Gould and co-workers have examined the pancreatotoxic and nephrotoxic effects of these nitriles in rats extensively (366-268). The toxicity of l-cyano-3,4-epithiobutaneto rats was confirmed in a separate study (269). E. GlycosidesAffectingReproduction(Isoflavones) The mammalian female steroid hormones estrone, estradiol, and estriol have been detected in at least 20 species of higher plants (270, 271). However, the estrogenic isoflavone glycosides, including the biosynthetically related coumestan aglycones, are much more abundant and they occur at much higher concentrations (270, 272). Structural features of the isoflavone aglycone that link the phytoestrogens to steroid estrogens are the sterically opposite and equidistant phenolic hydroxyls (7,4’) on the A and B rings attached to the pyran. This is readily evident with daidzein, genistein, and coumestrol, but with biochanin A and formononetin the 4’ hydroxyl is methylated. However, for bioactivation, O-demethylation can occur in the rumen or the liver (1 3, 272). The resultant unique configuration of the phytoestrogen aglycone permits specific binding to the protein receptor that initiates the estrogenic response (273). The phytoestrogens have a much lower affinity for the receptor and hence much less estrogenic potency than the steroid estrogens. For example, coumestan affinity for the receptor was approximately 5% and isoflavone affinity was less than 1% of the binding capacity of estradiol in vitro (274). Similarly, when uterine enlargement was measured with the mouse bioassay, estrone was 200 times more potent than coumestrol and the steroid was at least 7000 times tnore potent than the isoflavones (275). Metabolic studies on phytoestrogens largely have been conducted with aglycones (13). It is well established that formononetin is reduced to equol(7,4’-dihydroxyisoflavan) in sheep rumen contents and that equol is a more potent estrogen than formononetin, biochanin A, or daidzein (274). To our knowledge, the hydrolysis and the absorption of the isoflavone glycosides have not beenexamined. The isoflavones can occur as glucosides and as 6-0-malonylglucosides (276). The majority of the studies on the effects of phytoestrogens in livestock deal with sheep grazing legume pastures. The reproductive problems were first encountered over 50 years ago with the advent and establishment of subterranean clover (Trifolium subtermzeum) on pastures in westem Australia. A dramatic decrease in the fertility of sheep was noted. The failure to conceive was attributed to a change in the viscosity of the cervical mucus and the resultant impaired passage of spermatozoa. The condition was accompanied by cystic glandular hyperplasia of the cervix and uterus and lactation in nonpregnant ewes and wethers. The pathophysiology of the disorder has been reviewed by Davies (277). Clinical signs of reproductive disorders diminished with the introduction of new cultivars of clover that were low in formononetin, but a temporary infertility still prevails among ewes exposed to phytoestrogen-containing pastures. Continued exposure can lead to permanent infertility (273). Reproductive disorders on alfalfa pastures usually are associated with increases in coumestrol concentrations resulting from fungal infections, often referred to as a phytoalexin response (278). Coumestrol and related coumestans decrease the ovulation rate in ewes, hence the reproductive loss. The bioactivation of formononetin in the rumen contents of cattle has been demonstrated (279), but there is no satisfactory explanation for the reduced susceptibility of cattle, as compared to sheep, to the effects of phytoestrogen-containing pastures (280).

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Immunological approaches for the prevention of phytoestrogenic disorders have been partially successful under experimental and field conditions (162, 281).

F. GlycosidesInducingSpecificDisorders 1. Carcinogens The carcinogenicity of Cycas species to such laboratory animals as rats, mice, hamsters, and monkeys has been recognized for almost 30 years (282), but the cycads rarely induce tumors in livestock. Cycasin, the unique glycoside of the cycads, releases on hydrolysis the carcinogenic agent MAM, which can induce renal, hepatic, and intestinal tumors that are either benign or malignant (241, 282). It was postulated that MAM was metabolized to such an alkylating intermediate as diazomethane or methyldiazonium hydroxide, which are capable of nucleic acid methylation (282). It was also shown that MAM acetate can be oxidized in vitro with horse liver alcohol dehydrogenase, presumably to form the aldehydic form of MAM, methylazoxymethanal (MAMAL) (247). The alkylating activity of MAM was attributed to the methylcations (CH,’) that were generated from the unstable oxidation product MAMAL (247). Refer also to Sec. III.C.2. In contrast to the cycads, bracken fern (Pter-idim aquiliuunz) is carcinogenic to both laboratory animals and livestock. The carcinogen was identified as ptaquiloside, a novel sesquiterpene glycoside (283). Mutagenic p-coumaroyl esters of ptaquiloside also have been detected (138). The prominent feature of “bracken poisoning” in cattle is depressed bone marrow activity that results in leukopenia, thrombocytopenia, and hemorrhages of the urinary bladder that give rise to hematuria (284). In rats, the disease is manifested primarily in the ileum and cecum, in which the tumors are expressed as adenomas, adenocarcinomas, and sarcomas (283). The incidence of intestinal tumors was not reduced significantly when bracken was fed to germfree rats (285). In fact, there was a higher incidence of sarcomas in germfree rats; it was suggested that intestinal microflora of conventional rats might be involved in the degradation of the bracken carcinogen. In ruminants, the aglycone of ptaquiloside might be generated by microbial P-glucosidases of the rumen. Data on the stability of the aglycone at neutral pH are not available, but it is known that the glycoside is very unstable at pH greater than 9 or at pH less then 3 (138, 283). Thus, the stability range of ptaquiloside and its 2-hr half-life at pH 2 (283) should permit passage of the glycoside through the gastrointestinal tract of mammals. It also is possible that the aglycone is absorbed from the rumen in cattle, and because it is nonionic, the glycoside might be absorbed from the upper regions of the digestive tract in monogastric mammals (28). To our knowledge, the mechanism of ptaquiloside carcinogenicity has not been elucidated. Bracken is cyanogenic and hence may contain endogenous P-glucosidases that could affect the fate of the carcinogenic glycoside in damaged or macerated plant tissue. The p-hydroxypolystyrene glycosides of bracken also are implicated as possible carcinogens (283). Bracken also contains thiaminases (239).

2. Calcinogens The primary manifestations of vitamin D deficiency are rickets in infancy and osteomalacia in adulthood, generally characterized by decreased concentrations of calcium and phosphorus in bone (253). Hypervitaminosis D results from an intake of the vitamin considerably in excess of requirements and it leads to calcification of soft tissues such as joints, lungs, arteries, and kidneys. Several plant species, particularly in the Solanaceae, contain

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glycosides of vitatnin D, and the sterol aglycones can induce vitamin D intoxication or calcinosis, reminiscent of hypervitaminosis D. The 3-@glycosides are hydrolyzed by microbial enzymes of the Iumen and intestine (146, 152). Dietary or endogenous vitamin D3 is hydroxylated at CZ5,and stored in the liver. It is activated further in the kidney by hydroxylation at C,. This active, hormonal form of vitamin D is required for the synthesis of calcium carrier proteins involved in the transport of the cation from the intestine under conditions of calcium deprivation (286). The dihydroxylated, active form of vitamin D occurs in glycosidic forms in Solmurzl glnucophyllzm and Cestrurn diumurn. Consequently, consutnption of this exogenous form of vitamin D results in excess absorption of calcium and phosphate from the intestine. Other plant species such as T~isetlrm~c~~lescens also are calcinogenic (146), but the active principles have not been identified. Symptoms of calcinosis in both livestock and laboratory animals have been reviewed comprehensively by Weissenberg (146). There is no known antidote for acutely affected anitnals and the calcification persists in tissues.

3. Other Glycosides The 1970- 1980 decade saw the isolation and characterization of hypoglycemic agents from species of the Compositae such as Atmcfylis, Xanthium, and Wedelia. The toxic, diterpenoid glycosides frotn these sources were respectively named atractyloside, carboxyatractyloside, and wedeloside (126,287,288). Theglycosides can block the mitochondrial ADP/ATP energy-carrier system and the resultant cellular dysfunction is characterized by inhibited oxidative phosphorylation, accelerated anaerobic glycolysis, lactate production, and glycogenolysis. Clinical signs of poisoning in livestock include acute depression, weakness, and convulsions, and the accompanying pathological changes include nephrosis, gastric irritation, hepatic necrosis, and marked hypoglycemia (126). Antidotes for these toxic glycosides are not available. A cun-ent andcomprehensive review on the biochemistry and toxicology of atractyloside is available (289). Melilotoside, coumarinic acid P-D-glucoside, is the bound form of coumarin that can be present in high concentrations in sweet clover (Melilotus species). When the cisglucoside is hydrolyzed by endogenous glucosidase during plant tissue disruption, the aglycone undergoes a spontaneous lactonization to form free coumarin and gives off the characteristic odor of new-mown hay. The glycoside also occurs in other legumes and grasses (290, 291). Sweet clover poisoning is associated with moldy hay or silage in which enzymes of fungal origin can further metabolize the cinnamates to dicoumarol, a potent anticoagulant (292, 293). The precursor of dicoumarol is probably mold-produced 4-hydroxycoumarin and not coumarin itself (293). Cattle are most susceptible and signs of poisoning include lethargy, anemia, and the development of subdermal swelling in response to internal hemorrhaging, which is the cause of death (291, 292). Another coumarin glycoside, aesculin (7-hydroxycoumarin 6-P-glucoside), is believed to be the poisonous principle in horse chestnut (Aescrhs hippocnstunzm) (238). In hamsters, clinical signs of horse chestnut poisoning included depression, abdominal pain, ataxia, and dehydration (294). “Low-coumarin” cultivars of sweet clover are available commercially (295), but large forage-crop and soil-development areas still are seeded to “high-coumarin” Melilotus and thus the problem of sweet clover poisoning persists (296-298). The mycotoxin dicoumarol, a vitatnin K antagonist, interferes with the synthesis of thrombin, which is required for fibrin formation and blood clotting. The induced vitamin deficiency can be

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ameliorated with increased vitamin K supplies, especially vitamin K, given intramuscularly (299). Cyanomethylenecyclohexyl P-D-glucosides such as simmondsin and simmondsin felulate (300) are the toxic principles in jojoba meal, a by-product derived from the oilseed shrub Sirmlonclsia cnlifornicn and a potential source of protein for livestock. Poor weight gains and poor feed intake were reported for rats on diets formulated with jojoba meal or formulated with pure simmondsin (301). However, simmondsin administered to mice intraperitoneally showed little toxicity and itwas suggested that the nitrile-containing aglycone, not the intact glycoside, exerts the toxic effect (301,303). Similar noncyanogenic but nitrile-containing glucosides have been reported in Lithosyerrnum yurpUreo-caeruleu17z (303) and Thlictrzm species (304). Nitriles derived from glucosinolates are discussed in Sec. 1II.D. Finally, it should be noted that the cathartic agents in sennas (Cassia species) are anthraquinone glycosides (13, 305). Faba beans (Vicia.fabn) contain hemolytic glycosides that are also pyrimidine derivatives. These are considered in Sec. 1V.B in the discussion of human poisoning.

IV. ASPECTS OF HUMANPOISONING A.

The Potential for Transfer of Toxic Glycosides or Aglycones to Milk or Meat Products

Toxic glycosides occur in many feeds and poisonous forages that are consumed by livestock. The toxicants can have acute or chronic effects on the health of animals, in which case the carcass is not suitable or acceptable for human consumption. However, when morbidity is not plainly evident, as in the case of subacute or possibly chronic poisoning, it is possible that toxic glycosides or aglycones enter the human food chain through consumption of milk, meat, or their by-products. This section reviews the potential for transfer of these toxicants from livestock, mainly ruminants, to humans. Aliphatic nitrotoxins occur in poisonous species of Astragalus that are abundant on rangelands in the temperate regions of North and South America (306, 307). They also are found in a number of cultivated legumes (308-310). Both types of forages may be consumed readily by grazing livestock. In cattle and sheep, 3-nitropropionic acid is the lethal metabolite formed in the biotransformation of nitropropyl glucosides or nitropropanoyl glucose esters (31, 193). The half-life of 3-nitropropionic acids was estimated to be 22-1 16 min in circulating blood in cattle and 10-27 min in sheep (31 1). The decay of the acid in the circulatory system was always accompanied by nitrite formation in vivo, suggesting a precursor-product relationship (195, 196, 3 11). The rapid metabolism of the acid to inorganic nitrite suggests that the accumulation of 3-nitropropionate in tissues and the transfer of the nitroacid to food is unlikely. However. relatively large quantities of the acid were detected in the brain when nitropropionate was injected in rats (312). The acid can be excreted and detected in the urine of cattle when it is given intravenously (3 13). Cyanogenic glycosides occur in a number of cultivated forages and also are present in commonly browsed shrubs (9, 75, 314). HCN is formed in the lethal biotransformation of these glycosides by ruminal microorganisms. Free HCN, which is very soluble and volatile, is absorbed rapidly from the forestomachs (21), but it binds very rapidly to serum albumin (223,224),methemoglobin (3 15), and other macromolecules. Consequently, free

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HCN is detected only at very low levels in circulating blood. For example, in sublethal to lethal doses of cyanogenic glycosides, the blood levels of HCN were less than 1 part per million (ppm) (21, 316). Therefore, the transfer of free HCN from livestock to food products is unlikely. However, HCN (including bound HCN) is detoxified rapidly to thiocyanate (223), a well-recognized goitrogen that may be transferred to milk (254). Many cardiac-glycoside-containing species are indigenous to Africa but some have spread throughout the world as cultivated ornamentals, and others have escaped from gardens to become established weeds (94). Milkweeds that are native to North America are unpalatable to livestock but they often are foraged during conditions of drought or when the range has been overgrazed (94, 238). In contrast to the rapid metabolism of nitrotoxins and cyanogens, ingested cardenolides and bufadienolides probably are eliminated very gradually. There is no information on the rate of elimination of native cardiac glycosides in ruminants. In humans, however, it is known that the average half-life for digoxin is 1.7 days (3 17) and it has been detected in breast milk (94). Digitoxin, which is less polar, has an even greater half-life of 7 days (317). It is possible, therefore, that cardiac glycosides could enter the food chain if animals are slaughtered before these toxins are eliminated completely from tissues. Major feedstuffs, including alfalfa, clover, guar, sunflower, and lupine, contain triterpenoid saponins (103) but, unlike the cardiac glycosides, saponins, in most cases, are not acutely toxic. Saponins produce antinutritional effects such as reduced palatability and growth inhibition in nonruminant livestock (239) and only rarely are they lethal to ruminants (232, 233). In contrast to the cardiac glycosides, saponins are not absorbed readily from the gastrointestinal tract and hence their low oral toxicity to mammals (103, 104). Both types of glycosides are polycyclic, but saponin aglycones typically have five fused rings and cardenolides have four with an unsaturated lactone ring attached at C-17. The sugar substitutions and the structures of the aglycones well may determine the differences in permeability of the two types of toxicants. There also may be considerable variation in the extent to which saponins from different sources are absorbed. For example, the saponin aglycones of Parziculn species may be absorbed readily since their aglycone skeletons were detected in the bile ducts of sheep (234, 235). Cycasin and ranunculin yield unstable and reactive aglycones in the forestomachs of ruminants. These agents primarily cause inflammation of the gastrointestinal tract. Other organs also may be affected indirectly, but in ruminants it is unlikely that the aglycones are bioactive in tissues beyond the digestive tract. Important vegetable oils are obtained from the extraction of rapeseed and other cruciferous seed and the residual seed meal, containing glucosinolates, often is used for animal feedstuffs (121, 254). The use of low-glucosinolate rapeseed meal (canola meal) for livestock feed is becoming very widespread (239). Tookey et al. (254) and Fenwick et al. (121) reviewed studies on the transfer of such goitrogens as goitrin and thiocyanate to humans through cows’ milk. However, it was notestablished clearly and unambiguously that the occurrence of endemic goiters in humans could be attributed directly to the ingestion of milk from cows consuming cruciferous forages. It is clear, though, that off flavors and taints in milk can be attributed to the transfer of glucosinolate breakdown products (33, 121). One such example is allylthiocyanate (ATC), the mustard oil of stinkweed (Thlaspi nwerzse), which has a distinctly unpleasant, garlic-like odor (121). Unlike allylisothiocyanate, ATC is not significantly metabolized in rumen fluid and therefore it could be rapidly absorbed from the forestomachs ( 318). The transfer of isoflavone phytoestrogens from livestock to the human food chain is not viewed as a hazard since the levels

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of endogenous steroid estrogens are relatively high in humans compared to levels in other mammals (272). Bracken fern is widely distributed as a pasture weed and it may bereadily consumed by cattle and sheep, especially at early stages of growth or when other feed is scarce. Ptaquiloside, the major carcinogen of bracken (283), apparently is transferred to cows’ milk since bone marrow activity was impaired in animals consuming milk from brackenfed cows (3 19, 320). Milk consumption from dairy cattle grazing bracken or fed brackencontaining hay could pose a human health hazard. The potent anticoagulant dicoumarol has been detected at less than 6 ppm (wet weight) in the tissues of cattle suffering from sweet clover poisoning (296). However, dicoumarol showed a low oral toxicity to monogastric laboratory animals (293). At this time, there is no additional information on the potential transfer of other toxic glycosides, aglycones, or their metabolites from milk or meat products to humans.

B. Review of Incidents of Acute and Chronic Poisoning and Potential Risks Nitrotoxins do not usually occur in human foodstuffs, but a potential public health problem may develop with fermented foods in which 3-nitropropionic acid can occur as a mycotoxin from fungal sources such as Aspergillrls o p m e (321), A. soyne (322), and species of Arthrinium (207). Recently, the Arthrirliurn species were traced as the source of the nitroacid that causes deteriorated sugarcane poisoning in China, an acute and sometimes fatal syndrome of food poisoning mainly affecting the central nervous system (refer to Sec. 1II.A). The fruit of the karaka tree, which contains karakin, has been used for food (see Sec. 1I.A). Cyanogenic glycosides have a nutritional impact on a number of food sources, mostly in the tropics, where sorghum, bamboo, lima beans, and especially cassava are major components of the human diet (75, 314, 323). In Africa, goiter and a syndrome known as tropical ataxic neuropathy have been attributed to chronic cyanide intoxication resulting from protein-poor cassava diets. The subject of chronic cyanide uptake by humans has been reviewed comprehensively by Poulton (324). Cassava continues to be implicated in acute cyanide poisoning (325-327). Peracute and fatal cyanide poisonings occasionally have resulted from the ingestion of amygdalin-containing seeds, notably bitter almonds, apricot kernels, and chokecherry seeds, all of which contain hydrolytic enzymes for cyanide release (324, 328, 329). Ingestion of laetrile, a commercial preparation of amygdalin once advocated for the treatment of cancer, also has been implicated as the cause of at least two cases of fatal cyanide poisoning (330, 331). Because of the large oral doses of laetrile, sufficient cyanide probably was generated by the action of P-glucosidases associated with enteral microflora. Cardiac glycosides occur in a wide variety of tropical and subtropical plants and ingestion of these plants, especially by children, is a frequent cause of acute poisoning (332, 333). The oleanders (Neriunz species) are popular decorative shrubs with a worldwide distribution and they are probably the most notorious of the cardenolide-containing plants. Curative potions of oleander have a reputation for fatal and nonfatal poisoning (334, and oleander wood used as a cooking skewer can be lethal (94). Significant quantities of triterpenoid saponins occur in over 30 species of food plants, including such food staples as chick-peas and soybeans and such condiments as ginseng, nutmeg, and sage (103). However, most saponins are absorbed poorly from the gastrointes-

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tinal tract. When intoxication does occur in humans, the symptoms may be limited to severe gastroenteritis, usually resulting from excessive multiple exposures. A number of saponins have been implicated as skin irritants and allergens and their distribution in houseplants was reviewed in 1990 (243). Zamias and cycads have a long history of use as sources of food and medicine for humans (335, 336). Early explorers in Australia suffered from violent emesis and catharsis after the ingestion of raw Cycas or Macr-ozcrmia nuts (335). Chronic as wellas acute poisoning, manifested with seizures and coma, is attributed to the inadequate removal of cycasin, macrozamin, and other azoxyglucosides from cycad seeds or from unprocessed cycad flour (243). A degenerative motor neuron disease has been linked to the chronic ingestion of cycad seed and to its use as a topical medicine (336). Cases of ingestion of ranunculin-containing plants by humans are poorly documented. Shearer concluded that the blistering action of protoanemonin on the buccal mucous membranes would inhibit mastication to the extent that it would preclude the ingestion of sufficient plant material to cause acute toxicity (252). However, the irritant effects of protoanemonin to the skin are well documented; they include erythema and edema progressing to vesication (243). The crucifers are an economically important group of plants in ternls of cultivated sources of human foods and condiments. For example, cabbage, cauliflower, turnip, radish, watercress, and broccoli are all in the Cruciferae family. Such condiments as horseradish and the various mustards are also crucifers and their flavor is determined by glucosinolate breakdown products (121), which can be toxic at higher concentrations. Horseradish, for example, has been singled out as a condiment that may behazardous to humans, especially if the raw, unprocessed root is ingested (243). Mastication initiates autolysis with the resultant release of relatively large quantities of mustardoils, including allylisothiocyanate (AITC), a potent irritant of the skin and digestive system. At high concentrations, AITC is also a diaphoretic and lachrymatory agent (121, 243). However, AITC is also the most familiar of the mustard oils and is synthesized as a food additive to impart the distinctive flavor of mustard to a variety of foods. At normal and moderate rates of intake, AITC is not considered to be a health hazard to humans (337). The potential antinutritional effects of glucosinolates, including their potential goitrogenicity, have been considered by a number of authors (33, 121, 254) but toxicological data on long-term exposure to cruciferous vegetables, either raw or cooked, are not available at this time. Soybean and soybean products, long used as food staples in theOrient, are becoming increasingly popular as health foods in developed countries, as are alfalfa sprouts. However, in contrast to other phytoestrogen-containing foods (272), alfalfa and soybean contain much higher levels of isoflavones or coumestans (338, 339). Toxicological data are not available at this time on the effects of long-term exposure to these estrogenic and antiestrogenic glycosides and their aglycones (e.g., daidzein, genistein, and coumestrol). In Japan, the fronds of young bracken fern, or “fiddleheads,” are used as human food, usually after the plant material is processed in boiling water with woodash or sodium bicarbonate. Bracken is also pickled in salt and then washed with water. The content of ptaquiloside, the major carcinogen of bracken, apparently is reduced but not eliminated by these treatments (340). Tumor incidence was 78.5% in rats fed unprocessed bracken, butit decreased to 25%, lo%, and 4.7% for the ash, bicarbonate, and salt treatments, respectively (340). Thus, the carcinogenicity of bracken could be reduced mrkedly by the treatments, but weak carcinogenic activity still was retained in the treated bracken. Bracken usage in human diets definitely is not advocated (239).

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Calcinogenic glycosides usually do not occur in human foodstuffs and they are not viewed as a human health hazard. In fact, plant preparations containing the glycosides have therapeutic applications in both human and veterinary medicine (146j. Numerous cases of accidental or purposeful poisonings have been attributed to the rhizomes of AfrncQlisgumnzifei-a (birdlime thistle) and Cnllilepsis laureoln (oxeye daisy), which contain the hypoglycemic glycosides atractyloside and the more toxic carboxyatractyloside (126, 341). Pathological conditions seen in humans are similar to those in livestock and laboratory animals and they include convulsions, cerebral edema, gastric irritation and hemon-hage, nephrosis, and hepatocellular necrosis ( 136). Faba beans (broad bean, Vicia faba) contain the hemolytic glycosides vicine and convicine, which are also pyrimidine derivatives. The biological activity of the aglycones divicine and isouramil in humans and laboratory animals has been reviewed comprehensively by Marquardt and associates (190, 191). In short, the aglycones are potent reducing agents generating hydrogen peroxide under aerobic conditions. Stores of reduced glutathione, which are essential for cell membrane integrity, are depleted concomitantly. The supply of glutathione is replenished rapidly in normal red blood cells. However, if cells are deficient in glucose-6-phosphate dehydrogenase, the required reducing power for glutathione regeneration is not available and the accumulated H202can cause irreversible cell damage. The hemolytic disease in humans is known as favism; the epidemiology of the disorder has been reviewed by Mager et al. (342).

REFERENCES 1. Hosel, W. (1981). Glycosylation and glycosidases. In The Biochertristry of Plmts. Vol. 7, Secondmy Plmt Products (E. E. Conn, ed.), Academic Press, New York, pp. 725-755. 2. Evidente, A., Capasso, R., Cutignano, A., Taglialatela-Scafiti, O., Vurro, M., Zonno, M. C., and Motta, A. (1 998). Ascaulitoxin, a phytotoxic bis-amino acid N-glucoside from Aschochytn caulinn. Ph?ltoclrenristr?,48: 1 131-1 137. 3. Balke, N. E., Davis, M. P., and Lee, C. C. (1987). Conjugation of allelochernicals by plants: glucosylation of salicylic acid by Averln sntivn. In Allelochenricals: Role irz Agricralt~tr-eand Forestry, ACS Synlposium Series 330, American Chemical Society, Washington, D.C., pp. 214-227. 4. Spencer, K. C. (1987). Specificity of action of allelochernicals: diversification of glycosides. In Allelochenricnls: Role in Agriculture nnd Forestry, ACS Sytnposium Series 330, American Chemical Society. Washington, D.C., pp. 275-288. 5. Bell, E. A. (1981). Thephysiological role(s) of secondary (natural) products. In The Biochemistry of Plmts, Vol. 7, Secorrdm? Plant Products (E. E. Conn, ed.), Academic Press, New York, pp. 1-19. 6. Rosenthal, G. A., and Janzen, D. H. ( 1979). Her-bi~~ores, Their hrtemction Mith Secorrdary Plarrt Metabolites, Academic Press, New York. 7. Gershenzon, J. (1984). Changes in the levels of plant secondary metabolites under water and nutrient stress. In Receilt Advames in Phytochemistry, Phytochenzical Adaptatiom (B. N. Timmerman, C. Steelink, and F. A. Loewus, eds.), Plenum Press, New York, pp. 273-320. 8. Williams, M. C., and James, L. F. (1983). Effects of herbicides on the concentration of poisonous compounds in plants: a review, Anzer. J. Vet. Res., 44:2420-2422. 9. Majak, W., McDiarmid, R. E., Hall, J. W., and Cheng, K.-J. (1990). Factors that determine rates of cyanogenesis in bovine ruminal fluid in vitro, J. Anim. Sci., 68:1648-1655. 10. Dey, P. M., and Campillo, E. D. (1984). Biochemistry of the multiple forms of glycosidases

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11. 12. 13. 14.

15. 16.

17. 18. 19.

20.

21. 22. 23. 24. 25. 26. 27. 28.

29. 30. 31. 32. 33. 34.

335

in plants. Tn Advances in Enzyntology rind Reluted Areas of Molecular Biology, Vol. 56 (A. Maister, ed.), J Wiley, New York, pp. 141-250. Schmidt, E. S., Newton, G. W.. Sanders, S. M., Lewis, J. P.,and Conn. E. E. (1978). Laetrile toxicity studies in dogs, JAMA, 239:943-947. Adewusi, S. R. A., and Oke, 0. L. (1985). On the metabolism of amygdalin. 1. The LD, and biochemical changes in rats, Cun. J. Physiol. Phnrrmcol., 63:1080-1083. Scheline, R. R. (1991). Hurzdbook of Marrtnrnlian Metabolisnl of Plant Compounds, CRC Press. Boca Raton. F1. Conn, E. E. (1979). Cyanogenic glycosides. In Internutional Revie” of Bioclzerrzistl?, Vol. 27, Biochentistry of Nutrition (A. Neuberger and T. H. Jukes, eds.), University Park Press, Baltimore, Md.. pp. 21-32. Majak, W. (1999). Unpublished data. Gault, H., Kalra, J., Ahmed, M., Kepkay, D.. and Barrowman, J. (1980). Influence of gastric pH on digoxin biotransformation, 1. Tntragastric hydrolysis. Clin. Plzal-rrtacol. Ther.. 27: 1621. Hobson, P. N. (1988). The Runten Microbial Ecosystem, Elsevier Appl. Sci., New York. Majak, W., and Cheng, K.-J. (1984). Cyanogenesis in bovine rumen fluid and pure cultures of rumen bacteria, J. Anim. Sci., 59:784-790. Majak, W. and Cheng, K.-J. (1987). Hydrolysis of the cyanogenic glycosides amygdalin, prunasin and linamarin by ruminal microorganisms, Can. J. Anim. Sci., 67: 1133-1 137. Majak. W. (1991). Biotransformationoftoxicglycosidesby ruminal microorganisms.In Natural Toxins: Chenzistp, Toxicology and Safety (R. F. Keeler, N. B. Mandaya, and A. T. Tu, eds.), Alaken, Fort Collins, Colo., pp. 86-103. Majak, W.,Udenberg, T.. Clark, L. J., and McLean, A. ( 1980). Toxicity of Saskatoon serviceberry to cattle, Can. Vet. J., 21:74-76. Sakata, M., Yoshida, A., Yuasa, C., Sakata.K., and Haga, M. (1987).Toxicity of D.L-mandelonitrile-P-D-glucoside, “prulaurasin” in rat, J. Toxicol. Sci., 12:47-55. Wolin, M. J. (1981). Fermentation in the rumen and human large intestine, Sciewe. 213: 1463-1468. Williams, A. G., and Strachan, N. H. (1984). The distribution of polysaccharide-degrading enzymes in the bovine rumen digesta ecosystem, Crarr: Microbiol., 10215-220. Williams, A. G., Withers, S. E., and Coleman, G. S. (1984). Glycoside hydrolases of rumen bacteria and protozoa, Cwr. Micl-obiol., 10:287-294. Rowland, T. R., Mallett, A.K., Bearne, C.A., and Farthing. M. J. G. (1986). Enzymeactivities of the hindgut microflora of laboratory animals and man, Xenobiotica, 16519-523. Frakes, R. A., Sharnla, R. P., and Willhite, C. C. (1986). Comparative metabolism of linamarin and amygdalin in hamsters, Food Cltern. Toxicol., 24:417-420. Edwards, V. T., Jones, B. C., and Hutson. D. H. (1986). A comparison of the metabolic fate of phenol, phenyl glucoside and phenyl 6-0-malonyl glucoside in the rat. Xenobioticu, 16: 801-807. Maduagwu, E. N., and Umoh, I. B. (1986). Biliary excretion of linamarin in the Wistar rat after a single dose, Biocltent. Plmrnucol., 35:3003-3006. Maduagwu, E. N. (1989). Metabolism of linamarin in rats, Food Chem Toxicol., 27:451454. Majak, W., and Pass, M. A. (1989). Aliphatic nitrocompounds. In Toxicants of Plant Origin, Vol. 2,G/ycosides (P. R. Cheeke, ed.), CRC Press, Boca Raton, Fl., pp. 143-159. Majak. W.. Pass. M. A.. and Madryga. F. J. (1983). Toxicityof miserotoxin and its aglycone (3-nitropropanol) to rats, Toxicol. Lett., 19:171-178. Fenwick, G. R., Heaney, R. K., and Mawson. R. (1989). Glucosinolates. In Toxicants of Plant Origin, Vol. 2. Glycosides (P. R. Cheeke,ed.). CRC Press,Boca Raton, Fl., pp. 2-41. Majak. W.. McDiarmnid, R. E., Benn, M. H.,and Willms. W. D. (1991). Autolysis of Thluspi al-vense in bovine rumen fluid, Phytochemistry, 30: 127- 129.

336

Majak and Benn

35. Macholz, R., Kujawa. M., Schulze, J., Lewerenz, H. J., and Schnaak, W. (1985).The metabolism of some xenobiotics in germ-free and conventional rats, Arch. Toxicol.. 8 (Suppl.):373376. 36. Takechi, M.. and Tanaka, Y. (1992). Structure-activity relationships of synthetic methyl oleanolate glycosides. Plzytoclzemistry 3 1:3789-379 1. 37. Takechi, M., and Tanaka, Y. (1990).Structure-activity relationships of the saponin a-hederin. Plzytochemistty, 29:45 1-452. 38. Seiber, J. N.. Lee, S. M., and Benson, J. M. (1983). Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae). In Handbook of Natural Toxins. Vol. 1 , Plmt and Fu~rgalTo.vim (R.F. Keeler and A. T. Tu, eds.), Marcel Dekker, New York, pp. 43-83. 39. Baker. D. C., Keeler, R. F., and Gaffield, W. (1991). Toxicosis from steroidal alkaloids of Solmw?z species. In Hmdbook of Ncrtzrrcrl Toxins, Vol. 6, To.ricology of Plurlt and Furlgal Tosins (R. F. Keeler and A. T. Tu, eds.), Marcel Dekker. New York, pp. 71-82. 40. Matsumoto, Y., Oshako, M., and Sakata, R. (1991). Permeability of glycosides through human erythrocyte membranes, Clzem. Plzarm. Bull.. 39: 1346- 1348. 41. Puls. W., Keup, U., Krause, H. P., Muller. L., Schmidt, D. D., Thomas, G., and Truscheit, E. (1980). Pharmacology of a glucosidase inhibitor. Front. Hormorze Res., 7235-247. 42. Niwa, T., Inouye, S., Tsuruoka, T., Koaze. Y., and Niida, T. (1970). “Nojirimycin” as a potent inhibitor of glucosidase, Agr. Biol. Chenl., 34:966-968. 43. Evans, S. V., Fellows, L. E.. Shing, T. K. M.. and Fleet, G. W. J. (1985). Glycosidase inhibition by plant alkaloids which are structural analogues of monosaccharides. Phytochenlistp, 24:1953-1955. 44. Durham, P. L.. and Poulton, J. E. (1989). Effect of castanospermine and related polyhydroxyalkaloids on purified myrosinase from Lepidtrnz sutilwn seedlings, Plarlt Physiol., 90: 48-52. 35. Lizotte, P. A., and Poulton, J. E. (1988). Catabolism of cyanogenic glycosides by purified vicianin hydrolase from squirrel’s foot fern (Davallia tricllomunoides Blume), Plcrnt Physiol., 861322-323. 46. Caron, G., and Withers, S. G. (1989). Conduritol aziridine: a new mechanism-based glucosidase inactivator, Biochern. Biophys. Res. Commm., 163:495-499. 47. Field, R. A., Haines, A. H., Chrystal. E. J. T., and Luszniak. M. C. (1 991). Histidines, histamines and imidazoles as glycosidase inhibitors, Biocllent. J., 273:885-889. 48. Tong. M. K., Papandreou. G., and Ganem. B. (1990). Potent, broad-spectrum inhibition of glycosidases by an amidine derivative of D-glucose, J. Am. Chew. SOC., 1126137-6139. 49. Nash, R. J., Watson, A. A., Winters, A. L., Fleet. G. W. J.. Wormald, M. R., Dealler, S., Lees, E.. Asano, N., and Molyneux, R. J. (1998). Glycosidase inhibitors in British plants as causes of livestock disorders. In Toxic Plants arzd Other Natural Toxicants (T. Garland and A. C. Barr, eds.), CAB International, New York, pp. 276-284. 50. Molyneux, R. J., and Ja1nes.L.F. ( 1992).The chemistry ofpyrrolizidine and indolizidinealkaloids and their occurrence in toxic range plants. In Poisonous P h t s (L. F. James, E. M. Bailey, Jr., P. R. Cheeke, and M. P. Hegarty, eds.), Iowa State Univ. Press. Ames. pp. 198-203. 51. Molyneux. R. J., McKenzie, R. A., O’Sullivan, B. M., and Elbein, A. D. (1995). Identification of the glycosidase inhibitors swainsonine and calystegine B, in weir vine [Ipomoea sp. Q6 (Afs Ccrlobrtr)]and correlation with toxicity, J. Not. Prod.. 58:878-886. 52. Molyneux. R. J., Pan, Y. T., Goldmann. A., Tepfer, D. A., and Elbein, A. D. (1993). Calystegins. a novel class of alkaloid glycosidase inhibitors, Arch. BiocAern. Bioplys., 304:81-88. 53. Goldstein, J. L., and Swain, T. (1965).The inhibition of enzymes by tannins, Plzytochemistn, 4:185-192. 54. Haslatn, E. (1974). Polyphenol-protein interactions, Biocherrz. J., 139985-288. 55. Bae, H. D., McAllister, T. A., Yanke, J., Cheng, K.-J., and Muir. A. D. (1993). Effect of condensed tannins on endoglucanase activity and filter paper digestion by Fibrobncter succirlogeizes S85, Appl. Emiror1. Micr-obiol., 592132-21 38.

Glycosides

337

56. McAllister, T. A., Bae, H. D., Yanke, L. J., and Cheng, K.-J. (1994). Effect of condensed tannins from birdsfoot trefoil on endoglucanase activity and the digestion of cellulose filter paper by ruminal fungi, Can. J. Microbiol., 40298-305. 57. Connor. H. E. ( 1977). The Poisorzozts Plarzts of New Zealand, Government Printer, Wellington, New Zealand, p. 61. 58. Skey, W. (1 872). On the bitter substance of the karaka nut, Trans. NZ Imt., 4:316-321. 59. Finnegan, R. A.. and Stephani, R. A. (1968). The structureof karakin, Lloydia, 33:491-492. 60. Moyer, B. G., Pfeffer, P. E., Valentine, K. M., and Gustine, D. L. (1979). 3-NitropropanoylD-glucopyranoses of Coqnocarpus laevigatus, Phytochernistry, 8: 111- 114. 61. Majak, W.. and Benn, M. (1994). Additional esters of 3-nitropropanoic acid and glucose from the fruit of the New Zealand Karaka tree, Coqvzocarpus laevigatus, Plzytochemistq, 35:901-903. 62. Stermitz, F. R., Norris, F. A., and Williams, M. C. (1969). Misertoxin, a new naturally occurring nitro compound, J. Ant. Chem. SOC.,91:4599-4600. 63. Sternlitz, F. R., Lowry, W. T., Norris, F. A., Buckeridge, F. A., and Williams, M. C. (1972). Aliphatic nitro compounds fromAstragalus species, Phytochemistry, 11:1117- 1124. 64. Majak, W., and Benn, M. H. (1988). 3-Nitro-I-propyl-P-gentiobioside from Astragalus miser var. serotiruts, Phytochertzistry, 27: 1089- 109 1. 65. Benn, M. H., and Majak, W. (1989). 3-Nitro-l-propyl-P-~-laminaribioside from Astragalus miser var. serotirzus, Phytochernistn, 282369-237 1. 66. Long, M., Benn, M., Majak, W., and McDiamid, R. (1992). 3-Nitropropyl glycosides of Astragalus miser var. serotinus, Plzytoclzernistry, 3 1:321-323. of misertoxin 67. Majak, W., and Bose, R. J. (1974). Chromatographic methods for the isolation and detection of aliphatic nitro compounds, Phytochemistp, 13: 1005-1010. 68. Matsunloto, H., Unrau, A. M., Hylin, J. W., and Temple, B. (1961). Spectrophotometric determination of 3-nitropropanoic acid in biological extracts, Arznl. Clzewz., 33: 1442-1444. 69. Stemitz, F. R., and Yost, G. S. (1978). Analysis and characterization of nitro compounds from Astragalus species. In Effects of Poisorlous Plants 011 Livestock (R. F. Keeler, K. R. Van Kampen, and L. F. James, eds.), Academic Press, New York, pp. 371-378. 70. Williams, M. C., Stermitz, F. R.,and Thomas, R. D. (1975). Nitro compoundsin Astragalus species, Phytocheruistnv, 14:2306-2308. 71. Robinson, M. E. (1930). Cyanogenesis in plants, Biol. Rev., 5:126-144. 72. Dilleman, G. (1958). Compos6s cyanogenetique. Hatzdbuclz der P~an~eizplzysiologie. Vol. 8, Der StickstofjcLlnuatz (W. Ruhland, ed.), Springer-Verlag, Berlin, pp. 1050-1075. 73. Conn, E. E. (1980). Biosynthesisof cyanogenic glycosides. In Cyanide in Biology, (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, eds.), Academic Press, London, pp. 182- 196. 74. Seigler, D. S. (1981). Cyanogenic glycosides and lipids: Structural types and distribution. In Cyanide in Biology (B. Vennesland,E. E. Conn, C. J. Knowles, J. Westley, andF. Wissing, eds.). Academic Press, London, pp. 133-143. 75. Tewe, 0. O., and Iyayi, E. A. (.1989). Cyanogenic glycosides. In Toxicants ofplant Origin, Vol. 11, Glycosides (P. R. Cheeke, ed.), CRC Press, Boca Raton, Fla., pp. 43-60. 76. Poulton, J. E. (1990). Cyanogenesis in plants, Plant Physiol., 92401-405. 77. Janz, E. R..and Inoka Uluwaduge. D. (1997). Biochemical aspects of Cassava (Manihot esczdenta Cranz.) with a special emphasis on cyanogenic glucosides-a review, J. Natl. Sci. Courz. Sri Larlka, 25:l-24. 78. Conn, E. E. (1988). Biosynthetic relationship among cyanogenic glycosides, glucosinolates, and nitro compounds. InBiologically Active Natural Products. Potential Use in Agricztlture (H. G. Cutler, ed.), ACS Symposium Series 380, American Chemical Society, Washington, DC, pp. 143-154. 79. Ettlinger, M., andEyjblfsson, R.( 1972). Revisionof the structureof the cyanogenic glucoside triglochinin, J. Clzem. SOC. Clzem.Cornnzzln., 572-573.

338 80.

81.

82. 83.

84. 85. 86.

87. 88. 89. 90. 91.

92. 93.

94. 95.

96. 97. 98. 99. 100. 101. 102.

Majak and Benn Kim, H. S., Jeffrey, G. A.. Parke, D., Clapp. R. C., Coburn, R. A., and Long, L.. Jr. ( 1970). The x-ray crystallographic determination of the structure of gynocardin, J. Chern. SOC.Chenz. Conlmltn. 38 1-382. N'ahrstedt, A. (1975). Die isomerisierung von Amgydalin und Homologen, Arch. Pluzrrn., 308~903-910. Eyj6lfsson, R. (1970). Recent advances in the chemistry of cyanogenic glycosides, Fortsch. Cllern. Org. NcrturstofSe, 8:74-108. Mao, C. H.. and Anderson. L. (1965). Cyanogenesis in Sorghunz Iwlgare. TI. Mechanism of the alkaline hydrolysis of dhurrin (p-hydroxymandelonitrile glucoside), J. Org. Chew., 30: 603-607. Nambisan. B. (1994). Evaluation of the effect of various processing techniques on cyanogen content reduction in cassava, Acta Hort., 375:193. Sanni, M. 0..Sobaminwa, A. O., Eyinla. C. M., and Rossling, H. (1994). Safety aspects of processing Cassava to Gari in Nigeria, Acta Hort., 375227. Nahrstedt. A. (1981). Isolation and structure elucidation of cyanogenic glycosides. In Cyanine in Biology (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, eds.), Academic Press, London, pp. 145-181. Waterman, P. G., and Gray, A. J. (1987). Chemical systematics. Nut. Prod. Rep., 3:175-203. Spencer, K. C., and Seigler. D. S. (1985). Cyanogenic glycosides and the systematics of the Flacourtiaceae, Biochem. Syst. Ecol.. 1342 1-43 1. Spencer. K. C., Seigler, D. S., and Fraley, S. W. (1985). Cyanogenic glycosides of the Turneraceae, Biochenl. Syst. Ecol., 13:433-435. Chida, N., Yamada, K.. and Ogawa. S. (1991). Total synthesis of simnondsin, J. Chem SOC. Clzenz. Commn., 588-590. Abbott, T. P., Nakamnia, L. K., Buchholz, G., Wolf, W. J., Palmer, D. M., Gasdorf, H. J., Nelson, T. C., and Kleiman. R. (1991). Processes for making animal feed and protein isolates from jojoba meal, J. Agric. Food Chem.. 39:1488-1493. Fieser, L. F., and Fieser, M. (1959). Steroids, Rheinhold, New York, pp. 727-809. Stoll, A., and Jucker, E. (1955). Phytosterine, Steroidsaponine und Herzglykoside. In Moderne methoden der PJlcrnzenanrrlyse (K. Paech and M. V. Tracey. eds.), Springer-Verlag. Berlin, pp. 141-271. Joubert, J. P. J. (1989). Cardiac glycosides. In Toxicants of Plant Origin, Vol. 11, Glycosides (P. R. Cheeke, ed.), CRC Press, Boca Raton, Fla., pp. 63-96. Seiber, J. N., Lee, S. M., McChesney, M. M., Watson, T. R., Nelson, C. J., and Brower, L. P. ( 1985). New cardiac glycosides (cardenolides) from Asclepicrs species. In Plant Toxicology (A. A. Seawright, M. P. Hegarty, L. F. James, and R. F. Keeler, eds.), Queensland Poisonous Plants Committee, Yeerongpilly, pp. 427-437. Krenn, L., and Kopp. B. (1998). Bufadienolides from animal and plant sources, Phytoclzenlisti?, 4811-39. Skou, J. C. (1986). William Withering-the man and his work. In Cardiac Glycosides 17851985 (E. Erdmann, K. Greef, and J. C. Skou, eds.), Steinkopf Verlag, Darmstadt, pp. 1-10. Wagner, H., Bladt, S., and Zgainski, E. M. (1984). Plant Drug Analysis, Springer Verlag, Berlin. Marston, A.. and Hostettmann, K. (1991). Modern separation methods. Nut. Prod. Rep., 8: 391-413. Robien, W.. Kopp, B.. Schabl, D., and Schwarz, H. (1987). Carbon-13 NMR spectroscopy of cardenolides and bufadienolides, Prog. NMR Spectrosc., 19: I 3 1- 181. Xu, J.. Takeya, K., and Itokawa, H. (1990). Pregnanes and cardenolides from Periploca sepiunl. Plzytoclzenzistn. 29:344-346. Martin, R. A., Lynch, S. P., Schmitz, F. J., Pordesimo, E. O., Toth, S., and Horton. R. Y. (1 991). Cardenolides from Asclepias asperulusubsp. capriconzu and A. viridis, Phytochernistry, 3013935-3939.

Glycosides

339

103. Oakenfull, D., and Sidhu, G. S. (1989). Saponins. In Toxicants of Plant Origin, Vol. 11, Glycosides (P. R. Cheeke, ed.), CRC Press, Boca Raton, Fla. pp. 97-141. 104. Price, K.R., Johnson, I. T., and Fenwick, G. R. (1987). The chemistry and biological significance of saponins in foods and feeding stuffs, Crit. Rev. Food. Sci. Nutr., 26:27-135. 105. Hostettmann. K., and Marston, A. (1995). Saponins, Cambridge University Press, Cambridge. 106. Masood, M., Minocha, P. K., Tiwari, K. P., and Srivastava, K. C. (1981). Narcissiflorine. narcissiflorinine and narcissifloridine, three triterpenoid saponins from Ailentone narcissijlora, Phytochenlistry, 20: 1675-1 679. 107. Tiwari, K.P.. and Minocha, P. K. (1980). Pavophylline, a new saponin from the stem of Palyonin zeylandica, Plzytochernistry, 19:701-704. 108. Mahato, S. B., and Nandy, A. K. (1991). Triterpenoid saponins discovered between 1987 and 1989, Phytochemistry, 30: 1357- 1390. 109. Mahato, S. B.. Sarkar, S. P., and Poddar, G. (1988). Triterpenoid saponins, Plzytochenlistry, 27:3037-3067. 110. Chandel, R. S., and Rastogi. R. P. (1980). Triterpenoid saponins and sapogenins: 19731978, Ph?ltoc~lentist~?,. 19:1889-1908. 111. Agarwal, S. K., and Rastogi, R. P. (1974). Triterpenoid saponins and their sapogenins, Phytochenlistn, 132623-2645. 112. Mahato, S. B., Gangley, A. N.. and Sahu, N. P. (1982). Steroid saponins, Phytoclzemistry, 21 1959-978. 113. Griffiths, R. A., Watson, A. A., Kim, H., Asano. N., Sharp, H. J., Jones, M. G., Wormald, M. R.. Fleet, G. W. J., and Nash, R. J. (1996). The isolation and identification of the 3-0-pD-glucopyranoside of 1a,2p, 3a,6a-tetrahydroxy-nor-tropane(calystegine Bl), Tetrahedron Lett., 373207-3208. 114. Tschesche, R., and Wulff, G. (1973). Chemie and Biologie der Saponine, Fortsch. Chenl. Org. Naturstofle, 30:46 1-606. 115. Ngao, T., Tanaka, R., Shimokawa, H., and Okabe, H. (1991). Studies on the constituents of Aster scaber Thunb. 11. Structures of echinocystic acid glycosides isolated from the roots, Cllenl. Pharrw Bull. Jpn., 39:1719-1725. 116. Percival, G. C., and Dixon, G. R. (1997). Glycoalkaloids. In Handbook of Plant and Fmgal Toxicnuts (J. P. F. D’Mello, ed.) CRC Press, Boca Raton, Fla., pp. 19-35. 117. Phillips, B. J., Hughes, J. A., Phillips, J. C., Walters, D. G., Anderson, D. and Tahourdin, C. S. M. (1996). A study of the toxic hazard that might be associated with the consumption of green potato tops, Food Clzern. Toxicol., 34:438-439. 118. Kjaer, A. (1 960). Naturally derived isothiocyanates (mustard oils) and their parent glucosides, Fortsclt. Cllem. Org. Natzsrstofle, 18:122- 176. 119. Ettlinger, M. G., and Kjaer, A. (1968). Sulphur compounds in plants. In Recent Advances in Phytochemistr?, Vol. 1 (T. J. Mabry, R. E. Alston, and V. C. Runeckles, eds.). AppletonCentury-Crofts, New York, pp. 59-144. 120. Underhill, E. W. (1980). Glucosinolates. In Encyclopedia of Plant Plzysiology, New Series, Vol. 8. Secondaiy Plant Products (A. Pirson and M. H. Zimmerman. eds.), Springer-Verlag, Berlin, pp. 493-5 11. 121. Fenwick, G. R., Heaney, R. K., and Mullin, W. J. (1983).Glucosinolates and their breakdown products in food and food plants, Crit. Rev. Food Sci. Nldtr., 18:123-201. 122. Astwood, E. B., Greer, M. A., and Ettlinger, M. G. (1949). ~-5-Vinyl-2-thioxazolidone,an antithyroid compound from yellow turnips and brassica seeds, J. Biol. Chenz., 181:121- 130. 123. McGregor, D. I., Mullin, W. J., and Fenwick, G. R. (1983). Analytical methodology for determining glucosinolate composition and content, J. Assoc. OflAnal. Cltenl., 66:825-849. 124. Kjaer, A. (1974). The natural distribution of glucosinolates: a uniform class of sulphur-containing glucosides. In Nobel Syrr1posium, Vol. 25, Chenlisty in Botanical Class(ficatiorl (G. Bendz and J. Santesson, eds.), Academic Press, London, pp. 229-231.

340

Majak and Benn

125. Santi, R.,and Luciani, S. (eds.) (1978)Atrnctyloside., Chentistry, Biochemistryarzd Plznrmacology. Piccin Medical Books, Padova. Italy, pp. ix-xii. 126. Cole, R. J., Cutler, H. G., and Stuart, B. P. (1989). Carboxyatractyloside. In Toxicants sf PInnt Origin, Vol. 11, Glycosides (P. R. Cheeke, ed.),CRC Press, Boca Raton, Fla., pp.253363. E., Meier, U., and Wiedenfeld, H. ( 1 997). Toxic diterpene 127. Wernery, V.. Bourauel, T.. Roeder, glycosides from Iphionn auclteri, Fitoterapin, 68:278-280. 128. Lewis,I. A. S., MacLeod. J. K., and Oelrichs, P. B.(1981).The toxic extractivesfrom Wedelia asperima. 11. The structure of wedeloside, a novel diterpenoid aminoglycoside, Tetrahedron, 37:4305-43 11. 129. MacLeod, J. K., Lewis. T. A. S., Moeller, P. D. R., and Oelrichs, P. B. ( 1990). The toxic extractives from Wedelia asperima. 111. Structures of two naturally occurring rhanmosyl analogues of wedeloside, J. Nat. Prod., 53:1256-1261. An enzyme immunoassay for atractylo130. Bye, S. N.. Coetzer,T. H. T.,and Dutton, J. F. (1990). side, the nephrotoxin of Cnllilepsis latlreola (impila), Toxicon, 25:997-1000. 131. Aeschbach, R., Kusy. A., and Maier, H. G. (1982). Diterpenes of coffee. I. Atractyligenin, Z. Lebensm. Unters. Forsclz., 175:337-341 (in German). Chenz. Ber., 132. Obermann, H.,and Spiteller, G. (1976). Die Strukturen der “Kaffeatracyloside,” 109:3450-3461. 133. Obermann, H.. Spiteller, G.,and Heyer, G.A. (1973). Struktur eines Menschenham isolierten CI,,-terpenoids-2P-hydroxy-l 5-oxoatractylan-4a-carbonsaure, Chem. Ber., 106:3506-35 18. 134. Evans, I. A., and Mason, J. (1965). Carcinogenic activity of bracken. Nature, 208:913-914. 135. Niwa, H., Ojika, M., Wakarnatsu, K.. Yamada, K., Hirono, I., and Matsushita, K. (1983). Ptaquiloside. a novel norsesquiterpene glucoside from bracken, Pteridiu~?~ aqrlilinzm var. lntiusculwn, Tetrahedron Lett., 24:4 117-4 120. 136. Niwa, H., Ojika,M., Wakamatsu, K., Yamada, K., Ohba, S., Saito, Y., Hirono, I., and Matsushita, K. (1983). Stereochemistry of ptaquiloside, a novel norsesquiterpene glucoside from bracken, Pteridiurn aquilinum var. latirlsculum, Tetrahedron Lett., 25:537 1-5372. 137. Fukuoka. M.. Kuroyanagi, M., Yoshihara. K.,and Natori, S. (1987). Chemicaland tclxicological studies on bracken fern, Pteridiurn aqzdinum var. latizlsculurn.11. Structures of pterosins, sesquiterpenes having 1-indanone structures, Chern. Plzarm. Bull. Jpn., 262365-3385. 138. Saito, K., Nagao, T., Matoba, M., Koyama, K.. Natori. S., Murakanu, T.,and Saiki, Y. (1989). Chemical assay of ptaquiloside, the carcinogen of Pteridi~maquilirrurn, and the distribution of related compounds in the Pteridaceae. Plzytocltemistty, 28:1605-1611. 139. Saito, K., Nagao, T., Takatsuki, S.. Koyama, K., and Natori, S. (1990). The sesquiterpenoid carcinogen of bracken fern, and some analogues, from the Pteridaceae, Phytochenzistn, 29: 1475-1479. 140. Koyama, K., Takatsuki, S., and Natori, S. (1991). Dennstoside A, an analogue of ptaquiloside, from Dennstaedtia scnbra, Phytochemistry, 30:2080-2082. 141. Agnew, M. P., and Lauren, D. R. (1991 ). Determination of ptaquiloside in bracken fern (Pteridilm esculentran), J. Clzromatogr., 538~462-468. 142. Hirono, T. ( 1989). Carcinogenic bracken glycosides. In Toxicants of Plant Origin. Vol. 11, Glycosides (P. R. Cheeke, ed.), CRC Press, Boca Raton, Fla., pp. 239-251. 143. Smith, B. L. (1997). The toxicity of bracken fern (genus Pteridium) to animals and its relevance to man, In Handbook of Plant and Fzrngnl Toxicants (J. P. F. D’Mello. ed.), CRC Press, Boca Raton. Fla., pp. 63-76. 144. Jarvis, B. B., Wang, S., Cox, C., Rao, M. M., Valsa, P., Varaschin, M. S. and Barros, C. S. (1996). Brazilian Baccharis Toxins: livestock poisoning and the isolation of macrocyclic trichothecene glucosides, Natural Toxins, 4:58-7 1. 145. Kuti, J.O., Jarvis, B. B., Mokhtari. N.,and Bean, G. (1990). Role of plant-produced trichothecenes in sexual reproduction of two Brazilian Bncchnris species, J. Chem. Ecol., 16:34413453.

Glycosides

34 1

146. Weissenberg. M. (1989). Calcinogenic glycosides. In Toxicants of Plant Origin, Vol. 11, Glyosides (P. R. Cheeke, ed.), CRC Press, Boca Raton, Fla., pp. 201-238. 147. Wasserman, R. H. (1975). Active vitamin D-like substances in Solanum nzalaco.a?lon and other calcinogenic plants, Nutr. Rev.. 33: 1-5. 148. Rambeck. W. A., Wieser. H., and Zucker, H. (1987). A vitamin D3 steroid hormone in the calcinogenic grass Triseturn.flavescerts, Z. Natu~orsch.Teil. C, 42:430-434. 149. Wasserman, R. H.. Henim, J. D., Haussler, M. R., and McCain, T. A. (1976). Calcinogenic principle of Solanum ?nalerco.xylon:evidence thatit is a 1,3,5-dihydroxyvitamin D,-glycoside, Science, 194:853-855. 150. Haussler, M. R., Wasserman, R. H., McCain, T. A., Peterlik, M., Bursak,K. M., and Hughes, M. R. (1976). 1,25-Dihydroxyvitamin D3 glycoside: identification of a calcinogenic principle of Solcrnlrm malacoxylorz, Life Sci., 18: 1049-1056. 151. Esparza, M. S., Vega, M., and Boland. R. L. (1982). Synthesis and composition of vitamin D3 metabolites in Solcrnlanr ntalacodxylon,Biochim. Biophys. Acta, 79:633-640. 152. Boland, R. L., Skliar, M. I.. and Norman, A. W. (1987). Isolation of vitamin D3 metabolites from Solamrrn rnalacoqdon leaf extracts incubated with ruminalfluid, Planta Med., 53:161164. 153. Vidal, M. C., Lescano, W., Avdolov, and R., Puche, R. C.(1 985). Partial structure elucidation of the carbohydrate moiety of 1,25-dihydroxycholecalciferol glycoside isolated from Solanuln glaucophylll~nzleaves. Turrialbn, 35:65-70. 153. Hughes, M. R.. McCain.T. A.. Chang. S. Y.. Haussler, M.R.. Yillareale, M.. and Wasserman, R. H. (1977). Presence of 1,25-dihydroxy-vitamin D3 glycoside in the calcinogenic plant Cestrnml diw-num, Ncrture, 268:347. 155. Weissenberg, M., Levy, A.. and Wasserman, R. H. (1989). Distribution of calcitriol activity in Solantim glaucophyllum plants and cell cultures, Plzytochemistn, 28:795-798. 156. Weissenberg, M., Maoz, A., Levy. A., and Wasserman, R. H. (1988). Radioimmunoassay for rapid estimation of vitamin D derivatives in calcinogenic plants, Planter Med., 54:6365. 157. Harborne. J. B., Mabry, T. J., and Mabry, H. (eds.) (1975). The Flavonoids, Chapman & Hall, London. 158. Harborne, J. B., and Mabry, T. J. (eds.)(1982). TheFlavonoids.Advances in Research, Chapman & Hall, London. 159. Harborne, J. B. (ed.) (1988). The Flavonoids. Advances Since 1980, Chapman & Hall, London. 160. Spencer, K. (1988). Glycosides: the interface between plant secondary and insect primary metabolism. In Biologically Actille Natural Products. Potential Use in Agriculture (H. G. Cutler, ed.), ACS Synlposiunl Series 380, American Chemical Society, Washington, DC,pp. 403-416. 161. Reichardt, P. B.. Clausen, T. P., and Bryant, J. P. (1991). Role of phenolic glycosides in plant-herbivore interactions. In Handbook of N ~ ~ t u r aToxins, l Vol. 6, Toxicology of Plant crnd Fungctl Con~pouncls(R. F. Keeler and A. T. Tu, eds.), Marcel Dekker. New York, pp. 313-333. of phyto-oestrogen toxicity. In Plant Toxicol162. Cox, R. I. (1 985). I~nmunophysiological control ogy (A. A. Seawright, M. P. Hegarty, F. L.James, and R.F. Keeler, eds.), Queensland Poisonous Plants Conunittee. Yeerongpilly, pp. 98-108. of Plant Foodshfls 163. Liener, I. E. (1980). Miscellaneous toxic factors. In Toxic Constit~~ents (I. E. Liener, ed.), Academic Press, New York, pp. 429-467. 164. Barz. W., and Koster, J. (1981). Turnover and degradation of secondary (natural) products. In The Biochenlistry of Plants, Vol. 7, Secondary PlantProducts (E. E. Conn, ed.), Academic Press, New York, pp. 35-84. 165. Wartburg, A. V., Angliker,E., and Renz,J. (1957). Lignanglucoside ausPodoplzyllnnl peltatunr. 7 Mitteilung uber mitosehemmende Naturstoffe, Helv. Chim. Acta. 40: 1331- 1357.

342

Majak and Benn

166. Damayanthi, Y. and Lown, G. W. (1998). Podophyllotoxins: current status and recent developments, Czrrr. Med. Clzem., 5205-252. 167. Murray, R. D.H., Mendez, J.. and Brown, S. A. (1982). The Nntural Coun~nrins:Occurrence, Chemistly and Biochemistry, Wiley, Chichester. 168. Colegate, S. M., Dorling, P. R., Huxtable, C. R., Skelton, B. W., and White, A. B. (1985). Stypandrol, a toxic binaphthalene tetraol isolated from Svpnndra inlbricuta, Aust. J. Cllen?., 38:1233-1241. 169. Barrerham, T., Cooke, R. G., Duewell, H.. and Sparrow, L. G. (1961). Colouring matters of Australian plants. VIII. Naphthalene derivatives from Dimella species. Aust. J. Cheuz.. 14: 637-632. 170. Harborne, J. B. (ed.) (1990). Methods in Plant Biochenzistn, Vol. 1, Plant Phenolics, Academic Press. London. r Resoncrnce of Flavonoids, Elsev171. Agarwal. P. K. (ed.) ( 1989). Carbon13 N z ~ l e a Mngtletic ier, Amsterdam. 172. Markham, K. R. (1983). Techniques of Flavonoid Identification, Academic Press, London. 173. Mabry. T. J., Markham, K. R., and Thomas, M. B. (1970). The Systematic Identification of Flmyonoids, Springer-Verlag, New York. 174. Gaydou, E. M. (1997). Flavones. In Handbook of Plant and Fzrrlgal Tosicmts (J. P. F. D’Mello, ed.), CRC Press, Boca Raton, Fla., pp. 99-108. 175. Asahina. Y., and Fujita, A. (1922). Anemonins, Acta Plzytochim. (Tokyo), 1:l-42. 176. Hill, R., and van Heyningen, R. (195 1). Ranunculin: the precursor of the vesicant substance of the buttercup, Biochem. J., 49332-335. 177. Tschesche. R., Welmar, K., Wulff, G., and Snatzke, G. (1972). Uber Folgeprodukte einer unbekannten genuinen Vorstuffe des Ranunculin in Ranunculaceen, Chew. Ber., 105290300. 178. Tschesche, R., Wirth. W., and Welmar. K. (1981). 5-Hydroxylevulinic acid, a new intermediate in the biosynthesis of protoanemonin, Phytochemist~-y,20: 1835-1839. 179. Bai, Y., Benn. M. H., Majak, W.. and McDiarmid, R. E. (1996). Extraction and HPLC determination of ranunculin in species of the buttercup family, J. Agric. Food Chem. 4422352238. 180. Bonara, A., Dall’Olio, G., and Bruni. A. (1985). Separation and quantitation of protoanemonin in Ranunculaceae by normal- and reversed-phase HPLC, Plnntcl Med., 5:364-367. 181. Moriarty, R. M., Romain, C. R., Karle, I. L., and Karle, J. (1965). Thestructure of anemonin, J. Am. Clzenz. SOC.,87:3251-3252. 182. Ruijgrok, H. W. L. (.1966). The distribution of ranunculin and cyanogenic compounds in the Ranunculaceae. In Con?parative Phytochernist?y (T. Swain, ed.), Academic Press, London, pp. 175-186. 183. Bonora, A., Dall’Olio, G., Donini, A., and Bruni, A. (1987). An HPLC screening of some Italian Ranunculaceae for the lactone protoanernonin, Phytochenristp, 26:2277-2279. 184. Beaglehole, J. C. (ed.) (1965). The Endevour Jozrrnal o f J . Banks, 1768-1771, Vol. 3, Angus & Robertson, Sydney, p. 127. 185. Nishida, K., Kobayashi. A.. and Nagahama. T. (1955). Cycasin, a toxic glycoside of Cycas revoluta. I. Isolation and structure of cycasin, Bull. Agric. Chern. Soc. Jpn., 19:77-84. 186. Langley, B. W., Lythgoe, B., and Riggs, N. V. (1951). Macrozamin. Part 11. The aliphatic azoxy structure of the aglycone part, J. Chewl. SOC..2309-2316. 187. Nagahama, T. (1964). Studies on new cycasins, new glycosides of cycads, Bull. Fnc. Agr. Kagoslzinza Unil?.,14:1-49 (in Japanese). 188. Matsumoto, H., and Higa, H. H. (1966). Studies on methylazoxymethanol, the aglycone of cycasin: methylation of nucleic acids in vitro, Bioclzem. J., 98:20C-22C. 189. Moretti, A., Sebato, S., and Gigliano, G. S. (1983). Taxonomic significance of methylazoxymethanol glycosides in the cycads, Phytochemistry, 22: 1 15-1 17. 190. Marquardt, R. R. (1989). Vicine, convicine and their aglycones-divicine and isouramil. In

Glycosides

191.

192.

193.

194.

195. 196. 197.

198. 199.

200. 201.

202.

203.

204.

205.

206.

207.

208. 209.

343

Toxicailts of Plant Origin, Vol. 11, Glycosides (P. R. Cheeke. ed.), CRC Press. Boca Raton, Fla., pp. 161-200. Marquardt. R. R., Wang, N. and Arbid, M. S. (1997). Pyrimidine glycosides. In Handbook of Plant and Fztngal Toxicants (J. P.F. D’Mello, ed.) CRC Press, Boca Raton, Fla., pp. 139155. Pitz. W. J.. Sosuloki, F. W., and Hogge, L. R. (1980). Occurrence of vicine and convicine in seeds of some Vicicr species and other pulses, Can. Inst. Food Sci. Techrlol. J.. 13:3539. McDiarmid, R. E.. Pass, M. A., Benn, M. H.. and Majak, W. (1991). Biotransformation of 3-nitropropanol by alcohol dehydrogenase. In Poiso~rousPlcuzts (L. F. James, R. F. Keeler, E. M. Bailey, Jr., P. R. Cheeke, and M. P. Hegarty, eds.), Iowa State University Press, Ames. pp. 131-136. Pass, M. A., Muir, A. D., Majak, W., and Yost, G. S. (1985). Effect of alcohol and aldehyde dehydrogenase inhibitors on the toxicity of 3-nitropropanol in rats, Toxicol. Appl. P h r ~ l z a C O ~ . , 78~310-315. Pass, M. A., Majak. W., Muir. A. D., and Yost, G. S. (1984). Absorption of 3-nitropropanol and 3-nitropropionic acid from the digestive system of sheep, To.uicol. Lett., 23:l-7. Majak, W., Pass, M. A., Muir, A. D., and Rode, L. M. (1984). Absorption of 3-nitropropanol (miserotoxin aglycone) from the compound stomach of cattle. Tosicol. Lett.. 23:9-95. Alston, T. A., Mela, L., and Bright. H. J. (1977). 3-Nitropropionate, the toxic substance of Irdigofertr, is a suicide inactivator of succinate dehydrogenase, Nrrrl. Acad. Sci. Proc., 74: 3767-377 1. Coles, C. J.. Edmondson, D. E.. and Singer, T. P. (1979). Inactivation of succinate dehydrogenase by 3-nitropropionate. J. Biol. Clrem., 254:5161-5167. Porter, D. J., and Bright, H. J. (1980). 3-Carbanionic substrate analogues bind very tightly to fumerase and aspartase, J. Biol. Clzerir., 255:4772. Schloss, J. V., and Cleland, W. W. (1982). Inhibition of isocitrate lyase by 3-nitropropionate, a reaction intermediate analogue, Biochemist)?, 21 :4420-4427. Gould, D. H.. and Gustine, D. L. (1982). Basal ganglia degeneration. myelin alterations, and enzyme inhibition induced in mice by the plant toxin 3-nitropropanoic acid, Neuropathol. Appl. Neurobiol., 8:377-393. Gould, D. H., Wilson, M. P., and Hamar, D. W. (1985). Brain enzyme and clinical alterations induced in rats and mice by nitroaliphatic toxicants, Toxicol. Lett., 27:83-89. Hamilton, B. F., and Gould, D. H. (1987). Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage, Acta Newopntlzol. (Bed.),72286-297. Hamilton, B. F., and Gould, D. H. (1987). Correlation of morphologic brain lesions with physiologic alterations and blood-brain impairment in 3-nitropropionic acid toxicity in rats, Acta Ne~tropatl~ol. (Bed.), 74:67-74. Majak, W., Udenberg, T., McDiarmid. R. E., and Douwes, H. (1981). Toxicity and metabolic effects of intravenously administered 3-nitropropanol in cattle, Ca12. J. Artim. Sci., 61:639637. Benn, M. H.. McDiarmid, R. E., and Majak, W. (1989). In vitro biotransformation of 3nitropropanol (miserotoxin aglycone) by horse liver alcohol dehydrogenase, To.vico1. Lett., 47:165-172. Liu, X. J., Luo, X. Y., and Hu, W. J. (1989).Arthr-iniuntspp. and the etiology of deteriorated sugarcane poisoning. In Mycotoxins a d Plzycoto-Tins (S. Natori, K. Hashimoto. and Y. Ueno, eds.), Elsevier Sci. Publ., B. V. Amsterdam, pp. 109-118. Ming, L. M. (1995). Moldy sugarcane poisoning-a case report with a brief review, Clirr. Tosicol., 33:363-367. Handton, B. F., Gould, D. H.. and Gustine. D. L. (1998). History of 3-nitropropionic acid: occurrence and role in human and animal disease. In Mitochondrial Inhibitors arzd Newode-

344

210.

211.

212.

213. 214. 215.

216.

217.

218. 219.

220. 221. 222. 223. 224.

225.

226.

227.

228.

Majak and Benn generati$-eDisorders (P. R. Sandberg, H. Nishino, and C. V. Borlongan, eds.), Humana Press, Totowa, N.J. (in press). Ludolph, A. C., He, F., Spencer, P. S.. Hammerstad, J.. and Sabri. M. ( 1991). 3-Nitropropionic acid-exogenous animal toxin and possible human striatal toxin, Can. J. Neltrol. Sci., 18:492-498. Brouillet, E., Jenkins, B. G., Hyman, B. T., Ferrante. R. J., Kowall, N. W., Srivastava, R., Roy, D. S., Rosen, B. R.. and Beal, M. F. (1993). Age-dependent vulnerability of the striatum to the mitochondrial toxin 3-nitropropionic acid, J. Neurocl?em., 60:356-359. Beal, M. F., Brouillet, E., Jenkins, B. G., Ferrante, R. J., Kowall, N. W., Miller, J. M., Storey, E., Srivastava, R., Rosen, B. R., and Hyman. B. T. (1993). Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid, J. Neurosci., 13:4181-4192. Wullner, U., Young, A. B., Penny,J. B., and Beal, M. F. (1994). 3-Nitropropionic acid toxicity in the striatum, J. Neurochem., 63:1772-1781. Pass, M. A., Carlisle, C. H.. and Reuhl, K. R. (1994). 3-Nitropropionic acid toxicity in cultured murine embryonal cells, J. Nat. Toxins. 2386-394. Pass, M. A. (1994). Toxicity of plant-derived aliphatic nitrotoxins. In Plant-Associated Tosins (S. M. Colegate and P. R. Dorling. eds.), CAB International. Wallingford, Oxon, UK, pp. 541 -545. Majak, W., Stroesser, L., Hall, J. W., Quinton, D. A., and Douwes, H. E. (1996). Seasonal grazing of Columbia milkvetch by cattle on rangelands in British Columbia, J. Range Manage., 49223-227. Majak, W., Hunter. C., and Stroesser. L. (1998). Tolerance in cattle to timber milkvetch (Astrclgalmmiser var. serotinus). In Toxic Plants a17d Other.Natural Toxins (T. Garland and A. C. Barr, eds.), CAB International, New York, pp. 239-242. Majak, W., Johnson, D. L., and Benn, M. H. (1998). Detoxification of 3-nitropropionic acid and karakin by Inelanopline grasshoppers, Plzytochentistnl, 49:419-422. Pass, M. A., Majak, W., and Yost, G. S. (1988). Lack of a protective effect of thiamine on the toxicity of 3-nitropropanol and 3-nitropropionic acid in rats, Can. J. Anim. Sci., 68315-320. Maricle, B.. Tobey, J., Majak, W.. and Hall, J. W. (1996). Evaluation of clinicopathological parameters in cattle grazing timber nilkvetch, Can. Vet. J., 37:153-156. Gonzilez, E. R. (1982). Cyanide evades some noses. overpowers others, JAMA, 2489211. Way. J. L. (1984). Cyanide intoxication and its mechanism of antagonism, Annu. Rev. Pharmacol. Toxicol., 24~45 1-48 1. Vennesland, B., Castric, P. A., Conn, E. E., Solomonson, L. P., Volini, M., and Westley. J. (1982). Cyanide metabolism, Fed. Proc., 41:2639. Westley. J. (1981). Cyanide and sulfane metabolism. In Cyanide i n Biology (B. Vennesland, E. E. Conn, C. J. Knowles. J. Westley, and F. Wissing, eds.), Academic Press, New York, pp. 61-76. Burrows, G. E., and Way, J. L. (1979). Cyanide intoxication in sheep: enhancement of efficacy of sodium nitrite. sodium thiosulfate and cobaltous chloride, Am. J. Vet. Res., 40:613617. Benson, J. M., Seiber, J. N., Keeler, R. F., and Johnson, A. E. ( 1975). Studies on the toxic principle of Asclepias eriocnrpa and Asclepias labriformis. In Effects of Poisonous Plcrnts on Livestock (R. F. Keeler, K. R. Van Kampen. and L. F. James. eds.). Academic Press, New York, pp. 273-284. Gold, H., Kwit, N. T.. Shane, S. J., and Dayrit. C. (1969). The role of the sugar component in cardiac glycosides on their action in patients with auricular fibrillation and congestive failure, J. Clin. Pharrnacol., May-June:148-154. Lacarelle, B., Rahmani, R.. de Sousa, G., Durand, A.. Placidi, M., and Cano, J. P. (1991). Metabolism of digoxin, digoxigenin digitoxosides and digoxigenin in human hepatocytes and liver microsomes, Fundarn. Clin. Pl1ar.rnacol., 5567-552.

Glycosides

345

229. McKenzie, R. A., Franke, F. P., and Dunster, P. J. (1987). The toxicity to cattle and bufadienolide content of six Bryoplyll~cmspecies, Azrst. Vet. J., 64298-301. 230. Corrigall, W., Moody, R. R., and Forbes, J. C. (1978). Foxglove (Digitalis yurpureu) poisoning in farmed red deer (Cenws elaphus),Vet. Rec., 102:119- 122. 231. Joubert. J. P., and Schultz, R. A. (1982). The treatment of Mortrea polystaclrya (Thunb) KerGawl (cardiac glycoside) poisoning in sheep and cattle with activated charcoal and potassium chloride, J. S. Ajk Vet. Assoc.. 53249-253. 232. Williams, M. C. (1978). Toxicity of saponins in alfrombrilla (Dqwzrrria arennl-ioicles),J. Range Mmage., 31:182-184. 233. Williams. M. C.. Rodewijk, J. C. M., and Olsen. J. D. (1984). Intoxication in cattle. chicks and hamsters from seed of the palo santo tree (Bzd~esia snrmientii), Vet. Rec., 115:646-648. 234. Holland, P. T., Miles. C. O., Mortimer, P. H.. Wilkins. A. L., Hawkes, A. D., and Smith, B. L. (19911. Isolation of the steroidal sapogenin epismilagenin from the bile of sheep affected by Panicum dickotomiJorzcm toxicosis, J. Agric. Food Chem., 39: 1963-1965. 235. Patamalai, B., Hejtmancik. E., Bridges, C. H., Hill, D. W., and Camp, B. J. (1990). The isolation and identification of steroidal sapogenins in kleingrass. Vet. Hztwt. Toxicol., 3 2 3 14318. 236. George, A. J. (1965). Legal status and toxicity of saponins, Food Cosmet. Toxicol., 3235-91. 237. Nishikawa. M., Nojima, S., Akiyama, T., Sankawa, U., and Inoue, K. (1984). Interaction of digitonin and its analoks with membrane cholesterol. J. Biocltenl.. 96: 1231-1239. 238. Kingsbury. J. M. (1964). Poisonous Plants of the United Stutes ar7d Cmada. Prentice-Hall, Englewood Cliffs, N.J. 239. Cheeke. P. R. (1998). Notural Toxicants in Feeds, Forages arid Poisonous Plmts. 2nd ed.. Interstate Publishers Inc., Danville, Ill. 240. Gestetner, B., Birk, Y., and Tencer, Y. (1968). Soybean saponins-fate of ingested soybean saponins and the physiological aspect of their hemolytic activity, J. Agric. Food Clrenz., 16: 1031-1035. 241. Hooper, P. T. (1978). Cycad poisoning in Australia-etiology and pathology. In Eflects of Poisonozu Plants on Livestock (R. F. Keeler. K. R. Van Kampen, and L. F. James, eds.), Academic Press. New York, pp. 337-347. 242. Hooper. P. T. (1983). Toxins inducing psychic or neurotoxic effects. In Hmrdbook of Narzrral Toxins, Vol. 1. Plant and Fzrngd Toxins (R. F. Keeler and A. T. Tu, eds.). Marcel Dekker, New York. pp. 463-471. 243. Spoerke, D. G.. Jr., and Smolinske, S. C. (1990). Tosicih, of Houseplants. CRC Press, Boca Raton. Fla. 234. Charlton, T. S., Marini, A. M.. Markey, S. P., Nostag, K., and Duncan, M. W. (1992) Quantification of the neurotoxin 2-amino-3-(1nethylamino)-propanoicacid (BMAA) in Cycadales, Plytocllemistly, 3 1:3429-3432. 245. Wogan, G. N., and Busby, W. F., Jr. (1980). Naturally occurring carcinogens. In Toxic Constituents of Plant FoodstltfiT (I. E. Liener, ed.), Academic Press, New York, pp. 329-369. 246. Spatz, M.. Smith, D. W. E., McDaniel, E. G.. and Laqueur, G. L. (1967). Role of intestinal microorganisms in determining cycasin toxicity, Proc. SOC. Exp. Biol. Med., 124:691. 247. Feinberg, A., and Zedeck. M. S. (1980). Production of a highly reactive alkylating reagent from the organospecific carcinogen methylazoxymethanol by alcohol dehydrogenase, CancerRes., 40:4446-4450. 248. Benn. M. H., and Kazmaier, P. (1972). Methylation with (z)-methyl-ONN-azoxymethanol: the nature of the reactive species, J. Chenr. SOC.Chenz. Conmun., 887-888. 249. Benn. M. H., and Yelland, L. J. (1968). Ranunculin, Can. J. Chsnr., 46:729-732. 250. Olsen, J. D., Anderson, T. E., Murphy, J. C.. and Madsen, G. (1983). Bur buttercup poisoning of sheep, J. Am. Vet. Med. Assoc., 183538-543. 251. Hidiroglu, M., and Knutti, H. J. (1963). The effects of green tall buttercup in roughage on the growth and health of beef cattle, Cm. J. Anir~z.Sci., 43:68-71.

346

Majak and Benn

252.

Shearer, G. D. (1938). Some observations on the poisonous properties of buttercups, Vet. J., 94122-32. Swenson, M. J. (ed.) (1977). Duke’s Physiology of Domestic Animals, Cornel1 University Press. Ithaca, N.Y. Tookey, H. L., VanEtten, C. H., and Daxenbichler. M. E. (1980). Glucosinolates. In Toxic Comtitrcerzts of Plant Foodstufls (I. E. Liener. ed.), Academic Press, New York, pp. 103143,. Greer, M. A. (1962).The natural occurrence of goitrogenic agents, Rec. Prog. Hormone Res., 181187-219. Oginsky, E. L., Stein, A. E., and Greer, M. A. (1965). Myrosinase activity in bacteria as demonstrated by the conversion of progoitrin to goitrin. Proc. SOC.ESP. Med., 119:360-364. Tani, N., Ohtsuru, M.. and Hata, T. (1974). Isolation of myrosinase producing microorganism, Agr. Biol. Cltenz., 38:1617-1622. Lanzani, A., Piana, G.. Piva, G., Cardillo, M., Rastelli. A.. and Jacini, G. (1974). Changes in Brassica rzapus progoitrin induced by sheep rumen fluid, J. A m Oil Clzenr. SOC.,51517. Palop, M. L., Smiths, J. P.. and Brink, B. (1995). Degradation of sinigrin by Lactobacillzts agilis strain R16, lilt. J. Food Microbiol., 26:219-229. Kennaleguen, A., Smith, R. A., and Yong, C. W. (1989). Acute mustard seed toxicosis in beef cattle, Can. Vet. J., 30524. Semalulu, S. S.. and Rousseaux, C. G. (1989). Suspected oriental mustard seed (Brassica j m c e a ) poisoning in cattle, Can. Vet. J . , 30595-596. Cole, R. A. (1976). Isothiocyanates, nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae. Phvtochenzistn, 15:759-762. Smith, R. A., and Crowe, S. P. (1987). Fanweed toxicosis in cattle: case history, analytical method, suggested treatment and fanweed detoxification, Vet. Hwn. Toxicol., 29: 155- 156. Daxenbichler. M. E., VanEtten. C. H., and Wolff. I. A. (1968). Diastereomeric episulfides from epi-progoitrin upon autolysis of Crambe seed meal, Phytochernist~~~, 7:989-996. Kirk. J. T. 0.. and MacDonald, C. G. (1974). l-Cyano-3,4-epithiobutane:a major product of glucosinolate hydrolysis in seeds from certain varieties of Brassica canzpestris, Phytoc h e m i s t ~ 13261 ~, 1-2615. Van Steenhouse, J. L., Fettman, M. J., and Gould, D. H. (1991). The effect of glutathione depletion by buthionine sulphoximine on 1-cyano-3.4-epithiobutanetoxicity. Food Clzem. Tosicol., 29:153-157. Gould. D. H., Fettman, M. J., Daxenbichler, M. E., and Bartuska. B. M. (1985). Functional and structural alterations of the rat kidney induced by the naturally occurring organonitrile 2S-l-cyano-2-hydroxy-3.4-epithiobutane, Toxicol. Appl. Phnr~nacol..78: 190-201. Wallig, M. A.. Gould, D. H.. and Fettman, M. J. (1988). Selective pancreatotoxicity in the rat induced by the naturally occurring plant nitrile l-cyano-2-hydroxy-3-butene, Food Chenr. Toxicol., 26:137-147. Dietz, H. M., Panigrahi, S., and Harris, R. V. (1991). Toxicity of hydrolysis products from 3-butenyl glucosinolate in rats, J. Agric. Food Clzeln., 39:311-315. Farnsworth, N.R., Bingel, A. S., Cordell. G. A., Crane. F. A., and Fong. H. H. S. (1975). Potential value of plants as sources of new antifertility agents 11, J. Plzann. Sci., 64:717754. Heftman, E. (1975). Steroid hormones in plants, Lloydia, 38:195-205. Adams, N. R. (1989). Phytoestrogens. In Tosicam of Plant Origin, Vol. IV, Pherrolics (P. R. Cheeke, ed.), CRC Press, Boca Raton. Fla.. pp. 23-51. Jordan, V. C., Mittal. S., Gosden, B., Koch, R., and Leiberman. M. E. (1985). Structureactivity relationships of estrogens, Emiron. Health Perspect., 61 :97-110. Shutt, D. A., and COX,R. I. (1972). Steroid and phyto-estrogen binding to sheep uterine receptors in vitro, J. Endocrinol., 52299-310.

253. 254.

255. 256. 257.

258.

259. 260. 261. 262. 263. 264. 265.

266.

267.

268.

269. 270.

271. 272. 273. 374.

Glycosides

347

275. Livingstone, L. (1978). Forage plant estrogens, J. Tosicol. Environ. Hecdth, 4301 -324. 276. Beck. A. B.. and Knox. J. R. (1971). The acylated glycosides from subterranean clover and red clover, Aust. J. Chem., 24:1509-1518. 277. Davies, L. (1 987). Limitations to livestock production associated with phytoestrogens and bloat. In Temperate Pcrsttwes. Their Production, Use arzcl Managentent (J. L. Wheeler, C. J. Pearson, and G. E. Roberts, eds.). Aust. Wool Corp. Tech. Publ., pp. 446-456. 278. Olah, A. F.. and Sherwood, R. T. (1971). Flavones, isoflavones and coumestans in alfalfa infected by Ascochytcr irtzpwfecta, Pl1ytopcrthology,61 :65-69. 279. Dickinson, J. M., Smith, G. R., Randel. R. D., and Pemberton, I. J. ( 1988). In vitro metabolism of formononetin and biochanin A in bovine rumen fluid, J. Arzinz. Sci., 66:1969-1973. 280. Lundh, T. J.-0, Pettersson. H. I., and Martinsson, K. A. (1990). Comparative levels of free and conjugated plant estrogens in blood plasma of sheep and cattle fed estrogenic silage, J. Agric. Food Chenz., 38:1530-1534. 281. Cox, R. I. (1978). Plant estrogens affecting livestock in Australia. In EJjcects of Poisonous Plcrrzts on Livestock (R. F. Keeler, K. R. Van Kampen, and L. F. James. eds.), Academic Press, New York. pp. 451 -464. 252. Kinghom, A. D. (1983). Carcinogenic and cocarcinogenic toxins from plants. In Handbook of Natwal Toxins, Vol. 1. Plant am! Fwlgcrl Toxins (R. F. Keeler and A. T. Tu,eds.), Marcel Dekker, New York, pp. 239-298. 283. Hirono, I. (1986). Carcinogenic principles isolated from bracken fern, CRC Crit. Relq. Tosicol., 17:1-22. 283. Hirono, I., Kono, Y., Takahashi, K.. Yamada, K., Niwa, H.. Ojika, M.. Kigoshi, H., Niiyama, K.. and Uosaki, Y. (1984). Reproduction of acute bracken poisoning in a calf with ptaquiloside, a bracken constituent, Vet. Rec.. 115:375-378. 285. Sumi, Y., Hirono, I., Hosaka, S., Ueno. I., and Miyakawa, M. (1981). Tumor induction in germ-free rats fed bracken (Pteridium aqzdinum), Cancer Res., 41 250-252. 286. Wassernlan, R. H. ( 1978). Thenature and mechanism of action of the calcinogenic principle of Solanrun rnalacoxylon and Cestrztnr diunzunz and a comment on Trisetwn j h m c e n s . In ERects of Poisonous Plants O H Lityestock (R. F. Keeler, K. R. Van Kampen, and L. F. James, eds.), Academic Press, New York. pp. 545-553. 287. Craig, J. C., Jr., Mole, M. L., Billets, S.. and El-Feraly, F. (1976). Isolation and identification of the hypoglycemic agent, carboxyatractylate. from Xantllizolt stt-mzarizlnr,Plzytochernistly, 15:1-178. 288. Oelrichs, P. B., MacLeod. J. K.. and Klingenberg, M. (1992). Chemistry and toxic effects of the active compounds from Weclelicr asperrinla. In Poisonous Plcuzts (L. F. James, R. F. Keeler, E. M. Bailey, Jr., and M. P. Hegarty, eds.), Iowa State University Press. Ames, pp. 252-287. 289. Obatomi, D. K., and Bach, P. H. (1998). Biochemistry and toxicology of the diterpenoid glycoside atractyloside. Food Chenz. Toxicol., 36:335-346. 290. Haskins, F. A.. and Gorz. H. J. (1963). Glucosides of coumarinic acid and o-coumaric acids in tonka bean. Science, 139:496. 291. Pritchard, D. G.. Markson, L. M.. Brush, P. J., Sawtell. J. A. A.. and Bloxham, P. A. (1 983). Haemorrhagic syndrome of cattle associated with the feeding of sweet vernal (Ailtllo.~crntlzztriz odomtwz) hay containing dicoumarol, Vet. Rec., 113:78-84. 292. Kingsbury. J. M. (19SO). One man's poison, Bioscience, 30:171-176. 293. Scheel, L. D. (1978). Mycotoxicosis in cattle. In Mycotoxic Fungi, Mycotoxirls, Mycoto.yicosis, Arz Emyclopeclic Handbook. Vol. 2 (I. D. Wylie and L. B. Morehouse, eds.). Marcel Dekker, New York, pp. 121-142. 294. Williams, M. C., and Olsen, J. D. (1984). Toxicity of seeds of three Aesczrlzts spp. to chicks and hamsters, Am. J. Vet. Res.. 45:539-542. 295. Goplen, B. P. (198 1). Norgold-A low coumarin yellow blossom sweetclover, Can. J. Plant Sci., 61:1019-1021.

348

Majak and Benn

296. Blakely. B. R. (1985). Moldy sweet clover (dicoumarol) poisoning in Saskatchewan cattle, Can. Vet. J.. 26:357-360. 297. Benson. M. E.. Casper, H. H.. and Johnson, L. J. (1981 ). Occurrence and range of dicoumarol concentrations in sweet clover, Ant. J. Vet. Res., 422014-2015. 298. Puschner, B., Galey, F. D., Holstege, D. M., and Palazoglu, M. (1998). Sweetclover poisoning in dairy cattle in California. J. Ant. Vet. Med. Assoc. 212:857-859. 299. Alstad, A. D., Casper, H. H., and Johnson, L. J. (1985). Vitamin K treatment of sweet clover poisoning in calves, J. Am. Vet. Med. Assoc., 187:729-731. 300. Elliger, C. A., Waiss, A. C., Jr.,and Lundin. R. E. (1974). Cyanomethylenecyclohexyl glucosides from Sinzmondsin californiccr, Phvtochenristn, 132319-2320. 301. Booth, A. N., Elliger, C. A., and Waiss, A. C., Jr. (1974). Isolation of a toxic factor from jojoba meal, Life Sci., 15:1115-1120. 302. Verbiscar, A. J., Banigan, T. F., Weber, C. W.,Reid, B. L., Trei, J. E., Nelson, E. A., Raffauf, R. F., and Kosersky, D. (1980). Detoxification of jojoba meal, J. Agric. Food Chenz., 28: 57 1-578. 303. Sosa, A., Winternitz, F., Wylde, R., and Pavia, A. A. (1977). Structure of a cyanoglucoside of Litltospermum I’urpzlreo-caerulerIrn, Phytochemistry, 16:707-709. 304. Wu, J., Fairchild, E. H., Beal, J. L., Tomimatsu, T., and Doskotch, R. W. (1979). Lithospermoside and dasycarponin, cyanoglucosides from Thalictntm, J. Nnt. Prod., 42500-5 11. 305. Crawford, L., McDonald, G. M., and Friedman, M. (1990). Composition of sicklepod (Cassia obtusifolin) toxic weed seeds, J. Agric. Food Cltem., 382169-2175. 306. Williams, M. C., (1982). 3-Nitropropionic acid and 3-nitro-1-propanol in species of Astrrrgalus, Can. J. Bot., 60:1956-1963. 307 Williams. M. C.. and G6mez-Sosa, E. (1986). Toxicnitrocompounds in species of Astragalus (Fabaceae) in Argentina, J. Range Manage.. 39:341-344. 308. Williams, M. C. (1983). Toxic nitrocompounds in Lotus, Agron. J., 75:520. 309. Gustine, D. L. (1979). Aliphatic nitrocompounds in crownvetch: a review, Crop Sci.,l9: 197203. 3 10. Aylward, J. H., Court, R. D., Haydock, K. P., Strickland, R. W., and Hegarty, M. P. (1987). Zrzdigoferrr species with agronomic potential in the tropics. Rat toxicity studies, Azrst. J. Agric. Res., 38: 177-1 86. 31 1. Muir, A. D., Majak, W., Pass, M. A., and Yost. G. S. (1984). Conversion of 3-nitropropanol (miserotoxin aglycone) to 3-nitropropionic acid in cattle and sheep, Tosicol. Lett., 20: 137-141. 3 12. Matsumoto, H., Hylin, J. W.. and Miyahara, A. (1961). Methemoglobinemia in rats injected with 3-nitropropanoic acid, sodium nitrite. and nitroethane, Tosicol. AppI. P h w 7 ~ c o I . 3: , 493-499. 313. Majak, W., and McDiarmid. R. E. (1990). Detection and quantitative determination of 3nitropropionic acid in bovine urine, Tosicol. Lett., 50213-230. 3 14. Nartey, F. (1981). Cyanogenesis in tropical feeds and foodstuffs. In Cyarticle i n Biology (B. Vennesland. E. E. Conn. C. J. Knowles, J. Westley. and F. Wissing, eds.), Academic Press. New York, pp. 115- 132. 315. Lundquist, P., Rosling, H.. and Sorbo, B. (1985). Determination of cyanide in whole blood, erythrocytes and plasma, Clin. Chem., 3 1 5 91-595. 3 16. Majak, W. (1992). Metabolism and absorption of toxic glycosides by ruminants, J. R m g e Manage., 45:67-71. 317. Hoffman, B. F., and Bigger, J. T., Jr. (1980). Digitalis and allied cardiac glycosides. In The Phcrmzacological Basis of Tlzercryeutics (L. S. Goodman and A. Gilman, eds.), Macmillan. New York, pp. 729-760. 318. Majak, W. (1992). Stability of allylthiocyanate and allylisothiocyanate in bovine ruminal fluid, Toxicol. Lett., 63:75-78. 319. Evans, I. A., Jones, R. S., and Mainwaring-Burton, R. (1972). Passage of bracken fern toxicity to milk, Nature, 237:107-108.

Glycosides

349

320. Pamukcu. A. M., Erturk. E., YalGiner, S.. Milli, U.. and Bryan. G. T. (1978). Carcinogenic and mutagenic activities of milk from cows fed bracken fern (Pteridiurtt a q ~ d i r m ~Carzcer ). Res., 38:1556-1560. 321. Iwasaki, T.. and Kosikowski. F. V. (1973). Production of P-nitropropionic acid in foods, J. Food Sci., 38:1162-1165. 322. Kinosita, R., Ishiko, T., Sugiyama, S., Seto, T., Igarasi. S., and Goetz, I. E. (1968). Mycotoxins in fermented food, Cancer-Res.. 28:2296-2311. 323. Cook. R. D., and Coursey, D. G. (1981). Cassava: A major cyanide-containing food. In Cymzide in Biology (B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley. and F. Wissing, eds.). Academic Press, New York. pp. 93-114. 324. Poulton, J. E. (1983). Cyanogenic conlpounds in plants and their toxic effects. In Handbook of Ncrtztral To,xins, Vol. 1, Plarzt a i d Furlgcrl Tosirzs (R. F. Keeler and A. T. Tu, eds.), Marcel Dekker, New York, pp. 117-157. 325. Aregheore, E. M., and Agunbiade, B. S. (1991). The toxic effects of cassava (Mnnihot escw lerzta Crantz.) diets on humans: a review, Vet. H w l . To.yico1.. 33:274-275. 326. Espinoza, 0. B., Perez. M., and Ramirez, M. S. (1992). Bitter cassava poisoning in eight children: a case report. Vet. H~rm.Toxicol., 34:65. 327. Akintonwa, A., and Tunwashe, 0. L. (1992). Fatal cyanide poisoning from cassava-based meal. H m z . E.xp. Toxicol., 11:37-39. 328. Holzbecher. M. D., Moss, M. A.. and Ellenberger, H. A. (1984). The cyanide content of laetrile preparations, apricot, peach and apple seeds, Clirz. Tosicol., 22341-347. 329. Lewis, J. P. (1977). Laetrile, West. J. Med., 127:55-62. 330. Sadoff. L., F L K ~ K.. S . and Hollander. J. (1978). Rapid death associated with laetrile ingestion, JAMA, 239: 1532. 331. Humbert. J. R., Tress, J. H., and Braico, K. T. (1977). Fatal cyanide poisoning: accidental ingestion of amygdalin, JAMA, 238:482. 332. Cheung, K., Hinds, J. A., and Duffy, P. (1989). Detection of poisoning by plant-origin cardiac glycoside with the Abbott TDx analyzer, Clirz. Chen1., 35295-297. 333. Radford, D. J.. Gillies, A. D., Hinds. J. A., and Duffy, P. (1986).Naturally occurring cardiac glycosides. Med. J. Aust.. 144540-544. 333. Blum, L. M., and Rieders, F. (1987). Oleandrin distribution in a fatality from rectal and oral Ner-iztrtz olecrrzcler-extract administration, J. Amlyt. To.yico1.. 11:219-221. 335. Hall, W. T. K. (1987). Cycad (zamia) poisoning in Australia, Aust. Vet. J., 64: 149-151. 336. Spencer. P. S.. Ohta, M., and Palmer. V. S. (~1987).Cycad use and motor neurone disease in Kii peninsula of Japan, Lancet, 2:1462-1463. 337. Dunnick, J. K.. Prejean, J. D., Haseman, J., Thompson, R. B., Giles, H. D., and McConnell. E. E. (1982). Carcinogenesis bioassay of allyl isothiocyanate. Funcl. Appl. Tosicol., 2: 114120. 338. Knuckles, B. E.. deFremery. D., and Kohler. G. 0. (1976). Coumestrol content of fractions obtained during wet processing of alfalfa, J. Agric. Food Chem., 24:1177-1180. 339. Wang. G.. Kuan. S. S., Francis, 0. J., Ware, G. M.. and Carmen, A. S. (1990). A simplified HPLC method for the determination ofphytoestrogens in soybean and its processed products, J. Agric. Food Chem., 38:185-190. 340. Hirono. I.. Shibuya. C.. Shimizu, M., and Katsumasa, F. (1972). Carcinogenic activity of processed bracken used as human food, J. Nnt. Cancer. Z m t . , 48: 1245-1249. 341. Candy. H. A., Pegel, K. H., Brookes, B., and Redwell. M. (1977). The occurrence of atractyloside in Callilepis lrruereolcr. Phytochemistry, 16:1308- 1309. 342. Mager, J.. Chevion, M., and Glaser, G. (1980). Favism. In Toxic Comtitzlerlts of Plcrrzt Foodstuffs (I. E. Liener. ed.), Academic Press, New York, pp. 265-294.

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7 Analytical Methodology for Plant Toxicants Alister David Muir Agric~tlturem e 1 Agri-Food Cemadrr, Scrskatoow ScrskntcheM~an, Canada

I. IntroductionandNew 11. Alkaloids A. B. C. D. E. F. G. H. I. J. K. L.

Technologies352

353

Pyrrolizidine alkaloids 354 Piperidine alkaloids 357 Pyridine alkaloids 358 Indole,tryptamine,P-carboline,andrelatedalkaloids358 Quinolizidine alkaloids 360 Steroid alkaloids 36 1 Diterpene alkaloids 364 Indolizidine alkaloids 365 Tropane alkaloids 365 Isoquinoline alkaloids 366 Peptide alkaloids 367 Other alkaloids 368

111. Glycosides 368 Cyanogenic A. glycosides 368 B. Glucosinolates 369 C. Phenolic glycosides 370 D. Saponins and cardiac glycosides E. Nitropropanol glycosides 374 F. Other glycosides 375 IV. Proteins,Peptides,and

371

Amino AcidAnalogs

Lectins A.(hemagglutinins) B. Enzymes 377 Amino C. acid analogs 377 Anlines 378 D. E. Selenium accumulators

376

376

379

Isoprenoids V. 379 Sesquiterpene A. lactones B. Diterpenes 379 C. Other terpenes and hydrocarbons

379 380

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Muir

352 VI. VII.

Oxalates, Nitrates, Sulfides, and Organofluorine Compounds Mycotoxins

A. B. C. D. E. F. G. H. I. J.

Aflatoxins 382. Acidic mycotoxins (citrinin, ochratoxin A) Cyclopiazonic acid 383 Fumonisins 383 Furanoterpenes 384 Muscimol and ibotenic acid 384 Orellanine 384 Patulin 385 Trichothecenes (vomitoxin) 385 Zearaleone 386

References

1.

381

381 382

386

INTRODUCTIONAND NEW TECHNOLOGIES

The classification of compounds associated with cases of animal and human poisoning is often subjective. In many instances, compounds are deemed to be toxic by association. They are often the major compound found in a plant known to be poisonous and related compounds have been shown to cause similar symptoms of poisoning. Additional information on techniques for analysis of plant toxins also can be found in earlier reviews of poisonous plants (1 ). Some plant constituents (e.g., cyanogenic glycosides) are not particularly toxic in the form in which they exist in the plant; however, on ingestion they undergo transformations to more-toxic principles. Therefore, when looking for toxins in manlmalian systems, the metabolites of the toxin are often the only detectable compounds. These metabolites formed in mammalian systems were reviewed by Scheline (2). With alkaloids, there is often considerable variability within a species in the level and even in the composition of the toxic fractions, depending on location, stage of growth, and environmental conditions (3-5). This can cause problems in toxicological investigations if the analytical procedure is designed to target a specific compound. An essential key to identification of a plant toxin is correct identification of the plant responsible for toxicity. When livestock are suspected of having been poisoned by consumption of a poisonous plant, an examination of the rumen or stomach contents often will afford a clue as to the plant consumed. A second key to quick identification is the availability of authentic standards of the suspected toxins. Without standards for comparison, less-sophisticated tests usually will only confirm identification to the chemical group and not the specific toxin. In this chapter. methods are summarized for analysis of toxins of plant origin that are poisonous to mammals and that have been implicated in animal or human poisoning. Also included are methods for toxic fungal metabolites that are produced by fungi that infect plants and plant and animal products where these compounds are known to affect human or animal health. Since the analytical resources available vary significantly from laboratory to laboratory, these methods are grouped by analytical technique [e.g., thinlayer Chromatography (TLC), high-perfornlance liquid chromatography (HPLC), and gas chromatography (GC)] and the best methodologies are indicated if appropriate. If solvent ratios are cited for TLC and HPLC solvent combinations, only one has been included as

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an example. Experimental variation of the ratio often will be required to resolve different component mixtures. See Ref. 6 for a compilation of TLC methods for many of the compounds listed in this chapter. In addition to classic extraction techniques described in this chapter, supercritical fluid extraction likely will assume greater significance for toxicological and forensic research as instrumentation becomes available more widely. Lead acetate precipitation was a technique widely used in the past to remove polyphenolic material from plant extracts prior to the purification of tnost of the compounds listed in this chapter but now is used less often. In addition to standard TLC and open-column chromatography, such techniques as droplet countercurrent chromatography (DCCC), rotation locular countercurrent chromatography (RLCC) (7), centrifugal TLC (Chromatotron), and the use of reverse-phase packing material in low-pressure columns often are substituted for classic techniques. Many compounds implicated in mammalian poisoning, including many of the alkaloids, canbe analyzed directly by gas chromatography-mass spectrometry (GC-MS). However, a number of these toxins and their metabolic or hydrolysis products are too refractory for GC analysis. For these samples, the alternatives are direct insertion probe mass spectrometry (DIPMS) and liquid chromatography-mass spectrometry (LC-MS). DIPMS of methanolic extracts has been used effectively to identify toxins implicated in animal poisonings (8, 9). Recent advances in tandem mass spectrometry (MS/MS), LCMS, and LC-MS/MS now often allow identification of alkaloids and many other compounds with a minimum of plant sample treatment. Matrix-assisted laser desorption ionization (MALD1)-MS instruments, which are also now commercially available, offer exciting possibilities for detecting toxins in small fragments of plant material recovered from the stomach of poisoning victims. As LC-MS and LC-MS/MS instruments and the capiliary electrophoresis equivalents become less expensive and more common, methods using these instruments will become the preferred techniques for analysis of most of these toxic compounds. Fourier transform-infrared (FT-IR) spectroscopy also may become a useful detection system for both GC and HPLC analysis in the future, particularly for compounds that do not have strong ultraviolet (UV) absorptions. Flame ionization detectors (FIDs) or evaporative light scattering detectors (ELSD) for HPLC also can beused to improve the sensitivity for compounds with weak UV chromophores.

II. ALKALOIDS For extraction and purification of alkaloids, standard alkaloid workup methods are applicable unless otherwise stated. Plant samples are extracted with methanol (MeOH) or ethanol (EtOH). The alcohol is removed by evaporation and the resulting residue dissolved in dilute acid [0.2-1 N (normal) HISOj or HCl]. At this stage, the extract can be partitioned against a nonpolar solvent, usually hexane or petroleum ether, to remove lipids and pigments. Some procedures also call for partitioning of the acid aqueous phase against ethyl acetate (EtOAc) to remove other compounds that might interfere with alkaloid detennination. This is followed by alkalinization (pH 8-10) of the aqueous residue with ammonia (NH,OH), sodium carbonate (Na2C03),or sodium hydroxide (NaOH) and extraction of the alkaloids into chloroform (CHCl?) or dichloromethane (CH2C12). Partial purification of the alkaloid fraction also often can be achieved by increasing the pH of the aqueous phase in increments and extracting the alkaloids at each step with

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CHCl,. Alkaloids present in the rumen of a poisoned animal sometimes can be isolated by application of the methods appropriate for extraction from the plant itself. However, mild extraction conditions applied to rumen contents often lead to intractable emulsions. Alkaloids can be recovered from rumen fluid by basification with 1 N NaOH prior to extraction with CHC13. This can lead to the production of artifacts that must be cataloged by extraction and purification of the suspected toxin under the same conditions (8). Dragendorff reagent in its many forms and iodoplatinate are used widely for the detection of alkaloids on paper and TLC chromatograms. A significant number of nonalkaloid, nontoxic compounds also react with these reagents, particularly Dragendorff reagent (10). Other alkaloid reagents include Mayer’s (HgClJKl), Marme’s (CdlJKl), Wagner’s (lJKl), and iodine vapors. The GC analysis of alkaloids was recently reviewed by Dagnino and Verpoorte (1 1). A.

PyrrolizidineAlkaloids

Pyrrolizidine alkaloids (PAS) (Table 1) are the toxic principles causing livestock and human poisoning associated with the consumption of members of the Senecio, Crotnlurin, Heliotropium, Triclzodesnzcr, Cyoglossuwz,Amsinckia. and Echium genera ( 1). Most hepatotoxic pyrrolizidine alkaloids are esters of the unsaturated amino alcohol bases retronecine and heliotridine (1, 12, 13) and usually are stored as N-oxides (14). Plant samples should be frozen and freeze-dried as soon as possible. Since PA N-oxides are difficult to chromatograph ( 5 ) and not all N-oxides partition into CHCl, (15), extracts should be divided; the PA N-oxides in half the acidic aqueous fraction are reduced with Zn dust to the corresponding tertiary alkaloid or with Na2S204if quantitative reduction is desired (16) and the balance partitioned into CHC13 and processed by standard alkaloid workup. PAS can also be isolated from small plant samples (less than 10 g fresh weight) by extraction in 0.05 MH2SO4.After centrifugation, the extract is made basic with NH40H, applied to a Extrelut (Merck) column, and the PAS are eluted with CH,Cl?. Pyrrolizidine alkaloids have also been extracted from seeds by supercritical fluid extraction utilizing 10% ethanol (17) or 5% methanol (MeOH) (18) in carbon dioxide. Investigators should also look for the presence of dehydroretronecine, the putative toxic metabolite of pyrrolizidine alkaloids (19), or the corresponding S-conjugated pyrrolic metabolites (20) when examining samples obtained from poisoned animals. Pyrrolizidine alkaloids have no useful UV absorption or fluorescence for facile detection in extracts or during chromatography (15). 1. Spectrophotometric, Immunoassay, and Nuclear Magnetic Resonance Methods PA N-oxides can be detected spectrophotometrically after conversion to corresponding pyrroles by heating with acetic anhydride in diglyrne (21, 23). PAS themselves must be oxidized to the N-oxide by reaction with hydrogen peroxide (containing 2-4% sodium pyrophosphate) before conversion to the pyrrole. Ehrlich reagent (2% 4-dimethylaminobenzaldehyde in EtOH containing 2% boron trifluroide etherate) reacts with pyrroles to produce a purple chromophore (22). Naturally occurring pyrroles and indoles also will react with Ehrlich reagent without the oxidation step and saturated PAS of the otonecine group do not form pyrroles. Addition of ethanolic ascorbic acid prior to the addition of the Ehrlich reagent prevents polymerization and enhances color development (33-25). The conversion to pyrroles also may be achieved by reaction with 5% alkaline sodium nitroprusside (24). Ehrlich reaction is nonspecific and can give only relative results since

is s 0

3 3

Table 1 Toxic Pyrrolizidine Alkaloids Heliotridine esters Heliosupine Lasiocarpine Heliotrine Echinatine Rinderine Europine Heliotridine 7-Angel ylheliotndine

9 Retionecine esters

Retrorsine Seneciphylline Jacobine Senecionine Riddelliine Latifoline Monocrotaline Echinlidine Spectabiline Symphytine Lycopsamine Fulvine Echiumine

Sincanlidine Retronecine Dicrotaline Usaraiiline Junceine Senecivernine Nilgirine Heliospathine (-t) 7-Acetyllycopsanline Uplandicine Syiniand itie Crispatine

Supinidine esters

Otonecine esters

Crotanecine esters

He1eurine Cyanaustine

Otonecine Retusamine

Anacrotine Madurensine

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unfractionated samples usually contain mixtures of alkaloids that form colored complexes of varying absorptivities (13, 23). Enzyme-linked imtnunosorbent assay (ELISA) procedures have been developed for detection of PAS (26, 27). Antibodies were developed to detect the necine base retronecine. Retronamine and monocrotaline appear to be the haptens of choice for development of broad-spectrum cross-reactivity for detection of PAS. Polyclonal antibodies have also been developed to recognize monocrotaline (28). Recently monoclonal antibodies against a retrorsine cojugate have been developed (29); however, these antibodies do not recognize all of the tnajor toxic PAS nor do they recognize PAS in their naturally occuring N-oxide forms. Nuclear magnetic resonance (NMR) spectroscopy can be used to estimate the total unsaturated pyrrolizidine alkaloids in a sample by determining the intensity of the vinyl H-2 signal (30, 31). 2. TLC and Paper Chromatographic Methods Suitable TLC systems for analysis of PAS on silica gel include CHC13:MeOH: 25% NH40H (85 : 14 : 1) (32), CHC13:diethylamine (9: l), toluene :EtOAc : diethylatnine (5 :3 : 3 ) (16), CHCl :acetone :EtOH :NH40H (5 : 3 : 1 : 1) (33), and EtOAc :acetone :EtOH : NH,OH (5 :3 : 1 : 1) (34). Silica gel impregnated with 0.1 M NaOH (MeOH) or lithium chloride (35) tnay also be useful. N-oxides can be separated on silica gel using 17-butanol: H20:CH3COOH (4:5: 1) (33) or CHC13:MeOH:25% NH4OH:n-pentane (82:14:2.6:20, v/v) (36). The modified Ehrlich reagent procedure described above may also be used as a TLC spray reagent. The conversion to pyrroles is achieved by spraying with 10% acetic anhydride in benzene. TLC plates also may be sprayed with 1% 0-chloranil (tetrachloroo-benzoquinone), which oxidizes PAS directly to pyrroles prior to spraying with Ehrlich's reagent (37); however, a spray with acetic anhydride still is necessary to convert N-oxides to pyrroles. Chloranil (1% in toluene) can also be used directly to visualize PAS. This reagent is more specific than Dragendorff reagent (38). Iodine vapor also can be used to detect PAS on TLC, but this is one of the least-specific reagents (39). The iodoplatinate reagent (10) is a useful detection reagent for paper chromatography (PCj, but is less effective on TLC (33).

3. GC, GC-MS, MS, and HPLC Methods Capillary GC is the preferred method for analysis of underivatized PAS. Suitable capillary colutnn coatings for GC and GC-MS include BPI methyl silicone (51, DB-1 (16), DB-5 or DB-17 (40), cpSil 5 or SE-30 (41), OV-101 (14), OV-1 (18), and SE-54 (42). Some pyrrolizidine alkaloids are subject to thermal decomposition during GC analysis, particularly esters of echimidinic acid (43). When derivatization is required, trimethylsilyl (TMS) (44), butylboronate (43), alkylboronate, and trifluoroacetyl derivatives (45) give the best results. However, no single derivatization procedure is universally applicable (45). For GC-MS, 6% OV-101-coated packed columns may be used (46). Total alkaloid concentration can be determined by hydrolysis of an alkaloid mixture with base to retronecine, followed by formation of the bis(heptafluorobutyrate) derivative (46). Chemical ionization MS (Cl-MS) utilizing ammonia is also a useful technique (47). Chromatography on reversed-phase (RP) or NH2 HPLC columns also may be used to resolve and quantify PAS. Monocrotaline, usaramine, and their corresponding N-oxides were resolved on deactivated C-18 columns eluted with a complex gradient of acidic potassium dihydrogen phosphate (KH,PO,), triethylamine, trifluroacetic acid, H 2 0 ,acetonitrile (CH3CN), and tetrahydrofuran (THF) with UV detection at 205 nanometers (nm) (48). Methanol extracts of plant material containing PA N-oxides were resolved on C-18

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columns eluted isocratically with MeOH: 15 mM KHzPOj (pH 7.5) (1 :2) with UV detection at 209 nm (16). Adjusting the ratio of MeOH to buffer and buffer pH can permit analysis of free PA bases (49, 50). PAS also can be resolved by ion-pair chromatography on C-18 columns eluted with a gradient of CH,CN and 0.1-0.2 M Na2POj containing 8 mM 1-heptanesulfonic acid Na-salt and 0.1 mM EDTA (pH 2.6) (51). HPLC separation of N-oxides also has been achieved using NH2 columns eluted with CH3CN:H20(92: 8) (52). Radially compressed CN columns eluted with MeOH:H20 0.01 M dibutylamine phosphate (pH 3.4) will separate all classes of PAS with the N-oxides and free bases having equal retention times (31). PAS also can be resolved on PRP- 1 styrene-divinylbenzene columns eluted with CH3CN:NH40Hgradients, but the high pH affects the stability of PAs (53,54), or on PLRP-S columns eluted with CH3CN:H 2 0gradients and subsequent LC-MS analysis (19).

B. PiperidineAlkaloids Piperidine alkaloids (Table 2) are the toxic principles causing livestock poisonings associated with the consumption of poison hemlock (Conium maculntunz). Some of these alkaloids are volatile. Piperidine alkaloids can be extracted from dried plant material by grinding the material in 10% NH7 in water and extracting the alkaloids into peroxide-free ether. The alkaloids then are partitioned into 2% H2S04.The acidic aqueous phase then is adjusted to pH 8.5 and the alkaloids partitioned into CHCl?. 1. TLC and SpectrophotometricMethods Conium alkaloids canbe separated on activated silica gel using CHCl?:EtOH: 25% NHjOH (18 :2 :2) (55) or acetone: diethyl ether: NH40H (50: 50 : 3) (56) as the eluting solvents. Piperidine alkaloids can be resolved on silica gel TLC eluted with CHC13:MeOH (7 :3) and visualized with Dragendorff reagent. Suitable TLC systems for piperidine alkaloids on silica gel include CHC13:MeOH (1 : 1) or MeOH: NHjOH (97 :3) and CHC13: MeOH (4: 1) for alumina plates (57). Total alkaloid concentration can be determined spectrophotometrically by reaction with bromothymol blue (58). Gamma coniceine may be determined in the presence of other piperideines by reaction with sodium nitroprusside (58).

2. GC and HPLC Methods GC can be used to quantify conium alkaloids. Columns packed with Celite 70-80 coated with silicone elastomer (59), silica gel coated with PEG 4000 and KOH (60), and Chromosorb 80-100 coated with SE-30 (61) all have been used successfully. RP-HPLC analysis can be achieved on RP-18 columns eluted with a CH3CN:0.07 M triethylamine (pH 7.56) gradient (63). Table 2 ToxicPiperidineAlkaloids Coniine y-Coniceine N-methylconiine Lobelanine

Conhydrine Pseudoconhydrine a-Coniceine Lobeline

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PyridineAlkaloids

Nicotine is a highly toxic alkaloid found in Nicotinw species both cultivated and wild. Other toxic pyridine alkaloids associated with livestock poisoning include anabasine, anatabine, nornicotine, and ricinine. Pyridine alkaloids can be extracted from freeze-dried plant material with CH,C12: 1 N HC1 (1 : 1). The acidic aqueous phase is recovered, adjusted to pH 11, and the alkaloids extracted into CH,C12 (63). Tobacco alkaloids can be extracted from dried samples in 25 mM sodium phosphate buffer (pH 7.8) or from fresh samples with 0.1% 1 N HC1 in 40% aqueous MeOH (64). Anabasine also can be recovered from EtOH :NH40H (98 :2) extracts (65). 1. Paper Chromatographic and TLC Methods Tobacco alkaloids can be resolved by paper chromatography developed in tert-amyl alcohol: 0.2 M acetate buffer (pH 5.6) (1 : 1) (66). Pyridine alkaloid mixtures can be resolved by two-dimensional (2D) TLC on silica gel impregnated with KOH and developed first in CHC13:MeOH ( 100 :20) and then in CHC17:diethyl ether: THF (80 : 15 :5 ) in the second dimension (67), or on silica gel PF254 developed with toluene :acetone: MeOH: NHjOH (28: 6: 2:1) (68). Pyridine alkaloids can be visualized by Dragendorff reagent, iodoplatinate, or by spraying with 1% benzidine in EtOH and exposure to BrCN vapor (67). 2. GC and HPLC Methods A large number of GC column coatings have been used to determine nicotine and related alkaloids (62). Pyridine alkaloids chromatograph best on columns treated with a strong base (69). Recently, the following capillary column coatings have been used successfully to analyze for nicotine alkaloids : SE-30 (70), cross-linked Me-silicone (68), Carbowax 20M and dimethylsilcone (71), or amine deactivated (72). Suitable packed-column coatings include 3% OV-22 (71), 3970 SP 2250DB (73), or 1.5% SE-30 (74). Nitrogen-phosphorus detectors (NPDs) can be used to increase sensitivity (71, 72). Pyridine alkaloids can be resolved by normal, reversed-phase, and cation-exchange HPLC. Normal-phase separations include Si 50 eluted with CH2Cl1:diisopropyl ether: MeOH :NHjOH (62 : 30 :7.9 :0.1) (75). Reverse-phase separations include C- 18 columns eluted with 40% aqueous MeOH containing 0.2% H;PO, (pH 7.25 with triethylamine) (64), a0.07 M triethylamine (pH 7.56 with H3POj):CH3CNgradient (73), or C-8 columns eluted with a MeOH: NH4Cl-NHjOH(0.02 M, pH 9.2) gradient (76). lon-pair chromatography on C-18 columns eluted with 2 mM NaH2P04containing 0.25 mM sodium octyl sulfate :(CHjCN:MeOH; 3 : 1) (pH 3.0 with HjP04) (92: 5 :7.5) also has been tried (77). Cation-exchange separations were achieved on Partisil-1OSCXcolumns eluted with a gradient of MeOH: 0.3 M NaOAc (pH 4.5) and 1% triethylamine (78). Nicotine and related alkaloids also can be resolved on a beta-cyclodextrin column eluted with CH3CN in 1% aqueous triethylammonium acetate (79). Most systems employ UV detection, but some use electrochemical detection (77, 80).

D. Indole, Tryptamine, P-Carboline, and Related Alkaloids Indole, tryptamine, P-carboline, and related alkaloids are not related biosynthetically but traditionally have been considered as a group (Table 3). Ergot alkaloids are probably the most-important toxic indole alkaloids (81). Strychnine alkaloids can be extracted by conventional extraction procedures (82) or with CHC13from plant material moistened with

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Table 3 Toxic Indole, Tryptamine, P-Carboline,andRelatedAlkaloids P-Carboline

Clavine

alkaloids alkaloids alkaloids Indole Tryptamine alkaloids Gramine 5-Methoxydimethyltryptamine 5-Hydroxydimethyltryptamine Gelsemine Isoharmine Strychnine tryptamineN-Methyl 5-Methoxy-N,NBrucine dimethyltryptamine Novacine Dimethyltryptamine Vindoline Lolitrem B Serotonin Paxilline Ergonovine

Harmine

I Chanoclavine Agroclavine

Elymoclavine Harmaline

50% NH,OH: 20% Na2CO? (1: 1). The CHCl , extract is purified further by acid-base partitioning (83). Alternative extraction solvents for indole alkaloids include CHC13: MeOH: NH,OH (26: 33 : 1) (82), MeOH: NHIOH(99.5 :0.5) (84), or CHCl, :MeOH (2: 1) with purification on C-18 solid-phase cartridges (85). For HPLC analysis, 1% NH40H in MeOH extracts may be analyzed directly (86). Gelsemine and strychnine have very similar UV absorption spectra and similar color reactions. Ergonovine is light sensitive and precautions should be taken accordingly (87). 1. TLC andImmunologicalMethods TLC systems for strychnine alkaloids include acetone : i-PrOH :5.5% NH,OH (45 :35 :20), EtOAc : i-PrOH :NH,OH (1 00 :2 : 1), Iz-BuOH :0.1 M HC1: 7.4% aqueous (aq) K,Fe(CN)6 (100: 15 : 34), or CHCll: diethylamine (9 : 1) (88) with visualization using Dragendorff reagent, cerium sulfate in H,SO,, or 0.2 M Fe2Cl?in 35% aqueous HClO, (89,90). Gramine can be determined as a fluorescent derivative on silica plates after development in PrOH: EtOAc :NH,OH :2-ethoxy-EtOH (60 : 15 :3 :5 ) (91, 92). Tryptamines can be chromatographed on silica gel with BuOH :HCOOH :H 2 0 ( I6 : 1 : 3) or iso-PrOH :EtOAc :NHjOH (60: 15:3) (82), or on cellulose with BuOH: 2 N HC1 (1 : 1, upper phase) (82). The ergot alkaloid ergonovine can be detected by an ELISA assay using an antibody that recognizes a broad spectrum of ergot alkaloids (93). 2. GC,HPLC,andCapillaryElectrophoreticMethods Strychnine alkaloids can be resolved by GC on a 5% SE-52-coated packed column (94), and gramine and tryptamine alkaloids can be resolved on 2% OV-17 columns (95). Gramine can be determined directly by HPLC analysis of plant extracts (86). A number of procedures have been developed for HPLC analysis of strychnine-type alkaloids (C-18, CH,CN:H,O; 25: 75) (85); however, the only column that appears to separate all of the alkaloids found in Stlychrzos species are yPorasil columns eluted with CHC13:cyclohexane:diethylamine (60:40: 1) (96). Hypersil columns eluted with MeOH:2 M NH,OH: 1 M NH4N01(27 :2 : 1) also can be used (97). Cation-exchange columns eluted with H3B03: CH,CN:n-propanol gradients (98) and Si 60 columns eluted with 1% diethylamine in diethyl ether have been used (99). Beta-carbolines may be resolved by chromatography on ODS columns eluted with MeOH: CHC13: 10% NH,OH (200: 800:13) (100). Ergonovine can be chromatographed on C-18 columns eluted with CH3CN:0.01 M ammonium

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carbonate (pH 7.8) (3 :7) with fluorometric detection (87). Many of these alkaloids can also be detected using electrochemical detection (80). The ergot alkaloids have also been successfully quantified by capillary electrophoresis using p- and y-cyclodextrin :urea : poly(viny1 alcohol) :phosphate buffer (10 1).

E. QuinolizidineAlkaloids Quinolizidine alkaloids (Table 4) are responsible for a significant number of livestock poisonings. Alkaloid concentrations and types can v a y within a species collected from different areas (102). Quinolizidine alkaloids can be extracted by classical alkaloid extraction procedures (103). Alkaloids also may be extracted from plant material in 2 N HCl. The resulting homogenate then is made alkaline with 25% NH40H and applied to Extrelut solid-phase extraction cartridges and the alkaloids eluted with CH2C12(104). Plants suspected of containing esters of sparteine- or lupanine-type alkaloids should be processed as fresh satnples or frozen in liquid nitrogen prior to freeze drying as these esters are vulnerable to enzymatic hydrolysis (105). Extraction of quinolizidine alkaloids also may be achieved by refluxing air-dried material with CHC13 (106) and acid-base purification followed by chromatography on silica gel columns eluted with CHC13:cyclohexane :diethylamine (14 :4 : 1). Other methods were summarized by Leonard ( 1 07). 1. TLC, Spectrophotometric,andImmunoassayMethods Suitable TLC solvent systems for silica gel include MeOH :28% NH40H (13 1 :2), CHC13: 28% NH40H (85 : 15 : 1) (108), benzene: diethylamine (7 : 3) (102), CH2C12:MeOH: 28% NHjOH (90:9 : 1) (109), diethyl ether :MeOH :NH40H (100 : 10 : 1) (1 10) and, for alumina, benzene: acetone:MeOH (34: 3 : 3) (109). TLC detection is achieved by Munier’s modification of Dragendorff’s reagent (1 11) or by12K1 (102, 108, 1 12). Total quinolizidine alkaloid content may be determined spectrophotometrically by using Reifer’s reagent (KI/ I.) at 830 nm (1 13), by titration with tetrabromophenolphthalein ethyl ester (1 14,115), or bromocresol purple (1 14). Polyclonal antibodies have been prepared that recognize quinolizidine alkaloids of the lupanine type with a 6-lactam group (1 16), but a number of

Table 4 ToxicQuinolizidineAlkaloids Lupinine Cytisine typetype (bicyclic) type Tetracyclic (-) Sparteine (-) j3-Isosparteine N-Acetylcytisine (-) 17-Oxosparteine (-) Aphyllidine

Retamine (+) Lupanine (+ )- a-Isolupanine Lupanoline Nuttalline Anapyrine (?) Thermopsine Multiflorine

(tricyclic) (-)Angustifoline Lupinine Epilupinine Cytisine

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important quinolizidine alkaloids including cytisine and sparteine were not recognized by these antibodies. 2. GC,GC-MS,and MS Methods GC-MS and capillary GC-MS are now the methods of choice for analysis of quinolizidine alkaloids (1 13) as these compounds are thermostable and volatile under typical GC conditions, with capillary GC the standard option for routine analysis (1 17). Tables of TLC data, GC retention indices, and MS ions of the more-common quinolizidine alkaloids have been published (1 12, 1 18, 119). In most cases, the underivatized alkaloids may be analyzed, but TMS derivitization improves the sensitivity for some alkaloids (120). Some isomers may require preparative TLC separation before GC as their isomers are not resolved by GC (112). Suitable capillary column coatings include 3% OV-17 (1 10, 121), 3% OV-210 (102), CP-Si1 5 (122), SE-30 (105), DB-1, and SE-52 (119). Use of an NP detector increases sensitivity by a factor of at least 10 over an FID (105). Assignment of mass spectral fragments of quinolizidine alkaloids was revised in 1984 (123). Another new technique that offers interesting possibilities for toxin analysis is laser desorption MS. This technique has been used to locate quinolizidine alkaloids in stem tissue (124) and conceivably could be developed as a technique to analyze fragments of plant tissue recovered from the digestive tract.

3. HPLCMethods Quinolizidine alkaloids can be resolved by either normal- or reversed-phase HPLC. Normal-phase separations include Si 100 and Si 60 columns (1 25, 126) eluted with combinations of MeOH: Et,O:H - 0 :25% NH,OH (25 :75 :4 :3) (109) or pPorasil columns eluted with CHCl3:MeOH:NH4OH (100: 10:1) or CH2C12:MeOH:NH,0H (500:25:1) (127). Reverse-phase separations include C-18 columns eluted with CH3CN: 5mM KH-PO, (pH 5.5) (1 :9) (126) or 20% MeOH and 20% isopropanol in 0.01 M phosphate buffer (pH 6.4) (1 28). Sparteine derivatives can be separated on Spherisorb S5 CN RP columns eluted with CH3CN:MeOH :phosphate buffer (pH 2.5) (25 : 32 :43) (129). F. Steroid Alkaloids Methodology for analysis of toxic glycoalkaloids (Table 5) (130) was reviewed in detail by Coxon in 1984 (131). Glycoalkaloids can be extracted with a weak acidic solution (3% CH3COOH, 5% trichloroacetic acid in MeOH, or 2% CH,COOH in MeOH) (132), MeOH: CCI, (2: 1 or j, MeOH:CHC13(2: 1) (133) and the alkaloids precipitated by ammonia at pH 10 at 70°C (134). The aglycones can be liberated from the glycoalkaloids by reaction with 2 M HC1 in CC14 for 3 hr. The CCl, phase is recovered and the aqueous phase extracted with CHC13 for complete recovery of the aglycones. The organic phases are washed with 1 M NH,OH and reduced to dryness prior to analysis (135). The mostefficient extraction of glycoalkaloids from the potato occurs with THF:H20:CH7CN: CH,COOH (50: 30 :20: 1) (1 36). C ,,-solid-phase-extraction cartridges have also been used to clean up samples prior to analysis (137). Recently Isolute trifunctional endcapped (TFEC) C-18 SPE cartridges were shown to give better recoveries (138). 1. TLC,Spectrophotometric, and ImmunoassayMethods Suitable silica gel TLC solvents include i-PrOH :HCOOH :H 2 0 (73 :3 :24j, MeOH : EtOAc :NH,OH (5 :4 : l), CH,C12:MeOH :H,0 (70 :26 :4) (139), and MeOH :CHC13: 1% aqueous NH,OH (2 :2 : 1, lower phase) (140, 141). Glycoalkaloids can be visualized using

Table 5 Toxic Steroid Alkaloids Solanidine type

GIycoside

Aglycone ______

a-Solanine a-Chaconine p-Solanine

Solanidine

Spirosolane type GIycoside ~

a-Solamargine P-Solamargine a-Solasonine

Jerveratiuni type

Aglycone

G1ycoside

Aglycone

Other steroidal alkaloids

Cycloposine Pseudojervine

Cyclopanline Jervine

Veratranline Zygacine Zy gadenine

~

Solasodiene Solasodine

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8-anilinonaphthalene-1-sulfonate(142), Carr-Price reagent (SbC13 in CHC1,) (143, 144), 25% SbCl, in glacial acetic acid (141), or anisaldehyde:H2S04(144). Zygacine and zygadenine can be detected and quantified on TLC by fluorescence scanning after spraying of silica gel plates with MeOH :H2S04(9: 1) (145). Classic spectrophotometric methods include the Marquis reagent (146), Clarke’s reagent (137, 147, 148), and SbC13HC1(149, 150). Total glycoalkaloids also may bedetermined as their aglycones after H2S04hydrolysis by a nonaqueous titration of the nitrogen content using MeOH containing 0.067% bromophenol blue and 10% phenol (133, 150), or titration with p-toluenesulphonic acid and tetrabromophenolphthalein (15 1). Initial radioimmunoassay (RIA) procedures for glycoalkaloids have been superseded by less-expensive ELISA procedures (148, 152, 153) utilizing antisera highly specific for conmon potato alkaloids. Samples for ELISA analysis were extracted in MeOH :H 2 0:CH3COOH (94 :6 : 1) and diluted with PBSTween (phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20) prior to analysis. The antisera will detect a-chaconine, a-solanine, solanidine, and demissidine, but does not react with solasodine, tomatidine, or tomatine. Monoclonal antibodies are now replacing polyclonal antibodies in these ELISA assays (154). Antibodies have now been developed for atomatine for ELISA assays (155). 2. GC,GC-MS, and MS Methods The steroid aglycones can be resolved by GC and GC-MS as TMS or permethylated derivatives, but not all glycosides can be resolved (156). Capillary columns coated with CP-Si15 or CP-Si1 19CB allow direct analysis of the underivatized aglycones(l35). Other suitable capillary column coatings include Quadrex 007 methylsilicone (SE-30 equivalent) (157). Suitable packed-column coatings include 10% SE-30 (135), 5% OV-17 (150), and 3% OV-1 ( 156). More recently, advances in MS have resulted in rapid methods for quantitation of glycoalkaloids including one using matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) (158).

3. HPLCMethods Steroid alkaloids can be separated by HPLC on normal-, reversed-phase C-18, reversephase NH2,and carbohydrate columns, and by ion-pair chromatography on C-18 columns. Normal-phase separations for glycosides include Porasil eluted with acetone :12-hexane (2 : 1) or 97% aqueous acetone (159,160). The aglycones may be resolved on Zorbax-Si1 eluted with n-hexane: Me0H:acetone (1 8 : 1 : 1) (161). Aglycones and glycosides can be resolved on C-18 columns eluted with MeOH: 0.01 M Tris (75 :25), but glycosides with the same number of sugars are not resolved well (162). However, the type of C-18 column can have a significant effect on resolution (138). The effect of different C-18 columns (CH;CN: 100 mM ammonium phosphate) on the resolution of potato glycoalkaloids was recently evaluated (163) and increasing column acidity was found to improve resolution. Glycoalkaloids have been analyzed successfully on NH2columns eluted with THF: 0.34% aqueous KH,P04:CH3CN (50:25:25), EtOH:CH3CN:0.005M KH,PO, (3:2: 1) (164), or THF:CH3CN:H20:MeOH (55:30: 10:5) (165). Solasonine and solaxnargine can be resolved on a carbohydrate column eluted with MeOH : i-PrOH (30 :70) (162) or CH3CN: pentanesulfonic acid: triethanolamine (83 : 17 :0.01) (166), while THF: H 2 0 :CH3CN (56: 14:30) also can be used to resolve other glycoalkaloids (160, 162). Glycoalkaloids also have been resolved on Radial-Pak silca columns eluted with CH7CN:H,0 (77.5: 22.5, containing 0.01% ethanolamine, pH 4.0 with H3P04)(167), and ODs-hypersil columns using a similar solvent system (137, 168). Ion-pair chromatography systems have been

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employed for aglycones, including C-18 columns eluted with 0.017 M (0.5% w/v) aqueous sodium dodecyl sulfate (SDS): CH,CN (15: 85) (169). Extraction of glycoalkaloids with dilute acetic acid in the presence of an ion-pairing reagent (1-heptanesulfonic acid) (136, 170), followed by HPLC analysis on a C-&coated spherical silica column eluted with 0.29% ammonium phosphate in 50% aqueous CH3CN, appears to provide a simple and economical method for glycoalkaloid analysis. Pulsed amperometric detection can also be used to detect glycoalkoids (171) in addition to the standard UV detection.

G. Diterpene Alkaloids C19and diterpene alkaloids are the principal poisonous components in a number of range plants often implicated in livestock poisoning (Table 6). Solvent extraction (MeOH or 89% EtOH) from dried plant material is the standard procedure (172). Defatting with hexane or petroleum ether may be required when extracting seed (173, 174). Na2C03 should be used to alkalinize extracts to avoid creation of artifacts (175). Open-column and flash-column chromatography on silica gel or alumina (C6H6:CH2C11)are standard preparative chromatographic procedures (176). 1. TLC Methods Suitable TLC systems include silica gel eluted with CHCl, :MeOH (9 : 1) (177), cyclohexane :CHCl : diethylamine (5 :4 : 1) (178), cyclohexane :acetone :diethylamine (7 : 1 : 1) (179), or alumina eluted with EtOAc :diethyl ether (20: 80) or acetone:hexane (10: 90) (176). Centrifugal TLC has been used to separate diterpene alkaloids (180, 181).

2. GC, MS, and HPLC Methods Diterpene alkaloids can be chromatographed underivatized on SE-30 capillary columns (182). Chemical ionization MS (CI-MS) using ammonia hasbeen used effectively to characterize diterpene alkaloids (47). However, GC is not suitable for quantitation of anthranilic-acid-esterified lycoctonine diterpenes, including methyllycaconitine (183). Reverse-phase and ion-pair HPLC columns give better resolution than normal-phase columns (62, 184). Suitable columns include Micropak MCH-5 eluted with 4-mM sodium

Table 6 ToxicDiterpeneAlkaloids

C I q aconitine type Veatchine

C 19 lycoctonine type

Aconitine Atisine Lycoctonine Mesaconitine 13-0-acetate Delcosine Hetisine Garryfol Jesaconitine Ajaconine Ganyine Methyllycaconitine 3-Acetylaconitine Geyelidine Delsoline Hypaconi tine Geyerine Deltaline Lipoaconitine Anthranoyllycoctonine 13-Acetylhetisine Delphinine Tricornine Pseudaconitine Browniine Aconifine Delpheline Lappaconitine Glaucedine Delphidine Glaucenine Bicoloridine Glaucerine

Czc,type atisine

C2()veatchine type i ne

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hexane-sulfonate in 0.1 % aqueous orthophosphoric acid: CH,CN (8 :92) (185) and ODS columns eluted with THF and phosphate buffer (pH 2.7) containing 0.01 M Na hexanesulfonate (186). Ammonium carbonate (1.7 mM) canbe substituted for the phosphate buffer (1 87).

H. lndolizidineAlkaloids Consumption of plants containing the indolizidine alkaloids swainsonine, swainsonine Noxide, and castanospermine results in the condition known as locoism (188). Dried plant material can be extracted in EtOAc in a soxhlet. The water-soluble fraction obtained after evaporation of the EtOAc is eluted from Dowex 50W-X8 with dilute ammonia. The compounds are purified further by adsorption on CM-Sepharose CL-6B (pH 5.0) and elution with a NaCl gradient (0-1 M) in 10 mM NaOAc buffer (189). Extraction also may be achieved in hot 70% MeOH, followed by chromatography on strong cation-exchange columns ( 190). The MeOH extract also can be partitioned against petroleum ether (1 91). An equal volume of saturated NaCl then is added to the MeOH fraction and adjusted to pH 10. Swainsonine partitions into CH,Cl, or CHCZ?. The acetate derivatives then can be prepared by reaction with acetic anhydride in pyridine and the resulting acetates analyzed by GC or GUMS. Swainsonine N-oxide is insoluble in CHCl,; this can be used to separate the N-oxide from swainsonine (192). 1. TLC andEnzymeAssayMethods Castanospermine does not react with Dragendorff reagent, and swainsonine reacts only weakly; however, they can be made to react with Ehrlich’s reagent by converting them to pyrroles (10% acetic anhydride in benzene) (193). This reaction can be accomplished on a silica gel plate after development in CHC13:MeOH: NH,OH: H 2 0(35 : 13 : 1 : 1) ( 193). The N-oxide fot-rns generate more intense colors (194). The crude extracts also can be analyzed by TLC (191) with visualization by I2vapor, iodoplatinate spray, or ninhydrin (192). Swainsonine may be quantified enzymatically using jack bean a-mannosidase or mouse liver homogenate and 4-methylu1nbelliferyl-a-~-mannopyranosideas the substrate (1 89). 2. GC and HPLC Methods GC capillary analysis of the TMS derivatives (bis-trimethylsilyltrifuoroacetarnide)on an SE-30 column yields a single peak for swainsonine (195). Swainsonine can be detected in ethanol extracts by derivatization with bis-(trimethylsily1)trifluoroacetamide (BSTFA). Castanospermine should be derivatized with HMDS-TMCS (hexamethyldisilazane-trimethylchlorosilane) (194). Swainsonine also can be determined by GC as its acetate derivative on3% OV-17 or Macrobore DB-5 packed columns (191). Castanospermine and swainsonine can be resolved by HPLC on a cation-exchange column eluted with 10 mM HCl with pulsed amperometric detection (196).

1.

TropaneAlkaloids

Tropane alkaloids, including atropine, hyoscyamine, hyoscine, scopolamine, meteloidine, norhyoscyamine, and cocaine, are the dominant toxic principles found in members of the Dnturn, Hyoscyamus. Solnnclra, and Scopolia genera. Atropine can be isolated from biological samples by treating the sample with NaOH in CH2C12;the alkaloids are recov-

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ered from the CH2C12fraction. Tropane alkaloids also may be extracted with aqueous Ca(OH),. The unfiltered extract is applied to a column of diatomaceous earth and the alkaloids eluted with peroxide-free diethyl ether. The alkaloids then are partitioned into 0.1 M NH2S04,the acidic aqueous fraction adjusted to pH 10, and the alkaloids extracted into diethyl ether (197). Extracts also may be prepared by extraction in 1% NH40H in MeOH and subjected to the standard alkaloid workup (198). Hyoscyamine and scopolamine appear to be extractable frotn freeze-dried samples by a range of solvents ranging from dilute acid to basic CHC13 (199). Atropine is very fragile and care must be exercised to avoid hydrolysis to tropane. 1. TLC, Paper Chromatographic, Spectrophotometric, and Immunoassay Methods Tropane alkaloids can be chromatographed on silica gel PF254 with toluene: acetone: MeOH:NH,OH (28:6:2: 1) (62.9, 1,1,1,-C,H,C13:diethylamine (9: 1) (197), or CHC13: acetone:MeOH:28% NH40H (73: 10: 15 :2) (198). Chromatography can also be achieved by alumina TLC (acetone :EtOH, 1 : 1) or paper chromatography (petroleum ether: CH3COOH:n-amyl alcohol: H,O; 1 :3 :3 :3) (200). Tropane alkaloids can be visualized on TLC plates with 4-dimethylaminobenzaldehyde dissolved in EtOH : 8N H,S04 (1: 1) (1971, or by Dragendorff reagent followed by sodium nitrite (201). Atropine can be determined spectrophotometrically by reaction with y-dimethylaminobenzaldehyde or 2% citric acid in acetic anhydride:EtOH (9: 1) (202). Radioimmunoassays have been developed for scopolamine and related tropane alkaloids (203) but the antisera do not react with atropine and only weakly with hyoscyamine (204). Recently, monoclonal antibody ELISA assays for scopolamine were developed (205,206). A radioimmunoassay for atropine and hyoscyamine (207, 208) and, more recently, ELISA assays for atropine have been developed (209).

2. GC,GC-MS, and HPLC Methods Tropane alkaloids may be analyzed by capillary GC and GC-MS using cross-linked methyl silicone coatings (68). Atropine, scopolamine, and hyoscyamine can be chromatographed on SE-30 (74, 202) or 3% OV-17-coated packed columns. Tropane alkaloids can be analyzed by reverse-phase HPLC chromatography on ODs-120a columns eluted with CHICN:H 2 0(2: 8 v/v containing 6 mM H1P04,pH 2.7,50°C) (210),or on C-18 columns eluted with 0.2% H3P04(pH 7.25 with diethylamine) and MeOH (85: 15) (204), or 3% CH,COOH:MeOH (75 :25) (21 l), with UV detection at 215 nm (210) or 229 nm (204). Hyoscyamine and scopolamine can also be separated on Novapack C-18 columns eluted with 12.5% aqueous CH3CNcontaining 0.3% H3P04adjusted to pH 2.2 with triethylamine (199). Ion-pair chromatography can be achieved by eluting with CH3CN: 10 mM SDS (pH 3.3 with H3P04)(2: 3) (212) or 0.066 M Na3P04(pH 3.5) and MeOH (48:52) containing 17.5 mM SDS (213). Atropine can be quantified after hydrolysis to tropic acid and formation of the fluorescent 4-bromomethyl-7-methoxycoumarin derivative, which can be chromatographed on C-18 eluted with CH3CN:0.01 M NH4P04 (67:33) (214). Scopolamine can also be detected by electrochemical detection, but atropine gives only a relatively weak signal (80). J.

lsoquinoline Alkaloids

Isoquinoline alkaloids include protopine, protoberberine, aporphine, berberine, sanguinarine, and (+)-isocorydine, as well as several drugs of abuse (codeine and morphine).

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Isoquinoline alkaloids can be extracted by standard alkaloid extraction procedures and purified by Sephadex LH-20 chromatography. 1. TLC, HPTLC, and Immunoassay Methods TLC systems include silica gel eluted with C6H,:acetone:NH,0H (100: 100: 10) (315), EtOAc:C6H,:n-PrOH:MeOH:EtNH, (1 :8:2:2:1.5) (216j, BuOH:CH,COOH:H,O (7: 1 :2), or CHC13:MeOH:CH3COOH (25:10: 1) (217). Isoquinoline alkaloids can be analyzed by high-performance TLC (HPTLC) on silica gel or silica gel NH, plates eluted with rz-BuOH:H 2 0:NH40H (8 : 1: l), CH3Cl:MeOH :NH,OH (14 :7 : 1), ;I-BuOH: HOAc :H,O (4 : 1 : l), 0.05 M NaOAc in MeOH :H 2 0:dioxane (5 :4 : 1j. or CHClj :MeOH: NH,OH (14: 4: 1) (318). Visualization usually is achieved with Dragendorff reagent or iodoplatinate reagent (219). A radioimmunoassay (RIA') method was developed for the isoquinoline alkaloids codeine and morphine, but cross-reactivity with isoquinoline alkaloids implicated in livestock poisoning was not demonstrated (220). 2. GC and HPLC Methods Sanguinarine can be analyzed by GC on Diazolid-ZS-packed columns (219). Protopine, berberine, and dihydrosanguinarine can be analyzed by RP-HPLC using H,O: MeOH: CH,CN gradients containing an organic amine (221). Isoquinoline alkaloids can be resolved by ion-pair reverse-phase HPLC on a Lichrospher 100 CVH column eluted with a gradient of Me0H:THF (1 : 1) against 0.8% aqueous sodium lauryl sulfate and 0.6% citric acid: CH3CN(100: 80) (222). These alkaloids also can be resolved on C-18 columns eluted with H20: CH3CN:H3P04 (98: 3 :0.01) (223) or CH3CN:H20:triethylamine (40: 60 :0.1 ) (224) or on normal-phase columns eluted with hexane :CH,C12:triethylamine (30 : 6:2:2) (224). UV and electrochemical detection can be used (80). K. PeptideAlkaloids Ergopeptine alkaloids (ergovaline and ergotamine) are toxic components resulting from endophyte fungal attack on grasses, particularly fescue and ryegrass (81). They may be extracted with basic chloroform and the extracts purified by SPE on silica gel (225). Ergovaline may also be extracted from infected seed with aqueous lactic acid (226). Care should be exercised when extracting ergopeptine alkaloids as these compounds are extremely susceptible to photolytic and air oxidation and epimerization (81). Use of the organic binder used in silica gel TLC plates as a cleanup column can significantly increase the sensitivity of HPLC and MS-MS techniques (227). 1. TLC and Immunological Methods Ergot alkaloids including the peptide alkaloids can be separated by silica gel TLC (CH,C12:isopropyl alcohol; 92: 8) (81, 228) and detected by their fluorescence under UV. or by visualization with van Urk's reagent (p-N,N-dimethylaminobenzaldehyde) followed by 1% NaNO: (229). A competitive inhibition enzyme linked immunosorbent assay (CIELISAj can be used to detect peptide alkaloids when phenylalanine is part of the cyclol peptide (230). 2. HPLC and Capillary Electrophoretic Methods Ergotamine can be chromatographed on polymeric reversed-phase columns eluted with CH3CN:2.6 mM ammonium carbonate (7 :3) with fluorescence detection (235) or Zorbax C-18 columns eluted with an CH3CN:0.1 N atnmoniunl acetate buffer (pH 7.6) gradient

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Perloline Perlolidine Sesbanimide A Demecolcine Arecoline

Arecain Buxene Chelidonine Buphanine Dioscorine

(23 l), while ergovaline can be chromatographed on C- 18 columns eluted with a gradient of CH?CN:ammonium carbonate :MeOH (81). Recently a method for capillary electrophoresis determination of ergotamine was reported (232). L. OtherAlkaloids

A number of toxic alkaloids with unrelated chemical structures are listed in Table 7, including some conlpounds that are responsible for significant livestock losses. Colchicine is susceptible to acid hydrolysis and undergoes autooxidation in the presence of light (233). Lycorine can be extracted with 1% H2SO4(234) and crystallized from alkaline aqueous solutions or by conventional alkaloid extraction procedures (235j. Sesbanimide A is a relatively unstable compound requiring special precautions for quantification (236). Ground seeds were exhaustively extracted with methanol in a soxhlet. The methanol extract then was diluted to 50% with water and lipids were extracted into hexane. Sesbanimide A was recovered from the aqueous methanol fraction by extraction into CH2C12. Lycorine can be chromatographed on alkaline silica gel TLC layers developed in CHCl?: MeOH (19 : 1) (235). Dioscorine can be chromatographed on silica gel (CHCl?: EtOH: NH40H (100: 10:0.05) (237). Dioscorine also can be quantified using Mayer’s reagent or silicotungstic acid in a spectrophotometric assay (238). Sesbanimide was determined in extracts by chemical ionization-MS-MS using isobutane as the ionizing gas (236). Lycorine can be determined by chromatography on C- 18 columns eluted with CHjCN:0.01 M ammonium carbonate (47 : 53) (234). Colchicine and related compounds can be resolved on C- 18 columns eluted with CH3CN:MeOH :0.22 M phosphate buffer (pH 6.0)( 16 :5 : 79) (239, 240). Perloline can be resolved by HPLC (241). 111.

GLYCOSIDES

A.

CyanogenicGlycosides

Cyanogenic glycosides (Table 8j are characterized by the enzymatic release of hydrogen cyanide (HCN) (242). They can be extracted with hot EtOH and purified by chromatography on charcoal eluted with C6H6:EtOH(1 : 1) and polyamide eluted with water (243). Table 8 CyanogenicGlycosides Triglochinin Prunasin Amygdalin Dhurrin Hydrangin Tetraphyllin A and B

Gynocardin Linamarin Lotaustralin Sambunigrin Acacipetalin Linustatin

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1. Spectrophotometric Methods Most cyanogenic glycosides can be assayed for release of HCN after enzyme hydrolysis (244). The cyanide usually is trapped in alkali. The Guignard test using sodium picrate paper may be used todetect cyanide in plant tissue containing 50 pg HCN per gram (245). Cyanide also can be detected using Feigl-Anger test papers (246). Cyanide released after enzyme hydrolysis can be measured spectrophotometrically by the Lambert method (reactionwith acidic succinimidelN-chlorosuccinimide and barbituric acid/pyridine) (247249) with improved reproducibility at 0°C (250). Tetramethylbenzidine can be substituted for the benzidine reagent, an established carcinogen, also widely used to determine HCN (251). Potentiometric (252), fluorometric assays (253), and ion chromatographic methods (254) for HCN also are available. 2. GC,GC-MS, and HPLC Methods Cyanogenic glycosides can be analyzed after PSTFA derivatization on 2% OV-17-coated packed columns (255) or by capillary GC on SPB-5-coated columns after derivatization with N, N-bistrimethylsilyltrifluoroacetamideand trimethyl-chlorosilane (256). Cyanogenic glycosides can be analyzed by RP-HPLC on C- 18 columns eluted with 4% CH3CN in water using refractive index or UV detection (256).

B. Glucosinolates Glucosinolates or goitrogenic glycosides, including progoitrin, goitrin (L-5-vinyl-2-thioxazolidone), glucobrassicin, neoglucobrassicin, and sinigrin, occur widely in food plants and are responsible for a number of toxic effects (257). See Ref. 258 for a listing of naturally occurring glucosinolates. Careful extraction of plant material is essential for accurate measurement of glucosinolate concentrations since the hydrolytic enzyme myrosinase, which is present in plant tissue, can hydrolyze the glycoside rapidly if it is not inactivated. Fresh samples can bemacerated in boiling MeOH (259). If processing fresh tissue is not possible, then samples should be quick frozen (-40°C) (260) or freeze dried (261); however, care must be taken to ensure frozen samples do not thaw or freeze-dried samples do not contact moisture. For determination of glucosinolates in seeds, the Swedish tube method involving extraction with petroleum ether to first remove the oil is preferred (262). All of the hydrolysis products of tnyrosinase action on glucosinolates have been used to quantify these compounds in plant tissues with varying degrees of success (263). Samples for HPLC analysis may be concentrated and freed frotn interfering compounds by absorption on to and elution from C18 SPE columns (264). 1. EnzymeAssay and Spectrophotometric Methods Enzyme assays can be used to measure the glucose released by myrosinase hydrolysis of glucosinolates utilizing hexokinase and glucose-6-phosphate dehydrogenase (spectrophotometric) (265, 266) or glucose oxidase and peroxidase (265, 267). The glucose-6-phosphate dehydrogenase assay is more sensitive; however, it requires a kinetic measurement to achieve precision (268), while the glucose oxidase and peroxidase approach suffers from inhibition of the peroxidase activity by phenolics present in the extracts. This limitation can beremoved by substituting a hydrogen-peroxide-specific electrode for the peroxidase enzyme to measure the hydrogen peroxide generated by the glucose oxidase reaction (269, 270). Glucosinolates can be determined as thymol derivatives following purification

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on DEAE-Sephadex (37 1.272) or as thiourea derivatives following myrosinase hydrolysis to their isothiocyanate (273j; however, not all glucosinolates form stable isothiocyanates. 2. GC, Capillary Electrophoretic, and HPLC Methods The glucose released by myrosinase hydrolysis following ion-exchange purification of the glucosinolates can be measured by GC of the TMS derivative (374). Isothiocyanates released by myrosinase hydrolysis can be determined by GC (275, 276); however, not all glucosinolates produce volatile isothiocyanates on hydrolysis (263). Desulfation with the enzyme sulfatase can release glucosinolates adsorbed on a DEAE-Sephadex column (277), and the desulfated glucosinolate then converted to volatile TMS derivatives and analyzed by GC (260, 278). Desulfated glucosinolates can be analyzed by RP-HPLC (279, 280), while intact glucosinolates can be chromatographed by ion-pair chromatography on C- 18 columns (28 1)eluted with MeOH: 0.005 M tetrabutylanlmonium sulfate (364). Unknown glucosinolates can be identified by negative ion electrospray MS after ion pair chromatography using volatile buffers (383). Glucosinolates can also be separated and detected as their fluorescently labeled acid hydrolysis products by capillary electrophoresis (283).

C. PhenolicGlycosides Coumestans, particularly coumestrol, have pronounced estrogenic effects, while the furanocoumarins (Table 9) primarily have phototoxic effects (284). Isoflavones have pronounced estrogenic effects, with genistein being the most potent (285, 286). Furanocoulnarins can be extracted with diethyl ether from aqueous plant extracts and purified by adsorption onto C- 18 and silica solid-phase extraction cartridges to remove polar and nonpolar impurities (287). Isoflavonoid extraction procedures vary according to the source of material and generally involve extraction with an organic solvent (EtOH, MeOH, benzene, CHC13,or EtOAc) followed by partitioning between organic and aqueous solvents (288). Extracts may be purified further by colutnn chromatography on silica gel, polyclar, or Sephadex LH-20. Anthraquinones can be extracted from dried material into MeOH: CHCl: (1 : 1) (289). Quinone aglycones can be extracted from plants in diethyl ether while the glycosides extract into aqueous EtOH (288). Gossypol can be extracted in EtOH:H20:diethyl ether:CH;COOH (715:285:200:0.2) (290). Supercritical fluid extraction using C 0 2is becoming increasingly popular, while crude extracts can be fraction-

Table 9 Toxic Coumarins, Coumestans, Isoflavonoids.andPolyphenolic Furanocoumarins Xanthotoxin Bergapten Trisoralen Anthraquinones Sophanol Physcion Chrysophanol Naphthalenes Gossypol Stypadrol

Coumestans phenolics Other Tsoflavones Coumestrol Formononetin 4’-O-Methylcoumestrol Genistein Repensol Biochanin A Lucernol Calycosin Trifoliol Pratensein 3’-Methoxycoumestrol Pseudobaptigenin Sativol Daidzein Medicagol

Compounds Safrole 1’-Hydroxysafrole Isosafrole Estragole Sinapic acid Sinapine Hydroquinone Hypericin Usnic acid

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ated by DCCC (291). Anthracenones can be recovered by CHC13 extraction and precipitated by addition of hexane and purified by column chromatography on silica gel (292). 1. PC, TLC, and Spectrophotometric Methods Isoflavonoids can be separated by paper chromatography utilizing BAW (12-BuOH: HOAc: H 2 0 ; 41::5 upper layer) and 15% HOAc. These compounds can be detected under UV light and detection can be enhanced with NH1 fumes. TLC on silica-gel plates developed with CHC17:MeOHmixtures is astandard technique. Isoflavones also can be detected by spraying with diazotized p-nitroaniline or Naturstoff reagent (288). Anthraquinones can be resolved by TLC on silica gel developed in diethyl ether:EtOAc :petroleum ether (1.5 : 1.5 :6) (289) or ChHh:EtOAc (4 : 1) (291), with visualization by spraying with 510% KOH in MeOH (288, 289) or I:. Gossypol can be detected spectrophotometrically by reaction with aniline to form dianilinogossypol (290,293). Sinapine can be determined directly in purified satnples by measuring the UV absorption (294). Furanocoumarins can be detected by their UV fluorescence on TLC on silica plates developed in CHC13 and by inhibition of the germination of spores of Cladosporium cucurnerirmrn (295). 2. GC,GC-MS, and HPLC Methods Furanocoumarins can be determined by GC and GC-MS analysis on SPB-1 capillary columns (287) or quartz OV-1 columns (295). HPLC analysis of furocoumarins can be achieved on Ultrasphere columns eluted with CHC13 (287), on C-18 columns (296) or MCH-10 columns (297) eluted with CH3CN:H20,or on silica columns eluted with EtOAc :CHOOH: CHC13(0.1 :0.1 :99.8) (298), or hexane:THF (299). Coumestans and furocoumarins can be resolved on C-18 columns eluted with Me0H:H:O (65 :35) (300,301). HPLC analysis is the method of choice for quantitative analysis of isoflavones. Many different procedures exist, but the majority utilize C-18 columns eluted with MeOH: H 2 0 (302), CH3CN:dilute CH3COOH, or MeOH: dilute HCOOH gradients (288). Normalphase chromatography using hexane:CHC13:MeOH and hexane :THF: CHXOOH also may be employed. Gossypol can be quantified by chromatography on C- 18 columns eluted with 0.1% H;PO, in MeOH: H 2 0 (9: 1) (303). Usnic acid can be chromatographed on C18 columns eluted with CH3CN: KH2P04 gradients (304).

D. SaponinsandCardiacGlycosides The saponins and cardiac glycoside toxins can be divided into four groups depending on the aglycone: cardiac glycosides, steroidal saponins, triterpenoid saponins (Table lo), and basic steroidal saponins (Table 5). Distribution of steroidal saponins and analytical techniques were reviewed in 1982 (305). The cardiac glycosides, which can be divided into two groups, the cardenolides and the bufadienolides, are frequently the cause of poisoning in both humans and animals (306, 307). Cardiac glycosides tend to be heat labile and one or more of the sugars can be lost during the extraction process unless enzymatic hydrolysis is inhibited (308). Isolation methods include cold extraction with EtOAc, followed by evaporation to dryness with the resulting syrup partitioned between 95% MeOH and petroleum ether. The cardiac glycosides are recovered from the MeOH fraction and purified by column chromatography on silica gel (309). Finely ground material can be soaked in 0.2 M CH3COOH,extracted with CHC13,the filtered extract treated with Na2C03,partitioned against 50% Et0H:citrate buffer (pH 3.25), and the cardenolides partitioned in CHC13 (3 10). Cardiac glycosides also can be extracted with phosphate-buffered saline (10 mmol,

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pH 7.4) (31 l), with methanol (3 12). or with 95% ethanol (3 13). The resulting extracts were partitioned against petroleum ether and/or benzene, and the cardenolides extracted into CHCl?. The CHC13fraction then is purified by flash column chromatography on silica gel (314) or reversed-phase packing (315). Triterpenoid and steroidal saponins can be extracted into 80% EtOH (316), concentrated to the aqueous phase, partitioned against ether and EtOAc, and extracted into watersaturated n-butanol. The butanol fraction can be purified further by chromatography on silica gel (CHC13:MeOH :H 2 0 ,70 :30 : 3). Some triterpenoid saponins will partition into the EtOAc phase. The triterpene or steroidal aglycone component can be released by a number of techniques, including acid hydrolysis with 2% HClO, (317) or 1 N HISO., in dioxane H,O (1 : 3) (318), or enzymatic hydrolysis (319). Production of artifacts during hydrolyses is an ever-present problem. Saponins also can be isolated by extraction with a sterol solution (320). Digoxin-type cardenolides also may be purified by removal of other glycosides as phenylboronate derivatives (321). Centrifugal TLC also can be used to fractionate extracts (314). The presence of saponins in a plant extract often can be indicated by the formation of a stable foam and by their hemolytic activity. 1. Spectroscopic, Titrimetric, and Immunoassay Methods Interpretation of spectrophotometric assays for quantitation of saponin concentrations in plant extracts requires some knowledge of the identity of the saponins involved. The Lieberman-Burchard reagent (322) can be used to quantify the sapogenin content and distinguish between triterpenoid (pink/purple) and steroidal saponins (blue-green); however, it requires knowledge of the sugar content to permit accurate calculation of the saponin content. This reagent does not react with rnedicagenic acid. Medicagenic acid can be determined in the presence of other sapogenins by titration with KOH in the presence of thymolphthalein (323). Saponins also can be detected by their ability to inhibit the fungus Trichodennn viricle (324, 325). The Molisch test also can be used to detect saponins. Total cardenolides can be determined spectrophotometrically (326). The presence of cardenolides in plant tissue also may be determined by reaction with 2,2’,4,4’-tetranitrodiphenyl (TNDP), followed by addition of 10%NaOH (327). Radioimmunoassay methods now are available for some cardiac glycosides, including digoxin, digitoxin, and ouabain, in serum and are the preferred methods of analysis; these techniques often are specific for the glycoside, and subsequent metabolism of the compound in mammalian systems can result in an underestimation of the cardiac glycoside (328). Commercial digoxin immunoassay kits have been available for a number of years, but the highly specific antibodies used in these kits may make them unsuitable for detecting other cardenolides implicated in human and animal poisonings (3 1l), although some of these kits give sufficient cross-reactivity to be useful in investigating poisoning cases.

2. TLC Methods The sapogenin fraction can be characterized by silica-gel TLC utilizing the following solvents: CHCl?:MeOH (9 : 1), EtOAC :iso-PrOH :H 2 0 (65 :23 : 12), hexane :EtOAc (1 : 1) (329), EtOAc: CHCl3 (9 1) (312), EtOAc: CH3COOH: H20 (7 2 2) (330), 01’ EtOAc iso-PrOH :n-BuOH :H 2 0 (8 :4 :2 :2) (3 18). Compounds yield characteristic colors upon spraying with H2S0., (3 17) or MeOH: acetic anhydride :H2S04(50 :5 :5 ) (330) and heating. Cardenolides can be resolved on silica-gel plates developed with EtOAc :MeOH (97 : 3) (3 12, 33 1) or CHCl?:MeOH :formamide (94: 6 : 1). They can be visualized by spraying with 0.4% 2,4,2’,4’-tetranitrodiphenylin toluene followed by 10% KOH in 50% MeOH

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(314). Visualization also can be achieved by spraying with 3,5-nitrobenzoic acid (or 3 5 dinitrobenzene) followed by heating and spraying with 10% NaOH and 95% EtOH saturated with 2,4-dinitrophenylsulfone(or 70% EtOH saturated with 1,2-naphthoquinone-4sulfonic acid) (332), or with 1% aqueous picric acid followed by 10% NaOH (332). Glycosides can be detected by spraying with 0.5% p-anisaldehyde in H,SOJHOAc (2: 98) (120°C ) (331), which reacts with the carbohydrate component (3 15, 318). Saponins may be chromatographed on silica gel (IZ-BUOH :EtOH :H20,7:2 :5), CHC13:MeOH : H 2 0 (65 : 35 : 19) (329), silica pretreated with oxalic acid (CHC13:MeOH: H 2 0 , 65 :35 : 10 lower phase), or C-18 HPTLC (MeOH:H,O, 3 :2) (333). Cardenolides also may be determined fluorometrically by reacting with 0.065 M H201, 0.1%L-ascorbic acid in MeOH, and concentrated HC1 (331). The hemolytic activity can be assessed by coating a developed TLC plate with a suspension of erythrocytes in isotonic buffer with gelatin. Saponins appear as white spots against a red background (334, 335). 3. GC, GC-MS, MS, and HPLC Methods Structural identification of saponins is achieved by analysis of the carbohydrates and aglycones (sapogenin) generated by acid hydrolysis (336). The structure of the sugars can be determined by mild acidic or alkaline hydrolysis of permethylated glycosides with GC analysis on 5% neopenthylglycol adipate polyester on Chromosorb W (302). GC-MS likely will be the method of choice in the future. GC analysis of the TMS derivatives on 1% SE-30- or OV-1-coated columns also can be used (337). Sapogenins can be analyzed as BSTFA derivatives on 3% OV-1-coated packed columns (333). In the absence of authentic standards, high-resolution MS of acetate or methyl ester derivatives or GC-MS of the TMS derivatives is required for identification. Since most triterpene glycosides are not UV active, UV detection at 210, refractive index or evaporative light scattering detectors (ELSD) must be used for HPLC analysis of underivatized samples. Reversed-phase HPLC on C- 18 columns eluted with MeOH : H20:HCOOH (1500:500: 1) (317) or CH3CN:H20 (35:65) (312) can be used to analyze saponin and cardiac aglycones. The intact saponins and bufadienolides also can be separated by RP-HPLC chromatography on RP-8 and C-18 columns eluted with aqueous MeOH (338, 339) or CH3CN:H20gradients (313, 340, 341). Saponins also can be separated by normal-phase chromatography on silica columns eluted with EtOAc :COOH: H 2 0(10: 2 :2), pet-ether: EtOH (342), or Aquasil SS 452N aqueous silica columns eluted with CHCl,: MeOH: H,O (60: 12: 1) (343), or on hydroxyapatite columns eluted with CHjCN: Hz0(343).

E. NitropropanolGlycosides Toxic aliphatic nitrocompounds occur in higher plants as either glycosides of 3-nitropropano1 or glucose esters of 3-nitropropionic acid (344). The glycoside miserotoxin can be extracted with hot 80% EtOH and purified on polyamide (345) or deactivated coconut charcoal (346, 347). Such esters as karakin, coronarian, and hiptagin can be extracted with acetone and purified on silica-gel columns (348). Recently, centrifugal TLC has been utilized successfully to purify nitropropanol-containing glycosides. 1. TLC and SpectrophotometricMethods Miserotoxin can be chromatographed on Avicel cellulose (BEW) and visualized by spraying with 2 M NaOH: EtOH (1 : 1) and overspraying with diazotized y-nitroaniline (345).

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The aglycones can be chromatographed on silica-gel TLC (CHC13:acetone, 1 : 1 containing 1% H 2 0 ) (349). Aliphatic nitrotoxins can be detected by reaction with diazotized y-nitroaniline or diazotized sulfanilic acid (345). These compounds also may be quantified by measurement of the nitrite ion released after alkaline hydrolysis. However, in biological fluids, miserotoxin and nitropropanol gave poor yields of nitrite (347). Miserotoxin also can be quantified by reaction with diazotized p-nitroaniline after the plant extract is purified ona charcoal column (347,350). Glucose esters of nitropropionic acid can be resolved on silica-gel TLC in EtOAc :CHC13:HCOOH (89 : 10 : 1) or CHC13:acetone :HCOOH (50 : 50 : 1) (35 1).

2. HPLCMethods The aliphatic nitro aglycones can be determined quantitatively in plant, physiological solutions, and urine samples by RP-HPLC on MCH-5 columns eluted with 0.15% orthophosphoric acid (352, 353). The glycosides also can be analyzed by HPLC using the same solvent system (350) or H20:CH3CN:MeOH(354). F. Other Glycosides A number of plant toxins with a range of chemical structures are grouped together in this section because they occur as glycosides (Table 11). Consutnption of l-a,25-dihydroxycholecalciferol glycoside (1,25-dihydroxyvitamin D 3 ) causes an induced vitamin D toxicity (355). Several diterpene glycosides, including carboxyatractyloside and atractyloside, have been implicated in animal poisonings (356). Vicine and convicine, when hydrolyzed to their respective aglycones, are the agents responsible for favism (357). Protoanemonin can be recovered by steam distillation (358) and the distillate purified by hexane extraction (359). The glycoside ranunculin can be isolated by extraction with dilute acid (360) and purified on charcoal or celite (361); however, extraction with metha-

Table 11 Other ToxicSubstances Glycoside

Methylazoxy methanol

Azoxyglycosides Cycasin

Naphthalenes

Anthracenones Anthraquinones Other toxic glycosides

Aglycone

Macrozamin Neocycasins A-E Podophyllotoxin-,P-D-glucoside 5-Methoxypodophyliotonin a-Peltatin-,P-D-glucoside P-Peltatin-P-D-glucoside Aloin Frangulins A and B Chrysophanein Rmunculin Aucubin (iridoid glycoside) Syringin Vicine Convincine Carboxyatractyloside 1 a,25-Dihydroxycholecalciferolglycoside

Podophyllotoxin a-Peltatin P-Peltatin Aloe-emodin Emodin Chrysophaic acid Protoanemonin

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no1 was recently shown to be the preferred method for extraction of ranunculin (362). Cycasin is readily hydrolyzed by dilute acid. Vicine and convicine are best extracted in aqueous trichloroacetic acid solution. Addition of acetone and centrifugation results in a supernatant containing vicine and concicine that can be selectively precipitated by adjustment of the pH (357). Extraction and purification of l-a,25-dihydroxycholecalciferolglycoside is difficult and identification still is based largely on bioassays and identification of the aglycone and carbohydrate moieties. A RIA is available for the aglycone (363). Carboxyatractyloside can be extracted in water from plant tissue previously extracted with hexane, CHCl?, and EtOH. The aqueous extract is flash evaporated and the residue extracted with THF. After removal of traces of THF, the insoluble glycoside is recovered from MeOH and recrystallized as the K salt, which then is hydrolyzed to release the aglycone (364). Carboxyatractyloside also may be extracted in acetone and recovered as the potassium salt (356). Vicine and convicine can be resolved by 2D-TLC on cellulose developed in EtOH, followed by MeOH: NHIOH:H 2 0 (14: 1 : 5) in one direction and then MeOH: 0.02 M phosphate buffer (pH 5.8j (7 : 3). The UV-absorbing spots can be eluted and quantified spectrophotometrically (365). Vicine and convicine react with Folin-Ciocalteu reagent, yielding a blue color similar to that produced by tyrosine or tryptophan (366). Extracts must be treated with ether, copper carbonate, and alumina to remove interfering compounds (367). The preferred methods of analysis of vicine and convicine are RP-HPLC on C-18 columns eluted with 0.05M H,PO, (368) or water (369) and NH2 columns eluted with H 2 0 :CH,CN (3 :7) (370). Ranunculi11 can bedetected on Silica TLC plates developed in either CHCl?:MeOH: H 2 0 (65 : 35 : 10, lower phase) or CH3CN:H,O (9 : 1j by visualization with anisaldehydesulfuric acid-acetic acid (362). Protoanemonin can be quantified by HPLC on C- 18 columns eluted with CH;CN: H 2 0 (2: 8) (358) or on normal-phase (Si-60) columns eluted with 11-hexane: acetone:CH2C12: CHCl, (70: 1O:lO:lO)using UV detection at 258 nm (359, 371). Both protoanemonin and ranunculin can be quantified on NH2-10 columns eluted with CH3CN:H 2 0 (9 : 1) (362). H'NMRcan be used to quantify small quantities of ranunculin (372j. Atractyloside and carboxyatractyloside can be resolved and quantified by HPLC on C-8 columns eluted with a 0.05% TFA :CH,CN gradient and detected by evaporative light scattering (373).

IV. PROTEINS, PEPTIDES, AND AMINO ACID ANALOGS

A.

Lectins(Hemagglutinins)

Lectins (Table 12) and proteins or glycoproteins of nonimmune origin are capable of specific recognition and reversible binding to carbohydrate components of complex carboTable 12 ToxicLectins Absin Jatrophin (curcin) Momordin Phoratoxin Ricin

Robin Viscumin Robitin Concanavalin A Avidin

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hydrates. Lectins can be precipitated from aqueous or dilute acetic acid extracts by (NH4):S04 and recovered by dialysis (374). Lectins may also be extracted in 1% NaCl (373, or with saline phosphate (376) or Tris-HC1 buffer (377). The lectin fraction then is subjected to chromatography on Sephadex GlOO and DEAE cellulose equilibrated in 0.02 M phosphate buffer (378). Affinity columns utilizing carbohydrates or erythrocyte membranes linked to an inert matrix also may be used to purify lectins (376). Since lectins are proteins, classical protein extraction and isolation techniques also are applicable. Lectins are assayed routinely by their ability to cause agglutination of blood (377, 379). RIA methods have been developed for Dolichos biflorus lectins (380). Small animals such as mice and rats also often are used to assess toxicity of lectin preparations (377). Lectins can be characterized by their electrophoretic patterns (374).

B. Enzymes Consumption of large amounts of plant material containing thiaminase, an enzyme that destroys vitamin B I , often leads to cerebrocortical necrosis in ruminants (381). Assays for thiaminase I involve measurement of the destruction of thiamine by determination of residual thiamine by reaction with diazonium salt (382), by the fluorometric thiochrome method, or by release of hydrogen ions. Edwin and Jackman used I T - and 'H-thiamine as a substrate for thiatninase activity; however, this method is not applied readily to crude extracts (383).

C. Amino Acid Analogs Amino acid analogs and nonprotein amino acids (Table 13) are responsible for a number of deleterious effects, including neurolathyrism and osteolathyrism, in mammalian systems (384,385). Toxic nonprotein amino acids usually can be extracted from plant material

Table 13 Toxic Nonprotein Amino Acidsand Amino AcidAnalogs Hypoglycin A (2-amino-4.5-1nethanohex-5-enoic acid) p-N-Oxalylamino-L-alanine (BOAA) = ~-N-oxalyl-~-a-~-,6-diaminopropionic acid (ODAP) = L-3-oxalylanlino a-propionic acid (OAP) 2-(3-An~ino-3-carboxypropyl)-isoxazolin-5-one P-Cyano-L-alanine (BCNA) y-Glutamyl-P-cyano-L-alanine P-N-(y-L-Glutamyl) amino propionitrile (BAPN) L-a-y-Diaminobutyric acid (DABA) Minlosine 3,4-Dihydroxyphenylalanine(DOPA) L-Azetidine-2-carboxylic acid L-a-Amino-P-methylene-cyclopropyl propionic acid Canavanine Indospicine Djenkolic acid a-Amino-P-oxalylaminopropionicacid S-Methylcysteine sulfoxide

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in acidified aqueous alcohol, with purification by ion-exchange chromatography, PC, TLC, electrophoresis, or ionophoresis. Hypoglycin can be recovered by chromatography of concentrated starch-free extracts on a strongly acidic cation-exchange resin. The resin is washed with 0.1 N HC1 followed by water and the hypoglycin is eluted with 0.5 M ammonia (386). Proteinase and amylase inhibitors, including bromelain, papain, and trypsin inhibitors (387), occur in a wide range of plants and can be found in seeds, fruits, and vegetative parts. As these inhibitors rarely are involved in poisoning cases, readers are referred to recent reviews, including Ref. 388. 1. PC, TLC, and GC and Spectrophotometric Methods Amino acids and analogs can be detected on PC and TLC with ninhydrin. Isoxazolinone derivatives may be separated by PC with BuOH:CH3COOH:H,0 (60: 15: 20) (389). Canavanine can be determined fluorometrically (390). Mimosine can be determined by an FeClj assay (391, 392). BOAA can be determined by electrophoresis followed by reaction with 1% ninhydrin (384, 393), or by automated amino acid analyses (394). GC on 3% OV-1 of TMS derivatives of hypoglycin is possible if few other amino acids are present (386).

2. HPLC Methods Recent developments in precolumn derivitization HPLC provide mechanisms for analysis of these amino acids (395). 0-Phthalaldehyde (OPT) (396), phenylisothiocyanate (Pic0 Tag)(PTC) (397), orthophthaldehyde (OPA) (3981, and 5-dimethylamino-1-naphthalene sulfonyl chloride (DANSYL) (386) derivatives can be prepared and resolved on RP C18 columns using UV or fluorometric detection. Mimosine can be quantified by RP ionpair HPLC (0.01 M Na octyl sulfate containing NaNO?) (399) or by chromatography on C-18 (0.2% H3P04)(400). Hypoglycin cochromatographs with leucine and isoleucine by PC and TLC, but DANSYL (401) and PTC (402) derivatives can be separated. Some of these compounds may be determined directly using electrochemical detection (403).

D. Amines Toxic phenylethylamines, including tyramine, N-methyl-p-phenethylamine, hordenine, pphenylethylamine, and galegine, occur in a number of plant species (404). Amines can be extracted in dilute HC1 (pH 2.0). After pigment removal by lead acetate precipitation and alkalinization (pH 8.0) of the extract, the amines can be extracted into EtOAc and recovered as their hydrochlorides (405). Galegine can be extracted into hot MeOH: H 2 0 (1 : 1) (406) and purified by partitioning into 1.2-BuOH. 1. TLC, PC, and Spectrophotometric Methods Phenylethylamines may be visualized on TLC silica plates (n-BuOH: CH3COOH:H 2 0 , 75 :15: 10; or, i-PrOH :CH3COOH:H10,75 : 15: 10) (405) with ninhydrin, fluorescarnine, DANSYL chloride (free-amino groups), Dragendorff reagent (methylated amino groups), o-dianisidine, or Folin reagent (hydroxylated amines) (404). Galegine can be chromatographed on silica gel in CHC13:MeOH:CH3COOH:H20(13:5:1:1) or EtOH:1N NH40H (4: 1) (406). Hordenine may be determined spectrophotometrically and alkaloid samples as its diazo derivative (520 nm) (407). Hordenine also can be dansylated directly on TLC plates (91, 92).

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Table 14 Selenium-Containing Amino Acids Selenocystine Methylselenocysteine Selenocystathionine y-L-Glutamyl-Se-methylseleno-L-cysteine

Selenohomocystine Methylselenomethionine y-Glutamylselenocystathione y-Glutamylselenocystathione I1

2. GC and HPLC Methods Amines may be separated by GC without derivitization. 9-Phenylethylamine can be determined by HPLC as DMEQ-COC1 (3,4-dihydro-6,7dimetl~oxy-4-methyl-3-oxoq~~inoxaline-2-carbonyl chloride) derivatives by chromatography on a RPTSK ODs- 120T column eluted with CH2CN:SO r n M ammonium acetate (33 :67) (408).

E. SeleniumAccumulators Plants accutnulating selenium usually store the selenium as Se-amino acids (Table 14) or as selenate. Selenium-containing compounds in plants and their biological effects have been reviewed (409). For extraction procedures, see Sec. 1V.C. Selenium content can be determined as Se by atomic absorption spectroscopy after suitable ashing of the plant sample or by fluorometry (410).

V.

ISOPRENOIDS

A.

SesquiterpeneLactones

Sesquiterpene lactones, including ptaquiloside, hymenoxon, helenalin, tetradymol, and parthenin. exhibit a range of biological properties, including cytotoxic effects, while others are potent allergens (41 1). Hymenoxon can be extracted into EtOAc (412). Ptaquiloside is unstable toacid (pH < 3.0), base (pH > 9.0). light, and heat (413. 414). It can be extracted in cold water and the extract freeze dried. The resulting powder is dissolved in MeOH and chromatographed on silica (CHCl?:MeOH, 10: 1) (413-415). Ptaquiloside also may be extracted in water at room temperature and purified using XAD-2 resin or polyamide columns (416). The ptaquiloside is recovered into water-saturated ;]-butanol and a crystalline material is recovered by concentration of the 1.1-butanol fraction. Ptaquiloside and related compounds can be analyzed by 2D-TLC on silica gel developed with C6Ho:acetone ( 3 :7) and C6H, :EtOAc (1 : 1j and scanned with a TLC densitometer (126). Sesquiterpenes can be visualized on TLC plates using vanillin ory-dimethylaminobenzaldehyde (417). Hymenoxon can be quantified by GLC (3% OV-17) using flavone as an internal standard (418). Hymenoxon and ptaquiloside can bequantified by RP-HPLC analysis using 45-60% MeOH in water (412, 414).

B. Diterpenes Esters of the diterpene alcohols phorbol, resiniferonol, and ingenol are responsible for a range of severe ophthalmic and dermatological effects and cause severe poisoning in live-

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Table 15 Toxic Diterpenes Phorbol esters (croton oil) 4-Deoxy-4-a-phorbol esters Ingenol esters Tigliane esters Simplexin Grayanotoxin I (andromedotoxin, acetylandromedol, or rhodotoxin) Grayanotoxin I1 (A1"(18"andrornedenol or anhydroandronledol) Grayanotoxin I11 (andromedol) Grayanotoxin IV, XIV

Daphnetoxin Mezerein Prostratin (diterpene acetatej Wedeloside 12P-Acetoxyhuratoxn

stock (Table 15) (419). Some of the most-toxic diterpenes belong to the group known as grayanotoxins (420). There is some confusion as to the exact identity of some of these compounds, but it seems likely that andromedotoxin and grayanotoxin I are the same compound. Diterpene alcohol esters can be extracted with MeOH or 95% EtOH (421) and purified using droplet countercurrent chromatography (419). Acetone also can be used to recover these compounds from plant extracts. Grayanotoxins can be extracted with EtOH (422) or MeOH:H,O (420). Grayanotoxins can be detected by TLC on silica gel (C6H6:MeOH: CH,COOH, 18 : 6 : 1; toluene: EtOAc :HCOOH, 5 :4 : 1; or, acetone: CHC13, 1: 1) (420) by spraying with y-anisaldehyde: H2S04:MeOH (1 : 1 : 18) (422) or antimony tricholoride in CHC13 (420). Diterpene alcohol esters can be chromatographed on silica with CHC13:EtOAC (2: 3 ) or EtOAC :MeOH (10: 1) and visualized with H,SO, (423).

C. Other TerpenesandHydrocarbons A number of aliphatic hydrocarbons are extremely toxic, including any with a cyclopropene moiety. Unsaturated higher alcohols such as cicutoxin are highly toxic. In addition to the compounds listed in Table 16, a large number of thiophene acetylenic compounds cause photodermatitis-type reactions in humans and animals. Other terpenes, including furanosesquiterpenes, norsesquiterpenes, and triterpene acids, have toxic properties. Many of the compounds in this group are light sensitive in a purified state. They can be extracted Table 16 Other Toxic Isoprenoids Tetranortriterpenes Meliatoxins A,. A:, B,. B? Furanosesquiterpenes Myomontanone (-)-Ngaione Triterpenes Lantadene A, B Trematol

Hydrocarbons Tanacetin Erucic acid Sterculic acid Malvalic acid Cyclopropenoid fatty acids Cicutoxin Cicutol Aethusanol A Oenanthotoxin Crepenynic acid

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with ether or hexane and purified by partitioning against 20% NaOH or extracted directly into MeOH (424). They can be crystallized from benzene :CCll :petroleum ether mixtures (425). Nagaione can be recovered from plant material by steam distillation (426), or ether, CHCl?, MeOH extraction (427). Meliatoxins can be extracted into EtOH, concentrated to the aqueous phase, and partitioned into Et,O (428). Meliatoxins can be separated by TLC on silica (CHC17:11-BuOH,96 :4) (428). Cicutoxin and related cotnpounds can be analyzed by GC-MS on3% SP2100-DB packed columns (424). Nagione can be chromatographed on QF-1, XFl150, and UC-W98 (429). Lantadene A can be chromatographed by RP-HPLC on C-18 eluted with CH3CN:H,O gradient (430).

VI. OXALATESyNITRATES,SULFIDES,AND ORGANOFLUORINE COMPOUNDS Oxalates exist in plants as either soluble acid salts (potassium or sodium oxalate) or as insoluble salts of calcium and magnesium. A number of disulfide compounds (e.g., dimethyl disulfide) accumulate to high levels in some plants, causing toxic effects. Several plants can accumulate inorganic fluorides, which are stored as fluoroacetate and related compounds that can be recovered from acetone :water extracts (43 1). Soluble oxalate concentrations are determined by extraction in distilled water. Total oxalates are determined after extraction in 0.1% HCl (432). Many plants can accumulate toxic levels of nitrate that can be extracted from plant tissues in water or buffer solutions (433). The enzyme assay procedure 590 from Sigma can be used to determine oxalate concentrations (434). Oxalate also can bedetermined by reaction with o-phenylenediamine to form 2,3-dihydroxyquinoxalene,which then can be determined by HPLC on a C-8 column eluted with water (435). A number of classic spectrophotometric methods are available for nitrate and nitrite analysis (436,437). Nitrate-specific electrodes also can be used to quantify nitrate levels (438, 439), but interference from other ions necessitates extensive sample preparation prior to analysis. Recent developments in ion columns for HPLC now permit rapid quantitative analysis of nitrate levels in plant samples (433,440). Disulfides present in plant extracts and oils can be quantified by GC (441). Alkyl disulfides in plant samples also canbe analyzed by RP-HPLC on C-8 and C-18 columns (442, 443). Fluoroacetate compounds can be chromatographed on cellulose in MeOH :NH40H: pyridine :H 2 0 (95 : 3 : 1 : 1) and visualized with Nile blue (444). Fluoroacetate compounds can be determined as 2,3,4,5,6-pentafluorobenzylbromide derivatives by GC on columns coated with 7% OV-210 and 3.5% OV-101 or 5% SE-30 (431, 444). Fluoroacetate compounds can be determined by HPLC on C- 18columns (445) or NH. columns eluted with MeOH :0.05 M KH2P04(pH 4.0) (431,444). Fluoroacetate and other fluoride-containing toxins can be assayed using ion-selective electrode assays (446).

VII.

MYCOTOXINS

Mycotoxins can be separated by TLC (447,448) and detected using Besthorns hydrazone reagent (449) or A1C17 (448). As mycotoxins occur in a wide range of sample matricies, extraction and sample cleanup is often problematic and does not always remove interfering compounds. Recent advances in methods for aflatoxins and trichothecenes were re-

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viewed by Gilbert (450). Recent advances in immunoaffinity chromatography (45 1j have solved some but not all of these problems. Many combination methods have been developed to allow simultaneous determination of more than one class of mycotoxins (447, 452-455), which is particularly important when samples may contain more that one class of mycotoxin.

A.

Aflatoxins

Aflatoxins can be extracted with CH,CN containing KC1 (447). The extracts are defatted and the aflatoxins partitioned into CHCl, from aqueous CHjCN. Aflatoxins can also be extracted by supercritical fluid extraction (SFE) (456,457). Anumber of different cleanup procedures can be employed (455) prior to TLC or HPLC analysis. 1. TLC and ImmunologicalMethods A number of commercial screening kits are currently available for determination of aflatoxins inplant matrices (450) including both ELISA-type and immunoaffinity column systems; however, many of these kits are incompatible with the most effective extraction systems for the isolation of aflatoxins. This is not a problem for liquid samples such as milk (458, 459). TLC on silica can be used to detect a range of aflatoxins using UV light to detect their fluorescence (447). Typical solvent systems include hexane :THF :EtOH (70 :20 : 10) (460), ether: MeOH :H 2 0(95 :4 : 1) (460j or combinations of toluene :EtOAc : formic acid (455). 2. HPLC and MS Methods Reverse-phase HPLC analysis is widely used to detect and quantify aflatoxins using fluorescence detection; however, the fluorescence of some aflatoxins is solvent dependent (450). This can be addressed by postcolumn iodination (461) or bromination (462) in an electrochemical cell, or by precolumn derivatization with TFA (457). Typical HPLC solvent systems include MeOH:H,O (40: 60) (463); CH,CN:H,O (459), and H20:CHjCN: isopropyl alcohol (8 : 1 : 1j (457). Recently the addition of cyclodextrins to the HPLC mobile phase significantly increased the fluorescence response with a corresponding increase in sensitivity (463). Aflatoxins can also be detected in crude extracts using MS-MS (464).

B. AcidicMycotoxins(Citrinin,OchratoxinA) Acidic mycotoxins can be extracted with CHC13:0.1 M H,PO, (9: 1) (465). Ochratoxin A can also be extracted with 3% acetic acid in CHjCN (466) or 5% NaHC03:MeOH (6: 13) and subsequently partitioned into CHCl; from acidic aqueous MeOH (467). Acidic CHCl, or combinations of acidic CHC1, and MeOH are also commonly used (468). Sample cleanup can be achieved by passage over a strong anionic exchange SPE (466), by adsorption on to and subsequent elution from a bicarbonate-celite column (467), by liquid-liquid defatting (469), or by immunoaffinity chromatography (451, 469-472), depending on the sample matrix. Processing of foods and handling of samples may cause decomposition and/or rearrangement of these acidic mycotoxins such as the recently observed conversion of citrinin to citrinin H1, a compound that is 10 times more cytotoxic than the parent compound (473).

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1. TLC and ImmunologicalMethods Enzyme immunoassay methods for detection of citrinin (474, 475), and ochratoxin A (476-478) have been developed although subsequent confirmation byan independent method is desirable. 2. GC, GC-MS, andHPLCMethods Although several HPLC methods using UV absorbance detection have been reported for the analysis of citrinin and ochratoxin A, the preferred method is to utilize the natural fluorescence of these molecules (479, 480) with c,hromatography achieved on C-18 columns eluted with MeOH: H 2 0 (70: 30) containing 0.001 M tetrabutylammonium hydroxide as an ion-pairing agent, CH3CN:H20:0.2M K2HP04containing 3 mM cetyltrimethylammonium bromide (CTA) (50 :47 :3) (48 l), or elution with MeOH: isopropanol (9: 1): H3P04(pH 2.1) gradient (477). Ochratoxin A can be quantified by RP-HPLC eluted with MeOH:H20:acetic acid (30:70: 1) (482), or CH3CN:5% acetic acid ( 3 5 : 65) (467). Postcolumn addition of acid can improve the sensitivity (480) or, alternatively, these compounds can be detected by determination of the time-resolved luminescence of their lanthanide chelates (479). Confirmation of the presence of ochratoxin A at sub-ppb levels can be obtained by GC-MS analysis of the 0-methyl ester after HPLC analysis (483). Citrinin has also been successfully chromatographed on normal-phase (rz-hexane:CHC1,; 6:4) (484), and reverse-phase columns (485, 486). The chromatographic methods and sample preparation methods for ochratoxin A were recently reviewed by Valenta (468).

C.

CyclopiazonicAcid

Cyclopiazonic acid is a toxic indole tetarnic acid produced by many species of Aspergillus and Penicillium and can be extracted from plant (487) or animal tissues (488) with CHC13: MeOH. Cyclopiazonic acid can be detected on TLC (487) or HPTLC plates (489) by spraying with Ehrlich's reagent although this is not particularly sensitive or selective (490, 491). Cyclopiazonic acid may also be quantified by ligand exchange liquid chrornatography on reversed-phase ODS columns (488). A more sensitive normal-phase HPLC method (EtOAC :2-propanol : 25% aqueous ammonia; 55 :20 :5 ) (492) is available; however, recent developments in ELISA suggest that this will be the preferred method (490) and both polyclonal and monoclonal antibodies have been prepared (493, 494).

D. Fumonisins Fumonisins can be isolated from EtOAc-extracted plant material by extraction with MeOH:H,O (3: 1) (493, or by supercritical fluid extraction (496). Fumonisins can also be extracted from plant samples with CH3CN: H,O (1 : 1) (497), diluted with H 2 0 and selectively eluted from C- 18 SPE columns with mixtures of CH3CN:H 2 0 (498). Fumonisin B can be extracted from blood or urine by absorption onto Bond-Elut SAX SPE columns and eluted with 5% acetic acid in MeOH (499). 1. TLC and ImmunologicalMethods Fumonisins can be estimated by silica gel TLC (CHC13:MeOH :acetic acid (6 :3 : 1)) with visualization using y-anisaldehyde reagent (495), or by chromatography on RP-TLC plates

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developed in MeOH: 4% aqueous KC1 (3 :2) with visualization by spraying successively with 0.1 M Na borate buffer, fluorescamine, and 0.01 M boric acid and examination under long-wave UV light (497). Immunoassays are now the preferred method of detection for screening programs. ELISA assays using monoclonal antibodies have been developed (500, 501) and kits are commercially available (502, 503). 2. GC-MS, CapillaryElectrophoretic,and HPLC Methods Although GC-MS methods were initially used to detect these compounds, hydrolysis and derivativation was required and these methods have largely been replaced by HPLC and, more recently, LC-MS methods (504). Fumonisins are usually quantified as OPA derivatives by isocratic C-18 RP-HPLC (0.05 M K2POJ:CH,CN; 61 : 39) with fluorimetric detection (498). The major limitation of this method is the relative instability (4 min) of the OPA derivative. Fumonisins can also be determined quantitatively by RP-HPLC of their maleyl (505), or (9-fluorenylmethyl)chloroforn~ate(FMOC) derivatives (506). Recently 6-aminoquinolyl N-hydroxysuccinimidylcarbamate (AccQ-Fluor) derivatization has been demonstrated to produce stable fluorescent fumonisin derivatives suitable for HPLC analysis (507). Chromatographic methods for determination of fumonisins were recently reviewed by Shephard (504). Underivatized fumonisins can also be detected by ELSD after RP-HPLC (CH3CN:0.025% TFA) (508) or by electrospray LC-MS (508-5 10). Fast-atom bombardment MS-MS can also be used to detect fumonisins (509). Futnonisins can also be quantitated as their fluoroescein isothiocyanate derivatives by capillary electrophoresis ( 5 11).

E. Furanoterpenes 4-Ipomeanol, 1,4-ipomeadiol, and ipomeanarone can be extracted with 3% NaCl in MeOH and quantified by GC on 10% UC-W98 on 177-149 pm Gas-Chrom Q (512). An approximate concentration can also be determined spectrophotometrically by reaction with Ehrlich’s reagent ( 5 13) [ 10% p-dimethylamino benzaldehyde in EtOH and 40% ( v h ) aqueous H,SO,]; however, 1-ipomeanol forms a highly unstable chromophore with Ehrlich’s reagent and is not quantifiable using this technique (512). Furanoterpenes can also be separated by TLC on silica gel plates developed in MeOH: ChHh(1 :9) (512) or EtOAc: 12hexane ( 3 :8) (514).

F.

Muscimolandlbotenicacid

Muscimol and ibotenic acid can be chromatographed on RP-HPLC columns eluted with aqueous 0.005 M octylammonium o-phosphate (pH 6.4) with UV detection at 230 nm (515).

G. Orellanine Orellanine can be detected in mushroom extracts by TLC on cellulose plates eluted with n-butanol: acetic acid: H - 0 (4: 1 :5) with visualization using UV light (366 nm) (516). Orellanine can also be detected on agarose electrophoresis gels by irration with UV light (312 nm) (516). Quantitation is possible by RP-HPLC (517).

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H. Patulin Patulin appears to be mosteffectively extracted by EtOAc with subsequent sample cleanup by extraction with sodium carbonate (518), silica column chromatography (519), or SPE on C-18 columns (520). To detect very low levels a diphasic dialysis procedure has been developed (521). Patulin can be chromatographed on silica gel plates developed in toluene :EtOAc :CHCl?:90% CH3COOH (70 :50 :50 :20) with visualization using 0.5% 3methyl-2-benzothiazolinone-hydrazonehydrochloride (448, 52 1). 1. HPLCMethods Unlike most of the mycotoxins, patulin has significant UV absorption and can be determined by RP-HPLC on C- 18 columns eluted with a gradient of 2% aqueous acetic acid against 30% MeOH in 2% aqueous acetic acid (520). Alternative chromatography systems include 1% THF (519) or 1% CH3CN in water (522). Extraction with EtOAc, treatment with sodium carbonate and chromatography on C-18 columns eluted with HzO, H20: THF, or H 2 0 :CH3CN is now the recognized method (5 18); however, in some sample matrices where high levels of tannins and other phenolics are present, additional sample preparation and gradient Chromatography may be required (523).

1.

Trichothecenes(Vomitoxin)

Trichothecenes, including deoxynivalenol (DON) and nivalenol (NIV), can be extracted polar solvents ranging in polarity from MeOH: H 2 0(1 :9) to CH3CN:H 2 0(84: 15)(524). Sample cleanup can be achieved using charcoal: celite columns that are commercially available (450, 524) or C-18-alumina columns (525). 1. TLC,Fluorometric,Immunological, and BioassayMethods TLC methods have been widely used for detection and quantification of trichothecene mycotoxins (524,526). Typical TLC solvent systems include CHC13:acetone :isopropanol (8 : 1 : 1) using A1C13-impregnated silica gel plates and fluorescence detection (524), or EtOAc: ethyl ether (1 : 1) and a postdevelopment A1Cl3 spray (527). RIAs (for total type A and total DON-related trichothecenes) and ELISA assays (for T-2 toxin and deoxynivalenol) have been developed (528). Recently a competitive direct (CD) ELISA for deoxynivalenol was developed and is now commercially available (529, 530). Trichothecenes may also be detected by a yeast bioassay (53 1). Recently a fluorometric method for deoxynivalenol was developed in which DON wasreacted with zirconyl nitrate and ethylenediamine in methanol to give a fluorescent derivative that could be measured in a fluorometer (532).

2. GC,GC-MS,andHPLCMethods Trichothecenes can be detected and quantified by GC and GC-MS of their TMS or heptafluorbutyrl (HFB) derivatives (533, 534). Alternative derivatization reagents include trimethylsilylimidazole-trimethylchlorosilane(TMSI-TMCS) (525). Electron capture detection is employed for theHFB (535) and TMSI-TMCS derivatives (525). RP-HPLC (MeOH: H,O; 14 : 86) analysis of the underivatized trichothecenes with UV detection can be used for samples with moderate levels (536, 537); however, some form of postcolumn derivatization is often employed to increase sensitivity (450). Typical HPLC solvents include MeOH:H,O (538), and CH,CN:H,O (535) gradients.

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J. Zearaleone Zearaleone can be coextracted with most of the mycotoxins in aqueous methanol solvents and purified by SPE or pH controlled liquid-liquid partitioning (453). Detection and quantitation can be achieved by TLC (539). Spraying the plate with AlC13 and heating can enhance the fluorescence of zearaleone (455). Several RP-HPLC methods (MeOH:H20; 58 :42)have been described (540) using fluorescence detection. Recently the inclusion of P-cyclodextrin in the HPLC mobile phase allowed simultaneous detection of trichothecenes in samples also containing zearaleone (453).

REFERENCES 1. Cheeke, P.R., and Shull, L.R. (1985). Natwal To.yicants in Feeds and Poisonous Plmts. AVI Publishing Company, Westport, Conn. 2. Scheline, R.R. (1991). Handbook of Munzmaliun Metabolism of Plalzt Conlpounds. CRC Press, Boca Raton, Fla. 3. Kim, I.-C., Balandrin, M.F., and Kinghorn, A.D. (1982). Reinvestigation of the alkaloids of Lupzrs sericeus Pursh. Identification of a new natural product, 10-17-dioxo-~-isosparteine. J. Agric. Food Chem., 30:796-798. 4. Johnson, A.E., Molyneux, R.J., and Merrill, G.B. (1985). Chemistry of toxic range plants. Variation in pyrrolizidine alkaloid content of Senecio. Amsirzckia, and Crotulariu species. J. Agric. Food Cltenl., 33~50-55. 5. Ray, A.C., Williams, H.J., and Reagor, J.C. (1987). Pyrrolizidine alkaloids from Senecio lortgilobtrs and Sececio glabellrls. Phptochemist~,26243 1-2433. 6. Wagner, H., Bladt, S., and Zgainski, E. (1984). Plant Drug Analysis. Springer-Verlag, Berlin. 7. Roeder, E., Breitmaier, E., Birecka, H., Frohlich, M., and Badzies-Cronlbach. A. (1991). Pyrrolizidine alkaloids of Heliotropium spathulatunl. Phytocltemistp. 30: 1703-1706. 8. Smith. R.A. (1989). Comments on diagnosis of intoxication due to Ta.xus. Vet. Hunr. Toxicol., 31:177. 9. Smith, R.A., and Lewis, D. (1991). Death camas poisoning in cattle. Vet. H u m Toxicol., 33: 615-616. 10. Baerheim Svendsen, A., and Verpoorte, R. (1983). Chromutographv of alkaloids, Part A. Thin-Layer Chrornatogruphy. Jour11a1of Clu-onzutography Library. Vol. V23A. Elsevier, Amsterdam. 11. Dagnino, D.. and Verpoorte, R. (1994). Gas chromatography in the analysis of alkaloids. In Moden? Methods of Plant Amlysis (Linskens, H.F. and Jackson, J.F. eds.). Springer-Verlag, Berlin, pp. 115-135. 12. Edgar, J.A., Jago, M.V., Outteridge, A., Peterson, J.E., and Smith, L.W. (1976). Hepato- and pneumotoxicity of pyrrolizidine alkaloids and derivatives in relation to molecular structure. Chenr. Biol. Interact., 12299-324. 13. Mattocks, A.R. (1986). Chemistry am1 Toxicology of Pprrolizidine Alkuloids. Academic Press, New York. 14. Hartmann, T., and Zimmer, M. (1986). Organ-specific distribution and accumulation of pyrrolizidine alkaloids during the life history of two annual Senecio species. J. Plmt Physiol., 122167-80. 15. Bull. L.B.. Culvenor, C.C.J., and Dick, A.T. (1968). The Pyrroli:idine Alkaloids.Their Chemistt?, Pathogenicity and Other Biological Properties. American Elsevier, New York. 16. Hartmann. T., and Toppel. G. (1987). Senecionine N-oxide, the primary product of pyrrolizidine alkaloid biosynthesis in root cultures of Sertecio vulgaris. Phytoclzemistry, 36: 1639-1643. 17. Schaeffer, S.T.,Zalkow, L.H., and Teja. A.S. (1989). Extraction of monocrotaline from 0 - 0 -

Analytical Methodology for Plant Toxicants

18.

