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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Whyatt, R. M., Camann, D. E., Kinney, E L., Reyes, A., Ramirez, J., Dietrich, J., Diaz, D., Holmes, D., and Perera, E E (2002). Residential pesticide use during pregnancy among a cohort of urban minority women. Environ. Health Perspect. 110, 507-514. Whyatt, R. M., Barr, D. B., Camann, D. E., Kinney, E L., Barr, J. R., Andrews, H. E, Hoepner, L. A., Garfinkel, R., Hazi, Y., Reyes, A., Ramirez, J., Cosme, Y., and Perera, E P. (2003). Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns. Environ. Health Perspect. 111, 749-756. Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. E, Garfinkel, R., Hoepner, L. A., Diaz, D., Dietrich, J., Reyes, A., Tang, D., Kinney, E L., and Perera, E E (2004). Prenatal insecticide exposures and birth weight and length among an urban minority cohort. Environ. Health Perspect. 112, 1125-1132. Wu, Y. J., Harp, E, Yan, X. R., and Pope, C. N. (2003). Nicotinic autoreceptor function in rat brain during maturation and aging: Possible differential sensitivity to organophosphorus anticholinesterases. Chem.-Biol. Interact. 142, 255-268. Yanai, J., Vatury, O., and Slotkin, T. A. (2002). Cell signaling as a target and underlying mechanism for neurobehavioral teratogenesis. Ann. N. Y. Acad. Sci. 965, 473--478. Yanai, J., Beer, A., Huleihel, R., Izrael, M., Katz, S., Levi, Y., Rozenboim, I., Yaniv, S. E, and Slotkin, T. A. (2004). Convergent effects on cell signaling mechanisms mediate the
actions of different neurobehavioral teratogens: Alterations in cholinergic regulation of PKC in chick and avian models. Ann. N. Y. Acad. Sci. 1025, 595-601. Yildiz, D., Ercal, N., and Armstrong, D. W. (1998). Nicotine enantiomers and oxidative stress. Toxicology 130, 155-165. Zahalka, E. A., Seidler, E J., Lappi, S. E., McCook, E. C., Yanai, J., and Slotkin, T. A. (1992). Deficits in development of central cholinergic pathways caused by fetal nicotine exposure: Differential effects on choline acetyltransferase activity and [3H]hemicholinium-3 binding. Neurotoxicol. Teratol. 14, 375-382. Zahalka, E. A., Seidler, E J., Lappi, S. E., Yanai, J., and Slotkin, T. A. (1993). Differential development of cholinergic nerve terminal markers in rat brain regions: Implications for nerve terminal density, impulse activity and specific gene expression. Brain Res. 601, 221-229. 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. Zurich, M. G., Honegger, E, Schilter, B., Costa, L. G., and Monnet-Tschudi, E (2000). Use of aggregating brain cell cultures to study developmental effects of organophosphorus insecticides. NeuroToxicology 21, 599-605. Zurich, M. G., Honegger, E, Schilter, B., Costa, L. G., and Monnet-Tschudi, F. (2004). Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos. Toxicol. Appl. Pharmacol. 201, 97-104.
CHAPTER ~
/n Vitro Models for Testing Organophosphate-lnduced Neurotoxicity and Remediation EVELYN TIFFANY-CASTIGLIONI, VIJAYANAGARAM VENKATRAJ, YONGCHANG QIAN, AND JAMES R. WILD Texas A&M University, College Station, Texas
and Backschies, 1996). Their widespread availability, supertoxicity, and continuing use in agricultural, urban, and industrial applications pose environmental risks, as well as a challenge for remediation and detoxification. OP neurotoxicants also comprise a major legacy of the U.S. and former Soviet Union military stockpiles of chemical nerve agents, including tabun, sarin, soman, and VX. OP compounds cause potent neurotoxicological effects in mammals and birds that can be immediately acute and/or delayed onset, depending on the enzymes inhibited as well as other less well-understood factors. Our laboratory and others are developing and evaluating cell and tissue culture (in vitro) models with which to explore the mechanisms of OP-induced neurotoxicity. In vitro testing for the characterization of OP neurotoxicity has several overlapping goals, the foremost of which are to develop cell and tissue culture models that
I. I N T R O D U C T I O N This chapter describes and compares the reported toxic effects of various types of organophosphate (OP) neurotoxicants on cell and tissue culture models (i.e., in vitro models), that represent the constituents of the central nervous system (CNS). Like several environmental neurotoxicants, OP compounds affect multiple cell types, employ various mechanisms of toxic action, produce sublethal functional impairment to cells at low concentrations, and are nonessential from a primary metabolic standpoint. Individual cells are the basic functional unit in which the toxic actions of chemicals can be deduced, and cell and tissue culture models excel at providing controlled environments for exposing known cell types to toxicants and directly measuring their biological effects. This chapter summarizes existing studies on OP-induced neurotoxicity in vitro and critically evaluates the strengths, weaknesses, and further opportunities offered by such studies. A brief overview of concepts of in vitro neurotoxicology is presented, followed by analysis of neuronal and glial effects of OP compounds in vitro that may be related to neurotoxicity, such as esterase inhibition, neurite extension, and several additional biochemical end points. The novel use of cell cultures to test for enzymatic remediation of OP neurotoxicity is described as a new area for investigation. Recommendations for future studies that would increase the value of in vitro studies are also proposed.
9 Distinguish between compounds that induce delayed-onset neuropathy and those that induce acute neurotoxicity only 9 Expose additional toxicities that may be obscured in vivo 9 Identify and elucidate mechanisms that underlie the developmental neurotoxicities of OP compounds 9 Serve as rapid and discriminating systems for screening the potential toxicity of new OP compounds 9 Provide rapid screening systems for identifying potential prophylactic agents, survival therapies, or environmental remediators
A. Neural Cell Types as Interdependent Targets for OP Toxicity
II. WHY AND HOW ORGANOPHOSPHATEINDUCED N E U R O T O X I C I T Y IS S T U D I E D
The nervous system is composed of four major cell types that interact in complex, dynamic structural and biochemical contexts to generate organ function. In the CNS, these cell types are neurons (cells that generate action potentials), astrocytes (cells that maintain metabolic and ionic
IN VITRO
OP neurotoxicants have been used as insecticides and as chemical warfare agents for more than 60 years (Dingeman and Jupa, 1987; National Research Council, 1993; Chandler 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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SECTION IV 9O r g a n T o x i c i t y
homeostasis), oligodendrocytes (myelinating cells), and microglia (monocyte-derived cells). In the peripheral nervous system (PNS), Schwann cells rather than oligodendroglia make myelin. Neurotoxic processes encompass more than cytotoxic effects, because toxicants usually produce sublethal, functional impairments at low or moderate exposure levels. Various cells may demonstrate different sensitivities to toxicants as well as present different developmental windows of vulnerability. Furthermore, a neurotoxicant that alters the activities of a particular cell type also induces secondary changes in the interactions between that responsive cell and other cell types, and those responses can be developmental or physiological in nature. OP neurotoxicants used as pesticides and chemical warfare agents induce neurotoxicity by pathways involving the inhibition of specific esterases as well as esteraseindependent pathways (Casida and Quistad, 2004). OP compounds cause acute toxicity and organismal death by inhibiting acetylcholinesterase (ACHE), the enzyme necessary for the hydrolytic cleavage of acetylcholine into choline and acetic acid (O'Malley, 1997; Solberg and Belkin, 1997). In addition, OP compounds such as the insecticide mipafox (N,N'-diisopropyl phosphorodiamide fluoridate) and the industrial agent tri-o-cresyl phosphate also produce a delayed-onset, latent neurotoxicity with peripheral neuropathy and spastic paraparesis from nerve degeneration 2 or 3 weeks postexposure. This delayed neurotoxic action, termed OP-induced delayed neuropathy (OPIDN), is irreversible and independent of cholinesterase inhibition but related to inhibition or aging of neuropathy target esterase [neurotoxic esterase (NTE)] (Barrett et al., 1985). Other types of neurotoxicity that are more difficult to define may also result from OP exposure. For example, chronically exposed humans (farm workers and their children, pesticide applicators, sheep and cattle dip applicators, and workers in pesticide
manufacturing industries) may not show any serum AChE inhibition; however, they may exhibit latent neural and psychiatric symptoms, which include weakness, disturbed gait, cyanosis and tremors, anxiety, apprehension, impaired memory and concentration, depression, and psychotic reactions (Stephens et al., 1995; Salvi et al., 2003). Furthermore, OP compounds are increasingly recognized as developmental neurotoxicants that have both neuroanatomical and behavioral sequelae (Eskenazi et al., 1999; Tang et al., 2003; Ruckart et al., 2004). The mechanisms of nonneuropathic toxicity from chronic exposure and developmental neurotoxicity are poorly understood; however, these are areas in which in vitro models offer particularly promising avenues for toxicity testing.
B. The Promises and Shortcomings of in Vitro Models for Neurotoxicity Testing The issues and concepts related to neurotoxicity testing in vitro have been reviewed elsewhere (Harry et al., 1998; Pentreath, 1999; Tiffany-Castiglioni, 2004). A comparison of the features of in vitro vs in vivo systems is summarized in Table 1. The chief utility and probable value of in vitro systems is that they may provide improved screening efficiency for potential neurotoxicants and a better understanding of the mechanisms of action of the toxicants. As in vitro systems become standardized and more thoroughly characterized, they can be expected to result in decreased animal use. Furthermore, in vitro systems offer the opportunity to ethically and more readily obtain quantitative experimental data from human samples. On the other hand, in vitro neurotoxicity systems face intrinsic and extrinsic impediments to their utility, many of which resist effective resolutions. For example, in vitro cell systems typically exhibit phenotypic variations induced as epigenetic artifacts of culture conditions: They usually
TABLE 1. Comparison of Major Features of in vitro and in vivo Systems for Neurotoxicity Testing In Vitro systems
Selected cell types, pure or mixed, and simulated cell-cell interactions Molecular and cellular end points as indicators of toxicity Defined extracellular environment, usually static Direct interactions of toxicant with test cells Reduced costs, such as reduced requirement for test chemicals Ready availability of human cell cultures Limited potential for differentiation and aging Genetic uniformity; variants can be tested by transfection with engineered cDNA or siRNA sequences
In Vivo systems
Heterogeneous cell-cell interactions within normal histoarchitecture Morphological, physiological, behavioral, and cognitive end points Systemic endocrine and nervous control Metabolism and organ/tissue distribution of toxicants High costs Restriction of experimental toxicant testing to nonhuman animals Models of appropriate developmental stage and age can be tested Genetic polymorphisms
CHAPTER 22 9In Vitro Neurotoxicity Testing lack normal heterogeneous cell-cell interactions, they are often developmentally immature, and they do not exhibit toxic effects over an adequate period of time to be comparable to mammalian developmental periods or life spans. Furthermore, the experimental design must deal with uncertainties in defining and administering toxicologically relevant exposures in vitro. There is also a need to assess the insensitivity and the irrelevance of the end points tested. In addition to these intrinsic limitations, in vitro systems have two major extrinsic weaknesses: the lack of standardized analytical metrics and methods for comparing results across studies and laboratories, as well as the lack of benchmark criteria for correlating in vitro to in vivo studies. A wide range of in vitro approaches are examined in this chapter in order to gauge the strengths and weaknesses of extant, in vitro models. Cell and tissue culture preparations available for toxicity testing range from clonal, immortalized cell lines to organotypic excised tissue masses. Ideally, the experimental questions asked should be thoughtfully matched to the types of culture systems used to yield the most reproducible and meaningful results possible. In practice, the questions are often reduced to anecdotal observations as the end points or indicators of toxicity measured. Atterwill and Meakin (1990) offered the following useful classification of indicators of neurotoxicity in vitro: (1) cell viability or death, (2) generic cell functions (respiration, ion transport, Ca 2+ homeostasis, protein and DNA turnover, and oxidative stress responses), (3) differentiated cell functions (axonal transport, synapse function, myelination, cell-cell signaling, differentiated enzyme activities, and neurotransmitter uptake and metabolism), and (4) toxicant characteristics (uptake, accumulation, release, and metabolism). Clonal cell lines afford the investigator consistency in cell homogeneity and ease of preparation but lack heterotypic cell interactions and the subtle phenotypic variability normally found between cells within a tissue. Therefore, the clonal cell line cultures are often used to study viability and generic cell functions. If they do possess differentiated molecular properties that participate in a toxic mechanism, such clonal cell lines can also be used to examine toxicantspecific responses and mechanism. Primary cell cultures, which consist of dissociated cells freshly isolated and grown from tissues of origin, maintain some degree of intercellular differentiation and variability as they would in an organ or tissue, even among cells of the same type, such as astrocytes. Primary cultures thus provide more accurate models than clonal cell lines for the study of differentiated cell functions. However, primary cultures are more difficult to prepare, and their genetic cell functions may show confounding experimental variability within a population due to "minor" cellular heterogeneity. Organotypic preparations are slices or explants of tissue removed from an organ with as little trauma as possible in order to preserve both tissue structure and function. These preparations may thwart
317
conventional biochemical analysis of cell homogenates because the contributions of individual cell types cannot be distinguished. However, organotypic cultures are valuable for probing more complex functions and responses with sophisticated interactive laser cytometry, morphological analysis, and electrophysiology. Intermediate in biological complexity between clonal cell lines and organotypic cultures are mixed preparations in which heterogeneous cell interactions are simulated or reestablished, such as reaggregated cell cultures. A brief summary of the types of cell and tissue cultures is provided in Fig. 1.
FIG. 1. Types of in vitro systems. Representative types of cell and tissue cultures are shown clockwise in order of increasing heterogeneity and cytoarchitectural complexity. The adherent monolayer consists of cells that grow in a single layer attached to a culture surface, such as the inner surface of a dish. Neuroblastoma and glioma cell lines, as well as primary neural cultures, typically are cultured in this manner. The bicameral system allows for two cell types to share the same culture medium without being in direct contact with each other. One cell type is in the lower dish and the other is grown in a well with a semipermeable membrane as its bottom surface, and the well is inserted into the dish. Models for the blood-brain barrier are typically prepared in this manner, or two cell types may be grown on opposite sides of the membrane by inverting it during the time the first cell type is seeded and becomes attached. The reaggregated suspension culture consists of clumps of heterogeneous cell types derived from mechanically or enzymatically dispersed fetal or embryonic brain cells. Reaggregation is stimulated by rotary shaking. Organotypic hippocampal cultures may be prepared from transverse hippocampal slices typically 250-500 IxM thick. The tissues are maintained on a porous membrane at the interface between the medium and air. Such cultures survive days to weeks without loss of normal cytoarchitecture. Two electrodes are shown in the hippocampal culture drawing: the lower electrode stimulates neurons in the CA3 region and a recording electrode is located in the CA1 region of pyramidal neurons.
318
SECTION IV 9O r g a n T o x i c i t y
Selected representatives of the many types of in vitro OP studies are briefly described in Table 2, in which they are categorized according to the cell or tissue culture model used. Examples of the most relevant experimental details, findings, and implications are summarized for each of the selected studies in order to simultaneously highlight the emerging consensus findings regarding the utility and meaning of the in vitro studies and the elusiveness of patterns among the studies. The difficulty in comparing different in vitro studies is that the experimental designs are usually carried out under nonequivalent conditions or involve single, anecdotal studies for the cell type and/or end points to be measured. Although these conditions are useful for the examination of limited questions about the cell type or types, they obstruct rigorous comparisons between studies.
C. OP Exposure Regimens in Vitro One of the most difficult validation problems facing in vitro neurotoxicology is the consistent, repeated presentation of relevant exposure regimens to the toxicant. This subject has been reviewed previously with regard to lead (TiffanyCastiglioni and Qian, 2004, 2005) and has been addressed by Rocha et al. (1996) for paraoxon, a model non-OPIDN neurotoxicant. Ideally, basal conditions, such as the developmental stage of the cell culture at the time of exposure, the duration and intensity of exposure, and the biological availability of the toxicant to the cells, should be reproduced in subsequent uses of the in vitro system. Some of the approaches that have been used by in vitro neurotoxicologists to replicate these requirements are summarized in Table 2. Early developmental stages can be more readily modeled than older systems, as will be described for differential effects of OP compounds on younger vs older mixed neural cell cultures (Monnet-Tschudi et al., 2000). One example was observed in the modeling of neurite extension during the various phases of response to the presence of human nerve growth factor (NGF) with and without OP neurotoxicant exposure (Hong et al., 2003). Although these studies were quite successful with a reaggregate culture and a clonal cell line, respectively, the effects of OP compounds on the mature or aging nervous system in culture remain to be explored. Either single or repeated exposures to neurotoxins can also be carried out in cell culture, as illustrated in Table 2; however, the incremental effects or the sporadic nature of OP exposure are typically ignored in vitro. Biological availability for neurotoxic responses is a complex problem for in vitro toxicology, because the availability of OP compounds to target cells is dependent on many variable factors. OP compound availability in vivo is dependent on genetic polymorphisms of serum proteins, such as paraoxonase-1, as well as on age and other serum protease activities (Costa et al., 2003; Sklan et al., 2004)
that have not been explored in vitro. In addition, the antiesterase activity of OP compounds is quite flexible, requiring the presence of a phosphoryl triester bond that is subject to nucleophilic attack, but this bond can also be provided by a phosphonofluoridate bond as found in type G chemical warfare agents or by a phosponotioate bond as found in type V agents. Some OP pesticides, such as the oxon forms of parathion, malathion, and chlorpyrifos, are protoxicants that must be dethiolated to their oxon forms in order to inhibit esterases. This reduction is accomplished by P450 enzymes, which are abundant in liver but low or absent in neural cell cultures. Pairs of OP compounds in their protoxicant and antiesterase forms have been compared in cell cultures, with the finding that the protoxicant forms inhibit AChE and NTE after multiple but not single exposures (Barber and Ehrich, 2001). The biological availability of OP compounds in animals is also dependent on the dynamics of transport across the blood-brain barrier by an OP neurotoxicant directly or by its neurotoxic metabolites. These dynamics include its levels in interstitial fluid in the brain and its potential for interaction with brain cells, neither of which is well understood. Even when a known total amount of an OP compound is added to a culture, its biological availability is decreased by such factors as the presence of serum in the medium (Qiao et al., 2001), which detract from the achievement of precise, relevant exposure conditions in vitro. When evaluating results from in vitro studies of OP toxicity, it is useful to compare halfmaximal inhibitory concentrations (IC50 values) for AChE and NTE inhibition and half-maximal effective concentrations (ECs0 values) for cytotoxicity to other end points measured within the same culture system and experimental paradigm. Thus, several research groups report the concentrations necessary to inhibit AChE and NTE in their culture systems as reference points for comparisons to the concentrations necessary to affect other end points, such as nonesterase enzyme activities, cytoskeletal protein levels, and neurite extension (Henschler et al., 1992; Ehrich et al., 1997; Schmuck and Ahr, 1997; Li and Casida, 1997, 1998). However, because temperature and time of exposure can affect IC50 values, these factors should be considered when comparing studies. In general, cytotoxic levels in vitro are higher than those required to inhibit ACHE, NTE, or neurite extension.
III. E F F E C T S O F O P COMPOUNDS
ON NEURONS Neurons are responsible for the perception of sensory stimuli and the coordination of cellular, tissue, and organismal responses to stimuli from the environment. Among the possible effects of neurotoxicants on neurons are several general responses: apoptosis or necrosis of neuronal stem cells in both the developing and the mature brain, impaired
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SECTION IV- O r g a n Toxicity
neuronal migration (a secondary effect of damage to radial glia), and impaired synaptogenesis or altered synaptic function. Damage to neurons may produce developmental and/or late-onset cognitive dysfunction. An examination of Table 2, which describes most of the in vitro OP neurotoxicity studies presented in the literature, reveals that most studies have been carried out with clonal cell lines rather than primary cell cultures or tissue cultures. Four neuronal and one glial clonal cell line have been used for OP neurotoxicity testing in vitro. The characteristics of the neuronal cell lines that have made them useful for this purpose are briefly reviewed next, followed by selected comparisons with the results generated by studies in primary cultures.
A. Cell Lines and Primary Cultures Used in OP Neurotoxicity Testing Neuron-like cell lines are attractive models for exploring in vitro neurotoxicology because they proliferate rapidly in their undifferentiated state, and many of them can be induced to develop properties of more mature neurons (i.e., differentiate) with stimulants such as dibutyryl cyclic AMP (dBcAMP), retinoic acid, or NGE Several neuronal cell lines have been studied for more than 30 years and found to have neurobiological properties; these cells thus provide well-characterized models for specific toxicologic investigations. Mouse, rat, and human cell lines are commonly used for in vitro neurotoxicity testing because of their stability and hardiness. Three mouse neuroblastoma cell lines, cloned independently from the C1300 tumor, have been used in OP studies as neuronal cell models: NB2a (Klebe and Ruddle, 1969), NB41A3 (Augusti-Tocco and Sato, 1970), and N-18 (Amano et al., 1972). These cell lines proliferate rapidly, but are capable of developing properties of differentiated neurons. All three cell lines are characterized by a pseudotetraploid karyotype, a neuroblastoid morphology, a stable cadre of neurotransmitter enzymes and receptors, and the capacity to extend neurite-like processes. NB2A and NB41A3 clones display electrogenic membrane responses (Yogeeswaran et al., 1973). The NB2a (Harvey and Sharma, 1980; Flaskos et al., 1999; Fowler et al., 2001; Sachana et al., 2001; Axelrad et al., 2002, 2003) and N-18 cell lines (Henschler et al., 1992; Schmuck and Ahr, 1997) have been used in several OP toxicity studies because of their ability to extend neurite-like processes upon treatment with dBcAMP (Shea et al., 1991). Both cell lines express the 200-kDa neurofilament protein, which is the heavy subunit of neurofilaments localized to the cytoskeleton of neurons that provides a useful biochemical marker for neuronal differentiation. The NB41A3 cell line expresses AChE and NTE activities and can be induced to differentiate with retinoic acid. The AChE activities are considerably lower than the parallel AChE activities in mouse or hen brain. Specific activities of NTE correlate with those of mouse
brain (Ehrich and Veronesi, 1995), and this cell line has been used for comparisons of neurotoxic OP effects on murine vs human cells (Ehrich et al., 1995, 1997; Ehrich and Correll, 1998). PC12 cells, a neuron-like cell line established from a transplantable rat adrenal pheochromocytoma (Greene and Tischler, 1976), have been a neuron model in many neurobiological and neurochemical studies (Biocca et al., 1983; Levi et al., 1985). These cells have been used in several studies of OP neurotoxicity (Flaskos et al., 1994; Bagchi et al., 1995; Li and Casida, 1997, 1998; Song et al., 1998; Crumpton et al., 2000; Das and Barone, 1999; Qiao et al., 2001) because they express NGF receptors and become morphologically differentiated in response to NGE PC12 cells were originally characterized as resembling immature noradrenergic cells (Greene and Tischler, 1976). In addition, PC12 cells contain ACHE; synthesize, store, and release acetylcholine (ACh); and fail to synthesize epinephrine (Greene and Tischler, 1976; Green and Rein, 1977). The cell line also expresses monoamine oxidase type A (MAO-A) but not MAO-B. PC12 cells genetically expressing MAO-B display a dose-dependent increase in sensitivity to the toxicant MPTP (Wei et al., 1996). Ehrich and colleagues (Nostrandt and Ehrich, 1992, 1993; Veronesi and Ehrich, 1993) selected the SY5Y human neuroblastoma cell line as a model system for OP toxicity testing based on relatively high activities of NTE and AChE compared to other cell lines. After establishing that mipafox-induced NTE inhibition in SY5Y cells is similar to that of chicken brain, these investigators (Nostrandt and Ehrich, 1993) showed that mipafox-induced inhibition of NTE in SY5Y cells is decreased by aldicarb, a competitive inhibitor of NTE that protects hens from the development of OPIDN (Johnson, 1982; Lotti et al., 1993), and verapamil, a Ca 2+ channel blocker that reduces OPIDN effects in hens (E1-Fawal et al., 1990). Furthermore, NTE is inhibited in SY5Y cells by OP compounds that cause OPIDN in the hen test model, including DFP, phenyl saligenin cyclic phosphate, and tolyl saligenin phosphate. In contrast, several OP compounds that do not cause OPIDN in vivo, including paraoxon, parathion, malaoxon, malathion, dichlorvos, fenthion, and fenitrothion, do not significantly inhibit NTE in SY5Y cells (Ehrich et al., 1994). Thus, it is possible to use this cell line to examine the molecular mechanisms that distinguish acute and delayed neurotoxic effects of OP compounds. Ehrich et al. (1995) further determined NTE and AChE activities in SY5Y cells differentiated with retinoic acid or NGF after treatment with paraoxon, DFP, parathion, and mipafox. SY5Y cells differentiated with retinoic acid show similar AChE and NTE inhibition by DFP, paraoxon, and parathion compared to either undifferentiated or NGFdifferentiated cells. However, mipafox-induced inhibition is much lower in retinoic acid-differentiated cells. These findings suggest that SY5Y cells differentiated with NGF
CHAPTER 22 9In Vitro Neurotoxicity Testing would be a potentially valuable model for investigating OP-induced "neuropathy." In such a model system, the underlying mechanisms of OPIDN might also be examined, and these mechanisms might be distinguished from those associated solely with acute neurotoxicity. The cell line has been further studied as a model for OPIDN by our laboratory (Hong et al., 2003; Cho and Tiffany-Castiglioni, 2004). Several other types of culture systems have been used on a more limited basis to observe neuronal responses to OP compound exposure, including primary cultures and whole embryo cultures. These models exhibit more differentiated properties than those present in cell lines. Electrophysiological properties of synapses have been studied in primary cultures of neurons from fetal rat hippocampus, revealing several noncholinesterase-related effects of OP compounds. Acute exposure to low paraoxon levels increases the frequency of miniature postsynaptic currents mediated by GABA or glutamate in hippocampal neurons. These presynaptic effects are not mediated by actions on muscarinic or nicotinic receptors and are cholinesterase independent (Rocha et al., 1996). In the same culture system, the nerve agent VX directly interacts with presynaptic muscarinic receptors to block action potential-dependent release of GABA. VX also blocks the action potential-dependent release of glutamate, facilitating the action potential-independent release of both GABA and glutamate by cholinesterase-independent actions (Rocha et al., 1999). Long-term hippocampal slice cultures have not been examined for OP-induced neurotoxicity, although acutely isolated slices have been used to demonstrate that sarin inhibits the evoked release of GABA (Chebabo et al., 1999). In an interesting use of oocytes transfected to express the rat OL4[~2 nicotinic acetylcholine receptor, several OP compounds have been shown to interact directly with this receptor at concentrations several orders of magnitude lower than those that inhibit AChE (Smulders et al., 2004). Bovine adrenal chromaffm cells, which are of neuroepithelial origin, have been studied as an alternative model for neuronotypic cells because they have high carboxyl esterase activities, particularly NTE (Sogorb et al., 1996; Quesada et al., 2004). In these cells, acute treatment with a high concentration of triphenyl phosphite, but not paraoxon or diisopropyl phosphorofluoridate, reduces adenosine incorporation into ATP in mitochondria (Knoth-Anderson et al., 1992). Acute exposure to a high level of mipafox decreases depolarization-induced noradrenaline release and inhibits voltage-dependent calcium channel (VDCC) activity and nicotinic ionic currents. These results suggest that VDCC and nicotinic receptors are targets for mipafox leading to catecholamine release (Guti6rrez et al., 1996). These effects would be of interest to study at lower and repeated exposures to OP compounds. Whole rat embryos have been used in one study to morphologically examine the effects of OP compounds
329
on neuroepithelial development. At levels of chlorpyrifos (14 txM) that are below those causing dysmorphogenesis, the neuroepithelium of embryos shows increased apoptotic cell death, inhibition of mitosis, and displacement of mitotic cells. This model therefore appears to be sensitive to the detection of more subtle developmental abnormalities than those typically associated with teratogenesis (Roy et al., 1998).
B. Noncholinergic and Nonantiesterase End Points and Mechanisms As mentioned previously, OP compounds induce neurotoxicity by inhibition of AChE and NTE. Unlike whole organisms such as vertebrates, cells in culture do not require AChE activity for survival. This circumstance creates two operational conditions in vitro: (1) Cultured cells appear insensitive to cytotoxicity because molar concentrations in plasma that are lethal are not lethal in culture, and (2) noncholinergic mechanisms of toxicity (those that do not involve the inhibition of AChE and buildup of synaptic levels of ACh) become the focus of study in vitro. Increasing in vivo evidence on the importance of noncholinergic and nonantiesterase mechanisms, however, indicates that the latter is not a case of "looking under the lamppost" but a fortuitous circumstance in which cell culture models can be of particular value (Rocha et al., 1996, 1999; Schuh et al., 2002). Similarly, inhibition of esterases in general, including NTE, is not necessary for the induction of several biochemical alterations in cell and tissue cultures. It is of great interest that some studies listed in Table 2 show that protoxicant (nonesterase-inhibiting) forms of OP compounds, such as tri-o-tolyl phosphate (Carlson and Ehrich, 1999, 2001; Carlson et al., 2000), parathion (Carlson et al., 2000), and chlorpyrifos (Bagchi et al., 1995; Song et al., 1998; Crumpton et al., 2000; Das and Barone, 1999; Garcia et al., 2001; Schuh et al., 2002), exert biological effects on neural cells, albeit usually at high concentrations. Many in vitro studies suggest that the toxic effects of OP compounds on neural cells are unrelated to the inhibition of AChE because they occur at concentrations lower than those needed to inhibit ACHE. Some alterations are also uncorrelated with NTE inhibition. In vitro effects of OP compounds that appear to be affected via nonantiesterase and noncholinergic mechanisms include alterations in DNA synthesis (Song et al., 1998; Qiao et al., 2001), apoptosis (Carlson et al., 2000), transcription factor binding and expression (Crumpton et al., 2000; Garcia et al., 2001), signaling molecules and pathways (Garcia et al., 2001; Schuh et al., 2002; Hong et al., 2003), mitochondrial integrity (Knoth-Anderson et al., 1992; Carlson et al., 2000; Hong et al., 2003), stress responses (Garcia et al., 2001; Sachana et al., 2001), expression of cytoskeletal proteins (Schmuck and Ahr, 1997; Carlson and Ehrich, 2001; Sachana et al.,
330
SECTION IV. O r g a n T o x i c i t y
2001; Cho and Tiffany-Castiglioni, 2004), and major neurophysiological events such as synaptic or receptor function (Rocha et al., 1996, 1999; Smulders et al., 2004).
C. Neuronal Morphology and Differentiation It is interesting to note that all of the neuronal cell lines tested, including mouse, rat, and human clones of diverse origins, have been found to apparently distinguish OP compounds that cause OPIDN from those that do not, based on shortened neurites or alterations in the neuronal cytoskeleton (Henschler et al., 1992; Li and Casida, 1998; Fowler et al., 2001; Hong et al., 2003; Massicotte et al., 2003; Cho and Tiffany-Castiglioni, 2004). The observation that OPIDN-inducing compounds inhibit the initiation, outgrowth, or maintenance of neurites in cultured cells may have functional relevance to the phenomenon of dying-back neuropathy observed in vivo. Alternatively, or in addition, such changes in morphology may be relevant to changes in dendritic branching patterns observed in developing rat brains exposed to low or moderate levels of paraoxon (Santos et al., 2004). Three cell culture studies provide sufficient ranked numerical data for mipafox, chlorpyrifos or chlorpyrifosoxon, leptophos, and tri-o-cresyl phosphate to enable a comparison of relative potencies of the OP compounds on neurite extension and compare them with the in vivo hen model. The cell culture studies employed different treatment regimens, cell lines, and methods for assessing OP effects on neurite extension. In two studies, N-18 or NB2a mouse neuroblastoma cells were treated for 2 weeks with OP and then differentiated with dBcAMP for 6 days in the continued presence of OR In the case of N-18 cells, lengths of 50 processes per test group were measured microscopically and the ICs0 for inhibition of neurite extension was calculated (Henschler et al., 1992). In the case of NB2a cells, an enzyme-linked immunosorbent (ELISA) assay was used to measure reductions in levels of the neuronal cytoskeleton protein NF200 in lieu of a morphological assay, and the ECs0 was calculated (Schmuck and Ahr, 1997). In another study, PC 12 rat pheochromocytoma cells were differentiated with NGF in the absence of OP compounds for 5 days and then exposed to OP for 48 hr. Neurite lengths were measured microscopically (Li and Casida, 1998). Despite different treatment regimens and assays for neurite extension, the ranked order of potency for OP neurotoxicity is similar between the PC12 and NB2 cells in these studies, in which the order of potency is tri-o-cresyl phosphate > mipafox > chlorpyrifos or chlorpyrifos-oxon. Leptophos has a similar ECs0 as chlorpyrifos in NB2a; its effects were not measured in PC12 cells. In contrast, in N-18 cells, the order of potency is different: tri-o-cresyl phosphate, mipafox, and chlorpyrifos have similar ICs0 values and both are nearly three orders of magnitude lower than that of leptiphos. None of
these cell culture studies yielded the same rank order of potency for induction of OPIDN in hens in vivo, which is mipafox > leptophos > tri-o-cresyl phosphate > chlorpyrifos (Johnson, 1977). The lack of agreement between the hen animal model and rodent cell cultures suggests species or toxicokinetic differences because the mouse and rat are much more resistant to OPIDN than is the hen. The lack of agreement between two of the cell culture studies cannot be resolved without a standardized treatment regimen and method of assessment of neurite extension.
IV. E F F E C T S O F O P C O M P O U N D S ON GLIA Neuronal function and nervous tissue structure require the participation of neuroglia, or glial cells. The three main types of glia in the CNS are astroglia, oligodendroglia, and microglia. Astroglia possess active antioxidant systems that respond to stress and injury in the brain (Sagara et al., 1993; Bolanos et al., 1995; Tanaka et al., 1999; TiffanyCastiglioni and Qian, 2005). Astrocytes also integrate and modulate neuronal synaptic transmission through intrinsic signaling properties. These cells exhibit Ca 2+ excitability, possess functional neurotransmitter receptors that regulate intracellular Ca 2+ concentrations, demonstrate the ability to propagate Ca 2+ oscillations to neighboring cells through gap junctions, and facilitate the release of neuroactive transmitters to neurons (Araque et al., 2001; Carmignoto, 2000; Bezzi and Volterra, 2001). Radial glia and Bergmann glia, two specialized types of astroglia, provide scaffolding for neuronal migration during development (Rakic, 2003), Astroglia and radial glia may respond to toxicants by disruption of radial glial scaffolding in the developing nervous system, gliosis or glial activation, or altered metabolism. Oligodendroglia and Schwann cells myelinate axons in the CNS and PNS, respectively. Toxic effects on oligodendroglia may include demyelination, apoptosis succeeded by proliferation, and loss of oligodendroglial progenitor cells (Tiffany-Castiglioni et al., 2004). Microglias, which are of mesenchymal origin, mediate inflammatory responses in the CNS (Streit et al., 1988). Microglia have not been studied as primary targets for OP-induced neurotoxicity, and OP effects on other glia have received much less attention than neurons. The possibility that glia are targets for chlorpyrifos-induced neurotoxicity has been reviewed by Garcia et al. (2002).
A. C6 Rat Glioma Cell Line In vitro studies on OP-induced neurotoxicity effects on glia
have been conducted with the C6 rat glioma cell line as well as several primary culture types. C6 cells were derived from a rat brain tumor induced by N-nitrosomethylurea (Benda et al., 1968). These cells display properties of
CHAPTER 22 9In Vitro Neurotoxicity Testing oligodendrocytes, such as the expression of glucocorticoid receptors and the induction of glycerol phosphate dehydrogenase by glucocorticoids (de Vellis and Brooker, 1972; Beaumont, 1985), and astrocytes, such as the expression of glial fibrillary acidic protein (Bissell et al., 1975; Pishak and Phillips, 1980). Cells treated with dBcAMP extend thin cytoplasmic processes in a response believed to model either differentiation or glial reactivity, with the latter indicated by interleukin-6 production (Hamprecht et al., 1973; Slegers and Joniau, 1996). Cells induced to differentiate with norepinephrine or dBcAMP also express S-100 protein, a glial marker (Labourdette and Mandel, 1980; van Eldik and Zimmer, 1987). C6 cells are capable of producing and secreting NGF, which is stimulated by isoproterenol activation of [3-adrenergic receptors (Schwartz et al., 1977). In addition, C6 cells appear to express monoamine oxidase B (MAO-B) because (-)-deprenyl, a specific MAO-B inhibitor that has been used as an effective anti-Parkinsonian drug, can reduce GFAP expression. The results for the C6 cells are suggestive but not conclusive validation that glial cells are sensitive to OP neurotoxicants. Short-term exposure to chlorpyrifos at noncytotoxic levels arrests DNA synthesis in undifferentiated C6 cells and elicits reactive oxygen species formation in C6 cells differentiated with dexamethasone (Garcia et al., 2001). Furthermore, C6 cells are more sensitive than PC12 cells to inhibition of DNA synthesis by chlorpyrifos and other AChE inhibitors (Qiao et al., 2001). These results suggest that glia offer development-related targets of chlorpyrifos-induced toxicity. If so, the ramifications for chlorpyrifos exposure during brain development are significant because of the interdependence of neurons and glia during gliogenesis, neuronogenesis, neuronal migration, and synaptogenesis. However, other end points do not show greater sensitivity of C6 cells than neuronal cells to multiple exposures to OP compounds that do and do not induce OPIDN. For example, both dBcAMP differentiated N-18 mouse neuroblastoma cells and C6 rat glioma cells show a similar depression of "neurite-like" extension after 3-week exposures to chlorpyrifos or other OPIDN-inducing compounds. In general, strong inducers of OPIDN markedly depress cytoskeletal protein levels in both cell lines. In conjunction with NTE/AChE ratios, both cell lines appear to be able to detect the induction of OPIDN (Schmuck and Ahr, 1997).
B. Primary Culture Models Containing Glia In contrast to some of the previously mentioned results suggesting glial sensitivity to OP-induced neurotoxicity, studies with primary cultures have not detected either astroglial or oligodendroglial sensitivity to several OP compounds tested. The phosphorylation of the CaZ+/cAMP response element binding protein (pCREB) is a very sensitive response of primary embryonic rat cortical neurons
331
to chlorpyrifos and its metabolites chlorpyrifos-oxon or trichloropyridinol (Schuh et al., 2002). For example, pCREB levels are elevated at OP concentrations much lower than needed for AChE inhibition. However, chlorpyrifos exposure does not elevate pCREB in similarly exposed astrocyte cultures. The astrocyte cultures were treated in medium containing serum, whereas the neurons were in serum-free medium. One study showed that serum and albumin protect against chlorpyrifos toxicity in C6 and PC12 cultures (Qiao et al., 2001), which may therefore be a confounding factor in this study. Further study is needed on both genetic and differentiated end points in culture models in order to compare the relative sensitivities of glia and neurons to OP-induced neurotoxicity. Reaggregate cultures of dispersed cells from embryonic or fetal brain show developmental expression patterns that can be monitored for neuronal, astroglial, and oligodendroglial proteins in order to assess relative effects of toxicants on cell types (Zurich et al., 2004). Two such studies have been carried out with OP compounds. Funk et al. (1994) showed that brief exposure of chick brain reaggregate cultures to diisopropyl phosphorofluoridate (100 txM) or paraoxon (1 IxM) results in NTE and AChE inhibition patterns similar to those of the hen in vivo over 1 or 2 weeks. However, at the same OP compound concentrations, activity of the oligodendroglial enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) is not affected. This result has been confirmed in reaggregate cultures from fetal rat telencephalon and extended to include astrocytes (Monnet-Tschudi et al., 2000). In the rat culture study, activities of neuronal, oligodendroglial, and astroglial enzymes were monitored after a 10-day exposure to chlorpyrifos, parathion, or their oxon derivatives during immature vs differentiated developmental stages. Irrespective of culture age, AChE was inhibited at much lower concentrations than other neuronal enzymes (CHAT and glutamic acid dehydrogenase). Furthermore, activities of glutamine synthetase in astrocytes and CNP in oligodendrocytes were unchanged by treatment with OP compounds, except at concentrations of chlorpyrifos-oxon or paraoxon exceeding by 10- to 1000-fold those that reduce neuronal enzyme activities. These results indicate a much greater sensitivity of neuronal markers than glial markers to OP exposure. The latter result is in agreement with the finding that CNP activities are unchanged in the CNS of hens treated orally with an OPIDN-inducing dose of tri-o-tolyl phosphate (Luttrell et al., 1988).
C. OP Effects on Astrocyte-Endothelial Interactions Cells of the CNS directly interact with other cell types, notably the capillary endothelial cells that comprise the blood-brain barrier (BBB). Astrocytes are essential for the maintenance of B BB structural integrity and permeability
332
SECTION IV. O r g a n T o x i c i t y
characteristics (Janzer and Raft, 1987; Rubin et al., 1991). The BBB should be considered in two respects when discussing neurotoxicity: (1) the transport of toxicants across it to the brain parenchyma and (2) the direct effects of toxicants on the integrity of the barrier. Astroglial participation in OP-induced neurotoxicity has been explored in cell culture models of the BBB. Yang et al. (2001) measured the transendothelial permeability of chlorpyrifos across monolayers of the rat brain endothelium-4 (RBE4) cell line. When grown in astrocyte-conditioned medium, RBE4 cells showed a decreased permeability to [14C]-chlorpyrifos compared to those grown in control medium. This result was the first to demonstrate the positive effect of astroglial-derived soluble factors on reducing chlorpyrifos permeability across the BBB. In a second study, Parran et al. (2005) created an artificial BBB by culturing adult bovine microvascular endothelial cells (BMEC) and neonatal rat cortical astrocyte primaries on opposite surfaces of collagen-coated semipermeable membrane inserts. In this model, when chlorpyrifos was added to the BMEC side, it was metabolized by the endothelial cells and inhibited their carboxylesterase and cholinesterase activities. Furthermore, chlorpyrifos and its metabolites could cross the artificial B BB and reduced its electrical resistance. Astrocytes are a potential target for chlorpyrifos toxicity in this model. These two studies on astroglia-endothelial interactions in the BBB draw attention to the need to assess this important aspect of astrocyte physiology and OP-induced neurotoxicity.
V. FRONTIERS A. Prophylactic Detoxification of OP Compounds Assessed in Vitro OP neurotoxicants present major challenges for environmental remediation and for the protection of subjects who may be exposed to these compounds, such as farm workers and their children, pesticide applicators, and military or civilian personnel who may be exposed to chemical warfare agents. Although OP compounds are degraded environmentally, certain conditions, such as low pH, temperatures below 20 ~ and reduced natural light, allow them to persist in the environment. OP-based nerve agents have elicited worldwide attention because of the disarmament treaty of 132 countries in 1993 and the immediate need for a method to destroy these chemicals. Currently, the United States and Russia are seeking appropriate means to destroy large stockpiles of aging nerve agents. Incineration has not been practical because of operational safety and potential environmental impact concerns. Chemical neutralization of nerve agents, such as the application of sodium hydroxide, does not accomplish complete destruction and produces highly caustic and hazardous by-products. Therefore, biodegradation of OP compounds has been considered to be
a potentially reliable alternative method; however, exploration of alternatives has not been undertaken. The National Research Council (1993) recommended evaluation and potential development of this technology as a practical solution, but the current commitment of funds to the destruction of chemical warfare stockpiles by incineration appears to preclude altemative technology development that would address the primary destruction of OP neurotoxicants. A cell culture model possessing easily measured relevant toxicological end points would be a useful tool for evaluating enzymes and processes for the toxicological inactivation of OP compounds. Studies in our laboratory have begun to evaluate SY5Y cells for testing the potential of genetically engineered OPH to functionally biotransform OP neurotoxicants. In an initial study, interactive laser cytometry was used to compare the transient disruption of intracellular Ca 2+ (Ca2+i) homeostasis by paraoxon and its hydrolysis products (Hong et al., 2003). The highest paraoxon concentration used (1.8 mM) slightly exceeded the ICs0 (1.6 mM) for inhibition of AChE by paraoxon in SY5Y cells differentiated with retinoic acid but was below the ECs0 (2.6 mM) for cytotoxicity (Ehrich et al., 1997). Comparison of basal Ca2+i levels in cells treated acutely with paraoxon or OPH-hydrolyzed paraoxon revealed that OPH-hydrolyzed paraoxon produced qualitatively similar effects to those of paraoxon, indicating that the hydrolysis products, primarily p-nitrophenol, are no more toxic than paraoxon. Because OPH-hydrolyzed paraoxon has an effect like that of paraoxon, the effect on basal Ca2+i levels is likely not related to esterase inhibition. However, paraoxon invokes a sharp, transient increase in Ca 2+i levels, whereas hydrolyzed paraoxon invokes a slower, more sustained phase that persisted for 400 sec, suggesting some possible destabilization of the cell membrane by the hydrolysis products. Ca2+i homeostasis as an end point in the previously mentioned experiment was both cumbersome and insensitive. Therefore, we have examined other end points. The short-term effects of four OPH-treated OP compounds on AChE and NTE activities were measured in retinoic aciddifferentiated or undifferentiated SY5Y cells. This study revealed that there were delayed effects of OPH-treated paraoxon or mipafox on neuronal cytoskeletal proteins in NGF-differentiated cells. The anti-AChE activity of paraoxon (up to 3 ~M) and anti-NTE activity of mipafox (up to 250 ~zM) in SY5Y cells were prevented by biodegradation with OPH. Anti-AChE activities of mipafox, methyl parathion, and demeton-S were partially ameliorated, depending on OP concentration. Intracellular amounts of the 200-kDa neurofilament protein (NF200) were unchanged after treatment with OPH-treated or buffer-treated paraoxon, as expected, because this end point is insensitive to paraoxon. However, NF200 levels increased unexpectedly in cells treated during late differentiation with OPH-treated mipafox. This finding suggests the existence of a threshold concentration of mipafox below
CHAPTER 22 9In Vitro Neurotoxicity Testing
which SY5Y cells compensate for toxicity by cellular regeneration because biotransformation by OPH was incomplete and 4 IxM mipafox remained in the treatment medium. These results indicate that OPH can be used to enzymatically degrade OP compounds and remediate their neurotoxic effects in vitro. Furthermore, AChE and NTE were suitable detectors for OPH amelioration (Cho et al., 2005).
B. Extrapolation of in Vitro Results to Whole Animals and Humans Cell and tissue culture studies on OP-induced neurotoxicity are valuable if they provide valid insights into processes that occur in vivo. The extrapolation of in vitro results to animals and human populations requires careful attention to four elements of experimental design in vitro (Fig. 2). First, concentrations in vitro must be valid and comparable to toxicological doses. Unfortunately, little information is available on the brain concentrations of OP compounds that are
Operative dose to target cell:
9Bioavailability 9Metabolism in cell 9Cell defense
Functional endpoints:
9More sensitive than cytotoxicity 9Of potential value for screening and
I Phenotype of target cell: 9OP targets (enzymes, structural
~ J
~--I
proteins,
homeostatic mechanisms)
9Cell type 9Developmental stage
environment of target cell: I 9Secondary damage J to dependent cells [ 9Protection by other
remediation studies
FIG. 2. Critical interacting variables in the extrapolation of benchmark doses from in vitro to in vivo systems. In vitro models provide useful systems for dissecting the cellular and molecular mechanisms of adverse in vivo effects of OP compounds. They also help identify subtle biological effects of OP compounds that may be present in vivo but are masked by acute cholinergic effects or have gone unnoticed. Ideally, in vitro findings should be readily extrapolative to in vivo systems but, practically, this ideal has not been realized in neurotoxicology. The interacting variables that influence toxicity, as illustrated here, must first be better understood, and they present major challenges for cellular neuroscience and toxicology. Operative dose, for example, is influenced by the tissue context of a cell, such as the ability of other cells and tissues to metabolize or transport the OP compound. Toxicity is dependent on the cell having vulnerable OP targets, balanced against its ability to control the damage induced, which may be influenced by developmental maturity. The domino effects of secondary toxicity to cells that interact with the damaged cells must also be understood. Functional end points are key measurements for the extrapolation of in vitro findings to in vivo systems because they offer a rational possibility for the comparison of identical mechanistic events.
333
associated with nonacute OP-induced neurotoxicity. For protoxicants, such as parathion and chlorpyrifos, the concentrations of biologically active toxicant should be integrated into the experimental design. Some studies have shown biological effects of protoxicants on neurons in culture. This information could reveal either desulfuration capacities in the cultures or protoxicant mechanisms of neurotoxicity that were previously unrecognized. Second, test targets should be selected for their toxicological value and plausibility. The most sensitive culture models should be selected in order to detect toxicant concentrations that have a biological effect. The sensitivities of target cells could be enhanced through the use of genetically engineered or transfected cells that express targets of interest and their polymorphisms. Third, OP compound exposure regimens must be valid. Exposures may be short term or long term, depending on the type of toxicity being modeled. However, although acute toxicity induced by inhibition of AChE is a sensitive marker of OP exposure in culture, more attention should be paid to longer-term and repeated, intermittent, noncytotoxic (nonlethal) exposures. Superimposed on long-term exposure regimens is the necessity for cultures to differentiate and age as they would in vivo. Fourth, the end points must be biologically plausible. As discussed, neurotoxicity of a compound is not always represented solely by its cytotoxicity, because functional alterations may be harmful, neutral, or beneficial. The following are suggested strategies for in vitro testing of OP compounds: 9 Evaluate the active metabolite(s) for secondary toxicity. 9 Determine and test at toxicologically relevant concentrations. 9 Use cell and tissue culture models that possess appropriate OP compound targets and relevant OP compound-metabolizing systems. 9 Include neurotransmitter receptors, signaling molecules, and metabolic enzymes. 9 Test functional biochemical end points in addition to cytotoxicity. 9 Test in multiple culture systems of different biological complexities.
VI. C O N C L U S I O N S
AND FUTURE
DIRECTIONS Information generated from in vitro studies is beginning to yield an improved understanding of the effects of OP compounds on neuronal and glial cells. Among the general conclusions from these studies and parallel in vivo studies are the following: 9 OP compounds affect neurons at toxicologically relevant concentrations in vitro. 9 Most OP compound effects in vitro are unrelated to the inhibition of AChE and many are unrelated to NTE
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activity, which suggests other nonesterase mechanisms of neurotoxicity. 9 Neurons are more sensitive to OP-induced biochemical damage or cytotoxicity than are astroglia and oligodendroglia, but developmental stages in the context of fetal bases for adult neurological disease need to be fully considered. 9 OP compounds should be tested at pharmacologic concentrations, although the repeated or sporadic nature of clinical levels is difficult to reproduce in culture. The relevance of high concentrations is not clear and should be carefully considered. 9 New assays based on the use of molecular markers have great potential to be more powerful and toxicologically relevant than conventional in vitro assays. Novel in vitro assays that would address the effects of OPIDN inducers on molecular markers of nervous tissue structure and function in differentiating neuronal and glial cells should continue to be explored. These culture systems are being developed with the concept that a battery of highly sensitive assays can be assembled that give a predictable set of responses (i.e., a signature that is unique for classes of OP compounds).
Acknowledgments The authors thank Dr. Marion Ehrich for her critical review of the manuscript and numerous helpful suggestions. The authors' work was supported by National Institutes of Health grants P42 ES04917, P30 ES09106, and T32 ES07273 and by ATSDR grant U61/ATU684505.
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Electrophysiological Mechanisms in Neurotoxicity of Organophosphates and Carbamates TOSHIO NARAHASHI Northwestern University Medical School, Chicago, Illinois
I. I N T R O D U C T I O N
of their medical use and OP nerve gases such as sarin, tabun, and VX because of the military concern. Therefore, although the primary purpose of this chapter is the electrophysiological mechanism of action of insecticidal OPs and CMs, noninsecticidal CMs and nerve gases are included to some extent because these data provide useful information for understanding the mechanism of action of insecticides. This chapter focuses on the mechanism of action of OP/CM insecticides as studied by electrophysiological techniques that are expected to provide the functional aspect of the toxic action. Survey of the literature has shown that data are quite sketchy and fragmental, and there are not any systematic long-term studies on this subject. The electrophysiological effects could be due to the direct action on the receptor or the indirect action via the sustained presence of ACh in the synaptic left as a result of ChE inhibition. The effects vary greatly depending on the kind of OPs/CMs, the dose, and the method of administration. There have been many descriptive or phenomenological studies of the effects of OPs/CMs on action potential conduction and synaptic transmission. Some studies that do not contribute to our knowledge of the mechanism of action are not included in this chapter. Many review articles have been published dealing with the mode of action of OPs and CMs. However, many of them do not address the mechanistic aspects or electrophysiological aspects. Readers who wish to obtain more comprehensive general aspects of OPs/CMs should consult the following articles: O'Brien (1967), Fukuto (1979), Jones et al. (1979), Woolley et al. (1979), Murphy et al. (1984), Chambers and Chambers (1989a,b), Bardin et al. (1994), and Koelle (1994).
There is general agreement that organophosphate (OP) and carbamate (CM) insecticides inhibit cholinesterase (ChE) and that this action is an important mechanism by which neurotoxicity is produced. The simplest notion of the ChE theory states that the accumulation of acetylcholine (ACh) at synaptic junctions as a result of ChE inhibition causes excess stimulation, thereby causing toxicity. However, additional effects not through ChE inhibition have been suspected since the introduction of parathion in 1944 (O'Brien, 1967). A variety of approaches have been used to elucidate the mechanism o f action of insecticides on insects and mammals. Electrophysiology is the most straightforward to characterize changes in the function of the nervous system. Ligand binding methods have been used to determine the site of binding of chemicals at the target receptor or channel but cannot follow the fast time course of changes. Behavioral techniques are sensitive to subtle changes in animal behavior caused by insecticide intoxication, but these do not elucidate the detailed mechanism of action. Histological approaches can find structural changes caused by insecticides, but these are usually questioned whether the observed changes are the cause of intoxication or the result of intoxication. Recently, molecular biological techniques such as point mutations of receptor amino acids have proven effective in locating the target site on the receptor/channel; however, these approaches remain descriptive at the molecular level and cannot elucidate the true molecular mechanism. Because OP/CM insecticides have been used extensively since parathion was developed, their mechanism of action has been the subject of intense investigations. Most of these mechanistic studies focused on ChE inhibition and metabolism in insects, mammals, and plants. Regarding the mechanism of functional changes, two groups of anti-ChEs other than insecticidal anti-ChEs were also studied extensively-that is, CMs such as physostigmine and neostigmine because
Toxicology of Organophosphate and Carbamate Compounds
II. E F F E C T S O N A C T I O N P O T E N T I A L CONDUCTION Parameters associated with the conduction of action potentials in the peripheral nerve can be measured relatively 339
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SECTION IV. O r g a n T o x i c i t y
easily both in vivo and in vitro. Daily administration of trichlorfon to rats caused some changes in action potentials recorded from the sciatic nerve (Averbook and Anderson, 1983). Both the duration and the time to peak amplitude were shortened progressively over a 3-day period, and the rate of rise of the action potential was increased. The relative refractory period was shortened. In contrast, parathion had no effect on the action potential. However, acute exposure to trichlorfon did not cause any change in these action potential parameters. It was suggested that changes in nerve excitability may be a sensitive indicator of neurotoxicity. Nerve conduction in organophosphorus-induced delayed neuropathy (OPIDN) was studied using adult white leghorn hens treated with tri-2-o-cresyl phosphate or di-n-butyl2,2-dichlorovinyl phosphate (Robertson et al., 1987). Refractoriness was decreased in the tibial nerve but it was increased in the sciatic nerve, and the strength-duration threshold was elevated for both nerves. However, it is not known how these changes are related to the mechanism of OPIDN.
III. N E U R O M U S C U L A R
TRANSMISSION
In general, synaptic transmission is more vulnerable to drug action than action potential conduction. Thus, many electrophysiological studies of insecticide action were devoted to changes in synaptic transmission and various parameters associated with it. Soon after development of OP insecticides such as parathion, extensive electrophysiological studies were commenced for the purpose of elucidating the physiological mechanism of action of OPs/CMs on the insect nervous system. These studies were conducted almost at the same time as nonmedical CM anti-ChEs were examined. Many of these studies conducted in the late 1940s through the early 1960s involved vertebrate neuromuscular junctions and insect synaptic transmission. The effects of OPs/CMs on insect synapses are described later. Physostigmine, neostigmine (prostigmine), and diisopropyl fluorophosphate (DFP) were found to increase the endplate potential (EPP) duration and at high concentrations decreased the EPP amplitude of the frog (Eccles and McFarlane, 1949). Thus, this study clearly showed a postsynaptic block caused by the CMs. Presynaptic as well as postsynaptic actions were also demonstrated using rat diaphragm preparations in which paraoxon increased spontaneous miniature EPP (MEPP) frequency and half-decay time, but it caused neuromuscular block (Laskowski and Dettbarn, 1979). Rats injected with DFP showed neuromuscular weakness. In vitro experiments demonstrated that DFP prolonged the half-decay time of the MEPPs and nerve-evoked EPPs but had no effect on the amplitude of MEPPs and the quantal content of EPPs (Maselli and Soliven, 1991). oL-Tubocurarine had an antagonistic effect. It was concluded that the sustained endplate depolarization was
responsible for neuromuscular weakness of rats intoxicated with DFE However, it is not clear whether the direct action of DFP on the endplate is involved in addition to the effect caused by ChE inhibition. Involvement of the direct action on the endplate was indicated by the focal recording of miniature endplate currents (MEPCs) from frog sartorius muscle (Deana and Scuka, 1990). Neostigmine at 3 txM initially lengthened the decay phase of MEPCs and increased the amplitude of MEPCs probably due to ChE inhibition, but later it suppressed the amplitude and shortened the decay phase, suggesting a direct effect on the endplate. Tetanic contractions (Wedensky inhibition) induced by repetitive nerve stimulation in phrenic nerve-diaphragm preparations of mice was faded by 0.5-2 txM neostigmine (Chang et al., 1986). The fade was brought about by failure to elicit muscle action potentials, which was due to endplate depolarization and a decrease in transmitter release. Both effects were attributed to ACh accumulation as a result of ChE inhibition. Data that negate direct action on the muscle ACh receptor were also obtained using methamidophos, an OP insecticide (Camara et al., 1997). Despite its weak anti-ChE activity with an IC50 of --20 txM, ChE inhibition was long lasting, consistent with forming a covalent bond with the enzyme. Methamidophos increased the amplitude and prolonged the decay phase of nerve-evoked EPPs and MEPPs, but it did not affect transmitter release. There was no effect on whole cell currents induced by ACh, glutamate, or GABA in cultured hippocampal neurons. Thus, it was concluded that methamidophos acted as a selective anti-ChE. Albuquerque and associates conducted extensive studies of the mechanism of action of anti-ChEs at neuromuscular junctions and concluded that in addition to the effects via ChE inhibition, there were direct actions on the nAChRs. These studies are summarized in several papers (Albuquerque et al., 1984, 1985a,b, 1986, 1987, 1988, 1989). Furthermore, they showed that the antidotal effect of oximes is not simply due to carbamylation and reactivation of ChE but also due to the direct effect on the nACh receptors (Albuquerque et al., 1988b). Although their studies dealt with noninsecticidal OP compounds, such as pyridine-2-aldoxime (2-PAM), 1-(2-hydroxyiminomethyl1-pyridino)-3-(4-carbamoyl- 1-pyridino)-2-oxapropane (HI-6), and 1, l'-oxybis(methylene) bis-4-(1,1-dimethylethyl)-pyridinium (SAD-128), and CMs such as physostigmine, the data are worth summarizing because the studies are among the most extensive mechanistic studies. At the frog endplate, ( - ) physostigmine at low concentrations (0.2-2 txM) increased the peak amplitude of EPC and prolonged the decay phase of EPC. However, at high concentrations, the EPC peak amplitude was decreased, the EPC decay was accelerated, and single-channel lifetime was shortened (Shaw et al., 1985). DFP, dimethylphosphoramidocyanidic acid ethyl ester (tabun), O-ethyl S-2
CHAPTER 23 9Electrophysiological Mechanisms in Neurotoxicity diisopropylaminoethyl-methyl phosphonothiolate (VX), and ( - ) physostigmine interacted with pre- and postsynaptic regions of the glutamatergic neuromuscular synapse of locust muscle (Idriss et al., 1986). These agents initiated spontaneous EPSPs and muscle action potentials. Thus, this site is a new target of these anti-ChEs. In addition to ( - ) physostigmine, enantiomer (+) physostigmine also protected agonist lethality and myopathy caused by sarin, an effect most certainly not dependent on ChE inhibition but due to direct block of nACh receptors (Kawabuchi et al., 1988). The ChE reactivators 2-PAM and HI-6 increased the open probability of frog endplate channels that were activated by ACh. The oximes reduced mean channel open time and burst duration. EPC amplitude was increased by both of these oximes but depressed at their high concentrations. Thus, this study showed a direct interaction of 2-PAM and HI-6 with the nACh receptors (Alkondon et al., 1988). The nonoxime bispyridinium SAD-128 decreased the EPC peak amplitude and prolonged the EPC decay. At the single-channel level, SAD-128 reduced the mean open time and produced a blocked state evidenced as an additional phase in the closed time distribution. SAD-128 block of nACh receptors may underlie its efficacy in counteracting lethal effect of OP compounds (Alkondon and Albuquerque, 1989).
IV. S Y N A P T I C T R A N S M I S S I O N Studies on the effects of OPs/CMs have been performed with various preparations, including brain slices, cultured neurons, acutely isolated neurons, neuroreceptors expressed in a host cell, and neuronal cell lines. The electrophysiological techniques utilized are extracellular recording, intracellular recording, and patch clamp. Similar to the studies of neuromuscular functions, various compounds were tested. Thus, it is extremely difficult to obtain a unified picture regarding the mechanism of action of OPs/CMs on synaptic transmission. Many studies on the effects of OPs in brain slices have used slices containing only the hippocampus (Lebeda and Rutecki, 1985; Sarvey and Williamson, 1985; Williamson and Sarvey, 1985). In the evoked field potential, soman and other OPs induced a second population spike and spontaneous discharges. These effects are presumably caused by increases in ACh levels in the slice due to inhibition of ChE. However, there have been some suggestions of actions other than ChE inhibition (Lebeda and Rutecki, 1985; Williamson and Sarvey, 1985). A brain slice model has been developed that contains the entorhinal cortex as well as the hippocampus (Wood and Tattersall, 2001). The preparation allows the study of the spread of seizure discharges with the limbic system and the development of prolonged, sustained discharges that are rarely seen in the hippocampal slice preparation. In this preparation, 1 p.M soman induced a second
341
population spike in the evoked field potential in the CA1 or CA3 region, and a late repetitive discharge was also observed (Wood and Tattersall, 2001). Diazepam blocked these discharges. Although AP5 and MK-801, which are NMDA antagonists, had little or no effect on the discharges, CNQX and DNQX, which are AMPA/kainate antagonists, abolished the discharges. Effects of any chemicals on brain slice preparations could be caused by a direct or an indirect action. Therefore, the observed effects could be complicated. For example, 0.3-1 nM sarin decreased the amplitude of GABA-mediated postsynaptic currents in the CA1 pyramidal layer of the rat hippocampal slices. However, glutamatergic postsynaptic currents were not affected. It turned out that the observed effects were mediated by a direct interaction of the OP with muscarinic ACh receptors present on presynaptic GABAergic neurons and unrelated to ChE inhibition (Chebabo et al., 1999). Sarin had no effect on the amplitude or kinetics of GABA- or glutamate-mediated miniature postsynaptic currents, indicating that it does not interact with GABA or glutamate receptors. Another study using brain slices illustrates the difficulty in interpreting the data. Dichlorv0s exerted the opposite effect on somatosensory evoked potentials and hippocampal evoked population spikes (Papp et al., 1996). Only the effects on hippocampus could be explained in terms of an increased cholinergic activity. Friedman et al. (1998) studied the effects of several OPs and CMs on hippocampal slices using both electrophysiology and reverse-transcript polymerase chain reaction. DFP 1 txM or pyridostigmine 1 mM increased the mRNA levels of c-Fos and AChE and decreased the mRNA levels of choline acetyltransferase and vesicular ACh transporter. DFP, pyridostigmine, carbamylcholine, and physostigmine increased the amplitude of population spikes. When low stimulus intensities were used, pyridostigmine facilitated the second response but suppressed the response later. Both phases were prevented by atropine, indicating the dependence on muscarinic receptor activation. However, it remains to be determined how the changes in mRNA transcripts are related to electrophysiological changes. Chronic effects of sublethal injection of paraoxon on transmitter release were studied using intracellular recording of MEPPs and EPPs (Thomsen and Wilson, 1986). Tolerance was not due to a decrease in postsynaptic sensitivity but due to presynaptic changes. Transmitter release was suppressed due to a decrease in the transmitter store and mobilization ability. Thus, depression of quantal release of transmitter accounts for behavioral tolerance. Low-level, chronic dichlorvos treatment of rats caused alterations in all parameters measured, which included electrocorticogram, sensory cortical evoked potentials, conduction velocity, and refractory periods of peripheral nerve (D6si and Nagymajt6nyi, 1999). These parameters are sensitive biomarkers of the exposure at low-dose levels. Brain ChE was inhibited only at high doses.
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SECTION IV. O r g a n
Toxicity
Coho salmon was highly sensitive to CMs and the effects of three CMs on the electroolfactogram were studied (Jarrard et al., 2004). The electroolfactogram was sensitive to brief exposure to two CMs: The effective nominal concentration for a 50% reduction (ECs0) in electroolfactogram amplitude was 10.4 i~g/liter for carbofuran and 1.28 #g/liter for the anti-sapstain IPBC. However, the fungicide mancozeb was less potent with an ECs0 of 2.05 mg/liter. The sensitivity of salmon olfactory neurophysiology to pesticides appears to be not only due to ChE inhibition but also due to other unknown mechanisms. Single neurons are a convenient material for detailed analysis of drug-receptor interactions. Sympathetic ganglion neurons of bullfrogs were used for the study of action of VX (Heppner and Fiekers, 1992). The amplitude of excitatory postsynaptic potentials (EPSPs) was increased, the membrane was depolarized, the input resistance was reduced, and the duration of the spike after hyperpolarization was shortened. The observed increase in neuronal excitability may be due to the decrease in after-hyperpolarization. The effects of
A Control
VX on cultured hippocampal neurons were also studied (Rocha et al., 1999). At a low concentration of 0.01 nM, VX decreased the amplitude of evoked GABAergic currents, and at higher concentrations (> 1 nM) it decreased the amplitude of glutamatergic currents as well. The VX effect on GABAergic currents was prevented by atropine, indicating an action via the cholinergic system. In the presence of tetrodotoxin, VX (->10 nM) increased the frequency of GABA- and glutamate-mediated miniature postsynaptic currents. This is unrelated to ChE inhibition and is due to alteration of transmitter release. Detailed patch clamp analyses were performed for nACh receptors of PC12 cells (Nagata et al., 1997). Carbaryl showed a biphasic effect: At 0.1 #M it greatly potentiated carbachol currents, and at 0.3-3 #M it suppressed the current. Single-channel experiments indicated that both carbaryl and neostigmine increased short closures or gaps during channel opening, decreased the mean open time and burst duration, but caused no change in single-channel conductance (Fig. 1). These effects appear to be exerted by direct
C 1 I~ carbaryl
close open -r. . . . . . . . . . .
~-':'~
....
~-'-~.~-'~-'=-~~ L v ~ ~
B 101~1 neostigmine
,,-"r-"'," r '"T-L~r,,,,-,.-,,-,
'' .... .---&-r~':;-:=-~--;'~
D 10 I~ carbaryl.
50 ms , , ,
FIG. 1. Single-channel currents induced by application of ACh and coapplications of ACh and neostigmine or carbaryl to cell-attached membrane patches of PC12 cells clamped at a membrane potential 40 mV more positive than the resting potential. (A) Currents induced by 30 ~M ACh occurred during brief isolated openings or during longer openings interrupted by a few short closures or gaps. (B) Coapplication of 30 ~M ACh and 10 I~M neostigmine. Channel openings occurred in bursts separated by brief closures or gaps. (C) Coapplication of 30 ~M ACh and 1 ~M carbaryl. Channel openings were separated by brief closures or gaps. (D) Coapplication of 30 ~M ACh and 10 ~M carbaryl. Channel openings were further separated by brief closures or gaps and occurred in bursts. A-D were obtained from different membrane patches. From Nagata et al. (1997).
I
5pA
CHAPTER 23 9Electrophysiological Mechanisms in Neurotoxicity
FIG. 2. Increase in synaptic after-discharge and eventual block during exposure to thiol-methyldemeton in cockroach nerve preparation. Postsynaptic action potentials were evoked by presynpatic nerve stimulation (A1 and A2). After-discharges were greatly intensified 8, 13, and 16 min after application of 500 txM thiol-methyldemeton and synaptic transmission was eventually blocked (B3, 21 min). Time marker of 20 msec applies to A2, A3, B 1, and B2, and 1-msec time marker applies to A1 and B3. From Narahashi and Yamasaki (1960).
343
block of nACh receptors. For the study of identified subunits of receptors, Xenopus oocyte expression is a powerful approach. The effects of OPs were studied on the OL4~2 nACh receptors expressed in oocytes (Smulders et al., 2004). Parathion-ethyl, chlorpyrifos, and disulfoton inhibited ACh currents in micromolar concentrations. Binding experiments showed that OPs noncompetitively interact with nACh receptors. Reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It appears that OPs interact directly with the OL4~2 nACh receptor, causing suppression. These two studies clearly indicate that OPs/CMs can directly block nACh receptors. Extensive studies were conducted on the effects of anti-ChE OPs and CMs on synaptic transmission of cockroaches. Stimulation of presynaptic cercal nerve evoked postsynaptic responses comprising the initial large action potentials followed by after-discharges with smaller amplitudes (Fig. 2, A1 and A2). After treatment with OPs or CMs, after-discharges were greatly increased in amplitude and prolonged in duration (Fig. 2, A3, B1, and B2), but synaptic transmission was eventually blocked (Fig. 2, B3). However, the effect reappeared soon, and this block-recovery cycle was repeated many times in the continuous presence of anti-ChEs (Roeder et al., 1947; Roeder, 1948; Roeder and Kennedy, 1955; Twarog and Roeder, 1957; Yamasaki and Narahashi, 1958, 1960; Narahashi and Yamasaki, 1960; Heppner et al., 1987). Obviously, such intense after-discharges are responsible for hyperactivity of insects intoxicated with anti-ChEs. The mechanism of hyperexcitatory action of anti-ChEs was further studied by recording the EPSPs from cockroach ganglia (Yamasaki and Narahashi, 1958, 1960). This was accomplished by placing an external electrode on the sixth abdominal ganglion while the other external electrode was in contact with the nerve cord between the second and third
FIG. 3. Augmentation and prolongation by physostigmine of the excitatory postsynaptic potential recorded from the sixth abdominal ganglion of the cockroach by external electrodes. Recording was made in urethane to block discharges of action potentials. (A) Control; (B) 8 min after application of 15 ~M physostigmine; (C) 50-Hz time marker for records A and B; (D) 12 min after application of physostigmine; 50-Hz time marker. Voltage calibration in C, 0.5 mV, applies to all records. From Narahashi (1965).
