Gupta, Toxicology of Organophosphate and Carbamate Compounds This book brings together the expertise of leading scientists from around the world on the complex toxicology of anticholinesterase compounds (Organophosphates and Carbamates). It provides the most up-to-date and in-depth knowledge on various aspects of OP and CM compounds, including their use, classification, mechanism-based toxicity, and prophylactic and therapeutic measurements. Anticholinesterase compounds constitute the largest number of chemicals that are primarily used as insecticides in agriculture, industry, and around the home/garden. Some OPs (nerve agents) have been used both in chemical warfare and terrorist attacks; while other OPs and CMs have been recommended as therapeutic agents in human and veterinary medicine. Many chemicals of both classes are extremely toxic and lack selectivity, thus their inadvertent and accidental use continues to pose a threat to human and animal health, aquatic systems, and wildlife. These are some of the contributing factors that make this class of agents so very important and will make this book a crucial reference work for every researcher dealing with these agents. 9 Extensively covers pesticides, nerve agents, therapeutic drugs, and flame retardants 9 Describes epidemiology of the world's major disasters involving Organophosphates and Carbamates 9 Covers animal, human, aquatic, and wildlife toxicity of Anticholinesterases 9 Insights into in-depth cholinergic and noncholinergic mechanisms of toxicity 9 Describes recent advancements in cholinesterases, paraoxonases, carboxylesterases, oxidative stress, endocrine disruption, cardiac and pulmonary toxicity, and carcinogenesis 9 Provides in vitro and in vivo models for neurotoxicity testing 9 Integrates knowledge of studies in lab animals and humans 9 Offers risk/safety assessment and national/international guidelines for permissible levels of pesticide residues 9 Describes management of Anticholinesterase poisoning in humans
"SCIENCE IS THE GREAT ANTIDOTE TO THE POISON OF ENTHUSIASM AND SUPERSTITION" Adam Smith (1723-1790)
TOXICOLOGY OF ORGANOPHOS PHATE AND CARBAMATE C O M P O U N D S Edited by RAMESH C. GUPTA
AMSTERDAM PARIS
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Elsevier Academic Press 30 Corporate Drive, Suite 400; Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald's Road, London WC1X 8RR, UK This book is printed on acid-free paper. Copyright 9 2006, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail:
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Working together to grow libraries in developing countries www.elsevier.com I www.bookaid.org I www.sabre.org
Dedicated to My Beloved parents, the late Chandra Gupta and Triveni Devi Gupta, My Beloved wife, Denise, and Dear daughter, Rekha
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Contents
Contributors Foreword Kai M. Savolainen
xv
Pharmacokinetics & Metabolism 9. Physiologically Based Pharmacokinetic Modeling of O r g a n o p h o s p h o r u s and Carbamate Pesticides Charles Timchalk
Uses, A b u s e s , & E p i d e m i o l o g y 1. Introduction Ramesh C. Gupta
10. Metabolism of O r g a n o p h o s p h o r u s and Carbamate Pesticides Jun Tang, Randy L. Rose, and Janice E. Chambers
2. Classification and Uses of O r g a n o p h o s p h a t e s and Carbamates Ramesh C. Gupta 3. Therapeutic Uses of Cholinesterase Inhibitors in Neurodegenerative Diseases Randall L. Woltjer and Dejan Milatovic 4. Coadministration of Mernantine with Acetylcholinesterase Inhibitors. Preclinical and Clinical Evidence Andrzej Dekundy
1 1. Interspecies Variation in Toxicity of Cholinesterase Inhibitors Stephanie J. Garcia, Michael Aschner, and Tore Syversen
127
145
25
Esterases, Receptors, M e c h a n i s m s , & Tolerance Development
35
12. Structure and Function of Cholinesterases Zoran Radic and Palmer Taylor
5. Cholinesterase Inhibitors as Chemical Warfare Agents: Community Preparedness Guidelines AnnettaWatson, KulbirBakshi, Dennis Opresko, RobertYoung,VeroniqueHauschild, and Joseph King
47
6. O r g a n o p h o s p h a t e s and the Gulf War Syndrome Linda A. McCauley
69
7. The Bhopal Accident and Methyl Isocyanate Toxicity Daya R. Varma and Shree Mulay
79
8. Global Epidemiology of Organophosphate and Carbamate Poisonings Tetsuo Satoh
103
13. Cholinesterase Pharmacogenetics Roberta Goodall 14. M e t h o d s for Measuring Cholinesterase Activities in Human Blood Elsa Reiner and Vera Simeon-Rudolf 15. Interactions of O r g a n o p h o s p h o r u s and Carbamate C o m p o u n d s with Cholinesterases Lester G. Sultatos 16. Structure, Function, and Regulation of Carboxylesterases Masakiyo Hosokawa and Tetsuo Satoh
89
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161
187
199
209
219
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Contents
17. Noncholinesterase Mechanisms of Central and Peripheral Neurotoxicity: Muscarinic Receptors and Other Targets David A. Jett and Pamela J. Lein 18. Paraoxonase Polymorphisms and Toxicity of O r g a n o p h o s p h a t e s Lucio G. Costa, TobyB. Cole,Annabella Vitalone, and Clement E. Furlong 19. Tolerance Development to Toxicity of Cholinesterase Inhibitors Frode Fonnum and Sigrun Hanne Sterri
233
247
257
21. Developmental Neurotoxicity of Organophosphates: A Case Study of Chlorpyrifos Theodore A. Slotlcin
24. Behavioral Toxicity of Cholinesterase Inhibitors Philip J. Bushnell and Virginia C. Moser 25. Peripheral Nervous System Effects and Delayed Neuropathy Angelo Moretto and Marcello Lotti 26. Intermediate S y n d r o m e in O r g a n o p h o s p h a t e Poisoning Jan L. De Bleecker
30. Dermal Absorption/Toxicity of O r g a n o p h o s p h a t e s and Carbamates Jim E. Riviere
399
411
423
271 32. Reproductive Toxicity of O r g a n o p h o s p h a t e and Carbamate Pesticides Suresh C. Sikka and Nilgun Gurbuz
447
293
22. In Vitro Models for Testing Organophosphate-lnduced Neurotoxicity and Remediation 315 Evelyn Tiffany-Castiglioni,VijayanagaramVenkatraj, Yongchang Qian, and James R. Wild 23. Electrophysiological Mechanisms in Neurotoxicity of O r g a n o p h o s p h a t e s and Carbamates Toshio Narahashi
29. Approaches to Defining and Evaluating the Inhalation Pharmacology and Toxicology Hazards of Anticholinesterases Harry Salem and Bryan Ballantyne
31. Local and Systemic Ophthalmic Pharmacology and Toxicology of O r g a n o p h o s p h a t e and Carbamate Anticholinesterases Bryan Ballantyne
O r g a n Toxicity 20. Central Nervous System Effects and Neurotoxicity Carey N. Pope
28. Pulmonary Toxicity of Cholinesterase Inhibitors 389 Corey J. Hilmas, Michael Adler, and StevenI. Baskin
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33. Placental Toxicity of O r g a n o p h o s p h a t e and Carbamate Pesticides Olavi Pelkonen, Kirsi V~ihiikangas,and Ramesh C. Gupta
463
34. Endocrine Disruption by Organophosphate and Carbamate Pesticides 481 Shigeyuki Kitamura, Kazumi Sugihara, and Nariaki Fujimoto 35. Organophosphates, Carbamates, and the Immune System Raghubir P. Sharma
495
347
N o n s p e c i f i c Toxic Effects 361
371
27. Cardiovascular Toxicity of Cholinesterase lnhibitors 381 Csaba K. Zoltani, G. D. Thome, and Steven I. Baskin
36. Oxidative Stress in AnticholinesteraseInduced Excitotoxicity Wolf-D. Dettbarn, Dejan Milatovic, and Ramesh C. Gupta 37. DNA Damage, Gene Expression, and Carcinogenesis by O r g a n o p h o s p h a t e s and Carbamates Manashi Bagchi, ShirleyZafra, and Debasis Bagchi
511
533
Contents 38. Temperature Regulation in Experimental Mammals and Humans Exposed to Organophosphate and Carbamate Agents Christopher J. Gordon, Cina M. Mack, and Pamela J. Rowsey
44. WHO/FAO Guidelines for CholinesteraseInhibiting Pesticide Residues in Food P. K. Gupta
ix
643
549
Aquatic Life & Wildlife 39. Occupational Toxicology and Occupational Hygiene Aspects of Organophosphate and Carbamate Anticholinesterases with Particular Reference to Pesticides Bryan Ballantyne and Harry Salem
45. Aquatic Toxicity of Carbamates and Organophosphates Arun K. Ray and Manik C. Ghosh 567
46. Toxidty of Organophosphorus and Carbamate Insecticides Using Birds as Sentinels for Terrestrial Vertebrate Wildlife Spencer R. Mortensen
657
673
Risk A s s e s s m e n t & Regulations 40. Public Health Impacts of Organophosphates and Carbamates Daphne B. Moffett 41. Cumulative Effects of Organophosphorus or Carbamate Pesticides Stephanie Padilla 42. Federal Regulations and Risk Assessment of Organophosphate and Carbamate Pesticides Anna B. Lowit 43. Regulatory Considerations in Developmental Neurotoxicity of Organophosphorus and Carbamate Pesticides Susan L. Makris
Analytical & Biomarkers 599
607
47. Analysis of Organophosphate and Carbamate Pesticides and Anticholinesterase Therapeutic Agents Anant V. Jain 48. Biomarkers of Organophosphate Exposure Oksana Lockridge and LawrenceM. Schopfer
681
703
617 Therapeutic M e a s u r e s 49. Management of Organophosphorus Pesticide Poisoning Timothy C. Marrs and J. AllisterVale
715
633 Index
735
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Contributors
MICHAEL ADLER Neurotoxicology Branch, Pharmacology Division, USAMRICD (US Army Medical Research Institute of Chemical Defense), APG-AE, MD
STEPHANIE J. GARCIA Wake Forest University School of Medicine, Department of Pharamacology, Winston Salem, NC ROBERTA GOODALL Cholinesterase Investigation Unit, Department of Clinical Biochemistry, North Bristol Trust, The Lewis Laboratory, Southmead Hospital, Bristol, UK
MICHAEL ASCHNER Department of Pediatrics, Vanderbilt University, Nashville, TN DEBASIS BAGCHI Interhealth Research Center, Benicia, CA, and School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE
CHRISTOPHER I. GORDON US Environmental Protection Agency, Neurotoxicolgy Division, Research Triangle Park, NC MANIK C. GHOSH Department of Animal Physiology, Bose Institute, Calcutta, India
MANASHI BAGCHI Interhealth Research Center, Benicia, CA, and School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE
P. K. GUPTA Toxicology Consulting Services Inc., Bareilly, India
KULBIR S. BAKSHI National Academy of Sciences, Committee on Toxicology, Washington, DC
RAMESH C. GUPTA Toxicology Department, Breathitt Veterinary Center, Murray State University, Hopkinsville, KY
BRYAN BALLANTYNE Charleston, WV
NILGUN GURBUZ Department of Urology, Tulane University Health Sciences Center, New Orleans, LA
STEVEN I. BASKIN Biochemical Pharmacology Branch, Pharmacology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD
VERONIQUE HAUSCHILD Office of Emergency Management, US Environmental Protection Agency, Washington, DC COREY I- HILMAS Neurotoxicology Branch, Pharmacology Division, USAMRICD (US Army Medical Research Institute of Chemical Defense), Aberdeen Proving Ground-AE, MD
PHILIP J. BUSHNELL US Environmental Protection Agency, Neurotoxicology Division B 105-04, Research Triangle Park, NC JANICE E. CHAMBERS Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS
MASAKIYO HOSOKAWA Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Chiba, Japan
TOBY B. COLE University of Washington, Seattle, WA LUCIO G. COSTA University of Washington, Department of Environmental and Occupational Health Sciences, Seattle, WA
ANANT V. JAIN Toxicology Section, The University of Georgia, College of Veterinary Medicine, Athens Diagnostic Laboratory, Athens, GA
]. L. DE BLEECKER Ghent University Hospital, Neurology Department, Ghent, Belgium
DAVID A. JETIr National Institutes of Health, NINDS, Bethesda, MD
ANDRZEJ DEKHNDY Merz Pharmaceuticals GmbH, Preclinical Research and Development, Frankfurt Am Main, Germany
IOSEPH KING US Army Environmental Center, Aberdeen Proving Ground, MD
WOLF-D. DETI'BARN Vanderbilt University School Medicine, Department of Pharmacology, Nashville, TN
of
SHIGEYUKI KITAMURA Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
FRODE FONNUM Group of Molecular Neurobiology, Department of Biochemistry, Institute of Basal Medicine, University of Oslo, Oslo, Norway
PAMELA J. LEIN Oregon Health & Science University, Portland, OR OKSANA LOCKRIDGE University of Nebraska Medical Center, Eppley Institute, Omaha, NE
NARIAKI FUJIMOTO Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
MARCELLO LO'I'i'I Universita di Padova, Departimento di Medicina Ambientale e Sanita Pubblica, Padova, Italy
CLEMENT E. FURLONG University of Washington, Seattle, WA xi
x ii
Contributors
ANNA B. LOWIT US Environmental Protection Agency, Office of Pesticide Programs, Washington, DC
PAMELA I. ROWSEY School of Nursing, University of North Carolina, Chapel Hill, NC
CINA M. MACK Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC
HARRY SALEM USA SBCCOM, Edgewood CB Center, Aberdeen Proving Ground, MD
SUSAN L. MAKRIS US Environmental Protection Agency, Office of Research and Development, National Center for Environmental Assessment, Washington, DC
TETSIlO SATOH Laboratory of Biochemical Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, and HAB Research Laboratories, Ichikawa, Chiba, Japan
TIMOTHY C. MARRS University of Central Lancashire, UK and National Poisons Information Service, (Birmingham Center, UK)
KAI SAVOLAINEN Finnish Institute of Occupational Health, Department of Industrial Hygeine & Toxicology, Helsinki, Finland
LINDA A. Mr School of Nursing, Office of Nursing Research, University of Pennsylvania, Philadelphia, PA
LAWRENCE M. SCHOPFER University of Nebraska Medical Center, Eppley Institute, Omaha, NE
DEIAN MILATOVIC University of Washington, Seattle, WA
RAGHIIBIR P. SHARMA The University of Georgia, Department of Physiology & Pharmacology, College of Veterinary Medicine, Athens, GA
DAPHNE B. MOFFETY CDR US Public Health Service, Agency for Toxic Substances and Disease Registry, US Department of Health and Human Services, Atlanta, GA ANGELO MORETrO Universita di Padova, Departimento di Medicina Ambientale e Sanita Pubblica, Padova, Italy SPENCER IL MORTENSEN Syngenta Crop Protection, Inc., Ecological Sciences, Greensboro, NC VIRGINIA C. MOSER US Environmental Protection Agency, Neurotoxicology Division, Research Triangle Park, NC SHREE MILLAY Department of Medicine, McGill University, Montreal, Canada TOSHIO NARAHASHI Northwestern University, The Feinberg School of Medicine, Department of Molecular Pharmacology and Biological Chemistry, Chicago, IL DENNIS OPRESKO Toxicology and Hazard Assessment Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN STEPHANIE PADILLA US Environmental Protection Agency, Neurotoxicolgy Division B 105-06, NHEERL, Office of Research and Development, Research Triangle Park, NC
SURESH C. SIKKA Tulane University Health Sciences Center, New Orleans, LA VERA SIMEON-RHDOLF Institute for Medical Research and Occupational Health, Ksaverska cesta 2, Zagreb, Croatia THEODORE A. SLOTKIN Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC SIGRIIN HANNE STERRI Division for Protection, Norwegian Defence Research Establishment, Institutt Veien, Kjeller, Norway KAZUM! SUGIHARA Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan LESTER GRANT SULTATOS UMDNJ, Pharmacology and Physiology, Newark, NJ
Department
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TORE SYVERSEN Norwegian University of Science & Technology, Department of Neuroscience, Trondheim, Norway ]I.IN TANG Cerep Inc., Redmond, WA
OLAVI PELKONEN University of Oulu, Department of Pharmacology and Toxicology, Oulu, Finland
PALMER TAYLOR University of California at San Diego, Department of Pharmacology, La Jolla, CA
CAREY N. POPE Oklahoma State University, Department of Physiological Sciences, College of Veterinary Medicine, Stillwater, OK
GEORGE THORNE US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD
YONGCHANG QIAN Department of Integrative Biosciences, Texas A&M University, College Station, TX
EVELYN C. TIFFANY-CASTIGLIONI Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX
ZORAN RADIC University of California at San Diego, Department of Pharmacology, La Jolla, CA
CHARLES TIMCHALK Center for Biological Monitoring and Modeling, Pacific Northwest National Laboratory, Richland, WA
ARHN K. RAY Department of Animal Physiology, Bose Institute, Calcutta, India
KIRS! VAH,~,KANGAS University of Kuopio, Department of Pharmacology and Toxicology, Kuopio, Finland
ELSA REINER Institute for Medical Research and Occupational Health, Ksaverska cesta 2, Zagreb, Croatia
I. ALLISTER VALE National Poisons Information Service (Birmingham Centre) and West Midlands Poisons Unit, City Hospital, Birmingham, UK
IlM E. RIVIERE North Carolina State University, College of Veternary Medicine, Center for Chemical Toxicology Research & Phamacokinetics, Raleigh, NC RANDY L. ROSE Department of Molecular and Environmental Toxicology, North Carolina State University, Raleigh, NC
DAYA R. VARMA Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada VIJAYANAGARAM VENKATRAJ Department of Integrative Biosciences, Texas A&M University, College Station, TX
Contributors ANNABELLA VITALONE University of Bad, Bad, Italy ANNETrA WATSON Toxicology and Hazard Assessment Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN JAMES rc WILD Department of Biochemistry and Biophysics, Center for Environmental and Rural Health, Texas A&M University, College Station, TX
xiii
RANDALL L. WOLTIER Department of Pathology, Division of Neuropathology, University of Washington, Seattle, WA ROBERTYOUNG Toxicology and Hazard Assessment Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN SHIRLEY ZAFRA Interhealth Research Center, Benicia, CA CSABA K. ZOLTANI US Army Research Lab, MD
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Foreword KAI M. SAVOLAINEN Finnish Institute of Occupational Health, Helsinki, Finland
Consumers are protected by the setting of acceptable daily intake values. In the European Union, a separate communitylevel directive, 91/414/EC, is currently under revision for plant protection products according to which the safety of all pesticides is assessed. Similar pieces of legislation, rigorously enforced by health and other authorities, are in place in other industrialized countries. In the United States, the use of pesticides is regulated by the Federal Insecticide, Fungicide and Rodenticide Act, which originated in 1947, and the Food Quality Protection Act passed by the U.S. Congress in 1996 (Ecobichon, 2001). In developing countries, the situation is often much worse, and the information provided in this book is badly needed. There is a clear need to resort to OP and CM insecticides use in many developing countries since they are situated in climates that in addition to favoring the growth of crops also provide optimal breeding conditions for insects capable of destroying crops or causing communicable diseases such as malaria (Ecobichon, 2001). However, because there is such a lack of knowledge and no adequate legislation and regulations, OPs and CMs and other toxic pesticides are often misused, the protective measures are largely inadequate, and safe handling of crops is often inappropriate. In developing countries, there is also a lack of adequate infrastructure, including regulatory authorities, to enforce regulations and thus protect individuals who come into contact with pesticides, even when legislation and regulations are theoretically in place and the educational system does provide the necessary knowledge base to assess risks associated with the use of these Compounds (Rantanen et al., 2004). The literature on OPs and CMs is seemingly exhaustive (Ecobichon, 2001; Krieger, 2001). However, because ACHEinhibiting insecticides, as well as other pesticides, require marketing permission, most of the descriptive and much of mechanistic toxicology research has been carried out by the companies that manufacture these compounds. These data are often not publicly available, and much information
Highly toxic acetylcholinesterase (AChE)-inhibiting pesticides, organophosphates (OPs) and carbamates (CMs), are intensively used throughout the world and continue to be responsible for poisoning epidemics, especially in developing countries (e.g., Central American countries) (Wesseling et al., 2005). In industrialized countries, highly toxic OPs and CMs, and other toxic pesticides, are usually much better controlled, and the likelihood of occupational and other poisonings is relatively small. However, there is a continuous need to carefully assess the risks caused by the exposure and use in the occupational environment to workers as well as generally to consumers and other exposed groups. Although the literature on OP and CM insecticides is seemingly exhaustive and systematic, this is not the case. For example, there is really no truly comprehensive and in-depth analysis available on the toxicological data of the common AChE-inhibiting compounds, OPs and CMs. This book promises a welcome improvement by providing comprehensive coverage of the toxicology of these important group of pesticides. To my knowledge, this book is the first in-depth analysis of data on toxicology, risk assessment, and management, as well as the importance of OPs and CMs to society. I believe that this book will soon be on the bookshelves of researchers in academia and industry and risk assessors and managers within regulatory authorities. However, it would also be beneficial if we could convey the information contained in this book to the attention of policymakers and political and industrial decision makers and in this way multiply its impact in society and preserve human health. In this context, there are some issues that merit special consideration and that are relevant to this book in the field of toxicology and risk assessment of AChE inhibitors. In industrialized countries, highly toxic pesticides, including OPs and CMs, are regulated and controlled in the work environment via occupational exposure limits and by restrictions or bans on the use of the most toxic compounds. xv
xvi
Foreword
regarding toxicity of OPs and CMs is missing in the open literature. Therefore, there is a plethora of studies exploring mechanisms of acute and long-term toxicity of these compounds in experimental animals and man, but other areas are covered only vaguely, which means in effect that there is a very inconsistent database. In practice, this means that when an "old" AChE-inhibiting insecticide or another old pesticide is subjected to reevaluation for remarketing permission, the data available to the risk assessors are far from complete and additional studies are often required. Alternatively, the data may be old or scattered throughout the open literature and, hence, laborious to find. This book will improve the situation remarkably for evaluating the OP and CM pesticides. To carry out a comprehensive analysis of the often inconsistent and incomplete database on OPs and CMs is often a highly demanding task for risk assessors and decision makers. The situation is especially difficult if one wishes to combine analytical thinking with professional risk assessment and management that also takes into consideration societal impact, perception of risks, and the significance of these compounds to society. The editor, authors, and publisher of this book have decided to face this challenge by publishing this unique book dealing in an in-depth manner with various aspects of the toxicology of OPs and CMs. As indicated previously, there is a plethora of original papers and reviews on the toxicology of this group of compounds, especially on the mechanisms of immediate toxic actions of these compounds. Much of this information is based on studies dealing with AChE-inhibiting warfare agents, such as soman, tabun, and sarin (Savolainen, 2001). Although this information is important for protecting the general public from terrorist attacks, an even more important goal is to create a comprehensive systematic database on the toxicity of these compounds to allow a reliable and exhaustive assessment of their risks to workers and consumers and other exposed individuals. This book is unique in being able to provide a thorough assessment of all aspects of toxicology and risk assessment of OP and CM AChE-inhibiting compounds. The list of authors of the book is impressive - - the editor is to be congratulated for bringing together such a unique group of experts from various fields of OP and CM toxicology and risk assessment. The book is divided into nine sections that deal with different aspects of OP and CM toxicology and risk assessment. I am especially pleased by Section I, with its chapters on therapeutic uses, community preparedness, and epidemiology of OP and CM compounds. In Section III, noncholinesterase mechanisms of central and peripheral neurotoxicity, paraoxonase polymorphisms, and the development of tolerance are topics of special interest. The main body of the book (Section IV) discusses organ toxicity in especially interesting chapters dealing with in vitro testing, reproduction, placental toxicity, endocrinology, and effects on the immune system. In
Section V, special-interest areas are covered, and items of personal interest to me are those dealing with oxidative stress, DNA damage and gene expression, and occupational toxicology and hygiene. Special merits of the book, covered in Section VI, are the chapters that deal with in-depth risk assessment and risk management. These issues are crucial to ensure that the research conducted on the toxicology of OPs and CMs actually has an impact on human health and society at large. Another merit of the book is found in Section VII, in which issues dealing with ecotoxicology are reviewed in the context of human toxicology. Ultimately, human toxicology and ecotoxicology of OPs and CMs are interrelated and inseparable. A novel topic is introduced in Section VIII, a discussion on biomarkers of OP exposure. This topic has direct relevance both to human exposure and to effective assessment and, therefore, it holds major potential for prevention of risks induced by OPs. This book would not be a comprehensive presentation on issues relevant to OPs and CMs without a chapter on management of OP poisonings, and fortunately this issue is well covered in Section IX. The book is an extremely welcome addition to the literature on the toxicology and risk assessment of OPs and CMs. The chapters mentioned previously are personal choices and issues that I consider to be of special interest. As a whole, the book provides a thorough and analytical coverage of issues important in the field, without omitting anything of importance, and emphasizes new issues that have not been assessed in previous textbooks or reviews. Thus, it is a credit that this book has achieved its goal, to review the toxicology of OPs and CMs, in an indepth and comprehensive manner, including important and novel issues such as placental and reproductive toxicology and the effects of these compounds on the immune system. This book will be intensively used by not only scientists and teachers in academia, scientists in the industry, and regulators and decision makers but also students, who should be encouraged to study and learn from its wisdom.
References Ecobichon, D. J. (2001). Toxic effect of pesticides. In Casarett and Doull's Toxicology. The Basic Science of Poisons (C.D. Klaassen, Ed.), 6th ed., pp. 763-810. McGraw-Hill, New York. Krieger, R. (Ed.) (2001). Handbook of Pesticide Toxicology. Principles, 2nd ed. Academic Press, San Diego. Rantanen, J., Lehtinen, S., and Savolainen, K. (2004). The opportunities and obstacles to collaboration between the developing and developed countries in the field of occupational health. Toxicology 198, 63-74. Savolainen, K. (2001). Understanding the toxic actions of organophosphates. In Handbook of Pesticide Toxicology. Principles (R. Krieger, Ed.), 2nd ed., pp. 1013-1042. Academic Press, San Diego. Wesseling, C., Corriols, M., and Bravo, V. (2005). Acute pesticide poisoning and pesticide registration in Central America. Toxicol. AppL Pharmacol., 207, $697-$705.
Uses, Abuses, & Epidemiology
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CHAPTER
|
Introduction
RAMESH C. GUPTA Murray State University, Hopkinsville, Kentucky
By the turn of the 21st century, the development and use of organophosphate (OP) and carbamate (CM) compounds were greater than ever before. This trend will most likely continue because scientists are discovering new applications for these compounds. Both OPs and CMs are inhibitors of acetylcholinesterase (AChE) enzyme, which terminates the action of the neurotransmitter acetylcholine (ACh). Compounds with strong AChE-inhibiting potential are used as toxicants (e.g., pesticides and nerve agents), whereas those with weak ACHEinhibiting potential are used as prophylactic agents against nerve agent poisoning or as therapeutic agents in conditions such as glaucoma, myasthenia gravis, and Alzheimer's disease. In addition, some of these compounds are used as flame retardants, whereas others are misused in intentional and malicious poisonings. Currently, AChE-inhibiting compounds constitute the largest class of pesticides used in both industrialized and developing nations. The history and evolution of OPs and CMs is very interesting and intriguing. The earliest documentation of the synthesis of an OP compound, tetraethyl pyrophosphate, was in 1854 when Phillipe de Clermont presented a report to the French Academy of Sciences. Nearly 80 years later, in 1932, Lange and Kruger described the synthesis of dimethyl and diethyl phosphorofluoridate and noted that inhalation of their vapors produced dimness of vision and a choking sensation. Probably, these observations led Gerhard Schrader, a chemist, to the exploration of the OP class of compounds while he was engaged in the development of insecticides for I. G. Farbenindustrie. One of the earliest OP insecticides synthesized by Schrader was parathion, which is still commonly used worldwide. Prior to World War II (WWII), under the authority of the German Ministry of Defense, his priority shifted from insecticides to chemical warfare agents. The result was the development of diisopropyl phosphorofluoridate and then the development of considerably more toxic OP compounds of the G series (tabun, satin, and soman), which were intended to be used as nerve gases/agents. In the 1950s, agent VX was synthesized in the United Kingdom with a potency manyfold greater than nerve agents of the G series. At the same time, a compound of the V series (VR), with a supertoxicity, was synthesized by the Soviet military. In 1950, two separate accidental exposures occurred at Toxicology of Organophosphate and Carbamate Compounds
Dugway Proving Ground, wherein workers developed signs and symptoms, and laboratory evidence confirmed mild nerve agent exposure in a test area 3 days after a satin test. Since the 1980s, these agents have been used in wars and by dictators and terrorists. For example, sarin was used in Iraq against Kurdish villages in 1988 and in the Tokyo subway attacks in Japan in 1994 and 1995. Currently, many countries possess these deadly nerve agents, and in the current world situation the possibility exists that these compounds may be misused as chemical weapons of mass destruction (CWMD). In fact, OP nerve agents have received increasing attention as concerns about chemical warfare have intensified since the terrorist attack in the United States on September 11, 2001. After WWII, thousands of OP derivatives were synthesized worldwide in a search for compounds with species selectivity and less toxicity that could be used as insecticides more safely. In 1950, malathion was synthesized, and it has been the most popular insecticide for more than 50 years for use against certain insects, especially mosquitoes and medflies. Malathion has always been considered one of the safest OPs. However, in 1976 in Pakistan, out of 7500 spray men, 2800 became poisoned and 5 died from isomalathion that was produced during storage of formulated malathion. Many such poisoning incidences have occurred in the past with several other OPs due to their accidental or inadvertent use. Today, more than 100 OPs are in use for a variety of purposes, such as protection of crops, grains, gardens, and public health. Although OP insecticides are less toxic than the nerve agents, the illness they produce clinically resembles that produced by nerve agents. The knowledge of autonomic pharmacology, especially the cholinergic system, enabled us to understand the mechanism of toxicity of OPs and CMs. Subsequently, CMs such as aldicarb were synthesized based on the knowledge of ACh chemistry. These compounds were found to be the most toxic insecticides of the anti-AChE class. In the early 1950s, several nucleophilic agents (hydroxylamine, hydroxamic acid, and oximes) were developed as antidotes for reactivation of inhibited AChE against OPs. With a thorough understanding of the chemistry of ACh, AChE, and OPs, pralidoxime was synthesized, which was found to have 1 million times greater potency than hydroxylamine for reactivation of inhibited Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
4
S ECTI O N I 9Uses, Abuses, & Epidemiology
ACHE. Today, more than a dozen oximes are available, some of which are more effective against OP nerve agents and others are more effective against OP pesticides. The history of CMs is somewhat older than the history of OPs. In 1840, the Calabar bean (ordeal poison), from a perennial plant Physostigma venenosum, was brought to England from a tropical part of West Africa, where it was used for witchcraft. Approximately 25 years later, physostigmine (eserine alkaloid) was isolated by several investigators and used to treat glaucoma. Almost 50 years later, an aromatic ester of carbamic acid, neostigmine, was synthesized and used in the treatment of myasthenia gravis. It was not until the 1960s and 1970s that carbamates (esters of carbamic acid) were synthesized for pesticidal use. Today, CMs are preferred for pesticide use over OPs because some OPs have been found to be extremely toxic, whereas others induce delayed neuropathy in humans and animals. Carbaryl was the first CM compound used as an insecticide. Whereas OPs are irreversible ACHE inhibitors and extremely toxic, CMs are reversible ACHE inhibitors and therefore considered relatively less toxic. Although based on acute toxicity, some of the CMs, such as aldicarb, carbofuran, and many others, are extremely toxic. In 1984, an estimated 400,000 people were exposed to a toxic methyl isocyanate gas (used in the production of CM pesticides) that leaked from the Union Carbide plant in Bhopal, India. From this catastrophic incident, approximately 8000 humans and 4000 animals died. In 1985, an unprecedented outbreak of aldicarb poisoning occurred in which approximately 2000 California residents became sick due to consumption of contaminated melons. Since the early 1980s, both OPs and CMs have been used for multiple purposes, such as pesticides (crop and grain protection, indoors and around homes) and in veterinary (ectoparasiticides and endoparasiticides) and human medicine (in neurodegenerative diseases such as Alzheimer's disease). In human medicine, some of these compounds are also prescribed in myasthenia gravis and glaucoma and as prophylaxis to combat anticipated nerve agent poisoning. Like OPs, thousands of CMs have been synthesized, but not more than two dozen compounds have been used practically. In terms of volume, currently the use of CMs exceeds the use of OPs. OPs and CMs are the most commonly used pesticides throughout the world. This is partly due to their lack of residue persistence in the environment and in exposed individuals and also due to lesser resistance development in insects compared to the organochlorine pesticides. From the public health standpoint, in today's world the use of pesticides is a must rather than an option. For example, sporadic incidences of West Nile virus are reported in many countries, whereas malaria is still a major problem in developing countries. In both cases, the common vector is the mosquito. Without the use of pesticides against vectors of diseases, the impact on human and animal health would be devastating and the economic loss would be enormous. On the one hand, the world is greatly benefited from the use of
pesticides; on the other hand, pesticides are major contributors to environmental pollution. Many OPs and CMs are extremely toxic, and the majority of them lacks species selectivity and so, because of their global use, they constantly pose a threat to the environment, human and animal health, aquatic systems, and wildlife. It is important to note that OP and CM pesticides are encountered in intentional poisonings in humans and malicious poisonings in animals. Today, carbofuran is the pesticide most often associated with accidental and malicious poisoning in companion and domestic animals because of its widespread availability and extreme toxicity. Depending on the magnitude, frequency, and length of exposure, these compounds can produce minor health effects, such as mild discomfort or chest pain, or effects as severe as paralysis, coma, and death. The World Health Organization estimates that approximately 3 million people worldwide suffer from acute pesticide poisoning annually. By employing in vivo and in vitro models, these compounds are known to produce a variety of toxicological effects on the central nervous system, peripheral nervous system, cardiovascular, ocular, neurobehavioral, immunological, reproductive, placental, cutaneous, and other body systems, in addition to endocrine disruption, oxidative stress, and carcinogenesis. With the advent of sophisticated technologies, highly sensitive potentiometric and amperometric biosensors have been developed for qualitative and quantitative detection and monitoring of chemical warfare agents and OP and CM pesticides. Essentially, these biosensors aid in chemical and food safety, environmental monitoring, and agricultural production. During the past few years, investigators in the field of anticholinesterases have realized the need for a comprehensive compendium that can provide in-depth knowledge on various aspects of these compounds, including their use, toxicity, safety, regulations, and prophylactic and therapeutic measurements. This reference book, which is a collective work of approximately 100 subject experts from many countries, offers a plethora of cutting-edge knowledge on various aspects of OPs and CMs. The book is organized into nine sections with a total of 49 chapters. The editor and authors have made every effort to cite every important work in the field and avoid any duplication, but the possibility of some omissions and duplications certainly exists. Since OPs and CMs are used worldwide in agriculture, in gardens, in and around homes and offices, in therapeutic applications, in intentional and malicious poisonings, and possibly as CWMD, the book is intended for students and teachers; toxicologists; physicians; public health personnel and administrators; risk and safety assessors; local, state, federal, and international pesticide regulators and policy makers; industrial and agricultural watchdog groups; and medical, veterinary, and environmental advocacy groups. The editor truly appreciates the hard work and sincere efforts of each author, without which this book would not have been possible.
CHAPTER
Classification and Uses of O r g a n o p h o s p h a t e s and Carbamates RAMESH C. GUPTA Murray State University, Hopkinsville, Kentucky
I. INTRODUCTION Organophosphates (OPs) are a large class of chemicals. Since World War II, an estimated several thousand OPs have been synthesized for various purposes. The majority of these compounds are used as pesticides, whereas others are used as nerve agents, flame retardants, and parasiticides in veterinary medicine. Different OP compounds have structural similarities within classes. All OPs definitely share one thing in common: They all have a phosphorus atom and a characteristic phosphoryl bond ( ~ O ) or thiophosphoryl bond (P=S). Essentially, OPs are esters of phosphoric acid with varying combinations of oxygen, carbon, sulfur, or nitrogen attached. Of course, the chemistry of these compounds is much more complex and classification is somewhat confusing. In fact, complexity in classification of OPs arises due to different side chains attached to the phosphorus atom and the position at which the side chains are attached. More than 50 years ago, the Anglo-American system reached an agreement to adopt an "international nomenclature" instead of individual systems from four countries (British, Swedish, German, or American). However, none of the systems has ever been universally accepted. Compared to OPs, carbamate (CM) pesticides are of relatively recent origin and constitute another important group of pesticides. In addition to their use as pesticides, CMs are used as drugs of choice in human medicine against Alzheimer's disease, myasthenia gravis, and glaucoma and in veterinary medicine as parasiticides. Classification of CMs is simpler than classifying OPs. Some CMs have structural similarity with the neurotransmitter acetylcholine (ACh), and therefore they cause direct stimulation of ACh receptors, in addition to acetylcholinesterase (ACHE) inactivation. Although thousands of CMs have been synthesized, only a few dozen have practical utility. The classification of OPs and CMs presented in this chapter is based on their chemical structures and intended use or any syndrome they produce. Toxicology of Organophosphate and Carbamate Compounds
II. ORGANOPHOSPHATES Currently, there are hundreds of OP compounds in use, which are derivatives of phosphoric, phosphonic, or phosphinic acid. Throughout this chapter and the book, the term organophosphate is used as a generic term to include all the organic compounds containing phosphorus. These compounds are classified based on side chains and other elements attached to the phosphorus atom.
A. Types of Organophosphates There are at least 13 types of OPs, which are briefly presented in Table 1. The OPs that are derivatives of phosphoric or phosphonic acid possess anticholinesterase activity, unlike those that are derivatives of phosphinic acid. There are some OP compounds that do not conform to the structural requirement as shown in Table 1, but they possess anti-AChE activity. Usually, OP compounds have two alkyl substituents and an additional substituent group (leaving group), which is more labile to hydrolysis than the alkyl groups (Marrs, 1993). It is important to note that phosphorothioates (P---S) possess minimal or no anticholinesterase (anti-AChE) activity and require desulfuration to the analogous oxon before acquiring anti-AChE activity. Also, not all OPs exert anti-AChE activity, and therefore they are of low toxicity. For example, S,S,S-tributyl phosphorotrithioate and S,S,S-tributyl phosphorotrithioite (merphos), which are used as defoliants, and glyphosate and gluphosinate, which are used as herbicides, are of low mammalian toxicity.
B. OP Pesticides The majority of OP compounds are used as pesticides, and chemical descriptions for commonly used compounds are given in Table 2. Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
6
S ECTI O N I 9 Uses, Abuses, & Epidemiology
TABLE 1. Types of Organophosphates a Type
Chemical structure
Examples
O
Chlorfenvinphos Dichlorvos Monocrotophos Tri-o-cresyl phosphate Trichlorfon
Phosphates
II
RO~P~OR
I
OR O
Phosphonates
II
ROmPeR
I
OR O
Phosphinates
Glufosinate
II
R~P~R
I
OR
Phosphorothioates
(S--)
S
Bromophos Diazinon Fenthion Parathion Pirimiphos-methyl EPN Leptophos
II I
RO~P~OR OR
Phosphonothioates
(S--)
S
II I
ROmPeR OR
Phosphorothioates (S-substituted)
O
Demeton-S-methyl Echothiophate
II
RS~P~OR
I
OR
Phosphonothioates (S-substituted)
O
VX
II
RS~P~R
I
OR
Phosphorodithioates
O
II
RS~P~SR
or
I
OR S
II
RS~P~OR
I
OR O
Phosphorotrithioates
Azinphos-ethyl Azinphos-methyl Dimethoate Disulfoton Malathion Methidathion DEF (tribufos)
II
RS~P~SR
I
SR O
Phosphoramidates
II
/R
I OR
\R
RO ~ P ~ N
Phosphoramidothioates
S
II
/R
RO ~ P ~ N
I OR o
or
/a
I
\R
OR
Methamidophos Isofenphos
\R
II
RS ~ P ~ N
Fenamiphos
(continues)
CHAPTER 2 9Classification of OPs and CMs
7
TABLE 1. (continued) Type Phosphorofluoridates
Chemical structure O II RO~P~F
Examples Diisopropyl phosphorofluoridate (DFP)
I
OR
Phosphonofluoridates
O II
RO~P~F
I
R
Cyclosarin Sarin Soman
aAdapted fromMarrs (1993).
C. OP Nerve Agents/Gases Nerve agents of the OP group include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX. Soman, sarin, and cyclosarin are phosphonofluoridates, and VX is a phosphonothioate. Whereas soman has four isomers (C + P - , C - P - , C + P+, and C - P+), sarin and VX each have two isomers. V X is a mixture of two enantiomers resulting from the chiral center at the phosphorus atom, designated as P(+) and P ( - ) . There are significant differences in the reported toxicity and AChE inhibition rates of these isomers of nerve agents. Toxicological significance of stereoisomerism for other OPs, which have the potential of inhibiting ACHE, has been described (Battershill et al., 2004). This makes the ability to distinguish between them desirable for toxicological studies or the development of antidotal therapies (Benschop and De Jong, 1988; Smith, 2004; Ku~a and Kassa, 2004). Based on acute toxicity, VX is the most toxic compound among all the nerve agents. OP nerve agents are extremely toxic and have been used in wars and by terrorists on several occasions. They irreversibly inhibit the enzyme AChE at its active site. People who work at military sites where these nerve agents are stored may potentially be exposed. Soldiers and military personnel can be exposed to these compounds during war, and the general population can be exposed by accidental release from a military storage facility and during their transportation or destruction. For further details about OP nerve agents, see Chapter 5.
D. OPs Causing Delayed Neurotoxicity/ Neuropathy OP compounds that produce delayed neurotoxic effects are esters of phosphorus-containing acids. More than 35 years ago, tri-o-cresyl phosphate (TOCP) was known to produce delayed neurotoxic effects in humans and chickens characterized by ataxia and weakness of the limbs, developing 10-14 days after exposure (Johnson, 1969). This syndrome is called OP-induced delayed neuropathy (OPIDN). TOCP and certain other compounds have minimal or no anti-AChE
properties; however, they cause phosphorylation and aging (dealkylation) of a protein in neurons called neuropathy target esterase, and subsequently lead to OPIDN. Today, many compounds, such as diisopropyl phosphorofluoridate, N,N'diisopropylphosphorodiamidic fluoride (mipafox), tetraethyl pyrophosphate, paraoxon, parathion, o-cresyl saligenin phosphate, and haloxon, are known to produce this syndrome. For details of OPIDN syndrome, see Chapter 25.
E. OPs Causing Intermediate Syndrome OP insecticide-induced intermediate syndrome (IMS) was reported for the first time in human patients in Sri Lanka in 1987 (Senanayake and Karalliede, 1987), Since then, this syndrome has been diagnosed in OP-poisoned patients in South Africa (1989), Turkey (1990), Belgium (1992), the United States (1992), Venezuela (1998), France (2000), and elsewhere. IMS is usually observed in individuals who have ingested a massive dose of an OP insecticide either accidentally or in a suicide attempt. IMS is clearly a separate clinical entity from acute toxicity and delayed neuropathy. A similar syndrome has also been observed in dogs and cats poisoned maliciously or accidentally with massive doses of certain OPs. OPs that are known to cause IMS include bromophos, chlorpyrifos, diazinon, dicrotophos, dimethoate, fenthion, malathion, merphos, methamidophos, methyl parathion, monocrotophos, omethoate, parathion, phosmet, and trichlorfon. These compounds and IMS are discussed further in Chapter 26.
F. OPs Used as Flame Retardants Several OPs are used as fire retardants. Chemical structures of three commonly used compounds are as follows: 1. Tris (2-chloroethyl) phosphate (TCEP) O = P - (OCH2CH2C1)3 2. Tris (2-chloropropyl) phosphate (TCPP) O = P - (OCH2CHC1CH3)3 3. Tris (1,3-dichloroisopropyl) phosphate (TDCPP) O = P - (OCH(CH2C1)2)3
TABLE 2. A Brief Chemical Description of Commonly Used OP Pesticides ~
Chemical (CAS No.)
Chemical structure
Chemical name/ empirical formula
Molecular weight
Oral LD50 in rat (mglkg)
Dermal LD50 in rabbit (mglkg)
Acephate (30560- 19- 1)
0,S-dimethyl acetylamidothiophosphate C4Hl$rJ03PS
183.17
866
>2000
Azinphos-ethyl (2642-71-9)
0,O-diethyl S-[(4-0XO-1,2,3-benzotriazin3(4H)-yl) methyl] dithiophosphate
345.38
13
250
317.32
5
220
c12H16N303PS2 Azinphos-methyl (86-50-0)
0,O-dimethyl S-[(~-OXO1,2,3-benzotriazin3(4H)-yl) methyl] dithiophosphate ClOH12N303PS2
Bromophos (2104-96-3)
0-(4-bromo-2, 5dichlorophenyl) 0,O-dimethyl thiophosphate CgHgBrC1203PS
366.00
1600
2188
Cadusaphos (95465-99-9)
S,S-di-sec-butyl 0-ethyl dithiophosphate
270.40
391
143
342.87
6
22
c1OH2302PS2
Carbophenothion (78619-6)
S-{ [(4-chlorophenyl)thio]methyl} 0,O-diethyl dithiophosphate CllH16C102PS3 -i-
Chlorethoxyphos (54593-83-8)
Chlorfenvinphos (470-90-6)
0,O-diethyl 0-(1,2,2, 2-tetrachloroethyl) thiophosphate C6H11C1403PS
2-chloro-l-(2,4-dichloro-pheny1)
336.00
1.8
12.5
359.57
12
3200
350.59
135
2000
322.53
94 1
2000
362.77
13
314.27
125
vinyl diethyl phosphate
C12H14C1304P
Chlorpyrifos (2921-88-2)
0,O-diethyl
0-(3,5,6-trichloropyridin-2-y1)
thiophosphate CgHllC13N03PS
Q
Chlorpyriphos-methyl (5598-13-0)
0,O-dimethyl
0-(3,5,6-trichloropyridin-2-y1)
thiophosphate C7H7C13N03PS
Coumaphos (56-724)
Crotoxyphos (7700-17-6)
O-(3-chloro-4-methyl-2-oxo-
2H-chromen-7-yl) 0, 0-diethyl thiophosphate C14H16C105PS
1-phenylethyl (2E)-3-[(dimethoxyphosphory1)oxy]but-2-enoate
385
C14H1906P
(continues)
TABLE 2. (continued)
0
oI " \
0 -r
~ ~
r~
0-(4-~yanophenyl) 0,O-dimethyl thiophosphate CgHloN03PS
243.22
610
o
0,O-diethyl 0-[2-(ethy1thio)ethyll thiophosphate (C~H=JO)~PSOC~H~SC~H~
258.34
0,O-diethyl 0-(2-isopropyl6-methylpyrimidin-4-yl) thiophosphate
304.35
300 CD
379
220.98
25
~
59
237.19
22
~
223
229.26
250
400
r,.)
!
~ u
I 0
Z
~
c 12H21N203PS
0
co
"~.
d
2,2-dichlorovinyl dimethyl phosphate C4H7C1204P
m -r
o
ff \ I .0 O--n--O
0
o
0 y
II \
0
,.k, oi
Y-~
-1-
_~ o--z
oco
( 1 0 - 3-(dimethylamino)-
1-methyl-3-oxoprop-1-en-1-yl dimethyl phosphate o
I 0
z
0
~#o
C8H16N05P CD
0
0
C5H12N03PS2
.o
0,O-dimethyl S-[2-(methylamino)-2-oxoethyl] dithiophosphate ~ Eo~z
"I0 I "l-
00
m
I / O--n--O
z~(O
0/ \ "1I o-o o-I-
0 -I-
o
~9 o I
Dimethoate (60-51-5)
,-~
-r
\ /0
..%\ ~
o
o "1-
0
,.c:l~ 0
0
Dicrotophos (141-66-2)
9
o
-r"
0
0
#
0 I'T"
o ~ i"
"~
Dichlorvos (62-73-7)
8
o-J
/ \0
l..
"0 "T"
O" -r
I
e~
Diazinon (33341-5)
2.5
O0
co
=7~
5LJ
s 7
oV
N
i0 0
L
800
,Z
co
0 - - o ~:
o~
o
~o
e~
Demeton-0 (806548-3)
Dermal LD50 in rabbit (mgntg)
x:
Cyanophos (2636-26-2)
Chemical name/ empirical formula
=
Oral LD50 in rat (mg/kg)
0
"~ .=
Molecular weight
Chemical structure
@
Chemical (CAS No.)
"I0
274.40
2
o
~<~0
=
0
I'~ t-,,l
~
0,O-diethyl S-[Z-(ethylthio)ethyl] dithiophosphate
-r
Disulfoton (298-04-4)
6
o
'm"
%
0 -n-O
/
C8Hl 902ps 3 / no c-r
,.~
c,N
o ~
.~
r.~
C9H2204P2S4
-r
o
no
0
o---~
t-,,I t,~
915
27
0-{4-[(dimethylamino) sulfonyl]phenyl} 0,O-dimethyl thiophosphate
%
"r"
15.3
384.48
~
"=
--
00~j
~
o~%
,
oo t-~
v.,
co
"~
-~,
I
O,O,O',O'-tetraethyl S,S'-methylene bis(dithioph0sphate)
0
I
t"q
Ethion (563-12-2)
35
c,,r
325.34
t'N
~ 0
/
~
9
2730
9
o~
II
0 ,.=
o,~0
c1OH 16N05PS2 <0
r~
O--Z--O
~'~ I
" " -S
~~
i.
Famphur (52-85-7)
co
,-
o 0
Z - - O
11
4~
0
~~
!
~ ,,,,i
-r"
co
I
t'-,l
Ethyl 3-methyl-4-(methylthio) phenyl isopropylamidophosphate
-r o--~
~
r~ t'-,l
Fenamiphos (22224-92-6)
303.36
e~
o
I
"1-
II
0--~---0
0 co "r
c13H22N03PS
c0
\
no
t'N
277.23
o
I"~ t"q
~
250
1300
r a= c~ 9
=
.,.w
0,O-dimethyl 0-(3-methyl-4-nitrophenyl) thiophosphate CgH12N05PS
o0 "r"
o
u
II
co
-r
0
0--13---0
I
C"]
I o/ o,,
-r
o 4
o
""~
Fenitrothion (122-14-5)
0
~'-b (continues)
TABLE 2. (continued) Chemical (CAS No.) Fenthion (55-38-9)
Chemical structure
Chemical name/ empirical formula 0,O-dimethyl 0-[3-methy1-4-(methylthio) phenyl] thiophosphate
Molecular weight
Oral LD50 in rat (mgfltg)
Dermal LD50 in rabbit (mg/kg)
278.33
255
330
246.33
8
25
257.21
365
>1000
169.07
4300
>5000
198.16
2000
>4000
263.08
3925
>5010
c1OH 1503Ps2
Fonofos (944-22-9)
0-ethyl S-phenyl ethylphosphonodithioate C 1OH15ops2
Formothion (2540-82-1)
S-{ 24formyl (methyl)
amino]-2-oxoethyl) 0,O-dimethyl dithiophosphate C6H12N04PS2
Glyphosate (1071-83-6)
N-(phosphonomethy1)-glycine
Glufosinate ammonium (77182-82-2)
2-amino-4-[hydroxy(methyl) phosphoryl]butanoicacid ammoniate
C3HgNO5P
CSH 15N204P Glyphosine (2439-99-8)
N,N-bis(phosphonomethy1) glycine
C4H1 1NO8P2
Isazophos (42509-80-8)
Isofenphos (25311-71-1)
O-(5-chloro-l-isopropyl-1H-l,
313.75
40
>3100
345.40
32
162
330.36
885
4000
2,4-triazol-3-y1)0,O-diethyl thiophosphate CgH17ClN303PS
Isopropyl2- { [ethoxy (isopropylamino)phosphorothioyl] oxy ]benzoate C15H24N04PS
Malathion (12 1-75-5)
Diethyl 2- [(dimethoxyphosphorothioyl) thio]succinate c1OH1906ps2
3-
w
Methamidophos (10265-92-6)
0,s-dimethyl amidothiophosphate C2HgN02PS
141.13
13
110
Methidathion (950-37-8)
S-[(5-methoxy-2-oxo-1,3, 4-thiadiazol-3(2H)-yl)methyl] 0,O-dimethyl dithiophosphate
302.33
25
200
263.21
9
63
224.15
3
16
C6HllN204PS3 Methyl parathion (298-00-0)
0,O-dimethyl 0-(4-nitrophenyl) thiophosphate C8H1floSPs
Mevinphos (7786-34-7)
Methyl (2E)-3-[(dimethoxyphosphoryl)oxy]but-2-enoate C7H1306P
(continues)
TABLE 2. (continued) Chemical (CAS No.) Monocrotophos
(6923-224)
Chemical structure
Chemical name/ empirical formula Dimethyl (I@- 1 -methyl-3-(methylamino)3-oxoprop- 1 -en- 1-yl phosphate
Molecular weight
Oral LDsO in rat (mg/kg)
Dermal LDs0 in rabbit (mg/kg)
223.16
8
354
213.19
50
1400
C7H14N05P Omethoate ( 1 1 13-02-6)
0,O-dimethyl S-[2-(methylamino)-2-oxoethyl] thiophosphate C~HI~NO~PS
Paraoxon
Diethyl4-nitrophenyl phosphate
(31 1 4 5 - 5 )
Parathion
(56-38-2)
275.19
1.8
291.26
3
320.36
200
C10H14N06P
0,O-diethyl 0-(4-nitrophenyl) thiophosphate
6.8
C10H14N05PS
Phenthoate
(2597-03-7)
Ethyl [(dimethoxyphosphorothioyl)thio] (pheny1)acetate
4000
C12H1704PS2
Phorate
(298-02-2)
0,O-diethyl S-[(ethylthio)methyl] dithiophosphate C7H1702PS3
260.38
1.6
2.5
Phosmet (732-1 1-6)
Phosphamidon (13 171-21-6)
Phoxim (14816-18-3)
S-[( 1,3-dioxo-1,3-dihydro-
2H-iso-indol-2-yl)methyl] 0,O-dimethyl dithiophosphate c11 12N04PS2 (lZ)-2-chloro-3-(diethylamino)1-methyl-3-oxoprop-1-en-1-yl dimethyl phosphate C10H19ClNO.jP
Phenylglyoxylo-nitrileoxime, 0,O-diethyl phosphothioate
317.32
147
3160
299.69
15
125
289.30
1845
1126
c12H15N203PS
u1
Profenofos (41 198-08-7)
0-(4-bromo-2-chlorophenyl) 0-ethyl S-propyl thiophosphate C11H15BrC103PS
373.63
400
472
Propetamphos (31218-834)
Isopropyl (2E)-3-{ [(ethylamino)(methoxy) phosphorothioyl]oxy} but-2-enoate
281.31
82
2300
298.30
65
340
c1OH20N04PS
Quinalphos (13593-03-8)
0,O-diethyl 0-quinoxalin-2-yl thiophosphate C12H15N203PS
(continues)
TABLE 2. (continued) Chemical structure
Chemical name/ empirical formula
Oral LD50 in rat (mg/kg)
Dermal LD50 in rabbit (mg/kg)
~..=
Molecular weight
O
Chemical (CAS No.)
~
1250
2000
o~
0,O,O,0-tetraethyl dithiodiphosphate
322.32
5
-
I
322.45
107
820
288.43
1.6
o
o
321.55 ,.4
0,O-dimethyl 0-(2,4,5-trichlorophenyl) thiophosphate C8&$1303PS o
~
oo
Ronnel (299-84-3)
o ,~
~
.~
~
.c
o
~ I "~ oo
Sulfotepp (3689-24-5)
~
C8H2005P2S2
~
oo
9, ~
.~
~
~o
0-[4-(methylthio)phenyl]
~
0-ethyl
"~Y
~o ~ 0 0
r~
Sulprofos (35400-43-2)
I~
o
S-propyl dithiophosphate 0
12H1902PS3
~
o
o6
S- [(tert-butylthio)methyl]
~
16
9
c~
Terbufos (13071-79-9)
o
I
0,O-diethyl dithiophosphate r,.)
C9H2102PS3
83
280
630
>2100
oo
313.32
o
-'
0,O-diethyl O-(l-phenyl-lH-1,2, 4-triazol-3-yl) thiophosphate
oo
I
oo
O
o~
O
Triazophos (2401747-8)
~
c12H16N303PS
c~
A
257.44
~-~
0
N
Dimethyl (2,2,2-trichloro-1-hydroxyethyl) phosphonate C&$1304P -~o
'-~
Trichlorfon (52-68-6)
~
o
CHAPTER 2 9Classification of OPs and CMs These compounds are haloalkyl phosphates and act as flame retardants when added to polymers. They are used extensively in important commercial products, such as textiles, building materials, and packaging materials (Aston et al., 1996). TCEP is used as a flame retardant additive in such products as polyurethane and polyisocyanurate foams, carpet backing, flame retardant paints, lacquers, coatings, resins, and adhesives (U.S. Environmental Protection Agency, 1988; Green, 1993). TCPP and TDCPP are also used as flame retardant additives in flexible plastic products such as polyurethane foam. These compounds are used worldwide; as a result, contamination of river and lake water has been noted in several countries, including Japan, Canada, and the United States, and in Europe. Toxicity tests have shown these compounds to be toxic to aquatic organisms, and they are a concern in terrestrial ecosystems following chronic exposure. Mammalian toxicity is of less concern since these compounds do not possess anti-AChE activity.
III. CARBAMATES The CM compounds are esters of carbamic acid. Unlike OPs, CM compounds are not structurally complex. CMs are used as pesticides in agricultural crops and gardens, as therapeutic drugs in human medicine (Alzheimer's disease, myasthenia gravis, glaucoma, and in prophylaxis of OP nerve gas poisoning), and in veterinary medicine (as parasiticides).
A. CM Pesticides The volume of CMs used exceeds that of OPs because they are considered to be safer than OPs. Some of the commonly used N-methyl carbamate insecticides are shown in Table 3. For other CMs, readers are referred to previous publications (Kidd and James, 1991; Tomlin, 1997).
IV. THIOCARBAMATES The thiocarbamates include a wide variety of fungicides, such as ferbam, mancozeb, maneb, and thiram. The thiocarbamates are also used as herbicides and include butylate, S-ethyl dipropylthiocarbamate, pebulate, metham, molinate, cycloate, and vernolate. Their acute toxicity to humans is generally considered to be low, but they can be irritating to the skin and eyes. Inhalation of spray mist or dust from these pesticides may cause throat irritation, sneezing, and coughing.
17
V. AChE INHIBITORS IN HUMAN MEDICINE A. Alzheimer's Disease Acetylcholinesterase inhibitors (AChEIs) have been widely recognized as an effective treatment for Alzheimer's disease (AD). These compounds prevent the breakdown of ACh by inhibiting AChE in the brain regions (cortex and hippocampus) relevant to AD, thereby slowing the development of cognitive impairments and neurodegeneration and alleviating the symptoms of AD. The most widely studied AChEIs are carbamates, such as physostigmine and tacrine. Physostigmine was isolated from the Calabar bean and its structure was elucidated by Stedman and coworkers in 1925. Tacrine belongs to the first generation of AChEIs because it was the first drug approved for the treatment of AD. However, physostigmine's very short duration of action and tacrine's frequent dosing along with the need for monitoring liver enzymes (hepatotoxicity) have made the use of these compounds obsolete. One of the many tacrine derivatives, ameridine, is still under clinical trial study in Japan (Sugimoto et al., 2002). For many years, the OP compound trichlorfon (metrifonate), a slow-release reservoir of dichlorvos, which has a racemic mixture of two isomers, was used in the treatment of mild to moderate AD. However, metrifonate was found to cause serious respiratory complications and therefore was discontinued for treatment of AD. Currently, it is labeled as a mutagen, carcinogen, and possibly teratogen, and therefore it is used only as an investigational drug. The use of second-generation AChEIs in the treatment of AD has been found to be promising for rectifying cholinergic deficits. It is well established that the cholinesterases, particularly butyrylcholinesterase (BuChE), are associated with the pathogenesis and progression of AD (Guillozet et al., 1997; Darvesh et al., 2003). Therefore, CMs such as rivastigmine and eptastigmine, which are pseudoirreversible AChEIs and interact with the catalytic as well as regulatory anionic sites of the enzyme, have received a great deal of attention (Cuadra et al., 1994). Rivastigmine is derived from physostigmine and overcomes the deficiencies of physostigmine (Sugimoto et al., 2002). A list of these compounds also includes a novel carbamate compound TV3326 (N-propargyl-(3R) aminoindan-5-yl)-ethyl methyl carbamate), which is not only an AChE inhibitor but also an MAO inhibitor (Weinstock et al., 2002). Another derivative of physostigmine is phenserine (a phenylcarbamate compound), which is a potent and highly selective AChE inhibitor with a >50-fold activity compared to BuChE. Phenserine has been tested in clinical trials for the treatment of AD, although no clinical data have been released. Ganstigmine (CHF 2819) is another novel orally active AChEI developed for the treatment of AD. It is a selective inhibitor of AChE (>115 times greater than against BuChE). Ganstigmine is also more selective for inhibition
TABLE 3. A Brief Chemical Description of Commonly Used CM Pesticides Chemical (CAS No.) Aldicarb ( 116-06-3)
Chemical structure/ empirical formula
Chemical name
(1E)-2-methyl-2-(methylthio)propanal 0-[(methyl-amino)carbonyl] oxime
Molecular weight 190.26
Oral LD50 in rat (mg/kg) 0.9
Dermal LD50 in rabbit (m€m)
5
C7H14N202S Aminocarb (2032-59-9)
4-(Dimethylamino)-3-methylphenyl methylcarbamate
208.26
30
275
223.23
34
566
410.53
138
>2000
422.87
340
4200
201.22
307
2000
CllH16N202 Bendiocarb (2278 1-23-3)
2,2-Dimethyl- 1,3-benzodioxo1-4-y1 methylcarbamate CllH13N04
L
OC,
Benfuracarb (82560-54-1)
2,3-Dihydro-2, 2-dimethyl-7-benzofuranyl N-[n[2-(ethylcarbonyl) ethyl]N-isopropyl sulfenamoyl]N-methylcarbamate C20H30N205S
BPMC (37 66-8 1-2)
2-sec-Butylphenyl N-methylcarbamate C12H17N02
Carbaryl (63-25-2)
1-Naphthyl methylcarbamate
C12H1lNO2
Carbofuran (1563-66-2)
2,2-Dimethyl-2, 3-dihydro-1-benzofuran-7-yl methylcarbamate
221.25
8
2550
380.55
209
>2000
225.31
200
1000
301.34
10,000
2000
193.24
450
225.31
15
C12H15N03
Carbosulfan (55285-14-8)
2,3-Dihydro-2, 2-dimethyl-7-benzofuranyl[(di-buty1amino)thioJmethyl carbamate C20H32N203S
Croneton (29973-1 3-5)
2-[(Ethylthio)methyl]phenyl methylcarbamate
CllH15N02S
Fenoxycarb (7 2490-0 1-8)
Ethyl [2-(4-phenoxyphenoxy)ethyl] carbamate C17H1gN04
Isoprocarb (2631-40-5)
Methiocarb (2032-65-7)
2-Isopropylphenylmethylcarbamate C11H15N03
3,5-Dimethyl-4-(rnethylthio)phenyl
2000
methylcarbarnate
CllH15N02S
(continues)
TABLE 3. (continued)
17
Dermal LD50 in rabbit (mg/kg) o o
162.21
"~..=
~
, _
o
"1-
co -r Ox- r Z
/
ko
O9
o--~
Methyl (lQ-N-{ [(methylamino) carbonylloxy ] ethanimidothioate
5000
C5H10N202S
268
i
165
0o c'q
~
-
3-Methylphenyl methylcarbamate o
Metolcarb (1129-4-5)
Oral LD50 in rat (mg/kg)
0
i.}
r,.)
Chemical name
=
~9-~
Methomyl (16752-77-5)
Molecular weight
=
e ~-=
Chemical structure1 empirical formula
Chemical (CAS No.)
.
~
C9H1I N 0 2
I ~
It~
0
219.26
~--
o o
5000
~
=~
I 0
C12H1SN202
r
Z --0
15
,--
/ Z -I-
0 09 -r
222.28
~
.
4-(Dimethylamino)-3, 5-dimethylphenyl methylcarbamate
~
0
o
Mexacarbate (315-184)
o
1-
Oco
20
'
"1~
710
=
o
~Z
5
o
-r 0
Methyl 2-(dimethyl-amino)N - { [(methyl-amino)carbonyl]oxy ) 2-oxoethan-imidothioate ~
Z
0
(-q
/
O9
1-
Oxamyl (23135-22-0)
z
CO
I
0
~0
"1-
C7H13N303S
"1-
>500
A
147
o o
238.29
,~
!
!
I
o z
/
"i-
o
o~
c1lH 1g N 4 0 2
4
r
O0
2-(Dimethylamino)-5, 6-dimethyl-pyrimidin-4-y l dimethy lcarbamate .. =
.0 0
Pirimicarb (23 103-98-2)
O9
-r 0
o
Z--O I
Promecarb (2631-37-0)
3-Isopropyl-5-methylphenyl methylcarbamate
207.27
61
>loo0
209.24
95
> 1000
193.24
125
>2000
179.22
542
179.22
384
C12H17N02
Propoxur (114-26-1)
2-Isopropoxyphenyl methylcarbamate CllH15N03
Trimethacarb (12407-86-2)
3,4,5-Trimethylphenyl methylcarbarnate CllH17N02
N XMC (2655-14-3)
3,s-Dimethylphenyl methylcarbamate CIOH13N02
Xylylcarb (2425-10-7)
3,4-Dimethylphenyl methylcarbarnate CgH10N402
22
S ECTI O N I 9Uses, Abuses, & Epidemioloooy O
H3~
O
~
CH3
H3C~O
OH3 DONEPEZIL
OH3
RIVASTIGMINE OH
O o"
H 3 c ~ O ~ "CH3 GALANTAMINE
H3C
/0
~CH3
NH2
OH
CI
CI
TRICHLORFON
TACRINE H3
O NH
~ O
~N
.H
.-
FIG. 1. Chemical structures of compounds indicated in Alzheimer's disease.
EPTASTIGMINE
of central (brain) AChE than peripheral (heart) AChE (Racchi et al., 2004). Chemical structures of some of the compounds that have been used in the past or are currently in use are shown in Fig. 1. These compounds are discussed in detail in Chapters 3 and 4.
B. Myasthenia Gravis From a pharmacological standpoint, postsynaptic disorders are treated with cholinesterase inhibitors (AChEIs), such as neostigmine, physostigmine, and pyridostigmine. However, AChEIs represent only symptomatic therapy, and they are of little aid in most cases of moderate to severe or progressive myasthenia gravis (MG), particularly if there is oropharyngeal or respiratory muscle involvement. Therefore, use of AChEIs as the mainstay of therapy for MG has been deemphasized. In the past, three commonly used AChEIs were physostigmine, pyridostigmine, and galantamine. Currently neostigmine, pyridostigmine, and ambenonium are the standard anti-AChE compounds used in the symptomatic treatment of MG for cholinergic crisis (Fig. 2). These compounds presumably counteract MG by compensating for lost ACh receptors through elevation of neurotransmitter
levels, resulting in increased neuromuscular transmission and improved muscular strength.
C. Glaucoma Physostigmine (eserine) and echothiophate (phospholine) are the two AChE-inhibiting compounds indicated for glaucoma. Both compounds are known to exert ocular side effects.
D. Urine Voiding Dysfunction Impairment of destrusor muscle contractibility appears to be one of the causes of voiding dysfunction in both men and women. The destrusor muscle becomes weak due to many factors, including aging, prostate hypertrophy, diabetes mellitus, and multiple sclerosis. AChE-inhibiting carbamates, such as physostigmine, distigmine bromide, and neostigmine bromide, seem to have the potential for correcting the problem. A study conducted on guinea pigs suggested that a novel anti-AChE compound, TAK-802, may be useful in the treatment of voiding dysfunction associated with impaired destrusor contractility (Nagabukuro et al., 2004).
CHAPTER 2 9Classification of OPs and CMs
H3C~ CH3
23
CH3 I H3C~~OyN~cH3 H3c/N~cH3
NEOSTIGMINE
H3C
__,,
+
c,
o
PYRIDOSTIGMINE
H3C~ 7
c,_ L...CH o/ /
N
CI.
I. CH
,30. c,
1
0
AMBENONIUM E. OP Nerve Agent Poisoning During the Persian Gulf War (Operation Desert Shield/Storm) in 1990, military personnel received a reversible AChE inhibitor, pyridostigmine bromide, as a prophylactic measure to combat anticipated deadly OP nerve agent exposure. Due to severe side effects, the drug has been discontinued for application in the setting of such military action (Keeler et al., 1991).
VI. ACHE INHIBITORS IN VETERINARY MEDICINE
A. Anthelmintics Six OP compounds have been used as anthelmintics in domestic animals: dichlorvos, trichlorfon, haloxon, coumaphos, naphthalophos, and crufomate. The first two were used in horses and the latter four in ruminants. These compounds affect parasites by ACh accumulation attributed to AChE inhibition, leading to interference with neuromuscular transmission and subsequently paralysis, followed by expulsion of parasites from the animal's body (Reinemeyer and Courtney, 2001).
H3C~N+---CH3
OH
EDROPHONIUM
FIG. 2. Chemical structures of anti-AChE compounds indicated in myasthenia gravis.
phosmet, pirimiphos-methyl, ronnel, tetrachlorvinphos, and trichlorfon. Unlike OPs, only two CMs (carbaryl and propoxur) are recommended for the control of ectoparasites. The mechanism of action of CMs is similar to that of OPs, except CMs reversibly inhibit ACHE.
VII. CONCLUSIONS Both OPs and CMs are synthesized compounds. Within each class, the chemicals have some similarities and some differences. Because of the differences, these compounds produce varying degrees of cholinergic and noncholinergic effects, and as a result, they have different applications. Although the majority of these chemicals are used as pesticides, some are used as chemical weapons of mass destruction. In addition, many of these compounds are used as therapeutic drugs in human and veterinary medicine. It is expected that in the future many more new OPs and CMs will be synthesized and novel applications will be discovered.
Acknowledgments I thank Mrs. Debra A. Britton and Mrs. Denise M. Gupta for their assistance and support in the preparation of this chapter.
B. Ectoparasiticides Currently, more than a dozen OP compounds are used as ectoparasiticides in veterinary medicine. These compounds are known to cause paralysis and death of insects by virtue of irreversible AChE inhibition and subsequent accumulation of ACh. These compounds include chlorfenvinphos, chlorpyrifos, coumaphos, cythioate, diazinon, dichlorvos, ethion, famphur, fenthion, malathion,
References Aston, L. S., Noda, J., Seiber, J. N., and Reece, C. A. (1996). Organophosphate flame retardants in needles of Pinus ponderosa in the Sierra Nevada Foothills. Bull. Environ. Contam. Toxicol. 57, 859-866. Battershill, J. M., Edwards, E M., and Johnson, M. K. (2004). Toxicological assessment of isomeric pesticides: A strategy for
24
S E CTI O N I 9 Uses, Abuses, & Epidemiology
testing of chiral organophosphorus (OP) compounds for delayed polyneuropathy in a regulatory setting. Food Chem. Toxicol. 42, 1279-1285. Benschop, H. P., and De Jong, L. P. A. (1988). Nerve agent stereoisomers: Analysis, isolation and toxicology. Acc. Chem. Res. 21, 366-374. Cuadra, G., Summers, K., and Giacobini, E. (1994). Cholinesterase inhibitor effects on neurotransmitters in rat cortex in vivo. J. Pharmacol. Exp. Ther. 270, 277-284. Darvesh, S., Walsh, R., Kumar, R., et al. (2003). Kinetic properties of human acetylcholinesterase and butyrylcholinesterase in the presence of drugs for Alzheimer's disease. Alzheimer Dis. Assoc. Disord. 17, 117-126. Green, J. (1993). A review of phosphorus-containing flame retardants. J. Fire Sci. 10, 470-487. Guillozet, A. L., Smiley, J. E, Mash, D. C., and Mesulam, M. M. (1997). Butyrylcholinesterase in the life cycle of amyloid plaques. Ann. Neurol. 42, 909-918. Johnson, M. K. (1969). Delayed neurotoxic action of some organophosphorus compounds. Br. Med. Bull. 25, 231-235. Keeler, J. R., Hurst, C. G., and Dunn, M. A. (1991). Pyridostigmine used as a nerve agent pretreatment under wartime conditions. J. Am. Med. Assoc. 266, 693-695. Kidd, H., and James, D. R. (1991). The Agrochemicals Handbook, 3rd ed. Royal Society of Chemistry Information Services, Cambridge, UK. Ku6a, K., and Kassa, J. (2004). Oximes-induced reactivation of rat brain acetylcholinesterase inhibition by VX agent. Hum. Exp. Toxicol. 23, 167-171. Marrs, T. C. (1993). Organophosphate poisoning. Pharmacol. Ther. 58, 51-66. Nagabukuro, H., Okanishi, S., and Doi, T. (2004). Effects of TAK-802, a novel acetylcholinesterase inhibitor, and various
cholinomimetics on the urodynamic characteristics in anesthetized guinea pigs. Eur. J. Pharmacol. 494, 225-232. Racchi, M., Mazzucchelli, M., Porrello E., Lanni, C., and Govoni, S. (2004). Acetylcholinesterase inhibitors: Novel activities of old molecules. Pharmacol. Res. 50, 441-451. Reinemeyer, C. R., and Courtney, C. H. (2001). Chemotherapy of parasitic diseases. In Veterinary Pharmacology and Therapeutics 8th ed., (H. R. Adams, Ed.), pp. 947-979. Blackwell, Ames, IA. Senanayke, N., and Karalliede, I. (1987). Neurotoxic effects of organophosphorus insecticides: An intermediate syndrome. N. Engl. J. Med. 316, 761-763. Smith, J. R. (2004). Analysis of the enantiomers of VX using normal-phase chiral liquid chromatography with atmospheric pressure chemical ionization-mass spectrometry. J. Anal. Toxicol. 28, 390-392. Sugimoto, H., Iimura, Y., and Yamanishi, Y. (2002). The new generation of acetylcholinesterase inhibitors: In Mapping the Progress of Alzheimer's and Parkinson's Disease (Y. Mizuno, A. Fisher, and I. Hanin, Eds.), pp. 193-198. Kluwer/Plenum, New York. Tomlin, C. D. S. (1997). A World Compendium. The Pesticide Manual, 1 l th ed. British Crop Protection Council, Farnham, Surrey, UK. U.S. Environmental Protection Agency (1988). Twenty-third report of the interagency testing committee to the administrator: Receipt of report and request for comments regarding priority list of chemicals. Fed. Reg. 53, 46244-46272. Weinstock, M., Poltyrer, T., Bejar, C., Sagi, Y., and Youdin, M. B. H. (2002). TV3326, a novel cholinesterase and MAO inhibitor and Parkinson's disease. In Mapping the Progress of Alzheimer's and Parkinson's Disease (Y. Mizuno, A. Fisher, and I. Hanin, Eds.), pp. 193-198. Kluwer/Plenum, New York.
CHAPTER
Therapeutic Uses of Cholinesterase Inhibitors in N e u r o d e g e n e r a t i v e Diseases RANDALL L. WOLTJERAND DEJAN MILATOVIC University of Washington, Seattle, Washington
for AD is the passage of time, which is currently inconveniently recapitulated in the laboratory setting, it is likely that we will continue to learn more about these diseases through the further characterization of clinical responses of AD and, increasingly, other age-related dementias to such therapies. This chapter describes the rationale for such therapies in AD, aspects of their application to clinical disease, and what we have learned and may continue to learn from the effects of these drugs in neurodegenerative disease.
I. I N T R O D U C T I O N The most common form of age-related neurodegenerative disease in the United States is Alzheimer's disease (AD), with approximately 4 million Americans afflicted; in the European Union, approximately 3 million people suffer from the disease. Furthermore, as the average life span continues to increase, it is anticipated that AD and other age-related neurodegenerative diseases will become increasingly major public health concerns. Already, the annual cost of disease (including paid and unpaid caregiver costs, as well as losses in productivity due to illness and premature mortality) of patients with AD in the United States alone has been estimated to be $100 billion (Leifer, 2003), and the prevalence of AD is expected to increase by approximately three-fold by the middle of this century. This represents a potentially staggering cost, both monetarily and in terms of lost human potential, to developed societies. A detailed understanding of the pathogenesis of AD, which might be useful in the rational development of strategies to prevent or treat disease, is not yet available, although substantial progress has been made in this area, especially in the past decade. Also, there is reason for considerable optimism, particularly regarding the possibility of treatments that may prevent or at least delay the onset of AD. However, AD is a complex disorder, with both genetic and environmental factors that affect the risk of disease, cognitive and neuropsychiatric manifestations that may vary between patients and over time, and, as is becoming increasingly apparent, only a part of a spectrum of age-related neurodegenerative disease that is still in the process of clinical, pathologic, and biochemical definition. The historical lack of an ideal animal model for AD has necessitated that at least some of this definition has derived from empirical observations of the effects of putative therapeutic agents, such as the acetycholinesterase inhibitors (AChEIs). Because the major nongenetic risk factor Toxicology of Organophosphate and Carbamate Compounds
II. THE CHOLINERGIC HYPOTHESIS Early investigations on postmortem tissue demonstrated a reduction of choline acetyltransferase (CHAT) activity and of cholinergic neurons in the basal forebrain of patients affected by AD (Davies and Maloney, 1976; Perry et al., 1977; Whitehouse et al., 1981). Decreases in presynaptic cholinergic neurons are also observed in the cerebral cortex and hippocampus as AD progresses (Bartus et al., 1982; Coyle et al., 1983). These observations led to the formulation, approximately 25 years, ago, of the "cholinergic hypothesis," which states that loss of cholinergic function in the cerebrum contributes significantly to cognitive dysfunction in AD (Barms, 2000). In addition to CHAT, cholinesterases, particularly butyrylcholinesterase (BuChE), have been associated with the pathogenesis and progression of AD (Guillozet et al., 1997; Darvesh et al., 2003). Based on these findings, it has been hypothesized that cholinesterase inhibitors that inhibit both AChE and BuChE stabilize disease progression better than those that inhibit only AChE in AD patients (Ballard, 2002; Giacobini, 2000). Importantly, the cholinergic hypothesis does not necessarily stipulate that cholinergic deficiency initiates or contributes to the progression of disease, although we deal with this possibility later. However, the goal of any medical hypothesis is to produce new, effective therapies for 25
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
26
S ECTI O N I 9Uses, A b u s e s , & E p i d e m i o l o g y
disease; in this light, the cholinergic hypothesis, which has brought several compounds with significant efficacy to the clinical treatment of AD, has been regarded as one of the successes of modern neuropharmacology (Bartus, 2000). We will next discuss the origins of the cholinergic hypothesis, the uses of AChEIs in the therapy of AD, determinations of their clinical efficacy, and what the use of these compounds has taught us about the disease.
III. C H O L I N E R G I C F A I L U R E , NEUROPATHOLOGICAL
CORRELATES,
AND THE SYMPTOMS OF AD Impairments in AD occur in both memory and other areas of cognition, such as language or visuospatial awareness. In addition, neuropsychiatric symptoms including psychosis and mood alterations such as depression, apathy, and agitation may present during the course of dementia, (Lyketsos et al., 2001). These perturbations reflect structural and neurochemical alterations in brain regions such as the hippocampus and cerebral cortex that house normal functions of memory and cognition. Hippocampal and cortical neurons are innervated by cholinergic afferents from the basal forebrain, the site of the nucleus basalis of Meynert, which contains approximately 80% of the cholinergic neurons of the central nervous system and is characterized by marked atrophy in advanced AD (Cummings and Back, 1998). In experimental rodent models, lesions of cholinergic pathways result in impairments in the performance of memory tasks (Dunnett et al., 1987). Taken together, these observations are consistent with the idea that cholinergic failure may contribute strongly to symptoms of AD. The cholinergic hypothesis has also been strengthened by observations of correlations between cholinergic failure and the degree of clinical dementia. Interestingly, however, cholinergic failure and dementia have also been found to correlate with the extent of extracellular senile plaques in brain tissue (Arendt et al., 1985; Etienne et al., 1986; Perry et al., 1978). Senile plaques are one of the pathologic hallmarks of AD; another characteristic lesion, the intracellular neurofibrillary tangle, is found in a topographically organized pattern in the gray matter of the limbic system and progressively in the neocortex in AD. Although this association has invited speculation that cholinergic failure may contribute substantially to the pathogenesis of AD, historically this has not been widely believed to be the case based on several observations. The first involves studies of patients with amnestic mild cognitive impairment (MCI), a condition of memory loss that is widely considered a paradigm of preclinical AD (Petersen, 2000; Petersen et al., 2001). In MCI, levels of ChAT have been reported to be increased, with a subsequent decrease to normal levels with the onset of clinical dementia (DeKosky et al., 2002; Fr61ich, 2002). In advanced AD,
the loss of cholinergic neurons results in up to a 90% reduction in the activity of CHAT, which is needed for the synthesis of the neurotransmitter ACh, and ACh levels decrease by 90%, especially in the cerebral cortex and hippocampus (Murphy et al., 1998). Furthermore, although cholinergic failure is most pronounced late in the course of AD, anticholinesterase therapies (as described later) have been best characterized and appear most efficacious in mild to moderate dementia, although studies on the use of AChEIs for longer term disease are ongoing. Finally, the pattern of memory loss in AD, in which more recent memories are lost first, with loss of older memories later, is not well recapitulated by models that invoke only neuronal loss, but it can be accounted for if changes in cholinergic activity that occur in MCI are viewed as responses to other instigators of brain dysfunction in AD (Small et al., 2001; Small, 2004).
IV. DETERMINATION OF T H E E F F E C T S O F A C h E I S O N S Y M P T O M S I N AD The effects of AChEI therapy on AD have proven to be modest, and the determination of the significance of effects of this magnitude required the development of standardized tools of measurement that encompass important aspects of AD symptomatology. Commonly used instruments include the Mini-Mental Status Examination (MMSE) (Folstein et al., 1975), which assays cognition with the use of 11 questions that produce a single score ranging from 0 (severe impairment) to 30 (no impairment). The second tool is the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-cog) (Rosen et al., 1984), designed to determine cognitive function in AD. This test also consists of 11 items that assess cognitive functions that are typically impaired specifically in AD, with an output score ranging from 0 (no impairment) to 70 (marked impairment); the average rate of score increase in patients with AD is 7-11 points per year (Kramer-Ginsberg et al., 1988; Stern et al., 1994). Neuropsychiatric symptoms described in Section III can likewise be quantified with the use of the Neuropsychiatric Inventory (Cummings et al., 1994) or a noncognitive subscale of the Alzheimer's Disease Assessment Scale (ADASnoncog). Recently, there has been an enlargement of the scope of assessment to include effects on so-called functional symptoms. Functional decline in AD involves the loss of ability to perform activities of daily living, such as the simple arithmetic of basic finances, driving, or using household tools such as the telephone, with progression of losses to the point that patients in a terminal state are no longer able to feed or bathe themselves. These losses increase the burden on patient caregivers, are highly correlated with the decision to place patients in institutional facilities, and contribute significantly to the financial burden of disease. Functional losses are also quantifiable through such tools as the
C H A P T E R 3 9Therapeutic Uses of AChE inhibitors
Alzheimer's Disease Functional Assessment and Change Scale and the Progressive Deterioration Scale (Reisberg et al., 1986).
V. A BRIEF HISTORY OF THE USE OF AChEIs IN AD AChEIs initially came to the attention of investigators concerned with dementias with the recognition of their capacity to enhance cognition in animals with scopolamineinduced amnesia (Bartus, 1978). With the discovery that ACh was depleted in the hippocampus (Smith and Swash, 1978) and initial reports of effects of physostigmine on cognition in both normal subjects and AD patients (Davis et al., 1978; Muramoto et al., 1979; Peters and Levin, 1979; Smith and Swash, 1979), the stage was set for trials of a variety of AChEIs as potential therapeutic agents in AD. Some drugs, such as galantamine, had long been used for other indications. Two compounds, velnacrine and eptastigmine, were halted in development due to the association of blood dyscrasias with their use. More typical limitations of a variety of agents center on drug tolerability, the response rate to a tolerated dose, and intersubject variability. Overall, the response of AD patients to AChEI therapy has been characterized as modest, with 3 or 4 point decreases in ADAS-cog scores compared with the yearly rate of cognitive decline in untreated controls. The U.S. Food and Drug Administration (FDA) uses a 4-point improvement in ADAS-cog scores as a criterion for a clinically significant response to t h e r a p y - an improvement also used by many investigators to define rate of response, which is typically 30-40% of AD patients undergoing treatment. The FDA has approved four AChEIs for the treatment of AD; in fact, these are currently the clinical mainstays of AD therapy.
A. Tacrine
NH2
Tacrine
In 1993, tacrine (aminoacridine, competitive unselective reversible inhibitor of cholinesterases) was the first anticholinesterase drug to receive FDA approval for the treatment of mild to moderate AD. The benefits of treatment were evident in end points that used ADAS as well as general clinical impressions, with 10-26% of recipients of high doses showing measurable improvement over the course of 30 weeks (Knapp et al., 1994). However, only a minority of patients were able to tolerate the maximally effective dose
27
(160 mg/day) due to hepatotoxicity manifested as asymptomatic transaminase elevations, as well as nausea and vomiting, diarrhea, and anorexia. Other limitations of tacrine, such as a relatively short half-life (2-3 hr) and significant interindividual variation in clearance rates that necessitated drug titration and plasma determinations, motivated the search for novel agents. It is available under the trade name Cognex, but is rarely prescribed.
B. Donepezil o
2
H3C // H3C~O Donepezil
Donepezil, a piperidine-based rapidly reversible noncompetitive AChEI, was approved by the FDA for treatment of cognitive dysfunction in AD in 1996. The drug was developed specifically for pharmacotherapy of AD (Bryson and Benfield, 1997; Barner and Gray, 1998) and has been found to have selectivity for brain AChE over peripheral forms of the enzyme (Kosasa et al., 2000). Donepezil is highly selective for ACHE, with significantly lower affinity for BuCHE. Major advantages over tacrine are its long half-life (70 hr) and uniform dosing in patients with renal or hepatic impairment. In the course of a study of the effects of 24 weeks of treatment with ADAS-cog, MMSE, and other end point determinations, cognitive function was found to be significantly improved by 12 weeks. The drug was generally well tolerated at low (5 mg/day) doses, with a slight but statistically significant increased incidence of diarrhea and vomiting at 10 mg/day that nevertheless resolved spontaneously without reduction in dosage (Rogers and Friedhoff, 1998; Rogers et al., 1998). The drug is marketed under the trade name Aricept.
C. Rivastigmine
0
~
H3C~N'~O I CH3
CH3 N~CH 3
CH3
Rivastigmine
Rivastigmine, approved by the FDA in 2000, is a noncompetitive pseudo-irreversible carbamate AChEI that was
28
SECTION I 9Uses, A b u s e s , & Epidemioloooy
selected for study based on its high affinity for brain AChE compared to peripheral forms of the enzyme (Weinstock et al., 1992, 2004; Enz et al., 1993). Rivastigmine selectively inhibits monomeric ACHE, especially in the cortex and hippocampus, and is thereby thought to facilitate cholinergic neurotransmission by slowing the degradation of ACh released by functionally intact cholinergic neurons (Polinsky, 1998; Ibach and Haen, 2004). The mechanism of action of rivastigmine differs from that of donepezil because donepezil is an AChE-selective inhibitor, whereas rivastigmine is a dual inhibitor of both AChE and BuChE. Furthermore, unlike donepezil, rivastigmine is bound more tightly to the active center of AChE than a naturally occurring choline ester. Rivastigmine was shown to improve cognition as determined by ADAS scores, participation in activities of daily living, and global evaluation scores in patients with mild to moderate AD in a multicenter trial of 28 weeks' duration (Rosler et al., 1999). As with donepezil, most side effects were found to be gastrointestinal and transient in nature. The drug, titrated up to 12 mg/day with two or three times daily dosing, yielded ADAS improvements in 24% of treated patients versus 16% for placebo controls. It is marketed under the trade name Exelon.
VI. T H E P E R S I S T E N C E O F A C h E I EFFICACIES The initial results for the previously discussed agents were generally based on trials of 6 months' duration or less. These and subsequent studies have indicated that treatment of AD with AChEIs tends to lead to an improvement in cognition that is maintained for up to approximately 1 year. This is typically followed by declines in cognition, but to levels that still improved relative to those of untreated controls. Several studies, typically continuations of trials under open-label conditions, demonstrate benefit of up to several years' duration with FDA-approved AChEIs (Knopman et al., 1996; Lilienfeld and Parys, 2000; Rogers et al., 2000; Doody et al., 2001; Rockwood et al., 2001; Tariot, 2001). The efficacy of a variety of agents with widely varying structures but that all share anticholinesterase properties, as well as the continuation of clinical efficacy of these drugs into the stage of AD that is characterized neurochemically by cholinergic failure, and in some instances initiated in more severely affected AD patients (Feldman et al., 2001), is widely seen to support the cholinergic hypothesis of AD.
VII. P U T A T I V E E F F E C T S O F A C h E I s O N AD P A T H O G E N E T I C M E C H A N I S M S
D. Galantamine OH O"--
H3c/O~ ~'~/~'~ N "CH3 Galantamine
Galantamine is a phenanthrene alkaloid that was initially isolated from the common snowdrop, Galanthus niralus. Galantamine acts as a rapidly reversible, competitive AChEI. The agent has been used for approximately 40 years in the treatment of myasthenia gravis and in reversal of pharmacologic neuromuscular blockade; hence, considerable familiarity with its pharmacokinetic and toxicologic properties existed prior to its approval by the FDA for treatment of symptoms of AD in 2001. Several trials have demonstrated efficacy in the treatment of cognitive symptoms in AD at a dose of up to 32 mg/day, as determined by ADAS scores and other end points (Rainer, 1997; Raskind et al., 2000; Tariot et al., 2000). Nausea appears t o b e the most common side effect. Stimulation of nicotinic receptors has been proposed to be an additional mechanism of action of galantamine, and there is evidence that such an effect may be relevant to AD (Dajas-Bailador et al., 2003). Galantamine is marketed under the trade name Reminyl.
Although the clinical indication of AChEIs is limited to symptomatic therapy for AD, increasing experience with their use, and the persistence of drug efficacy in particular, has led some to suggest that these agents may alter the natural history of disease (Farlow et al., 2000, 2003; Doraiswamy et al., 2002; Erkinjuntti et al., 2003; Stefanova et al., 2003). It has been hypothesized that normal activation of neurons in aging and during the course of AD may lead to preservation of neuronal function and/or promotion of survival of remaining neurons; this "cognitive reserve" or "use it or lose it" hypothesis has been proposed to account for the effects of education and other socioeconomic factors on the risk of development of cognitive impaLrment in general and AD in particular in a variety of populations (Yu et al., 1989; Brayne and Calloway, 1990; Moritz and Petitti, 1993; Stem et al., 1995; Evans et al., 1997; Hall et al., 2000; Qiu et al., 2001; Salemi et al., 2002; Karp et al., 2004). Additional proposed interactions of AChEIs with specific aspects of steps that are widely believed to be important in the pathogenesis of AD have been reviewed (Lane et al., 2004) and are discussed next.
A. Effects of AChEIs on Amyloid 13 Peptide Approximately 5% of cases of AD are attributable to mutations in known genes that act in an autosomal dominant fashion. Each of these is related to the metabolism of the amyloid precursor protein (APP) and its cleavage products, most prominently amyloid [3 (A[3). The remaining 95% of
CHAPTER 3 9Therapeutic Uses of AChE Inhibitors cases appear as sporadic disease with a relatively late onset and a complex etiology attributable to interactions between aging, environmental, and other genetic factors (Munoz and Feldman, 2000; Lahiri et al., 2004). The pathologic similarities between inherited and sporadic forms of AD, however, have led to the "amyloid hypothesis" that increased production or accumulation of A[3, with its subsequent aggregation and accumulation in cerebrum as senile plaques, provides the pathogenetic foundation for all forms of AD (Hardy and Selkoe, 2002). Efforts to understand the effects of A[3 have focused on its aggregation and reorganization to form progressively insoluble structures. This has been attributed to the presence in A[3 of an unstable domain that can readily adopt multiple conformations, some of which are prone to form aggregates that may propagate as filamentous structures with decreased solubility that are deposited in cerebrum as amyloid. This propensity appears to be key to the pathologic actions of A[3 because the uniform effect of genetic mutations in familial forms of AD is the promotion of A[3 aggregation and insolubility, either via alterations in the structure of A[3 (in the case of mutations in APP) or by increasing the relative amount of the more amyloidogenic 42-residue species relative to the 40-residue species (in the case of mutations in presenilin 1 or 2, which determine the carboxy-terminal cleavage of the A[3 peptide) (Marjaux et al., 2004). One effect of AChEIs may be mediated by ACh receptor-mediated activation of protein kinase C and mitogen-activated protein kinase pathways (Haring et al., 1998; Beach et al., 2001; Beach, 2002). Such activation promotes an alternate cleavage of APP that produces nonamyloidogenic forms of the peptide. Conceivably, the persistence of ACh in the context of AChEI treatment may promote this process. In addition to A[3, senile plaques contain a host of other proteins (Liao et al., 2004), including ACHE, which has been proposed to serve as a nucleating factor in the deposition of A[3 with reduced solubility, or otherwise promote the toxicity or aggregation state of A[3 in vivo (Inestrosa et al., 1996; Alvarez et al., 1997; De Ferrari et al., 2001; Rees et al., 2003). Other studies indicate that A[3 complexed with AChE is more toxic than A[3 species alone (Alvarez et al., 1998). A number of AChEIs have been reported to inhibit at least partially A[3 aggregation in the presence of AChE (Bartolini et al., 2003; Piazzi et al., 2003); the significance of this observation and its validity in the case of clinically widely used AChEIs remain unknown.
29
of kinases, especially glycogen synthase kinase-3 (GSK3). Kinase activities described previously that are promoted by ACh not only affect APP metabolism but also are associated with decreases in the activity of GSK3, leading to decreased tau phosphorylation and conceivably to a reduction in tangle generation (Forlenza et al., 2000). Consistent with this mechanism, a rivastigmine-treated AD group showed no change in cerebrospinal fluid (CSF) levels of tau after 1 year, whereas significant increases were observed in untreated patients (Stefanova et al., 2003). However, the CSF tau content of patients treated with tacrine resembled that of controls more closely, making interpretation of these results difficult.
C. Effects of AChEIs on Cerebrovascular Parameters Recently, the coexistence of vascular lesions and those of AD in cerebrum of patients with so-called mixed dementia have received increasing attention, along with the possibility of heretofore unanticipated interactions between pathophysiological factors (Langa et al., 2004). Some authors have suggested that factors that lead to the development of senile plaques and neurofibrillary tangles may be promoted by cerebrovascular disease (de la Torte, 2002; Honig et al., 2003; Casserly and Topol, 2004); conversely, deposition of amyloid in vessel walls compromises cerebrovascular function and, in the extreme, promotes the risk of hemorrhagic stroke. Interestingly, AChEI therapy for mixed dementia yields favorable, but modest, clinical results similar to those observed in the treatment of AD (Kumar et al., 2000; Erkinjuntti et al., 2002) and in some studies demonstrates efficacy in the treatment of dementia associated with cerebrovascular disease alone (Erkinjuntti et al., 2004). As in the case of AD, cholinergic deficiency has been demonstrated in vascular dementia and has been attributed to ischemic injury to cholinergic neurons, as supported by rodent models of ischemic injury (Togashi et al., 1994). However, effects of ACh on the vascular endothelium may account for a portion of both the interaction of AD and vascular dementia and the efficacy of AChEIs in both of these conditions. ACh at this site mediates the release of nitric oxide, a vasodilator that may account for increases in cerebral blood flow and glucose metabolism upon treatment with AChEIs (Minthon et al., 1993; Harkins et al., 1997; Lojkowska et al., 2003). Conceivably, such improvements could influence the natural history of vascular dementia and AD.
B. Effects of AChEIs on Tau According to the amyloid hypothesis, abnormalities involving A[3 peptides also lead to changes in the organization of tau to produce neurofibrillary tangles, the lesion in brain tissue that is more closely associated with the presence of clinical AD. Deposition of tau in tangles is enhanced by tau hyperphosphorylation, which can be accomplished by a variety
VIII. AChEIS IN T H E T R E A T M E N T OF O T H E R D E M E N T I A S Dementia associated with Parkinson's disease (PDD) and dementia with Lewy bodies (DLB) together comprise the second most common form of age-related dementia after
30
SECTION 1 9 Uses, Abuses, & E p i d e m i o l o g y
AD. A recent body of literature suggests that AChEIs may be of utility in the treatment of these disorders, which have also been reported to be associated with cholinergic deficit (Aarsland et al., 2004). This deficit, as in the case of AD, has been attributed to basal forebrain degeneration, and this may be sufficient to account for any observed clinical efficacy of AChEIs. However, the interactions of AChE with A[3 in senile plaques described previously suggest other possibilities as well. Many neurodegenerative diseases are characterized by the presence of a variety of proteinaceous aggregates that are associated with neurotoxicity. As in the case of amyloid plaques, upon further examination these aggregates have been found characteristically to be constituted of numerous proteins that may or may not be predicted based on sequence structure analysis (Yoon and Welsh, 2004). Although the presence of AChE has not been reported in Lewy bodies of PDD or DLB, its association with these lesions would suggest the possibility of a more generalized role of AChE in the pathogenesis of age-related dementias. Whether treatments with AChEIs alter the course of these dementias, and, if so, by what mechanism, awaits further clinical studies and the further molecular characterization of these disorders.
IX. C O N C L U S I O N S Effective preventive and therapeutic strategies for AD and other age-related neurodegenerative diseases are the most pressing need in modem clinical neurological practice. Remarkable advances in our insight into the pathogenesis of AD in particular hold forth the prospect that manipulation of pathways of amyloid peptide synthesis, posttranslational processing, aggregation, degradation, interactions with other macromolecules, or the relation of A[3 to oxidative, inflammatory, or neuroexcitatory processes that may promote or be promoted by its presence in the cerebrum may profoundly alter the incidence or progression of AD in the future. The multifactorial nature of the etiology of sporadic AD seems to imply a likelihood that the most effective treatment strategies will target several or many of these processes. Indeed, many models of the progression of AD invoke self-reinforcing cycles of cerebral damage that may be checked in part at any one of a number of steps but that may be best approached by therapeutic strategies that target multiple aspects of disease pathogenesis. Definitive experimental evidence for a role of ACh in the pathogenesis of human neurodegenerative disease may prove elusive and perhaps become unambiguously manifest only in the context of cotreatments with AChEIs and agents that target other aspects of disease. In the meantime, the symptomatic effects alone of AChEIs dictate that they will remain important treatments for AD and, in all likelihood, for an expanding list of age-related dementing illnesses.
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Erkinjuntti, T., Skoog, I., Lane, R., and Andrews, C. (2003). Potential long-term effects of rivastigmine on disease progression may be linked to drug effects on vascular changes in Alzheimer brains. Int. J. Clin. Pract. 57, 756-760. Erkinjuntti, T., Roman, G., and Gauthier, S. (2004). Treatment of vascular dementia--Evidence from clinical trials with cholinesterase inhibitors. J. Neurol. Sci. 226, 63-66. Etienne, E, Robitaille, Y., Wood, E, Gauthier, S., Nair, N. E, and Quirion, R. (1986). Nucleus basalis neuronal loss, neuritic plaques and choline acetyltransferase activity in advanced Alzheimer's disease. Neuroscience 19, 1279-1291. Evans, D. A., Hebert, L. E., Beckett, L. A., Scherr, E A., Albert, M. S., Chown, M. J., Pilgrim, D. M., and Taylor, J. O. (1997). Education and other measures of socioeconomic status and risk of incident Alzheimer disease in a defined population of older persons. Arch. Neurol. 54, 1399-1405. Farlow, M., Anand, R., Messina, J., Jr., Hartman, R., and Veach, J. (2000). A 52-week study of the efficacy of rivastigmine in patients with mild to moderately severe Alzheimer's disease. Eur. Neurol. 44, 236-241. Farlow, M., Potkin, S., Koumaras, B., Veach, J., and Mirski, D. (2003). Analysis of outcome in retrieved dropout patients in a rivastigmine vs placebo, 26-week, Alzheimer disease trial. Arch. Neurol. 60, 843-848. Feldman, H., Gauthier, S., Hecker, J., Vellas, B., Subbiah, E, and Whalen, E. (2001). Donepezil MSAD Study Investigators Group. A 24-week, randomized, double-blind study of donepezil in moderate to severe Alzheimer's disease. Neurology 57, 613-620. Folstein, M. E, Folstein, S. E., and McHugh, E R. (1975). "Minimental state?' A practical method for grading the cognitive state of patients for the clinician. J. Psychiatr. Res. 12, 189-198. Forlenza, O. V., Spink, J. M., Dayanandan, R., Anderton, B. H., Olesen, O. E, and Lovestone, S. (2000). Muscarinic agonists reduce tau phosphorylation in non-neuronal cells via GSK-3beta inhibition and in neurons. J. Neural. Transm. 107, 1201-1212. Fr61ich, L. (2002). The cholinergic pathology in Alzheimer's diseasemDiscrepancies between clinical experience and pathophysiological findings. J. Neural. Transm. 109, 1003-1014. Giacobini, E. (2000). Cholinesterase inhibitors: From the Calabar bean to Alzheimer therapy. In Cholinesterases and Cholinesterase Inhibitors (E. Giacobini, Ed.), pp. 181-226. Martin Dunits, London. Guillozet, A. L., Smiley, J. E, Mash, D. C., and Mesulam, M. M. (1997). Butyrylcholinesterase in the life cycle of amyloid plaques. Ann. Neurol. 42, 909-918. Hall, K. S., Gao, S., Unverzagt, E W., and Hendrie, H. C. (2000). Low education and childhood rural residence: Risk for Alzheimer's disease in African Americans. Neurology 54, 95-99. Hardy, J., and Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics. Science 297, 353-356. Hating, R., Fisher, A., Marciano, D., Pittel, Z., Kloog, Y., Zuckerman, A., Eshhar, N., and Heldman, E. (1998). Mitogenactivated protein kinase-dependent and protein kinase C-dependent pathways link the ml muscarinic receptor to beta-amyloid precursor protein secretion. J. Neurochem. 71, 2094-2103. Harkins, S. W., Taylor, J. R., Mattay, V., and Regelson, W. (1997). Tacrine treatment in Alzheimer's disease enhances cerebral
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Epidemiology
blood flow and mental status and decreases caregiver suffering. Ann. N. Y. Acad. Sci. 826, 472-474.
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Lojkowska, W., Ryglewicz, D., Jedrzejczak, T., Minc, S., Jakubowska, T., Jarosz, H., and Bochynska, A. (2003). The effect of cholinesterase inhibitors on the regional blood flow in patients with Alzheimer's disease and vascular dementia. J. Neurol. Sci. 216, 119-126. Lyketsos, C. G., Breitner, J. C., and Rabins, E V. (2001). An evidence-based proposal for the classification of neuropsychiattic disturbance in Alzheimer's disease. Int. J. Geriatr. Psychiatr. 16, 1037-1042. Marjaux, E., Hartmann, D., and De Strooper, B. (2004). Presenilins in memory, Alzheimer's disease, and therapy. Neuron 42, 189-192. Minthon, L., Gustafson, L., Dalfelt, G., Hagberg, B., Nilsson, K., Risberg, J., Rosen, I., Seiving, B., and Wendt, E E. (1993). Oral tetrahydroaminoacridine treatment of Alzheimer's disease evaluated clinically and by regional cerebral blood flow and EEG. Dementia 4, 32-42. Moritz, D. J., and Petitti, D. B. (1993). Association of education with reported age of onset and severity of Alzheimer's disease at presentation: Implications for the use of clinical samples. Am. J. Epidemiol. 137, 456-462. Munoz, D. G., and Feldman, H. (2000). Causes of Alzheimer's disease. Can. Med. Assoc. J. 162, 65-72. Muramoto, O., Sugishita, M., Sugita, H., and Toyokura, Y. (1979). Effect of physostigmine on constructional and memory tasks in Alzheimer's disease. Arch. Neurol. 36, 501-503. Murphy, M. E, Sramek, J. J., Kurtz, M. M., et al. (1998). Optimizing the Development of the Next Generation of Compounds for Alzheimer's Disease. Greenwich Medical Media, London.
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CHAPTER ' 4
Coadministration of M e m a n t i n e with A c e t y l c h o l i n e s t e r a s e Inhibitors: Preclinical and Clinical Evidence
ANDRZEJ DEKUNDY Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany
in vivo showed that such a noncontingent activation of this receptor type also leads to impairment of neuronal plasticity (learning), which can be restored by therapeutic concentrations of memantine (Danysz and Parsons, 2003). These preclinical results were further confinned in multiple clinical trials. Memantine has been approved by the Food and Drug Administration as the first drug for moderate to severe AD and is also available in Europe. After the antidementia potential of memantine was widely acknowledged, it became obvious that possible effects of coadministration of this drug with the clinically available AChE inhibitors required investigation. This chapter presents the preclinical and clinical profile of memantine and discusses results of animal and human studies on its coadministration with various classes of AChE inhibitors.
I. INTRODUCTION The pathogenesis of Alzheimer's disease (AD) as a neurodegenerative disease involves more than one mechanism. Multiple studies in the past two decades revealed that degeneration of cholinergic nuclei localized in the basal forebrain occurs in the course of this disease. Impairment of the cholinergic system, which projects into large areas of the limbic system and the neocortex, is followed by disturbance of attentional processes and cognitive decline (Terry and Buccafusco, 2003). Consistent with these findings, some compounds that increase cholinergic neurotransmission were found to be effective in AD. Of the various cholinomimetic drug classes, only reversible acetylcholinesterase inhibitors (AChEIs) have been successfully used in AD patients (for review, see Ibach and Haen, 2004). The first compounds of this class tested in clinical trials were physostigmine (Stem et al., 1988; Thal et al., 1989) and tacrine (Summers et al., 1986). However, because of the short duration of action of the former and hepatotoxicity of the latter drug, their use in AD patients has been largely abandoned (Ibach and Haen, 2004). Tacrine was the first drug approved specifically for the treatment of AD (1993). This was followed by the development of compounds with improved specificity and tolerability: donepezil (1996), rivastigmine (2000), and galantamine (2001). Aside from the cholinergic hypothesis, it is generally agreed that compromised neuronal energy metabolism may occur in AD. Continuous mild activation of N-methylD-aspartate (NMDA) receptors under such conditions may render the cells susceptible to subsequent damage. Indeed, the NMDA antagonist memantine was shown to be efficient in animal models relevant to human neurodegenerative diseases and dementia. Preclinical studies in vitro and Toxicology of Organophosphate and Carbamate Compounds
II. PROPERTIES OF M E M A N T I N E Memantine (1-amino-3,5-dimethyl-adamantane hydrochlofide, ClzHz0N.HC1) was first synthesized in the early 1960s by Eli Lilly & Company as a derivative of amantadine, an anti-influenza agent. It was intended to become a hypoglycemic agent, but it was found to be void of such activity (Gerzon, 1963). Instead, the amino group in the three-ring (adamantane) structure (Fig. 1) has been shown to bind close to the magnesium site in the NMDA receptor channel (Blanpied et al., 1997; Sobolevsky et al., 1998; Kashiwagi et al., 2002). Memantine is a colorless to white crystalline substance readily soluble in water. Its oral bioavailability is nearly 100%. Memantine readily crosses the blood-brain barrier, and while in organism it shows linear, dose-proportional pharmacokinetics and weak protein-binding properties. 35
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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S ECTI O N I 9Uses, A b u s e s , & E p i d e m i o l o g y NH2
H3C
IV. C L I N I C A L U S E O F M E M A N T I N E
CH3
FIG. 1. The chemical structure of memantine.
Memantine's elimination half-life is approximately 60-80 hr; it is excreted mainly unchanged through the kidneys and undergoes limited metabolism in the liver. The concentrations of memantine in the cerebrospinal fluid (CSF) were shown to correlate well with its serum levels, with a CSF:serum ratio of 0.52 (Kornhuber and Quack, 1995). Memantine has also been shown to have minimal inhibitory effect on CYP450 enzymes (Periclou et al., 2004).
III. M E C H A N I S M OF MEMANTINE
OF ACTION
Serendipitous observation of beneficial effects of aminoadamantanes in Parkinson's disease patients undergoing antiviral therapy led to the conclusion that these compounds may possess dopaminomimetic or possibly cholinolytic properties. The fact that administration of clinically relevant doses of memantine results primarily in NMDA receptor antagonism was discovered only in the late 1980s (Bormann, 1989). Memantine is a specific, uncompetitive, voltagedependent NMDA receptor antagonist with moderate affinity, strong voltage dependency, and rapid blocking/ unblocking receptor kinetics (Fig. 2; Danysz et al., 2000). Under normal physiological resting conditions, magnesium ions block the NMDA receptor channels to inhibit calcium entry to the cell. Compromised neuronal energy metabolism is likely to occur in certain pathologic states, including AD. Continuous mild activation of NMDA receptors under such conditions may impair the magnesium block, rendering the cells more susceptible to subsequent damage (Greenamyre et al., 1988; Harkany et al., 2000; Winblad et al., 2002). Under such conditions, the moderate receptor affinity and fast blocking/unblocking kinetics give memantine a unique pharmacological profile. Memantine is able to prevent the pathogenic calcium influx caused by continuous mild activation by low-level glutamate. On the other hand, memantine allows the physiological activation of the NMDA channels by high concentrations of glutamate, a phenomenon necessary for synaptic plasticity underlying normal learning and memory (Parsons et al., 1999b). Thus, memantine can be compared to a "noise reduction system," filtering the necessary physiological "signal" from the unwanted "noise" disturbing the normal function (i.e., increasing the "signal-to-noise" ratio) (Danysz et al., 2000).
Subsequent to approval in Europe (2002), memantine was approved as the first drug for moderate to severe AD in the United States (2003). To date, seven double-blind, parallel group, placebo-controlled, randomized trials have been published in which memantine was administered as monotherapy to patients suffering from primary dementia (Ditzler, 1991; G6rtelmeyer and Erbler, 1992; Pantev et al., 1993; Winblad and Poritis, 1999; Wilcock et al., 2002; Orgogozo et al., 2002; Reisberg et al., 2003). The early, relatively small trials suggested memantine to be capable of exerting positive effects in unspecified dementia (Ditzler, 1991; G6rtelmeyer and Erbler, 1992). In a first trial applying recent methods, Winblad and Poritis (1999) assessed the clinical efficacy and safety of memantine in severe primary dementia in 166 patients, 49% of the Alzheimer type and 51% of the vascular type, after 12 weeks of treatment. In the prespecified AD subgroup, a total of 79 nursing home residents were randomized to receive either a conservative dose of 10 mg memantine (single daily dose) or placebo. Despite the small sample size, memantine had statistically significant favorable effects in all of the three main domains m that is, cognitive, functional, and globalmas measured by the cognitive subscore of the Rating Scale for Geriatric Patients [BGP (Beurteilungsskala ftir Geriatrische Patienten), based on the Stockton Geriatric Rating Scale], the care dependence subscore of the BGP, and the Clinical Global Impression of Change, respectively. Regarding the safety profile, no significant differences between treatment groups were observed (Graham et al., 2003). In the study of Reisberg et al. (2003), 252 outpatients with moderate to severe AD were randomly assigned to receive placebo or 20 mg of memantine daily for 28 weeks. Of these, 72% completed the study and were evaluated at week 28. Seventy-one patients discontinued treatment prematurely (42 taking placebo and 29 taking memantine). The primary efficacy variables were the Clinician's Interview-Based Impression of Change Plus Caregiver Input (CIBIC-Plus) and the Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory modified for severe dementia (ADCSADLl9 ). The secondary efficacy end points included the Severe Impairment Battery (SIB) and other measures of cognition, function, and behavior. Patients receiving memantine had statistically significantly better scores than those receiving placebo in cognitive functions and global outcomes as measured by the CIBIC-Plus, ADCS-ADL19, and SIB. Consistent with previous results, the frequency of adverse effects was similar to that for placebo (Reisberg et al., 2003). The results of these two trials provide further support to the hypothesis that memantine treatment leads to functional improvement and reduces care dependence in severely demented patients.
CHAPTER 4
9M e m a n t i n e and AChE Inhibitors
FIG. 2. (A) Schematic representation of glutamatergic synapse and the major ionotropic glutamate receptors, AMPA and NMDA. (B) The principle of synaptic plasticity in the central nervous system is detection of the relevant signal over the existing background noise. Such a signal, once detected, may lead to a long-lasting alteration in synaptic strength. NMDA receptors play a central role in such alterations and an endogenous "noise suppressant" is magnesium. Physiological mechanisms are fully capable of keeping very low glutamate levels in the synaptic cleft under normal conditions. (C) In neurodegenerative diseases such as Alzheimer's disease, contributing factors (e.g., malfunctioning uptake into the astroglia) lead to sustained enhanced concentrations of glutamate in the synaptic cleft and resulting partial depolarization. Under such conditions, magnesium is no longer capable of suppressing the "noise." This leads to malfunctioning of signal detection (cognitive functions) and, with time, to damage of vulnerable neurons. Reproduced from Danysz et al. (2000), copyright FP Graham Publishing Company.
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38
S E CTI O N I
9Uses, Abuses,
& gpidemiology
Two independent, placebo-controlled clinical trials conducted on approximately 900 patients with diagnosed mild to moderate vascular dementia (NINCDS-AIREN) showed that memantine (20 mg daily) significantly improved cognition relative to placebo as assessed by the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-cog) with no deterioration in global functioning and behavior. Memantine was considered well tolerated and safe (Wilcock et al., 2002; Orgogozo et al., 2002). Peskind et al. (2004) studied 403 patients older than 50 years of age with a diagnosis of mild to moderate AD. Patients were randomized to memantine 10 mg twice a day or to placebo and followed for 24 weeks. Over the course of 24 weeks, the patients taking memantine were shown to significantly improve in cognition (ADAS-cog) and remained improved compared to baseline at 24 weeks. In contrast, patients receiving placebo experienced small but significant steady declines in cognitive function over the 24-week study. Moreover, memantine was shown to significantly reduce societal as well as caregiver costs. Caregiver time for memantine-treated AD patients was assessed by means of the Resource Utilisation in Dementia Scale (Wimo et al., 1998). A significant reduction in mean monthly caregiver time by approximately 52 hr was demonstrated. In addition, a significant difference was found in the institutionalization status at week 28, favoring memantine treatment over placebo (Wimo et al., 2003). This finding may also indirectly point to the clinical efficacy of memantine. For more extensive reviews of clinical efficacy and safety trials, see Winblad and Jelic (2003), M6bius (2003), and Wilcock (2003).
discontinuations due to an adverse effect compared to placebo (7.4 vs 12.4%, respectively). Results of several postmarketing surveillance studies (PMS) of memantine in "dementia syndrome" are in accordance with the results of clinical trials. Rieke and Glaser (1996) followed 1420 patients treated with memantine (10-20 mg/day) for more than 1 year. At the end of the observation period, physicians appraised memantine as very well or well tolerated in 94% of cases. The most frequently reported adverse effects were restlessness (1.3%), nausea (0.9%), dizziness (0.8%), and fatigue/ tiredness or sleep disorders (0.4%). In another study of 531 care-dependent patients treated with memantine (up to 30 mg/day for a mean of 44 days), the drug was also well tolerated; 3% of patients reported adverse effects (Rt~ther et al., 2000). Noteworthy, a recent postmarketing surveillance study in 158 patients (median memantine dose, 20 mg/day), suffering mainly from Alzheimer's disease (77%) or vascular dementia (9%), and concurrently treated with memantine and AChE inhibitors revealed that the combination therapy was very well (56%) or well (42%) tolerated (Hartmann and M6bius, 2003). In both placebocontrolled and PMS trials, the nature of the memantine side effect profile appears to remain unaffected, irrespective of monotherapy or combination therapy with AChEIs. Based on the results of the previous studies, memantine may be considered a safe and well-tolerated drug. This is consistent with animal data showing that the therapeutic index for memantine is markedly higher than that for other NMDA antagonists (e.g., MK-801) (Parsons et al., 1999b).
VI. P R E C L I N I C A L P H A R M A C O L O G Y OF MEMANTINE V. S A F E T Y O F M E M A N T I N E The safety database of clinical trials with memantine comprises thousands of patients. The data derive from clinical trials of memantine carried out on patients suffering from primary dementia or other neurological disorders (e.g., peripheral neuropathy, Parkinson's disease, multiple sclerosis, and spasticity). A total of 2297 patients have been exposed to memantine, with 1244 patients receiving placebo, in 27 clinical trials. In double-blind, placebocontrolled dementia trials, comparable numbers of memantine patients and placebo patients experienced adverse effects, with dizziness, confusion, headache, and constipation among the most frequently reported. Most adverse effects were considered mild or moderate in severity and not related to the trial drug (M6bius et al., 2004). In a combination therapy trial, memantine was administered to patients receiving continuous donepezil therapy (Tariot et al., 2004). Daily doses of memantine (20 mg, added to donepezil) were very well tolerated, as indicated by fewer
A. In Vitro Profile of Memantine In in vitro studies, memantine displayed a Ki of approximately 1 IxM in NMDA competitive binding assays utilizing [3H]MK-801 in human and rat brain tissue (Komhuber et al., 1991; Bresink et al., 1995). Memantine is relatively selective for the [3H]MK-801 binding site. Memantine was shown to possess 5-HT3 receptor antagonistic properties in the low-micromolar range. Memantine's ability to block nicotinic receptors is approximately 6-10 times weaker than that to inhibit NMDA receptors (Danysz et al., 1997). Electrophysiological studies revealed an uncompetitive, voltage-dependent blockade of NMDA receptors with median inhibiting concentration (IC50) values of 1-3 txM (Parsons et al., 1993, 1999b). As mentioned previously, the hallmark of memantine that makes it unique among NMDA antagonists is its pronounced voltage dependence and rapid blocking/unblocking kinetics. It has been shown that memantine can block the toxicity of glutamate to cultured cortical neurons with an ICs0 of 1.4 txM (Parsons
CHAPTER 4 9Memantine and AChE Inhibitors et al., 1999a). On the other hand, hampering long-term potentiation requires much higher concentrations of memantine (ICs0 = 11.6 txM) (Frankiewicz et al., 1996; Frankiewicz and Parsons, 1999). This profile makes memantine capable of blocking pathological, but not physiological, activation of NMDA receptors. Unlike memantine, other NMDA receptor antagonists, such as MK-801 or ketamine, were shown to block physiological activation at their "therapeutic" doses (Parsons et al., 1999b). Neurofibrillary degeneration, considered to be a hallmark of AD and related tauopathies, is a consequence of abnormal hyperphosphorylation of tau protein. This in turn may be related to a decrease in protein phosphatase (PP-2A) activity found in the AD brain. Interestingly, memantine was shown to inhibit and reverse PP-2A inhibition-induced abnormal hyperphosphorylation and accumulation of tau in organotypic culture of rat hippocampal slices (Li et al., 2004).
B. In Vivo Profile of Memantine Memantine has been demonstrated to exert protective effects in animal models relevant to human neurodegenerative diseases and dementia. Given as a food supplement for 10 days prior to acute intrahippocampal exposure to quinolinic acid, memantine prevented excitotoxic damage to hippocampal neurons (Keilhoff and Wolf, 1992). When delivered subcutaneously (sc) by osmotic minipump (20 mg/kg/day), it prevented the occurrence of short-term memory deficits induced by chronic intracerebroventricular (icv) administration of quinolinic acid, as measured in T-maze (Misztal et al., 1996), and it protected against the loss of cholinergic neurons of the nucleus basalis of Meynert resulting from inflammation caused by chronic icv infusion of lipopolysaccharides (Willard et al., 2000). Furthermore, continuous infusion of memantine (15 mg/kg/day, sc) protected against pathological changes and learning deterioration (measured by the T-maze test) induced by intrahippocampal injection of [3-amyloid (Miguel-Hidalgo et al., 2002). Memantine also exerted direct effects on learning, demonstrating the capability to restore the learning impairment in radial maze caused by lesioning the entorhinal cortex (Zajaczkowski et al., 1996) or direct activation of NMDA receptors (Zajaczkowski et al., 1997). Many patients with AIDS are likely to develop dementia. Macrophages infected with HIV are believed to release toxins producing neuronal damage, a phenomenon diminished by NMDA antagonists (Giulian et al., 1993). Indeed, memantine has been demonstrated to prevent neuronal degeneration caused by gp 120, the HIV envelope glycoprotein (Muller et al., 1992). Like many other NMDA antagonists, memantine has been shown to exert dose-dependent protective effects in the rat model of transient forebrain ischemia (Seif el Nasr et al., 1990). Memantine provided neuroprotection in the model of transient MCA occlusion after a single injection
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of 20mg/kg (Chen et al., 1998). At a daily dose of 20 mg/kg, memantine enhanced the ischemic tolerance induced by transient ischemia (Parsons et al., 2001). NMDA receptors have been implicated in sensitization of central pain pathways in neuropathic pain (Dubner and Ruda, 1992), and N M D A antagonists have been demonstrated to exhibit analgesic properties in a range of animal models of pain (Parsons et al., 1999b). Accordingly, memantine (10 mg/kg, ip) has been shown to block and reverse thermal analgesia in chronic constriction injury in the rat, a model of painful neuropathy (Eisenberg et al., 1995). Memantine (25-75 mg/kg, po or im) has been shown to be capable of reducing mechanical allodynia induced by a spinal nerve ligation in macaque monkeys (Carlton et al., 1994, 1998). Memantine was also shown to inhibit morphine selfadministration (Semenova et al., 1999) and to attenuate morphine place preference (Popik and Danysz, 1997; Popik et al., 2000), suggesting its potential effectiveness in treating morphine dependence. Moreover, memantine inhibited tolerance to the analgesic action of this opioid (Popik et al., 2000). Relapse drinking of ethanol in rats has been selectively inhibited by continuous sc infusion of memantine (Holter et al., 1996). Higher doses of memantine have been demonstrated to exert antiparkinsonian-like activity in animal models, such as haloperidol-induced catalepsy, reserpine-induced sedation, and rotation in rats with a unilateral lesion to the nigrostriatal system (Danysz et al., 1994; Karcz-Kubicha et al., 1999). Memantine also displayed a clear-cut, dose-dependent antidepressant-like activity in the Porsolt test (Moryl et al., 1993). For a detailed review of the preclinical profile of memantine, see Danysz et al. (1997, 2000).
VII. I N T E R A C T I O N O F M E M A N T I N E
WITH AChEIs A. Experimental Studies 1. IN VITRO STUDIES The first study to specifically address the issue of possible interference of memantine with AChE inhibition by drugs approved for the treatment of AD was an in vitro study performed by Wenk et al. (2000). The authors assessed the effects of preincubation of homogenates of the striata of untreated rats with memantine (1 or 5 IxM) on the inhibition of AChE activity produced by a clinically relevant concentration of galantamine, donepezil, and tacrine. Additionally, an irreversible organophosphate (OP) AChE inhibitor, diisopropyl fluorophosphate (DFP), was used as a positive control (McLean et al., 1992). Memantine alone, even at concentrations exceeding the therapeutically relevant ones, was not found to be capable of inhibiting AChE activity. The enzymatic inhibition by galantamine, tacrine, and
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S ECTI 0 N I 9Uses, Abuses, & Epidemioloooy
donepezil was unaffected by memantine. However, memantine attenuated the inhibition produced by DFP, consistent with an earlier report investigating the nerve gas soman, an another irreversible OP inhibitor of AChE (McLean et al., 1992). It was suggested that the inhibitory effects of therapeutically relevant concentrations of memantine upon actions of AChEIs in vitro may be restricted to the irreversible ones (Wenk et al., 2000). McLean et al. (1992) demonstrated findings consistent with those of Wenk et al. (2000), demonstrating that memantine reduced AChE inhibition in crude brain homogenates by soman but not by an anionic site inhibitor (edrophonium) or a peripheral site inhibitor (decamethonium). The authors suggested that memantine may bind to some other, not yet characterized, modulatory site to protect ACHE. Enz and Gentsch (2004) attempted to determine in vitro whether memantine was able to prevent AChE inhibition by rivastigmine in a rat striatal preparation. After an initial preincubation period of 10 min, rivastigmine dose dependently inhibited AChE with an IC50 of 32 + 2 IxM. Memantine by itself did not inhibit the enzyme unless very high (millimolar) concentrations were used. After the 10-min preincubation, rivastigmine at a concentration of 30 txM caused approximately 45% inhibition of AChE in the rat striatal homogenate. Memantine (1, 5, 10, 50, or 100 IxM) did not influence the inhibition caused by rivastigmine, irrespective of whether it was applied before or after the AChE inhibitor (Enz and Gentsch, 2004). 2. IN VIVO STUDIES In vitro studies give important insight into the possible mechanisms of interaction of memantine. However, it is extremely important to prove whether and to what extent the results of in vitro experiments reflect the in vivo situation. Indeed, in a recent series of in vivo experiments, Gupta and colleagues investigated whether memantine may interact with inhibition of AChE activity by reversible (donepezil and rivastigmine) and irreversible (DFP and metrifonate) AChEIs in rat brain regions affected in AD (i.e., the cortex and the hippocampus). In preliminary experiments, a dose of each AChEI causing 40-50% inhibition of brain AChE was found. This level of AChE inhibition is well within the range previously shown to result from administration of therapeutic doses of donepezil and rivastigmine (Ibach and Haen, 2004). Then, in time course experiments, the time of maximal AChE inhibition was determined for each of the AChEIs studied. Further experiments per aimed to clarify whether memantine may interfere with AChE inhibition induced by donepezil, rivastigmine, metrifonate, or DFP (administered at the dose causing 40-50% AChE inhibition and twice that dose), measured at the time of maximum AChE inhibition. Memantine at a dose of 10mg/kg (i.e., two to four times greater than the therapeutically relevant dose) was
administered 15 min prior to donepezil (0.75 or 1.5 mg/kg), rivastigmine (0.35 or 0.7mg/kg), metrifonate (55 or 110 mg/kg), or DFP (1.5 or 3.0 mg/kg). All drugs were administered intraperitoneally (ip). Rats were sacrificed at the time of maximal AChE inhibition determined in the time course study (i.e., 15 min after donepezil, 30 min after rivastigmine or metrifonate, and 60 min after DFP) to determine AChE activity in the brain region homogenate. Neither memantine nor most of the AChEIs produced any toxic signs at any time during the study; however, metrifonate did produce muscle tremors at 110 mg/kg. Each AChEI studied produced a dose-dependent inhibition of ACHE. Memantine did not cause AChE inhibition in any brain area. The findings from those interaction studies revealed that memantine prevented AChE inhibition produced by DFP or metrifonate in both brain areas and at both dose levels. Memantine also prevented metrifonateinduced tremors. Finally, memantine did not interact with AChE inhibition by therapeutically used AChEIs, donepezil and rivastigmine, at either dose level. It was concluded that memantine can be applied concurrently with donepezil or rivastigmine (Gupta and Dekundy, 2005; Gupta et al., 2004; Dekundy et al., 2004). Enz and Gentsch (2004) compared AChE inhibition (measured ex vivo) following a 21-day chronic, oral administration of 6 Ixmol/kg rivastigmine or a combination of equimolar doses of rivastigmine and memantine (6 txmol&g perorally of either of the two compounds). Final administration of a drug or vehicle occurred 2 hr prior to decapitation of the rats. It was found that memantine, even at high brain levels of approximately 3500pmol/g, as achieved following the oral 6 txmol/kg dose, was unable to attenuate the inhibitory effect of rivastigmine on AChE activity in the rat brain. Following coadministration of memantine and rivastigmine for 21 days, AChE inhibition remained practically unaltered in comparison with that of the rivastigmineonly group. Whereas there are relatively few in vivo studies exploring a possible interaction of memantine with clinically available AChEIs, much more data have been published on the effects of memantine in animals treated with OP or carbamate (CM) insecticides used in agriculture or the household as well as in rodents experimentally intoxicated with the OP nerve gas soman. Pretreatment with a single dose of memantine (18 mg/kg, sc) alone or in combination with atropine (16 mg/kg, sc) prevented motor limbic seizures induced by a single sublethal dose of soman without causing any signs of sedation or ataxia. After seizure onset, memantine in combination with atropine, but not atropine alone, abolished the somaninduced seizures. AChE activities in the cortex, stem, striatum, and hippocampus were markedly reduced by soman but not by memantine, atropine, or a combination thereof. Preadministration of memantine and the combination of memantine and atropine in vivo significantly protected
CHAPTER 4 9Memantine and AChE lnhibitors AChE from inhibition by soman. However, when given after onset of soman-induced seizures, memantine and/or atropine did not reactivate ACHE, although seizures were controlled (McLean et al., 1992). Likewise, rats administered a single dose of an OP methyl parathion (5 mg/kg, ip) showed signs of hypercholinergic toxicity, including convulsions and depression of AChE activities in the cortex, stem, striatum, and hippocampus. Pretreatment with memantine (18 mg/kg, sc) 60 min and atropine (16 mg/kg, sc) 15 min before methyl parathion administration completely prevented the expected toxic signs and attenuated the induced inhibition of ACHE. Also, when given therapeutically, this combined treatment completely reversed the clinical evidence of methyl parathion toxicity and reduced AChE inactivation (Gupta and Kadel, 1990). Very similar results were shown in rats injected with a sublethal acute dose of the carbamate insecticide carbofuran (1.5 mg/kg, sc) (Gupta and Kadel, 1989). The results of these two studies suggest that memantine may counteract the acute toxicity of methyl parathion and carbofuran by protection of AChE from inhibition and rapid reactivation of inhibited ACHE. Additionally, memantine may increase rapid elimination of carbofuran and methyl parathion (Gupta and Kadel, 1989, 1990). A similar combination of memantine and atropine has been shown to reduce toxicity and AChE inhibition produced by other CM insecticides (i.e., aldicarb, methomyl, and oxamyl) (Gupta and Kadel, 1991; Gupta, 1994b). Interestingly, memantine was also demonstrated to exert beneficial effects in myotoxicity related to anticholinesterase intoxication. For instance, pretreatment with memantine (18 mg/kg, sc) together with atropine (16 mg/kg, sc) prevented necrotic lesions in skeletal muscles following administration of sublethal doses of different nerve agents (soman, satin, tabun, or VX). Attenuation of muscle AChE inhibition by anticholinesterases was implicated in the protective effects of memantine (Gupta and Dettbarn, 1992a). The beneficial effects of such antidotal treatment were shown in myotoxicity produced by administration of DFP (Gupta et al., 2002) and carbofuran (Gupta and Goad, 2000). Oxidative stress has been implicated in the pathology of myotoxicity, and pretreatment with memantine and atropine was demonstrated to prevent and/or reverse the increase in nitric oxide synthase and the decrease in high-energy phosphates induced by administration of DFP or carbofuran. These mechanisms may contribute to the well-documented favorable effects of memantine in AChEIinduced myopathology (Gupta and Goad, 2000; Gupta et al., 2002; Milatovic et al., 2005). On the other hand, Dai et al. (2004) studied the protective effect of memantine in dichlorvos-poisoned rats. Dichlorvos was applied at a dose of 25 mg/kg (ip) to three groups of rats, subsequently treated with memantine at doses of 5, 15, and 45 mg/kg. The activity of AChE and binding capacity of the NMDA receptor with [3H]MK-801 were determined 16 hr after dichlorvos injection. The low
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(5 mg/kg) and intermediate (15 mg/kg) doses of memantine were demonstrated to protect against the downregulation of NMDA receptor in rat brain. However, only the higher (45 mg/kg) dose of memantine slightly alleviated dichlorvos poisoning symptoms. The AChE activities in both blood and brain of memantine-treated groups were not significantly different from those in the dichlorvos-only group. The authors concluded that the observed protective effects of memantine in the dichlorvos-induced poisoning did not involve recovery of AChE activity (Dai et al., 2004).