19. 20.

21. 23. 23. 24.

25. 26.

27.

28.

29.

30.

31. 32. 33. 34. 35. 36.

37.

387

tnluris syectclblis using supercritical carbon dioxide and carbon dioxide-ethanol mixtures. Biotech. Bioeng., 34:1357-1365. Bicchi, C., Rubiolo, P., Frattini, C., Sandra, P., and David, F. (1991). Off-line supercritical fluid extraction and capillary gas chromatography of pyrrolizidine alkaloids in Serzecio species. J. Nntw-nl Prod., 54:941-945. Couet, C.E., Hopley, J., and Hanley, A.B. (1996). Metabolic activation of pyrrolizidine alkaloids by human, rat and avocado microsomes. Toxicon, 9:1058-1161. Mattocks, A.R., and Jukes, R. (1990). Recovery of the pyrrolic nucleus of pyrrolizidine alkaloid metabolites from sulphur conjugates in tissues and body fluids. Chew. Biol. Intercrct., 75:225-239. Mattocks, A.R. (1968). Spectrophotometric determination of pyrrolizidine alkaloids-some improvements. A d . Chenz., 30: 1749-1750. Mattocks, A.R. (1967). Spectrophotometric determination of unsaturated pyrrolizidine alkaloids. A n d . Cheuz., 39:443-447. Mattocks, A.R., and White, I.N.H. (1970). Estimation of metabolites of pyrrolizidine alkaloids in animal tissues. Arznl. Chenz., 38:529-535. Mattocks, A.R., and Bird, I. (1983). Pyrrolic and N-oxide metabolites formed from pyrrolizidine alkaloids by hepatic microsomes in vitro: relevance to in vivo hepatotoxicity. Chenz. Biol. Interact.. 43:209-222. Mattocks, A.R., and Jukes. R. (1987). Improved field tests for pyrrolizidine alkaloids. J. N n t 1 4 ~Prod., ~l 50: 161-166. Bober. M.A., Kurth, M.J., Milco, L.A., Roseman, D.M., Miller, R.B.. and Segal, H.J. (1991). A pyrrolizidine alkaloid enzyme-linked immunososbent assay detection strategy. In Inmunoassays for Trace Chemical Analysis (M. Vandeslaam, L.H. Stanker, B.E. Watkins. and D.W. Roberts, eds.). American Chemical Society, Washington, DC, pp. 176-183. Roseman, D.M., Wu, X., Milco, L.A., Bober, M., Miller, R.B., and Kurth, M.J. (1992). Development of a class-specific competitive enzyme-linked immunosorbent assay for the detection of pyrrolizidine alkaloids in vitro. J. Agric. Food Chew., 40:1008-1014. Roseman, D.M., Wu, X., and Kurth, M.J. (1996). Enzyme-linked inmunosorbent assay detection of pyrrolizidine alkaloids: Immunogens based on quaternary pyrrolizidinium salts. Bioconjugate Chern., 7: 187-195. Ziindorf, I., Weidenfeld, H., Roder, E., and Dingermann. T. (1998). Generation and characterization of monoclonal antibodies against the pyrrolizidine alkaloid retrorsine. Plantcr Med.. 64:259-263. Molyneux, R.J., Johnson, A.E., Roitman, J.N., and Benson, M.E. (1979). Chemistry of toxic range plants. Determination of pyrrolizidine alkaloid content and composition in Senecio species by nuclear magnetic resonance spectroscopy. J. Agric. Food Clzen?.,27:494-499. Molyneux, R.J. (1988). The chemistry of pyrrolizidine and indolizidine alkaloids and their role as constituents of toxic range plants. Rev. Lntinoanzer. Quint., 19:135-141. Sharma, K.K., Khajuria, G.S.. and Aral, C.K. (1965). Thin-layer chromatography of pyrrolizidine alkaloids. J. Chrorit., 19:433-434. Mattocks, A.R. (1967). Detection of pyrrolizidine alkaloids on thin-layer chromatograms. J. Chrom., 27:505-508. Mattocks, A.R.. and Nwude. N. (1988). Pyrrolizidine alkaloids from Crotcrlariu Iachnosemn and C. nnragutensis. Phytochemist?, 27:3289-3291. Huizing, H.J., and Malinge. T.M. (1981). Ion-pair adsorption chromatography of pyrrolizidine alkaloids. J. Chrom., 205:218-222. Hartmann, T., Ehmnke, A., Eilert, U.. von Borstel. K.. and Theuring, C. (1989). Sites of synthesis, translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris. Plcmta. 177:98-107. Molyneux, R.J., and Roitman, J.N. (1980). Specific detection of pyrrolizidine alkaloids on thin-layer chromatograms. J. Clzronz., 195:412-415.

388

Muir

38. Huizing, H.J., De Boer, F.. and Malingre, T.M. (1980). Chloranil, a sensitive detection reagent for pyrrolizidine alkaloids on thin layer chromatograms. J. Chrom., 195:407-411. 39. Chalmers, A.H., Culvenor, C.C.J., and Smith, L.W. (1965). Characterisation of pyrrolizidine alkaloids by gas, thin-layer and paper chromatography. J. Chrom., 20:270-277. 40. Witte, L.. Ernst, L., Adam, H., and Hartmann, T. (1992). Chemotypes of two pyn-olizidine alkaloid-containing Senecio species. Plzytoclzemistry, 3 1:559-565. 41. Wink, M.. Witte, L., Schiebel, H.-M., and Hartmann, T. (1980). Alkaloid pattern of cell suspension cultures and differentiated plants of L~lpinuspolFphyllus. Plrrzta Med., 38238245. 42. Luthy, J., Zweifel, U., Karlhuber, B., and Schlatter, C. (1981). Pyrrolizidine alkaloids of Senecio alpirzus L. and their detection in feedingstuffs. J. Agric. Food Clzem., 29:302-305. 43. Culvenor, C.C.J., Edgar, J.A., and Smith, L.W. (1981). Pyrrolizidine alkaloids in honey from Echiwn plantngirteum L. J. Agric. Food Chenr., 29:958-960. 43. Winter, C.K., Segall. H.J.. and Jones. A.D. (1988). Determination of pyrrolizidine alkaloid metabolites from mouse liver microsomes using tandem mass spectrometry and gas chromatography/mass spectrometry. Bionzed. Enlliron. Mass Spectrorn., 15:265-273. 45. Edgar, J.A. (1985). Gas chromatography of pyrrolizidine alkaloids. In Plant Toxicology (A.A. Seawright, M.P. Hegarty, L.F. James, and R.F. Keeler, eds.). Dominion Press-Hedges & Bell, Melbourne, pp. 227-234. 46. Deinzer, M.L., Arbogast, B.L., Buhler, D.R., and Cheeke, P.R. (1982). Gas chromatograhic determination of pyrrolizidine alkaloids in goat’s milk. A n d . Chem., 54: 1811-18 14. 47. McCoy, J.W., Roby, M.R.. and Sternlitz, F.R. (1983). Analysis of plant alkaloid mixtures by ammonia chemical ionization mass spectrometry. J. Nrrturcrl Prod., 46:894-900. 48. Conner. W.E., Roach. B., Benedict, E., Meinwald, J., and Eisner, T. (1990). Courtship pheromone production and body size as correlates of larval diet in males of the arctiid moth Utetheisa onzatri-r. J. Chem. Ecol.. 16:543-552. 49. Segall, H.J. (1979). Reverse phase isolation of pyrrolizidine alkaloids. J. Liquid Chrom., 2: 429-436. 50. Segall, H.J. (1979). Preparative isolation of pyrrolizidine alkaloids derived from Sertecio vulgaris. J. Liquid Chronlr. 2: 1319- 1323. 51. Hartmann, T.. Sander, H.. Adolph, R., and Toppel, G. (1988). Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio ~wlgnris. Plants, 175182-90. 52. Wagner, H., Neidhardt, U., and Tittel. G. (1981). DC- und HPLC-analyse der pyrrolizidinN-oxid-alkaloide von Synphyti r d i x . Planta Men., 412:232-239. 53. Ramsdell, H.S., and Buhler, D.R. (1981 1. High-performance liquid chromatographic analysis of pylrolizidine (Senecio) alkaloids using a reversed-phase styrene-divinylbenzene resin column. J. Chrom.. 210:154-158. 54. Kedzierski. B., and Buhler, D.R. ( 1986). Method for determination of pyrrolizidine alkaloids and their metabolites by high-performance liquid chromatography. Anal. Biochem., 1525965. 55. Moll, F. (1963). Dunnschichtchromatographie von Schierlingsalkaloiden und anderen piperidinbasen. Arch. Plzarm (Weinheinl),296:205-207. 56. Stahl, E., and Schmitt. W. (1975). TAS-Verfahren und therrnofraktographie zur schnellanalyse von alkaloiddrogen. Arch. Plzarnt (Weinlzeinz), 308:570-578. 57. Fairbairn, J.W., and Ali, A.A.E.R. (1968). The alkaloids of hemlock (Coninnt mncdnturn L.). 111. The presence of bound forms in the plant. Plzytochemist~~, 7:1593-1597. 58. Cromwell, B.T. (1956). The separation, micro-estimation and distribution of the alkaloids of hemlock (Coniztnz ~nncdnturnL.). Bioclzern. J.. 64:259-266. 59. Fairbairn, J.W., Shellard. E.J., and Talalaj, S. (1963). Gas liquid chromatographic estimation of the alkaloids of Coniurn rncrczdatu?nL. Plantn Med., 11:92-97. 60. Moll, F. (1962). Gaschromatographische trennung von piperidinbasen. Nntwwiss., 49:450.

Analytical Methodology for Plant Toxicants

389

61. Lloyd, H.A., Fales, H.M., Highet, P.F., VandenHeuvel, W.J.A., and Wildman, W.C. (1960). Separation of alkaloids by gas chromatography. J. Am. Clzem. SOC.,82:3791. 62. Verpoorte, R., and Baerheim-Svendsen, A. (1984). Cl~ron~atography of Alkaloids Part B: Gas-Liquid Chromatography arid High-Petformance Liquid Chromatography, Jo~trnalof Chronlatograplzy Library. Vol. 23B. Elsevier, Amsterdam. 63. Saitoh, F., Noma, M., and Kawashima, N. (1985). The alkaloid contents of sixty Nicotiana species. Phytochemistry, 24:477-480. 64. Saunders, J.S.. and Blume, D.E. (1981). Quantitation of major tobacco alkaloids by highperformance liquid chromatography. J. Chront.. 205: 147- 154. 65. Keeler, R.F., and Crowe, M.W. (1985). Anabasine, a teratogen from the Nicotiana genus. In Plant ToxicoZogy (A.A. Seawright, M.P. Hegarty, L.F. James, and R.F. Keeler, eds.). Dominion Press-Hedges & Bell, Melbourne, pp. 324-333. 66. Tso. T.C.. and Jeffrey, R.N. (1953). Paper chromatography of alkaloids and their transformation products in Maryland tobacco. Arch. Biochem. Biophys., 43:269-285. 67. Fejer-Kossey, 0. (1967). The separation of ten tobacco alkaloids by thin layer chromatography. J. Cltrom., 31:592-593. 68. Leete, E., Endo, T., and Yamada, Y. (1990). Biosynthesis of nicotine and scopolamine in a root culture of Duboisia leichltardtii. Phytoclzemist~y,29: 1847- 1851. 69. Jacob, P., Wilson, M., and Benowitz. N.L. (1981). Improved gas chromatographic method for the determination of nicotine and cotinine in biological fluids. J. Chrom., 222:61-70. 70. Manceau, F., Fliniaux, M.-A.. and Jacquin-Dubreuil, A. (1989). Ability of a Nicotiana pltrntbaginifdia cell suspension to demethylate nicotine into nornicotine. Phytocherrtistry. 28: 267 1-2674. 71. Thompson, J.A.. Ho. M.. and Petersen, D.R. (1982). Analysis of nicotine and cotinine in tissues by capillary gas chromatography and gas chromatography-mass spectrometry. J. Chront.. 231:53-63. 72. Davis, R.A. (1986). The determination of nicotine and cotinine in plasma. J. Chronz. Sci., 24~134-141. 73. Piade, J.J., and Hoffmann, D. (1980). Chemical studies on tobacco smoke. LXVII. Quantitative determination of alkaloids in tobacco by liquid chromatography. J. Liquid Cltrom.. 3: 1505-1515. 74. Griffin, W.J.. Brand, H.P., and Dane, J.G. (1975). Analysis of Duboisia myoporoides R. Br. and Dzrboisirt leichltardtii F. Muell. J. Pharm. Sci., 64:1821-1825. 75. Horstmann, M. (1 985). Simple high-performance liquid chromatographic method for rapid determination of nicotine and cotinine in urine. J. Chrom., 344:391-396. 76. Cundy, K.C.. and Crooks, P.A. (1984). High-performance liquid chromatographic method for the determination of N-methylated metabolites of nicotine. J. Chrorrz., 306:291-301. 77. Mousa, S., van Loon. G.R., Houdi, A.A., and Crooks, P.A. (1985). High-performance liquid chromatography with electrochemical detection for the determination of nicotine and N-methylnicotinium ion. J. Chront., 347:405-410. 78. Sudan, B.J., Brouillard, C., Strehler. C., Strub, H., Sterboul, J., and SainteLaudy. J. (1984). Determination of nicotine in allergenic extracts of tobacco leaf by high-performance liquid chromatography. J. Chrorn., 288:415-422. 79. Seeman, J.I., Secor, H.V., Armstrong, D.W., Ward, K.D., and Ward, T.J. (1989). Separation of homologous and isomeric alkaloids related to nicotine on a P-cyclodextrin-bonded phase. J. Chronl., 483:169-177. 80. Lin, L.A. (1 993). Detection of alkaloids in foods with a multi-detector high-performance liquid chromatographic system. J. Chrorrz., 632:69-78. 81. Porter, J.K. (1995). Analysis of endophyte toxins: Fescue and other grasses toxic to livestock. J. Arlimal Sci., 73:871-880. 82. Majak, W., and Bose, R.J. (1977). Further characterization and quantitative determination of 5-~nethoxy-N-methyltryptamine in Phalnris arzrndinacea. Pltytocher~istq~. 16:749-752.

390

Muir

83. Bisset, N.G., and Choudhury, A.K. (1974). Alkaloids and iridoids from Stpchnos rzz4.x-vomica fruits. Phytoclzemistry, 13265-269. 84. TePaske, M.R., and Powell, R.G. (1993). Analysis of selected endophyte-infected grasses for the presence of loline-type and ergot-type alkaloids. J. Agric. Food Cltem.. 41:22992303. 85. Hayakawa, J., Noda, N.. Yamada, S., and Uno, K. (1984). Studies on physical and chemical quality evaluation of crude drugs preparation. 11. Analysis of pharmaceutical preparations including Nus vomica extracts by high performance liquid chromatography. Yakugaku Zasslzi, 10457-61. 86. Muir, A.D., Westcott, N.D., and Hinks. C.F. (1992). New quantitative high-performance liquid chromatography method for analysis of granline in cereal leaf tissue. J. Agric. Food Chent.. 40:1836-1838. 87. Cox, S.K.,van Manen, R.T., and Oliver, J.W. (1992). Determination of ergonovine in bovine plasma samples. J. Agric. Food Cltem., 40:2164-2166. 88. Phillipson, J.D., and Bisset, N.G. (1970). Correlations between Rf values on thin-layer chromatograms and chemical structure of some tertiary Strychnos alkaloids. J. Clrrorn., 48:493508. 89. Bisset, N.G., and Phillipson, J.D. (1976). TheAsian species ofStqchnos. Part IV. The Alkaloids. Lloydia. 39263-325. 90. Baser, K.H.C.. and Bisset, N.G. (1982). Alkaloids of Sri Lankan Strychnos nus-vomica. Phytochemistry, 21:1423-1429. 91. Majak, W., McDiarmid, R.E., Powell, T.W., Van Ryswyk, A.L., Stout, D.G., Williams, R.J.. and Tucker, R.E. (1979). Relationship between alkaloids in reed canarygrass (Phnlaris nrurzdirzacea), soil moisture and nitrogen fertility. PZarztCeZl Erzvirorz., 2:335-340. 92. Majak, W.. McDiarmid, R.E., and Bose, R.J. (1978). TLC luminescence of gramine and related indole alkaloids in Plrctlaris nrunclinacea. Phytoclzemistiy, 17:301-303. 93. Shelby, R.A., and Kelly, V.C. (1991). Detection of ergot alkaloids in tall fescue by competitive immunoassay with a monoclonal antibody. Food Agric. ZrmzzmoZ., 3: 169-177. 94. Bisset, N.G., and Fouchk, P. (1968). Gas-Liquid chromatography of tertiary Stqdrnos alkaloids. J. Chront., 37:172-180. 95. Duynisveld. G.W.. Slominski, B.A., Wittenberg, K.M., and Campbell. L.D. (1990). Alkaloid content of reed canarygrass PlzaZaris arurtdirraceaL. as determined by gas-liquid chromatography. Can. J. Plant Sci., 70: 1097-1 104. 96. De, B., and Bisset, N.G. (1991). Separation of Stnchnos nux-vonrica alkaloids by high-performance liquid chromatography. J. Clzrom., 587:318-320. 97. Dennis, R. (1984). The rapid estimation of strychnine in tincture of nux llomica BP by highperformance liquid chromatography. J. Phanrz. Phnrmacol., 36:332-333. 98. Twitchett, P.J. (1975). Analysis of illicit diamorphine preparations by high-pressure liquid chromatography. J. Clzronz., 104:205-210. 99. Verpoorte, R., and Baerheim Svendsen, A. (1975). High-speed liquid chromatographic separation of some Stryclrnos alkaloids. J. Chrortz., 109:441-442. 100. Ayoub, M.T., and Rashen, L.J. (1991). Isoharmine, a P-carboline alkaloid from Pegartzm hamala seed. Plzytochernistly, 30: 1046-1047. 101. Frach, K., and Blaschke, G. (1998). Separation of ergot alkaloids and their epimers and determination in sclerotia by capillary electrophoresis. J. Clzrom., 808:247-252. 102. Kinghorn, A.D.. Selim, M.A., and Smolenski, S.S. (1980). Alkaloid distribution in some new7 world Lupinus species. Phytochemistry, 19:1705-1710. 103. Wink, M., and Hartmann, T. (1982). Diurnal fluctuation of quinolizidine alkaloid accumulation in legume plants and photomixotropic cell suspension cultures. 2. Naturforsch., 37c: 369-375. 104. Wink, M. (1983). Wounding-induced increase of quinolizidine alkaloid accumulation in lupin leaves. Z. Naturforsch.. 38c:905-909.

Analytical Methodology for Plant Toxicants

391

105. Wink, M.. Schiebel, H.M., Witte, L., and Hartmann. T. (1982). Quinolizidine alkaloids from plants and their cell suspension cultures. Plantu Med., 44:15-20. 106. Van Wyk. B.-E., and Verdoorn, G.H. (1989). Chemotaxonomic significance of alkaloids in the genus Lebeckia. Bioclzem. Syst. Ecol., 17:225-229. 107. Leonard, N.J. (1960). Lupin alkaloids. In The Alkaloids, Cl~emist~y and Plzysiology (R.H.F. Manske, ed.). Academic Press, New York, pp. 253. 108. Cho, Y.D., and Martin, R.O. (1971). Resolution and unambiguous identification of micrograms amounts of 22 lupin alkaloids by sequential use of thin-layer and gas-liquid chromatography and mass spectrometry. A n d . Biochem., 44:49-57. 109. Murakoshi. I., Kidoguchi, E., Nakamura, M., Haginiwa, J., Ohmiya, S., Higashiyama, K., and Otomasu, H. (1981). (-)-Epilamprolobine and its N-oxide. lupin alkaloids from Sophora tomentosa. Phytochenzistty, 20: 1725-1730. 110. Balandrin, M.F.. and Kinghorn, A.D. (1981). Tetrahydrophombifoline, a further constituent of Lupinus Oscar-lznuglltii and L. truncatus. J. Natural Prod., 44:495-497. 111. Munier, R. (1953). SCparation des alcalGdes de leurs N-oxydes par microchromatographie sur papier. B~dl.SOC.Chim Biol., 35:1225-1231. 112. Kinghorn, A.D., and Balandrin, M.F. (1984). Quinolizidine alkaloids of the leguminosae: structure types, analysis, chemotaxonomy and biological activities. In Alkaloids: Clzernicul and Biological Perspectives (S.W. Pelletier, ed.). Wiley, New York, pp. 105. 113. Reifer, I., and Niziolek, S. (1959). A nephelometric microdetermination of lupine alkaloids. Bull. L'Acad. Polon. Sei. CI. 11, 71485-489. 114. Von Baer, D., Reimeroles. E.H., and Feldeim. W. (1979). Metoden zur bestimmung der chinolizidinakaloide in Lupinus nlrtatbilis. Z. Lebensm. Unters. Forsclz., 169:27-3 1. 115. Cundiff, R.H., and Markunas, P.C. (1955). Determination of nicotine, nornicotine, and total alkaloids in tobacco. Anal. Chem., 27: 1650-1658. 116. Greirson, B.N., Allen, D.G., Gare, N.F., and Watson, I.M. (1991). Development and application of an enzyme-linked immunosorbent assay for lupin alkaloids. J. Agric. Food Cltem., 392327-233 1. 117. Priddis. C.R. (1983). Capillary gas chromatography for lupin alkaloids. J. Chrom., 261:95101. 118. Wink, M., Witte, L.. Hartmann. T., Theuring, C., and Colz, V. (1983). Accumulation of quinolizidine alkaloids in plants and cell suspension cultures: genera Lupinus, Cytisus, Baptisia, Genistu, Laburnum, and Sophora. Plunta Med., 48253-257. 119. Wink, M.. and Witte, L. (1984). Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap, leaves and fruits of Lupinus albus L. Planta. 161: 5 19-524. 120. Harris, D.J. (1987). Silylation: An improved and more accurate method for analysis of lupin alkaloids by capillary gas chromatography. Proc. 4th Int. Lupin Cor$, 329. 121. Keller, W.J., and Zelenski, S.G. (1978). Alkaloids from Lupinus argenteus var. stenoplzyllus. J. Pharm. Sci., 67:430-43 1. 122. Wink M., Hartmann, T., Witte, L., and Schiebel, H.M. (1981). The alkaloids patterns of cell suspension cultures and differentiated plants of Baptisia australis and their biogenetic implications. J. Natural Prod., 44: 14-20. 123. Majchrzak-Kuczynska, U., WiewiCirowski, M.. and Wyrzyliewicz, E. (1984). Mass spectrometry of bis-quinolizidine alkaloids: lactams of sparteine and a-isosparteine. Org. Muss Spectrom., 19:600-604. 124. Wink, M., Heinen, H.J., Vogt, H., and Schiebel, H.M. (1984). Cellular localization of quinolizidine alkaloids by laser desorption mass spectrometry (LAMMA 1000). Plant Cell Rep.. 3:230-233. 125. Saito, K., Yamazaki, M., Takamatsu, S., Kawaguchi, A., and Murakoshi, I. (1989). Greening induced production of ( +)-lupaninein tissue culture of Thennopsis lupinoides, Phytochemist)?, 28:2341-2344.

392

Muir

126. Saito. K.. Kobayashi, K., Ohmiya, S., Otomasu. H., and Murakoshi, I. (1989). Analysis of lupine alkaloids in plants by high performance liquid chromatography. J. Chon?.,462:333340. 127. Hatfield, G.M., Keeler, W.J., and Rankin, J.M. (1980). Quinolizidine alkaloids of Clathrotropis bruchypetala. J. Naturul Prod., 43:164-167. 128. Wink, M. (1987). Quinolizidine alkaloids: biochemistry. metabolism, and function in plants and cell suspensioncultures. Planta Med., 53:509-514. 129. Moncrieff, J. (1990). Simultaneous determination of sparteine and its 2-dehydro and 5-dehydro metabolites in urine by high-performance liquid chromatography with electrochemical detection. J. Chrom., 529: 194-200. 130. Keeler, R.F. (1986). Teratology of steroidal alkaloids. In Alkaloids: Chemical and Biological Perspectives (S.W. Pelletier, ed.). Wiley-Interscience, New York, pp. 389-425. 131. Coxon. D.T. (1984). Methodology for glycoalkaloid analysis. Am. Potato J.,61: 169-183. 132. King, R.R. (1980). Analysis of potato glycoalkaloids by gas-liquid chromatography of alkaloid components. J. Assoc. 08 Anal. Chenz.. 63:1226-1230. 133. Fitzpatrick, T.J.. and Osman, S.F. (1974). A comprehensive method for the determination of total potato glycoalkaloids. Am. Potato J., 51:3 18-323. 133. Bushway, R.J., Wilson, A.M., and Bushway, A.A. (1980). Determination of total glycoalkaloids in potato tubers using a modified titration method. Am. Potato J., 57:561-565. 135. van Gelder, W.M.J. (1985). Determination of the total C3,-steroidal alkaloid composition of Solmum species by high-resolution gas chromatography. J. Chron?.,33 1:285-293. 136. Bushway, R.J., Bureau. J.L., and King, J. (.1986).Modification of the rapid high-performance liquid chromatographic method for the determination of potato glycoalkaloids. J. Agr-ic. Food Chem., 34:277-279. 137. Hellenas, K.-E. (1 986). A simplified procedure for quantification of potato glycoalkaloid in tuber extracts by HPLC: comparison with ELISA and a colorimetric method. J. Sci. Food Agric., 37:776-782. 138. Edwards, E.J., and Cobb, A.H. (1996). Improved high-performance liquid chromatographic method for the analysis of potato (So/anunztuberoszrnr)glycoalkaloids. J.Agric. Food Cltem., 4412705-2709. 139. Eltayeb, E.A., and Roddick, J.G. (1985). Biosynthesis and degradation of a-tomatine in developing tomato fruits. Plzytochemistn, 24:253-257. 140. Boll, P.M. (1962). Alkaloidal glycosides from Solanum dulcantclra. Acta Chem. Scand., 16: 1819-1830. 141. Zitnak. A., and Filadelfi-Keszi, M.A. (1988). Isolation of P?-chaconine, a potato bitterness factor. J. Food Biochem., 12:183-189. 142. Gitler, C. (1972). Use of NAS to detect phospholipids and apolar molecules in chromatograms. Arzal. Bioclzem., 50:334-325. 143. Cam F.H., and Price, E.A. (1926). LXIV. Colour reactions attributed to vitamin A. Bioclzem. J., 20:497-501. 144. Shih, M.-J.. and Kuc, J. (1974). a-and p-solamarine in Kennebec Solanum tzlberoswz leaves and aged tuber slices. Plrytochenzistly. 13:997-1000. 145. Majak. W., McDiarmid, R.E., Cristofoli, W.,Sun, F., and Benn, M. (19923. Content of zygacine in Zygadenus venerrosus at different stages of growth. Plzytochemistn, 3 1:341734 18. 146. Wu, M.T.. and Salunkhe, D.K. (1976). Devices for improving color development in glycoalkaloid determination. J. Food Sci., 41:220-222. 147. Sachse, J., and Bachmann, F. (1969). Uber die alkaloid bestimmung in SolanLtm tuberosum L. Z. Lebensnz. Ulrters. Forsch., 141262-374. 148. Morgan. M.R.A.. Coxon, D.T., Bramham, S., Chan, H.W.S., van Gelder, W.M.J., and Alison, M.J. (1985). Determination of the glycoalkaloid content of potato tubers by three methods including enzyme-linked imrnunosorbent assay. J. Sci. Food Agric., 36:282-288.

Analytical Methodology for Plant Toxicants

393

149. Wierschowski, P., and Wierschowski, Z. (1961). Colorimetric determination of solanine and solanidine with antimony trichloride. Chem. Anal. (Warsaw), 6579-585. 150. Coxon, D.T., Price, K.R., and Jones, P.G. (1979). A simplified method for the determination of total potato glycoalkaloids in potato tubers. J. Sci. Food Agric., 30:1043-1049. 151. Ruiz, L.P. (1976). A rapid screening test for lupin alkaloids. NZ J. Agric. Res.. 20:51-52. 152. Morgan, M.R.A., McNerney, R., Matthew, J.A., Coxon, D.T.. and Chan, H.W.-S. (1983). An enzyme-linked immunosorbent assay for total glycoalkaloids in potato tubers. J. Sci. Food A g k . , 34:593-598. 153. Ward, C.M.. Franklin, J.G., and Morgan, M.R.A. (1988).Investigations into the visual assessment of ELTSA end points: Application to determination of potato total glycoalkaloids. Food Addit. Contantin., 5:621-627. 154. Plhak, L., and Sporns, P. (1994). Development and production of monoclonal antibodies for the measurement of solanidine potato glycoalkaloids. Am. Potato J., 71:297-313. 155. Barbour, J.D., Kennedy, G.G., and Roe, R.M. (1991). Development of an enzyme linked immunosorbent assay for the steroidal glycoalkaloid a-tomatine. In Pesticides and Their Future: Toxicological Studies of Risks and Benejts (E. Hodgson, R.M. Roe, and N. Motoyama, eds.). North Carolina State University, Raleigh, pp. 289-303. 156. Herb, S.F., Fitspatrick, T.J., and Osman, S.F. (1975). Separation of potato glycoalkaloids by gas chromatography. J. Agric. Food Cltern., 23:520-523. 157. Keeler, R.F., Baker, D.C., and Gaffield, W. (1990). Spirosolane-containing Solnrzum species and induction of cogenital craniofacial malformations. Tosicon, 28:873-884. 158. Abell, D.C., and Spoms, P. (1996). Rapid quantitation of potato glycoalkaloids by matrixassisted laser desorptionllonization time-of-flight mass spectrometry. J. Agric. Food Clzem., 4412293-2296. 159. Hunter, T.R., Walden, M.K.. Wagner, J.R., and Heftmann, E. (1976). High-pressure liquidchromatography of steroidal alkaloids. J. Chrom., 119:223-226. 160. Bushway, R.J., Barden, E.S., Bushway, A.W., and Bushway. A.A. (1979). High-performance liquid chromatographic deparation of potato glycoalkaloids. J. Chrorn., 178:533-541. 161. Hunter, I.R., Walden, M.K.. and Heftmann, E. (.1980).High performance liquid chromatography of Solarzlrr~and Veratrum alkaloids. J. Clzronl., 198:363-386. 162. Crabbe, P.G.. and Fryer, C. (1980). Rapid quantitative analysis of solasodine, solasodine glycosides and solasodiene by high-pressure liquid chromatography. J. Chrom., 187:87-100. 163. Friedman, M., and Levin. C.E. (1992). Reversed-phase high-performance liquid chromatographic separation of potato ‘glycoalkaloids and hydrolysis products in acidic columns. J. Agric. Food Chein., 409157-2163. 164. Kobayashi, K., Powell, A.D., Topyoda, M., and Saito, Y. (1989). High-performance liquid chromatographic method for the simultaneous analysis of a-solanine and a-chaconine in potato plants cultured in vitro. J. Chrom., 462357-364. 165. Bushway, R.J. (1982). High performance liquid chromatographic separation of potato glycoalkaloids using a radially compressed amino column. J. Chronz., 247: 180-187. 166. Cham, B.E.. and Wilson, L. (1987). HPLC of glycoalkaloids from Solmum sodomaem. Plarzta Med., 5359-62. 167. Eldridge. A.C., and Hockridge, M.E. (1983). High-performance liquid chromatographic separation of eastern black nightshade (Solnnuw ptycantl~urrl)glycoalkaloids. J. Agric. Food Chem., 31:1218-1220. 168. Percival, G., Dixon. G.R., and Sword, A. (1996). Glycoalkaloid concentration of potato tubers following exposure to daylight. J. Sci. Food Agric., 71 :59-63. 169. Ghazi, M., and Matthees, D.P. (1 989). Use of ion-pairing reversed-phase liquid chromatography in separation of solanidine and solasodine. J. Clzem. Ecol., 15:2661-2666. 170. Carman, A.S., Kuan. S.S.,Ware, G.M., Francis. O.J., and Kirschenheuter, G.P. (1986). Rapid high performance liquid chromatographic determination of the potato glycoalkaloids a-solanine and a-chaconine. J. Agric. Food Chem., 34:279-282.

394

Muir

in toma171. Friedman, M., Levin, C.E.,and McDonald, G.M. (1994). a-Tomatine determination toes by HPLC using pulsed amperometric detection. J. Agric. Food Chenz., 42:1959-1964. 172. Pelletier, S.W.. Kulanthaivel, P., and Olsen, J.D. (1989). Alkaloids of Delphirrizm barbeli. Pllytoclzentistry, 28:1521-1525. 173. Pelletier, S.W., Joshi, B.S., and Desai, H.K. (1985). Techniques for isolation of alkaloids. In Adlyarzces in Medicinal Plant Research (A.J. Vlietinck and R.A. Donmisse, eds.). Wissenschaftliche Verlag, Stuttgart, pp. 153. 174. Grina, J.A., Schroeder, D.R., Wydallis, E.T., Stermitz, F.R.. Melman,J., and Capinera, J.L. (1986).Alkaloidsfroin Delphirziurn geyeri, three new C,,-diterpenoidalkaloids. J. Org. Chem., 51:390-394. 175. Aiyar, V.N., Benn, M.H., Huang, Y.Y ., Jacyno, J.M., and Jones, A.J. (1978). The alkaloids of Delphiniurn brownii. Plzytochernistr?,. 17: 1453-1454. and Olsen, J.D. (1981). Isolation and structure 176. Pelletier, S.W., Daily, O.D., Mody, N.V., elucidation of the alkaloids of Delphinium glaucescens Rybd. J. Org. Chem., 46:3284-3293. of Delplzin177. Kulanthaival, P., Benn, M., and Majak, W. (1986). The Cly-diterpenoid alkaloids ium bicolor. Pllytochemistt?, 25 : 1511- 1513. 178. Katz, A.. and Staehelin, E. (1979). DC-Untersuchung der Euroaischen Acorzitizm lrapellus gruppe. Plrni-nz. Acta Helv., 54253-265. 179. Chen, Y.-Z., and Wu, A. (1990). Diterpenoid alkaloids from Delphirrium winklerianunr. Phytochemistry, 29: 1016-1019. Pelletier. S.W. (1986). Centrifugal chromatography of djter180. Desai, H.K., Trumbull, E.R.. and penoid alkaloids. Use of aluminium oxide containing two fluorescent materials. J. Clzrom., 366:439-444. 181. Desai, H.K., Joshi, B.S., Panu, A.M., and Pelletier, S.W. (1985). Separation of diterpenoid alkaloid mixtures using the Chromatotron. J. Clrrorn., 322:223-227. 182. Manners, G.D.,and Ralphs, M.H. (1989). Capillary gas chromatography of Delylziniurn diterpenoid alkaloids. J. Clzrom., 466:427-432. 183. Manners, G.D., Pfister, J.A., Ralphs, M.H., Panter. K.E., and Olsen, J.D. ( 199 1). Larkspur chemistry: toxic alkaloids in tall larkspur. J. Range Manage., 45:63-67. 184. Verpoorte, R., Verziji. J.M., and Baerheim Svendsen, A. (1984). Reversed-phase ion-pair chromatography of alkaloids on dodecylsulphonic acid and cetrimide (hexadecyltrimethylammonium)-impregnated C Is columns. J. Chrom., 283:401-405. 185. Majak, W., McDiarmid, R.E., and Benn, M.H. (1987). Isolation and HPLC determination of methyllycaconitine in a species of low larkspur(Delplzi?liunz nuttallinrzzr?lz). J. Agric. Food Chem., 35:SOO-802. 186. Hikino, H., Konno, C., Watanabe. H., and Ishikawa, 0. (1981). Determination of aconitine alkaloids by high-performance liquid chromatography.J. Chi-on?.,21 1:123-128. 187. Kulanthaivel, P., and Pelletier. S.W. (1987). High-performance liquid chromatographic analysis of Clp-diterpenoid alkaloids.J. Chrorn., 402:366-370. 188. Molyneux, R.J.. and James, L.F. (1982). Loco intoxication: indolizidine alkaloids of spotted locoweed (Astragalzls lerztiginosus). Science, 21 6: 190-121. 189. Colegate, S.M., Dorling, P.R., and Huxtable, C.R. (1979). A spectroscopic investigation of swainsonine: an a-mannosidase inhibitor isolated from Swairzsona canescens. Aust.J. Clzent., 3212257-2264. 190. Davis. D., Schwarz. P.. Hernandez, T., Mitchell, M., Warnock, B.,and Elbein, A.D. (1 986). Isolation and characterization of swainsonine from Texas locoweed (Astmgrrlus emoq’nnzls). J. Cell Biol., 103:A76. 191. Stahr, H.M., and Martin, P.J. (1989). Analytical methods for swainsonine and slaframine. In Swainsorrine and Related Glycosidase Znhibitoi-s (L.F. James,A.D. Elbein, R.J. Molyneux, and C.D. Warren, eds.). Iowa State University Press, Ames, pp. 118-124. 192. Elbein, A.D., and Molyneux, R.J. (1987). The chemistry and biochemistry of simple indolizidine and related polyhydroxy alkaloids. In Alkaloids: Chemical and Biological Perspec-

Analytical Methodology for Plant Toxicants

193. 194.

195.

196.

197.

198. 199.

300. 201.

202.

203. 204. 305.

206.

207. 208. 309.

210.

395

tives (L.F. James, A.D. Elbein, R.J. Molyneux, and C.D. Warren, eds.). Wiley, New York, pp.1-54. Molyneux, R.J., Campbell, B.C., and Dreyer. D.L. ( 1990). Honeydew analysis for detecting phloem transport of plant natural products. J. Clzent. EcoZ., 16: 1899-1909. Molyneux, R.J., James, L.F., Panter, K.E., and Ralphs, M.H. (1989). The occurrence and detection of swainsonine in locoweeds. In Swwinsorrine and Related Glycosidase Inhibitors (L.F. James, A.D. Elbein, R.J. Molyneux, and C.D. Warren, eds.). Iowa State University Press, Ames, pp. 100-117. Molyneux, R.J., James, L.F., andPanter, K.E. (1985). Chemistry oftoxic consituents of locoweed (Astragalns and 0.xytropis) species. In Plant Toxicology (A.A. Seawright, M.P. Hegarty, L.F. James, and R.F. Keeler, eds.). Dominion Press-Hedges & Bell, Melbourne, pp. 366-278. Donaldson, M.J.. Broby, H., Adlard, M.W., and Bucke, C. (1990).High pressure liquid chromatography and pulsed amperometric detection of castanospernline and related alkaloids. Plgltochern. Anal., 1:18-21. Jaziri, M., Legros, M., Homes. J.. and Vanhaelen, M. (1988). Tropine alkaloids production by haily root cultures of Datura strirmonium and Hyoscayums ]tiger. Phytochenaistry, 27: 4 19-430. Ishitnaru, K.. and Shimomura, K. (1989). 7P-Hydroxyhyoscyamine fromDuboisia myopol-0ides-D. Leiclthar-dtiihybrid and Hyoscyarltzrs albus. Phvtochentistly, 28:3507-3509. Fliniaux. M.-A., Manceau, F.. and Jacquin-Dubreuil, A. (1993). Simultaneous analysis of 1hyoscyamine, I-scopolamineand&tropicacidinplantmaterialby reversed-phase highperformance liquid chromatography. J. Cltront.. 644: 193-1 97. Ghani, A. (1986). The site of synthesis and secondary transformation of hyoscyamine in Solandm gmndij?ora. Ph~tocltenlistry,35:6 17-61 9. Puech, A., Jacob, M., and Grandy, G. (1972). Mise em Cvidence des alcaloides de la Belladone et de leurs d6rivCs par chromatographic en couchesminces. Emploi d’un rCvClateur de sensibilitb accrue. J. Cht-om.. 68:161-165. Karawya, M.S., Abdel-Wahab, S.M.. Hifnawy, M.S., and Ghourab, M.G. (1975). Colorimetric andgas-liquid chromatographic determinationofatropine-hyoscyamineandhyoscine (scopolanline) in solanaceous plants. J. Assoc. Off Anal. Chew., 58:884-887. Weiler, E.W., Stockgt, J., and Zenk, M.H. (1981). Radioimmunoassay for the quantitative determination of scopolamine. Plzytoclzenzistp, 20:2009-2016. Savary, B.J., and Dougall, D.K. (1990). Scopolamine radioimmunoassay for tissue cultures of Datura. Phytochentisti?, 29: 1567-1569. Kikuchi, Y., Irie, M., Yoshimatsu. K., Ishimaru, K., Shimomura, K.. Satake, M., Sueyoshi, S., Tanno, M.. Kanliya, S., Sawada, J.-I., and Terao, T. (1991). A monoclonal antibody to scopolamine and its use forcompetitive enzyme-linked inmunosorbent assay. Pltytoclzemistl?, 3013273-3276. Hagemann, K., Piek. K., Stockigt. J.. and Weiler, E.W. (1992). Monoclonal antibody-based enzyme immunoassay for the quantitative determination of the tropane alkaloid, scopolamine. Plnnta Med., 58~68-72. Wurzburger, R.J., Miller, R.L., Boxenbaum, H.G., and Spector. S. (1977). Radioimmunoassay of Atropine in plasma. J. Phcrmacol. ESP. T l w . , 303:435-441. Lehtola, T., Huhtikangas, A., and Virtanen, R. (1982). Application of radioimmunoassay to the quantitation of hyoscyamine in plant material. Planta Men., 45:237-239. Fliniaux, M.A., and Jacquin-Dubreuil, A. (1987). A competitive solid-phase enzyme immunoassay for the evaluation of tropane alkaloids in plant material, using anti-DL-tropic acid antibodies. Planta Med., 53:87-90. Okuda, T.. Nishida, M., Satneshima, I., Kyoyama, K.. Hiramatsu. K., Takehara, Y.. and Kohriyama, K. (1991). Determination of atropine in biological specimens by high-performance liquid chromatography. J. Chrom., 567: 141-149.

396

Muir

21 1. Paphassarang, S., Raynaud, J., Godeau, R.P.,and Binsard, A.M. (1985). Analysis qualitatives et quantitatives de la hyoscyamine-atropine et de la scopolamine dansles teintures mb-esde SolanacCes par chromatographie liquide haute performance. J. Chrom., 319:412-418. 21 2. Shimotnura, K., Sauerwein, M., and Ishimaru. K. (1991). Tropane alkaloids in the adventitious and hairy root cultures of solanaceous plants. Phytoc?~emistry, 302275-2278. 213. He, L.-Y., Zhang, G.-D., Tong, Y.-Y., Sagara,K., Oshima, T., and Yoshida. T. (1 989). Reversed-phase ion-pair high-performance liquid chromatographic separation and determination of tropane alkaloids in Chinese solanaceous plants. J. Chronr., 381:328-433. 214. Li, S., and Khalil, S.K.W. (1990).An HPLC method for determinationof atropine in human plasma. J. Liquid Chrom., 13:1339-1350. and Schiff, 215. Lopez, J.A., Laurito, J.G., Brenes, A.M., Lin, F.-T., Sharaf, M., Wong, L.K., P.L. (1990). Aporphinoid alkaloidsof Guattei-ia olivifovris and G. tonduzii. Phytochemistry, 2911899-1901. 216. Ikuta, A., and Itokawa, H. (1982). Berberine and other protoberberine alkaloids in callus tissue of Thalicti-unl minus. Phytochenzistry, 31: 1419-1421. 217. Yamada, Y.. and Sato, F. (1981). Production of berberine in culturedcells of Coptisjaponica. Phytochemistry, 20545-547. 218. Chattopadhyay, S.K., Ray, A.B., Slatkin, D.J.. and Schiff, P.L. (1983). Quaternary alkaloids of Thalictrum foliolosunr. Phytochewistv. 222607-2610. 219. Yoshikawa, T., and Furuya. T. (1985). Morphinan alkaloid production by tissues differentiated from cultured cells of Papaver somniferurtr. Plnllta Men., 51: 110-1 13. 220. Wieczorek, O., Nagakura, N., Sund, C., Jendrzejewski,S., and Zenk, M.H. (1986). Radioirnmunoassay determinationof six opium alkaloidsand its application to plant screening. Phytochemistry, 25:2369-2646. 221. Han, L.-F., Nowicky, W.,and Gutmann, V. (1991). Reversed-phase high-performance liquid chromatographic separation of tertiary and quaternary alkaloids from Chelidonizrn?mjus L. J. Chrorn., 543:123-128. 222. Bonora, A., Tosi, B., Dall’Olio, G., and Bruni, A. (1990). Quaternary alkaloids in rhizomes of Ranunculus sei-bicus. Phytochemistry, 292389-2390. 223. Tanahashi, T., and Zenk, M.H. (1990). New hydroxylated benzo[c]phenanthridine alkaloids from Eschsclzoltzin caliornica cell suspension cultures. J. Natural Prod., 53:579-586. 224. Hutin, M., Oztekin, A., Cavi, A., and Foucher, J.P. (1983). Analyse des alcoloi’des du Papaver yilosurn par chromatographie liquid haute performance: application i la sCparation d’ipimkres. J. Chi-om.. 265:139-142. 225. Craig, A.M., Bilich, D., Hovermale, J.T., and Welty, R.E. (1994). Improved extraction and HPLC methods for ergovaline from plant material and rumen fluid. J. Vet.Diagrz.Imyest.. 61348-352. 226. Testereci, H., Garner, G.B., Rottinghaus, G.E., Cornel, C.N., and Andersen. M.P.A. (1990). Method for large scale isolation of ergovaline from endophyte-infectedtall fescue seed (Fescuca nrundinacea). In Proc. Int. Synyx on Acrenloiziunr/Grass Irlteractions. Nos 3, 1990, NeMl Orleans, L.A. (S.S. Quisenberry, and R.E. Joost, eds.). Louisiana Agricultural Experimental Station, Baton Rouge, pp. 100. 227. Rottinghaus, G.E., Schultz, L.M., Ross, P.F., and Hill, N.S. (1993). An HPLC method for the detection of ergot in ground and pelleted feeds. J. Vet. Dingn. Invest., 5:242-247. 228. Porter, J.K., Bacon, C.W.,and Robbins, J.D. (1979). Ergosine. ergosinine,and chanoclavine I from Epichloe l y d u h . J. Agric. Food Chenr., 27:595-598. 229. Sprince, H. (1960). A modified Ehrlich benzaldehyde reagent for detection of indoles on paper chronlatograms. J. Cltrorn., 3:97-98. 230. Shelby. R.A.. and Kelley, V.C. (1990). An immunoassay for ergotamine and related alkaloids. J. Agric. Food Cherrz., 38: 1130-1 134. 231. Yates, S.G., and Powell. R.G. (1988). Analysis of ergopeptine alkaloids in endophyte-infected tall fescue. J. Agric. Food Chem.. 36:337-340.

Analytical Methodology for Plant Toxicants

397

232. Aboul-Enein, H.Y., and Bakr, S.A. (1997). Simultaneous determination of caffine and ergotamine in pharmaceutical dosage formulation by capillary electrophoresis. J. Liq. Chrom. Re/. Technol., 20:47-55. 233. Cook, J.W., and Loudon. J.D. (1952). Colchicine. In The Alkaloids, Chemistr?, and Physiology (R.H.F. Manske, and H.L. Holmes, eds.). Academic Press, New York, pp. 261329. 234. Evidente, A., Iasiello, I., and Randazzo, G. (1983). Rapid quantitative analysis of lycorine by reversed-phase high-performance liquid chromatography. J. Chronz., 281 :362-366. 235. Ali, A.A., Kating, H., Frahm, A.W., El-Moghazi, A.M., and Ramadan, M.A. (1981). Two nonhydroxylated alkaloids in Crinzm arrqustrcnt. Phytochemistnj, 20: 1121-1 123. 236. Powell. R.G., Plattner. R.D., and Suffness, M. (1990). Occurrence of sesbanimide in seeds of toxic Sesbrrirzn species. Weed Sci., 38:148-152. 237. Leete, E.. and Pinder, A.R. (1972). Biosynthesis of dioscorine. Phytochemistly, 11:32193224. 238. Willaman, J.J., Fenske. C.S., and Correll, D.S. (1953). Occurrence of alkaloids in dioscorea. Science, 118:329-330. 239. Klein, A.E., and Davis, P.J. (1980). Determination of colchinine and colchiceine in microbial cultures by high-performance liquid chromatography. Anal. Chenr., 522432-2435. 240. Klein, A.E., and Davis, P.J. (1981). Retention behaviour of selected colchicine derivatives on reversed-phase high-performance liquid chromatographic systems. J. Chrom., 207:247251. 241. Lepom, P., and Robowsky, K.D. (1985). Determination of the alkaloid perloline in grasses by high-performance liquid chromatography. J. Chronz., 322:261-264. 242. Montgomery, R.D. (1980). Cyanogens. In Toxic ComtitrcelltsofPlarlt Foodstz&s (I.E. Liener, ed.). Academic Press, New York, pp. 143-160. 243. Majak, W., Bose, R.J., and Quinton, D.A. (1978). Prunasin, the cyanogenic glycoside in Amelnnclzier-rclnifolin. Phytochemistry, 17:803. 244. Cooke, R.D. (1978). An enzymatic assay for the total cyanide content of cassava (Manilot esculerttcr Crantz). J. Sci. Food Agric., 29:345-352. 345. Guignard, L. (1907). Sur les quanti& d’acide cyanhydrique fournies par le Phnseolzrs lzlrzcrtzrs L. cultivi sous le climat de Paris. Bull. Sci. Pharmacol., 14:556-557. 246. Tantisewie, B., Ruijgrok, H.W.L., and Hegnauer, R. (1969). Die verbreitung der blausaure bei den cormophyten. 5. Mitteilung: Uber cyanogene verbindugen bei den parietales und bei einigen weiteren sippen. Pharm. Weedblnd. 104:1341-1355. 247. Lambert, J.L., Ramasamy, J.. and Paukstelis. J.V. (1975). Stable reagents for the colorimetric determination of cyanide by modified Konig reactions. A i d Chenr., 47:916-918. 248. Haskins, F.A., Gorz, H.J., and Hill, R.M. (1988). Colorimetric determination of cyanide in enzyme-hydrolyzed extracts of dried sorghum leaves. J. Agric. Food Chem, 36775-778. 249. Sharma, A.. and Thibert, R.J. (1985). The effect of barbituric acid concentration in the spectrophotometric determination of cyanide and thiocyanate by the pyridine-barbituric acid method. Mikroclzim. Acta ( Weirz), 1:357-363. 250. Brooke, B.M., McDiarmid, R.E., and Majak, W. (1988). The cyanide potential in two varieties of Amelanchier alnifolia. Can. J. Plant Sci., 68:543-547. 251. Williams, K.J., Rosenstein, R., and Smith, R.P. (1985). Substitution of tetramethylbenzidine for benzidine in cyanide analysis. Clin. Clzenz. Acta, 145:113-1 18. 252. Association of Official Agricultural Chemistry (1945). Methods ofAnalysis. 6th ed. AOAC, Washington, DC. 253. Morgan, R.L., and Way, J.L. (1980). Fluorometric determination of cyanide in biological fluids with pyridoxal. J. And. Toxicol.. 4:78-81. 254. Chadha. R.K., Lawrence, J.F., and Ratnayake, W.M.N. (1995). Ion chromatographic determination of cyanide released from flaxseed under autohydrolysis conditions. Food Addit. Contarnin., 12:527-533.

398

Muir

255. Mkpong, O.E., Yan, H., Chism, G., and Sayre, R.T. (1990). Purification, characterization, and localization of linamarase in cassava. Plarzt Physiol., 93: 176-181. 256. Frehner, M., Scalet, M., and Conn, E.E. (1990). Pattern of the cyanide-potential in developing fruits. Implications for plants accumulating cyanogenic Inonoglucosides (Phaseolus lurzatus) or cyanogenic diglucosides in their seeds (Linum zasitatissimum, Prunzas anzygdcrlus). Plant Plysiol., 94:29-34. 257. Fenwick, G.R., Heaney, R.K., and Mullin, E.J. (1983). Glucosinolates and their breakdown products in food and food plants. CRC Crit. Re\!. Food Sci. Nzltr., 18:123-201. 258. Tookey, H.L., VanEtten, C.H., and Daxenbichler, M.E. (1980). Glucosinolates. In Toxic Constituents of Plant Foodstzlffs (LE. Liener, ed.). Academic Press, New York, pp. 103142. 259. Van Etten, C.H., Daxenbichler, M.E., Peters, J.E., and Tookey, H.L. (1966). Seed meal hydrolysis. Variation in enzymatic degradation products from the major thioglucosides in Crambe abyssinica and Brassica napus seed meals. J. Agric. Food Chenz., 14:426-430. 260. Heaney. R.K., and Fenwick, G.R. (1980). Analysis of glucosinolates in Brassica species using gas chromatography, direct determination of the thiocyanate ion precursors, glucobtassicin and neoglucobrassicin. J. Sci. Food Agric., 3 1:785-793. 261. Mullin, W.J., and Sahasrabudhe, M.R. (1977). Glucosinolate content of cruciferous vegetable crops. Can. J. Plant Sci.. 57:1227-1230. 262. Troeng, S. (1955). Oil determination of oilseed. Gravimetric routine method. J. Am. Oil Clzenr. Soc., 32: 124-126. 263. McGregor, D.I., Mullin, W.J.. and Fenwick, G.R. (1983). Review of analysis of glucosinolates: analytical methodology for determining glucosinolate composition and content. J. Assoc. O f A n d . Cltern., 66:825-849. 264. Betz, J.M., and Fox, W.D. (1994). High-performance liquid chromatographic determination of glucosinolates in Brassica vegetables. In Food Pl1ytochemicalsfor Cancer Prevention. I. Fruits and Vegetables (T. Huang, T. Osawa, C. Ho,and R.T. Rosen, eds.). ACS. Washington, DC, pp. 181-196. 265. Lien, K.A. (1970). Methods for quantitative determination of seed-glucosinolates of Brassica species and their application in plant breeding of rape low in glucosinolates content. Z. Pfanzen:ucht., 63: 137- 154. 266. Lien, K.A.. and Schon, W.J. (1969). Quantitative determination of glucosinolate in half seeds of Brassica species. Angew. Botanik.. 43:87-92. 267. VanEtten. C.H.. McGrew, C.E., and Daxenbichler, M.E. (1974). Glucosinolate determination in cruciferous seeds and meals by measurement of enzymatically released glucose. J. Agric. Food Chenr., 22483-487. 268. Gardrat, C.. and Prevot, A. (1987). Dosage des glucosinolates dam lesgraines et les tourteaux de Colza par enzymeatique. Rev. Franc. Corps. Gras., 34:457-461. 269. Koshy, A., Bennetto, H.P., Delaney, G.M., MacLeod, A.J.. Mason, J.R.. Stirling, J.L.. and Thurston, C.F. (1988). An enzyme biosensor for rapid assay of glucosinolates. And. Lett., 21 :2177-2194. 270. Wang, X., and McGregor, D. (1991). Application of enzyme immobilization to achieve rapid, low cost glucosinolate analysis of seed and meal of canola/rapeseed. In Proc. GCIRC 8th Int. Rapeseed Congress (D.I. McGregor, ed.). Organizing Committee 8th Int. Rapeseed Congress, Saskatoon, pp. 850-855. 271. Brzezinski, W.. and Mendelewski, P. (1984). Determination of total glucosinolate content in rapeseed meal with thymol reagent. 2. PJan,-en,-uclzt., 93: 177-1 83. 272. Tholen, J.T., Shifeng, S., and Truscott, R.J.W. (1989). The thymol method for glucosinolate determination. J. Sci. Food Agric., 49: 157-1 65. 273. Wetter. J.T., and Youngs. C.G. (1976). Thiourea-UV assay for total glucosinolate content in rapeseed meals. J. A m Oil Chem SOC., 53:162-164. 274. Olsson, K., Theander, O., and Aman, P. (1980). Deternlination of total glucosinolate content

Analytical Methodology for Plant Toxicants

275. 276.

277. 278.

279.

280. 281. 282.

283.

284. 285. 286. 287.

288. 289. 290. 291. 292.

293. 294. 295.

399

in rapeseed and turnip rapeseed meals by gas chromatography. Swedish J . Agric. Res., 6: 225-239. Youngs, C.G., and Wetter, L.R. (1967). Microdetermination of the major individual isothiocyanates and oxazolidinethione in rapeseed. J. Ant. Oil Chem SOC..44551-554. Appelqvist. L.A.. and Josefsson. E. (1967). Methods for quantitative determination of isothiocyanates and oxazolidinethiones in digests of seed meals of rape and turnip rape. J. Sei. Food Agric., 181510-519. Thies, W. (1979). Detection and utilization of a glucosinolate sulfohydrolase in the edible snail, Helix ponzatia. Nattwwissen, 66:364-365. Thies, W. (1978). Quantitative analysis of glucosinolates after their enzyamtic desulfation on ion exchange columns. In Proc. GCZRC 5th bzt. Rapeseed Congress. Organizing Committee 5th Int. Rapeseed Congress, Malmo, pp. 136-139. Minchinton, I., Sang, J., Burke. D., and Truscott, R.J.W. (1982). Separation of desulphoglucosinolates by reversed-phase high-performance liquid chromatography. J. Chro-onz., 237: 141-148. Sang, J.P., and Truscott, R.J.W. (1984). Liquid chromatographic determination of glucosinolates in rapeseed as desulfoglucosinolates. J . Assoc. Off Anal. Clzern., 67:829-833. Helboe. P., Olsen, O., and Sorensen, H. (1980). Separation of glucosinolates by high-performance liquid chromatography. J. Chronz.. 197:199-205. Zrybko, C.L., Fukuda, E.K., and Rosen. R.T. (1997). Determination of glucosinolates in domestic and wild mustard by high-performance liquid Chromatography with confirmation by electrospray mass spectrometry and photodiode-array detection. J. Chronz., 767:4352. Karcher, A., and El Rassi. Z. (1998). High performance liquid phase separation of glycosides. I1 Capillary Electrophoresis of the fluorescently labelled acid hydrolysis products of glucosinolates-profiling of glucosinolates in white and red cabbages. J. Liq. Chom. Rel. Technol., 21~1411-1432. Murray, R.D.H., Mkndez, J., and Brown, S.A. (1982). The Natural Counzarins. Wiley. ChiChester. Livingston, L. (1978). Forage plant estrogens. J. To.vicol. Environ. Health, 4:301-324. Somolenski, S.J., Kinghorn, A.D., and Balandrin, M.F. (1981). Toxic constituents of legume forage plants. Econ. Bot., 35:321-355. Trumble, J.T., Dercks, W.. Quiros, C.F., and Beier, R.C. (1990). Host plant resistance and linear furanocoumarin content of Apiunz accessions. J. Econ. Entoi?zol.,83:519-525. Williams, C.A.. and Harborne, J.B. (1989). Isoflavonoids. In Methods in Plant Biochenzistn? (P.M. Dey. and J.B. Harborne, eds.). Academic Press, London, pp. 421-449. Ma, X., Chen, Y., and Hui. R. (1989). Analysis of anthraquinones in Rheunt frar7:erhrchii Munt (rhubarb) by thin-layer chromatography. Clzroi~zrrtogrylzin,27:465-466. Smith, F.H. (1968). Estimation of free gossypol in cottonseed meal and cottonseed meats: modified method. J. Am. Oil Chem SOC.,45:903. Inoue, K., Nayeshiro, H., Inouye, H., and Zenk, M. (1981). Anthraquinones in cell suspension cultures of Morinda citrifolin. Phytochemist~y.20: 1693-1700. Dreyer, D.L., Arai, I., Bachman, C.D., Anderson, W.R.. Smith, R.G., and Davies, G.D. (1975). Toxins causing noninflammatory paralytic neuronopathy. Isolation and structure elucidation. J. Am. Chem. Soc.. 97:4985-4990. Sherwood, F.W. (1926). Studies on gossypol: the gossypol and D-gossypol content of some North Carolina cottonseed meals. J. Agric. Res., 327934300. Legueut, C.,Hocquemiller, R., and Cave, A. (1981). Determination of sinapine. Ann. Phnnn. Fr., 39~557-561. Ataga. A.E., Epton, H.A.S., and Frost, R.R. (1993). Effect of virus infection on the concentration of furanocoumarins in celery (Apium graveolens L. var. duke Mill. D.C.). Physiol. Mol. Plat1t Pclth., 421161-168.

400

Muir

296. Zobel, A.M., and Brown, S.A. (1990). Seasonal changes of furamocoumarin concentrations of leaves of Heracleum Iamtmz. J. Chem. Ecol.. 16:1623-1634. 297. Ceska, O., Chaudhary, S.K., Warrington, P.J.,and Ashwood-Smith, M.J. (1986). Furocoumarins in the cultivated carrot, Damus carota. Plzytochemist~y,25:81-83. 298. Beier, R.C.,Ivie, G.W.. Oertli, E.H., and Holt, D.L.( 1983). HPLC analysis of linear furocoumarins (psoralens)in healthy celery(Apium grm7eolensT.). Food Chenz. Toxic., 21:163-165. 299. Trumble, J.T., Miller, J.G., Ott, D.E.,and Carson, W.C. (1992). Seasonal patternsand pesticidal effects on the phototoxic linear furanocoumarins in celery, Apiunzgrn\7eoZem L. J. Agric. Food Clzem., 40:1501-1506. 300. Lookhart, G.L. (1980). Analysis of coumestrol. A plant estrogen, in animal feeds by highperformance liquid chromatography. J. Agric. Food Clzern.. 28:666-667. 301. Baumann. U.. Dick, R., and Zimmerli, B. (1988). Orientierende untersuchung zum vorkommen von furocoumarinen in pflanzlichen Iebensmitteln und kosnletika.Mitt. Gebiete Lebebsum. Hyg., 79:112-129. 302. Patroni, J.J., Collins, W.J., and Stern, W.R. (1982). Quantitative analysis of the isoflavone phyto-oestrogens genistein, formononetin and biochanin A, in subterranean clover leavesby high-performance liquid chromatography. J. Clzrom., 247:366-368. 303. Nomeir, A.A., and Abou-Donia, M.B. (1982). Gossypol: high-performance liquid chromatographic analysis and stability in various solvents. J. A m Oil Clzem SOC..59:546-549. 304. Gagliardi, L., Amato, A., Basili, A., Cavazzutti, G., Gattavecchia, E., and Tonelli. D. (1985). Determination of preservatives in cosmetic products by reversed-phase high-performance liquid chromatography. J. Clu-onl., 325:353-358. 305. Mahato, S.B., Ganguly, A.N., and Sahu, N.P. (1982). Steroid saponins. Phytoclremist~,21: 959-978. 306. Benson. J.M., Seiber, J.N., Keeler, R.F., and Johnson, A.E. (1978). Studies on the toxic principle of Asclepias eriocarpa and Asclepias labriformis. In Effects of Poisonom Plants oiz Lillestock (R.F. Keeler, K.R. van Kampen, and L.F. James, eds.). Academic Press. New York, pp. 273-284. 307. Hollman, A. (1985). Plants and cardiac glycosides. Br. Heart J., 54258-261. 308. Seiber, J.N., Lee, S.M., and Benson, J.M. (1983). Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae). In Handbook of Natural Toxins, Vol 1. Plant and Fungal Toxins (R.F. Keeler, and A.T. Tu, eds.). Marcel Dekker, New York, pp. 43-83. 309. Steyn, P.S., van Heerden. F.R., Vleggar, R., and Anderson, L.A.P. (1986). Bufadienolide glycosides of the Crassulaceae. Structureand sterochemistry of orbicusides A-C, novel toxic metabolites of Cot)lledon orbiculata. J. Chem. SOC.Perkin 1:1633-1636. 3 10. Enslin, P.R., Naudk, T.W.. Potgieter, D.J.J., and van Wyk, A.J. (1966). 1a,2a-Epoxyscillirosidine, the main toxic principle of Homeria glauca (Wood and Evans) N.E.Br. Tetrahedron, 2213213-3220. 311. Cheung, K., Hinds, J.A., and Duffy, P. (1989). Detection of poisoning by plant-origin cardiac glycoside with the Abbott TDx analyzer. Clin. Chenz., 35295-297. 3 12. Paper, D.. and Franz, G. (1989). Glycosylation of cardenolide aglycones in the leaves of Neriunl oleander. Planta Med., 55130-34. 313. Enson, J.M., and Seiber, J.N. (1978). High-speed liquid chromatography of cardiac glycosides in milkweed plants and monarch butterflies. J. Chrom, 148:521-527. 3 14. Martin, R.A., Lynch,S.P., Schmitz, F.J., Pordesimo, E.O., Toth,S., and Horton, R.Y. (1991). Cardenolides fromAsclepias nsperula subsp. capricornu and A. siridis. Phytochemistv, 30: 3935-3939. 315. Price, K.R., and Fenwick, G.R. (1984). Soyasaponin I. a compound possessing undesirable taste characteristics isolated from the dried pea(Pisurn satilwnz L.). J. Sci. Food Agric., 35: 887-892. 3 16. Basu, N., and Rastogi, R.P. (1967). Triterpenoid saponins and sapogenins. Phytochemistry, 6: 1249-1270.

Analytical Methodology for Plant Toxicants

401

3 17. Besson, V., Lavaud, C., Massiot, G., Le Men-Oliver, L., Bianchi, S., Flament, P.,and Dattee, Y. (1989). Medicagenic acidand saponins in callusand suspension culturesof alfalfa. Phytochentistly, 28:1379-1380. 318. Gestetner, B., Birk, Y., and Bondi, A. (1966). Soyabean saponins VI. Composition of carbohydrate and aglycone moietiesof soya bean saponin extractand of its fractions.Phytoclzemistry, 5~799-802. 3 19. Chandel, R.S., and Rastogi, R.P. (1980). Triterpenoid saponinsand sapogenins: 1973-1978. Phytochemistty, 19:1889-1908. 320. Gestetner, B., Assa, Y., Henis, Y., Birk. Y., and Bondi, A. (1971). Lucerne saponins. IV. Relationship between their chemical constitution, and haemolytic and antifungal activities. J. Sci. Food Agric., 22:168-172. 321. Szeleczy, Z., and Ling. T. (1980). Purification and separation of digoxin by removal of accompanying glycosides as phenylboronate derivatives. Planta Med., 40:320-327. 322. Gestetner, B.. Birk, Y., Bondi, A.,and Tencer, Y. (1966). Soyabean saponins. VII. A method for the determination of sapogenin and saponin contents in soya beans. Phytochemistry, 5 : 803-806. 323. Tencer, Y., Shany, S., Gestetner, B., Birk, Y., and Bondi, A. (1972). Titrimetric method for determination of medicagenic acid in alfalfa (Medicago sativa). J. Agric. Food Chem., 20: 1149-1151. 324. Birk. T. (1969). Saponins. InToxic Constituents of Plant Foodstufs (I.E. Leiner, ed.). Academic Press, New York, pp. 169-210. 325. Livingston, A.L., Whitehead, L.C., and Kohler, G.O. (1977). Microbiological assay for saponin in alfalfa products. J. Assoc. Of Anal. Chem., 60:957-960. 326. Brower, L.P., and Moffitt, C.M. (1974). Palatability dynamics of cardenolides in monarch butterfly. Nature (Lond.), 246:280-283. 327. Sady, M.B.,and Seiber, J.N. (1991).Field test for screeningmilkweed latex for cardenolides. J. Natural Prod., 54:1105-1107. 328. Joubert, J.P.J. (1989). Cardiac glycosides. In Toxicants of Plant Origin (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 61-96. 329. Patamalai, B., Hektmancik, E., Bridges, C.H., Hill, D.W., and Camp, B.J. (1990). The isolation and identification of steroidal sapogenins in Kleingrass. Vet. Hum. Toxicol., 32:314-318. 330. Oleszek, W., Jurzysta, M., Ploszynski, M., Colquhoun, I.J., Price, K.R., and Fenwick, G.R. (1992). Zahnic acid tridesmoside and other dominant saponinsfrom alfalfa (Medicago sativa L.) aerial parts. J. Agric. Food Chenz., 40:191-196. 331. Blum, L.M., and Rieders. F. (1987). Oleandrin distribution in a fatality from rectaland oral Neriun~oleander extract administration. J. Anal. Toxicol., 11:219-221. 332. Duffey, S.S., and Scudder, G.G.E. (1972). Cardiac glycosides in North American Asclepiadaceae, a basis for unpalatability in brightly coloured Hemiptera and Coleoptera. J. Imect Plzysiol., 18:63-78. 333. Price, K.R., Curl, C.L., and Fenwick, G.R. (1986). The saponin contentand sapogenol composition of the seed of 13 varieties of legume. J. Sci. Food Agric., 37: 1185-1 191. and Stawinski,T.M.(1977).Mikrobestimmung von 334. Smoczkiewica,M.A..Nitschke,D., spuren von saponingglykosiden in “nicht-saponin fuhren dem” pflanzen material. Mikrochima. Acta, 11597. 335. Oakenfull, D., and Sidhu, G.S. (1989). Saponins. InToxicants of Plarlt Origin (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 97- 141. 336. Kulshreshta, M.J., Kulshreshta, D.K., and Rastogi, R.P. (1972). The triterpenoids. Phytochentistry, l l :2369-238 l . Toxic Constituentsof Plant Foodstufls(LE. Liener, 337. Birk, Y., and Peri, I. (1980). Saponins. In ed.). Academic Press, New York, pp. 161-182. and Dunster, P.J. (1987). The toxicity to cattle and bufadienol338. McKenzie, R.A., Franke, F.P., ide content of six Bryophyllum species. Aust. Vet. J., 64:298-301.

402

Muir

339.

Kitagawa, I., Yoshikawa, M.. Hayashi, T., and Taniyama, T. (1984). Quantitative determination of soyasaponins in soybeans of various origins and soybean products by means of high performance liquid chromatography. Ynkugnku Zasshi, 104275. Oleszek, W., Jurzysta, M., Price, K.R., and Fenwick, G.R. (1990). High-performance liquid chromatography of alfalfa root saponins. J. Chrom., 519: 109-1 16. Damon, B., Dorsaz,A.-C., and Hostettmann, K. (1 984). High-performance liquid chromatography of oleanane saponins. J. Chrortr., 314:441-446. Ireland, P.A., and Dziedzic, S.Z. (1985). Analysis of soybean sapogenins by high-performance liquid chromatography. J. Chrom., 325275-281. Okuyama, T., Takata, M., Takahashi, K., Ishikawa, T., Miyasaka, K., and Kaneyama, N. (1 989). High-performance liquid chromatographic analysis of naturally occuning glycosides and saponins. J. Cl~rom.,466:390-398. Majak, W., Cheng, K.J., Muir, A.D., and Pass, M.A. (1985). Analysis and metabolism of nitrotoxins in cattle and sheep. In Plant To.xicology (A.A. Seawright, M.P. Hegerty, L.F. James, and R.F. Keeler, eds.). Queensland Poisonous Plants Committee, Yeerongpilly, pp. 346-452. Majak, W., and Bose, R.J.(1974). Chromatographic methods for the isolation of miserotoxin and detection of aliphatic nitro compounds. Phytochemistry, 13:1005-1010. Benn, M.H., and Majak, W. (1989). 3-Nitro-l-propyl-beta-~-~ aminaribioside from Astragnllts miser var. serotirlus. Plzytochernistt?, 28:2369-237 1. Majak, W., Lindquist, S.G., and McDiarmid, R.E. (1983). A simple method for purification and determination of miserotoxin. J. Agric. Food Clzent.. 3 1 :650-652. Gnanasunderam, C.. and Sutherland, O.R.W. (1986). Hiptagin and other aliphatic nitro esters in Lotus yeduilculatw. Phvtochen~ist?~, 25:409-4 10. Benn, M.H., McDiannid, R.E., and Majak, W. (1989). In-vitro biotransformation of 3-nitropropanol (miserotoxin aglycone) by horse liver alcohol dehydrogenase. Toxicol. Lett., 47: 165-172. Quinton, D.A., Majak, W., and Hall, J.W. (1989). The effect of cattle grazing on the growth and miserotoxin content of Columbia milkvetch. J. Rnrzge Manage., 42368-371. Majak. W., Benn, M.. McEwan, D., and Pass, M.A. (1992). Three nitropropanoyl esters of glucose from Im1igofe1-nlinnnei. Phytochemistty, 3 12393-2395. Muir, A.D., and Majak, W. (1984). Quantitative determination of 3-nitropropionic acid and 3-nitropropanol in plasma by HPLC. To.xico1. Lett., 20:133-136. Majak. W., and McDiarmid, R.E. (1990). Detection and quantitative determination of 3nitropropionic acid in bovine urine. Tosicol. Lett., 50:213-220. Long, M.. Benn, M., Majak, W., and McDiamid, R. (1992). 3-Nitropropyl glucosides of Astragalus ntiser var. serotinus. Phytochenzistq, 3 1:321-323. Wasseman, R.H. (1978). The nature and mechanism of action of the calcinogenic principle of Solnnto?r mnZaco.xylonand Ccestrum diur~rum,and a comment on Trisetumflnvescens. In ZZfects of Poisonozls Plants on Livestock (R.F. Keeler, K.R. Van Kampen, and L.F. James, eds.). Academic Press, New York, pp. 545-553. Danieli, B., Bombardelli, E., Bonati. A., and Gabetta, B. (1972). Structure of the diterpenoid carboxyatractyloside. Phytochernist?, 11:3501-3504. Marquardt, R.R. (1989). Vicine. convicine, and their aglycones- divicine and isouramil. In Toxicants of Plant Origin (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 161-200. Bonora, A.. Botta, A.. Menziani-Andreoli. E., and Bruni, A. (1 988). Organ-specific distribution and accumulation of protoanemonin in Rnntlnculus jcaria. Biochenl. Plzysiol. Pflancerz. 183: 443-447. Bonora, A., Dall’Olio, G., Donini, A., and Bruni, A. (1987). An HPLC screening of some Italian Ranunculaceae for the lactone protoanemonin. Phytochentistry, 26:2277-2279. Hill. R., and Van Heyningen, R. (1951). Ranunculin: the precursor of the vesicant substance of the buttercup. Biochem. J., 49:332-335.

340. 341. 342.

343.

344.

345. 346. 347. 348. 349.

350. 351. 352. 353. 354. 355.

356. 357. 358.

359. 360.

Methodology Analytical

Toxicants for Plant

403

361. Benn. M.H., and Yelland, L.J. (1968). Ranunculin. Can. J. Clzem., 46:729-732. 362. Bai, Y., Benn, M.H., Majak, W., and McDiarmid, R. (1996). Extractionof HPLC determination of ranunculin in species of the buttercup family. J. Agric. Food Chem., 44:2235-2238. 363. Weissenberg, M.. Maoz,A., Levy, A.. and Wasserman, R.H. (1988). Radioimmunoassay for rapid estimation of vitamin D derivatives in calcinogenic plants. PIanta Med., 54:63-65. 364. Craig. J.C., Mole, M.L., Billets, S., and El-Feraly, F. (1976). Isolation and identification of the hypoglycemic agent carboxyatractylate fromXarztlzitm strtrmarizrm. Phytochemistry, 15: 1178. 365. Olsen, H.S., and Andersen. J.H. (1978). The estitnationof vicine and convicine in fababeans (Vicia faba L.) and isolated fababean proteins. J. Sci. Food Agric., 29:323-33 1. 366. Mager, J., Chevion,M., and Glaser, G. (1980). Favism. In Toxic Constituerztsof Plant Foodstzrfls (LE. Liener, ed.). Academic Press, New York, pp. 265-294. 367. Higazi, M.I., and Read, W.C.A. (1974). A method for the determination of vicine in plant material and in blood. J. Agric. Food Chert!., 22570-571. 368. Marquardt. R.R., and Frohlich, A.A. (1981). Rapid reversed-phase high-performance chromatographic method for the quantitation of vicine, convicine and related compounds. J. Chronz., 208:373-379. 369. Lattanzio, V.. Bianco, V., and Mercier, C. (1982). High-performance reversed-phase liquid chromatography (HPLC) of favism-inducing factors in Vicia fuba L. Experientia, 38:789790. 370. Quemener, B., Gueguen, J., and Mercier, C. (1982). Deternhation of vicine and convicine in fababeans by high pressure liquid chromatography. Can. I m t . Food Sci. Technol. J., 15: 109-1 15. 371. Bonora, A., Dall’Olio, G., and Bruni, A. (1985). Separation and quantitation of protonaemonin in Ranmculaceae by normal- and reversed-phase HPLC. Plant0 Med., 51:364-367. 372. Nachman, R.J.. and Olsen, J.D. (1983). Ranunculin: a toxic constituent of the poisonous range plant bur buttercup (Ceratocephalus testiculatus). J. Agric. Food Clleltt., 31: 13581360. 373. Calmes, M., Crespin, F., Maillard, C., Ollivier, E., and Balansard, G. (1994). High-performance liquid chromatographic determinationof atractyloside and carboxyatractyloside from Atmctylis gurnmifera L. J. Clrront., 663:l 19-122. 374. Wei, H.C., Hartman,F.C., Pfuderer, P.,and Kuang Yang, W. (1974).Purification and characterization of two major toxic proteins from seeds of Abrus precatorius. J. Biol. Chent., 249: 3061-3067. 375. Khan, A.H., Hashmi, M.A.,and Khan, M.I. (1961). Studieson Abrus yrecatorizrs Linn. Part I. Isolation and toxic properties of “abrulin,” a protein fraction from the seeds. Pak. J. Sci. Iad. Res., 4:53-56. 376. Ochoa, J.-L.,and Kristiansen, T. (1978). Stroma:As an affinity adsorbent for non-inhibitable lectins. FEBS Lett., 90:145-148. 377. Olsnes, S., Stirpe, F., Sandvig, K., and Pihl, A. (1982). Isolation and characterization of viscumin, atoxic lectin fromVisczvn album L. (mistletoe).J. Biol. Clzern., 257: 13263-1 3270. 378. Khang, N.Q., Jean-Luc, G., and Johan. H. (1990). A blood group A specific lectin from the seeds of Crotalarin striata. Biochim. Biophys. Acta, 1033:210-213. 379. Grant, G.. More, L.J., McKenzie, N.H., Stewart, J.C., and Pusztai, A. (1 983). A survey of the nutritional and haemagglutination properties of legume seeds generally available in the UK. Br. J. N m . , 50:207-214. 380. Talbort, C.F., and Etzler, M.E. (1978). Development and distribution of Dolichos bij7orus lectin as measured by radioimmunoassay. Plant Plzysiol., 61 :847-850. 381. Aplin, T.E.H., Steele.P., and Nottle, M.C. (1983).Toxic Ferns I n Western Australia. Department of Agsiculture, South Perth. Western Australia. 382. Melnick, D., and Field, H. (1939). Chemical determination, stability and form of thiamine in urine. J. Biol. Chenr.. 130:97-107.

404

Muir

383. Edwin, E.E., and Jackman, R. (1974).A rapid radioactive method fordetermination of thiaminase activity and its use in the diagnosis of cerebrocortical necrosis in sheep and cattle. J. Sci. Food Agric., 25:357-368. 384. Padmanaban, G. (1980). Lathyrogens.In Toxic Comtituents of Plant Foodstufls (I.E. Liener, ed.). Academic Press, New York, pp. 239-263. In ToxicSubstances in CropPlants (J.P.F. 385. D’Mello,J.P.F.(1991).Toxicaminoacids. D’Mello, C.M. Duffus, and J.H. Duffus, eds.). Royal Chemical Society, Cambridge,pp. 2248. 386. Kean, E.A. (1989). Hypoglycin. In Toxicants of Plant Origin. Vol. 3. Proteins cmd Anfirlo Acids (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 229-262. 387. Roy, D.N. (1981). Toxic aminoacids and proteins fromLathyrzds plants and other leguminosus species: a literature review. Nutr. Abstr. Rev. Ser. A: Hum.Exp., 51:691-707. 388. Xavier-Filho, J., and Campos, F.A.P. (1989). Proteinase inhibitors. In Toxicants of Plant Urigirl (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 1-28. 389. Lambein, F.,and Van Parijs, R. (1974).New isoxazolinone aminoacids from Lnthyrus odoratus. Biochem. Biopkys. Res. Commun., 6 1:155. assay of some alfalfa varieties(Medicago 390. Natelson, S,, and Bratton, G.R. (1984). Canavanine sativa) by fluorescence: practical procedure for canavanine preparation.Microchem. J., 29: 26-45. 391. Matsumoto, H., and Sherman, D.G. (1951). Arapid colorimetric method forthe determination of mimosine. Arch. Biochem. Biophys., 33:195-200. Leuclzenn in leaves. 392. Megarrity, R.G. (1978). An automated colorimetric method for mimosine J. Sci. Food Agric., 29: 182- 186. Associations of amino acidsand related compounds 393. Bell, E.A., and Tirimanna, A.S.L. (1965). in the seeds of forty-seven species of Vicia: their taxonomic and nutritional significance. Biochem. J., 97:104-111. of the neurotoxin P-N-oxalyl-a$-diamio394. Kuo, Y.-H., and Lambein, F. (1991). Biosynthesis propionic acid in callus tissue of Lathyrus sativus. Phytochemistry, 30:3241-3244. of free 395. Furst, P., Pollack, L., Graser, T.A., Godel, H., and Stehle, P. (1989). HPLC analysis amino acidsin biological material-an appraisal of four pre-column derivatizationmethods. J. Liquid Chrom., 12:2733-2760. 396. Roy, D.N., and Spencer, P.S. (1989). Lathyrogens. In Toxicants of Plant Origin, Vol. 3, Proteirzs and Amino Acids (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 169-201. 397. Cohen, S.A., and Strydom, D.J. (1988). Amino Acid analysis utilizing Phenylisothiocyanate derivatives. Anal. Biochem., 174:l-16. 398. Acamovic, T., and D’Mello, J.P.F. (1990). HPLC analysis of canavanine and canaline in Cannvnlia ensiformis, and in excreta and serum of chicks. J. Sci. Food Agric., 50:63-77. 399. Acamovic, T., D’Mello, J.P.F.,and Fraser, K.W. (1982).Determination of mimosine and 3hydroxy-4( lH)-pyridone in Leucaena, avian excreta and serum using reversed-phase ionpair high performance liquid chromatography.J. Clzronz., 236: 168-179. 400. Tangendjaja, B., and Willis, R.B.H. (1983). Analysis of mimosine and 3-hydroxy-4-(1H)pyridone in plasma by high-performance liquid chromatography.J. Chrom., 265: 143-144. 401. Abrahams, D.E., and Kean, E.A. (1969). HypoglycinA in biological material: its detection and assay. West Indian Med. J., 18: 147-151. 402. Sarwar, G.,and Botting, H.G. (1994).Reversed-phase liquid chromatographicdetermination of hypoglycin A (HG-A) in canned Ackee fruit samples. J. AOAC Int., 77:1175-1179. 403. Dou, L., Mazzeo, J., and Krull, I.S. (1990). Determination of amino acids, peptides and proteins by HPLC combined with electrochemical detection.Biochromatography, 5:74-96. 404. Smith, T.A. (1977). Phenethylamine and related compounds in plants. Phytochemistn, 16: 9-18. 405. Camo, B.J., and Novell, M.J. (1966). The phenylethylamine alkaloids of native range plants. Ecot1. Bot., 201274-278.

Analytical Methodology for Plant Toxicants

405

406. Eichholzer, J.V.. Lewis, I.A.S.. MacLeod, J.K., Oelrichs, P.B.,and Valley, P.J. (1982). Galegine and anew dihydroxyalkylacetamide fromVerbesina enceloiodes.Plzytochemistnl, 21 : 97-99. 407. Coulman, B.E., Woods, D.L., and Clark, K.W. (1977). Distribution within the plant, variation with maturity and heritability of gramine and hordenine in reed canarygrass. Can. J. Plant Sci., 57:771-777. 408. Ishida, J., Yamaguchi, M., and Nakamura, M. (1990). High-performance liquid chromatographic determination of P-phenylethylamine in human plasma with fluorescence detection. Anal. Biochenr., 184:86-89. 409. Whanger, P.D. (1989). Selenocompounds in plants and their effects on animals. In Toxicants of Plrcnt Origin, Vol. 3, Proteins and Amino Acids (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 141-167. 410. Olsen, O.E., Oalmer, IS., and Cary. E.E. (1975). Modification of the official fluorometric method for selenium in plants. J. Assoc. 08 Anal. Chem., 58:117-121. 41 1. Rodriguez, E., Towers, G.H.N., and Mitchell, J.C. (1976). Biologicalactivities of sesquiterpene lactones. Phytoche~nistry.15:1573-1580. 412. Kim, H.L., and Calhoun, M.C. (1986). Quantitation of hymenoxon and related toxicants in plants samples.In Sheep Goat Wool Mohair. Texas Ag. Exp.Stn., College Stn, TX, pp. 21-22. 413. Saito, K., Nagao, T., Matoba, M., Koyama, K., Natori, S.,Murakami, T., and Saiki, Y. (1989). Chemical assay of ptaquiloside, the carcinogenof Pteridium aquilinum, and the distribution of related compounds in the Pteridaceae.Phytochemistry, 28: 1605- 1611. 414. Niwa, H., Ojika, M., Wakamatsu, K., Yamada, K., Hirono, I., and Matsushita, K. (1983). Ptaquiloside, a novel norsesquiterpene glucoside from bracken, Pteridium aquilinum var. latiusculum. Tetrahedron Lett., 24:4117-4120. 415. Hirono, T. (1989). Carcinogenic bracken glycosides. In Toxicants of Plant Origin, VoZ. 2, Glycosides (P.R. Cheeke, ed.). CRC Press, Boca Raton, Fla., pp. 239-251. 416. Agnew. M.P.,and Lauren, D.R. (1991). Determination of ptaquiloside in bracken fern(Pteridium esculentunt). J. Chronz., 538:462-468. 417. Picman, A.K., Ranieri, R.L.. Towers, G.H.N.. and Lam, J. (1980). Visualization reagents for sesquiterpene lactones and polyacetylenes on thin layer chromatograms. J. Chrom., 189:187198. 418. Hill, D.W.. Kim, H.L., and Camp, B.J. (1979). Quantitative analysisof hymenoxon in plant tissue. J. Agric. Food Chem., 27:885-887. 419. Kinghorn, A.D. (1985). Skin-irritant and tumor-promoting compounds of plants of the Euporbiaceae. In Plant Toxicology (A.A. Seawright, M.P. Hegarty, L.F. James, and R.F. Keeler, eds.). Dominion Press-Hedges & Bell, Melbourne, pp. 357-366. and analysis 420. Scott, P.M., Coldwell, B.B., and Wiberg, G.S. (1971). Graynotoxins. Occurrence in honey and a comparison of toxicities in mice. Food Cosmet. Toxicol., 9:179-184. of Australian Pimelea spp. 421. Freeman, P.W.,E., R., and Taylor, W.C. (1979). The constituents I. The isolation and structure of the toxin of Pirnelea simplex and P. trichostachya Form B responsible for St. George disease of cattle. Aust. J. Chenz., 32:2495-2506. 422. Burke, J.W., and Dostotch, R.W. (1990). Highfield 'H- and "C-NMR assignments of grayanotoxins I, IV, and XIV isolated from Knlmia angust(fo1ia. J. Natural Prod., 53: 131-137. of croton 423. Marshall, G.T., and Kinghorn, A.D. (1984). Short-chain phorbol ester constituents oil. J. Am. Oil Chew SOC.,61 :1220-1225. 424. Smith, R.A., and Lewis, D. (1987). Cicuta toxicosis in cattle: case history and simplified analytical method. Vet. Hum. Toxicol, 29:240-241. and Trippett, S. (1953). Oenanthotoxinand cicutoxin. 425. Anet, E.F.L.J., Lythgoe, B., Silk, M.H., Isolation and structures. J. Chem. Soc., 155:309-322. 426. Hegarty, B.F., Kelly, J.R., Park, R.J., and Sutherland, M.D. (1970). Triterpenid chemistry. XVII. (-)-Ngaione, a toxic compound of Myoporurn deserti. The absolute configuration of (-)-ngaione. Aust. J. Chem., 23:107-117.

406 427.

428. 429. 430.

431. 432. 433.

434. 435. 436.

437. 438. 439. 440.

441. 442.

443.

444.

445. 446.

447.

448.

Muir Ng, J.C., Venzke, B.N.. and Seawright, A.A. (1985). Furanoid urinary metabolites of (-1ngainone in the rat. In Plant To.uko1ogy (A.A. Seawright, M.P. Hegarty. L.F. James, and R.F. Keeler. eds.). Dominion Press-Hedges & Bell, Melbourne, pp. 512-51 3. Oelrichs, P.B.. Hill, M.W.. Valley, P.J., MacLeod, J.K.. and Molinski, T.F. (.1983). Toxic tetranortriterepenes of the fruit of Melia uzedarach. Phytoclzentistry, 2253 1-534. Wood, G.. and Huang,A. (1975). The detection and quantitative determination of ipomeamorne in damaged sweet potatoes (Zponzoea batatas). J. Agric. Food Chem.. 23239-241. Pass, M.A., Pollitt, S., and Gossem, M.W. (1985). The pathogenesis of Lantrma poisoning. In P l m Toxicology (A.A. Seawright, M.P. Hegarty. L.F. James. and R.F. Keeler, eds.). Dominion Press-Hedges & Bell, Melbourne, pp. 487-494. Allender, W.J. (1990). Determination of sodium furoacetate (compound 1080) in biological tissues. J. Annlyt. To.xico1.. 14:45-49. Dickle, C.W.. Gerlach, M.L., and Hamar. D.W. (1989). Koclziascopcrria oxalate content. Vet. Hum. Toxicol., 31240-242. Muir. A.D.. and Soroka, J.J. (1992). HPLC-Ion chromatographic analysisof nitrate in plant tissues and its application to sweetclover (Melilotrts spp.) seedlings. J. Agric. Food Chen?., 40: 1602- 1605. Diagnostics. S., Procedure 590. Sigma Chemical Company, St. Louis. Martz, F.A., Weiss, M.F., and Belyea, R.L. (1990). Determination of oxalate in forage by reverse-phase high pressure liquid chromatography. J. Dai? Sci.. 73:474-479. Cataldo, D.A., Haroon. M., Schrader, L.E.. and Youngs, V.L. (1975). Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Comnzun. Soil Sci. Plaizt Anal., 6:71-80. Tanaka, A., Nose, N., and Iwasaki, H. (1982). Spectrophotometric determination of nitrate in vegetable products using 2-sec-butylphenol. Aiztdyst, 107:190- 194. Paul, J.L., and Carlson, R.M. (1968). Nitrate determination in plant extracts by the nitrate electrode. J. Agric. Food Chem., 16:766-768. Singh, J.P. (1988). A rapid method for determination of nitrate in soil and plant extracts. Plant Soil, 110:137-139. Kalbasi, M., and Tabatabai, M.A. (1985). Simultaneous determination of nitrate, chloride, sulfate, and phosphate in plant material by ion chromatography. Comnzun. Soil Sci. Plarzt And., 16~787-800. Gupta, D., and Joshi, B.C. (1983). Gas chromatographic analysis of low molecular weight alkyl disulfides. Chem. Anal. (Warsaw), 28:469-473. Moeckel, H.J., Freyholdt, T., Weiss, J., and Molnar. I. (1983). The HPLCof divalent sulfur. Elemental sulfur rings and nonionic sulfur compounds. Pmct. In Aspects Mod. High Pe$orrnance Liq. Chonzatogr. Proc. (I. Molnar, ed.). de Gruyter, Berlin, pp. 161-185. Lawson, L.D., Wang, Z.Y.,and Hughes, G.B. (1991). Identification and HPLC quantification of the sulfides and dialk(en)yl thiosulfinates in commercial garlic products. Plunta Med.. 57: 363-370. Allender, W.J. (1990). The determination of sodium monofluroacetate (compound 1080) in formulation and technical samplesby high pressure liquid chromatography. J. Liquid Clzront., 13:3465-3471. Kramer, H.L. (1984). Liquid chromatographic determination of sodium fluroacetate (compound 1080) in meat baits and formulations. J. Assoc. Of Anal. Chen?., 67:1058-1061. Livanos, G., and Milham, P.J. (1984). Fluoride ion-selective electrode determination of sodium monofluroacetate in meat baits and formulations. J. Assoc. OffAnal. Chem.. 67:lO12. Stoeloff. L., Nesheim, S., Yin, L.. Rodricks, J.V., Stack, M., and Campbell, A.D. (1971). A multimycotoxin detection method for aflatoxin zearaleone, sterigmatocystin and patulin. J. Assoc. OffAnal. Chenz., 5:91-97. Gimeno, A. (1979). Thin layer chromatography determination of aflatoxins, ochratoxins,ste-

Analytical Methodology for Plant Toxicants

449.

450. 451. 452.

453.

454.

455.

456.

457.

458.

459.

460.

461.

462.

463.

464. 465.

466.

407

rigmatocystin, zeralenone, citrinin, T-2 toxin, diacetoxycirpenol, penicillic acid, patulin and penitrem A. J. Assoc. Qf A n d . Cllem., 62:579-585. Reiss. J. (1973). N-rnethylbenzthiazolone-2hydrazone (Besthorns hydrazone) as a sensitive spray reagent for patulin. J. Clzronz.. 86: 190-191. Gilbert, J. (1993). Recent advances in analytical methods for mycotoxins. Food Addit. Contamin., 10:37-48. Scott, P.M., and Trucksess. M.W. (1997). Application of imnlunoaffinity columns to rnycotoxin analysis. J. AOAC Int., 80:941-949. Scudamore, K.A., Hetmanski, M.T., Nawaz, S., Naylor, J., and Rainbird, S. (1997). Determination of mycotoxins in pet foods sold for domestic pets and wild birds using linked-column immunoassay clean-up and HPLC. Food Addit. Conturnin., 14:175-186. Seidel, V., Poglits, E., Schiller, K., and Lindner, W. (1993). Simultaneous determination of ochratoxin A and zearalenone in maize by reversed-phase high-performance liquid chromatography with fluorescence detection and P-cyclodextrin as mobile phase additive. J. Chronl., 635:227-235. Dunne. C., Meaney, M., Smyth, M., and Tuinstra, L.G.M.T. (1993). Multimycotoxin detection and clean-up method for aflatoxins, ochratoxin and zearalenone in animal feed ingredients using high-performance liquid chromatography and gel permeation chromatography. J. Clzrorn., 6'9229-235. Soares, L.M.V. (1 992). Multi-toxin TLC methods for aflatoxins, ochratoxin A, Zearalenone and sterigmatocystin in foods. In Modern Metlzods of Plant Arzalysis (H.F. Linskens, and J.F. Jackson, eds.). Springer-Verlag, Berlin, pp. 327-1538. Wu. P.-Y., Yen, Y.-H., and Chiou, R.Y.-Y. (1995). Extraction of aflatoxin from peanut meal and kernels by C02-methanol using a highly pressurized fluid extraction process. J. Food Prot.. 58:800-803. Taylor, S.L., King, J.W., Richard, J.L., and Greer. J.I. (1993). Analytical-scale supercritical fluid extraction of aflatoxin B I from field-inoculated corn. J. Agric. Food Clzem., 41:910913. Mortimer, D.N., Glibert, J., and Shepherd. M.J. (1987). Rapid and highly sensitive analysis of aflatoxin M , in liquid and powdered milks using affinity column cleanup. J. Clzl-om.,407: 393-398. Tuinstra, L.G.M.T.. Roos, A.H., and van Trijp, J.M.P. (1993). Liquid chromatographic determination of aflatoxin M in milk powder using immunoaffinity columns for cleanup: Interlaboratory study. J. AOAC Int., 76:1248-1254. Femindez, A., Verde, M.T.. Gascbn, M., Ramos, J.J., and Gbmez, J. (1994). Aflatoxin and its metabolites in tissues from laying hens and broiler chickens fed a contaminated diet. J. Agric. Food Chem., 65:407-414. Tuinstra, L.G.M.Th., and Haasnoot. W. (1983). Rapid determination of aflatoxin B in Dutch feedingstuffs by high performance liquid chromatography and post-column derivatization. J. Clll-0111.,282~457-462. Kok, W.T.. van Neer. C.H.T., Traag. W.A., and Tuinstra, L.G.M.Th. ( 1986). Determination of aflatoxins in cattle feed by liquid chromatography and post column derivatization with electrochemically generated bromide. J. Chi-om.. 36723 1-236. Cepeda, A.. Franco, C.M., Fente, C.A., Viizquez, B.I., Rodriguez, J.L., Prognon, P., and Mahuzier, G. (1996). Postcolumn excitation of aflatoxins using cyclodextrins in liquid chromatography for food analysis. J. Clzronz.. 721:69-74. Plattner, R.D., Bennett, G.A., and Stubblefield. R.D. (1984). Identification of aflatoxins by quadrupole mass spectrometry/mass spectrometry. J. Assoc. OffAnal. Chem., 67:734-738. Nesheim, S., Stack, M.E.. Trucksess, M.W., Eppley, R.M., and Krogh, P. (1 992). Rapid solvent-efficient method for liquid chromatographic determination of ochratoxin A in corn, barley. and kidney: collaborative study. J. AOAC Int., 75:481-487. Biancardi, A.. and Riberzani, A. (1996). Determination of ochratoxin A in cereals and feed

408

467. 468. 469. 470.

471.

472.

473.

474. 475. 476.

477.

478. 479.

480.

481.

482.

483.

484.

Muir by SAX-SPE clean upand LC fluromimetric detection. J. Liq. Clzronz. Rel. Technol., 19: 2395-2407. Studer-Roher, I., Dietrich, D.R., Schlatter, J., and Schlatter, C. (1995). The occurrence of ochratoxin A in coffee. Food Cltem. Toxicol., 5:341-355. Valenta, H. (1998). Chromatographic methods for the determination of ochratoxin A in animal and human tissues and fluids. J. Chront., 815:75-92. Solfrizzo, M., Avantaggiato, G.,and Visconti, A. (1998). Useof various clean-up procedures for the analysis of ochratoxin A in cereals. J. Chrom., 815:67-73. Sharman, M., MacDonald, S., and Gilbert, J. (1992). Automated liquid chromatographic determination of ochratoxin A in cereals and animal products using immunoaffinity column clean-up. J. Chrorn., 603:285-289. Nakajima,M.,Terada, H., Hisada, K., Tsubouchi, H., Yamamoto, K., Uda, T., Itoh, Y . , Kawamura. O., and Ueno, Y. (1990). Determination of ochratoxin A in coffee beans and coffee products by monoclonal antibody affinity chromatography. Food Agric. Imnunol., 2: 189-195. Pittet, A., Tornare, D., Huggett, A., and Viani, R. (1996). Liquid chromatographic determination of ochratoxin A in pure and adulterated soluble coffee usingan immunoaffinity column cleanup procedure. J. Agric. Food Chent., 44:3564-3569. E., and Kitabatake, N. (1993). Formation of a new toxic Trivedi, A.B.. Hirota, M., Doi, compound, citrininH1, from citrinin on mild heating in water. J. Chent. Soc. Perkirt, 1:21672171. Abramson, D., Usleber, E., and Miirtlbauer, E. (1996). Determination of citrinin in barley by indirect and direct enzyme immunoassay. J. AOAC Int., 79:1325-1329. Abramson, D., Usleber, E., and Miirtlbauer, E. (1995). An indirect enzyme immunoassay for the mycotoxin citrinin. Appl. E n v i r o ~Microbiol., 612007-2009. Clarke, J.R., Marquardt, R.R., Oosterveld. A., Frohlich, A.A., Madrid, F.J., and Dawood, M. (1993). Developmentof a quantitative and sensitive enzyme-linked immunosorbent assay for ochratoxin A using antibodies from the yolk of the laying hen. J. Agric. Food Clzeln., 41:1784-1789. Clarke, J.R., Marquardt, R.R., Frohlich, A.A., and Pitura, R.J. (1994). Quantification of ochratoxin Ain swine kidneysby enzyme-linked immunosorbent assay using a simplified sample preparation procedure. J. Food Prot.. 57:99 1-995. Gyongyosi-Horvith, A., Barna-Vetr6, I., and Solti, L. (1996). A new monoclonal antibody detecting ochratoxin A at the picogram level. Lett. Appl. Microbiol., 22:103-105. Vizquez, B.I., Fente, C., Franco, C., Cepeda, A., Prognon, P., and Mahuzier, G. (1996). Simultaneous high-performance liquid chromatographic determinationof ochratoxin A and citrinin in cheese by time-resolved luminescence using terbium. J. Chrow?.,727:185-193. Franco, C.M., Fente, C.A., Vazquez, B., Cepeda, A., Lallaoui, L., Prognon, P., and Mahuzier, G.(1996).Simple and sensitivehigh-performanceliquidchromatography-fluorescence method for the determination of citrinin. Application to the analysis of fungal cultures and cheese extracts. J. Chrom., 723:69-75. Terada, H., Tsubouchi, H., Yamamoto, K., Hisada, K., and Sakabe, Y . (1986). Liquid chromatographic determination of ochratoxin A in coffee beans and coffee products. J. Assoc. OffAnal. Chewz., 69:960-964. Beker, D.,and Radic, B. (1991). Fast determination of ochratoxin A in serum by liquid chromatography: comparison with enzymic spectrofluorimetric method. J. Chrom., 570:441445. Jiao, Y . , Blaas, W., Ruhl, C., and Weber, R. (1992). Identification of ochratoxin A in food samples by chemical derivatization and gas chromatography-mass spectrometry. J. Chronz., 5951364-367. Zimmerli, B., Dick, R.,and Baumann, U. (1989). High-performance liquid chromatographic

Analytical Methodology for Plant Toxicants

485. 486.

487. 488.

489. 490. 491. 492.

493.

494. 495.

496. 497.

498. 499.

500.

501.

502.

503.

409

determination of citrinin in cereals usingan acid-buffered silica gel column.J. Chrom., 462: 406-410. Betina, V. (1989). Chromatographic methods as tools in the field of mycotoxins. J. & - o m . , 4771187-233. Vail, R.B., and Homann, M.J. (1990). Rapid and sensitive detection of citrinin production during fungal fermentation using high-performance liquid chromatography.J. Clzrom., 535: 317-323. Lansden, J.A. (1986). Determinationof cyclopiazonic acid in peanuts and corn by thin layer chromatography. J. Assoc. Of Anal. Chenl., 69:964-966. Norred, W.P., Cole, R.J., Dorner, J.W., and Lansden, J.A. (1987). Liquid chromatographic determination of cyclopiazonic acid in poultry meat. J. Assoc. OffAnal. Chem., 70:121123. Dorner, J.W., Cole, R.J., Erlington, D.J., Suksupath,S., McDowell, G.H., and Bryden, W.L. (1994). Cyclopiazonicacid residues in milkand eggs. J. Agric. Food Chem., 42: 1516- 15 18. Hahnau, S., and Weiler, E.W. (1991). Determination of the mycotoxin cyclopiazonic acid by enzyme immunoassay. J. Agric. Food Chem., 39: 1887-1891. Le Bars. J. (1979). Cyclopiazonic acid production by Penicillium camemberti Thom and natural occurrence of this mycotoxin in cheese. Appl. Emiron. Microbiol., 38:1052-1055. Goto, T., Shinshi, E., Tanaka, K., and Manabe, M. (1987). Analysis of cyclopiazonic acid by normal phase high-performance liquid chromatography. Agric. Biol. Chern., 5 1 :258 1 2582. Huang, X., and Chu, F.S. (1993). Production and characterization of monoclonal and polyclonal antibodies against the mycotoxin cyclopiazonic acid. J. Agric. Food Chem., 41:339333. Hahnau, S.. and Weiler, E.W. (1993). Monoclonal antibodies for the enzyme immunoassay of the mycotoxin cyclopiazonic acid. J. Agric. Food Chem., 41:1076-1080. Plattner, R.D., Norred. W.P., Bacon, C.W.,Voss, K.A., Peterson, R., Shackelford. D.D.,and Weisleder. D. (1990). A method of detection of fumonisins in corn samples associatedwith field cases of equine leukoencephalomalacia. Mycologin, 82:698-702. Selim, M.I., El-Sharkawy, S.H., and Popendorf, W.J. (1996). Supercritical fluid extraction of fumonisin B l from grain dust. J. Agric. Food Clzenz., 44:3224-3229. Rottinghaus, G.E., Coatney, C.E.,and Minor, H.C. (1992). A rapid, sensitive thin layer chromatography procedure for the detection of fumonisin B and B?. J. Vet. Diagn. Invest., 4: 326-329. Hopmans, E.C., and Murphy, P.A. (1993). Detection of fumonisins Bl, B?, and B, and hydrolyzed fumonisin B I in corn-containing foods. J. Agric. Food. Chem., 41: 1655-1 658. Shephard, G.S., Thiel, P.G., and Sydenham, E.W. (1992). Determinationof fumonisin B I in plasma and urine by high-performance liquid chromatography. J. Clzrom., 574:299-304. Azcona-Olivera, J.I., Abouzied, M.M., Plattner, R.D.,and Pestka, J.J. (1992). Productionof monoclonal antibodies to the mycotoxins fumonisins B B,. and B3. J. Agric. Food Chenz., 40:531-534. Abouzied, M.M., Askegard, S.D., Bird, C.B., and Miller, B.M. (1996). Fumonisins Veratoxo. A new rapid quantitative ELISA for determination of fumonisin in food and feed. In Fumorzisins in Food (L.S. Jackson, J.W. DeVries, and L.B. Bullerman, eds.). Plenum Press, New York, pp. 135-144. Dreher, R.M., and Usleber, E. (1996). Comparison study of a fumonisin enzyme immunoassay and high-performance liquid chromatography. In Immunoassays for Residue Analysis (R.C. Beier and L.H. Stanker, eds.). ACS, Washington, DC, pp. 341-348. Trucksess, M.W.,and Abouzied, M.M. (1996). Evaluationand application of immunochemical methods for fumonisin B in corn. In Zntrtzunoassays for Residue Analysis (R.C. Beier and L.H. Stanker, eds.). ACS, Washington, DC, pp. 358-367.

410

Muir

504. Shephard, G.S. (1998). Chromatographic determination of the fumonisin mycotoxins. J. ChrOl??.,815131-39. 505. Cawood, M.E., Gelderblom, W.C.A., Vleggaar, R., Behrend, Y., Thiel, P.G., and Marasas, W.F.O. (1991). Isolation of the fumonisin mycotoxins: a quantitative approach. J. Agric. Food Chem., 39:1958-1962. 506. Holcomb, M., Thompson, H.C., and Hankins, L.J. (1993).Analysis of fumonisin B I in rodent feed by gradient elution HPLC using precolumn derivatization with FMOC and fluorescence detection. J. Agric. Food Cltenl., 41:764-767. 507. Veliizquez, C., van Bloemendal, C., Sanchis, V., and Canela, R. (1995). Derivation of fumonisins B I and B? with 6-aminoquinolyl N-hydroxysuccinimidylcarbamate. J. Agric. Food Chen?., 43:1535-1537. 508. Wilkes, J.G.. Chruchwell, M.I., Billedeau, S.M., Vollmer, D.L., Volmer, D.A., Thompson. H.C., and Lay. J.O. (1996). Determination of underivatized fumonisin B, and related compounds by HPLC. In Fur~zonisinsin Food (L.S. Jackson, J.W.DeVries, and L.B. Bullerman, eds.). Plenum Press, New York, pp. 93-103. 509. Korfmacher, W.A., Chiarelli, M.P., Lay,J.O., Bloom, J., and Holconlb, M. ( 199 1).Characterization of the mycotoxin fumonisin B comparison of thermospray, fast-atom bombardment and electrospray mass spectometly. Rapid Conmlun. Mass Specti-om., 5:463-468. 5 10. Churchwell. M.I., Cooper, W.M., Howard, P.C.. and Doerge, D.R. (1997). Determination of fumonisins in rodent feed using HPLC with electrospary mass spectrometric detection. J. Agric. Food Chem., 452573-2578. 511. Maragos, C.M. (1995). Capillary zone electrophoresis and HPLC for the analysis of fluorescein isothiocyanate-labelled fumonisin B J. Agric. Food Clzem., 43:390-394. 512. Clark, C.A., A., L., and Martin, F.A. (1981). Accumulation of furanoterpenoids in sweet potato tissue following inoculation with different pathogens. Phytopathology, 71:708-711. 513. Hyodo, H., Uritani, I., and Akai, S. (1969). Production of furanoterpenoids and other compounds in sweet potato root tissue in response to infection by various isolates of Ceratocystis j k h - i a t a . Phytoputhol. Z.. 65:332-340. 5 14. Oguni, I.. Oshima, K., Imaseki, H.. and Uritani, I. (1969). Biochemical studies on the terpene metabolism in sweet potato root tissue with black rot; effect of C10- and C15 terpenols on acetate-2- I4C incorporation into ipomeamarone. Agric. Bioi. Chenz., 33:50-62. 515. Gennaro, M.C., Giacosa, D., Gioannini. E., and Angelino, S. (1997). Hallucinogenic species in Amanita muscaricr. Determination of muscimol and ibotenic acid by ion-interaction HPLC. J. Liq. Ch-om. Rel. Technol., 20:413-424. 5 16. Oubrahim, H., Richard, J.-M., Cantin-Esnault, D., Seigle-Murandi, F., and TrCcourt, F. (1997). Novel methods for identification and quantification of the mushroom nephrotoxin orellanine. Thin-layer chromatography and electrophoresis screening of mushrooms with electron spin resonance determination of the toxin. J. Clzron?.,758:145-157. 517. Cantin. D., Richard, J.-M., and Alary, J. (1989). Chromatographic behaviour and determination of orellanine, a toxin from the mushroom Cortinarins orellanus. J. Cl?ronz.,478231237. 518. Brause. A.R., Trucksess, M.W.. Thomas, F.S., and Page. S.W. ( 1996). Determination of patulin in apple juice by liquid chromatography: collaborative study. J. AOAC Zw., 79:451-355. 5 19. Rovira, R., Ribera, F., Sanchis, V., and Canela. R. (1993). Improvements in the quantitation of patulin in apple juice by high-performance liquid chromatography. J. Agric. Food Cher~r., 41:214-216. 520. Bartolomi, B., Bengoechea, M.L., PCrez-Ilzarbe, F.J., Hernindez, T., Estrella, I., and G6mezCordovis, C. (1994). Determination of patulin in apple juice by high-performance liquid chromatography with diode-array detection. J. Chrom., 664:39-43. 521. F’rieta, J., Moreno, M.A., Blanco, J.L., Suiirez, G., and Dominguez. L. ( 1992). Determination of patulin by diphasic dialysis extraction and thin-layer Chromatography. J. Food Plot., 5 5 : 1001-1002.

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41 1

522. Goknlen, V., and Acar, J. (1996). Rapid reversed-phase liquid chromatographic determination of patulin in apple juice. J. Chrorn., 73053-58. 523. Herry, M.P., and Lemitayer, N. (1996). Liquid chromatographic determination of patulin in French apple ciders. J. AOAC Znt., 79: 1107-1 110. 524. Trucksess, M.W., Nesheim, S., and Eppley. R.M. (1983). Thin layer chromatographic determination of deoxynivalenol in wheat and corn. J. Assoc. OffArzrrl. Clzem., 67:40-43. 525. Tacke, B.K., and Casper, H.H. (1996). Determination of deoxynivalenol in wheat. barley, and malt by column cleanup and gas chromatography with electron capture detection. J. AOAC Int., 79:472-475. 526. Wolf-Hall. C.E., and Bullerman, L.B. (1996). Comparison of thin-layer chromatography and an enzyme-linked inmunosorbent assay for detection and quantification of deoxynivalenol in corn and wheat, J. Food Prot., 59:438-440. 527. Eppley, R.M., Trucksess, M.W.. Nesheim, S., Thorpe, C.W., Wood, G.E., and Pohland, A.E. (1984). Deoxynivalenol in winter wheat: thin layer chromatographic method and survey. J. Assoc. OffA n d . Cllenz., 67:43-45. 528. Park, J.J., and Chu. F.S. (1996). Assessment of inmunochemical methods for the analysis of tricothecene mycotoxins in naturally occurring moldy corn. J. AOAC Int.. 79:465-471. 529. Sinha, R.C., Savard, M.E.. and Lau, R. (1995). Production of monoclonal antibodies for the specific detection of deoxynivalenol and 15-acetyldeoxynivalenol by ELISA. J. Agi-ic. Food Cl~ent.,43: 1740-1743. 530. Sinha. R.C., and Savard, M.E. (1996). Comparison of immunoassay and gas chromatography methods for the detection of the mycotoxin deoxynivalenol in grain samples. Cm. J. Plant Pothol., 181233-236. 531. Madhyastha, M.S., Marquardt, R.R., Frohlich, A.A., and Borsa, J. (1994). Optimization of yeast bioassay for trichothecene mycotoxins. J. Food Prot., 57:490-495. 532. Malone. B.R.. Humphrey, C.W., Romer. T.R., and Richard, J.L. (1998). One-step solid-phase extraction cleanup and fluorometric analysis of deoxynivalenol in grains. J. AOAC bzt., 81: 448-452. 533. Ware, G.M., Carman, A., Francis, 0..and Kuan, S. (1984). Gas chromatographic determination of deoxynivalenol in wheat with electron capture detection. J. Assoc. OffAnal. Chern., 67:73 1-734. 533. Scott, P.M., Kanhere. S.R.. and Tarter. E.J. (1986). Deternlination of nivalenol and deoxynivalenol in cereals by electron-capture gas chromatography. J. Assoc. OffA ~ n l Cl~em., . 69: 889-893. 535. Walker, F.. and Meier. B. (1998). Determination of the Fusarium mycotoxins nivalenol, deoxynivalenol. 3-acetyldeoxynivalenol, and 15-0-acetyl-4-deoxynivalenolin contaminated whole wheat flour by liquid Chromatographywith diode array detection and gas chromatography with electron capture detection. J. AOAC Znf.. 81:741-748. 536. Visconti, A.,and Bottalico, A. (1983). Detection of Fusarium trichothecenes (nivalenol. deoxynivalenol, fusarenone and 3-acetyldeoxynivalenol) by high performance liquid chromatography. Chi-omntogmphia, 17:97-100. 537. Lauren, D.R.. and Greenhalgh, R. (1987). Simultaneous analysis of nivalenol and deoxynivalenol in cereals by liquid chromatography. J. Assoc. OffAnal. Chern., 70:479-483. 538. Trucksess. M.W., Ready, D.E., Pender, M.K., Ligmond, C.A.. Wood, G.E.. and Page, S.W. (1996). Determination and survey of deoxynivalenol in white flour. whole wheat flour, and bran. J. AOAC Int.. 79:883-887. 539. Wilson, D.M., Tabor, W.H., and Trucksess, M.W. (1976). Screening method for the detection of aflatoxin. ochratoxin, zearalenone, penicillic acid, and citrinin. J. Assoc. OffA m l . Clzem., 59: 125-1 27. 530. Ware, G.M.. and Thorpe, C.W. (1978). Determination of Zearalenone in corn by high pressure liquid chromatography and fluorescence detection. J. Assoc. OffAnal. Clzem.. 61: 10581062.

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Medical Management and Plant Poisoning Robert H. Poppenga University of Penmylvarzicr, Kerznett Square. Pennsylvania

I. Introduction 41 3 11. General Treatment of Plant Intoxication 414 A. B. C. D.

Principles of management 414 Paradigm for initial management 414 Gastrointestinal decontamination 415 Enhancing toxin elimination 416 E. Antidotes 416

111. Specific Plant Toxins 419 A. B. C. D.

Nitrate 419 Cyanide 419 Phytoestrogens 436 Photodynamic agents E. L-Tryptophan 437

436

IV. Prevention 437

A. General principles 437 B. Feed aversion and rumen inoculation References 438

1.

437

INTRODUCTION

Recognition of plant intoxications depends on familiarity with potentially poisonous plants in the environment of the animal, knowledge of seasonal variations in concentrations of plant toxins, management practices that predispose to intoxication, and adequate physical examination and laboratory testing of sick animals. It is imperative that there is evidence of plant ingestion, either from examination of the environment or from detection of plant material in the gastrointestinal tract of dead animals, and positive identification of suspect plants according to genus and species. Thorough inspection of representative samples of feedstuffs such as hay, haylage, and green chop is important to detect possible contamination from toxic plants. Clinical signs and lesions should be compatible with those known to occur following ingestion of the plant under suspicion. It is often not possible to detect 413

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specific plant toxins in samples collected from affected animals, although laboratory confirmation of some intoxications such as those due to ingestion of nitrate- or cyanidecontaining plants are widely available from veterinary diagnostic laboratories. II. GENERAL TREATMENT OF PLANT INTOXICATION Once a tentative or confirmed diagnosis of a plant intoxication is made, appropriate steps can be taken to treat affected animals. The ease with which decontamination strategies or antidote administration can be accomplished is quite variable and depends on such factors as the location and number of affected animals, the expected rapidity of onset and duration of clinical signs, and availability and cost of sufficient quantities of decontamination, antidotal, or therapeutic agents. If large numbers of animals are affected, it is necessary to attempt to determine which individuals are likely to benefit from treatment so that effort is not wasted treating animals likely to die despite intervention. Thus, every situation is different and no single treatment approach is the best. A.

Principles of Management

Once a determination has been made that an animal has been exposed to a plant toxin or is intoxicated, a general approach to case management should adhere to the following principles: (1) stabilize vital signs (this may include administration of an antidote if sufficient information concerning a specific toxinexposure is immediately available), (2) obtain a history and clinically evaluate the patient, (3j prevent continued systemic absorption of the toxin, (4) administer an antidote if indicated and available, ( 5 ) enhance elimination of absorbed toxin, (6) provide symptomatic and supportive care, and (7) closely monitor the patient (Beasley and Dorman, 1990; Shannon and Haddad, 1998). It is recommended that the above principles be followed in sequence with modifications made depending on the circumstances of the case. For example, there may not be anantidote for a given plant toxin or a way to significantly enhance its elimination once systemically absorbed. In asymptomatic, recently exposed animals, it is important to attempt to determine the severity of exposure to the toxicant. Such an assessment will assist the clinician in choosing the appropriate sequence of management steps to follow. For example, ifan animal has recently ingested an amount of a toxic plant that is well below reported toxic doses (less than or equal to 1/ 10 of an LD5,,), then close monitoring for several days may be sufficient. Ingestion of higher doses may warrant administration of an adsorbent such as activated charcoal (AC) with or without a cathartic followed by close monitoring in the hospital or other controlled environment. In many situations, even where there is a strong suspicion of intoxication, no specific toxin can be identified and no exposure determined. Alternatively, a known toxin may have been ingested, but no information is available concerning its toxicity to the particular animal species exposed. In such situations, extrapolation of toxicity data from other species maybe all that is possible. In many plant intoxications, it is difficult to precisely determine ingested amounts and appropriate treatment strategies should be based on conservative estimates. Ultimately, the advice to ''treat the patient and not the toxicant" is sound. B. Paradigm for InitialManagement Specific approaches to stabilization of vital signs are discussed more thoroughly in standard veterinary medical texts. Briefly, attention should be paid to maintaining a patent airway

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Poisoning

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and providing adequate ventilation, maintaining cardiovascular function with attention to appropriate fluid andelectrolyte administration, maintaining acid-base balance, controlling central nervous system signs such as seizures, and maintaining body temperature. In some situations, it is important to administer an antidote quickly. For example, in suspected nitrate intoxication, administration of methylene blue may be critical to control life-threatening signs before proceeding with subsequent management steps. Once vital signs are stable, a thorough history should be obtained while the animal is being further evaluated. If blood or urine samples are obtained for clinical evaluation, appropriate portions should be set aside for possible toxicological testing.

C. GastrointestinalDecontamination Gastrointestinal decontamination (GID) is a critical component of case management. Appropriate and timely decontamination may prevent the onset of clinical signs or significantly decrease the severity or shorten the course of intoxication. GID consists of three components: (1) gastric evacuation, (2) administration of an adsorbent and (3) catharsis (Shannon and Haddad, 1998). 1. GastricEvacuation Approaches to gastric evacuation include induction of emesis with emetics and gastric lavage (GL). GL can be employed in those cases in which gastric evacuation is indicated but administration of an emetic is contraindicated (presence of seizures, severe depression or coma, loss of normal gag reflex, hypoxia, or species affected is unable to vomit). Induction of emesis is contraindicated in ruminants and horses since they do not vomit. In a conscious animal, GL may require tranquilization or anesthesia. Airway protection is necessary whenever GL is performed. As large a gastric tube as possible with terminal fenestrations is introduced into the stomach or rumen. Tube placement is confirmed by aspiration of gastric contents or air insufflation with a stethoscope placed over the stomach. Tepid tap water or normal saline (5-10 ml/kg) is introduced into the stomach or rumen with minimal pressure application and is withdrawn by aspiration or allowed to return via gravity flow. The procedure is repeated until the last several washings are clear; numerous cycles may be required. AC (+-cathartic) can be administered via the tube just before its removal. The initial lavage sample should be retained for possible toxicological analysis. Rumenotomy with manual removal of rumen contents is an option in an extremely valuable animal that has ingested a plant toxin whose onset of action is somewhat delayed. This is not practical when large numbers of animals are exposed or affected or when rapidly acting plant toxins (for example nitrate, cyanide, or Delphinium spp. alkaloids) are ingested. 2. Administration of Adsorbents Realistically, the only adsorbent routinely used in veterinary medicine is AC. AC is likely to be an effective adsorbent for most plant toxins although the adsorptive capacity of AC for most has not been determined. AC is available as a powder, an aqueous slurry, or combined with cathartics such as sorbitol. AC given repeatedly is effective in interrupting enterohepatic recycling of a number of toxins and the continued presence of AC in the gastrointestinal tract may allow the tract to serve as a sink for trapping toxin passing from the circulation into the intestines. There is little hazard to repeated administration of AC, although cathartics should be given only once. Other possible adsorbents include bentonite

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clay and aluminum silicates such as kaolin. Their efficacy has not been established for most plant toxins but is likely to be considerably less than that of AC. Mineral oil is not an effective adsorbent for use in plant intoxications.

3. Cathartic Both saline (sodium sulfate or magnesium sulfate or citrate) and saccharide (sorbitol) cathartics are available for use. In theory, cathartics hasten the elimination of unabsorbed toxin via the stools. In general, cathartics are safe, particularly if used only once. However, repeated administration of magnesium-containing cathartics can lead to hypermagnesemia manifested as hypotonia, altered mental status, and respiratory failure. Also, repeated administration of sorbitol can cause fluid pooling in the gastrointestinal tract, excessive fluid losses via the stool, and severe dehydration. Mineral oil has a laxative effect and can be used when saline or saccharide cathartics are not available. However, mineral oil is not as effective in inducing catharsis and it will interfere with the adsorptive capacity of AC if given concurrently. Table 1 lists the most commonly used GID agents and appropriate dosages. In recent years, a critical reappraisal of GID approaches in human intoxications has occurred that is relevant for the management of intoxicated animals (Perry and Shannon, 1996). There has been a movement away from gastric evacuation (induction of emesis or GL) followed by the administration of an adsorbent toward administration of only the adsorbent, especially in mild to moderate intoxications. Early administration of AC alone has been shown to be as efficacious as the combination of gastric evacuation followed by AC. Therefore, from a practical, logistical, and economic standpoint, initial use of AC in lieu of gastric evacuation is recommended in most situations involving large animals. The case for or against the inclusion of a cathartic with AC is less clear-cut but the administration of a single dose of a cathartic along with the initial dose of AC is currently recommended. Those AC formulations that include a cathartic such as sorbitol should be administered only once followed by AC alone if repeated doses are indicated. D. EnhancingToxinElimination Facilitating the removal of absorbed toxicants by pH manipulation of urine has not been investigated for most plant toxins and therefore no recommendations can be given. It is unlikely to be practical in most situations. Figure 1 provides a paradigm for initial management of an intoxicated patient. Critical factors that govern appropriate management choices include severity of toxin exposure, time since exposure, and the type of toxin to which the animal was exposed. In determining the severity of exposure one must consider several factors including the inherent toxicity of the plant, the amount of plant ingested, the species and age of the animal exposed, and the presence of underlying disease conditions resulting in liver and/or kidney dysfunction.

E. Antidotes Antidotes should be administered if indicated and available. Due to the cost associated with keeping a full range of antidotes on hand and unavailability of several, many veterinarians may not have ready access to all of those that are clinically useful. Table 2 lists antidotes that should be available for case management. It is recommended that a source

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.r(

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? 2

Medical Management and Plant Poisoning

U

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Y

W

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-

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418

Ingestion

, Observation & Monitonng

Moderate

(Spccicr. Age, Undcrlytng

.

Organ Irnpainncnl)

'Contraindications for induction of emesis include loss of consciousness or severe depression, loss of gag reflex, seizures, hypoxia or species involved unable to vomit.

Figure 1 Treatment algorithm for initial management of plant intoxications. AC = activated charcoal.

Table 2 Antidotes for the Most Common Plant Intoxications Antidote

Indication

Dosage

Vitamin K,

Anticoagulants

Sodium thiosulfate

Cyanide

Sodium nitrite Methylene blue

Cyanide Nitrateshitrites

Cattle, calves, horses, swine, sheep, and goats with acute hypoprothrombinemia and hemorrhage: 0.5-2.5 mg/kg b.w. IV not to exceed 10 mglmin in mature animals and 5 mg/min in newborn or young animals; for nonacute hypoprothrombinemia: same dose given IM or subQ; treatment may be needed for several days (Plumb, 1995) 30-40 mg/kg b.w. of a 20% solution IV, may be repeated 16 mg/kg b.w. of a 1% solution N,given only once 4-30 mg/kg b.w. of a 1% solution in distilled water IV

oning Plant

and Management Medical

419

such as a human hospital or pharmacy be identified for obtaining less commonly used antidotes prior to their need. In several states, antidote depots have been established to provide quick access to sufficient quantities of decontamination agents or antidotes to treat a large number of animals. Several useful antidotes such as sodium thiosulfate, sodium nitrite, and methylene blue are not commercially available and/or approved for use in food-producing animals. They should be used only if a valid veterinarian-client-patient relationship has been established and appropriate withdrawal times are followed. There are no withdrawal times following the use of sodium thiosulfate or sodium nitrite but there is a 180-day withdrawal time following the use of methylene blue due to its mutagenic and carcinogenic potential (Lynn Post, FDA-CVM, personal communication). Fortunately, many intoxicated patients will recover if attention is paid to appropriate symptomatic and supportive care.

111.

SPECIFICPLANTTOXINS

Some potential plant toxins such as nitrate and cyanogenic glycosides are found in a number of plant species. These are discussed below on a toxin-specific basis. Plant specific treatment recommendations are discussed in Table 3. A.

Nitrate

There are a number of common forage plants that can accumulate a toxic concentration of nitrate including Zea nznys (corn), Sorghum Iznlepeme (Johnson grass), Averza sntivn (oats), and Medicago sativa (alfalfa). In addition, many plants in the Amaranthaceae, Chenopodiaceae, Compositae, Cruciferae, Solanaceae, and Poaceae families can accumulate toxic concentrations under appropriate environmental conditions (Pfister, 1988). Nitrate intoxication is primarily of concern in ruminants due to the reduction of nitrates to nitrites in the rumen and subsequent oxidation of hemoglobin to methemoglobin following nitrite absorption, thus interfering with oxygen delivery to tissues. Owing to the rapidity of onset of clinical signs following ingestion of toxic amounts of nitrate, treatment is generally not administered sufficiently early to prevent significant morbidity and mortality. However, in those situations where treatment can be given, methylene blue is antidotal. It is administered as a 1% solution in distilled water at 4-30 mg/ kgbody weight IV (Haliburton, 1998). Methylene blue accepts electrons for NADPH reductase in the blood and accelerates the conversion of methemoglobin to hemoglobin. Nitrate intoxication prevention strategies include testing suspect forage prior to feeding, allowing animals to adapt to forages containing substantial nitrate concentrations, proper cutting of forages high in nitrate to avoid feeding lower portions of the plant which contain the highest concentrations, and ensiling forages that are otherwise unacceptable for hay or green chop use (Pfister, 1988).

B. Cyanide Many plants contain cyanogenic glycosides including Linurn spp. (flax), Prunus spp. (cherries, chokecherries, apricots, peaches), Sorghum spp. (sorghum, Sudan grass, Johnson grass), Triglochit2 spp. (arrowgrass), Trifolium repem (white clover), and Zen mays (corn) among others. Hydrolysis of the glycosides, releasing free cyanide, occurs via the action

Table 3 Treatment and Prevention Strategies for Individual Toxic Plants Scientific name Abrus precafurirts

Acacia berlandieri

Common name Rosary pea. precatory bean Guajillo

Toxin Abnn (lectin) Sympathonlimetic anlines, tyramine, N-methyl tyramine, Nmethyl- P-phenylethylamine

Acer rubiirm and perhaps other Acer SPP.

Red maple

Unknown

Aconitum napellurn

Monkshood

Aconitine and related alkaloids

Aesciilus glabra

Ohio buckeye, horse chestnut

Aesculin (saponin glycosides)

Agave lechugirilla

Lechuguilla

Steroidal sapongenin

Agrosteenznin githago Alliiim spp.

Corn cockle Onion

Saponin Dipropyl disulfide, dipropenyl disulfide

Treatmenta GJD, symptomatic: demulcents, balanced electrolyte solutions Chronic intoxication,GID not effective, need for prolonged supportive care often makes treatment impractical unless valuable animal GID, symptomatic: blood transfusion, diuresis with balanced electrolyte solutionto prevent hemogloginuric nephropathy, methylene blue should be considered if si,onificant methemoglobinemia is present (Divers et al.. 1982) GID. syniptomatic: atropine for bradycardia, other antidysrhythmic agents depending on the dysrhythnlia present GID, symptomatic: demulcents, balanced electrolyte solutions; supportive: segregate and protect from self-trauma GID unlikely to be useful, symptomatic: avoid direct sunlight,corticosteroids for pruritis, antibiotics for 2" skin infections, treat underlying liver dysfunction GID, symptomatic: demulcents GID. symptomatic: blood transfusion, diuresis with balanced electrolyte solution to prevent hemogloginuric nephropathy.

Specific prevention strategiesh None None

Avoid access to trees or fallen limbs by animals, especially horses; since wilting leaves are the most toxic, make sure any fallen limbs are removed from the environment of the animal as quickly as possible None

None

None

None Feed cattle cull onions in combination with other vegetable wastes or feeds to minimize problems

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Table 3 Continued Scientific name

Comion name

Toxin

Treatment’

Galegn officinalis

Goatsrue

Galegine (guanidine compound)

GID, symptomatic: treat pulmonary edema with diuretics; atiticholinergics such as atropine may be helpful

Gelseririuni senipervireiis

Yellow jessamine

Gossypium spp.

Cotton

Gelsemine, gelsenunine (indole alkaloids) Gossypol

GID, symptomatic: balanced electrolyte solution Delay in onset of clinical signs makes GID ineffective, syrnptomatic: treatment often futile

Specific prevention strategies” Intense efforts to eradicate the plant have been undertaken; sheep can at least partially adapt to the plant (Keeler et al., 1986) None Randomly test cottonseed for free gossypol content; calves, lambs, and other young ruminants less than 4 months of age should have I 1 0 0 ppm free gossypol in their ration (Morgan, 1998); growing steers and heifers no more than 200 and 900 ppm in cottonseed meal (CSM) and whole cottonseed (WCS), respectively; young developing bulls-150 ppm CSM and 600 ppm WCS; mature bulls during breeding season--200 pprn CSM and 900 WCS; mature cows-600 ppm CSM and pptn 1200 WCS: adult dairy animals should not be fed more than 5-6 pounds whole cottonseed daily; high intakes of iron, protein, or calcium are protective

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GID if acute ingestion of large quantities, symptomatic; GID ineffective with chronic syndrome; often, severe chronic liver damage makes treatment futile; consider runien reinoculation if loss of lumen motility Delayed intoxication, GTD probably not effective, need for prolonged supportive care often makes treatment impractical unless valuable animal GID, symptomatic.

Lantuna cainara

Lantana, large-leaf lantana

Lantadene A and B (polycyclic triterpinoids)

Lathyrus spp.

Singletary pea, wild pea, vetchling

P- Amino propionitrile

Lobelia spp.

Cardinal flower, Indian tobacco Leucaena

Lobeline (alkaloid) Miniosine (nonprotein amino acid)

Delayed onset of clinical signs makes GTD ineffective: if goiter present, can attempt iodine administration

Lupine, bluebonnet

Lupinine (alkaloid)

GID, symptomatic: control seizures

Anagyrine

Some affected calves can live for prolonged periods

Leucaeiza leucoceplznla

Lupinus spp.

None

None Rurninal inoculation with microbes able to detoxify 2,3DHP, a microbial breakdown product of mimosine, has shown pronuse (Hainmond et al.. 1989) Avoiding pastures during the seed pod stage of plant growth. Avoiding lupine-containing pastures from the 40th to the 100th day of gestation in cattle will minimize birth defects; also, avoiding pastures during the seed pod stage of plant growth

Table 3 Continued Scientific name

Conmon name

Toxin

Meliloti4s oficirialis and M . alba

Yellow and white sweetclover

Coumarin glycosides

Neriimz olearider

Oleander

Cardiac glycosides

Nicotiana spp.

Tobacco

Nicotine and other similar akaloids

Anabasine (alkaloid): teratogen

Treatmentd Delayed onset of clinical signs makes GID less important; vitamin K, is antidotal; cattle, calves. horses, swine, sheep, and goats with acute hypoprothrombinemia and hemorrhage: 0.5-2.5 mg/kg b.w. IV not to exceed 10 nig/min in mature animals and 5 mg/min in newborn or young animals; for nonacute hypoprothrombinetnia: same dose given IM or subQ (Plumb, 1995); treatment may be needed for several days GID, symptomatic: treat cardiac dysrhthymias (atropine, lidocaine, propanolol depending on specific dysrhthymia),potassium administration if animal hypokalenlic (Knight, 1988); digoxin-specific antibody fragments may be effective but cost prohibitive in most cases GID, symptomatic: control seizures. control bradycardia and possible muscaritiic effects with atropine. balanced electrolyte solutions, respiratory supPO* No treatment of affected offspring

Specific prevention strategiesh Proper curing of hay and proper ensiling of haylage to prevent mold growth

None: make sure clippings are disposed of properly

None

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Table 3 Continued Scientific name

Common name

Sophorn secuizd(fiot-rr

Mescal bean

Sorghum spp.

Sorghum. Sudan grass, milo, Johnson grass

Toxin Cytisine and other quinolizidine alkaloids Cyanogenic glycosides

P-Cyanoalanine

Taxiis spp.

Yew

Taxine (alkaloid)

Triglochiri spp.

Airowgrass

Cyanogenic glycosides

Veratruni califot-riiciirri and V. iiride

Hellebore, false hellebore

Steroidal alkaloids causing acute hypotension

Treatmenta

Specific prevention strategiesh

GID, symptomatic

None

Rapid onset of clinical signs and death often precludes GID; SOdium nitrite given at 16 nig/kg TV followed by 20% sodium thiosulfate at 30-40 tng/kg IV; repeat sodium thiosulfate only Delayed onset of clinical signs makes GID ineffective: remove from pasture. symptomatic: antibiotics for cystitis: recovery, if it occurs, may be prolonged Rapid onset of clinical signs and death often precludes GID but it should be considered, syniptomatic: atropine Rapid onset of clinical signs and death often precludes GID; sodium nitrite given at 16 mg/kg IV followed by 20% sodium thiosulfate at 30-40 nig/kg IV; repeat sodium thiosul fate only Rapid onset of clinical signs often precludes GID, symptomatic: balanced electrolyE solutions, vasopressors

Test forage for cyanide when environrnental conditions are conducive to glycoside forrimtion

Avoid grazing anitnals on sorghum pasture

None: make sure clippings are disposed of properly

Test forage for cyanide when environmental conditions are conducive to glycoside fonnation

None

2

Teratogenic steroidal alkaloids. jervine. cyclopamine. and cycloposine

Vicin villosrr and other Vicia spp.

Hairy vetch

Unknown

Xmtlziirrtt strunrnriinti

Cocklebur

Carboxyatractiloside (glycoside)

Xnritlzocephalurii

Western broomweed

Saponin

Zuniia

Cycads Death carnus

See Cycns spp. Zygadenine or zygacine (steroidal alkaloid)

Zygadenus

No treatment

Delayed onset of clinical signs makes GTD effective; niorbidity in a group of animals is relatively low GID, symptomatic: balanced electrolyte solutions, deniulcents GID, symptomatic: demulcents and antidiarrheals; treat retained placentas following abortion See Cycns spp. Rapid onset of clinical signs and death often precludes GID but it should be considered, symptomatic: balanced electrolyte solutions, vasopressors

Avoid grazing by pregnant ewes between days 14 and 30 of gestation. toxicity is lost after frosting. thus delaying breeding until after the first frost is preventive Avoid plant during vigorous growth phase (late spring)

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None

Standard gastrointestinal tract decontamination protocols should be followed when possible and practical. Supportive care should always be given and should include general measures such as avoidance of stress. provision of good quality water and feed in appropriate quantities, and provision of appropriate shelter and bedding. Bedding IS especially important in recumbent animals. Although there are no specific prevention strategies for many plant intoxications, general strategies include limiting or preventing access to the plant. appropriate pasture management and stochng rates, provision of adequate feed and water. and use of herbicides to control undesirable plants. All feed sources should be checked carefully for weed or weed seed Contamination. GID = gastrointestinal decontamination.

436

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of P-glycosidases in the plant tissue following plant damage due to maceration, wilting, frost, or drought. Systemically absorbed cyanide binds to cytochrome oxidase, which interferes with electron transport and thus cell respiration. As withnitrate exposure, the rapidity of onset of clinical signs following intoxication rarely allows intervention sufficiently early to prevent significant morbidity and mortality. Treatment of cyanide intoxication is directed toward removing cyanide from cytochrome oxidase. Sodium nitrite is administered at 16 mg/kg body weight IV to form methemoglobin, which helps pull cyanide from cytochrome oxidase (Galey, 1996). Subsequent or coadministration of sodium thiosulfate (20% solutionj at 30-40 mg/kg body weight IV provides substrate for the enzyme rhodanase to form thiocyanate, which is rapidly excreted via the urine. Repeated administration of sodium nitrite is not recommended although sodium thiosulfate can be administered more than once.

C. Phytoestrogens Phytoestrogens are found in many plants especially legumes such as Medicugo sativa (alfalfa), Trifoliuvz subte1.1-c~neun1 (subterranean clover), and Tr-ifoliumpraterzse (red clover) (Adams, 1995). The most important phytoestrogens are the isoflavones (biochanin A and formononetin) and coumestans (coumestrol). They are estrogenic receptor agonists and cause reproductive impairment when ingested in sufficient amounts. Sheep are particularly sensitive to their effects. The only effective treatment of affected animals is to remove them from the source of exposure. Reproductively sensitive animals should have limited access to yhytoestrogen-containing plants. Field-curing forage as hay can reduce phytoestrogen content by as much as 70% (Kallela, 1980)perhaps allowing otherwise problematic forage to be used without adverse consequences.

Dm PhotodynamicAgents Photosensitization is a syndrome that results from the interaction of an ingested photodynamic chemical and ultraviolet light within skin capillaries. Several plants contain photodynamic chemicals, including Hypeiiczcnz p e ~ o r u t z m(St. John’s wort), Fagopyr-um escu/e??tl4?ll(buckwheat), Ammi Imjus (Bishop’s weed), Cympterns ~ ~ ~ t s o(spring ~ l i i parsley), and Coopel-icr pendmccrlntcr(giant rain lilyj. Ingestion of these plants causes primary photosensitization. Photosensitization also occurs when phylloerythrin, a photodynamic agent and a metabolite of dietary chlorophyll produced by microbes in the gastrointestinal tract, accumulates within the systemic circulation. This occurs when normal biliary function is impaired. Such impairment follows ingestion of a number of hepatotoxic plants (Agave lechuglrilln, Kochin scopur-in.Lcuztntw cnmnm, Nolina texcuzn, Pnniczmz spp., Tetmdy7zio spp., and Tribulzls terrestris) and mycotoxins such as sporedesmin and phomopsins (Rowe, 1989) and is termed secondary or hepatogenous photosensitization. Treatment of affected animals involves removal from responsible plants, protection of animals from direct exposure to sunlight, provision of nutritious feed, prevention of secondary skin infections and fly strike, and general supportive therapy. Anti-inflammatory steroids may be indicated to help control pruritus early in the course of the syndrome and antibiotics may be needed to treat bacterial skin infections. In cases of hepatogenous photosensitization, underlying reasons for liver dysfunction should be determined and treated appropriately.

Medical Management and PlantPoisoning

E.

437

L-Tryptophan

An acute adult respiratory distress syndrome (ARDS) occurs in cattle when they are moved from poor grazing conditions to lush pastures. L-tryptophan, found in the lush pasture plants, is degraded in the rumen toindoleacetic acid, which is subsequently decarboxylated to 3-methylindole. Following absorption it is activated by P-450 enzymes in Clara cells and type 1 pneumocytes in the lung to form a reactive metabolite that causes cell damage. This results in sudden onset of respiratory distress (Smith, 1996). Adult beef cattle are most often affected. Treatment is often unrewarding for plant-induced ARDS. Forced removal from pastures can cause further losses and is generally not recommended as it does not seetn to alter the number of animals ultimately affected (Blood et al., 1983). Many palliative treatments such as antihistamines, corticosteroids, epinephrine, and atropine have been tried but efficacy is unproven and the stress of handling may exacerbate clinical signs and mortality. Flunixin tneglumine at 0.5-1.1 mg/kg IV or IM SID to BID and furosemide at 0.4-1 mg/kg IV or IM BID are recommended if treatment is necessary. Many less severely affected animals recover without treatment. Prevention is thebest approach. Gradual adaptation tolush pastures is recommended. Alternatively, prophylactic treatment involving the administration of monensin or lasalocid at 200 mg/head/day beginning 1 day and 6 days, respectively, before pasture change and continuing for 10 days is effective (Smith, 1996). Ionophores are not useful after the onset of clinical signs.

IV. PREVENTION

A.

GeneralPrinciples

Obviously, prevention of plant intoxications is preferred to treating exposed or intoxicated animals. The following general principles should be followed to minimize the incidence of intoxication: (1) when possible, animals should be gradually acclimated to newenvironments containing potentially toxic plants and they should be provided withadequate alternative feeds to limit initial consumption of poisonous plants, (2) overgrazing or withholding of water should be avoided, (3) animals should be provided with an adequate diet commensurate with their physiological or production status to minimize consumption of less desirable and potentially toxic plants, (4) animals should not be allowed access to areas recently treated with herbicides such as 2,4-D since the palatability of some poisonous plants is enhanced following treatment, (5) chopped or ground forages should be inspected carefully for the presence of extraneous plants or weeds, as animals are less able to sort or discriminate undesirable from desirable forage plants, (6) avoid access to areas infested with poisonous plants during holding, transport, or unloading of animals, and (7) know the poisonous plants specific to the locality where animals are kept. B. FeedAversionandRumenInoculation Other preventive approaches include induction of feed aversion and rumen inoculation with microbes capable of detoxifying specific toxins. Conditioned aversion to ingestion of palatable but toxic plants such as Delphirziurn spp. using an emetic such as lithium chloride may prove useful in some situations (Olsen and Ralphs, 1986; Ralphs, 1992). In

438

Poppenga

ruminants, the introduction of microbes able to detoxify plant toxins has been investigated as a way to prevent intoxication due to Le~rcnemlel~ocepl?crla(Hammond et al., 1989), and it maybe possible to protect against intoxication with pyrrolizidine alkaloids, oxalates, and fluoroacetate (Gregg, 1995). A commercial product is available that contains propionibacteria able to efficiently metabolize and detoxify nitrate (Haliburton, 1998). Such approaches may allow for more flexibility in utilizing toxic plant-infested ranges or pastures or poorer quality forages but they do not substitute for good pasture management and feeding practices.

REFERENCES Adams, N.R.: Detection of the effects of phytoestrogens on sheep and cattle. J. Arzim. Sci., 1995; 73: 1509. Albretsen, J.C., Khan, S.A., Richardson J.A.: Cycad palm toxicosis in dogs: 60 cases (1987-1997). JAVMA, 1998;213:99. Beasley, V.R., Dorman, D.C.: Management of toxicoses. In: Beasley, V.R. (ed.). Veterinary Clinics of North America: Toxicology of Selected Pesticides, Drugs, and Cltenticnls. Philadelphia, W.B. Saunders Co., 1990, pp. 307-337. Blood. D.C., Radostitis, O.M., Henderson. J.A.: Veterirzm-y Medicine. 6 ed. London, Bailliere Tindall, 1983, pp. 1255-1261. BUITOWS, G.E., Edwards, W.C., Tyrl, R.J.: Toxic plants of Oklahoma: coffeeweeds and sennas. OVMA, 198234: 101. Divers, T.J., George, L.W.. George, J.W.: Hemolytic anemia in horses after the ingestion of red maple leaves. JAVMA, 1982:180:300. Edwards, W.C., Burrows, G.E., Tyrl. R.J.: Toxic plants of Oklahoma: milkweeds. OVMA. 1984;3: 74. Galey, F.D.: Plants and other natural toxicants. In: Smith, B.P. (ed). Large Aizin~rl Interm1 Medicine. 2nd ed. St. Louis, Mosby. 1996, pp. 1877-1902. Galey, F.D., Hullinger, P.J., McCaskill, J.: Outbreaks of stringhalt in northern California. Vet. H w x Toxicol., 1991;33:176. Gregg, K.: Engineering gut flora of ruminant livestock to reduce forage toxicity: progress and problems. Trefzds Biotech., 1995;13:418. Haliburton, J.C.: Nitrate poisoning associated with the consumption of forages or hay. In: Howard. J.L., Smith. R.A. (eds.). Current Vete~-inmyTherapy 3. Philadelphia. W.B. Saunders Co., 1998, pp. 275-279. Hammond, A.C., Allison, M.J., Williams, M.J., Prince, G.M., Bates. D.B.: Prevention of leucaena toxicosis of cattle in Florida by ruminal inoculation with 3-hydroxy-4-(1H)-pyridone-degrading bacteria. Am. J. Vet. Res.. 1989;50:2176. James, L.F., Molyneux, R.J., Panter, K.E., Gardner, D.R.. Stegelmeier, B.L.: Effect of feeding ponderosa pine needle extracts and their residues to pregnant cattle. Cornell Vet., 1994;84:33. Kallela, K.: The estrogenic effect of silage fodder. N o d . Vet. Med.. 1980:32:180. Keeler. R.F., Johnson, A.E., Stuart, L.D.. Evans, J.O.: Toxicosis from and possible adaptation to Gdega oficinalisin sheep and the relationship to Verbesim encelioides toxicosis. Vet. H z m ~ Toxicol., 1986;28:309. Knight, A.P.: Rhododendron and laurel poisoning. Conperzd. Contin. Ed., 1987a;9:F26. Knight. A.P.: Larkspur poisoning. Compend. Contin. Ed., 1987b:9:F60. Knight. A.P.: Oleander poisoning. Conrpend, Corztin. Ed., 1988;10:262 Morgan, S.E.: Gossypol. In: Howard, J.L., Smith, R.A. (eds.). Curl-em Veterinaiy Therapy4. Philadelphia. W.B. Saunders Co.. 1998, pp. 245-247.

Medical Poisoning Management Plant and

439

Murphy, M.J., Reagor, J.C., Ray, A.C., Rowe, L.D.: Bovine abortion associated with ingestion of hu arzgzlstifolia (narrowleaf sumpweed). Proc. AAVLD, 1983, pp. 161-166. Olsen, J.D., Ralphs, M.H.: Feed aversion induced by intraruminal infusion with larkspur extract in cattle. JAVMA, 1986;47: 1829. Olson,C.T., Keller,W.C., Gerken. D.F., Reed, S.M.: Suspected tremetolpoisoninginhorses. JAVMA, 1983;185:1001. Panter, K.E., Baker. D.C., Kechele, P.O.: Water henlolock (Cicuta douglasii) toxicosis in sheep: pathologic description and prevention of lesions and death. J. Vet. Diagw Irzvest., 1996;s: 474. Perry, H.. and Shannon, M.: Emergency department gastrointestinal decontamination. Peclirttr: Arm., 1996;25:19. Pfister, J.A.:Nitrateintoxicationofruminantlivestock. In: James.L.F..Ralphs, M.H., Nielsen, D.B. (eds).The Ecoloy? crrd Ecorzornic Impact of Poisorlom Plarzts 011 Livestock Production. Boulder, Colo., Westview Press, 1988, pp. 233-260. Plumb, D.C.: Veterinrrg Drug Hctitdbook. 2nd ed. Ames. Iowa State University Press, 1995, pp. 496-499. Pluxnlee, K.H., VanAlstine. W.G., Sullivan, J.M.: Japanese pieris toxicosis of goats. J. Vet. Diagn. Im*est., 1992;4:363. Ralphs, M.H.: Continued food aversion: training livestock to avoid eating poisonous plants. J. Range Mcutage.. 1992;45:46. Ralphs, M.H., Graham, D.. Molyneux, R.J., James, L.F.: Seasonal grazing of locoweeds by cattle in northeastern New Mexico. J. R a q e Mcrnnge. 1993;46:416. Rowe, L.D.: Photosensitization problems in livestock. In: Burrows, G.E. (ed.). Veterinary Clinics oj’ North Airzericcr Food Animal Practice: Clinical Toxicology. Philadelphia, W.B. Saunders CO., 1989,pp.301-324. Salles. MS., Barros, C.S.L.. Lemos, R.A.. Pilati, C.: Perirenal edema associated with Amaranthus spp. poisoning in Brazilian swine. I7et. Hwl. To.ukol., 1991;33:616. Shannon, M.W., and Haddad, L.M.: The emergency management of poisoning. In: Haddad, L.M. et a]. (eds.). Clinical Mmager~rentof Poisoitirrg mdDt-rcg Overdose.Philadelphia, W.B. Saunders Co., 1998, pp. 2-31. Smith, J.A.: The interstitial pneumonias. In: Smith, B.P. (ed.). Large Anirncrl Znrerrzal Medicine. 2nd ed. St. Louis, Mosby, 1996, pp. 656-667. Wikse, S.E., Leathers, C.W.. Parish, S.M.: Disease of cattle that graze turnips. Comyerzd. Contin. Ed., 1987;9:F112.

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9 Plant Toxicants and Livestock: Prevention and Management Michael H. Ralphs U.S. Depcrrtmerlt of Agriculture, Logarz, U t d

I.

Introduction

441

11. Plant/Animal/Environnlental Interactions

A. Ecology of poisonous plants B. Plant factors 443 C. Animal factors 446 D. Environment 448 E. Management factors 449

442

442

111. Traditional Management to Prevent Poisoning IV. Future Prevention Technology

A. B. C. D. E. V.

452

Toxin binding 452 Vaccines 452 Microbial breakdown of toxin 453 Behavioral modification 454 Control of poisonous plants 454

Conclusions Appendix References

1.

45 1

455 456 462

INTRODUCTION

Preventing livestock poisoning from plants is, theoretically, a simple matter of either removing the animal from the infested area or controlling the plant in the pasture. However, land ownership and use patterns often require that the infested areas be grazed, and controlling the plant may not be practical from a logistical, economical, or environmental standpoint. In practice, preventing livestock poisoning is very complicated. The interactive factors of the ecology of the particular plant species, its palatability relative to the other species in the plant community, the specific toxin and its level within the plant, animal condition and propensity to consume the plant, environmental influences on both the plant and the animal, and management factors imposed on the entire system, all interact to determine whether an animal consumes enough of a particular species to be poi44 1

Ralphs

442

soned. These factors and interactions must be identified and understood to manage livestock on ranges or pastures containing toxic species. Once they are understood, the following strategy will prevent most livestock poisoning (Krueger and Sharp, 1978; Everist, 1981): 1. 2. 3. 4. 5.

Identify the poisonous plants on your range or pasture. Learn when these plants are most toxic and present a threat. Know when livestock are most likely to eat these plants. Learn the clinical signs and symptoms of poisoning for each plant. Devise grazing strategies that will restrict access to poisonous plants when they are likely to cause poisoning.

Many papers, bulletins, and books have been written describing poisonous plants, their clinical signs of poisoning, and some nonspecific treatments of poisoned animals. This chapter will concentrate on managing livestock, pastures, and rangeland to avoid the grazing conditions when poisoning is likely to occur. Management is the key to prevent livestock poisoning.

II. PLANT/ANIMAL/ENVIRONMENTALINTERACTIONS A.

Ecology of PoisonousPlants

Poisonous plants have been a major problem to the livestock industry, particularly in the Western United States, northern Mexico, and Canada. The great geological diversity of landforms and climatic extremes create the habitats for a great variety of toxic plants that livestock are exposed to in grazing these extensive rangelands. Furthermore, most Western ranges were overstocked by livestock until the mid 1930s, resulting in degradation of the native plant communities. Retrogression following tnisuse was the greatest single factor contributing to livestock poisoning (Stoddart et al., 1949): desirable forage species decreased while less palatable brush, weedy, and poisonous species increased or invaded. Livestock were forced to eat the poisonous species because of the shortage of desirable forage. Much of the early range management literature blamed poisonous plant problems on poor range conditions (Marsh, 1913, 1958; Stoddart and Smith, 1955). The level of management on most rangelands improved during the last 60 years resulting in marked improvement in range condition (Box and Malechek. 1987) and an accompanying decline of widespread catastrophic livestock losses to poisonous plants. Good range and pasture management is the surest and most economical means of reducing livestock loss to poisonous plants (Schuster, 1978): desirable forage species are encouraged, while undesirable and poisonous species are suppressed, and animals are provided abundant nutritious forage. Yet, some poisonous plant problems remain, even on good-condition rangeland and pastures. Some poisonous species are major components of climax plant communities (tall larkspur, Veratrum, water hemlock, bracken fern, chokecherry, ponderosa pine, oak, and black walnut). Although many poisonous increaser species have declined with better management, they are still components of plant communities and fluctuate with changing precipitation patterns (locoweed, lupine, death camas, sn'akeweed, threadleaf groundsel. low larkspur, redstem peavine, western bitterweed, twin leaf senna, white snakeroot). Many of the introduced and alien invader species that threaten rangelands and pastures are poi-

Livestock Plant Toxicants and

443

sonous (Hdogeton, Saint-John's-wort, poison hemlock, tansy ragwort, houndstongue, African rue, leafy spurge, yellow star thistle, and the knapweeds). On the other hand, many important forage, pasture, and range species contain toxic compounds that are capable of poisoning livestock under some conditions (alfalfa, clovers, tall fescue, sorghum and Sudan grass, reed canary grass, Klein grass, Kikuyu grass, annual and perennial ryegrass).

B. PlantFactors 1. Palatability A poisonous plant remains harmless until it is consumed. Luckily, many of the most toxic poisonous plants are also very unpalatable (whorled and horsetail milkweed, oleander, and jimson weed). Bate-Smith (1972) suggest that bitterness is a characteristic of many toxins, and Garcia and Hankins (1974) contend that natural aversions to bitter substances have been acquired by a wide variety of species through natural selection to enhance the survival of the species (see below). Poisoning occurs when hungry animals are forced to eat these plants, or they are mixed in with other feed or forages. Animals seldom graze these types of plants if other desirable forage is available. High toxin levels in some plants contribute to their lack of palatability. Reed canary grass (Plzalnr-isamrzclinnceae)is a hardy, high-yielding forage species in temperate regions throughout the world, but it contains varying levels of tryptamine alkaloids that can cause Phakrr-is staggers. Alkaloid levels ranged from 0.03% of dry matter (DM) in palatable strains, and increased up to 0.4% in unpalatable strains (Hagman et al.. 1975; Marten et al., 1976). L~~pirzus species contain several quinolizidine alkaloids that are toxic. Sweet varieties of lupin (seeds consumed by humans and foliage by livestock) contain alkaloid levels less than 0.1%, while bitter varieties had alkaloid levels greater than 0.4% (Keeler and Gross, 1980). Livestock (Forbes and Burton, 1960), rabbits (Waller and Nowacki, 1978), and thrips (Forbes and Beck, 1954) avoid eating bitter varieties of lupin. However, L. szdphzrr-eus was quite palatable to cattle (1.25% total alkaloids in foliage and 4.2% in seed pods), compared to L. ler~opl~yllus (.1.38% total alkaloids in foliage), which was not eaten at all (Panter et al., 1997. Apparently other factors besides total alkaloids influenced palatability. Species of Cr-otolar-iawere used in the southeastern United States as forage legumes. The toxic species C. spectnbilis contains high levels of pyrrolizidine alkaloids, and is veryunpalatable to livestock (Becker et al., 1935). Total and toxic levels of diterpenoid alkaloids in tall larkspur (Delylzinizm bar-heyi)were negatively correlated to palatability in sheep, but not in cattle (Pfister et al., 1996).

2. Toxin All plant species contain secondary compounds and many of these compounds can cause poisoning under certain conditions (Hegarty, 1981; McDonald, 1981). Identification of toxic plant species, their particular toxin, and the concentration of the toxin in the plant at various growth stages is the first step in managing livestock to avoid poisoning. Once the toxin level is known, the manager can assess the risk of poisoning for grazing infested areas at a particular time. Western larkspur species contain over 40 diterpenoid alkaloids, but the toxicalkaloid methyllycaconitine (MLA) is responsible for most of the toxicity in cattle, resulting in muscle fatigue, respiratory failure, and rapid death (Manners et al., 1995). Concentrations of toxic alkaloids in tall larkspur decline over the growing season (Ralphs et al., 1997),

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but cattle will not eat larkspur in its early growth stages when alkaloid levels are high. Based on this information, Pfister et al. (1997) proposed a grazing strategy to graze cattle on larkspur ranges early in the season to utilize the associated forage while it was still nutritious, remove the cattle during the toxic window (Fig. 1) when larkspur begins to flower and becomes palatable, and then return cattle to larkspur areas late in the season during the pod stage when toxicity is low. Lupinus species contain several quinolizidine alkaloids that cause acute respiratory failure in sheep. Concentrations range from 0.6 to 2.4% (Davis, 1982). A few species (L sulphureus, L. sericeus, L cuudutus, L laxifolrus,L lutifolius) contain the teratogenic alkaloid anagyrine (Keeler, 1976; Panter et al., 1997), and other species (L.formosus, L urbustus) contain teratogenic piperdine alkaloids (Panter et al., 1998). Both types of alkaloids have been shown to cause crooked calf disease in cattle (Shupe et al., 1967). Keeler et al. (1977) suggested that information on toxin concentration in the plant and susceptible period in the animal could be used to reduce the risk of crooked calf disease. Alkaloid concentration in whole plant material is fairly low in the early vegetative growth stages, then increases and peaks prior to bud formation, and then declines (Fig. 2). However, alkaloid concentration in seed pods can be high. Keeler et al. (1977) suggested avoiding lupine areas when the toxin is high and cows are in early gestation (days 40-100) when the fetus is susceptible to deformities. Senecio species contain several pyrrolizidine alkaloids (PA) that cause chronic liver damage. Young cattle and horses are particularly susceptible to PAS, succumbing to liver failure several weeks to many months after consuming the plant. Specific alkaloids vary in toxicity and concentration within the plants (Johnson et al., 1983, resulting in varying risks of intoxication (Johnson et al., 1989). Alkaloids accumulate over time and reach peak concentration during the reproductive stage of the plant (Fig. 3). Avoid grazing Senecio-infested areas when they are most toxic. Some plants, such as Asrrugulus species, contain several toxins. Nitrotoxins (3-nitropropanol or propionic acid) were found in 52% of 506 species and varieties sampled in North America, and ranged in levels from 2 to 25 mg NO/g of plant material (Williams and Bameby, 1977). Nitrotoxin levels are low during early vegetative growth, increase until buds are formed, and then decline after flowering (Majak et al., 1976). Other Astrugu-

Low,

i K’ I

VeglBud

’’

- p i -

Flower

Pod

I

PhenologicalStage

Figure 1 Toxicity and palatability of tall larkspur (Delphinium burbeyi) over the growing season and the resulting toxic window where the risk of poisoning is greatest (Pfister et al., 1997a).

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5-30

6-13

445

6-27

7-11

7-25

8-8

8-22

Date Figure 2 Total alkaloid concentration in Lzlpinus caudatus over the growing season in western Wyoming (Keeler et al., 1976). Error bars are standard error of the mean.

h s species can accumulate selenium (James et al., 1981j. Several species and varieties of Astrugulus and its closely related genus Oqtropis contain the locoweed toxin swainsonine (Molyneux and James, 1982). Woolly locoweed (A. ~~zullissi~~zus) contains higher levels of swainsonine and is much less palatable than white locoweed (0.sericen) (Marsh, 1909; Ralphs et al., 1993). Swainsonine levels in locoweeds are fairly low (Molyneux et al., 1994) and do not fluctuate greatly through the growing season (Ralphs and Molyneux, 1989). Poisoning depends on the amount of locoweed consumed, not the concentration of swainsonine in the plant. Swainsonine does remain in the dry senescent plants, thereby continuing the risk of poisoning until the plants wither away. Several species of deathcamas (Zigadems spp.) are toxic to both livestock and humans. The fleshy, tuberous root is most toxic, but foliar parts also contain steroidal alkaloids. There is also a wide range of toxicity among species with lethal doses ranging from 4 to 71 g of plant material/kg of weight (Marsh and Clawson, 1922; Panter et al., 1987). Toxin levels may also differ between sites or soil types. Toxicity of broom snakeweed (Glctien-ezia sm-othme)is higher on sandy soils, compared to heavy calcareous soils (Dollahite and Anthony, 1957). Timber milkvetch (Astlmgalns miser var. seriforus) contains higher concentrations of nitrotoxins when growing in open sun, compared to growing under forest canopy (Majak et al., 1974). Toxic alkaloid concentrations in low larkspur (Delphirzium rzz~ttcrllinr~z~nz) were greater at higher elevations (Majak and Engelsjord, 1988).

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1979 1980 1981 1982

”

-

0 FEB

MAR

APR

MAY

JUN

JUL

AUG

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Figure 3 Pyrrolizidine alkaloidconcentration in theadleafgroundsel Sonoita, Arizona from 1979 to 1982 (Johnson et al., 1985).

OCT

(Senecio lo~zgilobzas)at

C. Animal Factors Animals learn the value of food through postingestive feedback (Provenza, 1995). If the feedback is positive (i.e., nutrient replenishment that satisfies body requirements), preference for that food is enhanced and animals seek it out. If feedback is negative [gastrointestinal (GI) distress or activation of emetic system], hedonic shifts occur that lower the palatability of that food and cause animals to avoid it (Garcia et al., 1985). Poisoning occurs when there is a breakdown in an animal’s learning system (Provenza et al., 1992): (1j when the toxin circumvents the emetic system (neurotoxins in lupine and hemlocks); (2) when the negative consequences are delayed, such as in chronic poisoning from locoweed and species containing pyrrolizidine alkaloids; (3) when animals are unable to distinguish changes in toxicity of a plant that they have been eating; or (4) when animals are placed in a new environment and have not had time to differentiate between nutritious and toxic feed. Animals also have physiological mechanisms to detoxify poisonous compounds (Freeland and Janzen, 1974). Different species and individuals have varying abilities to bind the toxin or break it down in the GI tract before it enters the body. Animals also have varying abilities to metabolize and excrete toxins. Some animal species are simply able to tolerate certain toxic compounds. Poisoning occurs when more toxin is ingested than the body is able to compensate. Animal species differ in their grazing behavior and preference for particular plants. Native wildlife species appear more resistant than livestock to poisoning from native plants, presumably because of the evolutionary processes of selecting foods and developing preferences that would avoid harmful substances (Laycock, 1978). Basson et al.

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(1984) concluded that large wild herbivores in South Africa were generally more resistant to poisonous plants than were domestic ruminants, but they were not immune. In North America, elk (Wolf and Lance, 1984) and antelope (L.F. James, personal observation) have been poisoned by locoweed, and tule elk were poisoned by poison hemlock (Jessup et al., 1986). Differences in susceptibility or grazing behavior of different species or classes of livestock can be utilized to manage poisonous plant-infested ranges. Stocker cattle are less affected by high levels of selenium or teratogenic compounds that would cause low fertility or birth defects in breeding animals. Sheep and goats are less susceptible to oak poisoning than are cattle (Basden and Dalvi, 1987) and goats have been used as a biological tool to suppress several species of oak (reviewed by Brock, 1988). Cattle are less likely to eat woolly paper flower (Psilostrophe tqetina) than are sheep in west Texas. Woolly paper flower contains sesquiterpene lactones, which are highest in the plants' early growth. Cattle can graze areas heavily infested with woolly paper flower early in theseason and thenallow access to sheep after the paper flower has matured (Hart et al., 1998). Historically, grazing allotments on high mountain rangelands where tall larkspur is abundant were allocated to sheep (Aldous, 1917). Sheep are more resistant to the toxic larkspur alkaloids than cattle, tolerating four times more plant material than cattle at similar stages of intoxication (Olsen, 1978). The binding affinity of the toxic alkaloid methyllycaconitine (MLA) at the neuromuscular junction is substantially lower in sheep (Stegelmeier et al., 1998). Sheep have also been used to graze larkspur areas before cattle to reduce its availability or acceptability to cattle coming later (Ralphs and Olsen, 1992). Sheep and goats are more resistant to PAS found in Senecio spp. than are cattle and horses (Cheeke, 1998). This may be due to the combination of ruminal microbial breakdown of PA (Johnston et al., 1998) or enzyme detoxification in the liver (Cheeke, 1994; Cheeke and Huan, 1998). Sheep have beenused as a biological toolto control tansy ragwort (Senecio jctcobuea) (Sharrow and Mosher, 1982). Though tolerant of some toxic plants, sheep may be more susceptible to poisoning of other plants because of their grazing behavior and management. The gregarious nature of sheep causes them to graze together in bands, thus increasing grazing pressure and reducing forage selectivity. Furthermore, in some areas, range sheep are moved about by a herder who controls when and where the band is to be moved to new forage. These factors, along with their propensity to select forbs, may explain why sheep are poisoned more often by orange sneezeweed (Helenimz hooyesii),bitterweed (H~wzono-xys odomta), lupine ( L ~ ~ pspp.), i ~ sdeathcarnas (Zigcrdenus spp.), and Halogeton qlomerntus than are cattle. A wise herder will avoid infestations of these plants and avoid the conditions that may cause poisoning. Condition of the grazing animal is often the most important factor influencing poisoning. Hungry animals graze less discriminatorily and are more likely to consume toxic amounts of poisonous plants. Animals in poor condition are less able to withstand the stress of poisoning (Everist, 1981). Lactating females require a higher intake level to meet their greater physiological requirements; thus they maygraze longer and less discriminatorily than other animals. Many toxins are passed through the milk and thus poison the offspring (Panter and James, 1990). For example, tremetol in white snakeroot (Eupntorium rugosuvzz)and rayless goldenrod ( H n y l o p c p p s heterophvllus) is excreted in the milk and poisons the offspring, often before the mother is affected. Lactation is a significant route of excretion of tremetol.

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Pyrrolizidine alkaloids in Senecio, Crotalaria,Heliotroyiurzl, Eclziurn, Anuiizckia, Syzyhytzml, Cyrzoglossrrm;piperidine alkaloids in Coniurrl and Nicotiana: quinolizidine and piperidine alkaloids in L~4pirzus;the indolizidine alkaloid swainsonine in AstragalLrs and 0q)tropis locoweeds: and sesquiterpene lactones in bitterweed (Hvmeno.xy odorata) and rubberweed (Hyrzzerzo.xys ricllardsoni) are all excreted in the milk. The photosensitizing furocoumarin psoralen in Dutchman's breeches (Than.lnosma texarza) and other primary photosensitizing compounds in Wright's buckwheat (Eriogorzz41n wrightii) are also passed through the milk to the offspring (Hart et al., 1998). Lactating females should not be allowed access to these plants. The unborn fetus is susceptible to teratogens from many plants that may not affect the dam: the quinolizidine alkaloid anagyrine in several species of Lz~pirzus:piperdine alkaloids ammodendrine, N-methyl ammodendrine, and N-acetyl hystrine in Lrlpirzus.forI ~ O S Zand ~ S L. arhrstus; coniine and gamma-coniceine in poison hemlock (Conirnn ~ K I I Intrrm); anabasine in tobacco (Nicotiana tnbacum and N. glmca); and the steroidal alkaloids jervine, cyclopamine, and cycloposine in Verntrunz ccllifornic~4r1z (Keeler, 1991). The damage is insidious and difficult to trace back to the causative plant. Keeler (1978) suggested several management strategies to avoid birth defects by altering grazing so that periods of high teratogen levels in the plant did not coincide with susceptible periods of insult in the fetus.

D. Environment The type and amount of toxic compounds in plants are influenced by the relative availability of nutrient resources in the soil, external needs for chemical defense, and the internal demands for growth and reproduction (optimal defense theory) (Rhoades, 1979). The resource availability theory of plant defense suggests that availability of nutrients in the environment is the major factor influencing the type and amount of defense compounds (Coley et al., 1985). In less fertile environments where plants grow slowly, carbon-based defenses develop that deter herbivory (i.e., lignin, tannins, terpenes). In resource-rich habitats, plants grow rapidly and contain low levels of highly mobile toxins, such as alkaloids and cyanogenic glycosides. Nitrogen (N) istaken up early in the growing season in excess of the plants needs for growth and is available to be synthesized into N-based toxic defense compounds (Mooney et a1., 1983). The carbonhutrientbalance theory further suggests that environmental stresses may increase defense compounds (Bryant et al., 1983, 1992). If light becomes limiting to plants growing in nutrient-rich environments (i.e., shade or cloudy weather), the decline in photosynthesis may limit carbohydrates for growth and carbon-based defenses, but nutrient uptake continues, leaving excess N that could be shunted to N-based defense compounds such as alkaloids. By the same mechanism, water stress may increase toxin concentration. Water stress or mild drought increased toxin concentrations in the following plants (Gershenzon, 1984): cyanogenic glycosides in sorghum, sudangrass, white clover, arrowgrass (Trigloclzin nlaritinza), and serviceberry (Ardurzchier spp); alkaloids in poison hemlock, lupine, and Senecio species; and glucosinolates, phenolics, and terpenoids in other plants. Sesquiterpene lactones in Western bitterweed also increase during drought (Hart et al., 1998). Fertilizers or elevated nitrogen in soil increase soluble nitrogen co~npoundsin plants that form amino acids and other toxin precursors. Weather changes may alter concentrations of toxins and cause normally innocuous forages to become highly toxic. Frost damages cell membranes, which allow cyanogenic

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glycosides to be reduced to hydrocyanic acid in some forages (i.e., sorghum, sudangrass, Johnsongrass, arrowgrass, and chokecherry) (Kingsbury, 1964). Continuous cloudy weather can increase accumulation of nitrates in plants capable of taking up toxic levels (Everist, 1981). Acute poisoning from reed canarygrass ismost common in foggy or cloudy weather and during early morning hours (Everist, 1981). Nitrotoxin levels in timber milkvetch (Astrngalus miser var. serootinus) increase following rainfall, due to increased rate of plant metabolism (Majak et al., 1977). Weather changes may also affect animal grazing behavior. Decreasing barometric pressure prior to storms may reduce selective grazing. Sheep increased consumption of horsebrush (Tetradymin spp.) following spring rain or snowstorms, resulting in severe outbreaks of secondary photosensitization on desert ranges (Johnson, 1982a). Cattle consume more larkspur and are poisoned during or shortly after summer thunderstorms than during good weather (Ralph et al., 1994). Increasing snow cover and decreasing temperatures caused cattle to increase consumption of ponderosa pine needles (Pfister and Adams, 1993), which can cause abortions in cattle. Knowledge of the effects of weather on toxin concentrations will allow managers to avoid grazing livestock in infested areas during these conditions of higher risk. Weather patterns may also affect population cycles of poisonous plants. Population outbreaks of spotted locoweed (Astrngnlus le~?tiginosusvar. wahweaperzsis) occurred every 6-8 years in the Henry Mountain area of southeast Utah, and corresponded to warm, wet autumns, followed by above-average precipitation in the spring (Ralphs and Bagley, 1988). Large livestock losses accompanied these outbreaks. An outbreak of red stem peavine (Astr~@us emo~yanus)covered the entire region of eastern New Mexico and west Texas in 1975, following abundant fall precipitation (Williams et al., 1979). Broom snakeweed (Gutierrezin snrotf~rne)populations die off during droughts in the Southwest, but seeds germinate and establish dominant stands once rains resume (Pieper and McDaniel, 1989). Bitterweed (Hyrleno.ly odorcm)is a winter annual and dominates calcareous soils throughout the southwest when winter precipitation is high (Taylor and Ralphs, 1992). During cool, wet springs, low larkspur ( D e l y h h i ~ m nzrttnllinnum) and death camas (Zigadenus spp.) are more abundant on foothill rangeland in the western United States, and plains larkspur (D. geyeri) is abundant on shortgrass prairies of eastern Wyoming and north central Colorado. Sacahuista (Nolina texmn), a perennial desert shrub of the Liliaceae family, is generally considered a good forage plant, providing bulk reserve forage for grazing during winter. Its blooms and fruits are very palatable, but cause liver damage, resulting in secondary photosensitization. Heavy blooms only occur every 5 or 6 years (Hart et al., 1998). All animals should be denied access to sacahuista in those years while blooms and fruits are abundant.

E. ManagementFactors Poisoning of livestock is more commonly the result of management mistakes, overstocked ranges, or poor condition animals, rather than the presence of the plants concerned (Sperry et al., 1964). Most poisonous plants are eaten because the animal is hungry and the poisonous plant happens to be available. Many of the large catastrophic losses of livestock to poisonous plants resulted from management mistakes. Entire bands of sheep were poisoned by Halogeton glonzerntus after having been transported by train or truck, or trailed to desert ranges, and then released to graze in heavily infested areas (Kingsbury, 1964).

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Large losses occurred under similar conditions with Lupine species (Kingsbury, 1964), death camas (Panter et al., 1987), and greasewood (Srrrcobntus ver~niczrkatzrs)(Bowns, 1988). These losses could have been prevented by avoiding areas infested by these poisonous plants, or feeding animals before they were released into infested areas. Shortage of feed is an important factor compelling livestock to consume toxic anlounts of poisonous plants. Many of the early reports of poisoning were confused with starvation (Marsh, 1913). Forage availability is under direct control of the manager, who determines season of use, range readiness, stocking rates. and length of grazing season and provides salt, minerals, and supplemental feed when necessary. Tactical management decisions such as proper stocking rate, combinations of animal species to be grazed, and grazing system used can minimize livestock death losses to poisonous plants (Taylor and Ralphs, 1992). Many poisonous forbs and shrubs begin growth before grasses or other desirable forage in early spring (low larkspur, plains larkspur, deathcamas, locoweed, broom snakeweed, and oakbrush). The early vegetative growth of these plants is usually most toxic and the new green growth is appealing to livestock having been fed dry hay during the winter. In years when these plants are abundant, stockmen should delay turnout until range readiness when desirable grasses are available. Deep-rooted perennial forbs (sneezeweed, locoweed, lupine, and Senecio) remain green after grass matures in the fall. Reduced palatability of mature forage or shortage of native grass may make these green poisonous forbs appealing. Livestock should be removed from these ranges when desirable forage is properly utilized. On year-long ranges, forage availability becomes limiting during winter and early spring. Semidesert locoweed species, Western bitterweed, rayless goldenrod, and broom snakeweed either germinate in fall or remain green over winter and start rapid growth early in spring. Dry, dormant grass or shortage of feed makes this green growth appealing to livestock. Stockmen should supplement to satisfy protein requirements and keep animals in good condition, and should deny access to these poisonous plants until green grass is available. Grazing systems may provide flexibility to manage around specific plants, or their rigidity may compound poisoning problems. Strict adherence to rest-rotation grazing has contributed to locoweed poisoning by forcing cattle to uniformly graze all forage, including locoweed in the heavy-use pasture (James et al., 1969; Ralphs et al., 1984). Short-duration grazing requires intensive management and may have the flexibility to reduce poisoning problems or intensify them. If animals are rotated rapidly and “cream” the pasture, toxic plants are likely to be avoided. If there has been little or no regrowth in successive rotations, the lack of other desirable forage may force livestock to consutne toxic plants. Concentrating livestock in high-intensity grazing systems can reduce the abundance of palatable poisonous plants that cause chronic poisoning. Concentrating large numbers of anitnals in a small area causes them to rapidly remove the toxic species before any one animal becomes severely intoxicated. This has been recommended for control of darling pea ( S ~ r ~ r i m o nspp.) a in Australia (Everist, 1981), to remove the palatable seedpods of white locoweed (Ralphs, 1987), and to remove several varieties of timber milkvetch before poisoning occurs (James et al., 1980). Sheep can also be allowed to graze woolly paperflower (Psilostrohe trtgetina)for 2 weeks before symptoms appear (Hart et al., 1998). The Mel-rill three-herd, four-pasture, deferred-rotation grazing system (using cattle, sheep, and goats grazing in common) eliminated livestock loss to bitterweed, oakbrush,

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and sacahuista (Nolirzn te.um1) poisoning during a 20-year period in west Texas (Merrill and Schuster, 1978). Success of this common-use grazing system was attributed to reduced grazing pressure on the respective animal’s preferred forage and improved range condition, which increased the available forage and allowed greater diet selection. Salt and mineral supplements are commonly recommended to reduce livestock loss to poisonous plants. However, studies have shown that mineral supplements have no direct protective activity against tansy ragwort poisoning (Johnson, 1982b), lupine-induced birth defects (Keeler et al., 1977), locoweed poisoning (James andVan Kampen, 1974), or larkspur poisoning in cattle (Pfister and Manners, 1991). The purported value of mineral supplements may be the result of better management exercised by stockmen concerned about reducing losses to poisonous plants (Cheeke, 1998). A balanced salt and mineral supplement is important to maintain animal health.

111.

TRADITIONALMANAGEMENT TO PREVENT POISONING

Marsh (19 16) made some general management recommendations to reduce poisonous plant losses. These original principles have been rephrased, expanded, and adapted to specific situations (Huffman and Couch, 1942; James and Cronin, 1974: Dwyer, 1978; Schuster, 1978; Krueger and Sharp, 1978; James et al., 1980; Ralphs and Sharp, 1988). 1. Do not turn hungry livestock onto areas infested with poisonous plants. Hunger causes animals to eat plants they will normally avoid. Ensure adequate forage is always available. Do not overgraze a pasture. Remove livestock when the range is properly utilized. 2. Do not turn out too early in the spring. Many poisonous plants start growth early and are consumed before desirable grasses are available. 3. Cautiously introduce animals to poisonous plant-infested areas. Feed animals before moving into infested pastures. New, inexperienced animals are most likely to be poisoned. 4. Use the range or pasture when poisonous plants are least toxic. Toxin concentration is generally highest in early growth and again when the plant sets seed. 5. Graze the kind and class of animal that is least affected by particular poisonous plants when possible. 6. Maintain range in good condition to prevent invasion or increase of noxious and poisonous weeds and provide a variety of palatable and nutritious forage. 7. Control poisonous plants if livestock losses are severe enough to justify the cost of treatment. Selective herbicides are available for control of most poisonous plants. Consult your county agent for state and local recommendations. Biological control may be helpful in keeping some exotic poisonous plants in check. Plowing and seeding-improved forages may be feasible in suitable areas. 8. Consult with your veterinarian for specific treatment of poisoned animals. Recommendations for specific plants in local areas can be found in reference texts and state experiment station bulletins.

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IV. FUTUREPREVENTIONTECHNOLOGY John Edgar (1998j, research leader of the poisonous plants research unit, CSIRO, in Australia, summarized the current state and future direction of poisonous plant research: “Most of the economically important poisonous plants and widespread causes of livestock poisoning have been identified, the natural chemical toxicants responsible have been characterized, and the mechanisms of poisoning determined. In many cases, management and avoidance of poisoning have been taken as far as is practical, and poisoning still occurs at an unsatisfactory level. There is a growing expectation that cures to these poisonings are forthcoming.” He listed three areas of promising research: toxin binding, vaccines, and microbial breakdown of toxins. I would like to add to this list modification of grazing behavior and herbicide and biological control of poisonous plants.

A.

ToxinBinding

Activnfed chcrr-coal is a universal antidote due to its capacity to adsorb many toxicants in the GI tract. It should be considered as one of the first treatments of poisoned animals. However, problems with delivery in field grazing situations limit its potential as a preventive measure. It is nonspecific and could cause nutritional deficiencies if used over extended periods. Clay minerals have the capability to bind molecules of certain sizes and configurations. Aluminosilicates possess chemical adsorption properties that have been used to bind aflatoxins in stock feed (Edgar, 1998; Schell et al., 1993). Clinoptilolite, a naturally occurring zeolite clay (Bachman et al., 1992), and bentonite (Pulsipher et al., 1994; DugarteStavanja et al., 1997) were tested to determine if they would bind to the locoweed toxin swainsonine. They were somewhat effective in vitro, but they were ineffective in alleviating locoweed toxicosis in vivo in sheep. Cyc1ode.utrirl.s (CD) are cyclic oligomers of glucose with cylindrical hydrophobic cavities surrounded by hydrophilic margins that can encapsulate certain small toxins. Stewart and May (1998) reported CDs bonded strongly with corynetoxins, which cause annual ryegrass toxicity. CDs were successful in preventing toxicity in sheep grazing toxic annual ryegrass pastures. However, CDs formed only weak bonds with pyrrolizidine alkaloids, and did not protect sheep from Echiurn poisoning (Anderton et al., 1994).

B. Vaccines

Vaccines against low-molecular-weight natural toxins have not generally been successful (Edgar, 1994). The immune system has difficulty recognizing small toxins and effector mechanisms are not available to degrade them. However, some vaccines have shown promise. Than et al. (1998) reported a vaccine that gave significant levels of protection to sheep exposed to corynetoxins from annual ryegrass in Western and South Australia. Payne et al. (1992) developed a vaccine against lupinosis, a liver disease of livestock caused by phomopsin mycotoxins produced in aftermath of annual lupine. This vaccine has been refined (Than et al., 1994), fieldtested (Allen et al., 1994). and is ready for commercial development (Edgar et al., 1998). Vaccines against ergot alkaloids in tall fescue, which cause fescue foot and summer slump, are also being developed (Filipov et al., 1998; Rice et al., 1998).

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MicrobialBreakdown of Toxin

Rumen microbes can be considered the first line of active defense against plant toxins (Allison, 1978). Several toxins have been detoxified by specific rumen microbes (reviewed by Allison, 1978; Craig and Blythe, 1994): toxic proteins such as ricin in castor bean; the amino acid mimosine in Leucnencr; nitro toxins in Astrcrgalus species; nitrate and nitrite in many forage plants; oxalic acid in Halogeton; phytoestrogens in clovers; gossypol in cottonseed meal; pyrrolizidine alkaloids in Senecio and Heliotropiun? species; toxic sulfur compounds in Brassica species; thiaminase, a thiamine-degrading enzyme in bracken fern; and many mycotoxins. Several rumen microbes have also been identified that metabolize or reduce selenium (Rasmussen and James, 1994). Allison and Rasrnussen (1992) also discussed the potential of manipulating rumen microbe populations to enhance their detoxification abilities. James and Butcher (1972) reported that resistance to oxalates could be increased by feeding sheep increasing quantities of Hedogeton, which increased the rumen bacteria (0-xalobacterformigenes) that metabolized oxalates (Allison et al., 1985). Ruminants can adapt to high-nitrate diets (reviewed by Allison, 1978; Allison and Rasmussen, 1992). Cattle may also adapt to nitrotoxins in timber milkvetch. Several strains of nitrotoxin-metabolizing bacteria have been isolated (Anderson et al., 1998). One new strain of bacteria, NPOH1, was successfully inoculated in cattle, which significantly increased the detoxification rate beyond the natural adaptation. Feeding supplemental protein and nitroethane, a relatively innocuous analog of the toxin (Majak et al., 1998), can also enhance nitrotoxin-degrading bacteria. These supplements have been successfully fed in molasses blocks during field-grazing trials and show promise in preventing poisoning from timber milkvetch. Australian researchers (Lanigan, 1976) found a rumen bacterium (Peptococcus heliotrirzreducarzs) thatwould degrade pyrrolizidine alkaloids from Heliotropiurrz europaeum. Lanigan et al. (1978) attempted to modify the rumen environment to favor this bacterium by inhibiting methanogenic bacteria that competed with P. heliotri~zreducn~?.~ for free hydrogen. This approach failed because the specific antimethanogen was poisonous to the sheep. However, new technology is being developed that may provide long-term antinlethanogenic activity (McCrabb et al., 1997, as reported in Edgar, 1998). It may also be possible to transfer toxin-degrading microbes from resistant animals to susceptible animals. Jones (1985) reported the first case of transferring toxin-degrading microbes from one animal species to another. Leucaet~aIeucoceptzda is a vigorous palatable tropical shrub that is used for forage, but contains the toxic atnino acid mimosine. Ruminants in tropical areas where Lewaerza is native are not poisoned by this shrub. Transfer of detoxifying bacteria from resistant goats in Hawaiiand Indonesia to susceptible goats in Australia was successful in preventing mimosine poisoning (Jones and Megarrity, 1986). These bacteria have subsequently been transferred from goats to cattle in Australia, and from resistant cattle in the Virgin Islands to susceptible cattle in Florida (Hammond et al., 1989, as reported in Craig and Blythe, 1994). Sheep are more resistant to pyrrolizidine alkaloids than are cattle and this resistance is thought to be due in part to differing microbial populations. Johnston et al. (1998) reported a consortium of four rumen microbes in sheep that degraded toxic pyrrolizidine alkaloids found in tansy ragwort. They are in the process of determining whether this consortium can be transferred to cattle and whether it can survive in sufficient numbers to provide protection from PA toxicosis. Allison and Rasmussen (1992) stated that it is common to find bacteria or other

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microbes that will degrade specific toxins, but it is rare that these microbes can survive, compete, and increase in the rumen environment. It may be more promising to genetically manipulate the existing adapted microbes with the genes required for detoxification. F~uoroacetate is a very acute toxin in several indigenous Australian plants. Gregg et al. (1996) isolated a gene that encodes fluoroacetate dehalogenase, which degrades the toxin, and transferred this gene into a rumen bacterium, Bugiivibrio Jibrisohws. However, field testing was halted because of the remote threat of resistance being transferred to feral animals, which could endanger the survival of threatened indigenous fluoroacetate-producing plant species (reported by Edgar, 1998). Research is also continuing to find microbes that are capable of detoxifying corynetoxins, the causative agents of annual ryegrass toxicity (Payne et al.. 1994; Stuart et al., 1994).

D. BehavioralModification Cattle have been trained to avoid eating the poisonous plants tall larkspur (Ralphs, 1997), white locoweed (Ralphs et al., 1997), and ponderosa pine (Pfister, 1999) through the process of conditioned taste aversion (Ralphs and Olsen, 1998). The specific plant is offered to the animals, and then they are dosed with lithium chloride, a potent emetic, to induce GI distress. The animals associate the taste of the plant with the induced illness and subsequently avoid eating the plant. Aversions are strong and last indefinitely if animals are not compelled to resample the plant through hunger or peer pressure of other animals eating the plant (Ralphs, 1997). If averted animals taste the target plant without any adverse feedback, they will continue eating and eventually extinguish the aversion. Therefore, averted animals must by grazed separately to maintain the aversion.

E. Control of PoisonousPlants Removing a poisonous plant frotn a pasture or grazing area is the surest means of eliminating the risk of poisoning. Plowing a pasture and reseeding to adapted forage species may be feasible if there is little desirable forage. Selective herbicide control offers the most direct and immediate tool to eliminate specific plants. In addition, production of desirable forage may increase when competition from the target plant is removed. Herbicide control recommendations for important poisonous plants of the Western states have been published (Ralphs et al., 1991; Whitson, 1994) and specific recommendations for control of poisonous plants and other weeds in local areas can be obtained from county extension agents or agricultural advisors. Biological control may provide long-tem suppression of introduced poisonous plants if population densities of the specific biocontrol agent can be maintained. However, biological control seldom provides a total solution to weed problems. Saint-John’s-wort (Hypericum pelforcrtur~~) was introduced tu California around 1900, and by 1951 had infested over 800,000 ha. It was one of the most serious poisonous plants to sheep, causing primary photosensitization. The Klamath weed beetle (Clzrysolirza qucrclrigerr~irzc~) was found to be host-specific to Saint-John’s-wort. It was introduced in 1951 and almost eliminatedtheweed in areas wherethe beetle is adapted (Huffaker and Kennett, 1959). A problem remains where Saint-John’s-wort grows at higher elevations and cooler temperature than the beetle can survive. Tansy ragwort is one of the most serious noxious weeds and poisonous plant prob-

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lems in the Pacific Northwest, as it invades cultivated pastures, rangelands, and forests. Since 1960, three insects have been introduced from tansy ragwort's natural habitat in Europe: the cinnabar moth ( T ~ ~ rjacobaen), ia the ragwort flea beetle (Longitcmzls jacobaen), and the ragwort seed fly (Peyohvle~yiaseneciella) (Coombs et al., 1991). They have been successful in keeping tansy ragwort populations in check in the mesic regions along the coast, but have been unsuccessful in controlling it in the higher elevations and drier areas east of the Cascade Mountains. Annual ryegrass toxicity is caused by the bacterium Clawhxter toxicus, which produces corynetoxin. The bacteria occurs in galls in the seed heads of the grass, and is introduced to the grass by a parasitic nematode, A~rgrrir?njhest.Controlling this intermediate host may eliminate the disease. Opportunities for biological control include fungal antagonists, nontoxic Clcrvibncter. spp., and nonadhesive A. Jimesto populations to which C. tosicus cannot adhere (Riley, 1994). There is some potential for biological control of native poisonous plants. Populations of woolly locoweed die off in part due to the root-feeding weevil Cleonidius ti-ivittatus (Pomerinke et al., 1995; Thompson et al., 1995), but the weevil will notprevent locoweed population buildup. Three insects offer potential in controlling broom snakeweed: the redlegged grasshopper (Hesperotettix vir-idis),the leaf-tier S y z ~ 0 ~ 7lynosymxl, a and therootborer Crossidius prdchellus (Thompson and Richman, 1993). Further research is necessary to manage the populations of these potential biocontrol insects. A nontraditional approach hasbeen proposed using a native insect, the larkspur mirid (Hopplomachusafigur-atus),to reduce the risk of cattle poisoning from talllarkspur. The insect feeding damages larkspur leaves and reproductive heads and renders larkspur unpalatable to cattle (Jones et al., 1998). Cattle will not eat mirid-damaged larkspur plants (Ralphs et al., 1997); thus the risk of poisoning is greatly reduced. Research is continuing in transplanting the mirids and manipulating their population to increase their level of damage.

V.

CONCLUSIONS

Many of the management recommendations to prevent poisoning evolved from early USDA researchers, such as C. D. Marsh, A. B. Clawson, H. Marsh, W. T. Huffinan, W. Binns, L. F. James, and others i n state experiment stations who spent their time in the field observing the conditions under which poisoning occurred. Preventive management strategies were the practical result of their observations. Much of this early work was compiled by Kingsbury (1964) in his classic book Poisonous PImrts of the United States nrzd C m c l d ~Peter ~ . Cheeke (1998) has recently compiled a comprehensive list of natural toxicants in forages and poisonous plants. Recent research at the USDA/ARS Poisonous Plant Lab has further documented the toxin levels in specific poisonous plants and grazing behavior toward those plants, described the conditions under which poisoning occurs, and developed grazing strategies to avoid these conditions. Future technology such as toxin binding, vaccines, microbial breakdown of toxins, and aversive conditioning may further reduce the risk of poisoning. Finally, controlling poisonous plants with herbicides offers an immediate solution to poisoning plants if it is practical, economical, and environmentally acceptable.

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REFERENCES Aldous, A.E. 1917. Eradicating tall larkspur on cattle ranges in the national forests. USDA Fnmers Bull. 826. Allen, J.G., K.A. Than. J.A. Edgar, G.H. Doncon, G. Dragicevic, and V.H. Kosmac. 1994. Field evaluations of vaccines against lupinosis, pp. 427-432. In: S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford. Oxon, UK. Allison, M.J. 1978.The role of ruminal microbes in the metabolismof toxic constituents from plants. pp. 101-1 18. Zn: R.F. Keeler, K.R. Van Kampen, and L.F. James (eds.), Effectsof Poisonous Plants on Livestock. Academic Press. New York. Allison, M.J., K.A. Dawson. W.R. Mayberry, and J.G. Foss. 1985. O-~nlobncter-.for-i~zigens: oxalatedegrading anaerobes that inhabit the gastrointestinal tract. Arch. Micr-obiol. 141: 1-7. Allison, M.J., and M.A. Rasmussen. 1992. The potential for plant detoxification through manipulation of the rumen fermentation, pp. 367-376. In: L.F. James, R.F. Keeler, E.M. Bailey Jr.. P.R. Cheeke, and M.P. Hegarty(eds.). PoisonousPlants: Proceedings of the third international symposium. Iowa State Univ. Press. Ames. Anderson, R.C., W. Majak, M.A. Rasmussen, and M.J. Allison. 1998. Detoxification potential of a new species of ruminal bacteria that metabolize nitrate and naturally occurring nitrotoxins, pp. 154-158. In: T. Garland and A.C. Bair (eds.), Toxic Plantsand Other Natural Toxicants. CAB International, Okon UK. Anderton, N., J.J. Gosper, and C. May. 1994. The inclusion of pyrrolizidine alkaloids by s- and bcyclodextrins. Aust. J. Chem. 47:853-857. Bachman, S.E.. M.L. Galyean, G.S. Smith, D.M. Hallford, and J.D. Graham. 1992. Early aspects of locoweed toxicosis and evaluation of a mineral supplement or clinoptilolite as dietary treatments. J. Anirrz. Sci. 70:3125-3132. Basden, K.W.,andR.R.Dalvi.1987.Determinationof total phenolics in acorns from different species of oak trees in conjunction with acorn poisoning in cattle. Vet. H m . Toxicol. 29: 305-306. Basson, P.A., A.G. Norval. J.M. Hofmeyr, H. Ebedes, R. Anitra Schultz. T.S. Kellerman, and J.A. Minne.1984.Antelopesandpoisonousplants,pp.695-701. In: 13thWorld Congress on Diseases of Cattle. Hoeclzst Phnr-n?.,Johannesburg. Bate-Smith, E.C. 1972. Attraction and repellents in higher animals. Zn: J.B. Harborne (ed.). Phytochemical Ecology. PI-oc.Plzytochem. SOC.8:45-56. Academic Press, New York. Beasley, V.R.. D.C. Dorman, J.D. Fikes. S.G. Diana, and V. Woshner. 1997. A Systems Affected Approach to Veterinary Toxicology. Univ. Illinois. Champaign. Becker. R.B., W.M. Neal, P.T.D. Arnold, and A.L. Shealy. 1935. A study of the palatability and possible toxicity of 11 species of Ci-otolnr-in,especially of C. spectnbilis Roth. J. Agric. Res. 50191 1-932. Beier,R.C..andJ.O.Norman.1990. The toxicfactor in white snakeroot:identity, analysis and prevention. Vet. Hzalz. Toxicol. 32. (Suppl.):81-88. Binns, W., L.F. James, J.L. Shupe,and G. Everett. 1963. A congenital cyclopian-type malformation in lambs induced by maternal ingestion of a range plant, Verntrzmz crrltfonziczan.Am. J. Vet Res. 24:1164-1175. Bowns,J.E. 1988. The importance of poisonousplants asforages inthe intermountain region. pp.377-390. Zrz: L.F.James.M.H. Ralphs, and D.B. Nielsen (eds.),The Ecologyand Economic Impact of Poisonous Plants on Livestock Production. Westview Press. Boulder. co. Box, T.W., and J.C. Malechek. 1987. Grazing on the American rangelands. Proc. Western Section American Society of Animal Science 38: 107- 115. Brock, J.H. 1988. Biological control in brush/weed management programs. Rangelands 10:32-34. Bryant, J.P., F.S. Chapin. and D.R. Klein. 1983. Carbodnutrient balance in boreal plants in relation to vertebrate herbivory. Oikos 40:357-368.

Plant Toxicants and Livestock

463

Bryant, J.P., P.B. Reichardt, and T.P. Clawson. 1992. Chemically mediated interactions between woody plants and browsing mammals. J. Rarzge Mmcrge. 45: 18-24. Burrows, G.E., R.J. Tyrl. D. Rollins, T.R. Thedford, W. McMurphy, and W.C. Edwards. [no date.] Toxic plants of Oklahoma and the Southern plains. Oklahoma Coop. Ext. Ser. E-868. Cheeke, P.R. 1994. The role of the liver in the detoxification of poisonous plants. pp. 28 1-286. Zrz: S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford, Oxon, UK. Cheeke, P.R. 1998. Natural Toxicants in Feeds, Forages and Poisonous Plants. Interstate Publishers, Danville, IL. Cheeke, P.R., and J. Huan. 1998. Species differences in bioactivation and detoxification of pyrrolizidine alkaloids, pp. 559-563. Irz: T. Garland and A.C. Barr (eds.), Toxic Plants and Other Natural Toxicants. CAB International, Wallingford, Oxon, UK. Clawson, A.B., and E.A. Moran. 1937. Toxicity of arrowgrass for sheep and remedial treatment. USDA Tech. Bull. 580. Coley. P.D., J.P. Bryant, and F.S. Chapin. 1985. Resource availability and plant antiherbivore defense. Science 230:895-899. Coombs. E.M.. E.B. Bedell, and P.B. McEvoy. 1991. Tansy ragwort (Seweciojucobaea):importance, distribution, and control in Oregon. pp. 419-428. Irz: L.F. James. J.O. Evans, M.H. Ralphs, and R.D. Child (eds.), Noxious Range Weeds. Westview Press. Boulder, CO. Craig, A.M., and L.L. Blythe. 1994. Review of ruminal microbes relative to detoxification of plant toxins and environmental pollutants. pp. 462-468. In: S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford, Oxon, UK. Davis, A.M. 1982. The occurrence of anagyrine in a collection of Western American lupines. J. Range Manage. 3 5 8 1-84. Dollahite, J.W.. and W.V. Anthony. 1957. Poisoning of cattle with Gtierrezia vzicrocephala, a perennial broomweed. J. Am. Vet. Med. Assoc. 130525-530. Dollahite, J.W., G.T. Housholder, and B.J. Camp. 1966. Oak poisoning in livestock. Tex. Agr. Exp. Stcr. B d l . B-1049. Doran, C.W., and J.T. Cassady. 1944. Management of sheep on range infested with orange sneezeweed. U.S. Dept. Agr. Circ. 691 : 1-28. Dugarte-Stavanja, M., G.S. Smith, T.S. Edrington, and D.M. Hallford. 1997. Failure of dietary bentonite clay. silent herder mineral supplement, or parenteral banamine to alleviate locoweed toxicosis in rats. J. Arzim. Sci. 75: 1867-1875. Dwyer, D.D. 1978. Impact of poisonous plants on western U.S. grazing systems and livestock operations, pp. 13-22. Zrz: R.F. Keeler, K.R. Van Kampen, and L.F. James (eds.), Effects of Poisonous Plants on Livestock. Academic Press, New York. Edgar, J.A. 1994. Vaccination against poisoning diseases, pp. 421-426. Zit: S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford, Oxon, UK. Edgar, J.A. 1998. Treatment and prevention of livestock poisoning: where to from here? pp. 21 1214. In: T. Garland and A.C. Barr (eds.), Toxic Plants and Other Natural Toxicants. Cab International, Wallingford, Oxon. UK. Edgar, J., K. Than, N. Anderton, A. Payne, Y. Cao, A. Michalewicz. P. Cockrum, P. Stewart, J. Baell, and J. Allen. 1998. Towards a commercial vaccine against lupinosis, pp. 196-204. Zrl: T. Garland and A.C. Barr (eds.). Toxic Plants and Other Natural Toxicants. Cab International, Wallingford, Oxon, UK. Everist, S.L. 1981. Poisonous Plants of Australia, pp. 10-55. Angus & Robertson, Sydney. Filipov, N.M.. F.N. Thompson, H.S. Hill, D.L. Dawe, J.A. Stuedemann, J.C. Price, and C.K. Smith. 1998. Vaccination against ergot alkaloids and the effect of endophyte-infected fescue seed in diets of rabbits. J. Arlim. Sci. 76:2456-2463. Forbes, I., and E.W. Beck. 1954. A rapid biological technique for screening blue lupine populations for low alkaloid plants. Agrorz. J . 46:528-529.