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SECTION I V . O r g a n T o x i c i t y
abdominal ganglia. This arrangement allowed us to record changes in the membrane potential of the sixth abdominal ganglion as well as the action potentials. In the presence of urethane to step discharges, and EPSP could be elicited by presynaptic stimulation (Fig. 3A). The EPSP was augmented in amplitude and prolonged in duration after application of 15 IxM physostigmine (Fig. 3B). These effects were potentiated with time, and eventually a very large and greatly prolonged EPSP could be recorded (Fig. 3D). Since the synapses in the sixth abdominal ganglion were shown to be cholinergic, the effect of physostigmine was obviously due to accumulation of ACh as a result of ChE inhibition. Simultaneous measurements of postsynaptic responses and ChE activity were performed using the cockroach nerve cord, including the sixth abdominal ganglion intoxicated with parathion, thiol-demeton, thiol-methyldemeton, or thiono-methyldemeton (Yamasaki and Narahashi, 1960; Narahashi and Yamasaki, 1960). At the time when synaptic facilitation occurred, ChE was partially inhibited.
V. CONCLUSIONS OPs and CMs stimulate and then suppress synaptic and neuromuscular transmission. The effects are due partly to the inhibition of ChE and partly to the direct effect on the ACh receptors. The degree and time course of these effects varied considerably depending on the kind of OPs/CMs, dose, methods of administration, and the species of animals. However, the basic mechanism of action on mammals and insects appears to be the same.
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9Electrophysiological Mechanisms in Neurotoxicity
physiological and transcriptional responses in hippocampal slices. J. Physiol. Paris 92, 329-335. Fukuto, T. R. (1979). Effect of structure on the interaction of organophosphorus and carbamate esters with acetylcholinesterase. In Neurotoxicology Insecticides and Pheromones (T. Narahashi, Ed.), pp. 277-295. Plenum, New York. Heppner, T. J., and Fiekers, J. E (1992). VX enhances neuronal excitability and alters membrane properties of Rana catesbeiana sympathetic ganglion neurons. Comp. Biochem. Physiol. C 102, 335-338. Heppner, T. J., Drewes, C. D., and Coats, J. R. (1987). Electrophysiological effects of paraoxon on the giant interneurons in the American cockroach, Periplaneta americana. Pesticide Biochem. Physiol. 28, 57-66. Idriss, M. K., Aguayo, L. G., Rickett, D. L., and Albuquerque, E. X. (1986). Organophosphate and carbamate compounds have preand postjunctional effects at the insect glutamatergic synapse. J. Pharmacol. Exp. Ther. 239, 279-285. Jarrard, H. E., Delaney, K. R., and Kennedy, C. J. (2004). Impacts of carbamate pesticides on olfactory neurophysiology and cholinesterase activity in coho salmon (Oncorhynchus kisutch).Aquat. Toxicol. 69, 133-148. Jones, S. W., Sudershan, E, and O'Brien, R. D. (1979). Interaction of insecticides with acetylcholine receptors. In Neurotoxicology of Insecticides and Pheromones (T. Narahashi, Ed.), pp. 259-275. Plenum, New York. Kawabuchi, M., Boyne, A. E, Deshpande, S. S., Cintra, W., Brossi, A., and Albuquerque, E. X. (1988). Enantiomer (+) physostigmine prevents organophosphate-induced subjunctional damage at the neuromuscular synapse by a mechanism not related to cholinesterase carbamylation. Synapse 2, 139-147. Koelle, G. B. (1994). Pharmacology of organophosphates. J. Appl. Toxicol. 14, 105-109. Laskowski, M. B., and Dettbarn, W. D. (1979). An electrophysiological analysis of the effects of paraoxon at the neuromuscular junction. J. Pharmacol. Exp. Ther. 210, 269-274. Lebeda, E J., and Rutecki, P. A. (1985). Characterization of spontaneous epileptiform discharges induced by organophosphorus anticholinesterases in the in vitro rat hippocampus. Proc. West. Pharmacol. Soc. 28, 187-190. Maselli, R. A., and Soliven, B. C. (1991). Analysis of the organophosphate-induced electromyographic response to repetitive nerve stimulation: Paradoxical response to edrophonium and D-tubocurarine. Muscle Nerve 14, 1182-1188. Murphy, S. D., Costa, L. G., and Wang, C. (1984). Organophosphate insecticide interaction at primary and secondary receptors. In Cellular and Mechanism Neurotoxicology (T. Narahashi, Ed.), pp. 165-176. Raven Press, New York. Nagata, K., Huang, C. S., Song, J. H., and Narahashi, T. (1997). Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels. Brain Res. 769, 211-218. Narahashi, T. (1965). Mode of action of insecticides. In Shin Noyaku Soseiho (Creation of New Insecticides), ed. by (R. Yamamoto and T. Noguchi, Eds.), pp. 169-209. Nankodo, Tokyo. Narahashi, T., and Yamasaki, T. (1960). Nervous and cholinesterase activities in the cockroach as affected by demeton and methyldemeton. Studies on the mechanism of action of insecticides (XVIII). Jpn. J. Appl. Ent. Zool. 4, 64-69.
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O'Brien, R. D. (1967). Insecticides: Action and Metabolism. Academic Press, New York. Papp, A., Gyorgyi, K., Nagymajtenyi, L., and Desi, I. (1996). Opposite short-term changes induced by an organophosphate in cortical and hippocampal evoked activity. Neurobiology 4, 431-440. Robertson, D. G., Schwab, B. W., Sills, R. D., Richardson, R. J., and Anderson, R. J. (1987). Electrophysiological changes following treatment with organophosphorus-induced delayed neuropathy-producing agents in the adult hen. Toxicol. Appl. Pharmacol. 87, 420429. 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-independent actions. Toxicol. Appl. Pharmacol. 159, 3140. Roeder, K. D. (1948). Organization of the ascending giant fiber system in the cockroach (Periplaneta americana). J. Expl. Zool. 108, 243-261. Roeder, K. D., and Kennedy, N. K. (1955). The effect of certain tri-substituted phosphine oxides on synaptic conduction in the roach. J. Pharmacol. Exp. Ther. 114, 211-220. Roeder, K. D., Kennedy, N. K., and Samson, E. A. (1947). Synaptic conduction to giant fibers of the cockroach and the action of anticholinesterases. J. Neurophysiol. 10, 1-10. Sarvey, J. M., and Williamson, A. M. (1985). Diazepam and barbiturates antagonize the effect of an organophosphate anticholinesterase in the rat hippocampal slice. Proc. Med. Def. Biosci. Rev. 5, 421-423. Shaw, K.-E, Aracava, Y., Akaike, A., Daly, J. W., Rickett, D. L., and Albuquerque, E. X. (1985). The reversible cholinesterase inhibitor physostigmine has channel-blocking and agonist effects on the acetylcholine receptor-ion channel complex. Mol. Pharmacol. 28, 527-538. Smulders, C. J., Bueters, T. J., Vailati, S., van Kleef, R. G., and Vijverberg, H. P. (2004). Block of neuronal nicotinic acetylcholine receptors by organophosphate insecticides. Toxicol. Sci. 82, 545-554. Thomsen, R. H., and Wilson, D. E (1986). Chronic effects of paraoxon on transmitter release and the synaptic contribution to tolerance. J. Pharmacol. Exp. Ther. 237, 689-694. Twarog, B. M., and Roeder, K. D. (1957). Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. Ent. Soc. Am. 50, 231-237. Williamson, A. M., and Sarvey, J. M. (1985). Effects of cholinesterase inhibitors on evoked responses in field CAI of the rat hippocampus. J. Pharmacol. Exp. Ther. 235, 448-455. Wood, S. J., and Tattersall, J. E. (2001). An improved brain slice model of nerve agent-induced seizure activity. J. Appl. Toxicol. 21(Suppl. 1), $83-$86. Woolley, D. E., Chernobieff, J. R., and Reiter, L. W. (1979). Effects of parathion on the mammalian nervous system. In Neurotoxicology of Insecticides and Pheromones (T. Narahashi, Ed.), pp. 155-181. Plenum, New York. Yamasaki, T., and Narahashi, T. (1958). Synaptic transmission in the cockroach. Nature 182, 1805-1806. Yamasaki, T., and Narahashi, T. (1960). Synaptic transmission in the last abdominal ganglion of the cockroach. J. Insect Physiol. 4, 1-13.
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CHAPTER
24
Behavioral Toxicity of C h o l i n e s t e r a s e Inhibitors I
PHILIP J. BUSHNELL AND VIRGINIA C. MOSER U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
systemic toxicity caused by inhibition of cholinesterase activity and of treatment of acute poisoning from OPs followed subsequently (Minton and Murray, 1988; Marrs, 1993). Two reviews focusing on the behavioral effects of OPs were published in the early 1990s (Annau, 1992; D'Mello, 1993). It is interesting that these two reviews diverged greatly on the degree of concern warranted by the existing data. Thus, Annau concluded, "in all species examined and at all ages, exposure to these compounds can have deleterious and long-lasting, perhaps irreversible consequences." In contrast, D'Mello concluded that "very little confidence can be placed in existing descriptions of the behavioral effects of anticholinesterases (anti-ChEs) in humans," "data from animal experimental studies [yields] information [that] is relatively superficial," and "therefore, although much information is available, surprisingly little is known of the behavioural changes induced by anti-ChEs." Despite these differing conclusions, both authors called for improved and more systematic research into this contentious issue. The field has remained rife with energetic debates and contradictory conclusions, driven by the opposing concerns for agricultural benefit and public safety. The current debate centers around determining whether and under what conditions these compounds produce longlasting effects in humans. These questions have been examined descriptively in human clinical and epidemiological studies and experimentally in animal models. Reviews have flourished along with the debate; their authors have begun to organize the existing data in ways that bring the question in focus by classifying observed effects (acute vs long term) and attempting to categorize degrees of intoxication that may (or may not) be associated with each sort of effect. Section II reviews the acute behavioral effects of OP and carbamate (CM) pesticides in humans and animals. We describe the classic behavioral signs and symptoms experienced by humans poisoned with these compounds and illustrate ways in which animal models have been used
I. I N T R O D U C T I O N Because of the importance of acetylcholine (ACh) as a neurotransmitter in the mammalian nervous system, chemicals that inhibit acetylcholinesterase (ACHE) can exert profound effects on behaviors mediated by the cholinergic system. These effects may be either beneficial or detrimental, depending on the dose and degree of inhibition of the regulatory enzyme and the physiological condition of the treated individual. For example, the cholinesterase inhibitors tacrine and metrifonate have been used to treat the cognitive decline associated with aging under the assumption that insufficient cholinergic tone in the central nervous system (CNS) impairs cognitive function. Nonetheless, the widespread use of cholinesterase-inhibiting compounds as pesticides has generated concerns regarding their effects on public health. These concerns center around the acute adverse effects of high doses of these agents, how these effects may change with repeated exposure, and the possibility of long-term consequences of chronic, low-level exposure to them. This chapter examines these issues, focusing on the behavioral effects of cholinesteraseinhibiting pesticides as indicators of the impact of exposure to these beneficial products on public health. The effectsof these compounds have been the subject of many reviews since Gershon and Shaw (1961) described the anxiogenic effects of exposure to organophosphates (OPs) in humans. Early experimental work on these compounds in the 1970s focused on the mechanisms of action and tolerance to OPs (reviewed by Costa et al., 1982; Russell and Overstreet, 1987). More general reviews of the
1This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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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to characterize them and to understand the mechanisms by which they are produced. In Section III, we review studies on persistent effects of exposure to cholinesterase inhibitors. The human epidemiological literature is examined for evidence that persistent behavioral effects of exposure can be measured in populations with histories of acute poisoning and/or chronic ongoing exposure. We then review behavioral data from animal models designed to characterize the behavioral changes caused by experimental treatments with cholinesteraseinhibiting pesticides and identify similarities with the literature on humans exposed occupationally to these compounds. Age-related differences in sensitivity to cholinesterase inhibitors are reviewed in Section IV. A considerable research effort has been expended to characterize the increased sensitivity of the young animal to these pesticides. We present data comparing the sensitivity of young and adult animals to the neurochemical and behavioral effects of these compounds and discuss the factors that appear to mediate differences in response during development.
II. A C U T E E F F E C T S
OF CHOLINESTERASE INHIBITORS The signs and symptoms of acute exposure to cholinesterase inhibitors are consistent with overstimulation of cholinergic receptors in the central and peripheral nervous systems due to the blockade of AChE and subsequent buildup of ACh. This includes stimulation of muscarinic receptors at effector organs and in the CNS, as well as stimulation and then desensitization of nicotinic receptors at autonomic ganglia and skeletal muscle (Taylor, 1985). The result is a dose-dependent mix of physiological, neuromuscular, and behavioral signs and symptoms. Most of these changes may be detected with measures of unconditioned behaviors (e.g., observational evaluations and tests of motor function); detection and characterization of more subtle changes require tests of conditioned behavior (e.g., tests of learning, memory, attention, and affect) in both human subjects and laboratory animals.
A. Changes in Unconditioned Behaviors Case reports of accidental and intentional poisoning with cholinesterase-inhibiting pesticides have identified common signs and symptoms of acute exposure in humans (Brown and Brix, 1998; Hayes, 1982; Morgan, 1989; O'Malley, 1997). Mild poisonings often present as flu-like symptoms headache, fever, sweating, respiratory congestion, nausea, vomiting, and abdominal cramping. Somewhat higher exposures produce more pronounced symptoms as well as dizziness, ataxia, gastrointestinal distress (including diarrhea and polyuria), muscle weakness, excessive salivation and lacrimation, constriction of the pupils (miosis), and
muscular twitching and fasciculations. Severe poisoning produces bronchospasm, irregular heartbeat, loss of reflexes, progressing to convulsions and coma in extremis. Table 1 lists the incidence of a range of symptoms reported in banana plantation workers (Wesseling et al., 2002), which clearly indicates that signs of mild toxicity are most often reported. Curiously, the frequency with which these signs are reported does not correlate well with the degree of cholinesterase inhibition, possibly due to differences in the sampled tissue (e.g., plasma, erythrocytes, whole blood, and serum), sampling time after exposure, assay method, specific pesticide involved, and the wide variation of cholinesterase activity in the human population (Bobba et al., 1996; Lessenger and Reese, 1999; Nouira et al., 1994). Given the nature of case reports, it is often difficult to identify which specific pesticide(s) may be involved, and it is even more difficult to specify the level of exposure. Such issues are not as pertinent to animal studies, for which the chemical, dose, and time course can be specified accurately. General descriptions of the effects of cholinesterase inhibitors in laboratory animals resemble the signs evident in humans, including ataxia, twitching and fasciculations, miosis, salivation, diarrhea, lacrimation, muscle weakness, depressed responses, and tremors (Ehrich et al., 1993; Mattsson et al., 1996; Moser, 1995; Moser et al., 1988, 1997; Nostrandt et al., 1997; Pope et al., 1991). Because all cholinergic receptors are involved, the autonomic signs of miosis, salivation, and hypothermia likely reflect muscarinic stimulation, whereas the tremors, gait changes, muscle weakness, and motor activity likely reflect suppressed nicotinic responsiveness from prolonged activation and desensitization of nicotinic receptors (Taylor, 1985). Other effects also appear to be cholinergically mediated, including decreased tail pinch response (indicating antinociception) (Iwamoto and Marion, 1993; Koehn and Karczmar, 1978; Pedigo et al., 1975), mouth smacking or chewing (Kelley et al., 1989; Rupniak et al., 1990; Salamone et al., 1986, 1990), and hypothermia (Gordon, 1994). To compare the acute effects of cholinesterase inhibitors in humans and animals more explicitly, we listed some of the measures used in animal studies and tallied the number of studies that showed effects on each measure. The data were taken from seven different chemicals, some tested more than once, to yield 19 dose-response curves (Mattsson et al., 1996; Moser, 1995, 1999; Moser et al., 1988, 1997; Nostrandt et al., 1997). The resulting incidence of signs is listed in Table 1, alongside the corresponding human sign. Some end points that are often reported in humans (e.g., lightheadedness, weakness, and salivation) correspond to signs that are often observed in rats as well. Some human signs cannot be measured in rats (e.g., nausea and sweating) and likewise there are some measures used in animals that either have no direct correlation to the human condition (e.g., antinociception) or were not reported in the
CHAPTER 24 9Behavioral Toxicity
TABLE 1.
349
Incidence of Signs and Symptoms of Acute Poisoning with ChE lnhibitors a
Human sign/symptom
Incidence (%)
Lightheadedness Nausea General weakness Abdominal pain Excessive sweating Salivation Headache Vomiting Blurred vision Muscle twitching Muscle cramps Difficulty breathing Involuntary loss of urine Seizures
94 91 91 89 83 79 78 75 75 67 56 51 6 3
Corresponding animal sign Ataxia, incoordination
Incidence (%) 100
b
Lowered activity, weakness
79
Salivation
84
Miosis, pupil response Twitching, fasciculations
74 68
Polyuria Convulsions, seizures Lacrimation Decreased tail pinch response Hypothermia
16 11 58 89 100
aHuman signs and symptoms are reported by 81 Costa Rican banana plantation workers (Wesseling et al., 2002). Corresponding animal signs are compilation of data from 19 studies with seven different ChE inhibitors. b indicateslack of corresponding end point.
specific paper cited (e.g., lacrimation). Although drawing conclusions with this comparison must be tempered with caution, it is clear that the animal model predicts the acute signs of toxicity of cholinesterase-inhibiting pesticides remarkably well. Rodents observed in test batteries reveal a wide range of signs in response to poisoning with pesticides and other chemicals. Domain analysis (Moser, 1991) shows that different cholinesterase-inhibiting pesticides tend to produce similar toxicities overall (Ehrich et al., 1993; Mattsson et al., 1996; Moser, 1995; Moser et al., 1988, 1997; Nostrandt et al., 1997). For example, all produce marked changes in the measures that evaluate autonomic function, activity levels, and neuromuscular ability. At high dose levels, tests of excitability and sensorimotor responsiveness are altered. A closer evaluation of the profile of behavioral effects, however, shows differences across pesticides in terms of the effects that appear at the lowest dose ("critical effects"). In one study, the critical effects of cholinesterase inhibitors varied by pesticide, and the slopes and maximum values of the dose-response curves varied for these effects (Moser, 1995, 1999). For example, comparing doses of aldicarb and methamidophos that produced similar levels of cholinesterase inhibition revealed that all
doses of aldicarb produced behavioral signs, but only the highest methamidophos dose was effective (Moser, 1999). This dissociation between behavioral effects and cholinesterase inhibition suggests the existence of other mechanisms of action, as proposed in the literature (e.g., direct actions on the receptor and altered reuptake of choline; McDaniel and Moser, 2004). The behavioral effects of cholinesterase-inhibiting pesticides may be at least partially mediated by other mechanisms (reviewed in Pope et al., 1995; Pope, 1999). These other actions may influence a pesticide's overall toxicity by modifying or enhancing the consequences of cholinergic overstimulation since some of these actions are observed at levels at or above those required to inhibit ACHE. For example, some OPs have been shown to act at the c i s - m e t h y l d i o x o l a n e sensitive population of muscarinic receptors and alter presynaptic choline uptake and ACh release (Bakry et al., 1988; Jett et al., 1991; Katz and Marquis, 1989; Liu and Pope, 1998; Silveira et al,, 1990; Van Den Beukel et al., 1997; Ward and Mundy, 1996; Ward et al., 1993). Furthermore, pesticides differ with respect to these noncholinesterase actions (Chaudhuri et al., 1993), which could provide an explanation for the different behavioral profiles observed. However, the precise role of these
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Organ Toxicity
differing influences has not been delineated. The implications of this dissociation include (1) that the degree of cholinesterase inhibition will not always predict the functional effect of a compound, and (2) that some of the low correspondence between cholinesterase activity and behavioral effect in human studies may be related to the specific inhibitors involved.
B. Changes in Conditioned Behaviors The clear and compelling importance of cholinergic neurotransmitter pathways in mediating sensory, motor, and cognitive functions in animals suggests that these functions should be sensitive to disruption by cholinesteraseinhibiting pesticides. Sensory and motor deficits, including general weakness, muscle twitching, blurred vision, tingling in the extremities, and difficulty breathing (Richter et al., 1992; Wessseling et al., 2002), are consistent with this supposition. On the other hand, complaints of cognitive impairment (e.g., confusion or forgetfulness)do not appear in the symptomatology of acute intoxication with these agents (Richter et al., 1992; Wessseling et al., 2002). In fact, assessment of choice behavior in animals after single doses of rapidly acting pesticides has not revealed evidence for cognitive impairment. Injection of rats with carbaryl or propoxur did not affect response accuracy in two tests of working memory - - delayed response (Heise and Hudson, 1985a) and continuous nonmatching (Heise and Hudson, 1985b). These negative data contrast with reductions in trial initiation caused by these compounds as well as with clear disruptions of working memory after injection of scopolamine in these tests. Similarly, a single injection of DFP in rats trained in a delayed matchingto-position/visual discrimination task showed no evidence of impaired working or reference memory (Bushnell et al., 1991). On the other hand, treatments that cause prolonged inhibition of cholinesterase activity have been shown to impair cognitive functions in rats. Prolonged inhibition of cholinesterase activity can also be achieved after a single subcutaneous injection of the OP chlorpyrifos (Pope et al., 1992). By this route, chlorpyrifos inhibits cholinesterase activity for 7 or 8 weeks and also causes significant downregulation of muscarinic receptors that recover in parallel with recovery of cholinesterase activity (Bushnell et al., 1994). In addition, this treatment impaired delayed matching accuracy (a measure of attention and working memory) and reduced response speed, but it spared discrimination accuracy (a measure of reference memory) (Bushnell et al., 1994). These effects suggest that prolonged inhibition of cholinesterase activity elicits compensatory changes in the CNS (e.g., downregulation of muscarinic receptors) that secondarily reduce the cognitive capacity of the animal. The behavioral effects of subcutaneous chlorpyrifos on delayed matching accuracy reported by Bushnell et al.
(1994) involved a parallel downward shift in the function relating matching accuracy to the delay between the sample presentation and the animals' choice. In other words, accuracy did not decrease faster as a function of delay in the treated rats: thus, these animals did not forget the sample information faster than controls, but they encoded it less accurately. This pattern suggests a deficit in attending to the sample information. To explore the potential effect of chlorpyrifos on attention, Bushnell et al. (2001) dosed rats with chlorpyrifos and quantified their behavior with a visual signal detection method that was designed to assess sustained attention (Bushnell, 1999). In addition, to evaluate the relative sensitivity of this operant test and a standard neurobehavioral screen, the animals were also tested with a functional observational battery plus motor activity. Acute treatment with chlorpyrifos transiently reduced accuracy and increased response time in the signal detection test, confirming that acute chlorpyrifos can impair attention. The signal detection task was not more sensitive to the effects of chlorpyrifos than was the neurobehavioral battery, which detected differences in the motor, reactivity, and sensorimotor domains at the same doses that affected signal detection. Thus, although the signal detection method did not provide an increase in sensitivity to chlorpyrifos exposure, it did specify the cognitive deficit resulting from the exposure. Two studies suggest that OPs may also be acutely anxiogenic in rats, a finding consistent with long-standing observations that have associated pesticide exposure with anxiety, depression, and suicide in humans, both after long-term exposure (Gershon and Shaw, 1961; Metcalf and Holmes, 1969; London et al., 2005) and associated with acute exposure (Richter et al., 1992; Stephens et al., 1995). First, S~inchez-Amate et al. (2001) reported that rats dosed with chlorpyrifos explored open arms in an elevated plusmaze less than did controls, in the absence of signs of acute toxicity. This pattern of behavior was also observed after injection with pentylenetetrazol, an anxiogenic drug, and the converse pattern of exploration was observed after injection with diazepam, an anxiolytic drug. Second, the same group reported that chlorpyrifos shares interoceptive stimulus properties with pentylenetetrazol in rats, as evaluated with a drug discrimination procedure, indicating that the internal cues generated by CPF resemble those of the anxiogenic drug (S~inchez-Amate et al., 2002). Thus, these animal models confirm reports of anxiety in acutely exposed humans. This evidence also suggests the importance of assessing affect in further work with OPs in animals and in humans. Whereas the time course of recovery from acute poisoning in humans depends on the chemical and the level of exposure, acute cholinergic signs of intoxication with cholinesterase inhibitors typically abate by 24 hr after treatment in rats. Reactivation of the AChE enzyme is much slower than this time course indicates, suggesting that
CHAPTER 24 9Behavioral Toxicity behavioral recovery cannot be due solely to resynthesis of the enzyme (Bushnell et al., 1994; Carr and Chambers, 1991; Moser and Padilla, 1998; Reiter et al., 1973). The mechanisms underlying this recovery have not been thoroughly explained, but they probably include changes in cholinergic receptor density as well as learned compensations associated with behavioral tolerance (Bushnell et al., 1994; Young and Goudie, 1995). In summary, these observations indicate that the acute signs of cholinesterase inhibition in humans are generally well understood and can be modeled usefully in rats. The functional domains affected by these compounds reflect the immediate effects of overstimulation of muscarinic and nicotinic cholinergic receptors in the central and peripheral nervous systems. These effects are well documented and serve as a basis for addressing the more vexing questions about the potential effects of repeated exposure to cholinesterase inhibitors and the potential for persistent effects after acute poisoning and after long-term, subclinical exposures. Future work would also profit by including assessments of affect induced by treatment with these compounds.
III. E F F E C T S O F R E P E A T E D E X P O S U R E TO C H O L I N E S T E R A S E I N H I B I T O R S
A. Clinical and Epidemiological Studies in H u m a n s Studies on the behavior of people occupationally exposed to cholinesterase inhibitors have yielded a range of findings that suggest that persistent behavioral deficits can follow from frank poisoning with these agents. Whether permanent deficits follow prolonged exposure to doses that do not induce signs or symptoms of poisoning ("subclinical" exposure) is far more controversial. This section considers epidemiological studies in this area and some animal studies that have addressed these issues experimentally. Kamel and Hoppin (2004) reviewed the epidemiological literature that explored potential neurotoxicity from chronic exposure to pesticides, including OPs, CMs, fungicides, and fumigants. Of 39 studies reported between 1974 and 2003, 27 focused on OP and CM insecticides; of these, 25 studies reported some association between exposure to the insecticide and an effect. The domains most consistently affected by OPs and/or CMs were psychomotor function, symptoms and affect, and cognitive function (Table 2). Vibration sensitivity, balance, tremor, and nerve function were less frequently measured and less frequently affected when assessed. Thus, there is compelling evidence that acute poisoning from pesticides is frequently, but not inevitably, associated with persistent behavioral changes of some kind, and that alterations in affective, cognitive, and motor functions are most commonly observed.
351
TABLE 2. Neurobehavioral Effects of Chronic Exposure to Cholinesterase-lnhibiting Pesticides (OPs and CMs) in Humans (Derived from Kamel and Hoppin, 2004) and in Animals (Compiled from 15 Papers on Repeated Exposures in Animals)
Domain Human studies Psychomotor Symptoms and affect Cognitive Vibration sensitivity Balance Tremor Animal studies Motor activity Neuromotor Cholinergic signs Vestibular function Cognitive
No. positive/No, studies
8/11 13/19 10/15 4/10 1/4 0/2 9/13 7/13 9/13 4/9 5/8 worsened 2/8 improved
% positive
73 68 67 40 25 0 69 54 69 44 63 25
However, these reports included studies of people overtly poisoned with pesticides as well as people exposed but not clinically affected. Studies of agricultural pesticide applicators from the early 1990s suggested that overt poisoning episodes were necessary for induction of persistent cognitive sequelae of exposure. For example, Nicaraguan pesticide applicators with a history of hospitalization for treatment of poisoning with OPs showed long-term deficits in cognitive and motor functions assessed approximately 2 years after the poisoning episode (Rosenstock et al., 1991). Similar results were obtained from poisoned workers in California (Steenland et al., 1994). In contrast, the neuropsychological performance of agricultural workers applying pesticides ~under well-controlled conditions, with less than 15% inhibition of cholinesterase activity (Karr et al., 1992), did not differ from that of controls when examined with a standardized neurobehavioral test battery (Daniell et al., 1992). Thus, the evidence for probable long,term effects of poisoning with cholinesterase inhibitors is quite convincing because it has also been documented after mild poisoning in workers from a number of occupations using a variety of neurobehavioral end points (Stallones and Beseler, 2002; see also reviews by Jamal et al., 2002a,b; Kamel and Hoppin, 2004). The more troublesome issue involves potential longterm effects from subsymptomatic exposures. A series of studies of farmers in the United Kingdom who regularly dip their sheep into solutions of OP pesticides for the control of parasites raised concern about persistent effects of subsymptomatic exposure. Stephens et al. (1995) showed that "sheep dippers" performed more poorly on
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Toxicity
tests of sustained attention and information processing speed and were more vulnerable to psychiatric disorders than quarry workers who lacked exposure to pesticides. None of the farmers had experienced acute poisoning episodes, although they did experience symptoms of exposure during periods of dipping. Because the neurobehavioral tests were conducted 2 or more months after dipping, these effects were not related to recent pesticide exposure. In addition, a follow-up study showed that these effects of chronic exposure were not correlated with effects of acute exposure (Stephens et al., 1996). Subsequent studies of this population have focused on other effects, including sensory dysfunction (Beach et al., 1996; Jamal et al., 2002a) and peripheral neuropathy (Pilkington et al., 2001). As with the behavioral effects previously reported, a loss of somatotopic sensory acuity was related to the severity of symptoms during periods of acute exposure (Beach et al., 1996). Two studies of termiticide applicators in the United States support the conclusion that occupational exposure to OPs in the absence of frank poisoning episodes engenders risk of subclinical neurological dysfunction. Thus, commercial applicators of chlorpyrifos performed more poorly than unexposed controls on vestibulomotor tests and reported a higher frequency of symptoms of cognitive, emotional, and motor difficulties (Steenland et al., 2000). A small subgroup (eight men) who reported past poisoning episodes performed poorly on more tests than exposed men who did not report symptoms, confirming the additional risk of higher level exposure. In a separate follow-up analysis of subjects currently exposed by occupation, visual deficits were observed in addition to vestibular effects seen previously, suggesting a risk of other effects from acute exposure to the OP (Dick et al., 2001). These studies indicate that persistent neurological sequelae of exposure to OP pesticides can follow long-term exposure in the absence of acute poisoning episodes, although the severity of the sequelae appear to be related to the degree and currency of exposure, as assessed by symptom reporting (Jamal et al., 2002b) and careful analysis of subgroups stratified by recency of exposure and previous poisoning (Steenland et al., 2000; Dick et al., 2001). This conclusion is supported by reports of persistent deficits in other populations of exposed workers who did not require medical treatment for poisoning, including South African orchard workers (London et al., 1998) and Costa Rican banana workers (Wesseling et al., 2002). Recent evidence from large-scale studies of farm workers suggests that long-term exposure to pesticides may be associated with disorders of affect leading to suicide (London et al., 2005) rather than sensory, motor, or cognitive function per se. This finding is consistent with early studies of chronically exposed people (Gershon and Shaw, 1961; Metcalf and Holmes, 1969) and previously reported changes in the profile of effects after recovery from acute poisoning. That is, prospective studies of Nicaraguan
patients poisoned by OP pesticides showed that initial sensory, cognitive, and motor deficits tended to abate over a 2-year period and were replaced by an increase in neuropsychiatric symptoms even after mild poisoning episodes (Delgado et al., 2004; Miranda et al., 2002a,b). These studies suggest that further examination of affective disorders may be warranted for assessing the risk of exposure to OPs. A second study reported an increase in the prevalence of neurological symptoms in a large sample (-19,000) of U.S. pesticide applicators whose exposure was insufficient to cause acute symptoms (Kamel et al., 2005). In this study, the prevalence of nonspecific medical symptoms (i.e., symptoms not related to pesticide exposure) was associated with use of many pesticides, particularly OP insecticides, and was related to the degree of self-reported exposure. This study suggests that long-term, subsymptomatic exposure to OPs and other pesticides may exert important adverse effects on public health that escape detection in smaller studies. On the whole, however, epidemiological studies provide insufficient evidence to determine whether chronic, lowlevel exposure to cholinesterase-inhibiting insecticides, without acute poisoning episodes, results in persistent neurological or behavioral deficits in occupationally exposed humans (Kamel and Hoppin, 2004). Similarly, evidence for such effects in the general population is entirely lacking. Because of the difficulties of disentangling effects of chronic and acute exposures, as well as the impossibility of controlling all relevant variables in epidemiological studies, animal models have been developed to address the potential long-term effects of subsymptomatic exposure to these compounds.
B. Behavioral Effects of Repeated Exposure in Animals Perhaps because the question of the potential adversity of long-term, low-level exposure to cholinesterase-inhibiting pesticides in humans has only recently come into focus, few experimental animal studies of these compounds have addressed the issue. On the other hand, considerable work has been done to characterize the effects of repeated exposures, often with the goal of determining whether tolerance or sensitization occurs when exposure is prolonged or repeated. Some of this work bears on the question of the hazard of chronic exposure in humans, and a few studies have assessed the behavior of animals after the termination of dosing, when cholinesterase activity had returned to pretreatment levels. As noted previously, the neurobehavioral effects of chronic pesticide exposure in humans include psychomotor and cognitive functions, affect, vibration sensitivity, and balance. A survey of the animal literature revealed 15 papers on this topic in which exposure lasted at least 30 days;
CHAPTER 24 9Behavioral Toxicity these studies were classified according to outcome in each of several different domains (Table 2). Despite a lack of standardization in test measures and wide variation in route and duration of exposure, many of the effects can be combined into similar domains. For example, fighting reflex, ataxia, and balance beam behavior assess vestibular function, and grip strength, landing foot splay, and rotarod behavior assess neuromotor function (Moser, 1991). Motor activity, a commonly used and highly apical measure of nervous system function, was also analyzed. As Table 2 indicates, effects of cholinesterase inhibitors on activity and neuromotor changes were most commonly observed in those studies in which they were tested (Abdel-Rahman et al., 2004; Ivens et al., 1998; Llorens et al., 1993; Mattsson et al., 1996; Moser et al., 2005; Palumbo et al., 2001; Satin and Gill, 1998; Schulz et al., 1990; Sheets et al., 1997; Sobotka et al., 1986; Terry et al., 2003). These findings compare reasonably with the psychomotor alterations observed in humans and are also reported most frequently in human studies. Cholinergic signs in animals, and symptoms in humans, were also commonly observed (Bushnell et al., 1994; Drsi and Nagymajt6nyi, 1999; Ivens et al., 1998; Llorens et al., 1993; Mattsson et al., 1996; Maurissen et al., 2000; Moser et al., 2005; Sheets et al., 1997). Fewer studies evaluated vestibular function, but in both animals and humans, fewer than half of them showed adverse effects (Abdel-Rahman et al., 2004; Ivens et al., 1998; Moser et al., 2005; Sheets et al., 1997). Finally, cognitive function was worsened in a similar number of studies in humans and animals (Bushnell et al., 1994; Cohn and MacPhail, 1997; Llorens et al., 1993; Maurissen et al., 2000; Moser et al., 2005; Sarin and Gill, 1998). A few studies also reported improved cognitive function (Ivens et al., 1998; Palumbo et al., 2001), which likely reflects the memory-enhancing effects of low-level cholinesterase inhibition. This admittedly rough comparison suggests that animal models fairly represent the spectrum of effects reported in epidemiological studies of humans exposed to cholinesterase inhibitors and supports the contention that examination of the animal literature can provide insights into the hazard to be expected in human populations from long-term exposure to these compounds. As indicated, altered neuromotor function (decreased activity levels, impaired rotarod performance, slower responding, and decreased grip strength) corresponds to both the psychomotor and balance domains in Table 2. Effects of this nature have been observed duringtreatment with several pesticides administered in the diet, water, or parenterally, with durations ranging from 14 days to 4 months (Kobayashi et al., 1988; Llorens et al., 1993; Palumbo et al., 2001; Prendergast et al., 1997; Sheets et al., 1997; Schulz et al., 1990; Terry et al., 2003). In general, the largest and most prolonged motor effects are obtained during exposure to doses that markedly inhibit brain cholinesterase activity. On the other hand, when brain
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cholinesterase inhibition is absent, is slight (e.g., <20%), or has recovered, motor effects are absent. For example, propoxur slightly suppressed rotarod performance when testing took place immediately prior to a daily dose of the carbamate, at which time brain cholinesterase inhibition had nearly or completely recovered (Kobayashi et al., 1988). Similar findings were reported after cholinesterase activity had recovered after treatment with chlorfenvinphos (Socko et al., 1999). Whereas tolerance did not develop to the motor activity-decreasing effects of disulfoton (Llorens et al., 1993), the effects of chlorpyrifos were transient, suggesting that tolerance had developed (Mattsson et al., 1996). No neuromotor effects were observed with extended dietary exposures to triphenyl phosphate (Sobotka et al., 1986), parathion (Ivens et al., 1998), or chlorpyrifos (Moser et al., 2005). The domain of cognitive function in humans can be linked more readily to behavioral tests in animals. Alterations in these tests have been reported during and after repeated exposure to many OPs. Spatial learning in a water maze was impaired following dosing with disulfoton (Llorens et al., 1993), DFP (Prendergast et al., 1997; Upchurch and Wehner, 1987), chlorpyrifos (Terry et al., 2003), or methyl parathion plus endosulfan (Castillo et al., 2002). Other studies reported changes in spontaneous alternation after exposure to disulfoton or DFP (McDonald et al., 1988) or in active avoidance after dichlorvos exposure (Sarin and Gill, 1998). Working memory, assessed with a delayed match-to-position/visual discrimination procedure, was impaired by daily injections of DFP (Bushnell et al., 1991) or weekly injections of chlorpyrifos (Bushnell et al., 1994; but see also Maurissen et al., 2000). Learning, assessed by an operant repeated acquisition technique, was also impaired by chlorpyrifos (Cohn and MacPhail, 1997). Near the end of a year-long exposure, dietary chlorpyrifos with periodic challenges impaired learning and altered spatial strategies in a water maze (Moser et al., 2005). In some cases, cognitive deficits have also been observed after cholinesterase activity had ostensibly recovered following repeated exposures to an inhibitor. For example, reporting of time intervals was impaired by injections of DFP given after testing on three consecutive Fridays, and this impairment persisted for at least 6 weeks after the last treatment (Raslear et al., 1988). Rats that had been dosed daily with DFP for 2 weeks performed more poorly in a water maze both 2 days after dosing, (Prendergast et al., 1998) and 21 days after dosing, when ChE activity had returned to control levels (Prendergast et al., 1997). In contrast, in a similar experiment with chlorpyrifos, 14 daily doses caused behavioral impairment 4 days, but not 14 days, after the end of dosing (Terry et al., 2003). Similarly, an extensive study of the behavior of rats after chronic methamidophos showed no persistent effects on acquisition and retention of spatial navigation and repeated acquisition in a water maze (Temerowski and van der Staay,
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SECTION IV. O r g a n T o x i c i t y
2005). These contrasting results suggest that the mechanisms by which DFP, often used as a model OP, and actual pesticides affect behavior may differ, and that highly reactive OPs such as DFP and nerve gases may produce more persistent effects on the nervous system than less reactive pesticides. However, persistent effects of commercial pesticides have also been observed. Using an atypical dosing regimen, S~inchez-Santed et al. (2004) dosed rats twice with paraoxon or chlorpyrifos before and after 22 weeks of testing in a spatial delayed alternation (SDA) test of working memory, followed by spatial navigation tests in a water maze. Both OPs improved acquisition of the SDA task slightly. Chlorpyrifos, but not paraoxon, increased response latency in the water maze 50-52 weeks after the second dose of OP. Finally, 52-64 weeks after the second OP dose, neither treated group showed a preference for a location paired with an injection of amphetamine, whereas the control group did. In another study, a deficit in visual signal detection was observed in rats 3 months after the end of a year-long exposure to dietary chlorpyrifos with periodic high-dose challenges, whereas rats that received CPF either chronically or in periodic high doses did not differ from controls (Samsam et al., 2005). It should also be noted that some studies have shown improvements in learning associated with exposure to cholinesterase inhibitors. These effects in rats were either transient during exposure or observed at low doses or after recovery; the compounds studied include parathion (water maze; Ivens et al., 1998), chlorfenvinphos (active avoidance; Socko et al., 1999), and aldicarb (passive avoidance and water maze; Palumbo et al., 2001). These observations probably reflect the memory-enhancing properties of low levels of cholinergic stimulation, the basis for the therapeutic use of some cholinesterase inhibitors in the treatment of Alzheimer's dementia. Thus, evidence that subtle and adverse changes in behavior of rats persist beyond exposure to these pesticides is mixed, and when effects occur, they appear to depend on the OP tested. Beneficial effects on conditioned behaviors have also been reported. The question of whether prior poisoning is necessary for persistent effects remains open because the only rats in the Samsam et al. (2005) study that exhibited persistent effects had been exposed both chronically and to high-level doses of chlorpyrifos that elicited acute signs of poisoning, whereas S~inchez-Santed et al. (2004) observed persistent effects in rats that had not exhibited acute toxicity during treatment. Evidence from epidemiological studies regarding the affective domain (Gershon and Shaw, 1961; Metcalf and Holmes, 1969; Mearns et al., 1994; Kame! et al., 2005; London et al., 2005) indicates that this domain is a fruitful area for work with animal models. Clear acute anxiogenic effects of chlorpyrifos in the elevated plus-maze (S~inchezAmate et al., 2001) and in drug discrimination tests
(Sfinchez-Amate et al., 2002) have been reported (see Section I). In addition, a study of chronic dietary exposure to chlorpyrifos found evidence that long-term inhibition of cholinesterase activity may also be anxiogenic because water maze tests revealed significantly greater thigmotaxis in rats consuming chlorpyrifos in the diet compared to controls (Moser et al., 2005). Studies of affective responses to cholinesterase inhibitors should be designed to differentiate clearly between effects of ongoing exposure and effects that persist beyond exposure and recovery of cholinesterase activity.
IV. A G E - R E L A T E D D I F F E R E N C E S IN S E N S I T I V I T Y T O B E H A V I O R A L E F F E C T S Awareness of the unique sensitivity and susceptibility of the young to environmental chemicals has increased during the past decade. In this section, we consider only the primary response of the individual to direct pesticide exposure. The influence of cholinesterase-inhibiting pesticides on the developing nervous system is another key issue that is discussed in another chapter. Almost all of the comparative sensitivity studies in laboratory animals employed acute or short-term repeated dosing and evaluated the temporal responses to exposure. Early studies using lethal doses of pesticide reported that younger animals are often, but not always, more sensitive than adults (Brodeur and DuBois, 1963; Gaines, 1960, 1969; Gaines and Lindner, 1986; Goldenthal, 1971; Harbison, 1975; Lu et al., 1965). This increased sensitivity was highlighted with the publication of the National Research Council (1993) document, Pesticides in the Diets o f Infants and Children, which concluded that the database was inadequate for sound regulatory decisions. Public awareness has been further heightened by publications by environmental groups (Environmental Working Group, 1998; National Resources Defense Council, 1998). The 1996 Food Quality and Protection Act (Public Law 104-170, August 1996) mandated an additional 10-fold uncertainty factor to account for the potentially greater sensitivity of the young. These societal and regulatory pressures stimulated basic research to characterize changes in sensitivity to pesticides during development. During the past decade, considerable research has been directed toward defining age-related sensitivity differences to a small number of pesticides. Instead of lethality, these studies focused on measures of cholinesterase activity and behavior, ranging from motor activity and subtle signs of toxicity to scoring the SLUD (salivation, lacrimation, urination, and diarrhea) syndrome (Vidair, 2004). Chlorpyrifos, once one of the most widely used OPs, has also been one of the most thoroughly studied. Several studies indicate that young rats are up to seven-fold more sensitive than adult rats to an acute dose of chlorpyrifos, with marked changes
CHAPTER 24 9Behavioral Toxicity in sensitivity occurring from 5 to 20 days of age (Atterberry et al., 1997; Carr et al., 2001; Chakraborti et al., 1993; Moser and Padilla, 1998; Pope and Chakraborti, 1992; Pope and Liu, 1997; Pope et al., 1991; Zheng et al., 2000). For example, when comparing maximally tolerated doses (MTDs), 10-, 17-, and 27-day old rat pups were seven-, f'lve-, and twofold, respectively, more sensitive than adults to a single oral dose of chlorpyrifos (Moser and Padilla, 1998). Similar differences in sensitivity have also been documented for parathion, methyl parathion, diazinon, and aldicarb (Moser, 1999; Pope et al., 1991; Padilla et al., 2004). In contrast, methamidophos was equally potent in both young and adult rats (Moser, 1999). Because the magnitude of age-related sensitivity varies from chemical to chemical, generalization across pesticides will be difficult until the mechanisms underlying these differences are discovered. Three factors that may account for the age-related differences in sensitivity to pesticides are being explored: the dose level and the toxicokinetic and toxicodynamic factors that may differ in developing and adult animals. It has been suggested that sensitivity differences exist only at high dose levels, and that as the dose decreases, the age differences disappear (Pope and Liu, 1997). However, age-related differences in cholinesterase inhibition have been observed at both the MTD and doses of chlorpyrifos lower than those studied by Pope and Liu (Moser, 2000). Similarly, comparisons of cholinesterase inhibition produced by aldicarb showed a parallel shift of the doseresponse curves in the young, rather than the differences occurring only at the high doses (Moser, 1999). Resolution of this issue will be important for evaluating the age-related sensitivity of pesticides at environmentally relevant exposure levels. Age-related differences in kinetic parameters of detoxification can partially explain the increased sensitivity of the young to acute exposure to chlorpyrifos and other OPs (Mortensen et al., 1996; Moser et al., 1998; Padilla et al., 2000, 2004). Thus, in vitro assays show that differences in vivo are not due to intrinsic differences in sensitivity of the target enzyme (Mortensen et al., 1998). Furthermore, differences in liver microsomal metabolism, which mediates activation and/or inactivation of some pesticides, do not adequately explain the increased sensitivity (Benke and Murphy, 1975; Brodeur and DuBois, 1967). Differences in detoxification pathways correlate better with age sensitivity. B-esterases (e.g., carboxylesterases) and A-esterases bind to and/or hydrolyze, and thus detoxify, some cholinesteraseinhibiting pesticides (Jokanovic et al., 1996; Maxwell, 1992). These pathways are much less well developed in the young, and maturation of these systems tracks the decreasing sensitivity to acute exposure to chlorpyrifos and other OPs (Atterberry et al., 1997; Benke and Murphy, 1975; Brodeur and DuBois, 1967; Chanda et al., 1997, 2002; Mendoza, 1976; Mortensen et al., 1996, 1998;
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Moser et al., 1998; Sterri et al., 1985). Because these same detoxification enzymes are deficient in young children, these findings may apply to humans as well. The fact that the differential sensitivity of young animals is end point specific, however, shows that simple kinetic parameters will not provide a full explanation for all age-related differences. As mentioned previously, many cholinesterase-inhibiting pesticides act at other neuronal sites (e.g., muscarinic and/or nicotinic receptors) in addition to the AChE enzyme. Since the expression of these receptors and/or actions develops at different rates (Karanth and Pope, 2003; Tice et al., 1996), the age-related differences in the behavioral profile of a specific pesticide may be a function of the cholinergic receptors, if any, that it directly affects. Thus, whereas the toxicokinetic factors for different pesticides predict age-related differences in cholinesterase inhibition, it appears that toxicodynamic differences may have a greater influence on the behavioral effects. MTDs and cholinesterase inhibition are correlated for any particular pesticide and can explain age-related changes in sensitivity; however, some neurobehavioral studies report a dissociation between behavioral effects and cholinesterase inhibition. Depending on the pesticide tested, young rats may be more sensitive than adults to some behavioral effects, whereas young rats are either as sensitive or less sensitive than adults on other end points (Moser, 1999, 2000). For example, chlorpyrifos decreased motor activity in both young and adult rats, but the same level of cholinesterase inhibition in young rats resulted in much less activity depression than was seen in adult rats (Moser, 2000). Again, aldicarb decreased activity in the adult rat but did not alter activity in the young, even at near-lethal doses (Moser, 1999). In contrast, a similar degree of activity depression was observed in both young and adult rats with methamidophos. The finding that mature rats show greater CNS depression (e.g., decreased motor activity) and more muscarinic receptor-mediated effects (e.g., antinociception and lacrimation) differs from those of human case reports comparing anti-ChE poisonings in young and adult humans (Lifshitz et al., 1997; Sofer et al., 1989; Zwiener and Gingsburg, 1988). In those reports, poisoned children present with more CNS depression (coma and stupor) than adults, and nicotinic signs such as muscle fasciculations are rarely observed in the young. These case histories, however, report mostly poisonings by CMs, with only a few involving OP pesticides. Thus, both animal and human studies implicate pharmacodynamic factors in the differential responses to some, but not all, pesticides. These data underscore the need to characterize a full range of behavioral and neurochemical effects before making conclusions regarding age-related sensitivity differences. Recovery from behavioral effects of cholinesterase inhibitors is somewhat similar between the young and adult animals, which in both cases occurs much more rapidly
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SECTION I V .
Organ Toxicity
than recovery of cholinesterase activity (see Section II). The young, however, recover cholinesterase activity more rapidly than do adults (Moser and Padilla, 1998; Moser, 1999). This finding is attributed to faster protein synthesis in the developing animal (Lajtha and Dunlop, 1981), such that the inhibited enzyme is replaced with newly synthesized, active enzyme more rapidly. In summary, kinetic mechanisms can explain age-related differences in cholinesterase inhibition, but age-related differences in functional responses appear to involve changes in toxicodynamic processes that require further investigation before effects in young animals can be predicted from effects in mature animals. In addition, age-related differences in sensitivity to cholinesterase inhibitors depend critically on the behavioral end point measured, and caution is needed when extrapolating findings from one pesticide to another.
V. C O N C L U S I O N S Acute exposure to cholinesterase inhibitors produces a well-defined spectrum of behavioral effects in mammals that are generally well explained by overstimulation of cholinergic receptors in the central and peripheral nervous systems. Compensations for these neurochemical effects complicate the situation when exposure is prolonged or repeated. Acute poisoning can lead to persistent disorders in the cognitive, motor, sensory, and affective domains. The question of the potential long-term consequences of subclinical exposure remains unanswered. Animal models have been developed that reflect many of the signs and symptoms of exposure that are reported by humans, for both acute and chronic exposures, and have generated data critical for characterizing and understanding these effects. Reviews of this area continue to flourish, along with extreme views of the data. On the one hand, an expert panel concluded in 1999 that "long-term exposure to OP compounds does not cause problems in the peripheral or central nervous systems, unless poisoning is acute and severe" (Clegg and van Gemert, 1999). On the other hand, Jamal et al. (2002b) concluded the opposite: "The weight of current evidence is therefore very much in favor of the motion that chronic low-level exposure to OP produces neurotoxicity." As a rule, truth often lurks between extremes of interpretation, and in this area, it remains to be revealed.
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CHAPTER
~ 5
Peripheral Nervous System Effects and Delayed Neuropathy ANGELO MORETTO AND MARCELLO LOTYI Universitgt degli Studi di Padova, Padova, Italy
I. I N T R O D U C T I O N
days). In addition, the cleavage of one of the bound alkyl groups of the phosphoryl residue, known as aging, may subsequenfly occur. This leaves a negative charge at the residue attached to the active site. In this way, the enzyme becomes permanently inhibited (Aldridge and Reiner, 1972). The putative target of OPIDP is a neural protein with esteratic activity called neuropathy target esterase (NTE). It is thought that the mechanism of axonal degeneration is initiated by phosphorylation and subsequent aging of at least 70% of NTE (Johnson, 1990). Among OPs, with general structure R1R2P(O or S)X, that are capable of causing OPIDP, several factors determine their potential to inhibit NTE. For instance, phosphonates and phosphoroamidates are more potent than their homologous phosphates; increasing the carbon chain length of R1 and R2 also increases the inhibitory power. Characteristics that decrease this potency are bulky hydrophilic or nitrophenol groups at X and thioether linkages at X (Johnson, 1982). NTE inhibition/aging occurs within hours after dosing and NTE activity returns to normal well before the onset of clinical and morphological signs (Caroldi and Lotti, 1982). It has been shown that the 70% threshold inhibition is also required after repeated exposures. In fact, even cumulative doses higher than a single effective one failed to cause OPIDP when threshold of inhibition was not reached. Only when the doses and the dosing interval did not allow substantial resynthesis of the enzyme could the threshold be reached and OPIDP initiated (Lotti and Johnson, 1980). Studies on the sensitivity of the target enzymes of a variety of OPs showed that the comparative inhibitory power of OPs against hen acetylcholinesterase (ACHE) and NTE in vitro correlates with their comparative effects in vivo (i.e., death or delayed neuropathy). This correlation can be numerically expressed by the ratios ACHE ICs0/NTE IC50 in vitro and LD50/neuropathic dose in vivo. Moreover, comparison of the in vitro effects seen with hen and human enzymes indicated that the hen animal model well predicts the development of OPIDP in humans (Lotti and Johnson, 1978). Therefore,
The relevant effect of organophosphates (OPs) on the peripheral nervous system (PNS) is the organophosphate-induced delayed polyneuropathy (OPIDP). OPIDP is characterized by distal degeneration of long and large-diameter motor and sensory axons of both peripheral nerves and spinal cord. Several species, including man, are sensitive to OPIDP, and the animal model is the hen (Johnson, 1975a). OPIDP is caused by a single dose of some, but not all, OPs, and several reviews have summarized available experimental data on individual compounds (Johnson, 1975b, 1982; Lotti, 1992). Other OPs have been tested for their delayed neurotoxic potential by Tkachenko et al. (1992), Jokanovid (1993), Jokanovid et al. (1995), Abdelsalam (1999), Carrington (1989), Mortensen and Ladefoged (1992), Daugherty et al. (1996), and Moretto et al. (1994). Rodents are relatively resistant to OPIDP. In particular, clinical and morphological signs of OPIDP can be elicited in the rat by very high single or repeated doses of neuropathic OPs (Veronesi, 1984; Padilla and Veronesi, 1985; Moretto et aL, 1992a). Neuropathology not accompanied by clinical signs was observed in mice. The distribution of the lesions was slightly different from that observed in the rat (Veronesi et al., 1991). The clinical and morphological onset of OPIDP in hens generally occurs between 10 and 15 days after dosing, but depending on the dose, it can be as short as 6 or 7 days after poisoning. Full expression of clinical signs usually occurs within 3-5 days. In humans, the onset and the progression of the disease are generally somewhat slower.
II. M E C H A N I S M OF O P I D P OPs and carbamates (CMs) react covalenfly as pseudosubstrates at the catalytic center of a variety of serine hydrolases. Contrary to true substrates, the rate of hydrolysis of the carbamylated enzymes is slow (minutes to hours) and that of phosphorylated enzymes is extremely slow (hours to Toxicology of Organophosphate and Carbamate Compounds
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SECTION IV. O r g a n T o x i c i t y
ratios of inhibitory powers for AChE and NTE and/or that of lethal dose/neuropathic dose represent the key dose-effect relationships of each OR Consequently, they are among the fundamental criteria for the etiological attribution of cases of peripheral neuropathy in humans when sorting out various other differential diagnoses and etiologies. Although OPIDP hazard is shared by several OPs, the risk of developing OPIDP is actually rather small for existing commercial insecticides. In fact, premarketing toxicity testing in animals selects OPs with a cholinergic toxicity potential much higher than that which causes OPIDP (e.g., the ratios are <0.1 for the compounds in current use) (Moretto, 1999; Organisation for Economic Co-operation and Development, 1995): Consequently, neuropathic doses of these OPs will cause a severe cholinergic syndrome before the onset of clinical and morphological OPIDE Thus, OPIDP may develop in humans after very large exposures only (e.g., suicide attempts), causing unambiguous cholinergic toxicity. However, this is not the case for certain triaryl phosphates that do not cause cholinergic toxicity and have been used as hydraulic fluids, lubricants, or plasticizers (World Health Organization, 1990). Currently used triarylphosphates do not contain the neuropathic isomer tri-o-cresyl phosphate (Mackerer et al., 1999). OPs, as well as other non-OP inhibitors such as CMs and sulfonyl fluorides, also covalently react with NTE but cannot undergo the aging reaction. As a consequence, these inhibitors do not cause OPIDE and when given to experimental animals before a neuropathic OP, they protect from OPIDP when they occupy at least 30% of NTE active sites. Thus, the loss of NTE catalytic activity is not the mechanism leading to axonal degeneration (Johnson, 1990; Lotti et al., 1993). Hypotheses that have been proposed to explain the consequences of these OP-NTE interactions include either a loss of a nonesterase function of NTE that is essential for the axon or a gain of a toxic function of phosphorylated/aged NTE (Glynn, 1999). It has been shown that the relationship between the degree of NTE inhibition and the severity of OPIDP changes according to the compound involved. Whereas certain compounds cause OPIDP with a minimum of 70% NTE, others require a higher, almost complete, inhibition to cause OPIDE For this reason, it was proposed that all NTE inhibitors may have the potential to cause neuropathy, in analogy with pharmacological models of drug-receptor interactions (Lotti et al., 1993). According to this hypothesis, NTE inhibitors may have variable intrinsic activities to trigger the mechanism leading to axonal degeneration. Classic neuropathic OPs, such as diisopropyl fluorophosphate (DFP), are considered strong agonists since they cause OPIDP with 70% NTE inhibition, whereas nonageable inhibitors, such as CMs and sulfonyl fluorides, are considered partial agonists and therefore are among the weakest at initiating OPIDE requiting almost 100% NTE inhibition. Their weak agonist activity is also consistent with their protective effect when causing lower NTE inhibition.
Young animals are relatively resistant to OPIDE Although the pool of PV-esterases and NTE-specific activity show some age-related changes, OPs affect NTE in chicks as in hens (Moretto et al., 1991). The threshold level of inhibition required to trigger the initiation mechanism was found to be much higher (>90%) in young animals than in adults (Peraica et al., 1993). In addition, in young chicks (20 days old) OPIDP is mild and clinical signs show predominant spasticity rather than flaccidity, suggesting selective toxicity to the spinal cord. This was confirmed by histopathological lesions observed in spinal cord, but not in peripheral nerves, in 2-week-old chicks and by the earlier appearance of spinal cord lesions in 10-week-old chicks (Funk et al., 1994). Young animals recover from OPIDE The extent and timing of recovery depend on age and severity of the lesions, being shorter in younger and less severely affected animals. To explain the resistance of young animals, the hypothesis was made that they can compensate for a higher level of disruption because of their efficient repair systems (Lotti, 2002a). The events leading to axonal degeneration following NTE inhibition/aging are unknown, except for a deficit of retrograde axonal transport (Moretto et al., 1987). It was shown in hens that retrograde axonal transport is selectively impaired within a few days after poisoning, and the deficit progresses and reaches its maximum before the onset of OPIDE However, the cascade of events from NTE inhibition/aging to impairment of retrograde axonal transport and axonal degeneration is not understood.
III. STUDIES OF THE NTE PROTEIN NTE is present not only in neurons but also in a variety of nonneuronal tissues (Moretto and Lotti, 1988; Williams, 1983) but not in glial cells (Glynn et al., 1998). NTE from various species, including man, is a homolog of a protein required for brain development in Drosophila (Kretzschmar et al., 1997) and contains a domain that is highly conserved from bacteria to man (Lush et al., 1998). Studies conducted on the recombinant domain of NTE purified from bacterial lysates or by using differential inhibition with mouse brain NTE both in vitro and in vivo suggested that membrane lipids are putative cellular substrates of this enzyme (Van Tienhoven et al., 2002; Quistad et al., 2003; Quistad and Casida, 2004). In mammalian cell cultures, it was found that NTE is one of the enzymes that catalyze the deacylation of phosphatidylcholine to glycerophosphocholine. However, the relative contribution of NTE and calciumindependent phospholipase A2 plus lypophospholipases to this reaction has not been established (Zaccheo et al., 2004). This resulted in the hypothesis that NTE may be involved in intraneuronal membrane trafficking and lipid homeostasis. However, since this activity of NTE is inhibited in vitro by the nonneuropathic compound phenylpentyl
CHAPTER 25 9Delayed Neuropathy phosphinate (Zaccheo et al., 2004), it is obviously not essential for the maintenance of the axon and therefore not correlated with the initiation of axonal degeneration. The catalytic domain of NTE (NEST) associated with phosphatidyl choline liposomes facilitates transmembrane ionic conductance (Forshaw et al., 2001). The facilitation is partially inhibited by neuropathic OPs, such as DFP and phenyl saligenin phosphate, but not by nonneuropathic covalent NTE inhibitors, such as phenylpentyl phosphinate and phenylmethane sulfonyl fluoride (PMSF). Whether this effect has any relevance to the mechanism of OPIDP is unknown. NTE has an essential role in fetal development. Winrow et al. (2003) and Moser et al. (2004) showed that knockout Nte - / - mice died in utero probably as a result of failed placental development, whereas Nte +/- animals were viable and fertile. NTE mutation did not affect preimplantational growth; additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development (Moser et al., 2004). Conditional inactivation of the NTE gene in the mouse peripheral and central nervous systems resulted in elimination of NTE protein in the nervous system from embryonic day 11. Although this was compatible with embryonic nervous system development, these animals displayed neuronal degeneration and loss of endoplasmic reticulum in the hippocampus, thalamus, and cerebellum at 6 but not 2 weeks of age (Akassoglou et al., 2004). In summary, although there is compelling evidence of the involvement of NTE in OPIDP initiation, its role in axonal degeneration remains obscure.
IV. OTHER MECHANISTIC STUDIES Several studies have investigated the hypothesis that the development of OPIDP may involve protein kinase-mediated phosphorylation of cytoskeletal proteins. Most of this body of research on the phosphorylation of endogenous proteins was summarized by Abou-Donia (2003). However, it has provided equivocal results and, as a whole, little understanding of the molecular mechanism(s) of OPIDP considering, for instance, that results on endogenous phosphorylation of proteins were different according to the compound, neuropathic doses, and time of assay (Seifert and Casida, 1982; Patton et al., 1983, 1985, 1986; Hugghins and Richardson, 1999; Gupta and Abou-Donia, 1995; Gupta et al., 2000). Distinction should be made among the effects observed at different times during the development of OPIDP. Thus, early events include the altered expression of neurofilament subunits, which has been detected in hen spinal cord of animals treated with neuropathic doses of DFP as early as 1 day after dosing (Gupta et al., 2000). Middle-molecular-weight neurofilament protein expression was increased, whereas that of high- and low-molecular-weight neurofilament proteins
363
was decreased. Also, accumulation of these proteins was observed in the cytoskeletal inclusions in DFP-treated hen spinal cord. Although these changes were not present in the brain, a part of the central nervous system not affected by OPIDP, the pathophysiological significance of these changes remains unclear. Intermediate events include erratic changes of protein kinases A and C (Gupta and Abou-Donia, 2001), glyceraldehyde-3-phosphate dehydrogenase (Damodaran et al., 2002a), phosphorylated cAMP-response element binding protein (Damodaran et al., 2002b), and c~-tubulin expressions (Damodoran et al., 2001), which have all been detected during OPIDP development in the spinal cord of DFP-treated animals. Late changes include the increased Ca 2+ calmodulindependent autophosphorylation of hen brain and spinal cord proteins from animals paralyzed by TOCP treatment (Patton et al., 1986; Suwita et al., 1986). Several cytoskeletal proteins also seem to be affected. For instance, Ca 2+ calmodulin-dependent phosphorylation of several amino acids in tau proteins was enhanced by brain supernatants of DFP-treated animals. This effect may be associated with a small increase in Ca 2+ calmodulin-dependent protein kinase IIoL subunit mRNA expression that was observed 5-10 days after similar treatment (Gupta et al., 1998), followed by an equally small increase in the protein level (Gupta and Abou-Donia, 2001). The increase in phosphorylation of tau proteins may cause a decrease in tau microtubule binding, and it has been hypothesized that this would cause destabilization of microtubules and thus OPIDP (Gupta and Abou-Donia, 1998, 1999). Similarly, an increased phosphorylation of microtubule-associated protein-2 (Abou-Donia et al., 1993) and a small decrease in tubulin polymerization were observed in hen brain of DFP-paralyzed animals (Gupta and Abou-Donia, 1994). Moderate depletion of ATP was observed in peripheral nerves of hens with severe OPIDP (Massicotte et al., 2001). Most of these effects were measured 18-20 days after dosing, when animals were already paralyzed. Therefore, it is difficult to ascertain whether they were the cause or the consequence of axonal degeneration. Moreover, similar changes were observed in young rats (Choudhary et al., 2001), which are known to be resistant to OPIDP (Moretto et al., 1992a). Several studies investigated the possible role of calcium homeostasis disturbances in OPIDP pathogenesis, but results were inconsistent across various OPs and species (Suwita et al., 1986; E1-Fawal et al., 1989; Luttrell et al., 1993; Barber et al., 2001; Piao et al., 2003; Wu et al., 2003; Choudhary and Gill, 2001). Finally, several studies investigated various possible mechanisms in vitro (Pope et al., 1995; Sachana et al., 2001a,b; Fowler et al., 2001; Hong et al., 2003; Sales et al., 2004). However, results are very preliminary and do not add much to our understanding of OPIDP pathogenesis.
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SECTION IV. O r g a n
Toxicity
V. C L I N I C A L A S P E C T S O F O P I D P I N M A N Several thousand cases of OPIDP in humans caused by accidental ingestion of TOCP have been reported and reviewed by Inoue e t al. (1988). TOCP and related triaryl phosphates do not cause cholinergic toxicity and have been used as plasticizers, lubricants, and hydraulic fluids. Triaryl phosphates used as jet-engine lubricants contain very little, if any, TOCP (Mackerer e t al., 1999). In contrast with triaryl phosphates, many fewer cases of OPIDP have been convincingly attributed to OP insecticides, as summarized in Table 1. Clinical, electrophysiological, and histopathological details of these cases, and criteria for their inclusion in Table 1, have been discussed elsewhere (Lotti and Moretto, 2005). In addition to those reported in Table 1, cases of peripheral neuropathy likely caused by the CMs carbaryl, carbofuran, and metolcarb have been reported (Dickoff et al., 1987; Umehara e t al., 1991; Yang e t al., 2000). In these cases, the dose was very high, as judged by the severe cholinergic syndrome. Perhaps, the very effective antidotal and supportive treatment for cholinergic toxicity allowed a very high NTE inhibition in v i v o , similar to that associated with neuropathy in animals given repeated doses of CMs. In these animals, repeated dosing allowed some spontaneous reactivation of AChE between doses and, con-
TABLE 1.
sequently, the survival of the animals to doses causing almost complete NTE inhibition (Lotti e t al., 1993). Symptoms of OPIDP usually appear 2 or 3 weeks after a single dose; this delay depends on both the kinetic characteristics of the compound and the dose. After a high dose, it can be as short as 10 days, whereas the onset after poisoning by an OP with prolonged kinetics or at a relatively low dose may be up to 5 weeks. In the case of poisoning by insecticides, both cholinergic and intermediate syndromes have subsided before the onset of OPIDP (Lotti et al., 1984). Within a few days of the onset of symptoms, the full clinical expression of OPIDP is usually observed, and in the absence of further exposure, no progression of the disease occurs. The onset of symptoms and signs and their full development are more variable and less definable following repeated exposures to non-AChE triaryl phosphates. In fact, these compounds do not have AChE activity, and exposure may occur over several days without symptoms and the threshold of NTE inhibition may be reached as a consequence of a cumulative effect that overcomes the capability of the neurons to resynthesize NTE. The usual initial complaint is cramping musclepain in the lower limbs, followed by distal numbness and paresthesia. Progressive weakness then occurs, together with depression of patellar and Achilles reflexes. When severe,
Compounds Reported to Cause OPIDP in Humans a
Compound
Case reports in humans
Chlorpyrifos Dichlorvos
Lotti et al. (1986), Tracy and Gallagher (1990) Vasilescu and Florescu (1980), Wadia et al. (1985), Vasconcellos et al. (2002), Sevim et al. (2003) Moretto and Lotti (1998) Senanayake and Johnson (1982), McConnell et al. (1999), Eray et al. (1995), Moretto and Lotti (1998), Aygun et al. (2003) Bidstrup et al. (1953) Hierons and Johnson (1978), Vasilescu and Floreseu (1980), Johnson (1981) Vasilescu et al. (1984), Shiraishi et al. (1983), Niedzella et al. (1985), Csik et al. (1986) Jedrzejowska et al. (1980), De Kort et al. (1986) Inoue (1988) (review), Senanayake (1981) (in youngs), Goldenstein et al. (1988) (in youngs)
Isofenphos Methamidophos
Mipafox Trichlorfon
Trichlornat Triaryl phosphates
Phosphamidon/mevinphos
Chuang et al. (2002)
Reference for OPIDP in hens
Richardson (1995) Caroldi and Lotti (1981) Francis et al. (1985) Johnson (1981)
Barnes and Denz ( 1953) Johnson (1981)
Johnson (1975a,b) World Health Organization (1990) (review), Weiner and Jortner (1999) (review) Jokanovid et al. (1995)
aln addition, several unconvincing cases attributed to these compounds have been reported, such as those associated with chlorpyrifos (De Silva et al., 1994; Aiuto et al., 1993; Chattarjee and Sarma, 2003; Kaplan et al., 1993; Meggs, 2003), isofenphos (Catz et al., 1988), and methamidophos (Sun et al., 1998; De Haro et al., 1999). Several other OP insecticides have been reported to cause OPIDP. However, these reports are not convincing for several reasons, including lack of details and information, no evidence of OPIDP in experimental animals, and more likely alternative etiologies. Compounds involved in these case reports were fenthion (Aygun et al., 2003; Martinez-Chuecos et al., 1992), malathion (Monje Argiles et al., 1990; Dive et al., 1994; Rivett and Potgieter, 1987), mecarbam (Stamboulis et al., 1991), omethoate (Curtes et al., 1981), dimethoate (Sol~ Violhn et al., 1985; Sahin et al., 1994), parathion (De Jager et al., 1981; Alonso et al., 1983; Nisse et al., 1998;Aygun et al., 2003; Carod-Artal and Speck-Martins, 1999), and merphos (Fisher, 1977).