B. Clinical Studies Schmitt et al. (2004) noted that the agents of at least six classes are approved for clinical use or are being tested or ready for phase III clinical trials for the treatment of AD. The authors listed AChEIs, NMDA receptor antagonists, antioxidants (including Gingko biloba preparations), antiinflammatory agents, neurotrophic factors, and anti-amyloid agents. Although all of these approaches may differentially modify the course of the disease and thus provide a rationale for the use of combination therapy, only nine clinical studies have investigated the effects of a combination regimen on cognitive performance or AD, and only five of these followed a randomized, controlled design. The authors emphasized the great need for further welldesigned studies on combination therapy in AD. The first publication to address the issue of combination treatment with memantine was a PMS study conducted among German physicians who, during routine clinical practice, treated demented patients with memantine in combination with an AChEI (Hartmann and M6bius, 2003). Most of the 158 surveyed patients (mean age, 74 years) were diagnosed with AD. However, other dementias were also included. Memantine was prescribed at a wide range of daily doses (5-60 mg) but the median dose was 20 mg/day, as recommended. Most patients received concomitant donepezil (84%), although 15% received rivastigmine and 1% received concomitant tacrine. In nearly all patients (98%), the combination therapy was well tolerated over the 4-month average observation period, during which the study participants were maintained at stable doses of both antidementia agents. The six reported adverse drug reactions resolved without sequelae and without drug discontinuation. Of these six events, two were considered unrelated to either drug, two mild events were probably related to memantine (10 mg/day), and one moderately severe event was probably related to donepezil (10 mg/day); drug relationship was not assessed by the physician in one case. No changes in blood chemistry were reported in 81% of patients (in 16%, no laboratory analyses were performed). Global clinical status was judged as improved or stable in 93% of patients (54% improved and 39% stable) over the observation period; worsening was observed in only 6%. In addition, improved communication abilities
4 2.
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9Uses, Abuses,
& Epidemiology
and elevated mood were frequently reported by treating physicians. These findings suggested for the first time that memantine in combination with AChEIs is effective, safe, and well tolerated (Hartmann and M6bius, 2003). Results of the first randomized, double-blind, placebocontrolled, parallel group trial of memantine in patients receiving an AChEI have been published only recently (Tariot et al., 2004). In this study, the efficacy and safety of memantine were compared to placebo in 404 patients with moderate to severe AD already receiving stable treatment with donepezil. The study started with a 1- or 2-week single-blind placebo screening period followed by 24 weeks of double-blind treatment. A total of 322 patients (80%) completed the trial. Participants were randomized to receive memantine (starting dose 5 mg/day, increased to 20 mg/day; n = 203) or placebo (n = 201) for 24 weeks. In patients with moderate to severe AD already receiving donepezil (5 or 10 mg/day) for the immediate preceding 6 months and at a stable dose for at least 3 months prior to and during the study period, memantine resulted in significantly better outcomes than placebo on measures of cognition, activities of daily living, global outcome, and behavior. Patients treated with memantine/donepezil appeared to show improvement relative to baseline over the 24-week course of the study, whereas patients receiving placebo/ donepezil exhibited progressive cognitive decline over the same duration. The drug-placebo difference was statistically significant. In addition, memantine was found to be safe and well tolerated. An open-label, multiple-dose study in 24 healthy volunteers (aged 18-35 years) was aimed at determining whether an in vivo pharmacokinetic interaction exists between memantine and donepezil (Periclou et al., 2004). Furthermore, the effect of memantine coadministration on inhibition of red blood cell AChE activity by donepezil was examined. The subjects received memantine (10 mg, po) on day 1. Following a 14-day washout period, the subjects were administered donepezil at a single daily dose of 5 mg for 7 days, followed by 10 mg for 22 days. The last dose was administered concurrently with memantine 10mg on day 43. Pharmacokinetic and safety parameters were assessed. AChE inhibition was measured in red blood cells by radiolabeled enzyme assay following administration of donepezil alone or donepezil with a single memantine dose. Data from 19 subjects who completed the study indicated no significant pharmacokinetic interactions between a single dose of memantine and donepezil at steady state. Maximum inhibition of AChE activity from baseline by donepezil was not significantly altered by coadministration with a single dose of memantine. In addition, single memantine doses administered with donepezil at steady state were well tolerated (Periclou et al., 2004). The previously mentioned clinical studies indicate that memantine and donepezil can be safely coadministered, with no significant effects on the pharmacokinetic profile
of either drug and no significant alterations of donepezilinduced AChE inhibition. These findings support the potential for the combined use of memantine and AChEIs in patients with AD. Further controlled clinical studies are needed to confirm the available data and to extend them to other clinically available AChEIs.
VIII. M E C H A N I S T I C - B A S E D C L I N I C A L CONSIDERATIONS AD is a progressive neurodegenerative disease characterized primarily by memory loss, behavioral problems, and the inability to perform daily activities. AD is the most common cause of dementia, affecting 11% of the population between the ages of 80 and 85 years and 24% of the population over the age of 85 years (Grutzendler and Morris, 2001). In fact, in the next 50 years, the prevalence of AD is expected to quadruple, affecting an estimated 1 in 45 people. AD is characterized by neuronal loss, particularly affecting cholinergic neurons in the basal forebrain, which projects into the hippocampus and neocortex, the brain structures that play an important role in memory and cognitive function. The loss of cholinergic neurons results in a remarkable decrease in acetylcholine levels, especially in the cerebral cortex and hippocampus (Terry and Buccafusco, 2003). It is accepted that disturbances in the glutamatergic system also play an important role in dementia. Although glutamate mediates physiological neurotransmission, under certain conditions, prolonged and elevated glutamate levels lead to increased calcium influx into neurons and finally to neurodegeneration due to "excitotoxicity" (Rothman and Olney, 1986, 1995). This phenomenon may also take place in AD (Greenamyre et al., 1988; Harkany et al., 2000). Bearing in mind that disturbances of several neurotransmitter systems may underlie AD, future pharmacotherapy may combine drugs with different modes of action to optimize symptomatic treatment and to potentially slow disease progression. The first approved therapeutic principles comprise AChE inhibition and NMDA receptor antagonism. Clinically, AChE inhibitors may improve cognition by boosting brain acetylcholine level (Ibach and Haen, 2004). On the other hand, the first approved NMDA receptor antagonist, memantine, seems to exert its beneficial effects by improving glutamatergic neurotransmission and by protecting against glutamate-induced excitotoxicity (Jacobsson and Fowler, 1999; Parsons et al., 1999b; Danysz et al., 2000). It is conceivable that memantine combined with an AChEI may represent a fruitful path in the treatment of dementia by targeting both cholinergic and glutamatergic neurotransmission. However, many in vivo studies of animals have shown that memantine is able to attenuate AChE inhibition by several organophosphate or carbamate AChEIs, including
CHAPTER 4 9Memantine and AChE Inhibitors
reference substances, insecticides, and nerve agents (Gupta and Kadel, 1989, 1990; McLean et al., 1992; Gupta and Dettbarn, 1992; Gupta, 1994a,b; Gupta and Goad, 2000). The in vivo data are supported by in vitro studies (McLean et al., 1992; Antonijevic et al., 2002; Stojiljkovic et al., 2002). Although the doses of memantine used in these investigations exceeded the therapeutically relevant doses by several times, the results of the previous studies raised concerns about possible coadministration of memantine with AChEIs approved for the treatment of AD. This in turn triggered further research aimed at elucidating whether such interaction really does exist. In in vitro interaction studies, Wenk et al. (2000) showed that memantine does not influence AChE inhibition in rat brain induced by donepezil, tacrine, or galantamine. These findings have been strengthened by the observation that memantine is not able to prevent either rivastigmine- or donepezil-induced AChE inhibition both in vitro and in vivo (Gupta and Dekundy, 2005; Gupta et al., 2004; Dekundy et al., 2004; Enz and Gentsch, 2004). Nevertheless, studies have also confirmed the potential of the NMDA antagonist to prevent AChE inhibition by some irreversible inhibitors of this enzyme, namely metrifonate and DFP (Wenk et al., 2000; Gupta et al., 2004; Dekundy et al., 2004; Gupta and Dekundy, 2005). Numerous in vivo studies in rodents give clear evidence that memantine at doses up to 72 mg/kg does not cause AChE inhibition (Gupta and Kadel, 1990; Antonijevic et al., 2002; Stojiljkovic et al., 2002), a finding excluding the possibility of an interaction with memantine at the active center (anionic site) of ACHE. This would explain the lack of interaction of memantine with donepezil that binds to this site. On the other hand, rivastigmine is a pseudo-irreversible AChEI that binds to the esteratic binding site of the active center of the enzyme. Rivastigmine is bound firmly to the active center of ACHE, with a reported decarbamylation time for the rivastigmine-AChE complex of approximately 10 hr (Ibach and Haen, 2004). This finding implies that memantine does not interact with the esteratic binding site. It was suggested that the biochemical mechanism underlying the interaction of memantine with some AChEIs could be allosteric modulation of the active center of the enzyme, which in turn could lead to its hindrance and unavailability to AChEIs, such as DFP, methyl parathion, and nerve agents (Gupta and Kadel, 1990; McLean et al., 1992; Antonijevic et al., 2002; Stojiljkovic et al., 2002). However, this mechanism remains to be elucidated.
IX. C O N C L U S I O N S AND FUTURE DIRECTIONS Because memantine and AChEIs reduce dementia symptoms through distinct mechanisms of action (glutamate antagonism and cholinesterase inhibition, respectively),
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there is growing interest in investigating whether this combination therapy may improve therapeutic benefit in demented patients. Results of animal studies and the first clinical trials suggest good efficacy and tolerability of the combination of the NMDA receptor antagonist and AChEIs. Future clinical research will optimize this approach.
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unified glutamatergic hypothesis on the mechanism of action. Neurotoxicity, Icol. Res. 2, 85-97. Dekundy, A., Gupta, R., Danysz, W., and Quack, G. (2004). Lack of interaction of memantine with antidementia AChE inhibitors in rat brain: Mechanistic based therapeutic considerations. Toxicol. Appl. Pharmacol. 197, 366. Ditzler, K. (1991). Efficacy and tolerability of memantine in patients with dementia syndrome. A double-blind, placebo controlled trial. Arzneimittelforschung 41, 773-780. Dubner, R., and Ruda, M. A. (1992). Activity-dependent neuronal plasticity following tissue injury and inflammation. Trends Neurosci. 15, 96-103. Eisenberg, E., LaCross, S., and Strassman, A. M. (1995). The clinically tested N-methyl-D-aspartate receptor antagonist memantine blocks and reverses thermal hyperalgesia in a rat model of painful mononeuropathy. Neurosci. Lett. 187, 17-20. Enz, A., and Gentsch, C. (2004). Co-administration of memantine has no effect on the in vitro or ex vivo determined acetylcholinesterase inhibition of rivastigmine in the rat brain. Neuropharmacology 47, 408-4 13. Frankiewicz, T., and Parsons, C. G. (1999). Memantine restores long term potentiation impaired by tonic N-methyl-D-aspartate (NMDA) receptor activation following reduction of Mg 2+ in hippocampal slices. Neuropharmacology 38, 1253-1259. Frankiewicz, T., Potier, B., Bashir, Z. I., Collingridge, G. L., and Parsons, C. G. (1996). Effects of memantine and MK-801 on NMDA-induced currents in cultured neurones and on synaptic transmission and LTP in area CA1 of rat hippocampal slices. Br. J. Pharmacol. 117, 689-697. Gerzon, K., Krumkalns, E. V., Brindle, R. L., Marshall, E J., and Root, M. A. (1963) The adamantyl group in medicinal agents. 1. Hypoglycemic arylsulfonyl-N'-adamantylureas. J. Med. Chem. 122, 760-763. Giulian, D., Wendt, E., Vaca, K., and Noonan, C. A. (1993). The envelope glycoprotein of human immunodeficiency virus type1 stimulates release of neurotoxins from monocytes. Proc. Natl. Acad. Sci. USA 90, 2769-2773. G6rtelmeyer, R., and Erbler, H. (1992). Memantine in the treatment of mild to moderate dementia syndrome. A double-blind placebo-controlled study. Arzneimittelforschung 42, 904-913. Graham, S., Lee, G., M6bius, H. J., McDonald, S., and Winblad, B. (2003). Efficacy and tolerability of memantine in nursing home patients with moderate to severe dementia of the Alzheimer's type. 43rd annual New Clinical Drug Evaluation Unit (NCDEU) meeting, Boca Raton, FL, May 26-28, p. 224. Greenamyre, J. T., Maragos, W. E, Albin, R. L., Penney, J. B., and Young, A. B. (1988). Glutamate transmission and toxicity in Alzheimer's disease. Prog. Neuropsychopharmacol. Biol. Psychiatr. 12, 421-430. Grutzendler, J., and Morris, J. C. (2001). Cholinesterase inhibitors for Alzheimer's disease. Drugs 61, 41-52. Gupta, R. C. (1994a). Carbofuran toxicity. J. Toxicol. Environ. Health 43, 383-4 18. Gupta, R. C. (1994b). Mechanistic and clinical approaches in antidotal treatment with memantine and atropine against oxamyl and methomyl-acute toxicity. Ind. J. Toxicol. 1, 1-10. Gupta, R. C., and Dekundy, A. (2005). Memantine does not influence AChE inhibition in rat brain by donepezil or rivastigmine but does with DFP and metrifonate in in vivo studies. Drug Dev. Res., 64, 71-81.
Gupta, R. C., and Dettbarn, W.-D. (1992). Potential of memantine, d-tubocurarine, and atropine in preventing acute toxic myopathy induced by organophosphate nerve a g e n t s - Soman, satin, tabun and VX. Neurotoxicology 13, 649-661. Gupta, R. C., and Goad, J. T. (2000). Role of high-energy phosphates and their metabolites in protection of carbofuraninduced biochemical changes in diaphragm muscle by memantine. Arch. Toxicol. 74, 13-20. Gupta, R. C., and Kadel, W. L. (1989). Prevention and antagonism of acute carbofuran intoxication by memantine and atropine. J. Toxicol. Environ. Health 28, 111-122. Gupta, R. C., and Kadel, W. L. (1991). Novel effects of memantine in antagonizing acute aldicarb toxicity: Mechanistic and applied considerations. Drug Dev. Res. 24, 329-341. Gupta, R. C., and Kadel, W. L. (1990). Methyl parathion acute toxicity: Prophylaxis and therapy with memantine and atropine. Arch. Int. Pharmacodyn. Ther. 305, 208-221. Gupta, R. C., Milatovic, D., and Dettbarn, W.-D. (2002). Involvement of nitric oxide in myotoxicity produced by diisopropylphosphorofluoridate (DFP)-induced muscle hyperactivity. Arch. Toxicol. 76, 715-726. Gupta, R. C., Dekundy, A., Danysz, W., and Quack, G. (2004). Memantine does not interfere with donepezil or rivastigmineinduced inhibition of AChE in rat cortex and hippocampus. J. Neurochem. 90(Suppl. 1), 43. Harkany, T., Abraham, I., Timmerman, W., Laskay, G., Toth, B., Sasvari, M., Konya, C., Sebens, J. B., Korf, J., Nyakas, C., Zarandi, M., Soos, K., Penke, B., and Luiten, E G. (2000). Beta-amyloid neurotoxicity is mediated by a glutamatetriggered excitotoxic cascade in rat nucleus basalis. Eur. J. Neurosci. 12, 2735-2745. Hartmann, S., and M6bius, H. J. (2003). Tolerability of memantine in combination with cholinesterase inhibitors in dementia therapy. Int. Clin. Psychopharmacol. 18, 81-85. Holter, S. M., Danysz, W., and Spanagel, R. (1996). Evidence for alcohol anti-craving properties of memantine. Eur. J. Pharmacol. 314, R1-R2. Ibach, B., and Haen, E. (2004). Acetylcholinesterase inhibition in Alzheimer's disease. Curr. Pharm. Design 10, 231-251. Jacobsson, S. O., and Fowler, C. J. (1999). Dopamine and glutamate neurotoxicity in cultured chick telencephali cells: Effects of NMDA antagonists, antioxidants and MAO inhibitors. Neurochem. Int. 34, 49-62. Karcz-Kubicha, M., Lorenz, B., and Danysz, W. (1999). GlycineB antagonists and partial agonists in rodent models of Parkinson's disease--Comparison with uncompetitive N-methyl-D-aspartate receptor antagonist. Neuropharmacology 38, 109-119. Kashiwagi, K., Masuko, T., Nguyen, C. D., Kuno, T., Tanaka, I., Igarashi, K., and Williams, K. (2002). Channel blockers acting at N-methyl-D-aspartate receptors: Differential effects of mutations in the vestibule and ion channel pore. Mol. Pharmacol. 61, 533-545. Keilhoff, G., and Wolf, G. (1992). Memantine prevents quinolinic acid-induced hippocampal damage. Eur. J. Pharmacol. 219, 451-454. Kornhuber, J., and Quack, G. (1995). Cerebrospinal fluid and serum concentrations of the N-methyl-D-aspartate (NMDA) receptor antagonist memantine in man. Neurosci. Lett. 195, 137-139.
CHAPTER 4 9Memantine and AChE lnhibitors Kornhuber, J., Bormann, J., Hubers, M., Rusche, K., and Riederer, E (1991). Effects of the 1-amino-adamantanes at the MK-801-binding site of the NMDA-receptor-gated ion channel: A human postmortem brain study. Eur. J. Pharmacol. 206, 297-300. Li, L., Sengupta, A., Haque, N., Grundke-Iqbal, I., and Iqbal, K. (2004). Memantine inhibits and reverses the Alzheimer type abnormal phosphorylation of tau and associated neurodegeneration. FEBS Lett. 566, 261-269. McLean, M. J., Gupta, R. C., Dettbarn, W.-D., and Wamil, A. W. (1992). Prophylactic and therapeutic efficacy of memantine against seizures produced by soman in the rat. Toxicol. Appl. Pharmacol. 112, 95-103. Miguel-Hidalgo, J. J., Alvarez, X. A., Cacabelos, R., and Quack, G. (2002). Neuroprotection by memantine against neurodegeneration induced by beta-amyloid (1-40). Brain Res. 958, 210-221. Milatovic, D., Gupta, R. C., Dekundy, A., Montine, T. J., and Dettbarn, W.-D. (2005). Carbofuran-induced oxidative stress in slow and fast skeletal muscles: Prevention by memantine and atropine. Toxicology 208, 13-24. Misztal, M., Frankiewicz, T., Parsons, C. G., and Danysz, W. (1996). Learning deficits induced by chronic intraventricular infusion of quinolinic acid--Protection by MK-801 and memantine. Eur. J. Pharmacol. 296, 1-8. M6bius, H. J. (2003). Memantine: Update on the current evidence. Int. J. Geriatr. Psychiatr. 18, $47-$54. M6bius, H. J., St6ffier, A., and Graham, S. M. (2004). Memantine hydrochloride: Pharmacological and clinical profile. Drugs Today 40, 685-695. Moryl, E., Danysz, W., and Quack, G. (1993). Potential antidepressive properties of amantadine, memantine and bifemelane. Pharmacol. Toxicol. 72, 394-397. Muller, W. E., Schroder, H. C., Ushijima, H., Dapper, J., and Bormann, J. (1992). gpl20 of HIV-1 induces apoptosis in rat cortical cell cultures: Prevention by memantine. Eur. J. Pharmacol. 226, 209-214. Orgogozo, J. M., Rigaud, A. S., St6ffier, A., M6bius, H. J., and Forette, E (2002). Efficacy and safety of memantine in patients with mild to moderate vascular dementia: A randomized, placebo-controlled trial (MMM 300). Stroke 33, 1834-1839. Pantev, M., Ritter, R., and G6rtelmeyer, R. (1993). Clinical and behavioral evaluation in long-term care patients with mild to moderate dementia under memantine treatment. Z. Gerontol. Psychiatr. 6, 103-117. Parsons, C. G., Gruner, R., Rozental, J., Millar, J., and Lodge, D. (1993). Patch clamp studies on the kinetics and selectivity of N-methyl-D-aspartate receptor antagonism by memantine (1-amino-3,5-dimethyladamantan). Neuropharmacology 32, 1337-1350. Parsons, C . G., Danysz, W., Bartmann, A., Spielmanns, E, Frankiewicz, T., Hesselink, M., Eilbacher, B., and Quack, G. (1999a). Amino-alkyl-cyclohexanes are novel uncompetitive NMDA receptor antagonists with strong voltage-dependency and fast blocking kinetics: In vitro and in vivo characterization. Neuropharmacology 38, 85-108. Parsons, C. G., Danysz, W., and Quack, G. (1999b). Memantine is a clinically well tolerated N-methyl-D-aspartate (NMDA) receptor antagonistmA review of preclinical data. Neuropharmacology 38, 735-767.
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Parsons, C. G., Frankiewicz, T., Lazarewicz, J. W., and Gadamski, R. (2001). Memantine enhances ischemic tolerance. Paper presented at the 53rd annual meeting of the American Academy of Neurology, May 5-11, Philadelphia. Periclou, A. E, Ventura, D., Sherman, T., Rao, N., and Abramowitz, W. T. (2004). Lack of pharmacokinetic or pharmacodynamic interaction between memantine and donepezil. Ann. Pharmacother. 38, 1389-1394. Peskind, E. R., Potkin, S. G., Pomara, N., Ott, B. R., McDonald, S., Xie Y., and Gergel, I. (2004). Memantine monotherapy is effective and safe for the treatment of mild to moderate Alzheimer's disease: A randomized controlled trial. Int. J. Neuropsychopharmacol. 7(Suppl. 1), $207. Popik, E, and Danysz, W. (1997). Inhibition of reinforcing effects of morphine and motivational aspects of naloxoneprecipitated opioid withdrawal by N-methyl-D-aspartate receptor antagonist, memantine. J. Pharmacol. Exp. Ther. 280, 854-865. Popik, E, Kozela, E., and Danysz, W. (2000). Clinically available NMDA receptor antagonists memantine and dextromethorphan reverse existing tolerance to the antinociceptive effects of morphine in mice. Naunyn-Schmied. Arch. Pharmacol. 361, 425-432. Reisberg, B., Doody, R., St6ffier, A., Schmitt, E, Ferris, S., and M6bius, H. J. (2003). Memantine in moderate-to-severe Alzheimer's disease. N. Engl. J. Med. 348, 1333-1341. Rieke, J., and Glaser, A. (1996). Efficacy and tolerability of memantine in patients with dementia. Med. Welt. 47, 251-254. Rothman, S. M., and Olney, J. W. (1986). Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19, 105-111. Rothman, S. M., and Olney, J. W. (1995). Excitotoxicity and the NMDA receptormStill lethal after eight years. Trends Neurosci. 18, 57-58. Rtither, E., Glaser, A., B leich, S., Degner, D., and Wiltfang, J. (2000). A prospective PMS study to validate the sensitivity for change of the D-scale in advanced stages of dementia using the NMDA-antagonist memantine. Pharmacopsychiatry 33, 103-108. Schmitt, B., Bernhardt, T., Moeller, H. J., Heuser, I., and Fr61ich, L. (2004). Combination therapy in Alzheimer's disease: A review of current evidence. CNS Drugs 18, 827-844. Seif el Nasr, M., Peruche, B., Rossberg, C., Mennel, H. D., and Krieglstein, J. (1990). Neuroprotective effect of memantine demonstrated in vivo and in vitro. Eur. J. Pharmacol. 185, 19-24. Semenova, S., Danysz, W., and Bespalov, A. (1999). Low-affinity NMDA receptor channel blockers inhibit acquisition of intravenous morphine self-administration in naive mice. Eur. J. Pharmacol. 378, 1-8. Sobolevsky, A. I., Koshelev, S. G., and Khodorov, B. I. (1998). Interaction of memantine and amantadine with agonistunbound NMDA-receptor channels in acutely isolated rat hippocampal neurons. J. Physiol. 512(Pt. 1), 47-60. Stem, Y., Sano, M., and Mayeux, R. (1988). Long-term administration of oral physostigmine in Alzheimer's disease. Neurology 38, 1837-1841. Stojiljkovic, M. E, Maksimovic, M., Kilibarda, V., Antonijevic, B., Milovanovic, Z. A., Jokanovic, M., and Boskovic, B. D. (2002).
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Memantine treatment improves antidotal efficacy of atropine, HI-6 and diazepam in rats poisoned with soman. Proc. Chem. Biol. Med. Treat. Symp., 3rd, Spiez, Switzerland, May 7-12, 2000. Summers, W. K., Majovski, L. V., Marsh G. M., Tachiki, K., and Kling, A. (1986). Oral tetrahydroaminoacridine in long-term treatment of senile dementia, Alzheimer type. N. Engl. J. Med. 315, 1241-1245. Tariot, E N., Farlow, M. R., Grossberg, G. T., Graham, S. M., McDonald, S., and Gergel, I. (2004). Memantine treatment in patients with moderate to severe Alzheimer disease already receiving donepezil: A randomized controlled trial. J. Am. Med. Assoc. 291, 317-324. Terry, A. V., Jr., and Buccafusco, J. J. (2003). The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: Recent challenges and their implications for novel drug development. J. Pharmacol. Exp. Ther. 306, 821-827. Thal, L. J., Masur, D. M., B lau, A. D., Fuld, E A., and Klauber M. R. (1989). Chronic oral physostigmine without lecithin improves memory in Alzheimer's disease. J. Am. Geriatr. Soc. 37, 42--48. Wenk, G. L., Quack, G., M6bius, H. J., and Danysz, W. (2000). No interaction of memantine with acetylcholinesterase inhibitors approved for clinical use. Life Sci. 66, 1079-1083. Wilcock, G., M6bius, H. J., and St6ffier, A. (2002). A doubleblind, placebo-controlled multicentre study of memantine in mild to moderate vascular dementia (MMM500). Int. Clin. Psychopharmacol. 17, 297-305. Wilcock, G. K. (2003). Memantine for the treatment of dementia. Lancet Neurol. 2, 503-505.
Willard, L. B., Hauss-Wegrzyniak, B., Danysz, W., and Wenk, G. L. (2000). The cytotoxicity of chronic neuroinflammation upon basal forebrain cholinergic neurons of rats can be attenuated by glutamatergic antagonism or cyclooxygenase-2 inhibition. Exp. Brain Res. 134, 58-65. Wimo, A., Witthaus, E., Rother, M., and Winblad, B. (1998). Economic impact of introducing propentofylline for the treatment of dementia in Sweden. Clin. Ther. 20, 552-566. Wimo, A., Winblad, B., St6ffier, A., Wirth, Y., and M6bius, H. J. (2003). Resource utilization and cost analysis of memantine in patients with moderate to severe Alzheimer's disease. Pharmacoeconomics 21, 327-340. Winblad, B., and Jelic, V. (2003). Treating the full spectrum of dementia with memantine. Int. J. Geriatr. Psychiatr. 18, $41-$46. Winblad, B., and Poritis, N. (1999). Memantine in severe dementia: Results of the M-Best Study (benefit and efficacy in severely demented patients during treatment with memantine). Int. J. Geriatr. Psychiatr. 14, 135-146. Winblad, B., M6bius, H. J., and St6ffier, A. (2002). Glutamate receptors as a target for Alzheimer's disease~Are clinical results supporting the hope? J. Neural. Transm. Suppl., 217-225. Zajaczkowski, W., Quack, G., and Danysz, W. (1996). Infusion of (+)-MK-801 and memantine~Contrasting effects on radial maze learning in rats with entorhinal cortex lesion. Eur. J. Pharmacol. 296, 239-246. Zajaczkowski, W., Frankiewicz, T., Parsons, C. G., and Danysz, W. (1997). Uncompetitive NMDA receptor antagonists attenuate NMDA-induced impairment of passive avoidance learning and LTP. Neuropharmacology 36, 961-971.
CHAPTER 5
Cholinesterase Inhibitors a s Chemical Warfare Agents: Community Preparedness Guidelines ANNE'I-FA WATSON, 1 KULBIR BAKSHI,2 DENNIS OPRESKO, 1 ROBERT YOUNG, 1 VERONIQUE HAUSCHILD, 3,* AND JOSEPH KING4 1Oak Ridge National Laboratory, Oak Ridge, Tennessee 2National Research Council, Washington, DC 3Aberdeen Proving Ground, Maryland 4U.S. Army Environmental Center, Aberdeen Proving Ground, Maryland
Destruction; Department of the Army (DA), 1988; Carnes, 1989; Carries and Watson, 1989; Munro et al., 1994]. Previous domestic (civilian) guidance regarding airborne agent vapor exposure was largely confined to "no adverse effect" limits established by the Centers for Disease Control and Prevention (CDC) for agent demilitarization workplaces and the general public [U.S. Department of Health and Human Services (DHHS) 1988, 2003]. Although the CDC guidelines provided necessary criteria for standing operating procedures at stockpile sites, the need to address potential civilian acute vapor exposure for a range of toxicological end points, as well as potential civilian incidental ingestion exposure, remained a concern. In furtherance of international treaty and congressional requirements to eliminate risks associated with continuous storage, the U.S. Federal Emergency Management Agency (FEMA) and the Chemical Materials Agency (CMA) of the U.S. Department of the Army have collaborated under the Chemical Stockpile Emergency Preparedness Program (CSEPP) to provide technical emergency planning and preparedness support as well as assistance to agencies and authorities representing host communities located near CW agent munition stockpile sites. Such support is being employed to develop and prioritize emergency response actions for use in the event of a release during CW agent destruction and munition disposal activities. Nerve agents comprise munition fill at six of the eight unitary stockpile sites in the domestic United States: Anniston Army Depot (Anniston, AL), Blue Grass Army Depot (Richmond, KY), Newport Chemical Depot (Newport, IN), Pine Bluff Arsenal (Pine Bluff, AR), Deseret Chemical Depot (Tooele, UT), and Umatilla Chemical Depot (Umatilla,
I. I N T R O D U C T I O N Recent global events have focused attention on the potential threat of international and domestic chemical terrorism as well as the possibility of chemical warfare (CW) proliferation. The need for preparedness is highlighted by the well-documented domestic terrorist use of an anticholinesterase compound during the Tokyo subway incident of March 1995, when commuters received toxic inhalation and dermal exposures to a nerve agent deliberately released within subway cars and stations (Lillibridge, 1995; Morita et al., 1995; Okumura et al., 1996; Sidell, 1996). Emergency preparedness plans and priorities for accidental or intentional release of CW agents were under development long before the Tokyo incident and are now in place for a number of U.S. and international communities as a consequence of (1) congressional mandate to destroy the existing U.S. unitary chemical munitions stockpile (PL 99-145, the Department of Defense Authorization Act of 1986 and subsequent acts); (2) congressional directives to examine and perform safe disposal of nonstockpile CW materiel (PL 102-484, the Defense Authorization Act of 1993), and (3) the April 1997 entry into force of the international chemical arms control treaty banning manufacture, use, stockpiling, and transfer of chemical weapons [Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on Their
*Present address: U.S. Environmental Protection Agency, Washington, DC.
Toxicology of Organophosphate and Carbamate Compounds
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Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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9Uses, Abuses,
& Epidemiolooay
OR) (Carnes 1989; Army Chemical Materials Agency at www.cma.army.mil). "Unitary" munitions are loaded with undilute, finished CW agent, as opposed to "binary" munitions, in which agent precursors mix and react to form finished agent after the munition is fired. Non-stockpile chemical material (e.g., wastes from past CW agent disposal efforts, unserviceable munitions, contaminated containers, and "found rounds" that have been recently located after having been historically buried) are also undergoing disposal. Furthermore, any soil or groundwater that may be contaminated by potentially toxic agent residues at the sites where non-stockpile material has been found are remediated and subjected to the protocols of a formal cleanup program (Opresko et al., 1998, 2001; Bakshi et al., 2000). Responsible and efficient response during the crisis phase of a CW agent release as well as during the decontamination and restoration of potentially contaminated sites requires knowledge of key, agent-specific toxicological parameters to determine the most appropriate and protective responses. Such responses include, but are not limited to, determination of zones requiring shelter-in-place or evacuation, identifying geographic areas most likely to require immediate medical support, accurate diagnosis and treatment of agent intoxication, selection of protective clothing, and performance of site health risk assessments in support of installation restoration and site recovery. During release of a hazardous material, emergency phase decision making tends to focus on acute exposures to vapor plumes, and assessments generally do not center on "no effects criteria"; minimizing severe toxicological effects in populations most likely to be exposed is priority during this phase, and acute exposure guidelines are needed. In the case of site recovery and decontamination, exposure to potential residual CW agents in soil or water or on surfaces is a more critical concern (Opresko et al., 1998, 2001; Bakshi et al., 2000). Determining responsible cleanup standards in these efforts (as for other toxic substances) hinges on the existence of chronic reference doses so that public health may be properly safeguarded without defaulting to overly conservative actions (e.g., clean to "nondetect") that would divert limited resources without significant benefit. The necessary toxicological parameters have been examined and employed to develop guidelines for addressing both acute nerve agent vapor exposures [acute exposure guideline levels (AEGLs)] as well as incidental ingestion of residual nerve agents, such as on surfaces or in soils or water [reference doses (RfDs)]. These guidelines are in the process of being implemented by local, state, and federal agencies in preparation for possible accidental or intentional agent release. Fixed facilities and transportation carriers, as well as active or formerly used defense sites are being evaluated (Bakshi et al., 2000; Opresko et al., 1998, 2001, 2003; Krewski et al., 2004; Watson et al., 2006).
Development of these acute vapor and chronic ingestion CW agent exposure guidelines, initially performed to facilitate disposal of the U.S. stockpile of CW agent munitions as well as installation restoration or closure at sites where CW agents have historically been processed, has received new interest for homeland defense application since the events of September 2001. Due to their relatively recent finalization, there has been limited awareness among the scientific and emergency preparedness communities of the availability of these protective and technically sound guidelines derived under civilian protocols. As such, these guidelines allow agent-related public health issues to be considered within frameworks essentially equivalent to those involving more commonly encountered toxic industrial substances. This article represents the first time that the pertinent background, supporting logic, and guidelines for both acute vapor and chronic lifetime ingestion exposure to nerve agents have been published together in the same document. It is hoped that this wider distribution will encourage their greater use as the objective decision criteria for which they were intended.
II. HISTORY AND BACKGROUND A. Anticholinesterase Compounds as Threat Agents Sidell (1997) noted that a naturally occurring (botanical) anticholinesterase had long been known to west African tribal authorities, who employed the special properties of the Calabar bean ( P h y s o s t i g m a v e n e n o s u m ; Leguminosae) during local trials by ordeal during the 19th century (Koelle, 1975; Davis, 1985). At that time, the Calabar bean and its extracts were considered powerful "ordeal poisons." The active principle (physostigmine; C15H21N302) was isolated by various investigators in 1864 and 1865 (Koelle, 1975). Developments in synthetic chemistry begun during the mid-19th century resulted in the discovery of organophosphorous (e.g., tetraethyl pyrophosphate in 1854; Holmstedt, 1963) and carbamate (e.g., neostigmine in the 1930s) anticholinesterases and medicinal use of carbamates (e.g., for glaucoma; Koelle, 1975). By the mid-1930s, the expanding commercial chemical industry in Germany supported a significant research and development effort, which synthesized several organophosphate compounds with potent insecticidal properties. The first was tabun (in 1936; agent GA), the second was sarin (in 1937-1938; agent GB), followed by soman (in 1944; agent GD). Their toxicity attracted attention from the German Ministry of Defense for potential military application, and commercial production of tabun and sarin for weaponry use by the ministry was in progress at multiple German facilities by 1942 (Sidell, 1997; Harris and Paxman, 1982; Robinson, 1967). Robinson reported
CHAPTER 5 9Chemical Warfare Nerve Agent Guidelines that at least 12,000 tons of tabun, and smaller quantities of sarin, were produced by the German Ministry of Defense during World War II. Much smaller amounts of agent GD had been produced in German manufacturing facilities by the end of the war. There is no record of nerve agent weapon deployment by German forces during WWII (Sidell, 1997; Robinson, 1967). As a Cold War deterrent, nerve agents began to be manufactured and weaponized by the United States in the 1950s. By the time the U.S. CW agent production program was terminated by the Nixon presidential "Statement on Chemical and Biological Defense Policies" of November 1969 (National Security Decision Memorandum 35, see Stockholm International Peace Research Institute 1973), the U.S. stockpile of unitary munitions included bulk ("ton") containers, underwing spray tanks, projectiles, rockets, bombs, land mines, and rockets (Carnes, 1989; Sidell, 1997). All of the unitary munitions were filled with either one of three nerve agents (GA, GB, and VX) or vesicant (blister) agents (sulfur mustard agent or the organic arsenical, Lewisite). The U.S. CW stockpile was never employed, is obsolete, and is currently undergoing destruction and disposal by the CMA in compliance with the international treaty and to eliminate the risk of continued storage for these aging CW munitions. Of all the munitions stored in the U.S. unitary stockpile, it is generally recognized that the M-55 rockets (fully functional and each loaded with lethal concentrations of weapons-grade nerve agent GB or VX) pose the greatest risk of unintended release due to their potential for leakage (Carnes, 1989). A similar variety of nerve agent-containing unitary munitions is present in the CW agent stockpile of the former Soviet Union; this stockpile was also manufactured as a Cold War deterrent and is also scheduled for destruction. A disposal facility at Shchuch'ye, Russia, is under construction with U.S. assistance under the Cooperative Threat Reduction Program of the Army Chemical Materials Agency. Nevertheless, continued storage and safeguarding of the former Soviet CW munitions stockpile will be required until the facility becomes operational. Although there have been numerous instances of suspected nerve agent deployment during periods of civil unrest in various nations since WWII, there is little definite documentation of nerve agent deployment until the 1994-1995 incidents of chemical terrorism involving the nerve agent sarin in Japan. In both incidents, civilian populations were deliberately exposed to lethal satin concentrations by followers of a cult (Aum Shinrykyo) originally local to Japan but with members in other nations (Lillibridge, 1995; Morita et al., 1995; Okumura et al., 1996; Sidell, 1996). The first incident occurred in June 1994 in the central highland city of Matsumoto, where seven people died soon after exposure to a then unknown vapor later determined to be agent GB released into a residential area during the night
49
(Morita et al., 1995). The Matsumoto incident also resulted in 56 hospital admissions, as well as 253 cases in which the affected individuals sought medical consultation, plus reports of "mild symptoms" presented by 8 of 53 rescue personnel and 1 attending physician (Morita et al., 1995). Prompt deaths (n = 3) and those that occurred before arrival at the hospital (n = 4) appear to have been the result of respiratory insufficiency. At the time of the Morita et al. (1995) report, 1 patient remained "in a vegetative state because of anoxic encephalopathy." The second occurrence, widely known as "the Tokyo subway incident," took place on March 20, 1995. The same terrorist group responsible for the Matsumoto incident deployed individual sources of passive, evaporative release of nerve agent GB in each of five individual subway cars serving three separate subway lines during the morning commuter rush hour (Okumura et al., 1996; Sidell, 1996; Lillibridge, 1995). Of the 5510 people known to have been given medical attention, 8 died promptly; 4 more died later (hours to days). This "later" group included individuals who had initially presented with "critical" respiratory effects requiring mechanical ventilation and intensive care (Lillibridge, 1995). The total of 12 fatalities included commuters and subway transport employees receiving ocular, inhalation, or dermal exposures. Death appeared to be a result of respiratory insufficiency. On hospital day 28, a death occurred as a consequence of "severe hypoxic brain damage" sustained during the release incident (Okumura et al., 1996). This delayed fatality was a previously healthy woman 21 years of age who had presented without heartbeat or spontaneous respiration at the hospital but had been revived with CPR and treated with agent antidotes. Plasma and red blood cell cholinesterase had returned to normal within a period of days, but the patient eventually succumbed to hypoxic brain damage (Okumura et al., 1996).
B. Chemical and Physical Properties of Nerve Agents The standard threat nerve agents include the G series agents [GA (tabun), GB (sarin), GD (soman), GF (cyclosarin)] and nerve agent VX. These compounds are all toxic ester derivatives of phosphonic acid containing a cyanide (GA), fluoride (GD or GF), or sulfur (VX) substituent and are commonly termed "nerve" agents as a consequence of their anticholinesterase properties as well as their effects on both peripheral and central nervous systems (Table 1). The "G" series military nomenclature used by NATO member nations has historically been considered to be an abbreviation for "German," with the second letter of the code ("A," "B," etc.) identifying the order in which these compounds were found and analytically identified by Allied forces investigating materials found in captured German military facilities at the end of WWII (Sidell, 1997). Agent VX
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CHAPTER 5 was industrially synthesized in the United Kingdom in the early 1950s; the code letter "V" is a reported reference to "venom" (Sidell, 1997; Robinson, 1967). The G agents are all viscous liquids of varying volatility (vapor density relative to air between 4.86 and 6.33) with faint odors ("faintly fruity," "spicy," or "odor of camphor"). Agent VX is an amber-colored liquid with a vapor density of 9.2 and is considered odorless. As a consequence, nerve agent vapors possess little or no olfactory warning properties. The vapor pressures and acute toxicity of these agents are sufficiently high for the vapors to be rapidly lethal. Within the G series, GB is considered to be a greater vapor hazard than agent GD. Agent GA represents a smaller vapor hazard and is expected to present a relevant contact hazard. The vapor density of agent GF is intermediate between those of agents GA and GD. Agent VX, with a vapor density greater than that of any G agent under consideration, was deliberately formulated to possess a low volatility; VX is approximately 2000 times less volatile than nerve agent GB (DA, 1990a,b). As a consequence, agent VX is considered a persistent, "terrain denial" military compound with the potential to off-gas toxic vapor concentrations over a period of days following surface application. Although not readily volatile, VX vapors (if allowed to accumulate) are nevertheless considered more acutely potent than those of agent GB or the other G series agents (Mioduszewski et al., 1998).
III. T O X I C I T Y
A. Mechanisms Nerve agents were specifically designed and formulated to produce death, major injuries, or incapacitation to enemy forces during wartime. From a military standpoint, these agents are particularly effective due to their potency. All of the nerve agents under consideration are anticholinesterase compounds. By phosphorylating acetylcholinesterase (AChE), the nerve agents prevent deactivation of the neurotransmitter acetylcholine (ACh). Reactivation of inhibited cholinesterase by dephosphorylation is not possible once the nerve agent-cholinesterase complex undergoes "aging," which is thought to be the consequence of the loss of an alkyl or alkoxy group. Agent GD ages very rapidly, with a tl/2 (time required for 50% of the enzyme to become resistant to reactivation) of 1.3 min (Harris et al., 1978). The aging half-time for agent GA is 46 hr, as calculated from a rate constant of 2.5 x 10-4/min (De Jong and Wolring, 1978), and the tl/2 for agent GB has been reported to be 5 hr (Sidell and Groff, 1974). The complex formed between ChE and agent VX does not age significantly (half-life of approximately 48 hr) (Sidell and Groff, 1974).
Chemical Warfare Nerve Agent Guidelines
51
Although nerve agents exert toxic effects on the central and peripheral nervous systems indirectly through AChE inhibition (Koelle, 1975, 1981), nerve agents may also affect nerve impulse transmission by additional mechanisms at neuromuscular junctions (Somani et al., 1992) and at neurotransmitter receptor sites in the central nervous system (CNS). Rao et al. (1987) reported that VX caused an increase in ACh release at neuromuscular junctions in the frog by an interaction with the nicotinic ACh receptor-ion channel complex. Aas et al. (1987) reported alterations in muscarinic receptors in rat bronchi and lung tissue after subacute inhalation exposures to agent GD. In the CNS, nerve agents may act directly on muscarinic, nicotinic, and glutamate receptors. Bakry et al. (1988) reported that nanomolar concentrations of agent GD affected muscarinic ACh receptors that have a high affinity for [3H]-cis-methyldioxalane binding. Rocha et al. (1998, 1999) reported that in cultured rat hippocampal neurons, 0.01 nM VX reduced the evoked release of the neurotransmitter gamma-aminobutyric acid (GABA) and reduced the amplitude of evoked GABAergic postsynaptic currents. VX concentrations > 1 nM decreased the amplitude of evoked glutamatergic currents. In the presence of a Na § channel blocker, VX increased the frequency of GABA- and glutamate-mediated miniature postsynaptic currents m a Ca2+-dependent effect reported to be unrelated to cholinesterase inhibition (Rocha et al., 1999). Chebabo et al. (1999) reported that 0.3-1 nM of agent GB reduced the amplitude of GABA-mediated postsynaptic currents but had no effect on the amplitude of glutamatergic-mediated postsynaptic currents. The observed effect was thought to be due to the direct interaction of GB with muscarinic ACh receptors present on presynaptic GABAergic neurons. Chebabo et al. suggested that the selective reduction in the action potential-dependent release of GABA in the hippocampus may account for GB-induced seizures. Lallement et al. (1991a,b) suggested that GD-induced overstimulation of glutamatergic receptors contributed to maintenance of seizures. Although these data indicate that nerve agents may have direct effects on the nervous system unrelated to AChE inhibition, the in vitro data do not provide a means of relating electrophysiological alterations in rat hippocampal neurons or determining a dose conversion to an integrative whole body end point such as lethality. Neither do they allow qualitative/quantitative comparisons directly relevant to adverse effects. The induction of amplitude changes in postsynaptic currents for in vitro rat hippocampal neurons by nanomolar concentrations of nerve agent cannot presently be correlated to dose levels inducing multisystem failure and death. It should be further noted that the effects of nerve agents on GABAergic transmission in the CNS may have profound implications for behavioral effects in laboratory animals and humans and may also contribute to the induction of
52
S ECTI O N I 9Uses, A b u s e s , & Epidemioloooy
convulsions at higher doses (Bakshi et al., 2000). Nevertheless, given the current undefined application of noncholinergic data to whole body estimations, reliance on the primary assumption of anticholinesterase action is consistent with recognized opinion (Bakshi et al., 2000). Studies with cholinesterase inhibitors such as galantamine, which affect neuronal nicotinic ACh receptors and inhibit AChE in a manner similar to that reported for nerve agents, have shown that such compounds have therapeutic benefits for patients with mild to moderately severe Alzheimer's disease (Maelicke et al., 2001; Albuquerque et al., 2001). As such, these compounds may be helpful in stabilizing behavior in Alzheimer's patients by improving memory and cognitive and daily function. For further details, see Chapters 3 and 4. It is also understood that organophosphorus (OP) compounds interact with detoxification enzymes such as carboxylesterases (CarbE) and A-esterases, and that the degree of such interaction may alter the magnitude and extent of the toxic cascade following AChE inhibition (Pope, 1999; Pope and Liu, 2002). Studies indicate that full characterization of the OP-protective capabilities of CarbE requires assessment not only of the amount but also of the affinity exhibited by CarbE for the inhibitor, as well as the total CarbE activity unlikely to be inhibited (inhibitorresistant esterase activity) (Chanda et al., 2002). The detoxification potential of CarbE is multifaceted and is an area requiting further experimental characterization. B. Effects Nerve agents are toxic by all routes of exposure, and they exhibit a steep dose-response. In laboratory animals, high-concentration exposures cause convulsions that, if maintained, can result in irreversible brain damage as a consequence of hypoxia (Somani et al., 1992; Sidell, 1992). Brain lesions as well as myocardial degeneration have been observed in laboratory animals administered single doses of agent GB or GD sufficient enough to induce convulsions (Singer et al., 1987). Lethality following acute and high-dose exposures to nerve agents is attributed to anoxia resulting from respiratory paralysis, bronchoconstriction, and paralysis or weakness of accessory respiratory muscles (Somani et al., 1992). Detailed descriptions of nerve agent toxicity may be found in reviews by Bakshi et al. (2000), National Research Council (NRC) (1999), Mioduszewski et al. (1998), Opresko et al. (1998, 2003), Sidell (1997), and Munro et al. (1994). 1. NERVOUS SYSTEM Anticholinesterase effects of nerve agent exposure can be characterized as muscarinic, nicotinic, or CNS. Muscarinic effects occur in the parasympathetic system and, depending on the amount absorbed, can be expressed as conjunctival congestion, miosis, ciliary spasm, nasal discharge, increased
bronchial secretion, bronchoconstriction, anorexia, emesis, abdominal cramps, sweating, diarrhea, salivation, bradycardia, and hypotension. Nicotinic effects are those that occur in somatic (skeletal/motor) and sympathetic systems, and they can be expressed as muscle fasciculations and paralysis. CNS effects may be manifested as confusion, reflex loss, anxiety, slurred speech, irritability, forgetfulness, depression, impaired judgment, fatigue, insomnia, depression of central respiratory control, and death (Somani et al., 1992; Sidell, 1992, 1997; Sidell and Groff, 1974; Opresko et al., 1998; Bakshi et al., 2000). Minimal effects observed at low concentrations include miosis, a feeling of "tightness" in the chest, rhinorrhea, and dyspnea (Dunn and Sidell, 1989) (Table 2). 2. CHOLINESTERASECHANGES The activity of red blood cell cholinesterase (RBC ChE), as well as that of plasma cholinesterase (plasma ChE or butyrylcholinesterase), has been used to monitor exposure to, and recovery from, anticholinesterase pesticides as well as nerve agents. There is some historical evidence that RBC ChE can be as sensitive as brain ChE to the anticholinesterase effects of nerve agents; Grob and Harvey (1958) reported that in vitro concentrations producing 50% activity depression of brain ChE and RBC ChE were equivalent in the case of GA (1.5 X 10 -8 tool/liter) and comparable in the case of GB (3.0 X 10 -9 vs 3.3 X 10 -9 mol/liter). The results of in vivo animal studies conducted by Jimmerson et al. (1989) disagree, which is further reflected by the fact that blood ChE activity may not always be correlated with exposure or with signs and symptoms of toxicity (Holmstedt, 1959; Sidell, 1992, 1997) (Table 2). It is generally considered that systemic effects in humans following acute nerve agent exposures are likely when RBC ChE is inhibited by 75-80% (e.g., to 20-25% of normal activity levels) (Sidell, 1992). Nevertheless, it is well known that local signs and symptoms of the eye and nose in humans and animals (e.g., miosis and rhinorrhea) can occur in the absence of any measurable change from baseline ChE or CarbE activity in the blood (Harvey, 1952; Craig and Woodson, 1959; Sidell, 1992; see also Tables 2 and 3). Science policy published by the U.S. Environmental Protection Agency (EPA) Office of Pesticides serves to guide the use and application of cholinesterase inhibition data (EPA, 2000) and recommends a weight-of-evidence approach for evaluating anticholinesterase toxicity end points. Such an approach is consistent with that of Storm et al. (2000), who considered that the most defensible means of deriving inhalation exposure limits for anticholinesterase OPs should be based on weight of evidence, where first priority is given to clinical signs and physiological or behavioral effects in humans and animals followed by 9 Symptoms in humans 9 Central nervous system AChE inhibition
CHAPTER 5
9Chemical Warfare Nerve Agent Guidelines
53
TABLE 2. Human Experimental Data for GB Vapor (Single Exposures) a Concentration (mg/m 3)
Exposure duration
Ct (mg-min/m3)
0.05
20 min
1
0.05
20 min
1
0.06 0.06 0.3 0.5
20 min 40 min 0.5 min 30 min
1.2 2.4 0.15 15.0
0.6 2
1 min 2 min 10 min to 5 hr 10 min to 5 hr 2 min
0.6 4 3.13 13.85 8.38
2 min
41.4
4.19 (average)
20.7 (average)
Signs and symptoms Headache, eye pain, rhinorrhea, tightness in chest, cramps, nausea, malaise Threshold (< 1 mm pupil diameter decrease) to mild (1-2 mm pupil diameter decrease) miosis b in test subjects No reported effects Miosis; slight tightness in chest (n = 4) Rhinorrhea in 16/16; chest tightness in 7/16 Miosis, dyspnea, photophobia, 40% inhibition of RBC ChE, subclinical single fiber electromyography changes Miosis and slight tightness in chest Miosis; no other signs of ChE inhibition 50% pupil area decrement 90% pupil area decrement Average 47% inhibition of RBC ChE; no other effects (breathing rate, 5.6-8.4 liters/min through nose or mouthpiece) Average 49% inhibition of RBC ChE; no other effects (breathing rate, 47-65 liters/min through nose or mouthpiece)
Reference Harvey (1952) Johns (1952)
McKee and Woolcott (1949) McKee and Woolcott (1949) Fairley and Mumford (1948) Baker and Sedgwick (1996)
McKee and Woolcott (1949) Rubin et al. (1957) Callaway and Dirnhuber (1971) Callaway and Dimhuber ( 1971) Oberst et al. (1968)
Oberst et al. (1968)
aAdapted from Opresko et al. (2003) with permission by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC. bMild miosis defined by Johns (1952) as "decrease of 1 to 2 mm" in pupil diameter; reversible within 24 hr.
9 Peripheral nervous system AChE inhibition 9 Red blood cell AChE inhibition 9 Plasma ChE inhibition in humans and animals The EPA science policy guidelines generally consider blood ChE inhibition to be an imperfect measure due to the need for comparing individual baseline measurements and the fact that there is no fixed percentage of blood ChE activity change that can distinguish adverse from nonadverse effects (EPA, 2000; Storm et al., 2000). A number of investigations have noted the poor association between blood (RBC and plasma) ChE activity and nerve agent intoxication (Koelle, 1994; Sidell, 1992; Rubin and Goldberg, 1957; Mioduszewski et al., 2002a). Circulating ChE activity does not parallel tissue ChE activity, and minimal blood ChE activity has been observed in association with normal tissue function (Sidell, 1992). In the GB vapor exposure study of Mioduszewski et al. (2002a), "miosis was not correlated with, or even accompanied by, significant reduction of circulating ACHE, BuChE, or CarbE" as a consequence of nerve agent vapor whole body exposure to Sprague-Dawley (SD) rats. The findings of Mioduszewski et al. (2002a) for SD rats are consistent with those for human volunteers exposed to GB vapor in the study of
Rubin and Goldberg (1957). These results further document the fact that miosis alone, and in the absence of signs such as ChE or CarbE activity inhibition, is a local effect and reflects an exposure much less than that required to produce a systemic clinical effect. Thus, selection of a local effect such as miosis as a critical end point for guideline determination allows a greater margin of protection against the potential for agent exposures great enough to generate systemic effects. Although RBC ChE inhibition in the blood is considered an operationally acceptable surrogate for CNS inhibition, plasma ChE is more labile and is a less reliable reflection of enzyme activity change at neuroeffector sites (EPA, 2000; Young et al., 1999; California Environmental Protection Agency, 1998). As a consequence, plasma ChE activity inhibition is considered a biomarker of exposure and is rejected as a critical end point.
C. Minimal Potential for Delayed Neuropathy A continuing area of public concern regarding nerve agent exposure is the possibility of chronic neurological effects, particularly delayed neuropathy, given that neuropathic
54
SECTION I 9 Uses, A b u s e s , & E p i d e m i o l o g y TABLE 3.
Relative Potency Estimates for Agents GB and VX (Primary Sources Only) a
Species
End point
Units
Human
Inhalation Ct ChEs0
mg.min/m3
Human
Oral RBC ChEs0
Human
GB
VX
GB/VX ratio
References
42
--6.5
--6.5
txg/kg
10
2.4
4.3
ia/iv RBC ChEs0
p~g/kg
3
1.1
2.7
Mouse Guinea pig
im LDs0 iv LDs0
txg/kg txg/kg
204.81 24
13.07 28
15.7 0.9
Guinea pig Rat (female)
Subcutaneous L D s 0 Vapor ECs0 (miosis) b 10 min
txg/kg
41.26
6.89
5.99
GB: Oberst et al. (1968); VX: Bramwell et al. (1963) (estimated from tabulated data; not verifiable) GB: Grob and Harvey (1958); VX: Sidell and Groff (1974) GB: Grob and Harvey (1958); VX: Sidell and Groff (1974) Koplovitz et al. (1992) GB: Spruit et al. (2000); VX: Van der Schans et al. (2003) Koplovitz et al. (1992)
mg/m3
0.068
0.007
9.7
GB: Mioduszewski et al. (2002a); VX: Benton
60 min
mg/m3
0.020
0.002
10.0
240 min
mg/m3
0.012
0.001
12.0
GB: Mioduszewski et al. (2002a); VX: Benton et al. (2004) GB: Mioduszewski et al. (2002a); VX: Benton
ixmol/kg mg.min/m3
0.57 1.32
0.03 0.04
19 (9.9) c 33
Maxwell (1992) Callaway and Dirnhuber (1971)
mg.min/m3
2.71
0.23
11.8
Callaway and Dirnhuber (1971)
et al. (2004)
et al. (2004)
Rat Rabbit
Rabbit
Subcutaneous L D s 0 Vapor ECts0 (50% pupil area decrement) Vapor ECt90 (90% pupil area decrement)
aAdapted from Opresko et al. (2003) with permission by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC. bNo significant change in RBC ChE, BuChE, or CarbE activity from baseline. CRatio shown in parentheses based on grams per kilogram.
effects have been observed following high levels of occupational exposure to agricultural pesticides. Exposure to some OP anticholinesterase compounds results in delayed neurotoxic effects (ataxia, distal neuropathy, and paralysis), which have collectively been described as OP-induced delayed neuropathy (OPIDN). OPIDN is characterized by myelin sheath and axon degeneration and was once thought to be the consequence of inhibition and aging of neuropathy target esterase (NTE) (Abou-Donia, 1993; Ehrich and Jortner, 2002). With greater knowledge, the NTE theory has been replaced with one involving a noncholinergic, proteolytic mechanism in which the kinase-mediated protein Ca2+/calmodulin kinase II (activated by OP-induced phosphorylation) reacts with cytoskeletal proteins found in neurofilaments (De Wolff et al., 2002). The resulting proteolysis, accompanied by perturbed ionic gradients, cellular edema, and myelin debris, can generate neuropathy. A number of well-conducted studies employing EPA (1998) guidelines for experimental determination of delayed
neurotoxicity have been performed for the G agents and agent VX. The EPA protocol requires toxicological testing with the domestic hen, an OPIDN-sensitive laboratory animal.
1. G AGENTS Supralethal doses of G agents have produced delayed neuropathy in antidote-protected hens in v i v o (Gordon et al., 1983; Willems et al., 1984). Doses of 120 >< LDs0 for agent GA resulted in mild neuropathic signs, and delayed neuropathy was observed at 120-150 X LDs0 for GD in a single surviving hen but not for GD doses of 38 x LDs0; delayed neuropathy has also been observed in chickens administered agent GB at 30-60 X LDs0. In all these challenge tests, nerve agents were administered to adult hens previously protected from lethality by large antidote doses to allow survival long enough for neuropathic signs to develop (Gordon et al., 1983; Willems et al., 1984).
CHAPTER 5 9Chemical Warfare Nerve Agent Guidelines Serial exposure investigations for induction of delayed neuropathy have also been conducted. Signs indicative of delayed neuropathy have been observed in hens receiving serial subcutaneous injections of 1/10 LD50 of agent GB on each of 10 successive days (a total of 1 x LD50; Husain et al., 1995) and in mice exposed to GB vapors of 5 mg/m 3 (1/6 LDs0) for 20 min/day on each of 10 successive days (a total of 1.66 • LD50; Husain et al., 1993). Rats receiving daily gavage doses of GB for 90 days at the maximum tolerated (nonlethal) dose did not exhibit neuropathy (Bucci et al., 1991; Bucci and Parker, 1992). The fact that highly elevated (e.g., supralethal) doses are required before OPIDN manifests in a sensitive laboratory test animal indicates that delayed neuropathy in humans exposed to G agents would be a concern only for those individuals surviving single exposures ->30 • LDs0 or daily serial exposures that are cumulatively equivalent to >1 LD50. Survival following a single exposure to 30 • LD50 is unlikely. 2. AGENT VX Delayed neuropathy was not observed in three strains of antidote-protected chickens given a single sc dose of VX equivalent to 5-10 times the lethal dose. Furthermore, repeated supralethal im injections of VX (each injection being equivalent to 1.3 times the LDs0) for either 3 days per week over 30 days or 5 days per week over 90 days produced no signs of OPIDN (Goldman et al., 1988; Wilson et al., 1988). No evidence indicates that VX causes OPIDN in test animals or humans. In SD rats, continuous sc exposure via osmotic pump to a daily supralethal dose of VX equivalent to 1.3 times the sc LDs0 for 14 days is reported to generate myopathy in the soleus muscle (Lenz et al., 1996). Nevertheless, application of the Lenz et al. results for soleus muscle myopathy seems appropriate only for individuals who survive daily systemic exposures to multiple lethal or supralethal concentrations of agent VX, which is an extremely unlikely event.
D. Evaluation of Other Potential Effects Animal data from vapor, oral, and injection exposure studies for the G series nerve agents and agent VX indicate that these agents do not induce reproductive or developmental effects in mammals (Denk, 1975; LaBorde and Bates, 1986; LaBorde et al., 1996; Bucci et al., 1993; Bates et al., 1990; Schreider et al., 1984, 1988; Goldman et al., 1988; Mehl et al., 1994; Van Kampen et al., 1970). Neither agent GB nor agent VX was genotoxic in a series of microbial and mammalian assays (Goldman et al., 1987, 1988; Crook et al., 1983), whereas agent GA has been reported to be weakly mutagenic in similar cellular assays (Wilson et al., 1994). Experimental results indicate that agents GB, GA, and VX have no carcinogenic potential (Weimer et al., 1979; Bucci et al., 1992a,b; Goldman et al., 1988).
55
IV. REFERENCE DOSES AND ACUTE EXPOSURE GUIDELINE LEVELS The following sections summarize toxicological support and developmental rationale for the two primary criteria of interest to community decisionmakers managing a response to an intentional or accidental release of nerve agent(s) to the environment. Generally, the crisis phase of a release response focuses first on acute hazard management; assessment of chronic hazards (especially if liquid deposition has occurred during the chemical emergency) would be a subsequent priority. The first section outlines the development of nerve agent (chronic) reference doses due to the fact that remediation and restoration at existing and closing military sites was, and remains, a priority activity and because much of the underlying data and logic used to develop agent RfD estimates in 1998 (Opresko et al., 1998) formed the toxicological basis for development of nerve agent AEGLs in 2003 (Opresko et al., 2003).
A. Estimated Reference Doses A reference dose is "an estimate (with an uncertainty spanning perhaps an order of magnitude or greater) of a daily exposure level for the human population, including sensitive subpopulations, that is likely to be without an appreciable risk of deleterious effects" for chronic exposures (EPA, 1989). This parameter is to be independently derived from compound-specific laboratory or epidemiological data, and it is employed in estimating noncancer health risks at Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA) Superfund sites. As such, it is an essential component of the site risk assessment comparing potential levels of exposure for specific environmental pathways (for reviews of noncancer risk assessment guidelines and rationale, see Dourson, 1994; Cicmanec et al., 1996; Abernathy et al., 2004). Methods used to derive oral reference doses for chemical warfare agents follow standard EPA protocols (EPA, 1989; Dourson, 1994), employ appropriate toxicological data, and have undergone review for consistency by the NRC (Bakshi et al., 2000; Opresko et al., 1998, 2001). Because the EPA has not officially verified the derived values for nerve agents, they are identified and referenced as estimated RfDs (RfDe). For humans, the chronic exposure duration assumed for reference dose development is defined by EPA (1989) as lasting between 7 years (approximately 10% of a human lifetime) and a full lifetime. Calculation of an RfD requires use of animal or human toxicity data for a dose or exposure corresponding to a no-observed-adverseeffect level (NOAEL) or a lowest-observed-adverse-effect level (LOAEL) (EPA, 1989). The NOAEL is the exposure level at which there are no statistically or biologically significant increases in frequency or severity of adverse
56
SECTION I 9 Uses, A b u s e s , & Epidemioloooy
effects between the exposed population and its appropriate control. Effects may be produced at this level, but they are not considered to be adverse if they do not result in functional impairment and/or pathological lesions that affect the performance of the whole organism or that reduce an organism's ability to cope with additional challenge (EPA, 1994). The LOAEL is the lowest exposure level at which there are biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control (EPA, 1994). The NOAEL or LOAEL can then be adjusted by the application of a set of uncertainty factors (UFs) and a modifying factor (MF) as necessary (Dourson, 1994; Cicmanec et al., 1996; Dourson et al., 2004; Abernathy and Roberts, 1994; Abernathy et al., 2004), as illustrated in the following formula: RfO e =
NOAEL (or LOAEL) UFH • UFA • UFs • UFL • UFD • MF
where NOAEL = No-observed-adverse-effect level, expressed as milligrams chemical per kilogram body weight per day. LOAEL = Lowest-observed-adverse-effect level, expressed as milligrams chemical per kilogram body weight per day. UFH = Human to sensitive human. Intraspecies UF up to maximum of 10 to account for variation in sensitivity among humans. UFA -- Animal to human. Interspecies UF up to a maximum of 10 to be used when extrapolating from animal data to humans when human exposure data are inadequate or unavailable. UFs = Subchronic to chronic. A UF up to a maximum of 10 to be used when extrapolating from a less-thanchronic study to derive a chronic RfD e. UFL = LOAEL to NOAEL. A UF up to a maximum of 10 to be used when a suitable NOAEL is not available and when a LOAEL is substituted. U F D - - Incomplete to complete database. A "data gap" UF up to a maximum of 10 to be used when the available database does not include a specific study type (e.g., reproductive). MF = Modifying factor of >0 to 10 to be used when necessary to reflect a qualitative professional assessment of the critical study and the entire database. Default value for MF is 1.0. High confidence in an oral RfD can be achieved if the database is made up of at least one two-generation reproductive toxicity study, two chronic oral toxicity studies in two different species, and two developmental toxicity studies in different species. For compounds that pose only acute health hazards because low doses are degraded and/or excreted, chronic studies may not be as critical in deriving an RfD as special studies assessing specific end points, such as neurotoxicity (Cicmanec et al., 1996).
1. RfI) e DERIVATION FOR AGENT GB An example RfD e derivation for data-rich agent GB is provided here to illustrate the logic and protocol for deriving reference doses for nerve agents. Complete details of RfD e derivation for the other G agents and agent VX are documented in Bakshi et al. (2000) and Opresko et al. (1998,
2ool). a. Selection of Key Study The subchronic/chronic exposure evaluations conducted with GB consist of two reports: a 90-day study in which rats were given GB type I (Bucci et al., 1991) or GB type II (Bucci and Parker, 1992) by gavage and a 1-year study in which rats, mice, and dogs were exposed to GB by inhalation (Weimer et al., 1979). For the development of an oral RfD e, a study involving the same exposure pathway is preferred even though the exposure period may be less than chronic. Therefore, the subchronic rat gavage studies are considered to be more relevant than the inhalation studies for deriving this RfD e. Decreases in plasma and RBC AChE activity levels occurred in male and female CD rats dosed once per day, 5 days/week for 13 weeks with GB type II at 75, 150, or 300 Ixg GB/kg/day (Bucci and Parker, 1992). Inhibition of RBC ChE by GB type II was dose related for females in the two highest dose groups and for males in all dose groups. Maximum (48%) and significant (p <-0.05) RBC ChE activity inhibition from baseline occurred in week 7 for both high-dose males and females. At the low dose, female rat RBC ChE activity levels were never significantly reduced from baseline or control values. In contrast, male rat RBC ChE activity exhibited significant reductions from the control (p < 0.05) at week 1 and significant reductions from control and baseline (p-< 0.05) at weeks 3 and 7. By week 13, RBC ChE activity in the low-dose males was still depressed (by 16%) but had increased to the point that there was no longer any significant treatment difference when compared to the baseline or control (Bucci and Parker, 1992; Opresko et al., 1998). Effects on plasma ChE were not as great and were observed to be significant (p -< 0.05) only at the two highest dose groups, most consistently in the males (Bucci and Parker, 1992; Opresko et al., 1998). Because inhibition of blood ChE is an acceptable end point to use in identifying a NOAEL or LOAEL, the Bucci and Parker (1992) study on GB type II was employed for the derivation of an RfD e for agent GB (Bakshi et al., 2000; Opresko et al., 1998, 2001). b. RIDe Derivation The lowest test dose of 0.075 mg GB type II/kg/day, 5 days/week, resulted in a statistically significant (p -< 0.05) RBC AChE activity reduction in male rats at weeks 1, 3, and 7 but not at week 13 (Bucci and Parker, 1992); thus, this dose is considered an LOAEL. The LOAEL is adjusted to a 7 day/week exposure period by using a factor of 5/7 (i.e., 5/7 X 0.075 mg/kg/day = 0.054 mg/kg/day).
CHAPTER 5 9Chemical Warfare Nerve Agent Guidelines The RfD e is then calculated according to the following formula: RfO e =
57
reduced reproduction in the controls as well as in the dosed animals, a UFD of 3 was applied. No additional adjustment was considered necessary (e.g., MF = 1). Therefore,
0.054 mg/kg/day UFH X UF A X UFs X UF L X UF D X MF
where UFH = 10 (sensitive subpopulations), UF A = 10 (animal-to-human extrapolation), UFs = 3 (subchronic-tochronic extrapolation), UFL = 3 (LOAEL-to-NOAEL extrapolation), UFD = 3 (database), and MF = 1 (modifying factor, not needed). A UFH of 10 for variation in human sensitivity is needed because some individuals possess genetically or physiologically depressed levels of blood ChE or CarbE activity; such activity depression may make them especially susceptible to the effects of ChE inhibitors such as nerve agents (Chanda et al., 2002; Lehmann and Liddell, 1969; Harris and Whittaker, 1962; Sidell and Kaminskis, 1975; Davies et al., 1996; Morgan, 1989; Wills, 1972). There is ample evidence that humans are more sensitive to GB than laboratory rodents for this end point (Grob and Harvey, 1958; Bucci and Parker, 1992); thus, an uncertainty factor of 10 is used for animal-to-human extrapolation. An uncertainty factor of 3 has been used to extrapolate from a subchronic to chronic exposure. At the time when agent RfD estimates were developed, experimental studies were not available to ascertain whether or not adverse effects would occur following chronic exposures (Opresko et al., 1998; Bakshi et al., 2000). Such studies are planned and/or under way, and it is hoped that the value of this uncertainty factor can soon be validated. A LOAEL-to-NOAEL uncertainty factor of 3 has been used instead of 10 because the end point, ChE inhibition, was not associated with signs of toxicity in the critical study, and the LOAEL for this end point is considered a minimal LOAEL. The database for GB consists of two well-designed and well-conducted subchronic toxicity studies in rats (Bucci et al., 1991; Bucci and Parker, 1992); developmental studies in rats and rabbits (LaBorde and Bates, 1986; LaBorde et al., 1996); a multigenerational inhalation study in rats (Denk, 1975); delayed neuropathy studies in hens, rats, cats, and mice (Davies et al., 1960; Gordon et al., 1983; Husain et al., 1993, 1995; Bucci et al., 1991; Goldstein, 1989; Goldstein et al., 1987); and chronic inhalation studies in mice, rats, and dogs (Weimer et al., 1979). The principal study was well designed and well conducted, used a relevant exposure pathway, and examined the appropriate toxicological end points. In addition, there are substantial human data on acute and short-term exposures (Grob and Harvey, 1958; Sidell, 1992; Thienes and Haley, 1972). These studies support the use of ChE inhibition as the critical end point for deriving an oral RfD e and add confidence to the RfD e determination for agent GB. Because the multigenerational study was a pilot study and gave inconclusive results due to
RfDe --
0.054 mg/kg/day 10x 10x3x3x3x
1
RfD e = 0.00002 mg GB/kg/day 2. SUMMARY OF AGENT RfO e VALUES Application of the same protocol and logic for data characterizing chronic and subchronic toxicity of the other G agents and agent VX resulted in the reference dose estimates (and composite uncertainty factors) summarized in Table 4. Details of derivation can be found in Bakshi et al. (2000) and Opresko et al. (1998, 2001). These criteria are those selected by the Office of the Army Surgeon General (OTSG) as the most appropriate oral toxicity reference values for use in environmental risk assessments, and they represent the Army's current position (OTSG, 2000; Opresko et al., 2001). They are currently being applied on an Army-wide basis in calculating and implementing health-based environmental screening levels (U.S. Army Center for Health Promotion and Preventive Medicine, 1999; Office of the Assistant Secretary of the Army, 1999).
B. A c u t e E x p o s u r e G u i d e l i n e Levels AEGLs (expressed in units of mg/m 3 or ppm) are exposure limits for the general public that are designed to aid state and local government agencies in developing emergency response plans in the event of accidental or deliberate atmospheric release of extremely hazardous chemical substances. AEGL values for vapors of 22 hazardous compounds (including hydrogen cyanide, phosgene, and methyl isocyanate) have been published by the National Academy Press (NRC, 2000, 2002, 2003, 2004); others are in various stages of review. For each hazardous compound, guideline levels are developed for vapor exposure durations of 10 and 30 min, 1 hr, 4 hr, and 8 hr, as well as for three gradations of toxic effect severity. It is important to keep in mind that the A E G L concentrations represent effect levels. TABLE 4. Nerve agent VX GA GB GD
Summary of RfDe and Uncertainty Factor Values for Nerve Agentsa Composite uncertainty
Critical study
(mg/kg/day) 6E-7 4E-5 2E-5 4E-6
100 3000 3000 3000
Rice et al. (1971) Bucci et al. (1992a) Bucci and Parker (1992) Bucci et al. (1992b)
RfO e
aAdapted from Opresko et al. (1998, 2001).
58
SECTION I 9Uses, A b u s e s , & Epidemioloooy
AEGL-1 concentrations are the mildest effect category ("above w h i c h . . , the general population, including susceptible individuals, could experience notable discomfort, irritation, o r . . . nonsensory effects. However, the effects are not disabling and are transient and reversible upon cessation of exposure") (NRC, 2001, p. 35). Exposures below the A E G L - 1 concentration represent concentrations and exposure durations that could produce mild, transient, and nondisabling odor, taste, and sensory irritation (NRC, 2003).
AEGL-3 concentrations represent the most severe effect category ("above which .... the general population, including susceptible individuals, could experience life-threatening health effects or death") (NRC, 2001, p. 35). The point above the AEGL-3 concentration at which "level 3" effects would initiate for any given human exposure duration is not identified in the AEGL assessment protocol. In practice, and depending on the degree of conservatism incorporated during review of individual hazardous compounds subjected to the AEGL process, the AEGL concentration established for any given effect level can be less than the known experimental concentrations at which such toxicological effects have been observed to occur. Selection protocols for critical effects and studies, AEGL derivation, time scaling, use and selection of uncertainty and modifying factors, and a description of the lengthy and deliberative review process employed are all described in NRC (2001) as well as in papers by Krewski et al. (2004) and Bruckner et al. (2004). Development of AEGL values includes consideration of uncertainty factors (intraspecies and interspecies) as well as the need for any modifying factors. Because exposure-response data are usually not available for each AEGL-specific exposure duration (for any hazardous chemical and not just nerve agents; NRC, 2001), temporal extrapolation is employed in the development of AEGL values for some AEGL-specific time periods. The concentration-exposure time relationship for many systemically acting vapors and gases may be described by C n • t = k, where the exponent n ranges from 0.8 to 3.5 (Ten Berge et al., 1986). Haber's rule (C • t = k) is a special case of this principle, and it occurs when n = 1; Haber's rule has often been used for short-term exposure predictions involving a small set of highly toxic gases (NRC, 1993, 2001). For larger groups of structurally heterogeneous compounds, the more general relationship of C n • t = k applies (Ten Berge et al., 1986; NRC, 2001). As noted in the AEGL Standing Operating Procedures (NRC, 2001), toxicity data from human studies are preferred over those obtained from animal studies. Furthermore, and because AEGLs are intended as airborne exposure limits, inhalation toxicity data are preferred rather than ingestion, dermal or other exposure pathways (NRC, 2001). Human studies evaluated in the AEGL process must meet rigorous criteria for acceptance of human subject data in that subjects must provide informed consent and there must be evidence
that human studies were performed under appropriate clinical supervision (NRC, 2001). Once all review is completed and interim AEGL values for a specific compound are judged scientifically valid by the NRC Committee on Toxicology (Subcommittee on Acute Exposure Guideline Levels), AEGL values and logic are considered "final" and published by the NRC. Final AEGL values for nerve agents are published in NRC (2003; see Opresko et al., 2003). 1. A E G L DERIVATION FOR AGENT GB
The toxicological database for agent GB is robust and includes sufficient human data for direct derivation of AEGL-1 and AEGL-2 estimates as well as ample laboratory animal lethality data for directly deriving AEGL-3 values. a. AEGL-1 a n d A E G L - 2 Values
AEGL-1 values for agent GB were derived from a wellconducted study on adult female SD rats exposed whole body in a dynamic airflow chamber to a range of GB vapor concentrations (0.01-0.48 mg/m 3) for 10, 60, or 240 min (a total of 283 agent-exposed rats, of which 142 were female and 141 were male; Mioduszewski et al., 2002a). With the inclusion of range-finding experiments and controls, a total of 423 rats were tested in this study, which employed highly credible protocols for GB vapor generation and measurement. Analysis of pre- and postexposure rat pupil diameters allowed determination of ECs0 values for miosis (defined as a postexposure pupil diameter of 50% or less of the preexposure diameter in 50% of the exposed population). Blood samples collected from tail vein and heart at 60 min and 7 days postexposure indicated no significant change from preexposure baseline in monitored blood RBC ChE, BuChE, or CarbE activity. No other clinical signs were evident during the study. Gender differences (females more susceptible) were statistically significant at two of the three exposure durations. The ECs0 for miosis in female SD rats is thus a well-defined transient, reversible, and nondisabling animal end point in a susceptible gender. In terms of potential human effects, an ECs0 for miosis is not considered an adverse effect. This degree of miosis is the first measurable change, by modem and reproducible techniques, in the continuum of response to anticholinesterase compounds. In bright daylight or under bright lighting, a 50% reduction in pupil diameter would result in greater visual acuity among some members of the affected exposed population and no marked reduction in visual acuity for the majority of the affected population. During the Tokyo subway incident of GB release by domestic terrorists, people who experienced -->50% reduction in pupil diameter were able to self-rescue and to render aid to others. Data from GB vapor studies of nonhuman primates (marmosets, 5-hr exposures; Van Helden et al., 2001, 2002, 2003, 2004) and human volunteers (minimal and reversible effects of miosis, rhinorrhea, headache, etc. after a 20-rain
CHAPTER 5 9Chemical Warfare Nerve Agent Guidelines GB vapor exposure; Harvey, 1952; Johns, 1952) are considered secondary and supportive. Compared to the available human data, the miosis data derived from the Mioduszewski et al. (2002a) study on rats are a more reliable data set due to the contemporary and multiple analytical techniques employed for quantifying exposures and measuring miosis and to the experimental protocol incorporating sufficiently large test and control populations. With the additional knowledge that the ECs0 for miosis exhibited by rats in the study of Mioduszewski et al. is transient and reversible, the EC50 for miosis in female (susceptible gender) SD rats is well supported as an appropriate point of departure for estimating AEGL-1 values. The study by Mioduszewski et al. is thus the critical study for agent GB AEGL-1 estimates, with miosis data for marmosets (Van Helden et al., 2001, 2002, 2003, 2004) and human volunteers (Harvey, 1952; Johns, 1952) considered secondary and supportive. Opresko et al. (2003) ascertained that the miotogenic response of mammalian eyes to agent GB vapor exposure is similar across multiple species, including standard laboratory species (rabbits, rats, and guinea pigs), nonhuman primates (marmosets), and humans. As a consequence, the interspecies uncertainty factor for the critical AEGL-1 end point of ECs0 miosis is equal to 1. To accommodate known variation in human ChE and CarbE activity that may make some individuals susceptible to the effects of ChE inhibitors such as nerve agents, the intraspecies uncertainty factor was set equal to 10. A modifying factor is not applicable. ' Thus, the total uncertainty factor for estimating AEGL-1 values for agent GB is 10 (Table 5). AEGL-2 values for agent GB were derived from a human exposure study in which miosis, dyspnea, photophobia, inhibition of RBC ChE, and changes in single fiber electromyography (SFEMG) were observed in volunteers following a 30-min exposure to 0.5 mg/m 3 (Baker and Sedgwick, 1996). The SFEMG changes noted in the study were not clinically significant and were not detectable after 15-30 months. Baker and Sedgwick concluded that these electromyographic changes were persistent (>15 months), but that they were reversible and subclinical. Although not considered debilitating or permanent effects in themselves, SFEMG changes have been considered during the AEGL review as an early indicator of exposures that could potentially result in more significant effects. Selection of the SFEMG effect as a protective point of departure for determination of an AEGL-2 level has been considered appropriate given the steep dose-response toxicity curve of nerve agents (Aas et al., 1985, 1987; Mioduszewski et al., 2000, 2001, 2002b). The fact that AEGL-2 analyses for agent GB are based on the Baker and Sedgwick data from human volunteers precludes the use of an interspecies uncertainty factor. As in the case of the AEGL-1 estimations, a factor of 10 was applied for intraspecies variability (protection of susceptible populations exhibiting variable ChE and CarbE activity). A modifying factor is not
59
applicable. Thus, 10 is the total uncertainty factor for estimating AEGL-2 values for agent GB (Table 5). The temporal extrapolation used in the estimation of AEGL values for agent GB is based on a log-log linear regression of female SD rat miosis data following GB vapor exposures (Mioduszewski et al., 2002a) and a log-log linear regression of the LC01 lethality of GB to female SD rats (Mioduszewski et al., 2000, 2001, 2002b). Regression analysis of the LC01 values yields an n value of 1.93 with an r 2 of 0.9948, whereas regression analysis of the miosis data yields an n of 2.00 with an r 2 of 0.4335 (Opresko et al., 2003). Given that all mammalian toxicity end points observed in the data set for all nerve agents represent different points on the response continuum for anticholinesterase exposure, and that the mechanism of acute mammalian toxicity (ChE inhibition) is the same for all nerve agents, the experimentally derived n = 2 from the Mioduszewski et al. (2000, 2001, 2002a,b) rat lethality and miosis data sets has been used as the scaling function for all the nerve agent AEGL derivations. AEGL-1 and -2 values for other G agents and agent VX were derived from those of agent GB by a relative potency protocol (Opresko et al., 2003; see Table 3 for examples of end point-specific relative potency estimates for agent VX vs agent GB). This is considered a toxicologically acceptable approach given that all mammalian toxicity end points observed in the nerve agent data set represent different points on the response continuum for anticholinesterase effects, and that the principal mechanism of mammalian toxicity for the G agents and agent VX is ChE activity inhibition. As a consequence, target organ effects are expected to be identical but different in magnitude. Furthermore, there are no uncertainties regarding other toxic end points, such as reproductive or developmental effects or carcinogenicity. This concept has been previously applied in the estimation of G series nerve agent exposure limits by Mioduszewski et al. (1998). b. AEGL-3 Values AEGL-3 values for agent GB were derived from inhalation studies in which the lethality of GB vapor to female SD rats was evaluated for the time periods of 10, 30, 60, 90, 240, and 360 min (Mioduszewski et al., 2000, 2001, 2002b). Both experimental LC0m and LCs0 values were evaluated. The use of a rat data set resulted in selection of an inter' species uncertainty factor of 3; the full default value of 10 was not considered appropriate since the mechanism of toxicity in rats and humans is the same, and lethality is only one point on the response continuum for these anticholinesterase compounds. The full default value of 10 for intraspecies uncertainty was considered necessary to protect susceptible populations. Because a modifying factor is not applicable, the composite uncertainty factor for AEGL-3 determination for agent GB is equal to 30. For consistency with the AEGL-1 and -2 estimates described previously, AEGL-3 values for other G agents
60
SECTION
I 9 Uses, Abuses,
TABLE 5. Agent GA
GB
GD
GF
VXY
& Epidemiology
S u m m a r y of Final AEGL Values for N e r v e A g e n t s GA, GB, GD, GF, a n d VX (rag/m3) a
Classification
10 min
30 min
1 hr
4 hr
8 hr
AEGL- 1
0.0069
AEGL-2
0.087
AEGL-3
End point
0.0040
0.0028
0.0014
0.0010
Based on relative potency from GB. b ( E U F = 10)
0.050
0.035
0.017
0.013
Based on relative potency from GB. b ( E U F = 10)
0.76
0.38
0.26
0.14
0.10
Based on relative potency from GB. c ( E U F = 30)
AEGL- 1
0.0069
0.0040
0.0028
0.0014
0.0010
ECs0 for miosis observed in adult female SD rats receiving a range of GB vapor concentrations (0.01-0.48 mg G B / m 3) for 10-240 min (Mioduszewski et al., 2002a) and miosis data from supportive studies on marmosets (Van Helden et al., 2001, 2002) and humans (Harvey, 1952; Johns, 1952). ( E U F = 10)
AEGL-2
0.087
0.050
0.035
0.017
0.013
Miosis, dyspnea, R B C - C h E inhibition, single fiber electromyographic (SFEMG) changes in human volunteers receiving 0.5 mg G B / m 3 for 30 min (Baker and Sedgwick, 1996). ( E U F = 10)
AEGL-3
0.38
0.19
0.13
0.070
0.051
Experimental SD rat lethality data (LC01 and LCs0); whole body dynamic exposure to concentrations between 2 and 54 mg G B / m 3 for 3 - 3 6 0 min (Mioduszewski et al., 2000, 2001, 2002b). (EUF = 30)
AEGL-1
0.0035
0.0020
0.0014
0.00070
0.00050
Based on relative potency from GB. d ( E U F = 10)
AEGL-2
0.044
0.025
0.018
0.0085
0.0065
Based on relative potency from GB. d ( E U F = 10)
AEGL-3
0.38
0.19
0.13
0.070
0.051
Based on relative potency from GB. Supported by Wistar rat LCs0 from dynamic chamber exposures at 21 mg GD/m 3 for three time periods of -<30 min duration (Aas et al., 1985). e ( E U F = 30)
AEGL-1
0.0035
0.0020
0.0014
0.00070
0.00050
Based on relative potency from GB. d ( E U F = 10); Data from Whalley et al. (2004) are supportive.
AEGL-2
0.044
0.025
0.018
0.0085
0.0065
Based on relative potency from GB. d ( E U F = 10)
AEGL-3
0.38
0.19
0.13
0.070
0.051
Based on relative potency from GB. e ( E U F = 30)
AEGL-1
0.00057
0.00033
0.00017
0.00010
0.000071
Based on relative potency from GB.g ( E U F = 30), including MF of 3; Data from Benton et al. (2004) and Van der Schans et al. (2003) are supportive.
AEGL-2
0.0072
0.0042
0.0029
0.0015
0.0010
Based on relative potency from GB. h ( E U F = 30), including MF of 3; Data from Benton et al. (2004) and Van der Schans et al. (2003) are supportive.
AEGL-3
0.029
0.015
0.010
0.0052
0.0038
Based on relative potency from GB. / ( E U F = 100), including MF of 3; Data from Benton et al. (2004) and Van der Schans et al. (2003) are supportive.
aAdapted from Opresko et al. (2003) with permission by the National Academy of Sciences, courtesy of the National Academies Press, Washington, DC. bBased on relative potency equal to that of agent GB. CAgent GA is considered approximately one-half as potent as GB in causing lethality; thus, AEGL-3 values for GA are estimated by multiplying each time-specific AEGL-3 value for agent GB by a factor of 2. aAgents GD and GF are considered approximately twice as potent as agents GA and GB for causing miosis and are equipotent to each other. Thus, AEGL- 1 and AEGL-2 values are estimated by multiplying each time-specific AEGL- 1 or AEGL-2 value for agent GB by a factor of 0.5. eBased on a relative potency for lethality of GD = GF = GB and lethality data of Aas et al. (1985) (from which can be derived a 10-min AEGL-3 estimate of 0.27 mg/m 3 and a 30-min AEGL-3 value of 0.15 mg/m 3 and is thus supportive of the GD AEGL-3 estimate derived from relative potency). fBased on relative potency. Agent VX is considered approximately four times more potent than agent GB. gDerived from miosis effects noted in young adult female SD rats exposed to agent GB vapor at concentrations (0.010-0.48 mg/m3) for 10, 60, and 240 min (Mioduszewski et al., 2002a). VX concentration to achieve same end point estimated by relative potency adjustment. hDerived from transient effects noted in exercising human volunteers exposed to agent GB vapor at 0.5 mg-min/m3 for 30 min (Baker and Sedgwick, 1996). VX concentration to achieve same end point estimated by relative potency adjustment. /Derived from LC01 values for female SD rats exposed to GB vapor in dynamic exposure chamber (Mioduszewski et al., 2000, 2001, 2002b). VX concentrations to achieve same end point estimated by relative potency adjustment.
CHAPTER 5 9Chemical Warfare Nerve Agent Guidelines
61
nerve agent AEGL determination. For example, AEGL-1 values for GB are well below any human effect levels reported from controlled experimental studies, and human data points associated with the AEGL-3 line include non-life-threatening and nonsevere responses [Fig. 1; e.g., reversible dyspnea, miosis, and SFEMG, from Baker and Sedgwick (1996) and reversible headache, eye pain, chest tightness, and nausea from Harvey (1952)]. Animal toxicity comparison reveals that the AEGL-3 plot line is located considerably below points where lethal effects are recorded
and agent VX were derived from those of agent GB by a relative potency protocol (Opresko et al., 2003; Mioduszewski et al., 1998; see Table 3).
c. Comparison of AEGL Values with Experimental Data Comparison of AEGL values for agent GB with available human and animal toxicity data (Figs. 1 and 2) reveals the protective nature of each AEGL value for this agent as well as the conservative nature of the review process applied to
Human Data Nerve Agent GB 100.000 i
E] 1o.ooo -
EZ
No effect
D
1.ooo
Level 1
03
[]
E
Level 2
E
AEGL-3
O.lOO
A AEGL
O.OLO
............... ,~
o.ool
~-L-
~--'-"-"'------~ 60
120
FIG. 1. Category plot comparing AEGL concentrations (mg/m3) for agent GB with human toxicity data. Copyright 2006 from Journal of Toxicology and Environmental Health (Part B) by Watson et al. Reproduced by permission of Taylor & Francis, Inc., http://www.taylorandfrancis.com.
AEGL-1
180
240 Mintues
300
360
420
480
Animal Data Nerve Agent GB
1000.0000
100.0000
O
N o Effect
10.0000
9 9
03
,
< Level 1
@
1.0000
E E
Level 1 AEGL-3
0.1000
@ 1
Level 2
AEGL-2
0.0100
Some Lethality
0.0001
__
60
120
180
~
[
_ ~
240 Minutes
i
300
360
A
AEGL-1
D
0.0010
420
AEGL
I
480
FIG. 2. Category plot comparing AEGL concentrations (mg/m3) for agent GB with animal toxicity data. Lowest measurable effects such as observed EEG changes that are not associated with cognitive or behavioral effects are represented as "
62
S ECTI O N I 9Uses, Abuses, & Epidemiology'
for experimental animals (Fig. 2; Mioduszewski et al., 2000; Cohen et al., 1954) and overlaps no-effect concentrations for some species. The AEGL-1 plot line is associated with changes in marmoset visual evoked response and EEG that are not linked with cognitive or behavioral change and marmoset threshold miosis (Van Helden et al., 2001, 2002, 2003, 2004). These latter observations in a well-conducted study of nonhuman primates are of minor toxicological significance and further reflect the protective nature of the AEGL-1 determinations for agent GB. This protective nature of agent GB AEGL values also applies to any estimates derived from them (e.g., relative potency determinations for agents GA, GD, GF, and VX). 2. SUMMARY OF AEGL VALUES Following publication for comment in the Federal Register (66FR 21940; 2 May 2001) and extensive review, guideline concentrations for each cell of the matrix created by three AEGL levels of effect x 5 exposure durations for each of the 5 nerve agents (75 individual determinations) were finalized and published by the National Academy Press (Opresko et aL, 2003, as cited in NRC, 2003). These final AEGL values (and composite uncertainty factors) for the G agents and agent VX are summarized in Table 5. Details of derivation can be found in Opresko et al. (2003). The utility of these guidelines for CW agent emergency preparedness planning was recognized by CSEPP when FEMA and Army representatives adopted these final CW agent AEGLs to replace previous agent toxicity criteria for emergency response decision making (CSEPP, 2003). As of February 2003, standing CSEPP policy guidance for each of the communities hosting agent demilitarization facilities in the United States recommends application of AEGL-2 concentrations as the protective action level for evacuation or shelter-in-place and AEGL-1 concentrations as notification levels. Since publication of final AEGL levels by NRC (2003) and enactment of the CSEPP policy paper (CSEPP, 2003), multiple stockpile states and counties have incorporated the policy paper recommendations into their individual community emergency response plans and employed them in making regulatory decisions permitting agent munition disposal operations.
storage to adjacent communities in the United States and abroad and have been the subject of extensive emergency preparedness and response planning in preparation for stockpile demilitarization. In addition, active and closing military installations, as well as formerly utilized defense sites where CW agents were historically processed, pose separate remediation and installation restoration requirements. A key need for both community emergency preparedness and installation restoration is the availability of health-based exposure guidelines derived in a transparent manner by contemporary methods of data analysis. This chapter summarized such guidelines for acute vapor and chronic ingestion exposure to the nerve agents GA (tabun), GB (satin), GD (soman), GF (cyclosarin), and VX. The toxicological analysis and logic incorporated into estimation of reference dose (RfDe) and AEGL values for these nerve agents employs standard protocols already established by U.S. civilian authorities to govern management of Superfund sites and extremely hazardous substances unintentionally released during industrial accidents or chemical spills. In addition, both parameters are considered critical to maintaining compliance with international treaty obligations governing the stockpile and destruction of chemical weapons. The nerve agent RfD e and AEGL values have completed development and review and are now available for application to civilian and military defense purposes, such as shelter-in-place and evacuation protocols, recovery and remediation levels, analytical monitoring requirements, and protective clothing specifications. These guidelines are intended to preserve public health by not only characterizing potentially harmful exposure levels but by also identifying levels at which minimal or no toxic effects are expected. As a consequence, there is now a sound and health-based framework for not applying overly conservative measures (e.g., remediation to "nondetect") and to thus more efficiently allocate limited resources. The toxicological analyses, protocol and logic incorporated into nerve agent chronic RfD e and AEGL guideline development were documented, and their recent use by civilian and military authorities was summarized.
Acknowledgments V. C O N C L U S I O N S Chemical warfare nerve agents are potent anticholinesterase compounds deliberately formulated to induce debilitating effects or death during wartime hostilities. Due to their steep dose response, these agents have been used by military authorities of several nations to develop munitions (e.g., Germany during the Nazi era, the United States, and the former Soviet Union) and have been deployed by certain domestic terrorists (the Tokyo subway incident of 1995). Stockpiles of CW munitions manufactured as a Cold War deterrent decades ago pose inherent risks of continued
This work was prepared for two agencies of the U.S. Department of the Army: the U.S. Army Environmental Center under Interagency Agreement 2134-K006-A 1 and the U.S. Army Center for Health Promotion and Preventive Medicine under Interagency Agreement 2207-M135-A1. The Oak Ridge National Laboratory (ORNL) is managed and operated by UT-Battelle, LLC., for the U.S. Department of Energy under contract DE-AC05-00OR22725. The critical advice of Robert Ross, leader of the Toxicology and Hazard Assessment Group, Life Sciences Division, ORNL, in the preparation of the manuscript is acknowledged. During review of the nerve agent AEGL analyses by the NRC Committee on Toxicology, Subcommittee on Acute Exposure Guideline Levels, Dr. Daniel Krewski served as subcommittee
CHAPTER 5 chair; his leadership and wise counsel during this lengthy process are acknowledged with appreciation.
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cal/biological research program. National Security Council, Washington, DC. Oberst, E W., Koon, W. S., Christensen, M. K., Crook, J. W., Cresthull, E, and Freeman, G. (1968). Retention of inhaled satin vapor and its effect on red blood cell cholinesterase activity in man. Clin. Pharmacol. Ther. 9, 421-427. Office of the Army Surgeon General (2000). Chronic toxicological criteria for chemical warfare compounds, Memorandum MCHB-CG-PPM. U.S. Department of the Army, Office of the Surgeon General, Falls Church, VA. Office of the Assistant Secretary of the Army (1999). Derivation of health-based environmental screening levels (HBESL) for chemical warfare agents. Memorandum signed by Raymond J. Fatz, Deputy Assistant Secretary of the Army, 28 May 1999. Department of the Army, Office of the Assistant Secretary (Environment, Safety, and Occupational Health), Army Pentagon, Washington, DC. Okumura, T., Takasu, N., Ishimatu, S., Miyanoki, S., Mitsuhashi, A., Kumada, K., Tanaka, K., and Hinohara, S. (1996). Report on 640 victims of the Tokyo subway satin attack. Ann. Emerg. Med. 28, 129-135. Opresko, D. M., Young, R. A., Faust, R. A., Talmage, S. S., Watson, A. E, Ross, R. H., Davidson, K. A., and King, J. (1998). Chemical warfare agents: Estimating oral reference doses. Rev. Environ. Contam. Toxicol. 156, 1-183. Opresko, D. M., Young, R. A., Watson, A. E, Faust, R. A., Talmage, S. S., Ross, R. H., Davidson, K. A., King, J., and Hauschild, V. (2001). Chemical warfare agents: Current status of oral reference doses. Rev. Environ. Contam. Toxicol. 172, 65-85. Opresko, D., Watson, A., and Young, R. (2003). Nerve agents. In Acute Exposure Guideline Levels for Selected Airborne Chemicals, Vol. 3, pp. 15-300. National Academies Press, Washington, DC. Pope, C. (1999). Organophosphorous pesticides: Do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health (Part B) 2, 161-181. Pope, C., and Liu, J. (2002). Nonesterase actions of anticholinesterase insecticides. In Handbook of Neurotoxicology, (E. J. Massaro, Ed.), Vol. 1, pp. 29-43. Humana Press, Totowa, NJ. Rao, K. S., Aracava, Y., Rickett, D. L., and Albuquerque, E. X. (1987). Noncompetitive blockade of the nicotinic acetylcholine receptor-ion channel complex by an irreversible cholinesterase inhibitor. J. Pharmacol. Exp. Ther. 240, 337-344. Rice, G. B., Lambert, T. W., Haas, B., and Wallace, V. (1971). Effect of chronic ingestion of VX on ovine blood cholinesterase, DTC 71-512. Deseret Test Center, Dugway Proving Ground, Dugway, UT. Robinson, J. E (1967). Chemical warfare. Sci. J. 4, 33-40. Rocha, E. S., Chebabo, S. R., Santos, M. D., Aracava, Y., and Albuquerque, E. X. (1998). An analysis of low level doses of cholinesterase inhibitors in cultured neurons and hippocampal slices of rats. Drug Chem. Toxicol. 21(Suppl. 1), 191-200. Rocha, E. S., Santos, M. D., Chebabo, S. R., Aracava, Y., and Albuquerque, E. X. (1999). Low concentrations of the organophosphate VX affect spontaneous and evoked transmitter release from hippocampal neurons: Toxicological relevance of cholinesterase-dependent actions. Toxicol. Appl. Pharmacol. 159, 31-40.
CHAPTER 5 Rubin, L. S., and Goldberg, M. N. (1957). Effect of sarin on dark adaptation in man: Threshold changes. J. Appl. Physiol. 11, 439-444. Rubin, L. S., Krop, S., and Goldberg, M. N. (1957). Effect of satin on dark adaptation in man: Mechanism of action. J. Appl. Physiol. 11, 445-449. Schreider, J. P., Rowland, J. R., Rosenblatt, L. S., and Hendrick, A. G. (1984). The teratology effects of VX in rats, draft final report. Prepared by the Laboratory for Energy-Related Health Effects Research, University of California, Davis, for the U.S. Army Medical Bioengineering Research and Development Laboratory, Ft. Detrick, MD. Schreider, J. P., Remsen, J. E, and Shifrine, M. (1988). Toxicity studies on agent VX, Final report (AD A201397). Prepared by the Laboratory for Energy-Related Health Effects Research, University of California, Davis, for the U.S. Department of the Army, Medical Research and Development Command, Ft. Detrick, MD. Sidell, F. R. (1992). Clinical considerations in nerve agent intoxication. In Chemical Warfare Agents (S. M. Somani, Ed.), pp. 155-194. Academic Press, New York. Sidell, E R. (1996). Chemical agent terrorism. Ann. Emerg. Med. 28, 223-224. Sidell, E R. (1997). Nerve agents. In Medical Aspects of Chemical and Biological Warfare (E R. Sidell, E. T. Takafuji, and D. R. Franz, Eds.), pp. 129-179. Office of the Surgeon General, Walter Reed Army Medical Center, Washington, DC. Sidell, E R., and Groff, W. A. (1974). The reactivatibility of cholinesterase inhibited by VX and sarin in man. Toxicol. Appl. Pharmacol. 27, 241-252. Sidell, E R., and Kaminskis, A. (1975). Temporal intrapersonal physiological variability of cholinesterase activity in human plasma and erythrocytes. Clin. Chem. 21, 1961-1963. Singer, A. W., Jaax, N. K., Graham, J. S., and McLeod, C. G., Jr. (1987). Cardiomyopathy in soman and satin intoxicated rats. Toxicol. Lett. 36, 243-249. Somani, S. M., Solana, R. E, and Dube, S. N. (1992). Toxicodynamics of nerve agents. In Chemical Warfare Agents (S. M. Somani, Ed.), pp. 67-123. Academic Press, New York. Spruit, H. E., Langenberg, J. B., Trap, H. C., Van der Wiel, H. J., Helmich, R. B., van Helden, H. E, and Benschop, H. E (2000). Intravenous and inhalation toxicokinetics of sarin stereoisomers in atropinized guinea pigs. Toxicol. Appl. Pharmacol. 169, 249-254. Stockholm International Peace Research Institute (1973). CB Weapons today. In The Problem of Chemical and Biological Warfare, Vol. 2. Humanities Press, New York. Storm, J. E., Rozman, K. K., and Doull, J. (2000). Occupational exposure limits for 30 organophosphate pesticides based on inhibition of red blood cell cholinesterase. Toxicology 150, 1-29. Ten Berge, W. E, Zwart, A., and Appelman, L. M. (1986). Concentration-time mortality response relationship of irritant and systemically acting vapours and gases. J. Hazard. Materials 13, 301-309. Thienes, C. H., and Haley, T. J. (1972). Clinical Toxicology, pp. 95-115. Lea & Febiger, Philadelphia. U.S. Army Center for Health Promotion and Preventive Medicine (1999). Derivation of Health-Based Environmental Screening Levels for Chemical Warfare Agents: A Technical Evaluation.
9Chemical Warfare Nerve Agent Guidelines
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U.S. Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD. U.S. Department of Health and Human Services (1988). Final recommendations for protecting the health and safety against potential adverse effects of long-term exposure to low doses of agents GA, GB, VX, Mustard agent (H, HD, T), and Lewisite (L). Centers for Disease Control and Prevention. Fed. Reg. 53, 8504-8507. U.S. Department of Health and Human Services (2003). Final recommendations for protecting human health from potential adverse effects of exposure to agents GA (Tabun), GB (Sarin), and VX. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Fed. Reg. 68, 58348-58351. U.S. Environmental Protection Agency (1989). Risk assessment guidance for Superfund: Vol. 1. Human health evaluation manual (part A), EPA/540/1-89/002. Office of Emergency and Remedial Response, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1994). Methods for the derivation of inhalation reference concentrations and application of inhalation dosimetry, EPA/600/8-90/066F. Office of Health and Environmental Assessment, National Center for Environmental Assessment, Cincinnati, OH. U.S. Environmental Protection Agency (1998). Health effects test guidelines: OPPTS 870.6100. Acute and 28-day delayed neurotoxicity of organophosphorous substances, EPA 712-C98-237. Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2000). Office of Pesticide Programs science policy on the use of data on cholinesterase inhibition for risk assessment of organophosphorus and carbamate pesticides. Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC. Van der Schans, B. J., Lander, B. J., Van der Wiel, H., Langenberg, J. P., and Benshop, H. P. (2003). Toxicokinetics of the nerve agent (_)-VX in anesthetized and atropinized hairless guinea pigs and marmosets after intravenous and percutaneous administration. Toxicol. Appl. Pharmacol. 191, 48-62. Van Helden, H. P. M., Langenberg, J. P., and Benschop, H. P. (2001). Low level exposure to GB vapor in air: Diagnosis/ dosimetry, lowest observable effect levels, and performance incapacitation, Award No. DAMD17-97-1-7360, final report. Prepared by TNO Prins Maurits Laboratory, Rijswijk, The Netherlands, for the U.S. Army Medical Research and Materiel Command, Ft. Detrick, MD. Van Helden, H. P. M., Trap, H. C., Kuijpers, W. C., Groen, B., Oostdijk, J. P., Vanwersch, R. A. P., Philippens, I. H. C., Langenberg, J. P., and Benschop, H. P. (2002). Low level exposure to GB vapor in air: Diagnosis/dosimetry, lowest observable effect level, and performance-incapacitation. In Operational Medical Issues in Chemical and Biological Defense, Proceedings of the 75th Meeting of the Research and Technology Organisation, Estoril, Portugal, May 14-17, 2001, RTO-MP-075, AC/323 (HFM-060) TP/37. North Atlantic Treaty Organisation Research and Technology Organisation, Neuilly sur Seine Cedex, France. Van Helden, H. P. M., Trap, H. C., Oostdijk, J. P., Kuipers, W. C., Langenberg, J. P., and Benschop, H. P. (2003). Long-term, low-level exposure of guinea pigs and marmosets to satin
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vapor in air: Lowest-observable-adverse-effect level. Toxicol. Appl. Pharmacol. 189, 170-179. Van Helden, H. P. M., Trap, H. C., Kuipers, W. C., Oostdijk, J. E, Benschop, H. E, and Langenberg, J. P. (2004). Low-level exposure of guinea pigs and marmosets to sarin vapour in air: Lowest-observable-adverse-effect level (LOAEL) for miosis. J. App. Toxicol. 24, 59-68. Van Kampen, K. R., Shupe, J. L., Johnson, A. E., James, L. E, Smart, R. A., and Rasmussen, J. E. (1970). Effects of nerve gas poisoning in sheep in Skull Valley, UT. J. Am. Vet. Med. Assoc. 156, 1032-1035. Watson, A., Opresko, D., Young, R., and Hauschild, V. (2006). Development and application of acute exposure guideline levels (AEGLs) for chemical warfare nerve and sulfur mustard agents. J. Toxicol. Environ. Health. Part B. 9(2): (Inpress). Weimer, J. T., McNamara, B. E, Owens, E. J., Cooper, J. G., and van de Wal, A. (1979). Proposed revision of limits for human exposure to GB vapor in nonmilitary operations based on one-year exposures of laboratory animals to low airborne concentrations, ARCSL-TR-78056. U.S. Army Armament Research and Development Command, Chemical Systems Laboratory, Aberdeen Proving Ground, MD. Whalley, C. E., Benton, B. J., Manthei, J. H., Way, R. A., Jakubowski, E. M., Jr., Burnett, D. C., Gaviola, B. E, Crosier, R. B., Sommerville, D. R., Muse, W. T., Forster, J. S.,
Mioduszewski, R. J., Scotto, J. A., Miller, D. B., Crouse, C. L., Matson, K. L., Edwards, J. L., and Thomson, S. A. (2004). Low-level cyclosarin (GF) vapor exposure in rats: Effect of exposure concentration and duration on pupil size. ECBC-TR-407S (081004). U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD. Willems, J. L., Narcaise, M., and DeBisschop, H. C. (1984). Delayed neuropathy by the organophosphorous nerve agents soman and tabun. Arch. Toxicol. 55, 76-77. Wills, J. H. (1972). The measurement and significance of changes in the cholinesterase activities of erythrocytes and plasma in man and animals. Crit. Rev. Toxicol. 1, 153-202. Wilson, B. W., Henderson, J. D., Kellner, T. E, Goldman, M., Higgins, R. J., and Dacre, J. C. (1988). Toxicity of repeated doses of organophosphorous esters in the chicken. J. Toxicol. Environ. Health 23, 115-126. Wilson, B. W., Kawakami, T. G., Cone, N., Henderson, J. D., Rosenblatt, L. S., and Goldman, M. (1994). Genotoxicity of the phosphoramidate agent tabun (GA). Toxicology 86, 1-12. Young, R. A., Opresko, D. M., Watson, A. E, Ross, R. H., King, J., and Choudhury, H. (1999). Deriving toxicity values for organophosphorous nerve agents. A position paper in support of the procedures and rationale for deriving oral RfDs for chemical warfare agents. Hum. Ecol. Risk Assess. 5, 589-634.
CHAPTER
6
Organophosphates and the Gulf War Syndrome LINDA A. McCAULEY University of Pennsylvania, Philadelphia, Pennsylvania
following Iraq's invasion of Kuwait on August 2, 1990. There was a rapid buildup of Coalition forces primarily from the United States, Britain, and Canada. The conflict, referred to as Operation Desert Storm, began on January 17, 1991, when the U.S.-led Coalition air forces attacked Iraqi targets, and intense ground warfare was of a remarkably short duration, lasting from February 24 toFebruary 28, 1991. During the postcombat period, military posts were dismantled, munitions depots in Iraq were inspected and destroyed, and by August 1991 all U.S. service members who participated in the ground war had returned to the United States. The military conflict was viewed as a major success with few casualties; however, in the months following the military conflict, disturbing reports emerged from military units of unexplained health problems that were persistent and worsening. By 1992 and 1993, these complaints had caught the attention of scientists, the U.S. govemment, the Department of Defense, and the Department of Veterans Affairs. Reports of unexplained illness also emerged from other countries involved in the GW. The environmental exposures during the GW were numerous and potentially toxic to human health. Troops were exposed to temperature extremes; the threat of chemical warfare, including detection alarms sounding; combat stress; severe sand storms; insects, including sand flies carrying leishmaniasis; petrochemicals; oil well smoke; pesticides; munitions combustion products; depleted uranium; pyridostigmine bromide; and biological warfare agent vaccines (anthrax, plague, and botulinum toxoid vaccine). Approximately 35-45% of troops who served in Desert Storm received the CM anticholinesterase drug PB, a soman antidote enhancer commonly used in the treatment of myasthenia gravis. Not all servicemen who were dispensed packets of PB were compliant in taking them as ordered. Approximately 150,000 U.S. troops received anthrax vaccine and 8000 received botulinum toxoid vaccine (Presidential Advisory Committee, 1996a). Controversy emerged regarding the possible etiology of the multisymptom complexes seen in ill veterans, with many
I. I N T R O D U C T I O N More than a decade after the end of Operation Desert Storm in 1991, a large number of veterans who served during the war continue to experience an array of chronic health symptoms, including memory loss, fatigue, cognitive problems, somatic pain, skin abnormalities, and gastrointestinal difficulties. Since reports of unexplained illness among veterans of the 1991 Gulf War (GW) first appeared, organophosphate (OP) and other chemical exposures were believed by many to have contributed to the symptoms that were being reported. Now, more than 15 years after this military conflict, some scientists strongly believe the unexplained illness can be attributed to military combat and stress-related conditions, whereas others contend that OPs and carbamates (CMs) are implicated. In addition to possible exposure to chemical warfare agents, veterans were also potentially exposed to the OP insecticide chlorpyrifos and non-OP insect repellants such as N,N-diethyl m-toluamide (DEET) and the CM pyridostigmine bromide (PB) as a nerve agent antidote enhancer. This chapter examines the OP and CM exposures and health effect associations found in epidemiological studies and the methodological issues inherent in these study designs. Animal studies contributing to the conception model of how OPs and CMs mechanistically resulted in neurological damage are reviewed. The Khamisiyah detonations of nerve gas stores, the military incident in which there was overwhelming evidence of exposure to chemical warfare agents, is discussed. Finally, this chapter discusses current research that continues to strive to uncover the link between OP and/or CM exposure and unexplained neurological disease.
II. H I S T O R I C A L P E R S P E C T I V E The first U.S. troops arrived in the southwest Asia theater of operations in August 1990 during the preparatory period referred to as Operation Desert Shield, the period immediately Toxicology of Organophosphate and Carbamate Compounds
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scientists and advisory groups believing that the symptoms were related to the rapid deployment and combat stress. Although combat stress diagnoses such as posttraumatic stress disorder were not seen in excess, there was intense psychological stress associated with the severe environmental conditions and the ever-present threat of chemical warfare.
III. C H A L L E N G E S W I T H E X P O S U R E ASSESSMENT AMONG GULF WAR VETERANS Many investigators argued that the symptoms of the unexplained illnesses could be related to environmental exposure, and the role of exposure to insecticides, repellents, and PB was examined in most epidemiological studies of Gulf War veterans. Distribution and use of nonpersonal-use pesticides applied to military compounds were strictly controlled by the military in the theater of operations and restricted to certified personnel or contractors. Although the actual shipment amounts of personal-use insecticides and repellents were kept in military records, no records are available on personal use, but the use of insecticide repellent, insecticide spray (permethrin) applied to uniforms, and unauthorized use of flea collars did frequently occur. There was frequent combination use of these substances. Area fogs were regularly sprayed in camp areas for vector control. A 2003 Department of Defense report indicated that 37 individual pesticide ingredients were probably used during the GW (Table 1) and
that a potential link with illness in GW veterans could not be eliminated (U.S. Department of Defense, 2003). In the first decade after the conflict, most investigations of the role of chemicals and other exposures were evaluated through surveys of self-reported health symptoms and exposures (Fukuda et al., 1998; Iowa Persian Gulf Study Group, 1997; Spencer etal., 2001; Steele, 2002; Unwin et al., 1999). The symptoms reported in these study populations were remarkably similar. The veterans also reported a large array of environmental exposures, many of which occurred simultaneously. The lack of objective data on environmental and other exposures resulted in severe limitations in linking adverse health outcomes detected by epidemiologic studies to specific exposures and risk factors (Presidential Advisory Committee, 1996a). No biomarker of exposure has been available in epidemiological studies of this cohort and records of exposures do not exist. McCauley et al. (1999) first reported on the significant methodological issues associated with the dependence on self-reported exposure data. Using the time of employment and records of when PB, vaccines, and other exposures could have occurred, these investigators found significant overreporting of exposures during the Desert Shield and Desert Storm time periods. For example, 8.6% of surveyed veterans who were deployed to Southwest Asia only during noncombat periods reported that they took PB tablets. Twenty-eight percent of veterans deployed only during the months preceding combat reported that they believed they had been exposed to chemical warfare agents. Forty-seven percent of veterans who served after the combat period was over
TABLE 1. Products Used during the Gulf War Identified by the Deployment Health Support Directorate as Pesticides of Potential Concern a Pesticide DEET, 33% cream/stick DEET, 75 % liquid Permethrin, 0.5% spray d-Phenothrin, 0.2% aerosol Methomyl, 1% crystals Azamethiphos, 1% crystals Dichlorvos, 20% pest strip Chlorpyrifos, 45% liquid Diazinon, 48% liquid Malathion, 57% liquid Propoxur, 14.7% liquid Bendiocarb, 19% liquid Chlorpyrifos Malathion, 91% liquid Lindane, 1% powder
Class
Purpose
Application method
Repellnt Repellnt Repellnt Area spray Fly bait Fly bait Pest strip Sprayed liquid Sprayed liquid Sprayed liquid Sprayed liquid Sprayed powder Fog Fog Delouser
Repel flies and mosquitoes Repel flies and mosquitoes Repel flies and mosquitoes Knock down, kill flies and mosquitoes Attract and kill flies Attract and kill flies Attract and kill mosquitoes Kill flies, mosquitoes, flying insects Kill flies, mosquitoes, flying insects Kill flies, mosquitoes, flying insects Kill flies, mosquitoes, flying insects Kill flies, mosquitoes, flying insects Kill flies, mosquitoes Kill flies, mosquitoes Kill lice'
By hand to skin By hand to skin, uniform, netting Sprayed on uniforms Sprayed in area Placed in pans outside latrines, tents Placed in pans outside latrines, tents Hung in tents, working area, dumpsters Sprayed in comers, cracks, crevices Sprayed in comers, cracks, crevices Sprayed in comers, cracks, crevices Sprayed in comers, cracks, crevices Sprayed in comers, cracks, crevices Large area fogging Large area fogging Dusted on prisoners, also for personal use
aFrom the U.S. Departmentof Veterans Affairs (2004).
CHAPTER 6 9Gulf War Syndrome (during the cleanup period) also believed they were exposed to chemical or biological warfare agents. Test-retest reliability estimates indicated inconsistency in the frequency and rate of self-reported exposures. Veterans were found to have poor recall reliability for use of insecticide cream, taking more than three PB pills per day, reports of use of insecticide cream or sprays, and the belief that they had been exposed to chemical warfare agents. Nevertheless, the majority of reports with associated links between exposure and illness were dependent on self-reported exposures, although some investigations included physical examinations of veterans and verification of self-reported health information (Bourdette etal., 2001; Coker etal., 1999; Joseph, 1997; Murphy, 1999). Tests that were used in these clinical studies did not indicate any major differences between symptomatic and nonsymptomatic veterans. These negative clinical studies and the frequency of mental health diagnoses among symptomatic veterans led to many reports that the symptom patterns were suggestive of posttraumatic stress syndrome or other combat-related mental health problems (Hyams et al., 1996; Presidential Advisory Committee, 1996a; Storzbach et al., 2001; Vlahov and Galea, 2004).
IV. T H E R O L E OF P Y R I D O S T I G M I N E B R O M I D E IN G U L F W A R I L L N E S S Packets of 21 PB tablets were distributed to personnel in the GW for use as a nerve gas antidote enhancer. Personnel were instructed to take one 30-mg tablet three times per day at 8-hr intervals. PB is routinely administered to patients with myasthenia gravis in the United States at doses up to 17-fold greater than suggested for the short-term pretreatment against chemical warfare agents (Presidential Advisory Committee, 1997). The use of PB for protection in the event of an imminent attack with chemical warfare agents was not Food and Drug Administration (FDA) approved, and the FDA granted waivers of informed consent during military exigencies. Cook et al. (1992) conducted one of the few clinical trials of PB use among military populations and described the side effects that were most likely. Keeler et al. (1991) reported that approximately one-half of U.S. soldiers who took PB pills reported side effects associated with the medicine, the most common being anticholinesterase effects such as vision problems, headache, nausea, and abdominal cramping. Veterans report that use of PB was frequently discontinued due to the uncomfortable side effects. In 2000, the Institute of Medicine reported that there is inadequate evidence to determine whether an association exists between PB and long-term adverse health effects. Assessment of PB use has been included in all epidemiological studies of GW illness. Haley et al. (1997) reported an association between symptoms while taking PB and the likelihood of having GW illness. Although Spencer et al. (2001) also found this association, further analysis found that any
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combination of symptoms, regardless of their specificity with PB side effects, was strongly related to the likelihood of having unexplained illness after the war. When PB is examined as a singular exposure, it is almost consistently associated with the risk of having GW unexplained illness. However, PB exposure always occurred in combination with other environmental exposures during the GW. The possibility of a synergistic reaction with other exposures cannot be disregarded.
V. I N C R E A S E D T O X I C I T Y F R O M CHEMICAL COMBINATIONS Some investigators have postulated that the unexplained illness among GW veterans is related to the interaction of stress, PB, and OP exposures. The interaction of PB and insecticides has received considerable attention (Table 2). PB is a rapidly reversible inhibitor of acetylcholinesterase (ACHE) and blocks the binding of agents to ACHE. Investigators have hypothesized that the insect repellent DEET and the insecticide permethrin can access and damage the nervous system if peripheral cholinesterase binding sites are occupied by PB (Abou-Donia et al., 1996a,b; McCain et al., 1997). Multiple animal studies have helped elucidate potential pathways of neurological damage from low-level exposure to OPs and CMs. A review of 28 animal studies was conducted by the Department of Veterans Affairs Research Advisory Committee on GW veterans' illness (2004). These studies consistently demonstrate synergistic effects of different combinations of GW-related exposures. Contrary to previous assumptions, both animal a n d human studies suggest that exposure to these agents at levels too low to produce acute symptoms can result in chronic adverse effects on the nervous and immune systems. Controlled studies on rodents and hens administered single or repeated doses of DEET with or without PB and an OP or pyrethroid insecticide suggest that the toxic potency of these substances may differ when they are administered in various combinations. Some investigators have suggested that the combination of chemical exposures affects the ability of one or more of the substances to penetrate the blood-brain barrier; however, this has not been a consistent finding (Buchholz et al., 1997). Haley and Kurt (1997) began an intensive research program that has linked clinical syndromes among GW veterans with self-reported combinations of chemical warfare agents, PB, pesticides, and DEET. However, McCauley et al. (2001) found that in a multivariate analysis of a case-control study of GW veterans, combined self-reported PB use and insecticide use were not associated with an increased risk of unexplained illness. The possibility that stress induces increased susceptibility to chemical agents was reported by Kaufer et al. (1998) and Friedman et al. (1996). From animal studies, Friedman et al.
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TABLE 2. Study
Animal Studies Evaluating Synergistic Effects of Gulf War-Related Exposures a
Animal model
Exposures studied
Major finding(s)
Abou-Donia et al. (1996a)
Hen
PB, DEET, permethrin
Neurotoxicity greater when two exposures combined, further enhanced when all three exposures combined
Abou-Donia et al. (1996b)
Hen
PB, DEET, chlorpyrifos
Combined exposures enhanced inhibition of brain ACHE, BuChE, and NTE, neurologic dysfunction, and neuropathologic lesions
Baynes et al. (1997)
Rodent, pig skin
DEET, permethrin, carbaryl
DEET does not enhance dermal absorption of permethrin
Buchholz et al. (1997)
Rat
DEET, permethrin
PB ingestion resulted in lower central nervous system levels of permethrin
McCain et al. (1997)
Rat
PB, permethrin, DEET
Significantly greater lethality from combinations of exposures than single exposures
Chaney et al. (2000)
Rat
DEET, PB
DEET + PB significantly inhibited brain AChE activity but not peripheral AChE activity
van Haaren et al. (2000)
Rat
PB, permethrin
Serum permethrin levels were increased by coexposure to PB, but the combination did not affect behavioral responses
Hoy et al. (2000a,b)
Rat
PB, DEET, permethrin
Permethrin in combination with either PB or DEET affected locomotion rates in male but not female rats
Abou-Donia et al. (2001a)
Rat
PB, DEET, permethrin
Combined exposures at physiologically relevant doses led to neurological and behavioral deficits and alterations in brain AChE receptors
Abou-Donia et al. (2001b)
Rat
DEET, permethrin
Combined dermal exposures decreased blood-brain barrier permeability in cerebral cortex and produced impaired sensorimotor performance
Abu-Qare and Abou-Donia (2001a)
Rat
Satin, PB
Combined exposures led to increases in urine levels of markers of oxidative stress
Abu-Qare and Abou-Donia (200 lb)
Rat
DEET, permethrin
Single dermal dose of combined exposures produces significant increase in mitochondrial release of cytochrome c
Abu-Qare et al. (2001)
Rat
PB, DEET, permethrin
Oral PB combined with dermal application of DEET resulted in maximum urine levels of markers of oxidative stress
Peden-Adams et al. (2001)
Mouse
DEET, PB, JP-8 jet fuel
Exposure combination altered selected immunological end points, including delayed hypersensitivity and suppression of IgM response, but did not affect other immune measures
van Haaren et al. (2001)
Rat
PB, permethrin, DEET
Small doses of chemicals disrupted behavioral responses,with synergistic effects observed in some measures
Abdel-Rahman et al. (2002)
Rat
Restraint stress, low-dose PB, DEET, permethrin
Combined exposures disrupted blood-brain barrier in some brain areas and led to increased neuronal death
Baynes et al. (2002)
Pig skin
PB, permethrin
PB significantly enhanced dermal absorption and distribution of permethrin
Vogel et al. (2002)
Mice
DFP, PB, permethrin, parathion
PB reduced DFP binding; permethrin and parathion increased DFP binding in the brain
Abou-Donia et al. (2003)
Rat
Stress, PB, DEET, permethrin
Combined exposure to physiologically relevant doses of chemicals caused destruction of testicular germ cells; effect enhanced by stress
Husain and Somani (2003)
Mouse
Satin, PB, exercise
Exercise augmented synergistic effects of chemical exposures on AChE activity and NTE activity in various tissues (continues)
CHAPTER 6 9Gulf War Syndrome
TABLE 2.