464

Ralphs

Forbes, T., and G.W. Burton. 1960. Blanco blue lupine: a sweet variety insured against bitter mixtures. Georgia Agric. Res. 2:3. Freeland, W.J., and D.H. Janzen. 1974. Strategies in herbivory by mamals: the role of plant secondary compounds. Am. Nntur. 1 08:269-289. Galey, F.D.,V.R. Beasley, D. Schaeffer, and L.E. Davis.1990. Effect ofaqueousblackwalnut (Jzrglans nigra) extract on isolated equine vessels. Am. J. Vet. Res. 51:83-88. Galey, F.D.,D.M. Holstege, B.J. Johnson. and L. Siemens. 1998. Toxicity and diagnosis of oleander (Nerizm oleander)poisoning in livestock, pp. 215-219. 1 ~T.: Garland and A.C. Barr (eds.), Toxic Plants and Other Natural Toxicants. Cab International, Wallingford, Oxon, UK. Garcia, J., and W.G. Hankins. 1973. The evolution of bitter and the acquisition of toxiphobia. pp. 1-12. I t ? : D. Denton (ed.), Fifth International Sytnposium on Olfaction andTaste. Melbourne, Aust. Garcia. J., P.A. Lasiter, F. Bermudez-Rattoni, and D.A. Deems. 1985. A general theory of aversion learning, pp. 8-21. In: N.S. Braveman and P. Bronstein (eds.), Experimental Assessments and Clinical Applications of Conditioned Food Aversions. A m . NY Acnd. Sci. 443. Gardner, D.R.,K.E. Panter,L.F.James, and B.L. Stegelmeier.1998.Abortifacienteffectsof lodgepole pine (Pirzus corltortcr) and coInn1on juniper (Jznziperus cominunis) on cattle. Vet. H u m Tosicol. 40260-263. Gershenzon, J. 1984. Changes inthelevelsofplantsecondarymetabolites under waterand nutrient stress. pp.273-320. IH: B.N. Tirnmermam. C. Steelink, andF.A. Loewus (eds.), RecentAdvances in Phytochemistry; Phytochemical Adaptationsto Stress. Plenum Press, NewYork. Gregg, K., G. Allen, and C. Beard. 1996. Genetic ~nanipulationof rumen bacteria: from potential to reality. Arm. J. Agric. Res. 47247-256. Hagtnan, J.L., G.C. Marten, and A.W. Hovin. 1975. Alkaloid concentration in plant parts of reed canarygrass of varying maturity. Crop Sci. 15:31-33. Hammond. A.C.. J.J. Allison, M.J. Williams, G.M. Prine. and D.B. Bates. 1989. Prevention of leucaena toxicosis of cattle in Florida by ruminal inoculation with 3-hydroxy-4-(I h)-pyridonedegrading bacteria. Anr. J. Vct. Res. 502176-2180. Hart. C.R., A. McGinty, and B.B. Carpenter. 1998. Toxic Plant Handbook: Integration Strategies for West Texas. Tems Agr. Ext. Set-. Bull. 6072. Hegarty. M.P. 1981. Deleterious factors in forages affecting animal production, pp. 133-150. IT[: J.B. Hacker (ed.), Nutritional Limits to Animal Production from Pastures. Common. Agr. Bureaux, Slough UK. Herron, J.W., and D.E. LaBore. 1979. Some Plants of Kentucky Poisonous to Livestock. Keiztucky Coop. Ext. Set-. ID-2. Hill, R.J. andD. Folland. 1986. Poisonous Plants of Pennsylvania.P e m . Dept. Agr. Bur. Plarzt h d . , Harrisburg. PA. Huffaker, C.B., and C.E. Kennett. 1959. A ten year study of vegetation changes associated with biological control of klamath weed. J. Rurzge Manage. 12:69-83. Huffman. W.T.. and J.F. Couch. 1942. Plants that poison livestock, pp. 354-373. h z : Keeping Livestock Healthy. USDA Yearbook of Agric. Hulbert, L.C., and F.W. Oehme. 1968. Plants Poisonous to Livestock. Kansas State Univ. Press, Manhattan. Huddleston. E.W.. and R.D. Pieper. 1989. Snakeweed: Problems and Perspective-Ecology. Management. Biological Control. Toxicology. New>Mex. Agr. Esy. Sta. Bd1. 751. James.L.F..andJ.E.Butcher.1972.Halogetonpoisoningofsheep:effect of highleveloxalate intake. J. Arziwt. Sci. 35:1233-1238. James. L.F.. and E.H. Cronin. 1974. Management practices to minimize death losses ofsheep grazing on Halogeton-infested range. J. Range Manage. 27:423-426. James.L.F., W.J. Hartley.andK.R.VanKampen.1981. Syndromes of Astrcrgcrlzu poisoningin livestock. J. A m Vet. Men. Assoc. 178:146-150.

Plant Toxicants and Livestock

465

James, L.F.. R.F. Keeler, A.E. Johnson. M.C. Williams, E.H. Cronin, and J.D. Olsen. 1980. Plants Poisonous to Livestock in the Western States. USDA/SEA Agr. Zrlfo. Bull. 415. James, L.F.,R.E. Short, K.E.Panter,R.J.Molyneux.L.D. Stuart, and R.A. Bellows.1989. Pine needle abortion in cattle: a review and report of 1973-1984 research. Cornell Vet. 79:39-52. James, L.F., and J.L. Shupe. 1984. Selenium poisoning in livestock. Rangelands 6:64-67. James. L.F., and K.R. Van Kampen. 1974. Effect of protein and mineral supplementation on potential locoweed (Astragalrrs spp.) poisoning in sheep. J. Anr. Vet. Med. Assoc. 164: 1042-1043. James, L.F.. K.R. Van Kampen, and G.B. Staker. 1969. Locoweed (Astragalus lentigirtoszcs)poisoning in cattle and horses. J. Am. Vet. Med. Assoc. 155:525-530. Jessup, D.A., H.J. Boermans, and N.D. Kock. 1986. Toxicosis in tule elk caused by ingestion of poison hemlock. J. Am. Vet. Med. Assoc. 189: 1 173-1 175. Johnson, A.E. 1982a. Toxicologic aspects of photosensitization in livestock. J. Ncd. Curzcer Znst. 69:253-258. Johnson, A.E. 1982b. Failure of mineral-vitamin supplements to prevent tansy ragwort (Senecio jacobaen) toxicosis in cattle. Am. J. Vet. Res. 43:718-723. Johnson, A.E.. R.J. Molyneux, and G.B. Merrill. 1985. Chemistry of toxic range plants. Variation in pyrrolizidine alkaloidcontent of Senecio, Amsinckia, and Crotalnria species. J. Agric. Food Clrenr. 3350-55. Johnson, A.E., R.J. Molyneux,andM.H.Ralphs.1989. Senecio: a dangerous plant for manand beast. Rangela~zds11:26 1-264. Johnston, W.H., A.M. Craig, L.L. Blythe, J. Hovermale,and K. Walker. 1998.Pyrrolizidine alkaloid detocification by and ovine ruminal consortium and itsuse as a ruminal supplement in cattle. pp. 185-190. In: T. Garland and A.C. Barr (eds.). Toxic Plants and Other Natural Toxicants. CAB International, Wallingford. Oxon, UK. Jones, R.J. 1985. Lezccaena toxicity and the ruminal degradation of mimosine. pp. 111-1 19. In: A.A. Seawright, M.P. Hegarty, L.F. James, and R.F. Keeler (eds.). Plant Toxicology. The Queensland Poisonous Plants Committee, Yeerongpilly, Queensland. Jones, R.J., and R.G. Megarrity. 1986. Successful transfer of DHP-degrading bacteria from Hawaiian goats to Australian ruminants toovercome the toxicity of Lmcaena. Aust. Vet. J. 63259-262. Jones, W.A., M.H. Ralphs, and L.F. James. 1998. Use of a native insect to deter grazing and prevent poisoninginlivestock.pp.23-28. IH: T. Garland and C.A. Barr (eds.), Toxic Plants and Other Natural Toxicants. CAB International, Wallingford. Oxon, UK. Keeler, R.F. 1976. Lupin alkaloids from teratogenic and nonteratogenic lupins. 111. Identification of anagyrine as theprobable teratogen by feeding trials. J. Toxicol. Envirorz. Heultlr 1:887-898. Keeler, R.F. 1978. Reducing incidence of plant-caused congenital deformities in livestock by grazing management. J. Range Mancrge. 31 :355-360. Keeler, R.F. 1991. Congenital malformations caused by poisonous plants. C o n p Pathol. Bull. 23: 1-6. Keeler, R.F., D.C. Baker, and W. Gaffield. 1991. Solanum alkaloids, pp. 607-636. In: R.P. Sharma and D.K. Salunkhe (eds.), Mycotoxins and Phytoalexins. CRC Press, Boca Raton, FL. Keeler,R.F.,E.H.Cronin.andJ.L. Shupe. 1976. Lupin alkaloids from teratogenicandnonteratogenic lupins. IV. Concentration of total alkaloids. individual major alkaloids, plant part and stage of growth and their relationship to crooked calf disease. J. Toxicol. Environ. Health 1~899-908. Keeler, R.F., and R. Gross. 1980. The total alkaloid and anagyrine contentsof some bitter and sweet selection of lupin species used as food. J. Elrviron. Patlzol. Toxicol. 3:333-340. Keeler, R.F., L.F. James, J.L. Shupe, and K.R. Van Kampen. 1977. Lupine-induced crooked calf disease and a management method to reduce incidence. J. Range Manage. 30:97-102. Kingsbury, J.M. 1964. Poisonous plants of the United States and Canada. Prentice-Hall,Englewood Cliffs, NJ. Krueger, W.C., and L.A.Sharp. 1978. Management approachesto reduce livestockloss frompoisonous plants on rangeland. J. Range Manage. 31:347-350.

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Lanigan, G.W. 1976. Peptococcus Ileliotrir2redllcarzs, a cytochrome-producing anaerobe which metabolises pyrrolizidine alkaloids. J. Microbiol. 94: 1- 10. Lanigan, G.W., S.L. Payne, andJ.D.Peterson.1978.Antimethanogenicdrugsand Heliotropizrnl etrropaerun poisoning in penned sheep. Allst. J. Agric. Res. 29: 1281- 1292. Laycock. W.A. 1978. Coevolution of poisonous plants and large herbivores on rangelands. J. Range Manage. 31:335-342. Majak. W., and M. Engelsjord. 1988. Levels of a neurotoxic alkaloid in a species of low larkspur. J. Rcrnge Mcrnage. 41224-226. Majak. W., C. Hunter, and L. Stroesser. 1998. Tolerance in cattle to timber milkvetch (Astragchs miser var. serotinrls) due to changes in rumen microbial populations, pp. 239-242. Zn: T. Garland and A.C. Bary (eds.), Toxic Plantsand Other Natural Toxicants. CAB International, Wallingford, Oxon, UK. Majak. W.A., R.E. McDiarmind, J.W. Hall, and A.L. Van Ryswyk. 1980. Seasonal variation i n the cyanide potential of arrowgrass (Treglochin rncrritima). Can. J. Plcmt Sci. 60: 1235- 1241. Majak, W., A. Mclean, T.P. Pringle, and A.L. Van Ryswyk. 1974. Fluctuations in miserotoxin concentration of timber milkvetch on rangelands in British Columbia. J. Range Manage. 27: 363-366. Majak, W., P.D. Parkinson, R.J. Williams, N.E. Looney, and A.L. Van Ryswyk. 1977. The effect of light and moisture on columbia milkvetch toxicity in lodgepole pine forests. J. Range Mamge. 30:423-427. Majak, W., L. Stroesser, J.W.Hall, D.A. Quinton, andH.E. Douwes. 1996.Seasonalgrazingof Columbia milkvetch by cattle on rangelands in British Columbia. J. Range Manage. 49223227. Majak. W., R.J. Williams,A.L.VanRyswyk,andB.M. Brooke. 1976. The effectofrainfallon columbia milkvetch toxicity. J. Range Mcrizcrge. 29:28 1-283. Manners, G.D., K.E. Panter, and S.W. Pelletier. 1995. Structure-activity relationships of norditerpenoid alkaloids occun-ing in toxic larkspur (Delphiniltm) species. J. Nut. Prod. 58:863-869. Marsh, C.D. 1909. The Locoweed Disease of the Plains. USDA Bur. Aninl. I d . Bull. 112. Marsh, C.D. 1913. Stock Poisoning Due to Scarcity of Food. USDA Farmers Bull. 536. Marsh, C.D. 1916. Prevention of Losses of Livestock from Plant Poisoning. USDA Farmers Brdl. 720. Marsh, C.D. 1929. Trembles. USDA Farmers Brlll. 1593. Marsh, C.D., and A.B. Clawson. 1922. The Stock-Poisoning Death Camas. USDA Famzers Bull. 1273. Marsh, C.D.,and A.B. Clawson. 1930. Mountain Laurel (Kcrbnialatifolia)and Sheep Laurel (Kcrlrnin angrrstifolia) as Stock-Poisoning Plants. USDA Tech. Bd1. 219. Marsh, H.D. 1958. Newsoms Sheep Diseases, 2nd ed. Williams & Wilkins, Baltimore. Marten, G.D..R.M. Jordan, andA.W. Hovin. 1976.Biologicalsignificanceofreed canarygrass alkaloids and associated palatability variation to grazing sheep and cattle. Agron. J. 68:9099 14. McCrabb. G.J., K.T. Berger, T. Manger, C.May and R.A. Hunter. 1997. Inhibiting methane production in Brahman cattle by dietary supplementation with a novel compound and its effects on growth. Aust. J. Agric. Res. 48:323-329. McDonald, I.W. 1981. Detrimental substances in plants consumed by grazing ruminants, pp. 349378. Zrz: F.H.W.Morley (ed.), WorldAnimal Science:Grazing Animals.Elsevier,New York. McKenzie. R.A. 1991. Bentonite as therapy for Lcriztana calmv-a poisoning of cattle. Aust. Vet. J. 681146-148. Merrill, L.B., and J.L. Schuster. 1978. Grazing management practices affect livestock losses from poisonous plants. J. Range Maizcrge. 3 1:35 1-354. Molyneux. R.J.. and L.F. James. 1982. Loco intoxication: indolizidine alkaloids of spotted locoweed (Astragallu lentiginoslu).Science 21 6: 190-1 96.

Plant Toxicants and Livestock

467

Molyneux, R.J., L.F. James, M.H. Ralphs. J.A. Pfister. K.E. Panter, and R.J. Nash. 1994. Polyhydroxy alkaloid glycosidase inhibitors from poisonous plants of global distribution: analysis and identification, pp. 107-112. In: S.M. Colgate and P.R. Dorling (eds.), Plant Associated Toxins. CAB International, Wallingford, Oxon, UK. Mooney, H.A., S.L. Gulmon. and N.D. Johnson. 1983. Physiological constraints on plant chemical defenses, pp. 21-36. In: P.A. Hedin (ed.), Plant Resistance to Insects. ACS Symposium Series No. 208, American Chemical Society, Washington, DC. Nelson, P.D., H.D. Mercer. H.W. Essig. and J.P. Minyard. 1982. Jimson weed toxicity in cattle. Vet. Hum. Toxicol. 24:321-325. Nsimba-Llubaki, M.. and W.J. Peumans. 1986. Seasonal fluctuations of lectins in bark of elderberry (Sanlbucus nigra), and black locust (Robinia pseudoacacia). Plant Plrysiol. 80:747-751. Olsen, J.D. 1978. Tall larkspur poisoning in cattle and sheep. J. Am. Vet. Med. Assoc. 173:762765. Olsen. J.D., T.E. Anderson, J.C. Murphy, and G. Madsen. 1983. Bur buttercup poisoning of sheep. J. Am. Vet. Med. Assoc. 183:538-543. Panter, K.E., D.R. Gardner, C.C. Gay, L.F. James, R. Mills, J.M. Gay, and T.J. Baldwin. 1997. Observations of Lupin~tssulpl~ureus-induced crooked calf disease. J. Range Manage. 50: 587-592. Panter, K.E., D.R. Gardner, and R.J. Molyneux. 1998. Teratogenic and fetotoxic effects of two piperidine alkaloid-containing lupines (L.for~nosz4sand L. arhstus) in cows. J. Not. Toxins 7:131-140. Panter, K.E., and L.F. James. 1989. Death camas early grazing can be hazardous. Rangelands 11: 147-149. Panter, K.E., and L.F. James. 1990. Natural plant toxicants in milk: a review. J. Anim. Sci. 68:892904. Panter, K.E., R.F. Keeler, and D.C. Baker. 1988. Toxicosis in livestock from the hemlocks (Conium and Cicuta spp.). J. Anim Sci. 662407-2413. Panter, K.E., R.J. Molyneux, R.A. Smart, L. Mitchell, and S. Hansen. 1993. English yew poisoning in 43 cattle. J. Am. Vet. Med. Assoc. 202:1476-1377. Panter, K.E., M.H. Ralphs, R.A. Smart, and B. Duelke. 1987. Death camas poisoning in sheep: a case report. Vet. Hunz. Toxicol. 29:45-48. Pass, M.A. 1991. Poisoning of livestock by Lantana plants, pp. 297-31 1. IH: R.F. Keeler and A.T. Tu (eds.), Toxicology of Plant and Fungal Compounds. Vol. 6. Marcel Dekker, New York. Payne, A.L., P.A. Cockrum. and J.A. Edgar. 1994. Metabolic transformation of corynetoxin and tunicamycin by Alternaria ulternutc~,pp. 445-449. In: S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins-Agricultural, Phytochemical and Ecological Aspects. CAB Tnternational, Oxon. Payne A.L., K.A. Than, P.L. Stewart, and J.A. Edgar. 1992. Vaccination against lupinosis, pp. 234238. In: L.F. James, R.F. Keeler, E.M. Bailey Jr.. P.R. Cheeke, and M.P. Hegarty (eds.). Poisonous Plants: Proceedings of the third international Symposium. Iowa State Univ. Press, Ames. Pfister, J.A. 1999. Food aversion learning to reduce cattle consumption of ponderosa pine needles. J. Range Munuge. (in review). Pfister, J.A., and D.C. Adam. 1993. Factors influencing pine needle consumption by grazing cattle during winter. J. Range Mrrrzuge. 46:394-398. Pfister, J.A., D.R. Gardner, and K.W. Price. 1997a. Grazing risk on tall larkspur-infested ranges. Rangelards 19:12-15. Pfister, J.A., and G.D. Manners. 1991. Mineral salt supplementation of cattle grazing tall larkspurinfested rangeland during drought. J. Range Manage. 44: 105-1 11. Pfister, J.A., G.D. Manners, D.R. Gardner, K.W. Price, and M.H. Ralphs. 1996. Influence of alkaloid concentration on acceptability of tall larkspur (Delplliniunt spp.) to cattle and sheep. J. Chem. EcoI. 22: 1147-1 168.

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Ralphs

Pfister, J.A., M.H. Ralphs. G.D. Manners, K.E. Panter. L.F. James, B .L. Stegelnleier. and D.R. Gardner. 1993. Tall larkspur poisoning 11 cattle: current research and recommendations. Rangelarzds 15:157- 160. Pfister, J.A., M.H. Ralphs. G.D. Manners, K.W. Price, and L.F. James. 1997b. Early season grazing by cattle of tall larkspur-infested rangeland. J. Range Mrrrqe. 50:391-398. Pieper, R.D., and K.C. McDaniel. 1989. Ecology and management of broom snakeweed. pp. 1-12. Irz: E.W. Huddleston and R.D. Pieper (eds.), Snakeweed: Problems and Perspectives, New Mexico Agr. Exp. Stcr. Bull. 751. Pomerinke, M.A., D.C. Thompson, and D.L. Clason. 1995. Bionomies of Clemidizrs trivittcrtzls (Coleoptera: Curculionidae): native biological control of purple locoweed (Rosales: Febaceae). Emiron. Erltomol. 34: 1697- 1702. Provenza, F.D., J.A. Pfister. and C.D. Cheney. 1992. Mechanisms of learning in diet selection with reference to phytotoxicosis in herbivores. J. Rurzge Mlznage. 45:36-45. Provenza, F.D. 1995. Positive feedback as an elementary determinant of food preference and intake in ruminants. J. RerrzgeMcrnage. 482-17. Pulsipher, G.D., M.L. Galyean, D.M. Hallford, G.S. Smith, and D.E. Kiehl. 1994. Effects of graded levels of bentonite on serum clinical profiles, metabolic hormones, and serum swainsonine concentrations in lambs fed locoweed (O.~~rropis sericerr). J. Aninz. Sci. 72:1561-2569. Ralphs. M.H. 1987. Cattle grazing white locoweed: influence of grazing pressure and palatability associated with phenological growth stage. J. Range Manage. 10330-332. Ralphs. M.H. 1997. Persistence of aversions to larkspur i n naive and native cattle. J. Range Mcrrzage. 501367-370. Ralphs, M.H., and V.L. Bagley. 1988. Population cycles of Wahweap milkvetch on the Henry Mountains and seed reserve in the soil. Great Basin Ncrtuml. 48:531-547. Ralphs, M.H., D. Graham, M.L. Galyean. and L.F. James. 1997. Creating aversions to locoweed in naive and familiar cattle. J. RcrrzgeMarzcrge. 50:361-366. Ralphs, M.H.. D. Graham, R.J. Molyneux, and L.F. James. 1993. Seasonal grazing of locoweeds by cattle in northeastern New Mexico. J. Range Mcrnage. 46:416-430. Ralphs, M.H., and L.F. James. 1998. Locoweed grazing. J. Nrrtrrml Tosim 8:47-51. Ralphs. M.H., L.F. James, D.B. Nielsen, and K.E. Panter. 1984.Management practices reduce cattle loss to locoweed on high mountain range. Rangelands 6:175-177. Ralphs, M.H., D.T. Jensen, J.A. Pfister, D.B. Nielsen, and L.F. James. 1994. Storms influence cattle to graze larkspur. J. Rarzge Manage. 47:275-378. Ralphs, M.H.. W.A. Jones, and J.A. Pfister. 1997. Damage from the larkspur mirid deters cattle grazing of larkspur. J. Range Manage. 50:371-373. Ralphs, M.H., G.D. Manners. J.A. Pfister. D.R. Gardner, and L.F. James. 1997. Toxic alkaloid concentration in tall larkspur species in the western U.S. J. Range Manage. 50:497-502. Ralphs, M.H., and R.J. Molyneux 1989. Livestock grazing locoweed and the influence of swainsonine on locoweed palatability and habituation, pp. 39-49. h z : L.F. James, A.D. Elbein. R.J. Molyneux, and C.D. Warren (eds.), Swainsonine and Related Glycosidase Inhibitors, Iowa State Univ. Press. Anles. Ralphs, M.H., and J.D. Olsen. 1992. Prior grazing by sheep reduces waxy larkspur consunlption by cattle. J. Range Manage. 45: 136-139. Ralphs, M.H., and J.D. Olsen. 1998. Conditioned food aversion: a management tool to prevent livestock poisoning, pp. 223-326. In: T. Garland and A.C. Barr (eds.), Toxic Plants and Other Natural Toxicants. CAB International. Wallingford, Oxon, UK. Ralphs, M.H., and L.A. Sharp. 1988. Management to reduce livestock loss from poisonous plants, pp. 391-405. In: L.F. James, M.H. Ralphs. and D.B. Nielsen (eds.), The Ecology and Economic Impact of Poisonous Plants on Livestock Production. Westview Press. Boulder, Co. Ralphs, M.H., T.D. Whitson, and D.N. Ueckert. 1991. Herbicide control of poisonous plants. Rcrrzgelands 13:73-77.

Plant Toxicants and Livestock

469

Rasmussen, M.A., and L.F. James. 1994. Selenium metabolism in the rumen, pp. 512-515. In: S.M. Colgate and P.R. Dorling (eds.), Plant Associated Toxins. CAB International, Wallingford, Oxon, UK. Rhoades, D.F. 1979. Evolution of plant chemical defense against herbivores, pp. 1-55. In: G.A. Rosenthall and D.H. Janzen(eds.), Herbivores, Their Interaction with Secondary Plant Metabolites. Academic Press, New York. Rice, R.L.. G.C. Schurig, and D.J. Blodget. 1998. Evaluation of physiologic indices in mice vaccinated with protein-ergotamine conjugats and fed an endophyte-infected fescue diet. Am. J. Vet. Res. 59:1258-1262. Riley, I.T. 1993. Opportunities forbiological control of the toxigenic Anguirzcr/CZavibucter association in annual ryegrass, pp. 439-444. I n : S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford, Oxon, UK. Rowe, L.D.1991. Cassia-induced myopathy,pp.335-351. h a : R.F.KeelerandA.T. Tu (eds.), Handbook of Natural Toxins-Toxicology of Plant and Fungal Compounds. Marcel Dekker, New York. Schell, T.C., M.D. Lindemann. E.T. Kornegay. D.J. Blodgett, and J.A. Doen. 1993. Effectiveness of different types of clay for reducing the detrimental effects of aflatoxin-contaminated diets on performance and serum profiles of weanling pigs. J. Arrinz. Sci. 7 1: 1226. Schuster, J.L. 1978. Poisonous plant management problems and control measures on U.S. rangelands, pp. 23-34. In: R.F. Keeler, K.R. VanKanlpen, and L.F. James (eds.). Effects of Poisonous Plants on Livestock. Academic Press, New York. Sharrow, S.H., and W.D. Mosher. 1982. Sheep as a biological control agent for tansy ragwort. J. Rarzge Manage. 35:480-451. Shupe, J.L., W. Binns, L.F. James, and R.F. Keeler. 1967. Lupine, a cause of crooked calf disease. Am. Vet.Mecl. Assoc. 151: 198-203. Sperry, O.E., J.W. Dollahite, G.O. Hoffman, and B.J. Camp. 1964. Texas Plants Poisonous to Livestock. Texas Agr. Exp. Sta. B-1028. Stegelmeier, B.L., K.E. Panter, J.A. Pfister. L.F. James, G.D. Manners, D.R. Gardner, M.H. Ralphs, and J.D. Olsen. 1998. Experimental modification of larkspur (Delpkinizrm spp.) toxicity, pp. 205-210. In: T. Garland and C.A. Barr (eds.). Toxic Plants and Other Natural Toxicants. CAB International, Wallingford, Oxon, UK. Stewart, P.L., and C. May. 1998.Protective effect of cyclodextrins against tunicaminyluracil toxicity in rats and sheep, pp. 179-184. I n : T. Garland and C.A. Barr (eds.). Toxic Plants and Other Natural Toxicants. CAB International, Wallingford, Oxon, UK. Stoddart. L.A., A.H. Holmgren. and C.W. Cook. 1949. Important Poisonous Plants of Utah. Utah Agr. Exp. Sta. Special Report No. 2. Stoddart,L.A.,and A.D. Smith. 1955. RangeManagement, 2ndEd. McGraw-Hill, NewYork. pp. 234-257. Stuart, B.P.. R.J. Cole. and H.S. Gosser. 1981. Cocklebur (Xantlziurtr strro~lariunrvar. strzrntcrrizrm) intoxication in swine: review and redefinition of the toxic principle. Vet. Prrthol. 18:368383. Stuart, S.J., A.L. Payne, D. Reyes,F. Ashton. and J.A. Edgar. 1994. Detoxification of annual ryegrass toxins by Sphirzgobcrcterium rrrrdtiwrzmr, pp. 451-456. I I I :S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford. Oxon, UK. Taylor. C.A., Jr., and M.H. Ralphs. 1992. Reducing livestock losses from poisonous plants through grazing management. J. Rmge Manage. 45:9- 12. Tennant, B., S.G. Dill. L.T. Glickman. E.J. Mirro, J.M. King, D.M. Polak. M.C. Smith, and D.C. Kradel. 1981. Acute hemolytic anemia, methemoglobinema, andHeinz body formation associated with ingestion of red maple leaves by horses. J. Am. Vet. Med. Assoc. 179: 143-1 50. Than, K.A., Y.Cao.A. Michalewicz, and J.A.Edgar. 1998. Development of a vaccineagainst annual ryegrass toxicity, pp. 165-168. In: T. Garland (ed.), Toxic Plants and Other Natural Toxicants. CAB International, Wallingford. Oxon, UK.

470

Ralphs

Than, K.A., N. Anderton, P.A. Cockrum, A.L. Payne, P.L. Stewart, and J.A. Edgar. 1994. Lupinosis vaccine: positive relationship between anti-phomopsin IGG concentration and protection in Victorian fieldtrials, pp. 433-338. In: S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford, Oxon, UK. Thompson, D.C., J.L. Knight, T.M. Sterling, and L.W. Murray. 1995. Preference for specific varieties of woolly locoweed by a specialist weevil, Cleonidius tritittcrtus (SAY). SouthHlest. EirtoItlol. 20:325-333. Thompson. D.C., and D.B. Richman. 1993. A grasshopper that only eats snakeweed, pp. 18-19. In: T.M. Sterling, and D.C. Thompson (eds.), Snakeweed Research Updates and Highlights. New Mexico State Univ. Ag. EX^. Strr. Res. Rep. 673. Throp, F, G.S.Harshfield, L.W. Durrell, and C. G. Barr. 1940. Osytenia acersoa-a plant poisonous to livestock. J. Am. Vet. Med. Assoc. 96:97. Ueckert, D.N., and M.C. Calhoon. 1988.Ecology and toxicology of bitterweed (Hyrnerzo-xysodorata. pp. 131-144. Zn: L.F. James, M.H. Ralphs. and D.B. Nielsen (eds.). The Ecology and Economic Impact of Poisonous Plants on Livestock Production. Westview Press, Boulder, CO. Waller, G.R.. and G.W. Nowacki. 1978. Alkaloid Biology and Metabolism in Plants. Plenum Press. New York. Walter, M., and M. Engelsjord. 1988. Levels of a neurotoxic alkaloid in a species of low larkspur. J. Rcrnge Munage. 41 224-226. Whitson, T.D. 1994. Control of poisonous plants in the Western United States, pp. 468-472. Zrr: S.M. Colegate and P.R. Dorling (eds.), Plant-Associated Toxins. Cab International, Wallingford, Oxon, UK. Williams, M.C., and R.C. Barneby. 1977. The occurrence of nitro-toxins in North America Astragcrlzrs (Fabaceae). Brittortia 29:310-326. Williams. M.C., L.F. James, and B.O. Bond. 1979. Emory rnilkvetch (Astragalus emonanus var. ernoryarzus) poisoning in chicks, sheep, and cattle. Am. J. Vet. Res. 40:403-406. Williams, M.C., and J.D. Olsen. 1984. Toxicity of seeds of three Aesczrlzts spp. to chicks and hamsters. Am. J. Vet. Res. 45:539-542. Wolf. G.J.. and W.R. Lance. 1984. Locoweed poisoning in a New Mexico elkherd. J. Range Manage. 37159-63.

10 Aspergillus Zofia Kozakiewicz CABI Bioscience, Eghnm. Surrej: England

I.

Introduction 471

11. Aspergillus Mycotoxins

A. B. C. D. E. F. G.

472

Aflatoxins 472 Sterigmatocystin 374 Ochratoxin and related compounds Cyclopiazonic acid 475 Patulin 475 Citrinin 475 Neurotropic toxins 476

111. Aspergillosis

474

477

A. Human infections B. Animal infections

477 478

IV. Descriptive Morphology and Terminology 479 V.

Species Group Descriptions A. B.

VI.

480

Species identification and their importance Species descriptions 48 1

481

Control and Prevention 492 A. Factors influencing spoilage of foodstuffs and mycotoxin production B. Reducing mycotoxin levels in foodstuffs 495 References 497

1.

492

INTRODUCTION

The large number of species that constitute the genus Aspergillus occupy a wide spectrum of habitats in thehuman environment. As a consequence, many have become economically important in either harmful or beneficial roles. Their presence leads to deterioration and spoilage of many foodstuffs, while exposure to their spores frequently results in allergic reactions, often of a serious nature. In addition, mycotoxins (metabolic by-products of deterioration) are highly poisonous to the consumer, animal or human. Their beneficial roles relate to industrial processes, particularly to production of acids, vitamins, enzymes, 471

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and certain oriental fermented foods products. Correct identification of the species is therefore of paramount importance. Aspergillus is a hyphomycetous genus. Although first described by Micheli (l), Link (2) is considered to be the validating author. Three major monographs have been produced (3-5). Indeed, the concepts proposed in Ref. 5 still are accepted and followed widely. However, some groups have proved to be problematic and have been further investigated (6-1 1). These problems derive mainly from the widely accepted but highly subjective use of color and physical appearance of growing colonies in identification (12). Thus in recent years more modern techniques have been introduced to improve the taxonomy of Aspergillus. These include: isoenzyme patterns (13- 17), ubiquinone systems (1 8), molecular techniques (1 9-25), serology (26, 27), secondary metabolites (28, 29), and scanning electron microscopy (30-32). In view of the increasing emphasis on modern techniques in Aspergillus taxonomy and the need to develop a consensus viewpoint amongst taxonomists, a special workshop was organized in 1985 in the Netherlands with a view to formulating workable criteria (33). A direct result of this workshop was the formation of the International Commission on Penicillizrnz and Aspergillm (ICPA). It consists of 12 members from seven countries, some with a morphological approach and other specialists from the fields of physiology and biochemistry. ICPA plans to carry out collaborative taxonomic studies, revise culture collection names, develop taxonomic techniques, and other activities. They have recently completed a list of "Names in Current Use" both for Aspergillus and Per~icillirrm(34). A second and third workshop were held in 1989 and 1997, respectively (35, 36). Publications from these workshops herald a new era in our understanding of these two important genera (33, 35, 36).

II. ASPERGILLUS MYCOTOXINS Certain species of Aspergillus produce chemical substances that cause toxic symptoms when food containing them is ingested by humans or animals. These chemicals are called mycotu.rim and their toxic effects in animals are known as n?ycoto.uicoses (5). Mycotoxins can be: carcinogenic, teratogenic, tremorgenic, hemorrhagic, and dertnatitic (37). Most are hepatotoxins, nephrotoxins, and neurotoxins. The Aspergillr4s species are saprophytes and are regularly encountered on poorly dried cereal grains, groundnuts, rice, and cottonseed, which make up over 90% of our food (37). Some of the more important toxigenic Aspergillus species and their toxins are listed in Table 1. However, while many of these toxins have been isolated on occasion from human and animal foodstuffs, only a few are genuinely considered to be a serious threat (38). These include: aflatoxins, sterigmatocystin, cyclopiazonic acid, and ochratoxin A. Only brief outlines can be given here, but for more detailed descriptions of toxins and their effects on human and animal foods see Refs. 39-41. Methods that prevent contamination and growth of mycotoxigenic Aspergillus species in foodstuffs are discussed in Section VI.

A.

Aflatoxins

Aflatoxins are produced by certain strains of A. j m w s and A. pcrrnsiticus together with a newly described species: A. I Z U I ~ all ~ ~members US, of the Section F h i . Natural formation

Aspergillus

473

Table 1 Important Mycotoxin-ProducingSpecies of Aspergillus Mycotoxin Aflatoxin Citrinin Cyclopiazonic acid Fumitoxins Kojic acid Nidulotoxin Ochratoxin Patulin Physcions Sterigmatocystin Territrems

Species

A. J l c l v w , A. parasiticm, A. nomius A. terreus A. Jln\ws, A . tamarii A. jimigatus A. flallus, A. no mil^^, A. parasiticus, A. tawarii A. versicolor, Emericella rridulans A. ochraceus A. claLutus. A. terseus Eurotiurn anlstelodmti, E. cheldieri, E. repem, E. rubrum A. llersicolor, E. Flidrllms A. terr-eeus

of aflatoxin is more prevalent in tropical and subtropical areas, but it does occur in temperate and cooler climates, particularly in on-farm storage (38). Although highest levels of aflatoxin have been found in oily products, particularly groundnuts, cottonseeds, and corn (38), they also are isolated from ordinary foodstuffs. There are approximately 18 aflatoxins produced, of which aflatoxin B I, B,, G I , and G, are naturally occurring, the rest being metabolic products of animal systems. Aflatoxin B 1 is the most toxic. 1. Chemistry Aflatoxins are fluorescent heterocyclic compounds characterized by dihydrodifurano or tetrahydrodifurano moieties fused to a substituted coumarin moiety. Aflatoxin B ,: C 17Hll-0h, molecular weight (MW) 3 12, melting point (m.p). 268269°C; bright blue fluorescence under ultraviolet (UV) 360 nanometers (nm) Aflatoxin B?: CI7Hl4O6, MW 314, m.p. 286-289°C; bright blue fluorescence under UV 360 nnl. Aflatoxin G , :CI7HlLO7, MW 328, m.p. 244-246°C; blue-green fluorescence under UV 360 nm. Aflatoxin G2:C 17H1407, MW 328, m.p. 230°C; blue-green fluorescence under UV 360 nm.

2. Toxicology The effects of aflatoxin on animals depend on the species, age, sex, nutritional condition of the animal, dosage level, frequency, and composition of the diet (37). Aflatoxins are mutagenic, carcinogenic, teratogenic, and acutely toxic to most domestic and experimental animals. Aflatoxin B I is the most toxic, attacking the liver and causing acute hepatotoxicity at levels that are half the LDFolevel (38). Toxicity of G I , B2, and G2 is 50% 30%, and lo%, respectively, that of B , (38). Cirrhosis of the liver, caused by long-term feeding on diets containing aflatoxin, has been demonstrated in pigs, cattle, fish, and birds (43), and there is some evidence of human liver tumors, cirrhosis (43), hepatitis, and kwashiorkor (74). Primary mycotoxicoses caused by regular low-level intake of aflatoxin-contaminated feed have been demonstrated. These include low weight gain, reduced feed intake, poor

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feed conversion, and poor milk yield (44). Aflatoxin consumption also causes poor resistance to various diseases in domestic animals and increased susceptibility to others (45). There is some evidence of teratogenic effects (protrusion of intestines, open eyes) in mice, hamsters, and rats (46). B. Sterigmatocystin Sterigmatocystin is closely related to aflatoxin, and is produced by members of Sections Versicolores (A. versicolor group) and Nid~larztes(A. r?idularzs group). There are some reports that A. ustus and certain members of the A. glaucus group produce sterigmatocystin (38), but these are probably misidentifications (47). The mycotoxin has been found in cereals, rice, various nuts, green coffee beans, and cheese (37, 74). 1. Chemistry Structurally sterigmatocystin is related to aflatoxin, being characterized by a xanthone moiety fused to a dihydrodifurano or tetrahydro moiety. Sterigmatocystin is ayellow crystalline compound, with an empirical formula of C 18H120h, MW 324, m.p. 246°C; and orange-red fluorescence under UV 360 nm.

2. Toxicology Sterigmatocystin is acutely toxic, albeit at dose levels higher than for aflatoxin. The reason for this is thought to be themuch-reduced absorbability of sterigmatocystin from the digestive tracts of test animals (48). However, sterigmatocystin is still highly toxic and has the potential to cause human liver cancer (45). Its presence in food should therefore not be taken lightly. It can also cause tumor growths at the sites of application.

C. OchratoxinandRelatedCompounds Ochratoxin A was first isolated from A. ochreleeus (Section Circumduti),by van der Merwe et al. (49). Other members of the Section Circumcluti, together with Per~icilliunz verrucosum, also produce the mycotoxin (47). The production of ochratoxin A by P. w”ucosunz appears to be confined to temperate regions (38). It is isolated worldwide at significant levels from cereals, green coffee beans, and compound animal feedingstuffs and at lower levels i n beer and vine fruits. Other less important metabolites are the methyl and ethyl esters of ochratoxin A and the dichloro derivative ochratoxin B and its esters (38). 1. Chemistry Ochratoxins are composed of a 3,4-dihydromethylisocournarin moiety linked by way of the 7-carboxy group to L-P-phenylalanine by an amide bond. Ochratoxin A has an empirical formula of C2(,HISO6NCl. MW 403, m.p. 94-96°C; and green fluorescence under UV 360 nm.

2. Toxicology Ochratoxin A attacks the kidney and liver in animals, causing nephropathy, enteritis, and immunosuppressive symptoms (38). It has also been implicated in human Balkan endemic nephropathy (50), although more recent work (51) would indicate the fungal species to be a Petu’cilliz41n(P.nurantiogriseum) (51). Ochratoxins B and C, viomellein and xanthomegnin produced by A. ochruceus, A. s~dphurez~s, A. ostinnus, and A. rrlelleus (47), and

Aspergillus

475

viriditoxin produced by A. viridinutam and A. brevipes (Section Furnignti) have shown some toxicity to test animals, but their effects in animal feedstuffs are still unclear (52). D. CyclopiazonicAcid The mycotoxin CPA is produced by A. j h w s and A. tanmrii (Section Flrvi). It is worldwide in distribution, occurring on naturally contaminated agricultural raw materials and compound animal feedingstuffs (38). 1. Chemistry CPA is an indole tetramic acid. Its empirical formula is C2(,H2"OjN2,MW 336, m.p. 245246°C.

2. Toxicology After ingesting CPA-contaminated feeds, test animals display severe gastrointestinal upsets and neurological disorders (53). Organs affected include liver, kidney, heart, and digestive tract, which show degenerative changes and necrosis (54). The occurrence of aflatoxin and CPA in corn and peanuts contaminated with A. flcI1"s suggests that synergism may be involved (37).

E. Patulin Patulin is produced by A. terreus and A. clalatus. The latter species is particularly important in themalting industry where conditions of high temperature and humidity are particularly suitable for growth of A. clcrvatzas (55). A. terreus is commonly isolated from airtight storage (56). 1. Chemistry Patulin is a crystalline material [4-Hydroxy-4-H-furo-[3,2-c] pyran-2(6H]-one), with an empirical formula C7Hh04,MW 154, m.p. 1 10-1 11°C.

2. Toxicology Patulin has been shown to be carcinogenic when subcutaneously injected into rats (38). It is also acutely and chronically toxic to chicks, producing hemorrhages in the digestive tract (38). However, patulin production by A. terrezts has beendemonstrated only in laboratory conditions and not in the field (41).

F. Citrinin Citrinin like patulin is produced by A. clcrvcrtus and A. terreus. 1. Chemistry Citrinin [(3R-tra1~s)-4,6-Di-hydro-8-hydroxy-3,4,5-tri1nethyl-6-oxo-3H-2-benzopyran-7carboxylic acid], is a yellow crystalline compound with an empirical formula C ,?Hl1O5, MW 250, m.p. 175"C, which fluoresces yellow under UV 360 nm. 2. Toxicology When fed to experimental animals (rats, mice, and guinea pigs), citrinin is an acute nephrotoxin, causing renal lesions with degeneration and necrosis of the tubular epithelium (37).

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Again there is no evidence that citrinin produced by A. terreus is isolated in the growing cereal crop, since the mycotoxin has been produced artificially in the laboratory (47).

G. Neurotropic Toxins Neurotropic mycotoxins attack the central nervous system (CNS) and are termed tremorgenic. Other Aspel-gillus toxins, such as aflatoxin, patulin, and cyclopiazonic acid, can affect the nervous system, but this is not their primary function (38). There is very little information regarding their toxicity in the field, since most experiments have been conducted in the laboratory (57), but A. fi4rrligatz~~, a prolific producer of tremorgens, is common in badly heated produce (38). Further research would seem wan-anted. 1. Fumitremorgens The fumitremorgen toxins are produced by A. fi4migcrtus and A. caespitoszrs (Section Versicolores). Chenzistry The toxins are characterized as cyclic dipeptides with as a 6-O-methyl indole moiety. The empirical formula is Ci2H4,07Ni,MW 579, m.p. 206-209°C. The UV absorption is typical of indole derivatives with two absorption peaks at 224-233 nnl and 275-295 nrn. Toxicology When fed to laboratory mice and rats, the toxins caused tremors and death in 70% of the animals tested (58).

2. Verruculogen Verruculogen is also produced by A. fimigatus and A. ccmpitoszrs. Chenlisty The structure is the same as for fumitremorgen A, but with a different side chain. The empirical formula is C27Hi307N7, MW 5 11, m.p. 233-235°C. Toxicology Verruculogen is tremorgenic to laboratory-fed mice and 1-day-old chicks (58), and appears to work by inhibiting the alpha-motor cells of the anterior horn (59). 3. Aflatrem The toxin aflatrem is produced by some strains of A. jlavus. Chemistry Aflatrem is a-a-dimethylallyl-paspalinine, with an empirical formula C37H3g0,N,MW 501, m.p. 222-224°C. Tosicologg The toxin aflatrem causes tremors when fed to laboratory mice, guinea pigs, and rats (58).

4. Territrems Telritrem A, B, and C are produced by A. tel-reus. Toxicology Depending on the dosage and the administrative route used, territrems cause variable responses from mild tremoring to convulsive muscle spasm (38). 5. Aspergillic Acid This is produced by A. jlczvus (59j. Chemistr? Aspergillic acid is ([3-sec-butyl-6-isobutyl-2-hydroxy-2(lH)-pyrazinone]), with an empirical formula CI2H2-,O2N2, MW 223, m.p. 97-99°C. Toxicology The toxin causes severe convulsions in mice, followed by death (38).

Aspergillus

111.

477

ASPERGILLOSIS

Aspergillosis is primarily a pulmonary disease, although through dissemination other organs may become affected (60). The common species involved is A. fionigntus. This is a thermotolerant species, and is commonly isolated from spontaneously heated products and organic substrates. Such sources are: ventilation systems, dust created during renovation and maintenance work, bird excreta in air ducts, moldy litter, and feedstuffs (61). Other important species include: A. j k ~ \ w A. , terreus, A. niger, and A. nidulnns. Factors that cause the growth of all these species in humans include susceptibility through various chemotherapy treatments, prolonged use of antibiotics (particularly the broad-spectrum drugs), and certain surgical operations such as open-heart techniques (62). Immunosuppressed patients are particularly susceptible (61). Several Aspergillus species have been isolated from AIDS patients, but aspergillosis is not considered to be an important disease in this syndrome (63). Infections are worldwide in distribution, although prevalence does appear to be seasonal with autundearly winter the dominant season (64). Only a brief summary of Aspergillus species isolated from humans and animals can be given here. For a more comprehensive account the reader is directed to Smith’s excellent book on the subject (61).

A.

Human Infections

Aspergilll~sinfections in humans are primarily pulmonary. These include farmer’s lung disease, aspergillomas, and invasive aspergillosis. A. firrnigntus is the causal agent. However, other Aspergillus species can infect eyes, ears, skin, nails, bones, and the gastrointestinal tract (Table 2). 1. Farmer’s Lung Disease Farmer’s lung disease is anacute, chronic, and sometimes fatal disease caused by continual exposure to the fungal spores in hayand other moldy produce, A. fiunigatusis the principal

Table 2 Aspergillus Species AssociatedwithHuman Disease/lesion

Diseases Species

A. candidus, A. corneus, A. jlallus, A. firnrigatus. A. glalmls (group), A. niger, A. niveus, A. sydowii, A. t e r r e ~A. , versicolor, E. rliclul a m , N . jscher-i Nasal tissue A. conicus, A. j m ~ c s A. , jmTipes, A. fitmigatus, A. g h c w (group), A. rliger, A. niveus, A. ocIIroceus, A. terreus. A. \.ersicolor, E. nidlrlrrrls Centralnesvous system A. candidrrs, A. fimigcrtus, A. glmcus (group), A. nigcr, A. sJdo\t*ii,A. terreeus, A. versicolor. E. nicldans Intestines A. filnligatus. A. terreus Eyes codidus, A. .fla~ws, A. A. jirmigatlrs, A. niger. A. syc1o”ii Bones/joints A. julipes. fimigatus, A. A. sydoulii, E. rlidlrlam Skin A. condidus. A. .fl~vus,A. ftrnligemrs,niger, A. A . terreus Nails A. candicllcs, A. j o ~ w s A. , f h i g a t m ,niger, A. A. terreus, E. nidlrlcrrrs Ears .flmipes, A. Jlrr\~rs, A. A. fianigntus, A. niger. E. nidlrlails Pulmonary

~

Solrrc-e: From Ref. 61.

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Kozakie

species, but A. f l c ~ vand ~ ~A. rliger are also implicated (65). It occurs in more temperate regions and particularly in high-rainfall areas. Although the disease is most prevalent in the farming community, office workers have also suffered due to the presence of spores in air-conditioning units (66). Three stages have been described for the disease: (1) an acute stage, found in harvesters and threshers, exposed initially to an overwhelming concentration of spores, (2) a subacute stage, found in silo and grain-mill workers who have long-spaced periods of exposure, and (3) a chronic form, found in silo and grain-mill workers who have lowlevel but constant exposure (65). Symptoms include fever, chills, and general malaise, which last for only a few days and may resolve if there are no further exposures. Obstruction of the air passages is the major feature of chronic farmer's lung disease. The disease is debilitating (shallow breathing, chronic cough), and is often ultimately fatal. 2. Aspergilloma Aspergilloma is a disease caused by Aspergillzrs species growing and forming a tumorlike mycelial mass in a pulmonary cavity (62). Aspergillomas develop secondarily, usually in cavities formed by an abscess, bronchial carcinoma, or tuberculosis. Once these infections have been cleared by antibiotics, an aspergilloma may develop. Initially, health is not impaired, but ultimately fever and persistent coughing appear. A chest x-ray reveals a crescent-shaped growth

3. InvasiveAspergillosis Invasive aspergillosis is usually found at autopsy or after lung resection. A. funzigcrtus usually develops in pure culture within the pulmonary parenchyma and often ruptures into the surrounding arterioles, which thrombose (62). 4. Infections of theEyes,Ears,andNose Injury to the cornea of the eye and the accumulation of debris in the ear, particularly in the damp tropics, allows colonization and infection of these two organs. A. fiunigntzrs and A. fziger are most commonly isolated in both mycotic keratitis (eye infections) and infections of the external ear, with A. niger predominant in the latter case (63). A. j l c w u , A. j h i g a t r ~and ~ , A. ~zidulcrrrshave been isolated from nasal cavities (61). 5. ReproductiveOrgans There are implications that Aspergillus species can infect reproductive organs following uterine instrumentation and long-term use of intrauterine devices (67). 6. Dermatological Infections A. terreelrs, A. rriger, A. j?c~vzrs,A. ccmlidm, A. ~idulnr~s, and members of the A. g l n ~ t c z ~ ~ species group (Section Aspergillus) have been isolated from skin and nails (61). 6. AnimalInfections

Aspergillosis in animals is primarily a respiratory infection, as it is in humans. It is worldwide in distribution, and has been reported in nearly all domestic animals and birds, and in some wild animals (61). Contributory factors are still unclear, but stress of captivity and immaturity are indicated.

Aspergillus

479

The most common infection is that of the respiratory tract in birds. But Aspergillus species also cause mycotic abortion in cattle, guttural pouch lesions in horses, and nasal and systemic lesions in dogs (61). Veterinary aspergillosis will be discussed only briefly here, but more details can be found in Smith (61). 1. PulmonaryDiseases A. firnzigatus is thedominant species causing severe pulmonary problems in poultry (chickens and turkeys), lambs, and occasionally cows. Outbreaks of aspergillosis can cause 90% mortality in chickens and turkeys, with newly hatched chicks being particularly vulnerable (known in this instance as brooder pnezrrno~zin)(61). The source of infection is usually moldy straw and litter. Straw used for bedding was also the problem in a case of pulmonary aspergillosis in lambs but, although 50% of the animals displayed symptoms only four died (68). Numerous cases in cattle have also been reported (61).

2. Horses Guttural pouch mycosis has been demonstrated in stabled horses in the United Kingdom, Canada, United States, Denmark, Sweden, Switzerland, and Australia (67). The source of infection is presumed to be moldy feed. Species isolated have been A . j h i g a t u s and A. rzidzrlms. Aspergillus species have also been associated with equine keratomycosis (69).

3. Dogs A. frlrrligcrtzds is the causal agent of aspergillosis of the nasal cavity in dogs (61). Disseminated disease, with A. terreus being the causal agent, has also been reported on numerous occasions (61). German shepherds appear to be particularly vulnerable (70). A. deflectus was also isolated from disseminated aspergillosis in another case involving German shepherds (71). 4. Eggs The infection of incubating eggs by A. jimigatrlsis another economic problem. Penetration of the egg by germinating conidia occurs at the time of laying. It is believed that modern techniques of large-scale egg production are to blame for these extensive outbreaks of aspergillosis (61).

IV. DESCRIPTIVEMORPHOLOGYANDTERMINOLOGY Aspergillus is an anamorphic (asexual) genus, characterized by the production of spores (conidia) on a specialized structure termed the conidiophore (Fig. 1). Minter et al. have proposed a terminology based on function (72); this is followed here. Diagnostic characteristics are colony color, head shape, size, shape, and ornamentation of conidia. Some species of Aspergilhs reproduce sexually. These teleomorphic states belong to the Ascomycete family Trichocomaceae and are characterized by the production of ascospores within an ascus, which in turn is contained within an ascoma. Ascospore size, shape, color, ornamentation, and presence of equatorial crest or flanges are important diagnostic characteristics for the identification of teleomorphic species. Four teleomorphic genera are considered and are discussed under their respective anamorph sections.

Kozakiewicz

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anidiogcnxrs cell

c e l l s u p p o r t i n g conidiogenous

omidiophore:

wollen q e x

midiophore:

rpedian part

II

Ii

Figure 1 Terms recommended to describe the different conidiogenous structures in Aspergillus.

V.

SPECIESGROUPDESCRIPTIONS

Raper and Fennell assigned species into species groups according to comnlon characteristics, and considered the form of the conidiogenous structures to be a key feature for species group recognition ( 5 ) . Accordingly, groups were assigned to one of three classes: those with one series of conidiogenous cells (uniseriate), those with two series (conidiogenous cells plus supporting cells) (biseriate), and those in which both conditions are found (Table 3). However, recent changes in the taxonomy of A.~pyergillushave divided the genus into six subgenera (73j, each containing one or more sections. Sections correspond tothe groups of Raper and Fennell (5). Only those sections that have medical or veterinary importance will be discussed here.

Table 3 Aspergillzts Sections Accordingto Structure of Conidiogenous Cells Uniseriate sectionJ Niclulal?tes Clnvrrti Wentii Versicolores Restricti Frtriligrrti Terrei Aspergillus (A. glrruclrs group)

.I

Biseriate section

Both sections Flmi

Flmipedes

Nigri Cnndicli Circlmtdati (A. ocl~rrrce~rs group)

Sections comespond to the specles groups of Ref. 5 .

Aspergillus

A.

481

SpeciesIdentificationandTheirImportance

In identifying an Asyergillzrs species, its section (species group) relationship should be determined first. A key to the 14 sections discussed here is provided (see Table 4j. Primary emphasis has been placed on the arrangement of the conidiogenous cells. Secondary emphasis is on the shape and pigmentation of the conidial heads, with further separations based on presence or absence of ascomata, Hiille cells (thick-walled cells of no known function), and pigmentation of the conidiophore. All species are grown on Czapek Dox agar (Difco, Detroit, MI) unless otherwise stated.

B. SpeciesDescriptions 1. Section Aspergillus (Aspergillus glaucus Group) Diagnostic features Species in the A. gluucus group possess a teleomorph (sexual stage) named Eur-otiun?.Eurotim is a well-defined genus characterized by the formation of yellow to orange ascomata. The Asper-gillus anamorphs (asexual stage) possess bluish-green radiate conidial heads producing uniseriate conidiogenous cells, which in turn produce large barrel-shaped spinose conidia. Species of the A. gluucns group are xerophilic and require low water activity media [Malt extract Agar (MEA) (Oxoid, Basingstoke, UK) or Czapek Dox agar plus 20% sucrose] in order to grow.

Table 4 1

2 (1) 3 (2) 4 (3)

5 (1)

6 (5) 7 (6)

8 (7) 9 (7) 10 ( 5 ) 11 (10) 13, (11)

13 (13,)

Key to Sections Conidiogenous cells strictly uniseriate Conidiogenous cells biseriate or uniseriate and biseriate Conidial heads clavate Conidial heads not clavate Conidial heads radiate. ascomata present Conidial heads not radiate, ascomata absent Conidial heads columnar, conidia echinulate Conidial heads columnar, conidia spinose Conidigenous cells strictly biseriate Conidiogenous cells uniseriate and biseriate Ascomata present Ascomata absent Conidial heads columnar Conidial heads not columnar Conidial heads white to fawn, conidiophores brown Conidial heads cinnamon, conidiophores not brown Conidial heads radiate, dull brown, conidiophores brown Conidial heads radiate, green, conidiophores not brown Conidial heads yellow-green Conidial heads not yellow-green Conidial heads ginger-brown Conidial heads not ginger-brown Conidial heads globose, black Conidial heads globose, not black Conidial heads globose. white Conidial heads globose, yellow to ochraceus

2 5 Clavati 3 Aspergillus 4 Restricti Funrigcrti 6 10 Nidrdantes 7 8 9 Flcrvipecles Te ]-re i Usti Versicolores Flmi 11 Werltii 12 Nigri 13 Cmdidi Circlrrdati

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Kozakiewicz

Although about 20 species belong to this group, only four species are common: E. amstelodumi, E. rubrum, E. repens, and E. chevalieri. Species differentiation is based on ascospore ornamentation, that is, smooth or rough convex walls, presence or absence of a longitudinal furrow, and equatorial crest or flange. Both E. repens and E. rubrum have smooth, convex walls, no equatorial crest, but the former has a hint of a furrow and the latter a definite furrow. E. amstelodumi has rough convex walls, a prominent furrow, and rounded crests and E. chevulieri has smooth walls and thin prominent crests resembling a pulley (Fig. 2). Toxins Eurotium species have been reported as producing such carcinogenic mycotoxins as aflatoxin, sterigmatocystin, and ochratoxin A (47). However, such toxin production has not been reproduced under laboratory conditions, and most Eurotium species are considered to be benign fungi (74). Medical aspects Although they are not considered to be important pathogens of humans or animals, Eurotium species have been isolated from human paranasal tissue, eyes, and nails (61). Occurrence All species of Eurotium are xerotolerant, and will therefore be found on foods containing little free water (e.g., stored cereals, flour, nuts, jams, jellies, fruit

Figure 2 Scanning electron microscopy micrographs illustrating ascospore ornamentation in Eurotium species: (A) E. repens; (B) E. rubrum; ( C ) E. chevalieri; (D) E. amstelodami.

Aspergillus

483

cakes, cheesecake, filled chocolates, smoked bacon, dried salted fish, and dried meats) (74, 75).

2. Section Restricti (Aspergillus restrictus Group) Diagnostic featzwes Raper and Fennel1 recognized five species (5), but Pitt and Samson accepted three (76): A. caesiellus, A. yerzicillioides, and A. restrictus, of which the last two are common. However, these species are very xerophilic (requiring MEA plus 40% sucrose) and are often missed during isolation because inappropriate media have been used. Diagnostic characteristics are dark-green, columnar, uniseriate conidial heads bearing large elliptical spinose conidia. Conidial heads of A. restrictus are particularly long and often twisted columnar shapes, whereas those of A. yerzicillioides are radiate when young, and loosely columnar with age. Toxins Although A. restrictus produces mitogillin, species in the Section Restricti are not considered to be important mycotoxin producers (47). Medical aspects A. yerzicillioides has been implicated in human diseases, particularly in the tropics, causing lobomycosis and tinea pedis (5, 61). Another species in this section, A. conicus, is the causal agent of the tropical disease known as tokelau (5, 61). Occurrence Reports of A. yenicillioides are rare, probably due to the use of inappropriate isolation media. The species has been isolated from various dried foods such as flour, cured meats, spices particularly chilies and black pepper, nuts, dried fruit, and fish (74). A. restrictus has been isolated more frequently from a variety of such foods as cereals, pulses, spices, health foods, dried and cured meats and fish (74), coffee beans, and maize (75).

3. Section Fumigati (Aspergillus fumigatus Group) Diagnostic featzwes Species of the A. f m i g a t u s group are recognized by rapidly growing, dark-smoky-green colonies, bearing uniseriate short, columnar conidial heads. Conidiophores often are pigmented green, as are the swollen apex and conidiogenous cells (Fig. 3A). Conidia are small, globose, and delicately roughened. Some species in the group have a teleomorph Neosnrtory, of which N. fischeri is the most common. It is characterized by rapidly growing colonies, sparsely sporulating columnar heads with soft-walled white to cream-colored ascoma, containing rough walled ascospores with two sinuous equatorial crests or flanges. Toxins A. Jtrrnigatusproduces a large number of mycotoxins: fumiclavines, fumigatin, fumitoxins, fumitremorgins, verruculogen, gliotoxin, and tryptoquivalins (47). Gliotoxin is the most important of these, causing numerous mycotoxicoses (55). Evidence suggests that the toxinis important in the invasion of animal and bird lungs by A. jhzigntus (74). N. fischeri also produces fumitremorgens and verruculogen (74). Medical aspects A. jknzigatus is a human and animal pathogen, being the causal agent of the disease known as Aspergillosis (see Sec. 111). Occurrence Species inthe A. $migatus group are thermotolerant. A. fumigntus occurs on produce undergoing heating. Therefore, it is commonly isolated from heated cereals and in cocoa beans both during and after fermentation (5,47). It has been isolated also from spices, pulses, soybeans, copra, nuts, and meat products, particularly in the tropics (74). The ascospores of N. Jischeri are heat resistant. As a consequence, N . Jischeri is commonly isolated from pasteurized foods (e.g., canned fruits, fruit juices, purees, and syrups)(74).

Kozekiewicz

484 -a

*

Figure 3 Light micrographs illustrating conidial head shapes in Aspergillus: (A) A. fumigatus (columnar); (B) A. clavatus (clavate); (C) A. versicolor (radiate);(D) A. rerreus (columnar).

4. Section Clavati (Aspergillus clavatus Group) Diagnostic features The Clavari section is a small group, of which A. clavatus is the commonest. Growth is rapid, velvety, with grayish-green uniseriate heads producing smooth, elliptical conidia. The group is characterized by its clavate-shaped swollen apex (Fig. 3B). Toxins A. clavatus produces the toxins patulin, ascladiol, tryptoquivaline, and a derivative of patulin: cytochalasin E (47). The last two are known as potent mycotoxins (59). Medical aspects A. clavatus may cause allergic alveolitis in humans (77). Tryptoquivalines in a moldy rice sample were implicated in the death of a child (74). A. clavatus is also impIicated in mycotoxicoses of ruminants (77).Patulin has been recorded as causing ill thrift in animals (74). Occurrence A. clavatus is commonly isolated from malting barley in which it is a serious problem (79). It also has been isolated from cereals, red peppers, pecan nuts, and health foods (74).

Aspergillus

485

5. Section Nidulantes (Aspergillus nidulans Group) Diagnostic featzcres Species in the A. nidulans group possess a teleomorph, Emericelln, which is distinguished by Hulle cells surrounding black ascomata (white when young), bearing red-purple, lenticular, smooth-walled ascospores with two equatorial pleated but entire crests. The Aspergillus anamorph bears biseriate, deep-green, short columnar heads with a distinctive short, sinuous, brown-pigmented conidiophore. Conidia are globose and rough. The colony reverse is usually deep red-purple. There are about 20 species in the group, of which only E. rzidulnns is common in food. It can be distinguished by the ornamentation of the ascospores, which are red, smooth walled, and bear two equatorial crests or flanges. Toxins E. rzidrrlarzs produces sterigmatocystin (a precursor of aflatoxin), nidulotoxin, and emestrin (47, 73). Medical aspects E. niduZurzs has been isolated from human skin, nails, and nasal cavities, and guttural pouch mycosis in horses (61). It has also been isolated from AIDS patients. Occzcrrence Although not a common food-borne species, it has been isolated from cereals, nuts, soybeans, beans, spices, meat, and chocolate (74). 6. Section Versicolores (Aspergillus versicolor Group) Diagnostic features Species are slow growing, with dull-green, radiate, biseriate heads bearing small, globose, spinose conidia (Fig. 3C). Conidiophores are colorless, or sometimes pigmented brown, and Hiille cells are occasionally present. Although there are over 20 species in the group, only A. versicolor and A. sydowii are common. A. versicolor. can show a range of colony color, from pale green to yellow-green, gray-green, buff, or even pink; reverse is deep red to plum, and the spinose conidia turn emerald green when mounted in lactophenol. The turquoise colony color of A. sydowii easily distinguishes it from A. versicolor. It is mistaken easily for a Penicillium at first sight, until examined with a stereomicroscope. Dwarfed heads can also be found in nearly all isolates. Colony reverse is very deep red. Toxins A. versicolor, like E. izidrrlms, produces sterigtnatocystin and nidulotoxin (47). A. sydowii is not known to produce any mycotoxins. Medical aspects A. sydowii appears to be more pathogenic, and is isolated from keratomycosis and human bones and joints (61). Both species have been isolated from human lungs and the central nervous system (61). Occzcrrence A.wrsicolor is distributed widely in cereals, oilseeds, feedstuffs, bread, cereal products, nuts, dried and cured meats, cheese (39, 74), and black and white peppercorns (39). It is also commonly isolated from airtight storage (47). A. sydowii appears to have a preference for drier foods such as nuts, spices, beans and soybeans, cured meats, and health foods (73). It has been found on cereals such as barley, wheat, corn, and flour, but not as frequently as A. w”icoloI‘ (74). 7 . Section Usti (Aspergillus ustus Group) Diagnostic features Usti is a small group, of which only A. ustrls is important. Distinguishing features are floccose, moderately fast growth, brown-gray to dull gray colonies, bearing small, biseriate radiate to loosely columnar heads. Conidiophores are short, smooth, and brown, with small, globose, echinulate conidia. The reverse is yellow, with irregular to elongate (sausage-shaped) Hulle cells sometimes present.

486

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Toxins Some isolates of A. ustus can produce austamid, austdiol, austins, and austocystins (80). Medical aspects A. rrstus is not known to be pathogenic. Occurrence A. rrstus is not a serious spoiler of food, but has been isolated from cereals, soybeans, flour, peanuts, nuts, and health foods (74).

8. Section Terrei (Aspergillus terreus Group) Diagnostic features The Terrei have velvety to floccose growth, and biseriate, cinnamon to tan, compactly columnar conidial heads, bearing very small, smooth-walled conidia (Fig. 3D). Two varieties have been described: A. terreus var. qfricnnrrs and A. terreus var. nureus (5). The former possesses yellow to buff-colored sclerotial-like masses on MEA, and the latter bright yellow mycelium with conidiophores over 500 p m long. Toxins Isolates of A. terreeus are able to produce citrinin, patulin, terrein, territrems, and citreoviridin. However, these have been produced under laboratory conditions, and it is unclear whether this is the case under natural conditions (47). Medical aspects A. terrerrs is commonly isolated from human slun and particularly nails (61). It is now being isolated from AIDS patients. A. terreus is the usual etiological species in disseminated disease in dogs. The organs affected are spleen, kidney, and bone (61). Occurrence A. tel-reeus commonly is isolated from airtight stored cereals (56) and a variety of nuts (hazelnuts, peanuts, walnuts, pistachios, and pecans) (74). It also has been isolated from barley, flour, pasta, and flour products, pulses, soybeans, meat, and products (74). 9. Section Flavipedes (Aspergillus flavipes Group) Diagnostic features The Flnvipedes have velutinous to slightly granular growth, with small, white, biseriate columnar heads. Conidiophores are brown, bearing small, globose, smooth-walled conidia. A teleomorph rarely develops, but according to Wiley and Simmons (81), the ascomata are yellow, surrounded by Hulle cells, and produce smoothwalled ascospores with an indistinct longitudinal furrow. The teleomorph is Fenlzellin jkwipes. Two other anamorphic species are found in this group: A. rziveus and A. carr~eus. The former bears white, columnar conidial heads, but has an unpigmented conidiophore, while the latter bears fawn-colored columnar heads, also with unpigmented conidiophores. Toxins A. jluvipes is reported to produce flavipin and flavipucins, while A. carr~eus produces citrinin (47). Medical aspects Both A. cunzeus and A. rziveus have been implicated in invasive pulmonary aspergillosis, while A. Jlnvipes and A. niveus have been isolated from paranasal tissues and orbits (61). In addition, A. jlnvipes has been isolated from human bones and joints and has been implicated in otomycosis (61). Occurrence A. j h i p e s has been reported in fruits and cassava in the tropics and in European wheat (74). 10. Section Wentii (Aspergillus wentii Group) Diagnostic features Section Wer~tiiisa small group, of which A. werztii is the most common. Distinguishing features are floccose growth bearing sparsely produced, large, globose, ginger-colored, uniseriate and biseriate conidial heads, the biseriate condition predominating. Conidiophores can be several millimeters long. Conidia are globose to elliptical, smooth to very rough.

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Toxins Emodin, wentilacton, physicons, P-nitropropionic acid, and numerous other metabolites are produced by A. werztii (47, 80). Only emodin is of mycotoxic significance. Medical aspects A. wentii is reported to cause toxicity problems in animals (47, 55). Occurrence A. werztii is particularly common on dried fish (74), but it is also found in cereals, dried beans, peanuts, pistachios, soybeans, and cured meats (39, 74, 75). 11. Section Flavi (Aspergillus flavus Group) This group has undergone intense study since Raper and Fennell’s classification ( 5 ) ,using a wide variety of techniques ranging from traditional morphology (9), DNA homology (23, restriction fragment length polymorphistns (RFLPs) (21, 22, 82), ubiquinones (17) to SEM (10, 12, 31), with species being removed from (17, 31) and added to the group (9, 83). Despite this intense scrutiny, basic concepts of the group remain the same. Distinguishing features are yellow-green to jade-green to yellow-brown to brown colonies, with uniseriate and biseriate, radiate to columnar conidial heads. Conidiophores are colorless, usually roughened, and conidia are globose to subglobose, finely roughened to spinose to echinulate. Species discussed here include A. j a v u s and A. parmiticus, A. otyzcre and A. sojae (two important species used in food fermentations), and A. nomius a recently described species (43). Despite the fact that A. tamarii has been removed into a separate group (31), it will be discussed here for convenience.

a. Aspergillus javus Diagnosticfeatures Colonies of A. j m u s are yellow-green; conidial heads are predominantly biseriate, radiate or columnar; conidiophores are coarsely roughened up to 1 tnm in length; and conidia are globose to subglobose or spinose (Fig. 4A). Sclerotia are sometimes produced. Toxins Aflatoxin B I and cyclopiazonic acid (CPA) are produced (47). Aflatoxin is both a hepatotoxin and hepatocarcinogen, and is described as the most potent liver carcinogen so far recognized (84). Aflatoxins are both acutely and chronically toxic to both humans and animals. Regulatory limits for both foods and feeds therefore have been set in most countries (85). Aflatoxin levels in fungal spores also can be high (74). Medical aspects A. j a w s is particularly prevalent in hot, dry geographic regions where it causes invasive lesions of the orbit. It has also been isolated from human skin, nails, bones, the central nervous system, and lungs (61). While there is no evidence that aflatoxin-containing spores ingested into the lungs can cause cancer, nevertheless aflatoxin is readily absorbed into the body, with detrimental effects on the alveolar macrophage function (74). In addition, ingestion of aflatoxin-contaminated foods can result in depression of cell-mediated immunity, increasing the risk and severity of diseases such as kwashiorkor (a protein energy malnutrition) (74). Aflatoxin contaminated foods and feeds cause hemorrhaging of the gastrointestinal tract, prolonged blood clotting time, and immunosuppressive activity (84). Occurrence A. j h v u s is particularly common on such oil seeds as peanuts. In addition, A. Jlnvz4s is of economic importance on corn and cottonseed crops (74). Cereals are another common source, as are spices, cereal products such as pasta, flour, bread, corn grits, dairy products (milk, cheese), rapeseed, olives, betel nuts, and nuts (74).

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Figure 4 Scanning electron microscopy micrographs illustrating spore ornamentation: (A) A. JIL7\ws;(B) A. pmsiticus.

b. Aspergillus ynrnsiticm Diagnostic features Colonies of A. ynrnsiticus are velutinous, jade green (a much richer green, deeper in shade than in A. .fl~wu.s);conidial heads are predominantly uniseriate (although about 10% can be biseriate) and radiate; conidiophores are roughened, usually less than 400 urn in length; and conidia are globose and echinulate (Fig. 4B). Toxins Aflatoxin B I and G , , but not cyclopiazonic acid, are produced consistently (44). Medical aspects Toxic effects are as for A. jlavus.However, it is important to note that all isolates of A. pat-asiticus are toxigenic (not all isolates of A. j h w s produce toxins), and that aflatoxins are produced in much higher concentrations. Occurrence A. pnrnsiticus is prevalent in tropical and subtropical regions where it is found on peanuts and corn. Other commodities include various nuts, soybeans, and processed tneat products (74). c. Aspergillus ol-yzae Diagnostic features The A. or-yzne species is considered to be the “domesticated” form of A. j h r l s and therefore is not dissimilar in morphology. Colonies are floccose,

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489

seldom green, but are brown-green to brown; conidial heads predominantly are biseriate, radiate; conidiophores are rough, 2 mm and up to 4-5 mmin length; conidia globose, spinose, and variable in size, but usually 5-6 pm in diameter (larger than conidia of A. flu1ws).

Toxins A. olvzne is not known to produce any toxins. Medicalaspects None known. Occzrrrence A. o p m e is isolated only from fermented foods, where it is of major economic importance, being used in the Far East for the production of soy sauce, koji, miso, hamanatto, and other Oriental foods and flavorings (39, 74). d. Aspergillus sojae Diagnostic featzlres The A. sojne species is considered to be the “domesticated” form of A. parasiticus. It differs in its colony color, which turns olive-brown in age, slightly larger conidia, and floccose growth. Toxirts Toxins for A. sojae are not known. Medical aspects There are noknown medical aspects. Occurrence Like A. olyzae the A. sojne species is used in and isolated from fermented food products in Asia (39, 74). e. Aspergillus non~irrs Diagnostic features The A. rzornius species resembles A. javus except that sclerotia (when produced) are smaller and vertically elongate, unlike those of A. flnvus, which are larger and spherical. Toxins A. nomius produces aflatoxin B I and GI, and is the only member of the section Flavi to produce nominine (44j. Medical aspects There are no known medical aspects for A. nomius. Occurrence Isolations of A. nornius to date include those from wheat and turmeric (44), maize, peanuts, soybeans, cassava, and black beans (74).

J: Aspergillus tanm-ii Diagnostic features Colonies of A. tmnnrii are brown-green to brown; conidial heads are predominantly biseriate, large, and radiate; conidiophores are roughened; and conidia are large, globose, and echinulate with a distinct double wall. Toxirts A. tmnnrii produces cyclopiazonic acid (CPA) (47). It has also been implicated in “kodua poisoning,” where high levels of CPA and fumiclavine produced by A. tcrrrlnrii were found in kodo millet seed (74). Medical aspects The A. tclnzarii species has been isolated from human nails (61). Occurrence A. tcmcrrii has been isolated from various nuts (peanuts, walnuts, hazelnuts, pistachiosj (74), coffee, nutmeg, cacao (9), yams, soybeans, dried beans, smoked and dried fish (74), cured hams, and salamis (34). 12. Section Nigri (Aspergillus niger Group) Raper and Fennel1 recognized approximately 12 species in this group (5). However, this group too has been the subject of intense study in recent years. Techniques include morphology (6), SEM (31), and RFLPs (23, 36, 37j. Several species are distinct, but there still remains a species complex based around A. niger, and thegroup will remain problematical. However, basic group concepts remain the same, with A. niger and A. jcryonicus the most commonly isolated species.

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Distinguishing characteristics are rapidly growing, purple-black to black colonies bearing predominantly biseriate, globose conidial heads. Conidiophores smooth and uncolored. Conidia are globose to elliptical to horizontally flattened, and are rough with irregular ridges and bars. Sclerotia are produced by some isolates. Diagnostic features A. lziger possesses biseriate black heads, and globose conidia with very rough walls bearing prominent ridges or color bars (Fig. 5A). Toxins A. niger produces malformins and naphthopyrones (47). In addition, recently certain isolates have been shown to produce ochratoxin A (74). Medical aspects A. niger is a serious human pathogen, particularly in tropical areas where it is found in human lungs, paranasal tissues, the central nervous system, skin, nails, keratomycosis, and otomycosis (61). Occurrence A. niger is more prevalent in hotter climates, in which it has been isolated frequently from various nuts (peanuts, hazelnuts, cashews, walnuts, pecans, pistachios), various cold stored fruits, yams, figs, raisins, onions, garlic, copra, maize, dried fish, spices, meat products (741, rapeseed oil (39), and desiccated coconut (41). a. Aspergillus jcryorzicus Diagnosticfeatures A. juponicus possesses uniseriate purple-brown heads with globose to elliptical spinose conidia (Fig. 5B).

Figure 5 Scanning electron microscopy micrographs illustrating ger: (B) A. jcryonicus.

spore ornamentation: (A) A Izi-

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Toxins A. jnponicus produces E-64 (47j. Medical aspects A. jcryonicus is not known to be pathogenic. Occurrence A. japonicus is not isolated frequently from food, but this may be due to misidentifications of A. rziger. It has been found on green coffee berries and monkey apple (75). 13. Section Circumdati (Aspergillus ochraceus Group) Circzrmdcrti is a large group comprising 15 species, of which only A. ochrnceus and A. mellezrs are common. Distinguishing features are pale yellow to orange-yellow to ochraceus or buff-colored colonies, bearing large, biseriate, globose conidial heads. Conidiophores are faintly colored yellow and coarsely roughened. Conidia are small, globose, and smooth to delicately roughened. Sclerotia are frequently produced and vary in size, shape, and color, ranging from white to yellow to vinaceous to brown and black. Diagnostic features A. oclzracezrs produces spreading ochraceus colonies, with a reddish-purple reverse. Conidia are small, globose, and delicately roughened. Sclerotia are often produced, but take several weeks to appear; they are white to pink at first, darkening to vinaceus or lavender with age. Toxins Several members of the section Circunzdati, including A. oclzmceus, produce ochratoxin A, xanthomegnins, and penicillic acid (47). Medical aspects A. ochraceus has been isolated from allergic human pulmonary aspergillosis and paranasal tissues (61). Ochratoxin A attacks animals primarily in the liver and kidneys, where it causes nephropathy, enteritis, and immunosuppressive symptoms (41). Ochratoxin A is also implicated in Balkan endemic nephropathy (50),although it would appear that P. wm"Icosz4YyI may be thecausal agent. Renal failure due to inhalation of ochratoxin A has been reported. This was produced by A. ochraceus growing in a granary that had been shut for several months (74). Occurrence A. ochraceus has been isolated from a wide variety of foods and in particular from smoked and salt dried fish (74j. Nuts are also a major source (pecans, peanuts, walnuts, pisatchios, hazelnuts) (74). Other products include cereals, cereal products, soybeans, beans, dried fruits, cheese, and spices (74, 75).

a. Aspergillus melleus Diagnosticfeatures A. mellerrs differs from A. ochrnceus in that it produces smaller, paler conidial heads, and numerous small, yellow sclerotia 400-500 p n in diameter. Toxins The toxins produced by A. welleus are the same as those for A. ochraceus. Medica2 aspects A. tnelleus is not known to be a pathogen, but toxicosis is the same as for A. oclzrncezu (4 1). Occurrence A. melleus has been isolated from peanuts and citrus fruit (75). 14. Section Candidi (Aspergillus candidus Group) Diagnostic features Candidi is amonotypic group. The species is distinguished by white to cream-colored, predominantly biseriate, globose heads, which appear wet or slimy in young conidial heads. Conidiophores are colorless, smooth, usually 200-500 p n in length, but can be 1000 pm. Conidia are subglobose to globose, smooth walled. Sclerotia are present in some isolates, and are cream at first, then becoming purple to black. Toxins Terphenyllin, xanthoascin, candidulin, chloroflavonins, kojic acid, and 3nitropropionic acid are produced by A. carzdidus, with only kojic acid of notable significance (74).

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Medical aspects A. cnrdidus has been isolated from human eyes, skin, nails, and central nervous system (61). It has also been implicated in mycotoxicoses (55). Occzcrrence A. carzdidus is of common occurrence in cereals and cereal products (74). in particular cereals stored under controlled atmospheres (56) and acid-preserved cereals (80). Other isolations have been from rice (39), various nuts (peanuts, hazelnuts, walnuts, pecans), processed meats, dried fish (74), spices (79), and fats (75).

VI. CONTROL AND PREVENTION The oldest method of food preservation is probably drying. Salting, drying, and occasionally smoking were the only methods used until recent times, the reason being that if food is dried improperly, fungi will grow. Members of the genus Aspergillrrs are well known as spoilers of food and stored products. They cause deterioration of the product in appearance, quality, and food value. They also produce harmful mycotoxins. Only a brief summary can be given here. For more detailed reviews of Aspergillrrs in food and stored products see Refs. 31, 39, 40, 74, 86, and 87.

A.

Factors Influencing Spoilage of Foodstuffs and Mycotoxin Production

The factors influencing spoilage of foodstuffs and mycotoxin production are both physical and chemical. According to Pitt (74, 87), eight factors are involved: (1) water activity (moisture of the product), (2) pH, (3) temperature, (4) gas tensions (usually oxygen and carbon dioxide levels), ( 5 ) consistency of the product, (6) nutrient status, (7) specific solutes, and (8) use of preservatives. The production of a fungal metabolite or mycotoxin requires the previous growth of a fungus (88). Thus, formation of mycotoxins is influenced not only by environmental and nutritional factors, but also by the previous growth history of the fungal species. The factors that influence fungal growth indicated above also will exert an influence on mycotoxin production.

1. Water Activity Since the publication of Scott concerning the effect of water content (measured as water activity, a , )on the growth of microorganisms (89), a , has been adopted by food microbiologists to describe the relationship between moisture in food and the ability of fungi to grow in the food. Water activity is equal to equilibrium relative humidity (ERH) and is expressed as a decimal (75% ERH is equivalent to 0.75 a,). It is probably the most important factor governing food spoilage. Like all fungi Aspergillrrs species require water for growth. Unlike Penicillium species, which require water activities over 0.80, Aspergillus species can tolerate a wide range of a, (Table 5). The most xerotolerant is Eurotiwn 11nlophilicun?,a member of the Section Asper-gillzas (A. glnucus group). It is capable of growing at 0.65 a,. The cutoff point of lowest tolerable water activity for most fungi is considered to be 0.70 a,. Other xerotolerant Aspergillus species include members of the Sections Restricri and Aspergillus. As the water activity increases, the Aspergillus flora changes, with A. j h u s requiring the highest a,\. Thus, for safe storage, a, should be less than 0.65.

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Table 5 Water Activities for the Growth of Aspergillus species Water activity (a,)) Species

Minimum

Optimum

Ewotiurn nnlstelodnmi E. chevdieri E. echimtlcrtum E. hcrlopl1ilicutn E. herbariorm E. reperrs E. rrrbrwrl E. xeroplrilicwl A. petricillioides A. restrictus A. ccrrzrlihs A. ochr.crceus A. rnellelrs A. rliger A. werztii A. tlidularzs A. terreus A. clavrrtus A. j7or.jforrrlis A. pseuclocle$ectus A. sydolvii A. versicolor A. $crvus 4. oryzne A. prrnrsiticlrs Neosartoryrr jscheri A. j h i g a t n s

0.70-0.78 0.72-0.78 0.70-0.74 0.65-0.70 0.70-0.74 0.72-0.74 0.73-0.77 0.74-0.78 0.76 0.70-0.76 0.74-0.79 0.76-0.84 0.86 0.74-0.85 0.73-0.84 0.79-0.83 0.74-0.82 0.88 0.88 0.84-0.88 0.76-0.79 0.71-0.79 0.7 1-0.74 0.74 0.7 1 0.84 0.84-0.86

0.93-0.96 0.93-0.95 0.93-0.95 0.93-0.95 0.93 0.93-0.96 0.95

~~

-

0.9 1-0.93 0.82-0.93 0.98 -

0.98 0.94-0.96 0.96-0.98 0.95-0.97

-

0.96 -

0.98

0.97 0.97 ~~~~

Source: From Lacey and Magan ( 1991 ), Fungi In cereal grains: Thelr occurrence and water and temperature relationships.In Cered Grain Mycotosns, Furzgi atld Quulih in D n i n g and Storage (J. Chelkowski, ed.). Elsevier, Amsterdam, p. 77.

2. pH Most Aspergillus species are affected little by pH and can grow over a broad range, pH 3-8, with an optimum growth around pH 5. However, A. Iziger has been shown to tolerate pH 2 at high water activities (74). Since most foods have a neutral pH, this is not one of the more important factors for Aspergillus growth.

3. Temperature Aspergillus species can grow over a wide range of temperatures, from -8°C (A. glaucrrs) to 55°C (A. jhigntrrs).However, the optitnun1 temperatures for growth are between 20°C and 35°C (40). Thus, Aspergillus species will tend to be found in more tropical conditions in which the combination of high temperatures and low a,,, will cause deterioration of foods and other stored products (87).

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Some Aspergillus species are heat resistant and cause considerable problems in pasteurized food. Notable among these are the ascospores of Neoscwtolya jscher-i, which survived boiling distilled water for 60 min (74). A. fiu7ligntus is the most thermophilic of the Aspergillus species, with minimum growth at 12"C, optimum at 4Oo-42"C, and maximum growth at 55°C (74). It is the causal agent of spontaneous heating in grain that is stored damp (5). However, thermotolerant fungi, species able to grow at both moderate and high temperatures, are a bigger problem. A. j h w s and A. uiger, capable of growing between 8°C and 4S°C, are the most destructive of all fungi (71). 4. GasComposition In general, a reduction in oxygen and an increase in carbon dioxide concentrations can reduce the growth of fungi (74). However, many storage fungi (e.g., Aspergillus) are capable of growing at low oxygen levels, particularly in grain stored at high moisture. High concentrations of carbon dioxide usually are more inhibitory but other factors such as temperature and water activity do influence the degree of this inhibition (87). Growth of most Aspergillus species is reduced considerably in atmospheres of 1% 01,with A. ccrrldidzts and Ezrr-otium species proving the most tolerant. However, at levels of 0- 1% oxygen in a nitrogen atmosphere, growth is inhibited only slightly with Eurotirrm repens growing at 60-90% of the control rate (87). In atmospheres of 100% nitrogen "good" growth was observed in A. fziger and A. jkrligatus (91). This is because atmospheres high in nitrogen are effective only because of their low oxygen content, since nitrogen itself has little inhibitory effect (87). Laboratory tests using storage fungi held at 0.95-0.98 a, and 23°C showed that concentrations of carbon dioxide above 15% were required to halve the linear growth rate (90). The most sensitive species were En1ericellc1nidularls,A. firmigntus, and A. versicolor. However, the situation is somewhat different under storage conditions. Growth of A. .fln~ws in peanuts stored at high moisture levels was reduced in 80% C02/20% 02,but not in peanuts atlow water activity (0.70 a,) in 82% CO-, stored for 12 months (87). This indicates that, although growth is inhibited, spores of A. flaws remain viable at low water activities. Maintaining high concentrations of carbon dioxide requires a well-sealed storage system, which is costly. Fumigation, used to control insects, now is being used as an alternative, cheaper method for control of fungal growth. Studies using phosphine have shown that it has little effect on nongrowing fungi, but may prove useful against actively growing fungi (87).

5. Consistency of theProduct In general, filamentous fungi prefer a solid substrate and yeasts prefer liquid substrates for growth (74). Yeasts cause more spoilage in liquid products since they are single-celled organisms, and can consequently disperse more readily in a liquid. In addition, they prefer anaerobic conditions, which liquid substrates provide. By contrast, filamentous fungi, such as Aspergillus, prefer a firm substrate for growth together with an available source of oxygen. 6. NutrientStatus The nutritional status of food is adequate to allow growth of fungi (74). Fungi prefer food high in carbohydrates. They are capable of assimilating any food-derived carbon source,

Aspergillus

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and most (with the exception of some Pelzicillium species) can break down any nitrogen source (74). It has been observed that certain mycotoxins are produced in greater quantities on one particular commodity rather than another (88). Thus, aflatoxin is recorded most frequently from such oil seeds as groundnuts and other edible nuts, as well as cereals, particularly those, such as millet, maize, and sorghum, grown in warmer climates. Aflatoxin is not found to any great extent in soybeans. Laboratory studies on nutritional requirements for mycotoxin production sometimes can explain these field requirements. For example, zinc appears necessary for aflatoxin biosynthesis. The fact that soybeans contain phytic acid, a zinc-chelating agent, may account for soybeans being relatively aflatoxin free (88). 7. Specific Solute Effects To acertain extent, fungal growth under conditions of reduced water availability has been discussed in the section on a,. But, the presence of different solutes in food can affect the growth of fungi. Growth of E. nmstelodmni and E. ckevaliei-i is 50% faster at 0.96 a, when that a, is controlled by glucose rather than magnesium, sodium chloride, or glycerol (74, 89).

8. Use of Preservatives Preservatives are used to increase the shelf life of food (74), but with changes in attitude to food additives, most stored products destined for human consumption are free from preservatives. However, propionic acid at levels of up to 0.30% (w/w) is still added to flour to prevent fungal growth (41). Chemical preservation of stored crops is conm~onon small farms with limited resources for drying, but since the germinability of the grain is lost after treatment, such grain is only suitable for animal feed (41). In most cases, propionic acid is the preferred choice. Dosage levels depend on the moisture content of the grain and, if applied correctly, have proved most affective in controlling fungal growth during storage. However, incorrectly applied doses of the acid have caused aflatoxin production in barley in both Sweden and the United Kingdom (88). Indeed, laboratory studies have shown that propionic acid can enhance aflatoxin production by A. Jmws at concentrations that can partially inhibit growth (88).

B. ReducingMycotoxin Levels inFoodstuffs Once a foodstuff has become contanlinated with mycotoxins, it can be removed either by diverting the foodstuff from the commercial food chain or by using a detoxification procedure. Detoxification may occur naturally during food processing or a deliberate detoxification treatment may be used. Only a brief description can be given here. For more details, see Refs. 92-95. 1. Irradiation The use of irradiation for reduced microbial contamination in foods and feeds is likely to increase. Studies over 25 years ago showed that 350 kilorads (krad) were sufficient to inhibit the growth of Aspergillus (88). However, a number of studies have shown that cultures grown from irradiated spores of A. J%IW and A. pnrnsiticus have given enhanced levels of aflatoxin production compared to controls (88). There have been similar reports for ochratoxin A production by A. ochrnceus (88). This probably is due to irradiation

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causing changes in the biochemical pathways of these species, leading to more efficient syntheses of mycotoxin (96). Ultraviolet (UV) radiation appears to be more successful. Reports indicate that at a pH of less than 3 or greater than 10, UV radiation is effective against aflatoxin B (96). However, the limiting factor in this technique is the narrow range of wavelengths used in the experiments (96). Solar radiation also has been tested, but the production of new degradation compounds of unknown toxicity limits the successful application of this technique (96). 2. Heating Heat treatments already are used during the processing of feedstuffs to destroy microorganisms and insects, inactivate enzymes, and improve their quality, palatability, and digestibility (96). Mycotoxins therefore should degrade during such manufacturing processes. However, it is impossible to predict the extent of the mycotoxin reduction since degradation depends on the concentration of mycotoxins, the extent of binding between mycotoxin and food constituents, heat penetration, and other forms of protection (96). Aflatoxins have been found in such finished food products as tortillas (92). Citrinin, which often co-occurs with ochratoxin A, is more heat sensitive than ochratoxin A, but more work needs to be done on the toxicity and occurrence of breakdown or bound products (92). Ochratoxin A also survives most food processing.

3. Ammoniation Many chemical treatments have been tested for the removal of mycotoxins from feedstuffs. These include treatments with sodium hypochlorite, sodium and potassium hydroxide, hydrogen peroxide, sodium bisulfite, fomlaldehyde, and ammonia, used as either a gas or ammonium hydroxide (97). Citrinin and patulin are generally decomposed completely by ammonia, but sterigmatocystin remains stable, and there are conflicting reports regarding ochratoxin A (92). Aflatoxins are degraded readily by ammonia (92). Successful treatments have been conducted for peanut meal, peanut products, and maize (97). Indeed, detoxification using ammonia is used on many farms in the United States (92). To date, only ammoniation and reaction with sodium bisulfite have received industrial attention. In the United States ammoniation is used in cottonseed and corn products. In the EU imported ammonia-treated peanut meal for animal feed is an accepted feature. 4. Absorbents Several absorbents, such as clay, charcoal, asbestos, silicas, xeolites, and aluminosilicates, have been used to remove aflatoxins from aqueous solutiona (92). The anticaking agent hydrated sodium aluminosilicate (HSCAS) has been added successfully to the aflatoxincontaminated diets of chicks and pigs with no adverse effects on the animals (92). However, these organic materials can sequester and immobilize aflatoxin in the gut of livestock and poultry, preventing normal uptake in the blood and hence transport to target organs such as the liver (98). At least one of these products is already on the market (Sorb-it, Anitox-Corp.). 5. Sorting In general, mycotoxins are confined to only a small proportion of individual seeds and kernels. It should be possible, therefore, to remove such seeds or kernels mechanically. Techniques for sorting color and other visual characteristics have been used for removing

Aspergillus

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aflatoxic-contaminated products (99). Such techniques include hand sorting on a conveyor belt, electronic color sorting, and fluorescence under UV light (99). The fluorescence technique is used commercially for sorting peanuts, almonds, cottonseeds, and figs. 6. Biotransformation In processes similar to those used in bioremediation techniques, it has been shown from studies in the Far East that several microorganisms, especially Rhizopus species and Neurospora sitophiln, can degrade aflatoxin in peanuts by solid substrate fermentation. Aflatoxin B I was almost totally degraded with only a small residual amount of toxic intermediates (100).

7. Dilution of ContaminatedFoodstuffs Finally, dilution of contaminated grain with sound grain may reduce the toxicity of feed for farm animals and lower mycotoxin levels in foods destined for human consumption (92). Such mixing will occur normally during storage, transport, and processing of the foodstuff.

REFERENCES 1. Micheli, P. A. (1 729). NOIU Plarztnrzm Genera, Florence. Italy [in Latin]. 2. Link, H. F. (1809). Observationesinordinesplantarunlnaturales, GessellsclzarftNcrtllrforscllender Freuncle ,-u Berlin Mugazin, 3: 1. 3. Thorn. C., and Church. M. B. (1926). The Aspergilli, Williams & Wilkins, Baltimore. 4. Thorn, C., and Raper, K. B. (1945). h!k7iZl4Ci/ sf the Aspergilli, Williams & Wilkins, Baltimore. 5. Raper, K. B., and Fennell, D. I. (1965). The G e m s Aspergillus, Williams & Wilkins, Baltimore. 6. Al-Musallam,A. (1980). Revision of the black Aspergillus species, thesis, University of Utrecht, Utrecht. 7. Christensen, M. (1982). The Aspergillus oclzmceus group: two new species from western soils and a synoptic key, Mycologia, 74910. 8. Christensen, M.. and States, J. S. (1982). Aspergillus izidularls group: Aspergillus ~zavahoensis. and a revised synoptic key, Mycologia, 74:226. 9. Christensen, M. (1981). A synoptic key and evaluation of species in the Aspergillns ja174s group, Mycologia, 73: 1056. 10. Kozakiewicz, Z. (1982). Theidentity and typification of Aspergillus parmiticus, Mycotcmon. 15293. 11. Frisvad, J. C., and Samson, R. A. (1990). Chemotaxonomy and morphology of Aspergillus jiln~igcrtusand related taxa. InModen1 Concepts in Penicilliton arzd Aspergillns Classificcrtion (R. A. Samson, and J. I. Pitt, eds.), Plenum Press, New York, p. 201. 12. Kozakiewicz, Z. (1 986). New developments jn the accurate identification of aspergilli in stored products. In Spoilage m d Mycotoxins of Cereals ard Other Stored Products (B. Flannigan, ed.), Z~tterrmtionalBiodeteriomtion 32. CAB International, Wallingford, UK, p. 115. 13. Nealson, K. H.. and Garber, E. D. (1967). An electrophoretic survey of esterases, phosphatases. and leucineamino-peptidases inmycelial extracts of species of Aspergillus, Mycologia, 59330. Garber, E. D. (1973).A geneticstudy of electrophoreticallyvariant 14. Kurzeja,K.C.,and extracellular amylolytic enzymes of wild-type strains of Aspergilllts nidzrlrrzs, Can. J. Gen. Cytol.. 15975. and closely 15. Cruickshank, R. H., and Pitt, J. I. (1990). Isoenzymepatterns in As~~ergillusJkr~~us

498

16.

17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27.

28.

29.

30.

31. 32.

Kozakiewicz related taxa. In Modem Concepts in Perzicilliurlr and Aspergillrrs ClassiJicatiorz(R. A. Samson, and J. I. Pitt, eds.), Plenum Press, New York, p. 259. Sugiyama, J., and Yamatoya, T. (1990). Electrophoretic comparison of enzymes as a chemotaxonomic aid among Aspergillus taxa (1). Aspergillus sects. Ornati and Crerrrei. In Moclerrr Concepts irl Penicillium and Aspergillm Clnssijicatiorl (R. A. Samson. and J. I. Pitt, eds.), Plenum Press, New York, p. 385. Yamato, T., Sugiyama, J., and Kuraishi, H. (1990). Electrophoretic comparison of enzymes as a chemotaxonomic aid among Aspergillzw taxa (2). Aspergillus sect. Flavi. In Modern Concepts in Penicillium and Aspergillns Clmsijication (R. A. Samson, and J. I. Pitt, eds.), Plenum Press. New York, p. 395. Kuraishi, H., Tto, M.. Tsuzaki, N.. Katayama, Y., Yokoyama, T., and Sugiyama, J. (1990). Ubiquinone systems as a taxonomic tool i n Aspergillus and its teleomorphs. In Modenz Cow cepts in Penicilliw?l crnd Aspergillus Classijicntiorz (R. A. Samson,and J. I. Pitt, eds.), Plenum Press, New York, p. 407. Kozlowski, M.,and Stepien, P. R. (1982). Restriction enzyme analysis of mitochondrialDNA of members of the genus Aspergillus as an aid in taxonomy. J. Gen. Microbiol. 128:471. Mullaney, E. J.. and Klich. M. A. (1990). A review of molecular biological techniques for systematic studies of Aspergillus and Penicilliwn. In Modern Concepts in Penicillir4it1 arzd Aspergillus Classification (R. A. Samson, and J. 1. Pitt, eds.), PlenumPress.New York, p. 301. Moody, S. F., and Tyler, B. M. (1990a). Restriction enzyme and mitochondrial DNA of the Aspergillus jlnvus group, Aspergillus jarus. Aspergillus parmiticus and Aspergillus nomius. Appl. Em7iron. Microbiol.. 56:2441. Moody, S. F., and Tyler, B. M. (1990b). Use of nuclear DNA restriction length polymorphisms to analyze the diversity of the Aspergilluskflalxsgroup, As~?ergillus~cllsrs, Aspergillus parmiticus and Aspergillus Ilonlius. Appl. Em7iron. Microbiol., 562453. Kusters-van Someren, M.A., Samson, R.A., and Visser. J. ( 1991). The use of RFLP analysis in classification of the black Aspergilli: reinterpretation of the Aspergillus niger aggregate, Cwr. Genet., 1991. Croft. J. H.. Bhatacherjee, V.. and Chapman. K. E. (1990). RFLP analysis of nuclear and mitochondrial DNA and its use in Aspergillus and Penicillium systematics. In Modem Concepts irz Penicillilrm andAspergillus Clcrss(jicatiorz(R. A. Samson, and J. I. Pitt, eds.), Plenum Press, New York, p. 309. Kurtzman, C. P., Smiley, M. J., Robnett, C. J., and Wicklow, D. T. (1986).DNA relatedness among wild and domesticated species in the Aspergillus j h w s group, Mycologirr. 78:955. Polonelli, L., Castagnola. M., D’Urso, C., and Morace, G. (1985). Serological approaches for identification of Aspergillus and Penicillium species. In Ad\wzces in Penicilliunz ctnd Aspergillus Systenlatics (R. A. Samson, and J. I. Pitt, eds.). Plenum Press, New York, p. 267. Sekon, A. S., Standard, P. G., Kaufman, L., and Garg, A. K. (1985). Value of exoantigens for grouping Aspergilli. IX. Internatiorzcrl Congress of the Interncrtiorzal Society for Human and Aninla1 Mycology, 125. Frisvad, J. C. (1985). Secondary metabolites as an aid to Emericella classification. In Advances in Perlicillimz and Aspergillus Systematics (R. A. Samson, and J. I. Pitt, eds.), Plenum Press, New York, p. 437. Frisvad, J. C. ( 1989). The connectionbetween the Penicillia and Aspergilli and mycotoxins with special emphasis on misidentified isolates, Arch. Emiron Contam. Tosicol., 18:452. Udagawa, S., and Takada, M. (1985). Contribution to our knowledge of Aspergillus teleomorphs: some taxonomic problems. In Adlmces in Penicilliu~?land Aspergillns Systematics (R. A. Samson, and J. I. Pitt. eds.), Plenum Press, New York, p. 329. Kozakiewicz, Z. (1989). Aspergillus species onstoredproducts, Mycol. Pap., 161. CAB International, Wallingford, UK, pp. 1 - 188. Samson, R. A., Nielsen, P. V., and Frisvad, J. C. (1990). The genusNeosartmya: differentia-

Aspergillus

33. 34.

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

48. 49. 50.

51. 52. 53. 54.

55.

499

tion byscanning electron microscopy and mycotoxin profiles. In Modern Conceptsin Penicill i m am1 Aspergillus Classification (R. A. Samson, and J. I. Pitt, eds.), Plenum Press, New York, p. 455. Samson, R. A., and Pitt, J. I. (1985). Advances in Penicillium and Aspergillus Systematics, Plenum Press, New York. Pitt. J. I., and Samson, R. A. (1993). Species names in current use in the Trichocomaceae, (Fungi, Eurotiales). InNames ill Ctrrr-entUse irr the Fantilies Trichocornaceae, Cladorziaceae. Pinaceae (W. Greuter. ed.), Koeltz Scientific Books, Konigstein, Regnuwt vegetabile 128: 13. Samson, R.A., and Pitt, J. I. (1990). Modem Corlcepts in Penicilliunt and Aspergillus Classijicatio~t,Plenum Press, New York. Samson, R.A.. and Pitt, J. I. (in press). Integration of Modern Taxonomic Methodsfor Penicillium and Aspergillus Classificatiou Methods, Harwood Academic Publishers, Amsterdam. Davis, N. D., and Diener, U. L. (1987). Mycotoxins. In Food and Beverage Mycology (L. R. Beuchat, ed.). Van Nostrand Reinhold, New York. Smith, J. E., and Ross, K. (1991). The toxigenicaspergilli. In Mycotoxins arzd Animal Foods (J. E. Smith, and R. S. Henderson, eds.), CRC Press, Boca Raton, FL. Beuchat, L. R. (1987). Food and Belverage Mycology, Van Nostrand Reinhold, New York. Chelkowski, J. (1 99 1).Cereal Grain Mycotoxins, Frmgi and QualiQ ill Dlying arzd Storage, Elsevier, Amsterdam. Smith, J. E., and R. S. Henderson. (1991). Mycotoxins and Animal Foods, CRC Press, Boca Raton, FL. Newborn, P. M., and Butler, W. H. (1969). Acute and chronic effects of aflatoxin on the liver of domestic and laboratory animals: a review, Carlcer Res., 29236. World Health Organisation. (1979). Emironmental Health Criteria. II, Mycoto.xins, WHO, Geneva. Bryden, W. L. (1982). Aflatoxins and animal production: an Australian perspective, Food Tech. Aust., 34:216. Richard, J. L., and Thurston, J. R. (1986). Diagl1osis of Mycotoxicoses, Martinus Nijhoff, Boston. Arora, R. G., Frolen, H.. and Nilsson, A. (1 981). Interference of mycotoxins with prenatal development of the mouse, Acta Vet. Scand., 22:534. Frisvad, J. C.. and Samson, R. A. (1991). Mycotoxins produced by species of Penicilliurtt and Aspergillus. In Cered Grain Mycotoxins, Fmgi and Quality irt Dving and Storage (J. Chelkowski, ed.). Elsevier, Amsterdam, p. 441. Dickens, F.. Jones, H. F. H., and Waynforth, H. B. (1966). Oral, subcutaneous and intratracheal administration of carcinogenic lactones and related substances, Br. J. Cancer, 20: 134. van der Merwe,K. J., Steyn, P. S.. Fourie, L., Scott, D. B.,and Theron, J. J. (1965). Ochratoxin A, a toxic metabolite produced by Aspergillus ochracetrs Wilh., Nature (Lond.), 205: 11 12. Pepeljnak, S., and Cvetnik, Z. (1988). Ochratoxigenicity of Aspergillus oclzraceus strains from nephropathy and anephropathy areas in Yugoslavia, Proceedings of Jpn. Assoc. Mycotoxicol., Supplement 1:69. MacGregor, K. A., and Mantle,P. G. (1991). Nephrotoxic fungi in a Yugoslavian community in which Balkan nephropathy is hyperendemic, Mycol. Res., 95:660. Vleggaar, R., and Steyn, P. S. (1980). The biosynthesis of some miscellaneous mycotoxins. In The Biosynthesis of Mycotosirls (P. S. Steyn, ed.), Academic Press, New York, p. 395. Nishie, K., Cole, R. J., and Dorner, J. W. (1985). Toxicityand neuropharmacology of cyclopiazonic acid, Food Chent. Toxicol., 23:831. Norred. W. P., Morrissey, R.E., Riley, R. T., Cole, R. J., and Dorner, J. W. (1985). Distribution, excretion and skeletal muscle effectsof the mycotoxin (14C) cyclopiazonic acid rats, in Food Chent. Toxicol., 23:831. Moreau, C. (1979).MOU/dS, tosirls and food, translated by M. 0. Moss, J. Wiley, Chichester.

500

Kozakiewicz

56. Ceynowa, J. (1986). Mykologische Untersuchungenand luftdicht gelagertem Getriede. Dissertation, Christian-Albrechts Universitat, Kiel. 57. Ciegler, A. (1975). Mycotoxins: occurrence, chemistry. biological activity, Lloydia, 38:21. metabolites (mycotoxins) produced by fungi colonizing cereal 58. Golinski, P. (1991). Secondary grain in store-structure and properties. In Cereal Grcrin Mycotoxins, Fu~zgiand Qualih in Dtyirzg and Storage (J. Chelkowski, ed.j, Elsevier, Amsterdam, p. 355. 59. Cole, R. J., and Cox, R. H.( 1981). Hrrndbook of Toxic Fungal Metabolites, Academic Press, New York. 60. Austwick. P. K. C. (1965). Pathogenicity. In The Genus Aspergillus (K. B. Raper, and D. I. Fennell, eds.), Williams & Wilkins, Baltimore. p. 82. 61 Smith, J. M. B. (1989). Opportmistic Mycoses of Mall and Other Aninzals, CAB Tnternational, Wallingford, UK. 62. Grigoriu, D., Delacretaz. J., and Borelli, D. (1987).Medical Mycology. Hans Huber. Toronto. 63. Pervez, N. K., Kleinerman, J., Kattan, M., Freed, J. A., Harris, M. B., Rosen, M. J.. and Schwartz, I. S. (1985). Pseudomembranous nectrotizing bronchial aspergillosis. A variant of invasive aspergillosis in a patientwith hemophilia andacquired immune deficiency syndrome, Am. Rev. Respir. Dis., 131:961. 64. Mullins, J., Harvey, R., and Seaton, A. ( 1976). Sources andincidence of airborne Aspergillus jianigatus Fres.. Clin. Allergy, 6209. 65. Rippon, J. W. (1982). Medical Mycology, 2d ed.. W. B. Saunders, Philadelphia. 66. Banaszak, E. F., Thiede, W. H. (1970). Hypersensitivity pneumonitis due to contamhation of an air conditioner, N. Engl. J . Med., 283:271. 67. Kostelnik, M. D., and Fremount, H.N. (1976). Mycotic tubo-ovarian abscess associated with the intrauterine device, Am. J. Obstet. Gpecol., 125272. 68. Young, N. E. (1970). Pulmonary aspergillosis in the lamb, Vet. Rec., 86:790. 69. Coad, C . T., Robinson, N. M., and Wilhemus, K. R. (1985). Antifungal sensitivity testing for equine keratomycosis, Am. J. Vet. Res., 46:676. 70. Day, M.J., Penhale, W. J., Eger, C.E., Shaw. S. E., Kabay, M. J., Robinson, W. F.. Huxtable, C. R. R., Mills, J. N., and Wyburn, R. S. (1986). Disseminated aspergillosis in dogs, Aust. Vet. J., 6355. 71. Yu, V. L., Muder, R. R., and Hurt,J. J. (1986). Significance of isolation of Aspergillus from the respiratory tract in diagnosis of invasive pulmonary aspergillosis, Ant. J. Med.. 81949. 72. Minter, D. W., Hawksworth, D. L., Onions, A. H. S., and Kozakiewicz, Z. (1985). Descriptive terminology of the conidiogenous structures in Aspergillus and Perzicillium. In Advawes in Perzicilliunz and Aspergillus Systematics (R. A. Samson, and J. I. Pitt, eds.), Plenum Press. New York, p. 71. 73. Gams, W., Christensen, M., Onions, A. H. S.. Pitt, J. I., and Samson. R. A. (1985). Tnfrageneric taxa of Aspergillus. In Advances ilt Pe~zicilliuma d Aspergillus Systematics (R. A. Samson, and J. I. Pitt, eds.). Plenum Press, New York, p. 55. 74. Pitt, J. I., and Hocking, A. D. (1997). Furlgi and Food Spoilage. 2d ed., Chapman & Hall. London. 75. Ccrtalogne of the Culture Collection of the International Mycological Institute. (1992). 10th ed., CABI, Wallingford. 76. Pitt, J. I.. and Samson, R. A. (1990). Taxonomy ofAspergillus Section Restricta. In Modenz Concepts in Perlicilliunt and Aspergillus Clnss$cation (R. A. Samson. and J. I. Pitt, eds.), Plenum Press, New York, p. 249. 77. Channell, S., Blyth, W., Lloyd, M., Weir. D. M., Amos, W. M. G.. Littlewood, A. P., Riddle, H. F. V., and Grant. I. W. B. (1969). Alveolitis in malt workers, &. J. Med., 38:351. 78. Gilmour, J. S., Inglis. D. M., Robb, J., and Maclean. M. (1989). A fodder mycotoxicosis of ruminants caused by contamination of a distillery by-product with Aspergillus clavatus, Vet. Rec., 124:133. 79. Flanningan, B., Day,S. W., Douglas, P. E.. and MacFarlane, G. B. (1984). Growthof myco-

Aspergillus

80.

81. 82.

83.

84. 85. 86. 87.

88.

89. 90. 91. 92.

93. 94. 95.

96. 97.

98. 99.

100.

501

toxin-producing fungi associated with malting of barley. In Toxigenic Fwlgi-their To.xins and Health Hazard (H. Kurata, and Y. Ueno, eds.). Elsevier, Amsterdam, p. 52. Frisvad. J. C., and Filtenborg, 0 (1995). Mycotoxin production by food-borne fungi.In htroduction to food-bornefruzgi (R. A. Samson, van Reenen-Hoekstra, E. S., Frisvad, J. C. and Filtenborg, 0..eds.), 4th Edit. CBS, Baarn, p. 251. Wiley, B. J.. and Simmons, E. G. (1973).New species and new genus of Plectomycetes with Aspergillus states, Mycologin, 65:934. Klich, M. A., and Mullaney, E. J. (1987). DNA restriction enzyme fragment polymorphism as a rapid differentiation of Aspergillus jhws from Aspergillus onzae, Expt, Mycol., 11 : 170. Kurtzman, C. P., Horn, B. W.,and Hesseltine, C. W. (1987).Aspergillus nomius, a new aflatoxin-producing species related to Aspergillus j m u s and Aspergillus tnmnrii, Antorlie Ivan LeeLwedzoek 53: 147. Hayes, A. W. (1980). Mycotoxins: a review of biological effects and their role in human diseases, Clin.Tosicol., 17:45. Van Egmond, H. P. (1989). Aflatoxin M: occurrence, toxicity, regulation. In Mycotoxins in Dniry Products (H. P. Egmond ed.), Elsevier, London, p. 1 I . Samson, R. A., van Reenen-Hoekstra, E. S., Frisvad, J. C. and Filtenborg, 0. (eds.) (1995). I n t r o d ~ ~ t i oton Food-borne Fwlgi. 4th ed., CBS, Baarn. Champ, B. R., Highley. A. D., Hocking, A. D., and Pitt, J. I. (eds.) (1992). Fungi and Mycotosirrs in Stored Products, ACIAR, Canberra. Moss, M. 0. (1991). The environmental factors controlling mycotoxin formation. In Mycotoxins and Arziwcrl Foods (J. E. Smith and R. S. Henderson, eds.) CRC, Boca Raton, FL, p. 37. Scott, W. J. (1957). Water relations of food spoilage micro-organisms, Ad)!.Food Res., 7: 83. Niles. E. V., Norman, J. A., and Pimbley, D. (1985). Growth and aflatoxin production of Asper.gillusjavus in wheat and barley. Tram. Br. Mycol. SOC.,84:259. Tabak, H. H., and Cook, W. B. (1968). Growth and metabolism of fungi in an atmosphere of nitrogen, Mycologirr, 60: 115. Scott, P. M. (1991). Possibilities of reduction or elimination of mycotoxins present in cereal grains. In Cereal Grain Mycotoxins, Fungi and Qlraliv in D~yirrgand Storage (J. Chelkowski. ed.), Elsevier, Amsterdam, p. 529. Muller, H.-M. (1983). A survey of methods of decontaminating mycotoxins. T. Physical methods. Arzirn. Res. Dev., 18:70. Muller, H.-M. (1983). A survey of methods of decontaminating mycotoxins. 11. Chemical methods and reactions with components of feedstuffs, Anim. Res. Dev., 19:7. Park, D. L., Lee, L.S., Price, R. L., and Pohland, A. E. (1988). Review of the decontamination of aflatoxins by ammoniation: current status and regulation, J. Assoc. Ofs Anal. Clrenz., 71: 685. Samarajeewa, U. (1991). In situ degradation of mycotoxins by physical methods. In Mycotoxins arldAnirtza1 Foods (J. E. Smith,and R. S. Henderson, eds.), CRC, Boca Raton, FL, p. 785. Pemberton, A. D., and Simpson, T. J. (1991). The chemical degradation of mycotoxins. In Mycotoxins anclAtrirna1Foods, (J. E. Smith, and R. S. Henderson, eds.), CRC. Boca Raton, FL, p. 797. Smith, J. E., and Solomons, G.L. (1 994). Mycotoxins i n Hzman Nutrition and Health. AgroIndustrial Research Division, European Commission Directorate-General XII, pp. 1-300. West, D. I., and Bullerman. L. B. (1991). Physical and chemical separation of mycotoxins from agricultural products. In Mycotoxins and Anirnal Foods, (. J. E. Smith,and R. S. Henderson, eds.), CRC, Boca Raton, FL, p. 777. Medlock, V. F. P. (1996).Fungal toxins in Foods and Feeds. SOC.Gen. Microbiol. Qu., 233): 71.

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11 Claviceps and Related Fungi Gretchen A. Kuldau Pennsylvania State University, University Park Pennsylvania

Charles W. Bacon U.S. Depastment of Agricrclture, Athens, Georgia

I. Introduction 503 11. Biology 505

A. Claviceps species 505 B. Grass endophytes 510 111. ErgotAlkaloids and OtherToxins514 A. Ergot alkaloids 5 15 B. Tremorgenic toxins 16 5 C. Miscellaneous toxins 5 16 IV. Toxicity Syndromes 517 A. Claviceps toxicity 517 B. Neoephodium-induced toxicities 18 5 C. Symptoms of Balmsin toxicity and relatedfungi520 D. Pnspalum staggers 521 V.Control and Management521 A. Claviceps 521 Grass B.endophytes 522 VI. Future Prognosis 523 References 524

1.

INTRODUCTION

The family Clavicipitaceae (Ascomycetes, order Hypocreales) is characterized by perithecia embedded infleshy stromata and the production of filiform ascospores (sexual spores), in long cylindrical asci with a thickened apex (1). All members are either obligate biotrophs of grasses and grass allies, or pathogens of arthropods and fungi in the genus Elaphonzyces. The family Clavicipitaceae is a relatively small homogeneous grouping of approximately 200 known fungal species of which the genus Claviceps is the mostinfamous. Species of Claviceps and their toxins (ergot alkaloids) are historically the first re503

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corded groups of extrinsic biological antiquality factors reported in mankind‘s food. The fungus is very well known for its toxicity in rye and rye products, but it also affects wheat, barley, sorghum, and other small grain. Epidemics that killed and crippled hundreds of thousands of people in Europe date back to the time of the Caesars and continued to ravage Europe until the end of the Middle Ages. Ergotism as a distinct fungus-related plant disease was first established by Von Munchhausen in 1764 (2) but it was not until 1853 that Tulasne named the fungus and established its life cycle (3). The chemical nature and biological effects of the toxins produced by Clmliceps species were established in the middle of the nineteenth century (4, 5). This species is a pathogen of grasses and their relatives, particularly cereals, where it infects and parasitizes the developing reproductive organs of grasses. Shortly after this discovery, improvements in agricultural practices, tnilling techniques, and grading of cereal grains have all but eliminated the potential risks of ergotism. However, the disease still occurs, primarily in developing countries. The recent outbreak of ergotism on sorghum (6) in all countries where this grain is grown indicates the constant threat of cereal grasses to epidemics from this fhgus. During the latter half of the 20th century another discovery revealed that the family Clavicipitaceae consisted of other species of fungi, grass endophytes, including species of Neo@phodiurtz, Bdcrrzsicr, and others, that produced identical toxins on forage grasses, causing livestock diseases identical to ergotism. These fungi. however, are unlike the species of Clcrviceps in being endophytic, living within the plants’ reproductive and vegetative parts. Studies on endophytic members established that ergot alkaloid biosynthesis was maintained throughout evolution of most species in the family that are parasitic on plants. The internal colonization of grasses serves to distinguish the endophytic group from the rest of the Clavicipitaceae. Further, endophytic associations are natural, only a few are pathogenic, while most are mutualistic and ecologically essential for infected grasses to survive the stresses from herbivory and competition. Thus, management practices used to control Clmiceys species will not work for the grass endophytes, creating problems primarily in the livestock industry. Human and livestock toxicity problems are associated with Clmiceps spp. as these species are parasitic on cereal grain used for human food. Only livestock toxicity is associated with endophytic fungi as they are associated with forage grasses. In both groups, the resulting toxicity problems are biological antiquality factors to the grass. This chapter concerns the plant-associated members of the family, which comprise the genera Atkimorlellrl, Brrlarzsin, Clnviceps,Echil~odothis,Epicldoe(Neotyphodium), Myr-iogenospor.a, and Prrr-epichloe (7-9). Endophytes under the name Neohphoc l i m replace Acr-emorziLm (lo), the earlier name proposed for this section of fungi (1 1). Our focus will be on Clcnliceps, Epichloe, Neoh’yhodiurn, Bnlrrnsin, and Myr-iogenosyom as these are the genera with the greatest direct impact on humans and livestock. The association of all members of this family on a very wide range of grain crops and forage grasses has produced a myriad of toxicity problems for humans and livestock. This chapter is concerned with the wide expression of ergotism, reflective of all ergot alkaloid producing fungi of the Clavicipitaceae. We will present information that suggests that the grass hosts and the environment are also factors that influence the final toxicity observed. This is one of the first known families of fungi in which most members produce. almost exclusively, one class of mycotoxins: ergot alkaloids. The primary objective of this chapter is to discuss both Clnviceps and grass endophytes in terms of their

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biological differences and similarities, as well as their toxicities, toxins, and management for either toxin production or control of fungal growth.

BIOLOGY II. Fungal species of theClavicipitaceae can bedifferentiated on the basis of their morphology and growth habit as well as by the details of their life history, which will be discussed in this section. Epiclzloe and its asexual relatives inthe genus Neotyphodium (10) are exclusively endophytic and produce small ovoid conidia (asexual spores) (12, 13). In contrast, the genus Balnnsiu has endophytic and epiphytic members that produce large needle-shaped conidia, which is referred to as the ephelis state (7, 14, 15). Mvriogenospor-cr is exclusively epibiotic and also produces an ephelis state (16, 17). Clcwiceps spp. are also considered epibiotic since they produce a localized infection within the plant ovary. This genus produces only small ovoid conidia termed sphacelia that are remarkably similar to the Neotyphodiunl state of the Epiclzloe species (18).

A.

ClavicepsSpecies

Members of the genus Clcrviceps are found on all continents. Over 30 species of Clcrviceps are described in the literature and new species have been designated recently (Tables 1 and 2) (4, 19-21). In addition, there are numerous mentions of unidentified species suggesting that the actual number of biological species of Clnviceps is likely greater than what is currently described. However, all descriptions thus far are based on morphological characters and have not used the biological species concept. Such approaches are hampered by the fact that C. purprrrecr (Fig. 1A) and probably all Clnviceps spp. are homothallic; i.e., both mating types are found in one individual (22). Of the species described, several are of agronomic concern. A major concern is C. purpurea, which has a very wide host range including both forage grasses and the cereal grain crops rye, wheat, oats, and barley (Table 3). The wide host range of C. purpui-ecr appears to be the exception as most species are limited to a particular genus or tribe of grasses (Table 2j (4). For example, C. pnspedi is a pathogen of grasses in the genus Pnspalwn (Dallis grasses and related species) and has a worldwide distribution because Dallis grasses are used extensively for forage. Others include Clcrvicepsfrrsiforrnis,an important pathogen of pearl millet in India and Africa (23), C. c!fricnnn (Fig. lB), and C. sorghi are pathogens of sorghum that have recently become globally distributed (6). Further, there are also species of Clcrviceps that infect nongraminaceous plants in the Cyperaceae and Junceae (Table 1). Clnviceps species infect the floral ovary of their hosts the end result of which is a hardened fungal resting structure, the sclerotium or ergot, which replaces the seed. In the field, dark Clcrviceps sclerotia can be seen on the seed heads of grasses (Fig. 1). Ascospores (sexual spores) or conidia (asexual spores) germinate on the stigma and style of open florets and the fungal hyphae grow intercellularly down the style and into the ovary where a dense mycelium forms within a few days of infection (24-36). The cells of the ovary are destroyed and the fungus establishes a foot structure at the base of the ovary (2426). This fungal structure has been observed to extend hyphae into the vascular tissue of the plant to tap nutrients and is distinct from the sclerotium, which develops distal to it

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Table 1 The World’s Species of Clnviceps,aother than C. purpurea, and their Hosts Species Claliceps anrlulatn Langdon C. africam Frederickson, Mantle and De Millicno C. citrirln Pazoutova, Fucikousky, Leyva-Mist Flieger C. cinerea Griffiths C. cynodorltis Langdon C. cyperi Loveless C. diadema (Moller) Diehl

C. digitariae Hansford C. jnvelln (Berk. and Curt.) Petch C. jlsiforrrris Loveless C. gigantic1 Fuentes, Isla. Ullstrup, and Rodriguez C. glabra Langdon C. grohii Groves C. hirtella Langdon C. inconspicua Langdon C. junci J.F. Adams

C. litoralis Kawatani C. lutea Moller C. nzaximerzsis Theis C. microspora Tanda C. nigricnns Tul. C. orthocladae (P. Henn.) Diehl C. paspnli Stevens and Hall C. phnlaridis J. Walker C. plahtricha Langdon C. pusilla Ces. C. queemlnrzdicn Langdon C. ramrnculoides Moller C. sorghi McRae C. sdcatn Langdon C. tripsaci Stevens and Hall C. dearla P. Henn. C. viridis Padwick and Azmatullah C. yarzagnwaensis Tangashi C. Lizarziae (Fyles) Pantidou

Hosts Edalia j h ~ z . Sorglnm bicolor Distichlis spicata Hilaria vttutica, H. cenchroides, and Meliccc sp. Cynodo~ldactylon Cypenu rotundus, C. esculentus Icchantllus rieclelii, I. vertillatus, and other Ichnnthus species Digitaria diagonalis, D. Gazertsis, and other Digitnria SPP. Pnnicunz spp. Perlnisetlrrn fyphoides Staph. and Hubbasd Zea m a y Digitnria longijlorcr Carex angustior Mackenzie, Carex stipatcc Muhl., C. arencrria L., and other Carex spp. Eriocldoa pseudoacrotricha Hyparrhenia jilipendrdu Juncus glaucus Ehrh., J. nodosus L.. and other Junc~ls SPP. Several Elyrnlu spp. and Hosdeunr spp. Several species of Paspalunr Panicnm maximum Eccoilopus cotdifer Eleocllaris palustris R.Br., other Eleocharis spp., Capillipediurn spp., and Scirpus spp Several Orthoclade species and Panicurn species Paspalurn dilatmt and 17 other Puspcrlun~spp. Phalaris spp. Ischaenrm australe Parlicuvn maxinlunt, Hyparrhenia spp., and Botkrioclzloa SPP. Thenzeda australis, T. aIwraca, Vetiverin jilipes, Soreghum leocladlrm Several Setaria spp. Sorghum +p” Pers. Brachiaria spp. and Urochloa sp. Tripsacurn dachloides (L.) Panicum spp. Oplismerzus compositus Zoysia japonica Steudel Zizania aquatica L. and Z. yalustris L.

List compiled according to Brady (206), except for the species C. africana (20). C. cirrina (21), C. cyperi (207). C. filsifomis (129). and C. sorgki (20).

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Table 2 HostIndex for Claviceps Purpuread Grass tribeh

Genera

Agrostideae Andropogoneae Aveneae Chlorideae Festuceae Hordeae Phalarideae Zizanieae (' C. purpurea occurs on more than 600 grass species, representing 40 genera of grasses (208).

Tribes and species defined according to Hitchcock (209).

(25). The establishment of hyphae at the site of the ovary is referred to as the sphacelial stage. One of the hallmarks of the sphacelial stage is the production of honeydew, a sugarand conidia-filled exudate that emanates from the infected florets. The production of honeydew begins as early as 4 days after infection and can be copious, as in the case of C. crfriccrnn, or sparse, as in the case of C. pcrspcrli (6, 24). Sugar composition of honeydew varies among species and is determined by the fungus (27, 28). The honeydew contains abundant conidia, which are produced on the surface of the developing fungal sphacelium. The honeydew is thought to play at least three roles in the life history of C. purpuren: it attracts insects, which then vector the conidia embedded in it to other plants, the honeydew flow forces the conidia out of the florets, and the high concentrations of sugars in the honeydew may act as a self-inhibitor that maintains conidial dortnancy since germination is inhibited in honeydew (27, 29). The honeydew state is highly variable. In contrast to C. purpureen, the sphacelial conidia produced by C. cfricnrza and C. paspnli do germinate in the honeydew and give rise to secondary conidia that protrude above the surface of the honeydew droplets on their phialides (24, 30). Indeed, in the species C. tripscrci there is no honeydew; it is replaced with tnasses of dry fusoid to lunulate conidia. Epidemiological studies suggest that the secondary conidia of C. africnrza are important in dissemination of the fungus and may explain this species' ability to spread quickly over large geographic areas (30, 31). Ergot alkaloids have not been found in the honeydew exudates. The sclerotium is the site of ergot alkaloid production, which begins at the very moment of the formation of the sclerotial cells (32, 33). To emphasize the accumulation at this stage, ergot is historically used as a synonym for the sclerotium as well as the toxic alkaloids it contains. This morphological shift to sclerotial cells also occurs in laboratory culture (33). The sclerotium is initiated from a meristematic region adjacent to the foot, with development proceeding such that the more distal regions mature first (25, 26). Triglycerides also accumulate in the mature sclerotium (29). The role of ergot alkaloids in the biology of Claviceps is entirely unknown; however, a proposed role in protection of the sclerotia against insect herbivory may be drawn from work dealing with toxicity of ergot alkaloids to insects (34). Further, mammalian toxicity of the alkaloids tnight serve

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k Figure 1 Claviceps and grass endophytes: (A) ergot sclerotia of C. purpurea on rye; (B) honeydew state of C. africana on sorghum; (C) enlargement of (B) showing copious production of honeydew of C. africana (arrows); (D) Balansia henningsiana, arrows, on broomsedge grass; (E) Epichloe typhina on Sphenopholis ohrusata showing conidial state (left arrow) and sexual state (right arrow); (F) light micrograph of endophytic hyphae of Neotyphodium coenophialum, the endophyte of tall fescue.

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Table 3 Species of Endophytic and Epiphytic Species of Grass Endophytes species

Endophytic BalarzsicP Balansia aristidae (Atk.) Diehl; B. cla,iceps Spreg.; B. epiclzloe (Wesse) Diehl; B. gndvae J.F. White; B. granulosa (Chardon) White & Reddy; B. hemrirzgsiana (Moell.) Diehl; B. rzigricmu (Spreg.) Seaver; B. obtecta Diehl; B. sclerotica (Pat.) Hohn; B. strcrngulans (Mont.) Diehl Epichloe’ Epichloe amarillans J.F. White; E. bcrcorzii J.F. White; E. brachyelytri Schardl & Leuchtm.; E. bronticola Leuchtm. & Schardl; E. clarkii J.F. White; E. elyrrli Schardl & Leuchtm.; E. festuccre Leuchtm.. Schardl & Siegel, E. glyceriae Schardl & Leuchtm., E. syhutica Leuchtm. & Schardl, E. typlzina (Fr.) Pers.. Parcrepichloe‘ Parepichloe cwoclontis (Syd.) White & Reddy, P. barnbustle (Pat) White & Reddy, P. oplisrneni (P. Henn.) White & Reddy, P. scrsae (Hara) White & Reddy; P. sclerotica Pat., P. volkensii (P. Henn.) White & Reddy

species Balarzsia crspertata; B. mtbiens Moell.; B. andropogorris Lyd. & Butl.: B. crsclerotica P. Henn. Diehl; B. clavzrlo Berk. & Curt.; B. c?peraceurn Berk. & Curt.; B. cyperi Edg.; B. discoidea P. Henn.; B. gigas Racib.; B. pallidcl Wint.; B. pilultreforrnis (Berk. & Curt.) Diehl Myriogenosporcr Myriogenosporo crtranrentoscr (Berk. & Curt.) Diehl; M. lirzearis J.F. White & Glenn Atkirlsorlelln Atkiruonella hy~>oxylor~ (Pic.) Diehl A. texensis (Diehl) Leuchtmann & Clay

.’ According to Diehl (7). White et al. (3101, and Reddy et al. (21 1). According to White (212), Schardl and Leuchtmann (313). and Leuchtmann and Schardl (213). According to White and Reddy (9).

to discourage birds and small animals from consuming dormant ergot sclerotia in the field, similar to the mechanism proposed for sclerotia of species of Aspergillus (35). Mature sclerotia of C. p r y u r e a are characterized by a hard texture, darkly pigmented outer rind, and lighter-colored internal region (25). The sclerotia compete with developing seeds on the same plant for photosynthate, complete their development prior to that of the seed, and are generally larger than a mature seed often protruding well beyond the glumes of the floret (36). Mature C. purpurea sclerotia are resting structures and fall to the ground where they lay dormant over the winter. After a dormant period, sclerotia germinate and produce one to several ascostromata. Germination of C. yu~purecrand C. yaspnli sclerotia depends on a period of cool temperatures (24). The absolute time and temperature requirements vary from species to species and the requirement for cold may be absent for tropical and subtropical members of the genus such as C. c$ricann and C. sorglli. Perithecia are embedded in the capitulum of the stromata and the ascospores are forcibly discharged from the ostiole of the perithecium (24, 37). In C. purpurea and C. yasynli, these ascospores initiate the primary round of infection each season (24). Sclerotia of C. qfricann have been germinated experimentally for study but the importance of ascospores in disease initiation of this species is dubious since it is based on field observations (20).

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Disease incidence and the concomitant accumulation of alkaloid-filled sclerotia depends on infection of the flowers. Generally any factor that increases the time flowers are open and not pollinated increases the opportunity for infection because flowers that have been pollinated quickly become resistant to infection (38). Male sterile lines that have seen increasing use for hybrid seed production are particularly susceptible. Protogynous flowers in which the stigmas emerge well before anthesis are also more susceptible. Cool, overcast conditions at the time of flowering favor infection largely because flowers are open longer and pollen develops more slowly (39, 40).

B. Grass Endophytes 1. Epichloe and Neotyphodiurn Species The genus Eyichloe (Fig. 1E) in contrast to Clmiceys is exclusively endophytic (Table 3). Epichloe is heterothallic, meaning that the two mating types are found in separate individuals (41). Currently, there are 10 biological species of EpichZoe (Table 3) (42), all of which are found in cool season grasses in the subfamily Pooidae, Tribe Festuceae (Table 3 ) .Significant forage grasses infected by Epichloe and its asexual Neogphodiurr1 relatives are the fescues (Festucn species) and perennial ryegrass (Loliuru species) (Table 4). Infections are often asymptomatic as these species grow intercellularly in the stems and leaf sheaths of their hosts (Fig. 1F). Observations of stained leaf sheath material of infected plants reveal convoluted intercellular hyphae (Fig. 1F) often arrayed in parallel with the vascular tissue of the host (12, 13, 43). Epichloe species are sexually reproducing fungi that elaborate a white stroma that surrounds the developing grass inflorescence preventing flowering (choke disease) (Fig. 1E). Stroma that have been mated by contact with conidia of the opposite mating type becotne tan as the perithecia develop. Ascospores are forcibly discharged from the perithecia embedded in the stroma and are able to infect nearby grass flowers (44). However, sexual reproduction does not occur under all conditions, so some Epiclzloe infections are asymptomatic. In such asymptomatic infections, normal flowering can occur and the fungus is transmitted to the next generation of grass through the seed. Neohphodiunz is a completely asexual genus of eight species whose closest relatives are Epiclzloe species (10,45). In fact, many Neotyyhodiunz species arose through interspecific hybridization between EpiclzZoe species and Neo~phocliurnspecies (46,47). Because Neovphodizml endophytes do not produce stromata, grasses infected with them are entirely asymptomatic, and the only means of dissemination of this species is through the seed or through vegetative propagation of its host. Like Epicldoe, it grows intercellularly in the sterns and leaf sheaths of grasses (Fig. 1F). EyichZoe and Neo1yphocliz4rr1are found primarily in the sterns and leaf sheaths of their hosts with less incidence in the leaf blades. This distribution of N. coerzopl~ialzrn~ in tall fescue is reflected in relative alkaloid concentrations in the two plant parts (48), and a study documenting N. lolii and the alkaloid lolitrem B (Table 5) in perennial ryegrass showed similar results (49). Neotyplzodiunl and ergot alkaloids have also been observed in roots of tall fescue but the level of alkaloids is low and their presence in this organ is not of much consequence for grazing animals (50, 51). Epichloe and Neo~yhoclirn~ infections confer a number of beneficial effects on their hosts and the ergot alkaloids and other secondary metabolites produced in these associations are thought to mediate many of these positive effects. Generally, endophyte-infected grasses show increased competitiveness and better stand maintenance (52, 53). Field observations and experimental analyses have repeatedly indicated that grasses infected with

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Table 4 Distribution of Neotyphodiltnl, Bnlansia, and Related Fungi Among the World's Major Cereal and Forage Cropsa ~~

Tribe/species Agrostideae Agrostis alba and other spp. Alopecurus pt-atensis Phelurlz pratense Sporobolus Stipa comnta Aveneae Arrherzatheiwm elatium Andropogoneae Andt-opogon scopariml S o r g h m wlgt-e and other spp. Chlorideae Bouteloua spp. Chlot-is gajvmam Cylodorz dactylon Festuceae Dactylis glonzernta EIyrms spp. Ei-ngrostis spp. Festucn spp. Loliuril spp. Poa spp. Paniceae Axonopus ofinis Parzicrm spp. Paspalml spp. Setaria italica

name Common

~

~

Endophyte genus

Bent grasses Meadow foxtail Timothy Dropseed Needle grass

Neotypllodium Neotyplzodiunt' Neotyphoditm Balnrzsia Neotyphodiwn

Tall oatgrass

Neohphodiunlb

Blue stern Forage sorghums

Bolarzsia Balamicr

Grama grasses Rhodesgrass Bermuda grass

Bnlarlsin Bnlamia Balamia Balamia

Orchard grass Wildryes Lovegrasses Fescue grasses Ryegrasses Bluegrasses

Neotyphodiwn" Neohphodiunf Bdarlsia Ncotyphodiunl Neotypltodium Neotyphodium"

Carpet grass Panic grasses Dallis grasses Foxtail millet

Balansia Balamia Balansia Balarzsia

spp. spp. spp. spp.

<'Themajor forage grasses are cornpilatlons from Hover et al. (215). Bula et al. (216). and Crowder (317). The distributionof fungi 1s from Diehl(7). Brady (206). Kohhneyer and Kohlmeyer (218). and White (219). This endophytic species IS Eyichloe ~ y h i n a( A . ~ p h i n l r m ) .

Neohphodium endophytes have increased drought tolerance compared to uninfected members of the same species (52, 54-56). Endophyte-infected grasses also deter or otherwise negatively affect a variety of insect pests although many of these interactions are specific to particular endophyte grass combinations (34, 57-59). The alkaloids most often associated with insect deterrence are the pyrrolizidines and extracts of endophyte-infected tall fescue containing loline alkaloids produced a significant feeding deterrent when offered to aphids on uninfected fescue (60). Nematode levels in tall fescue vary and this variation may be attributable to different levels of endophyte infection (61). In addition, greenhouse studies show that feeding endophyte-infected tall fescue reduces reproduction in some species of nematodes (62). Susceptibility to fungal pathogens can be reduced by endophyte infection as well. For example, the number of seedlings of endophyte-infected tall fescue surviving Rhizoctonia zeae seedling blight disease was significantly higher than for uninfected seedlings (63). The tall fescue endophyte N. coerzoyhialur~zproduces the plant hor-

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Table 5 Ergot Alkaloids and Toxins Produced by Clavicipitaceous Species and Their Allies Claviceps species

Agroclavine Elymoclavine Ergosine Ergosinine Festuclavine Lysergol Fumigaclavine A Chanoclavines (1,II.III) Costaclavine Penniclavine Isopenniclavine Setoclavine Isosetoclavine

9,lO-dihydroxysetoclavine Molliclavine Pyroclavine Isochanoclavine I Lysergene Lysergine Ergonovine Ergotamine Ergotaminine 8a-Hydroxyergotamine Ergocristine Ergocristinine Ergostine Ergostinine Ergometrine Lysergic acid amide Lysergic acid-L-valinemethyl ester Isolysergic acid Ergocornine a-Ergoktyptine P-Ergokryptine Sa-Hydroxy-a-ergokryptine Ergosine P,P-Ergoannam Sa-Hydroxyerginine 8a-Hydroxyergine Ergobasine Ergobasinine Ergoladinine Also includes the Epichloe species.

Balarzsia species Elymoclavine Ergonovine Ergonine Chanoclavine I Isochanoclavine I Agroclavine Dihydroelymoclavine 6,7-Secoagroclavine Penniclavine Erytho 1-(3-indoly)propane1.2,3-triol Threo 1-( 3 indo1y)propane12,3-triol 3-Indole acetic acid 3-Indole ethanol 3-Indole acetamide Methyl-3-indole-carboxylate Ergobalansine Ergobalansinine

Neo@phodiunl species3 Ergovaline Ergonovine Ergosine Chanoclavine I Peramine Indole acetic acid Ergosterol Ergosinine Cyclopentanoid sesquiterpenoids3 Ergonovine Caffeic acid” p-Coumaric acid p-Hydroxybenzoic acid Loline N-Methyloline N-Acetylloline N-Formylloline N-Acetylnorloline Paxilline Lolitrem A Lolitrem B Lolitrem C Lolitrem D

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mone indole acetic acid (IAA) in vitro: however, it is not known if in vivo IAA production mediates any of the beneficial effects the endophyte affords tall fescue (64). Many factors affect accumulation of ergot alkaloids in grasses infected with Epiclzloe or Neotyphodium endophytes. Seasonal variation is seen in tall fescue ergot alkaloid and pyrrolizidine alkaloid accumulation. These are high in fall and spring and may be due to some of the variables discussed below (65-67). Water-stressed tall fescue infected with N. coenophialzrrr~contains higher levels of ergopeptine and pyrrolizidine alkaloids than plants grown under adequate water conditions (48, 68). High application rates of nitrogen or phosphorus also increase ergot alkaloid levels in endophyte-infected tall fescue (65, 67-69). Plants subjected to'repeated defoliation by clipping had lower levels of ergot alkaloids than nonclipped plants (70). Ergot alkaloid biosynthesis is a function of the endophyte as evidenced by its ability to produce these compounds in vitro and isolates producing differing levels have been identified (71, 72). Recently, the in vitro production of pyrrolizidine alkaloids by N. ur~cirzuturnwasshown ( 7 3 , extending the biochemical competence of the Neotyphoclium species to include this class of alkaloids. However, plant genotype also plays a role in determining alkaloid levels in infected plants (74, 75).

2. Balansia Species At present 21 species are included in the genus Bnlcmsia (Table 3) and most of these species have a tropical or subtropical distribution (7, 76). The number of species of endophytes in this genus is expected to increase since several endophytes have not been delineated, or placed in other endophytic genera (77). Species of Balmsin (Fig. 1D) are also found in other tropical and subtropical regions of the world such as Asia (7). This group elaborates white to brown and black conidial and perithecial stromata on various aboveground parts of its hosts (Fig. 1D) (7, 8). The hosts include forage grasses (Table 4) and a few sedges. Like Epiclzloe, the mating system in at least one species, B. epichloe, is heterothallic (78). Emergent reproductive structures occur seasonally, usually in the late summer and early fall. For the remainder of the year these infections are asymptomatic. In contrast to Diehl's assumption that all Bnlarzsia species are endophytic (79), epibiotic species have also been described (15) (Table 3). Some Bulnnsin grass associations result in sterility of the host (80). Ergot alkaloids produced by Balnnsin spp. are distributed throughout the plant as they are inNeotyplzodizrm-infected species. Another similarity between Eyicldoe and Bnlarzsin species is that plant growth hormones are produced, as, for example, B. epiclzloe has been shown to produce the plant hormone IAA in vitro (81). It appears that Bnlurzsin infections of grasses and sedges have some beneficial aspects for the plant as is the case for Epichloe- and Neotyphoclirm-infected grasses. For example, negative effects on insect herbivores and reduced fungal pathogen load have been documented in Balnr.rsia-infected plants (82, 83). The only epibiotic member of the Bularzsin-Epichloe group with agronomic impact is M~~iogerzospora (16, 84). Morphological and molecular phylogenetic analysis indicate that itis taxonomically allied with the Bnlnnsicr group (10, 16,451. This fungus is observed in the field by its brown and black stromata, which line the adaxial surfaces of leaf blades, causing leaf curling, mummification of inflorescences, and the tips of the blades to stick together (16, 17). This last phenomenon gives the disease the name of tangle top. It is commonly found on Bahia grass (Pnspalunl rlotutum) in the Southeastern United States although it has many other hosts (16, 84). The mode of infection and mating system of Myriogenosporcl are not known.

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The graminicolous Clavicipitaceae are partners in complex biotrophic associations and interactions with their hosts. These interactions are universally characterized by the production of ergot alkaloids and several classes of other alkaloids, all of which appear to form the basis, in several cases, for enhanced fitness benefits for the plant partner. However, the ergot alkaloids and related toxins are also the reason for the mammalian toxicoses associated with these fungi.

111.

ERGOT ALKALOIDSAND OTHER TOXINS

The content of ergot alkaloids and other toxins in sclerotia and in endophyte-infected plants (Table 5 ) varies with the species of Clmiceps, Bnlansin, and Neoiyphodirrn? (4, 80). Clcrliceps and related grass endophytes are parasitic on thousands of plants that are cultured in almost every country of the world, and hundreds of these include agriculturally important species and cultivars. All the world's cereals are hosts of Clnviceps, including wheat, corn, sorghum, barley, rice, rye, oats, and millet. The sclerotia from species of Claliceps show considerable variation relative to alkaloid content and there is no correlation between grass host species and the capacity to produce ergot alkaloids either quantitatively or qualitatively. Alkaloid content in sclerotia depends on the genotype of the fungus and may vary with differing nutritional context of the host (85). Strains of Clmiceps have been separated into chemical races with as few as three to as many as 16 (4). These races are distinguished on thebasis of their major ergot alkaloid and/or host. Individual sclerotia may or may not contain ergot alkaloids, which may reflect either fungal genetics or environmental interaction. The ergot alkaloids include many of those produced by the grass endophytes (Table 5), as well as several others produced by species of Clrrviceps; see, for example, the cotnpilation of Bove (4). The situation of ergot alkaloid accumulation is complicated in endophyte-infected grasses. Individuals within a population of endophyte-infected grasses will vary because of the process of natural selection under herbivory. Long-term herbivory, and specific types of herbivory (insect or mammalian), will lead to subpopulations of toxic grasses, each chemically defined and based on its specific mixture of deterring and toxic compounds. As most endophytes are maternally transmitted, seed-sown pastures will reflect this diversity. This may serve to confound any potential cattle toxicities initially, but as cattle grazing within a location continues, the seed of toxic and deterring individual plants will be consumed the least, resulting in these individuals self-seeding, producing more of the toxic types. Years may be required for the establishment of a population high in endophyteinfected grasses to be toxic to cattle. In areas where there are no pressures from herbivory, there should be a mixture of ecotypes, including individuals totally devoid of insect and mammalian toxins. The major groups of toxins chemically identified with antiquality factors of infected forage grasses are the ergot alkaloids and tremorgenic neurotoxins and these may be monitored to determine the percent distribution within a location. The degree of chemical expression by an endophytic fungus depends not only on the biochemical competence of the fungus (86, 87) but also on the genotype of each plant (88). The environmental factors of soil nitrogen (54, 89, 90) and moisture (91). pH, and phosphorus (69, 92) also interact to affect the accumulation of ergot alkaloids in grasses. Further, there is a fungus-grass genotype interaction since ergovaline content of one genotype of infected tall fescue increases with increased leaf area while this relationship is

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not observed in other genotypes (93). This raises the issue as to whether the controlling mechanisms are within each plant, or an interaction between the two. By inserting endophytes into a common tall fescue genotype and by conducting genetic studies between high- and low-ergovaline-producing infected plant genotypes (88, 94), it has been documented that at least the plant can regulate the expression of ergovaline production by the endophyte. In this regard, regulation may well reflect the individual variation of toxin precursors and primary metabolites released in the apoplasm from the plant. The apoplasm is the intercellular location where nutrients are utilized by the fungi for growth and synthesis of ergot alkaloids and other toxic secondary metabolites. Individual grass genotypes are expected to vary in terms of concentration of nutrients within the apoplasrn and this should reflect the concentration of ergot alkaloids.

A.

Ergot Alkaloids

Ergot alkaloids are 3,4-substituted indoles derivatives that may be divided into the clavine ergot alkaloids and the peptide ergot alkaloid or acid amide-type derivatives of lysergic acid (Fig. 2). These two groups of ergot alkaloids differ from each other in the presence (ergopeptide bond) or absence (clavine) of a peptide bond and attached amino acids. More than 40 different ergot alkaloids have been identified from ergot sclerotia (Table 5). The ergot alkaloids found in species of Claviceps and endophyte-infected grasses are both the ergopeptine and clavine types (65, 71, 95-97). The same type and relative proportions of ergot alkaloids have been shown to be produced in culture, in the sclerotium, or ill plnnta (4, 29, 65, 80, 98). There is considerable controversy over the biological activity of each ergot alkaloid, but it is generally agreed that the ergopeptine alkaloids are more active than the clavine alkaloids. The predominant ergot alkaloids found in the Bnlcrrzsiainfected grasses are of the clavine types, while the ergopeptide ergot alkaloids are found as the dominant ergot alkaloid in the Neo~~honiurlz-infected grasses (Table 2). Both types of ergot alkaloids are biosynthetically derived from the same precursors, usually the simple clavine alkaloids (99), and there are biotypes of endophytes and biochemical races of

COR

Lysergic acid derivatives

R = Tripeptide or smaller units

Clavine alkaloids

R=H=OH

Figure 2 General formulas for the two types of ergot alkaloids.

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Clcwiceps that reflect this variation (4, 87, 100). The concentration of ergot alkaloids and tremogens in endophyte-infected grasses varies in concentration from 0.01 to 3.0 pg/g of plant (dry weight) (65, 97, lOl), which depends on the fungal genotype, the season, and the physiological status of the plant (67, 87), and the nature of the grass-fungus interaction (88, 94). The total alkaloid content in Claviceps sclerotia ranges from 0.01 to 0.05% (4). This content does not relate to the size of the sclerotium or ploidy level of the host, which if tetraploid will produce sclerotia three times the weight of sclerotia from diploid plants (102). However, small sclerotia tend to have lower alkaloid content than larger ones harvested from the same host (103). Human and livestock toxicity is due to these alkaloids. B. TremorgenicToxins The tremorgenic neurotoxins, commonly called the lolitrems, have been isolated only from endophyte-infected perennial ryegrass. This group of toxins is found in N. lolii-infected perennial ryegrass, is apparently absent from N. coellopkinluill-infected tall fescue (104, 105), and has notbeen examined in the Balcuzsia-infectedgrasses. This class of compounds is considered responsible for ryegrass staggers of sheep (106. 107), and consists of four biologically active compounds, all containing a complex indole isoprenoid ring system (107). The major lolitrem is lolitrem B, which ranges from 3 to 25 pg/g dry weight in perennial ryegrass herbage, and a smaller concentration also occurs in ryegrass seed (101). The lolitrems. unlike theergot alkaloids, have not been isolated from cultures of the fungus N. lolii, but its indole isoprenoid precursor, paxilline, has, indicating that it is synthesized by the fungus in culture (104, 108). Thus, it is unknown if the fungus produces only paxilline that is converted to the lolitrems by the plant, or if, under culture or other conditions, there is an incomplete synthesis by the fungus. The latter might be the case, as other fungi can make tremorgenic neurotoxins, structurally related to the lolitrems, in culture (109) and otherwise (1lo), which suggests that theN. lolii might becapable of synthesizing the lolitrem molecule in vivo but not in vitro.

C. Miscellaneous Toxins There are a variety of chemically diverse compounds (Table 5 ) in Clnviceps and endophyte-infected grasses, which, in the case of grass endophyte, were possibly one of the driving forces in evolution that led to the successfhl establishment of the associations and/ or the production of these bioprotective compounds. With the exception of the class of ergot alkaloids, there is at present no identified consistent and specific chemical that would suggest that there are other evolutionary biochemical relationships within the two broad types of fungi found in the Clavicipitaceae. The loline alkaloids are pyrrolizidine bases that are found in endophyte-infected tall fescue in concentrations as high as 0.8% of the dry weight of tall fescue plants (111). These alkaloids have recently been isolated from cultures of the fungus (1 12). Belesky et al. (113), determined that the concentration of loline alkaloids reflected the extent of infection within the population of endophyte-infected grass. A limited number of studies suggest that the loline alkaloids are only mildly toxic (1 14, 1 151. especially when that toxicity is compared with that of the usual pyrrolizidine alkaloids (116). Two of the forms present in infected tall fescue, N-acetyl and N-formyl lolines, are considered more toxic than the other forms reported in this grass. However, these compounds may act synergistically or potentiate the activity of other toxins in infected tall fescue, as suggested from

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their effects on insect herbivory (57j. Thelolines are being tested for a role in the reducing drought stress observed in endophyte-infected tall fescue grasses (1 12). Other compounds reported in infected grasses that may impose toxicity problems to livestock include 3-indole acetic acid, indole ethanol and related simple indoles (104, 117j, peramine (1 18), the tetraenone steroid (119), the ergosterol (120), phenolic acid derivatives (121), and sesquiterpenes (122j. The effects of some of these on insects, laboratory animals, and fungi suggest toxicity (109, 120, 123). while the toxicities of others have not been established.

IV. TOXICITYSYNDROMES A.

Claviceps Toxicity

1. Human Human consumption of bread made from ergotized flour produced epidemics of ergotism as far back as the 10th century (4). Since C. purpurea infects most of the cereal grains used as human food, this is most likely the species of concern for human toxicity. However, there are specific species that are also found on maize and small grains used for human consumption, including C. ufriccrnn, C. gignntia, and C. sorghi (Table l), but human toxicity has not been documented for these species. Bove (4) presents a very comprehensive account of ergotism in human history, which will not berepeated. Mortality of humans with ergotism varied between 10 and 20% and with treatment mortality canbe reduced. Children are more susceptible to ergotism than adults. Most cases of ergotism are related to ergotized flour that is used to make all types of breadstuffs, soups, dumplings, and other foods. Ergotism is rare in areas where cereal grains are not a staple, and in those areas it is usually a disease of the poor who do not bother to separate sclerotia from grain either from ignorance or to prevent waste of what is considered valuable foodstuffs. The most recent documented case of ergotism in the modern period occurred in the city of Pont St. Esprit, France in 1951. The following is a summary of this account (124). Ergotized rye was milled and the flour sold to a baker who made bread and sold it to approximately 200 people. The first symptoms appeared within 6-48 hr after consumption of the bread. The disease developed more quickly in children, but it also left them more quickly. Most patients recovered after several days. Doctors reported that one man plunged out of the window screaming hysterically that he was a jetplane. Another woman jumped out of another window to escape the flames she imagined engulfed her. Other sufferers insisted that they were being chased by wild beasts. Most victims had to be subdued. All patients had a disagreeable body odor, weak pulse, muffled heart sounds, low body temperature, digestive disorders with burning sensations throughout the entire digestive tract, and some reported vomiting and dian-hea. Severe cases included three deaths, one or both leg amputations, with severe convulsions that lasted for a month. Barger (125) described two types of ergotism: convulsive and gangrenous. These two types of ergotism occur in humans, livestock, and poultry. Each apparently is produced by the distinctly different major alkaloids present in the sclerotium produced by specific genotypes of Claviceps species. Convulsive ergotism is characterized by mild to severe convulsion of the muscles resulting in spasms in the face, vocal cords, esophagus, and the diaphragm. In nonfatal cases convulsive ergotism might last 6-8 weeks, with convales-

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cence taking several months. Gangrenous ergotism is characterized by a burning sensation followed by swollen and inflamed limbs that is followed by a blackening and shrinking of the affected part, which becomes mummified and dried. Finally the affected part is lost, usually by surgery. The affected part may include legs, arms, or fingers. The atnount of alkaloids present in the flour in the Pont St. Esprit case was not determined. However, correlations of grain inspection and medical statistics show that the disease occurs when there is 1% or more of ergot in the cereal grain, 7% causing fatal poisoning. In the epidemics occurring within the 10th through the 18th centuries the amount of ergot in the grain is estimated to have exceeded 30% (4, 125). There is still a low concentration of ergot alkaloids in today’s commercial flour (126), but the concentrations found do not appear to constitute a health hazard for the general population. However, the toxicity of low doses of ergot alkaloids to children and other more susceptible adults has not been determined. Ergocristine was the major alkaloid detected in commercial wheat and rye flour samples with concentrations up to 62 1-18 reported (136). Tolerances of ergot alkaloids are established in most countries but vary with grain types. Wheat and rye in the United States are considered “ergoty” if they exceed 0.3% and 0.1% ergot, respectively (127). In Canada the highest grade for all grain for milling (human consumption) must be completely free of ergot (128). 2. Livestock and Poultry Ergotism in livestock and poultry still occurs in spite of modern agricultural practices. This occurs primarily because of unknowingly grazing livestock on Claviceps-infected grasses or feeding infected seed to livestock. All species of Clmiceps are expected to play a role in the toxicity of animals since more than one species infect the major forage grasses and seed used for animal feed. However, the species C. pur‘pur’en,C. pasyuli, C. jksjfomis, C. ufricancr, C. cynodontis, C. phalm-idis, and C. sorghii infect major forage species and should be considered as causes of ergotism in livestock and poultry. The signs of ergotism in livestock and poultry include gangrene, abortion, convulsions, agalactia, lameness, and ataxia (129-132). The range of effects depends on the type and concentration of ergot alkaloids in the sclerotia, with mild signs including infertility, poor circulation, poor lactation, reproductive cycling, and other physiological depressed changes. In cattle, ingesting sclerotia has been reported to cause reduced feed intake and weight gain, decreased milk production, gangrene of the feet, tail, and ears, convulsions, trembling gait, palsy, and long hair (131, 133-135). In sheep mild to severe gangrene, reproductive problems, and similar signs as those reported in cattle are observed (129). In swine, diets containing 0.5% milled ergot sclerotia caused decreased feed consumption and reduced weight and growth rate with lesions in the stomach, intestine, liver (135), and agalactia (1 29). Ergotism in poultry is rare, and the level of ergot alkaloid was critical to the final toxicity signs reported, but in general included poor feed conversion ratios, poor growth rates, poor feathering, loss of coordination, liver damage, congested kidneys, and, in broilers and ducks, mortality (5, 136). Other symptoms include necrosis of the womb, wattles, and tongue (136).

B. Neofyphodium-InducedToxicities 1. Tall Fescue Historically, endophyte-infected tall fescue and perennial ryegrass were associated with poor animal performance problems and toxicities. The most common of these are fescue

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toxicosis and ryegrass staggers (105, 137, 138). Plants with detements to ruminant herbivory should have a competitive edge over plants without such a mechanism; thus selection would favor endophyte-infected grasses over uninfected plants. The degree of toxicity to ruminant herbivory is expected to reflect the amount of toxin contained within grasses. The amount of toxin within a plant at a given location varies both qualitatively (65, 91, 97j, and quantitatively (67, 80, 113, 139) but fluctuates seasonally (67, 113, 140). Both genotypes of the grass and fungus affect the final expression of ergot alkaloid content (75, 141, 142). Seasonal variation and fluctuation of endophyte-infected tall fescue (143) within a location is expected to affect variation in animal performance similarly (144-146). Cattle consuming N. coelzoyhinlunz-infected tall fescue may show either severe or mild symptoms. Cattle showing severe tall fescue toxicosis resemble those showing classic ergotism and include gangrene of the extremities and a slight nervousness or palsy in the flank region. These symptoms, commonly referred to as fescue foot, are usually observed under cool temperatures. During the early studies of toxicity on this grass, these were the only signs recognized (138). Fescue foot was observed to affect only a few animals within the herd, and toxicity from tall fescue was not considered a serious economic threat, especially since it was so sporadic. When the endophyte and the recognition of mild or subclinical effects on cattle were revealed (147). it became apparent that infected tall fescue affected all animals within a herd and imposed severe economic losses ( 148). The syndrome resulting from the subclinical effects is often referred to as fescue summer toxicosis or the summer slump as it is more evident in the warm periods of the year. Cattle with mild or subclinical symptoms exhibited reduced weight gain and feed intake, reduced reproductive efficiency, reduced milk production, low heat tolerance or hyperthermia, increased respiration rate, and reduced circulating prolactin and serum cholesterol (149). Cattle affected with fescue summer toxicosis are further characterized as having low plasma melatonin (150), rough and long hair coats that last well into spring-summer (149), and decreased tolerance to light (1 5 1, 152). Cattle grazing infected tall fescue seek shade or stand in water, and graze in the cooler periods of the day or at night. Another aspect of tall fescue summer toxicosis in cattle is the idiosyncratic development of fat necrosis, which has been associated with high blood cholesterol of animals grazing infected tall fescue (153). Tall fescue summer toxicosis can be alleviated when the feed of cattle is changed, but average daily gains are still depressed if cattle are moved from infected tall fescue to feedlots for finishing during warm weather. The greatest economic impact to the cattle industry is from the tall fescue summer toxicosis, which is estimated to impact the production of over 8 million beef cows and approximately 700,000 horses in the United States yearly. This results in an annual loss of approximately $750 and $793 million (148, 154). According to Crawford et al. (144), average daily gain of cattle was reduced by 45.5 g for each 10% increase in endophyte infestation level. The toxicity effects can be reduced by diluting the overall pasture infestation level with another forage such as clover (species of Trifolium) (146). In another study (145), the average daily gains were depressed when the percentage of endophyte infestation levels was adjusted to 22% by the addition of clover. This depression continued up to the 35% infection level, but no further reductions in average daily gain and beef production were observed when the infection was increased to 81%, the highest level used (146). Horses grazing on N. coerzoyhialur~~-infectedtall fescue have very specific symptoms, particularly reproductive and neuroendocrine effects. These symptoms are, as in

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cattle, related to toxicity induced by ergot alkaloids and can be prevented by the daily administration of perphenazine, a synthetic dopamine antagonist (1 55). There has been no report of the gangrenous aspect, fescue foot, occurring in horses. The endophyte lowers circulating progesterone and prolactin levels in brood mares, which also have extended gestation periods, retained, mineralized, and thickened placentas, agalactia, and deliver foals that are dysmature, weak, or stillborn (156-158). These symptoms are, as in cattle, related to toxicity induced by ergot alkaloids and can be prevented by the daily administration of perphenazine, a synthetic dopamine antagonist (150). Sheep grazing on Neotyphodium-infected tall fescue have lower circulating prolactin, cholesterol, and as much as a 59% reduction in milk production (159, 160). Ewes have lowered conception rates, but unlike sheep grazed on perennial ryegrass, the growth rate and feed intake are not reduced (1 52, 160). 2. Perennial Ryegrass Toxicity from perennial ryegrass infected with N. lolii affects tnainly sheep (161, 162), but cattle, horses, and deer also are affected. This disease is a neuromuscular disorder and is referred to as ryegrass staggers (161). The disorder occurs sporadically and primarily inNew Zealand and southern Australia. It is distinct from the grass staggers (tetany) disease, which is induced by a magnesium deficiency. It has been shown experimentally that sheep develop symptoms of ryegrass staggers within 7-14 days of being placed on toxic pastures (105). As is true of the infected tall fescue, the syndrome in sheep is manifested in a variety of symptoms, including severe clinical symptoms of head nodding, trembling of the neck and shoulder muscles, swaying while standing, staggering, and a stilted gait with collapse if overprodded (161). Similar tetanic spasms are observed in cattle grazing perennial ryegrass; however, they may either collapse or assume a sitting posture if excited. These severe symptoms are associated with the level of endophyte (163), increased amounts of dead basal dry matter in ryegrass stands, slow plant growth rate, or overgrazing (161). There is a 2-10% loss of animals to ryegrass staggers, which can account for at least half of the total profits. Subclinical effects include reduced average daily weight gains, depressed prolactin blood levels in ewes (164,1651, and reduced testosterone levels in rams (159).

C. Symptoms of Balansia Toxicity and Related Fungi The BcrZarzsia species are mainly associated with invasive and rangeland grass species, e.g., species of Sporobolus, Ardropogor1, Agrostis, Clnrzrogrostis, Clzloris, Eragrostis, and Pmicunz (Table 4).Detailed studies of their effects on grass quality and cattle performance have not been conducted. Since the grasses infected by species of Bnlcrrzsia contain ergot alkaloids similar to those contained in grasses infected by species of Neotyphodiurn and Claviceps (Table 5) (65, 71, 95, 139, 166), effects and modes of action ofBcrlmzsiainfected grasses on livestock should be the same as those discussed above (167, 168). Ergotism has been reported in livestock consuming both Neoh,phodiurw and Bulansiainfected grasses (168-170). Experimental feeding of the culture medium of one species, B. epichloe, reduced total prolactin concentrations in lactating Holstein cows (171). Also, there was a decrease in the milk-induced rise in serum prolactin, although there was no effect on milk production (171). This work was not only the first to establish that serum prolactin concentration was affected in cattle consuming an infected grass, but it also established that the ergot alkaloids produced by the fungus were the active toxins. There

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has been no documented case of ergotism or livestock toxicity due to ingesting Myriogenosporcl- or Atlcirlsonella-infected grasses. However, the data indicate that all members of this family are competent in ergot alkaloid production; thus the presence of these two species should be suspected if livestock grazing on grasses infected by them are exhibiting performance problems. D.

Paspalurn Staggers

The Dallis grasses (Pnspnlzrrn spp.) are found primarily in the tropical sections of the world. These grasses are parasitized specifically by C. ynspnli, and at least 18 species of Paspnlurlz are reported as being hosts for this species (Table 2). In addition to producing the ergot alkaloids, this species also produces the tremorgenic substances collectively referred to as the paspalitrems (A, B, and C), and paspalinine that are similar in structure to the lolitrems produced by the Neotyphorliz,w species (Table 5). These substances are also produced in the sclerotia; thus, livestock toxicity results from ingesting C. pnspcrliinfected seed heads. Ingested seed heads produce a neurological disease called paspalum staggers in cattle and sheep with clinical signs including tremors, incoordination, and ataxia, somewhat resembling the convulsive type of ergotism. However, the neurological disease is not caused by and is distinct from similar-appearing conditions caused by ingestion of ergot alkaloids ( I 35). Pcrspnlzr~nstaggers has been reported to occur in Australia, Italy, New Zealand, Portugal, South Africa, and the United States. Paspalurn staggers is caused by one of several diterpene indole-type compounds referred to as paspalinine and paspalitrems, of which paspalitrem B is the most biologically active (107, 172).

V.

CONTROL AND MANAGEMENT

A.

Claviceps

Control of Clnviceps infection in cereal grain requires strong management practices that include removal or mowing of weed species that can serve as alternate hosts, use of a 1year crop rotation, alternating with a noncereal grain, deep plowing (1 ft or more) of ergotized fields (4), and use of ergot-resistant cultivars, or cultivars with appropriate characteristics that will lessen Clnviceps species infection. In areas prone to ergotism, cytoplasmic male sterile lines of grain should be avoided, as they have been shown to be very susceptible to several species of Clcnliceps, including wheat, barley, sorghum, and pearl millet (173, 174). None of these recommendations are effective control measures. The major barrier to effectiveness of control is the lack of resistance to the disease in the more desirable cultivars, and in the case of C. purpurerr large numbers of alternate weed hosts that are susceptible to infection serve as inoculum sources. Deep burial of sclerotia and mowing of flowers off of adjacent weed species will decrease inoculum spread, but only briefly. Both conidia and ascospores are wind disseminated over considerable distances, still ensuring infections (31). The use of systemic fungicides has had contradictory results. Triadimefon and benomyl have been used for control on buffelgrass and male sterile wheat and barley, respectively (175, 176). The results from both studies suggest that protective chemicals must be placed on the ovary during the 10- to 15-day period of susceptibility, but since plants produce fertile tillers over a considerable length of time this would entail several sprays to make sure all the ovaries are protected. Fungicides have also been used as a soil drench

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to prevent ascocarp formation and subsequent inoculum increase (177). Of the three fungicides tested, sodium azide was the best suppressor of ascocarp formation. However, the use of such pesticides will increase the cost of producing a crop, and will not prevent the wind-disseminated ascospores from adjacent regions. Biological control with species of Fusariun~and other species have been shown to work experitnentally (178) but we are not aware of any field applications for their effectiveness under field conditions. B. Grass Endophytes The controls of grass endophytes are completely different from those recommended for control of Clmiceps infection. Since we are dealing with an endophytic fungus, the use of crop rotation and mowing would not retnove the fungus from perennial grasses since the leaves are also infected. Systemic fungicides at their recommended levels do not work as the removal of endophytes from grasses for experimental use requires severalfold higher concentration, and is administered in water solutions over an extended time period of days (43). This procedure while effective is also phytotoxic; the grass requires several months to recover frotn this severe treatment. The fungus can be removed from the seed by storing it for a year or more, resulting in death of the fungus, and the resulting pasture generated from such seed will be endophyte-free. As presented above, removal of the fungus will result in loss of the grass in most locations due to stresses of grazing, heat, drought, and others. Currently, research is underway in which natural occurring Neohphodiun~species are identified, removed from their hosts, and reinserted into desirable forage grasses (179181). The ecological benefits are maintained while the livestock toxins are absent. Another approach utilizes molecular technology to knock out the undesirable gene or genes in the desirable but otherwise toxic endophyte, reinsert it into forage grasses. and the resulting grass is considered surrogately transformed (182). In theory such surrogately transformed grasses can be tailored to specific environmental conditions. Surrogate transformation of grasses promises to be a very useful strategy for both turf and pasture grasses (18 1). This technology is a long way off but Considerable progress has been made as required for experimental approaches (183- 187). There are millions of acres of endophyte-infected pastures that cause a considerable economic loss due to poor weight gains and reproductive problems (148). To use these pastures several strategies have been proposed. Pasture management options used to reduce the impact of endophyte-infected grasses on livestock include dilution with other forages such as a combination of planting legumes such as the clovers, along with another grass such as Bermuda grass. Other options include the use of various additives such as the plant growth regulator mefluidide, the vitamin thiamine (1 88), zearanol and aluminosilicate (1 54), activated carbon, anthelminthic compounds ( 189), ensiling endophyte-infected forages (190), energy supplementation (191), and copper and selenium (156, 192). The usefulness of these additives has been reviewed previously (154). Ammoniation of endophyte-infected hay reduces the toxicity signs observed in cattle and may prove to be an effective procedure to render tons of endophyte-infected seed and hay usable (193, 194) that are currently presenting a disposal problem. Physiological studies of cattle grazing endophyte-infected and -free pastures indicated that the basic mechanism centers around a dopamine response (159). This dopaminergic effect has been demonstrated as an essential feature of cattle grazed on endophyteinfected grasses. Thus, various pharmacological agents, dopamine (D2) antagonists, have

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been tried to alleviate cattle grazing ergot alkaloid-containing forages. One of these antagonists, metoclopramide, has been fed to cattle while grazing ergot alkaloids in endophyteinfected tall fescue and caused an increase in gains, indicating a positive response (35, 195). However, when fed to sheep metoclopramide was not as dramatic (196). Another dopamine antagonist, domperidone, reversed the depressive effects of ergovaline, the major alkaloid in endophyte-infected tall fescue (197). This drug also was positive in mares whose results suggest that it is an effective treatment for equine fescue toxicosis (198). Another technique that may find some usefulness in protecting cattle grazing endophyteinfected grasses is passive immunizations. Steers were immunized by infusion of ergot alkaloid-specific mouse tnonoclonal antibodies (199). The results indicated that cattle were protected from the adverse effects of endophyte-infected tall fescue ( 1 54).

VI.

FUTUREPROGNOSIS

Predictions of future incidence of ergot alkaloid-contaminated food and feed are difficult to make because of changing and uncertain conditions. Ergot alkaloid-infected material, and associated fungi may occur in unexpected places and at unexpected times. Several factors suggest that continued vigilance in assessing for the presence of these mycotoxins and associated fungi is warranted. Our culrent level of sampling in germ plasm collections for asymptotic Epiclzloe and Neotphodirnn endophytes that tnay be used in breeding efforts is limited (59). As a consequence, endophytes may be unknowingly introduced into forage grasses or grain crops. Given the potential harmful and beneficial effects of these organisms, informed use of thetn is prudent. Current belief is that grain crops are not infected with seed-transmitted endophytes. However, surveys of wild barley species from germ plasm collections have found the Neohphodizlm-type endophytes. Some of these produce ergot alkaloids such as ergovaline (200, 201), and recently a report of a NeoQphodiurzl-like endophyte was isolated frotn space-grown wheat (202). The challenges of growing plants in space may be compounded by the presence of previously undetected endophytes. While nothing is yet known about the alkaloid profile of the wheat endophyte, it points out our limited understanding regarding distribution of these fungi. Changing agricultural practices are likely to influence incidence of ergot alkaloid toxicities. For example, incidence of C. prqmrea may be affected by practices such as less frequent mowing of grass borders of grain fields, increased use of male sterile lines, and a move toward no-till agriculture (19). An outbreak of ergotism in dairy cattle fed contaminated barley occurred in Iowa in 1996 and weather conditions were cited as an influential factor but the need to examine other influential factors was etnphasized (203). Dramatic changes in the global distribution of a disease can occur through the inadvertent introduction of contaminated material. C. africann was only known in Asia and Africa before 1995 but spread rapidly from contaminated sorghum in the mid- 1990s to South America in 1995 and Australia in 1996. It then moved through Central America reaching North America by 1997 (6). Many questions about the life cycle and toxicity of C. ufricarzu sclerotia in these new locations need to be answered. Global climate change may also affect the distribution and incidence of these fungi. While there is still debate about the magnitude of global climate change, there is little question that it is occurring and will continue to occur (204). As climatic zones shift, geographical regions may see introduction of grasses with their associated clavicipitaceous

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fungi, and regions that retain their flora maysee changes in toxin levels due to environmental effects such as drought. The outcome of such new grass-endophyte-environment interactions is unclear. The plant-associated Clavicipitaceae have probably impacted humans since the beginnings of agriculture and will undoubtedly continue to do so (4). Negative impacts have included crop losses and illness and loss of life in humans and livestock. However, some of the ergot alkaloids produced by these fungi have found pharmaceutical use historically and currently in both developed and traditional cultures, and the NeoQphodiunl endophytes have been used to improve turf and forage grasses (4, 205). As we continue to learn more about the fascinating endophytic section of the Clavicipitaceae, our goal will be to avoid their toxicity but utilize their beneficial aspects for increased forage performance. It is clear that this family is universally present on the planet, has produced a negative impact on human food and feedstuffs throughout civilization, and will continue to do so unless modern techniques of science are used to minimize harmful components produced by species of this family.

ACKNOWLEDGMENTS

We thank Gary Odvody, Texas A&M Research and Extension Center, Corpus Christi, Texas, for the use of his prints of C. nfricann, and D. M. Hinton for her assistance and use of the remaining prints used in Figure 1.

REFERENCES 1. Webster, J. (1980). Introduction to Fungi. Cambridge University Press, Cambridge. 2. Von Munchhausen. 0. (1763). Der Hcrusvnter-, Part 1, p. 332. Hannover. 3. Tulasne, L.R. (1853). Memoire sur 1’Glumacees. A m . Sci. Natur. Bot. 20:5-56. 4. Bove. F.J. (1970). The Stor?? of Ergot. S. Karger, New York. 5. Tanret, C. (1875). Sur la presence d’un nouvel alcaloide, l’ergotinine, dans le seigle ergote. Corttpt. Rend. Acad. Sci. 81:896. 6. Bandyopadhyay, R., Frederickson, D.E.. McLaren, N.W.. Odvody, G.N. and Ryley, M.J. (1998). Ergot: a new disease threat to sorghum in the Americas and Australia. PZartt Dis. 821356-367. 7. Diehl, W.W. (1950). Balarlsia and Balarzsiue in Arttericcr. In USDA Agric. Monograph 4 (U.S. Government Printing Office, ed.). U.S. Govt. Print. Office, Washington, DC, pp. 182. 8. White, J.F.. Jr. (1994). Taxonomic relationships among the members of the Balansieae (Clavicipitales). In Biotechnology of Endophytic F m g i of Gmsses (C.W. Bacon and J.F. White, Jr., eds.). CRS Press, Boca Raton, FL. pp. 3-20. 9. White, J.F., Jr. and Reddy, P.V. (1998). Examination of structure and molecular phylogenetic relationships of some graminicolous symbionts in genera Epichloe and Par-epiclzloe.Mycologia 90:226-234. 10. Glenn, A.E.. Bacon, C.W., Price, R. and Hanlin, R.T. (1996). Molecular phylogeny of Acrentorziwn and its taxonomic implications. Mycologicr 88:369-383. 11. Morgan-Jones, G. and Gams, W. (1982). Notes on Hyphomnycetes. XLI. An Endophyte of Festuca alvrdinacea and the anamorph of Epichloe hphina, new taxa in one of two new sections of Acrernonium. Mycotaxon 15:3 11-3 18.

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12. Sampson, K. (1933). The systemic infection of grasses by Epichloe vphina (Pers.) Tul. Trans. Br. Mycol. SOC.18:30-47. 13. Bacon, C.W., Porter, J.K., Robbins, J.D. and Luttrell, E.S. (1977). Epiclzloe fyphina from toxic tall fescue grasses. Appl. Environ. Microbiol. 34:576-58 1. 14. Leuchtmann, A. and Clay, K. (1988). Atkinsonella hypo,x-ylort and Balansia cyperi, epiphytic members of the Balansiae. Mycologia 80: 192-199. 15. Clay, K. and Frentz, I.C. (1993). Bnlarzsia pilzdaeformis, an epiphytic species. Mycologia 85:527-534. 16. Luttrell, E.S. and Bacon, C.W. (1977). Classification of Myriogenosporn in the Clavicipitaceae. Can. J. Bot. 55:2090-2097. 17. Rykard, D.M., Luttrell, E.S. and Bacon, C.W. (1982). Development of the conidial state of Myriogerzospora atramentosa. Mycologia 74:648-654. 18. Rykard, D.M., Luttrell, E.S. and Bacon, C.W. (1984). Conidiogenesis and conidiomata in the Clavicipitoideae. My-ologia 76: 1095- 1103. 19. Tudzynski, P., Tenberge, K.B. and Oeser, B. (1995). Claviceps purpurea. In Pathogenesis and Host Specifciv in Plant Diseases: Histopathological, Biochemical, Genetic and Molecular Bases (U. Singh, R. Singh and K. Kohmoto, eds.). Pergamon, Tarrytown, NY, pp. 161186. 20. Frederickson, D.E., Mantle, P.J. and de Milliano, W.A.J. (1991). Claviceps nfiicana sp. nov.; the distinctive ergot pathogen of sorghum in Africa. Mycol. Res. 95:llOl-1107. 21. Pazoutova. S., Fucikovsky. L., Leyva-Mir, S.G. and Flieger, M. (1998). Claviceps citrina sp. nov., a parasite of the halophytic grass Distichlis spicata from Mexico. Mycol. Res. 102: 850-854. 22. Esser, K. and Tudzynski, P. (1978). Genetics of the ergot fungus Clrwiceps purpurea. Theor. Appl. Genet. 53:145-149. 23. Thakur, R.P. and King, B. (1992). Ergot of pearl millet. In Plant Diseases of International hyortalzce. Vol. 1. Diseases of Ceretrls and Pulses (U.S. Singh, A.N. Mukhopadhyay, J. Kumar and H.S. Chaube, eds.). Prentice-Hall, Englewood Cliffs, NJ, pp. 302-315. 24. Luttrell, E.S. (1977). The disease cycle and fungus-host relationships in dallisgrass ergot. Ph>~topntl~ology 67: 1461- 1468. 25. Luttrell, E.S. (1980). Host-parasite relationships and development of the ergot sclerotium in Claviceps purpurea. Call. J. Bot. 58:942-958. 26. Shaw, B.J. and Mantle, P.J. (1998). Host infection by Clavicepspurpurea. Trans. Br. Mycol. SOC.75177-90. 27. Mower, R.L. and Handcock, J.G. (1975). Mechanisms of honeydew formation by Clcrviceps species. Can. J. Bot. 532826-2834. 28. Mower, R.L. and Hancock, J.G. (1 975). Sugar composition of ergot honeydews. Can. J. Bot. 53:2813-2825. 29. Taber, W.A. (1985). Biology of Claviceps. In Biology of Industrial Microorganism (A.L. Demain and N.A. Solomon, eds.). Benjamin/Cummings Publishing Company, London, pp. 449-486. 30. Frederickson, D.E. and Mantle, P.G. (1989). Secondary conidiation of Splrncelin sorghi on sorghum, a novel factor in the epidemiology of the ergot disease. Mycol. Res. 93:497502. 31. Frederickson, D.E., Mantle, P.G. and de Milliano, W.A.J. (1993). Windborne spread of ergot disease (Claviceps africartn) in sorghum A-lines in Zimbabwe. Plant Pntlzol. 42:368-377. 32. Ramstad, E. and Gjerstad, G. (1955). The parasitic growth of Claviceps purpurecr (Fries) Tulasne on rye and its relation to alkaloid formation. J. Am. Pharrn. Assoc. 44:741. 33. Didek-Brumec. M., Gaberc-Porekar, V. and Alacevic, M. (1996). Relationship between the Claviceps life cycle and productivity of ergot alkaloids. Crit. Rev. Bioteclznol. 16257-299. 34. Siegel, M.R., Latch, G.C.M., Bush, L.P., et al. (1990). Fungal endophyte-infected grasses: alkaloid accumulation and aphid response. J. Clrenz. Ecol. 16:3301-3315.

526

Kuldau and Bacon

35. Wicklow, D.T., Vesonder, R.F., McAlpin, C.E., Cole, R.J. and Roquebert, M.-F. ( 1989). Examination of Stilbotharlzrlizlllz togoeme for Aspergillm j h l u s group mycotoxins. Mycotaxon 34:249-252. 36. Bacon, C.W. and Luttrell, E.S. (1982). Competition between ergots of Claviceps pzlrpuren and rye seed for photosynthates. Phytopatlzolop 72: 1332-1336. 37. Colotelo, N. and Cook, W. (1977). Perithecia and spore liberation of Claviceps pwpuren: scanning electron microscopy. Can. J. Bot. 55: 1257-1259. 38. Futrell, M.C. and Webster, O.J. (1 965). Ergot infection and sterility in grain sorghum. Plnrzt Dis. Rep. 49:680-683. 39. Watkins, J.E. and Littlefield, L.J. (1976). Relationship of anthesis in Waldron wheat to infection by Claviceps purpurea. Trans. Br. Mycol. SOC. 66362-363. 40. Anahosur, K.H. and Patil, H.S. (1982). Effect of date of sowing on the incidence of ergot of sorghum. Indiurr Plzytopatl?ol.35:507-509. 41. White, J.F., Jr. and Bultman, T.L. (1987). Endophyte-host associations in forage grasses. VTII. Heterothallism in Epichloe t?,plzina. Am. J. Bot. 74: 1716-1722. 42. Schardl, C.L.. Leuchtmann, A., Chung, K.-R., Penny, D. and Siegel, M.R. (1997). Co-evolution by common descent of fungal symbionts (Epichloe spp.) and grass hosts. Mol. Biol. EIJO~. 141133-143. 43. Bacon. C.W. and White, J.F., Jr. (1994). Stains, media, and procedures for analyzing endophytes. In Biotechnology of Erdophytic Fungi (C.W. Bacon and J.F. White, Jr., eds.). CRC, Boca Raton, FL. pp. 47-56. 44. Chung. K.-R. and Schardi, C.L. (1997). Sexual cycle and horizontal transmission of the grass symbiont, Et’ichloe typhinu. Mycol. Res. 101:295-301. 45. Kuldau, G.A., Liu, J.-S., White, J.F., Siegel, M.R. and Schardl, C.L. (1997). Molecular systematics of Clavicipitaceae supporting monophyly of genus Epichloe and form genus Ephelis. M~’c010gia89143 1-441. 46. Schardl, C.L., Leuchtmann, A., Tsai. H.-F., Collett, M.A., Watt. D.M. and Scott, D.B. (1994). Origin of a fungal symbiont of perennial ryegrass by interspecific hybridization of a mutualist with the ryegrass choke pathogen, Epiclzloe pplzirzu. Gemtics 136:1307- 1317. 47. Tsai, H.-F., Liu, J.-S., Staben, C., et al. (1994). Evolutionary diversification of fungal endophytes of tall fescue grass by hybridization with Epichloe species. Proc. Nutl. Acad. Sci. USA 9 1:2542-2546. 48. Belesky, D.P., Stringer, W.C. and Hill, N.S. (1 989). Influence of endophyte and water regime upon tall fescue accessions. I. Growth characteristics. Ann. Bot. 63:495-503. 49. Keough, R.G., Tapper,B.A. and Fletcher, R.H. (1996). Distributions of the fungal endophyte Acremoniun? lolii, and of the alkaloids lolitrem B and peramine, within perennial ryegrass. N.Z. J. Agric. Res. 39:121-127. 50. Azevedo, M.D. and Welty, R.E. (1995). A study of the fungal endophyte Acreinolzizcnl coenophialum in the roots of tall fescue seedlings. Mycologia 87289-297. 51. Lane, G.A., Ball, O.J.P., Davies, E., and Davidson, C. (1997). Ergovaline distribution in perennial ryegrass naturally infected with endophyte. In Neotyphodiztm/Grass Interactions (C.W. Bacon and N.S. Hill, eds.). Plenum Press, New York. pp. 65-67. 52. Read, J.C. and Camp, B.J. (1986). Theeffect of the fungal endophyte Acremoniurn coeizophialzm in tall fescue on animal performance, toxicity, and stand maintenance. Agron. J. 78: 848-850. 53. Hill, N.S., Belesky, D.P. and Stringer, W.C. (1991). Competitiveness of tall fescue as influenced by Acrentoniunz coenophiahm. Crop Sci. 3 1 :185-190. 54. Arechavaleta, M., Bacon, C.W., Hoveland, C.S. and Radcliffe, D.E. (1989). Effect of the tall fescue endophyte on plant response to environmental stress. Agrom J. 81:83-90. 55. Hill, N.S., Pachon, J.G. and Bacon, C.W. (1 996). Acremoniurn coenophialurn-mediated short- and long-term drought acclimation in tall fescue. Crop Sci. 36:665-672. 56. Malinowski, D., Leuchtmann, A., Schmidt, D. and Nosberger, J. (1998). Growth and water

Claviceps

57.

58. 59.

60. 61. 62. 63. 64, 65. 66. 67. 68.

69.

70. 71. 72. 73. 74. 75.

76.

77.

78.

527

status in meadow fescue (Festttca pmtensis) is affected by NeoQplzoclizou and PhiaIopl1orcr endophytes. Agron. J . 89:673-678. Siegel, M.R., Latch, G.C.M., Bush. L.P., et a]. (1991). Alkaloids and insecticidal activity of grasses infected with fungal endophytes. J. Chem. Ecol. 16:3301-3315. Latch. G.C.M. (1993). Physiological interactions of endophytic fungi and their hosts. Biotic stress tolerance imparted to grasses by endophytes. Agl-i. Eco. Emiron. 44:143-156. Clement, S.L., Kaiser, W.J., and Eichenseer. H. (1994). A c ~ - e ~ ~ o n endophytes izm in germplasms of major grasses and their utilization for insect resistance. In Bioteclznologyof Erzdophytic Fzozgi of Grosses (C.W. Bacon and J.F. White, eds.). CRC Press, Boca Raton, FL. pp.185-199. Johnson, M.C., Dahlman, D.L., Siegel, M.R., et al. (1985).Insect feeding deterrents i n endophyte-infected tall fescue. Appl. Envirow Microbiol. 49:568-571. Pederson. J.F. and Rodriguez-Kabana, R. (1984). Differences among nematode populations in tall fescue pastures in north, central, and south Alabama. Crop Sci. 24:819-821. Kimmons, C.A., Gwinn, K.D. and Bernard, E.C. (1990). Nematode reproduction on endophyte-infected and endophyte-free tall fescue. Plartt Dis. 74:757-761. Gwinn, K.D. and Gavin, A.M. (1992). Relationship between endophyte infestation level of tall fescue seed lots and Rl~i,-octonier zene seedling disease. Plant Dis. 76:911-914. De Battista, J.P., Bacon, C.W.. Severson, R., Plattner, R.D. and Bouton. J.H. (1990). Indole acetic acid production by the fungal endophyte of tall fescue. Agl-on. J. 82878-880. Lyons, P.C., Plattner, R.D. and Bacon,C.W. (1986).Occun-ence of peptide andclavine ergot alkaloids in tall fescue. Scierzce 232487-489. Belesky, D.P.. Devine, O.J.. Pallas, J.E., Jr. and Stringer, W.C. (1987).Photosynthetic activity of tall fescue as influenced by a fungal endophyte. Pltotosyntlzetica 21:82-87. Belesky, D.P., Stuedemann, J.A. and Wilkinson, S.R. (1988).Ergopeptine alkaloids in grazed tall fescue. Agrolz. J. 80:209-212. Arechavaleta, M., Bacon, C.W., Plattner, R.D., Hoveland. C.S. and Radcliffe, D.E. (1992). Accumulation of ergopeptide alkaloids in symbiotic tall fescue grown under deficits of soil water and nitrogen fertilizer. Appl. Envirow Microbiol. 58:857-861. Malinowski. D.P., Belesky, D.P., Hill, N.S., Baligar, V.C. and Fedders, J.M. (1998). Influence of phosphorus on the growth and ergot alkaloid content of Neor;\phodizlnz coenophinlum-infected tall fescue (Festuccl at-wzdinacecr Schreb.). P l m t Soil 19853-61. Belesky, D.P. and Hill, N.S. (1997). Defoliation and leaf age influence on ergot alkaloids in tall fescue. Ann. Bot. 79:259-264. Porter. J.K., Bacon, C.W.andRobbins,J.D. (1979). Ergosine,ergosinine,chanoclavine I from Epicltloe typhim. J. Agl-ic. Food Chent. 27:595-598. Bacon, C.W. (1988). Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids. Appl. Emiron. Mici-obiol. 53:2615-2618. Blankenship, J.D., Williinson, H.H., and Schardl. C.L. (1998).Protective alkaloids expression in grass-endophyte mutualisms. Pkytopatllology 88:sS. Agee, C.S. and Hill, N.S. (1994). Ergovaline variability in Acl-enzoiziunz-infected tall fescue due to environment and plant genotype. Crop Sci. 34:221-226. Roylance, J.T., Hill, N.S. and Agee, C.S. (1994). Ergovaline and peramine production in endophyte-infected tall fescue: independent regulation and effects of plant and endophyte genotype. J. Chew. Ecol. 202171 -2183. White, J.F., Jr. and Glenn. A.E. (1994). A study of two fungal epibiontsof grasses: structural features, host relationships, and classification in the genus Myriogtwospol-a (Clavicipitales). Ant. J. Bot. 81 1216-223. White, J.F., Jr., Marrow, A.C.. and Morgan-Jones, G. (1990). Endophyte-host associations in forage grasses. XII. A fungal endophyte of Tricllacllrze insdcrris belonging to Pseudocercosporella. Mycologia 82218-226. White, J.F., Jr. and Owens, J.R. (1992). Stromaldevelopment and mating system of Balamia

528

79.

80. 81. 82.

83.

84.

85. 86. 87.

88. 89.

90.

91.

92. 93.

94. 95. 96. 97.

9s. 99.

Kuldau and Bacon epicllloe, a leaf-colonizing endophyteof warm-season grasses. Appl. Environ. Mict-obiol. 58: 513-519. Leuchtmann, A.. Schardl, C.L. and Siegel, M.R.(1994).Sexual co~npatibilityand taxonomy of a new species of Epicllloe symbiotic with fine fescue grasses. Mycologicr 86:802-812. Bacon. C.W., Lyons, P.C.. Porter, J.K. and Robbins, J.D. (1986).Ergot toxicity from endophyte-infected grasses: a review. Agrotz. J. 78:106-116. Bacon. C.W. and De Battista,J. (1991 ). Endophytic fungiof grasses. In Ahnnces ir1 Applied Mycology (D.K. Arora, ed.). Marcel Dekker. New York, pp. 231-256. Hardy, T.N., Clay, K. and Hammond, A.M., Jr. (1985).Fall armyworm (Lepidoptera: Noctuidae): Alaboratory bioassay and larval preference study for the fungal endophyte of perennial ryegrass. J. Econ. Efltonlol. 78:571-575. Clay, K., Cheplik. G.P. and Marks. S. (1989). Impact of the fungus Bnlrrrzsia herzningsiartn in Pnnicwtl agrostoides: frequency of infection, plant growth and reproduction, resistance to pests. Oecologia 80374-380. characterization Glenn, A.E., Rykard. D.M., Bacon, C.W. and Hanlin, R.T. (1998). Molecular of Myriogenospot-cr atrrrnrentosn and its occurrence on some new hosts. Mycol. Res. 102: 483-490. Kybal, J. (1963). Response of ergot fungus to the nutrition available from the host plant. Phytopathology 53:363. Bacon, C.W., Porter, J.K. and Robbins, J.D. (1975). Toxicity and occurrence of Bnlcrrzsin on grasses from toxic fescue pastures. Appl. Microbiol. 29:553-556. Bacon, C.W. (1985). A chemically defined medium for the growth and synthesis of ergot alkaloids by species of Balmsin. Mycologia. 77:413-423. Hill, N.S., Parrott, W.A. and Pope, D.D. (1991). Ergopeptide production by endophytes in a common tall fescue genotype. Agron. Abst. 3 1: 1545-1547 (abstract). Gaynor, D.L. and Hunt, W.F. (1983). The relationship between nitrogen supply. endophytic fungus, and Argentine stem weevil resistance in ryegrass. In Proc. N.Z. Grassland Assoc.. pp. 257-263. Lyons, P.C.. Evans, J.J. and Bacon, C.W. (1990). Effects of the fungal endophyte Acremoniml coenophinlurtr on nitrogen accumulation and metabolism in tall fescue. Plant Physiol. 92726-732. Arechavaleta, M., Bacon, C.W., Plattner, R.D., Hoveland, C.S. and Radcliffe, D.E. ( 1992). Accumulation of ergopeptide alkaloids in endophyte-infected tall fescue grown under deficits of soil water and nitrogen fertilizer. Appl. Em-iron. Microbiol. 58:857-861. Belesky, D.P. and Fedders. J.M. (1995). Tallfescue development in response toAcremorzium coenophinlunr and soil acidity. Crop Sci. 35:529-533. Hill, N.S., Stringer, W.C., Rottinghaus, G.E., Belesky, D.P., Parrott, W.A. and Pope. D.D. (1990). Growth,morphological, and chemical component responses of tall fescue to Acrenlor t i m coenoplzialunz. Crop Sci. 30: 156- 161. Agee, C.S. and Hill. N.S. (1992). Variability in progeny from high- and low-ergovaline producing tall fescue parents. Agr-oil. Abst. 3 2 8 2 (abstract). Porter, J.K., Bacon, C.W. and Robbins, J.D. (1979). Lysergic acid amide derivatives from Bnlansirl epichloe and Bnlansia claviceps (Clavicipitaceae). J. Not. Prod. 42309-3 14. Porter. J.K., Bacon. C.W., Robbins, J.D. and Betowski, D. (1981). Ergot alkaloid identification in Clavicipitaceae systemicfungi of pasture grasses. J. Agric. Food Chem. 29:653-657. Yates, S.G., Plattner, R.D. and Garner, G.B. (1985). Detection of ergopeptine alkaloids in endophyteinfected, toxicKy-31 tall fescue by mass spectrometry/mass spectrometry. J. Agric. Food Cltem. 33:719-722. Riggs, R.K. and Chapman. R.A. (1968). Infectivity of alkaloid production by some isolates of Claviceps prulmrea. Phytopatllology 58:54-55. Floss, H. (1976). Biosynthesis of ergot alkaloids and related compounds. Tetrahedrolz 32: 873-912.

Claviceps

529

100. Bacon, C.W. and Siege1 M.R. (1988). The endophyte of tall fescue. J. Prod. Agric. 1:4555. 101. Gallagher, R.T., Smith, G.S. and Sprosen, J.M. (1987). Distribution and accumulation of lolitrem B neurotoxin in perennial ryegrass plants. Proc. 4th Anim. Sci. Congress. Hamilton, New Zealand. von auf tetraploidem roggen gewachsenen mutterkorn102. Deufel, J. (1952). Der Alkaloidgehalt sklerotien. Nntl(ru1issenshaftert 39:433. 103. Silber. A. and Bischuff. W. (1954). Die konstanz des alkaloidgehaltes bei verschiedenen rassen von mutterkorn. Phar-mazie 9:46. 104. Christopher, W.M. and Mantle, P.G. (1987). Paxilline biosynthesis by Acrernorziztnz loline, a step toward defining the origin of lolitrem neurotoxins. Phytopathology 26:969-971. 105. Fletcher, L.R. (1982). Observation of ryegrass staggers in weaned lambs grazing different ryegrass pastures. N.Z. J. Exp. Agric. 10:203-207. 106. Gallagher, R.T., Smith, G.S., Di Menna, M.E. and Young, P.W. (1982). Some observations on neurotoxin production in perennial ryegrass. N.Z. Vet. J. 30:203-204. 107. Gallagher, R.T., Hawkes, A.D., Steyn, P.S. and Vleggaar. R. (1984). Tremorgenic neurotoxins from perennial ryegrass causing ryegrass staggers disorder of livestock: structure and elucidation of lolitrem B. J. Chenz. SOC.Chenz. Contrmn. 614-6 16. 108. Weedon, C.M. and Mantle, P.G. (1987). Paxilline biosynthesis by Acre~noniumloline; a step towards defining the origin of lolitrem neurotoxins. PhFtochemistv 26:969-97 1. 109. Lanigan, G.W., Payne, A.L. and Cockrum, P.A. (1979). Production of tremorgenic toxins by Perticillitm janthinellunz Biourge: a possible etiological factorin ryegrass staggers.Aust. J. E.xp. Biol. Med. Sci. 57:31-37. 110. di Manna, M.E.. Mantle, P.G.and Mortimer, P.H. (1976). Experimental productionof staggers syndrome in ruminants by a tremorgenic Penicillium from soil. N.Z. Vet. J. 24:45-51. 111. Bush, L.P., Cornelius, P.C., Buckner, R.C.,et al. (1982). Association of N-acetyl loline and N-formyl loline with Epichloe ~ p h i n ain tall fescue. Crop Sci. 22:941-943. of grass-fungal 112. Bush. L.P., Wilkinson, H.H.and Schardl, C.L. (1997). Bioprotective alkaloids endophyte symbioses. Plant Ph?~siol.114:1-7. 113. Belesky, D.P., Robbins, J.D., Stuedemann, J.A.. Wilkinson, S.R. and Devine. O.J. (1987). Fungal endophyte infection-loline derivative alkaloid concentration of grazed tall fescue. Agron. J. 79217-220. Tall Fescue (R.C. Buck114. Bush, L.P., Bolling,J., and Yates, S.G. (1979). Animal disorders. In ner and L.P. Bush, eds.). American Society of Agronomy, Maidson, WI, pp. 247-292. of endophyte infection. plant 115. Eichenseer, H., Dahlman, D.L.and Bush, L.P. (1991). Influence age and harvest interval on Rhopcrlosiphzcnz pndi survival and its relation to quantity of Nformyl and N-acetyl loline in tall fescue. Entomol. Exp. Appl. 6029-38. 116. McLean, E.K. (1970). The toxic actions of pyn-olizidine (Senecio) alkaloids. Pharm Rev. 221429-483. 117. Porter. J.K.. Bacon, C.W.. Robbins.J.D.,Himmelsbach,D.S. and Higman,H.C.(1977). Indole alkaloids from Balamia epichloe (Weese). J. Agric. Food Chenz 25:88-93. 118. Rowan, D.D., Hunt, M.B. and Gaynor, D.L. (1986). Peramine, a novel insect feeding deterrent from lyegrass infected with the endophyte Acremonium loliae. J. Chem. SOC.Chem. Commtaz. 935-936. of endophyte toxins: Fescueand other grasses toxicto livestock. 119. Porter, J.K. (1995). Analysis J. Anirtt. Sci. 73:871-880. and Diener, U.L. (1986).Steriodmetabolites of 120. Davis,N.D.,Clark,E.M.,Schrey.K.A. Acrenzoniurrz coenophialunl, an endophyte of tall fescue. J. Agric. Food Chem 34: 105- 108. 121. Koshino, H., Terada, S., Yoshihara, T., et al. (1988). Three phenolic acid derivatives from stromata of Epiclzloe hphirza on Pldeunz pmtense. Phytochenristr?) 27: 1333-1338. 122. Yshihara, T., Togiya, S., Koshino, H., et al. (1985). Three fungitoxic sesquiterpenes from stromata of Epicltloe typhina. Tetra. Lett. 26:555 1-5554.

530

Kuldau and Bacon

123. Dowd, P.F.. Cole. R.J. and Vesonder, R.F. (1988).Toxicity of selected tremogenic mycotoxins and related compounds to Spotkopter-ofr-zlgil)el.daand Heliothis :eo. J. Arztibiot. 61:18681872. 134. Gabbai, D., Lisbonne, D. and Pourquier. D. (1951). Ergot poisoning at Pont St. Espirit. BY. Med. J . 1:650-679. 125. Barger, G. (1931). Ergot arzd Er-gotisnr.Gurney & Jackson, London. 126. Scott. P.M. and Lawrence. G.A. (1980). Analysis of ergot alkaloids in flour. J. Agric. Food Chem. 38:1258-1261. 127. U.S. Department of Agriculture, (1977). U.S, Standards of Triticale. Fed. Reg. 42 F.R.. February16. 128. Zeleny, L. (1971). Criteria of wheat quality. In W?1eat-Clzer?ristp m d Teclwology (Y. Pomeranz, ed.). American Assoc. Cereal Chemists, St. Paul, MN. sp. nov.. the causal agent of an agalactia ofsows. 129. Loveless, A.R. (1967). Clo~licef~sfirsiJfbrI~zi~ Trat1s. Br. M ~ c o SOC. ~ . 50: 15-1 8. 130. Mantle, P.G. (1967). Growth of Atkinsonello hypoxylolt in pure culture. Trans. BY. Mycol. SOC.501497-506. 131. Woods, A.J., Jones, J.B. and Mantle. P.G. (1966). An outbreak of gangrenous ergotism in cattle. Vet. Rec. 78:742-749. 132. Greatorex, J.C. and Mantle, P.G. (1973). Experimental ergotjsm in sheep. Res. Vet. Sci 15: 337-346. 133. Drink, J.C. (1955). Ergot poisoning in cattle. Vet. J . 10:14. 134. Greatorex. J.C. and Mantle, P.G. (1974). Effectof rye ergot on the pregnant sheep. J. Reprod. Fertil. 37:33. 135. Mantle, P.G. (1969). The roleof alkaloids inthe poisoning ofmammalsbysclerotiaof Clmyiceps spp. J. Stored Prod. Res. 5237-244. 136. O’Neil, J.B. and Rae, W.J. (1965). Ergottolerancein chicks andhens. Poult~ySci. 44: 1304. 137. Cunningham, I.J. (1943). A note on the cause of tall fescue lameness in cattle. N.Z. J. Sci. Teclznol. 24: 167b-178b. 138. Yates, S.G. (1962). Toxicity of tall fescue forage: a review. ECOIZ. Bot. 16295-303. 139. Rowan. D.D. and Shaw, G.J. (1987). Detection of ergopeptine alkaloids in endophyte-infected perennial ryegrass by tandem mass spectrometry. N.Z. Vet. J . 35:197-198. 140. Fluckiger, E., Doepfner. W., Marks, M. and Niederer, W. (1976). Effects of ergot alkaloids on the hypothalmic-pituitary axis. Postgrad. Med. J. 52:57-61. 141. Richard, J.L., Meerdink, G., Maragos. C.M., et al. (1996). Absence of detectable fumonisins in the milk of cows fed Fzuariunz prolifemtzon (Matsushima) Nirenberg culture material. Mycoprrthologicr 133: 123-1 26. 142. Hill, N.S.. Parrott, W.A. and Pope. D.D. (1991). Ergopeptine alkaloid production by endophytes in a cotnmon tall fescue genotype. Crop Sci. 31:1545-1547. 143. Thompson. R.W., Fribourg. H.A. and Reddick, B.B. (1989). Sample intensity and timing for detecting Acremolzizlnz coe~zophialun~ in tall fescue pastures. Agron. J . 81 :966-971. 144. Crawford, R.J., Forwood, J.R., Belyea, R.L. and Garner, G.B. (1989). Relationship between level of endophyte infection and cattle gains on tall fescue. J. Prod. Agric. 2: 147-151. 135. Chestnut, A.B., Fribourg, H.A., McLaren, J.B., et al. (1991). Effects of Ac/-emorrirmrcoenophialum infestation. bermudagrass. and nitrogen fertilizer or clover on steers grazing tall fescue pastures. J. Prod. Agric. 4:208-313. 146. Chestnut, A.B., Fribourg, H.A., McLaren,J.B., et al. (1991). Effect of endophyte infestation level and endophyte-free tall fescue cultivar on steer productivity. Tenn. Farnz Home Sci. 160:38-44. 147 Schmidt, S.P.. Hoveland, C.S., Clark, E.M., et al. (1982). Association of an endophytic fungus with fescue toxicity in steers fed Kentucky 31 tall fescue seed or hay. J. Anirrr. Sci. 55: 1259-1263.