CHAPTER 25 9Delayed Neuropathy symptoms and signs of neuropathy appear in the arms and forearms. Physical examination reveals wasting and flaccid weakness of distal limb muscles, especially in the legs. Objective evidence of sensory loss is usually less severe or even absent (Moretto and Lotti, 1998). Signs include a characteristic high-stepping gait associated With bilateral footdrop. Quadriplegia with foot- and wristdrop as well as pyramidal signs are observed in the most severe cases. A detailed clinical description of a case series of TOCP poisoning can be found in Susser and Stein (1957). Functional recovery occurs with time in less severe cases, with most distal involvement and sparing of spinal cord axons. Otherwise, pyramidal and other signs of central neurological involvement may become more evident, and spastic ataxia may be a permanent outcome of severe OPIDP (Morgan and Petrovich, 1978; Susser and Stein, 1957). The very few cases of OPIDP reported in young individuals indicate that they recover completely, even from severe lower and upper limb involvement (Senanayake, 1981; Goldenstein et al., 1988). At onset, the electrophysiological examination is characterized by reduced amplitude of the compound muscle potential after supramaximal stimulation of motor nerves, increased distal latencies, and normal or slightly reduced nerve conduction velocities. Over a few days, unexcitability of the nerve ensues in severe cases. Signs of denervation of the affected muscles with increased insertional activity, spontaneous activity (fibrillation potentials and positive sharp waves), and reduced interference pattem are observed at electromyography (Lotti et al., 1986; Sevim et al., 2003; Vasconcellos et al., 2002; McConnell et al., 1999; Senanayake and Johnson, 1982; Wadia et al., 1985; Vasilescu and Florescu, 1980; Shiraishi et al., 1983; Vasilescu et al., 1984; Moretto and Lotti, 1998). These findings are consistent with distal axonal degeneration, observed when biopsies of the sural nerve were performed (Shiraishi et al., 1983; Vasilescu et al., 1984; Jedrzejowska et al., 1980; De Kort et al., 1986; Lotti et al., 1986; Chuang et al., 2002). The very few data available on spinal cord histopathology show a distribution of the lesions similar to that seen in animals, with involvement of the distal pyramidal tract and the proximal columns of Goll (fasciculus gracili medullae spinalis) (Aring, 1942, as reported by Susser and Stein, 1957). Observational studies aimed at detecting mild peripheral neuropathy or changes in peripheral nerve functions have been performed on individuals with varying long-term, lowlevel exposures to OPs, including different occupational exposures such as those occurring in sheep dip farmers and exposures during the first Gulf War. These studies were reviewed by Lotti (2002b). It was concluded that they suffered from a number of limitations. For instance, they did not accurately assess exposure, and reported changes in peripheral nerves were usually mild and inconsistent, sometimes reversible, and sometimes apparently irreversible because they were observed long after cessation of exposure.
365
Understanding these changes is difficult because of the lack of histopathology, follow-up data, and an experimental model for such peripheral nerve changes that seem different from classic OPIDE In addition, electrophysiological results were usually examined together on a group basis and correlation with clinical data was almost always missing. Finally, since these pesticides are far better inhibitors of AChE than NTE, they are expected to cause peripheral neuropathy at doses that inevitably cause cholinergic toxicity, irrespective of the type of exposure.
VI. PROMOTION OF OPIDP AND OTHER AXONOPATHIES IN EXPERIMENTAL ANIMALS Certain nonneuropathic OPs and CMs, as well as other esterase inhibitors such as sulfonyl fluorides, exacerbate toxic and traumatic axonopathies when administered in combination (Pope and Padilla, 1990; Lotti et al., 1991; Moretto et al., 1992b, 1993; Johnson and Read, 1993). This phenomenon was called promotion of axonopathies when it was first observed while studying OPIDP (Pope and Padilla, 1990; Lotti et al., 1991). Several of these compounds are NTE inhibitors as well, and they protect from OPIDP when given before and promote OPIDP when given after the neuropathic OE However, other inhibitors of esterases, but not of NTE (e.g., paraoxon and various sulfonyl fluorides), did not promote OPIDP when given at maximum tolerated doses (Lotti et al., 1991; Osman et al., 1996). The type and distribution of histopathological lesions in promoted animals did not differ from those observed in animals affected by classical OPIDE This indicates that promoters exacerbate existing damage and do not affect areas, neurons, or axons that are not typically involved in OPIDP (Pope et al., 1992, 1993; Randall et al., 1997; Moretto et al., 2001). A number of observations led to the hypothesis that promotion may involve the compensation/repair mechanism(s) of the nervous system (Lotti, 1995). For instance, promotion is not specific to OPIDP since other axonopathies of toxic (e.g., 2,5-hexanedione) (Moretto et al., 1992b) as well as traumatic origin (Moretto et al., 1993) were also promoted by PMSE Promotion was found to be less effective in chicks, in which repair mechanisms are believed to be more efficient (Peraica et al., 1993). Moreover, since promotion also occurred when the promoter was given up to a few days before the toxic or traumatic neuropathic insult, the mechanism involved in promotion is not activated by the neuropathic lesion/insult to the nerve but appears to be already present in healthy axons (Moretto et al., 1993, 1994). The molecular target of promotion is unknown, but there is evidence that it is not NTE (Moretto et al., 1994; Lotti et al., 1995). Nevertheless, all promoters tested so far are NTE inhibitors (Lotti et al., 1995; Osman et al., 1996; Lotti and Moretto, 1999; Moretto et al., 2001). Initial experiments
366
SECTION IV. O r g a n T o x i c i t y
aimed at the identification of the target of promotion searched for PV esterase activities in peripheral nerve after exclusion of NTE but were not successful (Milatovic et al., 1997). Upon reconsideration of the operational definition of NTE, a PV esterase activity with a sensitivity to inhibition by mipafox lower (IC50, --200 IxM) than that of NTE (IC50, -7 ~ , was found and called M200. Inhibition of this activity correlated with promotion when several compounds were tested (Moretto et al., 1996). This activity had approximately the same sensitivity to inhibition by mipafox of a soluble activity in the peripheral nerves identified by Escudero and Vilanova (1997) by separation with a Sephacryl S-300 column. This activity was also inhibited by promoters (Nicolli et al., 2002). Further purification of this fraction by ion-exchange chromatography identified a fraction apparently containing one 80-kDa protein with esteratic activity that, when tested with a limited number of compounds, was inhibited by promoters (Nicolli et aL, 2002). A mechanism different from that of typical OPIDP was suggested to explain the neuropathy occurring after promotion because the pattern of neurofilament subunit alterations found in animals, in which OPIDP was promoted (Xie et al., 2001, 2002), was different from that observed in animals with typical OPIDP (Gupta et al., 2000). However, because of the lack of concurrent controls, it is difficult to ascertain if such differences between promoted and classical OPIDP are real, considering that changes were often erratic over time and it was difficult to distinguish whether they were causally related or secondary to neuropathy.
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CHAPTER 25
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the central nervous system of hens treated with diisopropylphosphorofluoridate (DFP). Neurochem. Int. 40, 371-379. Daugherty, W., Biles, R., Jortner, B., and Ehrich, M. (1996). Subchronic delayed neurotoxicity evaluation of jet engine lubricants containing phosphorus additives. Fundam. Appl. Toxicol. 32, 244-249. De Haro, L., Arditti, J., Davide, J. M., and Jouglard, J. (1999). Intoxication au methamidophos: Toxicit6 neurologiqu6 immediate et retard6e; Apropos de deux observations. Acta Clin. Belgica. (Suppl.)l, 64-67. [in French] De Jager, A. E. J., van Weerden, T. W., Houthoff, H. J., and De Monchy, J. G. (1981). Polyneuropathy after massive exposure to parathion. Neurology 31, 603-605. De Kort, W. L. A. M., Savelkoul, T. J. F., Sindram, J. W., and Jennekens, E G. I. (1986). Delayed neurotoxicity'na intoxicatie met organofosforverbindingen. Ned. Tijdscher. Geneesked. 130, 1896-1898. [in Dutch] De Silva, H. J., Sanmugtanathan, P. S., and Senanayake, N. (1994). Isolated bilateral recurrent laryngeal nerve paralysis: A delayed complication of organophosphorus poisoning. Hum. Exp. Toxicol. 13, 171-173. Dickoff, D. J., Gerber, O., and Turovsky, Z. (1987). Delayed neurotoxicity after ingestion of carbamate pesticide. Neurology 37, 1229-1231. Dive, A., Mahieu, R., van B inst, R., Hassoun, A., Lison, D., De Bisschop, H., Nemery, B., and Lauwerys, R. (1994). Unusual manifestations after malathion poisoning. Hum. Exp. Toxicol. 13, 271-274. E1-Fawal, H. A. N., Jortner, B. S., and Ehrich, M. (1989). Effect of verapamil on organophosphorus-induced delayed neuropathy in hens. Toxicol. Appl. Pharmacol. 97, 500-511. Eray, D., Kamer, U. M., and Ozden, A. (1995). Organophosphateinduced delayed polyneuropathy. J. Trop. Ped. 41, 189. [Letter] Escudero, M. A., and Vilanova, E. (1997). Purification and characterization of naturally soluble neuropathy target esterase from chicken sciatic nerve by HPLC and Western blot. J. Neurochem. 69, 1-8. Fisher, J. R. (1977). Guillain-Barr~ syndrome following organophosphate poisoning. J. Am. Med. Assoc. 238, 1950-1951. Forshaw, P. J., Atkins, J., Ray, D. E., and Glynn, P. (2001). The catalytic domain of human neuropathy target esterase mediates an organophosphate-sensitive ionic conductance across liposome membranes. J. Neurochem. 79, 400-406. Fowler, M. J., Flaskos, J., Mclean, W. G., and Hargreaves, A. J. (2001). Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells. J. Neurochem. 76, 671-678. Francis, B. M., Metcalf, R. L., and Hansen, L. G. (1985). Toxicity of organophosphorus esters to laying hens after oral and dermal administration. J. Environ. Sci. Health B 20, 73-95. Funk, K. A., Henderson, J. D., Liu, C. H., Higgins, R. J., and Wilson, B. W. (1994). Neuropathology of organophosphateinduced delayed neuropathy (OPIDN) in young chicks. Arch. Toxicol. 68, 308-316. Glynn, P. (1999). Neuropathy target esterase. Biochem. J. 344, 625-631. Glynn, P., Holton, J. L., Nolan, C. C., Read, D. J., Brown, L., Hubbard, A., and Cavanagh, J. B. (1998). Neuropathy target
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CHAPTER ~ 6
Intermediate Syndrome in Organophosphate Poisoning JAN L. DE BLEECKER Ghent University Hospital, Ghent, Belgium
There was no distinct pattern in the development of the symptoms, but they disappeared in a characteristic sequence in the seven survivors. Cranial nerve palsiesm palatal, facial, and external ocular, in that ordermwere the first to recover. Then followed improvement of respiratory function and proximal limb muscle strength, and neck flexion was the last to recover. The time to recovery ranged from 5 to 18 days. One methamidophos-poisoned patient developed delayed neuropathy. The causative organophosphate was identified in nine patients: four fenthion, two dimethoate, two monocrotophos, and one methamidophos. Standard biochemistry and cerebrospinal fluid examination were normal. Cholinesterase (ChE) assays were not available. Electromyography (EMG) showed normal motor and sensory nerve conduction velocities and normal needle myography. Tetanic stimulation of the abductor pollicis brevis muscle 24-48 hr after the onset of IMS showed a marked fade at 20 and 50 Hz. A train of four stimuli applied at 2 Hz produced no changes in the amplitude of the compound muscle action potential (CMAP). CMAPs are the motor responses recorded with surface electrodes over a muscle after stimulation of its motor nerve. Treatment was mainly symptomatic. Atropine did not seem to influence the course of IMS. No definite mechanism of IMS was identified, but the authors wondered whether the necrotizing myopathy induced by acute organophosphate poisoning in patients (De Reuck and Willems, 1975; Wecker et al., 1986; Tattersall, 1990) and in experimental animals ( A r i ~ n s et al., 1969; Dettbarn, 1984; Inns et al., 1990; De Bleecker et al., 1991, 1992b, 1998) might underlie the selective muscle weakness.
I. I N T R O D U C T I O N In 1987, Senanayake and Karalliedde in a landmark paper reported 10 patients who developed facial, proximal limb, and respiratory muscle weakness. Because this symptom complex occurs in the interval between the acute cholinergic crisis and the possible development of a delayed motor neuropathy called organophosphate-induced delayed neuropathy, they termed this new entity the intermediate syndrome (IMS). The acute cholinergic crisis is due to inhibition of acetylcholinesterase (ACHE) which is clinically the most important (Grob, 1963), and the delayed neuropathy has been linked to inhibition of a separate esterase termed the neuropathy target esterase or neurotoxic esterase (Johnson, 1975; Abou-Donia and Lapadula, 1990). The pathogenesis of IMS was unclear and the question arose whether or not IMS bore a separate structure-activity relationship (De B leecker et al., 1992a; Marrs, 1993).
II. O R I G I N A L D E S C R I P T I O N Senanayake and Karalliedde (1987) observed 10 patients of Asian origin who were admitted with a well-defined cholinergic crisis. Following treatment with atropine and oximes in conventional doses, the initial outcome was favorable, but the patients went on to develop an IMS usually 1-4 days after the cholinergic poisoning. The most threatening symptom was the rather sudden occurrence of respiratory distress, often requiring reintubation and positive-pressure ventilation. Variable degrees of weakness of muscles innervated by several cranial nerves were present in 8 patients. All subjects had weakness of neck flexors and of proximal limb muscles. In all but 1 patient, the tendon reflexes were absent or markedly reduced. There was no sensory impairment. Transient dystonic movements appeared in two fenthion-poisoned victims. Two patients died on hospitals day 3 and 5 from respiratory failure, and another patient died on day 15 because of a technical failure. Toxicology of Organophosphate and Carbamate Compounds
III. P R E V I O U S R E P O R T S Several patients reported before the recognition of IMS by Senanayake and Karalliedde (1987) can in retrospect be related to this syndrome. The largest cohort was probably presented by Wadia et al. (1974) for diazinon poisoning. 371
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372
SECTION IV. O r g a n T o x i c i t y
These authors divided the signs and symptoms into type I (those present on admission) and type II (those appearing 24 hr after onset of poisoning). Type I signs included impaired consciousness and fasciculations and were responsive to atropine therapy. Type II signs were very much like IMS and comprised proximal limb weakness, areflexia, and cranial nerve palsies. Type II signs were not influenced by atropine. Some type I patients developed type II signs after an initial recovery. Thirty-six of 200 consecutive patients developed type II signs and 15 died from respiratory paralysis. In 1966, Clarman and Geldmacher-von Mallinckradt successfully managed a fenthion-poisoned patient with relapse of respiratory paralysis a few hours after initial improvement. Atropine and obidoxime (Toxogonin) administration ultimately appeared to be successful in this patient. In another fenthion-poisoned victim, sudden respiratory insufficiency necessitating artificial ventilation occurred 72 hr after ingestion (Dean et al., 1967). This patient was simultaneously restless, sweating, salivating, and had profuse fasciculations. Strength was only slightly decreased in all limbs, and he could lift his trunk from the bed. Combined atropine and oxime (PAM) treatment was successful in rapidly controlling the muscarinic signs. In contrast, it took 7 days before spontaneous breathing returned. The authors pointed to the discrepancy between respiratory (intercostal, bulbar, and diaphragm muscles) and nonrespiratory muscle strength as the most striking feature in their patient. A Belgian group reported severe relapses of an initial cholinergic crisis 31 and 72 hr and 7, 12, 13, and 16 days after ingestion of fenthion, despite atropine (not on days 2 and 3) and oxime administration (Mahieu et al., 1982). Biochemical investigations disclosed that disappearance or reappearance of cholinergic signs coincided with the initial decrease and subsequent increase of free ChE inhibitor in serum. In all those cases of fenthion poisoning, the initial cholinergic symptoms started less than 1 hr after oral ingestion and were moderately intense. Relapse of cholinergic symptoms and unconsciousness has also been reported following dimethoate intoxication (Molphy and Rathus, 1964). A tractor driver with subacute dicrotophos poisoning probably through both skin contact and inhalation recovered from a moderate cholinergic crisis after atropine and pralidoxime treatment. He further improved when atropine dosage was tapered, but on day 7 after the last exposure, he relapsed with prominent respiratory paralysis. Response to drug treatment, if any, was not mentioned. The ultimate outcome was favorable (Perron and Johnson, 1969). Gadoth and Fisher (1978) reported a victim of malathion ingestion who, 20 hr after a mild and apparently welltreated cholinergic crisis, needed urgent reintubation for sudden respiratory insufficiency. This patient had vomiting, diarrhea, and muscle cramps at the same time. A progressive paresis developed, and after 50 hr a neurological
evaluation revealed complete respiratory paralysis, bilateral ptosis, miosis, orbicularis oculi muscle fasciculations, and external ophthalmoplegia. Deep tendon reflexes were absent. Repeated doses of atropine and obidoxime gave no improvement. The ultimate outcome was favorable.
IV. FURTHER DELINEATION OF THE INTERMEDIATE SYNDROME A. Experimental Animal Studies Our group compared in Wistar rats the acutely toxic organophosphate (OP) paraoxon with fenthion, one of the agents frequently involved in human IMS. The clinical symptoms, the occurrence of muscle fiber necrosis and histochemical assessment of neuromuscular junction AChE activity in muscle biopsies, biochemical assessment of brain AChE activity, and EMG parameters including repetitive nerve stimulation at various frequencies were studied at various time points. Marked differences in the clinical course of the cholinergic crisis were noted between paraoxon and fenthion poisoning, regardless of the route of administration (De Bleecker et al., 1994b) (Fig. 1). Paraoxon provoked an acute, severe, and short-lasting cholinergic crisis lasting less than 24 hr. In contrast, fenthion poisoning produced a gradual increase of cholinergic signs lasting several days. All surviving animals were symptom-free after 1 week. Fasciculations peaked within the first days of fenthion poisoning, and they gradually decreased and eventually disappeared when the rats got weaker. A single poisoning with these OPs turned out to be a useful model to compare acute and chronic types of poisoning in animals, but many clinical signs and symptoms relevant to the clinical presentation of IMS in humans cannot be ascertained in rodents. Histochemically determined end-plate AChE and spectrophotometrically determined brain AChE activity closely paralleled each other (Fig. 1). In paraoxon poisoning, AChE inhibition was severe between 1 and 3 hr after subcutaneous injection and gradually recovered within 24 hr. In fenthion-poisoned animals, a slowly progressive decline in AChE activity occurred, with maximal inhibition after 8 days. At that time, most animals had no residual weakness or fasciculations. The necrotizing myopathy began soon after the onset of the cholinergic symptom complex in both paraoxon and fenthion poisoning, with a nadir of affected muscle fibers being reached after 24-48 hr. The necrotizing myopathy did not get worse after a further decline in AChE activity in the fenthion-poisoned animals. The myonecrosis appeared to be a monophasic event related to the initial decline in endplate AChE activity. The severity of the myopathy was similar in paraoxon- and in fenthion-poisoned rats with similar severity of the fasciculations.
CHAPTER 26 9Intermediate Syndrome
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FIG. 1. Time course of symptoms and AChE activity in paraoxon and fenthion poisoning in rats. Evolution of clinical signs (fasciculations and weakness) and end-plate and brain AChE activity in subcutaneous paraoxon and fenthion poisoning. Clinical signs are expressed as a semiquantitative score from 0 to III; control rats scored 0 at all times. The average value for a whole cohort of rats is plotted. Histochemically determined end-plate AChE activity is rated from 0 to 8; controls scored 7 or 8. Brain AChE activity, determined with Ellman's spectrophotometrical method (Ellman et al., 1961), is expressed as the percentage activity compared to the mean activity (100%) of the control rats at each time interval. For details, see De Bleecker et al. (1994b).
Repetitive activity after single motor nerve stimulation and decrement after repetitive nerve stimulation were the major E M G f i n d i n g s in either type of poisoning (Fig. 2) (Besser et al., 1989a,b, 1992; De Bleecker et al., 1994a; Van Dijk et al., 1996; Singh et al., 1998). Repetitive activity is defined as at least one negative deflection occurring immediately after the initial normally biphasic compound muscle action potential (for review, see Bowman et al., 1986) (Figs. 2A and 2B). CMAP amplitude decrements provoked by repetitive nerve stimulation occurred only in weak rats with severe end-plate AChE inhibition. The smallest amplitude occurred at the second response followed by a gradual increase in the subsequent responses (decrement-increment phenomenon) (Fig. 2B), or the amplitude decreased progressively toward the last response (decrement phenomenon) (Fig. 2C). The decrement-increment phenomenon preceded the decrement phenomenon and occurred at a slightly less severe degree of AChE inhibition. Conclusions from the animal studies were the following: 1. The AChE inhibition by a single sc fenthion poisoning is much prolonged. 2. The necrotizing myopathy is determined by the initial decline in end-plate AChE activity and is not further aggravated by sustained inhibition. 3. Decrement-increment and decrement responses on EMG are related to severe AChE inhibition and emerge at a slightly different severity of AChE inhibition. These data argued against the monophasic necrotizing myopathy being the cause of IMS and suggested that
persistent AChE inhibition was the prime mechanism. We went on to test this hypothesis in patients. B. O b s e r v a t i o n s in P a t i e n t s
Consecutive OP-poisoned patients admitted to our institution were prospectively studied. The protocol included a standard neurological examination at frequent prefixed time intervals, serum ChE and erythrocyte AChE activity, urinary OP metabolite excretion, and EMG with repetitive nerve stimulation. Some patients underwent a muscle biopsy. Eight out of 19 patients developed clinical signs and symptoms of IMS. Some of them had short relapses of muscarinic symptoms as well. No clinical, electromyographic, biochemical, or other differences were noted between patients with or without symptom-free interval or those with or without short relapses of muscarinic signs and symptoms. OPs such as fenthion (n = 1), dimethoate (n = 1) (De Bleecker et at., 1992c), and methyl-parathion (n = 5) (De Bleecker et al., 1992e) apparently carried a higher risk of IMS. However, we also noted a protracted IMS in a parathion-poisoned man with severe renal and hepatic failure, indicating that IMS is not restricted to a few OP agents (De Bleecker et al., 1992d). Table 1 lists the OPs most frequently involved in IMS (cases cited in this chapter). Prospective studies on the relative incidence of IMS with certain OP agents are needed to avoid the biases inherent to case reports. The clinical signs of IMS were weakness in the territory of multiple cranial nerves (diplopia, dysphagia, and facial diplegia), sudden respiratory distress necessitating
374
SECTION I V . O r g a n T o x i c i t y
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FIG. 2. Electromyographic findings in organophosphate-poisoned rats. (A) Response to two subsequent stimuli at 2 Hz administered 2.5 hr after subcutaneous injection of paraoxon. Note the prominent repetitive activity after the first stimulus (top line). After the second stimulus, the repetitive activity is less marked (bottom line). The initial CMAP amplitudes are normal. (B) Decrement-increment phenomenon in response to 50-Hz repetitive nerve stimulation 3 hr after subcutaneous paraoxon injection. The odd responses are displayed on the top line and the even on the bottom line. The second response has the smallest amplitude, with gradual but incomplete amplitude recovery at the subsequent responses. Note that the marked repetitive activity at the first response is entirely abrogated at the second response and does not recur. The sharp initial deflection preceding each CMAP represents the stimulation artifact. (C) Decrement phenomenon in response to 50-Hz repetitive nerve stimulation 9 days after subcutaneous injection of fenthion. There is a gradual decrease in amplitude and area toward the ninth response. The repetitive activity disappears completely at the second response. The odd responses are displayed on the top line and the even responses are on the bottom line. For methodological details, see De Bleecker et al. (1994a).
reintubation, neck and proximal limb muscle weakness, and depression of the deep tendon reflexes. Fasciculations were not part of IMS but coincided with the appearance of muscarinic signs. The duration of IMS varied from a few days to several weeks. The outcome was good. No patients developed clinical or EMG features of delayed polyneuropathy (De Bleecker et al., 1993). All patients had severe AChE inhibition during the entire period of IMS. OP metabolite excretion was also prolonged. Consecutive EMG findings in the evolution of IMS were decrement, decrement-increment, increment, and eventually normal repetitive nerve stimulation studies. The
EMG normalized before the last neurological symptom (i.e., fatigable external ocular muscle weakness) had disappeared (De Bleecker et al., 1993). In the four patients who underwent muscle biopsy, a few necrotic muscle fibers were noted, but these were too sparse to explain severe muscle weakness and were no more frequent in IMS than in non-IMS patients. We concluded from the prospective patient study that 1. IMS is not rare, and although it is more likely to occur with certain OP agents, it is not restricted to these agents. Toxicokinetic factors such as high lipid
CHAPTER 26 9Intermediate Syndrome in Organophosphate Poisoning
TABLE 1. Causative Organophosphates in Intermediate Syndrome OP
No. of cases
Fenthion Dimethoate/omethoate Parathion Methyl-parathion Malathion Monocrotophos/dicrotophos Methamidophos Diclorvos/triclorvos Chloropyrifos Diazinon (Wadia et al., 1974) Total no. of cases
11 6/5 8 5 5 2/1 1 1/1 1 36 47 + 36
solubility of certain OPs (Davies et al., 1975), but also impaired systemic functions (cardiovascular, hepatic, and renal) (Betrosian et al., 1995) with slower than normal OP metabolism and excretion, can probably all contribute to the development of an IMS (Table 1). 2. The syndrome invariably coincides with sustained AChE inhibition and is not due to muscle fiber necrosis. No separate structure-activity relationship is involved.
375
3. When viewed together, the clinical and EMG features are best explained by combined pre- and postsynaptic impairment of neuromuscular transmission (De Wilde et al., 1991) (see also Section V). 4. IMS is not related to incipient delayed neuropathy.
V. C O M P A R I S O N W I T H O T H E R H U M A N DISEASES WITH IMPAIRED NEUROMUSCULAR TRANSMISSION The clinical, electromyographic, microelectrophysiological, and morphological features of a number of acquired or congenital human disorders of neuromuscular transmission have been well characterized. Comparison of IMS with some of these syndromes is helpful in interpreting some of the observations in IMS patients. Besides practical problems involved in studies of end-plate-rich human intercostal muscle biopsies, the changing dynamics of the end-plate AChE inhibition in the course of OP poisonings make IMS less amenable to many electrophysiological and morphological studies than more frequent or stable disorders of neuromuscular transmission. Table 2 summarizes the main clinical and EMG characteristics of some well-known disorders of neuromuscular transmission. Myasthenia gravis is an autoimmune disorder with postsynaptic impairment of neuromuscular transmission due to complement deposition at the ACh receptor
TABLE 2. Main Characteristics of Some Human Disorders of Neuromuscular Transmission Myasthenia gravis
Lambert-Eaton myasthenic syndrome
Disorder Botulinum toxin poisoning
Site of defect
Postsynaptic
Presynaptic
Presynaptic
Clinical findings
Fatigable
Fatigable
Extraocular, bulbar, facial, respiratory, proximal limb
Proximal limb
Fatigable, descending Extraocular, head and neck, trunk and proximal limb
Normal Yes
Decreased Poor or absent
Decreased Absent
Normal No Decrement at low frequency
Small No Increment at high frequency
Some small No Increment at high frequency
Distribution of muscle weakness
Tendon reflexes Response to AChE inhibitors Electromyography CMAP amplitude Repetitive activity Repetitive nerve stimulation
Intermediate syndrome
Congenital AChE deficiency
Pre- and postsynaptic Fatigable
Pre- and postsynaptic Fatigable
Extraocular, bulbar, facial, neck, respiratory, proximal limb Decreased Absent or adverse
Extraocular, bulbar, facial, neck, proximal limb Decreased Absent
Normal Yes Decrement at low frequency; decrementincrement at intermediate/high frequency
Normal Yes Decrement at low and high frequency
376
SECTION IV. O r g a n
Toxicity
sites. In the Lambert-Eaton myasthenic syndrome, antibodies against the presynaptic voltage-sensitive calcium channels cause reduced release of ACh quanta from the motor nerve terminal. In botulinum toxin poisoning, the ACh release from the nerve terminal is reduced by impaired'fusion of presynaptic vesicles with the presynaptic membrane. The distribution of the weakness and the obvious fatigability o f some muscle groups in IMS are very similar to those of generalized autoimmune myasthenia gravis. Areflexia or hyporeflexia, on the other hand, is more typical of presynaptic disorders. Along the same line, the EMG findings of IMS are a combination of those observed in pre- and postsynaptic disorders of neuromuscular transmission. In some IMS patients with severe weakness, persistent decrements at low stimulation frequencies were found, similar to severe myasthenia gravis. In most patients, a decrement-increment phenomenon with the smallest amplitude at the second stimulus occurred at intermediate or high-frequency stimulation only. This is more typical of presynaptic disorders. However, a severely reduced amplitude of the first CMAP after a single nerve stimulus, which is the most typical and constant EMG feature of the Lambert-Eaton myasthenic syndrome, has never been noted. The decrement-increment phenomenon, with the smallest amplitude being elicited at the second stimulus, appears to be rather unique and typical of IMS. Taken together, the data for IMS are compatible with a combined pre- and postsynaptic disturbance of neuromuscular transmission, presumably each contributing to a different proportion along the evolution of IMS. A congenital myasthenic syndrome with synaptic AChE deficiency, resulting from a mutation in the collagenic tail subunit of asymmetric ACHE, has been described by Engel (Engel et al., 1977; Ohno et al., 1998). The clinical syndrome in these patients strongly resembles IMS. They have persistent fatigable weakness of external ocular, facial, palatal, masticatory, neck, and proximal limb muscles and depressed deep tendon reflexes. EMG shows repetitive compound muscle action potentials after single nerve stimulation and decrements at slow and rapid rates of repetitive nerve stimulation. Detailed microelectrophysiological and morphological studies have revealed combined pre- and postsynaptic abnormalities of neuromuscular transmission (Table 2).
VI. LATER IMS REPORTS In the years following the initial description, heightened awareness led to IMS being increasingly diagnosed. Unfortunately, the true incidence remains unknown due to ascertainment bias and lack of prospective studies using a prespecified set of diagnostic criteria in larger series of patients.
He et al. (1998) found IMS in 21 of 272 cases (7.7%). The causative OPs were parathion, omethoate, dimethoate, diclorvos, and some pesticide mixtures. Persisting blood AChE inhibition was a constant finding, and decrements were noted in weak patients at 20 or 30 Hz repetitive nerve stimulation. The case fatality rate was 19%. Choi and Quinonez (1998) reported a case of IMS in malathion poisoning. Two more malathion poisonings were reported from Morocco (Benslama et al., 1998, 2004). Karademir et al. (1990) observed IMS in a young female poisoned with trichlorfon and the carbamate proxyfur and in a fenthion-poisoned female, both with complete recovery. Two pregnant fenthion-poisoned women were successfully managed by Karalliedde et al. (1988). An ethyl parathionpoisoned woman developed both IMS and delayed distal polyneuropathy (Nisse et al., 1998). A single typical IMS case was seen after suicidal ingestion of chlorpyrifos (Guadarrama-Naveda et al., 2001). All these cases have largely confirmed previous clinical, biochemical, and electromyographic observations and have added little to the further understanding of the disease mechanisms or the development of more rational or individualized treatment approaches.
VII. FURTHER STUDIES ON IMS PATHOGENESIS A few investigators undertook in vivo electrodiagnostic studies in man (Baker and Sedgwick, 1996; Singh et al., 1998). Baker and Sedgwick performed an elegant study using single-fiber EMG, a more sensitive technique than repetitive motor nerve stimulation to detect subtle changes in neuromuscular transmission. In sarin-poisoned healthy volunteers, they found small changes in single-fiber EMG at 3 hr and 3 days after exposure to a small dose of satin sufficient to cause a 60% reduction of red blood cell ACHE. These people had no clinical neuromuscular signs or symptoms. These investigators and others (Norman, 1990) concluded from studies of volunteers that reversible subclinical changes indicating a nondepolarizing block could be found to a variable extent in all study participants and suggested that similar mechanisms were probably involved in IMS patients. Based on observations of a fenthion-poisoned IMS patient, the same group proposed downregulation of postsynaptic ACh receptors as a possible explanation for the clinical and electrophysiological abnormalities in that patient (Sedgwick and Senanayake, 1997). The current data do not explain why not all OP-poisoned patients with a similar severity of intoxication develop IMS. Interindividual differences in the safety factor of neuromuscular transmission, the degree and duration of end-plate AChE inhibition, and conformational changes of the ACh receptors in the postsynaptic membrane due to the precedent cholinergic crisis may play a role. Different
CHAPTER 26 9Intermediate Syndrome in Organophosphate Poisoning affinity of different OPs for nicotinic versus muscarinic cholinesterases or selective distribution of some OPs to muscle have also been proposed (Benson et al., 1992). Other groups have studied long-term changes in neurotransmission in experimental animals as an approach to elucidate the pathogenesis of IMS. A study by Dongren et al. (1999) on dimethoate-poisoned rats confirmed the higher sensitivity of single-fiber EMG compared to repetitive nerve stimulation to detect subtle changes in neuromuscular transmission after OP poisoning. Other investigators described a number of delayed changes in brain and neuromuscular neurotransmission in mice exposed to low doses of diazinon for a short period (de Blaquibre et al., 2000). A Japanese group used quantitative reverse transcriptionpolymerase chain reaction to study mRNA expression of a number of neuromuscular transmission proteins after a single dose of disulfoton (O,O-diethyl S-2-ethylthioethyl phosphorodithioate) in rats (Matsuda et al., 2000). They found decreased AChE and nicotinic ACh receptor (nAChR) mRNA expression with a nadir at 12 hr after oral poisoning, but the decrease persisted for nearly 30 days. These studies implicate alterations at the transcriptional level as part of the mechanisms of downregulation of AChE and nAChR. The relevance of these various animal data for the pathogenesis of IMS in patients remains to be determined.
VIII. T H E R A P E U T I C C O N S I D E R A T I O N S A definite therapeutic regimen that successfully treats or ideally prevents IMS is not available. In fact, a high index of suspicion allowing early diagnosis and intensive care facilities for respiratory monitoring, tracheal intubation, and artificial ventilation, when necessary, are the best guarantees for a favorable outcome. Since prolonged AChE inhibition at the neuromuscular junction is involved in IMS pathogenesis, all efforts have to be made to prevent further inactivation and aging and to enhance reactivation of this enzyme. The efficacy of oxime therapy to improve neuromuscular transmission in patients has been demonstrated by electrophysiological techniques (Besser et al., 1995). Benson et al. (1992) suggested that IMS may be an artifact of insufficient oxime therapy. Early high-dose oxime administration in severely poisoned patients is therefore logical (Besser et al., 1995; Tush and Anstead, 1997; Thiermann et al., 1999) but does not always prevent IMS from developing (Willems et al., 1992, 1993). Several centers currently prefer bolus dosing followed by continuous infusion, but controlled studies on dosage and dose regimens that would best prevent or treat IMS are lacking. It also remains to be studied whether different oximes (pralidoxime, obidoxime, and H-oximes) confer a differential therapeutic benefit for the treatment and prevention of the IMS entity as such. Likewise, it is unknown whether the usage of oximes with different reactivation
377
potential for AChE inhibition by different OPs (e.g., powerful reactivation by HI-6 of soman-inhibited AChE vs refractoriness to HI-6 of paraoxon-inhibited ACHE) results in any better clinical outcomes in patients (Worek et al., 1996). Glycopyrrolate, a quaternary ammonium compound, has been used with good results in a case of IMS after malathion poisoning (Choi and Quinonez, 1998). As expected, this predominantly antimuscarinic agent positively influenced bronchorrhea in this patient, but it is not expected to be of much value for the prevention or treatment of IMS symptoms strictu sensu.
IX. D I A G N O S T I C C R I T E R I A Based on our observations and the cases reported from experienced centers throughout the world, we propose the following IMS diagnostic guidelines" 1. Persistent acquired inhibition of red blood cell AChE through OP or possibly carbamate poisoning. 2. Fatigable weakness of the extraocular, bulbar, facial, respiratory, neck flexor, and proximal limb muscles. The weakness occurs 1-4 days after a cholinergic crisis, with or without a symptom-free interval. Short relapses of muscarinic signs and symptoms do not exclude the diagnosis. 3. Absent or depressed deep tendon reflexes without other identifiable cause (e.g., preexistent neuropathy). 4. EMG abnormalities indicating pre- or postsynaptic impairment of neuromuscular transmission are not constant. Decrement studies may reveal decrement, decrement-increment, or abnormal increment. Single-fiber EMG is likely to be more sensitive, but further prospective studies are required to determine whether single-fiber EMG changes are constant and/or specific and whether they are of use for diagnosis or therapeutic monitoring, i:
X. F U T U R E S T U D I E S The clinical recognition of IMS is no longer problematic for most medical workers in the field, and considerable progress toward the elucidation of the underlying mechanisms has been made in the 10 years following the initial description. A number of pertinent questions remain unanswered. Most important, why do not all patients with a similar severity and duration of anti-ChE poisoning develop IMS? May genetic factors or the metabolic or hormonal circumstances at the time of poisoning confer differential susceptibility to this syndrome? Which are the precise pre- and postsynaptic factors that reduce the safety factor of neuromuscular transmission in IMS? Do these factors change over time during the course of IMS? Which factors
378
SECTION IV- O r g a n T o x i c i t y
differ between the various OPs or carbamates, and are these differences explanatory for the different incidence of IMS with some poisons? Does postsynaptic membrane damage with decreased numbers or altered conformation of postsynaptic ACh receptor channels play a role? What are the microelectrophysiological characteristics of neuromuscular transmission in IMS patients? Possible approaches to solve these questions include studies of chronically poisoned rodents, with detailed morphological and electrophysiological studies on repeated muscle biopsies in the same animal. The morphological studies should include estimations of the number of ACh receptor sites by quantitative electron microscopy and immunocytochemistry at the light and electron microscopic levels, as well as measurement of the number of 125I-labeled ot-bungarotoxin binding sites. EMG studies, including decrement studies at various frequencies of nerve stimulation and single-fiber EMG, should be complemented and correlated with in vitro electrophysiology studies on the same biopsy tissue. The latter should combine conventional microelectrode studies to determine miniature end-plate potential, miniature end-plate current, and evoked quantal release, with noise analysis and single-channel patch clamp recordings. Identical studies could be done on end-plate-rich intercostal muscle biopsies obtained from IMS patients. Ethical and practical considerations have to be considered at this point. The precise incidence of IMS and the relative incidence of IMS with different OPs are unknown. All studies are case reports or suffer from a number of other biases. Prospective studies using a set of diagnostic criteria are needed to avoid these biases. Finally, the best therapeutic algorithm is still undefined. Is IMS preventable by optimal oxime therapy, as suggested by Benson et al. (1992), or is optimal oxime therapy beyond our reach in many patients, as suggested by Willems and coworkers ( 1992, 1993) ?
References Abou-Donia, M. B., and Lapadula, D. M. (1990). Mechanisms of organophosphorus ester induced delayed neurotoxicity: Type 1 and type 2. Annu. Rev. Pharmacol. Toxicol. 30, 405-440. Ari~ns, A. T., Meeter, E., Wolthuis, O. L., and Van Benthem, R. M. J. (1969). Reversible necrosis at the end-plate region in striated muscles of the rat poisoned with cholinesterase inhibitors. Experientia 25, 57-59. Baker, D. J., and Sedgwick, M. E. (1996). Single fibre electromyographic changes in man after organophosphate exposure. Hum. Exp. Toxicol. 15, 369-375. Benslama, A., Moutaouakkil, S., Mjahed, K., E1 Moknia, M., Lahbil, D., and Fadel, H. (1998). Syndrome intermrdiaire lors d'une intoxication aigu~ par le malathion. Presse Med. 27, 713-715. Benslama, A., Moutaouakkil, S., Charra, B., and Menebhi, L. (2004). Le syndrome intermrdiaire des intoxications aigu~s par
les insecticides organophosphorrs. Ann. Fr. Anesth. Rdanim. 23, 353-356. Benson, B., Tolo, D., and Mclntire, M. (1992). Is the intermediate syndrome in organophosphate poisoning the result of insufficient oxime therapy? Clin. Toxicol. 30, 347-349. Besser, R., Gutmann, L., and Weilemann, L. S. (1989a). Inactivation of end-plate acetylcholinesterase during the course of organophosphate intoxications. Arch. Toxicol. 63, 412-415. Besser, R., Gutmann, L., Dillmann, U., Weilemann, L. S., and Hopf, H. C. (1989b). End-plate dysfunction in acute organophosphate intoxication. Neurology 39, 561-567. Besser, R., Vogt, T., Gutmann, L., Hopf, H. C., and Wessler, I. (1992). Impaired neuromuscular transmission during partial inhibition of acetylcholinesterase: The role of stimulus-induced backfiring in the generation of the decrement-increment phenomenon. Muscle Nerve 15, 1072-1080. Besser, R., Weilemann, L. S., and Gutmann, L. (1995). Efficacy of obidoxime in human organophosphorus poisoning: Determination by neuromuscular transmission studies. Muscle Nerve 18, 15-22. Betrosian, A., Balla, M., Kafiri, G., Kofinas, G., Makri, R., and Kakouri, A. (1995). Multiple systems organ failure from organophosphate poisoning. Clin. Toxicol. 33, 257-260. Bowman, W. C., Gibb, A. S., Harvey, A. C., and Marshall, I. G. (1986). Prejunctional actions of cholinoceptor agonists, and of anticholinesterase drugs. In New Neuromuscular Blocking Agents (D. A. Kharkevic, Ed.), pp. 141-170. Springer-Verlag, Berlin. Choi, P.-L., and Quinonez, L. (1998). The use of glycopyrrolate in a case of intermediate syndrome following acute organophosphate poisoning. Can. J. Anaesth. 45, 337-340. Clarman, M., and Geldmacher-von Mallinckradt, M. (1966). Uber eine erfolgreich behandelte akute orale Vergiftung durch Fenthion und dessen Nachweis in Mageninhalt und Ham. Arch. Toxikol. 22, 2-11. Davies, J. E., Barquet, A., Freed, V. H., Haque, R., Morgrade, C., Sonneborn, R. E., and Vaclavek, C. (1975). Human pesticide poisonings by a fat-soluble organophosphate insecticide. Arch. Environ. Health 30, 608-613. Dean, G., Coxon, J., and Brereton, D. (1967). Poisoning by an organophosphorus compound: A case report. S. A. Med. J. 41, 1017-1019. de Blaqui~re, G. E., Waters, L., Blain, P. G., and Williams, F. M. (2000). Electrophysiological and biochemical effects of single and multiple doses of the organophosphate diazinon in the mouse. Toxicol. Appl. Pharmacol. 166, 81-91. De Bleecker, J., Willems, J., De Reuck, J., Santens, E, and Lison, D. (1991). Histological and histochemical study of paraoxon myopathy in the rat. Acta Neurol. Belg. 91, 255-270. De B leecker, J. L., De Reuck, J. L., and Willems, J. L. (1992a). Neurological aspects of organophosphate poisoning. Clin. Neurol. Neurosurg. 94, 93-103. De Bleecker, J. L., Van Den Abeele, K. G., and De Reuck, J. L. (1992b). Variable involvement of rat skeletal muscles in paraoxon-induced necrotizing myopathy. Res. Commun. Pathol. Pharmacol. 75, 309-322. De Bleecker, J., Van Den Neucker, K., and Willems, J. (1992c). The intermediate syndrome in organophosphate poisoning: Presentation of a case and review of the literature. Clin. Toxicol. 30, 321-329.
CHAPTER 26
9Intermediate Syndrome in Organophosphate Poisoning
De B leecker, J., Vogelaers, D., Ceuterick, C., Van Den Neucker, K., Willems, J., and De Reuck, J. (1992d). Intermediate syndrome due to prolonged parathion poisoning. Acta Neurol. Scand. 86, 421-424. De Bleecker, J., Willems, J., Van Den Neucker, K., De Reuck, J., and Vogelaers, D. (1992e). Prolonged toxicity with intermediate syndrome after combined parathion and methyl parathion poisoning. Clin. Toxicol. 30, 333-345. De Bleecker, J., Van Den Neucker, K., and Colardyn, E (1993). Intermediate syndrome in organophosphorus poisoning: A prospective study. Crit. Care Med. 21, 1706-1711. De B leecker, J. L., Van Den Abeele, K. G., and De Reuck, J. L. (1994a). Electromyography in relation to end-plate acetylcholinesterase in rats poisoned by different organophosphates. NeuroToxicology 15, 331-340. De B leecker, J., Lison, D., Van Den Abeele, K., Willems, J., and De Reuck, J. (1994b). Acute and subacute organophosphate poisoning in the rat. NeuroToxicology 15, 341-348. De Bleecker, J. L., Meire, V. I., and Pappens, S. (1998). Quinidine prevents paraoxon-induced necrotizing myopathy in rats. NeuroToxicology 19, 833-838. De Reuck, J., and Willems, J. (1975). Acute parathion poisoning: Myopathic changes in the diaphragm. J. Neurol. 208, 309-314. Dettbarn, W.-D. (1984). Pesticide induced muscle necrosis: Mechanisms and prevention. Fundam. Appl. Toxicol. 4, S 18-$26. De Wilde, V., Vogelaers, D., Colardyn, E, Van Den Neucker, K., Vanderstraeten, G., De B leecker, J., De Reuck, J., and Van Den Heede, M. (1991). Postsynaptic neuromuscular dysfunction in organophosphate induced intermediate syndrome. Klin. Wochenschr. 69, 177-183. Dongren, Y., Tao, L., and Fengsheng, H. (1999). Electroneurophysiological studies in rats of acute dimethoate poisoning. Toxicol. Lett. 107, 249-254. Ellman, G. L., Courtney, D. K., Andres, V., Jr., and Featherstone, L. M. (1961). A new and rapid colorimetric method for the determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Engel, A. G., Lambert, E. H., and Gomez, M. R. (1977). A new myasthenic syndrome with end-plate acetylcholinesterase deficiency, small nerve terminals, and reduced acetylcholine release. Ann. Neurol. 1, 315-330. Gadoth, N., and Fisher, A. (1978). Late onset of neuromuscular block in organophosphorus poisoning. Ann. Intern. Med. 88, 654-655. Grob, D. (1963). Anticholinesterase intoxication in man and its treatment. In Cholinesterases and Anticholinesterase Agents (G. B. Koelle, Ed.), pp. 989-1027. Springer-Verlag, Berlin. Guadarrama-Naveda, M., Calderon de Cabrera, L., and MatosBastidas, S. (2001). Intermediate syndrome secondary to ingestion of chlorpiriphos. Vet. Hum. Toxicol. 43, 34. He, E, Xu, H., Qin, E, Huang, J., and He, X. (1998). Intermediate myasthenia syndrome following acute organophosphates poisoning - - An analysis of 21 cases. Hum. Exp. Toxicol. 17, 40--45. Inns, R. H., Tuckwell, N. J., Bright, J. E., and Marrs, T. C. (1990). Histochemical demonstration of calcium accumulation in muscle fibres after experimental organophosphate poisoning. Hum. Exp. Toxicol. 9, 245-250.
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Johnson, M. K. (1975). The delayed neuropathy caused by some organophosphate esters: Mechanisms and challenge. Crit. Rev. Toxicol. 3, 289-316. Karademir, M., Erttirk, E, and Koqak, R. (1990). Two cases of organophosphate poisoning with development of intermediate syndrome. Hum. Exp. Toxicol. 9, 187-189. Karalliedde, L., Senanayake, N., and Ariaratnam, A. (1988). Acute organophosphorus insecticide poisoning during pregnancy. Hum. Toxicol. 7, 363-364. Mahieu, E, Hassoun, A., Van Binst, R., Lauwerys, R., and Deheneffe, Y. (1982). Severe and prolonged poisoning by fenthion. Significance of the determination of the anticholinesterase capacity of plasma. Clin. Toxicol. 19, 425-432. Marrs, T. C. (1993). Organophosphate poisoning. Pharmacol. Ther. 58, 51-66. Matsuda, H., Seo, Y., Kakizaki, E., and Takahama, K. (2000). Changes in mRNA expression levels of synaptic- and target tissue-specific proteins after organophosphate exposure. Legal Med. 2, 55-63. Molphy, R., and Rathus, E. M. (1964). Organic phosphorus poisoning and therapy. Med. J. Aust. 2, 337-340. Nisse, P., Forceville, X., Cezard, C. C., Ameri, A., and MathieuNolf, M. (1998). Intermediate syndrome with delayed distal polyneuropathy from ethyl parathion poisoning. Vet. Hum. Toxicol. 40, 166-168. Norman, J. (1990). Neuromuscular blockade. In A Textbook of Anaesthesia (A. R. Aitkenhead and G. Smith, Eds.), pp. 211-224. Churchill Livingstone, London. Ohno, K., Brengman, J. M., Tsujino, A., and Engel, A. G. (1998). Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme. Proc. Natl. Acad. Sci. U S A 95, 9654-9659. Perron, R., and Johnson, B. B. (1969). Insecticide poisoning. N. Engl. J. Med. 281, 274-275. Sedgwick, M. E., and Senanayake, N. (1997). Pathophysiology of the intermediate syndrome of organophosphorus poisoning. J. Neurol. Neurosurg. Psychiatr. 62, 201-202. Senanayake, N., and Karalliedde, L. (1987). Neurotoxic effects of organophosphorus insecticides. An intermediate syndrome. N. Engl. J. Med. 316, 761-763. Singh, G., Mahajan, R., and Whig, J. (1998). The importance of electrodiagnostic studies in acute organophosphate poisoning. J. Neurol. Sci. 157, 191-200. Tattersall, J. E. H. (1990). Effects of organophosphorus anticholinesterases on nicotinic receptor ion channels at adult mouse muscle endplates. Br. J. Pharmacol. 101, 349-357. Thiermann, H., Szinicz, L., Eyer, F., Worek, E, Eyer, P., Felgenhauer, N., and Zilker, T. (1999). Modern strategies in therapy of organophosphate poisoning. Toxicol. Lett. 107, 233-239. Tush, G. M., and Anstead, M. I. (1997). Pralidoxime continuous infusion in the treatment of organophosphate poisoning. Ann. Pharmacother. 31, 441-444. Van Dij~:, J. G., Lammers, G. J., Wintzen, A. R., and Molenaar, E C. (1996). Repetitive CMAPs: Mechanisms of neural and synaptic genesis. Muscle Nerve 19, 1127-1133. Wadia, R. S., Sadagopan, C., Amin, R. B., and Sardesai, H. V. (1974). Neurological manifestations of organophosphorus insecticide poisoning. J. Neurol. Neurosurg. Psychiatr. 37, 841-847.
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Organ Toxicity
Wecker, L., Mrak, R. E., and Dettbarn, W. (1986). Evidence of necrosis in human intercostal muscle following inhalation of an organophosphate insecticide. Fundam. Appl. Toxicol. 6, 172-174. Willems, J. L., Langenberg, J. P., Verstraete, A. G., De Loose, M., Vanhaesebroeck, B., Goethals, G., Belpaire, F. M., Buylaert, W. A., Vogelaers, D., Colardyn, F. (1992). Plasma concentrations of pralidoxime methylsulphate in organophosphorus poisoned patients. Arch. Toxicol. 66, 260-266.
Willems, J. L., De Bisschop, H. C., Verstraete, A. G., Declerck, C., Christiaens, Y., Vanscheeuwyck, and P., Buylaert, W. A., Vogelaers, D., and Colardyn, E (1993). Cholinesterase reactivation in organophosphorus poisoned patients depends on the plasma concentrations of the oxime pralidoxime methylsulphate and of the organophosphate. Arch. ToxicoL 67, 79-84. Worek, E, Kirchner, T., Backer, M., and Szinicz, L. (1996). Reactivation by various oximes of human erythrocyte acetylcholinesterase inhibited by different organophosphate compounds. Arch. Toxicol. 70, 497-503.
CHAPTER ~ 7
Cardiovascular Toxicity of Cholinesterase Inhibitors CSABA K. ZOLTANI, 1 G. D. THORNE, z AND STEVEN I. BASKINz 1U.S. Army Research Laboratory, Aberdeen Proving Ground, Maryland 2U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland
I. I N T R O D U C T I O N
There are two types of smooth muscle, visceral and multiunit. Visceral smooth muscle (e.g., muscle that lines the intestines) functions much like cardiac muscle in that it forms a functional syncytium in which several muscle cells receive a signal and respond as one unit. Vascular smooth muscle is of the multiunit type, which functions in smaller independent muscle groups much like skeletal muscle. Both types are highly dependent on circulating compounds such as hormones and exogenous chemicals. There is little nerve enervation and regulation of contractility is maintained within the multiunit cellular groups, which makes the muscle highly sensitive to circulating hormones and chemical compounds such as acetylcholine (ACh). This makes vascular smooth muscle regulation by OPs potentially distinct from that of cardiac muscle. This difference is exacerbated by the many different receptor and receptor subtypes that populate a particular vascular branch. It has been reported that cardiac and vascular smooth muscles exhibit varying degrees of parasympathetic modulation and vasodilatation following cholinergic stimulation depending on muscle type and muscarinic receptor subtype (Tsukahara et al., 1989; Alonso et al., 1991; Harvey and Belevych, 2003). Although nerve-dependent signaling, and therefore activation of said receptors, is minimal in these tissues, ACh can have a direct effect on vessel wall physiology by activating endothelial cells, resulting in an increased release of nitric oxide (NO). NO triggers the production of vasodilatory cyclic compounds such as cGMP in smooth muscle cells. The end response is an endothelium-dependent depression in vascular contractility. The primary mechanism of conventional OP-containing chemical nerve agents is inhibition of cholinesterase and the concomitant accumulation of ACh. Logically, vascular smooth muscle contractility will be altered if substantially high levels of circulating ACh remain. Freeman et al. (1986) reported that the disruption of ACh signaling
Organophosphates (OPs) cause histopathological changes in cardiac tissue and, depending on the dosage, can seriously affect the functioning of the heart. In severe cases of OP intoxication, the heart goes into ventricular fibrillation (VF). Some details of the OP-modulated cellular processes are still unknown. Progress in the development of antidotes against OP-caused cardiovascular toxicity depends on detailed parsing of the underlying cellular processes. Such an approach requires insight into how muscle responds to the presence of toxic insult and a description of the changes initiated. Extending the work of Baskin and Whitmer (1991), this chapter addresses these issues by discussing the effect of acetylcholinesterase (ACHE) inhibition on contractile tissue and the results obtained by the use of high-performance computer simulation of the cellular processes to lend better insight into the modulation of electrophysiology by OPs. The results suggest the nature of pharmacological approaches that need to be considered to treat OP toxicity.
II. E F F E C T O F C H O L I N E S T E R A S E I N H I B I T O R S ON C O N T R A C T I L E TISSUE Vascular smooth muscle is inherently distinct from the other contractile muscle types, skeletal and cardiac. The contractile machinery is less organized and usually appears as sporadic bundles rather regular striations. The result is an inherent functional difference in the modulation of contractility and response to chemical threats by compounds such as OP agents. However, relatively little work has been performed to investigate the regulation of vascular smooth muscle by OP compounds and chemical warfare agents in general. 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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SECTION IV. O r g a n Toxicity
cascades by nerve agent exposure could have detrimental effects on the function of these vascular systems and may potentially contribute to major cardiovascular complications and minimize systemic recovery. In this study, the OP anticholinesterase soman potentiated electrophysiological effects of ACh stimulation in guinea pig atrium. Presumably, such an augmentation of the ACh signaling cascade would occur if the circulatory vessels experienced a similar chemical threat. The effect of anticholinesterase compounds has been investigated in some pulmonary smooth muscle airway systems (Filbert et al., 1992; Shibata et al., 1998; Tsuda et al., 2001). These studies focused on evaluating pulmonary performance after therapy for neurodegenerative diseases such as myasthenia gravis and the effect of compounds on OP-activation contractions. The investigation paradigm utilized large amounts of compound applied directly to the isolated tissue. Although compelling, these experiments misrepresent the in vivo processing of the compound and induction of physiologic responses that could affect vascular function. Similar studies have been performed with isolated rabbit hearts demonstrating purported toxic and vasodilatory effects of soman and sarin poisoning (Preston and Heath, 1972). Maintenance of cardiovascular and systemic vascular muscle function involves the regulation of several pathways, including activation and deactivation cascades, calcium homeostasis, and force development. The ultimate output of these processes is maintenance of systemic or localized blood flow. There is evidence indicating an altered cardiac and systemic vascular function following nerve agent poisoning. For example, reduced skin blood flow has been demonstrated following AChE inhibition with pyridostigmine (Stephenson and Kolka, 1990). This study utilized human subjects to measure various cardiovascular parameters following pyridostigmine ingestion and is one of very few studies that provide evidence of potential vascular consequences of systemic chemical agent circulation. Calcium is an integral part of the regulation of vascular smooth muscle physiology. It is involved in both receptormediated signaling cascades and the activation of contractile machinery. The electrical events required to maintain vessel tone rely heavily on the modulation and control of the smooth muscle cellular calcium concentration. Soman has been shown to disrupt calcium uptake mechanisms (Hu et al., 1991), potentially upsetting overall calcium homeostasis. Furthermore, large concentrations of soman (0.1 IxM) impair electrical field stimulation of canine trachea, presumably altering force development (Filbert et al., 1992). These findings are some of the only evidence indicating a mechanistic level of influence of OP-type compounds on vascular smooth muscle. Future investigations will need to clarify any influence these anticholinesterase compounds may have on other regulators of smooth muscle contractility, such as calcium-independent
tone maintenance controlled through phosphorylation of actin-myosin cross-bridges. It is imperative to gain a better understanding of how vascular smooth muscle, specifically the critical functional pathways, may be affected by OP chemical agents. Studies have been conducted within the context of the cardiovascular system that imply an acute alteration of vascular smooth muscle function. Whether this alteration has long-term ramifications in terms of irreversible detriment to functionally significant vascular function remains to be determined.
III. I N S I L I C O
APPROACHES TO AN
UNDERSTANDING OF OP-INDUCED CARDIAC TOXICITY OPs, the anticholinesterase constituents of nerve agents, bind to sites usually occupied by ACHE, preventing hydrolysis of ACh and resulting in ACh overload and overstimulation of the cardiac tissue. The ancillary ion concentration imbalances lead to membrane current derangement, QT prolongation of the ECG, ST-wave abnormalities, conduction system delays, and acidosis. Cell swelling and necrosis of the damaged cells activate normally dormant membrane currents while blocking others. Overviews of the effect of OPs on the cardiovascular system have been provided by Baskin and Whitmer (1991) and Bar-Meir et al. (2001). Considerable new insights into the cellular mechanisms controlling the electrophysiology of the tissue have become available with the use of computer codes run on high-performance, parallel computers (Zoltani and Baskin, 2000, 2002; Zoltani et al., 2004) that permit detailed simulation of ongoing processes at the cellular and subcellular levels. It is now possible to simulate the effect of the presence of threat agents on the homeostasis of the cell. Mathematical models of cell functioning affected by threat agent-caused modulations are presented in this chapter. Included and evaluated for the first time are the cell swelling-activated currents. Interpretation of the action potential (AP) and electrocardiograph morphology changes in terms of the cationic and anionic state variables in turn suggests therapeutic intervention strategies.
A. Signatures of OP Presence in the Cardium 1. HISTOPATHOLOGICALCHANGES A starting point for this analysis was the identification of the major hist0pathological changes caused by OP. OP deposition in the heart is dose and route of administration dependent and spatially heterogeneous. The damage to the tissue is focal, with pericapillary hemorrhage, micronecrosis, and patchy fibrosis (Kiss and Fazekas, 1982, 1983) resulting in decreased conduction and altered repolarization dynamics. Pimentel and Carrington da Costa (1992) reported myocardial ultrastructural morphological
CHAPTER 27 9Cardiovascular Toxicity changes, including "swollen forms, fragmentation, or lysis of cristae." Povoa et al. (1997) also noted myocardial necrosis from OP intoxication. Opie (1998) noted the "formation of amorphous dense bodies in the mitochondria, possibly derived from local lipids so that ATP cannot be produced and energy deficit becomes disastrous." Roth et al. (1993) noted that lesions in the presence of OP are primarily seen in the left ventricular region. Tryphonas et al. (1996) also noted histopathologic changes in somanaffected Sprague-Dawley rats. Abraham et al. (2001) remarked on lesions in cardiac tissue and attributed QT prolongation to blockade of the Ito potassium channel. Soman exposure of macaques studied by Britt et al. (2000) caused heart lesions consisting of myocardial degeneration and necrosis. For the OP-produced ACh overload of the tissue, Yang et al. (1996) found notable regional differences in the action of ACh, with the greatest being in subepicardial cells of the canine left ventricle and considerably less in the subendocardial cells. ACh activates IKACh in subepicardial cells but not in subendocardial cells. Balazs and Ferrans (1978) noted granular swelling of the sarcoplasm and loss of nuclei in cardiac tissue upon exposure to adrenergic [3-receptor agonists and vasodilating agents. One of the effects is the accumulation of calcium in the mitochondria, impairing its function. Limaye (1966) reported necrosis, swelling, and extensive subepicardial ecchymoses localized in the left atrium and the upper portion of the back of both ventricles. The significance of the histopathological changes, especially the cell volume modulation and necrosis of affected cells, resides in the changed and evolving electrical state of the tissue. Besides altered current dynamics, several membrane currents, dormant under normal conditions, are activated, transforming the ion flow aggregate of the cell and revamping the electrophysiology of the tissue. 2. MODULATION OF ELECTROPHYSIOLOGY-RELATED MARKERS a. Ion Concentration Changes
First, OP causes increased sympathetic tone resulting in cholinergic flooding. OP binds covalently to the active site of serine residue on ACHE, thereby preventing the acetylation of AChE by ACh. Perturbation of the oxygen uptake of the tissue ensues due to interference with the ACh interaction with the M2 myocardial receptors (Thomsen a n d Baskin, 1988). Interference can cause complete heart block due to increased outward K + conductance and a reduction of inward/Ca. The ICs0 of ACh was estimated to be 30 nM. The cholinergic effect is noticeable at approximately 50% AChE inhibition. Overload of ACh decreases /Ca(L) and also inhibits the background current, IK1. Second, external potassium concentration change [K+]o, due to inhibition of the sodium-potassium pump, contributes to the derangement of homeostasis. Third, ancillary mitochondrial calcium overflow [Ca 2+] affects the inotropic
383
characteristic of the tissue. Fourth, sodium concentration change [Na +]i occurs due to inhibition of the sodium pump (Tobin et al., 1973; Baskin and Whitmer, 1991) and affects the depolarization of the tissue. b. Homeostatic Modulation Caused by Activation of M e m b r a n e Currents
Cell swelling, coupled with energy availability changes, activates several currents that are dormant under homeostatic conditions: 1. IKAChis present in the sinoatrial (SA) node and the atria. Under pathological conditions, this current is also active in the ventricles, although not as prominently as in the atria (Sakmann et al., 1983; Dobrzynski, 2001,2002). 2. Icl,sw is activated by cell swelling (Baumgarten and Clemo, 2003; Nilius and Droogmans, 2003; Wright and Rees, 1998; Tseng, 1992; d'Anglemont de Tassigny et al., 2003). 3. IKATPactivates when the ATP concentration declines and in response to an overflow of ACh. 3. OP-CAUSED CHANGES IN THE AP AND THE ELECTROCARDIOGRAM
There is still a lack of unanimity of agreement on the details of the cellular processes leading to OP-caused cardiotoxicity. Exterior markers have been catalogued. Kotev and Paliev (1968) were one of the first to remark on the electrocardiographic changes caused by the presence of OP in cardiac tissue. It is well established that increased sympathetic tone and activity cause myocardial damage that leads to QT prolongation, ultimately culminating in VE QT prolongation, especially in the context of genetically and pharmacologically induced variety, has been extensively studied (Antzelevitch and Shimizu, 2002; Tan et al., 1995). Under the effect of OP, AP prolongation occurs when repolarization channels are blocked. These electro-chemical processes are marked by hypoxemia, acidosis, and electrolytic derangement (Ludomirsky et al., 1982; Saadeh et aL, 1997; Karki et al., 2004) and ultimately result in the development of VE Ischemia and OP toxicity share elevated free fatty acid (FFA) and catecholamine levels. FFA puts great demand on oxygen uptake. The electrocardiograms (ECGs) of affected people also show ST elevation and T-wave abnormalities. As the end result, OP shortens the cycle time of the AP and lessens the amplitude of the mainly sodium currentcontrolled depolarization spike. The dome of the AP is affected by OP-caused changes in the /to, I~, and Ii~s currents. Block of the IKr current lengthens the QT interval of the ECG, reminiscent of LQT2 caused by a defect in the H E R G gene. M cells of the ventricular wall prolong QT more than do the epicardial or endocardial cells. Drug-induced/Ks block, such as produced by azimilide (Zoltani and Baskin, 2002), contributes to LQT2 (Viskin,
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SECTION IV- O r g a n T o x i c i t y
1999). Reducing net outward current IK or /to can extend the AP dome of myocytes and counteract the development of VE Class III drugs (e.g., sotalol) or class IA drugs that block sodium channels (tetrodotoxin) can also lead to LQ prolongation. Hypokalemia prolongs AP and leads to the onset of torsades de pointes.
B. Numerical Simulation of OP-Caused Toxicity The L-R model of the ventricular AP (Luo and Rudy, 1994), modified to account for several additional currents that are dormant under usual, nonpathological conditions, was the starting point for the calculation of the electrophysiology of the ventricular myocytes in the presence of OE OPs cause cell swelling, which in turn activates Icl,sw membrane current (Baumgarten and Clemo, 2003; Nilius and Droogmans, 2003). The I-V relationship of the Icl,sw used in these calculations is based on the experimental work described in Vandenberg et al. (1997). Icl,sw current density is in the range of 2-10 pA/pF in ventricular myocytes and somewhat higher in atrial myocytes. ACh overload activates IKACh. Quantification of the muscarinic potassium current l ~ c h has been developed by Osterrieder et al. (1980), Egan and Noble (1987), and Demir et al. (1999). Additional factors affecting the AP of the myocytes and the morphology of the ECG are the OP-affected and reduced/Ca(L), blocked I~, accumulation of [K +]o, and [Ca2+]i overload. A series of calculations were carried out to explore the effect of these changes on the markers of the electrophysiology. The simulation outcomes are a function of the locations where the observations are made since the epicardial, M, and endocardial cells of the ventricular wall layer respond differently. The simulation was run on ventricular tissue in which, under pathological conditions, all of the mentioned currents are active. It should be noted that ACh affects the SA node and atrial currents also under nonpathological conditions. The calculations were carried out on a string consisting of 165 cells. A stimulus traversed the string, starting at one end consisting of the endocardial type and transiting to the M cells and subsequently the epicardial cells. Each of the regions consisted of 57 cells and the conductivity in each of the regions was adjusted, following Viswanathan and Rudy (2000), to reflect the nature of the cell type. The stimulus duration was 2 msec and the amplitude 200 mA/cm 2.
C. Cardiotoxic Observations The homeostatic simulated ECG of cardiac tissue is shown in Fig. 1. The AP of a ventricular epicardial cell is shown in the inset. In OP-affected cardiac tissue, the fast potassium current is diminished. The effect of a 90% block of this membrane current, I~, is shown in Fig. 2. A 50% decline in the calcium current, /Ca(L), with I ~ unblocked, shows a similar behavior (Fig. 3).
Histopathological data indicate that OP-affected cardiac tissue displays swelling. Several otherwise dormant membrane currents, Icl,sw and IACh, are activated. In these circumstances, the cycle length of the AP is sharply reduced (Fig. 4). In these simulations, the ACh concentration was 0.1 IxM. OP also causes a steep rise in the potassium concentration external to the cell. The simulated ECG (Fig. 5) shows a radically altered morphology. The T wave, representing the repolarization of the ventricles, is missing; indeed, repolarization no longer takes place in the usual sense. Blocking the cell swelling-activated membrane current (Fig. 6) restores qualitatively the morphology of the ECG, although the T wave has considerably higher amplitude and base than under baseline conditions. Blocking the AChactivated membrane current (the dashed curve in Fig. 7) only marginally improves the morphology. Return to baseline conditions (the lower solid curve in Fig. 7) requires reactivation of the membrane pumps, thereby restoring the exterior potassium concentrations. It is clear that the cell swelling-activated membrane current plays a central role in the modulation of homeostatic conditions in OP intoxication. Cell swelling and ACh overflow activate two usually dormant currents. The changes in the ECG and the AP (Figs. 4 and 5) are startling. IKACh current was calculated based on the ONT (Osterrieder-Noma-Trautwein) formulation (Osterrieder et al., 1980), which was also used by Shumaker et al. (1990) for atrial tissue. The concentration of the ACh in the tissue was taken as 1.0 IxM. When an OP-caused increase in [K+]o (8.0mM) is included in the model simulation, the changes are profound (Fig. 5), Under severe OP intoxication, all of these effects are active and the changes in the morphology of both the ECG and the AP are remarkable. Pharmacological intervention, blocking OP-activated but normally dormant currents, reverses some of the modulations (Figs. 6 and 7), pointing to possible therapeutic approaches.
D. Prospects for Pharmacological Intervention Until recently, atropine (Kiss and Fazekas, 1983) and anisodamine (Yang et al., 1991) were the drugs of choice for OP-affected cardiac toxicity. Atropine and diazepam also block the development of cardiac lesions. However, Kiss and Fazekas (1983) noted that arrhythmias were aggravated for atropine therapy in doses larger than 100mg. Based on animal models (Ying et al., 2003), anisodamine is less likely to cause arrhythmia while exhibiting ACh antagonism. It also shows muscarinic block while prolonging QT intervals of the ECG (Yang et al., 1991). Pharmacological interventions to overcome OP-caused cardiac toxicity rely on the effect of the introduced
CHAPTER 27
9Cardiovascular Toxicity
385
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386
SECTION
Organ Toxicity
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I
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,
,
,
[ms]
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FIG. 6. Pharmacological intervention, such as blocking of lCL,sw by an anionic blocker (dashed curve), can help to restore the tissue to homeostatic condition from that of the OP-affected condition (solid curve).
I
i
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therapeutic agent that acts on ionic concentrations, channels, and membrane currents. In addition, pretreatment with huperzine (Lallement et al., 2002) has been found to be effective. However, the widely used antidote, atropine, can also be toxic because it can cause VE Therefore, the strategy of choice is chemical blocking of OP-activated currents, such as Icl,sw or IKACh, and restoration of function of pumps and exchangers. Such an approach reconstitutes the morphology of the AP of cardiac myocytes and the ECG to baseline form.