73
(continued)
Study
Animal model
Exposures studied
Riviere et al. (2003)
Pig skin
Scremin et al. (2003)
Rat
DEET, PB, DFR permethrin, sulfur mustard PB, satin
Abdel-Rahman et al. (2004a)
Rat
Malathion, DEET, permethrin
Abdel-Rahman et al. (2004b)
Rat
Stress, PB, DEET, permethrin
Abou-Donia et al. (2004)
Rat
PB, DEET, permethrin
Olgun et al. (2004)
Mouse
Lindane, malathion, permethrin
Major finding(s) DEET absorption enhanced by PB, mustard, and DFP PB reduced delayed neurological and behavioral effects of sarin but did not reverse delayed effects on brain muscarinic receptors Combination of pesticides induced neurobehavioral deficits and neuron degeneration, with no overt signs of neurotoxicity Combined exposures produced damage to areas of the brain associated with motor and sensory function, learning and memory, and coordination Combined exposure produced sensorimotor deficits and altered brain AChE activity levels Exposure of thymus cells to combinations of chemicals resulted in significantly higher levels of apoptosis and necrosis
aFrom the U.S. Departmentof VeteransAffairs (2004). found that the crossing of PB through the blood-brain barrier is increased more than 100-fold in stressed animals. Exposing animals to increased stress apparently increases access of anticholinesterases and circulating viruses to the brain. However, this finding has not been replicated in other animal models (Lallement et al., 1998; Sinton et al., 2000).
VI. C H E M I C A L W A R F A R E E X P O S U R E Prior to and during the GW, the threat of enemy use of chemical warfare agents against Allied troops was seriously considered and troops were issued Mission Oriented Protective Posture (MOPP) protection, PB pill packets, and syringes of atropine. Several types of chemical warfare agent detectors were deployed; however, all these detectors and warning systems detected only nerve agent concentrations that would cause acute symptoms or death, not subclinical concentrations (Presidential Advisory Committee, 1996b). Detectors were reported by veterans to frequently alarm. Veterans would use their MOPP protection, only to be told that there was a false alarm. Not surprisingly, a large number of veterans in repeated epidemiological studies reported the belief that they were exposed to low levels of nerve agents. In general, the nerve gas warning systems used during the GW have been viewed as inadequate to protect military personnel from low levels of exposure and recommendations have been made to improve this surveillance system (Presidential Advisory Committee, 1996a). The most definitive potential exposure to OP agents occurred during the aftermath of the GW with the detonation of Iraqi munitions at the Bunker 73 Khamisiyah site in Iraq. Munitions containing 8.5 metric tons of sarin/cyclosarin
were detonated on March 4, 1991. On March 10, additional rockets were destroyed in a pit at Khamisiyah. Evidence of chemical warfare release during these detonations was considered overwhelming and exposure was assumed for nearby troops (Presidential Advisory Committee, 1996a). The Presidential Advisory Committee on Gulf War Veterans' Illnesses reported that the intelligence community clearly possessed information prior to and during the GW that constituted reasonable cause for concern that Khamisiyah was a chemical munitions storage facility. Current evidence indicates that this knowledge was obtained at least as early as the mid-1980s. However, no serious effort was made to examine the possibility of chemical warfare agent exposure to U.S troops at Khamisiyah until late 1995. When the detonations were made public in 1996, multiple projects modeling the atmospheric dispersion of the chemical warfare agents were begun. As a result of the modeling, more than 100,000 troops were believed to be potentially exposed and received notification to seek medical attention and register in the health registries that had been established at the Department of Defense and the Department of Veterans' Affairs (Walpole and Rosttker, 1997). A General Accounting Office (GAO) report concluded that the number of troops potentially exposed to chemical warfare agents as a result of the Khamisiyah detonations may have been underestimated and that the epidemiological studies using the plume modeling estimates are likely to be unreliable (GAO, 2004). The possibility of long-term health effects associated with low-dose exposure to chemical warfare agents has been a controversial issue. A 1982 National Academy of Sciences report could not rule out the possibility of longterm effects. The Japanese terrorist attacks with sarin during the 1990s provided evidence of the long-term effects of
74
S E CTI O N I
9Uses, Abuses,
& Epidemioloooy
toxic exposures to sarin, but individuals who did not present with evidence of acute toxicity at the time of the attacks have not been followed in longitudinal studies (Murata et al., 1997; Nakajima et al., 1999). The Department of Veteran Affairs Research Advisory Committee (2004) reviewed the animal studies of chronic effects of low-level satin exposure and concluded that low-dose sarin exposure is associated with chronic indicators of both neurological and immunological impairment. These animal studies report a number of effects, including decreased immune function, downregulation of muscarinic receptors in the hippocampus, chronic depression of AChE activity, memory loss, and cognitive changes and persistent changes on electroencephalograph readings in different animals. The disclosure from the Department of Defense of a definitive release of nerve gas agents at Khamisiyah, the animal studies indicating the potential for residual effects from low-dose exposure to these agents, and the prevalence of multisymptom complexes among GW veterans all point to the need for more studies of this phenomenon.
VII. HEALTH OUTCOMES ASSOCIATED WITH H U M A N EXPOSURE TO O R G A N O P H O S P H O R U S CHEMICALS A growing body of research indicates that veterans deployed in Desert Storm have increased health complaints compared to veterans who were either not deployed or deployed to other conflicts. Most often, investigators have found that selfreported health symptoms are significantly associated with diverse and multiple exposures rather than just PB- or UPassociated exposures (Iowa Persian Gulf Study Group, 1997; Spencer et al., 2001; Steele, 2002; Unwin et al., 1999). The possibility of residual neurological symptoms associated with the Khamisiyah exposure was investigated using a computer-assisted telephone survey of 2918 GW veterans from Oregon, Washington, California, North Carolina, and Georgia to evaluate the prevalence of self-reported symptoms (2001) and medical diagnoses and hospitalizations (2002) among a subsample of troops in close proximity to the Khamisiyah site and comparison groups of veterans deployed and nondeployed to the Southwest Asia theater of operations. Veterans who had participated in or witnessed the demolition were more likely to report historical or extant symptoms than were veterans from other military units. Five current neurological symptoms were reported in excess: tingling or burning sensations of the skin, changes in memory, difficulty sleeping, persistent fatigue, and depression. A subsequent factor analysis of this study sample found that in addition to the previously mentioned symptoms, veterans who witnessed the Khamisiyah detonations were also more likely to report dysesthesia (Shapiro et al., 2002). McCauley et al. (2002) reported that troops reported to be within 50 km of the Khamisiyah site did not differ from
other deployed troops with regard to reports of any medical conditions or hospitalizations in the 9 years following the GW. Other investigators have also studied the health of veterans who were exposed to chemical agents at Khamisiyah. Gray et al. (1999) also failed to detect an increase in hospitalization among active military personnel; however, when the atmospheric modeling of the dispersion of the chemical warfare agents was improved, they found an increased risk of hospitalization for dysrhythmias among exposed personnel (Smith et al., 2003).
VIII. EMERGING RESEARCH ON NEUROLOGICAL DISEASE IN GULF WAR VETERANS Although initial studies of GW veterans focused on selfreported symptoms and exposures, recent reports have investigated specific diseases and biological mechanisms for the neurological deficits seen in GW veterans. An advisory panel of the Department of Veterans Affairs concluded that the growing body of evidence indicates that an important component of GW veterans' illnesses is neurological in character. The most sobering indication of neurological disease among GW veterans comes from two studies indicating an increased risk of amyotrophic lateral sclerosis, commonly referred to as ALS or Lou Gehrig's disease, at approximately twice the rate of comparison populations in the years since the GW (Haley, 2003; Homer et al., 2003). Haley collected cases of ALS diagnosed from 1991 through 1998 from military registries and a publicity campaign in 1998. Diagnoses were established from neurologists' medical records. The expected incidence was estimated from the age distribution of GW veterans, weighted by age-specific death rates of the U.S. population. During the 8 years postwar, the incidence among veterans younger than age 45 increased from 0.93 cases/year in 1991 to 1.57 cases/year in 1998 and the observed incidence increased from one to five cases per year. Homer et al. conducted a nationwide epidemiologic case study to ascertain all occurrences of ALS for the 10-year period since August 1990 among active duty military and mobilized reservists who served in the GW. The ALS diagnosis was confirmed by medical record review. These investigators found a significant elevated risk of ALS among all deployed personnel (RR = 2.15, 95% confidence interval, 1.38-3.36). Although the number of cases is small, these study results indicate a need to closely monitor this military cohort for further ALS diagnoses. These reports are of serious concern; however, neither group of authors attempted to speculate if UP and CM exposure contributed to this increased risk. The initial clinical studies of GW veterans were largely negative, and no pathology could be detected using routine physical examinations and diagnostic tests (Bourdette et al., 2001; Fukada et al., 1998; Haley et al., 1997). However, in recent years Haley and others have begun to integrate
CHAPTER 6 9Gulf War Syndrome objective measures of neurological pathology and impairment using specialized neuroimaging techniques, tests of autonomic nervous system function, and audiovestibular testing (Haley et al., 2000a,b, 2004; Roland et al., 2000). These physiological findings have not been widely replicated by other investigators but findings are intriguing. Haley et al. (2000b) found physiological measures of functional brain mass to differ between ill GW veterans and matched veteran controls. As part of the MRS study reported previously, Haley et al. (2000a) also assessed the level of central dopamine activity in the basal ganglia of ill GW veterans and controls and found evidence suggesting an injury of dopaminergic neurons in the basal ganglia. A separate team of investigators reported the findings of a study using MRS that indicated that GW veterans have evidence of neuronal damage in the hippocampus (Menon et al., 2004). Vojdani and Thrasher (2004) reported significant immune alterations in veterans 2-8 years after participation in the GW but did not attribute these findings to the specific action of OPs or CMs. Three investigative teams have explored the possibility of autonomic nervous system disorders in GW veterans. GW veterans have been found to have abnormal responses to tilt-table testing when compared to healthy controls (Davis et al., 2000). Haley et al. (2004) found other indications of autonomic dysfunction, including heart rate blunting during sleep. Peckerman et al. (2000) also reported blunted cardiovascular response among ill GW veterans. Other investigations of neurophysiological assessments have been less consistent. Audiovestibular assessments have only been studied by Roland et al. (2000). Study findings on peripheral nerve function have been inconsistent (Jamal et al., 1996; Sharief et al., 2002). Multiple investigators have examined the potential role of polymorphisms in veterans with unexplained illness, but the results have been mixed (Haley et al., 1999; Mackness et al., 2000; Hotoph et al., 2003). Haley et al. reported that the most severely symptomatic GW veterans exhibited particularly low activity of paraoxonate (PON1) type Q, the type that would be most active in neutralizing nerve gases. Mackness et al. found that veterans' decreased capacity to metabolize OP chemicals may have contributed to their likelihood of developing GW illness. Hotoph et al. found that PON1 activity, which is a major determinant of OP's toxicity in humans, was significantly decreased in British veterans deployed to the GW compared to nondeployed veterans. Low PON1 activity is associated with several diseases with an inflammatory component; however, a link with OP exposures during the GW has not been established.
IX. C O N C L U S I O N S Fifteen years after the GW, the etiology behind the large number of veterans who have unexplained illnesses remains a mystery. Perhaps the role of OP and CM chemicals in
75
the risk of unexplained illness will never be delineated. The lack of objective data on exposure and the inability of routine physical examinations and tests to detect subtle neurological dysfunction compound the inability to find answers to this problem. A large number of scientists and clinicians believe that the unexplained illness is not chemical in origin but results from a psychological reaction to the war trauma, although there is little objective evidence to support this assumption. What is clear is that the symptoms are disabling and more than $1 billion has been spent in the United States on health evaluations and research on this phenomenon (Gray, 2004). As recently as 2004, a scientific advisory panel review recommended increased studies on the chronic health effects of low-dose exposures to OP substances (U.S. Department of Veterans Affairs, 2004). Most important, the GW syndrome has resulted in many improvements in the surveillance of environmental exposures that occur during military deployments and has increased the number of research studies aimed at finding effective treatments for the many veterans who still suffer from unexplained illness.
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Randall, B., Herwaldt, B. L., Mawle, A. C., and Reeves, W. C. (1998). Chronic multisymptom illness affecting Air Force veterans of the Gulf War. J. Am. Med. Assoc. 20, 981-988. Gray, G., Smith, T., Knoke, J., and Heller, J. (1999). The postwar hospitalization experience of Gulf War veterans possibly exposed to chemical munitions destruction at Khamisiyah, Iraq. Am. J. Epidemiol. lg0, 532-540. Gray, G. C., Gackstetter, G. D., Kang, H. K., Graham, J. T., and Scott, K. C. (2004). After more than 10 years of Gulf War veteran medical evaluations, what have we learned? Am. J. Prev. Med. 26, 443-452. Haley, R. W. (2003). Excess incidence of ALS in young Gulf War veterans. Neurology 61, 750-756. Haley, R. W., and Kurt, T. L. (1997). Self-reported exposure to neurotoxic chemical combinations in the Gulf War: A crosssectional epidemiological study. J. Am. Med. Assoc. 277, 231-237. Haley, R. W., Kurt, T. L., and Hom, J. (1997). Is there a Gulf War syndrome? Searching for syndromes by factor analysis of symptoms. J. Am. Med. Assoc. 277, 215-222. Haley, R. W., Billecke, S., and La Du, B. N. (1999). Association of low PON1 type Q (type A) arylesterase activity with neurologic symptoms complexes in Gulf War veterans. Toxicol. Appl. Pharmacol. 157, 227-233. Haley, R. W., Fleckstein, J. L., Marshall, W. W., McDonald, G. G., Kramer, G. L., and Petty, E (2000a). Effect of basal ganglia injury on central dopamine activity in Gulf War syndrome: Correlation of proton magnetic resonance spectroscopy and plasma homovanillic acid levels. Arch. Neurol. 57, 1280-1285. Haley, R. W., Marshall, W. W., McDonald, G. G., Daugherty, M. A., Petty, E, and Fleckenstein, J. L. (2000b). Brain abnormalities in Gulf War syndrome: Evaluation of 1H MR spectroscopy. Neuroradiology 215, 807-817. Haley, R. W., Vongpatanasin, W., Wolfe, G. I., Bryan, W. W., Armitage, R., Hoffman, R. E, Petty, E, Callahan, T. S., Charuvastra, E., Shell, W. E., Marshall, W. W., and Victor, R. G. (2004). Blunted circadian variation in autonomic regulation of sinus node function in veterans with Gulf War syndrome. Am. J. Med. 117, 469-478. Homer, R. D., Kamims, K. G., Feussner, J. R., Grambow, S. C., Hoff-Linquist, J., Harati, Y., Mitsumoto, H., Pascuzzi, R., Spencer, E S., Tim, R., Howard, D., Smith, T. C., Ryan, M. A. K., Coffman, C. J., and Kasarskis, E. J. (2003). Occurrence of amyotrophic lateral sclerosis among Gulf War veterans. Neurology 61, 742-749. Hotoph, M., Mackness, M. I., Nikolaou, V., Collier, D. A., Curtis, C., David, A., Durrington, E, Hull, L., Ismail, K., Peakman, M., Unwin, C., Wessely, S., and Mackness, B. (2003). Paraoxonase in Persian Gulf War veterans. J. Occup. Environ. Med. 45, 98-405, 668-675. Hoy, J. B., Comell, J. A., Karlix, J. L., Schmidt, C. J., Tebbett, I. R., and van Haaren, E (2000a). Interactions of pyridostigmine bromide, DEET, and permethrin alter locomotor behavior in rats. Vet. Hum. Toxicol. 42, 65-71. Hoy, J. B., Comell, J. A., Karlix, J. L., Tebbett, I. R., and van Haaren, E (2000b). Repeated coadministration of pyridostigmine bromide, DEET, and permethrin alter locomotor behavior of rats. Vet. Hum. Toxicol. 42, 72-76. Husain, K., and Somani, S. (2003 January 18). Delayed toxic effects of nerve gas satin and pyridostigmine under physical
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stress in mice. J. Burns Surg. Wound Care (online journal). Available at www.journalof burns.com. Hyams, K. C., Wignall, E S., and Roswell, R. (1996). War syndromes and their evaluation: From the US Civil War to the Persian Gulf War. Ann. Intern. Med. 125, 398-405. Institute of Medicine (2000). Gulf War and Health: Volume 1 m Depleted Uranium, Pyridostigmine Bromide, Sarin, Vaccines. National Academy Press, Washington, DC. Iowa Persian Gulf Study Group (1997). Self reported illness and health status among Gulf War veterans. J. Am. Med. Assoc. 277, 238-245. Jamal, G. A., Hansen, S., Apartopoulos, E, and Peden, A. (1996). The "Gulf War syndrome." Is there evidence of dysfunction in the nervous system? J. Neurol. Neurosurg. Psychiatr. 60, 449-451. Joseph, S. (1997). The Comprehensive Clinical Evaluation Program Evaluation Team: A comprehensive clinical evaluation of 20,000 Persian Gulf War veterans. Mil. Med. 162, 149-155. Kaufer, D., Friedman, A., Seidman, S., and Soreq, H. (1998). Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature 393, 308-309. Keeler, J. R., Hurst, C. G., and Dunn, M. A. (1991). Pyridostigmine used as a nerve agent pretreatment under wartime conditions. J. Am. Med. Assoc. 266, 693-695. Lallement, G., Foquin, A., and Baubichon, D. (1998). Heat stress, even extreme, does not induce penetration of pyridostigmine bromide into the brain of guinea pigs. Neurotoxicology 19, 759-766. Mackness, B., Durrington, P. N., and Mackness, M. I. (2000). Low paraoxonase in Persian Gulf War veterans self-reporting Gulf War syndrome. Biochem. Biophys. Res. Commun. 276, 729-733. Martin J. Deputy Director, Persian Gulf Veterans' Illnesses Investigative Team, Department of Defense, testimony before the Presidential Advisory Committee on the Gulf War Veterans' Illnesses, April, May, July, and August, 1996. McCain, W. C., Lee, R., Johnson, M. S., Whaley, J. E., Ferguson, J. W., Beall, E, and Leach, G. (1997). Acute oral toxicity study of pyridostigmine bromide, permethrin and DEET in the laboratory rat. J. Toxicol. Environ. Health 50, 113-124. McCauley, L. A., Joos, S. K., Spencer, E S., Lasarev, M. R., Shuell, T., and other members of the Portland Environmental Hazards Research Center (1999). Strategies to assess validity of self-reported exposures during the Persian Gulf War. Environ. Res. A 81, 195-205. McCauley, L., Rischitelli, G., Lambert, W., Lasarev, M., and Sticker, D. (2001). Symptoms of Gulf War veterans possibly exposed to organophosphate chemical warfare agents at Khamisiyah, Iraq. Int. J. Occup. Environ. Health 7, 79-89. McCauley, L. A., Lasarev, M. R., Sticker, D., Rischitelli, D. G., and Spencer, E S. (2002). Illness experience of Gulf War veterans possibly exposed to chemical warfare agents. Am. J. Prev. Med. 23, 200-206. Menon, E M., Nasrallah, H. A., Reeves, R. R., and Ali, J. A. (2004). Hippocampal dysfunction in Gulf War syndrome. A proton MR spectroscopy study. Brain Res. 1009, 189-194. Murata, K., Arak, S., Yokoyama, K., Okumura, T., Ishimatsu, S., Takasu, N., and White, R. E (1997). Asymptomatic sequelae to acute satin poisoning in the central and autonomic nervous system 6 months after the Tokyo subway attack. J. Neurol. 244, 601-606.
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Murphy, E M. (1999). Gulf War syndrome. Br. Med. J. 318, 274-275. Nakajima, T., Ohta, S., Fukushima, Y., and Yanagisawa, N. (1999). Sequelae of satin toxicity at one and three years after exposure in Matsumoto, Japan. J. Epidemiol. 9, 337-343. National Academy of Sciences (1982). Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1 i Anticholinesterases and Anticholinergics. National Academy Press, Washington, DC. Olgun, S., Gogal, R. M., Jr., Adeshina, E, Choudhury, H., and Misra, H. E (2004). Pesticide mixtures potentiate the toxicity in murine thymocytes. Toxicology 196, 181-195. Peckerman, A., LaManca, J. J., Smith, S. L., Taylor, A., Tiersky, L., Pollet, C., Korn, L. R., Hurwitz, B. E., Ottenweller, J. E., and Natelson, B. H. (2000). Cardiovascular stress responses and their relationship to symptoms in Gulf War veterans with fatiguing illness. Psychosom. Med. 62, 509-516. Peden-Adams, M. M., Eudaly, J., Eudaly, E., et al. (2001). Evaluation of immunotoxicity induced by single or concurrent exposure to N,N-diethyl-m-toluamide (DEET), pyridostigmine bromide (PYR), and JP-8 jet fuel. Toxicol. Ind. Health 17, 192-209. Presidential Advisory Committee on Gulf War Veterans' Illnesses (1996a, December). Final Report. U.S. Government Printing Office, Washington, DC. Presidential Advisory Committee on Gulf War Veterans' Illnesses (1996b, February). Interim Report. U.S. Government Printing Office, Washington, DC. Presidental Advisory Committee on Gulf War Veterans' Illnesses (1997, October). Special Report. U.S. Government Printing Office, Washington, DC. Riviere, J. E., Baynes, R. E., Brooks, J. D., Yeatts, J. L., and Monteiro-Riviere, N. A. (2003). Percutaneous absorption of topical N,N-diethyl-m-toluamide (DEET): Effects of exposure variables and coadministered toxicants. J. Toxicol. Environ. Health A 66, 133-151. Roland, P. S., Haley, R. W., Yellin, W., Owens, K., and Shoup, A. G. (2000). Vestibular dysfunction in Gulf War syndrome. Otolaryngol. Head Neck Surg. 122, 319-329. Scremin, O. U., Shih, T.-M., Huynh, L., Roch, M., Booth, R., and Jenden, D. J. (2003). Delayed neurologic and behavioral effects of subtoxic doses of cholinesterase inhibitors. J. Pharmacol. Exp. Ther. 304, 1111-1119. Shapiro, S. E., Lasarev, M. R., and McCauley, L. (2002). Factor analysis of Gulf War illness: What does it add to our understanding of possible health effects of deployment? Am. J. Epidemiol. 156, 578-585. Sharief, M. K., Priddin, J., Delamont, R. S., et al. (2002). Neurophysiologic analysis of neuromuscular symptoms in UK Gulf War veterans: A controlled study. Neurology 59, 1518-1525. Sinton, C. M., Fitch, T. E., Petty, E, and Haley, R. W. (2000). Stressful manipulations that elevate corticosterone reduce blood-brain barrier permeability to pyridostigmine in the rat. Toxicol. Appl. Pharmacol. 165, 99-105. Smith, T. C., Gray, G. C., Weir, J. C., Heller, J. M., and Ryan, M. A. (2003). Gulf War veterans and Iraqi nerve agents at Khamisiyah: Postwar hospitalization data revisited. Am. J. Epidemiol. 158, 457-467. Spencer, P. S., McCauley, L. A., Lapidus, J. A., Lasarev, M., Joos, S. K., and Storzbach, D. (2001). Self-reported exposures
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and their association with unexplained illness in a populationbased case-control study of Gulf War veterans. J. Occup. Environ. Med. 43, 1041-1056. Steele, L. (2002). Prevalence and patterns of Gulf War illness in Kansas veterans: Association of symptoms with characteristics of person, place, and time of military service. Am. J. Epidemiol. 152, 992-1002. Storzbach, D., Campbell, K. A., Binder, L. M., McCauley, L., Anger, W. K., Rohlman, D. S., and Kovera, C. A. (2001). Psychological differences between veterans with and without Gulf War unexplained symptoms. Psychosom. Med. 62, 726-735. Unwin, C., Blatchley, N., Coker, W., Ferry, S., Hotopf, M., Hull, L., Ismail, K., Palmer, I., David, A., and Wessely, S. (1999). Health of U.K. servicemen who served in Persian Gulf War. Lancet 353, 169-178. U.S. Department of Defense, Office of the Special Assistant to the Under Secretary of Defense (Personnel and Readiness) for Gulf War Illnesses, Medical Readiness, and Military Deployment (2003). Environmental exposure report: PesticidesmFinal report. Available at www.gulflink.osd.mil. Accessed March 20, 2005. U.S. Department of Veterans Affairs Research Advisory Committee on Gulf War Veterans' Illnesses (2004, September 4). Scientific progress in understanding Gulf War veteran's illnesses: Report and recommendations. Available at www.ngwrc.org. Accessed March 20, 2005. Research Advisory Committee on Gulf War Veterans' Illnesses (2004, September). Scientific progress in understanding Gulf
War Veterans' illnesses: Report and recommendations. Available at www.ngwrc.org. Accessed March 20, 2005. U.S. General Accounting Office (2004, June). Gulf War illnesses: DOD's conclusions about U.S. troops' exposure cannot be adequately supported, GAO-04-159. Available at www.gao.gov. Accessed March 20, 2005. van Haaren, E, Cody, B. Hoy, J. B., et al. (2000). The effects of pyridostigmine bromide and permethrin, alone or in combination, on response acquisition in male and female rats. PharmacoL Biochem. Behav. 66, 739-746. van Haaren, E, Haworth, S. C., Bennett, S. M., et al. (2001). The effects of pyridostigmine bromide, permethrin, and DEET alone, or in combination, on fixed-ratio and fixed-interval behavior in male and female rats. Pharmacol. Biochem. Behav. 69, 23-33. Vlahov, D., and Galea, S. (2004). War and anxiety disorders. Epidemiology 15, 129-130. Vogel, J. S., Keating, G. A., and Buchholz, B. A. (2002). Protein binding of isofluorophate in vivo after coexposure to multiple chemicals. Environ. Health Perspect. 110, 1031-1036. Vojdani, A., and Thrasher, J. D. (2004). Cellular and humeral immune abnormalities in Gulf War veterans. Environ. Health Perspect. 112, 840-846. Walpole, R., and Rostker, B. (1997, September). Modeling the chemical warfare agent release at the Khamisiyah pit. Central Intelligence Agency and Department of Defense, Washington, DC. Available at www.gulflink.osd.mil.
CHAPTER 7
The Bhopal Accident and Methyl Isocyanate Toxicity DAYA i~ VARMA AND SHREE MULAY McGill University, Montreal, Quebec, Canada
subject of several books (Eckerman, 2005; Everest, 1985; Lapierre and Moro, 2001; Morehouse and Subramaniam, 1986; Sufrin, 1985); reviews (Bucher, 1987; Dhara and Dhara, 1995, 2002; Dhara and Gassert, 2002; Dhara and Kriebel, 1993; Mehta et al., 1990; Sriramachari, 2004; Sriramachari and Chandra, 1997; Varma, 1986; Varma and Guest, 1993; Varma and Varma, 2005); commentaries (Anonymous, 1984a,b; Crabb, 2004; Anonymous, date a,b; Lepkowski, 1985; Marwick, 1985; Nemery, 1996; Sharma, 2005); documentary films, such as "Bhopal beyond Genocide" by the Cinemart Foundation (1986) and "Bhopal: The Search for Justice" by White Pine Picture and the National Film Board of Canada (2004); and a play by Rahul Varma titled "Bhopal" in Canada (2002) and "Zahrili Hawa" (poisonous gas) in India (2003). This chapter discusses acute and chronic toxicities of MIC and the possible mechanisms. Although the effect of the accident on the environment, the consequences of consuming polluted underground water, the cause of the accident, legal aspects of the tragedy, and the issues of compensation and rehabilitation of the population are extremely important, these are not covered in this chapter. These can be found elsewhere (Eckerman, 2005; Everest, 1985; Lapierre and Moro, 2001; Sufrin, 1985; Varma, 1986). Instead, the chemistry and toxicity of MIC are situated in relation to other members of the isocyanate family, all of which are toxic. Also, the cyanide-sodium thiosulfate controversy is briefly discussed.
I. I N T R O D U C T I O N Bhopal is a picturesque historic city surrounded by two large lakes and hills. It is also the home of the world's worst industrial diaster. Just past midnight on December 2, 1984, nearly 30 metric tons of methyl isocyanate (MIC) spewed from the Union Carbide Pesticide plant within a matter of 45-90 min, killing more than 2500 people within 2 days and more than 15,000 thereafter. Nearly 200,000 suffer from long-term adverse effects of MIC. In Bhopal, MIC used to be stored in steel tanks for the manufacture of the carbamate pesticide carbaryl (Sevin). The disaster was caused by the entry of water into the MICcontaining tank No. 610. The exothermic reaction between water and MIC converted liquid MIC into vapor, generating enough heat and pressure to burst open the vent. MIC vapor was discharged in the atmosphere, and because it was heavier than air it descended on the densely populated areas; people were awakened by a choking sensation and eye irritation (Varma, 1986). The reluctance of the Union Carbide West Virginia operation to speedily divulge the identity of the noxious chemical compounded the catastrophe. In panic, people ran as fast as they could to escape the poison; as a result, they inhaled more than they would have had they laid o n t h e ground. The confusion between the terms "isocyanate" and "cyanide" and the erroneous popular notion that nothing is more toxic than cyanide only increased the anxiety of a frightened population. When the identity of the culprit gas was finally revealed, people were told that MIC is just an irritant but not fatal. However, on the morning of December 3, 1984, Bhopal was a city of death and of dying people; neither animals nor trees were spared, only the houseflies survived the gas. More than 20 years later, Bhopal is a city of lingering pain. Given the magnitude of the tragedy, it is not surprising that the Bhopal disaster drew the attention of the media and the scientific community throughout the world and it has been the Toxicology of Organophosphate and Carbamate Compounds
II. T H E S O D I U M T H I O S U L F A T E CONTROVERSY Whether or not hydrogen cyanide (HCN) was the cause of death following the disaster in Bhopal and whether or not people should be treated with sodium thiosulfate (NTS) became a controversial issue, involving the medical profession, the Indian Council of Medical Research (ICMR), 79
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the press, nongovernmental organizations, and the Indian Supreme Court (Varma, 1986). How and why did the HCN-NTS controversy arise? The first message from the Union Carbide West Virginia plant implied that HCN had leaked and people should be given the cyanide antidote NTS (Varma, 1986; Varma and Varma, 2005). A generous quantity of NTS was donated by a German toxicologist, who erroneously believed that the antidote could work days after the ingestion of the poison. Cherry-red blood (characteristic of cyanide poisoning) at autopsy, reports of dramatic beneficial effects of NTS injections, and increased urinary levels of the cyanide metabolite NTS, coupled with the popular notion that nothing is more poisonous than cyanide, lent credence to claims that the Bhopal population was poisoned by HCN. The ICMR recommended, and the Supreme Court ordered, the use of NTS as an antidote. It is very possible that the mixture of gases in Bhopal contained some cyanide. However, if the principal culprit gas was HCN, almost all deaths would have occurred in less than 3 hr and not between 4 and 48 hr as was the case. Moreover, even if most of the escaping MIC was transformed into HCN, which somehow managed to descend to the ground even though its relatively lower density than air does not favor such a descent, its concentration could not be high enough (>30 ppm) to kill so many people. Cyanide is not known to cause long-term health effects; it is a very fast killer, but if the dose is not high enough it spares the victim without causing long-term disability (Goldstein et al., 1968). On the other hand, some benefit from NTS injections days or months after the accident is very possible since NTS can improve tissue oxygenation by neutralizing cyanide normally present in the blood of smokers and in people living in polluted areas (Sriramachari, 2004; Varma, 1986, 1989; Varma and Varma, 2005). Moreover, MIC can cause cherry-red blood by interacting with hemoglobin (Sriramachari, 2004). Experimental studies noted the ineffectiveness of NTS against MIC (Alarie et al., 1987; Bucher et al., 1987; Nemery et al., 1985a; Varma et al., 1988); however, lack of antagonism between NTS and MIC does not rule out the presence of HCN in Bhopal.
III. T H E I S O C Y A N A T E F A M I L Y Isocyanates were first synthesized in 1849 and are primarily used for the manufacture of polyurethane resins; their production increased after World War II. Most of the commercially used isocyanates are mono- or diisocyanates. All isocyanates are toxic and their toxicity is greater following inhalation than following oral ingestion. MIC is the smallest member of the family and the most toxic (Varma, 1986). The general structure of isocyanates is R-N ~ - C - - O ; these are highly reactive members of the heterocumelene family and the presence of adjacent double bonds confers high reactivity to isocyanates by cumulative action. The molecular weight,
LC50 (mg/m3), and ceiling (mg/m 3) for MIC and commonly used diisocyanates are, respectively, as follows: MIC, 57, 12, and 0.05; hexamethylene diisocyanate, 168, 385, and 0.14; toluene diisocyanate (TDI), 174, 350, and 0.14; isophorone diisocyanate, 222, 260, and 0.18; and diphenylmethane diisocyanate, 250, 370, and 0.20. As early as 1956, more than 100 cases of illness and 4 cases of death due to TDI were recorded (Baader, 1956). If an isocyanate causes both sensory and pulmonary irritation, as observed for MIC (Alarie et al., 1987; Ferguson et al., 1986), its toxicity is greater than if it only causes sensory irritation (Weyel et al., 1982). Isocyanates can cause direct irritant effects on lungs, eyes, skin, and exposed mucosa as well as nonpulmonary effects; indirect toxicity of diisocyanates is secondary to lung damage and immunologic sensitization (Adams, 1970; Anonymous, 1966; Brugsch and Elkins, 1963; Hama, 1957; Karol et al., 1987; Munn, 1965). Diisocyanate toxicity may range from temporary reversible to long-term irreversible damage (Axford et al., 1976) as well as delayed appearance of complications (Le Quesne et al., 1976). With most isocyanates, with the exception of MIC, direct effects ensue a few hours after exposure. It should be noted that despite similarities, there are differences in the toxicity of different isocyanates (Anonymous, 1966; Munn, 1965).
IV. P H Y S I C O C H E M I C A L CHARACTERISTICS OF METHYL ISOCYANATE MIC (CH3-N = C = O) is the smallest member of the isocyanate family and the most toxic. MIC is a flammable colorless and odorless liquid; its properties and dispersion profile in Bhopal are presented in Table 1. The extremely
TABLE 1. Physicochemical Properties of Methyl Isocyanate (MIC) and Dispersal in Bhopal Molecular mass Specific gravity (water = 1) at 20 ~ Vapor density (air = 1) Boiling point (~ Vapor pressure (mbar) at 4 ~ Vapor pressure (mbar) at 20 ~ Vapor pressure (mbar) at 31 ~ Bhopal area exposed to MIC (km2) Estimated mean MIC concentration in Bhopal (ppm)a Estimated range of MIC concentration in Bhopal (ppm)a
57.05 0.96 1.97 39.1 267 464 800 40 27 0.12-86.5
aData on MIC concentration in Bhopal are from Dhara and Dhara (2002) and Varma(1986).
CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity high vapor pressure of MIC can contribute to its high toxicity (Rye, 1973). MIC vapor settled on the ground level because it is heavier than air. The boiling point of MIC (39.1 ~ is lower than the daytime temperature in Bhopal on certain summer days. The concentration of MIC in Bhopal was well above its lethal concentration as determined in later studies (Bucher et al., 1987; Varma, 1987). The Occupational Safety and Health Administration of the United States has set an 8-hr exposure limit for MIC of 0.02 ppm (0.05 mg/m3), which is in conformity with other estimates (Alarie et al., 1987). A portion of MIC also degraded into nearly 20 products, including monomethylamine, dimethylamine, trrimethylamine, dimethylurea, trimethylurea, dimethylisocyanate, trimethyl isocyanate, trimethylbiuret, CO2, and cyanide; reaction between the products produced urethane, amides, and carbamates (Andersson et al., 1985; D'Silva et al., 1986; Vardarajan et al., 1985). It is very likely that some of these degradation products, which have a longer biological half-life than the parent compound, augmented the toxicity of MIC and may even account for some of the long-term effects. In any case, it is not possible to determine if the adverse effects of MIC observed in Bhopal were influenced by the degradation products. Also, the generation of other products in unspecified relative concentration in the MIC storage tank and possibly outside must have been influenced by several environmental factors. Factors such as temperature, humidity, and pollution in Bhopal, relatively long and unknown period of storage of MIC in tank No. 610 (MIC leaked from this tank), mixing of different batches of MIC in tank No. 610, reaction with an unknown quantity of water, and other specifics of the disaster could determine the generation of degradation product. Therefore, it is not possible to replicate the events of Bhopal at a test site as being planned (Crabb, 2004), and such an expensive operation, even if safe, will not yield any useful information.
V. GENERAL FEATURES OF MIC TOXICITY MIC can interact with a large number of inorganic and organic molecules and it is rapidly (minutes) degraded in aqueous media (Brown et al., 1987). The high chemical reactivity of MIC constituted the basis for the claim by Union Carbide physicians during a press conference in Bhopal on December 14, 1984, that MIC will be destroyed upon contact with body surface and will not produce any systemic effects (Varma, 1986); notwithstanding the high chemical reactivity of MIC and a short (2 min) biological half-life (Brown et al., 1987), subsequent studies (Bhattacharya et al., 1988; Ferguson et al., 1988; Karol and Kamat, 1988; Karol et al., 1987) disproved this claim. For example, administration of [14C]MIC by inhalation or injection into laboratory animals resulted in widespread
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distribution of 14C, including to the brain and conceptus (Bhattacharya et al., 1988; Ferguson et al., 1988). Also, MIC-induced myelotoxicity (Hong et al., 1987), complement activation (Kolb et al., 1987), chromosomal aberration (Shelby et al., 1987), and anti-MIC antibody formation in animals and Bhopal residents (Karol and Kamat, 1988; Karol et al., 1987) imply transport of intact MIC molecule to internal organs. Note that the half-life of a number of clinically used peptides is in the range of 2-5 min. MIC can cause N-carbamoylation of several end-terminal amino acids of tissue proteins (Sriramachari, 2004). Isocyanates can react with hydroxyl, sulfhydryl, and imidazole groups under physiologic conditions (Brown et al., 1987), which would imply that a wide variety of physiological functions can be altered following exposure to MIC, for which now there is scientific evidence (Dhara and Dhara, 2002; Sriramachari, 2004; Varma and Guest, 1993; Varma and Varma, 2005). The ability of MIC to increase the affinity of hemoglobin for oxygen (Lee, 1976) and inhibit mitochondrial respiration (Jeevaratnam et al., 1992) can lead to tissue hypoxia. Unlike organophosphates, MIC is not a potent inhibitor of cholinesterases (Brown et al., 1987), and its main toxic effects cannot be attributed to be a result of an inhibition of cholinesterase. A glutathione conjugate of MIC, S-(N-methylcarbamoyl) glutathione, has been isolated from rat tissues injected with MIC (Pearson et al., 1990; Slatter et al., 1991). Cysteine and glutathione conjugates of MIC are cytotoxic and can carbamoylate DNA and proteins. Isocyanate-glutathione conjugates can release isocyanate at the cell membrane (Bruggeman et al., 1986; Guest and Varma, 1994; Guest et al., 1992; Pearson et al., 1991).
VI. TOXICITY OF MIC IN ANIMAL MODELS At the time of the Bhopal accident, there was a single report on the toxicity of MIC in rats, mice, guinea pigs, rabbits, and human volunteers (Kimmerle and Eben, 1964). Exposure of animals to 1-23 ppm MIC for 1-4 hr caused severe irritation and pulmonary edema; mortality was biphasic such that animals died within hours and several days after up to 18 days. Exposure of humans to 2-4 ppm MIC caused lacrimation and irritation of the nose and throat and higher concentrations forced volunteers to leave the area (Kimmerle and Eben, 1964). The paucity of data on MIC at the time of the Bhopal accident and extensive toxicity data on diisocyanates (Anonymous, 1966; Varma, 1986) prompted the prestigious journal L a n c e t (Anonymous, 1984a) to comment, "In a year's time we will have learned a lot more about methyl i s o c y a n a t e - at an appalling price." Indeed, we did learn a lot in a few years' time. The U.S. National Toxicology
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Program and the National Institute of Environmental Health Sciences, as well as the Indian Council of Medical Research, released funds for MIC toxicity studies. It should nonetheless be noted that most of the post-Bhopal animal studies (Alarie et al., 1987; Bucher et al., 1987; Dodd et al., 1987; Fowler et al., 1987; Nemery et al., 1985b), reviewed by others (Bucher, 1987; Nemery et al., 1987; Varma and Guest, 1993; Dhara and Dhara, 2002), merely confirm the results of the 1964 study by Kimmerle and Eben published in German, which was not taken note of by isocyanate toxicologists and even by the Union Carbide authorities. In some of the post-Bhopal studies, rats, mice, and guinea pigs were exposed to 1-30 ppm MIC for 1-3 hr to simulate the Bhopal situation (Alarie et al., 1987; Ferguson et al., 1986; Varma 1987; Varma et al., 1987, 1988, 1990); other studies used excessively high concentrations (25-3500 ppm) of MIC (Dodd et al., 1987; Fowler et al., 1987), which have little bearing on the Bhopal episode. Toxic effects of MIC degradation products have also been investigated (Guest and Varma, 1991, 1992, 1993; Guest et al., 1992, 1994). There is a significant correlation between the effects of MIC in animal models and those in the Bhopal population, especially with regard to the mortality pattern. For example, both in animal models and in Bhopal, there is a lag period between the exposure and onset of death. Also, effects of MIC on pulmonary function, eyes, reproductive function, and immune response in animals models broadly mimic clinical toxicity in Bhopal. Fetal malformations reported in Bhopal have not been observed in animal models. Unfortunately, the initial enthusiasm for laboratory investigation subsided rather quickly, and very few studies were done to determine the long-term toxicity of MIC in animal models.
A. General Effects of MIC and Mortality in Animal Models All experimental studies noted signs of restlessness, lacrimation, discharge from the nose and mouth, and respiratory distress during the exposure of animals to MIC (Alarie et al., 1987; Bucher et al., 1987; Dodd et al., 1987; Varma et al., 1988). Exposure to MIC produced a marked decrease in body weight and food intake; although animals started gaining weight after 2 or 3 days, body weights did not approach those of control animals for as long as 2 weeks (Varma et al., 1988). Mortality during and soon after exposure to MIC (minutes) is rare, even at concentrations as high as 3500 ppm (Dodd et al., 1987). At MIC concentrations of 10-30 ppm, animals die several hours and days after exposure; deaths occurred either within the first 2 days or after a lag period of 1 week, and by 20 days postexposure, approximately 80% of exposed animals had died (Bucher et al., 1987; Varma et al., 1988). The biphasic death pattern has also been reported in Bhopal, although it is difficult to translate 8-20 days in the life of a rat to that for humans. Mortality in mice exposed to 40 ppm MIC could be reduced by prior treatment with gluco-
corticoids but not by sodium thiosulfate and atropine (Varma et al., 1988); both these agents were used by Indian doctors in Bhopal. Subcutaneous injection of MIC into rabbits caused a decrease in arterial pressure and lactic acidosis (Jeevarathinam et al., 1988).
B. Pulmonary Toxicity of MIC in Animals MIC was found to be both a sensory and a pulmonary irritant; animals died of pulmonary damage and tissue hypoxia (Alarie et al., 1987; Dodd et al., 1987; Nemery et al., 1985b), and a decrease in lung function persisted in surviving animals (Alarie et al., 1987; Bucher et al., 1987; Boorman et al., 1987; Nemery et al., 1985b; Stevens et al., 1987; Tepper et al., 1987). Guinea pigs were more susceptible to the lethal effects of MIC than rats (Dodd et al., 1987), which points to the limitation of animal studies in terms of mortality in Bhopal. Exposure to MIC also produced degenerative damage to olfactory epithelium in rats and mice (Uraih et al., 1987) and an increase in lung weight suggestive of proliferative changes (Bucher et al., 1987; Varma et al., 1987).
C. Reproductive and Other Nonpulmonary Toxic Effects of MIC in Animals The exposure of mice to 9 and 15 ppm MIC on day 8 of gestation was found to cause resorption of more than 80% of implants (Varma, 1987; Varma et al., 1987, 1990), which was associated with a decrease in plasma progesterone to nonpregnant levels (Varma et al., 1990). Fetal toxicity seemed unrelated to maternal toxicity since suppression of pulmonary edema by dexamethasone or injections of progesterone did not increase fetal survival; also, exposure of embryos to MIC in culture caused a concentration-dependent decrease in growth (Varma et al., 1990). Repeated exposure of pregnant mice from day 14 to day 17 of gestation to 3 ppm MIC also caused significant fetal and neonatal deaths (Schwetz et al., 1987). Lens opacity (Harding and Rixon, 1985) and corneal damage (Salmon et al., 1985) in rats exposed to MIC have been reported, although negative data also exist (Gupta et al., 1987). Immunosuppression in rats exposed to MIC was found by Dwiwedi et al. (1988) but not by Tucker et al. (1987). In addition, myelotoxicity (Hong et al., 1987), inhibition of cell cycle in lymphocytes (Conner et al., 1987), and changes in chromosome structure (Shelby et al., 1987) have also been reported.
D. Toxicity of MIC Metabolites in Rats and Mice Injections of 5 mmol/kg body weight of trimethylamine into mice from day 1 to day 17 of gestation caused maternal mortality, decreased fetal body weight, and fetal deaths; no such effect was observed with monomethylamine and
CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity dimethylamine, although all three methylamines were toxic to mouse embryos in culture (Guest and Varma, 1991). Also, trimethylamine inhibited macromolecular synthesis in embryos and caused neural tube defects (Guest et al., 1992, 1994). Interestingly, administration of trimethylamine into pregnant mice from day 6 to day 15 of gestation led to a selective decrease in the body weight of male fetuses and their growth for up to 8 weeks (Guest and Varma, 1993). S-(N-methylcarbamoyl) glutathione, a product generated by MIC (Pearson et al., 1990; Slatter et al., 1991), also caused a dose-dependent (0.1-2 mM) decrease of mouse embryo size in culture (Guest et al., 1992).
VII. C L I N I C A L T O X I C I T Y O F M E T H Y L
ISOCYANATE IN BHOPAL A. Mortality in Bhopal "The dead and dying arrived by the truckload, others came by rickshaw or were carried by relatives. For some the effort of the journey itself proved too much, and they died soon after arrival," wrote Sutcliffe (1985), a British medical student who arrived at Bhopal on the day before the accident to start her elective. By the early morning of December 3, nearly 50,000 patients had reached the three major hospitals. Soon, Bhopal became a city of death (Varma, 1986). The exact number of people who died within 48 hr of the accident is not known and perhaps will never be known. A conservative estimate puts the death toll at nearly 2500 people soon after the accident and more than 15,000 in the following days and months (Mehta et aL, 1990).
B. Pulmonary Toxicity Clinical evaluations by different investigators (Bhargava et al., 1987; Kamat et al., 1985, 1987, 1992; Misra and Nag, 1988; Misra et al., 1987; Patel et al., 1987; Sharma and Gaur, 1987) based on physical examination, review of patient charts, and radiological findings strongly suggest that the most common and severe problem in Bhopal was caused by damage to lungs. Predominant symptoms in the victims were cough, expectoration, chest pain, and breathlessness, and physical findings included necrotizing lesions in almost all parts of the respiratory tract, edematous lungs, consolidation, hemorrhage, bronchopneumonia, and bronchiolitis (Dhara and Dhara, 2002). Follow-up studies of 903 patients with radiological records found that approximately 25% had radiological and 39% had functional abnormalities 2-4 months after the disaster (Dhara and Dhara, 2002; Nemery, 1996). A survey conducted 10 years after the disaster also found respiratory symptoms in 81% of 474 exposed residents compared to 38% in a control cohort of 76 subjects (Cullinan et al., 1996). Spirometry of 74 subjects (Cullinan et al., 1997) revealed significant decreases
83
in 1-sec forced expiratory volume, forced vital capacity, and forced expiratory flow between 25 and 75%. These findings are in conformity with those reported by other investigations involving a sizable number of subjects (Kamat et al., 1992; Vijayan and Sankaran, 1996). Kamat et al. also noted a worsening of respiratory complications after a lag period of 1 year. Persistent airway hyperreactivity after a single exposure to a chemical irritant has been termed reactive airways dysfunction syndrome (RADS) (Brooks et al., 1985); whether or not Bhopal victims suffer from RADS needs to be established (Nemery, 1996). A study by Avashia et al. (1996) (Avashi was the medical director of the Union Carbide Institute at the West Virginia plant in 1984) concluded that prolonged low exposure of workers to MIC did not cause any pulmonary complications; the level of exposure was not quantified. However, this study has little bearing on the situation in Bhopal, in which the population was exposed to high concentrations of MIC. Indeed, there are reports that minor leaks of MIC were not uncommon in the Bhopal plant, which workers used to detect because of watering in their eyes (Varma, 1986). It would be of interest to locate workers who used to work in the Union Carbide plant and monitor their lung functions. The data of Avashia et al. (1996) reinforce a pharmacological principle that no chemical produces detectable toxicity at all doses.
C. Ocular Toxicity Irritation in the eyes and profuse lacrimation were the second most common symptoms in the Bhopal population; these were apparent even in areas farther from the plant, implying that irritation of the eyes can occur with much lower concentration than is needed for other complications. Major findings included ocular burning, watering, pain, and photophobia (Andersson et al., 1984, 1985, 1988; Dwivedi et al., 1985). Follow-up studies conducted 9 months to 2 years after the accident revealed persistent eye watering, itching, redness, photophobia, burning, Bitot spots, and even corneal opacity (Andersson et al., 1986, 1990; Khurrum and Ahmad, 1987; Raizada and Dwivedi, 1987). Given the poor living conditions of the majority of victims, the propensity for infection has been found to further complicate chronic ocular lesions (Crabb, 2004; Dhara and Dhara, 2002).
D. Reproductive Toxicity A survey involving 865 pregnant women from 3270 families within 1 km of the pesticide plant found that 43.8% did not deliver live babies; of the 486 live births, 14.2% died within 30 days, compared with an infant death rate of approximately 3% in the two preceding years (Varma, 1987, 1991). A study of 18,978 households also found that
84
S ECTI O N I 9Uses, A b u s e s , & Epidemioloooy
miscarriage rates were 23.6% in MIC-exposed area compared with 5.6% in an unaffected area of comparable sociological background (Bhandari et al., 1990). A decrease in fetal and placental weight following exposure to MIC in Bhopal has also been recorded (Kanhere et al., 1987).
E. Neurologic and Behavioral Toxicity Psychological problems resulting from the sheer horror of the accident could be anticipated and have been documented (Srinivasamurthy and Isaac, 1987). In addition, neuroses, anxiety state, and exacerbation of preexisting neurological problems have also been documented (Sethi et al., 1987). Kamat et al. (1992) did a follow-up of their original cohort of 113 subjects for up to 2 years and noted significant neurological problems, such as anxiety and depression, in a substantial number of subjects. Cognitive impairment in the MIC-exposed population 1 year after the accident has also been reported (Misra and Kalita, 1997).
F. Carcinogenic, Cytogenetic, and Immunotoxic Effects A significantly higher frequency of chromosomal aberrations (gaps, dicentrics, rings, and altered configurations) in MIC-exposed compared to a control population 3 years after the accident has been documented (Ghosh et al., 1990). Lymphocytes from gas-exposed victims revealed chromosomal aberrations (Goswami, 1986; Saxena et al., 1988). The carcinogenic potential of MIC is equivocal (Dikshit and Kanhere, 1999). Nevertheless, scientists in Bhopal anticipate a high rate of cancer in the coming years, as is the experience in smokers (Crabb, 2004). However, to relate an increase in cancer to MIC exposure would require follow-up of a large number of Bhopal residents and of people throughout India living under similar conditions. Anti-MIC antibodies belonging to IgG, IgM, and IgE classes have been detected in animals and Bhopal residents exposed to MIC (Karol and Kamat, 1988; Karol et al., 1987; Kamat et al., 1992). Although these data clearly indicate that a hapten as small as MIC interacted with some native protein to generate antibodies, the health implications of these findings need to be established; for example, could they cause RADS (Nemery, 1996)?
G. Effects on the Progeny of Victims Although long-term effects of the Bhopal disaster on different organ systems have received some attention as stated previously, there is much less information on the effect, if any, of exposure to MIC on the progeny. The long-term health monitoring program undertaken by the ICMR was terminated in 1994, and it is not known whether its mandate extends to the progeny of survivors. Anecdotal reports suggest a high rate of birth defects, such as syndactyly (fused
digits) and pigeon chest among the children of MICexposed parents (Crabb, 2004). Ranjan et al. (2003) conducted a study in the summer of 2001 on the growth patterns of boys and girls born before the disaster, soon after the disaster to exposed parents, or exposed to MIC in utero; the study included 104 families with 68 girls and 73 boys, with 71 of the adolescents exposed to the gases (mean age, 16.9 years) and 70 unexposed cohorts (mean age, 16.7 years). This study found that the height and mid-arm circumference of boys exposed to gases as infants or in utero or born to exposed parents were significantly less than those of comparable cohorts; in the case of boys exposed in utero or born to exposed parents, body weight, height, mid-arm circumference, as well as head circumference were less relative to control cohorts. The maximal decrease in height (13.5 cm) was for boys exposed in utero. No such effect on girls was noted in these studies. It is interesting that an earlier study in mice found that injections of MIC metabolite trimethylamine into pregnant mice caused selective retardation of male pups along with a decrease in serum testosterone (Guest and Varma, 1993). Preliminary unpublished data suggest that although the height of girls was not affected, they do have menstrual problems. The data of Ranjan et al. (2003) showing MIC exposure-linked growth retardation of boys need not be genetic. It would be of interest to follow the growth pattern of the next generation.
VIII. C O N C L U S I O N S The exposure of the Bhopal population to MIC caused thousands of deaths and long-term effects of varying severity in nearly 200,000 survivors. Although chemically induced acute respiratory distress syndrome was probably the cause of acute deaths, the magnitude and the underlying mechanisms of long-term effects with the exception of pulmonary complications have yet to be identified. Animal studies corroborate some of the clinical findings, but they neither offer an explanation nor disclose the underlying mechanism of long-term effects. Soon after the accident, the highly respected journal N a t u r e (Anonymous, 1984b) commented, "The anguish vividly carried round the world by the television cameras seems not to have matured into anger, even hysteria, there would have been had the accident occurred on the edge of a European city or in Connecticut [headquarters of Union Carbide]." Could this be the reason why the concern of the scientific community in the developed countries was short-lived? It is safe to say that the full dimension of the toxicity of a chemical (and also its therapeutic potential) cannot be predicted from its chemical structure but can be approximated by careful and painstaking studies. Such an enquiry into MIC would be well deserved not because it will disclose an astounding
CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity
rational therapeutic approach (as often demanded by activists of nongovernmental organizations, who fail to recognize that the treatment, for example, of obstructive lung disease is the same regardless of what led to it), but because it will be a fitting conclusion to the speculative assertions one way or another. Ten years after the Bhopal disaster, Dhara and Kriebel (1993) felt a need for a "sound epidemiology"; the demand is still valid 20 years later. Also, there is a merit to the demand for the state control of hazardous corporate operations (Varma and Varma, 2005) and global monitoring of potentially toxic material (Sriramachari and Chandra, 1997).
Acknowledgments The studies of the authors, cited herein, were supported by the Canadian Institutes of Health Research a n d its predecessor, Medical Research Council of Canada, and by the Faculty of Graduate Studies, McGill University. The studies in Bhopal were facilitated by volunteers and the Sambhavana Clinic.
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CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity Lee, C. K. (1976). Methylisocyanate as an antisickling agent and its reaction with hemoglobin S. J. Biol. Chem. 251, 6226-6231. Lepkowski, W. (1985, December 2), Bhopal, Indian city begins to heal but conflicts remain. Chem. Eng. News, 18-32. Le Quesne, P. M., Axford, A. T., McKerrow, C. B., and Parry, J. A. (1976). Neurological complications after a single severe exposure to toluene diisocyanate. Br. J. Ind. Med. 33, 72-78. Marwick, C. (1985). Bhopal tragedy's repercussions may reach American physicians. J. Am. Med. Assoc. 253, 2001-2013. Mehta, P. S., Mehta, A. S., Mehta, S. J., and Makhijani, A. B. (1990). Bhopal tragedy's health effects: A review of methyl isocyanate toxicity. J. Am. Med. Assoc. 264, 2781-2787. Misra, U. K., and Kalita, J. (1997). A study of cognitive functions in methyl isocyanate victims one year after Bhopal accident. Neurotoxicology 18, 381-386. Misra, U. K., and Nag, D. (1988). A clinical study of toxic gas poisoning in Bhopal, India. Indian J. Exp. Biol. 26, 201-204. Misra, N. P., Pathak, R., Gaur, K. J. B. S., Jain, S. C., Yesikar, S. S. Manoria, P. C., Sharma, K. N., Tripathi, B. M., Asthana, B. S., Trivedi, H. H., Sharma, V. K., Malhotr, Y., Verma, A., Bhargava, D. K., and Batni, G. (1987). Clinical profile of gas leak victims in acute phase after Bhopal episode. Indian J. Med. Res. 86 (Suppl.), 11-19. Morehouse, W., and Subramaniam, M. A. (1986). The Bhopal Tragedy: What Really Happened and What It Means for American Workers and Communities at Risk. Council of International & Public Affairs, New York. Munn, A. (1965). Hazards of isocyanates. Ann. Occup. Hyg. 8, 163-169. Nemery, B. (1996). Late consequences of accidental exposure to inhaled irritants: RADS and the Bhopal disaster. Eur. Respir. J. 9, 1973-1976. Nemery, B., Dinsdale, D., and Sparrow, S. (1985a). Methyl isocyanate: Thiosulphate does not protect. Lancet 2, 1245-1246. Nemery, B., Dinsdale, D., Sparrow, S., and Ray, D. E. (1985b). Effects of methyl isocyanate on the respiratory tract of rats. Br. J. Ind. Med. 42, 799-805. Nemery, B., Dinsdale, D., and Sparrow, S. (1987). The toxicity of inhaled methyl isocyanate in experimental animals: A review of studies published less than two years after the Bhopal disaster. Bull. Eur. Physiopathol. Respir. 23, 315-322. Patel, M. H., Kolhatkar, V. P., Potdar, V. P., Shekhavat, K. L., Shah, H. N., and Kamat, S. R. (1987). Methyl isocyanate survivors of Bhopal: Sequential flow volume loop changes observed in eighteen months' follow-up. Lung (India) 2, 59-65. Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D. H., Grillo, M. P., and Baillie, T. A. (1990). S-(N-methylcarbamoyl)glutathione: A reactive S-linked metabolite of methyl isocyanate. Biochem. Biophys. Res. Commun. 166, 245-250. Pearson, E G., Slatter, J. G., Rashed, M. S., Han, D. H., and Baillie, T. A. (1991). Carbamoylation of peptides and proteins in vitro by S-(N-methylcarbamoyl)GSH and S-(N-methylcarbamoyl)cysteine, two electrophilic S-linked conjugates of methyl isocyanate. Chem. Res. Toxicol. 4, 436-444. Raizada, J. K., and Dwivedi, E C. (1987). Chronic ocular lesions in Bhopal gas tragedy. Indian J. Ophthalmol. 35, 453-455. Ranjan, N., Sarangi, S., Padmanabhan, V. T., Holleran, S., Ramakrishnan, R., and Varma, D. R. (2003). Methyl isocyanate exposure and growth patterns of adolescents in Bhopal. J. Am. Med. Assoc. 290, 1856-1857.
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Rye, W. A. (1973). Human responses to isocyanate exposure. J. Occup. Med. 15, 306-307. Salmon, A. G., Kerr Muir, M., and Andersson, N. (1985). Acute toxicity of methyl isocyanate: A preliminary study of the doseresponse for eye and other effects. Br. J. Ind. Med. 42, 795-798. Saxena, A. K., Singh, K. P., Nagle, S. L., Gupta, B. N., Ray, P. K., Srivastav, R. K., Tewari, S. P., and Singh, R. (1988). Effect of exposure to toxic gas on the population of Bhopal. IV. Immunological and chromosomal studies. Indian J. Exp. Biol. 26, 173-176. Schwetz, B. A., Adkins, B., Harris, M., Moorman, M., and Sloane, R. (1987). Methyl isocyanate: Reproductive and developmental toxicology studies in Swiss mice. Environ. Health Perspect. 72, 149-152. Sethi, B. B., Sharma, M., Trivedi, J. K., and Singh, H. (1987). Psychiatric morbidity in patients attending clinics in gas affected areas of Bhopal. Indian J. Med. Res. 86(Suppl.), 45-50. Sharma, D. C. (2005). Bhopal: 20 years on. Lancet 365, 111-112. Sharma, P. N., and Gaur, K. J. B. S. (1987). Radiological spectrum of lung changes in gas-exposed victims. Indian J. Med. Res. 86(Suppl.), 39--44. Shelby, M. D., Allen, J. W., Caspary, W. J., Haworth, S., Ivett, J., Kligerman, A., Luke, C. A., Mason, J. M., Myhr, B., Tice, R. T., Valencia, R., and Zeiger, E. (1987). Results of in vitro and in vivo genetic toxicity tests on methyl isocyanate. Environ. Health Perspect. 72, 183-187. Slatter, J. G., Rashed, M. S., Pearson, P. G., Han, D. H., and Baillie, T. A. (1991). Biotransformation of methyl isocyanate in the rat: Evidence of GSH conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities. Chem. Res. Toxicol. 4, 157-161. Srinivasamurthy, R., and Isaac, M. K. (1987). Mental health needs of Bhopal disaster victims and training of medical officers in mental health aspects. Indian J. Med. Res. 86(Suppl.), 51-58. Sriramachari, S. (2004). The Bhopal gas tragedy: An environmental disaster. Curr. Sci. 86, 905-920. Sriramachari, S., and Chandra, H. (1997). The lessons of Bhopal [toxic] MIC gas disaster scope for expanding global biomonitoring and environmental specimen banking. Chemosphere 34, 2237-2250. Stevens, M. A., Fitzgerald, S., M6nache, M. G., Costa, D. L., and Bucher, J. R. (1987). Functional evidence of persistent airway obstruction in rats following a two-hour inhalation exposure to methyl isocyanate. Environ. Health Perspect. 72, 89-94. Sufrin, S. C. (1985). Bhopal: Its Setting, Responsibility and Challenge. Ajanta, New Delhi. Sutcliffe, M. (1985). My student elective: An eyewitness in Bhopal. Br. Med. J. 290, 1883-1884. Tepper, J. S., Wiester, M. J., Costa, D. L., Watkinson, W. P., and Weber, M. E (1987). Cardiopulmonary effects in awake rats four and six months after exposure to methyl isocyanate. Environ. Health Perspect. 72, 95-103. Tucker, A. N., Bucher, J. R., Germolec, D. R., Silver, M. T., Vore, S. J., and Luster, M. I. (1987). Immunological studies on mice exposed subacutely to methyl isocyanate. Environ. Health Perspect. 72, 139-141. Uraih, L. C., Talley, E A., Mitsumori, K., Gupta, B. N., Bucher J. R., and Boorman, G. A. (1987). Ultrastructural changes in the nasal mucosa of F344/N rats and B6C3F1 mice following an acute exposure to methyl isocyanate. Environ. Health Perspect. 72, 77-88.
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SECTION I 9 Uses, Abuses, & E p i d e m i o l o g y
Vardarajan, S., Doraiswamy, L. K., Ayyangar, N. R., Iyer C. S. E, Khan, A. A., Lahiri, A. K., Muzumdar, K. V., Mashelkar, R. A., Mitra, R. B., Nambiar, O . G. B., Ramachandran, V., Sahasrabudhe, V. D., Sivaram, S., Sriram, M., Thyagarajan, G., and Venkataraman, R. S. (1985). A Scientific Enquiry into the Methyl Isocyanate Leak in Bhopal. Council of Scientific and Industrial Research, New Delhi. Varma, D. R. (1986). Anatomy of the methyl isocyanate leak in Bhopal. In Hazard Assessment of Chemicals (J. Saxena, Ed.), pp. 233-289. Hemisphere, Washington, DC. Varma, D. R. (1987). Epidemiological and experimental studies on the effects of methyl isocyanate on the course of pregnancy. Environ. Health Perspect. 72, 153-157. Varma, D. R. (1989). Hydrogen cyanide and Bhopal. Lancet 2, 557-558. Varma, D. R. (1991). Pregnancy complications in Bhopal women exposed to methyl isocyanate vapor. J. Environ. Sci. Health A26, 1437-1447. Varma, D. R., and Guest, I. (1993). The Bhopal accident and methyl isocyanate toxicity. J. Toxicol. Environ. Health 40, 513-529.
Varma, D. R., Ferguson, J. S., and Alarie, Y. (1987). Reproductive toxicity of methyl isocyanate in mice. J. Toxicol. Environ. Health 21, 265-275. Varma, D. R., Ferguson, J. S., and Alarie, Y. (1988). Inhibition of methyl isocyanate toxicity in mice by starvation and dexamethasone but not by sodium thiosulfate, atropine and ethanol. J. Toxicol. Environ. Health 24, 93-101. Varma, D. R., Guest, I., Smith, S., and Mulay, S. (1990). Dissociation between maternal and fetal toxicity of methyl isocyanate in mice and rats. J. Toxicol. Environ. Health 30, 1-14. Varma, R., and Varma, D. R. (2005). The Bhopal disaster of 1984. Bull. Sci. Technol. Soc. 25, 37--45. Vijayan, V. K., and Sankaran, K. (1996). Relationship between lung inflammation, changes in lung function and severity of methyl isocyanate exposure in victims of the Bhopal tragedy. Eur. Respir. J. 9, 1997-1982. Weyel, D. A., Rodney, B. S., and Alarie, Y. (1982). Sensory irritation, pulmonary irritation and acute lethality of polymeric isocyanate and sensory irritation of 2,6-toluene diisocyanate. Toxicol. Appl. Pharmacol. 64, 423-430.
CHAPTER
Global Epidemiology of Organophosphate and Carbamate Poisonings TETSUO SATOH Chiba University, Chiba, Japan
I. I N T R O D U C T I O N
chronic OP-induced neuropsychiatric disorder. The first evidence of this type of syndrome (delayed psychopathologic-neurologic lesions) was reported by Spiegelberg (1963), who worked on the production and handling of highly toxic nerve gases in Germany during World War II. Using the characteristic symptomatology, patients could be classified into two distinct groups. The first and largest group was characterized by persistently lowered vitality and ambition; defective autonomic regulation leading to cephalalgia and gastrointestinal and cardiovascular symptoms; premature decline in potency and libido; intolerance to alcohol, nicotine, and various medicines; and an impression of premature aging. The second group, in addition to the previously mentioned symptoms, showed one or more of the following: depressive or subdepressive disorders of vital function, cerebral vegetative (syncopal) attacks, slight or moderate amnestic or demential effects, and slight organoneurologic defects. These symptoms developed and persisted for 5-10 years following exposure to these most toxic OPs during the war years. The controversial paper of Gershon and Shaw (1961), who performed a study of 16 cases of pesticide applicators exposed primarily to OP insecticides for 10-15 years, reported a wide range of persistent signs of toxicity (Ecobichon, 2001). Although the results of other studies have been equivocal in their support of such an array of long-term signs and symptoms, there is a persistent recurrence of the symptomatology in a number of anecdotal and documented reports (Ecobichon, 1994; Marrs, 1993; Jamal, 1997). This chapter describes the mechanism of toxicity of OPs, intoxication and fatality rates associated with OPs, clinical aspects of OP intoxication, treatment of poisoning, and epidemiological studies of OP poisoning throughout the world.
Organophosphorus compounds (OPs) are a diverse group of chemicals, including insecticides (malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, and others) and nerve gases (soman, satin, tabun, and VX). OPs were first synthesized in the early 1800s, and in 1934, Lange in Berlin and Schrader (a chemist at Bayer AG) in Germany investigated the use of OPs as insecticides. However, the German military prevented the use of OPs as insecticides and instead developed an arsenal of chemical warfare agents (i.e., tabun, sarin, and soman). In 1941, during World War II, OPs were reintroduced worldwide for agricultural use, as originally intended. Serious poisonings due to misuse of OP insecticides have been reported for more than four decades. OPs are some of the most widely used insecticides in the world, and the agents comprising these insecticides have a common mechanism of action. Although the structures are diverse in nature, the mechanisms by which the OP insecticides elicit their toxicity are identical and are associated with the inhibition of the nervous tissue acetylcholinesterase (ACHE) (Chambers and Levi, 1992). The classical picture of anticholinesterase (anti-ChE) insecticide intoxication, first described by DuBois (DuBois, 1948; DuBois et al., 1949), has become more complicated in recent years due to the recognition of additional and persistent signs of neurotoxicity not previously associated with these chemicals. First and frequently associated with exposure to high concentrations of the insecticides are effects that may persist for several months following exposure involving neurobehavioral, cognitive, and neuromuscular functions (Marrs, 1993; Ecobichon, 1994, 1998; Jamal, 1997). Jamal described this phenomenon as a Toxicology of Organophosphate and Carbamate Compounds
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S ECTI O N I 9Uses, A b u s e s , & E p i d e m i o l o g y
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II. M E C H A N I S M O F T O X I C A C T I O N S OF OPs
A. AChE Inhibition The primary mechanism of action of OP pesticides is inhibition of ACHE. AChE is an enzyme found in the central nervous system (CNS) and the peripheral nervous system, and its normal physiologic action is to metabolize acetylcholine (ACh), a neurotransmitter. OPs inactivate AChE by phosphorylating the serine hydroxyl group located at the active site of ACHE. The phosphorylation occurs by loss of an OP leaving group and establishment of a covalent bond with ACHE. Although anti-ChE insecticides have a common mode of action, there are significant differences between OP and carbamate (CM) insecticides. The reaction between OP and serine residue at the active site in AChE results in the formation of a transient intermediate complex that partially hydrolyzes with the loss of the Z substituent group, leaving a stable, phosphorylated, and largely unreactive inhibited enzyme that can be reactivated only at a very slow rate (Fig. 1). With many OP insecticides, an irreversibly inhibited enzyme is formed, and the signs and symptoms of intoxication are prolonged and persistent. Without intervention, toxicity will persist until sufficient quantities of "new" AChE are synthesized 20-30 days later to destroy efficiently the excess ACh. The nature of the substituent groups at X, Y, and Z plays an important role in the specificity for the enzyme. Introduced OP insecticides (acephate, temephos, dichlorvos, etc.) are less tenacious inhibitors of nervous tissue ACHE, with the phosphorylated enzyme being more readily and spontaneously dissociated. In contrast, CM insecticides, which attach to the reactive site of ACHE, undergo hydrolysis in two stages. The first stage is the removal of the X substituent (an aryl or
Organophosphorus Ester XO E-OH
+
\
YO /
p
0 #. \Z
xo ~
+
O II X O C - NHCH 3
XO =
E-OH
YO ~ +
Carbamate Ester E-OH
\
E-O-P=O
\
YO /
p
0 9 \ OH
ZH
O II ~- E- O - C - NHCH 3 +
+
~ E-OH
O It + H O - C - NHCH 3
XOH
FIG. 1. Inhibition of AChE by organophosphorus and carbamate insecticides. The interaction between an organophosphorus or carbamate ester with the serine hydroxyl groups in the active site of acetylcholinesterase (E-OH). The intermediate, unstable complexes formed before the release of the "leaving" groups (ZH and XOH) are not shown. The dephosphorylation or decarbamylation of the inhibited enzyme is the rate-limiting step in forming free enzymes. Reproduced with permission from Ecobichon (2001).
alkyl group) with the formation of a carbamylated enzyme. The second stage is the decarbamylation of the inhibited enzyme with the generation of free, active enzyme (Fig. 1). CMs are poor substrate for serine-containing enzymes such as ACHE. A number of OP esters (phosphate, phosphonate, and phosphoramidate), chemical warfare agents (sarin, soman, and tabun), and a few other compounds, such as DFP, mipafox, and leptophos, have the ability to bind strongly to the active site of AChE and neuropathy target esterase to produce an irreversibly inhibited enzyme by a mechanism known as aging. The aging process is dependent on the size and configuration of the alkyl (R) substituent, with the potency of the ester increasing in the order of diethyl, diisopropyl, and dibutyl for such analogs as DFP and mipafox (Aldridge and Johnson, 1971).
B. Pesticide-Induced Oxidative Stress Abdollahi et al. (2004) reviewed the oxidative stress caused by pesticides. The widespread use of pesticides in public health and agricultural programs has caused severe environmental pollution and health hazards, including cases of severe acute and chronic human poisoning (Ellenhom et al., 1997; Abdollahi et al., 1997; Jalali et al., 2000; Pajoumand et al., 2002). The toxic pesticides have become an integral part of the ecosystem, although many of them are extremely toxic to mammals and other nontarget creatures. However, the implications of pesticide residues for human health have yet to be comprehensively documented. Free radicals play an important role in the toxicity of pesticides and environmental chemicals. Pesticides may induce oxidative stress by excess generation of free radicals, especially reactive oxygen species and reactive nitrogen species, and alteration in antioxidants and the scavenging system, causing lipid peroxidation (Banerjee et al., 1999; Dettbarn et al., 2001; Gupta et al., 2001a,b, 2002; Etemadi-Aleagha et al., 2002). The toxicology of various pesticides is noted in Table 1. Several studies have demonstrated oxidative stress induced by OPs in rats (Gultekin, 2000; Gupta et al., 2001a,b; Verma, 2001; Akhgari et al., 2003) and humans (Banerjee et al., 1999; Ranjbar et al., 2002; Dantoine et al., 2003). Lipid peroxidation is also evident in rat brains (Verma, 2001) and human erythrocytes (Gultekin, 2000; Dantoine et al., 2003). OP-induced seizures have been reported in association with oxidative stress (Gupta et al., 2001a,b). It has also been shown that the acute tubular necrosis that accompanies OP toxicity is related to reactive oxygen species and lipid peroxidation (Poovala et al., 1999). Gupta (2004) reviewed the importance of brain regional heterogeneity in relation to cholinergic and noncholinergic (oxidants/antioxidants) determinants, with particular reference to OP and CM pesticides and OP nerve agents.
CHAPTER 8 9Epidemiology of OP and CM Poisonings
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TABLE 1. Toxicological Characteristics of Organophosphate Poisoninga
Examples Diazinon, malathion, parathion, chlorpyrifos, dichlorvos
Site of toxicity
Major acute symptoms
Irreversible inhibition of red blood cell cholinesterase, acetylcholinesterase, plasma cholinesterase
Mild: fatigue, headache, blurred vision, dizziness, numbness of extremities, nausea, vomiting, excessive sweating and salivation, tightness in chest Moderate: weakness, difficulty talking, muscular fasciculations, miosis Severe: unconsciousness, flaccid paralysis, moist rales, respiratory difficulty, cardiac arrhythmias, cyanosis
aAdapted from Ellenhornet al. (1997). III. I N T O X I C A T I O N A N D F A T A L I T Y R A T E S
A. Mortality and Morbidity Worldwide mortality, studies report mortality rates of 3-25%. The compounds involved most frequently are malathion, dichlorvos, trichlorfon, and fenthion/malathion. Mortality rates depend on the type of compound used, amount ingested, general health of the patient, delay in discovery and transport, insufficient respiratory management, delay in intubation, and failure to wean off ventilatory support. Signs and symptoms of OP poisoning can be divided into three broad categories, including: muscarinic effects, nicotinic effects, and CNS effects. Complications include respiratory distress, seizures, and aspiration pneumonia. Respiratory failure is the most common cause of death.
B. Age Emerson e t al. (1999) reported that men aged 30-50 years were more likely to attempt suicide with OPs. In the study, 68 of 69 patients were men. Agarwal (1993) found that most OP poisonings occur in patients aged 21-30 years. The male-to-female ratio in the study was 2.1:1. Both Emerson et al. and Agarwal reported that accidental poisoning was more likely in children than in adults.
IV. C L I N I C A L A S P E C T S OF OP INTOXICATION
A. Physical Furtado and Chan (2004) reported that as the vital signs showing depressed respiratory rate, bradycardia, and hypotension are common. Hypothermia also can be observed. Paralysis due to OP intoxication is categorized into three types. Type I involves acute paralysis secondary to persistent depolarization at the neuromuscular junction. As the type II intermediate syndrome, was described in 1974, with an
incidence of 8-49%. It develops 24-96 hr after resolution of acute commonly cholinergic poisoning symptoms and manifests as paralysis and respiratory distress. This syndrome involves proximal muscle groups, with relative sparing of distal nuscle groups. Various degrees of cranial nerve palsies are also involved. Neuromuscular transmission defect and toxin-induced muscular instability play a role in intermediate syndrome. Intermediate syndrome persists for 4-18 days, can require intubation, and can be complicated by infections or cardiac arrhythmias. Type III involves OP-induced delayed polyneuropathy (OPIDN), occurs 2 or 3 weeks after exposure to large doses of certain OPs. Distal muscle weakness with relative sparing of the neck muscles, cranial nerves, and proximal muscle groups characterize OPIDN, and recovery can take up to 12 months. Neuropsychiatric effects associated with chronic OP intoxication include impaired memory, confusion, irritability, lethargy, and psychosis. Ophthalmic effects due to direct ocular exposure to OPs include optic neuropathy, retinal degeneration, defective vertical smooth pursuit, myopia, and miosis. Respiratory effects, including muscarinic, nicotinic, and central effects, contribute to respiratory distress in acute and delayed OP toxicity. Muscarinic effects, such as bronchospasm and laryngeal spasm, can lead to airway obstruction. Nicotinic effects can lead to weakness and paralysis of respiratory oropharyngeal muscles. Central effects can lead to cessation of respiration. Rhythm abnormalities include sinus tachycardia, sinus bradycardia, extrasystoles, atrial fibrillation, ventricular tachycardia, and fibrillation. Other cardiovascular effects include hypertension, hypotension, and noncardiogenic pulmonary edema. Gastrointestinal manifestations such as nausea, vomiting, diarrhea, and abdominal pain are the first to occur after OP exposure.
B. Cholinesterase Inhibition as an Extensively Used Biomarker Laboratory diagnosis of OP poisoning is based on the measurement of cholinesterase activity. Both erythrocyte and plasma cholinesterase levels can be used. Urinary
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S E CTI O N I 9 Uses, Abuses, & Epidemiology
p-nitrophenol can be measured in parathion poisoning. Depressed cholinesterase levels only confirm the diagnosis of OP poisoning retrospectively. Measurement of red blood cell (RBC) and plasma ChE levels prior to treatment with 2PAM is of immense value. RBC AChE represents the AChE found in CNS gray matter, RBCs, and peripheral nerve, tissue, muscle, and brain. Plasma ChE is synthesized in the liver and eventually circulates in the blood plasma. It is found in the CNS white matter, pancreas, and heart. RBCChE is the more accurate of the two measurements, but plasma ChE is easier to assay and is more widely available. Mild poisoning is defined as depression of ChE activity to 20-50% of normal. Moderate poisoning occurs when activity is 10-20% of normal. Severe poisoning occurs at less than 10% of ChE activity. Small short-term exposures can depress ChE activity to very low levels with minimal symptoms. Levels do not always correlate with clinical illness. The level of ChE activity is relative and is based on population estimates. Neonates and infants are baseline level that are lower than those in adults. Because most patients do not know their baseline level, the diagnosis can be confirmed by observing a progressive increase in the ChE value until it plateaus over time. Falsely depressed levels of plasma ChE are observed in cases of liver dysfunction (e.g., cirrhosis), low protein conditions (e.g., malnutrition), neoplasia, and infectious hypersensitivity reactions. In addition, the use of drugs such as succinylcholine, codeine, and morphine renders falsely depressed plasma ChE levels. The first and second trimesters of pregnancy and genetic deficiency of plasma ChE are other causes. Other laboratory findings include leukocytosis with a normal differential consistent with a stress reaction, increased hematocrit from hemoconcentration due to fluid losses, anion gap acidosis due to poor tissue perfusion and hyperglycemia with hypokalemia, and hypomagnesemia due to catecholamine excess. Electrocardiogram (ECG) findings include prolonged QTc interval (most commonly up to 67%), elevated ST segments, inverted T waves, and prolonged PR interval (Chung et al., 1996).
TABLE 2.
C. Extremely Sensitive B i o m a r k e r of OP Exposure A novel biomarker of OP insecticide exposure, egasyn, is an accessory protein of [3-glucuronidase ([3G) in the liver microsomes (Swank and Paigen, 1973). Medda et al. (1987) reported that egasyn has esterase activity and it is one of the carboxylesterase (CarbE) isozymes. Later, Hosokawa and coworkers (1987, 1990) purified three CarbE isozymes (RL 1, RL2, and RH1) from rat liver microsomes, and egasyn was identified as RL2 isozyme. In 1998, Satoh and Hosokawa proposed a new classification of CarbE isozymes based on the molecular homology of the individual esterase isozymes. Figure 2 summarizes the localization of the egasyn-[3G complex and dissociation and release of [3G from liver microsomes into plasma. When animals as well as humans are intoxicated with OP, the OP is incorporated into the liver microsomes and metabolized to form the corresponding oxon. In order to study the release of [3G from the liver to plasma, rats and hamsters were treated with bis-p-nitrophenylphosphate, which is a specific CarbE inhibitor, and plasma [3G activities were determined. As shown in Table 2, plasma [3G activities
FIG. 2. Schematic representation of the release of [3-glucuronidase ([3G) from hepatocytes to blood by OP administration in animals. EG, egasyn; OP, organophosphate; ER, endoplasmic reticulum.
Changes in 13-Giucuronidase Activity after Administration of Insecticides in Ratsa
Time after administration (hr) Insecticide (mg/kg) EPN (30) Fenitrothion (100) Phenthoate (8.0) Carbaryl (200) Phenothrin (100) Allethrin (100)
3 3.42 4.22 0.15 4.56 0.042 0.050
__+0.43** (114.0) b _____0.51"* (114.0) _____0.07 (5.1) _____0.50** (152.0) __+0.005 (1.4) __+0.006 (1.6)
5 4.16 3.81 0.60 3.32 0.051 0.060
+ 0.51"* (138.6) • 0.42** (117.0) + 0.09* (20.1) __+0.53** (110.5) +__0.005 (1.7) __+0.006 (1.6)
24 0.05 0.04 0.04 0.03 0.03 0.040
_ 0.006 _ 0.007 _ 0.006 --- 0.007 +- 0.006 _ 0.006
(1.5) (1.3) (1.3) (1.0) (1.0) (1.3)
aNormal value of plasma [3-glucuronidase activity for control rats is 0.034 ___0.02 unit/ml. bNumbers in parentheses indicate the fold change from nontreated rats. Significantly different from nontreated rats: *p < 0.05, **p < 0.01.
CHAPTER 8 9Epidemiology of OP and CM Poisonings TABLE 3. Inhibition of Plasma Cholinesterase and Increase in Plasma 13-Glucuronidase after EPN Treatment in Ratsa
Dose (mg/kg) None 1.0 5.0 10.0 30.0
Plasma cholinesterase activity (unit/liter)
Plasma 13-glucuronidase activity (unit/ml)
29.5 + 3.66 (1.00)b 31.1 _+ 4.23 (1.05) 20.0 _+ 3.41 (0.65) 18.4 + 2.20* (0.62) 14.9 + 2.48* (0.50)
0.03 + 0.01 (1.00) 0.95 _+ 0.10" (31.7) 2.89 + 0.54** (96.3) 3.46 _+ 0.55** (115.3) 4.51 + 0.37** (150.3)
aAnimals were sacrificed 5 hr after EPN administration.Valuesrepresent mean __+SE from three to five rats. bNumbers in parentheses indicate the fold change from nontreated rats. Significantly different from nontreatedrats: *p < 0.05,**p< 0.01.
were increased after oral administration of EPN, fenitrothion, and carbaryl, but not pyrethroids, in a timedependent manner and reached maximum at 2.5 hr after administration. To compare the extent of plasma ChE inhibition, which is widely used as the marker of OP intoxication, and increase in plasma [3G activities, rats were treated with EPN and sacrificed 5 hr later. As shown in Table 3, plasma ChE activity was decreased to approximately 50% of the normal level after EPN administration, whereas plasma [3G activity was significantly increased approximately 150 times that of control. This indicates that the increase in plasma [3G activity is much more susceptible to exposure to OP insecticides than ChE inhibition. A similar increase in plasma [3G activities is observed in carbaryl intoxication but not in the case of pyrethroids. The reason for insensitivity of pyrethroids to OP seems to be due to the rapid metabolism by the enzyme to form the inactive metabolites. In summary, the increase in blood [3G activity after OP exposure is a much more sensitive and rapid biomarker than blood ChE inhibition.
V. T R E A T M E N T
OF POISONING
In order to secure the patient's airway, intubation is necessary in cases of respiratory distress from laryngospasm, bronchospasm, or severe bronchorrhea. Regular monitoring of neck muscle weakness, respiratory rate, arterial blood gas, and mental status is required to assess progression or decompensation. The tidal volume initiated by the patient can be used as a measure of disease severity in those who are intubated. Administration of atropine should be withheld until a cardiac monitor and a defibrillator are in place and the patient's airway is secured. Atropine can precipitate ventricular fibrillation in hypoxic patients. Continuous cardiac monitoring and an ECG are necessary. Electrical pacing is the treatment
93
of choice for ventricular tachycardia associated with a prolonged QTc. Atropine can reverse some cardiac manifestations. Electrolyte abnormalities may cause dysarrhythmias. Health care providers must avoid contaminating themselves while handling patients. Personal protective equipment, such as neoprene or nitrile gloves and gowns, should be used when decontaminating patients because hydrocarbons can penetrate nonpolar substances, such as latex and vinyl. Charcoal cartridge masks should be used for respiratory protection when decontaminating patients. The eyes of patients should be irrigated with ocular exposures using isotonic sodium chloride solution or lactated Ringer's solution. Morgan lenses can be used for eye irrigation. Activated charcoal (0.5-1gq4h) is used for gastric decontamination. Sorbitol can be used; however, many patients have increased gastrointestinal motility following OP poisoning. The mainstay of medical therapy in OP poisoning is atropine or glycopyrrolate 2-PAM, and diazepam which can be used for seizure control. In 1992, De Silva and Wijewickrema studied the treatment of OP poisoning with atropine and 2-PAM and, later in the same year, with atropine alone. They found that atropine seemed to be as effective as atropine plus 2-PAM in the treatment of acute OP poisoning. The controversy continued when other authors observed more respiratory complications and higher mortality rates with the use of high-dose 2-PAM. Low dose (1-2 g) 2-PAM is the current recommendation.
VI. E P I D E M I O L O G I C A L STUDIES OF OP-INDUCED POISONING More than 1.5 million tons of pesticides is manufactured every year. Most pesticides are hazardous. Extremely hazardous pesticides are easily available in many developing countries, and pesticide poisoning remains a serious problem worldwide. Internationally, pesticide poisonings are the most common mode of suicide in some developing countries (e.g., Sri Lanka). The World Health Organization (1999) estimated that more than 500,000 people died from self-harm in Southeast Asia and the west Pacific during 2000, and pesticides are among the most important methods of self-harm (Eddleston et al., 2002). In addition, 3 million pesticide poisoning cases occur worldwide every year, with 220,000 deaths, most of which are intentional.
A. India and Australia In a study of OP poisoning in India, Agarwal (1993) found that 67.4% of patients had suicidal intentions, 16.8% of the poisonings were caused by occupational exposures, and 15.8% of patients were poisoned accidentally. An
S E CTI O N I 9 Uses, Abuses, & Epidemiolooay
94
3.9% o f p o i s o n i n g s are due to insecticides. A c c o r d i n g to the E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , cases r e p o r t e d in C a l i f o r n i a w i t h d o c u m e n t e d pesticide e x p o s u r e h a v e b e e n
A u s t r a l i a n study o f O P p o i s o n i n g p e r f o r m e d b y E m e r s o n et al. ( 1 9 9 9 ) f o u n d that o n l y 3 6 % o f patients h a d suicidal intentions c o m p a r e d to 6 5 - 7 5 % in d e v e l o p i n g countries.
p u b l i s h e d (Table 4).
B. U n i t e d S t a t e s C. J a p a n
The American Association of Poison Control Centers' " N a t i o n a l I n c i d e n c e R e p o r t " indicates that pesticide injuries r a n g e f r o m 7 0 , 0 0 0 to 80,000 annually. N a t i o n w i d e ,
T A B L E 4.
T h e National R e s e a r c h Institute o f Police Sciences ( N R I P S ) has published the annual report o f the epidemiological
Cases Reported in California with D o c u m e n t e d Pesticide Exposure S u m m a r i z e d by the Type of Illness and the Type of Pesticide, 2 0 0 2 a
Antimicrobials c
Type of illness b
Occupational d
Cholinesterase inhibitors c
Nonoccupational d
Occupational
Nonoccupational
Other pesticides c
Occupational
Nonoccupational
Total
Systemic Systemic with respiratory and topical effects
27
3
11
1
27
56
125
Systemic with respiratory effects
27
26
17
13
27
32
142
Systemic with topical effects
7
0
20
6
50
37
120
Systemic only
7
17
44
29
54
54
205
Respiratory with topical effects
18
9
4
1
18
52
102
Respiratory only
31
51
3
5
10
19
119
Eye only
94
14
10
3
198
85
404
Skin only
40
2
10
2
27
4
85
2
1
3
0
7
1
14
2
4
21
10
8
64
109
255
127
143
70
426
404
1425
Respiratory
Topical
Eye and skin
Asymptomatic Asymptomatic
Total
aData from the California Department of Pesticide Regulation, Pesticide Illness Surveillance Program (www.cdpr.ca.gov). Documented pesticide exposure includes cases classified as definitely, probably, or possibly related to pesticide exposure as well as cases of documented exposure that did not lead to symptomatology. Definite: High degree of correlation between the pattern of exposure and resulting symptomatology. Requires both medical evidence (such as measured cholinesterase inhibition, positive allergy tests, and characteristic signs observed by medical professional) and physical evidence. Probable: Relatively high degree of correlation exists between the pattern of exposure and the resulting symptomatology. Either medical or physical evidence is inconclusive or unavailable. Possible: Some degree of correlation evident. Medical and physical evidence is inconclusive or unavailable. bCategorization of the types of symptoms experienced. Systemic: Any health effects not limited to the respiratory, skin, and/or eye. Cases involving multiple illness symptom types including systemic symptoms are included in the systemic category. Respiratory: Health effects involving any part of the respiratory tree. Topical: Health effects involving only the eyes and/or skin. This excludes outward physical signs (miosis and lacrimation) related to effects on internal bodily systems. These signs are classified under Systemic. Asymptomatic: Exposure occurred but did not result in illness/injury. Cholinesterase depression without symptoms falls in this category. CType of pesticide is based on functional class. Antimicrobials: Pesticides used to kill or inactivate microbiological organisms (bacteria, viruses, etc.). Cholinesterase inhibitors: Pesticides known to inhibit the function of the cholinesterase enzyme. Other pesticides: Any pesticide that is not an antimicrobial or cholinesterase-inhibiting pesticide. dOccupational or nonoccupational: The relationship between the illness/injury and the individual's work. Occupational: Work related. The individual was on the job at the time of the incident. This includes both paid employees and volunteers working in a similar capacity as paid employees. Nonoccupational: Not work related. The individual was not on the job at the time of the incident. This category includes individuals on the way to or from work (before the start or after the end of their workday).
CHAPTER 8 9Epidemiology of OP and CM Poisonings
FIG. 3.
The incidence of various toxicants in Japan.
studies on criminal and suicidal events. The toxicants studied are carbon monoxide, cyanide and its derivatives, medicines, alkaloids, and pesticides, including paraquat and miscellaneous. As shown in Fig. 3, the numbers of the incidence of pesticide poisoning are approximately 22-30% of the total incidences between 1991 and 1996. The incidence of pesticide poisoning has been gradually declined during this time. This seems to be influenced by the decrease in the incidence of paraquat poisoning, which has extremely high toxicity. According to the NRIPS, there are many more cases of OP intoxication of OP than CM (Table 5). D. T a i w a n
Unlike the prevalence of drug poisoning exposures in most Western countries, pesticide poisoning exposures are numerous in Taiwan. Epidemiological studies of the
TABLE 5.
Mortality Rate of OP-Intoxicated Patients in Japan a Year
Insecticide
2000
2001
Organophosphate Malathion DDVP MEP Miscellaneous b Carbamates c Paraquat Miscellaneous d
31 39 40 127 129 293 74
37 27 42 123 105 216 55
aData from the National Research Institute of Police Science, Japan (2002) (www.nrips.go.jp). 9Fenitrothion, glyphosate,EPN, dipterex, diazinone, and acephate. CMethomyland others. dChlorpicrine and others.
95
National Poison Center in Taiwan indicate that the fatality rate is much higher in Taiwan than in most Western countries, with 5.65% of all poisoning exposures resulting in death. There were 6872 cases of pesticide intoxication among 223,436 total cases of various intoxications from 1985 to 1993 (Table 6), and these accounted for approximately 22-30% of the total incidences, from 1991 to 1996. The incidence of pesticide poisoning gradually declined during this time. Extremely toxic pesticides are readily available and pesticide poisoning is common. OP insecticides (n = 1854) were the most common offending agents, followed by the OP herbicide glyphosate (n = 983), pyrethrins and pyrethroids (n = 936), paraquat (n = 892), and CM insecticides (n -- 721). There were a total of 1325 fatalities (19.3%). Pesticide poisonings (n = 875) accounted for 60% of all fatalities during the same study period. Among them, paraquat was the leading cause of death (n = 485, 54.4%). As shown in Table 7, paraquat is the leading cause of death in Taiwan, followed by the OP herbicide glyphosate, amphetamine, and CMs, most of which are pesticides. Insecticides (5217 patients, 46.3%) accounted for nearly half of the poisoning exposures. In Taiwan, 11,269 patients were intoxicated with acute pesticide poisoning, and male patients (63.9%) outnumber female patients. After adjusting for measured baseline characteristics, gender is not associated with the risk of death among patients with acute pesticide poisoning' OPs were more toxic than CMs. Suicide (7511 patients, 66.7%) was the most common intent of exposure. The oral route (8930 patients, 79.2%) was the most common route of exposure. Table 8 shows increased age, suicide attempt, oral exposure, and concurrent exposure with a higher risk of fatality. In terms of age differences, people 19-39 years old comprised the largest number of injured patients. Acute pesticide poisoning mainly involved adults, and the mean age of patients was 43.3 _ 18.9 years. Acute pesticide poisoning was associated with a very high case fatality rate (14.5%), and there was a wide range of differences in clinical severity among various pesticides. E. T h a i l a n d
Poisoning is considered a serious problem in Thailand. According to the Division of Epidemiology of the Ministry of Public Health, overall morbidity due to poisoning from 1990 to 1995 was 30 per 100,000 population, with 300 deaths per year. The morbidity rate of reported pesticide poisoning in the country was 10 per 100,000 population. The National Environmental Board reported that there were 380 deaths of 5458 reported cases of pesticide poisoning in 1985. The incidence of poisoning at Ramathibodi Hospital, one of three medical schools in
SECTION I 9 Uses, Abuses, & Epidemioloooy
96
TABLE 6.
Distribution of Reported Human Poisoning Exposures to Poison Control Center in Taiwan, 1985-1993 Children
TABLE 7. Substance
Adults
Total
Category
No.
%
No.
%
No.
%
Pesticides Drugs Cleaning substances Solvents Animal bites and stings Rodenticides Cosmetics Insect repellents Chinese herds CO and toxic gases Food-borne toxins Hydrocarbon Plants Miscellaneous
633 1886 410 285 172 148 246 251 89 89 49 63 1376
10.9 32.4 7.1 4.9 3.0 2.5 4.2 4.3 1.9 1.5 1.5 0.8 1.1 23.7
6239 4874 1196 736 781 658 383 257 319 190 190 168 126 1366
35.4 27.7 6.7 4.2 4.4 3.7 2.2 1.5 1.8 1.1 1.1 1.0 0.7 7.8
6872 6760 1606 1021 953 806 629 508 430 279 279 217 189 2742
29.3 28.8 6.9 4.4 4.1 3.4 2.7 2.2 1.8 1.2 1.2 0.9 0.8 11.7
Total
5812
100.0
17624
100.0
223436
100.0
Clinical Severity by Pesticides and Fungicides among 11,269 Patients with Acute Poisoning in Taiwan Symptomless, No. (%)
Mild, No. (%)
Moderate, No. (%)
Severe, No. (%)
Death, No. (%)
Total, No. (%)
Paraquat Glyphosates Other herbicides Organophosphates Carbamates Pyrethrin and pyrethroids Mixed insecticides Other insecticides Fungicides Others
71 (5.3) 178 (10.3) 116 (15.3) 111 (4.2) 33 (4.3) 147 (11.3)
259 (19.3) 1168 (67.4) 508 (67.1) 1293 (49.2) 483 (63.6) 906 (69.4)
144 (10.7) 224 (12.9) 69 (9.1) 547 (20.8) 128 (16.8) 156 (12.0)
43 49 22 335 54 55
(3.2) (2.8) (2.9) (12.7) (7.1) (4.2)
825 (61.5) 115 (6.6) 42 (5.6) 344 (13.1) 62 (8.2) 41 (3.1)
1342 (11.9) 1734 (15.4) 757 (6.7) 2630 (23.3) 760 (6.7) 1305 (11.6)
8 (5.0) 52 (14.3) 106 (13.9) 161 (11.1)
69 (43.4) 236 (65.0) 526 (68.8) 907 (62.4)
23 (14.5) 45 (12.4) 82 (10.7) 157 (10.8)
13 (8.2) 16 (4.4) 15 (2.0) 116 (8.0)
46 (28.9) 14 (3.9) 36 (4.7) 113 (7.8)
159 (1.4) 363 (3.2) 765 (6.8) 1454 (12.7)
Total
983 (8.7)
6355 (56.4)
1575 (14.0)
718 (6.4)
1638 (14.5)
Bangkok, was 200 cases per year, which accounted for 4.1% of patients in the medical ward. At one provincial hospital at Nakornrachsima, the medical in-patient admission rate for OP poisoning alone was 2.8%, and OP poisoning was ranked the eighth most common illness requiring hospitalization. The Ramathibodi Poison Center (RPC) was established in 1996 under the auspices of the Faculty of Medicine, Ramathibodi Hospital. In the year of 2000, the RPC Toxic Exposure Surveillance System was supported by the Royal
11269
Thai Government W H O Collaboration Program. The system collects human exposures having consultation to RPC. The definition of terms in the system is adopted or modified from the IPCS INTOX data management system and the American Association of Poison Control Center data collection system. According to the RPC, in 2001, the total number of pesticide poisonings was 834. Insecticide exposure was 50.2% of all pesticide exposures, followed by herbicide (20.9%), rodenticide (15.5%), and miticide 6.7% (Table 9).
CHAPTER 8 9Epidemiology of OP and CM Poisonings TABLE 8.
Age Distribution of Fatal and Nonfatal Cases with Acute Pesticide Poisoning in Taiwan No. of fatal cases (n = 1638) (%)
No. of nonfatal cases (n = 9631) (%)
Age (years)
~18 19-29 30-39 40-49 50-59 60-69 -->70 Unknown
64 (8.2) 327 (15.9) 271 (12.3) 209 (12.4) 239 (13.8) 233 (15.4) 259 (23.3) 36 (19.2)
716 (91.8) 1725 (84.1) 1926 (87.7) 1481 (87.6) 1497 (86.2) 1283 (84.6) 851 (76.7) 152 (80.9)
Oral 1552 (17.4) Inhalation 22 (1.5) Skin/mucous membrane 18 (1.2) Other/unknown 63 (26.9)
7378 (82.6) 1473 (98.5) 1439 (98.8) 171 (73.1)
Route of exposure
Chemicals used as miticides were usually similar to insecticides. Among the 653 cases of insecticide exposure, the most common ones were exposure to CMs (25.7%), OPs (29.3%), and pyrethroids (19.1%). Products that were a combination of two or three-insecticides accounted for 9.5%. Organochlorine exposure was 6.6%. In herbicide exposure, paraquat, Chloroacetanilide, and chlorophenoxy compounds comprised 24.4, 8.4, and 9.3% respectively. In terms of final medical outcome after insecticide exposures, 714 of 821 patients (87%) were followed until completion; 92 of 821 patients (11.2%) died, 7.3% developed major effect, 4% developed moderate effect, 34.1% had only minimum effect, and 30.3% did not have any effect. Paraquat, methomyl, endosulfan, methyl parathion, and monocrotophos were the major substances responsible for fatality in this group.
VII. S A R I N V I C T I M S IN T O K Y O On March 20, 1995, a terrorist attack using sarin occurred on the Tokyo subway. The sarin was mixed in organic solvent and vaporized in the closed compartment of a train. Many people inhaled the sarin gas and collapsed. Eventually, 12 people died and more than 5000 were injured (Suzuki et al., 1995; Masuda et al., 1995; Nozaki et al., 1995). However, neither sarin nor its hydrolysis products were detected in the blood of almost all the sarin victims. It was concluded that satin passed through the blood-brain barrier and became distributed in brain tissue. These facts suggest that various brain
97
regions may show differing degrees of vulnerability to it. In 1998, Masuda and associates performed judicial autopsies on four sarin victims, and they detected sarin hydrolysis products bound to AChE on the erythrocyte membranes of all four victims. The plasma ChE activity of patients 2-4 was extremely low compared to the average activity of normal samples. The AChE activity in the brain cortices and blood of normal control subjects was 110.0 _ 8.1 U/g wet tissue and 5.00 ___ 1.20 U/ml, respectively. The brain AChE activity of all the victims was significantly lower than that of normal subjects. The AChE activity in the blood of patients 1 and 2 was low (89.8 and 90.0% decrease, respectively), whereas that of patients 3 and 4, who received PAM in the hospital, had partly recovered (32.8 and 24.4% decrease, respectively). In 1998, Masuda et al. attempted to detect the sarin hydrolysis products in the cerebellums of the victims, which had been stored in formalin fixative for 2 years. Sarin-bound AChE was solubilized~ purified by immunoaffinity chromatography, and digested with trypsin. The sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion and detected by gas chromatography mass spectrometry. Using these procedures, methylphosphonic acid was detected. However, no isopropyl methylphosphonic acid was detected in the formalin-fixed cerebellums of the four sarin victims for approximately 2 years, probably because the isopropoxy group of sarin underwent chemical hydrolysis during storage. The procedureS used here are useful for the forensic diagnosis of poisoning by protein-bound, highly toxic agents such as sarin.
VIII. C O N C L U S I O N S OPs cause serious intoxication in both acute and chronic exposure. The epidemiological data in Taiwan and Thailand show that paraquat intoxication causes the most serious irreversible damage in patients. Although the mortality rate slightly decreased during the past decade, pesticide poisoning is still associated with a high mortality rate in Taiwan. Legislation to control the availability of extremely hazardous pesticides and further innovation in "effective" therapeutic measures are required to reduce the high mortality rate that accompanies acute pesticide poisoning. Increased use of a poison control center service may be helpful in reducing the risk of pesticide-related fatalities. In the 1995, 12 people died by terrorist attack using sarin, one of the most toxic OPs on the Tokyo subway, 12 people died. This is an example of acute intoxication of OP, in which some people are still hospitalized. Plasma 13G activity is a novel biomarker of OP exposure. Plasma 13G is rapidly and significantly increased after OP exposure, and this is much more sensitive to OP exposure than ChE inhibition.
98
SECTION I 9 Uses, Abuses, & E p i d e m i o l o g y TABLE.9
The most common of pesticides related in human poison exposure categorized by major group classification
Major/subgroup classification
1. Insecticide Carbamate Organophosphate Pyrethroid Combined Organochlorine Repellant Others Unknown 2. Herbicide Glycine Bipyridyl Others Chloroacetanilide Chlorophenoxy Unknown 3. Rodenticide Zinc phosphide Wafarin Long-acting anticoagulant Unknown 4. Miticide Combined Carbamate Pyrethroid Unknown Arsenic troxide Organochlorine Organophosphate 5. Plant hormone & growth regulator 6. Acaricide 7. Mollusicide 8. Poison dog 9. Fungicide 10. Synergist 11. Poison bird 12. Unknown pesticide Total
Number
419 102 86 92 57 30 11 17 24 174 74 41 22 18 16 3 129 51 66 5 7 56 23 12 10 6 2 2 1 19 12 8 5 5 2 2 3 834
%
50.2 24.3 20.5 22 13.6 7.2 2.6 4.1 5.7 20.9 42.5 23.6 12.6 10.3 9.2 1.7 15.5 39.5 51.2 3.9 5.4 6.7 41.1 21.4 17.9 10.7 3.6 3.6 1.8 2.3 1.4 1 0.6 0.6 0.2 0.2 0.4
Most common products or substances
Methomyl (63 events), Carbofuran (19 events) Parathion methyl (44 events), Methamidophos (10 events) Cypermethrin (36 events) Endosulfan (28 events)
Glyphosate (74 events) Paraquat (41 events) Quizalofop-p-tefuryl (5 events), Fenoxaprop-p-ethyl (4 events) Alachlor (11 events) 2,4-D (16 events)
100
Acknowledgments The author acknowledges the following coworkers for their significant contributions to this chapter: Dr. Anna Fan (Environmental Protection Agency, Sacramento, CA), Dr. Jou-Fang Deng (Veterans Administration Hospital, Taipei, Taiwan), Dr. ChenChang Yang (Veterans Administration Hospital, Taipei, Taiwan), Dr. Songsak Srianujata (Mahidol University, Bangkok, Thailand), and Dr. Takemi Yoshida (Showa University, Tokyo).
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Etemadi-Aleagha, A., Akhgari, M., and Abdollahi, M. (2002). A brief review on oxidative stress and cardiac diseases. Middle East Pharmacol. 10, 8-9. Furtado, M., and Chan, L. (2004, August 7). Toxicity, organophosphate, eMedicine, 1-12. Gershon, S., and Shaw, E H. (1961). Psychiatric sequelae of chronic exposure to organophosphorus insecticides. Lancet 1, 1371-1374. Gultekin, E (2000). The effect of organophosphate insecticide chlorpyrifos-ethyl on lipid peroxidation and antioxidant enzymes (in vitro). Arch. Toxicol. 74, 533-538. Gupta, R. C. (2004). Brain regional heterogeneity and toxicological mechanisms of organophosphates and carbamates. Toxicol. Mechan. Methods 14, 103-143. Gupta, R. C., Milatovic, D., and Dettbarn, W.-D. (2001a). Depletion of energy metabolites following acetylcholinesterase inhibitor-induced epilepticus: Protection by antioxidants. Neurotoxicology 22, 271-282. Gupta, R. C., Milatovic, D., and Dettbarn, W.-D. (2001b). Nitric oxide modulates high-energy phosphates in brain regions of rats intoxicated with diisopropylphosphorofluoridate or carbofuran: Prevention by N-tert-oL-phenylnitrone or vitamin E. Arch. Toxicol. 75, 346-356. Hosokawa, M., Maki, T., and Satoh, T. (1987). Multiplicity and regulation of hepatic microsomal carboxylesterases in rats. Mol. Pharmacol. 31, 579-584. Hosokawa, M., Maki, T., and Satoh, T. (1990). Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277, 219-227. Jalali, N., Pajoumand, A., Abdollahi, M., and Shadnia, S. (2000). Epidemiological survey of poisoning mortality in Teheran during 1997-1998. Toxicol. Lett. Suppl. 116, 309. Jamal, G. A. (1997). Neurological syndromes of organophosphorus compounds. Adverse drug reaction. Pralidoxime (1 gm single bolus dose vs. 12 gm infusion) in the management of organophosphorus. Toxicol. Rev. 16, 133-170. Marrs, T. C. (1993). Organophosphate poisoning. Pharmacol. Ther. 58, 51-66. Masuda, N., Takatsu, M., Norinami, H., et al. (1995). Satin poisoning in Tokyo subway. Lancet 345, 1446-1447. Masuda, Y., Nagano, Y., Takatori, T., et al. (1998). Detection of the satin hydrolysis product in formalin-fixed brain tissues of victims of Tokyo subway terrorist attack. Toxicol. Appl. Pharmacol. 150, 310-320. Matkevich, V. A., Simonenkov, A. P., Ostapenko, L., Li, N., et al. (1995, May/June). Use of serotonin adipinate in acute oral poisoning. Anest. Reanimatol., 16-20. Medda, S., Takeuchi, K., Devore-Carter, D., et al. (1987). An accessory protein identical to mouse egasyn is complexed with rat [3-glucuronidase and is identical to rat esterase-3. J. Biol. Chem. 262, 7248-7253. Nozaki, H., Aikawa, N., Shinozawa, Y., et al. (1995). Satin poisoning in Tokyo subway. Lancet 345, 980-981. Pajoumand, A., Jalali, N., Abdollahi, M., and Shadnia, S. (2002). Survival following severe aluminum phosphide poisoning. J. Pharm. Pract. Res. 32, 297-299. Poovala, V. S., Huang, H., and Salahudeen, A. K. (1999). Role of reactive oxygen metabolites in organophosphateinduced renal tubular cytotoxicity. J. Am. Soc. Nephrol. 10, 1746-1752.
1 O0
S ECTIO N I 9 Uses, Abuses, & Epidemiology
Ranjbar, A., Pasalar, E, and Abdollahi, M. (2002). Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorus pesticide manufacturing workers. Hum. Exp. Toxicol. 21, 179-182. Satoh, T., and Hosokawa, M. (1998). The mammalian carboxylesterases: From molecules to functions. Annv. Rev. Pharmacol. Toxicol. 38, 257-288. Satoh, T., and Hosokawa, M. (2000). Organophosphates and their impact on the global environment. Neurotoxicology 21, 223-227. Spiegelberg, U. (1963). Psychopathologische-neurologische spat und dauerschaden nach geweblicher Intoxikation durch
Phosporsaeureester (alkylphosphate). Proc. 14th Int. Congr. Occup. Health Exerpta Med. Found. Int. Congr. Ser. 62, 1778-1780. Suzuki, T., Morita, H., Ono, K., et al. (1995). Sarin poisoning in Tokyo subway. Lancet 345, 980. Swank, R. T., and Paigen, K. (1973). Biochemical and genetic evidence for a macromolecular [3-glucuronidase complex in microsomal membranes. J. Mol. Biol. 77, 371-389. Verma, R. S. (2001). Chlorpyrifos-induced alterations in levels of thiobarbituric acid reactive substances and glutathione in rat brain. Indian J. Exp. Biol. 39, 174-177.
Pharmacokinetics & Metabolism
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CHAPTER 9
Physiologically Based Pharmacokinetic Modeling of Organophosphorus and Carbamate Pesticides CHARLES TIMCHALK Pacific Northwest National Laboratory, Richland, Washington
insecticides in animals and humans and for the assessment of risk. The approach will not entail a comprehensive review of the extensive literature but, rather, a focused presentation highlighting important principles using specific examples for these classes of insecticides. OP and CM insecticides constitute two large families of pesticides that share a common mode of insecticidal and toxicological action associated with their ability to inhibit the enzyme AChE within nerve tissue (Ecobichon, 2001a,b; Sultatos, 1994). A major difference between these two insecticidal classes relates to the rate of AChE reactivation, which is measured in minutes for CMs versus days or even weeks for OP insecticides (Tobia et al., 2001; Ecobichon, 2001b). A comparison of the chemical structures for OP and CM insecticides is illustrated in Fig. 1. OP insecticides are structurally related, pentavalent phosphorus acids (Fig. 1, I). The "leaving group" (R1) is released upon phosphorylation of ACHE, R 2 and R3 are most commonly alkyloxy groups that are more stable constituents, whereas "X" represents either a sulfur or oxygen in association with a phosphate (H. W. Chambers et al., 2001; Chambers, 1992; Mileson et al., 1998). Carbamates (Fig. 1, II) are N-mono or-dimethylated carbamic acids, with R1 representing a range of alkyl or aryl substitutions that can result in substantial modification of the physiochemical properties of the CM (Ecobichon, 2001a,b).
I. I N T R O D U C T I O N Pharmacokinetics has and will continue to play an important role in assessing organophosphorus (OP) and carbamate (CM) insecticide dosimetry, biological response, and risk in humans exposed to these agents. These two major classes of pesticides share a common toxicological mode of action associated with their ability to target and inhibit acetylcholinesterase (ACHE). Pharmacokinetics is associated with the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. Pharmacokinetic studies provide important data on the amount of toxicant delivered to a target site as well as species-, age-, and gender-specific and dose-dependent differences in biological response. These studies have been conducted with OP and CM insecticides in multiple species, at various dose levels, and across different routes of exposure to understand how in vivo kinetics contributes to the observed toxicological response. Pharmacokinetic studies with these insecticides are also useful to facilitate extrapolation of dosimetry and biological response from animals to humans and for the assessment of human health risk. In this regard, physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models are being utilized to assess risk and understand the toxicological implications of known or suspected exposures to various OP and CM insecticides. In this chapter, a number of examples are presented that illustrate the utility of pharmacokinetic studies to address human health concerns associated with these important insecticides.
A. Mode of Action OP and CM insecticides have a high affinity for binding to and inhibiting the enzyme ACHE, an enzyme specifically responsible for the destruction of the neurotransmitter acetylcholine (ACh) within nerve tissue (Wilson, 2001; Ecobichon, 2001b). Since the cholinergic system is widely distributed within both the central and peripheral nervous systems, chemicals that inhibit AChE are known to produce
II. B A C K G R O U N D This chapter focuses on the application of pharmacokinetic principles and in particular the development of PBPK/PD models to better understand the toxicology of OP and CM Toxicology of Organophosphate and Carbamate Compounds
103
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
104
SECTION
i
II
ii
/
0
II
X
R ,,H
9Pharmacokinetics & Metabolism
RI.-.O--..C ..... R 1
N~-H
I
CH 3
o
II
R 1- - - O ~ ~ N
I
CH 3
CH 3 F I G . 1. Genera] structure for organophosphorus (I) and carbamate (II) insecticides.
a broad range of well-characterized symptoms (for review, see Savolainen, 2001). A comparison of the AChE inhibition dynamics for the interaction of ACh, carbaryl (CM), or chlorpyrifos-oxon (OP) with AChE is illustrated in Fig. 2. All three substrates have relatively high affinities for AChE and will readily complex with the enzyme; however, the rates of hydrolysis and reactivation of AChE following carbamylation or phosphorylation of the active site will be drastically slower than for the hydrolysis of the acetylated enzyme (Ecobichon, 2001b). Specifically, the turnover time for the neurotransmitter ACh is on the order o f - 1 5 0 Ixsec, whereas the carbamylated enzyme tl/2 for hydrolysis is substantially slower (-15-30 min). The phosphorylated enzyme is highly stable (tl/2 -days), and further dealkylation of the phosphorylation group produces an "aged" AChE enzyme that is irreversibly inhibited (Taylor, 1980; Murphy, 1986; Ecobichon, 2001b; Sogorb and Vilanova, 2002).
B. Organophosphorus and Carbamate Pharmacokinetics Pharmacokinetics is the evaluation of those processes associated with the ADME of drugs and xenobiotics. The ADME of OP and CM insecticides in both animals and humans has been studied (Timchalk et al., 2002a, 2005; Poet et al., 2004; Tos-Luty et al., 2001; Wu et al., 1996; Moody and Franklin, 1987; Tomokuni et al., 1985; Nolan et al., 1984). Like all chemical contaminants, these insecticides can gain entry into the body, and based on the detection of low levels of metabolites in urine within human populations, there is good evidence for widespread although low-level exposures (Aprea et al., 1999; Shealy et al., 1997; Hill et al., 1995; Brouwer et al., 1993). These exposures can come from numerous sources, including ingestion of pesticide residues on foods or accidental as well as intentional ingestion of insecticides (Drevenkar et al., 1993). Dermal exposure represents a relevant route, particularly during the mixing, loading, and application of insecticides or from skin contact with contaminated
surfaces (Knaak et al., 1993). Likewise, inhalation of airborne insecticide is feasible either during the spraying of or from exposures associated with chemical drift (Vale and Scott, 1974). Once the pesticide arrives at a portal of entry, it is available for absorption, and based on the bioavailability for a given insecticide and exposure route, a systemic dose of the parent compound will enter the circulation. Although localized portal of entry metabolism (i.e., lung, intestines, and skin) is feasible (Poet et al., 2003), the bulk of the metabolic activation as well as detoxification reactions occur within the liver (Sultatos et al., 1984a,b; Sultatos, 1988). It is likewise clear from both tissue distribution and partitioning studies that OP and CM insecticides are generally well distributed in tissue throughout the body (Tos-Luty et al., 2001; Wu et al., 1996; Tomokuni et al., 1985). Finally, due to the extensive metabolism (see Section II, C) little if any parent insecticide is available for excretion; however, more stable degradation metabolites are readily excreted in the urine and are of potential utility as biomarkers of exposure (Colosio et al., 2002; Shealy et al., 1997; Iverson et al., 1975; Mticke et al., 1970).
C. Insecticide Biotransformation 1. ORGANOPHOSPHATES A more detailed overview of the metabolism of OP insecticides can be found in Calabrese (1991), Jakanovic (2001), Sogorb and Vilanova (2002), and Knaak et al. (2004). The three major classes of OP insecticides are the phosphorothionates, phosphorodithioates, and the phosphoroamidothiolates (Chambers, 1992; Mileson et al., 1998; J. E. Chambers et al., 2001). Phosphorothionate insecticides such as chlorpyrifos are weak inhibitors of ACHE, but once they undergo metabolic activation (desulfation) to their corresponding oxygen analogs (oxon), they become extremely potent inhibitors. This enhanced toxicity is due to the oxon having a higher affinity and potency for phosphorylating the serine hydroxyl group within the active site of AChE (Mileson et al., 1998; Sultatos, 1994). The toxic potency is dependent on the balance between a delivered dose to the target site and the rates of bioactivation and/or detoxification (Calabrese, 1991). In Fig. 3, the thionophosphate pesticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2pyridyl]-phosphorothioate) is utilized for illustration purposes, and based on a common mode of action, this scheme is readily extended to other structurally related organophosphorus insecticides. As previously mentioned, phosphorothionates do not directly inhibit AChE but must first be metabolized to the corresponding oxygen analog (chlorpyrifos-oxon) (Mticke et al., 1970; Iverson et al., 1975; Murphy, 1986; Sultatos, 1994). Activation to the oxon-metabolite is mediated by cytochrome P450 mixed function oxidases (CYP450)
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106
SECTION II 9Pharmacokinetics
& Metabolism
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primarily within the liver, although extrahepatic metabolism has been reported in other tissues including the brain (Guengerich, 1977; Chambers and Chambers, 1989). In the case of chlorpyrifos, oxidative dearylation produces both 3,5,6-trichloro-2-pyridinol (TCP) and diethylthiophosphate and represents a competing detoxification pathway that is mediated by hepatic CYP450 (Ma and Chambers, 1994). These initial activation/detoxification reactions are believed to share a common phosphooxythiran intermediate and represent critical biotransformation steps required for toxicity (Neal, 1980). Differences in the ratio of activation to detoxification are associated with chemical-, species-, gender-, and age-dependent sensitivity to organophosphates (Ma and Chambers, 1994). Hepatic and extrahepatic (i.e., blood and tissue) A-esterases, such as PON-1, effectively metabolize chlorpyrifos-oxon, forming TCP and diethylphosphate. Likewise, B-esterases, such as carboxylesterase (CarbE) and butyrylcholinesterase (BChE), that are also well distributed across tissues can metabolize the oxon; however, these B-esterases become irreversibly bound (1:1 ratio) to the oxon and thereby become inactivated (Chanda et al., 1997; Clement, 1984). Studies on both humans and rodents indicate that the primary metabolite TCP can likewise undergo additional glucuronide or sulfate conjugation (Bakke et al., 1976; Nolan et al., 1984).
2. CARBAMATES The metabolism of CM insecticides has been previously reviewed (Ecobichon, 1994, 2001a; Sogorb and Vilanova, 2002) and can involve oxidative metabolism,
LCH
FIG. 3. Metabolic scheme for the metabolism of chlorpyrifos and the major metabolites chlorpyrifos-oxon, trichloropyridinol ( a n d conjugates), diethylphosphate, and diethythiophosphate. Adapted with permission from Timchalk et al. (2004).
ester hydrolysis, and conjugation reactions. The extent of metabolism is compound specific and highly dependent on the nature and position of substituent groups on either oxygen or nitrogen (Ecobichon, 2001a). For illustration, the metabolism of the CM insecticide carbaryl is presented in Fig. 4. The major pathway for detoxification of CM involves the hydrolysis by nonspecific esterases such as CarbE (Sogorb and Vilanova, 2002). Enzymatic hydrolysis results in the formation of oL-naphthol and methyl-carbamic acid, which is unstable and rapidly decomposes to COe and monomethylamine. The oL-naphthol can be further conjugated with sulfate or glucuronide prior to excretion (Chin et al., 1979a,b,c). Additionally, oxidative metabolism forming either ring or side chain hydroxylation is anticipated for carbaryl (Hodgson and Levi, 2001) but is of potentially less importance than esterase-mediated metabolism. Pharmacokinetic studies provide important data on the amount of toxicant delivered to a target site as well as species-, age-, and gender-specific and dose-dependent differences in biological response. The objective of this chapter is to further illustrate the utility of pharmacokinetics to address health concerns associated with cholinesterase inhibiting insecticides and, more specifically, to focus on the development, validation, and potential application of PBPK/PD models. These PBPK and PBPK/PD models can be used as a quantitative tool for integrating our understanding of dosimetry and biological response for these important classes of chemical insecticides.
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates
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III. PHARMACOKINETIC PRINCIPLES Studies on the pharmacokinetics of a xenobiotic provide critically useful insights into the toxicological response associated with a given agent. An important application of pharmacokinetics within toxicology has been to estimate risk by predicting the amount of absorbed dose under realistic exposure conditions (Clewell, 1995). Toxicology studies are designed to provide a quantitative assessment of toxicity based on what the chemical agent does to test animals, whereas pharmacokinefics focuses on what the animal (or human) does to the chemical. Clearly, toxicity and pharmacokinefics are integrally related since the extent of absorption, retention, metabolic activation, or detoxification is ultimately responsible for delivering a dose to a target tissue resulting in observed effects. Pharmacokinetic models are critically important tools that, if used correctly, can quantitatively establish a unifying model that describes dosimetry and can be related to biological response across exposure routes, species, and chemical agents. The further linkage of pharmacokinefics with pharmacodynamics (i.e., measure of response) is particularly useful for OP and CM insecticides since they share a common mode of action through their capability to inhibit AChE activity (Mileson et al., 1998). Bioanalytical methods for quantifying dosimetry have been developed to measure the parent compound and the active (i.e., oxon) or inactive metabolites. It is also feasible to link dosimetry with biologically based PD response based on a common mode of action (i.e., AChE inhibition). In general, pharmacokinetic modeling approaches can be characterized as empirical or physiologically based, and both types of models have been applied to understand the toxicological
FIG. 4. Metabolic scheme for the oxidative and B-esterase metabolism of carbaryl to the ring and side chain hydroxylated methyl carbamates and the major metabolite oL-naphthol.
response to AChE-inhibiting insecticides in multiple species (Wu et al., 1996; Tomokuni et al., 1985; Pena-Egido, 1988; Brimer et al., 1994; Gearhart et al., 1990; Sultatos, 1990).
A. Compartmental Pharmacokinetic Models For a more detailed discussion of the application of pharmacokinetic modeling approaches in toxicology, including the fundamental principles, see Renwick (1994) and Dix (2001). Compartmental models have been extensively utilized to assess bioavailability, tissue burden, and elimination kinetics in various species including humans. Pharmacokinetics is concerned with the time course by which a chemical is absorbed into the systemic circulation, distributed throughout the body, altered through metabolic transformation, and eliminated. Compartmental models are empirical and as such consider the organism as a single or multicompartment homogeneous system. The number and behavior of the compartments are primarily determined by the equations chosen to describe the time course data and not the physiological characteristics of the organism (Krishnan and Andersen, 2001). In these models, the net transfer between compartments is directly proportional to the difference in chemical concentration between compartments. However, the rate constants associated with this transfer cannot be experimentally determined (Srinivasan et al., 1994). Compartmental models range from a simple well-mixed single compartment to more complicated multicompartments that are used to describe the blood and/or plasma time course of a chemical or drug. These approaches have been utilized to model the pharmacokinetics of OP and CM insecticides and their major metabolites
108
SECTION 1I Pharmacoldnetics 9 & Metabolism
(Braeckman et al., 1983; Nolan et al., 1984; Drevenkar et al. 1993; Wu et al., 1996). Although compartment modeling is extremely useful for interpolation within the confines of the test species and experimental conditions (i.e., exposure routes and dose levels), these models are limited in their capability to extrapolate across dose, species, and exposure routes (Krishnan and Andersen, 2001). To enable extrapolation, PBPK models have emerged as an important tool that has seen broad applications in toxicology and, more specifically, in human health risk assessment (Andersen, 1995, 2003; Krishnan and Andersen, 2001; Mason and Wilson, 1999; Clewell and Andersen, 1996; Leung and Paustenbach, 1995). If there is a fundamental understanding of how target tissue dosimetry modulates a pharmacological or toxicological response, then it is feasible to extend these models to incorporate pharmacodynamics (Andersen, 2003).