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148. Stuedemann, J.A. and Hoveland, C.S. (1988). Fescue endophyte: history and impact on animal agriculture. J. Prod. Agric. 1:39-44. 149. Yates, S.G. (1983). Tallfescue toxins. In Hnltdbook ofNntlmdly Occurring Food Toxicants (M. Recheigel. ed.). CRC Press, Boca Raton, FL. pp. 249-273. 150. Boling, J.A., Bunting, L.D., Davenport, J.L., Van Der Veen, K.M., Meeking, N.W., Bradley. N.M. and Kohls, R.E. ( 1989). Physiological responses of cattle consuming tall fescue to environmental temperature and supplemental phenothiazine. J. Anim. Sci. 672377-2385. 151. Hemken, R.W., Boling, J.A.. Bull. L.S. and Hatton, R.H. (1981). Interaction of environmental temperature and anti-quality factors on the severity of summer fescue toxicosis. J. Anim Sci. 58:710-713. 152. Bond, J., Powell, J.B., Undersander, D.J., Moe, P.W., Tyrell, H.F. and Oltjen. R.R. (1984). Forage composition and growth and physiological characteristics of cattle grazing several varieties of tall fescue during summer conditions. J. Arzirn. Sci. 59:584-593. 153. Stuedemann, J.A., Rumsey, T.S.. Bond, J., et al. (1985). Association of blood cholesterol with occurrence of fat necrosis in cows and tall fescue summer toxicosis in steers. Am. J. Vet. Res. 46:1990-1995. 154. Stuedemann, J.A. and Thompson. F.N. (1993). Management strategies and potential opportunities to reduce the effects of endophyte-infested tall fescue on animal performance. In Proc. Second Interrzntiontrl Symposium on Acremoilium/Grass Interactions: Plenary Papers (D.E. Hume, G.C.M. Latch and H.S. Easton. eds.). AgResearch, Palmerston North, pp. 103-1 14. 155. Ireland, F.A., Loch, W.E., Anthony, R.V. and Worthy, K. (1989). The use of bromocriptine and perphenazine in the study of fescue toxicosis in pregnant pony mares. Eq. Nzrtr. Plzysiol. synzp. Proc. 201. 156. Monroe, J.L.. Cross, D.L., Hudson, L.W., Hendricks, D.M., Kennedy, S.W. and Bridges, W.C. (1988). Effects of selenium and endophyte-contaminated fescue on the performance and reproduction in mares. Equine Vet. Sci. 8:148-153. 157. Putnam, M.R., Bransby. D.I.. Schumacher, J., et al. (1991). Effects of the fungal endophyte Acrertlonium coenophinlzun in fescue on pregnant mares and foal viability. Am. J. Vet. Res. 5212071-2074. 158. McCann, J.S., Caudle, A.B., Thompson, F.N., Stuedemann, J.A.. Heusner, G.L. and Thompson, D.L., Jr. (1992). Influence of endophyte-infected tall fescue on serum prolactin and progesterone in gravid mares. J. Anirn. Sci. 70:217-223. 159. Henson, M.C.. Piper. E.L. and Daniels, L.B. (1987). Effects of induced fescue toxicosis on plasma and tissue catecholamine concentrations in sheep. Dorn. Aninr. Endocrinol. 4:7- 15. 160. Bond, J., Lynch, G.P.. Bolt. D.J., Hawk, H.W., Jackson. C. and Wall, R.J. (1988). Reproductive performance and lamb weight gain for ewes grazing fungus-infected tall fescue. Ntrtr. Rep. h t . 37:1099-1102. 161. Keogh, R.G. (1873). Induction and prevention of ryegrass staggers in grazing sheep. N.Z. J. Exp. Agr-ic. 155-57. 162. Byford. M.J. (1979). Ryegrass staggers in sheep and cattle. N.Z. J. Agr-ic. Res. May 1979: 65. 163. Hannah, S.M., Paterson, J.A., Williams, J.E., Kerley, M.S. and Miner, J.L. (1 990). Effects of increasing dietary levels of endophyte-infected tall fescue seed on diet digestibility and ruminal kinetics in sheep. J. Aninz. Sci. 68: 1693-1701. 164. Stilham, W.D.. Brown, C.J., Daniels. L.B.. Piper, E.L. and Fetherstone, H.E. (1982). Toxic fescue linked to reduced nlilk output in ewes. Arkansas Farm Res. 31:9. 165. Fletcher, L.R. and Barrel, G.K. (1984). Reduced liveweight gains and serum prolactin levels in hoggets grazing ryegrasses containing Lolizm endophyte. N.Z. Vet. J. 32:139-140. 166. Bacon, C.W., Porter, J.K. and Robbins, J.D. (1981). Ergot alkaloid biosynthesis by isolates of Balnnsin epichloe and B. henningsinnn. Ctm. J. Bot. 592533-2538. 167. Witters, W.L.. Wilms. R.A. and Hood, R.D. (1975). Prenatal effects of elymoclavine administration and temperature stress. J. Aninz. Sci. 41: 1700-1704.

532

Kuldau and Bacon

165. Bailey, V. (1903). Sleepy grass and its effect on horses. Science 17:392-393. 169. Hance. H.F. (1 876). On a mongolian grass producing intoxication in cattle. J. Bot. 1421021 2. 170. Nobindro, U. (1934). Grass poisoning among cattle and goats in Assam. Indian Vet. J. 10: 235-236. 171. Wallner, B.M., Booth, N.H., Robbins. J.D., et al. (1983). Effect of an endophytic fungus isolated from toxic pasture grass on serum prolactin concentrations in the lactating cow. Anr. J. Vet. Res. 43:1317-1322. 172. Cole, R.J., Dorner, J.W., Lansden, J.A., et al. (1977). Pnspalwt staggers: isolation and identification of tremorgenic metabolites from sclerotia of Clcrlicepspnspcrli Stevens et Hall. Tetrahedron Lett. 21:235-238. 173. Thakur, R.P. and Rao, V.P. (1989).Ergot susceptibility in relation to cytoplasmic male sterility in pearl nlillet. Plcritt Dis. 73:676-678. 174. Thakur, R.P., Rao, V.P. and King, S.B. (1991). Tnfluence of flowering event factors in cytoplasmic male sterile lines and f l hybrids on infection by Clnllicepsfirsiforilzis in pearl millet. Plnrrt Dis. 75: 1217- 1222. 175. Craig, J. and Hignight, K.W. (1991). Control of ergot in buffelgrass with triadimefon. Plant Dis. 75:627-629. 176. Puranik, S.B. and Mathre, D.E. (1971). Biology and control of ergot on male sterile wheat and barley. Pltytopcrtltology 61 :1075-1079. 177. Hardison. J.R. (1977). Chemical control of ergot in field plots of Lolizm perenne. Plant Dis. Rep. 61:835-848. 178. Mower, R.L. and Hancock, J.G. (1 975). Biological control of ergot by Fustrrium. Phjltopntho log^ 65:5-10. 179. Scott, B. and Schardl, C. (1993). Fungal symbionts of grasses: evolutionary insights and agricultural potential. Trends Microbiol. 1:196-200. 180. Schardl, C.L. and Phillips, T.D. (1997). Protective grass endophytes: where are they from and where are they going. Plant Dis. 81:430-438. 181. Bacon. C.W.. Richardson, M.D. and White, J.F., Jr. (1997). Modification and uses of endophyte-enhanced turfgrasses: a role for molecular technology. Crop Sci. 37: 1415-1425. 152. Murray, F.R., Latch. G.C.R.I. and Scott, D.B. (1992). Surrogate transformation of perennial ryegrass, Lolium perelme, using genetically modified Acrenronium endophyte. Mol. Gen. Genet. 233: 1-9. 183. Tsai, H.-F., Siegel, M.R. and Schardl, C.L. (1 992). Transformation of Acrernonium coenoplrialum, a protective fungal symbiont of the grass Festucn arundinacecr. C u m Genet. 22399406. 184. Tsai, H.F., Wang, H., Gebler. J.C., Poul ter, C.D. and Schardl, C.L. ( 1995). The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step forergot alkaloid biosynthesis. Biochem. Biophys. Res. Comntun. 2 16: 119- 125. 185. Schardl, C.L. and Siegel, M.R. (1992). Genetics of Epichloe hphina and Acremoniu~ncoenoyltiahir. In Acreliroiziliril/Grrrss lnterctctions (S.S. Quisenberry and R.E. Joost. eds.). Elsevier Science Publishers. Amsterdam. 156. Tsai, H.F., Wang. H., Gebler. J.C., Poul ter, C.D. and Schardl, C.L. ( 1995). The Clnviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step forergot alkaloid biosynthesis. Bioclzern. Biophys. Res. Co~nrnun.216: 1 19-125. 187. Schardl, C.L. (1994). Molecular and genetic methodologies and transformation of grass endophytes. In Biotechnology of Endophytic Fungi o f Grasses (C.W. Bacon and J.F. White, Jr., eds.). CRC Press, Boca Raton. FL, pp. 151-165. 158. Lauriault, L.M., Dougherty, C.T., Bradley, N.W. and Cornelius, P.L. (1990). Thiamin supplementation and the ingestive behavior of beef cattle grazing endophyte-infected tall fescue. J. Aninz. Sci. 68:1245-1253. 189. Bransby. D.J., Holliman, J., and Eason, J.T. (1993). Tvermectin could partially block fescue

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191.

192.

193.

194.

195. 196.

197.

198. 199.

200. 201.

202.

203. 204. 205.

206.

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toxicosis. In Proc. 1993 American Forage and Grasslmd Cortrzcil Meeting (.W. Faw and G.A. Pederson, eds.). American Forage and Grassland Council, March29-3 1. Georgetown, TX, pp. 81-84. Jackson, J., Sorgho,Z. and Hatton, R.H. (1988). Effectof nitrogen fertilization or urea addition and ensiling as large round bales of endophyte infected tall fescue on fescue toxicosis when fed to dairy calves. Nutr. Rep. Int. 37:335-345. Tucker, C.A., Morrow, R.E., Gerrish, J.R., et al. (1989). Forage systems for beef cattle: effect of winter supplementationand forage system on reproductive performanceof cows. J. Prod. Agric. 2217-221. Coffey, K.P., Moyer,J.L.. Lomas. L.W., Smith, J.E., La Rue, D.C. and Brazle, F.K.(1992). Implant and copper oxide needles for steers grazing Acrenzonium coenophialum-infected tall fescue pastures: effectson grazing and subsequent feedlot performance and serum constituents. J. Aninz. Sci. 70:3203-3214. Kerr, L.A., McCoy, C.R., Boyle, C.R. and Essig, H.W. (1990). Effects of ammoniation of endophyte infected tall fescue on serum prolactin concentration and rectal temperature in beef cattle. Aut. J. Vet. Res. 51 :76-78. Chestnut, A.B., Fribourg, H.A., Cochran, M.A. and Anderson, P.D. (1990). Effects of anmoniating Acrenzoniunt coenophinlr~nl infested fescue hay on signs of fescue toxicosisin sheep. In Proc. International Symposircnz Acrenzoniunz/Grass Interactions (S. Quisenben-y and R. Joost, eds.). LSU Press, New Orleans, LA, pp. 212-215. Lipham. L.B., Thompson, F.N., Stuedemann, J.A. and Sartin, J.L. (1989). Effects of metoclopramide on steers grazing endophyte-infected fescue. J. Anim Sci. 67: 1090-1097. Aldrich, C.G., Rhodes, M.T., Miner, J.L., Kerley, M.S. and Paterson, J.A. (1993). The effects of endophyte-infected tall fescue consumption and use of a dopamine antagonist on intake, digestibility, bodytemperature, and bloodconstituents in sheep. J. Anint. Sci. 71:158163. Redmond, L.M., Cross, D.L., Strickland, J.R. and Kennedy, S.W. (1994). Efficacy of domperidone and sulphide as treatments for fescue toxicosis in horses. Am. J. Vet. Res. 55:722729. Cross, D.L., Redmond, L.M. and Strickland. J.R. (1995). Equine fescue toxicosis: signs and solutions. J. Anim. Sci. 73:899-908. Thompson, F.N.,Hill, N.S., Dawe, D.L. and Stuedemann, J.A. (1993). The effects of passive immunization against lysergic acid derivatives on serum prolactin in steers grazing endophyte-infected tall fescue. In Proc. Second International Symposium Acrenzonium/Gr-assIntemctions (D.E. Hume, G.C.M. Latch and H.S. Easton, eds.). AgResearch, Palmerston North, NZ, pp. 135- 137. Wilson, A.D., Clement, S.L., Kaiser. W.J. and Lester, D.G. (1991). First report of clavicipitaceous anamorphic endophytes in Hordeum species. Plant Dis. 75915. TePaske, M.R., Powell, R.G. and Clement, S.L. (1993). Analyses of selected endophyteinfected grasses forthe presence of loline-type and ergot-type alkaloids.J. Agric. Food Clzent. 4112299-2303. Bishop, D.L., Levine, H.G., Kropp, B.R. and Anderson, A.J. (1997). Seedborne fungal contamination: consequences in space-grown wheat. Phvtopatlzology 87: 1125-1 133. Munkvold, G.P., Carso, T. and Thoreson, D. (1997). Outbreak of ergot (Clal~icepspurpurea) in Iowa barley, 1996. Plant Dis. 81 :830. Mahlman, J.D. (1997). Uncertainties in projections of human-caused climate warming. Science 278:1416-1417. Plowman, T.C., Leuchtmann, A., Blaney,C. and Clay, K. (1990). Significanceof the fungus Balansia cyperi infecting medicinal speciesof Cyperzrs (Cyperaceae) from Amazonia.Econ. Bot. 44:452-462. Brady. L.R. (1962). Phylogenetic distribution of parasitism by Clmiceps species. Lloydia 25: 1-36.

534

Kuldau and Bacon

207. Loveless, A.R. (1967). A new species of Claliceps on Cyperaceae. Truns. Br. Mycol. SOC. 50: 19-22. 208. Sprague, R. (1950).Diseuses of Cereals and Grasses i n North Alnericu (Fungi Except Snluts rrrd Rusts). Ronald Press, New York. 209. Hitchcock, A.S. (1951). MaMl4~/of the Grasses of the Urzitecl States. U.S. Dep. Agr. Misc. Pub]. 200, Washington, D.C. 21 0. White, J.F., Jr., Reddy. P.V., Glenn, A.E. and Bacon, C.W. ( 1997). An examination of structural features and relationships in Bulrrrlsia subgenus Dothichloe. Mycologirr 89:408-419. 211. Reddy, P.V.. Bergen, M.S.. Patel, R. and White, J.F., Jr. (1998). An examination of tnolecular phylogeny and morphology of the grass endophyte Balansirr claliceps and similar species. Mycologin 90: 108-1 17. 212. White, J.F., Jr. (1993). Endophyte-host associations in grasses. XIX. A systematic study of some sympatric species of Epicldoe in England. Mvcologia 85:434-455. 213. Schardl. C.L. and Leuchtmann, A. (1999). Three new species of Epichloe symbiotic with North American grasses. filwologiu 91 :95- 107. 214. Leuchtmann, A. and Schardl. C.L. (1998). Mating conlpatibility and phylogenetic relationships among two new species of Epicltloe and other congeneric European species. Mycol. Res. 103:1169-1 182. 215. Hoover, M.M., Hein, M.A., Dayton, W.A. and Erlanson, C.O. ( 1948). The main grasses for farm and home. In Grass, the Yerrrbook of Agriczdture 1948 (U.S. Department of Agriculture, Washington, DC pp. 639-700. 21 6. Bula, R.J., Lechtenberg, V.L. and Holt, D.A. (1977). Potential of the world’s forages for ruminant animal production. In Potential sf Temperate Zone Czdtivrrted Foruges (Winrock International Livestock Research and Training Center, Morrilton, AR, pp. 7-28. 217. Crowder. L.V. (1977). Potential of the world’s forages for ruminant animal production. In Potential of Tropical Zones Czdtivcrted Forages (Winrock International Livestock Research and Training Center, Monilton, AR, pp. 49-78. 218. Kohlmeyer, J. and Kohlmeyer. E. (1974). Distribution of Epichloe typlzirm (Ascomycetes) and its parasilic fly. Mycologin 66:77-86. 219. White. J.F., Jr. (1987). The widespread distribution of endophytes in the Poaceae. Plarzt Dis. 71:340-342.

12 Fusarium Walter F. 0. Marasas Medical Resecrl-cll Colc?zcil, Tvger-berg,South Afiicn

I. Introduction 535 11. Toxigenic Fusarium Species

536

537 A. Fusar-iunt syorotrichioides B. Flrsmiwrl gramirzearunt 537 C. Fusariltrtr morzilifonne 537 111. Mycotoxins

538

A. Trichothecenes 538 B. Zearalenone 540 C. Moniliformin 541 D. Fusarin C 541 E. Fumonisins 541 IV.

V.

Associated Foodborne Diseases

542

A. Alimentary toxic aleukia B. Scabby grain intoxication C. Esophageal cancer 551

542 545

Conclusions References

557 558

I. INTRODUCTION The genus Fzrsarizrrn Link contains many important plant pathogens as well as mycotoxinproducing species. This combination results in situations in which diseased host plants infected by pathogenic as well as toxigenic Fusarium species becotne toxic to human and animal consumers. In this context, the most important host plants of toxigenic Fzrsarizwz species are cereal grains, including corn (Zea mays L.), wheat (Triticum aestivurn L.), barley (Hordeurn vzrlgcrre L.), oats (Avena sativa L.,), rye (Secale cereale L.), rice (Oryza sntivcr L.), sorghum (Sorghum species), and millets (Penniseturn, Pmiczrnz, and Setaria species). Fz~sariummycotoxins have also been found in foodstuffs other than cereals, such as groundnuts (Amchis hypogea L.), tomatoes (Lycopersicurn esculerztur~zMill.,),potatoes (Solcrnurn tlrberosu~~z L.), and others, but it is questionable whether such contaminated foodstuffs ever have been implicated in human disease. All of the above cereal grains, 535

Marasas

536

however, are used both as ingredients of animal feeds and as human dietary staples. Consequently, the assessment of the risk to human health of foodborne Fusarium mycotoxins in cereal grains should take cognizance of the mycotoxicoses known to be caused in animals by F~lsnrilml-infectedgrains. The most important toxigenic Fusarium species, the mycotoxins produced by them and that are known to occur naturally in cereal grains, the mycotoxicoses caused by them in animals. and the human diseases that have been associated with these foodborne Fusnriurn mycotoxins are reviewed in this chapter.

II.

TOXIGENICFUSARIUM SPECIES

At least 20 Fusarium species belonging to 10 sections of the genus have been reported in the literature to be toxigenic (1, 2). The three most important toxigenic Fz4sarium species, with respect to the production of mycotoxins in cereal grains that are known to cause field outbreaks of mycotoxicoses in animals and are most probably associated with foodborne diseases of humans (1, 3-7), are: Fusarium sporotriclzioides Sherbakoff Fusnrium grnmirzecrrum Schwabe Fusnriunl morzil(forme Sheldon Several other Fusarium species also are known to produce the same mycotoxins as these three most important ones and consequently also may be involved in the etiology of veterinary and human mycotoxicoses. In addition, some other Frrsnrizm species are known to produce high concentrations of other mycotoxins in cereals. Although these mycotoxins have not been positively identified as the causes of naturally occurring mycotoxicoses, they are potentially dangerous to human and animal health. In view of these two considerations, the following 13 Fuscrriun~species also are mentioned in this review: Fuscrrium yoae (Peck) Wollenw. Fzlsnrirm a v e ~ m e u m(Fr.) Sacc. Fusarium equisefi (Corda) Sacc. Flrscrrium nczrmi~zcrtzln~Ell. and Ev. sensu Gordon Fusnl-ium snrnbucinum Fuckel Fusnrium c~dr11orun.1 (W. G. Smith) Sacc. Fzlsnrizrnl croohvellerzse Burgess, Nelson, and Toussoun Fusarium proliferntum (Matsushima) Nirenberg Fusnrium subgluti1zarzs (Wollenw. and Reink.) Nelson, Toussoun, and Marasas Fusarium globosum Rheeder, Marasas. and Nelson Fusnrizrrn fhqmirwn Klittich, Leslie, Nelson, and Marasas F~uariumo.xysporunl Schlecht-.emend. Snyder and Hansen Fusarium rzygnmni Burgess and Trimboli All ofthe above toxigenic Fusariurn species can be isolated from plant tissues, cereal grains, or feeds and foods containing cereal grains by either direct plating or dilution plating on agar media (2). Single conidial cultures of Fusnrirrnz colonies isolated in this way can then be identified according to the system proposed by Nelson et al. (3). The main distinguishing characteristics (2) and typical habitats (1) of the three most important toxigenic Fzlsarirrm species are discussed below.

Fusarium

A.

537

Fusariumsporofrichioides

Sectiorl: Sporotrichiella Synonym: F. sporotrichiella Bilai, F. tricirlcturn (Corda) Sacc. emend Snyd. and Hans. pro pcrrte. Teleomorph: Unknown. Czrlturcrl cl~nrc~teristics: Colony diameter 5-6 cm at 25°C and3-4 cm at 30°C after 72 hr; aerial mycelium floccose, white to carmine red; undersurface carmine red. Conidiophores: Monophialides and polyphialides. Microco~~ida: Present, abundant, and of two types (i.e., spindle-shaped and globose), ovoid, or napiform; 0- to 2-septate. Macroconidin: Produced in orange sporodochia, falcate, curved with thewalls parallel through most of their length; basal cell not distinctly pedicellate; apical cell curved, tapering; 3- to 5-septate. Chlnrnydospores: Present, abundant, formed singly or in pairs, chains, or clumps. Habitat: Soil and a wide variety of hosts, particularly cereals and grasses and including cereals that overwintered under snow, in such temperate to cold areas of the world as northern Europe, Russia, northern United States, and Canada. Associatecl~foo~lbor~ze diseases: F. sporotrichioides and the trichothecenes T-2 toxin and diacetoxyscirpenol produced by it are associated with hemonhagic syndromes in animals and with alimentary toxic aleukia (ATA) in humans.

B. Fusariumgraminearum Section : Discolor S y n o ~ ~ y ~F.n sr:o s e m Lk. emend. Snyd. and Hans. pro parte, F. rosezm Lk. emend. Snyd. and Hans. “Gramir~ear~~r~~.” Teleornorph: Gibberella zeere (Schw.) Petch. Cultlrral characteristics: Colony diameter 5-6 cm at 25°C and 2-3 cm at 30°C after 72 hr; aerial mycelium floccose, white becoming carmine red and tinged yellow; undersurface carmine red. Conidiophores: Monophialides only. Microcorzidicr: Absent. Mncrocolzidicr: Produced in orange sporodochia, falcate straight or curved with the ventral wall almost straight and the dorsal wall smoothly arched, thick-walled; basal cell distinctly pedicellate; apical cell elongated, curved, slightly constricted; 5- to 6-septate. Clnrnydospol-es:Present, sparse, formed slowly in pairs or short intercalary chains. Habitat: Distributed worldwide as a serious plant pathogen that causes root, crown, stern, and ear rot and head blight (scab) of cereals and many other hosts. Associated foodborne diseuses: F. gramirzee1rum and zearalenone produced by it cause hyperestrogenism in animals. In addition, the two trichothecenes deoxynivalenol and nivalenol are associated with emetic and food refusal syndromes in animals and with scabby grain intoxication (SGI) in humans. C.

Fusariummoniliforme

Syr1oqw1s: F. monilijorme Sheldon emend. Snyd. and Hans. pro parte, F. verticillioides (Sacc.) Nirenberg.

Marasas

538

Teleourorplr: Gibber-elln.fujikur-oi (Sawada) Wollenw. C l ! h - d clmracteristics: Colony diameter 3-4 cm at 25°C and 3-4 cm at 30°C after 72 hr; aerial mycelium floccose to powdery, white to purple, undersurface purple. Conidiophores: Monophialides only. Microcor?iclin:Present, abundant, produced in chains and false heads, ovoid or clavate with a truncate base, 0- to 1-septate. Mncroconidia: Produced in orange sporodochia by some strains, slightly curved or straight with thedorsal and ventral walls almost parallel, thin-walled; basal cell pedicellate, apical cell curved and tapering, 3- to 5- to 7-septate. Clcrnlydosyores: Absent. Hclbitclt: Occurs worldwide as a pathogen of corn and is one of the most prevalent fungi associated with corn grain intended for human and animal consumption. Associated foodborne diseases: F. moniliforrne and the fumonisin B mycotoxins produced by it cause leukoencephalomalacia in horses and pulmonary edema in pigs, and are associated with esophageal cancer (EC) in humans.

111.

MYCOTOXINS

Fusarirun species produce a stunning array of secondary tnetabolites with widely divergent biological effects. The most important naturally occurring Fusarirm mycotoxins from the viewpoint of foodborne diseases of animals and humans are discussed below.

A.

Trichothecenes

Among the large family of trichothecenes produced by Fllsariunz species, four occur naturally in cereal grains and cause mycotoxicoses in animals and possibly humans: the type A trichothecenes T-2 toxin (T-2) and diacetoxyscirpenol (DAS), and the type B trichothecenes deoxynivalenol (DON) and nivalenol (NIV). Thetrichothecenes are difficult to analyze by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) because they do not exhibit ultraviolet (UV) fluorescence (8). Consequently gas chromatography (GC) combined with mass spectrometry (MS) using selected ion monitoring (SIM) is the recommended technique for analyses of trichothecenes in cereals, foods, and feeds (8). Alternatively, capillary GC with electron-capture detection (ECD) can be used. In our laboratory, the method of choice to detect the type A trichothecenes T-2 and DAS separately is the method of Sydenham and Thiel(9), and for the typeB trichothecenes DON and NIV, the method of Scott et al. is used (10). 1. T-2 Toxin T-2 toxin [4p,15-diacetoxy-3a-hydroxy-8a-(3-1nethylbutyryloxy)12,13-epoxytrichothec9-eneI was isolated and characterized first in 1968 by Bamburg et al. from cultures of F. sporotrichioides (also called F. tricirzctunz) strain T-2 (1l), which originally was obtained from corn in France. The most important producer of T-2 is F. sporotrichioides, which frequently is referred to incorrectly in the literature as F. tricirzctrm (1, 5 , 7 , 12). The highest T-2-producing strains of F. sporotrichioides are reportedly those isolates from overwintered cereals implicated in the etiology of thehuman hemorrhagic disease alimentary toxic aleukia (ATA) in Russia (13-18).

Fusarium

539

Other Fzrsariun? species that produce T-2 are F. pone, including isolates from overwintered cereals implicated in the etiology of ATA in Russia (13-17); F. gmrninea~.lanr (19-24); F. ncunzimtzrrn (1, 25); and F. snnzbzrc~inur~~ (26, 27). In contrast to the T-2producing isolates of F. spot-otriclzioides (and F. pone) from cereals overwintered under snow in the northern hemisphere, high T-2-producing isolates of a Fusnrirrn~species resembling F. acumimztunz but distinct in several characteristics were obtained from oats and barley in South Africa in the southern hemisphere (25). The ability of this fungus to produce T-2 has been confirmed (28) and it was subsequently described as a separate taxon, F. acumii?ntrrm subspecies arnleninczun Forbes, Windels, and Burgess (29). 2. Diacetoxyscirpenol Diacetoxyscirpenol (4~,15-diacetoxy-3a-hydroxy-12,13-epoxytrichothec-9-ene) (DAS) was isolated and characterized first in 1961 from cultures of F. equiseti CMI 35100 by Brian et al. (30). The most important producers of DAS are F. equiseti ( 1, 21, 3 1) and F. pone (1, 12j, including isolates from overwintered cereals implicated in the etiology of ATA in Russia (32). It seems that, in contrast to F. sporotrichioicles, F. pone is a good producer of DAS but a poor producer of T-2 (1, 5). Other Fzrscrrizm species that produce DAS in culture are F. acunri~atu~tz (25); F. sporotrichioides (18,23); F. granzi~rectrum(23, 24); F. c r o o l ~ ~ ~ e l l(33); e ~ s eand F. sambucirlzrm (24, 26, 27, 34). In particular, some cultures of F. sarnbz~inzrrn,isolated from potato tubers in Iran and referred to as F. s ~ d ~ d ~ z ~ rSchlecht., emr are high producers (35).

3. Deoxynivalenol Deoxynivalenol (3a,7a,l5-trihydroxy-12,13-epoxytrichothec-9-e1~-8-one) (DON) was isolated and characterized in 1972 from scabby barley infected by Frrsarizrnz species in Japan by Morooka et al. (36) and from moldy corn refused by pigs in the United States by Vesonder et al. (37). Subsequently F. gmminearzun (also called F. rosezrrn) No. 117, which was isolated from the scabby barley (38),and F. grami;~enrunzNRRL 5833, which was isolated from the moldy corn, were also shown to produce DON in culture 09-41), The most important producer of DON is F. grc~mineururrr(1, 5). In Japan where scabby barley and wheat infected by F. grnminenrum have been implicated in scabby grain intoxication (SGI) in humans, many isolates of F. gr-czrni/zecrrz{r~ from scabby cereals have been reported to produce DON in culture (31, 42-44). The production of DON by cultures of F. g~-nminecrrzm.~ also has been demonstrated in many other parts of the world in isolates of this fungus from either wheat and barley (23, 45-49) or corn (39-41, 505 3 ) naturally contaminated with DON. Isolates of F. grcrmiflenrcrnz that produce DON in culture also have been obtained from many other sources (1, 54, 5 5 ) . See the section on nivalenol below for a discussion of the existence of DON and NIV chemotypes in F. grcrmirzenrzrm. Another Fz4sariunr species that produces DON is F. culnrorzrm (40, 41, 51, 56-60). 4. Nivalenol Nivalenol (3a,4P,7a, 15-tetrahydroxy-12,13-epoxytrichothec-9-en-8-one) (NIV) was isolated and characterized first in 1968 from cultures of F. sporotricllioides (also called F. Izilynle) strain Fn-2B, which was originally obtained from scabby wheat in Japan by Tatsuno et al. (61,62). Another isolate of F. sporotricllioicks that has beenreported to produce NIV is also a Japanese isolate, F. sporotrichioides (also called F. eyisplmrin) Fn-M (21, 63). Nucleotide sequencing of strain Fn-2B indicated that it is genetically more closely

540

Marasas

related to F. poae than F. sporofrichioides (64). However, O’Donnell (65) considers the above two nivalenol-producing isolates from Japan to represent a new species of Fusarium, F. hyushuense O’Donnell and Aoki (66). The most important producer of NIV is F. grmninecrrum (1, 5, 2.0, 21, 63). In Japan where scabby barley and wheat infected by F. graminenrum have been implicated in SGI in humans, many isolates of F. gramirlearurn from scabby cereals have been reported to produce NIV in culture (31,42,43). According to Ichinoe et al. (43). isolates of F. ylamirzeurmz from barley and wheat grain in Japan can be divided into two chemotaxonomic groups, the NIV chemotype, which produces NIV (and fusarenon-x), and the DON chemotype, which produces DON (and 3-acetyldeoxynivalenol). None of the isolates analyzed by Ichinoe et al. produced both NIV and DON (43). The existence of NIV and DON chemotypes in F. grmzinenrzun has been confirmed in single-ascospore isolates from perithecia of Gibberello a w e in Italy (67) and in F. gmnzinenr-unz isolates from wheat grain in Australia (68) and Korea (69). In South Africa, all the F. grnrtzir7earum isolates from corn analyzed were the NIV type (70) as was also the case in Australia (68). However, all the isolates from wheat in South Africa were the DON type (70). In Argentina (48j, the United States, and Canada (23, 71) only the DON type has been reported among F. gratnimcrrzm isolates from either corn or wheat. Other Fusarim1 species that produce NIV are F. pone (44) and F. croohwellerzse (33, 70, 72, 73).

Zearalenone B. Zearalenone [6-( 1O-hydroxy-6-oxo-tra~~s1-undecenyl)-P-resorcylic acid p-lactone] (ZEAj was isolated and characterized first in 1962 from cultures of F. graminenrum NRRL 2830 obtained from corn implicated in a field outbreak of hyperestrogenism in pigs in the United States by Stob et al. (74). The most important producer of ZEA is F. gr-ctnzinenrunl, which frequently is referred to incorrectly in the literature as F. roseurn (1, 5). Isolates of F. graminenrzur1 that produce large amounts of ZEA in culture have been obtained from moldy corn implicated in porcine hyperestrogenism in the United States and elsewhere (41, 50, 75-82). Isolates of F. gmmir1earrun from scabby barley and wheat in Japan (42, 43), Canada (23, 45.46), India (59), and South Africa (70) also have been shown to produce ZEA in culture. Isolates of F. graminearzmz that produce ZEA in culture also have been obtained from many other sources (1, 54, 55, 68, 83, 84). Other Fzmrizm species that produce ZEA are F. cz~lmorzrm(57, 59, 60, 85, 86), including isolates from cereals implicated in outbreaks of estrogenic and/or infertility syndromes in animals (56, 87, 88), and F. croolwellerrse (33, 70, 73). Several other Fzisarizrm species, including F. sporotr-ichioides and F. eqlriseti, have also been reported to produce small amounts of ZEA in culture (1, 5). The official method of the Association of Official Analytical Chemists (AOAC) for the determination of ZEA in corn (89) is based on TLC with visual detection under UV light (8). This is a rapid screening method and is not as sensitive as ZEA-dedicated procedures such as the HPLC method with fluorescence detection (90), which is currently the method of choice for the analysis of feeds and foods for ZEA and its naturally occurring derivative zearalenol.

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541

C. Moniliformin Moniliformin (potassium or sodium salt of 1-hydroxycyclobut-1-ene-3,4-dione) (MON) was isolated first in 1973 from cultures of F. nloniliforn~eNRRL 5860, which originally was obtained from corn kernels in the United States by Cole et al. (91). This strain soon lost the ability to produce MON (92j and numerous isolates of F. rnorliliforn~esubsequently were shown to be nonproducers of MON (1, 5, 93). It appears that F. nlo~ziliforme is not an important producer of MON, although some isolates apparently have the ability to produce small amounts (93). In contrast to F. r~orziliforme.many, if not most, other Fuscrrirrm species have been shown to produce MON in culture (1, 5 , 57, 93, 94). These MON-producing Fzrsnrirrrn species include F. sporotrichioides, F. nverlnceurn, F. equiseti, F. crczrrnirlatunz, F. czhzorum, F. szrbgl~rtirznrzs,F. proliferatur~l,and F. o.~ysyorzm(24, 53. 57, 95-100). Among Fusarium isolates from corn, most isolates of F. sz~bglutirzarzsproduce MON, whereas most isolates of F. nzoniliforme do not (93). By far the highest producers (up to 33 g/kg) of MON, however, belong in two uncertain taxa referred to as Bakcrrzae strains from rice and F. rzygcrmai strains from millet (94). The taxonomic position of these two uncertain taxa is being reevaluted. An analytical method for MON based on HPLC and ion-pair chromatography on a reverse-phase column (101) was used to demonstrate the natural occurrence of MON in corn for the first time (53). This procedure also is suitable for the analysis of culture material containing high levels of MON (93,94,96), but a more-sensitive HPLC procedure for the determination of MON in cereals, foods, and feeds has been developed (102).

D. Fusarin C Fusarin C (methyl-[ 1R-[la(2E,3E,5E,7E,9E),4a,5a]]-2-ethylidene1 1-[4-hydroxy-4-(2hydroxyethyl)-2-oxo-6-oxa-3-azabicyclo[3.1.0]hex-1-yl]-4,6,10-trimethyl-l l-ox0-3,5,7, 9-undecatetraenoate) (FUS C) was first isolated in 1981 from F. rnorlilifome strain M- 1 in the United States by Wiebe and Bjeldanes (103). Subsequently, this compound was isolated from cultures of F. r~~or~ilifornze MRC 826, which originally was obtained from corn in an area with a high esophageal cancer (EC) rate in Transkei in southern Africa, and the structure was elucidated by Gelderblom et al. (104, 105). The most important producer of FUS C is F. morziliforme (103-1 10). Among Frrsnrirtm isolates from corn, most isolates of F. morzilifor~~e analyzed thus far produced FUS C, whereas none of the F. szrbglzltitzarls isolates did (106, 107). Other Fz~sarizm species that produce FUS C are F. grclnzirzenrnrzl (106, 111): F. culmorunl (99, 111); F. croolr7r~elle~se (33 j; F. syorotrichioides, F. pone, F. s a ~ ~ ~ b ~ cand i m F. u ~aver~rcer~1n , (1 11). The HPLC method of Gelderblonl et al. (106) can be used for the analysis of FUS C in Fusnriznn cultures as well as that naturally occurring in corn.

E. Fumonisins The funlonisins (diesters of propane- 1,2,3-tricarboxylic acid and 2-amino- 12,16-dimethylpolyhydroxy-icosanes) were isolated first in 1988 from cultures of F. rnonizifornze MRC 826, which originally was obtained from corn in a high-EC-rate area in Transkei in south-

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542

ern Africa by Gelderblom et al. (1 12),and the structures were elucidated by Bezuidenhout et al. (1 13).Approximately 20 fumonisin analogs have been isolated thus far and characterized (112-121 j. Three of these, fumonisin B, (FBI),fumonisin B2 (FB?),and fumonisin B3 (FB,), are the major fumonisins produced in culture by F. nlorzilifornze and occurring naturally in corn and corn-based foods and feeds. The most important producer of fumonisins is F. nzor~ilifor~~le (1 12. 116-125). Most isolates of F. morlilifor-rtzefrom corn analyzed to date have been found to produce fumonisins. Other Fusarizrrr~species that produce furnonisins are F. proliferatzm (120, 123, 125), F. ryyanlcri (120, 123, 126), F. crr1thopllilwn, F. dlrmini, F. mpiforme (126), F. tllcrpsi~llrrzl (127j, and F. globosza9z (128). There are also unconfirmed reports in the literature about fumonisin production by the following three fungi: F. polyyllinlidiczrrzz (129); F. 0 . y syorzrm (130, 13 1); and Alterrzaricr crltermta f.sp. lycopersici (132). The development of analytical methods for the quantification of naturally occurring levels of fumonisins was difficult because they do not possess any chromophores and therefore do not absorb UV or visible light nor do they fluoresce (124). Theoriginal HPLC method for the determination of FBI and FB-, in culture material of F. rnor?il(folaleinvolved UV detection of their maleyl derivatives (1 12). Although this method has been used successfully for the analysis of cultures containing relatively high levels (up to 17.9 g/kgj of FBI and FB2 (133), more sensitive methods were required for the determination of fumonisins in naturally contaminated corn samples. The HPLC technique of Shephard et al. (1 34), which involves fluorescence detection of the o-phthaldialdehyde (OPAj derivatives, proved to be a suitable method and has been evaluated in international collaborative studies (135, 136). Consequently, the method of Shephard et al. (134) as modified by Sydenham et al. (137) is recommended for the quantitative analysis of FBI, FB?, and FB, in corn-based foods and feeds (138-141).

IV. ASSOCIATED FOODBORNEDISEASES A.

AlimentaryToxicAleukia

1. Background Outbreaks of alimentary toxic aleukia (ATA) (also called septic angina and endemic panmyelotoxicosis) that caused the death of hundreds of thousands of people occurred in Siberia during the closing years of World War 11. Cereal grains (wheat, rye, barley, oats, and millets) that could not be harvested because of shortages of workers were allowed to overwinter under snow in the fields. The grain that was harvested in the spring, following alternating freezing and thawing conditions, was consumed by humans in the form of bread because of acute food shortages and proved to be extremely toxic (1, 5 , 6, 14- 16, 142-152). 2. Clinical SignsandPathologicalChanges The characteristic clinical signs of ATA are nausea, vomiting, necrotic oral lesions, dermatitis, bloody diarrhea, and extensive hemorrhages in manyorgans. Gross lesions are characterized by hemorrhages in the brain, lungs, heart, musculature, and the entire digestive tract. Necrotic lesions are found in the mouth, throat, pharynx, esophagus, and along the entire gastrointestinal tract. The lymph nodes are enlarged and edematous. The bone marrow is severely depleted, and damage to the hematopoietic and lymphopoietic systems is

Fusarium

543

reflected in drastic hematological abnormalities, particularly leukocytopenia and thrombocytopenia, and immunosuppression. Histopathologically, the disease is characterized by hemorrhage, thromboses, and necrosis in various organs: atrophy of the lymph nodes, thymus, and spleen: devastation of the bone marrow to the extent that the myeloid tissue appears empty; sepsis; and severe hematological abnormalities. The pathological changes characterized by cellular destruction and karyorrhexis in the hematopoietic and lymphopoietic systems are said to be radiomimetic, that is, similar to the effects of radiation from an atomic explosion (15, 16, 142148). The massive hemorrhage that reportedly occurs in human victims of the chemicalwarfare agent known as yellow rain has been attributed to Fzrsnrium mycotoxins similar to those that caused ATA in Russia (153-155). Many of the characteristic features of ATA also have been recorded in human cancer patients treated with DAS (anguidinej as a chemotherapeutic agent (156-160). These include nausea, vomiting. diarrhea, fever and chills, headaches, skin erythema with burning sensation; such neurological signs as confusion, somnolence, disorientation, hallucinations, and psychomotor seizures; and hematological abnormalities such as leuko- and thrombocytopenia and myelosuppression. 3. AssociatedFungi and Mycotoxins Abundant circumstantial evidence suggests that the outbreaks of ATA in Russia were caused by T-2 (and possibly DAS) produced by F. syorotriclzioides (and possibly F. yocre) colonizing overwintered cereal grains. Both F. syorotrichioides and F. yoae have low optimal temperatures (6-12°C) for toxin production, and maximal toxicity in culture is obtained under conditions of alternating freezing and thawing (13- 17). Russian scientists implicated the steroidal glycosides sporofusarin and poaefusarin, and their aglycones sporofusariogenin and poaefusariogenin, isolated from F. syor-otrichioides and F. pone, respectively, as the fusariotoxins responsible for ATA (16 1, 162j. However, the striking pathological similarities between ATA and the lesions caused in experimental animals by a group of Fusarium mycotoxins known as trichothecenes led to suggestions that ATA more likely was caused by these trichothecenes than by the steroidal compounds implicated by the Russian scientists (149, 163). Further evidence for this hypothesis was provided by the reports that F. sporotr-ichioides NRRL 3510 isolated from millet involved in ATA (20) and “authentic toxic isolates of F. pone and F. sporotrichioides obtained from the USSR’’ (164) produced several trichothecenes, including T-2, in culture. Moreover, the major toxic component of an authentic sample of poaefusarin isolated from F. poae in Russia was identified as T-3 (165, 166). Findings that more than 95% of F. syorotrichioicles and F. yoap strains from cereals associated with clinical ATA produced T-3 in culture, together with the reproduction of the characteristic pathological changes of ATA in cats with crystalline T-2, have led to the conclusion that T-2 primarily was responsible for the outbreaks of ATA in Russia (13-17, 167, 168). This conclusion would be strengthened considerably if it were possible to detect T-2 toxin (and DASj in samples of toxic overwintered cereals that were associated with the actual outbreaks of ATA, if such samples are still in existence anywhere. Both T-2 and DAS have been reported to occur naturally in the range of 0.05-5.0 pg/g in feed samples associated with outbreaks of hemorrhagic syndromes in animals (56, 169-175). The limited amount of published information available on the natural occurrence of T-2 and DAS in feeds and foods maybe due to the chemical difficulties in analyzing for these trichothecenes, which requires such sophisticated procedures as GC/

544

Marasas

MS and capillary GC (8). However, many authors have employed such sophisticated procedures to screen for T-2 and DAS with negative results (175- 182). These negative findings imply that T-3 and DAS may not be commonly occurring natural contaminants of cereal grains. 4. AnimalModels Cultures of F. sporotrichioides (and F. pone) isolated from the toxic overwintered grain associated with outbreaks of ATA proved to be highly toxic to a variety of experimental animals. These cultures caused many of the characteristic clinical signs and pathological changes of ATA in experimental animals, including cats, cattle, dogs, guinea pigs, horses, monkeys, mice, pigeons, pigs, poultry, rabbits, rats, and sheep (15, 16, 142-148, 183). The typical lesions of ATA were reproduced successfully for the first time by Russian scientists during the 1940s in cats dosed with toxic overwintered grain infected by F. sporotrichioides and with pure cultures of the fungus (15, 147, 148, 168, 183). Several authors have subsequently confirmed that a satisfactory experimental model of ATA can be obtained in cats dosed with culture material of F. sporotrichioides (14-16, 142-148, 168) or with crystalline T-2 isolated from F. sporotrichioides (14, 167, 168, 184). A particularly convincing model of ATA was also obtained in monkeys dosed with toxic grain, according to Rubenstein (148). The monkeys dosed with toxic grain vomited, had diarrhea, and lost appetite. Within 20-30 days petechial hemorrhages developed on the skin and hematological examination revealed progressive leukopenia, relative lymphocytosis, anemia, and thrombocytopenia-all characteristic findings of ATA in humans (145). Some of these characteristic clinical signs and pathological changes of ATA have also been induced in monkeys with pure DAS (185), semipurified T-2 toxin (186), and pure T-2 toxin (187). Further support for the hypothesis that ATA is a mycotoxicosis caused by T-2 and DAS produced in cereals by F. spomtrichioides (and F. pone) is provided by sporadic outbreaks of hemorrhagic syndromes in animals. The disease is associated with the ingestion of moldy cereals, particularly corn (moldy corn toxicosis), and is characterized by necrotic oral lesions, bloody diarrhea, hemouhagic gastroenteritis, extensive hemorrhages in many organs, and immunosuppression in such animals as cattle, pigs, and poultry (1, 5 , 6, 149, 150, 163, 166, 169, 188-195). The most toxic fungus isolated from cereals implicated in outbreaks of hemorrhagic syndromes in many parts of the world proved tobe F. syorotriclzioicles (1). Many of the characteristic clinical signs and pathological changes observed in field outbreaks of hemorrhagic syndromes are very similar to those of ATA and have been reproduced experimentally with cultures, extracts of cultures, and/or pure T-2 and DAS isolated from cultures of toxic strains of F. spor-otrichioides (1). These findings leave little doubt that hemorrhagic syndromes such as moldy corn toxicosis in animals are naturally occurring animal models of ATA in humans. No convincing evidence has been presented that either T-2 or DAS is carcinogenic in experimental animals, and the International Agency for Research on Cancer (IARC) concluded in 1993 that “toxins derived from Fz~snrizm~ sporotrichioides are not classifiable as to their carcinogenicity to humans (Group 3)” (196). Both of these compounds, as well as several other trichothecenes, are nonmutagenic to Scrlrrzorwllct in the Ames test (197-200). On the other hand, it has been claimed that T-2. causes chromosomal aberrations in cultured human peripheral blood lymphocytes (201) and causes basal cell hyperplasia, dysplasia, and increased mitoses in cultured human fetal esophagus (202).

Fusarium

545

On the basis of these results, Hsia et al. (202) suggested a “possible relation” of T-2 with human esophageal cancer (EC). However, T-2 could not be detected in corn and wheat from Linxian County, a high-incidence area of EC in northern China ( 180, 203), or in corn from Transkei, a high-incidence area in southern Africa (18 1). A variety of non-dose-dependent tumors have been reported in rats administered T-2 alone or in combination with nicotinamide (204). In contrast, no papillomas were induced in mice by the repeated topical application of either T-2 (205,206)or DAS (206). Chronic feeding experiments with T-2 in trout and rats (205) and mice (207-209) and with DAS in rats (185) also failed to induce papilloma or carcinoma. Benign pulmonary and hepatic adenomas were increased significantly in mice fed a diet containing 3 yg/g T-2 for 16 months (2091, but no carcinoma was found. It is concluded that there is little or no evidence, on the basis of either frequency of natural occurrence in human foodstuffs or experimental carcinogenesis, for the oftenrepeated claims in the literature that T-2 and DAS may be carcinogenic to humans (201, 202, 204, 210-218j.

5. Control It is concluded from abundant circumstantial evidence discussed above that ATA is a mycotoxicosis caused by the type A trichothecenes T-2 and DAS produced in cereals by F. sporotr-ichioides (and F. pone). It follows that moldy cereals, and particularly cereal grains overwintered under snow, should not be consumed by humans or animals. Thus it may seem that ATA is only of interest to medical historians and has been “eradicated” in Russia by “intensive education programs in rural communities’’ (219). However, F. sporotr-ichioides has not been eradicated and undoubtedly will continue to produce trichothecenes in cereal grains under favorable climatic conditions. It is interesting that Russia is the only country in the world where a legal tolerance level for T-2 (100 yg/g in grains) has been proclaimed (220). There is aneed for continued vigilance to prevent a recurrence of outbreaks of ATA, particularly under conditions of political instability and famine in countries with cold climates in the Northern Hemisphere.

B. ScabbyGrainIntoxication 1. Background Outbreaks of scabby grain intoxication (SGI) of humans attributed to the consumption of bread made from cereal grains affected by head-blight or scab caused by F. graminenr’unz have been reported under a variety of names in different countries since the 19th century. In 1891 Woronin described an outbreak of Tnumelgetreide (staggering grain) intoxication in eastern Siberia and implicated bread made from scabby rye (221). Subsequent outbreaks in Russia following the consumption of “drunken” or “intoxicating” bread were recorded during seasons when widespread epiphytotics of head-blight occurred in association with abnormally wet and cool seasons (222, 223). Similar cases of human intoxication due to scabby barley also have occurred in China (224) and in Korea (225j. In Japan, sporadic epiphytotics of crkcrkubi-byo (red mold disease or scab) of wheat, barley, oats, rye, and rice caused by F. gr-nnzirzenrz~rncan affect more than one-third of the national production and are frequently associated with outbreaks of a human mycotoxicosis (63, 151, 152, 176, 226).

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During 1987, several thousand people in the Kashmir Valley of India were affected by a gastrointestinal disorder. Epidemiological investigations together with laboratorybased studies implicated the consumption of bread made from scabby wheat (227). 2. Clinical Signs andPathologicalChanges The characteristic clinical signs of foodborne SGI in humans are anorexia, feelings of fullness of the abdomen, abdominal pain, nausea, vomiting, irritation of the throat, diarrhea, bloody stools, headache, vertigo, shivering chills, pyrexia, reduction in body weight, and physical weakness (63, 151, 152, 176, 221-227). The clinical and epidemiological aspects of the outbreak of SGI in the Kashmir Valley in India were described in detail by Bhat et al. (227). The number of persons affected reached “several thousands” and came from all age groups and socioeconomic strata. The disease was characterized by mild to moderate abdotninal pain or a feeling of fullness of the abdomen within 15 min to 1 hr of consuming local bread made of refined wheat flour or unleavened homemade bread prepared from wheat flour bought at the local market. All 97 affected persons interviewed had the above characteristic symptoms of abdominal pain or a feeling of fullness of the abdomen. In addition 63% of them noticed irritation of the throat, 39% had diarrhea, 5% had blood in thestools, and 7% had vomited. Other minor symptoms included nausea and flatulence. Such secondary infections as upper respiratory tract infections were reported in children who had consumed considerable amounts of wheat preparations for more than a week. The disease normally lasted as long as the incriminated bread was being consumed, generally 1-2 days. Notable events preceding the disease outbreak were unseasonable rains at the harvest time of wheat in Kashmir resulting in a large portion of the standing wheat crop becoming rain damaged and blighted. It is evident that some of the clinical signs of SGI are similar to those of ATA, thus suggesting common etiological factors. Although the pathological changes of SGI have never been described. it appears to be a much milder, nonfatal syndrome that lacks the characteristic hemorrhage and severe hematological abnormalities of ATA. In the case of SGI, the disease may in fact be self-limiting because of the reduced food intake due to the feeling of fullness soon after consumption of the incriminated bread (237). 3. AssociatedFungiandMycotoxins Convincing circumstantial evidence suggests that SGI is caused by the trichothecenes DON and NIV produced by F. gr.mrzinetrr.um (and F. cdmor-m) in scabby cereal grains, primarily wheat, barley, and rye (1). Proof that these two trichothecenes occur naturally in grain samples actually incriminated in outbreaks of the disease recently was provided following outbreaks of SGI in Shanghai, China in 1976 (224), in Korea in 1983 (225) and in Kashmir, India in 1987 (227, 228). A human toxicosis with the typical clinical signs of SGI (nausea, dizziness, vomiting, abdominal pain, and diarrhea) in Zhejiang, China has, however, been attributed to moldy rice colonized by “Frrsariu~nheterosyorlrnl and F. grami11e~lr-14~~’ ’ and contaminated with T-2 toxin at levels of 0.18-0.42 pg/g (229). The scabby wheat from Shanghai contained 9.76 yg/g DON and 0.74 pg/g NIV, as well as 0.03 yg/g ZEA (224). In scabby barley from Korea, the levels of DON, NIV, and ZEA were 0.003-5.840, 0.017-3.002, and 0.002-1.581 yg/g, respectively (225). Analyses of samples of wheat, whole-wheat flour, and refined-wheat flour implicated in the outbreak in Kashmir revealed the presence of DON (0.3 1-8.38 pg/g) and NIV (0.030.1 yg/g) as well as T-2 (0.55-4.0 pg/g) and 3-acetyldeoxynivalenol (0.64-2.49 pg/g)

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(228). The production of DON in culture by two isolates of F. gran1iizearrrm and four isolates of F. cr{lmorum from the wheat and wheat flour from Kashmir has also been demonstrated (59). In Japan, where outbreaks of SGI caused by scabby cereal grains occur sporadically, the presence of DON and NIV has not been demonstrated (to my knowledge) in samples from actual outbreaks of the disease. However, the natural occurrence of DON (0.02-7.3 pg/g) and NIV (0.020-7.0 pg/g) in scabby barley and wheat infected predominantly by F. grcminearunz in Japan is well documented (44, 176, 226, 230, 231). In the Japanese samples of barley and wheat analyzed by these authors, DON and NIV frequently were found to co-occur and sometimes occurred with low levels of ZEA. In culture, however, Ichinoe et al.found that isolates of F. grnnzinenrum from barley and wheat grains could be divided into two chemotaxonomic groups, the NIV chemotype, which produces NIV (and fuserenon-x), and the DON chemotype, which produces DON (and 3-acetyldeoxynivalenol) (43). No cross-production of the two types of trichothecenes was observed in any of the 114 isolates analyzed, but both chemotypes produced ZEA. These results are in agreement with those of Yoshizawa et al., who found that 46.5% of F. grcmi~enrz~m isolates from southern Japan were the NIV type and 44.8% the DON type (42). In central Japan, however, 93% of the isolates were the NIV type and only 7% the DON type (31), whereas in northern Japan only the DON type has been found (44). In contrast to these results that F. graminenrum isolates from Japan produce either DON or NIV but never both, Yoshizawa et al. found two of 61 strains that produced both (42) and the production of both DON and NIV by some Japanese isolates of F. grelnzinecmm recently has been confirmed (71). On the basis of their findings, Yoshizawa et al. concluded that the natural occurrence of DON and NIV in wheat and barley in southern Japan could be attributed to infection of these cereals by strains of F. grnnzi~zecrrumcapable of producing either DON, NIV, or both (42). Differences in the geographical distribution of DON and NIV chemotypes of F. granzinearum have important implications with respect to the natural contamination of cereals with the trichothecenes produced by them. In southern Japan (Kagawa), where the DON and NIV chemotypes are represented almost equally (42, 43), approximately equal levels of DON and NIVhave also been found in most wheat and barley samples analyzed (176). In central Japan (Saitama), where the NIV chemotype predominates (31,43), higher levels of NIV than DON have been reported in some samples of wheat and barley (331). In northern Japan (Hokkaido), only the DON chemotype of F. grnminearzrm has been isolated from scabby wheat (44j, and the levels of DON occurring naturally in wheat and barley were approximately 20-fold higher than those of NIV (230). The low level of NIV present in scabby wheat in Hokkaido has been attributed to the production of this trichothecene by F. poae (44, 232). Another Fuscrrium species isolated from scabby cereals in Japan that produces NIV (together with T-2 and fusarenon-x), is F. sporotrichioicles (also called F. niwle) Fn-2B ( 1, 61, 62). This trichothecene-producing strain originally was referred to as F. nivale, was subsequently identified as F. sporotriclzioides by Marasas et al. (1) and as F. pone by Mu16 et al. (64), but is considered by O’Donnell (65) to be a distinct, new species of Fusnrirun, F. kydzuerrse (66). Yet another Fusarium species that has been reported to produce NIV in culture is F. croohwellense (33, 70, 72, 73, 232). Taxonomically F. crookwellense is related closely to F. graminenruln and F. crdmorum and easily can be confused with F. graminenrum (233), particularly since it also is associated with scabby wheat in South Africa (234),

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Japan (232), and Poland (235) as well as with ear rot of corn in Poland (236). The data on the worldwide contamination of cereals by NIV and DON (231) have to be interpreted in the light of DON production by the DON chemotype of F. grcrrrIinenrm1 and by F. culmo~.u~~z, and NIV production by the NIV chemotype of F. grcminearutzI, F. yoae, F. syorotr-ichioides (or F. kyushuense), and F. croohvellerzse. The worldwide contamination of cereal grains intended for human consumption, including wheat, barley, oats, rye, and corn, with DON, NIV, and ZEA has been demonstrated unequivocally (231). In addition to Japan, this also applies to other countries where outbreaks of SGI have been recorded, i.e., Russia (231, 237, 238), China (180, 23 1, 237), Korea (225, 231, 2391, and India (227, 228). The accumulated data revealed definite geographical differences in the level and frequency of DON and NIV in wheat and barley. Similar to the situation in southern Japan, levels of NIV tended to be higher than DON in cereal grains from Russia (23 1, 237) and Korea (225, 23 1, 239). Inmost other countries, such as Argentina, Canada, China, Germany, and Poland, the pattern of contamination was similar to that in northern Japan, that is, levels of DONhigher than NIV (23 1). In the United States, however, cereals frequently contain DONand ZEA (175, 177, 178). but notNIV (55, 240). The same applies to Canada (47, 241) with the exception of one report of trace amounts of NIV in wheat (242). In South Africa, DON and NIV have been reported to co-occur in scabby wheat, with levels of DON 9.3 times higher than those of NIV and no ZEA could be detected in any of the samples (243). The occurrence of DON, NIV, and ZEA has also been reported in homegrown corn from high-EC-risk areas in Transkei, southern Africa (53, 181, 244). Subsequently, it was claimed that corn consumed in a high-risk area for EC in China was contaminated more frequently and at higher levels with DON, NIV, and ZEA than in a low-risk area (180, 203, 245, 246). Contrary results with respect to the incidence of F. gr-nrnirrenrwnr and these three mycotoxins, however, have been obtained in high- and low-EC-risk areas in Transkei (181, 247). Moreover, there is little or no evidence from animal models (see next section) that any of these three F. grnnlirleumrn mycotoxins is carcinogenic. 4. Animal Models Sporadic field outbreaks of feed refusal in pigs, sometimes associated with vomiting, are caused by the infection of cereals, particularly corn and barley, by F. grumirlennrrr~in the midwestern United States, Japan, Korea, and elsewhere (37, 39, 150, 151, 177, 225, 348j. The reduced palatability of the scabby grain is reflected in decreased weight gains and slower growth rates of pigs and is associated with nausea and emesis in animals forced to eat the grain by starvation. These feed-refusal and emetic syndromes appear to be the animal equivalents of SGI in humans. Outbreaks of feed refusal and emesis in pigs occur in certain seasons in association with epiphytotics of barley and wheat scab or ear rot of corn caused by F. grcrminenr-unl. Such outbreaks occurred in the midwestern United States during 1928 (249), 1965 (250), 1972 (37, 39), and other years. Massive epiphytotics in the United States and Canada during 1991- 1996 have resulted in scab of wheat and barley being referred to as a “reemerging disease of devastating impact” (251). Analysis of the climatic conditions associated withthe 1965 and 1972 outbreaks revealed that optimal conditions for infection of corn by F. gl-nnrinetrrzrn~were at least 9 days of rain and a mean temperature below 25OC during silking of corn (252). Low temperatures with concomitant high rainfall and humidity during the summer of 1980 in Ontario, Canada also favored the infection of

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wheat by F. grn~ninearzlmand resulted in outbreaks of feed refusal, emesis, and death in pigs (10, 45-47, 241, 253, 254). The devastating outbreaks of wheat and barley scab in the United States and Canada during 1991- 1996 were also associated with very high rainfall as well as other factors such as increases in minimum tillage and short rotation intervals (25 1). The emetic principle, referred to as vomitoxin, was isolated and characterized by Vesonder et al. from naturally infected corn that caused vomiting in pigs in 1972 in Ohio (37). The same trichothecene was isolated from Fusnriunz-infected barley in Japan in 1972 and referred to as Rd toxin by Morooka et al. (36). This compound was called deoxynivalenol (DON) by Yoshizawa and Morooka (38), who determined that the structure was identical to that proposed for vomitoxin (37). The level of DON in the corn from Ohio was reported to be 40 yg/g by Vesonder et al. (39), who also demonstrated that pure DON caused feed refusal as well as emesis in pigs. On the basis of these findings, these authors concluded that DON wasresponsible for the feed refusal as well as the emetic syndromes and that pigs will refuse corn containing high levels of DON, but will consume sufficient quantities of corn containing lower levels to elicit the emetic response (39). The same situation could apply in outbreaks of SGI in humans such as the one in India in which all affected persons reported a “feeling of fullness of the abdomen” (equated with food refusal), but only 7% vomited (227). Feed refusal as well as emesis have been reproduced with pure DON experimentally in pigs (39, 226, 255-258). DON also has caused feed-refusal activity in mice and rats (226, 255, 259) and emetic activity in cats, dogs, and ducklings (199, 226, 260, 261). Other clinical signs and pathological changes reported to be caused by DON include radiomimetic cellular damage in the actively dividing tissues of cats (260), extensive ecchymotic hemorrhaging throughout the carcass and irritation of the upper gastrointestinal tract in chickens (262), and hemorrhage and necrosis of the gastrointestinal tract, bone marrow, and lymphoid tissues in mice (263). The oral LD5”of DON in mice is 78 mg/kg (263). DON has been reported to occur naturally at levels ranging from 0.025 to 50.5 pg/g in corn and other feedstuffs associated with field outbreaks of the feed-refusal and emetic syndromes in pigs in many countries (37, 39,45-47, 51, 52,56, 177, 240,254, 256, 261). The occurrence of DON, together with NIV and ZEA, in wheat or barley incriminated in outbreaks of SGI in hutnans in China (224), Korea (225), and India (227, 228). also has been reported. The co-occurrence of DON with NIV and ZEA in corn, barley, and wheat has been found worldwide (see “Associated Fungi and Mycotoxins,” Sect. IV.B.3). This raises the question whether feed refusal and emesis are dose-dependent responses to DON alone (39), or whether NIV and/or ZEA also is involved. Feed refusal in pigs in fact has been demonstrated to be much greater for naturally infected corn than for feeds with equal concentrations of pure DON, thus indicating the involvement of such additional factor(s) as other mycotoxins or synergism between mycotoxins (256-258, 264). A possible other mycotoxin is NIV, which is much more toxic than DON to mice (oral LD5”is 38.9 mg/kg) (265), cultured cells (266, 267), and rabbit skin (268). NIV causes emesis in ducklings at much lower concentrations than DON (269). NIV also causes the characteristic effects of trichothecenes in mice, including feed refusal, changes in the bone marrow, and severe leukopenia (265, 270). Thus, NIV may contribute to the feedrefusal and emetic effects of naturally contaminated cereals. However, corn contaminated with DON and ZEA and refused by pigs in the United States did not contain NIV (177, 240).

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Another possible candidate is ZEA, but ZEA is not a feed-refisal factor in mice and does not enhance the feed-refusal activity of DON in mice (259). Aslight enhancement by ZEA of the effects of DON in pigs, however, has been reported (271). Even so, it is unlikely that ZEA is involved in the feed-refusal and emetic syndromes in animals or SGI in humans. It should be kept in mind, however, that ZEA occurs worldwide in cereal grains infected with F. gt-crmirzecrrrrm (1, 175, 182, 231, 254, 272) and causes an estrogenic syndrome in pigs and other animals characterized by vulvar and mammary swelling, uterine hypertrophy, nymphomania, pseudopregnancy, and infertility in females and mammary swelling, testicular atrophy, and reduced libido in males (1, 6, 75-82, 166, 254, 273276). Similar clinical signs of hyperestrogenism in humans have been attributed to foodborne ZEA in Puerto Rico (277,278), in southern Africa (279,280) and in Hungary (281). In Puerto Rico, an outbreak of precocious pubertal changes in thousands of young children was characterized by premature pubarche, prepubertal gynecomastia, and precocious pseudopuberty and it was claitned that ZEA or a derivative was present in the blood of some of these patients (277, 278). In southern Africa, gynecomastia with testicular atrophy in rural males has been associated with the occurrence of ZEA in homegrown corn used as the staple diet (279, 280). In Hungary, cases of early thelarche in children have been associated with the presence of ZEA in “health foods’‘ and in the sera of patients (281). DON is not mutagenic (198, 282) or teratogenic (283, 284), but chronic toxicity and carcinogenicity data are not available. NIV has been reported to be mutagenic in yeast cells (631, and it has been claimed that both NIV and DON are genotoxic in mammalian cell cultures (245), but NIV is not carcinogenic in mice (265). ZEA is not mutagenic in the Arnes test (197, 198) and is not carcinogenic in Fisher rats (285) or in Wistar rats (286, 287). In B6C31 mice, however, ZEA caused an increased incidence of pituitary adenomas in males and females with the possible progression to malignancy indicated by the presence of three pituitary carcinomas (285). No cervical carcinomas were present in these mice. On the basis of these data, the IARC concluded in 1993 that there is inadequate evidence for DON and NIV and limited evidence for ZEA with respect to carcinogenicity in experimental animals, and that the “toxins derived from Fusnl-iunzgl-~11rzi1zecrrzrrn,F. crrlnzorzrnz and F. croohvellemse are not classifiable as to their carcinogenicity to humans (Group 3)” (273). Consequently the numerous suggestions in the literature that ZEA may be responsible for the high incidence of human cervical cancer in Africa (288-290) and for the variable incidence of neoplasms in the sex organs of humans (210-217) are not supported by the IARC evaluation.

5. Control The best way to avoid clinical signs of SGI (and possibly of hyperestrogenism) in humans is elimination from the human food chain of scabby cereal grains infected by F. grmniwar’zrm and other toxigenic Fmarirmz species and contaminated with DON, NIV, and ZEA. This is easier said than done and various ways of avoiding the problem are in use such as cultural practices, grading regulations, and processing (264). The most promising method to control scab is the breeding of cereals resistant to F. gt-unzirwnrzrnz (251, 264). Tolerance levels for DON in wheat have been established in Canada, the United States, Romania, and Russia (220). The guideline tolerance levels in Canada are 2.0 pg/g in uncleaned soft wheat used for nonstaple foods, except that intended for use in infant food when the level is 1.0 pg/kg. In the United States, the tolerance levels for DON are

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on an advisory level with no legal force and are 2.0 pg/g in wheat intended for milling for human consumption and 1.0 pg/g in finished wheat products (220). Following the outbreak of wheat scab in the United States i n 1993, the U.S. Food and Drug Administration (FDA) guideline “level of concem” of 2.0 pg/g DON in harvested wheat grain was scrapped, but the guideline of 10 pg/g in finished wheatflourwas maintained (251). This change in the FDA “level of concern” shifted the economic consequences of DON contamination from the buying point of wheat grain to the selling point of wheat flour (25 1). A risk-assessment study of ZEA in Canada was done in 1987 by Kuiper-Goodman et al. (254). A tentative tolerable daily intake (TDI) of 0.1 pg/kg body weight/day was calculated. The estimated daily intake (EDI) of ZEA from corn products in young children (aged 1-4 years) was calculated as 0.05-0.10 pg/kg body weightlday. Although this value is very close to the tentative TDI, no adverse health effects were anticipated from exposure to ZEA in corn in Canada. Consequently, no regulatory action was recommended. Tolerance levels for ZEA, however, have been established in Brazil (0.2 pg/g in corn) and in Russia (1 .O pg/g in grains, fats, and oils), according to Van Egmond (220).

C.

EsophagealCancer

1. Background The etiology, epidemiology, pathology, cytology, diagnosis, and treatment of carcinoma of the esophagus in humans is a vast topic that is not within the scope of this chapter to review (291-294). Suffice it to say that the cause or causes of esophageal cancer (EC) still are unknown, the enigmatic geographical distribution of EC among widely different population groups remains unexplained, the methods of early detection are poorly developed and the prognosis remains poor, and EC remains one of the most important causes of cancer death in many population groups in different parts of the world. The geographical distribution of EC is enigmatic because the disease occurs worldwide with a marked variation in incidence, even within relatively small geographical areas (295, 296). In particular, extremely high incidence rates of more than 50 per 100,000 per annum have been recorded in three areas of the world, i.e., the southwestern districts of Transkei in southern Africa (295, 297, 298), Linxian County of Henan Province in northern China (296, 299,300), and the Caspian littoral of Iran (301-303). In the United States, the age-adjusted incidence rate of EC is less than 5 per 100,000 per annum, but the rate in blacks is 3.5 times higher than that of whites (304). Among blacks in the United States, the highest incidence and mortality rates of EC occur in a relatively small geographical area around Charleston, South Carolina (305-307). Many possible etiological factors in EC have been proposed and no attempt is made to review this voluminous literature here. Attention is focused on only one possible factor, i.e., the contamination of corn with fumonisins produced by F. rnonil{fome. This does not imply that the author considers foodborne fumonisins as the sole cause of EC. To the contrary, I fully subscribe to the multifactorial and multistage hypothesis (308) for EC and believe that totally different factors may be involved in different parts of the world. In addition to the well-established role of smoking and drinking in developed countries, the involvement of specific vitamin and trace element deficiencies in the high-risk populations with a very poor socioeconomic status in Africa, China, Iran, and the United States is considered to be very important (218, 303, 309-321).

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In the high-risk population in Transkei, the coexistence of deficiencies in vitamin A, B,,, E, and folic acid (315, 316) and selenium (317) with exposure to fumonisins in the corn staple diet (181, 247, 322-325) is well established. The consumption of corn as a dietary staple and/or as a home-brewed alcoholic beverage has been implicated as a risk factor for EC in Africa (181, 309, 312, 322-334), China (299, 300, 318, 335), Italy (336, 337), and the United States (307). This association is with corn (maize, Zea rmys L.) and not sorghum as was assumed erroneously by Morton (338). The contamination of corn with F. morzilifornze and fumonisins, and the implications for human and animal health, are discussed in this chapter.

2. Clinical SignsandPathologicalChanges The high mortality rate of EC is related to the fact that symptoms first appear when the patient cannot swallow, and by that time, the tumor is large, invasion of the esophagus and surrounding tissues is advanced, and prognosis is poor (294). Some progress has been made relatively recently in the early detection of EC by the cytological examination of esophageal ballon and brush biopsies (339-344). 3. Associated Fungi andMycotoxins One of the most prevalent fungi associated with corn intended for human and animal consumption all over the world is F. rzlonilifome (1-3, 247, 328, 345). This fungus was described first (as Oospora verticillioicles Saccardo) in 1881 when it was isolated from moldy corn kernels implicated as the cause of pellagra in Italy (3). The same fungus was incriminated in 1904 as the cause of moldy corn toxicosis of animals in the United States and described as Fz4saril.m monilifornze by Sheldon (346). Thus F. nzorziliforme has been implicated by association as the cause of human and animal diseases since its original description in the 19th century. However, scientific proof that F. monil(forme is highly toxic and carcinogenic and presents a serious threat to human and animal health was obtained only during the latter part of the twentieth century. An important role in the elucidation of the medical and veterinary relevance of F. modifornze was played by a tnultidisciplinary research team at the South African Medical Research Council, in collaboration with scientists at the Veterinary Research Institute, Onderstepoort, South Africa, and the Council for Scientific and Industrial Research (CSIR), Pretoria, South Africa. This research, which culminated in the isolation and characterization of the fumonisins and the detnonstration that these metabolites of F. monilifor-me are important foodborne toxins and carcinogens, is reviewed briefly in this section. Our involvement with the toxicology of F. mo11iZ2:formecommenced in July 1970 when some horses in South Africa died of a neurotoxic disease, known as leukoencephalomalacia (LEM), characterized by liquefactive necrotic lesions in the white matter of the cerebral hemispheres (347). At that time, it was known that field outbreaks of LEM in horses and donkeys occurred regularly in many countries (e.g., Argentina, China, Egypt, and the United States) and that thousands of horses had died of the disease in the United States during some seasons (1). Although it was demonstrated as early as 1902 that the disease was caused by the consumption of moldy corn (348), the specific causative organism of LEM was still unknown in July 1970. F. n~onilifor-nzewas isolated as the predominant fungus from the corn associated with the field outbreak in South Africa during 1970. We had started to dose pure cultures of this fungus to horses when Professor B. J. Wilson of the University of Tennessee reported atthe International Sytnposium on Mycotoxins in Human Health in Pretoria

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during Septetnber 1970 that he had reproduced LEM experimentally with an Egyptian isolate of F. modiforme in a donkey (349). The causative role of F. nlorziliforme in equine LEM subsequently was confirmed with several South African isolates of the fungus and the pathognomonic pathological changes were described in detail (347, 350, 351). We also established that the characteristic brain lesions of LEM were induced by relatively small doses of F. molzilifolme culture material whereas higher doses resulted in a fatal hepatosis. The occurrence of bile duct proliferation, increased numbers of mitotic figures, multinucleated hepatocytes, and large, bizarre, hyperchromatic nuclei in thelivers of these horses (347, 350) were the first indications that F. nzorziliforme may be a carcinogenic fungus. Subsequently, we became involved in a study of the possible role of fungal toxins in the etiology of human EC in the Transkei region of South Africa. The incidence rate of EC in men as well as women in the southern part of Transkei is among the highest in the world, whereas the rate in the northern part of Transkei is moderate to low (297, 298). The staple diet in both areas is homegrown corn. Detailed mycological analyses of homegrown corn samples intended for human consumption from different EC rate areas in Transkei during six seasons over the period 1976-1989 revealed a statistically highly significant correlation between the incidence of F. moniliform in corn and EC rate (247, 328, 329). In these studies, the correlation was between F. monilifornze and EC rate based on death certificate cancer registry data. The association also was shown to exist between F. modiforme contatnination of corn and premalignant esophageal cytological changes in living individuals (329). In addition to the obvious epidemiological advantages of studying living individuals rather than death certificate statistics in the model, the development of esophageal brush cytological screening methods has presented real possibilities of the early diagnosis and treatment of EC in remote areas in southern Africa (343). This possibility has been enhanced further by recent findings that deficiencies of specific vitamins (vitamins A, BI2. E, and folic acid) (315, 316) and trace elements (selenium) (3 17) in the blood of highrisk populations in Transkei also are correlated with esophageal cytological abnormalities. In continuing investigations on the toxicology of F. rnonillfot-me isolates from corn associated with field outbreaks of LEM in horses, isolates from corn in high-EC-risk-areas in Transkei were included. One of these Transkeian isolates, designated F. rnorziliforrne MRC 826, soon was found to cause LEM experimentally in horses (352) and to be highly hepato- and cardiotoxic to rats and other experimental animals, including primates (353355). In 1984, culture material of F. morzillfome MRC 826 was proven to behepatocarcinogenic in rats and to cause primary hepatocellular carcinoma as well as cholangiocarcinoma (356, 357). Although chemical investigations on the mycotoxin(s) produced by F. nzolzillforuze were commenced in South Africa in July 1970, the chemical nature of the metabolite(s) responsible for LEM still hadnotbeen elucidated whenthe fungus was shown to be carcinogenic in 1984. The isolation and chemical characterization of the mycotoxin(s) and carcinogen(s)produced by F. nlonilifornze then became a matter of paramount importance. The urgency of the matter was accentuated even further when researchers in the United States reported that corn implicated in field outbreaks of equine LEM and naturally infected by F. rnonilifonne also was hepatocarcinogenic in rats (358). The pathological changes in these rats were identical to those that previously had been described in rats fed pure cultures of F. vnorziliforvne MRC 826 (356). This provided evidence that the unidentified carcinogen(s) produced by F. monilifornw not only was present in culture

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material of F. wzorzilfonrze MRC 826, but also occurred naturally in corn in the United States. The taxonomic confusion surrounding F. rnorzilifonne has resulted in chaos in the literature regarding the mycotoxins produced by this species (1, 3, 359). As an example, the highly toxic metabolite moniliformin (MON) was isolated first from F. rnoniliforrne by Cole et al. i n the United States (91). We subsequently found, however, that only a small percentage of F. moizilifonne isolates produced small amounts of MON and that none of the isolates that have been used in the successful experimental reproduction of LEM, including F. nzoniliforv?e MRC 826, produced this compound (93). Although we have demonstrated the natural occurrence of MON in Transkeian corn (53) as well as in corn from the United States implicated in LEM and known to be hepatocarcinogenic in rats (360), the MON in these corn samples was probably produced by F. subglutinnns (361) rather than F. modiforme. We could conclude from the nonproduction of MON by F. monilifon~zeMRC 826 that this compound was not involved in the etiology of LEM and was not responsible for the hepatocarcinogenicity of this isolate in rats. This agreed with our previous findings that MON is not mutagenic to Sctlrnorzella t?,phi~nzuizmin the Ames test (198). The carcinogenic culture material of F, rnorzilifonne MRC 826, however, was highly mutagenic to S. typhimzlrizmz (104). Consequently, intensive efforts were made to isolate and characterize the mutagen from the culture material. These efforts resulted in the structural elucidation of a potent mutagen, fusarin C (FUS C), produced by F. rnoniliforrne MRC 826 (105). Since most mutagens are also carcinogens, the findings that FUS C occurred naturally in Transkeian corn (106) as well as in hepatocarcinogenic corn from the United States (360) strongly suggested that FUS C was the carcinogen produced by F. nzoniliforme that had eluded us for so long. This, however, was not thecase and FUS C proved to be noncarcinogenic in rats (362). Thus, the search for the elusive F. rnoniliforrne carcinogen continued. These efforts finally met with success in 1988 when the chemical nature of the carcinogen was unraveled. Fumonisin B, (FBI)and fumonisin B2 (FB,), novel mycotoxins with cancer-promoting activity in rats, were isolated from cultures of F. rnonilifornze MRC 826 by Gelderblom et al. (112) and their structures elucidated (1 13). In addition to its cancer-promoting activity in a short-term bioassay in rat liver (1 12, 122), pure FBI subsequently has been shown to cause LEM in horses (363, 364), pulmonary edema in pigs (365). and liver cancer in rats (366). The optimal conditions for the production of FB in culture have been defined (1 33) and a sensitive HPLC analytical method for the detection of FBI, FB?, and FB, has been developed (134, 137) and tested in international collaborative studies (1 35, 136). This method was used to demonstrate the natural occurrence of FBI and FB2 in corn-based feeds associated with LEM (124, 134); in feeds associated with animal mycotoxicoses in Brazil (367); in field-trial corn in Argentina (368); in South African commercial corn (369) and export corn (370): in homegrown corn from different EC rate areas in Transkei (18 1, 247, 322): and in corn-based commercial human foodstuffs in South Africa, the United States, and other countries (138, 371). The presence of FB, and FB-,in commercial corn-based foodstuffs from Charleston, South Carolina, which is a high-incidence area of EC in the United States, also was demonstrated (371). In addition to F. ~nordiforn~e (1 12, 116-125), the production of fumonisins by seven other Fuscrr-ium species has been reported, i.e., F. proliferaturn (120, 123, 125), F. nygnvrzni (120, 123, 126),F. ar~tl~ophilunz (126), F. cZlnrnirzi (126), F. napifornze (126), F. t/~npsinlr~n

Fusarium

555

(127, 372), and F. globosum (128, 373). In addition to FBI and FB2, the structures of

approximately 18 additional fumonisin analogs also have been elucidated (1 14- 12.1). Methods also were developed to detect FBI and FBI in plasma, urine, and feces (374, 375) and applied in toxicokinetic studies in rats (376-378). Radiolabeled (IT) FBI was prepared for the first time by spiking a growing culture of F. nzoniliforme MRC 826 with ~-[methyl-I~C] tnethionine (379, 380) and the purified “C-labeled FBI was used to detemine the fate of the major portion of the FBI dosed to rats (381) and vervet monkeys (382, 383, 384). The cancer-initiating potential of FBI and FB2 was screened in rat liver and the lack of genotoxicity confirmed in DNA repair assays in primary hepatocytes (385, 386). Structure-activity (387) and dose-response (388) relationships and cancer-promoting potential (389) have been determined, mitoinhibitory effects (390) and effects on lipid synthesis (391) and fatty acid composition of lipids (392) described, and possible mechanisms of action of the fumonisins proposed (333, 393). Progress was made in the determination of F. rnorzilifome levels in different commercial corn cultivars under different climatic conditions and the selection of cultivars resistant to F. rnonilifornze (394-397). Finally, the implications of naturally occurring levels of fumonisins in corn for human and animal consumption have been reviewed (33 1,398) and risk assessment is in progress (333, 334). These findings that the fumonisins produced by F. modifortne occur naturally as foodborne toxins and carcinogens in the staple diet of people at high risk for EC as well as in commercial corn products in several countries have caused worldwide interest in these tnycotoxins. Particularly in the United States, the death of large numbers of horses and pigs during 1989 and 1990 due to the ingestion of commercial mixed feeds containing fumonisin-contaminated corn of the 1989 crop (see the next section, “Animal Models”) triggered a great deal of interest in and research on the fumonisins (182, 332, 399-41 1). Several comprehensive reviews of different aspects of the fumonisins have been published (1 38, 33 1-334, 41 2-419). The IARC in 1993 concluded that the “toxins derived from Fzwu-ium modifornze are possibly carcinogenic to humans (Group 2B)” (420). The need certainly exists to establish tolerance levels for fumonisins in foods and feeds as soon as possible to protect human and animal health (334). In the high-incidence area of EC in northern China, an association between F. moniliforrne contamination of corn and the incidence of EC has also been found (218, 299, 300, 335). A survey of Fusnt-iunz toxins in corn used as the staple diet in Linxian County during 1989 revealed the presence of DON. 15-acetyldeoxynivalenol, NIV, and ZEA, but the satnples were not analyzed for fumonisins (1 80). High levels of FBI (up to 155 pg/g) were reported in 1994 in moldy corn from high-EC-risk areas (Linxian and Cixian Counties) in China (421). Contradictory results have been reported in comparative studies of fumonisin levels in corn from high- and low-risk areas of EC in China. Corn samples collected in Linxian during 1989 (246) and 1995 (203) were again shown to contain high levels of FBI and FB?, but the mean levels were not significantly different from those found in a low-risk area (Shangqiu County) in Henan Province. In contrast, corn samples also collected during 1995 in Linxian and other high-risk areas in China were found to be twice as frequently contaminated with fumonisins at levels approximately three times higher than in samples from low-risk areas (422). Significantly higher levels of FBI have also been reported in corn samples from a high-risk area for primary liver cancer in China than in corresponding samples from a low-risk area (423). It is interesting that the COoccurrence of fumonisins and aflatoxins has been reported in corn from China (421-423) and elsewhere (424-428).

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The natural occurrence of FUS C in corn from Linxian and the production of this metabolite by a local isolate of F. rnoniliforrne has been reported (108). In one study in China, FUS C was reported to induce papillomas and carcinomas of the esophagus and forestotnach in mice and rats (429). Chinese researchers have also claimed that several carcinogenic nitrosamines are produced in corn bread inoculated with isolates of F. n1o1ziliforme and incubated with the addition of NaNO? and that such corn bread causes papillomas and early carcinomas in the forestomach of rats (218, 299). These results have not been confirmed elsewhere. The association between high levels of F. monilifome and funlonisins in corn and high risk for EC that has been established in Transkei and China (and in corn products from a high-incidence area in South Carolina, in the United States) does not exist in the high-incidence area of Iran in which the staple diet is wheat and the most prevalent fungus associated with the wheat is Altemnria nlternafa (Fr.) Keissler (301-303, 309). Thus, it seems that if the fumonisins produced in corn by F. mozdiforrne are foodbome carcinogens involved in the etiology of EC in Transkei, China, and the United States, other factors must be involved in Iran. There are unconfirmed reports by Chinese researchers that A. alternata cultures can induce carcinoma of the forestomach and esophagus in rats (182). Interestingly, the Carcinogenic fumonisins produced by F. moniliform structurally are very similar to the AAL toxins, a group of host-specific phytotoxins produced by A. alternata f. sp. lycopersici (1 13,430). There is also an unconfirmed report that this fungus produces FBI (132). The biological effects of FBI and AAL toxins are very similar in plants (431,432) and cultured mammalian cells (433). but it has not yet been established whether AAL toxins can induce LEM in horses and/or hepatocarcinoma in rats. The examination of wheat from the high-incidence area of EC in Iran for A. nlternatcr metabolites related to fumonisins and AAL toxins is clearly indicated. 4. Animal Models Certain asymmetrical methyl-alkylnitrosamines are site-specific esophageal carcinogens in rats and a useful model for the study of human EC has been developed in rats treated with N-methyl-N-benzylnitrosamine (MBZN) (434). In humans, squamous cell carcinoma is by far the most common and important EC, and MBZN also induces multiple squamous cell neoplasms as well as papillomas of the esophagus in rats (434). This model has been used inter alia to study nutritional effects on MBZN-induced esophageal carcinogenesis in rats (310, 312, 313). EC has not been reproduced experimentally in animals with either culture material of F. nzonilifol-meor pure fumonisins, although both the culture material (356, 357) and FBI (366) have been proven to cause primary hepatocellular carcinoma and cholangiocarcinoma in rats. Claims by researchers in China that corn bread inoculated with F. moniliforme and incubated with NaN02 caused papillomas and early carcinomas in rats have not been confirmed (218, 299). F. wolziliforme and the fumonisins exhibit a remarkable species-specific variation in the target organ(s) affected in different animal species, such as the brain in horses (350, 352, 363, 364), the lungs in pigs (352, 365), and the liver in rats (352, 353, 356, 357, 366). The acute and chronic toxic effects, and target organ(s) in humans, of course, are unknown. Thus, the epidetniological evidence for the involvement of futnonisins i n the etiology of EC has not yet been supported by experimental evidence that these metabolites of F. morziliforme can cause EC in an animal model.

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Field outbreaks of two animal diseases that are known to be caused by FBI, LEM in horses and pulmonary edema in pigs, occur sporadically in different parts of the world, e.g., in the United States in 1989 and 1990 (406-410). The occurrence of these outbreaks confirms that FBI (and FB.) occurs naturally in corn used in animal feeds at sufficiently high levels to cause mycotoxicoses in animals. This conclusion is supported by chemical analytical data on the levels of FBI (up to 330 pg/g) and FB? (up to 23 pg/g) in the incriminated feeds (124, 367, 398, 407, 409, 410). Similar high levels of FB, (up to 1 17.5 pg/g) and FB-, (up to 22.9 pg/g) also have been detected in homegrown corn consumed by people at high risk for EC i n Transkei (181, 247, 322, 398). One can assume that corn containing such high levels of FBI and FB-, as that used for human consumption in Transkei will cause LEM when fed to horses and pulmonary edema when fed to pigs. However, during 25 years of observations in Transkei, I have never encountered a case of LEM in a horse, or of pulmonary edema in a pig, or of EC in any animal. The only explanation that I can offer for this anomaly is that animals in Transkei are not fed significant amounts of homegrown corn, which is used mainly for human consumption as porridge or as home-brewed beer. This apparently is different from thesituation in Linxian County in northern China, where chickens belonging to people at high risk for EC have been reported to develop squamous cell carcinomas in the esophagus (296). It would be interesting to determine experimentally whether FBI can cause carcinoma of the esophagus in chickens. Recent findings that the disruption of sphingolipid metabolism in several animal species (41 I), including nonhuman primates (435). is an indicator of fumonisin exposure are very promising with respect to the use of changes in the sphingosine: sphinganine ratio as a biomarker of fumonisin exposure in humans.

5. Control The natural occurrence of fumonisins produced by F. rnonilifotww in corn and the threat to humans and animals of such contaminated corn have been demonstrated beyond doubt. The implicated corn samples range from moldy homegrown corn in Africa to commercial corn products on supermarket shelves in the United States. Alarmingly high levels of FBI and FB? have been found in corn consumed by people at high risk for EC. Thus, it is imperative that the fumonisin levels in corn-based human and animal foods and feeds should be reduced. Approaches to achieve this goal should include the selection of corn cultivars that have low levels of seedborne F. morziliforn~eassociated with the grain, breeding and/or genetic engineering of resistant corn cultivars (418), amendments to corn-grading regulations in corn-producing countries to discriminate against kernels infected with F. m o d i .forme, physical (436) and chemical (437, 438) decontamination, and the establishment of tolerance levels. The urgency of the establishment of scientifically sound and economically realistic tolerance levels for fumonisins in foods is attested to by the recent outbreak of a foodborne disease of humans attributed to the consumption of moldy corn and sorghum contaminated with furnonisins (427). V.

CONCLUSIONS

The involvement of feedborne Fz~st~ri~rrr? mycotoxins in hemorrhagic, feed-refusal, emetic, estrogenic, neurotoxic, and pulmonary diseases in animals and the association of food-

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borne F~rsnriurnmycotoxins with ATA, SGI, and EC in humans have been reviewed in this chapter. Whereas the roles of T-2, DAS, DON, NTV, ZEA, and FBI in the causation of veterinary mycotoxicoses have been proven experimentally, this is much more difficult to establish in the case of human diseases attributed to foodborne Fzwu-iurn toxins. In the case of human diseases, one has to make use of circumstantial and epidemiological data together with chemical analytical results on the natural occurrence of Fusarium toxins in incriminated foodstuffs and toxicological data in animal models. The literature on the three human syndromes for which sufficient data are available to implicate foodborne FLrsarirm toxins in their etiology hasbeen reviewed. Several other human foodborne diseases that have been attributed to Fzlsni-ium mycotoxins in the literature (e.g., pellagra, Kashin-Beck disease, Keshan disease, and idiopathic cardiopathy) have not been included because the associations are based on insufficient data. A discussion of the carryover of Fusarium toxins from feeds into animal products such as meat, milk, and eggs has not been included because this is considered to be a very minor vehicle of human exposure to foodborne F ~ ~ s m - i ztoxins, m with the possible exception of ZEA and its metabolites in bovine milk. If the new era of research on foodborne diseases caused by mycotoxins was introduced by the aflatoxins in the 1960s, the twentieth century closed with worldwide attention focused on the fumonisins. The next chapter to be written on foodborne diseases caused by Fruariun? mycotoxins may well be called ‘ ‘Fumonisins-Foodborne Fz~sctriumToxins for the Third Millennium.’’

REFERENCES 1. Marasas, W.F.O.. P.E. Nelson, and T.A. Toussoun, Toxigenic Fusrrriunl Species. Identih crnd Mvcotoxicology, Pennsylvania State University Press, University Park, 1983. 2. Nelson. P.E., T.A. Toussoun. and W.F.O. Marasas, Fusuriunt Species: An Illzrstmted M n m n l for IdentiJication, Pennsylvania State University Press, University Park, 1983. 3. Marasas. W.F.O.. Fuseu-iunz monil(forme: a mycotoxicological miasma. In Mycotoxins curd Plzycotoxins. P.S. Steyn and R. Vleggaar (Eds.), Elsevier, Amsterdam, 1986, pp. 19-28. 4. Marasas. W.F.O., Medical relevanceof mycotoxins insouthernAfrica, Microbiol. Alint. NUtT., 6:1-5 (1988). 5. Marasas, W.F.O., Toxigenic fusaria. In Mycotoxins crnd Arzimcrl Foods, J.E. Smith and R.S. Henderson (Eds.). CRC Press, Boca Raton. FL, 1991, pp. 119-139. 6. Marasas, W.F.O., and P.E. Nelson, Myotosicology. Introduction to the Mycology, Plcm Pcrthology, Chemistry, Tosicologv and Pathology of Nuturally Occurring Mycotoxicoses in Animals and Man, Pennsylvania State University Press, University Park. 1987. 7. Marasas, W.F.O., P.E. Nelson, and T.A. Toussoun, Taxonomy of toxigenic fusaria. InTt-ichothecenes crnd Other Mycotoxins, J. Lacey (Ed.), Wiley. New York, 1985, pp. 3-14. 8. Steyn. P.S.. P.G. Thiel. and D.W. Trinder, Detection and quantification of mycotoxins by chemical analysis. In Mycotoxins and Animal Foods, J.E. Smith and R.S. Henderson (Eds.), CRC Press. Boca Raton, FL. 1991, pp. 165-221. 9. Sydenham, E.W.,and P.G. Thiel,The simultaneousdetectionofdiacetoxyscirpenoland T-2 toxin in fungal culturesand grain samples by capillary gas chromatography, Food Addir. Corztai7l.. 41277-284 (1987). 10. Scott, P.M., S.R. Kanhere, and E.J. Tarter, Determination of nivalenol and deoxynivalenol in cereals by electron-capture gas chrotnatography, J. Assoc. 08 Anal. Chern., 69:889-893 (1986).

Fusarium

559

11. Bamburg, J.R., N.V. Riggs, and F.M. Strong, The structures of toxins from two strains of Fusariurn tricincturir, Tetralwdron. 24:3329-3336 (1968). 12. Logrieco, A., J. Chelkowski, A. Bottalico, and A. Visconti, Furtherdata on specific trichothecene production by Fusariunt sect. Sporotrichiella strains, Mycol. Res.. 94:587-589 (1 990). 13. Yagen, B., and A.Z.Joffe, Screeningof toxic isolates of Fusariuvt poae and Fl4Saril11n sporotricltioides involved in causing alimentary toxic aleukia, Appl. Emiron. Microbiol., 32:423427 ( I 976). 14. Yagen, B., A.Z. Joffe, P. Horn, N. Mor, and 1.1. Lutsky, Toxins from a strain involved in A.T.A. In Mycotosiru in Human and Aniinal Health, J.F. Rodricks, C.W. Hesseltine, and M.A. Mehlman (Eds.), Pathotox Publishers, Park Forest South. IL. 1977. pp. 329-336. 15. Joffe, A.Z.. Fusariunt poae and Fusnrium sporotrichioides as principal causal agents of alimentay toxic aleukia. In Mycotoxic Fungi, Myotoxins, Mycotoxicoses, An Encylopedic Handbook, Vol. 3, T.D. Wyllie andL.G. Morehouse (Eds.), MarcelDekker.NewYork. 1978, pp. 21-86. 16. Joffe, A.Z., Fusnrium Species, Their Biology and Toxicology. Wiley, New York, 1986. 17. Joffe, A.Z., and B. Yagen. Comparative study of the yieldof T-2 toxin produced byFlrsariurn pone, F. sporotrichioides and F. sporotrichioides var. tricinctwn strainsfrom different sources. Mycopathologia, 60:93-97 (1977). 18. Marasas, W.F.O., B. Yagen, E. Sydenham,S. Combrinck, and P.G. Thiel. Comparative yields ofT-’2 toxin and related trichothecenes from five toxicologically important strains of Fz4scrriwt sporotrichioides, Appl. Enliroiz. Microbiol., 53:693-696 (1 987). 19. Burmeister, H.R., J.J. Ellis, and C.W. Hesseltine, Survey for fusaria that elaborate T-2 toxin, Appl. Microbiol., 23:1165-1166 (1972). 30. Ueno,Y.,N. Sato, K. Ishii, K. Sakai, and M. Enomoto, Toxicologicalapproachesto the metabolites of fusaria. V. Neosolaniol, T-2 toxin and butenolide, toxic metabolites of Fusclrirtm sporotriclzioides NRRL 3510 and Fusarium pone 3287, J p . J. Exp. Men., 42:361472 (1972). 21. Ueno. Y., N. Sato, K. Ishii, K. Sakai. H. Tsunoda, and M. Enomoto. Biological and chemical detection of trichothecene mycotoxins of Fuscrrizlnt species, Appl. Microbiol., 25:699-704 (1973). 22. Ueno, Y., M. Sawano, and K. Ishii, Production of trichothecene mycotoxins by Fusar-iurn species in shake culture, Appl. Microbiol.. 30:4-9 (1975). 23. Miller. J.D., A. Taylor, and R. Greenhalgh. Production of deoxynivalenol and related compoundsinliquid culture by Fusariunt grmninecrrum, Cart. J. Mict-obiol., 39:l171-1178 (1983). 24. Hussein, H.M., M. Baxter, I.G. Andrew, and R.A. Franich. Mycotoxin production by Fusarium species isolated from New Zealand maize fields. Mycopcrthologia, 1 13:35-40 (1991). 25. Rabie. C.J., E.W. Sydenham. P.G. Thiel, A. Lubben. and W.F.O. Marasas, T-3 toxin production by Fusarium ncunrincrtum isolated from oats and barley, Appl. EIzviron. Microbiol.. 52: 594-596 (1986). 26. Thrane, U., and U. Hansen, Chenlical and physiological characterization of taxa in the Fusctrizm snn~bt~+zttm complex, Mycopatltologia. 129:183- 190 ( 1995). 27. Altomare, C., A. Logrieco, A. Bottalico, G. Md6. and A. Evidente, Production of type A trichothecenes and enniatin by Frmrium scrntbucinum Fuckel sensu Into. Mycopathologia, 129:177-181(1995). Cultural and toxigenic variability 28. Logrieco, A., C. Altomare, A. Moretti, and A. Bottalico, in FLwu-iunt cmsminntto,l, Mycol. Res., 9 6 5 18-523 (1 992). 29. Burgess, L.W., G.A. Forbes, C. Windels,P.E. Nelson, W.F.O. Marasas, and K.P. Gott, Characterization and distribution of Fusariurzl aclmincltum subsp. armeniacum subsp. nov., Mycologin, 85:119-124 (1993). 30. Brian, P.W., A.W. Dawkins. J.F. Grove, H.G. Hemming, D. Lowe, and G.L.F. Norris, Phytotoxic compounds produced by Fuscwiwt equiseti, J. ESP. Bot., 12:1-12 ( 196 l).

560

Marasas

31. Suzuki. T., M. Kurisu, Y. Hoshino, M. Ichinoe, N. Nose, Y. Tokumasu, and A. Watanabe, Production of trichothecene mycotoxins of Fusarizwl species in wheat and barley harvested in Saitama Prefecture, J. Food Hyg. SOC.Jpn., 21:43-49 (1980). 32. Lafont. P., C. Lafarge-Frayssinet. J. Lafont. G. Bertin.and C.Frayssinet, Metabolites toxiquesde Fzrsaritrm,agents del’aleucimie toxiquealimentaire.Ann.Microbiol. (Znst. Pasteur), 128B:215-220 (1977). 33. Vesonder, R.F., P. Golinski, R. Plattner, and D.L. Zietkiewicz, Mycotoxin formation by different geographic isolates of Fusari~/r~z croohdlerzse, Mycopthologin, 113: 11- 14 (1991 ). 33. Sigg. H.P., R. Mauli. E. Flury, and D. Hauser. Die Konstitution von Diacetoxyscispenol, Hehl. Clzinl.Artcr. 48:963-988 (1965). 35. Steyn. P.S., R. Vleggaar, C.J. Rabie, N.P.J. Kriek. and J.S. Harington, Trichothecene mycotoxins from Fusmizm sulphlrrew1. Plzytocl~emistn,17:949-95 1 (1 978). 36. Morooka, N., N. Uratsuji. T. Yoshizawa, and H. Yamamoto, Studieson the toxic substances in barley infected with FLrsurirm spp. (in Japanese), J. Food Hyg. Soc. Jpn., 13:368-375 (1 972). emetic principle from Fusariunz37. Vesonder, R.F., A.Ciegler, and A.H. Jensen. Isolation of the infected corn, Appl. Microbiol., 26:1008-1010 (1973). 38. Yoshizawa, T., and N. Morooka. Deoxynivalenol and its monoacetate: new mycotoxins from Fzmwiurt? rosezm and moldy barley, Agric. Biol. Clwnr., 37:2933-2934 (1973). 39. Vesonder, R.F., A. Ciegler. A.H. Jensen, W.H. Rohwedder, and D. Weisleder, Co-identity of the refllsal and emetic principle from Flrsarizlnz-infected corn, Appl. Environ. Microbiol., 3 1:380-285 (1 976). 40. Vesonder,R.F.,A. Ciegler, and A.H. Jensen, Production of refusal factors byFusariunz strains on grains. Appl. Envirm. Microbiol., 34: 105-106 (1977). 41. Vesonder, R.F.. J.J. Ellis. and W.K. Rohwedder. Elaboration of vomitoxin and zearalenone by F~rsarizmisolates and the biological activity of Fztsar.iun~-produced toxins, Appl. Erztyirorz. Microbiol., 42:1132-I 134 (1981). 42. Yoshizawa, T.,Y. Matsuura, Y. Tsuchiya, N. Morooka. K. Kitani, M. Ichinoe, and H. Kurata, On the toxigenic fusaria invading barley and wheat in the southern Japan, J. Food HI3g. SOC. Jyn., 20:21-26 (1979). 33. Ichinoe. M., H. Kurata, Y . Sugiura, and Y. Ueno, Chemotaxonomy of Gibberelltz zeae with special referenceto production of trichothecenes andzearalenone, Appl. Environ. Microbiol., 46: 1364- 1369 ( 1983). 44. Tanaka, T., A. Hasegawa, S. Yamamoto, M. Toyazaki,Y. Matsuda, Y. Sugiura. and Y. Ueno, Production of mycotoxins by Fusarizm isolates from scabby wheat harvested in Hokkaido, Japan, Proc. J ~ n 7 Assoc. . Mycotoxicol., 25:31-33 (1987). 45. Neish,G.A.. and H. Cohen, Vomitoxinandzearalenoneproductionby Fzrsuriztm gmnzinenrzcm from winter wheat and barley in Ontario. Can. J. P l m t Sci., 61:811-815 (1981). 46. Neish. G.A., E.R. Farnworth,R. Greenhalgh, and J.C. Young, Observations on the occurrence of Fusarium species and their toxins in corn in Eastern Ontario, Can. J. Plant Pathol., 5: 11-16 (1983). 47. Trenholm, H.L., W.P. Cochrane. H. Cohen. J.I. Elliot, E.R. Farnworth, D.W. Friend, R.M.G. Hamilton, G.A. Neish. and J.F. Standish, Survey of vomitoxin contamination of the 1980 Ontario white winter wheat crop: results of survey and feeding trials, J. Assoc. Qft: Anal. Chenz., 66:92-97 (1983). 48. Faifer,G.C.. M.S.De Miguel.andH.M. Godoy, Patternsofmycotoxinproductionby Fzrsarimzgrawzinearvnlisolated from Argentine wheat, Mycoputhologia,109:165-170 (1990). 49. Leonov, A.N.. G.P. Kononenko, and N.A. Soboleva, Production of DON-related trichothecenes by Fusarium granzineanon Schw. from Krasnodarski & - a iof the USSR. Mycoto.t-i~z Res., 6:54-60 (1990). 50. Marasas, W.F.O., N.P.J. Kriek. S.J. van Rensburg, M. Steyn, and G.C. van Schalkwyk, Oc-

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55.

56. 57.

58.

59.

60. 61. 62.

63.

64. 65. 66. 67.

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70.

561

currence of zearalenone and deoxynivalenol, mycotoxins produced by Fzuariunt granrirtearum Schwabe. in maize in southern Africa, S. Afr: J. Sci., 73:346-349 (1977). Pathre, S.V., and C.J. Mirocha, Analysis of deoxynivalenol from cultures of Fztsarirsuz species, Appl. Emiron. Microbiol., 35:992-994 (1978). Vesonder, R.F., and A. Ciegler, Natural occurrence of vomitoxin in Austrian and Canadian corn, Eur. J. Appl. Microbiol. Biotechnol., 8:237-240 (1979). Thiel, P.G., C.J. Meyer, and W.F.O. Marasas, Natural occurrence of moniliformin together with deoxynivalenol and zearalenone in Transkeian corn, J. Agric. Food Chenz., 30:308312 (1982). Greenhalgh. R., G.A. Neish, and J.D. Miller, Deoxynivalenol, acetyl deoxynivalenol, and zearalenone formation by Canadian isolates of Fzrsnriz4nz graminearzm on solid substrates, Appl. Ewiron. Microbiol., 46:625-639 (1983). Mirocha, C.J., H.K. Abbas, C.E. Windels, and W. Xie, Variation in deoxynivalenol, 15acetyldeoxynivalenol. 3-acetyldeoxynivalenol, and zearalenone production by Fusarium grarninearzrm isolates, Appl. Envirow. Microbiol., 55:1315-1316 (1989). Mirocha, C.J.. S.V. Pathre, B. Schauerhamer, and C.M. Christensen, Natural occurrence of Fz~sariunztoxins in feedstuff, Appl. Environ. Microbiol., 32:553-556 (1976). Marasas, W.F.O., L. Leistner. G. Hofmann, and C. Eckardt, Occurrence of toxigenic strains of Fzasarizm in maize and barley in Germany, Eur. J. Appl. Microbiol. Biotechnol., 7289305 (1979). Palmisano, F., A. Visconti, A. Bottalico, P. Lerario. and P.G. Zambonin, Differential pulse polarography of trichothecene toxins: detection of deoxynivalenol in corn, Ancrlyst. 106:992998 (1981). Ramakrishna, Y., R.V. Bhat, and V. Ravindranath. Production of deoxynivalenol by F w sarizanl isolates from samples of wheat associated with a human mycotoxicosis outbreak and from sorghum cultivars, Appl. Emiron. Microbiol., 552619-2620 (1989). Snijders, C.H.A., and J. Perkowski, Effects of head blight caused by Fzrsarium czdnzo~z4rn on toxin content and weight of wheat kernels. Pltytopatlzology, 80:566-570 (1990). Tatsuno, T., M. Saito, M. Enomoto, and H. Tsunoda, Nivalenol, a toxic principle of Fzrsnrizm nilwle, Chern. Pltnrrn. Bull., 16319-2520 (1968). Tatsuno, T., Y. Fujimoto, and Y. Morita, Toxicological research on substances from Fusariurlz nivale. 111. The structure of nivalenol and its monoacetate, Tetrahedron Lett., 33: 2823-2826 (1969). Ueno, Y.. Y. Ishikawa, M. Nakajima, K. Sakei, K. Ishii, H. Tsunoda, M. Saito, M. Enomoto. K. Ohtsubo, and M. Umeda, Toxicological approaches to the metabolites of fusaria.1. Screening of toxic strains, Jpn. J. Exp. Men., 41:257-272 (1971). Muli, G.,A. Logrieco, and A. Bottalico. Fz~sarizcnzsp. FN-2B: A controversial strain genetically close to Fusnrizlm poae, Mycotoxin Res., 11:53-58 (1995). O’Donnell, K., Phylogenetic evidence indicates the important mycotoxigenic strains Fn-2, Fn-3, Fn-2B and Fn-M represent a new species of Fzrsarizsn~,Mycotoxins, 45: 1- 10 (1997). Aoki, T., and K. O’Donnell, Fztsariurn kyshuense sp. nov. from Japan, Mycoscience, 39: 1-6 (1 998). Logrieco, A., A. Bottalico, and C. Altomare, Chemotaxonomic observations on zearalenone and trichothecene production by Gibberella zeac from cereals in southern Ttaly. Mycologia, 80:892-895 (1988). Blaney, B.J., and R.L. Dodman, Production of the mycotoxins zearalenone, 4deoxynivalenol and nivalenol by isolates of Fzssariunz grc~ntinear~rm Groups 1 and 2 from cereals in Queensland, Aust. J. Agric. Res., 39:21-29 (1988). Lee, U.S., H.S. Jang, T. Tanaka, N. Toyasaki, Y. Sugiura, Y.J. Oh, C.M. Cho. and Y. Ueno, Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates, Appl. Envir-on. Microbiol., 52: 1258-1260 (1986). Sydenham, E.W., W.F.O. Marasas, P.G. Thiel, G.S. Shephard, and J.J. Nieuwenhuis, Produc-

Marasas

562

71.

72.

73.

74. 75. 76. 77. 78. 79. 80.

81.

tion of mycotoxins by selected FLtsarizm granti~zearunzand F. crookwellense isolates. Food Addit. Corttant.. 8:31-41 (1991). Sugiura, Y., Y. Watanabe, T. Tanaka, S. Yamamoto. and 1’. Ueno, Occurrence of Gibberela zeae strains that produce both nivalenol and deoxynivalenol, Appl. Emiron. Microbiol., 56: 3047-3051 (1990). Lauren, D.R..A.Ashley,B.A., Blackwell, R. Greenhalgh,J.D. Miller,andG.A.Neish, Trichothecenes produced byFuscrrilrm croohlellense DAOM 19361 1. J. Agric. Food Chem., 35~884-889(1987). Golinski, P., R.F. Vesonder, D. Latus-Zietkiewicz, and J. Perkowski. Formation of fusarenone x. nivalenol, zearalenone, a-trans-zearalenol, P-trans-zearalenol, and fusarin C by F w sariwtz crooh’ellense. Appl. Emiron. Microbiol., 53:2147-2148 (1988). Stob, J.. R.S. Baldwin, J. Tuite, F.N. Andrews, and K.G. Gillette, Isolation of an anabolic, uterotrophic compound from corn infected with Gibberelle zecre, Nature, 196: 1318 (1962). Mirocha, C.J., and C.M. Christensen, Oestrogenic mycotoxins synthesized by Fusarium. In Mycotoxins, I.F.H. Purchase (Ed.), Elsevier, Amsterdam, 1974, pp. 129-148. Mirocha, C.J.,and S.V. Pathre. Mycotoxins-their biosynthesis in fungi: zearalenonebiosynthesis, J. Food Prof., 32:821-824 (1 979). Mirocha. C.J., C.M. Christensen, and G.H. Nelson, Estrogenic metabolite produced by Fzrsarilrm gr.anzinearwr in stored corn. Appl. Microbiol., 15:497-503 (1 967). Mirocha, C.J., C.M. Christensen, and G.H. Nelson, Toxic metabolitesproducedbyfungi implicated in mycotoxicoses, Biotech. Bioeng., 10:469-482 (1968). Mirocha, C.J.. C.M. Christensen. and G.H. Nelson,Physiologic activity of some fungal estrogens produced by Fmcrrium. Cancer Res., 28:23 19-2322 (1968). Mirocha, C.J., C.M. Christensen, and G.H. Nelson, F-2 (zearalenone) estrogenic mycotoxin from Fz~sariur~r. In Microbial To.xins, Vol. VII, S. Kadis,A. Cjegler, and S.J. Ajl (Eds.), Academic Press, New York, 1971, pp. 107-138. Kotsonis, F.N., E.B. Smalley, R.A. Ellison, and C.M. Gale, Feed refusal factors inpure cultures of Fusarizrnt r o s e ~ o“granzinearmt~, ~ Appl. Microbiol., 30:362-368 (1975). Hidy. P.H., R.S. Baldwin, R.L., Greasham, C.L., Keith. and J.R. McMullen. Zearalenone and some derivatives: production and biological activities, A h . Appl. Microbiol., 2 5 9 - 8 2 (1977). Abbas. H.K.. and C.J. Mirocha, Production of fusarenon-s, nivalenol. and zearalenone by Gibberella zeae isolates and their toxicity in fibroblasts and rats, Mycotoxin Res., 4:67-74 (1 988). Windels, C.E., C.J. Mirocha, H.K. Abbas, and W. Xie, Perithecium production in Fztsari~rtn grcrnzirzearwz populations and lack of correlation with zearalenone production, Mvcologia, 8 1 1272-277 (1989). Bacon, C.W., J.D. Robbins, andJ.K. Porter, Media for identification of Gibberella Ceae and production of (F-3) zearalenone, AppI. Eiziirow Mici-obiol., 33:445-449 (1977). Caldwell, R.W., J. Tuite, M. Stob, and R. Baldwin, Zearalenone production by Fusnrizrm species, Appl. Microbiol.. 20:31-34 (1970). Roine, K., E.-L. Korpinen, and K. Kallela, Mycotoxicosis as a probable cause of infertility in dairy cows. A case report, N o d . Vet. Med., 23:628-633 (1971). Gross, V.J., and J. Robb, Zearalenone production in barley, A m . Appl. Biol.. 80:211-216 (1975). Williams, S., Oficial Methods ofAna!\:sis ofthe Association of OfJicicrl Analytical Chemists, AOAC, Arlington, VA, 1984. Bagneris. R.W., J.A. Gaul, and G.M. Ware, Liquid chrolnatographic determination of zearalenone and zearalenol in animal feeds and grains. using florescence detection, J. Assoc. Of Allal. Chertt., 69:894-898 ( 1986). Cole, R.J., J.W. Kirksey, H.G. Cutler,B.L. Doupnik, and J.C. Peckham. Toxin from Fl4sarizm monilifontze: effects on plants and animals, Science. 179:1324-1326 (1973). ”

82.

83.

84.

85. 86. 87.

88. 89. 90.

91.

Fusarium

563

92. Springer, J.P., J. Clardy, R.J. Cole, J.W. Kirksey, R.K. Hill, R.M. Carlson, and J.L. Isidor, Structure and synthesis of moniliformin, a novel cyclobutane microbial toxin, J. Am. Chem. SOC.,96:2267-2268 (1974). 93. Marasas, W.F.O.,P.G. Thiel, C.J.Rabie. P.E. Nelson, and T.A. Toussoun, Moniliformin production in Fzrsaritrnz section Liseola, Mycologia, 78:242-247 (1986). 94. Marasas, W.F.O., P.G.Thiel,E.W.Sydenham, C.J. Rabie, A. Lubben, andP.E.Nelson, Toxicity and moniliformin production by four recently described species of Fusnriun? and two uncertain taxa, Mycopathofogirr, 113 :191- I97 ( 1991). 95. Bottalico, A.. A. Visconti, and M. Solfrizzo, Production of moniliformin by FLrsariwn species, in Italy, Phytopathol. Medit., 21:105-106 (1982). 96. Rabie, C.J.. W.F.O. Marasas, P.G. Thiel, A. Lubben, and R. Vleggaar, Monilifor~ninproduction and toxicity of different Fusariunr species from southern Africa, Appl. Environ. Microbiol., 43:517-521 (1982). 97. Lamprecht, S.C., W.F.O. Marasas, P.G. Thiel, D.J. Schneider, and P.S. Knox-Davies, Incidence and toxicity of seedborne Fzrsariwn species from annual Medicngo species in South Africa, Phytopathology, 76: 1040- I042 (1 986). 98. Vesonder, R.F., Moniliformin production by culturesof Flrsariwn monil(fornte var. subglutinuns isolated from swine feed, Mycopathologia, 95: 149-153 (1986). 99. Scott, P.M., H.K. Abbas, C.J. Mirocha, G.A. Lawrence, and D. Weber, Formation of moniliformin by Fz~wrinnrsporotrichioirles and Fusariwn culnrorum, Appl. Emiron. Microbiol., 531196-197 (1957). 100. Farber, J.M., G.W. Sanders,G.A. Lawrence, and P.M. Scott, Productionof moniliformin by Canadian isolates of Fzwu-iLlnz, Mycopnthologia, 101:187-190 (1988). 101. Thiel, P.G.,Determinationof monilifonnin by high-performance liquid chromatography, J. Envirorz. Patltol. To.xicol. Oncol., 10:162-165 (1990). 102. Scott, P.M., and G.A. Lawrence, Liquid chromatographic determination and stability of the F w n - i m mycotoxin, moniliformin in cereal grains,J. Assoc. Ojf Anal. Chem., 70:850-853 (1987). 103. Wiebe, L.A., and L.F. Bjeldanes, Fusarin C. a mutagen from Fztsarizc~nmonilifornze grown on corn, J. Food Sci.. 46:1424-1426 (1981). 104. Gelderblom, W.C.A., P.G. Thiel, K.J. Van der Merwe, W.F.O. Marasas, and H.S.C. Spies. A mutagen produced by Frwariunt modiforme, Toxicon, 21 :467-473 (1983). 105. Gelderblom. W.C.A., W .F.O.Marasas, P.S.Steyn, P.G. Thiel,K.J. Van der Merwe, P.H. Van Rooyen, R. Vleggaar, and P.L. Wessels, Structure elucidation of fusarin C, a mutagen produced by Fztsarizrrn modiforme. J. Chern. SOC.Chern. Commun ., 1984: 122- 124 (1984). 106. Gelderblom, W.C.A., P.G. Thiel, W.F.O. Marasas, and K.J. Van der Merwe, Natural occurrence of fusarin C, a mutagen produced by Fusarium monil(fonne, in corn, J. Agr. Food Chem., 32:1064-1067 (1984). 107. Farber. J.M., and G.W. Sanders, Fusarin C production by North American isolates of Fusarizlnl rnoniliforme, Appl. Errvirorz. Microbiol., 51:381-384 (1986). Fzr108. Cheng. S.J., Y.Z. Jiang, M.H. Li, and H.Z. Lo, A mutagenic metabolite produced by srtriunz moniliforrne isolated fromLinxian county. China,Carcinogenesis, 6:903-905 (1985). 109. Bacon, C.W., D.R. Marijanovic, W.P. Norred, and D.M. Hinton, Production of fusarin C on cereal and soybean by Frcsariunr rnoniliforme, Appl. Emiron. Microbiol., 552745-2748 (1989). I. Li, Productionof fusarin Cmycotoxin by Fusarium monili110. Tseng, T.-C., C.-S. Chung, and forme isolates of Taiwan, Bot. Bzdl. Acrrd. Sin., 3 1: 169- 174 (1990). 111. Farber, J.M., and G.W. Sanders, Production of fusarin C by Fusarizrnr spp., J. Agric. Food Clzenz., 34:963-966 (1986). 112. Gelderblom, W.C.A., K. Jaskiewicz, W.F.O. Marasas, P.G. Thiel, M.J. Horak, R. Vleggaar, and N.P.J. Kriek. Fumonisins-novel mycotoxins with cancer promoting activity produced by Fusarium ntoniliforme, Appl. Emirow Microbiol., 54: 1806-181 1 (1988).

564

Marasas

113. Bezuidenhout, S.C., W.C.A. Gelderblom, C.P. Gorst-Allman, R.M. Horak, W.F.O. Marasas, G. Spiteller, and R. Vleggaar, Structure elucidationof the fumonisins, mycotoxins fromFlasariunr nronil(fot-me,J. Cltem. SOC.Clrent.Comrrtlln., 1988:743-745 (1988). and W.F.O. Mara114. Cawood, M.E., W.C.A. Gelderblom, R. Vleggaar, Y. Behrend. P.G. Thiel, sas, Isolation of the funlonisin mycotoxins-a quantitative approach, J. Agric. Food Chern., 39:1958-1962 (1991). 115. Gelderblom, W.C.A., W.F.O. Marasas, R. Vleggaar, P.G. Thiel, and M.E. Cawood, Fumonisins: isolation, chemical characterization and biological effects, Mycopathologia, 117:1116 (1992). and R.G. Powell, Anew fumoni116. Plattner, R.D., D. Weisleder, D.D. Shackelford. R. Peterson, sin from solid cultures of Fusarium rnorziliforrne,Mycoprrtlzologia. 117:23-28 (1992). 117. Branham, B.E., and R.D. Plattner, Isolation and characterization of a new fumonisin from liquid cultures of Fusrrrium monilifot-me, J. Nat. Prod., 56: 1630-1 633 (1993). 118. Plattner, R.D., Detectionof fumonisins produced inF~rsariumvzonilijorrrle cultures by HPLC withelectrosprayMSandevaporativelightscatteringdetectors, Nat. Toxins, 3:294-298 ( 1995). 119. Musser, S.M., M.L. Gay, and E.P. Mazzola, Identification of a new series of fumonisins containing 3-hydroxypyridine, J. Nat. Prod., 59:970-972 (1996). 120. Musser, S.M., and R.D. Plattner. Fumonisin composition in cultures of Fz~sariumnroniliforme, Fusrtrium pi-olifercltunt,and Fzasariunr n?)gunri,J. Agt-ic. Food Clzenl., 45: 1 169-1 173 ( 1997). 121. MacKenzie, S.E., M.E. Savard. B.A. Blackwell, J.D. Miller, and J.W. ApSimon, Isolation of a new fumonisin from Fzcsariutn rnoniliforme grown in liquid culture, J. Nut. Prod., 61: 367-369 (1998). 122. Gelderblom, W.C.A., W.F.O. Marasas, K. Jaskiewicz, S. Combrinck, and D.J. Van Schalkwyk, Cancer promoting potential of different strains of Fusarium nzonilifontze in a shortterm cancer initiation/promotion assay. Carcinogenesis, 9:1405-1409 ( 1 988). 123. Thiel, P.G., W.F.O. Marasas. E.W. Sydenham, G.S. Shephard, W.C.A. Gelderblom, and J.J. Nieuwenhuis, Survey of fumonisin production by Fusariurzl species, Appl. Emiron. Microbiol.. 57:1089-1093 (1991). 124. Thiel, P.G., G.S. Shephard, E.W. Sydenham. W.F.O. Marasas. P.E. Nelson, and T.M. Wilson, Levels of fumonisin B, and B, in feeds associated with confirmed cases of equine leukoencephalomacia, J. Agric. Food Clzenz., 39: 109-1 1I (199 1). 125. Ross, P.F., P.E. Nelson, J.L. Richard, G.D. Osweiler, L.G. Rice, R.D. Plattner. and T.M. Wilson, Production of fumonisins by Fusarium moniliforrne and Fzrsariwn proliferaturn isolatesassociatedwithequineleukoencephalomalaciaandpulmonaryedemasyndrome in swine, Appl. Environ. Microbiol., 56:3225-3226 (1990). 126. Nelson, P.E., R.D. Plattner, D.D. Shackelford, and A.E. Desjardins, FumonisinB, production by Fusariurn species other than F. nzonilifornre in Section Liseola and by some related species, Appl. Ewiron. Microbiol., 58:984-989 (1992). 127. Klittich, C.J.R., J.F. Leslie, P.E. Nelson, and W.F.O. Marasas,Fusarilrnl tlrapsinunt (Gibberella thapsina): a new species in section Liseola from sorghum, Mycologia. 89:633-652 (1997). 128. Sydenham, E.W., G.S. Shephard, S. Stockenstrom, J.P. Rheeder, W.F.O. Marasas, and M.J. Van der Merwe, Production of fumonisin B analogues and related compounds by Fz{sarium globosunl, a newly described species from corn. J. Agric. Food Cltem., 45:4004-4010 (1 997). 129. Abbas, H.K., and C.M. Ocamb, First report of production of fumonisin B, by Fztsaritrtn pol~plzialicu~n collected from seeds of Pintrs sti-obrls, Plarrt Dis., 79:642 ( I 995). 130. Abbas, H.K., C.M. Ocamb, W. Xie, C.J. Mirocha,and W.T. Shier, First reportof fumonisin B,, B?, and B3 production by Fusariunz o.xysporurn var. redolens, Plant Dis., 79:968 (1995). 131. Seo, J.-A., J.-C. Kim, and Y.-W. Lee, Isolation andCharacterization of two new type C fumonisins produced by Fusarium O . X ~ ~ S ~ O Y U J. F I INot. , Prod., 59: 1003- I005 ( 1996).

Fusarium

565

132. Chen. J., C.J. Mirocha. W. Xie, L. Hogge, and D. Olson, Productionof the mycotoxin fumonisin B, by Alternaria alternata fsp. lycopersici, Appl. Environ. klicrobiol., 58:3928-3931 (1 992). 133. Alberts, J.F., W.C.A. Gelderblom, P.G. Thiel, W.F.O. Marasas, D.J. Van Schalkwyk, and Y. Behrend, Effects of temperature and incubation period on the production of fumonisin B, by Fusariunr moniliforrne, Appl. Environ. Microbiol., 56: 1729-1733 (1990). 134. Shephard. G.S.. E.W. Sydenham, P.G. Thiel. and W.C.A. Gelderblom, Quantitative determination of fumonisins B, and B2 by high-performance liquid chromatography with fluorescence detection, J. Liq. Clzront., 13:2077-2087 (1990). 135. Thiel, P.G., E.W. Sydenham, G.S. Shephard, and Van D.J.Schalkwyk, Studyof the reproducibility characteristics of a liquid chromatographic method for determination of fumonisins B, and B2 in corn: IUPAC collaborative study, J. AOAC h t . , 76361-366 (1993). 136. Sydenham, E.W., G.S. Shephard, P.G. Thiel. S. Stockenstrom, P.W. Snijman, and D.J. Van Schalkwyk, Liquid chromatographic determination of fumonisins B,, B2, and B3 in corn: AOAC-IUPAC collaborative study. J. AOAC Znt., 79:688-696 (1996). 137. Sydenham.E.W.,G.S.Shephard, and P.G.Thiel,Liquidchromatographicdetermination of fumonisins B,. B2, and Bi, in foods and feeds, J. AOAC h t . , 75:313-318 (1992). 138. Shephard. G.S.. P.G. Thiel, S. Stockenstrom, and E.W. Sydenham. Worldwide survey of fumonisin contamination of foods and feeds, J. AOAC Int., 79:671-687 (1996). 139. Sydenham, E.W., G.S. Shephard, P.G. Thiel, C. Bird, and B.M. Miller, Determination of fumonisins in corn: evaluationof competitive immunoassay and HPLC techniques,J. Agric. Food Cllenz., 44:159-164 (1996). 140. Sydenham, E.W., S. Stockenstrom, P.G. Thiel, J.P. Rheeder, M.B. Doko, C. Bird, and B.M. Miller, Polyclonal antibody-based ELISA and HPLC methods for the determination of fumonisins in corn: a comparative study, J. Food Protect., 59:893-897 (1996). 141. Thiel, P.G., E.W. Sydenham, and G.S. Shephard, Thereliability and significance of analytical data on the natural occurrence of fumonisin in food. In Frc?nonisins in Food, L.S. Jackson, J.W. De Vries, and L.B. Bullerman (Eds.), Plenum Press, New York, 1996, pp. 145-151. 142. Joffe, A.Z., Toxin production by cereal fungi causing toxic alimentary aleukia in man. In Mycotoxins in Foodstufls, G.N. Wogan (Ed.), MIT Press. Cambridge, MA, 1965. pp. 7785. 143. Joffe, A.Z., Alimentary toxic aleukia. In Microbial Toxins, Vol. VII, S. Kadis, A. Ceigler, and S.J. Ajl (Eds.), Academic Press, New York, 1971, pp. 139-189. 144. Joffe, A.Z.. Toxicity of Fusariuln porre and Fusarium sporotrichioides and its relation to alimentary toxic aleukia. In Mycotoxins, I.F.H. Purchase (Ed.), Elsevier, Amsterdam 1974, pp. 229-262. 145. Mayer, C.F., Endemic panmyelotoxicosis in the Russian grain belt. Part 1. The clinical aspects of alimentay toxic aleukia (ATA). A comprehensive review, Milit. Surg., 113:173189 (1953). 146. Mayer, C.F., Endemic panmyelotoxicosisin the Russian grainbelt. Part 11. The botany, phytopathology, and toxicology of Russian cereal food, Milit. Surg., 1 13295-315 (1953). 147. Rubenstein. Yu.I., The effect of the cultivation conditions on toxin formation in Fusarium sporotriclziella Bilai. In Mycotoxicoses of Man and Agricultural Aninmls, V.I. Bilai (Ed.), English translation, JPRS 7434, U.S. Joint Publications Research Service, U.S.ofDept Commerce, Washington, DC, 1960, pp. 66-73. 148. Rubenstein, Yu.I., Food fusariotoxicoses. In MJrotoxicoses of Man and Agricultuval Animals, V.I. Bilai (Ed.), English translation, JPRS 7434, U.S. Joint Publications Research Service, U.S. Dept of Commerce, Washington, DC, 1960, pp. 89-110. 149. Bamburg, J.R., and F.M. Strong, 12,23-Epoxytrichothecenes.In Microbial Toxins, Vol. VII, S. Kadis, A. Ciegler, and S.J. Ajl (Eds.), Academic Press, New York, 1971, pp. 207-292. 150. Smalley, E.B., and F.M. Strong, Toxic trichothecenes. InMycotoxins, I.F.H. Purchase (Ed.), Elsevier, Amsterdam, 1974, pp. 199-228.

566

Marasas

151. Ueno. Y., Trichothecenes: Overview address. In Mycotosim in Hzcnran and Animal Health, J.V. Rodricks, C.W. Hesseltine,and M.A. Mehlman (Eds.), Pathotox Publishers. Park Forest South, IL. 1977, pp. 189-207. 152. Ueno. Y. (Ed.), Ti.icllotl~ecenes-Chemical, Biological ami Toxicological Aspects.Kodansha Ltd., Tokyo, 1983. 153. Rosen, R.T., and J.D. Rosen. Presence of four Fusarium mycotoxins and synthetic material in “yellow rain.” Evidence for the use of chemical weapons in Laos, Bionted. Mass Spectrom., 2443-450 (1 982). 154. Mirocha, C.J.. R.A. Pawlosky. K. Chatterjee, S. Watson, and W. Hayes, Analysis for Fusariun? toxins in various samples implicatedin biological warfarein southeast Asia,J. Assoc. Oft: Ancrl. Chem., 66, 1485-1499 (1983). 155. Heyndrickx, A., N. Sookvanichsilp, and M. Van den Hegde, Detectionof trichothecene mycotoxins (yellow rain) in blood, urine and faeces of Iranian soldiers treated as victims of a gas attack, A?-ch.Belg. Suppl., 1984:143-146 (1984). and P.H.Wiernik.Phase I1 evaluation of anguidine(NSC156. Diggs,C.H..M.J.Scoltock, 141537) for adenocarcinomaof the colon or rectum,Cancer Clin. Trials Winter,1978:297299 (1978). and B. Hoogstraten. Phase I evalua157. Goodwin, W., C.D. Haas, C. Fabian, I. Heller-Bettinger, tion of anguidine (diacetoxyscirpenol. NSC-141537), Cancer, 4223-26 (1 978). 158. Belt. R.J., C.D. Haas, U. Joseph, W. Goodwin. D. Moore,and B. Hoogstraten, Phase I study of anguidine administered weekly, Cmcer Treat. Rep., 63:1993-1995 (1979). 159. DeSimone, P.A., F.A. Greco, and H.F. Lessner. Phase I evaluation of a weekly schedule of anguidine, Cuncel- Treat. Rep., 63:2015-2017 (1979). 160. Yap, H.Y., W.K. Murphy,A. DiStefano, G.R. Blumenschein,and G.P. Bodey, PhaseTI study of anguidine in advanced breast cancer, Cancer Trent. Rep., 63:789-791 (1979). 161. Olifson, L.E., The chemical activity of some speciesof fungi which effect the grainof cereals. In Mycotoxicoses of Man and Agriczhrd Anirmls, V.I. Bilai (Ed.), English translation, JPRS 7434. U.S. Joint Publications Research Service, U.S. Dept of Commerce, Washington, DC, 1960, pp. 58-66. 162. Olifson, L.E., S.M. Kenina,and V.L. Kartashova, Examinationof the fractional composition of the lipid complex of cereals infected by the microscopic fungus Ft~sariumsporotrichiellcr Bilai (in Russian), Prikl. BioWt. Mikrobiol., 14:630-634 (1978). English translation inAppl. Bioclzem. Microbiol., 14:488-491 ( 1979). 163. Bamburg, J.R., F.M. Strong, and E.B. Smalley. Toxins from moldy cereals, J. Agric. Food Chem., 17:443-450 (1969). 164. Szathmary, Cs.1.. C.J. Mirocha, M. Palyusik, and S.V. Pathre. Identification of mycotoxins produced by species of F~rscrl-iw~l and Strrclqhm-ys obtained from eastern Europe, Appl. Enliron. Microbiol., 32579-584 (1976). 165. Mirocha, C.J., and S.V. Pathre, Identification of the toxic principlein a sampleof poaefusarin, Appl. Microbiol., 26:719-724 (1973). 166. Mirocha, C.J., S.V. Pathre, and C.M. Christensen, Chemistry of Fusnriurrz and Studtybottys mycotoxins. In Mycotoxic Fungi, Mycotosins, Mycotoxicoses, An Encyclopedic Handbook, Vol. 1, T.D. Wyllie and L.G. Morehouse (Eds.), Marcel Dekker,New York, 1977, pp. 365420. 167 Lutsky. I.I., and N. Mor, Alimentary toxic aleukia (septic angina, endemic panmyelotoxicosis, alimentary hemorrhagic aleukia), T-2 toxin-induced intoxication of cats, Aril. J. Patltol., 104~189-191 (1981). 168. Lutsky, I.I., N. Mor. B. Yagen, and A.Z. Joffe, The role of T-2 toxin in experimental alimentarytoxicaleukia:atoxicitystudy in cats. Toxicol. Appl. Pharmacal.. 43:lll-124 (1978). 169. Hsu, I.C., E.B. Smalley, F.M. Strong,and W.E. Ribelin, Identificationof T-2 toxin in moldy corn associated with a lethal toxicosis in dairycattle, Appl. Microbiol., 24:684-690 (1972).

Fusarium

567

170. Pathre. S.V., and C.J. Mirocha, Assay methods for trichothecenes and review of their natural occurrence. In Mycotoxins i n Human ctnd Animal Health, J.V. Rodricks. C.W. Hesseltine, and M.A. Mehlman (Eds.). Pathotox Publishers, Park Forest South, IL, 1977, pp. 229-253. 171. Pathre, S.V., and C.J. Mirocha. Trichothecenes: Natural occurrence and potential hazard. J. Am. Oil Clzem. SOC..56:820-823 (1979). 172. Abramson. D., J.T. Mills, and B.R. Boycott, Mycotoxins and mycoflora in animal feedstuffs in western Canada, Can. J. Conlp. Med., 47:23-26 (1983). 173. Cirilli, G., Trichothecene problems in Italy. In Trichotlzecenes-Cl?eilzical, Biological arid Toxicological Aspects, Y. Ueno (Ed.), Kodansha Ltd.. Tokyo, 1983, pp. 254-258. 174. Szathmary, C.I., Trichothecene toxicoses and natural occurrence in Hungary. In Trichotlzecenes-Chemical,BiologicalandTosicologicalAspects, Y. Ueno (Ed)., Kodansha Ltd., Tokyo, 1983, pp. 229-250. 175. Jelinek, C.F., A.E. Pohland, and G.E. Wood, Review of mycotoxin contamination, J. Assoc. 08 Anal. Cl1enz., 72:223-230 (1989). 176. Yoshizawa, T., Red-mold diseases and natural occurrence in Japan. In TriclzothecenesChewical, Biological crnd Toxicological Aspects, Y. Ueno (Ed.), Kodansha Ltd., Tokyo, 1983, pp. 195-209. 177. Cotk L.M., J.D. Reynolds, R.F. Vesonder, W.B. Buck, S.P. Swanson. R.T. Coffey. and D.C. Brown, Survey of vomitoxin-contaminated feed grains in midwestern United States, and associated health problems in swine, J. Am. Vet. Med. Assoc., 184:189-192 (1984). 178. Hagler, W.M.. K. Tyczkowska, and P.B. Hamilton, Simultaneous occurrence of deoxynivalenol. zearalenone. and aflatoxin in 1982 scabby wheat from the midwestern United States, Appl. Emiron. Microbiol., 37:151-154 (1984). 179. Blaney, B.J., C.J. Moore, and A.L. Tyler, The mycotoxins 3-deoxynivalenol, zearalenone and aflatoxin in weather-damaged wheat harvested in 1983-1985 in south-eastern Queensland, Alrst. J. Agric. Res., 38:993-1000 (1987). 180. Luo, Y., T. Yoshizawa, and T. Katayama, Comparative study on the natural occurrence of Fusariurtz mycotoxins (trichothecenes and zearalenone) in corn and wheat from high- and low-risk areas for human esophageal cancer in China. Appl. Enlliron. Microbiol., 56:37233726 (1990). 181. Sydenham, E.W., P.G. Thiel, W.F.O. Marasas, G.S. Shephard, D.J. Van Schalkwyk. and K.R. Koch, Natural occurrence of some Fztsuriurn mycotoxins in corn from low and high esophageal cancer prevalence areas of the Transkei, southern Africa, J. Agric. Food Clzem., 38: 1900-1903 (1990). 182. Scott. P.M., Mycotoxins, J. Assoc. 08 Anal. Chern., 74:120-128 (1991). 183. Sarkisov, A.K., General characterization of the alimentary mycotoxicoses of agricultural animals. In Mycotoxicoses of Mall and Agricultural Animals, V.I. Bilai (Ed.), English translation, JPRS 7433, U.S. Joint Publications Research Service, U.S. Dept of Commerce. Washington, DC, 1960, pp. 155-163. 184. Sato, N.. Y. Ueno, and M. Enomoto. Toxicological approaches to the toxic metabolites of fusaria. VIII. Acute and subacute toxicities of T-2 toxin in cats, J. Plzarmacol., 25263-270 (1975). 185. Stiihelin. H.. M.E. Kalberer-Rusch, E. Signer, and S. Lazary, Uber einige biologische Wirkungen des Cytostaticum Diacetoxyscirpenol, Ar.~-neinzittelforschullg.18:989-994 (1968). 186. Rukmini, C., J.S. Prasad. and K. Rao, Effects of feeding T-2 toxin to rats and monkeys. Food Cosnlet. Toxicol., 18:267-269 (1980). 187. Kriek, N.P.J., and W.F.O. Marasas, Trichothecene research in South Africa. In Trichotlzecenes-Chenzicnl Biological a d ToxicologicalAspects, Y. Ueno (Ed.), Kodansha Ltd., Tokyo, 1983. pp. 273-284. 188. Forgacs, J.. and W.T. Carll, Mycotoxicoses, A h . Vet. Sci., 7273-282 (1962). 189. Forgacs, J., H. Koch. W.T. Carll. and R.H. White-Stevens. Mycotoxicoses. I. Relationship of toxic fungi to moldy feed toxicosis in poultry, Alian Dis., 6:363-380 (1962).

568

Marasas

190. Marasas, W.F.O., and E.B. Smalley, Mycoflora, toxicity and nutritive value of mouldy maize. Onderstepoort J. Vet. Res.. 39:l-10 (1972). 191. Smalley, E.B., T-2 toxin, J. Am. Vet. Med. Assoc., 163:1278-1281 (1973). 192. Ribelin, W.E., Trichothecene toxicosis in cattle. In Mycotoxic Fungi. Mycotoxirzs, Mycotoxicoses. An E~~cyclopedic Handbook, Vol. 2, T.D. Wyllie and L.G. Morehouse (Eds.), Marcel Dekker, New York, 1978, pp. 36-45. 193. Joffe, A.Z.. Fusarium toxicosis in poultry. In Mycotoxic Fungi, Mycotoxins, Mycotoxicoses. An Encyclopedic Handbook, Vol. 2. T.D. Wyllie and L.G. Morehouse (Eds.), Marcel Deklier, New York, 1975, pp. 309-321. 194. Kurmanov, I.A.. Fusariotoxicosis in cattle and sheep. In Mycotoxic Fzcngi, Mycotoxins, Mycotoxicoses. Arz Encyclopedic Handbook, Vol. 2, T.D. Wyllie and L.G. Morehouse (Eds.), Marcel Dekker, New York, 1978, pp. 85-110. 195. Kurmanov, LA., Fusariotoxicosis in chickens in the U.S.S.R. In Mycotoxic Fungi. Mycotoxins, Mycotoxicoses. Air Emyclopeclic Hadbook. Vol. 2, T.D. Wyllie and L.G. Morehouse (Eds.), Marcel Dekker, New York. 1978, pp. 322-326. 196. International Agency for Research on Cancer (IARC), Toxinsderived from Frrsrrritrnz sporotrichioides: T-2 Toxin, IARC Monograplzs or1 the Eldrscrtion of the Carcinogenic Risks to Humuns. Some Natrarolly Occurring Substcrnces: Food I t e m , Heterocyclic AronznticArlzirzes altd Myotoxins. 56:467-488 (1993). 197. Kuczuk, M.H., P.M. Benson, H. Heath, and A.W. Hayes, Evaluation of the mutagenic potential of mycotoxins using Sulnrorzellrr ~pl~imuriurn and Smcharonzyces cerelisiae, Mutat. Res., 53:ll-20 (1978). 198. Wehner, F.C., W.F.O. Marasas, and P.G. Thiel, Lack of mutagenicity to Salnlonella hplzinzuriwz of some Fusarizm mycotoxins. Appl. Envir-on. Microbiol., 35:659-663 (1978). 199. Ueno, Y., Mode of action of trichothecenes, Pure Appl. Clzenz., 39:1737-1745 (1977). 200. Ueno, Y., K. Kubota. T. Ito, and Y. Nakamnura, Mutagenicity of carcinogenic mycotoxins in Salmonella t?phiwturium, Crrncer Res., 38:536-542 (1978). 201. Hsia, C.-C., Y. Gao, J.-L. Wu, and B.-L. Tzian, Induction of chromosome aberrations by Fzmwilatz T-2 toxin in cultured human peripheral blood lymphocytes and Chinese hamster fibroblasts, J. Cell. Plzysiol. Strppl., 4:65-72 (1986). 202. Hsia. C.-C., B.-L. Tzian, and C.C. Harris, Proliferative and cytotoxic effects of Fusarirlnz T-2 toxin in cultured human fetal esophagus, Carcinogenesis. 4: 1101-1 107 ( 1 983). 203. Gao, H.-P., and T. Yoshizawa, Further study on Ftrsrrritrriz nlycotoxins in corn and wheat from a high-risk area for human esophageal cancer in China, Mycotoxins, 45:5 1-55 (1997). 204. Schoental, R., A.Z. Joffe. and B. Yagen, Cardiovascular lesions and various tumors found in rats given T-2 toxin, a trichothecene metabolite of Fusar-izon, Cancer Res.. 39:2179-2189 (1979). 205. Marasas, W.F.O., J.R. Bamburg, E.B. Smalley, F.M. Strong, W.L. Ragland, and P.E. Degurse, Toxic effects on trout, rats, and mice of T-2 toxin produced by the fLmgus Fzlsariunr tricinctum (Cd.) Snyd. et Hans., Toxicol. Appl. Plzarmacof., 15:471-482 (1969). 306. Lindenfelser, L.A., E.B. Lillehoj, and H.R. Burmeister, Aflatoxin and trichothecene toxins: skin tumour induction and synergistic acute toxicity in white mice, J. Ncrtl. Cancer Inst., 52: 113-1 16 (1974). 207. Ohtsubo, K., and M. Saito, Chronic effects of trichothecenes. In klycotoshs ill Humrrrz and Arzimul Health, J.V. Rodricks. C.W. Hesseltine, and M.A. Mehlman (Eds.). Pathotox Publishers, Park Forest South, IL, 1977, pp. 255-262. 20s. Ohtsubo. K.. Chronic toxicity of trichothecenes. In Triclzotllecerzes-Clzentical, Biological nrzd To.xicological Aspects, Y. Uello (Ed.), Kodansha Ltd., Tokyo. 1983, pp. 171-176. 209. Schiefer, H.B., C.G. Rousseaux, D.S. Hancock, and B.R. Blakley, Effects of low-level long-term oral exposure to T-2 toxin in CD-1 mice. Food Cherrr. 70xico/., 25593-601 (1987). 210. Schoental, R., The role of nicotinamide and of certain other modifying factors in diethylni-

Fusarium

211. 212. 213.

214. 215. 216. 217.

21 8.

219.

220.

221. 222. 323. 223.

325.

226.

227.

228. 229. 230.

231.

569

trosamine carcinogenesis. Fusaria mycotoxins and “spontaneous” tumors in animals and man, Ccrncer, 40: 1833- 1840 (1977). Schoental. R., Mycotoxins in food and the variations in tumor incidence among laboratory rodents, Nzrtr. Cancer, 1 :13-14 (1978). Schoental, R, Fusarium mycotoxins in the aetiology of neonatal abnormalities, cardiovascular and sexual disorders and tumours in man and animals, Toxicon, 17:164 (1979). Schoental, R., The role of Fztsariunz mycotoxins in the aetiology of tumours of the digestive tract and of certain other organs in man and animals, Front. Gastrointest. Res., 4:17-24 (1979). Schoental, R.. Cancer of the digestive tract and mycotoxins of Fusarium species, Lnncet, 593 (September 13, 1980). Schoental, R., Relationships of Fz~sar-izt~n mycotoxins to disorders and tumors associated with alcoholic drinks, Nrrtr. Cancer, 2:88-92 (1980). Schoental, R., Relationships of Fztsarium toxins to tumours and other disorders in livestock, J. Vet. Phanrlcrcol. Ther., 4: 1-6 (1981). Schoental, R.. Fzrsar-i~~nz mycotoxins and the effects of high-fat diets. Nutr. Cancer, 35762 (1981). Li, M.X.. and S.J. Cheng, Etiology of carcinoma of the esophagus. In Carcinonta of the Esophagus am! Gcrstr-ic Cardicr, G.J. Huang and Y.K. Wu (Eds.), Springer-Verlag, Berlin, 1984. pp. 31-51. Leonov. A.N.. Current view of the chemical nature of factors responsible for alimentary toxic leukia. In Mycotoxins in H~rmnnand Animrl Hecrlth, J.V. Rodricks, C.W. Hesseltine, and M.A. Mehlman (Eds.), Pathotox Publishers, Park Forest South, IL. 1977, pp. 323-328. Van Egmond. H.P., Current situation on regulations for mycotoxins. Overview of tolerances and status of standard methods of sampling and analysis, Food Addit. Contam., 6:139-188 (1989). Woronin, M.. Uber das “Taumelgetreide” in Sud-Ussurien, Bot. Zeitschr., 49:81-93 (1891). Shapovalov. M., Intoxicating bread, Phytopathology, 7:384-386 (1917). Dounin, M., The fusariosis of cereal crops in European Russia in 1923, Phytopat~~o/ogy, 16: 305-308 (1926). Ueno. Y.. T. Tanaka, A. Hasegawa, Z.-H. Hu, and D.D. Xu. Deoxynivalenol, nivalenol and zearalenone in scabby wheat from Shanghai, China. J. Food Hyg. SOC.Jpn., 37: 180-182 (1 986). Lee. U.-S.. H.-S. Jang, T. Tanaka, A. Hasegawa, Y.-J. Oh. and Y. Ueno, The coexistence of the Fmarizrm mycotoxins nivalenol, deoxynivalenol and zearalenone in Korean cereals harvested in 1983, Food Addit. Conturn., 2185-192 (1985). Yoshizawa, T., and N. Morooka, Trichothecenes from mold-infested cereals in Japan. In Mycotoxins in Human and Animal Health, J.V. Rodricks, C.W. Hesseltime. and M.A. Mehlman (Eds.), Pathotox Publishers, Park Forest South, IL. 1977, pp. 309-321. Bhat, R.V., Y. Ramakrishna, S.R. Beedu, and K.L. Munshi, Outbreak of trichothecene nlycotoxicosis associated with consumption of mould-damaged wheat products in Kashmir Valley, India, Lancet, 35-37 (January 7. 1989). Ramakrishna, Y., R.V. Bhat, and S. Vasanthi, Natural occurrence of mycotoxins in staple foods in India. J. Agr-ic. Food Chem., 38:1857-1859 (1990). Wang, Z.-G., J.-N. Feng. and Z. Tong, Human toxicosis caused by moldy rice contaminated with Fzlsar-itlnl and T-2 toxin, BioMed. Emiron. Sci., 6:65-70 (1993). Tanaka, T., H. Hasegawa, Y. Matsuki. Y. Matsui. U.-S. Lee, and Y. Ueno, Co-contamination of the Fusar.ium mycotoxins, nivalenol, deoxynivalenol, and zearalenone in scabby wheat grains harvested in Hokkaido, Japan, J. Food Hyg. SOC.Jpn., 26:519-522 (1985). Tanaka, T., A. Hasegawa, S. Yamamoto, U.-S. Lee, Y. Sugiura, and Y. Ueno, Worldwide Contamination of cereals by the Fzlsar-izm mycotoxins nivalenol, deoxynivalenol, and zearalenone. 1. Survey of 19 countries, J. Agric. Food Chern., 36:979-983 (1988).

570

Marasas

232. Sugiura, Y., K. Fukasaku. T. Tanaka. Y. Matsui. and Y. Ueno, Fusariurn poae and Fusari~a~n crooh-ellense,fungi responsible for the natural occurrence of nivalenol in Hokkaido, Appl. Emiron. Microbiol., 59:3334-3338 (1993). 233. Burgess, L.W.. P.E. Nelson. and T.A. Toussoun, Characterization. geographic distribution and ecology of Fusariur~zcroohlellense sp. nov., Trans. Br. M y o l . SOC.,79:497-505 (1982). South 234. Scott, D.B., E.J.H. de Jager, and P.S. Van Wyk. Head blight of irrigated wheat in Africa, Pltytoplzylactica, 20:317-319 (1988). 235. Kwasna, H., and J. Chelkowski, Occurrence of Fusarium crooXwelleme in Poland. Acta M ~ c o ~24:73-177 ., (1989). in corn earsnaturally 236. Visconti, A., J. Chelkowski. M. Solfrizzo, and A. Bottalico, Mycotoxins infected with Fusarium grmnirzearum and F. crooh~ellerzse,Can. J. Plant Pathol., 12: 187189 (1990). 237. Ueno. Y., U.-S. Lee, T. Tanaka, A. Hasegawa, and Y. Matsuki, Examinationof Chinese and U.S.S.R. cereals for the Fusarium mycotoxins, nivalenol, deoxynivalenol and zearalenone. Toxicon, 24:618-621 (1986). Pimenova, L.P. Zakharova, and N.I. Muzy238. Tutelyan. V.A., K.I. Eller. V.S. Sobolev. V.V. chenko, A survey of the occurrence of deoxynivalenol in wheat from 1986-1988 harvests in the USSR, Food Addit. Contam., 7:521-525 (1990). 239. Lee, U.-S.. H.-S. Jang, T. Tanaka, A. Hasegawa, Y.-J. Oh, C.-M. Cho. Y. Sugiura, and Y. Ueno, Further survey on the Fusarium mycotoxins in Korean cereals. Food Addit. Contarn., 31253-261 (1986). 240. Abbas, H.K., C.J. Mirocha, and J. Tuite. Natural occurrence of deoxynivalenol, 15-acetyldeoxynivalenol, andzearalenonein refusal factor corn stored since 1972,Appl. Emirow Microbiol., 51:841-843 (1986). 241. Trenholm, H.L., W.P. Cochrane, H. Cohen, J.I. Elliot, E.R. Famworth,D.W. Friend, T.M.G. Hamilton, J.F.Standish.and B.K. Thompson, Survey ofvomitoxin contamination ofthe 1980 Ontario white winter wheat crop. Results of survey and feeding trial, J. Assoc. Of Anal. Chent., 66:92-97 (1983). 242. Tanaka, T., A. Hasegawa, S. Yamamoto, Y. Sugiura, and Y. Ueno, A case report on a nlinor contamination of nivalenol in cereals harvested in Canada, Mycopathologia, 101: 157- 160 (1988j. 243. Sydenham, E.W., P.G. Thiel, W.F.O. Marasas, and J.J. Nieuwenhuis, Occurrenceof deoxynivalenol and nivalenol in Fusarirtm gramineorum infected undergrade wheat in South Africa, J. Agric. Food Clzent., 37:921-926 (1989). 234. Marasas, W.F.O., S.J. van Rensburg, and C.J. Mirocha, Incidence of Fusariunz species and the mycotoxins, deoxynivalenol and zearalenone, in corn produced in esophageal cancer areas in Transkei, J. Agric. Food Chenz.. 27: 1108-1 112 (1979). 245. Hsia, C.C., J.L. Wu. X.Q. Lu, and Y.S. Li. Natural occurrence and clastogenic effects of nivalenol. deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol,andzearalenone in corn from a high-risk area of esophageal cancer. Cancer Detect. Prevent.. 13:7986 (1988). 246. Yoshizawa, T., A. Yamashita, and Y. Luo. Fumonisin occurrence in corn from high- and low-risk areas for human esophageal cancer in China, Appl. Ewiro~z.Microbiol., 60:16261629 (1994). 247. Rheeder, J.P., W.F.O. Marasas, P.G. Thiel, E.W. Sydenham, G.S. Shephard, and D.J. Van Schalkwyk, Fzuuriurn nrorriliformd and furnonisins in corn in relation to human esophageal cancer in Transkei, Pltytopatl~olop,82:353-357 (1992). 248. Kurtz. H.J., and C.J. Mirocha, Refusal factor and emetic syndrome of swine. In Mycotoxic Fuigi, Mycotosiru, Mycotoxicoses. An Encyclopedic Hcmdbook, Vol. 2, T.D. Wyllie and L.G. Morehouse (Eds.), Marcel Dekker, New York, 1978,pp. 265-268. 249. Mundkur, B.B., Some preliminary feeding experiments with scabby barley, Phytopathology, 24:1237-1243 (1934).

Fusarium

571

250. Curtin, T.M., and J. Tuite. Emesis and refusal of feed in swine associated with Gibber-el/ci zeae-infected corn. Life Sei., 5:1937-1944 (1966). 251. McMullen, M., R. Jones. and D. Gallenberg, Scab of wheat and barley: a reemerging disease of devastating impact, Plalzt Dis.. 81:1340-1348 (1997). 352. Tuite, J., G. Shaner, G. Rambo, J. Foster. and R.W. Caldwell, The Gibberella ear rot epidemics of corn in Indiana in 1965 and 1972, Cereal Sei. Todq,. 19:238-231 (~1974). 253. Sutton, J.C., W. Baliko, and H.S. Funnel, Relation of weather variables to incidence of zearalenone in corn in southern Ontario, Ccrn. J. Plant Sci., 60: 149-155 (1980). 254. Kuiper-Goodman, T.. P.M. Scott, and W. Watanabe, Risk assessment of the mycotoxin zearalenone, Regul. Tosicol. Phnrrnacol., 7253-306 (1 987). 255. Vesonder. R.F., A. Ciegler, H.R. Burmeister. and A.H. Jensen, Acceptance by swine and rats of corn amended with trichothecenes, Appl. Erniron. Microbiol., 38344-346 (1979). 256. Forsyth, D.M., T. Yoshizawa, N. Morooka. and J. Tuite, Emetic and refusal activity of deoxynivalenol to swine, Appl. Emiron. Microbiol., 34547-552 (1977). 257. Friend, D.W.. H.L. Trenholm. B.K. Thompson, P.S. Fiser, and K.E. Hartin, Effect of feeding diets containing deoxynivalenol (vomitoxin)-contaminated wheat or corn on the feed consumption, weight gain, organ weight and sexual development of male and female pigs. Cm. J. Alzinl. Sci.. 66:765-775 (1986). 258. Foster, B.C., H.L. Trenholm.. D.W. Friend, B.K. Thompson, and K.E. Hartin. Evaluation of different sources of deoxynivalenol (vomitoxin) fed to swine, Ccrn. J. Alzirn. Sci.. 66: 11491154 (1986). 259. Burmeister, H.R., R.F. Vesonder. and W.F. Kwolek, Mouse bioassay for Fusarizm tnetabolites: rejection or acceptance when dissolved in drinking water, Appl. Emiror). Microbiol., 39:957-961 (1980). 260. Ueno, Y.. K. Ishii, N. Sato, and K. Ohtsubo. Toxicological approaches to the metabolites of fusaria. VI. Vomiting factor from moldy corn infected with Fmw-izanr spp., Jpn. J. E.y. Med., 44:123-127 (1973). 261. Ishii, K., Y. Ando, and Y. Ueno, Toxicological approaches to the metabolites of fusaria. IX. Isolation of vomiting factor from moldy corn infected with Fzrsarizmz species, Cl?eri?.Pl~nr~rz. Bull., 23:2162-2164 (1975). 362. Huff, W.E.. J.A. Doerr, P.B. Hamilton. and R.F. Vesonder. Acute toxicity of vomitoxin (deoxynivalenol) in broiler chickens, Poulr. Sci.. 60:1412-1414 ( 1981). 263. Forsell, J.H.. R. Jensen, J.-H. Tai, M. Witt, W.S. Lin, and J.J. Pestka, Comparison of acute toxicities of deoxynivalenol (vomitoxin) and 15-acetyldeoxyninivalenol in the B6C3F1 mouse, Food Chenz. Toxicol., 35:155-162 (1987). 264. Scott, P.M., H.L. Trenholm, and M.D. Sutton (Eds.), Mycoto.virzs:A Ccrrrcrdicrn Perspectille. National Research Council of Canada, Publ. No 22848, Ottawa, Canada, 1955, pp. 1-155. 265. Ryu. J.C., K. Ohtsubo, N. Izumiyama, K. Nakamura, T. Tanaka, H. Yamamura, and Y. Ueno, The acute and chronic toxicities of nivalenol in mice. Fztrzdam. Appl. Tosicol.. 11:38-47 (1988). 266. Ryu, J.C.. F. Tashiro, N. Konzaki, and Y. Ueno. Toxicological studies on nivalenol. I. Lethal toxicity in mice and cytotoxicity to cultured cells, PI-oc. Jpn. Assoc. Myotosicol., 24:3134 (1986). 267. Ueno. Y ., M. Nakajima, K. Sakai, K. Ishii, N. Sato, and M. Shimada, Comparative toxicology of trichothec mycotoxins: Inhibition of protein synthesis in animal cells, J. Biochenz., 74: 285-396 (1973). 268. Ueno, Y., Y. Ishikawa. K. Amakai, M. Nakajima, M. Saito. M. Enomoto, and K. Ohtsubo, Comparative study of skin-necrotizing effect of scirpene metabolites of fusaria, Jlm. J. Exp. Men.. 40:33-38 (1970). 269. Sato, N., and Y. Ueno, Comparative toxicities of trichothecenes. In Mycoto.rim in H w z m crncl Anirrzcrl Health, J.V. Rodricks, C.W. Hesseltine, and M.A. Mehlman (Eds.), Pathotox Publishers. Park Forest South, TL, 1977, pp. 295-321.

572 270. 271.

272. 273.

274.

275.

276. 277. 278. 279. 280. 281.

282.

283. 284. 285.

286.

287.

288. 289.

Marasas Ryu, T.C.,K. Ohtsubo, N. Izumiyama, M. Mori, T. Tanaka, and Y. Ueno, Effects of nivalenol on the bone marrow in mice, J. Toxicol. Sci.. 12:ll-21 (1987). Trenholm, H.L.. D.W. Friend, B.K. Thompson, and D.B. Prelusky, Effects of zearalenone and deoxynivalenol combinations fed to swine, Proc. Jplz. Assoc. Mycotoxicol. Special Issue, 1:101-102 (1988). ,Gilbert. J., Current views on the occurrence and significance of Fzrsarizrm toxins, J. Appl. Bacteriol. Syntp. Szypl.. 89s-98s (1989). International Agency for Research on Cancer (IARC), Toxins derived from Fusarium granzinearum. F. C L I ~ W Z O and ~ L I F. ~ croohwellense: zearalenone, deoxynivalenol, nivalenol and fusarenone X, IARC Morzogmphs 011 the E\dtation of the Carcinogenic Risks to H m a n s : Some Nnturally Occurriq Substances: Food Items, Heterocyclic Aromatic Amines m d Mycotoxins, 56:397-444 (1993). Kurtz, H.J.. and C.J. Mirocha, Zealenone (F-2) induced estrogenic syndrome in swine. In Mycotoxic Fungi, Mycotoxins, Mycotoxicoses. An Encyclopedic Hcmdbook, Vol. 2, T.D. Wyllie and L.G. Morehouse (Eds.j. Marcel Dekker. New York. 1978, pp. 256-268. Mirocha. C.J., S.V. Pathre, and C.M. Christensen, Zearalenone. In Mycoto;lins in Hzmn~t and Aninzal Health, J.V. Rodricks, C.W. Hesseltine, and M.A. Mehlrnan (Eds.). Pathotox Publishers, Park Forest South, IL, 1977, pp. 345-364. Nelson, G.H., C.M. Christensen, and C.J. Mirocha, Fusarizan and estrogenisrn in swine, J. Am. Vet. Med. Assoc.. 163:1276-1277 (1973). Sienz de Rodriguez, C.A., Environmental hormone contamination in Puerto Rico, N. Engl. J. Med.. 310:1741-1742 (1984). Sienz de Rodrigues, C.A., A.M. Bongiovanni, and L. Conde de Borrego. An epidemic of precocious development in Puerto Rican children, J. Pediatr., 107:393-396 (1985). Campbell, G.D.. Sporadic clinical human mycotoxicosis. A widespread new unitarian pathology? Nlcti: Hetrlth. 721-33 (1990). Campbell. G.D., Trichothecene mycotoxicosis-a new entity? S. Afi-. Med. J., 80:361-362 (1991). Szuets, P.. A. Mesterhazy. G.Y. Falkay, and T. Bartok, Early telarche symptoms in children and their relations to zearalenon contamination in foodstuffs, Cereal Res. C O ~ I ? I U25:429II., 336 ( 1997). Rogers, C.G., and C. Hiroux-Metcalf, Cytotoxicity and absence of mutagenic activity of vomitoxin (4-deoxynivalenol) in a hepatocyte-mediated mutation assay with V79 Chinese hamster lung cells, Cancer Lett., 20:29-35 (1983). Morrisey, R.E.. Teratological study of Fisher rats fed diets containing added vomitoxin, Food Cherlz. Toxicol., 22:453-457 (1984). Khera, K.S., C. Whalen, and G. Angers, A teratology study on vomitoxin (4-deoxynivalenol) in rabbits, Food Chem. Toxicol., 24:421-424 (1986). National Toxicology Program (NTP). Carcinogenesis Bioassay of Zearnlenone in F344hV Rats mzd B6C3F1 Mice, NTP Tech. Rep. Ser. No. 235, Dept. Health Human Serv., Research Triangle Park, NC, 1982. Becci, P.J., W.D. Johnson, F.G. Hess, M.A. Gallo. R.A. Parent, and J.M. Taylor, Combined two-generation reproduction teratogenesis study of zearalenone in the rat, J. Appl. Toxicol., 2:201-206 (1982). Becci, P.J., K.A. Voss. F.G. Hess, M.A. Gallo, R.A. Parent, and K.R. Stevens, Long-term carcinogenicity and toxicity study of zearalenone in the rat, J. Appl. To.xico1.. 2247-254 (1982). Martin. P.M.D., Fungi associated with common crop products and their significance, S. Afi-. Med J.. 48:2374-2378 (I 974). Martin, P.M.D., and G.A. Gilman,A Corzsideratiorz of the Mycotoxin Hypothesiswith Special Reference to the kl~cojiorcr of Maize, Sorghum, Wheat and Grounclnuts, Rep. Trop. Prod. Inst., G10, Tropical Products Institute, London. 1976, pp. 1-112.

Fusarium

573

290. Martin, P.M.D.. and P. Keen, The occuuence of zearalenone in raw and fermented products from Swaziland and Lesotho, Sabour-auclia. 16: 15-22 (1978). 291. Pfeiffer, C.J. (Ed.), Curzcer of tlze Esophagus, CRC Press, Boca Raton, FL, 1982. 292. Huang, G.J.,andY.K.Wu(Eds.), Curcirzomn of the Esophagtrs a d Gastric Cardia, Springer-Verlag. Berlin, 1984. 293. Drake, M.. Gnstro-Esoplmgeal Cytology, S. Karger, Basel. 1985. 294. Day, D.W., Biopsy Pathology of the Oesophngus, Stomuch a d Duodemnz, Chapman & Hall, London, 1986. 295. Rose, E.F.. Esophageal cancer in the Transkei. J. Natl. Ccrrzcer Zrzst., 5 1 :7-16 (1973). 296. Liu, B.Q., and B. Li, Epidemiology of carcinoma of the esophagus in China. In Ccrrcirlorm of the Esophagus a d Gastric Cardia, G.J. Huang and Y.K. Wu (Eds.), Springer-Verlag, Berlin, 1984, pp.1-24. 297. Jaskiewicz, K.. W.F.O. Marasas, and F.E. Van der Walt, Oesophageal and other main cancer patterns in four districts of Transkei, 1981-1984, S. Afr. Med. J., 7227-30 (1987). 298. Makaula. N.A., W.F.O. Marasas. F.S. Venter, C.J. Badenhorst, D. Bradshaw, and S. Swanevelder, Oesophageal and other cancerpatterns in four selected districts of Transkei. southern Africa, A f . J. Health Sci.. 3:ll-15 (1996). 299. Li, M.H., P. Li. and P.J. Li, Research on esophageal cancer in China, Adv. Ccrncer Res., 33: 174-249 (1980). 300. Yang, C.S., Research on esophagealcancer in China: areview. CancerRes., 40:2633-2644 (1980). 301. Knlet, J., and E. Mahboubi, Esophageal cancer in the Caspian littoral of Iran: initial studies, Science. 175:846-853 (1972). 302. Joint Iran-International Agency for Research on Cancer Study Group, Esophageal cancer J. Natl. studies in the Caspian littoral of Iran. Results of population studies-a prodrome, Cuncer Inst., 59:1127-1138 (1977). 303. Hormozdiari, H.. N.E. Day, B. Aramesh. and E. Mahboubi. Dietary factors and esophageal cancer in the Caspian littoral of Iran, Cancer Res., 35:3493-3498 (1975). 304. National Cancer Institute (NCI), Cancer Statistics RevieM11973-1987, National Institutes of Health Publication, No. 90-2789, 1990. 305. Fraumeni. J.F., and W.J. Blot, Geographic variation in esophageal cancer mortality in the United States, J. Chronic Dis., 30:759-767 (1977). 306. O’Brien. P.H., E.F. Parker, and H.B. Gregory, Epidemiology and treatment of carcinoma of the esophagus in South Carolina, a high-risk area in the U.S. In Cancer of the Esoplzaglrs, Vol. I., C.J. Pfeiffer (Ed.), CRC Press. Boca Raton, FL, 1982. pp. 65-80. 307. Brown, L.M., W.J. Blot. S.H. Schumann, V.M. Smith, A.G. Ershow, R.D. Marks. and J.F. Fraumeni, Environmental factors and high risk of esophageal cancer among men in coastal South Carolina, J. Ncltl. Cmcer Inst., 80:1120-1125 (1988). esophagus. In Curcinoma of the Esophngus a d Gastric 308. Xia. A.J., Carcinogenesis in the Cardia, G.J. Huang and Y.K. Wu (Eds.), Springer-Verlag, Berlin. 1984. pp. 52-76. 309. Van Rensburg, S.J., Epidemiologic and dietary evidence for aspecific nutritional predisposition to esophageal cancer. J. Natl. Cmcer Inst., 67243-251 (1981). 3 10. Van Rensburg, S.J., D.B. du Bruyn, and D.J. Van Schalkwyk. Promotion of methylbenzylnitrosamine-induced esophageal cancer in rats by subclinical zinc deficiency, Nutr. Rep. h t . . 22:891-899 (1980). 31 1. Van Rensburg, S.J., A.S. Benadk. E.F. Rose,and J.P. du Plessis. Nutritional status of African populations predisposed to esophageal cancer, Nutr. Cancer-,4:206-216 ( 1983). 312. VanRensburg, S.J., J.M. Hall, and D.B. du Bruyn, Effects ofvarious dietary staples on esophageal carcinogenesis induced in rats by subcutaneously administered N-nitrosomethylbenzylamine. J. Natl.. Cmzcer-Zmt., 75561-566 (1985). 313. Van Rensburg, S.J., J.M. Hall, and P.S. Gathercole, Inhibition of esophageal carcinogenesis in corn-fed rats by riboflavin, nicotonic acid, selenium. molybdenum, zinc and magnesium. N m . Cnncel-, 8:163-170 (1986).

574 314.

315.

3 16.

317.

318.

319.

320.

321.

333.

323. 323.

325.

326.

327.

328.

329.

Marasas Kibblewhite. M.G.. S.J. Van Rensburg, M.C. Laker. and E.F. Rose, Evidence for an intimate geochemical factor in the etiology of esophageal cancer, Emiron. Res., 33370-378 (1983). Van Helden, P.D., A.D. Beyers, A.J. Bester, and K. Jaskiewicz, Esophageal cancer: vitamin and lipotrope deficiencies in an at risk South African population, Nzctr. Cancer, 10247-255 (1987). Jaskiewicz, K.. W.F.O. Marasas, C. Lazarus,A.D. Beyers, and P.D. VanHelden, Association of esophageal cytological abnormalities with vitamin and lipotrope deficiencies in populations at risk for esophageal cancer. Arzticcrncer. Res., 8:711-716 (1988). Jaskiewicz, K., W.F.O. Marasas, J.E. ROSSOUW, F.E. Van Niekerk, and E.W.P.,Heine, Selenium and other mineral elements in populations at risk for oesophageal cancer, Cancer. 62: 2635-2639 (1988). Li, J.-Y., A.G. Ershow. Z.-J. Chen, S. Wacholder. G.-Y. Li, B. Li. and W.J. Blot, A casecontrol study of cancer of the esophagus and gastric cardia in Linxian. Znt. J. Curzcer, 43: 755-761 (1989). Li, J.-Y., P.R. Taylor. B. Li.S. Dewsay, G.-Q. Wang, A.G. Ershow, W. Guo, S.-F. Liu, C.S. Yang, Q. Shen, W. Wang, S.D. Mark, X.-N. Zon, P. Greenwald, Y.-P. Wu, and W.J. Blot. Nutrition intervention trials in Linxian. China: multiple vitamin/mineral supplementation, cancer incidence, and disease-specific mortality among adults with esophageal dysplasia, J. N d . Cancer Irzst.. 85: 1493-1398 (1993). Blot, W.J., J.-Y. Li, P.R. Taylor, W. Guo, S. Dawsey, G.-Q. Wang. C.S. Yang, S.-F. Zheng, M. Gail, G.-Y. Li, Y. Yu, B.-Q. Liu, J. Tangrea. Y.-H. Sun, F. Liu, J.F. Fraumeni, Y.-H. Zhang, and B. Li, Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations. cancer incidence, and disease-specific mortality in the general population, J. Nntl. Cnrzcer Zrzst., 85: 1483-1492 (1993). Rheeder. J.P., W.F.O. Marasas. M.P.W. Farina,G.R. Thompson, and P.E. Nelson, Soil fertility factors in relation to oesophageal cancer risk in areas in Transkei, southern Africa, Ezu. J. Cnncer Plrverzt., 3:49-56 (1994). Sydenham, E.W., W.C.A. Gelderblom, P.G. Thiel, and W.F.O. Marasas, Evidence for the natural occurrence of furnonisin B,, a mycotoxin produced byFusnrizalr r~roniliforme,in corn. J. Agric. Food Clrenz., 38:285”90 ( 1990). Marasas, W.F.O., Occurrence of Fzrscrriunz rnorziliforrtre and fumonisins in maize in relation to human health (Editorial), S. Afi-. Mecl. J.. 83:382-383 (1993). Marasas. W.F.O.. G.S. Shephard, E.W. Sydenham, and P.G. Thiel. Worldwide contamination of maize with fumonisins: food-borne carcinogens produced by Fuscrrium moniliforrne. In Cereal Scieme crnd Teclrnology: Iqvact on cr CIzuilgirzg Africcr, J.R.N. Taylor, P.G. Randall, and J.H. Viljoen (Eds.), CSIR, Pretoria, 1993, pp. 791-805. Marasas. W.F.O., P.G. Thiel. W.C.A. Gelderblom, G.S.Shephard. E.W. Sydenham. and J.P. Rheeder, Fumonisins produced by Fztscmirrn nronilifonne in maize: foodborne carcinogens of Pan African importance, Aft-. Neudrtter- Ormp. Henlth Snfeo. Suppl., 2/93: 1 1- 18 ( 1993). Cook, P.. Cancer of the oesophagus in Africa. A summary and evaluation of the evidence for the frequency of occurrence. and a preliminary indication of the possible association with the consumption of alcoholic drinks made from maize, Br. J. Ccrncer, 25:853-880 (1971). Marasas. W.F.O., Mycotoxicological investigations on corn produced in esophageal cancer areas in Transkei. In Ccrrzcer of the Esophagus, Vol. I.. C.J. Pfeiffer (Ed.), CRC Press, Boca Raton. FL, 1982, pp. 29-40. Marasas. W.F.O.. F.C. Wehner, S.J. Van Rensburg, and D.J. Van Schalkwyk, Mycoflora of corn produced in human esophageal cancerareas in Transkei, southern Africa, Phytopcrtlzolo g ~71~792-796 , (1981). Marasas. W.F.O.. K. Jaskiewicz, F.S. Venter, and D.J. Van Schalkwyk, Fuscrrium moniliforme contamination of maize in oesophageal cancer areas in Transkei, S. Afr-. Mecl. J.. 74: 110-1 14 (1988).

Fusarium

575

330. Van Rensburg, S.J., E.S. Bradshaw, D. Bradshaw, and E.F. Rose, Oesophageal cancer in Zulu men, South Africa: a case-control study, Br. J. Cancer, 51:399-405 (1985). 33 1. Marasas, W.F.O., Fumonisins: Their implications for human and animal health, Not. To-yim, 3:193-198 (1995). 332. Marasas, W.F.O., Fumonisins: History, worldwide occurrence and impact. In Fz4rnonisins in Food, L.S. Jackson, J.W. de Vries, and L.B. Bullerman (Eds.), Plenum Press, New York, 1996, pp.1-17. 333. Gelderblom. W.C.A.. S.D. Snyman,S. Abel, S. Lebepe-Mazur, C.M. Smuts, L. van der Westhuizen,W.F.O.Marasas, T.C. Victor, s. Knasmuller,and W. Huber,Hepatotoxicityand carcinogenicity of the fumonisins in rats. A review regarding mechanistic implications for establishing risk in humans. In Furnortisins in Food, L.S. Jackson, J.W. de Vries, and L.B. Bullerman (Eds.), Plenum Press, New York, 1996, pp. 279-296. 334. Marasas, W.F.O., Risk assessment offumonisins produced by Fusarium ntonilifor1ue in corn. Proceedings of the Fifth European Fzmriunr Seminar, Szeged. Hungary, Cereal Res. Corw I H L I I I , . , 25:399-406 (1997). low incidence 335. Zhen, Y .Z., Isolation and culture of fungi from thecereals in five high and three counties of esophageal cancer in Henan Province (China). Zhorzghzra Zhorzglia Zashi, 6:2729 (1984). 336. Rossi, M., E. Ancona. G. Mastrangelo, D. Solimbergo, P. Paruzzolo, G. Assarini. P. Sorrentina, and A. Peracchia, Epidemiologic findings in esophageal cancer in the Veneto region. Mirzenw Med., 73:1531-1540 (1982). 337. Franceschi, S., E. Bidoli, A.E. Barbn, and C. La Vecchia, Maize andriskof cancers of the oral cavity, pharynx, and esophagus in north Italy, J. Natl. Carlcer Inst.. 82:1407-1411 (1990). 338. Morton, J.F.. Maize and risk of cancers of the oral cavity. pharynx and esophagus in Northeastern Italy, J. Ncltl. Cancer hst., 83:138 (1991). 339. Nabeya, K., Markers of cancer risk in the esophagus and surveillance of high-risk group. In Precuncerozls Lesions of the Gastrointestinal Tract, P. Sherlock, B.C. Marson. and L. Barbara (Eds.). Raven Press, New York, 1983, pp. 71-75. 340. Crespi, M., N. Munoz, A. Grassi. S. Qiong, W.K. Jing, and L.S. Jien, Precursor lesions of oesophageal cancer in a low-risk populations in China: comparison with high-risk populations. Int. J. Cancer, 34599-602 (1984). 341. Shen, Q.,Diagnostic cytology and early detection. In Carcinontn of the Esophagus and Costric Carrlicr., G.J. Huang and Y.K. Wu (Eds.), Springer-Verlag. Berlin, 1984, pp. 155-190. 342. Munoz, N.. M. Lipkin, M. Crespi, J. Wahrendorf. A. Grassi, and L. Shik-Hsien, Proliferative abnormalities of the oesophageal epithelium of Chinese populations at high and low risk for oesophageal cancer, h t . J. Cmcer, 36: 187- 189 (1985). in 343. Jaskiewicz, K., F.S. Venter,andW.F.O.Marasas,Cytopathologyoftheesophagus Transkei, J. Natl. Ccrncer-Inst.. 79:961-967 (1987). 334. Dawsey, S.M., Q. Shen, R.K. Nieberg,S.-F. Liu, S.A.English, J. Cao, B. Zhou, G.-Q. Wang, K.J. Lewin, F.-S. Liu, M.J. Roth. and P.R. Taylor, Studies of esophageal balloon cytology in Linxian. China, Cancer Epid. Biontar-k. Pre\>.,6:121-130 (1997). 345. Marasas, W.F.O., N.P.J. Kriek, V.M. Wiggins, P.S. Steyn, D.K. Towers, and T.J. Hastie. Incidence, geographical distribution, and toxigenicity of Fusariunt species in South African corn, Phytopathology. 69:1181-1185 (1979). n. sp.). Agric. Exp. Str7. Nebraska 17th 346. Sheldon, J.L., A cor11 mold (Fzwarium nto11i1ifoi-n~ A m . Rep., 1904, pp. 23-32. 347. Kellerman, T.S., W.F.O. Marasas, J.G. Pienaar, and T.W. Naude, A mycotoxicosis of EquiFusar-iunzmorliliforrne Sheldon, Onderstepoort J. Vet.Res.. 39:205-208 daecausedby (1972). 348. Butler, T., Notes on a feeding experiment to produce leucoencephalitis in a horse with positive results, Ant. Vet. Rev., 26:748-751 (1902).

576

Marasas

349. Wilson, B.J., Recently discovered metabolites with unusual toxic manifestations. In Mycotoxirzsirz Hwnarz Health, I.F.H. Purchase (Ed.), Macmillan, London, 1971, pp. 223-229. 350. Marasas, W.F.O.. T.S. Kellerman, J.G. Pienaar, and T.W. Naude, Leukoencephalomalacia: a mycotoxicosis of Equidae caused by Fzwarium n?oniliforrtleSheldon, OrzderstepoortJ. Vet. Res., 43:113-122 (1976). 351. Pienaar, J.G., T.S. Kellerman, and W.F.O. Marasas, Field outbreaks of leukoencephalomaverticillioides in South Africa. J. S. lacia in horses consuming maize infected by Fustrriu~~ Aj?. Vet. Assoc.. 5221-23 (1981). 352. Kriek, N.P.J., T.S. Kellerman, and W.F.O. Marasas. A comparative study of the toxicity of FLrsarium verticillioides ( = F. morziliforrne) to horses, primates. pigs. sheep and rats. Onderstepoort J. Vet. Res., 48:129-131 (1981). 353. Kriek, N.P.J.. W.F.O. Marasas, and P.G. Thiel, Hepato- and cardiotoxicity of Fusariurrl verticillioides (F. moniliforrrre) isolates from southern African maize, Food Cosmet. Tosicol.. 19: 447-456 (1981). 353. Jaskiewicz, K., W.F.O. Marasas. and J.J.F. Taljaard. Hepatitis in vervet monkeys caused by Fusnriunl monilifonne. J. Comp. Patllol., 97:281-291 (1987). 355. Fincham. J.E., W.F.O. Marasas, J.J.F. Taljaard, N.P.J. Kriek, C.J. Badenhorst. W.C.A. Gelderblom, J.V. Seier, C.M. Smuts, M. Faber, M.J. Weight, W. Slazus, C.W. Woodroof, M.J. Van Wyk, M. Kruger, and P.G. Thiel, Atherogenic effects in a non-human primate of Fz4scrrium nzonil(forme cultures added to a carbohydrate diet, Atl~erosclerosis,94: 13-25 (1992). 356. Marasas, W.F.O., N.P.J. Kriek, J.E. Fincham, and S.J. Van Rensburg, Primary liver cancer and oesophageal basal cell hyperplasia in rats caused by Fusarium rrzorliliforn~e. Irlt.J. Carlcel-. 34:383-387 (1984). 357. Jaskiewicz, K., S.J. Van Rensburg, W.F. Marasas, and W.C. Gelderblom, Carcinogenicity of Fzrscrrizrrn moniliforrr1e culture material in rats, J. Natl. Cnncer Irlst., 78:321-325 (1987). 358. Wilson. T.M., P.E. Nelson, and C.R. Knepp, Hepatic neoplastic nodules. adenofibrosis, and cholangiocarcinomas in male Fischer rats fed corn naturally contaminated with Fzrsarizrnl nloniliforme, Car-cinogenesis. 6: 1155-1 160 (1985). 359. Mirocha, C.J., H.K. Abbas, and R.F. Vesonder, Absence of trichothecenes in toxigenic isolates of Fustrrizo~lmorziliforme. Appl. Em~iro~z. Mici-obiol., 56:520-525 ( 1990). 360. Thiel, P.G., W.C.A. Gelderblom, W.F.O. Marasas. P.E. Nelson, and T.M. Wilson, Natural occurrence of moniliformin and fusarin C in corn screenings known to be hepatocarcinogenic in rats, J. Agr-ic. Food Cltem., 34:773-775 (1986). 36 1 Kriek. N.P.J., W.F.O. Marasas, P.S. Steyn, S.J. Van Rensburg, and M. Steyn. Toxicity of a moniliformin-producing strain of Fusarium ~~oniliforme var. subglzrtimms isolated from maize, Food Cosnlet. Tosicol., 15:579-587 (1977). 362. Gelderblom. W.C.A., P.G. Thiel, K. Jaskiewicz, and W.F.O. Marasas, Investigations on the carcinogenicity of fusarin C-a mutagenic metabolite of Frrsarizrm nzonilifornle, Can"nogenesis, 7:1899-1901 (1986). 363. Marasas, W.F.O., T.S. Kellerman, W.C.A. Gelderblom, J.A.W. Coetzer. P.G. Thiel, and J.J. Van der Lugt, Leukoencephalomalacia in a horse induced by fumonisin B, isolated from Fusarimr monilfol-r?~e, Ordel-stepoort J. Vet. Res., 55: 197-203 ( I 988). 364. Kellerrnan, T.S.. W.F.O. Marasas. P.G. Thiel, W.C.A. Gelderblom, M. Cawood, and J.A.W. Coetzer, Leukoencephalonlalacia in two horses induced by oral dosing of fumonisin B,. Onderstepoort J. Vet. Res., 57:269-275 (1990). 365. Harrison, L.R., B.M. Colvin. J.T. Green, L.E. Newman. and J.R. Cole, Pulmonary edema and hydrothorax in swine produced by fumonisin B,, a toxic metabolite of Fztscrriunl ~nonili.fomle. J. Vet. Diagn. Invest., 2217-221 (1990). 366. Gelderblom, W.C.A., N.P.J. Kriek. W.F.O. Marasas. and P.G. Thiel, Toxicity and carcinogenicity of the Fusrrrizm nzonilifor~~le metabolite, fumonisin B,, in rats, Ccrrcinogenesis, 12: 1247-1251 (1991).

Fusarium 367.

368.

369.

370.

371.

372.

373. 374.

375. 376. 377. 378. 379.

380.

381. 382.

383.

384. 385. 386.

577

Sydenham, E.W., W.F.O. Marasas. G.S. Shephard, P.G. Thiel, and E.Y. Hirooka, Fumonisin concentrations in Brazilian feeds associated with field outbreaks of confirmed and suspected animal mycotoxicoses, J. Agric. Food Clzen~.,40:994-997 (1992). Sydenharn, E.W., G.S. Shephard, P.G. Thiel, W.F.O. Marasas, J.P. Rheeder, C.E. Peralta Sanhueza, H.H.L. Gonzalez, and S.L. Resnik, Fumonisins in Argentinian field trial corn, J. Agric. Food Chern., 41:891-895 (1993). Rheeder, J.P., E.W. Sydenham, W.F.O. Marasas, P.G. Thiel, G.S. Shephard, M. Schlechter, S. Stockenstrom, D.W. Cronje, and J.H. Viljoen, Fungal infestation and mycotoxin contamination of South African commercial maize harvested in 1989 and 1990, S. Afr. J. Sci., 91: 127-131 (1995). Rheeder, J.P., E.W. Sydenham, W.F.O. Marasas. P.G. Thiel. G.S. Shephard, M. Schlechter, S. Stockenstrom, D.E. Cronje, and J.H. Viljoen. Ear-rot fungi and mycotoxins in South African corn of the 1989 crop exported to Taiwan. Mycopathologia. 127:35-41 (1994). Sydenham. E.W., G.S. Shephard, P.G. Thiel, W.F.O. Marasas, and S. Stockenstrom, Fumonisin contamination of commercial corn-based human foodstuffs, J. Agric. Food Clzem., 39: 2014-2018 (1991). Leslie, J.F., W.F.O. Marasas, G.S. Shephard, E.W. Sydenham, S. Stockenstrom, and P.G. Thiel, Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and F mating populations of Gibbel-ellcr~4jikul.oi (Fusnrium wonilifornze), Appl. Erniron. Microbiol., 62:1182-1187 (1996). Rheeder, J.P., W.F.O. Marasas, and P.E. Nelson, Fusarizun globosuln, a new species from corn in southern Africa, Mycologin. 88:509-513 (1996). Shephard, G.S., P.G. Thiel, and E.W. Sydenham, Determination of fumonisin B, in plasma and urine by high-performance liquid chromatography, J. Clzronlnt. Bio-Med. Appl., 574: 299-304 (1992). Shephard, G.S., P.G. Thiel, and E.W. Sydenham, Liquid chromatographic determination of the mycotoxin fumonisin B? in physiological samples, J. Chomat. A , 692:39-43 (1995). Shephard. G.S., P.G. Thiel, and E.W. Sydenham, Initial studies on the toxicokinetics of fumonisin B, in rats, Food Chenz. Toxicol., 30:277-279 (1992). Shephard. G.S., P.G. Thiel, E.W. Sydenham, and J.F. Alberts, Biliary excretion of the mycotoxin fumonisin B, in rats, Food Chem Toxicol., 32:489-491 (1994). Shephard, G.S., P.G. Thiel, E.W. Sydenham. and P.W. Snijman, Toxicokinetics of the mycotoxin fumonisin B2 in rats, Food Clzenl. Toxicol., 33:591-595 ( 1995). Alberts, J.F.. W.C.A. Gelderblom, R. Vleggaar, W.F.O. Marasas, and J.P. Rheeder. Production of [ ' T ] fumonisin B, by Fzrscrrizon nzonilijiornze MRC 826 in corn cultures, Appl. Environ. Microbiol., 592673-2677 (1993). Cawood, M.E., W.C.A. Gelderblom, J.F. Alberts, and S.D. Snyman, Interaction of IT-labelled fumonisin B mycotoxins with rat hepatocyte cultures, Food Chem. Toxicol., 32:627632 (1994). Shephard, G.S., P.G. Thiel, E.W. Sydenham, J.F. Alberts, and W.C.A. Gelderblom, Fate of a single dose of the lJC-labelled mycotoxin fumonisin B, in rats, Toxicon, 30:768-770 (1992). Shephard, G.S., P.G. Thiel, E.W. Sydenham, J.F. Alberts, and M.E. Cawood, Distribution and excretion of a single dose of the mycotoxin fumonisin B, in a non-human primate, TOXicon. 32:735-741 (1994). Shephard, G.S.. P.G. Thiel, E.W. Sydenham. R. Vleggaar, and J.F. Alberts, Determination of fumonisin B, and identification of its partially hydrolysed metabolites in the faeces of non-human primates, Food Cllem. Toxicol., 3223-29 (1994). Shephard, G.S., P.G. Thiel, E.W. Sydenham, and M.E. Savard. Fate of a single dose of IT-labelled fumonisin B, in vervet monkeys, Nat. Toxins, 3:145-150 (1995). Gelderblom, W.C.A., E. Semple. W.F.O. Marasas, and E. Farber, The cancerinitiating potential of the fumonisin B mycotoxins, Carcinogenesis, 13:433-437 (1992). Knasmuller, S.. N.Bresgen, K. Fekadu. V. Mersch-Sundermann, W. Gelderblom. E. Zohrer,

578

387.

388. 389.

390.

391.

392.

393. 394.

395.

396. 397.

398.

399.

400.

401.

403.

403.

304.

Marasas and H. Eckl, Genotoxic effects of three Frrsurirm mycotoxins, fumonisin B,, moniliformin and vomitoxin in bacteria and in primary cultures of rat hepatocytes. Mutcrt. Res., 391:3948 (1997). Gelderblotn, W.C.A., M.E. Cawood, S.D. Snyman, R.Vleggaar. and W.F.O. Marasas, Structure-activity relationships of fumonisins inshort-term carcinogenesis and cytotoxicity assays. Food Chenr. Toxicol., 31:407-414 (1993). Gelderblom, W.C.A., M.E. Cawood, S.D. Snyman, and W.F.O. Marasas, Fumonisin B, dosimetry in relation to cancer initiation in rat liver, Carcinogenesis, 15:209-214 (1994). Gelderblom. W.C.A., S.D. Snyman, S. Lebepe-Mazur, L. Van der Westhuizen,N.P.J. Kriek, and W.F.O. Marasas, The cancer-promoting potential of fumonisin B, in rat liver using diethylnitrosamine as a cancer initiator, Cancer Lett.. 109:lOl-108 (1996). Gelderblom. W.C.A., S.D. Snyman, L.Van der Westhuizen,and W.F.O. Marasas, Mitoinhibitory effect of fumonisin B, on rat hepatocytes in primary culture, Carcinogenesis, 16:62.5631 ( 1995). Gelderblom, W.C.A., C.M. Smuts, S. Abel, S.D. Snyman. M.E. Cawood, L. Van der Westhuizen, and s. Swanevelder, Effect of fumonisin B, on protein and lipid synthesis in primary rat hepatocytes, Food Clzem. Toxicol., 34:361-369 (1996). Gelderblom, W.C.A., C.M. Smuts, S. Abel, S.D. Snyman, L. Van der Westhuizen. W.W. Huber, and S. Swanevelder. Effect of fumonisin B, on the levels and fatty acid composition of selected lipids in rat liver in vivo, Food Chenz. To.yicol., 35:647-656 (1997). Riley, R.T.. K.A. Voss, H.-S. Yoo, W.C.A. Gelderblom, and A.H. Merrill, Mechanism of fumonisin toxicity and carcinogenesis, J. Food Prot., 57:638-645 (1994). Rheeder, J.P., W.F.O. Marasas, P.S.Van Wyk, W. duToit.A.J. Pretorius, andD.J.Van Schalkwyk, Incidence of Fusar.iunz and Diplodio species and other fungi in naturally infected grain of South African maize cultivars, Phytoyhylacticcr, 2297-102. (1990). Rheeder. J.P.. W.F.O. Marasas, P.S. Van Wyk, and D.J. Van Schalkwyk, Reaction of South African maize cultivars to ear inoculation with Fusorium ruonilifornre, F. gmrninecrrunz and Diplodiu maydis. Phytoyllylacticrr. 2.3213-2.18 ( 1990). Rheeder. J.P., W.F.O. Marasas, and P.S. Van Wyk, Fungal associations in corn kernels and effects on germination, Phytopmtllology, 80:13 1 134 (1990). Rheeder, J.P.. W.F.O. Marasas, andD.J.Van Schalkwyk, Incidenceof Fusarium and Diplodia species in naturally infected grain of South African maize cultivars: a follow-up study, Phytophylactica. 25:43-48 (1993). Thiel, P.G., W.F.O. Marasas, E.W. Sydenham,G.S. Shephard. andW.C.A. Gelderblom, The implications of naturally occurring levelsof fumonisins in corn for human and animal health, Mycoprrtlzologia, 117:3-9 (1992). Norred, W.P., R.D. Plattner, K.A. Voss, C.W. Bacon, and J.K. Porter, Natural occurrence of fumonisins in cornassociated with equine leukoencephalomalacia (abstract),To.xicologist, 9 2 5 8 (1989). Norred, W.P., C.W. Bacon,J.K. Porter, and K.A. Voss. Inhibition of protein synthesis in rat primary hepatocytes by extracts of Fzwarium moniliforrne-contaminated corn, Food Chem. Tos~coZ.,25:89-94 (1990). Voss. K.A.. W.P. Norred, R.D. Plattner, and C.W. Bacon, Hepatotoxicity and renal toxicity in rats of corn samples associated with field cases of equine leukoencephalomalacia, Food Chenz. To.uico1.. 27:89-96 (1990). Voss. K.A., R.D. Plattner, C.W. Bacon, and W.P. Norred, Comparative studies of hepatotoxicityand fumonisins B, and B2 contentofwaterand chloroform/methanolextracts of Flssar-irsnz nzoniliforrtre strainMRC826culturematerial, Myoycrttzologia. 11281-92 (1990). Jackson, M.A., and G.A. Bennett, Production of fumonisin B , by Fusarimz r~or~ilifonne NRRL 13616 in submerged culture, Appl. Erwirorr. Microbial., 56:2296-2298 (1990). Plattner, R.D., W.P. Norred. C.W. Bacon. K.A. Voss, R. Peterson, D.D. Shackelford. and

Fusarium

405,

406.

407.

308,

409.

4 10.

41 1. 412. 413.

414. 415. 416. 417. 418.

579

D. Weisleder, A method of detection of fumonisin in corn samples associated with field cases of equine leukoencephalomalacia, Mycologia, 82:698-702 (1990). Vesonder, R., R. Peterson, R. Plattner, and D. Weisleder, Fumonisin B,: Isolation from corn culture and purification by high performance liquid chromatography, Mycotoxin Res., 6:8588 (1990). Wilson. T.M.. P.E. Nelson, W.F.O. Marasas, P.G. Thiel. G.S. Shephard, E.W. Sydenham, H.A. Nelson, and P.F. Ross, A mycological evaluation and in vivo toxicity evaluation of feed from 41 farms with equine leukoencephalomalacia, J. Vet. Diagrz. Znllest., 2352-354 (1990). Wilson, T.M., P.F. Ross, L.G. Rice, G.D. Osweiler, H.A. Nelson, P.L. Owens, R.D. Plattner, C. Reggiardo, T.H. Noon. and S.W. Pickerell, Fumonisin B, levels associated with an epizootic of equine leukoencephalomalacia, J. Vet. Dingrz. Invest., 2213-216 (1990). Nelson, P.E., R.D. Plattner, D.D. Shackelford, and A.E. Desjardins, Production of fumonisins by Frut-criunz nrorzilifornzc. strains from various substrates and geographic areas, Appl. Enviroll. Microbiol., 572410-2412 (1991). Ross, P.F., L.G. Rice, R.D. Plattner, G.D. Osweiler. T.M. Wilson, D.L. Owens, H.A. Nelson, and J.L. Richard, Concentrations of fumonisin B in feed associated with animal health probl e m , Mycopcrthologia, 114:129-135 (1991). Ross, P.F., L.G. Rice, J.C. Reagor, G.D. Osweiler, T.M. Wilson, H.A. Nelson, D.L. Owens, R.D. Plattner, K.A. Harlin, J.L. Richard, B.M. Colvin, and M.I. Banton, Fumonisin B, concentrations in feeds from 45 confirmed cases of equine leukoencephalomalacia, J. Vet. Diagn. Znl’est., 3:238-241 (1991). Wang, E.. W.P. Norred, C.W. Bacon. R.T. Riley, and A.H. Merrill, Inhibition of sphingolipid biosynthesis by furnonisins, J. Biol. Clzem., 266: 13486-14490 (1991). Merrill. A.H., E. Wang, D.G. Gilchrist, and R.T. Riley, Fumonisins and other inhibitors of de novo sphingolipid biosynthesis. Adv. Lipid Res., 26215-234 (1993). Nelson. P.E., A.E. Desjardins. and R.D. Plattner, Fumonisins, mycotoxins produced by F11sariurn species: biology. chernistly and significance, Annu. Rell. Phytopathol., 3 1233-252 (1993). Norred, W.P., Fumonisins-mycotoxins produced by Fusariunz moniliforme, J. Toxicol. En\iron. Hecrlth, 38:309-328 (1993). Riley, R.T.. W.P. Norred, and C.W. Bacon, Fungal toxins in foods: recent concerns, Arzn~c. Rev. Nutr., 13:167-289 (1993). Miller, J.D., Fungi and mycotoxins in grain: Implications for stored product research, J. Stored Prod. Res.. 31:l-16 (1995). Dutton, M.F., Fumonisins. mycotoxins of increasing importance: their nature and their effects, Pharrnacol. Tlzer., 70:137-161 (1996). Munkvold, G.P., and A.E. Desjardins, Fumonisins in maize. Can we reduce their occurrence? Plant Dis., S1:556-565 (1997). Riley. R.T., K.A. Voss. W.P. Norred. R.P. Sharma. E. Wang, and A.H. Memill, Fumonisins: mechanism of mycotoxicity, Rev. Med. Vet.. 139:617-626 (1998). International Agency for Research on Cancer (IARC), Toxins derived from Fusnriurn moniliforrne: fumonisins B, and B, and fusarin C, IARC Monographs oiz the Evaluation of the CarcirzogerlicRisks to H~~nzans: Some Nrttural/y Occurring Substrrrrces:Food Items arzd Constituents, Heterocyclic Aronratic Amines and Mvcotoxirzs, 56:435-466 (1993). Chu, F.S., and G.Y. Li, Simultaneous occurrence of fumonisin B, and other mycotoxins in moldy corn collected from the People’s Republic of China in regions with high incidences of esophageal cancer, Appl. Envirorz. Microbiol., 60:847-852 (1994). Zhang, H., H. Nagashima, and T. Goto, Natural occurrence of mycotoxins in corn, samples from high and low risk areas for human esophageal cancer in China, Mycotoxins, 4429-35 (1997). Ueno, Y., K. Iijima, S.-D. Wang, Y. Sugiura, M. Sekijima, T. Tanaka, C. Chen, and S.-Z.

,

9

419. 420.

421.

422.

423.

580

424. 425.

426.

427.

328.

429.

430.

431.

433.

433.

434.

435.

436.

437.

438.