-1
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,
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IV. C O N C L U S I O N S DIRECTIONS
AND FUTURE
Computer simulations of OP-affected cellular processes can add considerable new insights into the mechanisms responsible for the modulation of cardiac electrophysiological processes. The identification of the importance of cell swelling-activated membrane current is a case in point. It was shown that inhibition of chloride transport can change overshoot of action potential and thus it can stop the AP
CHAPTER 27 firing. Process simulations can quantify the nature and magnitudes of the incurred changes and the deviation from the normal conditions. There is a need for additional cellular experimental data on membrane currents and ion concentrations in the various cellular compartments under pathological conditions. Such data will make the simulation of the cardiac cycle of the whole heart, from the start of systole to termination of diastole, possible and suggest pharmacological intervention strategies to overcome the effect of OP on cardiac tissue.
Acknowledgments We acknowledge the use of the computer assets of the Major Shared Resource Center of the U.S. Army Research Laboratory and the advice of Dr. John Pormann of Duke University on some software issues.
References Abraham, S., Oz, N., Sahar, R., and Kadar, T. (2001). QTc prolongation and cardiac lesions following acute organophosphate poisoning in rats. Proc. West Pharmacol. Soc. 44, 185-186. Alonso, M. J., Arribas, S., Marin, J., Balfagon, G., and Salaices, M. (1991). Presynaptic M2-muscarinic receptors on noradrenergic nerve endings and endothelium-derived M3 receptors in cat cerebral arteries. Brain Res. 567, 76-82. Antzelevitch, C., and Shimizu, W. (2002). Cellular mechanisms underlying the long QT syndrome. Curr. Opin. Cardiol. 17, 43-51. Balazs, T., and Ferrans, V. J. (1978). Cardiac lesions induced by chemicals. Environ. Health Perspect. 26, 181-191. Bar-Meir, A., Grubstein, A., Givoni, S., and Tadmor, B. (2001). Organophosphates "Continuous injury t o the heart" Harefuah 140, 764-769. Baskin, S. I., and Whitmer, M. P. (1991). The cardiac toxicolology of organophosphorous agents. In Principles of Cardiac Toxicology (S. I. Baskin, Ed.). CRC Press, Boca Raton, FL. Baumgarten, C. M., and Clemo, H. E (2003). Swelling-activated chloride channels in cardiac physiology and pathophysiology. Prog. Biophys. Mol. Biol. 82, 25-42. Britt, J. O., Martin, J. L., Okerberg, C. V., and Dick, E. J. (2000). Histopathologic changes in the brain, heart, and skeletal muscle of rhesus macaques, ten days after exposure to soman. Comp. Med. 50, 133-139. d'Anglemont de Tassigny, A., Souktani, R., Ghaleh, B., Henry, P., and Berdeaux, A. (2003). Structure and pharmacology of swelling-sensitive chloride channels, Icl,swell- Fundam. Clin. Pharmacol. 17, 539-553. Demir, S. S., Clark, J. W., and Giles, W. R. (1999). Parasympathetic modulation of sinoatrial node pacemaker activity in rabbit heart: A unifying model. Am. J. Physiol. 276, H2221-H2244. Dobrzynski, H., Marples, D. D. R., Musa, H., Yamanushi, T. T., Henderson, Z., Takagishi, Y., Honjo, H., Kodama, I., and Boyett, M. R. (2001). Distribution of the muscarinic K + channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart. J. Histochem. Cytochem. 49, 1221-1234.
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Dobrzynski, H., Janvier, N. C., Leach, R., Findlay, J. B., and Boyett, M. R. (2002). Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells. Am. J. Physiol. Heart Circ. Physiol. 283, H615-H630. Egan, T. M., and Noble, S. J. (1987). Acetylcholine and the mammalian "slow inward" current: A computer investigation. Proc. R. Soc. London B 230, 315-337. Filbert, M. G., Moore, D. H., and Adler, M. (1992). Relaxation of soman-induced contracture of airway smooth muscle in vitro. Drug Chem. Toxicol. 15, 203-215. Freeman, S. E., Dawson, R. M., and Keeghan, A. M. (1986). Modification of the effects of muscarinic agonists by reversible and irreversible anticholinesterase compounds in the guinea pig atrium. J. Mol. Cell Cardiol. 18, 231-239. Harvey, R. D., and Belevych, A. E. (2003). Muscarinic regulation of cardiac ion channels. Br. J. Pharmacol. 139, 1074-1084. Hu, C. Y., Hsu, C. H., and Robinson, C. P. (1991). Effects of soman on calcium uptake in microsomes and mitochondria from rabbit aorta. J. Appl. Toxicol. 11, 293-296. Karki, P., Ansari, J. A., Bhandary, S., and Koirala, S. (2004). Cardiac and electrocardiographical manifestations of acute organophosphate poisoning. Singapore Med. J. 45, 385-389. Kiss, Z., and Fazekas, T. (1982). Organophosphate poisoning and complete heart block. J. R. Soc. Med. 73, 138-139. Kiss, Z., and Fazekas, T. (1983). Organophosphate poisoning and complete heart block. J. R. Soc. Med. 76, 85. Kotev, G., and Paliev, B. (1968). Electrocardiographic changes in dogs exposed to soman and the effect of the antidote nemicol-3. Eksp. Med. Morfol. 7, 240-244. Lallement, G., Demoncheaux, J. P., Foquin, A., Baubichon, D., Galonnier, M., Clarencon, D., and Dorandeu, E (2002). Subchronic administration of pyridostigmine or huperzine to primates: Compared efficacy against soman toxicity. Drug Chem. Toxicol. 25, 309-320. Limaye, M. R. (1966). Acute organophosphorus compound poisoning. J. Indian M. A. 47, 492-498. Ludomirsky, A., Klein, H. O., Sarelli, P., Becker, B., Hoffman, S., Taitelman, U., Barzilai, J., Lang, R., David, D., DiSegni, E., and Kaplinsky, E. (1982). Q-T prolongation and polymorphous ("torsades de pointes") ventricular arrhythmias associated with organophosphorus insecticide poisoning. Am. J. Cardiol. 49, 1654-1658. Luo, C., and Rudy, Y. (1994). A dynamic model of the cardiac ventricular action potential: I. Simulations of ionic currents and concentration changes. Circ. Res. 74, 1071-1096. Nilius, B., and Droogmans, G. (2003). Amazing chloride channels: An overview. Acta Physiol. Scand. 177, 119-147. Opie, L. H. (1998). The Heart Physiology, from Cell to Circulation, 3rd ed. Lippincott Williams & Wilkins, Philadelphia. Osterrieder, W., Noma, A., and Trautwein, W. (1980). On the kinetics of the potassium current activated by acetylcholine in the SA node of the rabbit heart. Pfluegers Arch. 386, 101-109. Pimentel, J. M., and Carrington da Costa, R. B. (1992). Effects of organophosphates on the heart, In Clinical and Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.). Butterworth-Heinemann, Oxford. Povoa, R., Cardoso, S. H., Luna, F. B., Ferreira, E C., Ferreira, M., and Ferreira, C. (1997). Organophosphate poisoning and myocardial necrosis. Arq. Bras. Cardiol. 68, 377-380.
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Preston, E., and Heath, C. (1972). Depression of the vasomotor system in rabbits poisoned with an organophosphate anticholinesterase. Arch. Int. Pharmacodyn. Ther. 200, 245-254. Roth, A., Zellinger, I., Arad, M., and Atsmon, J. (1993). Organophosphates and the heart. Chest 103, 576-582. Saadeh, A. M., Farsakh, N. A., and A1-Ali, M. K. (1997). Cardiac manifestations of acute carbamate and organophosphate poisoning. Heart 77, 461-464. Sakmann, B., Noma, A., and Trautwein, W. (1983). Acetylcholine activation of single muscarinic K + channels in isolated pacemaker cells of the mammalian heart. Nature 303, 250-253. Shibata, O., Tsuda, A., Makita, T., Iwanaga, S., Hara, T., Shibata, S., and Sumikawa, K. (1998). Contractile and phosphatidylinositol responses of rat trachea to anticholinesterase drugs. Can. J. Anaesth. 45, 1190-1195. Shumaker, J. M., Clark, J. W., Giles, W. R., and Szabo, G. (1990). A model of the muscarinic receptor-induced changes in K +current and action potentials in the bullfrog atrial cell. Biophys. J. 57, 567-576. Stephenson, L. A., and Kolka, M. A. (1990). Acetylcholinesterase inhibitor, pyridostigmine bromide, reduces skin blood flow in humans. Am. J. Physiol. 258, R951-R957. Tan, H. L., Hou, C. J. Y., Lauer, M. R., and Sung, R. J. (1995). Electrophysiologic mechanisms of the long QT interval syndromes and torsades de pointes. Ann. Intern. Med. 122, 701-714. Thomsen, R. H., and Baskin, S. I. (1988). Effects of AF-DX 116, a cardioselective M-2 muscarinic antagonist on the negative inotropic action of acetylcholine. FASEB J. 2, A789. Tobin, T., Akera, T., Baskin, S. I., and Brody, T. M. (1973). Calcium ion and Na + + K+-ATPase: Its mechanism of inhibition and identification of the E1-P intermediate. Mol. Pharmacol. 9, 336-349. Tryphonas, L., Veinot, J. P., and Clement, J. G. (1996). Early histopathologic and ultrastructural changes in the heart of Sprague-Dawley rats following administration of soman. Toxicol. Pathol. 24, 190-198. Tseng, G. (1992). Cell swelling increases membrane conductance of canine cardiac cells: Evidence for a volume-sensitive C1 channel. Am. J. Physiol. 262, C 1056-C 1068. Tsuda, A., Shibata, O., Saito, M., Hashimoto, S., Iwanaga, S., Makita, T., and Sumikawa, K. (2001). A dose-response study
of anticholinesterase drugs on contractile and phosphatidylinositol responses of rat trachea. Anesth. Analg. 92, 100-105. Tsukahara, T., Hongo, K., Kassell, N. E, and Ogawa, H. (1989). Characterization of muscarinic cholinergic receptors on the endothelium and the smooth muscle of the rabbit thoracic aorta. J. Cardiovasc. Pharmacol. 13, 870-878. Vandenberg, J. I., Bett, G. C., and Powell, T. (1997). Contribution of a swelling-activated chloride current to changes in the cardiac action potential. Am. J. Physiol. Cell Physiol. 273, C541-C547. Viskin, S. (1999). Long QT syndromes and torsades de pointes. Lancet 354, 1625-1633. Viswanathan, P. C., and Rudy, Y. (2000). Cellular arrhythmogenic effects of congenital and acquired long-QT syndrome in the heterogeneous myocardium. Circ. Res. 101, 1192-1198. Wright, A. R., and Rees, S. A. (1998). Cardiac cell volume: Crystal clear or murky waters? A comparison with other cell types. Pharmacol. Ther. 80, 89-121. Yang, E, Zhang, B. H., and Hong, N. (1991). Anti-arrhythmia and vegetative nervous system effects of anisodamine. Acta Pharmacol. Sinica 12, 173-176. Yang, Z. K., Boyett, M. R., Janvier, N. C., McMorn, S. O., Shui, Z., and Karim, E (1996). Regional differences in the negative inotropic effect of acetylcholine within the canine ventricle. J. Physiol. (London) 492, 789-806. Ying, X. Y., Zhong, Y. X., and Ruan, J. X. (2003). Effect of anisodamine on the toxicokinetics of soman in rats. Toxicology 191, 28. Zoltani, C. K., and Baskin, S. I. (2000). Simulation of acetylcholine cardiac overload caused by soman, a cholinesterase inhibitor. In Proceedings of Comparative Cardiology 2000 (A. Murray, Ed.), Vol. 27, pp. 243-246. IEEE Press, Piscataway, NJ. Zoltani, C. K., and Baskin, S. I. (2002). Organophosphate-induced toxicity: Computer study of reentry in atrial tissue. In Proceedings of Comparative Cardiology 2002 (A., Murray, Ed.), Vol. 29, pp. 509-512. IEEE Press, Piscataway, NJ. Zoltani, C. K., Baskin, S. I., and Platoff, G. E. (2004). ECGs and metabolic networks: An in silico exploration of cyanide-caused cardiac toxicity. In Pharmacological Perspectives of Toxic Chemicals and Their Antidotes (S. J. S. Flora, J. A. Romano, S. I. Baskin, and K. Sekhar, Eds.). Narosa, New Delhi.
CHAPTER
~8
Pulmonary Toxicity of Cholinesterase Inhibitors COREY HILMAS, MICHAEL ADLER, AND STEVEN I. BASKIN U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland
that they originated in Germany. The A through F designation was based on their chronological order of synthesis. The first nerve agent to be synthesized was tabun (GA) in 1936 by Gerhard Schrader, a chemist at I. G. Farbenindustrie interested in developing OP compounds as insecticides (Harris and Paxman, 1982). This was followed by sarin (GB), named after the four scientists involved in its development (Schrader, Ambrose, Rudriger, and van der Linde) (Harris and Paxman, 1982; Sidell, 1997). The third nerve agent, soman, was synthesized by Richard Kuhn in Germany in 1944 and was termed GD rather than GC since the latter acronym had already been established in the medical literature. Cyclosarin (GF) was the fourth to be synthesized, but interest in this nerve agent declined in favor of the other OP compounds. The fifth agent (VX) was named for being venomous and was synthesized at Porton Down, England, in 1952. Due to their high toxicity in mammals and volatile nature, some of these fluoride (sarin and soman) and nitrile (tabun) containing OP compounds were further tested, manufactured, and stockpiled by the German military during World War II, but they were never deployed. Some experts believe that Hitler, a victim of a chlorine gas attack during World War I, disliked poison gas and would only use these agents as a last resort. Others speculate that the German High Command mistakenly believed the Allies had developed the nerve agents simultaneously and feared Allied retaliation as the Axis retreated. Nevertheless, tons of nerve agents in munitions were synthesized and stockpiled in Germany during World War II that the United States and Great Britain were not aware of at the time. German tabun production facilities, able to synthesize 100 tons a month, were in place near the end of the war (Saunders, 1957). The former Soviet Union captured an entire nerve agent production facility late in the war and moved it back to Russia, where it started to manufacture and stockpile these agents (Robinson, 1971). Allied forces found that the AChEI nerve agents were 15- to 100-fold more potent than the chemical agents used in World War I.
I. I N T R O D U C T I O N The lungs are a major organ system of entry into the body and a target for the toxic effects of organophosphorus (OP) compounds, potent inhibitors of the enzyme acetylcholinesterase (ACHE). In general, ACHE inhibitors (ACHEIs) were developed for a variety of indications, including military, medical, and insecticide applications. Nerve agents, OP chemicals with remarkable toxic activity, were first developed by Germany prior to World War II. Whereas nerve agents were produced primarily for military deployment, other cholinesterase inhibitors were used for treating conditions such as myasthenia gravis and as pretreatments for nerve agent exposure. As powerful inhibitors of ACHE, these compounds exhibit profound toxicity on multiple organ systems. This chapter discusses respiratory and pulmonary toxicity through direct inhalation of ACHEIs and indirect effects on all aspects of respiration through systemic toxicity. OP nerve agents can be disseminated as liquids or aerosols and are toxic by oral, dermal, or inhalational exposure. The lungs are one of the first organs affected following contact with aerosols and vapors. Lung toxicity by ACHEIs is due to the following: (1) parasympathetic muscarinic effects leading to increased glandular secretion throughout the respiratory tract and alveoli, (2) bronchoconstriction from contraction of airway smooth muscle, (3) nicotinic effects on respiratory muscles in the thorax and accessory muscles of the neck causing labored breathing and eventually flaccid paralysis, and (4) central effects resulting in a decrease in respiratory drive.
II. H I S T O R I C A L P E R S P E C T I V E The modem age of chemical warfare began during the past century with the development of the present-day vesicants and AChEIs (or OP class nerve agents). There are five OP compounds recognized as nerve agents, designated GA, GB, GD, GF, and VX by their North Atlantic Treaty Organization military abbreviation. The "G" series are named for the fact
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SECTION IV. Organ Toxicity
Since OPs were not used in World War II, the majority of cases of OP toxicity have come from accidental exposure in laboratories and agricultural exposure to OP insecticides. The earliest reported incident of OP toxicity from inhalation came from the laboratory of Willy Lange at the Friedrich-WilhelmsUniversity. In the early 1930s, Lange and his student, Gerde von Krueger, prepared dialkyl monofluorophosphates and noted their toxic fumes (Holmstedt, 1963; Sidell, 1997). They described the aromatic vapors as leading to dyspnea and laryngeal edema minutes later, followed by a lucid interval, diplopia, and photophobia. The symptoms of toxicity were noted to last several hours before subsiding. Similar clinical pictures have been reported for the OP compounds DFP (Grob et aL, 1947), satin and tabun (Grob and Harvey, 1953, 1958; Krop and Kunkel, 1954), as well as parathion (DuBois et al., 1949). Detailed clinical signs and symptoms have also been described in case reports of accidental exposures to G agents (tabun, sarin, and soman) (Craig and Comblath, 1953; Craig and Freeman, 1953; Sidell, 1974) and VX (Freeman et al., 1956; Lubash and Clark, 1960; Sidell, 1967).
toxicity to mammals at doses less than 1 mg/kg (Saunders, 1957; O'Brien, 1960). O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX), an agent that is less volatile than the G agents, is the most potent AChEI. The first OP synthesized was B ladan, shown to be tetraethyl pyrophosphate (TEPP). The most studied of the OP compounds is diisopropyl fluorophosphate (DFP), originally synthesized by the British, who found it to be more potent than eserine (Adrian et al., 1947). The phosphorothioates and phosphorodithioates, shown in Table 1 of Chapter 2, were developed when the nerve agents were found to be too toxic and volatile for use in agriculture. These OP insecticides contain a P-S-alkyl and/ or a P - S group in their structure. The best known member of this class is parathion, the most widely used insecticide at one time and responsible for more cases of accidental poisoning and death than any other OP compound. The activation and conversion of this weak AChEI (parathion) to the more active and potent form (paraoxon) was demonstrated to take place in the liver (Diggle and Gage, 1951; Gage, 1953).
III. STRUCTURES OF OPs IV. RESPIRATORY PHYSIOLOGY The general formula and chemical structures of most OP compounds discussed in this chapter are shown in Table 1 of Chapter 2. The nerve agents tabun, sarin, and soman were the most potent compounds in the class, causing lethality to animals in the submilligram range. Their chemical structures are shown in Fig. 1. Originally developed for the agricultural industry, they contain either an F or CN substituent group and display
The respiratory system can be viewed as two components acting in tandem to facilitate gas exchange, namely the conducting and respiratory portions. The conducting portion supplies the lungs with warmed air on inhalation and allows gases to escape on exhalation, whereas the respiratory portion provides for actual gas exchange between the air and blood. The conducting portion consists of a
O 02H50
iiP ~ C N I N(CH3)2
Tabun (GA) Ethyl N-dimethylphosphoramido-cyanidate
O
il i CH3
(H3C)2HCO~ p~
Sarin (GB) Isopropyl methylphosphonofluoridate OH3
O
C2H50
ii I O02H5
..... P ~ O
O
IIP OC2H5 I OC2H5
Tetraethyl pyrophosphate (TEPP)
I
CH3
H
I
CH3
Soman (GD) Pinacolyl methylphosphonofluoridate H3C
O
I II HC~O~P~F I I CH 3 O H3C~C
HC~O-----P~F
O
ii I OCH2CH3
H3C~P~S
CH 3
Diisopropyl fluorophosphate (DFP)
\ CH~CH / CH2CH2--N \
CH~CH 3
H3C O-ethyl- S-(2-diisop ropylam inoethyl) methylphosphonothiolate (VX)
FIG. 1. Chemical structures of OP compounds and their common abbreviations.
3
CHAPTER 28 9Pulmonary Toxicity of AChEIs branching system of airways, including the nasal cavities, nasopharynx, larynx, trachea, mainstem bronchi, progressing to even smaller bronchi and eventually to bronchioles. This elaborate system of pipes conducts air into and out of the lungs as a result of respiratory movements of the thoracic intercostal musculature and diaphragm. Air reaches the alveolar ducts and finally the alveoli, the site of gas exchange. The most important factor of respiratory physiology to consider for pulmonary toxicity by AChEIs is the airway resistance through the conducting portion. Airflow through the conducting system from the trachea and mainstem bronchi to the small bronchioles can be characterized as airflow through a series of straight tubes or laminar flow. Jean Poiseuille, a French physician and physiologist, described the volume flow rate through straight circular tubes by the following equation, known as Poiseuille's law (West, 1995; Guyton and Hall, 2005): F = P'rrr4/8nl
where n is the coefficient of viscosity, P is the pressure difference across the length 1 of the tube, r is the radius of the tube, and F is the volume flow rate. Since the resistance to flow R is driving pressure P divided by flow F using the analogy of Ohm's law, we arrive at the following relationship for flow resistance R. R = 8nl/'rrr 4
When applied to airways, if the airway radius decreases by half its original diameter as a result of bronchoconstriction, the airway resistance increases 16-fold. In actuality, the airflow is a mixture of laminar and turbulent flow because the airways must branch to progressively smaller bronchi as they reach the lung periphery. The major site of airway resistance lies in the medium bronchi because the vast number of smaller airways negates any effect they might impose on airway resistance. Airway smooth muscle tone is under the control of the autonomic nervous system. Whereas sympathetic stimulation of adrenergic receptors causes bronchodilation, parasympathetic activity via acetylcholine (ACh) release causes bronchoconstriction. Excess ACh at smooth muscles surrounding airways due to ACHE inhibition by OPs produces significant increased airway resistance that is readily characterized on inspiratory and expiratory auscultation of the lungs (West, 1995).
V. C O N T R O L O F V E N T I L A T I O N Since the end result of OP-induced toxicity from lethal doses through inhalation or other routes is asphyxia secondary to respiratory failure, a brief summary of the mechanisms involved in control of ventilation is provided. Although ACHEIs affect several aspects of respiration, a detailed review of respiratory
391
physiology and ventilation can be found elsewhere (West, 1995; Guyton and Hall, 2005). The elements of the respiratory control system are the following: chemoreceptors, peripheral sensors, and central sensors, which monitor various measures of respiration to inform the brain; the effector muscles of respiration, which allow for ventilation; and the respiratory control centers in the brain, which integrate the information from the chemoreceptors and regulate the effector muscles. The lungs contain specialized receptor sensors: the pulmonary stretch receptors, the juxtacapillary or J receptors, and the irritant receptors. The J receptors, found in the alveolar walls close to the capillaries, are very sensitive to chemicals monitored in the pulmonary circulation. Receptor activation in this group leads to rapid, shallow breathing patterns and apneic episodes mediated through the vagus nerve. Furthermore, an increase in the interstitial fluid volume of the alveolar wall will activate these receptors, suggesting a possible role in late stages of OP toxicity after the onset of pulmonary edema. Clinical evidence of an abnormal pattern of breathing (Cheyne-Stokes respiration) has been noted to occur in patients exposed to nerve agents or pesticides. This is an unusual periodic breathing pattern characterized by long periods of apnea interspersed with episodes of hyperventilation (Taylor, 1996). This pattern is due to respiratory insufficiency, hypoxemia, and brain damage and is a grave clinical sign as the tidal volume gradually waxes and wanes prior to death. Direct brain damage from OPs and hypoxemia from respiratory insufficiency secondary to pulmonary congestion and flaccid paralysis of respiratory muscles probably contribute to this respiratory pattern. In addition, the lungs contain irritant receptors located between airway epithelial cells. These groups are activated by noxious fumes, gases, smoke, and dust, inducing a bronchoconstriction reflex that is thought to be responsible for the onset of asthma attacks. The effector muscles of respiration include diaphragmatic, intercostal, abdominal, and accessory muscles of respiration (e.g., sternocleidomastoid). They are very sensitive to the toxic effects of AChEIs. Accessory muscles of respiration are not a major contributor to the work of breathing under normal conditions, but they come into play during periods of labored breathing. OP compounds are toxic to these muscles of respiration through inhibition of ACHE, leading to an excess of ACh, excessive stimulation of nicotinic cholinergic synapses, and eventual flaccid paralysis. Subsequent expansion of the chest wall to inflate the lungs will not occur and respiration will cease. The respiratory centers are neuronal groups found primarily in the medulla and pons of the brain stem. The medullary respiratory center comprises a dorsal and ventral group, located in the reticular formation of the medulla below the fourth ventricle. They are believed to be central targets for OP toxicity through an unknown mechanism. Damage to these neuronal control centers will affect inspiration and expiration.
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S ECTI O N IV 9Organ Toxicity
VI. E V I D E N C E O F P U L M O N A R Y TOXICITY Nerve agents, extremely potent chemicals, are esters of phosphonic acid. A C t [the concentration C of agent vapor or aerosol in air (mg/m 3) multiplied by the time t of exposure (in minutes)] of 2 or 3 mg.min/m 3 of sarin is enough to produce symptoms in man (Johns, 1952). Derived from Haber's rule, the product of Ct is a constant such that this Ct for sarin can be attained with an exposure to a concentration of 2 or 3 mg/m 3 for 1 min or a concentration of 0.05-0.075 mg/m 3 for 40 min. Only a few milligrams of VX, absorbed through the skin, will cause clinical signs and symptoms of toxicity (Bowers et al., 1964; Craig et al., 1977). The initial signs and symptoms of exposure to small quantities of agent vapor are discussed later. Larger amounts will undoubtedly lead to loss of consciousness, seizure activity, respiratory and cardiac arrest, and death. Clinical effects are evident within minutes of exposure (Ward, 1962), and after a large exposure (Ct of 10-200 mg.min/m3), depending on the agent, death is inevitable in 10-15 min without medical intervention. After exposure to a sublethal amount on the skin (1-3 mg), the onset time for clinical effects is typically 1 or 2 hr (Bowers et al., 1964; Craig et al., 1977). The initial effect is usually vomiting, followed by muscular weakness. A lethal amount on the skin (10 mg) in the case of VX, the most toxic by percutaneous absorption, will cause clinical effects within several minutes and death soon thereafter.
A. Clinical Signs and Symptoms We have learned valuable information regarding the signs and symptoms observed after mild to moderate OP intoxication as it relates to the respiratory system. In most of these clinical cases, the exposure Ct is unknown. One study (Craig and Freeman, 1953) analyzed clinical cases of 53 individuals exposed accidentally to tabun (4 cases) or sarin (49 cases). Although miosis and rhinorrhea were the two most consistent signs of toxicity, occurring in 91 and 58% of reported cases, respectively, respiratory symptoms were recorded in 77% of cases of mild to moderate exposure. These cases were considered mild to moderate since symptoms were not severe enough to require atropine therapy, although atropine was administered in several cases. The types of respiratory symptoms described by patients were not consistent and included coughing, wheezing, increased exertional dyspnea, dyspnea at rest, inability to breathe deeply, and a sensation of pressure in the throat or chest. Rhinorrhea was shown to be accompanied by hyperemia of the nasal mucosa, persisting for the duration of clinical signs and reported symptoms. Since the nose is part of the upper airway, we consider rhinorrhea as part of pulmonary toxicity. Auscultation of the chest revealed prolongation of the expiratory phase and wheezy breath sounds (lung field
location unreported) for 5 days following exposure. In addition, the earliest symptom was recorded, and in the majority of cases, chest pressure and rhinorrhea were noted first, taking place 5 - 2 0 m in after exposure. Miosis and dim vision occurred next, with onset beginning 15-60 min after exposure. With regard to respiratory symptom severity, exertional dyspnea, wheezing, and cough became more severe over time, with no consistent pattern for regression of respiratory symptoms. Chest tightness, appearing early in the course of intoxication, was the first to disappear, and exertional dyspnea, reported later among cases, persisted the longest among all respiratory symptoms. Furthermore, an earlier onset for respiratory symptoms noted after exposure predicted a more uncomfortable and prolonged clinical course. This was associated with the suspected scenario in which the concentration was high and duration short. In contrast, individuals believed to have experienced more prolonged but lower concentrations of the agent developed symptoms more slowly. Among all cases, there was no correlation between cholinesterase activity and the clinical signs and symptoms, and no differences in symptoms were noted between those exposed to tabun or sarin. In contrast to GA and GB, VX contains a low vapor tension, resulting in absorption through the skin to produce systemic toxicity rather than inhalation via the respiratory tract. However, there is clinical evidence for pulmonary toxicity after dermal exposure to such agents. For example, case reports of accidental VX poisoning in humans have indicated evidence of respiratory toxicity from liquid agents without copious nasal secretions (Freeman et al., 1956). Since rhinorrhea is a hallmark of inhalational toxicity, this suggests an indirect toxicity of the respiratory system through a systemic route. In addition, respiratory symptoms of chest tightness were late appearing, beginning 24 hr after initial exposure and lasting 3 days, unlike the early respiratory symptoms experienced by patients exposed to the more volatile GA and GB agents (Craig and Cornblath, 1953; Craig and Freeman, 1953). At autopsy, evidence of pulmonary toxicity is quite pronounced, independent of the route of administration. Elevation of the diaphragm and collapse of the lungs are common findings. Further observations indicate pulmonary ischemia and congestion (O'Brien, 1960).
B. Nasal Airway Since the nasal passages constitute the initial conducting portion of the respiratory tract, we consider toxicity of the nasal mucosa here. The anterior aspect of the nose is the area most prone to toxic effects by any inhalants because it is the initial primary site of deposition of highly volatile vapors and particles (Stott and McKenna, 1984; Dahl and Bechtold, 1985). Rhinorrhea can be an early warning indicator of recent OP exposure because it is very common in case histories after inhalational exposure and is the earliest clinical sign, along with miosis, for the diagnosis of an acute exposure.
CHAPTER
Rhinorrhea or "runny nose" occurs because of muscarinic receptor activation from excess ACh secondary to inhibition of ACHE. Rhinorrhea usually occurs minutes after an exposure and secretions tend to be thin, clear, and serous in nature. Thick, rubbery secretions may be seen after atropine treatment. Even small amounts of OP vapor can set off a profound rhinorrhea, but the symptoms are typically dose related. One case report demonstrates the abundant rhinorrhea one might experience after exposure to inhaled vapors of sarin. The rhinorrhea was described by the patient to be like a "leaking faucett" (Sidell, 1997). A general increase in secretions from any glands, including nasal mucosa, intestinal, and salivary, can be triggered from dermal or inhalational exposure. From numerous clinical cases, it is known that low Cts will produce a triad of effects on the eyes, nose, and airways. Not only will bronchoconstriction in the airways contribute to dyspnea but also goblet cells and other secretory cells of the nasal mucosa and bronchi will contribute to the dyspnea experienced from OP exposure. Only during moderate exposure will one see deficits in ventilation, copious secretions, and severe dyspnea. Severe exposure will produce cyanosis, loss of consciousness, and convulsions.
C. Trachea and Bronchi Although the extent of signs and symptoms varies with the type of OP agent, concentration, time of exposure, and route of administration, the predominant signs of exposure include constriction of the airways and increased secretions, leading to various degrees of dyspnea (Taylor, 1996). Similar to rhinorrhea, low levels of exposure to nerve agents such as sarin at a Ct of 5-10 mg.min/m 3 will produce respiratory discomfort in the majority of patients, primarily due to bronchoconstriction (Sidell, 1997). The severity of pulmonary complaints will increase as the concentration of the agent or the time of exposure to the agent increases. Although pulmonary function studies have yielded mixed results on the importance of bronchoconstriction in subjects exposed to various low levels of satin (Cts up to 19.6 mg.min/m 3) (Clements et al., 1952), it is clear that pulmonary changes in airway resistance and tracheobronchial secretions are heard clinically upon auscultation of the lungs (Sidell, 1997). It is important to realize how sensitive lungs are to low levels of nerve agent vapors or aerosols because even a Ct exposure of 5 mg.min/m 3 of satin will produce signs and symptoms of toxicity (Marrs et al., 1996). Patients exposed to sufficient levels of OP compounds that are toxic to the respiratory system will indicate vague symptoms of chest tightness or pressure but show striking pulmonary signs on physical examination. Combinations of wheezing (expiratory, inspiratory, or both), rales, and rhonchi have all been reported in clinical cases. The pulmonary effects begin within seconds after inhalation. If the amount inhaled is large, the patient will exhibit signs of severe dyspnea, poor ventilation, cyanosis, and loss of consciousness.
28
9 Pulmonary Toxicity of AChEis
393
Although generalized systemic effects are sometimes present depending on the inhaled dose, local effects on the airways are always typically present and are the earliest symptoms recorded following inhalation of nerve agent vapors or aerosols (Craig and Freeman, 1953; Vojvodic, 1981). The airways are particularly vulnerable to the toxicity of AChEIs, considering that inhaled gases will be taken up at a rate of 8-14 breaths/min for an average adult human male. The involuntary smooth muscle that surrounds the airways of the bronchial tree is the target for AChEI activity, leading to bronchoconstriction with subsequent wheezing. Airway smooth muscles contain numerous excitatory cholinergic inputs (Suzuki et al., 1976) and a relative resistance to muscarinic receptor desensitization and muscle fatigue, making them highly vulnerable to AChE inhibition. One study investigating anti-ChE-induced constriction of isolated canine tracheal smooth muscle showed that a very low soman concentrations (10-9M) can increase the amplitude and prolong the half-relaxation time of contractions elicited by electric field stimulation (Adler et al., 1992). This was true for all AChEIs examined, including three OP (soman, satin, and paraoxon) and three carbamate (CM) ChE inhibitors (physostigmine, neostigmine, and pyridostigmine). Contractures of canine airway smooth muscle were detected when AChE activities were reduced by more than 52% (Fig. 2). When contractures are plotted as a function of AChE inhibition for the OPs and CMs, there is a linear rise in contracture amplitudes as AChE inhibition increases. This demonstrates that contracture depends on the degree of AChE inhibition and not on the nature of the inhibitor, suggesting that it is mediated by ACh accumulation. Another experiment compared the ability of two different oximes, pralidoxime (2-PAM) and HI-6, to relax soman- and sarin,induced contractures in canine tracheal smooth muscle (Fig. 3). For nerve agents that undergo rapid aging (soman), 2-PAM was unable to
100
o o o
,&
80 o ,,,_, tO
60
.E_ 40
0
~
~ ~ " O
X t~
20
Q Soman O Sarin A Paraoxon
0. .Physosti . . gmine ,8, Neostigmine Q Pyridostigmine
A 0
50
I
60
I
70
I
80
I
90
I
100
% AChE Inhibited
FIG. 2. Contractures plotted as a function of ChE inhibition for three OP and three carbamate AChEIs. Each symbol represents a single muscle strip on which both tension and AChE activity were determined.
394
SECTION IV- Organ Toxicity
E. Central Respiratory Center
FIG. 3. Relaxation of soman- or satin-induced contractures in isolated canine tracheal smooth muscle strips by the oximes HI-6 and 2-PAM. Incubation times for HI-6 (100 IxM) or 2-PAM (1 raM) were 30 min, and the oximes were added 15 min after soman (0.1 p.M) or satin (0.1 IxM) exposure. The symbols represent the mean +__SE of data from four to six muscle strips.
sufficiently reactivate ACHE. Surprisingly, the best known AChE reactivator, HI-6, was unable to reactivate AChE inhibited by soman in airway smooth muscle. Both oximes demonstrated an ability to reactivate AChE in the presence of satin, but HI-6 treatment led to more relaxation of satininduced contractures.
D. Bronchioles and Alveolar Cells One study of satin effects on rat lungs indicated increased cellular proliferation in the lungs with interstitial thickening 4 days after satin exposure (Pant et al., 1993). Signs of respiratory bronchiole damage, loss of alveolar spaces, and evidence of lung consolidation occurred 16 days after satin exposure. A typical combination therapy consisting of atropine, diazepam, and pralidoxime prevented these lung changes. There was significant interest in the toxic effects of trialkylphosphorothioates, contaminants formed during synthesis and storage of P=S phosphorothioate pesticides, because of their ability to produce pulmonary toxicity. Although these are classified as OPs (Clothier et al., 1981), these weak AChEIs do not produce cholinergic changes at doses causing visible lung pathology (Dinsdale, 1992). Trimethyl phosphorodithioate (OSSMeO), a representative of this class, was shown to produce selective type I alveolar pneumocyte damage in rats within 12 hr after oral administration of a lethal dose followed by consolidation of the lungs and alveolar edema (Dinsdale and Verschoyle, 1988). In addition, OSSMeO was shown to cause distortion of rat Clara cells of the bronchiolar epithelium. Similar changes in rat Clara cells, namely hypertrophy, distortion, and cell death, were demonstrated 24 hr after exposure to sublethal doses of OSSMeO (Imamura et al., 1983).
OP compounds affect important respiratory control centers in the brain stem. Although it was known early on that AChEIs cause death by respiratory failure (Modell et al., 1946; Freeman and Himwich, 1949), the majority of the actions were thought to be systemic-related effects on bronchi and respiratory muscles. Central nervous system effects of AChEIs on respiration were not considered a major mechanism until Douglas and DeCandole showed that anticholinesterases caused depression of the respiratory center (DeCandole and Douglas, 1949; Douglas, 1950). The first demonstration of decreased output from the respiratory center was shown by Krivoy and Marrazzi (Krivoy and Marrazzi, 1951; Krivoy et al., 1951). They showed that the output of the respiratory center, recorded as phrenic nerve potentials, was sensitive to the systemic effects of toxic DFP levels. It was also shown that recovery from this central respiratory depression could occur spontaneously or be induced with atropine. Further studies demonstrated similar findings in cats using TEPP (Douglas and Matthews, 1952) and in rabbits, cats, and monkeys using satin (Holmes, 1952, 1953).
VII. MECHANISM OF RESPIRATORY FAILURE FROM OP TOXICITY Although it was known that asphyxia from respiratory failure was the cause of death from OP intoxication, it was unclear which component (muscle paralysis, bronchoconstriction, or central respiratory drive) played a greater role. Respiratory failure was shown to be mostly due to failure of the central respiratory drive by Rickett and colleagues (Rickett, 1981; Ricket et al., 1986). They administered 1 LD50 of soman, satin, tabun, or VX every 15 min into a cat until the onset of respiratory arrest. Disruption of the normal firing pattern of the medullary respiratory-related neurons ensued first, followed by changes in phrenic nerve activity, diaphragmatic electromyogram, diaphragm contraction, and airflow. During respiratory arrest, the diaphragm muscle was still able to contract tetanically at 100 Hz for 500 msec, but the medullary respiratory-related units and the phrenic nerve stopped firing. An early technical report by DeCandole et al. (1953) showed similar findings of the importance of a central component in respiratory failure, but this depended on the species and the OP agent. DeCandole et al. investigated seven compounds against nine mammalian species and concluded that central respiratory failure predominated, but this depended on the species, the drug used, and the dosage administered. For example, central failure appeared to be the sole cause of respiratory arrest in monkeys. There were also differences between the two studies. Whereas the later study by Rickett et al. in cats showed the importance of central respiratory drive, the DeCandole report
CHAPTER 28 9Pulmonary Toxicity of AChEIs hinted at bronchoconstriction as the predominant feature occurring earliest in cats. Another study comparing the effects of bronchoconstriction in dogs and monkeys injected intravenously with satin (Johnson et al., 1958) supported the earlier findings (DeCandole et al., 1953) of weak bronchoconstriction in monkeys, but canine airway smooth muscle showed significant sensitivity to satin. In another study involving rabbits exposed to sarin, loss of central respiratory drive and neuromuscular block of diaphragmatic muscles were shown to be responsible for respiratory failure (Wright, 1954). From these studies, it can be concluded that central failure of respiration is most likely the predominant cause of death, which is aided by weakening of respiratory muscles and airway obstruction from increased secretions and bronchospasm. Furthermore, respiratory failure has been shown to precede significant cardiovascular depression (Wright, 1954; Rickett et al., 1986; Sidell, 1997), strengthening the importance of respiratory mechanisms as the primary cause of death.
VIII. T H E R A P E U T I C S T R A T E G I E S
FOR OP INTOXICATION OP nerve agent toxicity is due to their irreversible inhibition of the enzyme ACHE, present at all known cholinergic synapses (Taylor, 1996). AChE limits the duration of the activity of ACh and thus prevents its accumulation at synaptic junctions. Inhibition of AChE results in excessive stimulation of cholinergic synapses, which leads to bronchoconstriction, laryngospasm, muscle weakness, convulsion, and death (Ho and Hoskins, 1987). The standard U.S. military therapy for intoxication by OP compounds consists of administering atropine to antagonize excessive muscarinic stimulation and 2-PAM to reactivate the inhibited ACHE. For the nerve agents that undergo rapid aging, such as soman, 2-PAM is inadequate since AChE becomes resistant to reactivation within several minutes of exposure (Berman and Decker, 1986). In this case, the only practical strategy is to protect a critical pool of AChE from irreversible inhibition by pretreatment with the CM pyridostigmine bromide (PB) (Gordon et al., 1978; French et al., 1979). One study showed that when used as a pretreatment against the nerve agent soman, PB could rescue primates from respiratory failure and death (Kluwe et al., 1987). Low doses of PB have been shown to be effective in protecting against soman toxicity when combined with atropine and 2-PAM (Caldwell et al., 1989; Dawson, 1994; Marino et al., 1998). Since pyridostigmine does not enter the blood-brain barrier, peripheral protection of diaphragmatic muscle function and airway musculature is paramount to its mechanism. Although pretreatment with PB followed by treatment with atropine and an oxime represents a rational strategy for protection against soman exposure, newer pretreatment candidates for central
395
protection against OP toxicity would be useful therapeutic adjuncts.
IX. ORGANOPHOSPHATE USE AND ASTHMA Epidemiological studies have linked OP exposure to wheezing and symptoms related to a hyperactive airway (Deschamps et al., 1994; O'Malley, 1997; Salam, 2004). The establishment of this link is interesting because asthma prevalence has been increasing in the United States. OP insecticide use has increased in agrarian communities (Fenske et al., 2002; Koch et al., 2002) as well as urban populations (Lu et al., 2001; Berkowitz et al., 2003). Therefore, it is not surprising that the largest increase in asthma prevalence has occurred among youth in nonrural populations (Hartert and Peebles, 2000). The proposed mechanism of action to explain OP insecticide effects on asthma has been AChE inhibition, leading to excess ACh, resulting in activation of M3 muscarinic receptors on airway smooth muscle and subsequent bronchoconstriction (Roffel et al., 1990, 1994; Coulson and Fryer, 2003). Studies indicate that the OP insecticide chlorpyrifos induces airway hyperreactivity by a different mechanism in guinea pigs (Fryer et al., 2004; Lein and Fryer, 2005). It was shown that the OP insecticide chlorpyrifos could induce bronchoconstriction in guinea pig airways, and this was independent of AChE inhibition. Autoinhibitory M2 muscarinic receptors on parasympathetic nerves supplying airway smooth muscle prevent airway hyperreactivity (Minette and Barnes, 1988), but chlorpyrifos, parathion, and diazinon inhibit this M2 receptor population.
X. C O N C L U S I O N S AChEIs are toxic to multiple organ systems, but the main cause of death is through pulmonary toxicity. OP nerve agents cause toxicity to multiple aspects of breathing, including inhibition of central respiratory drive, constriction of airway smooth muscle leading to bronchospasm and bronchoconstriction, increased airway secretions, and neuromuscular block of diaphragmatic and intercostal muscles. Airway constriction, bronchospasm, and increased airway and nasal secretions compound the dyspnea experienced by the patient, but they can indicate early signs of mild OP exposure. Although death by asphyxiation is predominantly due to central respiratory failure, fatigue and flaccid paralysis of muscles responsible for expanding the chest wall in order to inflate the lungs are contributors to respiratory arrest, leading to hypoxemia, convulsions, brain damage, and death.
396
SECTION I V . Organ Toxicity
References Adler, M., Moore, D. H., and Filbert, M. G. (1992). Effects of anticholinesterases on airway smooth muscle. In Clinical & Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 149-155. ButterworthHeinemann, Oxford. Adrian, E. D., Feldberg, W., and Kilby, B. A. (1947). The cholinesterase inhibitory action of flurophosphonates. Br. J. Pharmacol. 2, 56-58. Berkowitz, G. S., Obel, J., Deych, E., Lapinski, R., Godbold, J., Liu, Z., Landrigan, P. J., and Wolff, M. S. (2003). Exposure to indoor pesticides during pregnancy in a multiethnic, urban cohort. Environ. Health Perspect. 111, 79-84. Berman, H. A., and Decker, M. M. (1986). Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonyl acetylcholinesterase. Electrostatic control of enzyme topography. J. Biol. Chem. 261, 10646-10652. Bowers, M. B., Goodman, E., and Sim, V. M. (1964). Some behavioral changes in man following anticholinesterase administration. J. Nervous Mental Dis. 138, 383-389. Caldwell, R. W., Lowensohn, H. S', Chryssanthis, M. A., and Nash, C. B. (1989). Interactions of pyridostigmine with cardiopulmonary systems and their relationships to plasma cholinesterase activity. Fundam. Appl. Toxicol. 12, 432-441. Clements, J. A., Moore, J. C., Johnson, R. P., and Lynott, J. (1952). Observations on airway resistance in men given low doses of GB by chamber exposure, MLR Report No. 122. Medical Laboratories Research, Edgewood Arsenal, MD. Clothier, B., Johnson, M. K., and Reiner, E. (1981). Interaction of some trialklphosphorothiolates with acetylcholinesterase. Characterisation of inhibition, aging and reactivation. Biochim. Biohys. Acta 660, 306-316. Coulson, E R., and Fryer, A. D. (2003). Muscarinic acetylcholine receptors and airway diseases. Pharmacol. Ther. 98, 59-69. Craig, A. B., Jr., and Comblath, M. (1953). Further clinical observations on workers accidentally exposed to "G" agents, MLR Report No. 234. Medical Laboratories Research, Edgewood Arsenal, MD. Craig, A. B., Jr., and Freeman, G. (1953). Clinical observations on workers accidentally exposed to "G" agents, MLR Report No. 154. Medical Laboratories Research, Edgewood Arsenal, MD. Craig, E N., Cummings, E. G., and Sim, V. M. (1977). Environmental temperature and the percutaneous absorption of a cholinesterase inhibitor, VX. J. Invest. Dermatol. 68, 357-361. Dahl, A. R., and Bechtold, W. E. (1985). Deposition and clearance of a water-reactive vapor, methylphosphonic difluoride (difluoro), inhaled by rats. Toxicol. Appl. Pharmacol. 81, 58-66. Dawson, R. M. (1994). Review of oximes available for treatment of nerve agent poisoning. J. Appl. Toxicol. 14, 317-331. DeCandole, C. A., and Douglas, W. W. (1949). The mechanism of respiratory arrest after GB poisoning. Porton Technical Paper No. 149. DeCandole, C. A., Douglas, W. W., and Evans, C. L. (1953). The failure of respiration in death by anticholinesterase poisoning. Br. J. Pharmacol. Chemother. 8, 466-475. Deschamps, D., Questel, E, Baud, E J., Gervais, P., and Dally, S. (1994). Persistent asthma after acute inhalation of organophosphate insecticide. Lancet 344, 1712.
Diggle, W. M., and Gage, J. C. (1951). Cholinesterase inhibition in vitro by O,O-diethyl O-p-nitrophenyl thiophosphate (parathion, E 605). Biochem. J. 49, 491-494. Dinsdale, D. (1992). Pulmonary toxicity of anticholinesterases. In Clinical & Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 156-166. Butterworth-Heinemann, Oxford. Dinsdale, D., and Verschoyle, R. D. (1988). Comparative toxicity of two trialkylphosphorothioates to rat lung and the effects of atropine sulphate pretreatment. Arch. Toxicol. Suppl. 12, 432-434. Douglas, W. W. (1950). An effect of atropine in GB poisoning in the cat. Porton Technical Paper No. 195. Douglas, W. W., and Matthews, P. B. C. (1952). Acute TEPP poisoning in cats and its modifications by atropine and hyoscine. J. Physiol. 116, 202. DuBois, K. P., Doull, J., Salerno, P. R., and Coon, J. M. (1949). Studies on the toxicity and mechanisms of action of p-nitrophenyl diethyl thionosphosphate (parathion). J. Pharmacol. Exp. Ther. 95, 79-91. Fenske, R. A., Lu, C., Barr, D., and Needham, L. (2002). Children's exposure to chlorpyrifos and parathion in an agricultural community in central Washington State. Environ. Health Perspect. 110, 549-553. Freeman, A. M., and Himwich, H. E. (1949). DFP: Site of injection and variation in response. Am. J. Physiol. 156, 125. Freeman, G., Hilton, K. C., and Brown, E. S. (1956). V poisoning in man. CWL Report No. 2025. Chemical Warfare Laboratories, Edgewood Arsenal, MD. French, M. C., Wetherell, J. R., and White, P. D. (1979). The reversal by pyridostigmine of neuromuscular block produced by soman. J. Pharm. Pharmacol. 31,290-294. Fryer, A. D., Lein, P. J., Howard, A. S., Yost, B. L., Beckles, R. A1, and Jett, D. A. (2004). Mechanisms of organophosphate insecticide-induced airway hyperreactivity. Am. J. Physiol. Lung Cell. Mol. Physiol. 286, L963-L969. Gage, J. C. (1953). A cholinesterase inhibition derived from O, O-diethyl O-p-nitrophenyl thiophosphate in vivo. Biochem. J. 54, 426-430. Gordon, J. J., Leadbeater, L., and Maidment, M. P. (1978). The protection of animals against organophosphate poisoning by pretreatment with a carbamate. Toxicol. Appl. PharmacoL 43, 207-216. Grob, D., and Harvey, A. M. (1953). The effects and treatment of nerve gas poisoning. Am. J. Med. 14, 52-63. Grob, D., and Harvey, J. C. (1958). Effects in man of the anticholinesterase compound satin (isopropyl methyl phosphonofluoridate). J. Clin. Invest. 37, 350-368. Grob, D., Lilienthal, J. L., Harvey, A. M., and Jones, B. E (1947). The administration of di-isopropyl fluorophosphates (DFP) to man. I. Effect on plasma and erythrocyte cholinesterase; General systemic effects; Use in study of hepatic function and erythropoiesis; And some properties of plasma cholinesterase. Bull. Johns Hopkins Hosp. 81, 217-244. Guyton, A. C., and Hall, J. E. (2005). Textbook of Medical Physiology, 1 lth ed. Saunders, Philadelphia. Harris, R., and Paxman, J. (1982). A Higher Form of Killing. Hill & Wang, New York. Hartert, T. V., and Peebles, R. S., Jr. (2000). Epidemiology of asthma: The year in review. Curr. Opin. Pulm. Med. 6, 4-9. Ho, I. K., and Hoskins, B. (1987). Biological and pharmacological aspects of neurotoxicity from and tolerance to organophosphorus cholinesterase inhibitors. In Handbook of
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Toxicology (T. J. Haley and W. O. Bemdt, Eds.), pp. 44-73. Hemisphere, Washington, DC. Holmes, R. (1952). The mechanism of respiratory failure in the rabbit poisoned with GB. Porton Technical Paper No. 275. Holmes, R. (1953). The cause of death from acute anticholinesterase poisoning in the rabbit, cat and monkey. Porton Technical Paper No. 356. Holmstedt, B. (1963). Structure-activity relationships of the organophosphorus anticholinesterase agents. In Cholinesterases and Anticholinesterase Agents (G. B. Koelle, Ed.). SpringerVerlag, Berlin. Imamura, T., Gandy, J., and Fukuto, T. R. (1983). An impurity of malathion alters the morphology of rat lung bronchiolar epithelium. Toxicology 26, 73-79. Johns, R. J. (1952). The effects of low concentrations of GB on the human eye, MRL Report No. 100. Medical Research Laboratory, Edgewood Arsenal, MD. Johnson, R. E, Gold, A. J., and Freeman, G. (1958). Comparative lung-airway resistance and cardiovascular effects in dogs and monkeys following parathion and sarin intoxication. Am. J. Physiol. 192, 581-584. Kluwe, W. M., Chinn, J. C., Feder, E, Olson, C., and Joiner, R. (1987). Efficacy of pyridostigmine pretreatment against acute soman intoxication in a primate model. In Proceedings of the Sixth Medical Chemical Defense Bioscience Review, Report No. AD B121516, pp. 226-234. U.S. Army Medical Research Institute for Chemical Defense, Aberdeen Proving Ground, MD. Koch, D., Lu, C., Fisker-Andersen, J., Jolley, L., and Fenske, R. A. (2002). Temporal association of children's pesticide exposure and agricultural spraying: Report of a longitudinal biological monitoring study. Environ. Health Perspect. 110, 829-833. Krivoy, W. A., and Marrazzi, A. S. (1951). Evaluation of the central action of anticholinesterases in producing respiratory paralysis. Fed. Proc. 10, 316. Krivoy, W. A., Hart, E. R., and Marrazzi, A. S. (1951). Further analysis of the actions of DFP and curare on the respiratory center. J. Pharmacol. Exp. Ther. 103, 351. Krop, S., and Kunkel, A. M. (1954). Observations on pharmacology of the anticholinesterases sarin and tabun. Proc. Soc. Exp. Biol. Med. 86, 530-533. Lein, E J., and Fryer, A. D. (2005). Organophosphorus insecticides induce airway hyperreactivity by decreasing neuronal M2 muscarinic receptor function independent of acetylcholinesterase inhibition. Toxicol. Sci. 83, 166-176. Lu, C., Knutson, D. E., Fisker-Andersen, J., and Fenske, R. A. (2001). Biological monitoring survey of organophosphorus pesticide exposure among preschool children in the Seattle metropolitan area. Environ. Health Perspect. 109, 299-303. Lubash, G. D., and Clark, B. J. (1960). Some metabolic studies in humans following percutaneous exposure to VX, CRDL Report No. 3003. Chemical Research and Development Laboratory, Edgewood Arsenal, MD. Marino, M. T., Schuster, B. G., Brueckner, R. E, Lin, E., Kaminskis, A., and Lasseter, K. C. (1998). Population pharmacokinetics and pharmacodynamics of pyridostigmine bromide for prophylaxis against nerve agents in humans. J. Clin. Pharmacol. 38, 227-235. Marts, T. C., Maynard, R. L., and Sidell, E R. (1996). Chemical Warfare Agents, Toxicology and Treatment. Wiley, New York. Minette, P. A., and Barnes, P. J. (1988). Prejunctional inhibitory muscarinic receptors on cholinergic nerves in
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CHAPTER ~ 9
Approaches to Defining and Evaluating the Inhalation Pharmacology and Toxicology Hazards of Anticholinesterases HARRY SALEM l A N D BRYAN BALLANTYNE z 1U.S. Army Chemical and Biological Center, Aberdeen Proving Ground, Maryland 2Charleston, West Virginia
I. I N T R O D U C T I O N
physically in a liquid state of variable vapor pressure and thus present differing vapor inhalation hazards (Table 1). When anti-ChEs are generated as dusts or liquid aerosols, the degree of respiratory tract exposure (total dose, depth of penetration, and distribution) will depend mainly on the particle size. The effects of inhaled anti-ChEs may, for descriptive purposes, be considered as local pharmacological effects on tissues within the respiratory tract, local toxicological effects on the respiratory tract, and systemic pharmacological and toxic effects that occur following absorption of anti-ChE into the systemic circulation. The nature of toxic effects also depends on whether exposure is by a single (acute) exposure or by repeated exposures. The acute lethal toxicity of anti-ChEs by inhalation exposure is usually the result of a combination of both local anti-ChE effects on the respiratory tract and systemic effects from absorbed anti-ChE. Acute lethal toxicity can be numerically expressed as either timed LCs0 (i.e., the concentration of material in the exposure atmosphere, calculated from the analytically measured exposure concentration-mortality data, that will be lethal to 50% of the species exposed for a set exposure time; e.g., mg m -3 for an exposure of x hours) or as the inhalation exposure dose (concentration • exposure time; CT), which is lethal to 50% of the exposed species, the L(CT)50. This is expressed as the product of exposure time and concentration (e.g., mg min m-3). The former method of citing lethality data is preferred, providing the exposure time is kept constant for the various exposure concentrations, since it gives a directly useable value for lethal hazard evaluation and permits a ready comparison between different materials. In the case of L(CT)50 values, however, this does not
The extensive use of anticholinesterases (anti-ChEs) as pesticides and their application in the form of dusts, land-based sprays, and by aerial spraying means that anti-ChE pesticide exposures by inhalation of the vapor and/or aerosols are frequent. Additionally, inhalation of vapor may be encountered in the intended lethal use of organophosphates (OPs) in chemical warfare operations and terrorist situations (Petrolanu et al., 2005). Inhalation studies are designed to determine (1) the nature, onset, and duration of adverse effects and hazards from acute, short-term repeated and long-term repeated respiratory exposure; (2) factors influencing the development of effects; and (3) the efficacy of antidotal treatment. They are also designed to aid in the development of protective and precautionary measures, including recommendations on appropriate airborne concentrations to ensure safe working conditions, such as Threshold Limit Values (American Conference of Governmental Industrial Hygiene, 2005) and Acute Emergency Guideline Levels (Hartmann, 2002). The development of pharmacological and potential short- and long-term toxicological effects from exposure to OP and carbamate (CM) anti-ChEs by the inhalation route of exposure depends on various factors, of which the most important a r e the physicochemical properties of the anti-ChE, whether exposure is to undiluted material or a formulation, and whether the material is physically and intentionally dispersed in the atmosphere for in-use application. Depending on the nature and required use of the material and its formulation, exposure by the inhalation route may be to vapor, liquid aerosol, or dust. Most neat CMs are solids of very low vapor pressure and inhalation hazards are generally low. The majority of OPs are Toxicology of Organophosphate and Carbamate Compounds
399
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
400
SECTION IV. Organ Toxicity TABLE 1.
Representative Physical Properties of Anti-ChEs Relevant to Assessing Vapor Inhalation Hazards a
Anti-ChE Carbamate Aldicarb Carbaryl Carbofuran Methomyl Propoxur Organophosphate DFP Diazinon Dichlorvos Fenamiphos Malathion Parathion
Physical state
Vapor pressure (mmHg)
X X • x •
10 -5 10 -3 10 -5 10 -5 10 -6
(25 (26 (33 (25 (20
Water solubility (g dl-1)
Solid Solid Solid Solid Solid
9.8 5.0 2.0 5.0 6.5
~ ~ ~ ~ ~
0.6 (25 ~ 0.012 (30 ~ 0.07 (25 ~ 5.8 0.2 (25 ~
Liquid Liquid Liquid Solid Liquid Liquid
0.6 (20 ~ 1.4 • 10 -4 (20 ~ 1.2 x 10 -2 (20 ~ 7.5 X 10 -7 (30 ~ 4 x 10 -5 (30 ~ 3.8 X 10 -5 (20 ~
1.5 (25 ~ 0.004 (20 ~ 1.0 0.04 0.014 0.002
aData from Dodd (1992) and the World Health Organization (1986).
give a direct index of hazard for a given exposure time. The value cited could result from a variety of combinations of reciprocally related exposure times and concentrations (i.e., high concentration with short exposure time vs lower concentration with longer exposure period). Clearly, arithmetic manipulation of lethal inhalation exposure values could result in the calculation of a value for lethal concentration that in practice is too low to cause lethality at the corresponding exposure period to give the cited L(CT)50 value. Some have assumed that the inhalation exposure dosage (CT) for a given toxic end point is a constant that holds for a wide range of the reciprocal concentration values leading to this supposedly constant value. This is a gross misuse of the data resulting from a misunderstanding of factors governing toxic responses. For example, whereas in a limited time frame (usually a short time span) the constancy of CT may hold for local respiratory injury, outside this short time frame the relationship often does not hold for even direct injury to the respiratory tract. The inapplicability of CT as a constant is seen most obviously with those inhaled materials that become absorbed into the systemic circulation and then exert systemic toxicity. In such cases, toxicity will depend on the interaction of a variety of processes, which include the mechanism of uptake from the respiratory tract, tissue biodistribution, metabolism (metabolic activation/detoxification), the mechanism of toxicity in target organs, and elimination. Many of these processes are efficient at low concentrations but become saturated at higher concentrations, resulting in nonlinearity among concentration, exposure time, and target organ toxic effect (Hext, 1999). This is seen most obviously with the lethal toxicity of hydrogen cyanide vapor exerting its effects by absorption of inhaled material and subsequent systemic
toxicity (Ballantyne, 1994; Ballantyne and Salem, 2005). The variability in the inapplicability of L(CT)50 data across a wide range of conditions can be seen from some of the materials shown in Table 2. For example, with fensulfothion for both 1- and 4-hr exposures, the L(CT)50 values are within a close range of 113-118 m g h r m - 3 ; this accords
TABLE 2. Examples of Timed LC5o Values and Corresponding L(CI')5o Values for Rats Exposed to Several Organophosphates ~
Anti-ChE Carbamate Methomyl Propoxur Organophosphate DFP Diazinon Dichlorvos Fenamiphos Fensulfothion Mevinphos Parathion Satin Soman Tabun
LCs0 (mg -3)
Time
L(CT)50
(hr)
(mg hr m -3)
300 1440
4 1
1200 1440
360 3500 15 110-175 91-100 113 29.5 128 84 10.6 21 304
0.16 4 4 1 4 1 4 1 4 0.5 0.32 0.16
60 1400 600 110-175 364-400 113 118 128 336 5.3 6.7 51
aData from Dodd (1992) and the World Health Organization (1986).
CHAPTER 29 9Inhalation Toxicity of Anti-ChEs with the 1-hr LC50 being approximately four times greater than the 4-hr LCs0. However, with fenamiphos the 1-hr L(CT)50 values range from 110 to 175 mg hr m -3, whereas the 4-hr L(CT)50 values range from 364 to 400 mg hr m-3; thus, prediction of the 1-hr LC50 from the 4-hr data and vice versa would not be accurate and reliable.
II. F A C T O R S I N F L U E N C I N G T H E INHALATION AND RESPIRATORY TRACT DISTRIBUTION OF MATERIALS
A. Vapors The degree of respiratory tract exposure to vapors depends on a variety of factors, including vapor pressure of the material, vapor concentration in inspired air, the duration of exposure, breathing rate, and respiratory minute volume. Materials with peripheral sensory irritant properties will cause a decrease in breathing rate that will influence respiratory minute volume and hence inspired dose (Ballantyne, 1999, 2005). Differential distribution in the respiratory tract is largely dependent on the water solubility of the material. Substances of high water solubility may be absorbed in the upper respiratory tract and very little material may reach the deeper lung tissue. On the other hand, materials of low water solubility may reach the deep lung. Thus, the concentration of inspired material and its relative water solubility may affect the concentration gradient of material between the nasal cavity and alveolus and, therefore, the differential distribution and consequent local effects within the respiratory tract. For mixed inspired atmospheres (vapor and particulate), vapor may be adsorbed on the particles and the deposition pattern will then be determined by the influence of particle deposition. As discussed in detail later, the distribution of metabolizing capability within the respiratory tract may determine regional toxicity and uptake within the tract.
B. Aerosols An aerosol is a two-phase system consisting of finely divided particulate material suspended in a gaseous phase. The condensed particulate material may exist as a liquid, a solid, or a combination of the two. The most significant factor that determines the depth of penetration in the lung and the differential deposition of the material in the respiratory tract is particle size; this does not refer to physical size of the particles but, rather, to aerodynamic size. The aerodynamic diameter of a particle is defined as the diameter of a sphere of unit density having the same settling velocity as the particle being considered. The relationship between aerodynamic diameter and physical diameter is broadly covered by the following equation (Hext, 1999): Aerodynamic diameter = physical diameter • (density) ~
401
Thus, for example, a particle of 1 lxm physical diameter and density 4 g cm -3 will have an aerodynamic behavior in air similar to a unit density particle 2 txm in diameter. There may be species variations in the relation between the degree of penetration and differential deposition (Raabe et al., 1977; Schlesinger, 1985), which are related to factors such as differences in the regional anatomy and physiology (notably airflow pattern), of the respiratory tract. Thus, in the rat, an obligate nose breather with a complex turbinate system, there will be filtration of fine particles that would be expected to reach the alveolar region in man. In relation to airflow pattern, this is a function of volume and crosssectional diameter of the airway. As particles suspended in air pass through the dichotomous branching of the airway passages, the velocity of particles will decrease since the same volume of inspired air is passing through increasing surface area. This decrease in velocity continues as penetration becomes deeper in the respiratory system. Inhaled particles deposit in the respiratory tract by four processes: impaction, gravitational settlement, Brownian diffusion, and interception. Impaction occurs where there is a change in the direction of the airstream and is associated with both velocity and directional change. It thus occurs predominantly in the upper respiratory tract. Particles with aerodynamic size >0.5 Ixm may deposit by impaction. Gravitational settlement occurs principally in the lower respiratory tract, where velocities are low. Brownian diffusion occurs with particles <0.5 Ixm that are subject to bombardment by gas molecules and thus acquire random movement in air and may contact respiratory tract walls. This type of depositional mechanism occurs with the low airflow velocities in the smaller bronchioles and alveoli. Interception occurs where there is a change in airflow direction with irregularly shaped particles (e.g., fibers) resulting in partial wall contact and deposition. Additionally, generated aerosols in inhalation studies may carry an electrostatic charge, and this can enhance the fraction and site of deposition after inhalation by particle-particle charge interaction and/or particle-respiratory tract charge interaction. The fraction of an atmosphere that can enter the respiratory tract orally and nasally is referred to as the inhalable fraction (International Standards Organization, 1995). Particles having aerodynamic diameters of < 100 txm will deposit in the nose or mouth, and those of smaller aerodynamic size will penetrate further into the respiratory tract, with the depth of penetration related principally to the aerodynamic diameter. Those reaching the alveoli are defined as the respirable fraction; in general, the upper size range for respirable particles is 10 txm. This measured fraction is often referred to as the TM10mthat is, the mass concentration of a particulate aerosol as determined with sampling instruments having a 50% cutoff point of size sampling at 10 txm. However, the majority of particles that will deposit in the alveoli are of aerodynamic diameter 2.5 lxm, and most particles larger than this will deposit higher in the respiratory tract. Thus, TM2.5 is often used as a basis for defining high risk of alveolar deposition (Hext, 1999).
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SECTION IV. O r g a n T o x i c i t y
An approximation of the inhaled dose of aerosol can be obtained by reference to plots of deposition efficiency as a function of particle size, which shows total and regional deposition curves for particles of varying sizes in the species of interest. An accurate assessment of dose for vapor and aerosol atmospheres requires chemical analysis of tissues, or exposure to radioisotopically labeled materials, in conjunction with measurement of respiratory physiology variables.
III. L A B O R A T O R Y I N V E S T I G A T I O N A L ASPECTS OF INHALATION TOXICOLOGY For assessing adverse effects (pharmacological and toxicological), and thus potential hazards, from the inhalation of anti-ChEs, both acute and repeated exposure studies are necessary. The acute studies are usually designed principally to determine lethal potential, possible long-term sequelae from a single exposure (e.g., neurobehavioral), effects on biomonitors of exposure (notably ChE activity), and the effectiveness of therapeutic countermeasures. Repeated exposure studies are designed to determine the potential for both cumulative and long-term anti-ChE and general toxic effects by sequential repetitive exposures; thus, the degree and sophistication of monitoring are usually greater than for acute studies. Overall, the design and conduct of studies by exposure via the inhalation route require many factors to be taken into account, principal of which are the following: 1. The reason for conducting the study, which is often related to the potential end use of the anti-ChE (e.g., for pesticides, a determination of all potential occupational hazards, factors influencing toxicity, safe working conditions, protective and precautionary measures, and therapeutic measures; for chemical warfare purposes, an assessment of the lethal potential of the anti-ChE and thus its threat capability and, secondary to this, considerations of protective and antidotal measures). 2. The method for generating the required atmospheric dispersion of anti-ChE (e.g., as vapor or aerosol). Highly volatile and/or reactive liquids may have to be metered from stainless-steel cylinders (Doddet al., 1986). For generation of liquid aerosols of respirable size, the Laskin nebulizer is suitable for one-component test materials (Drew et al., 1978); and for generation of dust particles, the Wright dust feeder is commonly used. There are several reviews that discuss in detail the generation of test atmospheres (Hext, 1999; Hinds, 1982; Liu, 1976; Miller et al., 1988; Phalen, 1996; Snellings and Dodd, 1990). 3. The method(s) for analyzing the exposure atmosphere. Sampling atmospheres from the exposure system can be conducted continuously or sequentially and periodically for measurement of test material concentration, and
4.
5.
6.
7.
with aerosols for measurement of particle size. The concentration of anti-ChE in the exposure atmosphere is usually measured by chemical analysis of samples for vaporsmtypically by gas chromatography, highperformance liquid chromatography, gas chromatographymass spectrometry, and infrared gas analysis. For liquid aerosols or dusts, gravimetric analysis is often used. The particle size distribution in liquid aerosols and dusts can be determined by various methods, including sedimentation, impaction, microscopy, and velocimetry. Details are found in Hext (1999), Hidy (1984), Hinds (1982), Rabbe et al. (1988), and Vincent (1995). It needs to be decided whether exposures should be by whole body or by a nose-only technique. Many OPs may have significant percutaneous absorption during a whole body exposure that could lead to complications in the interpretation of the findings of the study. Also, there may be absorption of anti-ChE through the gastrointestinal tract following preening. Exposure systems and chamber construction and design/operation have been considered in detail in several reviews (Hext, 1999; Leong, 1981; MacFarland, 1983). Monitoring for toxic and/or pharmacological effects. For acute studies, often a preliminary to repeated exposure investigations, monitors for anti-ChEs are frequently limited to clinical signs, ChE measurements, gross pathology, and determination of a timed LCs0 value. For repeated exposure studies with anti-ChEs, more extensive and detailed monitoring is required and should include at least the following: clinical signs (including neurobehavioral), body weight, food/water consumption, hematology, blood chemistry (including sequential ChE measurements), urinalysis, ophthalmic examinations (see Chapter 31), and gross and microscopic pathology. Laboratory safety requirements. Escape of potent anti-ChE into the laboratory environment should be avoided by chamber design to prevent leaks into the atmosphere, including negative pressure differential with respect to the exterior. Protective clothing (including respiratory protective equipment), in the event of a leak or for handling residually contaminated animals, and appropriate first aid equipment and antidotes should be readily available. Local contamination of the environment should be prevented by appropriate effluent filter and stack design.