B. Physiologically Based Pharmacokinetic Models Unlike compartment modeling approaches, PBPK modelS utilize biologically meaningful compartments that represent individual organs, such as liver and kidney, or groups of organ systems (i.e., well perfused/poorly perfused) (Mason and Wilson, 1999). The general model structure is based on an understanding of comparative physiology and xenobiotic metabolism, a chemical's physical properties that define tissue partitioning, the rates of biochemical reactions determined from both in vivo and in vitro experimentation, and the physiological characteristics of the species of interest (Krishnan and Andersen, 2001). PBPK models have been developed to describe target tissue dosimetry for a broad range of environmental contaminants, such as solvents, heavy metals, and pesticides, including organophosphate insecticides (Poet et al., 2004; Timchalk et al., 2002b; O'Flaherty, 1995; Gearhart et al., 1990; Sultatos, 1990; Corley et al., 1990; Andersen et al., 1987). A number of reviews have been published on the development, validation, application, and limitations of PBPK models in human health risk assessment (Andersen, 1995, 2003; Krishnan and Andersen, 2001; Frederick, 1995; Clewell, 1995; Leung and Paustenbach, 1995; Clewell and Andersen, 1996; Slob et al., 1997; Mason and Wilson, 1999). 1. PBPK MODEL STRUCTUREAND PARAMETERS A generalized compartmental structure for a PBPK model is illustrated in Fig. 5. The model includes compartments for key tissues associated with the absorption, disposition, metabolism, or elimination of the chemical of interest, and it is based on a general understanding of species-specific anatomy/physiology (Krishnan and Andersen, 2001). Additional compartments can be included to describe potential target tissues for dosimetry or additional metabolically active tissues; for example, a brain compartment can
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be included for those chemicals that target the nervous system. The remaining tissues are accounted for by lumping them into either slowly or rapidly perfused compartments. The model is parameterized to describe species-specific anatomy and physiology (i.e., organ volumes, cardiac output, and blood flow), tissue composition (i.e., water vs. lipid content), and biochemical processes (i.e., protein binding and metabolism). In addition, pharmacodynamic models require the inclusion of additional parameters to adequately describe the dynamic response. To illustrate the application of this modeling approach to OP and CM insecticides, PBPK models that also incorporate a PD component to describe AChE inhibition following exposure to the phosphorothionate insecticides (e.g., chlorpyrifos and diazinon) in rodents and humans are described (Poet et al., 2004; Timchalk et al., 2002b). These models are based on the conceptual structure developed by Gearhart et al. (1990) to describe AChE inhibition following an acute exposure to diisopropyl fluorophosphate (DFP). Although CM-specific models have not been developed, in principle the generalized structure for OP insecticides can be used as a starting point. 2. ORGANOPHOSPHATE-SPECIFICMODEL STRUCTURE A diagram of the PBPK and PD model structure for thionophosphorus OP insecticides is illustrated in Fig. 6. The conceptual representation of the PBPK/PD model for these insecticides is based on the anatomical and physiological characteristics of the rat and human. The major determinants of insecticide disposition include absorption rates, tissue partitioning, plasma protein binding, CYP450 metabolism, and esterase binding and hydrolysis (Poet et al., 2004; Timchalk et al., 2002b). The PBPK/PD model allows for the simulation of differing exposure scenarios, such as acute oral "garage," chronic dietary, and dermal. A lung compartment was not included in this model since inhalation exposure to
CHAPTER 9
9 PBPK/PD O r g a n o p h o s p h a t e s
and Carbamates
109
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FIG. 6. Physiologically based pharmacokinetic and pharmacodynamic model used to describe the disposition for a parent phosphorothionate insecticide, its oxon metabolite, associated leaving groups, and B-esterase inhibition in rats and humans following oral (gavage and dietary) and dermal exposures. The shaded tissue compartments indicate organs in which B-esterase (ACHE, BuChE, and CarbE) enzyme activity is described. Model parameter definitions: QC, cardiac output (liters/hr); Qi, blood flow to i tissue (liters/hr); Ca, arterial blood concentration (l~mol/liter); Cao, arterial blood concentration of oxon (txmol/liter); Cv, pooled venous blood concentration (l~mol/liter); Cvi, venous blood concentration draining i tissue (l~mol/liter); Cvio, venous blood concentration of oxon draining i tissue (l~mol/liter); SA, surface area of skin exposed (cm2); KP, skin permeability coefficient (cm/hr); Kzero, zero (txmol~) rate of absorption from diet; Fa, fractional absorption (%); KaS and KaI, first-order rate constants for absorption from compartments 1 and 2 (per hr); KsI, first-order rate constant for transfer from compartment 1 and 2 (per hr); Ke, first-order rate constant for elimination of metabolite from compartment 3; Km(1-4), Michaelis constant for saturable processes (txmol/liter); Vmax(1-4), maximum velocity for saturable process (txmol/hr). Adapted with permission from Timchalk et al. (2002b).
chlorpyrifos and diazinon is not considered a major exposure route; however, a lung compartment was used in the DFP model due to the greater volatility of this OP agent (Gearhart et al., 1990). In these models, physiological and metabolic parameters were scaled as a function of body weight according to the methods of Ramsey and Andersen (1984). The CYP450-mediated activation and detoxification was limited to the liver and was linked to the oxon models that incorporated equations to describe A-esterase (PON-1) metabolism in both liver and blood. The CYP450
activation/detoxification and PON-1 detoxification were all described as Michaelis-Menten processes. Oxon metabolites bind with and inhibit B-esterases, including ACHE, BuChE, and CarbE. Whereas binding to AChE is associated with acute neurotoxicity, binding to BuChE and CarbE is suggested to be without adverse physiological effect and as such these represent detoxification pathways (Pond et al., 1995; Clement, 1984; Fonnum et al., 1985). Interactions of the oxon with B-esterases were modeled as second-order processes occurring in the liver, blood [plasma and red
1 10
S ECTI O N I I 9P h a r m a c o k i n e t i c s & M e t a b o l i s m TABLE 1.
Physiological Parameters for Rats and Humans Used in the PBPK models for Chlorpyrifos and Diazinon a
blood cells (RBCs)], diaphragm, and brain. The B-esterase enzyme levels (micromoles) were calculated based on the enzyme turnover rates and enzyme activities (Maxwell et al., 1987). A balance between the bimolecular rate of inhibition and the rate of cholinesterase regeneration and aging determined the amount of free cholinesterase. In this model, the major metabolites of chlorpyrifos and diazinon [TCP or isopropyl-methylhydroxypyrimidine (IMHP), respectively] were formed by CYP450 metabolism of the parent compounds or PON-1 and B-esterase hydrolysis of oxons. The pharmacokinetics of the metabolites was described using a simple one-compartment model. These PBPK/PD models developed for the OP insecticides are fairly complex and are data intensive; thus, to adequately develop and validate these models generally requires extensive experimentation to support model parameterization and validation. The following section provides a more detailed description of some of the experimental approaches used to develop and validate these models. 3. ORGANOPHOSPHATE-SPECIFIC MODEL PARAMETERS
The physiological parameters utilized to develop the PBPK/PD models for the OP insecticides chlorpyrifos and diazinon are presented in Table 1. In general, many of these parameters have been previously measured in both animals and humans (Brown et al., 1997; Arms and Travis, 1988). However, it is important to recognize, particularly as one attempts to address questions concerning age-dependent or disease state-specific pharmacokinetics, that appropriate physiological parameters may not be available. An initial first approximation should utilize available parameters, recognizing the potential uncertainty, but it should also be understood that the experimental determination of new physiological parameters may need to be considered. One primary physiochemical parameter is the partitioning coefficient (PC), which is needed to describe the distribution of the chemical of interest between the body fluids and tissues (Knaak et al., 2004; Krishnan and Andersen, 2001). There are a number of approaches to experimentally determine PCs, including in vitro vial equilibrium,
Rat
Human
Body weight (kg) Tissues % of body weight Blood Brain Diaphragm Fat Liver Rapidly perfused Slowly perfused Cardiac output (liters/hr) Tissues % of cardiac output Brain Diaphragm Fat Liver Rapidly perfused Slowly perfused Skin
0.25
70
6 1.2 0.03 7 4 4 78 5.4
7 2 0.03 21 3 4 63 347.9
3 0.6 9 25 42.6 14 5.8
11.4 0.6 5.2 23 40 14 5.8
aAdapted from Timchalk et al. (2002b) with permission.
equilibrium dialysis, and ultrafiltration as well as in vivo tissue vs. blood concentration determinations (Krishnan and Andersen, 2001). For many of these insecticides, the simple vial equilibrium method, developed by Gargas et al. (1989), and in vivo methods cannot be utilized for PC determination due to the lack of volatility of these chemicals and their rapid metabolism. Therefore, ultrafiltration and equilibrium dialysis are the methods of choice (Knaak et al., 2004) and have been used to determine PCs for several OP insecticides (Jepson et al., 1994; Sultatos et aL, 1990). For example, the time course for the distribution of fenitrothion in buffer and homogenate following equilibrium dialysis is illustrated in Fig. 7 using the approach described by Kousba and
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I
m
~E~ 0.2 n- t ' - 0.1
0
). . . . . . . . . . . . . . . . . . . . .
lml l
i
i
0
20
40
i
i
60 80 Time (min)
" . . . . . . . .
(T~
i
i
1O0
120
i
140
FIG. 7. Gas chromatography ratio counts for the time course of fenitrothion distribution in buffer and liver homogenate following equilibrium dialysis at 37 ~ The homogenate:buffer partitioning was calculated using data from 120 min (2 hr). The values represent the mean +_ SD for five determinations per time point.
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates
Sultatos (2002). For fenitrothion, equilibrium was achieved by --60 min and was clearly stable during 120 min of dialysis (37 ~ Based on these results, it was feasible to experimentally determine both tissue:blood and tissue: buffer partitioning and calculate a liver:blood PC. Poulin and Krishnan (1995, 1996) also developed an algorithmbased approach for determining PCs using the chemical's octanol:water partitioning and the tissue lipid content. This approach was utilized to calculate PCs for chlorpyrifos, diazinon, and their oxon metabolites, and these are presented in Table 2. As previously suggested, the metabolism of phosphorothionate insecticides is complex, involving CYP450s, esterase hydrolysis, and conjugation reactions (see Fig. 3). A number of in vivo and in vitro approaches have been utilized to determine metabolic parameters (for review, see Krishnan and Andersen, 2001); however, for these nonvolatile insecticides in vitro cellular and subcellular metabolizing systems are most appropriate for obtaining metabolic rate constants. For a more detailed review of the metabolism of organophosphorus insecticides and the integration of these data into PBPK models, see Knaak et al. (2004).
TABLE 2.
The in vitro kinetic results used to determine metabolic parameters for the hepatic CYP450 and PON-1 metabolism of chlorpyrifos and diazinon in rats are illustrated in Fig. 8, and the metabolic rate constants (Km and Vmax) used to describe the nonlinear metabolism in the PBPK model are presented in Table 2. These data illustrate that the major CYP-mediated hepatic metabolites for both chlorpyrifos and diazinon (Figs. 8A and 8B) are the inactive pyridinol (TCP) and pyrimindinol (IMHP) metabolites, whereas both oxons are relatively minor metabolites (Poet et al., 2003). Likewise, the hepatic metabolic detoxification of chlorpyrifos- and diazinon-oxon by PON-1 (Fig. 8C) was nonlinear. The metabolic rate constants were described by Michaelis-Menten equations by fitting the data using nonlinear regression (Poet et al., 2003), and the resulting parameter estimates were used in the development of the PBPK/PD models. To develop a PD model for cholinesterase-inhibiting compounds, the steady-state levels (Ixmol) of B-esterase enzymes (ACHE, BuChE, and CarbE) were determined for the various tissues (e.g., brain, blood, liver, and diaphragm) based on the rates of enzyme synthesis (zero-order) and degradation (first-order) (Gearhart et al., 1990). The
Partition Coefficients and Metabolic and Protein-Binding Parameters Used in the PBPK Models for Chlorpyrifos and Diazinon a
Parameter Partition coefficient Brain/blood Diaphragm/blood Fat/blood Liver/blood Rapidly perfused/blood Slowly perfused/blood Skin/blood Metabolic constant CYP450 Parent-to-oxon (liver) K m (p.mol/liter)
[ixmol/(hr kg)] CYP450 Parent-to-detox. (liver) Vmax
K m (ixmol/liter)
Vmax [Ixmol/(hr kg)] PON,1 oxon-to-metab. (liver) Km (txmol/liter) Vmax [ixmol/(hr kg)] PON-1 oxon-to-metab. (blood) K m (p, m o l / l i t e r )
[txmol/(hr kg)] Plasma protein binding (%) Vma x
111
Chlorpyrifos
Chlorpyrifos-oxon
Diazinon
Diazinon-oxon
33 6 435 22 10 6 6
26 4.9 342 17 8.1 4.9
28 5 360 18 8 5 5
2.86 80
m m
25 14
24 273
~ --
200 180
~
240 74,421
~ ~
270 63,000
250 57,003 98
m ~ 89
270 63,000 89
--
~ 97
aAdaptedfrom Poet et al. (2004) and Timchalket al. (2002b) with permission.
10 2 120 7 2 2 5
1 12
S ECTI O N I I 9P h a r m a c o k i n e t i c s & M e t a b o l i s m
A
B CPF
DZN 3.0
3.0 2.4 1.8
1.8
1.2
1.2
o~ 0.6
E
E 0.0 o E v
t'--
0.6 --r i
i
i
i
30
60
90
120
9IMHP
2.4
TCP
9
o DZN-Oxon ~
o CPF-Oxon
#
0.0
150
100
200
Substrate Concentration (pM)
J
~
T
I
I
I
I
300
400
500
600
700
800
900
Substrate Concentration (pM)
"O O "O OL
2500
(1> .--
[] D Z N - O x o n
O
2000 -
9C P F - O x o n
TCP
1500 1000 500 []
0
0
I
I
I
I
200
400
600
800
I
I
I
I
I
I
1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2000
Substrate Concentration (l.tM)
FIG. 8. The in vitro CYP450-mediated metabolism of (A) chlorpyrifos (CPF) and (B) diazinon (DZN) to their oxon and trichloropyridinol (TCP) and isopropyl methyl hydroxypyrimidine (IMHP) metabolites and ( C ) PON-l-mediated metabolism of CPF-oxon and DZN-oxon in rat liver microsomes. Adapted with permission from Poet et al. (2003).
cholinesterase turnover rates, enzyme activity, and degradation rates that were used to calculate steady-state cholinesterase tissue levels for the dynamic models are presented in Table 3. Maxwell et al. (1987) provided an initial estimate of the amount of ACHE, BuChE, and CarbE in tissues from control rats using the following equation:
E s t e r a s e (txmol) =
E n z y m e activity E n z y m e t u r n o v e r rate
These estimates were then utilized for both the rat and the human dynamic models (Timchalk et al., 2002b). It is important to note that since the PD model is highly dependent on the estimates of these enzyme levels, additional experimental measurements, particularly in humans, may be warranted (Knaak et al., 2004). The enzyme degradation rates for ACHE, BuChE, and CarbE were initially based on the first-order loss of rat brain AChE (Wenthold et al., 1974) as described by Gearhart et al. (1990). As a first
approximation, the synthesis and loss rates for BuChE were set the same as for ACHE; however, for CarbE the rates were optimized with the PD model to fit CarbE inhibition data from Chanda et al. (1997) (Timchalk et al., 2002b). In developing PD models for cholinesterase inhibiting insecticides, an important consideration is to adequately characterize the type of B-esterases (i.e., AChE vs BuChE) that are present in a given tissue since across species there are marked quantitative differences in the amounts and types of tissue B-esterases present (Timchalk et al., 2002b). For example, rat plasma cholinesterase is the sum of both plasma AChE and BuChE activity (Maxwell et al., 1987), whereas in humans plasma cholinesterase is exclusively BuChE (Oak Ridge National Laboratory, 2000). To characterize tissue cholinesterase activity, the specific inhibitors of AChE (BW284C51) and BuChE (iso-OMPA) activity can be used in combination with differing substrates, such as acetylthiocholine (ATC) or butyrylthiocholine (BTC). Although AChE and BuChE can both hydrolyze ATC, only BuChE can hydrolyze BTC (Lassiter et al., 1998; Chuiko, 2000). Therefore, a combination of specific enzyme
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates
1 13
TABLE 3. Cholinesterase Parameters for Pharmacodynamic Models in Ratsa Parameters Enzyme turnover rate (pLmol substrate hr-1/pLmol active site) AChE BuChE CarbE Enzyme activity [ixmol/(kg/hr)] AChE Brain Diaphragm Liver Plasma BuChE Brain Diaphragm Liver Plasma CarbE Brain Diaphragm
1.17 E +7 3.66 E +6 1.09 E +5
4.4 E +5 7.74 E +4 1.02 E +4 1.32 E +4 4.68 E +4 2.64 E +4 3.0 E +4 1.56 E +4 6.0 E +3 3.18 E +5
Liver Plasma Enzyme degradation rate (hr -1) AChE Brain Diaphragm Liver Plasma BuChE Brain Diaphragm Liver Plasma CarbE , Brain Diaphragm Liver Plasma
1.94 E +6 4.56 E +5
0.01 0.01 0.1 0.1 0.01 0.01 0.1 0.1 7.54 E -4 0.001 0.001 0.0033
aAdapted from Timchalk et al. (2002b) with permission. Initial parameters from Maxwell et al. (1987).
inhibitors and substrates makes it possible to quantitatively determine the specific types of cholinesterase activities present within a given tissue. This is clearly illustrated in Fig. 9, in which Kousba et al. (2003) compared the in vitro cholinesterase activity in rat brain (--100% ACHE), plasma (-50:50% AChE:BuChE), and saliva using both ATC and BTC as substrates with and without specific cholinesterase inhibitors. The results strongly suggested that >95% of the saliva cholinesterase activity in the rat was associated with BuChE. This type of experimental approach can be utilized to determine tissue-specific cholinesterase activity for PD model development in a broad range of tissues obtained from both animals and humans. The biomolecular inhibition rate constant (Ki) describes both the affinity and the rate of cholinesterase phosphorylation and is an indicator of inhibitory potency (Kousba etal., 2004; Kardos and Sultatos, 2000; Amitai et al., 1998; Carr and Chambers, 1996). A typical Ki determination is illustrated in Fig. 10 for the in vitro inhibition of rat BuChE with chlorpyrifos-oxon. In this example, the Ki was determined by incubating BuChE with varying concentrations of chloropyrifos-oxon (0.25-5 nM); the maximum inhibition ranged from 10 to 90% during a 7- to 30-min incubation period (Kousba et al., 2003). The slopes obtained from this analysis were then analyzed by linear regression to calculate a Ki (Fig. 10B). Similar in vitro approaches have been used to calculate the spontaneous first-order reactivation
rate constant (Kr) as is illustrated in Fig. 11. In this example, the reactivation rate (0.078 hr -1) following in vitro incubation of brain AChE with paraoxon was obtained from a linear regression of the terminal slope of the percentage of AChE inhibition (Kousba et al., 2004; Levine and Murphy, 1977). A summary of the biomolecular inhibition rate constants, reactivation, and aging rates for ACHE, BuChE, and CarbE inhibition with chlorpyrifos- and diazinon-oxon that were utilized in the PBPK/PD models is presented in Table 4. The extent and rate of B-esterase inhibition and recovery are dependent on the amount of available enzyme, the Ki, and the amount of time the B-esterase is exposed to the oxon (Vale, 1998). The amount of available B-esterase binding sites (txmol) follows the order CarbE > > BuChE ~ AChE (Maxwell et al., 1987), whereas the Ki rates for both chlorpyrifos- and diazinonoxon follow the order BuChE > > AChE > CarbE. Since chlorpyrifos and diazinon have the same diethylphosphate group responsible for phosphorylating the enzyme active site, it was anticipated that the rates of ChE reactivation and aging would be similar. Also, since experimental data were available only for ACHE, for modeling purposes it was assumed that BuChE and CarbE would have similar rates of reactivation and aging (Poet et al., 2004; Timchalk et al., 2002b). The development and application of PBPK modeling for human health risk assessment is not without its challenges
SECTION II 9 Pharmacotinetics & Metabolism
1 14
A 100
ChE activity using BTC substrate
~
0.014
3
0.012
0.55 nM
E 0.010 O
o t~
0.OO8
r"~l
O
~
1:13
:~ 0.004 0
10
I.IJ t-
0.006<
o~
0.002
ill
o.ooo Control
iso-OMPA
BW284C51
0
0.1
-5 nM
0.40
!
0.5
I-I plasma
ChE activity using ATC substrate
[] Saliva
o.o4o 0.035 O.030
.--
4.5 4
Ki = 8.83 nM-lh -1 R2 = 0.9753
~ 3.
E 3 o 2.5 X
0.o25 o.020
1.1_
a..
1.5
=> 9
0.010
~
0.5
O
0.005 0.000
.# o.o15 <
nM
0.20 0.3 Incubation time (h)
9 Brain
"~" .m E a O E
nM
.
0 Control
iso-OMPA
BW284C51
011
012
1/Slope
013
014
015
FIG. 9. In vitro determination of total ChE activity described as (A) butyrylthiocholine (BTC) and (B) acetylthiocholine (ATC) hydrolysis rates in tissues following iso-OMPA (BuChE inhibitor) or BW284C51 (ACHE inhibitor) incubation (15 min) with brain, plasma, and saliva samples obtained from naive adult male rats. The data represent the m e a n _ SD for three determinations. Adapted from Kousba et al. (2003).
FIG. 10. (A) In vitro rat BuChE activity (%BuChE activity) described as the rate of acetylthiocholine (ATC) substrate hydrolysis as a function of oxon concentration for different incubation periods (mean _+ SD of triplicate samples). (B) Final Ki determination plot. Each symbol represents a specific slope obtained from a given oxon concentration (see Fig. 10A). For both data sets, the lines represent the best fit from linear regression analysis. Adapted with permission from Kousba et al. (2003).
and limitations. Before a model can be used to assess risk, a determination must be made concerning the model's capability to accurately predict dosimetry and biological response (Frederick, 1995). Second, PBPK/PD models are data intensive, so to adequately develop and validate a model generally requires extensive experimentation to support model parameterization and validation (Clewell, 1995). In the case of OP and CM insecticides, despite the large number and diversity of studies that have been performed,
many of the biological parameters needed for model development and validation are unavailable (Knaak et al., 2004). Nonetheless, these models have been developed for a limited number of insecticides and are being utilized as tools for integrating the complex biological response associated with exposure to these agents. The following sections highlight the capability of these models to accurately predict dosimetry and dynamic response in both animals and humans.
100 -_
K r = 0.078 h -1
t"
e
.O m ..Q .m ct"-
,,i
9
9
9
9
9
10=
O
O
cO
<
~
_ 1
0
I
I
!
!
5
10
15
20
Incubation Time (hr)
FIG. 11. In vitro determination of rat brain AChE spontaneous reactivation (Kr) following incubation with paraoxon. The line represents the best fit for the terminal portion of the curve, where Kr equals the slope of the line. Adapted with permission from Kousba et al. (2004).
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates
1 15
TABLE 4. PBPK Model Parameter Estimates for Bimolecular Inhibition, Reactivation, and Aging a Parameter Bimolecular inhibition rate (IxM hr)- 1 AChE BuChE CarbE Enzyme reactivation rate (hr- 1) AChE BuChE CarbE Enzyme aging rate (hr -1) AChE BuChE CarbE
Chlorpyrifos-oxon 243 2000 20
Diazinon-oxon 525 1700 0.5
1.43 E -2 1.43 E -2 1.43 E - 2 1.13 E -2 1.13 E - 2 1.13 E - 2
aAdapted from Poet et al. (2004) and Timchalket al. (2002b) with permission.
IV. P B P K / P D M O D E L D E V E L O P M E N T AND VALIDATION
A. In Vivo Validation i n A n i m a l s Model refinement and validation for both the chlorpyrifos and the diazinon PBPK/PD models was accomplished by conducting a series of in vivo pharmacokinetic and pharmacodynamic studies in the rat and by evaluating the capability of the model to accurately simulate in vivo data published in the literature. The experimental details are fully described in Timchalk et al. (2002b) and Poet et al. (2004). In brief, these studies involved an acute oral exposure to chlorpyrifos or diazinon and the blood time course of the parent compounds and metabolites was determined, as well as the time course for the cholinesterase inhibition in several tissues. Representative results and model simulations are presented in Fig. 12 and 13 for the pharmacokinetic and pharmacodynamic response in rats following comparable oral doses (50 and 100 mg/kg) of chlorpyrifos and diazinon, respectively. The overall response was fairly comparable for these two insecticides, and the models reasonably simulated both dosimetry and the dose-dependent cholinesterase inhibition. These results are very consistent with a fairly rapid oral absorption for both insecticides and subsequent metabolism and distribution of the active oxon metabolites. Figure 14 illustrates the capability of the diazinon PBPK/PD model to simulate rodent dosimetry data from the open literature and the capability of the model to accommodate alternative exposure routes (Poet et al., 2004). In these examples, the time course of diazinon in plasma and cholinesterase inhibition in tissues (i.e., blood,
brain, and liver) were determined in rats following oral, intravenous (iv), or intraperitoneal (ip) acute administration (Wu et al., 1996; Tomokuni et al., 1985). As is seen in Fig. 14A, iv administration resulted in a more biphasic kinetic profile, with the terminal response paralleling the kinetics following oral administration (Poet et al., 2004). The inhibition kinetics (Figs. 14B and 14C) following ip administration (Tomokuni et al., 1985) indicated a slightly greater inhibition of RBC AChE than plasma cholinesterase. This was inconsistent with the model, which predicted a more comparable maximum inhibition for plasma and RBC, although the model reasonably simulated the enzyme recovery rates. These simulations illustrate that the lack of model fit may be related to a number of confounders, including route-dependent differences in exposure, straindependent differences in sensitivity, or even analyticaldependent differences in methodology used to measure enzyme activity. Nonetheless, the model provides a reasonable simulation of the experimental results, but more important, a lack of complete concordance does provide additional opportunity for further experimentation and model refinement.
B. In Vivo Validation in H u m a n s The primary objective associated with the development of a PBPK/PD model is to predict with some confidence dosimetry and dynamic response in humans. Confidence in the predictive capability of these models is enhanced by validating the model against carefully conducted controlled human pharmacokinetic/pharmacodynamic studies
1 16
SECTION II 9 P h a r m a c o k i n e t i c s & M e t a b o l i s m
A 10 ~
Blood Dosimetry
II
,_1 0
E
0.1
::L
0.01 0.001 ~
o
8
2o
24
Time (hr)
C 100 ~
100 ~:~ ~..-.-~::~:.~:.;~.... ..-.:~
Plasma ChE
80 .i-,
t- 60 O o "5 40
: 60 O o "5 40 o--e 20
~
0
0
Brain AChE
--O 80 _
20 i
i
4
8
i
i
12 Time (hr)
i
16
i
20
o
24
0
4
8
1
'
Time (hrs)
;0
24
FIG. 12. (A) Blood time course of chlorpyrifos (CPF), (B) plasma ChE inhibition, and (C) brain AChE inhibition in rats following oral administration of 50 (gray) or 100 (black) mg CPF/kg of body weight, respectively. The lines represent the model simulation of the experimental data. Adapted with permission from Timchalk et al. (2002b).
A 10
Blood Dosimetry
._1 O
E
::I.
0.11 0
i
4
~}
i
12 Time (hr)
C 100
s tO
o
-
Plasma BuChE
~,;,.
80
o
O
i
'~
20
24
100 :~' i~;:
-~ 80 t-
60
i
16
Brain AChE
60
,.iO
!i!i!i~:
~ 40 0
a.
20 o
!1 0
i
4
i
8
1'2 Time (hr)
i
16
!
20
i
24
0
~,
!
8
1'2 Time (hr)
16
2'0
2~4
FIG. 13. (A) Plasma time course of diazinon (DZN), (B) plasma BuChE inhibition, and (C) plasma AChE inhibition in rats following oral administration of 50 (gray) or 100 (black) mg DZN/kg of body weight, respectively. The lines represent the model simulation of the experimental data. Adapted with permission from Poet et al. (2004).
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates
1 17
A ~
~
Oral
.
._1 O
E :=L
............. :i~ii.................................................... ~i~;
9 :==== %.;
0.1 Time (hr)
120
120
10o ~ 8o
o
-5
Plasma ChE
'~ 100
9
9
60 4o
~o
80
"5
60
O
9
9 9
RBC AChE
~ 40
~. 20
~_ 2o 0
6
12 Time (hr)
0
i
24
18
i
0
6
i
12 Time (hr)
or against available exposure and dosimetry data obtained from biomonitoring or clinical evaluations (Wilks and Woolen, 1994; Woollen, 1993). For chlorpyrifos, controlled human pharmacokinetic studies have been conducted to facilitate biomonitoring and have also been utilized to further validate the PBPK/PD model (Nolan et al., 1984; Timchalk et al., 2002b). The time course of chlorpyrifos and the major metabolite trichloropyridinol in blood of a volunteer who ingested a capsule containing chlorpyrifos at a dosage of 2 mg/kg is presented in Fig. 15. Although both chlorpyrifos and the major metabolite trichloropyridinol were readily detected in the blood, the blood levels for chlorpyrifos were approximately two orders of magnitude
!
i
18
24
FIG. 14. (A) Plasma time course of diazinon (DZN) in rats following intravenous (iv) and oral administration of 10 (gray) and 80 (black) mg DZN/kg of body weight, respectively (data from Wu et al., 1996), (B) inhibition of plasma ChE, and (C) RBC AChE in rats after ip dosing with 100 mg DZN/kg of body weight. Experimental data from Tomokuni and Hasegawa et al. (1985). Adapted with permission from Poet et al. (2004).
less than for the metabolite, consistent with the model simulations and the known rapid metabolism of chlorpyrifos to trichloropyridinol (see Fig. 8). Likewise, the pharmacodynamics has also been evaluated in human volunteer studies, in which the time course of plasma BuChE inhibition kinetics following a single oral (0.5 mg/kg) or dermal (5 mg/kg) dose of chlorpyrifos were evaluated (Nolan et al., 1984) and are presented in Fig. 16. In this example, the amount of available plasma cholinesterase enzyme and the rate of enzyme recovery were optimized to fit the
Dermal 5 mg/k
Oral 0.5 mg/kg
100 1.E+01 -I_,,,,._ ,, ,,
,r ~
1. E + 00 - ~ " ~ ~ - - - - - I .E-02 -~ E : I.E-03 I .E-04 I .E-05
0
TCP
~
-~
80
o o ,.i.o
60
.4--' C"
"~ CPF
40 20 0
50
I
100 Time (Hrs)
i
150
i
0
200
FIG. 15. Experimental data (symbols) and model simulations (lines) for the plasma concentration of trichloropyridinol (TCP) and chlorpyrifos (CPF) in a volunteer administered CPF as an oral dose of 2 mg CPF/kg of body weight. Adapted with permission from Timchalk et al. (2002b).
200
400 Time (hrs)
600
i
800
FIG. 16. Experimental data (symbols) from Nolan et al. (1984) and model simulations (lines) of the plasma ChE inhibition in human volunteers administered an oral or dermal dose of chlorpyrifos. The time course data represent the mean _+ SD for five male volunteers. Adapted with permission from Timchalk et al. (2002b).
1 18
S E C T I O N II
9Pharmacoldnetics & Metabolism
plasma cholinesterase inhibition time course (oral). The model parameters were then held constant and the model was used to predict the plasma cholinesterase inhibition for the dermal exposure. In this case, the model predicted a maximum inhibition o f - 9 0 % of control, which was comparable to the observed 87% seen with the experimental data (Timchalk et al., 2002b). To further validate the capability of the model to reasonably describe the chlorpyrifos blood pharmacokinetics, the time course of chlorpyrifos in serum obtained from an individual who ingested a concentrated formulation of chlorpyrifos (Drevenkar et al., 1993) was simulated and the results are presented in Fig. 17. In this particular case, the subject was a young man who had consumed a commercial insecticide that contained chlorpyrifos. The subject was admitted to the hospital within --5 hr of the ingestion, and blood samples were repeatedly taken and analyzed for both chlorpyrifos and the oxon metabolite. Although Drevenkar et al. reported that oxon was not detectable in any of the samples, chlorpyrifos was readily measured (1-10 Ixmol/liter blood) as late as 15 days postexposure. As shown in Fig. 17, the PBPK model reasonably simulated the serum chlorpyrifos time course, and the predicted dosage is well within the range to elicit acute toxicity (>150 mg/kg). These PBPK/PD models provide a reasonably good prediction of dosimetry and biological response, but it is important to recognize that the predictive capabilities of these models are limited by the adequacy of the parameters and limitations of the experimental data (Timchalk et al., 2002b). However, it is anticipated that these basic structures can be used as a starting point for the development of other OP and CM insecticide models. Once validated, these models can then be used to understand complex mixture interactions, sensitive subpopulations, and the role of metabolic polymorphisms in altering dosimetry and biological response.
100 10
::t,.
0.1 0.01
0
5'0
100
1;0
2;0
250
300
3;0
4;0
Time (hrs) FIG. 17. Time course of chlorpyrifos (CPF) in the serum of a single poison victim who orally ingested a commercial insecticide product containing CPF (data from Drevenkar et al., 1993). The symbols represent observed data, whereas the line represents the model prediction. Adapted with permission from Timchalk et al. (2002).
V. SENSITIVE SUBPOPULATIONS: CHILDREN AND METABOLIC POLYMORPHISMS A. Children's Sensitivity There is currently a significant focus on and concern over the potential increased sensitivity of infants and children to the toxic effects of chemicals. The importance of this issue is highlighted by the National Research Council's report, "Pesticides in the Diets of Infants and Children," and the passage of the Food Quality Protection Act. It is recognized that children are not just "small adults" but, rather, a unique subpopulation that may be particularly vulnerable to chemical insult. Age-dependent changes in a child's physiology (i.e., body size, blood flow, and organ functions) and metabolic capacity (i.e., phase I and II metabolism) may significantly impact their response to a chemical insult, resulting in either beneficial or detrimental effects (Makri et al., 2004; Ginsberg et al., 2004; Johnson, 2003; Miller et al., 1997). Clear variability in the capacity to detoxify environmental chemicals has been established in both animals and humans. However, the current risk assessment paradigms may not adequately consider the implications of these differences on the risk to infants and children. Numerous studies have demonstrated that juvenile animals are more susceptible to the acute high-dose effects of OP insecticides than adults (Brodeur and DuBois, 1963; Benke and Murphy, 1975; Harbison, 1975; Gaines and Linder, 1986; Pope et al., 1991; Pope and Liu, 1997; Moser and Padilla, 1998). This greater sensitivity has primarily been attributed to the lack of complete metabolic competence during neonatal and postnatal development (Benke and Murphy, 1975). Several studies provide important perspective on this age- and dose-dependent sensitivity to OP insecticides. Collectively, they suggest that the age-dependent sensitivity in neonatal animals is associated with a lower CYP450 dearylation (detoxification) capacity and an age-dependent lower PON-1 and CarbE-mediated oxon hydrolysis capacity in neonates relative to adult animals (Atterberry et al., 1997; Li et al., 1997; Mortensen et al., 1996). These findings in animals are in agreement with observations in which newborn and young humans have lower metabolic capacity for CYP450 and PON-1 activity compared to adults (Johnson, 2003; Augustinsson and Barr, 1963; Mueller et al., 1983). The application of PBPK/PD modeling offers a unique opportunity to integrate age-dependent changes in metabolic activation and detoxification pathways into a comprehensive model that is capable of quantifying dosimetry and response across all ages (for review, see Corley et al., 2003). In this context, PBPK models are being extended to the modeling of chemical exposure in developing children (Price et al., 2003; Clewell et al., 2004) and in developing/neonatal animals (Fisher et al., 1990; Byczkowski et al., 1994; Sundberg et al., 1998).
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates
1 .E + 01 : 1 .E
+00
However, as illustrated in Fig. 19, the magnitude of brain AChE inhibition is clearly age- and dose-dependent, and although dramatically inhibited in neonatal rats (10 mg/kg response), the adults appear to be refractory to any AChE inhibition at these same dose levels. Although these simulations illustrate the ability to scale age-dependent changes within a species, the different time scales between development in neonatal animals and human infants create uncertainty in extrapolation across species (Ginsberg et al., 2004).
TCP
1
,
~
1.E-01 =. .E-02
CPF
1.E-03 1.E-04
0
5
1'0
1'5 Time (hrs)
2;
2;
3;
FIG. 18. Experimental data (symbols) and model simulations (lines) for the blood concentration of trichloropyridinol (TCP) and chlorpyrifos (CPF) in postnatal day 5 rats given an oral dose of 1 mg CPF/kg of body weight (Domoradski et al., 2004). Values represent the mean + SD for four or five animals per time point.
B. Genetic Polymorphisms A number of human and experimental animal studies have demonstrated a wide range of variability in the metabolism of drugs and xenobiotics (Mackenzie et al., 2000; Tucker, 2000; Jones et al., 1995; Gonzalez and Gelboin, 1993). A human genetic polymorphism in the PON-1 detoxification of several OP insecticides, including the active metabolite of chlorpyrifos, chlorpyrifos-oxon, has been well established, resulting in the expression of a range of PON-1 enzyme activity within a segment of the population (Cowan et al., 2001; Furlong et al., 1998; Eckerson et al., 1983; Geldmacher-von Mallinckrodt et al., 1983). The PBPK/PD model for chlorpyrifos was used to assess the potential contribution of the human PON-1 (chlorpyrifos-oxonase) polymorphism on chlopyrifos dosimetry (Timchalk et al., 2002a), based on the distribution of chlorpyrifos-oxonase activities reported in human serum (Davies et al., 1996). A comparison of the dose-dependent
Based on the potential sensitivity of children to OP insecticides, there is a need to develop quantitative models that can be used to assess the risk associated with exposure in infants and children. The PBPK/PD model for chlorpyrifos has been modified to scale both the metabolism and the esterase levels based on the age of the animal. The time course for chlorpyrifos and the major metabolite trichloropyridinol in postnatal day 5 (PND 5) rats is illustrated in Fig. 18, and the time course of AChE inhibition as a function of dose and age is presented in Fig. 19. It is of interest that even in PND 5 animals, CYP450 activity is adequate to metabolize chlorpyrifos to trichloropyridinol and the PBPK/PD model provides a reasonable simulation.
PND 5 Brain AChE
PND 12 ChE
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j
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o
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l mg/kg
1 mg/kg
Y 10mg/kg T/
80 60
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Time (hrs)
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20
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Adult Brain AChE
PND 17 ChE
-5100 , , - - - ~ , 8o
30
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oo 6o "~ 4o 2o o
10 mg/kg 0
10
20
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FIG. 19. Experimental data (symbols) and model simulations (lines) for the brain AChE response in neonatal (PND 5 to PND 17) and adult rats given chlorpyrifos (CPF) as oral doses of 1 or 10 mg CPF/kg of body weight. Values represent the mean _ SD for four or five animals per time point.
1 20
SECTION II 9P h a r m a c o k i n e t i c s & M e t a b o l i s m
TABLE 5. Comparison of Theoretical Brain-oxon AUC Calculated Utilizing Monte Carlo Simulation for the QQ, OR, and RR PON 1 Polymorphism in Humans Following a Single-Dose Exposure to Chlorpyrifos (CPF) at Different Doses a Theoretical brain CPF-oxon (AUC) b CPF dose (mg/kg) PON1 QQ (low) Mean _+ SD CV
Maximum Minimum QR (medium) Mean _+ SD CV Maximum Minimum RR (high) Mean _+ SD CV
0.005
0.05
0.5
5.0
0.06 _____.001 (1.2) c 24% 0.15 0.03
0.63 __ 0.16 (1.31) 26% 1.66 0.36
11.6 + 6.54 (1.63) 56% 72.9 4.64
616 _____333 (1.72) 54% 4350 245
0.05 + 0.01 (1.0) 17% 0.10 0.04
0.57 + 0.19 (1.19) 18% 1.06 0.36
9.37 __+3.16 (1.31) 34% 32.0 4.73
492 __ 158 (1.38) 32% 1600 245
0.05 __+0.01 20% 0.09 0.03
0.48 -+- 0.10 20% 0.99 0.29
7.11 __ 2.45 34% 26.7 3.57
358 __ 123 34% 1310 171
Maximum Minimum
aAdapted from Timchalk et al. (2002a) with permission. bAUC = (txmoles liter -1 hr -1) x 10 -6. These theoretical AUCs are based on model predictions; at these dose levels, the CPF-oxon brain concentration is below the limits of quantitation (Timchalk et al. 2002b). cValues in parentheses represent the ratio of AUC for the QQ and QR relative to the RR polymorphism.
theoretical brain chlorpyrifos-oxon area under the concentration curve for the various chlorpyrifos-oxonase polymorphisms is presented in Table 5. These simulations suggest that the response is relatively insensitive to changes in chlorpyrifos-oxonase activity at low doses (-5 txg/kg); however, with increasing dose (-0.5-5 mg/kg) chlorpyrifos-oxonase status may be an important determinant of sensitivity (Timchalk et al., 2002a). A simulation of the dose response for plasma cholinesterase activity in humans following acute exposure to a broad range of chlorpyrifos doses is presented in Fig. 20. Timchalk et al. (2002a) proposed that other B-esterase detoxification pathways (e.g., plasma cholinesterase) may
100
-~ 80
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0.001
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~
/
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,
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adequately compensate for lower chlorpyrifos-oxonase activity; hence, an increased sensitivity to low chlorpyrifoso-xonase is not observable until nontarget esterases have been appreciably depleted. Pharmacokinetics and, in particular, the application of PBPK/PD modeling have been shown to be extremely useful approaches for dosimetry and biological response extrapolation for the assessment of human health risk from chemical exposures. The utilization of PBPK/PD modeling to address OP and CM insecticide toxicity issues is particularly intriguing since these models can be used to assess the health consequences of both interindividual (i.e., age and gender) and extrinsic factors (i.e., multiple exposure routes, chemical/drug interactions, and variable exposure rates) that may significantly modify the toxicological response.
.
.
.
.
.
.
.
.
.
I
VI. S U M M A R Y A N D C O N C L U S I O N S
.
O.1 Dose (mg/kg)
1
'
. . . . . . .
1
10
FIG. 20. Simulation of peak plasma BuChE inhibition doseresponse in humans following an acute exposure to a broad range of chlorpyrifos doses. Adapted with permission from Timchalk et al. (2002a).
This chapter has illustrated the applications of PBPK/PD modeling to assess OP and potentially CM insecticide dosimetry, biological response, and risk in humans exposed to these insecticides. Pharmacokinetics is concemed with the quantitative integration of absorption, distribution, metabolism, and excretion and can be used to provide insight into the toxicological responses associated with
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates these insecticides. Since OP and CM insecticides share a common mode of action through their capability to inhibit AChE activity, it is feasible to develop pharmacokinetic strategies that link quantitative dosimetry with biologically based PD response modeling. Pharmacokinetic studies that have been conducted in multiple species, at various dose levels, and across different routes of exposure have provided important insight into the in vivo behavior of these insecticides. The development and application of PBPK/PD modeling for these insecticides represent a unique opportunity to quantitatively assess human health risk and to understand the toxicological implications of known or suspected exposures. Validated PBPK/PD models can be used to consider the potential variability in human response associated with both interindividual (i.e., age, gender, and polymorphism) and extrinsic variability (i.e., exposure routes and rates and single vs. multiple exposures).
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12 2
S E CTI O N I I 9Pharmacokinetics & Metabolism
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SECTION II 9Pharmacoldnetics & M e t a b o l i s m
Murphy, S. D. (1986). Toxic effects of pesticides. In Casarett and Doull's Toxicology, the Basic Science of Poison (C. D. Klaassen, M. O. Amdur, and J. Doull, Eds.), 3rd ed., pp. 519-581. Macmillan, New York. Neal, R. A. (1980). Microsomal metabolism of thiono-sulfur compounds, mechanisms and toxicological significance. In Reviews in Biochemical Toxicology (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), Vol. 2, pp. 131-172. Elsevier-North Holland, New York. Nolan, R. J., Rick, D. L., Freshour, N. L., and Saunders, J. H. (1984). Chlorpyrifos: Pharmacokinetics in human volunteers. Toxicol. Appl. Pharmacol. 73, 8-15. Oak Ridge National Laboratory (2000). Appendix G: Inhibition of cholinesterases and an evaluation of the methods used to measure cholinesterase activity. J. Toxicol. Environ. Health A 59, 519-526. O'Flaherty, E. J. (1995). PBK modeling for metals. Examples with lead, uranium and chromium. Toxicol. Lett. 82/83, 367-372. Pelekis, M., and Krishnan, K. (1997). Assessing the relevance of rodent data on chemical interactions for health risk assessment purposes: A case study with dichloromethane-toluene mixture. Regul. Toxicol. Pharmacol. 25, 79-86. Pena-Egido, M. J., Rivas-Gonzalo, J. C., and MarinoHernandez, E. L. (1988). Toxicokinetics of parathion in the rabbit. Arch. Toxicol. 61, 196-200. Poet, T. S., Wu, H., Kousba, A. A., and Timchalk, C. (2003). In vitro rat hepatic and intestinal metabolism of the organophosphate pesticides chlorpyrifos and diazinon. Toxicol. Sci. 72, 193-200. Poet, T. S., Kousba, A. A., Dennison, S., and Timchalk, C. (2004). Physiologically based pharmacokinetic/pharmacodynamic model for the organophosphate pesticide diazinon. Neurotoxicology 25(6), 1013-1030. Pond, A. L., Chambers, H. W., and Chambers, J. E. (1995). Organophosphate detoxification potential of various rat tissues via A-esterase and aliesterase activity. ToxicoL Letr 78, 245-252. Pope, C. N., and Liu, J. (1997). Age-related differences in sensitivity to organophosphorus pesticides. Environ. Toxicol. Pharmacol. 4, 309-314. Pope, C. N., Chakraborti, T. K., Chapman, M. L., Farrar, J. D., and Arthun, D. (1991). Comparison of the in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides. Toxicology. 68, 51-61. Poulin, P., and Krishnan, K. (1995). An algorithm for predicting tissue: Blood partition coefficients of organic chemicals from n-octonol:water partition coefficient data. J. Toxicol. Environ. Health 46, 117-129. Poulin, P., and Krishnan, K. (1996). Molecular structure-based prediction of the partition coefficients of organic chemicals for physiological pharmacokinetic models. Toxicol. Methods 6, 117-137. Price, K., Haddad, S., and Krishnan, K. (2003). Physiological modeling of age-specific changes in the pharmacokinetics of organic chemicals in children. J. Toxicol. Environ. Health A 66, 417-433. Ramsey, J. C., and Andersen, M. E. (1984). A physiologically based description of the inhalation pharmacokinetics of styrene in rats and humans. Toxicol. Appl. Pharmacol. 73, 159-175.
Renwick, A. G. (1994), Toxicokinetics-pharmacokinetics in toxicology. In Principles and Methods of Toxicology (A. W. Hayes, Ed.), 3rd ed., pp. 101-148. Raven Press, New York. Savolainen, K. (2001). Understanding the toxic actions of organophosphates. In Handbook of Pesticide Toxicology (R. I. Krieger, Ed.), Vol. 2, pp. 1013-1041. Academic Press, San Diego. Shealy, D. B., Barr, J. R., Ashley, D. L., Patterson, D. G., Jr., Camann, D. E., and Bond, A. E. (1997). Correlation of environmental carbaryl measurements with serum and urinary 1-naphthol measurements in a farmer applicator and his family. Environ. Health Perspecr 105(5), 510-513. Sogorb, M. A., and Vilanova, E. (2002). Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis. Toxicol. Letr 128, 215-228. Srinivasan, R. S., Bourne, D. W. A., and Putcha, L. (1994). Application of physiologically based pharmacokinetic models for assessing drug disposition in space. J. Clin. Pharmacol. 34, 692-698. Sultatos, L. G. (1988). Factors affecting the hepatic biotransformation of the phosphorothioate pesticide chlorpyrifos. Toxicology 51, 191-200. Sultatos, L. G. (1990). A physiologically based pharmacokinetic model of parathion based on chemical-specific parameters determined in vitro. J. Am. Coll. Toxicol. 9(6), 611-619. Sultatos, L. G. (1994). Mammalian toxicology of organophosphorus pesticides. J. Toxicol. Environ. Health 43, 271-289. Sultatos, L. G., Basker, K. M., Shao, M., and Murphy, S. D. (1984a). The interaction of the phosphorothioate insecticides chlorpyrifos and parathion and their oxygen analogues with bovine serum albumin. Mol. Pharmacol. 26(1), 99-104. Sultatos, L. G., Shao, M., and Murphy, S. D. (1984b). The role of hepatic biotransformation in mediating the acute toxicity of the phosphorothionate insecticide chlorpyrifos. Toxicol. Appl. Pharmacol. 73, 60-68. Sultatos, L. G., Kim, B., and Woods, L. (1990). Evaluation of estimations in vitro of tissue/blood distribution coefficients for organothiophosphate insecticides. Toxicol. Appl. Pharmacol. 103, 52-55. Sundberg, J., Jonsson, S., Karlsson, M. O., Palminger Hallen, I., and Oskarson, A. (1998). Kinetics of methylmercury and inorganic mercury in lactating and nonlactating mice. Toxicol. Appl. Pharmacol. 151, 319-329. Taylor, P. (1980). Anticholinesterase agents. In Goodman and Gilman's The Pharmacology of Therapeutics (A. Goodman Gilman, L. S. Goodman, and A. Gilman, Eds.), 6th ed., pp. 100-119. Macmillan, New York. Timchalk, C., Kousba, A., and Poet, T. S. (2002a). Monte Carlo analysis of the human chlorpyrifos-oxonase (PON1) polymorphism using a physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model. Toxicol. Lett. 135, 51-59. Timchalk, C., Nolan, R. J., Mendrala, A. L., Dittenber, D. A., Brzak, K. A., and J. L Mattsson (2002b). A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for the organophosphate insecticide chlorpyrifos in rats and humans. Toxicol. Sci. 66, 34-53.
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates Timchalk, C., Poet, T. S., Hinman, M. N., Busby, A. L., and Kousba, A. A. (2005). Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat. Toxicol. Appl. Pharmacol. 205, 31-42. Timchalk, C., Poet, T. S., Kousba, A. A., Campbell, J. A., and Lin, Y. (2004). Noninvasive biomonitoring approaches to determine dosimetry and risk following acute chemical exposure: Analysis of lead or organophosphate insecticide in saliva. J. Toxicol. Environ. Health A 67, 635-650. Tobia, A. J., Pontal, E G., McCahon, E, and Carmichael, N. G. (2001). Aldicarb: Current science-based approaches in risk assessment. In Handbook of Pesticide Toxicology (R. I. Krieger, Ed.), Vol. 2, pp. 1107-1121. Academic Press, San Diego. Tomokuni, K., Gasegawa, T., Hirai, Y., and Koga, N. (1985). The tissue distribution of diazinon and the inhibition of blood cholinesterase activities in rats and mice receiving a single intraperitoneal dose of diazinon. Toxicology 37, 91-98. Tos-Luty, S., Tokarska-Rodak, M., Latuszynski, J., and Prezebirowska, D. (2001). Dermal absorption and distribution of 14C-carbaryl in Wistar rats. Ann. Agric. Environ. Med. 8(1), 47-50.
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Tucker, G. T. (2000). Advances in understanding drug metabolism and its contribution to variability in patient response. Ther. Drug Monit. 22(1), 110-113. Vale, J. A. (1998). Toxicokinetic and toxicodynamic aspects of organophosphate (OP) insecticide poisoning. Toxicol. Lett. 102-103, 649-652. Vale, J. A., and Scott, G. W. (1974). Organophosphate poisoning. Guy's Hosp. Gazette 123, 12-25. Wenthold, R. J., Mahler, H. R., and Moore, W. J. (1974). The halflife of acetylcholinesterase in mature rat brain. J. Neurochem. 22, 941-943. Wilks, M. F., and Woollen, B. H. (1994). Human volunteer studies with nonpharmaceutical chemicals: Metabolism and pharmacokinetic studies. Hum. Exp. Toxicol. 13(6), 383-392. Wilson, B. W. (2001). Cholinesterases. In Handbook of Pesticide Toxicology (R. I. Krieger, Ed.), Vol. 2, pp. 967-986. Academic Press, San Diego. Woollen, B. H. (1993). Biological monitoring for pesticide absorption. Occup. Hyg. 37(5), 525-540. Wu, H. X., Evereux-Gros, C., and Descotes, J. (1996). Diazinon toxicokinetics, tissue distribution and anticholinesterase activity in the rat. Biomed. Environ. Sci. 9, 359-369.
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Metabolism of Organophosphorus and Carbamate Pesticides JUN TANG 1,* RANDY L. ROSE, z AND JANICE E. CHAMBERS 3 1 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 2North Carolina State University, Raleigh, North Carolina 3Mississippi State University, Mississippi State, Mississippi
most important phase II enzymes include glutathione transferases, glucuronosyl transferases, sulfotransferases, and acetyl transferases. More often than not, these metabolic processes are detoxication reactions. However, there are some cases in which metabolism through either phase I or phase II can make the chemical more reactive than the parent compound. As will be shown later, this is particularly true in the case of OP compounds, in which the conversion of a P---S moiety to a P---O group can result in increasing toxicity substantially. Usually, however, metabolic events that increase the water solubility of a chemical cause significant reductions in its biological half-life by making it more readily excreted. This chapter provides an overview of the characteristics of enzymes catalyzing pesticide metabolism and common metabolic pathways of OP and CM pesticides. Also, it presents a recent trend of using human tissue for the in vitro study of pesticide metabolism, which may provide more relevant data to human health.
I. I N T R O D U C T I O N Because of the lipophilic nature of biological membrane systems, nonpolar compounds readily pass through the plasma membrane and are absorbed. Once entering into the body, these lipophilic compounds are difficult to excrete without being transformed to more polar metabolites. Therefore, metabolism, the enzyme-mediated processes that transform compounds to more hydrophilic metabolites, is one of the most important determinants of pesticide persistence in the body and subsequent toxicity to the organism. Generally, the study of xenobiotic metabolism focuses on the recognition of enzymes involved and identification of metabolites generated as well as the understanding of the effects of xenobiotics on the metabolic enzymes. Several families of metabolic enzymes, often with wide arrays of substrate specificity, are involved in organophosphorus (OP) and carbamate (CM) pesticide metabolism. These metabolic enzymes are often divided into two distinct groups, referred to as phase I and phase II enzymes. Phase I enzymes introduce a polar reactive group onto the molecule, making it more water soluble while also increasing the possibility for further metabolism by phase II enzymes. Enzymes typically involved in phase I metabolism include cytochrome P450-dependent monooxygenases (CYPs), flavin-containing monooxygenases (FMOs), and hydrolases. Phase II enzymes often conjugate the polar groups introduced by phase I enzymes (e.g., OH, N = O , and epoxide groups), to introduce more bulky hydrophilic substituents, such as sugars, sulfates, or amino acids, into the molecule. This substantially increases the water solubility of the chemical, making it more easily excreted. The
II. X E N O B I O T I C M E T A B O L I Z I N G ENZYMES
A. Phase I E n z y m e s Phase I metabolizing enzymes include the CYP and FMO enzymes, esterases, and epoxide hydrolases, among others. These enzymes and the reactions they catalyze have been described in detail previously (Hodgson and Goldstein, 2001; Parkinson, 2001). The CYP and FMO families are perhaps the best characterized because they are capable of catalyzing a wide range of reactions with broad substrate specificity. These enzymes are located in the endoplasmic reticulum of the cell and have been studied in many organs and tissues.
*Current address: Cerep, Inc., Redmond, Washington
Toxicology of Organophosphate and Carbamate Compounds
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1. THE CYTOCHROME P450-DEPENDENT MONOOXYGENASE SYSTEM The prefix CYP denotes the most important superfamily of xenobiotic metabolizing enzymes (XME) known. Originally described as a single protein, there are now known to be more than 1900 CYPs widely distributed throughout animals, plants, and microorganisms. The Web site, http ://drnelson.utmem.edu/CytochromeP450.html, catalogs the nomenclature and discovery of CYPs. Although many organs and tissues express CYP and other enzymes, in vertebrates the liver is the primary organ of xenobiotic metabolism. Portals of entry also tend to be high in CYP content, including the skin, nasal mucosa, lung, and gastrointestinal tract. CYPs have also been found in skin, kidney, adrenal cortex and medulla, placenta, testes, ovaries, fetal and embryonic liver, corpus luteum, aorta, blood platelets, and the nervous system. The name cytochrome P450 derives from the characteristic difference spectrum that has an absorbance maximum at 450 nm when the reduced cytochrome binds to CO. This CO difference spectrum is also commonly used as a quantitative estimate of CYP content. Other difference spectra, formed in response to different organic and inorganic ligands, have been used for several years in the characterization of CYPs. CYP oxidation reactions involve a complex series of steps that have been well defined (Rose and Hodgson, 2004). The initial step involves the binding of substrate to oxidized CYP, followed by a one-electron reduction catalyzed by NADPH cytochrome P450 reductase to form a reduced cytochrome-substrate complex. The next several steps involve interaction with molecular oxygen, the acceptance of a second electron from NADPH cytochrome P450 reductase or NADH cytochrome b5 reductase, followed by the subsequent release of water and the oxygenated product of the reaction. This complicated reaction sequence results in the transfer of one atom of molecular oxygen to the substrate while the other atom is reduced to water. A system of nomenclature utilizing the prefix CYP has been devised for the genes and cDNAs corresponding to the different forms, although P450 is also used as a prefix for the protein products. The original nomenclature system, proposed in 1987, was updated in 1996 (Nelson et al., 1996). This system designates cytochrome P450 genes as CYP (or Cyp in the case of mouse genes). Arabic numerals are then added to CYP to denote gene families, followed by a letter to designate a subfamily. Individual isoforms are identified by the use of a second Arabic numeral following the subfamily designation. Polymorphic isoforms are indicated using an asterisk followed by an Arabic numeral. In the absence of subfamilies, or if there is only one gene within the family or subfamily, the letter and/or the second numeral may be omitted (e.g., CYP17). The name of the gene is italicized, whereas the protein (enzyme) is not. As many as 18 CYP families have been identified in mammals. Families 1-3 are primarily responsible for xeno-
biotic metabolism. Some gene families (including subfamilies and even isoforms) may be found in several species (e.g., CYP1A1). In other cases, genes have diverged from species to species, and no exact analogue is found in various species (e.g., the CYP2C subfamily). Even though there may be some level of gene identity among species, gene products do not necessarily share similar substrate specificity due to differences at the binding site. The abundance of CYP subfamilies in particular organs is also different from species to species (e.g., the most abundant CYP subfamily in rat liver is CYP2C, whereas in human liver it is CYP3A). Reactions catalyzed by CYPs require several elements and generally, although not always, result in oxygenated metabolites. Successful in vitro reactions using microsomal tissues require NADPH and oxygen as essential cofactors. Purified CYP isoforms require a source of lipid, such as phosphatidylcholine, and the necessary coenzyme NADPH cytochrome P450 reductase. In some cases, other microsomal components, such as cytochrome bs, may also be required for optimal metabolic activity. 2. THE FLAVIN-CONTAINING MONOOXYGENASE Like the CYP family, ravin-containing monooxygenases (FMOs) are membrane-bound metabolic enzymes that are capable of metabolizing a wide array of substrates. Like CYPs, these enzymes also utilize NADPH and 02, although NADPH cytochrome P450 reductase is not essential. Substrates that are metabolized by FMOs generally include soft nucleophiles (e.g., organic compounds with nitrogen, sulfur, phosphorus, or selenium heteroatoms). Known pesticide substrates include phorate, fonofos, coumaphos, and methiocarb. Although most metabolism by FMOs results in detoxication, there are some examples of substrates that are activated by FMO oxidation, particularly in the case of substrates involving sulfur oxidation. 3. ESTERASES Many pesticides have ester bonds, amide bonds, or substituted phosphate ester groups that are susceptible to hydrolysis by esterases. Enzymes with carboxylesterase (CarbE) and amidase activities are found in both microsomal and soluble fractions from a variety of tissues. Although functionally ester and amide groups are different, no CarbE has been found that does not have hydrolytic activity toward the corresponding amide and vice versa. Because of the large numbers of esterases in many tissues and fractions, as well as the large number of substrates that they are capable of hydrolyzing, classification of esterases has been difficult. One traditional classification scheme derived by Aldridge (1953) divides esterases into the A-, B-, and C-esterases based on their behavior toward phosphate triesters such as paraoxon. In this scheme, A-esterases, also known as arylesterases, are capable of hydrolyzing esters derived from aromatic compounds, including many organophosphate oxons. Mammalian A-esterases are calcium dependent.
CHAPTER 1 0 9Metabolism of Anti-ChE Pesticides B-esterases, although capable of binding to the same compounds, are inhibited by them as a result of their phosphoryl groups binding to the enzymes. C-esterase, or acetylesterases, prefer acetyl esters as substrates and do not bind to the organophosphate substrates. Many esterases have also been classified on the basis of their metabolic activities toward a variety of artificial substrates. This classification scheme, however, is unsatisfactory because esterases are highly promiscuous in their substrate specificity and have high potential for many polymorphic forms as well as variations in glycosylation and other posttranslational modifications that may vary activity. A more useful method involves molecular identification of the genes followed by analysis of evolutionary divergence (Satoh and Hosokawa, 1998). Methods have been published that allow the classification of two types of esterases, the carboxylic ester hydrolases (CEHs) and the phosphoric triester hydrolases (PTEHs) (Anspaugh and Roe, 2004). The CEHs contain the B-esterases, which are inhibited by organophosphates. B-esterases include many other esterases, such as CarbE, acetylcholinesterase (ACHE), cholinesterases (ChE), arylesterases, sterol esterases, insect juvenile hormone esterases, and others. The determination of A-esterases uses a protocol for the detection of PTEHs. The PTEH assay allows for the identification of tWO subclasses of esterases, the A-esterase (known as aryldialkylphosphatase) and diisopropyl fluorophosphatase. Both these enzymes metabolize OP compounds. More detailed descriptions of CarbE and paraoxonases and their interactions with OPsiand CMs can be found in Chapters 16 and 18 of this book, respectively. B. Phase II E n z y m e s As described previously, the role of phase II enzymes is to add bulky hydrophilic substituents to xenobiotics to make them more easily excreted. Although some xenobiotics can be directly attacked by conjugating enzymes, most require biotransformation by phase I enzymes prior to conjugation. Such biotransformation produces many of the substituents that are preferred by conjugating enzymes that include hydroxyl, amino, carboxyl, epoxide, sulfhydryl, and halogen groups. Because conjugation generally adds bulky polar substituents to the molecule, products are highly water soluble and easily excreted. Thus, conjugation products are most often less toxic than the parent compound, although there are many examples in which conjugation products are ultimate toxicants. There are two types of mechanistic reactions involved in conjugations. In the first (type I), the xenobiotic reacts with a reactive conjugating ligand to produce a conjugation product. In the second (type II), the substrate is activated and then combines with the conjugating ligand to form the conjugated product. Type I products include sulfates and glycosides, whereas type II products are often products of glutathione or amino acid conjugation. More detailed reviews relating to
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conjugation pathways can be found in Rose and Hodgson (2004) and LeBlanc and Dauterman (2001). 1. GLUTATHIONETRANSFERASES The glutathione transferases are soluble dimeric proteins that are composed of identical subunits, although some forms are heterodimers. These enzymes are present in most tissues and are primarily cytosolic (approximately 95%), although a small percentage are found in the endoplasmic reticulum (approximately 5%). The cytosolic glutathione S-transferases (GSTs) are divided into six families or classes: alpha, kappa, mu, pi, sigma, and theta. The alpha, mu, and pi GST classes are primarily responsible for most of the catalytic activity associated with liver detoxication of xenobiotics. Glutathione is a necessary high-energy cofactor for GST, because it is composed of three amino acids: glycine, cysteine, and glutamic acid. Because glutathione is a nucleophile, it is possible for glutathione to directly interact with electrophilic substituents in the absence of enzyme to form conjugation products. The relatively high concentration of glutathione in liver (approximately 10 mM) certainly would facilitate such interactions; however, the stereoselective nature of many glutathione conjugates and the relatively high levels of GST enzyme suggest that many reactions are catalyzed by the enzyme. In vivo products resulting from glutathione conjugation may be excreted intact in the bile or may be converted to mercapturic acids in the kidney and excreted in the urine. GSTs catalyze a wide variety of reactions. The groups attacked include epoxides, haloalkanes, nitroalkanes, alkenes, methyl sulfoxide derivatives, and aromatic haloand nitro- compounds. Because many of these electrophiles are reactive and capable of binding to critical nucleophiles, including proteins and nucleic acids, conjugation represents an important detoxication reaction. 2. GLUCURONOSYLTRANSFERASES The most common form of conjugation found in the plant and animal kingdom is glycosylation. In plants and many invertebrates, the major form of glycosylation involves glucosidation, which transfers the glucose from uridine diphosphate glucose to xenobiotics. In vertebrates, the major conjugative pathway is glucuronidation, which transfers glucuronic acid from uridine diphosphate glucuronic acid (UDPGA) to xenobiotics. The enzyme catalyzing glucuronidation is the uridine diphosphate glucuronosyltransferase. Glucuronidation reactions predominantly take place in the liver, intestinal mucosa, and kidney, although other organs and tissues possess lower amounts of the enzyme. The enzyme is primarily found in microsomal tissues. Most glucuronide conjugates are excreted in the urine or bile but may be hydrolyzed by [3-glucuronidases prior to excretion. Substrates suitable for glucuronide conjugation contain one of many functional groups, including phenols, aliphatic alcohols, carboxylic :acids, hydroxylamines, thiophenols,
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thiols, thiocarbamic acids, aromatic amines, and hydroxylamines. As is true for many other conjugating enzymes, glucuronidation generally produces detoxication products; however, there are some examples in which glucuronide conjugation results in greater toxicity. 3. SULFOTRANSFERASES The sulfotransferase (SULT) enzymes belong to a superfamily of enzymes that catalyze sulfation reactions. The sulfation reaction involves high-energy inputs from the cell because two molecules of ATP are necessary for the synthesis of one molecule of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The amino acids cysteine and methionine are the major sources of sulfate required for PAPS synthesis. Cellular concentrations of PAPS are considerably less than those of UDPGA and glutathione, limiting the capacity for sulfation. Hydroxyl groups of phenols, alcohols, and N-substituted hydroxylamines are the most common groups attacked by SULT. Sulfation of thiols and amines has also been reported. In many species, sulfation and glucuronidation are competing for the same substrates. At lower substrate concentrations, sulfation generally provides for the most efficient substrate conjugation, whereas at higher substrate levels the glucuronidation reaction assumes greater importance. Sulfate conjugates are highly water soluble and are readily excreted. Although these reactions generally detoxify xenobiotics, many sulfate conjugates have been implicated in carcinogen activation, prodrug processing, cellular signaling pathways, and the regulation of many endogenous chemicals, including thyroid hormones, steroids, and catechols (Rose and Hodgson, 2004). 4. ACETYLTRANSFERASES Acetyltransferases catalyze the addition of acetyl groups to xenobiotics containing amino or hydroxyl groups. Two types of acetyltransferases are involved. The first involves the addition of acetyl CoA to the xenobiotic and the second involves the activation of a xenobiotic to form an acyl CoA intermediate, which then reacts with an amino acid to form an amino acid conjugate. Several types of amino groups can be biotransformed by acetylation, including arylamino, aliphatic amino, oL-amino, hydrazine, and sulfonamide groups. The addition of the acetyl group generally masks the amine group with a nonionizable acetyl moiety, producing xenobiotics that are less water soluble than the parent compound. Despite this, in some cases acetylation still facilitates urinary excretion.
III. M E T A B O L I S M O F
ORGANOPHOSPHORUS A N D CARBAMATE PESTICIDES It is not sufficient to evaluate toxicological significance by monitoring parent compounds alone because many metabolites of OP and CM pesticides have similar or
greater toxicity. Decreasing parent compound concentration does not necessarily mean reduction of toxicity (Montesissa et al., 1994). It is important to identify metabolites and understand the metabolic pathways. The study of metabolism will also elucidate which enzymes are involved in the biotransformation of pesticides and at what rate. It has been shown that metabolic enzymes from different species or even individual humans have different substrate specificities and/or activities toward pesticides; this may be the reason for interspecies and/or intraspecies differences. Synergism, potentiation, antagonism, or other effects of exposure to chemical mixtures can also result from interactions of different pesticides with a common enzyme that metabolizes either of them, causing inhibition or activation of metabolic rate. An understanding of the metabolic pathways and enzymes involved will provide the insight to evaluate the uncertainties of inter- and intraspecies extrapolation and mixture effects in risk assessment and, therefore, to ensure safe use of pesticides.
A. Metabolism of OP Insecticides The OP insecticides, frequently called colloquially organophosphates (OPs), are a widely used and readily metabolized group of pesticides. It is their chemical instability to metabolism (and also environmental degradation) that led to their displacement of the more chemically and metabolically stable organochlorine insecticides (i.e., DDT and the chlorinated cyclodienes) that were the insecticides of choice during the 1950s and 1960s. With the concerns of the late 1960s regarding the environmental persistence and the bioaccumulation of these organochlorine insecticides, the OPs came into prominence because of their lack of persistence and despite the high acute toxicity levels of some of these. The OP insecticides, as discussed elsewhere in this book, have as a primary mechanism of acute toxicity the inhibition of the critical and widespread nervous system enzyme ACHE. However, the anticholinesterase (anti-ChE) potencies do not correspond with the acute toxicity levels (Chambers et al., 1990), indicating that metabolism is an important factor in determining the overall toxicity level. The OP insecticides evolved from the chemical technologies of World War II, which were used to develop the antiChE nerve agents. The OP insecticides bear some chemical resemblance to these nerve agents but are generally less toxic, often require metabolic activation to display anti-ChE activity and therefore are slower to act, and usually have more complex chemical substituents. The OP insecticides are characterized by a pentavalent phosphorus atom bound to three organic substituents by single bonds (frequently through an oxygen) and bound to an oxygen or sulfur by a coordinate covalent bond (usually depicted in print as a double bond). One of the organic
CHAPTER 1 0 9Metabolism of Anti-ChE Pesticides substituents is called the "leaving group" because it is bound through the least chemically stable of the three single bonds, and this is the substituent that "leaves" as the compound phosphorylates its target enzyme; this is also the substituent that is removed from the central phosphorus when the compound is hydrolyzed or oxidatively cleaved. The leaving group is usually the most complex substituent chemically, frequently involving an aromatic or heterocyclic ring. The other two organic substituents are frequently simple alkoxy groups, such as methoxy or ethoxy. The history, chemistry, and toxicity of the OP insecticides have been reviewed in the following references: Eto (1961), Fest and Schmidt (1982), Chambers and Levi (1992), H. W. Chambers et al. (2001), J. E. Chambers et al. (2001), and Ecobichon (2001). The OP insecticides display a broad range of acute toxicity levels, and these can be found in Meister (1990). This section provides an overview of the metabolic pathways most commonly seen for compounds within the OP insecticide class. However, the reader is referred to Aizawa (1982, 1989), J. E. Chambers et al. (1995, 2001), and Dikshith (1991) for more detailed summaries of the metabolic pathways for individual insecticides. 1. PHASE I METABOLISM: OXIDATION As summarized previously, the phase I reactions are involved in adding or uncovering a reactive or functional group on a xenobiotic and are usually the initial steps of metabolism. Because of the variety of types of atoms present in OP insecticides (e.g., C, O, P, N, S, and C1) and the variety of groups present (e.g., acids, alcohols, esters, and ethers), there are many opportunities for phase I reactions. Oxidations mediated by CYPs and FMOs and hydrolyses mediated by A-esterases are among the most prominent phase I reactions. In addition, the stoichiometric destruction of OPs as they phosphorylate target and nontarget serine esterases is an important mechanism of detoxication. These reactions are discussed next. a. Desulfuration Although many of the insecticides in other chemical classes are toxic in their original parent form, this is not true for many of the OP insecticides, especially those of the phosphorothionate configuration, characterized by a P = S group. There are many OP insecticides that have been or are currently widely used that are phosphorothionates; examples include p~athion, methyl parathion, chlorpyrifos, and diazinon. The insecticides possessing a P - - S group are usually not very potent anti-ChEs, and they require bioactivation to their P = O metabolites, called oxons, in order to display appreciable anti-ChE potency. The oxons of some phosphorothionates, such as paraoxon and chlorpyrifos-oxon, are approximately 1000-fold more potent than their parent insecticides, parathion and chlorpyrifos (Forsyth and Chambers, 1989). This bioactivation is mediated by CYPs through an attack on the sulfur by oxygen to create a theorized unstable phosphooxythiiran intermediate (a three-membered ring composed of
131
P, O, and S) that subsequently decomposes to the oxon (P---O) metabolite plus an active form of S (S:) (Neal, 1980). Desulfuration can also be catalyzed by FMOs, where the attack by oxygen is on the phosphorus; this FMO-mediated desulfuration occurs only with phosphonates in which at least one P-C bond is present, such as in fonofos (Smyser and Hodgson, 1985; Smyser et al., 1985). AS mentioned previously, the oxon is the active AChE inhibitor, so it is the metabolite that is responsible for acute toxicity to the nervous system. However, the S: is reactive in situ and is capable of damaging surrounding proteins, including the CYPs. This destruction leads to a lack of linearity in the desulfuration reaction in in vitro assays. Because of the removal of the S from the OP molecule, this reaction has been termed desulfuration, and the reaction is essential for many of the OP insecticides to display neurotoxicity. Figure 1 illustrates the desulfuration of chlorpyrifos to its toxic metabolite chlorpyrifos-oxon as an example of this critical bioactivation reaction. b. Dearylation A reaction parallel to the desulfuration reaction is the CYPmediated dearylation reaction that arises from the same putative unstable phosphooxythiiran intermediate described previously. In this instance, instead of the removal of the S when the phosphooxythiiran ring decomposes, the leaving group is oxidatively cleaved, with the leaving group and the dialkyl phosphorothionate (or dialkyl phosphate) as products. Although this does not appear to be a classic oxidation, it is mediated by CYP since it requires NADPH and oxygen. Since it is the result of the phosphooxythiiran intermediate, the dearylation of the oxons does not occur. The dearylation reaction is a detoxication. Because dearylation and desulfuration compete with one another, there will be a ratio of bioactivation to detoxication occurring following CYP metabolism of phosphorothionates. This ratio varies with the particular CYP (Levi et al., 1988; Tang et al., 2001). The dearylation reaction of chlorpyrifos is also illustrated in Fig. 1. c. Dealkylation T h e desulfuration/dearylation reactions described previously are probably the most common of the CYP-mediated reactions on OP insecticides. However, the CYPs can also mediate the oxidative removal of one of the alkoxy groups, leaving, for example, a monoalkyl phosphorothionate and the aldehyde (Appleton and Nakatsugawa, 1972). Since this reaction will prevent subsequent desulfuration, the dealkylation reaction is a detoxication. The demethylation of methyl parathion to monomethyl parathion and formaldehyde is illustrated in Fig. 2. d. Oxidation of Leaving Group Substituents As indicated previously, the leaving groups contain a variety of chemistries, many of which are susceptible to CYP-mediated oxidations. The leaving groups can undergo oxidations such as hydroxylations. From a toxicological standpoint, these
132
SECTION II. P h a r m a c o k i n e t i c 5 & M e t a b o l i s m
O
N
CI
/ c, ~-"~~.-~-.c,
H5C2
I
HO
CYP
s~
c,
H502--O
\p~S(O)
~/~OH
Desulfuration 0 \\
H502~ / P / o
o
C2H5
N
,oYY /
H5C2
CI
c
Dearylation
/
~
CI
c,~-'~~.~c, Hydrolysis
H5C2~O
\ ,//o
I
C2H5
HO
~
CI
c,
OH
FIG. 1. Metabolism of chlorpyrifos.
.s
~
~o\..//- I I ..1
H3C /P'~O..-~.,~" H3C--O
No2
,q
Demethylation
0 II CH2
+
i~~-------~NO2
~o\~i I I ..1
H3C
/V~O,.,.'~'...,.,..~"
HO
FIG. 2. Demethylationof methylparathion. reactions are capable of making the molecule more polar, more hydrophilic, and more amenable to phase II conjugations; therefore, these reactions promote excretion.
and subsequently to the sulfone; the conversion of the sulfoxide to the sulfone is mediated only by CYP and not by FMO. These are bioactivation reactions. The sulfoxidation of phorate is shown in Fig. 3.
e. Sulfoxidation
One of the most noteworthy reactions of the FMOs within the OP insecticides is the FMO-mediated sulfoxidation of the sulfur ether in phorate or phorate-oxon; this reaction can also be catalyzed by CYP (Levi and Hodgson, 1992). This sulfur can be oxidized twice--to the sulfoxide initially
2. PHASE I METABOLISM: HYDROLYSIS
Because OPs are esters of phosphoric acid or phosphorothioic acid, they are susceptible to hydrolysis by the A-esterases. One of the most prominent of the OP insecticides, malathion, contains two carboxylic acid esters and is
CHAPTER 1 0 9Metabolism of Anti-ChE Pesticides
s,..s..s.,
O/P v C2H5 I O--C2H5 02H5
~
s,..s.
0
O/P v C2H5 I O--C2H5 02H5
therefore susceptible to hydrolysis by the CarbEs. Although not a true catalytic hydrolysis, the phosphorylation of serine esterases by organophosphates cleaves the compound, so the phosphorylation reaction accomplishes a stoichiometric hydrolysis. The hydrolytic reactions, both catalytic and stoichiometric, are very important detoxication reactions in the metabolism of the OP insecticides. a. A - E s t e r a s e s
As described previously, the A-esterases are calcium-dependent hydrolases capable of hydrolyzing OPs. The phosphorothionates do not serve as substrates, so only parent insecticides possessing a P---O group or the oxons can be hydrolyzed by the A-esterases. They are therefore capable of hydrolyzing and detoxifying the oxon metabolites of the phosphorothionates. The A-esterases hydrolyze the phosphate to the dialkyl phosphate and the leaving group, thereby destroying the anti-ChE activity of the compound. The hydrolysis of chlorpyrifos-oxon is illustrated in Fig. 1. Because of this action, they are a potentially significant route of detoxication of the oxons following oxon formation by CYP-mediated desulfuration. The A-esterases are also called paraoxonases, named for the hydrolysis of the active metabolite of parathion (i.e., paraoxon). The A-esterases display low affinity for many phosphates and oxons and a high affinity for only a very few OP compounds, most notably chlorpyrifosoxon and diazoxon, the active metabolites of the insecticides chlorpyrifos and diazinon, respectively (Chambers et aL, 1994; Furlong et al., 1989; Pond et al., 1996, 1998). These two oxons are hydrolyzed quite readily, and A-esterase hydrolysis is probably a major contributing factor to the moderate toxicity levels of these two insecticides. Despite the name of paraoxonase, the A-esterases have a low affinity for paraoxon, so it is unclear how significant the A-esterases are in detoxifying paraoxon and most of the other phosphates and oxons. However, these affinities are usually estimated in relatively dilute tissue homogenates. In intact tissue, they may be more able to hydrolyze even the low-affinity substrates. Aesterase-mediated hydrolysis of relatively low, more realistic concentrations of paraoxon was observed when high liver or serum concentrations were used in in vitro assays (Tang and Chambers, 1999). This result stands in contrast with the need to use high paraoxon concentrations to demonstrate A-esterase-mediated hydrolysis when using relatively dilute tissue suspensions (Chambers et aL, 1994). b. C a r b o x y l e s t e r a s e s
The CarbEs hydrolyze carboxylic acid esters. Such esters are rarely encountered within the OP insecticides, but one very important and widely used insecticide, malathion,
133
O 0~ \ 0 I 0--C2H5 02H5
FIG. 3. Sulfoxidation of phorate.
contains two such esters. The CarbEs readily hydrolyze one or both of these esters to form the oL- or the [3-monoacid or the diacid metabolites, all three of which are incapable of being bioactivated to an active anti-ChE. These CarbE-mediated hydrolyses are extremely important contributors to the very low vertebrate toxicity of malathion, which is one of the least acutely toxic insecticides within the OP insecticide class. Malathion is one of the most selective of the OP insecticides because mammals readily hydrolyze malathion, but insects are capable of little hydrolysis. Malathion hydrolysis by CarbEs is illustrated in Fig. 4. In addition to their active catalytic hydrolysis of malathion, the CarbEs are also important contributors to the stoichiometric detoxication of many of the oxons, even those that are low affinity for the A-esterases. The oxons and phosphates generally have a high affinity for the serine esterase ACHE, which is the molecular target for the acute toxicity, as mentioned previously. However, the oxons and phosphates also frequently have a high affinity for other serine hydrolases, such as the CarbEs. The phosphorylation of CarbEs stoichiometrically destroys the oxons or phosphates, releasing the leaving group. This phosphorylation occurs at relatively low concentrations of oxons in some cases (Chambers et al., 1990) and undoubtedly represents an important defense for the oxon as it is generated in the liver. CarbEs in the liver and serum can protect the target AChE from inhibition by destroying the oxons before they have an opportunity to circulate to the nervous system. However, because this detoxication is stoichiometric and not catalytic, it is saturable and would be limited in its efficacy if the OPs were present in high concentrations (Tang and Chambers, 1999). It should be noted that there are several routes, both catalytic and stoichiometric, that yield the leaving group as a product. The CYP-mediated dearylation reaction on phosphorothionates, the A-esterase-mediated hydrolysis of oxons and phosphates, and the stoichiometric phosphorylation of serine esterases (CarbEs, ACHE, and butyrylcholinesterase) all yield the leaving group as a product. Therefore, the appearance of the leaving group as a product in experiments, either in vitro or in vivo, does not indicate which pathway was involved. In in vitro experiments, the pathway could be elucidated by knowing whether the substrate was the phosphorothionate (dearylation) or the oxon, whether there was a dependency on NADPH (dearylation) or on calcium (A-esterases), and whether the process was cofactor independent and saturable (serine esterase phosphorylation).
SECTION
134
/" s
H3C-O
II.
Pharmacokinetics
s
& Metabolism
0 II
H3C-O II ~ C H 2 C - O H "P-S----~ H3C-O/ C-OmC2H 5 II O
o II
O II ~CH2"C-OH
s
H3C-O.II
~1-0~02H5 0
H3C_O/P S---~Cl -OH 0
s
H3C-O.II
O II
//
~CH2C-O~C2H5
H3G_o/P-S~<~C_OH II O 3. PHASE II METABOLISM The intact insecticides and the metabolites described previously are susceptible to phase II conjugation reactions, and sulfate and glucuronide conjugates are frequently formed. These conjugates are water soluble and readily excreted. Since many of the leaving groups that have been formed by the several phase I processes discussed previously have functional groups, such as hydroxyl groups, that are very amenable to conjugation, the leaving groups are certainly prime substrates for the phase II pathways. The phase II metabolites would not be anti-ChE and therefore would be detoxified products. Another possible phase II reaction with the OP insecticides is a glutathione-mediated dealkylation, which is most likely with the methoxy compounds (Sultatos, 1992). However, although theoretically possible, the relevance of this reaction in vivo is not clear. B. M e t a b o l i s m of C M s Although CMs contain a diverse array of structures, they are all derivatives of carbamic acidmHOC(O)NH2. Whereas carbamic acid cannot exist in its free form because of its rapid decomposition into carbon dioxide and ammonia, ester derivatives of carbamic acid are quite stable. Adding an aryl ester to carbamic acid, in combination with substituting a methyl group for one of the hydrogen atoms associated with the nitrogen, results in the formation of compounds with insecticidal activity. Although many of the first CM insecticides were structurally related to physostigmine, the most potent CM, aldicarb, was developed to mimic acetylcholine based on the mode of action for CM inhibition of ACHE. CMs can also have herbicidal or fungicidal activity if one of the nitrogen-attached protons is replaced by an aro-
FIG. 4. Hydrolysisof malathion.
matic group or by a benzimidazole. Examples include the herbicide propham and fungicides diethiofencarb and benomyl. The mechanisms underlying herbicidal and fungicidal activities bear no relationship to anticholinesterase activity (Schlagbauer and Schlagbauer, 1972). Although most CMs have two oxygen groups within the carbamic acid moiety, sulfur can be substituted for either or both oxygens to form thio- or dithiocarbamates. Because of the great varieties of structures, the metabolic pathways for CMs can be very complicated. Although products of hydrolysis or oxidation are ready for conjugation, many hydrolysis products will be further oxidized, or oxidation products still retaining the ester bond will be hydrolyzed. An overview of the metabolic pathways most commonly seen for compounds within the CM pesticide class is provided here. However, the reader is referred to Knaak (1971), Ryan (1971), Schlagbauer and Schlagbauer (1972), and Kuhr and Dorough (1976a,b) for the history and chemistry of the CM pesticides and more detailed summaries of the metabolic pathways for individual insecticides. 1. PHASE I METABOLISM: HYDROLYSIS Hydrolysis of CMs results in cleavage of the ester bond, generating phenol, oxime, or enol, as well as methyl- or dimethylcarbamic acid, which rapidly decomposes into carbon dioxide and methyl- or dimethylamine. The hydrolysis rates in mammals are higher than those in insects or plants because of the abundance of esterases in the former. This metabolic difference between mammals and insects is responsible for the selective nature of CM toxicity to insects. Most CM insecticides can form significant amounts of hydrolytic products in vivo in various tested animals. It is worth noting that the exhaled radiolabeled carbon dioxide, which indicates cleavage of the ester bond, can result from hydrolysis of either the parent compounds or their
CHAPTER 10 9Metabolism of Anti-ChE Pesticides oxidation products. The substituents on the ring have a major influence on the rate of carbamate hydrolysis (Krishna and Casida, 1966). Monomethylcarbamates with p a r a substituents in the ring are more likely to be hydrolyzed. 2. PHASE I METABOLISM: OXIDATION Studies by Hodgson and Casida (1960, 1961) were the first investigations of CM oxidation by microsomal NADPH-dependent enzymes. There is evidence that oxidation is the more important route for some CM metabolism, not only in a pharmacokinetic but also in a toxicological perspective because, unlike hydrolysis that usually generates detoxication products, oxidative metabolites often retain the CM ester bond and can sometimes have more potent anti-ChE activity (Oonnithan and Casida, 1968). The oxidation of CMs is quite complicated. There are many hypothetical sites for CM oxidations, including hydroxylation, dealkylafion, and sulfoxidation (Kuhr and Dorough, 1976a). a. Hydroxylation Hydroxylation is the most common oxidation reaction on CMs. This reaction usually occurs on the alkyl side chain or on the aromatic ring but not directly on the carbamoyl nitrogen atom (Kuhr and Dorough, 1976a). The first CM oxidation study observed hydroxylation of the N-methyl group (Hodgson and Casida, 1960, 1961). Indeed, N-methyl hydroxylation is one of the most common pathways in CM metabolism. The activity of hydroxylation reactions of N-alkyl groups decreases with increasing alkyl chain length. The hydroxylated methyl group of N,N-dimethylcarbamate can undergo N-dealkylation, yielding formaldehyde. However, the hydroxylated methyl group of monomethylcarbamates, such as methyl hydroxycarbaryl, is quite stable (Ryan, 1971) and rarely undergoes further dealkylation or carboxylation.
135
Besides the N-methyl group, alkyl substituents in other parts of CM molecules can also be hydroxylated. For example, the alkyl chain in chlorpropham can be hydroxylated and then further carboxylated (Bobik et al., 1972). The alkyl side chain on aromatic tings of CMs, such as metolcarb and butacarb, can also be hydroxylated (Kuhr and Dorough, 1976b; Kulkarni and Hodgson, 1980). Further oxidation of the hydroxylated methyl groups to carboxylic acids is also observed (Fig. 5). Hydroxylation of saturated tings has similar characteristics to alkyl hydroxylation (Ryan, 1971). The dominant oxidation metabolite of carbofuran in mice and rats is 3-hydroxy carbofuran, which is further oxidized to 3-keto carbofuran (Metcalf et al., 1968; Dorough, 1968). N-methyl hydroxylation is a minor pathway for carbofuran metabolism and aromatic ring hydroxylation is not found in mammals (Gupta, 1994). However, liver microsomes from some fish can generate significant amounts of the N-methyl hydroxylation product of carbofuran (Gill, 1980). In mouse, rat, and human liver microsomal studies, carbofuran is mainly metabolized to 3-hydroxy carbofuran, which is slowly oxidized by CYP to 3-ketocarbofuran. 3-Ketocarbofuran is not stable and is immediately hydrolyzed nonenzymatically to 3-keto-7phenol (Fig. 6) (Usmani et al., 2004a,b). Ring hydroxylation is another typical reaction of CM metabolism catalyzed by NADPH-dependent microsomal enzymes (Strother, 1972). Examples of ring hydroxylation include carbaryl and propham (Fig. 7). Carbaryl is hydroxylated at the 4-, 5-, and 6-positions (Dorough and Casida, 1964; Leeling and Casida, 1966; Sullivan et al., 1972), whereas 4-hydroxylation of the ring is the predominant pathway for propham metabolism (Bend et al., 1971). Ring hydroxylation may involve an epoxide intermediate (Hodgson and Goldstein, 2001). For instance, formation of 4- and 5-hydroxycarbaryl may result from the rearrangement of 3,4-epoxide and 5,6-epoxide, respectively; formation of 5,6-dihydro-5,6-dihydroxycarbaryl may result from
Chlorpropham
O
~ l ~ ./OH3 O NH
I
CH3 Metolcarb
~ O
O
O
/CH3 NH
CH2OH
O~U"~NH_J"CH3
COOH
FIG. 5. Alkyl oxidation carbamates.
of
136
SECTION II- P h a r m a c o k i n e t i c s
& Metabolism
H3C~NH
H3C~NH
H3C~NH
o, o
o1-.o
HaC~ ( H3C" )
HAG"
HaC~ H3C" ~ O
HO
hydration of 5,6-epoxide; and formation of glutathioneconjugated hydroxycarbaryl may involve glutathione conjugation of the epoxide (Kulkarni and Hodgson, 1980).
FIG. 6. Oxidationof carbofuran.
rarely occurs on methyl groups attached to carbamoyl nitrogens in monomethylcarbamates. c. S-Oxidation S-oxidation, or sulfoxidation, occurs on thioether moieties of CMs to form sulfoxides, which sometimes can be further oxidized to sulfones. Although the thioether structure can also be S-dealkylated (Hodgson and Goldstein, 2001), this reaction seldom occurs with thioether groups of CMs. Both CYP and FMO catalyze sulfoxidation, but only FMO results in highly stereoselective products (Tynes and Hodgson, 1983, 1985; Buronfosse et al., 1995). Sulfoxidation products are the major
b. Dealkylation Dealkylation usually occurs on O- or N-alkyl groups in CMs. The reaction may involve an unstable oxidized intermediate and the alkyl group is detached and becomes an aldehyde (Kulkami and Hodgson, 1980). O- and N-dealkylations are illustrated in Fig. 8. In N,N-dimethylcarbamates, one of the N-methyl groups can undergo N-demethylation after hydroxylation. N-demethylation
HN/CH3
HN/CH3
I
I
o/C~o
o/C~o
I
OH
carbaryl
HN~CH3 I
o/C~o
o/C~o HO H OH \ H3C~cH3 0-0
Propham
OH
O\\
H3C, /~CH 3
0-0
FIG. 7. Ring hydroxylation of carbamates.
CHAPTER 1 0 9Metabolism of Anti-ChE Pesticides
H3C~NH
H3C~
137
H3C~
NH
NH
H,O OH, H,o oH, H3c/N~cH3
H3C
~CH3 NH2
/NH
H3C"
Zectran O O
H3C
O ~ ~
H3C
~ -CH3
o
HO
H3C
FIG. 8. Dealkylation of carbamates.
Propoxur
CH3 . H3C~/SI HaG~ .,C., ..N~ /L, NH~ ~O / "~CH \ CH3
CH3 H3C,, /C__ ~NH
O~.~~
..N /C ~ O / ~CH \
O
OH3
1
o H3c \ \ / C H a \ /S H3C,~ /C.. ..N~. /C \ \ ~NH "~O/ "~'CH \ O CH3 FIG. 9. Sulfoxidationof aldicarb. metabolites for CMs with a thioether group, suggesting that S-oxidation is more active than N-methyl- and/or ring hydroxylation. The metabolism of aldicarb is a good example of sulfoxidation (Fig. 9). The major metabolite of aldicarb in vivo and in vitro is aldicarb sulfoxide, which is much more toxic than the parent compound (Kuhr and Dorough, 1976b; Pelekis and Krishnan, 1997). FMO is a primary contributor to aldicarb sulfoxidation (Montesissa et aL, 1994).
3. PHASE II REACTIONS Most polar functional groups added to CMs by phase I reactions can accept the glucuronyl moiety from UDPGA to form glucuronide conjugates or sulfate from PAPS to form sulfate conjugates, which are then excreted in the urine (Dorough, 1979). For instance, glucuronide and sulfate conjugates of phase I metabolites of carbaryl, such as 1-naphthol, 4-hydroxycarbaryl, and 5,6-dihydro5,6-dihydroxycarbaryl, are found in urine from animals treated with carbaryl (Knaak, 1971; Sullivan et al., 1972). Glutathione conjugation reactions may involve the opening of epoxide rings (Kuhr and Dorough, 1976b; Kulkami and Hodgson, 1980). The toxicological importance of the interaction of glutathione with epoxides is that glutathione can neutralize electrophilic epoxides, thereby preventing them from binding to nucleophilic groups of macromolecules, such as proteins and nucleic acids. The conjugation of epoxidized urethane by glutathione illustrates the importance of this reaction in prevention of carcinogenesis. The carcinogenic potential of urethane (ethyl carbamate) has been linked with vinyl carbamate epoxide, which can form a DNA adduct, ethenoadenosine (Dahl et al., 1978; Park et al., 1993). Urethane can be oxidized by CYP2E1 to form epoxide via a vinyl intermediate (Guengerich et al., 1991). Glutathione inhibits formation of ethenoadenosine (Kemper et al., 1995) by either the spontaneous or GST-catalyzed conjugation with vinyl carbamate epoxide. Besides conjugation, glutathione also involves reduction reactions, such as reducing disulfiram to diethyldithiocarbamate (Hart and Faiman, 1993). Acetylation and methylation are two phase II pathways that differ from others by masking the functional
138
SECTION
II.
Pharmacokinetics
& Metabolism
0
H2N acetylation
S--O
H3C
~'~
O
O--CH 3
S--O
O
O--CH 3
O
0
H2N acetylation
H3C
/s-o 0
groups and generating less water-soluble products (Parkinson, 2001). The major substrate for acetylation is an aromatic amine but not an aliphatic amine. Some CMs undergo acetylation or methylation. The CM herbicide asulam and its metabolite, sulfanilamide, can be acetylated to their aromatic amine group (Fig. 10) (Heijbroek et al., 1984). Methylation involves broader functional groups, such as phenols, catechols, aliphatic and aromatic amines, and sulfhydryl groups (Parkinson, 2001). Thiocarbamates such as disulfiram are one group of CMs that are involved in methylation. As mentioned previously, disulfiram is reduced by glutathione to diethyldithiocarbamate. This metabolite can be methylated at the sulfhydryl group to the diethyldithiocarbamate methyl ester, which can further undergo desulfuration to remove thiono sulfur and sulfoxidation to oxidize the other sulfur in the thioester bond (Fig. 11) (Hart and Faiman, 1993). These methylated products can inhibit aldehyde dehydrogenase, which is the mechanism that produces the alcohol'deterring ability of disulfiram. Some thiocarbamate herbicides, such as EPTC (S-ethyl N,N-dipropylthiocarbamate), and soil fumigants, such as metam, can also form methylated metabolites at sulfhydryl groups, which display aldehyde dehydrogenase inhibition activity (Quistad et al., 1994; Staub et al., 1995). This S-methylation reaction is catalyzed by thiol S-methyltransferase using S-adenosylmethionine as the methyl donor (Weisiger and Jacoby, 1979).
\
~ --
/s-o 0
FIG. 10. Acetylationof asulam.
animals and humans are generally similar in physiology, there are some differences in metabolic pathways and activities toward pesticides. Extrapolation of animal data to humans brings uncertainties. Direct in vitro studies using human tissues and comparing the results with animal data will reduce or redefine these uncertainties. In vitro studies involving human metabolism of pharmaceuticals have been routine for many years and are gaining popularity for agricultural chemicals as well (Hodgson, 2003). This effort is aided by the increased availability of human tissues and recombinant human enzymes. Microsomes, $9,
Sk
~C~S
.S
C2H5
/ NmC2H5 H502
GSH
I Methylation O
~-s / \ H5C2-N\ OH3 02H5
0
Desulfuration
0 /
Most toxicological studies of OPs and CMs, including the metabolic studies described previously, are performed in laboratory animals or animal tissues. Although laboratory
C2H5
Disulfiram
Sulfoxidation
IV. In Vitro Metabolic Studies of OPs and CMs Using Human Tissues
k~C~SH / H5C2-N\
H5C2-N\
\
02H5
OH3
FIG. 11. Metabolismof disulfiram.
/ ~ \
H5C2-N\
02H5
CH3
CHAPTER 1 0 9Metabolism of Anti-ChE Pesticides and other preparations of human tissues with high metabolic activity, such as the liver and intestine, can be easily obtained from several vendors. Recombinant human phase I and phase II enzymes are also commercially available. The methods used for in vitro metabolic and interaction studies have been standardized and can easily be adopted for high-throughput screening (Bjornsson et al., 2003). Because metabolic studies of pesticides have been extensively conducted in animals in vitro and in vivo, in vitro-in vivo correlations have been established in animals. By comparing in vitro data between humans and animals and assuming the in vitro-in vivo correlations observed in animals also exist in humans in a similar manner, one may be able to predict in vivo human metabolism of pesticides using in vitro human pesticide metabolism data. Therefore, the parameters obtained from the in vitro studies, such as Krn (Michaelis-Menten constant), Vmax (maximal reaction velocity), intrinsic clearance (Vmax/Km), and Ki (inhibition constant), can be integrated into physiologically based pharmacokinetic models (see Chapter 9) to predict in vivo plasma concentrations and tissue dosimetry, potential metabolic interactions with other xenobiotics, and differences between laboratory animals and humans or within human populations. The metabolic stability of laboratory animals and humans can be compared using in vitro techniques. In vitro studies using liver tissue showed that humans have a similar ability to detoxify chlorfenvinphos as rabbits, which are not very sensitive to the acute toxicity of the insecticide (Hutson and Logan, 1986). Metabolism of chlorpyrifos is less active in human liver microsomes than in rat and mouse liver microsomes (Tang et al., 2001), suggesting that less chlorpyrifos-oxon is generated in a short period of time in humans than in rodents. Similarly, human liver microsomes are less active than rodent liver microsomes in metabolism of carbaryl and carbofuran (Tang et al., 2002, Usmani et al., 2004a). A study of molinate, a CM herbicide, metabolism in liver microsomes and liver slices indicated that human tissues are more capable than those of rats to form the detoxified ring hydroxylation metabolite, whereas rats are more active in the generation of the toxic sulfoxide metabolite (Jewell and Miller, 1999). Following the identification of metabolic pathways using human liver microsomes, reaction phenotyping studies may be conducted to determine which CYP isoforms are responsible for the metabolism. The active CYP isoforms can be identified using any of the following assay systems, either alone or in combination: (1) metabolic activity using recombinant CYP isoforms, (2) determinations of metabolic activity using microsomes in combination with specific inhibitors or antibodies, and (3) correlation between metabolic activity and individual CYP activities using microsomes from single donors.
139
Several popular OP and CM pesticides have been studied. Studies of parathion, chlorpyrifos, diazinon, azinphosmethyl, and malathion by several groups have indicated that CYP1A2, -2B6, and -3A4 are the major metabolic enzymes responsible for the desulfuration reaction, which activates OP insecticides (Butler and Murray, 1997; Mutch et al., 1999; Sams et al., 2000; Tang et al., 2001; Kapper et al., 2001; Buratti et al., 2003, 2004). CYP3A4 has low affinity toward these pesticides and may play a significant role in desulfuration at high levels of exposure, whereas CYP1A2 and -2B6 are very active at low pesticide concentrations, which are more likely to occur in potential human exposures (Buratti et al., 2003, 2004). A similar preference of pathways by CYP isoforms is also observed in chlorpyrifos metabolism (Tang et al., 2001). The ratio of desulfuration versus dearylation of chlorpyrifos is 3.38 for CYP2B6 but only 0.14 for CYP2C19, suggesting that the former isoform favors desulfuration, whereas the latter is more likely to generate the dearylation product of chlorpyrifos. CYP3A4 has similar activity toward both pathways. People with a higher activity of CYP2C19 and lower activities of CYP2B6 and -3A4 may be less sensitive to the acute toxicity of chlorpyrifos because of less chlorpyrifos-oxon generated. This selectivity of the reaction pathway suggests that CYP not only involves the addition of oxygen to form the phosphooxythiiran intermediate (Chambers, 1992) but also influences the rearrangement of the oxidized molecule. It is possible that the selectivity of CYP2C19 may be specific for each individual OR Although several studies have reported that CYP2C19 is more active in dearylation than desulfuration of several phosphorothionates (Buratti et al., 2003; Tang et al., 2001; Vittozzi et al., 2001), it has been reported that CYP2C19 is also highly active in the desulfuration of diazinon (Kappers et al., 2001). In humans, many CYP isoforms are involved in three major pathways of carbaryl metabolism--4-, 5-, and methyl hydroxylationmthat do not share a common intermediate (Tang et al., 2002). Different isoforms show different activities toward these pathways. Among active CYP isoforms, CYP1A1 and -1A2 have the greatest ability to form 5-hydroxycarbaryl, CYP3A4 and -1A1 are the most active in generation of 4-hydroxycarbaryl, and CYP2B6 is the primary isoform for methyl hydroxylation of carbaryl. Fewer CYP isoforms are involved in carbofuran metabolism, which has only one major metabolic pathway (Usmani et al., 2004a). CYP3A4 is the predominant isoform responsible for carbofuran oxidation. The metabolic activity toward carbofuran in human liver microsomes correlates very well with CYP3A4 activity and can be significantly inhibited by ketoconazole, a CYP3A4-specific inhibitor. A study of in vitro metabolism of thioether containing OP and CM pesticides indicated that the CYP2C family is highly responsible for sulfoxidation (Usmani et al., 2004b). Polymorphisms of several human CYP isoforms have been observed, including CYP2C19 and -3A4 (Demorais et al., 1994; Dai et al., 2001). These genetic defects affect
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SECTION I I . P h a r m a c o k i n e t i c s
& Metabolism
metabolism of chlorpyrifos (Tang et al., 2001; Dai et al., 2001). The activity of chlorpyrifos metabolism in polymorphic isoforms of CYP2C19 is significantly lower than that in wild-type isoforms. Polymorphisms of CYP3A4 have various effects on chlorpyrifos metabolism; some alleles display lower activity than the wild type, some display higher activity, and others display the same activity as the wild type. Similarly, changes in XME activity due to exposure to alcohol, tobacco, drugs, and some occupational chemicals may also alter the metabolism of pesticides catalyzed by these enzymes, increasing blood concentrations of pesticides or their active metabolites and prolonging the clearance process. Therefore, certain human subpopulations may be more vulnerable to pesticide toxicity due to high frequency of defective CYP alleles or repeated exposure to XME inducers or inhibitors. In vitro studies can also be used to investigate metabolic interactions between chemicals. Using human tissues, especially human liver microsomes and hepatocytes, potential inhibitory and inductive effects of OPs and CMs on XME can be identified. There are two common inhibitory effects: irreversible and reversible. Pesticides in the phosphorothionate category, such as parathion and chlorpyrifos, can irreversibly inhibit or inactivate CYP activity (Buffer and Murray, 1997; Tang et al., 2002; Usmani et al., 2004a). The mechanism underlying this irreversible inhibition or inactivation involves the release of sulfur during the desulfuration process, resulting in the formation of a free radical that binds to the heme moiety of CYP (Neal, 1980). Competitive inhibition is the m o s t common metabolic interaction because any two compounds or pathways catalyzed by a common enzyme can exclude each other from binding the enzyme active site and inhibit each other's metabolism. Chlorpyrifos desulfuration competitively inhibits carbaryl methyl hydroxylation because CYP2B6, which is highly active in the former pathway, is the predominant CYP isoform responsible for the latter pathway (Tang et al., 2002). The inhibition constant (Ki) of chlorpyrifos was calculated to be 2 ~M based on its inhibition of carbaryl metabolism. An in vitro study using human liver fraction also demonstrated the ability of chlorpyrifos-oxon and carbaryl to inhibit hydrolysis of trans-permethrin, a pyrethroid insecticide (Choi et al., 2004). Similarly, OP and CM pesticides have the potential to interfere with the metabolism of endogenous compounds, such as testosterone, which are catalyzed by the same XME (Usmani et al., 2003, 2004a). Although in vitro methods provide a clear way to understand mechanisms of effects, one needs to be cautious when interpreting in vitro data and extrapolating them to the in vivo situation. It is important to scale the in vitro assay concentration to in vivo doses before drawing a conclusion based on in vitro results. In vitro studies tend to use high concentrations to display effects. Those concentrations may be higher than lethal doses of pesticides. Therefore, many effects observed in vitro at high concentrations may not be relevant in vivo.
Acknowledgment We thank Dr. Ernest Hodgson for reviewing the manuscript and many helpful suggestions.
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Interspecies Variation in Toxicity of Cholinesterase Inhibitors STEPHANIE J. GARCIA, 1 MICHAEL ASCHNEIL z AND TORE SYVERSEN3 1Wake Forest University Health Sciences, Winston Salem, North Carolina, USA 2Vanderbilt University Medical Center, Nashville, Tennessee, USA 3Norwegian University of Science and Technology, Trbndheim, Norway
I. I N T R O D U C T I O N
II. V A R I O U S S P E C I E S A R E DIFFERENTIALLY AFFECTED
Cholinesterase (ChE) inhibition by organophosphorus compounds (OPs), and to a lesser extent carbamates (CMs), has been studied extensively as the primary mechanism of toxicity for these broad-spectrum insecticides. Although several factors must be considered in toxicology testing of anticholinesterases (anti-ChEs), including the specific compound in question, species, and age, as well as level and duration of exposure, this chapter focuses on interspecies variability. Various species respond differently to anti-ChEs; response, recovery, and reversal depend on both the species affected and the compound. Abundant studies exist on various species, from the intended target group (insects) to animals vulnerable to unsolicited effects, including fish, amphibians, birds, and mammals. These interspecies comparisons seek to improve species selectivity and to extrapolate toxicity testing between unrelated species for regulatory purposes. First, in order to improve selectivity for target species, it would be helpful to exploit the differences between species. Second, similarities among unrelated species provide insight into conserved mechanisms of action and toxicity. The extrapolation of toxic effects from animal testing to humans may impact human health and safety by providing a basis for setting reference dose levels. Currently, uncertainty factors are applied to account for interspecies variability from experimental animals to humans as well as intraspecies variability to account for the sensitive individuals within a species. Adequate extrapolation among species may help minimize or refine animal toxicity testing. With these goals in mind, this chapter discusses how various species differ in response to anti-ChEs and proposes potential causes, focusing on pharmacokinetics and pharmacodynamics (i.e., how the body handles the compound and the mechanism of action of the compound). Toxicology of Organophosphate and Carbamate Compounds
Both the central nervous system (CNS) and the peripheral nervous system (PNS) contain sites of action of anti-ChEs. OP and CM pesticides inhibit acetylcholinesterase (ACHE), the enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh) into acetate and choline. The ACh accumulating in the synaptic cleft binds to both muscarinic and nicotinic cholinergic receptors, leading to cholinergic toxicity. The resultant cholinergic hyperstimulation produces a range of intoxication signs. Early symptoms are the result of parasympathetic stimulation with indicators such as bradycardia, miosis, diarrhea, urination, lacrimation, and salivation. Overstimulation of the neuromuscular junctions may result in muscle twitching and, at higher doses, paralysis. CNS symptoms include hypothermia, tremors, headache, and convulsions, with death occurring from depression of respiratory centers in the brain (Ecobichon, 2001a). The discussion of interspecies variability focuses on the more widely studied OPs, but the principles are applicable to both OPs and CMs. Species differ quantitatively and qualitatively in their response to anti-ChEs. This is evident in variable doses that cause death in 50% of a test population (LDs0)(Table 1) as well as the levels that yield no effect (NOEL), no adverse effect (NOAEL), the lowest level an effect is observed (LOEL), and the lowest level an adverse effect is observed (LOAEL). To adequately compare OP :effects across species, the route and duration of exposure should be similar since absorption and distribution are influenced by the route of administration. These are key factors in determining the body burden of the compound. Moreover, similar end points must be compared across species [e.g., LDs0, the effective dose for 50% (ED50) AChE inhibition in blood or brain, cholinergic receptor binding, or behavioral tests]. 145
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
14 6
SECTION II 9Pharmacokinetics & Metabolism
TABLE 1. Compound
LD5o Values Are Species, Compound, and Exposure Dependent Species
LDso (mg/kg)
Route
References a
Comparing species
Aldicarb
Mouse
0.3
Oral
1, 2
Aldicarb Aldicarb
Rat Rabbit
0.46 0.46
Oral Oral
1,3
Aldicarb
Guinea pig Chicken
1 8
Oral Oral
Mouse
108
Oral
1, 3
Carbaryl
Rat Guinea pig
230 250
Oral Oral
1
Carbaryl Carbaryl
Dog Rabbit
250 710
Oral Oral
1,3
Carbaryl
Monkey
1000
Oral
1, 3
Aldicarb Carbaryl Carbaryl
1, 3 1,3 1
1, 3
1, 3
Carbaryl
Pig
1500
Oral
1,3
Chlorfenvinphos
10-25 150-200 125-250
Oral Oral Oral
4
Chlorfenvinphos Chlorfenvinphos
Rat Mouse Guinea pig
Chlorfenvinphos
Rabbit
500-1000
Oral
4
Chlorfenvinphos Chlorpyrifos
Dog Chicken
> 5000 25.4
Oral Oral
4
Chlorpyrifos Chlorpyrifos
Mouse
60
Oral
Rat Guinea pig
82-155 504
Oral Oral
7, 8
1000 8.4
Oral Oral
1
Diazinon
Rabbit Chicken
Diazinon
Mouse
17
Oral
1
Diazinon
Rat Rabbit Guinea pig
250-285 143 250
Oral Oral Oral
7, 8
Chlorpyrifos Chlorpyrifos
Diazinon Diazinon
4 4
1,5 1, 6 1 1
1 1
Diazinon
Pig
320
Oral
1
Parathion
Cat Pigeon
0.93 1.33
Oral Oral
1
3-7 2.1
Oral Oral
7,8
Parathion
Rat Duck
Parathion
Human
3
Oral
1
Parathion Parathion Parathion
Dog Quail Donkey
3 4 5
Oral Oral Oral
1
Parathion Parathion
Mouse Guinea pig
5 8
Oral Oral
Parathion Parathion
Rabbit Chicken
10 10
Oral Oral
Parathion Parathion
1 1 1 1 1 1,9 1 1
Comparing compounds
Aldicarb Satin
Rat
0.46
Oral
1
0.55 3-7
Oral Oral
1
Parathion
Rat Rat
7,8
Chlorfenvinphos
Rat
10-25
Oral
4
(continues)
CHAPTER 1 1 9Interspecies Variation in Anticholinesterases
TABLE 1. Compound Comparing compounds (Continued) Methyl parathion Dichlorvos Chlorpyrifos Carbaryl Diazinon Tri-o-cresyl phosphate Malathion Comparing route of exposure Aldicarb Aldicarb Aldicarb Aldicarb Aldicarb Carbaryl Carbaryl Carbaryl Carbaryl Carbaryl Parathion Parathion / Parathion Parathion Parathion Parathion Satin Sarin Sarin Sarin Sarin Sarin
147
(continued)
Species
LD5o (mg/kg)
Route
References a
Rat Rat Rat Rat Rat Rat Rat
14-24 56-80 82-155 230 250-285 1160 1000-1375
Oral Oral Oral Oral Oral Oral Oral
7, 7, 7, 1, 7, 1 7,
Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat
0.28 0.46 0.47 0.67 2.5 18 64 230 1400 4000 3-7 3.8 2 6 9 6.8 0.039 0.103 0.108 0.218 0.55 2.5
ip Oral iv sc Dermal iv ip Oral sc Dermal Oral iv ip im sc Dermal iv sc im ip Oral Dermal
1 1 1 1, 11 1, 7 1 1, 12 1, 10 1 1 7, 8 1 1 1, 13 1 1, 14 1, 15 1, 16 1, 17 1, 15 1 1
8 8 8 10 8 8
al, CCOHS (2004); 2, Fahmy et al. (1970); 3, Ecobichon (2001b); 4, Hutson and Hathway (1967); 5, Sherman et al. (1967); 6, E1-Sebae et al. (1978); 7, Gaines (1969); 8, Storm et al. (2000); 9, Klimmer and Pfaff (1955); 10, Weiss and Orzel (1967); 11, Natoff and Reiff (1973); 12, Brodeur and Dubois, (1963); 13, Grob and Harvey (1958); 14, Gaines (1960); 15, Fleisher et al. (1963); 16, Brimblecombe et al. (1970); 17, Schoene et al. (1985).
Standardized laboratory guidelines are necessary for conducting various tests because even within the same species, different tests can lead to LOAELs that differ by up to 100-fold (Burbacher et al., 1990; Vidair, 2004). For an extensive review on tabulating NOAELs and LOAELs for a variety of end points in different species exposed to physostigmine, parathion, or diisopropyl fluorophosphate (DFP), see Raffaele and Rees (1990). NOAELs and LOAELs for neurotoxic endpoints generally are lower than LDsos for each of the compounds evaluated. However, the most sensitive endpoint differed among substances, reit-
erating compound-dependent effects when considering species variability (Raffaele and Rees, 1990). Table 1, compiled predominately from the R e g i s t r y o f Toxic Effects o f C h e m i c a l S u b s t a n c e s database [Canadian Centre for Occupational Health and Safety (CCOHS), 2004], summarizes some LDs0s for various compounds and species exposed via different routes, with the most sensitive (lowest LDs0 ) species and route listed accordingly. The LDs0, of course, depends on the compound in question, as is illustrated for rats exposed orally to 10 different compounds. It is generally accepted that birds are extremely
148
SECTION
II
9Pharmacokinetics
& Metabolism
susceptible to acute ChE inhibition by OPs, whereas fish and amphibians are relatively resistant and rodents are intermediate (Wallace, 1992). On the other hand, this is just a generalization and species sensitivity also depends on the compound exposure, as illustrated in Table 1 and by Johnson and Wallace (1987). Generally, birds are less sensitive than rodents to aldicarb and carbaryl but more sensitive to chlorpyrifos and diazinon, whereas mammals seem to be more resistant to chlorfenvinphos, carbaryl, and diazinon. For parathion, there is no obvious trend, with birds, rodents, and mammals (including human) intermixed (Table 1). Although rodents are more sensitive to parathion and paraoxon than sunfish and bullheads, fish in general are more sensitive to malathion and malaoxon compared to rodents (Johnson and Wallace, 1987). The greater sensitivity of rodent brain AChE to inhibition by paraoxon may contribute to the greater toxicity of parathion and paraoxon in rodents compared to fish, whereas the differences in malathion sensitivity may be due to greater carboxylesterase activity in rodents than in fish. Clearly, no hard and fast rules can explain interspecies variability to effects of antiChEs, but discussion of the pharmacokinetics and pharmacodynamics will shed some light on the contributing factors.
III. W H Y A R E S P E C I E S D I F F E R E N T I A L L Y AFFECTED? A. P h a r m a c o k i n e t i c s
A compound's action or effect within the body over a period of time is regulated by pharmacokinetics: absorption, distribution, metabolism, and elimination. These processes differ across species and provide some insight into species variability following exposure to anti-ChEs. In addition to species-dependent effects, signs and symptoms of ChE inhibition by OPs or CMs depend on the compound, dose, route, frequency and duration of exposure, as well as the time of observation relative to the time of peak effect (Table 1). Therefore, it is difficult to compare species across studies in which any one of these factors is not consistent, and the reader must bear this in mind when reviewing the literature for species-related differences. 1. ABSORPTION AND ELIMINATION Because the physiological response of an organism is dependent on the steady-state concentration of the compound at the site of action, differences in the relative rates of absorption and elimination of a compound in various species account, in part, for some of the interspecies variability of anti-ChEs. Oral exposures are subject to first-pass hepatic metabolism, which produces metabolites more or less toxic than the parent compound (Storm et al., 2000). Thus, to compare the effects of any compound across species, the same route of administration is required as well as the same endpoint. In the case of aquatic and
nonaquatic animals, presumably the lethal concentration that causes death in 50% of an aquatic population (LCs0) is sometimes correlated to the LDs0. Pharmacokinetic information provides insight into NOAEL and NOEL differences among exposure routes. For example, dichlorvos is rapidly absorbed, metabolized, and eliminated following both oral and inhalation exposures, possibly explaining its equivalent potency to inhibit red blood cell (RBC) AChE when exposure occurs via either route (Storm et al., 2000). On the other hand, malathion appears to be eliminated more rapidly following inhalation exposure compared to oral exposure, providing some context for the apparently higher inhalation NOEL, where RBC AChE is the endpoint (Storm et al., 2000). Inhibition of RBC AChE is not itself considered an adverse effect, although it is often measured as an indicator for the adverse inhibition of brain ACHE; thus, "NOEL" is used to describe this effect. In addition, clearance of the carbamate carbaryl is slower in rats than in hens, potentially contributing to the greater sensitivity of brain AChE inhibition in rats (Ehrich et al., 1992, 1995). Furthermore, dogs excreted this vinyl phosphate more rapidly than rats, reflective of the higher LDs0 in dogs versus rats (Table 1) (Hutson and Hathway, 1967; Natoff, 1971). Although differences in the relative rates of absorption and elimination explain some of the variability among species, they are insufficient to account for all interspecies variabilitymfor example, the insensitivity of fish compared to rodents to acute parathion toxicity (Hodson, 1985; Wallace, 1992). Nevertheless, in general, the LDs0 was lowest in rats following intravenous (iv) administration of an anti-ChE and highest following dermal exposure with intraperitoneal (ip) and oral LDs0s on the low end and subcutaneous (sc) LDs0s on the high end. The absorption rates depend on exposure route (iv > ip > sc > oral > dermal) and seem to correlate with acute toxicity (iv -> ip > oral > sc > dermal) (Table 1). 2. DISTRIBUTION Physiological response depends on bioavailability. The concentration of nonspecific binding sites affects the amount of compound that is free to bind target receptors and thus can serve as a reservoir to decrease the effective levels of anti-ChEs (Lauwerys and Murphy, 1969; Sultatos et al., 1984; Wallace, 1992). Plasma proteins such as albumin and soluble aliesterases bind OPs, decreasing toxicity. For instance, soluble aliesterases bind to the active metabolite of parathion, paraoxon, to limit toxicity (Chambers and Chambers, 1990; Lauwerys and Murphy, 1969; Wallace, 1992). As evidence, Lauwerys and Murphy demonstrated that tri-o-cresyl phosphate (TOCP), which inhibits nonspecific binding of OPs, potentiated paraoxon toxicity in rats. Additionally, albumin promotes nonenzymatic hydrolysis of OP esters, resulting in inactivation of the compound (Sultatos et al., 1984). Hutson and Hathway (1967) found that the blood concentration of chlorfenvinphos in dogs was one-fifth of that found in rats at similar
C H APT ER 1 1 oral dose levels and that it is taken up more readily by the brain of rats than dogs at similar blood levels, consistent with greater sensitivity of rats to acute toxicity (Table 1). A species comparison among rats, guinea pigs, and marmosets (primates) found that blood soman levels differed among these species following intravenous administration (Benschop and De Jong, 1991). This interspecies variability is probably due to decreasing amounts of binding sites (rats > guinea pigs > primates) that compete with ACHE, leading to increasing "toxicoavailability" (primates > guinea pigs > rats) (Benschop and De Jong, 1991). Contrary to these findings, guinea pigs were more sensitive to acute soman toxicity than rats following subcutaneous administration (Maxwell e t al., 1987). Since OPs are hydrophobic compounds, the lipid composition of an organism governs their hydrophobic partitioning (Wallace, 1992). The protective blood-brain barrier (BBB) is permeable to these pesticides. Likewise, the high lipid content of the brain results in a disproportionate amount of accumulation in the brain and partially explains TABLE 2.
Interspecies Variation in Anticholinesterases
149
the neurotoxicity of OPs. Mammalian P-glycoproteins are cell membrane ATP-dependent transporters expressed at blood-tissue barriers (e.g., BBB and blood-placenta barrier) that pump hydrophobic drugs out of the cell, protecting the brain and fetus (Fromm, 2004). I n v i v o studies suggest that chlorpyrifos oxon is a substrate for P-glycoprotein (Lanning e t al., 1996). Interspecies differences in P-glycoprotein expression have the potential to influence distribution of OP substrates; indeed, there are intraspecies differences within humans and rodents, as well as age-dependent and tissue-dependent differences in expression of this protein (Fromm, 2004; Rosati e t al., 2003; Warrington e t al., 2004). 3. METABOLISM Most studies comparing species variability focus on metabolism; activation and deactivation of the anti-ChEs within the body depend on several factors that vary among species. The amounts and activities of enzymes involved in metabolism must be considered (Table 2). For example, liver
Basal Enzyme Activity/Level in Various Species
Species
Site
Value
References a
Rabbit Guinea pig Mouse Human Rat Mouse Minnow Trout
Serum Serum Serum Serum Brain Brain Brain Brain
-300 nmol/ml x min -2900 nmol/ml x min -4800 nmol/ml x min --4800 nmol/ml X min 37.7 nmol/min/mg protein 49.9 nmol/min/mg protein 178.3 nmol/min/mg protein 88.8 nmol/min/mg protein
1 1 1 1 2 2 2 2
Carboxylesterase Carboxylesterase Carboxylesterase Carboxylesterase
Rabbit Human Guinea pig Mouse
Serum Serum Serum Serum
-300 nmol/ml x min -300 nmol/ml x min -2000 nmol/ml x min -3200 nmol/ml X min
1 1 1 1
Paraoxonase Paraoxonase Paraoxonase Paraoxonase Enzyme level Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450
Guinea pig Mouse Human Rabbit
Serum Serum Serum Serum
- 100 nmol/ml x min -300 nmol/ml X min -300 nmol/ml X min -3400 nmol/ml x min
1 1 1 1
Chicken Cat Human Rat Mouse Rabbit Hamster Guinea pig
Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes
0.13 nmol/mg protein 0.34 nmol/mg protein 0.28-0.53 nmol/mg protein 0.84 nmol/mg protein 1.1 nmol/mg protein 1.1 nmol/mg protein 1.26 nmol/mg protein 1.45 nmol/mg protein
3 4 5, 6 7 7 7 7 7
Enzyme activity Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase
al, Kaliste-Korhonenet aL (1996); 2, Johnson and Wallace (1987); 3, Lasker et al. (1982);4, Kato (1966); 5, Nelson et al. (1971); 6, Pelkonen et al. (1973); 7, Chhabraet al. (1974).
150
SECTION II 9Pharmacokinetics & M e t a b o l i s m
microsomal cytochrome P450 levels and ChE, carboxylesterase, and paraoxonase (PON1) activities ,vary across species (Table 2) (Chhabra et al., 1974; Kato, 1966; Lasker et al., 1982; Nelson et al., 1971; Pelkonen et al., 1973). In a study to predict toxicity of OPs to four avian species, LDs0 correlated with rate of OP activation and brain AChE inhibition in the brain and serum carboxylesterase inhibition, supporting the contribution of OP metabolism to acute toxicity (Thompson et al., 1995). a. Organophosphate Activation CMs generally are not activated prior to AChE interaction. However, to inhibit ACHE, cytochrome P450 enzymes metabolize some OPs (e.g., chlorpyrifos, parathion, and malathion) to their more potent oxon via oxidative desulfuration (deBethizy and Hayes, 2001). Conversely, methyl parathion is highly toxic to rats, whereas its oxon is a relatively nonpotent inhibitor of brain AChE (Chambers, 1992). Parathion is metabolized to the more potent paraoxon, so a lower rate of activation--as in fish--is consistent with relative resistance to acute toxicity. On the other hand, a greater rate of activation in rodents compared to birds suggests that rodents are more sensitive, but this is not consistent with the general finding that birds are more sensitive to acute OP toxicity compared to rodents (Chambers, 1992; Hitchcock and Murphy, 1971; Thompson et al., 1995; Wallace, 1992; Wallace and Dargan, 1987). Thus, although differences in the rate of activation among species account for some species variability, this is not the definitive parameter in determining interspecies variability. b. Organophosphate Deactivation Although the rate of OP activation influences the toxic bioavailability of the oxon, the rate of deactivation is important in the protection of the organism. Both A- and B-esterases deactivate OPs. Not only do the amounts and activities of the esterases vary according to species and tissue but also their affinities for OPs are species, tissue, and compound specific (Kaliste-Korhonen et al., 1996). The A-esterases, including oxonases and arylesterases, hydrolyze the parent compound or oxon metabolite to produce inactive metabolites. These enzymes are present in plasma and tissue and are not inhibited by the oxon (Karanth and Pope, 2000; Moser et al., 1998; Padilla et al., 2000; Vidair, 2004). In contrast, the B-esterases, including carboxylesterases, aliesterase, ACHE, and butyrlcholinesterase (BuChE), bind OPs stoichiometrically, are irreversibly inhibited, and reactivate very slowly (Karanth and Pope, 2000; Moser et al., 1998; Padilla et al., 2000; Vidair, 2004). An important A-esterase involved in the detoxification of OPs is PON 1, which hydrolyzes the OP oxon metabolite. Accordingly, injected PON1 protects against OP poisoning in rodents (Cowan et al., 2001; Davies et al., 1996). PON1 is polymorphic in human populations, contributing to
intraspecies variability, whereas interspecies differences in PON1 activity are correlated with observed LDs0 values (Davies et al., 1996). In a very informative review, Wallace (1992) provides references to suggest that the sensitivity to acute cholinergic intoxication is inversely proportional to the serum arylesterase activity (Butler et al., 1985); likewise, the bimodal distribution of plasma A-esterases in humans may account for individual sensitivity to OP toxicity (Eckerson et al., 1983; La Du and Eckerson, 1984; Wallace, 1992). The plasma and tissue from fish, amphibians, birds, and mammals vary in A-esterase activity (Wallace, 1992). Fourteen species of birds tested have virtually undetectable plasma A-esterase activity, whereas rodents, sheep, and humans have abundant plasma arylesterase activity (Wallace, 1992). The low arylesterase activity in birds translates to a lower rate of paraoxon hydrolysis in their plasma and liver, correlating with the greater susceptibility of birds to OP intoxication (Walker and Mackness, 1987; Wallace, 1992). Interaction of OPs such as parathion and chlorpyrifos with carboxylesterase and aliesterase decreases the availability to interact with AChE to produce toxic effects, thus protecting against neurotoxicity (Storm et al., 2000). Conversely, inhibition of serum and lung carboxylesterases by cresylbenzodioxaphosphorin oxide potentiates the in vivo toxicity of some OPs (soman, satin, tabun, and paraoxon, but not dichlorvos or DFP) in rats and mice (Clement, 1984; Maxwell, 1992) and eliminates the soman LDs0 interspecies variability in rats, mice, guinea pigs, and rabbits (Maxwell et al., 1987). Moreover, Kaliste-Korhonen (1996) reported that human serum does not detoxify OPs as well as serum of rodents (mouse and guinea pig), making man potentially more sensitive to OPs than rodents. This is potentially related to the lower basal activity of carboxylesterase in man compared to mice and guinea pigs (Table 2) (KalisteKorhonen et al., 1996). Within animal classes, there are species and temporal variations in activity, level, and forms of carboxylesterases and ChEs, as noted in avian carnivore versus omni/herbivore serum B esterases (Thompson, 1993). Thus, it is challenging to generalize trends among classes such as birds, fish, and mammals for different compounds. Although it is tempting to offer a simplistic proposal regarding the role of OP deactivation and toxicity based on these findings, the relationship between hydrolyzing activity and in vivo sensitivity does not hold true for all OPs or for all species. Indeed, the lower arylesterase activity in fish compared to rodents is inconsistent with the relative resistance of fish to OP toxicity (Wallace, 1992; Wallace and Dargan, 1987). Although rodent susceptibility to OPs is generally intermediate between birds and fish, the activity of activating and deactivating enzymes is greater in rodents compared to these other species, making generalizing statements impractical. A number of other factors besides A- and B-esterase activities influence physiological responses to OPs
CHAPTER 1 1 among species; however the importance of the differences of these enzymes to determine interspecies variability remains controversial (Wallace, 1992).
B. Pharmacodynamics In addition to differences in pharmacokinetics, species also differ in their biochemical and physiological response to
lnterspecies Variation in Anticholinesterases
anti-ChEs; that is, the mechanism of action, in this case AChE inhibition, is subject to species-specific modification. 1. ACETYLCHOLINESTERASEINHIBITION Whereas CMs react with the serine group on ACHE, yielding carbamylation of the serine hydroxyl group, OPs phosphorylate the serine hydroxyl group (Fig. l a). Carbamylation of AChE is reversible and typically
A Organophosphorus
Ester
0 (or S) R10~
II
x
,
I
i
.....~ii;~'~i'~..... i~i~i:i~, OH .....~!ii~ii~~.ii~. i. . . . .:~,~i!i~.i~.i.i~..,~ ....~iii~ !!,,i~,~,~J~,~,/,i~ ~ i l i ~ ~,~,~,~,~,~ii~i ~,~;....... i~,i~,~ ...~,..~.',!i~ i~ i i~,ii........ i~
......
Acetylcholinesterase
Organophosphorus Ester + Free Acetyicholinesterase
Reversible Complex[ O
0 RIO~]I R20
~P~X
ka
,. R 1 0 ~
p[I_ _ x
R20
\
+ OH .......