Marasas Yu. Fumonisins as a possiblecontributory risk factor forprimary liver cancer: a3-year study of corn harvested in Haimen, China,by HPLC and ELISA, Food Che?n.Toxicol., 35:11431150 (1997). Chamberlain, W.J.. C.W. Bacon. W.P. Norred, and K.A. Voss, Levels of fumonisin B, in corn naturally contaminated with aflatoxins, Food Chenl. Toxicol., 31 :995-998 (1993). Wang, D.-S.,Y.-X. Liang, N.T. Chau, L.D.Dien, T.Tanaka, and Y. Ueno,Natural cooccurrence of Fztsariztnt toxins and aflatoxin B, in corn for feed in North Vietnam, Ncrt. Toxins, 3:445-449 ( 1995). Yamashita. A., T. Yoshizawa, Y. Aiura, P.C. Sachez, E.I. Dizon, R.H. Arim, and Sardjono, Fzrsnrium mycotoxins (fumonisins, nivalenol, and zearalenone) and aflatoxins in corn from southeast Asia, Biosci. Biotech. Bioclzeut.. 59: 1804-1 807 (1 995). Bhat. R.V.. P.H. Shetty, R.P. Amruth. and R.V. Sudershan, A foodborne disease outbreak due to the consumptionof moldy sorghum and maize containing fumonisin mycotoxins,Clin. To.vicol., 35249-255 (1997). Shetty, P.H.. and R.V. Bhat, Natural occurrence of fumonisin B, and its co-occurrence with aflatoxin B, in Indian sorghum, maize and poultry feeds, J. Agric. Food Chenr.. 45:21702173 (1997). Li. M.-X., Y.-Z. Jian, N.-J. Han, W.-G. Fan,J.-L. Ma, and L.E. Bjeldanes, Fusarin C induced esophageal and forestomach carcinoma in mice and rats, chin. J. Oncol., 14:27-29 (1992) [in Chinese]. Shephard, G.S., P.G. Thiel, W.F.O. Marasas, E.W. Sydenham, and R. Vleggaar. Isolation and determination of AAL phytotoxins from corn culturesof the fungus Alterrlaria rrlternrrtcr f.sp. lycopersici, J. Cltroonlcrt., 641:95-100 (1993). Mirocha. C.J., D.G. Gilchrist, A. Martensen, H.K. Abbas. J. Plasencia, and R.F. Vesonder, Production of toxins with equivalent host specific activity, fumonisins and AAL. by nonpathogens of tomato (abstract), Phyfopcrthology, 80:1004 ( 1990). Lamprecht.S.C., W.F.O. Marasas, J.F. Alberts, M.E. Cawood. W.C.A. Gelderblom, G.S. Shephard, P.G. Thiel, and F.J. Calitz, Phytotoxicity of fumonisins and TA-toxin to corn and tomato, Phytopathology, 84:383-391 (1994. Shier. W.T., H.K. Abbas, and C.J. Mirocha, Toxicity of the mycotoxins fumonisins B , and B2and Altermria alternatcr fsp. lycopersici toxin (AAL) incultured mammalian cells,Mycopcrthologia, 116:97-104 (1991). Stinson, S.F.. R.A. Squire, and M.B. Sporn, Pathology of esophageal neoplasms and associated proliferative lesions induced in rats by N-methyl-N-benzylnitrosamine, J. N d .Cancer Illst., 61:1471-1475 (1978). Shephard, G.S., L. Van der Westhuizen. P.G. Thiel, W.C.A. Gelderblom, W.F.O. Marasas, and D.J. Van Schalkwyk, Disruption of sphingolipid metabolism in non-human primates consuming diets of fumonisin-containing Fzrscrrizm nloniliforme material, Toxicon, 34:527533 ( 1996). Sydenham. E.W., L. Van der Westhuizen, S. Stockenstrom, G.S. Shephard, and P.G. Thiel, Fumonisin-contaminated maize: physical treatment for the partial decontamination of bulk shipments, Food Addit. Contmz., 11:25-32 (1 994). Sydenham, E.W., S. Stockenstrom, P.G. Thiel, G.S. Shephard, K.R. Koch, and W.F.O. Marasas, Potential of alkaline hydrolysis for the removal of fumonisins from contaminated corn, J. Agr-ic. Food Cllem.. 43:1198-1201 (1995). Sydenham. E.W.. P.G. Thiel. G.S. Shephard, K.R. Koch. and T. Hutton. Preparation and isolation of the partially hydrolysed moiety of fumonisin B,, J. Agric. Food Chem., 4324002405 (1995).

13 Penicillium John 1. Pitt Food Science Australia, North Ryde, New South Wales. Alrstralicr

I. Introduction 581 11. Significant Perzicillizrn?Mycotoxins582 A. Citreoviridin 582 B. Citrinin 583 Cyclopiazonic C. Acid 584 D. Ochratoxin 584 A E. Patulin 585 F. Penitrem A 585 G. Penicillizrm roqueforti toxins: PR toxin and roquefortine586 H. Secalonic acid D 586 111. ThePrincipalToxigenic

Penicilliunz Species586

References 588

1.

INTRODUCTION

The common foodborne fungal genus Penicillium produces a very wide range of toxic metabolites. A major review in 1981 ( 1) recognized 42 such compounds, and no less than 85 Penicillium species were reported to be toxigenic. This list, drawn principally from two sources (2, 3) was by no means complete. A major problem in understanding the impact of PerzicilZiuln toxins on human health has been persistent inaccuracy in identification. The literature on Penicillirr~nmycotoxins, and the species producing them, includes contributions from many sources. The emphasis in the majority of papers has been on chemistry or toxicology rather than mycology. From the literature, the impression is gained that toxin production by Per~iciIZiumspecies lacks specificity, i.e., that each toxin can be produced by several species. Once a species name appears in the literature as a producer of a particular toxin, it has all too often been accepted as correct without adequate checking and verification, so large lists of putative toxigenic species have built up. For example, citrinin has been reported to be produced by at least 22 species (1 -3). It is doubtful whether more than three of these are correct (4, 5). Misidentifications, even by experts, have also been common: of 12 PenicilZiz,~nz species reported in the literature to produce trenlorgenic toxins, only two were found 581

Pitt

582

tobe accurate (6). A great improvement in our awareness of the genuinely toxigenic species has resulted from a major revision of species mycotoxin associations in Perzicillizm (5). Other considerations must also be assessed if we are to understand the real significance of particular Per~icilliz~m mycotoxins in human or animal health. In particular, are the compounds included in toxin lists (1, 7) really toxic? Second, do they occur naturally and, hence, may affect human and animal health, or are they merely laboratory curiosities? Studies of toxicity, while far from complete, provide guidance on the first question, but answers to the second require more detailed studies of the occurrence and distribution of the toxigenic species. It has become increasingly clear that particular fungi, and hence particular mycotoxins, may be quite specifically associated with particular substrates, and with particular geographical regions. Some highly toxic compounds appear to be of little practical significance because the species producing them are uncommon, or are not found in foods. This chapter provides an up-to-date summary of current knowledge on significant PenicilZi1rm mycotoxins and the species producing them.

II. SIGNIFICANT PENICILLIUM MYCOTOXINS Table 1 lists nine mycotoxins, produced by 16 Pe~ziciZliunzspecies, that the author considers to be significant, or to have potential significance, in human health. Relative toxicities of the various compounds are shown for comparative purposes: for this reason, toxicities in a standard test animal (mouse) and by a standard route of administration (oral) have been provided where possible. The list of species shown as producing each toxin results from the author’s own experience and is undoubtedly conservative. As pointed out earlier, literature reports that list a wide range of species producing each toxin must be treated with great caution. Some general notes follow on the various toxins and the species that produce them. A.

Citreoviridin

The role of citreoviridin in the human disease acute cardiac beriberi has been well documented (8). Acute cardiac beriberi was a common disease in Japan in the second half of the 19th century. Symptoms were heart distress, labored breathing, nausea, and vomiting, followed by anguish, pain, restlessness, and sometimes maniacal behavior. In extreme cases progressive paralysis leading to respiratory failure occurred. It is notable that victims of this disease were often young, healthy adults. In 1910 the incidence of acute cardiac beriberi suddenly decreased: this coincided with implementation of a government inspection scheme that dramatically reduced the sale of moldy rice in Japan (8). Acute cardiac beriberi has been principally associated with P. citreorzigrwn (synonyms P. citreoviride, P. toxiccrriunz), a species that sometimes occurs in rice, is less common in other cereals, and is rarely found in other foods (9, 10). Recent studies (1 1, 12) have failed to find more than occasional infections of P.citreor~igmmin Southeast Asian rice. The threat of citreoviridin toxicosis, at least in advanced Asian countries, appears now to be verylow. However, the possibility cannot be discounted that P. citreorligr~m still occurs in less developed countries of Africa or Asia, where adequate rice drying systems and controls are not yet in place.

Penicillium

583

Table 1 Significant Mycotoxins Knownto Be Producedby Penicillircm Species Mycotoxin

Toxicity, LD5(I"

Citreoviridin

Mice, 7.5 mg/kg i.p. Mice, 20 mg/kg oral

Citrinin

Mice, 35 mg/kg i.p. Mice, 110 mg/kg oral

Cyclopiazonic acid

Rats, 2.3 mg/kg i.p. Male rats, 36 mg/kg oral Female rats, 63 mg/kg oral

Ochratoxin A Patulin

Young rats, 22 mg/kg oral Mice, 5 mg/kg i.p. Mice, 35 mg/kg oral

Penitrem A

Mice. 1 mg/kg i.p.

PR toxin

Mice, 6 mg/kg i.p. Rats, 115 mg/kg oral Mice, 340 mg/kg i.p. Mice. 42 mg/kg i.p.

Roquefortine C Secalonic acid D a

producingb Species Penicillitrm citreonigrum Dierckx Eupenicilliwn oclu-osahoneurn Scott & Stolk P. citt-inml Tholn P. expcrnsum Link P. vert-ucosum Dierckx P. camembet-ti Thorn P. comnzune Thom P ch~ysoge~zzln~ Thom P crustosunz Thom P. griseofirlvum Dierckx P lzirsutum Dierckx P, Iiriclicaturn Westling P, verrucoswn P. espnnsum P. vztlpinunz (Cooke & Massee) Seifert & Samsonc P. griseofid~wnz P. roquefot-ti Thom P. crmtosum Thom P. glanclicola (Oudem.) Seifert & Samsond P. roqtreforti P. roqueforti P. osalicrm Currie & Thom

Refs. 1 and 2. Dosing: i.p., intraperitoneal injection; s.c., subcutaneous injection: i.v., intravenous injection. Ref. 14, and personal observations. Now the accepted name for P. clariforme Bainier (Ref. 68). Now the accepted name for P. gmrzcdntum Bainier (Ref. 68).

Citreoviridin is also produced by EupeniciZZiunz ochroscrZmorzeum (anamorph P. ochl.osnlmonerlnz),which is a relatively uncommon, though widespread species usually associated with cereals (9). The reported formation of citreoviridin by this species in standing U.S. corn is potentially of concern (13j, but E. och~osnZmoneu~~~ was not found in Southeast Asian corn (11, 12). The ecology of this fungus/commodity/mycotoxin relationship remains to be fully elucidated. B. Citrinin Primarily recognized as a metabolite of P. citrinum, citrinin has been reported from more than 120 other species ( 1 -3). However, apart from P. citrinuru, only P. expnnsunr and P. verrucosum are certain producers (4, 14). These three species are among the more commonly occurring Penicillia, and citrinin appears to be abundantly produced in nature (9). Citrinin is a significant renal toxin affecting monogastric domestic animals such as pigs and dogs (15, 16), and is also an important toxin in domestic birds.

584

Pitt

The importance of citrinin in human health is more difficult to assess. When ingested alone, i.e., in the absence of other toxins, significant toxicity appears unlikely. However, citrinin is produced in nature along with ochratoxin A by some isolates of P. verrucoszlrn (1 7), and the possibility of synergy between these toxins must not be discounted. P. viridiccrturn was originally reported to be the main producer of citrinin in Scandinavian barley (17), but this is now known to be incorrect, as the species involved is correctly identified as P. verrucosu~~z (1 8). C.

CyclopiazonicAcid

Cyclopiazonic acid (CPA) is unusual for the range of fungi producing it: Table 1 lists seven common Penicilliunz species, and it is also produced by Aspergillus-flavus. Hence it is of common occurrence in the environment. It has been detected in naturally contaminated corn, peanuts, and other foods and feeds (19). CPA is quite toxic to chickens (2Oj, but appears to be of less concern in humans or other mammals (21). The most common Penicillizmt species producing CPA was usually reported as P. cyclopizrrn (now a synonym of P. nrlrclr?tiogriseurz);however, isolates producing CPA and previously assigned to P. cyclopiurn, P. nurmtiogriseurn, or P. puberulum are correctly identified as P. cornrnurle (22). Apart from Aspergillusflavus, P. conznzune appears to be the most common natural source of CPA, as it frequently grows on cheese in storage. It has been shown (22) that P. conznz1rne is the wild ancestor of the domesticated species P. carnemberti, important in the production of mold-ripened cheeses. Nearly all known isolates of P. cnnzernberti also produce CPA (23j, but apparently not under commercial cheese manufacturing conditions (24). The search for nontoxigenic P. cnnzernber-ti strains suitable as cheese starter cultures has continued (25). D. Ochratoxin A Ochratoxin A (OA) is the most important toxin produced by a Penicillium species. OA was originally described as a metabolite of Aspergillus ochrnceus (26, 27). It was shown subsequently that production of OA by A. oclzrnceus is not common: in one study only two of 33 isolates were found to be producers (28). Later, OA (and sometimes citrinin) was reported to be produced by P. viridicntum (29), and this view prevailed for more than a decade. A complicating factor was that some isolates identified as P. viridicatlm were found not to produce OA, but rather produced another, unrelated, group of toxic metabolites (30). Eventually it became clear that isolates regarded as P. viridicnturn but producing ochratoxin and citrinin were more correctly classified in a separate species, P. verrucoswn (18). The majority of P. verrucosum isolates are now known to be OA producers: in a comprehensive study, 48 of 84 isolates produced OA in the laboratory, and six of these same isolates produced citrinin (18). P. ~ ~ e r n ~ o soccurs u r n commonly in cereals grown in cool, temperate climates, ranging across northern and central Europe, Canada, and northern Asia. The occurrence of this species in cool, temperate cereal crops has two important consequences: ochratoxin A is present in European, Canadian, and Japanese cereal products, especially bread and flour-based foods, and in animals that eat cereals as a major dietary component. Ochratoxin A was detected in Danish pig meats 25 years ago (3 1, 32) and its implications for human and animal health were recognized at the same time. As bread and other cereal products

Penicillium

585

and pig meats are major components of European and other diets, the further consequence is that many people from the cool temperate zone have shown appreciable concentrations of ochratoxin A in their blood (33-37). OA has immunosuppressive, embryonic, and carcinogenic effects. It plays a major role in the etiology of nephritis in Scandinavian pigs, a serious problem in animal health (17, 31, 32). Moreover, because OA is fat soluble and not readily excreted, it accumulates in the depot fat of affected animals, and from there is ingested by humans eating pork, bacon, and other pig meats. Because OA is also cotnmonly consumed in cereals and cereal products, it must be considered to be a human health risk in cool temperate zones. However, the extent of this risk has been difficult to assess. It has been suggested that OA is a causal agent of Balkan endemic nephropathy, a kidney disease with a high mortality rate in certain areas of Bulgaria, Yugoslavia, and Romania, and which has symptoms consistent with OA poisoning (37, 38). However, proof of this association has not been demonstrated, and it is now considered that other, less well-characterized Penicillium mycotoxins may be responsible (39, 40). This major toxicosis still awaits resolution. As noted above, P. verrucosum is common only in cool climates. Where OA has been found to occur in foods from tropical or subtropical regions, Aspe~-gillusspecies are the likely source: either A. ochruceus or closely related species, or A. carbonmius (1 1, 12, 41, 42). E. Patulin Patulin was once considered to be a relatively important mycotoxin (43), but early reports of carcinogenicity (44j have not been substantiated. The most important Perzicilliurn species producing patulin is P. expnsum, best known as a fruit pathogen, but also of widespread occurrence in other fresh and processed foods (10). P. e.xpnnsunz produces patulin as it rots apples and pears, and the use of rotting fruit in juice or cider manufacture can result in high concentrations of patulin, up to 350 pg/L (45) or even 630 pg/L (46) in the resultant juice. Levels in commercial practice are usually much lower than this and, given that patulin appears to lack any known chronic toxic effects in humans, its presence is perhaps of little concern (47). It is probably more important as an indicator of the use of poor-quality raw materials in juice manufacture than as a cause of disease or illness. However, a number of countries have set a limit for patulin of 50 pg/L in apple juice and other apple products (48).

F. Penitrem A Chemicals capable of inducing a tremorgenic (trembling) response in vertebrate animals are regarded as rare-except for fungal metabolites, where at least 20 such compounds have been reported (49, 50). Tremorgens are neurotoxins, which in low doses cause no apparent adverse effects to animals, which are able to feed and function more or less normally while sustained trembling occurs, even over periods as long as 18 days (51). However, relatively small increases in dosage (five- to 20-fold) can be rapidly lethal (52, 53). The presence of tremorgens is exceptionally difficult to diagnose postmortem because no discernible pathological effects are produced. Several tremorgenic mycotoxins are produced by Penicillium species, the most important being the highly toxic penitrem A. Verruculogen, equally toxic, is not produced

586

Pitt

by species of common occurrence in foods. Less toxic Penicilliunz tremorgens include fumitremorgen B, paxilline, verrucosidin, and janthitrems (5). The taxonomy of Pelzicillizm species producing penitrem A has been particularly confusing. Early literature described penitrem production by P. cyclopiurn, P. palitms, P. yuberldzm, P. nzarterzsii, and P. crz~~tosum (49, 54-56). Itwas concluded (6) that this confusion was due to misidentification and that the only common foodborne species producing penitrem A is P. crustosum. Nearly all isolates of P. crustosm produce penitrem A at high levels, so the presence of this species in food or feed is a warning signal (6, 10, 14, 57). Although the tremorgenic toxicosis produced by penitrem A in animals is well documented, the response in humans is unclear. The acute toxicity of penitrem A to animals is such as to preclude experimental dosing to humans. Circumstantial evidence suggests that penitretn tnay produce an emetic effect, which would render serious toxic effects much less likely (58).

G.

Penicilliumroqueforti Toxins: PR Toxin and Roquefortine

P. royueforti as commonly accepted includes two distinct varieties, which are sometimes considered to be two separate species (59). P. roqueforti var. royueforti is widely used as a starter culture for mold-ripened cheeses, while P. royzreforti var. cczmeum occurs in meats and silage (60). P. roqueforti var. roqueforti produces PR toxin, P. royueforti var. cc1rrzez4mproduces patulin, and both varieties produce roquefortine (60). Because of their potential occurrence in staple foods, PR toxin and roquefortine are of considerable significance from the human viewpoint. PR toxin is unstable in cheese in storage, but roquefortine has been isolated from finished products (2). Extensive searches for nontoxic strains for use as cheese starter cultures have so far been largely unsuccessful (25, 61, 62). Roquefortine has also been shown to be produced by P. crustoszm~ (58) and P. ch~ysogerzun~ (14,63). All three species are common in foods, so the presence of roquefortine in cheese and other foods is to be expected. No occurrences of disease from PR toxin or roquefortine have been reported in association with cheese (10). However, roquefortine has been implicated in a case of human intoxication from moldy beer, invaded by P. crzrstosurn (58). H.SecalonicAcid

D

Secalonic acids are dimeric xanthones produced by a range of taxonomically distant fungi (1). Secalonic acid D is produced as a major metabolite of P. oxalicurn and has significant animal toxicity (64, 65). It has been found in nature, in grain dusts (66), at levels of up to 4.5 mg/kg. The possibility that such levels can be toxic to grain handlers should not be ignored.

111.

THE PRINCIPAL TOXIGENIC PENICILLIUM SPECIES

The principal toxigenic PeniciZlium species are shown in Table 2, which is arearrangement of Table 1. The majority of species produce a single toxin in a well-defined one-to-one

Penicillium

587

Table 2 Inlportant Toxigenic Penicillium Species Species ~~~

~

Penicillic acid Verrucosidin

P. citreonigrwn

Viomellein Xanthomegnin Cyclopiazonic acid Cyclopiazonic acid Roquefortine Citreoviridin Citrinin Cyclopiazonic acid Cyclopiazonic acid Penitrem A Roquefortine Citrinin Patulin Cyclopiazonic acid Citreoviridin Secalonic acid D Patulin PR toxin Roquefortine Citrinin Ochratoxin A Cyclopiazonic acid Viomellein Xanthomegnin

__________~

P. cyclopium Westling P. verrucosum var. cyclopium (Westling) Samson et al. P. pubesulunl Bainier P. nlnrtensii Biourge

P. citreoviride Biourge P. toxicnriwtl Miyake P. puberulrm Bainierb P. terrestre‘

P. coprnbiferunz Westling

Only selected synonyms are given. i.e., ones appearing commonly in the llterature on mycotoxins. Senslt Ref. 9. Sensrr Ref. 69.

relationship. However, some species produce more than one toxin: for example, P. expansum, P. c r z ~ ~ t o ~P. u ~roqueforti, n, and some others make three, while P. nurnntiogriseum makes four. Looking at the tabulated information from another viewpoint, it can be seen that some toxins are made by more than a single species, e.g., penitrem A and patulin by four species, and cyclopiazonic acid by seven. Some toxin molecules, e.g., citrinin and patulin, are very simple, while others, notably the penitrems, are very complex, with years of work needed to elucidate their structures. The overall picture of Penicillium and its associated mycotoxins is one of great complexity and diversity. Only one of the 14 species in Table 2, Euyerzicillium odzrosnlmoneurn, has a sexual (Ascomycete) stage. In general, species of Euye~zicilliumand Tdnromyces, the other Ascomycete genus associated with Penicillizrm, are nontoxigenic. Perhaps this reflects their primary and predominant ecological niche in soil, an environment where any toxins produced would be rapidly leached or dispersed. All of the other species in Table 2 belong

588

Pitt

among the common species in the genus (67). More than 50% of commonly isolated Penicillilrm species produce one or more genuinely toxic compounds (5). Of the mycotoxins produced by Penicillinm species, only OA appears likely to be carcinogenic. Most concern about Penicillium toxins lies with their acute toxicity and, in some cases, teratogenicity. Citreoviridin, citrinin, OA, patulin, and penitrem A have all been shown to exist in significant amounts in the natural environment and to have influenced the health of humans or other animals. Moreover, the other compounds listed are also genuinely toxic, and the species producing them are mostly widespread in foods. The involvement of PenicilZiu~zztoxins in human nephritis in northern Europe is highly probable, and in endemic nephropathy in eastern Europe, likely. Much more research is needed into both these disease syndromes. Evaluation of the significance of low concentrations of specific Perzicilliwn mycotoxins in human or animal health is also urgently needed. Information about many Pelzicilliurrz toxins is still fragmentary at best. Reasons include low overt toxicity (i.e., a lack of readily recognized symptoms) and the paucity of routine analytical methods, both mycological and chemical, suitable for foods, feeds, and animal tissues. Despite this, it appears certain that animal health at least is affected by low level ingestion of Penicilliunz toxins, resulting in reduced fertility, increased susceptibility to infectious diseases, reduced feed conversion, and ill thrift. The potential involvement of mycotoxins produced by Penicillium species in low level disease in humans undoubtedly warrants continued research.

REFERENCES 1. Cole, R. J., and Cox, R. H. 1981. Handbook of Toxic Fungal Metabolites. New York: Academic Press. 2. Scott, P. M. 1977.Penicillium mycotoxins. In Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, an Encyclopedic Handbook. Vol. 1. Mycotoxigenic Fungi, eds. T.D. Wyllie and L. G. Morehouse. pp. 283-356. New York: Marcel Dekker. 3. Leistner, L., and Pitt, J. I. 1977. Miscellaneous Penicillium toxins. I n Mycotoxins in Human and Animal Health, eds.J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, pp. 639-653. Park Forest South, IL: Pathotox Publishers. 4. Frisvad, J. C. 1987. High performance liquid chromatographic determination of profiles of mycotoxins and other secondary metabolites. J. Chromatogr. 392333-347. 5. Pitt, J. I., and Leistner. L. 1991. Toxigenic Penicillium species. I n Mycotoxins and Animal Foods, eds. J. E. Smith and R. S. Henderson, pp. 81-99. Boca Raton, FL: CRC Press. 6. Pitt, J. I. 1979. Penicillium crustosrtn~and P. sinlylicissimrw~,the correct names for two common species producing tremorgenic mycotoxins. Mycologia 17: 1166- 1 177. 7. Frisvad, J. C. 1986. Taxonomic approaches to mycotoxin identification. Zrr Modern Methods in the Analysis and Structural Elucidation of Mycotoxins, ed. R. J. Cole, pp. 415-457. Orlando, FL: Academic Press. 8. Uraguchi, K. 1969. Mycotoxic origin of cardiac beriberi. J. Stored Prod. Res. 5:227-236. 9. Pitt, J. I. 1979. The GenusPenicillim and Its Teleomorphic States Eupenicillirml and Tuluronlyces. London: Academic Press. 10. Pitt, J. I., and Hocking, A. D. 1997. Fungi and Food Spoilage. 2nd ed. London: Blackie Academic and Professional. 11. Pitt, J. I., Hocking, A. D., Bhudaswami, K., Miscamble, B., Wheeler, K. A., and TanboonEk, P. 1994. The normal mycoflora of commodities from Thailand. 2. Beans, rice and other commodities. Int. J. Food Microbiol. 23:35-53. 12. Pitt, J. I., Hocking, A. D., Miscamble, B. F., Dharmaputra, 0. S., Kuswanto, K. R., Rahayu.

Penicillium

13. 14. 15.

16. 17.

18. 19. 20.

21.

22.

23. 24. 25.

26. 27.

28.

29. 30.

31. 32.

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E. S., and Sardjono. 1998. The mycoflora of foodcommodities from Indonesia.J. Food Mycol. 1:41-60. Wicklow, D. T., and Cole, R. J. 1984. Citreoviridin in standing corn infested by Elrpenicilliunr ocltrosrrlrironezrm.Mycologia 76:959-961. El-Banna, A. A., Pitt, J. I.. and Leistner, L. 1987. Production of mycotoxins by Penicillizm species. Syst. Appl. Microbiol. 10:42-46. Friis, P., Hasselager, E., and Krogh, P. 1969. Isolation of citrinin and oxalic acid from Perlicilliunr liridicnturn Westling and their nephrotoxicity in rats and pigs. Acta Pathol. Microbiol. Scand. 77:559-560. Carlton, W. W., Sansing. G.. Szczech. G. M., and Tuite, J. 1974. Citrinin mycotoxicosis in beagle dogs. Food Cosmet. Toxicol. 12:479-490. Krogh, P.. Hald, B., and Pedersen, E. J. 1973. Occurrence of ochratoxin A and citrinin in cereals associated with mycotoxic porcine nephropathy. Acta Pathol. Microbiol. Scand.. Sect. B, 81:689-695. Pitt, J. I. 1987. Penicillium \tridicatunt, Penicilliur~ \ ~ r r ~ r c o s z ~and r t ~production , of ochratoxin A. Appl. Environ. Microbiol. 53266-269. Morrissey, R. E.. Norred, W. P., Cole, R. J., and Dorner, J. 1985. Toxicity of the mycotoxin. cyclopiazonic acid. to Sprague-Dawley rats. Toxicol. Appl. Pharmacol. 77:94-107. Dorner, J. W., Cole, R.J., Lomax, L. G., Gosser, H. S., and Diener,U. L. 1983. Cyclopiazonic acid production byAspergillusj7~rwsand its effect on broiler chickens. Appl. Environ. Microbiol. 46:698-703. Van Rensburg, S. J. 1984. Subacute toxicity of the mycotoxin cyclopiazonic acid. Food Chem. Toxicol. 2993-998. Pitt, J. I., Cruickshank, R. H., and Leistner, L. 1986. Perricilliut~c o n t ~ w mP. , cantembertii, the origin of white cheese moulds, and the production of cyclopiazonic acid. Food Microbiol. 3:363-371. Still, P., Eckardt, C.. and Leistner, L. 1978. Bildung von Cyclopiazonsaure durch Perricillizrrn cnnzembertii-isolate von Kase. Fleischwirtschaft 58:876-878. Le Bars, J. 1979. Cyclopiazonic acid production by Pe~zicilliur~ camenzberti Thorn and natural occurrence of this mycotoxin in cheese. Appl. Environ. Microbiol. 38:1052-1055. Leistner. L.. Geisen, R.,andFink-Gremmels, J. 1989.Mould-fermented foods of Europe: hazards and developments. Zrr Mycotoxins and Phytotoxins '88, eds. S. Natori, K. Hashimoto, and Y. Ueno, pp. 145-154. Amsterdam: Elsevier Science Publ. Van der Merwe. K. J., Steyn. P. S.. and Fourie, L. 1965. Ochratoxin A, a toxic metabolite produced by Aspergillus ochrncezrs Wilh. Nature (Lond.) 205:1112-1113. Steyn. P. S. 1971. Ochratoxin and other dihydroisocoutnarins. I n Microbial Toxins, Vol. 6, Fungal Toxins, eds. A. Ciegler, S. Kadis and S. J. Ajl. pp. 179-205. New York: Academic Press. Natori. S.. Sakaki. S.. Kurata, H.. Udagawa, S., Ichinoe, M., Saito, M., and Umeda, M. 1970. Chemical andcytotoxicitysurveyontheproductionofochratoxinsandpenicillicacidby Aspergillus ochr-riceus Wilhelm. Chem. Pharm. Bull. 182259-2268. Van Walbeek, W., Scott,P. M.. Harwig, J., and Lawrence, J. W. 1969. Pelticillirm viridicntunz Westling: a new source of ochratoxin A. Can. J. Microbiol. 15:1281-1285. Stack, M. E., Eppley, R.M., Dreifuss, P. A., and Pohland, A. E. 1977. Isolation and identification of xanthomegnin, viomellein, rubrosulphin, and viopurpurin as metabolites of Penicillizrm viridicatrm. Appl. Environ. Microbiol. 33:35 1-355. Krogh, P., Hald, B., Englund,P., Rutqvist, L.. and Swahn, 0. 1974. Contamination of Swedish cereals with ochratoxin A. Acta Pathol. Microbiol. Scand., Sect. B, 82:301-302. Krogh, P., Axelsen. N. H., Elling, F., Gyrd-Hansen, N., Hald, B.,Hyldgaard-Jensen. J.. Larsen, A. E., Madsen, A., Mortensen, H. P., Mgller, T.. Petersen, 0. K.. Ravnskov. U., Rostgaard, M.. and Aalund, 0. 1974. Experimental porcine nephropathy. Acta Pathol. Microbiol. Scand. Sect. A, Suppl. No. 246:l-21.

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33. Breitholtz-Emanuelsson, A., Olsen, M., Oskarsson, A., Paltninger, I., and Hult, K. 1993. Ochratoxin A in cow’s milk and in human milk with corresponding human blood samples. J. AOAC Int. 76:842-846. 34. Scott, P. M., Kanhere, S. R., Lau, B. P-Y., Lewis, D.A., Hayward, S., Ryan, J. J.. and KuiperGoodman, T. 1998. Survey of Canadian human blood plasma of ochratoxin A. Food Addit. Contam. 15:555-562. 35. Ueno, Y.. Maki, S.. Lin, J., Furuya. M., Sugiura, Y., and Kawamura, 0. 1998. A 4-year study of plasma ochratoxin A in a selected population in Tokyoby immunoassay andimmunoaffinity culumn-linked HPLC. 36. Zimmerli, B., and Dick. R. 1995. Determination of ochratoxin A at the ppt level in human blood, serum.milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup methodology and Swiss data. J. Chromatogr. B 666:85-99. 37. Krogh, P., Hald, B., Plestina, R., and Ceovic, S. 1977.Balkan(endemic)nephropathyand foodborne ochratoxin A: preliminary results of a survey of foodstuffs. Acta Pathol. Microbiol. Scand., Sect. B.. 85238-240. 38. Austwick, P. K. C. 1981. Balkan nephropathy. Practitioner 225:1031-1038. 39. Macgeorge. K. M., and Mantle, P. G. 1990. Nephrotoxicity of Penicillizrrn rrztrmztiogriseurtz and P. conmzuw from an endemic nephropathy area of Yugoslavia.Mycopathologia 112: 139145. 40. Mantle, P. G., and Macgeorge, K. M. 1991. Nephrotoxic fungi in a Yugoslav community in which Balkan nephropathy is hyperendemic. Mycol. Res. 95:660-664. 41. Pitt. J. I., Hocking, A. D., Bhudaswami, K., Miscamble. B.. Wheeler, K. A., and TanboonEk, P. 1993. The normal mycoflora of commodities from Thailand. 1. Nuts and oilseeds. Int. J. Food Microbiol. 20:211-226. 42. Heenan,C. N., Shaw, K. J.. and Pitt, J. I. 1998.Ochratoxin A productionby Aspergillus carbonrrrizrs and A. niger isolates and detection using coconut cream agar. J. Food Mycol. 1: 63-72. 43. Stott, W. T., and Bullerman, L. B. 1975. Patulin: a mycotoxin of potential concern in foods. J. Milk Food Technol. 38:695-705. 44. Dickens, F., and Jones, H. E. H. 1961. Carcinogenic activity of a series of reactive lactones and related substances. Br. J. Cancer 15:85-100. 45. Brackett, R. E., and Marth, E. H. 1979. Patulin in apple juice from roadside stands in Wisconsin. J. Food Prot. 428624363. 46. Watkins, K. L., Fazekas. G., and Palmer, M. V. 1990. Patulin in Australian apple juice. Food Australia 42:438-439. 47. Mortimer, D. N.,Parker, I., Shepherd, M. J., and Gilbert, J. 1985. A limited survey of retail apple and grape juices for the mycotoxin patulin. Food Addit. Contam. 2:165-170. 48. Van Egmond, H. P. 1989. Current situation on regulations for mycotoxins. Overview oftolerances and status of standard methods of sampling and analysis. Food Addit. Contam. 6: 139188. 49. Wilson. B. J., Wilson, C. H.. and Hayes, A. W. 1968. Tremorgenic toxin from PerzicilZitm cyclopiztnz grown on food materials. Nature (Lond.) 220:77-78. 50. Cole. R. J. 1981. Tremorgenic mycotoxins: an update. In Antinutrients and Natural Toxicants in Foods, ed. R. L. Ory, pp. 17-33. Westport, CT: Food and Nutrition Press. 51. Jortner, B. S.. Ehrich, M., Katherman. A. E., Huckle, W. R.. and Carter, M. E. 1986. Effects of prolonged tremor due to penitrem A in mice. Drug Chem. Toxicol. 9: 101-1 16. 52. Hou. C. T.. Ciegler, A., and Hesseltine, C. W. 1971. Tremorgenic toxins from Penicillia. 11. A new tremorgenic toxin, tremortin B, from PerziciZZiunzpnlitmzs. Can. J. Microbiol. 17:599603. 53. Cole, R. J., Kirksey. J. W., Moore, J. H., Blankenship, B. R., Diener, U. L., and Davis, N. D. 1972. Tremorgenic toxin from Penicillilrm \wrzrczrlosztm. Appl. Microbiol. 24248-250.

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54. Ciegler. A. 1969. Tremorgenic toxin from Perzicillizrrrl ycrlitcrrls. Appl. Microbiol. 18: 128-129. 55. Ciegler, A.. and Pitt, J. I. 1970. Survey of the genus Perlicillizrnz for tremorgenic toxin production. Mycopathol. Mycol. Appl. 42: 1 19-124. 56. Wilson, B. J., Hoelunan, T., and Dettbarn, W. D. 1972. Effects of a fungus tremorgenic toxin (penitrem A)on transmission in rat phrenic nerve-diaphragm preparations. Brain Res. 40:530544. 57. El-Banna, A. A., and Leistner, L. 1988. Production of penitrem A by Perzicillizm crzlstoszun from foodstuffs. Int. J. Food Microbiol. 7:9-17. 58. Cole. R. J., Dorner, J. W., Cox, R. H., and Raymond, L. W. 1983. Two classes of alkaloid mycotoxinsproduced by Pelzicillizrrn crzastoszrm Thom isolated from contaminatedbeer. J. Agric. Food Chern. 31:655-657. 59. Boysen, M., Skouboe, P.. Frisvad. J., and Rossen, L. 1996. Reclassification of the Per1icillizcrn roqzreforti group into three species on the basis of molecular genetic and biochemical profiles. Microbiology 142541-549. 60. Frisvad, J. C., and Filtenborg, 0. 1989. Terverticillate Penicillia: chemotaxonomy and mycotoxin production. Mycologia 81 :837-861. 61. Leistner, L. 1984. Toxigenic Penicillia occurring in feeds and foods: a review. Food Technol. Aust. 36:404-306, 413. 62. Medina, M., Gaya, P.. and Nunez, M. 1985. Production of PR toxin and roquefortine by Perzicillizrm ropeforti isolates from Cabrales blue cheese. J. Food Prot. 48:118-121. Penicillizrm 63. El-Banna,A. A., Fink-Gremmels, J., andLeistner,L.1987.Investigationof chryogerzurlz isolates for their suitability as starter cultures. Mycotoxin Res. 3:77-83. R. F. 1980. Production and biological activity of 64. Ciegler, A., Hayes, A. W., and Vesonder. secalonic acid D. Appl. Environ. Microbiol. 39285-287. D 65. Sorenson, W. G., Green, F. H. Y., Vallyathan, V., and Ciegler. A. 1982. Secalonic acid toxicity in rat lung. J. Toxicol. Environ. Health 9:515-525. 66. Ehrlich. K. C., Lee, L. S., Ciegler, A., and Palmgren. M. S. 1982. Secalonic acid D: natural contaminant of corn dust. Appl. Environ. Microbiol. 43: 1007-1008. 67. Pitt, J. I. 1988.Laboratory Guide toCommon Perzicilliz4m Species. 2nd ed. North Ryde, N.S.W.: CSTRO Division of Food Research. 68. Seifert, K. A., and Samson, R. A. 1985. The genus Corenrizml and the synnematous Penicillia. I n Advances in Perzicillizm and Aspergillus Systematics, eds. R. A. Samson and J. I. Pitt. pp. 143-154. New York: Plenum Press. MD: Williams & 69. Raper, K. B., and Thom, C. 1949. A Manual of the Penicillia. Baltimore. Wilkins.

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14 Foodborne Disease and Mycotoxin Epidemiology Sara Hale Henry U.S. Food arid Drug Administration, Washington, D.C.

F. Xavier Bosch Llobregat Hospital, Barceloiza, Spain

I. Introduction

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594 TI. Ergot 111. Aflatoxins

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A.Relationshipbetweenaflatoxin exposureand humandisease 597 B. Acutehepatitis, kwashiorkor, andReye’s syndrome597 C. Epidemiology of primary livercancer598 D. Vaccinationagainst HBV as a preventive measure against livercancer599 E. Epidemiological studies ondietaryaflatoxins andlivercancer599 F. Epidemiological studies that considered HBV, HCV, and aflatoxin in relation to liver 602 cancer G. Epidemiological studies including aflatoxins in countries that are low risk for liver 603 cancer H. Epidemiological studies that used biomarkers of exposure to aflatoxins including studies ongeneticsusceptibilitytoaflatoxins 603 I. Hematochromatosis-an additional risk factorforlivercancer609 J. Discussion and conclusions 609 IV. Trichothecenes 6 1 1 A. Relationship to disease in V.

Fumonisins6

humans 612

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A. Relationship to disease in humans VI.

Patulin

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A. Relationship to

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VTI. Zearalenone 6 18 A. Relationship to disease VIII.

Ochratoxin

in humans 619

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I. INTRODUCTION Mycotoxins are toxic metabolites produced by certain fungi growing on agricultural commodities in the field and/or during storage. Mycotoxin occurrence on commodities susceptible to mold infestation is largely unavoidable; environmental factors such as temperature, humidity, and extent of rainfall during the preharvest, harvest, and postharvest periods influence mycotoxin production. Thus the incidence of mycotoxin contamination of a particular food crop can vary from region to region, country to country, and year to year. Levels of contarnination of a commodity with mycotoxin(s) vary withgeographic location, agriculture, and agronomic practices and the susceptibility of the plants to fungal invasion during all phases of growth (Wood and Trucksess, 1998). It has been estimated that approximately 25% of the world's food crops are affected by mycotoxins annually (Council for Agriculture Science and Technology, 1989); hence, the potential for human consumption ofmycotoxin-contaminated food leading to chronic or acute illness is not insignificant. Mycotoxins may produce or be associated with various forms of toxicity in humans and susceptible animals, including acute toxicity, teratogenicity, mutagenicity, and/or carcinogenicity (Wood and Trucksess, 1998). Classic epidemiological studies have been conducted for only a few mycotoxins; other less rigorous studies report on co-occun-ence of a mycotoxin and a particular disease in humans. The mycotoxins most commonly found in naturally contaminated foods include ergot, aflatoxins, trichothecenes, fumonisin, ochratoxins, zearalenone, and patulin. The relationships between these mycotoxins and human foodborne disease will be discussed in this chapter.

II. ERGOT An excellent review of the properties of ergot has been conducted by the International Programme on Chetnical Safety (IPCS) (1990). Ergot is the common term for sclerotia of fungal species within the genus Clcrviceps, particularly C. pur-pur-en.Florets of grasses and cereal are infected with the fungi; the florets are replaced with compact fungal structures or sclerotia, 2-20 mm long, strongly colored, and often purple-black. These sclerotia contain a large number of biologically active alkaloids, as well as amino acids, carbohydrates, lipids, and pigments. Cereals most commonly contaminated with ergot from C. pm-pwea are rye, wheat, triticale (the cross-breed between wheat and rye), barley, oats, and sorghum. Consumption of the sclerotia by humans and animal results in toxicosis, called ergotism (IPCS, 1990). The alkaloids of ergot are derivatives of lysergic acid and are divided into three groups: those derived from lysergic acid, e.g., ergotamines; those derived from isolysergic acid, e.g., ergotamine; and those derived from dimethylergoline (clavines), e.g., agroclavine (IPCS, 1990). Generally, it is recommended that animals not be given feed containing more than 0.1 % ergot (expressed on a weight basis). An estimate of human exposure to total ergolines of 5.1 pg/person in Switzerland has been performed. based on average concentrations of total ergolines found in wheat flour, rye flour, and "by-products." Cleaning or milling processes remove the sclerotia. Various food processing techniques of flours containing ergolines have been shown to produce 50-10096 reduction in total ergolines.

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Epidemics of ergotism have been described in recorded history, being most numerous between the 9th and 18thcenturies. Two types of disease have been noted: gangrenous and convulsive ergotism. In gangrenous ergotism, the affected part, e.g., arm or leg, shrank, became mummified and dry, and the gangrene gradually spread upward. In the second type of convulsive ergotism, the whole body was attacked by general C O I ~ V U I S ~ ~ ~ S , which returned at intervals of a few days. The IPCS reported that the latest outbreaks of ergotism in Europe occurred in the United Kingdom and the USSR in 1926-28 (IPCS, 1990). Sources did not agree on whether an outbreak with symptoms both bizarre and atypical in France in 1951 was due to ergot or another toxicant (Louria et al., 1970). The IPCS (1990) reported an ergometrine-related outbreak of ergotism in Ethiopia in 1978. The episode occurred after 2 years of drought and involved the locally grown barley, the staple food, which became dominated by wild oats heavily contaminated with C. purpurea sclerotia. The amount of grain or ergot consumed by the affected individuals was unknown; the grain consisted of 0.75% ergot by weight and ergometrine was detected in the sclerotia by thin-layer chromatography. A total of 93 cases of ergotism was reported including 47 deaths; the ma1e:female ratio was 2.5: 1, while more than 80% of affected persons were between 5 and 34 years of age. Examination of 44 cases revealed ongoing dry gangrene, feeble or absent peripheral pulses, swelling of limbs, desquamation of the skin, loss of one or more limbs, weakness, burning sensation nausea, votniting, and diarrhea. A total of 50-60 infants and young children also died from starvation due to failure of the mothers to lactate. The IPCS (1990) also reported several clavine-related outbreaks in India following ingestion of ergot from C. jk(forrnis in bajra or pearl millet. Symptoms included nausea, vomiting, and giddiness, followed by drowsiness and prolonged sleepiness; there were no signs of veno-occlusion. The most recent outbreak occurred in 1975 in the state of Rajasthan. The amount of grain consumed by affected individuals was not reported: pearl millet frotn affected villages contained 15-174 g ergot/kg, resulting in a contamination of the grain with 15-199 mg total ergolinedkg. Pearl millet from villages with no cases of intoxication contained 1-38 g ergot/kg with a total ergoline content of 15-26 mg/kg. The number of households studied was too srnall for no-effect levels of ergot to be calculated, but it was suggested that the intake of 28pg total ergolinedkg body weight would be nontoxic.

111.

AFLATOXINS

The scientific literature on aflatoxins in the past30 years includes more than 3000 research articles. A key recent publication in the aflatoxin field was the book The Toxicology of Aflatoxins: Humcw Henlth, Veterinnq and Agricultural Signijkance (Eaton and Groopman, 1994). Eaton and Gallagher (1994) wrote a review of the mechanisms of aflatoxin carcinogenesis. An extensive review of aflatoxin toxicology, carcinogenesis, and the relationship of aflatoxin consumption to human liver cancer was preformed by the Joint FAO/ WHO Expert Committee on Food Additives in 1997 (JECFA, WHO/FAO, Food additive series no. 40, 1998). Aflatoxins (AF) B1, B2, G1, and G2 are mycotoxins that may be produced by three molds of the Aspergillus species: A. f l n ~ ~ uA.s ,parasiticus, and A. nomizls, which contaminate plants and plant products. Aflatoxin M1 (AFM1) and M2, the hydroxylated metabolites of AFB 1 and AFB2, may be found in milk or milk products obtained from livestock

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that has ingested contaminated feed. Of these six aflatoxins, AFBl is the most frequent one present in contaminated samples; AFB2, G1, G2 are generally not reported in the absence of AFB1. Most of the toxicological data relate to AFB 1. Dietary intake of aflatoxins arises mainly from contamination of corn and peanuts and their products (WHO, 1998). An excellent review on the cellular interactions and metabolism of aflatoxin has been published by McLean and Dutton (1995). Gorelick (1990) has written a review comparing metabolism of aflatoxin by different species; Guengerich et al. (1996) have discussed the involvement of cytochrome P-450, glutathione-S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 (AFB1) and the relevance to risk of human liver cancer. A wide variety of vertebrates, invertebrates, plants, bacteria, and fungi are sensitive to aflatoxins, but the range of sensitivity is wide for reasons not yet fully understood (Cullen and Newberne, 1994). Two important factors in species and strain variation of sensitivity are (1) the proportion of AFBl that is metabolized to the 8,9-epoxide relative to other metabolites that are considerably less toxic and (2) the relative activity of phase I1 metabolism, which forms nontoxic conjugates and inhibits cytotoxicity. The 8,9-epoxide of AFB1 is short-lived, but highly reactive, and is believed to be the principal mediator of cellular injury (McLean and Dutton, 1995). Formation of DNA adducts of AFBl epoxide is well characterized (Cullen and Newberne, 1994). The primary site of adduct formation is the N7 position of the guanine nucleotide. An excellent review by Massey et al. (1995) covers the biochemical and molecular aspects of mammalian susceptibility to AFB 1 carcinogenicity. Important considerations include: (1 ) different mechanisms for bioactivation of AFB 1 to its ultimate carcinogenic epoxide metabolite; (2) the balance between bioactivation to and detoxification of the epoxide; (3) the interaction of AFB 1 epoxide with DNA and the mutational events leading to neoplastic transformation; (4) the role of cytotoxicity in AFBl carcinogenesis; (5) the significance of nonepoxide metabolites in toxicity; and (6) the contribution of mycotoxin-unrelated disease processes. The aflatoxins are among the most potently mutagenic substances known; literature in this area is extensive. AFBl covalently binds to DNA and efficiently induces G-to-T transversions; codon 249, one site in p53, is a striking hot spot for AFBl mutagenesis (Cullen and Newberne, 1994). Aflatoxins are very toxic, particularly to the liver, and their acute toxicity, particularly in some species of fowl, was instrumental to their discovery. The LDSUof AFBl varies from 0.34 mg/kg bw in the Pekin duckling, to 0.81 in the rainbow trout, to about 1 in most species of rats tested, to 10 in the hamster, to >150 in the male CFW mouse (Roebuck and Maxuitenko, 1994). Aflatoxins exert adverse effects on immunocompetence; this area has received less attention by researchers than it deserves (Cullen and Newberne, 1994) Studies in poultry, swine, guninea pigs, and cattle have been done; other species have been neglected. It is particularly important to study the effects of long-term exposure to low levels of AFB 1, such as would be encountered in environmental situations. Also, exposure to mixed mycotoxins such as AFBl and toxins produced by members of the Fusarium family of molds as well as to combinations of AFB 1 and other environmental contaminants, e.g., pesticides, should be studied. Aflatoxin impairs the cellular and humor immune system, making animals more susceptible to bacterial, viral, fungal, and parasitic disease (Miller and Wilson, 1994) Since animals consunling aflatoxin often succomb to infectious disease, aflatoxin as the initiating factor often is overlooked. Examples of aflatoxin’s effects on the immune system in ani-

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mals include the following. Aflatoxin exposure has been shown to reduce the protective effects of vaccines against Marek’s disease virus in chicken and P. rnultocida in turkeys. Aflatoxin has been shown to affect immunoglobin synthesis in chickens and mice, to reduce complement levels in guinea pigs, chickens, and pigs, to impair phagocytosis in chicken, and others. (Hall and Wild, 1994). The most important toxicological characteristics of the aflatoxins drawn from the voluminous literature may be summarized as follows: Aflatoxin B 1is the most toxic and most carcinogenic form. The acute toxicity correlates well to the susceptibility to hepatic cancer induced by AFB 1. Rats are generally more sensitive than mice. The LD50 varies within species by strain, sex, age, route of administration, nutritional status of the animal, and concurrent composition of the diet. Males are generally more susceptible to acute and carcinogenic effects than females. Large doses of AFBl lead to liver failure and death in animals. Smaller doses lead to cell death and regeneration. Single doses of AFB 1 do not cause liver cancer without extraordinary measures, but chronic exposure very effectively produces liver cancer. Long-term administration of AFBl in animals induces metabolic enzymes that are involved in biotransformation of aflatoxin: the metabolic fate of AFB 1 in an animal never exposed may be different from that in an animal chronically exposed. AFM1, the metabolite that occurs in milk when dairy cattle are fed AFB l-contaminated feed, is about an order of magnitude less toxic than AFB 1. A.

RelationshipBetweenAflatoxinExposureand Human Disease

Most of the information on disease outcomes related to aflatoxin exposure has been derived from animal models. Aflatoxin exposure occurs at highest levels in societies where health statistics may beundeveloped; studies in such areas begin with theidentification of persons suffering with diseases possibly associated with aflatoxin, rather thanwith the careful accurate measure of aflatoxin . uposure. The immunosuppressive effects of aflatoxin in humans have not been examined thoroughly. These effects may be particularly important in view of the high rates of infection seen in children in developing countries that have high levels of exposure to aflatoxin: the immune suppressive effects of aflatoxin may also be important in explaining the marked geographical variations seen in responses to some vaccines. (Hall and Wild, 1994). B. Acute Hepatitis,Kwashiorkor,andReye’sSyndrome

Extensive epidemiological work has been done primarily with regard to liver cancer and aflatoxin exposure (Hall and Wild, 1994); other diseases that have been less extensively studied are acute hepatitis, kwashiorkor, and Reye‘s syndrome. Aflatoxin has been implicated in several outbreaks of acute hepatitis, e.g., in India and Kenya. Although causality has not been clearly established in such situations, the known measurable high levels of aflatoxins in foods and the liver histology are highly persuasive. Long-term follow-up is needed in future outbreaks: biological exposure mark-

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ers such as serum adducts should be of great value in estimating exposure (Hall and Wild, 1994). Protein-energy malnutrition (usually defined to include all disorders attributable to lack of protein and calories, including marasmus and kwashiorkor) affects millions of children worldwide. Corn, cassava, and peanuts, which are common staples in tropical countries, are among the commodities that are most susceptible to aflatoxin contamination. Hendrickse (1991) has pointed out that kwashiorkor is limited to the tropics and has an increased incidence in the wet season, which canalso be said of aflatoxin exposure. Studies in Kenya and Sudan have demonstrated high levels of urinary excretion of aflatoxin in affected children compared with marasmic or normal children (Hall and Wild, 1994). However, much of the information from these studies is anecdotal; defining the clinical condition of kwashiorkor is difficult, the number of cases is small and not always controlled for age, and markers used to assess exposure to aflatoxin are subject to the same biases that will be discussed later in the investigation of aflatoxin and liver cancer. That is, it is uncertain if the elevated level of aflatoxin in body fluids or tissues found in some studies precedes and promotes the development of kwashiorkor or, alternatively, if the metabolic disturbances present in kwashiorkor impair the degradation and excretion of ingested aflatoxin (De Vries et al., 1990; Jelliffe et al., 1992).

C. Epidemiology

of Primary Liver Cancer

Liver cancer is prevalent in some of the developing parts of the world. It is frequent in China, Southeast Asia, and sub-Saharan Africa. In sotne of these regions, like the Qidong area in Southem China, liver cancer is the first cause of death from cancer among men. It is relatively common in Japan and in the countries in the Mediterranean basin and it is rare in the Americas and Northern Europe. Pockets of high-risk populations have been described in the Amazonian basin, among Eskimos, and in special populations like renal transplant patients. The incidence of liver cancer is consistently higher in men than in women with a sex ratio ranging from 2 to 3 in most countries. Within countries, further variation in incidence rates is observed across cancer registries, men showing greater variation than women. Worldwide, the incidence of liver cancer in men and women shows a strong correlation. Migration from high-risk areas to lower-risk areas tends to reduce the risk to the levels of the host country, and this is observable within the first and second generations. The etiology of primary liver cancer is now largely understood. In both scenarios, viral infections to hepatitis B (HBV) or C virus (HCV) are associated with liver cancer in a range of 65-100% of cases. In low-risk countries HCV predominates, and the relevant other factors are alcohol, tobacco, and use of oral contraceptives. In high-risk areas HBV predominates, and aflatoxins play a role, although quantification of this role has been difficult. The evidence points to a synergistic interaction between HBV or HCV and aflatoxins i n the etiology of liver cancer; there is some debate as to the degree of independence of aflatoxins as etiological agents in humans. It is noteworthy that the large majority of the available epidemiological studies including data on aflatoxin exposure are based on high-risk countries where both HBV and aflatoxins are highly prevalent. Since the nature of the interaction at low levels of exposure is unknown, extrapolation of results from these available studies in high-risk areas to other settings is fraught with difficulties.

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In addition to these established factors, studies have identified other factors that may modulate the incidence of the disease. Risk factors identified are the consumption of contaminated drinking water (such as pond water), liver flukes, severe malnourishment, alcohol, smoking, and use of oral contraceptives. Protection from liver cancer has been reported for diets rich in retinol, riboflavin, and protein. In addition, associations have been reported between liver cancer and testosterone levels in blood, HLA types, and certain polymorphisms in some of the glutathione-S-transferase and cytochrome P-450 metabolic regulatory genes (Bosch, 1995; Bosch and Munoz, 1991; IARC, 1994; Thomas et al., 1991).

D. Vaccination Against HBV as a Preventive Measure Against Liver Cancer In 1983 the World Health Organization (WHO) proposed as a medium-term objective trials of immunization against HBV to prevent liver cancer. A recently published study in Taiwan (Chang et al., 1997j has described the rigorous application of universal imtnunization against hepatitis B and the prevention of the carrier state in children; these data provide further evidence of a direct causal relationship between HBV and liver cancer. The immunization program against hepatitis B in Taiwan, an area of hyperendemic infection and moderate to high aflatoxin exposure, reduced the rate of HBV carriage in 6-year-old children from about 10% in the period 1981-1986 to between 0.9% and 0.8% in the period 1990-1994. The drop in the rate of carriage occurred as the proportion of infants immunized against hepatitis B increased from 15% of children born to mothers at high risk during the earlier period to 84-94% of all newborn infants during the later period. This significant reduction in the prevalence of hepatitis B surface antigen was accompanied by a decline in the average annual incidence of hepatocellular carcinoma in children 6-14 years of age, from 0.70 per 100,000between 1981 and 1986 to 0.57 between 1986 and 1989 and 0.36 between 1990 and 1994. The incidence of hepatocellular carcinoma in children 6-19 years old fell even more dramatically, from 0.52 among those born between 1974 and 1984 to 0.13 among those born between 1984 and 1986. As the investigators pointed out, since the incidence of hepatocellular carcinoma in Taiwan peaks in the sixth decade of life, it may take 40 years or longer to see an overall decrease in the rate of hepatocellular carcinoma as a result of the vaccination program. E. Epidemiological Studies on Dietary Aflatoxins and Liver Cancer Yeh et al. (1989) examined the roles of the hepatitis B virus and AFB 1 in the development of primary hepatocellular carcinoma (PHCj in a cohort of 7917 men 25-64 years old in southern Guangxi, China, where the incidence of PHC is among the highest in the world. After accumulating 30,188 person-years of observation, Yeh et al. found 149 deaths, 76 (51%) of which were due to PHC. Ninety-one percent (69 of 76) were Hbsag+ at enrollment into the study in contrast to 23% of all members of the cohort. Three of the four patients who died of liver cirrhosis were also Hbsag+ at enrollment. There was no association between Hbsag positivity and other causes of death. To estimate AFBl exposure, between 1978 and 1984, the Fusui Liver Cancer Institute regularly sampled and tested staple foods consumed in the counties of southern Guangxi for contamination by AFB 1. Twice a year, samples of raw foods were collected from

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all over the region and analyzed for AFBl content by TLC. An estimated mean level was computed for each commune as follows. The yearly amount consumed of a given raw foodstuff was multiplied by the average AFBl content as determined from tested samples of raw foodstuffs. These cross-product terms were then summed over all staple foods, and the resultant figure was divided by the total population to obtain an estimated intake per person per year. These population-based levels of AFB 1 were correlated with mortality rates of PHC among members of the cohort by the communes from which the subjects were derived. When estimated AFBl levels in the subpopulations were plotted against the comesponding mortality rates of PHC, a positive and almost perfectly linear relationship was observed. On the other hand, the prevalence of HBsAg was very high and homogeneous across the study areas (range 21.6-24.7%), and therefore, no significant association was observed when the prevalence of Hbsag positivity in the subpopulations was compared with their corresponding rates of PHC mortality. The authors conclude that despite the “crudeness” of their exposure estimate (i.e., population-based instead of personal exposure assessments), it is reasonable to conclude that AFBl seems to play a role in the unusually high rates of PHC in southern Guangxi. The population prevalence of Hbsag is extraordinarily high in this study population, with close to one in every four adult men a positive carrier of HBV. Primary infection occurs very early in this high-risk population, possibly through vertical transmission from carrier mothers to infants during the perinatal period, based on a survey of serum Hbsag in children 1-9 years old in a county adjacent to Fusui. Even though most cases of liver cancer in this study did not have histopathological confirmation, the authors indicate that probably all were PHCs. The Yeh et al. (1989) paper is an important study showing that in a region where HBV is highly prevalent, and PLC is common, the HBV carriers are at very high risk. It further indicates that in an area of high AFB 1 exposure, the PLC mortality rates are higher than in areas of lower AFB 1 exposure. However, the study has the general limitations of correlation studies in which ( I ) exposure to AFB 1 is estimated from raw foodstuffs available to populations and attributed to individuals, (2) the correlation between PLC and AFB 1 was not adjusted for any of the possible confounders such as HCV, alcohol, tobacco, or nutritional status as shown in Taiwan, (3) HBV exposure may have been underestimated owing to lack of use of PCR methodology, and (4) Hbsag prevalence was measured in a 25% sample of the cohort and attributed to the region. Campbell et al. (1990) conducted a comprehensive cross-sectional survey in the People’s Republic of China of possible risk factors for primary liver cancer (PLC) to include 48 survey sites, an approximately 600-fold aflatoxin exposure range, a 39-fold range of HCC mortality rates, a 28-fold range of hepatitis B virus surface antigen (Hbsag+) carrier prevalence, and estimation of exposures for a large number of other nutritional, dietary, and life-style features (Campbell et al., 1990). PLC mortality was unrelated to aflatoxin intake, but was positively correlated with Hbsag+ prevalence, plasma cholesterol, frequency of liquor consumption, and mean daily intake of cadmium from foods of plant origin. The authors commented on the lack of an association between aflatoxin exposure and PLC mortality in this China study in view of the findings of most previous investigations. The absence of an aflatoxin-PLC association was consistent with a similar lack of association of PLC mortality with the consumption of the two foods most commonly contaminated with aflatoxin-corn and moldy peanuts. In contrast to the lack of an associ-

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ation with aflatoxin, PLC mortality was highly correlated with HBsAg+ prevalence, and not with past HBV infection, as assessed by the prevalence of antibody to the HBV core protein. Campbell et al. offered several explanations for the finding of a nonassociation between aflatoxin intake and PLC mortality, which contrasts with the finding of other previous studies. First, Chinese might respond differently to aflatoxin, perhaps because of unique genetic or environmental characteristics. This is unlikely given the previously shown positive association between aflatoxin intake and PLC mortality in Chinese subjects (Yeh et al., 1985, 1989) and because major ethnic differences in risk for other cancers are greatly reduced or eliminated after migration to new environments. The lack of an effect in this study may have occurred because measurement of aflatoxin exposure during the survey period was not representative of past intakes when the cancers were forming. However, a similar limitation existed for all other Chinese studies; this study is more reliable, Campbell et al. stated, because it is based on urinary aflatoxin metabolite excretion, which directly represents and integrates actual consumption over a day or so. In addition, aflatoxin contamination rates in a county in the Guangxi Autonomous Region were relatively stable during the years 1972-1983. A third line of reasoning suggests that aflatoxin may not be a significant human carcinogen, in the opinion of the authors. The present study has greater statistical power and more comprehensive range, diversity, and inclusiveness of risk factors than other previous studies. Humans may also be resistant to aflatoxin carcinogenesis, a finding that is supported by in vitro aflatoxin studies on species of varying resistance (Booth et al., 1981). Humans may also be further refractory when consuming lower-protein diets; whereas acute toxicity of aflatoxin is increased in protein-malnourished children (Hendrickse et al., 1982). Campbell et al. pointed out that data from animal studies have shown that when animals were fed either lower levels of animal protein (5-1096) or the same level (20%) of plant protein after completion of aflatoxin dosing, development of preneoplastic lesions and tumors was markedly inhibited (Appleton and Campbell, 1983; Schulsinger et al., 1989). Protein in theChinese diet is primarily of plant or fish origin as compared to protein in the U.S. diet, which is primarily of animal origin (Food and Nutrition Board, 1989; Chen et al., 1990). Campbell et al. continued with a critique of previous aflatoxin epidemiology studies and offered the following model to explain the etiology of PLC. The vast majority of individuals who are susceptible to PLC are mostly those who are persistently infected with HBV. Within this HBsAg+ population, additional risk is contributed chiefly bynutritional and dietary practices that enhance liver cell proliferation, such as diets containing significant amounts of animal protein. Aflatoxin may act as a carcinogenic initiator, but contributes only a very small proportion of the initiating activity routinely exposing the liver. Therefore, Hbsagf is a necessary but insufficient cause of PLC, aflatoxin is an unnecessary and insufficient cause, and sustained nourishment causing liver cell proliferation (and elevated plasma cholesterol) is a necessary and insufficient cause for Hbsag negative carriers, but a necessary and sufficient cause for Hbsag positive carriers. Why is PLC so much more common in undernourished and impoverished societies? PLC may be more common because HBsAg+ carriers are more common. In evaluating the significance of this study by Campbell et al. (1990), a number of issues, both statistical and nonstatistical, should be considered. For example, PLC rates were determined for the years 1973-1975 and the biochemical analysis (covariate ascer-

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tainment) was conducted in 1983. With regard to the statistical analysis presented in the paper, there is some indication that the sample data do not adequately satisfy the normality assumptions upon which the univariate correlation and multiple regression analyses are based. Finally, the urinary aflatoxin measurements were of total aflatoxin metabolites, which have been shown not to correlate well with levels of AFBl consumed (Wild et al., 1992; Groopman et al., 1993).

F. Epidemiological Studies that Considered HBV, HCV, and Aflatoxin in Relation to Liver Cancer Viral hepatitis is a major worldwide public health problem. It is estimated that over 300 million individuals are chronically infected with HBV and perhaps 100 million with HCV. Chronic infection with either virus has been linked to cirrhosis and liver cancer. HBV is prevalent in the developing parts of the world, and HCV is emerging as a major cause of hepatocellular cancer in Japan and western societies (Bosch, 1995). The identification of HCV in the last decade has been a major step forward in the understanding of the origins of liver cancer and on the quantification of the proportion of cases related directly to viral infections. Epidemiological studies are largely consistent in showing a strong association between carriers of anti-HCV and PLC. The specific potential to induce PLC by each of the HVC types and variants of types as well as the impact of other factors from the host and the environment still requires further research. High estimates of the relative risk for carriers of anti-HCV are also consistent in areas of HBV endemicity. The risk linked to HCV is independent of HBV and persons who are carriers of both HBsAg and anti-HCV are at a very high risk of developing liver cancer. HCV is likely to be the major cause of liver cancer in countries at low/intermediate risk like the United States and Europe. The epidemiological evidence of the carcinogenicity of HCV has been reviewed and endorsed by an international group under IARC leadership (IARC Monograph no. 59, 1994). To evaluate the limitations of standard serological methods in assessing exposure to HBV and HCV, a comprehensive project was organized. Serum samples (12 = 503) and liver tissue ( n = 80) from patients with hepatocellular carcinoma were collected from six European countries and the specimens were analyzed in a single research leaboratory for hepatitis markers. Testing for the standard HBV markers was performed using radioimmunoasaay, and HCV markers were assessed by second- or third-generation enzymelinked immunosorbent assay (ELISA). The presence of HBV DNA in serum or tissue was assessed by PCR amplification followed by Southern blot. Testing for HCV RNA included a standardized RT-PCR system and a simple restriction fragment length polymorphismbased assay to identify the three major genotypes and the two subtypes (a,b) of type 1. In addition, testing for hepatitis G (GBV-C/HGV RNA) was performed using a standard LCRx assay and confirmed with ad hoc PCR. Among 19 patients who were HBsAg positive, 82% and 91% were also positive for HBV DNA. However, among the HBsAg liver cancer cases, 33% and 47% were also HBV DNA positive. HBV DNA was also found in 7/9 patients who were HBsAg negative and HBsAg positive or in 51/203 patients who showed negativity to anyof the HB markers. These results, which are consistent with previous results from various investigators, established that in countries with low levels of exposure to HBV, between one-third and one-half of the liver cancer cases are in fact related to HBV. Similar results were obtained

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for HCV. Among 40 cases that were anti-HCV positive, 89% and 75% were HCV-RNA positive in serum and in tissue. Among the anti-HCV negative, 7% and 26% were HCVRNA positive. Of these liver cancer patients, 7%)were HGV/GB-C RNA positive, ranging from 0% among the HBV DNA positive and 14% among the cases negative to both HBV DNA and HCV RNA. Overall, close to 15% of the liver cancer cases were negative for any of three viral markers tested (Brechot et al., 1998). This study strongly suggests that previous epidemiological studies based on standard serology for HBV and HCV systematically underestimate the risk and the derived attributable fraction. The role of other hepatotropic viruses (HGV or TTC viruses) remains to be established although the evidence of anassociation with liver cancer is marginal (Tagger et al., 1997). G.EpidemiologicalStudiesIncludingAflatoxinsin Countries that Are Low Risk for Liver Cancer

In countries where liver cancer is rare and aflatoxin exposure is low, most etiological studies on liver cancer have not considered AF as a risk factor. The populations with higher exposures are the workers occupationally exposed to grain dust in the animal feed processing plants. In studies conducted in the Nordic countries in Europe, Sweden, Denmark, and the Netherlands, and in the United States, AF has been isolated from dust samples and an excess of mortality of several such cohorts has been documented for liver cancer (risk 2.4-fold expected rates), liver and biliary tract cancer (risk 2.5-fold expected rates), lung cancer and lymphomas (risk 1.5-3-fold expected rates) (Hayes et al. 1984; Alavanja et al., 1987; Olsen et al., 1988). Some of these studies did not evaluate other relevant exposures such as hepatitis infection and alcohol. It is of interest that few studies are available on liver cancer in Latin America. In this geographically large region of the world, climate encourages mold growth on agricultural products and consumption of corn is part of the staple food in many countries. Yet liver cancer is rare in these populations as is HBV infection. If that is the case, Latin America would be anideal field to investigate the occurrence of liver cancer i n populations exposed to AF as the central risk factor.

H. Epidemiological Studies that Used Biomarkers of Exposure to Aflatoxins Including Studieson Genetic Susceptibility to Aflatoxins Biomarkers have been developed and are being introduced in epidemiological studies with the purpose of increasing the accuracy of the assessment of exposure to aflatoxins. Various biomarkers have been developed, including urinary total aflatoxins, aflatoxin adducts in urine, aflatoxin albumin adduct in serum, aflatoxin adducts in liver cancer tissue, and more recently, p53-specific mutations in liver cancer specimens. Other studies are investigating genetic polymorphisms in some key genes involved in the metabolism of aflatoxin that may introduce some variability in the response to aflatoxin (Groopman et al., 1994; Wild et al., 1996; IARC, 1997). The use of the major aflatoxin-nucleic acid adduct, AFB-N7-guanine, in urine as a biomarker was enhanced by the finding that this metabolite is excreted exclusively in urine of exposed rats, thus simplifying pharmacokinetic considerations. The aflatoxin-albumin adduct in serum has also been examined as a biomarker of exposure; because of the longer

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half-life in vivo of albumin compared to the urinary AFB-N7-guanine, the serum albumin adduct can integrate exposures over longer time periods. Data from human exposure studies have shown that the excretion of the urinary aflatoxin nucleic acid adduct and formation of the serum albumin adduct are highly correlated. In the rat, validation studies for the dose-dependent excretion of urinary aflatoxin biomarkers were done in rats following a single exposure to AFB 1; excellent linear correspondence between oral AFB 1 dose and excretion of AFB-N7-guanine in urine was shown (Scholl et al., 1995). Aflatoxin metabolites in urine or adducts in serum can be a useful tool to evaluate exposure, but with the currently available methods remain relatively short-term exposure markers: biomarkers are of much less use in predicting long-term or lifetime human exposure. As such, they reflect poorly the natural pattern of exposure to aflatoxin (i.e., seasonality, manual sorting of foodstuffs, age at exposure, etc.); therefore, it is not surprising that studies conducted using aflatoxin biomarkers as markers of exposure show conflicting results just by chance. At present, it is not known how the functional status of the liver or the coexistence of other risk factors for liver cancer may affect the different biomarkers that are being proposed for epidemiological studies. Few studies have described the natural history of these markers in patients with chronic liver disease including chronic hepatitis and liver cirrhosis, conditions that usually precede liver cancer by months or years. (Wild et al., 1993; Wang et al., 1996a) In this circumstance, the interpretation of the findings is complicated since the aflatoxin biomarker may be confounded by the presence of some risk factors, (e.g., HBV, HCV, alcohol), the presence of some protective factors (e.g., retinol, in the diet), or the presence of liver disease (i.e., chronic active hapatitis B infection). The best evidence of an interaction between HBV and alfatoxin in the causation of human liver cancer is the cohort study in Shanghai (Ross et al., 1992; Qian et al., 1994; Yuan et al., 1995). This is an ongoing prospective study of 18,244 middle-aged men in Shanghai, China. Assays for urinary AFB1, its metabolites AFPl and AFMl, and DNA adducts have been undertaken to assess the relationship between aflatoxin exposure and liver cancer. After 35,299 person-years of follow-up, 22 cases of liver cancer had been identified. For each case, five or 10 controls were randomly selected from cohort members without liver cancer on the date the disorder was diagnosed in the case and matched to within 1 year of age, within 1 month for sample collection, and for neighborhood of residence. Each subject provided a blood sample and a urine sample. A positive result was defined as the presence of at least 1 ng of an individual aflatoxin compound in the sample. HBsAg surface antigen was measured by a standard radioimmunoassay method. Subjects with liver cancer were significantly more likely than were controls to have detectable concentrations of any of the aflatoxin compounds: the strongest association was for AFP1. Positivity for HBsAg was strongly associated with risk of liver cancer. The authors concluded that their results are based on too few cases to give a reliable estimate of attributable risk, but they estimated that up to 50% of cases of liver cancer in Shanghai may be due to aflatoxin exposure. In further follow-up of the Shanghai study, Qian et al. (1994) reported on 70,000 person-years of follow-up and 55 cases of HCC. Levels of urinary AFB 1 and the oxidative metabolites, including the major aflatoxin nucleic acid adduct, aflatoxin-N7-guanine,were determined for 50 of the 55 identified cases of HCC. A total of 267 controls were matched against the 50 cases as above. A nested case-control analysis showed a highly significant association between the presence of at least one of the urinary aflatoxin metabolites, serum HBsAg positivity, and HCC risk. Risk was especially elevated in individuals who were

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positive for both of these biomarkers. However, the number of liver cancer cases in which the interaction was explored is small (i.e., 13 cases of AFB 1 positive and HBsAg negative), and there is room for misclassification of cases in relation to their viral exposure. Thus risk estimates become unstable and the barely significant increase in the odds ratios for the AFBl exposed may be easily lost if only two-thirds of cases now considered HBV negative turn out to be HBV (or HCV) positive. On the other hand, a cohort analysis using all 55 cases of HCC revealed no statistically significant association between HCC risk and dietary aflatoxin consumption as determined frotn the in-person food frequency interview combined with the survey of market foods in the study region, adding additional uncertainty of the value of the biomarkers used. HCV prevalence was low in this cohort (Yuan et al., 1995). A number of problems with the Shanghai study have been discussed by Campbell (1994). The HCC risk putatively attributed to aflatoxin in the Ross et al. study appears to be accounted for mostly by the urinary AFB-N7-guanine adduct; instead this risk could also be caused by factors that enhance enzymatic activation of AFBl by the hepatic P450 enzyme system to produce more AFB-N7-guanine. These enzyme-inducing factors could readily be nutritional, especially those also are associated with elevated plasma cholesterol. This interpretation would be inaccord with theecological study by Youngman et al. (1992) in 48 survey counties in China that the most significant and robust determinants of HCC risk were elevated cholesterol levels and HBsAg positivity, not aflatoxin. This finding is given further plausibility by animal experiments (Preston et al., 1976; Hu et al., 1994). These experiments have shown thatinvivo activation of AFBl to form hepatic DNA adducts could be markedly enhanced by a modest elevation in the intake of animal protein. This same modest animal protein intake that markedly elevates AFBl activation also markedly increased the overexpression of a hepatitis B virus transcript in rmce. However, there is an overwhelming amount of experimental data across species and in experimental models demonstrating the potency of AFB 1 as a carcinogen and mutagen (IARC Monogr., 1993). There is also evidence that humans have the metabolic capacity to activate AFB 1 to the same DNA-damaging products that occur in animal models. Although HBV is a well-established major risk factor for liver cancer: there is at least a 58-fold variation in liver cancer incidence across regions of the world where the prevalence of hepatitis B viral markers is comparable. The Shanghai study using aflatoxin-specific biotnarkers and HBV markers has provided the first direct evidence in human studies that aflatoxins are major risk factors for HCC and that a synergistic interaction between HBV and aflatoxin exposure occurs. The Campbell Chinese study is an ecological study and, as such, may not be suited to demonstrate cause and effect. In addition, a questionnaire administered to the Shanghai subjects showed no difference in daily intake of animal protein between liver cancer cases and their matched controls, or between HBsAg positive cases and controls (Ross et al., 1992). Groopman et al. (1993) have stated that, based on urinary measures ofAFB-N’guanine and dose-response characteristics of persons living in China and the Gambia, (1) levels of daily urinary excretion of ford aflatoxin rrwranbolires are unrelated to risk of aflatoxin-induced disease; and (2) the AFB-N7-guanineadduct in urine is agood, noninvasive, short-term biomarker for determining both aflatoxin exposure and risk of genetic damage in target organs. However, the Shanghai study is clearly limited for purposes of quantitative risk assesstnent of the risk to humans of aflatoxin exposure (Qian et al., 1994). No dose-

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dependent association between the dietary aflatoxin index and either liver cancer risk or biomarker status was found. This is due at least in part to the facts that urinary levels of aflatoxin accurately reflect intake levels of the past 24 hours and dietary assessment is inadequate to reflect lifetime aflatoxin exposure. An unexplained observation was the rather marked decline in the prevalence of unmetabolized AFBl with longer follow-up. Urinary adducts were measured with inadequate precision; a patient was scored “positive” or “negative” if an adduct was detected. Levels of adducts were not considered, nor was the fact that one measurement represents one “snapshot” out of a lifetime. One might question whether or not theincreased excretion of aflatoxin-DNA adducts represents the activity of a diseased liver rather than a causal relationship. Exposure to AFBl may be expected to fluctuate greatly on a day-to-day basis (as a result of varying behavior and AFBl concentrations): exposure for each individual was evaluated at a single time point. This study strongly suggests that aflatoxin exposure in the presence of a persistent HBV infection increases the risk of liver cancer; it is less convincing in the conclusion that AFB 1 is capable of independently inducing liver cancer and provides no useful quantitative data on aflatoxin. Follow-up of this cohort will provide much needed clarification on the roles of aflatoxin and HBV in liver cancer. In a study by Wild et al. (1993), blood samples were collected over a 1-month period from 117 children aged 3-4 years residing in Kuntair or Kerr Cherno in the Upper Niumi District of the Gambia. Samples were analyzed for aflatoxin-albumin (AF-alb) adducts, markers of HBV infection, liver enzymes (serum alanine aminotransferase, ALT) as markers of liver damage and glutathione-S-transferase MI genotype. All buttwo children showed detectable serunl AF-alb with levels ranging from 3.2 to 250 pg AFBl-lysine equivalent/mg albumin. There was a statistically significant positive correlation between AF-alb and ALT. HBV carriers showed moderately higher levels of AF-alb than noncarriers, but the difference was not statistically significant and the association between AFalb and ALT was unchanged when the HBV carriers were excluded from the analysis, suggesting that factors other than HBV infection contributed to the association. The null glutathione-S-transferase M l genotype was infrequent in this population and was notassociated with any difference in AF-alb adduct levels compared to glutathione-S-transferase M1-positive persons. However, the percentage of individuals with the nullgenotype varied significantly between ethnic groups. The association between AF-alb and ALT could be a result of the hepatotoxicity of aflatoxin, but the data are also consistent with the hypothesis that liver damage resulting from HBV and/other factors can alter aflatoxin metabolism resulting in an increased binding to cellular macromolecules including DNA. The authors recommended more study of this hypothesis. Srivatanakul et al. (1991) conducted a case-control study on hepatocellular carcinoma in Thailand using the (AF-alb) adduct as a marker of recent exposure to aflatoxin. HBV exposure and anti-HCV were assessed using standard methods. HBV was the predominant risk factor; neither AF-alb nor HCV was associated with liver cancer. This study lacked sufficient statistical power to detect aflatoxin or HCV as independent risk factors. In addition, AF-alb samples were taken from liver cancer cases; levels or kinds of aflatoxin metabolites might have been affected by illness. Hatch et al. (1993) conducted a survey in eight areas in Taiwan with a gradient in the estimates of exposure to aflatoxin and in the incidence of PLC. Exposure to aflatoxin was assessed using urinary tests, and a regression model was used to predict aflatoxin urinary metabolites using mortality due to PLC as a predictor (as well as five other vari-

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ables). The conclusion of the study was that aflatoxin played an independent role in PLC in Taiwan. Monoclonal antibodies recognizing the stable imidazole ring-opened form of the major N7-guanine aflatoxin B1-DNA adduct have been used in competitive ELISA and indirect immunofluorescence assays to quantitate adduct levels in liver tissue. Santella et al. (1993) developed methods in AFB 1-treated animals, and then applied these to paired tumor and nontunlor liver tissues of hepatocellular carcinoma patients from Taiwan. An avidin-biotin complex staining method was also used for the detection of HBsAg and HBxag antigens in liver sections. A total of eight (30%) HCC samples and seven (26%) adjacent nontumor liver tissue samples from Taiwan were positive for AFBl-DNA adducts. For HBsAg, 10 (37%) HCC samples and 22 (81%) adjacent nontumorous liver samples were positive, and nine (33%) HCC samples and 11 (41%) adjacent nontumor liver samples were HBsAg positive. No association with AFB1-DNA adducts was observed for HBsAg and HBxag. The authors concluded that these results were compatible with the conclusion that HBV and AFBl do not act synergistically in the genesis of HCC, but called for further investigation to define the relationship between HBV and AFB1. Wang et al. (1996a) conducted studies in Qidong, China, where liver cancer accounts for 10% of all adult deaths and both HBV and AFBl exposures are common. Serum samples were collected during a longitudinal study designed to measure aflatoxin molecular biomarkers in residents of Daxin Township, Qidong City, China. In this study, the temporal modulation of aflatoxinadduct formation with albumin over multiple lifetimes of serum albumin was examined in both HBV-positive and HBV-negative people in two periods: September-December 1993 (wave 1) and June-September 1994 (wave 2). During the 12week monitoring period of wave 1, 120 persons (balanced by gender and HBV status) provided a total of 792 blood samples. AFB1-albumin adducts were detected in all but one of the serum samples. During wave 2, 103 individuals from wave 1 provided 396 blood samples collected monthly over wave 2. Using linear regression models, the mean aflatoxin-albumin adduct levels increased during the 12 weeks of wave 1 and decreased over the 4 months of wave 2. Neither HBV status nor gender modified either the baseline mean or the temporal trend. High-pelformance liquid chromatography confirmation was done on a subset of serum samples, and the results showed an excellent association between the immunoassay data and high-performance liquid chromatography. The investigators concluded that AFB1-albumin is a sensitive and specific biomarker for assessing exposure to AFBl in the Qidong population. The rate of turnover of these adducts is similar to that of the blood protein. The half-life of albumin in normal persons is about 14-20 days, but there is some information to indicate that persons with serious liver diseases have a much more variable turnover time. In this study, an eightfold range of adduct formation existed among individuals within a cycle. Such factors as liver disease may account for the lack of tracking shown in this study between HBV status and adducts. The authors urged the need to follow up on this investigation with studies that have more frequent and longer sampling intervals for albumin adducts. Wang et al. discussed the data supporting the hypothesis that HBV enhances aflatoxin metabolism to genotoxic derivatives. This study did not seem to support this hypothesis nor did previous results in adult populations in West Africa and Taiwan. HBV may affect the metabolism of aflatoxin in children at a time when hepatocytes are maximally dividing; more data are needed in this regard.

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Wang et al. (1996b) investigated the carcinogenic effect of aflatoxin exposure in Taiwan. A total of 56 cases of HCC diagnosed between 1991 and 1995 were identified and individually matched by age, sex, residence, and date of recruitment to 220 healthy controls from the same large cohort in Taiwan. Blood samples were analyzed for hepatitis B and C virus markers and for aflatoxin-albumin adducts. Urine was tested for aflatoxin metabolites. Information was obtained about sociodemographic characteristics, habitual alcohol drinking, cigarette smoking, and diet in a structured interview. HBsAg carriers had a significantly increased risk for HCC. After adjustment for HBsAg serostatus, the matched odds ratio (Orm) was significantly elevated for subjects with high levels of urinary aflatoxin metabolites. When stratified into tertiles, a doseresponse relationship with HCC was observed. The Orm for detectable aflatoxin-albumin adducts was not significant after adjustment for HBsAg serostatus. HBsAg-seropositive subjects with high aflatoxin exposure had a higher risk than subjects with high aflatoxin exposure only or HBsAg seropositivity only. The OR for developing HCC was found to increase in the presence of anti-HCV alone, HBsAg alone, and both anti-HCV and HBsAg. There was a poor correlation between aflatoxin-albumin adducts and urinary metabolites in the same controls, although both were related to HCC risk. The investigators suggested that environmental aflatoxin exposure may enhance the hepatic carcinogenic potential of hepatitis B virus, expressed concern about the small sample size in their study, and urged the mounting of a large-scale study to evaluate the effect of aflatoxin exposure on Hbsag noncarriers. Olubuyide et al. (1993) screened for the presence of Hbsag and aflatoxin in the sera of 100 nonhospitalized individuals from the rural population of Igobo-Ora and 89 nonhospitalized individuals from the urban population of Ibadan, Nigeria. Controls were 31 healthy British Caucasians who had not traveled to the tropics or subtropics in the 6 months before the venipuncture. Forty-nine percent of rural subjects and 47% of urban subjects were consistently and reproducibly seropositive for Hbsag (as determined by the ELISA test). Two of the former subjects and five of the latter were positive for both HBV DNA (as measured by spot hybridization) and Hbsag. Total aflatoxin levels were less than 17 pglml in the British controls; serum levels of aflatoxins greater than this were detected in 8% of rural subjects and in 9% of urban subjects. The types and amounts of aflatoxins and the amounts of aflatoxins found were so widely dispersed that it was not possible to draw any conclusions about differences in types and amounts of aflatoxins between rural and urban populations. The authors intend to follow their subjects to determine their propensity to develop HCC. An ongoing study designed to evaluate the role of aflatoxin apart from the role of HBV attempts to calculate absolute risk (incidence) of liver cancer among HBsAg carriers under different environmental conditions, namely the expected lifetime exposure to aflatoxin (Evans et al., 1998). The study was organized as a set of three independent cohorts of male HBsAg carriers in Senegal, in Haimen City in China, and among HBsAg carriers in the United States (largely of Asian origin). The initial observations already indicated that the risk of liver cancer (incidence or mortality) in some areas in China was significantly higher (2-3 fold) than in the West Coast in Africa. This was in contrast with the prevalence of HBsAg, which was only moderately higher in Senegal (20% vs. 16%). and with the expected level of exposure to aflatoxin (also higher in the African setting). The initial results indicated that the risk of liver cancer in these three cohorts was dramatically higher in China (878 per 100.000 py) than in Senegal (68 per 100,000 py) or among U.S. HBV carriers (330 per 100,000 py). Theresults are clearly in contrast with

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the expectations under the hypothesis of a strong interaction between aflatoxin and HBV chronic infection in the origin of liver cancer. One of the original findings of the study describes differences in host reponse to the viral infection in these populations. Among Hasbro carriers in China, viral replication, as indicated by HBV DNA detection by Southern blot, remains between 25 and 30%across all age groups. In contrast, among Senegalese HBsAg carriers, there is strong decrease in HBV DNA rates with age, from 14% in the age group 20-29, to 3% in the 30-49 age group, and undetectable levels after age 50. U.S. HBsAg carriers also showed a strong decline with age from 37% in the 20-29 age group to 5% in the age group above 50. Prolonged high-titer virus production correlated with several parameters of liver damage and may be a determinant of the high rate of liver cancer among Chinese populations. Studies on aflatoxin markers are in progress. These comparisons of biomarkers among concurrent cohorts of HBV carriers have begun to reveal factors that could explain the difference in risk given persistence of HBV exposure and suggest that host response factors may be important. 1.

Hematochromatosis-An Additional Risk Factor Liver Cancer

for

Hemochromatosis and the subsequent iron deposits in tissues is a risk factor for liver cancer, liver cirrhosis (Hsing et al., 1995; Yang et al., 1998), and perhaps lung cancer and colorectal cancer (Stevens et al., 1998; Knecht et al., 1994). Iron deposits are also related to heart failure, diabetes, impotence, and arthritis. In addition, the extent of hereditary hemochromatosis (HH) is grossly underdiagnosed in the population and current estimates on the prevalence of HH in blood donors may be as high as 0.8-1% among men and 0.3-0.5% among women (Edwards et al., 1988; Mclaren et al., 1995). One of the few studies available that explored the relationship between liver cancer, aflatoxin, and HHwas performed in South Africa, a region with high exposure rates to viral infections and aflatoxin. In this relatively small study, hepatitis viruses were evaluated using conventional serological assays and aflatoxin exposure was assessed using AFalbumin adducts in serum. After adjustment for the relevant factors, individuals with iron overload (a good marker of HH) were at a 10-fold increased risk of liver cancer. HBV chronic carriers werer at a 33-fold increased risk, HCV antibodies increased the risk by sixfold (nonsignificant) and alcohol consumption by twofold (nonsignificant). Aflatoxinalbumin adducts were not related to the risk of liver cancer (Mandishona et al., 1998). In summary, HH is now recognized as a host factor potentially invoolved in the etiology of liver cancer in,most populations irrespective of the background prevalence of the other risk factors. Epidemiological studies are needed to evaluate the possibility of risk factor interactions to further clarify the relative attributable fractions and modulate the prevenetive strategies. J.

DiscussionandConclusions

One may conclude as follows from this extensive discussion of the voluminous data on HBV, aflatoxin, liver cancer, and other risk factors. Aflatoxins are among themost potent mutagenic and carcinogenic substances known. A large body of literature suggests that a bulky nutagenic compound such as

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aflatoxin causes a cell to become tumorigenic by reacting rapidly with DNA to give DNA adducts. These aducts or their breakdwon products then cause carcinogenic mutations. Sensitivity to aflatoxin carcinogneic effects varies widely among species. Many isoforms of the P-450 enzymes are able to‘biotransform AFB 1 to DNA-ginding mutagenic compounds. Differences in P-450 isofrom activities, due either to genetic polymorphisms or to environmental alteration in exporesion, may be important contributors to human susceptibility to AFB 1 (Massey, 1995). For example, damaged liver cells (by HBV, HCV, nutritional status, etc.) may metabolize AFBl differently than undamaged cells (CamusRadon et al., 1996). Animal studies suggest that glutathione-S-transferase-catalyzed detoxification is a very crucial factor in susceptibility to AFB1; humans appear to lack significant GSTmediated protection against AFB 1. Most AFB metabolism studies have been conducted in vitro; the exclusion of enzymes and cofactors for competing metabolic pathways restricts quantitative comparisons of metabolism between species (Gorelick, 1990). Taking the extensive body of metabolic data as a whole, it is not possible at the present time to describe quantitatively a species-dependent effect of metabolism on AFB 1. Differing sensitivity to AFB 1 between species may be at least paritally attributed to metabolic differences. The currently available studies utilizing aflatoxin biomarkers do not provide a reliable quantitative measure of long-term aflatoxin exposure in humans. Aflatoxin has not been shown to be an independent carcinogen in humans. With regard to controlling aflatoxin in commodities in world trade and reducing possible related human liver cancer risks: The carcinogenic potency of aflatoxins in HBsAg+ individuals is substantially higher than the potency in HBsAg- individuals. Thus, reduction of the intake of aflatoxin in populations with a high prevalence of HBsAg+ individuals will have greater impact on reducing liver cancer rates than reductions in populations with a low prevalence of HBsAg+ individuals. If the findings from the Shanghai cohort (Ross et al, 1992; Qian et al., 1994) are correct (despite small numbers of cases and the possibility of misclassification of viral status due to methodological problems), then aflatoxins. carcinogenic potency is greater in HBsAg+ individuals. A cautionary note is sounded by the data of Evans et al. (1998). Aflatoxin may not significantly modify the risk of a HBV carrier. Basic virological observations, such as DNA persistence at high levels, suggest the importance of genetic differences in handling HBV exposure. Carcinogenic potency of the aflatoxins may be reduced, and consequently liver cancer risk, by vaccination against hepatitis B, which will reduce the prevalence of carriers. Analyses of selected aflatoxin regulatory standards in foods (10-20 pg/kg) indicated that ( 1) populations with a low prevalence of HbsAgf individuals and/or with a low mean intake (less than 1 ng/kg bw/day) are unlikely to exhibit detectable differences in population risks for standards in this range; (2.) populations with a high prevalence of HBsAg+ individuals and high mean intake of aflatoxin would benefit from reduction in aflatoxin intake. Here, ‘‘detectable‘’ refers to

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an aflatoxin-induced change in liver cancer rates that exceeds the year-toyear variability around the current incidence and mortality. The degree of “detectability” is dependent on the quality of the data available on historical trends in incidence and mortality.

IV. TRICHOTHECENES The trichothecene mycotoxins are a large family of toxic metabolites produced by molds belonging to the Fr~snriumand other genera including Trichoderma, Trichotheciznrz, Myrotheciurrz, and Stachybotlys. Currently, 148 trichothecenes, having the same basic tetracyclic scirpenol ring system, have been described. These mycotoxins contaminate some cereals, particularly corn, harvested under rainy conditions or stored under bad conditions (IPCS, 1990). The trichothecenes are associated with several pathological outbreaks in humans and farm animals; alimentary toxic aleukia (ATA), a leukopenia that occurred in eastern Europe during World War I1 has been described in the literature. The more toxic members of thefamily are immunosuppressive and also inhibit synthesis of cellular macromolecules in animals (Mekhancha-Dahel et al., 1990). The trichothecenes that have been shown to contaminate food or feed are deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), and T-2 toxin; less commonly found derivatives include 3-acetoxyDON, 15-acetoxy-DON, fusarenon-X, and HT-2 toxin. The most common trichothecene in food and animal feed is DON, often found with lesser amounts of NIV (IPCS, 1990). It is beyond the scope of this chapter to review the toxicology of trichothecenes; a summary is presented below (IPCS, 1990; Rotter et al., 1996). Acute systemic effects result from the more potent trichothecenes (e.g., T-2 and DAS) when administered orally parenterally or by inhalation to rodents, pigs, and cattle. Cytotoxic effects, as shown for example by T-2, include necrosis of the intestinal crypt epithelium and of lymphoid and hematopoietic tissues after oral, parenteral, or inhalation exposure. Trichothecenes are toxic for actively dividing cells, by impairing protein synthesis or causing dysfunction of cellular membranes. Hematological and coagulopathic abnormalities result from T-2 toxin and DAS exposure. Suppression of cell-mediated and humoral immunity results from exposure to T-2, DON, and DAS, including decreased resistance to secondary infection by bacteria and viruses. Immunostimulatory effects occur, which appear to involve impairment of normal regulatory mechanisms, such as superinducing IL-1 production by macrophages, stimulation of T helper cytokines, etc. Elevation in total serum IgA, but concurrent decreases in total IgM and IgG, occur. Teratogenicity is exhibited (T-2 and DON in the mouse). Weak clastogenic activity of T-2 is demonstrated in some assays, but there is general lack of mutagenic activity. No evidence of carcinogenicity has been shown in long-term animal studies of those trichothecenes studied.

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A.

Relationship to DiseaseinHumans

Historically, trichothecenes or Fusarium-related disease outbreaks have been reported in various countries. A human disease known as alimentary toxic aleukia (ATA) occurred in the USSR in 1931-47 and was reported to be related to the ingestion of overwintered grain invaded by toxic Fzrsurizm species (particularly Fuscrrium poae and F. sporotrichioides) (IPCS, 1990). Pathological changes included necrotic lesions of the oral cavity, esophagus, and stomach and a pronounced leukopenia; the primary lesion was bone marrow hypoplasia and aplasia. The disease was fatal in a high proportion of cases. The clinical symptoms reported in ATA, as well as the identified occurrence of Fzcm-ium in foodstuffs, suggest that it might have been associated with mycotoxins, identified years later in fungal culture of Fzrsurium species under laboratory conditions, possibly T-3 or wortmannin. From Korea and Japan in the period 1946-63, scabby grain toxicosis was reported in humans as well as farm animals. Common clinical symptoms were nausea, vomiting, diarrhea, and abdominal pain, followed by recovery within a few days. Fusarium species, especially F. granrirzinrurn, were isolated from suspected cereals (IPCS, 1990). Both ATA and scabby grain toxicosis have not been studied in epidemiological studies; features of these human diseases were similar to trichothecene toxicosis as observed in experimental animals, notably symptoms caused by DON and NIV, DAS, and T-2 toxin. Recent outbreaks of trichothecene-related disease have been reported in Japan (red mold disease), India (DON toxicosis), and China (fusariotoxicosis) (Rotter et al., 1996). Two outbreaks have been reported in some detail by the IPCS (1990). In the Chinese outbreak of 1984-85, approximately 463 of 600 persons who consumed moldy corn and scabby wheat reported symptoms including nausea, vomiting, abdominal pain, diarrhea, dizziness, and headache with an onset of 5-30 min. Pigs and chickens fed the mold cereal were also affected. DON was detected at a range of 0.34-0.93 mg/kg and zearalenone at a range of 0.004-0.59 mg/kg; T-2 toxin and NIV were not found (IPCS, 1990). In an Indian outbreak in 1987, illness was ascribed to consumption of bread made from flour that had molded in storage following unseasonal rains. Fuscrriurn species were grown from the wheat. A total of 97 of 224 persons were affected with symptoms including abdominal pain, throat irritation, diarrhea, blood in stools, and vomiting, with a time of onset of 15 min to 1 hr. In 12 of 24 wheat flour samples, DON (0.35-8.4 mg/kg); AcDON (0.64-2.5 mg/kg); NIV (0.03-0.1 mg/kg); and T-2 toxin (0.55-0.8 mg/kg) were found: quantitative estimation of these toxins was obtained using HPLC and TLC, but no rigorous confirmation of identity was undertaken (Bhat et al., 1989; IPCS, 1990). Leon-S. et al. (1996) have suggested that mycotoxins, particularly DON, NIV, and fumonisin B 1, contaminating foods may have a role in chronic idiopathic spastic paraparesis (CHISPA). Such patients are usually found in hot and humid regions, where foods are likely to be contaminated by fungi. Eleven of 12 CHISPA patients from southwestern Colombia were shown to have DON, fumonisin B 1, and NIV in their urine; none had toxic metabolites belonging to general Aspergillus or Peuicillium. The authors suggested that these mycotoxins could interfere with metabolic pathways involving sphingolipids (e.g., fumonisins), blocking the N-acetyltransferase enzyme, producing apoptosis and spastic paraparesis in animals. These mechanisms may be involved in the development of similar but seronegative clinical pictures frequently found simultaneously in these endemic areas for CHISPA, resembling those in other diseases associated with viral infections and

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described in places with similar environments to those described above. DON and NIV have consistently been found in staple food from such locations as Kyushu Island, Japan, also an endemic area for CHISPA. The association of Fuscrrizm toxins with human esophageal cancer has been reported in China and Africa (Luo et al., 1990; Chu and Li, 1994; Ueno et al., 1997; Sydenham et al., 1990; Rheeder et al., 1992). In one Chinese study (Luo et al., 1990) the natural occurrence of Fusnrizm toxins was studied in 47 corn and 30 wheat samples collected in 1989 from Linzian and Shangquiu Counties in Henan Province, People’s Republic of China. Linxian County is the highest-risk area for esophageal cancer; its annual death rate is 132 per 100,000, 8.4 times higher than that of Shangquiu County, a low-risk area in Henan province, about 250 km southeast of Linxian. DON, ac-DON, NIV, and zearalenone (ZEA) were found in corn samples; DON, NIV, and ZEA were found in wheat samples. Compared with Shangquiu corn, the incidence and mean level of DON in Linxian were 2.4 and 5.8 times higher, respectively, and thoseof ac-DON were 16.3 and 2.6 times higher, respectively. Although trichothecenes levels in wheat were significantly lower than those in corn, the level of DON in Linxian wheat was 3.3 times higher than in Shangquiu wheat. The authors emphasized that studies on esophagitis and esophageal cancer in Linxian have suggested that esophageal carcinogenesis is most likely a multistage and multifactorial process in which deficiencies of certain vitamins and trace elements, high levels of nitrates and nitrites in food and drinking water, Cundidu esophagitis, and Fusnriurn mycotoxins might be involved. In another Chinese study by Chu and Li (1994), a total of 3 1 corn samples were collected from households in the counties of Cixian and Linxian, where high incidences of esophageal cancer have been reported, and analyzed for FB 1, aflatoxin, and total trichsthecene mycotoxins. Fumonisins were found at lower levels in the corn without visible mold and higher levels were found in the visibly molded corn. Levels of aflatoxin in the samples were low: high levels of total type A trichothecenes were also found in the moldy corn samples. Immunochromatography of selected samples revealed that these samples contained T-2 toxin, HT-2 toxin, iso-neosolaniol, MAS, and several other type-As. Ueno et al. (1997) also analyzed 40 corn samples collected from agricultural stocks for human consumption in Haimen and Penlai, high- and low-risk areas for primary liver cancer (PLC). Samples were analyzed by high-pressure liquid chromatography (HPLC), gas-liquid chromatography (GLC), ELISA for fumonisins B1 and 2 (Fbs), aflatoxins, and trichothecenes. Levels and rates of contamination of Fbs and DON were significantly higher in Haimen than in Penlai. The authors postulated that trichothecenes, such as NIV, may promote AFB 1-initiated hepatocarcinogenicity, as these authors have demonstrated in rats. In one study in South Africa (Sydenham et al., 1990), moldy and healthy corn samples were collected from two opposing human esophageal cancer prevalence areas of the Transkei, southern Africa, during 1985 and screened mycologically. In southern Africa, the esophageal cancer rate is highest in the southwestern districts of theTranskei (Kentani), while the rate in the northeastern region (Bizana) is relatively low. Moldy corn samples were analyzed for the presence of several Fusarium mycotoxins, including DON, DAS, moniliformin (MON), NIV, T-2 toxin, ZEA, fumonisins B 1 (FB1) and B2 (FB2), and tricarballylic acid (TCA), a compound present in the structures of the fumonisins. Unmolded corn samples were also screened for the presence of FB 1 and FB2. In the moldy corn samples high concentrations of DON, MON, NIV, ZEA, FB 1, and FB2 were observed. Significantly higher levels of both FBI and FB2 were present in the healthy corn

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samples from the high-esophageal-cancer-rate area than in corresponding samples from the low-rate area. The authors pointed out that an interrelationship has been demonstrated between the fungal contamination of corn collected from the Transkei and the mycotoxins produced by the separate Fz~snriurrzspecies. In addition, moldy corn samples in areas of the Transkei are heavily contaminated with a number of Flrsarizun mycotoxins that can enter the human food chain. Rheeder et al. (1992) collected homegrown corn samples from areas with high and low rates of human esophageal cancer in the Transkei for six seasons from 1976 to 89. In most rural Transkei households, ears of corn judged to be free of most mold or “good” by the householder are stored either indoors on the floor of a hut or outdoors in wooden cribs. Visibly moldy corn ears are kept separate form the good corn and used for animal feed and for brewing corn beer. Moldy corn is widely believed to enhance the flavor of the beer. The most consistent difference in the mycoflora of the corn kernels was the significantly higher incidence of F. moniliforme in corn from high- vs. low-rate areas. The fumonisins were the only mycotoxins to show a highly significant difference between the two esophageal cancer areas. Rotter et al. (1996) have pointed out that DON-induced dysregulation of IgA production is strikingly similar to human IgA nephropathy (Berger’s disease’),which is the most common glomerulonephritis worldwide. DON-exposed mice show kidney mesangial IgA accumulation and hematuria, hallmarks of this human autoimmune disease, in addition to in vitro and in vivo IgA hyperelevation. Up to 3 months after withdrawal of DON from the diet, elevated serum IgA, mesangial IgA, and hematuria persist in the mice. The potential of DON and other trichothecenes to superinduce cytokines may be related to some of its toxic effects. Acute trichothecene poisoning in hutnans and animals has been described as multisystem shock-like syndrome, including dermal irritation, nausea, vomiting, diarrhea, hemorrhage, and hematological lesions, such as leukopenia and anemia; these effects could be mediated by cytokines (Rotter et al., 1996). To summarize the data linking trichothecenes and human disease, the epidemiological data, although not rigorous, points toward trichothecene- or specifically DON-contaminated grain products as the causative factor of the human toxicosis (Rotter et al., 1996). The association of Fusclriurn toxins with cancer, specifically with esophageal cancer, has been reported in China and Africa, as discussed above. In the Transkei, the area in Africa known to have the highest rate of esophageal cancer, the natural contamination of corn with DON was much higher than in low-incidence areas. However, a subsequent study was not able to establish a correlation between the proportion of mold-contaminated and healthy kernels infected with F. grcuzzinecwum, contamination with DON and zearalenone, and the rates of esophageal cancer, despite a correlation between F. rnonil[fornze in corn and cytological abnormalities in the esophagus of infected individuals (Marasas et al., 1988). Recently, the presence of fumonisin, produced by F. rnorzil[forme,has been implicated as a possible contributing factor in esophageal cancer (Norred and Voss, 1994). The most likely conclusion retarding trichothecenes and esophageal cancer is that this cancer, like most human cancers, has a complex etiology and probably involves multiple factors, such as presence of fungi, other environmental chemical, poor nutritional status, low economic status, poor food storage conditions, etc. The importance of nutritional status on esophageal cancer was clearly shown recently. Two recent randomized nutrition intervention trials, conducted in Linxian, China in conjunction with the National Cancer Institute, showed a 13% reduction in cancer death rate and a 9% reduction in total death rate when diets were supplemented with beta carotene, vitamin E, and selenium (Blot et

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al., 1995). Major cancer types affected were stomach and esophageal; these two types of cancer make up 87% of all deaths from cancer in this population. In addition, based on its 1993 evaluation, the World Health Organization (WHO, 1993) concluded that the evidence for the carcinogenicity of toxins derived from F. granzinearum in humans or for DON in experimental animals is inadequate .

V.

FUMONISINS

One of the most prevalent seedborne fungi associated with corn (Zea wzays L.) worldwide, Fzlsnriun? w?onilifome Sheldon, produces fumonisins. These mycotoxins were first isolated from cultures of F. rnorzil[fornze strain MRC 826 at the South African Medical Research Council in 1988; earlier reports in the literature, going back to 1881, from various countries had implicated corn contaminated with F. monilifornre in incidents of toxicosis in farm animals, including leukeoencephalomacia (LEM) in horses and porcine pulmonary edema syndrome (PES). Fumonisins B1 and B2 were isolated and their structures elucidated in 1988 (Marasas, 1996). Fumonisin B1 has been demonstrated to have the following toxicological effects in animals, but has not been demonstrated to be mutagenic or genotoxic: LEM in horses Pulmonary edema syndrome (PES) in pigs Hepatotoxicity and liver cancer in rats Initiator and promotor of cancer in rat liver Cytotoxicity in mammalian cell cultures Phytotoxicity Sphingolipid biosynthesis inhibition Possible association with esophageal cancer in humans

A.

Relationship to Disease in Humans

Bhat et al. (1997) reported an outbreak in 1995 when unseasonal rains damaged the maize and sorghum crops harvested in a few villages of the Deccan plateau in India. Human consumption of those grains resulted in a foodborne disease outbreak characterized by abdominal pain, borborygmus, and diarrhea. For field studies, the recommendations made by the International Programme on Chemical Safety (IPCS) for establishing chemical etiology of specific diseases as a basis for their prevention were followed. The nature of the outbreak was investigated according to World Health Organization guidelines. A rapid epidemiological survey was conducted in the affected villages and a detailed house-tohouse survey in selected villages. Persons in 27 of 50 villages surveyed were affected; disease was seen only in households and subjects consuming the rain-damaged moldy sorghum or maize. The poorest of the poor segment of the population (small landholders of agricultural laborers), though aware of the mold damage of the grains, were forced to consume unleavened bread prepared from such grains owing to nonavailability of good grains at the household level and lack of purchasing power to buy grains from the market. The mycological profile indicated that Fusarium sp., Aspergillus sp., and Altelnnria sp. were the dominant mycoflora in sorghum, while Fzrsnrium sp. and Aspergillus sp. were the dominant mycoflora in maize. Among the various mycotoxins, fumonisin Bl (FBZ)

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was the most important mycotoxin detected; levels of aflatoxin detected were extremely low. Levels of FB I in samples collected from affected households were 0.14-7.8 mg/kg, whereas samples collected from unaffected households showed 0.07-0.36 mg/kg fumonisin B 1. A more accurate estimate of fumonisin exposure in affected individuals was not possible. The disease was self-limiting and disappeared when alternate food sources became available. Diarrhea was reproduced in day-old cockerels fed an incriminated maize sample continuing 8 mg/kg FB1. Very high fumonisin concentrations have been reported from homegrown maize in Transkei (1 18 mg/kg; Rheeder et al., 1992) and China (150 mg/kg; Chu and Li, 1994). Lower levels have been reported from northern Italy (150-3760 mg/kg FB 1; Pascale et al., 1995). There is indirect evidence for fumonisin effects on humans from epidemiological studies. Studies by Rheeder et al. (1992) and Syndenham et al. (1990) on the possible relationship between esophageal cancer and corn contaminated with mycotoxin(s) in the Transkei have been discussed in the section on trichothecenes. Inrural areas of the Transkei, maize porridge is the staple diet (up to 100% of calories). Adults also consume beer made from moldy maize deliberately selected by the housewife; such beers could contain a fumonisin concentration of 30 mg/L beer. In northeast Italy, Pordenone Province has the highest mortality for oral, pharyngeal, and esophageal cancer in Italy and is among the highest in Europe (Franceschi et al., 1990). In this area risk factors identified include alcohol and tobacco usage; significant associations with corn consumption were found for oral, pharyngeal, and esophageal cancer. Most corn is locally produced and eaten as cooked cornmeal (polenta). Twenty corn samples grown between 1993 and 1994 and analyzed for FB1 contained 150-3760 ng/ kg FBI (Pascale et al., 1995); these levels are at least an order of magnitude lower that FB 1 levels found in the Transkei corn. Consumption of polenta based on recall data taken between 1991 and 1994 hasbeen estimated as 97 g/week for males and 87 g/wk for females; in Milano and Genoa, consumption was 20 g/wk, decreasing to about 4.5 g/ week in Roma/Latina. Data on occurrence of fumonisin-producing Fusa~-iunzspp. on corn grown in northern Italy are lacking. Marasas (1995) noted that some of the highest levels of FB1 and FB2 in commercial corn-based products recorded to date (0.17-2.4 pg/g of FB 1 and FB2 combined) occurred in samples of cornmeal and corn grits purchased from retail outlets in the United States. Seven of these samples were from a supermarket in Charleston, South Carolina, which has the highest incidence of esophageal cancer among blacks in the United States, as pointed out by Marasas. Abnormal climatic conditions occurred in many corn-producing areas in 1989-90; high levels of fumonisins were observed in corn, particularly in the corn screenings from the affected areas. When these corn screenings were incorporated into commercial feeds, large numbers of horses died due to LEM and pigs due to PES. Corn is a staple food in parts of China including Linxian and Cixian Counties in Henan Province (Chu and Li, 1994). As has been pointed out in the trichothecene section, mortality for esophageal cancer for males in the low-risk areas ranges from 26 to 361 100,000 and 76 to 161/100,000 in the high-risk areas. F. nzoniliforrne has been reported to be in higher prevalence on corn in the high-risk areas in some papers (Chu and Li, 1994; Luo et al., 1990). Ueno et al. (1997) reported that levels of fumonisins in a survey of three crop-years of corn samples (240 total samples) are significantly higher in Haimen (Jiangsu Countyj, a high-risk area for liver cancer, than in Penlai (Shandong Province), a low-risk area.

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Samples were also contaminated with aflatoxins and DON. Ueno et al. postulated that fumonisins may be a promoting factor for liver cancer, as has been demonstrated in experimental animals (Gelderblom et al., 1988). However, Yoshizawa et al. (1994) collected 27 samples from households with esophageal cancer and 20 samples from similar families in a low-risk area. Similar concentrations of fumonisin were found in both sets of samples (ca. 890 pg/kg). When Chu and Li (1994) collected 3 1 samples from households in the same areas, the visibly moldy samples contained 18-155 mg/kg fumonisins and the not visibly moldy samples contained 20-60 mg/kg. These Chinese studies demonstrate the difficulty of estimating human exposure to a putative carcinogen; human cancers may be expected to have a long latency period and hence present-day estimates of exposure to a carcinogen do not necessarily reflect longterm exposures. In addition, sampling of a food crop in a certain geographic area must be planned to give a statistically valid representation of the levels of the contaminant in question. None of the Chinese studies is a true epidemiological study. One can conclude that fumonisins are toxic to humans at high enough levels, but their relationship to human esophageal or liver cancer has not been definitively proven. However, as Marasas (1 995) has urged, the contatnination of corn with futnonisins represents a real threat to human and animal health, particularly in Africa where corn is the main dietary staple of millions of people.

VI.

PATULIN

Patulin is a mycotoxin produced by fungi belonging to several genera including Perzicilliurn, Aspergillus, and Byssochlclmys. Patulin can occur in many molding fruits, grains, cheeses, and other foods; the major source of patulin contamination is from Perzicilliunz expansum growth, which is encountered under certain types of apple spoilage such as “blue mold” on fruit with surface damage. The degree of contamination correlates with the degree of spoilage; patulin generally does not occur far from spoiled tissues. Patulin is not destroyed by heat and is stable at an acid pH. Its content is reduced by prolonged storage, the action of ascorbic acid, the action of sulfites and high temperature, alcoholic fermentation, an alkaline medium, and the presence of sulfhydryl groups such as those from cysteine and glutathione (WHO, 1990). The toxicological characteristics of patulin are summarized briefly as follows (WHO, 1990):

LDSoin mice of 15-35 mg/kg, depending on mode of administration. Cytotoxic effect, including antibiotic, antifungal, and antiprotozoal properties. Mechanisms of action: demonstrates effects on membrane permeability, disorganizes cytoplasmic microfilaments, inhibits in vitro several enzymes including RNA and DNA polymerase, affects DNA directly by effects on transcription and translation. Mutagenicity testing results mixed; classic mutagenicity tests such as the Ames test generally negative. Sister-chromatid exchange and chromatid breaks, isochromatid breaks, chromatid translocation tests positive.

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Carcinogenicity tests negative in three long-term-exposure rodent studies. When administered subcutaneously, produced sarcomas probably due to local irritation effect. Fetotoxicity and embryotoxicity shown in rodents at doses overtly toxic to the mothers. A.

Relationship to Disease in Humans

When patulin was evaluated at the 35th Joint FAO/WHO Expert Committee on Food Additives (JECFA) meeting in 1990, a maximum provisional tolerable weekly intake (PTWI) of 7 g/kg body weight/week was proposed. Patulin has been tested as an antibiotic for treatment of the comnlon cold in humans. Application was through the nasal route. A summary of two reports by WHO (1990) stated that it was not possible to determine from the information given as to which clinical tests were performed to support the author’s assertion that no ill effects were observed. The absence of reported cases of hutnan disease associated with patulin exist in the face of ample opportunity for exposure. In particular, apples and apple products have been consumed in large quantities historically, but there are no reports in the literature to date of patulin intoxication of humans.

VI. ZEARALENONE Zearalenone was firstisolated in 1962 from cultures of the fungus Fusarium grmnimm-uru; it is also produced by F. crookwellense, F. crmorzm, and F. semitecturn. The compound is produced commercially as an intermediate in the preparation of zeranol (alpha-zearalenol) by submerged fermentation; zeranol is used as a growth promoter in beef cattle, feedlot lambs, and suckling beef calves (WHO, 1993). Zearalenone, one of the most widely distributed Fz4asariurn mycotoxins, is associated primarily with corn, but also occurs in wheat, barley, and sorghum. The toxicological characteristics of zeralenone maybe summarized as follows (Kuiper-Goodman et a1., 1987): Estrogenic and anabolic activity demonstrated in studies in various species (rodents, rabbits, pigs, monkeys). Major effects are on reproduction, including reproductive organs and their function, leading to hyperestrogenism. A noadverse effect level (NOAEL) of 0.06 mg/kg bw has been estimated for the pubertal pig, the most sensitive species tested. Greater amounts of alpha-zearalenol, the more estrogenic metabolite, are formed in humans and the pig compared to rodents. Biological half-life of these substances was longer in humans than in other species tested. Binding of zearalenone to estrogen receptors was approximately 20-fold lower than that seen with 17-beta-estradiol in several assays. Bioassays in mice and rats are considered to be “positive evidence of carcinogenicity” by the National Cancer Institute/National Toxicology Program. IARC placed zearalenone in the category of “limited evidence of carcinogenicity.” Did not induce SOS error-prone DNA repair in E. coli; differential toxicity shown in repair-deficient and -proficient Bacillus strains. Did not induce mutation in

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S. typl?imwium or mitotic crossing over in S. cel-evisiae. Induced sister chromatid exchange, chromosomal aberration, and polyploidy in Chinese hamster cells.

A.

Relationship to DiseaseinHumans

Several ecological studies have examined the relathionship between exposure to Fusarium toxins and esophageal cancer; most of the studies refer to mixtures of many toxins from many species of fungi on corn. The studies by Marasas et al. (1988), Marasas (1995), and Syndenhaln et al. (1990) in the Transkei, South Africa with moldy corn and esophageal cancer have been discussed elsewhere in this chapter. No analytical epidemiological studies are available that address the carcinogenicity of Fmarirml toxins or zearalenone alone.

VIII. OCHRATOXIN Ochratoxin A was first isolated from a culture of Aspergillus ochruceus Wilh. in 1965 (WHO, 1993). However, there has been disagreement on the taxonomy of Aspergillus and Penicillium for some years; an international conference harmonized the taxonomy in 1990. Ochratoxin A is now generally agreed to be produced by one species of Penicilliunl, P. verrucosurn, and atnong the aspergilli, primarily A. ocl.raraceus.Grains contaminated with P. verl-ucosum probably contain also citrinin and penicillic acid as well as ochratoxin A. Ochratoxin A occurs in manycommodities, such as grains, coffee beans, peas, beans, feeds, copra, rice, pork kidneys, and sausage, and smoked meat products, such as bacon and ham. In 1990, a WHO/FAO Joint Expert Committee on Food Additives reviewed the literature on ochratoxin A and recommended a provisional tolerable weekly intake (PTWI) of 112 ng/kg bw (WHO, JECFA, 1990). This PTWI was reconfirmed based on the lowest observed adverse effect level for kidney damage in pigs and a safety factor of 500 at the JECFA 44th meeting in 1995, resulting in 14 ng/kg bw/day, when rounded off. The toxicological characteristics of ochratoxin A may be sutnmarized as follows (Castegnaro et al., 1990): Acute toxicity varies frotn 3.4 to 30 mg/kg bw depending on species. In rats, females are more sensitive following oral administration of OA. Kidney, followed by liver, is primary target organ. Kidney tubular functions are impaired; capacity to concentrate urine is reduced. Immunological effects in rodents include reduced plasma fibrinogen, factors 11, VII, and X, and reduced thrombocyte and megakaryocyte counts. In chickens lymphocytopenia developed and levels of serum immunoglobulins (IgA, IgC, and IgM) were reduced. In mice myelotoxicity developed. Embryotoxic effects in mice and rats treated with 3-5 mg/kg bw included increased prenatal mortality, decreased fetal weight, various fetal malformations. No OA activity in short-term tests in vitro, in bacteria, yeast, or cultured cells. DNA single-strand breaks observed in renal and hepatic tissues of mice and rats treated with single or chronic doses of OA. OA is carcinogenic in mice and rats, inducing kidney tumors, predominantly in male animals. Liver tumors also observed in male and female mice.

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OA inhibits protein synthesis and tRNA synthetase inboth microorganisms and mammalian cells. OA may induce DNA-single-strand breaks by producing reactive oxygen species and enhancing lipid peroxidation in microsomes. A.

RelationshipBetweenOchratoxinandHuman Disease

Acute gastrointestinal poisoning from food contaminated with high levels of OA has been described (IARC, 1993). A fatal chronic renal disease was identified in the 1950s in certain geographically limited areas of Bulgaria, Yugoslavia, and Romania (IARC, 1993). In 1963, this disease was recongnized as a new nosological entity and was referred to as Balkan endemic nephropahty (BEN). BEN is a bilateral, noninflammatory, chronic nephropathy, in which the kidneys are extremely reduced in size and weight and show diffuse cortical fibrosis extending into the corticomedullary junction, hyalinized glomeruli, and severely degenerated tubules. Clinically, the characteristics are progressive hypercreatininemia, hyperuremia, and normo- or slightly hypochromoic anemia, with generally nonelevated blood pressure. Castegnaro et al. (1990) and Tatu et al. (1998) have summarized the reasons implicating ochratoxin (or other mycotoxins) in the etiology of BEN and the associated urinary tract tumors (UTT). 1. BEN has a similar morphology and progressive course to the endemic porcine nephropathy observed in many countries and caused by ingestion of OA. 2. The kidneys, and to a lesser extent liver, are the major target organs for toxicity and induction of neoplasnls by OA in several mammalian species. 3. OA impairs the immune function in experimental animals as has been observed in patients with BEN and subjects living in the endemic areas. 4. OA when added to rat liver microsomes greatly enhances the rate of NADPH- or ascorbate/Fe”-dependent lipid peroxidation, as measured by malondialdehyde formation; lipid peroxidation is similarly enhanced in kidney microsomes by OA. As Fe” enhances lipid peroxidation, higher levels of Fe’+, managanese, and chromium have been reported to occur in BEN-endemic villages. These heavy metals may act as cofactors by forming complexes with OA, leading to an increase in oxidative damage. 5. Environmental contamination by OA (and citrinin) of cereal and beans has been found to be more frequent in endemic than in control areas and also in affected than in nonaffected families. 6. Case-control studies have revealed more frequent and higher blood and urine contamination of OA in patients with BEN and/or UTT in the Balkan area and also in Tunisia. A BEN-like nephropathy in Tunisia allegedly related to OA has recently been described (Maaroufi, et al., 1995a, b). In this regard, a recent study is worthy of further discussion because exposure to OA has been well documented from food analyses, serum analyses, urine analyses, and OA-DNA adducts in tissues from patients with kidney tumors (Nikolov et al.. 1996). Analysis of the age-adjusted incidence of UTT in four populations characterized as very high and high risk for BEN ( n = SSOO), low risk (11 = 23,000), and populations unaffected

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by BEN (11 = 239,000), indicated a 10-fold difference in the incidence of cancers of the kidney, pelvis, ureters, and urinary bladder. Taking the unaffected population as the reference group, populations in the high and very high incidence areas of BEN showed an adjusted relative risk for cancers of the kidney and ureters of 8-29 in men and 27-34 in women. For cancers of the urinary bladder, the relative risks were 2-4 in men and 6-1 1 in women. The geographic correlation observed between BEN and UTT inthis study stongly suggested a common etiology with OA was a relevant factor. In addition to the fact that exposure to OA was well documented, UTT tumors are rare enough so that a 10-fold excess paired with strong geographic clustering is difficult to attribute to misclassification/underdiagnosis biases or to chance. However, the implication of OA as sole risk factor for BEN and/or UTT has not been proven. The following factors should be considered (Castegnaro et al., 1990j: 1. No direct epidemiological proof for a causal relationship between mycotoxins and BEN has yet been presented. Complex exposures to mycotoxins usually occur in conjunction with other poorly characterized environmental agents. Epidemiological studies at the individual level are required; both these and future laboratory studies should take into consideration the involvement of multiple risk factors. 2. In some studies, data collected on environmental contamination relate to periods after the time when the disease was likely to have begun. 3. It is not clear how differences in food storage or other dietary practices, as opposed to or in combination with individual susceptibility, can explain the unusual clustering of BEN and UTT. Villages afflicted in the past continue to be afflicted today, while nonendemic villages, sometimes located in close proximity to afflicted villages, have remained free of BEN. 4. BEN may be caused by long-term exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds leaching into the well drinking water from low-rank coal underlying or proximal to the endemic settlements. These organic compounds have been observed in well water in endemic villages, but not in nonendemic villages. (Tatu et al., 1998). 5. A viral etiology for BEN is unlikely, but has not been ruled out. For example, there may be a relationship between Bolivian hemon-hagic fever, an arenaviral hemorrhagic fever disease spread by rodents, and BEN (Tatu et al., 1998). 6. Phenotypic and genotypic differences in a number of activating and detoxifying enzymes have frequently been linked to individualized susceptibility to certain forms of cancer: the association between the efficiency of oxidative metabolism of debrisoquine and the risk for developing BEN and/or UTT, individuals suspected of having BEN,health controls from the endemic regions, and healthy controls from nonendemic regions. The extensive metabolizer phenotype was encountered in most of the BEN cases and subjects with BEN and UTT (Tatu et al., 1998).

REFERENCES Alavanja, M. J., Rush, G. A., Stewart, P. and Blair, A. (1957). Proportionatemortalitystudy workers in the grain industry. J. Natl. Cancer Inst., 75:247-252.

of

622

Henry and Bosch

Appleton, B. S. and Campbell, T. C. (.1994). Micronuclei, chromosomal aberrations and aflatoxinalbumin adducts in experimental animals after exposure to aflatoxin B1. Mutat. Res. 333: 6 1-67. Bhat, R. V.. Ramakrishna. Y., Beedu, S. R., and Munshi, K. L. (1989). Outbreak of trichothecene mycotoxicosis associated with consumption of mould-damaged wheat products in Kashmir Valle. India. Lancet, Jan. 7, 1989, 35-37. Bhat, R. V., Shetty, P.H., Anmth. R. P., and Sudershan, R. V. (1997). Afoodborne disease outbreak due to the consumption of moldy sorghum and maize containing fumonisin mycotoxins. Clin. TOX. 35(3):249-255. Blot, W. J., Li, J.-Y.. Taylor, P. R., Guo, W., Dawsey, S. M.. and Li, B. (1995). The Linxiantrials: mortality rates by vitamin-~nineralintervention group. Am. J. Clin. Nutr. 63(suppl.):1424S6s. Booth. S. C., Bosenberg, H.. Garner. R. C., Herzog, P. J., and Norpoth, K. (1981). The activation of aflatoxin B1 in liver slices and in bacterial mutagenicity assays using livers from different species including man. Carcinogenesis 2: 1063-1068. Bosch, F. X. (1995). HCV and livercancer: the epidemiological evidence.In: Kobayaski, K., Purcell, R.. Schimotohno, K., and Tabor, E. ed. Hepatitis C virus and its involvement in the development of hepatocellular carcinoma. Princeton Scientific Publishers, Princeton, NJ, pp. 15-25. Bosch. F. X. and Munoz. N.(1991). Hepatocellular carcinoma in the world:epidemiologci questions. In: Tabor, E., Di Bisceglie. A. M.. and Purcell, R. H. eds. Etiology. pathology, and treatment of hepatocellular carcinoma in North America. Gulf Publishing Company, Houston, pp. 3554. Brechot, C. et al. (1998). Impact of HBV, HCV, and GBV-C/HGV on hepatocellular carcinomas in Europe: results of a European concerted action. J. Hepatol. 29(2):173-183. Campbell, T. C. (1994). Correspondence re: Qian et al., a follow-up study of urinary markers of aflatoin exposure and liver cancer risk in Shanghia. People’s Republic of Chian. Cancer Epidemiol Biomarkers Prev. 3:3-10, 1994. and c . c.Harris, Solving theviral-chemical puzzle of human liver acrcionogeneiss. Cancer Epidemiol. Biomarkers Prev. 3: 1-2. Cancer Epidemiol. Biornarkers Prev. 3:519-521. Campbell, T. C., Chen, J., Liu, C.. Li, J. and Parpia, B. (1990). Nonassociation of alfatoixn with wprimary liver cancer in a cross-sectionalecologicalsurveyinthePeople’sRepublicof China. Cancer Res. 50:6882-6893. Camus-Radon, A.-M., Raffali, F., Bereziat, J.-C., McGregor, D., Konstandi, M.. and Lang, M. A. (1996). Liverinjury and expression ofcytochrome P450: evidencethat regulation ofCYP2A5 is different from that of other major xenobiotic metabolizing CYP enzymes. Toxicol. Appl. Pharmacol.. 138:140-148. Castegnaro, M. Chermozemsky, I. N.. Hietanen, E., and Bartsch. H. (1990). Are mycotoxins risk factors for endemic nephropathy and associated urothelial cancer? Arch. Geschwulstforsch. 60(4):295-303. Chang, M.-H., Chen. C.-J., Lai. M.-S., Hsu, H.-M.. Wu, T.-C..Kong.M.-S.,Lian.D.-C.. Shau, W.-Y., and Chen, D.-S.( 1997). Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. N. Engl. J. Med. 336(26):1855-1859. Chen, J., Campbell, T. C., Li,J. and Peto, R. (1990). Diet. lifestyle and mortality in China: a study of the characteristics of 65 Chinese counties. Joint publication of Oxford University Press (Oxford, England), Cornell University Press (Ithaca, NY), and China People’s Medical Publishing House (Beijing, China). Chu, F. S. and Li, G. Y. (1993). Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People’s Republic of China in regions with high incidences of esophageal cancer. Appl. Environ. Microbiol. 60(3):847-852. Council for Agriculture Science and Technology (1989). Mycotoxins: Economic and Health Risks. Council for Agriculture Science and Technology Task Force Report 1 16. Ames. IA. Cullen, J. M. and Newberne, P. M. (1994). Acute hepatotoxicity of alfatoxins. In: The Toxicology of Aflatoxins (D. L. Eaton and J. D. Groopman. eds.) Academic Press, New York, pp. 3-21.

Disease Foodborne

and Mycotoxins

623

De Vries, H. R.. Maxwell, S. M., and Hendrickse, R. G. (1990). Aflatoxin excretion in children with kwashiorkor or marasmic kwashioror-a clinical investiation. Mycopathology 110: 1-9. Eaton, D. L. and Groopman, J. D. (1994). The Toxicology ofAflatoxins.Academic Press, San Diego. Edwards, C. Q. et al. (1988). Prevalence of hemochromatosis among 11,065 presumably healthy blood donors. N. Engl. J. Med. 318(21):1355-1362. Evans, A. A. et al. (1998). Geographic variation in viral load among hepatitis B carriers with differing risks of hepatocellular carcinoma. Cancer Epidemiol.Biomarkers Prev. 7(7):559-565. FoodandNutritionBoard (1989). Recommendeddietaryallowances, 6th ed. NationalResearch Council, National Academy of Sciences, Washington. DC, Chapter 6. pp. 52-77. Franceschi. S., Bidoli, E., Buron, A. E., andLa Vecchia, C. (1 990). Maizeand risk of the oral cavity. pharynx and esophagus in northeastern Italy. J. Natl. Cancer Inst. 82:1407-1411. Gelderblom, W. C. A., Jackiewicz, K., Marasas, W. F. O., Thiel, G., Horak. R. M.. Vleggaar, R.. and Kriek, N. P. J. (1988). Fumonisins-novel mycotoxins with cancer promoting activity produced by Fusarizoiz nlonilijbrme. Appl. Environ. Microbiol. 54: 1806- 18 11. Gorelick, N. J. (1990). Risk assessment for aflatoxin. I. Metabolism of aflatoxin B1 by different species. Risk Anal. 10(4):539-559. Groopman. J. D., Wild, C. P., Hasler, J., Chen, J., Wogan, G. N., and Kensler, T. W. (I 993). Molefular epidemiology of aflatoxin exposures: validation of aflatoxin-N-7-guanine levels in urine as a biomarker in experimental rat models and humans. Environ. Health Perspect. 99: 107113. Groopman, J. D., Wogan, G. N., Roebuck, B. D., and Kensler, T. W. (1994). Molecularbiomarkers for aflatoxin and their application to human cancer prevention. Cancer Res. 54(Suppl.) 1907s1911s. Guengerich, F. P., Johnson, W. W., Ueng, Y.-F., and Shimade. T. (1996). Involvement of cytochrome p450, gluytathione S-transferase, and epxoide hydrolase in the metaabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ.Health Perspect. 103(3):557562. Hall. A. J. and Wild, C. P. (1994). Epidemiology of aflatoxin-related diseases. In: The Toxicology of Aflatoxins. (D. L. Eaton and J. D. Groopman, eds.) Academic Press, New York. pp. 233258. Hatch, M. C., Chen. C. J., Levin, B., Ji. B. T., Yang. G. Y., Hsu, W. S., Wang, L. W., and Santella, R. M. (1993). Urinary aflatoxin levels. hepatitis B virus infection and hepatocellular carcinoa in Taiwan. Tnt. J. Cancer 54:93 1 - 9 3 . Hayes, R. B., van Niewenhuize, J. P., Raatgever, J. W., and ten Kate, F. J. W. (1984). Aflatoxin exposures in the industrial setting: an epidemiological study of mortality. Food Chem. Toxicol. 22:39-43. Hendrickse. R. G., Coulter, J. B. S.. Lamplugh, S. M., MacFarlane, S. B. J.. Williams, T. E.. Omer. M. J. A., and Suliman, G. I. (1982). Aflatoxin and kwashiorkor: a study in Sudanese children. Br. Med. J. 285:843-846. Hendrickse, R. G. (1 991). Kwashiorkor: the hypothesis that incriminates alfatoxin. Pediatrics 88(2): 376-379. Hsing, A. W. et al. (1995). Cancer risk following primary hemachromatosis: a population-based cohort study in Denmark. Tnt. J. Cancer 60(2):160-162 1995. Hu, J.-F.. Chisari F. V.,and Campbell,T.C.(1994). Modulatingeffectofdietaryproteinon transgene expression in hepatitis B virus (HBV) trangenic mice. Proc. Am. Assoc. Cancer Res. 35:104. IARC (1993). Some naturally occurring substances: food items and constituents. heterocyclic aromatic amines and mycotoxin. International Agency for Research on Cancer, Lyon, pp. 245395. (IARC Monographs on the Evaluation of Carcinogenic Risks of Chemicals to Humans, Volume 56). IARC (1994). Hepatitis viruses. International Agency for Research on Cancer, Lyon (TARC Monographs on the Evaluation of Carcinogenic Risks of Chemicals to Humans, No. 59).

624

Henry and Bosch

IARC (1997). In: Toiniolo. P., Boffetta. P., Shuker, D.. Rothman, N., Hulka, B.. and Pearce, H. ed. Application of biomarkers in cancer epidemiology. International Agency for Reserach on Cancer, Lyon, pp. 25 1-264 (IARC Scientific Publications No. 142.). IPCS (1990).Selected Mycotoxins: Ochratoxins, Trichothecenes, Ergot Environmental Health Criteria 105. JECFA (1998). Safetyevaluation of certain food additives. WHO Food Additive Seried 40. International Program on Chemical Safety, World Health Organization, Geneva. Jelliffe, D. B. et al. (1992). Causation of kwashiorkor: toward a multifactorial consensus. Pediatrics 90(1 part1):110-113. Knecht, P. et al. (1994). Body iron stores and risk of cancer. Int. J. Cancer 56(93):379-382. Kuiper-Goodman, T., Scott, P. M., and Watanabe, H. (1987). Risk assessment of the mycotoxin zearalenone. Reg. Toxicol. Pharmacol. 7:253-306. Leon-S., F. E., Carpintero, M., Gaffga, N.. Ocampa, L. and Bayona, J. (1996). Mycotoxins in myelopathies of man. Lancet 348(9033):1039. Louria. D. B., Smith,J. K., andFinkel G.C.(1970).Mycotoxinsother thanaflatoxins: tumorproducing potentialandpossiblerelationtohumandisease.Ann.N.Y.Acad. Sci. 174(2): 583-59 1. Luo, U., Yoshizawa, T., and Katayama, T. (1990). Comparative study on the natural occurrence of F ~ ~ u - i u mycotoxins n? (trichothecenes and zearalenone) in corn and wheat from high- and low-risk areas for human esophageal cancer in China. Appl. Environ. Microbiol.56(13,): 3723-3726. Maaroufi, K., Achour, A., Betbeder, A. M., Hammani. M., Ellouz, F., Creppy. E. E., and Bacha, H. (1995a). Foodstuffsand human blood contamination by the mycotoxin ochratoxin A: correlation with chronic interstitial nephropathy in Tunisia. Arch. Toxicol. 69:552-558. Maaroufi, K.. Achour, A., Hammami. M.. el May, M., Betbeder. A. M., Ellouz, F., Creppy, E. E., and Bacha, H. (1995b). Ochratoxin A in human blood in relation to nephropathy in Tunisia. Hum. Exp. Toxicol. 14:609-614. Mandishona, E., et al. (1 998). Dietary iron overload as a risk factor for hepatocellular carcinoma in Black Africans. Hepatology 27: 1563-1566. Marasas. W. F. O., Jaskiewicz, K., Venter, F. S., and Van Schalkwyk, D. J. (1988). Fmwiuuz tlzoniliforme contamination of maize in oesophageal cancer areas in Transkei. S. Afr. Med. J. 74: 110-114. Marasas, W. F. 0. (1995). Fumonisins: their implication for human and animal health. Nat. Toxins. 31193-198. Marasas, W. F. 0. (1996). Fumonisins: history, world-wide occurrence and impact. Adv. Exp. Med. Biol. 392:1 - 17. Massey, T. E., Stewart, R. K., Daniels, J. M., and Liu, L. (1995).Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B 1carcinogenicity. Proc. SOC. Exp. Biol. Med. 208: 213-227. Mclaren, C. E. et al.( 1995). Prevalence of heterozygotes for hemochromatosisin the white population of the United States. Blood 86(5):2021-2027. McLean. M. and Dutton, M. F. (1 995). Cellularinteractions and metabolism of aflatoxin: an update. Pharmacol. Ther. 65:163- 192. Mekhamacha-Dahel, C., Lafarge-Frayssinet, C., and Frayssinet, C. (1990). Immunosuppressive effects of four trichothecene mycotoxins. Food Addit. Contam. 7(suppl. l):S94-S96. Miller, D. M. and Wilson, D.M. (1 994). Veterinary disease related to alfatoxins In: The Toxicology of Aflatoxins (D. L.Eaton and J. D. Groopman. eds.) Academic Press. New York pp. 27-41. Nikolov. I. G., Petkova-Bocharova, D., Castegnaro,M., Pfohl-Leskowicz, A., Gill. C.. Day, N., and Chernozemsky. I. N. (1996). Molecular and epidemiological approaches to the etiology of urinary tract tumors in an area with Balkan endemic nephropathy. J. Environ. Path. Toxicol. Oncol. 1X2-4):201-207. Norred, W. P. and Voss, K. A. (1994). Toxicityand role of fumonisins in animal disease and human esophageal cancer. J. Food Prot. 57:522-527.

Foodborne Disease and Mycotoxins

625

Olsen, J. H., Dragsted, L., and Autrup, H. (1988). Cancer risk and occupational exosure to aflatoxin in Denmark. Br. J. Cancer 58:392-3396. Olubuyide, I. 0.. Maxwell, S. M., Akinyinka, 0. 0..Hart, C. A.,Neal, G. E., and Hendrickse, R. G. (1993). Hbsag and aflatoxin in sera of rural (Igbo-Ora) and urban (Tbadan) populations in Nigeria. Afr. J. Med. Med. Sci. 22:77-80. Pascale,M., Doko, M. B., andVisconti, A. (1995). Determination of fumonisins inpolentaby high performance liquid chromatography., In: Atti 2" Congress0 Nazional di Chimica degli Alimenti, Giardini-Naxos. Italy. pp. 1067-1071. (in Italian). Preston, R. S., Hayes, J. R., and Campbell, T. C. (1976). The effect of protein deficiency on the in vivo binding of aflatoxin B1-induced hepatic preneoplastic lesions. J. Natl. Cancer Inst. 81: 1241-1245. Qian, G.-S., Ross, R. K., Yu, M. C., Yuan, J.-M., Gao, Y.-T.. Henderson, B. E., Wogan, G. N., and Groopman. J. D. (1994). A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People's Republic of China. Cancer Epidemiol. Biomarkers Prev. 3:3333-10. Rheeder, J. P., Marasas, W. F. 0..Thiel, P. G., Sydenham, E. W., Shephard. G. S., and Van Schalkwyk, D.J. (1992). Fusariunz nzorzil(formeand fumonisins in cornin relation tohuman esophageal cancer in Transkei. Phytopathology 82353-357. Roebuck, B. D. and Maxuitenko. Y. Y. (1994). Biochemical mechanisms and biological implications if the toxicity of aflatoxin as related to aflatoxin carcinogenesis. In: The Toxicology of Aflatoxins (D. L. Eaton and J. D. Groopman, eds.) Academic Press. New York, pp. 2741. Ross, R. K., Yuan, J.-M., Yu, M. C., Wogan. G. N., Qian, G.-S., Tu, J. T., Groopman, J. D., Gao, Y.-T., and Henderson, B. E. (1992). Urinary aflatoxin biolnarkers and risk of hepatocellular carcinoma. Lancet 339(8799):943-946. Rotter, B. A., Prelusky, D. B., and Pestka, J. J. (1996). Toxicology of deoxynivalenol (vomitoxin). J. Toxicol. Environ. Health 48:l-34. Santella. R. M., Zhang. Y.-J., Chen, C.-J., Lee, C.-S., Haghighi, B., Hang, G.-Y., Wang, L.-W., and Feitelson, M. (1993). Immunohistochemical detection of aflatoxin-adducts and hepatitis B virus antigens in hepatocellular carcinoma and nontumorous liver tissues. Environ. Health Perspect. 99: 199-202. Scholl, P., Musser, S. M., Kensler, T. W., and Groopman, J. D. (1995). Molecular biomarkers for alfatoxin and their application to human liver cancer. Pharmacogenetics 5:s171-S 176. Schulsinger, D. A., Root, M. M., and Campbell, T. C. ( 1989). Effect of dietary protein quality on development of aflatoxin B1-induced hepatic preneoplastic lesions. J. Natl. Cancer Inst. 81: 1241-1245. Srivatanakul, P., Parkin, D. M., Khlat, M., Chenvidhya, D.. Chotiwan. P., Tnsiripong, S. L., Abbe', K. A. andWild, C. P. (1991). Liver cancer in Thailand. TI. A case-control study of hepatocellular carcinoma. Int. J. Cancer 8:3229-332. Stevens, R. G. et al. (1998). Body iron stores and the risk of cancer. N. Engl. J. Med. 319( 16): 1047-52. Sydenham, E. W., Thiel, P. G., Marasas, W. F. O., Shephard, G. S., Van Schalkwyk, D. J., and Koch, K. R. (1990). Natural occurrence of some Flwwiur?l mycotoxins in corn from Low and high esophageal cancer prevalence areas of the Transkei, SouthernAfrica. J. Agric. Food Chem. 38:1900-1903. Tagger, A. et al. (1997). A case-Control study on GB virus C/hepatitis G virus infection and hepatocellular carcinoma. Brescia HCC study. Hepatology 26(6): 1653-1657. Tanaka, N.. Chiba, T., Matsuzaki,Y., Osuga, T., Aikawa, T., and Mitamura,K. (1993). High prevalence of hepatitis B and C viral markers in Japanese patients with hepatocellular carcinoma. Gastroenterol. Jpn. 28(4):547-553. Tatu, C.A.. Orem, W. H., Finkelman,R. B. and Feder, G.L. (1998). Theetiology of Balkan endemic nephropathy: still more questions than answers. Environ. HealthPerspect. 106(11):689-700. Thomas, D. B. (1991). Exogenous steroid hormones and hepatocellular carcinoma. In: Tabor, E.,

626

Henry and Bosch

Di Bisceglie, A. M., and Purcell, R. ed. Etiology, pathology and treatment of hepatocellular carcinotna. PPC-Gulf Publishing Company, Houston, TX, Chapter 6, pp. 77-91. Ueno, Y.. Iijima. K., Wang, S.-D., Sugiura, Y., Sekijima, M., Tanaka. T., Chen, C., and Yu. S.-Z. (1997). Fumonisins as a possible contributory risk factor for primary liver cancer: a 3-year study of corn harvested in Haimen. China, by HPLC and ELTSA. Fd. Chem. Toxicol. 35: 1133- 1 150. Wang. J.-Ss., Qian. G.-Ss., Zarba. A.,He, X.. Zhu, Y.-R., Zhang. B.-C., Jacobson, L., Gange, S. J.. Munoz, A., Kensler, T. W., and Groopman, J. D. (1 996a). Temporal patternan of alfatoxin-albumin adducs in hepatits B surface antigen-positive and antigen-negative residents of Daxin, Qidong County, People’s Republic of China. Cancer Epidemiol. Biomarkers Prev. 5:253-261. Wang, L. Y., Hatch, M., Chen. C.-J.. Levin, B.. You. S.-L., Lu, S.-N., Wu, M.-H., Wu. W.-P., Wang. L.-W., Wang, Q., Huang, G. T., Yang, P.-M., Lee. H.-S., and Santella. R. M. (1 996b). Aflatoxin exposure and risk of hepatocelular carcinoma in Taiwan. Int. J. Cancer 67:620625. Wild, C. P., Hudson, G. J.. Sabbioni, G.. Chapot, B., Hall. A. J., Wogan. G. N., Whittle, H., Montesano, R.. and Groopman, J. D. (1992). Dietary intake of aflatxoins and the level of albuminbound aflatoxin in peripheral blood in the Gambia, West Africa. Cancer Epidemiol. Biomarkers Prev. 129-234. Wild. C. P., Fortuin, M., Donato, F., Whittle, H. C.. Hall, A. J., Wolf. C. R.. and Monetesano, R. (1993). Aflatoixn, liver enzymes and hepatitis B virus infection in Gambian children. Cancer Epidemiol. Biomarkers Prev. 2555-561. Wild, C. P., Hasegawa, R., Barraud, L., Chutimataewin. S.. Chapot, B., Ito, N., and Montesano, R. (1 996). Aflatoxin-albumin adducts: a basis for comparative carcinogenesis between animals and humans. Cancer Epidemiol. Biomarkers Prev. 5:179-189. Wood, G. E. and Trucksess, M. W. Regulatory control programs for mycotoxin-contaminated food. In: K. Sinha and D. Bhatnager, eds. Mycotoxins in Agriculture and Food Safety, Marcel Dekker, New York, 1998, p. 461. World Health Organization, JECFA (1990). Toxicological evaluation of certain food additives and contaminants., IPCS. WHO Food Additives Series no. 26:143-165. World Health Organization ( 1993). Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monogr. Eval. Carcinogen. Risks Hum. 56:397-444. Yang, Q. et al. (1998). Hemochromatosis-associated mortality in the United States from 1979 to 1962: an analysis of multiple-cause mortality data. Ann. Intern. Med. 129(11):946-953, 1998. Yeh, F. S., Mo, C.-C., and Yen, R.-C. (1985). Risk factors for hepatocellular carcinoma in Guangxi, People’s Republic of China. Natl. Cancer Inst. Monogr. 69:47-48. Yeh, F. S.. Yu. M. C., Mo. C.-C., Luo, S., Tong. M. J., and Henderson. B. E. (1 989). Hepatitis B virus, aflatoxin, and hepatocellular carcinoma in Southern Guangxi, China. Cancer Res., 49: 2506-2509. Youngman, L. D. and Campbell. T. C. (1992).Inhibition of aflatoxin B1 -induced gamma-glutamyltranspeptidase positive (GGT-t) hepatic preneoplastic foci and tumors by low protein diets: evidence that altered GGT+ foci indicate neoplastic potential. Carcinogenensis 13(9):16071613. Yoshizawa, T., Yamashita, A. and Luo. Y. (1994). Fumonisin occurrence in corn from high- and low-risk areas for human oesophageal cancer in China. Appl. Environ. Microbiol. 60:16261629. Yuan, J. M.. Ross. R. K.. Stanczyk, F. Z., Govindaragan, S., and Gao, Y. T. (1995). A cohort study of serum testosterone and hepatocellular carcinoam in Shanghai, China. Tnt. J. Cancer 63: 49 1-493.

15 Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Koshinsky Iwestigell, Alameda, California

Adrienne Woytowich and George G. Khachatourians University of Saskatclte~twn,Saskatoon, Saskatcltewun, Canada

I. Introduction 628 11. Mycotoxin-Mycotoxin 629 A. Aflatoxin B1 629 Cyclopiazonic B. acid 629 Ochratoxin C. A 630 D. Fusariotoxins 630 E. Summary 630 111. Mycotoxin-Other Organisms A. Aflatoxin B1 Ochratoxin B. 632 A Fusariotoxins C. 632 D. Summary 632

630

630

IV. Mycotoxin-Plant Components 632 V.

Mycotoxin-Heavy Metals 633

A. Aflatoxin B1 Citrinin 633 B. Ochratoxin C. A Fusariotoxins D. E. Summary 634

633 634 634

VI. Mycotoxin-Ethanol 634 VII. Mycotoxin-Sorbents 634 A. Aflatoxin B1 Fusariotoxins B. 635 Other C.mycotoxins 635 D. Summary 635

635

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VIII. Mycotoxin-Other Chemicals 636 A. Aflatoxin I31 636 B. Citrinin 637 C. Cytochalasin E 637 D. Ochratoxin A 637 E. Fusariotoxins 637 F. Other Inycotoxins 638 G. Summary 638 IX. Mycotoxin-Environment 638 A. Aflatoxin B1 638 B. Ochratoxin 639 C. Fusariotoxins 639 D. Summary 639 X.

Conclusions 639 References 640

1.

INTRODUCTION

Feed contaminated with multiple mycotoxins is regularly implicated in mycotoxicoses (1, 2). Numerous suspected mycotoxicoses occur when the detected levels of mycotoxins are far below the levels needed to induce symptotns under laboratory conditions (2). The inability of single mycotoxins to mimic disease development under laboratory conditions also occurred in plants (3). A probable reason for this discrepancy is the interactions that occur between mycotoxins themselves and/or interactions that occur between mycotoxins and other factors. These interactions can cause presumed "noninhibitory" levels of mycotoxins to have deleterious effects. Several authors warn of synergistic interactions between fungal metabolites that affect humans, animals (4), and plants (5). However, the science of understanding and predicting mycotoxin interactions is in its infancy. This chapter surveys mycotoxin interactions and may help elucidate the context(s) in which mycotoxin exposure is a serious concern. In 1994 a similar work surveyed the interactions that occur between mycotoxins (6). While this chapter brings that survey up to date, it concentrates on the interactions that occur between mycotoxins and other factors such as exercise, hepatitis infection, or cadmium consumption. A review of how interactions are examined is appropriate. The reader is referred to specific articles and reviews for more detailed discussion (6-19). The general approach to studying the interaction that occurs between two treatments involves observing the effect of treatments individually and the effects of the same treatments together. From the effects of the individual treatments a prediction is made regarding the effect of the treatments together. This prediction is based on either addition or multiplication of the individual effects. The observed effect of the combined treatment is compared to the predicted effect of the combined treatment. From this comparison the interactions between treatments are classified as either more inhibitory than predicted, about as inhibitory as predicted, or less inhibitory than predicted. Interactions between inhibitory treatments are described with variations of the terms spzergisrn, crdditive, or crntagollism, respectively. To avoid implying a mechanism of determining the predicted effect, the term additive

Mycotoxicoses

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irztermtion is avoided. When the observed effect of the combined treatment is about as inhibitory as predicted, the term zero irlteractiorz is used. If the interaction is between very different treatments, these terms become less descriptive and even cumbersome. In these cases, variations of the terms i~lci-eased/decretrsedthe toxic effect, protected from, restored, inhibited, or enhanced are used. Finally, it is rare that the two treatments have the same type of interaction for all parameters examined. Thus the type of interaction between two treatments describes an overall sense of the interaction. Dramatic exceptions to this are noted.

II. MYCOTOXIN-MYCOTOXIN Fungi can simultaneously produce multiple mycotoxins (20), multiple fungi often infect food or feed, and contamination of food and feed by multiple mycotoxins is often observed. In food and/or feed aflatoxin B 1 has been found with fumonisins (21,22), cyclopiazonic acid (23), or ochratoxin A (24). Aflatoxin B1 and ochratoxin A have been found in human breast milk in Sierra Leone (25). Beer from Africa contained aflatoxin B1 and zearalenone (26). Corn from the Asian tropics contained fumonisins, aflatoxins, nivalenol, and zearalenone (27). Ochratoxin A and citrinin were found in barley from the United Kingdom (22). In Korea beer was found contaminated with deoxynivalenol and nivalenol (28). Corn samples from China contained multiple trichothecenes (29). Many maize and maize products contained a mixture of trichothecenes, fumonisins, and moniliformin (30). With the frequent occurrence of multiple mycotoxins an understanding of the interactions that occur between them is desperately needed. We do not need to understand the molecular mechanism of the interactions. However, we do need to know the mycotoxin combinations that have a synergistic interaction and thus are particularly dangerous. A.

Aflatoxin B1

Aflatoxin B 1 and kojic acid interacted synergistically and increased toxicity to Spodoptera Zitfordis larvae and adult sterility (31). In chick embryo assays, aflatoxin B1 and ochratoxin A had a synergistic interaction when the occurrence of physical abnormalities was measured and zero interaction when mortality was measured (32). In chicks, the interaction between aflatoxin B1 and moniliformin was zero to slightly antagonistic (33). In turkey poults, the interaction between aflatoxin B 1 and fumonisin B was zero too slightly antagonistic (34, 35). In rats, nivalenol and aflatoxin B1 interacted antagonistically when the aflatoxin B 1-DNA adduct concentration was measured (36). In pigs, there was generally zero interaction between aflatoxin B 1 and fumonisin B1. However, when liver disease was examined, the interaction was synergistic (37j. Aflatoxin B1 and either aflatoxin B2 or ochratoxin interacted synergistically when liver cancer was examined (38). B. Cyclopiazonic Acid Cyclopiazonic acid and patulin interacted antagonistically and decreased lipid peroxidation, calcium influx, and blebbing in tissue culture (39). In chicks, cyclopiazonic acid and T-2 toxin generally interacted synergistically. However, the interaction was antagonistic when body weight wasmeasured and zero when efficiency of feed utilization or mortality was measured (40).

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C.

OchratoxinA

In renal cortical cubes isolated from swine kidneys, ochratoxin A and citrinin interacted synergistically when ion transport or protein synthesis was measured. With all other parameters there was zero interaction (41). In chicks, there was zero interaction between ochratoxin A and diacetoxyscirpenol (42). In turkey poults, there was zero interaction between ochratoxin A and fumonisin (43). In pigs, there was zero interaction between ochratoxin A and T-2. toxin (44) or zearalenone (45).

D. Fusariotoxins In yeast, the interaction between T-2 toxin and either HT-2 toxin (46) or vermcarin A (47) or between deoxynivalenol and nivalenol was synergistic. The interaction between deoxynivalenol and T-2 toxin was antagonistic (46). In maize embryos, deoxynivalenol and zearalenone interacted synergistically and inhibited root and shoot elongation (48). In yolk sacs, fusaric acid and fumonisin B1 interacted synergistically (49). T n chick embryo assays, there was zero interaction between zearalenone and deoxynivalenol (50). In chick toxicity assays, there was zero interaction between fumonisin and either moniliformin (5 1) or T-2 toxin (52). In chicks, the interaction between deoxynivalenol and tnoniliformin (53) or fumonisin (52) was zero to antagonistic. T-2 toxin and diacetoxyscirpenol interacted synergistically and inhibited egg production in laying hens. However, with most other parameters there was zero interaction (54). In turkey poults, there generally was zero interaction between T-2 toxin and fumonisin (55). In turkey poults, the interaction between fumonisin and either diacetoxyscirpenol (43) or moniliformin (56) was zero to slightly antagonistic. In immature swine, deoxynivalenol and fusaric acid interacted synergistically and decreased weight gain (57). In growing pigs, deoxynivalenol and fumonisin B 1 had zero to synergistic interaction (58).

E. Summary As observed before (6, 18) two mycotoxins generally have zero or an antagonistic interaction. In poultry, the fusariotoxins generally have zero interaction; the combinations are neither more nor less toxic than expected. Swine are very sensitive to synergistic interactions occurring between mycotoxins. The notable situations where a synergistic interaction occurs are between: (1) deoxynivalenol and other fusariotoxins in swine, (2) diacetoxyscirpenol and T-2 toxin in laying hens, and (3) aflatoxin B1 and fumonisin B 1 in swine.

111.

MYCOTOXIN-OTHER ORGANSIMS

A.

Aflatoxin B1

Exposure to mycotoxins increases stress and may leave animals more susceptible to infection. In quail chicks, there was a synergistic interaction between aflatoxin B 1 and coccidia when weight gain (59) or mortality rate (60) was measured. The addition of Sacchcrromyxs cerevisiae to aflatoxin B 1-containing diets restored to normal the body weight, organ weight, and serum profiles compared to chicks fed diets containing aflatoxin B1

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alone (61). In calves, there was zero interaction between infection with flukes and aflatoxin B 1consumption (62). Epidemiological studies indicate that exposure to aflatoxin B l is one of many risk factors for the development of hepatocellular carcinoma (HCC). A 32-year study with nonhuman primates confirmed that aflatoxin B1 is a potent hepatocarcinogen (63). In humans, HCC is cornmon, difficult to treat, and lethal (64). Epidemiological surveys suggest that aflatoxin B 1and hepatitis virus B (HBV) interact synergistically and increase the frequency of liver cancer (65). In human chronic HBV carriers, the level of urinary aflatoxin B1 metabolites increased along with the risk of developing HCC (66). HBV carriers have a significantly higher frequency of aflatoxin B 1-DNA adducts (67). A study of over 18,000 people provides convincing evidence that there is a synergistic interaction between aflatoxin B1 and HBV when the incidence of HCC is examined (68, 69). The theory that aflatoxin B1 and hepatitis synergistically increase the risk of HCC is supported experimentally. The incidence of HCC was significantly higher in tree shrews infected with HBV and exposed toaflatoxin B l compared to shrews exposed to each treatment individually (70). The woodchuck hepatitis virus (WHV) is closely related to human HBV. Woodchucks that carried WHV were fed aflatoxin B 1. These woodchucks had a significantly higher incidence of HCC compared to WHV carriers not consuming aflatoxin B 1. HCC was not detected in noncarriers receiving aflatoxin B 1 (71). Elegant studies were performed with transgenic mice that have HBV DNA integrated into their genome. HCC developed only in transgenic mice fed aflatoxin B 1. There were no tumors in nontransgenic mice fed aflatoxin B 1 or in transgenic mice not fed aflatoxin B1 (72). These studies strongly support the idea that exposure to HBV and aflatoxin B1 interact synergistically and increase the occurrence of HCC. Further research has investigated the basis of this interaction. The synergistic interaction between aflatoxin B 1and HBV may be due to increased mutations at codon 249 of the p53 tumor-suppressing gene or altered p53 expression. In humans, the codon 249 mutation occurred in HBV-infected individuals. Additional aflatoxin B1 exposure marginally increased codon 249 mutations (73). The p53 expression was similar in tree shrews with HCCs induced by aflatoxin B 1 and HBV infection compared to tree shrews with HCCs induced by aflatoxin B 1 alone (74). Other reports indicate that a cotnbined aflatoxin exposure and HBV infection effectively causes mutations in p53 (75). Thus, the interaction between HBV and aflatoxin B1 when alterations of the p53 tumor-suppressing gene are measured is uncertain. The synergistic increase in HCC with HBV and aflatoxin B l exposure may be due to enhanced metabolic activation of aflatoxin B l in the presence of HBV. The aflatoxin B 1activation rate was significantly higher in livers from WHV-infected woodchucks compared to livers from noninfected animals (76). However, aflatoxin B 1activation was lower in liver microsomes isolated from woodchucks chronically infected with WHV compared to liver microsomes from WHV-free woodchucks. The extent of the reduction depended on the severity of the hepatitis (77). Thus, it is not clear if there is increased or decreased activation of aflatoxin B l with HBV infection. Perhaps the increased rate of HCC with aflatoxin B1 and HBV exposure is due to aflatoxin B 1suppressing cell-mediated immunity. Impaired immunity would allow persistence of HBV in the liver, and may lead to an increased HCC incidence (78). Inclusion bodies occurred more frequently. were more prominent, and appeared earlier in chicks fed aflatoxin B l and inoculated with inclusion body hepatitis virus compared to chicks not receiving aflatoxin B1 (79).

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An alternative theory for the mechanism of the synergistic interaction is that aflatoxin B1 causes alteration in p53 and that HBV proliferate in hepatocytes with this alteration (75). Thus, while it is clear that aflatoxin B l and HBV interact synergistically and increase the rate of HCC, the mechanism of this interaction remains unknown. B. OchratoxinA In chicks, inclusion body hepatitis virus increased the effect of ochratoxin A on liver and kidney phosphatase activity (SO). Additionally, inclusion bodies were more frequent, more prominent, and appeared earlier than in chicks not receiving ochratoxin A (79). In chicks, there was zero interaction between ochratoxin A consumption and Snlmorzella infection for most parameters examined. However, when mortality or decrease in body weight was measured, the interaction was synergistic (8 1).

C.

Fusariotoxins

In mice, T-2 toxin and Schzomdln lipopolysaccharide interacted synergistically and dramatically increased the mortality rate (82). Diacetoxyscirpenol and Sdmo~zellcrinteracted synergistically and increased mortality if the diacetoxyscirpenol was administered after infection. There was zero interaction if the diacetoxyscirpenol was administered before infection (83). D. Summary Most organisms interact with mycotoxins to produce a more toxic effect. It is clear that exposure to aflatoxin B1 and HBV interact in a synergistic manner and increase the risk of HCC. The mechanism of this interaction is unknown but is being investigated. In most situations there was a striking increase in mortality when the animals were given a mycotoxin and infection with Schorzelle~or coccidia. The yeast S. cerevisiae protected chicks from aflatoxicosis.

IV. MYCOTOXIN-PLANTCOMPONENTS Plants produce many compounds to protect themselves. These products often alter the effects of mycotoxins. Indole-3-carbinol, a vegetable secondary metabolite, can either enhance or inhibit aflatoxin B 1-induced carcinogenesis. The effect depends on whether exposure is sequential or simultaneous (84). In rats, indole-3-carbinol protected against the development of preneoplastic lesions when administered with aflatoxin B 1. If the indole-3carbinol was administered after aflatoxin B 1, there was no protective effect (85). Similarly, in trout feeding indole-3-carbo1 prior to or along with aflatoxin B1 inhibited the carcinogenic response (86). However, if the indole-3-carbinol was given after the aflatoxin B l , the tumor incidence increased (87j. Several plant compounds inhibit the mutagenicity of aflatoxin B 1. Turmeric (88), chlorophyllin (89), aqueous extracts of various Chinese medicinal herbs (90), ethanol exIracts, Cassia senna concentrate (9 l), or xanthophylls from marigolds (93) significantly reduced the mutagenicity of aflatoxin B 1in Snlrnonellcr mutagenicity assays. Feeding rats aflatoxin B l and garlic, curcumin. or ellagic acid reduced the number of liver cancer

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indicators (88). In trout, beta-naphthoflavone (86) or chlorophyllin (89) reduced aflatoxin B 1-related carcinogenisis. Crocetin (93), aqueous extracts of Scutellrrr-in barbcrtcl (90), or chlorophyllin (89) reduced aflatoxin B 1-DNA adduct formation. Caffeine did not alter the mutagenicity of aflatoxin B 1 in a Snlmonella mutagenicity assay (94). In unscheduled DNA synthesis assays the antigenotoxic agent eugenol did not alter the effects of aflatoxin B1 (95). Other plant compounds increase the mutagenicity of aflatoxin B 1. Various Nigerian food additives increased the mutagenicity of aflatoxin B1 in SnlmorzeZla mutagenicity assays (96). Caffeine increased the genotoxicity of aflatoxin B1 in tissue culture (97). In mice, coadministration of an antioxidant and a flavonoid increased activation of aflatoxin B1 (98). In trout, cyclopropene fatty acids increased the aflatoxin B1-induced carcinogenic response (86, 99). ScrcogZottis gcrbollelzsisbark extract increased the binding of aflatoxin B1 to DNA (100). In chicks, neither beta-carotene nor canthaxanthin overcame the growth-depressing effects of aflatoxin B 1 ( 101). Aflatoxin B 1 and tannic acid or high-tannin sorghum had zero interaction when chick growth and feed conversion ratios were measured (102). There is no striking compartmentalization of the interaction of various plant compounds with aflatoxin B1. There was zero interaction when growth or feed conversion was measured. When carcinogenic responses are measured, the interaction ranges from antagonistic to synergistic depending on the compound and 011 the order of exposure. However, most plant secondary metabolites decrease the mutagenicity/carcinogenicity of aflatoxin B 1.

V.

MYCOTOXIN-HEAVYMETALS

A.

Aflatoxin B1

Indian childhood cirrhosis (ICC) is characterized by copper accumulation in the liver. It has been suggested that mycotoxins interact synergistically with copper to cause ICC (103). Copper increased the mutagenicity of aflatoxin B 1 in a Salrtlonella mutagenicity assay (104). In guinea pigs receiving aflatoxin B 1 and copper, the liver injury was more frequent and more severe compared to aflatoxin B 1or copper treatment alone (105). Additionally, the combined treatment resulted in decreased biliary copper excretion (106). The addition of cadmium to feed significantly reduced the toxicity of aflatoxin B1 to pigs (107). Selenium relieves the aflatoxin B 1-mediated immunosuppression of human peripheral blood lytnphocytes (lOS), protected turkeys from aflatoxicosis (109), and decreased the aflatoxin B 1-induced liver necrosis in rats (1 10). Both selenium deficiency and selenium excess reduced the binding of aflatoxin B 1 to DNA (1 1 1). Selenium deficiency protects rats from aflatoxin B 1-induced hepatotoxicity ( 112).

B. Citrinin Iron reversed the inhibitory effects of citrinin on growth of the bacterium Neisseria rnelzingitidis (1 13) and on mitochondrial lipid peroxidation (1 14). In rats, chromate interacted synergistically with citrinin and increased urine excretion (1 15). Chromate and citrinin interacted synergistically and depressed organic ion transport (1 16).

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C.

OchratoxinA

Iron reversed the growth inhibitory effects of citrinin on the bacterium N. nzenirzgitidis (1 13). Vanadium further reduced the ochratoxin A-inhibited performance of chicks (1 17).

D. Fusariotoxins The growth inhibitory effects of zearalenone on the bacterium N. menir~gifidiswere not reversed with iron (1 13). Selenium protected rats from acute toxicosis caused by either T-2 toxin or deoxynivalenol (1 18, 119). Zinc did not alter the effects of T-2 toxin on mice. Interestingly, T-2 toxin decreased absorption of zinc in animals fed low-zinc diets and increased absorption of zinc in animals fed high-zinc diets (120).

E. Summary

Generally the addition of cadmium, zinc, iron, or selenium is either beneficial or has no effect. Copper, chromate, and vanadium increased the toxicity of aflatoxin B1, citrinin, and ochratoxin A, respectively. These studies investigate the interaction between mycotoxins and high levels of heavy metals. The more common problem is metal deficiency. Iron deficiency affects over 2100 million people (121). Specific studies should be designed to explain the interaction between mycotoxins and nutrient deficiency.

VI.

MYCOTOXIN-ETHANOL

The diets of 90 individuals with primary liver cancer were compared to the diets of 90 age-sex-matched controls. Aflatoxin B 1and alcohol consumption likely interacted synergistically and increased the risk of developing primary liver cancer (122). In numerous studies with rats, the interaction between aflatoxin B1 and ethanol was synergistic. Aflatoxin B1 and ethanol interacted synergistically and increased the amount of subcellular damage in hepatocytes ( 123), increased the covalent binding of aflatoxin B 1to DNA (100, 124, 125), increased hepatotoxicity (125). increased the severity of liver necrosis (126, 127), and increased lysosome lipid peroxide levels (128). They interacted synergistically and decreased the liver ATP content, mitochondrial respiratory enzyme activity (128), and hepatic glutathione (129). There was zero interaction when hepatic fat accumulation was examined (126, 130). Thus, the overall interaction between aflatoxin B1 and ethanol is synergistic; the negative effects are increased by treatment with the combination.

VII.

MYCOTOXIN-SORBENTS

There is no definitive way in which complete detoxification of food and feed contaminated with mycotoxins can be achieved. Thus new methods to eliminate mycotoxicoses are sought. Inorganic sorbents or activated charcoal are effective at removing many mycotoxins in vitro.

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A.

635

Aflatoxin B1

Hydrated sodium calcium aluminosilicate clay (HSCAS) binds aflatoxin B1 in vitro and in vivo (13 1). The addition of HSCAS to the diets of chicks (132- 134) or rats (135) reduced the toxicity of aflatoxin B 1. The addition of HSCAS to the diet of goats reduced the levels of aflatoxin M1 in the milk (136). When the diet of chicks contained T-2 toxin and aflatoxin B 1, the HSCAS was not as effective in reducing the toxic effects (134). When the diet of chicks contained aflatoxin Bl and either diacetoxyscirpenol (132) or ochratoxin A (137), the addition of HSCAS did not reduce the toxicity. HSCAS does not bind ochratoxin A, T-2 toxin, or fumonisin B 1 (138,139). The protective effect of HSCAS against aflatoxicosis was enhanced if an antioxidant such as selenium, methionine, or vitamin E was also included in the diet of chicks (140). Superactivated charcoal marginally reduced the toxicity of aflatoxin B1 to chicks (141). However, not all clays and mineral additives have the desired protective effect (142). Clinoptilolite is commonly added to animal feed to decrease the toxicity of aflatoxin B 1. In pregnant rats, the maternal liver lesions were more severe in animals receiving aflatoxin B1 and clinoptilolite together compared to those receiving aflatoxin Bl alone. This suggests that the clinoptilolite may interact with dietary components that ordinarily reduce aflatoxicosis (143). The inorganic sorbents S1, S2, and S3 had little effect in reducing the toxicity of aflatoxin B 1to chicks (144).

B. Fusariotoxins In mink HSCAS alleviated some of the reproductive toxicity of zearalenone. However, it had noeffect on thehyperestrogenic effects of zearalenone ( 145). A mixture of metoclopramide, activated charcoal, magnesium sulfate, dexamethasone sodium phosphate, sodium bicarbonate, and normal saline decreased the toxicity of T-2 toxin to swine. The activated charcoal and magnesium sulfate were thought to be the critical components of the mixture (146). Asoap and water wash reduced skin lesions on swine topically exposed to T-2 toxin. A superactivated charcoal paste was less effective (147). The addition of HSCAS (1341, superactivated charcoal (141), T-bind (148), or other inorganic sorbents (144) had no effect on the toxicity of T-2 toxin to chicks. Addition of HSCAS had no effect on the toxicity of diacetoxyscirpenol to chicks (132).

C. OtherMycotoxins Acidic phyllosilicate clay, neutral phyllosilicate clay, HSCAS, or clinoptilolite did not reduce the overall toxicity of cyclopiazonic acid to chicks (149). HSACS did not reduce the toxic effects of ochratoxin A to chicks (137).

D. Summary It is well established that several sorbents are effective in reducing the toxic effects of aflatoxin B I . As expected, selenium and HSCAS were very effective in reducing aflatoxicosis. However, some additives are not effective and may even increase the mycotoxicosis. Generally the sorbents are not effective in reducing toxicity of nonaflatoxin mycotoxins.

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VIII. MYCOTOXIN-OTHERCHEMICALS A.

Aflatoxin B1

Numerous compounds increased the mutagenicity/carcinogenicity of aflatoxin. The phenolic antioxidants, butylated hydroxyanisole (l50), butylated hydroxytoluene (150, 15l), tetrandrine (152), and benzo[e]pyrene (153) increased the mutagenicity of aflatoxin B1 in Snlnzorzella mutagenicity assays. In transgenic mice, phorone, a glutathione-depleting agent, increased aflatoxin B1 mutagenicity fourfold in the liver. There was no significant increase in the mutation frequency in the kidneys, lung, or intestine (154). L-Buthionine sulfoximine enhanced aflatoxin B 1-induced liver cancer in rats (155) and increased the binding of aflatoxin B1 to DNA (156). Most carcinogens interacted synergistically with aflatoxin B 1and increased the incidence of HCC (38, 157). The peroxisome proliferator nafenopin interacted synergistically with aflatoxin B1 and increased the number and size of liver tumors in rats. The effect was more pronounced in male than female rats (158). Cystine supplementation accelerated the emergence of aflatoxin-induced liver tumors in rats (159). Dehydroepiandrosterone (DHEA) is a steroid with protective effects against cancer, obesity, diabetes, and cardiovascular disease. In trout, aflatoxin B1 and DHEA synergistically increased the liver tumor incidence, multiplicity, and size (160). Thyroideum POLFA increased the carcinogenicity of aflatoxin B l to rat kidneys (161). Other compounds such as selenite (94), or propyl gallate (150) did not alter the mutagenicity of aflatoxin B 1 in Salmorzelln mutagenicity assays. A few carcinogens had zero interaction with aflatoxin B 1 when the cancer incidence was measured (162). Aflatoxin B 1mutagenicity can be inhibited. Aflatoxin B 1and other known carcinogens had an antagonistic interaction when naked DNA was sequenced after exposure. Subsequent exposures did not increase sequence alterations (163). Tiamulin decreased the aflatoxin B1-induced mutation frequency in reporter bacteria (164). While butylated hydroxyanisole and butylated hydroxytoluene increased the mutagenicity of aflatoxin B 1 in Scrlnzorlelln mutagenicity assays (150), they inhibited the initiation of aflatoxin B 1induced HCC in rats (165). A few carcinogens, such as diethylstilbestrol or hexachlorocyclohexane, interacted antagonistically with aflatoxin B1 and decreased the incidence of HCC (38). Many compounds increase the toxicity of aflatoxin B 1. 2,3,7,8-Tetrachlorodibenzoy-dioxin (TCDD) interacted synergistically with aflatoxin B 1,increased aflatoxin epoxidation, and decreased growth in human epidermal tissue culture (166). Aflatoxin B1 interacted synergistically with tetracycline when bacterial growth was measured (167). In chicks, aflatoxin B 1 and the organophosphate pesticide malathion interacted synergistically (168, 169). In rats, the interaction between aflatoxin B1 and cortisol was synergistic when mortality rate was measured (170). In chickens, aflatoxin B 1reduced the effectiveness of the anticoccidial monensin ( 171). In chickens, aflatoxin B 1and high concentrations of gizzerosine, a component of fishmeal, interacted synergistically when lethality was measured ( 172). Other compounds have no effect or reduce the toxic effects of aflatoxin. In rat hepatocyte suspension culture, aflatoxin B 1 and oxytetracycline interacted antagonistically (173). Aflatoxin B 1 interacted antagonistically with penicillin when bacterial growth was measured ( 167). The antioxidant coenzyme QlO in combination with L-carnitine reduced the bacterial growth inhibitory effects of aflatoxin B1 (174). When polyvinylpolypyrrolidone and aflatoxin B1 were given to chicks there was a striking reduction in toxicity ( 175). Geniposide and aflatoxin B 1 interacted antagonistically and decreased hepatotoxic-

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ity in rats (176). In mice, flunixin normalized the aflatoxin-induced increase in tryptophan levels and vitamin E normalized the aflatoxin-induced decrease in catecholamine levels (177). However, vitamin E did not prove beneficial against aflatoxicosis in growing pigs (178).

B. Citrinin In rats, citrinin and diethylmaleate interacted synergistically to increase glutathione depletion and inhibit renal tubular organic ion transport in kidney slices. Citrinin and hexachloro- 1,3-butadiene had zero interaction when glutathione depletion was measured (1 79). Citrinin increased the rate of tumor formation induced by either N-(3,S-dichloropheny1)succinimide or N-nitrosodimethylamine (180). C.Cytochalasin

E

Macrophages treated with either concanavalin A, phytohemagglutinin, or wheat germ agglutinin all released more superoxide anions when pretreated with cytochalasin E (18 1). Cytochalasin E and vincristine interacted synergistically and increased DNA fragmentation ( 182). Cytochalasin E sensitized P-glycoprotein overexpressing human breast cancer cells to daunomycin, vinblastine, and actinomycin D. Other cytochalasins did not sensitize the overexpressing cells (183).

D.

Ochratoxin A

In chicks, vitamin E counteracted the toxic effects of ochratoxin A. This was not observed with vitamin C (184). However, in mice, vitamin C counteracted the ochratoxin A-induced mitotic chromosome abnormalities (185). Pretreatment of mice with vitamin A, C, or E significantly reduced the genotoxicity of ochratoxin A (186). In rats, cholestyramine reduced the amount of ochratoxin A excreted in the urine, increased the amount of ochratoxin A excreted in the feces, and decreased the bioavailability of ochratoxin A in the blood ( 187). In pregnant rats, phenylalanine significantly reduced the number of ochratoxin Ainduced fetal malformations (188). The addition of phenylalanine to the diets of chickens ameliorated some of the toxic effects of ochratoxin A (189). In mice, coinjection of phenylalanine reduced the mortality associated with ochratoxin A from 100% to 0%. This protective effect was dramatically reduced if the phenylalanine was administered after the ochratoxin A (190). Aspartame reduced the toxicity of ochratoxin A. In contrast to phenylalanine, the protective effect was observed even when the rats were given ochratoxin A for several weeks before being given aspartame ( 191 ). In renal proximal tubules isolated from rabbits, ochratoxin A-induced cell injury and death were increased with lowered extracellular pH (192). In bacteria, coenzyme QlO in combination with L-carnitine reduced the growth inhibitory effects of ochratoxin A (174).

E. Fusariotoxins In chicks, vitamin E counteracted the toxic effects of T-2 toxin (193, 194). This was not observed with vitamin C (193). In rats, vitamin C or vitamin E protected against acute

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I

toxicosis caused by either T-3 toxin or deoxynivalenol (118, 119). In bacteria, coenzyme QlO in combination with L-carnitine reduced the growth inhibitory effects of T-2 toxin ( 174). Emetine ( 195) or cycloheximide ( 196) reduced T-2 toxin-mediated inhibition of protein synthesis. In chickens, T-2 toxin significantly reduced the effectiveness of the anticoccidials, lasalocid (197), or monensin (198). In a yeast bioassay, polymyxin B sulfate or polymyxin B nonapeptide increased the toxicity of T-2 toxin or deoxynivalenol (199). In rats, the diacetoxyscirpenol-mediated reduction in the frequency of methylbenzylnitrosamine-induced esophageal cancer may be due to an overall decrease in growth rate (200, 201). The combination had zero interaction when activity of a DNA repair enzyme was measured (202). In mice, tryptophan did not increase the toxicity of deoxynivalenol (203). Isoproterenol reduced fusaric acid-induced aggressiveness in rats (204). Higher potassium levels increased the fusaric acid-induced release of noradrenaline from the brain stem of rats (205). In rats, fumonisin did not alter the effects of methylbenzylnitrosamine on the esophagus (206). In swine and mink, the hyperestrogenic effects of zearalenone were not reduced by treatment with tamoxifen, an estrogen antagonist (207). Pretreatment of mice with vitamin E reduced the genotoxicity of zearalenone ( I 85). Estradiol enhanced the effect of zearalenone on prostacyclin production in umbilical cord cells (208).

F. OtherMycotoxins In human blood lymphocytes, cysteine increased the frequency of patulin-induced sisterchromatid exchanges (209). Pentobarbitol decreased rubratoxin A-related toxicity to mice (2 10). TCDD increased the toxicity of sterigmatocystin to human epithelial cells (21 1). Dimethylnitrosamine, a component of bacon and tobacco smoke, increased sterigmatocystin-induced carcinogenesis in rats (2 12).

G. Summary In the various assay systems over half of the colhpounds tested increased either the mutagenicity/carcinogenicity or toxicity of aflatoxin B 1. Thus a system’s ability to cope with aflatoxin can be easily perturbed and the deleterious effects manifested. Most compounds decrease the toxicity of ochratoxin A. Most noticeable is the protective effect of aspartame even if given several weeks after the ochratoxin exposure. Most compounds decrease the toxicity of trichothecenes. Treatment with vitamins seems to be especially effective.

IX. MYCOTOXIN-ENVIRONMENT A.

Aflatoxin B1

The interaction between dietary protein levels and aflatoxin B1-induced changes in the liver is well documented. Significant liver damage occurred in young rhesus monkeys fed aflatoxin B 1 and a low-protein diet compared to animals fed a high-protein diet (213). In rats, the aflatoxin B 1-induced liver damage was found to be different in animals consuming the low-protein or high-protein diet. However, 50% of the animals on the high-protein diet developed cancer compared to 0% of the animals on the low-protein diet (214). Vari-

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om other studies support the data that there is an increased aflatoxin-induced tumorigenic response with increased dietary protein (160, 215-220). The aflatoxin-induced precancerous loci were very sensitive to the dietary protein level. In rats, switching from a highprotein diet to a low-protein diet caused a regression in the lesions to preexposure levels. Refeeding the high-protein diet caused the lesions to reappear (221). The risk of aflatoxin-induced HCC is greater in male than in female rats (215). Sex differences altered the ability of aflatoxin to bind to DNA. However, dietary protein levels exerted a stronger effect on this ability. Rats were subjected to endurance training for 14 weeks before being treated with aflatoxin B 1. In these physically fit rats, the aflatoxinB -induced 1 hepatotoxicity increased (222).

B. Ochratoxin In chickens, treatment with salted drinking water, low temperatures and high humidity decreased the survival time after exposure to ochratoxin A (223). Extra protein added to the diets of chicks reduced the toxic effects of ochratoxin A. Generally supplementation with higher concentrations of protein was more effective (224). C.

Fusariotoxins

Some low-protein diets and T-2 toxin caused anemia in mice (225). In chickens, an increase in the amount of dietary fat increased the toxic effect of diacetoxyscirpenol. This may be due to the high-fat diet promoting cellular absorption of diacetoxyscirpenol(226). D. Summary Environments that place additional stress on animals generally increase the toxicity of mycotoxins. Interestingly, environments with high-fat diets or increased exercise also increased the toxicity of mycotoxins. Numerous studies indicate that high-protien diets increase the occurrence of aflatoxin-induced cancers.

X.

CONCLUSIONS

This chapter elucidates the context in which mycotoxin exposure is a serious concern. These situations are: (1) Swine are particularly sensitive to synergistic interactions occurring between mycotoxins. (2) Most chemical treatments interact with aflatoxin B1 in a manner that increases the deleterious effects of aflatoxin exposure. Especially striking are the increased risk of HCC with exposure to aflatoxin andHBV and the increased liver toxicity with exposure toaflatoxin and alcohol. ( 3 ) High dietary protein increases the carcinogenicity of aflatoxin. This chapter also elucidates treatments that reduce the inhibitory effects of mycotoxins. Generally the addition of cadmium, zinc, iron, selenium, or vitamins reduced the inhibitory effects of mycotoxins. Notable protective effects occurred with aspartame and ochratoxin, S. cerevisiae and aflatoxin, and HSCAS and aflatoxin. In contrast to chemical treatments, most plant secondary metabolites decrease the carcinogenic/mutagenic effects of aflatoxin.

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Five years ago we noticed six areas in the study of interactions between mycotoxins that needed further research. At this point these can be refined to four areas that need additional study. Many of these observations also occur when the interactions between a mycotoxin and another treatment are examined. First, there can be striking differences between male and female animals. Generally the mycotoxin interaction is more toxic to male animals (215). Second, the order of exposure is important. The interaction of a mycotoxin with another treatment tnay be synergistic or antagonistic depending on whether the treatments were sequential or simultaneous (190). Third, research needs to expand and include the interactions of mycotoxins with other fungal metabolites and the interactions with more complex combinations. Based on this work and the previous work (6), tnost mycotoxin combinations have zero interaction. However, under field conditions with additional stress factors, the toxicity of these mycotoxins could be altered to adversely affect health and perfortnance. This must be understood before we relax our concern of the deleterious interactions that can occur between mycotoxins. Fourth, inorganic sorbents are effective in reducing aflatoxicosis. However, their effectiveness in combination with other treatments that increase aflatoxicosis needs to be determined. For example, there needs to be a study to investigate the effectiveness of the inorganic sorbents in reducing the carcinogenicity of aflatoxin in high-protein diets or with exposure to HBV.

REFERENCES 1. Prathapkumar, S. H.. Rao, V. S.. Paramkishan. R. J., and Bhat, R. V. (1997). Disease outbreak in laying hens arising from the consumption of fumonisin-contaminated food. BI: Poult. Sci., 38~475-479. 2 . Abramson D.. Mills, J. T., Marquardt, R. R., and Frohlich, A.A. (1997). Mycotoxins in fungal contaminated samples of animal feed from western Canada, 1982-1994. Cm. J. Vet. Res., 61:49-52. 3. Desjardins, A. E., Plattner, R. D., Nelsen, T. C., and Leslie. J. F. (~1995).Genetic analysis of fumonisin production and virulence of Gibberella jirjikzuoi mating population A ( Fztscrriunz moniliforrne) on maize (Zetr n g w ) seedlings. Appl. Enliron. Microbiol., 61:79-86. 4. Miller, J. D. ( 1993). The toxicological significance of mixtures of fungal toxins in food. A@. Ne\jdett. O C C I IHealth. ~. Sajep Sllppl., 232-38. 5. McLean, M. (1996).The phytotoxicity of Fusarium metabolites: an update since 1989. MJICOpcrtlzologiu. 133:163-179. 6. Koshinsky. H. A., and Khachatourians, G. G. (1993). The effect of mycotoxin combinations. In Foodborne Disease Hcrrzdbook: Diesaease Caused by Viruses, Parasites a d Fungi (Y. H. Hui, J. R. Gorham. K. D. Murrell. and D. 0. Cliver. eds.). Marcel Dekker, New York, pp. 363-520. 7. Calabrese, E. J. (1991). Multiple Clzenzicnl Irzteractions. Lewis Publishing, Chelsea, MI. 8. Chou, T.-C., and Rideout, D. C. (199 1 ). Syzergism and Arltago~lisr~z in Chenzothercp. Academic Press, New York. 9. King, T. C., and Krogstad. D. J. (1983). Spectrophotometric assessment of dose-response curves for single antimicrobial agents and antimicrobial combinations. J. Irfect. Dis., 147: 758-764. 10. Loewe, S. (1953). The problem of synergism and antagonism of combined drugs. Ar,-neinz.Forsch., 3:285-290. 11. Chou, T.-C., and Talalay. P. (1983). Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Ad17. E q w z e Reg.. 22:27-55.

Mycotoxicoses

641

12. Chou, J., and Chou, T.-C. (1985). Dose-Effect Analysis with Microcomputers. Elsevier, Amsterdam. 13. Koshinsky, H. A., and Khachatourians, G. G. (1992). Trichothecene synergism, additivity andantagonism:thesignificanceof the maximallyquiescent ratio. Nat. Toxins, 1:3847. 14. Tallarida, R. J., Porreca, F., and Cowan, A. (1989). Statisticalanalysis of drug-drug and sitesite interactions with isobolograms. Life Sci., 45:947-961. 15. Suhnel, J. (1990). Evaluation of synergism or antagonism for the combined action of antiviral agents. Antivirtrl Res., 13:23-40. 16. Brunden, M. N., Vidmar, T. J., and McKeam, J. W. (1988). Drug Interactioll am! Lethality Analysis. CRC Press, Boca Raton, FL. 17. Plunlmer, J. L., and Short, T. G. (1990). Statistical modeling of the effects of drug combinations. J. Phnrm. Meth., 23297-309. 18. Groten, J. P., Tajima, O., Feron, V. J., and Schoen, E. D. (1998). Statistically designed experiments to screen chemicalmixtures for possible interactions. Environ. Health Perspect., 106s: 1361-1365. 19. Chen. D. G., and Pounds, J. G. (1998). A nonlinear isobologram model with box-cox transformation tobothsides forchemical mixtures. Environ. HealthPerspect., 106S313671371. 20. Lisker, N., Michaeli, R., and Frank, Z. R. (1993). Mycotoxigenic potential of Aspergillus Jlavus strains isolated from groundnuts growing in Israel. Mycopathologin, 122: 177-1 83. 21. Yoshizawa, T., Yamashita, A., and Chokethaworn, N. (1996). Occurrence of fumonisins and aflatoxins in corn from Thailand. Food Addit. Contant., 13:163-168. 22. Scudamore, K. A., Hetmanski, M. T., Chan, H. K.. and Collins, S. (1997). Occurrence of mycotoxins in raw ingredients used for animal feedingstuffs in the United Kingdom in 1992. Food Addit. Contam., 14:157-173. 23. Balachandran. C., Parthasarathy, K. R. (1996). Occurrence of cyclopiazonic acid in feeds and feedstuffs in Tamil Nadu, India. Mycopathologia, 133:159-162. 24. Nakajima, M., Tsubouchi, H.. Miyabe, M., and Ueno, Y. (1997). Survey of aflatoxin B-1 and ochratoxin A in commercialgreen coffee beans by high-perforrnance liquidchromatography linked with immunoaffinity chromatography. Food Agric. hrnwzol., 9:77-83. 25. Jonsyn, F. E.. Maxwell, S. M., and Hendrickse. R. G. (1995). Ochratoxin A and aflatoxins in breast milk samples from Sierra Leone. Mycopathologin, 131:121-126. brew26. Scott, P. M. (1996). Mycotoxinstransmitted into beer from contaminated grains during ing. JAOAC. 79:875-882. Aiura, Y., Sanchez, P.C., Dizon, E. I., Arim. R. H., and 27. Yamashita,A.,Yoshizawa,T., Sardjono. (1995). Fusarium mycotoxins (fumonisins, nivalenol, and zearalenone) and aflatoxins in corn from Southeast Asia. Biosci. Biotecltnol. Biochem., 59: 1804-1807. 28. Shim, W. B., Kim, J. C., Seo, J. A., and Lee, Y. W. (1997). Natural occurrence of trichothecenes and zearalenone in Korean and imported beers. Food Addit. Contam., 14: 1-5. 29. Chu, F. S., and Li, G. Y. (1994). Simultaneous occurrenceof futnonisin B 1 and other mycotoxins in moldy corn collected from the People's Republic of China in regions with high incidences of esophageal cancer. Appl. Enviro~t.Microbiol.. 60:847-852. 30. Scudamore K. A., Nawaz, S.. and Hetmanski, M. T. (1998). Mycotoxins in ingredients of animal feeding stuffs. 11. Determination of mycotoxins in maize and maize products. Food Addit.Contnm., 15:30-55. 31. Sadek, M. M., Crailsheim, K., and Azab, S. G. (1996). The chemosterilizingactivity of some mycotoxinsandtheirinfluenceonthe development and survival of Spodoptera littoralis (Boisd.) (Lep., Noctuidae). J. Appl. Entonzol., 120:53-61. 32. Edrington, T. S., Harvey, R. B., and Kubena, L. F. (1995). Toxic effectsof aflatoxin B1 and ochratoxin A, alone and in combination, on chicken embryos. Bull. Environ. Contnnz. TOXicol., 54:33 1-336.

642

Koshinsky et al.

33. Kubena, L.F., Harvey, R. B.. Buckley. S. A., Edrington, T.S., and Rottinghaus, G. E. (1997). Individual and combined effectsof moniliformin present in Fltsarizrri?~rjikuroiculture material and aflatoxin in broiler chicks. Poult. Sci.,76265-270. 34. Weibking, T. S., Ledoux. D. R., Bermudez, A. J., and Rottinghaus, G. E. (1994). Individual and combined effects of feeding Fwarizlrn rnoniliforrrze culture material. containing known levels of fumonisin B1, and aflatoxin B l in the young turkey poult. Poult. Sci., 73:15171525. 35. Kubena, L. F, Edrington, T. S., Kamps-Holtzapple, C., Harvey, R. B., Elissalde, M. H., and Rottinghaus, G. E. (1995). Effectsof feeding fumonisin B-1present in Fzrsarinn~nzorzilifot-me culture material and aflatoxin singly and in combination to turkey poults. Poult. Sci., 74: 1295-1303. 36. Yabe. T., Hashimoto, H., Sekijima, M., Degawa, M.,Hashimoto, Y., Tashiro, F., and Ueno, Y. (1993). Effects of nivalenol on hepatic drug-metabolizing activity in rats. Food Chenz. Toxicol., 31:573-581. 37. Harvey, R. B., Edrington, T. S., Kubena, L. F., Elissalde, M. H.,and Rottinghaus, G. E. (1995). Influence of aflatoxin and fumonisin B-1-containing culture material on growing barrows. Anz. J. Vet. Res., 56:1668-1672. 38. Berger, M. R. (1995). Synergismand antagonism between chemical carcinogens.In Chemical Indztction of Cancer. Moddatiorr and Cornbination Effects (J. C. Arcos, ed.). Birkhauser, pp. 23-49. 39. Riley, R. T., and Showker, J. L. (1991). The mechanism of patulin's cytotoxicity and the antioxidant activity of indole tetramic acids. Toxicol. Appl. Pharrnacol.. 10: 108-126. 40. Kubena, L. F., Smith, E. E., Gentles, A., Harvey. R. B., Edrington, T. S.. Phillips. T. D., and Rottinghaus, G. E. (1994). Individual and combined toxicity of T-2 toxin and cyclopiazonic acid in broiler chicks. Poult. Sci., 73:1390-1397. N., Gantt, 0. O., and Friedman, L. (1994). Interaction of citrinin 41. Braunberg, R. C., Barton, C. and ochratoxin A. Nat. Tosirrs, 2: 124-13 1. 42. Kubena. L. F., Harvey, R. B., Edrington, T. S., and Rottinghaus. G. E. (1994). Influence of ochratoxin A and diacetoxyscirpenol singly and in combination on broiler chickens. Poult. Sci., 73~408-415. 43. Kubena, L. F., Edrington, T. S., Harvey, R. B., Phillips, T. D., Sarr, A. B.. and Rottinghaus. G. E. (1997). Individual and combined effects of fumonisin B l present in Fusarium morziliforme culture material and diacetoxyscirpenol or ochratoxin A in turkey poults. Poult. Sci., 76256-263. 44. Harvey, R. B., Kubena. L. F., Elissalde, M.H., Rottinghaus, G.E., and Corrier, D.E. (1994). Administration of ochratoxin A and T-2 toxin to growing swine. Am. J. Vet. Res., 55:17571761. 45. Lusky, K., Tesch, D., Gobel, R., and Haider. W. (1997). Effectsof OTA and ZEA on animal health and residue behaviour of pigs. Tierarctl. Urmchau, 52:212-221. 46. Madhyastha, M. S., Marquardt. R. R., and Abramson, D. (1994). Structure-activity relationships and interactions among trichothecene mycotoxins as assessed by yeast bioassay. Tosicon, 32:1147-1152. 47. Jones, J., Koshinsky,H. A., and Khachatourians, G. G. (1995). Effectof T-2 toxin and verrucarin A in combination on k'luyeromyes nzarxinnus. Nar. T o . h s , 3: 104- 108. 48. McLean, M. (1996). The phytotoxicity of selected mycotoxins on mature, germinating Zea mays embryos. Mycopathologia. 132: 173- 183. 49. Bacon. C. W.. Porter, J. K., and Norred, W. P. (1995). Toxic interaction of fumonisin B1 and fusaric acid measured by injection into fertile chicken egg. Mycopathologia, 129:2935. 50. Vesely, D.. and Vesela, D. (1995). Embryotoxic effectsof a combination of zearalenone and vomitoxin (4-dioxynivalenole) on the chick embryo. Vet. Men., 40279-281. 51. Javed, T., Bennett, G.A., Richard, J. L., Dombrink-Kurtzman, M. A., Cote, L.M., and Buck.

Mycotoxicoses

52.

53.

54. 55.

56.

57.

58.

59. 60. 61.

62.

63.

64. 65. 66.

67.

68.

643

W. B. (1993). Mortality in broiler chicks on feed amended with Fusarium yrol$eratum culture material or with purified fumonisin B l and moniliformin. Mycopathologia, 123: 171184. Kubena, L. F.. Edrington, T. S., Harvey, R. B.. Buckley, S. A., Phillips, T. D., Rottinghaus, G. E., and Casper, H. H. (1997). Individual and combined effects of furnonisin B1 present in Fz4sarizmr nzonil(fornzeculture material and T-3 toxin or deoxynivalenol in broiler chicks. Poult. Sci., 76:1239-1247. Harvey. R. B., Kubena. L. F.. Rottinghaus, G. E.. Turk. J. R.. Casper. H. H.. and Buckley, S. A. ( 1997). Moniliformin from Fusariurtr fig%h-oi culture material and deoxynivalenol from naturally contaminated wheat incorporated into diets of broiler chicks. Avian Dis., 41: 957-963. Diaz, G. J., Squires. E. J., Julian, R. 3.. and Boermans, H. J. (1994). Individualand combined effects of T-2 toxin and DAS in laying hens. Br. Poult. Sci., 35:393-405. Kubena, L. F., Edrington, T. S., Kamps-Holtzapple. C., Harvey, R. B.. Elissalde. M. H., and Rottinghaus, G. E. (1995). Influence of fumonisin B1, present in Fzrsari~mmolliliforltle culture material. and T-2 toxin on turkey poults. Polllt. Sci., 74:306-313. Bermudez, A. J., Ledoux, D. R.. Rottinghaus.G. E., and Bennett, G. A. (1997). Theindividual and combined effectsof the Fzlsari~rrtzmycotoxins moniliformin andfumonisin B 1 turkeys. in Avian Dis., 41:304-311. Smith, T.K., McMillan, E. G., and Castillo J. B. (1997). Effect of feeding blends of Fusnrizan mycotoxin-contaminated grains containing deoxynivalenol and fusaric acid on growth and feed consumption of swine. J. Aninz. Sci., 75:2184-2191. Harvey, R. B., Edrington, T. S., Kubena, L. F., Elissalde, M. H., Casper, H. H.. Rottinghaus, G. E., and Turk, J. R. (1996). Effects of dietary fumonisin B1-containing culture material, deoxynivalenol-contaminated wheat, or their combination on growing balrows. Am. J. Vet. Res., 57:1790-1794. Rao, J. R., Sharma, N. N., and Johri, T. S. (1995). Influence of dietary aflatoxin on Eimeria uzura infection in Japanese quail (Cotunzix coturnix japonica). Vet. Parnsitol., 56: 17-22. Awadalla S. F. (1998). Influence of dietary aflatoxin on the severity of coccidial infection in quails. J. Egypt. SOC. Pnrasitol., 28:437-447. Stanley. V. G.. Ojo, R., Woldesenbet, S.. Hutchinson, D. H., and Kubena, L. F. (1993). The use of Saccharonzyces cerevisiae to suppress the effects of aflatoxicosis in broiler chicks. Poult. Sci., 72:1867-1872. Osuna, O., Edds, G. T. and Blankespoor, H. D. (1977). Toxic effects of aflatoxin B1 in male Holstein calves with prior infection by flukes (Fasciola hepatica). Am.J. Vet. Res., 38:341349. Thorgeirsson, U. P., Dalgard, D. W., Reeves, J., and Adamson, R. H. (1994). Tumor incidence in a chemical carcinogenesisstudy of nonhuman primates. Regul. Toxicol. Pharnrncol.. 19~130-151. Paraskevopoulos, J. A. (1994). Management options for primary hepatocellular carcinoma: an overview. Acta Oncol., 33:895-900. Grassman, J. (1997). Acquiredrisk factors andsusceptibility to environmental toxicants. Emiron. Toxicol. Plzarmncol., 4909-217. Wu, M.-W., Lien, J.-P.. Chiu, Y.-H., Santella. R. M., Liaw. Y.-F., and Chen. C.-J. (1997). Effectofaflatoxinmetabolismand DNA adduct formationon hepatocellular carcinoma among chronic hepatitis B calriers in Taiwan. J. Hepatol., 27:320-330. Yu, M. W., Chiang, Y. C., Lien, J. P., and Chen, C. J. (1997). Plasma antioxidant vitamins, chronic hepatitis B virus infection and urinary aflatoxin B1-DNA adducts in healthy males. Carcilzogenesis, 18:1189-1194. Ross.R.K.,Yuan, J. M., Yu, M. C., Wogan, G. N.. Qian,G. S., Tu, J. T.. Groopman, J. D.. Gao. Y. T., and Henderson, B. E. (1992). Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet. 339:943-946.

644

Koshinsky et al.

69. Qian, G. S., Ross, R. K., Yu. M. C., Yuan. J. M., Gao, Y. T.. Henderson, B. E., Wogan, G. N., and Groopman, J. D. (1994). A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai. People’s Republic of China. Cancer Epiclerrriol. Biontarkers Prev., 3:3-10. 70. Yan, R. Q., Su, J. J., Huang, D.R., Gan, Y. C., Yang. C., and Huang, G. H. (1996). Human hepatitis B virus and hepatocellular carcinoma 11. Experimental induction of hepatocellular carcinoma in tree shrews exposed to hepatitis B virus and aflatoxin B1. J. Cancer Res. Clin. Orlcol., 122:289-295. 71. Bannasch, P., Khoshkhou. N. I.. Hacker, H. J.. Radaeva, S., Mrozek, M., Zillmann,U., KoppSchneider, A.,Haberkorn. U.. Elgas, M., Tolle. T., and others. (1995). Synergistichepatocarcinogenic effect of hepadnaviral infection and dietary aflatoxin B-1 in woodchucks. CancerRes., 55:3318-3330. 72. Sell, S., Hunt. J. M., Dunsford, H. A., and Chisari, F. V. (1991). Synergy between hepatitis B virus expression and chemical hepatocarcinogens in transgenic mice. Cmcer Res., 51 : 1278-1385. 73. Lunn, R. M., Zhang, Y. J.. Wang, L. Y.. Chen. C. J., Lee, P. H., Lee, C. S.. Tsai, W. Y., and Santella, R. M. (1997). p53 mutations, chronic hepatitis B virusinfection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan. Cancer Res., 57:3471-3477. 74. Su. J., Qin, G., Yan, R., Huang. D.. Yang, C., Huang, G., and Lotlikar, P. D. (1997). Expression of p53 gene in hepatocellular carcinomas induced by aflatoxinB 1with or without human hepatitis B virus in tree shrews. E.yp Mol. Med., 39: 177-183. 75. Greenblatt, M. S., Bennett, W. P., Hollstein. M., and HaIris, C. C. (1994). Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cmzcer Res., 54:4855-4878. 76. De Flora, S., Hietanen, E., Bartsch. H., Camoirano, A., Izzotti, A., Bagnasco, M., and Millman, I.( 1989). Enhanced metabolic activation ofchemical hepatocarcinogensin woodchucks infected with hepatitis B. Cnrcinogelresis, 10: 1099-1 106. 77. Gemechu-Hatewu, M., Platt. K.-L., Oesch. F.. Hacker, H.-J., Bannasch. P., and Steinberg, P. (1997). Metabolic activation of aflatoxin B-1 to aflatoxin B-l-8,9-epoxide in woodchucks undergoing chronic active hepatitis. Zrrt. J. Cancer, 73:587-591. 78. Ayoola, E. A. (1984). Synergism between hepatitis B virus and aflatoxin in hepatocellular carcinoma. ZARC Sci. Prrbl., 63:167-179. 79. Sandhu, B. S.. Singh, H., and Singh, B. (1995). Pathological studies in broiler chicks fed aflatoxin or ochratoxin and inoculated with inclusion body hepatitis virus singly and in concurrence. Vet. Res. Contntun., 1927-37. 80. Singh, H., Singh, B., Roy, K. S., and Singh, B. (1994). Histochemical-histoenzymic studies in broiler chicks fed aflatoxin, ochratoxin and inoculated with inclusion body hepatitis virus singly and in concurrence. Mycopathologia, 125: 119-128. 81. Elissalde, M. H.. Ziprin. R. L., Huff, W. E.. Kubena, L. F.. and Harvey, R. B. (1994). Effect of ochratoxin A on Srrl~~zonell~~-challenged broiler chicks. POLdt. Sci., 73: 1241- 1248. 82. Tai. J. H. and Pestka, J. J. (1988). Synergistic interaction between the trichothecene T-2 toxin and Salrnonelln !yphinw-iunr lipopolysaccharide in C3H/HeN and C3H/HeJ Inice. Toxicol. Lett., 34:191-200. 83. Bottex. C., Martin, A. and Fontanges, R. (1990). Action of a mycotoxin (diacetoxyscirpenol) on theinunune responseof the mouse-interaction with animmunomodulator (OM-89).Irnnzzrnopharrmcol. bnmunotosicol., 12:311-325. 84 Dashwood, R. H., Fong, A. T., Williams. D. E., Hendricks, J. D. and Bailey, G. S. (1 991). Promotion of aflatoxin B 1 carcinogenesisby the natural tumor modulator indole-3-carbinol: influence of dose, duration, and intermittent exposure on indole-3-carbinol promotional potency. Cancer Res., 51:2362-2365. 85. Manson.M. M., Hudson. E. A., Ball,H. W., Barrett,M. C., Clark. H. L., Judah.D. J., Verschoyle R. D., and Neal, G.E. (1998). Chemopreventionof aflatoxin B1-induced carcino-

Mycotoxicoses

86.