IV. R E S P I R A T O R Y T R A C T L O C A L PHARMACOLOGICAL EFFECTS Transepithelial penetration of the nasal, laryngeal, and tracheobronchial mucosa by inhaled anti-ChEs will cause an accumulation of ACh at the parasympathetic M-cholinergic
CHAPTER 2.9 9Inhalation Toxicity of Anti-ChEs neuroeffector junctions; this will result mainly in rhinorrhea, excessive tracheobronchial secretions, and bronchoconstriction, with the latter presenting clinically as a tightening sensation in the chest, wheezing, breathing, and dyspnea. The effects of inhaled ChE inhibitors (dichlorvos, fenamiphos, methamidophos, parathion, and propoxur) on bronchial tonus and ChE activity were studied in the rat by Pauluhn et al. (1987). It was found that bronchospasm to anti-ChEs by inhalation exposure in the absence of ACh provocation did not occur at toxicologically significant doses of the anti-ChEs. An increase in lung resistance was obtained only after ACh provocation. Plasma ChE activity was a more sensitive index of exposure than the provocation test. Inhalation exposure to OPs such as soman reduces the contraction of bronchial smooth muscle induced by cholinergic stimulation (Aas et al., 1987). Thus, a 45-min exposure to 8.51 mg m-3 soman inhibited AChE activity of the bronchial smooth muscle by 85% and reduced the contraction induced by ACh and carbachol by 70 and 80%, respectively (Aas et al., 1987). Anti-ChE activity is not shown in vitro by P=S phosphorothiates (e.g., parathion and malathion), but in vivo anti-ChE activity is the result of oxidative desulfuration of the molecules of the oxons (De Matteis, 1989); they are produced by microsomal mixed function oxidases, particularly in the liver but also to some extent in the lungs.
V. L O C A L R E S P I R A T O R Y T R A C T TOXICITY Local toxicity to the respiratory tract resulting from inhalation of anti-ChEs may be due in some cases to a direct effect of the inhaled material on the tract. For example, repeated inhalation exposure of rats to the carbamate benomyl [methyl-l(butylcarbamoyl)-2-benzimidazole carbamate] for 6 hr a day, 5 days a week resulted in olfactory epithelial degeneration by 45 days, with no observed adverse effect level of 10 ppm for males and 50 ppm for females (Warheit et al., 1989). However, when benomyl was fed to rats in the diet at up to 15,000 ppm for 32 days, no histopathological lesions were seen in the nasal epithelium, indicating that the olfactory degenerative changes were most likely the result of local respiratory toxicity and not a consequence of a systemic effect (Hurtt et al., 1993). In contrast, some respiratory toxic effects may be systemically mediated. For example, overdosage with physostigmine has resulted in pulmonary edema (Cumming et al., 1968). As noted previously, the anti-ChE activity of P = S phosphorothioates is due to their oxidative desulfurafion to oxons. The cytochrome P450-dependent monooxygenase system also metabolizes P--S phosphorothioates to produce reactive metabolites that may inactivate P450 (De Matties, 1989). In cells with a high level of monooxygenase activity, the reactive metabolites may cause hydropic degeneration
403
(Seawright et al., 1976). Although pulmonary cell injury has apparently not been reported after dosing with P = S phosphorothioates (Dinsdale, 1992), microsomes from the lungs of rats treated with these materials show a marked reduction of P450 activity (Verschoyle and Aldridge, 1987). The metabolism of these phosphorothioates by P450 is more marked in the lung than in the liver, resulting in a selective inhibition of pulmonary P450 activity, which may protect the lung from injury (Verschoyle and Dinsdale, 1989). Trialkyl phosphorothioates are potential contaminants formed during the synthesis and/or storage of P--S phosphorothioates, and they may exacerbate the anti-ChE activity of P--S phosphorothioates. Many trialkyl phosphorothioates can cause fatal lung injury at doses that induce only minor cholinergic effects. Studies on the acute oral toxicity of O,S,S-trimethyl phosphorothioate showed that it had an LDs0 of 26 mg kg -1, with deaths occurring 3 or 4 days after resolution of cholinergic effects, and it was associated with labored breathing and a two- or three-fold increase in lung weight (Umetsu et al., 1981; Verschoyle et al., 1980). Lethal oral doses of O,S,S-trimethyl and triethyl phosphorothioates were found to cause injury to type 1 pneumocytes, which was apparent within 24 hr and became more severe after 24 hr. This was followed by a proliferative response of the type 2 cells and an influx of monocytes and macrophages and the development of alveolar edema (Dinsdale et al., 1982; Verschoyle and Cabral, 1982). Early injury of type 1 pneumocytes has been confirmed by the detection of a depression in the accumulation of [3H]putrescine in lung slices from rats dosed with O,S,S-trimethyl phosphorothioate (Nemery et al., 1987). The independence of pulmonary toxicity and anti-ChE activity produced by trialkyl phosphorothioates is indicated by the fact that pulmonary toxicity decreases and anti-ChE activity increases as molecular weight increases (Ali and Fukuto, 1982). In addition to causing injury to alveolar type 1 pneumocytes, some trialkyl phosphorothioates also cause changes in the bronchiolar epithelium, notably to the Clara cells. There is initially (within a few hours) morphological distortion of the Clara cells, and after approximately 3 days they appear hypertrophied and decreased in number (Imamura et al., 1983). Rat Clara cells are more sensitive to O,O,S-trimethyl phosphorothioate than O,S,S-trimethyl phosphorothioate, but injury to Clara cells is probably not a critical factor in the delayed deaths caused by trialkyl phosphorothioates (Dinsdale and Verschoyle, 1988).
VI. S Y S T E M I C E F F E C T S O F I N H A L E D ANTI-ChEs Anti-ChEs may be absorbed from the respiratory tract into the systemic circulation and subsequently exert pharmacological and toxic effects after inhalation as vapor, dust, or
404
SECTION IV. O r g a n
Toxicity
aerosol. Absorption of inhaled material can occur through the nasal and tracheobronchial mucosa, pulmonary alveolar capillaries, and, to a limited extent, from the gastrointestinal tract following the swallowing of contaminated mucosa from the nasal passages or coughed from the lungs.
A. Factors Influencing the Respiratory Tract Absorption of Inhaled Anti-ChEs The principal factors determining the inhalation exposure dosage, and hence the degree of absorption of anti-ChEs from the respiratory tract, are physicochemical characteristics of the anti-ChE, inhaled concentration, duration of exposure, breathing characteristics (rate and volume), pulmonary blood flow, and the anatomy of the respiratory tract, including alveolar surface area. However, the nature of inhaled materials may influence the breathing pattern of the exposed test animals (e.g., with peripheral sensory irritant materials there may be a significant decrease in the breathing rate that will influence the respiratory minute volume and hence the absorbed dose of inhaled material) (Ballantyne, 1999, 2005). With inhaled anti-ChEs, the induced local bronchoconstriction may influence both the inhaled and the absorbed dose of the material. The extent to which the physicochemical properties of the inhaled material may affect the breathing pattern, and thus the inhaled and absorbed dose of material, can be measured during the actual exposure by the use of appropriate monitoring (Hext, 1999). Vapors or aerosols that are water soluble may be absorbed rapidly in the upper respiratory tract, and at low concentrations in the inspired air there may be complete absorption in these regions. With increasing concentration of inspired material, a gradient will develop between the nasal cavity and alveolus. With water-insoluble material, there may be penetration to the deeper lung regions. Also, the potential for some regions of the respiratory tract to metabolize inhaled materials (e.g., due to the presence of cytochrome P450 monooxygenase enzymes) may influence both the regional uptake of vapor and its toxicity; the metabolite may be locally toxic and/or systemically toxic if absorbed into the circulation (Dahl and Lewis, 1993; Hadley and Dahl, 1982, 1983). The concentration of anti-ChE in the inspired air is a determinant of a diffusion process that continues until equilibrium is attained. Vapor diffusion rate is mainly a function of molecular weight and solubility of inhaled material; absorption generally increases with a decrease in molecular weight (MW) and increase in (water) solubility (Table 1). Blood to alveolar air partition coefficients can be estimated from the fractional composition of water (-80%) and lipids (--0.5%) in blood (Dodd, 1992). As the blood-air partition coefficient increases, the rate of uptake of material into the bloodsteam increases, and correspondingly greater amounts of material are absorbed. In general, inhibition of metabolizing enzymes causes a higher blood concentration for a
given inspired anti-ChE concentration, and induction of metabolizing enzymes lowers blood concentration (Dahl et al., 1988). Species variations in the respiratory uptake and absorption of inhaled vapor occur and probably are mainly the result of differences in breathing rate (Chang et al., 1981), metabolism (Bond et al., 1986), and respiratory tract anatomy (Morris et al., 1986). Within limits, dosimetry modeling can be used for extrapolation of data from one species to another by taking into consideration chemical reactivity, diffusion, solubility, and physiological and regional anatomical factors (E E Miller et al., 1978; E J. Miller et al., 1985, 1989).
B. Systemic Effects of Absorbed Anti-ChEs The effects exerted systemically by OPs and CMs following their absorption by the respiratory tract may be descriptively classified as anti-ChE pharmacological and toxic effects and as toxicological effects generally unrelated to the anti-ChE activity of the absorbed material. They may be a result of activity of the absorbed material and/or following systemic metabolism, although (in contrast with the oral route) there is limited first-pass hepatic metabolism. On the functional respiratory complex, the anti-ChE effects will cause an initial stimulation followed by paralysis of the N-cholinergic innervated respiratory muscles and paralysis of the brain respiratory center, both of which will further impair breathing, which is already compromised by the local mediation of anti-ChE effects on the respiratory tract. Additionally, the rapid respiratory tract absorption of potent anti-ChEs, such as soman and DFP, may also exert systemic effects on the parasympathetic innervation of the bronchial smooth muscle and glandular tissues (Scimeca et al., 1985). Classical pharmacological effects may be seen systemically in other tissues following absorption of anti-ChEs by the inhalation route, including miosis, ciliary muscle spasm, hyperemia of the conjunctivae, nausea, vomiting, abdominal cramps, diarrhea, hyperhydrosis, bradycardia (followed by tachycardia), bradyarrhythmias, weakness, fatigue, fasciculations, muscle cramps, anxiety, restlessness, headache, tremor, confusion, and convulsions. Following absorption of neurotoxic anti-ChE OPs, there may be development of the intermediate syndrome and inhibition of neurotoxic esterase activity leading to classical delayed-onset demyelinating peripheral polyneuropathy (Bajgar, 2004; Husain et al., 1993). There is increasing interest in possible long-term and persistent central nervous system (CNS) effects following exposure to OPs, particularly neurobehavioral and psychiatric symptoms and signs (Bajgar, 2004a,b; Bajgar et al., 2004; Kamel et al., 2005; Kassa et al., 2001a,b, 2004a) that may be neurotoxic, possibly compounded by hypoxic encephalopathy (Newmark, 2004). Specific neurobehavioral effects of concern include hallucinations, mood change, disturbance of personality, memory loss, and cognitive dysfunction (Dahlgren et al., 2004; Kassa et al., 2002). Low concentrations of potent OP anti-ChEs may be
CHAPTER 29 9Inhalation Toxicity of Anti-ChEs associated with significant decreases in ChE activity in various regions of the CNS. For example, rats exposed to sarin vapor at concentrations of 1.25 and 2.5 p,g liter -1 for 60 min showed significant decreases in erythrocyte ACHE, plasma butyrylcholinesterase (BuCHE), and frontal cortex and pontomedullary ACHE activities. There was a linear relationship between plasma sarin concentration and erythrocyte and frontal cortex ACHE activities and an exponential relationship between plasma satin and BuChE activity in plasma and pontomedullary regions (Sevelova et al., 2004). Neuropsychological evaluation is essential in the clinical evaluation and follow-up of people exposed to anti-CHE agents, notably to OPs. The lethal inhalation toxicity of anti-ChEs is usually due to a combination of local and systemically mediated cholinergic effects on the respiratory tract, systemic cholinergic effects on the respiratory muscles, effects on the CNS regulation of breathing, and other systemically mediated cholinergic adverse effects. For any given anti'ChE agent, there may be significant species differences in susceptibility to the lethal inhalation toxicity of anti-ChEs. This is shown for DFP vapor in Table 3, with the monkey being the most sensitive species. For more lethal OPs, the range of relative variability in toxicity [expressed as L(CT)50)] is smaller
TABLE 3. Inhalation Lethal Toxicity of DFP Vapor to Several Species a Species
L(CT)50 (mg min m -3)
Mouse R~ Guinea pig Rabbit Dog Monkey
5900 2800 8000 8000 5000 800
aData fromDuBois (1963). TABLE 4.
than for less lethal OPs, as demonstrated in Table 4, which shows that the phosphorofluoridates are less toxic and have a wider species variability in lethal toxicity than the cyanidates and phosphonofluoridates. The higher toxicity to all species is probably mainly a result of both their greater inherent potency as anti-ChEs and their less effective rate and extent of detoxification (DuBois, 1963). The alkyl pyrophosphates, such as TEPP, have the ability to inhibit ChEs in vivo and in vitro and to produce central and peripheral cholinomimetic effects. However, pyrophosphoric acid derivatives in which amide groups replace the alkoxy links [e.g., octamethyl pyrophosphortetramide (OMPA)] differ functionally from the alkyl pyrophosphates. Thus, OMPA produces peripheral M-cholinergic and skeletal muscle N-cholinergic effects typical of OPs but does not have actions on the CNS. Also, although small doses of OMPA given in vivo (5 mg kg -1, ip) produce inhibition of ChE activity (with the exception of brain ChE), OMPA concentrations >0.01 M are required to cause 50% inhibition of ChE activity in vitro. This indicates the need for metabolic activation of OMPA for effective anti-ChE activity, and several studies have shown the conversion of OMPA to an active anti-ChE metabolite (Aldridge and Barnes, 1952; Casida, 1956; Casida et al., 1956; Davison, 1955; DuBois, 1963; Tsuyuki et al., 1955). The requirement for metabolic activation of OMPA explains the difference between its anti-ChE activity in vitro and in vivo and the latency to onset of cholinergic signs in vivo. For comparison, the antiChE actions of TEPP and OMPA are respectively as follows: ip LDs0 in rats, 0.65 and 8.5 mg kg-1; and I50 for in vitro ChE inhibition, 4 • 10 -9 and 1 • 10 -2 M (DuBois, 1963). Systemic noncholinergic toxicity that may be encountered clinically or experimentally includes the following: 1. Altered immune function (Kassa et al., 2003, 2004b,c,d). This occurs at cholinergic symptomatic or nonsymptomatic inhalation exposure doses, and it includes a decrease in CD3 cells, an increase in CD19 cells,
Lethal Inhalation Toxicity of Several OP anti-ChEs to Mice, Monkeys, and Rats by 10-min Exposures to the Vapor a 10-min L(CT)50 (mg min m -3)
OP Ethyl-N-dimethylphosphoroamidocyanidate (tabun) Isopropyl methylphosphonofluoridate (satin) Isopropylethyl phosphonofluoridate Dimethyl fluorophosphonate Diethyl fluorophosphonate DFP aData from DuBois (1963). bNA, not available.
405
Mouse
Monkey
Rat
380 250 330 2600 8200 59OO
250 150 200 NAb NA 8OO
300 300 260 4000 10,500 28OO
406
2. 3. 4. 5.
SECTION I V -
Organ Toxicity
decreased CD4 T lymphocytes, increased phagocytosis by peritoneal macrophages, and lymph proliferation. Nephrotoxicity involving glomerular and tubular damage (Mohssn, 2001; Wedin, 1992). Myonecrosis (Ballantyne and Marrs, 1992; Futagami et al., 2001). Cardiovascular toxicity (Ballantyne and Marrs, 1992; Karki et al., 2004). Hepatotoxicity (Seawright et al., 1976).
VII. C O M B U S T I O N T O X I C O L O G Y OF ANTI-ChEs Fires involving materials treated with anti-ChEs or bulk pesticides stores (CM and OP) may release both the unaltered anti-ChE and thermal decomposition products. Also, certain phosphorus-based fire retardants that are subjected to heat and flame in a fire may result in the generation of anti-ChEs and other toxic materials. Thus, although fire retardants may slow the rate of fire progression, their involvement in a conflagration may result in an increase in the toxic potency of combustion products (Purser, 1992). For example, polyurethane foams treated with a trimethylol propane polyol base containing phosphorus-based retardants formed a highly neurotoxic combustion product [trimethylolpropane phosphate (TMPP); Petajan et al., 1975]. According to Smith and Ledbetter (1971), OPs are unstable when heated and decompose, and it is thus unlikely that substantial decomposition would occur in a fire, although some evaporation may take 'place and be a source for potential OP exposure. In a simple experiment, and probably unrealistic from a practical standpoint, solutions of malathion in xylene and kerosene (0.1 g ml-l) were burned. The maximum malathion concentrations in the gaseous products were 10 lxg m -3 for xylene and 4 Ixg m -3 for kerosene. Decomposition products included dimethyl fumarate, malathion isomers, and isomers of dimethyl phosphorodithioate. From this and other work, it was concluded that for solvents with boiling points below the pesticide decomposition temperature, the pesticide yield increased with the boiling point of the solvent, whereas for other solvents the yield decreased with boiling point since the rate of decomposition increased with temperature faster than the vapor pressure. They found that under the conditions of the experiment, 90-99% of malathion and 85-98% of parathion decomposed before evaporation and that this would substantially reduce the amount released during actual fires (Purser, 1992). CM residues are unlikely to occur in fires because the majority are solids with low vapor pressures and are unlikely to be readily volatilized. Also, the carbamic ester linkage is probably thermolabile, and without this there will be no anti-ChE activity (Murphy, 1986). Some OP flame retardants may be directly neurotoxic; a typical example is tri-o-cresyl phosphate (Abou-Donia,
1981). Lhomme et al. (1984) investigated the effects of pyrolytic and oxidative thermal decomposition of trimethyl, triethyl, and triphenyl phosphates. The trialkyl phosphates were thermolabile with scission at the C-O bond at 200-300 ~ yielding phosphorus pentoxide and various aliphatic products, mainly methane and ethane, under pyrolytic conditions and CO with traces of aldehydes under oxidation. Triphenyl phosphate was more thermally stable, decomposing only above 600 ~ with scission of both P-O and C-O bonds. All phosphorus was recovered as phosphoric acid, resulting from hydrolysis of the phosphorus pentoxide. This work indicates that phosphate esters are readily thermally destroyed to yield inorganic phosphorus oxides and acid. The major inhalation hazard would be anticipated to be from phosphorus pentoxide, a pulmonary irritant with a 1-hr LCs0 of 1.22 mg m -3 (Ballantyne, 1981,1988). In the previous studies, the OPs were decomposed alone, but when fire retardants are added to materials the inorganic phosphate may be released to combine with other chemicals such as alcohols in the solid or vapor phase to form new phosphate esters that may survive in the cooling smoke or char (Purser, 1992). An example in the solid phase is intumescent coatings containing ammonium polyphosphate and pentaerythritol. On heating, ammonia and water are evolved with the formation at 250 ~ of a bicyclic phosphate. This material may be neurotoxic, as is the caged bicyclic phosphate ester TMPP (Petajan et al., 1975). However, when rats were exposed to the thermal decomposition products of lubricants containing pentaerythritol and tricresyl phosphate, no neurotoxic signs were seen (Wyman et al., 1987). The previous study indicates that OP esters can be formed during the thermal decomposition of materials treated with phosphorus-based retardants. Phosphine, a potent lung irritant and cause of pulmonary edema, has also been identified in the thermal decomposition products from fire retardant materials (Stevenson and Guest, 1987). Fire retardants used as additives to polyurethane foams usually give a greater yield of common toxic products, often with more unusual molecules. This is illustrated by a typical comparison of test atmospheres from flaming samples (600 ~ of untreated and fire-retarded thermoplastic polyurethane foam (Purser, 1992). The untreated sample burned cleanly, producing relatively little CO (350 ppm), HCN (11 ppm), and smoke (0.07 OD m-3), and it was slightly irritant (RDs0 = 4.0mg liter-l); in contrast, the fire-retarded sample had intermittent flaming, produced significantly higher CO (4200 ppm), HCN (77 ppm), and smoke (1.23 OD m-3), and was numerically 20 times more potent as a peripheral sensory irritant (RDs0 = 0.2 mg liter-I). Other studies indicate that under nonflaming or early flaming conditions, the toxic potency of fire-retarded polyurethane foams can be greater than that of nonretarded materials as a result of increased yields of toxic products such as CO, HCN, and isocyanates (Braun et al., 1987;
CHAPTER 29 9Inhalation Toxicity of Anti-ChEs
Purser, 1992). Other studies have shown that fire-retarded polyesters and cottons have similar combustion characteristics as those for fire-retarded polyurethane foams, indicating that fire retardant treatment of these fabrics also reduces combustion efficiency and gives increased yields of toxic products (Braun and Levin, 1986; Kallonen et al., 1985). There has been much interest in the possibility of the formation of a highly potent neurotoxic caged bicyclophosphoms ester during the combustion of materials treated with phosphorus-containing fire retardants. This was first reported by Petajan et al. (1975), and the substance was identified as TMPP (4-ethyl-l-phopha-2,6,7-trioxabicyclo(2.2.2)octane-l-oxide). It was discovered when rats were exposed to thermal decomposition products from a rigid polyurethane foam consisting of an isocyanate and a longchain polyol that react to form urethane groups of the polymer. The rigid foam was formulated from a propoxylated trimethylolpropane polyol (MW 340) and polymethylene polyphenyl isocyanate. It was tested untreated and with the addition of O,O-diethyl N,N-bis(2-hyroxymethyl)aminomethylphosphonate. Exposure to the untreated foam had no effect, but rats exposed to the foam containing 4% retardant developed focal seizures immediately postexposure that developed to grand mal seizures after 4 3 - 7 0 imn . With 8% retardant, rats developed myoclonic jerks that proceeded to status epilepticus and death. Thermal decomposition of the foam released the propoxylated trimethylolpropane polyol adduct that decomposed to form trimethylolpropane, which combined with reactive phosphorus species from the retardant in the smoke to form mainly TMPP. In addition to TMPP, which has a 4-ethyl group in the molecule, the 4-methyl homologue was also detected during the combustion of polyurethane foams. TMPP is a member of a series of highly neurotoxic molecules, the bicyclophosphorus esters, whose effects were first recorded by Gage (1970). Their basic mechanism of action is by antagonism of ~/-aminobutyric acid (GABA) by allosteric binding to GABA receptors. Thus, the convulsant action of the bicyclophosphorus esters is by blocking the neuroinhibitory effect of GABA rather than from excess ACh activity by an inhibition of synaptic ACHE. As a result, the convulsive activity is not accompanied by signs of parasympathetic overstimulation or by N-cholinergic-mediated skeletal muscle paralysis. In rats, barbiturates are antidotal at 25-50 mg kg-1 ip after oral convulsive doses of bicyclophosphorus esters (Kimmerle et al., 1986). The influence of stearic effects on the acute toxicity of bicyclophosphate esters is shown in Table 5 for different 4-alkyl side groups (Bellet and Casida, 1973), and the acute inhalation toxicities for aerosols of trimethyl propane phosphate and phosphite are shown in Table 6 (Kimmerle et al., 1986). Woolley and Fardell (1976) investigated the liberation of TMPP during thermal decomposition of flexible and rigid polyurethane foams. At 500 ~ under nitrogen TMPE yields were negligible for flexible
407
TABLE 5. lntraperitoneal LD50Values for Tricyclophosphate Esters Having Different 4-Alkyl Side Groups a
4-Alkyl side group CH3 C2H5 n-C3H4 Iso-C3H4 C4H9 HOCH2
LD50 (mg kg -1) 32.0 1.0 0.38 0.18 1.5 >500.0
aData from Bellet and Casida (1973).
TABLE 6. Acute Lethal Inhalation Toxicity of Aerosols of Trimethyl Propane Phosphate and Trimethyl Propane Phosphite to Male and Female Rats
1-hr LCs0 (mg liter -1) with 95 % confidence limits Gender
Phosphate
Phosphite
Male Female
0.037 (0.033-0.040) 0.030 (0.027-0.034)
0.015 (0.013-0.017) 0.015 (0.013-0.017)
aData from Kimmerleet al. (1986).
polyurethane foams and some rigid polyurethane foams, but other rigid foams containing trimethylol propane polyols gave significant yields. The maximum yield was 0.2% by mass when the foam was decomposed at 500 ~ under nonflaming conditions in air. It was calculated that the equivalent concentration in air would be 0.12-0.06 mg liter-1. The 1-hr LCs0 for TMPP has been experimentally determined to be 0.03-0.07 mg liter -1 (Kimmerle et al., 1986). It was concluded that along with other toxic materials, such as CO and HCN, in the atmosphere, the TMPP yield could make major contributions to the combustion toxicology of polyurethane foams (Purser, 1992).
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4 10
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IV 9 O r g a n T o x i c i t y
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CHAPTER ~ 0
Dermal Absorption/Toxicity of Organophosphates and Carbamates JIM E. RIVIERE North Carolina State University, Raleigh, North Carolina
provide the only available human data with which to perform dermal absorption assessment. Dermal toxicity of pesticides is thus usually synonymous with dermal absorption since the critical adverse effect is classical OP systemic neurotoxicity (e.g., cholinesterase inhibition). The major mechanism of direct pesticide toxicity to skin is generally related to immunological mechanisms, either directly with skin immune components after topical absorption or indirectly after systemic administratio n .
I. INTRODUCTION Topical exposure to organophosphate (OP) and carbamate (CM) pesticides remains an important route for exposure to humans and animals. As in many subdisciplines in toxicology and other sciences, interpretation of pesticide dermal absorption data is highly dependent on the model systems used to generate the data. This chapter discusses studies of pesticide absorption in the context of the experimental and theoretical approaches used to assess absorption. This field was reviewed by Baynes and Riviere (2001), which should be consulted for further details. This review highlighted the overarching importance of experimental design and subsequent interpretation of pesticide data reported in the literature since these factors often overshadow differences in absorption and subsequent toxicity between individual pesticides. Topical pesticide exposure remains an important health issue. Epidemics of pesticide poisoning following cutaneous exposure have also been reported for nonoccupational uses (Ferrer and Cabral, 1993). These cases often involved accidental contamination of infant clothing or talcum powder with pesticides (Martin-Bouyer et al., 1983). Large-scale residential exposure to methyl parathion with resulting OP-induced toxicity occurred in 1994 and 1996 in Ohio, Illinois, and Mississippi (Rubin et al., 2002). Dermal exposure may occur secondary to processing of agricultural products such as tobacco (Lonsway et al., 1997). Fatal human pesticide exposures continue to occur (Rosenthal, 2003). Dermal absorption is the probable route of entry in 65-85% of all cases of occupational exposure to pesticides (Galli and Marinovich, 1987). Similarly, spray or dusting of pesticides can result in direct deposition of 20-1700 times the amount deposited in the respiratory tract after inhalational exposure, a loading that would potentiate absorption and direct irritation to the skin. Anecdotal case reports, coupled with dermal exposure estimated from various direct and indirect dosimetric experiments in outbreaks, often Toxicology of Organophosphate and Carbamate Compounds
II. M E C H A N I S M OF ABSORPTION The skin is composed of two primary layers--the epidermis, which includes the outermost stratum corneum barrier and underlying viable keratinocytes, and the dermis (MonteiroRiviere, 1991). Skin is relatively impermeable to most aqueous solutions and ions; however, it may be permeable in varying degrees to a large number of more lipophilic drugs or xenobiotics such as pesticides. The stratum corneum cell layer in humans (10-50 lxm) and pigs (15 ~zm) is nonviable and is considered the rate-limiting barrier in percutaneous absorption of many drugs and pesticides (Monteiro-Riviere, 1991). It is axiomatic that a topically applied chemical must first traverse the stratum corneum barrier before it is capable of eliciting any toxicological or immunological effect on subsequent cell layers, making absorption the primary factor in assessing the dermal effects of pesticides. Chemical absorption pathways can hypothetically involve both intercellular and intracellular passive diffusion across the epidermis and dermis and/or transappendageal routes via hair follicles and sweat pores. Transappendageal pathways are considered to contribute very little to the dermal transport of most drugs compared to transport across the epidermis (Barry, 1991). Most available research has concentrated on the stratum corneum as the primary barrier to absorption, although the viable epidermis (80 txm 411
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in humans and 60 txm in pigs) and dermis (3-5 mm in humans) may also contribute significantly to the percutaneous penetration of specific chemical classes, for example, when the true barrier to absorption is not diffusional but, rather, metabolic. The term barrier is thus often used to denote either a physical structure (e.g., stratum corneum) or a biological process (e.g., diffusional resistance, metabolism, and vascular uptake) that retards absorption of topically applied chemicals. The accepted hypothesis for dermal absorption is that the dominant pathway for chemicals, including pesticides, to traverse the stratum corneum is through the intercellular lipids. Lipophilic compounds diffuse through this lipid milieu while polar molecules traverse the aqueous region of the intercellular lipids. This intercellular region, described as the mortar in the "brick and mortar" model of the stratum corneum (Elias, 1981), is considered the most likely path for absorption of lipophilic drugs. Although this model is conceptually simple, the actual physical chemical environment of the intercellular lipids is complex. It is filled with neutral lipids (complex hydrocarbons, free sterols, sterol esters, free fatty acids, and triglycerides) that make up 75% of the total lipids, as well as other polar lipids (Magee, 1991; Monteiro-Riviere et al., 2001). These intercellular lipids are also inextricably linked to the outer cellular membranes of the corneocytes, making a relatively complex and fluid structure that is often modeled as a simple homogeneous lipid pathway. Successive tape stripping, delipidization techniques, and use of epidermis from heat or chemical separation techniques have been used by investigators to demonstrate the dominant influence that the stratum corneum and the lipid domain holds on penetration of hydrophilic and lipophilic chemicals. Percutaneous absorption through the intercellular pathway of the stratum corneum is driven by passive diffusion down a concentration gradient described at steady state by Fick's law of diffusion (Roberts et al., 1999; Wester and Maibach, 1983; Riviere, 1999); Flux = [(D. PC. SA)/H] ( ~ ) where D is the diffusion coefficient, PC is the partition coefficient, SA is the applied surface area, H is membrane thickness (or, more precisely, the intercellular path length), and zXx is the concentration gradient across the membrane. Since in vivo blood or in vitro perfusate concentrations after absorption are negligible compared to applied surface concentration, 2tx reduces to the concentration (C). It is this relationship that allows the prediction of compound flux across the skin to be correlated to factors predictive of D and PC (e.g., octanol/water partition coefficients). Flux is expressed in terms of applied surface area, often normalized to cm 2. The t e r m ( D . PC//-/) is compound dependent and is termed the permeability coefficient (Kp), reducing the
determination of flux to Kp 20( or Kp. C, a first-order pharmacokinetic equation (dx/dt = kX). Rearrangement of this equation yields the primary method used to experimentally determine Kp: Kp = steady state flux/concentration It must be stressed that both transdermal flux and Kp are not only chemical dependent but also tightly constrained by the membrane system studied as well as the method of topical application (neat compound, vehicle, length of experiment, etc.). The PC that is integral to Kp is the PC between the surface or applied vehicle and the stratum corneum. Different vehicles will thus result in different PCs. Similarly, skin from different species may result in different PCs due to differences in the stratum corneum lipids and intercellular path lengths. Since passive diffusion is the primary driving force behind dermal absorption, physicochemical factors such as molecular weight and structure, lipophilicity, pKa, ionization, solubility, partition coefficients, and diffusivity can influence the dermal absorption of various classes of pesticides. In addition, penetration of acidic and basic pesticides will be influenced by the skin surface, which is weakly acidic (pH ~ 5), since only the uncharged moiety of weak acids and bases is capable of diffusing through the lipid pathway. Several of these factors (e.g., molecular weight and PCs) have been used to predict absorption of various drug classes (Potts and Guy, 1992; Cleek and Bunge, 1993; Bunge and Cleek, 1995). This approach has not been widely applied to pesticides. The first such relationship widely used to assess chemical absorption is that of Potts and Guy (1992): log
kp =
0.71 log
PCoctanol/wate r -
0.0061 MW - 6.3
(R 2 = 0.67) where MW is the molecular weight. This equation was subsequently modified (Potts and Guy, 1995) to relate kp to molecular properties of the penetrants as log
kp -
0.0256 MV - 1.72 ZoLH2 -- 3.93 Z~H2 -- 4.85
(R 2 = 0.94) where MV is molecular volume, ~ OLH2 is the hydrogen bond donor acidity, and ~ H 2 is the hydrogen bond acceptor basicity. The most promising approach is to further extend this rationale using linear free energy relationships (LFERs) to relate permeability to the physical properties of the penetrant under defined experimental conditions (dose, membrane selection, and vehicle). Geinoz et al. (2004) critically reviewed most such quantitative structure permeability
CHAPTER 30 9Dermal Toxicity of OPs and CMs relationships (QSPeR) applied to dermal absorption. Abraham's LFER model is representative of the dermal QSPeR approaches currently available (Abraham and Martins, 2004). This model was selected since it is broadly accepted by the scientific community as being descriptive of the key molecular/physiochemical parameters relevant to solute absorption across skin. This basic model can be written as log kp = c + a~aH2 + b~13H2 + savH2 + rR2 + vV~ where 'rrH2 is the dipolarity/polarizability, R 2 represents the excess molar refractivity, Vx is the McGowan volume, and the other parameters are as described previously. The variables c, a, b, s, r, and v are strength coefficients coupling the molecular descriptors to skin permeability in the specific experimental system studied. In order to incorporate mixture effects, our laboratory has been exploring the use of an additional term operationally called the mixture factor (MF), yielding log kp : c + mMF + a~]aH2 + bE[3H2 + s~rH2+ rR2 + vV~ The nature of the MF is determined by examining the residual plot (actual - predicted log kp) generated from the base LFER equation based on molecular descriptors of the permeants against a function of the physical chemical properties of the mixture/solvents in which they were dosed (Riviere, 2006; Riviere and Brooks, 2005). The literature on QSPeR is exhaustive and rapidly growing. The limitation of applying these approaches to pesticide absorption is the lack of large and comparable databases of pesticide dermal absorption, as well as the lack of availability of molecular descriptors for many pesticides. As discussed later, data suitable for large-scale analyses must be rigorously controlled relative to the species studied, the nature of the experiments (in vitro vs in vivo), dose, surface area, vehicle, and method of sample collection and analyses. When dermal absorption data are not analyzed using the models described previously, data are often expressed as percentage dose absorbed. This is conceptually correct if one assumes that permeability is unchanged across dose because it represents a first-order pharmacokinetic process. It is also appropriate when comparing experimental treatments (e.g., temperature and vehicle) using the same applied dose. However, in many cases, topical dosing results in applying substantial amounts of chemical compared to what can be absorbed across the skin. In soils, thick layering where most soil is not in contact with skin, and thus not able to reach its surface to partition into, suggests that most of an applied dose is not actually available for absorption. In such studies, only a monolayer of soil is actually in contact with the skin. Unlike in fluid or gel matrices, compound generally does not diffuse from soil
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layers not in contact with the skin, unless water is added as another vehicle. In these cases, accounting for the applied dose as total dose in a multilayer system overestimates the available dose that artificially reduces the calculated percentage dose absorbed. Caking of heavy dermal formulations results in a similar layering phenomenon. Similarly, dose may bind to the application device and not be available. In these cases, a large fraction of dose may not be thermodynamically driving the diffusion process. Finally, when the dose is applied in solution, saturation may result in precipitation of chemical. Rapid evaporation of a volatile vehicle may also precipitate pesticide, decreasing its availability for absorption. All of these factors lead to a phenomenon often seen in dermal absorption studies in which percentage dose absorbed decreases with applied dose. Conducting a study at high applied doses may underestimate absorption of lower applied doses and vice versa. Thus, comparing percentage dose absorbed across pesticides exposed at different doses often provides more information on applied dose than the nature of the individual pesticide absorption. Unfortunately, this phenomenon is repeatedly encountered in the pesticide examples discussed later and limits the value of information gained on individual pesticide behavior.
III. EXPERIMENTAL D E R M A L ABSORPTION MODELS Assessment of dermal absorption for any topically applied chemical, including pesticides, can be classified based either on a model's level of biological complexity (in silico, in vitro, and in vivo) or on the specific species studied (human, laboratory rodent, monkey, and pig). The goal of the research must also be taken into consideration: Is the work being conducted to study the mechanism of absorption (e.g., identify a specific mathematical model or assess the effect of a vehicle) or to quantitatively predict absorption in humans? Another perspective is whether the study is designed to examine a local effect in skin versus a systemic effect after absorption; in the first case, skin concentrations are important, whereas in the later case flux of chemical across skin is needed. These end points determine both the level of model used and the appropriate species to employ. Model systems and approaches in use today to assess dermal absorption have been reviewed (Riviere, 2005). The primary approach to assess dermal absorption is the in vitro diffusion cell. In this model, skin sections (full thickness, dermatomed to a specific thickness) are placed in a two-chambered diffusion cell in which receptor fluid is placed in a reservoir (static cells) or perfused through a receiving chamber (flow-through cells) to simulate cutaneous blood flow. Chemical may either be dosed under ambient conditions neat or dissolved in a vehicle (Franz and Bronaugh cells) or in water (side-by-side diffusion
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cells), resulting in finite versus infinite dosing conditions, respectively. Selection of the receptor fluid (e.g., saline and albumin-based media) is also critical since absorption is only detected if the penetrating compound is soluble in the receptor fluid. This is particularly important for many lipophilic pesticides, making studies ignoring this factor difficult to interpret. Many studies of pharmaceutical compounds use saline as the receptor fluid due to the hydrophilic nature of many drugs, a choice that falsely suggests minimal absorption for lipophilic pesticides since they are not soluble in the receptor fluid and thus cannot be detected as absorbed. Steady-state flux is measured in these models and permeability calculated using the previously discussed relationship. In addition to perfusate composition, the temperature of perfusate is also controlled, with pharmaceutical investigators suggesting that studies be conducted at 35~ to mimic the surface temperature of skin. These techniques have been exhaustively reviewed (Riviere, 2005; Bronaugh and Stewart, 1984, 1985). The second major approach used to assess dermal absorption is in vivo. Many pesticide absorption studies referenced in the literature are full mass balance studies in which compound is dosed on the surface of an animal and total excreta (urine, feces, and expired air) are collected and analyzed for parent pesticide or metabolites. Radiolabeled compounds are often employed in these studies. These data are usually expressed as percentage dose absorbed per unit of surface area exposed. This method is best adapted to laboratory rodent models. Dose may be applied occluded (evaporation of dose prevented) or nonoccluded (dose site open to ambient environment). In calculating absorbed dose, all chemical at the dose site must be segregated from other tissues that would reflect absorbed chemical. This usually involves gently washing nonabsorbed chemical with a soapy solution. When larger animals (e.g., pigs and primates) or humans are studied and total mass balance is not possible (e.g., cannot collect feces and expired air), the fraction of a systemically absorbed compound excreted in the urine must first be determined using parenteral dosing. In some classic studies, this parenteral route correction factor was conducted in monkeys (Feldmann and Maibach, 1974) under the assumption that systemic distribution, metabolism, and elimination of these pesticides are similar in man and primate. In pigs, separate parenteral injections have been made to determine fractional excretion by other routes (Carver and Riviere, 1989). Finally, for many pharmaceutical compounds, absorption may be assessed by determining the area under the curve of the plasma concentration-time profile, much as it is for determining bioavailability from oral routes of administration. There are several perfused skin preparations with an intact functional microvasculature. The perfused rabbit ear model, perfused pig ear model, in situ sandwich skin flap in athymic rats, and the hybrid rat-human sandwich flap have been developed (Pershing and Krueger, 1987), but each
intuitively has severe limitations. The isolated perfused porcine skin flap (IPPSF) is a unique ex vivo skin preparation developed in our laboratory that has many advantages over other ex vivo models and most in vitro systems. The major advantage of such a perfused system is that subsequent systemic influences on absorbed chemical are not present, but the tissue is fully functional with an intact microcirculation, unlike simpler in vitro models. In addition to having an intact functional cutaneous microcirculation, predictions from IPPSF studies have correlated well with in vivo absorption data for several drugs and insecticides (Riviere et al., 1986, 1995; Wester et al., 1998). IPPSFs are physiologically and biochemically viable and therefore can be used to assess cutaneous toxicity of topically applied chemicals (Monteiro-Riviere, 1993). The latter is most important because cutaneous toxicity as well as dermal absorption of various pesticide formulations can be assessed simultaneously. For some pesticides, in vivo and in vitro data are comparable. An excellent example is the dermal absorption of malathion (6.8% dose at 24 hr and 8.2% dose at 5 days) in human volunteers (Feldmann and Maibach, 1974; Maibach and Feldmann, 1974), which is comparable to in vitro absorption (8.77% dose at 24 hr) in human skin (Wester et al., 1996), in vivo absorption of malathion in Yorkshire pigs (5.2% at 6 days) (Carver and Riviere, 1989), as well as predicted IPPSF absorption (5.9%) (Williams et al., 1990). Recent approaches have concentrated on developing noninvasive methods for assessing exposure. These include using postdosing stratum corneum tape stripping (Rougier et al., 1985), in which after calibration to the classical in vivo models just presented, estimates of absorption or exposure can be obtained by searching for the test compound in tape strips obtained after washing nonabsorbed chemical from skin. This is also used by some workers to document whether exposure has occurred in a field situation (Nylander-French, 2000). The mechanistic basis that allows for this to be used is that a chemical diffusing through the skin must first partition into the stratum corneum. At equilibrium (approximately 30 min), the concentration in the stratum corneum tape strips is the driving concentration that determines how much absorption will occur. The assumption is made that chemical absorbed into the stratum corneum may be absorbed into the systemic circulation. The alternate noninvasive approach is to assay collected urine for parent pesticide or metabolites (e.g., p-nitrophenol for parathion) that would be indicative of systemic absorption. If timed urine collections are not employed, the observed values are often corrected by assaying for creatinine to compensate for altered urine volumes. There is general consensus that for most risk assessment needs, in vivo models are preferred since they most closely match the exposure scenario to be modeled. However, in vivo models are also the most variable due to the myriad
CHAPTER 30 9Dermal Toxicity of OPs and CMs points of potential interactions in the absorption process. Thus, studies designed to examine the mechanism of pesticide absorption tend to use in vitro models to reduce the level of biological complexity so that specific mechanistic hypotheses can be evaluated. However, it is often not clear if these findings are extrapolatable to in vivo exposures.
IV. SPECIES DIFFERENCES There is an important phenomenological component to selecting animal models that is related to what the studies are trying to predict: toxicity or a pharmacological effect. Absorption studies designed to assess systemic toxicity after topical application generally attempt to overestimate absorption, whereas those trying to predict pharmacological effect in humans tend to try to use a model with a closer correlation to transdermal flux in humans. If the goal is to compare relative absorption by rank order of permeability, then the use of specific models may not be as crucial as long as absorption is representative across species. As long as the same model system is used, this generally also holds for comparing specific treatment effects (e.g., vehicle and occlusion) on dermal absorption. A good example related to pesticides is the comparative absorption study conducted on mice (Shah et al., 1981), in which absorption across a wide variety of compounds was greater than 90% of applied dose. In contrast, absorption of similar pesticides in humans or pigs was generally well below 25% (Riviere, 1996). Paraquat and diquat are two compounds that provide insight into interspecies comparisons. They are hydrophilic pesticides with fixed charged cations and remain dissociated at all pH values. Very little paraquat or diquat would therefore be expected to be absorbed across the lipoidal stratum corneum barrier. However, percutaneous absorption of paraquat has resulted in systemic effects and deaths in humans (Smith, 1988). Studies have determined that the in vitro permeability constants for paraquat in various animal species (rat, hairless rat, nude rat, mouse, hairless mouse, rabbit, and guinea pig) are 40-1600 times greater than those in humans (Walker etal., 1983). Dermal absorption studies in human volunteers demonstrated approximately 0.25% absorption from multiple sites (Wester et al., 1984). One in vivo study on rats suggested that greater paraquat absorption (3.5%) was attributable to an occlusive dressing or due to differences in skin thickness between species (Chui et al., 1988). Like paraquat, very little diquat is absorbed (0.3%) in the human forearm in vivo (Maibach and Feldmann, 1974). Diquat absorption increased to 1.4% with occlusion and to 3.8% with damaged skin. Topical application of 3-, 24-, and 200-mg doses of paraquat to IPPSFs for 8 hr resulted in total penetration (skin deposition as well as absorption into perfusate) of 0.91, 1.09, and 0.50%, respectively (Srikrishna et al., 1992). These absorption data are comparable to the human in vivo data.
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Despite the limited amounts absorbed, they were sufficient to cause morphological and biochemical changes in the IPPSFs. This finding clearly indicates the importance of the phenomenological dilemma introduced earlier, in which although minimal systemic absorption and thus systemic toxicity would be expected to occur, sufficient paraquat was absorbed across the stratum corneum into the epidermis/ dermis to illicit a local dermatotoxic effect. The potential for local toxicological effects is present for topical dosing of these pesticides. The use of laboratory animal models may lead to overprediction of pesticide absorption in human skin, as shown for paraquat. Differences in permeability properties between human and laboratory animal skin can account for this overestimation. Inherent structural differences in skin biology, such as skin thickness and sebaceous secretions, make species-species extrapolation of dermal absorption data tenuous. Although the fundamental architecture of terrestrial mammalian skin is similar, well-documented differences in epidermal and dermal anatomy and physiology exist (Monteiro-Riviere, 1991; Monteiro-Riviere et al., 1990). It is plausible that a high density of hair follicles attenuates the thickness of the interfollicular epidermis, which may promote absorption. Alternatively, species differences in the size or width of epidermal cells would modulate path length across species, a critical parameter in the value of the permeability constant for a specific species. Species differences in stratum corneum lipid composition may be the overriding factor in determining the rate and extent of absorption. The skin of the domestic pig is functionally and structurally similar to that of humans (Monteiro-Riviere, 1991), supporting the observation that percutaneous absorption of toxicants through pig's skin mimics that through human skin. Studies have demonstrated that the range of percutaneous absorption of carbaryl, lindane, malathion, and parathion in pig skin in vivo (Carver and Riviere, 1989) or in vitro (Chang et al., 1994) is similar to that observed in humans (Feldmann and Maibach, 1974). This supports the close correlation of absorption across the IPPSF compared to in vivo humans previously discussed (Riviere et al., 1986, 1995; Wester et al., 1998). However, this was not the case when the permeability of hydrophilic chemicals (mannitol, water, and paraquat) and lipophilic chemicals (carbaryl, aldrin, and fluazifop-butyl) in pig ear skin was compared with human abdominal and rat dorsal skin (Dick and Scott, 1992). This study demonstrated that for hydrophilic chemicals, pig ear and rat skin overestimated permeability in human skin. It should be noted that pig ear skin is different from that of other body sites, being both thinner and closely associated with the underlying ear cartilage. Alternatively, confounding effects associated with using an in vivo model rather than the ex vivo IPPSF may weaken the inherent correlation between human and pig skin.
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Although permeability is generally higher in animal skin than in human skin for the lipophilic chemicals, permeability of carbaryl in human and pig skin was almost identical. Permeability of lipophilic chemicals in pig skin correlated better with data from human skin compared to permeability of hydrophilic chemicals. Bartek et al. (1972) demonstrated good agreement between human in vivo and pig in vivo dermal absorption data for lipophilic chemicals, such as butter yellow, DDT, haloprogin, lindane, and testosterone, and hydrophilic chemicals, such as caffeine, acetylcysteine, and cortisone. Further studies on nonhuman primates observed that lindane absorption from rhesus monkey forearm (18%) was approximately twice that for the ventral forearm in humans (9.3%) (Feldmann and Maibach, 1974) and for the ventral abdomen in pigs (7.7%) (Chang et al., 1994). Although all of these studies are taken into consideration for the majority of moderately lipophilic pesticides studied, the rank order of absorption is generally rabbits > rodents > weanling pig -- rhesus monkey -> human.
V. F A C T O R S A F F E C T I N G A B S O R P T I O N An additional variable that must be accounted for in dermal absorption studies that may overshadow the difference between chemicals is body site differences in absorption within a species. Regional variation in skin permeability at different body sites may be related to skin thickness, number of cell layers, cell size of the epidermis and stratum corneum, and distribution of hair follicles and sweat pores. Because of thick layers of stratum corneum, permeability in palmar and plantar skin is expected to be less than that in the scalp or forearm (Feldmann and Maibach, 1974). Data from several studies suggest that regional variation in vascular anatomy and blood flow should also be considered (Monteiro-Riviere et al., 1990; Qiao et al., 1993). Various studies have demonstrated regional variation in penetration of drugs and pesticides in pig skin (Qiao et al., 1993; Qiao and Riviere, 1995), rat skin (Bronaugh, 1985), rhesus monkey skin (Wester et al., 1980), and human skin (Wester et al., 1980, 1984), clearly demonstrating that regional variability is not limited to a single species. Qiao et al. (1993) demonstrated that parathion penetrated nonoccluded pig skin in the following order: back > shoulder > buttocks > abdomen; however, for occluded skin, the order was back > abdomen > buttocks > shoulder. This demonstrates the importance of experimental conditions for interpreting even carefully controlled comparative studies. Occlusion could mechanistically change the nature of both the partition coefficient and the diffusivity of a compound in skin, which could result in different mechanisms becoming rate limiting. Wester et al. (1994) demonstrated that pyrethrin absorption through human forearm was less than the predicted absorption in human scalp. This anatomical difference is somewhat consistent with lindane absorption
through the forearm (18%), forehead (34%), and palm (34%) of rhesus monkeys (Moody and Ritter, 1989). This anatomical range for lindane was also similar to that for dermal absorption of DEET (diethyl-m-toluamide) in rhesus monkeys (Moody et al., 1989). There is also significant data to suggest that dermal absorption of permethrin, aminocarb, DEET, and fenitrothion in monkey forehead is twice that in monkey forearm (Moody and Franklin, 1987; Moody et al., 1987; Sidon et al., 1988). In contrast, Moody et al. (1990, 1992) demonstrated no difference between the absorption of acid and amine forms of 2,4-D in rhesus monkey forearm and forehead or forearm and palm regions. The palmar absorption data are in conflict with the accepted dogma that absorption through palmar skin should theoretically be less than that in forearm skin because of the thickness of the stratum corneum in palmar skin (Maibach et al., 1971). It is proposed that because of the hydrophilic nature of 2,4-D-amine, absorption can occur through polar routes such as eccrine glands, which are more frequent in the palmar skin than in forearm skin. This anatomical difference does not explain the discrepancy with lindane, which is more lipophilic than 2,4-D and least likely to be absorbed via a polar route. Despite a 3-fold range in follicle area in the marmoset, no differences in absorption rates of paraquat, mannitol, water, and ethanol were observed between different body sites (Scott et al., 1991). However, among the different species examined in this study, there was an 80-fold range in follicle area, which correlated with observed differences in the rate of mannitol and paraquat absorption. The authors concluded that this correlation was only possible with relatively slowly absorbed test penetrants, such as paraquat and mannitol. Further work is needed to determine to what extent unique anatomical features at different body sites play a role in absorption and penetration of both lipophilic and hydrophilic pesticides. Another factor important in pesticide absorption is age. Topical application of a 2.5% chlorpyrifos spray to pigs resulted in almost uniform mortality when exposed at 3, 6, or 24 hr of age and no mortality at 36 hr (Scheidt et al., 1987). This study suggests that the barrier properties of newborn skin are not mature enough to prevent exposure immediately after birth. This could be secondary to the morphological (e.g., barrier integrity) or physiological (e.g., dermal blood flow) changes that occur as skin matures (Monteiro-Riviere and Stromberg, 1985; Monteiro-Riviere et al. 1991). Age is thus an important factor to assess when comparing absorption studies. Carbaryl is one of the most studied of all CM pesticides, and despite its low toxicity, it appears to penetrate human and animal skin more readily than most other pesticides. This increased permeability seen in most species, compared to most other pesticides, is most likely associated with its unique physicochemical characteristics. Almost complete penetration of carbaryl was observed when low-dose
CHAPTER 30 9Dermal Toxicity of OPs and CMs carbaryl (4 ixg/cm2) was dissolved in acetone and applied to the forearm and jaw angle of six human volunteers (Maibach et al., 1971; Feldmann and Maibach, 1974). The data from these studies demonstrated that 74% of the 24-hr applied dose was excreted in urine over 5 days. As discussed previously, percentage dose absorption for such a low applied concentration would be expected to be higher than for other doses. Utilizing deconvolution analysis of the same human data (skin-to-urine and blood-to-urine data), cumulative absorption over 5 days was estimated to be 63% of the applied dose, with 45% of this occurring 8 hr after onset of penetration (Fisher et al., 1985). When this analysis was performed with 11 other pesticides (e.g., parathion, aldrin, and diquat), more of the carbaryl dose was absorbed within 120 hr compared to other pesticides (0.3-20%). Only carbaryl had a lag time (3.5 hr) that was followed by rapid absorption. Approximately 50% of the 120-hr total absorption of parathion and dieldrin occurred in the first 4 hr. Therefore, although the absorption rate appeared to be less (due to the 3.5-hr lag time) with carbaryl compared to parathion in humans, the extent of absorption during 120 hr was greater for carbaryl (Shah et al., 1983; Hall et al., 1992). In a study comparing carbaryl absorption across formulations and vehicles, increased absorption was seen in some aqueous formulations compared to acetone (Baynes and Riviere, 1998), illustrating that formulation is an important variable that must be taken into consideration in dermal absorption studies. Knowledge of the vehicle or formulation in which the pesticide is dosed is crucial for interpreting dermal absorption studies. This is the primary confounding factor that makes comparisons across studies difficult. As discussed previously, the vehicle or complex formulation significantly affects the value of the partition coefficient, which in turn determines permeability. Vehicles are often not controlled in pesticide exposure studies, but they have repeatedly been shown to have a major effect on both the extent and the rate of pesticide absorption. This has been illustrated in our laboratory with carbaryl (Baynes and Riviere, 1998) as well as parathion (Qiao et al., 1996). In fact, co-administration of pesticides, in this case fenvalerate and parathion, modified absorption compared to single pesticide administration (Chang et al., 1995). Finally, first-pass biotransformation of topically applied pesticides may also occur, which complicates the comparison of the biological relevance of absorbed compound unless metabolism is specifically evaluated, a phenomenon clearly studied for parathion, in which both paraoxon and p-nitrophenol are produced by skin (Carver et al., 1990; Qiao et al., 1994). Insecticide efficacy, the stability of active ingredients, and programmed release of active ingredients from the vehicle/device are the most important characteristics controlled for when pesticides are formulated (Krenek and Rhode, 1988). Environmental Protection Agency (EPA) registration does not always require percutaneous absorp-
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tion studies. For this reason, more efficacy data are available in the literature compared to dermal absorption or pharmacokinetic data. Furthermore, most of the available pesticide absorption data pertain to binary mixtures (pesticide plus vehicle), which, as described previously, may confound cross-pesticide comparisons. However, technicalgrade formulations are complex mixtures of formulation additives, making risk assessment based on data from exposure to binary mixtures inappropriate. Pesticides are usually formulated to contain active and inactive or inert ingredients. The latter component(s) can enhance the rate and extent of absorption or slow the release of the active ingredient and thus reduce the rate and extent of absorption (Waiters and Roberts, 1993). These "inert" ingredients can be classified as adjuvants, surfactants, preservatives, solvents, diluents, thickeners, and stabilizers. These pesticide additives were first controlled by the Food and Drug Administration and now by EPA regulation 40 CFR 180.1001 and also TSCA and FIFRA (Seaman, 1990). This increasing list of inert ingredients, as well as the prohibitive cost to obtain 40 CRF 180.1001 clearance of new ones, strongly supports the need to evaluate the influence of current and novel additives on the toxicology and dermal absorption of active ingredients in pesticide formulations. Early work by O'Brien and Dannelley (1965) showed that compared to benzene and corn oil, acetone was best at enhancing absorption of carbaryl. Recent studies have also demonstrated the differential effects of solvent systems on the absorption of carbaryl, p-nitrophenol, and 2,4-D (Moody et al., 1992; Brooks and Riviere, 1996; Baynes and Riviere, 1998). Many studies have demonstrated that commercial formulations are more effective than simple solvents at enhancing pesticide absorption. Methyl parathion absorption in vitro in human skin at 24 hr was 1.3% in acetone but was significantly increased to 5.2% in a commercial formulation (Sartorelli et al., 1997). Likewise, in vivo dermal exposure studies of lindane in humans found approximately 60% with white spirits formulation and 5% with acetone vehicle (Dick et al., 1997a,b). In these latter experiments, more of the lindane dose (79%) remained on the skin surface at 6 hr with acetone than with white spirits formulation (10.5%) and significant levels of lindane accumulated in the stratum corneum with white spirits (30%) and with acetone (14.3%) at 6 hr. These findings strongly suggest that the white spirits formulation enhanced lindane penetration with respect to acetone vehicle. The in vitro studies on human skin also demonstrated a similar pattern, although only 18 and 0.3% of the dose was absorbed into the perfusate at 6 hr for white spirits formulation and acetone vehicle, respectively. Topical application of 1% commercial lotion of lindane in vitro in human skin and guinea pig skin resulted in absorption levels as high as 71.72 and 35.31%, respectively, at 48-hr exposure (Franz et al., 1996). Based on the previous discussion of partition coefficients and permeability, vehicle effects are not surprising.
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SECTION IV 9O r g a n T o x i c i t y
Vehicles that are capable of solubilizing the pesticide, but that also favor pesticide partitioning into the stratum comeum, will promote absorption. The effect seen is thus dependent on both the inherent solubility properties of the pesticide relative to the vehicle and the relative solubility of the pesticide in the vehicle versus the stratum corneum. This makes easy generalizations very difficult. Skin hydration can be increased by occlusion with high relative humidity or immersion conditions (e.g., swimming/ bathing). Although it was once assumed that hydration only affects dermal absorption of polar compounds, there is significant data that suggest that at high relative humidity, this hydration effect becomes more important for nonpolar molecules such as pesticides and is most likely secondary to an increase in diffusivity of the penetrating molecule (Behl et al., 1980). Under relative humidity conditions greater than 80%, parathion absorption at three different doses (4, 40, and 400 lxg/cm2) was significantly increased in pig skin in vitro by as much as two or three times the value under standard conditions of 60% relative humidity (Chang and Riviere, 1991, 1993). Occlusion can change dermal absorption by various mechanisms, including reducing evaporative loss of volatile pesticides from the skin surface; enhanced skin hydration, which increases the pesticide's permeability; alteration of the water content of the dosing site, which would reflect dosing in an aqueous vehicle; changes in cutaneous metabolism; dermal irritation; and altered cutaneous blood circulation (e.g., vasodilation). Occlusion can increase hydration of the stratum comeum from as little as 5-15% to as much as 50% (Bucks et al., 1989), thereby modulating the absorption profile for the pesticide. The practical impact of occlusion is seen when exposure occurs through bathing or swimming or, more often, when pesticides get into and under the clothing of workers, creating the ideal conditions for penetration and absorption into the skin. Similarly, many in vitro studies prehydrate skin samples, a requirement in side-by-side and Franz-type diffusion cells, which results in all findings representing flux through fully hydrated skin, a condition not seen in many human exposure scenarios. Prolonged full hydration of skin (e.g., >24 hr) may lend to epidermal degradation (Monteiro-Riviere et al., 1987). One in vivo study on pigs (Qiao et al., 1997) demonstrated that occlusion significantly enhanced pentachlorophenol (PCP) absorption in a soil-based mixture from 29 to 85% of dose and changed the shape of the absorption profile in blood and plasma. The study also suggested that occlusion enhanced metabolism of PCP and resultantly the 14C partitioning between plasma and red blood cells. Occlusion was kinetically linked to modification of cutaneous biotransformation of topical parathion (Qiao and Riviere, 1995). Occlusion enhanced cutaneous metabolism of parathion to paraoxon and to p-nitrophenol as well as the percutaneous absorption and penetration of both parathion and p-nitrophenol. This probably resulted in the
enhanced absorption that was seen. Occlusion also reduced parathion and p-nitrophenol levels in skin, but it increased p-nitrophenol and p-nitrophenol glucuronide in blood. Other in vivo studies showed that dermal occlusion significantly enhanced the rate and extent of parathion absorption in pigs in the abdomen (44 vs 7.5%), buttocks (48 vs 16%), back (49 vs 25%), and shoulder (29 vs 17%) (Qiao et al., 1993). Although significant anatomical site differences were observed with nonoccluded skin, these site differences were concealed with occluded skin. Recall that in vitro studies with parathion also demonstrated that occlusion increased absorption from 0.5-7.7% to 1-17% at doses ranging from 4 to 400 txg/cm2 (Chang and Riviere, 1993). Pesticides can be transferred from cotton fabric onto and through human skin as demonstrated in several studies (Snodgrass, 1992; Wester et al., 1996), although it should be recognized that these studies were often conducted under wet and/or occlusive conditions. Dermal absorption of malathion was 4% with ethanol wet fabric and 0.6% with 2-day-treated cotton sheets (Wester et al., 1996). However, malathion absorption was increased to 7% when the 2-day treated/dried cotton fabric was wetted with aqueous ethanol. In the same study, absorption of glyphosphate was 1.4% in water solution, 0.7% when applied as wet cotton sheets, and 0.1% when applied as 2-day-treated/ dried cotton sheets. Absorption increased to 0.4% when the 2-day dried cotton sheets were wetted with water to simulate sweating and wet conditions. Military uniforms have been impregnated with permethrin as a defense against nuisance and disease-bearing insects. Application of fabric impregnated with permethrin to the backs of rabbits resulted in a 3% migration to the skin surface, with 2% of the impregnant being absorbed and 1% remaining on the skin surface after 7 days of continuous skin contact (Snodgrass, 1992). These interactions have implications for agricultural workers during pesticide application in humid climates and for military personnel under desert combat conditions. Occlusion is a primary experimental variable that may impact the magnitude of dermal absorption fluxes seen. Comparing fluxes for pesticides dosed under occluded versus nonoccluded conditions, or at different sites from the same species, does not necessarily provide data on the comparative absorption of different compounds but, rather, on the overarching effect of application methods.
VI. C O N C L U S I O N S As can be clearly appreciated from the previous presentation, there are numerous experimental variables that have a major impact on the assessment of dermal absorption of pesticides. This was clearly seen with studies involving different methods (in vitro vs in vivo), different species, different doses, or application conditions such as occlusion.
C H A PT E R 3 0 9Dermal Toxicity of OPs and CMs A similar conclusion was derived from analysis of how data are used in making pesticide risk assessments, where the authors concluded that the inability to match a specific exposure scenario to available data (e.g., species and duration of dosing) led to overestimation of absorption and thus risk (Ross et al., 2000). The "take home" lesson from this chapter is that the experimental conditions under which pesticide absorption studies are conducted often overshadow differences between individual compounds, as can easily be appreciated in the classic studies comparing absorption of pesticides in mice (Shah et al., 1981) versus humans (Feldmann and Maibach, 1974). Any comparison can only be conducted if all possible variables are controlled. These variables include, but are not necessarily limited to, 9 Dose 9 Area of skin dosed 9 Application site 9 Vehicle, impurities, and other formulation additives 9 Length of study 9 Occlusion 9 Species 9 Age of animal 9 In vitro vs ex vivo vs in vivo system 9 Assay method The major challenge facing a critical assessment of pesticide absorption is that very few data sets, either across pesticides or for dosing factors within a specific pesticide, are available for analysis. This is the major limitation to compiling large databases of comparative pesticide absorption because the previous factors confound any analysis. Building such a comprehensive dermal absorption database of pesticides would also facilitate development of more robust QSPeR models that ultimately would facilitate the risk assessment process.
Acknowledgments I thank Dr. Ronald Baynes for his invaluable input and guidance in tutoring me on the fine points of experimentally assessing dermal pesticide absorption.
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in vivo percutaneous absorption studies: Influence of application time. J. Invest. Dermatol. 84, 66-68. Rubin, C., Esteban, E., Hill, R. H., and Pearce, K. (2002). I n t r o d u c t i o n - The methyl parathion story: A chronicle of misuse and preventable human exposure. Environ. Health Perspect. 110, 1037-1040. Sartorelli, P., Aprea, C., Bussani, R., Novelli, M. T., Orsi, D., and Sciarra, G. (1997). I n vitro percutaneous penetration of methyl-parathion from a commercial formulation through the human skin. Occup. Environ. Med. 54, 524-525. Scheidt, A. B., Long, G. G., Knox, K., and Hubbard, S. E. (1987). Toxicosis of newborn pigs associated with cutaneous application of an aerosol spray containing chlorpyfifos. J. Am. Vet. Med. Assoc. 191, 1410-1412. Scott, R. C., Corrigan, M. A., Smith, F., and Mason, H. (1991). The influence of skin structure on permeability: An intersite and interspecies comparison with hydrophilic penetrants. J. Invest. Dermatol. 96, 921-925. Seaman, D. (1990) Trends in the formulation of pesticides. Pestic. Sci. 29, 437-449. Shah, P. V., Monroe, R. J., and Guthrie, F. E. (1981). Comparative rates of dermal penetration of insecticides in mice. Toxicol. Appl. Pharmacol. 59, 414-423. Shah, P. V., Monroe, R. V., and Guthrie E E. (1983). Comparative penetration of insecticides in target and nontarget species. Drug Chem. Toxicol. 6, 155-179. Sidon, E. W., Moody, R. P., and Franklin, C. A. (1988). Percutaneous absorption of cis- and trans-permethrin in rhesus monkeys and rats: Anatomic site and interspecies variation. Toxicol. Environ. Health 23, 207-216. Smith, J. G. (1988). Paraquat poisoning by skin absorption: A review. Hum. Toxicol. 7, 15-19. Snodgrass, H. L. (1992). Permethrin transfer from treated cloth to the skin surface: Potential for exposure in humans. J. Toxicol. Environ. Health 35, 91-105. Srikrishna, V., Riviere, J. E., and Monteiro-Riviere, N. A. (1992). Cutaneous toxicity and absorption of paraquat in porcine skin. Toxicol. Appl. Pharmacol. 115, 89-97. Walker, M., Dugard, P. H., and Scott, R. C. (1983). In vitro percutaneous absorption studies: A comparison of human and laboratory species. Hum Toxir 2, 561-568. Waiters, K. A., and Roberts, M. S. (1993). Veterinary applications of skin penetration enhancers. In Pharmaceutical Skin Penetration Enhancement (K. A. Waiters and J. Hadgraft, Eds.) Dekker, New York. Wester, R. C., and Maibach, H. I. (1983). Cutaneous pharmacokinetics: 10 steps to percutaneous absorption. Drug Metab. Rev. 14, 169-205. Wester, R. C., Noonan, P. K., and Maibach, H. I. (1980). Variations in percutaneous absorption of testosterone in the rhesus monkey due to anatomic site application and frequency of application. Arch. Dermatol. Res. 267, 229-235. Wester, R. C., Maibach, H. I., Bucks, D. A. W., and Aufrere, M. B. (1984). In vivo percutaneous absorption of paraquat from hand, leg, and forearm of humans. J. Toxir Environ. Health 14, 759-762. Wester, R. C., Bucks, D. A., and Maibach, H. I. (1994). Human in vivo percutaneous absorption of pyrethrin and pipernyl butoxide. Food Chem Toxicol. 32, 51-53. Wester, R. C., Quan, D., and Maibach, H. I. (1996). In vitro percutaneous absorption of model compounds glyphosate and
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malathion from cotton fabric into and through human skin. Food Chem. Toxicol. 34, 731-735. Wester, R. C., Melendres, J., Sedik, L., and Maibach, H. I. (1998). Percutaneous absorption of salicylic acid, theophylline, 2,4-dimethylamine, diethyl hexylphthalic acid, and p-aminobenzoic acid in the isolated perfused procine skin
flap compared to man. Toxicol. AppL Pharmacol. 151, 159-165. Williams, P. L., Carver, M. P., and Riviere, J. E. (1990). A physiologically relevant pharmacokinetic model of xenobiotic percutaneous absorption utilizing the isolated perfused porcine skin flap (IPPSF). J. Pharm. Sci. 79, 305-311.
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Local and Systemic Ophthalmic Pharmacology and Toxicology of Organophosphate and Carbamate Anticholinesterases BRYAN BALLANTYNE Charleston, West Virginia
(extraocular) toxic or pharmacological effects. The various circumstances and conditions that determine the site and nature of effects are as follows:
I. I N T R O D U C T I O N Human subjects may be exposed to carbamate (CM) or organophosphate (OP) anticholinesterases (anti-ChEs) topically and/or systemically as a consequence of the wide range of their uses, which may result in exposures from deliberate, incidental, or accidental situations. These uses include the following:
1. Direct (local) contact of the substance with the external (visible) part of the eye (comea, conjunctivae, and palpebral surfaces); depending on the chemical and physical nature of the material, this may result in Injury and/or inflammation of the contaminated part of the eye and surrounding structures (local inflammation) A sensitization reaction (allergic conjunctivitis) A peripheral sensory irritant response Penetration of the substance through the surface structures, causing eye injury or pharmacological effect(s) deeper in the eye 2. Following local contact with the eye, there may be transport into the systemic circulation by absorption through pefiocular blood vessels, from the nasal mucosa after drainage into the nasal cavity through the nasolacrimal duct, or following swallowing into the alimentary tract. Materials with high toxic and/or pharmacological potency may exert systemic effects remote from the eye (extraocular) following such absorption (transocular systemic effects). 3. Materials (or their metabolites) absorbed through other routes of exposure may reach the eye after gaining access to the systemic circulation and exert toxic and/or pharmacological effects on the eye and its adnexa (systemic toxicity to the eye).
1. As pesticides in agriculture and horticulture, which may result in exposure through accidental contamination during their manufacture and in-use applications or incidental exposures through eating residues in treated crops (see Chapter 39). 2. Although the deliberate topical use of anti-ChE OP eye drops in the treatment of glaucoma has generally been replaced with other drug therapy having more effective and less toxic side effects, they are still used for producing short-term mydriasis in ophthalmic practice. Anfi-ChEs given by systemic dosing for conditions in which facilitation of cholinergic neuromuscular and central nervous system (CNS) cholinergic synaptic function is considered to be a therapeutic advantage, such as in Alzheimer's disease or parkinsonism. 3. Exposure may, and has, occurred in the context of the inhumane and vial use of potent OPs in chemical warfare, terrorism, and by some government "security" organizations for political assassinations. The eye may be a local target organ for toxic and/or pharmacological effects of anti-ChEs that are applied by direct (topical) contact to the eye or that reach the eye from the blood circulation. Additionally, the eye may be a route for absorption of anti-ChEs, which may result in distal Toxicology of Organophosphate and Carbamate Compounds
ChEs, both acetylcholinesterase (ACHE) and butyrylcholinesterase (BChE), are widely distributed in the eye. Activity is seen in the extraocular muscles, comeal 423
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epithelium, retina, choroid, and iris (Erickson-Lamy and Grant, 1992; Hikita et al., 1973). Many of the ocular pharmacological effects and some of the ocular toxic effects of anti-ChEs are attributable to their inhibitory effect on ChEs and the resultant cholinergic response, but some toxic effects may be related to other properties of the molecule that are not a function of its anti-ChE potency. Conjunctival hyperemia is characteristic of topical contamination of the eye, probably as a result of an anti-ChE activity (Chiou, 1992; Grant and Schuman, 1992). However, ChE inhibitors may produce varying types of corneal and ocular injury due to their chemical structure that is unrelated to anti-ChE activity, and for this reason it is necessary to undertake eye irritation studies on the OP or CM pesticides and medical products and their formulations. The nature of toxic and pharmacological effects produced by anti-ChEs as a result of their topical and/or systemic exposure of the eye, their investigation by laboratory in vitro and in vivo tests and by human clinical studies, and their practical and clinical implications are discussed in this chapter. For completeness, some reference is also given to cholinergic antagonists.