.....
k. ~
0
'i~,~!,ill~
i~i~i~
I Free Enzyme I
+
O
R,O~ II p
\
(+ XH)
O ...~iiil ... iii~i~i~i~!~i~]i~!i~ii~i~.i.i.i2..ii.l
..%i' ..
i!
OH
I Phosphorylated Enzyme [
R20 /
....:'!~' i!~i
~i~~i~
R IO ~
151
0 II II
R~o -~P-~
0 R10 ~ . II O- /
P
\
(+ ROH)
O .....~ii,,',i~~ii~,~:~ilii,~iii!ii,~,,~~i,.~i.... ....~;i~ifi!ii~i,, ii{iii,.~!~iii!~,......... :;!
I Aged Enzyme (irreversible) I FIG. 1. An organophosphorus ester associates with the serine-hydroxyl moiety at the acetylcholinesterase active site (A) to form a reversible complex that is then inhibited via phosphorylation. The rates of association and phosphorylation are governed by ka and kp. The phosphorylated enzyme is then either reactivated via hydrolysis or becomes aged (irreversibly inhibited) via dealkylation (B).
15 2
SECTION II 9Pharmacokinetics & M e t a b o l i s m
hydrolyzed within minutes, whereas the phosphorylation of AChE is irreversible and reactivation can take several hours or days (Stevens and Breckenridge, 2001). During ChE inhibition, the inhibitor compound associates with AChE to form a reversible complex; following phosphorylation, the enzyme is either dephosphorylated, resulting in spontaneous reactivation of the enzyme, or dealkylated, resulting in the irreversible aging of the inhibited enzyme. The rate of association is governed by the association constant ka, whereas the rate of phosphorylation is mediated by the constant/c o (Fig. l b). There is some dispute as to whether species differences are due to differences in ka only (Andersen et al., 1977; Forsberg and Puu, 1984) or to differences in both ka and kp (Johnson and Wallace, 1987; Kemp and Wallace, 1990; Wallace, 1992; Wang and Murphy, 1982). Rates of spontaneous reactivation and aging of phosphorylated AChE differ among species and contribute, in part, to species variability. Whereas bird AChE reactivates rapidly, fish or insect AChE reactivates slowly, if at all (Wallace, 1992; Wallace and Herzberg, 1988), contradicting the general tenet that birds are more sensitive than fish to AChE inhibition. Furthermore, paraoxon- or DFP-inhibited AChE from frogs or fish ages slower compared to rat, mouse, or chicken AChE (Andersen et al., 1972; Wallace, 1992; Wallace and Herzberg, 1988). Finally, somaninhibited AChE in humans reactivates slowly but ages rapidly compared to bovine or rat AChE (de Jong and Wolfing, 1984, 1985; Wallace, 1992), suggesting that humans are relatively susceptible to irreversible AChE inhibition by soman. Species differ in their rate of recovery from AChE inhibition; indeed, oximes used as an antidote to reactivate AChE show differential species susceptibility (Eyer, 2003). There are species- and compound-dependent differences in the sensitivity of AChE to inhibition. Because brain AChE inhibition is the primary mechanism of acute toxicity, the sensitivity.of brain AChE to in vitro inhibition is an important determinant of acute toxicity. This is evident in correlations between the concentration required to inhibit 50% of AChE in vitro (IC50) and the dose that results in 50% lethality in vivo (LD50) (Wallace, 1992). Sensitivity of brain AChE to inhibition, and not the total AChE activity, potentially influences species differences (Johnson and Wallace, 1987; Wallace, 1992). The enzyme activity of 15 mammalian species surveyed ranged from approximately 2 to 10 ixmol/min/g wet tissue, whereas avian AChE activity from 44 species ranged from 10 to 30 ixmol/min/g (Blakley and Yole, 2002). The higher AChE activity in birds compared to mammalians is counterintuitive since birds are generally more susceptible to acute cholinergic toxicity than rodents. On the other hand, fish (minnow and trout) AChE activity is higher than that of rats (Table 2) (Johnson and Wallace, 1987), corresponding to relative resistance of fish to acute toxicity. Table 3 lists effective doses for ChE inhibition across species, including humans, for comparison among species
and compound. It is evident that plasma ChE is more sensitive to inhibition in humans than in dogs for physostigmine and DFP. For the same compounds, brain ChE is more sensitive to inhibition in rats compared to dogs. Chicken brain ChE is more sensitive than rat brain ChE for DFP and dichlorvos, but sensitivities are reversed for malathion and carbaryl (Table 3) (Raffaele and Rees, 1990). Whereas monkey brain AChE is more sensitive than chicken brain AChE to inhibition by DFP (Wallace, 1992; Wang and Murphy, 1982) human and chicken brain AChE sensitivities to inhibition by various OPs are comparable (within 30%) (Lotti and Johnson, 1978; Wallace, 1992). Conversely, BuChE (also known as pseudocholinesterase) inhibition does not provide a reliable indication of species differences in acute toxicity (Ecobichon, 2001a; Wallace, 1992). Although RBC AChE inhibition is often used as an indicator of OP exposure, it is unlikely that RBC AChE inhibition plays a major role in mammalian species differences among various OPs. Based on a literature review of 30 OP pesticides, Storm and colleagues (2000) provide an extensive table of adjusted 8-hr inhalation RBC AChE inhibition NOELs from humans, monkeys, dogs, and rats. This review focuses on occupational exposures, so exposure data from oral studies and inhalation studies were adjusted to provide an equivalent 8-hr inhalation exposure. The available data indicate that most OPs are equally potent RBC AChE inhibitors in different mammalian species (Storm et al., 2000). The adjusted 8-hr inhalation RBC AChE inhibition NOELs for the pesticides evaluated vary by a factor of two or three across species in most cases, when the exposure durations are approximately equivalent, although a few exceptions are noted. One exception, trichlorphon, appears to be 10-40 times more potent in primates than in dogs or rats (Storm et al., 2000). A contributing factor to this large variability may be relatively greater binding to nonspecific esterases in rats and dogs compared to primates (Anzueto et al., 1986; Storm et al., 2000), indicating that pharmacokinetic factors influence pharmacodynamics. 2. ALTERATIONSIN ACETYLCHOLINESTERASE STRUCTURE In addition to differences in enzyme kinetics contributing to species differences in sensitivity to AChE inhibition, alterations in the AChE enzyme impact its sensitivity to inhibition. Various interspecies, and possibly intraspecies, structural variants of mammalian AChE are synthesized by alternative splicing and posttranslational modification (Grisaru et al., 1999; Vidair, 2004). The distribution and molecular forms of AChE in the blood exhibit interspecies differences (Skau, 1985), although the significance to toxicity is not well appreciated. Differences in the physical or molecular properties of AChE influence enzyme and inhibitor interaction. Binding affinity of the parent compound and/or its metabolite for AChE is a major determinant of compound potency and is
CHAPTER 1 1 9lnterspecies Variation in Anticholinesterases TABLE 3.
153
Cholinesterase Inhibition in Various Species Exposed to Different Compounds
Compound
Species
Site
Value (mg/kg)
% ChE inhibition
Physostigmine Physostigmine Diisopropyl fluorophosphate Diisopropyl fluorophosphate Diisopropyl fluorophosphate Diisopropyl fluorophosphate Dichlorvos Dichlorvos Mipafox Mipafox Malathion Malathion Carbaryl Carbaryl
Rat Dog Chicken Rat Rat Dog Chicken Rat Rat Chicken Chicken Rat Rat Chicken
Physostigmine Physostigmine Diisopropyl fluorophosphate Diisopropyl fluorophosphate
Human Dog Human Dog
References a
Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain
0.2 0.35 0.25 1.0 0.2 1.5 5 30 30 30 150 2000 160 300
40 40 63 66 17 12 26 21 81 76 53 56 47 60
1, 2 1, 3 4 4 1, 5 1, 3 4 4 4 4 4 4 4 4
Plasma Plasma Plasma Plasma
0.06 0.35 0.03 1.5
25 30 80 80
1, 2 1, 3 1, 6 1, 3
al, Raffaele and Rees (1990); 2, Becker and Giacobini (1988); 3, Paulet (1956); 4, Ehrich et aL (1995); 5, Chippendale et al. (1972); 6, Grob et aL (1947).
govemed by structural characteristics of AChE and the inhibitor (Storm et al., 2000). Wallace (1992) provides a detailed review of these properties. In brief, the steric properties and nucleophilic strength of the esteratic site, the presence and electronic strength of the anionic site, and the distance between the esteratic and anionic sites govern the interaction of the enzyme-inhibitor complex. The following properties confer relative resistance to AChE inhibition: (1) A smaller esteratic site, such as in fish, provides steric exclusion from the enzyme binding site; (2) weaker nucleophilic strength; (3) less effective allosteric regulation by the anionic site; and (4) a greater distance between the esteratic and anionic sites (Wallace, 1992). 3. NEUROTOXIC ESTERASE INHIBITION AND ORGANOPHOSPHATE-INDUCED DELAYED NEUROPATHY Exposure to some OPs produces a neuropathic response distinct from acute cholinergic toxicity, termed organophosphate-induced delayed neuropathy (OPIDN), and characterized by sensorimotor deficits affecting the distal extremities and reflecting selective degeneration of fibers in the spinal cord (CNS) and PNS (Cavanagh, 1963; Veronesi, 1992). Aside from species differences in acute cholinergic toxicity, there are also differences in OPIDN. Chickens are the sentinel species for studying OPIDN due to their sensitivity as a result of high accumulation rate and
low elimination rate relative to rodents (Abou-Donia, 1983). However, delayed neuropathy also is readily produced in cats and farm animals and is observed occasionally in humans as well (Veronesi, 1992). For purposes of human health risk, the cat may be a better model to extrapolate neurotoxicity results to humans because the exaggerated effects in chickens overestimate the risk to humans (Abou-Donia, 1983). Although rodents classically are considered insensitive to OPIDN due to their failure to develop hindlimb paralysis after exposure to neuropathic OPs, they do develop neuropathology and biochemical endpoints typical of OPIDN but without ataxia (Ehrich e t al., 1995; Veronesi, 1992). The species differences have been attributed to various notions, including differences in pharmacokinetics and qualitative differences in the target enzyme, generally accepted as neurotoxic esterase (NTE) (Abou-Donia, 1983; Hussain and Oloffs, 1979; Soliman e t al., 1982; Veronesi, 1992). NTE and AChE from both rat and chicken brain can be inhibited, albeit with varying sensitivities (Ehrich et al., 1995). Pretreatment with hepatic metabolic inhibitors increases rodent sensitivity to OPIDN, suggesting that interspecies differences in the metabolism of TOCP play a major role in neuropathy to this OP (Veronesi, 1992). Several species, including cats, chickens, humans, and rats, have been used to demonstrate that NTE inhibition can be used to predict neuropathic damage, distinct from
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SECTION II 9P h a r m a c o k i n e t i c s & M e t a b o l i s m
acute cholinergic toxicity predicted from AChE inhibition (Veronesi, 1992). Mice also are sensitive to delayed neuropathy characterized by spinal cord pathology 2 or 3 weeks after TOCP exposure (Veronesi, 1992; Veronesi et al., 1991). Interspecies differences in the time course of NTE inhibition and recovery contribute to the species variability in sensitivity to OPIDN, with birds exhibiting a greater and longer duration NTE inhibition than rats despite an equipotent exposure to TOCP (Veronesi, 1992). The faster recovery of NTE inhibition in rats is due to reduced aging of the inhibited enzyme and/or more rapid synthesis of NTE (Clothier and Johnson, 1980; Soliman et al., 1982; Veronesi, 1992). A subsequent study found that rat and chicken brain NTE were similar with respect to inhibitor sensitivities, pH sensitivity, and molecular weight but differed in specific activity; hen brain NTE has approximately twice the specific activity as rat brain NTE (Novak and Padilla, 1986; Veronesi, 1992). Because the most striking difference in OPIDN between rats and chickens is the resistance of the rat to hindlimb ataxia compared to the chicken, interspecies neuroanatomical differences underlying modes of locomotion potentially account for species variability in sensitivity to OPIDN (Barnes and Denz, 1953; Cavanagh, 1954; Veronesi, 1992). Species such as chickens and humans become ataxic when fewer CNS neural tracts are damaged compared to rodents (Veronesi, 1992). Tracts housing the largest and longest nerve fibers are the most vulnerable to OPIDN, regardless of species, so that different tracts are damaged in various species. Spinal cord damage seen in rats differs from that in hens. For example, in humans and cats the most severely affected descending tracts are the pyramidal tracts, which are absent in the bird (Veronesi, 1992). On the other hand, the most vulnerable tracts in the hen are scattered throughout the lateral, ventral, and dorsal columns, whereas the large-diameter sensory fibers terminating in the dorsal columns of the upper cervical cord are most affected in the rat (Abou-Donia, 1981; Cavanagh, 1954; Veronesi, 1992). Thus, when considering species variability to nonacute toxicity, other target enzymes such as NTE are important as well as differences in neuroanatomy that could alter the outcome of OP exposure.
C. Age- and Gender-Dependent Effects Age is also an important factor to consider, and it is discussed elsewhere in this book. It is worth mentioning briefly here because some of the species-specific factors that contribute to interspecies variability also play a role in age-dependent effects of anti-ChEs. Relevant to this discussion is that young animals are more susceptible to OP toxicity than adults (Pope and Chakraborti, 1992; Pope et al., 1991; Vidair, 2004). Activities of brain AChE and liver aliesterase increase with age in rats; thus, both target (ACHE) and protective (aliesterase) esterases become more
abundant. However, sensitivity of brain AChE to paraoxon and chlorpyrifos-oxon-mediated inhibition does not change with age (Atterberry et al., 1997). Moreover, the similar maximal brain ChE inhibition in both neonatal and adult rats exposed to methyl parathion, parathion, or chlorpyrifos, coupled with the faster ChE synthesis and recovery in neonates (Pope and Chakraborti, 1992; Pope et al., 1991), suggests that target enzyme sensitivity is not key in age-related effects. In contrast, the greater sensitivity of neonates to acute OP toxicity correlates with lower levels of enzymes involved in OP deactivation compared to adults in both rats and humans (Vidair, 2004). For example, newborns have low levels of PON1, predicting greater sensitivity than adults to oxons hydrolyzed by PON1 (Davies et al., 1996). Similarly, gender differences in the overall rates of detoxification contribute to the greater sensitivity of female rats to some OPs (parathion, methyl parathion, and chlorpyrifos) compared to male rats (Chambers et al., 1994; Ma and Chambers, 1994; Neal and Dubois, 1965; Storm et al., 2000). Fetal enzyme isoforms differ from adult isoforms, and the ratio of phase I (e.g., cytochrome P450, glutathione peroxidase, and carboxylesterase) to phase II (e.g., sulfotransferase, glutathione S-transferase, and N-acetyltransferase) enzyme activities tends to increase with age (deBethizy and Hayes, 2001; Vidair, 2004). Primate fetuses contain a more developed set of toxicant-metabolizing enzymes compared to rodent fetuses (Dorman et al., 2001; Gow et al., 2001; Nau, 1986; Vidair, 2004), suggesting that primates are better equipped to protect against toxicity, although this is not always an accurate prediction. Furthermore, plasma binding proteins are lower in experimental animals compared to humans and are lower in human infants compared to adults (Nau, 1986; Vidair, 2004). Based solely on this, adult humans are expected to be protected from circulating OPs, whereas experimental animals and human infants are not expected to be protected. A host of noncholinergic developmental effects of OPs also contribute to age-related differences in toxicity (Slotkin, 1999, 2004; Pope, 1999). For instance, neurotransmitters, including serotonin and norepinephrine, in addition to ACh act as trophic regulators during development and are altered following developmental chlorpyrifos exposure (Aldridge et al., 2004; Dam et al., 1999a,b; Dreyfus, 1998; Lauder and Schambra, 1999; Raines et al., 2001; Weiss et al., 1998). Additionally, signaling cascades (e.g., adenylyl cyclase-cyclic AMP-protein kinase A cascade) involved in the proliferation and differentiation are disrupted following chlorpyrifos administration (Garcia et al., 2001; Huff et al., 1994; Olivier et al., 2001; Song et al., 1997; Ward and Mundy, 1996; Yanai et al., 2002; Zhang et al., 2002). Undeniably, developmental events render young animals, including humans, potentially more vulnerable to neurotoxicity from toxicants. In some cases,
CHAPTER 1 1 9Interspecies Variation in Anticholinesterases these age-dependent differences parallel species-dependent differences, as is the case for variable enzyme levels and activities as well as sensitivity to AChE inhibition.
IV. C O N C L U S I O N S A N D F U T U R E DIRECTIONS It is challenging to tease out a definitive source of interspecies variability; pharmacokinetic and pharmacodynamic parameters contribute dynamically to the sensitivity, response, and recovery of various species. Metabolism of anti-ChEs clearly plays a key role not only in species differences but also in age-dependent differences, whereas absorption, distribution, and elimination influence the bioavailability of the compound for metabolism. The route of exposure to an OP (or CM) significantly impacts the rate of absorption into the body and target organs and thus contributes to the toxic effects. Although anti-ChEs exert a common mechanism of action by inhibiting cholinesterases in blood and brain, the target enzyme is subject to species deviations. Whereas sensitivity to AChE inhibition contributes to the ultimate response, basal enzyme activity probably does not play a significant role in this sensitivity. Rather, the interaction of AChE and specific compounds is the predominant factor influencing sensitivity to inhibition. Structural AChE variants exist that can be more or less amenable to a "fit" from a specific compound. Finally, neuroanatomical differences among species may contribute to manifestations of toxicity, as proposed for the resistance of rodents to OPIDN compared to chickens. Interspecies comparisons of noncholinergic mechanisms are not well appreciated. Future studies should exploit both the similarities and the differences among species to delineate factors that are most influential on speciesspecific responses and to elucidate mechanisms of action beyond ChE inhibition. Although mammals are used for Environmental Protection Agency guideline toxicity tests (rats, mice, rabbits, dogs, and primates), a new corps of nonmammalian experimental animals, such as in vitro systems, have the potential to prove useful for addressing specific mechanistic questions. Nematodes, sea urchins, zebrafish, and avian embryos are being considered as models of mammalian neurotoxicity because they offer one or more of the following: more rapid screening, simplistic neural and neurotrophic systems, real'time in vivo observations during development, and elimination of confounding factors arising from maternal-fetal interactions (Buznikov et al., 2001; Cole et aL, 2004; Grunwald and Eisen, 2002; Slotkin, 2004; Yanai et al., 2004). Through pharmacokinetic and pharmacodynamic studies, we are closer to understanding the interspecies variability in response to anti-ChE intoxication.
155
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Lotti, M., and Johnson, M. (1978). Neurotoxicity of organophosphorus pesticides: Predictions can be based on in vitro studies with hen and human enzymes. Arch. Toxicol. 41, 215-221. Ma, T., and Chambers, J. (1994). Kinetic parameters of desulfuration and dearylation of parathion and chlorpyrifos by rat liver microsomes. Food Chem. Toxicol. 32, 763-767. Maxwell, D., Brecht, K., and O'Neill, B. (1987). The effect of carboxylesterase inhibition on interspecies differences in soman toxicity. Toxicol. Lett. 39, 35-42. Moser, V., Chanda, S., Mortensen, S., and Padilla, S. (1998). Age- and gender-related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of esterase activities. Toxicol. Sci. 46, 211-222. Natoff, I. (1971). Organophosphorus pesticides: Pharmacology. Prog. Med. Chem. 8, 1-37. Natoff, I., and Reiff, B. (1973). Effect of oximes on acute toxicity of anticholinesterase carbamates. Toxicol. Appl. Pharmacol. 25, 569-575. Nau, H. (1986). Species differences in pharmacokinetics and drug teratogenesis. Environ. Health Perspect. 70, 113-129. Neal, R., and Dubois, K. (1965). Studies on the mechanism of detoxification of cholinergic phosphorothioates. J. Pharmacol. Exp. Ther. 148, 185-192. Nelson, E., Raj, E, Belfi, K., and Masters, B. (1971). Oxidative drug metabolism in human liver microsomes. J. Pharmacol. Exp. Ther. 178, 580-588. Novak, R., and Padilla, S. (1986). An in vitro comparison of rat and chicken brain neurotoxic esterase. Fundam. Appl. Toxicol. 6~ 464--471. Olivier, K., Jr., Liu, J., and Pope, C. (2001). Inhibition of forskolin-stimulated cAMP formation in vitro by paraoxon and chlorpyrifos oxon in cortical slices from neonatal, juvenile, and adult rats. J. Biochem. Mol. Toxicol. 15, 263-269. Padilla, S., Buzzard, J., and Moser, V. (2000). Comparison of the role of esterases in the differential age-related sensitivity to chlorpyrifos and methamidophos. Neurotoxicology 21, 49-56. Paulet, G. (1956). Cholinesterase activity and function of the respiratory centers. J. Physiol. (Paris) 48, 915-936. Pelkonen, O., Jouppila, P., and Karki, N. (1973). Attempts to induce drug metabolism in human fetal liver and placenta by the administration of phenobarbital to mothers. Arch. Int. Pharmacodyn. Ther. 202, 288-297. Pope, C., and Chakraborti, T. (1992). Dose-related inhibition of brain and plasma cholinesterase in neonatal and adult rats following sublethal organophosphate exposures. Toxicology 73, 35-43. Pope, C., Chakraborti, T., Chapman, M., Farrar, J., and Arthun, D. (1991). Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides. Toxicology 68, 51-61. Pope, C. N. (1999). Organophosphorus pesticides: Do they all have the same mechanism of toxicity? J. Toxicol. Environm. Health 2, 161-181. Raffaele, K., and Rees, C. (1990). Neurotoxicology dose/response assessment for several cholinesterase inhibitors: Use of uncertainty factors. Neurotoxicology 11, 237-256. Raines, K. W., Seidler, E J., and Slotkin, T. A. (2001). Alterations in serotonin transporter expression in brain regions of rats exposed neonatally to chlorpyrifos. Dev. Brain Res. 130, 65-72.
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Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 114-125. Butterworth-Heinemann, Oxford. Veronesi, B., Padilla, S., Blackmon, K., and Pope, C. (1991). Murine susceptibility to organophosphorus-induced delayed neuropathy (OPIDN). Toxicol. AppL PharmacoL 107, 311-324. Vidair, C. (2004). Age dependence of organophosphate and carbamate neurotoxicity in the postnatal rat: Extrapolation to the human. Toxicol. Appl. Pharmacol. 196, 287-302. Walker, C., and Mackness, M. (1987). "A" esterases and their role in regulating the toxicity of organophosphates. Arch. Toxicol. 60, 30-33. Wallace, K. (1992). Species-selective toxicity of organophosphorus insecticides: A pharmacodynamic phenomenon. In Organophosphates: Chemistry, Fate, and Effects (J. Chambers and P. Levi, Eds.), pp. 79-105. Academic Press, New York. Wallace, K., and Dargan, J. (1987). Intrinsic metabolic clearance of parathion and paraoxon by livers from fish and rodents. Toxicol. Appl. Pharmacol. 90, 235-242. Wallace, K., and Herzberg, U. (1988). Reactivation and aging of phosphorylated brain acetylcholinesterase from fish and rodents. Toxicol. Appl. Pharmacol. 92, 307-314. Wang, C., and Murphy, S. (1982). Kinetic analysis of species difference in acetylcholinesterase sensitivity to organophosphate insecticides. Toxicol. Appl. Pharmacol. 66, 409-419. Ward, T. R., and Mundy, W. R. (1996). Organophosphorus compounds preferentially affect second messenger systems coupled to M2/M4 receptors in rat frontal cortex. Brain Res. Bull. 39, 49-55. Warfington, J., Greenblatt, D., and von Moltke, L. (2004). The effect of age on P-glycoprotein expression and function in the Fischer-344 rat. J. Pharmacol. Exp. Ther. 309, 730-736. Weiss, L., and Orzel, R. (1967). Some comparative toxicologic and pharmacologic effects of dimethyl sulfoxide as a pesticide solvent. Toxicol. Appl. Pharmacol. 11, 546-557. Weiss, E., Maness, P., and Lauder, J. (1998). Why do neurotransmitters act like growth factors? Perspect. Dev. Neurobiol. 5, 323-335. Yanai, J., Vatury, O., and Slotkin, T. A. (2002). Cell signaling as a target and underlying mechanism for neurobehavioral teratogenesis. Ann. NYAcad. Sci. 965, 473-478. Yanai, J., Beer, A., Huleihel, R., Izrael, M., Katz, S., Levi, Y., Rozenboim, I., Yaniv, S., and Slotkin, T. (2004). Convergent effects on cell signaling mechanisms mediate the actions of different neurobehavioral teratogens: Alterations in cholinergic regulation of protein kinase C in chick and avian models. Ann. N. Y. Acad. Sci. 1025, 595-601. Zhang, H. S., Liu, J., and Pope, C. N. (2002). Age-related effects of chlorpyrifos on muscarinic receptor-mediated signaling in rat cortex. Arch. Toxicol. 75, 676-684.
Esterases, Receptors, Mechanisms, & Tolerance Development
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CHAPTER
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2
Structure and Function of C h o l i n e s t e r a s e s
ZORAN RADI(~ AND PALMER TAYLOR University of California at San Diego, La Jolla, California
much better bind generally bulky ligands, including covalent inhibitors isoOMPA and bambuterol and reversible inhibitor ethopropazine, all well-known specific inhibitors of ChEs (Vellom et al., 1993; Radi6 et al., 1993). The site of catalysis in both enzymes is buried-~20/k deep in the center of the globular catalytic subunit. Tissue distributions and physiological functions of the two enzymes in higher organisms are different. AChE is mainly found in the central nervous systems, neuromuscular junctions, and the hematopoietic system of vertebrates, and it plays a key role in cholinergic neurotransmission. BuChE is found in liver, blood serum, and kidney. Its primary physiological role is not completely clear, but it may be involved in hydrolysis of dietary esters. Mice lacking the AChE gene and activity have normal BuChE activities and can survive more than 1 year but are more sensitive to organophosphate (OP) inhibitors, suggesting that BuChE activity may substitute at least in part for AChE activity (Adler et al., 2004). The mammalian cholinesterase profile contrasts with that of Drosophila, which lacks BuChE and in which AChE knockout results in embryonic lethality (Greenspan et al., 1980). In humans and most other vertebrate species, only one gene encodes ACHE. Insects and worms can have multiple genes encoding up to two (insects) or four (worms) AChEs.
I. I N T R O D U C T I O N From the beginning of the past century until the early 1970s, assay of the cholinesterases was based mainly on their capacity to catalyze the hydrolysis of acetylcholine and on the selectivity of compounds that interfere with the catalytic reaction (Dale, 1914). In the late 1960s and early 1970s, inhibitors and substrates were found to bind to a remote site(s), thereby allosterically interfering with the catalytic reaction (Changeux, 1966; Aldridge and Reiner, 1969; Taylor and Lappi, 1975). Improvements in protein purification techniques identified the active center and sufficient sequence to clone the gene encoding the enzyme (Schumacher et al., 1986). Five years later, the first threedimensional structure of a cholinesterase was solved (Sussman et al., 1991). Those events led to rapid accumulation of structural information on cholinesterases in the years to come, resulting in the determination of at least 125 primary structures and 61 three-dimensional structures of cholinesterases available today. In this chapter, we summarize the available structural information in the context of the evolutionary and functional relationships of the cholinesterases and related od[3 hydrolase fold proteins.
II. C H O L I N E S T E R A S E
FAMILY
A. Primary Structure
OF ENZYMES
Catalytic subunits of both AChE and BuChE consist of a single 500- to 600-amino acid-long peptide. The first amino acid sequence of an AChE was determined by Schumacher and colleagues in 1986 by cloning cDNA for the enzyme from fish Torpedo californica, whereas the first BuChE sequence was determined by sequencing purified enzyme protein isolated from human serum (Lockridge et al., 1987). A search of the SwissProt database revealed approximately 125 full-length or nearly full-length cholinesterase sequences, including approximately 108 AChE and 17 BuChE sequences. More than half of these refer to proteins
Two structurally and functionally very similar, yet distinct enzymes form the family of cholinesterases (ChEs). Acetylcholinesterase (ACHE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) both catalyze acetylcholine (ACh) hydrolysis with similarly high efficiency and only differ in efficiency to catalyze the hydrolysis of carboxylic acid esters of larger acyl group size, such as butyrylcholine or benzoylcholine. Larger substrates are hydrolyzed much better by BuChE due to small but significant differences in their structure that also allows BuChE to Toxicology of Organophosphate and Carbamate Compounds
161
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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SECTION III. Esterases, R e c e p t o r s , M e c h a n i s m s , & Tolerance D e v e l o p m e n t
whose function was not established directly but was inferred from sequence comparisons. The largest numbers of deposited sequences are of insect cholinesterases (51 sequences), including aphids (16 sequences), flies (15 sequences), and mosquitoes (10 sequences), followed by 19 entries of mammalian sequences. Also numerous are parasite (17 sequences) and worm (16 sequences) entries. The distribution of species with known cholinesterase sequences reflects an interest in studying enzymes targeted by OP and carbamate (CM) pesticides. Some deposited AChE sequences originate from species that developed resistance to pesticides used to eradicate them, most notably insects such as, mosquitoes, and flies. Cholinesterase activities have been documented in a number of plants and algae (Gupta and Gupta, 1997; Gupta et al., 1998; Fletcher et al., 2004), but their sequences are not known. Among the simpler life-forms, recently resolved genomic sequences of fungus Neurospora crassa (Galagan et al., 2003) and virus Mimivirus (Raoult et al., 2004) revealed cholinesterase-like sequences, suggesting utilization of cholinesterase activity in simple life cycles of primitive organisms. Table 1 summarizes full-length amino acid sequences of fish (ACES_TORCA; i.e., T. californica), insect (ACES_DROME; i.e., Drosophila melanogaster, 36% identity with Torpedo), human (ACES_HUMAN, 55% identity with Torpedo ACHE), and mouse (ACES_MOUSE, 56% identity with Torpedo ACHE) AChEs; human and mouse BuChEs; as well as four cholinesterases of very simple organisms m fungi (Q6MGI2 and Q872U5, 21 and 20% identity with Torpedo ACHE, respectively), tunicata Oikopleura dioica (Q675X9, 14% identity with Torpedo ACHE), and virus (Q5UR02, 21% identity with Torpedo ACHE). Out of approximately 50 serine residues found in cholinesterase sequences, only one is directly involved and essential in catalysis, Ser200, as confirmed by site-directed mutagenesis (Gibney et al., 1990). It is found in all sequences in Table 1, including viral and fungal proteins. A detailed comparison shown in Table 2 includes all 125 currently available cholinesterase sequences. The active Ser200 is conserved in 124 sequences and shifted by one residue C-terminal only in human hookworm Necator Americanus AChE (Q8IT86). It is not clear whether this protein is catalytically active, but similar one-position shifts in the active serine, although N-terminal, are found in sequences of structurally related, but catalytically inactive, neuroligins, a family of neuroadhesive proteins from human, mouse, and rat sharing approximately 30% amino acid identity with ACHE. Besides Ser200, five more serines are very well conserved throughout the family: Ser91, Ser205, Ser212, Ser226, and Ser428. The amino acid fragment (195-205) around the active Ser200 is well conserved in most cholinesterases. Of the 11 residues of the fragment, Gly202 appears to be strictly conserved in all 125 sequences, whereas the pattern Gly-X-Ser200-X-Gly-X-X-Ser
can be found in 123 sequences; exceptions are the simple marine organism Oikopleura dioica (Q675X9) and worm N. Americanus ACHE. Interestingly, 13 of 15 deposited fly AChE sequences have, starting with Ser200, the conserved pattern Ser-x-x-Ser-Ser-Ser, and one of the sequences (Q8MXC9) has a Ser-Ser'Gly-Ser-Ser-Ser pattern. Residues His440 and Glu327 were identified as the remaining two elements of the catalytic triad. Site-directed mutagenesis showed that substitution of His440Gln in T. californica AChE yielded inactive protein (Gibney et al., 1990), and Glu327 was identified as part of the catalytic triad only upon resolution of the first three-dimensional structure of an AChE (Sussman et al., 1991). The two residues are conserved in 123 (Glu327) and 124 (His440) sequences, suggesting that Oikopleura and one of two fungal proteins (Q6MGI2) in which residues other than Glu and His are found may not be catalytically active. Several other glutamates and histidines are well conserved in the cholinesterase family. Glu92 is conserved in all 125 sequences, and Glu199, Glu443, and His209 are conserved in almost all of them, whereas positions 172, 297, and 397 have either Asp or Glu always conserved. Besides the catalytic triad, additional residues are known to be critical for maintaining the catalytic activity of cholinesterases. The oxyanion hole stabilizes carbonyl oxygen of ACh during hydrolysis. It is formed by protein backbone amide nitrogen protons of residues Glyl 18, Gly119, and Ala201, as suggested by the positioning of trifluoroacetophenone, an ACh transition state analogue, in the threedimensional structure of Torpedo AChE (PDB code 1AMN; Harel et al., 1996). Two of three residues are likely sufficient to form a functional oxyanion hole entity, and all 125 sequences in Table 2 have at least two of three oxyanion hole residues conserved. The most conserved is Glyll8 (in 124 sequences), followed by Ala201 (in 120 sequences). The Ala201 is substituted with serine in 5 sequences. Gly 119, is conserved in 115 sequences with serine substitution in 6 of the remaining 10 sequences. Two additional sequence fragments exceptionally well conserved throughout the cholinesterase family are required for catalytic activity, although their involvement in the catalytic process is not fully understood. Residues 91-95 with sequence SEDCL (Ser-Glu-Asp-Cys-Leu) are conserved in 115 of 122 residues, motif xEDCL is conserved in 120 of 122 sequences, and motif xExxL is conserved in all 122 sequences. Two anionic residues in that fragment, Glu92 and Asp93, are involved in a salt bridge and a hydrogen bond formation with Arg44 and Tyr96, respectively. Both are conserved in number of cholinesterases, particularly Arg44, which is absent only in viral and O. dioica AChEs out of 125 sequences. Substitutions of Glu92Gln, Glu92Leu, Glu93Val, and Arg44Glu in single-site Torpedo AChE mutants resulted in the complete loss of catalytic activity, indicating their important role in folding and maintaining the three-dimensional structure of a disulfide loop.
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SECTION III. E s t e r a s e s , R e c e p t o r s , M e c h a n i s m s ,
covering the AChE active center gorge (Bticht et al., 1994). Lastly, Ser226 is conserved in 122 of 125 sequences. Located in the spatial vicinity of the active Ser200 and the other two residues of the catalytic triad, it is positioned to be directly involved in catalytic reaction. Its substitution with asparagine, observed in the AChE of embryonic lethals in zebrafish (Behra et al., 2002), rendered zebrafish AChE inactive. Similarly, substitution of this serine with alanine in Torpedo and mouse AChEs yielded inactive mutants (Z. Radid, unpublished data). Serine at the homologous position is fully conserved in the structurally related carboxylesterase family and in several lipases (Stok et al., 2004). Ser247Ala and Ser247Gly mutants of rat carboxylesterase ES10, at a position two- and three-dimensionally equivalent to Ser226 of Torpedo ACHE, were found to be only 5- to 15-fold less active than the wild-type carboxylesterase in hydrolysis of the substrate p-nitrophenylacetate (Stok et al., 2004). Rat carboxylesterase ES10 shares 31% identity and 55% amino acid sequence similarity with Torpedo ACHE. Most cholinesterase sequences include at least seven cysteines. Six cysteines are usually involved in the formation of three intramolecular disulfide loops, and the seventh one, closest to the C-terminal end, is through intermolecular bond involved in the formation of covalent AChE homodimers (MacPhee-Quigley et al., 1986). Two of three intramolecular cysteine loops have 1} configuration exposed on the protein surface. Their sequence conservation is shown in Table 3. Out of a total of 125 ChE sequences shown in Tables 2 and 3, 120 are fully known in the fragment Cys67-Cys94. All of them contain two cysteines, potentially capable of forming a disulfide loop, whereas in the fragment Cys254-Cys265 117 ChEs out of 125 have the ability to make a disulfide loop. A high degree of conservation of a disulfide bond within fragment Cys67-Cys94, known as the "big ~ loop" or just "1"}loop," is consistent with its location on top of the active center gorge. It is the thinnest of walls that separate the gorge from the bulk solvent. Most insects have an additional one or two cysteines located next to 12 loops or between second and third loops that most likely remain unpaired. Several aromatic residues appear very well conserved in cholinesterases, including tyrosines 148, 420, 421, 130, and 334; phenylalanines 187, 197, 155, 45, 448, and 476; and tryptophanes 179, 114, 84, 233,432, 435, and 492. Insects carrying two AChE genes include mosquitoes and aphids, and their sequences are shown in Table 4 in parallel to T. californica and D. melanogaster sequences. Clearly, two sequences found in each insect appear significantly different. Intraspecies sequence comparisons yield only 36-40% identity, but interspecies comparison of like sequences within each cluster shows larger (60-99%) identity. Both clusters of sequences have approximately 40% identity with Torpedo ACHE. One of the two clusters (the lower one in the Table 4), however, shares significantly
& Tolerance Development
higher (56-71%) identity with Drosophila AChE than the 36-40% identity shared by the other cluster. Worms can carry up to four AChE genes, showing generally similar (approximately 40%) intraspecies identity, with interspecies similarity less pronounced than that observed in insects.
B. Secondary and Tertiary Structure Several fragments in sequences of cholinesterases are likely to form an oL-helical secondary structure, as confirmed by three-dimensional structures of both AChE and BuChE. Obtaining three-dimensional structures of ChEs, AChEs, and BuChEs was not trivial, however. The first studies on the three-dimensional structure of an AChE were done using AChE crystals obtained from protein purified from the electric organ of the freshwater eel (Electrophorus electricus) (Chothia and Leuzinger, 1975; Schrag et al., 1988), but insufficient quality of the electron diffraction data prevented its immediate solution. An abundant source of the enzyme and an improved purification scheme proved critical to obtain large (milligram) quantifies of the enzyme protein in a state more homogeneous than that possible for E. electricus AChE (Taylor et al., 1974). Consequently the first successful three-dimensional structure determination was done for AChE of a saltwater fish, T. californica, at 2.8 ~ resolution (original PDB code, lace; Sussman et al., 1991). Several years later, it was realized that in the active center of the solved AChE structure, the inhibitor decamethonium was bound, whose electron density was initially interpreted as an array of water molecules (Raves et al., 1997). Decamethonium, originally used in the purification protocol to elute AChE bound to affinity chromatography column, remained in a complex with AChE despite exhaustive dialysis. This results from a high concentration of AChE in the dialysis bag, exceeding the KD of decamethonium by several orders of magnitude. This fortunate coincidence was important for the successful solution of the T. californica AChE three-dimensional structure and indicated that the structure of a ligand-free AChE would be more difficult to obtain. Accordingly, the determination of crystal structures of mammalian AChEs from mouse (PDB code, l mah: Bourne et al., 1995) and man (PDB code, l b41; Kryger et al., 2000) was initially possible only in complex with the potent inhibitor, snake venom toxin fasciculin 2. The first ligand-free AChE structures were obtained later (Raves et al., 1997; Bourne et al., 2004), requiting the use of different inhibitors for enzyme elution. Drosophila melanogaster AChE is the only insect AChE with a known three-dimensional structure (Harel et al., 2000). Because of extensive glycosylation, crystallization of BuChE was more difficult. BuChEs typically have seven to nine N-linked oligosaccharides, instead of the three or four oligosaccharides found in AChEs. Only recombinantderived, partially deglycosylated BuChE containing five oligosaccharides instead of nine appeared sufficiently
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174
SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
FIG. 1. Three-dimensional structures of five ChEs. (A) Overlay of Ca backbone of all five structures. (B-F) Solvent-accessible surfaces of individual enzymes: (B) Torpedo ACHE, (C) mouse ACHE, (D) human ACHE, (E) Drosophila ACHE, and (F) human BuChE. Active center gorge is indentation in the center of each structure. Visualized by WebLabViewer software (Accelrys, San Diego). TABLE 5.
homogeneous for crystallization (Nachon et al., 2002; Nicolet et al., 2003). The protein backbones of the five mentioned ChE structures, four AChEs and one BuChE, overlay reasonably well, with only Drosophila AChE showing slightly larger deviations (Fig. 1A; Table 5). The common fold includes a twisted [3 pleated sheet (approximately 10 strands long) in the core of the enzyme molecule, surrounded by more than 12 longer and shorter oLhelices. Such unique distribution of elements of the secondary structure was recognized first in ChEs and lipases and then in a number of other proteins, and it was consequently termed the od[3 hydrolase fold (Cygler et al., 1993). Catalytically active residues in enzymes belonging to this fold are located almost in the center of a globular protein, at the bottom of a narrow, approximately 20/k deep active center gorge. The shape and size of the gorge are similar in Torpedo, mouse, and human AChEs, but despite similarity in the backbone fold, the respective volumes of gorges in human BuChE and Drosophila AChE are approximately 50-100% larger and 50% smaller than in mammalian and fish AChEs (Harel et al., 2000; Saxena et al., 1999). This significant structural difference~likely affects the ability of ChEs to effectively hydrolyze ACh (Fig. 2). Hydrolysis appears more efficient in the active center gorges of fish and mammalian AChEs than in either the smaller volume gorge of Drosophila .ACHE or the larger volume gorge of BuChE. To date, 61 cholinesterase structures have been deposited in the Protein Data Bank. Forty are of T. californica ACHE, nine of mouse ACHE, three of Drosophila ACHE, two of human ACHE, and seven of human BuCHE. Overlaying of all Torpedo ACHE structures reveals exceptional similarity in their protein backbone (Fig. 3A) and even in their side chain conformations (Fig. 3B). The mean value of the root mean square (RMS) deviation of the 40 Torpedo AChE structures from the alpha carbon trace of the highest resolution (1.8 ~), unliganded lea5 structure is only 0.26 _+ 0.10 ]k. Twelve
Structural Pairwise Overlays of Five Unliganded ChE Structures a RMS (Ca backbone) (~.)
ChE
Torpedo californica AChE Mouse AChE Human AChE
Drosophila melanogaster AChE Human BuChE
PDB ID No.
Resolution (A)
1ea5 lj06
1.8 2.4
lb41 1q09 lp0i
Torpedo californica AChE
Mouse AChE Chain A Chain B
Human AChE
Drosophila melanogaster AChE
Human BuChE
2.8 2.7
0 0.76 (chain A) 0.74 (chain B) 0.87 1.2
0.76 0 0.24 0.56 1.2
0.74 0.24 0 0.57 1.2
0.87 0.56 (chain A) 0.55 (chain B) 0 1.2
1.2 1.2 (chain A) 1.2 (chain B) 1.2 0
0.95 0.86 (chain A) 0.86 (chain B) 0.88 1.1
2.0
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aOnly Ca backbone atoms were used in pairwise alignments. Sequences were overlaid and RMS was calculatedusing SwissPDBViewerversion 3.7 software interface.
CHAPTER 1 2 9Structure and Function of Cholinesterases
175
hAChE
3 kcat
(10 ~ min 1)
TAChE
2 mAChE hBuChE DAChE I
I
I
I
I
0.01
0.1
1
10
100
A T C h (mM)
FIG. 2. Activity of ChEs (expressed as kcat) as a function of substrate acetylthiocholine (ATCh) concentration. Curves (except for DAChE) were calculated using Eq. (1) and literature values of catalytic constants: hAChE and hBuChE from Kaplan et al. (2001), TAChE from Radic et al. (1992), and mAChE from Radic et al. (1993). The DAChE curve was calculated using the equation and data from Stojan et al. (1998). All constants were determined at room temperature except for hAChE and hBuChE, which were determined at 27 ~ contributing to relatively higher kca t values compared to TAChE, mAChE, and DAChE. Unliganded AChEs are 0nly slightly more similar to the lea5 with a mean RMS value of 0.20 _+ 0.08 ,~ compared to an RMS of 0.28 _+ 0.10 A for the remaining 28 liganded structures. This is further emphasized in complexes of mouse ACHE, in which RMS variation between subunits of the same structure (mean RMS, 0.25/k) appeared even slightly larger than RMS deviation between the same protein chains of different structures (mean RMS, 0.21 ,~), indicating that the effect of packing identical subunits within the same structure perturbed the backbone conformation more than the presence of bound ligand. Even structures containing ligands forming a covalent bond with the AChE active serine did not differ in their RMS from noncovalent ones. The high degree of similarity and small RMS values found also for mouse and Drosophila AChEs and human BuChE suggest that binding of ligands to cholinesterases in general is not associated with large conformational changes of the protein backbone. However, dimensions of substrate molecules appear similar to or larger than the size of the AChE gorge that they need to traverse on the way to the active serine at its bottom. The fight entry of a substrate molecule into the narrow site of catalysis in the active center is thus possibly facilitated by a series of fast, small amplitude side chain motions, occurring repeatedly at time intervals far shorter than the catalytic cycle. The absence of structural evidence of large backbone movements in AChE structures may therefore result from evolutionary pressure to achieve and maintain their very fast catalytic tumover. Every AChE molecule (depending on the species) can tum over between 200 and 16,000 molecules of ACh every second or 1 molecule in as short as 63 Ixsec (Nolte
FIG. 3. Overlay of 40 Torpedo californica AChE structures using only CoL backbone atoms. (A) Trace of CoL backbone atoms with combined volume of all ligands found in structures represented as a solvent-accessible surface. (B) Representation of all enzyme atoms found in 40 overlaid structures. Structures were overlayed using SwissPDBViewer, software. Visualized by WebLabViewer software (Accelrys~ San Diego).
176
SECTION III. E s t e r a s e s ,
Receptors,
Mechanisms,
& Tolerance
Development
1. TOPOGRAPHY OF BINDING SITES IN C h E s
FIG. 4. Topography of binding sites in ChEs represented by selected residue side chains of Torpedo AChE structure. A molecule of ACh docked in the active center (taken from PDB entry 2ACE) and shown as solvent-accessible surface is given as a frame of reference. Binding sites are acyl pocket (residues 288 and 290), choline binding site (residues 84 and 330), and peripheral site (residues 70, 72, 121, and 279). Visualized by WebLabViewer software (Accelrys, San Diego).
et al., 1980). Continuous repetition of backbone movements would possibly require both more time and more energy invested per each hydrolytic cycle. These molecular motions have to be fast enough to allow diffusion-limited binding of relatively large ligands. Catalytic turnover of BuChEs is slower despite the much larger opening and larger volume of the active center gorge. Due to the absence of aromatic amino acid side chains lining the AChE gorge, approximately 20 water molecules found in either of the structures or substrate molecules may be retained along the gorge walls by hydrogen bonding and electrostatic interactions, This could inherently slow down catalysis, which may be even slower in the more water-accessible, and thus less hydrophobic, catalytic site of BuChE. Most of the volume of the AChE active center gorge seems to be well accessible to ligand binding judging from the combined volume of all ligands in active center gorges of 40 superimposed Torpedo AChE structures totaling approximately 1100/~3 (Fig. 3A) and filling practically 1000 ,&3 of the available gorge space (calculated by CASTp software; Liang et al., 1998).
In the immediate vicinity of catalytic triad residues at the base of the active center gorge, just above the active serine, is located the electrophilic oxyanion hole, which has the capacity to attract carbonyl oxygen of ACh and other substrates as well as phosphyl oxygen of covalent, OP inhibitors. Three backbone nitrogens well conserved throughout the cholinesterase family (Table 2) form the oxyanion hole, lending their amide protons for the interaction. In oxyanion holes of proteases, only two donor residues are involved. Site-directed mutagenesis of AChEs and use of substrates and inhibitors specific for interaction with either AChE or BuChE helped delineate specific locations of additional ligand binding sites within the enzyme gorge (Shafferman et al., 1992a; Vellom et al., 1993; Ordentlich et al., 1993; Radi6 et al., 1993). The acyl pocket and the choline binding site are located next to the active serine, at the base of the active center gorge (Fig. 4), controlling the size of ligands that can approach the site of catalysis. The space available for binding is generally smaller in AChEs in which several aromatic residues (14 in fish and mammalian AChEs; Sussman et al., 1991) line the walls of the gorge. In the acyl pocket, in-place of phenylalanines Phe288 and Phe290 found in AChEs smaller aliphatic residues are found in BuChEs, whereas in the choline binding site Phe330 (many AChEs have tyrosine at this position) in BuChEs is replaced by alanine (Table 6). The smaller residues and larger available space in BuChE enable preferential binding of the large substrates butyrylthiocholine and benzoylcholine in the acyl pocket and large inhibitors ethopropazine and isoOMPA in the choline binding site and acyl pocket, respectively. Some mutant insects (mosquitoes and flies) that developed resistance to pesticides have amino acid residues altered in this region in their AChEs, selectively preventing binding of pesticides to active serine while not compromising catalysis (Menozzi et al., 2004). In addition to the choline binding site and acyl pocket in the third binding domain of cholinesterases, the peripheral site is located at the rim of the gorge, approximately 14 /~ from the active serine. Formed by Tyr70, Tyrl21, and Trp 279, this aromatic cluster specifically binds cationic and aromatic inhibitors that are too large to enter the gorge, such as propidium, gallamine, or snake toxins fasciculins (Bourne et al., 1995, 2004), or long and slender bisquaternary ligands that extend from the bottom of the gorge, such as BW286c51, decamethonium, and a variety of bifunctional ligands including bis-tacrines, bishuperzines, and very high-affinity triazoles (Lewis et al., 2002; Bourne et al., 2004). In the vicinity of the aromatic cluster is Asp72, a specifically located anionic residue lending its stabilizing contribution to ligands binding to the -6 ~ proximal peripheral site and/or ,--8 A distal choline binding site. Devoid of an aromatic cluster in the peripheral site, BuChEs bind most bisquaternary and bifunctional inhibitors with three or four orders of magnitude lower
CHAPTER
T A B L E 6.
P e r i p h e r a l site I~
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Y
W
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W
Y
F
W
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MOUSE
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AMHUMANyt
Q86TM9
AMHUMAN2 AMONKEY AMONKEY ABCHICK
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RABIT
Q86YX9
r-
Y Y Y Y Y Y
Q67BCI
Y
Q67BC2
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CHICK
D D D D
T
D
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D
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061459
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Y
D
AF
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Y
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AHAG F I S H
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MYXGL
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061372
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BRARE
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ASNAKE
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097110
AIAPHID
Q6KAV3
TORMA BUNFA
ASPIDER
Q86CZ4
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QSUR02
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T F
Y
Y
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Q65Z60
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Y
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D Y
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D Y
D
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M
Y Y M M
Y Y M
Y Y
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Y
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W
W
W
L
F
W
F
W
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W
F
W
F
Y
W W
W W
W W
Y Y Y
Y
Y Y
Y
Y
M
W
L
W
M
W
M
W
M M
W W
I
L L
Y Y
Y Y
W
Y
L
Y
Y
W
Y
W
E
Y
Y
W
Y
Y
Q8MXC8
E
Y
W
Y
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Q8MVZ4
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Y
W
W
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W
W
W
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Y
F
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M
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Y
Y
W
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Q7YZP7
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W
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Y
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M
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W
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C
F
W
L
F F S
W
W
W
F
C
F
C
L
S
W
C
F
F G
W
W
G
L
F S
F
C
G
F
G
F
F
W
L
G
F
W
L
G
F
W
F
W
F F
F
F
W W
W
W
L L L
L
L
C
G G G G
G
E
W
L
G
W
Y
F
W
L
S
W
Y
W
Y
W
Y
Y
W
F
E
F
Y
L
Y
W
L
E
Y
W
Y
Y
W
L
W
Y
L
F
F
Y
Y
Y
E
W
V
W
Q95P20
Q8MXC4
Y
Y
L
AIFLY
AIFLY
F
Y
L
M
Y
E
Y
W
W
Y
E
W
M
W
ACES
DROME
Y
Y
Y
AIFLY
P91954
F
W
M
R
Y
Y
L
F
W
F
R
R
R
Y
F
R
F
W
-
R
R
E
F
Y
F
R
L
F
F
F
R
W
W
Y
F
F
R
L
Y
W
L
W
Q75VY0
AIFLY
W
W
M
AICULEX
E
W
F
F
R
W
W
W
L
QSRLH9
D
W
W
AIBUDWORM
I
W W
F
F
F F
F
W
W W
W
F
F
F
Y
~
W
F
Y
F
W
Y
L
W
W
W
W
Y
Y
L
W
Y
F
W
W
Y
Y
L
W
W
Y
F
W
W
F
~
Y
N
M
W W
Y
Y
Y
Y
T
F
Y
W
W
V
T
W
Y
E
W
AIAPHID
Y
G
Y
Y
-
Y
V
Y
D
-
F
V
W
D
I
W
Y
F
W
I
I
-
W
Y
F
F
W
W
Y
W
Y
F
Y
Y
D
Y
W
Y
W
L
A
Y
W
Y
W
-
K
V
Y
~
V
W
Y
V
oq
W
Y
W
0~
I
E
Q86QW5
T
W
D
D
Q8T7U9
AIBOLLWORM
Y
W
W
G
AIAPHID
LEPDE
Y
W
W
D
E
Y
e~
D
M
V
Y
Y
D
G
Y
D
D
ACELEGANS
Q9NDG9
Y
Y
Y
M
~
D
ACE4
ACELEGANS
CAEBR
r-
ACBRIGSAE ACBRIGSAE
Acyl pocket
C~
AM
ACES
Choline b i n d i n g site
(~
SwissProt code
AM
177
O
ID
AM
9S t r u c t u r e a n d Function o f C h o l i n e s t e r a s e s
Elements of ChE Primary Structures Involved in Ligand Binding as Found in A l i g n m e n t of 125 ChE Structures a Sequence
AM
12
W W
Y Y
Y Y Y
Y
F F F
F F F
W W
W
W
W W
L
L
L
L
L L
S
S S
S
S
G
(continues)
178
SECTION
III. s
R e c e p t o r s , M e c h a n i s m s , & Tolerance D e v e l o p m e n t TABLE
Sequence
Peripherai site O
C~
Q8MXC9
E
Y
Q7YWJ9
E
Y
ID
SwissProt code
AIFLY AIFLY
AIFLY
AIFLY AIFLY AIFLY AIFLY
AIFLY ~
AIHOPPER
AIHOPPER
Q8MXC5 QSMXC6 QSMXC7 Q7YZP8
Q5WIL0 QgNJH6
ANOGA
AIMOSQTO
ACES
CULPI
AIMOSQTO AIMOSQTO
AIMOSQTO AIMOSQTO AIMOTH AIMOTH AIMOTH
APBOOPHILUS
E
QSMU94
ACE1
AIMOSQTO
e.
Q95WV7
AIMOSQTO
AIMOSQTO
ACES
ANOST
Q5XL61
Q6A2E2
E
'
E E
E
E
E
I
I
E
Q7RTM0
E
Q8MZL2
Q5S579 QSMZM0
E
E
I
E
V
Q7RTL7
E
APFLUKE
Q71SU7
P
APTICK
Q6XR73
APCIONA
APFLUKE
APFLUKE APTICK
APTICK
Q7RTL6 Q71SU5 Q86GL8 061864 Q9NFKI
I
~'-
f'1
~
~
C'~
~
r
M
W
L
Y
W
Y
F
M
W
L
Y
W
Y
F
M
Y Y
Y
Y
D
I
c~
Y Y
D D
M M M
M M M
Y Y Y
D
Y
Y
M
Y
M
D Y
Y D
Choline binding site
,~
M
I
I
(",I
Y
Y
Q7RTL9 QgTXll
e.
E
061987
APCIONA
(continued)
6.
Y
M
M
Y
W
W W W W
W W W W W W
~
L
L
L L L L L
r
Y
Y
Y Y Y
Y Y
L
Y
I
Y
I I
Y Y
W
I
Y
W
L
Y
W
L
W W
W W
I
L
Y Y
Y
L
Y
L
Y
Y
M
W
L
Y
D
Y
W
L
Y
D
T
Q
W
D
Y
D
L
Y
L
D
Y
P
Q
Y
V
D
Q
Y
P
v
D D
D
Y Y Y
Y
D D
I
Y
Y
P
D
Y
L
L
T
Q
T
Y
Y Y
Qo
W
W W W
W W W W
W W W W
Y
Y Y
Y, Y
Y Y Y Y Y
Y Y
S
Y
L
S
Y
L
S
Y
F
W W
L
S
Y
W
L
G
F
W
L
S
X
W
L
S
F
W
L
S
X
W
L
G
F
W
L
G
F
W
C
E
F
W
C
E
F
W
C
E
F
W
C
E
F
F F F
F F F F
F
F F
W
Y
F
W
W W
W W W W W
W W
Y Y Y Y W
Y Y Y Y
Y
W
Y
W
W
w
W
L
F,.
W
~ o (x) o~
W
Y Y
(D
F
W W
Acyl pocket
F
F F F F
F
F F F F
F
F
F
W
C
E
F
W
L
G
F
W
L
G
F
W
L
G
F
W
L
G
F
W
C
~E
F
W
L
G
F
V
D
F
W
F
D
F
F
D
F
W
F
s
v
F
S
V
W
F
S
V
v
D
F
W
V
D
F
V
D
F
W
V
D
F
I
D
F
I
H
A
L
W W W
W
W
F
W
W
F
W
APTICK
Q6WVH4
V
D
Y
T
Q
Y
W
W
APTICK
Q9NFK2
C
Q
S
N
F
Y
P
Y
APTICK
Q9NFK3
A
Y
T
W
N
I
APTICK
Q6XR75
Y
T
W
N
I
G
Y
F
W
Y
W
W
APTICK APTICK APTICK APTICK
AW
Q9NFK4
G
Q6XR74
G
062563
G
V
S
D
ACE1
CAEBR
ACE1
AW
061378
AW
CAEEL
AW AW
A
D
W
W
D
F
S
Q6XPY6
T
T
w
F
D
P
Y
W
Y
W
Y
F
D N
G
S
Q967G8
T
S
F
D D D D
D D
N
Y
F
F
,W ' Y
F
P
P
Y
Y Y
W
W
W W
V
Y
P
Y
W
V
Y
W
S
P
Y
W
P
Y
D D D
F
N
F
A
F
T
T
W
N
T
F
S
T
W
W
D D
Y
T
W
W
T.
D
Y
Y
Y
W
AW
QSIT86
D
Q
A
W
T
AW
D
Q
D
Q86GL7
Qgu640
V
S
AW AW
A
Q
T
Q71JB7
Q
V
Q6QDP4
QgXYA9
A
G
T
Q6QDP5
Q
Q
061587
AW
AW
T
V
T
096529
V
Y
G
061371
AW
AW
D
045210
AW
AW
V
I
P
P P
P
P
Y
w
Y
W
Y Y
Y
W
W W W
W
N
Y
F
F
I
I I
F
W
F
W
Y
W
Y
W
F
Y Y
F
W F
G
G
W W
G
D
F
G
D
F
M
T
F
M
T
F W
M
E
W
A
D
F
W
M
T
F
W
M
S
F
W
M
T
F
A
D
F
D
F
W
W'
,~
W
M
W
W W
W
F
W
F
G
W
F
F
G
F
W
M
S
F
M
E
W
W
M
E
W
L,
T
F
(continues)
CHAPTER
12
179
9 S t r u c t u r e a n d Function of C h o l i n e s t e r a s e s
TABLE 6. (continued) Sequence
P e r i p h e r a l site
ID
O
Ol
C4
O~ ~,-
T
D
F
Y
-
-
M
W
D
M
Q
A
N
FELCA
N
D
Q
HUM.AN
N
SwissProtcode
AW
QgGPL0
BAMPHIOXUS
CHLI CHL2
BAMPHIOXUS
BAMPHYOXUS
m w
076998
BAMPHYOXUS
076999
BFUNGUS
Q6MGI2
BBCHICK
Q872U5
BM BM
BM
BRALA
CHLE
CHLE
~
CHLE CHLE
BM
CHLE
BM
MOUSE
PANTT
C H L E ,,RABIT
QgNIN9
BMRAT
QgJKCI
QgGKJ6
BMPIG
BOIKOPLEURA
Q675X9
A
L
HORSE
BMHORSE
-
L
Q90ZK8
BFUNGUS
BM
BRALA
e-
D
e.
-
D D D
~
F
F
E
N
G
K
L
Q
D
Q
V
Q
V
N
D
-
N G
-
D T
~ f~
f~ f~
P
Y
W
F
W
W
L
Y
-
Y
F
W
W
Y
F
W
G
G
G
-
A
F
V
D~
Ni
O f~
V
N
-D
~
W
D
N
~ f~
m
V
W
V
m
Y
V
Y
W
P
R
G
V
V
F
Y
K
V
T
Y
Y
G
A
Q
F
I
Y
F
A
V
T
F
W
L
S
W
L
S
W
L
F
W W
F
W
F
W
A
F
Q
Y
F
A
F
H
-
W
Q
L
F
W
F
W
F
A
Y
S
D
W
V
A
O O~
A
Y
V
W
A
e~
M
O0
W
W
F
W
O~
eq
00
D
A
Y
Y
M
00
A
,,W
V
Q
S
m
W
Y
L
V
F
W
A
Q
V
F
m
W
,Y
V
C
W
m
Y
V
N
-
W
Y
A
Q
~
Y
L
Q
R
Acyl pocket
f~ f~
W
N
D
Q
e~
C h o l i n e b i n d i n g site
F
W
W
Y
C C P
L L
I
W
S
V
L
S
R
I
V
S
A
I
S
L
L
e~
S
V I
V V V
S
V
N
Y
S
I
aone hundred eighteen AChE sequences are listed first, followed by 17 BuCfiE sequences. Sequences areroughly clustered as mammals, fish, insects, pests, worms, fungi, and virus. Dashes indicate missing sequence fragments. Sequences were aligned using BioEdit software interface (Hall, 1999).
affinity than AChEs (Radid et al., 1993), whereas the a f f i n ity of fasciculins is up to eight orders of magnitude weaker than in corresponding AChEs (Radid et al., 1994). The absence of the aromatic cluster does not critically influence ACh hydrolysis in BuChEs, and its substitution w i t h . aliphatic residues in AChEs does not seem to affect catalytic parameters; however, substitution of Asp72 has a pronounced effect on substrate Km in both AChEs and BuChEs (Shafferman et al., 1992b; Radid et al., 1993; McGuire et al., 1989; Masson et al., 1997). The BuChE variant containing Asp70Gly substitution (corresponding to the Asp72 position in Torpedo ACHE) is naturally occurring in the human population. Individuals with Asp70Gly mutation appear unable to hydrolyze the muscle relaxant succinyldicholine efficiently and thus experience life-threatening apnea lasting from a few minutes to several hours. Binding of ligands to the various sites in Torpedo and mouse AChEs seems to have a comparatively small effect on the overall fold of the enzyme structure (Figs. 5A and 5B), the exception is the largest peripheral site ligand fasciculin that consistently caused the largest RMS deviations of the Ca fold (Figs. 6A and 6B) due to more than a 1100 ~2 contact area in the complex. The increased RMS was thus the cumulative result of a larger number of small, sub-,~ngstrom shifts associated with residues of the large f~ loop (Cys67-Cys94). The loop was slightly, but noticably (Figs. 5A and 5B), pressured by fasciculin into the space of the active center gorge. The Phe330 of the active center choline binding site, however, consistently and sig -~ 1/
nificantly changes its side chain conformation in all structures recorded to date, both in Torpedo and mouse AChEs (in which it is replaced by Tyr). Its unique position at the base of the gorge allows it to regulate available space for ligand binding by rotating around its Ce~-C[3 bond (Figs. 5C and 5D). Tacrine and its derivatives in Torpedo, mouse, and Drosophila structures are stabilized by an aromatic "sandwich" between Trp84, which is always fixed in the same conformation at the very bottom of the gorge, and Phe330 (or its Tyr equivalents in mouse and Drosophila). The latter residue assumes conformation roughly parallel to Trp84, thereby closing access to the lower part of the gorge. When bisquaternary ligands, decamethonium and BW286c51 are bound, the position of the Phe330 ring is nearly perpendicular to Trp84. thus opening the full length of the gorge. In the immediate vicinity of Phe330 lies catalytic triad His440. It has been implicated that this histidine 'might be "mobile" during catalytic reaction m that is, that it can repetitively revert between two or more conformational states during catalysis. The structural evidence of one of these hypothetical states is given in the threedimensional structure of the "aged" VX phosphonylated T. californica AChE with the nerve agent VX (Millard et al., 1999) (Fig. 7A). The analogous crystal structure solved with VX phosphonylated and aged human BuChE, however, did not reveal any movements in the His of the catalytic triad (Nachon et al., 2005). The largest conformational change in the peripheral 'site was observed in the complex of the tightest binding triazole, the femtomoiar
180
S E C T I O N I I I . Esterases, Receptors, M e c h a n i s m s ,
& Tolerance D e v e l o p m e n t
FIG. 5. Variations in conformations of "ligand-bound" and free AChEs found in 40 Torpedo californica structures and 18 mouse AChE molecules extracted from nine structures. Ligands found in overlaid structures are presented as dark sticks. Slight movement of the 1~ loop covering bound ligands was found in complexes of both AChEs with fasciculin 2. (A) Torpedo californica overlay. (B) Mouse AChE overlay. Side chain conformers of (C) Phe330 in TorpedoAChE and (D) Tyr337 in mouse AChE found in overlaid structures. Visualized by WebLabViewer software (Accelrys, San Diego).
FIG. 6. Quantitative representation of variations in Ca traces represented as RMS values of (A) 42 Torpedo AChEs using the unliganded lEA5 structure as a frame of reference and (B) 18 mouse AChEs using the 1J06 chain a as a frame of reference. Structures were overlaid and RMS deviations calculated using SwissPDBViewer software. Bar patterns represent the type of ligand found in each structure.
CHAPTER 1 2 9Structure and Function of Cholinesterases
FIG. 7. Conformational changes of AChEs found in (A) Torpedo AChE-DFP aged adduct. DFP is covalently attached to the active Ser200. The gray ribbon and selected side chain sticks represent unliganded Torpedo AChE structure. Aged DFP-AChE adduct is shown as black ribbon and sticks. (B) Mouse AChE conformations of Trp286 found in the peripheral sites of triazole syn-TZ2PA6 liganded (two conformations) and unliganded or liganded with other ligands (16 conformations). Visualized by WebLabViewer software (Accelrys, San Diego).
inhibitor syn-TZ2PA6 and mouse AChE (Bourne et al., 2004). Trp286 (Trp279 in Torpedo) flips in that structure by approximately 120 ~ (Fig. 7B) to enable a tight fit of the phenantridinium moiety of syn-TZ2PA6 in another "aromatic" sandwich between Trp286 and Tyr72 (Tyr70 in Torpedo). A substantial change in the backbone conformation has been reported in only two AChE structures, in both cases on the same acyl pocket loop (Trp279-Ser291). In the structure of Torpedo AChE phosphorylated by diisopropyl fluorophosphate (DFP) (Millard et al., 1999), and then aged, DFP is partially dealkylated, having only one of two isopropoxy groups bound to phosphorus. To fit between two phenylalanines of the AChE acyl pocket (Phe288 :and Phe290), the remaining isopropoxy group had to distort its backbone, causing the extended cationic side chain of Arg289 to flip by almost 90 ~ (Fig. 7A), facing the entrance of the gorge and incoming ligands. An even larger acyl pocket loop conformational change was reported for the complex between Torpedo AChE and a bifunctional galanthamine derivative (Greenblatt et al., 2004). Positioning of the ligand in this complex caused the acyl pocket loop (Trp279-Ser291) to assume a severely disordered and poorly defined conformation that was not included in the crystal structure coordinates.
C. Quaternary Structure Genetic information encoding AChE protein as documented for variety of species is contained in one gene, except for some insects and w o r m s , carying multiple genes for AChEs or BuChEs. The myriad of molecular forms includes covalent homodimers, either soluble or through a glycophospholipidinositol anchor attached covalently to mostly erythrocyte membranes; homotetramers formed as noncovalent dimers of covalent
181
dimers; and "readthrough" monomers expressed only during development and in mouse brain under stress as well as heteromeric associations of tetramers with structural peptides ColQ and PRIMA found in mammalian muscle and brain, respectively (Massoulie et al., 1993; Taylor and Radi6, 1994; Massoulie, 2002). All molecular forms contain identical catalytic subunits. Slightly different are C-terminal sequences that dictate oligomerization and cellular disposition of subunits. Sequence divergence of C-terminal end is achieved through 3' alternative splicing of mRNA (Gibney et al., 1988). Additional alternative splicing at the mRNAs 5' end was reported in tick AChEs (Baxter and Barker, 1998; Xu et al., 2003) but without affecting the sequence of the mature protein. In the vertebrate nervous system, AChE is found in tetrameric form associated through C-terminal peptides forming a four-helix amphipathic bundle around ColQ or PRIMA peptides. The regularly spaced Trp residues at the C-terminal (WAT domain) intercalate between Pro residues of PRAD (proline-rich amphipathic domain) and ColQ or PRIMA as described in a structural study on the WAT/PRAD tetramerization domain (Dvir et al., 2004) showing four parallel helices of WAT supercoiled around the antiparallel helix of PRAD. The naturally occurring monomeric form of fetal bovine serum ACHE, unlike the naturally occurring tetrameric form of fetal bovine serum, has a modified C-terminal end sequence, thus compromising its formation of tetramers (Saxena et al., 2003).
III. ChE VARIANTS IN THE H U M A N POPULATION Numerous structural variants of BuChE in humans consisting of one amino acid substitutions or frameshift mutations have been well documented in the literature (Kalow, 2004). Individuals carrying structural variants of very low or no BuChE activity in their tissues appear normal and are at risk only if given the muscle relaxant succinylcholine in preparation for surgery. On the other hand, variability of AChE structure in the human population is only starting to be discovered. To date, only approximately 13 single nucleotide polymorphisms (SNPs) have been identified in the AChE encoding DNA in the human population (Hasin et al., 2004). Eight are synonymous and they do not alter the protein sequence, and five cause amino acid substitutions in mature AChE protein. Substitutions are positioned on the AChE surface (Fig. 8) and are not likely to affect its catalytic function, but the ability of monomers to associate with other molecules may be influenced. In particular, SNP resulting in substitution located close to the C-terminal end (Pro561Arg in the mature human AChE sequence) could influence tetramerization of AChE and its anchoring in nervous tissue.
182
SECTION III- E s t e r a s e s , R e c e p t o r s , M e c h a n i s m s ,
& Tolerance Development
strate activation, respectively. A unique and simple reaction scheme can describe both kinds of interactions: kcat E +
+
S ~
S
FIG. 8. Variants of AChE structure, resulting in amino acid substitutions of the mature protein, found in the human population (data from Hasin et al., 2004). Sites and types of substitutions are labeled on the gray ribbon, with a molecule of ACh shown as a gray surface, docked in the active center. The fifth identified substitution, Pro561Arg located at the very C-terminal end, is not shown because the known three-dimensional structure extends only through residue 542. Visualized by WebLabViewer software (Accelrys, San Diego).
A. Reaction of Cholinesterases with Substrates The kinetics of substrate hydrolysis by both AChEs and BuChEs deviates from Michaelis-Menten kinetics. As implicated in Fig. 2 for acetylthiocholine, only at substrate concentrations lower than 1 mM does the rise in enzyme activity follow Michaelis-Menten kinetics. At higher concentrations, AChE activity decreases, inhibited by the excess substrate, whereas BuChE activity increases, activated by the excess substrate. Hence, the terms substrate inhibition and substrate activation are respective hallmarks of catalysis by AChE and BuChE. Both phenomena can be simply described as a consequence of the formation of a ternary complex between the enzyme and two substrate molecules and thus as an allosteric phenomenon. The ternary complex in AChE has reduced or no activity compared to the Michaelis-Menten complex, whereas it appears more active in BuChE hydrolysis. It is important to emphasize that this is a substrate-specific phenomenon. Not all AChE and BuChE substrates exhibit substrate inhibition and sub-
ES +
>E+P
S
ssl SE
~
ssll +
S ~
~ S E S ~ S E +
P
where E and S stand for the free enzyme and substrate molecules respectively; ES is the Michaelis-Menten complex; SE is substrate bound to an allosteric, peripheral site on the enzyme; and SES is the ternary complex having one substrate molecule bound to the active center and the other to the allosteric, peripheral site. Kss is the dissociation constant for substrate bound to an allosteric, peripheral site, and kca t and bkca t a r e turnover numbers for the Michaelis-Menten complex and the ternary complex, respectively. Kss constants for ChEs are always larger than Km. Thus, the previous reaction scheme differs from the simple Michaelis-Menten scheme in the assumption that S can bind to more than one site on the enzyme, influencing the enzyme turnover by the factor b. When b = 1, the reaction kinetics is indistinguishable from Michaelis-Menten kinetics and binding of S to the allosteric, peripheral site appears kinetically "silent." When b < 1, the enzyme is inhibited by excess substrate as most frequently found for hydrolysis of ACh or acetylthiocholine by AChEs. The relationship between enzyme activity and the log of the substrate concentration (Fig. 2) is bell-shaped, indicating substrate inhibition. When b > 1, the enzyme activity is increased by excess substrate, as found for hydrolysis of ACh or acetylthiocholine by BuChEs, and the relationship between enzyme activity and the log of the substrate concentration appears as a double sigmoidal substrate activation curve (Fig. 2). The kinetic equation derived from the previous scheme summarizes these relationships as follows (CE Radi6 et al., 1993): v = v ( 1 + b[S]/Ks~)/[(1 +
[S]/K,~)(1 + Km/[S])]
(1)
where v is the enzyme activity at concentration of substrate IS], V is maximal activity of the enzyme, Km is the Michaelis-Menten constant, and Kss is the dissociation constant for substrate bound to the allosteric, peripheral site of the enzyme. Although the previous relationships hold for most ChEs, there are exceptions. Drosophila ACHE, for example, exhibits slightly more complex catalytic behavior in which some substrate activation is observed at low substrate concentrations in addition to substrate inhibition observed at millimolar substrate concentrations (Fig. 2) in the same reaction profile. In order to describe the reaction mechanism more precisely in that case, a more complex reaction
CHAPTER 1 2 9Structure and Function of Cholinesterases mechanism has to be assumed (Stojan et al., 1998, 2004). The molecular basis for this complexity is likely related to the fact that the geometry of the active center gorge in Drosophila AChE differs significantly from the gorge geometries of other AChEs and BuChEs.
B. Reaction of Cholinesterases with Inhibitors The vast majority of molecules that inhibit ChE activity are either reversible inhibitors or "progressive," "irreversible" covalent inhibitors.
1. REVERSIBLE INHIBITORS Reversible inhibitors form noncovalent complexes with the enzyme at the bottom of the active center gorge, at the peripheral site at its rim, or they span between the two sites. Association rates of most reversible inhibitors with ChEs are diffusion limited, and traversing the path leading to the base of the enzyme active center gorge does not slow down their entry. The binding equilibrium for those complexes is established rapidly, and only the magnitude of their dissociation rates controls their binding affinity, with dissociation constants usually found in the micromolar to nanomolar range (Radi6 and Taylor, 2001). A separate group of reversible ChE inhibitors m fasciculins, triazoles, and trifluoroacetophenonesmdissociate from enzyme very slowly (requiting hours, days, or weeks for complete dissociation) while maintaining very fast, diffusion-limited association rates. Affinities of these fight binding inhibitors are exceptionally high, with KD constants in the low pM and fM range. Fasciculins are a family of peptidic toxins from snake venom (Fasciculinl, Fasciculin2, and Fasciculin3) (Marchot et al., 1993), 61 amino acids long with a characteristic three-finger shape. The central finger enters halfway into the AChE active center gorge, blocking access to the active serine, whereas the bulk of the toxin molecule resides at the enzyme peripheral site (Bourne et al., 1995; Harel et al., 1995). The mechanism of inhibition combines steric blockade with allosteric components (Rosenberry et al., 1999; Radic and Taylor, 2001). Triazoles are a family of small molecule inhibitors designed by the "in situ" cycloaddition reaction between azide and acetylene building blocks in the AChE active center gorge (Lewis et al., 2002; Krasinski et al., 2005; Manetsch et al., 2004). The enzyme is exposed to a library of building blocks, and the tightest binding products pedominate in the gorge. This exceptional strategy for the design of high-affinity ligands yielded multiple inhibitors with femtomolar KD in a single round of screening from a small library of building blocks having high nanomolar to micromolar KDS. The triazole inhibitors are bifunctional, spanning the peripheral site and the active center (Bourne et al., 2004), but the triazole ring, formed in the cycloaddition reaction, contributes to binding affinity as a separate pharmacophore as well (Manetsch et al., 2004).
183
In contrast to the previously discussed noncovalent but slowly dissociating inhibitors, trifluoroacetophenones are reversible ChE inhibitors that covalently bind to the active serine but maintain the same structure upon dissociation from the enzyme (Nair et al., 1994). Their structure resembles "extended" reactive conformation of ACh, mimicking the transition state in its reaction with ChEs. Trifluoroacetophenones share three points of stabilization with the enzyme. In addition to the formation of a covalent bond between the active serine and carbonyl carbon of a trifluoroacetophenone, its carbonyl oxygen is stabilized in the oxyanion hole and the positive charge of quaternary nitrogen is stabilized through cation-xr interactions by aromatic residues of the choline binding site (Harel et al., 1996).
2. PROGRESSIVE, IRREVERSIBLE ChE INHIBITORS These inhibitors are substrates of ChEs that deacylate extremely slowly from the active serine, thus causing prolonged occupation of the enzyme active center and its inability to hydrolyze acetylcholine (Aldridge and Reiner, 1972). OPs and CMs are common irreversible inhibitors of ChEs and are frequently used as pesticides (Taylor, 2005). In some pesticides, such as malathion and parathion, specificity of inhibition of insect AChE is achieved by in vivo oxidation of the pesticide by cytochrome P450 forming the active oxon (malaoxon and paraoxon). Some OPs are also used as nerve warfare agents. Similar to pesticides, nerve agents are usually small neutral esters of organophosphoric or organophosphonic acid that are prone to be volatile and efficiently penetrate tissues. Upon initial phosphorylation or phosphonylation of the active serine, many OPs undergo an additional reaction called aging (Barak et al., 2000). Aging is a loss of an additional alkyl substituent group of an OP-inhibited enzyme that renders inhibitory OP moiety negatively charged (Aldridge and Reiner, 1972): AChE
0II + RI'O'P-O'Rz R3
0
aging
II > AChE-O-P-O-R 2 I OR 1 + R3
>
0
II -AChE-O-P-O +R z I OR1
hydrolysis ~ H 2 0
AChE +
O II HO-P-O-R 2 OR 1
Although catalytic activity of phosphorylated or phosphonylated enzymes can be restored by spontaneous deacylation in water or by using nucleophiles stronger than water, such as oximes, means of reactivating aged enzyme have not been demonstrated. Aging of nerve agents such as soman is very fast and occurs in minutes, whereas aging of pesticides such as paraoxon or ethyldichlorvos can take days to complete (Nachon et al., 2005; Aldridge and Reiner, 1972). Intensive and repeated application of pesticides can lead to the development of pesticide resistance in some insects, as documented for mosquitoes and flies (Hemingway et al.,
184
SECTION I I I - Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
References
FIG. 9. Locations of residue substitutions found in insecticideresistant insect strains mapped onto Drosophila AChE structure. Eleven side chain positions (compiled by Menozzi et al., 2004) are given as black surface contours. A molecule of ACh docked in the active center is shown as a gray surface. Visualized by WebLabViewer software (Accelrys, San Diego).
2004; Menozzi et al., 2004). One of the mechanisms involved in this process is the alteration of the insect AChE structure in such a way so as to preserve its ability to hydrolyze ACh but minimize its ability to react with OPs (Fig. 9). This is possible since planar geometry around the carbonyl carbon of ACh and tetrahedral geometry around phosphorus are sufficiently different, as are the overall sizes of pesticide and ACh molecules.
IV. C O N C L U S I O N S This chapter summarizes the latest structural information on the cholinesterase family of enzymes and discusses their catalytic properties. Alignment of 125 full-length or nearly full-length ChE sequences demonstrates a high degree of similarity conserved in the family from fungal and viral enzymes to man. Sixty-one reported three-dimensional structures are analyzed herein, illustrating the high degree of similarity in protein fold conformation irrespective of the presence of bound ligands, and this argues for the absence of large conformational changes in the protein interaction with both inhibitors and substrates. At the same time, a plentitude of small-amplitude backbone and selected side chain movements illustrate that rapid fluctuations in conformational states of ChEs prevail in solution and ligands have the capacity to select those conferring higher affinity and lower energy of the respective complex.
Adler, M., Manley, H. A., Purcell, A. L., Deshpande, S. S., Hamilton, T. A., Kan, R. K., Oyler, G., Lockridge, O., Duysen, E. G., and Sheridan, R. E. (2004). Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice. Muscle Nerve 30, 317-327. Aldridge, W. N., and Reiner, E. (1969). Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: One the formation of phosphorylated enzyme and the other analogous to inhibition by substrate. Biochem. J. 115, 147-162. Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates. North Holland, Amsterdam. Barak, D., Ordentlich, A., Kaplan, D., Barak, R., Mizrahi, D., Kronman, C., Segall, Y., Velan, B., and Shafferman, A. (2000). Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase. Biochemistry 39, 1156-1161. Baxter, G. D., and Barker, S. C. (1998). Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: Characterization and role in organophosphate resistance. Insect. Biochem. Mol. Biol. 28, 581-589. Behra, M., Cousin, X., Bertrand, C., Vonesch, J. L., B iellmann, D., Chatonnet, A., and Strahle, U. (2002). Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nat. Neurosci. 5, 111-118. Bourne, Y., Taylor, E, and Marchot, E (1995). Acetylcholinesterase inhibition by fasciculin: Crystal structure of the complex. Cell 83, 503-512. Bourne, Y., Kolb, H. C., Radi6, Z., Sharpless, K. B., Taylor, E, and Marchot, E (2004). Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation. Proc. Natl. Acad. Sci. USA 101, 1449-1454. Bticht, G., Haggstrom, B., Radi6, Z., Osterman, A., and Hjalmarsson, K. (1994). Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase. Biochim. Biophys. Acta 1209, 265-273. Changeux, J. E (1966). Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Mol. Pharmacol. 2, 369-392. Chothia, C., and Leuzinger, W. (1975). Acetylcholinesterase: The structure of crystals of a globular form from electric eel. J. Mol. Biol. 97, 55-60. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Doctor, B. E (1993). Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins. Protein Sci. 2, 366-382. Dale, H. H. (1914). The action of certain esters of choline and their relation to muscarine. J. Pharmacol. Exp. Ther. 6, 147-190. Dvir, H., Harel, M., Bon, S., Liu, W. Q., Vidal, M., Garbay, C., Sussman, J. L., Massoulie, J., and Silman, I. (2004). The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix. EMBO J. 23, 4394-4405. Fletcher, S. E, Geyer, B. C., Smith, A., Evron, T., Joshi, L., Soreq, H., and Mor, T. S. (2004). Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants. Plant. Mol. Biol. 55, 33-43.
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CHAPTER |
Cholinesterase Pharmacogenetics ROBERTA GOODALL North Bristol NHS Trust, Bristol, United Kingdom
Until recently, pharmacogenetic studies focused on BuChE rather than AChE because the latter, although considered more important physiologically, has not been regarded as a DME. AChE has also been assumed to be a much less polymorphic enzyme than BuChE, with any variants likely to have severe clinical consequences. However, recent research has shown that AChE may be more polymorphic than previously thought (Hasin et al., 2004), and it may also have pharmacogenetic relevance, particularly with respect to the anticholinesterase drugs used to treat Alzheimer's disease and dementia. The genetic variation that causes the differing pharmacological effects of cholinesterase drug targets may also have resonance in causing differences in how individuals respond to exposure to anticholinesterases, including organophosphate (OP) and carbamate (CM) pesticides. This chapter focuses on the pharmacogenetics of BuChE and the clinical aspects thereof but also considers the possible pharmacogenetic implications of AChE variants.
I. I N T R O D U C T I O N Pharmacogenetics is a field of medicine concerned with inherited variations in drug metabolizing enzymes (DMEs) that result in an altered ability to metabolize their target drugs. In a clinical setting this translates to the identification of the causes and prevention of adverse drug reactions (ADRs) responsible for millions of dollars in potentially avoidable medical costs worldwide. The term, originally proposed by Vogel in 1959, was brought into common usage by Werner Kalow in the early 1960s (Kalow, 1962a) with respect to the plasma form of cholinesterase, butyrylcholinesterase (BuChE; EC 3.1.1.8). BuChE is a DME responsible for hydrolysis of the muscle relaxant drugs suxamethonium and mivacurium as well as certain drugs of abuse, such as cocaine and heroin. Its physiological function is unknown. Deficiencies in the enzyme arise from genetic polymorphism as well as from secondary and physiological causes. Deficiency from any cause can lead to prolonged apnea and paralysis following administration of suxamethonium and mivacurium. A highly polymorphic enzyme, it provided an early model for pharmacogenetic studies, with biochemical phenotyping being used to identify individuals and families at risk of prolonged paralysis. Other such inherited deficiencies in DMEs were observed at approximately the same time, for example, with respect to N-acetyltransferase ("acetylator") status and the antituberculosis drug isoniazid (Evans, 1989), but Kalow's was the first applied use of the term that is now in common use in the biomedical community. The two cholinesterase enzymes, acetyl (ACHE) and butyryl (BuChE), although closely related, show differences both in their occurrence in the body (leading to their older vernacular names of erythrocyte, red cell, or true cholinesterase in the case of AChE and plasma or pseudocholinesterase for BuChE) and in their substrate specificity. AChE is more correctly called acetylcholine acetylhydrolase (EC 3.1.1.7), and BuChE is more correctly called acylcholine acylhydrolase (EC 3.1.18). AChE is present in most vertebrates in several molecular forms, whereas BuChE is present in only c0ae, the tetrameric "T" form (Massouli6, 2002). Toxicology of Organophosphate and Carbamate Compounds
II. B U T Y R Y L C H O L I N E S T E R A S E A. Clinical Aspects Clinical and laboratory interest in BuChE has a different emphasis in different countries. Its considered value in the clinical arena varies from its use as a marker of exposure to OP pesticides to its utility as a marker of liver "function" (with decreased activity indicating impaired hepatic synthesis) and its pharmacological actions and role as a DME. Although both AChE and BuChE are inhibited by anticholinesterase agents and by OPs, it appears that BuChE activity falls more rapidly following exposure to pesticides than does ACHE, and for this reason BuChE measurements can be used as a marker of occupational exposure (Barnes and Davies, 1951). A DME, BuChE is involved in the metabolism of drugs such as cocaine and heroin (Lockridge et al., 1980), several local anesthetics such as procaine (Lockridge, 1990), and in hydrolyzing the short-acting muscle relaxants suxamethonium (Bourne et al., 1952) and mivacurium (Cook et al., 1989). 187
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Clinically, the main area of concern is with a decrease in enzyme levels, usually manifested as a decrease in the enzyme activity measured in vitro. Such decreases in enzyme activity can arise from a variety of factors, either by causing a reduction in concentration of the enzyme or by directly affecting its action (usually by inhibition). There are occasions when an increase in activity or concentration is observed that although uncommon, will have an effect on the drug metabolizing properties of the enzyme. The following are some of the factors leading to a decrease in activity: Disease 9 Liver disease (Orpollo, 1978; Schmidt and Schmidt, 1993) 9 Renal failure (Phillips and Hunter, 1992) 9 Malnutrition (Umeki, 1993) 9 Certain malignant diseases (Kaniaris et al., 1979) Physiological changes 9 Age; newborns have low levels, which gradually rise to a peak at approximately 6 years of age. Levels then fall, with adult levels reached by puberty (Hutchinson and Widdowson, 1952). 9 Pregnancy; levels fall during the first trimester and the lowest levels occur around, and just after, parturition (Robertson, 1966; Blitt et al., 1977). Iatrogenic 9 Anticholinesterase drugs (e.g., neostigmine) 9 Oral contraceptive pill (Robertson, 1967) Toxic inhibition 9 Pesticides, particularly OPs and CMs 9 Dietary inhibitors, particularly solanaceous glycoalkaloids, found in members of the plant family Solanaceae, such as potatoes, tomatoes, and aubergine (eggplant) (Krasowski et al., 1997; McGehee et al., 2000) Genetic variants of the enzyme, the focus of this chapter Many of the causes of decreased activity have been well reviewed by Whittaker (1989) and Davis et al. (1997). B. B i o c h e m i c a l Genetics Evidence for the genetic variability of BuChE originally came in the 1950s from individuals experiencing unexpected effects following the use of the newly introduced muscle relaxant drug suxamethonium (succinylcholine or "scoline"). Suxamethonium, a dicholine ester of succinic acid, is a depolarizing muscle relaxant with an action at the motor end-plate similar to that of acetylcholine (Ziamis and Head, 1976). Its effect is to cause a short-lived cascarinic or paralyzing effect that allows the performance of certain procedures, such as intubation, required during general anesthesia and surgery. In normal circumstances, the drug's effects last only minutes due to its rapid hydrolysis by the action of BuChE. However,
soon after the drug's introduction into clinical practice, it became clear that in some patients the paralyzing effect lasted much longer, in some cases many hours. This meant that for the duration of the drug's effect, affected individuals were apneic (i.e., they were unable to breathe for themselves) and required artificial ventilation. This prolonged apnea was referred to as "scoline apnea," a term still used today. The consequences of such a reaction were that for some individuals, a minor procedure became a significant event, whereby the sufferer required artificial ventilation in an intensive care unit, and may even have been life threatening if no such facility was available. This phenomenon was found to be frequently associated with reduced BuChE activity (Evans et al., 1952), and investigations began into the nature of this relationship. In vitro tests of the serum of patients demonstrating scoline apnea showed a greatly reduced affinity for succinylcholine in comparison to serum from individuals with a normal response. In fact, in some of the patients tested, the affinity was so low that it failed to hydrolyze any of the injected succinylcholine in vivo (Kalow, 1962b). However, it became clear that not all cases of prolonged apnea caused by low enzyme levels could be attributed to decreased enzyme synthesis arising from liver disease or malnutrition because it occurred in patients in whom neither of these situations applied. The first observations of certain cases of BuChE deficiency being a familial condition seemed to indicate that it was inherited in a recessive manner (Lehman, 1956). However, Kalow (1956) reported studies on the serum of deficient individuals and their families that pointed to a codominant mode of inheritance and he applied the terms "usual" (U) and "atypical" (A) to the two forms of BuChE. By inhibiting the enzyme, in vitro, with known cholinesterase inhibitors, notably the local anesthetic dibucaine, before determining the activity Kalow demonstrated a trimodal distribution of residual activity. That of the usual form of the enzyme was inhibited by 80%, but that from affected individuals was inhibited by only 20%; a third group of individuals showed an intermediate degree of inhibition of approximately 60%. This was evidence that homozygotes for the atypical form had enzyme that was dibucaine resistant, whereas that of usual homozygotes was not. The intermediate level of inhibition represented heterozygotes for the condition. 1. INHIBITION STUDIES AND PHENOTYPES Kalow and Staron (1957) developed a method that could be used for the routine determination of BuChE type with the use of inhibition studies employing precise concentrations of dibucaine. With this first analytical tool, the practice of monitoring patients for genetic variations in DME arrived and the concept of pharmacogenetics was born. Testing of the close relatives of individuals known to have a deficient enzyme is an important aspect of pharmacogenetic studies because it is this knowledge that can prevent future ADRs in those family members. In fact, Lehman suggested such
CHAPTER 1 3 9Cholinesterase Pharmacogenetics testing and the provision of "warning letters" to be shown to anesthetists, in 1956. He also suggested that affected individuals may be at risk should their employment bring them into contact with anticholinesterases. This early history of BuChE investigation is of interest not only because of its seminal role in establishing pharmacogenetic study but also because the methods of inhibition studies to derive a dibucaine number (DN) are still in use today in the routine biochemical characterization of BuChE phenotype. Work using other inhibitors of the enzyme has shown that the situation is rather more complex than was originally discovered and that there are more than two variants of BuChE. For example, fluoride ion at particular concentrations also inhibits the action of usual BuChE and analysis in its presence allows the calculation of the fluoride number (FN). Some patients who had suffered a moderate degree of scoline apnea were found to have an enzyme that resisted inhibition by fluoride, with a lower FN than the normal enzyme, and so the fluoride-resistant variant was discovered. Determination of both DN and FN allowed further classification of an individual's BuChE type (Harris and Whittaker, 1961). 2. GENETIC VARIANTS
a. Qualitative and Quantitative---Decreased Activity Variants Immunological examination of the atypical enzyme showed that it is produced in normal amounts but is functionally i m p a i r e d - it is a qualitative variant (Eckerson et al., 1983). It appeared that the fluoride-resistant enzyme is also a qualitative variant (see Section II.C.4). Using the two types of inhibition numbers as an analytical tool, it was possible to identify people at risk of scoline apnea and search for possible carriers in their families. Such work revealed that there were other variants. In some of the families investigated for BuChE deficiency, anomalies were discovered in the pedigrees in that some women, defined as being homozygote for the atypical allele on the basis of their DNs, had children with a usual, as opposed to a UA, phenotype (Kalow and Staron, 1957; Harris et al., 1960). Parentage was not in dispute in either report, and so it was proposed that another variant must exist that had no cholinesterase activity and so exerted no effect on the DN - - a "silent" (S) variant. Further evidence for the existence of such a variant came from reports of apparently healthy individuals who had no measurable BuChE activity, homozygotes for the S variant (Hart and Mitchell, 1962; Liddell et al., 1962) who demonstrated severe sensitivity to scoline. Because the first descriptions of the S variants were of individuals with apparently no enzyme activity, S variants were thought to be quantitative variants. However, since those early days of investigation into BuChE, it has been demonstrated that the S variant phenotype is a heterogeneous one, with some individuals lacking functional enzyme altogether, whereas that produced by other people
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with this phenotype lacks the appropriate structure to hydrolyze the choline ester bond. This has been shown to be due to the fact that the silent phenotype is produced by more than one polymorphism (see Section II.C.2). Thus, some silent phenotypes are now known to be due to qualitative variants of the enzyme (Primo-Parmo et al., 1996). The use of the term silent in this context leads to a rather unfortunate terminological problem. The early work on discriminating and characterizing BuChE variants was done based on clinical, biochemical, and immunological studies and before the molecular nature of the gene was determined and accepted terminology for such molecular variation developed. The expression "silent variant" was coined to describe those enzyme variants that showed little or no enzyme activity; however, in molecular biology the term is more commonly used for a mutation or polymorphism that has no effect on either the gene product's function or its amino acid sequence. In the case of BuChE, the word silent refers to a biochemical phenotype, and such a variant is certainly not associated with a silent mutation in the gene. For example, when Whittaker et al. (1990) published their immunological studies on the heterogeneity of the silent variant they referred to "the silent gene," whereas now it is more likely to be referred to as a silent "variant" or "allele." It is important that the terms are appropriately used, and the temptation to refer to a mutation or polymorphism leading to a silent variant/phenotype as a silent mutation is avoided, especially when reporting results. Three more quantitative variants that cause a decrease in activity (i.e., functionally normal enzyme that is produced in decreased amounts) have also been described that show varying degrees of reduced activity. The first was described by Garry et al. (1976), who named it the James or J variant, and it is associated with a 66% reduction in enzyme activity. The Kalow or K variant, named in honor of Werner Kalow, is associated with an approximately 30% reduction in activity (Rubenstein et al., 1978). Of the named quantitative variants determined initially by phenotypic studies, the most recent addition is that described by Whittaker and Britten (1987). This was named the Hammersmith, or H variant because it was first identified in two cases presenting at the Hammersmith Hospital in London. The reduced activity in these variants from reduced concentration of the enzyme was thought to be due to changes in the production, stability, or half-life of the enzyme. The quantitative variants demonstrate usual inhibition numbers in vitro and can only be identified using a third inhibitor, usually the carbamate Ro 02-0683 that gives the RO number (RN), and then only when they are present in families segregating for the atypical variant. In such circumstances, they demonstrate unusual inhibition numbers due to the combination of effects of reduced activity and decreased inhibition. Heterozygotes with the usual enzyme cannot be distinguished from usual homozygotes (or from each other) by inhibition studies (Table 1); the lower activity is the only indication.
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TABLE 1. Characteristic Inhibitor Number Patterns Demonstrated by the Different Biochemical Phenotypes (Based on the Reference Ranges in Use in the Author's Laboratory) and the Expected Degree of Suxamethonium Sensitivity Inhibition Nos. (mean values) Phenotype
DN
FN
RN
Usual UA
81 62
62 51
97 72
Atypical UF AF FS, FF AK
17 74 48 63 81 53
26 50 31 39 61 45
16 96 62 93 97 60
AJ
40
40
48
UK, UJ, JKa
Suxamethonium sensitivity Rare Rare (except pregnancy) Severe Rare Moderate Moderate Variable Moderate (25% of cases) Moderate
aThese types cannot be unequivocally identified biochemically in a single sample and should be regarded as "usual" phenotypes. They are included here as separate types because they are often reported on the basis of low activity and so may have a slightly higher risk of suxamethonium sensitivity.
The difficulty in identification can be further exacerbated by other effects on enzyme concentration and by the lack of a universally accepted or standardized analytical methodology. Examples of the former include intercurrent illness and intraindividual variations due to age or pregnancy, as described in Section II.A. The problems arising from the latter occur mainly in a clinical setting, as opposed to a research or academic one. A variety of different substrates are used to determine enzyme activity and inhibitor numbers along with inhibitors other than dibucaine, fluoride, and Ro 02-0683. The effect of such variation has been demonstrated in a National External Quality Assessment Scheme (Proficiency Testing Scheme) in which the identification of certain phenotypes proves difficult when certain combinations of substrate and inhibitors are used (Goodall, 2004).
b. Increased Activity Variants Individuals have been described with enzymes that showed increased activity in vitro, with the increase apparently inherited. One of these was discovered as an electrophoretic variant containing an additional fifth, slow-moving band, to the four bands seen in normal sera (Harris et al., 1962, 1963). This variant was called C5 and demonstrated increased enzyme activity. It was believed for some time that the C5 enzyme was encoded by a second genetic locus (see Section II.C. 1), but Masson and associates (1990) clearly demonstrated that its production is not caused by a second BuChE gene. It now seems clear that C5 is created by the association of normal, tetrameric BuChE with another protein. Unlike other macro enzymes, such as creatine kinase and amylase,
in C5 BuChE the complexing protein has been shown not to be immunoglobulin (Akizuki et al., 2004) but its exact nature remains unclear. Also unlike other macroenzymes, which often have normal physiological function, the C5 variant does have anesthetic consequences, with C 5 + individuals demonstrating a shortened neuromuscular blockade following suxamethonium administration (Sugimori, 1986), indicating that larger doses are required to achieve the desired clinical effect. However, although it had been clear for some time that the pharmacogenetics of BuChE was not completely straightforward, the true complexity began to emerge when investigation began at the molecular (DNA) level and the structural bases of the variants were determined.
C. M o l e c u l a r Genetics 1. THE BUCHE GENE, B C H E The observation that some individuals have higher than normal enzyme activity led to the idea that BuChE was encoded by two separate loci (called E1 and E2), with E2 being responsible for the higher levels. However, using linkage and in situ hybridization studies, Arpargus et al. (1990) demonstrated that BuChE is encoded by a single gene locus only (BCHE), which was then localized on the long arm of chromosome 3 at 3q26 (Allerdice et al., 1991). The gene is minimally 73 kilobases long with four exons separated by three introns. The gene product is the BuChE subunit, four of which are needed to make the active enzyme. Exon 2 is large and codes for 83% of the subunit,
CHAPTER 1 3 9Cholinesterase Pharmacogenetics including the active triad covering the esteratic serine site at amino acid 198 (Lockridge et al., 1987). Examination of the gene, for mutations/polymorphisms causing the observed biochemical phenotypes, resulted in the isolation of those responsible for all of the most widely known variants. 2. POLYMORPHISMSAND MUTATIONS The first variants to be identified were identified by screening libraries of genomic DNA j for comparison with known biochemical phenotypes; for example, in the case of the A variant, by looking at those known to be homozygous for the U and A traits and comparing the DNA sequences. Whereas only one mutation has been found to cause the A (McGuire et al., 1989) and K phenotypes (Bartels et al., 1992a), two have been assigned to the F type (Nogueira et al., 1990) and many (in excess of 30) cause the S variants (Yen et al., 2003). Most of the variant alleles described so far are the result of single nucleotide polymorphisms (SNPs). Using the A variant as an example, the mutation giving rise to it is a change/at nucleotide (nt) 209, where an adenine residue is substituted by a guanine and the codon thus changes from GAT to GGT. This changes the aspartate at position 70 to glycine and is designated D70G. In the S variant genotypes, however, there are several different types of mutation/polymorphism:
9 Single base deletions producing premature stop codons, leading to truncated proteins 9 Single base insertions leading to reading frameshifts that change the amino acid sequence of the remaining part of the protein and may also lead to a premature stop codon 9 Combinations of a point change and an insertion, leading to a frameshift 3. MUTATION FREQUENCY The issue of the frequency of the various mutations/polymorphism is still not completely resolved, but as more molecular genetic studies have been performed and more polymorphisms discovered, some issues have become clear. The K variant polymorphism appears to be the most common overall, especially in European and North American populations (La Du et al., 1990). The allele frequency is approximately 0.13, making 1 person in 65 a homozygote for this variant and therefore approximately 1 in 4 a heterozygote or carrier. The allele frequency for the A variant has been calculated at 0.017 (McGuire et al., 1989), although it appears that an isolated A mutation is rare in the North American population and that there is strong linkage disequilibrium with the K variant (Bartels et al., 1992b). However, work in my laboratory indicates it is slightly less uncommon in the United Kingdom, and in other areas of the world it is linkage with the K variant that is rare. Ehrlich et al. (1994) showed that in the Ashkenazi Jewish populations in Israel, although the A variant was
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relatively common (approximately 5.8%), there were no occurrences of the K allele. A study of 1000 Iranians found that as many as 70-80% of the people studied carried at least one atypical allele (Vahdati-Mashhadian et al., 2004). This study used DN rather than molecular analysis to identify the variant, but this is appropriate in the case of the atypical variant because it does demonstrate a unique DN range. In contrast, some populations demonstrate a complete absence of certain variants. Acuna et al. (2003) described work that demonstrated that certain tribes of native Amerindians show a complete absence of the atypical variant allele, whereas others show a frequency attributable to the degree of European admixture that indicated that this allele was absent before the arrival of Europeans. The overall frequency of silent variant polymorphisms was estimated in Caucasian populations by Hodgkin et al. in 1965 to be 0.003 using nonmolecular methods; however, as with the atypical allele, the silent phenotype has been shown to be more common in some populations and ethnic groups than in others. The phenotype appears in approximately 1% of the Inuit people of western Alaska (Gutsche et al., 1967) and ~in 2% of people in certain areas of India (Rao and Gopalam, 1979). The frequency of the phenotype is somewhat higher in groups of patients selected because they have experienced scoline apnea. Yen et al. (2003) deternfined that in a group of 65 patients referred for genotyping following scoline apnea, 52 had primary hypocholinesterasemia, of which 8% had previously described silent variants and 12% had new or rare mutations. This is indicative of the probability that although the combined frequency of all the mutations leading to a silent phenotype may be higher than originally thought, the frequency of individual polymorphisms may be too low to accurately calculate. A search of the literature for details of the numerous silent variant polymorphisms reveals that many of them may be "private I' to single families. This may also be true in the case of the J phenotype because it appears that the mutation causing it has only been described in the original, extended family in which it was first identified. There are relatively high occurrences of multiple polymorphisms, with the possession of the common K variant certainly not precluding an individual from also carrying one or more of the other variant polymorphisms. Genuine silent variant mutations (i.e., those that do not affect the enzyme's amino acid sequence or function) have also been reported (PrimoParmo et al., 1996), indicating that care has to be exercised when extrapolating from genotype to phenotype. The high degree of heterogeneity means that the possibility of further mutations (and variants) is high, as demonstrated in the work of Yen et al. (2003) described previously. In addition to differences in the frequency of the variants in different populations, there are several reports of variants that appear to be restricted to certain ethnic groups and races. This has been most frequently reported in Japanese populations, particularly with respect to silent variants
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(Sudo et al., 1996; Maekawa et aL, 1997). Sudo et al. describe a fluoride-resistant variant that appears to be unique to the Japanese, whereas the polymorphisms producing this phenotype (F1 and F2) in North American and European populations are apparently absent. Therefore, there are definite ethnic and geographic differences in both gene frequency and allele linkage, a fact that has important consequences for those working in the clinical arena to identify the causes of scoline apnea in individual patients. 4. MOLECULAR BIOLOGY Not all DNA variations alter the function of a gene's protein product, and not all of those that do will exert the same effects. As with the types of polymorphisms found in BCHE, the effects of the variations are also somewhat heterogeneous. The likely effects of different polymorphisms can be extrapolated from knowledge of the structure of the gene product and from comparative studies with the corresponding genes of different species. The degree to which a particular codon or sequence is conserved across species and between similar proteins is a good indication of its importance and, therefore, of the likely severity of any variation. In the case of BuChE, the effects of the molecular changes on action can be explained for most of the variants, but for some they remain the subject of conjecture. a. Atypical (Dibucaine-Resistant) Variant The atypical variant shows reduced binding for the choline esters that bind to the anionic site of the enzyme. However, it has normal affinity for those neutral organic esters that interact primarily with the esteratic site. The aspartate at position 70, changed in the atypical variant, is located at the ligand binding site situated at the mouth of the active site gorge of the enzyme (Masson et al., 1997), indicating that the effect of the polymorphism is to change the binding, and therefore the access, of the substrate to the active site. This confirms the atypical variant as a qualitative, or low-affinity, variant. b. Fluoride-Resistant Variants That the fluoride-resistant variants exert an independent effect on the action of BuChE is demonstrated by the fact that individuals who are phenotypically heterozygous for the atypical and fluoride variants (AF) show a moderate degree of apnea, whereas UA heterozygotes do not (Viby-Mogensen and Hankel, 1981). By examining the pedigrees of known occurrences of the fluoride-resistant phenotype, Nogueira and associates (1990) identified two different SNPs (F1 and F2) leading to the variant in Caucasian populations but were unable to identify any linkage between them (i.e., they were mutually exclusive). The F1 SNP (T243M) affects the tripeptide recognition sequence for the glycosylation of the asparagine at position 241. Thus, the F1 polymorphism may lead to the loss of one of the nine carbohydrate chains present in the normal enzyme molecule, thereby reducing the molecular weight of the protein and altering its function.
The F2 SNP (G390V) leads to the loss of the glycine residue at position 390. Glycine 390 is important for substrate and ligand binding, and so the F2 variant is another lowaffinity (qualitative) variant, like the atypical but with less severe consequences. The Japanese fluoride-resistant variant, L330I, was demonstrated to have low activity when expressed in human fetal kidney cells and appears to be a low-affinity variant (Sudo et al., 1997). This is thought to be because leucine 330 is adjacent to the phenylalanine at position 329, close to the ligand binding site that is known to affect the enzyme's catalytic properties. c. Silent Variants As mentioned in Section II.C.2, there is a fair degree of heterogeneity in the polymorphisms producing the silent variant phenotype. The first to be described, and hence given the name S 1, arises from a frameshift at the glycine at position 117, where the codon GGT (gly) becomes GGAG (gly + 1) (Nogueira et al., 1990). This additional nucleotide shifts the reading frame in the gene, which leads to a premature stop codon 12 amino acids downstream. Consequently, only 22% of the normal mature protein is transcribed, and because the stop codon occurs upstream of the active site at serine 198, the gene product has no enzymatic activity. Other silent variants arise from straightforward single nucleotide polymorphisms; for example, a polymorphism that leads to no enzymatic activity is that for the $9 variant. An adenine-to-guanine change at nucleotide 198 (AGT ---> GGT) changes the active serine to a glycine (S198G) and therefore eliminates the enzyme's activity (Primo-Parmo et al., 1996). Unlike the S1 variant, however, $9 BuChE is produced in normal amounts, as judged by its immunoreactivity--it is simply inactive. d. Quantitative Variants Although some of the silent variants can be deemed quantitative, the term is usually reserved for the K, J, and H variants, the molecular bases of which were all published in the same year. As described in Section II.B.3.a, these variants all demonstrate reduced enzyme activity but cannot always be identified by normal biochemical phenotyping methods. Using pedigree analysis combined with DNA sequencing, Bartels et al. (1992b) determined that the K variant was caused by a SNP in exon 4 of the gene. A change at ntl615 (G--~ A) changes the alanine (GCA) at position 539 to a threonine (ACA) and the SNP is labeled A539T. The same group demonstrated that the enzyme produced had the same kinetic properties as the usual enzyme. It also exhibits the same heat stability as the usual enzyme, which argues against the theory that reduced molecular stability leads to the decrease in activity seen in this variant. Before the Bartels et al. study, that possession of this common polymorphism causes a reduction in activity (approximately 30%) had been well demonstrated (Whittaker and Brtitten, 1985) and shown to be due to the reduced number of circulating molecules (Rubenstein et al., 1978). How the A539T
CHAPTER 1 3 9Cholinesterase Pharmacogenetics change leads to this reduction remains unclear, although the alanine at 539 is well preserved across species. Given the high proportion of multiple polymorphisms in BCHE, and the degree of linkage, Bartels et al. proposed that the true cause of the decrease in activity may be another polymorphism/mutation elsewhere in the gene that is linked to A539T. However, such a mutation has not been reported. At the same time that they reported on the K variant, Bartels et al. (1992b) also described the SNP responsible for the J variant by investigating the pedigree of the extended family in whom Garry et al. (1976) had first reported the variant. They identified a SNP in exon 3, G497V, that segregated with the J phenotype, as determined by inhibition studies that included Ro 02-0683 inhibition, which is responsible for this variant. They also found that this SNP did not occur in isolation; it was always linked to the K variant in the same allele. Consequently, the J type is, in fact, JK. As with the K variant, Rubenstein et al. (1976) performed immunological studies on samples demonstrating the J variant phenotype and demonstrated a 66% reduction in circulating BuChE molecules (cf. the 33% reduction in K variant enzyme). It remains unknown whether the J variant produces the additional reduction, because it has never been found alone, or whether it acts in synergy with the K mutation. The third quantitative variant to be characterized was the H variant. This very rare variant is also only identifiable biochemically when present in a family segregating for the atypical variant. Jensen et al. (1992) demonstrated that the valine at position 142 is changed to a methionine (V142M). This amino acid is highly conserved in both cholinesterases in several species (Krejci et al., 1991), and its position in the folded protein indicates that it may be important in the folding and stability of the enzyme. There is overlap between some of the variants named silent and the H variant in that the 90% reduction in activity seen in the H variant is similar to the levels seen in some of the silent variants that do not result in the complete absence of function. This situation is indicative of the continuum of function and activity present in this enzyme due to the number and variety of genetic variants and the high number of multiple polymorphisms. When combined with the physiological changes that can occur, this can make interpretation of results in a clinical setting complex. 5. TERMINOLOGY Although DNA analysis has made it possible to identify specific BCHE polymorphisms, a routine clinical service for this is currently available in only a few centers, but it has identified difficulties in the previously accepted way of classifying patient samples by biochemical phenotypes. For example, it had been the practice to assign a phenotype on the basis of enzyme activity and inhibitor numbers with the assumption that a low activity and a usual pattern of inhibitor numbers indicated the presence of the silent variant and that the genotype was U/S (previously termed E lU E lS). The discovery of the K variant allele showed that not all lower activity samples would be due to the presence of
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silent variants. It is only possible to biochemically identify the probable presence of this allele in a family or pedigree study in which the atypical allele is also present. However, such was the confidence in biochemical phenotyping that extrapolation from such pedigrees was made to provide reference ranges for enzyme activity and to subdivide samples with usual inhibitor numbers into specific phenotypes, such as UU, US, UK, or UJ. There is still some difference of opinion on this practice; however, unless all possible secondary causes of reduced activity can be excluded, it appears to be a potentially dangerous practice and such samples should be classified as having only a usual or U phenotype (La D u e t al., 1991) At this point, a description of the terminology involved may be useful. The use of the word "phenotype" refers, in this instance, to a biochemical classification rather than a true clinical phenotype and is that which is determined by the pattern of inhibitor numbers as listed in Table 1. Therefore, any sample with a pattern that fits the ranges for a particular type by definition has that phenotype. To standardize the terminology, La Du et al. (1991) proposed a system of nomenclature that is in almost universal usage today. Phenotypes are written, in upper case, as a pair of letters representing the expected variants: U for usual, A for atypical, F for the fluoride-resistant types, and so on (e.g., UA and AF). The exception is the usual and atypical phenotypes, where both alleles cannot be predicted. Genotypes are represented in two ways. The shorthand version uses the same letters as phenotypes but separates the letters by a forward stroke to indicate the alleles on the two chromosomes (e.g., U/A). The formal terminology for genotypes gives the gene name, B C H E , followed by the DNA change responsible for any polymorphism using standard genetic notation. In the example, the U/A genotype becomes B C H E / B C H E * 7 O G , with the forward stroke separating the alleles and the asterisk indicating a change at position 70 to the amino acid glycine. Genotyping of individuals who have suffered prolonged paralysis following suxamethonium or mivacurium administration has shown that any biochemical phenotype can arise from several possible genotypes, and that although the majority of samples fall into the defined range for a particular phenotype, there is not always the expected correlation when the genotype is determined. Table 2 shows some of the reported genotypes associated with currently defined phenotypes. In general, sensitivity to short-acting muscle relaxants is dependent on enzyme activity. Anyone with a very low activity, from whatever cause, is likely to suffer a severely prolonged paralysis. However, there are some individuals with a reasonably well-preserved activity who experience a more moderate reaction, and these individuals may have functionally impaired enzyme due to the presence of one of the qualitative variants. A further group has no determinable abnormality, in either activity or phenotype, but suffers prolonged paralysis that appears to be BuChE dependent. It is in these groups that genotyping can be
19 4
S ECTI O N I I I 9Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t TABLE 2.
Heterogeneity of Biochemical Phenotypes Determined by Molecular Genotyping a
r
Phenotype Usual Atypical UA UF AK AF AJ
Genotypes U/U, U/S, U/K, K/K, K/S, K/Sc, U/S, U/Sc, U/JK, K/JK A/AK, AK/AK, A/S, AK/KS U/A, U/AK, K/AK, A/K U/F2, K/F2 A/K, K/AK, U/A A/F1, A/F2, AK/F2 A/K, AK/F 1, AJJK
aData compiled mainlyfrom the author's own laboratorybut also from the literature. particularly useful. In some such individuals, it has been shown that there is at least one variant that is capable of hydrolyzing the substrates used in the standard methods for laboratory determination of activity but cannot hydrolyze suxamethonium in vivo. This variant, called the scoline or Sc variant, can be identified by DNA analysis and demonstrates reduced activity when succinylcholine is used as the substrate in a biochemical assay (Greenberg et al., 1995). As more genetic analysis is performed, it is likely that either phenotyping will be replaced by activity measurements plus genotyping or the acquired data on phenotype:genotype correlations will allow more accurate reference ranges for phenotyping.
III. PHARMACOGENETICS OF ACETYLCHOLINESTERASE Because of its physiological importance, it has long been assumed that effective mutations in the AChE gene would have severely deleterious consequences. The AChE gene (ACHE) has been mapped to the long arm of chromosome 7 (Zelinski et al., 1991) and localized to 7q22 (Getman et al., 1992), and the sequence has been determined. The gene is composed of six exons and the repertoire of isoforms is the result of alternate splicing producing three distinct polypeptides (Li et al., 1991). Since then, some polymorphisms have been found, but until recently, none appeared to affect the enzyme's normal action. For example, Bartels et al. (1993) reported that a mutation in the coding region of the gene was responsible for the human YT blood group. The YT blood group is defined, as are all blood groups, by antigens on red blood cells that are recognized by specific antibodies. Immunological evidence had implicated the erythrocyte AChE as the location for the two YT group antigens, YT1 and YT2 (Spring et al., 1992). By sequencing all the AChE exons and the intron/exon junctions in individuals showing all three YT genotypes (homozygous YT1 and YT2 plus heterozygotes), Bartels et al. identified three point
mutations. Two of these did not alter the amino acid sequence of the mature ACHE. One was a genuine silent mutation that did not lead to an amino acid change and the second changed the proline at position 561 to arginine, but this residue is not part of the mature, active protein in the AChE anchored to erythrocyte membranes. The third mutation resulted in a histidine (His)-to-asparagine (Asn) change at position 322, a residue located on the surface of the molecule and segregated with the different blood types, with YT1 having His322 and YT2 having Asn322. The presence of this polymorphism, although having possible consequences in blood transfusion, was shown to have no effect on the catalytic properties of the enzyme (Masson et al., 1994). Therefore, although it was apparent that the YT polymorphism conferred no genetic disadvantage or disease state, its discovery did raise the question that other genetic variants may exist that may be disease associated or, as in the case of BuChE, have pharmacogenetic consequences. The use of anticholinesterases as therapeutic agents in the treatment of myasthenia gravis and Alzheimer's disease has shown variation in individual responses to these agents, including some individuals who suffered severe hypersensitivity. Such observations have prompted a closer look at possible pharmacogenetic variability in ACHE. It was known that certain anticholinesterases promote overproduction of one of the splice variants, the readthrough AChE-R, with the resultant increase in enzyme activity, and thus scavenging ability, conferring a short-term protection to exposure to such chemicals (Kaufer et al., 1998, 1999). Conversely, it was possible that an impaired ability for such induction could result in a hypersensitivity to anticholinesterases. Shapira et al. (2000) were able to identify two polymorphisms in the distal promoter region of the gene. One of these, a 4-base pair deletion, was discovered in a woman who had displayed acute hypersensitivity to pyridostigmine. The deletion was shown to abolish a binding site for hepatocyte nuclear factor-3, which resulted in a constitutive increase in basal AChE expression, which in turn impaired the antiAChE-induced overexpression thus causing the sensitivity to the drug. This deletion was also found in
CHAPTER 1 3 9Cholinesterase Pharmacogenetics
other individuals who had also suffered intoxication following a subacute dose of pyridostigmine. In none of the cases studied was any abnormality of BuChE seen. The second mutation disrupts a probable glucocorticoid response element, but the consequences of this mutation are yet to be elucidated; however, there is surely some kind of effect. There had been no reports of any significant degree of polymorphism in the A C H E coding sequence until Hasin et al. (2004) reported on highly detailed studies in a cohort of 96 unrelated individuals. They examined four different ethnic populations and identified 13 different SNPs, of which 10 were new and 5 were likely to produce amino acid substitutions. Their contention was that the previously held belief that AChE was not a polymorphic enzyme was due to a combination of the technical difficulties encountered in amplifying the gene using the polymerase chain reaction and the limitation of previous studies to small numbers of single ethnic groups. The study identified 15 different haplotypes and 5 apparently ethnospecific polymorphisms. Although none of the SNPs identified appear likely to alter the catalytic action of ACHE, 3 of them are within the mature protein, mapping to the external surface, and so may have antigenic effects. This work seems to support the theory that polymorphisms affecting the enzyme's function may be too deleterious for survival. Conversely, it also demonstrates that AChE is polymorphic and that further work is needed to search for variations that may account for some of the adverse reactions seen in response to the therapeutic anticholinesterase drugs.
195
atypical BuChE or with the AChE promoter deletion, was a possible contributing factor or cause of Gulf War syndrome 9and indicates that individuals with variant cholinesterases would not benefit from pretreatment with anticholinesterases (Soreq and Seidman, 2001). Conversely, "artificial" mutations have also been shown in experimental animals to apparently confer resistance to the toxic effects of certain OPs (Wang et aL, 2004). It has been demonstrated that treatment with wild-type (usual) human BuChE with fetal bovine serum AChE can confer protection against OP agents such as soman in primates (Doctor et al., 1993). Because anticholinesterase prophylaxis appears to provide protection against certain agents (e.g., soman) but to increase toxicity to others such as satin (Abu-Qare and Abou-Donia, 2002), the use of purified enzyme in such instances appears to have far less risk, although the requirements are currently too large for practical application. There are no reports of how such a prophylaxis is likely to work in the presence of variant BuChE, and this would appear to be a useful area of study, given the fact that atypical and other variant enzymes occur in relatively high frequencies in certain populations. The cholinesterases have long been known to be important physiologically and pharmacologically. However, recent expansion in the use of anticholinesterases and cholinesterase inhibitors, both therapeutically and as weapons, means that the effect of genetic variation may be more relevant than previously thought.
References IV. C O N C L U S I O N S The genetic variability demonstrated by BuChE leads to a wide spectrum of in vivo activity with respect to its main pharmacological substrates, which can have severe clinical consequences. The same variability also produces differences in the degree of inhibition by a range of different substances, which allows biochemical characterization (phenotyping) to be performed. Recently, it has also been shown that genetic variation in AChE occurs, which may account for differences in responses to the therapeutic use of anticholinesterases. It is probable that this genetic variation will have an effect on individuals' responses to OP exposure. Case histories of severe consequences when individuals with variant BuChE have been exposed to OPs have been reported (LoewensteinLichtenstein et al., 1995). There is one case in the literature of a family of farmers exposed to OP pesticides who demonstrated a de novo amplification of B C H E (Prody et al., 1989), indicating that the consequences of OP poisoning may not be confined to exposed individuals but may have long-term genetic consequences. Because of the risk of chemical attack, anticholinesterases were used prophylactically during the Gulf War. It has been hypothesized that this, combined in some individuals with
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9Esterases, Receptors,
Mechanisms, & Tolerance Development
Whittaker, M. (1989). Cholinesterase. Monographs in Human Genetics, Vol. 2. Karger, Basel. Whittaker, M., and Britten, J. J. (1985). Plasma cholinesterase variants: Family studies of the E1k gene. Hum. Hered. 35, 364-368. Whittaker, M., and Britten, J. J. (1987). E1k, a new allele at cholinesterase locus 1. Hum. Hered. 37, 54-58. Whittaker, M., Jones, J., and Brave, J. (1990). Heterogeneity of the silent gene for plasma cholinesterase. Immunological studies. Hum. Hered. 40, 153-158.
Yen, T., Nightingale, B. N., Burns, J. C., Sullivan, D. R., and Stewart, P. M. (2003). Butyrylcholinesterase (BChE) genotyping for post-succinylcholine apnea in an Australian population. Clin. Chem. 49, 1297-1308. Zelinski, T., Getman, D., Coghlan, G., and Phillips, S. (1991). Assignment of the YT blood group locus to chromosome 7q. Genomics 11, 165-167. Ziamis, E. J., and Head, S. (1976). Depolarizing neuromuscular blocking drugs. In Neuromuscular Junction (E. Ziamis, Ed.), pp. 1-18. Springer-Verlag, Berlin.
CHAPTER
/ 4
Methods for Measuring Cholinesterase Activities in H u m a n B l o o d ELSA REINER AND VERA SIMEON-RUDOLF Institute for Medical Research and Occupational Health, Zagreb, Croatia
I. I N T R O D U C T I O N
hydrolysis. When the substrate is acetylcholine or acetylthiocholine, EA is the acetylated enzyme E-O-C(O)CH3, P1 is choline or thiocholine, and P2 is the acetic acid. The constants kl, k-l, k2, and k3 are rate constants of the individual reaction steps. Acylation (Eq. 1) and deacylation (Eq. 2) take place on the hydroxyl group of serine, which is the key residue of the catalytic triad in the catalytic site of the enzyme. Cholinesterases also have a peripheral, allosteric site that can reversibly bind substrates and other ligands. This site has no catalytic activity. However, binding of a ligand to the site can cause either inhibition or activation of the enzyme. OPs and CMs are acylating inhibitors (ABs) of AChE and BuChE. Cholinesterases react with AB compounds in the same way as they react with substrates; that is, they acylate the hydroxyl group of serine in the catalytic site. However, there is a significant quantitative difference between substrates and AB compounds in the rates of the individual reaction steps. In the reaction with substrates, acylation and deacylation of the serine is very fast, whereas AB compounds quickly acylate the enzyme but very slowly deacylate from the enzyme, particularly when AB is an OP. The enzyme therefore stays acylated by AB compounds for a long time and cannot hydrolyze substrates during that time. Consequently, OP and CM compounds are inhibitors of cholinesterases. Acylation by AB compounds is termed progressive inhibition or irreversible inhibition (Eq. 3), and its time course follows (Eq. 4)"
Organophosphates (OPs) and carbamates (CMs) are toxic compounds primarily due to inhibition of the acetylcholinesterase (ACHE; EC 3.1.1.7) in the neural and neuromuscular synapses. Butyrylcholinesterase (BuChE; EC 3.1.1.8) and other serine esterases, such as carboxylesterase, trypsin, or chymotrypsin, are also inhibited by these compounds. Absorption of OPs and CMs can be detected by measuring the activity of cholinesterases in human blood, which contains both AChE and BuChE. Absorption of cholinesterase inhibitors can also be detected by determining the parent compound and/or its metabolite(s) in blood or urine. This approach is not generally applied, and no routine methods are available for these analyses. This chapter describes methods for determining AChE and BuChE activities and summarizes distribution profiles and interindividual variations of cholinesterase activities in human blood. The kinetics of interaction of cholinesterases with substrates, OPs, and CMs is briefly outlined.
H. I N T E R A C T I O N O F C H O L I N E S T E R A S E S
WITH SUBSTRATES, ORGANOPHOSPHATES, AND C A R B A M A T E S The hydrolysis of cholinesterase substrates proceeds in three steps: E+S
~
kl
k2
ES
~
EA+P1
E+AB
(1)
k3
EAB
~
In (eo / et) = ka" ab. t ,~ E + P2
(2)
(3)
(4)
where EAB is a Michaelis-type complex between AB and the enzyme; EA is the acylated enzyme - - that is, the phosphylated enzyme when AB is an OP compound and the carbamylated enzyme when AB is a carbamate; P1 is
where E and S are the enzyme and substrate, ES is the enzyme-substrate Michaelis complex, EA is the acylated enzyme, and P1 and P2 are the products of substrate Toxicology of Organophosphate and Carbamate Compounds
EA+P1
ka
k_l EA + H20
~
199
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
200
SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
the leaving group of AB; ka is the overall second-order rate constant of inhibition; eo and et are the enzyme concentrations at time zero and time t of inhibition, respectively; and ab is the concentration of AB. Deacylation of the inhibited enzyme (i.e., dephosphylation or decarbamylation) is termed spontaneous reactivation (Eq. 5), and its time course follows (Eq. 6):
EA+H20
~
E+P2
In (eao / eat) = kr" t
(5) (6)
where P2 is a phosphoric or carbamic acid, E is the reactivated enzyme, kr is the first-order rate constant of spontaneous reactivation, and eao and eat are the concentrations of the acylated enzyme at time zero and time t, respectively. Compounds with an oxime group in the molecule reactivate phosphylated cholinesterases much faster than water due to the powerful nucleophilicity of the oxime group. In the reactivation process, reactivated enzyme and phosphylated oximes are formed (Eq. 7), and the time course of reactivation follows (Eq. 8), where k' r is the second-order rate constant of oxime reactivation:
ktr EA + oxime
E + phosphylated oxime
In (eao / eat) = k'r" [oxime]- t
(7)
(8)
Due to their efficacy as reactivators, oximes are used as antidotes in the therapy of OP poisoning. Many publications extensively cover the structure and catalytic properties of cholinesterases (Giacobini, 2000; Inestrosa and Campos, 2004; Silman et al., 2004), the kinetics of interaction with ligands (Reiner and Radi6, 2000), and the role of oximes in OP poisoning (Eyer, 2003).