87.

88.

89.

90. 91.

92.

93.

94.

95.

96. 97.

98.

99.

100.

101. 102. 103.

645

genesis by indole-3-carbinol in rat liver-predicting the outcome using early biomarkers. Carcinogenesis, 19: 1829- 1836. Bailey, G., Taylor, M., Selivonchick, D., Eisele, T., Hendricks, J., Nixon, J., Pawlowski, N., and Sinnhuber. R. (1982). Mechanisms of dietary modification of aflatoxin B1 carcinogenesis. Basic Life Sci., 21:149-165. Bailey, G. S., Hendricks, J. D., Shelton, D. W., Nixon, J. E. and Pawlowski, N. E., (1987). Enhancement of carcinogenesis by the natural anticarcinogen indole-3-carbinol.J. Nutl. Cancer- Inst., 78:93 1-934. Soni, K. B., Lahiri, M., Chakradeo. P., Bhide, S. V.. and Kuttan, R. (1997). Protective effect of food additives onaflatoxin-induced mutagenicity and hepatocarcinogenicity. Cuncer Lett., 115~129-133. Dashwood, R., Negishi, T., Hayatsu, H., Breinholt V., Hendricks, J., and Bailey, G. (1998). Chemopreventive properties of chlorophylls towards aflatoxin B 1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout. Mzrtat. Res.. 399:245-253. Wong, B. Y., Lau. B. H., Tadi,P. P., and Teel. R. W. (1992). Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of aflatoxin B1. Mutat. Res., 279209-216. al-Dakan, A. A., al-Tuffail, M. and Hannan. M. A. (1995). Cassia senm inhibits mutagenic activities of benzo[a]-pyrene, aflatoxin B 1, shammaand methyl methanesulfonate. Phcrr-rnncol. Tosicol., 77:288-292. Gonzalez De Mejia,E., Ramos-Gomez, M. and Loarca-Pina. G. (1997).Antimutagenic activity of natural xanthophylls against aflatoxin B-1 in SulmonelZcr tylzinzuriurn. Emiron. Mol. Mutat., 30:346-353. Wang. C. J., Shiah, H. S., and Lin, J. K. (1991). Modulatory effect of crocetin on aflatoxin B1 cytotoxicity and DNA adduct formation in C3HlOT1/2 fibroblast cell. Cancer-Lett., 56: 1-10. Balansky, R. (1992). Effects of sodium selenite and caffeine on mutagenesis induced by Nmethyl-N-nitrosourea, N-methyl-N’-nitro-N-nitrosoguanidine and aflatoxin B 1in S. qphimzlrim!. Mutat. Res., 269:307-317. Rompelberg, C. J.. Evertz, S. J., Bruijntjes-Rozier, G. C., van den Heuvel, P. D., and Verhagen, H. (1996). Effect of eugenol on the genotoxicity of established mutagens in the liver. Food Chenl. Toxicol., 34:33-42. Osowole, 0. A.. Ogidi, H. J., and Uwaifo, A. 0. (1992). Influence of four Nigerian food additives on the mutagenicity of aflatoxin B1. Af. J. Med. Sci., 21:83-87. Kiefer, F. and Wiebel. F. J. (1998). Caffeinepotentiates the formation of micronuclei caused by environmental chemical carcinogens in V79 Chinese hamster cells. Toxicol. Lett., 9697~131-136. Sun, B. and Fukuhara, M. (1997). Effects of co-administration of butylated hydroxytoluene, butylated hydroxyanisole and flavonoids on the activation of mutagens and drug-metabolizing enzymes in mice. To.xicoZogy, 12261-72. Schoenhard, G. L., Hendricks, J. D., Nixon, J. E., Lee,D. J.. Wales, J. H., Sinnhuber, R. O., and Pawlowslu, N. E. ( 1981). Aflatoxicol-induced hepatocellular carcinoma in rainbow trout (Snhno gairdtreri) and the synergistic effects of cyclopropenoid fatty acids. Cancer Res., 41:lOll-1014. Okoye Z. S., and Neal, G. E. (1985). Enhanced ethanol-induced changes in disposition and toxic response to dietary aflatoxin B 1 dueto Sacoglottis gabonensis bark extract, aNigerian alcoholic beverage additive. Food Chem Toxicol., 36:679-689. Okotie-Eboh, G. 0.. Kubena, L. F., Chinnah, A. D., and Bailey, C. A. (1997). Effects of beta-carotene and canthaxanthin on aflatoxicosis in broilers. Poult. Sci., 76: 1337- 1341. Dale. N. M., Wyatt, R. D.. and Fuller, H. L. (1980).Additive toxicity of aflatoxin and dietary tannins in broiler chicks. Poult. Sci., 59:2417-2420. Tanner, M. S., and Mattocks, A. R. (1987). Hypothesis: plant and fungal biocides, copper and Indian childhood liver disease. Ann. Trop. Puediatr-., 7264-269.

646

Koshinsky et al.

104. Yourtee, D. M., Elkins, L. L.. Nalvarte E. L., and Smith, R. E. (1992). Amplification of doxorubicin mutagenicity by cupric ion. Tosicol. Appl. Plzarmncol., 116:57-65. 105. Schiller, F., Lippold, U., Heinze, R., Hoffmann, A,, and Seffner, W. (1998). Liver fibrosis in guinea pigs experimentally induced by combined copper and aflatoxin application. Exp. Tosicol. Pnthol.. 50:5 19-527. 106. Seffner, W., Schiller, F.. Lippold, U.. Dieter. H. H. and Hoffmann, A. (1997). Experimental induction of liver fibrosis in young guinea pigs by combined application of copper sulphate and aflatoxin B1. To.xicol Lett., 2161-172. 107. Osuna, 0.. and Edds, G. T. ( 1982). Toxicology of aflatoxin B 1, warfarin, and cadmium in young pigs: clinical chemistry and blood coagulation. Am. J. Vet. Res., 43:1387-1394. 108. Methenitou, G., Maravelias, C., Koutsogeorgopoulou, L., Athanaselis, S. and Koutselinis. A. ( 1996). Immunomodulative effects of aflatoxins and selenium on human peripheral blood lymphocytes. Vet. Hum. To.xicol., 38274-277. 109. Burguera, J. A., Edds. G. T. and Osuna, 0. (1983). Influence of selenium on aflatoxin B1 or crotalaria toxicity in turkey poults. Am. J. Vet. Res.. 44:1714-1717. 110. Milks, M. M., Wilt, S. R., Ali, I. I.. and Couri. D. (1985). The effects of selenium on the emergence of aflatoxin B 1-induced enzyme-altered foci in rat liver. Furrckrm. Appl. Tosicol., 5:320-336. 111. Chen, J., Goetchius. M. P., Campbell, T. C., and Combs, G. F. (1982). Effects of dietary selenium and vitamin E on hepatic mixed-function oxidase activities and in vivo covalent binding of aflatoxin B1 in rats. J. Nzm., 112:324-331. 112. Burk, R. F., and Lane, J.M. (1983). Modification of chemical toxicity by selenium deficiency. Fundam Appl. Toxicol., 3218-221. 113. Stormer, F. C. and Hoiby. E. A. (1996).Citrinin, ochratoxin A and iron. Possible implications for their biological function and induction of nephropathy. Mycopatlzologicr, 133:103107. 114. Ribeiro, S. M., Canlpello, A. P., Chagas, G. M. and Kluppel, M. L. (1998). Mechanism of citrinin-induced dysfunction of mitochondria. VI. Effect on iron-induced lipid peroxidation of rat liver mitochondria and microsomes. Cell Bioclzem Fzozct.. 16:15-20. 115. Haberman, P. J., Baggett, J. M. and Berndt, W. 0. (1987). The effect of chromate on citrinininduced renal dysfunction in the rat. Toxicol. Lett.. 38:83-90. 116. Baggett, J. M., and Berndt, W. 0. (1984). Interaction of potassium dichromate with the nephrotoxins, mercuric chloride and citrinin. To.xicology, 33: 157-169. 117. Kubena, L. F., Harvey. R. B.. Fletcher, 0. J., Phillips, T. D., Mollenhauer, H. H., Witzel, D. A., and Heidelbaugh, N. D. (1985). Toxicity of ochratoxin A and vanadium to growing chicks. Poult. Sci., 63:620-628. 118. Atroshi. F., Rizzo, A., Biese, I., Salonen. M.. Lindberg, L.-A., and Salonieme. H. (1995). Effects of feeding T-2 toxin and deoxynivalenol on DNA and GSH contents of brain and spleen of rats supplemented with vitamin E and C and selenium combination. J. Anint. Plzysiol. Arzim. Nzltr., 74:157-164. 119. Rizzo. A. F., Atroshi, F., Ahotupa. M., Sankari. S. and Elovaara, E. (1994). Protective effect of antioxidants against free radical-mediated lipid peroxidation induced by DON or T-2 toxin. Zentralbl. Vererimrmed A. 3 1:8 1-90. 120. Chanin. B. E., Hamilton, D. L., Hancock, D. S., and Schiefer, H. B. (1983). Biointeraction of dietary T-2 toxin and zinc in mice. Can. J. Pltysiol. Pitnnnncol., 62:1320-1336. 121. Combs. G. F., Welch, R. M., Duxbury, J. M., Uphoff, N. T., and Nesheim, M. C. (1996). Food-Based Approaches to Prellenting iklicromtrierzt iVlalmtrition. CIIFAD, Cornell, NY. 122. Bulatao-Jayme, J., Almero, E. M., Castro, M. C.. Jardeleza. M. T., and Salamat, L. A. (1983). A case-control dietary study of primary liver cancer risk from aflatoxin exposure. Irzt. J. Epidentiol.. 11:113-119. 123. Sahaphong, S., Toskulkao, C.. and Glinsukon, T. (1992). Enhanced hepatotoxicity of aflatoxin B1 in the rat by ethanol: ultrastructural changes. Toxicol. Lett., 61:89-98.

Mycotoxicoses

647

123. Toskulkao. C., and Glinsukon. T. (1986). Effect of ethanol on the in vivo covalent binding and in vitro metabolism of aflatoxin B1 in rats. Toxicol. Lett., 30:151-157. 125. Wang, C. J., Wang, S. W.. Shiah, H. S., and Lin. J. K. (1990). Effect of ethanol on hepatotoxicity and hepatic DNA-binding of aflatoxin B I in rats. Bioclzenz. Phormxol., 40:715-721. 126. Glinsukon. T., Taycharpipranai, S., and Toskulkao, C. (1978). Aflatoxin B1 hepatotoxicity in rats pretreated with ethanol. E.xperierztia. 34:869-870. 127. Toskulkao. C., Yoshida T.. Glinsukon T., and Kuroiwa. Y. (1986). Potentiation of aflatoxin B1 induced hepatotoxicity in male Wistar rats with ethanol pretreatment. J. Toxicol. Sci., 11:41-51. 128. Toskulkao, C. and Glinsukon, T. !1990). Hepatic mitochondrial function and lysosomal enzyme activity in ethanol-potentiated aflatoxin B1 hepatotoxicity. Toxicol Lett., 52: 179-190. 129. Toskulkao, C.. and Glinsukon, T. (1988). Hepaticlipid peroxidation and intracellular calcium accumulation in ethanol potentiated aflatoxin B1 toxicity. J. Phal.nzncobiodvrz.. 11: 191-197. 130. Toskulkao, C.. Lohakachonpan, P. and Glinsukon, T. (1991). Time-courseeffects of ethanol pretreatment on hepatic necrosis and fat accumulation induced by aflatoxin B1 in the rat. Toxicol. Lett., 55: 1-9. 131. Ramos, A. J.. and Hernandez, E. (1997).Prevention of aflatoxicosis in farm animals bymeans of hydrated sodium calcium aluminosilicate addition to feedstuffs: a review. Anirnol Feed Sci. Technol., 65: 197-206. 132. Kubena, L. F., Harvey, R. B., Huff, W. E.. Elissalde, M. H., Yersia, A. G., Phillips T. D.. and Rottinghaus, G. E. (1993). Efficacy of a hydrated sodium calcium aluminosilicate to Poult. Sci. 7251-59. reduce the toxicity of aflatoxin and diacetoxyscirpenol. 133. Abo-Norag, M.. Edrington, T. S., Kubena, L. F., Harvey, R. B., and Phillips, T. D. (1995). Influence of a hydrated sodium calcium aluminosilicate and virginiatnycin on aflatoxicosis in broiler chicks. Poult. Sci., 74:626-632. 134. Kubena, L. F., Harvey, R. B., Huff, W. E., Corrier, D. E., Phillips, T. D., and Rottinghaus, G. E. (1990). Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and T-2 toxin. Portlt. Sci., 69:1078-1086. 135. Sarr, A. B., Mayura, K., Kubena, L. F., Harvey, R. B., and Phillips, T. D. (1995). Effects of phyllosilicate clay on the metabolic profile of aflatoxin B1 in Fischer-344 rats. Toxicol. Lett.. 75:145-151. 136. Smith. E. E.. Phillips, T. D., Ellis, J. A., Harvey. R. B., Kubena, L. F., Thompson, J., and Newton, G. (1994). Dietary hydrated sodium calcium aluminosilicate reduction of aflatoxin M1 residue in dairy goat milk and effects on milk production and components. J. Aninz. Sci., 72~677-682. 137. Huff, W. E.. Kubena. L. F., Harvey, R. B., and Phillips. T. D. (1992). Efficacy of hydrated sodium calcium aluminosilicate to reduce the individual and combined toxicity of aflatoxin and ochratoxin. Poult. Sci., 71:64-69. 138. Galvano. M. (1997). Activated carbons: In vitro affinity for fumonisin B-1 and relation of adsorption ability to physicochemical paratneters. J. Food Protect., 60:985-991. 139. Galvano, F., Pietri, A., Bertuzzi, T., Piva, A., Chies, L. and Galvano, M. (1998). Activated carbons: In vitro affinity for ochratoxin A and deoxynivalenol and relation of adsorption ability to physicochemical parameters. J. Food Protect., 61 :469-475. 140. Nahm, K. H. (1995). Prevention of aflatoxicosis by addition of antioxidants and hydrated sodium calcium aluminosilicate to the diet of young chicks. Jplz. Poult. Sci., 32:117-137. 141. Edrington, T. S., Kubena. L. F. and Harvey, R. B., Rottinghaus, G. E. (1997). Influence of a superactivated charcoal on the toxic effects of aflatoxin or T-2 toxin in growing broilers. Polllt. Sci., 76:1205-1211. B1 on 142. Scheideler, S. E. (1993). Effects of various types of aluminosilicates and aflatoxin aflatoxin toxicity, chick performance, and mineral status. Pozrlt. Sci., 72:282-288. 143. Mayura, K., Abdel-Wahhab, M. A., McKenzie, K. S., Sarr, A. B.. Edwards, J. F., Naguib. K., and Phillips, T. D. (1998). Prevention of maternal and developmental toxicity in rats via

648

144.

145.

146.

147.

148.

149.

150. 151. 152.

153.

154. 155.

156.

157.

158.

159.

160.

Koshinsky et al. dietary inclusion of common aflatoxin sorbents: potential for hidden risks. TodricolSci., 41: 175-182. Bailey, R. H.. Kubena, L. F.. Harvey, R. B., Buckley, S. A., and Rottinghaus, G. E. (1998). Efficacy of various inorganic sorbents to reduce the toxicity of aflatoxin and T-2 toxin in broiler chickens. Poult. Sci., 77: 1623-1630. Bursian, S. J., Aulerich, R. J.. Cameron, J. K., Ames, N. K., and Steficek, B. A. (1992). Efficacy of hydrated sodium calcium aluminosilicate in reducing the toxicity of dietary zearalenone to mink. J. Appl. Toxicol., 12:85-90. Poppenga, R. H., Lundeen, G. R., Beasley, V. R., and Buck, W. B. (1987). Assessment of a general therapeutic protocol for the treatment of acute T-2. toxicosis in swine. Vet. Hzm. Toxicol., 2.9:237-239. Biehl, M. L., Lambert, R. J., Haschek, W. M., Buck, W. B., and Schaeffer, D. J. (1989). Evaluation of a superactivated charcoal paste and detergent and water in prevention of T-2 toxin-induced local cutaneous effects in topically exposed swine. Fzmdanz. Appl. To.xicoZ., 13~523-532. Kubena, L. F., Harvey, R. B., Bailey, R. H.. Buckley, S. A., and Rottinghaus, G. E. (1998). Effects of a hydrated sodium calcium aluminosilicate (T-Bind) on mycotoxicosis in young broiler chickens. POl4lt. Sci.. 77: 1502-1509. Dwyer, M. R., Kubena, L. F., Harvey, R. B.. Mayura, K., Sam, A. B.. Buckley, S.. Bailey, R. H., and Phillips, T. D. (1997). Effects of inorganic adsorbents and cyclopiazonic acid in broiler chickens. Poult. Sci., 76:1141-1149. Shelef, L. A., and Chin, B. (1980). Effect of phenolic antioxidants on the mutagenicity of aflatoxin B1. Appl. En~+-on.Microbiol., 40:1039-1033. Malkinson, A. M. (1983). Review: putative mutagens and carcinogens in foods. 111. Butylated hydroxytoluene (BHT). Emiron. Mutagen., 5:353-362. Whong, W. Z., Lu, C. H., Stewart. J. D., Jiang, H. X., and Ong, T. (1989). Genotoxicity and genotoxic enhancing effect of tetrandrine in Salmowelln typhinzurirtm. Mutat. Res.. 222: 237-244. Ma, X. F.. Gibbons, J. A., and Babish, J. G. (1991). Benzo[e]pyrene pretreatment of inmature, female C57BL/6J mice results in increased bioactivation of aflatoxin B1 in vitro. Toxicol. Lett., 59:5 1-58. Autrup, H.. Jorgensen, E, C.. and Jensen, 0. (1996). Aflatoxin B1 induced lac1 mutation in liver and kidney of transgenic mice C57BL/6N: effect of phorone. Mutagenesis, 11:69-73. Hiruma, S., Kimura, M., Lehmann, K., Gopalan-Kriczky. P.. Qin, G-Z.. Shinozuka, H.. Sato. K., and Lotlikar, P. D. (1997). Potentiation of aflatoxin B-1-induced hepatocarcinogenesis in the rat by pretreatment with buthionine sulfoximine. Cancer Lett., 113:103-109. Gopalan-Kriczky, P., Himma, S., and Lotlikar, P. D. (1994). Effect of glutathione levels on aflatoxin B-1-DNA binding in livers and kidneys of male rats and hamsters pretreated with buthionine sulfoximine and diethylmaleate. Cancer Lett., 7625-30. Angsubhakorn, S.. Bhamarapravati, N., Romruen, K., and Sahaphong, S. (1981). Enhancing effects of dimethylnitrosamine on aflatoxin B hepatocarcinogenesis in rats. bzt. J. Cancer. 28:62 1-626. Kraupp-Grasl, B., Huber, W., Putz, B., Gerbracht, U.. and Schulte-Hermann, R. (1990). Tumor promotion by the peroxisome proliferator nafenopin involving a specific subtype of altered foci in rat liver. Carum- Res., 50:3701-3708. Wells, P., Aftergood, L.. and Alfin-Slater. R. B. (1976). Effect of varying levels of dietary protein on tumor development and lipid metabolism in rats exposed to aflatoxin. J. Am. Oil Chenl. SOC.,53:559-562. Orner, G. A., Donohoe, R. M., Hendricks. J. D., Curtis, L. R.. and Williams, D. E. (1996). Comparison of the enhancing effects of dehydroepiandrosterone with the structural analog 16 alpha-fluoro-5-androsten- 17-one on aflatoxin B 1 hepatocarcinogenesis in rainbow trout. Fundnnz.Appl. Toxicol., 34: 132- 140.

Mycotoxicoses

649

161. Zawirska, B. (1991). Aflatoxin carcinogenesis in Wistarrats enhancedby thyroideum POLFA. Mater Med. Pol., 23:191-194. 162. Fears. T. R., Elashoff, R. M., and Schneiderman, M. A. (1989). The statistical analysis of a carcinogen mixture experiment. 111. Carcinogens with different targetsystems, aflatoxin B 1, N-butyl-N-~4-hydroxybutyl~nitrosamine, lead acetate, and thiouracil. Toxicol. Ind. Health, 5: 1-23. 163. Said, B., Ross, M. K., Salib, T., and Shank, R. C. (1995).Modulation of DNA adduct formation by successive exposuresof DNA to small and bulky chemicalcarcinogens. Carcinogenesis. 16:3057-3062. 164. De Groene. E. M., Nijmeijer, S. M., Horbach, G. J. M. J., and Witkamp, R. F. (1995). Tiamulin inhibits human CYP3A4 activity in an NIH-3T3 cell line stably expressing CYP3 A4 cDNA. Biochenl. Pharntacol., 50:77 1-773. 165. Williams, G. M.. and Iatropoulos, M. J. (1996). Inhibition of the hepatocarcinogenicity of aflatoxin B-1 in rats by low levels of the phenolic antioxidants butylated hydroxyanisole and butylated hydroxytoluene. Cancer Lett., 104:49-53. 166. Walsh, A. A., Hsieh, D. P., and Rice, R. H. (1992). Aflatoxin toxicity in cultured human epidermal cells: stimulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.Carcinogerlesis, 13: 2029-2033. 167. Uwaifo,A.,andBassir. 0. (1979). Interactionoftheaflatoxinswithclassicalantibiotics which inhibit Bacillus OrelYs (2611). Afi: J. Med. Med. Sci., 8:35-39. 168. Sodhi, S.. Brar, A. P. S., and Grewal, G. S. (1996). Biochemical studieson combined immunomodulatory effects of aflatoxin and tnalathion in broiler chicks. Znd. J. Poult. Sci., 31:8891. 169. Sodhi, S., Singh, H.. Grewal, G. S., Dhaliwal, A. S., and Brar, A. P. S. (1996). Effect of aflatoxin contaminated feed on resistance of young broiler chicks to malathion challenge. Ind. J. Poult. Sei., 31:92-95. 170. Chentanez, T., Patradilok, P., Glinsukon, T., and Piyachaturawat, P. (1988). Effectsof cortisol pretreatment on the acute hepatotoxicity of aflatoxin B l . Toxicol. Lett., 42:237-248. 171. Kalra, C. S., Gill, B. S., and Singh. H. (1996). Serum biochemical studies on interaction of aflatoxicosis and coccidiosis in poultry. Znd. Vet. J., 73:504-508. 172. Diaz. G. J. and Sugahara, M. (1 995). Individual and combined effectsof aflatoxin and gizzerosine in broiler chickens. Br. Poult. Sei., 36:729-736. 173. Blaude, M. N.. Goethals, F. M., Ansay, M. A., and Roberfroid, M. B. (1992). Synergism between aflatoxin B1 and oxytetracycline on fatty acid esterification in isolated rat hepatocytes. Toxicol. Lett.. 61 : 159-166. 174. Atroshi, F., Rizzo, A., Westermarck, T.. and Ali-vehmas, T. (1998). Effects of tamoxifen, melatonin, coenzyme QlO, and L-carnitine supplementation on bacterial growth in the presence of mycotoxins. Pharmacol. Res., 38:289-295. 175. Kiran, M. M., Demet, O., Ortatatli, M.. and Oguz, H. (1998). The preventive effect of polyvinylpolypy~~olidone on aflatoxicosis in broilers. AvI'a11Pathol., 27250-255. 176. Wang, C., J., Wang, S. W., and Lin, J. K. (1991). Suppressive effect of geniposide on the hepatotoxicity and hepatic DNA binding of aflatoxin B1 in rats. Cancer Lett., 60:95-102. 177. Weekley, L. B. (1991). AflatoxinB1alters central and systemic tryptophanandtyrosine metabolism: influence of immunomodulatory drugs. Metab. Bruin Dis., 6: 19-32. 178. Harvey, R. B., Kubena, L. F., and Elissalde. M. H. (1994). Influence of vitamin E on aflatoxicosis in growing swine. Am. J. Vet. Res., 55:572-577. 179. Baggett, J. M., and Berndt, W. 0. (1984).Renal andhepatic glutathioneconcentrations in rats aftertreatmentwith hexachloro-l,3-butadieneand citrinin. Arch. Toxicol., 56:4649. 180. Shinohara, Y.,Arai, M., Hirao, K., Sugihara, S., and Nakanishi, K. (1976). Combination effect of citrinin and other chetnicals on rat kidney tumorigenesis. Gnnrz, 67:147-155. 181. Kayashima, K., Onoue, K., Nakagawara, A., and Minakami. S. (1980). Superoxide anion-

650

182. 183. 183. 185. 186.

187.

188.

189.

190.

191. 192.

193. 194. 195. 196. 197. 198.

199.

200.

201.

Koshinsky et al. generating activities of macrophages as studied by using cytochalasin E and lectins as synergistic stimulants for superoxide release. Micl-obiol. InmuI~ol.,241449-461. Kolber, M. A., and Hill, P. (1992). Vincristine potentiates cytochalasin B-induced DNA fragmentation in vitro. Cancer Chemother. Phnl-mncol., 30:286-290. Zilfou, J. T.. and Smith, C.D. (1995). Differential interactions of cytochalasins with P-glycoprotein. Orlcol. Res., 7:435-443. Hoehler, D., and Marquardt, R. R. ( 1996). Influence of vitamins E and C on the toxic effects of ochratoxin A and T-2 toxin in chicks. Poult. Sci., 75:1508-1515. Bose, S.. and Sinha, S. P. (1994). Modulation of ochratoxin-produced genotoxicity in mice by vitamin C. Food. Chern. Toxicol., 32:533-537. Grosse, Y., Chekir-Ghedira, L., Huc, A., Obrecht-Pflumio, S., Dirheimer, G., Bacha, H., and Pfohl-Leszkowicz, A. ( 1997). Retinol, ascorbic acid and alpha-tocopherol prevent DNA adduct formation in mice treated with the mycotoxins ochratoxin A and zearalenone. CancelLett., 114:225-229. Madhyastha, M. S., Frohlich, A. A.. and Marquardt. R. R. (1 992). Effect of dietary cholestyramine on the elimination pattern of ochratoxin A in rats. Food Chem. Toxicol., 30:7097 13. Mayura, K., Parker, R., Bemdt, W. O., and Phillips, T. D. (1984). Ochratoxin A-induced teratogenesis in rats: partial protection by phenylalanine. Appl. Erwil-ow Microbiol., 48: 11 86-1 188. Bailey. C. A., Gibson, R. M., Kubena, L. F., Huff, W. E.. and Harvey, R. B. (1990). Impact of L-phenylalanine supplementation on the performance of three-week-old broilers fed diets containing ochratoxin A. 2. Effects on hematology and clinical chemistry. Poult. Sci.. 69: 420-425. Creppy, E., Schlegel, M., Roschenthaler, R., and Dirheimer, G. (1979). Preventive action of phenylalanine on acute intoxication of ochratoxin-A. C. R. Seances Acnd. Sci. D,289:91591 8. Creppy, E. E., and Baudrimont, I. A.-M., (1998). How aspartame prevents the toxicity of ochratoxin A. J. Toxicol. Sci., S2:165-172. Rodeheaver, D. P., and Schnellmann, R. G. (1993). Extracellular acidosis ameliorates metabolic-inhibitor-induced and potentiates oxidant-induced cell death in renal proximal tubules. J. Plznrnrncol. Exp. They., 265: 1355-1360. Hoehler. D., and Marquardt. R. R. (1996). Influence of vitamins E and C on the toxic effects of ochratoxin A and T-2 toxin in chicks. Poult. Sci., 75:1508-1515. Tobias, S.. Rajic, I., and Vanyi, A. (1992). Effect of T-2 toxin on egg production and hatchability in laying hens. Acta Vet Hung., 40:47-52. Leatherman, D. L., and Middlebrook, J. L. (1993). Effect of emetine on T-2 toxin-induced inhibition of protein synthesis in mammalian cells. J. Pharmacol. Exp. Ther., 266:741-748. Bergmann, F., Feinmesser, M., and Yagen, B. (1991).Central effects of cycloheximide alone and of its combination with T-2 toxin. Toxicol. Lett.. 57:l-9. Varga, I., and Vanyi, A. (1992). Interaction of T-2 fusariotoxin with anticoccidial efficacy of lasalocid in chickens. Int. J. Pnrasitol., 22:523-525. Vanyi, A., Salyi, G., Majoros. G.. Glavits, R., Sandor, G., and Bago, G. (1989). Interaction of T-2 fusariotoxin and monensin in broiler chickens infected with Coccidia. Acta Vet. Hzsrtg.. 37:327-333. Connolly. P., and Cony, J. E. (1990). Effect of polymyxin B nonapeptide and polymyxin B sulphate on trichothecene mycotoxin sensitivity of yeasts using a conductimetric instrument. Int. J. Food Micr-obiol., 10:73-89. Craddock. V. M., Sparrow, S. and Henderson, A. R. (1986). The effect of the trichothecene mycotoxin diacetoxyscirpenol on nitrosamine-induced esophageal cancer in the rat. Cancer Lett., 3 1:197-204. Craddock, V. M. (1987). Effect of the trichothecene mycotoxin diacetoxyscirpenol on nitros-

Mycotoxicoses

202.

203.

204.

205.

206.

207.

208.

209.

210. 211.

212. 213.

2 14. 215.

216. 217.

218.

219.

220.

651

amine-induced oesophageal cancer and on relevant enzymes in oesophagus and liver. ZARC Sci. Publ., 84266-269. Craddock, V. M. and Henderson A. R. (1987). Effect of the esophageal carcinogen methylbenzylnitrosamine and of a putative potentiating factor, a trichothecene mycotoxin. on 06-methylguanine-dna methyl transferase in rat esophagus and liver. Cancer Lett., 37:8186. Rotter. B. A., Prelusky D. B., and Thompson, B. K. (1996). The role of tryptophan in DONinduced feed rejection. J. Emiron. Sci. Herrlth. B.. 31:1279-1288. Troncone, L. R., and Tufik, S. (1990). Effects of selective adrenoceptor agonists and antagonists on aggressive behavior elicited by apomorphine, DL-dopa and fusaric acid in REMsleep-deprived rats. Physiol. Behav., 50:173-178. Fischer, H. D., Rudolph, E., Schmidt, J., Hilse, H., Oehme, P.. and Piesche, L. (1 980). Interaction at the synaptic level of fusaric acid with neurotransmitters. Acta Biol. Med. Ger.. 39: 935-940. Wild C. P., Castegnaro, M.. Ohgaki, H., Garren, L., Galendo, D.. and Miller, J. D. (1997). Absence of a synergistic effect between fumonisin B1 and N-nitrosomethylbenzylamine in the induction of oesophageal papillomas in the rat. Nut. Toxirls 5 : 126- 131. Yang, H. H., Aulerich, R. J., Helferich, W.. Yamini, B., Chou, K. C., Miller, E. R., and Bursian, S. J. (1995). Effects of zearalenone and/or tamoxifen on swine and mink reproduction. J. Appl. Toxicol., 15:223-232. Neuer, A., Seeger, H., Muck, A. O., and Lippert, T. H. (1994). The effect of zearalenone and 17 beta-estradiol on prostacyclin and thromboxane production in cell cultures from human umbilical cord veins. Prostaglandins Leukot. Essent. Fcrtn, Acids, 5 1:241-244. Cooray, R., Kiessling, K. H.. and Lindahl-Kiessling, K. (1982). The effects of patulin and patulin-cysteine mixtures on DNA synthesis and the frequency of sister-chromatid exchanges in human lymphocytes. Food Chem. Toxicol., 20:893-898. Hayes, A. W., and Ho. I. K. (1978). Interaction of rubratoxin B and pentobarbital in mice. J. Erzviron. Pathol. Toxicol., 1:49 1-500. Walsh, A. A., deGraffenried, L. A., and Rice, R. H. (1995). 2,3.7,8-Tetrachlorodibenzo-pdioxin sensitization of cultured human epidermal cells to carcinogenic heterocyclic amine toxicity. Carcirzogetzesis. 16:2187-2191. Terao, K.. Aikawa, T., and Kera, K. (1978). A synergistic effect of nitrosodimethylamine on sterigmatocystin carcinogenesis in rats. Food Cosntet. Toxicol., 16:591-596. Madhavan, T. V., Rao, K. S., and Tulpule, P. G. (1965). Effect of dietary protein level on susceptibility of monkeys to aflatoxin liver injury. Znd. J. Med. Res., 53:984-989. Madhavan, T. V., and Gopalan, C. (1968). The effect of dietary protein on carcinogenesis of aflatoxin. Arch. Patlzol., 85:133-137. Prince, L. 0..and Campbell, T. C. (1982). Effects of sex difference and dietary protein level on the binding of aflatoxin B 1 to rat liver chromatin proteins in vivo. Cancer Res.,4250535059. Appleton, B. S.. and Campbell, T. C. (1982). Inhibition of aflatoxin-initiated preneoplastic liver lesions by low dietary protein. Nzctr. Cancer, 3200-206. Appleton, B. S., and Campbell, T. C. (1983). Dietary protein intervention during the postdosing phase of aflatoxin B 1-induced hepatic preneoplastic lesion development. J. Natl. Cancer Illst., 70:547-549. Appleton, B. S., and Campbell, T. C. (1983). Effect of high and low dietary protein on the dosing and postdosing periods of aflatoxin B 1-induced hepatic preneoplastic lesion development in the rat. Cancer Res., 432150-2154. Dunaif, G. E., and Campbell, T. C. (1987). Relative contribution of dietary protein level and aflatoxin B 1 dose in generation of presumptive preneoplastic foci in rat liver. J. Natl. Cancer Illst., 78:365-369. Horio, F., Youngman, L. D., Bell, R. C., and Campbell, T. C. (1991). Thermogenesis, low-

652

Koshinsky ef al.

protein diets, and decreased development of AFBl-induced preneoplastic foci in rat liver. Nzltr. Cancer, 16:3 1-41. 221. Youngman, L. D., and Campbell, T. C. (1991). High protein intake promotes the growth of hepatic preneoplastic foci in Fischer#344 rats: evidence that early remodeled foci retain the potential for future growth. J. Nzrtr., 121:1454-1461. of endurance 222. Toskulkao, C., Ubolsakka, C., Temcharoen, P., and Glinsukon. T. (1996). Effect exercise training on aflatoxin B-1 hepatotoxicity in rats. J. Clirz. Biochenr. Nutr., 20:37-47. 223. Huff, W. E.. and Hamilton, P. B. (1975). The interactionof ochratoxin A with some environmental extremes. Poult. Sci., 54: 1659-1662. 224. Gibson, R. M., Bailey, C. A., Kubena. L. F., Huff, W. E.. and Harvey, R. B. (1989). Ochratoxin A and dietary protein. 1. Effects on body weight, feed conversion, relative organ weight, and mortality in three-week-old broilers. Poult. Sci., 68:1658-1663. 225. Hayes, M. A., and Schiefer, H. B. (1990). Synergistic effects of T-2 toxin and a low protein diet on erythropoiesis in mice. J. Emiron. Pathol. Toxicol. Oncol., 10:69-73. 226. Ademoyero, A. A., and Hamilton, P. B. (1991). High dietary fat increases toxicity of diacetoxyscirpenol in chickens. Poult. Sci., 70:2271-2274.

16 Analytical Methodology for Mycotoxins James K. Porter U.S. Department of Agricdture, Athens, Georgia

I. Introduction 653 11. Methodology Overview

654

111. Other Analytical Considerations655 IV. MethodsforMycotoxins655

A. Ergot and other alkaloids from Claviceps, Neotlnphoclium, Epicldoe, Balansia. and Myriogenosporrr 655 B. Fzrsariur?~metabolites 669 V.

Summary and Conclusions672 References 673

1.

INTRODUCTION

The term rnycotoxirz was originally designated for fungal metabolites primarily toxic to mammals and avians, but bioassays now include those toxic to most biological systems as well as bacteria, fungi, insects, reptiles, crustaceans, and plants. Nevertheless, this discussion will be mainly directed at the analysis of mycotoxins deleterious to animal and human health and that are produced by fungi growing in or on stored grain products, other agricultural commodities, forage crops, and pasture grasses. Accordingly, food safety, animal health and productivity, and human health problems identified with fungal-contaminated grains have concentrated chemical and biological research on the detection of mycotoxins produced by a variety of fungi. Aspergillus, Perzicilliurrl, Fusarium, Alternaria, and Claviceps-infected foods, feed products, and pasture grasses have been the focus of analytical methodologies for mycotoxin research (1- 14). Major toxicology problems associated with decreased weight gains, reproductive problems, poor feed conversion, and mortality in livestock and poultry have been traced to the above genera and their toxic metabolites (1-14). A variety of mycotoxins produced by this group of fungi also have been targeted as major health concerns in several human disorders related to the consumption of contaminated cereal grains (i.e., esophageal cancer, spontaneous abortions, agalactia, alimentary toxic aleukia, dermal toxicities, immune suppression, central nervous system disorders, hepatic and renal toxicities, nausea, vomiting, diarrhea, and precocious puberty in children, to mention a few) (3, 11653

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14). Therefore, the routine occurrence of these fungi and their toxic metabolites in corn, wheat, barley, oats, rye, sorghum, millet, other cereal grains, and agricultural commodities necessitates the most sophisticated isolation and identification methodologies. Owing to the diversity of mycotoxins that occur in food and feed products and their isolation and identification procedures, it is beyond the scope of these discussions to cover in detail all analytical methods employed for mycotoxin analyses. However, this chapter will focus on the various chemical procedures and methods used in the qualitative and quantitative analyses of the major mycotoxins in which definitive safety guidelines have been suggested for animal and human health. Consideration will be given to methods employed for the prominent mycotoxins in which guidelines are currently under assessment, and that toxicology research has more than emphasized a need for expedient identification andanalytical procedures. Minor examples will bereferenced to demonstrate some techniques used to separate certain mycotoxins from important plant metabolites. The overall discussion will concentrate on successful analytical methods that are convenient, economical, and that may berapidly conducted in most mycotoxin screening laboratories. Interests will be directed at procedures with minimum analytical variances and include the analysis of several mycotoxins within a single test battery. Aspergill~rs,P e ~ ~ i c i l l iF~rsorirrm, u ~ ~ ~ , Alterfzm-i.icl,and Clmiceys are not the only genera that produce tnycotoxins incongruent with health and the economics of production, but their structural diversity will serve as a general example and guide for mycotoxin procedures. In this review, emphasis will be placed on the analyses of both Claviceps and Fuscwiurr~mycotoxins, since these two genera produce the most structurally diverse and biologically active compounds. Additionally, Fuscrl-ium is reported as a mycoparasite on Clcrviceps (15, 16jand the possibility of synergistic toxicity among co-occurring mycotoxins has been raised (6, 10, 17-32). While Aspergillus, Perzicilliunz,and Alternaria produce numerous mycotoxins harmful to both anitnals and plants (1-5, 7-12) and also include some of the most common, aggressive saprophytes that are either plant pathogens or that induce nisi opportunistic diseases in many crops (4-7j, nominal historic references will be given to the analyses of these toxins. Nevertheless, individually, each genus represents a major economic and toxicological threat to plant production and animal and human health. The major mycotoxins associated with all of the above, their analytical methods, and references are discussed within the individual subsections, which should provide the reader with a better prospectus, compendium, and a more pragmatic approach to mycotoxin analyses. The discussions, tables, and figures are not exhaustive or totally inclusive, but rather are illustrative of the general methods for mycotoxin analyses.

II. METHODOLOGYOVERVIEW In general, either liquid-liquid or solid phase extraction, clean-up procedures with appropriate solvents, and chromatographic methods with authentic standards have been the techniques most often employed for the isolation, identification, and quantitative analysis of mycotoxins in food and feed products. Because of the environmental concerns with the amount of organic waste solvents resulting from extraction and chromatographic clean-up procedures, supercritical fluid extraction (SFC) has gained in popularity within the last decade (23). Column and thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC) along with ultraviolet (UV), flame ionization (FID), electron capture (ECD), and mass spectrometric (MS) detections and

*

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characterizations have been reported as methods of choice. Chemical derivatization prior to HPLC and GC analyses has been employed either to add a substituent chromaphore to the mycotoxin and thus make it more sensitive to UV and/or fluorescence detections or to make polar compounds more volatile for GC analyses (cf. references cited within text). Although nuclear magnetic resonance (NMR) spectroscopy (i.e., ‘H; “C, etc.), x-ray chrystalography, and circular dichroism have been (and still are) powerful analytical tools used for conformational and structural analyses of novel mycotoxins, instrumentation cost and operation restrict their use in routine screening and analytical procedures. However, the reader is referred to the excellent reviews on their uses for natural plant toxins and other phytotoxins and their applicability to mycotoxins in general (24-26). Structures, spectra, chemistry, and/or references to the same are also listed in Cole and Cox (37), Scott (4), Savard and Blackwell (28), Berde and Schild (14), and Turner and Aldridge (29).

111.

OTHER ANALYTICALCONSIDERATIONS

The above methodologies have their own advantages and disadvantages, but owing to the myriad number of natural compounds that comprise our food and feed products and the equal number of naturally occurring mycotoxins produced by fungi that contaminate them, the quantitative accuracy of all analytical methods is contingent upon the matrix from which the mycotoxins have been extracted. How samples are collected, handled, and stored to prevent secondary fungal and bacterial contamination from saprophytic organisms are other considerations. Since fungal infection and growth in forages, cereal crops, and stored grain products is notan omnipresent process, the mycotoxin concentrations willvary throughout a set of samples. Thus, sample collection representative of a grain bin, field, pasture, silo, etc. should be considered when preparing samples for extraction. Pre- and postharvest treatment and storage, sample handling, and geographic location are all important when dealing with other natural compounds that may interfere with the analysis. To complicate matters further, the analytical success of each procedure also depends on the stability ofthe mycotoxins during extraction, their ability to react withthe extraction media (Le., solvents, ion exchange resins, chromatographic supports, acids, bases, etc.), their susceptibility to chemical reactions with other compounds within the extracted magma, and their susceptibility to photolytic and air oxidation once extracted from their natural environment. These concerns underscore the significance of procedurally scrutinizing authentic standards and confirming their reproducible recovery from foods, feeds, forages, cereal crops, etc. with minimal analytical variability. They further emphasize the necessity that sample preparation, prior to extraction, results in a homogeneous mixture and the need for duplicate and/or triplicate analyses.

IV. METHODS FOR MYCOTOXINS A.

Ergot and Other Alkaloids from Claviceps, Neothyphodium, Epichloe, Balansia, Myriogenospora

1. HistoricalPerspective in Miniature The ergot alkaloids are probably the earliest recorded mycotoxins in which the analytical methodology took over 200 years to come abreast with their toxicology and pharmacology.

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The chemistry, biochemistry, and pharmacology of the ergot alkaloids from C h i c e p s spp.-infected cereal grains, grass seeds, and laboratory cultures have been the focus of analytical research since before the gangrenous and convulsive syndrome, known as ergotism (St. Anthony‘s fire), swept across Europe during the Middle Ages (10, 12-14). The subsequent isolation and identification of the active constituents from the black sclerotia (Le., ergot’)of C. purpurea and C. pnspali infecting wheat and rye began to unravel one of the most biologically significant group of mycotoxins associated with both analytical and toxicology investigations (13, 14, 30, 31). Currently, over 100 ergot alkaloids (and epimeric derivatives) have been identified as naturally occurring in infected cereal grains, forages, and pasture grasses (1 3, 14, 3033) and taxonomic listings reference over 30 species of Clen~iceps(13, 14, 34). A more detailed perspective of Clnviceps and related fungi is covered in Chapter 11 (pages 503524). Since grain regulations limit Clcniceps ( “ergot’ ’)-contaminated products from entering foods used for human consumption, ergotism is not an immediate threat to humans. Nevertheless, a prevalent and constant threat exists to domestic livestock from grains and forages infected with Clnlliceps, and subsequently, there may be anindirect risk to humans (35). Moreover, livestock grazed on “clavicipitaceous” endophyte (fungal)-infected pasture grasses suffer devastating production losses from the same ergot alkaloids (36-40). Classic “ergot toxicity,’’ not associated with the genus Claviceps, has been described in livestock grazed on endophyte-infected pastures: e.g., tall fescue toxicity in cattle (gangrenous fescue foot) and horses (agalactia in pregnant mares) and perennial ryegrass staggers in sheep and other livestock (convulsive ryegrass staggers) (41-45). Neo~yphodiumspp. (46-48), the asexual anamorph of Epichloe, the ergopeptine alkaloids, and the indolediterpenoid lolitrem alkaloids have been reported as the endophytes and alkaloids related with these two toxic syndromes in livestock (32, 33,42,43,45,49). Further, other genera of endophytic fungi (i.e., Epichloe, Balamin, Myriogenosporn) also have been related to similar atypical or idiopathic ergotism in livestock on pasture grasses (32, 43, 44, 49). Neotyphodiurn, Epichloe, Bnlmsia, Myriogenosporcl, and Clcwiceps all share corresponding evolutionary relationships and are taxonomically aligned within the Clavicipitaceae (34, 46, 50, 51); thus, it is not surprising to find these endophytes produce the same alkaloids with the analogous toxic syndromes in livestock grazed on infected pasture grasses (32, 33, 34,41, 43, and references cited therein). Prior to the discovery of the endophytegrass associations and production of ergot alkaloids by these fungi, only Clnviceps was recognized as the genus capable of producing the ergopeptine alkaloids. The chronicles surrounding classic ergotism (13, 14, 30, 31) and the reviews surrounding the clavicipitaceous endophytic fungi of pasture grasses (32-34, 41 -45‘) emphasize the importance of reliable, quantitative procedures and truly represent significant milestones in the development of chemical, biochemical, and pharmaceutical methods for mycotoxin analyses. These historical references are also excellent examples of the analytical problems associated with mycotoxins in general and underscore the importance of sample collection, handling, storage, and fungal identification prior to analytical methodology. The ergot alkaloids produced by Clnviceps spp. maybe divided into five major classes: the clavine, lysergic acid and simple lysergic acid amides, ergopeptine, and ergopeptam alkaloids (14,52-54) (Figs. 1-4). The ergopeptine and ergopeptam alkaloids may be further subdivided into the ergotamine, ergoxine, ergotoxine, and ergocristine groups depending on the substituents attached to positions C-2’ (Rl) and C-5’ (R2) of the tricyclic peptide portion of the molecule (Fig. 3; Table 1). The ergopeptine alkaloids are the ones

NOVINE

Analytical Methodology for Mycotoxins

657

HO%

\

N H CHANOCLAVINE I

‘H AGROCLAVINE

\

H ELYMOCLAVINE

H PENNICLAVINE

Figure 1 Ergotalkaloids:clavines

generally related to the gangrenous and convulsive ergotism and agalactia and spontaneous abortions in animals and humans (13, 14, 41, 49). Most recently however, C. nfricnrzn (a “relatively new species of ergot”) has globally threatened the sorghum industry (15) and the ergot alkaloids produced by this species deviate somewhat from those produced by C. yurpzu-eu, etc. C. nfricnntr produces primarily the dehydrogenated ergot alkaloids: dihydroergosine, dihydroelmoclavine, festuclavine, pyroclavine (Fig. 5), and other minor unidentified compounds (21, 56). The toxicity of these compounds is still in question, but a sixth class (or subclass preferably) of alkaloids may also be designated under the above for the dehydrogenated ergot alkaloids.

H

ACID

LYSERGIC

O

’

j

3

\

%

\

H

HOL

N H

LY‘SERGIC ACID METHYLCARBINOLAMIDE /

H O H NH> LYSERGIC ACID AMIDE

H Figure 2 Ergotalkaloids:lysergicacid.

H simple lysergicacidamides.

Porter

658

TRICYCLIC PEPTIDE PORTION 9, IO-ERGOLENE RING

9

A

/

BASIC ERGOPEPTINEALKALOID

Figure 3 Ergotalkaloids:

ergopeptine.

Another class of ergot alkaloids (i.e., ergobalansine; Fig. 6) has been isolated from cultures of Bnlnrlsin spp. (57j; these alkaloids are devoid of the proline moiety in the tricyclic peptide portion of the molecule (compare Fig. 3 vs. Fig. 5). Predicated on the biochemistry for the ergopeptines (14, 31, X), it would appear that two amino acids (e.g.. instead of three) are incorporated in the biosynthesis of this portion of the compound. The toxicity of this alkaloid has not been investigated, but because of the evolutionary relationship between Bnlarzsicl and Clcrviceps (34, 46, 50, 51), future studies will define if ergobalansine is prototypical or a prelude to a distinct class of ergot alkaloids analogous to the ergopeptines.

open

0

H‘ ERGOVALAM: R1=-Me; R2=-iso-Pr

Figure 4 Ergopeptam alkaloids.

Analytical Methodology for Mycotoxins

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Table 1 General Structure of the Ergot Peptide Alkaloids and Their Major Ions, Atomic Mass Units (amu), Resulting from Electron Impact (ET) (fragments A, B, C) and/or Isobutane Chemical Ionization (CI) (fragments AH, BH, and CH) Mass Spectrometry (see text for explanation) alkaloids Ergopeptine Ergotamine group (R, = -CH,) Ergotamine Ergosine beta-Ergosine Ergovaline Ergobine Ergoxine group (R, = -C,HS) Ergostine Ergoptine beta-Ergoptine Ergonine Ergobutine Ergotoxine group (R, = -i-Pr) Ergocristine alpha-Ergocryptine beta-Ergocryptine Ergocornine Ergobutyrine

R2

MW

CH AH

BH

-CH?Ph -i-Bu -sec-Bu -i-Pr -Et R2 -CH:Ph -i-Bu -sec-Bu -i-Pr -Et R? -CH2Ph -i-Bu -sec-Bu -i-Pr -Et

581 547 547 533 519

268 268 268 268 268

315 28 1 28 1 267 252

245 21 1 21 1 197 183

595 56 1 56 1 547 533

268 268 268 268 268

329 295 295 28 1 267

245 21 1 21 1 197 183

609 575 575 561 547

268 268 268 268 268

343 309 309 295 28 1

245 21 1 21 1 197 183

MW = molecular weight.

CH3

H’

H’

FESTUCLAVINE

PYROCLAVINE

H’ DMYDROELYMOCLAVINE

Figure 5 Festuclavine,pyroclavine,dihydroelymoclavinevia C. ufiicnnu.

Porter

660

Figure 6 Ergobalansine.

The ergopeptines, simple lysergic acid amides, and clavine alkaloids (Figs. 1-3) have been isolated from Neotyphodiuminfected tall fescue (58-62) and cultures of this fungus (63, 64), which is consistent with its evolutionary relationship with Clcrviceps (34, 46, 50, 51). Although ergovaline is the major ergopeptine alkaloid in N. coenoylzicdurninfected tall fescue (58-60,65), lysergic acid amide (or ergine; Fig. 2) can exist in concentrations approximately equal to that of ergovaline (66). Lysergic acid amide is now accepted as a true natural product, but it can, as well, result from the solvolytic cleavage of lysergylmethylcarbinolamide (Fig. 7 ) (31, 67). This is a classic example for compound reaction and/or stability after extraction from its natural matrix. Ergonovine (Fig. 2), another simple lysergic acid amide, also has been isolated from endophyte-infected fescue seeds (61), but this compound may be from CZnviceps contamination of the seeds (59) and further serves as an example of howsecondary infection (or Contamination) ofsamples

HLYSERGYLMETHYLCARBINOLAMIDE

LYSERGIC ACID AMIDE

Figure 7 Lysergic acidmethylcarbinolamide slovolytic cleavage to lysergic acid amide.

Analytical Methodology for Mycotoxins

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can obscure analytical results. Because ergonovine is found in conjunction with the ergopeptine alkaloids in Claviceps-infected wheatand rye (68), it is possible this alkaloid may also be present in endophyte-infected grasses. The clavine alkaloids chanoclavine(s), penniclavine, elymoclavine, and agroclavine (Fig. 1) also have been isolated from endophyte-infected tall fescue (60) and are precursors in the biosynthesis of the simple lysergic acid amides and the ergopeptines (14, 31, 32, 69). 2. Isolation and Identification Since the endophytic fungi of pasture grasses are systemic infections of their host versus the localize seed head infection of Clmiceps (34, 41, 42), the extraction of the ergot alkaloids from the two matrices illustrates how background materials influence the isolation and analytical processes. There are a variety of procedures for their extraction and isolation from endophyte-infected pasture grasses (i.e., tall fescue, perennial ryegrass, etc.), which represent variations on those employed for Clnviceps-infected grains (70,71). Current methods of choice involve extraction with either an aqueous tartaric or lactic acid solution; lactic acid appears to work best for the extraction of ergovaline from the infected fescue (72), partition chromatography with chloroform or methylene chloride at an appropriate pH, column cleanup procedures using either silica, alumina, or an ion exchange resin, and identification and analysis using cochromatography (TLC and/or HPLC) with ultraviolet or fluorescence detection. Mass spectrometry (MS) also has been employed for the identification and quantitative analysis of the ergot alkaloids. The ergot alkaloids are extremely susceptible to photolytic and air oxidation, hydration, and epimerization at the C-8 position of the ergolene ring (14, 32, 69) (Fig. 3). Epimerization of the C-8 position occurs in either acidic or basic conditions, and therefore, the isolation of the C-8 epimers (designated with the suffixal-inine, i.e., ergovaline vs. ergovalinine) occurs in most extraction procedures. Decomposition and epimerization may be minimized by working under subdued or yellow light, by concentrating extracts in vacuo at room temperature or less (i.e., 125"C), and by concentrating small volumes of extracts under a stream of nitrogen. Storing dried concentrates in amber vials under nitrogen at or below 0°C will help prevent further decomposition. Moubarak et al. (73) have successfully stored ergovaline at -4°C for up to 12 months. Furthermore, ergovaline decomposes rapidly when extracted from non-freeze-dried plant tissues (69). Thus, observing the correct precautions during sample collection, handling, and preparation is crucial for the isolation and quantitative analysis of the ergot alkaloids from Claviceps-infected grains and/or endophyte-infected grasses. The total concentration of ergot alkaloids in endophyte-infected tall fescue varies with the season and amount of nitrogen fertilization (60,65,74-76). Whether the relative concentration of the individual ergot alkaloids varies with these parameters is currently unknown and such factors should be considered (and recorded) prior to sample collection and analysis. 3. High-Performance Liquid Chromatography HPLC with either ultraviolet or fluorescence detection is the preferred method for routine screening and analysis of the ergopeptine alkaloids in endophyte-infected grasses (55, 59, 65, 66, 72, 73, 77-79). A rapid, simplified HPLC method for the analysis of the ergot alkaloids associated with N. coerzophialum-infected tall fescue has been developed (66). Extraction of infected seed or grass with alkaline methanol, followed by filtration, and direct HPLC analysis (fluorescence detection) with a mobile phase of either 60 or 70% alkaline methanol results in separation of ergovaline, ergovalinine, lysergic acid amide,

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and its isomer isolysergic acid amide (erginine). The use of gradient elution allows for the complete separation of complex mixtures of the ergot alkaloids (66). A unique preparative method for the isolation and purification of large quantities of ergovaline has been reported (73). This procedure involves a modification of previous methods (65, 72, 80) in which the infected seeds are extracted with a 5% aqueous lactic acid solution and the ergot alkaloids are adsorbed onto SM-2 Biobeads (BioRad, Hercules, CA). Extraction of the Biobeads with methanol, followed by an HPLC cleanup procedure using a C- 18 RP column (Vydac, Separations Group, Hesperia, CAI, and HPLC analysis using gradient elution with acetonitri1e:ammonium carbonate:methanol as the mobile phase results in pure ergovaline (295%). Zhang et al. (79) have employed an amberlite XAD-2 exchange resin for a rapid cleanup step after extracting infected fescue seed with a 5% lactic acid: methanol (4: lv/v) solution. Other HPLC methods (81) have been employed for the analysis of ergovaline/ergovalinine distribution in the leaves of fescue colonized by two different Neofhyphodium spp. Scott et al. (68) and Rottinghaus et al. (82) have reported additional extraction, cleanup, and HPLC procedures for the analysis of the ergopeptine alkaloids in cereal grains, flour, and feeds. 4. Thin-Layer Chromatography After the ergot alkaloids have been extracted into a suitable organic solvent, TLC on silica gel remains one of the most powerful tools for the analysis and identification of these compounds. The major advantages of TLC are that several samples may be analyzed at the same time, TLC does not involve expensive instrumentation, relatively small quantities of standards may be used, mg quantities of the individual alkaloids may be separated and purified with preparative TLC, and it compliments MS in the confirmatory identification and analysis of epimeric alkaloid mixtures (see below). Perellino et al. (54) have reported a TLC procedure on silica gel for the separation of most all of the known ergot alkaloids. When the TLC plates are developed in methylene chloride: isopropyl alcohol (92 :8 v/v) three times (drying the plates between ~uns),these alkaloids separate into the isolysergic acid group (i.e., -inirze epimers), the ergotoxine group, the ergoxine group, and a mixture of the ergotamine and clavine groups. Removal of the silica from the area of the plates consistent withthe known standards, extraction of the alkaloids from the silica using methanol: chloroform (1 :4 or 1 : 1 v/v) (54, 71), and rechromatography in chloroform: methanol (9 : 1 or 4 : 1 v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines) (63). Other solvent systems used for the TLC analysis of these compounds are listed in Table 2. Table 2 Solvent Systems3 Effectively Used for TLC on Silica Gel for the Separation and Identification of Ergot Alkaloids CH2C12:iso-PrOH (92: 8; 90 : 10; 75 :25) CHC17:MeOH (95:5; 90 : 10; 80 : 20) CHC&:MeOH (90: 10) in a saturated NH3 atmosphere CHC13:MeOH :NH3 (94 : 5 : 1) CHC13 :Et,NH (90 10) Benzene : dimethylformamide (86 : 5 : 13: 5 ) ' All systems (v/v) are in a saturated atmosphere. in tanks lined with Whatman No. 1 filter paper. 54. 63, 64, and 7 1.

Sowce: Refs. 21,

Analytical Methodology for Mycotoxins

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Visualization of the ergot alkaloids on a TLC plate may be accomplished with a hand-held UV light at 254 and 366 nm. The 9,lO-double bond in the wring of the ergolene portion of the molecule (Fig. 1) is conjugated with the indole nucleus and gives the ergopeptine alkaloids their characteristic bright, pale blue fluorescence (UV lambda max in methanol at approximately 315 and 242 nm). Those ergot alkaloids devoid of the 9,lOdouble bond give a characteristic dark blue absorption under 254 nm (UV lambda max in methanol at approximately 292,280, 275, and 222 nm) indicative of the indole nucleus. Spraying the TLC plates with a solution of p-N,N-dimethylaminobenzaldehyde(van Urk's reagent; 83) followed by spraying with a 1% sodium nitrite solution (water: ethanol, 1 : 1 v/v; 84) produces intense blue spots that are also characteristic of the ergot alkaloids. Colorimetric analysis at 590 nm of a crude alkaloid fraction relative to a known standard (i.e., ergonovine maleate; ergotamine tartrate) provides a method for quantitating total ergot alkaloids in crude extracts (85). Individual ergot alkaloids may then be identified and quantified by a combination of TLC, HPLC, and/or MS. 5. Mass Spectrometry Identification and quantitative analysis of the ergot alkaloids isolated from infected grains, grass, and laboratory cultures using MS include electron impact (ET) (63), chemical ionization (CI) (64, 86), and tandem mass (MS/MS) spectrometry (58, 60, 62, 70). Under lowresolution electron impact (70 eV), the ergopeptine alkaloids pyrolytically decompose into the lysergic acid amide, the cyclic peptide, and diketopiperazine fragments A, B, and C, respectively (Fig. 8). These fragments then undergo E1 and produce spectra characteristic of the ergolene ring and the peptide portion of the parent molecule. Fragments useful in the interpretation of these spectra occur at m/z+ 70, m/z+ 125, and m/z+ 154 atomic mass units (amu), which are characteristic of the proline moiety (63, 87). These ions, in combination with the lysergic acid amide ion at m/zf 267 amu (ion A, Fig. S), are indicative of an ergopeptine alkaloid. Using ergovaline for example, in addition to m/z+ 267 amu, the other two major fragments associated with ions B and C (i.e., fragments indicative of the methyl substituent at R1 and the isopropyl substituent at R2: Figs. 3 and 8; Table

Figure 8 Mechanism for the mass fragmentation of the erogopeptine allkaloids (EI; CI).

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1) occur at m/z+ 266 and m/z+ 196 amu, respectively. The major disadvantage with low resolution EIMS (70 eV)in the analysis of the ergopeptine alkaloids is the low abundance (i.e., 5 1%)of the molecular ion and the diagnostic fragment ion B when R2 is an alkyl substituent (i.e.. ethyl-, n-butyl-, isobutyl-, sec-propyl-, isopropyl-; Figs. 3 and 8; Table 1). Although fragments associated with theergolene nucleus are isobaric with those fragments related to the cyclic peptide (B) and diketopiperzine (C) portions of the parent molecule (64), ergovaline and its C-8 epimer ergovalinine produce a high abundance of ion m/z+ 196 amu (ion C) (63). Spectral interpretation of complex mixtures of the ergopeptines, however, becomes somewhat more difficult. Isobutane chemical ionization mass spectrometry (CIMS) has been employed in the identification of the ergopeptine alkaloids (64, 86). This method involves an ion-molecule reaction of the alkaloids in the presence of a reagent gas (isobutane). The ion-molecule reaction results in reduced fragmentations, as seen with low-resolution EIMS, produces simplified spectra, and thus circumvents interpretation difficulties of the more complex spectra and those resulting from mixtures of these alkaloids. Under isobutane CIMS, the lysergic acid amide (A), cyclic peptide (B), and diketopiperazine (C) molecules (Fig. 8) abstract a proton from a r-butyl cation (which is generated in the mass spectrometer) and the resulting ion-molecule reaction produces spectra containing three major ions represented by AH, BH, and CH in Fig. 8 and Table 1. These ions are 1+ amu greater than the parent fragments A, B, and C (58, 62, 64, 86). The major fragments used to identify most of the known ergopeptine alkaloids by this method are outlined in Table 1. Although ergovaline and its C-8 epimer ergovalinine produce the same three major fragments at m/z+ 268 (AH), m/z+ 267 (BH), and m/z+ 197 (CH) amu and cannot be distinguished by this method, both compounds (as with the other ergopeptines and their C-8 epimers) separate nicely using TLC and/or HPLC analysis (54, 59, 63, 65, 71, 82). Tandem mass spectrometry (62) also has been employed in the separation and analysis of ergopeptine alkaloids from both endophyte-infected tall fescue and perennial ryegrass ( 5 8 , 75, 88). In an overly simplified discription, MWMS (which also is conducted in the presence of a reagent and/or a target gas) uses one stage of mass separation to isolate the individual alkaloids of interest in a crude extract; depending on the ionization mode, this usually involves the molecular ion, a protonated molecular ion, or a molecular anion; a second stage of mass separation is then employed to analyze the product and/or daughter ions (also dependent on the reagent and/or target gas). The fragmentation mechanisms for the ergopeptine alkaloids utilizing MS/MS with isobutane as the reagent gas and argon as the target gas are analogous to that described for isobutane CIMS and are described i n detail (62). Major advantages of this system are that complex mixtures of these compounds can be analyzed without prior cleanup, only small samples of extracted material are needed, and isomeric species (i.e., ergosine vs. beta-ergosine; Fig. 3; Table 1) can be distinguished. However. MS/MS analysis requires expensive instrumentation not readily available to most laboratories, and like CIMS, this system cannot distinguish between epimeric ergopeptine alkaloids. Subsequently, MS/MS is not practical for routine screening of toxic or infected grasses. Field desorption mass spectrometry also has been used in the identification of the ergopeptine alkaloids (87) and provides extremely simplified spectra along with intense molecular ions. Recently, Porter et al. (21, 56) have used a combination of TLC and GUMS to analyze and quantitate the clavine alkaloids (dihydroelymoclavine, festuclavine, and pyroclavine; Fig. 5) extracted from C. afi-icann-infected sorghum. Infected sorghum was extracted with MeOH-H,O (70: 30 v/v) containing 1% NH40H at room temperature; the

Analytical Methodology for Mycotoxins

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aqueous solution was extracted with CHCll and the crude alkaloid material subjected to preparative TLC (silica gel) using CHC13-MeOH (9: 1 v/v) followed by CHC13-MeOH (95 :5 v/v) as the developing solvents. Fractions consistent with festuclavine and pyroclavine, dihydroelymoclavine, chanoclavine I, and dihydroergosine (i.e., Rf, visualized under UV 254; and giving a blue reaction to p-dimethylaminobenzaldehyde) were collected, eluted from the silica (CHC13-MeOH, 1: 1 v h ) , and the eluate concentrated to dryness (N2).Each alkaloid fraction was subjected to GUMS analysis neat and also after reaction with MSTFA. Festuclavine and pyroclavine could be analyzed neat (M' 240) and as their TMS-derivatives (M' 312; TMS-moiety attached to the indole-N; Fig. 9), whereas chanoclavine and dihydroelymoclavine were analyzed as their TMS-derivatives (M' 472 and M+ 400, respectively) (Figs. 9 and 10). With MSTFA, chanoclavine I gave a mixture of the di-TMS-(M' 400) and tri-TMS-derivatives (M+ 472) (Fig. 10); this mixture was quantitatively converted to a single compound by reaction with MBTFA, in which the substituent at position 6 [i.e., the CH3-N-H (disubstituted derivative) and the CH3-N-TMS (trisubstituted derivative)] was exchanged for CH,-N-COCF, (M+ 496) (cf. Fig. 10). The major amu(s) and fragmentation patterns for all TMS derivatives follows the analogous patterns as that for the parent alkaloid (EIMS, 70 eV), with the expected shift of 73 amu for each displaceable proton. Subsequent G U M S evaluation of costaclavine and agroclavine [neat (M+ 240 and M' 238) and as their TMS derivatives (M' 3 12 and M+ 3 lo), respectively], lysergol-TMS (M+ 398), elymoclavine-TMS (M' 398), and dihydrolysergamide-TMS (ndz' 324, M+-NH7)proved this method unequivocal for the identification and quantitation of this class of ergot alkaloids in the nanogram range using both SCAN and SIM modes. Dihydroergosine was analyzed using both TLC and HPLC.

6. Loline Alkaloids The loline alkaloids (Fig. 11) in N. coenophinlurn-infected tall fescue are produced by the fungus as a plant host-defense mechanism in response to insect herbivory and, except as a deterrent to insects, may not fall under the classic definition for mycotoxins (see

$ *

H

CH,

N T/MS

TMS

TMS-ESTUCI>AVINE

TMS-PYROCLAVINE

TMS TMS-DIHYDROELYMOCLAVINE

Figure 9 TMS-festuclavine, -pyroclavine, -dihydroelymoclavine alkaloids.

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666

($, CH2OH

H3

MSTFA

/

N

CH2OTMS CH7OTMS

/

m/z 256

( f

H' CH20TMS

MBTFA

N TMS TMS

m / z 400

& + +

S)CH,

/

N

TMS

dz472

ThS

Figure 10 TMS-chanoclavine I alkaloid.

above). This defense mechanism may involve interactions between both the endophyte and its host grass and, as with the ergot alkaloids, suggest a true symbiosis between the endophytes and their plant host (50, 89-91). Moreover, Petroski et al. (92) have suggested the lolines may be alleopathic, thus improving the ability of the infected grass to compete with other grasses. The chemistry, occurrence, and biological effects of the loline alkaloids and associated endophyte-grass interactions have been reviewed (89, 93). Capillary gas chromatography using either FID and/or MS detection is an established method for the analysis of the loline alkaloids (89, 92, 94). Extraction of ground seed (approximately 5 g) with methylene chloride :methanol :ammonia (75 :25 :0.5 v/v/v),

LOLINE

RI

R2

CH3

H

N-ACETYLLOLINE COCH3 CH3 N-FORMYLLOLINE CHOCH3 N-ACETYLNORLOLINE COCH3 H N-METHYLLOLINE CH3

Figure 11 Lolinealkaloids.

cH3

Analytical Methodology for Mycotoxins

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followed by filtration, provides extracts ready for GC/FID or GUMS analysis. Forage samples may be extracted analogously, but prior to analysis, a sulfonic acid solid phase cleanup step of the forage extracts is necessary to remove substances that interfere with the assay. The detection limit for N-acetylloline and N-formylloine is 10 ng. Tepaske et al. (94) have described a similar GUMS procedure for the analysis of the loline alkaloids in bovine urine and plasma. The loline alkaloids do not provide a well-defined molecular ion in the mass spectrum (70 eV), but their separation under GC conditions and characteristic mass fragmentation patterns (89) allow for their unequivocal identification and quantification. Petroski et al. (92) and Powell and Petroski (89) reported a method for the separation of the loline and the ergot alkaloids from endophyte-infected tall fescue whereby an alkaloidal extract is subjected to column chromatography using Sephadex LH-20. The loline alkaloids pass through the column and the ergot alkaloids are recovered by exhaustive elution of the LH-20 with methanol. Also, countercurrent chromatography for the separation of milligram quantities of N-methylloline, N-acetylloline, and N-formylloline (Fig. 11) from an endophyte-infected tall fescue seed has been reported (61). Total lolines (defined as N-formyl- and N-acetylloline) occurring in endophyte-infected tall fescue seed (3263 pg/g) and forage (1723 pg/g) were quantified using GC/FID (90). Concentrations of these alkaloids (as with the ergot alkaloids) in forages vary with season, the amount of nitrogen fertilization, grazing pressure, and the amount of insect herbivory (93,95, 96). 7. Paxilline and the Lolitrem Alkaloids from N. lolii-Infected Perennial Ryegrass Evidence suggests that the indole-isoprenoid lolitrems are almost as diverse as the ergot alkaloids. Paxilline and lolitrem B (Fig. 12) are the two major alkaloids associated with perennial ryegrass staggers in sheep, cattle, and other livestock (97-101). Studies involved with determining the biosynthesis of paxilline and lolitrem B resulted in the identification of alpha-paxitriol, lolitriol, and the lolitrems A, C, D, and E (102).These additional minor lolitrems may contribute to the overall toxicity of paxilline and lolitrem B. H

PAXILLINE

LOLITREM B

Figure 12 Paxilline and lolitrem B.

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Gallagher et al. (97) reported an isolation and screening method for lolitrem B in N. Mi-infected perennial ryegrass. One gram of oven-dried, milled grass was extracted with chloroform: methanol (50 ml; 2 : 1 v/v) for 1 hr. One milliliter of the extract was dried under nitrogen, reconstituted in methylene chloride (2 ml), and subjected to a cleanup step on silica. The eluent (100 pl)from the silica was then analyzed by HPLC on a Zorbax Silica column (DuPont, Wilmington, DE), using methylene chloride: acetonitrile (80 :20 v/v) as the mobile phase. Recovery of lolitrem B was 93-97% with detection limits at 500 pg (fluorescence detection). This HPLC method has been used to screen up to 80 samples per day. The amount of lolitretn B in the infected grass necessary to elicit the staggers syndrome is only 5 ppm.

8. Peramine Although peramine (Fig. 13) is a plant metabolite and the major insect deterrent in N. coelzophiaIlllll-infected tall fescue and N. lolii-infected perennial ryegrass (91, 103-106), its isolation is mentioned to exemplify a unique separation method for both peramine from lolitrem B using a two-phase extraction system. Freeze-dried, ground grass (100 mg) is first extracted with methano1:chloroform (3 ml) and followed by concurrent extractions with hexane and water (3 ml each). The aqueous and organic phases are separated by centrifugation. Lolitrem B was analyzed in the organic phase as previously reported (97), whereas peramine, after minor cleanup using ion-exchange chromatography, was analyzed in the aqueous phase by HPLC with a mobile phase of acetonitri1e:guanidinium formate (pH = 3.7). Recovery of peramine is 93-loo%, with detection limits at 1 yg/g of infected grass. Alternatively, peramine may be analyzed in the aqueous phase, after an ion-exchange cleanup step with two minicolumns connected in series. The first column (BioRad AG 2 X 8, 200-400 mesh) is in the hydroxide form and the second column (Analytichem Bond Elut CBA) is in the carboxylic acid form. After the aqueous phase is aspirated onto the columns, they are washed with 80% aqueous methanol (3 ml), the columns separated, and peramine eluted from ihe acid column with aqueous methanol and formic acid. Pera-

PERAMINE

Figure 13 Peramine.

Analytical Methodology for Mycotoxins

669

mine is then analyzed by TLC using chloroform :methanol :acetic acid :water (30 : 10 : 1 : 1 v/v/v/v) as the developing solvent (103). A rapid, sensitive, reverse-phase TLC method for the analysis and quantification of peramine in crude extracts of several endophyteinfected grasses has been reported (107) and the characteristic low-resolution mass fragmentation (70 eV) of peramine has been defined (106).

B. Fusarium Metabolites Another mycotoxin problem has been recognized throughout the world since: (1) F. rnoniliforme and the fumonisins (Fig. 14) were associated with equine leukoencephalomalacia (108-1 10); (2)the fumonisins were statistically associated with esophageal cancer in humans in certain areas dependant on corn as the staple diet (12, l l 1-1 17); and (3) the routine occurrence of the fumonisins and other Ftrsa~iun1toxins in corn, wheat, barley, rice, and other cereal grains (1 18- 121). Although Fz4sarium species and their toxic metabolites occur most prevalently in corn, wheat, and barley, they are also found in nuts, fruits, and vegetables, and in other nonfood items of economic importance (e.g., tobacco, cotton, forage grasses, alfalfa, red clover, flax) (119, 122). Corn, wheat, and barley comprise twothirds of the world cereal production and the numerous Fusarium species associated with animal and human health problems ( 1 3 , 124) warrant the most expedient and precise analytical procedures for their toxic metabolites. 1. Fumonisins The ubiquitous detection of the fumonisins in cereal grains and especially corn, rice, and corn and rice-based products indicates that the potential for human and animal exposure is a worldwide problem (6, 12, 109, 116- 120, 125). Several fumonisins have been identified from F. morzilifomle-infected grains and defined as fumonisin B, , B2, B3, B4, AB,, and AB2 (109, 120, 121). Fumonisin B, is the major metabolite found in nature and also

ACID

FUSARIC MONILIFORMIN

OH 0 I

ZEARALENONE

OH

OH

OH

DEOXYNIVALENOL

Figure 14 Fz4sarizlnz mycotoxins: moniliformin, fumonisin B,, fusaric acid, zearalanone. deoxynevalenol.

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in cultures and defined as the 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyicosane with a propane-l,2,3-tricarboxylatesubstituent at C-14 and C-15 positions (109, 120). Fumonisins B, and B, are the C10 and C5 deoxy analogs, respectively, whereas B4 is missing the hydroxyl moiety at both C-5 and C- 10. Fumonisin AB and AB2are the corresponding N-acetyl analogs of B1 and B?, respectively. Since their isolation and identification, numerous HPLC procedural variations have been reported (120-121, 125-135). Briefly, the fumonisins may be extracted with either methanol-water or acetonitrile-water followed by cleanup on an anion-exchange resin. The resin is then eluted with acetic acid-methanol, followed by derivatization of the extract with o-phthalaldehyde (OPA), and HPLC analysis with fluorescence detection (125). Variations of both derivatization (i.e., 9-fluorenylmethylchloroformate and 4-fluoro-7-nitrobenzofurazan (129, 133) and HPLC analysis (isocratic and gradient elution) have been employed (125-135). An improved preparative HPLC procedure for funonisin BI (B? and B3) (underivatized; using light-scattering detection) and the subsequent quantitative analysis have also been reported (127, 128); fumonisin B, purity was 195% with a recovery of >90%. Other preparative procedures using amberlite, XAD-2, silica gel, and CI8reverse phase-chromatography have been reported (136, 137). Meredith et al. (127, 128, 138) have further utilized centrifugal spinning TLC and analytical HPLC with fast-atom bombardment mass spectrometry for the preparative and quantitative analysis of the fumonisins, and Rottinghaus et al. (139) have reported a sensitive TLC method for the detection of fumonisin B, and B2. In studies directed at the removal of fumonisins from contaminated corn, Sydenham et al. (13 1) have reported the analysis of partially hydrolyzed fumonisin B I using a combination of column, thin-layer, and HPLC-electrospray MS to isolate, purify, and quantitate the corresponding aminopentol of this mycotoxin. Procedural analysis for another analog of fumonisin B, (i.e., the N-acetyl-C-15-keto-derivative)isolated from corn cultures of F. yrol(femtznzr has been reported (140), and Sydenham et al. (130) have compared a monoclonal antibody-based competitive direct enzyme-linked immunosorbent assay (CDELISA) with the HPLC determination of fumonisin in corn. The structurally related fumonisin-like compounds present in naturally contaminated corn may contribute to the differences between these two methods (i.e., CD-ELISA results were consistently higher than the HPLC results) and emphazises the importance of specific chemical analyses for the mycotoxins in question. The CD-ELISA however may still be used as an initial semiquantitative screening technique (130). Most recently, Meredith has reported a compendium on the isolation and characterization of the fumonisins and the reader is referred to this chapter (141). Then too, the co-occurrence and analysis of the fumonisins with other F~sm-irnntoxins (Le., zearalenone, deoyxnivalenol, and the other trichothecens) are referenced below.

2. Zearalenone Zearalenone (ZEN) and related metabolites are nonsteroidal estrogenic mycotoxins produced by several species of Fz~sariun~ (10, 142) that are routinely surveyed in cereal products (143) and directly related to hyperestrogenism and infertility in swine (10, 142) and precocious puberty in children (144). Bennett et al. (143) developed an HPLC method using UV or fluorescence detection with an average recovery of 82-100% for ZEN and its metabolite a-zearalenol. Doko et al. (125) employed an improved HPLC method in conjunction with TLC for the analyses of ZEN and the fumonisins in cereals and cerealbased foods from Africa, and Ryu et al. (145) have described a GUMS analytical method

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for the simultaneous detection and quantitation of naturally occurring ZEN and six major trichothecenes in barley and maize (see below). 3. Trichothecenes and Co-occurrence with Other Fungal Toxins Over 100 trichothecenes have been identified from Fusarium and other genera (Myrothecium, Trichothecium, Cephulosporiurn, Verticirnonsporium, and Stachybotqs) (145), but major analytical interests have concentrated on those of Type A and B (6) because of their diverse toxicological effects in both animal and human disorders and their routine occurrence in cereal grains and stored grain products ( 1-3,6, 10- 12, 145- 153). Although deoxynivalenol (DON; vomitoxin) is only one of several tricothecene mycotoxins produced by Fuscrrium spp. (F. nlorziliforrne, F. C14~?11O~l411Z, F. graminenrum, F. roseum, etc.), it is among the most frequent tricothecenes analyzed in cereal crops (10, 12). DON is a 12,13-epoxytricothecene associated with feed refusal (i.e., reduced weight gains and growth depression) and the emetic responses primarily in swine (10, 122, 154). Current food safety guideline recommendations for this mycotoxin in cereals used for humans is 2 ppm with no more than 1 ppm for foods used in infant formulations (1 1). Other structurally related tricothecenes routinely screened in cereals are 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15-ADON), 3,15-diacetyl-deoxynivalenol (3,15DADON), nivalenol (NIV), 4-acetylnivalenol (4-ANIV), 4,15-diacetyl-nivalenol (4,15DANIV), Fusarenon-X (F-X), T-2 toxin (T2). and HT-2 Toxin (HT-2) (145-150). Ryu et al. (145) have developed a GUMS procedure for the analysis of DON, 3ADON, NTV, F-X, T-2, HT-2, and ZEN in cereal grains that involves extraction of the pulverized cereal samples with actonitrile-water, defatted with n-hexane, followed by solid-phase extraction using a florisil column. After elution from the column with chloroform: methanol (9 : 1). the mycotoxins were derivatized with N,O-bis(trimethyl)acetamide/ trimethylchlorosilane and analyzed by GC (EC and/or MS) with a mean recovery of 913 % for DON, 3-Ac-DON, F-X, T-2 toxin, HT-2 toxin, NIV, and ZEN. Previous investigations (147) compared the extraction efficiency and quantitative recovery of DON and NIV from florisil and sep-pak-silica cartridge columns and their confirmation by GC-EC and GCMS analyses; the range of recovery for the two mycotoxins was 89-99% for DON and 23-99% for NIV. These investigators emphasize the critical points necessary for high recovery of the toxins with solvent selection for extraction, subsequent cleanup procedures, and also recovery of the mycotoxins from the extracts and the different commodities (i.e., wheat, barley, corn, etc.). Other studies (146) have outlined the GUMS conditions and analysis using selective ion monortoring (SIM) of the trimethylsilyl derivatives of DON, 3-ADON, 15-ADON, 3,15-DADON, NIV, 4-ANIV, and 4,15-DANIV extracted from rice cultures of F. grurninarium isolated from barley and corn. The detection limit of this procedure is reported at ca.10 ppb per mycotoxin. These authors emphasize the utility of the procedure for defining chemotaxonomic types of F. gruminarium (and other Fusnriutrl spp.), the significance of geographic location, and the spectrum of mycotoxins produced by the isolates. Furthermore, this report describes the co-occun-ence of ZEN (via HPLC analysis) by the tricothecene-producing isolates from corn (5 1.4%) and barley (31.3%) along with the regional differences in trichothecene production. Analysis of F. grc/mirzelrium- and F. crookwellense-infected maize by extraction (acetonitrile/water, 82/ 18 v/v), column cleanup (employing charcoal, celite, and aluminium oxide), and TLC analysis further demonstrated the co-occurrence of DON, 3- and 15-ADON, and NIV with ZEN and differentiated between the production of NIV instead of DON when ears are

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infected with F. croohvellense; ZEN accumulation occurred in cobs colonized by both species (150, 155, 156). Other Fusnriunz toxins that have provided unique problems with analytical procedures are: the fusarins (157, 158), beauverisin (159), fusaproliferin (160), fusarochromanone (161, 162), fusaric acid (10, 18, 20, 21, 1221, and moniliformin (27, 154, 163). The fusarins are pyrrolopolyketides of which fusarin C is mutagenic (157, 158); beauverisin is a cyclic hexadepsipeptide consisting of three N-methylphenylalanyl- and three 2-hydroxy-3-methylbutyric acid residues in a continuous-alternating sequence and is reported toxic to both mammalian and insect cell lines (159); fusaproliferin is a sesterterpen, toxic to both brine shrimp larvae and human B-lymphocytes cell line (160). Fusarochromanone, 2,2-dimethyl-5-a1nino-6-(3’-an~ino-4’-hydroxybutyryl)-chromo~~e, causes tibial dyschondroplasia in avian species and reduced hatchability in fertilized eggs (161, 162), while fusaric acid (5-butylpicolinic acid) is a common metabolite of several Fuscrrir4rz-l species (F. morzilifonne, F. o q s p o n ~ mF. , subglrltinnns, F. yrol(femtunz, F. solalzi, F. jidjikuroi, F. croolwellense, F. napiforme, F. lnteritium, F. thcrpsiszurn (10, 18, 20, 21, 122). Analysis of fusaric acid and its natural occurrence with other Fusnriunz toxins (i.e., ZEN, fumonisins, DON, other tricothecenes, and ergot alkaloids) has been reported (20, 21). Moniliformin (27, 154, 163) is an unusual cyclobutadione produced by a number of Fuscrri~rm isolates and toxicity data have been reported (27); it too has been isolated in conjunction with DON and ZEN (154). The simultaneous occurrence in foods and feeds of mycotoxins from Fusclriunz, Aspergillus, Pelzicillizm, Alternaria, and Clcrviceps has generated major concerns about the synergistic activity between the toxins and created a unique challenge in the screening, detection, and analysis of these compounds. In addition to the procedures outlined above for the isolation and identification of several analogs within a series of mycotoxins, Rava (148j employed a variety of GC/ECD, TLC, HPLC, and irnmunoassays to investigate the co-occurrence of DON, NIV, T-2 (analyzed by GC/ECD) (1 16, 164,165), ZEN,alternariol monomethyl ether (an Altermrin metabolite), and ochratoxin (a Penicillium toxin) (both analyzed by TLC) (166, 167), along with the fumonisin (analyzed by HPLC) (132) and aflatoxin B I (an Aspergillus metabolite, analyzed by immunoassay) (168). Analogously, Wang et al. (169) demonstrated the simultaneous analysis of NIV, DON, and T-2 (with GUMS), fumonisin BI, B2,and B3 (by HPLC with fluorescence detection), and aflatoxin B, (by ELISA). Frisvad (170) has reported a collective HPLC profile for Penicillium, Aspergillus, and Fusrrriurn polyketides, terpenes, and alkaloids; this procedure represents the separation of 134 secondary fungal metabolites with elution times between 1 and 34 min. For the individual analysis of most of the Pellicilliu~n, Asyergillu,~, and Alternaria toxins (ochratoxin, citrinin, patulin, penicillic acid, roquefortine, cyclopiazonic acid, verrucosidin, aflatoxins, sterigmatocystin, tenuazonic acid, alterariol, etc.), the reader is referred to Scott (4), Smith (5), Abramson (7), Panigrahi (8), Viscinti and Sibilia (9), Cole and Cox (27), Savard and Blackwell (28) and Turner and Aldridge (29).

V. SUMMARYANDCONCLUSIONS In addition to the discovery of mycotoxins in cereal grains and other foods and feeds, cheeses and milk, other dairy products, sausages, fruits and nuts, vegetables, and alcoholic beverages (i.e., beer, wine, and whisky) have all come under surveillance for mycotoxin contamination. Hence, a plethora of rapid, sensitive, and efficient mycotoxin screening

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kits have been developed, but these are only mentioned to give the reader an idea of other techniques available that would suggest mycotoxin contamination. Within the last decade, immunological techniques involving monoclonal and polyclonal antibodies, radioimmunoassay, and ELISA have gained wide acceptance with varying degrees of success depending on cross-reactivity and/or specificity of reactants and analytes, and a detailed prospectus is given in another chapter. The natural occurrence of saprophytic, parasitic, and endophytic plant fungi (both localized and systemic) and their evolutionary processes directed at species survival more than suggest an ecological justification for the production of previously referred to secondary metabolites or mycotoxins. Survival mechanisms (Le., physiological, reproductive, defensive, etc.) among these species, their economic significance to production, and their role in human and animal health underscores the importance of definitive analytical methodology for mycotoxins in our food and feed products. Environmental concerns to reduce the volume of herbicides and fungicides have precipitated a movement toward eliminating these practices and going to a no-till agricultural system. Subsequently, fungal infection and mycotoxin contamination of cereal grains, stored grain products, agricultural commodities, field crops, forages, and pasture grasses is a story without an end. However, with the continued judicious development of new analytical technology, advances in tnycotoxin research (i.e., the chemical isolation and identification and toxicology investigations) should provide avenues for understanding fungal-plant growth interactions and contribute to the developtnent of safer and more nutritious products for a global economy.

REFERENCES 1. Mycotoxic Fungi, Mycotoxins. Mycotoxicoses:An Encycolopedic Handbook. Vol.1. Mycotoxic Fmgi and Chemistry of Mycotoxins. T. D. Wyllie and L. G. Morehouse (Eds.). Marcel Defier, New York. 1977. An EncycolopedicHandbook.Vol. 2. 2. MycotoxicFungi,Mycotoxins.Mycotoxicoses: MJrotoxicoses of Domestic and Lnboratory Animals, Poult~y,and Aqrlatic Invertebrates and Vertebrates. T. D.Wyllie and L. G. Morehouse(Eds.).MarcelDekker. New York. 1977. 3. Mycotoxic Fungi, Mycotoxins, Mycotoxicoses:An Encycolopedic Handbook. Vol.3. Mycotoxicoses of Mail and Plcrnts: Mycotoxin Control and Regulatory Practices. T. D. Wyllie and L. G. Morehouse (Eds.). Marcel Dekker. New York. 1977. 4. Scott, P. M. 1994. Penicillirrr~zand Aspergillus toxins. Ch. 5, pp. 261-285. In: Mycotoxins irz Grain: Cor~~pozcnds Other Tlmn Aflatoxirl. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 5. Smith. J. E. 1997. Aflatoxins. Ch. 19. pp. 269-285. In: Handbook of Plant and Furrgal Toxicants. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton. FL. 6. D’Mello, J. P. F., Porter, J. K., MacDonald, A. M. C. and Placinta, C. M. 1997. Fusariunz mycotoxins. Ch. 20, pp. 287-301. In: Handbook of Plant and F~rngalTosicants. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton, FL. 7. Abramson, D. 1997. Toxicantsof the genus Penicillium. Ch. 21. pp. 303-317. In: Handbook of Plant arzd Fungal Toxicants. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton, FL. 8. Panigrahi, S. 1997. Altenzaria toxins. Ch. 32, pp. 319-337. In: Handbook of Plant am1 Fungal Tosicanrs. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton, FL. 9. Visconti, A. and Sibilia. A. 1994. Alternaria toxins. Ch. 7. pp. 315-336. In: Mycotoxim in Grairz:Compozrnds Other Than Aflatoxin. J. D. Miller andH. L. Trenholm (Eds.). Egan Press, St. Paul, MN.

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and Rotter, R. G. 1994. Toxicology of mycotoxins. Ch. 9, 10. Prelusky. D. B., Rotter, B. A. pp. 359-403. In: Mycotoxins in Grain: Corrtpounds Other Than AJlatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 11. Kuipe-Goodman, T. 1994. Preventionof human mycotoxicoses through risk assessmentand risk management. Ch. 12. pp. 439-469. In: Mycotoxins irt Grain: Cornpolrrzds Other. Than AJlatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 12. Beardall, J. M. and Miller, J. D. 1994. Diseases in humans with mycotoxins as possible causes. Ch. 14, pp. 487-539. In: Mycotoxins in Grain: Compounds Other Than Ajatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 13. Bove, F. J. 1970. The Stoiy of Ergot. S. Karger, New York. 14. Berde, B. and H. 0. Schild. 1978. Ergot Alkaloids and Related Contpounds. Handbook ESP. Pharmacology, Vol. 49. Springer-Verlag, New York. 15. Bandyopadhyay, R., Frederickson, D. E., McLaren, N. W. and Ryley, M. J. 1998. Ergot: a new disease threat to sorghum in the Americas and Austrailia. Plant Dis. 82356-367. C. Hancock, J. G. 1975. Biological control of ergot by Fusarzl16. Mower, R. L., Snyder, W. and ium. Phytopathology 65:5-10. 17. Bacon, C. W., Porter, J. K. and Norred, W. P. 1995. Toxic interaction of fumonisin B, and fusaric acid as measured by injection into fertile chicken eggs. Mycopatlrology. 129:29-35. 18. Bacon, C. W.. Porter, J. K., Norred. W. P. and Leslie, J. F. 1996. Production of fusaric acid by Fusarium species. Appl. Environ. Microbiol. 624039-4043. 19. Porter, J. K., Wray, E. W., Rimando. A. M.. Stancel, P. C., Bacon, C. W. and Voss, K. A. 1996. Lactational passage of fusaric acid from the feed of nursing dams to the neonate rat and effects on pineal neurochemistry in the F1 and F2 generations at weaning, J. Toxicol. Enriron. Health 50275-284. 20. Porter, J. K., Bacon, C. W., Wray, E. M. and Hagler, W. M. Jr. 1995. Fusaric acidFusariunl in rnonilifontte cultures, corn, and feeds toxic to livestockand the neurochemical effects in the brain and pineal glands of rats. Nut. Tosirts 3:91-100. 21. Porter, J. K., Bacon, C. W.. Kuldau, G., Wray, E. M. and Meredith, F. I. 1998. Alkaloids and other mycotoxins associated with ergot damaged sorghum. Proceedings National Conference on Sorgltum Ergot, Corpus Christi, TX, June 25-26, 1998. (c~http://MlwlMl.ars-griiz.Qov/ i~lav/coilfernc/pol?er.l2tnz). and biochemical interactionsof the fungal metabolites fusaric 22. Dowd, P. F. 1988. Toxicologic acid and kojic acid with xenobiotics in Heliotlu's zea and Spodopterct fiwgiperda (J. Smith). Pesticide Biochem Physiol. 32: 123-134. 23. Taylor, L. T. 1996.Supercritical Fluid Extraction: Techniques in Analytical Chernistry. Wiley, New York. 24. Byrne, L. T. 1993. Nuclear magnetic resonance spectroscopy: strategies for structural determination. Ch. 4, p. 75. In: Bioacti]*eNatural Products: Detection, Isolation, and Structurul Determination. S. M. Colegate and R. J. Molyneux (Eds.). CRC Press, Boca Raton, FL. 25. Wong, R. Y. and Gaffield. 1993. Determinationof three-dimensional structure and configuration of bioactive natural compoundsby X-ray crystallography. Ch.6. p. 125. In: Bioactise Natural Products: Detection, Isolation, and Structural Determination. S. M. Colegate and R. J. Molyneux (Eds.). CRC Press, Boca Raton. FL. 26. Gaffield, W. 1993. Determination of the absolute configuration of bioactive natural cotnpounds, utilizing exciton chirality circular dichroism. 7., Ch.147. In: Bioactive Natural Products: Detection, Isolation, and Structural Determination. s. M. Colegate and R. J. Molyneux (Eds.). CRC Press, Inc., Boca Raton, FL. 27. Cole, R. J. and Cox, R. H. 1981. Handbook of Toxic Fungal Metabolites. Academic Press, New York. 28. Savard, M. E. and Blackwell, B. A. 1994. Spectral characteristics of secondary metabolites from Fusarium fungi. Ch. 4, pp. 59-257. In: Mycoto.yins in Grain: Compo~~nds Other Than AJlatoxin. J. D. Miller and H. L. Trenholrn (Eds.). Egan Press. St. Paul. MN.

Analytical Methodology for Mycotoxins

675

29. Turner, W. B. and Aldridge, D. C. 1983. Fungal Metabolites. Academic Press, New York. 30. Hofmann. A. 1964. Die Mutterkornalkaloide. Enke Verlag, Stuttgart. 31. Floss, H. G. 1976. Biosynthesis of ergot akaloids and related compounds. Tetrahedron 32: 873-912. 32. Porter, J. K. 1994. Chemical constituents of grass endophytes. Ch. 8, pp. 103-123. In: Biotechnology of Endophytic Fungi of Grasses. C. W. Bacon and J. F. White, Jr. (Eds.). CRC Press, Boca Raton, FL. 33. Porter, J. K. 1997. Endophyte alkaloids. Ch. 4, pp. 51-62. In: Handbook of Plant and Fungal Toxicants. J. P. F. D'Mello (Ed.). CRC Press, Boca Raton, FL. 35. Thompson, F. N. and Porter, J. K. 1991. Tall fescue toxicoxes in cattle: could there be a public health problem. Vef. Hum. Toxicol. 3251-57. 36. Stuedemann, J. A. and Hoveland, C. S. 1988. Fescue toxicity: history and impact on animal agriculture. J. Prod. Agric. 1:39-44. 37. Hoveland, C. S. 1993. Importanceand economic significance of Acrenzonirrn? endophytes to performance of animals and grass plants. In: Acremonium/Grass Interactions. R. E. Joost and S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands. Agriculture, Ecosystems and Environment 44:3- 12. and Kerley, M. 1995. The effects of 38. Paterson, J., Forcherio, C., Larson, B., Samford, M. fescue toxicosis in beef cattle productivity. J. Anirn. Sci. 73:889-898. 39. Cheeke, P. R. 1995. Endogenous toxins and mycotoxins in forage grasses and their effects on livestock. J. Anim. Sci. 73:909-918. 40. Cross, D. L., Redmond, L. M. and Strickland, J. R. 1995. Equine fescue toxicosis: signsand solutions. J. Anim. Sci. 73:899-908. 41. Robbins, J. D., Porter, J. K. and Bacon, C. W. 1986. Occurrence and clinical manifestation of ergot and fescue toxicoses.In: Diagnosis ofMycotoxicoses. Ch. 6, pp 61-74. J.L. Richards and J. R. Thurston (Eds.). Martinus Nijhoff Publishers, Dordrecht, Netherlands. 42. Bacon, C. W., Lyons, P. C., Porter, J. K. and Robbins, J. D. 1986. Ergot toxicities from endophyte-infected grasses: a review. Agron. J. 78: 106-116. 43. Prestidge, R. A. 1993. Causes and control of perennial ryegrass staggers in New Zealand. In: Acremoniunz/Grass Interactions. R. E. Joost and S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands. Agriculture, Ecosystems and Environment 44:283300. 44. Cunningham, P.J., Foot, J. Z. and Reed, K. F. M. 1993. Perennial ryegrass(Loliumperenne) endophyte (Acrentoniunz lolii) relationships:theAustralianexperience. In: Acrentonium/ Grass Interactions. R. E. Joost and S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands.Agriculture, Ecosystents and Environment 44: 157- 168. In: Handbook of Naturally Occurring Food Toxicants. 45. Yates, S. G. Tall fescue toxins. 1983. pp. 249-273. M. Recheigl (Ed.). CRC Press, Boca Raton, FL. and Hanlin, R. T. 1996. Molecular phylogeny of Acrem46. Glenn, A.E., Bacon, C. W., Price, R. onium and its taxonomic implications. Mycologia 88:369-383. 47. Based on DNA sequence analyses,Acremonium coenophialu?n (Morgan-Jones & Gams), A. typhinunt (Morgan-Jones & W. Gams), A. lolii (Latch, Christensen& Samuels), A. chisosunt (J. F. White & Morgan-Jones), A. starrii (J. F. White & Morgan-Jones). and A. unicincrtum (W. Gams, Petrini & D. Schmidt) have been reclassified as: Neotyphodiurn coenopltialum-, N. typhinum-, N. lolii-, N. chisosum-, N.stcrrrii-, and N. unicinatum-Glenn, Bacon, & Hanlin comb. nov.. respectively. A dual system of nomenclature differentiates Neotyphodium and the sexually reproducing species classified in Epichloe. 48. Neotyphodiunt-Grass Interactions. C. W. Bacon and N. S. Hill (Eds.). 1997. Proceedingsof the Third International Symposium on AcremoniumlGrass Interactions, May 28-3 1, 1997, Athens, GA. Plenum Press, New York. 49. Porter, J. K. and Thompson, F. N. Jr. 1992. Effects of fescue toxicosis on reproduction in livestock. J. Anirn. Sci. 70:1594-1603.

676

Porter

and their metabolitesas extrinsic factors 50. Bacon, C. W. 1994. Fungal endophytes, other fungi, of grass quality. Ch. 8, pp. 318-366. In: Forage Qualio. Evnl~mtio~t a d Utilization. G. C. Fahey Jr. (Ed.). American Society of Agronomy, Madison, WI. of Epiclzloe typhirza and Acremo51. Schardle, C. L.and Siegel, M. R. 1993. Molecular genetics rzium coelzophialurn. In: AcrenzoniludGrass Interactions, R. E. Joostand S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands. Agriculture, Ecosystems and Enliroilment 44:169-185. 53. Perellino, N. C . , Malyszko, J., Ballabio, M., Gioia, B.and Minghetti, A. 1992. Direct biosynthesis of unnatural ergot alkaloids. J. Not. Prod. 55:424-427. 54. Perellino, N. C.. Malyszko. J., Ballabio, M., Gioia, B. and Minghetti.A. 1993. Identification of ergobine, a new natural peptide ergot alkaloid. J. Nat. Prod. 56:489-493. 55. Flieger, M., Wurst. M., Stuchlik, J. and Rehacek, Z. 1981. Isolation and separation of new natural lactam alkaloids of ergot by high performance liquid chromatography. J. Chronzatogr. 207:139-144. 56. Porter, J. K., Bacon, C. W., Meredith, F. I., Wray, E. M. and Wilson, J. P. 1998. G U M S analysis of the TMS-clavine alkaloids in Claviceps africana-infected sorghum from Georgia. 112'hAOAC International Annual Meeting and Exposition, Montreal, Quebec, Canada, Sept. 13-17, 1998. p. 65, A-201 (abstract). 57. Powell, R. G., Plattner. R. D., Yates, S. G., Clay, K. and Leuchtmann, A. 1990. Ergobalansine, a new ergot-type peptide alkaloid isolated from Cenchrus ecfzinatus (sandbur grass) infected with Balansia obtecta, and produced in liquid cultures of B. obtecta and Balansia cyperi. J. Nut. Prod. 53:1272-1279. 58. Yates, S. G., Plattner, R. D. and Garner, G. B. 1985. Detection of ergopeptine alkaloids in endophyte infected, toxic K-3 1 tall fescue by mass spectrometry/mass spectrometry.J. Agric. Food Chem. 33:719-722. 59. Yates, S. G. and R. G. Powell. 1988. Analysisof ergopeptine alkaloidsin endophyte-infected tall fescue. J. Agric. Food Chem. 36:337-340. 60. Lyons, P. C., Plattner, R. D. and Bacon, C. W., 1986. Occurrence of peptide and clavine ergot alkaloids in tall fescue grass. Science 232487-489. 61. Petroski, R. J. and Powell, R. J. 1991. Preparative separation of complex alkaloid mixture by high-speed countercurrent chromatography.In: Naturally Occurring Pest Bioregulmors. Paul A. Hedin (Ed.) Am. Chem. SOC. Symposium Series #449. p 326. 62. Plattner, R. D., Yates, S. G. and Porter, J. K. 1983. Quadrupole mass spectrometry/mass spectrometry of ergot cyclol alkaloids. J. Agric. Food Client. 31:785-789. 63. Porter, J. K., Bacon, C. W. and Robbins.J. D. 1979. Ergosine, ergosinine,and chanoclavine I from Epichloe typlzina. J. Agric. Food Chem. 27:595-598. 64. Porter, J. K., Bacon, C. W., Robbins.J. D. and Betowski, D. 1981. Ergot alkaloid identification in clavicipitaceae systemic fungi of pasture grasses. J. Agric. Food Clwm. 29:653657. 65. Rottinghaus, G. E.. Garner, G. B., Cornell. C. N. and Ellis, J. L. 1991. An HPLC method for quantitating ergovaline in endophyte-infected tall fescue: seasonal variationof ergovaline levels in stems with leaf sheaths,leaf blades and seed heads.J. Agric. Food Clzem. 39: 112115. 66. Shelby, R. A. and Fleiger. M. 1997. Analysisof ergot alkaloids in plantsand seeds of endophyte-infected tall fescue by gradient HPLC. Ch.50, pp. 271-273. In: Neot?;ol?odiroiz/Grass I~lterrrctions.C. W. Bacon and N. S. Hill (Eds.). Proceedingsof the Third International Symposium on AcrernoniunzlGrass Interactions, May 28-3 1, 1997, Athens, GA. Plenum Press, New York. 67. Groger. D., Erge, D. and Floss, H. G. 1968. Uber die herfkunft der seitkette D-lySergSaUreim methlcarbinolamide. Zeitschr. Nccturforsclt. B. 23B: 177-180. 68. Scott, P. M., Lombaert, G. A., Pellaers, P., Bacler, S. and Lappi, J. 1992. Ergot alkaloids in grain foods sold in Canada. J. AOAC Int. 75:773.

Analytical Methodology for Mycotoxins

677

69. Gamer, G. B., Rottinghaus, G. E., Cornell, C. N. and Testereci, H. 1993. Chemistry of compounds associated with endophyte/grass interaction: ergovaline- and ergopeptine-related alkaloids. Agric. Ecosyst. Erniron. 44:65-80. 70. Porter, J. K.. Bacon, C. W., Plattner, R. D. and Arrendale, R. F. 1987. Ergot peptide alkaloid spectra of Claviceps-infected tall fescue, wheat, and barley. J. Agric Food Clzem. 35:359-361. 71. Porter, J. K.. Bacon, C. W. and Robbins, J. D. 1974. Major alkalodis of a Claviceps isolated from toxic bermuda grass. J. Agric. Food Cl~ent.228384341. 72. Testereci, H., Garner, G. B., Rottinghaus, G. E., Cornel, C. N. and Andersen, M. P. A. 1990. Method for large scale isolation of ergovaline from endophyte-infected tall fescue seed (Festuca arzrndinacea). In: S.S. Quisenbeny and R. E. Joost (Eds.). Proc. Int. Symp.on AcremoniumlGrass Interactions, New Orleans, LA, Nov. 3, 1990. p 100. Louisiana Agricultural Experiment Station, Baton Rouge. 73. Moubarak, A. S., Piper, E. L., West, C. P. and Johnson, Z. B. 1993. Interaction of purified ergovaline from endophyte-infected tall fescue with synaptosomal ATPase enzyme system. J. Agl-ic. Food Cllenz. 41:407-409. 74. Belesky, D. P., Stuedemann, J. A., Plattner, R. D. and Wilkinson, S. R. 1988. Ergopeptine alkaloids in grazed tall fescue. Agron. J. 80:209-212. 75. Lyons, P. C., Evans, J. J. and Bacon, C. W. 1990. Effects of fungal endophyte Acrenzonizrm coenophialzrm on nitrogen accumulation and metabolism in tall fescue. Plant. Pltpiol. 92: 726-732. 76. Archavaleta, M., Bacon, C. W., Plattner, R. D., Hoveland, C. S. and Radcliffe, D. E. 1992. Accumulation of ergopeptide alkaloids in symbiotic tall fescue grown under deficits of soil water and nitrogen fertilizer. Appl. Emiron. Microbiol. 58:857-861. 77. Hill, N. S., Parrott, W. A. and Pope, D. D. 1991. Ergopeptine alkaloid production by endophytes in a common tall fescue genotype. Crop. Sci. 3 1:1545-1547. 78. Hill, N. S., Rottinghaus, G. E., Agee, C. S. and Schultz, L. M.. 1993. Simplified sample preparation for HPLC analysis of ergovaline in tall fescue. Crop Sci. 33:331-333. 79. Zhang, Q., Spiers, D. E., Rottinghaus, G. E. and Garner, G. B. 1994. Thermoregulatory effects of ergovaline isolated from endophyte infected tall fescue seed on rats. J. Agric. Food Chent. 42:954-958. 80. Scott, P. M. and Lawrence, G. A. 1980. Analysis of ergot alkaloids in flour. J. Agric. Food Cltem. 28:1258. 81. Christensen, M. J., Lane, G. A., Simpson, W. R. and Tapper, B. A. 1997. Leaf blade colonization by two Neotyphodium endophytes, and ergovaline distribution within leaves of tall fescue and meadow fescue. Ch. 23, pp. 149-151. In: C. W. Bacon and N. S. Hill (Eds.). Neot?lplzodiuliz/GI-nss Interactions. Plenum Press, New York. 82. Rottinghaus, G. E., Schultz. L. M., Ross, P. F. and Hill, N. S. 1993. An HPLC method for the detection of ergot in ground and pelleted feeds. J. Vet. Diagn. Intvst. 5:242. 83. Stahl, E. 1969. Tlzirt Layer Clzrornatograplty,A Laboratory Handbook, No. 73. pp. 127, 869. Springer-Verlag, New York. 84. Sprince, H. 1960. A modified Ehrlich benzaldehyde reagent for detection of indoles on paper chromatograms. J. Cltrontatogr. 3:97-98. 85. Michelon, L. E. and Kelleher, W. J. 1963. The spectrophotometric determination of ergot alkaloids. A modified procedure employing paradimethylaminobenzaldehyde. Lloydia 26: 192-201. 86. Porter, J. K. and Betowski, D. 1981. Chemical ionization mass spectrometry of the ergot cyclol alkaloids. J. Agric. Food Chent. 29:650-653. 87. Bianchi, M. L., Perellino, N. C., Gioia, B. and Minghetti, A. 1982. Production by Clmiceps purpurea of two new peptide ergot alkaloids belonging to a new series containing alphaaminobutyric acid. J. Nut. Prod. 45: 191-196. 88. Rowan, D. D. and Shaw, G. J. 1987. Detection of ergopeptine alkaloids in endophyte-infected perennial ryegrass by tandem mass spectrometry. NZ Vet. J . 35:197-198.

678

Porter

J. 1992.Theloline groupofpyrrolizidinealkaloids. In: 89. Powell,R.G. andPetroski,R. S. W. Pelletier (Ed.). The Alkaloids: Chemicaland Biological Persl>ectilyes. Vol. 8. pp. 320338. Springer-Verlag. New York. 90. Yates, S. G., Petroski, R. J. and Powell, R. G. 1990. Analysis ofloline alkaloids in endophyteinfected fescue by capillary gas chromatography. J. Agric. Food Clzem. 38:182-185. 91. Siegel, M. R., Latch, G. C. M., Bush, L. P., Fannin, F. F., Rowan, D. D., Tapper, B. A., Bacon, C.W. and Johnson. M. C.1900. Fungal endophyte-infected grasses: alkaloid accumulation and aphid response. J. Cltem. Ecol. 123301-3315. 92. Petroski, R. J., Dornbos D.L., Jr. and Powell, R. G.1990. Germination and growth inhibition of annual ryegrass (Loliunt mdtiJlorunz L.) and alfalfa (Medicngo sativa L.) by loline alkaloids and synthetic N-acetylloline derivatives. J. Agric. Food Clzerrz. 38:1716-1718. 93. Bush, L. P., Fannin, F. F., Siegel. M. R., Dahlman,D. L. and Burton, H. R. 1993. Chemistry, occurrence and biological effects of saturated pyrrolizidine alkaloids associated with endophyte-grass interactions. Agric. Ecosyst. Emiron. 44:8 1- 102. 94. Tepaske. M. R., Powell, R. G.and Petroski, R. J. 1993. Quantitative analysis of bovine urine and blood plasma for loline alkaloids. J. Agric. Food Chertz. 4 1 2 3 1-234. 95. Eichenseer, H.,Dahlrnan. D. D. and Bush, L. P. 1991.Influenceof endophyte infection, plant age and harvest interval on RhopaZosipl?uvz pndi survival and its relation to quantity of N-formyl and N-acetyl loline in tall fescue. Entomol. Esy. Appl. 6029-38. 96. Belesky, D. P.. Robbins, J. D., Stuedemann, J. A., Wilkinson, S. R. and Devine, 0. J. 1987. Fungal endophyte infection-loline derivativealkaloidconcentrationofgrazed tall fescue. Agron. J. 79:217-220. 97. Gallagher. R. T., Hawkes, A. D. and Stewart, J. M.. 1985. Rapid determination of the neurotoxin lolitrem B in perennial ryegrass by high-performance liquid chromatography with fluorescence detection. J. Cltromatogr. 321:217-226. 98. Weedon, C. M. and Mantle, P.M. 1987. Paxilline biosynthesisby Acrentorzizut loliae; a step toward defining the origin of lolitrem neurotoxins. Plzytochenzistv 26:969-971. 99. Miles, C. O., Wilkins, A. L.. Gallagher, R. T., Hawkes, A. D.,. Munday, S. C and Towers, N. R. 1992. Synthesis and tremorgenicityofpaxitrols and lolitriol: possiblebiosynthetic precursors of lolitrem B. J. Agric. Food Chem. 40234-238. 100. Fletcher, L. R., Garthwaite. I. and Towers. N. R. 1993. Ryegrass staggers in the absence of lolitrern B. In: D. E. Hume, G. C. M. Latch, and H. S. Easton (Eds.) Proc. 2nd Int. Symp. on Acrerrzortiur~t/Grass Interactions.Massey University, Feb. 4-6, 1993. p 119. AgResearch, Palmerston North, New Zealand. 101. Penn, J.. Garthwaite, I., Christensen, M. J., Johnson. C. M. and Towers, N. R. 1993. The importance of paxilline in screening for potentially tremorgenic Acrernortiun isolates. In: D. E. Hume. G. C. M. Latch, and H. S. Easton (Ed.) Proc. 2nd Int. Symp. on Acrentoniud Grass Interactions. Massey University, Feb. 4-6, 1993. p 88. AgResearch, Palmerston North, New Zealand. 102. Miles, C. O., Munday, S. C., Wilkins. A. L., Ede, R. M., Hawkes, A. D., Embling. P. P. and Towers, N. R. 1993. Large scale isolation of lolitrem B, structure determination of some minor lolitrems, and tremorgenic activities of lolitrem B and paxilline in sheep. In: D. E. Hume, G. C. M. Latch and H. S. Easton (Eds.). Proc. 2nd Int. Synlp. on Acremonium/Grass Interactions, Massey University, Feb. 4-6, 1993. p 85. AgResearch, Palmerston North,New Zealand. 103. Tapper, B. A., Rowan, D. D. and Latch, G. C. M. 1989. Detection and measurement of the alkaloid peramine in endophyte-infected grasses. J. Chromatogr. 463: 133-138. 104. Rowan, D. D. and Tapper, B. A. 1989. An efficient method for the isolation of peramine, an insect feeding deterrent produced by the fungus Acrmtonium lolii. J. Not. Prod. 52:193195. 105. Rowan, D. D. 1993. Lolitrems, peramine and paxilline: mycotoxins of ryegrass/endophyte interaction. Agric. Ecosyst. Erniron. 44: 103-122.

Analytical Methodology for Mycotoxins

679

106. Rowan, D. D.. Hunt, M. B. and Gaynor, D. L. 1986. Peramine, a novel insect feeding deterrent from lyegrass infectedwiththe endophyte Acrenroniunr loliae. J Chem. SOC.Clzem. COt?1ti1lc?z.935-936. 107. Fannin, F. F., Bush, L. P., Siegel, M. R. and Rowan, D. D. 1990. Analysis of peramine in fungalendophyte-infectedgrassesbyreversed-phasethin-layer chromatography. J. Cl11-0matogr. 503:288-292. 108. Kellerman. T. S., Marasas, W. F. O., Thiel, P. G., Gelderblom. W. C. A., Cawood, M. and Coetzer. J. A. W. 1990. Leukoencephalomalacia in two horses induced by oral dosing of fumonisin B1. Onderstepoort J. Vet. Res. 57:269-275. 109. Bezuidenhout, S. C., Gelderblom, W. C. A., Gorst-Allman, C. P., Horak. R. M., Marasas, W. F. 0..Spiteller, G.and Vleggaar, R. 1988. Structure elucidation of the fumonisins, mycotoxins from Fmariunz ~~orzilifornle. J. Chenr. SOC.Chem. Conrrmn. 743-745. 110. Norred, W. P. and Voss, K. A., 1994. Toxicity and role of fumonisins in animal diseases and human esophageal cancer. J. Food Protect. 57:522-527. 111. Gelderblom, W. C. A., Jaskiewicz, K., Marasas, W. F. O., Thiel, P. G., Horak,R. M., Vleggaar, R. and Kriek. N. P. J. 1988. Fumonisins-novel mycotoxins with cancer-promoting activity produced by Fusarium monilifornre. Appl. Errviron. Microbiol. 54: 1806- 18 11. 112. Youshizawa, T., Yamashita, A. and Luo, Y. 1994. Fumonisin occurrence in corn from highand low-risk areas for human esophageal cancer in China. Appl. Environ. Microbiol. 60: 1626. 113. Franceschi, S., Bidoli, E., Baron, A. E. and La Vecchia, C. 1990. Maize and risk of cancers of the oral cavity, pharynx, and esophagus in northern Italy. J. Cancer Inst. 821407. 114. Gelderblom, W. C. A., Kriek, N. P. J., Marasas, W. F. 0. and Thiel. P. G. 1991. Toxicity and carcinogenicity of theFusariwn moniliforme metabolite, fumonisin B 1, rats. in Carcinogenesis 12:1247-1251. 115. Marasas, W. F. O., Jaskiewicz, K., Venter, F. S. and van Schalkwyk, D. J. 1988. Fusarium monilifonne contamination of maize in oesophageal cancer areas in Transkei. S. A@. Med. J. 74:llO-114. 116. Thiel, P. G.,Marasas, W. F. 0.. Sydenham, E. W., Shephard,G. S. and Gelderblom, fumonisins in corn for W. C. A. 1992. The implicationsofnaturally-occurringlevelsof human and animal health. Mycopntlzologia 117:3-9. 117. Riley, R. T.. Voss, K. A.. Yoo, H.. Gelderblom. W. C. A. and Merrill,A. H. 1994. Mechanism of fumonisin toxicity and carcinogenesis. J. Food Protect. 57:638. 118. Riley, R. T., Norred, W. P. and Bacon, C. W. 1993. Fungal toxins in foods: recent concerns. Annu. Rev. Nzrtr. 13: 167-1 89. 119. Bacon, C. W. and Nelson, P. E. 1994. Fumonisin production in corn by toxigenic strains of Fmn-iunr lnorriliforme and Fusarimr proliferatum. J. Food Protect. 5 7 5 14-521. 120. Sydenham, E. W., Shephard, G. S., Theil, P. G., Marassas. F. O., Rheeder, J. P., Sanhueza, C. E. P., Gonzalez, H. L. and Resnik, S. L. 1993. Fumonisins in Argentinian field-trial corn. J. Agric. Food. Chem. 41:89 1. 121. Plattner, R. D., Norred, W. P., Bacon, C. W., Voss, K. A., Peterson, R., Shackelford, D. D. and Weisleder, D. 1990. A method of detection of fumonisins in corn samples associated with field cases of leukoencephalomalacia. Mycopatlzologia 82:698-702. 122. Drysdale, R. B. 1984. The production and significance in phytopathology of toxins produced by species of Fusar-iuw. In: The Applied Mycology of Fusarium, p. 95. M. 0. Moss and J. E. Smith (Eds.). Cambridge University Press, New York. Ch. 1, pp. 3- 18. 123. ApSimons, J. 1994. The biosynthetic diversity of secondary metabolites. In: Mycotoxins in Grain: Cortzpounds Other Than Ajlatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 124. Bacon, C. W.,Bennett,R.M., Hinton, D. M. and Voss, K. A.1992. Scanning electron microscopy of Fusarinnt moniliforrire within asymptomatic corn kernels and kernels associated with equine leukoencephalomalacia. Plalrt Dis. 76: 144-148.

680

Porter

125. Doko. M. B., Canet, C., Brown, N., Sydenham, E. W., Mpuchane, S. and Siame, B. A. 1996. Natural occurrence of fumonisins and zearalenone in cerals and cereal-based foods from Eastern and Southern Africa. J. Agric. Food Clzem. 44:3240-3243. 126. Gelderblom. W. C. A., Marasas, W. F. O., Thiel, P.G.. Veggaar, R. and Cawood, M. E. 1992. Fumonisins, chemical characterization and biological effect. Mycopathologia 117:11-1 4. 127. Meredith, F., Bacon C., Plattner, R. and Norred, W. 1996. Preparative LC isolation and purification of fumonisin B1 from rice cultures. J. Agric. Food Chem. 44:195-198. 128. Meredith, F., Bacon C.. Norred, W. and Plattner, R. 1997. Purification of fumonisin B2 isolated from rice culture. J. Agric. Food Chem. 45:3143-3147. 129. Holcomb, M., Thompson, H. C.. Jr. and Hankins, L. J. 1993. Analysis of fumonisisn B1 in rodent chow by gradient elution HPLC using precolumn derivatization with FMOC and fluorescence detection. J. Agric. Food Chem. 41:764-767. 130. Sydenham, E. W., Shepard, G. S., Thiel, P. G., Bird, C. and Miller. B. M. 1996. Determination of fumonisins in corn: evaluation of competitive inmunoassay and HPLC techniques. J. Agi-ic. Food Chem. 44: 159-164. 131. Sydenham, E. W.,Thiel, P. G., Shepard, G. S.. Koch, K. R. and Hutton, T. 1995. Preparation and isolation of the partially hydrolyzed moiety of fumonisin B 1. J. Agric. Food Chenr. 43: 2400-2405. 132. Sydenham E. W., Shepard, G. S. and Thiel, P. G. 1992. Liquid chromatographic determinations of fumonisins B,, B2, and B? in foods and feeds. J. Assoc. Off Anal. Chenz. Int. 75: 313-318. 133. Scott, P. M. and Lawrence, G. A. 1992. Liquid chromatographic determination of fumonisins with 4-fluoro-7-nitrobenzofurazan. J. Assoc. Off Anal. Clzem. 75:829-834. 134. Shepard, G. S.. Sydenham, E. W., Thiel, P. G. and Gelderblorn, W. C. A. 1990. Quantitative determination of fumonisins B1 and B2 by high-performance liquid chromatography with fluorescence detection. J. Liq. Chromatogr-. 13:2077-2087. 135. Stack M. E. and Eppley, R. M. 1992. Liquid chromatographic determination of fumonisins B1 and B2 in corn and corn products. J. Assoc. OffAnd. Chem. 75:834-837. 136. Cawood, M. E., Gelderblom, W. C. A., Vleggaar, R., Behrend, Y., Thiel, P. G. and Marasas, W. F. 0. 1991. Isolation of the fumonisin mycotoxins: a quantitative approach. J. Agric. Food C I I ~ I39: ? ~1958-1962. . 137. Vesonder, R., Patterson, R., Plattner, R. and Weisleder, D. 1990. Fumonisin B1: islolation from corn culture, and purification by high performance liquid chromatography. Myoto.xin Res. 85-88. 138. Meredith, F., Bacon C., Nolred,W. and Plattner, R. 1996. Isolation and purification of fumonisin B1 and B2 from rice culture. Ch. 10, p. 113-122. In: Fzlmonisins irz Food. L. Jackson (Ed.). Plenum Press, New York. 139. Rottinghaus, G. E., Coatney, C. E. and Minor, H. C. 1992. A rapid, sensitive thin layer chromatography procedure for the detection of fumonisin B, and B?. J. Vet. Dirrgn. Invest. 4:326-329. 140. Musser, S. M., Eppley, R. M., Mazzola, E. P., Hadden, C. E., Shockcor, J. P., Crouch, R. C. and Martin, G. E. 1995. Identification of an N-acetylketo derivative of fumonisin B1 in corn cultures of Fusarium prolifemtunz. J. Not. Prod. 58:1392-1397. 141. Meredith, F. I. 1999. Isolation and characterization of fumonisins. Vol. 311, pp. 361-373. In: Methods in Enzymology: Sphirlgolipid Metabolism and Cell Signaling. Y. A. Hannun and A. H. Merrill. Jr. (Eds.). Academic Press, Orlando, FL. 142. Haschek, W. M.and Haliburton, J. C. 1986.Fusarium nloniltforrneand zearalenone toxicoses in domestic animals: a review. Ch. 20, pp. 213-235. In: J. L Richards and J. R. Thurston, J. R. (Eds.). Diagnosis of Mycoto.vicoses. Martinus Nijhoff Publishers. Dordrecht, Netherlands. 143. Bennett, G. A., Shotwell, 0. L. and Kwolek, W. F. 1985. Liquid determination of a-zearalenol and zearalenone in corn: collaborative study. J. Assoc. Of Anal. Chem. 68:958-961.

Analytical Methodology for Mycotoxins

681

144. Kuiper-Goodman, T.. Scott, P. M.and Watanabe, H. 1987. Risk assessmentof the mycotoxin zearalenone. Regul. Toxicol. Pharnzacol. 7:253-306. 145. Ryu, J-C., Yang, J-S., Song, Y-S., Kwon, 0-S., Park, J. and Chang, I-M. 1996. Survey of natural occurrence of trichothecene mycotoxinsand zearalenone in Korean cereals harvested in 1992 using gas chromatography/mass spectrometry. Food Addit. Contam. 13:333-341. 146. Seo, J-A., Kim, J-C., Lee, D-H.and Lee, Y-W. 1996. Variations in 8-ketotrichothecenesand zearalenone production by Fusarium graminearum isolates from corn and barley in Korea. Mycopathologia 134:3 1-37. 147. Tanaka, T., Hasegawa. A., Matsuki, Y., Ishii, K., Ueno, Y. 1985. Improved methodology for the simultaneous detectionof trichothecene mycotoxins deoxynivalenoland nivalenol in cereals. Food Addit. Contam. 2125-137. 148. Rava, E. 1996. Mycotoxins in maize productsof the 1994/ 1995 marketing season. Mycotoxin Res. 1225-30. 149. Wang, D-S., Liang, Y-X., Chau, N. T., Dien, L. D., Tanaka, T. and Ueno, Y. 1995. Natural co-occurrence of Fusarium toxins and aflatoxin B1 in corn for feed in North Vietnam.Nat. Toxins 3:445-449. 150. Grabarkiewicz-Szczesna, J., Foremska, E. and Golinski. P. 1996. Distribution of trichothecene mycotoxinsin maize ears infected with F. gramirzearum and F. cookwellense. Mycotoxin Res. 12:45-50. 151. Rotter, B. A., Prelusky, D. B. and Pestka, J. J. 1996. Toxicology of deoxinivalenol (vomitoxin). J. Toxicol. Ernliron. Health 48: 1-34. 152. Rotter. B. A., Prelusky, D. B. and Thompson, B. K. 1996. The role of tryptophan in DONinduced feed rejection. J. Emyiron. Sci. Health B31(6):1279-1288. 153. Prelusky, D. B. 1994. The effect of deoxynivalenol on serotonergic neurotransmitter levels in pig blood. J. Emiron. Sci. Health B29(6):1203-1218. 154. Thiel, P. G., Meyer, C. J. and Marasas, W. F. 0. 1982. Natural occurrence of moniliformin together with deoxynivalenoland zearalenone in Transkeian corn.J. Agric. Food Clzem. 30: 308-312. 155. Snijders. C. H. A. and Perkowski, J. 1990. Effects of head blight caused by Fusariunz culmorum on toxin content and weight of wheat kernels. Phytopathology 80:566-570. 156. Perkowski, J., Kiecana, I. and Chelkowski. J. 1995. Susceptibility of barley cultivars and lines to Fmarizsm infection and mycotoxin accumulation in kernels. J. Phytopathol. 143: 547-555. 157. Miller, J. D., Savard. M. E., Sibilia. A.,Rapior, S., Hocking, A. D. and Pitt, J. I. 1993. Production of fumonisns and fusarins by Fusarium modiforrm from Southeast Asia.Mycologia 85:385-381. 158. Savard. M. E. and Miller, J. D. 1992. Characterization of fusain F, a new fusarin from Fusarium monilifonne. J. Nat. Prod. 55:64-70. 159. Gupa, S., Krasnoff, S. B., Underwood, N. L., Renwick, J. A. A. and Roberts. D. W. 1991. Isolation of beauvericin as an insect toxin from Fusarium semitectunt and Fusarium nroniliforme var. subglz4tinans. Mycopathologia 1 15:185-1 89. 160. Ritieni, A., Fogliano, V., Randazzo, G., Scarallo, A., Logrieco, A., Moretti, A., Mannina, L. and Bottalico, A. 1995. Isolation and characterization of fusaproliferin, anew toxic metabolite from Fusariwt proliferaturn. Nat. Toxins 3: 17. 161. Xie, W., Mirocha, C. J., Wen, Y., Cheong, W-J. and Pawlosky, R. J. 1991. Isolation and structure elucidation of four fatty acid derivatives of the mycotoxin fusarochromanone produced by Fusarium episeti. J. Agric. Food Chenz. 39:1757-1761. 162. Xu, Y., Mirocha, C. J. and Zie, W. 1993. Analysisby liquid chromatography of fusarochromanone (TDP-I) added to corn and wheat. J. Assoc. 08 Anal. Chem. Int. 77: 1 179-1 183. 163. Scott, P. M., Abbas, H. K.. Mirocha, C. J., Lawrence, G. A. and Weber, D. 1987. Formation of moniliformin by Fusarium sporotrichioides and Fusariurtz culrnorum. Appl. Emiron. Microbiol. 53: 196-1 97.

682

Porter

164. Scott, P. M., Kanhere, S. R. and Tarter, E. J. 1986. Determinationof nivalenol and deoxynivalenol in cereals by electron-capture gaschromatography.J. Assoc. Qfl Anal. Cltem. 64:961963. 165. Sydenham, E. W. and Thiel, P. G. 1987. The simultaneous determination of diacetoxyscirpenol and T-2 toxin in fungal cultures and grain samples by capillary gas chromatography. Food Addit. Contant. 4:277-284. 166. Sydenham, E. W., Thiel, P. G. and Marasas, W. F. 0. 1988. Occurrence and chemical determination of zearalenone and alternariolmonomethyl ether in sorghum based mixed feeds associated with an outbreak of hyperestrogenism in swine. J. Agric. Food Chent. 36:621625. 167. Roberts, B. S., Glancy, E. M. and Patterson, D.S.P. 1981. Rapid and economical method for determination of aflatoxin and ochratoxin in animal feedstuffs. J. Assoc. 08 Anal. Chent. 64:961-963. 168. Trucksess, M. W., Stack, M.E., Nesheim, S., Page, S. W. and Albert, R. A. 199 1. Immunoaffinity column coupled with solution fluorometry or liquid chromatography postcolumn derivatization for the determination of aflatoxins in corn, peanutsand butter: collaborative study. J. Assoc. 08 Anal. Chem. 74:81-88. 169. Wang, D-S., Liang, Y-X, Chau, N. T., Dien, L. D., Tanaka, T. and Ueno, Y. 1995. Natural co-occurrence of Fusariun? toxins and aflatoxin B1 in corn for feed in North Vietnam. Nat. Toxins 3:445-449. 170. Frisvad. J. C. 1987. High performance liquid chromatographic determination of profiles of mycotoxins and other metabolites. J. Chrornatogr. 392:333-347.

17 Mycotoxin Analysis: Immunological Techniques Fun S. Chu Urlillersi@of Wiscorzsin-Madisorz, Madison, Wisconsin

684 I. Introduction 11. GeneralConsiderations684 A. Availability of antibody and markers 685 B. Adequate method for the separation of free and bound toxin 685 C.Understanding the specificity of the antibodies used in the assay 685 D. Understanding the effect of matrix and solvent system on the assay 686 111. Radioimmunoassay 687 A. B. C. D.

Principles 688 Preparation of radioactiveligands used in the RIA of mycotoxins688 Separation of Ab-mycotoxincomplexfromfreemycotoxins688 Application of RIA formycotoxins689

IV. Enzyme Itnmunassay A. B. C. D.

689

General considerations and assay configurations 689 Direct competitive ELISA (dc-ELISA) 690 Indirectcompetitive ELISA (idc-ELISA or double-antibodyELISA) Considerations of sampletreatment in the ELISA692

691

V. ImmunoscreeningMethods693 VI. Complementation of Immunoassays with Chemical Methods 694 A. Immunoaffinity chromatography 694 B. Combination of immunoassay with other chemical methods

695

VII. Other Newly DevelopedImmunochemicalMethods696 A. Anti-idiotype antibody-based immunoassays 696 B. Immunoassay for mycotoxin-producing fungi 697 C. Development of biosensors 697 VIII. Concluding Remarks 699 References 700

683

684

1.

Chu

INTRODUCTION

Mycotoxins are low-molecular-weight, secondary metabolites produced by naturally occurring fungi (1-8). Since the discovery of aflatoxins in the early 1960s (1, 6), developments in the last three decades have disclosed many new fungal poisons that are attracting attention because of their high toxicity and their association with foods and animal feeds (1-8). The presence of mycotoxins in foods and feeds is potentially hazardous to human and animal health. To decrease the risk of human exposure to mycotoxins, a rigorous program has been established for monitoring these toxins in foods. Governments in most countries have established limits for the levels of a number of mycotoxins that are permissible in foods and feeds and official methods of analysis for many mycotoxins have also been established (9, 10). However, because only trace amounts of the toxin are present in the sample, analysis of mycotoxins in foods is difficult. Research attempting to develop more sensitive, specific, and simple methods for mycotoxin detection has been done over the years. Rapid progress in the area of mycotoxin analysis has been made during the last few years (1 1- 16). The progress of such research can be seen from the numerous papers cited by the "General Referee on Mycotoxins," which appears in every year's January/ February issue of the J o w m l of the Association of OfJicial Analytical Chemists, Intermtioml (JAOAC Int.) (e.g., 17-20), and in several recent reviews and books including a separate chapter in this book. As has been discussed in another chapter, extensive sample cleanup treatment is needed for most chemical methods for mycotoxin analysis and they are very time consuming and need expensive instruments. Although several biological methods are available, most of these methods are nonspecific and relatively insensitive. To overcome the difficulties encountered with the chemical and biological methods, new immunochemical methods have been developed (21-37). Within the last few years, wide application of immunoassays for mycotoxins has been noted (22, 24-28, 36, 37). There has been a rapid increase in publications in this area. For example, in the 1991 and 1994 AOAC mycotoxin-associated referee reports, as many as 25-28% of the cited publications on mycotoxin analysis were immunoassay-related articles. More than 30% ofpublications on the analytical methods for mycotoxins now involve immunochemical techniques that either serve as a cleanup step or are used directly as a screening method or for quantitation (17-20). Many immunoassay kits for mycotoxin analysis are also commercially available (14, 22, 24, 36, 37). The application of mycotoxin immunoassay is not limited to foods and feeds: it has been used as a sensitive approach to monitoring mycotoxins in body fluids and tissues or organs of humans and animals that have been exposed to the mycotoxins (14, 24, 36). Thus, a new dimension of methodology for mycotoxin analysis as well as a new tool for diagnosis of mycotoxicoses in humans and animals has emerged since we first initiated a research project to develop an immunoassay for mycotoxins in the early 1970s (21-23, 29, 31). In this chapter, the general principles and recent applications of immunoassay for mycotoxins will be discussed. For a detailed discussion on immunoassays and earlier literature, several of the most recent reviews should be consulted (14, 23, 26, 30-36). For the specificity of antibodies against various mycotoxins, see Chu (14, 24, 26, 28).

II. GENERALCONSIDERATIONS

Immunochemical methods are based on the specific interaction between antibodies and the toxin. However, mycotoxins are low-molecular-weight compounds and they are not

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685

immunogenic. Like most other natural products, mycotoxins must first be conjugated to a protein/polypeptide carrier before subsequent use in immunization for antibody production (14, 31, 23, 24, 26-28). Extensive studies have been done on the development of methods of conjugation of mycotoxins to a protein or polypeptide carrier and optimization of conditions for antibody production in rabbits and other animals (polyclonal antibodies). Newer innovative approaches for the coupling of mycotoxin to marcomolecule have been developed in recent years (28). With the advances in hybridoma technology, monoclonal antibodies against many mycotoxins were also made. Antibodies against almost all the important mycotoxins have been made available. Many types of immunoassays, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA), as well as several novel immunochemical screening tests, have been developed. Most of these methods are very sensitive, specific, and simple to operate. Specific antibodies have also been used as immunohistochemical reagents and to arm affinity columns that are used as a cleanup tool for analysis of mycotoxins by other methods. Before selecting appropriate immunoassay for mycotoxin analysis, the following criteria should be considered: A.

Availability of Antibody and Markers

Both monoclonal and polyclonal antibodies against mycotoxins have been generated. Whereas most polyclonal antibodies were generated in rabbits, useful antibodies have also been obtained from eggs of hen, goat, and pigs as well as mice (28). Antibodies against different mycotoxins are summarized in Table 1 (38-90). Because most immunoassays for mycotoxins are based on the competition of binding between unlabeled toxin in the sample and labeled toxin in the assay system for the specific binding sites of antibody molecules, a well-labeled mycotoxin (as a marker) is needed in the assay system, in addition to a specific cmtibody. Approaches for the preparation of conjugates for antibody production and for the preparation of markers have been reviewed by the author (24, 26, 28).

B. Adequate Method for the Separation of Free and Bound Toxin For accurate quantification of the interaction between antigen and antibody, a good method for the sepnrntion of free and bound forms of toxin is important (28, 36).

C. Understanding the Specificity of the Antibodies Used in the Assay Depending on the approaches that have been used for raising antibodies, the degree of cross-reactivity (speciJiciv)of these antibodies with their respective structural analogs varies considerably; thus, one must be familiar with the specificity of the antibody to be used in the assay system (24, 26, 27). The cross-reactivity of antibody is determined by an immunoassay where various structurally related analogs of mycotoxin at a wide range of concentrations are used to compete with the binding of the marker ligand with the antibody in the assay. The concentration at 50% inhibition (IC5")of the binding is generally used as the basis to calculate the relative cross-reactivity for each analog. A typical example of such a competitive RIA and enzyme-linked immunoassay for aflatoxins is shown in Fig. 1. The cross-reactivity of various antibodies against mycotoxins is generally described alone in the publications for the production of specific antibodies against mycotox-

Chu

686

Table 1 AntibodiesAgainst

Mycotoxins3

Mycotoxins AAL toxin Aflatoxins: B1, B2, G1, G2 Aflatoxin metabolites: B2a. Q1, M1 aflatoxicol, DNA adducts Citrinin Cyclopiazonic acid Ergot alkaloids Fusarochromanone Fumonisin Kojic acid Ochratoxin A Patulin Paxilline related PR-toxin Rubratoxin B Secalonic acid Sporidesmin Sterigrnatocystin Trichothecenes: DAS, DON, FX. DOVE, AcDON, NIV, Roridin A, T-2 toxin, and T-2 toxin metabolites (HT-2, T-2-tetraacetate 3’-OH-T-2, dep-T-2) Versicolorin A Zearalenone a

Type PAb mAb, pAb mAb, pAb PAb mAb, pAb mAb, pAb PAb mAb, pAb PAb mAb, pAb PAb mAb, pAb PAb PAb PAb mAb, pAb PAb mAb, pAb

PAb mAb, pAb

References for most mycotoxins: see Chu(14,34,26,27,28). Addltlonal selected references: AAL toxin (38-40). AFB (41, 42). citrinin (43, 34).cyclopiazonic acid (CPA, 45-47), DON and acetyl-DON (48-54), ergot alkaloids (55-59), fumonisin (Fm; 37.60-70), fusarochromanone (71 ), OA (72-741, patulin (75). paxilline related (76-78). sporidesmin (77-80). sterigmatocystin (ST: Sl-83),nivalenol (84), T-3 toxin (85, 86). versicolorin A (S7). zearalenone (ZE: 85, 88-90). AF,aflatoxin: DAS. diacetoxyscirpenol:DON.deoxynivalenol;AcDON,Acetyl-DON:DOVE,deoxyverrucarol: FX, fusarenon-X: OA, ochratoxin A: NIV, nivalenol; dep-T-2, deepoxide T-3 toxin;mAb,monoclonalantlbody:pAb,polyclonal antibodies.

ins; several reviews have been appeared (24,26,36). Whereas antibodies against mycotoxins are very specific for each specific mycotoxin used in the immunizations, antibodies reactive with a group of mycotoxins, including aflatoxins and trichothecenes, have been also been made (91-96). D. Understanding the Effect of Matrix and Solvent System on the Assay Because there is always a possibility of the presence of some structurally related compounds in the sample that may react with the antibody, the sample Inatrix should be tested

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LOG TOXIN CONCENTRATION (nglmL)

Figure 1 Examples of radioimmunoassay (RIA) and enzyme-linked immunosorbent (ELISA) of selected mycotoxins. (Top) The cross-reactivity of pAb with different aflatoxins using tritiated aflatoxin B1 as the marker. (Bottom) Typical standard curveof competitive-direct ELISA for selected mycotoxins and the respective mycotoxin-HRP conjugate was used as the marker in each case.

before the assay. In most of the immunoassays described below, sample cleanup is not necessary. Sample after extraction from the solid matirx could be directly used in the assay after appropriate dilution in the assay buffer. Nevertheless, the sensitivity increased after appropriate cleanup treatment.

111.

RADIOIMMUNOASSAY

The RIA procedure involves incubation of specific antibody simultaneously with a solution of unknown sample or known standard, and a constant amount of labeled toxin. After separation of the free and bound toxin, the radioactivity in those fractions is then determined. The toxin concentration of the unknown sample is determined by comparing the results to a standard curve that is established by plotting the ratio of radioactivities in the bound fraction and free fraction versus log concentration of unlabeled standard toxin.

Chu

688

A.

Principles

Radioimmunoassay involves the use of a radioactive marker, which competes with analyte in the sample for binding to an Ab. For RIA of large-molecular-weight antigen, either the antigen or antibody molecules can be labeled. It is also comtnon to use a radiolabeled second antibody, i.e., antibody against the primary antibody. In contrast, labeled mycotoxin is typically used in RIA of mycotoxins. Although RIA is simple and sensitive, it is limited by the need of a marker with high specific radioactivity, instruments for measuring radioisotopes, licenses for using radioactive materials, and disposal of radioactive materials. Because the radioactive marker has the same structural features as the compound to be analyzed, RIA provides good accuracy and is an effective method in the initial phase for screening of antibodies.

B. Preparation of Radioactive Ligands Used in the of Mycotoxins

RIA

In addition to the affinity constant of the Ab and Ag interaction, the specific activity of the radioactive ligand plays an essential role in the sensitivity of RIA. Although mycotoxins labeled with I4C, 'H, and '"1 have been used in RIA, the 'H-labeled toxins are most commonly used. Some high-specific-activity mycotoxins, including aflatoxin B 1(AFB 1) and ochratoxin A (OAj,are made commercially by a tritium exchange method (24). Other mycotoxins, including those in the trichothecene (TCTC) group, are made by reduction with high specific 'H-NaBH,. This was generally done by first oxidizing the secondary hydroxyl group and followed by reduction with'H-NaBH, (24j. Iodinated mycotoxin markers have also been made by first preparing a mycotoxin derivative containing a tyrosine, histamine, or tyramine. and then iodinating with "'1 using the standard methods such as Bolton-Hunter reagent, chloramine T, iodogen, lactoperoxidase, and iodo-beads, which are commercially available. For example, 3'-''sI-tyramine-AFB 1-0-carboxymethyl oxime (2300 Ci/nmol) was used in the RIA of both AFB 1 and AFM1 with high sensitivity (10 pg per assay) (31, 26, 32).

C. Separation of Ab-Mycotoxin Complex from Free Mycotoxins

Many methods have been used for the separation of the free and bound mycotoxins after incubation. In the earlier studies, methods such as equilibrium dialysis, amtnoniutn sulfate precipitation, precipitation with organic solvent, polyethylene glycol 6000, membrane filtration, dextran-coated charcoal, and albumin-coated charcoal have been used. As newer solid-phase matrix and more immunochemical reagents became available, antibodies could be coated noncovalently to a solid matrix such as polystyrene tubedbeads, microtiter plate, or modified nylon tubes or beads, or conjugated covalently to various matrices such as acetylbromo-cellulose, various types of Sepharose or agarose gels, controlled-size fineparticle magnetic gels, and others. Separation is achieved by filtration or centrifugation after the reaction. A second antibody has also been used to separate the free antigen and immunocomplex either by the formation of precipitate with the primary antibody or by coupling to a solid matrix (21, 26, 32).

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D. Application of RIA for Mycotoxins Although RIA was developed in the early phase of immunochemical studies, this method is still used in some laboratories. The method is very simple. In general, the antibody either in the solid-phase or in solution is incubated together with the radioactive-labeled mycotoxin and the sample solution for an appropriate time and then one of the methods to separate the free and bound mycotoxin is used. The radioactivity in the solution, usually the free-form fraction, is then determined. RIA has been used for the analysis of AFB 1 in corn, wheat, peanuts, milk, serum, and eggs as well as for deoxynivalenol (DON) in corn and wheat, OA in serum and kidney, nivalenol in barley, PR toxin in cheese, and T-2 toxins in corn, wheat, serum, and urine (21, 23, 24, 28). Generally, RIA can detect 0.25-0.5 ng of purified mycotoxin in each analysis when tritiated mycotoxins are used as the markers. However, because of the sample matrix interference, the lower limit for mycotoxin detection in food or feed samples is about 2-5 ppb. Higher sensitivity, 0.0040.1 ng/assay, can be achieved by using iodinated-mycotoxin marker. The sensitivity of RIA can also be improved by a simple cleanup procedure after extraction and by using radioactive markers of high specific activity (24). As newer solid-phase matrices and more immunochemical reagents became available, more efficient methods for the separation of free and bound toxin were developed (24). Thus, separation can be achieved by a simple filtration or centrifugation step. A RIA-based immunoassay kit for mycotoxin analysis is commercially available (33, 96).

IV.

ENZYMEIMMUNOASSAY

A.

General Considerations and Assay Configurations

Enzyme immunoassay (EIA) is ageneral term for the immunoassays involving use of an enzyme as a marker for the detection of immunocomplex formation. Whereas the general principle of EIA is similar to that of RIA, there is an amplification system present in this assay, and thus, EIA is more sensitive. Since no radioactive substances are used, this assay avoids the problems encountered in handling radioactivity. Enzyme labeling can be done by conjugation of enzyme to Ag or Ab via the periodate oxidation and subsequent reductive alkylation method or by cross-linking using glutaraldehyde. Some of the methods used in conjugation of hapten to proteins can also be used. Although horseradish peroxidase (HRP) and alkaline phosphatase are the two enzymes most commonly used, others, such as glucose-6-phosphate dehydrogenase coupled with oxidoreductase and luciferase, glucose oxidase, beta-galactosidase, and urease, have also been used (24, 26, 28, 37). Depending on whether or not the immunocomplex is separated from the free Ag, EIA is further divided in two types. One type is a homogeneolrs system, which is based on modification of enzyme activity occurring when Ab binds with the enzyme-labeled Ag/hapten in solution. No separation is necessary in this assay. This system, which is also called ewyme multiplied im~nunonssny(EMIT), has been used for analysis of some antibiotics and hormones in theclinical diagnosis area. Because modification of enzymatic activity generally is not significant, this system is not very sensitive (pg/ml to mg/ml range) and has not been widely used in food analysis. The other is a hete~-ogeneoussystem involving separation of free and bound Ag-Ab. In this system, either Ag or Ab is bound to the solid matrix noncovalently or conjugated to it covalently. Unreacted Ab or Ag/hapten is easily removed by washing or centrifugation.

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The term etz:yme-lird-ed i~nrr1ur~osol-bent assay (ELISA) is used for this type of assay and this system is widely used for analysis of a variety compounds, including mycotoxins. Solid phases such as tnicrotiter plates, cellulose, nylon beaddtubes, nitrocellulose membrane, polystyrene tubedballs, and modified magnetic beads have been used. In some cases, staphylococcal protein A or protein G is coated on the solid surface, entrapping the antibody for subsequent analysis. ELISA is further divided into two major types. One type is cotnpetitive ELISA (c-ELISA), which can be used for the analysis of both hapten and macromolecule; the other is noncompetitive sandwich ELISA, which is used only for divalent and multivalent Ag. c-ELISA is used most frequently for mycotoxin analysis; therefore, only this method will be discussed in detail. Depending on whether enzymelabeled Ag or Ab is used or whether Ab or Ag is coated to the solid phase, several types of competitive ELISA have been developed. Two major types, Le., direct competitive ELISA (dc-ELISA) and indirect conlyetitive ELISA (idc-ELISA), are used most commonly in mycotoxin analysis.

B. DirectCompetitive ELSA (dc-ELISA) In this assay, specific antibodies against mycotoxins are coated on the ELISA plate. The sample or mycotoxin standard solution is generally incubated simultaneously with enzyme-conjugate or incubated separately in two steps. The amount of enzyme bound to the plate is then determined by incubation with a chromogenic substrate solution. The resulting color/fluorescence, which is inversely proportional to the mycotoxin concentration present in the sample, is then measured instrumentally or by visual comparison with the standards. In this assay, the mycotoxin-enzyme conjugate (marker) and free mycotoxin compete for the same binding site on the solid-phase antibody. Although HRP is most commonly used as the enzyme for conjugation, other enzymes such as alkaline phosphatase and beta-galactosidase also have been used (14, 15, 21, 34, 26-28, 32-37). Excluding the time for sample preparation, dc-ELISA itself generally can be completed in 0.5-2 hr. In general, dc-ELISA is approximately 10-100 times more sensitive than RIA when purified standard is used and as little as 2.5 pgof pure mycotoxin can be measured. Since a cleanup step is usually not necessary, many samples can be analyzed within a relatively short period. dc-ELISA can detect 0.05-50 ppb of mycotoxins in foods and feeds (14, 15, 28). Like RIA, the sensitivity of ELISA is improved when a cleanup treatment is included in the assay protocol (24). Many examples could be cited. For example, Hongyo et al. (97) found a good correlation between the data obtained from a one-step ELISA of aflatoxin in corn with either HPLC or TLC, but the correlation between the ELISA data for AF in the mixed feed with HPLC and TLC was poor. In contrast, a good correlation was obtained when the mixed feed was subjected to column chromatography before ELISA. The efficacy of ELISA of fumonisin was also improved after a cleanup treatment of the samples (37, 63, 65, 98).We also found that the sensitivity of an ELISA of cyclopiazonic improved considerably when the samples were subjected to an immunoaffinity column (99). Owing to the use of better antibody and toxin-enzyme conjugates, the time required to run the ELISA has improved considerably. Thus, the entire ELISA procedure can be completed within 1 hr (24, 28, 33, 36, 37, 100). Better sensitivity can also be achieved by variation of substrate as well as by using amplification systems such as the biotinavidin interactions. For example, a more sensitive substrate, such as tetramethylbenzidine,

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has been used for immunoassays using HRP as the marker enzyme (63) and fluorescent substrates have also been used to improve sensitivity. Thus, it is not surprising that some systems can detect as low as 0.05 pg in each assay. To save antibodies, Pesavento and Carter (101) have covalently conjugated antibodies against aflatoxin to the chemically activated hydrophilic membrane in an ELISA plate, which can be regenerated a number of times for repeated aflatoxin analysis. dc-ELISA is one of the most common protocols currently being used for immunoassay of mycotoxins. The sensitivity of dc-ELISA for mycotoxins in different commodities is summarized in Table 2 (102- 118). C.

Indirect Competitive ELSA (idc-ELISA or DoubleAntibody ELISA)

In the indirect competitive (idc-ELISA), a mycotoxin-protein (or polypeptide) conjugate is first prepared and then coated to the microtiter plate before assay. The plate is then incubated with specific rabbit (or other type) antibody in the presence or absence of the homologous mycotoxin. The amount of antibody bound to the plate coated with mycotoxin-protein conjugate is then determined by reaction with goat anti-rabbit (or anti-other type) IgG-enzyme cotnplex (which is commercially available) and by subsequent reaction with the substrate. Thus, toxin in the samples and toxin in the solid phase compete for the same binding site with the specific antibody in the solution. The idc-ELISA has also

Table 2 Sensitivity of Direct, Competitive ELISA for Selected Mycotoxinsa Standard Detection range limits Mycotoxins AAL AFB/AFs AFM CPA DON 3-AcDON 15-Ac-DON Fm H-Fm OA T-2 Type A TCTC ZE

Fooddfeeds (nglassay) C C, Wh, P, Pb M, Ch C, P, MF C, Wh B Wh C, MF, M, BR C Wh, B, K C, Wh C C

0.0025- 1 0.0012-1 0.1-0.6 0.012-125 0.0002-0.4 0.002-0.025 0.0005-50 0.1-15 0.025-0.5 0.0025-0.2 0.0025-0.2 0.02-2.5

(pg/kg) or (pg/l) 1-10 (1)b 0.10 (0.01) (0.05-0.1) 1000 (10) 16 50-100 10-500 (5-10) 30 (1-2) 2.5-50 (1) 50- 100 50 (10)

Data and references: see Chu (14, 27, 28). Additional selected references: AAL (40); AFs (102); AFM (103); CPA (104); DON (105-107); 3-Ac-DON (54); 15-Ac-DON (53); Fm (66, 67.70, 108-1 14); OA (1 15-1 17); ZE (1 18). Values in parentheses are for samples that had been subjected to a cleanup treatment before immunoassay. B, barley; BR, beer; C, corn; Ch, cheese; Fm, fumonisin; H-Fm, hydrolyzed Fm; K. kidney; M, milk; MF. mixed feed;P. peanuts; Pb, peanut butter; Wh, wheat;OA,ochratoxin A; Tctc.trichothecenes;otherabbreviations,see Table 1.

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been widely used for analysis of a number of mycotoxins (14, 15, 21, 24, 27,28,36) with a sensitivity that is comparable to or slightly better than that of the direct ELISA in some cases. This type of ELISA requires less antibody (100 times less) and does not require preparation of a toxin-enzyme conjugate. However, it takes more analytical time (2 hr). To optimize the assay, selection of secondary antibody-enzyme conjugate in the idc-ELISA is important. In a recent study. for example, Okumura et al. (119, 120) found that their mAb-based ELISA for AFM1 was 50 times more sensitive in the HRP-labeled anti-mouse antibody than in the alkaline phosphate-labeled system. The sensitivity of idc-ELISA of ZE improved considerably when affinity-purified mycotoxin-conjugate was used as the coating antigen together with a flourescent substrate in the assay (118). To shorten the assay time for the idc-ELISA, two modifications were made by several investigators. One involved the conjugation of antibody to an enzyme, which is then used in the ELISA instead of a second antibody-enzyme conjugate, and the other involved premixing the antibody with the second antibody-enzyme conjugate before the assay (24, 36, 121, 122). The application and sensitivity of idc-ELISA for mycotoxins in different commodities are shown in Table 3 (123-128). D. Considerations of Sample Treatment in the ELISA In addition to the antibody affinity, efficacy of marker enzymes, enzyme substrate, and sample matrices, the presence of an extraction solvent system also greatly affects ELISA performance. Early studies showed that ELISAs could run in a system containing as much Table 3 Sensitivity of Indirect Competitive ELISA for Selected Mycotoxinsa ~~

Mycotoxins idc-ELISA AFs AFM DAS DON 3-Ac-DON Fm NIV OA ST T-2 HT-2 ZE Md-idc-ELISA AFB T-2

Foods/Feeds

C, P, Pb, F. Rs. S M c, w h c, Wh R C B B. K. MF, Wh

~

Standard limits Detectionrange (&assay)

M, S, U U C, W, MF

0.0002-1 0.0001-0.005 0.005-5 0.010-100 0.005-1 1-100 0.05-5 0.005-10 0.000 1-5 0.002-0.2 0.005-0.5 0.05-2.5