II. L O C A L O C U L A R I R R I T A N T E F F E C T S BY D I R E C T C O N T A C T
A. General Considerations Chemical injuries to the eye are common (Saunders et al., 1996). With anti-ChE pesticides the eye can become accidentally contaminated in the workplace (e.g., leakage in a manufacturing facility, and in the field/greenhouse there may be spray, droplet, or dust contamination), in the domestic environment (accidental spill, splash, or aerosol spray), or in transportation accidents. Many eye injuries result from misuse in the home, where protective and precautionary measures are often not strictly followed (e.g., eye protection). For example, in one study 84.4% of chemical eye injuries occurred in the home, and these were predominantly accidental exposures in children. In the same study, industrial eye accidents accounted for 14.2% of the eye accidents (Keres et al., 1987). With ophthalmic anti-ChE medication preparations, the eye is, by intended use, deliberately contaminated. The time to onset, severity, and duration of eye injury depend on a number of differing factors, all of which must be taken into account during the design, conduct, and interpretation of investigational laboratory studies and in considerations of in-use hazard and occupational hygiene. These include the chemistry and concentration of the active (anti-ChE) material, formulation (ingredients and concentrations), intended use pattern (e.g., liquid, aerosol, and dust), and likely mode and degree of exposure. In view of the likelihood for accidental and deliberate (medicinal) contamination of the eye, there is a need for
laboratory studies to be conducted in order to determine the eye injuring potential of active materials and formulations. The results of such studies should give practical information on the degree (severity) and nature of any induced eye injury; whether it is irreversible (permanent) or reversible (heals); if reversible, the duration for healing to occur; and whether first aid and medical treatment measures (e.g., eye irrigation) or the use of ocular anti-inflammatory agents are of therapeutic benefit. In many counties, there are mandated testing requirements for eye irritating potential; this covers domestic, agricultural, and industrial chemicals, including anti-ChE pesticides (Ballantyne and Marrs, 2004). There are several approaches to testing for eye irritation potential, which include the following: 1. In vivo direct eye irritation testing in which the material
to be tested is applied to the eye of an appropriate test species, and the eye is subsequently and periodically directly inspected for irritant (injurious) effects. Various ancillary techniques are available to allow a more precise evaluation of the nature and severity of the injury produced. 2. Because of the concern for possible severe discomfort to animals with conventional direct eye irritation testing, various in vivo alternatives have been developed to significantly reduce discomfort. These approaches in general employ lower concentrations (doses) and use noninvasive sensitive methods to detect (early) injury, often with provision for some degree of quantitation (in vivo irritant threshold approaches). 3. In vitro alternative methods, of differing degrees of specificity and reliability, have been developed in order to screen for eye irritating potential. These differing approaches use isolated ocular preparations and/or cytotoxicity assays. 4. Nonbiological models and computer-based analogy predictions have also been employed. Approaches to the various methods for assessing eye irritation potential are briefly summarized here. Details are discussed elsewhere (Ballantyne, 1999a; Ballantyne and Swanston, 1977; Green, 1999).
B. In Vivo Direct Eye Irritation Tests Topical contamination of the eye results in immediate contact with corneal epithelium. This tissue has not only high acetylcholinesterase (ACHE) activity but also choline acetyltransferase activity and the presence of the neurotransmitter acetylcholine (ACh) (Laties, 1969; Plestina and Piukovic-Plestina, 1978; van Alphen, 1957). These findings indicate the activity of a cholinergic physiology. Laties noted that OP anti-ChEs are bound in high concentration by the corneal epithelium.
CHAPTER 3 1 9Ocular Pharmacology and Toxicology Most studies are conducted as acute (single-exposure) investigations. For practical purposes, the formulation is normally tested, although for some registration and investigational needs the active ingredient alone is also tested. Ideally, the eye of an appropriate test species is exposed by the most likely in-use mode of contamination (e.g., droplet, aerosol spray, or vapor). However, many tests are routinely conducted by instilling a given volume (often 0.1 ml) into the inferior conjunctival sac of the test species (usually rabbit) and the eye is inspected sequentially and periodically to determine the nature, severity, time to onset, and duration of the ocular and periocular responses. The influence of varying dose on the effects produced can be studied by varying the concentration of material instilled (by serial dilution) and/or by reducing the volume of material instilled. The major ocular and periocular lesions that are searched for at the various inspection times are as follows: excess lacrimation, corneal injury (opacity/ulceration, vascularization, and area of involvement), blepharitis (hyperemia and thickness), inflammation of the conjunctivae and nictitating membrane (injection, congestion, chemosis, and sloughing), and iris (injection and congestion). For the purposes of convenient and standardized documentation of the effects, scoring systems for ocular lesions have been developed that give a numerical indication of the severity of lesions as a function of time; in this way, and in tabular or graphical form, the onset, duration, and resolution of individually noted lesions can be presented. This also permits a comparison of the eye irritating potential between different materials. Details of the conduct and scoring of local eye irritation tests have been discussed by Ballantyne (1999a) and Green (1999). In addition to macroscopic evidence for inflammation and injury, the eye is also examined for any pharmacological effects that may be produced (e.g., mydriasis and cycloplegia). With potentially potent biologically active materials, the test animals should be inspected for general (systemic) pharmacological and/or toxic effects following transocular absorption. Also, with some materials for which there may be occupational ocular exposure, or with medicinal eye drops, it may be required to perform studies on the peripheral sensory irritant potential of the material (Ballantyne, 1999b), In order to obtain a more precise evaluation of the presence and nature of eye injury in ocular irritation tests, the following ancillary procedures have been used. 1. FLUORESCEINSTAINING When the corneal epithelium is breached by injury, the barrier to fluorescein penetration is lost, and it can diffuse into the underlying corneal stroma. Thus, detection of the presence of fluorescein staining is valuable for the detection of early corneal injury. This is facilitated by examination under ultraviolet light or by using a blue filter in a slit-lamp biomicroscope.
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2. CORNEAL PERMEABILITY The detection of topically applied fluorescein in the aqueous humor can give an indication of whether corneal permeability has increased. This can be detected by the presence of a "fluorescein flare" in the aqueous humor when using a blue filter in the slit-beam path of a biomicroscope. Measurement of fluorescein has been used as the basis for the quantitative evaluation of corneal permeability (Maurice, 1967, 1968). Sulphorhodamine B is probably a better material for corneal permeability studies because of its lower lipid solubility at physiological pH, and hence decreased permeability with the undamaged cornea, and since the red wavelength emitted permits better quantifiable discrimination (Maurice and Singh, 1986). Using a fluorophotometer attached to a slit-lamp biomicroscope allows quantification of aqueous humor fluorescein and forms the basis for an objective test for eye injury, in which there is good agreement between fluorescein penetration and in vivo irritant potential (Easty and Mathalone, 1969; Etter and Wildhaber, 1985). 3. SLIT-LAMP BIOMICROSCOPY Slit-lamp examination of the eyes is valuable for detecting early and minimal changes in the cornea, and it permits a more precise evaluation of the structural integrity or otherwise of the cornea, iris, and lens and certain abnormalities in the aqueous humor, such as a flare due to the presence of excess protein (McDonald et al., 1972). A system for scoring was devised by Baldwin et al. (1973) for recording the effects and changes seen during biomicroscopy of the eye. 4. CORNEAL THICKNESS MEASUREMENT In vivo measurement of corneal thickness is an objective
approach for determining injury to the cornea, particularly during the early stages of the injury process. However, this approach may be more useful as an alternative method, rather than as an ancillary, for in vivo eye irritation studies. 5. CORNEAL EPITHELIUM HEALING RATE After injury of the corneal epithelium, reepithelialization occurs and the thickness of the cornea increases; however, with toxic substances there may be a retardation of the normal healing rate that forms the basis for a semiquantitive assessment of injury potential (Ubels et al., 1982). Combined, measurement of corneal thickness and healing rate can be used as an approach for the evaluation of eye irritants, notably with ophthalmic preparations that may be applied therapeutically to the injured eye. The method involves creating a standard epithelial wound using a trephine and then stripping the corneal epithelium. The material to be tested is then applied to the eye; the contralateral eye is also deepithelialized and used as a control. Corneal healing rate is assessed by fluorescein staining, photographing the injured area, and assessing its size
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by planimetry. Corneal thickness is measured before and sequentially following wounding of the epithelium. Several studies have demonstrated that chemicals of various categories delay the return to normal corneal thickness and retard epithelial healing rate (Fujihara et al., 1993; Green et al., 1989; Ubels et al., 1982). 6. HISTOLOGY At the end of the observation period for the progression and possible regression of eye Injury, it is valuable to remove some of the treated eyes for fixation and subsequent detailed histological observation in order to determine the completeness or otherwise of ocular healing. In some instances, adding additional animals allows for sequential sacrifice during the inspection period and thus permits a study of the pathogenesis of chemically induced ocular lesions. 9
.
III. A L T E R N A T I V E S T O C O N V E N T I O N A L IN VIVO E Y E I R R I T A T I O N S T U D I E S In attempts to replace standard eye irritation tests with other approaches for assessing eye injuring potential that cause significantly less discomfort to test animals and that do not involve deliberately inducing eye injury, several methods have been developed, some of which have markedly reduced discomfort, and others have been proposed as alternatives or replacements to conventional in vivo eye irritation methodology. They are briefly considered here.
A. In Vivo Alternatives Although still employing whole animals, these methodologies significantly reduce discomfort by using lower concentrations or smaller volumes of test material, and they increase the sensitivity for detection of early injury by the use of noninvasive objective techniques. Most also have the advantage of allowing a quantitative approach. 1. MEASUREMENTOF CORNEAL THICKNESS (PACHYMETRY) This is a particularly sensitive approach for the early detection of corneal injury. Corneal thickness, hydration, and transparency are interrelated, with corneal thickness and hydration being linearly related (Medbys and Mishima, 1966). Following acute ocular chemical injury, there is increased corneal thickness, indicating the development of edema in the corneal stroma, which may reflect damage to the epithelium, endothelium, or limbic blood vessels (MacDonald et al., 1983). Such damage facilitates the passage of water into the stroma, resulting in an increase in corneal thickness. Thus, corneal edema can result from corneal epithelial or endothelial damage, an increase in intraocular pressure, and inhibition of Na+-K + ATPase
(Chan and Hayes, 1985). Measurement of corneal thickness has several advantages: (i) Quantitative data are obtained that can be statistically analyzed for the comparative evaluation of different substances, (ii) it is a noninvasive method and causes a minimum of discomfort, (iii) increases in corneal thickness usually occur at concentrations below those that cause macroscopic evidence of corneal injury, and (iv) examination of the concentration-response curve allows for the prediction of irritant potential (Ballantyne, 1999a; Ballantyne et al., 1975, 1976). Corneal thickness can be measured by a simple optical device attached to a slit-lamp biomicroscope or by means of ultrasonic probe devices (pachymeters), both of which show good reproducibility (Chan and Hayes, 1985; Martins et al., 1992; Myers et al., 1998). There is a predictive correlation between an increase in corneal thickness and the potential for eye irritancy (Ballantyne et al., 1975, 1976; Burton, 1972; Chun and Ballantyne, 1997; Conquet et al., 1977; Jacobs and Martens, 1989; Martins et al., 1992). The technique of confocal microscopy through focusing allows the quantitation of depth and thickness of different tissue layers and structures of the cornea, and it permits a threedimensional reconstruction of corneal thickness (Li et al., 1996; Maurer and Jester, 1999). Using this approach, Maurer et al. (1997) assessed epithelial cell size, epithelial and corneal thickness, and depth of keratinocyte necrosis following the application of various solvents and determined that it may be used to differentiate different degrees of irritation. 2. MEASUREMENTOF INTRAOCULARPRESSURE Increased intraocular pressure (IOP) can result from causes that include obstruction of the aqueous humor outflow tract, an increase in aqueous humor production, and increasing aqueous humor solute concentration (Chan and Hayes, 1985), and it is a frequent finding when irritant materials are applied to the eye (Ballantyne et al., 1977). The magnitude and duration of the increase in IOP depend on the severity of the ocular irritant response (Ballantyne et al., 1972, 1977; Walton and Heywood, 1978). Most appropriate for measurement of IOP changes are tonometric methods, which involve either measuring the amount of corneal deformation produced by a standard force applied to the corneal surface (indentation tonometry) or measuring the force required to produce a standard degree of corneal flattening (applanation tonometry); details of these techniques were discussed by Ballantyne et al. (1977). Due to the occurrence of circadian variations of IOP in experimental animals, it is necessary to undertake measurements at similar times in control and treated animals in comparative investigations (Ballantyne et al., 1977; Moore et al., 1995). For continuous and long-term recording of IOP changes a technique has been described that involves the implantation of a miniaturized pressure transducer radiotelemeter into the cervical subcutaneous tissues, from which a cannula
CHAPTER 3 1 9Ocular Pharmacology and Toxicology is tunneled to the superior conjunctival sac and inserted behind the corneoscleral junction into a midvitreous position (Percicot et al., 1996; Schnell et al., 1995, 1996). This approach has been used to investigate the effect of antiglaucoma eye drops on rabbits in which chronic ocular hypertension was induced by a posterior chamber injection of oL-chymotrypsin (Percicot et al., 1996). An increase in IOP is usually measurable within a few minutes of topical application of an irritant to the eye, and the time for pressure to return to control values varies from a few hours to several days. The magnitude of the increase in IOP and its duration depend on the concentration of substance applied, its irritant potential, and its ability to penetrate the cornea to produce deeper structural and functional injury (Ballantyne, 1999a). The magnitude and duration of the changes in IOP and the slope on the dose-response curve can be used to predict the likely irritant potential of a material (Ballantyne, 1999a; Ballantyne et al., 1977). 3. CORNEAL PERMEABILITY MEASUREMENTS The measurement of effects of substances on corneal permeability to fluorochromes can be used as a means for the early detection of corneal injury and is a predictor of eye irritating potential (Ballantyne, 1999a; Etter and Wildhaber, 1985; Maurice and Brooks, 1995).
B. In Vitro Alternatives 1. USE OF ENUCLEATED EYES A method was described by Burton et al. (1981) involving the direct application of irritant test substance to the cornea of eyes removed immediately after sacrifice of rabbits and subsequent evaluation of the cornea for swelling, opacity, and fluorescein staining. The eye is mounted on a clamp in a temperature-controlled superfusion chamber with isotonic saline dripped onto the surface of the cornea. After an equilibrium period of 30-45 min, the test material is applied to the surface of the cornea and then washed off. Eyes are inspected macroscopically. Then, with a slit-lamp biomicroscope, the corneal thickness is measured and any fluorescein staining noted. The predictive value of the method has been confirmed in several studies (Commission of the European Communities, 1991; Koeter and Prinsen, 1985; Price and Andrews, 1985; Whittle et al., 1992). By using corneal thickness measurement, fluorescein retention, and corneal opacity as criteria for ocular injury, the suitability of the chicken eye as an enucleated model was confirmed by Prinsen and Koeter (1993) and Prinsen (1996).
2. ISOLATED CORNEA AND CORNEAL CELL PREPARATIONS Isolated corneas from bovine eyes were used by Gautheron et al. (1992) to assess eye irritancy. The corneas were placed in holders with compartments in front of and behind the mounted corneas in a temperature- and humidity-
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controlled chamber. Corneal injury was subjectively assessed by corneal opacity and measured by light transmission and fluorescein permeability from the anterior to posterior chamber of the corneal mount holder. A good assessment of ocular irritancy was obtained. Using a bovine corneal opacity-fluorescein preparation, Vanparys et al. (1993) tested 50 materials and found a 77% concordance using a four-level irritation classification system (non, mild, moderate, and severe irritation) and a 95% correct classification using a two-level classification (nonirritant and irritant). Cassidy and Stanton (1997) also obtained a correct classification using the opacity/permeability approach, and they found these two monitors to accord with corneal histology. Kruszewski et al. (1995, 1997) used a cell line grown from human corneal epithelial cells, which were grown as a three-dimensional culture on a collagen membrane. Effects on the preparation were measured using fluorescein retention. A good correlation with Draize injury scores was obtained. Schneider et al. (1997) described a complex in vitro corneal preparation prepared by establishing primary and subcultures of endothelial, stromal, and epithelial cells from fetal pig eye. A three-dimensional in vitro corneal model was then established in culture plates by sequentially adding endothelial, stromal, and epithelial cells. The cytotoxic response of the preparation was assessed by measuring mitochondrial activity using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Based on calculation of 50% inhibition of mitochondrial capacity (Es0 values), the investigators obtained a good correlation between the E50 values and Draize classifications for various substances.
3. ISOLATED LENS Based on the fact that the lens is embryologically derived from the same ectodermal source as corneal and conjunctival epithelium, Sivak et al. (1992) used an automated scanning laser system to monitor spherical aberration and transmission of the lens in culture. They believed that this method of measuring lens damage compared favorably with standard Draize scores. 4. NONOCULAR TISSUES The chorioallantoic membrane (CAM) of the hen egg is highly vascular and has been used as a tissue to determine whether its irritant response can be employed as a predictor of eye irritant potential. It is a borderline in vivo/in vitro system and does not conflict with ethical and legal standards (Leupke, 1985). The method involves removing shell from around the air cell on day 12 of incubation. The vascular CAM is exposed by removing the inner egg membranes, and the test substance is dripped onto the membranes. Blood vessels and albumin are scored for hyperemia, hemorrhage, and coagulation at 0.5, 2, and 5 min posttreatment. A good correlation with Draize scoring was found by Leupke (1985), and Bagley et al. (1989) showed the
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method to have high sensitivity, specificity, and to be of predictive value. However, Blein et al. (1991) found the method to be too sensitive with undiluted materials, but at 10-fold dilutions there was a good correlation with in vitro eye tests and under these conditions it was possible to differentiate mild, moderate, and severe irritants. A combined CAM-excised bovine eye (opacity/fluorescein stain/ epithelial detachment) test was used by Martins et al. (1992) to simulate corneal injury (bovine eye) and mucosal response (CAM). They obtained limited correlation, with slightly less than 40% of the substances tested agreeing with in vivo findings. In contrast, Van Erp and Weterings (1990) obtained a good correlation between the combined assay and in vivo findings, and Gilleron et al. (1996) obtained a sensitivity of 80% and concordance with the Draize test of 80.4%. Using other nonocular tissues, Muir et al. (1983) obtained a good correlation between hemolysis of bovine erythrocytes and blocking of spontaneous contractions of ileum, an in vivo eye irritation. However, this model is biologically and mechanistically remote from the pathogenesis of ocular irritation. 5. CYTOTOXICITYASSAYS Cytotoxicity assays measure loss of some cellular or intercellular structure and/or functions, including lethal cytotoxicity. They thus give an indication of the potential to cause cell and tissue injury and as such have been used by some investigators to predict tissue injury, including eye injury. Predictability may be variable for a variety of reasons, including the fact that cytotoxicity assay systems are continuously exposed to test material and lack the biological protective mechanisms of mucosae. The choice of screening cytotoxicity assay(s) should include consideration of past experience, likely mechanism for the irritant response, and chemistry of the substance tested. Cell lines that have been used in cytotoxicity assays include corneal epithelial cells, lung fibroblasts, Chinese hamster ovary (CHO) cells, canine renal cells, HeLa cells, and microorganisms. When nonocular-derived cell lines are used, investigators have attempted to rationalize their choice. For example, canine renal cells were used by Shaw et al. (1991) on the basis that integrity of corneal epithelial cells depends on tight and desmosomal junctions, which are also observed in renal canine cells. Thus, they can be used to detect chemicals that may cause an increase in transepithelial permeability (e.g., by using fluorescein leakage to detect injury to tight junctions and neutral red to detect renal cell membrane injury). A large number of cytotoxicity assays have been developed, as listed in Ballantyne (1999a), and only a few illustrative examples are given here. Crystal violet staining uses lethal cytotoxicity as the end point (Itagaki et al., 1991). Cultured HeLa $3 cells or SIRC cells (an established line of rabbit corneal epithelial cells) are incubated with serial dilutions of the test substance, and then crystal violet is used to stain residual viable cells. The
concentration of test substance causing a 50% inhibition of growth (IC50) is calculated. A good correlation has been found between IC50 and maximum in vivo eye irritation for various surfactants (Itagaki et al., 1991). A silicon microphysiometer (a light-addressable potential sensor device) has been used to indirectly measure the rate of production of acidic metabolites from cells in a biosensor flow chamber. The end point calculated is the MRDs0, which is the concentration of test substance required to reduce the metabolic rate by 50% (Brunner et al., 1991a,b). Using mouse fibroblasts, a good correlation was found between the MRDs0 and in vivo eye irritation [Bagley et al., 1994; Rougier et al., 1992 (r 2 = 0.81-0.91); Catroux et al., 1993a,b (r = 0.89)]. Using human epidermal keratinocytes, Brunner et al. (1991a,b) obtained good agreement between MRDs0 values and in vivo eye irritation findings. Photobacterium phosphoreum luminescence has been used to assess eye irritation. Luminescence is generated through a process linked to respiration by NADH and ravin mononucleotide (Bulich, 1979), with light emission being measured photometrically before and after addition of the test substance and an ECs0 calculated (i.e., a value of 50% reduction in light emission). Bagley et al. (1992) found that test substances with the greatest in vivo eye irritation potential had the lowest ECs0 value. The plasminogen activation assay is based on the release of plasminogen activator from primary rabbit corneal epithelial cells as a quantitative index of toxicity. A high correlation between plasminogen activation and known in vivo eye irritating potential was demonstrated by Bagley et al. (1994). In the neutral red uptake assay, mouse fibroblasts or CHO cells are exposed to the test substance and then to neutral red, the retention of which indicates cell viability. Bagley et al. (1992) noted that, in general, the concentration of test substance required to reduce neutral red uptake decreased as the in vivo determined irritant potential of the test substance increased. However, B lein et al. (1991) found that correlation with pH extreme materials was underestimated due to buffering in the incubation medium, and volatile material irritant potential was also underestimated, probably as a consequence of volatilization of the test substance. Several intertest comparisons of cytotoxicity assays have been undertaken. Sina et al. (1992) compared the following assay procedures: leucine incorporation (a general cytotoxicity test), MTT dye reduction (an indicator of mitochondrial damage), and neutral red uptake. They found that none of the end points accurately predicted in vivo eye irritating potential, but the MTT dye reduction method gave the overall best correlation. Christian and Diener (1996) noted that the neutral red uptake assay had merit for ranking potential ocular irritation, particularly the weaker irritants.
CHAPTER 3 1 9Ocular Pharmacology and Toxicology
C. Nonbiological Model Predictors In general, these have not been widely applied. One example is synthetic protein membranes, the use of which is based on the fact that protein denaturation may be a mechanistic function in corneal eye injury with some materials. The reactive component is a synthetic protein-globulin matrix. In one series, the predictability of eye irritating potential was 89%, and in another it was 93% (Soto and Gordon, 1990). 1. PREDICTIVE APPROACHES BASED ON ANALOGY The most commonly used approaches that are based on analogy are structure-activity relationships (SARs) and prediction based on known irritant potential (by in vivo testing) in other mucosal surface or skin. Sugai et al. (1990) used a quantitative SAR (QSAR) to analyze the correlations between chemical structure and eye irritation in rabbits. They claimed 86.3% accuracy in classifying substances with respect to eye irritation. Barratt (1997) described an eye irritation QSAR model for neutral organic compounds. Based on the perturbation of ion transport across the cell membrane being related to dipole moments of the causative substance, the model parameters chosen were log Pow (octanol-water partition coefficient) and the inertial axes Ry and Rz (representing the crosssectional area of the molecule). The results were stated to provide support for the validity of the QSAR model. However, it is hoped that product safety evaluation will not be based solely on QSAR (Ballantyne, 1999a). Many authors who have recommended analogy with skin irritants as a predictive basis for classifying eye irritation potential note that materials that have been shown to be skin irritants in vivo will also be irritants to mucosal surfaces, including those of the eye. However, the converse situation is of much more concern: That is, substances shown not to produce skin irritation should not be assumed to be devoid of irritancy to the eye. Indeed, several studies have shown that there is not a good correlation between skin and eye irritating potential and that some eye irritants are not skin irritants (Dalbey et al., 1993; Kennedy and Banerjee, 1992; Rhodes, 1987; Williams, 1984). There is no simple relationship between skin and ocular irritancy, and in some cases it may be very misleading to attempt to predict one based on the other (Ballantyne, 1999a; Daston and Freeberg, 1991; Williams, 1984). 2. GENERAL COMMENTS ON ALTERNATIVESTO OCULAR IRRITANCY TESTS No single alternative test can completely predict the eye irritating potential of all materials, and expectations for a uniform test should be discouraged (Christian and Diener, 1996; Hutak and Jacarusco (1996) Reinhardt, 1990). However, an appropriately tiered scheme may be valuable in significantly reducing in vivo eye irritation testing (Jackson
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and Rutty, 1985; Reihardt, 1990). Schemes should include the following sequential approach: (i) consideration of the physicochemical characteristic of the substance to be tested, along with a comparison of other substances in the same chemical class whose eye irritating potential has been established; (ii) use of appropriate mechanistically based cytotoxicity studies, established on the prediction for specific chemical substances; (iii) confirmation of the potential for corneal injury by using in vitro enucleated and/or corneal preparation; and (iv) in situations in which there will be deliberate eye contamination (e.g., ophthalmic medications), it may be necessary to confirm the absence of ocular injuring potential by carefully controlled in vivo studies with objective measurements.
IV. S P E C T R U M O F O C U L A R PHARMACOLOGICAL EFFECTS P R O D U C E D BY A N T I C H O L I N E S T E R A S E S As discussed previously, toxic and pathological effects produced on the eye may cover a wide variety of end points and mechanisms of production depending on a number of factors, including the physical properties and chemical structure of the material, the duration and frequency of dosing, the magnitude of individual doses and total dose, and the route of exposure and whether this involves first-pass metabolism leading to enhancement of toxicity or detoxification. Pharmacological effects due to anti-ChEs are a consequence of cholinergic effects resulting from the inhibition of AChE at preganglionic nicotinic (N-) and muscarinic (M-) synapses, postganglionic M-receptors, somatic motor end-plates, and CNS N- and M-receptors (Koelle, 1994). In the eye and its adnexa, cholinergic activity due to anti-ChE activity is seen as the following: 1. Miosis due to contraction of the sphincter pupillae and that may be associated with reduced visual field (Rengstorff, 1985, 1994). 2. Ciliary muscle spasm due to contraction of the ciliary sphincter muscle, with accommodative spasm, myopia, and reduced visual acuity. 3. Reduction of IOP due to contraction of the sphincter pupillae and ciliary muscle. 4. Excess tear production due to cholinergic stimulation of the lacrimal gland. 5. Changes in visual sensitivity, dark adaptation, and visual fields. 6. Extraocular muscle weakness. These effects produced by anti-ChEs may be of clinical value (e.g., lowering of IOP in cases of glaucoma). In addition to their potentially lethal effects caused by paralysis of respiratory muscles, bronchoconstriction, increased tracheobronchial secretions, and depression of the CNS respiratory center, exposure to low concentrations can
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SECTION IV. Organ Toxicity
cause ocular effects that may be harassing. For example, in the context of potential harassment in the battlefield, it has been noted that a very small amount of nerve agent vapor can produce miosis, eye pain or headache, nausea, and a general sensation of being unwell, which may decrease the willingness and ability of a soldier to perform a task (Sidell, 1994).
V. I N T R A O C U L A R INJURY, T O X I C I T Y , AND P H A R M A C O L O G I C A L E F F E C T S BY P E N E T R A T I O N O F S U B S T A N C E S T O P I C A L L Y A P P L I E D TO T H E O C U L A R SURFACES Depending on their chemical and physical properties, after contact with the surface of the eye anti-ChEs may penetrate deeper into the eye by passing through the cornea and/or sclera. Penetration from the anterior ocular surface was studied in rabbits by examining the autoradiographic distribution of topically applied [3H]diisopropyl fluorophosphate (DFP) and by measuring inhibition of tissue ChEs following the administration of ecothiopate iodide eyedrops (S-2-dimethylaminoethyl diethyl phosphorothiolate methiodide) (Laties, 1969). From the cornea, anti-ChEs diffuse across the aqueous humor in the anterior chamber to the iris, the papillary surface of the lens, and the aqueous outflow system. After passing through the sclera, anti-ChEs enter the ciliary body. In this way, topically applied anti-ChEs can have pharmacological actions and possible toxicity in deeper eye structures. Thus, corneal and scleral penetration of eyedrop anti-ChEs into the anterior aqueous chamber is a major pathway for the development of lens opacities in patients with glaucoma.
VI. T H E E Y E AS A T A R G E T F O R SYSTEMICALLY MEDIATED TOXIC AND P H A R M A C O L O G I C A L E F F E C T S After absorption of anti-ChEs through routes of exposure remote from the eye, the parent substance or metabolite(s) may reach ocular and periocular tissues through the systemic circulation. Depending on the nature of the antiChE or metabolite(s), toxic and/or pharmacological effects may be exerted on the eye. The potentially hazardous effect on the human eye from uncontrolled exposure of humans to pesticides, including anti-ChE OPs, was emphasized by the identification in the 1950s and 1960s, notably in children, of a possible correlation between a broad spectrum of ocular complications and exposure to fields sprayed with OP pesticides in the Saku region of Japan. The range of adverse effects (Saku disease) included blurring of vision, progressive myopia, astigmatism, blood ChE inhibition, altered electroretinography
(ERG), optic nerve atrophy, optic disk edema, and retinal degenerative changes (Dementi, 1994; Ishikawa, 1972, 1978; Ishikawa and Ohto, 1972; Ohto, 1974). The OP insecticides used near where there were reported cases of Saku disease included dichlorvos (DDVP; 2,2dichlorovinyl dimethyl phosphate), diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl-phosphorothioate), O-ethyl-O-p-nitrophenyl-thiophosphonate (EPN), fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate), fenthion (O,O-dimethyl O-4-methylthio-m-tolyl phosphorothioate), malathion (diethyl [(dimethoxyphosphinothioyl)thio] butanedioate), parathion (O,O-diethyl O-(4-nitrophenyl) phosphorothionate), parathion-methyl (O,O-dimethyl O-4-nitrophenyl phosphorothioate), and trichlorofon (dimethyl 2,2,2-trichloro-l-hydroxyethylphosphonate) (Boyes et al., 1994). Although no other studies have been reported outside Japan that document the spectrum of ocular pathology seen in Saku disease, and some consider that the Japanese population studies are not conclusive, several experimental studies on OPs appear to have substantiated much of the ocular toxicity reported in Saku disease, and several epidemiological studies on the affected populations did establish a reasonable probability of the association between an increased incidence of adverse ocular effects and the greater use of OPs in Japan (Dementi, 1994; Ishikawa, 1973; Ishikawa and Miyata, 1980; Tamura and Mitsui, 1975). However, the possibility exists that there is an increased susceptibility of Oriental populations to anti-ChE-induced ocular toxicology and pathology. The potential for the eye to be a target for substances (and metabolites) distributed systemically following extraocular absorption stresses the necessity for inspection of the eye to detect toxic or pharmacological effects in general acute or repeated dosing studies by all routes of exposure, particularly for chemicals to be used medicinally, as food additives, and those with a potential for exposure in industrial or domestic environments (Ballantyne, 1999a; Barnett and Noel, 1969). Where screening studies suggest a potential for systemic effects on the eye, particularly if there is deliberate exposure of humans to the test substance (e.g., medicinal product or food additive) or a high probability of accidental exposure (e.g., chemicals employed domestically), in definitive general toxicology studies there should be consideration of inclusion of a "satellite" group of animals used specifically to determine ocular toxic and/or pharmacological effects. Ophthalmoscopy, slit-lamp biomicroscopy, tonometry, corneal pachometry, light microscopy, histocytochemistry, transmission electron microscopy, scanning electron microscopy, angiography, ERG, visual evoked potentials, and electrooculography may be required (Ballantyne, 1999a). The U.S. Environmental Protection Agency requires ocular toxicity testing in support of registration of OP pesticides (Hamernik, 1994). Monitors include plasma/erythrocyte/retinal (and possibly brain)
CHAPTER 3 1 9Ocular Pharmacology and Toxicology ChEs; routine gross eye examination and indirect ophthalmoscopy; slit-lamp biomicroscopy; fundus observations; tonometry; ERG; determination of objective refractivity, pupillary response, and tracking; and ocular histology (with possible electron microscopy). In some cases, experimental animal studies may be unable to detect certain forms of systemic ocular toxicity, and these may only be seen in carefully controlled human studies, during detailed clinical examination of exposed humans during in-use applications, or in epidemiological studies. Examples include dyschromatopsia from styrene overexposure (Gobba et al., 1991); impaired or defective color vision following overexposure to styrene (Fallas et al., 1992), n-hexane (Raitta et al., 1978), or carbon disulfide (Raitta et al., 198!), and marked overexposure to toluene (Sch~iper et al., 2004). The potential for substances to produce pharmacotoxic effects on the eye as a component of systemic distribution is related to a variety of factors, includeing total exposure dose absorbed and determinants, notably frequency, magnitude, and duration of exposure dose; route of exposure, which will determine the pharmacokinetics and metabolism of systemic distribution; and the biotransforming capacity of the eye (Watkins et al., 1991). Notable pharmacological effects produced by OPs reaching the eye by systemic distribution include miosis, enhancement of accommodation, and reduction of IOP.
VII. THE EYE AS A PORTAL FOR SYSTEMIC TOXICITY (TRANSOCULAR SYSTEMIC PHARMACOTOXICITY) In general, most substances that are deliberately applied to, or accidentally come into contact with, the eyes are unlikely to be absorbed in significant amounts and produce systemic toxicity because of the small volume of material that can be accommodated on the surface of the eye or in the conjunctival sac. Additionally, the protective reflexes of excess lacrimation and blepharospasm will reduce the degree of ocular contamination (Ballantyne, 1999b). However, substances of high toxicity or pharmacological potency, including certain anti-ChE CMs and OPs, may be absorbed in amounts sufficient to produce systemic toxicity or pharmacological effects. Indeed, this has led to the suggestion of the possibility of transocular systemic pharmacotherapy with certain biologically active materials. For example, facilitated by absorption enhancers, glucagon may be readily absorbed by ocular instillation (Chiou and Li, 1993; Chiou et al., 1990; Pillion et al., 1992), and with insulin the blood glucose was markedly reduced in both normal and hyperglycemic rabbits and rats (Chiou and Chuang, 1989; Chiou et al., 1990; Pillion et al., 1991; Yamamoto et al., 1989). The absence of significant local toxicity from these procedures was confirmed in human volunteer studies on normo-
431
glycemic individuals having an ocular instillation of 50 Ixl of sterile saline solution containing various concentrations of porcine insulin, following which no anterior segment toxicity was seen by slit-lamp biomicroscopy (Bartlett et al., 1994).
A. Pathways of Absorption Substances coming into contact with the surface of the eye can be absorbed through conjunctival blood vessels, pass through the nasolacrimal drainage system into the nasal cavity and then through the naso- and oropharynx, and be swallowed. The relative contribution of ocular vascularity, nasal mucosa, and alimentary tract to the absorbed dose varies with the chemical species and its formulation. Absorption through the conjunctival blood vessels may be facilitated by materials also producing a conjunctival hyperemia. Absorption from the conjunctivae may also be modified by the rate of lacrimation, blepharospasm, and patency of the nasolacrimal duct. The large surface area and high vascularity of the nasal mucosa account for this site being a major component in the absorption of substances applied topically to the eye. It is well known that many pharmacologically active substances are absorbed following instillation into the nasal cavity as such (Chien, 1985), and for drugs with a significant first-pass metabolism by the peroral route, the nasal mucosa may offer a practical route for doing so. Hydrophobicity may be an important determinant for enhancing nasal mucosal absorption, whereas hydrophilicity may inhibit absorption (Duchateau et al., 1986; Hussain et al., 1980). The rate of drainage of substances from the conjunctival sac into the nasal cavity depends on a number of factors, including rates of lacrimation and blinking, both of which can be increased by irritant materials. Also, the volume of material is a determinant of the amount of materials transferred (Chrai et al., 1973). Since the drainage capacity of the eye exceeds the normal rate of lacrimation, drops instilled into the conjunctival sac may be rapidly transferred to the nasal cavity (Shell, 1992). Reducing the rate of lacrimal drainage may decrease systemic absorption. For example, Zimmerman et al. (1984) investigated the effects of eyelid closure and manual occlusion of the nasolacrimal duct on the absorption of the antiglaucoma drug timolol applied to the eye and on the permeability of the eye to topically applied fluorescein. It was found that with both eyelid and nasolacrimal duct occlusion, the plasma concentration of timolol was significantly decreased and anterior chamber fluorescein was increased. For timolol, Chang and Lee (1987) found that the nasal mucosa was approximately 2.5 times more effective than the conjunctival mucosa in contributing to total systemic absorption. That anti-ChEs applied topically to the eye may attain the alimentary tract and be absorbed there has been shown by several studies. For example, Wilensky et al. (1967) found that after ocular application of echothiophate or DFP, the greatest inhibition of ChE outside
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SECTION IV. Organ Toxicity
the eye was in the intestine. Also, anti-ChEs applied to the eye produced symptoms of intestinal pain and discomfort (Humphreys and Holmes, 1963).
B. Illustrative Examples Tropicamide [N-ethyl-N-(pyrid-4-ylmethyl)tropamide] is an M-receptor antagonist that produces transient mydriasis and cycloplegia. Because the paralysis of accommodation with 0.5-1.0% tropicamide is of short duration, its main use is in diagnostic examination or surgery of the eye requiring mydriasis of short duration. Although it has a low incidence of side effects, there have been reports of tropicamide causing a myasthenia gravis-like syndrome following topical application to the eye (Meyer et al., 1992). Plasma concentrations of tropicamide, and M-ACh receptor occupancy, were investigated following the conjunctival sac instillation of two 40 Ixl drops of 0.5% tropicamide in eight women (Vuori et al., 1994). Peak plasma concentrations of tropicamide were 2.8 + 1.7 ng m1-1 at 5 min postinstillation. There was rapid plasma clearance, with respective concentrations at 60 and 120 min of 0.46 + 0.51 ng m1-1 and <240 pg m1-1. Maximum tropicamide plasma muscarinic occupancy was approximately 8%. It is considered that the low affinity for M-receptors and the relatively negligible receptor occupancy explain the low incidence of side effects following the use of tropicamide eye drops. Cyclopentolate [2-dimethylaminoethyl oL-(1-hydroxycyclopentyl)-oL-phenyl acetate], a potent anticholinergic, is used in ophthalmic therapy to rapidly produce mydriasis and cycloplegia. Its systemic absorption is indicated by signs of CNS effects that develop within minutes of ocular instillation, including ataxia, dysarthria, disorientation, hallucinations, amnesia, and drowsiness (Awan, 1976; Shihab, 1980). In a study of 40 patients and a double blind with 35 participants, Birkhorst et al. (1963) demonstrated a statistically significant incidence of systemic reactions. Gastrointestinal effects were noted in premature twin neonates given cyclopentolate eyedrops for eye examinations after oxygen therapy; blood samples taken 24 hr postinstillation demonstrated plasma cyclopentolate concentrations of 2 and 22 txg m1-1 (Bauer et al., 1973). Echothiophate (S-2-dimethylaminoethyl diethyl phosphorothiolate methiodide) is an anti-ChE used in the treatment of glaucoma. Long-term use of echothiophate eyedrops can lead to an inhibition of erythrocyte AChE and serum BChE, which has raised the issue of the need for periodic blood ChE measurements in those receiving topical ocular anti-ChE therapy (Leopold, 1984). In a clinical study of 24 patients receiving echothiophate, a direct correlation was found between clinical effects (nausea, vomiting, diarrhea, abdominal pain, and weakness) and changes in blood ChEs (Ellis, 1985; Humphreys and Holmes, 1963). Diarrhea may be the first clinical indication of systemic toxicity from the transocular absorption of
echothiophate (Markman et al., 1964). More serious indications of transocular systemic cholinergic toxicity are vomiting, hyperhidrosis, muscle weakness, fasiculations, hypersalivation, seizures, miosis, bronchospasm, hypotension, bradycardia, and cardiac arrest (Alexander, 1981; Grant and Schuman, 1992; Hallet and Cullen, 1972; Hiscox and McCulloch, 1965; Leopold, 1984). In a case of severe echothiophate poisoning recorded by Manoguerra et al. (1995), the patient presented with profound muscle weakness, nausea, and dysphagia and had marked decreases in red blood cell (RBC) AChE and plasma BChE. There was spontaneous resolution of symptoms after discontinuation of echothiophate treatment. Asthmatic patients receiving ecothiopate may have an exacerbation of bronchospasm (Prakash and Rosenow, 1990), which may be accentuated by the coadministration of timolol (Gerber et al., 1990).
VIII. T O X I C A N D P H A R M A C O L O G I C A L
EFFECTS PRODUCED BY A N T I C H O L I N E S T E R A S E S AT S P E C I F I C OCULAR ANATOMICAL LOCATIONS AND ON OCULAR PHYSIOLOGICAL FUNCTIONS For materials subject to regulation by competent government authorities, it is generally required that the eye irritating potential by acute exposure to an appropriate physicochemical state or formulation of the substance should be determined. However, as noted previously, general studies on repeated exposure toxicity should also include provision to detect any potential for ocular pharmacological effects and for ocular injury at various anatomical sites, which may result in structural and/or physiological adverse effects. In particular, this should cover the likelihood for cataract formation, retinal pathology, optic nerve injury, and aqueous humor production and circulation. Some of these considerations are detailed here, together with examples of the effects of anti-ChEs on specific locations in the eye and adnexa.
A. Iris Miosis caused by exposure to an OP vapor usually starts during exposure but may not be complete for some minutes postexposure. The degree and duration of miosis are generally dose related. However, this may not follow as a general rule. Thus, certain concentrations of anti-ChEs in contact with the eye may cause extreme miosis but affect accommodation only moderately (Erickson-Lamy and Grant, 1992). For example, volunteers exposed to sarin vapor had marked miosis but only 2.3 D of induced accommodation compared with a potential maximum of 7-10 D (Moylan-Jones and Thomas, 1973). If severe, the inability of the pupil to dilate fully in darkness may persist for a few
CHAPTER 3 1 9Ocular Pharmacology and Toxicology weeks, and the affected individual may complain of "dim" or "blurred" vision and periocular pain or headache (Sidell, 1994; Rengstorff, 1985). Dim vision, or the inability to see well in dim light, is a common complaint of people exposed to OP vapor (Sidell, 1994). Stewart et al. (1968) found that after topical ocular instillation of satin (isopropyl methyl phosphonofluoridate) there was a decrease in light sensitivity that correlated with a decrease in pupil size, and they suggested that miosis alone was responsible for the decrease in light sensitivity. However, Rubin et al. found that topical application of satin did not decrease dark adaptation, but inhalation of the vapor with the eyes protected decreased dark accommodation in the absence of miosis (Rubin and Goldberg, 1958; Rubin et al., 1957). Also, systemic administration of atropine, which penetrates the blood-brain barrier, reversed this decrease (Rubin and Goldberg, 1958). These findings suggest that cholinergic mechanisms proximal to the pupil, such as those in the retina or visual pathways, are responsible for the visual changes. Miosis can be reversed with topical atropine or other cholinergic blockers, such as cyclopentolate, given following, but not before, OP exposure. However, this therapy causes blurred vision, which is not present before treatment (Moylan-Jones and Thomas, 1973). When miosis is maintained for long periods (weeks or months) by daily topical anti-ChE eyedrops, there is a gradual shallowing of the anterior chamber (Drance, 1969; Romano and Jackson, 1964). Thus, Wilkie et al. (1969) studied the effect of 0.125% echothiophate iodide on the axial depth of the anterior chamber and found that in the first 8 hr there was little change, but with daily administration over 8 weeks there was a gradual narrowing; the mean decrease in axial depth was 0.2 mm at 1 week and 0.44 mm at 8 weeks. None of the subjects developed glaucoma. After discontinuing echothiophate, the axial depth gradually returned to normal over 6 weeks. Rarely, iritis can occur from contact with anti-ChEs and is usually associated with conjunctival hyperemia. This complication responds to anticholinergic treatment (Aldridge et al., 1947), but in some cases the response to anticholinergics is slow despite treatment with mydriatics, corticosteroids, and 2-PAM (Becker et al., 1959). Iritis with the formation of posterior synechiae after long-term antiChE treatment has been reported (Kadin, 1963; Leopold, 1984; Werhahn and Schnarr, 1987). Morphological changes that have been documented to occur as a consequence of the repeated application of anti-ChEs in humans are iris cysts of the pupillary border (Grant and Schuman, 1992; Werhahn and Schnarr, 1987). Typically, they are multiple and 0.1-1.0 mm in diameter, and they may develop within 1-40 weeks (Abraham, 1954). Pupillary cysts can sometimes interfere with vision, particularly in association with extreme miosis (Swan, 1964; Werhahn and Schnarr, 1987). The simultaneous use of phenylephrine drops can enlarge the pupil slightly and reduce the tendency to form iris
433
cysts (Abraham, 1967; Chin et al., 1964; Ellis, 1985; Hill and Stromberg, 1962). Histologically, Straub and Conrads (1955) found that two posterior epithelial layers of the iris were separated in some places, forming fluid-filled cysts. This accords with the suggested pathogenesis of the cysts being a result of miosis pinching the iris pigment epithelium against the lens, with retention of fluid between the layers producing cysts (Swan, 1964).
B. Ciliary Body Topically applied to the eye, anti-ChEs can enhance the accommodative capacity of the eye. For example, in humans the near point of accommodation may move forward by 10 cm, and this requires a lens of - 4 D for correction. However, when the effort to accommodate is relaxed, the focus of the eye, and the visual acuity at distance, may rapidly return to normal (Aldridge et al., 1947; Upholt et al., 1956). The rate and completeness of recovery appear to be dose dependent (Erickson-Lamy and Grant, 1992). Enhancement of accommodation by anti-ChE eyedrops is a useful therapy for accommodative esotropia in children. In this condition, a relative weakness of accommodation and disproportionately strong convergence produce "crossed eyes" with a danger of emblyopia in one eye. Anti-ChE eyedrops can enhance accommodation so that focusing corresponds to the amount of convergence, assisting in alignment of the eyes (Erickson-Lamy and Grant, 1992). A further function of the ciliary muscle that can be potentiated by anti-ChEs is control of resistance to outflow of aqueous humor from the anterior chamber. In the condition of glaucoma, contraction of the ciliary muscle reduces the resistance to outflow and anti-ChE eyedrops potentiate this action (Becker et al., 1959; Bito, 1968). Suzuki and Ishikawa (1974) described ultrastructural changes in the ciliary body of dogs chronically dosed orally (by capsule) with ethylthiometon at a dosage of-~0.5 to -1.5 mg kg-1 per day, 5 days a week for 2 years. All dogs developed myopia within 1 year. Ultrastructural examination of the ciliary muscle cells showed membranous meshwork structures, which the investigators considered was the cause of the myopia. Tokoro et al. (1973) studied dogs dosed with ethylthiometon for 2 years at doses of 5, 10, and 15 mg per dog per day. Myopic changes were noted, the onset of which was related to the degree of erythrocyte AChE inhibition. Chronic peroral studies in dogs with the OP ethylthiometon at doses of 0.5-1.5 mg kg -1 per day resulted in decreases in refractive error and corneal curvature suggestive of myopia (Kawamura et al., 1974; Otsuka and Tokara, 1976), increased IOP, and decreased erythrocyte AChE (Tokoro et al., 1973). Beagle dogs were dosed orally (capsule) with fenitrothion twice a week at dosages of 10, 20, and 100 mg kg-1 per week for 1 year (Ishikawa and Miyata, 1980). Myopia was significantly increased in dosed versus control (nondosed) animals by 9 months of
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Organ Toxicity
dosing, and it was still present 1 year after the cessation of dosing. Lens thickness and lOP were also increased. Histology of the ciliary body showed muscle fiber swelling at all doses, which was morphologically similar to that described for ethylthiometon. Both RBC AChE and serum BChE were slightly decreased during the dosing period but returned to normal 3 months after the cessation of dosing. Ciliary body AChE was significantly reduced.
C. Aqueous Humor Production, lntraocular Pressure, and Glaucoma Aqueous humor is secreted by the ciliary processes and passes behind the ciliary muscle into the posterior compartment (chamber) of the aqueous chamber, and then it flows between the fibers of the suspensory ligament of the lens into the anterior aqueous chamber. It is reabsorbed into the canal of Schlemm. The trabecular meshwork of the canal is opened, and reabsorption of aqueous humor is facilitated, by contraction of the sphincter pupillae of the iris and the ciliary muscle, both of which have a cholinergic innervation. Thus, anti-ChE agents can pharmacologically cause an accumulation of ACh at the sphincters of the iris and ciliary body, resulting in their contraction, producing miosis and ciliary spasm with accommodation for near vision, and facilitating aqueous humor reabsorption and a decrease in lOP. The elevated IOP in glaucoma can be reduced with the use of anti-ChE drugs due to their ability to increase cholinergic activity in the sphincter muscles of the iris and ciliary body. They can therefore be used in the emergency treatment of acute narrow-angle glaucoma and in the longterm therapy of chronic wide-angle glaucoma (Koelle, 1994). They are generally not advised for the long-term treatment of narrow-angle glaucoma because they may produce hyperemia of the iris and trabecular network (Ellis, 1985). However, it was noted in several clinics that continued ophthalmic use of long-acting anti-ChE drugs resulted in lenticular opacities. Therefore, in general, they have been replaced by drugs operating through other mechanisms such as parasympathomimetic agents (e.g., pilocarpine), which cause contraction of the ciliary body, relaxation of the lens zonules, and forward movement of the lens (Hung et al., 1995; Yang et al., 1997), and [3-adrenergic receptor blocking agents (e.g., timolol), which decrease the rate of aqueous humor formation (Coakes and Brubacker, 1978; Wang et al., 1997). Although anti-ChEs reduce lOP in the normal and glaucomatous eye, a paradoxical transitory increase in IOP may occur in some patients due to the breakdown of the blood-aqueous barrier (Erickson-Lamy and Grant, 1992). In rabbits this response can be marked. Thus, application of physostigmine, neostigmine, or DFP to the rabbit eye initially causes hyperemia of the iris, an increase in IOP, and an increased capillary permeability, permitting entry of proteins that may cause an aqueous
humor flare (B~r~iny, 1947; Von Sallam and Dillon, 1947); this may be prostaglandin mediated (Erickson-Lamy and Grant, 1992). By repeated application, rabbits can be made tolerant to repeated topical application of anti-ChEs, resulting in decreased IOP and no inflammation (Becker et al., 1959). Angle-closure glaucoma is probably precipitated in eyes with a shallow anterior aqueous chamber and narrow angle, which can be accentuated to the point of angle closure by miosis and anterior movement of the lens. Some cases have been described in which young individuals developed angle-closure glaucoma (Franqois and Verbracken, 1977, 1978; Jones and Watson, 1967). In these cases, glaucoma was the result of the iris closing the anterior chamber angle and obstructing the outflow of aqueous humor. However, contraction of the ciliary muscle probably also played a role by loosening the lens zonules and permitting the lens and iris to move forward (Erickson-Lamy and Grant, 1992). Treatment with anticholinergic drugs was effective in these cases, probably as a consequence of tightening of the lens zonules resulting from ciliary muscle contraction.
D. The Lens and Cataractogenesis The lens may be subject to toxic effects as a result of materials applied topically to the eye reaching it by penetration through the cornea and sclera into the anterior chamber and/or reaching the eye through the systemic circulation. The mechanism of pathogenesis of cataractogenesis may be complex and vary between different classes of xenobiotics and their metabolites. Common etiological factors are changes in osmotic pressure resulting from electrolyte shifts and lens protein denaturation. Major mechanisms include oxidative stress, lens hydration, inhibition of Na+-K + ATPase, and proteolysis by Ca2+-dependent calpain activation. The potential for cataractogenesis may depend on whether exposure is pre- or postnatal. Prenatally, the developing lens has a blood supply that starts to degenerate on the first postpartum day, and the adult lens is avascular. Thus, access of substances to the lens in the adult is via the aqueous humor or, to a lesser degree, the vitreous humor. Thus, in the adult, the cataractogenic material or metabolite must be able to penetrate the blood-aqueous barrier. A variety of investigational techniques are available for studying lens morphology and toxicity, including various forms of microscopy, biochemical, and metabolic studies; these may be followed by magnetic resonance imaging and spectroscopy (Schliech et al., 1985), tryptophan fluorescence spectroscopy (Lerman and Moran, 1988), and scanning lens monitoring (Mitton et al., 1990). Biochemical changes in the lens that have been investigated include those of aldose reductase, sorbitol dehydrogenase,
CHAPTER 3 1 9Ocular Pharmacology and Toxicology phosphofructokinase, glutathione reductase, glutathione, NADH, NADPH, calcium, cholesterol, phospholipids, and protein (Chiou, 1992). As noted previously, the long-term use of anti-ChE OPs in the treatment of glaucoma results in changes in the transparency of the lens and the development of opacities (Chiou, 1999; Harrison, 1960; Muller et al., 1956). AntiChE drugs given perorally do not produce lens changes (Lieberman et al., 1971), which may be a function of high concentrations reaching the anterior chamber through the cornea and/or possibly related to metabolism after peroral dosing. The development of lens changes appears to be age related, with the elderly being the most susceptible and children showing changes only rarely (Axelsson and Nyman, 1970; Chamberlain, 1975; Harrison, 1960; Pietsch et al., 1972; Wolter and Lee, 1978). Slit-lamp biomicroscopy of the eye after several months of daily application of anti-ChE eyedrops demonstrates anterior and posterior subcapsular vacuoles or small opacities in approximately 50% of patients (Erickson-Lamy and Grant, 1992). Detailed descriptions of the lesions have noted that they consist of minute anterior subcapsular vacuoles in groups, very small anterior subcapsular wooly or mossy opacities in aggregates, often associated with nuclear sclerosis, and posterior subcapsular small vacuoles or opacities. In some cases, surgical cataract extraction has been required (Drance, 1969; Morton et al., 1969; Tarkkanen and Karjalainen, 1966). In this respect, it is relevant to note that Axelsson (1969) provided evidence that glaucoma per se does not result in cataracts. The cause of the development of lens changes in glaucoma patients treated with anti-ChE eyedrops is not fully understood, but some patients are resistant to the adverse effects of anti-ChE on the lens and maintain normal visual acuity and lens transparency after many years of treatment (DeRoetth, 1996; Drance, 1969; Shaffer and Hetherington, 1966). Also, eyes treated with pilocarpine before anti-ChEs are protected to some degree from the effects of anti-ChEs on the lens (Nordmann and Gerhard, 1969, 1970; Shaffer and Hetherington, 1966). Several experimental studies have been conducted on the possible pathogenesis of anti-ChE-induced lens opacities. In rabbit lens in culture, Michon and Kinoshita (1968a,b) found that concentrations of anti-ChEs sufficient to inhibit all lens ChE did not produce changes in transparency but at 1000 times this concentration caused anterior, posterior, and equatorial vacuoles to appear associated with increased lens permeability, increased Na + content, and a decrease in K +. Echothiophate produced these changes without altering lens metabolism, but decarium bromide caused anaerobic metabolism to predominate (Michon and Kinoshita, 1968a,b). A 50% inhibition of oxygen consumption was reported for the pig, rabbit, and human lens treated with paraoxon (Muller et al., 1956). Rabbit eyes chronically exposed to echothiophate iodide eyedrops do not show macroscopic lens changes, but HS_rkrnen and Tarkkanen
435
(1970, 1976) found a decrease in ATP and lactate but no change in glycogen, and Firth et al. (1973) found no change in lens glutathione. In calf cultured lens, echothiophate iodide (0.06-0.5%) produced no opacities but altered lens nucleotides and 32p incorporation. Daily application of 0.25% ecothiophate eyedrops to the primate eye produced anterior and posterior opacities within 2-14 weeks; the anterior opacities reached a maximum at 3 or 4 months, and the posterior opacities reached a maximum at 1.5-3 months (Kaufman and B~ir~iny, 1975; Kaufman et al., 1977). Pretreatment iridectomy increased the incidence of opacities, but addition of atropine to the echothiophate eyedrops reduced the number of opacities and delayed their development. It has also been demonstrated that echothiophate eyedrops applied daily to primates produced subcapsular lens opacities and also caused swelling of the anterior cortex of the lens (Albrecht and B~ir~iny, 1979; Philipson et al., 1979).
E. Retinal Toxicity In general repeated exposure toxicity studies, toxic effects on the retina may occasionally be detected by the use of ophthalmoscopy or histological examination of the eye. However, the sensitivity of ophthalmoscopy for the detection of retinal lesions can be low if not conducted by a person experienced in veterinary ophthalmology (Ballantyne, 1999a), and general pathologists reviewing tissue sections in general toxicology studies may not detect some of the subtle retinal cytological lesions that could be of toxicological significance. A variety of differing techniques are available to detect, or confirm, a retinotoxic effect. These include retinoscopy, pupillary light reflex, fluorescein fundus angiography, ERG, and various forms of microscopy. ERG is well established as a noninvasive procedure for investigating functional retinal defects (Armington, 1974; Shirao and Kawasaki, 1995). Maertins et al. (1993) found that ophthalmoscopic fundus examination for retinopathy did not yield characteristic correlates, but time- and dose-dependent effects were found with ERG and light microscopy, with indications of retinal changes occurring earlier and more distinctly with ERG. The retina has AChE and cholinergic activities (Hutchins, 1987). Choline uptake and its conversion to ACh in the rat and rabbit retina have been shown to be very active (Atterwill et al., 1975). As noted later, some studies indicate that retinal toxicity may be associated with antiChE activity, and it is of interest and possible relevance to note that the ERG responds to ACh and its agonists with an initial increase followed by a decrease in b-wave amplitude, and that anti-ChEs have similar effects. However, neurotransmission in the retina is complex, and at least five neurotransmitters have been identified: ~r acid, glycine, dopamine, idolamine, and ACh. Thus, the
436
SECTION I V .
Organ Toxicity
biochemical pathogenesis of retinal lesions may be complex. Boyes et al. (1994) noted that several studies have shown retinal degeneration in photoreceptors to the outer nuclear layers. Since these are without cholinergic innervation, they suggested that if OPs are producing this effect through anti-ChE activity, then they are probably doing so by an indirect method such as altering blood supply or distribution. Examples of retinal effects produced by antiChEs are considered below. Retinal detachment has been noted to be a complication of the treatment of glaucoma with miotics, especially anti-ChEs. Although a cause-effect relationship has been suggested, a definitive established relationship is unproven (Alpar, 1979; Beasley and Fraunfelder, 1979; EricksonLamy and Grant, 1992; Lemcke and Pischel, 1966). If this occurs, it probably involves an intense contraction of the ciliary muscle to produce retinal tears. Becker and Shaffer (1976) noted that patients with open-angle glaucoma may be predisposed to peripheral retinal degeneration. Lemcke and Pischel (1966) reported holes or horseshoe-shaped tears usually in the periphery of the retina, occasionally surrounded by pigment changes suggestive of preexisting chorioretinal adhesion. Retinal detachment has not been recorded as a complication of repeated dosing with antiChEs by routes of administration other than topical ocular in human cases or animal experiments. This had led to the suggestion that retinal detachment occurs only in eyes with preexisting retinal pathology. It may also imply that it is only by topical application that sufficiently high intraocular concentrations of anti-ChEs are attained. In an essentially environmental study, Hamm et al. (1998) investigated the effect of the OP pesticide diazinon on AChE activity and embryonic retinal cells in the teleost (Oryzias latipes); concentrations employed were 1.8 X 10 -5 , 4.4 x 10 -5 , and 8.8 X 10 -5 M. They found that diazinon significantly inhibited AChE activity in a dose-related manner in whole embryos and in homogenates of retinae. Histology showed that as the retina underwent differentiation into distinct cell layers, small foci of necrotic cells appeared within the inner nuclear layer and isolated individual pyknotic cells were seen in the ganglion layer. Quantitation of foci of necrotic cells by image analysis revealed a dose-related increase in the number and area of these lesions that was statistically significant at 8.8 x 10 -5 M diazinon. Enzyme histochemistry localized AChE activity to regions corresponding to those of sites of necrosis. The investigators also demonstrated that DFP produced large foci of necrotic retinal cells equivalent to those seen in diazinon treatment. Several studies have suggested that anti-ChEs can modify retinal physiology and function. Gazzard and Thomas (1975) analyzed the threshold luminance of the central visual fields of human volunteer subjects after exposure to satin vapor. They found that sarin raised the visual threshold, influencing cone more than rod function. Other studies
demonstrated that satin induced an elevation of the absolute scotopic threshold in humans (Rubin and Goldberg, 1957; Rubin et al., 1957). Carricabura et al. (1980, 1981) found that high doses of mevinphos and malathion alter the electrical properties of the retina by a direct action on photoreceptors (increase in lag time and reduced amplitude of the a-wave) and cause possible damage to the bipolar cells and/or ganglion neurons. In the cat, chlorfenviphos [2-chloro-l-(2,4-dichlorophenyl)vinyl diethyl phosphate] at dosages of 1-16 mg kg -1 increased a-wave amplitude and at doses >4 mg kg -~ increased the amplitude of the b-wave (Takeda et al., 1976). Von Bredow et al. (1971) reported that satin (10 mg in cat, intraperitoneally) caused an increase in the ERG b-wave, which was completely abolished by atropine. Imai (1974) showed that in the rat, four daily intramuscular injections (0.5 mg kg -1) of fenthion resulted in an increased amplitude and decreased latency and peak times of the ERG a- and b-waves. At higher doses (>25 mg kg -1) latency and peak times were increased and the amplitude of the a- and b-waves was decreased. Imai (1975a,b) demonstrated ERG changes in rats given an acute intramuscular injection of fenthion (5, 25, and 50mg kg-1). At the low dose, there was an increased amplitude of the a- and b-waves that persisted up to 10 days postinjection; thereafter, latency and peak times were lengthened and did not return to normal until 40 days postinjection. At the mid-dose, the a- and b-waves increased, and by postinjection day 4 the latency and peak times increased; the amplitudes recovered by 60 days, but the peak time remained increased. At the high dose, the a- and b-wave amplitudes were decreased and the latency and peak times were increased; no recovery of amplitudes was noted. Various studies, using both acute and repeated exposure dosing, have shown that OPs and, less frequently, CMs can be associated with effects on retinal ChE and the ERG and produce retinal histopathology and other ocular toxicity. Studies on several OPs in rats and dogs have suggested that they can produce abnormal ERGs and histopathological evidence of retinal degeneration (Boyes et al., 1994; Dementi, 1994). Studies on radiolabeled benomyl showed that it was concentrated in the mouse retina following systemic administration (Hellman and Latyea, 1990). Acute doses of parathion, malathion, and mevinphos (2-methoxycarbonyl-l-methylvinyl dimethyl phosphate) have produced ERG changes in mice (Carricabura and Lacroix, 1973; Carticabura et al., 1979, 1980, 1981). Fenthion at doses >50 mg kg-1 produced dose-dependent ERG changes, a decrease in retinal ChE activity, fundoscopic evidence of retinal degeneration, and histopathological changes in the pigmented epithelium and sensory retina (Imai, 1975a,b, 1977, 1978; Imai et al., 1973, 1983; Miyata et al., 1973). Details of some studies are presented here. The effects on the ERG and retinal AChE were studied in Wistar rats at acute subcutaneous dosages of
CHAPTER 3 1 9Ocular Pharmacology and Toxicology 0.005-500 mg kg -1. All rats dosed at 500 mg kg -1 died. At 50 and 100 mg kg -1, subnormal ERG activity was obtained; 25 mg kg -1 was a transitional dose (normal ERG), and at less than 25 mg kg -1 the ERG was supernormal. Minimal change was seen at the lowest dose of 0.005 mg kg -1, and thus a no observed effect level (NOEL) for the ERG was not obtained. Retinal and cerebellar AChE activity was inhibited in a dose-related manner in the 0.5-100 mg kg -1 range. Thus, ERG changes were detected at acute fenthion dosages below those causing inhibition of retinal AChE activity. Imai (1975a,b) also studied the effect of fenthion given acutely by subcutaneous injection at doses of 0, 5, 25, and 50 mg kg -1 to Wistar rats. At 5 mg kg -1, supernormal ERGs were recorded that increased until 10 days postdosing and decreased thereafter to normal by 2 months. A transitional ERG was conformed at 25 mg kg-1, and at 50 mg kg-1 a subnormal ERG was obtained that was still present at 66 days postdosing. Measured serum BChE activity decreased to approximately 15% 4 days postdosing. Retinal AChE activity was decreased for up to 49 days, and in the high-dose group the ERG changes corresponded with the retinal AChE recovery curve. Repeated exposure studies have been conducted with 50 mg kg -1 fenthion given subcutaneously every 4 days to Long-Evans black rats for 1 year (Imai, 1977; Miyata et al., 1979; Uga et al., 1979). Dosed animals showed signs of systemic toxicity (exophthalmos, shivering, and diarrhea). At 3 months, the ERG was found to be subnormal, and at 9 months the ERG a-waves disappeared. Fundoscopy revealed paleness of the papilla. Histology showed the disappearance of the retinal pigmented epithelial layer, outer and inner nodes, and outer granular layer. Ultrastructural examination showed complete disappearance of photoreceptor cells. Uga et al. (1976) studied the ultrastructure of the retina of dogs dosed with ethylthiometon at dosages of 0.5-1.5 mg kg -1 for 2 years. Degenerative changes were seen, principally in pigment epithelial cells in the area around the papilla, and myelin-like material was seen in retinal cells. Effects of fenthion on the retina of Long-Evans and Wistar rats were also studied by Imai et al. (1983) using a dosage of 50 mg kg -1 given subcutaneously twice a week for 1 year. ERGs were subnormal by 3 months and not recordable by 12 months. Retinal degeneration was seen in all Long-Evans (pigmented) rats and two-thirds of the Wistar rats at 1 year. Wistar rats dosed with chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) showed ERG changes of decreases in a- and b-wave amplitudes of more than 50% and increased latencies of 10-20%, which were associated with inhibition of plasma, erythrocyte, and brain ChE activities (Yoshikawa et al., 1990). Neuropathological lesions included optic nerve demyelination, partial necrosis of pigmented epithelial Cells, and cytoplasmic edema and degeneration of ciliary muscle myofilaments (Kono et al.,
437
1975; Mukuno and Imai, 1973; Mukuno et al., 1973). However, some repeated exposure studies have suggested that inhibition of retinal AChE may not be a major etiological factor in the production of retinal toxicity by anti-ChEs. For example, in a subchronic (6-month) study in which beagle dogs were given daily peroral doses of the OP ethyl parathion (2.4, 7.9, or 794 p~g kg -1 per day) by gelatin capsules, although plasma, erythrocyte, and retinal ChEs were inhibited at the high dose, there was no functional impairment of eye function during the 6-month period as assessed by ophthalmoscopy, slit-lamp biomicroscopy, refraction, tonometry, ERG, and ocular histology (Atkinson et al., 1996). In a chronic peroral study (1 year), beagle dogs were dosed with disufoton (O,O-diethyl S-2-ethyl thioethyl phosphorodithioate) in the diet with average daily consumptions of 0.0, 0.015, 0.121, and 0.321 mg kg -1 (Jones et al., 1999). ChE activity was measured in erythrocytes, plasma, brain, retina, cornea, ciliary body, and extraocular muscles. Neuroophthalmological monitors included task performance tests, gait and behavioral observations, ERG, refractivity, tonometry, and pachymetry. RBC, plasma, corneal, and brain ChEs were significantly decreased at 0.121 and 0.321 mg kg -1 per day, and ciliary body and retinal ChEs were significantly decreased at 0.321 mg kg -1 per day; the NOEL for ChE inhibition was 0.015 mg kg-1 per day. Despite the inhibition of ChE activity in various tissues, there were no adverse clinical neurological effects and no ophthalmologic findings by ERG, refractivity, tonometry, or pachymetry. It is of interest to note that a spontaneous retinal degeneration occurs in the rd mouse as a result of increased turnover of retinal cGMP phosphodiesterase (Farber et al., 1988). Perfused isolated cat eyes treated with inhibitors of phosphodiesterase activity cause ERG changes similar to those found in individuals with hereditary retinitis pigmentosa (Sandberg et al., 1987). These studies suggest the possibility, although theoretical, that OP-induced retinal degeneration may occur if a substantial inhibition of cGMP occurs in addition to inhibition of retinal ACHE. Tacrine (9-amino-l,2,3,4-tetrahydroacride hydrochloride hydrate) is a cholinesterase inhibitor of potential use in Alzheimer's disease, based on the ability to facilitate cholinergic function in the CNS via direct stimulation of M- and N-receptors through inhibition of AChE (Avery et al., 1997; Newhouse et al., 1997). Since ACh plays a fundamental role in visual function (Famiglietti, 1983; Hutchins, 1987; Ross et al., 1985), and because some visual symptoms and optic nerve degeneration occur in Alzheimer's disease patients (Hinton et al., 1986) andthe decrease in ACh found in the CNS may also occur in the retina (Strenn et al., 1991), it was considered appropriate by Alhomida et al. (2000) to investigate the effects of tacrine on human retinal AChE activity. In vitro studies on human retinal homogenates showed a concentrationdependent inhibition of AChE activity by tacrine, with an
438
SECTION IV. O r g a n T o x i c i t y
IC50 of approximately 45 nM. The Michaelis-Menten constant (Km) for the hydrolysis of acetylthiocholine iodide by retinal AChE was 0.12 mM, which increased in the presence of tacrine. The Vmax was determined to be 2.23 ~mol hr-1 mg protein-1 and was decreased by tacrine treatment. Dixon and Lineweaver-Burk plots, and their secondary replots, indicated that the inhibitory process of tacrine on human retinal AChE was a combination of competitive and noncompetitive processes. Several human clinical and epidemiological studies have drawn attention to a potential for, or a suggestion for, retinal toxicity from exposure to anti-ChEs. A cross-sectional study of fenitrothion sprayers in India found macular degeneration i n 16% of workers compared to 3% of controls (Misra et al., 1985). Morse et al. (1979) described ocular toxicity, including a 22% incidence of macular degeneration, in workers exposed to methomyl [S-methylN-(methylcarbamoyloxy) thioacetimidate] in a pesticide manufacturing plant. Fluorescein angiography suggested that the macular lesion was a consequence of a lesion in the pigment epithelium. In a cohort study of licensed pesticide applicators from Iowa and North Carolina, there was evidence that retinal degeneration was associated with CM insecticides (Kamel et al., 2000). Schoolchildren from Saku, Japan, demonstrated a 65% incidence of optic neuritis and/or chorioretinal atrophy (Ishikawa et al., 1971). In Japanese farmers chronically exposed to pesticides including OPs, the incidence of optic neuritis was increased (Imaizumi et al., 1971).