III. G E N E R A L O U T L I N E O F CHOLINESTERASE ACTIVITY
MEASUREMENTS IN HUMAN BLOOD AChE activity in human blood reflects the activity of synaptic ACHE, which is the target enzyme of anticholinesterases. However, many OPs or CMs inhibit plasma BuChE faster than erythrocyte ACHE, and the inhibition of plasma BuChE is for those compounds a more sensitive indicator of absorption. Consequently, more information about the exposure of an individual to OPs or CMs can be obtained if both AChE and BuChE are measured. Human blood contains BuChE in the plasma and AChE bound to the erythrocyte membrane. In order to determine the activity of each enzyme, erythrocytes and plasma either have to be separated or one has to apply selective substrates and/or inhibitors to assay only one enzyme in the presence of the other. There are no known substrates or inhibitors
that are fully selective for either AChE or BuChE. On the other hand, it is difficult to achieve full separation of erythrocytes from plasma without loss of some of the activity. Therefore, each approach to measuring AChE and BuChE activities in whole blood has its advantages and disadvantages, and any assay in whole blood is subject to some intrinsic error. BuChE activities are measured not only to evaluate inhibition by OPs or CMs but also to diagnose a wide range of physiological or pathological conditions reflected in BuChE activities. These measurements are done in serum or plasma and not in whole blood. Therefore, more data on BuChE than AChE activities are available, and more methods have been developed for the analysis in plasma or serum than in whole blood.
A. Collection and Storage of Blood Samples Blood samples are collected from finger pricks or earlobe pricks into capillaries and from venous blood into test tubes. Capillaries and test tubes should be heparinized (to prevent blood clotting) and dried (to prevent uncontrolled sample dilution). To prevent contamination of the samples by OPs and CMs during collection, the skin must be cleaned before sampling. Individuals exposed to anticholinesterases may have some unreacted OP or CM in the blood, and those treated with antidotes may also have some oximes in the blood. If cholinesterase activities cannot be measured immediately after sampling, some precautions should be undertaken to minimize changes in enzyme activities during storage of blood samples. Dilution of the sample will slow down further cholinesterase inhibition and further oxime reactivation because both reactions depend on the concentrations of the inhibitor and oxime (Eqs. 4 and 8). However, spontaneous reactivation will continue irrespective of dilution (Eq. 6). Because all reactions are temperature dependent, samples can be stored at a low temperature but not below zero if one wants to have nonhemolyzed erythrocytes. All three reactions are also pH dependent. Oximes reactivate when they are deprotonated, and lowering the pH therefore decreases rates of oxime reactivation. The pH profiles of inhibition and spontaneous reactivation depend on the structure of the inhibitor, but for the majority of compounds these rates decrease with decreasing pH. Blood samples can therefore be diluted with a buffer of pH between 5 and 6. If cholinesterase activities are measured in haemolysed blood, dilution with water is suggested, and these samples may be stored below zero.
B. Expression of Activities Activities of AChE and BuChE are expressed in micromoles hydrolyzed substrate per minute per volume of whole blood or plasma. AChE activities can also be expressed per
CHAPTER 1 4 9Methods for Measuring Cholinesterase Activities volume of packed erythrocytes, per hemoglobin content, or per iron content. Due to the wide interindividual variations of AChE and BuChE activities in human blood (see Section V), a single cholinesterase assay does not give information about the absorption of a cholinesterase inhibitor. To calculate the degree of cholinesterase inhibition due to absorption of an inhibitor, one has to know the AChE and BuChE activities before the exposure of an individual to OPs or CMs (preexposure activities).
IV. M E T H O D S
Enzyme activities are measured by determining either the decrease in substrate concentration or the increase in product concentration (i.e., the acid or the alcohol). These reactions are usually followed continuously over a time interval, which makes it ~possible to verify whether the increase or decrease in concentrations is linear over the time of assay. However, some methods are restricted to two measurements (end point methods) m one at the beginning and the other at the end of a specified time interval in which case the linearity of the reactions cannot be verified. To ensure a linear increase in product formation, the initial substrate concentrations should remain almost constant during the assay. On the other hand, this is not favorable if one measures the decrease of unhydrolysed substrate because the calculated activities are based on differences between two almost equal concentrations. Many methods have been developed for measuring cholinesterase activities, and readers are referred to several
TABLE 1.
comprehensive reviews that also cover some methods no longer in general use (Augustinsson, 1971; Evans, 1986; Hoffmann et al., 1999; Silver, 1974; Simeon, 1967; Whittaker, 1986; Wilson, 2001). Choline and thiocholine esters are good substrates of AChE and BuChE, and a selected list of compounds used for activity measurements is given in Table 1. Acetylcholine is the physiological substrate of ACHE, and BzCh is the suggested substrate for BuChE phenotyping. When activities are measured in whole blood that contains both AChE and BuChE, the following selective inhibitors are most often applied to differentiate between the two cholinesterases: ethopropazine, quinidine, and isoOMPA inhibit BuChE, and Huperzine A and BW2845c51 inhibit ACHE. iso-OMPA is an organophosphate, and the other compounds are reversible cholinesterase inhibitors. A. M e t h o d s B a s e d o n t h e D e c r e a s e in S u b s t r a t e Concentration
For the assay of ACh hydrolysis in whole blood, Winteringham and Disney (1964) introduced a radiometric method with [14C]ACh as substrate. This is an end point method intended to be used as a field method. Several modifications were later developed with [14C]ACh or [3H]ACh as substrates (Wilson, 2001), but no radiometric method was widely used. Disposal of radioactive waste material has been noted to be a problem. Another end point method for ACh concentrations is the spectrophotometric method of Hestrin (1949). ACh reacts with hydroxylamine to form acethydroxamic acid, which forms a colored complex with ferricions.
Structures, Names, and Abbreviations of Selected AChE and BuChE Substrates
Substrate
H3C-C(O)-OCH2CH2 N+ (CH3)3 X
Name and abbreviation -a
H3C-C(O)-SCH2CH2 N+ (CH3)3 XHsC2-C(O)-SCH2CH2N+ (CH3)3 XH7C3-C-C(O)'SCH2CH2N+ (CH3)3 XH5C6-C(O)-OCH2CH2 N+ (CH3)3 XCH2-C(O)-OCH 2 CH2N+ (CH3)3 I CH2-C(O)-OCH 2 CH2N+(CH3) 3 2XCH2-C(O)-SCH2 CH2N+ (CH3) 3 i CH2-C(O)-SCH2 CH2N+(CH3)3 2Xax- is an anion.
201
Acetylcholine ACh Acetylthiocholine ATCh Propionylthiocholine PTCh Butyrylthiocholine BTCh Benzoylcholine BzCh Succinyldicholine SuxCh Succinyldithiocholine SuxTCh
202
SECTION III. E s t e r a s e s ,
Receptors,
Kalow et al. (1956) introduced a spectrophotometric method for determining BuChE activities in plasma or serum by continuously measuring the decrease in BzCh concentration in the UV region. BzCh is a substrate of BuChE and is not hydrolyzed by ACHE. This substrate is used for phenotyping BuChE.
B. Methods Based on the Increase in Acid Concentration These methods are suitable for both AChE and BuChE and are applicable to any substrate because hydrolysis of an ester always results in acid formation. The increase in acid concentration can be measured potentiometrically by continuous titration with sodium or potassium hydroxide at a constant pH (pH-stat method) (Jensen-Holm et al., 1959), or one can measure the decrease in pH after a selected time of assay (ApH). The ApH method is an end point method introduced by Michel (1949), and it is used for routine or field measurements, pH can be measured colorimetrically with a pH indicator, which is a simple protocol.
C. Methods Based on the Increase in Choline Concentration Choline released during the hydrolysis of choline esters is oxidized with choline oxidase, and the formed hydrogen peroxide is reacted with peroxidase-phenol-4aminoantipyrine to give a chromofore measured at 500 nm (Abernethy et al., 1984, 1986, 1988; Faye and Evans, 1986). The method was developed for succinyldicholine and benzoylcholine as substrates and applied to identify patients sensitive to succinyldicholine, a neuromuscular relaxant used during anesthesia that is very slowly hydrolyzed by patients with the atypical BuChE variant. This method is not widely used, and sensitive patients are identified by phenotyping BuChE by other methods using BzCh (see Section IV.A) and, recently, succinyldithiocholine (a thio analogue of succinyldicholine) as substrates (Mosca et al., 2003).
D. Methods Based on the Increase in Thiocholine Concentration In 1961, a spectrophotometric method for the detection of low thiocholine concentrations was published by Ellman et al. The method is based on the reaction of thiocholine with the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). Due to its high sensitivity, reproducibility, and simplicity, this method became the most widely applied, and thiocholine esters the most widely used substrates, for measuring cholinesterase activities. Thiocholine released during hydrolysis reacts with the chromogenic DTNB to form the yellow dianion 5-thio-
Mechanisms,
& Tolerance
Development
2-nitrobenzoic acid (TNB). In the procedure of Ellman et al. (1961), the assay medium is 0.1 M phosphate buffer (pH 8.0) containing 0.33 mM DTNB. The enzyme activity (i.e., formation of TNB) is continuously measured at 412 nm for up to several minutes. The molar absorption coefficient (eM) of TNB reported by Ellman (1959) is 13,600M -1 cm -1 derived in 0.01 M phosphate buffer (pH 8.0) at unspecified temperature. This method has been applied to a variety of cholinesterase preparations, in different media at pH values other than 8, and over a wide temperature range. The activities were usually measured at 412 or 405 nm, and the eM stated previously was generally used for the calculation of cholinesterase activities. Since the method was published, slightly different eM values have been reported at 412 nm (Eyer et al., 2003). Furthermore, it has been suggested that in samples containing hemoglobin, activities should be measured at 436 nm (Worek et al., 1999). The absorbance of hemoglobin is high at 412 nm, which interferes with the assay, particularly when cholinesterase activities are low. Finally, it was shown that TNB reveals thermochromic properties" By increasing the temperature, the absorbance spectra of TNB are shifted toward longer wavelengths, whereas the absorbance maxima decrease (Worek et al., 1999). In reassessment studies, eM values were evaluated for temperatures between 10 and 50 ~ and wavelengths between 405 and 470 nm. Recommended eM values (M -1 cm -1) at 412 nm are 14,150 at 25 ~ and 13,800 at 37 ~ at 436 nm, the eM are 11,000 at 25 ~ and 11,280 at 37 ~ (0.1 M phosphate buffer, pH 7.4) (Eyer et al., 2003). Reassessment of eM values is required whenever experimental conditions are altered. DTNB reacts not only with thiocholine but also with other thiol compounds, thus producing TNB. When cholinesterase samples contain other thiol compounds, as in hemolyzed erythrocytes, DTNB should be added to the enzyme sample before the substrate. When the reaction of DTNB with the other thiols is completed, the substrate is added. Thiocholine esters react with oxime groups (oxymolysis), whereby thiocholine is produced (Primo~i~ et al., 2004; Skrinjarid-Spoljar et al., 1992). When samples contain oximes, the contribution of oximolysis should be measured in the absence of the enzyme and subtracted from the total thiocholine increase measured in the enzyme sample. However, when blood samples are taken from patients under oxime therapy, the samples may contain an unknown oxime concentration, and one has to be aware that due to oximolysis, the measured increase in thiocholine may lead to a false conclusion concerning enzyme activity.
E. Protocols Based on the Ellman Method Activities of BuChE are measured in plasma or serum at substrate concentrations in the millimolar range.
CHAPTER 14 9Methods for Measuring Cholinesterase Activities Thiocholine esters listed in Table 1 are good substrates of human BuChE, with activities decreasing in the order: PTCh > BTCh > ATCh. Activities in plasma (obtained from heparinized blood) are 4 or 5% lower than in serum (measured with ATCh and PTCh) (Reiner et al., 1996). ATCh is the preferred substrate of human ACHE. The hydrolysis of PTCh is slower, and that of BTCh is extremely slow. When AChE activities are measured in erythrocytes separated from plasma by centrifugation, they are measured either in unwashed erythrocytes or in erythrocytes washed with buffer or saline in order to remove residual plasma and residual inhibitors or oximes that may be present in the blood. Both separation from plasma and washing may cause some loss in activity. The pS curve for the hydrolysis of ATCh is bell-shaped with an optimum activity at approximately 1 mM ATCh, which is usually the suggested concentration for the assay. Several protocols have been published for measuring both AChE and BuChE in whole blood with no separation of the erythrocytes from plasma. These protocols require either more than one substrate or one substrate and a selective inhibitor (Reiner et al., 2000). Ethopropazine [(10-2-diethylaminopropyl) phenothiazine hydrochloride] is a reversible and almost selective inhibitor of BuChE (Meuling et al., 1992). The suggested concentration for the assay is 20 ~M ethopropazine, which inhibits 98% of the usual (UU) BuChE phenotype. However, the atypical (AA) phenotype and heterozygotes of the A variant are less inhibited (74-87%) (Simeon-Rudolf et al., 2001). Furthermore, at 20 txM ethopropazine, the AChE is also inhibited, although only 5-8% (Reiner et al., 2000; SimeonRudolf et al., 2001; Worek et al., 1999). Worek et al. (1999) published an improved method for AChE activities in whole blood with ATCh as substrate (0.45 mM) in the presence of 20 ~M ethopropazine, in 0.1 M phosphate buffer (pH 7.4). The activity is measured in hemolyzed whole blood at 436 nm and expressed per hemoglobin contents. The protocol proved very reliable even at very low AChE activities. For BuChE activities, the authors suggest a separate assay in plasma with BTCh as substrate (1.0 mM). Based on the previous protocol, Reiner et al. (2004) evaluated both AChE and BuChE activities in whole blood. The activities are measured in nonhemolyzed blood with ATCh as substrate (1.0 mM) in the absence (VT) and in the presence (VE) of 20 txM ethopropazine. The VE corresponds to the activity of ACHE, and the difference, VT-VE, corresponds to the activity of BuChE. The authors suggest that VE be corrected for the inhibition of AChE by ethopropazine. The authors consider this protocol suitable for screening both AChE and BuChE activities in whole blood (hemolyzed or nonhemolyzed) for routine and field tests. In the protocols of Wicki (1994) and Portmann and Hofmann (1998), activities are measured with two substrates
203
but no inhibitor. The activity of both enzymes in whole blood is first measured with ATCh (1.0 mM), and then BTCh (5.0 mM) is added to the same cuvette and the measurement continued. The calculation of AChE and BuChE activities is based on the assumption that BTCh is hydrolyzed only by BuChE, and that the addition of BTCh stops the hydrolysis of ATCh. In the protocol of Feaster et al. (2004), three substrates are used: ATCh, BTCh, and PTCh. The cholinesterase activities in whole blood are measured separately with each substrate, and the activities of AChE and BuChE are calculated by using the so-called sensitivity coefficients. These are determined prior to the assay by applying selective AChE and BuChE inhibitors to reference samples in order to evaluate the contribution of each enzyme to the hydrolysis of each of the three substrates.
F. Kits Based on the E l l m a n M e t h o d Several test kits have been produced for routine measurements of AChE and BuChE activities. Test kits comprise preweight reagents and instructions for dissolving the reagents before the assay. Test kits can be used either in automatic analyzers or in standard manual spectrophotometers or equipment modified with well microplate readers (Doctor et al., 1987). Several studies have reported on the validation of test kits (Wilson et al., 1995) using automatic and manual measurements. Lassiter et al. (2003) found good agreement between AChE activities measured with a Boehringer-Mannheim test kit on an automatic analyzer and a standard spectrophotometer with a 96-well microplate reader. Simeon (1989) studied the stability of dissolved reagents from two commercial test kits (Boehringer-Mannheim and Pliva) and reagents prepared in the laboratory. The three tested sets of reagents were equally stable at 4 ~ over several weeks. The demand for routine measurements of cholinesterase activities under field conditions resulted in the development of various field kits. The Test-mate field kit, which is currently widely used, is based on the Ellman method. It is a self-contained portable device comprising a set of reagents and instructions for use. The Test-mate kit measures AChE and BuChE activities and the hemoglobin content in a drop of blood; the activities are automatically normalized to 25 ~ (Wilson, 2001). Oliveira et al. (2002) tested three Test-mate kit models (on fetal bovine serum ACHE) and compared the results with those measured on a standard spectrophotometer with a 96-well microplate reader. The results from the three models showed a discrepancy, and there was also no good agreement between the results of the three models and those obtained from the standard photometric measurement. The authors strongly recommend the use of cholinesterase standards when activities are measured with field kits.
204
SECTION III. E s t e r a s e s ,
Receptors,
G. Cholinesterase Standards and Quality Control Measurements Several standards are available for intralaboratory and interlaboratory quality control studies, which also include day-to-day control of the precision and accuracy of withinrun and between-run imprecision of activity measurements. Commercially available standards also provide information on the activity of the standards for a given substrate at specified experimental conditions. Precinorm U and Precinorm S (Boehringer-Mannheim) are BuChE standards from lyophilized sera with activities within and below the normal range. Simeon (1989) compared the two standards with a pool of frozen native human sera and with a lyophilized US-NBS serum sample and concluded that a pool of native human sera, divided into aliquots and kept frozen, is a reliable and stable BuChE standard for quality control studies. Arrieta et al. (2003) produced a preparation from bovine red blood cell ghosts as a standard for AChE assays. The activity of this standard was stable at - 7 0 ~ for up to 3 years. Due to the widespread need for cholinesterase activity measurements, and for field kits, several attempts have been made to standardize and validate methods and protocols. Considerable effort toward this goal has been expended by the World Health Organization, the U.S. Environmental Protection Agency, and the Chemical and Biological Medical Treatment Symposia held regularly in Spiez, Switzerland, since 1994. So far, no general agreement has been obtained.
V. DISTRIBUTION PROFILES AND GENETIC VARIANTS OF HUMAN BLOOD CHOLINESTERASES Cholinesterase activities have a bell-shaped distribution profile, and the profile appears symmetrical for both AChE and BuChE. However, one statistical analysis of BuChE activities in plasma and serum showed that the distribution is slightly skewed toward high activities (Reiner et al., 1996). More data are available for BuChE activities in plasma or serum than for AChE activities in whole blood or packed erythrocytes. A selected list of data obtained with several commonly used substrates is given in Table 2. BuChE activities were usually measured in samples in which the BuChE was not phenotyped, but the activities presented in Table 2 most likely reflect the usual phenotype, which is most widely distributed. It follows from Table 2 that the range of activities is broad, and this indicates broad interindividual variations for both AChE and BuChE. The reported coefficients of variation (CV) are higher for BuChE than for AChE
Mechanisms,
& Tolerance
Development
activities, confirming larger interindividual variation for the BuChE. The intraindividual variations of AChE and BuChE activities (CV = 6-9%) are significantly smaller than the interindividual variations and are similar to variations reported for the day-to-day imprecision of measurements (Abernethy et al., 1988; Brock, 1990; Flegar-Me~tri6 et al., 1999). BuChE activities reflect, more than AChE activities, inherited genetic variants and some physiological conditions or some diseases. BuChE activities in serum are decreased in the last trimester of pregnancy. Impaired liver function and some other diseases decrease BuChE activities, whereas obesity and coronary arterial disease increase BuChE activities (Lockridge and Masson, 2000; Whittaker, 1986; Alc~ntara et al., 2002, 2003). BuChE activities also depend on age and sex. Children and adolescents (8-19 years) have significantly higher BuChE activities than adults. BuChE activities of women younger than 50 years are significantly lower than those of adult men; women older than 50 years have similar activity as men (Whittaker; 1986; Flegar-Me~tri6 et al., 1999). The BuChE is determined by a single B C H E gene that encodes the sequence of the enzyme. In addition to the wild-type allele producing the usual enzyme (UU), there are approximately 40 identified genetic variants resulting from mutations in coding regions of the B C H E gene. In populations screened throughout the world, atypical (A), fluoride-resistant (F), J, Kalow (K), H, and approximately 30 different silent variants were identified (G~itke et al., 2001; Liu et al., 2002; Lockridge and Masson, 2000; Primo-Parmo et al., 1996; Whittaker, 1986; Yen et al., 2003). The variants are characterized by different primary structures resulting either in different catalytic properties of the enzyme or in lower protein expression or partial or complete lack of catalytic capacity. All variants have lower activities than the usual enzyme. The BuChE variants give rise to 15 phenotypes that can be identified by standard biochemical methods coupled with family studies. The phenotypes can by identified by measuring activities with different substrates and by inhibition of the enzyme with dibucaine, sodium fluoride, dimethylcarbamate Ro 02-0683, and other inhibitors. The frequencies of the homozygous usual genotype (UU), atypical (AA), and silent (SS) are 1 person out of 1.3, 2500, and 110,000 individuals, respectively (Lockridge and Masson, 2000; Whittaker, 1986). Approximately 10% of Caucasians have the C5 + isozyme. This variant has an approximately 25% higher activity than the usual BuChE. It has been reported that farmers with the C5 + isozyme are more resistant to intoxication by anticholinesterase pesticides because BuChE is a scavenger of OPs and CMs (Akizuki et al., 2004; Fontoura-da-Silva and ChautardFreire-Maia, 1996; Lockridge and Masson, 2000). The biochemistry of BuChE variants and their clinical and toxicological significance are extensively described in
C H A P T E R 14
TABLE 2.
N 257 (M)
Substrate (mM) Temperature (~C) 1.0/ATCh (22-25)
151 (F) Eythrocytes d
205
AChE and B u C h E Activities in Male (M) and Female (F) Individuals Measured with Acetylthiocholine (ATCh), Butyrylthiocholine (BTCh), Propionylthiocholine (PTCh), and Benzoyicholine (BzCh) as Substrates a
Enzyme source Whole blood c
9M e t h o d s for M e a s u r i n g Cholinesterase Activities
30 (M)
0.50/ATCh
30 (F)
Activity range or mean 4_-_SD (Ixmol m i n - 1 m l - 1)
CV ( % )b
Reference
2.10-8.05
19
Wilhelm and Bradamante (1980)
2.45-6.65
19
8.71 + 1.39
16
8.54 + 1.24
15
Jones et al. (1991)
Whole blood e
894
5.4/ATCh
14.7 _+ 1.2
18
Wilson et al. (1998)
Serum BuChE-UU
295 (M + F)
5.0/ATCh (25)
0.82-4.93
23
Simeon-Rudolf et al. (1987)
0.050/BzCh (25)
0.33-1.54
23
Simeon-Rudolf et al. (1987)
Serum BuChE-UU
295 (M + F)
Simeon-Rudolf and Evans
4.0/PTCh (25)
Serum 506 (M + F)
3.89-8.13
18
BuChE-AA
83 (M + F)
0.52-2.50
33
BuChE-AMAS
341 (M + F)
0.30-2.10
38
BuChE-UU
Surina et al. (2004)
7.0/BTCh (30)
Serum
(2001)
BuChE-UU
359 (M + F)
3.99-16.9
21
BuChE-AS/AA
3 (M + F)
1.02-2.87
m
1.30-3.70
28
1.22-3.20
23
0.35-2.60
23
0.35-2.45
25
1.80-4.40
18
1.60-3.50
17
0.82-5.23
23
Simeon-Rudolf et al. (1987)
0.89-3.62
23
Simeon-Rudolf and Reiner (1991)
0.89-3.24
26
Serum
226 (M)
0.256/ATCh (25)
198 (F) Plasma
252 (M)
Serum
36 (M)
1.0/ATCh (22-25)
148 (F) 5.0/ATCh (25)
17 (F) Serum
180 (M)
5.0/ATCh (25)
154 (F) Plasma
134 (M)
von Prellwitz et al. (1976) Wilhelm and Bradamante (1980) Huizenga et al. (1985)
1.29-4.14 1.0/ATCh (25)
103 (F) Serum
89 (M + F)
1.0/ATCh (25)
0.92-3.24
23
Reiner et al. (1996)
Serum
226 (M)
6.0/BTCh (25)
2.27-7.40
28
von Prellwitz et al. (1976)
2.05-6.70
28
3.10-7.50
21
198 (F) Serum
221 (M)
20/PTCh (25)
173 (F)
von Prellwitz et al. (1976)
2.90-6.90
21
1.0/PTCh (25)
1.63-6.27
24
1.30-5.46
25
88 (M + F)
1.0/PTCh (25)
1.67-5.74
24
Reiner et al. (1996)
180 (M)
0.050/BzCh (25)
0.33-1.54
22
Simeon-Rudolf et al. 1987
0.35-1.1
23
Plasma
134 (M)
Serum
Serum
103 (F)
154 (F)
Simeon-Rudolf and Reiner (1991)
aData are from selected references. bCV is the coefficient of variation, i.e., the relative standard deviation of mean activities. CActivities measured in whole blood with 1.0 mM ATCh reflect primarily ACHE. CtMean + SD activity expressed as I~molmin-1 ml-1 packed erythrocytes. eMean _+ SD activity expressed as txmol min-1 mg-1 Hb. Activities measured in the presence of 0.2 mM quinidine, a selective BuChE inhibitor.
206
SECTION I I I . Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
several reviews (Evans, 1986; Lockridge, 1990; Lockridge and Masson, 2000; Pantuck, 1993; Whittaker, 1986). There are only a few reports on AChE polymorphism resulting from the mutations on the A C H E gene. It seems that these mutations have no impact on the catalytic properties of the enzyme but may affect protein stability and/or antigenicity (Bartels et al., 1993; Erlich et al., 1994; Hasin et al., 2004; Lockridge and Masson, 2000). However, their possibly different response to OPs and CMs has not been investigated.
VI. C O N C L U S I O N S AND F U T U R E DIRECTIONS OPs and CMs are used as pesticides, and some OPs are also potential warfare nerve agents. Pesticides are used worldwide, and warfare agents may also be used extensively. To detect absorption of anticholinesterases, a standardized protocol for measuring AChE and BuChE activities in human blood is needed that is simple in technical terms but reliable and well reproducible. This would enable a better comparison of data from different laboratories and an exchange of samples for analysis and confirmation of results. It seems that a protocol based on the Ellman method would meet the outlined requirements, but further studies including interlaboratory quality control are required, particularly when field methods are developed.
References Abernethy, M. H., George, E M., and Melton, V. E. (1984). A new succinylcholine-based assay of plasma cholinesterase. Clin. Chem. 30, 192-195. Abernethy, M. H., George, P. M., Herron, J. L., and Evans, R. T. (1986). Plasma cholinesterase phenotyping with use of visibleregion spectrophotometry. Clin. Chem. 32, 194-197. Abernethy, M. H., Fitzgerald, H. P., and Ahem, K. M. (1988). An enzymatic method for erythrocyte acetylcholinesterase. Clin. Chem. 34, 1055-1057. Akizuki, S., Ohnishi, A., Kotani, K., and Sudo, K. (2004). Genetic and immunological analysis of patients with increased serum butyrylcholinesterase activity and its C5 variant form. Clin. Chem. Lab. Med. 42, 991-996. Alc~ntara, V. M., Chautard-Freire-Maia, E. A., Scartezini, M., Cerci, M. S. J., Braun-Prado, K., and Picheth, G. (2002). Butyrylcholinesterase activity and risk factors for coronary artery disease. Scand. J. Clin. Lab. Invest. 62, 399-404. Alc~.ntara, V. M., Oliveira, L. C., Rea, R. R., Suplicy, H. L., and Chautard-Freire-Maia, E. A. (2003). Butyrylcholinesterase and obesity in individuals with the CHE2 C5 + and CHE2 C5- phenotypes. Int. J. Obesity 27, 1557-1564. Arrieta, D., Ramirez, A., DePeters, E., Bosworth, D., and Wilson, B. W. (2003). Bovine red blood cell ghost cholinesterase as a monitoring standard. Bull. Environ. Contam. Toxicol. 71, 447-452.
Augustinsson, K. B. (1971). Determination of activity of cholinesterases. Methods Biochem. Anal. 19, 217-273. Bartels, C. E, Zelinski, T., and Lockridge, O. (1993). Mutation at codon 322 in the human acetylcholinesterase (ACHE) gene accounts for YT blood group polymorphysm. Am. J. Hum. Genet. 52, 928-936. Brock, A. (1990). Immunoreactive plasma cholinesterase (EC 3.1.1.8) substance concentration compared with cholinesterase activity concentration and albumin: Inter- and intraindividual variation in a healthy population group. J. Clin. Chem. Biochem. 28, 851-856. Doctor, B. E, Toker, L., Roth, E., and Silman, I. (1987). Microtiter assay for acetylcholinesterase. Anal. Biochem. 166, 399-403. Ehrlich, G., Ginzberg, D., Loewenstein, Y., Glick, D., Kerem, B., Ben-Aft, S., Zakut, H., and Soreq, H. (1994). Population diversity and distinct haplotype frequencies associated with ACHE and BCHE genes of Israeli Jews from trans-Caucasian Georgia and from Europe. Genomics 22, 288-295. Ellman, G. L. (1959). Tissue sulfydryl groups. Arch. Biochem. Biophys. 82, 70-77. Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Evans, R. T. (1986). Cholinesterase phenotyping: Clinical aspects and laboratory applications. CRC Crit. Rev. Clin. Lab. Sci. 23, 35-64. Eyer, E (2003). The role of oximes in the management of organophosphorus pesticide poisoning. Toxicol. Rev. 22,
166-190. Eyer, P., Worek, E, Kiderlen, D., Sinko, G., Stuglin, A., SimeonRudolf, V., and Reiner, E. (2003). Molar absorption coefficients for the reduced Ellman reagent: Reassessment. Anal. Biochem. 312, 224-227. Faye, S., and Evans, R. T. (1986). Is succinyldicholine the substrate of choice for the measurement of cholinesterase activity? CRC Crit. Rev. Clin. Lab. Scie. 32, 1477-1480. Feaster, S. R., Gordon, R. K., and Doctor, B. E (2004). Assay for detecting, measuring and monitoring the activities and concentrations of proteins and methods for use thereof, U.S. patent, June 8, Patent No. 6,746,850 B2. Flegar-Me~tri6, Z., Surina, B., and Siftar, Z. (1999). Biological variation of human butyrylcholinesterase activity in a population from Zagreb, Croatia. Chem.-Biol. Interact. 119-120, 193-199. Fontoura-da-Silva, S. E., and Chautard-Freire-Maia, E. A. (1996). Butyrylcholinesterase variants (BCHE and CHE2 loci) associated with erythrocyte acetylcholinesterase inhibition in farmers exposed to pesticides. Hum. Hered. 46, 142-147. G/atke, M. R., Ostregaard, D., Bundgaard, J. R., Varin, E, and Viby-Mogensen, J. (2001). Response to mivacurium in patient compound heterozygous for a novel and a known silent mutation in the butyrylvholinesterase g e n e m Genotyping by sequencing. Anesthesiology 95, 600-606. Giacobini, E. (Ed.) (2000). Cholinesterases and Cholinesterase Inhibitors. Dunitz, London. Hasin, Y., Avidan, N., Bercovich, D., Korczyn, A., Silman, I., Beckmann, J. S., and Sussman, J. L. (2004). A paradigm for single nucleotide polymorphism analysis: The case of acetylcholinesterase. Hum. Mutat. 24, 408-416.
CHAPTER 1 4 9Methods for Measuring Cholinesterase Activities
Hestrin, S. (1949). The reaction of acetylcholin and carboxylic acid derivatives with hydroxylamine and its analytical application. J. Biol. Chem. 180, 249-261. Hoffmann, W. E., Solter, P. E, and Wilson, B. W. (1999). Clinical enzymology cholinesterases. In The Clinical Chemistry of Laboratory Animals (W. E Loeb and E W. Quimby, Eds.), 2nd ed., pp. 430-454. Taylor & Francis, Philadelphia. Huizenga, J. R., van der Belt, K., and Gips, C. H. (1985). The effect of storage at different temperatures on cholinesterase activity in human serum. J. Clin. Chem. Clin. Biochem. 23, 283-285. Inestrosa, N. C., and Campos, E. O. (Eds.) (2004). Cholinesterases in the Second Millenium. Biomolecular and Pathological Aspects. Universidad Cat61ica de Chile FONDAP Biomedicina, Santiago Chile. Jensen-Holm, J., Lausen, H. H., Milthers, K., and MOiler, K. O. (1959). Determination of the cholinesterase activity in blood and organs by automatic titration, with some observations on the method and remarks on the photometric determination. Acta Pharmacol. Toxicol. 15, 384-394. Jones, J. W., Whittaker, M., and Braven, J. (1991). Immunological assay of erythrocyte acetylcholinesterase. Clin. Chim. Acta 200, 175-182. Kalow, W., Genest, K., and Staron, N. (1956). Kinetic studies on the hydrolysis of benzoylcholine by human serum cholinesterase. Can. J. Biochem. Physiol. 34, 637-653. Lassiter, T. L., Marshall, R. S., Jackson, L. C., Hunter, D. L., Vu, J. T., and Padilla, S. (2003). Automated measurement of acetylcholinesterase activity in rat peripheral tissues. Toxicology 186, 241-253. Liu, W., Cheng, J., Iwasaki, A., Imanishi, H., and Hada, T. (2002). Novel mutation and multiple mutations found in the human butyrylcholinesterase gene. Clin. Chim. Acta 326, 193-199. Lockridge, O. (1990). Genetic variants of human serum cholinesterase influence metabolism of the muscle relaxant succinylcholine. Pharmacol. Ther. 47, 35-60. Lockridge, O., and Masson, P. (2000). Pesticides and susceptibile populations: People with butyrylcholinesterase genetic variants may be at risk. NeuroToxicology 21, 113-126. Meuling, W. J. A., Jongen, M. J. M., and van Hemmen, J. J. (1992). An automated method for the determination of acetyl and pseudo cholinesterase in hemolyzed whole blood. Am. J. Ind. Med. 22, 231-241. Michel, O. H. (1949). An electrometric method for the determination of red blood cell and plasma cholinesterase activity. J. Lab. Clin. Med. 34, 1564-1568. Mosca, A., Bonora, R., Ceriotti, R., Franzini, C., Lando, G., Patross, M.C., Zaninotto, M., and Panteghini, M. (2003). Assay using succinyldithiocholine as substrate: The method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose succinyldicholine sensitivity. Clin. Chem. Lab. Med. 41, 317-322. Oliveira, G. H., Henderson, J. D., and Wilson, B. W. (2002). Cholinesterase measurements with an automated kit. Am. J. Ind. Med. 2(Suppl.), 49-53. Pantuck, E. J., (1993). Plasma cholinesterase: Gene and variations. Anesth. Analg. 77, 380-386. Portmann, R., and Hofmann, W. (1998). Instructions for the test kit for the interlaboratory comparison test on measurements of
207
cholinesterase activity. Internal document of NC Laboratory, Spiez, Switzerland. Primo-Parmo, S. L., Bartels, C. E, Wiersema, A. E L., Innis, J. W., and La Du, B. N. (1996). Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene. Am. J. Hum. Genet. 58, 52-64. Primo,S, I., Od~ak, R., Tomi6, S., Simeon-Rudolf, V., and Reiner, E. (2004). Pyridinium, imidazolium and quinuclidinium oximes: Synthesis, interaction with native and phosphylated cholinesterases, and antidotes against organophosphates. J. Med. Chem. Defense, online at jmedchemdef.org. Reiner, E., and Radi6, Z. (2000). Mechanism of action of cholinesterase inhibitors. In Cholinesterases and Cholinesterase Inhibitors (E. Giacobini, Ed.), pp. 103-119. Dunitz, London. Reiner, E., Simeon-Rudolf, V., and Buntic, A. (1996). Cholinesterase activities in human serum/plasma and their distribution profiles. Period. Biol.v98, 119-120. Reiner, E., Skrinjari6-Spoljar, M., Sinko, G., and Simeon-Rudolf, V. (2000). Comparison of protocols for measuring of human blood cholinesterases by the Ellman method. Arh. Hig. Rada. Toksikol. 51, 13-18. Reiner, E., Bosak, A., and Simeon-Rudolf, V. (2004). Activity of cholinesterases in whole blood measured with acetylthiocholine as substrate and ethopropazine as selective inhibitor of plasma buryrylcholinesterase. Arh. Hig. Rada. Toksikol. 55, 1-4. Silman, I., Soreq, H., Anglister, L., Michaelson, D., and Fisher, A. (Eds.) (2004). Cholinergic Mechanisms. Function and Dysfunction. Taylor & Francis, London. Silver, A. (1974). The Biology of Cholinesterases. Frontiers of Biology, Vol. 36. North-Holland, Amsterdam. Simeon, V. (1967). Methods for cholinesterase activity measurements [in Croatian]. Arh. Hig. Rada Toksikol. 18, 29-39. Simeon, V. (1989). Measurement of the serum cholinesterase activity: Comparison of commercial and laboratory test reagents, enzyme standards and statistical processing of the results [in Croatian]. Arh. Hig. Rada. Toksikol. 40, 183-189. Simeon-Rudolf, V., and Evans, R. T. (2001). Interlaboratory study into the proficiency of attribution of human serum butyrylcholinesterase phenotypes: Reference values of activities and inhibitor numbers. Acta Pharm. 51, 289-296. Simeon-Rudolf, V., and Reiner, E. (1991). Phenotypes of human serum esterases reacting with organophosphates, carbamates and other esters. In Ecogentics. Genetic Predisposition to the Toxic Effects of Chemicals (P. Grandjean, Ed,). Chapman & Hall, London. Simeon-Rudolf, V., Bunti6, A., Surina, B., and Flegar-Me~tri6, Z. (1987). Cholinesterase phenotyping and distribution of activity of sera of 346 individuals. Acta Pharm. Jugosl. 37, 107-114. Simeon-Rudolf, V., Sinko, G., Stuglin, A., and Reiner, E. (2001). Inhibition of human blood acetylcholinesterase and butyrylcholinesterase by ethopropazine. Croat. Chem. Acta 74, 173-182. Skrinjari6-Spoljar, M., Franciskovic, L., Radi6, Z., Simeon, V., and Reiner, E. (1992). Reaction of imidazolium and pyridinium oximes with the cholinesterase substrate acetylthiocholine. Acta Pharm. 42, 77-83. Surina, B., Nosso, D., Siftar, Z., Flegar-Me~tri6, Z., and SimeonRudolf, V. (2004). Cholinesterase unit establishment and issuing of warning cards for carriers of suxamethonium
208
SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
sensitive serum butyrylcholinesterase variants. Coll. Antropol. 28(Suppl. 2), 297-304. von Prellwitz, W., Kapp, S., and Mtiller, D. (1976). Vergleich von Methoden zur Aktivit~it der Serumcholinesterasen (acylcholin-acethyhydrolase E.C.3.1.1.8) und deren diagnostische Wertigkeit. J. Clin. Chem. Clin. Biochem. 14, 93-97. Whittaker, M. (1986). Cholinesterase. In Monographs in Human Genetics (L. Beckman, Ed.), Vol. 11, Karger, Basel. Wicki, A. (1994). Monitoring of acetylcholinesterase and butyrylcholinesterase in human whole blood. In Proceedings of the Chemical and Biological Medical Treatment Symposium CBMTS, Spiez, Switzerland, pp. 1.18-1.24. Applied Science and Analysis, Portland (ME), USA. Wilhelm, K., and Bradamante, V. (1980). Blood cholinesterase activity in workers exposed to anticholinesterases, a ten year follow-up. Arh. Hig. Rada Toksikol. 31, 109-124. Wilson, B. W. (2001). Cholinesterases. In Handbook of Pesticide Toxicology (R. I. Krieger, Ed.), Vol. 2, pp. 967-985. Academic Press, San Diego. Wilson, B. W., Padilla, S., Sanborn, J. R., Henderson, J. D., and Billitti, J. E. (1995). Clinical blood cholinesterase
measurements for monitoring pesticide exposures. In Enzymes of the Cholinesterase Family (D. M. Quinn, A. B. Balasubramanian, B. E Doctor, and E Taylor, Eds.), pp. 329-386. Plenum, New York. Wilson, B. W., McCurdy, S. A., Henderson, J. D., McCarthy, S. A., and Billitti, J. E. (1998). Cholinesterase and agriculture. Humans, laboratory animals and wildlife. In Structure and Function of Cholinesterases and Related Proteins (B. E Doctor, E Taylor, D. M. Quinn, R. L. Rotundo, and M. K. Gentry, Eds.), pp. 539-546. Plenum, New York. Winteringham, E E W., and Disney, R. W. (1964). A radiometric method for estimating blood cholinesterase in the field. Bull. Worm Health Organization 30, 119-125. Worek, F., Mast, U., Kiderlen, D., Diepold, Ch., and Eyer, E (1999). Improved determination of acetylcholinesterase activity in human whole blood. Clin. Chim. Acta 288, 73-90. Yen, T., Nightingale, B. N., Bums, J. C., Sullivan, D. R., and Stewart, E M. (2003). Butyrylcholinesterase (BCHE) genotyping for post-succinylcholine apnea in an Australian population. Clin. Chem. 49, 1297-1308.
CHAPTER
15 Interactions of Organophosphorus and Carbamate Compounds with C h o l i n e s t e r a s e s
LESTERG. SULTATOS New Jersey Medical School, Newark, New Jersey
I. I N T R O D U C T I O N Cholinesterases are enzymes that are serine hydrolases that preferentially hydrolyze choline esters. Vertebrates are known to have two cholinesterases, referred to as acetylcholinesterase (ACHE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). These two cholinesterases have been differentiated historically on the basis of their substrate selectivity. AChE hydrolyzes acetylcholine (ACh) faster than butyrylcholine (BuCh) or propionylcholine, whereas BuChE metabolizes BuCh and propionylcholine faster than ACh. This difference in substrate selectivity is thought to arise from differences in acyl pocket structure, which in the case of BuChE allows access of larger substrates to the active site (Harel et al., 1992; Cygler et al., 1993; Kovarik et al., 2003). AChE is known to play a critical role in the termination of the action of ACh (Fig. 1) at synapses and neuromuscular junctions, whereas the exact function(s) of BuChE remains unclear. However, numerous potential roles for BuChE have been suggested, ranging from metabolism of lipoproteins (Kutty and Payne, 1994) to cell adhesion (Tsigelny et al., 2000), and the etiology of certain neurodegenerative diseases (Darvesh et al., 2003). Many organophosphorus (OP) and carbamate (CM) compounds exert their acute toxicity through inhibition of ACHE. These compounds include all of the OP and most of the CM pesticides, as well as certain chemical warfare agents. Additionally, a limited number of OP and CM drugs exert their therapeutic effects through inhibition of AChE (Taylor, 2001). Inhibition of this critical enzyme leads to an accumulation of ACh at synapses and neuroeffector junctions (Fig. 1), which in turn leads to a range of symptoms known as cholinergic crisis. Although inhibition of BuChE by these compounds has not been shown to result in a specific adverse event, BuChE is considered to represent an important nonenzymatic pathway for detoxification of
Toxicology of Organophosphate and Carbamate Compounds
@ ACh ACh
~
ACh 9 A + Ch ~ ~
ACh
ACh ACh
ACh
ACh
ACh -I~ A + Ch
,
FIG. 1. Role of AChE at cholinergic nerve endings. AChE catalytic subunits are represented by the shaded circles. Acetylcholine (ACh) stored in presynaptic terminals is released in response to depolarization of the nerve and diffuses across the synapse or junction to bind to and activate postjunctional cholinergic receptors. The postjunctional action of ACh is terminated primarily through its hydrolysis to acetate and choline by AChE. Inhibition of AChE by OP or CM compounds results in an accumulation of ACh within synapses and neuroeffectorjunctions, leading to cholinergic crisis. anticholinesterase compounds and is often used as a surrogate measure for inhibition of AChE elsewhere in the body.
II. I N H I B I T I O N OF C H O L I N E S T E R A S E S BY OPs A N D CMs The cholinesterases (ChEs) are serine hydrolases that catalyze the breakdown of ACh through an acyl-transfer, where water is the acceptor molecule to which the substrate acyl 209
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2 10
SECTION III 9Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
moiety is transferred (Walsh, 1979). A serine oxygen of the active site gorge in ChEs carries out a nucleophilic attack on the electrophilic carbon of the carbonyl group of ACh, resulting in an acetylated enzyme intermediate and the release of choline (Fig. 2) (Wilson et aL, 1950; Froede and Wilson, 1984; Quinn, 1987). Deacylation occurs when an attacking water molecule (hydroxyl ion) acts as a more effective nucleophile, thereby releasing acetate (Fig. 2) (Walsh, 1979). The inhibition of AChE and BuChE by OPs and CMs can be viewed in many ways as a reaction analogous to that of the hydrolysis of acetylcholine by these same enzymes (Aldridge and Reiner, 1972) (Fig. 2). The molecular interactions between certain OPs and AChE have been investigated utilizing a variety of techniques, and much is known regarding how OPs bind to and phosphylate ACHE. Ordentlich et al. (1996) reported that the acyl pocket of the active gorge in AChE (Phe295 and Phe297) (all numbers in this chapter refer to amino acid positions in human AChE or BuChE) participates in the positioning of an OP molecule for the in-line attack by the catalytic
serine (Ser203), with Phe297 more important for branched alkoxy substituents, which are larger in volume (Fig. 3). This positioning can be interpreted as analogous to the formation of a Michaelis complex. Ordentlich et al. concluded that the ability to form these Michaelis complexes is extremely important in determining the reactivity of OPs toward AChE (and presumably BuChE as well). In addition to the acyl pocket, the peripheral anionic site as well as other subsites play important roles in the stereoselectivity toward enantiomers of the chemical warfare agent VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (Ordentlich et al., 2004) (Fig. 3). Replacement of Asp74 of the peripheral anionic site with asparagines (D74N) nearly eliminated the stereoselectivity of AChE toward the p s - v x enantiomer (Ordentlich et al., 2004). The charged leaving group of p s - v x interacts with the acidic Asp74 to promote formation of the Michaelis complex, thereby accounting for, at least in part, the greater reactivity of the p s - v x enantiomer compared to pR-vx (Ordentlich et al., 2004).
OH S~r I
lb
[la
,RI?
0II /CH3 CH3"- (~'-O--CH2--CH2- N - - C H 3 t ~'CH3
t I
O
,,CH 3
CH 3- (~ + NO - CH 2- CH 2- N ~, CH 3 I
O
CH3
sgr I
2c
,R,O CH 3- N - C + H O - R 2 I
O R2-O ,,~ R1-O " I + HO-R 3
?
Ser !
I
J
3c
3b
R,,O
oii
CH 3 - N - C~...OH OH-
Set
o s~r
-
I
OH
R 1- O
o
,R10 CH 3 - N - C I
OH
s~r I
OH-
4c
4b
o
ii CH 3 - C
;~o
R=-O
Sgr I
i
4a
I,
J
S~r
3a
OH
I
J,~
J
s~r
g;
OS~r
2a
I
oii 1R2-O-O~ - O -R3 R
CH 3- N - C - O - R 2
I
CH3- CI O
[,c
Organophosphate
Carbamate
Acetylcholine
OH
O R2-O ,,J' R 1-O "P
OH
OH
S~r I
FIG. 2. The interaction of ACh, CM, and OP compounds with the active site serine of AChE and BuChE. Reaction 1 represents the formation of a stable Michaelis complex and the beginning of the nucleophilic attack of the serine. Reaction 2 represents the acylation of the active site serine, coupled with the release of the first reaction product or leaving group. Reaction 3 begins the nucleophilic attack of a hydroxyl ion, which leads to the regeneration of active enzyme (reaction 4).
CHAPTER 1 5 9Interactions of OP and CM Compounds with Cholinesterases Peripheral Anionic I Binding Site
S CH 3-O ii
c. -o
~p37~_. 0 , Oxyanion Hole Acyl Binding Site
RiO
/
-0""
...
I
Ser203
O CH2CO2C2H5
Malathion
IC5o= 2.9 mM
OR 2
~
HN
/
0
N. . . . ~ " .o
His447 Catalytic Triad
Glu334
FIG. 3. Schematic drawing of the active site gorge of ACHE, with the entry of an OP molecule. R] and R2 on the OP are usually identical alkyl chains, whereas R 3 is the leaving group. The catalytic triad consists of Ser203, His447, and Glu334. The acyl binding site is likely important in positioning the inhibitor for the nucleophilic attack from Ser203 (Ordentlich et al., 1996), whereas the oxyanion hole may polarize the P=O bond, thereby facilitating the nucleophilic attack (Ordenflich et al., 1998). Binding of ligand to the peripheral anionic site can lead to inhibition or activation (Masson et al., 2004). Additionally, the peripheral anionic site plays an important role in the stereoselectivity of AChE toward methylphosphonates (Ordentlich et aL, 2004).
Ordentlich et al. (1998) suggested that the oxyanion hole subsite (Glyl21, Gly122, and Ala204) may polarize the P--O bond (or the C = O bond in the case of carbamates) during formation of the Michaelis complex and therefore activate the phosphorus, thereby promoting a nucleophilic attach by the active site serine oxygen. This nucleophilic attack is made possible through a proton transfer from Ser203 (ACHE) or Ser226 (BuChE) to a nitrogen in the imidazole ring of His447 (ACHE) or His466 (BuChE), which in turn transfers a proton from the second nitrogen of the imidazole ring to the carboxyl group of Glu334 (ACHE) or Glu353 (BuChE; Fig. 3) (Soreq and Seidman, 2001). The serine is phosphylated, and the remaining half of the molecule (the leaving group) disassociates from the enzyme. Although the acetylated enzyme intermediate in the case of AChE hydrolysis is rapidly broken down by a water molecule, the phosphylated intermediate is usually very stable and is only slowly destroyed by an attacking water molecule. The presence of the phosphyl moiety covalently bound to the active site serine prevents AChE and BuChE from hydrolyzing ACh, thereby leading to the accumulation of this transmitter and cholinergic crisis. It should be noted that most OP insecticides are generally poor inhibitors of AChE and BuChE since they contain a P = S moiety. However, they are metabolically activated by cytochromes P450 to produce the oxygen analogs, also known as oxons, that are very potent anticholinesterase compounds (Fig. 4). The greater capacity of the oxygen
211
LD5o = 2600 mg/kg
CH 3-O
CH2CO2C2Hs Malaoxon
IC5o= 700 nM LDso = 308 mg/kg
FIG. 4. Comparison of the anticholinesterase capacity and acute toxicity of the OP malathion (dicarbethoxyethyl-O,Odimethyldithiophosphate) with its oxon, malaoxon (O,O-dimethyl S-1,2-bis(ethoxycarbonyl)ethyl phosphorothioate). It should be noted that ICs0s were determined under identical conditions. These data were taken from Eto (1974).
analogs to inhibit AChE and BuChE results from the greater electronegativity of oxygen compared to sulfur, and therefore greater polarization of the P = O linkage compared to the P--S linkage. This in turn leads to a more electrophilic phosphorus in the oxons, which greatly facilitates the nucleophilic attack by the oxygen on the active site serine residue of the enzyme. Consequently, it is generally assumed that the inhibition of AChE and BuChE following exposure to an OP insecticide occurs as a result of the oxygen analog and not the parent compounds. The CM anticholinesterases are thought to inhibit AChE and BuChE in a manner similar to that of OPs. However, instead of an electrophilic phosphorus, as in the case of the OPs, CMs contain an electrophilic carbonyl carbon, which undergoes nucleophilic attack by the active site serine oxygen (Fig. 2). The resulting carbamylated enzyme intermediate inhibits enzyme activity until a water molecule attacks the carbonyl carbon to reactivate enzyme and produce a carbamic acid derivative (Fig. 2). This rate of reactivation is considerably faster than that of phospylated enzyme, although it is not as rapid as reactivation of the acetylated intermediate.
III. KINETIC SCHEMES FOR AChE METABOLISM AND INHIBITION BY OPs AND CMs For many decades, it has been thought that the interactions of the anticholinesterase OP and CM compounds with AChE and BuChE are kinetically analogous to those of ACh. In the absence of binding of ACh to the peripheral anionic site of AChE (the validity of this assumption is discussed later), hydrolysis of ACh by AChE or BuChE can be described by a Ping Pong Bi Bi kinetic scheme (Fig. 5). In this scheme, ACh represents the first substrate, whereas water represents the second. Additionally, choline is the first product formed, and it is released before the second
21 2
S E CTI O N I!I 9 Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
AB+E
k~
~
k_ 1
k~
E"AB
~
k_2
E-A
+
H20~
k3
k_3
+
B
AB+E
k~
~
k-1
E..AB
AB+E
E +
A
k~
-->E-A-->
k3
E
+
+
B
A
k,
"-> E - A m > E +
+
B
A
KD = K_I l K1 Ki= K2 / KD FIG. 5. Kinetic models descriptive of the interaction of CMs and OPs with AChE and BChE. The top panel represents an Ping Pong Bi Bi kinetic mechanism, with the following parameter definitions: AB is the CM or OP; E is free, active cholinesterase; E . . AB is the Michaelis complex between enzyme and CM or OP; E-A is the carbamylated or phosphylated intermediate; B is the first product released (leaving group); kl is the association rate constant for the formation of the Michaelis complex; k-1 is the dissociation rate constant for the Michaelis complex; k2 is the rate constant for acylation of the active site serine; k-2 is the rate constant for the reverse acylation step (negligible for CMs and OPs); k3 is the rate constant descriptive of the reactivation of enzyme by water; and k_ 3 is the rate constant for the reverse reactivation reaction (also negligible for CMs and OPs). As described by Segel (1975), since water is always present in excess, the Ping Pong Bi Bi reaction mechanism reduces to an Ordered Uni Bi reaction mechanism (middle). Note that k_ 2 and k_ 3 have been eliminated in the middle panel. The bottom panel represents the ki scheme, derived by Main (1964) from the Ordered Uni Bi reaction mechanism.
substrate binds. The second product is acetate. This reaction is extremely fast, with a turnover number greater than 104 sec -1 (Quinn, 1987). Because water is always present in excess, the Ping Pong Bi Bi reaction scheme can be simplified to an Ordered Uni Bi kinetic scheme (Fig. 5), in which there is one substrate with two products that are formed in a specific order (Segel, 1975). Note that the kinetic interactions of substrates with AChE and BuChE are in reality more complex than portrayed in Fig. 5. Both cholinesterases have been shown to display substrate inhibition and activation, depending on the incubation conditions (Masson et al., 2004), probably as a result of the presence of a binding site separate from the active site, termed the peripheral anionic site (Changeux, 1966; Taylor and Radi~, 1994; Barak et al., 1995; Soreq and Seidman,
2001; Bourne et al., 2003). Furthermore, occupation of the peripheral anionic site of AChE by ACh in vivo likely occurs since the evoked release of ACh at terminal endings can produce local concentrations in narrow synaptic clefts in the millimolar range (Van Der Kloot and Molg6, 1994). Moreover, BuChE has been shown to display an oscillatory hysteretic approach to steady state with some substrates under certain incubation conditions by an unknown molecular mechanism (Masson et al., 2004). However, because these kinetic complexities do not appear to affect the basic mechanism of the hydrolysis of ACh by the cholinesterases as outlined in Figs. 2 and 5 but instead alter the rates at which these events occur, the Ordered Uni Bi scheme can be said to represent the minimal catalytic mechanism for the hydrolysis of substrate by cholinesterases (Barnett and Rosenberry, 1977). Alternatively, more complex, comprehensive kinetic models of cholinesterases have been suggested in order to account for the documented substrate inhibition and activation (Barnett and Rosenberry, 1977; Radi~ et al., 1993; Stojan et al., 1998; Rosenberry et al., 1999; Goli~nik, 2001), as shown in Fig. 6. In these schemes, binding to a peripheral site may alter events at the active site gorge by a variety of putative mechanisms that may affect one or more steps outlined in the kinetic scheme shown in Fig. 5. Note that in most cases, the molecular mechanisms resulting in inhibition or activation of enzyme are not completely understood. The interactions of CM and OP anticholinesterase compounds with AChE and BuChE have also been viewed as a Ping Pong Bi Bi kinetic mechanism that simplifies to an Ordered Uni B i kinetic mechanism since water is always present in excess. The primary difference between hydrolysis of ACh and inhibition by OP and CM compounds is the rate at which the acylated intermediates are reactivated by water to form the second product and free, uninhibited enzyme (reaction 3 in Fig. 2 and k3 in Fig. 5). In the case of ACh, the acetylated enzyme exists for only
AB+E
qb AB + AB--E
"--~,---E-.AB /<1 k;
~
k'l
*-4t'
>-E-A--->.E
+B
k~
+A
AB--E-'AB - - - ~ A B " E - A ~
+B
k:~
k4
AB-.E ~
+A
k-4
AB + E
FIG. 6. General kinetic scheme proposed for hydrolysis of ACh by AChE and BuChE. The minimal catalytic mechanism scheme (top line) is identical to that in Fig. 4. Binding to a modulatory site is represented in the bottom line by AB 9 9E. Those rate constants represented as k' designate steps that could be altered (either increased or decreased) as a result of ligand binding to the modulatory site. Adapted from Barnett and Rosenberry (1977) and Stojan et al. (1998).
CHAPTER 1 5 9Interactions of OP and CM Compounds with Cholinesterases a fraction of a second before a water molecule regenerates free enzyme and forms acetate. In contrast, spontaneous regeneration of a carbamylated enzyme intermediate usually requires several to many minutes depending on the structure of the carbamate moiety. For example, the halflife for the recovery of N-methylcarbamylated AChE is approximately 30 min (Fukuto, 1990). Still longer is the time required for the reactivation of a phosphylated enzyme intermediate formed by an OE The half-life for the recovery of a phosphylated intermediate can range from several hours to days, depending on the structure of the particular OE Moreover, certain phosphylated intermediates can undergo a process termed aging (see Section VI), after which regeneration cannot occur. Beginning with the Ordered Uni B i kinetic reaction scheme, Main (1964) derived a bimolecular inhibition rate constant, ki, which quantifies the inhibitory power of an OP toward AChE (the concept of k i can also be extended to BuChE). The k i is a function of both KD and k2 (Fig. 5) and has been considered the best estimate of inhibitory capacity of an OP (Fukuto, 1990). The larger the ki, the greater is the capacity of an individual molecule of OP to inhibit an individual molecule of enzyme. Although k i was derived based on OP interactions with ACHE, the term k i can be applied with the same meaning to the inhibition of enzyme by the CMs as well (O'Brien, 1968). Recent studies have raised questions about the accuracy of k i, and therefore the kinetic schemes in Figure 5, in describing the inhibitory capacity of AChE insecticides. Kardos and Sultatos (2000) reported that the k i for the OP paraoxon (and methyl paraoxon) changed over a wide range of inhibitor concentrations, suggesting that at low oxon concentrations an individual paraoxon molecule has a greater capacity to inhibit enzyme than an individual paraoxon molecule at high oxon concentrations. If viewed as an enzymatic reaction in which the oxon is hydrolyzed by ACHE, this phenomenon would be considered substrate inhibition. However, the stability of the phosphylated intermediate precludes the use of this terminology. The k i of an OP should be invariant, unless KD and/or k2 change as a function of oxon concentration (Fig. 5). Similar results were observed for chlorpyrifos oxon (Kousba et al., 2004). The authors suggested that these oxons may bind to a secondary site distinct from the active site and alter reactivity of the active site toward other oxon molecules through either steric hindrance or allosteric modification m a phenomenon that has been described in detail for occupation of the peripheral anionic site by certain ligands (Taylor and Radic, 1994). Consequently, just as the kinetic schemes in Fig. 5 represent the minimal catalytic mechanism for hydrolysis of substrate by ChEs, these same kinetic schemes probably represent the minimal inhibitory mechanism that describes, in part, the interactions of certain OPs with ACHE.
213
IV. S T R U C T U R E - A C T I V I T Y
RELATIONSHIPS The role of OP and CM structure in the inhibition of AChE has been studied intensively for decades. Structure-activity studies have often focused on the effects of chemical structure on reversible binding of inhibitor to enzyme, usually quantified by KD (Fig. 5) (formation of Michaelis complexes), and the phosphylation or carbamylation step, usually quantified by k2 (Fig. 5). Although investigators have sometimes debated the relative importance of reversible binding to the active site versus the phosphylation or carbamylation step in determining overall reactivity of inhibitor toward the enzyme, it is clear that both steps are critical in controlling inhibitor reactivity, and both steps are controlled by chemical structure and the structure of the active site gorge.
A. OPs For an OP to possess the capacity to phosphylate AChE or BuChE, the phosphorus must be electrophilic enough to accept a nucleophilic attack from the active site serine. As described by Fukuto (1990), structure-activity studies have found a direct relationship between the reactivity of the phosphorus atom and the capacity to phosphylate AChE and BuChE. As stated previously, the presence of a sulfur bonded to the phosphorus, such as can be found with most OP insecticides, renders the molecule a rather weak inhibitor, whereas replacement of the sulfur with an oxygen markedly increases the capacity to phosphylate AChE and BuChE due to the greater electron-withdrawing capacity of the oxygen (Fig. 4). The greater electronegativity of the oxygen therefore renders the phosphorus more electrophilic and more susceptible to a nucleophilic attack (Eto, 1974). Other substituents with varying electronwithdrawing capacity attached to the phosphorus will similarly affect anticholinesterase activity. The greater the electron-withdrawing capacity of the substituent, the greater the anticholinesterase activity of the OP (Eto, 1974). Systematic studies with paraoxon (diethyl p-nitrophenyl phosphate) and similar substituted phenyl phosphates have shown that their anticholinesterase activity is a direct function of their alkaline hydrolysis rates, which reflects phosphorus reactivity (Fukuto, 1990). The greater electron-withdrawing capacity of a particular substituent makes the P-O phenyl bond deficient in electron density and consequently makes the ester readily accept nucleophilic substitution on the phosphorus (Eto, 1974). Likewise, diethyl-substituted phenyl phosphates with little electron-withdrawing capacity has little or no anticholinesterase activity (Fukuto, 1990). Of equal importance to phosphorus reactivity are the steric properties of the OP with respect to the active site gorge of the enzyme. Unless an OP molecule can diffuse into the active site gorge and form a stable Michaelis complex,
2 14
S ECTI 0 N I II 9Esterases, Receptors, Mechanisms, & Tolerance Development
phosphylation will not have the opportunity to occur, regardless of phosphorus reactivity. Early studies demonstrated that the length of alkyl side chains was a critical determinant of anticholinesterase activity, with reduced inhibitory capacity associated with bulkier alkyl side chains (Fukuto and Metcalf, 1959; Hansch and Deutsch, 1966; Eto, 1974). These studies demonstrated that for anticholinesterase activity, the alkyl side chains should remain small (methyl or ethyl groups). In this regard, most OP insecticides possess either methyl or ethyl alkyl side chains, although some noninsecticidal OPs have isopropyl groups for side chains. Furthermore, the importance of steric properties was confirmed in recent studies in which specific AChE mutations that decreased the stability of Michaelis complexes of certain OPs decreased their anticholinesterase activity (Ordentlich et al., 1996). Additionally, the differences in the acyl pocket structure of AChE and BuChE allow the BuChE active site to better accommodate bulkier OPs such as iso-OMPA (N,N'diisopropyl phosphorodiamidic anhydride) and mipafox (N,N'-diisopropyl phosphorodiamidic fluoride). Iso-OOMPA has often been used as a selective inhibitor of BuChE experimentally (Austin and Berry, 1953; Harel et al., 1992). Although the acyl pocket is critical in determining the "fit" of OPs into the active site gorge, Ordenflich et al. (2004) documented the importance of the peripheral anionic site, as well as several additional sites, in determining the stereoselectivity of AChE toward enantiomers of methylphosphonates. B. C M s Extensive structure-activity relationships with CM insecticides have revealed general similarities with those of the OPs. Specifically, the anticholinesterase activity of a CM is primarily a function of the reactivity of the carbonyl carbon (rather than the phosphorus of OPs) and the ability of the CM to diffuse into the active site gorge and form Michaelis complexes (steric properties). Chemical modifications that reduce the electrophilic nature of the carbonyl carbon reduce the anticholinesterase activity, whereas the opposite is observed with substituents that increase the electrophilicity of this same carbon atom (Kuhr and Dorough, 1976; Fukuto, 1990). In contrast to the OPs, most (but not all) CMs can directly inhibit ChEs and do not require metabolic activation. In this regard, the reactivity of the carbonyl carbon for most methylcarbamate insecticides is so significant that the equilibrium constant for dissociation of the enzyme-inhibitor complex (Ko) correlated directly with the inhibitory capacity of the molecule (Fukuto, 1990). As is the case with the OPs, the steric properties of a CM modulate the access of the CM to the catalytic serine. Bulky substituents reduce access, whereas others increase access. For example, in phenyl N-methylcarbamates the placement of the substituent alkyl groups on the ring relative to the CM moiety influences anticholinesterase activity. Substitution at the 3-position produced the most active compounds,
probably because the distance between the CM group and the meta-substituent was most conducive to formation of a Michaelis complex (Khur and Dorough, 1976). Similary, additions of larger, bulkier substituents reduced anticholinesterase activity (Khur and Dorough, 1976). Only limited carbamate structure-activity relationship studies have been done with regard to comparisons of the inhibitory capacity toward AChE versus BuChE. However, based on structure-activity studies with five N-methyl carbamates, Loewenstein et al. (1993) concluded that the CM binding site on BuChE is less flexible than the same site on ACHE.
V. OXIME REACTIVATION OF I N H I B I T E D CHOLINESTERASES The classic studies of Wilson and Ginsburg (1955a,b) established that reactivation of phosphylated AChE could be accelerated by certain compounds more nucleophilic than water, such as N-methylhydroxylamine. Since that time, a variety of reactivators, termed oximes, have been synthesized for use in the treatment of subjects poisoned by OP anticholinesterases. In the United States, the oxime pralidoxime [N-methyl-(2-hydroxyaminoformylpyridinium)chloride] is the only reactivator approved for use (Fig. 7), whereas obidoxime (bis[4-hydroxyamino-
O
It R,-o.,p R1-O
O
3
O ~
S~r
11 I .,~--~ O-N=HCR2- O I,p R1-O O Sler
I
R1-O" I O
+
R2
Sir
Aged Form
t CH3
O
OH sler
I
R2- O " RI_O ~ P - O - N - H C -
I
CH3
FIG. 7. Reactivation and aging of phosphylated cholinesterase. Reaction 1 represents the nucleophilic attack of pralidoxime on the phosphate, followed by regeneration of free, active enzyme and the phosphylated oxime (reaction 2). Reaction 3 represents aging and therefore the formation of a monophosphylate ester that is resistant to reactivation by pralidoxime (and other oximes).
CHAPTER 1 5 9Interactions of OP and CM Compounds with Cholinesterases methyl-methylpyridinium] ether dichloride) is also available in other areas of the world (Kwong, 2002). Other experimental reactivators include HI-6 [1-(2-hydroxyiminomethyl1-pyridinium)-l-(4-carboxyaminopyridinium) dimethyl ether hydrochloride], TMB4 [1,1'-trimethylene bis(4-(hydroxyimino)-methyl) pyridinium dibromide], and LtiH6 [1,1'-(oxybismethylene) b i s ( 4 - ( h y d r o x y i m i n o ) m e t h y l ) p y r i dinium dichloride] (Rousseaux and Dua, 1989; Luo et al., 1999). Pralidoxime (and other reactivators) accelerates reactivation of phosphylated AChE as a result of a nucleophilic attack of the oxime on the phosphorus of the phosphylated enzyme to give phosphylated oxime and free, active enzyme (Fig. 7), provided that aging has not occurred (see Section VI). Aging greatly increases the resistance to reactivation, even by powerful reactivator oximes. The rate of reactivation of phosphylated enzyme by an oxime is dependent on the structure of the phosphyl moiety, the nature of the enzyme, and the structure of the oxime reactivator (Luo et al., 1998). Interestingly, certain phosphylated oximes have been shown to have the capacity to reinhibit reactivated ACHE, thereby reducing their potential as treatment of OP intoxication (Luo et al., 1998, 1999; Kideden et al., 2000). This inhibition can be prevented by certain drugs, such as edrophonium, that compete with the phosphylated oxime for the ACHE active site (Luo et al., 1998, 1999). Pralidoxime is not used for treatment of CM insecticide poisoning since it enhanced the toxicity of carbaryl (1-naphthyl N-methylcarbamate) (Harris et al., 1989). However, pralidoxime and the reactivator HI,6 reduced the lethality of the CM drug physostigmine in rats, providing a rational basis for the use of physostigmine or pyridostigmine pretreatment in conjunction with the standard therapy for exposure to the highly toxic nerve gases (Harris et al., 1989). Although the exact molecular interactions between phosphylated enzyme and oxime that lead to reactivation are not known, Ashon et al. (1995) and Wong et al. (2000), utilizing mutant forms of ACHE, identified a number of amino acid residues that are important in the reactivation of phosphylated ACHE. Wong et al. suggested that a major determinant of oxime-induced reactivation rates is simply the ability of the reactivator to "fit" into the active center gorge and gain access to the tetrahedral phosphorus. This fit is controlled by the structure of the oxime and the steric bulk of the intervening groups surrounding the tetrahedral phosphorus (Wong et al., 2000).
VI. AGING OF PHOSPHORYLATED CHOLINEsTERASE Following the phosphylation of serine at the active site of AChE or BuChE by certain OP compounds, a dealkylation of the phosphyl moiety can occur. This reaction, referred to
215
as "aging," results in the loss of an alkyl group from the phosphyl alkoxy substituent (Fig. 7), probably through the formation of a carbocationic transition state that forms a carbonium ion that rearranges to form alkene products (Smith and Usdin, 1966; Michael et al., 1967; Bencsura et al., 1995). This reaction is of great toxicological significance since the remaining negatively charged monophosphylate ester of serine is resistant to reactivation by nucleophilic oximes such as pralidoxime (Aldridge and Reiner, 1972; Worek et al., 1996). Consequently, the activity of aged cholinesterase in a patient poisoned with an anticholinesterase OP compound can only be regained through synthesis of new enzyme and not through treatment with the antidotal oximes. Thus, the recovery of aged red blood cell cholinesterase is longer than that of aged plasma cholinesterase since the turnover of red blood cells is much slower than the turnover of plasma cholinesterase (Mason, 2000; Mason et al., 2000). The rate of aging, and therefore the extent of aging, that occurs with phosphylated AChE or BuChE is a function of the chemical structure of the phosphyl moiety. Most OP insecticides have either two methoxy or two ethoxy side chains, and the methoxy compounds have been reported to age faster than the corresponding inhibitors with ethoxy side chains. For example, Worek et al. (1999) reported that the aging half-time of diethylphosphorylated AChE (31 hr) is approximately eight times longer than the aging halftime of dimethylphosphorylated ACHE. In contrast, aging of the nerve agents such as soman is markedly faster. Talbot et al. (1988) reported that the aging half-time for human red blood cell somanyl-AChE is approximately 1 min. Phosphylated BuChE has been reported to age at approximately the same rate or slightly faster than phosphylated AChE (Worek et al., 1999; Masson et al., 1997). Although the specific molecular events that lead to aging are not known with certainty, considerable progress has been made in understanding the details of aging of compounds such as soman and diisopropyl fluorophosphate through approaches combining site-directed mutagenesis, kinetic analyses, and molecular modeling studies. It is unlikely that aging of AChE or BuChE phosphorylated by OPs will be markedly different. In what has been referred to as a "push-pull" mechanism for aging, carbocation formation has been suggested to result from the strong negative electrostatic field of Glu202 (Kovach et al., 1997; Saxena et al., 1998). Shafferman et al. (1996) suggested that Glu202 and Phe338 contribute to aging by stabilizing the imidazolium of the catalytic triad His447, which has been proposed to act as an acid catalyst for the cleavage of the C-O bond (Ordentlich et al., 1993; Qian and Kovach, 1993). However, many other active site residues have also been implicated in the aging of somanyl-AChE, including Asp72, Trp86, Tyr133, Ser199, Glu202, and Glu450 (Shafferman et al., 1996; Masson et al., 1997).
21 6
S E CTI O N I I I
9Esterases, Receptors, Mechanisms,
In BuChE phosphylated by DFP, Trp82 seems to stabilize the carbonium ion released during aging, and Glu197 carboxylate appears to help stabilize the developing carbocation (Masson et al., 1997). Finally, Asp70 exerts allosteric control of dealkylation probably by altering the conformation state of Trp82 (Masson etal., 1997). As stated previously, the primary toxicological significance of aging is that aged phosphylated enzyme cannot be reactivated, even by the oximes. Resistance to reactivation has been attributed, at least in part, to two possible mechanisms. The first is an aging-induced conformation change of the inhibited enzyme, resulting in increased stability. Masson et al. (1997) suggested that such a change may result from the formation of a salt bridge between the catalytic protonated histidine and the negatively charged oxygen atom on the monophosphylate moiety. Second, aged ChEs may electrostatically repulse oximes as a result of the negatively charged oxygen of the monophosphylate moiety and the adjacent negatively charged Glul00 (Masson et al., 1997).
& Tolerance Development
inhibition profiles of wild-type and the atypical variant of BuChE toward five different N-methyl carbamates were not different. The atypical variant of BuChE they examined has approximately 30% lower activity toward succinylcholine than wild-type BuChE (Loewenstein et al., 1993; Neville et al., 1990). Finally, genetically engineered human BuChEs have been developed that are resistant to OP (and presumably CM) inhibition since the reactivation step by water (Fig. 2) is markedly accelerated in these mutants (Millard et al., 1995; Schopfer et al., 2004). Since human wild-type BuChE has been shown to act as a scavenger to protect against OP toxicity in animal models (Broomfield et al., 1991; Raveh et al., 1997), BuChE mutants with the capacity to hydrolyze OPs (and perhaps CMs) may eventually be utilized in the antidotal treatment for OP or CM intoxication.
References Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates. Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North-Holland,
VII. GENETIC VARIANTS Genetic variants of AChE and BuChE have been identified. However, unlike AChE variants, BuChE variants display varied substrate activities. Since the classic work of Kalow and coworkers, who first investigated this phenomenon (Kalow and Staron, 1957; Kalow and Dunn, 1959), more than 40 variants of BuChE have been identified (Darvesh et al., 2003). The wild-type BuChE allelic frequency has been reported to be approximately 85%, whereas two variants, termed K and the atypical form, have reported allelic frequencies of 10 and 13%, respectively (Darvesh et al., 2003). The allelic frequencies of the remainder of the identified variants are extremely low. Although most of the BuChE variants have not been fully characterized, it is known that they generally have reduced enzymatic activity compared to the wild type (La Du et al., 1990; Darvesh et al., 2003), and at least 12 have no activity at all (Primo-Parmo et al., 1996). The functional significance of BuChE variants in the metabolism of certain drugs, such as succinylcholine, has been well characterized (Darvesh et al., 2003). However, their functional significance with respect to OPs and CMs is less clear. BuChE is thought to serve as a nonenyzmatic pathway for the detoxification of OPs and CMs. Given the lower activities of BuChE variants, it has been proposed that certain individuals with reduced BuChE activity will have a reduced capacity to "scavenge" anitcholinesterase OPs and CMs and therefore may have a genetic predisposition to the adverse effects of the anticholinesterase OPs and CMs (Loewenstein-Lichtenstein et al., 1995; Fontoura-da-Silva and Chautard-Freire-Maia, 1996; Lockridge and Masson, 2000). Although this is a reasonable proposition, it should be noted that Loewenstein et al. (1993) reported that the
New York. Ashon, Y., Radic, Z., Tsigelny, I., Vellom, D. C., Picketing, N. A., Quinn, D. M., Doctor, B. P., and Taylor, P. (1995). Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes. J. Biol. Chem. 270, 6370-6380. Austin, L., and Berry, W. K. (1953). Two selective inhibitors of cholinesterase. Biochem. J. 54, 695-700. Barak, D., Ordentlich, A., Bromberg, A., Kronman, C., Marcus, D., Lazar, D., Ariel, N., Velan, B., and Shafferman, A. (1995). Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite. Biochemistry 34, 15444-15452. Barnett, P., and Rosenberry, T. L. (1977). Cataysis by acetylcholinesterase. J. Biol. Chem. 252, 7200-7206. Bencsura, A., Enyedy, I., and Kovach, I. M. (1995). Origin and diversity of the aging reactions in phophonate adducts of serine hydrolase enzymes: What characteristics of the active site do they probe? Biochemistry 34, 8989-8999. Bourne, Y., Taylor, P., Radi~, Z., and Marchot, P. (2003). Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site. E M B O J. 22, 1-12. Broomfield, C. A., Maxwell, D. M., Solana, R. P., Castro, C. A., Finger, A. V., and Lenz, D. E. (1991). Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates. J. Pharmacol. Exp. Ther. 259, 633-638. Changeux, J. P. (1966). Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing agents. Mol. Pharmacol. 2, 369-392. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Doctor, B. P. (1993). Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related enzymes. Protein Sci. 2, 366-382. Darvesh, S., Hopkins, D. A., and Geula, C. (2003). Neurobiology of butyrylcholinesterase. Nat. Rev. Neurosci. 4, 131-138.
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217
La Du, B. N., Bartels, C. E, Nogueira, C. R, Hajara, A., Lightstone, H., van der Spek, A., and Lockridge, O. (1990). Phenotypic and molecular biological analysis of human butyrylcholinesterase variants. Clin. Biochem. 23, 423-431. Lockridge, O., and Masson, E (2000). Pesticides and susceptible populations: People with butyrylcholinesterase genetic variants may be at risk. Neurotoxicology 21, 113-126. Loewenstein, Y., Denarie, M., Zakut, H., and Soreq, H. (1993). Molecular dissection of cholinesterase domains responsible for carbamate toxicity. Chem.-Biol. Interact. 87, 209-216. Loewenstein-Lichtenstein, Y., Schwarz, M., Glick, D., NorgaardPedersen, B., Zakut, H., and Soreq, H. (1995). Genetic predisposition to adverse consequences of anti-cholinesterases in "atypical" BCHE carriers. Nat. Med. 1, 1082-1085. Luo, C., Ashani, Y., and Doctor, B. E (1998). Acceleration of oxime-induced reactivation of organophosphate-inhibited fetal bovine serum acetylcholinesterase by monoquaternary and bisquaternary ligands. Mol. Pharmacol. 53, 718-726. Luo, C., Saxena, A., Smith, M., Garcia, G., Radic, Z., Taylor, E, and Doctor, B. E (1999). Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium. Biochemistry 38, 9937-9947. Main, A. R. (1964). Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science 144, 992-993. Mason, H. J. (2000). The recovery of plasma cholinesterase and erythrocyte acetylcholinesterase activity in workers after overexposure to dichlorvos. Occup. Med. 50, 343-347. Mason, H. J., Sains, C., Stevenson, A. J., and Rawbone, R. (2000). Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides. Hum. Exp. Toxicol. 19, 511-516. Masson, E, Fortier, E-L., Albaret, C., Froment, M.-T., Bartels, C. E, and Lockridge, O. (1997). Aging of di-isopropyl-phosphorylated human butyrylcholinesterase. Biochem. J. 327, 601-607. Masson, E, Goldstein, B. N., Debouzy, J.-C., Froment, M.-T., Lockridge, O., and Schopfer, L. M. (2004). Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate. Eur. J. Biochem. 271, 220-234. Michael, H. O., Hackley, B. E., Berkovitz, L., List, G., Hackley, E. B., Gillian, W., and Pankau, M. (1967). Aging and dealkylation of soman (pinacolylmethylphosphonofluoridate)-inactivated eel cholinesterase. Arch. Biochem. Biophys. 121, 29-34. Millard, C. B., Lockridge, O., and Broomfield, C. A. (1995). Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase. Biochemistry 34, 15925-15933. Neville, L. E, Gnatt, A., Padan, R., Seidman, S., and Soreq, H. (1990). Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations. J. Biol. Chem. 265, 20735-20738. O'Brien, R. D. (1968). Kinetics of the carbamylation of cholinesterase. Mol. Pharmacol. 4, 121-130. Ordentlich, A., Kronman, C., Barak, D., Stein, D., Ariel, N., Marcus, D., Velan, B., and Shafferman, A. (1993). Engineering resistance to "aging" of phosphylated human acetylcholinesterase: Role of hydrogen bond network in the active center. FEBS Lett. 334, 215• Ordentlich, A., Barak, D., Kronman, C., Ariel, N., Segall, Y., Velan, N., and Shafferman, A. (1996). The architecture of
21 8
SECTION 1II 9 Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors. J. Biol. Chem. 271, 11953-11962. Ordentlich, A., Barak, D., Kronman, C., Ariel, N., Segall, Y., Velan, B., and Shafferman, A. (1998). Functional characteristics of the oxyanion hole in human acetylcholinesterase. J. Biol. Chem. 273, 19509-19517. Ordentlich, A., Barak, D., Sod-Moriah, G., Kaplan, D., Mizrahi, D., Segall, Y., Kronman, C., Karton, Y., Lazar, A., Marcus, D., Velan, B., and Shafferman, A. (2004). Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of ACHE. Biochemistry 43, 11255-11265. Primo-Parmo, S. L., Bartels, C. E, Wiersema, B., van der Spek, A. E, Innis, J. W., and La Du, B. N. (1996). Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene. Am. J. Hum. Genet. 58, 52-64. Qian, N., and Kovach, I. M. (1993). Key active site residues in the inhibition of acetylcholinesterase by soman. FEBS Lett. 336, 263-266. Quinn, D. M. (1987). Acetylcholinesterase: Enzyme structure, reaction dynamics, and virtual transition states. Chem. Rev. 87, 955-979. Radi~, Z., Picketing, N. A., Vellom, D. C., Camp, S., and Taylor, P. (1993). Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32, 12074-12084. Raveh, L., Grauer, E., Grunwald, J., Cohen, E., and Ashani, Y. (1997). The stoichiometry of protection against soman and VX toxicity in monkeys pretreated with human butyrylcholinesterase. Toxicol. Appl. Pharmacol. 145, 43-53. Rosenberry, T. L., Mallender, W. D., Thomas, P. J., and Szegletes, T. (1999). A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate. Chem.-Biol. Interact. 119-120, 85-97. Rousseaux, C. G., and Dua, A. K. (1989). Pharmacology of HI-6 and H-series oxime. Can. J. Physiol. Pharmacol. 67, 1183-1189. Saxena, A., Viragh, C., Frazier, D. S., Kovach, I. M., Maxwell, D. M., Lockridge, O., and Doctor, B. P. (1998). The pH dependence of dealkylation in soman-inhibited cholinesterase and their mutants: Further evidence for a push-pull mechanism. Biochemistry 37, 15086-15096 Segel, I. H. (1975). Enzyme Kinetics. Wiley, New York. Schopfer, L. M., Boeck, A. T., Broomfield, C. A., and Lockridge, O. (2004). Mutants of human butyrylcholinesterase with organophosphate hydrolase activity: Evidence that HIS117 is a general acid base catalyst for hydrolysis of echothiophate. J. Med. Chem. Def. 2, 1-21. Shafferman, A., Ordentlich, A., Barak, A., Stein, D., Ariel, N., and Velan, B. (1996). Aging of phosphylated human acetylcholinesterase: Catalytic processes mediated by aromatic
and polar residues of the active centre. Biochem. J. 318, 833-840. Smith, T. E., and Usdin, E. (1966). Formation of nonreactivatible isopropylmethyl-phosphono-fluoridate-inhibited acetylcholinesterase. Biochemistry 5, 2914-2918. Soreq, H., and Seidman, S. (2001). Acetylcholinesterase - - New roles for an old actor. Nat. Rev. Neurosci. 2, 294-301. Stojan, J., Marcel, V., Estrada-Mondaca, S., Klaebe, A., Masson, P., and Fournier, D. (1998). A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase. FEBS Lett. 440, 85-88. Talbot, B. G., Anderson, D. R., Harris, L. W., Yarbrough, L. W., and Lennox, W. J. (1988). A comparison of in vivo and in vitro rates of aging of soman-inhibited erythrocyte acetylcholinesterase in different animal species. Drug Chem. Toxicol. 11, 289-305. Taylor, P. (2001). Anticholinesterase agents. In Goodman & Gilman's The Pharmacological Basis of Therapeutics (J. G. Hardman and L. E. Limbird, Eds.), 10th ed., pp. 175-191. McGraw-Hill, New York. Taylor, P., and Radi~, Z. (1994). The cholinesterases: From genes to proteins. Annu. Rev. Pharmacol. Toxicol. 34, 281-320. Tsigelny, I., Shindyalov, I. N., Bourne, P. E., Sudhof, T. C., and Taylor, P. (2000). Common EF-hand motifs in cholinesterases and neuroligins suggest a role for Ca 2+ binding in cell associations. Protein Sci. 9, 180-185. Van Der Kloot, W., and Molg6, J. (1994). Quantal acetylcholine release at the vertebrate neuromuscular junction. Physiol. Rev. 74, 899-991. Walsh, C. (1979). Enzymatic Reaction Mechanisms. Freeman, San Francisco. Wilson, I. B., and Ginsburg, S. (1955a). The reactivation of acetylcholinesterase inhibited by tetraethyl pyrophosphate and diisopropylfluorophosphate. J. Am. Chem. Soc. 77, 4286-4288. Wilson, I. B., and Ginsburg, S. (1955b). A powerful reactivator of alkyl phosphate-inhibited acetylcholinesterase. Biochim. Biophys. Acta 18, 168-170. Wilson, I. B., Bergmann, E, and Nachmansohn, D. (1950). Acetylcholinesterase X. Mechanism of the catalysis of acylation reactions. J. Biol. Chem. 186, 781-790. Wong, L., Radic, Z., Bruggermann, R. J. M., Hosea, N., Berman, H. A., and Taylor, P. (2000). Mechanism of oxime reactivation of acetylcholinesterase analyzed by chirality and mutagenesis. Biochemistry 39, 5750-5757. Worek, E, Kirchner, T., B~icker, M., and Szinicz, L. (1996). Reactivation by various oximes of human erythrocyte acetylcholinesterase inhibited by different organophosphorus compounds. Arch. Toxicol. 70, 497-503. Worek, E, Diepold, C., and Eyer, P. (1999). Dimethylphosphorylinhibited human cholinesterase: Inhibition, reactivation, and aging kinetics. Arch. Toxicol. 73, 7-14.
CHAPTER
116
Structure, Function, and Regulation of Carboxylesterases MASAKIYO HOSOKAWA 1 AND TETSUO SATOH l,z 1Chiba, Institute of Science, Chiba, Japan eHAB Research Laboratories, Ichikawa, Chiba, Japan
dog intestine was very weak and produced no appreciable active band in a disk electrophoresis coupled with staining of esterase activity. On the other hand, esterase activities were observed in the intestines of other species (human, rat, mouse, guinea pig, and rabbit) and found to produce a few active bands in an electrophoretic assay. Since pharmacokinetic and pharmacological data of ester prodrugs obtained from preclinical experiments using various animals are generally used as references for human studies, it is important to clarify the biochemical properties of each CarbE isozyme, such as substrate specificity, tissue distribution, and transcriptional regulation. Recent developments have included more detailed biochemical characterization of mammalian CarbE enzymes and genes, leading to a better understanding of the biochemical significance and physiological role of CarbEs. This chapter deals primarily with the characteristics and the molecular cloning of the individual, recently identified CarbE isozymes.
I. I N T R O D U C T I O N The mammalian carboxylesterases CarbEs comprise a multigene family whose gene products are localized in the endoplasmic reticulum (ER) of many tissues. These enzymes efficiently catalyze the hydrolysis of a variety of ester- and amide-containing chemicals as well as drugs (including prodrugs) to the respective free acids. They are involved in detoxification or metabolic activation of various drugs, environmental toxicants, and carcinogens. Carboxylesterases also catalyze the hydrolysis of endogenous compounds, such as short- and long-chain acyl-glycerols, long-chain acyl-carnitine, and long-chain acyl-CoA esters. We have reviewed the characteristics of CarbEs in relation to the metabolism of xenobiotics (Satoh and Hosokawa, 1998). Multiple isozymes of hepatic microsomal CarbE exist in various animal species (Hosokawa et al., 1990; Satoh and Hosokawa, 1998), and some of these isozymes are involved in the metabolic activation of certain carcinogens, as well as being associated with hepatocarcinogenesis (Maki et al., 1991). Mammalian CarbEs are members of an oL,[3-hydrolasefold family and are found in various mammals (Hosokawa et al., 1990; Satoh and Hosokawa, 1998; Satoh et al., 2002). The expression of CarbEs is ubiquitous, with high levels in the liver, small intestine, kidney, and lung. CarbEs show such a broad range of substrate specificity that they can be involved in detoxification or biotransformation of many kinds of drugs as well as endogenous fatty acid esters. It has been suggested that CarbEs can be classified into four major groups according to the homology of the amino acid sequence (Hosokawa et al., 1990; Satoh and Hosokawa, 1998; Satoh et al., 2002), and the majority of CarbEs that have been identified belong to the CES1 or CES2 family. It has also been shown that striking species differences exist (Inoue et al., 1979; Hosokawa et al., 1990, 1994; Prueksaritanont et al., 1996; Zhu et al., 2000). For example, Inoue et al. showed that esterase activity in the Toxicology of Organophosphate and Carbamate Compounds
II. R O L E O F C a r b E I S O Z Y M E S IN D R U G M E T A B O L I S M
CarbEs are members of an oL,[3-hydrolase-fold family (Satoh and Hosokawa, 1998; Satoh et al., 2002) and they show ubiquitous tissue expression profiles with the highest levels of CarbE activity present in liver microsomes in many mammals (Satoh and Hosokawa, 1998; Satoh et al., 2002). Drug-metabolizing enzymes that are present predominantly in the liver are involved in biotransformation of both endogenous and exogenous compounds to polar products to facilitate their elimination. These reactions are categorized as phase I and phase II reactions. CarbEs are categorized as phase I drug-metabolizing enzymes that can hydrolyze a variety of ester-containing drugs and prodrugs, such as angiotensin-converting enzyme inhibitors (temocapril, cilazapril, quinapril, and imidapril) (Takai et al., 219
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220
SECTION 1II. Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
1997; Mori et al., 1999; Furihata et al., 2004a), antitumor drugs (CPT-11 and capecitabine) (Satoh et al., 1994; Tabata et al., 2004), and narcotics (cocaine, heroin, and meperidine) (Pindel et al., 1997; Zhang et al., 1999). In this regard, it is thought that CarbEs are one of the major determinants for pharmacokinetics and pharmacodynamics of ester drugs or ester prodrugs (Fig. 1). Actually, it has been shown that dog CES 1 isozyme was involved in a pulmonary first-pass effect in the disposition of a propranolol ester prodrug (Imai et al., 2003). It has also been shown that the expression level of human CarbE isozyme was correlated with the conversion ratio of CPT-11 to SN-38, the active metabolite, which is thought to be a key step for the chemotherapeutic action of this antitumor drug (Pindel et al., 1997; Zhang et al., 1999; Ohtsuka et al., 2003; Sanghani et al., 2003).
The CarbEs and the UDP-glucuronosyltransferase (UGT) families, the catalytic domains of which are localized in the luminal sides of the ER membrane, are two major enzyme groups responsible for phase I and phase II reactions (Fig. 2). The hydrolyzed products of CarbEs are also substrates for UGT, such as SN-38 from CPT-11. Thus, CarbE-UGT interaction in the luminal sides of the ER membrane is important for drug metabolism. Furthermore, hydrolyzed products of CarbEs have two kinds of chemical properties; one is the alcohol or phenol, which are substrates for UGT, and the other is organic anions, which are substrates for organic anion transporter such as multidrug resistance-associated protein 2 (MRP2) (Fig. 2). In this regard, CarBEs are one of the major drug-metabolizing enzymes for enzyme-enzyme interaction and enzyme- transporter interaction.
III. C L A S S I F I C A T I O N
AND NOMENCLATURE OF CarbEs
FIG. 1. Role of CarbE isozyme in drug metabolism. CES, carboxylesterase; CYP, cytochrome P450; UGT, UDP glucuronosyltransferase; MDR, multidrug resistance; MRP2, multidrug resistance-associated protein 2.
According to the classification of esterases by Aldridge (1993), the serine superfamily of esterases (i.e., acetylcholinesterase, butyrylcholinesterase, and CarbE) falls into the B-esterase group. CarbEs have very wide substrate specificity. It is becoming increasingly clear that esterases tend to have broad and overlapping substrate specificity toward amides and esters. A single esterolytic reaction is frequently mediated by several kinds of enzymes. Studies on esterases, as with other enzymes concerned with xenobiotic metabolism, have afforded evidence of multiple forms. It seems almost impossible to classify these CarbE isozymes
FIG. 2. CarbE-UGT interaction in the luminal sides of the ER membrane and CES-transporter interaction in the cell.
CHAPTER 1 6 9Structure and Function of CarbEs based on their substrate specificity using the IUB, International Union of Biochemistry classification because the individual hydrolases exhibit properties of CarbE, lipase, or both. Mentlein et al. (1984) proposed to classify these hydrolases as unidentified CarbE (EC 3.1.99.1 to 3.1.99.x). Table 1 summarizes the sequence identities of CES isozymes based on human liver CarbE (Satoh and Hosokawa, 1998). These esterases are markedly similar in terms of characteristics such as substrate specificity. The amino acid sequences of the isozymes of these esterases are highly homologous. Based on the high homology and similarity of the characteristics, we tried to classify CarbE isozymes into four families; CES1, CES2, CES3, and CES4 (Table 1) (Satoh and Hosokawa, 1998). The CES1 family includes the major forms of CES isozymes (more than 60% homology of human CES). Thus, they can be divided into three subfamilies: CES1A, CES1B, and CES1C. The CES 1A subfamily includes the major forms of human CarbEs and the major isoforms of rat, dog, rabbit, and mouse CarbE. The CES 1B subfamily includes RL1, ML1, and hydrolases B and C, which catalyze the long-chain acyl-CoA hydrolysis. Members of the CES 1C family, which share homology with
221
human CarbEs, are similar to the CES 1B family members. However, these families are all secretory type CarbEs. In contrast, the CES2 family includes human intestinal CarbE (hCE2, CES HU3), rat RL4 (rCES2), and rat intestinal CarbE. Mouse ML3(mCES2), rabbit form 2, and hamster AT51 are mainly expressed in the small intestines. CES3 includes ES-male, whose functions are not clear. The CES-4 family includes the novel 46.5-kDa CarbE isozymes, which have a different structure from other CarbE families. ES 46.5-kDa from mouse liver (Watanabe et al., 1993) and amido hydrolase of monkey liver (Kusano et al., 1996) probably belong to this family. These groupings are similar to those derived from phylogenetic analysis (Fig. 3).
IV. S T R U C T U R E - A C T I V I T Y RELATIONSHIPS OF SUBSTRATES W I T H CES1 A N D C E S 2 F A M I L I E S It has been suggested that CarbEs can be classified into four major groups according to the homology of the amino acid sequence as described previously (Satoh and
TABLE 1. Classification and Nomenclature of the Carboxylesterases Gene
Trivial name
Species
Homology (%)
Gene
Trivial name
Species
CES1A1
Macrophage HUla hCE-1 Hbrl
Human Human Human Human
100.0 99.9 99.5 99.6
CES1 C1
CESIA2
HUlb HUMLCEA HUMCES MoCE31 D1 hBr2
Human Human Human Monkey Dog Human
99.3 94.1 93.3 92.9 79.7 79.0
Serum Rat ES 1 Rat CE Hydrolase S Es-N MS1
Rat Rat Rat Rat Mouse Mouse
69.6 68.9 68.9 68.9 65.2 65.2
hBr3 MH1 pl 6.1 Rabbitl MouseCE Egasin ES3(Egasin)
Human Mouse Rat Rabbit Mouse Mouse Rat
76.8 77.9 77.4 77.8 73.2 75.5 74.6
hCE-2 HSiCE HU3 Rabbit 2 ML3 AT-51
Human Human Human Rabbit Mouse Hamster
46.8 46.8 46.8 46.9 43.5 45.7
CES3A1
Rat CE4.6 Rat ICES CES RL4 ES-male
Rat Rat Rat Mouse
42.7 44.1 44.0 41.1
CES4A1
46.5K
Human
31.3
ES4 Hydrolase B Hydrolase C RL1 ML1
Rat Rat Rat Rat Mouse
67.9 67.0 67.0 66.0 63.0
CESIC2 CESIA3
CESIA4
CES2A1
CES2A2 CES2A3
CESIA5 CESIA6
CESIA7
CES1B1
CES1B2
CES2A4 CES2A5
Homology (%)
222
SECTION III. E s t e r a s e s ,
Receptors, Mechanisms, & Tolerance Development D1 (dog liver) hBr2 (human brain)
( l~
C
hBrl (human brain) hCE/HU1 (human liver) hCE-1/Macrophage/ HUMCARA/ Rabbit1 (rabbit liver) hBr3 (human brain) MH1 (mouse liver)
CES1A
RH1 (rat liver) Egasyn (mouse liver) ES3 (Egasin) Hydrolase C (rat liver) RL1 (rat liver) ES4 (rat liver) Kidney (rat kidney) ML1 (mouse liver) Es-N (mouse liver) MS1 (mouse serum) RS1 (rat serum) RL4/rCES2 (rat liver) ratlCES (rat intestine) AT51 (hamster liver)
CES1B
CES1C
CES2 I HSiCE (human intestine) I hCE-2/HU3 (human liver/intestine) Rabbit2 (rabbit liver) ES-male (mouse liver) 46.5 kDa (human liver)
CES3 CES4
FIG. 3. Phylogenetic tree of the carboxylesterase superfamily using a simple unweighted pair-group method of analysis dendrogram. Hosokawa, 1998), and the majority of CarbEs that have been identified belong to the CES1 or CES2 families. Studies have shown that there are some differences between these families in terms of substrate specificity, tissue distribution, immunological properties, and gene regulation (Satoh and Hosokawa, 1998). For example, the preferential substrates for CES 1 (also called hCE1, hCE, or CES HU1) (Kroetz et al., 1993; Satoh and Hosokawa, 1998), a human CES1 family isozyme, are thought to be compounds esterified by small alcohol, whereas those for hCE-2, a human CES2 family isozyme, are thought to be compounds esterified by relatively large alcohol (Table 2). For drugs of abuse, heroin shows the highest rates of catalysis by both enzymes. CES 1, but not CES2, hydrolyzed the methyl ester of cocaine and the ethyl esters of meperidine and delapril (Kroetz et al., 1993; Pindel et al., 1997; Takai et al., 1997; Satoh and Hosokawa, 1998; Takayama et al., 1998; Zhang et al., 1999). In contrast to the specificity of CES 1 for the methyl ester of cocaine, only CES2 hydrolyzed the benzoyl ester of cocaine. For the remaining substrates that could be hydrolyzed by both enzymes, CES2 exhibited higher catalytic efficiency than CES1 for heroin; enzymatic conversion of 6-acetylmorphine to morphine was not known before the isolation and characterization of CES2 (Kamendulis et al., 1996). We reported that mouse MH1, a mouse CES 1 family isozyme, also hydrolyzed the temo-
capril, which esterified a small alcohol, similar to the human CES 1 isozyme (Moil et al., 1999, Fuilhata et al., 2004a). On the other hand, we also reported that rat rCES2 (also called CES RL4), a rat CES2 family isozyme, hydrolyzed methylprednisolone hemisuccinate, which esterified a large alcohol, similar to the human CES2 family (Furihata et al., 2005). It has been suggested that although these two CarbE families exhibit broad substrate specificity for ester, carbamate, or amide hydrolysis, these CarbE isozymes do exhibit distinct catalytic efficiencies that correlate with the relative size of the substrate substituents versus that of the enzyme active sites. Knowledge of these substrate structure-activity relationships and the tissue distribution of CarbE isoenzymes is critical for predicting the metabolism and pharmacokinetics and pharmacodynamics of ester drugs or prodrugs.
V. S T R U C T U R E A N D C A T A L Y T I C M E C H A N I S M OF CarbE I S O Z Y M E S It has been shown that several proteins of the ER lumen have the common carboxy-terminal sequence KDELCOOH, and the structural motif is essential for retention of the protein in the luminal site of the ER through KDEL receptor bound to the ER membrane (Pelham, 1990; Tang and Kalow, 1995).
CHAPTER 1 6 9Structure and Function of CarbEs TABLE 2. Substrate
223
Structure-Activity Relationship of Substrates with CESI and CES2 Families Alcohol substituent
Cocaine (methylester)
Acyl substituent
N~CH3 0
CH3OH
Substrate specificity
CES1
II
0
Meperidine
CH3CH2OH
CES1
N
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0
Temocapril
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CES1
CPT- 11
CES2 > > > CES 1
HO
Heroin
HO
CH3COOH
CES2 > > > CES 1
HOOCmCH2mCH2mCOONa
CES2 > > > CES 1
O CHz
HoJ'--..~ Methylprednisolone 21-hemisuccinate
Korza and Ozols (1988) and Ozols (1989) established the primary structures of two microsomal esterases purified from rabbit liver and designated them as 60-kDa esterase form 1 and form 2. These two forms of CarbE have the consensus sequence of the ER retention tetra-peptide (HTEL or HIEL in the one-letter code) which recognized with the luminal side of the KDEL receptor. Robbi et al. (1990) reported cDNA cloning of rat liver pI 6.1 esterase (ES-10) and pI 5.5 esterase (ES-3, egasyn). This was the
first report to show that cDNA of liver CarbE has the consensus sequence of the ER retention tetrapeptide (HVELCOOH). Later, Robbi and Beaufay (1994) isolated a cDNA clone of another rat liver pI 5.5 esterase (ES-3, egasyn), which has the consensus sequence of the ER retention tetrapeptide (HTEL-COOH). In the case of mouse liver microsomal CarbE, the carboxyl-terminal amino acid sequence of clone Es-N is HTEHK-COOH, which differs from the consensus sequence of the ER retention signal.
224
SECTION III. E s t e r a s e s ,
Receptors,
Mechanisms,
The other clone encoded egasyn, an accessory protein of [3-glucuronidase in the liver microsomes (Medda et al., 1987). Egasyn is identical to CarbE, and it binds [3-glucuronidase via its CarbE active site. Thus, it contains the consensus sequence of the ER retention signal (HTELCOOH). Ovnic et al. (1991a) conducted genetic mapping and confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Shibata et al. (1993) found that the human CarbE gene spans approximately 30 kilobases (kb) and has 14 small exons. Many CarbEs have a signal peptide of 17-20 amino acid residues, including hydrophobic amino acid, for retention in the lumen of the ER (Fig. 4). In general, a bulky aromatic residue followed by a small neutral residue directly precede the cleavage site (von Heijne, 1983). Many CarbEs have four cysteines that may be involved in specific disulfide bonds. Among them, Cys98 is the most highly conserved residue in many CES isozymes (Fig. 4). Cygler et al. (1993) reported an important alignment of a collection of related amino acid sequences of esterase, lipase, and related proteins based on X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase. According to these authors, Ser203, Glu336, and His450 form a catalytic triad, and Gly124Gly125 may be part of an oxyanion hole. These residues are also highly conserved among CarbE isozymes. Thus, we performed mutation analysis (Satoh and Hosokawa, 1998). Site-specific mutation of Ser203 to The203, Glu336 to Ala336, or His450 to Ala450 greatly reduced the CES activity toward substrates. Therefore, the mutagenesis confirmed the role for Glu336 and His450 in forming a putative charge relay system with the active site Ser203 (Satoh and Hosokawa, 1998). Frey et al. (1994) found that the formation of low-barrier hydrogen bonds between His and Asp (Glu for CarbE) facilitates the action of nucleophilic attack by the [3-OH group of Ser on the acyl carbonyl group of peptide in chymotrypsin. The catalytic triad in the tetrahedral addition intermediate is stabilized by the low-barrier hydrogen bonds. According to their theory, we thought the low barrier hydrogen bond between Glu336 and His450 facilitates the action of nucleophilic attack by [3-OH group of Ser203 on the carbonyl group of substrate in CarbE (Figs. 5 and 6). In this mechanism, the His450 removes a proton from the Ser203 in the transition state for its addition to the acyl carbonyl group of substrate. In the tetrahedral intermediate, the formation of low-barrier hydrogen bonds between His450 and Glu336 and the transition state is stabilized by the low-barrier hydrogen bonds (Figs. 5 and 6). The low-barrier hydrogen bonds facilitated mechanism includes weak hydrogen bonds between the tetrahedral oxyanion and peptide N-H bonds contributed by Gly123 and Gly124, which stabilize the tetrahedral adduct on the substrate side of the transition state. Formation of the acyl-enzyme complex in the next step requires the removal of a proton from His450 so that the tetrahedral
& Tolerance
Development
intermediate is disrupted in the acyl-enzyme intermediate (Fig. 5). When the unbound portion of the alcohol group of the first product of substrate has diffused away, a second step occurs in which the deacylation step is essentially the reverse of the acylation step, with the water molecule substituting for the alcohol group of the original substrate. To clarify the catalytic mechanism of CarbE, mutation analysis of other structural domains, such as the sites of salt bridges, the substrate binding site, and glycosylation would be worthwhile. It is of interest that the sequences required for .the hydrolytic capability at the catalytic triad (Glu, His, and Ser) of CarbE, acetylcholinesterase, butyrylcholinesterase, and cholesterolesterase are highly conserved. This is a common structure of serine hydrolase superfamilies, which are responsible for the hydrolysis of endogenous and exogenous compounds. Furthermore, these elements are strongly conserved among orthologous CarbEs from mouse, rat, rabbit, monkey, and human. A three-dimensional model for human CarbE has been proposed based on the crystal structure coordinates of acetylcholine esterase and overlapping active sites with pancreatic lipase and CarbE (Alam et al., 2002). The modeled structure shares the overall folding and topology of the proteins identified in the published crystal structures of the rabbit (Bencharit et al., 2002) and human CarbE (Bencharit e t al., 2003a,b). CarbE has a threedimensional oL,[3-hydrolase fold that is a structural feature of all lipases (Wong and Schotz, 2002). In general, the structure of CarbE may be viewed as comprising a central catalytic domain surrounded by et,[3 and regulatory domains (Bencharit et al., 2002, 2003a,b). In essence, the oL,[3-hydrolase fold consists of a central [3 sheet surrounded by a variable number of oLhelices and accommodates a catalytic triad composed of Ser, His, and a carboxylic acid. The residues that comprise the catalytic domain of human CES 1 are very highly conserved among orthologous CES 1 proteins from different species (Fig. 4). This suggests that the catalytic function of these proteins is conserved across species. The catalytic triad is located at the bottom of a deep active site cleft in the molecule and comprises a large flexible pocket on one side of Ser203 and a small rigid pocket on the opposite side. The orientation and location of the active site provide an ideal hydrophobic environment for the hydrolysis of a wide variety of hydrophobic substrates. The small rigid active site pocket is adjacent to the oxyanion hole formed by Gly123-Gly124 and is lined by several hydrophobic residues (Bencharit et al., 2003a). Short acyl chains would be easily accommodated within the small rigid pocket. The larger flexible active site pocket is lined by several nonpolar residues and could accommodate larger or polycyclic molecules such as cholesterol. The large pocket is adjacent to a side door secondary pore that would permit small molecules (substrates and reaction products) to enter and exit the active site (Bencharit et al., 2003a). Longer acyl chains may be oriented for catalysis in
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CHAPTER 1 6 9Structure and Function of CarbEs
227
FIG. 6. A proposed mechanism of the formation of the tetrahedral intermediate. (A), Hydrolysis of substrate start with an attack by the oxygen atom of the hydroxy group of Ser203 on the carbonyl carbon atom of the substrate. (B), The carbon-oxygen bond of this carbonyl group becomes a single bond, and the oxygen atom acquires a net negative charge. The four atoms now bonded to the carbonyl carbon are arranged as in a tetrahedron. The formation of this transient tetrahedral intermediate from a substrate is made possibility by hydrogen bonds between the negative charged oxygen onion (called an oxyanion, and main-chain NH group (Fig. 5 and Fig. 6)). This site is called oxyanion hole.
such a way that they extend through the side door. Indeed, the presence of a hydrophobic residue at position 423 in mouse and 425 in human is necessary for efficient hydrolysis of hydrophobic substrates since mutation of Met present in position 423 of the related rat lung CarbE to lie increased the CarbE activity toward a more hydrophobic substrate without affecting activity toward short-chain esters (Wallace et al., 1999). According to the X-ray crystal structure of the human CES 1, this residue lines the flexible pocket adjacent to the side door (Bencharit et al., 2003a). Given the wide range of substrates that CarbEs are known to hydrolyze, the large flexible pocket confers the ability to hydrolyze many structurally distinct compounds, whereas the rigid pocket is much more selective with regard tO the substrates that may be accommodated.
VI. I N D U C T I O N O F CarbEs Much interest has been shown by both clinicians and researchers in the induction of expression of drugmetabolizing enzymes by chemicals, including medical agents, since this is one of the main reasons for drug-drug interaction that causes adverse effects and for the reduction in pharmacological potencies of drugs. Regarding CarbEs, it has been shown that rodent CarbE isozyme(s) is induced by phenobarbital (Hosokawa et al.,
1987), aminopyrine (Hosokawa et al., 1988), and peroxisome proliferators [clofibrate, di(2-ethylhexyl)phthalate, and perfluorinated fatty acids] (Hosokawa et al., 1994; Howarth et al., 2001; Furihata et al., 2003, 2004a). The mouse is one of the most widely used experimental animals in the process of drug development, and several mouse CarbE isozymes have been identified (Hosokawa et al., 1990; Ovnic et al., 1991a,b; Aida et al., 1993; Satoh and Hosokawa, 1998; Dolinsky et al., 2001; Xie et al., 2003; Furihata et al., 2003 2004a, 2005). However, information on the involvement of mouse CarbEs in drug metabolism is limited. We have reported that exposure of C57BL/6 mice to di(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator, through their diet resulted in a significant increase in the amount of CarbE protein concomitant with an increase in the level of hydrolytic activity toward xenobiotics in mouse liver microsomes (Hosokawa et al., 1994). We have also shown that one of the mouse CarbE isozymes induced by DEHP is mCES2/microsomal acyl carnitine hydrolase, a CES2 family isozyme (Furihata et aL, 2003). Our immunochemical study also suggested that mouse CES 1 isozymes were induced by DEHP treatment, but they remain to be identified. Recently, we identified a mouse CES 1 isozyme, mCES1, that was induced by DEHP. Purification, cDNA cloning, and baculovirus-mediated expression of mCES1 revealed that mCES 1 plays an important role in temocapril metabolism and that it belongs to the CES1A subfamily.
228
SECTION III. E s t e r a s e s ,
Receptors,
Collectively, our results show that mCES 1 is very similar to hCE-1. Therefore, mCES 1 is thought to be one of the critical determinants for pharmacokinetics and pharmacodynamic actions of ester prodrugs as well as ester drugs. This work provides Useful information for the study of metabolism and dispositions of ester prodrugs as well as ester drugs. Zhu et al. (2000) reported that dexamethasone caused a slight increase in human CES isozymes. Among the inducers, dexamethasone possesses a potent and interesting ability to affect CarbE expression in the rat liver. Hattori et al. (1992) reported that methylprednisolone hemisuccinate (MPHS) was hydrolyzed to methylprednisolone via CarbE in rat liver microsomes and that several clinically used glucocorticoids, including dexamethasone, caused a remarkable increase in the level of this hydrolytic activity. In contrast to the report of induction of CarbE activity, some researchers have shown that the level of microsomal p-nitrophenylacetate hydrolase activity was significantly decreased in rat liver microsomes. This apparent contradiction in the same animal is probably due to the different methods for determination of CarbE activity by different CarbE substrate. Therefore, it is hypothesized that the CarbE isozyme contributing to p-nitrophenylacetate hydrolysis in rat liver microsome is different from the one contributing to MPHS hydrolysis. It has been reported that dexamethasone decreased the levels of expression of rat CES1 isozymes (CES RH1, also known as ES-10 or hydrolase A, and CES RL1, also known as ES-4 or hydrolase B) in the rat liver and that the decrease in the expression levels of these enzymes was linked to the reduction in the level of p-nitrophenylacetate hydrolase activity (Furihata et al., 2005). On the other hand, rat CarbE isozymes responsible for MPHS hydrolysis in rat liver microsomes have not been identified. However, we identified a dexamethasone-induced CarbE isozyme that can hydrolyze MPHS in the rat liver and plasma as a member of the CES2 family, rCES2 (Furihata et al., 2005). The different biochemical properties of rCES2 from those of known rat CES1 isozymes, including its unique existence in plasma, will be useful for studies aimed at elucidating the functions of CarbEs in drug metabolism. In addition, we also identified the gene encoding rCES2 by cDNA cloning and functional expression in Sf9 cells. Since we demonstrated that the level of the corresponding mRNA expression was markedly increased, the identification of the coding gene is valuable for studies aimed at elucidating the molecular mechanisms by which dexamethasone induces rCES2 expression (Furihata et al., 2005).
VII. GENE STRUCTURE AND
REGULATION OF CarbE ISOZYMES Both the murine (Satoh and Hosokawa, 1998) and the human (Shibata et al., 1993; Langmann et al., 1997; Satoh et al., 2002) CES1 genes span approximately 30 kb and contain 14
Mechanisms,
& Tolerance Development
small exons. Recently, sequencing of the mouse and human genomes was completed, enabling detailed sequence comparisons. The previously published sequences of the individual exons, splice junctions, size of the introns, and restriction sites within the murine and human CarbE genes are consistent with theft respective genes sequenced by the mouse and human genome projects. Therefore, the organization of the CarbE gene is evolutionarily conserved in mice and humans. Previous studies have mapped the human carboxylesterase gene to chromosome 16 at 16q13-q22.1 (Zschunke et al., 1991; Kroetz et al., 1993). This region is syntenic to a region of mouse chromosome 8 at 8C5 (Zschunke et al., 1991). The murine CarbEs Es22 (Ovnic et al., 199 l a) and Es 1 (Ovnic et al., 1991b) have previously been mapped to chromosome 8. The completion of the mouse genome sequencing project unambiguously demonstrated that the murine CarbE gene was located on the minus strand of chromosome 8 at 8C5 in a cluster of six CarbE genes that spans 260.6 kb in total. These six CarbE genes are presumed to have originated from repeated gene duplications of a common ancestral gene that encoded a CarbE (Shibata et al., 1993) and subsequent evolutionary divergence occurred. Recent studies have shown that there are some differences between these families in terms of substrate specificity, tissue distribution, immunological properties, and gene regulation. Therefore, the 5'-flanking regions of the CES1 and CES2 genes were isolated from mouse, rat, and human genomic DNAs by polymerase chain reaction amplification. Two individual mouse CES genes (mCES MH1 and m C E S ML1) (Furihata et al., 2004a) and two individual human CarbE genes (CES H U l a and H U l b ) were found to belong to the CES1 family, and mouse m C E S 2 (Furihata et al., 2003), rat rCES2 (Furihata et al., 2004a), and human CES H U 3 genes were found to belong to the CES2 family. A TATA box does not precede the transcription start site of any of the CarbE promoters. CarbE promoters share several common binding sites for transcription factors among the same CarbE families, suggesting that orthologous CarbE genes have evolutionally conserved transcriptional regulatory patterns. Potential binding sites of CarbE promoters for transcriptional factors include Spl, Sp3, C/EBE USF1, NF-1, NF-KB, PPAR-~/, GR, SREBE HNF1, HNF3, and HNF4. In the case of human CES1 genes, we isolated two CarbE genes encoding human CES HU1, which were tentatively designated as CES H U l a and CES H U l b (Fig. 7). These genes are identical except for exon 1 and cis elements. Electrophoretic mobility shift assays and reporter gene assays demonstrated that SP1, C/EBE and NF-1 could bind to each responsive element of the CES H U l a promoter but that C/EBP could not bind to responsive elements of the CES H U l b promoter. On the other hand, the structure of the CES2 gene promoter was different from that of the CES1 gene promoter (Fig. 7). We have shown tissue expression profiles of mCES2 and parts of the mechanism by which transcription of the mCES2 gene is regulated (Furihata et al.,
CHAPTER 1 6 9Structure and Function of CarbEs
229
FIG. 7. Gene structure and 5' regulatory element of CES HUla (CESIA), CES HUlb (CESIA), and CES HU3 (CES2A) genes.
2004b). mCES2 is expressed in the liver, kidney, small intestine, brain, thymus, lung, adipose tissue, and testis. We have also shown that Spl, Sp3, and USF1 contribute to synergistic transactivation of the mCES2 promoter. Although the possibility of involvement of other transcription factors in the regulation of mCES2 gene expression cannot be ruled out and further studies are needed to elucidate the mechanism fully, our data indicate that Spl, Sp3, and USF1 are indispensable factors for transcription of the mCES2 gene. Our results have provided some clues for understanding the molecular mechanisms regulating mCES2 gene expression and represent an important step toward elucidation of physiological functions of mCES2 (Furihata et al., 2004b).
VIII. C O N C L U S I O N S AND F U T U R E DIRECTIONS Multiple CarbEs play an important role in the hydrolytic biotransformation of a vast number of structurally diverse drugs. These enzymes are a major determinant of the pharmacokinetic behavior of most therapeutic agents containing an ester or amide bond. There are several factors that influence CarbE activity, either directly or at the level of enzyme regulation. In the clinical field, drug elimination is decreased and the incidence of drug-drug interactions increases when two or more drugs compete for hydrolysis by the same CarbE isozyme. Exposure to environmental pollutants or to lipophilic drugs can result in induction of CarbE activity. Several drug-metabolizing enzymes, such as cytochrome P450, UGT and sulfotransferase have been extensively
studied to clarify the substrate specificity using molecular cloning and cell expersion system. Consequently, the novel findings obtained reveal that the substrate specificity of CarbE is, at least in part, explained by the differences in the nucleotide sequences of the individual CarbE isozymes. In addition, it is clear that membrane-bound type CarbE isozymes in microsomes are required to possess the KDEL tetrapeptide motif at the carboxy terminal of the molecule. Mammalian CarbE have been found to have acyl-glycerol, acyl-CoA, and acyl-carnitine hydrolyzing activities in vitro; however, physiological roles of CarbEs remain unclear. To clarify the substrate specificity of each CarbE isozymes, we have begun to study for search the substrate recognition site of each isozymes. In the future, we should be able to clarify the three-dimensional structure of CarbE by X-ray analysis and estimate the catalytic triad, oxyanion hole, salt bridge, substrate binding site, and so on. Also, we should be able to utilize in vivo experimental results to predict in vivo results. The substrate specificity of CarbE toward newly developed prodrugs under consideration may be examined using purified CarbE, mammalian cell expression systems and specific inhibitor. However, such in vitro experiments may not possible to predict in vivo results, except in particular cases. Therefore, we must obtain enough information for different pharmacokinetic parameters of prodrugs among mammalian species. We should clarify the inter-individual difference in human CarbE for the study of the prediction of pharmacodynamics. The expression levels of CarbE isozymes are extremely different in each liver. Thus, further investigations of the regulatory mechanism in CarbE may be able to clarify the cause of individual variation.
7_30
SECTION I I I . E s t e r a s e s , R e c e p t o r s , M e c h a n i s m s ,
References Aida, K., Moore, R., and Negishi, M. (1993). Cloning and nucleotide sequence of a novel, male-predominant carboxylesterase in mouse liver. Biochirr~ Biophys. Acta 1174, 72-74. Alam, M., Vance, D. E., and Lehner, R. (2002). Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: Identification of the catalytic triad and a glycosylation site. Biochemistry 41, 6679-6687. Aldridge, W. N. (1993). The esterases: Perspectives and problems. Chem.-Biol. Interact. 87, 5-13. Bencharit, S., Morton, C. L., Howard-Williams, E. L., Danks, M. K., Potter, E M., and Redinbo, M. R. (2002). Structural insights into CPT-11 activation by mammalian carboxylesterases. Nat. Struct. Biol. 9, 337-342. Bencharit, S., Morton, C. L., Hyatt, J. L., Kuhn, E, Danks, M. K., Potter, E M., and Redinbo, M. R. (2003a). Crystal structure of human carboxylesterase 1 complexed with the Alzheimer's drug tacrine: From binding promiscuity to selective inhibition. Chem. Biol. 10, 341-349. Bencharit, S., Morton, C. L., Xue, Y., Potter, E M., and Redinbo, M. R. (2003b). Structural basis of heroin and cocaine metabolism by a promiscuous human drug-processing enzyme. Nat. Struct. Biol. 10, 349-356. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Doctor, B. E (1993). Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins. Protein Sci. 2, 366-382. Dolinsky, V. W., Sipione, S., Lehner, R., and Vance, D. E. (2001). The cloning and expression of a murine triacylglycerol hydrolase cDNA and the structure of its corresponding gene. Biochim. Biophys. Acta 1532, 162-172. Frey, E A., Whitt, S. A., and Tobin, J. B. (1994). A low-barrier hydrogen bond in the catalytic triad of serine proteases. Science 264, 1927-1930. Furihata, T., Hosokawa, M., Nakata, E, Satoh, T., and Chiba, K. (2003). Purification, molecular cloning, and functional expression of inducible liver acylcarnitine hydrolase in C57BL/6 mouse, belonging to the carboxylesterase multigene family. Arch. Biochem. Biophys. 416, 101-109. Furihata, T., Hosokawa, M., Koyano, N., Nakamura, T., Satoh, T., and Chiba, K. (2004a). Identification of di-(2-ethylhexyl) phthalate-induced carboxylesterase 1 in C57BL/6 mouse liver microsomes: Purification, cDNA cloning, and baculovirusmediated expression. Identification of the cytosolic carboxylesterase catalyzing the 5'-deoxy-5-fluorocytidine formation from capecitabine in human liver. Drug Metab. Dispos. 32, 1170-1177. Furihata, T., Hosokawa, M., Satoh, T., and Chiba, K. (2004b). Synergistic role of specificity proteins and upstream stimulatory factor 1 in transactivation of the mouse carboxylesterase 2/microsomal acylcarnitine hydrolase gene promoter. Biochem. J. 384, 101-110. Furihata, T., Hosokawa, M., Fujii, A., Derbel, M., Satoh, T., and Chiba, K. (2005). Dexamethasone-induced methylprednisolone hemisuccinate hydrolase: Its identification as a member of the rat carboxylesterase 2 family and its unique existence in plasma. Biochem. Pharmacol., in press.
& Tolerance Development
Hattori, K., Igarashi, M., Itoh, M., Tomisawa, H., Ozawa, N., and Tateishi, M. (1992). Purification and characterization of glucocorticoid-inducible steroid esterase in rat hepatic microsomes. Specific induction by glucocorticoids of steroid esterase in rat hepatic microsomes and its release into serum. Biochem. Pharmacol. 43, 1929-1937. Hosokawa, M., Maki, T., and Satoh, T. (1987). Multiplicity and regulation of hepatic microsomal carboxylesterases in rats. Mol. Pharmacol. 31, 579-584. Hosokawa, M., Maki, T., and Satoh, T. (1988). Differences in the induction of carboxylesterase isozymes in rat liver microsomes by xenobiotics. Biochem. Pharmacol. 37, 2708-2711. Hosokawa, M., Maki, T., and Satoh, T. (1990). Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277, 219-227. Hosokawa, M., Hirata, K., Nakata, E, Suga, T., and Satoh, T. (1994). Species differences in the induction of hepatic microsomal carboxylesterases caused by dietary exposure to di(2ethylhexyl)phthalate, a peroxisome proliferator. Drug Metab. Dispos. 22, 889-894. Howarth, J. A., Price, S. C., Dobrota, M., Kentish, E A., and Hinton, R. H. (2001). Effects on male rats of di-(2-ethylhexyl) phthalate and di-n-hexylphthalate administered alone or in combination. Toxicol. Lett. 121, 35-43. Imai, T., Yoshigae, Y., Hosokawa, M., Chiba, K., and Otagiri, M. (2003). Evidence for the involvement of a pulmonary first-pass effect via carboxylesterase in the disposition of a propranolol ester derivative after intravenous administration. J. Pharmacol. Exp. Ther. 307, 1234-1242. Inoue, M., Morikawa, M., Tsuboi, M., and Sugiura, M. (1979). Species difference and characterization of intestinal esterase on the hydrolizing activity of ester-type drugs. Jpn. J. Pharmacol. 29, 9-16. Kamendulis, L. M., Brzezinski, M. R., Pindel, E. V., Bosron, W. E, and Dean, R. A. (1996). Metabolism of cocaine and heroin is catalyzed by the same human liver carboxylesterases. J. Pharmacol. Exp. Ther. 279, 713-717. Korza, G., and Ozols, J. (1988). Complete covalent structure of 60-kDa esterase isolated from 2,3,7,8-tetrachlorodibenzo-pdioxin-induced rabbit liver. J. Biol. Chem. 263, 3486-3495. Kroetz, D. L., McBride, O. W., and Gonzalez, E J. (1993). Glycosylation-dependent activity of baculovirus-expressed human liver carboxylesterases: cDNA cloning and characterization of two highly similar enzyme forms. Biochemistry 32, 11606-11617. Kusano, K., Seko, T., Tanaka, S., Shikata, Y., Ando, T., Ida, S., Hosokawa, M., Satoh, T., Yuzuriha, T., and Horie, T. (1996). Purification and characterization of monkey liver amidohydrolases and its relationship to a metabolic polymorphism of E6123, a platelet activating factor receptor antagonist. Drug Metab. Dispos., in press. Langmann, T., Becker, A., Aslanidis, C., Notka, E, Ullrich, H., Schwer, H., and Schmitz, G. (1997). Structural organization and characterization of the promoter region of a human carboxylesterase gene. Biochim. Biophys. Acta 1350, 65-74. Maki, T., Hosokawa, M., Satoh, T., and Sato, K. (1991). Changes in carboxylesterase isoenzymes of rat liver microsomes during hepatocarcinogenesis. Jpn. J. Cancer Res. 82, 800-806.
CHAPTER 1 6 9Structure and Function of CarbEs Medda, S., Takeuchi, K., Devore-Carter, D., von Deimling, O., Heymann, E., and Swank, R. T. (1987). An accessory protein identical to mouse egasyn is complexed with rat microsomal beta-glucuronidase and is identical to rat esterase-3. J. Biol. Chem. 262, 7248-7253. Mentlein, R., Suttorp, M., and Heymann, E. (1984). Specificity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase, palmitoyl-carnitine hydrolase, and nonspecific carboxylesterase from rat liver microsomes. Arch. Biochem. Biophys. 228, 230-246. Moil, M., Hosokawa, M., Ogasawara, Y., Tsukada, E., and Chiba, K. (1999). cDNA cloning, characterization and stable expression of novel human brain carboxylesterase. FEBS Lett. 458, 17-22. Ohtsuka, K., Inoue, S., Kameyama, M., Kanetoshi, A., Fujimoto, T., Takaoka, K., Araya, Y., and Shida, A. (2003). Intracellular conversion of irinotecan to its active form, SN-38, by native carboxylesterase in human non-small cell lung cancer. Lung Cancer 41, 187-198. Ovnic, M., Swank, R. T., Fletcher, C., Zhen, L., Novak, E. K., Baumann, H., Heintz, N., and Ganschow, R. E. (1991a). Characterization and functional expression of a cDNA encoding egasyn (esterase-22): The endoplasmic reticulum-targeting protein of beta-glucuronidase. Genomics 11, 956-967. Ovnic, M., Tepperman, K., Medda, S., Elliott, R. W., Stephenson, D. A., Grant, S. G., and Ganschow, R. E. (199 l b). Characterization of a murine cDNA encoding a member of the carboxylesterase multigene family. Genomics 9, 344-354. Ozols, J. (1989). Isolation, properties, and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase. J. Biol. Chem. 264, 12533-12545. Pelham, H. R. (1990). The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem. Sci. 15, 483-486. Pindel, E. V., Kedishvili, N. Y., Abraham, T. L., Brzezinski, M. R., Zhang, J., Dean, R. A., and Bosron, W. E (1997). Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin. J. Biol. Chem. 272, 14769-14775. Prueksaritanont, T., Gorham, L. M., Hochman, J. H., Tran, L. O., and Vyas, K. P. (1996). Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells. Drug Metab. Dispos. 24, 634-642. Robbi, M., and Beaufay, H. (1994). Cloning and sequencing of rat liver carboxylesterase ES-3 (egasyn). Biochem. Biophys. Res. Commun. 203, 1404-1411. Robbi, M., Beaufay, H., and Octave, J. N. (1990) Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum. Biochem. J. 269, 451-458. Sanghani, S. P., Quinney, S. K., Fredenburg, T. B., Sun, Z., Davis, W. I., Murry, D. J., Cummings, O. W., Seitz, D. E., and Bosron, W. E (2003). Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis. Clin. Cancer Res. 9, 4983-4991. Satoh, T., and Hosokawa, M. (1998). The mammalian carboxylesterases: From molecules to functions. Annu. Rev. Pharmacol. Toxicol. 38, 257-288.