B, P. Pb, C B

0.01-1.5 0.05-5

s, u, w

~~~~

~

~~

( u g k g ) or ( u g L )

0.25-5 0.005 300 1000 (10) 1 200 (50) (30) 0.06-50 (1) 0.01-5 (0.05) 5 (0.2-1 ) (0.5) 1-60

Data and references: see Chu (14. 37, 28). Additional selected references: AF( 123-125); Ac-DON (54):FmB (68,136): ST (83): T-3 (127.128). Values i n parentheses are for samples that had been subjected to a cleanup treatment before immunoassay. B. barley; C, corn; Ch, cheese: F, figs; K. kidney; M, milk; MF. mixed feed; P, peanuts; Pb, peanut butter; R. rye; Rs, raisins; S, serum; Wh, wheat; U, urine; Md, modified; other abbreviations. see Tables 1 and 2.

Mycotoxin Analysis

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as 20-30% of methanol (21, 24). Generally, samples contain 7-15% methanol in the phosphate buffer. In the ELISA of hydrolyzed fumonisin BI, Maragos and Miklasz (65) found that more dilution was necessary for corn samples extracted with acetonitrile than those extracted with methanol. This effect could be due to the solvent itself and also because more interfering materials were extracted by the acetonitrile. In a mAb-based ELISA for FmB1, we found that the presence of either 10% methanol or acetonitrile did affect the assay significantly (27, 28, 70). A number of studies have been carried out in recent years to investigate the efficacy of both direct and indirect immunoassays by comparing them with HPLC and TLC. Whereas good correlation has been found in most immunoassays (14, 21, 24, 28, 36), problems do exist for some other assays. For example, data obtained from immunoassay of Fnls were always higher than those obtained from chemical analysis (27, 112). This problem was attributed to the cross-reaction of the antibodies with some structurally related compounds. Once high-affinity antibodies became available, the nonspecific interaction was minimized (66-70). Collaborative studies for some ELISA protocols have been conducted. Several quantitative ELISA methods for the analysis or screening of mycotoxins have been adopted as first action by the AOAC (15-20).

V.

IMMUNOSCREENINGMETHODS

By shortening the incubation time and adjusting the antibody and enzyme concentrations in the dc- or modified idc-ELISA assay system, it is possible to do a quick screening test at certain toxin levels in less than 30 min (e.g., 20 ppb) (2, 14-16, 22, 24, 26, 37, 100, 130-134). Based on the same principle as the dc-ELISA, several other types of immunoscreening tests with sensitivity similar to ELISA were also developed. Rather than coating the antibody onto the tnicrotiter plate, the antibody is immobilized on a paper disk or other membrane (134-140), which is used directly as a strip (136) or mounted either on a plastic card (card screen test), a plastic strip (as dipstick; 141, 142), in a plastic cup (Cup test and Cite), or in a syringe (Cite probe and Idexx probe, 135). Antibody has also been coated on polystyrene beads (142, 143). The reaction is carried out on the wetted membrane disk. Thus, after reaction, the absence of color (or decrease in color), generally blue, at the sample spot indicates the presence of toxin in the sample. The reaction is generally very rapid and takes less than 10-15 min to complete. Like the above formats, dipstick-type enzyme imtnunoassay has been developed for quick screening of Penicillizm islnndicunz in rice grain (144) and 3-acetyl-DON (140) and T-2 toxin (145) in wheat. Abouzied and Pestka ( 146) immobilized different mAbs against AFB FmB and ZE, as multiple lines on the nitrocellulose strip and respective mycotoxin-peroxidase conjugates were used as the testing markers. Thus, this system can screen all three mycotoxins simultaneously with detection limits of 0.5,500, and 3 ng/mL for AFBl, FmB,,and ZE, respectively. Another screening test is the immunoaffinity method, which was originally designed for mycotoxins such as AF, OA, and ZE that fluoresce (14, 15, 21, 26, 28, 139, 147152). In this assay, sample extracts diluted in phosphate buffer are applied to the affinity columns in which specific antibody was covalently bound to the solid matrix. After washing to remove the unbound materials, specific mycotoxin is then eluted from the column with the appropriate solvent system and then subjected to other chemical analyses. For mycotoxins with native fluorescence such as AF, OA, and ZE, the toxin level in the eluate

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could be directly determined fluorometrically or be determined after derivatization to enhance the fluorescence (150-1 52). For fumonisin and DON screening, it is necessary to introduce a fluorophore to the materials eluted from the immunoaffinity column (IAC) (27, 36). The sensitivity to the IAC screening tests for AFB 1, FmB 1, OA, and ZE is 2 ppb, 1 ppm, 5 ppb, and 0.2 ppm, respectively. The application of various immunoscreening tests to mycotoxins has been summarized by Chu (14, 21, 24, 27, 28) and Pestka (36), and most of the screening tests are commercially available as kits (2, 15, 22, 36). All of the rapid-screening test kits pemlit monitoring of mycotoxins semiquantitatively and have been found to be effective in screening mycotoxins in the field by FSIGS (2, 36, 154). Other evaluations for the commercial kits also concluded that such kits could be used for screening tests (17-20, 28, 90, 155-158). Collaborative studies for some of these immunoscreening tests have been done, and sotne of them have been adopted by the AOAC as first action for screening for AFs in different commodities (1 6-20, 28, 36, 138, 152, 157). With the increasing availability of commercial immunoassay kits, the AOAC International has established a research institute to evaluate the performance of different kits (16-20).

VI.

COMPLEMENTATION OF IMMUNOASSAYSWITH CHEMICAL METHODS

A.

lmmunoaffinityChromatography

With the availability of antibodies against various mycotoxins, imtnunoaffinity columns (IACs) were made by conjugating antibodies to a solid-phase matrix. These columns are then used either in a screening test as discussed above or as a cleanup column for subsequent chemical analysis (14, 21, 26-28). IAC was first used in the RIA (159) and later for recovery of AFM from urine and milk samples (160) for subsequent analyses. Although earlier applications of this technique were primarily aimed at biological fluids (149- 151, 161), the IAC has gained wide application as a cleanup tool for a number of mycotoxins and is not limited to fluid samples (14,21,26-28, 162). IACs for a number of mycotoxins are also commercially available. Table 4 summarizes the most recent applications of IAC technology to mycotoxin analysis. It is apparent that once the contaminants are removed by specific IACs, the solution can be directly subjected to liquid chromatographic (LC) quantitation, either off-line or on-line in an automated system, or by fluorometry. IAC not only serves as a cleanup tool but also concentrates the mycotoxin from a large amount of sample. Thus, it lowers the detection limits. Sometime as low as parts-per-trillion of mycotoxins can be measured. A number of collaborative studies indicate that IAC is an efficient method for cleanup of mycotoxins (14, 153, 162, 169, 170). A combination of an IAC column and PsCD is considered the official method for screening and clean-up of AF in several commodities (152, 162). Automation involving the use of an affinity column and HPLC was developed for routine analysis of AFM in milk (163-165, 204), AFB in peanut butter (171), in peanuts and corn (206), and in other nuts (207) and OA in cereal and animal products (192) and fumonisin (158, 187, 208). With the increased use of IACs as a tool for cleanup for mycotoxin analysis, one issue is the cost of the column. Research on regenerating columns for repeated use has also been conducted over the years; some of the approaches have been reviewed by Scott and Trucksess (162). Using IAC for cyclopiazonic acid as the tnodel, we found that the key to the successful regeneration of IACs

ed

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Table 4 ImmunoaffinityChromatography of Mycotoxins AnalysisCommodities Mycotoxins AFM AFB AFB AFs AFL AFQ 1 AF-adducts AF-albumin CPA DON F I B . HFms OA OA ZE

ref. Md F. P Cff, P. nuts, figs, etc C.P,Ct,F,Pb,BR

Flh TLC HPLC/PsCD Fl/Br: HPLC/PCD

s, u

HPLC HPLC HPLC, ELISA HPLC, ELISA ELISA HPLC HPLC: LC/MS HPLC CE ELISA, HPLC, MS

U TAU S C, P, feed

c. w C, starch Cff, T. W. C, So Cff, c , so M, u, c

149, 163, 164. 204 166, 167 168-174 150,152,154, 174. 175 176 177 161, 178-182 182- 186 99 256 158,187-190 173, 191-196 197 198-203

Commodities tested: BR, beer. Cff, coffee; T, animal tissues: So, sorghum; other abbreviations, see Tables 1 and 3. Methods for final analysis: FI/Br and F1 represent fluorometric analysis of the solution eluted from the column with and without treatment with bromine solution, respectively.

rests on the equilibration time. One must equilibrate the IAC in the loading buffer for sufficient time (>4 hr) before reuse (99).

B. Combination of Immunoassay with Other Chemical Methods 1. Combination with HPLC and TLC With the availability of sensitive ELISA methods, this technique has proved effective as a postcolumn monitoring system for HPLC (209). This is especially useful for the analysis of compounds with no specific absorption, such as TCTCs and Fms. For example, in the analysis of various type A TCTC mycotoxins, the sample extract with no cleanup treatment was first subjected to HPLC with a C-18 reverse-phase column. Individual fractions eluted from the column were analyzed by ELISA using “generic” antibodies against group A TCTCs. This approach can not only identify each individual group A TCTC, but can also determine its concentration quantitatively. As little as 2 ng of T-2 toxin and related TCTCs as well as their metabolites can be monitored by this method. A combination of HPLC and ELISA technology proved to be an efficient, sensitive, and specific method for the analysis of TCTC (2 10-212) and other mycotoxins (8 1, 82, 2 13). In a recent study, we were able to determine FrnBl, FmB2, Fr-3, and AAL toxin TA simultaneously when two types of pAb, one against Fms and one against AAL toxins, were used in the postcolumn ELISA (40). The detection limit was 0.1 ng FmB 1 per tube (0.5 ml). Recovery of FmB1 added to ground corn in the 100-1000-ng/g range in this system was 78.8%. Analysis of extracts from cultures of three Alternaria nlternatu strains revealed that both FmB 1 and the AAL toxin TA were present, but their amounts varied considerably with the cultures tested.

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Likewise, immunoassay has been used in combination with TLC (213) in which the crude sample extract was applied to the TLC plate. After separation, each fraction in TLC was analyzed immunochemically. It is interesting that while this approach quantifies the known toxins, it is also capable of uncovering new mycotoxins with structures similar to those of the known mycotoxin. Miles et al. (214) were able to isolate paxinorol, a new paxilline derivative, when they monitored the TL chromatogram with ELISA using antibody specific to paxilline. 2. Combination of High-Performance TLC (HPTLC) with lmmunoblotting An approach called HPTLC-ELISAgram was introduced by Pestka (215). This method involves separation of mycotoxins using HPTLC, followed by blotting the chromatogram onto a nitrocellulose membrane coated with antibody, incubation with mycotoxin-enzyme conjugate, and a final incubation with substrate to develop the color. Although this method has good sensitivity, the need for a large amount of antibody limits its wide application. 3. Combination of Immunofluorescence and Capillary Electrophoresis (CE) Based on the competition between unlabeled FmB 1 (i.e., from a sample) and a fluoresceinlabeled FmB 1 reagent (FmB 1-FL) with the mAb, Maragos (2 16,217) used CE to separate the bound and free FmB 1-FL. In the assay, purified FmB 1-FL was subjected to CE. Addition of purified mAb to FmB 1-FL before separation resulted in the formation of mAbFmB 1-FL complex with resulting quenching of fluorescence and decrease in the intensity of the FmB1-FL peak. When a sample containing FmB 1 is present, it competes with FmB 1-FL for binding of the limited amount of mAb causing an increase in FmB 1-FL peak. The intensity of such increase is directly proportional to the amount of unlabeled FmBl present. The IC5" of unlabeled FmBl was highly dependent upon the antibody concentration and ranged from 58 to 4170 ng/ml (at 15-75 pg/ml of antibody). The method is rapid and requires only 6 min for cotnplete analysis of FmBl standard.

VII. A.

OTHERNEWLYDEVELOPEDIMMUNOCHEMICAL METHODS Anti-idiotypeAntibody-BasedImmunoassays

The development of immunochemical methods for tnycotoxin detection has led to a great demand for specific antibodies and related immunochemical reagents for the assay. An alternate approach to preparing imtnunochemical reagents is through generating anti-idiotype (anti-ID) antibodies (218). Anti-ID (Ab2j for large molecules have been well developed and have been applied to clinical diagnosis and ilnnlunotherapy (218). Recent studies have succeeded in generating Ab2 against a number of small-molecular-weight haptens, including mycotoxins AFs (219-221), FmB 1 (98), and T-2 toxin (222,223).Anti-idiotype antibodies for mycotoxins were demonstrated in different animal species after immunization with the affinity-purified original idiotype antibodies (Abl) (98, 219j. Ab2 were not only bound specifically to the original Ab1 but also were capable of being used as an immunogen in generating anti-anti-ID antibodies (Ab3). Whereas Ab2 could not be used as a mycotoxin-protein conjugate in the indirect ELISA for the determination of aflatoxin and T-2 toxin, an Ab2-based indirect ELISA has been established for Fm analysis (98).

Mycotoxin

Analysis697

Thus, these anti-idiotype antibodies are indeed the surrogates of mycotoxins. Most recently, a hybridoma cell line that generates monoclonal Ab3 was obtained in our laboratory (257). From the ID5"values, it is apparent that the Ab3 have similar characteristics to the original Ab 1. The availability of Ab2 and Ab3 for mycotoxins has provided a new generation of immunochemical reagents, which could be used for both therapeutic and analytical purposes. Ab2, a surrogate of the toxin, could be used as the immunogen in generating antibodies for the toxin (thus. a vaccine) and could also be used in the immunoassay. In an in vitro study of the effect of antibodies on the binding of aflatoxin to DNA, Hsu (224) found that Ab2 were capable of inhibiting the binding of aflatoxin B, to DNA, but the inhibitory effect was not as high as that of the Abl. 6. Immunoassay for Mycotoxin-ProducingFungi Other than mycotoxins, antibodies against specific fungi (144, 225-228) as well as several enzymes involved in the biosynthesis of aflatoxin(229-234) and trichothecenes (235,236) have been generated also. Five mAbs against Aspergillus~nvuswere recently produced by Candlish et al. (237) and those mAbs were used in the immunoassays for identification of A. flavus. Whereas we used the partially purified proteins for generating of antibodies against the enzymes in the aflatoxin biosynthetic pathways (229-234), Shapira et al. (238) used both the culture filtrate and two chimeric proteins, which were expressed in Escllerichin coli from genes ver- 1 and npn-2, as the antigens for generating the antibodies against toxic Aspergillus pnrnsiticus and A. .fkrvus. The pAb generated from the chimeric proteins were also specific for these fungi. These antibodies produced in different laboratories have been used for identifying specific fungi in foods, for studying the kinetics of enzymes involved in the biosynthesis of mycotoxins, and for cloning the genes that encode the enzymes for mycotoxin synthesis.

C. Development of Biosensors Although development of An/Ab-based biosensors for mycotoxin detection was initiated in the late 1980s, application of this technology only emerged in recent years. Nevertheless, some of the principles and methodology are useful for designing the biosensors. In the so-called "hit-and-run" assay (239,240) for T-2 toxin, a T-2 toxin column was equilibrated with a fluorescein isothiocyanate (F1TC)-labeled Fab fragment of IgG (anti-T-2 toxin). Samples containing T-2 toxin were injected into the column. The FITC-Fab that eluted together with the samples containing T-2 toxin was then determined in a standard flowthrough fluorometer. A similar approach in which ribonuclease-labeled Fab was used as the indicator was also reported (240). Another approach that may lead to the development of a biosensor is a homogeneous immunoassay for T-2 toxin, which involves the use of liposomes and complement (241). Whereas these methods were not as sensitive as the regular ELISAs, several biosensors with some modifications can be used for rapid screening of mycotoxins. 1. Time-Resolved Fluoroimmunoassay (FI) This method involves the use of Europium ion (Eu)-labeled antibodies and has a sensitivity similar to that of most ELISA methods with an ICsovalue of 0.2 ng AFB l/ml (123, 242). The same approach can be used for other mycotoxins.

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2. Fiberoptic lmmunosensor In this assay, mAb are covalently bound to an optical fiber and an evanescent wave effect was utilized to excite the flourescent-tagged toxin near the surface of the mAb-fiber as the tagged toxin bound to the fiber. In the presence of unlabeled toxin, it competes with the labeled toxin for binding with mAb and results in a decrease in signal. Thus, it is also a competitive assay and has been tested with T-2 toxin (243) and fumonisin (244). Using mAb against FmB 1 and FnlB 1-FITC, Thompson and Maragos (244) tested the feasibility of this system for analysis of FmB 1. The assay involves: (1) saturation of mAb binding sites by FmB 1-FITC, (2) competition of FmB 1 and FmB 1-FITC with displacement of the labeled toxin, and (3) resaturation of binding sites with FmB 1-FITC. The signal generated in the assay was inversely proportional to the concentration of FmB 1. This sensor has a working range of 10- 1000 ng of FmB l/ml, an IC5,)of 70 ng/ml, and a limit of detection of 10 ng/ml. These values compared favorably with those for currently available ELISA techniques. The methanol/water-extracted corn sample did not affect the sensor performance. 3. Automated Particle-Based lmmunosensor (API) This sensor is based on the kinetic exclusion assay. The system consists of a simple fluorimeter with a 1.5-mm-diameter glass capillary to serve as the flow cell within the final lens. Application of this system for the analysis of AFBl was studied by Strachan et al. (245). In the assay, the appropriate amount (ca. 100 beads) of polymethylmethacrylate beads (98 pm) coated with AFB1-BSA is pumped into the capillary and trapped on a filter. The sample or calibrated standard solutions that had been incubated with antibodies were then allowed to pass through for a period of 120 sec, followed immediately with FITC-labeled goat-anti-rabbit antibody (130 sec), and then finally washed with buffer to remove excess label. The fluorescence during each step of the reaction was recorded. The amount of antibody to the sample can be calculated by measuring the difference in voltage from the sensor during each step and at the end of the assay. This automated sensor can easily detect AFBl down to a level of 4 ng/g (4 ppb) in reference food materials in 8 min excluding the sample extraction step (<60 min to completej. Experiments with other aflatoxins indicate that only G(2) shows significant cross-reactivity (23%). 4. Miniaturized ELISA A miniaturized ion sensitive field effect transistor (1SFET)-ELISA system was recently developed by Yacoub-George et al. (246). This system consisted of a flowthrough setup with a pretreated activated fused silica capillary as a reaction cartridge to serve as a solid phase for the reversible idc-ELISA. Urease acts as enzyme marker to produces a pH shift that can be detected by the ISFET array. The lower detection limit is 10 pg/ml for T-2 toxin. The main advantages of the system are small sample volumes, short cycle times, and an excellent stability of the regenerable receptor layer.

5. Surface Plasmon Resonance Biosensor A surface plasmon resonance (SPR) immunosensor for the determination of FmBl was developed by Mullett et al. (247). pAb against FmB 1 were adsorbed onto a thin gold film substrate, which is coupled to a glass prism. The output beam of a planar light-emitting diode is focused through the prism to excite SPR at the surface of the gold film. When a sample containing FmBl is added to a cell on the outside of the gold film, the angular profile of reflected light intensity shifts. This changes the resonance angle and the reflected

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beam intensity at a selected angle, both of which are proportional to the FmB 1 concentration. After optimization of the antibody overlayer, a detection limit of 50 ng/ml isobtained for the direct assay with an analysis time under 10 min. Multiple sample additions and large-volume sample circulation can be used with the high-affinity antibodies to achieve lower detection limits.

VIII.

CONCLUDING REMARKS

From this review, it is apparent that immunoassay techniques have gained wide acceptance as analytical tools for mycotoxins. Antibodies against almost all the important mycotoxins are currently available. Sensitive, simple, and specific immunoassays have been established for the analysis of various mycotoxins in foods, feed, animal tissue, and body fluids. Several immunoassay protocols have been adopted as first action by the AOAC. Immunoscreening methods have been widely accepted as a simple method for screening aflatoxin and several other mycotoxins in several commodities and many immunoscreening kits are commercially available. The immunoaffinity method has also become popular as a cleanup method in conjunction with other chemical methods. Immunochemical methods have also been used in various toxicological studies including immunohistological examination of animal tissues (24, 26, 28, 248, 249), analysis of mycotoxin metabolites, and in epidemiological studies tofind a correlation between mycotoxin exposures and certain diseases in humans. Recent developments have also led to the production of antibodies against specific groups of fungi in foods that are used for the determination of specific molds that contaminate foods. Antibodies for the key enzymes involved in the biosynthesis of aflatoxin and trichothecenes have been raised and have been used effectively incloning the genes for some of these enzymes. Such antibodies could be used in the future for controlling toxin formation. Production of anti-idiotype and anti-anti-idiotype antibodies against several important mycotoxins has been demonstrated. These antibodies have been shown to be effective in the ELISA and could possibly be used as a vaccine in the future. Although most immunoassays are very effective, the sensitivity of some assays is still very low. Thus, the analyst should make a decision regarding whether a cleanup step is necessary to achieve a specific sensitivity. Improvements in the production of some immunoassay reagents and protocols are still needed. In most cases, the low antibody affinity is considered by this reviewer to be one of the major factors contributing to low sensitivity. Thus, future efforts should be directed to generating high-affinity antibodies for some mycotoxins, e.g., DON. Better labeling techniques, including fluorescent-labeled antibodies/mycotoxins, should also be tested to evaluate the possibility of achieving a more sensitive and rapid method for the toxin detection. With new approaches to the production of antibodies, including using anti-idiotype and anti-idiotype antibodies and structural modulation (250, 251), that have been tested or proposed, it is probable that better and high-affinity antibodies can be obtained in the near future. Several laboratories, including our own, have initiated work in cloning these antibodies. However, problems in selecting clones to generate useful antibodies remain to be solved. Hopefully, it will not be too long before we understand how these antibodies react with these mycotoxins; thus, a new generation of antibodies could be made available through point mutation. With newer methods being used in the production of hapten-proten conjugates (28), cloning of antibodies (28, 70, 252-254) as well as new developments in the biotechnology and bio-

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sensor areas (255), immunoassays for mycotoxins will be advanced to another new era. I hope that this review will not only provide some general principles for different immunoassay protocols but also help to generate more interest in using immunochemical methods in the analytical, diagnostic, and possibly therapeutic areas. I also hope that it willstimulate additional research in this rapidly progressing area to improve the methodology, to simplify the assay procedures, and to increase the sensitivity and specificity of the assays to alleviate matrix interference problems.

ACKNOWLEDGMENTS

This work was supported by the College of Agricultural and Life Sciences, the University of Wisconsin, Madison. Part of the work described in this contribution was supported by a Public Health Service grant (CA 15064) from the National Cancer Institute, a contract (DAMD17-86-C-6173j from the U.S. Army Medical Research and Development Command of the Department of Defense, a USDA grant (58-6435-1-116), and a USDA North Central Regional Project (NC-129). The author thanks Ellin Doyle and Barabra Cochrane for their help in preparing the manuscript.

REFERENCES 1. Busby, W. F. and Wogan, G. N. (1981) Aflatoxins. In Mycotoxins and N-Nitrosocoilt/7ou~lds. Em~ironnlerztulRisks. Vol. 11. Shank. R. C., Ed. CRC Press: Boca Raton. FL, pp. 3-45. 2. CAST (1989) Mycotoxins, Economic a r d Health Risks, Council of Agricultural Science and Technology (CAST), Task force report No. 116. CAST. Ames, IA, 92 pp. 3. Cole, R. J. and Cox, R. H. (1981) Hmdbook of Toxic Fruzgal Metabolites. Academic Press: New York, 937 pp. 4. Sharma, R. P. and Salunkhe, D. K. (1991) Mycotoxirr m d Phyocrle.uins in Human and Animal Health. CRC Press: Boca Raton. FL. 5. Bhatnagar, D., Lillehoj, E. B. and Arora. D. K. (1992). Hmdbook of AppliedMycology Vol. V. Mycotoxins i n Ecological System. Marcel Dekker: New York. 6. Eaton, D. L. and Groopman, J. D. (1 994) The Toxicologyof A~~rto.uirzs-Hz4rnnrz Health, Vererinanl am1 Agricdtzwal Significmce. Academic Press: New York, 544 pp. 7. Miller, J. D. and TrenhoIm, H. L. (1994) Mycotoxins in Grain: Cor~poztndsOther Tlml Ajatoxin. Eagan Press: St. Paul, MN. 552 pp. 8. Sinha, K. K. and Bhatnagar, D. (1998) Mycoto.xins in Agriculture m d Food Safely. Marcel Dekker: New York, 520 pp. 9. Van Egmond, H. P. (1989) Current situation on regulations for mycotoxins. Overview of tolerances and status of standard methods of sampling and analysis. Food Acldit. Coiltam. 6, 139-188. 10. FAO/WHO (1997). Worldwide regulations for mycotoxins- 1995. A compendium. F A 0 Food and Nutrition paper. # 63. F A 0 of UN, Rome, Italy. 11. Cole, R. J. (1986) Modern methods in the analysis and structural elucidation of mycotoxins. Academic Press: Orlando, FL. 471 pp. 12. Van Egmond, H. P. and Paulsch. W. E. !1986) Determination of mycotoxins. Pzrre Appl. Chem. 58. 315. 13. Chu. F. S. (1991) Detection and Determination of Mycotoxin. In Mycotoxill arrd Plzytoalexins ii?Hmrcrrz and Animal Health. Sharma. R. P., and Salunkhe, D. K.. eds. CRC Press: Boca Raton, FL.

Mycotoxin Analysis

701

14. Chu, F. S. ( 1995) Mycotoxin analysis. InAnalyzing Food for Nutrition Labeling and Hazardous Contamirzants. Jeon. I. J. and Ikins, W. G. eds. Marcel Dekker: New York, pp. 283332. 15. Wilson, D. M.. Sydenham, E. W., Lombaert, G. A., Trucksess, M. W.. Abramson, D. and Bennett. G. A., ( 1998) Mycotoxinanalyticaltechniques.In Mycotoxins in Agricu1tllr.e a d Food Scfety. K. K. Sinha and D. Bhatnagar, eds. Marcel Dekker: New York. pp. 135182. 16. QfJicial Methods of AnalJqsis(1990) 15th ed. Assoc. Off. Anal. Chem. (AOAC): Arlington, VA. 17. Trucksess. M. W. (1994) Mycotoxins. JAOAC Znt. 77. 135-142. 18. Trucksess, M. W. (1995) Mycotoxins. JAOAC Irzr. 78, 135-141. 19. Trucksess, M. W. (1997) Mycotoxins. JAOAC Int. 80, 119-126. 20. Trucksess, M. W. (1998) Mycotoxins. JAOAC Int. 81. 128-137. 21. Chu, F. S. (1986). Inmunoassay for mycotoxins. In Modern Methods in the A n d p i s m d Structural Elucidation of Mycotoxins. Cole, R. J.. ed., Academic Press: New York. pp. 207237. 22. Chu, F. S. (1990) Immunoassay for mycotoxins: current stateofthe art. commercial and epidemiological applications. Vet. Hum. Toxicol. 32(Suppl.), 42-50. 23. Chu, F. S. ( 1991) Current immunochemical methods for mycotoxin analysis. In Irrrmunocrssays for Trrrce Chemical Analysis: Monitoring Toxic Chemicals in Humans, Foods and the Environnzent. Vanderlaan, M., Stanker. L. H.. Watkins. B. E. and Roberts. D. W., eds. ACS Symposium Series 451. American Chemical Society: Washington, DC, pp. 140-157. 24. Chu, F. S. (199 1) Development and use of immunoassays in detection of the ecologically important mycotoxins. In Hcrmlbook of Applied Mycology. Vol. 5. Mycotoxins. Bhatnagar, D., Lillehoj, E. B.. and Axora, D. K., eds. Marcel Dekker: New York, pp. 87-136. 25. Chu, F. S. (1992) Recent progress on analytical techniques for mycotoxins in feedstuffs. J. Aninz. Sci., 70, 3950-3963. 26. Chu, F. S. (1 994) Development of antibodies against aflatoxins. InThe Toxicology of AJ7nto.virzs-Hm?rrn Health. Veterinary and Agricultui-rrl Significarzce. Chapter 21.Eaton,D.L.. Groopman. J. D., eds. Academic Press: New York, pp. 451-481. in Food. Jackson, 27. Chu. F. S. (1996) Immunochemical methods for fumonisins.In Frrr?~onisins L., DeVries, J. W. and Bullerman, L. B., eds. Plenum Press: New York, pp. 123-134. 28. Chu. F. S. (1996) Recent studies on immunoassays for mycotoxins. In Irmunoussays for Residue Amlysis: Food Snfev. Beier. R. C. and Stanker, L. H., eds. ACS Symposium Series book no. 621. American Chemical Society: Washington, DC, pp. 294-31 3. 29. Morgan, M.R. A. (1985) Newer techniques in food analysis-immunoassay and their application to small molecules. J. Assoc. Pub. Anal. 23, 59-63. 30. Pestka. J. J. (1988) Enhanced surveillance of foodborne mycotoxins by immunochemical assay. JAOAC 71. 1075-1081. 31. Morgan. M. R. C. (1 989) Mycotoxin immunoassays with special reference to ELISAs. Tetrahedron 35, 2237. 32. Fukal, L. and Kas. J. (1989) The advantages of inmunoassay in food analysis. Trerzds A I Z ~ ~ . Chenz. 8, 112-1 16. 33. Morgan. M. R. A. and Lee, H. A. (1990) Mycotoxinsand natural food toxicants. In Del,eloprnellt and Applicatiolz of Inzmmotrssay for Food Ancrlysis. Rittenburg. J. H.. ed. Elsevier: New York, pp. 143-170. 34. Martibauer, E., Dietrich.R.and Terplan, G. (1991) Erfahrungenbei der anwendung von in~lunoassaysnachweis von mykotoxinen in lebensmitteln. Arch. Lerlbelzsiizittellz!ldiene 32, 3-6. 35. Wilkinson, A. P., Ward, C. M.and Morgan, M. R.A. (1992) Immunologicalanalysisof mycotoxin. In Modertz Methods of Plant Analysis, Neb17 series. Plant Toxin Analysis. Linskens, H. F. and Jackson. J. F., eds. Springer-Verlag: Berlin, pp. 185-225.

702

Chu

36. Pestka, J. J. (1994) Application of immunology to the analysis and toxicity assessment of mycotoxins. Food Agric. Znzntunol. 6, 219-234. 37. Beier, R. C. and Stanker, L. H. (1996) Zm,zrrrzoassclys for Residue Analysis: Food Sqfet?,. ACS Symposium Series bookno. 621. American Chemical Society: Washington, DC, 1996, 528 pp. 38. Szurdoki, F.. Trousdale, E., Gee, S. J., Ward, B., Hammock, B. D. and Gilchrist, D. (1996) Development of an enzyme immunoassay for Alterrmfrr ulternata f.s.p. lycopersici toxins. In Zrnr~zzcnoassaysfor Residue Analysis: Food Sr$et;v. Beier, R. C. and Stanker. L. H., eds. ACS SymposiumSeries bookno. 621. American ChemicalSociety:Washington,DC, pp.330-340. 39. Szurdoki, F., Trousdale, E., Ward, B., Gee. S. J.. Hammock, B. D. and Gilchrist, D. G. (1996) Synthesis of protein conjugates and development of immunoassays forAAL toxins. J. Agric. Food Chern. 44, 1796-1803. 40. Yu, Feng-yih and Chu, F. S. (1998) Analysis of fumonisins and Alternoria alterrzatcr toxin by liquid chromatography-enzyme-linked immunosorbent assay. JAOAC Zrzt, 81, 749-756. 41. Hsu, K.-H. and Chu, F. S. (1992) Production and characterization of antibodies against aflatoxin in laying hens. Food Agric. Zrnnrz~rzol.4, 83-91. 32. Ward, C. M., Wilkinson, A. P., Bramham, S., Lee, H. A., Chan, W. S., Butcher, G. W., Hutchings, A. and Morgan, M. R. A. (1990) Production and characterization of polyclonal and monoclonal antibodies against aflatoxin B 1 oxime-BSA an in enzyme-linked immunosorbent assay. Mycotoxin Res. 6. 73-83. 33. Abramson, D., Usleber, E. and Martlbauer, E. (1995) An indirect enzyme immunoassay for the mycotoxin citrinin. Appl. Emiron. Mict-obiol. 61, 2007-2009. 44. Usleber, E., Martlbauer, E. and Abramson, D. (1996). Comparison ofdirectand indirect enzyme immunoassays for the detection of the mycotoxincitrinin. In Zmntunotrssaysfor Resih e AnalFsis: Food Sqfev. Beier, R. C. and Stanker, L. H., eds. ACS Symposium Series book no. 621. American Chemical Society: Washington, DC, pp. 322-329. 45. Hahnau, S. and Weiler, E. W. (1991) Determination of the mycotoxin cyclopiazonic acid by enzyme immunoassay. J. Agric. Food Chent. 39, 1887-1891. 46. Hahnau, S. and Weiler, E. W. (1993) Monoclonal antibodies for the enzyme immunoassay of the mycotoxin cyclopiazonic acid. J. Agric. Food Chenz. 41, 1076-1080. 47. Huang, X. and Chu, F. S. (1993) Production and characterization of monoclonal and polyclonal antibodies against the mycotoxin cyclopiazonic acid. J. Agric. Food Clzent. 41. 329333. 48. Mills, E. N. C., Alcock, S. M.. Lee. H. A. and Morgan, M. R. A. (1990) An enzyme-linked immunosorbent assay for deoxynivalenol inwheat, utilizing novel hapten derivatization procedures. Food Agric. Zr~trnunol.2, 109-1 18. 39. Abouzied, M. M., Azcona-Olivera, J. I., Yoshizawa. T. and Pestka, J. J. (1993) Production of polyclonal antibodies to the trichothecene mycotoxin 4, 15 diacetylnivalenol with carrieradjuvant cholera toxin. Appl. E?zvit-orz.Microbiol. 59, 1264-1268. 50. Nicol, M. J., Lauren, D. R., Miles, C. 0. and Jones, W. T. (1993) Production of a monoclonal antibody with specificity for deoxynivalenol,3-acetyldeoxynivalenoland 15-acetyldeoxynivalenol. Food Agric. I r m z w w l . 5. 199-209. 51. Usleber, E., Murtlbauer, E., Dietrich, R. and Terplan. G. (1991) Direct enzyme-linked inlmunosorbent assay for the detection of the 8-ketotrichothecene mycotoxins deoxynivalenol, 3 acetyldeoxynivalenol and 15 acetyldeoxynivalenol in buffer solution. J. Agric. Food Chenz. 39. 2091-2095. 52. Schmitt, K., Martlbauer, E.. Usleber, E., Gessler. R., Lepschy, J. and Abramson. D. (1996) Detection of acetylated deoxynivalenol by enzyme-linked inlmunosorbent assay. InZmrmrroassays for Residue Analpis: Food Safev. Beier, R. C. and Stanker, L. H., eds. ACS Symposium Series book no. 621. American Chemical Society: Washington, DC, pp. 314-321. 53. Sinha. R. C., Savard, M. E. and Lau. R. (1995). Production of monoclonal antibodies for

Mycotoxin Analysis

54.

55. 56.

57. 58.

59.

60.

61.

62. 63. 64.

65.

66. 67. 68.

69.

70. 71. 72.

703

the specific detection of deoxynivalenol and 15-acetyldeoxynivalenol by ELISA. J. Agric. Food Cheln. 43, 1740- 1744. Wang, N., Kierek-Jaszczuk, D.. Marquardt, D. R.; Frohlich, A. A. and Abramson, D. (1996). Use of a heterologous bridge coating antigen for the immunoassay of 3-acetyloxinivalenol in barley. J. Food Protect. 59, 525-433. Hill, N. S., Thompson, F. N., Dawe, D. L. and Stuedemann. J. A. (1994) Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue. Am. J. Vet. Res. 55, 419-424. Shelby, R. A. (1996) Detectingergot alkaloids by immunoassay. InIn~nz~~noassnysResifor due Analwis: Food Safety. Beier, R. C. and Stanker, L. H., eds., ACS Symposium Series book no. 621. American Chemical Society: Washington, DC, pp. 231-242. Shelby, R. A. and Kelley,V. C. (1990) An immunoassayfor ergotamineand related alkaloids. J. Agric. Food Chern. 38, 1130-1 134. Shelby, R. A. and Kelley, V. C. (1992) Detection of ergot alkaloids from Claviceps species in agricultural products by competitive ELISA using a monoclonal antibody.J. Agric. Food G e m . 40, 1090- 1092. Shelby, R. A., Bridgman, R. C., Smith, F. T. and Atigadda, V. R. (1998) Determination of ergovaline in tall fescue by a specific monoclonal antibody. Food Agric. In~nzunol.10, 339347. Azcona-Olivera, J. I., Abouzied, M. M.,Plattner, R.D., Norred, W. P. and Pestka,J. J. (1992) Generation of antibodies reactive with fumonisins B 1, B2, and B3 by using cholera toxin as the carrier-adjuvant. Appl. Environ. Microbiol. 58, 169-173. Azcona-Olivera, J. I., Abouzied, M. M., Plattner, R. D. and Pestka, J. J. (1992) Production of monoclonal antibodies to the mycotoxins fumonisin B1, B2, B3. J. Agric. Food Clzem. 40, 531-534. Fukuda, S., Nagahara, A., Kikuchi, M. and Kumagai, S. (1994) Preparation and characterization of antifumonisin monoclonal antibodies. Biosci. BiotechnoL Biochem. 58, 765-767. Usleber. E., Straka. M. and Terplan, G. (1994) Enzyme immunoassay for fumonisin B-1 applied to corn-based food. J. Agric. Food Clzern. 42, 1392-1396. Elissalde,M.H.,Kamps-Holtzapple,C.,Beier,R.C.,Plattner, R. D.and Stanker, L. H. (1995). Development of an improved monoclonal antibody-based ELISA for fumonisinB 13 and the used of molecular modeling to explain observed detection limits. Food Agric. I H I ~ H U 7, IIO 109-122. ~. Maragos, C. M. and Miklasz. S. D. (1995) Monoclonal antibody-based competitive enzyme linked immunosorbent assays for the hydrolysis product of fumonisin B 1(HFB 1). In Irnrnunotrssays for Residue Analysis: Food Safety. Beier. R. C. and Stanker, L. H., eds. ACS Symposium Series book no. 621. American Chemical Society: Washington, DC. pp. 349-357. Yeung, J. M., Prelusky, D. B., Savard, M. E., Dang, B. D. M. and Robinson, L. A. (1996) Sensitive immunoassay for fumonisin B-1 in corn. J. Agric. Food Clzem. 44. 3582-3586. Yu, Feng-yih and Chu, F. S. (1996). Production and characterization of antibodies against fumonisin B 1. J. Food Protect. 59, 992-997. Iijima, K., Kawamura, O., Wang, D. S., Liang, Y. X., Chen, G.. Yu, S. Z., Chau, N. T. and Ueno. Y. (1996) Development of highly sensitive immunosorbent assay for fumonisisns and its application for contaminated corn. Mycotoxins 42, 63-66. Newkirk, D.K.. Benson, R. W., Howard, P. C., Churchwell, M.I., Doerge, D.R. and Roberts, D. W. (1998). On-line inlmunoaffinity capture, coupled with HPLC and electrospray ionization mass spectrometry, for automated determination of fumonisins. J. Agric. Food Cl?er?Z. 46, 1677- 1688. Yu. Feng-yih andChu, F. S. (1999) Production and characterization ofmonoclonal antibodies against fumonisin B 1. Food Agric. Itnmunol. 11, 297-306. Yu, J., Wei, R. D., Mirocha, C. J. and Chu, F. S. (1990). Production and characterization of antibody against fusarochromanone. Food Agric. Irnmuzol. 2, 55-64. Clarke, J. R., Marquardt, R. R.,Frohlich,A.A.andPitura. R. J. (1994) Quantitiation of

704

73.

74. 75. 76.

77.

78. 79.

80.

81. 82.

83.

84. 85.

86.

87. 88.

89. 90.

91.

Chu ochratoxin A in swine kidneys by enzyme-linked immunosorbent assay using a simplified sample preparation procedure. J. Food Protect. 57, 991-995. Clarke, J. R., Marquardt, R. R., Frohlich, A. A.. Madrid, F. J. and Dawood, M. (1993) Development of a quantitative and sensitive enzyme-linked immunosorbent assay for ochratoxin A using antibodies from the yolk of the laying hen. J. Agric. Food Cllem. 41, 17841789. Breitholtz-Emanuelsson. A., Dalhammar, G.and Huh, K. (1992) Immunoassay of ochratoxin A, using antibodies against a new ochratoxin-albumin conjugate. JAOAC I m . 75, 824-829. McElroy, L. J. and Weiss, C. M. (1993) The productionof polyclonal antibodies against the mycotoxin derivative patulin hemiglutarate. Can. J. Microbiol.. 39, 861-863. Garthwaite, I.. Briggs, L. R., Miles, C. 0.. Munday. S. C. and Towers, N.R. (1991) The production of monoclonalantibodiestopaxilline.1990-1991Ann.ReportofMycotoxin Diseases Group. Ruakura Agric.Center, Ministry of Agriculture and Fisheries, New Zealand, pp.12-14. Garthwaite, I., Sprosen, J., Briggs, L., Collin. R. and Towers, N. R. (1994) Food quality on the farm: Immunological detection of mycotoxins inNew Zealand pastoral agriculture. Food Agric. Irwzwzol. 6, 123-129. Garthwaite, I., Shearer,S. and Towers, N.R. (1994) Effects of the grass matrix on inmunological assays for mycotoxins. Food Agric. Ir~znlurzol.6, 235-239. Briggs, L. R., Collin, R. G., Miles, C. 0. and Towers, N. R. (1991) Production of antibodies binding sporidesmin. 1999-1991 Ann. Report of Mycotoxin Diseases Group. Ruakura Agric. Center, Ministry of Agriculture and Fisheries, New Zealand, pp. 4-5. Briggs, L. R., Towers, N. R. and Molan. P. C. (1994). Development of an enzyme-linked immunosorbent assay for analysis of sporidesmin A and its metabolites in ovine urine and bile. J. Agric. Food Chern. 42. 2769-2777. Olson, J. J. and Chu, F. S. 1993. Immunochemical study of sterigmatocystin-DNA adducts produced in vitro. Food Agric. Inzntunol. 5, 3-12. Olson, J. J. and Chu, F. S. 1993. Urinary excretion of sterigmatocystin and retention of DNA adducts in liver of rats exposed to the mycotoxin: an immunochemical analysis. J. Agric. Food Cl1enz. 41, 602-606. Li. S. Z., Chen, P. Y ., Marquardt, R. R., Han, Z. K. and Clarke, J. R. ( 1996). Production of a sensitive monoclonal antibody to sterigmatocystin and its application to ELISA of wheat. J. Agsic. Food Chenl. 44, 372-375. Wang, C.-R. andChu. F. S. (1991) Productionand characterization ofantibodiesagainst nivalenol tetraacetate. Appl. Ewirorz. Microbiol. 57, 1026- 1030. Barna-Vetro, T.. Gyongyosi, A. and Solti, L. (1994) Monoclonal antibody-based enzymelinked immunosorbent assay of fusarium T-2 and zearalenone toxins in cereals. Appl. E m i ron. Microbiol. 60, 729-73 1. Minervini, F., Gyongyosi-Horvath, A.. Lucivero, G., Visconti. A.. Barna-Vetro. I. and Solti. L. (1994) In vitro neutralization of T-2 toxin toxicity by a monoclonal antibody. Ncrtz{t-al To.xirzs. 2, 1 11-1 14. Reynolds, G. and Pestka. J. J. (1991) Enzyme-linked inmunosorbent assay of versicolorln A and related aflatoxin biosynthetic precursors. J. Food Pro-otect.54, 105-108. Teshima, R.. Kasase, M.. Tanaka, T., Hirai,K.. Sato. M., Sawada, J. I., Ikebuchi, H., Ichinoe M. and Terao, T. (1990) Production and characterization of a specific monoclonal antibody against zearalenone. J. Agt-ic. Food Cllem. 38, 1618-1622. Teshima, R., Hirai,K.. Sato, M.,Ikebuchi. H.. Ichinoe. M. and Terao, T. (1990) Radioimmunoassay of nivalenol in barley. Appl. Emyiron. Microbiol. 56, 764-768. Bennett. G. A., Nelsen, T. C. and Miller, B. M. (1994)Enzyme-linked immunosorbent assay for detection of zearalenone in corn, wheat, and pig feed: collaborative study. JAOAC I w . 77, 1500- 1509. Fan, T. S. L., Schubring. S. L.. Wei, R. D. and Chu. F.S. (1988) Production and characteriza-

Mycotoxin Analysis

92. 93. 94.

95. 96. 97.

98.

99.

100.

101.

102.

103. 104.

105.

106.

107. 108. 109.

110.

705

tion of a monoclonal antibody cross-reactive with most group A trichothecenes. Appl. E m f row Micr-obiol. 54, 2959-2963. Wei, R. D.andChu.F. S. (1987) Productionandcharacterization of a genericantibody against group A trichothecenes. Anal. Biockem. 160, 399-408. Xu, Y. C., Zhang, G. S. andChu. F. S. (1988) Enzyme-linked immunosorbent assay for deoxynivalenol in corn and wheat. JAOAC 71, 945-949. Hefle, S. L. and Chu, F. S. (1990) Production and characterization of monoclonal antibodies cross-reactive with major aflatoxins. Food Agric. Inmzurzol. 4, 181-1 88. Zhang, G. S. and Chu. F. S. (1989). Production and characterization of a generic antibody against aflatoxins. Experienticr 45, 182- 183. Fukal. L. and Reisnerova, H. (1990). Monitoring of aflatoxins and ochratoxin-A in Czechoslovak human sera by immunoassay. Bull. Environ. Contam Toxicol. 44. 345-349. Hongyo, K.-I., Itoh, Y., Hifumi, E. and Takeyasu, A. (1992) Comparison ofmonoclonal antibody-based enzyme-linked irnmunosorbent assay with thin layer chromatography and liquid chromatography for aflatoxin B1 determination in naturally contaminated corn and mixed feed. JAOAC Int. 75, 307-312. Chu. F. S.. Huang, X. and Maragos, C. M. (1995) Production and characterization of antiidiotype and anti-anti-idiotype antibodies against fumonisin B1. J. Agric. Food Clzem. 43, 26 1-267. Yu. W., Dorner, J. W. and Chu, F. S. (1998) Inmunoaffinity column as clean-up tool for a direct competitive enzyme-linked immunosorbent assay for cyclopiazonic acid in corn, peanuts and mixed feed. JAOAC Int. 81, 1169-1 175. Chu. F. S.. Fan, T. S. L.. Zhang. G. S., Xu, Y. C., Faust, S. and McMahon, P. L. (1987) Improved enzyme-linked immunoassay for aflatoxin B I in agricultural commodities. JAOAC 70, 854-857. Pesavento, M. and Carter, B. (1993) Comparison of different solid phases for an ELISA based determination of aflatoxin B. In Use of Inznzobili,-ed Biological Co~~zpouwds. Guilbault G. G. and Mascini M., eds. Kluwer Academic Publishers: Dordrecht, pp. 443-452. Barnavetro, I., Solti, L., Szabo, E. and Gyongyosihorvath, A. (1997) Sensitive enzyme-linked immunosorbent assay for screening aflatoxin B-1 in food- and feedstuffs. Cered Res. Coinmun. 3. 29 1-292. Biancardi, A. ( 1997). Determination of aflatoxinm-1 residues in milk-a comparative assessment of elisa and IAC-HPLC methods. Indust. Aliment. 36, 870-876. Yu, W. J. and Chu, F. S. (1998) Improved direct competitive enzyme-linked immunosorbent assay for cyclopiazonic acid in corn, peanuts, and mixed feed. J. Agric. Food Clzem. 36, 1012-1017. Abramson, D., Clear, R. M.. Usleber. E., Gessler, R., Nowicki, T. W. and Martlbauer, E. (1998) Fusarium species and 8-keto-trichothecene mycotoxins in Manitoba barley. Cereal Cherll. 75, 137-141. Sinha, R. C. and Savard, M.E. (1996) Comparisonof immunoassay and gas chromatography methods for the detection of the mycotoxin deoxynivalenol in grain samples. Can. J. Plant Pnthol. 18, 233-236. Sinha, R. C. and Savard, M. E. (1997) Concentration of deoxynivalenol in single kernels and various tissues of wheat heads. Can. J. Plant Pathol. 19, 8-12. Maragos, C. M. and Richard, J. L. (1994) Quantitation and stability of fumonisins B-1 and B-2 in milk. JAOAC Int. 77. 1162-1 167. Shelby. R. A.. Rottinghaus, G. E. and Minor. H. C. ( 1994) Comparisonof thin-layer chromatography and competitive immunoassay methods fordetecting fumonisin onmaize. J. Agric. Food C l ~ e ~ t42, t . 2064-2067. Maragos, C. M., Plattner, R. D. and Miklasz, S. D. (1996) Determinationofhydrolyzed fumonisin (HFB1) in corn by cotnpetitive direct enzyme-linked immunosorbent assay. Food Addit. Contmt . I 3, 105- 113.

706

Chu

111. Dreher, R. M. and Usleber, E. (1996) Comparison study a fumonisin enzyme immunoassay and high-performance liquid chromatography. In Imntunoassa~s for Residue Analysis: Food Safety. Beier, R. C. and Stanker, L. H., eds. ACS, Symposium Series book no. 621. American Chemical Society: Washington, DC, pp. 341-348. 112. Pestka, J. J., Azcona-Olivera, J. I., Plattner, R. D., Minervini, F., Doko, B. and Visconti, A (1994). Comparative assessment of fumonisin in grain-based foods by ELISA, GC-MS, and HPLC. J. Food Protect. 1994, 57, 167-172. 113. Sydenham, E. W., Shephard, G. S., Thiel, P. G., Bird, C. and Miller, B. M. (1996). Determination of fumonisins in corn: Evaluation of competitive immunoassay and HPLC techniques. J. Agric. Food Chem. 44, 159-164. 114. Scott, P. M., Yeung, J. M., Lawrence and Prelusky, D. B. (1997). Evaluation of enzymelinked immunosorbent assay for analysis of beer for fumonisins. Food Addit. Contam. 14, 445-450. 115. Barnavetro, I., Solti, L., Teren, J., Gyongyosi, A., Szabo, E.and Wolfling, A. ( 1996) Sensitive ELISA test for determination of ochratoxin A. J. Agric. Food Clzem. 44, 4071-4074. 116. Frohlich, A. A., Marquardt, R. R. and Clarke, J. R. (1997). Enzymatic and immunological approaches for the quantitation and confirmation of ochratoxin a in swine kidneys. J. Food Protect. 60, 172-176. 117. Tapai, K., Teren, J. and Mesterhazy, A. (1997). Ochratoxin a in the sera of blood donors and ill persons. Cereal Res. Cormnun. 3, 307-308. 118. Tanaka, T., Teshima, R., Ikebuchi, H., Sawada, J. and Ichinoe, M. (1995) Sensitive enzymelinked inmunosorbent assay for the mycotoxin zearalenone in barley and Job’s-tears. J. Agric. Food Chem. 43, 946-950. 119. Okumura, H., Okimoto, J., Kishimoto, S., Hasegawa, A., Kawamura, O., Nakajima, M., Miyabe, M. and Ueno, Y. (1993) An improved indirect competitive ELLISA for aflatoxin M1 in milk powders using novel monoclonal antibodies. Food Agric. bnmunol 5, 75-84. 120. Okumura. H., Kawamura, O., Kishimoto, S., Hasegawa, A., Shrestha, S. M., Okuda, K., Obata, H., Okuda, H., Haruki, K., Uchida, T., Ogsawara. Y. and Ueno, Y. (1993) Aflatoxin M1 in Nepalese sera, quantified by combination of monoclonal antibody immunoaffinity chromatography and enzyme-linked immunosorbent assay. Carcinogenesis 14, 1233- 1235. 121. Itoh, Y.. Nishimura, M., Hifumi, E., Uda, T., Ohtani, K., Kawamura, O., Nagayama, S. and Ueno, Y. (1986) Detection of ochratoxin A and T-2 toxin by one step ELISA. Proc. Jpiz. Assoc. Mycotoxicol. 1986, 24, 25-29. 122. Sidwell, W. J. H., Chan, W. S. and Morgan, M. R. A. (1989) Reduction in assay time for a double antibody, indirect enzyme-linked immunosorbent assay applied to ochratoxin A. Food Agric. Irmunol. 1, 111- 118. 123. Bacigalupo, M. A., Ius. A.. Meroni, G., Dovis, M. and Petruzzelli, E. (1994) Determination of aflatoxin B-1 in agricultural commodities by time-resolved fluoroirnmunoassay and inmunoenzymometric assay. Annlyst 119, 2813-2815. 124. Sekhon, C. P. S., Kapur, J., Sodhi, S. S. and Jand, S. K. (1996) Qualitative and quantitative detection of aflatoxin B-1 in poultry sera by enzyme-linked immunosorbent assay. J. Biosci. 21, 471-476. 125. Pesavento, M., Domagala, S.. Baldini, E. and Cucca, L. (1997). Characterization of an enzyme linked immunosorbent assay for aflatoxin B-1 based on commercial reagents. Tdanta 45, 91-104. 126. Ueno et al., Ueno, Y., Iijima, K., Wang, S. D., Sugiura, Y., Sekijima, M., Tanaka, T.. Chen. C. and Yu, S. Z. (1997) Fumonisins as a possible contributory risk factor for primary liver cancer-a 3-year study of corn harvested in Haimen. China, by HPLC and ELISA. Food Chem. Toxicol. 35, 1143-1 150. 127. Xiao, H., Clarke, J. R., Marquardt, R. R. and Frohlich, A. A. (1995). Improved methods for conjugating selected mycotoxins to carrier proteins and dextran for immunoassays. J. Agric. Food Cltem. 43, 2092-2097.

Mycotoxin Analysis

707

128. Kierek-Jaszczuk, D., Marquardt, R. R. and Abramson, D. (1997). Use of a heterologous solid-phase antigen in an indirect competitive antibody-capture enzyme-linked immunosorbent assay for T-2 mycotoxin. J. Food Protect. 60, 321-327. 129. Abouzied, M. M., Askegard, S, Bird, C. and Miller, B. (1995). Fumonisin Veratox: a new rapid quantitive ELISA for determination of fumonisin in food and feed. In Funzonisins in Food. Jackson, L., DeVries. J. W. and Bullerman. L. B., eds. Plenum Press: New York. 130. Lee, L. S., Wall, J. H., Cotty, P. J. and Bayman, P. (1990) Integration of enzyme-linked immunosorbentassaywithconventionalchromatographicproceduresforquantitation of aflatoxin in individual cotton bolls, seeds, and seed sections. JAOAC 73, 581-584. 131. Park, D. L.. Miller, B. M., Hart, P., Yang, G., McVey, J., Page, S. W., Pestka, J. J. and Brown, L. H. (1989) Enzyme-linked immunosorbent assay for screening aflatoxin in cottonseed products and mixed feed: collaborative study. JAOAC 72, 326-332. 132. Park, D. L., Miller, B. M., Nesheim,S., Trucksess, M. W., Vekich, A., Bidigare, B., McVey, J. L. and Brown, L. H. (1989). Visual and semiquantitative spectrophotometric screening method for aflatoxin B in cornand peanut products: follow-up study.JAOAC 72, 638-643. 133. Patey, A. L., Shaman, M., Wood, R. and Gilbert, J. (1989) Determinationof aflatoxin concentrations in peanut butter by enzyme-linked immunosorbent assay (ELISA): study of three commercial ELISA kits. JAOAC 72, 965-969. 134. Schneider, E., Dietrich, R., Martlbauer, E., Usleber, E. and Terplan, G. (1991) Detection of aflatoxins, trichothecenes, ochratoxin Aand zearalenone by test strip enzyme immunoassay: a rapid method for screening cereals for mycotoxins. Food Agric. Immunol. 3, 185-193. 135. Beaver, R. W., James, M. A. and Lin, T. (1991) Comparison of an ELISA-based screening test with liquid chromatography for the determination of aflatoxin in corn. J. Assoc. Of Anal. Chenz. 74, 827-829. 136. Dorner, J. W. and Cole, R. J. (1989) Comparison of two ELISA screening tests with liquid Chromatography for determination of aflatoxins in raw peanuts. JAOAC 72, 962-964. 137. Stubblefield. R. D., Greer, 5. I., Shotwell, 0. L. and Aikens, A. M. (1991) Rapidimmunochemical screening method for aflatoxin B1 in human and animal urine. JAOAC 74, 530532. 138. Trucksess. M.W., Stack, M.E., Nesheim, S., Park, D. L. and Pohland, A. E. (1989) Enzymelinked immunosorbent assayof aflatoxins B B2, and GI in corn, cottonseed, peanuts, peanut butter, and poultry feed: collaborative study. JAOAC 72, 957-961. 139. Trucksess, M. W., Young, K., Donahue, K. F., Morris, D. K. and Lewis, E. (1990) Comparison of two immunochemical methods with thin-layer chromatographic methods for determi-’ nation of aflatoxins. JAOAC 73, 425-428. 140. Usleber, E., Schneider, E., Martlbauer, E. and Terplan, G. (1993) Two formats of enzyme immunoassay for 15-acetyldeoxynivalenol applied to wheat. J. Agric. Food Chem. 41,20192023. 141. Sashidhar, R. B. (1993) Dip-Strip method for monitoring environmental contamination of aflatoxin in food and feed-use of a portable aflatoxin detection kit. Environ. Herrlth Perspect. 101(Suppl. 3), 43-46. 142. Nieuwenhof, F. F. J., Hoolwerf, J. D. and van den Bedem, J. W. (1990) Evaluation of an enzyme-immunoassay for the determination of aflatoxin M1 in milk using antibody coated polystyrene beads. Milchwissensclzaj?1990, 45, 584-588. 143. Steimer, J., Hahn, G., Heeschen,W. and Bluthgen, A. (1988)On the enzyme-immunological detection of aflatoxin M1 in milk: development of a rapid aflatoxin M screening test using polystyrol beads as solid-phase. Milcheissenschafr 43, 772-776. 144. Dewey, F. M.. MacDonald, M. M., Phillips, S. I. and Priestley. R. A. (1990) Development for islundicum of monoclonal-antibody-ELISA and a DIP-STICK immunoassays Penicillium in rice grains. J. Gen. Microbiol. 136, 753-760. Fzmrium 145. DeSaeger, S. and VanPeteghem, C. (1996) Dipstick enzyme immunoassay to detect T-2 toxin in wheat. Appl. Environ. Microbiol. 62, 1880-1884.

708

Chu

146. Abouzied, M. M. and Pestka, J. J. (1994) Simultaneous of fumonisin B 1, aflatoxin B 1,and zearalenone by line immunoblot: a computer-assisted multianalyte assay system. JAOAC Int. 77. 495-501. 147. Candlish, A. A., Haynes, C. A. and Stimson, W. H. (1988) Detection and determination of aflatoxins using affinity chromatography. Int. J. Food Sci. Technol. 23, 479-485. 148. Candlish, A. A. G., Faraj, M. K., Harran, G. and Smith, J. E. (1991) Immunoaffinity column Chromatography for detecting total aflatoxins in experimental solutions. Biotechrzol. Tech. 5. 3 17-322. 149. Fremy, J. M. and Chu. F. S. ( 1989) Immunochemical methods of analysis for aflatoxin M1. In Mycotoxins in Drriry Proclucts. van Egmond, H. P., ed. Elsevier: Amsterdam, pp. 97126. 150. Groopman, J. D. and Donahue, K. F. (1988) Aflatoxin, a human carcinogen: determination in foods and biological samples by monoclonal antibody affinity chromatography. J. Assoc. Of Anal. Chew. 71, 861-867. 151. Hansen, T. J. (1990) Affinity column cleanup and direct fluorescence measurement of aflatoxin M, in raw milk. J. Food Protect. 53. 75-77. 152. Trucksess, M. W., Stack. M. E., Nesheim, S., Page, S. W., Albert. R. H., Hansen, J. T. and Donahue, K. F. (1991) Immunoaffinity column coupled with solution fluorometry or LC postcolumn derivatization for aflatoxins in corn, peanuts. and peanut butter: collaborative study. JAOAC 74, 81-88. 153. Garnder, C. R., Whattam, M. M., Taylor, P. J. L. and Stow, M. W. (1993)Analysis of United Kingdom purchased spices for aflatoxin using an immunoaffinity column clean-up procedure followed by high-performance liquid chromatographic analysis and post-column derivatisation with pyridinium bromide perbromide. J. Clzronzatogl-.648, 485-490. 154. Koeltzow. D. E. and Tanner, S. N. (1 990) Comparative evaluation of commercially available aflatoxin test methods. JAOAC 73, 584-589. 155. Dorner, J. W. and Cole, R. J. (1993) Performance of two immunochemical assays in the analysis of peanuts for aflatoxin at 37 field laboratories. JAOAC Int. 76, 637-643. 156. Goto. T. and Manabe. M. (1998) Application of ELISA to aflatoxin analysis: Results of preliminary tests of analysis kits on the market. Rep. Natl. Food Res. Zmt. 52, 53-59. 157. Trucksess, M. W. and Stack, M. E. (1994). Enzyme-linked immunosorbent assay of total aflatoxin-B 1, aflatoxin-B2, and aflatoxin-G1 in corn-follow-up collaborative study. JAOAC lilt. 77, 655-659. 158. Ware, G. M., Umrigar. P. P., Carman, A. S. and Kuan, S. S. (1994) Evaluation of Fumonitest immunoaffinity columns. Anal. Lett. 1994, 27, 693-715. 159. Sun, P. S., and Chu, F. S. (1977) A simple solid-phase radioimmunoassay for aflatoxin B, J. Food Safe5 1, 67-75. 160. Wu, S., Yang, G. and Sun. T. (1983) Studies on immuno-concentration and immunoassay of aflatoxins. J. Clin. Oncol. 5, 81-84. 161. Groopman, J. D., Hasler, J. A., Trudel, L. J., Pikul, A., Donahue P. R. and Wogan, G. N. (1992) Molecular dosimetry in rat urine of aflatoxin-N-7-guanine and other aflatoxin metabolites by multiple monoclonal antibody affinity chromatography and HPLC. Cancer Res. 52, 267-274. 162. Scott. P. M. and Trucksess. M. W. (1997) Application of irmnunoaffinity columns to mycotoxin analysis. JAOAC Znt. 80, 941-949 (review). 163. Farjam, A., van de Merbel, N. C., Lingeman, H., Frei, R. W. and Brinkman, U. A. T. ( 1991) Non-selective desorption of immuno precolumns coupled online with column liquid chromatography: detection of aflatoxins. Irzt. J. Emiron. A n d . Chem. 45, 73-87. 164. Farjam. A., van de Merbel, N. C., Niernan, H., Lingeman, H. and Th. Brinkman, U. A. (1992) Determination of aflatoxin M1 using a dialysis-based immunoaffinity sample pretreatment system coupled on-line to liquid chromatography-reusable immunoaffinity columns. J. Clwomatogr. 589. 141-149.

Mycotoxin Analysis

709

165. Biancardi, A. (1997). Determination of aflatoxinm-1 residues in milk-a comparative assessment of ELISA and IAC-HPLC methods. hdzdst. Aliment. 36, 870-876. 166. Candlish, A. A. G.. Snlith, J. E. and Stimson, W. H. (1989) Monoclonalantibody technology for mycotoxins. Biotechol. Ad!.7, 401-418. 167. Carvajal, M., Muljolland, F. and Garner, R. C. (1990) Comparison of the EASI-EXTRACT immunoaffinity concentration procedure with the AOAC CB method for the extraction and quantitation of aflatoxin B1 in raw ground unslunned peanuts. J. Chromatogr. 51 1. 379383. 168. Barmark, A. L. and Larsson, K. (1994) Immunoaffinity column cleanup liquid chromatographic determination of aflatoxins-an interlaboratory study JAOAC Int. 77, 46-52. 169. Patey.A. L., Sharman, M.and Gilbert, J. (1990) Determinationofaflatoxin B, levelsin peanut butter using an immunoaffinity column clean-up procedure: inter-laboratory study. Food Addit. Contcrm. 7, 515-520. 170. Patey, A. L., Sharman, M. and Gilbert. J. (1991) Liquid chromatographic determination of aflatoxin levels in peanut butters using an inmunoaffinity column cleanup method: international collaborative trial. JAOAC 74, 76-8 l . 171. Sharman, M. and Gilbert, J. (1991) Automated aflatoxin analysis of foods and animal feeds using imnmnoaffinity column cleanup and high-performance liquid chromatographic determination. J. Clzrov1atogr. 543. 220-225. 172. Kussak. A., Andersson, B. and Andersson, K. (1995) Determination of aflatoxins in airborne dust from feed factories by automated inlmunoaffinity column clean-up and liquid chromatography. in human urine by immunoaffinity colunm clean-up and liquid chromatography. J. ChI.o/~lc~togr.708. A 55-60. 173. Nakajirna, M., Tsubouchi, H.. Miyabe, M. and Ueno, Y . (1997) Survey of aflatoxin B1 and ochratoxin A in commercialgreen coffee beansby high-performance liquid chromatography linked with immunoaffinity chromatography. Food Agric. Inznrrlnol. 9, 77-83. 174. Holcomb, M. and Thompson, H. C. Jr. (1991) Analysis of aflatoxins (Bl, B2, GI and G2) in rodent feed byHPLC using postcolumn derivatization and fluorescence detection.J. Agric. Food CIzerrz. 39, 137. 175. Scott. P. M. and Lawrence, G. A. (1997) Determination of aflatoxins in beer. JAOAC I m . 80. 1229-1233. 176. Kussak, A.. Andersson, B.. Andersson, K. and Nilsson, C. A. ( 1998) Determination of aflatoxicolin humanurine by immunoaffinity columnclean-up andliquid chromatography. Chemosphere 36, 184 1 1848. 177. Kussak, A.. Andersson, B. and Andersson. K.(1994) Determination of aflatoxinQ(l) in urine by automated inmunoaffinity column clean-up and liquid chromatography. J. Chromntgr. B Biomed. Appl. 656, 329-334. 178. Groopman, J. D., Wild, C. P., Hasler, J., Chen. J., Wogan, G. N. and Hasler, T. W. (1993) Molecular epidemiology of aflatoxin exposures: validation of aflatoxin-N7-guanine levels in urine as a biomarker in experimentalrat models and humans. Emiron. Heultlr Perspect. 99, 107-1 13. 179. Hanison, J. C. and Garner, R. C. (1991) Immunological and HPLC detection of aflatoxin adducts in humantissues after an acute poisoning incident in S. E. Asia. Carcirzogerzesis 12. 74 1-733. 180. Harrison, J. C., Carvajal. M. and Garner, R. C. (1993) Does aflatoxin exposure in the United Kingdom constitute a cancer risk? Emiron. Health Perspect. 99, 99-105. 181. Vidyasagar, T.. Vyjayanthi, V.. Sujatha, N., Rao, B. S. and Bhat, R. V. (1997) Quantitation of aflatoxinB-1 -n-7-guanine adduct in urine by enzyme-linked immunosorbent assay coupled with immunoaffinity chromatography. JAOAC Int. 80, 1013-1022. 182. Sabbioni, G. and Sepai, 0. (1998) Determinationof human exposure of aflatoxins. In Mycoto.uins i n Agriczdture orrd Food Sofety. Sinha K. K. and Bhatnagar D. eds. Marcel Dekker: New York. pp. 183-226.

710

Chu

183. Sabbioni, G., Ambs,G.,Wogan. G. N. and Groopman, J. D. (1990) The aflatoxin-lysine adduct quantified by high-performance liquid chromatography from human serum albumin samples. Cclrcirzogerlesis 11, 2063-2066. 184. Wild, C. P., Jiang, Y. S.. Sabbioni, G., Chapot, B. and Montesano, R. (1990) Evaluation of methods for quantitation of aflatoxin-albumin adducts and their application to human exposure assessment. Carzcer Res. 50, 245. 185. Sheabar, F. Z., Groopman, J. D., Qian, G. S. and Wogan, G. N. (1993) Quantitative analysis of aflatoxin-albumin adducts. Carcinogenesis 14, 1203-1208. 186. Wild, C.P., Jiang, Y. Z., Allen, S. J., Hall, A. J. and Montesano, R. (1990)Aflatoxin-albumin adducts in human sera from different regions of the world. Ccrrcinogenesis 11, 271-2374. 187. Trucksess, M. W. andAbouzied, M. M. (1996) Evaluationand application of immunochemical methods for fumonisinB1 in corn. In Inlr?lurlonssaysfor Residue Analysis: Food Scrfev. Beier, R. C. and Stanker, L. H., eds. ACS Symposium Series book no. 621. American Chenical Society: Washington, DC, pp. 358-367. 188. Marogos, C. M. and Greer, J. I. (1997) Analysis of aflatoxin B 1 in corn using capillary electrophoresis with Laser-induced fluorescence detection. J. Agric. Food Chew. 45,43374341. 189. Marogos, C. M.. Bennett, G. A. and Richard, J. L. (1997) Affinity column clean-up for the analysis for fumonisins and their hydrolysis products in corn. Food Agric. Irnmzmol. 9, 312. 190. Duncan, K., fi-uger, S., Zabe, N., Kohn, B. and Prioli, R. (1998) Improved fluorometric and chromatographic methods for the quantification of fumonisins B-1, B-2 and B-3. J. Clzronzntogr. A 815, 41-47. 191. Nakajima, M.. Terada, H., Hisada, K., Tsubouchi, H.. Yamamoto, K., Uda, T., Itoh. Y.. Kawamura, 0. andUeno, Y. (1990) Determination ofochratoxin A in coffee beans and coffee products by monoclonal antibody affinity chromatography. Food Agric. hnturzol. 2, 189-195. 192. Sharman, M., MacDonald, S. and Gilbert, J. (1992) Automated liquid-chromatographyic determination of ochratoxin A in cereals and animal products using immunoaffinity column clean-up. J. Clzromatogr. 603, 285-289. 193. Pittet, A., Tornare. D., Huggett. A. and Viani. R. (1996) Liquid chromatographic determination of ochratoxin a in pure and adulterated soluble coffee using an immunoaffinity column cleanup procedure. J. Agric. Food Chew. 44, 3564-3569. 194. Scudamore, K. A. and MacDonald, S. J. (1998) A collaborative study of an HPLC method for detennination of ochratoxin A in wheat using immunoaffinity column clean-up. Food Addit. Corztcrm. 15, 401-410. 195. Zimmerli, B. and Dick, R. (1995) Determination of ochratoxin A at the ppt level in hunlan blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescencedetectionandimmunoaffinity columncleanup: methodologyand Swiss data. J. Clzronlatogr. B 666, 85-99. 196. Valenta, H. (1998) Chromatographic methods forthe determination of ochratoxxin A in animal and human tissues and fluids. J. Clzromatogr. A 815, 75-92. 197. Corneli, S. and Maragos, C. M. (1998) Capillary electrophoresis with laser-induced fluorescence - method for the mycotoxin ochratoxin A J. Agric. Food Cl?em. 46, 3162-3165. 198. Azcona, J. I., Abouzied, M. M. and Pestka, J. J. (1990) Detection of zearalenone by tandem immunoaffinity-enzyme-linked immunosorbentassayand its application to milk. J. Food Protect. 53, 577-580. 199. Bagnati, R. M., Oriundi, M. P., Russo, V.,Danese,M., Berti, F. and Fanelli, R. (1991) Determination of zeranol and beta-zearalanol in calf urine by inmunoaffinity extraction and gas chromatography-mass spectrometry after repeated administration of zeranol. J. Clzr-onmtogr. 564, 493-502. 200. Krska, R., Schuhmacher, R., Weingartner, J. and Grasserbauer, M. (1997) Modern sample

Mycotoxin Analysis

preparation techniques for the analysis of fusarium mycotoxins in cereals. Cereal Res. Corn25. 327-329. Schuhmacher. R., Krska, R.. Weingaertner, J. and Grasserbauer, M. (1997). Interlaboratory comparison study for the determination of the fusarium mycotoxins deoxynivalenol in wheat and zearalenone in maize using different methods. Fresenitrs J. A w l . Chem. 359. 510515. Schuhmacher, R., Krska, R., Grasserbauer, M., Edinger, W. and Lew, H. (1998) Immunoaffinity columns versus conventional clean-up-a method-comparison study for the determination of zearalenone in corn. Fresenius J. Anal. Chem. 360, 241-245. Visconti, A. and Pascale, M. (1998) Determination of zearalenone in corn by means of immunoaffinity clean-up and high-performance liquid chromatography with fluorescence detection. J. ChrotT1utogr. A 815, 133-140. Carman, A. S.. Kuan. S. S., Ware, G. M., Umrigar, P. P.. Miller, K. V. and Gurrero, H. G. (1996) Robotic automated analysis of foods for aflatoxins. JAOAC, Zrrt. 79, 456-464. Tuinstra. L. G. M. T., Roos. A. H. and Van Trijp, J. M. P. (1993). Liquid chromatographic determination of aflatoxin In( 1) in milk powder using immunoaffinity columns for cleanupinterlaboratory study. JAOAC Znt. 76, 124-1354. Urano. T., Trucksess, M. W. and Page, S. W. (1993) Automated affinity liquid chromatography system for on-line isolation, separation, and quantitation of aflatoxins in methanol-water extracts of corn or peanuts. J. Agric. Food Chenz. 41, 1982-1985. Niedwetzki, G., Lach, G. and Geschwill, K. (1994). Determination of aflatoxins in foods by use of an automatic station. J. Chronzatogr. A 661, 175-180. Jordan, L., Hansen, T. L. and Zabe, N. A. (1994) Automated mycotoxin analysis. Am. Lab. (March), 18-24. Kramer, P. M., Li. Q. X. and Hammock, B. D. (1994) Integration of liquid chromatography with immunoassay: an approach combining the strengths of both methods. JAOAC Int. 77, 1275-1287. Chu, F. S. and Lee, R. C. (1989) Immunochromatography of group A trichothecene mycotoxins. Food Agric. I m m n o l . 1, 127-136. Park, J. J. and Chu, F. S. (1993) Immunochemical analysis of trichothecenes produced by various fusaria. Mycopathologia 121, 179- 192. Park, J. and Chu. F. S. ( 1996) Assessment of Immunochemical methods for the analysis of trichothecene mycotoxins in naturally occurring moldy corn. JAOAC Int. 79, 365-371. Yu, J. and Chu, F. S. (1991) Immunochromatography of fusarochromanone mycotoxins. JAOAC 74, 655-660. Miles, C. 0..Wilkins, A. L., Garthwaite, I., Ede, R. M. and Munday-Finch, S. C. (1995). Application of immunological techniques to natural products chemistry: isolation and structure determination of paxinorol with the aid of the TLC-ELISAgram technique. J. Org. Chem. 60, 6067-6069. Pestka. J. J. (1991) High performance thin layer chromatography ELISAGRAM: Application of a multi-hapten immunoassay to analysis of the zearalenone and aflatoxin mycotoxin families. J. Imnrunol. Meth. 136, 177. Maragos, C. M. (1995). Capillary zone electrophoresis and HPLC for the analysis of fluorescein isothiocyanate-labeled fumonisin B 1. J. Agric. Food Clwm. 43, 390-394. Maragos, C. M. (1997) Detection of the mycotoxin fumonisin B-1 by a combination of immunofluorescence and capillary electrophoresis. Food Agric. Immtrrtol. 9, 147- 157. Nisonoff, A. (1991) Idiotypes: concepts and applications. J. Immurrol. 147. 2429-2438. Hsu. K. H. and Chu, F. S. (1994) Production and characterization of anti-idiotype and antianti-idiotype antibodies from a monoclonal antibody against aflatoxin B1 J. Agric. Food Chenz. 42, 2353-2359. Hsu. K. H. and Chu, F. S. (1995) Anti-idiotype and anti-anti-idiotype antibodies generated from polyclonal antibodies against aflatoxin B, . Food Agric. Imntrizol. 7 , 139-15 1. ifrUi2.

201.

202.

203.

204. 205.

206.

207. 208. 209.

210. 21 1. 212. 213. 214.

215.

216. 217. 218. 219.

220.

71 I

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Chu

221. Hsu. K. H. and Chu, F. S. (1995) Anti-idiotype and anti-anti-idiotype antibodies for a aflatoxin from laying hens. Food Agric. Inzinunol. 7. 163-174. 222. Chanh, T. C.,Huot, R. I., Schick, M.R. and Hewetson, J. F. (1989) Anti-idiotypic antibodies against a monoclonal antibody specific for the trichothecene mycotoxin T-2. Toxicol. Appl. Phnrrizacol. 100. 20 1-207. 223. Chanh, T. C., Kennedy. R. C. and Hewetson. J. F. (1992) Anti-idiotype vaccines in toxicology. Int. J. Clin. Lab. Res. 22, 28-35. 224. Hsu, K.-H. (1995) Idiotype, anti-idiotypeand anti-anti-idiotype antibodies for aflatoxin: production. characterization and application. Ph.D. dissertation,University of Wisconsin-Madison, Madison. 225. Lu, P., Marquardt. R. R., Frohlich, A. A. and Mills, J. T. (1994). Detection of PenicilliLm nrcrantiogriseum by ELISA utilizing antibodies produced against its exoantigens. Micr-obiol. UK 130, 3267-3276. 226. Lu, P., Marquardt, R. R. and Kierekjaszczuk, D. (1995) Immunochemical identification of fungi using polyclonal antibodies raisedin rabbits to exoantigens fromAspergillus oclzraceus. Lett. Appl. Microbiol. 20, 41-45. 227. Tsai, G. J. and Cousin, M. A. (1990) Enzyme-linked immunosorbent assay for detection of molds in cheese and yogurt. J. Dcri?y Sci. 73, 3366-3378. 228. Tsai, G. J. and Cousin, M.A. (1993)Partial purification and characterization of mold antigens commonly found in foods. Appl. Environ. Microbiol. 59, 2563-2571. 229. Lee, R. C., Cary. J. W., Bhatnagar, D. and Chu, F. S. 1995. Production and characterization of antibodies against norsolorinic acid reductase. Food Agric. Imnrlnol. 7, 21-32. 230. Liang, S. H., Wu, T. S.. Lee. R.. Chu, F.S. and Linz,J. E. (1997) Analysis of the mechanisms regulating the expression of the ver-I gene involved in aflatoxin biosynthesis. Appl. Emiron. Microbiol. 63, 1058-1065. 231. Lee, R. C. and Chu, F. S. (1999) Production and characterization of monoclonal antibodies against norsolorinic acid reductase. Food Agric. h l 1 7 l l l 1 2 0 ~ .11, 29-42. 232. Liu, B. H., Keller, N. P., Bhatnagar, D., Cleveland, T. E. and Chu, F. S. (1993) Production and characterization of antibodies against sterigmatocystin 0-methyltransferase. Food Agr-ic. Irnmtlnol. 5. 155-164. 233. Liu, B. H. and Chu. F. S. (1997) Production and characterization of monocloanl antibodies against sterigmatocystin 0-methyltransferase. Food Agric. Imnunol. 9, 167-176. 234. Liu, B. H., Brewer, J. F., Flaherty, J. E., Payne, G., Bhatnagar. D. and Chu, F. S. (1997) Immunochemical identification of AFLR, a regulatory protein involved in aflatoxin biosynthesis. Food Agric. Inrmunol. 9, 289-298. 235. Park, J. and Chu, F. S. (1996) Imlnunochemical studiesof an esterase from Fusarizrrn sporotrichioides. Food Agric. Iwr~r~cnol. 8, 4 1-49. 236. Hohn, T. M., McCormick. S. P. and Desjardins, A. E. (1993) Evidence for a gene cluster involving trichothecene-pathwaybiosyntheticgene in Fzrsarirm sporotrichioides. Cwr. Genet. 24. 291-295. 237. Candlish, A. A. G., Wibowo, M. S. and Smith, J. E. (1997) Immunoassay identification of AspergillusJlcrvususing monoclonal antibodies raised to the whole cell extracts. Bioteclznol. Tech. 11.21-23. 238. Shapira, R., Paster, N., Menasherov, M., Eyal,0..Mett. A., Meiron, T., Kuttin, E. and Salomon, R. (1997) Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Esclrericlzia coli. Appl. Environ. Microbiol. 63, 990-995. 239. Warden, B. A., Allam, K., Sentissi, A., Cecchini. D. J. and Giese, R. W. (1987) Repetitive hit-and run fluoroimmunoassay for T-2 toxin. Anal. Bioclzenz. 162. 363. 240. Warden, B. A., Sentissi, A., Ehrat, M., Cecchini, D. J.. Allam, K. and Giese, R. W. (1990). Chromatographic enzyme immunoassay for T-2 toxin. J. I~nmunol.Met??.131, 77. 241. Ligler, F. S., Bredehorst, R., Talebian. A., Shiver, L. C., Hammer. C. F.. Sheridan. J. Vogel.

Mycotoxin Analysis

242. 243.

244. 245.

246.

247. 248. 249.

250.

251.

252. 253.

254. 255. 256.

257.

713

P. C. and Gaber, B. P. (1987) A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes. monoclonal antibodies, and complement. Anal. Bioclzern. 163, 369-375. Degan. P., Montagnoli, G. and Wild. C. P. (1989) Time-resolved fluoroimmunoassay of aflatoxins. Clh. Cllenl. 435, 2308-2310. Williamson, M. L., Atha, D. H., Reeder, D. L. and Sundaram, P. V. (1989) Anti-T 2 monoclonal antibody immobilization on quartz fibers: Kinetics of inactivation an recognition of T-2 mycotoxin. A w l . Lett. 22, 803-816. Thompson, V. S. and Maragos, C. M. (1996) Fiber-optic immunosensor for the detection of fumonisin B-1. J. Agric. Food Chem. 44, 1041-1046. Strachan, N. J. C., John. P. G. and Millar, I. G. (1997) Application of an automated particlebased imrnunosensor for the detection of aflatoxin b-1 in foods. Food Agric. Immztnol. 3, 177-1 83. Yacoub-George. E., Wolf. H., Koch, S. and Woias, P. (1996). A miniaturized ISFET-ELISA system with a pretreated fused silica capillary as reaction cartridge. Sensors Actuators B Clrenl. 34. 429-434. Mullett, W., Lai, E. P. C. and Yeung, J. M. (1998) Immunoassay of fumonisins by a surface plasmon resonance biosensor. Anal. Bioclzem. 258, 161-167. Hsieh, L. L. and Hsieh, T. T. (1993) Detection of aflatoxin B 1-adducts in human placent and cord blood. Cancer. Res. 53, 1278-1280. Santella, R. M.. Zhang, Y. J., Chen, C. J., Hsieh, L. L.. Lee, C. S., Haghighi, B., Yang, G. Y., Wang, L. W. and Feitelson, M. (1993) Immunohistochemical detection of aflatoxin B 1-DNA adducts and hepatitis B virus antigens in hepatocellular carcinoma and nontumorous liver tissue. Envirow. Health Perspect. 99, 199-202. Beier, R. C., Elissalde. M. H. and Stanker, L. H. (1996) Molecular modeling studies of the fumonisin mycotoxins. In Irwnunoassays for Residue Analysis: Food Safe?. Beier, R. C. and Stanker, L. H., eds. ACS Symposium Series book no. 621. American Chemical Society: Washington, DC, pp. 368-385. Holtzapple, C. K., Carlin, R. J., Rose, B. G., Kubena, L. F. and Stanker, L. H. (1996) Characterization of monoclonal antibodies to aflatoxin ml and molecular modeling studies of related aflatoxins. Mol. Inzmunol. 33, 939-946. Tseng, T. C. J. (1994) Recent aspects of aflatoxin research in Taiwan. Toxic01 Toxin Re]?. 13, 229-241. Yuan, Q., Clarke, J. R.. Zhou, H. R., Linz, J. E., Pestka, J. J. and Hart. L. P. (1997)Molecular cloning. expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone. Appl. Emiron. Microbiol. 63, 263-269. Zhou, H. R., Pestka, J. J.and Hart, L. P. (1996) Molecular cloning and expression of recombinant phage antibody against fumonisin B-1. J. Food Protect. 59, 1208-1212. Freemantle, M. (1999) Downsizing chemistry. Chem Eng. News. (Feb. 22), 27-36. Yu, Wanjun and Chu, F. S. (1999) Rapid and sensitive tandem immunoaffinity column cleanup and high perfomlance liquid chromatography for the determination of filsarium mycotoxin deoxynivalenol in wheat and corn. Mycotoxins: Supplement '99. 182-187. Yu, Feng-yih and Chu, F. S. (1999) Production and characterization of a monoclonal antianti-idiotype antibody against fumonisin B-1. J. Agric. Food Clzem. 47, 4815-4820.

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18 Mushroom Biology: General Identification Features David G. Spoerke, Jr. Br-istlecone Enterprises, Denver, Colorado

I.

General Biology 715

11. GrowthHabitat/Requirements719 111. Identification of theMushroom720

A. B. C. D. E. F. G. H. I. J. K.

Mushroom keys 720 Mushroom photographs 721 Fieldguides and expertidentifiers721 Collectingmushroomsamplesforidentification721 Macroscopicphysicalstructures and characteristics722 Spore prints 725 Smells and tastes 727 Chemical keys 727 Microscopic characteristics 727 Other field tests orfactorsuseful in identification733 Preservingmushroomsfor later examination733

IV. Mushroom Look-Alikes 733

A. B. C. D. E. F. G. H.

Blue chanterellelhorn of plenty 733 Boletus edulislother Boletus species 734 ChanterelleslGomphus Jloccosus 734 ChanterelleslOnzphalotus olenriuslHygropkoropsis aurantiaca 734 False truffles (Hymenogastralesfamily) and Scleroderma 734 Lepiotcr rhacodeslChloroplzyllurn molybdites 734 Moreldfalse morels 734 Puffballslamanitaslsclerodermas 735 References 736

I. GENERAL BIOLOGY The term mushroom comes from the Greek word mykes. The study of mushrooms is mycology (mykes, meaning mushroom, and logos, meaning discourse). Actually it ismore correct according to Greek grammar to call the study mycetology (l), but this term is seldom used. Mushrooms are some of the most visible of the fungi, and have been studied 715

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Spoerke

by scientists even before the microscope was available. The “modern” study of fungi, after the discovery of the microscope by van Leeuwenhoek, is thought to have been greatly advanced by the Italian botanist Pier Antonio Micheli. He published, in 1729, the Nova Plantar-unz Genera, which included his studies on various mushrooms. Fungi are difficult to define and categorize. Fungi are neither plants nor animals exactly, having some of the characteristics of both, and characteristics found in neither. They reproduce both sexually and asexually, and may have cell walls composed of chitin, cellulose, or both (1). Most cell walls are multilaminate (2) and composed of fibrils consisting of polysaccharides, proteins, lipids, and other substances. They usually have threadlike bodies, true nuclei in their cells, reproduce by spores, and have no chlorophyll. Fungi play important roles in nature and in the lives of humans. They are responsible for human, animal, and plant diseases, destruction of organic matter such as food, leather, cloth, and paper, and are involved in the process of making bread, alcoholic beverages, cheeses, chocolate, organic acids, and a few drugs such as various antibiotics, ergot alkaloids, and vitamins. They are nature‘s great recyclers. The study of fungi usually includes the rusts, the stnuts, cup fungi, truffles, mildews, slime molds, yeasts, and mushrooms. Some fungi bear their spores on a large, visible structure called a mushroom. Mushrooms and toadstools are generally considered macromycetes of the order Agaricales. They are fleshy, occasionally tough, sporophores that have their spore-producing basidia on the surface of gills, pits, or tubes. The actual tissue of the mushroom is comprised of tightly packed dikaryotic hyphae. Other fungi, such as smuts, rusts, molds, and mildews, generally called micromycetes, usually bear their spores on mycelium filaments (3). There is no difference between mushrooms and toadstools. It has become popular to call macromycetes that are safe to eat nmshrooms, and those potentially poisonous, toadstools. In this chapter we will call all the macromycetes mushrooms. Actually, the mushroom is just the fruiting body of the actual fungus, which is a mycelium found in the growth substrate (e.g., earth, dung, wood) (4). The actual reason that a mushroom is formed is not clearly understood. Certainly factors such as light, moisture, temperature, nutrients, and aeration bear on this development. Studies have been done on various fungi (such as Agaricus brumesceru, Flar~zrnulirzavelutipes, Copr-inus stercol-ar-ius,Coprinus rcldiatm, Coprirzus lagopus, Clitocybe illldem, and Boletus rubi~zellus)in culture (5-17), but the exact relationship of these factors has not been determined. Often when mushrooms are pulled from a stump or the ground a cobweb-like substance may be seen hanging from the bottom. This material is the plant itself, the mycelium (18), not the “roots.” Thus, picking mushrooms is like picking an apple from a tree. It does the plant little harm. One should be careful to take only a small amount, or no part, of the earth or wood, so as not to hurt the actual plant. Taking a large part would be like breaking off an entire branch of an apple tree to pick just one apple. The life cycle of a mushroom starts with spores that germinate to form a mycelium, which in turn forms the mushroom primordia, which expands into the macroscopic structure we call a mushroom. The mushroom then produces new spores and the cycle repeats (19). Mushrooms plants (mycelium) do not contain chlorophyll; they obtain nutriments by being parasitic or saprophytic on other organisms. Often mushrooms develop special synlbiotic relationships with specific plants or classes of plants, trading nutrients to the benefit of both (20). This association is known as a mycorrhizal association (21). Hacskaylo (22) defined the relationship as a “physiologically well balanced reciprocal parasitism.” Literally, mycorrhizal means fungus roots (19) and refers to the root-like functions

Mushroom Biology

717

performed by some mushrooms associated with large plants, like trees. Knowing these mycorrhizal relationships may help identify a mushroom. At one time mycorrhizal relationships were thought to be rare, but it is now known that these relationships may be the rule rather than the exception. Not all relationships are equal. The amount a particular mycelium benefits the host varies considerably, but many plants would not be able to live in various hostile environments without their mycorrhizal fungi (22). The very fine mushroom hyphae may form endomycorrhizal associations where the hyphae grow intracellularly. penetrating the epidermal cells and cortical cells of the root hairs, or the association may be ectomycorrhizal. where the hyphae contact the smallest rootlets of the tree or plant and form a sheath-like covering of cells surrounding the rootlet. Endomycorrhizal associations occur most often with angiosperms (conifers). Ectomycorrhizal relationships often result in the tree root hairs being covered with a mantle of hyphae. Strands of hyphae extend out into the surrounding soil and collect nutrients for the planthree. Therootlet does not then develop root hairs and uses the hyphae instead. Ectomycorrhizal associations occur most often with plants such as willow, oak, beech, and pines (1. 23). This relationship develops all through the tree’s root system benefiting both plants. The fungus provides water, nitrogen, phosphorus, potassium, and other soluble minerals to the tree, while the tree provides carbohydrates (24). The fungi may also protect theplant rootlets from pathogens, provide growth hormones, and move carbohydrates (22). The mycelial hyphae are smaller and denser than those of the roots of the trees, allowing the tree (via the mycorrhizal relationship) to access many times the volume of soil and nutrients it normally could with just roots. Mycorrhizal fungi are often less capable of breaking down complex carbon compounds than true saprobes; thus the tree provides some of these nutrient needs (19). The specific planthee connection may vary indifferent parts of the country or the world. Some trees are able to form relationships with a wide variety of mushrooms. For example, the Douglas fir [Pseudotssugnnzellziesii (Mirb.) Franc01 is able to form relationships with almost 2000 species (25). Many keys contain this information as an identification aid. Knowing the type of tree or other plant in the area may help identify the mushroom. Mycorrhizal relationships play an important part in reforestation, reclamation of strip-mined areas, development of better producing forests, and introduction of various exotic plant species. When a forest with anextensive mycorrhizal associations iscut down, the mushrooms surrounding the roots of those trees die, and the mushrooms are not seen again until another, species-appropriate, forest is well established. This may take 20 years or more (19). For a more detailed description of the role of mycorrhizal relationships see Marks and Kozlowski (26), Hacskaylo (22), and Harley (27). A partial list of mycorrhizal associations is found in Table 1. The mushroom or fruiting body usually produces its spores (reproductive units) on the outside of microscopic, club-shaped structures call basidia. Mushrooms that reproduce this way are called Basidiomycetes. If the spores are produced inside microscopic, saclike cells called asci, they are called Ascomycetes (28). Millions of spores are produced by a single mushroom and are carried away to find a suitable growth site. Only a few find acceptable sites. The spore then puts out a germ tube, which then branches forming thread-like cells known as hyphae. If two spores from opposite strains (think of them as sexes) germinate near each other, they unite to form hyphae with two nuclei, which then grow rapidly into a network of these filaments called a mycelium or spawn. The ends of themycelium produce enzymes that helps to digest the substrate (18). Fungal hyphae grow only from the tip of the strand (1). A mushroom is formed after the mycelium has stored enough food and the environmental conditions are right. A group of hyphae form

Spoerke

718

Table 1 Examples of MycorrhizalAssociations ~~

species

~

