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.

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30.

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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

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5.

6.

7.

8. 9. 10. 11.

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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.

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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.

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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).

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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.

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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

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487. 488.

489. 490. 491. 492.

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494. 495.

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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.

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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.

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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.

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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.

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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.

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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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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.

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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.

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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

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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.

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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-

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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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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.

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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.

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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.’’

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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,

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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.

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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, damage