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Satoh, T., Hosokawa, M., Atsumi, R., Suzuki, W., Hakusui, H., and Nagai, E. (1994). Metabolic activation of CPT-11, 7-ethyl-10-[4(1-piperidino)-l-piperidino]carbonyloxycamptothecin, a novel antitumor agent, by carboxylesterase. Biol. Pharm. Bull. 17, 662-664. Satoh, T., Taylor, P., Bosron, W. E, Sanghani, S. P., Hosokawa, M., and La Du, B. N. (2002). Current progress on esterases: From molecular structure to function. Drug Metab. Dispos. 30, 488-493. Shibata, E, Takagi, Y., Kitajima, M., Kuroda, T., and Omura, T. (1993). Molecular cloning and characterization of a human carboxylesterase gene. Genomics 17, 76-82. Tabata, T., Katoh, M., Tokudome, S., Nakajima, M., and Yokoi, T. (2004) Identification of the cytosolic carboxylesterase catalyzing the 5'-deoxy-5-fluorocytidine formation from capecitabine in human liver. Drug Metab. Dispos. 32, 1103-1110. Takai, S., Matsuda, A., Usami, Y., Adachi, T., Sugiyama, T., Katagiri, Y., Tatematsu, M., and Hirano, K. (1997). Hydrolytic profile for ester- or amide-linkage by carboxylesterases pI 5.3 and 4.5 from human liver. Biol. Pharm. Bull. 20, 869-873. Takayama, H., Watanabe, A., Hosokawa, M., Chiba, K., Satoh, T., and Aimi, N. (1998). Synthesis of a new class of camptothecin derivatives, the long-chain fatty acid esters of 10-hydroxycamptothecin, as a potent prodrug candidate, and their in vitro metabolic conversion by carboxylesterases. Bioorg. Med. Chem. Lett. 8, 415-4 18. Tang, B. K., and Kalow, W. (1995). Variable activation of lovastatin by hydrolytic enzymes in human plasma and liver. 4. Eur. J. Clin. Pharmacol. 47, 449-451. von Heijne, G. (1983). Patterns of amino acids near signalsequence cleavage sites. Eur. J. Biochem. 133, 17-21. Wallace, T. J., Ghosh, S., and McLean Grogan, W. (1999). Molecular cloning and expression of rat lung carboxylesterase and its potential role in the detoxification of organophosphorus compounds. Am. J. Respir. Cell. Mol. Biol. 20, 1201-1208. Watanabe, K., Kayano, Y., Matsunaga, T., Yamamoto, I., and Yoshimura, H. (1993). Purification and characterization of a novel 46.5-kilodalton esterase from mouse hepatic microsomes. Biochem. Mol. Biol. Int. 31, 25-30. Wong, H., and Schotz, M. C. (2002). The lipase gene family. J. Lipid Res. 43, 993-999. Xie, M., Yang, D., Wu, M., Xue, B., and Yan, B. (2003). Mouse liver and kidney carboxylesterase (M-LK) rapidly hydrolyzes antitumor prodrug irinotecan and the N-terminal three quarter sequence determines substrate selectivity. Drug, Metab. Dispos. 31,21-27. Zhang, J., Burnell, J. C., Dumaual, N., and Bosron, W. F. (1999). Binding and hydrolysis of meperidine by human liver carboxylesterase laCE-1. J. Pharmacol. Exp. Ther. 290, 314-318. Zhu, W., Song, L., Zhang, H., Matoney, L., LeCluyse, E., and Yan, B. (2000). Dexamethasone differentially regulates expression of carboxylesterase genes in humans and rats. Drug Metab. Dispos. 28, 186-191. Zschunke, F., Salmassi, A., Kreipe, H., Buck, F., Parwaresch, M. R., and Radzun, H. J. (1991). cDNA cloning and characterization of human monocyte/macrophage sefine esterase-1. Blood 78, 506-512.
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CHAPTER
17
Noncholinesterase Mechanisms of Central and Peripheral Neurotoxicity: Muscarinic Receptors and Other Targets DAVID A. JETT1 AND PAMELA J. LEIN2 1National Institutes of Health, Bethesda, MD 2Oregon Health & Science University, Portland, OR
the brain (McDaniel and Moser, 2004). Perhaps some of the strongest evidence has been presented with AChE null mice in which cholinergic signs and lethality were comparable to those in wild-type mice (Duysen et al., 2001). Certain OP and CM pesticides interact directly with cholinergic receptors and alter their function. Importantly, these effects can occur at concentrations below those required to inhibit ACHE. Moreover, some compounds at extremely low doses may bypass the receptor altogether and target molecules that are involved in critical cellular functions. This chapter discusses the role of AChE inhibition in the toxicity of OP and CM pesticides, with particular emphasis on lower doses that are sublethal but induce changes in cholinergic receptors either by indirect modulation of ACh levels via AChE inhibition or by direct interaction with the receptor. Finally, case studies are presented that suggest that some of the toxicities associated with OP insecticides may not involve AChE or cholinergic receptors at all.
I. I N T R O D U C T I O N Organophosphorus (OP) and carbamate (CM) pesticides inhibit the catalytic function of acetylcholinesterase (ACHE; EC 3.1.1.7) by phosphorylating or carbamylating the esteratic site of the enzyme (Taylor, 1990). This effectively diminishes the capacity of the enzyme to catalyze its endogenous substrate acetylcholine (ACh). As a consequence, the hydrolysis of ACh is prevented, leading to accumulation of ACh in the synaptic cleft and overstimulation and subsequent desensitization of muscarinic and nicotinic ACh receptors. Acute poisonings are treated with atropine to inhibit central muscarinic effects, and an oxime, usually pralidoxime, is used to reactivate AChE. The consequences of the desensitization of cholinergic receptors by lethal exposures to OP and CM pesticides include respiratory failure and death; however, at sublethal doses that inhibit ACHE, exposure to these compounds may induce a compensatory downregulation of muscarinic receptors that allow for tolerance to some, but not all, of the toxic effects (Costa et al., 1982). In mammalian systems, it is believed that the acute toxicity of exposure to higher levels of OP and CM pesticides is derived from this anti-AChE activity and the subsequent parasympathomimetic effect. There is a general consensus that AChE inhibition represents a common mechanism of toxicity for OP pesticides (Mileson et al., 1998), and that unique affinities for other molecular targets in addition to AChE inhibition account for the range of toxicities among different OP compounds (Pope, 1999). However, it has become clear that the role of AChE inhibition in mediating toxicity following moderate and chronic low-level exposures to OP and CM pesticides is less certain, as suggested in subsequent sections of this chapter and by the lack of a good correlation between certain behavioral effects and the magnitude and regional selectivity of AChE inhibition in Toxicology of Organophosphate and Carbamate Compounds
II. C O R R E L A T I O N B E T W E E N CHOLINESTERASE INHIBITION AND NEUROTOXICITY The neurotoxicity of OPs has been extensively documented in accidental human poisonings, epidemiological studies, and animal models. From these studies, it is clear that OPs can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and host factors that influence susceptibility and sensitivity. These effects include acute cholinergic toxicity, a delayed ataxia known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), chronic neurotoxicity, and developmental neurotoxicity. As discussed in the following paragraphs, there is increasing evidence that OPs target molecules in 233
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S ECTI O N I II 9Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
addition to or other than AChE to give rise to this spectrum of neurotoxic effects. Most OP compounds cause acute toxicity following exposure to higher levels and this is believed to derive predominantly from AChE inhibition and the subsequent overstimulation of nicotinic and muscarinic receptors (Ecobichon and Joy, 1995). Thus, the symptoms of acute OP neurotoxicity, which include autonomic dysfunction (e.g., miosis and excessive secretions of the airways, excretory systems, salivary glands, and lacrimal glands), involuntary movements (tremors and convulsions), muscle fasciculations, and ultimately respiratory depression, are consistent with the initial stimulation and subsequent paralysis of cholinergic transmission that are observed following AChE inhibition, and they can be mostly reversed upon administration of agents that antagonize cholinergic receptors or reverse AChE inhibition (Ecobichon and Joy, 1995). However, several lines of evidence argue strongly for the involvement of molecular targets in addition to or other than AChE in acute OP neurotoxicity. This was first suggested by comparisons of the in vitro anticholinesterase potency of various OPs with their published LD50 values, which revealed a poor correlation between these parameters (Chambers, 1992). Thus, of six commonly used OP pesticides, methyl chlorpyrifos had the lowest acute toxicity as measured by the LD50 (-3 g/kg), yet its oxon was the most potent anticholinesterase in vitro. Although it is possible that the different ratios between in vitro and in vivo potencies of these OPs simply reflect differential rates of activation and deactivation in vivo, this seems unlikely since subsequent comparative in vivo neurotoxicity studies reported that different OPs gave rise to different toxicological profiles, even at doses that caused comparable AChE inhibition (Liu et al., 1999; McDaniel and Moser, 2004; Sheets et al., 1997). Perhaps the most convincing evidence for targets other than AChE in acute OP toxicity comes from studies of the AChE knockout mouse (Duysen et al., 2001; Li et al., 2000). Targeted deletion of four axons of the AChE gene reduces AChE expression by half in heterozygous (AChE +/-) mice and eliminates it entirely in nullizygous (AChE - / - ) mice. If AChE is the critical molecular target in OP acute neurotoxicity, then it might be predicted that neurotoxic effects following acute exposure to OPs would be attenuated or absent in mice nullizygous for the AChE gene relative to their wild-type littermates. However, experiments in which the OP nerve agent VX was administered to these mice subcutaneously revealed just the opposite: The LD50 value was 10-12 Ixg/kg in nullizygous, 17 Ixg&g in heterozygous, and 24 Ixg/kg in wild-type AChE mice (Duysen et al., 2001). Interestingly, the same cholinergic signs of toxicity were present in AChE - / - mice as in wild-type mice; however, wild-type, but not AChE - / - , mice were protected by pretreatment with atropine. This phenomenon is not unique to VX since similar observations were made following exposure of these mice to DFP (Li et al., 2000). These data, together with the comparative toxicity studies, strongly suggest that
mechanisms not mediated by AChE inhibition must be contributing to the acute neurotoxic effects of OPs. Another action of at least some OPs, which follows either single or repeated exposures, is a delayed effect known as OPIDN (Abou-Donia and Lapadula, 1990). OPIDN is characterized by a delayed onset of ataxia accompanied by a Wallerian-type degeneration of the axon and myelin in the most distal portion of the longest axon tracts in both the central nervous system (CNS) and the peripheral nervous system (PNS) (Cavanagh and Patangia, 1965). Early studies on the mechanisms of OPIDN focused on inhibition of AChE or butyrylcholinesterase by OPs; however, subsequent studies eliminated both esterases as targets for OPIDN (Aldridge and Barnes, 1966). Neurotoxic esterase (NTE) has since been proposed to be a critical molecular target in OPIDN because OP compounds that cause OPIDN preferentially inhibit its enzymatic activity (Johnson, 1969). However, inhibition of NTE is not sufficient for axonal degeneration (Abou-Donia, 2003; Abou-Donia and Lapadula, 1990), and reports that OPs exert neurotoxic effects in NTE knockout mice indicate that targets other than or in addition to NTE mediate OPIDN (Glynn, 2003). Potential alternate molecular targets include calcium/calmodulin-dependent protein kinase II (CaM kinase II). The evidence supporting this hypothesis, which has been reviewed by Abou-Donia (2003), includes observations that aberrant phosphorylation of cytoskeletal proteins is present in OPIDN and may be causally related to the OP-induced axonal degeneration and demyelination, and that CaM kinase II, which phosphorylates cytoskeletal proteins, is activated by OPs that cause OPIDN. There is increasing evidence that OPs may also cause a long-term, persistent chronic neurotoxicity following either a single acute high-dose exposure or repeated exposures to low-level, subclinical doses of OPs. The clinical and epidemiological data in support of OP-induced chronic neurotoxicity that is distinct from both cholinergic and OPIDN effects have been reviewed (Abou-Donia, 2003; Kamel and Hoppin, 2004; Ray and Richards, 2001) and are not discussed here. Chronic OP neurotoxicity presents with pathological lesions in both the PNS and CNS, but it is the latter that is primarily responsible for the presenting neurologic symptoms and changes in neurobehavioral performance, reflecting cognitive and psychomotor dysfunction. The most sensitive manifestation of chronic OP neurotoxicity is a general malaise lacking in specificity and related to mild cognitive dysfunction, similar to that described for Gulf War syndrome (Kamel and Hoppin, 2004). The mechanisms underlying these effects are not known, and the role of AChE inhibition is controversial (Abou-Donia, 2003; Kamel and Hoppin, 2004) and may vary depending on the exposure parameters. Chronic neurotoxicity subsequent to a single acute exposure to OPs may be triggered by AChE inhibition. It has been hypothesized that increased ACh levels following AChE inhibition activate glutamatergic neurons causing the release of glutamate, which ultimately results in excitotoxicity
CHAPTER 1 7 9Muscarinic Receptors and OP Neurotoxicity
via increased intracellular calcium and activation of nitric oxide synthase following NMDA receptor activation (de Groot et aL, 2001). The resultant necrosis in affected brain regions may thus contribute to the persistent neurologic and neurobehavioral deficits. There is support for this proposed mechanism of chronic OP neurotoxicity in that it has been demonstrated that nitric oxide synthesis inhibitors block OP-induced seizures (Aschner et al., 1999). However, the role of AChE inhibition in this sequence of events has yet to be established. In contrast, chronic OP neurotoxicity induced by repeated exposures to subclinical OP doses has been reported to occur in the absence of AChE inhibition (Abou-Donia, 2003; Kamel and Hoppin, 2004), suggesting that mechanisms other than anticholinesterase activity mediate the neurotoxic effects elicited by this exposure scenario. However, what these mechanisms are has yet to be established. A review of data relevant to this question suggests the intriguing possibility that repeated exposures to sublethal or subclinical doses of OPs increases apoptotic neuronal death via oxidative stress (Abou-Donia, 2003). Whether OP-induced apoptosis gives rise to long-term neurological and neurobehavioral deficits remains to be directly demonstrated. Another important neurotoxic sequel of OP exposure is developmental neurotoxicity. Since OP d e v e l o p m e n t a l neurotoxicity in both humans (Garry, 2004) and animal models (Izrael et aL, 2004) has been reviewed, we discuss only selected case studies to illustrate the conclusion that this neurotoxic effect of OPs can also occur independent of AChE inhibition. Initial studies indicated that the developing nervous system is far more sensitive to the acute cholinergic toxicity of OPs (Bushnell et al., 1991; Pope and Chakraborti, 1992), most likely due to age-related differences in the hepatic detoxification of the AChE-active metabolites of OPs (Atterberry et al., 1997; Benke and Murphy, 1975; Mortensen et al., 1996). However, subsequent studies
TABLE 1. Exposure period
235
suggested that OPs could also cause neurodevelopmental defects independent of AChE inhibition. Slotkin and colleagues reported decreased cell division and DNA and protein synthesis in the brains of animals treated with OPs and concluded that these effects are not mediated by AChE inhibition because they occur at doses that do not cause systemic cholinergic toxicity, and they are not reversed by cholinergic receptor antagonists (Campbell et al., 1997; Garcia et al., 2001; Johnson et al., 1998; Whitney et al., 1995). Jett et al. (2001) and Levin et al. (2002) demonstrated that exposure of developing rats to low levels of chlorpyrifos also impairs their cognitive function. We demonstrated that spatial learning in the Morris water maze was impaired in weanling rats exposed to chlorpyrifos during development (Table 1), and this cognitive deficit occurred in the absence of a significant effect on AChE or the downregulation of cholinergic receptors that typically accompanies AChE inhibition (Jett et al., 2001). Whether this OP interfered with neural development leading to cognitive impairment in the weanling rats is unknown. However, these in vivo studies are in agreement with in vitro studies indicating, for example, that chlorpyrifos inhibits axon outgrowth in primary neuronal cell cultures (Howard et al., 2005) or PC12 cells (Das and Barone, 1999) independent of AChE inhibition. The evidence indicates that although there is a correlation between AChE inhibition and the prevalence and/or severity of OP neurotoxicity, the spectrum of toxic effects elicited by any given OP cannot be attributed entirely to the inhibition of ACHE. If this is true, then OPs must react not only with AChE but also with other proteins, and it has been suggested that different OPs elicit different toxicological profiles because each interacts with a unique subset of molecular targets (Chiappa et al., 1995; Liu et al., 1999; Ray and Richards, 2001). In support of the hypothesis that OPs interact with proteins other than AChE, various OP
Performance in the Morris Water Maze in Chlorpyrifos-Exposed Weanling Ratsa Dosage (mg/kg)
N
Latency on last day of test (sec)
Time in training quadrant (sec) b
Swim speed (cm/sec) c
Preweaning
0 0.3 7.0
20 19 17
12.3 _+ 2.6 17.5 + 3.0 23.7 +_ 2.3*
13.8 _+ 1.2 12.2 +_ 1.0 7.7 _+ 1.0"
19.1 +_ 1.0 18.3 _+ 1.0 18.8 + 1.3
Postweaning
0 0.3 7.0
7 7 8
9.0 _+ 2.3 22.7 _+ 4.7* 22.3 _+ 4.4*
17.0 + 1.4 11.5 +_ 1.0" 12.2 _+ 1.4"
18.2 + 1.3 18.6 + 1.0 18.1 + 1.0
aRats in the preweaning group were injected before weaning on postnatal days 7, 11, and 16; rats in the postweaning group were injected after weaning on postnatal days 22 and 26. All rats were weaned on postnatal day 21 and tested from day 24 to day 28. Adapted from Jett et al. (2001). bin the probe test, rats were allowed to swim freely without the platform for 30 sec and the time spent in the quadrant of the pool that previously contained the platform during 5 days of prior training was recorded. CTotaldistance traveled during the probe test divided by 30 sec. *Value is different from control value at the 0.05 level of significance as determined by analysis of variance.
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S ECTI O N III 9Esterases, Receptors, M e c h a n i s m s , & Tolerance D e v e l o p m e n t
compounds have been shown to bond covalently to a wide variety of proteins, including other cholinesterases (Casida and Quistad, 2004); serine hydrolases such as serum, liver, and brain carboxylesterases (Ray and Richards, 2001); muscarinic and nicotinic receptors; the cannabinoid CB 1 receptor (Bomser et al., 2002); and albumin (Peeples et al., 2005). The ongoing challenge is to determine which of these molecular interactions is toxicologically significant.
III, DIRECT AND INDIRECT E F F E C T S O N M U S C A R I N I C RECEPTORS
A. Tolerance Mediated by Muscarinic Receptor Downregulation Virtually all cholinergic synapses can be affected by exposure to anticholinesterase compounds such as OPs and CMs. These include autonomic postganglionic parasympathetic nerve endings, sympathetic and parasympathetic ganglia, motor end plates of skeletal muscle, and, of course, various regions of the CNS. Hyperactivity at these synapses due to accumulation of ACh causes a variety of symptoms mediated by overstimulation of muscarinic and nicotinic receptors. If laboratory animals are pretreated with sublethal doses of OPs and then treated with a higher, more toxic dose of the same compound, they are able to tolerate this dose to a much greater extent than naive rats given the same toxic dose. This adaptive response is a subsensitivity phenomenon and has been termed "tolerance." Tolerance to anti-AChE insecticides was first described by Barnes and Denz (1951) when they noticed that rats survived a long-term feeding trial with the highly toxic parathion. Although there are several factors that determine the sensitivity of an organism, or even a specific tissue, to AChE inhibitors, it is widely believed that the compensatory mechanism for the development of tolerance to prolonged inhibition of AChE is the downregulation of muscarinic cholinergic receptors in response to the accumulation of ACh at the synapse. Two important observations led to the discovery that cholinergic receptors mediated tolerance to anti-AChE compounds. First was the observation that cross-tolerance occurred between OPs, CMs (Costa and Murphy, 1983b), and nicotinic (Costa and Murphy, 1983a) and muscarinic (Brodeur and Dubois, 1964; Costa et al., 1981; Schwab and Murphy, 1981) cholinergic agonists. Second was that attenuation of AChE inhibition did not correlate well with tolerance. In fact, AChE activity can remain significantly depressed throughout the exposure period after the development of tolerance (Chippendale et al., 1972; Sivam et al., 1983). Importantly, these data showed that the metabolic disposition or differences in target tissue distribution of the AChE inhibitor may not play as prominent a role in the development of tolerance. Since these early studies, decreases in muscarinic receptors have become recognized universally as a hallmark of
exposure to OPs. The evidence for CM pesticides is not as strong, partially due to the rapidly reversible inactivation of AChE by these compounds compared to OPs; however, some studies have shown that muscarinic receptors are regulated by exposure to CMs (Jones et al., 1998). Downregulation of muscarinic receptors has been demonstrated repeatedly in a variety of experimental paradigms with several OPs (Abdallah et al., 1992; Costa et al., 1982; Russell and Overstreet, 1987). We have shown that the degree of AChE inhibition correlates well with decreases in muscarinic receptors (Jett et al., 1993). Stamper et al. (1988) found that rats exposed to parathion during development did not show any signs of overt toxicity, possibly due to tolerance as indicated by decreases in AChE and muscarinic receptors. However, these rats exhibited spatial memory deficits in the radial arm and T-mazes. This was one of the first studies to suggest that "compensatory" changes in receptors may result in tolerance to some effects (e.g., lethal) but may in fact be an underlying mechanism for other more subtle effects such as memory impairment. In light of the prominent role these receptors play in cognitive function, it is not surprising that changes in the availability of muscarinic binding sites due to OP-induced downregulation have been associated with altered behavior. Muscarinic receptors are a well-characterized group of five subtypes (M1-M5) belonging to the seven-transmembrane family of receptors coupled to signal transduction systems. Generally, the M1, M3, and M5 subtypes couple to Gq/11 protein, which activates phospholipase C, and M2 and M4 to G~0, which inhibits adenylyl cyclase. These receptors are distributed widely throughout the nervous system and are involved in many central and peripheral functions. They are the target for many therapeutics as well, such as Alzheimer's disease drugs. Changes in these receptors as a result of exposure to anti-AChEs may be restricted to one or two subtypes, which may not be detected by nonselective methods of identification. We have observed that protein and mRNA of certain subtypes may be more affected by OP exposure than others (Jett et al., 1993, 1994), and other laboratories have corroborated these findings (Yagle and Costa, 1996) and pointed to the M2 subtype as being especially vulnerable to OPs (Bakry et al., 1988; Katz and Marquis, 1989; Silveira et al., 1990).
B. Cholinesterase-Independent Effects on Muscarinic Receptors In addition to their indirect effects on muscarinic receptors through AChE-mediated changes in ACh levels, many OP and CM pesticides can affect their expression and function directly (Bakry et al., 1988; Eldefrawi et al., 1992). Paraoxon, dichlorvos, and tetraethyl pyrophosphate (TEPP) were found to be noncompetitive antagonists of muscarinic receptors in bovine caudate nuclei labeled with [3H]quinuclidinyl benzilate ([3H]-QNB) at concentrations that had no effect on AChE activity (Volpe et al., 1985). The
CHAPTER 1 7 9Muscarinic Receptors and OP Neurotoxicity CMs physostigmine and neostigmine were also tested in this study, but only physostigmine both decreased the number of receptors and enhanced their affinity for [3H]-QNB. The OP chemical weapon VX and the therapeutic agent echothiophate were found to inhibit high-affinity [3H]cis-methyldioxolane ([3H]-CD) binding at concentrations comparable to those that inhibit AChE in rat brain and N1E115 neuroblastoma cells (Bakry et al., 1988). It was found that the active metabolite of the pesticide chlorpyrifos, chlorpyrifos-oxon, bound to muscarinic receptors in rat striatum identified with [3H]-CD noncompetitively with an IC50 value of approximately 22 nM and resulted in a covalent modification of the receptor (Huff et al., 1994). [3H]-CD binds to M2 receptors (Huff and Abou-Donia, 1994) with high affinity, and thus it was suggested that direct actions on a subset of muscarinic receptors, in addition to their actions on ACHE, could account for some of the toxicity of OP compounds. Inhibition of [3H]-CD binding by OPs was observed at nanomolar to micromolar concentrations, with Ko.5 values of 3, 10, 40, 100, and 800 nM for VX, soman, satin, echothiophate, and tabun, respectively (Bakry et al., 1988). Other studies confirmed that binding of ligands to muscarinic receptors is inhibited by OPs at concentrations far below those that inhibit ACHE, as low as the picomolar range (Katz and Marquis, 1989; Silveira et al., 1990). Functional studies examining the effects of OPs on signaling events downstream of muscarinic receptor activation further support the hypothesis that OPs can interact directly with M2 receptors. Activation of M2 and M4 receptors generally reduces the activity of adenylyl cyclase, which decreases cAMP production, whereas activation of M1, M3, or M5 receptors increases phosphoinositide-specific phospholipase C activity, which increases release of inositol triphosphate. A comparative study of paraoxon, malaoxon, and chlorpyrifosoxon in slice cultures of rat frontal cortex indicated that all three OPs inhibited cAMP formation in a concentrationdependent manner (Ward and Mundy, 1996). In contrast, none of these OPs affected either basal or carbachol-stimulated phosphoinositide turnover. These data suggest that OPs activate M2/M4 receptors through direct interactions and not as the result of increased levels of endogenous ACh consequent to ACHE inhibition. Other in vitro studies of rat striatum predicted and confirmed that, generally, OPs act to stimulate M2/M4 receptor function (Axelrad et aL, 2002; Huff and Abou-Donia, 1994; Jett et al., 1991), with the exception of apparent inhibitory effects of chlorpyrifos-oxon on prejunctional M2/M4 receptors (Axelrad et al., 2002).
C. Direct Action on Muscarinic Receptors in the Lung In the lung, many of the normal physiological processes that govern airway function are mediated by muscarinic receptors. Cholinergic nerves in the vagi regulate airway tone and reactivity. These nerves release ACh onto M3 muscarinic
237
receptors causing contraction of airway smooth muscle, resulting in bronchoconstriction, and vagally induced bronchoconstriction is limited by autoinhibitory M2 muscarinic receptors on parasympathetic nerves (Fryer and Maclagan, 1984; Minette and Barnes, 1988). Previous studies have shown that neuronal M2 receptors are dysfunctional in animal models of asthma (Fryer and Wills-Karp, 1991; Gambone et aL, 1994; Jacoby and Fryer, 1991) and in patients with asthma (Minette et al., 1989). Loss of M2 receptor function leads to increased release of ACh from parasympathetic nerves resulting in potentiation of vagally mediated bronchoconstriction, which contributes to airway hyperreactivity. In part, these highly critical functions mediated by the cholinergic system within the lung suggest that pesticide exposure may be a contributing factor underlying the increased incidence of childhood asthma in the United States and other industrialized nations. A number of clinical and epidemiological studies have linked exposure to OPs to airway hyperreactivity and other symptoms of asthma (Deschamps et al., 1994; Hoppin et al., 2002; O'Malley, 1997). Many of the current pesticides, especially those that children may come in contact with, are anticholinesterases that may have adverse effects on the cholinergic control of vagally induced bronchoconstriction. To test the hypothesis that asthma may be exacerbated by low-level exposure to OPs, airway hyperreactivity was measured in guinea pigs exposed to chlorpyrifos, a widely used OP pesticide (Fryer et al., 2004). Electrical stimulation of the vagus nerves caused frequency-dependent bronchoconstriction that was significantly potentiated in animals 24 hr or 7 days after a single subcutaneous injection of either 390 or 70 mg/kg chlorpyrifos, respectively (Fig. 1). Mechanisms by which chlorpyrifos may have caused airway hyperreactivity include inhibition of ACHE, dysfunction of M3 muscarinic receptors on airway smooth muscle, or inhibition of autoinhibitory M2 muscarinic receptors on parasympathetic nerves in the lung. AChE activity in the lung was significantly inhibited 24 hr after treatment with 390 mg/kg chlorpyrifos but not 7 days after injection of 70 mg/kg chlorpyrifos (Fig. 2). The observations that subchronic exposure to 70 mg/kg potentiated vagally induced bronchoconstriction in the absence of significant AChE inhibition and that acute exposure to eserine (250 Ixg/kg) significantly inhibited lung AChE but did not potentiate bronchoconstriction (Figs. 1 and 2) suggest that this effect on respiratory function was not mediated by AChE inhibition. Neuronal M2 receptor function was tested using the M2 agonist pilocarpine, which inhibits vagally induced bronchoconstriction in control animals. In chlorpyrifostreated animals, pilocarpine dose-response curves were shifted significantly to the fight, demonstrating decreased responsiveness of neuronal M2 receptors (Fig. 3). In contrast, chlorpyrifos treatment did not alter methacholineinduced bronchoconstriction, suggesting that chlorpyrifos does not alter M3 muscarinic receptor function on airway
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FIG. 3. Neuronal M2 receptor function was tested using pilocarpine. Increasing doses of pilocarpine inhibited vagally induced bronchoconstriction in a dose-related manner in animals 7 days after treatment with peanut oil vehicle (open circles), demonstrating the presence of functional M2 receptors. The effect of pilocarpine was shifted significantly to the fight in animals 7 days after treatment with 70mg/kg chlorpyrifos (CPF; triangles). In the animals treated with 390 mg/kg CPF (upsidedown triangles), pilocarpine did not inhibit vagally induced bronchoconstriction, indicating neuronal M2 muscarinic receptor dysfunction after 24 hr. Each point is the mean __ SEM of five animals. *Significantly different from control. Adapted from Fryer et al. (2004). smooth muscle. The effects of chlorpyrifos on M2 receptormediated airway hyperreactivity were later generalized to two other important OPs, parathion and diazinon, in this model system (Lein and Fryer, 2005). These data demonstrate that organophosphate insecticides can cause airway hyperreactivity in the absence of AChE inhibition by decreasing neuronal M2 receptor function. Observations that OPs generally activate M2 receptors in neural tissues (Axelrad et al., 2002; Huff and Abou-Donia, 1994; Jett et al., 199 l) are in contrast to our in vivo studies indicating that concentrations of parathion, chlorpyrifos, and diazinon that do not inhibit ACHE; they do, however, exert inhibitory effects on prejunctional M2 receptors on airway nerves (Lein and Fryer, 2005). A possible explanation is that agonistic effects of OPs on M2 receptors were observed using striatal and cortical slice cultures (Axelrad et al., 2002; Huff and Abou-Donia, 1994; Jett et al., 1991). Although there are M2 receptors on presynaptic nerves in both the striatum (Hersch et al., 1994) and the cortex (Levey et al., 1991), studies on knockout mice suggest that it is the M4, and not the M2, receptors that are functionally significant in inhibiting ACh release in these brain regions (Zhang et al., 2002). These observations raise the possibility that direct stimulation of muscarinic receptors by OPs in this tissue is mediated by M4 rather than M2 receptors. In contrast, M4 receptors are not expressed on parasympathetic nerves in the lung (Fryer et al., 1996). Furthermore, it
CHAPTER 1 7 9Muscarinic Receptors and OP Neurotoxicity is unlikely that OPs increase cAMP in the lungs because if this were the case, then OPs would mimic the effect of [3-agonists on airway smooth muscle, which also increase cAME leading to bronchodilation. The in vivo data of Lein and Fryer (2005) do not show either a reduction in airway tone or a decrease in acetylcholine-induced bronchoconstriction. Thus, is it unlikely that increased cAMP is contributing to airway tone in parathion-, chlorpyrifos-, and diazinon-exposed guinea pigs.
IV. I N T R A C E L L U L A R
SIGNALS
AND OTHER MOLECULAR TARGETS OF NEUROTOXICITY As indicated in the preceding discussion, there is evidence that OPs may directly modulate intracellular signaling pathways. We demonstrated that paraoxon inhibited the synthesis of cAMP in dissociated cells from the rat striatum and these effects were blocked by atropine, suggesting that it was mediated by the high-affinity interaction between paraoxon and an M2/M4 muscarinic receptor (Jett et al., 1991). However, we also found that paraoxon inhibited cAMP synthesis in cells dissociated from the rat submaxillary gland in an atropine-insensitive manner, suggesting that the response was not mediated by coupling to Gi/0 and a muscarinic receptor (Abdallah et al., 1992). In contrast, paraoxon effects on phosphoinositide turnover were minimal or completely absent in both submaxillary gland and striatum cells, demonstrating that this OP selectively targets cAMP signaling. These observations were later confirmed by other laboratories that demonstrated inhibitory effects of chlorpyrifos oxon, paraoxon, and malaoxon on cAMP synthesis, but not phosphoinositide turnover, in rat frontal cortex slices (Ward and Mundy, 1996). Chlorpyrifos-oxon was also found to inhibit cAMP synthesis in striatal dissociated cells (Huff et al., 1994) but in an atropine-insensitive manner, similar to our results with submaxillary gland cells. Chlorpyrifos-oxon was also shown to inhibit cAMP synthesis in NG108-15 cells and in Chinese hamster ovary (CHO) cells transfected with muscarinic receptor subtypes (Huff and Abou-Donia, 1995), but only at relatively high concentrations. Slotkin and colleagues (Song et al., 1997) suggested that cAMP signaling may also be a target for the developmental neurotoxicity of chlorpyrifos based on evidence that postnatal exposure in neonatal rats decreases adenylyl cyclase expression and function and alters cAMP levels under a variety of experimental manipulations. Conversely, prenatal exposure to chlorpyrilos produced regionally selective augmentation of adenylyl cyclase activity downstream of [3-adrenoreceptors (Meyer et al., 2003). Further studies indicated that the effects of both pre- and postnatal chlorpyrifos exposure on adenylyl cyclase signaling persist in the adult brain (Dresbach et al., 2004). Pope and colleagues similarly demonstrated that muscarinic receptor-mediated cAMP is modulated in the developing
239
brain by chlorpyrifos (Zhang et al., 2002), and that inhibitory effects of chlorpyrifos-oxon and paraoxon on forskolinstimulated cAMP formation in cortical slices from neonatal and juvenile animals vary across developmental stages and are only partially reduced by atropine (Olivier et al., 2001). Thus, there is a sufficient database demonstrating that OPs target the cAMP/PKA signaling pathway independent of effects on muscarinic receptors. Interestingly, the mechanism of action for the OP steroidogenesis inhibitor, diethylumbelliferyl phosphate, is also believed to be mediated through an interaction with the cAMP/PKA pathway (Choi et al., 1995). Although most studies have focused on OP modulation of the cAMP/PKA signaling pathway, there is evidence that OPs disrupt other major signaling pathways. As discussed previously, mechanistic studies of OPIDN suggest that OPs activate CaM kinase II (Abou-Donia, 2003). Studies of CHO (CHOK1) cells indicate that OPs may also activate extracellular signal-regulated kinase (ERK) signaling pathways (Bomser and Casida, 2000), possibly via increased levels of diacylglycerol (DAG) subsequent to OP inhibition of DAG lipase (Bomser et al., 2002). The significance of these observations to OP neurotoxicity is suggested by reports that chlorpyrifos induces apoptosis in cultured rat cortical neurons at concentrations that do not inhibit ACHE, and this effect can be blocked by pharmacological agents that inhibit ERK1/2 activation (Caughlan et al., 2004). Interestingly, this same study found simultaneous activation of ERK1/2 and p38 in cortical neurons treated with chlorpyrifos, but addition of p38 inhibitors augmented the proapoptotic effect of chlorpyrifos. It has been reported that chlorpyrifos also interferes with muscarinic receptormediated translocation of protein kinase C (PKC)-~/ and decreases the basal levels of both PKC-~/and PKC-[3II, the two isoforms known to be relevant to behavioral performance (Izrael et al., 2004). In summary, the experimental evidence supports the hypothesis that OPs modulate intracellular signaling pathways downstream of receptors and suggests that the diverse neurotoxic effects of many OPs may reflect their influence on multiple intracellular signaling pathways. Determining the specificity and sensitivity of effects on intracellular signals relative to defined neurotoxic end points remains a significant experimental goal in the field. The signaling pathways identified as potential targets in OP neurotoxicity can modulate gene expression via alterations in the expression levels or activational status of transcription factors. Thus, an important question is whether OPs can alter gene expression via effects on transcription factors. One transcription factor of considerable interest in OP neurotoxicity is Ca2+/cAMP response element binding protein (CREB), which is activated via phosphorylation by a variety of signaling pathways, including cAMP/PKA, MAP kinase/ERK, p38, and CaM kinase II (Lonze and Ginty, 2002). Numerous studies have indicated that CREB is critical to several forms of use-dependent synaptic plasticity and transcription-dependent
2 40
S ECTI O N I I I 9Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
forms of memory, and evidence supports a major role for CREB in cell survival and differentiation during brain development (reviewed by Lonze and Ginty, 2002; Shaywitz and Greenberg, 1999). Since impairments of brain development and memory function are two primary neurological effects observed in laboratory studies with OPs, we hypothesized that the mechanisms underlying these effects may include alteration of the expression or activational status of CREB. Western blot analyses of lysates from primary cultures of cortical neurons exposed to chlorpyrifos, chlorpyrifos-oxon (Fig. 4), or trichloropyridinol (TCP) indicated that all three OPs increased the level of the phosphorylated (activated) form of CREB (pCREB) without significantly changing levels of total CREB or oL-tubulin (Schuh et aL, 2002). Remarkably, pCREB in cortical neurons was elevated by 300-400% of control levels at extremely low concentrations (picomolar range for chlorpyrifos and TCP, and fentomolar range for the oxon metabolite). These OPs caused similar effects in primary cultures of hippocampal neurons but not in astrocytes. AChE activity and cell viability were not affected by OP concentrations that increased pCREB levels, and consistent with these observations, atropine did not block OP-induced phosphorylation of CREB, suggesting that the effects were occurring downstream of muscarinic receptors. The mechanism(s) by which OPs activate CREB is not known but is probably not mediated by OP effects on adenylyl cyclase activity, which are predominantly inhibitory. Chlorpyrifos and its oxon metabolite inhibit AChE by phosphorylation of the enzyme, and evidence that the oxon causes diethylphosphorylation of cardiac muscafinic receptors in vitro suggests the possibility that these OPs phosphorylate CREB directly (Bomser and Casida, 2001). However, direct phosphorylation of CREB cannot be the mechanism by which TCP induces increased pCREB because TCP does not contain a phosphorus atom. Possible mechanisms that have yet to be addressed experimentally include activation of CaM kinase II (Abou-Donia, 2003) or enhanced DAG signaling (Bomser et aL, 2002). Although OP-induced pCREB is the most sensitive transcription factor effect demonstrated to date, there is documentation of OP effects on other transcription factors important in neurodevelopment and synaptic plasticity. Thus, OPs elevate levels and activation of c-fos (Adamko et al., 1999; Gupta et al., 2000), cause developmental stage-specific changes in AP-1 and Sp 1 expression and DNA binding activity (Crumpton et al., 2000), and stimulate phosphorylation of c-Jun (Caughlan et al., 2004). What remains to be determined is the toxicological significance of these alterations in transcription factor expression and activity. The increasing number of reports that OPs cause developmental neurotoxicity at doses significantly below those that inhibit the catalytic activity of AChE have been widely interpreted to mean that OPs target molecules other than AChE. However, there is increasing evidence that AChE acts to promote axonal growth via a morphogenic domain that is separate from its enzymatic domain. This was first suggested by observations that in situ, many neuronal cell types, even
those that are neither cholinergic nor cholinoceptive, express AChE along axons and in axonal growth cones during periods of axonal outgrowth (Bigbee et al., 1999; Brimijoin and Koenigsberger, 1999). Subsequent studies demonstrated that transfection of Xenopus spinal neurons with mutated AChE
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CHAPTER 1 7 9Muscarinic Receptors and OP Neurotoxicity mRNA encoding a nonfunctional catalytic domain caused neurite outgrowth comparable to that of neurons transfected with intact wild-type ACHE, and both transfectants exhibit enhanced neurite outgrowth relative to nontransfected neurons (Stemfeld et al., 1998). Moreover, pharmacological agents can inhibit the morphogenic activity of ACHE without altering its catalytic activity (Dupree and Bigbee, 1994, 1996; Johnson and Moore, 1999; Munoz et al., 1999). These observations raise the possibility that OPs target ACHE, but the mechanism of developmental neurotoxicity involves disruption of the morphogenic function of ACHE, not inhibition of its catalytic activity (Bigbee et al., 1999; Brimijoin and Koenigsberger, 1999). If this hypothesis is correct, then it is predicted that OPs will inhibit axon outgrowth at concentrations that do not inhibit the enzymatic activity of ACHE, and that the potency of the axon inhibitory effects will be positively correlated with the level of ACHE expression along axons. We have tested these predictions using primary neuronal cell cultures and our findings support the hypothesis. When grown on poly-D-lysine coverslips in the absence of laminin, sympathetic neurons express AChE along axons (Rotundo and Carbonetto, 1987). Under these conditions, axonal growth is inhibited by chlorpyrifos and chlorpyrifosoxon at concentrations that do not inhibit the catalytic activity of ACHE; however, TCP, which is not known to interact with ACHE, has no effect on axon outgrowth in these neurons (Fig. 5) (Howard et al., 2005). When grown on laminin, neurons have significantly decreased levels of ACHE activity and protein, and this correlates with decreased sensitivity to the inhibitory effects of chlorpyrifos (CPF) on axon outgrowth. More convincing are preliminary observations that axonal growth in neuronal cultures derived from ACHE knockout mice, which is attenuated relative to axonal growth in neuronal cultures derived from AChE wild-type mice, is not at all affected by OP exposure (P. J. Lein, unpublished observations). The relationship of OP effects on axonal and dendritic growth to the cognitive deficits observed in animals exposed developmentally to OPs remains to be determined. The functional properties of the vertebrate nervous system are determined by the pattern of neural connections formed during development, and disruption of either the temporal or spatial aspects of axonal or dendritic growth can result in functional deficits (Barone et aL, 2000; Berger-Sweeney and Hohmann, 1997; Cremer et al., 1997, 1998; Patel et al., 2000). Whether OPs inhibit axon outgrowth in vivo has yet to be tested directly. However, developmental exposure to CPF and other OPs has been reported to alter brain morphometry in rodents. Administration of CPF (1-5 mg/kg/day, po) to dams from gestational day 6 though lactational day 11 reduced the height and anterior-to-posterior measurement of the cerebellum and decreased the thickness of the parietal cortex and hippocampal gyms in both male and female offspring on postnatal day (PND) 12 [U.S. Environmental Protection Agency (EPA), 2000a,b]. Dysmorphogenic effects were still
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FIG. 5. Inhibition of axon outgrowth by chlorpyrifos (CPF) and its oxon metabolite (CPFO). SCG neurons were treated with varying concentrations of CPF, CPFO, or TCP for 24 hr. Neurons were then fixed and immunostained with antibodies to PGP9.5 or NPY. Using Spot Imaging software, axonal growth was quantified with respect to the number of axons per neuron and total axonal length per neuron. None of these OPs altered the number of axons per neuron (data not shown); however, both CPF and CPFO decreased the total axonal length at concentrations as low as 0.001 IxM or 0.001 nM, respectively. TCP treatment did not decrease axon outgrowth in these neurons over the concentration range tested (0.0001-1 txM). Data are expressed as mean +__SEM (n = 45 per experimental condition). V, vehicle control cultures exposed to medium supplemented with DMSO diluted 1:1000. *Significantly different from vehicle control by analysis of variance, p < 0.05; **p < 0.001. Adapted from Howard et al. (2005). evident in females by PND 66 as decreased parietal cortex measurements and decreased thickness of the hippocampal gyrus (EPA, 2000a,b). Similarly, rat pups exposed to parathion (0.822 mg/kg/day, sc) from PND 5 through PND 20 exhibited thinning of the infrapyramidal and suprapyramidal mossy-fiber projections at midseptal levels as well as disruption of synaptogenesis in the molecular layer of the dentate gyms (Veronesi et al., 1990). It has been demonstrated that paraoxon (0.1 or 0.2 mg/kg/day, sc) administered from PND 8 through PND 20 did not alter dendritic arborization of CA1 pyramidal neurons of the PND 21 rat hippocampus but did alter spine density on basal dendrites (Santos et al., 2004). Since spine density is strongly influenced by afferent input (Centers for Disease Control and Prevention, 2003), these observations suggest that, similar to our observations of CPF effects on neuronal morphogenesis in cultured SCG neurons, paraoxon may inhibit axonal growth in the intact hippocampus.
V. CONCLUSIONS Inhibition of AChE and subsequent cholinergic dysfunction represents a common mechanism of toxicity after acute exposure to high levels of OP and CM pesticides. However, the range of toxicities among different compounds is broad,
242
S E CTI O N I I I
9Esterases, Receptors,
Mechanisms, & Tolerance Development
and the potency of a compound for the inhibition of AChE is not a good predictor of its toxicity. Furthermore, toxicities associated with repeated exposures to lower levels of OPs do not correlate well with AChE inhibition. These observations suggest that significant interaction of OPs and CMs with other critical molecules within the cell may be important in explaining some of the toxicity of these compounds not attributable to AChE inhibition. We presented evidence from our laboratories and others to support this hypothesis. Examples were presented with AChE null mice, direct interactions with muscarinic receptors and other molecules, impairment of cognitive development in weaning rats, OP-induced airway hyperreactivity mediated by M2 receptors, and direct effects on axonal growth in cell culture. These case studies provide strong evidence that noncholinesterase mechanisms are involved in the toxicity of OPs, and they have identified important gaps in our understanding of the mechanism of action of these important classes of insecticides.
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& Tolerance Development
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18
Paraoxonase Polymorphisms and Toxicity of Organophosphates LUCIO G. COSTA, 1,z TOBY B. COLE, 1 ANNABELLA VITALONE, 3 AND CLEMENT E. FURLONG 1 1University of Washington, Seattle, Washington; 2University of Parms, Parms, Italy; 3University of Bari, Bari, Italy
I. I N T R O D U C T I O N
synthesized primarily in the liver and a portion is secreted into the plasma, where it is associated with high-density lipoproteins (Sorenson et al., 1999; Deakin et al., 2002). PON1 received its name from its ability to hydrolyze paraoxon, its first and most studied substrate (Fig. 1). PON1 hydrolyzes the active metabolites of several other OP insecticides (e.g., chlorpyrifos-oxon and diazoxon) as well as nerve agents such as sarin, soman, and VX. The crystal structure for a recombinant PON1 has been elucidated (Harel et al., 2004). PON1 is a six-bladed [3-propeller, which in the central tunnel contains two calcium ions, one of which is essential for enzyme activity and the other for stability of PON1. Earlier studies indicated that the plasma paraoxonase activity in human populations exhibited a polymorphic distribution, and individuals with high, intermediate, or low paraoxonase activity could be identified (Eckerson et al., 1983; Mueller et al., 1983). Studies in the early 1990s led to the purification, cloning, and sequencing of human PON 1 (Gan et al., 1991; Furlong et al., 1991; Hassett et al., 1991) and the molecular characterization of its polymorphisms (Humbert et al., 1993; Adkins et al., 1993). Two polymorphisms were observed in the PON1 coding sequence: a Gln(Q)/Arg(R) substitution at position 192 and a Leu(L)/Met(M) substitution at position 55 (Humbert et al., 1993; Adkins et al., 1993). PON1 192 and 55 genotypes have been established in several populations utilizing a polymerase chain reaction (PCR) method. The polymorphism at position 192 has been the most studied, with gene frequencies of PON1Q192 ranging from 0.75 for Caucasians of Northern European origin to 0.31 for some Asian populations (Brophy et al., 2002). In addition to these two polymorphisms in the coding region of PON1, 13 polymorphisms have been found in the noncoding region of the PON1 gene (http://pga.gs.washington.edu), and 5 of these have been characterized (Brophy et al., 2001a; Leviev and James, 2000; Suehiro et al., 2000) (Fig. 2). The most significant of these
In 1946, it was found that certain organophosphorus (OP) insecticides could be enzymatically hydrolyzed by plasma (Mazur, 1946). Seminal studies by Aldridge (1953) indicated that A-esterases were capable of hydrolyzing OPs, whereas B-esterases [such as acetylcholinesterase (ACHE)] reacted with a single OP molecule and were thus inhibited by this "suicide reaction." Aldridge's proposal that an A-esterase hydrolyzed both phenylacetate and paraoxon was conclusively proven several decades later, when it was shown that recombinant paraoxonase/arylesterase catalyzed both activities (Gan et al., 1991). Studies in the late 1970s and early 1980s indicated that the plasma hydrolytic activity toward paraoxon was polymorphically distributed in human populations (Playfer, 1976; Eckerson et al., 1983; Mueller et al., 1983), suggesting a genetically based differential susceptibility to OP toxicity. In recent years, the molecular basis of the paraoxonase (PON1) activity polymorphisms (Humbert et al., 1993; Adkins et al., 1993) and their role in the toxicity of OP compounds (Costa et al., 2002) have been elucidated. Furthermore, novel important roles of PON1 in the metabolism of oxidized lipids and of certain drugs have also emerged, highlighting the importance of this enzyme in investigations of cardiovascular disease. These latter aspects of PON1 are not discussed in this chapter, but the reader is referred to some reviews (Durrington et al., 2001; Costa and Furlong, 2002; Mackness et al., 2002; Costa et al., 2003a; Draganov and La Du, 2004).
II. P O N 1 P O L Y M O R P H I S M S
PON1 is a member of a family of proteins that also includes PON2 and PON3, the genes of which are clustered in tandem on the long arms of human chromosome 7 (q21.22). PON1 is Toxicology of Organophosphate and Carbamate Compounds
247
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248
SECTION
S
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
III.
......
(EtO)2 - P - O
NO 2
Parathion
NO 2
Paraoxon
CYPs 0 (EtO)2-
- O
PON1
0
II Diethylphosphate
(EtO)2 - P - OH
+ p - Nitrophenol
FIG. 1. Activation of the OP parathion to paraoxon by cytochromes P450 (CYPs) and detoxication of paraoxon by PON1.
promoter region polymorphisms is that at position -108, with the -108C allele providing levels of PON1 approximately twice as high as those seen with the -108T allele (Leviev and James, 2000; Suehiro et al., 2000; Brophy et al., 2001b). The coding region polymorphisms of PON1 have been investigated for effects on the catalytic efficiencies of hydrolysis of specific substrates. The L/M polymorphism at position 55 does not affect catalytic activity but has been associated with plasma PON 1 protein levels, with PON 1MS5 being associated with low plasma PON1 (Blatter Garin et al., 1997; Mackness et al., 1998). However, this appears to primarily result from linkage disequilibrium with the low efficiency of the - 108T allele of the - 108 promoter region polymorphism (Brophy et al., 2002). The Q/R polymorphism at position 192 significantly affects the catalytic 7q21 -q22
centromere
-140 kb
G-909CG-832A G-162A_.___.7.~..G-126C ~-11108C, ! !
iiji
(CA). ~
A/G C/T
\ Glutamine (Q)/Arginine (R) at amino acid 192 FIG. 2. Gene structure of PON1. Note the two polymorphisms in the coding region (Q192R and L55M) and the polymorphisms in the 5' promoter region.
efficiency of PON1. Initial studies indicated that the PON1R192 allozyme hydrolyzed paraoxon more readily than PON1Q192 (Humbert et al., 1993; Adkins et al., 1993). Further studies indicated that this polymorphism was substrate dependent because the PON1Q192 alloform was found to hydrolyze diazoxon, sarin, and soman more rapidly than PON1R192 in vitro (Davies et al., 1996). In the case of diazoxon, however, recent studies have shown that under physiological conditions, both PON1 alloforms hydrolyze this compound with the same efficiency (Li et al., 2000). Complete resequencing of PON1 from 47 individuals, as part of the Environmental Genome Project, has led to the identification of more than 160 new single nucleotide polymorphisms, some in the coding regions and others in introns and regulatory regions of the gene (Jarvik et al., 2003). These polymorphisms have for the most part not been characterized but may affect splicing efficiency, message stability, or efficiency of polyadenylation. A few of them, however, have explained discrepancies found when comparing PON1 status and PCR analysis of codon 192 (Jarvik et al., 2003).
IIl. P O N 1 G E N O T Y P E / P H E N O T Y P E : P O N 1 STATUS Most studies investigating the association of PON1 polymorphisms with diseases have examined only the nucleotide polymorphisms (Q192R, L55M, and C-108T) with PCRbased assays. A functional genomic analysis, however, provides a much more informative approach because measurement of an individual's PON 1 function (plasma activity) takes into account all polymorphisms that might affect activity. This is accomplished through the use of a highthroughput enzyme assay involving two PON1 substrates (usually diazoxon and paraoxon) (Richter et al., 2004). This approach, in addition to providing a functional assessment of the plasma PON]92 alloforms, provides the plasma level of PON1 for each individual, thus encompassing the two factors that affect PON1 levels or activity (position 192 amino acid and plasma alloform levels). This approach has been referred to as the determination of PON 1 "status" for an individual (Richter and Furlong, 1999). The catalytic efficiency with which PON1 degrades toxic OPs determines the degree of protection provided by PON 1. In addition, higher concentrations of PON1 provide better protection. Thus, for adequate risk assessment, it is important to know PON1 levels and activity. In a given population, plasma PON 1 activity can vary up to 40-fold (Eckerson et al., 1983; Mueller et al., 1983; Davies et al., 1996; Richter and Furlong, 1999), and differences in PON1 protein levels up to 13-fold are also present within a single PON1192 genotype in adults (Davies et al., 1996). Studies investigating the role of PON1 in cardiovascular disease have indeed provided evidence that PON1 status (encompassing genotype and activity levels) is
CHAPTER 1 8 9Paraoxonase Polymorphisms a much better predictor of disease than PON1 genotype alone (Jarvik et al., 2000; Mackness et al., 2001).
IV. P O N 1 A N D O P T O X I C I T Y
A. Early Studies Although the ability of PON1 to hydrolyze a number of OP substrates in vitro has been well established for some time, evidence that the enzyme plays a role in modulating the toxicity of the same OPs in vivo has emerged more slowly. Initial indirect evidence was provided by comparison across animal species that differ in the levels of their plasma PON1 activity. Birds, which have very low PON1 activity (Machin, 1976; Brealey et al., 1980; Furlong et al., 2000), display a much higher sensitivity, compared to rats, to the acute toxicity of some OPs (e.g., pirimiphos-ethyl and diazinon) (Brealey et al., 1980). Among mammals, rabbits have a 5- to 20-fold higher plasma PON1 activity than rats (Aldridge, 1953; Zech and Zurcher, 1974; Costa et al., 1987) and are 4-fold less sensitive to the toxicity of paraoxon (Costa et al., 1987). Although several other factors, such as rates of absorption and distribution of OPs, rates of activation and of detoxication by other metabolic pathways, and intrinsic susceptibility of target enzymes, can affect the overall toxicity outcome, these initial observations provided some supporting, albeit indirect, evidence to the hypothesis that low serum PON1 activity would lead to an increased sensitivity to the acute toxic effects of OPs (Costa et al., 2002). A more direct approach was provided by studies in which exogenous PON1 was injected into rats or mice. The background and impetus for such an approach were provided by an early study by Main (1956), who showed that intravenous administration of partially purified PON1 from rabbit serum to rats protected them from the toxicity of paraoxon. Subsequent experiments in rats and mice utilizing pure rabbit PON1 (Furlong et al., 1991) confirmed and expanded this early observation. Administration (via the tail vein) of the enzyme to rats increased serum PON1 activity toward paraoxon 9-fold and that toward chlorpyrifos-oxon 50-fold (Costa et al., 1990). Thirty minutes after PON1 injection, rats were challenged with an acute dose of paraoxon or chlorpyrifos-oxon given by the iv, dermal, ip, or oral route at doses causing similar degrees of AChE inhibition in plasma, red blood cells, brain, and diaphragm. Four hours later, at sacrifice, AChE activity measurements indicated a much lower degree of inhibition in animals that had been pretreated with PON1. Protection was more evident in the case of chlorpyrifos-oxon and was more prominent in two target tissues for OP toxicity, brain and diaphragm (Costa et al., 1990). Of practical relevance was that a substantial protective effect was present when OP exposure occurred by the dermal route, which represents an important route of exposure for occupationally exposed workers.
249
An additional series of experiments was carried out in mice because much less purified enzyme is required for injection and this species was deemed to be ideal for studies involving genetically modified animals with altered PON1 levels. An initial experiment followed the same protocol previously utilized in rats and provided evidence that iv administration of pure rabbit PON1 increased serum chlorpyrifos-oxon activity 30- to 40-fold and protected animals from AChE inhibition by dermally applied chlorpyrifos:oxon (Li et al., 1993). As with iv injection of rabbit PON1, the increased serum PON1 activity was short lasting (t 1~2 = - 6 hr); further experiments were aimed at investigating other routes of administration for PON1. Administration of PON1 by the iv + ip route increased plasma enzyme activity toward chlorpyrifos-oxon 35-fold and extended the half-life to 30 hr. An even longer half-life, albeit with lower peak activity levels, was found when PON1 was given by the iv + im route (Li et al., 1993). Rabbit PON1 also provided protection against the toxicity of the parent compound, chlorpyrifos, when the OP exposure occurred 30 min after iv injection of PON1 (Li et al., 1993) or 24 hr after an iv + ip administration of PON1 (Li et al., 1995). Further experiments also showed that PON1, when given 30 min after dermal administration of chlorpyrifos, prevented the reduction of AChE activity in all tissues; when PON1 was injected 3 hr after chlorpyrifos, a protective effect was still seen in brain and diaphragm (Li et al., 1995). This series of studies indicated that by artificially increasing serum levels of PON1 (by injection of purified rabbit enzyme), it was possible to decrease the acute toxicity of certain OPs. Also of relevance is the finding that PON1 exerted a protective effect when given after OP exposure, suggesting a potential use in OP poisoning, possibly in combination with other conventional treatments. This series of experiments provided convincing evidence that high serum PON1 levels protect against the toxicity of specific OP compounds.
B. Studies on Genetically Modified Mice PON1 knockout and transgenic animals have provided important new tools to investigate the role of PON1 in modulating OP toxicity. PON1 knockout (PON1 - / - ) mice were produced by targeted disruption of exon 1 of the PON1 gene and have a normal appearance and body weight (Shih et al., 1998). Plasma from PON1 - / - mice has no detectable hydrolytic activity toward paraoxon and diazoxon, and very limited chlorpyrifos-oxonase activity. A similar pattern of activity is also found in liver, indicating that both plasma and liver PON1 are encoded by the same gene (Li et al., 2000). PON1 hemizygous mice (PON1 +/-) have approximately 40% of plasma and liver PON1 activity compared to wild-type mice (PON1 +/+). As expected, PON1 knockout mice did not differ from wildtype animals in their sensitivity to demeton-S-methyl, an
250
SECTION III. E s t e r a s e s ,
Receptors,
Mechanisms,
OP insecticide with a structure similar to malathion, which is not a substrate for PON1 (Li et al., 2000). As also predicted, PON - / - mice showed a dramatically increased sensitivity to chlorpyrifos-oxon and diazoxon (Shih et al., 1998; Li et al., 2000). PON1 +/- mice showed an intermediate sensitivity to diazoxon toxicity (Li et al., 2000). PON1 null mice showed only a slight increase in sensitivity to the toxicity of chlorpyrifos and diazinon (Shih et al., 1998; Li et al., 2000). The most surprising observation was that PON1 null mice did not show an increased sensitivity to paraoxon, the substrate after which the enzyme was named, despite having no paraoxonase activity in plasma and liver (Li et al., 2000). Additional experiments were designed to determine whether administration of exogenous PON1 to PON1 - / mice to restore serum PON1 would also restore resistance to OP toxicity. For this purpose, either human pure PON1Q192 or PON1R192 was injected, by the iv route, into PON1 knockout mice; the effects of various OPs on brain and diaphragm AChE activity were then determined. PON1R192 provided significantly better protection than PON1Q192 toward chlorpyrifos-oxon, a finding confirmed in a subsequent study by Cowan et al., (2001), who administered recombinant adenoviruses containing PON1-LQ or PON1-LR genes to BALB/c mice before challenge with chlorpyrifos-oxon. Both alloforms were equally effective in protecting against the toxicity of diazoxon, whereas neither PON1R192 nor PON1Q192 afforded protection against paraoxon toxicity (Li et al.,
2000). The results of these experiments in PON1 knockout mice prompted a reexamination of the in vitro catalytic efficiencies of the two human PON 1 alloforms under more physiological salt concentrations (lower NaC1 concentration). Results from kinetic analysis of substrate hydrolysis by purified human alloforms provided an explanation for the in vivo finding. In the case of chlorpyrifos-oxon, the catalytic efficiency of both PON 1 alloforms was very high, and it was higher for the PON1R192 alloform (Table 1). Catalytic efficiency was still high in the case of diazoxon,
& Tolerance
Development
albeit lower than with chlorpyrifos-oxon, but no alloformspecific difference was evident. With paraoxon, the PON1R192 alloform was much more efficient than the PON1Q192 alloform; however, its overall catalytic efficiency was too low to protect against exposure. This confirms the hypothesis (Chambers et al., 1994; Pond et al., 1995) that PON1 is not efficient at hydrolyzing paraoxon at low concentrations, suggesting that PON1 may not degrade paraoxon efficiently in vivo and that other pathways (e.g., cytochromes p450 and carboxylesterase) are primarily responsible for detoxifying paraoxon in vivo. Additional experiments carried out in PON1 transgenic mice (mice expressing either human PONQ192 or human PON1R192 on a knockout background and mice carrying the human PON1R192 on top of mPON1) provided further evidence for such conclusions. A transgenic mouse line that carries the human PON1R192 allele over its mouse PON1 was tested for its sensitivity to paraoxon. These mice, whose serum paraoxonase activity was 3.5-fold higher than that of wild-type mice, showed similar sensitivity to paraoxon as wild-type mice (Li et al., 2000). On the other hand, hPON1R192-TG mice (expressing human PON1R192 on a knockout background) were significantly less sensitive to the toxicity of chlorpyrifos-oxon than hPON1Q192-TG mice, despite having the same level of PON1 protein in liver and plasma (T. B. Cole et al., 2005). Altogether, these animal experiments allow the following conclusions with regard to the role of PON1 in modulating the toxicity of the three OP compounds tested. First, in the case of chlorpyrifos-oxon, both the level of expression and the Q 192R genotype are important determinants of susceptibility, highlighting the importance of assessing PON1 status in potentially exposed individuals. Second, with regard to diazoxon, protection or susceptibility is dictated primarily by the level of expression of PON1, independently of the Q192R genotype, again stressing the importance of knowing PON1 levels. Third, PON1 status does not appear to play a role in modulating sensitivity to paraoxon toxicity.
TABLE 1. in Vitro Catalytic Efficiency and in Vivo Protection of Human PON 119z Allozymesa Catalytic efficiency (Vmax]Km) OP
Chlorpyrifo s-oxon Diazoxon Paraoxon
Degree of protection b
Q192
R192
Q192
R192
152 75 0.7
256 77 6.3
1.5 3.2 0
4.2 3.7 0
aAdapted from Li et al. (2000). bBrain AChE activity (fold increase) following challenges with the OPs in PON1 knockout mice pretreated with either PON1Q192or PONlR192comparedto untreated PON1-/- mice.
CHAPTER 1 8 9Paraoxonase Polymorphisms
V. PON1 AND THE DEVELOPMENTAL T O X I C I T Y A N D N E U R O T O X I C I T Y O F OPs Although genetic determinants (i.e., the genetic polymorphisms discussed previously) play a primary role in determining an individual's PON1 status, the contribution of other factors in modulating PON1 activity may also be important. Environmental, pharmacological, dietary, and lifestyle factors, as well as certain disease conditions, have been found to influence PON1 activity (Costa et al., 2005). Age is also a major determinant of PON1 activity. Studies in rodents have shown that serum and liver PON1 activity is very low at birth and increases up to postnatal day 21, with a parallel increase in liver mRNA (Mortensen et al., 1996; Li et al., 1997; Moser et al., 1998; Karanth and Pope, 2000). A similar increase was also seen in transgenic mice expressing either the human PON1R192 or the PON1Q192 alloforms under the control of the human PON1 regulatory sequences, indicating conservation of the developmental regulatory elements between human and mouse PON1 (Cole et al., 2003). Studies on humans have also shown that serum PON1 activity is very low at birth and increases over time, reaching a plateau between 6 and 15 months of age (Augustinsson and BAIT, 1963; Ecobichon and Stephens, 1973; Mueller et al., 1983; Cole et al., 2003: Chen et al., 2003). Low PON1 activity during development could represent a relevant risk factor for increased susceptibility to the toxicity of certain OP insecticides. There is ample evidence that OP toxicity is influenced by age, with young animals being more sensitive than adults to the effects of acute exposure (Harbison, 1975; Pope and Liu, 1997; Moser et al., 1998). Although intrinsic differences in brain AChE do not account for the age-related differences in sensitivity, as indicated by in vitro studies (Benke and Murphy, 1975; Pope and Chakraborti, 1992), lower metabolic detoxication abilities of young animals appear to be a major determinant for their increased susceptibility (Benke and Murphy, 1975; Murphy, 1982). In particular, studies with chlorpyrifos have indicated that a lower hydrolytic detoxication by PON1 accounts for the differential age-related sensitivity in acute toxicity (Mortensen et al., 1996; Moser et al., 1998; Padilla et al., 2000). Studies on PON1 knockout mice have shown that they are significantly more sensitive to the effects of chlorpyrifos-oxon on brain AChE than wild-type mice of the same age (T. B. Cole et al., unpublished data). Exposure of PON1 knockout mice to chlorpyrifos-oxon between postnatal days 4 and 21, at doses causing an initial 10-50% brain AChE inhibition, caused significant, doserelated histopathological changes in the neocortex at 3 weeks of age (Cole et al., unpublished data; C. Pettan-Brewer et al., unpublished data). Thus, low PON1 levels during early development contribute to the greater sensitivity of young animals to the acute toxicity of certain OPs, and a low PON1 status (exemplified here by the PON1 - / - mice) further exacerbates the neurotoxic effects of OPs upon
251
repeated exposures. Microarray analyses are under way to examine the effects of OP exposures on gene expression in the mouse brain during development and the role of PON1 status in modulating the effects of exposure.
VI. CLINICAL EVIDENCE FOR A ROLE O F P O N 1 IN O P T O X I C I T Y The early observations that human serum PON1 activity presented a bimodal/trimodal distribution led to the hypothesis that low metabolizers may be more sensitive to the toxicity of OPs. The studies summarized previously have characterized the PON1 polymorphisms responsible for different catalytic activities and levels of expression, demonstrated the relevance of PON1 in modulating OP toxicity in various animal models, and indicated the importance of an individual's PON1 status. Direct confirmation in humans of the relevance of PON1 status in determining relative sensitivity to OP toxicity, however, is still elusive. Nevertheless, in recent years, some studies have started to investigate the role of PON1 in OP toxicity in humans and have provided some interesting findings. The 1995 terrorist attack in the Tokyo subway system that left 12 people dead and more than 5000 injured provided the opportunity to investigate the role of PON1 in modulating the toxicity of satin in humans (Suzuki et al., 1995). Satin is metabolized by PON1, and homozygotes for the PON1Q192 allele were found in an in vitro assay to hydrolyze sarin approximately 10 times better than individuals homozygous for the PONlR192 allele (Davies et al., 1996). The prevalence of the PONlR192 genotype in the Japanese population is 0.66 compared to 0.25-0.30 in various Caucasian populations (Brophy et al., 2002; Yamasaki et al., 1997). Thus, Japanese individuals may have been more prone to sarin toxicity because of the low sarinhydrolyzing ability of the PONlR192 allozyme. However, among 10 of the victims of the Tokyo attack, 7 expressed the PONl192Q genotype, with 6 Q/R heterozygotes and 1 Q/Q homozygote (Yamada et al., 2001). Thus, the genotype that confers high hydrolyzing activity toward satin did not appear to provide protection from acute satin poisoning. Several issues, however, need to be considered. First, only the Q192R genotype of those 10 individuals was analyzed, with no information on their PON1 status. In a Caucasian population, the range of sarinase activity among individuals with the QQ or QR genotype ranged from 0 to 758 U/liter (Davies et al., 1996). Second, exposure to satin in these 7 QQ or QR individuals was indeed massive, because it caused death instantly or, with one exception, in less than 48 hr (Yamada et al., 2001). Such high-dose exposure would be expected to overcome any potential protection afforded by the PON1Q192 genotype. Third, and most important, the catalytic efficiency of satin hydrolysis by PON1 is low; the situation is thus similar (albeit reversed)
252
SECTION III. E s t e r a s e s ,
Receptors,
to that of paraoxon, with one PON1192 alloform hydrolyzing satin with better efficiency but still not efficiently enough to provide protection. 9 A few studies have also investigated PON1 polymorphisms in U.S. and UK troops that were deployed in the Persian Gulf area in 1990 and 1991. Individuals who served in the Gulf War theater were potentially exposed to a wide range of biological and chemical agents, including sand, smoke from oil well fires, solvents, petroleum fuels, depleted uranium, anthrax and botulinum toxoid vaccinations, insecticides, pyridostigimine bromide, and nerve agents [Institute of Medicine (IOM), 2000, 2003]. A large number of these veterans have complained of a range of unexplained illnesses, including chronic fatigue, muscle and joint pain, loss of concentration, forgetfulness, and headachewsymptoms that are often referred to as Gulf War syndrome (IOM, 2000, 2003). PON1 genotypes and plasma enzyme activity were investigated in a group of 25 ill U.S. Gulf War veterans and 20 controls (Haley et al., 2000). PON1R192 homozygotes or PON1Q/R192 heterozygotes were more likely to have neurologic symptoms than individuals homozygous for PONQ192. Furthermore, low activity of the plasma PON1Q192 isoform appeared to better correlate with illness than the PON1 genotype or the activity levels of the PON1R192 genotype (Haley et al., 2000). This study suggests that low PON1 status may represent a risk factor for illness in Gulf War veterans, although such findings necessitate further confirmation in a larger population (Furlong, 2000). A similar study in a group of 152 UK Gulf War veterans, who self-reported the presence of symptoms associated with the Gulf War syndrome, yielded somewhat different results (Mackness et al., 2000). Plasma paraoxonase activity and levels of PON1 protein were lower in veterans than in a control group, and these decreases were independent of the PON1 genotype (Mackness et al., 2000). Thus, although in both studies a reduced plasma paraoxonase activity was found, in one case it was attributed to an overrepresentation of the low-activity PON1 isozyme (Haley et al., 2000), whereas in the other it was common to all PON1 genotypes (Mackness et al., 2000). Although the latter study suggests that this group of veterans may have a decreased capacity to hydrolyze some OP insecticides, such as chlorpyrifos-oxon, its significance is hampered by the lack of information on the extent of exposure to such compounds among veterans (Costa et al., 2003b). A third study compared PON1 genotypes and plasma paraoxonase activity in groups of UK veterans from the Persian Gulf War who were symptomatic by self-reporting (n = 115), healthy Persian Gulf War veterans (n = 95), symptomatic Bosnia peacekeeping veterans (n = 52), and symptomatic nondeployed military controls (n = 85) (Hotopf et al., 2003). No differences in genotype distribution or PON1 activity were found between healthy and ill Gulf War veterans. However, individuals who were deployed to the Gulf had 25-35% lower median PON1 values than the
Mechanisms,
& Tolerance
Development
other two groups, and these differences were not explained by differences in PON1192 genotypes between groups. Thus, PONll92 genotype and activity were not associated with Gulf War syndrome but appeared to be the result of deployment in the Persian Gulf. Possible explanations for such findings were the potential exposure of those who served in the Gulf to unknown agents that led to a long-term decrease in PON1 activity and/or an overrepresentation in those two groups of individuals with the -108T allele, which is associated with lower PON1 levels (Hotopf et al., 2003). The role of all three PON1 genes in modulating oxidative stress has yet to be evaluated. Possible relationships between exposure of sheep dippers to diazinon and chronic central and/or peripheral nervous system abnormalities have also been investigated (Cherry et al., 2002). Cases (n = 173) and controls (n = 234) were recruited among sheep dippers who had utilized diazinon. The allele frequency of the PONR192 polymorphism was 0.35 in cases vs 0.22 in controls, and diazoxonase activity was lower in cases than referents (Cherry et al., 2002). In a follow-up study in the same populations, serum activity toward paraoxon, diazoxon, and phenylacetate was measured, and no differences between cases and controls were found (Mackness et al., 2003). When the two groups were divided into quintiles according to the capacity of their serum to hydrolyze diazoxon, sheep dippers in the lowest quintile had a greater risk of reporting ill health than those in the other quintiles (Mackness et al., 2003). The authors of these studies concluded that these findings suggest that diazinon may have contributed to the ill health of sheep dippers because of a lower ability to detoxify diazoxon. Two additional studies investigated PON1 polymorphisms and activity in workers chronically exposed to OPs. In one study of 100 South African workers, symptoms consistent with chronic OP toxicity were significantly more likely among subjects with the QQ or QR genotypes than the RR genotype (Lee et al., 2003). However, no indication of which OP was involved in exposure was provided. In another study, a substantial, although not significant, decrease in PON1 paraoxonase activity was found in a group of greenhouse workers with long-term exposure to unspecified OPs (Hernandez et al., 2003). The authors suggested that chronic exposure to OPs may decrease PON1 activity, as had been previously seen in individuals acutely poisoned by OPs (Sozmen et al., 2002). The finding of low PON 1 activity in neonates (Cole et al., 2003) suggested that PON 1 levels may be even lower before birth, as indeed indicated by data showing a 24% lower activity in premature infants (33-36 weeks of gestation) compared to term infants, using phenylacetate as a substrate (Ecobichon and Stephens, 1973). In addition, an expectant mother with low PON1 status would be predicted not to be able to provide protection for her fetus against exposure to some OPs (Cole et al., 2003). Recent data have provided initial support for this hypothesis. Offspring of mothers with
CHAPTER 1 8 9Paraoxonase Polymorphisms
low PON1 activity exposed in utero to chlorpyrifos had significantly smaller head circumference compared to those born to mothers with high PON1 activity or those not exposed to chlorpyrifos (Berkowitz et al., 2004). Since small head size has been found to be predictive of subsequent cognitive ability, these findings suggest that prenatal exposure to chlorpyrifos may have even more detrimental long-lasting effects in offspring of mothers with low PON1 activity. These studies in human populations, although they have some shortcomings, are of interest because they attempt to address the issue of whether an individual's PON1 status may confer protection or increased sensitivity to the toxicity of specific OPs. More studies are clearly needed in which better data on the extent and nature of exposure and of the consequences of exposure are documented and PON1 status (position 192 genotype and phenotype) is determined.
VII. C O N C L U S I O N S Polymorphisms in the PON1 gene influence both the quality and the quantity of PON1 (i.e., PON1 status). Evidence provided by animal studies indicates that PON1 plays a relevant role in the metabolism of certain OPs and modulates their toxicity and developmental neurotoxicity. Careful in vitro studies carried out under physiological conditions, together with in vivo studies in various lines of PON1 transgenic mice, have been shown to be extremely useful in dissecting the functional significance of PON1. The influence of PON1 status on inferring susceptibility or protection toward OPs needs to be evaluated for each individual compound. It is somewhat ironic that the toxicity of paraoxon, the OP after which PON1 was named, is not significantly influenced by PON1 status. Although strong evidence indicates that PON1 levels and, in some cases, the Q192R polymorphism determine the efficiency with which an individual will detoxify a specific OP, further proof in human populations is still needed. In particular, studies are needed in which PON1 status is correlated with the degree of exposure and with signs and symptoms of toxicity. Animal studies have also pointed out the potential therapeutic use of PON1 in treating individuals for exposure to OP insecticides and/or nerve agents. The recent expression of active human PON1 in Escherichia coli (Furlong et al., unpublished data) and the elucidation of PON1 structure (Harel et al., 2004) provide the necessary breakthroughs for producing, recombinant variants that have catalytic efficiency sufficient for therapeutic applications.
Acknowledgments Research by the authors was supported by grants from the National Institutes of Health (ES04696, ES07033, ESl1387, ES09883, ES09601/EPA-R826886, and T32 AG00057).
253
References Adkins, S., Gan, K. N., Mody, M., and LaDu, B. N. (1993). Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: Glutamine or arginine at position 191, for the respective A or B allozymes. Am. J. Hum. Genet. 53, 598-608. Aldridge, W. N. (1953). Serum esterases I. Two types of esterase (A and B) hydrolyzing p-nitrophenyl acetate, proprionate and butyrate and a method for their determination. Biochem. J. 53, 110-117. Augustinsson, K. B., and Barr, M. (1963). Age variation in plasma arylesterase activity in children. Clin. Chim. Acta 8, 568-573. Benke, G. M., and Murphy, S. D. (1975). The influence of age in the toxicity and metabolism of methylparathion and parathion in male and female rats. Toxicol. Appl. Pharmacol. 31, 254-269. Berkowitz, G. S., Wetmur, J. G., Birman-Deych, E., Obel, J., Lapinski, R. H., Godbold, J. H., Holzman, I. R., and Wolff, M. S. (2004). In utero pesticide exposure, maternal paraoxonase activity, and head circumference. Environ. Health Perspect. 112, 388-391. Blatter Garin, M. C., James, R. W., Dussoix, E, Blanche, M., Passa, E, Froguel, E, and Ruiz, J. (1997). Paraoxonase polymorphism Met-Leu 54 is associated with modified serum concentrations of the enzyme. A possible link between the paraoxonase gene and increased risk of cardiovascular disease in diabetes. J. Clin. Invest. 99, 62-66. Brealey, C. B., Walker, C. M., and Baldwin, B. C. (1980). A-esterase activities in relation to the differential toxicity of pirimiphos-methyl to birds and mammals. Pestic. Sci. 11, 546-554. Brophy, V. H., Hastings, M. D., Clendenning, J. B., Richter, R. J., Jarvik, J. E, and Furlong, C. E. (2001a). Polymorphisms in the human paraoxonase (PON1) promoter. Pharmacogenetics 11, 77-84. Brophy, V. H., Jampsa, R. L., Clendenning, J. B., McKinstry, L. A., and Furlong, C. E. (2001b). Effects of 5' regulatory-region polymorphisms on paraoxonase gene (PON1) expression. Am. J. Hum. Genet. 68, 1428-1436. Brophy, V. H., Jarvik, G. E, and Furlong, C. E. (2002). PON1 polymorphisms. In Paraoxonase (PON1) in Health and Disease: Basic and Clinical Aspects (L. G. Costa and C. E. Furlong, Eds.), pp. 53-77. Kluwer, Norwell, MA. Chambers, J. E., Ma, R., Boone, J. S., and Chambers, H. W. (1994). Role of detoxication pathways in acute toxicity of phosphorothioate insecticides in the rat. Life Sci. 54, 1357-1364. Chen, J., Kumar, M., Chan, W., Berkowitz, G. S., and Wetmur, J. G. (2003). Increased influence of genetic variation on PON1 activity in neonates. Environ. Health Perspect. 111, 1403-1410. Cherry, N., Mackness, M. I., Durrington, P., Povey, A., Dippnall, M., Smith, T., and Mackness, B. (2002). Paraoxonase (PON1) polymorphisms in farmers attributing ill health to sheep dip. Lancet 359, 763-764. Cole, T. B., Jampsa, R. L., Walter, B. J., Arndt, T. L., Richter, R. J., Shih, D. M., Tward, A., Lusis, A. J., Jack, R. M., Costa, L. G., and Furlong, C. E. (2003). Expression of human paraoxonase during development. Pharmacogenetics 13, 1-8. Cole, T. B., Walter, B. J., Shih, D. M., Tward, A. D., Lusis, A. J., Timchalk, C., Richter, R. J., Costa, L. G. and Furlosy, C. E. (2005). Toxicity of chlorpyrifos and chlorpyrifos oxon in a
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SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
transgenic mouse model of the human paraoxonese (PON1) Q192R polymorphism. Pharmacogenet. Genomics 15, 589-598. Costa, L. G., and Furlong, C. E. (Eds.) (2002). Paraoxonase (PON1) in Health and Disease: Basic and Clinical Aspects. Kluwer, Norwell, MA. Costa, L. G., Richter, R. J., Murphy, S. D., Omenn, G. S., Motulsky, A. G., and Furlong, C. E. (1987). Species differences in serum paraoxonase correlate with sensitivity to paraoxon toxicity. In Toxicology of Pesticides: Experimental Clinical and Regulatory Perspectives (L. G. Costa, C. L. Galli, and S. D. Murphy, Eds.), pp. 263-266. Springer-Verlag, Heidelberg. Costa, L. G., McDonald, B. E., Murphy, S. D., Omenn, G. S., Richter, R. J., Motulsky, A. G., and Furlong, C. E. (1990). Serum paraoxonase and its influence on paraoxon and chlorpyrifos-oxon toxicity in rats. Toxicol. Appl. Pharmacol. 103, 66-76. Costa, L. G., Li, W. E, Richter, R. J., Shih, D. M., Lusis, A. J., and Furlong, C. E. (2002). PON1 and organophosphate toxicity. In Paraoxonase (PON1) in Health and Disease: Basic and Clinical Aspects (L. G. Costa and C. E. Furlong, Eds.), pp. 165-183. Kluwer, Norwell, MA. Costa, L. G., Cole, T. B., Jarvik, G. R, and Furlong, C. E. (2003a). Functional genomics of the paraoxonase (PON1) polymorphisms: Effect on pesticide sensitivity, cardiovascular disease and drug metabolism. Annu. Rev. Med. 54, 371-392. Costa, L. G., Cole, T. B., and Furlong, C. E. (2003b). Polymorphisms of paraoxonase (PON1) and their significance in clinical toxicology of organophosphates. J. Toxicol. Clin. Toxicol. 41, 37-45. Costa, L. G., Vitalone, A., Cole, T. B., and Furlong, C. E. (2005). Modulation of paraoxonase activity. Biochem. Pharmacol., 69, 541-550. Cowan, J., Sinton, C. M., Varley, A. W., Wians, E H., Haley, R. W., and Munford, R. S. (2001). Gene therapy to prevent organophosphate intoxication. Toxicol. Appl. Pharmacol. 173, 1--6. Davies, H., Richter, R. J., Kiefer, M., Broomfield, C., Sowalla, J., and Furlong, C. E. (1996). The human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin. Nature Genet. 14, 334-336. Deakin, S., Leviev, I., Gomeraschi, M., Calabresi, L., Franceschini, G., and James, R. W. (2002). Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high-affinity, saturable, desorption mechanism. J. Biol. Chem. 277, 4301-4308. Draganov, D. I., and La Du, B. N. (2004). Pharmacogenetics of paraoxonases: A brief review. Naunyn-Schmiedeberg's Arch. Pharmacol. 369, 78-88. Durrington, P. N., Mackness, B., and Mackness, M. I. (2001). Paraoxonase and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 21, 473-480. Eckerson, H. W., Wyte, C. M., and LaDu, B. N. (1983). The human serum paraoxonase/arylesterase polymorphism. Am. J. Hum. Genet. 35, 1126-1138. Ecobichon, D. J., and Stephens, D. S. (1973). Perinatal development of human blood esterases. Clin. Pharmacol. Ther. 14, 41-47. Furlong, C. E. (2000). PON1 status and neurologic symptom complexes in Gulf War veterans. Genome Res. 10, 153-155. Furlong, C. E., Richter, R. J., Chapline, C., and Crabb, J. W. (1991). Purification of rabbit and human serum paraoxonase. Biochemistry 30, 10133-10140. Furlong, C. E., Li, W. F., Richter, R. J., Shih, D. M., Lusis, A. J., Alleva, E., and Costa, L. G. (2000). Genetic and temporal
determinants of pesticide sensitivity: Role of paraoxonase (PON 1). Neurotoxicology 21, 91-100. Gan, K. N., Smolen, A. L., Eckerson, H. W., and LaDu, B. N. (1991). Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab. Dispos. 19, 100-106. Haley, R. W., Billecke, S., and LaDu, B. N. (2000). Association of low PON1 type Q (type A) arylesterase activity with neurologic symptom complexes in Gulf War veterans. Toxicol. Appl. Pharmacol. 157, 227-233. Harbison, R. D. (1975). Perinatal development of human blood esterases. Clin. Pharmacol. Ther. 14, 4147. Harel, M., Aharoni, A., Gaidukov, L., Brumshtein, B., Khersonsky, O., Meged, R., Dvir, H., Revelli, R. B. G., McCarthy, A., Toker, L., Silman, I., Sussman, J. L., and Tawfik, D. S. (2004). Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes. Nature Struct. Mol. Biol. 11,412-419. Hassett, C., Richter, R. J., Humbert, R., Chapline, C., Crabb, J. W., Omiecinski, C. J., and Furlong, C. E. (1991). Characterization of DNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence. Biochemistry 30, 10141-10149. Hernandez, A. E, Mackness, B., Rodrigo, L., Lopez, O., Pla, A., Gil, E, Durrington, P. N., Pena, G., Passon, T., Serrano, J. L., and Mackness, M. I. (2003). Paraoxonase activity and genetic polymorphisms in greenhouse workers with long term pesticide exposure. Hum. Exp. Toxicol. 22, 565-574. Hotopf, M., Mackness, M. I., Nikolau, V., Collier, D. A., Curtis, C., David, A., Durrington, P., Hull, L., Ismail, K., Peekman, M., Unwin, C., Wessely, S., and Mackness, B. (2003). Paraoxonase in Persian Gulf veterans. J. Occup. Environ. Med. 45, 668-675. Humbert, R., Adler, D. A., Disteche, C. M., Omiecinski, C. J., and Furlong, C. E. (1993). The molecular basis of the human serum paraoxonase polymorphisms. Nature Genet. 3, 73-76. Institute of Medicine (2000). Gulf War and Health: Vol. 1. Depleted Uranium, Pyridostigmine Bromide, Sarin, Vaccines. National Academy Press, Washington, DC. Institute of Medicine (2003). Gulf War and Health: Vol. 2. Insecticides and Solvents. National Academy Press, Washington, DC. Jarvik, G. P., Rozek, L. S., Brophy, V. H., Hatsukami, T. S., Richter, R. J., Schellenberg, G. D., and Furlong, C. E. (2000). Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON 1192 or PON 155 genotype. Arterioscler. Thromb. Vasc. Biol. 20, 2441-2447. Jarvik, G. P., Jampsa, R., Richter, R. J., Carlson, C. S., Rieder, M. G., Nickerson, D. A., and Furlong, C. E. (2003). Novel paraoxonase (PON1) nonsense and missense mutations predicted by functional genomic assay of PON1 status. Pharmacogenetics 13, 291-295. Karanth, S., and Pope, C. (2000). Carboxylesterase and A-esterase activities during maturation and aging: Relationship to the toxicity of chlorpyrifos and parathion in rats. Toxicol. Sci. 58, 282-289. Lee, B. W., London, L., Poulauskis, J., Myers, J., and Christiani, D. C. (2003). Association between human paraoxonase gene polymorphism and chronic symptoms in pesticideexposed workers. J. Occup. Environ. Med. 45, 118-122. Leviev, I., and James R. W. (2000). Promoter polymorphisms of human paraoxonase PON1 gene and serum paraoxonase
CHAPTER 1 8 9 Paraoxonase Polymorphisms activities and concentrations. Arterioscler. Thromb. Vasc. Biol. 20, 516-521. Li, W. E, Costa, L. G., and Furlong, C. E. (1993). Serum paraoxonase status: A major factor in determining resistance to organophosphates. J. Toxicol. Environ. Health 40, 337-346. Li, W. E, Furlong, C. E., and Costa, L. G. (1995). Paraoxonase protects against chlorpyrifos toxicity in mice. Toxicol. Lett. 76, 219-226. Li, W. E, Matthews, C., Disteche, C. M., Costa, L. G., and Furlong, C. E. (1997). Paraoxonase (PON1) gene in mice: Sequencing, chromosomal localization and developmental expression. Pharmacogenetics 7, 137-144. Li, W. E, Costa, L. G., Richter, R. J., Hagen, T., Shih, D. M., Tward, A., Lusis, A. J., and Furlong, C. E. (2000). Catalytic efficiency determines the in vivo efficacy of PON1 for detoxifying organophosphates. Pharmacogenetics 10, 767-779. Machin, A. E, Anderson, P. H., Quick, M. P., Woddel, D. E, Skibniewska, K. A., and Howells, L. C. (1976). The metabolism of diazinon in the liver and blood of species of varying susceptibility to diazinon poisoning. Xenobiotica 6, 104. Mackness, B., Mackness, M. I., Arrol, T., Turkie, W., and Durrington, P. N. (1998). Effect of the human serum paraoxonase 55 and 192 genetic polymorphisms on the protection by high density lipoprotein against low density lipoprotein oxidative modifications. FEBS Lett. 423, 57-60. Mackness, B., Durrington, P. N., and Mackness, M. I. (2000). Low paraoxonase in Persian Gulf War veterans self-reporting Gulf War syndrome. Biochem. Biophys. Res. Commun. 276, 729-733. Mackness, B., Davies, G. K., Turkie, W., Lee, E., Roberts, D. M., Hill, E., Roberts, C., Durrington, P. N., and Mackness, M. I. (2001). Paraoxonase status in coronary heart disease: Are activity and concentration more important than genotype? Arterioscler. Thromb. Vasc. Biol. 21, 1451-1457. Mackness, B., Durrington, P. N., Povey, A., Thomson, S., Dippnall, M., Mackness, M., Smith, T., and Cherry, N. (2003). Paraoxonase and susceptibility to organophosphorus poisoning in farmers dipping sheep. Pharmacogenetics 13, 81-88. Mackness, M. S., Mackness, B., and Durrington, P. N. (2002). Paraoxonase and coronary heart disease. Artheroscler. Suppl. 3, 49-55. Main, A. R. (1956). The role of A-esterase in the acute toxicity of paraoxon, TEEP and parathion. Can. J. Biochem. Physiol. 34, 197-216. Mazur, A. (1946). An enzyme in animal tissue capable of hydrolyzing the phosphorus-fluorine bond of alkyl fluorophosphates. J. Biol. Chem. 164, 271-289. Mortensen, S. R., Chanda, S. M., Hooper, M. J., and Padilla, S. (1996). Maturational differences in chlorpyrifos-oxonase activity may contribute to age-related sensitivity to chlorpyrifos. J. Biochem. Toxicol. 11, 279-287. Moser, V. C., Chanda, S. M., Mortensen, S. R., and Padilla, S. (1998). Age- and gender-related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of esterase activities. Toxicol. Sci. 46, 211-222. Mueller, R. F., Hornung, S., Furlong, C. E., Anderson, J., Giblett, E. R., and Motulsky, A. G. (1983). Plasma paraoxonase polymorphism: A new enzyme assay, population, family, biochemical and linkage studies. Am. J. Hum. Genet. 35, 393-408. Murphy, S. D. (1982). Toxicity and hepatic metabolism of organophosphate insecticides in developing rats. Banbury Rep. 11, 125-136.
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CHAPTER
19 Tolerance D e v e l o p m e n t to Toxicity of Cholinesterase Inhibitors FRODE FONNUM 1 AND SIGRUN HANNE STERRI2 1University of Oslo, Oslo, Norway 2Norwegian Defence Research Establishment, Kjeller, Norway
I. I N T R O D U C T I O N
of soman on AChE or by a more rapid de n o v o synthesis of AChE (Soli et al., 1980). Also, the rapid return from hypothermia seen after treatment of animals with AChE inhibitors could not be explained by recovery of AChE activity; it must be due to some form of developed tolerance (Gordon, 1997). Animal knockouts for AChE in both the peripheral and the central nervous system survive for up to 1 year (Xie et al., 2000). This can only occur because the animal develops tolerance to the absence of ACHE. Tolerance to AChE inhibitors has been noted using different forms of administration and in several species, such as mice, rats, guinea pigs, and man. Tolerance can develop in several ways. It often occurs due to receptor changes either in the number of receptors or by decreased affinity of the receptor molecule. However, it can also occur due to the presence of other proteins that can bind or inactivate the inhibitor and thereby make it less readily available. Examples are binding of the inhibitor to carboxylesterases (CarbEs), buturylcholinesterases (BuChEs), or other binding proteins such as albumin. In addition, tolerance can be achieved through more rapid metabolism of the OP compounds by OP-hydrolyzing enzymes such as paraoxonases (PONs) and somanases.
Organophosphates (OPs) inhibit the enzyme acetylcholinesterase (ACHE) and thereby increase the level of acetylcholine (ACh) in the synaptic gap, The acute toxic effects of OPs are due to accumulation of ACh at the muscarinic and nicotinic receptors. It has been noted by several investigators that during prolonged exposure to an OP, the physiological effects often diminish more than expected from the degree of AChE inhibition (Bushnell et al., 1991) or that repeated additions of OP give lower responses with time. Barnes and Denz (1954a,b) gave rats 50 ppm of parathion for several months and observed that after 3 months several of the effects of AChE inhibition, such as fasciculation, lacrimation, and salivation, disappeared, although the animals still occasionally showed tremors. Another example was shown by Rider et al. (1952), who pretreated animals with 0.5 mg OMPA/kg/day for 5 days and then noted that the animals did not show any toxic effects after a dose of 1 mg/kg/day for 41 days. Normally, animals died after having been exposed to such a dose for 5 days. Another example, guinea-pigs survived administration of 0.5 LDs0 daily of the irreversible inhibitor soman for 11 days (Sterri, 1981). Another example of tolerance to AChE inhibition was seen when treating the pupil with the irreversible inhibitor soman. After a single topical soman instillation into the conjunctival sac, there was an almost linear relationship between the reduction in AChE activity and pupillary diameter. Topical administration of soman at 24-hr intervals in doses capable of almost complete inhibition of AChE in the iris was accompanied by a reduced miotic effect of this drug. This was indicated by a reduced rate of the soman-induced pupillary constriction, a less pronounced reduction in pupillary diameter, and a more rapid return of the pupillary diameter to normal size. These observations were seen irrespective of inhibition of blood ACHE. The decrease in response to repeated administration could not be explained by a reduced inhibitory effect Toxicology of Organophosphate and Carbamate Compounds
II. M U S C A R I N I C
RECEPTORS
Muscarinic receptors (mAChRs) are very involved in the development of tolerance to OPs. Prolonged inhibition of AChE leads to a high concentration of ACh at the receptor site, and after a long exposure period the postsynaptic receptor responds by decreasing the number of available receptor molecules and therefore the response to ACh. In contrast, the presynaptic muscarinic autoreceptors respond rapidly to release of ACh by reducing successive release. Both responses will increase the tolerance to OP. There are five subtypes of mAChRs, m l - m 5 . Subtypes ml, m3, and m5 are coupled to the A subunit of the Gq/11 257
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258
SECTION III- E s t e r a s e s ,
Receptors,
class of G proteins. This activates phosphatidylinositolspecific phospholipase C, resulting in the production of diacylglycerol and phosphoinositol. On the other hand, subtypes m2 and m4 are negatively coupled to adenyl cyclase through A subunits of G protein Gi/0 (Bonner, 1989). The assay of mAChR is generally performed using binding of labeled QNB, which binds all subtypes. QNB can be used to assay mAChR both at the plasma membrane and in the cytoplasm when it is internalized. In contrast, N-methyl scopolamine (NMS) is water soluble and does not penetrate cell membranes; therefore, it measures only the mAChR at the plasma membrane and not when it is internalized. NMS binding has thus been found to be more sensitive than binding with QNB. In two cell lines, NG108-15 and SK-N-Sh, carbachol caused a loss in NMS binding, but the binding of QNB was unchanged by the same treatment, indicating internalization of the receptor (Baumgold et al., 1989). Subtype ml can be measured with labeled telencipine and m2 with AF-DX-384 and 4-DAMP.
III. POSTSYNAPTIC RECEPTORS Treatment with a cholinergic agonist for a prolonged time leads to a decrease in the mAChRs. This is common for G protein-linked receptors. The downregulation occurs by means of receptor internalization and degradation, a process that takes several hours. Downregulation of mRNA can also occur with G protein-linked receptors. For example, Chinese hamster ovary (CHO) cells stably transfected with m l subtype were exposed to the cholinergic agonist carbachol for 24 hr, resulting in a 66% decrease in mAChR binding and a 73% decrease in the ml mRNA level (Wang et al., 1990). Likewise, in cultured granule cells both subtypes m2 and m3 were reduced after carbachol treatment. This also resulted in a decrease in cAMP production (50%) and a reduction in the corresponding mRNAs by 30-50% (Fukamauchi et al., 1993). In contrast, the m4 subtype of neuroblastoma cell was reduced 30% by 24-hr carbachol treatment, but in this case there were no effects on the corresponding m4 mRNA. The hypothesis that tolerance to chronic treatment with OP anti-ChE may be due to an inactivation of the cholinergic receptor was first proposed by Brodeur and DuBois (1964). This was later shown to be one of the most important ways for a cell or an individual to obtain tolerance toward AChE inhibitors. Early, it was shown that there was a reduction in the number of mAChRs after AChE inhibition in the brain or peripheral nervous system (Ehlert et al., 1980; Schiller, 1979; Gazit et al., 1979). Some authors reported a decrease in both the number of mAChRs and the affinity to the ligand in ileum and striatum (Costa et al., 1981), but most authors reported only a decrease in the number of mAChRs in the brain.
Mechanisms,
& Tolerance
Development
The decrease in mAChRs depends on both the time of AChE inhibition and the degree of inhibition. Schwab et al. (1981) and Costa et al. (1981) showed that there were no immediate effects after acute inhibition with OP. Jett et al. (1994) injected parathion over 21 days, which resulted in 84-90% AChE inhibition in the brain without overt signs of toxicity. To their surprise, they found a reduction in ml subtype and m l mRNA in the frontal cortex and m4 subtype and m4 mRNA in striatum but no change in hippocampus mAChR, although AChE inhibition was similar in all three regions. They concluded that the hippocampus had a different feedback mechanism for regulation of mAChRs. Yagle and Costa (1996) studied the effect of disulfoton (2 mg/kg) administration for 2 weeks on proteins and mRNA levels of mAChR subtypes in brain tissue (ml and m2) and peripheral mononuclear cells (m3). AChE activity in the cerebral cortex was 19% of uninhibited activity after 14 days and 90% after 42 days. QNB binding was 72% of uninhibited activity after 14 days and 99% after 42 days. The m l subtype dominated in cortex, hippocampus, and striatum, whereas m2 dominated in medulla and cerebellum. In contrast, there was no difference between the effect in the different regions. Thus, after the treatment, ml and m2 mRNA were reduced approximately 25% in the hippocampus, whereas in the medulla m2 mRNA was reduced 19%. Subtype m3 was reduced 10% in the cortex but not in lymphocytes. After 4 weeks, only m2 mRNA was reduced 29% in hippocampus, but this may have been caused by the pathology of some hippocampal cells. Yagle and Costa (1996) treated rats with 1 mg/day DFP and 2 mg/day disulfoton for 14 days. In both cases, AChE inhibition was 71-77%. QNB binding in cortex, striatum, and hippocampus was reduced 16-28%, but there was no change in ligand affinities. These animals showed a reduction in performance when tested on a spontaneous alternation task in a T-maze. The authors linked the reduction in performance to the decrease in mAChRs. In mice exposed to parathion in the diet, there was a large variation in AChE inhibition due to intake of food (Jett et al., 1993). In these animals, there was a correlation between AChE inhibition and reduction in both QNB binding and 4-DAMP binding but no effect on the affinities for these ligands. The maximal reduction in binding for these was 58%. In contrast, NMS binding was also reduced to 58%, but this occurred at only 10% AChE inhibition. A correlation between ChE inhibition and QNB and AF-DX 384 binding in the cortex and striatum of young and adult rats was also found by Liu et al. (1999). They gave young rats (age 7 days) and adult rats (age 90 days) chlorpyrifos and methyl parathion for 7 days and analyzed for ChE inhibition and muscarinic binding at 1 and 7 days postexposure. With chlorpyrifos, which inhibited the young rats slightly better than the adults, they found a linear correlation between ChE inhibition (mainly ACHE) and QNB or AF-DX 384 binding in both young and adult rats.
CHAPTER 1 9 9Tolerance Development In both cerebral cortex and striatum, 80% inhibition of ChE gave resulted in an approximately 30% reduction in both QNB and AF-DX 384 binding. With methyl parathion, younger rats were inhibited significantly more than adults, but again there was a linear correlation between ChE inhibition and receptor binding. Eighty percent ChE inhition gave a 20% reduction of QNB and AF-DX384 in adult cortex and 40% reduction in young cortex. In striatum, 80% ChE inhibition gave 20 and 30% reductions in QNB and AF-DX384 binding, respectively, in the adult and 30 and 35% in the corresponding young rats. The tolerance to hypothermia observed after treatment with OP is regarded as due to a decrease of mAChRs in the temperature-regulated region of the brain (Overstreet and Yamamura, 1979; Russel et al., 1975). In summary, studies on AChE inhibition in the brain show that there is generally a correlation between AChE inhibition and a reduction in mAChR binding. In general, the degree of AChE inhibition is higher than that of reduction in receptor binding. Also, NMS, which can measure only extracellular mAChRs, tends to be more sensitive than the other antagonists that also measure internalized receptor molecules. The reduction in mAChRs is similar in different brain regions. In almost all cases examined, there was a reduction in the number of muscarinic binding sites and no effect on affinity to the binding sites after exposure to AChE inhibitors. A complex factor in the discussion of effect between OPs and mAChRs is the direct action between several OPs and mAChRs (Ward et al., 1993; Huff et al., 1994). Active OPs, such as paraoxon, malaoxon, and chlorpyrifosoxon, inhibit forskolin-stimulated cAMP formation. The OP seems to inhibit 1,3-dioxalan, which is a high-affinity ligand of the m2 subtype. The effect of OP on dioxalan binding occurs in the same concentration range as that for AChE inhibition. OPs have different effects on the binding of dioxalan; parathion decreases and chlorpyrifos increases binding. It has been suggested that this may explain why rats treated with the maximally tolerated dose of parathion exhibited a higher degree of acute toxicity than those with similar treatment with chlorpyrifos (Chaudhuri et al., 1993).
IV. M U S C A R I N I C A U T O R E C E P T O R S Autoregulation of ACh release seems to be an important function of presynaptic mAChRs (Molenaar and Polak, 1980; Nordstrom and Bartfai, 1980, 1981; Dolezal and Wecker, 1991; Kirkpatrick and Richardson, 1993). This is a common feature of G protein-linked receptors. The importance of the autoreceptor is demonstrated by the fact that the addition of atropin, a well-known cholinergic antagonist, to various brain slices or peripheral tissues increased the release of ACh. In contrast, the cholinergic agonist
259
carbachol inhibited the release of ACh (Nordstrom and Bartfai, 1980; Aas and Fonnum, 1986; Dolezal and Wecker, 1990). From studies using antagonists specific for m2 and m4, it was suggested that the regulation of ACh release from brain tissue is controlled by these two subtypes (Quirion et al., 1995). By use of antisense to the m2 receptor and by using a very specific m4 antagonist in microdialysis of hippocampus, it was shown that the responsible subunit in hippocampus is m2 (Kitachi et al., 1999). Studies on ACh release from brain slices from m2/m4 receptor single knockout mice confirmed that autoinhibition of ACh release is mediated primarily by the m2 receptor in the hippocampus and cerebral cortex but predominantly by the m4 receptor in the striatum (Zhang et al., 2002). The presynaptic autoreceptor reduces the i'elease of ACh and thereby reduces the cholinergic effects on the postsynaptic receptors. The mechanism of the inhibition of ACh release by stimulation of presynaptic G protein-coupled receptors in the striatum is due to a restriction of the activity of the high-voltageactivated N or P/Q calcium channels (Krejci et al., 2004.) This restriction takes place by liberated [3/~/subunits of the receptor, which interact with the activated calcium channels and inhibit calcium influx. The inhibition of ACh release by autoreceptors has therapeutic consequences in Alzheimer's disease. AChE inhibitors used in therapy can inhibit the release of ACh. A consequence may be that use of high-affinity muscarinic antagonists can prevent the inhibition of ACh release and facilitate learning and memory in experimental animals (Quirion et al., 1995).
V. N I C O T I N I C A U T O R E C E P T O R S Whereas muscarinic autoreceptors inhibit ACh release, nicotinic autoreceptors enhance ACh release. To identify the effect of this autoreceptor, it is necessary to add atropine to inhibit the muscarinic autoreceptor. Under such conditions, ACh (3-100 p~M) increased the release of ACh from brain synaptosomes (Wu et al., 2003). The nicotinic antagonist mecamylamine, but not nicotine, inhibited the release. In addition, several anti-ChEs have a direct effect on the nicotinic receptor (nAChR), although the consequences are not known. The most potent OPs to inhibit the a4b2 nAChR were disulfoton, parathion, methyl, parathio n, and fenthion. The oxidized OPs are the best AChE inhibitors and also the best to interact with the nAChR. Chlorpyrifosoxon, the active metabolite of chlorpyrifos, inhibited the release in vitro and also inhibited the release when given 96 hr before testing (Wu et al., 2003). The oxidized metabolites of both parathion and chlorpyrifos inhibit the nAChR by binding to a site different from the ACh site (Katz et al., 1997). This desensitizes the receptor. One could therefore argue that the effect of these OPs will be to postpone the symptoms of anti-AChE poisoning. The
260
SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
toxicological relevance of this effect lies in the fact that a number of neurotoxic effects cannot be readily explained by AChE inhibition. In this respect, it is interesting that farmers chronically exposed to OP pesticides have increased anxiety, a symptom related to brain nAChR. In fact, nicotinic agonists have been suggested for use in the treatment of Alzheimer's disease since they increase ACh release. Nicotinic agonists improve working memory performance in animals blocked by muscarinic antagonists (Quirion et al., 1995.) In postsynaptic nAChRs of the electric organ of Torpedo and muscle endplate, it has been shown that OPs such as echothiopate, DFP, and VX block the open ion channel of the receptor and desensitize the receptor (Bakry et al., 1988; Eldefrawi et al., 1988).
VI. ADENOSINE RECEPTORS Adenosine has been shown to have powerful inhibitory actions on the neuronal activity and on release of excitatory transmitters (Kirkpatrick and Richardson, 1993; Broad and Fredholm, 1996; Jin and Fredholm, 1997). Adenosine is released from both neurons and glia. At the vertebrate neuromuscular junction and in the electric tissue of the Torpedo, ATP is known to be coreleased with ACh from the synaptic vesicles. Since they interfer with ACh release, release of ATP or changes in the adenosine receptor can therefore be expected to be involved in tolerance to OP poisoning. Both endogenous and added ATP will modulate ACh release. Adenosine interacts with the A 1 and A2 receptors. The A1 receptor is widely distributed in the brain, whereas the A2 receptor is mainly localized to a dopaminerich region such as the striatum. In general, activation of the A1 receptor will reduce ACh release, whereas activation of the A2 receptor in the few regions to which it is linked in the cholinergic terminal will increase ACh release. In agreement, in perfused rat cortical slices, the A 1-selective agonist N6-cyclopentyladenosine (CPA) caused a dosedependent inhibition of ACh release, which was attenuated in the presence of the Al-selective antagonist 1,3-dipropyl8-cyclopentylxanthine (1 txM). In neostriatum, the evoked release of [3H]ACh was significantly enhanced by the A2a agonist CGS 21680 but decreased by the A1 agonist R-PIA. The effects of NECA, which is equipotent in both receptor subtypes, were dependent on the concentration used. The evoked release of [3H]-ACh was inhibited with high concentrations of NECA and enhanced by low concentrations. After evaluating drugs that may be able to prevent and counteract ACh accumulation during OP intoxication, it was concluded that adenosine receptor agonists are promising candidates (van Helden et al., 1998). The selective adenosine A1 receptor agonist CPA prevented the clinical symptoms that are directly associated with such intoxication. CPA (1 or 2 mg/kg) effectively attenuated the cholin-
ergic symptoms and prevented mortality in lethally tabunor satin-intoxicated rats. Intracerebral microdialysis studies revealed that during OP poisoning, CPA-treated animals showed a minor elevation of extracellular ACh concentrations in the brain relative to the baseline value, whereas an 11-fold increase in transmitter levels was observed in animals not treated with CPA (Bueters et al., 2003). In the motor endplate, the excitatory A2a receptors regulate the autofacilitation of ACh release by interaction with the presynaptic nAChR. This probably occurs through an adenylate cyclase cAMP-dependent mechanism (Correira-de-Sa and Ribeiro, 1994). Blockade of A2 receptors potentiated, and blockade of A1 prevented the effect of the presynaptic nAChRs on Ach release. The nAChR agonist dimethyl-4-phenylpiperazinium causes facilitation of ACh release when applied for a short time and inhibition when applied for a long time. This change from facilitation to inhibition is prevented if endogenous extracellular adenosine is removed.
VII. PRESYNAPTIC CHANGES The miniature endplate potential (MEPP) amplitude is determined by AChE activity, the sensitivity of the postsynaptic receptors, or the amount of ACh per quantum. It is not easy to differentiate between the three parameters. Several electrophysiological studies on MEPP frequency in diaphragms from DFP-treated animals suggest that changes take place presynaptically, which may imply adaptation to the regulation of transmitter release (Melchers and van Helden, 1990; Carlson and Dettbarn, 1983). During AChE inhibition, there is an increase in ACh in the brain. There is no strong evidence that the synthesis of ACh is reduced, and there is no change in choline acetyltransferase (CHAT) in brains of rats tolerant to DFP or paraoxon (Russel et al., 1975; Wecker et al., 1977).
VIII. T O L E R A N C E IN AChE KNOCKOUT MICE Inhibitors of ACHE, such as OPs, are fast acting and lethal; it is therefore a surprise that a knockout animal for AChE can survive for almost 1 year. The animal must therefore have developed maximal tolerance to a high concentration of ACh. Investigation of the peripheral nervous system using the phrenic nerve-hemidiaphragm showed that the knockout animal had developed markedly greater twist tensions and slower rise and relaxation time. To compensate for the absence of AChE activity, the size of the endplate was reduced. This may provide some compensation because less ACh is provided. The juctional folds of the endplate and the number of nAChRs were heavily reduced. This allowed smaller responses to ACh and also more ACh to diffuse out
CHAPTER 1 9 9Tolerance Development of the synaptic gap. BuChE activity was not increased nor had its general localization changed. Inhibition of BuChE activity led to larger responses, suggesting that the enzyme hydrolyzed the diffused ACh molecules and that this had a positive effect (Adler et al., 2004). In the central nervous system, AChE was also eliminated. However, there were no effects on the level and localization of C h A T or on the ACh vesicle transporter. There was an increase in the choline plasma membrane transporter probably to compensate for less choline being available as a consequence of reduced hydrolysis of ACh in the synaptic gap. There was a major reduction in mAChR number, including subtypes ml, m2, and m4, and there was also a dramatic internalization of the mAChR subtypes. There was no change in the number of 132 subunits of the nAChR. It therefore seems that the most important way to obtain tolerance in AChE knockout animals is by a reduction in receptors and not by regulating ACh synthesis (Mesulam et al., 2002; Volpicelli-Daley et al., 2003).
IX. ENZYMES A. Carboxylesterase A total exposure of several times the acute LDs0 dose over a prolonged period can be tolerated by rodents when exposed repetitively to OP compounds such as schradan (Rider et al., 1952), demeton (Barnes and Denz 1954a,b), disulfoton (Brodeur and Dubois, 1964; Stavinoha et al., 1969), DFP (Glow et al., 1966; Russel et al., 1975), paraoxon (Wecker et al., 1977), dichlorvos (Sterri, 1981), sarin (Fonnum and Sterri, 1981; Sterri, 1981), or soman (Sterri et al., 1980, 1981; Fonnum and Sterri, 1981; Sterri, 1981). For several of these compounds, the spontaneous reactivation of inhibited AChE may explain part of the tolerance. For soman, however, the tolerance cannot be related to any recovery of AChE since soman is a completely irreversible AChE inhibitor (Coult et al., 1966). The tolerance of rodents to repetitive exposure to soman or sarin can to a large extent be explained by the binding of soman or sarin to high amounts of CarbE in rodent plasma and the subsequent spontaneous recovery of that enzyme (Sterri et al., 1980, 1981; Fonnum and Sterri, 1981; Sterri, 1981). After repetitive administration of sublethal doses of soman or sarin in rat, guinea pig, and mice at several constant time intervals, the cumulative LDs0 increased when the time interval between doses increased, and in some cases 50% mortality was not reached. The tolerance seemed to be most pronounced in guinea pigs. Thus, a large majority of the guinea pigs could tolerate at least a cumulative dose of 5.5-fold acute LDs0 of soman administered as a 0.5 LDs0 injection every 24 hr for at least 11 days without exhibiting distinct symptoms of poisoning. When soman was administered every 4, 8, 12, or 24 hr, the investigators
261
noted a shift toward higher activity of AChE in rodent brain and diaphragm when the time interval between soman doses was increased. They also observed that after each soman dose, the inhibited CarbE in the plasma fully recovered within 24 hr. At this time, the BuChE activity in guinea pig plasma was partly restored to approximately half of full activity (Fonnum and Sterri, 1981; Sterri et al., 1981; Sterri, 1981). The results indicated that soman reached the bloodstream but was partially prevented from reaching the target AChE of other tissues due to a retention mechanism in the blood that was fully restored within 24 hr and therefore could be the plasma CarbE. Similar recovery of rat plasma CarbE following sarin intoxication was observed by Polak and Cohen (1969). The previous results seem to satisfy the first two of three criteria for effective removal of repetitively administered soman or satin by a scavenger present in the blood: 1. The reaction rate between plasma CarbE and soman or sarin is rapid enough to pick up the compound before it leaves the blood. 2. The plasma CarbE is spontaneously and sufficiently recovered before another dose of soman or satin is administered. 3. The plasma CarbE is present in a sufficient amount to manage each dose of soman or satin. The latter criterion may also be fulfilled since both sarin and DFP have been shown by radioactive labeling to combine with large amounts (micromolar concentrations) of the CarbE protein in plasma of guinea pig and rat (Myers, 1952; Goutier, 1956; Christen and Cohen, 1969; Polak and Cohen, 1970), in accordance with reports by Aldridge (1953) and Augustinsson (1959) on the abundance of CarbE protein in rodent plasma. It follows that the concentration of the CarbE protein in rodent plasma is critical for tolerance to both repetitive and single exposure of highly toxic ChE inhibitors. In accordance, the decreasing acute soman toxicity (increasing LDs0) observed in young rats between 5 and 31 days of age was found to be highly correlated with the increasing content of endogenous plasma CarbE of these rats (Fonnum et al., 1985; Sterri et al., 1985). Also, a linear correlation was seen between soman toxicity and changes in plasma content of CarbE in aging rats (Maxwell et al., 1988). In addition, young rats with a low content of endogenous CarbE in the plasma (14 days old) showed strongly reduced mortality when an exogenous CarbE protein partially purified from liver was intravenously added into the plasma before soman exposure (Fonnum et al., 1985). A scavenger function of rodent plasma CarbE able to prevent the highly toxic ChE inhibitors from reaching the AChE in the brain and diaphragm depends on the fact that CarbE, which is a B-esterase (Aldridge, 1953; Jansz et al., 1959; Aldridge and Reiner, 1972), is able to bind and thereby detoxify the ChE inhibitors at the active site
262
SECTION III. E s t e r a s e s ,
Receptors,
in equimolar proportions. The quantitative contribution by plasma CarbE to rodent tolerance for repetitive exposure to the most toxic ChE inhibitors may therefore be elucidated by pretreatment with specific inhibitors of CarbE. In both rats and guinea pigs pretreated with the CarbE inhibitor TOCP (tri-ortho-cresyl-phosphate) (Mendel and Myers, 1953), the estimated rate of detoxification of repetitively administered soman was reduced to one-seventh of the rate without TOCP treatment (Sterri, 1981; Fonnum and Sterri, 1981; Sterri and Fonnum, 1984), which means that 80-90% of the repetitively administered soman may be inactivated through binding to the TOCP-sensitive active site of CarbE. The CarbE inhibitors CBDP (2-[o-cresyl]4H-1,2,3-benzodioxa-phosphorin-2-oxide) (Casida et al., 1961; McKay et al., 1971) and iso-OMPA (tetraisopropylpyrophosphoramide) were used by Yang and Dettbarn (1998) and Dettbarn et al. (1999) to show the significant protection by plasma CarbE toward the toxicity of repetitive paraoxon exposure in rat. Also, Dettbarn et al. noted that the plasma CarbE recovered rapidly following inhibition by paraoxon but recovered slowly following inhibition by DFP, in accordance with the fact that CBDP or iso-OMPA treatment counteracted the tolerance for repetitive exposure to paraoxon but had no effect on the tolerance for repetitive exposure to DFE CarbE inhibitors have been used by several investigators to evaluate the tolerance against single exposure to highly toxic OP compounds. In mice, the LDs0 of soman was reduced to approximately 1/18 by pretreatment with 35-50 mg/kg CBDP (McKay et al., 1971; Boskovic, 1979), whereas in guinea pig and rat the acute LDs0 of soman was reduced to one-third by pretreatment with 100 mg/kg TOCP (Sterri, 1981; Fonnum et al., 1981; Sterri et al., 1981). The LDs0 of satin was reduced to approximately one-fifth by pretreatment with 40-50 mg/kg TOCP in rat (Myers, 1959; Polak and Cohen, 1969). From studies of the CBDP effect on LDs0 for several OP compounds, Maxwell (1992) reported significant protection by CarbE against acute toxicity of the highly toxic compounds soman, satin, tabun, and paraoxon but not against the less toxic compounds, such as dichlorvos and DFE Also, only a marginal effect of TOCP pretreatment on LDs0 for dichlorvos was observed (Sterri, 1981). This may be due to the fact that the less toxic ChE inhibitors lead to toxic concentrations in rodent blood that are much higher than the plasma concentration of CarbE. However, some of the less toxic OP compounds will be hydrolytically detoxified through the enzymatic activity of CarbE due to their carboxylic ester linkages, which leads to more than a 100-fold increase in the acute toxicity of malathion by pretreatment with the CarbE inhibitor TOTP (tri-orthotolyl-phosphate) (Murphy et al., 1976). The ability of rodents to tolerate the repetitive exposure to soman or satin seemed to be a stable and invariable quality throughout the experimental period. Thus, when
Mechanisms,
& Tolerance
Development
cumulative LDs0 values were plotted against the ratio between the cumulative LDs0 and the dosing regimen (single doses given divided by the time interval between them), a series of linear relationships were found (Fonnum and Sterri, 1981; Sterri, 1981; Sterri and Fonnum, 1984). The linearity existed for each of the rodent species m rat, guinea pig, and mouse m and for both the ip and the sc route of administration. The correlation coefficients were better than 0.93. The linearity indicates that there is no increasing tolerance acquired during the repetitive administration of soman or sarin. This is consistent with criterion (2), which states that the CarbE protein in rodent plasma acts as a scavenger that is constantly renewed for each of the repetitive doses of soman or sarin every 24 hr. Whether the very important recovery of soman-inhibited plasma CarbE for the rodent tolerance to repetitive exposure (Sterri et al., 1981) is due to spontaneous reactivation or de novo synthesis may be a matter of interest. Originally, the recovery of soman-inhibited CarbE of guinea pig plasma within 24 hr was proposed to be due to de novo synthesis (Sterri, 1981), which may be a general apprehension based on the fact that the other soman-inhibited B-esterases (ACHE and BuChE) exhibit rapid aging and do not reactivate spontaneously. However, in a study using commercial plasma CarbE from rat, Maxwell and Brecht (2001) observed that the soman-inhibited rat plasma CarbE does not undergo aging and will reactivate spontaneously. It was suggested by the same authors that the spontaneous reactivation may be due to a highly conserved histidine located in the active site of CarbE but not present in AChE and BuChE. The position of histidine is similar to that of the histidine introduced by Millard et al. (1995) to produce an OP-hydrolyzing mutant of human BuChE. Polak and Cohen (1969) concluded that the observed disappearance of 3ap-labeled sarin from rat plasma within 24 hr is in accordance with spontaneous reactivation of CarbE. That satin-inhibited rat plasma CarbE reactivated spontaneously was confirmed by Maxwell and Brecht (2001). Species differences between rodent plasma CarbEs with respect to reactivatability by DAM (diacetyl monoxime) after OP inhibition were reported by Myers (1959) and Cohen et al. (1971). Their results may indicate possible species differences with respect to spontaneous reactivation of OP-inhibited plasma CarbEs as well, which would lead to some different degree of tolerance to repetitive soman or sarin exposure between rodent species, as was observed between guinea pig and mouse for repetitive soman exposure (Sterri et al., 1981). The tolerance of rodents to repetitive doses of highly toxic OP compounds based on the criteria mentioned previously include the capacity of OP-inhibited rodent plasma CarbE to recover spontaneously. The tolerance to a single dose, although independent of CarbE recovery, is based on the same criteria, which established rodent plasma CarbE as a functional scavenger of significant importance for
CHAPTER 1 9 9Tolerance Development detoxification of highly toxic OP compounds such as the nerve agents satin and soman. The scavenger function of plasma CarbE is obviously of great importance for the understanding of soman detoxification in rodents, but it may be even more important for the understanding of the soman toxicity in both rodent and nonrodent species with lower plasma content of CarbE. Rat, guinea pig, and primates including man are species with high, medium, and minor (zero) concentrations of plasma CarbE, respectively (Myers, 1952; Aldridge, 1953; Christen et al., 1969; Christen and Cohen, 1969; Cohen et al., 1971). These species were included in a mathematical approach to describe the influence of the CarbE concept on soman toxicity (Sterri and Fonnum, 1989). The approach could in principle explain the differences in LDs0 of soman between species that possess different plasma CarbE concentrations, and it could in principle also explain the differences between the same species with respect to the effect of pyridostigmine prophylaxis toward soman poisoning. Thus, from the CarbE concept as presented by Sterri and Fonnum (1989), both the theoretical LDs0 of soman and the theoretical protection factor of carbamate prophylaxis could be predicted for rat, guinea pig, marmoset, and rhesus monkey (or man). The LDs0 and protection factor found agree with the experimental value for subcutaneous LDs0 of soman in rat (Sterri et al., 1980, 1985), guinea pig (Sterri et al., 1981), marmoset, and rhesus monkey (Dimhuber et al., 1979) as well as with the experimental protection factor of pyridostigmine prophylaxis against soman in the same rodent (Gordon et al., 1978) and primate species (Dimhuber et al., 1979). Based on general knowledge of experimental LDs0s of soman, this agreement suggests that rodent plasma CarbE may be a main scavenger for soman by both inhaled iv and sc administration, whereas by pc and ip administration there may be additional dermal and hepatic parameters of threshold for soman (Sterri, 1989). Plasma CarbE of rodents is nature's example of a functional scavenger for the highly toxic OP compounds, which might inspire investigators to use artificial scavengers to protect against such compounds in human. The CarbE scavenger concept therefore includes the important possibility to estimate the efficiencies of any artificial scavenger added into human plasma at adequate concentrations to counteract the toxicity of OP compounds, and in principle such estimates were calculated by Sterri (1989). A well-acting artificial scavenger may be achieved by the prophylactic administration of exogenous CarbE, ACHE, or BuChE into the bloodstream, which was demonstrated to efficiently protect against OP poisoning in rodent and primate (Fonnum et al., 1985; Wolfe et al., 1987; Raveh et al., 1989, 1997; Broomfield et al., 1991; Maxwell et al., 1991, 1992; Doctor et al., 1993). The protection against soman may be further enhanced by DAM treatment, provided that the chosen scavenger is a CarbE reactivated by DAM. One must take into account that the DAM reacti-
263
vatability of the various soman-inhibited CarbEs differed between species and between tissues (Fonnum et al., 1985; Sterri and Fonnum, 1987; Sterri, 1989).
B. Acetylcholinesterase and Butyrylcholinesterase The spontaneous reactivation of dimethoxy phosphorylated ChEs is much more rapid than that of the corresponding diethoxy or phosphonyl compounds (Burgen and Hobbiger, 1951; Aldridge and Reiner, 1972). In accordance, repetitive exposure at adequate time intervals to a dimethoxy OP at a sublethal dose may be highly tolerable due to the recovery of ACHE, as was observed for dichlorvos (Sterri, 1981). When 0.4 LDs0 of dichlorvos was administered to rats every 24 hr, marked symptoms of poisoning were seen within 10 min, but the animals recovered from the symptoms in 1 or 2 hr and a cumulative LDs0 dose was not achieved. The symptoms and recovery from symptoms correlated well with the time frame of inhibition and spontaneous reactivation of AChE as well as BuChE in different tissues. AChE activity was inhibited and recovered spontaneously to a similar extent in the target organs, brain and diaphragm, as in the blood, consistent with a minimal role of plasma CarbE for tolerance to acute or cumulative toxicity of dichlorvos (Sterri, 1981). In contrast, the tolerance to cumulative toxicity of soman or sarin is only marginally influenced by binding to plasma BuChE. This is due to the insufficient recovery of plasma BuChE observed after each dose of soman or sarin in the repetitive exposure experiments (Fonnum and Sterri, 1981; Sterri et al., 1981; Sterri, 1981) and the fact that the large concentrations (micromolar) of plasma CarbE have not been reported for plasma BuChE. Grubic et al. (1988) excluded any effects of plasma BuChE on tolerance to acute soman toxicity after using iso-OMPA to obtain differentiated inhibition of plasma BuChE and CarbE. Recovery of AChE activity by de n o v o synthesis has been reported to be vital for tolerance to repetitive exposure to DFP in rat, especially with respect to restoration of AChE activity in skeletal muscle (Gupta et al., 1986a; Gupta and Dettbarn, 1986; Dettbarn and Gupta, 1989). Also, modifications in the affinity of AChE for ACh in paraoxon-tolerant rats have been reported by Milatovic and Dettbam (1996), who observed a 20-25% increase in affinity (decrease in Km) for ACh by both the brain and the diaphragm ACHE.
C. Phosphoric Triester Hydrolases Aryldialkylphosphatase/paraoxonase (PON) (EC 3.1.8.1) and DFPase/somanase (EC 3.1.8.2) are enzymes hydrolyzing OP compounds with generally low affinity (Km, 0.1-10 mM) (Aldridge and Reiner, 1972). The plasma enzymes may therefore require relatively high concentrations of
264
SECTION I I I . E s t e r a s e s ,
Receptors,
OPs present in the blood to be able to effectively influence tolerance to OP toxicity, and they may be of interest m a i n l y with respect to tolerance to the less toxic OP compounds. This is in accordance with the fact that PON1 knockout mice were extremely sensitive to diazoxon but did not show increased sensitivity to paraoxon (Li et al., 2000). Also, following injection of exogenous PON1 to the knockout mice, Li et al. observed protection against diazoxon and chlorpyrifos-oxon, but not against paraoxon. The results of Karanth and Pope (2000) are consistent in this respect since they concluded that both PON and CarbE can be correlated with the acute sensitivity to chlorpyrifos and parathion in rat, but plasma CarbE may play a more pivotal role in the sensitivity to parathion. The observed tolerance to repetitive administration of soman in rat could not be explained by any increase in liver somanase during the 11-day experiment (Sterri et al., 1980). However, liver perfusion experiments with soman demonstrated 80-90% detoxification of soman during a single pass through the liver, and this was not due to inhibition of ChE or CarbE (Sterri et al., 1983). The high capacity of soman detoxification in liver may therefore mainly be due to the somanase activity, and it may explain the tolerance to acute toxicity of soman by ip administration compared to sc administration (Fonnum and Sterri, 1981).