F. Extraocular Muscles Extraocular muscles have the highest activities of AChE in the body (Hikita et al., 1973; Mukuno and Imai, 1973; Mukuno et al., 1973). Anti-ChE eyedrops in the treatment of glaucoma commonly cause brief (few minutes) eyelid twitching. Similar effects may be seen with systemic intoxication, which has been observed with tetraethyl pyrophosphate given intramuscularly in patients with myasthenia gravis (Grob and Harvey, 1949). Mukuno and Imai (1973) studied the extraocular muscles of ethylthiometon-dosed dogs. They found decreased ChE activity, and electron microscopically there was degeneration of nerve fiber bundles, neuromuscular junction, and muscle fibers, with mitochondrial degenerative changes. Hikita et al. (1973) studied ChE activity in the extraocular muscles of ethylthiometon chronically dosed dogs at 0 . 5 - 1 . 5 m g k g -1 per day. AChE and BChE were inhibited in the muscles in a dose-related manner. They also noted that retinal cholinesterase activity was decreased in a dose-dependent manner (3, 50, and 70% inhibition). Extraocular muscle effects have been reported as neuropathies from anti-ChE poisoning. Suicidal poisoning by the use of OPs has been reported to cause bilateral third or sixth paralyses in 8/200
patients and facial nerve paralysis in 12/200 patients (Wadia et al., 1974).
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Reproductive Toxicity of Organophosphate and Carbamate Pesticides SURESH C. SIKKA AND NILGUN GURBUZ Tulane University Health Sciences Center, New Orleans, Louisiana
expressed serious concerns about the estrogenic effects of environmental xenobiotic chemicals, such as polychlorinated biphenyls, dichlorodiphenyl trichloroethane (DDT), dioxin, and some pesticides, especially organophosphates (OPs) and carbamates (CMs) (Boris, 1965; Colborn et al., 1993; McLachlan and Arnold, 1996). The potential effects these chemicals may have on human health and ecological wellbeing include reproductive tract cancers, reduced fertility, and abnormal sexual development. Reproductive toxicity is expressed as alterations in sexual behavior and performance, infertility, and/or loss of the fetus during pregnancy. Exposure to chemical substances may include alterations to the female and male reproductive system; adverse effects on onset of puberty, gamete production and transport, reproductive cycle normality, sexual behavior, fertility, and parturition; and premature reproductive senescence or modifications in other functions that are dependent on the integrity of the reproductive systems of both female and male species (Colborn et al., 1993; Sharpe and Skakkebaek, 1993; Kelce et al., 1994; Kumar, 2004). Toxicants that target the male reproductive system can affect sperm production and their motion, morphology, and function, and can alter sexual behavior and performance. This can cause infertility, erectile dysfunction (ED), and reduced quality of life (Sikka, 1999a,b). Many of these chemicals mimic or otherwise disrupt the estrogens or the androgen balance in the body by binding to hormone receptors during fetal and neonatal development, and these are called "endocrine disruptors" or "gender benders." Newer tools for the detection of Y chromosome deletions have further strengthened the hypothesis that the decline in male reproductive health and fertility may be related to the presence of these toxic chemical compounds in the environment. The development of intracytoplasmic sperm injection (ICSI), a technique introduced in the early 1990s, is beyond doubt the most important recent breakthrough in the treatment of male infertility. This was made
I. I N T R O D U C T I O N Many environmentalists believe that the human species is approaching a fertility crisis based on evidence of the past 20-30 years that has shown disturbing trends in male reproductive health (Guillette et al., 1994). Many others, however, think that the available data are insufficient to deduce worldwide conclusions (Fisch et al., 1996). Although these assertions have been disputed, the fact remains that one in six couples have trouble conceiving, with males equally responsible for infertility. Reduced fertility in males is one of the major end points of reproductive toxicity and is the focus of this chapter. Interference with the action of androgen during development can cause male reproductive system abnormalities that include reduced sperm production and/or fertilizing capability. It is also possible that the genetic information of the sperm may potentially be altered prior to fertilization, which can result in birth defects in the offsprings (Parvinen et al., 1984). However, the evidence that such environmental chemicals cause infertility is still largely circumstantial. There are many missing links in the causal chain that would connect receptor binding to changes in reproductive health with decreased fertility. In the past decade, the occupational health and safety community has directed greater attention to pesticide exposures among workers and their families. Initiatives focused on minority workers, women, and the children of workers have also made pesticide exposure assessment a timely topic for scientific investigation and medical management. With discoveries of deformed frogs in Minnesota lakes and fertility problems in alligators found in Lake Apopka in Florida attributed to embryonic exposure to such pollutants (Guillette et aL, 1994), a myriad of environmental agents and pesticides have been classified as reproductive toxicants. This has been the subject of a number of reviews (Kavlock and Perreault, 1994; Sokol, 1994; Sikka, 1997; Lamb, 1997; Cheek and McLachlan, 1998). Several investigators have Toxicology of Organophosphate and Carbamate Compounds
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possible by many well-controlled clinical studies and basic scientific discoveries in the physiology, biochemistry, and molecular and cellular biology of the male reproductive system. Despite these developments, the etiology, role of environmental toxicants, diagnosis, and treatment of male factor infertility remain a real challenge. Scientists have investigated the mouse embryonic stem cell approach using a precise combination of cellular growth factors to grow stem cells into mature sperm in the laboratory. This may lead to new approaches in understanding the pathophysiology and treatment for male infertility since the factors needed to grow mouse stem cells would probably also foster the growth of human sperm.
II. B A C K G R O U N D Pesticides are a heterogeneous group of chemicals developed to control a variety of pests and are generally categorized according to the type of pest for which they have been shown to be efficacious. The term "pesticides" is a general name that includes many chemicals, mostly in the classes of insecticides (OPs, organochlorines, CMs, and pyrethroids) and herbicides (bipyridyl compounds). Other categories include fungicides, termiticides, rodenticides, algaecides, repellents, miticides, and wood preservatives. OP and CM pesticides are an integral part of modem agriculture, whereas many organochlorine pesticides have been withdrawn from such usage due to their persistence in the environment. The use of OPs and CMs has soared due to their availability and quick degradation in the environment. Although OPs and CMs degrade quickly and are much less persistent in the environment, they are much more toxic to mammals, especially at high doses. Because of their adverse health effects, various governmental agencies set limits on allowable levels of pesticide residues in foods, animal feeds, and the environment. In order to enforce these allowable levels, pesticides are monitored in various types of samples. Most exposures are through respiration or dermal routes, although humans and animals may be poisoned accidentally or maliciously. The field of pesticide exposure assessment is complex and challenging. Exposures occur through multiple routes and are highly variable. Risks associated with pesticide handling differ substantially for different activities and from those experienced by agricultural reentry workers. Different assessment and control strategies are needed for each population. Families of pesticide handlers can be exposed to pesticides, and consideration of their children as a vulnerable subpopulation will likely lead to changes in the agricultural workplace that will reduce exposures for workers and families alike. Pesticides, which are designed to interfere with the function of the insect nervous system, interfere with the function of the mammalian nervous system at higher doses. In fact, the fetus and neonate are more vulnerable than the adult to insult by such pesticides because the drug-binding capacity of fetal
serum and tissue protein is significantly lower than that of the adult animal. Since the pituitary-gonadal axis differentiates during both prenatal and postnatal phases, it is vulnerable to toxic insult during a relatively long period of development. This may result in persistent endocrine dysfunction in the mature offspring leading to reproductive problems (Cranmer et al., 1978). OP and CM insecticides are widely used in both agricultural and landscape pest control. The potential for human exposure to this class of compounds is significant since OPs have the properties of low bioaccumulation and high rate of biodegradation (Sarkar et al., 2000; Tamura et al., 2001). In the mid-1970s, it was determined that dibromochloropropane (DBCP) exposure impaired fertility in the absence of any other clinical signs of toxicity, suggesting that the reproductive system was the most sensitive target organ. Reduced fertility, embryo/fetal loss, birth defects, childhood cancer, and other postnatal structural or functional problems were the most common outcomes from such exposures (Sokol, 1994). However, the database for establishing safe exposure levels or risk assessment for such outcomes remains very limited. Declining semen quality is not the only indicator that human reproduction is at risk. In fact, a marked increase in the incidence of testicular cancer in young men has been associated with other abnormalities (including undescended testis, Sertoli cell-only pattern, and hypospadias) that cause poor gonadal function and low fecundity rates. The human male produces relatively fewer sperm on a daily basis compared with many of the animal species used for toxicity testing. A less dramatic decrease in sperm numbers or semen quality in humans can have serious consequences for reproductive potential. In fact, in many men older than age 30, the lower daily sperm production rate already places them close to the subfertile or infertile range (Working, 1988). Decreased semen quality (low sperm number, motility, and structure) during the past 50 years has been attributed to environmental toxicants, many of which act as "estrogens" (Sharpe and Skakkebaek, 1993). This "estrogen hypothesis" has inspired a number of debates and serious investigations (Sharpe and Skakkebaek, 1993; Kelce et al., 1994). Does that make men less fertile? After all, it takes only one sperm to fertilize an egg. Problems in the production, maturation, and fertilizing ability of sperm are the single most common cause of male infertility. Even if produced in adequate numbers, sperm can have poor motility, viability, morphology, stay immature, and lack acrosome and other characteristics that will prevent them from fertilizing an oocyte. An environmental agent may disrupt reproductive function in the male at several potential target sites, most prominent being pituitary and testes that are under the control of the hypothalamus (Fig. 1). A dramatic increase in our knowledge of reproductive toxicity and infertility has resulted from advances in the understanding of gonadal function and dysfunction. Although any discussion of gonadal function and toxicity is of special relevance to man,
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FIG. 1. Scheme showing HPG axis in the male and potential target sites of action of organophosphate and carbamate pesticides.
much of the understanding of such target sites has been obtained from research using animal species and various experimental models (Maitra and Sarkar, 1996).
III. M A L E R E P R O D U C T I V E AS T A R G E T S I T E
1. SEMINIFEROUSTUBULES The proliferation of the mesenchyme separates the sex cords from the underlying coelomic epithelium by the seventh week of fetal development. These sex cords become the seminiferous tubules that develop a lumen after birth.
TRACT
A. Composition of Testis The male gonads in human usually exist in pairs and are the sites of spermatogenesis and androgen production. Spermatozoa are the haploid germ cells responsible for fertilization and species propagation. There are paracrine and autocrine regulations in various compartments of testis that are under the endocrine influences from the pituitary and hypothalamus. The testes develop abdominally at the renal level and descend into the scrotum. Embryonically, the testes develop from the testicular ridge located at the rear of the abdominal cavity. In the last months of fetal life, they begin a slow descent, passing out of the abdominal cavity through the inguinal canal and into the scrotum. The descent is usually complete by the seventh month. Primitive germ cells migrate to the medial surface from the yolk sac, cause the coelomic epithelial cells to proliferate, and form the sex cords that lead to the formation of major components of the testis. Approximately 80% of the testicular mass consists of highly coiled seminiferous tubules within which spermatogenesis takes place. The remaining 20% consists of Leydig cells and Sertoli cells, whose main job is to establish normal spermatogenesis.
2. RETE TESTIS During the fourth month, sex cords become U-shaped, and the ends anastomose to form the rete testis, which provides communication with the epididymis. The primordial sex cells are referred to as prespermatogonia and the epithelial cells of the sex cords as Sertoli cells. 3. LEYDIG CELLS These cells arise from interstitial mesenchymal tissue between the tubules during the eighth week of human embryonic development. They are located in the connective tissue between the seminiferous tubules. Leydig cells are the endocrine cells in testis that produce testosterone from cholesterol via a series of enzymatic pathways and steroidal intermediates under the control of luetinizing hormone (LH) from pituitary. The effects of testosterone can be grouped into the following categories: 1. Effects on the reproductive system before birth: Before birth, testosterone secretion by the fetal testes is responsible for masculinizing the reproductive tract and external genitalia and for promoting descent of the testes into the scrotum. After birth, testosterone secretion ceases, and the testes and remainder of the reproductive system remain small and nonfunctional until puberty.
450
SECTIONIV. Organ Toxicity
2. Effects on sex-specific tissues after birth: At puberty, the Leydig cells start secreting testosterone once again, and spermatogenesis is initiated in the seminiferous tubules for the first time. Testosterone is responsible for the growth and maturation of the entire male reproductive system as well as the libido. Ongoing testosterone secretion is essential for spermatogenesis, for maintaining a mature male reproductive tract throughout adulthood. 3. Other related effects: These include the development of libido at puberty; maintenance of the sex drive in the adult male; controlling the secretion of LH by the anterior pituitary via feedback mechanisms; the development and maintenance of male secondary sexual characteristics; and general protein anabolic effects, including bone growth and induction of aggressive behavior. Low levels of testosterone and decreased libido lead to ED. 4, SERTOLI CELLS Within the testicles are cells that envelop the developing sperm during spermatogenesis. These cells form a continuous and complete lining within the tubular wall and establish the blood-testis barrier by virtue of tight junctions. The luminal environment is both created and controlled by these Sertoli cells, also called "nurse cells." These Sertoli cells have several functions, including the following: 1. Provide nourishment for the developing sperm cells 2. Destruction of defective sperm cells 3. Secretion of fluid that helps in the transport of sperm into the epididymis 4. Release of the hormone inhibin, which helps regulate sperm production Thus, many irregularities of spermatogenesis due to gonadotoxicity may reflect changes in function of the Sertoli cell population, and not necessarily by pathology in the germ cells. The differentiation of Sertoli cells and the formation of a competent blood-testis barrier are essential to the establishment of spermatogenesis during puberty in all species.
B. Spermatogenesis and Spermiation Spermatogenesis is a chronological process spanning approximately 42 days in the rodent and 72 days in man (Sikka and Naz, 2002). Spermatogenesis can be divided into three distinct phases: mitosis, meiosis, and spermiogenesis. The first phase is referred to as spermatogonial proliferation and renewal. During this period, relatively undifferentiated diploid spermatogonia, the immature germ cells, undergo several mitotic divisions to generate a large population of cells called primary spermatocytes. In the second phase, the spermatocytes go through the process of two meiotic divisions leading to the formation of the haploid germ cells, spermatids. In the third phase, the spermatids go through a complex series of cytological transformations and
Spermatogonium i Mitosis I"
~MeiosisI " Prophase of
J
Primary Spermatoc~e
I Meiosiselted ~compl
: nesis Spermatoge(Meiosis) /
SecondarySpermatocytes I Meiosis tl
Spermiogenesis
L_
Spermatids
FIG. 2. Scheme of mammalian spermatogenesis showing the premeiotic and meiotic stages of spermatogenesis and postmeiotic spermiogenesis with the development and maturation of the spermatozoa.
dedifferentiate to form stem cells that cyclically develop into highly specialized spermatozoa (Fig. 2). Spermiogenesis is the transformation of spermatids into the elongated flagellar germ cells capable of motility. The release of mature germ cells is known as spermiation. The germ cells comprise the majority of testicular volume, which can be appreciated easily as a smaller size if testicular damage has occurred. A significant characteristic of mitotic arrest is that the gonocyte becomes acutely sensitive to toxic agents that may completely eradicate germ cells while causing little damage to developing Sertoli cells, thus creating a Sertoli cell-only testis.
IV. P E S T I C I D E S AND M A L E REPRODUCTION Many pesticides that are termed estrogenic pollutants (e.g., from agricultural products and industrial chemicals) have significant environmental consequences due to their multiple routes of exposure, their widespread presence in the environment, and their ability to bioaccumulate and resist biodegradation. Table 1 lists such possible agents and the reproductive adverse events caused by these agents.
CHAPTER 3 2 9Reproductive Toxicity of Anticholinesterases
TABLE 1. Effects of Hormonal Disruptors on Male Reproduction a Class Environmental Organochemicals and pesticides
Agent
Adverse events
DBCP DDT PCBs Dioxins Methyl chloride
~ fertility, ~ libido, embryo fetal loss, birth defects, cancer, estrogenic effects, poor semen quality HPG axis, $ spermatogenesis, CNS effects, testicular damage Germ cell and Leydig cell damage, steroidogenesis, 1' ROS, poor sperm morphology, ~, antioxidants, sperm function, LPO, 1' cytokines
aAbbreviafions used: DBCP, dibromochloropropane; DDT, dichlorodiphenyltrichloroethane; HPG,hypothalamic-pituitary-gonadalaxis; ROS, reactive oxygenspecies;LPO, lipid peroxidation.
A. Agricultural and Industrial Chemicals Agricultural chemicals implicated in male reproductive toxicity include DDT, epichlorhydrin, ethylene dibromide, kepone, and the dioxins (Worton et al., 1977). DBCP, a nematocide widely used in agriculture, is a testicular toxicant and induces hypergonadotropic hypogonadism (Mattison, 1983; Potashnik and Yanai-Inbar, 1987). DDT, a commonly used pesticide, and its metabolites (p,p'-DDT and p,p'-DDE) have estrogenic effects in males by blocking the androgen receptors (Kelce et al., 1994; McLachlan and Arnold, 1996). The levels of serum free/bound toxicant will influence the androgen-blocking capacity (Mattison, 1983). The plasma/tissue concentration of an estrogenic toxicant depends on the detoxification and elimination mechanisms in the organism. These agents can disrupt the hypothalamic-pituitary-gonadal axis shown in Fig. 1, thus affecting the endocrine and reproductive functions (Table 1).
hydrolyzes the neurotransmitter acetylcholine (ACh). The use of CM pesticides exceeds the use of OP and organochlofine pesticides. Some of the CMs, including carbofuran, are extremely toxic to the central nervous system (CNS) (Table 2). Overexposure of individuals involved in the production, transportation, and end use of these CMs can result in serious adverse health effects due to adverse events in the CNS and other vital organs. Similarly, carbofuran elicits the sign of acute intoxication by virtue of the reversible inhibition (carbamylation) of AChE at the synapses and neuromuscular junctions (Gupta, 2004; Goad et al., 2004). Cholinesterase has been detected in almost all major systems of the mammalian body, including the white matter of the brain, vascular system, respiratory system, digestive system, urogenital system, and certain endocrine systems. AChE is an enzyme located mainly in the nervous system and in the motor end plates of the skeletal muscle. Cholinesterase inhibitors were shown to modify the pituitary thyroid and pituitary-adrenal axes and to affect prolactin levels. When AChE is inhibited by cholinesterase inhibitors such as OP compounds, ACh accumulates at synapses, and parasympathetic overstimulation and adrenal stimulation may occur (Guven et al., 1999). In the presence of an inhibitor of AChE, synaptic acetylcholine may increase to abnormally high concentrations, which is postulated to precipitate a "cholinergic crisis" that can be debilitating and possibly fatal (Padilla et al., 1994; Fulton and Key, 2001). Sublethal doses of these pesticides lead to alterations in reproductive performance (Maitra and Sarkar, 1995, 1996; Sarkar et al., 2000). OP compounds have multiple effects on the animal endocrine system, but studies on the human endocrine system are inconclusive (Guven et al., 1999). All of these agents have numerous other chronic effects, including carcinogenesis, delayed polyneuropathy, immunotoxicity, and endocrine, developmental, and reproductive toxicity.
TABLE 2. Potential Sites of Toxic Effects of Pesticide Poisoning
Class Chlorinated hydrocarbons
Organophosphates
V. M E C H A N I S M ( S ) O F A C T I O N OF PESTICIDES Carbamates
A. Central Nervous System Effects In mammals, the primary sites of action of OP pesticides are the central and peripheral nervous systems because they inhibit acetylcholinesterase (AChE), the enzyme that
451
Agents Methoxychlor, iindane, toxaphene, chlordane Diazinon, malathion, parathion, chlorpyrifos dichlorvos Aldicarb, carbaryl, carbofuran
Site of action Neurotoxin,CNS, kidney, liver
Irreversible inhibition of red blood cell acetylcholinesterase and plasma cholinesterase Reversible inhibition of red blood cell acetylcholinesterase and plasma cholinesterase
4 52
S E CTI O N IV
9Organ Toxicity
B. Effects on the H y p o t h a l a m i c - P i t u i t a r y - G o n a d a l Axis
Pesticides that are potential gonadotoxic agents can alter physiological control processes and affect the normal functioning of the reproductive system either by a direct chemical action of the agent on reproductive organs or indirectly via the metabolic products formed during the reaction process. These can interrupt the normal function of the male reproductive system at the level of the hypothalamic-pituitary axis; the testicular level; and/or by altering posttesticular events, such as sperm maturation, motility, and/or function. Any disruption of these events by toxicants may lead to hypogonadism, infertility, and/or decreased libido/sexual function (Wilson et al., 1990; Sikka, 1999a,b). The effect may be mild or severe, and the duration may vary from transient to severe dysfunction (Fig. 1). C. E n d o c r i n e - R e l a t e d Effects
Interactions involved in normal gonadal function and hormonal communication are very complex. Any of these loci may be involved mechanistically in a toxicant's endocrinerelated effect. Such impaired hormonal control may occur as a consequence of altered hormone synthesis, storage/release, transport/clearance, receptor recognition/binding, or postreceptor responses. 1. ALTERED HORMONE SYNTHESIS A number of pesticides possess the ability to inhibit the synthesis of various hormones by inhibiting specific enzymatic steps in the biosynthetic pathway. Some fungicides block estrogen biosynthesis by inhibiting aromatase activity. Some environmental estrogens and antiandrogens alter protein hormone synthesis induced by gonadal steroids. Both estrogen and testosterone have been shown to affect pituitary hormone synthesis directly or through changes in the glycosylation of LH and follicle-stimulating hormone (FSH) (Wilson et al., 1990). A decrease in glycosylation of these glycoproteins not only reduces the quantity but also reduces the biological activity of these hormones. Pesticides that mimic or antagonize the action of these steroid hormones could presumably alter glycosylation. 2. ALTERED HORMONE STORAGE AND/OR RELEASE Steroid hormones do not appear to be stored intracellularly within membranous secretory granules. For example, testosterone is synthesized by the Leydig cells of the testis and released on activation of the LH receptor. Thus, compounds that block the LH receptor or the activation of the 3',5'cyclic AMP-dependent cascade involved in testosterone biosynthesis can rapidly alter the secretion of this hormone. The release of many protein hormones is dependent on the activation of second messenger pathways, such as cAMP, phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5trisphosphate (IP3), tyrosine kinase, and intracellular calcium
[Ca2+]i . Interference with these processes will alter the serum levels and bioavailability of many hormones. 3. ALTERED HORMONE TRANSPORT AND CLEARANCE Hormones are transported from blood in the free or bound state. Steroid hormones are transported in the blood by specialized transport (carrier) proteins known as sex steroid hormone-binding globulin or testosterone-estrogen-binding globulin. Regulation of the concentration of these binding globulins in the blood is of practical significance because there may be either increases or decreases that could affect steroid hormone availability. For example, DDT analogs are potent inducers of hepatic microsomal monooxygenase activities in vivo (Sikka, 1999a). Induction of this monooxygenase activity by treatment with DDT analogs could possibly cause a decrease in testicular androgen as a result of enhanced degradation. Similarly, treatment with lindane (7-hexachlorocyclohexane) has been reported to increase the clearance of estrogen (Welch et al., 1971). 4. ALTERED HORMONE RECEPTOR RECOGNITION[BINDING Hormones elicit responses on their respective target tissues through direct interactions with either intracellular receptors or membrane-bound receptors. Specific binding of the natural ligand to its receptor is a critical step in hormone function. Intracellular (nuclear) receptors and response elements (such as those for sex steroids, adrenal steroids, thyroid hormones, vitamin D, and retinoic acid) regulate gene transcription in a ligand-dependent manner through their interaction with specific DNA sequences. A number of pesticides may alter this process by mimicking the natural ligand and acting as an agonist or by inhibiting binding and acting as an antagonist. The best known examples are DDT, some PCBs, alkylphenols (e.g., nonylphenols and octylphenols), methoxychlor, and chlordecone (Kepone), which can disrupt estrogen receptor function (Mueller and Kim, 1978; White et aL, 1994). The antiandrogenic action of the dicarboximide fungicide vinclozolin is the result of an affinity of this compound's metabolites for the androgen receptor (AR) (Sharpe and Skakkebaek, 1993). Interestingly, the DDT metabolite p , p ' - D D E has been found to bind also to the AR and block testosterone-induced cellular responses in vitro (Kelce et al., 1995). Some pesticides have the ability to interfere with steroid hormone receptors. These include chemicals such as the herbicide linuron (Gray et al., 1999c; McIntyre et al., 2000), metabolites of the fungicides vinclozolin (Gray et al., 1999b) and procymidone (Ostby et al., 1999), the insecticide methoxychlor (Gray et al., 1999a) and its metabolite HPTE (Maness et al., 1998), and the DDT metabolite p,p'DDE (Gray et al., 1999a). The structural diversity of these chemicals has heightened concern about the potential of other environmental chemicals to disrupt AR function and has led t o the development of models and strategies for predicting potential AR activity from chemical structure.
CHAPTER 32 9Reproductive Toxicity of Anticholinesterases Tamura et al. (2001) demonstrated that fenitrothion is a competitive antagonist of the human AR and can inhibit androgen-dependent tissue growth in vivo. Inhibition of androgen-dependent tissue growth in vivo occurred with a dose of fenitrothion (15 mg/kg) that was not associated with a significant decrease in blood AChE activity, which is often used as a biomarker for human exposure to OP pesticides. Fenitrothrion represents one of the more potent environmental AR antagonists identified to date. Structural similarities between fenitrothrion and other OP compounds make it likely that additional OP insecticides will have antiandrogenic activity. Indeed, the OP pesticide parathion has been shown to inhibit DHT binding to the AR in the rat ventral prostate. Fenitrothrion inhibits brain AChE activity and induces signs of cholinergic stress (Tamura et al., 2001, 2003). In addition, Sarkar et al. (2000) reported that quinalphos, an OP insecticide, decreased the reproductive ability in males by affecting the hypothalamic-pituitary-gonadal axis. It was reported that fenitrothion bound to AR and acted as androgen antagonist. Many of the pesticides classified as environmental estrogens can actually inhibit binding to more than one type of intracellular receptor. For example, o,p-DDT and chlordecone can inhibit endogenous ligand binding to the estrogen and progesterone receptors, with each compound having ICs0s that are nearly identical for the two receptors. Receptors for protein hormones are located on and in the cell membrane. When these hormones bind to their receptors, transduction of a signal across the membrane is mediated by the activation of second messenger systems. These include (a) alterations in G protein/cAMP-dependent protein kinase A (e.g., after LH stimulation of the Leydig cell), (b) phosphatidylinositol regulation of protein kinase C and inositol triphosphate (e.g., after GnRH stimulation of gonadotrophs and thyrotropin-releasing hormone stimulation of thyrotrophs), (c) tyrosine kinase (e.g., after insulin binding to the membrane receptor), and (d) calcium ion flux through various calcium channels and their activation. Potent pesticides can disrupt such signal transduction mechanisms and interfere with one or more of these processes with toxic implications.
453
5. ALTERED HORMONE POSTRECEPTORACTIVATION Once the endogenous ligand or an agonist binds to its receptor, a cascade of events is initiated indicative of the appropriate cellular response. This includes the response necessary for signal transduction across the membrane or, in the case of nuclear receptors, the initiation of transcription and protein synthesis. A variety of environmental compounds can interfere with the membrane's second messenger systems. For example, cellular responses that are dependent on the calcium ion flux through the membrane (and the initiation of the calcium/calmodulin-dependent cellular response) are altered by a variety of environmental toxicants. Interestingly, the well-known antiestrogen tamoxifen also inhibits protein kinase C activity (O'Brian et al., 1985). Alternatively, the phorbol esters are known to mimic diacylglycerol and enhance protein kinase C activity. Steroid hormone receptor activation (including estrogen, progesterone, and glucocorticoid receptors) can be modified by indirect mechanisms, such as a downregulation of the receptor (temporary decreased sensitivity to ligand) as seen after TCDD exposure (Safe et al., 1991; Safe, 1995). Consequently, because of the diverse pathways of endocrine disruption, any assessment must consider the net result of all influences on hormone receptor function and feedback regulation. D. M e t a b o l i c Effects of Pesticides
Mahgoub and E1-Medany (2001) demonstrated that chronic administration of methomyl oxyethanimidothioate (lannate), a CM insecticide with anti-ChE activity, led to a significant reduction in the level of testosterone hormone and a significant increase in the level of FSH, LH, and prolactin. In addition, the decrease in succinic dehydrogenase, acid phosphatase, and esterase enzyme activities in the methomyl-treated male rats significantly affected mitochondrial metabolism and caused spermatogenic arrest (Fig. 3). The levels of acid phosphatase enzyme activity increased significantly in the Leydig and spermatogenic cells after such treatment (Mahgoub and E1Medany, 2001). Acid phosphatase enzyme plays an important role in cell metabolism, autolysis, differentiation, and many
T Membrane depolarization 11C 'oc rome c ox 0asel
,
I~ Creatinine kinase
TO em nma, rane a rxn0swen0 sru0, 0
~
~Organophosphoru~ Compounds
~ Bcl-2 levels
Alteration of mitochondrial oxygen uptake and respiration
I1 phosphorglation Oxidative I
FIG. 3. The metabolic effects of organophosphorus pesticides on mitochondrial metabolism.
454
SECTION IV. Organ Toxicity
related processes. The increase in acid phosphatase enzyme activity may be explained on the basis of enhancement of cell membrane permeability with disturbance in the transphosphorylation process as a result of cellular degeneration. In the same study, an increase in oL-esterase activity was observed in the interstitial Leydig cells that affected lipid metabolism induced by cellular membrane degeneration after insecticide intoxication (Afifi et al., 1991). Kackar et al. (1999) found that chronic exposure to mancozeb (a CM pesticide) in male rats produced a significant testicular dysfunction, as indicated by a marked reduction in serum testosterone level and sperm count. Goad and coworkers (2004) also demonstrated decreased serum testesterone levels in carbofuran-treated male rats due to reduced biosynthesis and release. Benomyl is a carbendazim-derivated insecticide that primarily affects the testis by sloughing effects of the chemical on microtubules and intermediate filaments of the Sertoli cells. These effects spread to dividing germ cells and also lead to abnormal development of the head of elongated spermatids. At higher dosages, it causes occlusion of the efferent ducts, blocking the passage of sperm from the rete testis to epididymis. The mechanism of occlusion appears to be related to fluid reabsorption, followed by leukocyte chemotaxis, sperm granulomas, fibrosis, and often the formation of abnormal microcanals. The occlusion results in a rapid swelling of the testis and ultimately seminiferous tubular atrophy and infertility. Benomyl may induce permanent testicular damage and a decrease in sperm production (Hess and Nakai, 2000). Afifi and coworkers (1991) reported that the administration of dimethoate (an OP compound) to male rats for 65 consecutive days caused suppression of testicular function with significant reduction in testosterone level and the number of motile sperm due to direct toxic effects of the insecticide on testicular tissue. OP insecticides, apart from inhibiting AChE activity, bind to the lipid component of mitochondrial membranes and alter mitochondrial function (Afifi et al., 1991). Such changes also include mitochondrial membrane depolarization, membrane disruption and matrix swelling, alterations in mitochondrial oxygen uptake and respiration, and inhibition of oxidative phosphorylation. In addition, inhibition of mitochondrial carboxylesterase, creatinine kinase and succinate dehydrogenase, cytochrome c oxidase, and NADH:cytochrome c reductase has been reported following exposure to OP compounds. These compounds increase mitochondrial transmembrane potantial and Bcl-2 levels (Fig. 3) (Carlson et al., 2000). Depending on the diminished level of ATP, intracellular sodium will accumulate with consequent gain of water associated with cell swelling and dilatation of the smooth endoplasmic reticulum in Sertoli, spermatogenic, and Leydig cells (Dunnick et al., 1984). Ezeasor (1990) correlated the dilatation of the endoplasmic reticulum in Leydig cells with the reduction in cellular activity and diminished androgen synthesis.
In addition, chlorpyrifos-methyl (CPM), another OP insecticide, is known to cause toxicity in adrenal gland, such as vacuolation of the zona fasicolata, which leads to low adrenal testosterone output (Breslin et al., 1996). Interestingly, Kang et al. (2004) observed that the relative weight of the adrenal gland was increased by the treatment of CPM in male castrated rats but not in immature female rats, suggesting compensatory activation of the androgen synthesis pathway in the adrenal gland.
E. Effects of OPs on Oxidative Stress and Apoptosis "Oxidative stress" is a condition associated with an increased rate of cellular damage induced by oxygen and oxygen-derived free radicals commonly known as reactive oxygen species (ROS). Exposure to many types of environmental contaminants can enhance this oxidative process both by increasing generation of free radicals and by decreasing antioxidant potential and thus causing gonadal damage (Sikka et al., 1995). Banks and Soliman (1997) showed that methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) induces lipid peroxidation and glutathione depletion in rats due to a significant increase in serum hydroperoxides and a significant decline in hepatic reduced glutathione (GSH) levels. In addition, N,Ndiphenyl-p-phenylenediamine (DPPD) and 21-aminosteroid (U74389G) blocked benomyl-induced lipid peroxidation and GSH depletion, thus confirming that in vivo toxicity of benomyl may be associated with increased oxidative stress to cellular membranes and that some degree of protection against this toxicity could be afforded by antioxidants. Such disturbance in the balance resulting in increased oxidative stress can influence male infertility (Fig. 4). The possible mechanism for loss of testicular and sperm function due to high oxidative stress has been shown to involve excessive generation of ROS (Aitken and Clarkson, 1987). Several studies have confirmed that oxidative stress is induced by OPs in rats (Gultekin et al., 2000; Gupta et al., 2001 a; Akhgari et al., 2003) and humans (Banerjee, 2001; Ranjbar et al., 2002). Small doses of OP compounds cause delayed neuronal cell death that involves free radical generation. OPs that cause mitochondrial damage and dysfunction due to depletion of ATP and increased generation of ROS can cause fetal depletion of mitochondrial energy (ATP), induction of proteolytic enzymes, and DNA fragmentation, leading to apoptotic death (Akhgari et al., 2003). Abdollahi et al. (2004a,b) demonstrated that inhibition of cholinesterase activity by malathion, a commonly used OP, is accompanied by induction of oxidative stress, suggesting use of saliva sampling as an alternative to plasma in OP toxicity monitoring. The phospholipid component of cell membranes is suggested as a site of toxic action of OP compounds. Similarly, the generation of nitric oxide (NO) and reactive nitrogen species has been found to have an astounding range
CHAPTER 32 9Reproductive Toxicity of Anticholinesterases
Cellular Free Radical-Scavenging System Superoxide Dismutase Catalase Glutathione Peroxidase Vitamin E Carnitine Glutathione
Free Radical-Generating System Leukocytes and Inflammatory Components Toxicants Drug Radiation Immature Germ Cells
IT
It ~ T Protein Damage
Lipid Peroxidation
I
1
1
I
I Stress I T Biomembrane Damage
T Sperm Damage
l
455
INFERTILITY
of biological roles, including vascular tone, inflammation, and as a mediator of many cytotoxic and pathological effects. NO along with superoxide radicals induces endothelial cell injury, which may result in testicular dysfunction. This is primarily due to the vasoactive effects of NO- radical rather than the direct effects on testicular cells. Rao and coworkers (1999) investigated the effect of carbaryl, kepone, and malathion on NO synthase (NOS) activity in the rat brain. They showed that all three compounds inhibit NOS activity of rat brain in vitro in a concentration-dependent manner. In addition, they demonstrated that these insecticides inhibit calmodulin (CaM)-stimulated NOS activity without affecting the basal enzyme activity. According to their results, the inhibition of NOS activity by these insecticides may be due to their interaction with CaZ+/CaM (Rao et al., 1999). It is important to note that increased NOS activity, especially iNOS, due to OP exposure results in increased generation of NO free radical in inflalmmatory cells that usually combines with available superoxide ions to form highly toxic peroxynitrite radicals, resulting in increased oxidative insult and apoptotic damage (Fig. 5).
T
DNA Damage
FIG. 4. ROS generating and scavenging systems responsible for inducing oxidative stress to components of male reproductive axis leading to sperm damage and infertility.
Cytochrome c oxidase (COx), the terminal enzyme complex of the mitochondrial respiratory chain and the last site for ATP synthesis, has a critical function that if disturbed would seriously affect the energy production in the cell. Its dependency on the polyunsaturated phospholipid cardiolipin renders this enzyme especially vulnerable to peroxidative damage (Potashnik and Yanai-Inbar, 1987). Under conditions in which COx is well controlled, ROS formation is small; however, when the capacity of this enzyme is reduced, the risk for incomplete reduction of 02 and the formation of ROS increases (Staniek and Nohl, 2000). Lipid peroxidation, mitochondrial dyshomeostasis or dysfunction or damage, reduction of neuronal energy level, and reduced COx activity support the contention that AChEIs, such as diisopropyl phosphorofluoridate (an OP compound) and carbofuran (a CM compound) cause neuronal injury by excessive formation of ROS (Yang and Dettbarn, 1998; Gupta et al., 2001b). Carlson et al. (2000) observed significant OP compoundinduced increases in caspase-3 activation followed by increased nuclear fragmentation, characteristic of traditional apoptosis. High concentrations of PSP and TOTP (1 mM)
456
SECTION IV. Organ Toxicity
I Organophosphoruscompounds I
I
Mitochondrial damage/dysfunction I ATP depletion I
l Increasedgeneration of ROS
IReleaseof L-GlutamateI Stimulation of NMDA receptor sites I Activation of NOS I
1 I Increases of peroxynitrite I
I OXIDATIVESTRESS I I proteolytic Increases ~ enzymes
I DNA fragmentation I
Free radicals play an important role in toxicity of pesticides and environmental chemicals. Pesticide chemicals may induce oxidative stress, leading to generation of free radicals and alteration in antioxidants or the oxygen free radical scavenging system. This system includes SOD, CAT, gamma-glutamyl transpeptidase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, etc. (Datta et al., 1992; Banerjee et al., 1999; Sharkawy et al., 1994). Glucose-6-phosphate dehydrogenase catalyzes the initial step of the pentose phosphate pathway, whose most important function is the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH, which is used for the reduction of oxidized glutathione to the reduced state (GSH) and for the reduction of mixed disulfides of GSH and cellular proteins. This GSH is used for the detoxification of hydrogen peroxide (H202) and organic peroxides (Gurbuz et al., 2004). It has been indicated that the enzymes associated with such antioxidant defense mechanism are altered under the influence of pesticide and that lipid peroxidation is one of the molecular mechanisms involved in pesticide-induced cytotoxicity (Banerjee, 2001). It will be interesting to investigate the role of potential antioxidants in OP- and CM-induced toxicity.
FIG. 5. Mechanism of action of organophosphorus compounds on oxidative stress and apoptosis. VI. A S S E S S M E N T O F G O N A D O T O X I C I T Y induced caspase-3 activation and nuclear condensation. In addition, pretreatment with carbachol, cyclosporin A, protease inhibitors, and PMSF altered OP compound-induced caspase-3 activation and nuclear fragmentation. This suggested that multifunctional pathways such as muscarinic receptor activation or an increase in Bcl-2, mitochondrial permeability transition pore closure, receptor-mediated caspase activation, or serine protease activation were involved in OP-induced cytotoxicity (Carlson et al., 2000). The assumption that free radicals can influence male fertility has received substantial scientific support (Gagnon et al., 1991). The proposed mechanism for loss of testicular and sperm function due to oxidative stress has been shown to involve excessive generation of ROS (Aitken and Clarkson, 1987). Free radicals can damage DNA and proteins, either through oxidation of DNA bases (primarily guanine via lipid peroxyl or alkoxyl radicals) or through covalent binding to MDA resulting in strand breaks and cross-linking (Alvarez et al., 1987). ROS can also induce oxidation of critical SH groups in proteins and DNA, which will alter cellular integrity and function with an increased susceptibility to attack by toxicants. Oxidative stress is theoretically the result of an improper balance between ROS generation and intrinsic scavenging activities. Adequate levels of superoxide dismutase (SOD), catalase (CAT), and probably GSH peroxidase and reductase normally maintain the free radical scavenging potential in the testes. This balance can be referred to as oxidative stress status, and its assessment may play a critical role in monitoring testicular toxicity and infertility (Sikka, 1997).
Several methods are being evaluated for the assessment of the effects of toxicants on the male reproductive system. Essentially, any risk assessment usually has four components: hazard identification (Johnson, 1986), dose-response assessment (Mattison et al., 1990), human exposure assessment, and risk characterization (Cranmer et al., 1978). The hazard identification and dose-response data are developed from experimental animal studies that may be supplemented with data from in vitro studies. This information is then extrapolated and integrated to characterize and assess the risk to the human population. The most common approach to evaluate the effect of cytotoxic drugs on the testis has used testicular biopsy, semen analysis, and endocrine assessment of the hypothalamic-pituitary-testicular axis (Table 3). Research on testicular toxicology has been advanced significantly by the introduction of in vitro testing systems. In vivo systems, however, are still essential parts of the risk assessment process, and they are unlikely to be eliminated by in vitro models.
A. In Vitro Systems In vitro systems are uniquely suited to investigate specific
cellular and molecular mechanisms in the testis and thus improve risk assessment (Lamb and Chapin, 1993). These in vitro models can be used alone or in combination with each other to test hypotheses about testicular toxicity. A toxicant, its metabolites, the precursors, or selective inhibitors
CHAPTER 32 9Reproductive Toxicity of Anticholinesterases TABLE 3.
Potential site Testis Leydig cells
Sertoli cells
Seminiferous tubules
Evaluation of Effects of OP and CM in the Adult MaMa Effects
Evaluative tests
Necrosis LH/PRL, T biosynthesis/ secretion FSH/inhibin/ steroids, Sertoli/Leydig cell function
Weight, histopathology receptor analysis, RIA, in vitro production and hormone assay Receptor analysis, RIA, in vitro tests (coculture),bloodtestis barrier, morphology Germ cell count and % tubules without germ cells Spermatid counts and % tubules with luminal sperm Germ cell culture, morphology Histopathology, biochemical tests Pituitary cell culture, hypothalamus perfusion, histopathology, hormone challenge, accessory sex organ weights Spermatid counts, semen evaluation Hormones/ABP assays
Spermatogonial mitosis Spermatocyte meiosis
Epididymis
Spermatid differentiation Sperm maturation
Brain
Hypothalamicpituitary a x i s
Seminal fluid
Daily sperm production HPG a x i s
Blood
aAbbreviations used: LH, luteinizing hormone; PRL, prolactin; RIA, radioimmunoassay; FSH, follicle-stimulating hormone; ABP, androgen binding protein; HPG, hypothalamic-pituitary-gonadalaxis. can be individually administered to isolated cell types to evaluate specific toxicity mechanisms and to note the interaction of adjacent cell types. Numerous in vitro model systems are described in the literature, including Sertoli-germ cell cocultures (Gray, 1988), Sertoli cell-enriched cultures (Chapin et al., 1990; Steinberger and Clinton, 1993), germ cell-enriched cultures (Foster et al., 1987), Leydig cell cultures (Ewing et al., 1981), Leydig-Sertoli cell cocultures (Chapin et al., 1990), and peritubular and tubular cell cultures (Gray, 1988; Chapin et al., 1990). Use of these in vitro systems is the only way to directly compare human and animal responses and to screen a class of compounds for new product development. Although these in vitro systems are a valuable adjunct to the in vivo test system, they do not replace the in vivo data because they cannot provide all the facts essential for hazard assessment. Moreover, certain dynamic changes associated with spermatogenesis are diffi-
457
cult to model in vitro. For example, the release of elongated spermatids by the Sertoli cells (spermiation), which is commonly inhibited by boric acid and methyl chloride, can only be studied by specific in vivo systems.
B. In Vivo S y s t e m s In vivo methods are important tools to study the integrated male reproductive system. The complete in vivo assessment
of testicular toxicity involves multigenerational studies, now required by most regulatory agencies. These multigenerational studies have a complex design because testicular function and spermatogenesis are very complicated processes. The spermatogenic cycle is highly organized throughout the testis. In the rat, it requires 53 days. If a toxicant affects the immature spermatogonia, the effect may not be detectable as a change in mature sperm before 7 or 8 weeks. Effects on more mature germ cells would be detected sooner. To test the sensitivity of all stages of spermatogenesis, the exposure should last the full duration of the cycle. This cannot be achieved in vitro because germ cell differentiation and the physical relationship of stages within the tubules are lost in cell culture systems. The germ cells are entirely dependent on the Sertoli cells for physical and biochemical support. Complicated endocrine and paracrine systems control Sertoli cells, Leydig cells, and germ cells. Besides the loss of paracrine interactions, the altered metabolic activity of target or adjacent cells and difficulty in isolating and testing certain spermatogenic stages are other significant limitations of in vitro assessment of testicular toxicity (Soto et al., 1992). In addition, for accurate identification of stage-specific lesions of the seminiferous epithelium, critical evaluation of morphological structures is very important. Because germ cells are continuously dividing and differentiating, the staging of spermatogenesis has proven to be an extremely sensitive tool to identify and characterize even subtle toxicological changes. The effects of chronic sublethal doses ( 7 - 1 4 m g k g -1 daily for 15 days) of quinalphos were evaluated in adult male rats for changes in testicular morphology, circulatory concentrations of hormones (LH, FSH, prolactin, and testosterone), activities of AChE and angiotensin-converting enzyme, as well as metabolism of biogenic amines (dopamine, noradrenaline, and 5-hydroxytryptamine) in the hypothalamus and pituitary. It may be postulated that the initial effects of the pesticide are the result of increases in LH and testosterone concentrations, which affect spermatogenesis. Pesticideinduced inhibition of AChE in turn might increase concentrations of ACh in the pituitary and hypothalamus. This indicates that in pesticide toxicity, the hypothalamic-pituitary-gonadal axis is operational. Since many of the observed pesticide effects could be inhibited by estradiol, it is suggested that the pesticide acts directly on the gonadotrophins. In conclusion, quinalphos decreases fertility in adult male rats by affecting the pituitary gonadotrophins (Sarkar et al., 2000).
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SECTION I V .
Organ Toxicity
C. Sperm Nuclear Integrity Assessment Chemicals may disrupt the structural stability of sperm nuclei, which depend on their unique packaging during either spermatogenesis or sperm maturation. Decondensation of an isolated sperm nucleus in vitro can be induced by exposure to disulfide reducing agents, and the time taken to induce extensive decondensation (assay end) is considered to be inversely proportional to the stability of the sperm nucleus. Attention has focused on assessments of sperm morphology and physiology as important end points in reproductive toxicology testing (Darney, 1991). Structural stability of sperm nuclei varies by species, appears to be enhanced by the oxidation of protamine sulfhydryl to inter- and intramolecular disulfide bonds, and is a function of the types of protamine present. This "sperm activation assay" is also useful in the evaluation of some cases of unexplained infertility (Brown et al., 1995). Human sperm decondenses most rapidly, followed by that of the mouse and hamster, whereas rat sperm nuclei show a slower decondensation (Perreault et al., 1988). Other tests, called DNA stability assay or sperm chromatin structure assay (SCSA), use direct evaluation of sperm chromatin integrity and may provide information about genetic damage to sperm. A shift in DNA pattern (from double-stranded intact DNA to denatured singlestranded) can be induced by a variety of mutagenic and chemical agents and evaluated either by DNA flow cytometric analysis or by SCSA (Evenson et al., 1986; Evenson, 1989). A single cell gel electrophoresis (Comet) assay, which uses fluorescence intensity measurements by microscopy and image analysis, has also been developed (Brown et al., 1995). A shift in the DNA pattern can also be evaluated by acridine orange staining, in which doublestranded DNA is stained green and single-stranded DNA is stained red. Animals exposed to known mutagens demonstrate increased amounts of single-stranded DNA, indicating an increase in genetic damage (Evenson et al., 1991; Ulbrich and Palmer, 1995). DNA flow cytometry is a very useful tool that permits rapid, objective assessment of a large number of cells but may not be readily available. Comet assay, when combined with centrifugal elutriation, can provide a useful in vitro model to study differences in metabolism and the susceptibility of different testicular cell types to DNA-damaging compounds. Thus, new findings using these systems should lead to greater knowledge about why a chemical or class of chemicals can cause testicular toxicity.
VII. S C I E N T I F I C D E B A T E In the wake of media coverage on possible reproductive health and cancer concerns (Raloff, 1994), a few toxicologists have questioned whether these adverse health effects can be attributed to environmental endocrine disruption
(Stone, 1994; Safe, 1995). Arguments for a demonstrable link between hormone-disruptive environmental agents and human reproductive health effects are supported by the fact that many pesticides and other agents with estrogenic or antiandrogenic activity operate via hormone receptor mechanisms. However, in the few studies on suspected weak estrogens, such as the alkylphenols, approximately 1000-10,000 times or up to 106 more agent is required to bind 50% of the estrogen receptor than estradiol (White et al., 1994). Of course, crucial to risk assessment is the need to know how many receptors must be occupied before activation of a response can ensue. For some hormones, such as human chorionic gonadotropin, as little as 0.5-5% receptor occupancy is required for full activation of response. For other hormones (those that require protein synthesis for expression of effect), higher levels of receptor occupancy are needed. Fluctuations of hormone concentration and receptor activities, by design, absorb some environmental and physiological challenges to maintain homeostasis in adults. Only when the equilibrium control mechanisms are overwhelmed do the deleterious effects occur. An important question is whether homeostatic mechanisms are operative in the embryo and fetus. Some investigators (Soto et al., 1992) have proposed the use of in vitro assays to screen for estrogenic or other hormonal activity. Although steroid receptors bound to their ligand act as transcription factors for gene expression in the target tissue, simple in vitro screening assays based on binding to a receptor are not sufficient for measuring hormone activity.
VIII. C O N C L U S I O N S Humans have experienced increased incidences of developmental, reproductive, and carcinogenic effects caused by pesticides acting to disrupt the endocrine system, which regulates these processes. In contrast, the hypothesis that the reported increased incidence of human cancers and reproductive abnormalities and infertility can be attributed to an endocrine disruption phenomenon is called into question for several reasons. First, secretion and elimination of hormones are highly regulated by the body, and mechanisms for controlling modest fluctuations of hormones are in place via negative feedback control of hormone concentrations. Therefore, minor increases following dietary absorption and liver detoxification of these xenobiotics may be inconsequential in disrupting endocrine homeostasis. Second, low ambient concentrations of chemicals along with low-affinity binding of purported xenobiotics to target receptors probably are insufficient to activate an adverse response in adults. Whether the fetus and the young are capable of regulating minor changes to the endocrine milieu is uncertain. Finally, the data are not available for mixtures such pesticides that may be able to affect endocrine function. At the same time, in the case of
CHAPTER 32
environmental estrogens as endocrine disrupters, it is known that competition for binding sites by antiestrogens in the environment may moderate estrogenic effects of some chemicals. Clearly, more research to fill data gaps and to remove the uncertainty in these unknowns is needed. With few exceptions (e.g., DES), a causal relationship between exposure to a specific environmental agent and an adverse effect on human health operating via an endocrine disruption mechanism has not been established. Short-term screening studies could be developed and validated in an effort to elucidate the mechanism. Through controlled dose-response studies, it appears that these compounds (e.g., alkyl phenol ethylates and their degradation products, chlorinated dibenzodioxins and difurans, and polychlorinated biphenyls) can induce irreversible induction of male sex characteristics on females (imposex), which can lead to sterility and reduced reproductive performance. There is an urgent need to characterize all the factors involved in such pesticide-induced reproductive toxicity and to develop reliable animal models of testicular disease. No major advances have been made regarding the medical management of poor sperm quality. The application of assisted reproductive techniques such as Y-chromosome deletion evaluation followed by ICSI regardless of cause does not necessarily treat the cause and may inadvertently pass on adverse genetic consequences.
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CHAPTER
~
Placental Toxicity of Organophosphate and Carbamate Pesticides OLAVI PELKONEN, 1 KIRSI VAHAKANGAS, z AND RAMESH C. GUPTA3 1University of Oulu, Oulu, Finland 2University of Kuopio, Kuopio, Finland 3Murray State University, Hopkinsville, Kentucky
diseases, such as diabetes (Radaelli et al., 2003; Bjarneg~d et al., 2004) or hypertension (Sgambati et al., 2004; Ockleford et al., 2004), may have altered function, and these fetuses may be at an increased risk for toxic effects. This chapter provides some background information for the understanding and prediction of what pesticides can, in principle, do to the placenta and what the placenta does to pesticides. Also, a survey of methodologies employing human placenta as a tool to investigate and predict some aspects of developmental toxicity is presented. Finally, our current knowledge on the toxicokinetics and toxicodynamics of pesticides in the placenta and how these processes affect the fetus and pregnancy in general is reviewed.
I. I N T R O D U C T I O N During pregnancy, placenta serves many functions, including the production and release of hormones and enzymes; transport of nutrients and waste products; chemical information flow between mother and fetus; implantation; cellular growth and maturation; and, at the terminal phase of placental life, participation in delivery. The placental functions are carried out by two cell types, cytotrophoblasts and syncytiotrophoblasts, probably with overlapping tasks. These cells, together with vascular endothelial cells and connective tissue cells, form a layer of cells connecting and separating two genetically distinct individuals: the mother and the conceptus. In the assessment of placental toxicology of any foreign chemical substances, there are two major areas of concern: what the placenta does to xenobiotics and what xenobiotics do to the placema (Myllynen et al., 2005). In the former area; the major topics of concern are the entry and possible storage of substances in placental cells and through the placenta, aided perhaps by various transporters and efflux pumps; the distribution and binding of compounds in placental cells; and biotransformation of substances by intracellular enzymes. Metabolic activation and production of reactive intermediates by placental enzymes link these areas with toxicodynamics of placental toxicants. In the latter area, effects of compounds on placental blood flow and vasculature and the presence of membrane and intracellular receptors, enzymes, and other potential targets for foreign substances are important areas of inquiry for placental toxicity. The health of the placenta is a prerequisite for the health of the fetus. Consequently, any xenobiotic-induced damage to the placenta may cause damage to the fetus. Thus, it is very important to identify xenobiotics damaging the placenta and to elucidate their mechanisms of action associated with the toxicity. Furthermore, placentas of mothers with certain Toxicology of Organophosphate and Carbamate Compounds
II. S P E C I F I C A S P E C T S O F P L A C E N T A L TOXICITY STUDIES A. Species Differences in Placental Structure and Function Anatomically, the placental architecture is composed of cells derived from fetal membranes. Placentation differs among differentspecies and is therefore classified into four types: hemochorial (human, rat, and rabbit), endotheliochorial (cat and dog), syndesmochorial (sheep and ruminants), and epitheliochorial (pig and horse). The placenta has also been described as zonary in the dog, bidiscoid in the monkey, and multicotyledonary in the sheep. The placental thickness depends on the number of fetal and maternal cell layers. For example, the rat and rabbit have a single layer of cells, primates and humans have three layers, and pigs and horses have six layers. There are anatomical and qualitative physiological functional similarities and differences in placentas of different species that should be taken into consideration when studying placental toxicity. The human placenta is of 463
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464
SECTION IV. O r g a n T o x i c i t y
the hemochorial type, in which the fetal tissue is in direct contact with the maternal blood. The membrane separating the maternal and fetal compartments is thin and consists of the trophoblastic epithelium covering the villi, the chorionic connective tissue, and the fetal capillary endothelium. The average thickness of the barrier at term is 3.5 I~m, the average exchange area is approximately 11 m 2, and the placental blood flow rate is approximately 450 ml/min (Pacifici and Nottoli, 1995). It is well established that the intensity of passage of substances across the placenta is inversely proportional to the thickness of placental membranes. In hemochorial placenta, the intensity of exchange decreases at the end of gestation due to deposition of fibrinoid on the exchanging surface. Exchange involves not only physiological constituents but also substances or chemicals that represent a pathological risk for the placenta and/or fetus. Therefore, in assessing placental toxicology of chemicals, all physiological and functional variables should be taken into consideration. Note that the toxicological findings observed in the placenta of one species for a particular chemical should not be generalized to the placenta of another species. In other words, wide species variability exists in sensitivity to acetylcholinesterase (AChE)-inhibiting pesticide-induced placental toxicity. Furthermore, a wide interindividual variability for susceptibility exists during the gestation period.
TABLE 1.
Cell type
B. Life Span of the H u m a n Placenta The appreciation of the complexity of placenta as an organ has increased. During its lifetime of 9 months, it goes through a complicated development that requires highly proliferative and invasive capacities as well as an extremely tight control of the process (Chakraborty et aL, 2003). The expression of many factors is very different at different stages of pregnancy. For instance, the expression of Fas ligand, protecting placenta from cell-mediated immune response, decreases at term (Balkundi et al., 2000). Anatomical, physiological, and metabolic characteristics vary depending on the age of the placenta. Thus, it is important to study placentas from different stages of pregnancy. However, obtaining material for such studies is difficult for practical and ethical reasons. Since the placental thickness and the number of cell layers diminish toward the end of pregnancy, it is possible that the term placenta is the most sensitive to environmental agents. However, deposition of fibrinoid may be a counteracting factor. In term placenta, there are two cell layers (syncytium and endothelium) with basal laminae and connective tissue in between (Jinga et al., 2000). Information on the detailed cellular structure of the placenta is still emerging (Ockleford et al., 2004). Histologically, several cell types with their specialized functions can be recognized in the placental tissue (Table 1). Whereas some factors, such as endothelin,
Placental Cell Types and Their Functions a
Specific markers
Reported functions and characteristics
References
Anchorage and invasive functions, mononuclear, do not fuse Transplacental transfer, multinucleated endocrine functions
Ockleford et al. (2004), Old et al. (2004) Old et al. (2004), Malassine and Cronier (2002), Guyon et al. (2004)
Progenitor cells for syncytiotrophoblasts Hemostasis
Old et al. (2004), Guyon et al. (2004)
Extravillous trophoblast (EVT)
hPL, ErbB2, CAM
Syncytiotrophoblast (S cell)
hPL, ErbB 1, ErbB2, NPD kinase C
Villous cytotrophoblast (C cell)
ErbB 1, NDP kinase A and B Tissue factor Cytosolic PGES Cytosolic PGES vWf, ACE, BS-I lectin
Immunologic function Granular cytoplasm
Strong expression of vWf, ACE, UEA lectin, WGA lectin vWf, ACE
Long life span, higher proliferation than large placental vessels, rod-like cytoplasmic particles Granular cytoplasm
Jinga et al. (2000)
oL-Smooth muscle actin, [3-tropomyosin, h-caldesmon
Elongated cells in parallel rows
Leik et al. (2004)
Decidual cells Villous fibroblasts Villous macrophages Endothelial cells from large vessels Endothelial cells from villous microvessels Endothelial cells from umbilical cord Smooth muscle cells of arterial wall
aAbbreviations used: CAM, cell adhesion molecules; hPL, human placental lactogen; vWf, von Willebrand factor.
Lockwood et al. (2001) Meadows et al. (2004) Meadows et al. (2004) Jinga et al. (2000)
Jinga et al. (2000)
CHAPTER 33 9Placental Toxicity of OPs and CMs are produced by most of the cell types of the placenta (Chakraborty et al., 2003), others are strictly expressed by one cell type only, such as cytokeratin-7, which is specific for trophoblast (Oki et al., 2004). Nitric oxide, produced by endothelial cells, diffuses to vascular smooth muscle cells, where it regulates the vessel diameter by relaxing the smooth muscle cells (Bisseling et al., 2004). Such local mediators are important in the placenta because it lacks autonomic innervation.
III. G E N E R A L A S P E C T S O F P L A C E N T A L TOXICOKINETICS An attempt to encompass salient features of transplacental transfer and potential major factors governing various processes in the transfer is presented in Fig. 1. Our understanding of detailed placental toxicokinetic processes has advanced thus far because there is a need to understand the fate and effects of drugs in pregnant subjects (Ala-Kokko et al., 2000). Furthermore, studies on drugs use relatively high and fixed doses, obviating the analytical sensitivity problems. Studies on pesticides need to be performed at a somewhat more elementary level and only in experimental animals. Thus, extrapolation to the human in vivo situation, in which exposures are at a relatively low level, may pose considerable problems and uncertainties. Although some toxicokinetic processes (e.g., metabolism, plasma protein binding, and membrane permeation) can be studied in in vitro systems, these systems are often based on liver (e.g., metabolism), making extrapolation to the placental situation problematic.
465
A. Entry of Xenobiotics into Placental Cells A paradigm accepted practically without reservation until just a few years ago was that foreign chemicals are transferred through placental membranes by passive diffusion. The rate and extent of transfer were thought to be dependent on surface area, blood flow, concentration gradient across placental membranes, and physicochemical properties of a chemical, such as lipophilicity, molecular weight, and degree of ionization (Pacifici and Nottoli, 1995). Although this paradigm is still true to a certain extent, it has to be modified by the emerging appreciation of the role played by various transporters, which in some cases are able to also accept xenobiotic substances. Table 2 lists some general properties of transporters that affect the entry and transfer of at least a few xenobiotic chemicals. The demonstration of a chemical as a ligand for a particular transporter does not automatically guarantee that the particular transporter is important for the transfer of the compound in vivo. It is possible that passive transfer could still be far more important than active transport, depending on the relative importance of these two ways of transfer and on other factors such as concentration of the compound.
B. Distribution of Xenobiotics in the Placenta It has been demonstrated in a number of cases that some xenobiotic substances bind relatively tightly, although reversibly, to placental components. Good evidence on binding has been provided for several drugs (Ala-Kokko et al., 2000). In these cases, placenta serves as a kind of a sink, which has to be saturated before substances can be transferred along the concentration gradient to the opposite side of the placenta. In all of the examples mentioned by Ala-Kokko et al., it is not known which placental components are responsible for binding. However, it is a good guess that certain placental proteins and small molecular xenobiotics may have mutual affinity, which is then reflected as an attenuation of transplacental transfer.
C. Metabolism of Xenobiotics in the Placenta
FIG. 1. Scheme of potential factors in placenta that affect the transplacental transfer and ensuing effects on placenta and fetus. Although very little is known about the role of transporters, metabolizing enzymes, binding proteins, and cellular targets in pesticide kinetics and dynamics, there are a number of experimental tools available for the elucidation of these factors.
The presence of various classes of the xenobiotic-metabolizing enzymes in human placenta has been demonstrated convincingly. However, on closer inquiry one has to come to a conclusion that regarding the enzyme profile and metabolic capacity, placenta is far more restricted than the liver (Pasanen, 1999; Hakkola et al., 1998). First, the most important phase I enzymes, cytochrome P450 (CYP) enzymes, are relatively sparingly expressed in the placenta. It is clear that when the expression has been studied with sensitive reversetranscriptase polymerase chain reaction (RT-PCR) methods, some expression can be observed for many CYP enzymes belonging to families 1-3, which are mainly responsible for xenobiotic metabolism (Hakkola et al., 1996a,b). However, regarding the expression of proteins, only the expression of
466
SECTION I V .
TABLE 2.
Transporter
Organ Toxicity
Expression and Detection of MDR and MRP Proteins in Human Placenta during the First Trimester and at Term a
First trimester
Suggested transport function in human placenta
At term/location
MDR1
+
Brush-border membranes of absorptive cells
MRP1
+
Syncytiotrophoblast cells, trophoblasts, abluminal side of blood vessels in villi
MRP2
+
Apical syncytiotrophoblast membrane
MRP3
+
MRP5
+
Vascular endothelia with some evidence for expression in the apical syncytiotrophoblast mRNA+ (RT-PCR); Western blot +
MRP6-8
9
mRNA
Organic cation, steroid transporter; transfer of vinblastine, vincristine, and digoxin Organic anion, glutathione, and glucuronide conjugate transporter; transfer of unconjugated bilirubin to maternal circulation Promote the excretion of glutathione/ glucuronide metabolites in the maternal circulation? Promote the excretion of glutathione/ glucuronide metabolites in the maternal circulation? Nucleotide analog pump; transfer of unconjugated bilirubin to maternal circulation 9
aSee Pasanen (1999) and Myllynen et al. (2004). Semiquantitation: +, positive signal; - , negative results; ?, no data available.
CYP1A1 has been unequivocally demonstrated (Pasanen and Pelkonen, 1994). This CYP activity is also induced by maternal cigarette smoking and exposure to polychlorinated biphenyl and other Ah-receptor inducers. The presence of some major phase II enzymes at the activity level has been demonstrated. However, not much information is available on the expression of specific isoenzymes of, for example, UGT, GST, or SULT (see Table 3). What can be inferred from the results is that only a restricted set of isoenzymes is present in placenta, although individual activities can sometimes be relatively high.
IV. E X P E R I M E N T A L M E T H O D S U S E D TO STUDY THE PLACENTAL FATE OF XENOBIOTICS A. B a c k g r o u n d Human placenta differs more than any other organ between species (Faber, 1995; Leiser and Kaufman, 1994), which is a good reason to use human placental material to study placental functions. Also, since placenta is a large piece of tissue, usually thrown away after birth, it can be used for many purposesmfor example, as a model organ for human proteins and molecular pathways. Placenta and cord also contain stem cells for the fetus, which can be used for experimental as well as therapeutic purposes. Thus, the importance of human placenta in medicine and science has increased during the past 15 years, and it will continue to do so for the foreseeable future.
Individual variation of normal placenta occurs, and various conditions can cause placental dysfunction. For instance, processes such as intrauterine growth retardation (IUGR) cause increased apoptosis and possible changes in the structure of the placenta (Merchant et al., 2004). Much more research and development are needed before the placental functions can be controlled during such situations. Placenta, with its hormonal and xenobiotic metabolism, putative storage capacity of foreign compounds (Ala-Kokko et al., 2000), and rather poorly characterized extensive transport system, is an important target of studies. All different types of experimental systems, from isolated cells to whole tissue perfusion, have been used to study the placenta (Table 4). Since placenta is anatomically complex and highly polarized in its functions (Ganapathy et al., 2000; Cariappa et al., 2003), the method to be used has to be carefully considered based on the purpose of the studies. For a complete view of placental transporters (Ganapathy et al., 2000; Cariappa et al., 2003), it may be necessary to use more than one experimental system. Heikkila and coworkers (2002) demonstrated the integration of a gene construct in placental cells by combining placental perfusion and explant culture thereafter.
B. H u m a n Placental Perfusion M e t h o d For human placental perfusion, one cotyledon from term placenta after birth is usually used because the whole placenta is too large for practical equipment (Schneider and Proegler, 1988; Pienimaki et al., 1995; Ala-Kokko et al., 2000). Fetal
C H A PT E R 3 3 9Placental Toxicity of OPs and CMs
TABLE 3.
467
Xenobiotic-Metabolizing Enzymes Detected in Human Placenta According to Gestational Status a First trimester
At term
++
++
The only placental xenobiotic-metabolizing CYP form for which expression and inducibility have been demonstrated at all possible levels of detection
CYP1A2 CYP1B1
-
(+) (+)
Immunoreaction equivocal at term Marker activity negative No catalytic activity; immunodetection negative
CYP form
Cytochrome P450 (CYP) CYP1A1
CYP2B6
(+)
(+)
CYP2C
(+)
-
CYP2D6 CYP2E1
(+) (+)
(+)
CYP2F1 CYP3A4, 3A5, 3A7 CYP4B 1
Comments
No relevant enzymatic assays
No catalytic activity
+
+
Marker activity negative No function established
(+) +
(+) +
No function established
++ ?
(+) ++
Positive correlation with smoking status
++ ?
++ ?
Accounts for 85% of placental activity
++
++
No definite correlation with the chemical stress and EH expression at term
?
9
++
++
Identification of isoforms not certain; activities dependent on specific substrates
?
++
Obviously polymorphic
?
?
Several marker activities negative
UDP-glucuronosyl transferase (UGT) UGT1A UGT2B, 2B4, 2B7
Activity levels very variable
Glutathione transferase (GST) GSTPI-1 GSTA, M, T
Probably negligible contributions
Epoxide hydrolase (EH) EH mEH Sulfotransferase (ST, SULT) SULT N-acetyl transferase (NAT) NAT1 NAT2 Other drug-metabolizing enzymes NQO
?
++
QR NOS ADH ALDH
++ + ? ?
++ ++ ++ ++
No induction in vivo Effects of xenobiotics on placental NOS have not been studied
CYP19
++
++
Aromatase activity
aSee Pasanen (1999) and Myllynen et al. (2004). +, positive evidence at functional (activity) level; (+), positive evidence at mRNA level (RT-PCR) but no evidence of activity; - , negativeresults; ?, no data available.
artery and vein can be cannulated to simulate fetal circulation, whereas in the matemal side, circulation can be gained by placing cannulae through the decidual plate. This dual setting can be used for both once-through and recirculating experiments. The latter simulate normal physiological conditions and can be used to study xenobiotic metabolism in addition to transplacental transfer (Pienimaki et al., 1995). In dual perfusions, both transfer from the fetal compartment to the maternal or transfer from the maternal circulation to
the fetal can be studied. For short experiments, clear perfusion medium with plasma expander, glucose, and salts can be used. However, for longer experiments, cell culture medium is necessary (Heikkila et al., 2002). Perfusion up to 18 hr has been described in the literature (Miller et al., 1989; Boal et al., 1997; Heikkila et aL, 2002). Successful perfusion can be ensured by the production of placental hormones, use of oxygen and glucose, and transfer of antipyrine (Ala-Kokko et al., 2000). However,
468
S ECTI O N IV 9 O r g a n T o x i c i t y
TABLE 4.