~~~

~~~

Mushroom Amanita species Amanita bisporigem Amanita muscaria

Castnnopsis species Oak (Quercus) Aspen (Popuhts),pines (Pinus ponderosa, Pinus confora lutifolia, Douglas fir (Pseudotsuga tasifolia) Amanita pnntherinn Pines (Pinus ponderosa,Pinus contora latifolia), spruce (Picen engelmannii) Amanita rubexens Quereus species Amanita vngincrta Pinus species, Pseudotsuga species Amnnitn vernu Betula species, Pop~tlusspecies Armillaria ponderosa Pines (Pinus contorta and other Pinus species) Boletus edulis Pines (Pinus). Douglas fir (Pseudotsuga) Boletus zelleri Douglas fir (Pseudotsuga) Cnnthnrellus species Spruce (Picea abies),fir (Pseudotsuga) Clitocybe nuda Cottonwoods ( P O ~ L I ~ L I S ) Cortinarius ci~zna~~~oneus Douglas fir (Pseudotsuga) Cortinnrius collinitus Aspen and cottonwood (Popzdzrs) Dentinurn repandum Douglas fir (Pseudotsuga) Flammdirza velutipes Aspen (Populus), elm (Ulnzzls) Fuscoboletinus ochraceorosezts Western larch (Lari-xoccidentdis) Gonyhidirls glutinosus Douglas fir (Pseudotsuga) Gornphiclius rutilzls Lodgepole and ponderosa pine (Pinus) Gomphidius subroseus Firs (Abies) Gomphidius linicolor Lodgepole pine (Pinus) Hericium abietis Firs (Abies) HJdnum imbricatum Pinus species, Douglas fir (Pseudotsuga) Hydrophorous chrysodon Douglas fir (Pseudotsuga) Laccarin laccatn Pines (Pinus), Douglas fir (Pseudotsuga) Lactarius deliciosus Ponderosa pine (Pinus), Douglas fir (Pseudotsuga), Engelemann spruce (Picea) Lnctnrius tominosus Birch (Betula) Lnctarius r@ts Spruce (Picen),pines (Pinus),Douglas fir (Pseudotsllga) Leccirzum atrostipitatum Birch (Betula papyrifern) Leccinzrm aurantiacunl Aspen (Populus),lodgepole pine (Pinus) Marasrnius oreades Ponderosa pine (Pinus) Ompltalotus olearia Oak (Querczls) Pholiota squarrosa Birch (Betula), aspen (Populus) Pisolitlzus tinctorius Douglas fir (Pseudotsuga) Pleurotus ostreatus Aspen (Populus),cottonwood (Populus). maple (Acer) Rhi,-opogon rubescens Douglas fir (Pseudotsuga) Rztssulu enteticn Englemann spruce (Picea), Douglas fir (Pseztdotsztga) Russula foetens Douglas fir (Pseudotsuga) Russula sermrrpelirzn Douglas fir (Pseudotsuga) Scleroderma species Douglas fir (Pseudotsuga) Suillus americanus White pine (Pinus monticoloa) Suillw brevipes Pines (Pinus) Suillus gram4lntus Lodgepole pine, white pine, ponderosa pine (Pinus) Douglas fir (Pseudotsugn) Suillus tomelztosw Lodgepole pine, ponderosa pine (Pinus) Tricholoma jlm~ovirens Lodgepole pine (Pinus) Tricholoma nzagnivelare Pines (Pinus contorta and other Pinus species) Tuber gibbosum Douglas fir (Pseudotsuga menziessi)

Ref. 59 59 59 59 59 59 19 59 59 19, 59 59 59 59 59 59 46 59 59 59 59 58 59 59 59 59 59 59

46 59 59 59 59 59 59 59 59 59 59 59 46 59 59 59 59 19 19

Mushroom

Biology719

a concentrated knot that is the mushroom. Although mushrooms lack sex organs, they do reproduce sexually, the dividing of the chromosomes occurring in the basidia or asci, and the recombining in the early hyphae stage. A few brief comments should be made about the naming of mushrooms. As with other organisms, the scientific name is a binomial usually from Latin or Greek derivation. The first name is capitalized, is most often a noun, and is the genus name. The second name is the species, and is often an adjective, describing the mushroom. Terms such as var. or cult. are sometimes seen after the species names. These terms further divide a species into varieties or cultivars. Binomials are always italicized. The mushroom name is often followed by the name of the person who named it or an abbreviation of that name. Sometimes there are two names, one of which is in parentheses. The name in parentheses is that of the person who first gave the mushroom its name, but the current name is not the one originally given. The second personal name is that of the person who assigned the current name. For example, the name Aplurzes treleuseanus (Humphrey) Coker is the name given the fungus by Coker in 1923. Humphrey actually described the species first, in 1983, but called it Saprolegina treleaseana Humphrey, a name no longer used for this species (1). Although it seems confusing at first, the system actually helps identify species when two different investigators used different names for the same species.

II. GROWTHHABITAT/REQUIREMENTS Unlike green plants, sunlight is not required for fungal growth. It is required for spore formation in some species (29), and plays an important part in spore distribution. Many fungi are positively phototrophic, and will discharge their spores toward the light (30). The effect of sunlight on some species is localized and is not transferred through the mycelium to other, nonilluminated portions of the fungal body (thallus) (3 1). Sunlight does alter growth somewhat by changing the water content and temperature of the mushrootn substrate. Sunlight may also alter the color of the mushroom somewhat, confusing possible identification. The amount of wind at the growth site also alters the atnount of moisture, and thus the growth of the mushroom. Temperature is also important. While most fungi will grow between 0°C and 35"C, the optimum temperature is 20-30°C. While in a dormant state, fungi can withstand very low temperatures. Some cultures are stored in liquid nitrogen at - 196°C. There are also a few thermophilic mushrooms that may tolerate temperatures of 50-60°C (32). Fungi usually prefer to grow in an acidic medium, a pH of 6 being near optimum (I). Fungal growth is more or less spherical when nutrients are equal in all directions. In nature this is seldom true and most mushroom mycelia start more or less circular, and move out from a central point in relation to the nutrients available. Mushroom mycelia grow on a number of different materials (substrates), such as earth (terrestrial), dung (coprophagic), wood (lignicolous), and living organisms (parasitic) (33, 34). Few fungi that produce mushrooms are parasitic but a few genera (Cordyceps, Asterophora, Hlpomyces, Sepedonium, Spnmsisrcrdicata,Armillnrielln mellea)and some polypores grow on insects, trees, or other mushrooms (35,36). Most mushroom-producing fungi are saprophytic, meaning they grow on dead or decaying matter. Some, like Arnzillnrielln mellea, may be parasitic under some conditions and saprophytic under others (facultative parasites or facultative saprobes). Fungal organisms that must use live organisms or dead matter are obligate parasites or saprobes (37). Under favorable conditions the

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growth of fungal organisms is unlimited. Some colonies are known to be 400 years or more old (38). It is thought that some mycelia, but not the individual fungal cells, may be thousands of years old (1). Mushrooms do not have to absorb their own proteins, but are able to synthesize their own if given carbohydrates and other nutrients in some form. Most find maltose or glucose best. Most fungi also require various inorganic tninerals such as boron, carbon, copper, hydrogen, manganese, magnesium, molybdenum, nitrogen, oxygen, phosphorous, potassium, iron, sulfur, and zinc. Some fungi also require calcium. Excessive food is stored as lipids or glycogen (1). The substrate on which a fungus may exist is greatly influenced by what enzymes the hyphae are able to manufacture and secrete. Mushrooms can form mycorrhizal relationships with more than one type of organism (usually trees). As anexample, the edible mushroom Cmzthnrellus cibaris forms a relationship with beeches, firs, hornbeams, and oaks (39). Fairy rings are created as a mycelium grows out from a central point. The mushrooms sprout from the edges of the mycelium, thus producing a ring of mushrooms (40).

111.

IDENTIFICATION OF THE MUSHROOM

Excluding mushroom poisonings caused by a curious child biting into a mushroom, the most common cause of these poisonings is misidentification of mushrooms intended for the table. Mushroom identification is important and usually fairly difficult for the novice. Mushrooms should always be positively identified before they are eaten. If illness develops and mushrooms are suspected, mushroonl identification should be attempted by the physician or other qualified specialists. Proper identification of the mushroom species involved is important not only for the case being treated, but also to help evaluate symptom complexes and treatment measures for future cases (41). Below are listed several methods and characteristics used to help mushroom identification.

A.

Mushroom Keys

Mushroom keys are important aids in identification (42). To use the keys successfully it is necessary to know something about the various structures and the terminology used by mycologists. Keys utilize characteristics like spore color, substrate, flesh color, smell, taste, season of the year, spore size and shape, and macroscopic structural characteristics to determine identity. As an example, a key might start out by asking whether the mushroom is parasol-shaped with gills or spongy material on the underside, round, or some other asymmetrical shape. An investigator would obtain a spore print and determine shape and color of the print. The microscope is used to determine the shape and type of spores. The type of substrate material the mushroom arose from would be established, and the smell (if any) of the mushroom determined. Taste is not usually recommended as an initial evaluation method. Most keys require a sport print early on, but a different key was developed by Laessoe et al. (24). This key starts with the shape of the fruiting body, then some gross characteristics like pores/tubes, teeth, folded/pitted cap or gills under the stem, and utilizes spore color at a later stage. Spore color is still important, but not as early in the identification process.

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B. MushroomPhotographs Mushroom photographs are most often seen in field guides (see below), but may also be part of a collection by a club, an individual’s collection, or even part of a cookbook or lay journal article. The general mushroom structures may be shown with a photograph, but there is often a problem in finding a “standard” mushroom to illustrate the range of colors and shape alterations possible for a single species. Color photographs can be an aid to identification, but should never be the sole method used. Some books have color keys to help in the standardization of colors, but there are very few. In some ways books that have black-and-white photos are of greater value, since they force the key user to concentrate on structure and form and not so much on color (28). C.

FieldGuidesandExpertIdentifiers

Several groups may be able to provide assistance in identification. Some poison information centers have experts who may be of help, or they may know of someone in the community with this expertise. Universities also often have an expert, who can be of assistance. Many communities have mushroom clubs that have members with years of experience in identifying the local species. These individuals may have seen a single species, with its many color, shape, and texture differences over many years. This experience will aid them in identification. When further expert advice is not available, field guides may be of some value. There is always a risk in using a field guide. The species presented in the book are often not the same ones found locally and the mushroom in hand almost never looks exactly like the one in thephotograph. There are usually several mushrooms that are close approximations, some of which may be toxic, others harmless. Additional information may be required to help with the gross characteristics seen in the field guide photographs. A hand lens is helpful in identifying characteristics such as “warts,” hairs, or filaments on the mushroom or the attachment of gills to the stem of the mushroom. A microscope may be used to examine spores of the mushroom. Various stains are used to help identify certain genus or species characteristics. D. CollectingMushroomSamples for Identification When collecting mushroom samples for identification (either by another expert or for identification after a poisoning), lay individuals should be instructed to collect samples by digging up the entire mushroom, not just breaking it off at the surface from which it grows. Breaking off the mushroom may damage or obliterate structures necessary for identification. Specimens should be in separate containers (usually waxed paper) placed in a wicker basket or paper bag, with notes about the mushroom’s environment (43, 44). Information such as the types of trees it was near, what it was growing on (earth, dung, wood), the date of collection, collection site (i.e., Lost Park cemetery, Mt. Mushroom, Uncle John’s pasture), the weather conditions, abundance, mushrooms found nearby, location (hillside, streamside, pasture), any bruising reactions, and whether it was found singly, in rings, or in a large group. Many of the important identification characteristics can be lost by improper collection techniques. The collector can also miss the type of mushroom he is looking for. In the Rocky Mountains if an Amanitn muscarin is found, there is a good chance the place is right for the edible Boletus edulis (43). A sample of such a

Location:

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Table 2 Formfor Collecting Mushrooms ~~~~~

Collection Nearby date: Growing on: Altitude: Other nearby mushrooms:

trees: Single/groups:

The blank side of the paper used to record growing conditions may be used to make a spore print.

collection sheet is given in Table 2. The written side of thepaper is used to record growing conditions and collection facts; the blank side may be used to make a spore print by wrapping the paper and tnushrootn (gills down) in waxed paper. A well-prepared collector will have a pencil and notebook for writing down the above variables, a knife or dandelion digger for digging specimens, binoculars for scanning a hillside for likely specimens, a hand lens ( lox) for examining the mushrootns, waxed paper, a field guide, a tweezers for removing foreign objects or sampling part of the mushroom, and a collecting basket (not a plastic sack). Some additional items that maybe useful in the woods are a first aid kit, map, whistle, flashlight, fire starter, insect repellent, and some extra food (44). Many people like to take a camera with closeup lens as well. Sometimes a miniature tape recorder can be used to provide the information above instead of a lot of writing. Be sure to give the specimen a number that you can refer to in your dictation. Although it would seem to be common sense, it should be mentioned that mushrootns are generally delicate and should be handled as such. Bouncing them around in the back of a truck, throwing heavy mushrooms on top of fragile, stnaller specimens, leaving them in the heat of a car, or mashing together a number of species and specimens in one paper container is bound to make identification difficult. A few conservation tips for those collecting mushrooms (43). Gather only those mushrooms you are planning to keep for food or study. Try to keep the mushrootn substrate intact as much as possible-avoid digging up wide areas surrounding the mushroom or smashing apart a log. Try to return the gathering site to its original form asmuch as possible. As golfers would say, “Replace your divots.’’ If you find a beautiful specimen you do not intend to eat or study, take a picture rather than the tnushroom itself.

E. Macroscopic Physical Structures and Characteristics Mushrooms may be found singly or in groups whose stems are usually clustered together (cespitose) Below are some general characteristics that can be used to start with mushroom identification (45). 1. Color As mentioned above, color is one of the most noticeable aspects of mushrooms, but it also is highly variable and may be deceptive. Often the color is used as the primary feature for identification, leading to misidentifications. The color can depend on age, amount of light exposure, environmental dyes from other plants, mushrooms, or minerals, and the amount of oxidation. Color should be used as one of several characteristics that provide an identification. Examples are numerous. Take, for instance, Arrmzitn muscarin. Most mushroom books would describe this as a mushroom with a bright-red cap. Actually the

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cap may be orange, yellowish, or even white depending on the part of the country it is found, its age, and whether it has been growing under some other plant. The facts that A. rmscnricr also has white patches on its cap, a volva at its base, and a white spore print should also be used toconfirm the identification. Some mushrooms contain chemicals that change color as they oxidize. Thus cutting, bruising, or scarring a mushroom may elicit a color change. This is seen in some Agaricus, which bruise yellow, and some Boletus, which bruise red or blue.

2. Consistency Mushrooms do not have the same consistency or texture. Some may be more rubbery, droopy, brittle, woody, tough, corky, or spongy. Often keys will describe a mushroom using these terms. Mushrooms shaped like the grocery store mushroom have some common physical characteristics. The common names for these structures are used in this chapter, with the mycological terms in parentheses. There is usually a cap (pileus), which is most often an umbrella-shaped structure, with a stem (stipe) and base. Some species have a ring on the stem (annulus) and a cup surrounding the base (volva). Under the cap there may be gills (lamellae) or a sponge-like material (pores). On the cap there may be warts (universal veil remnants), an edge (margin), and many different textures. The flesh can be evaluated from several places on the mushroom.

3. Size It is obvious that some mushrooms are large (Boletus edulis), some are small (Mycenn species), and some intermediate in size (Agaricus bisyorus). Size is usually determined genetically. Within each of these genetic ranges there is a wide variation, which depends on amount of substrate, water, and the age of the mushroom. Many mushroom keys provide a size to be used as a general rule, but one should always take into consideration the amount of water and age when determining whether this mushroom fits into the general description. Some keys give the size in inches, others in centimeters. The conversion is 1 in. = 2.54 cm. 4. Shape The fruiting bodies of fungi have different general shapes. Some are ball-shaped, others cup-shaped, lobed, bracket-shaped, nest-shaped, crust-like, coral-shaped, club-shaped, pear-shaped, phallic-shaped, star-shaped, trumpet-shaped, or pestle-shaped (24). Various mushroom structures, like the cap, stem, gills, etc., come in different sizes, shapes, and forms. Knowing these variations can aid in identification.

5. Annulus The “ring” on the stem is what remains on the stem after the mushroom cap has opened up. In many species part of it remains circling the stem; sometimes some is attached to the edge of thecap. The presence of a ring, and the typeof ring, are identification characteristics. Chlol-ophyllurn molybdites has a thick, heavy ring that is easily movable on the stem. Some rings are sheath-like, pointing toward the cap. Others are very fragile and thin. An annulus can also be almost skirt-like on the stem.

6. Caps There are many different types of cap shapes, with varying degrees of each type. Although cap shape is an important characteristic, it is also important to realize that various environ-

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mental factors can alter the shape. Mushrooms that have to push through hard soil, those that grow around or through wood, or a cap that is in the sunlight only on one side may be deformed. The types of shapes include flat (plane), bell-shaped (campanulate), dented in the center where the stem attaches (depressed), funnel-shaped (infundibuliform), flat on the bottom and round on top (convex), knobby at the center of the top (umbonate), folded, or varying degrees of cone-shaped (conic) (18). The cap surface is often described and may be granular (granulose), hairy to scaly (fibrillose-scaly), shingle-like (imbricate), woolly (tomentose), hairy (hirsute), silky (fibrillose), pleated (plicate), striped (striate), mica-like (micaceous), net-like (reticulate), or patchy (aerolate). How the cap reacts to water may provide an identification clue. The cap surface may be slightly sticky to the touch (viscid), somewhat slimy (glutinous), or very slimy (covered with a thick, mucous-like slime). Cap edges (margins) also vary considerably. Slicing the mushroom in half allows for better visualization of the cap edge. Edges may be smooth (entire), scalloped (crenate), grooved (plicate), finely split (striate), less finely split (sulcate), randomly split or damaged (eroded or rimose), or toothed like icicles on a roof edge (appendiculate). The edges may also have different degrees of inward or outward rolling. If they are mainly straight across, they are called straight. If they curve inward, they are incurved; if greatly turned inward, they are called inrolled or involute. If the turn is out and up, they are called upturned. The, skin of the cap (cuticle or pellicle) is also used for evaluation by color changes, texture, and ease of removal, especially in some of the Boletaceae (46).

7. Flesh The flesh from the stem or cap may be evaluated for color, color changes, odor, taste, texture, and thickness (28). A bruised Lclctarius deliciosus stains a greenish color; some Boletus species bruise blue; some Agaricus species, yellow. 8. Gills The gills or lamellae may be different in color from the stem or the cap, have different shapes, and different attachments to the stem. Color may be white to gray, yellow, buff, brown, green, pink, red, purple, or black (47). The color of the gills often changes as the mushroom matures, and this change is important to recognize. It is most helpful to have both an older and younger specimen to confirm a change, if any. As an example, the meadow mushroom, Agaricus carnpestris, has first white, then pink, then brown gills as the spores on the gills mature. The type and arrangement of the gill's inner tissues, called trama, has been used for identification by Singer (48j, who described four basic types. Most tranla consists of elongated hyphen cells (plectenchymatous tissue), but in some genera, such as Lactcwius and Russula, there are also large globose cells called sphaerocysts distributed in the plectenchymatous tissue. How the gill is attached to the stem is determined by examining the underside of the cap near where the stem and gills come together (20). Free gills never actually touch the stem. Seceding gills start their edge against the stem, but then taper away from it. Decurrent gills are somewhat triangular and attached to the stem quite far down toward the base. An adnexed gill is attached to the stem by a narrower band of gill near the stem than in the rest of the gill, sort of widely attached. The gill looks like a knife that has been cut out to make a handle. A sinuate (or knotched) gill starts out at the edge, widens

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as it comes near the stem, but then becomes notched before touching the stem with as much width as it had in the middle of the gill (24). Gills that are wide apart are called distant. As the gills get closer and closer together they are termed subdistant, close, and crowded. Gills that start at the edge but only progress a short distance toward the stem are termed intermediate (36). Some gills are joined to a collar, some radiate from a central point, while others have a pattern much like a maze (24). The edge of the gill may also aid in identification. A gill may have a smooth edge (entire), a scalloped edge (crenate), a toothed edge (serrate), or be finely fringed (marginate). The texture of the gills may also be important. For example, the waxy feel of the gills of Hygrophorus species and the ease of peeling the gills away from PnxilZus and LeucopnxiZZus species are characteristic (49).

9. Pores Some mushrooms do not have gills under the cap; they have pores. Pores are the openings of vertically arranged tubes. The spores are formed on basidia along the inside of the tube, much like the surface of the gills. The spores are then discharged down the tube and out the pore. The underside of the cap has been described as a pincushion or sponge-like. The color, shape, size, depth, and arrangement of the pores are used in identification. The tube section (sponge-like material) is often easily removed frotn the rest of the cap material. 10. Stems There are several types of stems. Some are bulbous or abruptly bulbous (suddenly bigger at the base than at the top), tapered in either direction, equal (the same size from cap to ground), clavate (club-shaped, with the club handle up), and radicate (root-like at the base). Where the stem attaches to the cap is also important. If it attaches in the middle of the cap, it is central. If attached nearer the edge than the center, it is eccentric. If there appears to be little, if any, stem, and the mushroom is attached directly to the substrate (like puffballs and some tree fungus), the attachment is called sessile. A stem that attaches to the side is lateral. The surface of the stem is also described, with terms such as those used to describe the cap. The stem may also be cut longitudinally to see if it is solid or is hollow. The consistency of the stem is also important. When you break a stem, does it snap like a piece of chalk, splinter like bamboo, or crumble?

F. Spore Prints Spores are the “seeds” of the mushroom plant and cannot be seen with the naked eye. Spores differ in size, shape, and color. When masses of spores from a mushroom are viewed together, only the color can be seen with the naked eye. Different mushrooms have varying methods to distribute the spores. Many simply have the spores fall out of the pores or off the gills to be carried away by wind, water, or animals. Spores often pass through the gastrointestinal tract unharmed, thus spreading the plant and obtaining growth substrate at the same time. Some mushrooms, like the Coprinus species, simply dissolve (deliquesce) into a black ink that can stick to animals or be otherwise carried away. The color of the mushroom spores is an important characteristic for identification, since unlike the cap or flesh color, spore colors are constant for a specific species. In a

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mature mushroom an initial estimation of the spore color can be made by looking at the underside of the mushroom and examining the gills or pores, or by looking at various structures or plants under the mushroom cap where the spores may have fallen. Examples of such structures are another mushroom, leaves, pine needles, the surface of the ground, or the stem or annulus of the same mushroom. This is just apresumptive result and should be followed by an actual spore print. Spores come in all colors, from white to black, reddish-brown to green. Spore prints are usually essential for identification, but take several hours (usually 2-6) to develop (28). They should be used to confirm any initial impression as to spore color and should always be done when the mushroom is unidentified. A spore print is done by cutting off the stem of the mushroom at the level of the cap and placing it gills (or pores) down on a white piece of paper. The spores in a mature mushrootn will fall out of the sporulating structures and color the paper. If the mushroom has become convex and the gills arch away from the paper, the mushroom should still be placed gills down, but a bowl should be placed over the mushroom to minimize the spread of the spores and concentrate the spore pattern and color. A cover is also used if the mushroom would be expected to dry out quickly, thus not releasing an adequate number of spores. White paper is best used, even if the spore print is expected to be white. Colored paper may distort colored spore prints and mask any tinges of color in a seemingly white print. If the spore print is believed to be white, and the pattern of the print is important, a black sheet of paper may be used. In special circumstances where both the pattern and the true color are required, the mushroom may be placed half on white and half on black paper. A spore print can also be done with mushrooms that have pores (spongy bottoms). The opening of the tubes is placed down on the paper. Spore prints can also be taken from coral mushrooms, chanterelles, and sotne polypores (bracket fungi). Puffballs, and other mushrooms that keep their spores inside, cannot be evaluated this way. If a mushroom is too young to have developed spores, or is too old to be still discharging spores, a spore print may not be possible. When in the field, it is sometimes useful to place the mushroom over a white index card or a collecting information card and then wrap it tightly with waxed paper. By the time the foray is over, a spore print may have developed on the card. When determining spore color, try to be somewhat flexible in color determination. White, pink, purple-brown, black, and brown may be relatively easy to identify, but there are many variations such as clay, cream, chocolate-brown, mauve, and cinnamon-brown that may take some experience to differentiate. A yellowish print should not be expected to be bright yellow, and a blackish-purple is not likely to be outstandingly purple. When the mushroom itself is not available a spore print cannot be done, but spores may be examined for other characteristics like size, staining, and attachment to the gills (if a piece of the gill is available). Sometimes other sources of spores may be used for spore identification. This may include cooked material, gastric aspirate, or vomitus. The sample material may be strained, and a sample of the liquid then examined for spore size and shape. Various stains may be used to rule out potentially toxic species. Cell walls may be used to microscopically identify some mushroom spores. For example Coprinz,ls Zagopus has a six-layered cell wall ( S O ) , Coprimu ~nicnceushas five layers ( 5 1, 52), Coprinus stercor-arius has four layers, and both Psilocybe species (53) and Agaricus br-unnesceus have three layers (54). This requires a rather sophisticated microscopic evaluation, and such differentiation is not commonly available in field guides, but may be found in the mycological literature.

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Table 3 lists some of the common spore print colors. Field guides should be used for a more comprehensive list. If you are interested in identifying a specific set of mushrooms, write down a few macroscopic characteristics and thespore colors. As stated above, spore color can sometimes be seen at the site or taken with waxed paper during the foray.

G. SmellsandTastes Different mushrooms have quite a few different smells, many of which are listed in field guides or in identification keys. Some of these include almond, anise, bean, camphor, carrot, cherries, chlorine, chocolate, curry powder, garlic, goaty, grain, green corn, pears, phenol, linseed oil, radishes, soap, turnips, and yeast. An extensive list of various mushrooms may be found in Ref. 55. Not all people can identify these smells, and the terminology used to define a particular smell may not be consistent, so identification using scent only is questionable at best. The odors may be present in varying amounts during different stages of maturity. Taste may also aid in identification but should not be used as a first-line method. The number of mushrooms that may cause serious problems with only one bite is very small, but chewing on a potentially toxic mushroom is not recommended. If you have eliminated the more toxic mushrooms using some other method, tasting a small amount of the cap may be tried. If the more toxic mushrooms have not been eliminated, do not eat the mushroom. The following technique is safe for almost all mushrooms. Bite off a small amount and let it sit on the lip or tongue for a few seconds to evaluate the taste. The fragment bitten from the cap should then be spit out and the taste sensation evaluated (38). Mushroom flavors are listed in some keys and in some textbooks (55). Some of the easier tastes to identify are the hot, peppery taste of many Rzlsszrla and Lactar.ius species and the very bitter taste of some Hylnellum and H y d ~ u r nspecies. Other tastes described include sweet, bland, brand-like, musty, and mealy (49).

H. ChemicalKeys 1. Potassium Hydroxide Test (KOH) A 5-10% solution of KOH is used and placed on the cap. Various color changes may occur (55). 2. Metlzer’s Reagent This is an iodine solution used to stain spores. If the spores turn bluish-gray to bluishblack, they are amyloid spores. If’they turn brown or reddish-brown, they are dextroid. For many spores no change is noted. The reaction is generally noted within a few minutes. The change is most easily observed under a microscope, but if one is not available you might dust some spores on a glass slide that has Metlzer’s solution on it. Place the slide over a piece of white paper and wait for a color change. The reaction is hardest to see in spores that are already colored.

1.

MicroscopicCharacteristics

Various mushroom structures can be seen only with a microscope. Much of mushroom taxonomy has been described only with the aid of the microscope. Some of the characteristics that can be determined are

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Table 3 Examples of Mushroom Spore Colors Associated with Various Species Color

Genus

Black (or near black)

Bulgaria Chroogompllus Coprinus Gowpltidius Lacrymarin Panaeolus Psathyrelln Scleroderrlrn (purplish) Stropharicc Black (olive) Clzroogontphus Bluish Brown Agrocybe Astraeus Ganodermn Hebeloma Hydnellum Hydmm Inocybe Leccinunt Morchella (pale) Pmillus Phellinus Pholiota Rozites Sarcodon Scleroderma Suillus Tuber Tulostoma blunmle Brown (chocolate) Agaricus Geastrum Brown (dark or purplish) Agrocybe Calvaticl Coprinlrs Crepidotus Geoglossum Hypholorm Naenzatolonta Panacolina Panaeoltcs Pholiotu Psathyrelln Psilocybe Stropharia

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Mushroom Biology Table 3

Continued

Color

Genus Thelephora Trichoglossuw Tubaria Tuber

Brown (cinnamon, rusty, or reddish) Bolbitius Boletus Corzocybe Cortirlarizrs Crepiclotus Fistlrlirza Galerinn Ganoderma Gyroporus Hebelomn (pale) Kuehnerontyces Phueocollybia Pholiota Pholiotina Rozites Suillus Thelephora Brown (olive or ochre) Boletus Calvatia Clathrus Ljcopedon Phallus Philiota Pltylloporus Pdverobolettrs Suillus Vascellunl Brown (yellowish) Cahwtia Coltrichia Gynnopilus Inonotus Paxillus Phylloporus Suillus Tuber Gray (to nearly black or violet) Psilocybe Stropharin Gray to lavender Psilocybe Greenish Chlorophyllum Phaeolus Rnmaria (pale)

Spoerke Table 3 Continued Color

Genus

~

Lilac Laccaria Pleurotus Olive

Orange (cream to whitish) Collybia Orange (rusty or brownish)

Pink. reddish, or flesh color Chanzacecota Claudopus Clitocybe Clitopilus Craterellus cr3’ptoporus Entolorm Lactarius (pale) Lepiotn Lepista Leptorlia Leucougaricus (late) Mucrolepiota Nolarzea Pll$lotopsis Pluteus Raillaria Rhodotus palwttus Tvlopilus Volvariella Tan Violet Laccaria White (cream or buff) Albatrellus Anmzita Arnanitopsis Arnzilkria Arntillariella Ascoconrle Asterophora Auricularia Auriscalpiunl Bankera Bisporella

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Table 3 Continued Color

Genus Bjerkandera BondarzeMia Cmthurellus Crrtathelasma Chorociboriu Clalvariu Clnvariadelpltus Clavicorona Clavulina Clu~wlinopsis Clitocybe Collybia Cordyceps Coriolus Craterellus Creolophus Crinipellis Cystoderlnu Dentinurn Disciotis Duwzolttiniu Flammllirzn Folnes Geoporu Grijiolu Gyrowitra Helvella Hericium Hirschioporus Hohenbllelteliu Hydmnt Hygrocybe Hygrophoropsis Hygrophoius Laccaria Lacturilrs Luetiporus Lentinellus Lentinus Lenzites Leotiu Lepiota Lepista Leucoagricus Leucopaxillus Lirmcella Lyophyllunl Macrolepiotu Murasmius Melulroleucn

732 Table 3 Continued Color

Genus Mitruln Morcltella Mycena Neobdgaria Orilphalina Omphalotus Otidea Oudemamiella Panellus Pams Peziza Phaeolus Phellinus Phellodon Phlebia Phyllotopsis Pleurotus Pol?ozellus Polyporus Poria Rnmariopsis Russuln Rutstroenlia Sarcoscypha Schizoplyllunt Scutellinia Sparassis Sterewil Tarzetta Tremelln Tricholomn Tricllolomopsis Typhula Tyromyces Verpa Xero-onlpludina

Yellowish

Bjerkandera Calostonla Cantharellus Clitocybe Folnes Gornphus Gyroporus Lactarius Omphalina Pasillus Ranlaria . Russula A genus of tnushrooms may have species with several colors of spores, so a genus may be listed several times in the above list. Sorrrce: Refs. 18. 24. 28. 49. 57.

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Cap: Structure of the cap cuticle (surface skin). The best way to observe the cap cuticle is by cross-sectioning a piece of the cuticle and placing it on a slide with a drop of water. Gills: Orientation of the hyphae, shape and size of the cystidia or basidia. A thin cross-section of a gill is made with a razor blade and the slice is placed on a slide with a drop of water. Spores: Size (in microns), general shape, Metlzer’s reaction, bumps, protuberances, etc. To observe some spores under a microscope, scrape some of the spores from the spore print into a drop of water on a glass slide.

J. Other Field Tests or Factors Useful in Identification Altitude: May influence where mushrooms are found since it alters the amount of water, wind, and temperature. Altitude should be considered when identifying a species. Latex: Mushrooms in the genus Lacturius exude a milky substance when cut. The color of this latex can help identify the mushroom. A fresh mushroom should be used; one that is too old may be too dried out to produce the latex. Season: Few nlushrooms sprout in the winter of the Northern Hemisphere, but occasionally a fruiting of Flammulina velutipes can be found at the base of some trees (especially elms), even inwinter. Some mushrooms canbe found throughout the growing season, others grow only in a particular season. K. Preserving Mushrooms for Later Examination

Mushrooms do not last very long before deteriorating. Probably the best way to preserve them is by drying, although they will shrink and fade as the water leaves them. Before drying make sure a sport print has been obtained and a description of size, color, and shape of stem and cap. Microscopic characteristics should not change with drying. Drying should be done over low heat with plenty of air circulation. The mushrooms should be free of insects. If drying is done outside, the mushrooms should be covered to prevent invasion by insects. A home drying kit is ideal for such work. Another method of preserving mushrooms is to slice them and press them between the pages of a book or in a flower press. This will give a better idea of structure, but there will still be discoloration and shrinkage. Encasing the mushroom in plastic is also a possibility. Changes may occur using this method as well, depending on the amount of heat the resin creates as it hardens, the size of the mushroom, and the water content.

IV.

MUSHROOMLOOK-ALIKES

A.

BlueChanterelle/Horn of Plenty

The blue chanterelle (Polyocellus nzultiplex) is sometimes mistaken for the horn of plenty (Craterellus comucopioides) but since both are edible there is little danger. The blue chanterelle is spoon-shaped to fan-shaped, not hollow like the horn of plenty. The blue chanterelle is dark-blue to gray-violet, the horn of plenty is very dark-brown to black (19).

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B. Boletus edulislother Boletus Species Boletus edz-lrllishas a reticulated stalk as do Boletus calopus and Boletus coniferclrun1. The later two species have flesh that bruises blue and tastes very bitter. C. ChanterelleslGomphus floccosus The scaly chanterelle GonrphusJIoccosus is sometimes confused with the common (golden chanterelle) Ccrr~tl~cwellus cibarius. Gosnphus Jloccosus has a hollow, vase-like cap that is reddish-orange and scaly, which is not the case with the common chanterelle (19). D. ChanterellesIOmphalotus oleariuslHygrophoropsis aurantiaca Onzplznlotus olenl-ir~s(jack-o'-lantern) looks a lot like the chanterelles and is potentially toxic (34). Hygrophoropsis urirnsztiaccr is another look-alike that is not toxic, but is not a good edible either (24). E. False Truffles (Hymenogastrales Family) and Scleroderma Neither of these species is edible, so the number of people gathering then1 should be small, and the number of illness-producing cases small as well. 1. False Truffles (e.g., Rhizopogon) These mushrooms are often found underground. cut the mushroom in half from top to bottom. The spore mass maybe rubbery, spongy, cartilaginous, hard, or rarely slimy. Often very small chambers can be seen in the spore mass, but they do not look like a mushroom cap or stem. The spore mass is not powdery as in the puffballs They often have strong odors that attract animals (28). 2. Scleroderma These mushrooms are also often found at least partially underground. Cut the mushroom in half, top to bottom. The spore mass is evenly distributed and will change color from white to greenish, brown, or purple-black (56). The spore mass becomes powdery with age.

F. Lepiota rhacodeslChlorophyllum molybdites Lepiota rllncodes (edible) is similar to Clzlo~-oplzyllunzrnolybclites (poisonous). C. snolybdites has a green-gray spore print and is found primarily on lawns: L. rhncodes has a salmon-red to brown spore print and is found on the ground under trees. The flesh of L. rlmcodes also bruises yellow (19).

G. MorelslFalse Morels Morels are mushrooms that are actively sought for food by both amateurs and professional mushroom hunters. Unfortunately they can sometimes be mistaken for other species that

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are potentially toxic. Below are listed some of the differences between morels and these other species. 1. Morchella species (Morels) Sterzl: White to cream-colored, hollow, usually wider at the base. Cup: Conical in shape, with large pits surrounded by ridges. The color varies by species but is generally buff to gray to brown. The cap is attached to the stem either at the edges or partway down the stem. 2.

Helvella Species (False Morels) Stem: Some species have deep holes in the stem, but the stem is not hollow. It is somewhat “whitish” and may have folds or wrinkles running the length of the stem. Cap: Most often reddish-or yellowish-brown, wrinkled or folded (not pitted), and often saddle-shaped. The edge of the cap will often curve away from the stem.

3. Gyromitra Species (False Morels) Ston: May be white to brownish, and is hollow. It is often thick and short. Cap: May be saddle-shaped or more globular. It is very wrinkled and convoluted, but is not pitted. The cap is dark brown, ranging from reddish-brown to chocolate or blackish-brown. 4.

Verpa Species (False Morels) Sten?: Whitish, hollow. Cap: Deeply folded and wrinkled, the folds being deep enough that they become true pits. The cap is attached the stem at the top, not at the edges or partway down the stem.

H. Puffballs/Amanitas/Sclerodermas 1. Puffballs Cut the puffball in half from the top to the bottom (toward the side that was in the earth). The insides (gleba) should be smooth and unbroken and stay a white or cream color. All puffballs should be cut and examined before ingestion. 2. Inedible Puffballs Cut the puffball in half from the top to the bottom (toward the side that was in the earth). The insides (gleba) should be smooth and unbroken. If the insides develop yellow or greenish discoloration, the puffball should be discarded. This type often is bitter and indigestible. The same is true if the insides are brown-spore production has already started and the puffball is not likely to taste very good.

3. Amanita Buttons Cut the puffball or egg-shaped mushroom in half from the top to the bottom (toward the side that was in the earth). The insides (gleba) should be smooth and unbroken if it is a puffball; but if it is one of the potentially toxic Amanita species, you should be able to determine the outline of a cap and stem on the material inside the egg.

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4. Scleroderma (Earthballs) These mushrooms are generally puffball-shaped and -sized. When cut from the top to the bottom, the insides will first appear white, but over the course of a few minutes will change to greenish, brown, or purplish-black (56). The skin of the earthball is much thicker than that of a puffball and has only one layer. Earthballs are generally considered either inedible or potentially poisonous. Earthballs are commonly found partially underground (28j.

REFERENCES 1. CJ Alexopoulos, CW Mims. Introductory Mycology, 3rd ed. New York: Wiley, 1979. 2. JM Aronson The celI wall. In: GC Ainsworth, AS Sussman (eds.). The Fungi. Vol 1. New York: Academic Press, 1965. 3. G Lincoff, DH Mitchel. Toxic and Hallucinogenic Mushroom Poisoning. A Handbook for Physicians and Mushroom Hunters. New York: Van Nostrand Reinhold, 1977. 4. AH Smith: Mushrooms in Their Natural Habitat. New York: Hafner, 1949. 5. H Hagimoto, M Konishi. Studies on the growth of fruit body of fungi. I. Existence of a hormone active to the growth of fruit body in Agaricus bisporus. Bot Mag (Tokyo) 72: 359366,1959. 6. H Hagimoto, M Konishi. Studies onthe growth offruit body of fungi. 11. Activity and stability of the growth hormone in the fruit body of Agaricus bisporus. Bot Mag (Tokyo) 73: 283287, 1960. 7. HE Gruen. Endogenousgrowth regulation in carpophoresof Agaricus bisporus. Plant Physiol 38:652-666,1963. 8. HE Gruen. Growth and rotation of Flmrmulirta velutipes fruit-bodies and the dependence of stipe elongation on the cap. Mycologia 61: 149-166, 1969. 9. MA Rogers. Basidocarp maturation and ultrastructure of basidiospores in the fungusCopriws stercorrrritls. PhD dissertation, University of Texas, Austin, 1972. 10. MA Rogers. Photoresponses of Coprinus stercorrrriras: basidiocarp development and maturation. Mycologia 65: 907-913, 1973. 11. FI Eilers. Growth regulation of Coprinus racliatw. Arch Microbiol 96: 353-364, 1973. 12. GW Gooday. Controlof development of excised fruit-bodies and stipes of Coprims cinereus. Trans Br Mycol SOC 62: 391-399. 1976. 13. RJ Cox, DJ Niederpruem. Differentiation in Coprinus lagopus.111. Expansion of excised fruitbodies. Arch Microbiol 105: 257-260, 1975. 14. TR Matthews, DJ Niederpruem. Differentiation in Coprirzus lagopus. I. Control of fruiting and cytology of initial events. Arch Mikrobiol 87: 257-268, 1972. 15. TR Matthews, DJ Niederpruem. Differentiation in Coprims lagopus. 11. Histology and ultrastructural aspects of developing primordia. Arch Mikrobiol 88: 169-180, 1973. 16. ST Carey. Clitocybe illudens; its cultivation, chemistry, andclassification. Mycologia 66:95 1968, 1974. 17. DJ McLaughlin. Environmental control of fruit-body development in Boletus rubirzellus in axenic culture. Mycologia 62: 307-331, 1970. 18. D Biek. The Mushrooms of Northern California. Redding. CA: Spore Prints, 1984. 19. R Molina, T O’Dell, D Luoma, M Amaranthus. M Castellano. K Russell. Biology, Ecology. and Social Aspects of Wild Edible Mushrooms in the Forests of the Pacific Northwest: A Preface to Managing Commercial Harvest. General Technical Report PNW-GTR-309. Portland, OR: US Department of Agriculture Forest Service, Pacific Northwest Research Station, 1993. 20. AH Smith. The Mushroom Hunter’s Field Guide. Ann Arbor: University of Michigan Press. 1975.

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21. MH Wells, DH Mitchel. Mushrooms of Colorado and Adjacent Areas. Denver, CO: Denver Museum of Natural History, 1966. 22. E Hacskaylo. Mycorrhiza: the ultimate in reciprocal parasitism? Bioscience 22: 577-582, 1972. 23. FH Meyers. Distribution of ectomycorrhizae in native and man-made forests. In: GC Marks, T Kozlowski (eds). Ectomycorrhizae-Their Ecology and Physiology. New York: Academic Press,1973. 24. T Laessoe, A Del Conte, G Lincoff. The Mushroom Book.New York: DK Publishing, 1996. 25. JM Trappe. Selection of fungi for ectomycorrhizal inoculation in nurseries. Annu RevPhytopatho1 15: 203-222, 1977. and Physiology, New York: Aca26. GC Marks, TT Kozlowski. Ectomycorrhizae. Their Ecology demic Press, 1973. 27. JL Harley. The Biology of Mycorrhiza. London: Leonard Hill, 1969. 28. D Arora. Mushrooms Demystified. Berkley, CA: Ten Speed Press, 1979. 29. VW Cochrane. The Physiology of Fungi. New York: Wiley, 1958. 30. AHR Buller. Researches on Fungi. Vols 1-6: London: Longman, Green:vol7: Toronto: University Press, 1909-1950. 31. RD Koehn. Laboratory culture and ascocarp development of Podosordaria leporinrr. Mycologia 63: 441-458, 1971. 32. MR Tansey, TD Brock. The upper temperature limit for eukaryotic organisms. ProcAcad Natl Sci USA 69: 2426-2428, 1972. of Poisonous Fungi. A Handbook for Pharmacists, Doc33. A Bresinsky, H Besl. A Colour Atlas tors, and Biologists. London: Wolfe Publishing Company, 1990. 34. OK Miller Jr. Mushrooms of North America. New York: EP Dutton, 1987. 35. GH Heptig. Diseasesof Forest and Shade Treesof the United States. Forest Service Handbook 386. Washington DC: US Department of Agriculture, 1971. 36. AH Smith. A Field Guideto Western Mushrooms. Ann Arbor: University of Michigan Press. 1963. 37. S Dickinson. Studies on the physiology of obligate parasitism. IV. The formation on membranes of haustoria by rust hyphae and powdery mildew germ tubes. Ann Bot 13: 345-353. 1949. 38. EJ Butler, SG Jones. Plant Pathology. London: Macmillan, 1939. 39. U Nonis: Mushrooms and Toadstools. A Color Field Guide. New York: Hippocrene Books, 1982. 40. M Lange, FB Hora. A Guide to Mushrooms and Toadstools. New York: EP Dutton, 1964. 41. JW Groves. Edible and Poisonous Mushrooms of Canada. Ottawa, ON: Research Branch, Canadian Department of Agriculture, 1962. 42. WS Thomas. Field Book of Conmon Mushrooms. New York: GP Putnam’s Sons, 1948. 43. TM Flynn. Stalking the Wild Mushroom. Denver: Colorado Mycological Society, 1980. 44. P Stamets. Psilocybin Mushrooms of the World. An Identification Guide. Berkeley CA: Ten Speed Press, 1996. 45. D Largent. How to Identify Mushrooms (to Genus). Eureka, CA: Mad River Press, 1973. 46. AH Smith, HD Theirs. The Boletes of Michigan. Ann Arbor, MI: University Press, 1971. 47. RL Shaffer. Keys to Genera of Higher Fungi. 2nd ed. Ann Arbor: University of Michigan Press.1968. 48. R Singer. The Agaricales in Modern Taxonomy. 3rd ed. Weinheim: J Cramer, 1975. 49. GL Grimes. In: DG Spoerke, BH Rumack (eds). Handbook of Mushroom Poisoning. Boca Raton, FL: CRC Press, 1994. 50. CE Heintz, DJ Niederpruem. Ultrastructure of quiescent and germinated basidiospores and oidia of Coprinz4s lagopus. Mycologia 63: 745-766, 1971. 51. L-M Melendez-Howell. Ultrastructuredu pore gernlinatif sporal donsle genre Coprinus Link. Compt Rend Hebd Seances Acad Sci 263 Ser D: 717-720, 1966.

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52. L-M Melendez-Howell. Les rapports entre le germinatif sporal et la germination chez le Basidiomycetes en mircrosopie electronique. Compt Rend Hebd Seances Acad Sci 264 Ser D:12661269, 1970. 53. DL Stocks, WM Hess. Ultrastructure of dormant and germinated basidiospores of a species of Psilocybe. Mycologia 62: 176-191, 1970. 54. B Gruter, D Rast. Ultrastructure of the dormant Agaricus bisporus spore. Can J Bot 53: 20962101, 1975. 55. DG Spoerke. Mushroom Odors. In: DG Spoerke, BH Rumack (eds). Handbook of Mushroom Poisoning. Boca Raton, FL: CRC Press, 1994. 56. WC Coker. JN Couch. The Gastromycetes of the Eastern United States and Canada. New York: Dover Press. 1974. 57. G Kibby. Mushrooms and Other Fungi. New York: Smithmark Books. 1992. 58. E Danell. N Fries. Methods for isolation of Cunrlm-ellus species, and the synthesis of ectomycorrhizae with Picea albies. Mycotaxon 38: 141-148, 1990. 59. BH Rumack, E Salzman. Mushroom Poisoning: Diagnosis and Treatment. West Palm Beach, FL: CRC Press, 1978, p. 211.

19 Identification of Mushroom Poisoning (Mycetismus), Epidemiology, and Medical Management David G. Spoerke, Jr. Br-istlecone Enterprises, Denver, Colorcldo

I. Identification of Mushroom Poisoning

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A. Epidemiology 739 B. Diagnosis 740 C. Identification of the mushroom 743 D. Collecting mushroom samples 744 E. Field guides and expert identifiers 744 F. Various mushroom tests 745

11. Medical Management

747 A. Management by toxin class 747 B. Cyclopeptides (amatoxins) 749 C. Orellanine/orelline 753 D. Muscirnol/ibotenic acid 754 E. Monomethylhydrazine 756 F. Muscarine 758 G. Coprine 759 H. Psilocybin/psilocin 760 I. Gastrointestinal irritants 762 764 J. Other considerations 111. Summary 768 References 768

1.

A.

IDENTIFICATION OFMUSHROOM POISONING Epidemiology

There are approximately 100 reported cases of mushroom poisoning in the United States each year, although the number of actual cases is probably higher (1). Poisoning cases are more common in Europe, with over 200 cases of mushroom poisoning in Germany alone. Based on information from the National Poison Information Centre in Poland, only 739

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about 6-7% of poisonings in that country are due to plants and mushrooms (2, 3). One study in Italy exatnined 216 cases of supposed mushroom poisoning between 1975 and 1990. About 45% of the mushrooms involved were not identified, 12% were actually poisonous, and another 12% of cases were most likely due to food allergy or other reactions (4). In this chapter we will be dealing with mycetismus (mushroom poisoning causing distress resulting from consumption of a fungus) and very little with mycotoxicosis [fungal interaction and alteration of foodstuffs of humans or animals (293)l. Thechapter will deal primarily with intoxications by macromycetes (fungi that produce their spores on large, visible structures called mushrooms). Other fungi, like molds, are called miromycetes and bear their spores on mycelium filaments. Other types of fungi such as rusts, smuts, and mildews will generally notbe covered in this chapter; the focus is on mushrooms and “toadstools.”

B. Diagnosis Mushroom identification is difficult since there is no known single test that can differentiate a poisonous from an edible mushroom. At various times the lay public believed that blackening a silver spoon ( 5 ) ,blackening onions, peeling of the mushroom’s cap, animal ingestion, or gill color would help separate toxic from nontoxic mushrooms (6). In fact these “rules” are valueless. Table 1 lists a few of them. Table 1 Erroneous“Rules“forDistinguishing

Poisonous from Nonpoisonous Mushrooms

1. A toxic mushroom will turn a silver spoon black. 2. If an animal eats the mushroom. it is all right for a human to eat it. 3. Poisonous mushrooms grow among rusty nails. (Dioscorides) 4. Poisonous mushrooms grow in rotten rags. (Dioscorides) 5 . Poisonous mushrooms grow near snake holes, and inhale the snake’s poisonous breath. (Dioscorides) 6. Poisonous mushrooms grow near trees with noxious or poisonous fruits. (Dioscorides) 7. A mushroom whose cap does not peel is toxic. 8. A mushroom that congeals milk is poisonous. 9. A mushroom that turns onions blue is toxic. 10. A mushroom that turns parsley yellow is poisonous. 11. The closer the mushroom comes to the color of a fig, the better chance it is not poisonous. 12. Mushrooms that grow in meadows are safest and best. (Horace) 13. A toxic mushroom will have a loathsome aspect. (Pliny) 14. A poisonous mushroom has a dilute red color. (Pliny) 15. A poisonous mushroom has a livid-blue internal color. (Pliny) 16. A poisonous Inushroom has gaping cracks and a pale lip on the edge of the cap. 17. Poisonous mushrooms all have a livid color. 18. Mushrooms that stay hard after cooking are poisonous. 19. Cooking a poisonous mushroom with meat makes it safer. 20. Eating pears after n ” o o m s will neutralize any toxins in the mushroom. 21. Cooking a poisonous mushroom with pear stakes will neutralize the toxins. 22. Black and green mushrooms are poisonous. (Avicenna) 23. Mushrooms the colors of peacocks are poisonous (Avicenna) 24. A poisonous mushroom will cause the white of an egg to go green. Sorwce: Refs. 24, 284.

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The diagnosis of mushroom poisoning is difficult without some indication that a mushroom has been ingested. Mushrooms have a bad reputation with the lay public and often are blamed for illness due to some other cause. Mycophobia is common in the United States, with many misconceptions leading to fear of all mushrooms. The public may believe that once a poisonous mushroom has been ingested there is little that can be done to save a patient, or that antidotes are readily available and must be given quickly to be effective. Deaths from mushroom poisonings are known to be ‘‘quick and common”-even if we do not actually know anyone who was seriously injured by mushroom ingestion. The truth is far from these beliefs. People who gather mushrooms knowledgeably seldom, but may ( 5 ) , have serious intoxications, and if a potentially fatal mushroom is ingested there is often time to attempt treatment. The success of the treatment will depend on the type of mushroom and availability of any antidote or specific treatment. The majority of mushroom “poisonings” in the United States are exposures in children and involve various gastrointestinal irritants (7). When a suspected case of mushroom poisoning is being evaluated, identification of the mushroom is very important, as is the use of a competent mycologist to identify the species. When a patient presents with signs and symptoms of vomiting, abdominal pain, and diarrhea, many illnesses maybe causal, including common food poisoning. Food poisoning is probably one of the most common types of all poisonings. It is unclear why people eat spoiled mushrooms. Is it possibly because some people expect mushrooms to be slimy and odd smellinghasting and therefore do not note the spoiled food (8)? Exposure to mushrooms, especially in the summer months, should be part of any differential when evaluating cases involving these symptoms and an otherwise unknown cause. Although most exposures are seasonal, ingestion of mushrooms that were dried or canned during mushroom season may also produce poisonings. When considering the possibility of mushroom ingestion, a number of questions concerning the mushrooms or their surroundings should be asked to help determine the likelihood of a fungal intoxication. 1. Can the patient, patient’s relatives, or friend verify that a mushroom has been ingested, or is ingestion just a suspicion? 2. Does the patient commonly eat mushrooms picked in the wild? (Some toxins are cumulative and appear only after several meals, or with a delayed onset.) 3. Is it a time of year that wild mushrooms might be present? Take into consideration the modifying effects of moisture, altitude, and season. 4. An adult would ordinarily be able to tell you if there were mushrooms around, but a child or unconscious patient may not. Try to determine if the patient was in an area where mushrooms might be found. 5. Was the mushroom eaten raw or was it cooked? If cooked, how was it prepared-frying, parboiling, pickled? (Some types of cooking destroy the toxins of some mushrooms.) 6. Was more than one type of mushroom ingested? The patient may think that only one type was picked and eaten, but may not be able to accurately identify the species collected. You may need to have an expert examine the collecting basket, or to survey the site(s) of collection to see what is available. To the amateur, many mushroom types look the same. 7. In what conditions were the mushrooms when they were prepared for the meal? If the mushrooms were already spoiled, there is the possibility of food poisoning. The

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mushrooms may have been contaminated with other substances, such as insect larvae or chemicals. 8. What was the mushroom growing on (substrate)? Wood? Soil? Dung? Knowing the type of substrate aids in identification, and can rule out several toxin classes. It is important to carefully examine the site where the mushrooms were grown. A mushroom may appear to be growing on grass, but in fact is springing from submerged wood or dung. 9. What types of trees are in the area? (Certain mushrooms are found around or have a mycorrhizal relationship with certain trees.) 10. What do the general surroundings of the mushroom look like? Were the mushrooms found in a park where herbicides might have been sprayed on them; were they picked under trees that hadbeen sprayed with an insecticide; were they found in the woods growing over mine tailings? Many of these conditions may contribute to toxicity, even with nonpoisonous mushrooms. A number of questions concerning the patient are also important. 1. When were the mushrooms eaten? How long ago? It is best to get an actual time (e.g., 1 P.M. or 13:OO)rather than time since exposure ( e g , about 2 hr ago). Patients may erroneously recall the time since ingestion. Get the times of each meal if several meals were eaten in the last 1-2 days. Some mushrooms have a delayed onset and the actual toxicity may be due to a previous meal. 2. When did symptoms first occur? Again, an actual time is best. 3. When evaluating a young child or unconscious adult, it is beneficial to note whether there was any physical evidence that an ingestion might have occurred, such as fungal material in the mouth or on the lips and face. Did the mushroom leave stains or other evidence only on the hands, or on the face as well? Older children may be able to express verbally whether a mushroom has been ingested, but younger children are very unreliable. Obviously the history that any child states should be suspect and corroborated with other evidence. 4. Was the mushroom eaten at only one meal, or was it reheated and eaten over several meals? Answers to these questions might change the probability that food poisoning or cumulative-effect toxins may be involved. 5. Did the patient consume any alcohol in the past 24 h? Some mushrooms species alter alcohol metabolism like the drug disulfiram, and produce a similar reaction. 6. Did all persons who ate the mushroom become ill? If not, were there any differences in the amount they ate, how the mushrooms were prepared, or in any coingestants? 7. Are there some ill individuals who ate none of the mushrooms? (Possible other causes for the illness other than the mushrooms.) Were symptomatic persons exposed in some other manner, such as boiling or frying? In rare cases breathing the vapors given off by hydrazine-containing mushrooms produced illness. 8. Is the patient on any medication that might contribute to or in other ways alter or produce the symptoms? (For example, a cough medicine containing ethanol after ingestion of a Coprirzus mushroom.) 9. Has the patient ever had an allergic reaction to a mushroom? As usual, it is important to determine the symptoms of the patient, while trying not to overtreat based on a presumptive diagnosis without corroborating symptomatology. Good supportive care is often the mainstay of mushroom poisoning treatment. This includes maintenance of fluids and electrolytes, respiration, and cardiac function. For those

Identification of Mushroom Poisoning

743

mushrooms that produce a latent period of 6 h or more between ingestion and symptoms, kidney and renal function should be monitored to establish a baseline. Not all mushrooms are deadly. There is a wide range of toxicity from nontoxic to lethal with the ingestion of just one mushroom. The mythology of mushrooms is extensive. Some people believe that simply handling a toxic mushroom may cause poisoning. That is not the case. As stated above, mushroom toxicity cannot be determined by whether or not a silver spoon blackens, whether or not a silver coin darkens, or whether garlic will turn black or blue. Just because a mushroom exudes a milky latex does not mean it is a toxic species. Not all mushrooms that bruise are toxic, and whether the cap peels easily or not does not relate to toxicity. Preparation for food use, such as boiling, frying, parboiling, and salting, may detoxify some, but not all, toxic species. Like other foods, mushrooms maybe contaminated with bacteria, heavy metals, pesticides, herbicides, or other potentially toxic substances. Symptoms may also arise from food allergic reactions, condiments, or preservatives. An intense burning sensation in the abdomen may be due to capsicum or horseradish ingested with the mushroom, not the mushroom itself. Bacterial food poisoning is always a consideration, but if there has been a concomitant tnushroom ingestion it is possible that the food poisoning might be caused by or might mask symptoms from the mushroom. Identification of the mushroom species is important in these cases. If there is some doubt as to the identity of the mushroom, specimens should be saved in a paper, not plastic, bag. Plastic will make the mushrooms deteriorate more rapidly, making them more difficult to identify. Specimens should be refrigerated, but not frozen. If possible, a spore print should be initiated early (see below), so that the mycologist will not have to perform that function. Especially in those cases where an actual mushroom specimen isnot available, samples of gastric contents should be saved for possible spore identification. Diagnosis may also be confused by medications that the patient has taken. One example involves a case of suspected fungal muscarine poisoning complicated by the fact the patient was taking prescribed bethanecol (9).

C.

Identification of theMushroom

Once an actual ingestion is suspected, mushroom identification should be attempted. Proper identification of the mushroom species involved is important not only for the patient being treated, but also to help evaluate symptom complexes and treatment measures for future cases. Much of the information we have on mushroom toxicity was derived from trial and error by mycophagists (10). If a person ate a mushroom and got sick, it was a "toxic" mushroom. Improper identification may lead to erroneous reputations for various mushroom species. In the majority of potential mushroom poisonings the physician first seeing the patient will not have a positive identification of the mushroom. Not all mushrooms are toxic, and only a few are extremely dangerous. The object is to identify the specific mushroom species if possible, but if that cannot be done, try to rule out the more toxic species. If the patient has contacted you via telephone, a sample of the suspected mushroom should be brought to the health care facility. Even when a particular mushroom is suspected, it is a good idea to search the surrounding area for other species as well. Poisonous and nonpoisonous species may grow alongside one another. More than one type of mushrooms

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from a specific area may have been collected and ingested. When no particular mushroom is suspected, then samples of all mushrooms available should be brought with the patient.

D. CollectingMushroomSamples Lay individuals should be instructed to collect samples by digging up the entire mushroom, not just breaking it off at the surface from which it grows. Samples should be in separate containers with notes about the mushroom’s location, such as the types of trees it was near, what it was growing on (earth, dung, wood), and whether it was found singly or in a large group. Many of the important identification characteristics can be lost by improper collection techniques. Even experienced collectors can make mistakes. Obrien and Khuu (1 1) describe a fatality due to collection of Anznnita mushrooms by a woman with 30 years of self-taught experience. If the patient arrives at the health care facility without the mushroom, or mushroom poisoning is not suspected until after arrival, someone may need to return to the exposure site and search for representative samples while the patient is given emergency decontamination treatment. Collection of vomitus for spore identification may also be helpful (12). E. FieldGuidesandExpertIdentifiers If the health care provider is not an expert in identifying mushrooms, several groups may be able to provide assistance in identification. Some poison information centers have experts who may help with identification, or have located someone in the community with this expertise. Universities also often have an expert who can be of assistance. Many communities have mushroom clubs that have members with years of experience in identifying the local species. These individuals may have seen a single species, with its many color, shape, and texture differences, over many years. This experience will aid them in identification. A competent identifier will have an extensive knowledge of general mushroom features, variations in the local species, and the types of mushrooms that have produced illness in their area (13). When further expert advice is not available, field guides may be of some value. There is always a risk in using a field guide. The species presented in the book are often not the same ones found locally and the mushroom in hand almost never looks exactly like the one in the photograph. There are usually several mushrooms that are close approximations, some of which may be toxic, others harmless. Additional information may be required to help with the gross characteristics seen in the field guide photographs. A hand lens is helpful in identifying characteristics such as “warts,” hairs, or filaments on the mushroom, or aid in observing the attachment of gills to the stem of the mushroom. A microscope may be used to examine spores of the mushrooms. Various stains are used to help identify certain genus or species characteristics. For the health care provider it is often not necessarily imperative to identify the exact species. It is most important to rule out those mushrooms that may produce serious reactions. This includes those mushrooms in the cyclopeptide, orellanine, or monomethylhydrazine group. Unless these types of mushrooms have been ruled out, emesis at home without further investigations may result in a serious poisoning. It is important to have an idea if these types of mushrooms grow in your area. It may save considerable time in making a diagnosis. Mushrooms in the other groups may produce symptoms, but will seldom produce fatalities.

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745

F. Various Mushroom Tests Various presumptive tests are available to aid in detemining the toxic potential of mushrooms. As these tests are presumptive, they should be confirmed with analytical laboratory techniques whenever possible. These tests should never be used as the sole determinant of a particular type of intoxication. Many of them require special reagents that are not readily available. Consult your local mushroom societies and emergency rooms to find out which mushrooms are commonly involved with poisonings in your area; then store those reagents that may be useful in helping to identify these species. Some of the tests available are listed below; other tests and how the various mushrooms react can be found in various field guides. 1. Agaricus Test for Yellow Staining Many of theAgaricus species that stain yellow will cause gastroenteritis in sensitive individuals. A test for producing this staining rapidly is done by placing one drop of potassium hydroxide, or some other alkaline solution, on the stem (near the base is best) or cap. The yellow stain should occur rapidly if this is one of these species. A strong basic solution can be prepared at home by adding one teaspoonful of Drano crystals to a quarter cup of water. This solution should be kept well out of the reach of children, and may cause bums if it touches the skin. Another test used for differentiating the yellow-staining Agaricus species centers around the use ofHenry’s reagent. The test involves the use of thallium and various strong acids (14), and although it is stable indefinitely, obtaining thallium and storing a solution safely probably is not worth the risks. If Henry’s reagent is available, place a few drops on the mushroom cap’s skin using a dropper or glass rod. Agaricus awensis and Agaricus silvicola will develop a brick-red color (15). Some Cortincrrius species will also react to this reagent, developing a red color or a blue to violet color (Cortinarius heryeticus) (16). The Schaeffer reaction is also done on suspect Agaricus species. A line is drawn on the cap surface using aniline or aniline water. Using a 65% nitric acid solution next, a second line is drawn to cross the first. The test is positive if a bright orange color is seen where the two lines intersect (17). Unfortunately, Agaricus plclcomyces and Agaricus xarzthodernzis, potentially toxic species, do not react positively to the Schaeffer reaction.

2. Ammonium Hydroxide Test Different mushrooms will react to a 25-50% solution of ammonium hydroxide in distilled water. A drop or so is placed on the cap or cuticle. The flesh will turn various colors for the different mushrooms. For example, the edible Boletus edulis will turn red to dark pink on the skin, while Boletus celleri, whose edibility is unknown, will turn the flesh green. Suillus breviyes will develop purple skin, while the peppery flesh of Suillzts piperatus willbe wine red. Ammonium hydroxide test results are often listed i n field guides (16-18).

3. Chorophyllum Test Eilers and Barnard (19) described a rapid method of identifying spores of Chlorophyllurn molybdites from vomitus and stool samples. In this test, 10-50-ml aliquots of stool or vomitus are filtered through four layers of cheesecloth. The filtrate is centrifuged at 7000 X g for 10 min to sediment the spores. The supernatant is carefully removed by suction. The pellet produced is then resuspended in a small volume of water. A drop of this suspension is placed on a slide, which is gently heated until the liquid evaporates. The solids

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are then fixed to the slide. Several drops of acid fuchsin ( 19% acid fuchsin in distilled water) are placed on the fixed material and heated slowly until completely dry. Gently wash off any excess stain with water. Place the slide in the microscope for viewing. The basidiospore walls will appear red and have sufficient contrast to be discerned from other particles. This procedure permits differentiation of the spores of C. wolybdites from those of other toxic mushrooms. The cited article contains spore photographs, which aid in the identification process. Should the spores be immature, they will still have the same shape, size, and ataining characteristics as the mature green spores. 4. Green Vitriol Test Ferrous sulfate is used for this test. A crystal or two may be placed on the mushroom, or a drop or so of a 10% solution is added to a macerated mushroom or a broken portion of the cap. Usually the reaction, if any, is seen within a minute. This test has been used for various Boletus, Ceriomyces,Krombholzielna, L ~ C C ~ I ZRrlssula, U I I Z , and Suillus species (15, 16, 18).

5. Hydrochloric Acid Hydrochloric acid has also been used to aid in mushroom identification, but the reactions may differ depending on what part of the mushroom comes in contact with the acid. As an example, Boletus ch~yserzteror~ flesh may turn yellow, but pink is seen when placed on the skin. Suillus megnporims may produce orange when placed on the skin, a dark lilac on the flesh, and red on the tubes. It is best to use this reagent with a key for mushr o o m that includes all the various color possibilities (18). 6. Melzer’s Iodine Test This test is done on tnushroom spores to determine whether they are amyloid or dextroid. Melzer’s test is prepared by dissolving potassium iodide, 1 g in 1-2 ml of water, adding a half gram of iodine, and stirring until dissolved. An additional 18-19 ml of water is added, then 20 g of chloral hydrate. The solution is moderately stable, lasting for about a year (15). To perform the test, add a drop of the solution to a spore deposit on a glass slide. Observe the reaction under a microscope. A positive amyloid reaction is the development of a blue-black coloration. If the color developed is reddish-brown, the reaction is dextroid. Amyloid and dextroid reactions are commonly listed with mushroom descriptions and can help in identification. This reaction is especially useful if the actual mushroom is not available, and only gastric washings are available.

7. Orellanine Test A presumptive test to identify orellanine in a mushroom was reported by Schumacher and Holland (20). A fresh or dried mushroom is crushed in water, allowed to stand in water, and filtered. The filtrate is added to an equal amount of 3% ferric chloride hexahydrate dissolved in 0.5 normal hydrochloric acid. If orellanine is present, a dark gray-blue color should appear.

8. Pistillarin Test Pistillarin may be partially responsible for the gastrointestinal upset effects of Rnrnnrin (coral mushroom) species.

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Macerate part of the mushroom and mix with a 5% iron (111) chloride solution (17). A blue-green color will be seen if pistillarin is present (21). This test can also be used to differentiate some of the Cortincrrius species (22). 9. Spore Prints The color of the mushroom spores is an important characteristic for identification. In a mature mushroom, an initial estimation of the spore color can be tnade by looking at the underside of the mushroom and examining the gills or pores, or by looking at structures under the mushroom where the spores may have fallen. Examples of such structures are another mushroom, leaves, pine needles, the surface of the ground, or the stem of the same mushroom. This is just a presumptive result, and should be followed by an actual spore print. Spore prints are usually essential for identification, but take several hours to develop. They should be used to confirm any initial impression as to spore color. A spore print is done by cutting off the stem of the mushroom at the level of the cap and placing it gills (or pores) down on a white piece of paper. The spores in a mature mushroom will fall out of the sporulating structures and color the paper. If the mushroom has become convex and the gills arch away from the paper, the mushroom should still be placed gills down, but a bowl should be placed over the mushroom to minimize the spread of the spores and concentrate the spore pattern. When the rnushroom itself is not available, other materials may be used for spore identification. This may include cooked material or materials from gastric aspirate or vomitus. The sample material may be strained, and a sample of the liquid then examined for spore size and shape. Various stains may be used to rule out potentially toxic species.

10. Wieland Test One test to determine amatoxins (cyclopeptides) is called the Wieland test (Meixner test). This test is done indoors, away from excessive heat and sunlight. A drop is squeezed from a fresh mushroom onto a piece of pulp paper or newspaper. Circle the area moistened with the drop, then dry gently. A drop of concentrated hydrochloric acid is added to the circled spot. If the circle turns blue, it is presumptive of amatoxins. When doing the test, a drop of hydrochloric acid should be placed elsewhere on the paper to make sure this is not one of those newsprints that turns blue on exposure to hydrochloric acid. The reaction occurs between the indoles in the mushroom and the aromatic aldehydes released when the acid reacts with the lignan paper. False positives are possible with other indole-containing mushrooms (with such agents as psilocybin and bufotenine), certain terpenes, and by heating the spot to over 63 degrees. Lignin-free paper will give a false negative, as no aldehydes will. be released (23).

II. MEDICALMANAGEMENT A.

Management by ToxinClass

Treatment of poisonous mushroom ingestion has been a problem since ancient times. Nicander’s initial antidote was to treat with cabbage heads or with sections cut from around the winding stems of the rue plant or with old copper particles or with a pound of Clematis dust with vinegar. One treatment consisted of using the bruised roots of pyrethrum, adding a sprinkle of vinegar or soda, the leaf of cress, pungent mustard, and wine, all combined

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with ashes or the dung of domestic fowl. After ingestion, the patient should stick his right index finger down his throat to induce vomiting (24). Celsius recommended ingestion of radishes with vinegar or salt and vinegar. Galen also suggested ingestion of radishes and lye ashes mixed with rue or vinegar. Drinking vinegar and wormwood or eating pears immediately after eating mushrooms was also tried (24). Looking at the above ingredients, it is obvious most of these agents are emetics, many of them toxic emetics. Removing the offending mushroom from the stomach or nullifying the toxin is still a mainstay of therapy. All the effort used in obtaining an accurate history of ingestion is to help identify the specific mushroom. If the species has been positively identified, a specific plan of action can be developed based on the potential for toxicity. It should also be retnembered that there are individual reactions to the substances in mushrooms, and there are numerous cases where a mushroom that is eaten by most people is not tolerated by a particular individual. Thus, the first time a person eats a particular edible mushroom it is wise to eat only small amounts (25). Potentially poisonous mushrooms fall into different toxin classes. These classes may be grouped into those that cause cellular destruction of the liver and kidney, those that affect the autonomic nervous system, those that primarily affect the central nervous system, and those that cause gastrointestinal effects (26). If the mushroom cannot be identified, or identification is likely to take several hours, gastric decontamination is recommended. If the mushroom has not already produced considerable vomiting and diarrhea, consider emesis, gastric lavage, and/or activated charcoal depending on the patient’s clinical condition. Emesis is most effective if done within the first 30 min. The usefulness of causing a patient to vomit decreases beyond this time, unless the mushroom is suspected of decreasing gastric emptying time or intestinal tnotility. Emesis is usually not necessary with coprine-, psilocybin-, or orellanine-containing mushrooms. Coprine mushrooms often cause emesis, as do the gastrointestinal-irritant mushrooms. Psilocybin is absorbed rapidly and the effects are generally short-lived. It1 most cases symptoms due to orellanine-containing mushrooms occur a long time after the ingestion, making emesis worthless. As usual, ipecac should not be given to patients in whom seizures or coma are present or are likely to occur. The normal dose of syrup of ipecac hot the fluid extract) is 15-30 ml in an adult or child weighing about 100 lb (45 kg). Children 1-12 years old should be given an initial dose of 15 ml. If emesis does not occur within the first 30 min, a second 15-ml dose may be given. Ipecac should be followed by oral administration of 4-8 oz of water in adults and approximately 15-20 ml/kg in children. Although uncommon, complications of emesis include prolonged vomiting, esophageal tears, and aspiration. If improperly dosed, the emetine in ipecac may cause cardiac abnormalities. When considering lavage, evaluate whether the patient’s potential symptoms are worth the trauma. Esophageal perforation, laryngospasm, electrocardiographic changes, fall in mean PO,, and dysrhythmias have occurred during lavage. If seizures are present, they should be controlled prior to starting lavage. If seizure potential is present, adequate control measures should be available. In adults, the used of a large-bore tube for lavage (34 French or larger) is recommended. Adult patients generally are given 150-200 ml per wash; children 50-100 ml (27). Comprehensive studies on the adsorption of mushroom toxins by activated charocal are rare, but theoretically most of the toxins would lend themselves to adsorption. Patients should be monitored for electrolyte abnormalities when vomiting and diarrhea are extensive, and fluids and electrolytes should be replaced appropriately. Patients

Identification of Mushroom Poisoning

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who are suspected of ingesting liver and kidney-damaging toxins should have liver and kidney function baseline levels drawn. The various clinical effects presented may aid with the diagnosis, as may the time of onset of these effects. Symptoms that occur more than 6 hr post ingestion should alert the physician to the potential for some of the more serious types of toxins such as the cyclopeptides and the hydrazines. An intoxication primarily affecting the autonomic system in the first 20 min-2 hr may be due to coprine or muscarine. If the central nervous system (CNS) is primarily affected in that first 20 min-2 hr, ibotenic acid-mucimol or psilocybin groups should be immediately considered. Gastrointestinal effects may occur anywhere from 30 min to 3 hr (26). If signs and symptoms occur rapidly, this does not rule out later symptoms occurring. Ingestions of mixed types of mushrooms may create both early and late symptomatology. Signs and symptoms of mushroom intoxication fall into eight broad, general groups, listed below. Possible mechanisms of action and treatments to consider are also listed.

B. Cyclopeptides(Amatoxins) 1. Symptoms Although there have been numerous articles in the press about the hazards of eating unidentified wild mushrooms, people still take the chance. Ingestion of Amanita yhnZZoicles caused nine people to be hospitalized (two died) from December 28, 1996 to January 6, 1997 in California (27). Cooking does not destroy the toxins in these mushrooms (28), and even sotne edible mushrooms contain very small amounts of amatoxins (29). A. yhaZZoides is responsible for 95% of fatal poisonings (29, 30). Intoxications generally occur in four phases. During the first phase (latent period) few symptoms are seen. Next there is a gastrointestinal phase, then another latent period, and finally a hepatorenal phase. Initial symptoms occur after about 6-14 hr (range about 6-24 hr, mean 12.3 hr) but may rarely occur as late as 48 hr (31). There is some evidence that the longer the latent period, the less toxin is involved and the milder the intoxication (17). In phase 2, nausea, vomiting, and abdominal pain may be intense (32). A cholera-like diarrhea may follow, and the diarrhea may be bloody. These symptoms may persist for 2-3 days and result in dehydration, hypovolemia, acid-base abnormalities, and electrolyte imbalances (33). Volume changes may produce tachycardia, mild hypotension, leg cramps, and shock. This phase generally lasts 12-24 hr, but may last 2-4 days in some individuals (17). Phase 3 is seen over the next several days. A patient may seem to improve, but changes are occurring at the cellular level. Blood coagulation changes, and liver enzymes start to elevate. Hepatic damage starts about 36-48 hr post ingestion. Three to four days after ingestion, liver and kidney damage signs may appear and the patient worsens (34). In serious cases, jaundice, fulminant hepatitis, hepatic coma (35), encephalopathy, bleeding, and hypoglycemia may be present (36-38). There may be bleeding of the stomach and intestine, and a pressure-sensitive liver is often reported. There may be hemorrhagic diathesis and severe brain edema (39). Hepatic failure may lead to magnesium deficiency (40). Eye changes that have been reported include endophthalmitis (enucleated eye) and macular deposits (glaucoma eye) (41). Renal failure may be seen either in the first phase of vomiting and diarrhea (due to hypovolemia) or later in the hepatic phase (42). Oliguria or anuria may be present. If the damage can be reversed, patients generally recover slowly, requiring hemodialysis (43,

I

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44). Fatalities do occur, and death may be seen between 6 and 16 days (mean 8 days) (26, 45). Acute tubular necrosis is often reported (39). Amanita smithinna, usually found in the Pacific Northwest, is especially nephrotoxic. It can be mistaken for the edible pine mushroom (Tricholomn mngrzivelnre) (46). Abnormal laboratory values include prothrombin time (also thromboplastin time, Quick value), AST and ALT (the aminotransferases), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine, serum bilirubin, and serum glucose (17). Endocrine hormone abnormalities in this type of poisoning have been investigated (47). Significant abnormalities were found in glucose-controlling hormones, calcium hemostatis, and thyroid homeostatis. On admission, insulin, C peptide, and serum calcitonin concentrations may be elevated. Parathyroid hormone concentrations may increase with time, decreasing when the hypocalcemia resolves. Thyroxine levels may fall significantly. T3 concentrations were undetectable in three of four patients studied in one case report. Abnormal blood coagulation may occur in serious cases. Transfusion of blood products, FFP, cryoglobulins, and vitamin K may be required (48). Amatoxins can be detected in the blood and urine using a radioimmunoassay (49), but may not be detectable if the ingestion occurred some time earlier (50). Homann et al. (51) found amatoxins in the blood and urine of a 15-year-old boy within 90-120 min of ingestion. Based on these data, it appears that the amatoxins are rapidly absorbed and fairly rapidly excreted by the kidneys. Japanese workers have identified amatoxins and phalloidin in prepared food eaten by the victim, using high-performance liquid chromatography (52). Jaeger et al. (53) took fecal, gastroduodenal fluid, urine, plasma, and tissue samples from 45 Anzaflita mushroom-poisoned patients and found that the detection of amatoxins was time dependent. Amatoxins were usually detectable in the plasma before 36 hr and were found in the urine for about 4 days after the ingestion. Data on exposure of pregnant women to the amatoxins are limited. A 24th-weekgestational woman was exposed to A. yhdloides toxins, yet delivered a healthy infant (54). 2. Mechanism of Action There are two general types of toxins in these mushroom, the phallotoxins and the amatoxins (55, 56). These compounds are colorless. There are at least seven phallotoxins, the primary one being phallodin (35). Phalloidin binds to membrane actin, which causes disorders of liver cell function (57, 58). Both amatoxins and phallotoxins are toxic when given by injection. Amatoxins are also toxic by ingestion. There are at least nine different amatoxins, the main ones in A. yhnlloicles are alpha-amanitin and beta-amanitin (26). Amanitins are generally crystalline compounds, colorless, and soluble in polar solvents such as methanol and water. They have a definite structure-toxicity relationship. As an example, alpha-amanitin has a LDsOof 0.3 mg/kg, while amanullin and proamanullin have LDSo’s greater than 20 mg/kg (17). Amatoxins are stable in the gastrointestinal tract and are stable to boiling. Actually, amatoxins are found in a number of species in nontoxic amounts. Agaricus, Boletus,and Cnntharellus provide various examples (142,294). Amanitin inhibits one of two mammalian nuclear RNA polymerase B’s that are involved with DNA transcription to messenger RNA (59). This action results in inhibition of protein synthesis at the ribosomes (60, 61). When tested in animals, the first morphological changes noted were plasmalemma and cytoplasmic vacuoles containing blood-derived materials (62). There were also changes in enzyme activity, namely increased activity of glutamic pyuvic and oxaloacetic transaminases, and lysosomal hydrolyses (62).

Identification of Mushroom Poisoning

751

3. Treatment After decontamination and fluid and electrolyte balance have been accomplished, patients should be monitored for impending liver and kidney damage. Since amatoxins undergo enterohepatic circulation, repeated doses of activated charcoal may be useful in further decontaminating the bowel (63). If the patient arrives for treatment after the acute gastrointestinal phase has begun, intermittent gastrodueodenal aspiration and repeated doses of activated charcoal are recommended. Renal failure can often be avoided with appropriate fluid management (42). Acid-base status should be monitored and corrections made. Hepatic failure may produce hypoglycemia and coagulation abnormalities. Monitor glucose and administer corrective amounts of a 10% glucose solution. If clinical hetnorrhage is present, or if hypoprothrombinemia or hypofibrinogenemia is noted, fresh frozen plasma or vitamin K (50-100 mg/day) should be considered. Hepatic failure may require the standard treatments of bowel cleansing, reduced-protein diet, artificial ventilation, and possibly hemodialysis and hemoperfusion (64). Liver transplants have been done in severe cases and have become more common as transplant rejection procedures improve (65, 66). Transplantation should not be attempted without a specific recommendation of the liver transplantation team. Potential complications of liver transplant in an Alnarzita-poisonedpatient include preoperative and/or postoperative severe diarrhea, gastrointestinal hemorrhage, hypophosphatemia, bowel edema, lymphopenia, thrombocytopenia, and neutropenia (65). Early identification of potential transplantation candidates is imperative, but patient selection criteria and the time when a transplant should be done are indefinite. Pinson et al. (65) found that some indications for transplant included a markedly prolonged prothrombin time that was only partially correctable, and a combination of factors such as metabolic acidosis, hypoglycemia, hypofibrinogenemia, increased serum ammonia, and marked elevation in serum aminotransferase levels. Elevation of serum bilirubin levels and azotemia were not indications for transplant. Beckurts et al. (67) recommend transplant with a Quick value less than 20% over several days, serum creatinine concentration greater than 1.4%, even after correction for water and electrolyte abnormalities, serum bilirubin greater than 4.6%, and progressive encephalopathy. Nine et al. (292) found that the decision to transplant the liver depended on liver recovery in Amanita poisoning. They proposed several serum biomarkers that could be monitored. Studies done by Jaeger et al. (68), and Vesconi et al. (42), indicated that the only method to substantially enhance the elimination of amatoxins was forced diuresis. The possibility of renal failure complicates the use of this procedure. However, plasmapheresis has been used successfully as well. Russo et al. (69) describe a patient poisoned with A. phalloides who was given four treatments of plasma filtration exchanging 70-80% of the plasma volume. Improvement was seen after the first treatment, and renal- and livermonitoring parameters were stabilized by the end of the treatments. Unfortunately, this treatment was used on only one patient, not a series. Early charcoal hemoperfusion has also been tried with some success (70). Evaluating the usefulness of other treatments has been difficult. Not all treatments are available worldwide, and because of the potential fatal outcome of the intoxication, several substances are generally used simultaneously. Penicillin G, given early after an amatoxin exposure in doses of 300,000-1,000,000 units/day, has seemed to be beneficial (31). Animal experiments using penicillin have also shown a reduce amatoxin uptake by the liver. Rambousek et al. (71) used silymarin and high-dose penicillin G in treatment of a 7-year-old girl with a severe case of A. phnlloides poisoning (prothrombin time less than 10% and hepatic coma). The child responded

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favorably to the treatment. Penicillin is often given with a number of other treatments, making it difficult to evaluate its effectiveness. In a study of 10 cases, penicillin, 10 million units every 6 hr, was used in combination with gastric lavage, a duodenal tube to disrupt enteropathic circulation of the toxins, and charcoal hemoperfusion (48). The three patients who ate the most mushrooms developed moderate to severe liver and kidney damage. One patient developed severe coagulation abnormalities. All recovered within 3 months. Silymarin, which is derived from the milk thistle [Silybunz mnrianum (L.) Gaertn.], is another potential treatment (64, 72). Silymarin-containing products such as Legalon 70 (R) have also been shown to be hepatoprotective in animal models (73) and in human studies (3 1, 74-76). Milk thistle contains various flavanolignans such as silymarin. The ripe seeds contain 4-6% silymarin. The main three flavanolignans &e silybin (also known as silibinin), silychristin (also known as silychrisin), and silydianin (also known as silidianin) (77, 78). Silymarin’s mechanism of action is thought to be as a free radical scavenger and as an inhibitor of the penetration of the amatoxin into liver cells. The binding sites of both phalloidine and the amanitins are blocked and the liver’s regenerative powers are increased. Enteropathic circulation of these toxins is also thought to be blocked. Silymarin should be given within 48 hr of ingestion for optimum effect. The dose is 20-50 mg/kg/day (79) intravenously, or 1.4-4.2 g/day for 4 days orally. The oral form may not be useful if there is extensive vomiting or if activated charcoal is being given frequently (37). Several new products have been developed to increase the effectiveness and absorption of silymarin. Among these are silibinin dihemisuccinate, silibinin betacyclodextrine, and IdB 1016 (also called silipide), a complex of silybin and phosphatidylcholine (80, 81). Silymarin is not currently available in the United States, but is being used extensively in Europe. Many milk thistle products are on the herbal market in the United States, but they have not undergone extensive testing for treatment of mushroom ingestion. Although death rates for cyclopeptide poisoning differ greatly, they may reach up to 40-50%. With good supportive care, this can be reduced to 20-30% (65,74). In 1984, Hruby (75) reported a 12.8% death rate in 220 cases of Anzcrrlita poisonings treated between 1979 and 1982. When silymarin was used with penicillin therapy, the death rate reported was about 10% (76). Floersheim et al. (31) reported 45 deaths in a group of 205 patients who ate cyclopeptide-containing mushrooms. Of the 16 patients who received silybin, none died. Hruby et al. (74) used severe liver damage as a marker for treatment effectiveness. If silybin was given within 48 hr of the time of mushroom ingestion, it was shown to be an effective prophylactic measure (74). Another study looked at 45 deaths in a group of 205 patients who ate these mushrooms. Sixteen of these patients were given silybin, and none died (3 1). With severe liver damage as a marker, silybin given within 48 hr of the time of mushroom ingestion was showed to be an effective prophylactic measure (74). Sierralta et al. (82) studied cases of Amanita mushroom poisonings in Chile. Twentyfive of the 36 patients (69%) became ill withacute gastroenteritis, seven of these developed acute hepatitis, and one a massive upper gastrointestinal bleed. The latter patient died. Three patients with fulminant hepatic failure also died. Treatment was supportive with trials of silymarin and penicillin. Death rate in this study was 11% of all who ingested the mushroom and 16% of those who became sick. Thioctic acid (150 mg intravenously every 6 hr) was once used as a treatment for amatoxin poisoning (83, 103), and still is in some regions (1). Animal experiments have

Identification Poisoning of Mushroom

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failed to demonstrate a beneficial clinical effect (84), and it is seldom used today. It is not commercially available except as a technical-grade material. Since the amatoxins are somewhat concentrated in the bile (thus the enteropathic circulation), endoscopic biliary diversion has also been tried (85). 4. OtherExperimentalAntidotes High-dose cimetidine (equivalent to a dose of 8400 mg in a 70-kg human) was tested with some success in mice (86). At this time the use of cimetidine in human amatoxin poisoning needs more study and should be considered investigational (87). N-acetylcysteine (NAC) has also beentried experimentally and found useful by Locatelli et al. (88) but to be of little use by Schneider et al. (89). Recently kutkin, an iridoid glycoside mixture obtained from the root of the Indian plant Picrorhiza kurroa, has shown benefits similar to that of silymarin when tested in animals (31, 289). It has not yet been tested in humans. Experimentally produced antibodies comprised of amanitin bound to albumin have been tried as treatment in rabbits. Unfortunately, the antibodies proved to be more toxic than amanitin itself (90). Ethanol administration as a treatment for Amanita phalloides was tried in mice and decreased toxicity reported. Ethanol was given to mice 30 min before and 5 min after A. phalloides (91). The practical application of this timing in an actual ingestion is questionable and would need to be more intensively studied before it could be recommended. Chronic alcohol administration has no value. Animal studies indicated protection against phalloidin toxicity by the drugs rifampin, cysteamine, and phenylbutazone (92, 93) but human studies are lacking. Acubin, from the plant Aucubn japonica, has, in animal studies, shown a significant protective effect against alpha-amanitin. The dose of acubin had to be administered within 12 hr of receiving the amanitin (94, 95). Corticosteroids have been suggested as treatment for the massive hepatic necrosis caused by amatoxins and other causes. No specific studies have substantiated the various claims, but various authors have both recommended and condemned their use (96-102).

5. Examples of Mushrooms in This Group This group includes Amanita gernrnata, Amanita phalloides, Amanita proxima, Amanita smithinnu, Amanita verna, Amanita virosn, Galerim autumnalis, Lepiota helveola, and Lepiota relveola.

C. Orellanine/Orelline 1. Symptoms Orellanine is a nephrotoxin (104) found in some Cortinnrius species. Symptoms are not usually seen for some time after ingestion. This may be as little as 36 hr or up to 14 days (105). It appears that ingesting larger quantities of mushrooms shortens the time to first symptoms (106). Symptoms include nausea, paresthesias, anorexia, gastrointestinal disturbances, headache, an intense burning thirst, oliguria, and eventually renal failure. Renal function may return to normal slowly, or result in prolonged, intermittent dialysis. In the early case reports there was a 15% fatality rate, but with modern dialysis techniques, there should be few fatalities. Most of these exposures have occurred in Europe. It has been thought that North American species of Cortinarius did not contain orelline, but an abstract

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present to the AAPCC has raised sotne questions concerning some North American species. Renal histology after orellanine poisoning shows that there are nonspecific changes occurring, such as tubular dilatation and epithelial flattening. There are signs of interstitial edema and interstitial fibrosis (107). 2. Mechanism of Action The orellanus toxins are typical nephrotoxins and are blue-fluorescing. When sections of kidney are examined under the fluorescence microscope, there are fluorescing deposits in therenal tubules (108). The exact mechanism of toxicity is unknown (107), but light microscopy and electron microscopy show acute tubular necrosis, multinucleated tubular epithelial cells, and concentric medial muscular hyperplasia of the arteries. There may be displaced and abnormally structured action filaments at the edges of the cytoplasm in the epithelial cells of the kidney tubules (109).

3. Treatment When dealing with these exposures, often it is not known that a toxic mushroom has been ingested until after symptoms appear, making gastric decontamination measures, like emesis and activated charcoal, useless. Diagnosis can be done by fluorimetry (see below) or by identification of spores in leftover mushrooms. There is no specific antidote. Treatment is symptomatic and supportive. Extracorporeal hemoperfusion over resin filters should be considered immediately if the patient is seen within 1 week of ingestion (20). Charcoal hemoperfusion may be of some benefit if resin hemoperfusion is not available, but has not been studied. Hemodialysis may be required to correct the fluid and electrolyte imbalances caused by renal failure. Early hemodialysis may improve the prognosis in lifethreatening exposures (107). Hemodialysis alone was tried in one group of six patients. Only one of these patients returned to normal renal function and four required kidney transplants (1 10). When renal damage is extensive, the patient may never regain normal kidney function. In these cases lifelong hemodialysis or kidney transplant may be required. Rohrnloser et a]. (104) evaluated 10 cases in northern Italy. Kidney tissue, urine, and blood samples were tested using fluorimetry after thin-layer chromatography, to determine the presence of orellanine. Orellanine was found after a relatively long period in kidney biopsy material, but no toxin was found in the urine or blood. Based on their research, it appears that orellanine is rapidly distributed to the kidney, and is not found in blood or urine by the time symptoms are reported. The laboratory test is only effective using the kidney tissue (107). In experimental animals, it has been shown that when a single dose of cyclophosphamide 150 mg/kg was given at the same titne as an oral dose of an orellanine-containing mushroom, renal damage was prevented. It has not been tried on humans and seems to have little bearing on accidental poisonings. 4. Examples of Mushrooms in This Group This group includes Cortinarius orellanus, Cortirlcrrius syeciosissimls, and Cortinnrius gerztilis.

D. Muscimol/lbotenicAcid 1. Symptoms Poisonings with these mushrooms are fairly common. One study of Amcu~itnyctntherirzn, done in the PacificNorthwest, showed it to be the commonest cause of nonfatal poisonings,

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while another in Europe found it to represent 50% of all cases (111). Within 30-90 min of ingesting one of these mushrooms the patient may experience drowsiness, which is followed by giddiness, manic behavior, derangement of the senses, or relaxation-somewhat like alcohol intoxication (1 12, 113). About 7-10 mg is necessary for a CNS effect (112, 114). Nausea and vomiting may also be seen (1 15).Both cholinergic and anticholinergic effects may occur (279). Symptoms are generally maximal in 2-3 hr. Later the patient may become confused, disoriented, delirious, and have visual distortions and muscle spasms (1 16-1 18). Hallucinations, and rarely convulsions, may be seen (119). There are often alternating phases of drowsiness and agitation (130). Patients often become slowed down, lethargic, and have difficulty performing various tasks (121). In some cases, exaggerated physical activity, lasting several hours, may be seen. After this time the patient becomes drowsy again, and may sleep for several hours. In some patients this has been described as a “death-like” sleep where the patient is very difficult to arouse. The eyes may also be affected. These toxins produce anticholinergic symptoms such as mydriasis and cycloplegia. Paralysis of convergence was also reported in one patient (122). Children who are severely poisoned may experience convulsions and coma, resolving within 6-9 hr. Nausea and votniting are present in some cases, absent in others (26, 121). Usually blood pressure and cardiac rate are not much affected. Mortality rates are variable, ranging from 0 to 12 percent ( 17). Benjamin (123j found an onset of the CNS effects occurred in 30-180 min in the nine cases studied. Vomiting was rare, but seizures or myoclonic twitching occurred in four of the nine patients. Seizures were controlled with standard anticonvulsive therapy. Because of the potential for hallucinogenic activity, this type of mushroom is often abused as a psychoactive. (124). Anlanitn ~~uscnricr is especially known for its use as a hallucinogen and its use in some religious ceremonies (125). 2. Mechanism of Action Several toxins are involved, including ibotenic acid and muscimol. All of them are isoxazole derivatives. The concentration of each found in a specific mushroom is quite variable. Ibotenic acid is a derivative of glutamic acid and muscimol is a derivative of gamma amino butyric acid (GABA). Decarboxylated ibotenic acid is muscimol, decarboxylated glutamic acid is GABA. Ibotenic acid is readily converted to muscimol, complicating experiments where the two are analyzed. Almost any reaction in acidic solvents will decarboxylate at least some of the ibotenic acid and convert it to muscimol (121). Both animal experiments and hutnan cases support a theory that these agents act as false neurotransmitters. Muscimol exerts a strong CNS action, unlike the neurotransmitter it mimics, GABA. A total of 30-60 mg of ibotenic acid is required to produce a CNS effect; only about 6 mg of muscimol is required (112). When muscimol is given to rats, it affects brain serotonin, norepinephrine, and dopamine levels in a manner similar to LSD, psilocybin, and mescaline (1 13, 126). Ibotenic acid and muscimol appear to cross the blood-brain barrier, possibly by an active transport system (121). Ibotenic does not stay in the body long. Ingested ibotenic acid is generally excreted within 20-90 min (1 14). Effects do not correlate with excretion or blood levels. Peak clinical signs may still be present more than 5 hr after peak excretion. Anznrlitcryarztherincr contains stizolobic and stizolobinic acids, which are derived from 3,4-dihydroxyphenylalanine (DOPA) (127). To date no toxicological importance has

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been attributed to these compounds, which are also found in the edible beans of Stizolobium species (17).

3. Treatment There is no specific antidote or treatment. Care is generally symptomatic and supportive. At one time it wasthought that muscarine was found in these mushrooms to an appreciable extent, and atropine was administered. In fact muscarine is usually not present in significant amounts and the administration of atropine to prevent cholinergic symptoms simply adds to the atropinic effects of ibotenic acid and muscimol. It would take over 70 kg of most strains of Amanita musccwia to produce a muscarine-like poisoning (17). As is usually the case when dealing with biologicals, no rule is 100% true. There have been cases of A. nzuscarin poisoning in Michigan (128), Colorado, and Europe (129, 130) with sweating and salivation as additional symptoms. It appears there are certain strains of A. muscaria (but not yetA. pnntherina)that are able to synthesize sufficient muscarine to produce symptoms The delirium and manic phases can usually be managed without pharmacological intervention (13 1); the “coma” phase may require hospitalization for observation. 4. Examples of Mushrooms in This Group This group includes Arnarzita nzusccuia, Arnarlitcr pantherim, Anmnitn strobilifonnis, and Tricholornn rnuscnrium.

E. Monomethylhydrazine 1. Symptoms There is considerable confusion by laymen over whether the Gyromitra mushrooms are toxic. The incidence of symptoms after exposure varies from one part of the world to another. These mushrooms do contain a monomethylhydrazine precursor that is toxic, and they should be considered potentially toxic. Some of the differences in poisoning incidence may occur from variability in strains, or in methods of preparing the mushroom. In Europe the mushroom is generally considered poisonous, while in the Pacific Northwest of the United States, the lay public generally considers it edible. Still, the mushrooms in this U.S. area do contain the toxin, and poisonings have been reported (13 1). The gyromitrin concentration was investigated by Hatfield (13 1) and found to be about 0.006% (57 mg/ kg of fresh mushrooms). As with most biologics, the chemical content varies with species, varieties, season, and year. Symptoms are more likely to occur if (1) the mushrooms are ingested raw, (2) are poorly prepared, or (3) the water used in boiling is also ingested. Patients have also become intoxicated from inhalation of the vapors created during cooking. Cooking produces the toxic chemicals N-methyl-N-formylhydrazine and methylhydrazine. The methylhydrazine is usually lost if the cooking is done in an open area (132). Symptoms seen with these mushrooms are similar to those seen after overdoses with the drug isoniazid. Poisoning from monomethylhydrazine-containing mushrooms usually produces symptoms that are delayed 6-24 hr after ingestion of the offending mushrooms (133). There have been cases of symptoms occurring up to 53 hr post ingestion. The toxic (lethal) dose is only an estimate, but is estimated at being between 10 and 50 mg/kg (131). Fatality rates have varied between 14.5 and 34.5% (26, 134). The onset of gastrointestinal symptoms is often sudden. The patient may experience a full or bloated sensation, which

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is followed by vomiting and watery diarrhea. The gastrointestinal effects may last about 2 days (135). Abdominal cramping, abdominal pain, headache, and general lassitude often follow. Some patients experience jaundice (135, 136), methemoglobinemia, and hemolysis. Symptoms seen in later stages may include vertigo, loss of muscle coordination, seizures, and coma (including hepatic coma) (137, 138). Although nephrosis has been reported, this is most likely not due to a direct gyromitrin effect, but is secondary to the hemolytic effect. Anuria was seen in one fatality (135). Seizures, although uncommon, may occur late in a serious case (137). Hydrazines are volatile compounds that may cause poisoning by inhalation. Several such cases were reported in the European canning industry (139). Persons wishing to eat Gqu"yn&u species know to boil the mushroom and to discard the water. Parboiling Gyromitra esculur~tafor 10 min has been shown by gas chromatographic analysis to remove 99.5% of the available hydrazines (140). Because of their volatility, hydrazines can also be removed by drying. 2. Mechanism of Action The toxin gyromitrin is described chemically as N-methyl-N-formyl hydrazine. It is not very stable, and is hydrolyzed by boiling into the potentially toxic chemical monomethylhydrazine. Boiling these mushrooms reduced the hydrazine content to 0.53% of the original content (131). Mushrooms that were air-dried at 15-20°C for 5 days still contained about 45 mg/kg of mushroom. This is approximately 12% of the original content. If this drying is extended to 14 days, the content is reduced to about 6 mg/kg. The exact mechanism of hydrazine toxicity is not totally understood, but hydrazines act as vitamin B, (pyridoxine) antagonists by trapping pyridoxal phosphate as hydrazones or oximes (121). Many biological processes utilize pyridoxine. In animal experiments, hydrazines inhibited brain GABA (141), which may be the cause of the seizures seen. Pyridoxine is not the total answer; injection of pyridoxine will terminate some, but not all, of the symptoms.

3. Treatment After decontamination, the patient should be monitored for electrolyte imbalances, hemolysis, and methemoglobinemia. Pyridoxine may be an antidote for the neurological manifestations of poisoning by this mushroom. A dose of 25 mg/kg given as an infusion over 15-30 min has been recommended. Recurring neurological signs such as seizures and coma may necessitate additional doses, to a maximum of 15-20 g/day (33). Since the toxic dose of pyridoxine is unknown, large doses should be administered with caution and for limited periods. Albin et al. (141) described two cases where Gyromitra esculenta mushroom poisonings were treated with greater than 2 g/kg of parenteral pyridoxine over a 3-day period. The patients developed acute, profound sensory losses. Because these mushrooms may affect several systems, blood sugar, free hemoglobin, and liver and kidney function should be monitored. Patients should be observed for the development of methemoglobinemia, which may require the administration of methylene blue. 4. Examples of Mushrooms in This Group This group includes Gyromitra gigas, Gyromitra esculenta, Helvella elastica, Morchella species, Verpcr bohernica, Piziza bndia, and Gyromitm ir$du.

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F. Muscarine 1. Symptoms Signs and symptoms of cholinergic overdrive are usually seen sometime between 30 min and 2 hr after ingestion of these mushrooms. The symptom-free period appears to be directly related to the amount of fungus ingested and its muscarine concentration (142). For example, when high concentrations of mucarine are present, as little as 15-20 min may be required (143, 144). To produce symptoms these mushrooms usually contain a minimum concentration of 0.01% muscarine. The patient may experience blurred vision, pupil constriction, excessive perspiration, salivation and lacrimation, slowed heart rate, reduced blood pressure, increased peristalsis, congested pulmonary circulation, bronchorrhea, mild hypotension, urinary urgency, nasal discharge, and dyspnea (asthma-like breathing) (145, 146).The excessive perspiration, salivation, and lacrimation are diagnostic, and are generally seen only with this type of mushroom poisoning. Patients may also experience peripheral vasodilation and flushing, vomiting, abdotninal pain, and watery diarrhea (142, 147, 148).

2. Mechanism of Action Muscarine is readily absorbed and is distributed throughout the body. It contacts the parasympathetic nervous system by simple capillary diffusion (142, 148). Muscarine affects the portion of the peripheral nervous system that uses acetylcholine as the internerve chemical mediator. Muscarine binds efficiently to the acetycholine receptor and initiates what would ordinarily be acetycholine effects at certain sites (now called muscarinic sites) (149, 150). Unlike acetylcholine, acetylcholinesterase does not significantly affect muscarine, so the acetylcholine effects produced at these particular sites are not rapidly stopped. The specific target organs of muscarine are gland cells, cardiac nodal muscle fibers, and smooth muscle. Skeletal muscle is not affected. The ionic nature of muscarine does not allow it to cross the blood-brain barrier to any extent, and effects that are seen are peripheral, not central (151). Muscarine is not affected by cooking or by digestive enzymes (153). The toxic or lethal dose of muscarine-containing mushrooms varies depending on the concentration in the mushroom. There have been various estimates as to the lethal dose in humans. One text (26) estimates about 300 mg, while another suggest 180 mg (17). If one assumes that Irzocybe pntouillnrclii contains 0.33% muscarine, and that as little as 100-150 g of mushroom is a potentially lethal dose (153, thenthe lethal dose of muscarine would be about 330-495 mg. Regardless of the exact figure, muscarine doses over 150 mg are potentially very dangerous. 3. Treatment If the patient is seen soon after an ingestion, etnesis with syrup of ipecac is probably indicated. Activated charcoal (30-100 g) may also be useful. Since muscarine causes increased gastric tract peristalsis and diarrhea in many patients, administration of a cathartic is probably unnecessary. If life-threatening cholinergic symptoms occur, atropine should be administered. A test dose of 2 mg intravenously is standard in an adult and 0.05 mg/kg in a child. If no signs of atropinization occurs, additional atropine may be administered. The end-point is not dilation of pupils, but the cessation of secretions such as salivation and sweating (142, 143). 4. Examples of Mushrooms in This Group This group includes Clitocybe denlbntcr, Clitocybe tr.mcicoln, Inocybe lacera, Entolorm rhodopolizrrn, and Ilzocybe pudicn. Muscarine is found in a wide variety of Basidiomy-

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759

cetes. Except for some Inocybe and Clitocvbe species, there is usually less than 0.002% mucarine in the dry weight of the mushroom (17). hocybe species commonly contain muscarine. In one study by Malone et al. (154), 30 Irzocybe species were identified, and 29 were found to contain muscarine.

G. Coprine 1. Symptoms Symptoms are seen shortly after ingestion of an alcohol-containing product. For major effects, one of the coprine-containing mushrooms should have been eaten within the last 24-72 hr. The timing of the ingestion may be crucial. There is some evidence that simultaneous ingestion of the mushroom and alcohol may not have an effect (155). Although List and Reith (155) stated the mushrooms had to be cooked to activate the coprine, this has yet to be proven in humans. In mice, cooking is not necessary to activate the coprine (156). Flushing of the neck and face, a metallic taste, palpitation, tachycardia, paresthesias of the extremities, malaise, and a feeling of swelling of the hands may be seen at first. Nausea and vomiting also occur (157, 158). In these patients the blood pressure is generally within normal limits, but sometimes there will be mild hypotension, probably due to hypovolemia. The reaction is similar to that seen with disulfiram. Mayer et al. (159) reported a case of esophageal rupture caused by vomiting. One case of atrial fibrillation was seen, but arrhythmias are rare (157). Ingestion of the mushroom and the alcohol must both occur. If the above symptoms occur after the ingestion of alcohol, it is diagnostic for this type of mushroom poisoning. Laboratory tests for these patients include electrolytes when vomiting and diarrhea is extensive. There is no laboratory test for coprine that would be generally available in a hospital laboratory. Some Coprinus species also are active in the Ames test on bacterial strains. Substances in these mushrooms have caused severe testicular injuries in dogs and rats (160), but damage has not been reported in humans. 2. Mechanism of Action Initially it was thought that disulfiram was the agent responsible, but that has since been disproved and coprine is now thought to be the responsible agent (155, 161). Lindberg et al. (162) demonstrated in rats that there was acetaldehyde accumulation when coprine was present. Coprine is the common name for the gamma glutamyl conjugate of l-aminocyclopropanol. Coprine, like disulfiram, interferes with ethanol metabolism, resulting in the accumulation of acetaldehyde. When tested in vitro, coprine did not show inhibitory action against aldehyde dehydrogenase (163). This led Tottmar and Lindberg (164) to search for a metabolically activated compound. They found that 1-aminocyclopropanol was the active derivative.

3. Treatment Emesis is generally not recommended after ingestion of these mushrooms. There have been no studies evaluating the effectiveness of administering activated charcoal or a cathartic. If it is known that a coprine-containing mushroom has been ingested, and symptoms have not been initiated, the patient should avoid ingesting alcohol for at least 2448 hr. This should prevent toxicity from the interaction. It should be remembered that

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alcohol is not just a recreation beverage, but is found in other household products such as mouthwashes and medications. If symptoms have begun, emesis is not necessary, as vomiting is one of the primary symptoms. Hypotension is generally not serious enough to justify pharmacological intervention; administration of intravenous fluids is usually adequate. Intravenous 4-methylpyrazole given in a dose of 5 mg/kg is known to terminate disulfiram/alcohol reactions, and in theory will terminate coprine reactions as well (165). This chemical inhibits alcohol dehydrogenase and therefore decreases the acetaldehyde formation. It has not been tested in humans, and this is not a labeled indication for the drug. 4. Examples of Mushrooms in This Group Copr-inus atmmentcwius, Copr-inus micaceous,Coprims insigrlis, and Clitocybe clavipes are suspected of also containing the toxins.

H. Psilocybin/Psilocin 1. Symptoms A dysphoric, hallucinogenic state usually begins about 20-60 min after ingestion of psilocybin-containing mushrooms, but may start as late as 3 hr after ingestion (166). If the mushroom is prepared in a soup or tea, the onset may be even more rapid, starting in 510 min (167). Most individuals have effects lasting 2-4 hr, with the peak activity at about 1 hr (168, 169). The pyschoactive effects may last longer, and there are reports of effects up to 15 hr (170, 171). One patient who was placed on a psychiatric ward had hallucinations three successive nights after an ingestion (172). The effect seen may vary depending on dose as well as the mood, personality, and physiological state of the patient, and the setting in which the drug is ingested (25). Some nonpsychological effects have persisted for longer than just 2-4 hr. Examples include mydriasis (18 hr), hypertension (18 hr), and drowsiness (24 hr). The patient’s mood may vary from euphoria to apprehension (167). Visual hallucinations, sweating, yawning, flushing, incoordination, tremulous speech, paresthesias, intensified hearing, unmotivated compulsive movements, and laughter may be present. Nausea and vomiting occur in about 20% of reported cases (173). Seizures have been reported in just a few cases of ingestion by children (174). The mushroom thought to be responsible was Psilocybe baeocystis. Puppies intraperitoneally injected with 15 mg/kgof pure psilocybin developed seizures (174). Flashbacks have been known to occur after use of these mushrooms. In one study of 318 patients, 21 developed flashbacks up to 4 months post mushroom ingestion (173). No fatalities we noted in this study. Intravenous use of these mushrooms produces symptoms that differ slightly from those of oral abuse. Nausea, vomiting diarrhea, arthralgias, myalgias headache, fever, vesicular skin eruptions, and hypoxemia may occur (175). Laboratory tests may show renal and liver function test abnormalities, leukocytosis, and mild methemoglobinemia (about 5%) . Psilocybin is active at a dose of 200 pg. At a dose of near 4 mg the effects are moderate (body relaxation, pleasant intellectual state, detachment), but when the dose exceeds 6 mg and ranges up to 20 mg, the psychic effects are more drastic, resulting in space-time distortions, hallucinations, illusions, and changes in self-awareness (121). As

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might be expected, the amount of mushroom necessary to produce the hallucinogenic state varies not only by mushroom species, but by the patient who is ingesting the mushroom. Examples of effective doses are (167): 1 to 3 5-mm caps of Conocybe cyarzescens or 5 to 10 of the 2.5 mm caps 4 to 8 fresh specimens (size not stated) of Gynmophilus spectnbilis 20 to 40 fresh Psilocybe pelliculosn 1 g of dried Psilocybe semilancenta 10-20 fresh specimens, or 2-5 g of dried Psilocvbe s~tbbaltentus Street use of these mushrooms has led to adulteration of these and other mushrooms with other chemicals. Phencyclidine is a common adulterant. One study done on 886 street products tested between 1973 and 1983 that were thought to be psilocybin containing found that approximately 3 1% contained substances other than psilocybin, 37% had no drug at all, and 28% contained psilocybin without any adulterants (176). When a patient is being evaluated for an ingestion of street psilocybin, the possibility that other drugs may be involved, or that no drugs have been ingested, should always be investigated. 2. Mechanism of Action Psilocybin and psilocin are 4-substituted tryptamine derivatives. These substances are active if they reach the brain. Psilocin’s and LSD’s effects on serotonin in the brain are quite similar. There is evidence that the actions of these toxins are related to their strong structural similarity with serotonin. In fact, both psilocin and psilocybin have serotonergic properties in the periphery as well as centrally ( 121). The clinical psychic effects of psilocin (which is the dephosphorylated ester of psilocybin) are closely related to the level of psilocin in the brain. Brain levels are uniform within 30 min and drop to near zero within 4 hr (121). Psilocin is distributed (in the rat brain) to (highest to lowest concentration): neocortex, hippocampus, and thalamus (177).

3. Treatment Inducing emesis may not alter the clinical course after a typical ingestion with these mushrooms. Most cases are managed with supportive care (173). In those cases where children may have ingested large amounts ofthe mushrooms, emesis may be beneficial. Most patients can be “talked down’’ in a low-stimulus, quiet room with dim lights. If a panic reaction occurs, diazepam in normal therapeutic doses may be administered. Although chlorpromazine has been used to treat the hallucinations, phenothiazines are not generally recommended. Fever may be treated with therapeutic doses of antipyretics such as acetaminophen (children) or aspirin. 4. Examples of Mushrooms in This Group Psilocybin has been isolated from four genera: Psilocybe, Panneolus, Gyms1opilrrs, and Coszocybe. Some Stropharia species in the United States are thought to contain this substance, but documentation of psilocyin in European Stropharia species has not been forthcoming. The group includes Psilocybe blteocystis, Psilocybe cymescerzs, Panaeolus sphinctrinus, Coslocybe cyasropus, and Gymopilus neruginosus. Psilocybin-containing mushrooms are listed in Table 2. As these mushrooms are commonly used illicitly, many slang or street names have been derived for the various mushrooms. Attempts have been made to associate specific species with specific names, but this is guesswork at best. The street names are often interchanged by the lay public. Because many of these species stain blue when the flesh

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762 Table 2 Psilocybin-ContainingMushrooms Name and synonyms Purlaeollrs acuminntus (Schff. ex Secr) Quel Pameollrs i-ickenii Hora Pnnaeolus africnnus Ola'h Prrnaeolus antillnmm (Fries) Dennis Punaeollts phnlaenar-linl (Fr.) Quel Pnnaeollw sepulcrnlis Berk. Pnmeollrs solidipes Peck Parlneolus cmlbodginielrsis Ola'h & Heim Copelandin cnrnbotlginiensis (Ola'h & HeiIn) Singer & Weeks Poncreolus castnneifolius (Murrill) Ola'h Panueollrs cynescens Berkeley & Broorne Copelandin cyanescens (Berk. & Br.) Sacc. Copelandia pnpilionucea (Bull. ex Fr.) Bres. Purlaeollts jrnicola Fries Parweolus nter- (Lange) Kuhner & Romagnesi

Country North America, Europe Southern Africa North and South America

Cambodia, Asia, Hawaii

North and South America United States, Mexico, Brazil. Bolivia, Australia, Philippines, Mediterranean Africa, Americas, Europe

Sorrrce: Refs. 280-283.

is injured, the common names often contain the term "blue" (178). Some of these names are blue meanies, blue legs, bluing psilocybe, cone heads, dimple tops, gold tops, golden tops, liberty caps, 10s ninos, magic mushroom, Palenque mushroom, San Ysidro mushrooms, 'shrooms, and simple simons. 1.

GastrointestinalIrritants

1. Symptoms This group includes a variety of mushrooms and toxins. It is a "catchall" category for those cases where no specific toxin has been identified, or when gastroenteritis is the main symptom. There are few fatalities reported with the gastrointestinal mushrooms, but some have been reported in Europe (17). Symptoms of nausea, vomiting, abdominal pain, and dian-hea occur relatively soon after an ingestion. Weakness, dizziness, paresthesias, tetany, headache, and sweating have also been reported (179). Some of these neurological symptoms, such as paresthesias and tetany, maybe due to fear and hyperventilation (180). Often there is a mixture of toxic principles, resulting in a confusing clinical presentation. About 60% of the total mushroom poisonings in Japan (as of 1997) are due to the gastrointestinal irritant mushrooms Lampter-orzlycesjaponica, Rhodopllyllus rhodopolius,and Rlzodophyllns sinatus (18 1). Onset of these symptoms can be quite variable, but may be as short as 15 min or up to a few hours. Usually these symptoms resolve spontaneously within a few hours, but may last a day or two. There are almost never serious sequelae. A conlmon gastrointestinal irritant in North America is the jack-0-lantern mushroom, Onzphnlotus illuderz (previously called Clitocybe illudem). The toxin is thought to be illuden S (287). The mushroom On~phalotusolear-ius (previously Clitocybe olivascens) and Lnnzptet-ornyces japonicus also contain the toxin and have been reported as causing illness ( 1 82). Lethargy, weakness, and sensation of cold occurred in eight of 14 patients

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in one study. Vomiting (eight patients) and diarrhea (five patients) were also commonly reported (183), with a short latency period before symptom onset. In the United States, the most common symptoms are generally just nausea and vomiting (184, 185), while in Europe liver toxicity and severe muscarinic effects have been reported (179, 186). Treatment for U.S. cases is generally just rehydration and correction of any electrolyte abnormalities. Another common mushroom that causes significant nausea and vomiting is Cldorophylum nzolybdites. This is a gray-green spored mushroom that superficially looks like the meadow mushroom Agcrricus campestris. Vomiting can be extensive and sometimes hospitalization is required for fluid and electrolyte replacement (180). A case of hypovolemic shock was reported in a 6-year-old (286). Not all of the gastrointestinal reactions are due to mushroom toxins. Occasionally spoiled (contaminated by bacteria) mushrooms are ingested and cause illness. When evaluating cases of gastrointestinal mushroom “poisonings,” food poisoning should always be included in the differential diagnosis. 2. Mechanism of Action There are many mechanisms involved with these reactions; some are hypersensitivity reactions (see below), some are irritant reactions, some are due to enzyme deficiencies, and for many of the others the mechanism is unknown. An example of enyzme deficiency was reported by Bergoz (187) in a patient who developed abdominal pain and diarrhea after ingestion of Psalliota campestris due to a trehalose deficiency. The chemicals involved in the gastrointestinal irritant category are as diverse as the mechanisms. Albcrtrellus species have shown irritant monoterpenes (188). Gomphus toxicity is due to norcaperatic acid, an aconitase inhibitor (189). Hebelorrm clustzrlinifonne contains the cytotoxic triterpene hebelomic acid A (190), and Hebeloma virzosophyllum contains various cucurbitane triterpene glycosides (191). Laccaria arrzethystina (192) contains lectins that may contribute to the gastroenteritis in a manner similar to the lectins in castor bean intoxications. Rzlsszlla and Lactnrius species contain marasmane or lactarane sesquiterpenes (193, 194); Lmtiporus srdphul-eus and Meripilus species contains phenolethylamines like tyramine (17). It is unknown just what contribution all these compounds make to the gastroenteritis.

a. Anirmls. The generally held opinion of the lay public is that if an animal eats a mushroom, it must be safe, implying that an animal would not eat a toxic mushroom. Such is not the case. Animals become poisoned on fungi as well as humans, and ingestion of a species by an animal does not make it safe for human consumption. There are several examples. A 7-month-old pig ate a Scleroderma citrirzunl. Within 20 min theanimal started vomiting and by 1 h it was depressed, weak, recumbent, hypothermic, tachycardic, and tachypneic in addition to still vomiting. Treatment was unsuccessful, and the animal died within 5 h (195). Guinea pigs that ingested Omphalotzls olear-iusdeveloped fatty degeneration of the liver, kidney, and myocardium, as well as anemia and cerebral edema.

3. Treatment There are no specific antidotes; treatment is symptomatic and supportive. Emesis is generally notrecommended if the specific species is known and is just agastrointestinal irritant. If the patient is unsure of what was ingested, or if there is a possibility that a number of different species have been eaten, emesis and/or activated charcoal may still be recom-

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mended. The necessity of inducing emesis is also tempered by the extent of the vomiting induced by themushroom itself, and the stomach content return produced by that vomiting. The potential for fluid and electrolyte abnormalities should be considered, based on the patient’s preingestion status and the amount of vomiting and diarrhea. This is particularly important when dealing with the elderly, debilitated patients, or small children. 4. Examples of Mushrooms in This Group This group includes Agaricus xanthodermus, Amanita volvatcr, Boletus satanus, Chlol-0plryllum molybdites, Entoloma lividurn, Gomphus JEoccosus, Lampteromyces japonica, Rlzodophyllus rhodopolius, and Rhodophyllus sinatus.

J.

OtherConsiderations

1. HypersensitivityReactions Various types of hypersensitivity reactions may occur after exposure to mushrooms. These include gastrointestinal, dermal, and respiratory reactions. Reactions may be with fungi thought of as mushrooms, smuts, rusts, slime molds, or dry rot fungi (196). Many of the reactions in the gastrointestinal group of toxins above may actually be hypersensitivity reactions. The Agaricus species, which are considered edible by most persons, will produce stomach upset in selected individuals. This includes species such as A.cnlifomicus,A.anJensis,A. placosnyes, and A. xalzthodermus (197). Even A. bispolus, the mushroom most commonly found in grocery stores of the United States, has been found to produce short-lived gastrointestinal upset in some people who are particularly sensitive (198). Verpn species are toxic to some people, but not to others. When large amounts have been eaten, some patients become ill (199, 200). The same is true when a number of small meals of this mushroom are eaten over a short period (26). Dernzal reactions have occurred after exposure to several species of mushrooms. A delayed hypersensitivity has been noted to one of the most common mushrooms seen in the United States, Agaricus bisporus. A 3 1-year-old worker in a mushroom nursery developed eczema without respiratory symptoms or rhinitis. Symptoms developed whenever she handled the mushrooms, but disappeared whenever she used gloves. She crossreacted with the same species from other nurseries. Other workers were patch tested, but did not react (201). Nakamura (202-204) described 23 cases of dermatitis to Lerrtims edodes (shiitake) in 1977, 30 cases in 1985, 41 in 1986, and 51 in 1992. Eruptions generally appear as early as 6 h and as late as 2 days after eating mushrooms. The trunk is almost always involved, and (in decreasing frequency) the extremities, neck, face, and head also may become involved (205). The digestive system and mucous membranes are usually involved. Chills, fever, and productive cough often appear about 6 h after exposure to the mushrooms (206). The dermatitis usually lasts 2-14 days, the average being 8.5 days (205). Respiratory reactions are fairly common to fungi and fungal spores (207, 208). At least 100 species of fungal spores interact with humans. Contact may occur either by airborne spores or by those spores being attached to dust (209). Basidiomycetes spores are known causes of bronchial asthma and allergic rhinitis (210, 21 1). Asthmatics worry about pollen counts, but the fungal spore counts may exceed the pollen count by 1000fold (212), and produce asthmatic reactions in many patients. IgG to fungal spores exists

Identification of Mushroom Poisoning

765

in most human serum, and specific IgE has been noted in some atopic patients (213-215). Studies have been performed that show an increase in hospital admissions for allergyrelated incidents when basidiospore levels are increased (57, 94). Respiratory reactions to Agaricus, Alternaria, Aspergillus, and Cladosporium are fairly well known to most health care providers (290), but they are not mushroom producing fungi. Some other “mushroom”-type fungi that produce respiratory symptoms include Agaricus, Amanita, Armilluria, Boletus, Culvatin, Chlorophyllurn, Coprinus, Geastrum, Pleurotus, Scleroderma, and Trichoderma (212, 216, 217, 278). About 10% of the 1.52% of all allergic persons react to fungal allergens. The spores of these mushrooms are often 10 microns or less in size, allowing penetration into the lower parts of the lung (218). Some mushrooms that are often reported as a cause of respiratory reactions, especially in growers or workers, are Lentinus edodes (shiitake) (219, 220), Aspergillus glauca (221), Agaricus bisporus, various Pleurotus species, Pholiota nameko, and various puffballs. An illness associated with mushrooms grown for food is an extrinsic allergic alveolitis, commonly called mushroom grower’s lung (MGL) (222) or farmer’s lung. The clinical diagnosis of this condition can be supported by a positive provocation test (223). MGL is most commonly seen in workers who grow mushrooms commercially, but may occur in someone who is growing them at home. The reaction has occurred in individuals involved in several stages of the mushroom-growing process, including composting, picking and sorting, growing, spawning, and disposal of the mushroom growth medium (222, 224-228). Symptoms of this illness include general malaise, vomiting, headache, fever, chills, weight loss, myalgia, arthralgia, dyspnea, nonproductive cough, and shortness of breath (140, 229, 230). Findings include cyanosis bibasilar crackles, reduced lung volumes, hypoxemia, leukocytosis, elevated ESR, positive C-reactive protein, and bilateral diffuse reticulonodular shadows (231). The onset is usually slow, over several months. The symptoms continue and worsen while the patient is exposed to the allergen. Once exposure is discontinued, recovery generally takes 1-6 weeks. For some individuals it takes several months (229, 232). Mushroom farm workers have had cough, fever, chills, myalgia, malaise, and dyspnea after exposure to the edible mushroom Agaricus bisporus (233). Some of these cases have been misdiagnosed as flu, bronchitis, viral respiratory infection, pneumonia, or coronary insufficiency (233). Allergies were reported in eight workers exposed to various mushroom species they encountered as part of processing of dried mushrooms for soups (234). Pleurotus species have been identified as causing allergic reactions and asthma (235). Within 4-6 weeks of working with P. floridupatients developed fatigue, limb pain, headache, and somewhat later, coughing. Allergenicity was established by intradermal and prick tests (236). Pleurotus ostreeatus cap, mycelia, and spore extracts were examined by Weissman et al. (237); at least 27 precipitating antigens were found (238). Symptoms found after several weeks of work may include fever, chills, dyspnea, cough, muscle pain, headache, wheezing, diaphoresis, sputum production, and nausea (239). Allergic alveolitis is often reported (234), as is allergic asthma (240). A number of puffball species also contain allergens. Ibanez et al. (241) showed that Calvatia cyanthiformis, Geaster snccatum, Pisolithus tinctorius, and Scleroderma nreolntum all contain allergenic components. Seventy-eight patients were skin tested for allergenicity to various mushrooms; 20.5% were found allergic to Pucciniu cyilodontis (a rust), 15.4% to Stemonitis ferruginen (a slime mold), 15.4% to Fuligo septicn (a slime

766

Spoerke

mold), 15.4% to Lycogala epidendrum (a slime mold), 14.1% to Ustilago muydis(a smut), 14.1% to Chlorophyllurn molybdites (a mushroom) and to Podaxis pistillaris (a puffball) (217). Pholiota nameko, a commonly cultivated mushroom in Japan, is also responsible for a number of occupational cases of nonproductive cough, dyspnea on exertion, and fever. Indoor cultivation of this mushroom appears to increase the chance of developing the hypersensitivity pneumonitis (242). The diagnosis is often made by identifying a precipitin to Pholiota spores (243, 244) There is no specific treatment for this illness. Obviously exposure should be discontinued. In some cases corticosteroids have been used to alleviate symptoms. A typical dose may be 60 mg/day tapered over 3 weeks, then terminated (227). Corticosteroids should not be used to minimize symptoms while a patient continues exposure. In some, but not all, cases, face masks have given some relief, but they are not well tolerated by workers (232, 236, 245). 2. Other Toxin Types The eight-class scheme used above is useful clinically, but there are many different types of mushroom toxins. Some of these fall into the above categories, some do not fall into any of the above classes (except, possibly, miscellaneous gastrointestinal for some). A more general approach may be taken to the potential toxins found in mushrooms.

a. Amides. Glutamic acid with 1-aminocyclopropanol found in Coprinus species is a representative of this type of toxin. Another is 4-hydroxyaniline found in various Agaricus species (17). b. Anthraquinones. These are found in a number of plant and mushroom species. Their primary effect would be irritation of the gastrointestinal tract with subsequent diarrhea. These are octaketides formed via the acetate-malonate pathways of plant metabolism. A representative mushroom containing anthraquinones is Der~nocybesanquirzea (17).

c. Hemolytic Agelzts. These are found in some mushrooms. Aqueous extract of Pleurotus ostreatus, an edible mushroom, has a hemolytic agent called pleurotolysin. The sensitivity of the hemolysin is related to the amount of sphingomyelin content of the erythrocyte membrane. Inhibition of lysis from this agent is with liposomes prepared from cholesterol (246). d. Kornbuclm Ten. This is a beverage taken by some to improve health. It is sometimes called mushroom tea or Kombucha mushroom tea. In fact it is a mixture of fungi, yeasts, and bacteria allowed to ferment. Some adverse effects have been reported, including allergic reactions, jaundice, neck pain, vomiting, and head pain (247). It is difficult to determine what the causative agents might be, as the composition of Kombucha may differ from case to case. e. NitrogenHeterocvclics. These make up a large group of potential toxins, many of which are psychoactive. Ibotenic acid, muscimol, baeocystin, bufotenine, and psilocybin are representatives of isoxazoles and tryptoamines in this group (1 7). The Orellanus toxins are pyridine-type compounds found in this group (17).

of

Identification

Mushroom Poisoning

767

J: N-N Bond Compounds. These have been found to be carcinogenic or mutagenic. Representatives are: lyophylline found in Lyophvllumconnatum, agaritine in Agcrricus, and gyromitrin in Gyromitra species (17). Agcrricus bisporus contains very small amounts of the reactive aryl diazonium ion. This ion reacts with 2-naphthol. Streaking the stem of this mushroom with 2-naphthol produces a red color (248, 249). g. Oxolnns. These nitrogen-containing compounds have a five-membered ring possessing an oxygen. A representative chemical would be muscarine (see muscarine above) ( 17).

11. Peptides. The cyclic tryptophan-containing peptides (e.g., amatoxins) found in various Amanita and Galerinn species are some of the most dangerous toxins in mushrooms (17). i. Radioacthit?,. The Japanese have tested various fungi for radiocesium. The common edible mushrooms such as Lentinus edodes (shiitake), Fla~nmuli~cr velutipes (enoke), Pleurotus ostreatus (oyster), and Pholiota mmeko (Nameko) were found to have levels generally below 50 Bq/kg, and many samples were below 5 Bq/kg. The range of levels in all mushrooms tested was 3-1520 Bq/kg. Suillus gra~zulatus and Lactnrius hntsuc1cA-e were found to selectively uptake cesium rather than potassium from the soil (252). When Levztirzus edodes found in the marketplace was tested, concentrations of cesium 137 ranged from 6.7 to 73.9 Bq/kg. Little if any wasdetected in Flcmrmdinn veltipes, LvophvlZ m z aggregntum (shimeji), or Plezrrotus ostreatus (oyster) (253). These samples were also tested for cesium 134, but none was found. j . Rrlbratoxins. This toxic metabolite from Penicillium rubrum is known to have caused hepatitis in cattle and pigs. When tested in mice and hamsters, liver, spleen, and kidney damage resulted. Necrosis of the hepatocytes and renal tubular degeneration were histological findings (254, 255).

k. Terpenoids. Plants produce these compounds from mevalonic acid. Terpenoids are common in mushrooms. Onzplznlotus olecwius, Hypholowzn fksciculare, Lnctcrrius species, and Russuln species all contain sesquiterpenes or triterpenes (17). The toxicological significance of these agents is still being investigated.

I. Trichotlzecerzes. Fusnrizm species mycotoxins such as deoxynivalenol (DON) and T-2 are often found in cereal grains and may cause mycotoxicosis in both animals and humans. Although these toxins are not generally found in “mushrooms,” they are potent toxins interfering with protein synthesis, DNA and RNA synthesis, and producing hemolysis (256, 257). Other mycotoxins associated with poisoning in foodstuffs include aflatoxins, vomitoxin, fumonisin, gossypol, glucosinolates, ochratoxin A, and zearalenone (256, 258, 259). PotentialMutagenic,Carcinogenic,andAntineoplastic Actions of Mushrooms Agnricus mushroom species are also suspected of containing potentially carcinogenic Nnitrosamines (260, 291), aromatic diazonium ions (250, 261), and arylhydrazines (251). 3.

Spoerke

768

The clinical significance of these compounds in normal consumption has yet to be determined (262-266). Ling-Zhi (Gurzodenna lz~idurn)contains various water-soluble compounds that inhibit the growth of sarcoma 180 or fibrosarcoma and Lewis lung carcinoma when tested in mice (267). Chinese traditional medicine practitioners have used Ling-Zhi as an antitumor agent (268). Gyrornitru escdentu is known to contain up to 0.3% acetaldehyde methylformalhydrazone, which has had some carcinogenicity when tested in laboratory animals. Various other gyromitrins have also been studied for carcinogenic potential (269, 270). When Swiss mice were given intragastric weekly doses of 100 pg/g of gyromitrin G for a year, they developed lung, clitoral, forestomach, and prepubertal tumors (272). Both hydrazine and monomethylhydrazine, constituents of some ofthe Gyrornitru species, have been shown to be carcinogenic in test animals (273, 274). Various Lcrcturius species have been shown to have mutagenic potential. L. deliciosus and L. deterrimus contain lactaroviolin I1 and deterrol, substances that have been shown in the Ames test (275) to have mutagenic activity (17). L. rztjius and L. lzelvus have shown mild mutagenicity in the Ames test (276). Lentiws edodes, the edible shiitake mushroom, has been shown to contain a mitogenic lectin. Cases of mutagenicity in humans or animals have not been reported (277). The species Lvcopl7yllum cor~~zutum has been shown to contain a potentially mutagenic azoxy compound (17). No human cases of mutagenicity have been reported.

111.

SUMMARY

Diagnosis of mushroom poisoning is often through a combination of history, symptom presentation, and physical evidence. Identification using various macroscopic and microscopic characteristics is important in predicting outcome. Once it has been determined that a mushroom has been ingested, the symptom complex, combined with the time of onset, can aid in determining the type of mushroom ingested. Most treatments should start with decontamination with good supportive care. There are few actual antidotes.

REFERENCES 1. WF Piering, N Bratanow. Role of the clinical laboratory in guiding treatment of Antarzita virosa mushroom poisoning: report of two cases. Clin Chem 36: 571-574, 1990. 2. M Kotwica, A Rogaczewska. Causes of poisonings during the period 1990-1993. Przegl Lek 53: 329-333, 1996. 3. M Kotwica, A Jarosz, Z Kolacinski, A Rogaczewska. Sources of poisoning exposures in children during 1990-1995. An analysis of the National Poison Information Centre files. Int J Occup Med Environ Health 10: 177-186, 1997. 4. M Chiossi, P DiPietro, A Marchi, G Messi, G Gallone, MG Peisino. Mushroom poisoning in children. Vet Hum Toxicol 35: 331, 1993. 5. RC Gazzero, R D Goos. A case of nlushroom poisoning involving Amrnitrr virosa. McI1vainea 10:45-46, 1991. 6. JA Herrick. Mushrooms-to eat or not to eat. School Sci Math 48: 679-685, 1948. 7. JM McPartland, RJ Vilgalys, MA Cubeta. Mushroom poisoning. Am Fam Physician 55: 1805-1809, 1997.

Identification of Mushroom Poisoning

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8. R Molina, T O’Dell, D Luoma, M Amaranthus, M Castellano, K Russell. Biology, Ecology, and Social Aspects of Wild Edible Mushrooms in the Forests of the Pacific Northwest: A Preface to Managing Commercial Harvest. General Technical Report PNW-GTR-309. Portland, OR: US Department of Agriculture Forest Service, Pacific Northwest Research Station, 1993. 9. D Stallard, TE Edes. Muscarinic poisoning from medications and mushrooms. A puzzling symptom complex. Postgrad Med 85: 341-345, 1989. 10. DM Huffman, LH Tiffany, G Knaphus. Mushrooms and Other Fungi of the Midcontinental United States. Ames: Iowa State University Press, 1989. 11. BL Obrien, L Khuu. A fatal Sunday brunch: Amanita mushroom poisoning in a Gulf Coast fanuly. Am J Gastroenterol 91: 581-583, 1996. 12. DS Holsen, S Aarebrot. (Poisonous mushrooms, mushroom poisons, and mushroom poisoning. A review). Tidsskr Nor Laegeforen 117: 3385-3388, 1997. 13. M Shaw. Amateur opportunity. In: D Spoerke, B Rumack, eds. Handbook of Mushroom Poisoning. Diagnosis and Treatment. Boca Raton, FL: CRC Press, 1994. 14. DG Spoerke. Mushroom field tests. In: DG Spoerke, BH Rumack, eds. Handbook of Mushroom Poisoning. Diagnosis and Treatment. Boca Raton, FL: CRC Press, 1994. 15. M Lange, FB Hora. Collins Guide to Mushrooms and Toadstools. New York: William Collins & Sons, 1963. 16. M Bon. The Mushroomsand Toadstools of Britain and Northwestern Europe. Toronto: Hodder & Stoughton. 1987. 17. A Bresinsky, H Besl. A Colour Atlas of Poisonous Fungi. A Handbook for Pharmacists, Doctors, and Biologists. London: Wolfe Publishing Company, 1990. 18. HD Theirs. California Mushrooms. A Field Guide to the Boletes. New York: Hafner Press, 1975. 19. FI Eilers, BL Barnard. A rapid method for the diagnosis of poisoning caused by the mushroom Lepiota morgani. Am J Clin Pathol 60: 823-825, 1973. 20. TK Schumacher, K Holland. Mushroom poisoning caused by species of the genus Cortinurim, Fries. Arch Toxicol 53: 87-106, 1983. 21. W Steglich, B Steffan, K Stroech. M Wolf. Pistillarin, ein charakeristisdcher Inhaltsstoff der Herkuleskeule (Cla~~ariadelphus pistillaris) und einiger Ramaria-Arten (Basidiomycetes) Z Naturforsch39c:10-12,1984. 22. GH Lincoff. Audubon Society Field Guide to North American Mushrooms. New York: Alfred A Knopf, 1975. North American mushrooms 23. KF Lanlpe, MA McCann. Differential diagnosis of poisoning by with particular emphasis on A?,mlita pltalloides. Ann Emerg Med 16:956-962. 1987. 24. CG Ainsworth. Introduction to the History of Mycology. Cambridge: Cambridge University Press, 1976. 25. CJ Alexopoulos, CW Mims. Introductory Mycology. 3rd ed. New York: J Wiley, 1979. 26. G Lincoff, DH Mitchel. Toxic and Hallucinogenic Mushroom Poisoning. New York: Van Nostrand Reinhold, 1977. 27. BH Rumack. Symptomatic diagnosis and treatment of mushroom poisoning. In: D Spoerke, B Rumack, eds. Handbook of Mushroom Poisoning. Diagnosis and Treatment. Boca Raton, FL: CRC Press, 1994. 28. CE Becker, TG Tong, U Boerner et al. Diagnosis and treatment of Amanita yllnlloides-type mushroom poisoning. West J Med 125: 100-109, 1976. 29. H Faulstich. New aspects of Amanita poisoning. Klin Wochenschr 57: 1143-1 152, 1979. 30. DM Mitchel. Amanitu mushroom poisoning. Annu Rev Med 31: 5 1-57, 1980. 31. GL Floersheim, 0 Weber, P Tschumi, et al. Knollenblatterpilzvergiftung (Amanita phnlloides): pronostische Faktoren und therapeutische Massnahmen. Schweiz Med Wochenschr 112: 1164-1 177, 1982. 32. E Omidynia. R Rashidpourai, MT Qaderi, E Ameri. Mycetismus in Hamadan, of west Iran. Southeast Asian J Trop Med Public Health 28: 438-439, 1997.

770

Spoerke

33. JP Hanrahan. MA Gordon. Mushroom poisoning. JAMA 351: 1057-1061, 1984. 34. RE Tulloss, JE Lindgren. Amcrnitcr sr,rithinncr-taxonomy. distribution, and poisoning. Mycotaxon 35: 373-387, 1992. 35. C, RW Brennan. Arnarzitcr mushroom poisoning with recovery from coma: a case report. Ann Neurol 3: 177-179, 1978. 36. H G Bivens, R Bivens, R Lammer. Mushroom ingestion. Ann Emerg Med 14: 101-106, 1985. 37 GL Floersheim. Treatment of human amatoxin mushroom poisoning: myths and advances in therapy. Med Toxicol 2: 1-9, 1987. 38. P Sanz, R Reig, L Borras, J Martinez, R Manez, J Corbella. Disseminated intravascular coagulation and mesenteric venous thrombosis in fatal Anznrzirn poisoning. Hum Toxicol 7: 199-201. 1988. 39. V Fineschi. M DiPaola, F Centini. Histological criteria for diagnosis of Anzcrrtitcr phalloides poisoning. J Forensic Sci 41: 429-432, 1996. 40. B Oralewska. J Zawadzki, I Jankowska, K Popinska, J Socha. Disorders of magnesium homeostasis in the course of liver disease in children. Magnes Res 9: 125-123, 1996. 41. H Misiuk, M Magnowska-Wozniak. (Ocular complications in toadstool mushroom poisoning.) Klin Oczna 1: 59-60, 1996. 42. S Vesconi, M Langer. G Iapichino, et al. Therapy of cytotoxic lnushroom intoxication. Crit Care Med 13: 402-406, 1985. 43. Fukuuchi-Fumiko. Hida-Miho, Sato-Takeshi, Inokuchi-Sadaki, Sawada-Yusuke. A case with acute renal failure induced by acute mushroom ingestion (Arrrcrnitcr virostr) poisoning. Nippon Toseki Igakkai Zasshi 28: 1455-1460, 1995. 44. L deHaro, J Jouglard, J Arditti, J M David. Acute renal failure after Anrcrrtitu prosinrcr ingestion: experience of the poison centre of Marseilles. Nephrologie 19: 21-24, 1998. 45. CM Christensen. Molds, Mushrooms and Mycotoxins. Minneapolis: University of Minnesota Press, 1975, p 264. 36. AM Leathem, RA Purssell, VR Chan, PD Kroeger. Renal failure caused by mushroom poisoning. J Toxicol Clin Toxicol 35: 67-75, 1997. 47. MJ Kelner, NM Alexander. Endocrine hormone abnormalities in Arrmirn poisoning. Clin Toxicol 25: 21-37, 1987. 48. AS Genser, SM Marcus. Arnnrzitcr mushroom poisoning, an "outbreak" of 10 cases. Vet Hum Tosicol 29: 461-462, 1987. 49. 0 Bruggemann. M Meder, R Freitag. Analysis of amatoxins alpha-amanitin in toadstool extracts and body fluids by capillary zone electrophoresis with photodiode detection. J Chromatogr 744: 167-176, 1996. 50. SM Pond, KR Olsen. OF Woo. JD Osterloh. Amatoxin poisoning in Northern California, 1982-1983. West J Med 135: 304-209, 1986. 51. J Homann, P Rawer, H Bleyl, et al. Early detection of amatoxins in human mushroom poisoning. Arch Toxicol 59: 190-191, 1986. 52. Terada-Hisaya. Tsubouchi-Haruo, Abe-Masao, Miyabe Masaki. (Determination of amanitoxins in prepared food.) Nagoya-shi Eisei Kenkyu Shoho (Nagoya City Health Res Inst) 41: 8-10. 1995. 53. A Jaeger, F Jehl, F Flesch, P Saunder, J Kopferschmitt. Kinetics of amatosins in human poisoning: therapeutic implications. Clin Toxicol 3 1: 63-80, 1993. 54. F Jaros. M Kascak. [Incidence and tun-ent treatment of cytotoxic cyclopeptide (phalloidine) poisoning in pregnant women.] Vnitr Lek 42: 414-417, 1996. 55. Th Wieland & H Faulstich. Amatoxins, phallotoxins. phallolysin and antamanide. the biologically active components of poisonous Arnnrritl~mushrooms. Crit Rev Biochern 5: 185, 1978. 56. Th Wieland. Poisonous principles of InushrooIns of the genus Amnnitu. Science (NY) 159: 946. 1931.

Identification of Mushroom Poisoning

771

57. VM Govindan, H Faulstich, Th Wieland, B Agostini, W Hasselback. In vitro effect of phalloidin on plasma membrane preparation from rat liver. Naturwissenschaften 59: 521, 1972. 58. M Frimmer, R Kroker, J Prostendoerfer. The mode of action of phalloidin. Demonstration ofrapiddeformationofisolatedhepatocytesby scanning electronmicroscopy.NaunynSchmiedebergs Archs Exp Pathol Pharmacol 284: 395. RNA59. KH Seifart. LE Sekeris. a-amanitin, aspecific inhibitor of transcription by mammalian polymerase. Z Naturforsch 23b: 1538. 1969. 60. F Stirpe. L Fiume. Studies of the pathogenesis of liver necrosis by alpha-amanitin. Biochem J 105: 779, 1967. 61. FM Fouad, WD Marshall, PG Farrell, M Goldberg, G Ruhenstroth-Bauer. Inhibition by the nlushroonl toxins a-amanitin and phalloidin of hepatopoietin-induced %-thymidine incorporation into rat liver DNA and plasma protein production in hepatocyte cultures. Toxicon 25: 1265-1271,1987. 62. B Tuchweber, RSieck, W Trost. Prevention of silybin of phalloidin-induced acute hepatotoxicity. Toxicol Appl Pharmacol 51: 265-275. 1979. 63. LL Olesen. Amatoxin intoxication. Scand J Urol Nephrol 24: 23 1-234, 1990. 64. AI Sabeel. J Kurkus. T Lindholm: Intensive hemodialysis and hemoperfusion treatment of Amerrliter mushroom poisoning. Mycopathologia 131: 107- 113, 1995. 65. CW Pinson, MR Daya, KG Benner, RL Norton, KE Deveney, NL Ascher, JP Roberts, JR Lake, AG Kurkchubasche, JWRagsdale. JP Alexander, EB Keeffe. Livertransplantation for severe Anznnitn phnlloides mushroom poisoning. Am J Surg 159: 493-499. 1990. 66. JC Favarel-Garrigues, J Saric, G Janiver, F Wickers, B Masson, S Winnock, P Couzigou, D Lugrin, F Penouil, A deMascarel.Intoxicationphalloidiennegrave-transplantation hepatique: un recours possible (a props d’une observation. [Serious A. phalloicles poisoning-hepatic transplantation: a possible remedy (case report.)] J Toxicol Clin Exp 8: 5966.1988. 67. KT Beckurts, AHHolscher, CD Heidecke, TR Zilker. W Natrath, Siewert JR. (The role of liver transportation in the treatment of acute liver failure following AInurzitu phnlloides poisoning.) Dtsch Med Wochenschr 122: 351-355, 1997. 68. A Jaeger, F Jehl, F Flesch, et al. Toxicokinetics of alpha and beta amanitins in Ammitu p l d o i d e s poisonings. Communication: XI1 Congress of EAPCC, Brussels, August 1986. 69. GE Russo, S Giusti, M Maurici. M Bosco. E Vitaliano, MS Caramiello, B Bauco, CM DeMarco. V Marigliano. (Plasmapheresis and mushroom poisoning: report ofa caseof Alwvzita phnlloides poisoning.) Clin Ter 148: 277-280, 1997. 70. JP Wauthers, C Rossel, JJ Farquet. Anmzitcl phnlloides poisoning treated by early charcoal haernoperfusion. Br Med J 2: 1465, 1978. 71. V Rambousek, J Janda,M Sikut. (SevereAInnrlitn phnlloides poisoning in a 7-year-oldgirl). Cesk Pediatr 48: 332-333, 1993. 72. D Jacobsen. The relative efficacy of antidotes. Proceedings of the 5th World Congress for the World Federation of Associations of Clinical Toxicology Centers and Poison Control Centers, Nov 8-11, Taipei, Taiwan, 1995. 73. GL Floersheim. Treatment of experimental poisoning produced byextracts of Amunitn p h d loicles. Toxicol Appl Pharmacol 34: 499-500, 1975. 74. K Hruby, A Donner. G Csomos. H Thaler. Silibinin in the treatment of deathcap fungus poisoning. Hum Toxicol2:183.1983. 75. K Hruby. Forum 6: 23-26, 1984. 76. K Hruby. Tntensivmed 1987; 24: 269-274. Chim Farm 77. I Morelli. (Constituents of Silybunz mrricrmm and their therapeutic use.) Boll 117:258-267,1978. 78. FM Harnmouda, SI Ismall, NM Hassan, AK Zaki, A Kamel, H Rimpler. Evaluation of the cultivated under different agricultural conditions. silymarin content in Silybrtnz r~znrin~zz~m Planta Med 57 (Suppl 2): A29, 1991.

772

Spoerke

79. TR Zilker, NJ Felgenhauer, H Michael, A Strenge-Hesse. Grading of severity and therapy of 154 cases of Anzanita poisoning. Vet Hum Toxicol 35: 331, 1993. 80. M Conti, Porzio, S Malandrino: Activity of silipide against ethanol-induced liver damage in rats. Planta Med 57 (Suppl 2): A1 15-A1 16, 1991. and 81. R Carducci, MF Arnlellino, C Volpe, G Basile, N Caso, A Apicella, V Basile. (Silibinin acute poisoning with Arnarzita phalloides.) Minerva Anestesiol 62: 187-193, 1996. 82. A Sierralta, ME Jeria, G Figueroa, J Pinto, JC Araya, J San Juan, J Grinsbergs, E Valenzuela. Intoxicacion por callampas venenosasen la IX region. Rol de Amanita gemmata. (Mushroom poisoning in the IX region. Role of Amanita genunata.) Rev Med Chi1 122: 795-802, 1994. 83. BM Berkson. Thioctic acidsin treatment of hepatotoxic mushrooms (Phalloides)poisoning. N Engl J Med 300: 371, 1979. 84. GL Floersheim. Treatment of mushroom poisoning. JAMA 253: 252, 1985. Arnanita poisoning treated with endoscopic biliary diver85. C Frank. L Cummins. Case review: sion. J Emerg Nurs 13: 132-136, 1987. 86. SM Schneider, D Borochovitz,EP Krenzelok. Cinletidine protection against alpha-amanitin hepatotoxicity in mice: a potential model for the treatment of Autanitu phaZloides poisoning. Ann Emerg Med 16: 1136-1 140, 1987. 87, E Redd: Hepatotoxicity from mushrooms may be prevented by cimetidine. EM ACN Dec: 8-9,1986. 88. C Locatelli, A Travaglis, D Maccarini, GT Sala, P Vecchiarelli, ML Ruggerone. Ar?znnita poisoning: forced diuresis and intravenous N-acetylcysteine combine therapy. EAPCCT International Congress, Milan, Sept 25-28, 1990. of cimetidine to affect phallodin toxicity. 89. SM Schneider, GJ Vanscoy, EA Michelson. Failure Vet Hum Toxicol 3 1: 359, 1989. 90. H Faulstich, H Trischmann. Toxicity and inhibition of RNA polymerase by alpha-anlanitin bound to macromolecules by an azo linkage. Z Physiol Chem 354: 1395, 1973. 91. GL Floersheim, L Bianchi. Ethanol diminishes the toxicity of the mushroom Arzlarzitcr yhalloides. Experimentia 40: 1268-1269, 1984. 92. GL Floersheim. Antagonistic effects to phalloidin-149 amanitin and extracts of Antarzitn phcrlloides. Agents Actions 1971; 2: 142. 93. GL Floersheim. Rifampicin and cysteamine protect against the mushroom toxin phalloidin. Experientia 30: 1310-1312, 1974. 94. IM Chang, TLS Yun, YS Kim, JW Aln. Aucubin potential antidote for alpha-amanitin poisoning. Clin Toxicol 22: 77-85, 1984. 95. IM Chang, Y Yamaura. Aucubin: a new antidote for poisonous Anzurzita mushrooms, Phytother Res 7: 53-56, 1993. 96. PL Cavalli, R Ragni, G Zuccaro. Poisoning caused by mushrooms. Diagnostic problems. Arch Sci Med (Torino) 125: 690, 1968. 97. J Cheymol, A Pfeiffer. Arch Int Pharmacodyn Ther 79: 273, 1949. 98. T Dyk, J Piotriwska-Sowinska, T. Buczkowska. Pol Tyg Lek 24: 1005-1007, 1969. 99. M Gaultier, F Dauchy. Plzalloides poisoning. Actual clinical, pathogenic, and therapeutic instances. J Eur Toxicol. 6: 240, 1973. 100. SA Kuz’ Menko. (Uses of glucocorticoids and lipoic acid in toadstool poisoning.) Klin Med (Moscow) 52: 130, 1974. 101. L Wojciechowski. Folia Med Lodz 18: 5, 1973. 102. R Zulik, F Bako, J Budavari. Death-cap poisoning. Lancet 2: 228, 1972. 103. R Zulik, F Bako,A Kisban: (The useof thioctic acid in mushroom poisoningby death-cap.) Orv Hetil 114: 3077-3078, 1973. 104. M Rohmoser, M Kirchmair, E Feifel, A Valli, R Corradini, E Pohanka, A Rosenkranza, R Poder. Orellanine poisoning: rapid detection of the fungal toxin in renal biopsy material. J Toxicol Clin Toxicol 35: 63-66, 1997. 105. AIK Short, R Watling, MK MacDonald, et al. Arch Toxicol 53: 87-106, 1983.

Identification of Mushroom Poisoning

773

106. KF Lampe, J Ammirati. McIlvainea 9: 12-25, 1990. 107. S Horn, JH Horina, GJ Krejs, H Holzer, M Ratschek. End-stage renal failure from mushroom poisoning with Cortinarius orellanus: report of four cases and review of the literature. Am J Kidney Dis 30: 282-286, 1997. 108. I Bouska, R Rehanek, K Motycka, J Veselsky. Ceska Mykol 34: 188-190, 1980. 109. M Odonnell, S Fleming. The renal pathology of mushroom poisoning. Histopathology 30: 280-282, 1997. 110. A Heath, I Wickstrom, J Ahlem. Lancet 1: 155, 1980. 111. A Straus. Z Pilzkunde 21 : 37-41, 1949. 112. W Theobald, 0 Buch, AA Kunz, P Drupp, EG Stenger, H Heimann. [Pharmacological and experimental psychological studies with 2 components of fly agaric (Anttlnita nzuscaria).] Arzneim Forsch 18: 311, 1968. 113. PG Waser. (Pharmacological action of hallucinations.) Bull Schweiz Akad Med Wiss 27: 57, 1971. 114. WS Chilton. The course of an intentional poisoning. McIlvainea. 2: 17, 1975. 115. E Elonen,L Tarssanen, M Harkonen. Poisoning with brown fly agaric. Anzanita regalis. Acta Med Scand 205: 121- 123, 1 979. 116. JF Ammirati. JA Traquair, PA Horgen. Poisonous Mushrooms of the Northern United States and Canada. Minneapolis: University of Minnesota Press, 1985. 117. W Leonhardt. States of inebriation in Arnanita patherina poisoning. Nervenartzt 20: 181, 1949. 118. DG Spoerke, SE Spoerke, BH Rumack. Rocky Mountain high. Ann Emerg Med 14: 828829,1985. 119. K Tupalska-Wilczynska, R Ignatowicz, A Poziemski, H Wojcik, G Wilczynski. (Poisoning with spotted and red mushrooms-pathogenesis, symptoms, and treatment.) Wiad Lek 49: 66-7 1, 1996. 120. K Tupalska-Wilczynska, R Ignatowicz, A Poziemski, H Wojcik, G Wilczynski. (Anznnita patherina and Anznnita rnzfscaria poisonings-pathogenesis, symptoms, and treatment.) Pol Merkuriusz Lek 3: 30-32, 1997. 121. WS Chilton. Chemistry and mode of action of mushroom toxins.In: BH Rumack, E. Salzman, eds. Mushroom Poisoning: Diagnosis and Treatment. Boca Raton, FL: CRC Press, 1978. 122. E Gilad, Y Biger. Paralysisof convergence causedby mushroom poisoning.Am J Opththalmol 102: 124-125, 1986. 123. DR Benjamin. Mushroom poisoning in infants and children: the Amtrnita pantherirdmuscaria group. Clin Toxicol 30: 13-22, 1992. 124. JF Rold. Mushroom madness. Psychoactive fungi and the risk of fatal poisoning. Postgrad Med 79: 217-218, 1986. 125. S Hajick-Dobberstein: Soma siddhasand alchemical enlightenment: psychedelic mushrooms in Buddhist tradition. J Ethnopharmacol 48: 99-118, 1995. theinbrain under 126. P Koenig-Bersin,PG Waser, H Langemann, W Lichtensteiger. Monoamine the influence of muscimol and ibotenic acid. Psychopharmacologia 18: 1, 1970. 127. WS Chilton, CP Hsu, WT Zdybak. Stizolobic and stizolobinic acids inAmanita pantherinn Phytochemistry13:1179-1181,1974. 128. HD Fabing. Sci Mon 83: 232, 1956. 129. J Turk, V Matevzic, F Krejci. Zdrav Vestn 43: 329, 1974. 130. PG Waser. The pharmacologyof Antnnitcl muscaria. In: DH Efron, ed. Ethnopharmacologic Search for Psychoactive Drugs. U.S. Public Health Service Publication no. 1645, 1967. 131. GM Hatfield. Toxic mushrooms. In: AD Kinghorn, ed. Toxic Plants. New York: Columbia University Press, 1979. 132. B Toth. Syntheticand naturally occurring hydrazinesas possible cancer causing agents. Cancer Res 35: 3693-3697. 1975. 133. EF Guba. Wild Mushrooms, Food and Poison. Kingston, MA: Pilgrim Publishers, 1977.

774

Spoerke

of the turban top Gyromitcr (Helvelln)esculentu 134. S Franke, U Freimuth. P List. (On toxicology Fr.14. Sustances contained in mushrooms.) Arch. Toxicol 8: 268-282, 1967. 135. G Giusti, A Carnevale. A caseof fatal poisoning by Gyromitru esculentcr. Arch Toxicol. 33: 49-54,1974. 136. R Braun, J Kremer, H Rau. Renal function response to the mushroom poison gyromitrin. Toxicology 13: 187- 196. 1979. 137. D Michelot. Poisoning by Gyonritrcr esculentu. J Toxicol Clin Exp 9: 83-99, 1989. 138. D Michelot, B Toth. Poisoning by Gyromitru escdenta-a review. Appl Toxicol 11: 235243,1991. 139. H Tanimukai. Modification of paper and thin-layer chromatographic methods to increase sensitivity for detecting n-methylated indoleamines in urine. J Chromatogr 30: 155. 1967. 140. H Pyysalo. Tests for gyromitrin, a poisonous compoundin the false morel Gyrolnitm esczrlentcl. Z Lebensm Unter Forsch 169: 325. 1976. 141. RL Albin, JW Albers, HS Greenberg, et al. Acute sensory neuronopathy from pyridoxine overdose. Neurology 37: 1729-1732. 1987. 142. TD Eisen. Clin Toxicol Rev 10: 1-2, W88. 143. K Lampe. Paediatrician 6: 289-299. 1977. 144. DN Pegler, R Watling. Bull Br Mycol SOC16: 66-75, 1982. 145. H Lambert, A Larcan. Enc Med Chir 3: 6077, 1989. 146. KF Lampe. Toxic effects of plant toxins. In: Casarett and Doull’s Toxicology, 3rd ed. New York: Macmillan, 1986. 147. BH Rumack,ESalzmann. Mushroom Poisoning:Diagnosis and Treatment. West Palm Beach, FL: CRC Press, 1978. 148. PG Waser. Pharmacol Rev 13: 465-515, 1961a. 149. RW Baker. CH Chothia, P Pauling, TJ Petcher. Structure and activity of muscarinic stimulants. Nature 230: 439, 1971. 150. PG Waser. Experientia 17: 300, 1961. 151. AG Gilman, TW Rall. AS Nies, et al, eds. Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 8th ed. New York: Pergamon Press, 1990. 152. PJ Fraser. Br J Pharmacol. 12: 47-52, 1957. 153. M Svreck. The Hamlyn Book of Mushrooms and Fungi. New York: Hamlyn, 1983. 154. MH Malone, RC Robichaud. VE Tyler, LR Brady. Relative muscarinic potency of thirty Inor-ybe species. Lloydia 25: 231-237, 1962. 155. PH List, H Reith.The mushroom Coprinus ntrnmentcrrius and its tetraethylthiuram disulfidelike action. Arzneim Forsch 10: 34, 1960. 156. K Genest, BB Coldwell, D W Hughes. Effect of Coprinzrs ntrcrmentariuson the metabolism of ethanol in mice. J Pharm Pharmacol 20: 102, 1968. 157. MJ Caley, RA Clark. Cardiac arrhythmia after mushroom ingestion. Br Med J 2: 1633, 1977. 158. W A Reynolds, F H Lowe. N Engl J Med 272: 630-63 1, 1965. 159. HH Mayer, JE Herlocher, J Parisian. N Engl J Med 285: 1323, 1971. B Wickberg, E Zimerson, G 160. 0 Sterner, R Bergman, E Kesler, G Magnusson, L Nilsson, Zetterberg. Mutagens in larger fungi. I. Forty-eight species screened for mutagenic activity in Sulrnonellcr microsome assay. Mutat Res 101: 269-281, 1982. 161. JK Weir, VE Tyler. J Am Pharm Assoc Sci Ed 49: 426, 1960. 162. P Lindberg, R Bergman, G Wickberg. Isolation and structure of coprine, a novel physiologically active cyclopropanol derivative from Coprintls crtrnmenturizw and its synthesis via 1aminocyclopropanol. Chem Commun 1 : 946, 1975. 163. GM Hatfield, J P Schaumberg. Isolationand structural studiesof coprine, the disulfiram-like constituent of Coprinzrs ntrinzentcwius. Lloydia 38: 489, 1975. 164. 0 Tottmar, P Lindberg. Effects on rat liver acetaldehydrogenase in vitro and in vivo by coprine, the disulfiram-like constituentof Coprinus utrurtzenturizrs. Acta Pharmacol Toxicol 40: 476, 1977.

Identification of Mushroom Poisoning

775

165. AR Stowell. KO Lindros, P Salaspuro. Biochem Pharm 29: 783-787, 1980. 166. DJ McCormick, AJ Avbel, RB Gibbins. Non-lethal mushroom poisoning. Ann Intern Med 90: 332-335, 1979. 167. JW Allen, MD Merlin, KLR Jansen. An ethnomycological review of psychoactive agarics in Australia and New Zealand. J Psychoactive Drugs 23: 39-69, 1991. 168. K Lampe. Toxic Fungi. Annu Rev Pharmacol Toxicol 19: 35, 1979. J Psychedel Drugs 12: 79, 1980. 169. AT Shulgin. Profiles of psychedelic drugs. Psilocybin. 170. RR Mills, D Lasinskas, G Watkinson. Scot Med J 24: 31 6, 1979. 171. RE Young, S Hutchison, R Milroy, CM Kesson. Lancet 1: 213. 1982. 172. NR Peden. AF Bisset. KEC Macaulay, J Crooks, AJ Pelosi. Clinical toxicology of “magic mushroom ingestion.” Postgrad Med J 57: 543-545, 1981. 173. J Francis, VSG Murray. Review of inquiries made to NPJS concerning Psilocybe mushroom ingestion, 1978-1 981. Hum Toxicol 2: 349-352, 1983. 174. EL McCawley, RE Brummett, GW Dana. Proc West Pharmacol SOC 5:27-33, 1962. 175. SC Curry, MC Rose. Intravenous mushroom poisoning. Ann Emerg Med 14: 900, 1985. 176. TA Messinger. Pharnl Chem Newslett 13: 1, 1984. 177. A Hoft, H Eckert. Psychopharmacologia 16: 201, 1969. 178. R Watling. Bluing in Conocybe, Psilocybe and a Stropharia species and the detection of psilocybin. Lloydia 30: 151-157, 1967. 179. Z Maretic, ERussell. V Golobic. Twenty-five cases of poisoning by the mushroomPlewotus olearius. Toxikon 13: 379-381, 1975. 180. CW Smith. Mushroom Poisoning by Clzlorophyllran rttol?&dites in Hawaii. Hawaii Med J 39: 13-13,1980. 181. Gonmori-Kunio. The forensic toxicological study of mushroom poisoning. Chudoku 1997; 15: 3-15, 1997. 132. TC McMorris, S Moon, G Ungab, RJ Kerekes. Isolation of illudin S from the mushroom Owphalotus olivrrscens. J Nat Prod 52: 380, 1939. jack o‘lantern mushroom, 183. AL French. LK Garrettson. Poisoning with the North American Ort~phalotusiZludens. J Toxicol Clin Toxicol 26: 81-88, 1988. 184. TL Vanden Hoek, T Erickson. D Hryhorczuk, K Narasimhan. Jack o’lantern mushroom poisoning. Ann Emerg Med 20: 559-561, 1991. 185. FJ Seaver: Poisoning with Clitocybe illudens. Mycologia 3 1: 1 10, 1939. 186. Z Maretic. Poisoning by the mushroom Clitocybe olearia Maire. Toxikon 4: 262-267, 1967. 187. R Bergoz. Trehalose malabsorption causing intolerance to mushroom. Report of a probable case. Gastroenterology 60: 909-912, 1971. 188. J Vrkoc, M Budesinsky, L Dolejs. Phytochemistry 16: 1409-1411, 1977. 189. RA Carrano, MH Malone. Phannacologic study of norcaperatic and agaricic acids. J Pharm Sci 56: 1611, 1967. 190. M DeBernardi, G Gronza, MP Gianotti, G Mellerio. G. Vidari, P Vita-Finzi: Tetrahedron Lett24: 1635, 1983. 191. H Fujimoto, H Hagiwara, K Suzuki, MYamazaki. New toxic metabolites from a mushroom, Hebeloma \~inosophylhrnr.11. Isolation and structures of hebevinosides VI, VJT, VIII, IX, X. Chem Pharm Bull 35: 2254, 1987. 192. J Guillot, L Genaud, J Gueugnot. et al. Biochemistry 22: s365-s369, 1983. 193. H Anke, 0 Bergendorff, 0 Sterner. Assay of the biological activities of guaiane sesquiterpenoids isolated from the fruiting bodies of edible Lactarizrs species. Food Chem Toxicol 27: 393-397. 1989. 194. WA Ayer, L M Browne. Tetrahedron 37: 21 99, 1981. 195. FD Galey, JJ Rutherford TIT, K Wells. A case of Scleroderma citrirlunl poisoning in a mature Chinese pot-bellied pig. Vet Hum Toxicol 32: 329-330, 1990. 196. AW Frankland, MJ Hay. Dry rot as a cause of allergenic complaints. Acta Allergol4: 186200, 1951.

776

Spoerke

197. 198. 199. 200. 201.

D Arora. Mushrooms Demystified. Berkeley, CA: Ten Speed Press, 1979. DM Simons. The mushroom toxins. Del Med J 43: 177-1 87, 1971. JH Trestrail 111. McIlvainea 10: 36-39, 1991. VL Wells, PE Kempton. Lloydia 30: 258-268, 1967. MJ Korstanje, WJBM van de Staak. A case ofhandeczemaduetomushrooms.Contact Derm 22: 115-1 16, 1990. T Nakamura. Toxicoderma caused by shiitake (Lentinus edodes). Jpn J Clin Dermatol 3 1 : 65-68, 1977. T Nakamura, A Kobayashi. Toxikodermie durch den Speisepilz Shiitake (Lentirzus edodes) Hautarzt 36: 591-593, 1985. T Nakamura. Shiitake dermatitis. J p ~ Med l J 3237; 48-50, 1986. T Nakamura. Shiitake (Lentims edodes) dermatitis. Contact Derm 27: 65-70, 1992. K Tarvainen, JP Salonen. L Kanerva, T Estlander. H Keskinen. T Pantanen. Allergy and toxicodermia from shiitake mushrooms. J Am Acad Dermatol 24: 64-66, 1991. SM Hasnaim, JD Wilson, FJ Newhook. Fungi and disease. Fungal allergy and respiratory disease. NZ Med J 98: 342-336, 1985. BT Butcher, CE O’Neil,MA Reed, LCAltman, M Lopez. SB Lehrer. Basidiomycete allergy: measurement of spore-specific IgE antibodies. J Allergy Clin Immunol SO: 803-809. 1987. E Weeke, G Bendixen Microfungal allergy. Allergy 33: 131-133, 1979 (editorial). J Santilli Jr, WJ Rockwell,RP Collins. The significance of the spores of the Basidiomycetes (mushrooms and their allies) in bronchial asthma and allergic rhinitis. Ann Allergy 55, 469371,1985. KF Adams, HA Hyde, DA Williams. Woodlands as a source of allergens. Acta Allergol 23: 265-281,1968. RK Bush, JW Yunginger. Standardization of fungal allergens. Clin Rev Allergy 5: 3-21, 1987. PF Lehmann, E Reiss. Comparison by ELISA of serum anti-Ccmdida albicnrzs mannan IgG levels of a normal population and in diseased patients. Mycopathologia 70: 89-93, 1980. WJ Metzger, C Dorrnmey, D Robbins, HB Richerson. J Allergy Clin Immunol67: 1 1, 1981. MJ Schumacher, JK McClatchy, RS Farr, P Minden. J Allergy Clin Immunol 56: 39-53, 1974. HA Burge. Fungus allergens. Clin Rev Allergy. 3: 319-329, 1985. EH Giannini. WT Northey, CR Leathers. The allergenic significance of certain fungi rarely reported as allergens. Ann Allergy 35: 372-376, 1975. A Hebling. G Reese, WE Homer,SB Lehrer. (Current aspectsof fungal sporeallergy.) Schweiz Med Wochenschr 124: 885-892, 1994. J Sastre, MD Ibanez, M Lopez, SB Lehrer. Respiratory and immunological reactions among (Lentinus eclodes) mushroom workers. Clin Exp Allergy 20; 13-19, 1990. PCC van Loon, AL Cox, OPJM Wuisman, SLGE Burgers, LJLDvan Griensven. Mushroom worker’s lung. Detection of antibodies against shii-take (Lentimts edocles) spores antigens in shii-take. J Occup Med 34; 1097-1101, 1992. K Yoshida, M Ando, K Ito, T Sakata, K Arima, S Araki, K Uchida. Hypersensitivity pneumonitis of a mushroom worker due to Aspergillus glaucus. Arch Environ Health 45: 345-247. 1990. SD Lockey. Mushroom worker’s pneumonitis. Ann Allergy 33: 282-288. 1974. YJ Kamm, HT Folgering, HG van den Bogart, A Cox. Provocation tests in extrinsic allergic alveolitis in mushroom workers. Neth J Med 38: 59-64. 1991. J Belmchon, M Oury. Med Int 16: 162-1 71, 1981. J Hou. Chung Hua Chieh Ho Ho Hun Hsi Hsi Chi Ping Tsa Chih 8: 34-46, 1985. 0 Kondo. Jpn J Allergy 18: 8 1-85, 1969. MS Phillips. AA Robinson,TW Higenbottam. IM Calder. Mushroomconlpost worker’s lung. SOCMed 80: 673-677, 1987.

202. 203. 204. 205. 206. 207. 208. 209. 210.

21 1. 212. 21 3.

21 4. 215. 216. 217. 218. 219. 220.

221.

223. 223.

224. 225. 236. 227.

Identification of Mushroom Poisoning

777

228. A Sakula. Mushroom worker's lung. Br Med J 3: 708-710, 1967. 229. M Chen-Yeung, S Grzybowski, ME Schonell. Mushroom workers' lung.AmRevRespir Dis 105: 819-822, 1972. 230. CJ Stewart. Mushroom worker's lung-two outbreaks. Thorax 29: 252, 1974. 231. S Matsui, T Nakazawa, Y Umegae. M Mori. Hypersensitivity pneumonitis induced by shiitake mushroom spores. Intern Med 31: 1204-1206, 1992. 233,. JL Stolz, PH Arger, JM Benson. Mushroom worker's lung. Radiology 119: 61-63. 1976. 233. W Sanderson, G Kullman, J Sastre. S Olenchock, A O'Campo, K Musgrave, F Green, Outbreak of hypersensitivity pneumonitis among mushroom farm workers. Am J Indust Med 22: 859-872, 1992. 234. IS Symington, JW Ken-, DA McLean. Type 1 allergy in mushroom soup processors. Clin Allergy11: 43-47, 1981. 235. A Michils, P DeVuyst,N Nolard, G Servais, J Duchateau, JCYernault. Occupational asthma to spores of Plezwotrls conzucopiae. Eur Respir J 4: 1143-1 137. 1991. 236. KH Schulz, G Felten, BM Hausen. Allergy to the spores of PIeurorus.poriclrr. Lancet 1: 29, 1974. 237. DN Weissman, L Halmepuro, JE Salvaggio. SB Leher: Antigenic/allergenic analysis of Basidiomycetes cap. mycelia, and spore extracts. Int Arch Allergy Appl Immunol 84; 56-61, 1987. 238. WE Homer, MD Tbanez, V Liengswangworng, JE Salvaggio, SB Lelu-er. Characterization of allergens from spores of the oyster mushroom, Pleurotrls osrreatus. J Allergy Clin Immuno1 83,: 978-986, 1988. 239. A Cox, HTM Folgering, LJLD van Griensven: Extrinsic allergic alveolitis caused by spores of the oyster mushroom Pleurotrts ostreutru. Eur Respir J 1: 466-468, 1988. 240. WF Lang, DD Li, JH Zhou, W Chen, F Li. Relation of IL-2, TL-3. and IL-4 with allergic asthma induced by spores of mushroom (Pleurotus supidus). Biomed Environ Sci 6: 281285, 1993. 241. MDIbanez, WE Homer. V Liengswangswong. J Sastre. SB Lehrer.Identificationand analysisofbasidiosporeallergens from puffballs. J AllergyClin Immunol 82: 787-795. 1988. 242. K Konishi, T Mouiri, Y Kojima, E Chida, K Sugawara, K Abe, T Bando, M Ishii. M Tamura. (Three cases of hypersensitivity pneumonitis caused by inhalation of spores of Plzoliotu numeko and the background of the disease.) Nippon Kyobu Shikkan Gakkai Zasshi 32: 655661,1994. 243. M Inage, H Takahashi, H Nakamura, I Masakane, H Tomoike. Hypersensitivity pneumonitis induced by spores of Pl?oliotu rzanreko. Intern Med 35: 301-304, 1966. 244. M Tshii, A Kikuchi, K Kudoh. K Konishi, T Mohri, M Tamura, N Tomichi. Hypersensitivity pneumonitis induced by inhalation of mushroom (Pholiotu rzanleko) spores. Intern Med 33: 683-685, 1994. 245. DB Craig, RE Donovan. Can Med Assoc J 102: 1289- 1293, 1970. 246. AW Bernheimer, LSAvigad. A cytolytic protein from the edible mushroom Pleurotus ostreatrrs. Biochim Biophys Acta 585: 451-461. 1979. 247. R Srinivasan, S Smolinske, D Greenbaum. Probable gastrointestinal toxicity of Kombucha tea: is this beverage healthy or harmful? J Gen Intern Med 12: 643-634, 1997. 248. B Levenberg. Structure and enzymatic cleavage of agaritine, aphenylhydrazine of L-glutamic acid isolated from Agricacaeae. J Am Chem SOC 83: 503, 1961. 249. B Levenberg. An aromatic diazonium compound in the mushroom Agaricus bisporus. Biochim Biophys Acta 63: 212. 1965. 250. B Levenberg. An aromatic diazonium compound in the mushroom Agaricus bisporrrs. Biochim Biophys Acta 63: 212-214. 1962. 251. B Levenberg. Isolation and structure of agaritine, a gamma-glutamyl-substituted arylhydrazine derivative from Agaricaceae. J Biol Chem 239: 2267-2273, 1964.

778

Spoerke

252. Y Maramatsu. S Yoshida. M Sumiya. Concentration of radiocesium and potassium in basidiomycetes collected in Japan. Sci Total Environ 105: 29-39, 1991. 253. Y Kawamura. S Uchiyama. Y Saito. (Survey of radiocesium in domestic mushrooms on the market.) Eisei Shikenjo Hokoku 98-99, 1991. 254. JA Engelhardt, WW Carlton, AH Rebar, AW Hayes. Rubratoxin Bmycotoxicosisinthe Syrian hamster. Food Chem Toxicol 25: 685-695. 255. JA Engelhardt, WW Carlton, AH Rebar. AW Hayes. Rubratoxin B mycotoxicosis in the Swiss ICE mouse. Food Chem Toxicol 1988; 26: 459-466. 256. Y Keno. Mode of action of trichothecenes. Ann Nutr Alimen 31: 885-900, 1977. 257. AF Rizzo, F Atroshi, T Hirvi, H Saloniemi. The hemolytic activity of deoxynivalenol and T-2 toxin. Natural Toxins 1: 106-1 10, 1992. 258. WD Price. RA Lovell, DG McChesney. Naturally occurring toxins in food stuffs: Center for Veterinary Medicine perspective. J Anim Sci 71: 2556-2562, 1993. 259. MD Lindemann. DJ Blodgett, ET Kornegay, SG Schurig. Potential ameliorators of aflatoxicos in weaning/growing swine. J Anim Sci 71: 171 -178, 1993. 260. WS Chilton, CP Hsu. N-Nitrosamine of Agaricus silvaticus. Phytochemistry 13: 2291-2292, 1975. 261. B Toth, K Patil. HS Jae. Carcinogenesisof 4-(hydroxymethy1)-benzenediazonium ion (tetraflouroborate) of Agariczrs bisporzls. Cancer Res 41 2444, 198la. P 262. BL Pool-Zobel, P Schmezer, Y Sinrachatanant, F Kliagasioglu,KReinhart,RMartin. Klein, AR Tricker. Mutagenic and genotoxic activities of extracts derived from the cooked and raw edible mushroom Agal-iczrs bisporus. J Cancer Res Clin Oncol 116: 475-479, 1990. 263. B Toth, J Erickson. Cancer induction in mice by feeding of the uncooked cultivated mushroom of commerce Agaricus bisporzas. Cancer Res 46: 4007-401 11, 1986. 264. B Toth, D Nagel, K Patil, J Erickson, K Antonson. Tumor induction with N-acetyl derivative of 4-hydroxymethylphenylhydrazine, a metabolite of agaritine of Agcrricus bisporus. Cancer Res 21: 73, 1986. 265. B Toth. D Nagel. A Ross. Gastric tumorigenesis by a single dose of 4-(hydroxymethy1)benzenediazonium ion of Agaricus bispoms. Br J Cancer 46: 417-422. 1982. 266. C Papaparaskeva-Petrides, C Ioannides, R Walker. Contribution of phenolic and quinonoid structures in the mutagenicity of the edible mushroom Agaricz4s bisponls. Food Chem Toxicol31:561-567,1993. 267. E Furusawa. SC Chou, S Furusawa, A Hirazumi, Y Dang. Antitumor activity of Gnnoclema llrcidzrrn, an ediblemushroom. on interperitoneally implanted Lewis lung carcinoma synerin getic mice. Phytother Res 6: 300-304, 1992. 268. T Miyazaki, M Nishijima. Studies of fungal polysaccharides. XXVII. Structural examination of a water soluble antitumor polysaccharide of Ganoderrtra lucid~rm.Chem Pharm Bull 29: 3611-3616.1981. 269. B Toth, D Nagel. Tumors induced in mice by N-methyl-N-formylhydrazine of the false morel Gyrornitra escrllenta. J Natl Cancer Inst 60: 201-204, 1978. ethylidene gyromitrin and 270. A von Wright, A Niskanen, H Pyysalo. Mutagenic properties of its metabolitesinmicrosomalactivation tests andin host mediatedassay.MutatRes54: 167-173,1978. 27 1 A von Wright, H Pyysalo. A Niskanen. Quantitative evaluation of the metabolic formation of methylhydrazine from acetaldehyde N-methyl-N-formylhydrazone,the main poisonous compound of Gwornitm esculerra. Toxicol Lett 2: 261-265, 1978. 272. B Toth, D Nagel. Carcinogenesis of mycotoxins of two edible mushrooms. Lab Invest 44: 67A,1981(abstract). 273. B Toth, K Patil. JErickson. et al. Falsemorelmushroom Gyrorlritruesczrlenta toxin: Nmethyl-N-formylhydrazine carcinogenesis in mice. Mycopathologia 68: 121 - 128, 1979. 274. B Toth, JW Smith. DP Kashinath. Cancer inductioninmicewith acetaldehyde methylformazone of the false morel mushroom. J Natl Cancer Inst 67: 881-887. 1981.

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275. BN Ames. J McCann, E Yamasaki. Methods for detecting carcinogens and mutagens with the SrrI?~~onelZrlmar~alian microsome mutagenicity test. Mutat Res 3 1: 347-364. 276. A Knuutinen, A von Wright. The mutagenicity of Lactarius mushrooms. Mutat Res 103: 115-1 18, 1982. 277. KH Jeune, IJ Moon, MK Kim, SR Chung. Studies on lectins from Korean higher fungi. V. A mitogenic lectin from the mushroom Lerrtinus edodes. Planta Med 56: 592, 1990. 278. HA Hyde. Atmospheric pollen and spores in relation to allergy. I. Clin Allergy 2: 153-179, 1972. 279. Medical Letter: Mushroom poisoning. Med Lett Drugs Therap 26: 67-68. 1984. 280. GM Ola’h. A taxonomical and physiological study of the genus Pameolzo with Latin descriptions of the new species. Rev Mycol 33: 284-290, 1969. 281. GM Ola’h. Le genre Panaeolus: essai taxonomique et physiologique. Rev Mycol Men1 Hors Ser 19, 1969. 282. E Gerhardt. Panneolzls cycrnescens (Bk. & Br.) Sacc. and Pnnaeolus antiZlrr.r~n~ (Fr.) Dennis zwei Adventivarten in Mitteleuropa. Beitr Kennin. Pilze Mitteleur 3: 223-227, 1996. 283. MD Merlin, JW Allen. Species idenlification and chemical analyses of psychoactive fungi in the Hawaiian Islands. J Ethnopharmacol 40: 21-40. 1993. 284. SA Friedman. Celebrating the Wild Mushroom. A Passionate Quest. New York: Dood, Mead, 1986. 285. WPK Findlay. Fungi. Folklore, Fiction, and Fact. Eureka. CA: Mad River Press, 1982. 286. PH Stenklyft, WL Augenstein. Chlor.ophyZZunzmolybdites-severe mushroom poisoning in a child. J Toxicol Clin Toxicol 28: 159-168, 1990. 287. T Erikson, T Vanden Hoek, K Narasimhan, et al. Jack o’lantern mushroom poisoning. Vet Hum Toxicol 32: 368, 1990. 288. S Zevin, D Dempsey, K Olson. Anrmita phnlloidesmushroom poisoning-Northern California, January 1997. J Toxicol Clin Toxicol 35: 461-463, 1997. 289. GL Floersheim, A Bieri, R Koening, et al. Protection against Amanita phulloides by the iroid glycoside mixture of Picrorhiza kzrrron (kutkin). Agents Actions 29: 386-387, 1990. 290. J Salvaggio, JJ Seabury, E Schoenhardt. New Orleans asthma. V. Relationships between Charity Hospital asthma admission rates, semiquantitative pollen and fungal spore counts and total particulate aerometric sampling data. J Allergy Clin Immunol 48: 478-485, 1971. 291. K Matsumoto, M Ita,S Yagyu, H Ogiono, T Hirono. Carcinogenicity examination of Agaricus bisporzw, edible mushroom, in rats. Cancer Lett 58: 87-90, 1991. 292. JS Nine, TG Martin, MA Virji, MA Moraca, SM Schnieder, KN Rao. Serum biomarkers of hepatic regeneration in Anrmitu mushroom poisoning. Vet Hum Toxicol 36: 358, ~1994. 293. JL Richard, GA Bennett, PF Ross, PE Nelson. Analysis of naturally occurring mycotoxins in food stuffs and food. J Anim Sci 71: 2563-2574, 1993. 294. H Faulstich, M Cochet-Meihac. Arnatoxins in edible mushrooms. FEBS Lett 64: 73, 1976.

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20 Fungi in Folk Medicine and Society David G. Spoerke, Jr. Bristlecone Erlterprises. Demer, Colorcrdo

I. Introduction 11. History

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A. Early beliefs

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TIT. Mushroom Dyes 783 IV.

Mycophagy

V.Religious VI.

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or RecreationalMushroomUses

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Environmental Uses 784

VIT. Medical Uses

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A. Disease- or illness-causing fungi 785 B. Fungi used as treatments 786 C. Combination products 796 References

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INTRODUCTION

There has always been a sort of magical air about mushrooms-they seem to grow from nowhere, gain size rapidly, and almost as rapidly are destroyed. They arise from dark, often wet and “unhealthy” places and for some people have an aura of death and decay, magic and deviltry. The Greek physician Nicander wrote in Alexiphnnnncn that fungi were the “evil ferment of the earth’’ (1) and Pliny called them the mud and acrid juices of the moist earth. German and English herbalists of the 16th century called mushrooms imperfections of the earth, bastard plants, and “earthie excrescensces.” Although the modern study of mushrooms is only about 250 years old, the use of various fungi in medicine and religion dates from ancient times. Some of the oldest known religious artifacts are stones carved as mushrooms (2j. Mushrooms have many uses, such as a source of food, dyes, paper (3), and medicines, as well as objects of scientific interest, beauty, and art. The term “rnushroom’ ’ may have come from several sources (2): I . From the Greek for nzvkes. 2. From the Old French for mousseroll (growing out of moss). 3. From the Welsh for mnesl-hum (rhunz = bump, rimes = in the field) 781

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4. Anglo-Gallic terms for mushrooms included muscher-or?,muscher-oms, and ~nushr-umy

II. HISTORY Great civilizations are said to have been named after mushrooms; for example, the kingdom of Mycenae was said to have been named such by thirsty Greek hero Perseus who was delighted when he took up a mushroom (mykes) and drank water from it (4). Although mushrooms have been sought and studied for centuries, it was C. H. Peterson who changed the study of mushrooms into a science by developing a system of nomenclature. His work was carried on by the Swedish scientist E. Fries ( 5 ) . A.

Early Beliefs

1. Mushrooms:Source of Origination The ancient Romans thought mushrooms were created when the god Jupiter hurled lightning to the earth. The classical author Juvenal in his Satires associated truffle growth with thunder. Plutarch (Symposicm, Book 4, question 2) also argued that lightning produced truffles (I). Some Mexican and Guatemalan natives (6) as well as some Hindi (1) also believed that certain mushrooms (like Amnnitc~muscar-in)appear due to thunder and lightning (7). A Bohemian legend tells that when Jesus and Peter walked through a poor Czech village, they stopped to eat. Peter disobeyed Jesus’s order to eat only bread and salt and instead ate cakes. When caught by Jesus, Peter spat out the cakes and denied eating them. Jesus ordered him to pick up the crumbs, but they had sprouted into mushrooms, which Jesus gave to the poor. Ever since, mushrootns have been abundant, but not quite filling.

2. FairyRings Rings of mushrooms were once thought to occur due to the paths of dancing fairies, but actually they occur in arcs and circles because the tnushrooms sprout near the ends of the mycelial mass, often a circle. Inside the circle formed by the fairy ring there is often grass that is somewhat greener in color than the surrounding area. This is due to dying and disintegrating hyphae that are releasing nitrogenous substances to the grass. Mushrooms famous for producing fairy rings include Mnrasmirrs orendes and Chlor-ophyllummolybidites (8). 3. Foxfire The mycelia of some agarics are bioluminescent. Decaying wood penetrated by Amillar-ielln mdlea, or the gills of Onphnlotus olecrrius Cjack-0’-lantern mushroom), and the basidiocarp of Mycenn Zux-coeli will glow in the dark, creating an eerie glow in the woods at night.

111.

MUSHROOM DYES

Mushrooms have been used as dyes and inks throughout history. Both fresh and dried mushrooms can be heated (simmered) in water to release their color. Silk, cotton, wool, and other fabrics have been dyed using mushrooms. As with many organic dyes, adding

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chemicals called mordants aids in fixing the dye to the fabric. Particular mordants may also alter the color of the dye-for example, red, blue, purple, or gray may be obtained from Dermocybe plzoerzicen, depending on the mordant selection (2). Entire texts have been written discussing mushroom dyes and dye methods (9j. One advantage of mushroom dyes over some other vegetable dyes is they tend to fade less rapidly.

IV.

MYCOPHAGY

Yeasts are types of f h g i , and are used in fermentation to create alcoholic beverages. Fermentation was considered by the Egyptians to be a gift from the god Osiris. The Greek god Dionysus and the Roman god Bacchus were also attributed with the gift of fermentation (10). The Greek medical writer Dioscorides thought that even the most flavorful of mushrooms were unhealthy because they were indigestible and caused strictures and cholera (5). Mushrooms are not required in the human diet. If persons do not eat them, they readily obtain the nutrients with other vegetables. Mushrooms do compare favorably with other vegetables and most often are eaten for their different flavors (10). In Europe the sale of wild mushrooms is taken seriously and mushroom markets are inspected by certified inspectors. By regulation the mushrooms in the market must be in displays that are one layer thick and have all parts intact for identification. The sellers must be locals (2). Relatively few mushrooms are grown for food. The most common in the United States is Agaricus brzmzesce~zs (Agaricus bisporus), which is found in mostgrocery stores. Other popular commercial mushrooms are Lerztinus edodes (the shiitake mushroom of the Far East) and Volvariellcr volvacea (the Padi straw mushroom of India and the Far East). The Padi straw mushroom is grown on straw, the shiitake on logs, and A. bmnnescens on composted horse manure. Growing mushrooms is not as simple as distributing the appropriate mushrooms spores on a suitable substrate. A. brwznescer~sundergoes at least eight stages from basidiospore germination to packing and marketing (1 1, 12). Mushrooms found in private, managed forests are now being investigated both for the potential for harvest and for potential mycorrhizal associations with forest trees that allow those trees to grow faster and more efficiently (2).

V.

RELIGIOUS OR RECREATIONAL MUSHROOM USES

Mushrooms, like other plants, have been used in religious ceremonies and as intoxicants and hallucinogens. As early as the seventeenth century the mushroom Ammita musccrria was used by the Kamchadal and Koryak tribes of northeastern Siberia to produce violent, erotic intoxications. This mushroom has been linked to Vedic Soma (a god and a plant), and may have been the fungus used in India during various religious ceremonies (1). Before settling on A. nzuscaricr as the substance known as soma, Wasson et al. (13) examined a large number of possible sources, including the mintLagochihs inebriarzs, morning glory seeds, Clmiceps prr~puren(ergot), and the psilocybin mushroom Str-ophcrria cubemi, known also as Psilocybe cuberzsis (14). The Buddhist adepts from the second to the ninth centuries were also thought to have used A. nzuscaria for enlightenment (15j. For over 2000 years the mushroom ling chih (thought to be Ganoclerma lucidlrm) was the symbol

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for a magic mushroom. Various Mexican Indians, including the ancient Myans and Aztecs, have used divine tnushrooms (thought to be psilocybinin/psilocin-containing) in various religious ceremonies. Mushroonls stones and mushroom motifs have been found in Mayan temples (16, 17). The ethnomycology of mushrooms has been studied in a number of cultures by such men as Blasius Reko, Richard Schultes, Roger Heim, and R. Golden Wasson. It has been proposed that Psilocybe nzexiccrrza is the teolzanacntl (God's flesh) used as a sacrament by the Aztecs. The use of mushrooms in ceremonies decreased dramatically when missionaries came to the Aztecs. Mushroom stones were destroyed and ceremonies were actively discouraged. A description of how these mushrooms were used is given by Bernardino de Sahagun, a priest who spent 50 years studying the Aztec culture (18). Wasson et al. (13) have also postulated that Aristole, Plato, and Homer participated in mushroom ceremonies at the temple of Demeter, the goddess of agriculture, in the city of Eleusis.

VI.

ENVIRONMENTALUSES

Most fungi aid the environment by decomposing organic materials into more useful substances. However, fungi are now being used to specifically detoxify some troublesome materials in theenvironment or to substitute for some more toxic agent, such as a pesticide. Some examples are listed below. Coriolopsis polyzona: In vitro experiments using mushroom cultures have shown a 41% removal of polychlorinated biphenyls (PCB) from a growth medium. The chemicals thought to be degrading the PCBs were magnesium-dependent peroxidase, magnesiumindependent peroxidase, lignin peroxidase, and laccase ( 19). Hericirrn~cornlloides: Various fatty acids have been isolated from H. corcdloides and tested on the nematode Ctrenor-habditis elegans.A nematicidal and nematode-repellent effect was noted using a fatty acid mixture of linoleic acid, oleic acid, and palmitic acid (20). Phmer-ochaete chrysosporizmz: In vitro experiments using mushroom cultures have shown a 25% removal of polychlorinated biphenyls from a growth medium. The chemicals thought to be degrading the PCBs were magnesium-dependent peroxidase, magnesiumindependent peroxidase, lignin peroxidase, and laccase (19). Pleur-otrrs ostrentus: In vitro experiments, using mushroom cultures, have shown removal of polychlorinated biphenyls from a growth medium (19). P. ostretrtrrs has been grown on substrates contaminated with cadmium (35, 139, and 285 mg of cadmium per kilogram). The fruiting body (mushroomi of the fungus was notaltered due to the cadmium in the growth mediums. The amount of cadmium found in the fruiting bodies is related to the concentration of the growth medium. The amount stored was highest in the cap, ranging from 22 to 56 mg/kg (dry weight), rather than in the stems (13-36 mg/kgj. Ingestion of mushrooms grown on wastes containing cadmiutn may represent a risk to consumers, depending on the amount and frequency of ingestion and the concentration of the growth medium (21). The fruiting body of P. ostrentus is also known to contain an inhibitor of 3-hydroxy3-methylglutaryl-coenzyme A reductase-lovastatin (109). P. ostreatus was grown in a medium containing 14C phenanthrene for 11 days. Ninety-four percent of the phenanthrene was metabolized, 3% to carbon dioxide, and 52% to t~-crrzs-9,l0-dihydroxy-9,10-dil~ydrophenanthrene (23). It is thought that P. osteratus ini-

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tially oxidizes phenanthrene stereoselectively using a cytochrome P-450 monoxygenase, then later by an epoxide hydrolase-catalyzed hydration reaction. Pleurotus pulmorzarius: Various fatty acids have been isolated from this mushroom and tested on the nematode Caerzorhnbclitis elegcrns. A number of different compounds were found to be nematicidal, some at concentrations of 5-10 parts per million (20). P. pulmonnrir4s was tested in a manganese-enhanced liquid culture medium contaminated with atrazine at concentrations up to 300 pnol/L. When the culture medium was analyzed, increases in rz-dealkylatedand propylhydroxylated metabolites had accumulated, as had lipid peroxidation, oxygenate, and peroxidase activities. Cytochrome P-450 levels also increased. The manganese, with the mushroom, appeared to enhance the biotransformation of atrazine (23). Pleurotus sajor-caju: This mushroom will incorporate heavy metals from its substrates. Metals that were highly absorbed during testing were copper-2 (182 pg ml-1) and cadmium-2 (178 pg nd-1); the least absorbed was mercury-2 (12 pg ml-1). Lead was also taken up and reduced mycelial proteins and growth (34). This mushroom is also known to biodegrade the pesticide lindane (25). Stropharia rugosoannulnta: Extracellular manganese peroxidase from this mushroom rapidly converted the main intermediates of the explosive 2,4-trinitrotoluene (26, 27). Trametes versicolor: In vitro experiments using mushroom cultures have shown a 50% removal of polychlorinated biphenyls from a growth medium. The chemicals thought to be degrading the PCBs were magnesium-dependent peroxidase, magnesium-independent peroxidase, lignin peroxidase, and laccase (19).

VII. MEDICAL USES Although fungal diseases have been described from the time of the Indian Vedas, Sumarians, Greeks, and Romans, it was not until the 17th century that there was an association of fungi with various plant diseases. Only in the last 50 years has the pathogenicity of fungi been on a sound experimental basis (1). In 1835 Bassi published information showing the origins of muscardine disease in silkworms was due to a fungus (Beauveria bassiana) (1). Within a few years of Bassi’s publications fungal diseases in humans were identified by Johannes Lucas Schonlein and Robert Remark in Germany, and David Gruby in France (1j. Fungi have been known to both cause and cure disease. Mushrooms play an important part in many cultures. Molitoris (28) discusses the role of mushrooms in medicine, as a food source, and for religious purposes in Czechoslovakia. A.

Disease- or Illness-CausingFungi

Fungi that produce disease fall into three main categories: cutaneous infections, which occur primarily on the skin and produce allergic reactions or inflammation; subcutaneous infections, which are produced by low-virulence fungi that enter through wounds or insect bites; and systemic infections, caused by either true pathogenic fungi or opportunistic fungi attacking a patient with a compromised immune system (29-32). An illness such as toxicoplasmosis in an AIDS patient can be devastating. Dermatophytes are keratinolytic fungi that attack the skin’s outer layers. Around 50% of these fungi exist only on humans and nowhere else in nature. Ringworm is an

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example, as are Ccmclidia albicnns, Epidermophytoll JIoccosum, Trichophyton rubrum, tinea imbricata, Mict-osporunz audouinii, and Trichophytorl tonsurnns. Others fungi of this kind have reservoirs in other mammals. An example is Microsporum carzis in the cat. This fungus can move to humans or dogs but will die out after a few person-to-person transfers (29, 33). Subcutaneous diseases include chromoblastomycosis (caused by Phialophora vert-ucosa, Cladosporium carrioni,or Fonsecaea pedrosoi), entomophthoromycosis (caused by Bnsidiobolus raIzarurn),mycetoma (caused by Mcldurella mycetonzntis, Exophialajeanselmei, Pseudallescheriaboydii, and Leptosphaeriaserlegalensis), and sporotrichoisis (caused by Sporotlzrix scherzckii) (29). Examples of systemic infections include histoplasmosis (caused by Histoplasma cnpsulatum), coccidiodomycosis (caused by Coccidioidesimmitis), paracoccidiodomycosis (caused by Paracoccidioides brasiliensis), blastomycosis (caused by Blastonzyces dernlntitidis), canadidiasis (caused by Candida albicans),zygomycosis (caused by Rhizopus arrhizus and Rhizopus oryae, or species of Mucor, Rhizomucor, and Absidin), crytococcosis (caused by Cryptococcus neoforrz-~ans),aspergillosis (caused by various species of Aspergillus), pheohyphomycosis, and hyalohyphomycosis (caused by various Fusarium species) (29, 34). Systemic and topical fungal infections have been on the increase due to immunocompromised AIDS patients (35, 36). Fungi may also cause various allergic reactions (such as asthma) if eaten or if the spores are inhaled. Fungal spores (including molds) account for a significant number of allergic reactions and asthma. Van Leeuwen in Holland found that half of the asthmatics he tested had a positive reaction to fungi. This may be compared to a study by Hansen in Germany in 1928 that showed a 15% sensitivity, by Fraenkel in Germany in 1938 who showed a 16% sensitivity, and by Fraenkel in England in 1938 showing a 53% sensitivity (1).

B. FungiUsedasTreatments While only a few species are listed in European and Native American pharmacopoeias, Asian and European societies found many species to be useful (28). Many fungi have been used as medicine if you consider the microscopic fungi responsible for many of our antibiotics such as penicillin, streptomycin, and vancomycin (37). Much of America is mycophobic, and study of the macromycetes for medicine is not widespread. Eastern and Western societies have fundamental differences concerning what represents a medication and how to determine its efficacy. Eastern societies may use mushrooms for tonics, food, and restoratives. A total of 187 mushrooms were listed in a book on Chinese folk medicines (38). Listed below are some of the mushrooms that have traditionally been recommended as treatment, or are being investigated for medicinal use. Agaricrrs campestris: Rats fed this mushroom in their diets did not have altered plasma or liver cholesterol concentrations (39). Arnatlita nzuscaria: Sometimes used in various homeopathic medications under the name Agaricus m w a r i u s (1). The amount used in these homeopathic preparations is nlinute: 1 X g. This mushroom was also commonly used for various recreational and religious purposes. The mushroom contains minute amounts of muscarine. Muscarine nitrate was used before 1900 as a treatment of diabetes insipidus and night sweats (40). Aragekikurage: Japanese name, see Auricularia p o l p i c h a . Auricularia auricula-judae: Prized by the Chinese for its medicinal value (41). Old English herbalists used it for making a soothing drink (due to its gelatinous nature) that would help a sore throat (42).

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Auricularia polytricl?~~: This species, commonly called “black tree ears,” is used in Chinese medicine as a demulcent (51). Ying et al. (38) have also claimed the mushroom is 80-90% effective against Ehrlich carcinoma and sarcoma-180 in animal studies. The research is in Chinese. An anticoagulant has also been isolated from this species (1 8). Battarea yhcrlloides: A puffball used as a poultice for swellings and sores by the American Paiute Indians (46, 48). Bear’s Head: See Hericium erinncezls. Benuvericr bnssiann: This lower fungus is used in Chinese folk medicine as whitestiff silkworm. It is a fungus that parasitizes insects, which become covered with a white powder. Tablets made from the powder are used to treat diabetes, epilepsy, enuresis, and paralysis (51 ). There is little, if any, Western research to substantiate these claims. Beech Mushroom: See Hyysizygus tesszrlntus. Black Forest Mushroom: See Lelztirzuln edodes. Black Mushroom: See Lerztirzzrla edodes. Bovistu pila: A puffball used by the American Chippewa Indians to stop bleeding (46, 48). Bovista p l m b e a : A puffball used by American Indians in the Missouri River area as a hemostat (46, 48). Brick Top: See Hypholorna sublnteritiurn. Buna-shimeji: See Hypsi:ygzfs tessulatus. Cdvntin crnniforrnis: A puffball used by American Chippewa Indians as a hemostat and by the Ojibwe Indians to staunch nosebleeds (46). Ccrhwtin cycmthiforrnis (C. lilncina): The Indians in the Missouri River area used this puffball as a hemostat. The Makah Indians used it as a general tonic medicine (46, 48). Culvnticr gigantect: Contains calvacin, which is being investigated as a possible antitumor agent (45). The American Indians, who used it as a hemostatic agent, did not use this mushroom as an antitumor agent. In both in vitro and in vivo tests on this species, extracts have shown activity against influenza (51). Calvntia utriforrnis (Lycoperdon cnelnturn):This puffball was used by the Ahnsishinaubeg as a hemostatic agent (46). Chinese Mushroom: See Lentimda eclodes. Chinese Sclerotium: See Polyporws umbellcrtus. Chorei-maitake: See Polvporus z~rnbellat~~s. Claviceps prlryzlrea: Ergot is the sclerotium (a dense mass of fungal hyphae) of this fungus. Usually the sclerotium falls to the ground and sprouts the next spring as small, purplish-pink fruiting bodies (appearing like little purple drumsticks). Vast amounts of spores are released, which may land in the flower of rye plants. The spores germinate and the hyphae invade the ovary of the plant. The plant is now contaminated. Sometimes the sclerotium does not fall off, and is harvested along with the rye. Flour made from this material will contain the ergot and may cause ergotism. Ergot (actually a combination of at least 12 alkaloids, histamine, and ergosterol) produces uterine contractions (mentioned in theliterature as early as 1582) and vasoconstriction (5,51). Later, as its potential toxicity became more widely known, it was recommended only to staunch postpartum bleeding (107). Symptoms of overdose include hallucinations, convulsions, burning pain in the extremities, smooth muscle contraction, gangrene, and death. Ergot alkaloids are still used in the control of migraine headaches. Cloud Mushroom: Common name for Grifoln fi-o~ldosn.

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Coyrinus utrcmer1tarius: Animal studies (with rats) seem to indicate that coprine from this mushroom could be used similarly to disulfiram because of its effects on acetaldehyde dehydrogenase ( 104, 105). Coprinz./s comntus: Both a new antibiotic and an antineoplastic agent are thought to be contained in this mushroom, and are being researched (18). Cor-clyceps sinensis and other Corclyceps species: These fungi grows on the bodies of insects. Spores land on a caterpillar, moth, beetle, etc., and the germ tube penetrates the insect’s body and grows until the insect dies and gradually becomes mummified. The Chinese cited this drug in early pharmacopoeias and called it Hin tscro tom tchow (summer plant, winter insect). It was listed in the Essentials of Materia Medica by Wang Ang of the Qing Dynasty. It is also known as the “Chinese caterpillar fungus.” The Chinese used it as an invigorating tonic, for chronic coughing. anemia, and asthma (51). The ALW tralians used C. robertsii in tattooing (5). Yamaguchi et al. (106) subcutaneously implanted mice with lymphoma cells, then orally administered an extract of C. sirlensis. Reduced tumor size, prolonged survival time, increased macrophage chemotaxis, and phagocytic activity of macrophages were reported. Coriollrs versicolor: This mushroom is claimed to have antineoplastic effects, but when studied against five tumor cell lines and mouse peritoneal macrophages, no antineoplastic activity or cytoxicity was observed. The polysaccharide from the mushroom was used in vitro, at doses of 2.5-10 pg/ml. The peritoneal macrophages from mice fed the polysaccharide for 2 weeks showed increased nitrogen intermediates, reactive oxygen intermediates, and tumor necrosis factor. It was concluded that C. versicolor did have some immunomodulating effect by activation of defense cells (101). The polysaccharide (or polysaccharopeptide as some cite it) is called a biological response stimulator, which induces the host (usually experimental animals) to increase production of gamma-interferon, interleukin-2, and T-cell proliferation (98). Analgesic effects have been noted in mice given an intraperitoneal injection of the polysaccharide and then subjected to a hot-plate test (99j. Dcrlditzier corlcelltricn: The ascocarps were used in the past to prevent cramping (1). Dancing Butterfly Mushroom: Common name for Grifolct frorzdosn. Donku: See Lentinulcr eclodes. Ear Fungus: See Auriczrlerricr polytricha. Earth Maitake: See Polyporus umbellertzds. Elophonlyces grnr~rrlatus:Recommended in the past as an aphrodisiac (1). It was also part of a preparation called Bnlsn~~zzrs Apoplecticzrs used to increase breast milk secretion (~42). Enoki: See Flnrrrtzwlincr velrltipes. Enokidake: Japanese name for Flnr~zrnulir~n velutipes. Enokitake: Japanese name for F~nn11T114~i~~ velutipes. Fengweigu: Chinese for Pleurotus mjor-caju. Flcunnzulirzcr velutipes: This mushroom is also called enokitake or Collybia Ilelutipes. These mushrooms contain the polysaccharide flammulin (FVP, for F. velutipes polysaccharide). This polysaccharide was able to agglutinate human blood cells in vitro, and showed stimulatory activity toward human peripheral blood lymphocytes and suppression of systemic anaphylaxis reactions in mice (100). It has been tested against sarcoma 180 and Ehrlich carcinoma in animal models and found to be 80- 100% effective (38). Studies investigating the antineoplastic properties of this mushroom have been done by several investigators (102, 103). Anecdotal information concerning a lowered cancer ratein a group of Japanese who frequently eat enokitake is also being investigated.

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Fomes.fomerltarirls:Used by the Greeks in the fifth century B.C. and in the nineteenth century A.D. by individuals in Nepal and Lapland. The fruiting bodies were soaked in saltpeter (NaNO;), dried, and then when burned used to cauterize wounds (1). Fomes oficiualis:In ancient times thought to be a universal antidote used for a wide variety of illnesses including bruises, broken limbs, liver problems, asthma, dysentery, kidney diseases, sores, and stomach disorders, and as a purgative (1, 5). The drug was called agaricum and does contain an acrid resin that acts as a cathartic. It is also thought to act on the sebaceous glands, and to decrease night sweats in patients with tuberculosis. Overdoses of the mushroom may cause vomiting, diarrhea, respiratory paralysis, and death. F u n g ~ sambuci: s An absorbent mushroom said to take upfrom 9-12 times its weight in water. It has been used asa local treatment for conjunctivitis (42). It is most commonly found on elder trees. Furry Foot Collybia: See Flnmmzdirln velutipes. Garzodema llacidzrrn: This polypore has been recommended as a tonic and agent of good health and longevity in China and Japan (5). Willard (50) claims the mushroom is useful in treating almost anything from cancer, fatigue, liver toxicity, to blood diseases. This mushroom is called lirzg-chih in China and reishi in Japan, and causes heart rot in Japanese plum trees and Chinese hemlock (5). Himalayan guides find it useful as an aid in high-altitude sickness, and the Mayan Indians blend it in a tea usedto prevent communicable diseases (18). Unfortunately there have been few long-term, double-blind, placebocontrolled studies published in English that would substantiate these claims. Antineoplastic effects: The Chinese attribute it to have actions against cancer and immunomodulary effects, but some studies have shown that tumors in mice have grown faster when the mice were injected with G. lucidlrlr~extracts (52). Methanolic and water extracts were tested in mice against solid tumors (sarcoma 180). Both oraland intraperitoneal administration was used. The aqueous extract was effective for inhibition of tumor growth but the methanolic extract did not show activity. The active molecule had a molecular weight of greater than 10,000 daltons (47). Immunomodulary effects: Its reported immunomodulary effects have made it a prime “natural” remedy for treatment of HIV by herbalists. The immune-stimulating properties are due to a group of polysaccharides that are thought to stimulate he1per-Tcell production. The immunomodulating proteins have been studied by a number of authors (18, 52-54). Lipid effects: Ganoderic acids may help to lower cholesterol (55). Modulating effects on lipid levels have been studied by Kabir and Kmura (56). Blood effects: Ganoderic acids are thought to have an anticoagulant effect (55). Blood pressure: Modulating effects on blood pressure have been studied by Kabir and Kimura (56). Virility: The antler form of G. hrcidum has been given to men as a stimulant to increase sexual virility (18) and as an anti-inflammatory (57, 108). No studies could be found. Gnnodemcr tsugne: A water-soluble polysaccharide mixture from the mycelium of this mushroom was analyzed and found to contain 16 different polysaccharides, three of which had antineoplastic activity in a mouse model using sarcoma 180. All three were protein-polysaccharide glycans, and all three showed tumor inhibitory activity and increased mouse survival (58). In another study with similar design, seven purified polysaccharides with antineoplastic activity were identified. The greatest activity was inthe waterinsoluble fractions (60).

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Geastr.ur~ (Gemter)species: These puffballs were used as a general medicine by the Tewa Indians and as a prophylactic against infection on the navels of newborn infants. Golden Mushroom: See Flnr~mrrlimvelutipes. Golden Oyster Mushroom: See Pleurotzls citr.ir7opilecrtrrs Grlfolu frorldosn: The Developmental Therapeutics Program of the Anti-HIV Drug Testing System (National Cancer Institute) has done in vitro studies onthe powdered sulfated fruiting bodies of this mushroom. Significant activity against HIV was reported both in this study and in a similar study conducted by the National Institute of Health in Japan. The mushroom extract has activity approximately equal to that of AZT (18). An interesting fact is that the mycelium is not active, just the fruiting bodies. The mushroom extracts are now being tested against HIV in humans in several countries. The immunostimulatory activity is thought to be due to grifolan, a glucan found in both the fruiting bodies and the mycelium (61). Ohno’sgroup (61) found antitumor activity against murine solid tumor and sarcoma 180, showing effect in about 35 days. One-half to one-third of the mice tested had complete tumor regression. An alkali extract had greater activity than just a hot-water extract. Water extracts have been tested by Chinese and Japanese investigators and found to inhibit murine tumors by approximately 86% (38, 62). The mice were given 1-10 mg of the extract per kilogram of body weight for 10 days. No human studies could be found. The method by which the protein-bound polysaccharide (D fraction) stimulates the immune system is unknown, but there is some evidence that T cells may be increased or a decrease in these cells stopped (18). Other health care claims being investigated for this mushroom include lowering of high blood pressure and treatment of diabetes, high cholesterol, and chronic fatigue syndrome. Mice were given the carcinogen N-butyl-N’-butanolnitrosoamineevery day for 8 weeks. Control animals (10) all developed urinary bladder cancer, all had decreased killer cell cytotoxicity, and all showed reduced macrophage chemotaxic activity. Those fed G. fionclosct as part of their diet had a reduction in the number of tumors (7 of 15 mice) to 53.3%. Macrophage activity did not drop, but stayed at the same level, and theymaintained normal cytotoxicity of natural killer cells (63). Additional information may be obtained from Hishida et al. (64), Adachi et al. (65, 66), Yamada et al. (67), and Kabir and Kimura (56). Hedgehog Mushroom: See Hericium erir1aceus. Hen of the Woods: Common name for Grtfoln fior~closn. Hericizm erirluceus: The fruiting bodies of this mushroom are dried, powdered, and made into tablets or pills. Traditional medicine claims include treatment for ulcers, cancer, inflammations, and tumors of the alimentary system (38, 68). Hiratake: See Pleurotrts ostreatus. Himeoln ~ t ~ ~ r i c ~ r l c Was ~ - j zat~ ~one ~ ~time e : used to treat throat infections (1). It is astringent and is thought to hold water like a sponge (42). Hog Tuber: See Polyporus umbellutus. Houbi take: Chinese for Pleur-otrrs scrjor~-cc~r~. Hypholornct sublnteritizm (Fr.) Quel.: Contains possible antineoplastic agents. Ying et al. (38) found inhibition rates against sarcoma 180 and Ehrlich carcinoma to be 6070%. References are in Chinese. Hypsiz~grrstessulatus: In unpublished work done by Ikekawa (1 01) with the National Cancer Institute of Japan, it was found that this mushroom inhibited 100% of the tumors in a group of mice implanted with Lewis lung carcinoma. The dose was 1 g of

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mushroom per kilogram of mouse per day. Without the mushroom, the tumors continued to grow (18). Human studies could not be found. Hypsizygus ul~?zurizrs: Used in traditional Chinese medicine as treatment for stomach and intestinal disorders. Anecdotal reports hint at anticancer properties that should be scientifically investigated (18). Il’mak: See Plerlrotz,fscitrirlopileatm Kikurage: Japanese name, see Auriczllnria yolytricllu. Kumotake: Common name for Grifolu fiondosa. Kuritake: See Hyyholonzu sublnteritizm. Lawyer’s Wig: See Coprinus conzutus. Lentiuula edocles (Ler~tirlr~s): Is thought by the Chinese to have antiviral, antitumor, and hypocholesterolemic effects (69). It has a protein content of about 15.2% in the cap and about 11.4% in the stem (70). The water-soluble polysaccharide lentinan is found in this mushroom. It is composed of various glucopyranoside branches. Antineoplastic effects: Mice were given the carcinogen N-butyl-N’-butanolnitrosoamine every day for 8 weeks. Control animals (10) all (100%) developed urinary bladder cancer, all had decreased natural killer cell cytotoxicity, and all showed reduced macrophage chemotaxic activity. Those fed L. eclodes as part of their diet had a reduction in tumors to 9 of 17 mice (47%); macrophage activity did not drop, and they maintained normal cytotoxicity of natural killer cells (63). Lentinan is approved as an anticancer drug in Japan. Via luller cells and T cells, lentinan inhibited methylchloranthrene-induced fibrosarcoma and solid tumors of the sarcoma 180 type (71). Lichens: Lichens are a combination of an alga and a fungus. They have been used as medicines in Europe, mostly during the Middle Ages. Some species such as Usnea and Cladoniu contain the antibiotic usnic acid, which is effective against gram-positive bacteria. European commercial topical preparations containing usnic acid include Usniplant, Binan, and USNO (51, 72j. Ling-chi: Chinese name for Garzoderrncr lucidurn. Ling-chih: Chinese name for Gcrrlodernza lucidzr~r~. Ling-zhi: Chinese name for Gnrzoderrna lucidunz. Lion’s Mane: See Hericium erinncezls. Lobnrin yulrnonuria: A lichen used to treat lung infections (1). Lycoperdon yerlatum: These small puffballs were used as a hemostat by the Kiowa, Pawnee, Ponca, Omaha and Dakota Indians, and as a healing agent for sores by the Cherokee and Yuki Indians (46, 48). They were also used by peasants in Europe. Slices of the mushroom and other puffballs were often dried and kept in storage until needed as a natural ‘ ‘Band-Aid’’ (42j. Maitake: Comtnon name for Grifola .fiondosa. Mammentake: Common name for Gnnoclermu lrrcidurn. Maomuer: Chinese name, see Auricularia polytrichn. Maotou-Gauisan: Chinese name for Coyrirzzrs cornatus. Mo-er: Chinese name, see Auricularia yolytrichn. Mokurage: Japanese name, see Ar~ricularia polytrichu. Monkey’s Head: See Hericirm erirzacem. Morgnnella subincar~zata (Lycoperdon sz~bincnnlntun.~):Used by the Potawatomi Indians as a cure for headache (46). Mountain-Priest Mushroom: See Hericiurn erinnceus.

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Mu-er: Chinese name, see Azrriczrlaria yolytrichn. Muk Ngo: Chinese name, see Auricularia yolytricha. Mushrootn of Immortality: Common name for Gcrnoderrnn lrrcidrrn7. Mzrtims cminus: A stinkhorn fungus with the general shape of the dog penis, which through the Doctrine of Signatures was then attributed to have sex-stimulating powers. It was used similarly to Phnllus i~~yudiczrs (5). It does not possess aphrodisiac or increased fertility actions. Ncrematolomn srrblcrteritiunr: Synonym for Hypholomn sz4blrrteritirm. Nameko: See Pholiotcl imnleko. Nametake: See Flnmmlina velrrtipes. Oakwood Mushroom: See Lelztirmla edodes. Old Man’s Beard: See Hericiunl erinnceus. Oyster Mushroom: See Pleurotzrs ostrecrtus. Oyster Shelf See Pler4rotus ostrentus. Panacea Polypore: Common name for Gcrnoder~~zn lr~iclunz. Pasania: See LerItirurlt edodes. Peltigern ccrninn: A lichen that, when mixed with black pepper, was recommended in the past as a treatment for rabies (1). It does not have any effect on this disease. P l d l u s imprdicz~s:Recommended in the past as an aphrodisiac (1). It has thegeneral shape of the male penis and through the Doctrine of Signatures was then attributed to have sexual powers. Besides its use in tnan, it was also fed to cattle to increase their fertility. The fungus was burned and the ash applied to the genitalia of infertile women (5). PholiotcI mnzeko: The nameko mushroom is claimed by Chinese researchers to enhance resistance to infection by Stcryhylococcusbacteria. The aqueous and sodium hydroxide extracts are 60% and 90%, respectively, effective against sarcoma 180 in the murine model (1 8, 38j. Substantiating Western studies are lacking. Pleurotzrs citriuopilecrtzrs:Chinese research claims this mushroom may treat or cure pulmonary edema (38). A water-soluble polysaccharide from this mushroom was tested in mice with implanted sarcoma 180. Antineoplastic activity was noted (59). Pleurotrrs jloriclcr: Indian researchers have given this Pleut-otus species to rabbits and found total lipids, total cholesterol, and glycerides levels in the liver and serum to be decreased. Heart lipids were not affected. Both HDL cholesterol/total cholesterol and HDL/LDL cholesterol ratios increased (73). Plezarotrrs ostreentcrs: These oyster mushrootns do not play a major role as medicinal mushrooms, but their use has been expanding, and anecdotal reports have shown liver and kidney finction improvement. P. ostreatus is known to contain lipids, sterols, lipophilic vitamins, B vitamins, and monosaccharides, oligosaccharides, and polysaccharides. Some portions of the fungal fruiting body are being investigated for antineoplastic, immunomodulatory, antiviral, and hypercholesterolemic actions (74). A hydroxy (-OH)-radical scavenging activity has been shown experimentally (75). as has a serine protease and two metalloendopeptidases (76). Antineoplastic effects: Mice implanted with sarcoma 180 had their tumors inhibited by more than 60% in 1 month when their diet consisted of 20% mushroom. Mice were given the carcinogen N-butyl-N’-butanolnitrosoamine every day for 8 weeks. Control animals (10) all developed urinary bladder cancer, all haddecreased natural killer cell cytotoxicity, and all showed reduced macrophage chemotaxic activity. Those fed P. ostrentrrs as part of their diet had tumor reduction to 13 of 20 mice (35%); macro-

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phage activity did not drop, and they maintained normal cytotoxicity of natural killer cells (63). Mice were administered dermal dimethylhydrazine 20 mg/kg for 12 weeks. A diet containing 5% P. ostreentus was given in two ways. It was administered either during the whole trial, or during the next 21 days after the application of dimethylhydrazine. Those mice on the mushroom diet throughout the experiment had significantly less lymphoid hyperplasia foci and aberrant crypt foci. The incidence of tumors was not significantly altered. Mushroom administration throughout the experiment reduced the ornithine decarboxylase activity in the colon and liver (77). Cholesterol lowering effects: Bobek et al. (78) studied Syrian hamsters fed P. ostrentus as 2% of a standard diet. The animals were given 8 weeks of alcohol (15% aqueous solution) resulting in a 40-60% increase in their serum cholesterol, triacylglycerol, and phospholipid concentration density. If the mushroom was given with the alcohol, the increase did not occur, but fell below the level of control animals. Aqueous extracts, fungal fruiting bodies, and ethanolic extracts lowered serum cholesterol and triacylglycerols in another study done on hamsters (79). In another experiment on hamsters, a diet consisting of 44% of calories from fat and containing 52 mg of cholestero1/100 g of food created increased blood and liver cholesterol and triacylglycerol. Test animals were administered 2% P. ostrecrtus (dried whole mushroom) in the diet during a 6-month study period. The dried whole mushroom retarded the increase in cholesterol and triacylglycerol in both serum and liver during the entire 6 months; the very-low-density lipoproteins were a significant amount of the decrease. High-density lipoproteins were not affected (80). Ingestion of 5% P. ostreentzrs in the diet, with cholesterol, reduced the production and secretion of very-low-density lipoproteins in hypercholesterolemic rats (81). The lower levels of cholesterol were thought due to the effect on the lipoproteins mentioned above, and to decreased absorption and biosynthesis of cholesterol and increased catabolistn of cholesterol (82). Hyperlipoproteinemic and hyperglycemic rats were given a diet of 4% P. ostreentus and 0.1% cholesterol. After 2 months of this diet the diabetic rats had a lower basal and postprandial blood glucose level. Insulin levels were constant. The cholesterol levels were reduced by 40%, and the lipoprotein profile showed decreases in both the low-density and very-low-density lipoproteins. There was no effect on serum or liver triglycerides (84). Similar cholesterol-lowering effects (40%) were seen in hereditary hypercholesterolemic rats treated with P. ostreatus for 7 weeks (83). Rats were given a diet containing 5% powdered P. ostreutus and a diet of 0.3% cholesterol for 12 weeks. On this diet the rats developed hypercholesterolemia. Animals were evaluated in the last 29 days of the experiment and showed a 50% increase in the rate of degradation and excretion of cholesterol in tested rats. In these animals the mushroom diet prevented any progression of the hypercholesterolemia with a decrease of 38% in serum cholesterol and 25% in liver cholesterol (85). The mechanism of this hypocholesterolemic effect (at least in rats) is thought to be due to reduced absorption of cholesterol (dual-isotope plasma ratio method used) and to increased catabolism and excretion (86, 87). The mushroom diet also accelerated plasma and liver clearance of very-low-density lipoproteins in hypercholesterolemic rats (88) by reducing secretion and accelerating fractional turnover rate (89). Immunomodulatory effects: The glucans from this mushroom were given as premedication to immunized calves. The dose was 10 mg/kg. The level of delayed skin hypersensitivity and primary antibody response was greater thanthe initial level, and

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greater than that of controls. The glucans were determined to have an immunomodulatory effect in these animals (90). When the P. osterntus glucans were administered to mice, an increase in phagocytic activity by blood leukocytes was noted. The increase in phagocytic activity was significant only after injection of the glucan (91). Pleurot~ls pulmowr-ius:This mushroom has been used in experimental veterinary medicine to immobilize preparasitic nematode larvae (Ostertuginostertugi,Cooperia oncoplzora, Oesophctgostorrwn cjrrn~~ispiszulatr~m, and Cyuthostorzra species). The actual infective stages were less sensitive to the immobilization effect than the preparasitic stage (92). Plezarotru sajor-caju Antineoplastic effects: The protein-containing polysaccharides from this mushroom were extracted and tested in vitro for antitumor activity. Several glucans were shown to have antitumor effects (93). Blood pressure: When tested in rats, an intravenous aqueous extract was found to have hypotensive effects in a dose-related manner. A 25-mg dose decreased the mean systemic blood pressure from 110 mmHg to 70 mmHg. There was a minimum change in heart rate. There was a decrease of 50% in the glomerular filtration rate after 2 hr (94). Plerrrotus tuber-regiwn: The sclerotia of this mushroom is used in the tropics (such as Nigeria) by native peoples for fever, high blood pressure, stomach pain, constipation, and smallpox (95, 96). Polypores: The fruiting bodies, which are often bitter and tough (97), were used to help staunch bleeding. Polyporus an~zosus:See Polyporus oficirzcrlis. This mushroom is also thought to contain agaric acid. Polyporus anthelminticus: Called chu-tau by the Chinese, this mushrooms was recommended as a vermifuge (42). POlypOl.14S fomerztnrizrs: The inner part of this polypore was dug out, washed with alkali (like ashes), beaten until soft, and then applied to sores and wounds. Its actions were primarily mechanical. It has been used to staunch bleeding and has been found on oak and beeches of Southern Europe. It is known to contain potassium chloride, some nitrogenous agent, calcium sulfate, iron, and magnesium (42). Polyporus ig~zinrius:Used to make amadou, an agent that staunches bleeding (42). Polyporrrs meqinntus: Used as an agent to staunch bleeding (42). Po~yporusoflciualis: The larch or white agaric contains agaric acid (laricic acid, agaricinic acid), which was used to quell sweating. It was used in a preparation called tincture antiperiodica. This preparation acts as a counterirritant when applied to mucous membranes and is irritating to the stomach and intestines (40, 42). The powdered white agaric has a sweetish, burning, bitter taste, and faint odor. It contains about 50% resins when extracted with boiling alcohol (42). Polyporws squnn~oszrs:See Polyporus oflcinnlis. This mushroom is also thought to contain agaric acid. Polyporus szrcweolnrzs: See Polyporzrs oficimlis. This mushroom is also thought to contain agaric acid. Polyporus umbellcms:Also called Zhu Ling, this mushroom is said to have anticancer and immunomodulatory effects, but few studies have been conducted in the West. Antineoplastic effects: When taken orally or by intravenous injection, P. unzbellatus extracts have wide application for treatment of lung cancer in the Orient. Chinese physicians administer extracts from this fungus for the treatment of lung cancer, cervical cancer,

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esophageal cancer, gastric cancer, liver cancer, leukemia, breast cancer, and lymphosarcoma (43). Most of the animal work being done is on sarcomas. Jung-lieh (44) showed strong inhibitory activity in the murine model using sarcoma 180. In another study, mice were implanted with sarcoma 180 and given P. urnbellatus extract in a dose of 1 mg/kg body weight. Tumors were reduced by 50% when compared to control animals (18). Ying et al. (38) reported a 70% reduction in tumor weight in mice studied. Work by Miyaski also demonstrated antisarcoma activity (18). In unpublished work at the Beijing Institute of Materia Medica, a water extract was tested against lung cancer (sarcoma 180) in human patients after radiation therapy. Most patients who did not receive the extract died, while those who received the extract had marked improvement ( 18). Porn Porn: See Hericiurn erinnceus. Psilocybin/Psilocin-Containing Mushrooms: These have found limited experimental use in the treatment of mental disorders (1). There are no documented medical uses, but shamans have used them to diagnosis illnesses (18). Puffballs: Puffballs have been believed by some to have anticancer activity (45). Puffballs were once recommended as a sedative (in a tincture), but other than the alcohol in the tincture, puffballs are not known to have sedative properties (42). Wound healing: The spores of these mushrooms were used by American Indians on wounds to reduce the inflammation and infection (2). Many tribes, including the Navajo and the Mohegan, used the puffballs themselves to staunch bleeding (46). A poultice of puffballs was applied to sore breasts and abscesses by the Arikara, and to sores, burns, and pruritic areas by the Navajo. The spores have been used as a hemostat by the Canadian Kwakiutl Indians. The spores were alternated with spiderwebs to create a primitive bandage (46). Theuse of puffballs as hemostatic agents is not just a North American remedy, but can be found in the works of 19th-century Europe (46). Red Woodlover: See Hypholornn sublnteritiurn. Reishi: Japanese name for Ganoderma lucidum (see above). Sacodorz asprotus: Methanolic and water extracts were tested in mice against solid tumors (sarcoma 180). Both oral and intraperitoneal administration was used. The aqueous extract was effective for inhibition of tumor growth while the methanolic extract did not show activity. The active molecule has a molecular weight of greater than 10,000 daltons (47). Saiwai-take: Common name for Ganoderrnn lucidurn. Sarunouchitake: Common name for Ganoderma lucidum. Satyr’s Beard: See Hericiurn erinaceus. Shaggy Mane: See Coprinus comatus. Shiangu-gu: See Lerltirdn edodes. Shiangu-ku: See Lentinuln edodes. Shiitake: See Lentirlula edodes. Shirotamogitake: See Hypsizygus ulmnrius. Songshan Lingzhi: See Gnrzodernza tsugae. Straw Mushroom: See Plellrotus ostrentus. Tamogitake: See Pleurotus ostreatus. Tamo-motashi: See Hypsiqjgus tessulatus. Tree Ear: See Auricularia polytricha. Tree Jelly Fish: See Auricularia polytrichn. Tree of Life Mushroom: Common name for Gnnodermu lucidum.

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Tree Oyster: See Pleurotrls ostrentus. Truffles: Some species of truffles have been attributed with aphrodisiac powers. The Czech folk name for these truffles means "lamb's testicles.'' Early English medicine used some truffles in a formula to induce pregnancy ( 5 ) . Tsuchi-maitake: See Polyporus lrrnbellntus. Trrlostorncr brwmnle (T. pedunculatun~~) and Tulostomcr ccrmnpestre:The Ramah Navajo Indians used this puffball in infusions or poultices for healing sheep leg-bone breaks (46, 48). Umbrella Polypore: See Polvporus urnbellatus. Ustilngo nznyclis: Commonly called corn smut, the fungus that grows on corn silk was at one time recommended for treatment of lung hemorrhage, bowel hemorrhage, and as an emmenagogue (42). Velvet Foot: See Flammulirzn velutipes. Verpa Species: A somewhat phallus-shaped fungus, which through the Doctrine of Signatures was attributed to have sexual powers. It was used similarly to Phcrllus i1npudicus (5). It does not possess aphrodisiac or increased fertility actions. Volvcrriellcrvolvrrcecr: A protein with immunonlodulary activity has been purified from this edible mushroom. The active agent is a polypeptide. In an in vitro study measuring blast formation stimulatory activity this protein maximally stimulated human peripheral blood lymphocytes at a concentration of 5 pg/ml. The mechanism of action appears to be via cytokine regulation (49). Wild Boar's Dung Maitake: See Polyporus urnbellatus. Winter Mushroom: See Flmmzdirzn Iyelzrtipes. Wood Ear: See Alrriculcu-icr polytricha. Yamabiko Hon-shimeji: See Hypsizygus tessulatus. Yamabushi-take: See Hericiurn eri~nceus. Yuhuangmo: See Pleurotus citri~~opileatus. Yuki-motase: See Flmrrnulim velutipes. Yung Ngo: Chinese name, See Auricularia polytricha. Zhu Ling: See Polyporus umbellcltzrs. C. Combination Products

The "tension-easing pill" is a combination of mushrooms used to treat numb hands and feet, and problems with veins, tendons, and limb tetany. The mushrooms included are Anlmitcr crgglutimtcr, Boletus edulis, Lactmiusinsulsrrs, Lcrctnrius picim4s, Lcrctcwirrs pipemtzls, Lnctcu-ius vellererrs, Lenzites betulilla, P m u s concfzcrtus,Pulveroboletrw ravenelii, Rrrssula alutrrcecr, Russuln densifolia, Rrrssulcr -foetens, Rrlssda integrcms, Russ~dn11igram, and Thelephorcl vialis (38).

REFERENCES 1. CG Ainsworth. Introduction to the History of Mycology. Cambridge: Cambridge University Press. 1976. 2. R Molina, T O'Dell. D Luoma. M Amaranthus, M Castellano, K Russell. Biology, Ecology, and Social Aspects of Wild Edible Mushrooms in the Forests of the Pacific Northwest: A Preface to Managing Commercial Harvest. General Technical Report PNW-GTR-309. Port-

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13.

14. 15. 16.

17. 18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29. 30.

797

land Oregon: US Department of Agriculture Forest Service,Pacific Northwest Research Station, 1993. M Rice. Fine paper from mushrooms. Mushroom J Wild Mushrooming. 34: 21-22, 1991. JG Frazer. Pausanias’s Description of Greece. (Trans.) Vol 1. London: Macmillan, 1898. WPK Findlay. Fungi. Folklore. Fiction, and Fact. Eureka, CA: Mad River Press, 1982. B LOWIT.Amnnitct nzzrscarin and the thunderbolt legend in Guatemala and Mexico. Mycologia 66:188-190.1973. B Lowry. Hallucinogenic mushrooms in Guatemala. J Psychedelic Drugs 9: 123-125, 1977. EJ Butler, SG Jones. Plant Pathology. London: Macmillan. 1949. M Rice, D Beebee. Mushrooms for Color. Eureka. CA: Mad River Press, 1980. CJ Alexopoulos. CW Mims. Introductory Mycology, 3rd ed. New York: Wiley, 1979. WD Gray. The Use of Fungi as Food and in Food Processing. Cleveland, OH: CRC Press, 1979. p 113. WA Hayes, NG Nair. The cultivation of Agaricus bispor~sand other edible mushrooms. In: JE Smith, DR Beny, eds. The Filamentous Fungi. Vol 1. Industrial Mycology New York: Wiley, 1975, pp 212-248. RG Wasson, A Hofnlann. CAP Ruck. The Road to Eleutis: Unveiling the Secret of the Mysteries. Ethno-mycological Studies 4. New York: Harcourt Brace Jovanovich, 1978. TJ Riedlinger. Wasson’s alternative candidates for soma. J Psychoact Drugs 25: 149-156, 1993. S Hajcek-Dobberstein. Soma siddhas and alchemical enlightenment: psychedelic mushrooms in Buddhist tradition. J Ethnopharmacol 48: 99-1 18, 1995. SF Borhegi. Mushroom stones of middle America: a geographicallyandchronologically arranged distribution chart. In: VR Wasson, RG Wasson, eds. Mushrooms, Russians, and Histoly. Appendix to Vol 2. New York: Pantheon Books, 1957. SF Borhegi: Miniature mushroom stones from Guatemala. Am Antiq 26: 498-503. 1961. P Stamets.Psilocybin Mushrooms of the World. An Identification Guide. Berkeley, CA: Ten Speed Press, 1993. C Novotny, BR Vyas, P Erbanova, A Kubatova, V Sasek. Removal of PCB’s by various white rot fungi in liquid cultures. Folia Microbiol (Praha) 42: 136-140, 1997. M Stadler, A Mayer. H Anke, 0 Sterner. Fatty acids and other compounds with nematicidal activity from cultures of Basidiomycetes. Planta Med 60: 128-132. 1994. N Favero. G Bressa, PCosta. Response ofPleurotrrs ostrecrtusto cadmium exposure. Ecotoxicol Environ Safety 20: 1-6, 1990. L Bezalel, Y Hadar, PP Fu, JP Freeman. CE Cemiglia. Metabolism of phenanthrene by the white rot fungus Pleurotus ostrentus. Appl Environ Microbiol 62: 2547-2553, 1996. S Masaphy, Y Henis, D Levanon. Manganese-enhanced biotransformation of atrazine by the white rot fungus Plezrrotrls p h o n a l - i u s and its correlationwithoxidationactivity.Appl Environ Microbiol 62: 3587-3593, 1966. RP Purkayastha, AK Mitra, BBhattacharyya. Uptake and toxicological effects of some heavy metals on Plerrrotus snjor-cqju (Fr.) Singer. Ecotoxicol Environ Safety 27: 7-13. 1994. M Ariosy, N Kolankaya. Biodegradation of lindane byPlezrrotzrs snjo~”cajuand toxic effects of lindane and its metabolites on mice. Bull Environ Contam Toxicol 59: 352-359. 1997. K Scheibner, M Hofrichter. Conversion of aminonitrotoluenes by fungal manganese peroxides. J Basic Microbiol 38: 51-59, 1998. K Scheibner, M Hofrichter, A Herre. J Michels. W Fritsche. Screening for fungi intensively mineralizing 2,3,6-trinitrotoluene. Appl Microbiol Biotechnol 47: 452-454, 1997. HP Molitoris. Mushrooms in medicine. Folia Microbiol (Praha) 39: 91-98, 1994. B Kendrick. The Fifth Kingdom, 2nd ed. Newburyport MA: Mycologue Publications, Focus Texts, 1992. J Delacretaz, D Grigoriu, G Ducel. Color Atlas of Medical Mycology. Bern, Switzerland: Hans Huber, 1976.

798

Spoerke

31

CW Emmons, CH Binford, JP Utz,KJKwon-Chung. Medical Mycology.Philadelphia: Lea & Febiger, 1977. JW Rippon. Medical Mycology, 3rd ed. Philadelphia: WB Saunders, 1988. CG Ainsworth, PKCAustwick. Fungal Diseasesof Animals, 2nd ed. Slough: Commonwealth Agricultural Bureaux, 1973. H Vanden Bossche, DWR Mackenzie, G Cauwenberg. Aspergillzts and Aspergillosis. New York: Plenum Press, 1987. CK Campbell. CG White. Fungal infections in AIDS patients. Mycologist 3: 7-9, 1989. CJ Fichtenbaum, WG Powderly. Recurrent oral candidiasis in HIV-infected patients. Infect Med 11: 191-200.1994. M Hager. Antibiotics. Newsweek Extra 70-72, Winter 1997-1998. Ying, Jianzhe; Mao, Xialoan, etal: Icones of Medicinal Fungi from China. Beijing: Science Press,1987. AC Beynen. AM Fielmich, AG Lennmens, AH Terpstra. Farm-grown mushrooms (Agaricus carrpestris) in the diet of rats do not affect plasma and liver cholesterol concentrations. Nahrung 40: 343-345. TS Blair. Botanic Drugs. New York: Therapeutic Publishing Company, 1917. T Laessoe, ADel Conte. G Lincoff. The MushroomBook. New York: DK Publishing, 1996. M Grieve. A Modem Herbal. New York: Dover, 1971. L Bo, B Yun-sun. Fungi Pharmacopeia (Sinicaj. Oakland,CA: The Kinoko Company, 1980. C Jung-lieh. Experimental study of antitumor effect of an extract derived from Zhu-Ling (Polyporus mbellatzuj. Institute of Materia Medica, Academy of Traditional Chinese Medicine, Peking, China, 1983. K Cochran. Medical Effects. In: ST Chang, WA Hayes, eds. The Biology and Cultivation of Edible Mushrooms. New York: Academic Press, 1978. W Burk. Puffball usage among North American Indians. J Ethnobiol 3: 55-62, 1983. H Maruyama, K Yamazaki, S Murofushi, C Konda, TIkedawa. Antitumor activity of Sarcodon aspratus (Berk.) S Ito and Gculoderrncr llrcidzrnl (Fr.) Karst. J Pharmacobiodyn 12: 118123, 1989. W Burk. T Fitzgerald. Puffball usage’s among North American Indians. McIlvainea 5: 1417, 1981. HC Hsu, CI Hsu, RH Lin, CL Kao. JY Lin. Fip-vvo, new a fungal immunomodulatoryprotein isolated from Volval-iella volvacea. Biochem J 323: 555-565, 1997. T Willard. The Reishi Mushroom: Herb of Spiritual Potency and Medical Wonder. Vancouver, British Columbia: Sylvan Press, 1990. AA Birks. Fungi in folk medicine. McTlvaninea 10: 89-94, 1991. SC Jong, R Donovick. Antitumor and antiviral substances from fungi. Adv Appl Microbiol 34:183-362,1989. SC Jong, JM Birmingham,SH Pai. Immuno-modulatory substances offungal origin. J Immuno1 Immunopharmacol 1 l(3): 1991. M Hirotani T Furuya, M Shiro. A ganoderic acid derivative, a highly oxygenated lanostnetype triterpenoid from Ganoclerrrzalucidzrrrz. Phytochemistry 24: 2055-2061. 1985. A Morigawa, K Ditabatake. Y Fujimoto. N Ikekawa. Angiotensinconverting enzymeChem Pharmaceutical Bull33: 3025-3028, inhibitory triterpenes from Garzoclerrnn Iz~cihtn~. 1986. YM Kabir. S Kimura. Effect of shiitake (Lentinus edodes) and maitake (Grifola fiorzdosa) mushrooms on blood pressure and plasma lipids of spontaneously hypertensive rats. J Nutr Sci Vitamin01 33: 341-346, 1987. WB Stavonoha, T Weintraub, T Opham,A Colorado, R Opieda. J Slama. Study of the antiinflammatory activity of Ganoclet-rrzcr lrtcidwn. Proceedings of the AcadenMIndustry Conference (AIJC), Sapporo, Japan, August 18-20, 1990. J Zhang, G Wang, H Li, C Zhuang, T Mizuno, H Ito, H Mayuzumi, H Okamoto, J Li.

32. 33. 34. 35. 36. 37. 38. 39.

40. 41. 32. 43. 44.

45. 46. 47.

48. 49. 50. 51. 52. 53. 54. 55.

56.

57.

58.

Fungi in Folk Medicine and Society

59.

60.

61.

62. 63.

64. 65. 66. 67. 68.

69. 70. 71.

72. 73. 74. 75. 76.

77. 78.

79.

80.

799

Antitumor active protein-containing glycans from the Chinese mushroom songshan lingzhi. Gnnoderrm tsugne, mycelium. Biosci Biotechnol Biochem 58: 1202-12051, 1994. J Zhang. G Wang, H Li, C Zhuang, T Mizuno,H Ito, C Suzuki,H Okamoto, J Li.Antitumor polysaccharides from a Chinese mushrooms. "yuhuangmo," the fruiting body of Plezlrottls citrirropileatzts. Biosci Biotechnol Biochem 58: 1195-1201, 1994. G Wang, J Zhang, T Mizuno. C Zhuang, H Ito, H Mayuzumi, H Okamoto, J Li. Antitumor active polysaccharides from the Chinese mushroom Songshnn lingzhi, the fruiting body of Gnrzoderwn tsugne. Biosci Biotechnol Biochem 57: 894-900, 1993. N Ohno, K Iino, T Takeyama, I Suzuki, K Sato. S Oikawa. T Miyazaki, TYadomae. Structural characterization and antitumor activity of the extracts frommatted mycelium of cultured Grifolafiondosa. Chem Pharm Bull 33: 3395-3401, 1985. H Nanba. Maitake: the king of mushrooms. Explore 3: 44-46, 1992. S Kurashige. Y Akuzawa, F Endo. Effects ofLelztizrs edodes, Gr-ifolctfiondosrr, and Pleurotlw ostreatus administration on cancer outbreak,and activities of macrophages and lymphocytes in mice treated witha carcinogen,N-butyl-N-butanolnitrosoamine. Immunopharmacol Immunotoxicol 19: 175-183, 1997. I Hishida, H Nanba, H Kuroda. Antitumor activity exhibited by orally administered extract from fruitbodies of Grifola fiondosn (maitake). Chem Pharm Bull 36: 8 19-827, 1988. K Adachi, H Nanba, M Otsuka, H Kuroda. Blood pressure lowering activity present in the fruiting body of Grifola frorzdosn (maitake). Chem Pharm Bull 36: 1000-1006. 1988. K Adachi, H Nanba, H Kuroda. Potential of host-mediated antitumor activity in mice by beta-glycan obtained from Grifoln frorzdosrr (maitake). Chem Pharm Bull 35: 262, 1987. P Stamets. Gourmet and Medicinal Mushrooms. Berkeley, CA: Ten Speed Press, 1993, pp 377-378. G-L Chen. Studies on the cultivation and on the medicinal efficacy of Hericiunr erinacerss. Translation. The Edible Fungi Research Institute of The Shanghai Academy of Agricultural Science and Cultivation of Edible Fungus, France, 1992. A Weil. Mushrooms as food and medicines: a review. McIlvaninea 5: 2, 1982. J Vetter. [Mineral andamino acid contents of edible, cultivated shiitake mushrooms (Lentinus edodes).] Z Lebensm Unters Forsh 201: 17-19. 1995. G. Chihara. Antitumor and immunological properties of polysaccharides from fungalorigins. National Cancer Institute, Tokyo. Proceedings of the Tenth International Congress on the Science and Cultivation of Edible Fungi. France, p809. ME Hall Jr. The Biology of Lichens, 2nd ed. New York: Elsevier, 1974. M Bajaj, S Vadhera, AP Brar, GL Soni. Role of oyster mushroom (PlezrrotusJlorida) as hypocholesterolemic/antiatherogenic agent. Indian J Exp Biol 35: 1070-1075, 1997. L Opletal.(Phytotherapeuticaspectsofdiseasesofthecirculatorysystem. 2. The oyster mushroom and its potential.) Cesk Farm 43: 160-166. 1993. J Filipek,V Misik. Themushroom Pleurotus ostrecrtus exhibits OH radicals scavenging activity. Pharmazie 48: 868-869, 1993. N Dohmae, K Hayashi, K Miki, Y Tsumuraya, Y Hashimoto. Purification and characterization of intracellular proteases in Pleurotus ostrmtus fruiting bodies. Biosci Biotechnol Biochem 59: 2074-2080, 1995. P Bobek,S Galbavy, LOzdin. Effect ofoyster mushroom (Pler4rotusostrentzts) on pathological changes in dimethylhydrazine-induced rat colon cancer. Oncol Rep 5: 727-730, 1998. P Bobek. E Ginter, M Jurcovicova, LOzdin, D Mekinova. Effect of oyster fungus (.Pleurotus ostrentus) on serum andliver lipids of Syrian hamsters with a chronicalcohol intake. Physiol Res 40: 327-332, 1991. P Bobeck. L Ozdin, L Kuniak. Influence of water and ethanol extracts of theoyster mushroom (Pleurotus ostrentusj on serum and liver lipids of the Syrian hamster. Nahrung 37: 571575, 1993. P Bobek, E Ginter, L Kuniak, J Babala, M Jurcovicova, L Ozdin, JCerven. Effect of mush-

800

81.

82.

83.

84.

85. 86.

87.

88. 89.

90.

91.

92. 93.

94. 95. 96. 97. 98. 99.

100.

Spoerke room Plezwotzu ostreatus and isolated fungal polysaccharide on serum and liver lipids in Syrian hamsters with hyperlipoproteinema. Nutrition 7: 105-108, 1991b. P Bobek, L Ozdin. Oyster mushroom (Pleurotzrsostreatrrs)reduces the production and secretion of very low density lipoproteins in hypercholeserolemic rats. Z Ernahrungswiss 35: 249252, 1996. P Bobek, L Ozdin, L Kuniak, M Hromadova. [Regulation of cholesterol metabolism with dietary addition of oyster mushrooms(Pleurotus ostreatus)in rat hypercholesterolen~ia.] Cas Lek Cesk 136:186-190,1997. P Bobek, E Ginter,M Jurcovicova. L Kuniak. Cholesterol-lowering effectof the nlushroom Pleurotus ostrerrtzls in hereditary hypercholesterolemic rats. Ann Nutr Metab 35: 191-1 95, 1991a. V Chorvathova. P Bobek, E Ginter, J Klvanova. Effect of the oyster fungus on glycaemia andcholesterolaemia in rats withinsulin-dependentdiabetes.Physiol Res 42:175-179, 1993. P Bobek, L Ozdin, M Mikus. Dietary oyster mushroom (Plezlrotus ostrentzrs) accelerates plasma cholesterol turnover in hypercholeserolaeic rats. Physiol Res 44: 287-291, 1995. P Bobeck, L Ozdin,Kuniak. L Mechanism of hypocholesterolemic effect ofoyster mushroom (Pleurotzrs ostreutus) in rats: reduction of cholesterol absorption and increase of plasma cholesterol removal. Z Ernahrungswiss 33: 34-50, 1994. P Bobeck, L Ozdin, LKuniak. Effect of oyster mushroom (PIez4rotusostreatrrs)and its ethanolic extract in diet on absorption and turnover of cholesterol in hypercholesterole~nicrats. Nahrung 40: 222-224. 1996. P Bobek. L Ozdin. The mushroom Pleurotus ostreatus accelerates plasma very-low-density lipoprotein clearance in hypercholesterolemic rat. Physiol Res 43205-206, 1994. P Bobek. L Kuniak, L Ozdin. Themushroom Pletlrotus ostreatus reduces secretion andaccelerates the fractional turnoverrate of very-low-density lipoproteins in the rat. Ann Nutr Metab 37:142-145,1993a. S Paulik, V Bajova, G Benko. (Delayed hypersensitivity and the primary antibody reaction after administration of infectious bovine rhinotracheitis inactivated oil vaccine to calves premedicated with glucan.) Vet Med (Praha) 38: 395-402, 1993. S Paulik, Svrcek. J Mojzisova, A Durove. Z Beni, M Huska. The imn~unomodulatoryeffect of the soluble fungalglycan (Pleurotzrsostreecrtus)on delayed hypersensitivity and phagocytic ability of blood leukocytes in mice. Zentralbl Veterinarmed 43: 129-135, 1996. M Larsen, PNansen. Ability of thefungus Plezwotus puhnorlnritls to immobilize preparasitic nematode larvae. Res Vet Sci 51: 246-249. 1991. C Zhuang, T Mizuno, A Shimada. H Ito, C Suzuki, Y Mayuzumi, H Okamoto, Y Ma, J Li. Antitumor protein-containing polysaccharides from a Chinese mushroom Fengweigu or Houbitake, Plezrrotrrs scrjor-cc7ju (Fr.) Sings. Biosci Biotechnol Biochem 57: 901-906, 1993. SC Tam. KP Yip. KP Fung, ST Chang. Hypotensive and renal effects of an extract of the edible mushroom Pleurotus sajor-cuju. Life Sci 38: 1 155-1 161, 1986. R Singer. The Agaricales in Modern Taxonomy. Koenigstein, West Germany:Koeltz Scientific Books, 1986. BA Oso. Pleurotus tuber-regium from Nigeria. Mycologia 69: 271-279, 1977. OK Miller Jr. Mushrooms of North America. New York: EP Dutton. 1978. TB Ng. A reviewofresearchontheprotein-boundpolysaccharide(polysaccharopeptide (PSP) from the mushroom Coriolrrs versicolor. Gen Pharmacol 30: 1-4. 1998. TB Ng, WY Chang. Polysaccharopeptide from the mushroom Coriolus \versicolor possesses analgesic activity but does not produce adverse effects on femalereproductive or embryonic development in mice. Gen Pharmacol 29: 269-273, 1997. JL KO, CI Hsu, RH Lin, CL Kao. JY Lin. A new fungal immunomodulatory protein, FIPfve isolated from the edible mushroom Flanzmulina \~elutipesand its complete amino acid sequence. Eur J Biochem 228: 244-249. 1995.

in

Fungi

Folk Medicine and Society

801

101. WK Liu, TB Ng, SF Sze. KW Tsui. Activation of peritoneal macrophages by polysaccharopeptide from the mushroom Coriolus Ivrsicolor. Inmunopharmacology 26: 139- 146, 1993. 102. Q Zeng, J Zhao, Z Deng. The anti-tumor activity of Flammulina velutipes polysaccharide (FVP). Edible Fungi of China 10: 2. 1990 (Szechwan Institute of Materia Medica). 103. Z Qungian et al: Antitunlor activity of Flatnmulina velutipes polysaccharide (FVP). Edible Fungi of China 10: 11-15, 1991. 104. 0 Totttnar, P Linderg. Effects on rat liveracetaldehyde dehydrogenase in vitro and in vivo by coprine, the disulfiram-like constituent of Coprims utruntenturius. Acta Pharmacol Toxicol (Copenhj 40: 476-481, 1977. 105. BB Coldwell, K Genest,DW Hughes: Effects of Coprims nttmzentnrilrs on the metabolism of ethanol in mice. J Pharm Pharmacol 21: 176-179, 1969. 106. N Yamaguchi, J Yoshida, LJ Ren, H Chen, Y Miyazawa, Y Fujii. YX Huang, S Takamura, S Suzuki, S Koshimura. Augmentation of various immune reactivities of tumor-bearing hosts with an extract of Cordyceps sinemis. Biotherapy 2: 199-205, 1990. 107. A Hoffman. Ergot-a rich source of pharmacologically active substances. In: T Swain, ed. Plants in the Development of Modern Medicine.Cambridge: Harvard University Press, 1972. 10s. K Jones. Reishi, Ancient Herb for Modern Times. Issaquah, WA: Sylvan Press, 1992. 109. N Gunde-Cimerman, A Cirnerman. Pleurotus fruiting bodies contain the inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase-lovastatin. Exp Mycol 19: 1-6, 1995.

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Index

AAL toxins in leukoencephalomalacia, 556 AAPCC (see American Association of Poison Control Centers) Abortifacients, 73 Abortions, spontaneous, 653, 657 Abrin. 21 5, 420 Abrus yrecntorim, 215, 270, 420 treatment of ingestion of, 420 Absidin, 786 Abutilon theoplwasti, 6 1 Acacia berlandieri, 215, 420 treatment of ingestion of, 320 Acer. rubrm. 189, 420, 449 treatment of ingestion of, 420 Aceraceae. 189 Acetals, 300 15-Acetoxy-DON (see 15-Acetyldeoxynivalenolj Acetylcholine, 237 N-Acetylcysteine, 214 treatment for amatoxins, 753 3-Acetyldeoxynivalenol, 546-547. 61 1 15-Acetyldeoxynivalenol, 555, 61 1 N-Acetylcytisine, 21 9 Achillea nlillefolimz, 7 1 natural chemicals in, 72-73 Acifluorfen, 43 Ackoccrrzthera oblortgifolia, 1 9 1 Ackocantherin, 191 Aconitine. 227, 25 1, 420 Aconitm nuyellus. 227, 420 treatment of ingestion of. 420 Acremonium (see Neohplzodiunl) Actaea rubra, 227 treatment of ingestion of, 227

Acuba japonica, 753 Acubin, treatment of amatoxins in, 753 Adenia volkensii, 46 Adenostyles alliariae, 250 Adducts DNA, 105 RNA, 105 Adonidin, 227 Adonis serndis. 227 Aesculin, 212, 329, 420, 449 A e s c h s spp., 212, 339. 420, 449 glubrn, 212, 420 lziypocastmwn, 212. 329, 449 treatment of ingestion of, 420 Afghanistan, 82 Aflatoxin, 57, 83, 134, 472. 555, 594-61 1, 613. 617 biomarkers, 603-607 carcinogenicity, 597-61 1 genetic susceptibility, 603 glutathione-S-transferase detoxification, 610 hematochromatosis and liver cancer, 609 hepatitis, 598-6 10 human illness, 597-598 kwashiorkor, 597-598 metabolism, 596-597, 606, 610 mutagenicity, 596 sources, 596 species sensitivity. 596 toxicity, 596-598 Aflatoxin B 1, 595-597, 600. 602, 604-607, 609-610, 613. 629 binding with calcium aluminosilicate clay, 635

803

804 [Aflatoxin B 11 binding with clinoptolite, 635 carcinogenicity. 63 1-633, 635-636 growth suppression effects. 632 interaction with: 2,3,7,8-tetrachlorodibenzo-p-dioxin, 636 cadmium. 633 coenzyme QlO, 636 copper, 633 cystine, 636 dehydroepiandrosterone (DHEA), 636 diethylstilbestrol, 636 ethanol, 634 flukes, 63 1 flunixin. 636 geniposide. 636 gizzerosine, 636 hepatitis virus, 63 1 hexachlorocyclohexane, 636 indole-3-carbinol, 632 kojic acid, 629 malathion, 636 monensin, 636 n~oniliformin, 629 nafenopin, 636 oxytetracycline, 636 polyvinylpolypyrrolidone,636 propyl gallate, 636 Sacchnronyces cel-evisiae, 630 selenium, 633, 636 tetracycline, 636 Thyroideunl Polfa. 636 tiarnulin, 636 vitamin C, 636 liver effects, 638-639 mutagenicity. 632-633, 635-636 protein effects, 638-639 Aflatoxin B2, 595-596 Aflatoxin G1, 595-596 Aflatoxin G2, 595-596 Aflatoxin MI. 595, 597, 604 binding with calcium aluninosilicate clay. 635 Aflatoxin M3, 595 Aflatoxin-N7-guanine, 604-605 Aflatoxin P1, 604 Aflatrem, 476 Africa 69, 82. 85, 331, 332, 505. 523 Agalactia, 653, 657 Agaratine, 77, 78 Agaricus: crn9ensis,745, 763

Index [Agaricus] bisporvs (see Agaricus brunrzescens) brm17escms, 77-78, 716, 783 benzyl alcohol in, 134 hydrazine conlpounds from 79, 134 respiratory reaction, 764-765 spores. 726 toxin, 767 cdijor-rlicus, 764 carzzpestris, 763 medical use, 786 muscm-iI~s(see Anrcrrzitu mrrscurin) placovnyces, 745, 764 respiratory reactions. 765 sihicola 745 test for yellow staining, 745 santhodemis, 745, 764 Agal'e leclurguillci, 420, 436 Agglutin, 27 1 Aglycone(s), 250. 301 Agmatine, 285 Agroclavine, 594. 657, 661 Agrostermna githago, 200, 420 treatment of ingestion of. 200, 420 Ah receptor, 57 binding of, ICZ 59 Akakabi-byo, 545 Albania. 220, 252, 253 Albntr-ellus, 763 Alberta, 216, 250. 253 Aleul-itis fordii. 207 Alimentary toxic aleukia (ATA), 537-539, 542-546, 61 1-612 causes, 542-543 clinical signs, 542-543 control of, 545 in animals. 544 legal tolerance level, 545 Aliphatic nitrocompounds, 304 Aliphatic nitrotoxins. 300, 330 Alkaloids: P-carboline, 359 cardiac, 421, 425 causing death in livestock, 251 causing death in humans, 248 c h i n e , 359 definition of, 248 diterpene, 364, 425, 446 ergopeptine alkaloids. 367 ergot, 88, 358 ester. 222 glyco-, 222, 233, 234, 333 indole, 359, 326, 428

.

Index [Alkaloids] indolizidine. 215, 218, 446 isoquinoline, 209, 210, 225, 366, 423. 435 other, 368 peptide, 367,368 piperidine, 236, 357. 445. 447 potato, 132 pyridine, 423 pyrrolizidine, 69, 195, 197, 198, 223, 250, 252, 254, 354, 355, 421, 424, 433, 447, 516 quinolizidine, 216-219, 360, 433, 447 solanaceous, 234 steroid(a1). 362, 434, 435, 447, 448 teratogenic, 435 tropane, 233-234. 254, 422, 424, 433, 435 tryptamine, 359 A l l i m , 420 treatment of ingestion of, 420 Allylisothiocyanate (ATTC), 333 Allylthiocyanate (ATC). 326, 33 1 Alterrrnria: nltemntcr, 542. 556 respiratory reactions. 765 Amanitrr : bisporigem mycorrhizal associations, 7 18 buttons, 735 gernnrcrtn. 753 mwm-in: medical use, 786 mycorrhizal associations, 7 18 poisoning, 754-756 religious uses, 783 yantherina mycorrhizal associations, 7 18 poisoning by, 754-756 toxins, 754-755 phnlloides. 248 poisoning, 749-753 proxilrrn, 753 respiratory reactions, 765 ntbescem mycorrhizal associations, 7 18 srtlithicrnu poisoning, 750, 753 strobilifomis, poisoning by, 756 wrginatn mycorrhizal associations, 7 18 1Yer-m. 753 tnycorrhizal associations, 7 18 virosa, 753 1~o/vata,764 Amaranthaceae, 189, 419 Amnr-nnh~sretrojems, 189, 42 1 treatment of ingestion of, 190, 421

805 Amatoxin poisoning, 749-754 tnechanism of action, 750 symptoms, 749-750 treatment, 75 1-754 Amelnnchier nlnifolin. 46 American Association of Poison Control Centers (AAPCC). 1-2 history of, 2 American Conference of Governmental Hygienists, 46 American Revolution, 39, 195 American Southwest, 67 Amide mushroom toxins, 766 Amines, 100, 248. 378 nitrosation of, 100 pressor, 284 primary, 248 secondary, 248 sympathomitnetic. 420 tertiary, 248 toxic examples, 378 Amino acid analogs, 376, 377 examples of toxic, 377 selenium-containing, 379 I-Amino-D-proline, 283 3-Anlinopropionitrile (see P-Amino propionitrile) 0-Aminopropionitrile, 277, 429 Amnri nlajrls, 73, 436 compounds in, 74 Ammodendrine, 25 1 Ammonium hydroxide test, 745 Amirlckin, 354. 421 inten?ledia, 42 1 treatment of ingestion of, 421 Amygdalin, 48, 449 Amylase inhibitors, 268-269 nutritional significance of, 268 role in plant, 268 weight loss and, 268 Amyotrophic lateral sclerosis (ALS), 278 Annbrrem Jilos-aquae, 253 Anabasine. 234, 430 Anacardiaceae, 190 Anagyrine, 219, 251, 429. 447 Analytical methodology for plant toxins, 351-412 alkaloids, 353-368 diterpene, 364-365 indole, tryptamine, 0-carboline. and related. 358-360 indolizidine, 365

806 [Analytical methodology for plant toxins] [alkaloids] isoquinoline, 366 other, 368 peptide. 367 piperidine, 357-358 pyridine. 358 pyrrolizidine, 354-357 quinolizidine, 360-361 steroid, 361-364 tropane, 365 amines, 378 anions, 38 I glycosides, 368-376 cardiac, 371 cyanogenic, 368 glucosinolates, 369-370 nitropropanol. 374-375 other, 375 phenolic, 370 saponins, 371-374 triterpenoid, 372 isoprenoids, 379-381 sesquiterpene lactones. 379 ditel-penes, 379 other terpenes/hydrocarbons, 380 mycotoxins. 381-386 aflatoxins, 382 citrinin, 382 cyclopiazonic acid, 383 deoxynivalenol, 385 fumonisins, 383 furanoterpenes, 384 ibotentic acid, 384 muscimol. 384 ochratoxin A, 382 orellanine, 384 nivalenol, 385 patulin, 385 trichothecenes, 385 vomitoxin, 385 zearalenone, 386 new technologies for, 352-353 proteins, peptides, and amino acids, 376379 enzymes, 377 hemagglutins, 376 lectins, 376 selenium accumulators, 379 Analytical methodology for plant toxicants, 351-412 Anatabine. 234

Index Anatoxin A, 253 Andromedotoxins (see Grayanotoxins) Arlerrrone, 3 19 Animals, germ-free and lectins, 273 Anthocyanins, 60 Anthracenones, 229 Anthraquinone glycosides, 330 Anthraquinone mushroom toxins, 766 "Anticancer nutrients." 67 Antineoplastic effects of garlic and onion, 71 Antinutritional factors: protein-related, 257-298 saponins, 324 Apharzizomenon, 253 Apiaceae (see Umbelliferae), 105, 106, 110, 11 1, 236, 250, 252 linear furanocoumarins in. 105, 106 medicinal use, 106 Apizm, 108. 137 Apocynaceae, 191. 309 Apocywn. 42 1 treatment of ingestion of, 421 Aporphine, 366, 445 At-ucltis lnpogrtert, 54, 259, 266, 270, 535 Aragekiurage (see Aw-ictdaria polytricltrr) Arecu ctrteclrn, 124, 224 Arecoline, 224 Argerttone rnesicana, 225 Argentina, 3 15 Arginine. 283 Arisaerm atroruberts, 192 treatment of ingestion of, 193 Arnrillariu ponderosa, mycorrhizal associations, 7 18 Annillaria, respiratory reactions, 765 Armilluriella nzellea, 719 Aromatic diazonium ions, 767 Alran pilot potato, teratogenicity of, 98 Arthrinm, 33 1 Alylhydrazines. 767 Aryl hydrocarbon hydroxylase (Ah) system, 55 Asclepiadaceae, 194, 309, 3 10 Asclepicrs spp., 77, 194, 308, 322, 331, 421, 447 eriocarpa, 194 ltrbrjformis, 194 treatment of ingestion of, 194, 421 Asia, 69 Asomycetes, 503 Aspergillic acid, 476

Index Aspergillus, 83. 85, 97, 31 1, 471-502, 786 animal infections, 478, 479 control, 492 dermatological infections, 478 descriptive morphology and terminology, 479 group descriptions, 480 human infections, 477 important mycotoxin-producing species of, 473 infections of eyes, ears, and nose, 478 morphology, 479 preservatives, use of, 495 pulmonary diseases, 479 reducing mycotoxin levels in foodstuffs, 495-497 reproductive organs, 478 respiratory reactions, 765 Section Aspergillus, 481 Section Candidi, 491 Section Circrrntdati, 49 1 Section Clnvnti, 484 Section Flali, 487 Section Flavipedes, 486 Section Fwnigati. 483 Section Nidulantes, 485 Section Nigri, 489 Section Restricti, 483 Section Terrei, 486 Section Usti, 485 Section Versicolores, 485 Section Wentii, 486 species associated with human disease, 477 species descriptions, 481 Aspergillus, control of gas composition, 494 nutrient status, 494 pH, 493 preservatives, 495 temperature, 493 water activity, 492, 493 Aspergillus javus, 83, 85, 487, 595 aflatoxin B, 487 cyclopiazonic acid, 487 Aspergillus japorzicus, 490 Aspergillus nlellezrs, 49 1 Aspergillus mycotoxins in foodstuffs: absorbents, 496 ammoniation, 496 biotransformation, 497 dilution, 497

807 [Aspergillus mycotoxins in foodstuffs] heating, 496 irradiation, 495 reducing levels, 495 sorting, 496 Aspergilltrs rtiger, 489 Aspergillus Ilonzius, 489, 595 Aspergillus ocltraceus, 488, 619 Aspergillus onzae, 332, 488 Aspergillus parasiticus, 83, 488, 595 Aspergillus soja,, 332. 489 Aspergillus fantarii. 489 Aspergillus terreus, 85 Aspergilloma, 478 Aspergillosis, 477, 786 invasive, 478 Asteracae (formerly Compositae). 252. 3 13, 314, 329,419 Asterophora species, 7 19 Astragalus, 68, 215, 218, 281. 305, 330, 421, 446 argilloplzilis. 215 cnnadensis, 21 5 membranecus, 68 miser, 215 rncemosm. 215 treatment of ingestion of, 215, 420 types of toxin in, 215 variants: oblorlgifollrs. 305 seroti~zus,305 Atkinsonella, 504 Atractylis. 314, 329 gumntifera, 313, 334 Atractyloside, 3 13, 329 Atropa belladonna, 39, 232, 422 treatment of ingestion of, 231, 422 Atropine, 39, 232, 233, 249. 365, 422, 424 Auricularia: aw-icula-judne, 786 polytriclta, 786-788, 790, 792, 795-796 Australia, 194, 197, 252, 280, 521 rumen microorganism modification in, 280 Austria, 250 Avena satila, 210, 41 9 infection, 535 ATA (see Alimentary toxic aleukia) Azadirachtin, 224 AzadirachLa indica, 224 Azaserine, 26 1 Azoxyglucosides, 333

808 Brrccltaris cordfolin, 3 15 Bercteroides fragilis, 68 Balansia, 504-505, 508-509, 5 11-5 13, 656 animal toxicity. 520 description of, 5 13 distribution in world’s crops, 51 1 ergot alkaloids produced by, 5 12 factors altering ergot production, 512, 514 Iterzrzingsinna, 508 hosts, 5 11 symptoms of toxicity, 520 toxins in, 512 Balansine, 658 Balkan endemic nephropathy (BEN), 620 Bamboo shoots, 47 Bcrrzdlern sirqdicfolia, 270 Bangladesh, 276 Barley infections, 535, 545-547, 548-549 Basidiobolus rarlnrzlrrr, 786 Battnrerr plrcrlloides, 787 Bnuhinict purpuren alba, 270 Bay leaves, 71 Beans: dly, 103 lima, 103 pllaseolus l m n t m (lima), 46, 47 snap, 103 velvet, 62 vulgaris (kidney), 37 Bear’s head (see Hericium erinacezts) Berrzr19erirr bassinna, 785, 787 Beech mushroom (see Hypsizygtls tessulatzw) Beer, potential contamination by: aflatoxins, 86 biogenic amines, 284 estrogenic substances, 85, 134 nitrate levels in commercial, 102 N-nitrosocompounds, 102 Belgium. 84 BEN (see Balkan endemic nephropathy) Benzo[e]pyrene. 636 Benzyl alcohol. 134 Berberidaceae, 196 Berberine, 225, 366 Bergamot oil, 50 Bergapten, 52. 53, 105, 107, 108, 112 acidic fog and, 107 in the Fabaceae, Rutaceae, and Apiaceae, 106 Berger’s disease. 614 Bertel-on irlcann, 422 treatment of ingestion of, 422

Index Betel wlgaris. 54, 200 treatment of ingestion of, 200 var. ciela, 54 Bhutan, 252 Biochanin A, 67 Biogenic amines, 284, 285 Bishop’s weed (see Amnri rmjus) Black forest mushroom (see Lerltirtula edodes) Black mushroom (see Lentirzh edodes) Black tree ears (see Auric~daria polytrichn) Blnstomyces clermntitidis, 786 Blastomycosis, 786 Bligh, William, 231, 278 Bliglricr snpida, 231, 278 treatment of ingestion of. 231 Boletm: CnlOpl4S. look-alikes, 734 chrysenteron, hydrochloric acid test, 746 corzferarzm, look-alikes, 734 edulis, 77 ammonium hydroxide test, 745 look-alikes, 734 mycon-hizal associations, 71 8 green vitro1 test. 746 rubirzellus. 7 16 scltunus, 764 zelleri: ammonium hydroxide test, 745 mycorrhizal associations, 7 18 Boraginaceae 197, 249, 252 Boston 250 Bovista : piln, 787 plurnbea, 781 Bowman-Birk inhibitor, 261 amino acid sequence of. 261 destruction by heat, 265 Bracken, 314. 328, 332. 333 thiaminases in, 328 Brcrdyrhi,-obiztm jnponicuru. 63 Bmssicn, 54, 198, 326, 422 canlornpa, 54 hirta, 54 napus, 54 nigra, 54 olerocen: var. acephnla, 54 var. bottTtis, 54 var. capitnta, 54 var. gemmifern, 54, 55 var. rmpobrussiccl, 54

Index [Brrtssica] pekinensis. 54 r a p , 54 treatment of ingestion of, 422. Brassicaceae (formerly Cruciferae), 53, 198, 313, 326, 333,419 Brazil, 88. 252, 315 Breeding safe varieties vegetables, 108 Brick top (see HyAolonln sublateritium) Britain, 229 British Columbia, 305 Broccoli, glucosinolates in, 43 Brunfelsiu pancijorn var. jloribunda, 232 treatment of ingestion of, 233 Brussels sprouts, 54, 55 Bufadienolides. 202, 213, 23 I , 308, 33 1, 371 Bldo, 308 Bulgaria, 84 Buna-shimeji (see Hypsizygus tessulntus) Buthionine sulfoximine, 636 Butylated hydroxyanisole, 636 Butylated hydroxytoluene, 636 Buxaceae, 199 Buxene, 199 Buxus sempenirerrs, 199 Cabbage, 55 Cadaverine, 285 Caesalpinoideae, 189 Cnjarms cajan, 47, 259, 266 Caladiwn, 193 treatment of ingestion of, 193 Calcinogenic glycosides, 3 15, 334 Calcinogens, 328 Calcinosis. 3 15 Calcitrol (la,25-dihydroxy vitamin D7). 315 California, 51, 107, 191, 252 Callilepsis, 3 14 lunreoln, 334 Callosobruchus maculatus, 269 Caltha palnstris, 227. 445 treatment of ingestion of, 228 Calvacin, 787 Cuhtia: craniforntis, 187 cynnthiformis, 787 gignntea, 787 lilucina, 787 respiratory reactions, 765 utriformis, 787 Calystegine B, 310

809 Campanulaceae, 199, 253 Canada, 84, 87, 88, 231, 232, 325, 442 Canadian Association of Poison Control Centers (CAPPC), 10 Canmdin ensiformis, 259, 270, 272, 282. 283 Canavarine, 283 Cancer of the: bladder, 57 bowel, 50 breast. 56, 57 cervix, 57 colon, 56, 57 esophagus, 57, 88, 89, 134 gastric system, 56, 203 liver, 85, 89, 134 lung, 57 oral cavity, 57 ovary, 57 pancreas, 57 prostrate, 56, 84 stomach, 57, 133 Cancer, promotion of, 57. 90 Cundida albicnns, 786 Candidiasis, 789 Cannabinaceae, 199 Cannabinoids, 199 Cannabis sativa, 199 treatment of ingestion of, 200 Cunthnrellus. 77 look-alikes, 734 mycorrhizal associations, 7 18, 720 Caparidaceae. 3 13 Capaurine. 209 Capparales, 3 13 CAPPC (see Canadian Association of Poison Control Centers) Capsidol, 99 P-Carboline alkaloids, examples of, 359 Carboxyatractyloside, 196, 313, 329, 435, 445 Carcinogenicity modulation, 55 Carcinogens, 328 Cardenolides, 194, 308, 322, 33 1, 371, 42 1 Cardiac glycosides, 229, 232, 300. 308, 421, 425, 430, 447. 449 Cardiovascular glycosides, 321 Cnslimt gummifera, 3 13 Carotatoxin, 43 Caryophyllaceae, 200 Cassava (manihot), 48, 132, 307. 332

810 Cassirr spp.. 61, 69. 218, 330, 422 alrgustijiolia. 218 mriculata. 69 obtusifolia, 61 occidentnlis, 422 roemeriam. 448 treatment of ingestion of, 422 Crrstc~nosper~lrurIr alrstrale, 303 Catechin(s), 60. 62, 64, 66 Cauliflower, 55 Cauloplryll~rnrtlrnlictroides, 196 CDC (see Centers for Disease Control) Celery, 107 breeding project, 108 cultivars, 109 linear furanocoumarins in, 107 photosensitization by, 107 Centaurer1 spp.: reperzs, 423 solstitiatus, 423 treatment of ingestion of, 423 Centers for Disease Control (CDC), 105, 108 Central America, 523 Central nervous system disorders, 653 Centrifugal TLC, 353 Cephaeline, 230 Ceplrnelus ipecacuanha, 230 Ceratocephalrts testiculatus, 325, 445 Ceriowryces, green vitro1 test, 746 Ceroyithecus pygentllzts, 89 Cestfrm dilrrmrm 315,329, 423 treatment of ingestion of, 423 a-Chaconine, 93-98,133 Clrnnlrrcristcrfasiculata, 259 Chamomile, 71 Chanoclavine I. 657, 661 Chanterelle mushrooms, 734 Cheilanthes sieberi, 315 Chelidonium majrls, 225 Chenopodiaceae, 200, 419 Chenopodiltnr spp.: dbLt~1,200 anrbrosiodes, 77 treatment of ingestion of, 201 China 68,82, 83.85,101, 129, 320 Chinese caterpillar fungus (see Cordyceps sinensis) Chinese mushroom (see Lenti~rrdaedodes) Chinese sclerotium (see Polyporus z m bellntus) CHISPA (see Chronic idiopathic spastic paraparesis)

lndex Chlorogenic acid 43,45, 62, 91 Cl~loroyhylhonmolybdites, 220 chemical test. 745-746 look-alikes, 734 poisonings, 763 respiratory reaction, 765-766 treatment of ingestion, 220 Cholesterol, 324 Cholinesterase inhibitors, 93 Chorei-maitake (see Polyporrds unzbellatus) Chromoblastomycosis, 786 Chronic idiopathic spastic paraparesis (CHISPA). 613-613 toxic mechanism, 612 Chymotrypsin, 261 CiBarius spp., 77 Cicer crrietirzlan, 47, 359, 266 Cicuta spp., 236, 445 douglasii, 336, 423 i?ruculata,423 treatment of ingestion of, 236, 423 Cicutoxin, 236, 423. 445 Cimetidine, treatment for atnatoxins, 753 Citrinin, 83, 475, 629 carcinogenicity, 637 interaction with: chromate, 633 diethylmaleate, 637 hexachloro-l,3-butadiene.633 iron, 633 ochratoxin A, 630 Citrus fruits. 50-53, 132 concentrations of d-limonene in, 52 Citrus berganrin. 50 Cladosporiunr; carrioni, 786 respiratory reactions, 765 Claviceps, 87-88, 503-524, 656-657, afiicam, 505-509, 523, 657 animal toxicity, 517 nnnulata, 506 citrinu, 506 cinerea, 506 concentrations. 516 control of infection, 521 cynodorrtis. 506 cyperi, 506 devastation of Europe by, 50 diadenza, 506 digitnriae, 506 ergot alkaloids produced by. 512 factors altering ergot production, 512,514

660

Index

81 1

[Claviceps]

[Clitocybe]

Jlavella, 506 fusiformis, 505 gigantia, 506 glabra, 506 grohii, 506 ltirtella, 506 hosts, 514 human toxicity, 517 inconspicua, 506 infection by, 505, 509 junci, 506 litoralis, 506 livestock toxicity from, 518 lutea, 506 masimensis, 506 microspora, 506 nigricans, 506 ortlzocladae, 506 prtspali, 505-507, 509, 656 phalaridis, 506 platytricha, 506 purpurea, 87-88, 505-509, 523, 594, 656-657, 783, 787 pusilla, 506 queenslandica, 506 ranunculoides, 506 sorglzi, 505, 509 sulcata, 506 toxins in, 512 tripsaci, 506-507 uleana, 506 viridis, 506 world’s species of and hosts of, 506 yanagawaensis, 506 Clavicepitaceae, 503-505, 524 biology of, 505 Claviceps, infection by, 505 important genera in, 504 miscellaneous toxins in, 516 pyrrolizidine alkaloids in, 5 16 toxicity of, 517 Clavine alkaloids, 359, 515 examples of, 359 structures of, 5 15 Clematis spp., 228, 319 virginiana, 228 Clinical signs of glycoside poisoning, 320 Clitocybe: clavipes, 760 dealbata, 758 illudens, 716, 762

mycorrhizal associations, 7 18 olilpascens, 762 truncicola, 758 Clittoria ternaten, 259 Cloud mushroom (see Grifola frondosaj Cocaine, 365 Coccidiodomycosis, 786 Coccidioides imnritis, 786 Cocoa powder, 43 Codeine, 250, 366 Colchicine, 220, 221, 249 Colchium autunmale, 220, 253 Collybia vellrtipes (see FhnmuIina sehtipes) Colocasia anti quo run^, 193 Colombia, 28 1 Colorado, 71, 253 Comfrey, 70, 197, 250 Committee on Food Protection, 39, 136 Compositae (see Asteraceae), 252, 313, 314, 329,419 Concanavalin A tetramer 269, 271 y-Coniceine, 250, 252 Coniine, 236, 250, 252, 423 Conium maculatum, 236, 250, 252, 357, 423, 445 treatment of ingestion of, 423 Conocybe: cyanescens, 761 cyanopus, 76 1 Consumer Product Safety Commission (CPSC), 2 Convallamarin, 221 Convallaria majalis, 221, 423 treatment of ingestion of, 221, 423 Convallarin, 22 1 Convallotoxin, 221 Convicine, 219, 3 19 Convolvulaceae, 253, 3 10 Convovulus arvensis, 253 Convulsive ryegrass staggers, 656 Committee on Food Protection, 39, 136 Cooperia pedunculata, 423, 436 treatment of ingestion of, 423 Copelandia: cambodginiensis, 762 cyanescens, 762 papilionacea, 762 Coprine mushroom poisoning, 759-760 mechanism of action, 759 symptoms, 759 treatment, 759-760

812 Coprirw: atramentarius, 760, 788 comcctus, 788, 790. 795 insignis, 760 lagopus, 716. 726 micnceus, 726, 760 radiatus, 7 16 stercorarius, 7 16, 726 Coprophagic mushrooms, 719 Cordyceps, 719, 788 robertsii, 788 sinensis, 788 Coriolopsis polyzona, 784 Coriolrss Ilersicolor, 788 Corn, infection of, 535, 541-544, 548-549, 552-557 Corortilla spp., 305 Corticosteroids, treatment of amatoxins in, 753 Cortinarius: cinnamoneus, mycorrhizal associations, 718 collinitl~~, mycorrhizal associations, 7 18 gentilis, 754 herpeticus, 745 orellanus, 754 pistillarin test, 746 speciosissimus, 754 Corydalis spp., 209, 423 aurea, 209 caseana, 209 treatment of ingestion of. 423 Corynocarpaceae, 201, 305 Corynocarpus laevigatus, 201, 304 Cotyledon orbiculata, 202 Coumarins, 50 Coumestrins, 317 Coumestrol, 63, 66, 67, 317 CPSC (see Consumer Product Safety Commission) Crassulaceae 202 Craterellus cornucopioides, 733 Crofalaria spp., 69, 216, 25 1, 354, 424, 443 juncea, 69, 251 spectabilis, 21 6, 443 treatment of ingestion of, 423 verrucosa, 69 Cruciferae (see Brassicaceae) Cruciferous vegetables, reduction of cancers by consumption of, 57 Cryptococcosis, 786

Index Cryptococcw neoformnns, 786 Cucurbitaceae, 202 Cucurbitane triterpene glycosides, 763 Cupressm me~crocelrpc~, 449 Cuscohygrine, 254 Cyanlopsis tetlngonoloba, 259 Cyanide, 46, 41 9 potential of selected foods, 47 Cyanidin, 606 3 (or P)-Cyanoalanine, 277, 278, 434 Cyanogenesis, 46 Cyanogenic: compounds, 132 foods, 46 glycosides, 210, 230, 232, 300. 305, 330, 368,432,434.444 Cyanogens, 321 Cyanophyceae, 253 Cycadales, 319 Cycads, 49, 333 Cycasin, 49, 225. 3 19, 324, 328, 331, 333. 424 Cycas spp., 278, 319, 328, 333, 424 circinalis 278 treatment of ingestion of, 424 Cyclopamine, 435, 448 Cyclopeptide mushroom poisoning, 749754 mechanism of action, 750 symptoms, 749-750 treatment, 75 1-754 Cyclophosphamide, treatment of orellanine poisoning in, 754 Cyclopiazonic acid, 83, 472, 475, 629 interaction with: patulin, 629 T2 toxin, 629 Cyclopsine, 435 Cygnoglossum spp., 252, 354, 424, 447 oflcinale, 252, 424, 447 treatment of ingestion of, 424 CJwtopterzu watsonii, 436 treatment of ingestion of, 435 Cynodon dactylon, 210, 424 treatment of ingestion of, 210, 424 Cysteamine, treatment of amatoxins in, 753 Cytisine, 21 7, 21 9, 28 1, 284, 434 Cytisus scoparius, 2 16 Cytochalasin E, 637 carcinogenicity, 637 interactions with macrophages, 637 interactions with vincristine, 637

lndex Daidzein. 62-64, 3 17 Daidzin, 62, 317 Dnldirtin comentrica, 788 Dancing butterfly mushroom (see Grifola fiondosa) Daplzm ntezeru~tt,235 Daphnetoxin, 235 Daphnia magna, 43 DAS (see Diacetoxyscirpenol) Datura spp., 61, 233, 248. 249, 252, 365, 424, 445 irtoxia, 249 snuveolerts, 249 stramonium, 61, 233, 248, 424. 445 treatment of ingestion of, 424 Dehydroalanine. 284 Dehydrotremotone, 41 microsomal activation of, 41 Delphinidin, 62 DelphhiwI spp., 228, 253, 425, 437, 446 bnrbeyi, 253, 443, 446 bicolor, 228, 446 brownii, 228 geyeri, 446 glaucescens, 446 g l m c u ~ t446 ~, nuttallianw~l.228, 446 treatment of ingestion of, 228, 425 tricorne, 446 Demecolcine, 220 Demissidine, 93 Denmark (Danes), 250 Dennstaedtiaceae, 203 Dentirturn repandwn, mycorrhizal associations, 718 Deoxynivalenol (DON), 83-84, 87, 537, 539-540, 546-550, 555, 611-613, 617, 629 genotoxicity, 550 human IgA nephropathy, 614 in scabby grain intoxication, 546-549 interaction with: fumonisin, 630 nivalenol, 630 selenium, 634 T2 toxin, 630 tryptophan, 638 zearalenone, 630 LDso of. 549 mutagenicity, 550 sources, 539. 546 structures, 669

813 [Deoxynivalenol (DON)] teratogenicity, 550 tolerance level, 550 toxic concentrations, 6 12 Dernzocybe sanquinea, 766 Derris elliptica, 303 Deterrol. 768 Dhurrin, 21 1, 306 Diacetoxyscirpenol (DAS), 537-539, 543544, 611-612 animal poisoning, 544 binding with calcium aluminosilicate clay, 635 carcinogenicity, 544 concentration in feed grain, 543 dietary fat effects, 639 interaction with: ochratoxin A, 630 fumonisin, 630 Salmonella liposaccharide, 632 sources, 539, 543-544 Diallyl sulfide, 71 2.4-. Diaminobutyric acid, 277, 278 3-Diazotyramine 101 Dicentro spp.. 210, 445 cucullaria, 210, 445 exirtlia, 2 10 Dichapetalaceae, 203 Dichapetnlum c y n o s ~ m203 , Dicoumarol, 329, 332 Dieffeenbachia sequine, 193 treatment of ingestion of, 193 Dietary flavonoids, 61 Dietary nitrate intake, estimated, 102 Diethylstilbestrol (DES), 63, 66. 84 Digitalin, 308 Digitrdis purpurea, 232, 308, 425 Digitoxigenin, 191 Digitoxin, 232, 323 Dihydroelymoclavine, 660 1,25-Dihydroxycholecalciferolglycodide, 423 3,4-Dihydroxyphenylalanine, 280, 28 1 Di(2-hydroxypropyl) nitrosamine, 261 3,4-Dihydroxypyridine, 279 3,3’-Diindolylmethane, 56 induction of Ah receptor, 57 3,5-Dimethoxyphenol, 250 Dimethylergotamine, 594 Diplodia maydis, 212 Dioscoraceae, 204 Dioscorea spp., 204, 3 11

814 Dioscorine. 204 Diplodia toxin, 83 Dipropenyl disulfide, 420 Dipropyl sulfide, 420 Direct insertion probe mass spectrometry (DIPMS), 353 Diterpene(s), 425, 446 alkaloids, types of C I 4aconitine, 364 C I Ylycoctonine, 364 C:,, atisine, 364 veatchine, 364 Diterpenes, examples of toxic, 379-380 Diterpenoid glycosides, 3 13 Divicine, 3 19 Djenkolic acid, 280 Dolichis: biJorus, 259, 270, 272 lnblab, 259, 272 DON (see Deoxynivalenol) Donku (see Lmtinula edodes) Dopa, 281 Droplet countercurrent chromatography CDCCC). 353 Durarztn repens, 237 Ear fungus (Auricularia polytricha) Earthballs, 736 Earth maitake (see Polyporus urnbellatus) East Africa, 84 Ecgonine bases, 206 Echimidine,197 Echinodothis, 504 Echiumine,197 Eclzium spp., 197, 354 plantngineum, 197 Eggplant, 98 Egypt, 82 Elaphomyces, 503 grarzulatus, 788 ELEM (see Equine leukoencephalomalacia) Eleutherococcus spp., 31 1 ELISA, 689-693 Elymoclavine, 657, 66 1 Emetine, 230 Endemic pannlyelotoxicosis, 542 Endophytes (see Grass endophytes), 509 Endophytic fungi. 504-505, 509-513 control of, 522-523 infection benefits, 5 11 Endoscopic biliary diversion, treatment of amatoxins, 753

lndex England, 82. 250, 272 warning label on kidney beans in, 272, 273 Enoki (see Flammulina velutipes) Enokidake (see Flalnmulina velutipes) Enokitake (see Flamntulirm velutipes) Entoloma: lividurn, 763 rhodopoliwn. 758 Entomophthoromycosis, 786 Ephedra viridis, 204 Ephedraceae, 204 Ephelis state, 505 Ephidrine, 204 (-)-Epicatechin, 62 Epichloe, 504-505, 508, 510, 656 infection by, 510 typhinia, 508 Epidemiological studies, 56, 133 Epidermophyton jloccosurn, 786 Epinephrine, 64 Epiphytic fungi, 505, 509 Equine leukoencephalomalacia (ELEM), 88, 89, 90 Equisetaceae, 204 EquisetLrrn arvense, 204, 425 treatment of ingestion of, 205, 425 Equol, 63, 67 Ergocornine, 87 Ergocristine, 87, 656 Ergokryptine, 87 Ergonovine, 87, 657, 660-661 Ergopeptam alkaloids, 656 Ergopeptine alkaloids, 367, 656. 659 Ergosine, 87 Ergot alkaloids, 83, 86, 358, 367, 503-504, 507-508,511-512, 514,523, 594, 655-656, 658 analytical methods. 655, 661-665 animal toxicity, 5 17 and cattle, 88 concentrations. 5 16 extraction from sources, 661 factors altering production, 512, 514-515 future prognosis for, 523 high-performance liquid chromatography of. 661 human toxicity, 517 lolitrem toxins, 510, 5 16 mass spectrometry of, 663 and other toxins. 514 photographs of, 508

Index [Ergot alkaloids] produced by Balarlsin spp.. 512 produced by Claviceps spp., 5 12 produced by NeoQphodium spp., 512 production of, 507 sources, 594 structure, 658-659 synonyms, 507 thin-layer chromatography of, 662 Ergotamine, 87, 367, 656 Ergotism, 504. 594, 656 history of, 504, 594 animal toxicity, 504, 5 17 human toxicity, 517. 594 Ergotoxine. 656 Ergovaline, 367, 660 Ergoxine, 656 Ericaceae, 205 treatment of ingestion of, 205-206 Eriocarpin. 194 Erythroxylaceae, 206 Enthrina corallodendron. 272 Etvtllro.xylum coca, 206 Esophageal cancer, 537, 551-553. 556-557, 613-615, 619, 653 causes, 552 clinical symptoms, 552 control of fungal causes, 557 fumonisins in, 55 1-552 incidence, 6 13 in humans, 537 mineral deficiency, 551, 553 relation to mycotoxins. 551 vitamin deficiency and. 551-553, 614 Ester(s), 300 alkaloids, 222 Estradiol, 327 Estriol, 327 Estrogenic agents, 84 Estrone, 327 Ethanol, treatment of amatoxins in, 753 Ethiopia, 276 Eugenol, 43 Ei!patorilcm rtigosum, 39, 194. 425, 448 treatment of ingestion of, 195, 425 Euphorbia spp., 207, 425 marginata, 207 treatment of ingestion of, 207, 425 Euphorbiaceae, 207 European Alps, 3 15 Europine, 197 Exophialn jenrtselwlei, 786

815 Fabaceae (formerly Leguminosae). 105, 189, 215, 251. 305 Frrbn vulgaris, 259 Fagaceae, 209 Fagopyrin, 226 Fogopyrunt spp., 226, 436 escdentunt, 436 sagittaturn, 226 treatment of ingestion of, 226 Fairy rings, 782 Falcarinol. 427 False morels, 734-735 False truffles, 734 Favism, 319, 334 Fengweigu (see Pleurotus sajor-cnju) Fescue (endophyte-infected): costs of summer toxicosis, 519 other compounds in, 517 symptoms in cattle, 519 symptoms in horses, 5 19 toxicities of, 518 Fescue toxicosis, 5 18-520 Festucn spp., 88, 210, 510 armdinaceae, 88, 210 treatment of ingestion of, 21 1 Festuclavine, 660 Fetal abnormalites, induced by mycotoxins, 86 a-Fetoprotein, 67 Ficus ccwica, 112 dermatitis from, 112 Flags, 112 Flammulin, 788 Flarmulinn velutipes: medicinal uses, 788-790, 792, 796 mycorrhizal associations, 7 18 Flavanoligans, 752 Flavanolols, 60 Flavanones, 60 Flavones. 60 Flavonoid(s). 59 biological effects of, 64 cancer and, 66 dietary, 61 general structures of, 60 Flavonols, 60 Florida, 88, 3 15 Fluoroacetate (monofluoroacetate) ion 203. 216, 230 Folinerin, 191 Fowzes: fonzentarius, 789 oflcinalis, 789

816 Fonsecnea pedrosoi, 786 Food(s): cyanide potential of selected, 37 cyanogenic, 46 estrogenic agents in, 84 global perspective of safety of, 82 handlers and dermatitis, 108 linear furanocoumarins in selected food plants, 52 nitrate-rich,100 oxalate-rich, 113, 115-1 18 safety and public health hazard, 83 tannin-rich, 122. 125-129 toxicology of naturally occuring chemicals in. 37-186 Food poisoning from mushrooms, 763 Food refusal, 549-550 Formonoetin. 63, 327 Foxfire, 782 France. 82, 86. 129, 214. 220, 517 Free radical scavengers, 64 Fruits and vegetables (flavonoids), 59 Ftrligo respiratory reactions, 765 Fumareaceae, 209 Fumarocoumarins, limes and, 53 Fumitremorgens. 476 Fumonisin mycotoxins, 82, 88, 90, 134, 541-542. 555-557, 594, 613-617, 629, 669 analysis of, 90, 542, 555 animal disease, 615 human disease, 61 5 in esophageal cancer, 551, 555, 613-614, 616 interaction with: methylbenzylnitrosamine, 638 ochratoxin A, 630 sources of, 542 Fumonisin B biological activity of. 90 ELEM and, 90 Fumonisin B mycotoxins. 538, 542, 554, 613, 616, 669-670 analytical methods, 670 binding with calcium aluminosilicate clay, 635 clinical symptoms, 615 enzyme-linked immunosorbent assay, 670 extraction methods. 670 high-performance liquid chromatography, 670

Index [Fumonisin B mycotoxins] interaction between: fumonisin and deoxynivalenol, 630 fumonisin and diacetoxyscirpenol, 630 fumonisin and moniliformin, 630 fumonisin and T2 toxin, 630 fusaric acid and fumonisin B 1, 630 structures. 669 toxic concentrations, 616 Furrgus sardulci. 789 Furry foot Collybia (see Flamnztrlina velutipes) Fusarenon-x, 540, 547, 611 Fusaric acid, 669 structure, 669 Fusarin C, 89. 541 carcinogenicity, 554, 556 esophageal cancer, relation to, 541 source of, 541. 554 Fusariotoxicosis, 612. Fusarium, 85, 535-558, 61 1-612, 614-616. 618, 669, 786 crcunlirmturn, 536. 539. 541 a~1tltophilurtt,542. 554 avenacelm, 536, 541 crookweller~se.536, 539-540, 547-548. 550. 618 cll~tlorl~t~l, 536, 539-541, 546-548, 550, 618 cllamini, 542, 554 episphaerin, 539 equiseti, 536, 539-541 globoslrm, 536, 542 granlirzear~an,536-537, 539-540, 545550, 614-615, 618 infected grains, 536 lquslzuense, 540, 547-548 morzilifornze, 82, 85, 88-91, 536-537, 541-542, 551-556, 614-616 Izapiforme, 542. 544 r1ivale, 547 rlygamai, 536, 541 -542, 554 oxysporwn, 536, 541 -542 pone, 536, 539-540, 543-545, 547-548, 612 I’ol?,pkinlic(icurn, 542 prolifemtunl, 536, 541 -542. 554 roseum. 537, 539-540 sanlblrcinml, 536. 539 semitectwn, 6 18 sporotrichioides, 536-541. 543-545, 547, 612

Index [Fusnriurtz] subglutinrrns, 536, 541, 554 sdphureum, 539 thapsinunt, 536, 542, 554 tricinctum, 537-538 verticilliodes, 537 FUS C (see Fusarin C) F~ucoboletinus ochrmeoroseus,7 18 Galega oficinalis, 426 treatment of ingestion of, 426 Galegine, 196, 378, 426 Galerirza autumnalis, 753 Galiotoxin, 42 1 Gallic acid. 449 Gallotannins. 123, 129, 209, 432 Gangrenous fescue foot, 656 Ganoderma: lucidun1, 768, 783, 789, 791-792, 795 tsugrre. 789, 795 Garlic, 70, 133 Gas chromatography, 352 Gastrointestinal decontamination agents, common, 417 Gastrointestinal disorders, 653 Gastrointestinal mushroom poisoning, 762764 mechanism of action. 763 symptoms. 762-763 treatment, 763-764 Grrstrolobiunt grmdijlorum, 21 6 Geastrwn; medicinal uses. 789 respiratory reactions, 765 Gelsemine, 222, 426 Gelseminine, 426 Gelsemium semper-virens, 222, 426 treatment of ingestion of, 426 General structures of flavonoids, 60 isoflavonoids, 63 Genetic engineering, potential problems with, 136, 276 Genistein, 62, 63, 66 Genistin. 62. 64 Georgia, 84 Geraniin.123 Gerarliunz tlzurzbergii, 123 7-Geranoxycoumarin, 52 Germ-free animals and lectins, 273 Germany, 86, 114, 250 Gitalin, 232

817 Githagenin. 200 Gitoxin, 232 Gloriosa superha, 221, 249 Glucobrassicin, 55, 57, 369 P-Glucosidase(s), 62, 301, 328 inhibitors of, 303 Glucosinolates, 300, 312, 369, 422 in broccoli, 43 in Brussels sprouts, 54 in cruciferous vegetables, 54 L-Glutamic acid, 78, 283 Glutathione (GSH), 71 Glycine max, 54, 259, 270 Glycoalkaloids, 95, 222, 234, 433 potato tubers content of, 95 Glycosides: aliphatic nitrotoxins, 330 anthraquinone, 226, 330 calcinogenic, 3 15 carcinogens, 328 cardenolides, 322 cardiac, 194, 229, 232, 308. 331, 423, 430,447,449 cardiovascular, 32 1 clinical signs of, 320 coumarin, 329,430 cyanogenic, 210, 230, 232, 300. 305, 330, 368,432,434,444,449 1,25-dihydroxycholecalciferol,423 diterpenoid, 3 13 gastrointestinal effects, 323 glucosinolate, 312, 325 hepatic effects from, 323 human poisonings by, 330 methylazoxymethanol, 3 19 mode of action of, 320 mustard oils, 326 neurotoxic, 320 nitriles, 326 other, 329, 375 phenanthrene, 227 phenolic, 3 16, 370, 371 pyrimidine, 3 19 convicine. 3 19 vicine, 3 19 ranunculin, 318 reproduction consequences of, 327 saponin, 309 sesquiterpene, 314 specific disorders induced by, 328 steroid, 372 toxic, 330

Index

818 [Glycosides] transfer to meat products, potential, 330 transfer to milk, potential, 330 treatment of livestock after exposure to, 320 Glycosidic bond: hydrolysis of, 301 Goiter, 332 Goitrin, 102, 313 Goitrogen(s), 53, 313, 325 potential transfer to milk, 331 Goitrogenic compounds, plants containing, 54 Golden mushroom (see Flarnrnulina velutiyes) Golden oyster mushroom (see Pleurotus citrinopilentus) Gonzphidius: glutinosus, mycorrhizal associations, 718 rutilus, mycorrhizal associations, 718 subroseus, mycorrhizal associations, 7 18 vinicolor, mycorrhizal associations, 718 G o ~ ~ p hJloccosus us look-dikes, 734, 763764 Gossypetin, 64 Gossypim spp., 77, 223, 426 barbadense, 223 hirsutum, 223 treatment of ingestion of, 426 Gossypol, 43, 45, 223, 426 Grain, contamination of, by: Brassicaceae.198 Crotalaria spp., 216 ergot, 86, 87 flavonoids, 61, 65 Gramineae, 210 Grass endophytes, 510 control and management of, 522 endophytic spp., 509 epibiotic spp., 509 Graveolone,110 Grayanotoxins, 205-206, 428, 431, 432, 449 Green peppers, 99 Green vitriol test, 746 Grifola frondosn, 787, 790-791 Guam, 49, 278 Gutierrezin spp., 62, 67, 427 microcepltalt, 62, 448 sarothrae, 448 treatment of ingestion of, 427 Gymlocladus dioica. 2 16

Gynnopilus: aerugirlosus. 761 spectabilis, 761 Gynocardin, 306 Gypsogenin, 200 Gjpsophila spp., 311 Gyromitra: esculenta, 77, 80, 757, 768 compounds in. 80 hydrazine analogs in, 134 gigas, 757 injkla, 757 look-alikes, 734 poisoning by, 756-757 Gyromitrin, 79-8 1 Halogeton spp.. 201. 443 glomeratus, 201, 427, 445 treatment of ingestion of, 201. 427 Haplopappus spp.. 41. 427. 428, 448 heterophyllus (formerly Isocorna wrightii), 41, 428,448 temtisectus, 448 treatment of ingestion of, 427, 428 Hawaii 192, 279, 280 HCN (see Hydrogen cyanide) Head blight, 545 Hebeloma: crustulinifor~ne,763 vinosophyllun?. 763 Hebelomic acid A, 763 Hedeoma spp.. 77. 213 oblongifolia, 77 yulegioides, 2 13 treatment of ingestion of, 214 Hedern heli.x, 427 treatment of ingestion of, 427 Hedgehog mushroom (see Hericiunl erinaceus) Helenalin, 379 Heleniunz spp., 427 azttuntnale, 444 Izoopesii. 444 treatment of ingestion of, 427 Heleurine, 197 Heliotrine, 69, 70. 197 Heliotropiun? spp., 69, 197, 249, 354 amplexicartle, 197 eichwaldi, 249 lasiocarpurn, 69 Helleborus spp.: foetidus. 229 niger. 228

Index Helleborin, 229 Hellebrin, 229 Helleborus, 229, 3 19 Helvelln: elastica, 757 look-alikes, 734 poisoning, 756-757 Hemiacetals, 300 Hemolytic mushroom toxins, 766 Hen of the Woods (see Grifola frondosa) Herbal(s). 67 abortifacients, 74 adulterated preparations of, 68 with aminopyrine, 69 with phenylbutazone, 69 Asian medicinals, 68 mutagenic compounds in, 68 bay leaf, 71 diverse biological activities in, 69 medicines, 249 psychoactive substances in, 77 pyrrolizidine alkaloids in, 69 rosemary and sage, 74 teas, 71, 250 chamomile, 7 1 sassafras root, 71 yarrow, 71 Hericium: abietis, mycorrhizal associations, 718 cortrlloicles. 784 erinnceus, 790-792, 795-796 Hia tsao tom tchom, 788 High-performance liquid chromatography (HPLC), 352 Hippocastanaceae. 212 Hiratake (see Pleurotus ostreatus) Hirneola trwicula-judae, 790 Histamine, 237, 285 Histoplnsnln ctrpslllatw?t,786 Histoplasmosis, 786 Hog tuber (see Polyporus ~o?rbellat~s) Holart-henu mzticlyseiltericn, 69 Holland, 104 Honeydew, 507-508 Honey tainted by: atropine, 249 grayanotoxins, 206 Hordenine, 378 Hordeum vdgare (see Barley) Hormesis, 132 Houbitake (see Pleurotus sajor-caju)

819 HT-toxin, 61 1, 613 interaction with T2 toxin. 630 Human IgA nephropathy, 614 Human poisonings. by glycosides, 330 H w a crepitnns, 207 Hyalohyphomycosis, 786 Hyderestrogenism, 537, 540, 550 in animals, 537, 540, 550 in humans, 550 Hydmm irnbrictrtum mycorrhizal associations, 718 Hyclrtmgea macrophyllcr, 232 Hydrangin, 232 Hydrazine(s), 43, 79, 80, 81 LDso of, 80 Hydrazine carcinogenicity, 768 Hydrochloric acid test, 746 Hydrocyanic acid (see Hydrogen cyanide) Hydrogenated oils. role in cancer and aging. 44 Hydrogen cyanide (HCN), 46, 48, 49, 208. 305, 307, 321 3-Hydroxy-4( 1H)-pyridone (DPH), 21 7 Hydroxylubimin, 9 1 7-Hydroxy-6-methoxycoun~arin,50 4-Hydroxymethylphenyl hydrazine, 78 7-Hydroxymyoporone. 122 a-Hydroxynitriles. 32 1 Hydroxytremetone, 41 Hygrine, 254 Hygrophoropsis a~trcaztiaca.734 Hygroyhorous chr-ysodon, mycorrhizal associations, 7 18 Hymenovin, 444 Hymenoxin, 379, 444 Hvwleno.xvs spp., 427, 444 odoratti. 427, 444 riclzardsonii, 443 treatment of ingestion of, 427 Hyoscine, 233, 365 Hyoscyamine. 39, 233, 234, 365, 425, 445 Hyoscyanlus spp., 249, 365 Hypaconitine, 25 1 Hypericaceae. 2 12 Hypericin. 2 12 Hypericum performam, 212, 436 treatment of ingestion of, 213 Hypersensitivity reactions, 764-766 Hypocoeris rndicntn, 427 treatment of ingestion of, 427 Hypocreales, 503

820

Hypoglycins A and B, 231, 278, 279 structure of, 279 Hyplroloma sdhteritiunl, 790-792, 795 Hypomyces, 7 19 Hypsizygus: tessdrrtus, 787, 790, 795-796 ulrmrilts, 79 1. 795 Ibotenic acid poisoning, 754-756 symptoms of, 754-755 mechanism of action, 755 treatment, 756 ICZ (indolo[3.2-b]carbazolej, 57 Idaho, 253 IdB 1016, 752 Idiopathic cardiopathy, 558 11' mak (.see Pleztrottls citrinopileatus) Illuden S, 762 Incanine, 198 India, 82, 190, 249. 505 Indian childhood cirrhosis, 633 Indigofera spp., 282, 283. 305 spiccttu. 282, 283 3-Indole acetic acid, 517 Indoleacetic acid 104, 5 13 Indole alkaloids, examples of, 359 Indole-3-carbinol, 56, 632 interaction with aflatoxin B1, 632 Indole ethanol, 517 Indoles, simple, 517 Indolizidine alkaloids, 215. 21 8. 446 Indolo[3.2-b]carbazole (ICZ). 57 3-Indolylacetonitrile, 56 Indolylmethylglucosinolate, 55.56 enzymatic hydrolysis of, 56 Indolylmethyl isothiocyanate, 56 Indospicine, 283 Inhibitor(s): chymotrypsin. 262 of P-glycosidase, 303 elastase, 262 trypsin. 262 Inocybe: Lacera, 758 pudica, 758 Integrated breeding and environmental chernicals (IBEC) strategy, 108, 136 Intermedine.197 1.4-Ipomeadiol, 1 18, 121 Ipomeamarone,118,121 Ipomeanine, 118, 121

Index 1-Ipomeanol,118,121 4-Ipotneanol.118. 121-122,132, 134 Ipomoea spp., 62. 428 bntrttas, 118. 120 asthma and, 122 average concentration of lung toxin in, 120 chemicals isolated from, 119 effects of pathogens on chemicals in, 120 phytoalexins from stressed, 119 treatment of ingestion of, 428 Iridaceae. 213 Isocomrr wrightii (see Haplopappus heterophylllls) Isocorydine, 366 Isocupressic acid, 431, 449 Isoflavones, 327 Isoflavonoid, general structure, 63 Isoimperatorin, 110 Iso-neosolaniol, 613 Isopimpinellin, 52, 53, 107 acidic fog and. 107 Isoprenoids, examples of toxic, 380 Isoquinoline alkaloids. 209, 210, 225, 366, 423, 445, 437 Isosparteine, 2 16 Isothiocyanates, 55, 56, 198, 302 allyl, 55, 198 arylalkyl, 55 indolylmethyl, 56 Isouramil. 3 19 Ivu alcgmt(folia,428 treatment of ingestion of, 428 Jamaica, 278 Japan, 101, 103, 133, 251. 333 Jatsophn nuilti$da, 208 Jervine, 435 Jesaconitine, 25 1 Jimmyweed. 41 Jimsonweed, 62, 248 Jordan, 104 Juglandaceae, 2 13 Jlrglam spp., 53, 213, 428, 448 nigra. 213, 428, 448 regia, 54 treatment of ingestion of, 428 Juglone, 438 Juncaginaceae, 213 Jurrcue, claviceps infection by, 505 Jmiperus conrmulis 449

Index Kaempferol, 61, 62, 65, 66 Knlmia latifolia, 205, 428, 449 treatment of ingestion of. 428 Karakin, 201, 304 Kaminskicr humboldtiarln, 229, 428 treatment of ingestion of, 428 Kashin-Beck disease, 558 Kentucky, 250 Kenya, 46 Keshan disease, 558 Kkurage (see Auricularia polytricha) Koclria scoparin, 201, 428, 436 treatment of ingestion of. 428 Kojic acid, interaction with aflatoxin B1, 629 Kombucha tea, 766 Krombholziela, green vitrol test, 746 Kumotake (see Grqola frondosa) Kunitz trypsin inhibitor, 258 amino acid sequence of, 260 Kuritake (see H?lyholonrn sublateritiunz) Kutin, treatment for amatoxins, 752 Labdanes, 2.26 Labriforrnin, 194 Laburnwu anagyroides, 217 Lnccnria rrmethystina poisoning, 763 Laccaria laccata, mycorrhizal associations, 718 Lactarane sesquiterpenes, 763 Lactrtrius: deliciosus: medicinal use, 768 mycorrhizal associations, 718 deterrimus, 768 mutagenicity. 768 tonlirzostrs, 77 mycorrhizal associations, 7 18 trivialis, 77 ryfirs, 77 mycorrhizal associations, 7 18 Lactaroviolin 11, 768 Lactobacillus spp., 326 Lactones, sesquiterpene, 427, 444 Laetiporus sdphureus, 763 Laetrile, 48 Lagochilus irzebsians. 783 Lamiaceae, 2.13 Lanzpteromyces japonictr, 762, 764 Lantadenes A and B, 237, 429, 449 Lantana crtmara, 237, 429, 336, 449 treatment of ingestion of, 429 Lantanin, 449

821 Larkspur, western, 443 Lasiocarpine, 69, 70, 197 Lathyrism, 276 Lathyrus spp., 217, 259, 276, 429 cicera, 276 clynenunr , 276 lathyrism from, 276 odoratus, 259 and osteolathyrism, 277 sativus. 2 17, 259, 276 treatment of ingestion of, 429 Lauraceae, 2 14 Laurifoline, 230 Lawyer’s wig (see Coprirzus comatus) LD TO of chemicals from sweet potatoes, 120 of hydrazines. 80, 81 of ipomeamarone, 118 of prunasin, 302 Leccirzunz atrostipitatunz, mycorrhizal associations, 718 armiltiacunr. mycorrhizal associations, 718 green vitrol test, 746 Lectins, 269-276 analytical techniques for, 276 animals, tolerance of germ-free to, 273 erythroagglutinating(E), 269 examples of, 376 N-fixation bacteria and, 274 isolectins. 270 leukoagglutinating. 269 mode of action of, 272 nutritional significance of, 272 physiochemical properties of, 269, 270 role in plants, 274 sugar specificities. 270 Lecythis ollaria. 282 Ledrrw glarzdulosuv1, 205 Leeks, 104 Legalon 70. 752 Legumes: double-headed inhibitors in, sequence homology of, 262 genetic studies of, 266 germination effects. 266 traditional modes of preparation of, 266 Leguminosae (see Fabaceae) LEM (see Leukoencephalomalacia) Lenape, 95. 133, 249 Letts esculenta (culinaris), 259, 270

Index

822 Lentinan, 790 Lentir~ulaedocles, 79 1, 795 Lerltinus edodes, 764, 768, 783, 787-788, 79 1-792 Lepiota hel\~eola,753 Lepiota relwolrr, 753 Lepiotn rhncocles, look-alikes, 734 Leptine I, 93. 94, 96 Leptosphereria serlegalemis, 786 Leucnerln spp., 217, 279, 429, 437 glmccr , 2 17 leucocephala, 217, 279. 429. 437 treatment of ingestion of, 429 Leucoanthocyanins, 60 Leucothoe dcrrlisiae, 205 Leukemia, 86 Leukoencephalomalacia (LEM), 538, 552. 554, 615 in animals, 538. 553, 557, 615 clinical signs, 552-553 Levotetrahydropalmatine, 223 Liberia. 49 Lichen, 79 1 Lignicolous mushrooms. 719 Ligustrm 1wlgnt-e. 224 Liliaceae. 320, 249, 252. 309. 310 Limes, 53 Limettin, 5 1, 53 el-Limonene, 5 1 concentrations in citrus juices, 52 Linamarin. 48. 39, 306, 321 Linatine. 283 Lincoln, Abraham, 40 mother of. 195 Lincoln. Nancy Hanks 40 Linear furanocoumarins. 50. 97, 106 acidic fog and, 107 medicinal use of, 106 Ling-chi (see Gmodermn lrrcid14m) Ling-chih (see Gcrnoderm lucidunl) Ling-zhi (see Gmoderrncr /ltCidtll?t) Limn1 spp.. 53. 282, 419 usitntissirnum, 54, 282 Lion’s mane (see Hericiurn erinncezts) Lipoproteins: high-density (HOL), 70 low-density (LDL), 70 very-low-density (VLDL), 70 Liquid chromatography-mass spectronletry (LC-MS), 353 Liriontyzn trifolii, 108 Lithosperrmrm purp~rreo-caer.uler~m. 330

Lobnria pulntol~nrin,79 1 Lobelicr spp., 199, 253, 429 berlmdieri. 199, 253 crrrdinnlis, 199 injlatcr 199 siphilitica, 199 treatment of ingestion of, 429 Lobeline, 199, 429 Loganiaceae, 222 Loline alkaloids, 656, 665-666 N-acetyl, 516 analytical methods, 665-666 capillary gas chromatography, 666 column chromatography, 667 countercurrent chromatography, 667 extraction, separation, 667 N-formyl. 5 16 Lolitrem B, 667-668 analytical methods, 668 extraction from sources. 668 Lolitrems, 5 10, 5 16, 667 indol-isoprenoid lolitrems, 667 Loliunl spp., 510 Lorwhocarpus seriecw, 303 Loranthaceae, 223 Los ninos, 762 Lossen rearrangement, 302 Lotaustralin, 306 Lotus spp., 305 Lubimin. 9 1 Lung edema from Ipornoecr bntatns, 118 Lung toxins, 118. 121 Lupinine, 218, 429 Lltpimrs spp., 217. 251. 259, 429, 443, 447 cllbUS, 259 nrbustrus, 25 1 “edible,” 251 formosis, 25 1 lelacoph?*ll1ts.443 sulphuseus, 443 treatment of ingestion of, 218, 429 Lupus erythematosus, 43 Lycogala respiratory reactions, 766 Lycoperdotr: ccrelc~trm,787 perlntum, 79 I subincrrl.ilatun1, 79 1 Lyopersicon esculentutrr, 233 Lycophylline, 767 L ~ ~ o l ~ h ycot1t1~tLlT~1, l l ~ ~ ~ l767-768 Lycopodiaceae. 223 Lycopodium serratwn, 223

.

Index Lycopsamine, 197 Lyonia ligustrina, 205 Lysergic acid, 428, 594, 656-657 derivative structures, 5 15 Lysergic acid amides, 656-657, 660 Lysergic acid methyl-carbinolamide, 657 Lysine, 284 Lysinoalanine, 283, 284 Macrozamia spp., 319, 333 Macrozamin, 319, 333 Madurella nzycetonzatis, 786 Magic mushrooms, 762 Magnoflorine, 230 Maitake (see Grifola frondosa) Mallotus japonicus, 123 Malonyl-daidzin, 64 Malonyl-genistin, 64 Malphigiaceae, 305 Malvaceae, 223 Mammentake (see Ganoderrna lucidunz) Mandelonitrile, 306 Mandragora ojjicinarum, 233 Mandragorine, 234 Maniltot esculenta, 48, 208 Maomuer (see Auricularia polytricha) Maotou-gauisan (see Coprinus comatus) Marasmane sesquiterpenes, 763 Marasmius oreades, mycorrhizal associations, 718 Maximum tolerated dose (MTD), 135 Medicago sativa, 311, 317, 419, 436 Meixner test, 747 Melilotoside, 329 Melilotis spp., 329, 430 alba, 430 oficinalis, 430 treatment of ingestion of, 430 Mercurialis spp., 208 annua, 208 perennis, 208 Meripilus, 763 Mesaconitine, 25 1 Meteloidine, 365 P(3)-N-Methylaminoalanine,277, 278, 424 Methylazoxymethanol (MAM), 307, 319, 324 glycosides, 319 N-Methyl-beta-phenethylamine, 215, 248, 378 3-Methyl-2-butenylguanine, 196 S-Methyl cysteine sulfoxide. 422

823 N-Methyl-cytisine, 196, 219 Methylene blue, 419 P-(Methylenecyclopropyl) acetyl-coA, 279 P-(Methylenecyclopropyl) alanine, 279 P-(Methylenecyclopropyl) pyruvate, 279 N-Methyl-N-formyl hydrazine (MFH), 79, 81 4-Methylhydrazine, 79, 132 N-Methylhydrazine, 79-81, 134 Methyllycaconitine (MLA), 228, 248, 443 5-Methoxypsoralen (5-MOP), 110, 112 8-Methoxypsoralen (%MOP), 110, 112 4-Methylphenyl hydrazine (4-MPH), 78 Methylselenocysteine. 282, 379 Methylselenomethionine, 379 Melzer's iodine test, 746 Metroxylon spp., 225 treatment of ingestion of, 225 Mevinolin, 85 Mexico, 253, 442 Mezerein, 235 MFH (see N-Methyl-N-formyl hydrazine) Microcystis spp., 253 Microsomal activation and toxicity of dehydrotremetone, 41 Microsporum: audouinii, 786 canis, 786 Milk sickness, 39, 41, 195 Milk thistle, 752 Milkweeds, 331 Millets, 535 infection of, 535, 541 Mimosine, 217, 279. 429 goitrogenic effect of, 280 Mimosoideae, 189, 215, 217 Mineral balance and oxalate, 114 Miserotoxin. 215, 302, 421 Mixed function oxidases (MFOs), 55 Mode of action of glycosides, 320 Mo-er (see Auricularia polytriclza) Mokurage (see Auricularia polytricha) MON (see Moniliformin) Moniliformin (MON), 541, 554, 613, 629 analysis of, 541 interaction with: aflatoxin B1, 629 fumonisin, 630 mutagenicity, 554 source of, 541, 554 structure, 669 Monkey's head (see Hericium erinaceus)

824 [Monilifornlin (MON)] Monomethylhydrazine carcinogenicity, 768 Monomethylhydrazine poisoning, 756-757 mechanism of action, 757 symptoms, 756-757 treatment, 757 Moraea spp.: bipnrtita. 2 13 polystacltya, 2 13 treatment of ingestion of, 213 Morchella species look-alikes, 734 Morels look-alikes, 734-735 Morganelln subirtcarnatn, 791 Moringaceae, 313 Morntodicia cf~nrantia,202 Mormodin, 202 Morning glory seeds, 783 Morphine, 225, 250, 366 Mountain-priest mushroom (see Hericium erinaceus) Mozambique, 49 4-MPH (see 4-Methylphenyl hydrazine) MTD (see Maximum tolerated dose) Mucrrrzn deeringianum, 259 Mucor. 786 Mu-er (see Arrricularia yolytriclm) M Lngo ~ (see Auricularia yolytrichn) Muscarine, 248 Muscarine mushroom poisoning, 758759 mechanism of action. 758 symptoms, 758 treatment. 758 Muscazone, 248 Muscimol poisoning, 754-756 symptoms of, 754-755 mechanism of action, 755 treatment, 756 Mushroom, 77,132 carcinogenicity, 767 collections, 721-725, 744 dermatitis, 764 dyes. 782-783 features, 715, 722-727 field guides, 721, 744 growing factors, 716, 719 hydrazines in, 43.77 hypersensitivity reactions, 764-766 identification, 721-727, 733, 743-747 chemical field tests, 727, 745-747 microscopic characteristics, 727, 733 keys, 720 look-alikes, 733-736

Index [Mushroom] medical uses, 785-796 mutagenicity, 767-768 names, 719 of immortality (see Gnnodermr lucidum) origins, 782 photographs, 719 poisoning: diagnosis, 740-743 epidemiology, 739-740 statistics, 1-3 treatment, 747-768 spore print, 725-732, 747 substrates, 719 toxin removers, 784 Mustard oils, 326 Mutagenic activity, structural requirements for, 65 Mutirlus cartimas. 792 Mycelium, mushroom, 716 Mycetoma, 786 Mycological associations, 23-35 Mycophagy. 783 Mycorrhizal associations, 716-7 18, 720 Mycotoxicoses, 536, 62 Mycotoxins, 69, 81-84, 535, 557. 594, 628640, 653-655, 684-700 activated charcoal adsorption, 634 analytical methods: anti-idiotype antibody methods, 696697 biosensors, 697 enzyme immunoassay (ELISA), 689692 immunochemical analytical methods, 684-700 inmunochemical antibodies. 685-686 irnmunochemical/chemical methods, 694-696 immunoscreening methods, 693-694 overview, 654-655. 684, 699-700 radioimmunoassay of, 687-689 induction of fetal abnormalities by, 86 monitoring, 684 mycotoxin-mycotoxin interactions, 629630 Myoporaceae, 224 6-Myoporol, 122 Myoyoium lnetum, 224 Myricetin, 62, 64-66 My-iogenosporn, 504-505, 513, 656 Myristicin, 43, 45, 77, 110 Myrosin,198

Index NAC (see N-Acetylcysteine) Naenlatoloma srddateritiunl, 792 Nafenopin, 636 NAMA (see North American Mycological Association) Nameko (see PIzoliota nameko) Nametake (see Flarnmulina velntipes) Napthoquinines, 448 Narcissiflorine, 309 Nartheciurn ossifmgum, 221 National Academy of Sciences, 64 National Clearinghouse for Poison Control Centers, 2 National Institute for Occupational Safety and Health (NIOSH) 105, 108 National Research Council, 39 Nebraska, 250. 252 Neoglucobrassicin, 57, 369 Neoherculin, 230 NeoQphodium (formerly Acrentoniurn) 88, 504, 505, 508, 510, 523-524, 656. 660 animal toxicity, 5 18-5 19 coenophialurn, 88, 508, 51 1, 513, 519 distribution in world’s crops, 511 ergot alkaloids producedby, 5 12 factors altering ergot production. 512 hosts, 5 11 lolii. 516, 520 tall fescue, 5 18 toxicities induced by, 5 18 toxins in. 5 10-5 12 urzcinatunt, 5 12 Neriifolin, 192 Neriine, 191 Neriurtr spp., 191. 332, 430, 449 oleander, 191, 430. 449 treatment of ingestion of, 191, 430 Neroside, 191 Netherlands, the, 129 Neurotoxic glycosides, 320 Neurotoxins, 276 Neurotrophic toxins, 476 New England, 249 New Guinea, 48,122 New Mexico, 77 New Zealand, 122, 224, 250, 304, 521 Ngaione, 224 Nicotiana spp., 234, 249, 310, 358, 430 glauca, 249 phyolodes, 310 tabacllwl, 234 treatment of ingestion of, 191, 430 Nicotine, 234, 430

Nigeria, 49 Nightshades (see Solanaceae) NIOSH (see National Institute for Occupational Safety and Health) Nitrate (s), 100, 196, 200, 201, 210-212, 419, 421, 422, 428 children and, 105 estimated dietary intake of, 102 leeks and, 104 methemoglobinemia and. 105 plants and, 104 reactions with amines, 100 reduction of, 100 turnips and, 104 vegetables containing, 103 Nitriles, 326 Nitrite, 100, 196, 215 N-Nitroamines, 100 Nitro compounds, 320 Nitrogen heterocyclic n ” o o n 1 toxins, 766 3-Nitropropanol, 2 15 3-Nitropropanol (NPOH), 302, 320 3-Nitropropionic acid (NPA), 302, 320, 330 N-Nitrosoamines, 100 N-Nitroso compounds (NOC). 100 quantitation of. 101 l-Nitroso-3-indolyl-acetonitrile,101 N-Nitrosomethylamine, 102 N-Nitrosoproline, 101 NIV (see Nivalenol) Nitrotoxins, 446 Nivalenol (NIV), 83, 537, 539-540, 546549, 555, 611-612, 629 carcinogenicity, 550 genotoxicity, 550 in scabby grain intoxication, 546-549 interaction with deoxynivalenol, 630 LD50 Of, 549 sources, 539-540, 546 toxic concentrations, 612 N-N bond, mushroom toxins, 767 N-Nitrosamines, 767 NOC (see N-Nitroso compounds) Nolina texana, 436, 447 Nonprotein amino acids, examples of toxic, 377 Norcaperatic acid, 763 Norepinephrine, 64 Norhyoscyamine, 365 North America, 194, 25 1, 252, 331, 523 North American Mycological Association: epidemiology, role in, 1 reports, 11- 12

Index

826 North Carolina, 39 Nonvogonin, 65 Nutritional significance of trypsin inhibitors,

264 Oakwood mushroom (see Lentinula edodes) Oat infections, 535 Ochratoxin A. 83, 87, 472, 474 Ochratoxins, 594,619-621, 629 Balkan endemic nephropathy, 620 binding with calcium aluminosilicate clay,

635 carcinogenicity, 620 clinical effects, 619-620 environmental effects on animal survival,

639 interaction with: aspartame, 637 cholestyramine, 637 citrinin, 630 coenzyme QlO, 637 diacetoxyscirpenol, 630 hepatitis virus, 630 iron, 634 fumonisins, 630 vanadium, 634 vitamins, 637 malformations induced by, 637 protein effects, 639 sources, 619 tolerable weekly intake. 619 toxic dose, 619 toxic effects, 619 Oenunthe crocnta, 236 Oenanthotoxin, 236 Oklahoma, 252 Old man’s beard (see Hericium erirzaceus) Oleaceae, 224 Oleanders, 332 Oleandrin, 191. 449 Oleandroside, 191- 192 Oleoresins, 190 Ornphulotus illudens, 762 Onphrrlotus olearius: look-alikes, 734 mycorrhizal associations. 718 poisoning, 762-763 Onion, 61, 70, 133 Oosporu verticilliodes, 552 “Opium tea,” 250 Orellanine poisoning, 753-754 diagnosis, 754

[Orellanine poisoning] mechanism of action, 754 symptoms. 753 treatment. 754 Orellanine test, 746 Orelline poisoning (see Orellanine poisoning) Oscillntoriu spp., 253 Other glycosides, 329. 375 anthracenones, 375 anthraquinones, 375 azoxyglycosides, 375 naphthalenes, 375 Other toxic alkaloids, 368 Oubain, 191, 322 Overwintered grain, 543-544 Oxalate(s), 113, 189, 192-194, 200-201,

226,421,427-428,

432-433, 445,447

absorption of dietary, 114 content of selected foods and plants, 115-

118 mineral balance and, 114 renal failure from, 1 13 rhubarb and, 123 toxicity of, 113 p-N-Oxalyl-L-alpha-beta-diarninopropionic acid, 217 3-N-Oxalyl-2,3-diaminopropionic acid, 277,

278 Oxolan mushroom toxins, 767 Oxypeucedanin, 1 10 hydrate 110 Oqtenirr ucerosa, 445 O.x?ltropis spp., 21 8, 1, 44643 sericea, 218 treatment of ingestion of, 431 Oyster mushroom (see Pleui-otus ostrerrtus) Oyster shelf (see Pleurotus ostrentusj Padi straw mushroom, 783 Palenque mushrooms, 762 Palicourea marcgravii. 230 Palmae, 224 Panacea polypore (see Ganoderma lucidum) Pumeolus acurilinutus. 762 ufricanus, 762 antillarum, 762 uter. 762 cambodginiensis, 762 custuneifolius. 762 cyanescens. 762 jirnicola, 762 phnlaenarum, 762

Index [Panaeolus acuminatus] rickenii, 762 sepulcralis, 762 solidipes, 762 sphinctrinus, 761 Panicum spp., 21 1, 33 1, 436 coloratum, 21 1 dichotonlijlorum, 2 1 1 treatment of ingestion of, 21 1 sclzinzii, 21 1 Papaveraceae. 225, 250 Papaver somniferum, 225, 250 Papilonoideae, 189, 2 16 Paracelcus, 13 1 Paracoccidiodomycosis, 786 Paracoccidioides brasiliensis, 786 Parncolobactrum aerogenoides, 326 Paraepichloe spp., 504 Parasitic mushrooms, 719 Parepichloe, 504 Parsley, 1 10 linear furanocoumarins in, 110, 11 1 Parsleys (see Apiaceae) Parsnips, 112 linear furanocoumarins in, 1 12 detection of by scanning electron microscopy (SEM), 112 Parthenin, 379 Pasania (see Lentiaula edodes) Paspalurn, animal toxicity, 521 staggers, 52 1 Patulin, 83, 134, 475, 594, 617-618 carcinogenicity, 618 cytoxicity, 617 interaction with cyclopiazonic acid, 618 interaction with cysteine, 638 LDso, 617 mechanism of action, 617 mutagenicity, 617 sources, 617 teratogenicity, 6 18 Pavophylline. 309 Paxilline, 667 Pellagra, 558 Peltigera canina, 792 Penicillin, treatment for amatoxins, 75 1-752 Penicilliurn spp., 83, 97, 472 Penniclavine, 657, 661 3-Pentadecylcatechol, 190 Peptide alkaloids, examples of, 367 Peptide mushroom toxins, 767

827 Peptides, 376 Peramine, 517, 668 analytical methods, 668-669 extraction from sources, 668 Perennial ryegrass staggers, 656 Perilla frutescens, 43 1 treatment of ingestion of, 431 Perilla ketone, 431 Perloline, 2 10 Per-nettya spp., 206 Persea arnericana, 43 1 treatment of ingestion of, 431 Peru, 91 Peruviside, 192 PES (see Porcine pulmonary edema syndrome) Peshwar, 68 Phalaris arundinacene, 443 Phalloidin, 248 Phallus impudicus, 792 Phanerochaete chrysosporium, 784 Phaseolin, 43, 45 Phcrseolus spp., 47, 64, 259, 266, 268-270, 272, 275 aconitijorus, 259 angularis, 259 crureus, 259 coccineus, 259, 270 lunatus, 47, 259, 266, 270, 272 mungo (radiatus), 259 vulgaris, 47, 64, 259, 266, 268, 270, 272, 275 electron micrographs of rats fed diets of, 275 immunofluorescence micrograph of rats fed diets of, 274 Phenanthrene glycosides, 227 P-Phenethylamine, 248, 285, 378 Phenolic acid derivative, 517 Phenolic glycosides, 316, 370 coumestrans, 370 furanocoumarins. 370 isoflavones, 370 other, 370 Phenylbutazone, treatment of amatoxins in, 753 Pheohyphomycosis, 786 Phialophora verrucosa, 786 Philodendron cordatum, 194 Pholiota: nameko, 792 respiratory reactions, 765

Index

828

[Pholiota] squarrosa, mycorrhizal associations, 7 18 Phomopsin, 83 Phorone, 636 Photodermatitis, 108 Photosensitization, 105, 107 Physalis peruviana, 234 Physostignla venenosum, 218 Physostigmine, 218 Phytoalexins 43, 43, 91, 105, 118 plants containing, 42 Phytoestrogens 67, 331 isoflavone, 33 1 Phytolaccn nmericann, 43 1, 447 treatment of ingestion of, 431 Phytolaccin(e). 43 1, 447 Phytuberin, 9 1 Picrorhiza kurroa, 753 Pieris japonica, 206, 431 treatment of ingestion of, 431 Pinaceae, 226 Pinus spp., 226, 431, 449 contorta. 449 ponderosa, 226, 431. 449 treatment of ingestion of, 431 Piperidine alkaloids, 236, 357, 445 examples of toxic, 357 Pisatin, 45 Pisolithus respiratory reactions, 765 Pisoliths tinctorius mycorrhizal associations, 718 Pistillarin test, 746 Pissm satiwt1, 259, 270 Pithecolobium lobatum, 280, 28 1 Piziza badia, 757 Plant(s): fanlilies containing troublesome species. 189-241 families harmful to humans, 248 fanlilies harmful to livestock, 251 goitrogenic compounds in, 54 intoxications by,41 8 antidotes for. 418 treatment algorithm for, 418 treatments of, 420-435 nitrate levels in, 104 Plant poisoning, medical management of, 4 13-440 Plant toxicants, prevention and management of livestock, 441 -470 Pleurotus: citrinopileatus, 790-792, 796

[Pleurotus] Jlorida, 792 ostrentlis: medical uses, 790, 792-793, 795796 mycorrhizal associations, 718 toxin, 766 toxin remover, 784-785 pulrnonnrius, 785, 794 respiratory reactions. 765 sajor-caju, 785. 788, 790, 794 tuber-regiwn, 794 Poaceae, 419 Poaefusarin, 543 Poaefusariogenin. 543 Podaxis respiratory reactions, 766 Podophyllin, 197, 431 Podophyllotoxin, 317 Podophyllum peltatunl, 196, 43 1 treatment of ingestion of, 431 Poison centers, 1 call handling, 6 call types, 5-6 data collection, 6 education programs, 8 epidemiology, role in, 1 funding, 2 quality assurance, 7 references, 7 consultants. 7 industry, 7 POISINDEX(R), 7 texts, 7 regional centers. 3 criteria for, 3 service areas, 3 staff, 4 consultants, 5 managing director, 5 medical director, 4 poison information specialists, 5 U.S. centers, 12-23 websites, 11 Poisonous higher plants, 187-246 Poland, 86, 101 Polygonaceae. 226 Polyozellus multiple.v, 733 Polypeptides, 248 Polyporus: annosis (see Polyporlu oflcinalis) ant~lelmiilticus,794 fomentnrim. 794

Index [Polyporm] igniai-ius, 794 marginatus, 794 oflcinalis, 794 squamosus (see Polyporus oflcinalis) suaveolans (see Polyporus oflcinalis) unzbellcttus, 787-788, 790, 794-796 Porn porn (see Hericium erirrttceus) Porcine pulmonary edema syndrome (PES), 615 Portugal, 521 Potassium hydroxide test, 727 Potato, 535 alkaloids, 132 infection of, 535 white (see White potatoes) Precocious puberty, 653, 670 Pressor amines, 284 Proacacipetalin, 306 Procyanidins, 64 Progoitrin. 55, 369 Propionic acid, 277 Proscillaridin, 22 1 Protease inhibitors, 258-268 analytical techniques for, 267 biological properties of, 259 detoxification of, 264 effect of heat treatment on, 264-265 mode of action of, 263 nutritional significance of, 259, 264 physiological role in plants, 267 present in legumes, table of, 259 Proteins 259, 376 antinutritional factors related to, 259 Protoanemonin, 228, 318, 319, 324, 333, 375,432 Protoberberine(s), 366, 445 Protopine. 210, 225, 366, 445 Prunasin 46, 230, 306, 32 1. 449 LD50for, 302 Przr~zusspp., 229, 419, 432, 449 pensylllarzica. 229 serotinn, 229, 449 treatment of ingestion of. 432 virginiann. 229. 449 Pseudallescherin bo-vdii, 786 Pseudotropine, 254 Psilocybe baeocystis, 760-76 1 cubensis, 783 cynrzescens, 76 1 pellicsdostr, 761

829 [Psilocybe] semilancenta, 76 1 spores, 726 subbalteatus, 76 1 Psilocybin/psilocin poisoning, 760-762 mechanism of action, 761 symptoms, 760-761 treatment, 76 1 Psophocarpus tetragonolobus, 259, 266, 270. 272 Psoralen, 52, 53, 105, 107, 108, 112, 132 acidic fog and, 107 citrus fruits containing, 132 in the Fabaceae, Rutaceae, and Apiaceae. 106 Psychoactive substances, 77 Ptaquiloside, 203, 314, 315, 328, 332, 379, 431, 444 Pteridaceae, 3 15 Pteridium nquilimm, 203, 314, 328, 431, 444 treatment of ingestion, 203, 431 Pzrccinia, respiratory reactions to, 765 Puffballs, 735, 795 Pulegone, 214 Pulmonary edema in animals, 538 Pltrlica granrttunt, 123 Putrescine, 285 Pyroclavine. 660 Pyrrolizidine alkaloids, 69, 195, 197, 198, 223, 250, 252, 254, 354, 355. 321, 424, 433, 447, 516 crotanecine esters, 355 heliotridine esters, 355 otonecine esters, 355 retionecine esters, 355 supinidine esters, 355 Queensland. 216. 252 Quercetagenin, 64 Quercetin, 61, 62, 64-66 Quercus spp.. 209, 432, 449 treatment of ingestion of, 209, 432 Quinolizidine alkaloids, 216-219, 360, 434, 447 cytisine type, 360 lupinine type, 360 tetracyclic type, 360 Radioactivity in Inushrooms, 767 Rmnaria, pistillarin test and, 746 Ranunculaceae, 189, 227, 25 1, 253, 319

Index

830 Ranunculin, 227-228, 3 18, 324, 33 1, 444 Ranunculus spp., 229, 432, 444 repens, 229 treatment of ingestion of, 229, 432 Rapeseed, 55 “Rayless goldenrod” (see Haplopapp~rs heter-ophyllus) Rd toxin, 549 Red mold disease, 545, 6 12 Red scab, 545 Red woodlover (see Hypholonla sublater-itiunt) Reishi (see Garzoderrna lucidurn) Religious purposes of mushrooms, 783784 Resedaceae, 3 13 Respiratory hypersensitivity reactions. 764765 Retrorsine, 195 Rhamnaceae, 229 Rhmtnus catharticu, 229 Rheum spp.. 68, 226, 432 japonicum, 432 oficinale, 68 rhapouticum, 226 treatment of ingestion of, 432 Rhisitin, 9 1 Rhi:obiurn spp., 59, 274 Rhizoctonia leguminicoln, 97 Rhizornucor, 786 Rhizopogon nrbescens: look-alikes, 734 mycorrhizal associations, 7 18 Rhizopus: arr-hizl~s, 786 oryae, 786 Rhodanase, 50, 322. Rhododendron spp.. 206, 432 maximum. 206 treatment of ingestion of, 432 Rhodophyllus: rhodopolilts, 762, 764 sirtatus, 762, 764 Rhubarb, 123 RIA. 687-689 Rice. 535, 541, 546 infection of, 535, 531, 546 Ricin, 208, 433 Ricinis communis, 208, 270, 433 treatment of ingestion of, 433 Rifampin, treatment of amatoxins in, 753 Robin, 218, 433, 348

Robinia pseudoacacia, 218, 433, 448 treatment of ingestion of, 423 Romania, 84 Rosaceae, 229 Rosemary (see Rosmarinus oflcinalis) Rosnlarinus oficirralis. 74 compounds in. 75 Rota, 278 Rotation locular countercurrent chromatography (RLCC), 353 Rubiaceae, 230 Rubratoxin A: interaction with pentobarbital, 638 poisoning. 767 Rubratoxin B, 86 Rumen: anearobic microorganisms in, 301 fermentation, 301 Rumex spp.: acetoscr, 226 acetosella, 226 c r i s p s , 226 treatment of ingestion of, 227 Rusorine, 195 Russula: enzeticu, mycorrhizal associations, 7 18 jbetem, mycorrhizal associations, 7 18 green vitriol test, 746 xerampelina, mycorrhizal associations, 718 Rutaceae. 52. 105, 230 Ruta grulleolen, 74 Rutin, 66 Ruvoside, 192 Ruwenine, 195 Rwanda, 129 Ryegrass staggers, 519-520, 656 convulsive, 656 perennial. 656 Rye, 535, 548 infection of, 535, 548 Sucodon aspratm, 795 Safrole, 214 Sage (see Salvia oflcincrlis) Saiwai-take (see Gnnodermn lucidum) Salvia oficinalis, 74 compounds in, 76 Scmguinar-in canndensis. 225 Sanguinarine, 225, 366 Santalaceae. 23 1 San Ysidro mushrooms, 762

Index Sapindaceae, 23 1 Saponnrin ojficinalis, 433 treatment of ingestion of, 433 Saponin(s), 207, 300. 309, 323, 433 antinutritional factors and, 324 steroidal, 31 1 Saprophytic mushrooms, 7 19 Sarcobatus venniculntus, 433 Sarunouchitake (see Ganoderma lucidum) Sassafras spp., 7 1 rclbidium, 214 Satyr's beard (see Hericiunt erinaceus) Saudi Arabia, 249 Saussurea lappa, 68 Saxifragaceae, 232 Scabby grain intoxication, 537, 545-550, 612 control of, 550 clinical signs, 546, 612 in animals, 548-549 level of concern, 551 mycotoxins involved, 546-547 sources, 545-546 Scabby wheat (see Scabby grain intoxication) Scanning electron microscopy (SEM), 112 Schaeffer reaction, 745 Scilin maritima, 308 Scilliroside, 308 Sclerodernta spp., 718 look-alikes, 734, 736 poisonings, 763 respiratory reactions, 765 sclerotioriunz, 97, 105 Sclerotium, site of alkaloid production, 507 Scopolamine, 39, 233, 234, 365, 424, 445 Scopoletin, 50, 93 Scopolia spp., 365 Scrophulariaceae, 232, 309 Selenium accumulators, 215, 446 Selenoamino acids, 28 1 Selenocystathionine, 282 Selenocystine, 28 1, 282 Selenomethionine, 282 SEM (see Scanning electron microscopy) Ser~icarprts nnacardium, 190 Seltecio spp., 195, 252, 354, 433, 447 jacobea, 195, 447 lautus, 252 ntadagascariertsis, 252 treatment of ingestion of, 195, 433 Senna (see Cassia)

831 Sennosides, 21 8 Sepedonium. 7 19 Septic angina, 542 Serotonin, 285 Serum prolactin levels, 520 Sesbanin spp., 219. 433, 444 drummondi, 433, 444 punicia. 444 treatment of ingestion of, 433 ayesicaria, 2 19, 444 Sesbanine, 2 19 Sesquiterpene glycosides, 3 14 Sesquiterpene lactones, examples of, 379, 427, 444 Sesquiterpene toxins, 517 SGI (see Scabby grain intoxication) Sexangularetin, 65 Shaggy mane (see Coprims comntus) Shiangu-gu (see Lentinula edodes) Shiangu-ku (see Lerttinula edodes) Shiitake, 764, 783, 795 Shirotamogitake (see Hypsizygus ulrnnrius) Silypide, 752 Silybin, 752 Silybinin, 752 Silybinin betacyclodextrine, 752 Silybinin dihemisuccinate, 752 Silybum marianum, 752 Silychrisin. 752 Silydianin, 752 Silymarin, treatment for amatoxins, 752 Simmondsin: cnlifornica, 330 chinensis, 307 Simmondsin, 307, 330 Simmondsin ferulate. 330 Simple simons, 762 Sinalbin, 3 12 Sinapis: alba, 312 nigra , 3 12 Sincamidine, 197 Singapore,102 Sinigrin 55, 198, 312, 369 Skinunin laureda, 68 Socrates, 250 Solanaceae, 39, 91, 189, 232, 248, 252, 310, 328, 419 Solanaceous alkaloids, 234 Solandrn spp., 234, 365 Solanidine, 92, 98, 433

832 a-Solanine, 93-98, 133, 234, 235, 310, 433 Solcmm spp., 39, 96, 234, 235, 433, 447 clones of, 96 dulcanm-a, 234, 447 elaerrgnifoliunl. 97, 234, 447 glatrcopl~yllunr, 3 15,329 rzigruw, 39, 447 rostrum, 447 snrrachoides, 234 treatment of ingestion of, 235, 433 tuberoslam, 234. 249. 310 Solasodine, 98 Soma, 783 Songshan Lingzhi (see Ganodemu lucidrlm) Sophora secuui’ora, 219, 434 treatment of ingestion of, 434 Sorghum spp.. 21 1, 419 Itcrlpense, 2 11, 419 infection of, 535 treatment of ingestion of, 21 1 South Africa, 90, 194. 521 South America. 65, 281 Southeast Asia, 82 Soybean, protein nutritive value improved by heat, 258 Sparasis rcdiccrtn, 719 Sparta. 82 Sparteine, 21 6, 21 8 Specific disorders induced by glycosides, 328 Spermidine, 285 Spermine, 285 Spkenopholis obtustatcr, 508 Sphinganine N-acyltransferase, 90 Sphingolipids. 90 Sphingosine, 90 Spore print, 725-732, 747 Spores, mushroom, 7 17, 725-727 Sporofusarin, 543 Sporofusariogenin, 543 Sporotlzris sclzenckii. 786 Sporotrichoisis, 786 Sri Lanka 69, 83 Stcdq~botn~s, 6 11 toxin, 83 Staggering grain, 545 clinical signs, 545 sources, 545 “Starch blockers,” 268 Stemonitis respiratory reactions, 765 Sterigmatocystin, 83, 472, 474, 638 carcinogenicity, 638

lndex Steroid(a1) alkaloids, 434. 335, 447, 448 examples of jerveratrum type, 362 other types, 362 solanidine type, 362 spirosolane type, 362 teratogenic. 435 Steroid glycosides, examples of, 361 bufadienolides, 372 cardenolides, 372 Steroidal saponins, 3 11 Stizobolium deeringianmt, 259, 280, 281 Sticoboliun? spp., 756 Stizolobic acid, 755 Stizolobinic acid. 755 Straw mushroom (see Plet{rotus ostreatus) Streptomyces spp., 303 Stropharia cubemi. 783 Stropharia nrgosoarzmlata, 785 Structural requirements for mutagenic activity. 65 Strychnine, 133, 222 Stiychnos nzdx-llornica, 222 Shpandra imbricata, 3 17 Stypandrol, 317 Sulforaphane, 59 Sullius: americanzfs, mycorrhizal associations, 7 18 brevipes: ammonium hydroxide test, 745 mycorrhizal associations, 718 green vitriol test, 746 granulatus, mycorrhizal associations, 71 8 megaporinus. hydrochloric acid test, 746 piperatus, ammonium hydroxide test, 745 to~nentosus,mycorrhizal associations. 718 Sumatra, 280 Supercritical fluid extraction, 353 Swainsonine, 215, 218, 365, 421, 431, 446 Swainsonine-N-oxide, 2 18,365 Sweet potatoes (see also Ipomoea butcrtcrs), 118 Sulertia chiraitn, 68 Switzerland, 104 Symphytine, 197 Symph?~trmspp.. 70, 197, 250 offkinale, 70, 197 T-2 trichothecene toxin, 537-538, 543-544. 546, 612-613 adsorption, 635 animal poisoning, 544

Index [T-2 trichothecene toxin] binding with calcium aluminosilicate clay, 635 carcinogenicity, 544-545 cause of chromosomal aberrations, 544 in scabby grain intoxication, 546 interaction with: coenzyme QlO. 638 cycloheximide, 638 cyclopiazonic acid, 629 diacetoxyscirpenol, 630 emetine, 638 fumonisin, 630 HT-2 toxin, 630 lasalocid, 638 monensin, 638 Salmonella liposaccharide, 632 selenium, 634 vermcarin A, 630 vitamins, 637 zinc, 634 protein effects, 639 sources, 538-539, 543, 546 toxic concentrations, 612 Tamogitake (see Pleurotus ostreatus) Tamo-motashi (see Hypsizygus tessulatus) Tandem mass spectrometry (MS/MS), 353 Tannic acid, 449 Tannin(s), 122, 209, 449 effects of, 123 presumed biological reactions of, 130- 131 rhubarb and, 123 selected foods and, 125-1 29 Tanning booths, 107 bergapten and, 107 celery and, 107 linear furanocoumarins and, 107 psoralen and, 107 Tanzania, 49, 272 Taumelgetreide (see Staggering grain) Taxaceae, 235, 250, 253 Taxicatine, 250 Taxines, 235, 248, 434, 449 Trtsus spp., 235, 248, 253, 434, 449 baccata, 235, 250 cuspidatn, 235 treatment of ingestion of, 434 TCA (see Tricarballylic acid) Tea(s), 43, 71, 124, 249, 250 Teratogens, 97-98 Terminalin chebrda, 123 Terpenes, 427

833 Terpenoid mushroom toxins, 767 Terrestrial mushrooms. 7 19 Territrems, 476 Tetmdymia spp., 436 Tetradymol, 379 Tetraenone steroid, 5 17 Tetrahydropalmatine 209 Tetrandrine, 636 Teucrium: chamaedrys, 214 polium, 214 treatment of ingestion of, 214 Texas, 41, 86, 253 TGA (see Total gyycoalkaloid content) Thailand, 84 Thalictrum spp.. 330 Thanksgiving dinner menu, toxins in, 44 Theobromine, 43, 45 Theraeutic, 132 Thermopsine, 219 Thermopsis montann, 2 19 Thesium lineaturn, 23 1 treatment of ingestion of, 231 Thevetia peruviann, treat as for Nerium spp., 192 Thevetins A and B, 192 Thevetoxin,192 Thiaminases, 425, 444 in bracken, 328 Thin-layer chromatography (TLC), 352 Thioctic acid, 752-753 Thiocyanate(s), 50, 53, 55, 100 Thlaspi awense, 198, 326, 331 treatment of ingestion of, 198 Thrombin, 329 Thymelaeaceae, 235 Thyroxine, 326 Tiliroside, 66 Tigonin, 309 TMP (see 4,5’.8-Trimethylpsoralen) TMS-festuclavine, 665 TMS-pyroclavine, 665 TMS-dihydroelymoclavine, 665 a-Tomatine, 99, 233 Tomatoes. 99 infection of, 535 Total gyycoalkaloid content (TGA), 94, 96 Tovariaceae, 3 13 Toxic amino acids, 276-286 canavanine, 282 dihydroxyphenylalanine, 280 distribution of lathyrogenic factors in, 277

834 [Toxic amino acids] djenkolic acid, 280, 281 hypoglycin, 278 indospicine, 282 linatine, 282 lysinoalanine, 283 mimosine, 279 neurotoxins in, 276 selenoamino acids, 28 1 Toxic glycosides, 330 Toxicodendron spp., 190 diversilobium, 190 radicans, 190 vernix, 190 Toxicology: naturally occuring chemicals in food and, 37-186 organizations, 8-10 websites,11 Toxic plants, treatment and prevention strategies for individual, 420-435 Toxic Surveillance System, 6 Toxin activation: in lungs, 121 microsomal, 41 Toxins in lung, 120 Trametes versicolor, 785 Transgenic plants, potential protease inhibitor activity of, 267 Transkei, 88-90 Treatment of livestock exposed to glycosides, 320 Tree ear (see Auricularia polytricha) Tree jelly fish (see Auricularia polytricha) Tree of life mushroom (see Ganoderma lucidum) Tree oyster (see Pleurotus osteratus) Trembles, 39, 41 Tremetol (tremetone), 41, 194-195, 425, 427, 428, 448 Tremetone (see Tremetol) Tremorgenic neurotoxins (see Lolitrems) Tribulus terrestris, 238, 436 treatment of ingestion of, 238 Tricarballylic acid (TCA), 613 Trichoderma, 6 1 respiratory reactions, 765 Tricltoloma javovirens, mycorrhizal associations, 718 Tricholoma magnivelare, mycorrhizal associations, 718 Tricholorna muscarium, poisoning by, 756

Index Trichothecene toxins, 83, 537-538, 543, 594, 611-615, 629, 671, 767 analysis of, 538, 671-672 clinical effects, 61 1-614 cytotoxic effects, 61 1 GUMS methods, 671 sources, 611 teratogenic effects, 61 1 type B, 538 Trichothecium, 6 11 Tricodesma spp., 198, 354 incanum, 198 Tricodesmine, 198 Trifolium spp., 64, 317, 327, 436 yratense, 436 repens, 4 19 subterraneum, 64, 317, 327, 436 Triglochin spp., 213, 419, 434, 444 maritirna, 2 1 3 palustrus, 21 3 treatment of ingestion of, 434 Triglochinin, 213, 306 4,5’.8-Trimethylpsoralen (TMP), 107 TriseturnJlavescens, 329 Triterpenoid saponins, examples of, 372 Triticum aestivum (see Wheat) Tropane alkaloids, 62, 233-234, 254, 422, 424, 433 Tropine, 254 Tropinone, 254 Truffles, 796 Trypsin, 26 1 inhibitors, 258-268 destruction of, by heat, 264-265 L-Tryptophan, 3-methylindole from, 436 Tsuchi-maitake (see Polyporus unlbellatus) Tuber gibbosunt, mycorrhizal associations, 718 Tulostorrm: brumale, 796 carttpestre, 796 pedunculatum, 796 Turkey, 48, 112 Turkey X disease, 82 Turnip, 104 Tussilago falfara, 250 Tylecodon spp., 202 grandijoris, 202 ventricosus, 202 wallichii, 202 Tyramine, 43, 101, 285, 378 Tryptamine, 285 alkaloids, examples of, 359

Index Umbelliferae (see Apiaceae) Umbrella polypore (see Polyporus z m bellatus) United Kingdom, 61, 102, 120, 235 United Nations (UN) Food and Agricultural Organization (FAO), 83 United States, 40, 41, 46, 68, 69, 74, 77, 82, 84, 86. 88, 100, 102, 104, 133, 231, 442, 513, 521 Department of Agriculture (USDA), 93 Environmental Protection Agency (EPA), 59 Food and Drug Administration (FDA), 135 Uranium, 55 Urechitin,192 Urechitis suberectus, 192 Urechitoxin.192 Urginea spp.: maritima, 221 physodes , 22 1 sanguinea, 221 Urishiol.190 Urtica dioica, 237 treatment of ingestion of, 237 Urticaceae, 237 Ustilago: nznydis, 796 respiratory reactions, 766 Vegetables, nitrate content of, 103 Velvet foot (see Flanmulina velutipes) Venezuela, 249 Veratrum spp., 222, 249, 434, 442, 448 californicurn, 222, 434, 448 treatment of ingestion of, 434 viride, 222, 249, 434 Verbenaceae, 237 Verbesinin encelioides, 196 treatment of ingestion of, 196 Verpa: look-alikes, 734 medicinal uses, 796 poisoning by, 756-757, 764 Verpa bohemica, 757 Vermcarin A, interaction with T2 toxin, 630 Vermculogen, 476 Veterinary mycotoxicoses, 558 Vicia spp.: fnba, 219, 259, 266, 270, 280-281, 330, 334 villosa, 435 treatment of ingestion of, 435

835 Vicine, 219, 319 Vigna spp., 47, 259, 266, 267 aconitifoliurn, 266 unguiculata (sinensis), 47, 259, 266, 267 5-Vinyl-2-oxazolidinethione, 102 5-Vinyl- 1,3-0xazolidine-2-thione(progoitrin) (5-VOT), 55 5-Vinyl-2-thiooxazolidone, 198, 369 Violaceae, 305 Virgil, 233 Viscotoxin A, 223 Viscuwt album, 223 Voandzein subtermnca, 259 Volvariella volvacea, 783, 796 Vomitoxin, 549 Von Munchhausen, 504 Wedeli spp., 314, 329 asperrima, 3 13 Wedeloside, 329 West Africa, 49 West Berlin, 272 Wheat, infection of, 535, 546-549 White potatoes, 91 cholinesterase inhibition by solanine in, 93 glycoalkaloid(s) content, 93-95 table of, 92 teratogens in, 97 White snakeroot, 39-41 distribution map of, 40 Wieland test. 747 Wild boar’s dung maitake(see Polyporus umbellatus) Wine: home-made, 249 tyramine in, 43 Winter mushroom (see Flammulina velutipes) Wisconsin, 94 Wisteria jloribunda, 2 19 Wood ear (see Auricularia polytricha) Wortmannin, 612 Xanthiun? spp., 314, 329, 435, 445 strumariunt, 196, 435, 445 treatment of ingestion of, 196, 435 Xanthocephalum spp., 435 treatment of ingestion of, 435 Xanthotoxin, 52, 53, 105, 107, 108 acidic fog and, 107 in the Fabaceae, Rutaceae, and Apiaceae, 106

836 Yamabiko hon-shimeji (see Hypsizygus tessulatus) Yamabushi-take (see Hericiwn erirraceus) Yarrow (see Achillea millfoliunr) Yellow rain, 82, 543 Yuhuangmo (see Pleurotus citrinopileatus) Yuki-motase (see Flnmntulina velutipes) Yung ngo (see Auricularia polytridra) Zaire, 49 Zamia spp., 319, 333, 435 Zantlto.xylim clava-herculis, 230 ZEA (see Zearalenone) Zea mays (see also corn), 88, 211, 419 Zeranol, 618 Zearalenol, 66, 134 Zearalenone (ZEA), 66, 83, 86, 134. 537, 540, 546-550, 555, 594, 613-614, 618, 629, 670 analytical method, 540, 670 binding with calcium aluminosilicate clay, 635 biological activity, 6 18 carcinogenicity, 550, 614, 618

Index [Zearalenone (ZEA)] clinical signs, 550 estimated daily intake. 551 high-performance liquid chromatography, 670 interaction with: deoxynivalenol, 630 estradiol, 638 iron, 634 tamoxifen, 638 mutagenicity, 61 8-619 in scabby wheat disease, 546-550 sources, 540. 546, 618 structures, 669 tolerable daily intake, 551 Zhu ling (see Polyporus urnbellatus) Zigadenus spp., 222, 249, 252, 435, 445 granlineus. 222 treatment of ingestion of, 222, 435 Zwitterions, 248 Zygacine (zigadenine), 222, 435 Zygomycosis, 786 Zygophyllaceae 238