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Organ Toxicity
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CHAPTER
~ 0
Central Nervous System Effects and Neurotoxicity CAREY N. POPE Oklahoma State University, Stillwater, Oklahoma
choline acetyltransferase (CHAT), or high-affinity choline uptake (HACU) in suspected neuronal projection sites. Histochemical methods for AChE were also used, but it was recognized that this method was somewhat nonspecific because AChE was located in both cholinergic and noncholinergic neurons. Autoradiography using cholinergic receptor radioligands and immunocytochemical methods using antibodies to ChAT and nerve growth factor (NGF) receptor led to a detailed description of cholinergic pathways in the brain (Kasa, 1986; Springer et al., 1987; Woolf, 1991; Tohyama and Takatsuji, 1998). ACh released from cholinergic neurons activates nicotinic and muscarinic receptors on innervated cells. Of these two basic types of cholinergic receptors, muscarinic receptors predominate in the cerebral cortex. Five subtypes of muscarinic cholinergic receptors (ml-m5) have been recognized (Bonner, 1989). In addition to postsynaptic receptors, presynaptic cholinergic receptors are found on cholinergic neurons and participate in regulation of ACh release in a "feedback" control mechanism. In this regard, muscarinic autoreceptors are generally inhibitory for ACh release (Weiler, 1989; Feuerstein et al., 1992; Kitaichi et al., 1999; Zhang et al., 2002a), whereas nicotinic autoreceptors typically stimulate ACh release (Marchi and Raiteri, 1996; Marchi et al., 1999; Re, 1999; Wu et al., 2003). Cholinergic nuclei in the CNS are typically small, discrete regions that provide diffuse innervation to wide areas of the brain (Mesulam et al., 1983; Kasa, 1986; Woolf, 1991). Six major cholinergic cell nuclei and their innervating fibers have been characterized (Mesulam et al., 1983; Cooper et al., 2003; Lucas-Meunier et al., 2003). Table 1 shows these cholinergic nuclei and their principal projection areas.
I. INTRODUCTION Organophosphorus (OP) and carbamate (CM) insecticides can elicit profound alterations in central nervous system (CNS) function by inhibiting acetylcholinesterase (ACHE). With severe, lethal intoxications, the most important central action of many AChE inhibitors is respiratory depression. Neurobehavioral alterations are also commonly seen following AChE inhibition in the CNS. Although the effects of the OP and CM insecticides are generally elicited through AChE inhibition, some have been shown to interact with other macromolecules in the CNS, which may modify cholinergic toxicity or affect other signaling pathways. A well-characterized neurotoxic consequence of exposure to some OP toxicants unrelated to AChE inhibition is OP-induced delayed neurotoxicity, associated with inhibition of the enzyme neuropathy target esterase. Disruption of adhesion, neurite outgrowth, and other cellular actions by anticholinesterase (anti-ChE) potentially leading to neurodevelopmental toxicity has been reported by a number of laboratories. Some of these topics are covered in other chapters. This chapter focuses on prominent, overt effects elicited by AChE inhibition in the CNS. Issues concerning central effects of anti-ChEs stimulated by research on Gulf War illnesses (i.e., the neurotoxicity of the prophylactic drug pyridostigmine and the potential for chronic effects following low-level anti-ChE exposures) are also briefly reviewed. To appreciate the possible spectrum of effects of anti-ChEs due to disruption of acetylcholine (ACh) breakdown in the CNS, a review of the distribution of cholinergic neurons and pathways is important.
II. CHOLINERGIC NEURONS AND PATHWAYS IN THE CENTRAL NERVOUS SYSTEM
A. Cholinergic Basal Forebrain
Visualization of central cholinergic pathways was achieved through use of aspiration, electrolytic or radiofrequency lesions, and subsequent evaluation of changes in ACh, Toxicology of Organophosphate and Carbamate Compounds
The Chl-Ch4 cell groups comprise the cholinergic basal forebrain. In primates, approximately 10, 70, 1, and 90% of neurons within the Chl, Ch2, Ch3, and Ch4 cell groups, 271
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TABLE 1. Cholinergic Nuclei and Their Primary Projection Areas a
Nucleus Chl--medial septum Ch2--diagonal band of Broca, vertical limb Ch3--diagonal band of Broca, horizontal limb Ch4--nucleus basalis magnocellularis Ch5~pedunculopontine nucleus Ch6~laterodorsal tegmental nucleus
Projection areas Hippocampus, thalamus Hippocampus, thalamus Olfactory bulb, thalamus Cortex, amygdala, thalamus Thalamus, basal ganglia, pons medulla Thalamus, cortex, basal ganglia
aFromMesulamet al. (1983) and Tohyamaand Takatsuji(1998). respectively, are cholinergic in nature (Mesulam et al., 1983; Mesulam, 2000). As shown in Table 1, Chl and Ch2 are the predominant nuclei for cholinergic projections into the hippocampus. Ch3 neurons project to the olfactory bulb, whereas Ch4 neurons send cholinergic projections primarily to the cerebral cortex and amygdala. Within the hippocampus, the highest density of cholinergic fibers is seen in the molecular layer of the dentate gyms and within the CA2, CA3, and CA4 regions. In the amygdala, cholinergic innervation is highest in the central and basal lateral nuclei and less dense in the lateral nucleus, whereas the medial nucleus has essentially no cholinergic innervation. Throughout the cortex and hippocampus, NGF receptor and ChAT staining of axons is essentially equivalent. Thus, localization of NGF receptor on cholinergic fibers is considered an indicator of basal forebrain origin (Springer et al., 1987; Woolf et al., 1989). The human cortex receives dense cholinergic innervation from cells in the nucleus basalis of Meynert (Ch4; Selden et al., 1998). Analogous to rodent brain, two bundles of fibers (medial and lateral tracts) originate in the nucleus basalis and innervate the cerebral cortex and amygdala. ACHE, CHAT, and NGF receptors are expressed by these neurons.
B. Cholinergic Neurons of the Basal Ganglia The human striatum (caudate, putamen, olfactory tubercle, and nucleus accumbens), globus pallidus, subthalamic nucleus, red nucleus, and substantia nigra are densely innervated by cholinergic neurons (Mesulam et al., 1992; Zhou et al., 2003). Whereas the striatum contains primarily cholinergic intemeurons, the putamen has fibers that are positive for NGF receptor, indicating cholinergic basal forebrain origin. The striatum and globus pallidus also receive innervation from the Ch5 and Ch6 cell groups (Mesulam et al., 1983; Cooper et al., 2003). Cholinergic innervation to the red nucleus and subthalamic nucleus appears to originate primarily from Ch5 and Ch6 (Mesulam et al., 1992).
The neostriatum is composed of the caudate and putamen (Zhou et al., 2003). This region receives dopaminergic input from the substantia nigra pars compacta, ventral tegmental area, and sensorimotor cortex. Approximately 2% of neostriatal neurons are cholinergic in nature. The cholinergic intemeurons are much larger than any other neurons in the striatum. There is dense interaction among the cholinergic intemeurons and dopaminergic fibers from the substantia nigra pars compacta and ventral tegmental area. Ninety percent of the striatal neurons are medium spiny projection neurons that use GABA as a neurotransmitter and provide inhibitory output to the substantia nigra pars reticulata and globus pallidus. Together, these pathways are essential for control of coordinated movements. The ventral striatum is the ventral part of caudate and putamen that merges with the nucleus accumbens and olfactory tubercles (Zahm and Heimer, 1988; Martin et al., 1991). It receives input from the ventral tegmental area and, to a lesser degree, the substantia nigra pars compacta. The nucleus accumbens sends inhibitory innervation to the ventral tegmental area and receives excitatory input from the prefrontal cortex, hippocampus, and amygdala. The nucleus accumbens also innervates the basal forebrain nuclei and can influence arousal, attention, and cognition. The ventral striatum/nucleus accumbens appears critical for rewardbased learning and addictive behaviors (Kelley, 2004; Nestler, 2004). Dopaminergic fibers exhibit extensive branching and arborization, and volume transmission appears important in dopaminergic transmission within the striatum (Parent and Hazrati, 1994; Rice, 2000; Cragg et al., 2001). Striatal cholinergic interneurons also show extensive arborization: Dopaminergic and cholinergic fiber arborization is more extensive in the striatum than elsewhere in the mammalian brain (Yelnik et al., 1991). Furthermore, considerable overlap between dopaminergic and cholinergic cell markers is evident in the neostriatum, and cholinergic intemeurons within the striatum also appear to use volume transmission. Endogenous ACh release activates nicotinic receptors, potently activating striatal dopamine release (Zhou et al., 2001; Salminen et al.,
CHAPTER 20 9Central Nervous System Effects and Neurotoxicity 2004; Cao et al., 2005), whereas dopamine regulates ACh release in the striatum (Acquas and Di Chiara, 2001; Alcantara et al., 2003). Nicotinic ~2 subunit-receptor knockout mice show markedly less depolarization-induced dopamine release (Picciotto et al., 1998). Glutamatergic inputs from the thalamus can also influence cholinergic interneurons, increasing striatal ACh release (Baldi et al., 1995; Consolo et al., 1996; Pollack, 2001). It has been reported that striatal cholinergic interneurons fire in a variable but persistent manner, leading to pulses in striatal ACh release (Zhou et al., 2003; Wilson, 2005). Because of volume transmission, ACh in the extracellular space can diffusely activate nicotinic heteroreceptors on dopaminergic terminals, markedly elevating DA release throughout the striatum (Zhou et al., 2001, 2002; Zhang et al., 2002b). Due to the extensive arborization, a relatively small number of cholinergic interneurons can synchronize the activity of a large number of dopaminergic neurons. Muscarinic receptors have a role in cholinergic modulation of striatal output, ml and m4 receptors are located on medium spiny neurons (Yan et al., 2001; de Rover et al., 2002; Lin et al., 2004). Thus, during intense activation of cholinergic interneurons, extensive extracellular ACh levels can be reached. Nicotinic receptor activation acts rapidly on striatal output, but muscarinic receptor activation influences output on a relatively delayed time frame (Koos and Tepper, 2002; Zhou et al., 2003). In conditioned learning, firing of cholinergic interneurons is highly correlated with stimulus pairing. The dense arborization and connectivity of cholinergic and dopaminergic fibers therefore coordinates output over a large area and facilitates the cholinergic modulation of motor function, learning, memory, reward, and other processes. With conditioned movements, tonically active cholinergic interneurons stop firing, and then they increase firing after the transient pause in activity (Zhou et al., 2002; Reynolds et al., 2004; Wilson, 2005). These differential firing rates are acquired during behavioral conditioning and are lost with extinction. It is proposed that the pause or transient cessation of firing of the cholinergic interneurons initiates a sequence of events leading to change in medium spiny neuron output. Through this pathway, cholinergic interneurons appear integral to learning through the mesolimbic dopaminergic innervation.
C. Cholinergic Neurons of the Upper Brain Stem Cholinergic neurons in the upper brain stem (Ch5 and Ch6) contribute t o the reticular activating system (Kubin and Fenik, 2004). Ch5 is a group of cells in the pedunculopontine nucleus. The Ch6 group is located in the laterodorsal tegmental nucleus, with almost all cells in this nucleus being cholinergic (CHAT positive). These neurons primarily innervate the thalamus, but Ch5-Ch6 neurons also send
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projections to the cortex, basal forebrain, and extrapyramidal regions, including the striatum, globus pallidus, subthalamic nucleus, substantia nigra, and ponsmedulla. The Ch5 group is associated more with sensory processing and extrapyramidal motor control, whereas Ch6 is associated more with the limbic system. As noted previously, the majority of thalamic cholinergic projections appear to originate from the Ch5 and Ch6 nuclei. Some thalamic regions also receive cholinergic innervation from the basal forebrain. Using colabeling with ChAT and NGF receptor antibodies, however, the predominant cholinergic innervation of thalamus was shown to come from the brain stem (i.e., NGF receptor-negative fibers). The pedunculopontine nucleus (Ch5) is located in the dorsolateral part of pontomesencephalic tegmentum (Lee et al., 2000). It contains both cholinergic and noncholinergic (GABAergic and glutamatergic) neurons. As in other instances, the cholinergic neurons of the pedunculopontine nucleus are large cells (>20 ~m diameter). All Ch5 and Ch6 neurons express NADPH-diaphorase, whereas 80-90% contain substance E
D. Other Cholinergic Cells in the Central Nervous System A number of brain regions contain local cholinergic interneurons. Cholinergic interneurons are located within the caudate putamen, nucleus accumbens, olfactory tubercle, and the Islands of Calleja (Cooper et al., 2003; Wirtshafter and Osborn, 2004). Cholinergic neurochemical markers are densely concentrated in some of these areas. Cholinergic innervation of motoneuron collaterals to spinal Renshaw cells in recurrent inhibition of anterior horn cells has been known for decades. Some cortical regions also contain intrinsic cholinergic interneurons, often colocalized with vasoactive intestinal polypeptide (Kawaguchi, 1997; Bayraktar et al., 1997). Together, the widespread innervating pathways and local cholinergic interneuron networks comprise the central cholinergic system.
III. F U N C T I O N S A S S O C I A T E D W I T H CHOLINERGIC NEUROTRANSMISSION IN T H E C E N T R A L N E R V O U S S Y S T E M As briefly reviewed previously, a major theme for cholinergic systems within the CNS is the presence of discrete cholinergic cell nuclei providing widespread innervation to broad regions of the brain. All regions of the cerebral cortex receive intense and diffuse cholinergic innervation; thus, numerous cortical functions are modulated by cholinergic neurotransmission. Innervation from the Chl-Ch4 cell groups (the cholinergic basal forebrain) contributes to behavioral arousal (Jones, 2004; Boutrel and Koob, 2004).
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Basal forebrain innervation appears prominent in maintaining theta rhythm in the hippocampus, contributing to arousal-associated cortical electroencephalogram (EEG) activity. The muscarinic antagonist scopolamine can block the cortical P-300 EEG arousal response elicited by novel stimuli. Ch5 and Ch6 also modulate behavioral arousal (Moruzzi and Magoun, 1995; Kayama et al., 1992; Kayama and Koyama, 2003). Limbic and paralimbic regions of the cortex play a key role in cognitive function (Iversen, 1997; Easton and Parker, 2003). The substantial cholinergic innervation of these areas is thought to be important in memory, learning, and other functions, such as mood, affect, reward, and aggression. Experimental disruption of Ch4 innervation can cause memory deficits that can be reversed by cholinergic agonists (Wenk, 1997; Xiong et al., 1998; Wellman and Pelleymounter, 1999; Nieto-Escamez et al., 2002). The role of acetylcholine in long-term potentiation within the hippocampus may also be important in cholinergic regulation of cognition (Yamazaki et al., 2002; Anagnostaras et al., 2003; Ovsepian et al., 2004). Cholinergic innervation may also participate in plasticity and axonal sprouting (Adams et al., 2002). Sensory-limbic pathways appear to play prominent roles in a variety of behaviors involving emotion, motivation, and memory (Morgane et al., 2005). Lesions of the septohippocampal (Chl) pathway disrupt delayed nonmatching to sample performance in rats (Aggleton et al., 1992), whereas lesions of basal nucleus (Ch4) lead to passive avoidance, water maze, and radial arm maze performance deficits (Page et al., 1991; Winkler et al., 1995). These results suggest that the Chl pathway may contribute more to working memory, whereas the Ch4 pathway may have more of a role in reference memory. The cholinergic system also participates in attention/vigilance in both rodents and primates. Performance in the five-choice serial reaction task was markedly impaired by basalis (Ch4) lesions (Robbins et al., 1989). Infusion of the ACh synthesis inhibitor hemicholinium-3 disrupted attention in rats, and those performance deficits were reversed by the CM cholinesterase inhibitor physostigmine (Muir et al., 1992). Intracerebroventricular infusion of the muscarinic antagonist scopolamine in rhesus monkeys decreased attention performance (Callahan et al., 1993). In humans, the degree of enhanced performance in serial learning tasks by cholinergic agonists and, conversely, the degree of decreased performance by cholinergic antagonists were inversely related to baseline performance (i.e., the poorest performers were most susceptible to both cholinergic challenges) (Sitaram et al., 1978). The basal forebrain innervating pathways therefore appear to play a prominent role in these forms of cognition. Cholinergic neurons within the central reticular core influence sleep patterns (Jones, 1993). The Ch4 nucleus, sending fibers to the cerebral cortex, and the Ch5 cell group, innervating the thalamus and cortex, appear important in
sleep regulation (Tojima et al., 1992; Gilbert and Lydic, 1994; Haxhiu et al., 2003). ACh signaling appears important in switching the firing mode of thalamic neurons from EEGsynchronized sleep to that associated with the awake state and rapid eye movement (REM) sleep (Steriade, 2004). The Ch6 cell group is also involved in brain stem mechanisms associated with REM sleep (Rye, 1997). Increased firing of the pedunculopontine nucleus leads to REM sleep, and direct cholinergic stimulation of the pedunculopontine nucleus increases REM frequency. In contrast, slow-wave sleep is associated with decreased Ch5 activity. Respiration is under the control of cholinergic innervation. Inspiratory and expiratory centers in the pons medulla receive input from the Ch5 and Ch6 nuclei (Kubin and Fenik, 2004). In fact, the coregulation of sleep-wake cycles (as discussed previously) and respiratory output seems due to shared innervation from Ch5 and Ch6 (Haxhiu et al., 2003). These cholinergic neurons show tonic activation during both the awake state and REM sleep (Kayama et al., 1992). Furthermore, the Ch5 cholinergic neurons appear to have extensive cholinergic autoreceptor regulation (Leonard and Llinas, 1994; Rye, 1997). Muscarinic m l, m2, and m3 receptors and various subtypes of nicotinic receptors are expressed in the inspiratory and expiratory centers in the pons medulla, whereas mRNA for m2 receptors appears high in the Ch5 and Ch6 regions. Postsynaptic activation of m3 receptors in the inspiratory neurons and OL4~2 nicotinic receptors mediates inspiratory rhythm (Shao and Feldman, 2000, 2001, 2005). Direct administration of the cholinergic agonist carbachol into pontine respiratory centers depresses respiratory output (Kimura et al., 1990; Kubin et al., 1992; Fenik et al., 1998). Leonard and Lydic (1997) evaluated a role for nitric oxide (NO) in cholinergic regulation of both REM sleep and respiratory depression in cats. Local injection of the NO synthase (NOS) inhibitor NC-nitro-L-arginine (NLA) significantly reduced ACh release in the cholinergic neurons in the pedunculopontine nucleus and in the cholinergic innervated medial pontine reticular formation. Microinjection of NLA into the medial pontine reticular formation blocked REM sleep and respiratory depression elicited by microinjection of neostigmine. The duration but not the frequency of REM sleep was significantly decreased by NLA administration into the medial pontine reticular formation. Together, these findings suggest that Ch5 cholinergic regulation of awake-sleep states and respiratory control is coordinated, and that this pathway represents a primary site of action for anti-ChEs that could lead to both sleep/arousal disturbances and the prominent lethal action of respiratory depression. The influence of cholinomimetic agents on motor activity is generally attributed to the dense cholinergic network within the striatum (Gerber et al., 2001; Pollack, 2001). Cholinergic agents tend to produce involuntary movements (e.g., tremors) (Matthews and Chiou, 1979; Bymaster et al., 2003), whereas anticholinergic agents can be effective for
CHAPTER 20 9Central Nervous System Effects and Neurotoxicity treating some kinetic disorders, such as rigidity and bradykinesia of parkinsonism (Suchowersky, 2002). The globus pallidus, red nucleus, substantia nigra, and subthalamic nucleus, as well as the pendunculopontine cholinergic neurons, also participate in mediating the central effects of cholinergic agents on extrapyramidal function. Springer and Dill (1975) reported that unilateral injection of carbachol into the globus pallidus elicited dyskinesias and hypertonic contralateral hind limb movements, whereas scopolamine caused ipsilateral circling and hypotonia of the contralateral hind limbs. Carbachol injection into the neostriatum produced hyperkinetic dyskinesias and catalepsy, blocked by the simultaneous administration of scopolamine. Brudzynski and coworkers (1988) demonstrated that unilateral injection of carbachol into the pedunculopontine nucleus decreased motor activity in rats, whereas carbachol injection into sites adjacent to the pedunculopontine nucleus increased activity: Both changes were sensitive to atropine. Analgesia is also under cholinergic regulation (Furst, 1999; Eisenach, 1999). Centrally active muscarinic agonists can produce profound analgesia (Sheardown et al., 1997; Naguib and Yaksh, 1997). Similarly, nicotinic agonists (e.g., epibatidine) can elicit marked analgesia (Bannon et al., 1998; Genzen and McGehee, 2005). Using m2 and m4 receptor knockouts, both spinal and surpraspinal roles for ACh in analgesia have been demostrated (Wess et al., 2003). Cholinergic stimulation of the pedunculopontine nucleus causes antinociception: Nicotine has a potent antinociceptive effect when injected directly into the pedunculopontine nucleus (Iwamoto and Marion, 1993). As briefly reviewed, cholinergic innervation is widespread in the CNS. Alteration of cholinergic neurotransmission in the CNS by anti-ChEs could therefore affect a number of functions, including arousal, cognition, sleep-wake cycles, respiration, motor activity, and analgesia.
IV. C H O L I N E R G I C N E U R O T R A N S M I S S I O N ACh is released by cholinergic neurons in both the central and the peripheral nervous system (Pope, 1999; Cooper et al., 2003). Figure 1 shows a schematic diagram of the cholinergic synapse: Fig. la represents the "normal" condition, whereas Fig. l b shows the consequences of extensive AChE inhibition. ACh is synthesized in cholinergic nerve terminals by the action of the synthetic enzyme, CHAT, using the cofactor acetyl coenzyme A and choline. ACh molecules are packaged into synaptic vesicles by the vesicular ACh transporter. Upon terminal depolarization, synaptic vesicles fuse with the plasma membrane, and their contents, including ACh, are released into the synaptic cleft. In the synapse, ACh molecules can interact with postsynaptic cholinergic receptors (of two major subtypes ~ muscarinic and nicotinic cholinergic receptors) to alter the postsynaptic cell's function. Muscarinic receptors are G protein-coupled
275
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FIG. 1. The cholinergic synapse. (A) An action potential depolarizes the presynaptic terminal, leading to synaptic vesicle fusion and release of acetylcholine molecules (~) into the synapse. The acetylcholine molecules have a finite time available to bind to and activate the muscarinic (Mus) or nicotinic (Nic) cholinergic receptors on the postsynaptic cell membrane. Acetylcholinesterase (ACHE), one of the fastest enzymes in the body, rapidly hydrolyzes acetylcholine molecules yielding choline (D) and acetic acid (G), efficiently regulating the extent of cholinergic receptor activation. A proportion of the choline released by hydrolysis of acetylcholine is taken back into the presynaptic terminal for resynthesis of transmitter by high-affinity choline uptake (HACU). (B) The consequences of extensive AChE inhibition by a carbamate or organophosphorus anticholinesterase. With acetylcholinesterase inhibited (AChE-I), acetylcholine molecules accumulate in the synapse, leading to persistent activation of cholinergic receptors and a net increase in cholinergic signaling through the postsynaptic cell. Although presynaptic muscarinic autoreceptors can be activated, inhibiting further acetylcholine release, this adaptative mechanism is not sufficient to prevent excessive activation of postsynaptic cholinergic receptors and consequent alteration of postsynaptic cell function.
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receptors that modulate second messenger (e.g., cAMP and inositol triphosphate) formation in the postsynaptic cell but can also modulate ion flux (e.g., potassium efflux). Nicotinic receptors are ligand-gated ion channels that primarily increase sodium influx into the postsynaptic cell. ACh can also interact with presynaptic cholinergic receptors (again, of the two major subtypes muscarnic and nicotinic receptors). These presynaptic cholinergic receptors utilize the same basic signaling pathways as postsynaptic cholinergic receptors to modulate presynaptic terminal function (e.g., ACh release) in a "feedback" manner. AChE is located strategically within the synapse to exert tight control over cholinergic neurotransmission. Because AChE is one of the most active of all enzymes (each molecule can hydrolyze approximately 5000 molecules of ACh per second) (Cooper et al., 2003), ACh molecules have a very short half-life in the synapse and a consequent transient ability to activate cholinergic receptors. Upon hydrolytic cleavage of the ACh molecule, choline and acetic acid are released into the synaptic space, after which choline is transported effectively back into the presynaptic terminal by a HACU process. ACh is therefore partially recycled, increasing metabolic efficiency.
V. O V E R V I E W O F T H E C E N T R A L NERVOUS SYSTEM TOXICITY OF O R G A N O P H O S P H O R U S A N D CARBAMATE CHOLINESTERASE INHIBITORS OP and CM insecticides disrupt cholinergic neurotransmission by inhibiting AChE (for review, see Ecobichon, 2001). Sufficient inhibition of AChE within the synapse prevents the efficient breakdown of ACh molecules, leading to accumulation of ACh within the synaptic region and persistent stimulation of the cholinergic receptors. The mechanism of cholinergic toxicity of OP and CM anti-ChEs can be chemically differentiated based on the nature of the modification of the active site serine of AChE (i.e., phosphorylation vs carbamylation; Fukuto, 1990; Mileson et al., 1998), but they essentially share a common mechanism of toxicity initiated by impairment of ACh breakdown. Although in some cases there can be important differences in the rate of onset, duration, types of signs and symptoms, etc. following exposure to an OP or CM inhibitor, and even differences among anti-ChEs within each class, the underlying biochemical and toxicological sequence of events following AChE inhibition is essentially the same for inhibitors that enter the CNS. Inhibition of AChE leads to a net accumulation of synaptic ACh levels, with persistent activation of cholinergic receptors on postsynaptic cells. Under normal conditions, this shift to enhanced cholinergic receptor activation leads to functional signs and symptoms of cholinergic
toxicity (Ecobichon, 2001). Many of the classical signs of cholinergic toxicity associated with OP or CM insecticide poisoning are peripheral in nature; that is, they are a consequence of AChE inhibition in the peripheral nervous system (PNS). The well-recognized syndrome of excessive salivation, lacrimation, urination, and defection (abbreviated by the acronym SLUD), bradycardia, and miosis are principally the result of AChE inhibition, ACh accumulation, and excessive stimulation of muscarinic receptors in the PNS. Other common signs of cholinergic toxicity (e.g., muscle fasciculations, tachycardia, and hypertension) involve overstimulation of nicotinic receptors in the PNS. These peripheral signs of cholinergic toxicity are discussed in more detail in Chapter 26.
VI. T O X I C I T Y D U E T O I N H I B I T I O N OF AChE IN T H E C E N T R A L N E R V O U S SYSTEM
A. Central Signs of Anti-ChE Intoxication in Humans With severe OP insecticide intoxication, central signs are always noted (Finkelstein et al., 1988; Moretto, 1998; Rousseau et al., 2000). Lee and Tai (2001) reported on the expression of clinical signs in 23 patients requiting intensive care after malathion intoxication in Singapore. Seventy-eight percent had toxic signs within 24 hr of exposure, suggesting CNS involvement (altered consciousness and hyperthermia), whereas 13% developed full convulsions. Verhulst and colleagues (2002) studied 54 pediatric cases of anticholinesterase intoxication in South Africa. CNS complications included coma (31%), seizures (30%), and respiratory failure requiring ventilation (35%). Lifshitz and coworkers (1997) reported clinical signs following CM insecticide (methomyl or aldicarb) intoxication in adults and children. The median age was 2.5 years (n = 36) and 22 years (n = 22), respectively. Coma/stupor and hypotonia were observed in all 36 children, but no adults presented with these CNS signs. Miosis was observed in only 55% of the children but in almost all (92%) of the adults. Fasciculations were noted in only 6% of the children but in 83% of the adults. A retrospective evaluation of CM (methomyl and aldicarb) and OP insecticide (parathion, fenthion, malathion, and diazinon) poisoning in children was also reported by this same group (Lifshitz et al., 1999). Thirty-six CM and 16 OP pesticide cases were studied (median age, 2.8 years). Predominant symptoms were CNS depression and severe hypotonia. Similar to their previous report, all CM and OP insecticide poisoned children showed stupor/coma and hypotonia, whereas only 55-56% showed the classic peripheral sign of miosis. Other peripheral signs (e.g., diarrhea and fasciculations) were even less consistent. Thus, signs of anti-ChE intoxication involving
CHAPTER 20 9Central Nervous System Effects and Neurotoxicity
the CNS are common; children may exhibit more central signs and less peripheral signs than adults following either CM or OP pesticide exposure.
B. Central Effects of OPs and CMs on Regulation of Respiration Some CM cholinesterase inhibitors are quaternary compounds and therefore penetrate the blood-brain barrier (BBB) with great difficulty (Taylor, 2001). Thus, central effects of these inhibitors, at least those mediated through inhibition of ACHE, are generally considered to be minimal. Most OP inhibitors are nonpolar and therefore freely pass through the BBB. Respiration is under cholinergic control (Kubin and Fenik, 2004). With ChE inhibitors that pass the BBB and enter the CNS, the depression of central respiratory control centers in the pons medulla is considered a primary event leading to death (Foutz et al., 1987; Finkelstein et al., 1988; Tsao et al., 1990; Goswamy et al., 1994; Sungur and Guven, 2001). Takahashi and coworkers (1991) studied the comparative effects of lethal doses of the OP insecticides chlorfenvinphos and dichlorvos on respiration and heart function. Intravenous or oral administration of both elicited classical signs of cholinergic toxicity and marked inhibition of AChE activity. In all cases, respiration ceased prior to heart failure and appeared to be the primary cause of death. Using the potent OP nerve agents (soman, sarin, tabun, and VX), Rickett and coworkers (1986) demonstrated that one of the first signs of respiratory distress was disruption of the normal firing of respiratory-related neurons in the medulla, followed by alteration of phrenic nerve activity, diaphragm electromyography (EMG), and diaphragm contractions. Soon after soman exposure in guinea pigs, the EMG peaked during the early or middle inspiratory burst, but later the EMG peaked at the end of the inspiratory burst (Chang et al., 1990). Some inspiratory bursts also persisted into the expiratory phase. Diaphragm contractions persisted throughout the decline of respiratory function, even after complete respiratory arrest, again suggesting a primary central component to respiratory failure following anti-ChE intoxication. Thus, for anti-ChEs that cross the BBB, central respiratory depression may be the primary cause of death with lethal exposures. Dickson and coworkers (2003) evaluated central and peripheral actions in respiratory depression following high-dose dichlorvos exposure in rats. Dichlorvos elicited profound fasciculations but no seizures. If only peripherally acting anticholinergics (glycopyrrolate or ipratropium) were administered, respiratory arrest and 100% lethality were noted within 10 min of dichlorvos dosing. In contrast, pretreatment with the anticonvulsant diazepam significantly improved survival. Peripherally active anticholinergics given together with diazepam further improved survival, suggesting both central and peripheral mechanisms in the central
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respiratory depression by dichlorvos. Furthermore, these findings illustrate the therapeutic benefits of diazepam in anticholinesterase intoxication, even in the absence of seizures and convulsions. The Ch5 (pedunculopontine nucleus) and Ch6 (laterodorsal tegmental nucleus) groups innervate medullary and pontine respiratory centers and may play a critical role in respiratory depression following anti-ChE exposure (Haxhiu " et al., 2003). Kok (1993) proposed that respiratory depression following anti-ChE exposure involves alteration of REM sleep pathways also mediated by Ch5 and Ch6 innervation. As noted previously, the Ch5 and Ch6 nuclei send cholinergic projections to the hindbrain, where respiratory control centers are located. Respiratory depression following anti-ChE exposure may be mediated by the same hindbrain cholinergic fibers that inhibit skeletal muscles during REM sleep (REM-associated atonia). Bursts of myoclonic contractions are also seen during REM sleep, primarily in the hindlimbs. During REM sleep, there are increased numbers of irregular contractions of the diaphragm and changes in inspiratory bursts, somewhat similar to that seen after anti-ChE exposure. Phrenic nerve activity (innervating the diaphragm) sometimes ceases altogether during REM sleep (Sieck et al., 1984). Local injection of neostigmine into the pontine reticular formation in cats promotes REM sleep (Baghdoyan et al., 1984). Carbachol injection in cats caused decreased respiratory frequency (Kimura et al., 1990). Thus, anti-ChE intoxication and REM sleep exhibit some common characteristics (e.g., EEG desynchronization, general cortical activation, atony, and respiratory depression) potentially through disruption of common Ch5 and Ch6 cholinergic signaling pathways.
C. Seizures, Neuropathology, and Blood-Brain Barrier Alterations Following Anticholinesterase Exposure It has been known for decades that anti-ChEs can cause seizures and convulsions in humans and laboratory animals. Many of the experimental studies evaluating seizures and neuropathology with anti-ChE exposures have focused on OP nerve agents. Petras (1981) reported neuropathological changes in rat brain following acute soman exposure (79-115 Ixg/kg) 15-28 days after exposure. Interestingly, the neuropathology was essentially the same in treated rats that did not exhibit seizures. Kadar and colleagues (1992) compared seizures in rats caused by soman (95 Ixg/kg, im), diisopropyl fluorophosphate (DFP; 1.8 mg/kg, im), or pentylenetetrazol (100 mg/kg, sc). Severe signs of toxicity were noted with all three toxicants, but neuropathological lesions were seen only following soman exposure. Lesions were primarily noted in piriform cortex, hippocampus, frontal cortex, and dorsolateral thalamus but pathology later progressed and expanded to other brain regions. Hippocampal CA1 neurons showed damage within 7 days after exposure, whereas CA3 neurons
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were affected only later (1 month after dosing). Of interest, Kadar et al., reported neuropathology following a lower dosage of soman, devoid of classic signs of cholinergic toxicity. It was concluded that two neuropathological processes were initiated by soman: an early phase involving necrosis in CA1 neurons, frontal cortex, and piriform cortex that occurred within a few hours to a few days and a protracted second pathological phase involving the CA3 region and septum, progressing over weeks to months. In a follow-up study, Kadar and coworkers (1995) studied time-dependent expression of neuropathology in rats following sarin (95 ~g/kg) exposures. Lesions were found in the hippocampus, piriform cortex, and thalamus as well as other sites in approximately 70% of sarin-treated rats early after exposure. Some regions (e.g., septum and hypothalamus) were only affected at later time points, however, again suggesting delayed neuropathology. Neuropathological changes were noted in cerebral cortex, dentate gyrus, Purkinje cells of the cerebellum, and CA1 and CA3 regions of the hippocampus 24 hr after sarin exposure (100 p~g/kg; Abdel-Rahman et al., 2002). Baille and coworkers (2001) reported that only soman-treated mice that exhibited seizures showed neuropathological changes. Tabun, sarin, soman, VX, VR, and cyclosarin were all capable of causing seizures and neuropathology (most extensive in the amygdala) in guinea pigs (Shih et al., 2003), which could be prevented by pharmacological blockade of the seizure activity. Kim et al. (1999) showed that DFP (9 mg/kg in rats pretreated with both pyridostigmine and atropine methylnitrate to block peripheral signs of toxicity) caused severe seizures, early necrosis (within 1 hr after exposure), and delayed (after 12 hr) apoptosis in rats. In contrast, Veronesi and coworkers (1990) reported that neuropathology could occur in the absence of seizures in rats repeatedly exposed to the OP insecticide fenthion. McDonough and colleagues (1995) reported that 98% of soman-treated rats exhibiting seizures also showed moderate to severe neuropathology. When seizures were pharmacologically blocked within 10 min of onset, no neuropathology was noted. If seizures were allowed to progress, seizure duration was correlated with severity of neuropathology. It is well-known that excessive levels of extracellular glutamate and stimulation of glutamate receptors lead to neurotoxicity and pathology (Sloviter and Dempsey, 1985; Mayer and Westbrook, 1987). It was concluded from these and other studies (Shih et al., 1991) that neuropathology in rats following OP anti-ChE intoxication is a consequence of prolonged seizures, elicited by excess ACh accumulation following AChE inhibition but maintained by subsequent release of glutamate and excitotoxicity. Pazdernik and coworkers (2001) used deoxyglucose labeling to demonstrate that soman initiated seizures in the striatal-nigral pathway and then subsequently spread to limbic areas. Early pathological changes were noted (within 24 hr) in rats that showed seizures. In contrast, indicators of
oxidative stress (reduced glutathione and increased synthesis of heme-oxygenase and metallothionein) were not noted until days later, suggesting delayed oxidative stress. The authors proposed that early excitotoxic damage was followed by delayed oxidative damage. Comparative studies using soman, paraoxon, physostigmine, and DFP indicated that seizure activity was required to elicit neuropathology. Although diisopropylphosphorofluoridate and physostigmine were unable to elicit seizures in control rats, if LiC1 (5 mEq/kg) was given 24 hr prior to DFP or physostigmine challenge, seizures were induced. Individuals with lower seizure thresholds may therefore be more sensitive to neurotoxicity and subsequent neuropathology from exposure to anti-ChEs. OP and CM insecticides can also elicit seizures and convulsions (Gupta and Kadel, 1990; Dekundy et al., 2001, 2003; Gupta et al., 2001a,b). Dekundy and coworkers (2001, 2003) reported dose-dependent induction of seizures in mice treated with dichlorvos, chlorfenvinphos, or methomyl. The nicotinic antagonist mecamylamine (1 mg/kg), but not atropine, reduced only seizures induced by methomyl. Interestingly, the NMDA antagonists MK-801 (1 mg/kg) and 3-((R,S)-2-carboxypiperazin-4-yl)-propyl- 1-phosphonic acid (10mg/kg) prevented dichlorvos- and chlorfenvinphosinduced seizures while having no effect on seizures caused by methomyl. Combined treatment with both mecamylamine and MK-801 was effective at blocking seizures by all three insecticides. Although muscarinic receptor antagonism is the mainstay of anticholinesterase treatment, these findings suggest combined muscarinic, nicotinic, and NMDA receptor antagonism may provide increased therapeutic benefits in amelioration of seizures. Gupta and colleagues (200 l a,b) compared seizure induction by an OP (DFP, 1.25 mg/kg) and CM (carbofuran, 1.25 mg/kg) anti-ChE in rats. Citrulline (as an indicator of NO generation) and high-energy phosphates were measured in selected brain regions (frontal cortex, amygdala, and hippocampus). Concurrent increases in citrulline (three- to seven-fold) and substantial reductions in ATP (36-60%) and phosphocreatine (25-53%) were noted within 30 min to 1 hr after exposure. Because antioxidants were noted to block changes in citrulline and high-energy phosphates, and in some cases the induction of seizures, the results suggested that oxidative stress may contribute to seizures elicited by OP and CM anti-ChEs. The B BB normally regulates the entry of charged compounds into the brain (de Vries et al., 1997; Hawkins et al., 2002; Ge et al., 2005). Some studies suggest that anti-ChEs can alter B BB permeability. Rats exposed to soman or paraoxon combined with the peripherally acting (polar) AChE inhibitor phospholine iodide displayed significantly greater inhibition of brain AChE than rats exposed to either soman or paraoxon only (Ashani and Catravas, 1981). It was concluded that the greater brain AChE inhibition was due to increased entry of phospholine iodide into the brain
CHAPTER 20 9Central Nervous System Effects and Neurotoxicity due to disruption of BBB integrity by the other toxicants. In rats exposed to 0.9 • LDs0 of soman, leaky capillaries in the brain were noted using brain uptake of the vital dye Evans blue, horseradish peroxidase, and [3H]hexamethonium (Petrali et al., 1991). Using Evans blue uptake to monitor BBB integrity, soman (85-95 Ixg/kg) increased B BB permeability, but only in rats that exhibited seizures (Carpentier et al., 1990). In soman-treated rats that did not exhibit seizures (n = 17), no neuropathology and no B BB leakage were noted. In rats exhibiting seizures, 18/27 had neuropathology and B BB leakage. Carpentier and colleagues (2001) reviewed five studies with soman treatment in rats showing prolonged shifts in cortical EEG power toward the delta band. These EEG changes occurred within the first hours of seizures and were correlated with later development of neuropathlogical changes. Neuropathology was not observed in soman-treated animals without shifts in EEG. Increased delta activity could therefore be a predictor of soman-induced neuropathology. Effects of CM and OP pesticides on BBB permeability have also been studied. Sinha and Shukla (2003) compared the species-dependent effects of dichlorvos (5 or 6.1 mg/kg/ day for 3 days) and carbofuran (0.5 or 0.2 mg/kg/day for 3 days) on BBB permeability in rats and mice. Uptake of sodium fluorescein into the brain following systemic injection was used as a marker of BBB integrity. Whereas rats showed no increase in fluorescein uptake with either pesticide treatment, fluorescein uptake was markedly increased in mice with exposure to diazinon or carbofuran. It should be noted that seizures were not induced in the mice with either treatment regimen. Song and coworkers (2004) studied the interactive effects of paraoxon and pyridostigmine on BBB permeability in rats. A subclinical dose of paraoxon (0.1 mg/kg, sc) causing approximately 50% inhibition of brain ChE increased BBB permeability, using horseradish peroxidase accumulation as a marker. A high dose of the CM anti-ChE pyridostigmine (30 mg/kg, po) caused cholinergic toxicity and extensive inhibition of blood ChE, but it had no effect on brain ChE activity. When pyridostigmine was given 1 hr prior to paraoxon, there was no additive toxicity and paraoxon's effect on the BBB was apparently blocked. The authors concluded that paraoxon could increase BBB permeability at an acutely nontoxic (no seizures or classic signs of cholinergic toxicity) dosage and that pyridostigmine, although having no apparent effect on brain ChE, could prevent paraoxon's effect on the B BB. Seizures were noted in two Alzheimer's patients being treated with metrifonate (Piecoro et al., 1998). In both cases, seizures occurred following cessation of concurrent therapy with antimuscarinic agents. Thus, interaction between any agents that have antimuscarinic activity and anticholinesterases used for treatment of Alzheimer's disease may increase sensitivity to seizures. Together, these findings suggest that both CM and OP anti-ChEs, under some conditions, can elicit seizures,
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increase B BB permeability, and potentially lead to neuropathology. Some studies indicate that BBB alterations and neuropathology occur only with severe cholinergic toxicity and seizures. In contrast, some reports indicate that BBB alterations and neuropathology may occur in the absence of classic signs of cholinergic toxicity or seizures. Regardless of the conditions required to influence BBB permeability, the induction of seizures and convulsions with anti-ChE intoxication potentially leads to irreversible CNS damage. In order to prevent or minimize damage caused by seizures, an anticonvulsant (e.g., diazepam) is recommended in cases of severe intoxication to prevent further CNS complications (Marrs, 2003).
D. Gulf War Illnesses and the CM Anti-ChE Pyridostigmine Upon returning from the Gulf War of 1991, thousands of soldiers exhibited a variety of signs and symptoms that were collectively referred to as Gulf War illnesses (Morgan and DaSilva, 1995; Goldstein et al., 1996; Jamal et al., 1996; Haley et al., 1997; Landrigan, 1997; Gray et al., 2004). The CM anti-ChE pyridostigmine was used by the military as a prophylactic agent to minimize the adverse health effects of exposure to nerve agents. Although pyridostigmine is a quaternary compound and thus its entry into the CNS is thought to be severely restricted by the intact B BB (Taylor, 2001), the possible central effects of pyridostigmine have been extensively studied during the past decade. A number of conditions, including xenobiotic exposure, physical stress, immune deficits, and radiation, are known to transiently increase BBB permeability (Frequin et al., 1992; Stahel et al., 2000). Physical stressors, including forced exercise, heat stress, and immobilization, have previously been reported to increase BBB permeability (Sharma et al., 1992, 1995; Esposito et al., 2001). Previous studies suggested that pyridostigmine was well tolerated in humans under different stress conditions (Prusaczyk and Sawka, 1991; Wenger and Latzka, 1992; Arad et al., 1992; Wenger et al., 1993). However, Friedman and coworkers (1996) published a pivotal paper describing a marked increase in brain anti-ChE potency of pyridostigmine in mice following forced swimming stress. Thus, a reasonable hypothesis for some Gulf War illnesses was that wartime stressors increased the access of pyridostigmine into the brain and led to unexpected CNS effects (Abou-Donia et al., 1996; Friedman et al., 1996; Hanin, 1996). A number of laboratories have since evaluated the possible modulation of pyridostigmine entry into the brain by various stressors. Friedman and colleagues (1996) reported that forced swimming reduced the dosage of pyridostigmine estimated to cause 50% brain AChE inhibition by greater than 100-fold. Following forced swimming, pyridostigmine also increased brain c-Fos and AChE mRNA levels. A number of subsequent papers found contrasting results, however.
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Lallament and coworkers (1998) found no evidence of increased entry of pyridostigmine into guinea pig brain under heat stress. Obviously, differences in the type of stressor and species differences in response to stressors could contribute to such diverse findings. However, Grauer and colleagues (2000) found little evidence of enhanced pyridostigmine entry into brain in mice with forced swimming stress mimicking those conditions in the work of Friedman and coworkers. Using two strains of rats and three different stress conditions (immobilization, forced swimming, and combined immobilization/swimming), Sinton and colleagues (2000) reported a slight decrease in brain AChE inhibition by pyridostigmine in stressed rats. A number of other studies from different laboratories have found relatively little evidence of increased toxicity or increased brain AChE inhibition with pyridostigmine treatment under either acute or prolonged stress (Servatius et al., 2000; Kant et al., 2001; Tian et al., 2002; Song et al., 2002; Shaikh and Pope, 2003; Shaikh et al., 2003). However, some reports have noted minor increases in brain regional AChE inhibition by pyridostigmine with some stress conditions (Beck et al., 2001, 2003). Combined exposure to restraint stress and pyridostigmine along with other chemicals (the insect repellant DEET and the pyrethroid permethrin) was reported to influence BBB permeability and lead to a number of neuropathological and neurochemical deficits (Abdel-Rahman et al., 2002, 2004). Furthermore, stress may influence peripheral actions of pyridostigmine and potentially affect CNS functions through indirect mechanisms (Johnson Rowsey et al., 2002). Servatius and Beck (2005) reported that relatively low dosages of pyridostigmine (0.1 or 1 mg/kg, ip) affected contextual learning using acoustic startle responses and either visual or olfactory cues. Metabolites of some stress hormones can modulate CNS function (Morris and Amin, 2004; Ueno et al., 2004). Although the majority of studies that have evaluated the possible modulation of pyridostigmine neurotoxicity by stress do not support the hypothesis that stress enhanced pyridostigmine entry into the CNS, stressors including pyridostigmine may alter CNS function indirectly through the activity of peripherally derived, centrally active signals.
E. Extrapyramidal Effects of OP Anti-ChEs A number of case studies have reported induction of extrapyramidal motor effects in patients following severe anti-ChE intoxication (Muller-Vahl et al., 1999; Shahar and Andraws, 2001; Arima et al., 2003; Brahmi et al., 2004). Joubert and Joubert (1988) reported two OP poisoning cases (unspecified toxicants) with choreiform movements. One patient exhibited choreiform movements in all limbs, but these were more extensive in the arms. Five months after exposure, the patient was depressed, listless, and had episodic grimacing and chorea. The movement disorder improved with haloperidol therapy. The other patient had
chorea of the right arm and leg and protrusion of the tongue. Dyskinesia in this patient disappeared by 10 days after intoxication. In another case, a 17-year-old female attempted to commit suicide with chlorpyrifos (Shahar and Andraws, 2001). The patient originally exhibited severe cholinergic signs of toxicity and was treated with atropine and toxogonin, but her condition deteriorated into a comatose state with respiratory depression requiting ventilation. By the sixth day after intoxication, extrapyramidal signs, including mask face, cogwheel rigidity, choreoathetosis, excessive drooling, and rigid posture with resting tremor, had developed. Treatment with a dopaminergic agonist (amantidine, 100 mg, three times per day) led to marked improvement. She was subsequently discharged and was virtually asymptomatic on follow-up evaluation (9 months later). In a retrospective study of patients (N = 633) with acute anti-ChE intoxication, three exhibited transient neuromuscular problems, including intermittent dystonia, rigidity, and tremor (Hsieh et al., 2001). Two of those patients developed mask face, with a duration ranging from 1 to 2 months. All three patients had initially exhibited severe signs of cholinergic toxicity including respiratory failure. Extrapyramidal signs occurred within 4 days of intoxication. Monocrotophos and methamidophos were involved in two of the cases, but the anti-ChE involved in the third case was not identified. Bhatt and colleagues (1999) reported five patients exhibiting symptoms of an acute akinetic rigidity syndrome following OP insecticide intoxication. All showed a poor clinical response to levodopa. Four of the five patients recovered completely without treatment, and the other patient was lost to follow-up. Of interest, one patient reportedly experienced repeated episodes of extrapyramidal signs upon reentry into the home where the pesticides had been applied. An 81-year-old female attempted suicide with dichlorvos (Arima et al., 2003). Following severe signs of cholinergic toxicity and respiratory depression requiting ventilation, rigidity and neck/upper limb tremor were noted on day 7 after exposure, increasing gradually in severity. Upon neurological examination on day 9, bradykinesia, tremor in the face and extremities, and rigidity were noted. Treatment with the antimuscarinic biperiden led to marked improvement of extrapyramidal signs. The patient was discharged after 47 days and was asymptomafic on subsequent follow-up. Thirty-seven tobacco workers from Brazil exposed to OP insecticides were clinically evaluated after a 3-month application season, and some (25/37) were subsequently evaluated 3 months after the cessation of pesticide use (Salvi et al., 2003). At the time of the first evaluation, all subjects had been exposed to OP pesticides within 24 hr. None (0/37) had a history of acute intoxication within the past year. Significant extrapyramidal signs and symptoms were present in 12 patients. Although extrapyramidal signs improved during the no-exposure period, 10 patients still exhibited some motor difficulties after removal from pesticide exposure.
CHAPTER 20 9Central Nervous System Effects and Neurotoxicity Four patients exhibited extrapyramidal signs following severe dichlorvos intoxication (Brahmi et al., 2004). All patients were comatose and had respiratory depression requiring mechanical ventilation. Extrapyramidal signs, including upper and lower limb dystonia, resting tremor, cogwheel rigidity, and hyperreflexia, were noted between 5 and 15 days after admission. Bromocriptine treatment led to progressive improvement with complete recovery. Together, these reports of extrapyramidal signs, generally (but not always) following severe anti-ChE intoxication, suggest that under some conditions anti-ChE intoxication can elicit parkinsonian signs. Considering the dense overlapping of cholinergic and dopaminergic fibers in the striatum as discussed previously and the loss of dopaminergic innervation in this brain region associated with Parkinson's disease, it is not surprising that extensive alteration of cholinergic transmission with severe anti-ChE intoxication may elicit extrapyramidal motor dysfunction. Moreover, there are a number of reports suggesting that exposure to agrochemicals in general may increase the risk of Parkinson's disease (Le Couteur et al., 1999; Ritz and Yu, 2000; Priyadarshi et al., 2001; Kamel and Hoppin, 2004). Baldereschi and coworkers (2003) reported an increased risk of Parkinson's disease in pesticide applicators, whereas other studies report higher risk with cumulative pesticide exposures (Fall et al., 1999; Baldi et al., 2003). Some studies have failed to find a link between pesticide exposure and Parkinson's disease, however (Behari et al., 2001; Kuopio et al., 1999; Taylor et al., 1999). All cases of extrapyramidal motor deficits to date appear to involve OP toxicants. Obviously, the role of anti-ChEs in extrapyramidal dysfunction, both transient motor changes associated with pesticide intoxication and classical parkinsonian signs associated with neurodegenerative disease, is a topic requiting further study.
E~ Long-Term Central Effects following Low-Level Anti-ChE Exposures Interest in possible long-term effects of low-level anti-ChE exposures was further stimulated by reports of neurological deficits (e.g., attention deficits, memory difficulties, and sleep disorders) in soldiers returning from the Gulf War (Jamal et al., 1996; Sillanpaa et al., 1997; Rosenberg and Paty, 2000). Besides intentional exposure to pyridostigmine, exposure to other anti-ChEs (i.e., OP nerve agents in munitions or OP or CM insecticides used in and around military installations) was also possible. Because there were few reports of overt cholinergic toxicity during the Gulf War, and those were apparently associated with pyridostigmine overdose (Almog et al., 1991), attention to the potential long-term effects of low-level anti-ChE exposures was increased. A number of epidemiological studies have associated subtle, long-term neurobehavioral and neuropsychological sequelae with past overt OP or CM anti-ChE intoxication
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(Savage et al., 1988; Rosenstock et al., 1991; McConnell et al., 1994; Steenland et al., 1994; Wesseling et al., 2002). Findings in these studies included reduction in some tests of cognitive function, but alterations were typically too subtle to be detected by a general neurological examination. Interestingly, individuals requiting hospitalization showed greater neurological deficits (Steenland et al., 1994; Colosio et al., 2003) and would most assuredly have been given atropine in the hospital setting. Although atropine is of proven benefit for reducing muscarinic receptor-mediated signs of toxicity, its central actions could potentially lead to undesirable neurochemical consequences. Atropine is a potent nonselective muscarinic antagonist shown in a number of studies to increase ACh release by blocking presynaptic muscarinic autoreceptors (Weiler, 1989; Feuerstein et al., 1992; Pope et al., 1995; Zhang et al., 2002a). Although atropine has beneficial efficacy against cholinergic toxicity through blocking postsynaptic muscarinic receptors in both the CNS and the PNS, blockade of muscarinic autoreceptors could lead to an increase in ACh release in the CNS, potentially leading to enhanced excitation of nicotinic receptors and modulation of the release of other neurotransmitters. This could be particularly important in areas such as the striatum where dense overlap of cholinergic fibers with dopaminergic neurons exists, where volume transmission is thought to be prominent, and where nicotinic receptors are prominent in modulation of dopamine release. Ray and Richards (2001) reviewed possible effects of chronic, low-level exposures to anti-ChEs. They proposed that any chronic effects of low-level OP exposures would likely occur through a mechanism independent of AChE inhibition. Any serine hydrolase could be sensitive to antiChEs due to the nucleophilic nature of the serine residue present in these hydrolytic enzymes. For example, Richards and colleagues (2000) reported that acyl peptide hydrolase was highly sensitive to both DFP and dichlorvos, and they proposed that inhibition of this hydrolase could be involved in low-dose cognitive effects of these toxicants. Desi and Nagymajtenyi (1999) reported electrophysiological changes in rats following long-term oral exposure to dichlorvos (0.98, 1.96, or 3.92 mg/kg/day for 4, 8, or 12 weeks). Electrocorticogram and cortical evoked potentials were disrupted after 8-12 weeks of exposure, even at dosages devoid of brain ChE inhibition. Van der Staay and coworkers (1996) reported improved cognition following DFP, dichlorvos, and metrifonate, but not eserine or paraoxon, exposure in rats at dosages lower than needed to inhibit ACHE. A number of non-AChE macromolecules have been shown to be affected directly and selectively by different anti-ChEs; thus, interaction with these additional sites could potentially contribute to chronic effects of anti-ChEs (Pope, 1999; Pope et al., 2005). Maizlish and coworkers (1987) compared neurobehavioral function in pest control workers having low-level, short-term (median, 39 days) exposure to diazinon to that in matched
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nonapplicators. Testing for concentration, eye-hand coordination, pattern recognition, visual memory, and finger tapping was conducted before and after work. Only symbol-digit pairing speed was significantly reduced in the exposed group, whereas 18 other end points were not different among applicators and nonapplicators. It should be noted that response latency and accuracy were not related to a marker of insecticide exposure, urinary diethylthiophosphate levels. Fielder et al. (1997) reported slowing of reaction time in fruit tree farmers exposed to OP insecticides. Ames and colleagues (1995) studied pesticide applicators that had never sought medical attention for anti-ChE intoxication, comparing performance in those that did or did not have a history of significant reduced blood ChE activity. Of 27 tests used, only 1 showed a difference between the two groups, and in that case those with a history of blood ChE inhibition actually had improved performance (serial digit test). These findings suggested that AChE inhibition without overt signs of toxicity would not lead to long-term neurological deficits. London et al. (1997) also detected few neurological or neurobehavioral differences in pesticide applicators occupationally exposed to OP insecticides compared to matched controls. Long-term effects of chronic low-dose exposures have thus been less consistently reported than chronic sequelae following overt, acute intoxications. Steenland (1996) reviewed findings of altered neurological function in humans following low-level anti-ChE exposures and concluded that the clinical relevance of the neurological decrements, if present, may be minimal. Brown and Brix (1998) noted that antiChE exposures are often difficult (or impossible) to quantify in epidemiological or clinical cases and thus characterized the degree of exposure based on reported initial signs and symptoms as (1) high-level (associated with overt cholinergic poisoning), (2) intermediate-level (associated with "threshold" signs, such as miosis, rhinorrhea, and blood ChE inhibition), and (3) low-level (associated with no cholinergic signs or symptoms and no detectable blood ChE inhibition). Based primarily on reports of Steenland and colleagues (1994) and Ames and coworkers (1995), Brown and Brix concluded that exposure thresholds for inducing chronic, long-term neurological sequelae were at or above grade 2 (intermediatelevel) exposure. They also concluded that long-term sequelae noted with intermediate- or high,level exposures were subtle, undetectable on an individual basis (i.e., only detectable as population effects), and, importantly, not associated with low-level exposures devoid of cholinergic signs or blood ChE inhibition. The World Health Organization Neurobehavioral Core Test Battery was used to evaluate neuropsychiatric function in female greenhouse workers (Bazylewicz-Walczak et al., 1999). Evaluations were done both before and after a spraying season in which anti-ChEs were used in greenhouse operations, and performance was compared in exposed and
matched, nonexposed subjects. Exposed workers demonstrated significant indicators of anxiety, depression, fatigue, and reductions in reaction time and motor "steadiness," but no seasonal differences were noted. It was concluded that long-term exposure to low-level anti-ChEs could be associated with adverse neurobehavioral/neuropsychological effects. Jamal (2002) argued that the cumulative evidence for chronic effects following low-level OP anti-ChEe exposure was convincing and proposed a new syndrome, chronic OP-induced neuropsychiatric disorder, to account for such neurological sequelae. In a review of previous experimental and clinical studies, Jamal concluded that there were consistent findings of altered neuropsychiatric function in 5 studies using experimental animals, 7 case studies without controls, and 15 of 19 cases studies with controls. In fact, it was concluded that the profile of cognitive dysfunction was similar in people exposed to OP anti-ChE, regardless of the number of exposures or severity of exposure. In contrast, Rusyniak and Nanagas (2004) came to a remarkably different conclusion m that there was no conclusive link between prior exposure to OP anti-ChEs and chronic neurologic or neuropsychiatric sequelae. They concluded that the neuropsychiatric differences previously reported and characterized as memory and concentration deficits, difficulties in problem solving, depression, and anxiety were too subtle for detection by routine neurological examination; that EEG changes had unclear clinical relevance; and that no pathological mechanism could explain the various symptoms. Even in patients with past acute intoxications requiring hospitalization, "no clear pattern" of impairment was evident. Thus, although overt intoxication with anti-ChEs may elicit long-term, subtle changes in neurologic function that are difficult to detect on neurological examination, the potential for low-level exposures to lead to chronic neurological changes is even less certain and a matter of diverse opinion.
VII. C O N C L U S I O N S Cholinergic neurons are widely distributed throughout the CNS. When a cholinergic terminal depolarizes, ACh is released as an intercellular signal to affect adjacent cells, in some cases both directly through synaptic transmission and indirectly through volume transmission. There is widespread innervation of the CNS by ACh-releasing fibers, derived either from distant cell nuclei or from local interneurons within a particular region. ACHE, one of the most efficient enzymes in the body, is strategically located to efficiently degrade the ACh released following cholinergic neuron depolarization, thereby rapidly terminating the signaling process.
CHAPTER 20 9Central Nervous System Effects and Neurotoxicity Because of the widespread innervation of the CNS by cholinergic neurons, CM and OP anti-ChEs, by prolonging the action of ACh and disrupting the dynamic relationship between neurotransmitter synthesis, release, and degradation, can alter a number of CNS functions. Although peripherally mediated actions (e.g., secretions in the airways) contribute to respiratory dysfunction, depression of respiratory control centers of the pons medulla is considered a primary basis for lethality with severe intoxication by many anti-ChEs. Seizures and convulsions, with regional loss of neurons and alteration in the integrity of the BBB integrity, can also be the consequence of extensive inhibition of AChE in the CNS. A number of case reports suggest that extrapyramidal motor deficits may be a consequence of acute anti-ChE intoxication, and epidemiological studies indicate that exposure to agrochemicals in general may be associated with Parkinson's disease, a disorder with extrapyramidal complications. Overt neurobehavioral as well as more subtle neuropsychiatric changes may occur with anti-ChE intoxication, but the nature of requisite exposures and the extent and/or duration of such changes in humans are a matter of varied interpretation. It is indisputable that CM and OP anti-ChEs can elicit severe toxicity via inhibition of AChE activity, and for many anti-ChEs, AChE inhibition in the CNS contributes to the spectrum Of clinical signs and symptoms of anti-ChE intoxication. One could assume that long-term neurological/neuropsychiatric changes could occur in individuals who have experienced acute intoxication with anti-ChEs or any severe poisoning episode. The trauma and stress associated with the classic signs and symptoms of acute anti-ChE intoxication (e.g., muscle fasciculations, excessive secretions in the airways and excretory systems, and dyspnea) could understandably have persistent effects on an individual that might be expressed as subtle neurological or neuropsychiatric changes. In reviewing the neurobehavioral effects of anti-ChEs, D'Mello (1993) noted that a source of error in the reporting of clinical signs and symptoms of chemical toxicity lies in the "expectations of patients and monitoring physicians." Thus, the knowledge and recognition of being poisoned by an anti-ChE (particularly with OP insecticides due to their widely known similarity to chemical warfare "nerve gases") could potentially have a long-lasting neurological impact. In this regard, Riddle and coworkers (2003) reported that although all evidence indicated that chemical warfare agents were not used during the Persian Gulf War, and that no confirmed symptoms of chemical warfare agent exposure were reported at any time before, during, or after the war, some Gulf War veterans believe that chemical weapons were indeed used. These authors concluded that the psychological effects of chernical warfare, whether real or perceived, could lead to persistent adverse health consequences. Hunt and coworkers (2004), studying the
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relationship between "symptom-belief," self-reporting of symptoms, and observed functional impairments in Gulf War veterans, concluded that illness beliefs could indeed impact clinical outcomes. Servatius and Beck (2005) investigated the interaction between interoceptive stressors and learning and suggested that aversive properties of even mild stressors could influence contextual learning and such influence could be long-lasting. They proposed that such higher order processing could be the basis for the generation and maintenance of subtle, nonspecific symptoms. Although inhibition of AChE in the CNS can undeniably lead to neurotoxicity, the demonstrated capacity of these toxicants to induce definitive chronic, neurological sequelae in the presence or absence of overt cholinergic toxicity remains unclear. It must be appreciated, however, that establishing a relationship between a chemical exposure and a given adverse health consequence becomes markedly more difficult as the incidence of toxicity decreases, as the time between exposure and adverse effect increases, and with more subtle, less overt forms of toxic responses. Complicating this challenge could be the selective interaction with a diversity of other macromolecules in addition to AChE that might influence cholinergic signaling or affect other neurological processes independent of cholinergic transmission (Pope et al., 2005), further contributing to difficulties in the hazard evaluation of low-level anti-ChE exposures. Finally, although CM and OP anti-ChEs have clearly recognized toxic potential, they have been used therapeutically for a number of clinical conditions (Taylor, 2001). The efficacy of the CM anti-ChE physostigmine for treatment of glaucoma was realized in the late 19th century, and the OP anti-ChE echothiophate is still used for this condition. Other ChE inhibitors have been used to treat myasthenia gravis, gastrointestinal atony, and other peripheral disorders. A series of ChE inhibitors, including tacrine, donepezil, galantamine, and rivastigmine, have been approved for treatment of Alzheimer's disease. In all cases, the therapeutic strategy for their use is based on the same sequence of events that leads to cholinergic toxicity m that is, increasing the persistence of synaptic ACh by blocking its degradation, leading to a net increase in cholinergic receptor activation. With clinical conditions having deficient cholinergic receptor activation, increasing the persistence of synaptic ACh by inhibiting AChE can at least partially counteract clinical signs of disease. In Alzheimer's disease, studies suggest that some anti-ChEs m a y have other actions aside from increasing synaptic ACh levels (e.g., disruption of [3-amyloid deposition) that can impair the progression of the disease (Racchi et al., 2004; Inestrosa et al., 2005). The CM and OP anti-ChEs thus continue to be important economic poisons, environmental contaminants, and therapeutic agents, and they are of concern for chemical terrorism, due.in large part to their capacity to affect a variety of CNS functions.
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Developmental Neurotoxicity of Organophosphates: A Case Study of Chlorpyrifos THEODORE A. SLOTKIN Duke University Medical Center, Durham, North Carolina
I. I N T R O D U C T I O N
when chlorpyrifos exposure grossly exceeds the threshold for systemic signs of intoxication (Mileson et al., 1998; Richardson et al., 1993). Nevertheless, examining the typical or expected exposures of pregnant women or children to chlorpyrifos after routine applications reveals unexpectedly high levels. Although the reference dose for all sources of chlorpyrifos is 3 p.g/kg/day (Davis and Ahmed, 1998), exposures after routine application may actually exceed this level by one or two orders of magnitude (Fenske et al., 1990; Gurunathan et al., 1998). In infants with high mouthing behavior, total exposure in a 2-week postapplication period can be as high as 3-5 mg/kg (Gurunathan et al., 1998). The common presence of young children either as agricultural workers or in conjunction with parents employed in field work renders unacceptable exposures equally likely. Indeed, pregnant women living in agricultural or inner-city environments with asymptomatic exposures show residues above the accepted safety level (Whyatt et al., 2002, 2003) and significant decreases in plasma cholinesterase activity (De Peyster et al., 1993). An analysis of meconium indicated fetal chlorpyrifos concentrations routinely reaching 8 mg/liter (Ostrea et al., 2002). Because the acute toxicity and lethality of organophosphates result from inhibition of cholinesterase, it is often assumed that this end point is both necessary and sufficient for risk assessment (Albers et al., 1999; Clegg and van Gemert, 1999a,b; Mileson et al., 1998). Accordingly, a great deal of attention has been paid to inhibition of fetal or neonatal cholinesterase after organophosphate exposure as an index of developmental toxicity. For chlorpyrifos, there is approximately a 10-fold difference between the LDs0 in adult rats and that in 1-week old rats and still another order of magnitude difference between 1-week-old and 1-day-old animals (Pope et al., 1991; Pope and Chakraborti, 1992; Whitney et al., 1995); thus, chlorpyrifos is nearly 100-fold
In 2000, the U.S. Environmental Protection Agency announced restrictions on the residential use of chlorpyrifos, followed by the cancellation of its registration (U.S. Environmental Protection Agency, 2002). At the time, chlorpyrifos was by far the most widely applied organophosphate insecticide, and its worldwide use continues unabated. The driving force behind this radical change in regulatory policy was the recognition that chlorpyrifos exerts untoward effects on nervous system development at far lower exposures than those that elicit signs of systemic intoxication so that unwarranted effects on the fetal or neonatal brain could occur in the absence of any recognition that exposure had taken place (Landrigan et al., 1999; Landrigan, 2001; Pope, 1999; Rice and Barone, 2000; Slotkin, 1999, 2004b; Weiss et al., 2004). Furthermore, as discussed later, the effects of chlorpyrifos on brain development involve mechanisms other than cholinesterase inhibition, with many of these exhibiting thresholds below that required for the detection of exposure by measurement of cholinesterase activity. Accordingly, this chapter addresses the case of chlorpyrifos as a specific example of how knowledge of the systemic toxicity of an organophosphate in adults can be misleading with regard to its potential to act as a developmental neurotoxicant. We also raise the issue of how to approach similar actions of other organophosphates or, indeed, other agents that may have adverse effects on brain development. The organophosphates replaced the more environmentally damaging organochlorine insecticides and chlorpyrifos rapidly became the most heavily used organophosphate because of its ease and persistence of application and because of its lessened propensity to elicit delayed-onset peripheral neuropathies; indeed, these generally appear only Toxicology of Organophosphate and Carbamate Compounds
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more toxic to newborn rats than to adults. Some of the differences may be pharmacokinetic and/or pharmacodynamic, as reflected by differential rates of development of cholinesterase compared to enzymes that break down chlorpyrifos or chlorpyrifos-oxon (Pope et al., 1991; Pope and Chakraborti, 1992). These differences are not specific to chlorpyrifos but are shared by a wide variety of organophosphates. Chlorpyrifos accumulates to a greater extent in the fetal brain than in the adult brain, with metabolite patterns indicating a 2-fold difference in concentration (Hunter et al., 1998). Nevertheless, these pharmacokinetic disparities are relatively minor compared to the major difference in neonatal vs adult toxicity (Atterberry et al., 1997). Furthermore, it is unlikely that differential effects on cholinesterase account for the effects in the fetus or neonate: Cholinesterase inhibition recovers far more quickly in developing animals (Chakraborti et al., 1993), reflecting the rapid de novo synthesis of cholinesterase molecules in the growing organism. Evidence from several laboratories suggests that, instead, the underlying differences actually pertain to events and mechanisms that are specific to the developing brain. In part, these may involve contributions from cholinesterase that are novel, nonenzymatic functions of the protein, not predicted by organophosphate-induced inhibition of hydrolytic activity (Koenigsberger et al., 1997, 1998). Cholinesterase is present in developing axons, and axonogenesis is faulty in cholinesterase knockouts (Koenigsberger et al., 1997, 1998). Cholinesterase inhibitors, including compounds other than organophosphates, have the ability to elicit growth cone collapse in developing neurons in vitro, albeit only at concentrations well above those necessary to inhibit enzymatic activity (Saito, 1998). Nevertheless, it is increasingly evident that the untoward effects of chlorpyrifos on brain development represent a unique repertoire of mechanisms exclusive of effects on cholinesterase, and we will elaborate how these processes contribute to adverse effects on the immature nervous system.
II. M E C H A N I S M S U N D E R L Y I N G DEVELOPMENTAL NEUROTOXICITY Besides the effects on cholinesterase activity or on nonenzymatic functions of cholinesterase, a compelling body of evidence indicates that chlorpyrifos (and potentially other organophosphates) affects brain development by a series of mechanisms rather than a single process, as summarized in a number of reviews (Barone et al., 2000; Pope, 1999; Slotkin, 1999, 2004b). As a general principle, these are all a reflection of the unique role of neurotransmitters and their associated cellular signaling cascades in the developing brain (Fig. 1). In maturity, neurotransmitters serve to communicate information across the synapse, but during development these same small molecules, acting through
the same receptors and signal transduction cascades as in the adult, serve as trophic factors that control the fate of their respective target cells (Dreyfus, 1998; Hohmann, 2003; Lauder, 1985; Whitaker-Azmitia, 1991). Thus, at an early stage of cell development, a given neurotransmitter signal may activate the genes required for replication of the target cell, whereas at a later stage the same transmitter and receptor signal may initiate the transition from replication to differentiation. At even later stages, neurotransmitter input is essential to communicate the signals for appropriate modeling of the brain, including axonogenesis, synaptogenesis, and apoptosis. Accordingly, any factor that interferes with transmission of the appropriate neurotransmitter signal at the proper time, or that elicits incorrect timing or intensity of stimulation, is likely to send the "wrong" signal to the target cell. Depending on the agent and the timing of application, the cellular end point can thus comprise deficits in cell numbers, programmed death of the wrong neural cells, alterations in patterns of innervation or synaptic activity, or the incorrect programming of the ability of the cell to respond to future inputs. Chlorpyrifos, through a diverse combination of mechanisms, acts on all these processes (Slotkin, 1999, 2004b; Yanai et al., 2002, 2004). Because chlorpyrifos, via its reactive metabolite chlorpyrifos-oxon, inhibits cholinesterase to prevent the breakdown of acetylcholine, an initial view of the potential impact of chlorpyrifos on neural development could thus focus on enhancement of cholinergic cell signaling as a primary mechanism for neurotoxicity. Certainly, acetylcholine is one of the transmitters that provides neurotrophic input, regulating the replication, differentiation, and migration of its target cells (Bachman et al., 1994; Hohmann et al., 1988, 1991). This provides, for example, the basis of the neurobehavioral teratology of direct cholinergic agonists, such as nicotine (Levin and Slotkin, 1998; Slotkin, 1992, 1998, 1999). However, as already discussed, inhibition of cholinesterase cannot explain the entire spectrum of the neurobehavioral teratology of chlorpyrifos. It is therefore important to note that chlorpyrifos also exhibits direct effects on both muscarinic and nicotinic cholinergic receptors (Betancourt and Carr, 2004; Bomser and Casida, 2001; Casida and Quistad, 2004; Chaudhuri et al., 1993; Gupta, 2004; Howard and Pope, 2002; Huff et al., 1994, 2001; Huff and Abou-Donia, 1995; Katz et al., 1997; Slotkin et al., 2004; Smulders et al., 2004; Song et al., 1997; Ward and Mundy, 1996; Wu et al., 2003), as discussed elsewhere in this book, Nor are these effects limited to cholinergic receptors: Chlorpyrifos also alters the expression and function of receptors for serotonin (Aldridge et al., 2003), one of the essential neurotrophic factors in mammalian brain development (Azmitia, 2001; Hamon et al., 1989; Lauder, 1985; Weiss et al., 1998; Whitaker-Azmitia, 1991, 2001). Further downstream from the receptors, we and others have found that chlorpyrifos, as well as chlorpyrifos-oxon, can
CHAPTER 21
9Developmental Neurotoxicity of Organophosphates
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FIG. 1. Multiple mechanisms underlying the developmental neurotoxicity of chlorpyrifos. During development, neurotransmitter stimulation of target cell receptors and the resulting activation of signaling pathways controls the expression of genes that determine cell fate (Dreyfus, 1998; Hohmann, 2003; Lauder, 1985; Whitaker-Azmitia, 1991). At early stages of development, signals may promote cell replication, whereas later in development the same neurotransmitter, operating on the same receptors and signaling pathways, may be responsible for expression of genes controlling differentiation, growth, or apoptosis. Early input also programs the future responsiveness of the cell, priming signaling pathways and receptor expression to achieve the appropriate response to subsequent stimuli. Chlorpyrifos affects all these processes through a variety of mechanisms (Slotkin, 1999, 2004b; Yanai et al., 2002, 2004). Inhibition of acetylcholinesterase (ACHE) by chlorpyrifos (CPF)-oxon increases the concentration of acetylcholine, resulting in inappropriate and mistimed overstimulation of cholinergic receptors. Chlorpyrifos also interacts directly with nicotinic (Gupta, 2004; Katz et al., 1997; Slotkin et al., 2004; Smulders et al., 2004; Wu et al., 2003) and muscarinic (Betancourt and Carr, 2004; Bomser and Casida, 2001; Casida and Quistad, 2004; Chaudhuri et al., 1993; Howard and Pope, 2002; Huff et al., 1994, 2001; Huff and Abou-Donia, 1995; Song et al., 1997; Ward and Mundy, 1996) cholinergic receptors and alters the expression and function of receptors for other neurotransmitters (Aldridge et al., 2003, 2004, 2005) and of signaling intermediates such as adenylyl cyclase and G proteins (Casida and Quistad, 2004; Meyer et al., 2003, 2004a,b, 2005; Olivier et al., 2001; Song et al., 1997). Through oxidative stress (Bagchi et al., 1995; Crumpton et al., 2000b; Garcia et al., 2001; Gupta, 2004; Jett and Navoa, 2000; Karen et al., 2001; Qiao et al., 2005) and direct effects on nuclear transcription factor expression and function (Crumpton et al., 2000a; Dam et al., 2003; Garcia et al., 2001; Schuh et al., 2002), chlorpyrifos also alters the basic intracellular mediators of cell differentiation.
interact with signaling intermediates such as G proteins and adenylyl cyclase (Casida and Quistad, 2004; Meyer et al., 2003, 2004a,b, 2005; Olivier et al., 2001; Song et al., 1997), as well as protein kinases (Buznikov et al., 2001b; Caughlan et al., 2004; Izrael et al., 2004), thus altering neurotrophic signals arising from multiple inputs. Finally, chlorpyrifos may interact directly with cellular energetics or the nuclear transcription factors necessary for cell replication and differentiation (Bagchi et al., 1995, 1996; Crumpton et al., 2000a; Dam et al., 1998; Johnson et al., 1998; Schuh et al., 2002; Song et al., 1997, 1998; Whitney et al., 1995), including the generation of oxidative stress (Bagchi et al., 1995; Crumpton et al., 2000b; Garcia et al., 2001; Gupta, 2004; Jett and Navoa, 2000; Karen et al.,
2001; Qiao et al., 2005). The latter mechanism is particularly interesting in that low concentrations of reactive oxygen species promote the differentiation of neural cells (Katoh et al., 1997), whereas high concentrations are overtly neurotoxic (Guan et al., 2003). The fact that the developmental neurotoxicity of chlorpyrifos resides in multiple mechanisms has important implications for the window during which the immature brain is vulnerable to this toxicant. Specifically, with a wide variety of cellular targets, adverse effects are likely to be elicited over a broad developmental period, displaying a shifting spectrum of cellular and synaptic targets and consequent neurobehavioral deficits. Within 48 hr of exposure to concentrations in the range of those found in fetal
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meconium (Ostrea et al., 2002), rat embryos cultured at the neural tube stage of development show gross structural abnormalities characterized by disordered columnar organization of the brain primordium, widespread apoptosis, widening of intercellular spaces, and disruption of mitotic activity (Roy et al., 1998) (Fig. 2). At the intracellular level, cytotoxicity is also evident from cytoplasmic vacuolation and nuclear fragmentation. Notably, these adverse effects, which remain detectable down to concentrations an order of magnitude below those found in human fetal meconium, occur in the absence of any evidence of general fetotoxicity, growth impairment, or teratogenic alterations, reinforcing the susceptibility of the developing brain to chlorpyrifos. If these effects resulted from cholinesterase inhibition and consequent cholinergic hyperstimulation, then one would expect to find a specific relationship between the neural damage and the development of cholinergic systems. However, the enzyme that synthesizes acetylcholine, choline acetyltransferase, is not detectable in the developing brain until after the neural tube stage (Lauder and Schambra, 1999). Chlorpyrifos may interfere with the aforementioned nonenzymatic functions of acetylcholinesterase (Brimijoin and Koenigsberger, 1999). Moreover, given the rapid resynthesis of new cholinesterase molecules in the fetus, one would expect to see a continuation of the adverse outcomes as a linear sequence of events from the inception of the damage. Instead, even a few days later, in late gestation, there is little biochemical evidence of cell loss or alterations
FIG. 2. Chlorpyrifos effects on brain development in cultured rat embryos at the neural tube stage (Roy et al., 1998). Embryos were exposed for a 48-hr period beginning at 9.5 days of gestation. (Left) The forebrain neuroepithelium of control embryos displays a bipolar pseudostratified epithelium, apical and basal processes containing a granular nucleus and inactive heterochromatin (n), as well as normal mitotic figures (m) located toward the internal limiting membrane. (Right) The neuroepithelium from a chlorpyrifos-exposed embryo exhibits extensive cell death (b) and extracellular bodies (arrowheads). A large cell (a) with multiple apoptotic condensations is also visible. Scale bars = 20 txm. Over a much larger cohort (>40 embryos per treatment), there was no evidence of gross dysmorphogenesis or changes in developmental landmarks, aside from the disruption of cell development in the neuroepithelium.
in indices of cell size or neuritic outgrowth after prior exposure during the neural tube stage (Qiao et al., 2002), and the deficits in neural cell numbers, synaptogenesis, and synaptic communication emerge later, in adolescence and young adulthood (Qiao et al., 2004). Instead of a direct effect mediated by cholinesterase, these delayed-onset effects suggest that the early prenatal chlorpyrifos exposure alters the signals that program neural and synapfic development, leading to a widening cascade of adverse consequences that appear only after a period of apparent normality. These effects are most compatible with targets involving the signaling cascades that program neural cell development. In a similar manner, chlorpyrifos exposure of rats in late gestation elicits initial morphological alterations commensurate with apoptosis and disruption of cell acquisition (Lassiter et al., 2002; White et al., 2002) that resolve rapidly as evaluated with biochemical indices of cell number and growth (Qiao et al., 2002). As with treatment during the neural tube stage, this later phase also produces cellular, synaptic, and behavioral anomalies that emerge later in development, with the important additional feature of sexselective effects (Aldridge et al., 2004, 2005; Garcia et al., 2002, 2003; Levin et al., 2001, 2002; Qiao et al., 2003a; Raines et al., 2001), as discussed later in the presentation of long-term outcomes of developmental exposure. Similarly, chlorpyfifos exposure during early postnatal periods, corresponding to late gestational or early neonatal stages in humans (Dobbing and Sands, 1979; Rodier, 1988), elicits both immediate and delayed neurotoxicity, culminating in neural cell damage and loss, anomalies of synaptic development, and corresponding behavioral deficits (Aldridge et al., 2004, 2005; Campbell et al., 1997; Dam et al., 1998, 1999, 2000; Johnson et al., 1998; Levin et al., 2001; Meyer et al., 2005; Rhodes et al., 2004b; Roy et al., 2004; Slotkin et al., 2001a, 2002; Song et al., 1997, 1998). In all these cases, it is critical that the delayed-onset deficits after chlorpyrifos exposure are evoked at doses either below the threshold for detectable cholinesterase inhibition during and after the corresponding exposure period or below the 70-80% inhibition required for systemic toxicity (Clegg and van Gemert, 1999a; Garcia et al., 2002, 2003; Icenogle et al., 2004; Levin et al., 2001, 2002; Qiao et al., 2002, 2003a, 2004). Again, this points to the pivotal role played by other, noncholinergic mechanisms underlying the adverse effects of chlorpyfifos in the developing brain. In fact, a number of the noncholinergic mechanisms have been explicitly elucidated in both in vivo and in vitro chlorpyfifos treatment models. Exposure during developmental windows ranging from the neural tube stage through the second postnatal week in rats elicits both immediate and delayed-onset alterations in cell signaling mediated through adenylyl cyclase (Aldridge et al., 2004, 2005; Meyer et al., 2003, 2004b, 2005; Olivier et al., 2001; Song et al., 1997). This second messenger system mediates the
CHAPTER 21 9Developmental Neurotoxicity of Organophosphates actions of numerous neurotransmitters and hormones, and its end product, cyclic AMP, is a primary modulator of cell differentiation (Groussin and Bertherat, 1998; Shaywitz and Greenberg, 1999; Stachowiak et al., 2003). The types of effects elicited by chlorpyrifos are "heterologous"; that is, chlorpyrifos elicits alterations in the expression and function of adenylyl cyclase as well as the G proteins that couple neurotransmitter and hormone receptors to the enzyme (Aldridge et al., 2004, 2005; Meyer et al., 2003, 2004b, 2005; Olivier et al., 2001; Song et al., 1997). Accordingly, chlorpyrifos shifts the entire spectrum of cyclic AMP-mediated responses, thus altering the fate of multiple neural cell populations rather than targeting one specific cell or transmitter type. We tested this relationship by challenging neonatal rats with an acute, subtoxic dose of chlorpyrifos and found an immediate decline in the mitotic index in a wide variety of brain regions, including one (cerebellum) that is very sparse in cholinergic projections (Whitney et al., 1995). Indeed, there was no regional selectivity of the effect, with cholinergically enriched regions (brain stem and forebrain) showing essentially the same response. In fact, we could elicit the same effect by injecting minute doses of chlorpyrifos directly into the cerebrospinal fluid, thus bypassing the hepatic conversion to chlorpyrifos-oxon, the active metabolite that inhibits cholinesterase. Regional selectivity corresponding to cholinergic innervation emerged only at later stages, corresponding to the development of the majority of cholinergic projections (Dam et al., 1998; Whitney et al., 1995). Nevertheless, the earlier, noncholinergic stage is critical to the net effects of chlorpyrifos exposure. Repeated administration of chlorpyrifos in these earlier phases elicits persistent inhibition of DNA synthesis (Dam et al., 1998); neural cell loss (Campbell et al., 1997); suppressed expression of the genes, macromolecules, and transcription factors required for differentiation (Crumpton et al., 2000a, Dam et al., 2003; Johnson et al., 1998; Schuh et al., 2002); and eventual shortfalls of synaptic function and corresponding behaviors (Aldridge et al., 2004; Dam et al., 1999, 2000; Garcia et al., 2003, Icenogle et al., 2004; Levin et al., 2001, 2002; Meyer et al., 2003, 2004a; Qiao et al., 2003a, 2004; Slotkin et al., 2001a, 2002). Again, all these effects involve exposure levels that are below the threshold for overt toxicity (Song et al., 1997; Whitney et al., 1995). Thus, these results indicate how chlorpyrifos acts through multiple mechanisms, including noncholinergic effects, with a decided shift in the balance of these mechanisms as development proceeds. In vitro systems have proven useful to characterize many of the noncholinergic contributors to the developmental neurotoxicity of chlorpyrifos. Major studies have used PC12 rat pheochromocytoma cells (Bagchi et al., 1995; Crumpton et al., 2000a,b; Das and Barone, 1999; Li and Casida, 1998; Qiao et al., 2001, 2003b, 2005; Song et al., 1998). In the native state, these cells behave like neuronal
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precursors and undergo replication; however, with addition of nerve growth factor, PC12 cells develop neuritic projections and differentiate into distinct cholinergic and catecholaminergic phenotypes, eventually resembling neurons morphologically, physiologically, and biochemically (Berse and Blusztajn, 1997; Greene and Tischler, 1976; Greene and Rukenstein, 1981; Tischler and Greene, 1975). Using undifferentiated PC12 cells, we were able to recapitulate the antimitotic effects of chlorpyrifos using cholinergic antagonists to demonstrate that the mechanisms were unrelated to cholinergic activation (Song et al., 1998). As was true in the developing brain in vivo, prolonged chlorpyrifos exposure during differentiation resulted in persistent inhibition of DNA synthesis and profound cell loss. Adverse effects on differentiation were confirmed by inhibition of neurite growth and by interference with the expression and function of the transcription factors required for differentiation, all of which were again unrelated to cholinesterase inhibition (Crumpton et al., 2000a; Das and Barone, 1999; Garcia et al., 2001; Li and Casida, 1998; Schuh et al., 2002; Song et al., 1998). Similar models have identified a host of potential signaling pathways through which chlorpyrifos produces its adverse effects on neural cell replication and differentiation, including the adenylyl cyclase-cyclic AMP-protein kinase A cascade (Garcia et al., 2001; Huff et al., 1994; Olivier et al., 2001; Schuh et al., 2002; Song et al., 1997; Ward and Mundy, 1996; Yanai et al., 2002; Zhang et al., 2002), protein kinase C (Bomser et al., 2002; Buznikov et al., 2001b; Yanai et al., 2002), and actions on transcription factors such as c-fos, p53, AP-1, Spl, and CREB (Crumpton et al., 2000a; Dam et al., 2003; Garcia et al., 2001; Schuh et al., 2002). The main point is that the outcome, developmental neurotoxicity elicited by chlorpyrifos, involves a family of mechanisms involved in neural cell development, not a single mechanism. Neural cell development continues in the brain over a prolonged period spanning fetal and neonatal life and, indeed, continues into adolescence (Rakic et al., 1994; Slotkin, 2002; Spear, 2000). Among the later occurring events, the generation of glial cells is of prominent importance. Glia are responsible for nutritional, structural, and homeostatic support for neurons, protecting neurons from oxidative damage, and guiding axons to their targets during development so that proper glial development is essential to the architectural modeling of the brain (Aschner et al., 1999; Barone et al., 2000; Guerri and Renau-Piqueras, 1997; Morita et al., 1999; Tacconi, 1998). Given the multiple mechanisms by which chlorpyrifos can affect neural cell development, it is critical to note that this agent also targets glia and, specifically, developing glia appear to be more sensitive to chlorpyrifos than are neurons, as evaluated both in vivo and in vitro (Garcia et al., 2001, 2002; Monnet-Tschudi et al., 2000; Qiao et al., 2001; Roy et al., 2004; Zurich et al., 2004). Given the later developmental
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timetable for glia compared to neurons, glial targeting is likely one of the reasons for the prolonged vulnerability of the developing brain to chlorpyrifos, especially for postnatal stages. Chlorpyrifos administration in vivo inhibits DNA Synthesis and causes brain cell loss during gliogenesis (Campbell et al., 1997; Dam et al., 1998; Whitney et al., 1995), and major functional defects appear with exposure confined to peaks of glial development (Campbell et al., 1997; Dam et al., 1999; Monnet-Tschudi et al., 2000; Slotkin, 1999; Song et al., 1997). Glial targeting has also been confirmed in aggregating brain cell cultures, again without any relationship to cholinesterase inhibition (Monnet-Tschudi et al., 2000). In fact, the specific loss of glia after developmental chlorpyrifos exposure is a particularly unusual feature (Roy et al., 2004, 2005). Neurotoxicants typically evoke reactive gliosis, increasing glia at the expense of neurons (O'Callaghan, 1988, 1993), an effect opposite that seen with chlorpyrifos (Roy et al., 2004, 2005). The simultaneous damage to neurons and glia likely plays a role in the wide window of vulnerability to chlorpyrifos and to the eventual pattern of neurobehavioral deficits. The developmental neurotoxicity of chlorpyrifos thus comprises mechanisms that produce a continuously shifting target in terms of types of cells involved and the developmental processes that are compromised so that vulnerability involves periods as early as the neural tube stage and as late as childhood or, potentially, adolescence. In the next section, we explore the long-term synaptic and behavioral consequences of chlorpyrifos exposure during different critical developmental periods.
III. L O N G - T E R M O U T C O M E S
OF DEVELOPMENTAL EXPOSURE Given the multiple mechanisms and targets for the developmental neurotoxicity of chlorpyrifos, and the correspondingly wide window of vulnerability of developing neural cells, synaptic and neurobehavioral defects can similarly be expected to occur regardless of whether exposure occurs in utero or in early postnatal life. Chlorpyrifos-induced neurobehavioral anomalies have been demonstrated for exposures targeted to the neural tube stage (Icenogle et al., 2004), in late gestation (Levin et al., 2002), and through postnatal stages of terminal differentiation and axonogenesis (Carr et al., 2001; Dam et al., 2000; Levin et al., 2001; Moser and Padilla, 1998; Moser, 2000; Tang et al., 2003). In all these cases, quantitative perturbations in the numbers of neurons and glia, neuritic projections, and other cellular features are readily demonstrable (Aldridge et al., 2004, 2005; Campbell et al., 1997; Garcia et al., 2003; Meyer et al., 2004a, 2005; Qiao et al., 2003a, 2004; Rhodes et al., 2004b; Richardson and Chambers, 2003, 2004; Roy et al., 2004; Slotkin et al., 2001a, 2002). However, the clearest
demonstrations of long-term alterations are evident from examination of biomarkers of synaptic activity and their corresponding behaviors.
A. Acetylcholine Most attention has been focused on cholinergic systems, as a logical extension of the assumed underlying cholinergic nature of the actions of chlorpyrifos and other organophosphates, although obviously this is not the case for the mechanisms underlying developmental neurotoxicity. Cholinergic synaptic integrity and function are typically monitored with two biomarkers, choline acetyltransferase activity and [3H]hemicholinium-3 binding to the high-affinity, presynaptic choline transporter. Choline acetyltransferase is a constitutive measure of cholinergic innervation (Aubert et al., 1996; Happe and Murrin, 1992; Navarro et al., 1989; Slotkin et al., 1990b; Zahalka et al., 1992, 1993) but is unreactive to changes in nerve impulse activity, whereas hemicholinium-3 binding is directly responsive to neuronal activity (Klemm and Kuhar, 1979; Simon et al., 1976). Thus, the comparative changes in choline acetyltransferase and hemicholinium-3 binding permit discrimination between effects on synaptic outgrowth as distinct from synaptic activity (Aubert et al., 1996; Happe and Murrin, 1992; Navarro et al., 1989; Slotkin et al., 1990b; Zahalka et al., 1992, 1993). In general, chlorpyrifos treatment during any of the four different developmental stages elicits only minor changes in choline acetyltransferase activity when assessed throughout the various brain regions that possess significant cholinergic innervation (Dam et al., 1999; Qiao et al., 2003a, 2004; Rhodes et al., 2004b; Richardson and Chambers, 2003, 2004; Slotkin et al., 2001a). In contrast, there are marked reductions in hemicholinium-3 binding, suggesting that the main effect of chlorpyrifos is to elicit a lasting reduction in cholinergic synaptic activity (Qiao et al., 2003a, 2004; Rhodes et al., 2004b; Richardson and Chambers, 2004; Slotkin et al., 2001a). This occurs in the absence of any compensatory upregulation of cholinergic receptors (Qiao et al., 2003a, 2004; Rhodes et al., 2004b) so that the deficits in cholinergic tone represent a true loss of synaptic function. Superimposed on this general deficit are important considerations of regional differences, critical exposure periods, and sex selectivity. Developmental exposure of rats to chlorpyrifos lowers hemicholinium-3 binding in adolescence through adulthood in basically all regions possessing cholinergic projections (Dam et al., 1999; Qiao et al., 2003a, 2004; Rhodes et al., 2004b; Richardson and Chambers, 2003, 2004; Slotkin et al., 2001a). However, by far the greatest and most consistent effects are seen in the hippocampus, in which cholinergic activity plays a pivotal role in learning and memory. Indeed, this region is adversely affected regardless of when exposure occurs, displaying significant decrements
CHAPTER 21 9Developmental Neurotoxicity of Organophosphates in cholinergic activity with treatment regimens ranging from exposure during the neural tube stage to exposure during late postnatal development. The synaptic deficits are accompanied by subtle morphological alterations involving preferential loss of glia, swelling of neuronal and glial perikarya, and thinning of the CA1 and CA3 regions (Roy et al., 2005). In contrast to the targeting of the hippocampus at all stages of development, cerebrocortical projections show peak sensitivity centered around late gestation, whereas those in the striatum are targeted primarily by exposure in early gestation or during postnatal phases of axonogenesis/synaptogenesis; again, these are accompanied by subtle morphological alterations that are revealed by quantitative assessment of the number and size of glia and neurons (Roy et al., 2004). The adverse effects on cholinergic tone also display peak sensitivity in late gestation and the early neonatal period, with generally lesser effects in earlier or later developmental phases. Interestingly, essentially the same results are obtained for acetylcholine systems when chlorpyrifos is given continuously throughout gestation (Richardson and Chambers, 2004), indicating that the net effect actually reflects that targeted to the sensitive window in the immediate perinatal period. Furthermore, in the continuous exposure study, chlorpyrifos was given by a different route (oral gavage instead of injection) and with a different vehicle (corn oil instead of dimethylsulfoxide), but the results were virtually identical. In turn, this implies that issues such as route of exposure or vehicle are relatively unimportant in the actual neurotoxic outcome as long as the concentration of chlorpyrifos reaching the fetus during the critical window of sensitivity exceeds the threshold for developmental neurotoxicity. Indeed, preliminary studies on different routes of administration indicate that an injection route using dimethylsulfoxide vehicle may actually produce lower peak plasma levels of chlorpyrifos and the consequent propensity to elicit systemic toxicity than seen with oral gavage (Domoradzki et al., 2004). The neurochemical findings are supported by parallel measurements of behavioral performance that reflect hippocampal function. After chlorpyrifos exposure during the neural tube stage (gestational days 9-12 in the rat), adult rats tested for learning and memory in the 16-arm radial arm maze show deficits in working and reference memory performance, as well as slowed learning (habituation) in the figure-eight maze, superimposed on a general pattern of locomotor hyperactivity (Icenogle et al., 2004). The deficits actually appear as a slowing of the learning process because the treated animals eventually do learn each of the tasks. However, they do so through "abnormal" synaptic mechanisms that overcome the deficits in cholinergic function. Accordingly, when control animals are challenged with a muscarinic cholinergic receptor antagonist such as scopolamine, they show a characteristic increase in the incidence of working and reference memory errors. In contrast, the
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chlorpyrifos-exposed animals do not display an increase in errors, indicating that the delayed learning that occurs in this group involves recruitment of alternative neurotransmitter pathways that are not ordinarily involved in this task (Icenogle et al., 2004). When the chlorpyrifos exposure occurs in the most sensitive window for developmental neurotoxicity, either in late gestation (gestational days 17-20) or during the early neonatal period (postnatal days 1-4), even greater deficits in learning and memory are revealed, with the additional feature of sex selectivity (Dam et al., 2000; Levin et al., 2001, 2002). For late gestational exposure, adverse effects are more prominent in females, whereas the early postnatal period tends to target the males to a greater extent. Nevertheless, the chlorpyrifos-exposed animals eventually do learn the tasks, albeit at a slower rate than normal, again using abnormal noncholinergic pathways as revealed by scopolamine challenge. Finally, when treatment is delayed until later in postnatal life (days 11-14), both the magnitude of behavioral effects and the degree of sex selectivity begin to wane, reflecting the closure of the critical window of highest susceptibility (Dam et al., 2000; Levin et al., 2001). The emergence of sex-selective effects gives important clues as to the mechanisms contributing to behavioral alterations that emerge later in life. Basically, the sex differences are not apparent with exposure during neurulation (gestational days 9-12) but only when exposure occurs during the commencement of sexual differentiation of the brain in late gestation through the early neonatal period (McCarthy, 1994; Mong and McCarthy, 1999), and they wane in the subsequent period. Chlorpyrifos lacks sufficient estrogenic activity to account directly for these effects (Andersen et al., 2002; Vinggaard et al., 2000), and although it interferes with testosterone catabolism at high doses (Usmani et al., 2003), this is not likely to be a critical factor during the specific developmental window in which exposure occurs. Conceivably, though, with exposures above the threshold for systemic toxicity, chlorpyrifos might evoke secondary endocrine alterations (Guven et al., 1999), but considering that the neurochemical and behavioral effects are elicited at exposures below the threshold for cholinesterase inhibition or any associated signs of systemic effects (Dam et al., 2000; Qiao et al., 2002; Slotkin, 1999, 2004a,b; Song et al., 1997), any contribution to sex differences in neurobehavioral outcomes would be marginal at best. Instead, given that the critical period corresponds to the sexual differentiation of the brain, which specifically involves the cyclic AMP pathway (Auger, 2003), one of the primary signaling targets of developmental chlorpyrifos exposure, it is most likely that the adverse effects of chlorpyrifos on brain cell development contribute to changes in sexual differentiation and resultant sex-related outcomes. In any case, the results with chlorpyrifos indicate the importance of distinguishing both neurochemical and behavioral outcomes in males and females as opposed to
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the not atypical practice of examining only males or of combining results from both sexes without discrimination.
shift to regions with 5-HT cell bodies. With even later exposure, the effects generally wane. The net pattern of effects, in terms of both the critical window of highest sensitivity and the emergence and disappearance of sex selectivity, thus resembles that seen for cholinergic systems, likely reflecting a common origin in the basic mechanisms by which chlorpyrifos perturbs neural cell development. The global upregulation of presynaptic and postsynaptic proteins involved in serotonergic neurotransmission is a pattern that typically reflects deficient synaptic communication (Abu-Roumi et al., 1996; Steingart et al., 1998; Yanai et al., 2002, 2004), and we have confirmed this conclusion with additional behavioral studies modeled on impaired 5-HT function as seen in animal models of depression. The chlorpyrifos-exposed animals display anhedonia, sex-selective augmentation of risk-taking behavior, and impaired learning and memory. With regard to the latter, an acute challenge with ketanserin, a 5-HT2 receptor blocker, increases the rate of working and reference memory errors in the 16-arm radial maze in the chlorpyrifos group but not in controls, indicating that the chlorpyrifosexposed animals depend on an abnormal, serotonergic mechanism to learn the task. This resolves one of the issues raised by the findings for cholinergic deficits, where the chlorpyrifos group was unresponsive to inhibition of the normal learning pathway as revealed by scopolamine challenge. Instead, the exposed animals learn the task through serotonergic mechanisms that are not ordinarily called into play. Thus, chlorpyrifos exposure during development elicits long-lasting changes in neurochemistry and behaviors related to monoamine systems, with changes evident over a wide range of exposure periods but with the greatest effects centered around the perinatal period. Serotonergic dysfunction is involved in appetitive and affective disorders so that these results are consonant with suggestions that at least some of the incidence of these
B. O t h e r N e u r o t r a n s m i t t e r s
Given the ability of chlorpyrifos to disrupt brain cell development by multiple mechanisms, it would not be surprising to find long-term alterations in neurotransmitter systems other than acetylcholine or in their associated behaviors. Although these have been far less studied, there is significant knowledge about effects on monoamine systems. Exposure of neonatal rats to chlorpyrifos has only sporadic effects on basal levels of norepinephrine or dopamine measured in adolescence or adulthood, but it profoundly suppresses norepinephrine turnover, an index of noradrenergic synaptic activity (Slotkin et al., 2002). Effects on dopamine turnover show regionally selective activation (striatum) or suppression (cerebral cortex). However, equally important, chlorpyrifos exposure results in total inactivation of cholinergic inputs to catecholamine systems: Animals exposed to chlorpyrifos are incapable of releasing norepinephrine or dopamine in response to cholinergic challenge, connoting a permanent desensitization of cholinergic inputs (Slotkin et al., 2002). These effects thus closely resemble the cholinergic defects revealed by scopolamine challenge in behavioral tests, as already described. Even more dramatic effects of developmental exposure to chlorpyrifos have been reported for serotonin (5-HT) systems (Aldridge et al., 2003, 2004, 2005; Raines et al., 2001). With exposure during neurulation, chlorpyrifos elicits lasting, global elevations in the expression of 5-HTIA and 5-HT 2 receptors and the 5-HT presynaptic transporter. The later gestational treatment (gestational days 17-20) elicits much larger effects that display selectivity for regions with 5-HT nerve terminals and that are preferential for males (Fig. 3). Similar receptor upregulation is seen after early postnatal (days 1-4) exposure, but the targets Chlorpyrifos Treatment on G D 1 7 - 2 0 - 1 mg/kg
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FIG. 3. Effects of prenatal chlorpyrifos exposure on expression of serotonin (5-HT) receptor 5-HT1Aand 5-HT2 subtypes and the presynaptic 5-HT transporter in adulthood (Aldridge et al., 2004). Pregnant rats were given 1 mg/kg of chlorpyrifos daily on gestational days 17-20, a regimen devoid of systemic toxicity and below the threshold for inhibition of fetal brain cholinesterase activity (Qiao et al., 2002). At 60 days of age, animals in the chlorpyrifos group display regionally selective upregulation of all the 5-HT-related synaptic proteins, with much greater effects in males.
CHAPTER 21 9Developmental Neurotoxicity of Organophosphates disturbances may have contributions from environmental neurotoxicant exposures (Slikker and Schwetz, 2003; Toschke et al., 2002; von Kries et al., 2002). On the surface, it seems unlikely that these effects by themselves would trigger such multifactor diseases, but certainly they might confer additional risk factors that act in concert with other contributors. As an additional consideration, the newest generation of antipsychotic and antidepressant drugs specifically act as antagonists at 5-HT2 receptors (Anttila and Leinonen, 2001; Poyurovsky et al., 2003; Tyson et al., 2004). The fact that developmental exposure to chlorpyrifos shifts learning and memory tasks to serotonergic systems dependent on 5-HT2 receptor mechanisms suggests that prior exposure to this pesticide may increase the incidence of cognitive and other adverse side effects of such medications, essentially creating a subpopulation in which use of these drugs may be problematic.
C. Cell Signaling Fetal or neonatal chlorpyrifos treatment not only alters cell signaling during and immediately after the exposure period (Meyer et al., 2003; Song et al., 1997) but also results in persistent or later emerging effects on the expression and function of signaling components such as adenylyl cyclase and G proteins (Aldridge et al., 2005; Meyer et al., 2004a, 2005). Again, since these elements are shared by numerous neurotransmitter and hormonal stimuli, alterations at this level of organization produce heterologous changes in signals originating in totally disparate inputs. The details are complex and have been presented elsewhere (Aldridge et al., 2005; Meyer et al., 2004a, 2005), so only a brief summary is given here. Exposure of developing rats in any of the four critical periods (gestational days 9-12 or 17-20 and postnatal days 1--4 or 11-14) elicits significant changes in adenylyl cyclase signaling in a wide variety of brain regions in adulthood. In keeping with earlier results, the most sensitive stage spans late gestation through the early neonatal period, displaying robust changes and sex selectivity. These types of effects almost certainly contribute to behavioral anomalies spanning the many neurotransmitter systems and receptors that converge on cyclic AMP as a second messenger. However, the importance transcends developmental neurotoxicity since the same signaling pathways operate in the periphery, controlling critical cardiovascular and metabolic functions. Indeed, we reported on abnormalities of neural and hormonal signals participating in hepatic metabolism after fetal or neonatal chlorpyrifos exposure and one of the defects involved hypersensitivity to inputs that foster glucose formation and release, with the effects taking place primarily in males (Meyer et al., 2004b). Since then, we have performed additional studies and found robust elevations in serum cholesterol and insulin, again restricted to males. These results, which resemble the risk factors for atherosclerosis and diabetes,
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reinforce the concept that chlorpyrifos-induced anomalies in cell signaling have effects beyond those in the central nervous system. Indeed, deficiencies in immune function also appear after developmental chlorpyrifos exposure, again affecting males to a greater extent than females (Navarro et al., 2001). The targeting of peripheral systems involved in cardiovascular, metabolic, and immune function thus extends the effects of chlorpyrifos to include some of the factors proposed in the "Barker hypothesis," which originally drew a connection between fetal growth retardation and the subsequent incidence of coronary artery disease and diabetes (Barker, 2003; Phillips, 2002). A significant literature exists on the long-term consequences of factors such as prenatal stress and related hormonal changes (Dodic et al., 1999, 2001; Nyirenda and Seckl, 1998), and there are suggestions that environmental toxicants may play an important contributory role in such disorders as hypertension, diabetes, and obesity (Power and Jefferis, 2002; Slikker and Schwetz, 2003; Toschke et al., 2002). Thus, although most studies on the developmental effects of chlorpyrifos are appropriately directed toward neurotoxicity, recent findings suggest that similar adverse effects may emerge from long-term alterations in cell signaling in peripheral systems.
IV. V U L N E R A B L E S U B P O P U L A T I O N S Given the virtually ubiquitous exposure of the human population to organophosphates, it is important to consider whether there are specific subpopulations that might be especially vulnerable to the developmental neurotoxicity of these agents. Polymorphisms that affect the activity of cholinesterase or paraoxonase (Berkowitz et al., 2004; Costa et al., 1999, 2003a), as reviewed in other chapters in this text, clearly can influence the net actions of organophosphates by affecting their toxicodynamics and toxicokinetics. Although gene knockouts have been generated for both cholinesterase and paraoxonase (Behra et al., 2002; Costa et al., 2003b; Duysen et al., 2001), to date there are no studies on the developmental neurotoxicity of organophosphates in these animal models. Of necessity, these types of alterations influence the systemic toxicity of organophosphates so that any augmentation of developmental neurotoxicity would be likely to represent a parallel shift in the dose-effect relationship for both effects rather than a specific targeting of the developing brain. Nevertheless, developmental differences in paraoxonase expression are likely to contribute to the greatly increased systemic toxicity of organophosphates in the immature organism (Cole et al., 2003; Costa et al., 2003a,b; Furlong et al., 2000). The multiple mechanisms by which chlorpyrifos perturbs brain development raise a different issue for vulnerable subpopulations. The cellular targets, signaling
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Toxicity
pathways, and transcription factors involved in the developmental neurotoxicity of chlorpyrifos are shared by numerous other, unrelated drugs or chemicals so that prior or concurrent exposure to these agents could sensitize the brain to organophosphate-induced developmental neurotoxicity (Slotkin, 1999, 2004b; Yanai et al., 2002, 2004). Although scant attention has been paid to this possibility, two scenarios have been explored in the research literature: pharmacotherapies used in the treatment of preterm labor (Aldridge et al., 2005; Meyer et al., 2005; Rhodes et al., 2004b) and nicotine exposure during pregnancy (AbdelRahman et al., 2003; Abou-Donia et al., 2003; Qiao et al., 2003b, 2005), which of course mirrors maternal tobacco use during pregnancy. These particular combinations share the advantages of representing a likely combination of exposures while at the same time allowing for the translation of findings in animal models into readily researched, documentable human parallels. Approximately 20% of all pregnant women in the United States experience preterm labor (Berkowitz and Papiernik, 1993), and as a result, up to 1 million cases are treated, often for periods of weeks, with [3-adrenoceptor agonist drugs to arrest labor, most notably terbutaline (Goldenberg, 2002; Guinn et al., 1998; Haram et al., 2003; L a m e t al., 1998). Terbutaline crosses the placenta to activate fetal [3-adrenoceptors (Auman et al., 2001; Bergman et al., 1984; Garofolo et al., 2002; Kudlacz et al., 1989; Navarro et al., 1991; Slotkin et al., 2001b), thus contributing to metabolic, cardiovascular, and neurobehavioral alterations that may emerge later in life (Bey et al., 1992; Caritis et al., 1988; Feenstra, 1992; Fletcher et al., 1991; Hadders-Algra et al., 1986; Lenselink et al., 1994; Pitzer et al., 2001; Queisserluft et al., 1996). In the developing rat, terbutaline alters neural cell replication and differentiation, synaptogenesis, and expression of synaptic proteins involved in neurotransmission, culminating in aberrant architectural organization of the cerebellum, hippocampus, and somatosensory cortex (Garofolo et al., 2003; Morris and Slotkin, 1985; Rhodes et al., 2004a,b; Slotkin et al., 1989, 1990a, 2003). Curiously, we found that the mechanisms underlying the adverse effects of terbutaline on brain development share some homologies with those of chlorpyrifos (Rhodes et al., 2004a,b; Slotkin, 1999, 2004a,b; Yanai et al., 2002), particularly the targeting of the adenylyl cyclase signaling cascade (Meyer et al., 2003, 2004a,b; Slotkin et al., 2001b, 2003; Song et al., 1997). Given the widespread use of terbutaline in pregnancy and the nearly ubiquitous exposure of infants and children to chlorpyrifos (Aprea et al., 2000; Fenske et al., 2000; Heudorf et al., 2004; Perera et al., 2004; Wessels et al., 2003; Whyatt et al., 2002, 2003), the convergence of their developmental neurotoxicant mechanisms on a common signaling pathway raises the possibility that the therapy of preterm labor may create a subpopulation that is sensitized to subsequent neurotoxic effects of organophosphate insecticides.
We tested that possibility in three separate long-term studies in developing rats, concentrating on treatment scenarios that simulate the sensitive periods for the use of terbutaline in preterm labor and peak exposures to chlorpyrifos in the postnatal period. One study was aimed at determining the effects on cellular development and cholinergic synaptic activity (Rhodes et al., 2004b) and is summarized here, a second delineated alterations in cell signaling (Meyer et al., 2005), and the third concentrated on serotonergic synaptic function (Aldridge et al., 2005). All three showed positive interactions between the two treatments. Biomarkers of brain cell number, cell size, and neuritic projections were affected by either agent alone, with patterns consistent with neuronal and neuritic damage accompanied by later emerging reactive gliosis. Notably, however, the combined exposure augmented these effects by both additive and synergistic mechanisms. For cholinergic synaptic systems, choline acetyltransferase was affected only by combined exposure to both terbutaline and chlorpyrifos, and synaptic activity markers (hemicholinium-3 binding and muscarinic receptor binding) showed impairment that was more severe after combined treatment than with either agent alone. Thus, the results are consonant with the potential for terbutaline treatment to sensitize the developing brain to subsequent chlorpyrifos exposure. Smoking during pregnancy also provides a clear-cut instance of fetal exposure to an acknowledged developmental neurotoxicant, with nicotine being one of the major factors that damage the developing brain (Levin and Slotkin, 1998; Slotkin, 1998, 1999, 2004b). Nicotine shares the specific feature of cholinergic stimulation with chlorpyrifos, although noncholinergic components obviously also contribute to elements that are unique to the latter. In addition, nicotine shares with chlorpyrifos the ability to elicit oxidative stress (Abreu-Villa~a et al., 2005; Gitto et al., 2002; Guan et al., 2003; Qiao et al., 2005; Yildiz et al., 1998), an issue of particular importance in light of the fact that the developing brain has higher metabolic demand but lower reserves of protective enzymes and antioxidants (Gupta, 2004) and, relative to the adult brain, is deficient in glia, which ordinarily protect neurons from oxidative molecules (Tanaka et al., 1999). The potential thus exists for an interaction between smoking during pregnancy and concurrent or subsequent organophosphate exposure. Despite the obvious importance of this issue, little work has been done. A single report of nicotine treatment of pregnant rats concurrently with minute exposures to transdermal chlorpyrifos failed to find any worsening of morphologic outcomes in response to combined treatment (Abdel-Rahman et al., 2003). However, the effects of the nicotine regimen in the study were sufficiently large to have obscured any additional contribution of chlorpyrifos, and the study did not address the critical targets of synaptic function and behavior. In vitro studies on transformed neural cell lines surprisingly found that nicotine protects
CHAPTER 21 9Developmental Neurotoxicity of Organophosphates neural cells from antimitotic and oxidative effects of chlorpyrifos (Qiao et al., 2003b, 2005), in keeping with prior reports showing that nicotine can promote neurotrophic responses that preserve neurons in the face of several other toxicants (Jonnala and Buccafusco, 1998; Li et al., 2000; Miao et al., 1997; Tohgi et al., 2000; Utsugisawa et al., 2002). However, it is not clear whether these phenomena will override the potentially damaging effect of the two agents in vivo. Obviously, this is an area for further work.
V. OTHER ORGANOPHOSPHATES OR DEVELOPMENTAL NEUROTOXICANTS: THE NEED FOR HIGH-THROUGHPUT SCREENING In contrast to the large body of work on the developmental neurotoxicity of chlorpyrifos, scant work has been done on other organophosphates. Despite U.S. regulatory decisions restricting its use, parathion exposure remains common in agricultural communities throughout the world (Fenske et al., 2002). With chronic exposure to frankly toxic doses, it inhibits protein synthesis in the fetus (Gupta et al., 1984). At lower exposures, it shows elevated toxicity in the immature organism, a selectivity that, as for chlorpyrifos, is not dependent on cholinesterase inhibition per se (Atterberry et al., 1997) but rather reflects differences in synaptic adaptations to exposure (Howard and Pope, 2002; Karanth and Pope, 2003; Liu et al., 1999). Neural culture systems have successfully recapitulated the adverse effects of parathion on neurodevelopment and shown its dissociation from mechanisms involving cholinesterase inhibition (Monnet-Tschudi et al., 2000; Zurich et al., 2000). In terms of developmental neurotoxicant actions, chronic postnatal parathion treatment at doses well above the threshold for cholinesterase inhibition and systemic toxicity produces hippocampal damage (Veronesi and Pope, 1990). For another organophosphate, diazinon, studies of in vitro model systems or lower organisms also confirm its potential to elicit developmental neurotoxicity (Axelrad et al., 2002; Morale et al., 1998; Qiao et al., 2001). Indeed, organophosphates all target cholinesterase and cholinergically related events, but they may also impair neural cell replication and are highly likely to differ in their other actions on developing neurons (Flaskos et al., 1994; Qiao et al., 2001). For example, the rank order for direct interaction of organophosphates with the same nicotinic cholinergic receptors that are the target for nicotine-induced developmental neurotoxicity is unrelated to their potencies as cholinesterase inhibitors (Smulders et al., 2004). We need to develop new strategies for the detection of developmental neurotoxicant actions of organophosphates or other suspected agents. What is critical about many of the targets for chlorpyrifos is that they are all involved in signaling elements that represent the convergent, final
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pathways for multiple receptor types; these kinds of heterologous alterations may therefore explain the widespread occurrence and delayed onset of many of the effects of developmental exposure to a wide variety of related and unrelated agents (Slotkin, 1999, 2004b; Yanai et al., 2002, 2004). In the past, organophosphates, along with other cholinesterase inhibitor insecticides such as carbamates, were grouped together because of their purported common mechanism of cholinesterase inhibition (Mileson et al., 1998). Now that it is evident that there are other mechanisms for disruption of brain development, the underlying assumption of a common mechanism and summation of effects of different compounds is no longer tenable (Pope, 1999; Slotkin, 1999, 2004a,b). Unfortunately, we may therefore have to consider each individual compound as a separate entity. In turn, this raises the issue of how we can begin to evaluate so many different compounds with potentially overlapping, but often distinctly different, mechanisms and outcomes. A global in vivo approach seems almost impossible: There are new organophosphates generated almost every year, as well as related and unrelated pesticides, and hundreds of new chemical entities that have the potential to elicit developmental neurotoxicity. Accordingly, we need to consider whether model systems using an in vitro approach, or lower organisms, might enable a high-throughput screen for developmental neurotoxicants. These issues were covered in reviews (Slotkin, 2004a,b) from which the following discussion has been adapted. Neural cell cultures represent one of the most promising approaches to high-throughput screening of suspected developmental neurotoxicants. Data obtained from PC12 cells, a cell line that recapitulates the mechanisms of neuronal cell replication and differentiation that are the specific targets for chlorpyrifos, were already presented (Bagchi et al., 1995; Crumpton et al., 2000a,b; Das and Barone, 1999; Li and Casida, 1998; Qiao et al., 2001, 2003b, 2005; Song et al., 1998). Along with C6 cells (Garcia et al., 2001; Li and Casida, 1998; Qiao et al., 2001; Schmuck and Ahr, 1997), which are gliotypic, these cell culture models have been used for rapid screening of antimitotic and cytotoxic effects of organophosphates and their active and inactive metabolites encompassing different phases of neural cell development. In each case, the culture models successfully recapitulate the mechanisms and outcomes identified with in vivo mammalian exposures. For example, inhibition of DNA synthesis can be evaluated with as little as a 1-hr exposure to organophosphates (Garcia et al., 2001; Qiao et al., 2001, 2003b, 2005; Song et al., 1998), including the independence of the effects from cholinergic mechanisms (inability of cholinergic antagonists to block the effect) or from cholinesterase inhibition (lower activity of chlorpyrifos-oxon or nonorganophosphate cholinesterase inhibitors), sharing of the effects by another organophosphate (diazinon), greater sensitivity of glial (C6)
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Toxicity ANOVA: Treatment, p < 0.0001 Treatment x Cell Type, p < 0.0001
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FIG. 4. Inhibition of DNA synthesis in neuronotypic PC12 cells and gliotypic C6 cells elicited by a 1-hr exposure to chlorpyrifos and related compounds and metabolites (Qiao et al., 2001). All compounds were present at a concentration of 30 IxM and all values are statistically significant compared to the corresponding control. Note the greater sensitivity of C6 cells, connoting the preferential vulnerability of glia vs neurons. Also, chlorpyrifos is more effective than its oxon, despite the fact that the latter is the agent that evokes irreversible cholinesterase inhibition. Chlorpyrifos is also more effective than physostigmine, a nonorganophosphate cholinesterase inhibitor. Diazinon, another organophosphate, also elicits inhibition of DNA synthesis with the same preference for C6 cells vs PC12 cells as seen for chlorpyrifos. Trichloropyridinol, the supposedly inactive metabolite of chlorpyrifos, possesses slight but significant antimitotic activity, which may be of functional significance given its much higher bioaccumulation in the fetal brain (Hunter et al., 1998).
vs neuronal (PC12) cells, and numerous other features (Fig. 4). Importantly, uniform cell lines point the way to the future use of microarrays for establishing transcriptional and proteomic "fingerprints" for developmental neurotoxicity. One of the major impediments to the application of microarray technology to neurotoxicity is the heterogeneity of brain tissues, which results in effects targeted to a specific neural cell population being "washed out" by the higher amounts of mRNA or proteins from unaffected cells. This problem is obviated in uniform cell cultures, which also offer the advantage of being able to correlate transcriptional changes with specified phenotypic outcomes. At the same time, there are drawbacks to the use of PC 12 and C6 cell lines or related culture systems. Although of neural origin, these are transformed cells and thus may display features that are not shared by actual neuronal or glial cells (Slotkin, 2004a). Furthermore, the use of a single cell population cannot detect developmental defects that originate in cell-to-cell interactions, such as glial guidance of axonal development or glial protection of neurons from oxidative stress. Considering potential alternatives, primary neuronal cultures also have numerous disadvantages: Neurons do not replicate in culture, making examination of antimitotic effects impossible; primary cultures are
nonuniform, containing a variety of cell types that undergo differentiation into various phenotypes, all at different rates; and primary cultures are difficult to obtain, maintain, or standardize so that their application to high-throughput screening is virtually out of the question. Approaches using micromass or other mixed cell cultures have also proven useful in identifying mechanisms and targets for organophosphate-induced developmental neurotoxicity (Cosenza and Bidanet, 1995; Monnet-Tschudi et al., 2000; Zurich et al., 2004), but for these the same factors operate as those for primary neuronal cultures, rendering them unlikely to be suitable for high-throughput approaches. Similarly, screening procedures using whole mammalian embryos, although valuable for the determination of mechanisms and outcomes of individual agents, are likely to be too cumbersome for high-volume, automated application (Roy et al., 1998) or may be unable to distinguish between developmental neurotoxicity and general fetotoxic actions (Greenlee et al., 2004). One potential solution is to combine a high-throughput screening approach using cell culture systems to identify the most worrisome candidates and the use of lower organisms for a secondary screen. At a higher level of organization, nonmammalian models represent important tools for these
CHAPTER 21
9Developmental Neurotoxicity of Organophosphates
types of studies. Invertebrates such as the sea urchin utilize neurotransmitter molecules as morphogens during embryonic development (Shmukier and Buznikov, 1998), with molecules such as acetylcholine and the monoamines playing distinct roles in the proper morphological assembly of the embryo. Of critical importance is that these morphogenic properties operate through the corresponding neurotransmitter receptors and signaling pathways that parallel those involved in mammalian brain development. Accordingly, sea urchins can be used to screen for developmental neurotoxicity of agents such as the organophosphates (Buznikov and Rakic, 2000; Buznikov et al., 2001a,b, 2003). It was found that, just as in the developing mammalian brain, the sea urchin model readily distinguishes between direct effects of chlorpyrifos and indirect effects mediated through cholinesterase inhibition. Defects appear only when the embryonic genome is turned on and the maternal genome is turned off, as would be expected from interference with gene transcription involved in cell differentiation, again resembling the course of events for adverse effects on brain development (Buznikov et al., 2001b). The advantage, of course, is that the sea urchin produces thousands of virtually identical embryos, and that the morphological abnormalities are readily visualized in the live organism under light microscopy (Fig. 5). The zebrafish has also undergone active examination as a nonmammalian surrogate for evaluation of developmental toxicants (Grunwald and Eisen, 2002; Moens and Prince, 2002). This organism undergoes complete embryonic development in a matter of days, and the transparency of the organism lends itself to light microscopy with the live embryo. Transgenic zebrafish models offer the opportunity to trace the specific development of the nervous system or any other organ system, using
FIG. 5. Morphological changes in sea urchin embryos after exposure to chlorpyrifos in the late blastula 1 stage (Buznikov et al., 2001b). Chlorpyrifos exposure results in phenotypic expression of pigmented cells forming a "mushroom-like" extralarval cap. Whereas chlorpyrifos or the related organophosphate, diazinon (Morale et al., 1998), evoke these abnormalities, chlorpyrifosoxon and nonorganophosphate cholinesterase inhibitors do not.
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simple microscopy in live fish (Behra et al., 2002; PerzEdwards et al., 2001). The zebrafish model also enables the testing of behavioral outcomes, because they can be trained to perform tasks encompassing learning and memory, and a clear demonstration of adverse behavioral effects of embryonic chlorpyrifos exposure has been reported (Arthur and Levin, 2001; Levin et al., 2003). Again, the zebrafish can be bred in the thousands and thus lends itself to rapid development of high-throughput screening. Finally, the availability of genomic arrays for both the sea urchin and the zebrafish may render these models particularly useful for developing a transcriptional fingerprint for developmental neurotoxicity (Grunwald and Eisen, 2002; Moens and Prince, 2002; National Human Genome Research Institute, 2004). Avian species can also provide a model for secondary screening that is much closer to mammalian species than are the sea urchin or zebrafish. Chick eggs are readily available and administration of suspected neurotoxicants into the yolk sac is readily performed, enabling a strict relationship to be established between dose and effect and bypassing confounding factors such as maternal toxicity. Furthermore, since the chick is entirely self-sufficient at hatching, there is no need to consider differential effects of toxicant exposures on maternal care or nursing, and unlike rodents, there are no "litter effects" (Spear and File, 1996). Along with batteries of neurochemical and morphological evaluations, cognitive behaviors can readily be assessed in newborn and developing chicks. To date, one study in which the developmental neurotoxicity of chlorpyrifos was assessed in a chick model has been performed, which successfully demonstrated how this species can be used to evaluate both neurochemical mechanisms and behavioral deficits of organophosphate exposure (Yanai et al., 2004). It is evident that avian species may provide a valuable tool for future screening purposes. Nonmammalian and in vitro models are thus particularly useful for hazard identification, for uncovering the mechanisms by which alterations occur, and for potential application to the rapid evaluation of large numbers of suspected developmental neurotoxicants. Nevertheless, it is important to note that all the new approaches lack several essential elements. Although they can help rank order the potential of a series of agents to elicit developmental neurotoxicity and point to the likely outcomes in mammalian species, none can substitute totally for the identification of relevant end points in an intact mammalian model or humans, nor can they mimic the pharmacokinetic and dosing issues that are essential to regulatory decisions. Obviously, the key use of the new methodologies will be to direct more traditional approaches toward the most likely and sensitive end points with which to make mammalian assessments. Nevertheless, given the clear-cut need to screen large numbers of organophosphates, other pesticides, or thousands of suspected developmental neurotoxicants, a high-throughput approach will enable us to focus on those compounds that require the most detailed scrutiny.
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SECTION IV. O r g a n T o x i c i t y
VI. C O N C L U S I O N S A case study of the developmental neurotoxicity of chlorpyrifos reveals important lessons for future work and for the human health relevance of animal and in vitro studies. First, the known mechanisms for systemic toxicity in the adult prove to be inadequate and misleading in evaluating the developmental neurotoxicity of chlorpyrifos and likely for the other organophosphates as well. However, there is no reason to single out the organophosphates in this regard and, indeed, it is highly likely that other potential neurotoxicants will exert adverse effects on brain development that reflect the unique mechanisms operating during formation and organization of the nervous system rather than representing a simple extrapolation of their effects in the mature organism. In turn, this indicates the need for highthroughput, rapid screening technologies to replace the cumbersome, one-by-one approach for neurotoxicants that is currently in place. Second, there is no one mechanism for the adverse effects of chlorpyrifos on nervous system development but, rather, a family of mechanisms, thus attacking multiple stages of brain cell maturation, disparate regions and cell types, and encompassing a wide 9window of vulnerability. Third, adverse effects on general aspects of cell signaling extend the types of alterations that can be expected to include signals required for peripheral functions so that agents acting as developmental neurotoxicants may also affect cardiovascular, metabolic, immune, or endocrine function as equally relevant end points: An approach evaluating only neural actions and corresponding behaviors may miss critical targets of the toxicant. Fourth, there may be predisposing factors that create subpopulations that are especially vulnerable to developmental neurotoxicants, including not only obvious genetic polymorphisms but also prior exposures to drugs and chemicals that target similar processes in brain cell development. Although this has been studied in detail for only one such scenario (terbutaline used in preterm labor), it is highly likely that a wide variety of agents may contribute to the creation of vulnerable subpopulations. Fifth, assessments of developmental neurotoxicants or of any toxicant need to take sex differences into account. Finally, recent work has addressed the essential issue of whether these kinds of studies really relate to the human condition. Following the restriction on home use of chlorpyrifos (U.S. Environmental Protection Agency, 2000, 2002), organophosphate residues in pregnant women and newborns in high-exposure groups began to decrease (Whyatt et al., 2004). The decline is directly correlated with a corresponding improvement in birth weight and length (Whyatt et al., 2004). Studies leading to the identification and control of developmental neurotoxicants can thus have a very real effect on public policy and human health.
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