Examples of Experimental Methods and Their Characteristics Used for Placental Studies
Method
Characteristics
Examples of studies used for
References
Pienimaki et al. (1995), Ala-Kokko et al. (2000), Myllynen et al. (2004) Heikkila et al. (2002), Polliotti et al. (1996) Heikkinen et al. (2000)
E x vivo perfusion of a placental cotyledon
Short-terma dual recirculating
Mimics physiological circulation
Transfer and metabolism of drugs
Long-term dual recirculating
Mimics physiological circulation Concentration of studied compound constant in perfusion medium
Integration of gene construct Transfer of infectious agents Transfer of physiological compounds and drugs
Include gap junctions
Microbial invasion
Short-term dual once-through
Explant cultures Villous explants
VEGF in preeclampsia Primary cell cultures Isolated extravillous trophoblasts (EVT) Cultured trophoblasts
Placental endothelial cells from microvessels Isolated membranes Microvillous membranes (MVM)
Basal membranes
Trophoblast cell lines BeWo
Jeg-3
Jar
Endothelial cell line HPEC-A1
Nishimura et al. (2004) Lecuit et al. (2004) Ahmad and Ahmed (2004)
Express markers specific for EVT Grow in monolayer, express corticotrophin-releasing factor Express markers specific for EC, Life span 45-50 pd
Hormonal effects on invasive potential Placental hormone secretion
Petraglia et al. (1989)
Isolation procedure
Jinga et al. (2000)
H/K ATPase, P-gp
Localization of proteins
MRP l, more susceptible to proteolytic cleavage than MVM
Localization of proteins
Nagashige et al. (2003), Jansson (2002), Johansson et al. (2004) Jansson (2002), Nagashige et al. (2003), Johansson et al. (2004)
Cytotrophoblasts, no differentiation to syncytium Derived from BeWo, form large multinucleated syncytia Resemble early placental trophoblasts, form syncytia
Ca uptake
Wadsack et al. (2003), Moreau et al. (2001)
Cholesteryl ester uptake
Wadsack et al. (2003)
Cholesteryl ester uptake
Wadsack et al. (2003)
Establishment and characterization
Schutz et al. (1997)
SV-40 transfected, express endothelial markers
Oki et al. (2004)
aUp to 6 hr.
comparison of various methods led Pienimaki and coworkers (1995) to conclude that the most useful method is to follow the leak from the maternal side to the fetal side. In the histology, typical change in the dual recirculating perfusion system after organ perfusion is tissue edema (V~ih~ikangas, 1981; Pienimaki et al., 1995). However,
compared to placental tissue explants, the viability of cells can be retained much better (Di Santo et al., 2003). Although human placental perfusion is the most tedious method to study placental transfer and metabolism, there are several good reasons to use it (Ala-Kokko et al., 2000). It is the only method that retains fully the structure of the
CHAPTER 33 9Placental Toxicity of OPs and CMs placenta. Interspecies differences in placental anatomy and physiology cause difficulties in species extrapolation. Ethical aspects are easier than in in vivo human studies. Also, it is self-evident that new drugs and toxic chemicals cannot be studied in vivo in humans. For instance, studies on the transplacental kinetics of drugs such as methadone are important but difficult, if not impossible, in vivo. Ex vivo placental perfusion has provided valuable information on the kinetics and putative mechanisms in the case of methadone (Nejhayeva et al., 2005).
469
cal for endothelial cells), and UEA lectin (but not BS-I lectin, typical of large placental vessels). The cultures growing in the monolayer represent two cell types with different cell organelle distribution (Jinga et al., 2000). The primary arterial cells isolated by Leik and coworkers (2004) were grown from explants of placental arteries by placing the vessel lumen down. Cells with uniform morphology appeared within 1 week and grew to confluency within 4 weeks. After gaining confluency, they expressed smooth muscle myofilament proteins and lacked expression for fibroblast-specific antigen.
C. Placental Explant Cultures Placental tissue can be retained as explant cultures (Siman et al., 2001; Merchant et al., 2004), although Di Santo and coworkers (2003) have shown that the trophoblast viability is very restricted despite culture conditions. Explants from normal and IUGR pregnancies were cultured by Merchant and coworkers to study matrix metalloproteinases. Placental explants can also be cultured after placental perfusion and have been used to show integration of a gene construct (Heikkila et al., 2002). Explant cultures have also been used to isolate specific cell types from the placenta. Leik et al. (2004) grew arterial smooth muscle cells from cultured small pieces of placental arteries from chorionic plate. Within 1 week, cells with uniform morphology expressing proteins similar to human aortic smooth muscle cells but clearly different from fibroblasts or endothelial cells grew out of the explants. These cells can be used as a general model for human arterial smooth muscle cells (Leik et al., 2004).
D. Primary Placental Cells Primary trophoblast cells (and also most cell lines of trophoblast origin) ~do not grow into confluent monolayers in culture (Cariappa et al., 2003). This inhibits studies on polarized transport of nutrients and other compounds. They are difficult to culture because of contamination by other cell types and poor viability (Choy et al., 2000). Villous and extravillous cytotrophoblasts differ in their function and characteristics. As studied by immunofluorescence, Ockleford and coworkers (2004) have shown a much higher cytokeratin expression in extravillous cytotrophoblasts compared to villous cytotrophoblasts. Placental venous endothelial cells have different characteristics depending on which part of the vasculature they have been isolated from. Isolation, purification, and culture of primary human placental endothelial cells (HPECs) from microvessels on the venous side were reported by Jinga and coworkers (2000). To gain pure cultures of HPECs, trypsin perfusion of the placental cotyledon was followed by Percoll gradient and sequential trypsinization of cultures. These cultures represent pure HPEC form microvessels by expressing typical patterns of markers: ACE, von Willebrand factor (typi-
E. Cell Lines of Placental Origin Immortalized trophoblast cell lines can be produced by transfection. Choy and coworkers (2000) were able to significantly increase the transfection efficiency by modifying a transfection protocol utilizing poly-L-ornithine. Choriocarcinoma cells resemble invasive trophoblasts morphologically and are used as a model to study trophoblast functions (Wadsack et al., 2003). Several choriocarcinoma cell lines exist (Table 2). Immortalized BeWo cells, which grow as a monolayer, are widely used because of their useful characteristics (van der Ende et al., 1990). Morphologically, they resemble normal trophoblasts and have similar biochemical marker enzymes. Interestingly, Cariappa and coworkers (2003) have shown that their characteristics are dependent on the material on which they are grown. Some of the growth factors typically expressed by these cells are only expressed when the cells are cultured on porous filters and not when cultured on plastic. The expression of these factors thus requires polarized membranes. BeWo cells appear to be an effective model for placental calcium transport (Moreau et al., 2001) but not as good as Jar or Jeg3 cells for selective cholesteryl ester supply to placental cells (Wadsack et al., 2003). This stresses the importance of comparing several models for any new studies to find the most suitable one.
V. P L A C E N T A L T O X I C O K I N E T I C S O F
ORGANOPHOSPHATES AND CARBAMATES From a search of the literature on pesticides and placental toxicokinetics, it is obvious that not much specific information is available. Most studies are on the effects of pesticide administration during pregnancy on experimental animals, mainly rabbits, rats, and mice, in which the fetal outcome and sometimes the presence of residues in the fetal tissues have been observed (Gupta, 1995). Naturally, if specific effects in the fetus, such as inhibition of cholinesterase or compound-related residues in the fetus and the placenta, have been observed, it is relatively safe to make the conclusion that the pesticide has been transferred from the mother to the fetus. Otherwise, very little direct information is available.
4 70
S ECTI O N IV 9Organ Toxicity
It is also important to stress that almost all studies have been performed in experimental animals and the extrapolation to human pregnancy should be made with caution.
A. Entry and Disposition of Organophosphates and Carbamates in the Placenta 1. THE PLACENTAAS A BARRIER FOR ANTICHOLINESTERASE PESTICIDES The placenta is a lipid membrane that permits bidirectional transfer of substances between maternal and fetal compartments. Essentially, the placenta is the entry (not a barrier) through which the fetus is exposed to xenobiotics, including organophosphate (OP) and carbamate (CM) pesticides. The two most common factors involved in transplacental transfer of common toxicants are physicochemical properties of the chemical and the type of placenta. In general, any chemical with a molecular weight < 1000 readily crosses the placenta, and most OPs and CMs have a molecular weight <500. Therefore, these chemicals are not restricted from reaching the fetus. It should be noted that chemical properties such as lipophilicity, polarity, and degree of ionization can affect the rate of placental transfer. The second factor that predominantly influences the transplacental transfer of chemicals is the type of placenta. For instance, the complex multilayered placenta of higher animals can make it more difficult for chemicals to gain access to the fetus compared to the simpler choriovitelline or chorioallantoic type of placenta (Welsch, 1982; Juchau, 1995; Ala-Kokko et al., 2000). Also, other factors, including maternal-fetal chemical gradient, uterine and umbilical blood flow, and protein binding, may affect the rate and extent of transplacental transfer and maternal-fetal equilibrium of a pesticide. Direct or indirect evidence for placental transfer of pesticides has been obtained from residue analysis of these compounds and/or their metabolites in the placenta, umbilical cord, embryo/fetus, or the specific biochemical and morphological changes induced by a compound of this class in the placenta/fetus. For a detailed account of placental transfer of pesticides and their metabolites, see Gupta (1995) and Gupta and Sastry (1999). Further indirect evidence for placental transfer of pesticides has also been substantiated by measuring the cholinesterase inhibition in fetal tissues (Cambon et al., 1979, 1980; Gupta et al., 1985; Gupta, 1995). Thus, it seems that anticholinesterase pesticides cross the placenta and adversely affect the developing fetus. Only one study was found in the literature in which the transplacental transfer of a pesticide was examined in human placental perfusion. Parathion, an OP, has been shown to cross the placenta in this experimental model by Benjaminov and coworkers (1992). However, fairly little is known of the placental transporters, even the best known P-glycoprotein, and pesticide transport across the placenta (Abu-Qare et al., 2003). Bain and LeBlanc (1996) studied the effect of 38 pesticides on
the transport of doxorubicin in human MDRl-transfected mouse melanoma cells. Most OPs and some organochlorine and other pesticides inhibited the transport of doxorubicin, whereas none of the CM pesticides did so (Table 5). Only one organochlorine pesticide, endosulfan, was slightly transported by P-glycoprotein, 2. METABOLISMOF PESTICIDESBY PLACENTAL ENZYMES A number of papers on in vitro metabolism of organophosphorothioates by P450 enzymes have demonstrated that usually CYP1A2, CYP2B6, CYP2C8, and CYP3A4 participate to a variable extent in the metabolism and metabolic activation of several OPs (Mutch e t al., 2003; Buratti et al., 2003, 2005). Especially CYP2B6 and, to a lesser extent, CYP3A4 seem predominant in metabolic activation. However, these enzymes are expressed to a very low extent, if at all, in human placenta; thus, placental metabolism is probably insignificant. The metabolism consequently occurs in maternal liver (and maybe some other maternal tissues) and placenta and fetus are exposed to metabolic products. Whether they are further metabolized in the placenta remains to be studied. Many CMs are metabolized in human liver by P450 enzymes. For example, at least CYP1A1/2, CYP2B6, and CYP3A4 participate in the metabolism of carbaryl (Tang et al., 2002). However, as depicted in Table 3, only CYP1A1 is definitely demonstrated to be functionally expressed in human placenta and thus has a potential to hydroxylate carbaryl. This potential has not been studied experimentally. Chlorpyrifos is metabolized to an oxon metabolite by CYP2B6, but this enzyme is not expressed in human placenta; thus, it is questionable whether chlorpyrifos is metabolized in the placenta. Transplacental transfer and pharmacokinetics of parathion methyl after dermal application were studied in pregnant rats (Abu-Qare et al., 2000). Both the parent drug and the oxon metabolite were transferred to the fetus, although concentrations were somewhat lower than in maternal tissues. It is clear that the fetus is exposed to a relatively high concentration of parathion methyl even after dermal exposure, and this exposure leads to a significant degree of cholinesterase inhibition (Abu-Qare and Abou-Donia, 2001) also in placental tissue (Benjaminov et al., 1992).
VI. P L A C E N T A L T O X I C O D Y N A M I C S OF O R G A N O P H O S P H A T E S AND C A R B A M A T E S In principle, chemicals are capable of causing disturbances in placental functions at many different levels and via variable mechanisms. Potential mechanisms of action include interference of transport of essential nutrients and substances by inhibiting specific transport proteins, inhibition of steroid
CHAPTER 33 TABLE 5.
9Placental Toxicity of OPs and CMs
471
Placental Toxicokinetics of Selected Organophosphate and Carbamate Insecticides P-glycoprotein interaction a
Bromophos Chlorpyrifos
nk a Inhibits
Chlorthiophos Leptophos Phenamiphos Phosmet Imidan Malathion Mevinphos Parathion Parathion-methyl Quinalphos Dicapthon Aldicarb Carbaryl Carbofuran Oxamyl Primicarb Propoxur
Inhibits Inhibits Inhibits Does not inhibit nk nk Does not inhibit Inhibits nk nk Inhibits Does not inhibit Does not inhibit nk nk nk Does not inhibit
Metabolism b
nk CYP1A2, CYP2B6, CYP3A4 nk nk nk nk nk CYP1A2, CYP2B6 nk CYP3A CYP3A nk nk nk CYP1A1 nk nk nk nk
Transfer to the fetus c
A, B nk nk nk nk nk A,B nk nk A,B A,B,D A nk A A,B A nk A nk
aInhibition of P-gp-mediated effiux of doxorubicin in human MDR1 gene transfected cells (Bain and Lablanc, 1996). No studies available on placental experimental systems. bAssignment of CYP enzymes on the basis of hepatic or recombinant enzyme studies. For the expression of CYP enzymes in placenta, see text. For references, see text. CEvidence for transfer: A, transfer evident on the basis of effects to the fetus; B, transfer evident on the basis of compound-related material in the fetus; C; transfer studied in perfused placenta; D; transfer studied in maternal-placental-fetal system. For references, see text. ank, not known.
hormone synthesis, disturbances in placental redox status, and interference with protein or nucleic acid synthesis or function (Myllynen et al., 2004). However, because the topic of this review is OP and CM pesticides, which have prominent anticholinesterase activities, the effects on the placental cholinergic system are discussed in more detail. Obviously, these effects have been a priority area of investigation.
A. Placental Susceptibility to Anticholinesterase Pesticides
Depending on the duration, frequency, and level of exposure, anti-AChE pesticides and related compounds can adversely affect one or all three components of the maternal-placental-fetal unit. The outcome of placental toxicity to these pesticides can be influenced by three major factors: maternal toxicity, placental transfer, and placental-fetal metabolism. Evidence suggests that the pesticide residue is present not only in the exposed mother
but also in placenta, cord blood, embryo, and fetus, thus strongly indicating that placenta and fetus are potentially exposed. (see Section V). Furthermore, the placenta is enriched with proteins and thereby may bioconcentrate pesticide residue by means of protein binding and then releasing them into the placental circulation, embryo, and fetus. From rat studies, there is evidence that placenta may be a target of direct toxic effect by OP pesticides (LevarioCarrillo et al., 2004). More than one mechanism exists by which toxicants are concentrated in the placenta and fetal tissues in greater quantifies than in maternal tissues. In general, the large placental surface area comes in contact with a relatively large volume of maternal blood (required for normal placental function), leading to a high exposure. Under physiological conditions, the fully formed placenta plays an important role in the maintenance of nutrition to the fetus and in the secretory and regulatory functions that are essential for the maintenance of pregnancy. All supplies (oxygen, water, electrolytes, nutrients, hormones, and antibodies) to
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the embryo and fetus must pass through the placenta. By having an active, albeit restricted, metabolic capability, the placenta can modulate the potency of OPs and CMs. The placenta can probably convert certain OPs of the thioate or dithioate group to their oxon forms, which are many times more potent AChE inhibitors and thus more toxic. In such circumstances, the placenta can be a determining factor for fetal toxicity. In addition, it is well known to have cholinesterases [AChE and butyrylcholinesterase (BuChE)], the key target enzyme for the toxicity of OPs and CMs. By having cholinesterases, drug-metabolizing enzymes, and oxidative enzymes, the placenta is vulnerable to damage by anti-AChE pesticides. The placenta is also an organ in which anti-AChE pesticides can induce oxidative stress. The health of the fetus is dependent on the health of the placenta because anti-AChE pesticides encounter the placenta before reaching the fetus. Therefore, anti-AChE pesticide-induced damage to placenta may result in equally severe damage to the fetus. Most pesticide-induced biochemical or morphological effects observed during the prenatal period persist during postnatal development. All of these factors place the placenta in a unique position in which its own health is as vulnerable as the health of the mother or conceptus. Note that placental susceptibility to OPs and CMs may vary depending on gestational age.
B. Effects of Anti-AChE Pesticides on the Placental Cholinergic System It is well recognized that an active cholinergic system exists in the placenta of some species and not in others, and there is evidence that a cholinergic system may have more than one function in the placenta. The presence of ACh in the human placenta was demonstrated as early as 1933 (Chang and Gaddum, 1933). Many studies have confirmed that the human placenta, in contrast to many nonprimate placentae, synthesizes and stores large amounts of ACh (Sastry, 1997; Leventer et al., 1982; Welsch and Wenger, 1980; Sastry et al., 1976). Placental ACh is synthesized in the cytoplasm of the syncytiotrophoblast (Satyanarayana, 1986; Sastry et al., 1976) and released from the placenta into both maternal blood (Olubadewo and Sastry, 1978) and fetal circulation (Sastry et al., 1973). The released ACh from human placental villi resembles that from the nerve in several aspects (Olubadewo and Sastry, 1978). Isolated human placental villi contain 167 nmol ACh/g wet weight tissue. The amount of ACh reported to be present in human placenta at term (100-200 nmol ACh/g wet weight) is considerably greater than that found in various regions of the brains of humans and laboratory animals (0.5-30 nmol/g wet weight) (McIlwain and Bachelard, 1971). In the placenta, ACh-like activity is highest in membranes, medium in the cotyledons, and minimal in the cord. The content of ACh-like activity in the placenta varies as a function of gestational age. The highest concentration of ACh is found at
approximately 20-22 weeks of gestation, and very little is found after parturition (Sastry et al., 1976). A marked decline occurs soon after delivery of the infant and expulsion of the placenta. The ACh concentration decreases in aged/termed placenta and syncytial degeneration. Approximately 95% of ACh-like activity in the human placenta is in a bound form, with most of the ACh localized possibly within vesicles in the villus tissue. The concentrations of ACh in floating villi and the basal plate are approximately 3.2 and 2.1 times higher than that in the chorionic plate (Sastry et al., 1976). Hebb and Ratkovic (1962) investigated term placentas for the existence of a cholinergic system [using choline acetyl transferase (CHAT) as a marker] in several species, including monkey, mongoose, lemur, horse, cow, sheep, goat, pig, hamster, cat, rabbit, guinea pig, and rat. Only monkey placentas contained CHAT. The content of ACh and Ch in term placentas from monkey (Macaca mulata) has been investigated in detail by Welsch (1977). At 150 days of gestation, the rhesus monkey placenta synthesizes approximately 4 txmol ACh/g wet tissue/hr. Literature abounds showing that the two species in which the placental cholinergic system has definitely been found are the two higher primates, man and monkey. The finding that no ChAT (an enzyme needed in ACh synthesis) is found in placentas from several other species may be due to species differences in the molecular forms and the synthesis of the enzyme, which may vary from species to species. The neurotransmitter function of ACh is well established, but placenta is not innervated. In the context of human placental function, ACh has been assigned many major roles: (1) to act as a local hormone related to uterine contractions and mechanisms associated with birth (Hierman, 1941), (2) the control of permeability and transport (Rowell and Sastry, 1978; Koelle, 1969), (3) regulation of blood flow and fluid volume in placental vessels (Sastry, 1997), (4) the release of placental hormones such as chorionic somatomammotrophin (Harbison et al., 1976), (5) involvement in the mechanism of human parturition (Brennecke et al., 1998), and (6) to act as a local messenger molecule (Sastry, 1997). ACh seems to play a vital role in the maturation of the human placenta. Furthermore, Sastry and Sadavongvivad (1979) demonstrated that intracellular rather than extracellular ACh is important in the control of prostaglandin release because bound placental ACh does not seem to be available for hydrolysis by cholinesterases. In essence, most of the physiological roles postulated relate to endogenously released ACh. These roles relate to the actions of ACh in the placental vessels, syncytiotrophoblast and its maturation and development, and modification of uterine function during labor. ACh may also regulate trophoblastic channels, fluid balance, and osmotic pressure and therefore may influence contractile properties of myofibroblasts in the placenta. For further details on the roles of ACh, see Sastry (2000, 1997) and King et al. (1991).
CHAPTER 33
In early studies, several investigators examined the effects of exogenously injected ACh on the perfused human placenta. Raghavan and Sastry (1970) found that endogenous ACh is released in considerable amounts into the perfusion medium, and the placental vasculature is already dilated, making it difficult to demonstrate the vasodilatory effects of exogenously injected ACh. Weak vasodilation or vasoconstriction was enhanced by physostigmine (a reversible AChE-inhibiting CM) and abolished by a muscarinic receptor blocker, atropine. Exogenously administered ACh caused vasodilation or vasoconstriction in a minority of perfused human placentas; usually no effect was observed (Sastry, 1991). Furchgott (1983) demonstrated that the presence of intact endothelial cells is necessary for the relaxation effect of ACh on isolated blood vessels. From this investigation, two basic facts were established: (1) Muscarinic receptors that are activated by ACh to release endothelium-derived relaxing factor (EDRF) are present on endothelial cells, and (2) EDRF mediates the ACh-induced relaxation of vasculature smooth muscles. The variation in placental ChAT activity as a function of gestational age is similar to that of ACh. ChAT activity peaks at approximately 16-20 weeks of gestation, and a fourfold decrease in activity is seen at parturition (Harbison et al., 1975). The variation in the pattern of AChE activity as a function of gestation age is similar to that of ACh and CHAT. The development of the placental cholinergic system, as indicated by ACh, CHAT, and ACHE, follows the development of the syncytiotrophoblast during the first 6 months of pregnancy (Tuchmann-Duplessis et al., 1972). The reasons for the decrease in ACh, CHAT, and AChE concentrations in the term placenta are yet to be explained. Finally, it needs to be described how the function of ACh is terminated in placenta, which includes (1) hydrolysis of ACh by placental cholinesterases; (2) release of ACh into maternal and fetal circulations, diffusion, and hydrolysis by maternal and fetal circulations; and (3) reuptake of released ACh by placental trophoblast. The degradation products of ACh (Ch and acetate) may be reutilized in the ACh synthetic pathway of placenta, as they are in autonomic nerve endings (Sastry, 2000). 1. OP- AND CM-INDUCED ALTERATIONS IN THE PLACENTAL CHOLINERGIC SYSTEM
Exposure of pregnant women to an OP, CM, or combination of both can cause severe damage to the mother, placenta, and fetus. The placenta and fetus, by having AChE and other cholinergic elements, remain susceptible to OPs and CMs (Simone et al., 1994; Sastry, 1993; Cambon et al., i979; Koshakji et al., 1974). Inhibition of AChE and BuChE activities can be used as a marker of exposure to an OP or CM, and AChE can also be used as a marker of effects. These compounds readily cross the placental barrier and can act on the cholinergic and noncholinergic components of the developing nervous system and other vital organs
9Placental Toxicity of OPs and CMs
473
(Gupta et al., 1985, 1984). Following prenatal exposure to OPs (quinalphos, dicrotophos, methyl parathion, and others) significant inhibition of AChE has been demonstrated in maternal, placental, and fetal tissues of rats and mice (Srivastava et al., 1992; Gupta et al., 1985; Bus and Gibson, 1974). Abu-Qare and Abou-Donia (2001) measured activities of AChE and BuChE enzymes in placenta of timed rats following a single cutaneous injection of methyl parathion (10mg/kg), diazinon (65 mg/kg), or both. Inhibition of placental AChE or BuChE activity occurred 12 and 1 hr, respectively, following a single injection of methyl parathion, corresponding to activities of 63 and 54% of control. Diazinon or a mixture of diazinon and methyl parathion inhibited AChE and BuChE to 80% of control within 24 hr of application. The activity of both enzymes recovered to >90% of control after 96 hr of dosing. The authors found that diazinon, as a less toxic insecticide compared to methyl parathion (LDs0, 455 vs 67 mg/kg), caused less inhibition of AChE than that caused by methyl parathion alone or in combination with methyl parathion. Similar findings have been reported for CMs, including aldicarb, carbaryl, carbofuran, and pirimicarb (Cambon et al., 1980, 1979; Declume and Derache, 1977). These studies revealed AChE inhibition as the major biochemical mechanism of toxicity. Furthermore, subchronic prenatal exposure to methyl parathion in rats resulted in altered postnatal development of brain AChE and ChAT activities and selected subtle alterations in behavior (Gupta et al., 1985). To our knowledge, there are no systematic studies that have examined the effects of OPs and CMs on the human placental cholinergic system. However, since OPs and CMs are known to inhibit AChE activity, it is expected that exposure of a pregnant woman to these compounds can lead to enhanced levels of ACh, and thereby ACh can influence other components of the cholinergic system in the placenta. 2. PLACENTAL NEUROTOXIC ESTERASE
Neurotoxic esterase (NTE), which is a membrane protein, was discovered in chicken brain in relation to tri-ocresylphosphate-induced neuropathy (Johnson, 1974). In later studies, NTE, which is the putative target for OP-induced delayed neuropathy (OPIDN), was found in the neural tissue of a large number of different vertebrate species (Gurba et al., 1981). The same authors found NTE in preparations of human placenta similar to that found in the avian and human brain (Gurba and Richardson, 1983). The human placenta may be an ideal source of material for further purification and characterization of NTE, although in the context of OPIDN it appears to be insignificant in the placenta. 3. ACh-INDUCED RELEASE OF PLACENTAL HORMONES
Human placenta has been recognized as a source of chorionic gonadotropins and steroid hormones. Since the development of the placental cholinergic system follows
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the development of the syncytiotrophoblast, it is interesting to determine the release of steroid hormones by ACh. Although the cytotrophoblasts, the source of gonadotropins in the placenta, are fully developed in the first 3 months of gestation, some cytotrophoblastic cells remain in full-term human placenta (Sastry, 1997). Harbison et al. (1976) reported that ACh stimulates placental release of chorionic somatomammotrophin. ACh increases the release of immunoreactive corticotrophin-releasing factor from human placental cell cultures in a dose-related manner, and its effect is reversed b y the cholinergic receptor antagonists atropine and hexamethonium.
C. Effects of Pesticides on Endogenous Steroid Synthesis Endocrine effects of pesticides have been known for some time. They can both interact with steroid hormone receptors and interfere with hormone synthesis. During pregnancy, the placenta is an important organ for the synthesis of steroid hormones, and it contains two important rate-limiting enzymes: CYPllA1, which catalyzes cholesterol side chain cleavage, and CYP19, an aromatase for the production of estrogens. CYP11A1 seems to be a rather stable and selective enzyme not affected to a great extent by environmental chemicals. On the other hand, CYP19 has a relatively nondiscriminatory binding site and a large number of inhibitors have been synthesized, some of which are in use in endocrine cancers. Regarding most OPs and CMs, it is not known whether they are inhibitors or activators of either one of these two important enzymes. Andersen and coworkers (2002) studied the endocrine effects of 24 pesticides in use in Danish greenhouses and showed that many had multiple effects. For instance, fenarimol acted as an estrogen agonist and androgen antagonist and, in addition, inhibited aromatase activity in human placental microsomes. Prochloraz inhibited aromatase even more drastically, and chlorothalonil more than 50% of the original activity. Others, such as methomyl and primicarb, weakly stimulated the placental aromatase. One molecular target of the endocrine effects in the placenta may be intracellular calcium homeostasis since estrogenic pesticides change calcium handling by trophoblasts and this effect may be endocrinally controlled (Derfoul et al., 2003).
D. General Effects on Protein and Nucleic Acid Synthesis by AChEs Placenta is a potentially important source of tissue for molecular studies in humans and animals because it is large, readily available, and responsive to environmental pollutants, including anti-AChE pesticides. Some of these pesticides are known to cause alterations in protein synthesis, DNA damage, mutagenesis, carcinogenesis, and teratogenesis. OPs have been shown to adversely influence protein
synthesis in both in vivo and in vitro studies (Marinovich et al., 1996; Koelle et al., 1977; Welsch and Dettbarn, 1971; Clouet and Waelsch, 1963). Gupta et al. (1984) examined the subchronic effects of methyl parathion administered throughout the period of organogenesis on in vivo protein synthesis in embryonic, fetal, and maternal tissues. The specific activity of [14C]valine in the free amino acid pool and protein-bound pool was significantly reduced in discrete regions of the maternal brain, maternal viscera (day 19), and placenta. The inhibitory effect of methyl parathion on net protein synthesis was dose dependent, greater on day 19 than on day 15 of gestation, and significantly greater in fetal than in maternal tissues. It is interesting to note that the inhibitory effect on protein synthesis was most pronounced at a dose causing overt maternal toxicity. Marinovich et al. also demonstrated that a mixture of OPs, including diazinon, dimethoate, and azinophos, was more toxic to protein synthesis than any of the single compounds alone. There are few studies in which OPs and CMs have been examined for their effects on embryonic development. It has been postulated that alkylation of nicotinamide adenine dinucleotide (NAD +) coenzymes by OPs may be a major factor in the induction of carcinogenesis (Schoental, 1977). In another study, malathion caused alterations in the levels of RNA, glycogen, sulfated mucopolysaccharides, and calcium in the developing tibiotarsus (Ho and Gibson, 1972).
VII. ABNORMAL PREGNANCY OUTCOMES DUE TO ANTICHOLINESTERASES Exposure to pesticides of the anticholinesterase class during pregnancy can cause several deleterious effects, such as stillbirths, fetal deaths, spontaneous abortions, low birth weights, and malformations at birth. Several OPs and CMs are known to exert direct embryocidal/fetocidal effects (Jayatunga et al., 1998a,b; Speilmann and Vogel, 1989; Deacon et al., 1980). Both OPs and CMs are known to readily cross the placenta and act on the cholinergic and noncholinergic components of the developing nervous system and other vital organs (Gupta and Sastry, 1999; Gupta et al., 1985; Gupta, 1995). The developing organism appears to be much more susceptible than the adult to the induction of functional neural deficits by OPs and CMs. The effects of maternal exposure on the fetus can vary from the production of severe anomalies to nondetectable differences from nontreated controls. Published studies have shown indications of elevated reproductive risk and exposure to pesticides, but the epidemiological evidence does not allow any clear inference to be drawn (Nurminen, 1995). Pesticides of both classes have been demonstrated to have potential for embryotoxicity, embryolethality, fetotoxicity, and teratogenesis. In general, these effects vary
CHAPTER 33 9Placental Toxicity of OPs and CMs depending on the particular OP or CM involved. Embryolethality is encountered so often that the expression of teratogenesis is rarely seen. In humans, infants born to mothers exposed to oxydemeton-methyl or mevinphos (both OPs) showed cardiac defects (ventricular and atrial septal defects), stenosis of the pulmonary artery and a patent ductus arteriosus, bilateral optic nerve colombo mass, microphthalmia of the eye, cerebral and cerebellar atrophy, and facial anomalies (Romero et al., 1989; Ogi and Hamada, 1965). In some studies, OPs have been shown to produce developmental alterations in rats, mice, hamsters, and rabbits. In a classical study, Khera (1979) discovered polydactyly in fetuses of cats treated with dimethoate (12 mg/kg/day) during days 14-22 of gestation. De Castro et al. (2000) reported that methamidophos, at the no-maternal toxicity dose (1 mg/kg, po), given to female rats during gestation (days 6-15) produced no lethal effect on embryos and did not cause congenital malformations at term; however, the embryo-fetal maturation process appeared to be affected. In a similar study conducted on rats with six OPs (tribufos, oxydemeton-methyl, azinophos-methyl, fenamiphos, isofenphos, and fenthion), Astroff and Young (1998) found no effects in fetuses at dose levels that elicited significant maternal effects. Among CM pesticides, carbaryl has been studied in detail for placental toxicity. Like OPs, CMs have a greater potential for embryolethality and fetotoxicity that precludes an expression of teratogenicity. A study conducted in beagle dogs showed dystocia due to atonic uterine musculature and evidence of terata in 21 of 181 pups. Fetal abnormalities included abdominal-thoracic fissures with varying degrees of intestinal agenesis and displacement, brachygnathia, ecaudate pups, failure of skeletal formation, and superfluous phalanges (Smalley et al., 1968). Carbaryl exposure during organogenesis produced terata in guinea pigs but not in hamsters and rabbits (Robens, 1969). Jayatunga et al. (1998a) demonstrated that exposure to carbofuran during early pregnancy in rats causes pregnancy loss and disruptions to neonatal development. In a detailed study, Jayatunga et al. (1998b) examined the antigestational effects of carbofuran (0.2, 0.4, and 0.8 mg/kg, po) on pregnancy outcome in rats following midterm exposure (days 7-14). Carbofuran was found to be injurious to the reproductive outcome because the findings revealed fetal deaths (in terms of postimplantation loss, viable uterine implants, litter index, and fetal survival ratio) and impaired prenatal development (size of uterine implants and interembryonic distance) and postnatal development (cranial length, craniosacral length, body weight, and time taken for the appearance of fur and opening of the eyes). In addition, there was a tendency for the pregnancy period to be prolonged and for female-biased litters to be produced. These effects of carbofuran appear to be mediated mostly via potentiation of postimplantational losses due to embryo/fetal toxicity. These authors observed a marked increase in the number of dead conceptuses at laparotomy
475
and by the pronounced reduction in the numbers of litters and of live pups at parturition. It is likely that the embryo/fetocidal action of carbofuran arose both directly and indirectly (via maternal toxicity). Carbofuran is estrogenic (Goad et al., 2004; Jayatunga et al., 1989a,b), and high levels of estrogens can induce fetal death by direct action (O'Neill et al., 1985). At birth, a predominance of female pups can possibly be attributed to the estrogenic activity of carbofuran and to its ability to inhibit food consumption. CMs such as propoxur and carbofuran have not been proved to be teratogenic. For further details on alterations in pregnancy outcomes following exposure to OPs and CMs, see Gupta and Sastry (1999) and Gupta (1995).
VIII. C O N C L U S I O N S AND F U T U R E
DIRECTIONS OP and CM pesticides are widely used and can expose pregnant mothers. The placenta is thus one of the targets for these pesticides, but both also convey deleterious effects to the developing fetus. Although pesticide-induced fetotoxicity has been studied in experimental animals and adverse effects have been observed, there is very little information concerning the toxicokinetic behavior of these pesticides (i.e., penetration, protein binding, distribution, and metabolism) in human placenta. Also, data on toxicodynamic mechanisms and consequences to the developing fetus mainly concern experimental animals, and human data are very scanty. In general, it is of considerable importance to elucidate the role of the placenta in contributing to developmental effects and fetotoxicity. Although there are large gaps in our knowledge of the placenta and pesticides, a variety of tools are available with which we may in principle obtain useful information for assessing the risks of pesticides to the placenta and developing fetus.
Acknowledgments Kirsi V~ih~ikangas is a member of the EU project CHILDRENGENONETWORK (QLRT-2001-02198). The research of Olavi Pelkonen has been supported by the DRUG2000 technology programme of the National Technology Agency of Finland (TEKES).
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CHAPTER ~4
Endocrine Disruption by Organophosphate and Carbamate Pesticides SHIGEYUKI KITAMURA, KAZUMI SUGIHARA, AND NARIAKI FUJIMOTO Hiroshima University, Hiroshima, Japan
However, investigations of incidents involving misuse of OP and CM pesticides have revealed virtually no endocrine-disrupting activity of these chemicals (Flickinger et al., 1984). The putative endocrine-disrupting effect of OPs and CMs in wildlife is largely a prediction based on data obtained from laboratory animals. The influence of these pesticides on human health is also discussed here, based on observed endocrine-disrupting actions of OPs and CMs in vitro and in laboratory animals in vivo.
I. I N T R O D U C T I O N Many organophosphate (OP) and carbamate (CM) pesticides are used throughout the world to protect crops from insects and to protect farm and domestic animals from endo- and ectoparasites. They act as pesticides by inhibition of acetylcholinesterase (ACHE). OPs bind irreversibly with ACHE, whereas CMs bind reversibly. OPs and CMs are nonvolatile and chemically stable, but they are easily decomposed by carboxylesterase to nonactive metabolites. Because these pesticides, including their metabolites, are polar and water soluble, they are believed not to be bioconcentrated in aquatic species. Although certain OPs and CMs have been reported to accumulate to some extent in fish and invertebrates (Tsuda et al., 1990, 1996; Pereira and Hostettler, 1993; Deneer, 1994; Kitamura et al., 2000; Tilak et al., 2004), the risk of bioaccumulation is low due to rapid metabolism in the body and degradation in the environment. Indeed, fenthion sulfoxide, a metabolite of fenthion, was rapidly excreted but fenthion was moderately persistent in the body of fish, with a half-life of approximately 10 days after exposure to the pesticide (Kitamura et al., 2000). Estrogenic and/or antiandrogenic activities are exhibited by a number of pesticides, especially organochlorine pesticides. However, some OP and CM pesticides are also reported to be positive in in vitro assay systems. Antiandrogenic compounds so far reported include metabolites of vinclozolin, linuron, iprodione, chlozolinate, procymidone, flutamide, p,p'-DDE, a metabolite of p,p'-DDT, and ketoconazole (Kelce et al., 1995; Wong et al., 1995; Gray et al., 1999; Ostby et al., 1999; Lambright et al., 2000; McIntyre et al., 2000). Many pesticides, including OPs and CMs, have been found to bind to androgen receptor (AR) and to act as antagonists of AR. Pesticides such as DDT and methoxychlor are well known to act as endocrine disruptors (Guillette et al., 1994). Toxicology of Organophosphate and Carbamate Compounds
II. E N D O C R I N E D I S R U P T I O N BY OP AND CM I N S E C T I C I D E S IN VITRO
A. Endocrine-Disrupting Action of OPs I n V i t r o Many organochlorine insecticides can interact with estrogen, androgen, and progesterone receptors (Table 1) (Kelce et al., 1995; Vonier et al., 1996; Gray et al., 1999; Ostby et al., 1999; Lambright et al., 2000). Many results from in vitro screening tests indicate that tested OPs are not estrogenic. Sonnenschein and Soto (1998) showed that neither malathion nor parathion is estrogenic in the estrogen proliferative screening assay (E-screen assay) using MCF-7 human breast cancer cells. Chen et al. (2002) reported that some pyrethroid pesticides were positive in estrogen screening assay, E-screen assay, estrogen receptor (ER) competitive binding assay, or pS2 expression assay. However, all OP pesticides (phoxim, malathion, monocrotophos, dimethoate, and opunal) examined were negative in the concentration range of 1 X 10-11 to 1 X 10 - 6 M in these estrogen assays. Fenitrothion did not interact with ER in recombinant yeast expressing the human estrogen receptor (Sohoni et al., 2001). Fenthion and its oxidized products, fenthion sulfoxide and fenthion sulfone, did not 481
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
482
S E C T I O N I V . Organ T o x i c i t y
TABLE 1.
Endocrine-Disrupting Action of OPs In Vitro
Estrogenic and antiandrogenic OPs CI C2H501P- O
CI
c,~ ~_~ NO 2
Br
bromophos-ethyl
II H3CH2CCHNH- P - O
C2H50
'CH3
butamifos
CI C2H50\S_ O _ _ ( ~ C I C2H50IP dichlofenthion S
C2HsO/
-0"--0"--
No2
EPN
ethion
C2H50\S_o._(~ CI C3H7SIP Cl prothiofos S
CH30-P-O CH30
C2H50\S S II II ,,OC2H 5 P-SCH2S --p C2HsO i ,,OC2H5
c.,c.=~ OH3cH20/P
CH3 CHNH-~- O - ~ CH3-~ C2H50
--CH3
COOCH-.cH 3
isofenphos CI
~
~'N
quinalphos
leptophos
CI CH3 CI
tolclofos-methyl Estrogenic OPs
C2H50\S O - ~ C l 03H7SIPCI prothiofos CH3CH20\S/P-O .--~/ ~ CH3CH20 ~N quinalphos
S CI H3CO.IIH3CO,,P 0 ~--(~~Br Cl bromophos-methyl 1110ClcHs~ClBr S
Qo
P,,
leptophos
c.,o\~ c ~ CH30/P-O
CH3
Cl
tolclofos-methyl 1170 "OC2H5
CN
cyanofenphos (continues)
CHAPTER 34
(continued)
TA BLE 1.
CI C2H50\S_ O _ ~ C I C2H50IP
C2HsO/ - O ' - - ~
dichlofenthion
CH3CH20
9 Endocrine Disruption by OPs a n d CMs
NO2
C2H50\S SII ,,OC2H5 P-SCH2S--p C2HsOI "OC2Hs
EPN
P- O
NO2
s
Br CI
ethion
C2H50
bromophos-ethyl
s
H - P- O
C2H5~11 CH3
C2Hs/P-O~
butamifos
CH30\ S /p_ O._~T/CH3 CH30 N~,~ N~"C2H5 "C2H5
S
c.~.c..._~_o_0 CH3"" C2H50
"CH33 COOCH~CH
isofenphos
pirimiphos-methyl
isoxathion
CH30\ S
-S-CH CH30IP "COOC2H 5 phenthoate
N Cl C2H50\S_ O ~ C I
C2HsO~P Cl chlorpyrifos
Antiestrogenic OPs
C2H50\ S C2H501P- O ' - ~ -
NO2
parathion
Antiandrogenic OPs
c.~o..~, o-~c. CH30~P-
N CI
CI chlorpyrifos-methyl
S-c~-
CH30"II -O CH30..P
CH3 NO2
fenitrothion
~- -C~-
CH3
CH30" CH30,. O
SCH3
fenthion 9
(continues)
483
484
SECTION I V . Organ Toxicity
TABLE 1. (continued)
CI
H3CO\sll C~ P-S-CH3-CH3CO1 O
CI
C2H501 -O---~'-NO 2
anilofos
C2H50\SII C2H501P-O
EPN
NO2
parathion
H3co\S_0.__~__NO2 H3COIP
methyl parathion
o- c.
C3H5sIP-
prothiofos
CH30\S C ~ CH30/P-O CI
CH3
tolclofos-methyl NO 2
CH3CH2CH20\S iL~cH3 CH3CH2CH201P-S-CH2"CO
C2H50\S S / P-SCH2S--pIIsOC2H5 C2H50 "OC2H5
piperophos
ethion
C2HsO\s
O,~,O~cl
C2HsO ~ P - S - CH 2 - N - " ~
CI C2H50\~10--~CI C2H501P-
phosalone
I1,,O Br QP"o 3 CI leptophos
S CH3~CHNH-~-O---~ CH3"" C2H50 .'CH3 COOCH,,CH3
dichlofenthion
C2HsO\!_O~~~_Br C2HsOI Cl bromophos-ethyl
H3CH2CCHNH-P-O C2H50 II
CH3
butamifos
H3CO\S H3co/P- O ' - ~
CN
cyanophos
CH3CH20\S/p-o. _ ~ / N ~ CH3CH20 ~N quinalphos
CH30\IO I
CH30~P-O ' - - ~ C HNO2
isofenphos
show estrogenic or antiestrogenic activity in ERE-luciferase reporter-transfected MCF-7 cells (Kitamura et al., 2003b). Nishihara et al. (2000) also reported that no estrogenic activity of EPN, ethyl parathion, fenitrothion, fensulfothion, fenthion, malaoxon, malathion, or methidathion was observed at a higher concentration, 1 x 10-4M, even though 17-[3-estradiol (E2) was active at 3 x 10-1~ in a yeast two-hybrid assay. Vitellogenin (a biomarker of estrogens in fish) assay of OPs in fish also gave a negative result. Fenthion did not have estrogenic activity, based on
MEP oxon
the finding that levels of vitellogenin were not enhanced in the blood of male or female goldfish (Carassius auratus) kept in water containing fenthion or its oxidation products, fenthion sulfoxide and fenthion sulfone (3 mg/liter), for 5 days (Kitamura et al., 1999). Although parathion did not activate estrogen- or progesterone-responsive reporter genes in breast MCF-7 and endometrial (Ishikawa) cancer cells except for weak activation of a progesterone-responsive gene, the OP decreased Ee- or progesterone-induced activity (Klotz
CHAPTER 34 9Endocrine Disruption by OPs and CMs et al., 1997). Andersen et al. (2002) tested 22 pesticides, including 4 0 P s and 4 CMs, for effect on proliferation of MCF-7 cells and interference with the activation of ER and AR. They reported that chlorpyrifos and tolclofosmethyl exhibited estrogenic activity in cell proliferation assay and transactivation assay using MCF-7 cells in the concentration range of 1 x 10 -5 to 5 x 10 -5 M. At higher concentrations, there was a decreased response due to cytotoxicity. These investigators further examined the effect of 9 positive estrogenic pesticides (prochloraz, fenarimol, endosulfan, dieldrin, tolclofos-methyl, chlorpyrifos, pirimicarb, propamocarb, and methiocarb) on ER-oL and -[3 mRNA levels in the mammalian cancer fibroblast MCF7BUS cells using on-line reverse-transcriptase polymerase chain reaction (Grunfeld and Bonefeld-Jorgensen, 2004). The OPs listed previously interfered with the ER-oL and ER-[3 mRNA steady-state levels. In particular, coexposure to tolclofos-methyl and E 2 significantly increased ER-oL and -[3 mRNA levels. Kojima et al. (2004) examined the estrogenic activity of 56 OP pesticides in in vitro reporter gene assay using Chinese hamster ovary (CHO) cells. They reported that 16 OPs showed affinity for hER-oL. Prothiofos, bromophos-methyl, tolclofos-methyl, quinalphos, leptophos, cyanofenphos, dichlofenthion, EPN, ethion, and bromophos-ethyl showed affinity for ER-oL and ER-[3, but butamifos, isoxathion, pirimiphos-methyl, isofenphos, phenthoate, and chlorpyrifos lacked affinity for ER-[3. Butamifos showed the highest activity (20% of the activity of E2 at 6.7 X 10-7M) among positive compounds for ER-oL. A 10-fold higher concentration of o,p'-DDT was required for activity, but the concentration corresponded to that of methoxychlor. In this study, no antiestrogenic activity was observed with any of the pesticides tested. Chlorpyrifos-methyl has weak affinity for estrogen, progesterone, and androgen receptors in yeast transformants that express human steroid hormone receptors and steroid hormone response elements (Jeong et al., 2001). Androgenic activity has not been observed in any pesticide tested. However, antiandrogenic activity of OPs has been observed in vitro. Parathion inhibits dihydrotestosterone (DHT) binding to AR in rat ventral prostate (Shain et al., 1977). Kojima et al. (2004) reported that 19 OPs (fenitrothion, anilofos, EPN, prothiofos, parathion, methyl parathion, tolclofos-methyl, piperophos, ethion, butamifos, phosalone, dichlofenthion, fenthion, cyanophos, leptophos, bromopho-ethyl, quinalphos, isofenphos, and MEP oxon) showed inhibitory effects on the androgenic activity of DHT in a reporter gene assay using CHO cells transfected with hAR. Fenitrothion showed the highest activity, and the activity of DHT was inhibited by 20% at 1.8 X 10 -7 M and completely abrogated at 1 • 10 -5 M. The activity was higher than that of p,p'-DDE. Fenthion showed antiandrogenic activity against the androgenic activity of DHT in a reporter assay using NIH3T3 cells, but fenthion sulfoxide was negative (Kitamura et al., 2003b). In the literature,
485
fenitrothion and EPN, as well as fenthion, but not fensulfothion, trichlorfon, or malathion, are reported to be antiandrogenic. Fenitrothion was the most active. In our study, it was found that fenthion is markedly inactivated after oxidative metabolism by mixed function oxidase (MFO). In contrast, fenthion sulfoxide was activated by rat liver preparations. This is due to interconversion between fenthion and fenthion sulfoxide by rat liver preparations (Kitamura et al., 2000, 2003a). Tamura et al. (2001) reported that fenitrothion competitively inhibited DHT-dependent human androgen receptor activation in HepG2 cells. The potency of fenitrothion was approximately 8- to 35-fold higher than those of p,p'-DDE and linuron, well-known environmental antiandrogens, and approximately 50% of that of flutamide, which is a pharmaceutical antiandrogen (Maness et al., 1998; Mclntyre et al., 2000). Antiandrogenic activity of methyl parathion was also seen in a preliminary study. Sohoni et al. (2001) observed activity of fenitrothion in recombinant yeast expressing the human androgen receptor. The structure of fenitrothion closely resembles that of flutamide. Fang et al. (2003) reported that methyl parathion, parathion, and triphenyl phosphate were positive in a comparative binding assay to AR. A SAR study suggested that hydrophobic interactions of these pesticides are important for AR binding. Currently, the significant structural differences between antiandrogenic and non-antiandrogenic OP insecticides are difficult to understand. Further study of the structural requirements for estrogenic and antiandrogenic activities and of the mechanisms of action of OP insecticides is needed.
B. Endocrine-Disrupting Action of CMs In Vitro Some CM pesticides show endocrine-disrupting action, whereas others do not (Table 2). A CM pesticide, carbaryl, lacks binding affinity for estrogen receptor from rat uterus (Blair et al., 2000). Estrogenic activity of methomyl was not observed at 2 x 10 -4 M (Nishihara et al., 2000). Bendiocarb, carbofuran, and carbaryl are not estrogenic in E-screen~ assay (Sonnennschein and Soto, 1998). In contrast, Klotz et al. (1997) reported that aldicarb, baygon, bendiocarb, carbaryl, methomyl, and oxamyl weakly activated estrogen- or progesterone-responsive repo~er genes in breast MCF-7 and endometrial (Ishikawa) cancer cells at a concentration of 1 X 10 -7 M. However, these CMs (1 x 10 -7 M) decreased estrogen- or progesterone-induced reporter gene activity induced at the concentration of 1 x 10 -9 M to approximately one-third. In contrast, Kojima et al. (2004) examined the estrogenic activity of 22 CM pesticides and reported that methiocarb has an agonistic effect on hER-oL and ER-~. Furthermore, they found that the activity against ER-[3 was higher than that against ER-oL. In contrast, Andersen et al. (2002) reported that methiocarb exhibits estrogenic activity in cell proliferation assay and transactivation assay using MCF-7 human breast cancer cells. Methiocarb was estrogenic in the range of 1 X 10 -5
486
S E C T I O N IV 9 Organ Toxicity
T A B L E 2.
Endocrine-Disrupting Action of CMs In Vitro
Estrogenic and antiandrogenic CMs H3C
HaCS~}'-OCONHCH3 HaC methiocarb Estrogenic CMs
.30
O-CO_N'CH3 H3C,,,~ N "CH3 H 3 c / ~ N ~ " N--CH3 "CH 3
pirimicarb
H3C~ H3c~NCH2CH2CH2NHCOOCH2CH2CH
propamocarb OCONHCH3
CH3
I
H3CS~--(~/~'- OCONHCH3 H3C
methiocarb
OCONHCH3
CH3SNCCH = NOCONHCH3 I CH3 aldicarb
baygon
bendiocarb
C H 3 ( ~ NOCONHCH3 SCH3
H3C~ NCOC= NOCONHCH3 H3C ~ SCH3
methomyl
oxamyl
OCONHCH3
carbaryl Antiestrogenic CMs
OCONHCH3
CH3
I
CH3SNCCH-- NOCONHCH3 I CH3
aldicarb
OCONHCH3
O" CH3 baygon
bendiocarb
CH3C~NOCONHCH 3 SCH3
H3C~ NCOC=NOCONHCH3 H3C ~ SCH3
methomyl
oxamyl
OCONHCH3
carbaryl
(continues)
CHAPTER 34 9Endocrine Disruption by OPs and CMs
TABLE 2. Antiandrogenic CMs
.30
methiocarb
(continued)
~2SCON"C2H5 "C2Hs
H3CS~-'~/~-OCONHCH3 H3C
487
CI thiobencarb
Aromatase-stimulators
~2SCON "C2H5 *'C2H5 CI primicarb
H3C~, H3C" NCH2CH2CH2NHCOOCH2CH2CH 3 propamocarb
to 5 x 10 -5 M in MCF-7 cell proliferation and ER transactivation assays. Some chemicals are known to potentiate the estrogenic activity of E2. Primicarb and propamocarb exhibited no estrogenic response when tested alone but potentiated the activity of E2 (10 nM) in the range of 5 X 10 -7 to 5 X 10 -5 M in ER transcription assay. Grunfeld and Bonefeld-Jorgensen (2004) examined the effect of estrogenic CMs primicarb, propamocarb, and methiocarb on ER-oL and -[3 mRNA levels. They reported that pirimicarb weakly decreased the ER-oL mRNA level, whereas propamocarb and methiocarb had no effect. In contrast, chlorpyrifos weakly increased the ER-[3 mRNA level. Hofmeister and Bonefeld-Jorgensen (2004), using the same cell line, found that prochloraz had the potential to downregulate the expression of ER-oL and ER-[3 mRNAs as well as E 2, and the effect was abolished by cotreatment with the ER antagonists ICI 182 and 789. Methiocarb had no effect on the ER-oL mRNA level, whereas it increased the ER-[3 mRNA level. Andersen et al. (2002) reported that methiocrab acted as an AR antagonist as well as an ER agonist. Methiocarb inhibited the androgenic response to R1881 at the concentration of 20 IxM in transactivation assay using CHO cells. Birkhoj et al. (2004) examined the antiandrogenic activity of methiocarb alone or combined with other commonly used pesticides. This pesticide inhibited R1881-induced transcriptional activation (IC25, 5.8 ixM) in in vitro AR reporter assay using CHO cells, but it was negative in the in vivo Hershberger assay using castrated rats. Kojima et al. (2004) reported that methiocarb and thiobencarb inhibited the androgenic activity of DHT in CHO cells transfected with hAR at 2.8 X 10 -6 and 9.4 x 10 -6 M (IC20), respectively. When combined with four other pesticides (deltamethrine, prochloraz, simazine, and tribenuron-methyl), there was an additive effect in in vitro assay, but no significant effect was
seen in vivo except for a 25% reduction in the weight of the levator ani/bulbocavernosus muscle. Furthermore, urea-type pesticides propanil, linuron, and diuron, which are the main herbicides used throughout the world, have the ability to bind to AR (Cook et al., 1993; Bauer et al., 1998). Indeed, linuron is structurally related to the nonsteroidal antiandrogen flutamide, and its ICs0 for competition at the AR is approximately 3.5 times that of flutamide. The activity of its metabolite, 3,4-dichloroaniline, is higher than that of linuron.
C. Disruption of Steroidogenesis by OPs and CMs In Vitro Some pesticides may have an ability to modulate the effects of potent natural steroid hormones. Interaction with hormone receptors is one possible mechanism of hormonedisrupting action. Activation or inhibition of enzyme activities involved in steroid hormone synthesis may also alter endogenous hormone levels. Andersen et al. (2002) examined the effect of pesticides on CYP 19-aromatase, which catalyzes the conversion of C19 steroids to estrogens, and showed that the OPs tested had no effect on the activity but pifimicarb and propamocarb enhanced the activity at the concentration of 50 p~M. They also examined serum levels of testosterone, LH, T4, and prolactin after administration of pesticides and reported that methiocarb significantly reduced luteinizing hormone (LH) levels compared to those in testosterone-treated animals. Vinggaard et al. (2000) reported that chlorpyrifos, tetrachlorvinphos, and linuron did not affect aromatase activity. In contrast, a urea-type fungicide, prochloraz, inhibited the aromatase activity of human placental microsomes and human JEG-3 chofiocarcinoma cells (Mason et al., 1987; Vinggaard et al., 2000;
488
SECTION IV. O r g a n T o x i c i t y
Andersen et al., 2002). Linuron decreased accessory sex organ weights in sexually immature and mature rats treated with linuron. In linuron-treated mature rats, serum E 2 and LH levels were increased (Cook et al., 1993). Bisson and Hontela (2002) reported that diazinon (OP) and mancozeb (CM) inhibited ACTH- and dibutyryl-cAMP-stimulated cortisol secretion in adrenocortical cells of rainbow trout at concentrations of 50-500 IxM. The capacity of mancozeb to disrupt adrenal steroidogenesis was much greater than that of diazinon. They also suggested that the activity is due to ethylene thiourea, a degradation product in the medium.
III. E N D O C R I N E - D I S R U P T I N G A C T I V I T Y O F O P s A N D C M s IN A N I M A L S A. O P Pesticides 1. ESTROGEN- AND ANDROGEN-RELATED ENDOCRINE EFFECTS Quinalphos exhibited estrogen-like action in vaginal cornification and increased the uterine wet weight (uterotrophic assay; Clark et al., 1980) in immature and ovariectomized mature rats by the treatment of 1 mg/kg/day, sc, for 3 days, but the activity was weaker than that of aldrin (Chatterjee et al., 1992). However, Sohoni et al. (2001) reported that fenitrothion (15 mg/kg/day for 3 days) did not have estrogenic activity in the immature mouse uterotrophic assay. Chlorpyrifos-methyl, the most commonly used OP pesticide throughout the world, was also nonestrogenic in immature rat uterotrophic assay when given at 250 mg/kg/day for 20 days (Kang et al., 2004). Regarding antiandrogenic activity, fenthion was positive in vivo in the Hershberger assay (Hershberger et al., 1953) using castrated male rats (Kitamura et al., 2003a). When fenthion (25-50mg/kg/day) was subcutaneously dosed together with testosterone propionate (0.5 mg/kg/day) for 7 days, the effect of testosterone on the weights of the prostate and seminal vesicles was significantly suppressed. In contrast, both positive and negative results on the antiandrogenic activity of fenitrothion have been reported (Ashby and Lefevre, 2000; Sunami et al., 2000; Tamura et al., 2001; Sohoni et al., 2001). Antiandrogenic activity of fenitrothion has been reported in the Hershberger assay using castrated male rats (Tamura et aL, 2001). When fenitrothion was administered by gavage at 15-30 mg/kg/day for 7 days together with testosterone propionate (50 rag/day, sc), the tissue weights of the ventral prostate, seminal vesicle, and levator ani plus bulbocavernous muscles were significantly decreased compared to those of corn oil-dosed castrated male rats. The study concluded that the activity of fenitrothion is comparable in potency to that of the pharmaceutical antiandrogen flutamide. Curtis (2001) featured the previous result in parallel with multiple measurements of neurotoxicity in "Toxicological Highlights" of
Toxicological Sciences. However, Ashby and Lefevre (2000) reported a negative result in the peripubertal male rat assay, which is an altemative to the Hershberger assay. Fenitrothion (15 mg/kg/day) failed to cause a significant delay in preputial separation of peripubertal male rats. Furthermore, their group reported a negative result for fenitrothion in the Hershberger assay using castrated male rats (Sohoni et al., 2001). Sunami et al. (2000) also reported that it showed no antiandrogenic activity in the Hershberger assay in castrated male rats. They found that fenitrothion (0.075-3 mg/kg/day for 5 days) significantly suppressed cholinesterase activities in the brain and erythrocytes but did not decrease the androgenic activity of testosterone. Unfortunately, they did not conduct a higher dose experiment, despite using a 100 mg/kg/day dose of p,p'-DDE as a positive control. Later experiments in vitro showed that fenitrothion had the highest antiandrogenic activity among OPs tested (Kitamura et al., 2003b; Kojima et al., 2004). The significant antiandrogenic activity of fenitrothion in vitro supports the in vivo findings of antiandrogenic properties. Regarding anticholinesterase activity, OPs are metabolically activated, but OPs seem to exhibit endocrinedisrupting activity. In fact, fenthion lost its antiandrogenic activity after metabolic transformation to fenthion sulfoxide. On the contrary, some pesticides may be metabolically activated. Although chlorpyrifos-methyl has weak or no affinity for estrogen and androgen receptors in in vitro assay (Jeong et al., 2001), chlorpyrifos-methyl showed antiandrogenic activity when given by gavage at 50 mg/kg/day for 10 days in the Hershberger test using rats (Kang et al., 2004). This OP may be metabolically activated in the animal body. It may be necessary to conduct ER or AR affinity tests in the presence of the metabolic system of MFO. To investigate whether fenthion has an impact on androgen-dependent sexual differentiation, the effects of in utero exposure were further examined in rats (Turner et al., 2002). Pregnant Sprague-Dawley (SD) rats were administered fenitrothion by gavage from gestation day 12 to day 21. In male offspring maternally exposed to it at 25 mg/kg/day, a reduction in anogenital distance was evident on postnatal day (PND) l, although the effect was transient, and no abnormalities in male organ development were noted at PND 100. Administration of fenitrothion at high dosages (55 mg/kg for 3 days) has been reported to reduce testicular cytochrome P450 without affecting cytochrome b5 or NADPH-cytochrome c reductase (Closet al., 1994). A decrease in serum testosterone levels was also detected at this dosage. The ability of parathion to interfere with the metabolism of androgen, as well as the uptake, in male accessory organs in rats was described in the 1960s and 1970s (Kupfer, 1967; Schein and Thomas, 1976; Thomas and Schein, 1974). The oral administration of parathion (1.3-5.2 mg/kg/day) caused significant alterations in the metabolism of testosterone in mice. An early study suggested that this compound
CHAPTER 34 9Endocrine Disruption by OPs and CMs competitively inhibits androgen binding to its receptor (Schein et al., 1979). Parathion was reported to interfere with mouse spermatogenesis, which may be related to its prenatal toxicity in mammals. An organ culture study of mouse testis revealed that parathion directly suppresses normal testicular differentiation (Rojas et al., 1998). An increase in apoptosis in spermatogonia by this pesticide was also reported (Bustos-Obregon et al., 2001). Quinalphos is also known to have adverse effects on the testis and male accessory glands in rats (Ray et al., 1991, 1992). When Wistar rats were given this compound at 0.25 mg/kg/day (ip) for 26 days, massive degeneration of germ cells in the testis, a reduction in the sperm count, and a decrease in plasma follicle-stimulating hormone (FSH) level were evident (Ray et al., 1992). Since administration of human chorionic gonadotrophin partially prevented these toxicological effects, quinalphos probably acts on the hypothalamuspituitary axis to suppress gonadotrophin release. High-dose administration of quinalphos, however, may impact differently on the endocrine system in rats. Treatment of SD rats with sublethal doses at 7-14 mg/kg/day for 15 days resulted in increased serum LH/FSH and testosterone levels, along with a decrease in testicular weight and severe disruption of spermatogenesis. Malathion shows a relatively high toxicity to fish. Morphological abnormalities in African catfish larvae exposed to malathion were observed (Lien et al., 1997). Diazinon has no hormone-like activity but shows endocrinedisrupting potential in fish. In dispersed adrenocortical cells of rainbow trout, this compound suppressed cortisol secretion in response to ACTH in vitro (Bisson and Hontela, 2002). When bluegill fish were exposed to diazinon at 60 Ixg/liter in water, serum E 2 became undetectable within 24 hr and there was morphological disruption of ovarian follicles (Maxwell and Dutta, 2005). The antiandrogen mechanism of OPs is clearly based on their ability to bind to AR in competition with testosterone or to bind to ER as agonists. However, other mechanisms are also involved. Chlorpyrifos, for instance, induced gene expression of gonadotropin-releasing hormone in a hypothalamic cell line, GT1-7, suggesting that it may have an impact on the hypothalamic-pituitary-gonad axis (Gore, 2002; Kojima et al., 2004). It has also been reported that chlorpyrifos may interfere with testosterone metabolism, potentially leading to hormonal imbalance (Usmani et al., 2003). 2. THYROID HORMONE-RELATED ENDOCRINE EFFECTS Malathion can disrupt thyroidal activity. In young adult rats, administration of malathion at 60 ixg/rat/day for 21 days resulted in decreased serum T3 and T4 and increased thyroid-stimulating hormone levels (Akhtar et al., 1996). In tadpole, malathion affects development. A significant delay of growth was observed in tadpoles in water
489
containing 1 mg/liter malathion, suggesting a decrease in thyroid function (Fordham et al., 2001). In the freshwater catfish, Clarias batrachus, malathion decreased serum T3 level but accelerated T4 synthesis in the pharyngeal thyroid. Extrathyroidal conversion of T4 to T3 was also inhibited (Sinha et al., 1991b, 1992). Rawlings et al. (1998) reported that chlorpyrfos and dimethoate decreased serum T4 levels in ewes treated with these chemicals at 12.5 and 0.2 mg/kg, respectively, three times a week for 43 days. They also demonstrated that chlorpyrifos increased the serum cortisol concentration, and dimethoate decreased the basal LH concentration. The teleost fish, Channa punctatus (Bloch), exposed to 2-4 ppm cythion in water developed hypertrophy and hyperplasia in the follicular epithelium and a reduction of colloid content in the thyroid (Ram et al., 1989). A significant reduction of thyroidal iodine uptake was also observed. Treatment of bullfrog tadpoles (Rana catesbeiana) with malathion significantly delayed developmental progression (Fordham et al., 2001). Malathion inhibited T3 binding to transthyretin but did not bind to the ligand-binding domain of thyroid hormone receptor [3 in a study using recombinant of Japanese quail transthyretin and thyroid receptor (Ishihara et al., 2003). This affinity for transthyretin may account for the effect of malathion on serum thyroid hormone levels in vivo. For other OP pesticides, no data are available regarding interaction with thyroid hormone receptor, transthyretin, or other thyroid hormone binding proteins. B. C M Pesticides 1. ESTROGEN- AND ANDROGEN-RELATED ENDOCRINE EFFECTS The mechanisms of the toxicity of CMs to testis and ovaries are not clear. The toxicity may be due to a direct effect on the ovary or the hypothalamus-pituitary-ovarian axis, causing hormonal imbalance. However, carbaryl and carbofuran have been reported to affect the function and morphology of the reproductive organs as well as serum estrogen levels in rats and fishes. When a high dose of carbaryl was orally administered to male rats (100 mg/kg body weight, 5 days a week), marked histopathological changes in the testes were seen, with degeneration of spermatogenic cells (Pant et al., 1995). These effects were associated with declines in epididymal sperm count and percentage sperm motility and increased abnormal sperm morphology. Adult male Wistar rats fed laboratory chow containing 1.0, 6.3, or 203 ppm benomyl exhibited decreased ejaculate sperm counts, decreased testicular weight, and a lowered male fertility index (Barnes et al., 1983). In catfish, carbaryl exposure produced a reduction in the number of oocytes, deformity in oocytes at different stages, and a reduction in the gonadosomatic index (Kulshrestha and Arora, 1984). Carbofuran seems to be more toxic. When female Swiss mice were orally administered
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with this chemical at 1 mg/kg body weight/day, there was a significant decrease in the number of estrous cycles and in the duration of the phases of each cycle. There was a significant decrease in the number of healthy follicles (Baligar and Kaliwal, 2002). Rats dosed with carbofuran (1.5 mg/kg, sc) showed transient endocrine disruption; that is, the levels of progesterone, cortisol, and E2 were significantly increased, whereas the levels of testosterone were decreased. No significant change occurred in T3 or T4 (Goad et al., 2004). In fish, treatment with 0.5-1 mg/liter of carbofuran inhibited oocyte maturational processes in females (Chatterjee et al., 1997) and caused deleterious testicular changes, included necrosis of Leydig cells (Ram et al., 2001). In contrast, an impairment of the duration of estrous cycles in rats treated with mancozeb was observed (Mahadevaswami et al., 2000). Molinate, a thiocarbamate herbicide, showed testicular toxicity after metabolic activation because the sulfoxide, a major metabolite generated by testis microsomes, exhibited more potent toxicity (Jewell et al., 1998).
2. THYROID HORMONE-RELATED ENDOCRINE EFFECTS CMs disrupt serum thyroid hormone levels in rodents and fishes. In a study of SD rats fed aldicarb at 10ppb, metribzin at 10 ppm, or methomyl at 1 ppm in drinking water (6-16 weeks), serum thyroxin levels increased significantly (Porter et al., 1999). In catfish, carbaryl exposure (12 mg/liter) for 96 hr suppressed serum T4 but elevated serum T3 levels, whereas exposure (5 mg/liter) for 16 days decreased both T3 and T4 (Sinha et al., 1991a). Rawlings et al. (1998) reported that carbofuran caused a significant increase in serum thyroxin level, and triallate increased the basal LH level compared with that of control ewes. In the teleost fish, C. punctatus, treatment with carbaryl at 1.7 ppm for 30 days also caused a decline in T4 and an elevation in T3 (Ghosh et al., 1989). In the same species, long-term exposure (6 months) to carbofuran at 4.5 ppm led to histological abnormalities of the thyroid gland, including hypertrophy, hyperplasia, and degeneration of follicular epithelial cells, and a reduction in colloid content along with retardation of thyroid function. Another study also found a greater susceptibility of the younger group to chronic toxicity of carbofuran compared with adults in this species (Ram, 1988). Mancozeb can disrupt thyroid function, and its degradation product, ethylene thiourea, is causally related to thyroid cancer in animals (Steenland et al., 1997; Chhabra et al., 1992). The mechanisms of disruption of thyroid function have not been established. Direct interaction of CMs with thyroid hormone receptors or serum thyroid binding proteins seems unlikely but has not been investigated. The adverse histophysiological alterations in the thyroid and changes in serum thyroid hormone levels may be a result of CMs directly interfering with the iodide transporter or
the organic iodine formation process in the thyroid gland, or they may occur as a result of disruption at any point in the hypothalamus-pituitary-thyroid axis.
IV. I N F L U E N C E O F O P s A N D C M s ON HUMANS Human exposure to single endocrine-disrupting chemicals is generally considered to be low, and the compounds are much less potent than the natural hormones. However, humans are expected to be exposed to a mixture of potential endocrine-disrupting chemicals. Hence, potential additive or synergistic effects have to be considered to assess the human risk. Furthermore, in vivo potency is often different from in vitro potency since the mechanisms of hormonal action of chemicals may not be adequately reflected by the in vitro assays. OPs and CMs are widely used in agriculture and for pest control. OPs such as chlorpyrifos have been found in human blood in Spain (Pitarch et al., 2003), in human urine in the United States (Olsson et al., 2003), and in human breast milk in India (Sanghi et al., 2003), together with organochlorine pesticides. There are many opportunities for human exposure. Because the pesticides are believed to be readily absorbed through the skin, monitoring is an essential component of assessment of exposure. Cocker et al. (2002) reported that whereas in nonoccupationally exposed people the urinary alkyl phosphates do not exceed 72 lxmol/mol creatine, occupationally exposed people excrete alkyl phosphates in the urine at the average level of 122 ixmol/mol creatine. However, the authors indicated that the levels of alkyl phosphates in workers are unlikely to cause a significant reduction in blood cholinesterase activity. In a volunteer study of exposure to chlorpyrifos, diazinon, propetamphos, and diazinon, almost all of these pesticides were found to be excreted after an oral dose, but large amounts were recovered in the skin surface after a dermal dose (Cocker et al., 2002; Garfitt et al., 2002). Tuomainen et al. (2002) reported that after application of malathion in greenhouses, the level of the pesticide in urine of workers reached a maximum at 6 or 7 hr and then rapidly decreased. Colosio et al. (2002) measured the ethylenethiourea level in urine of workers in vineyards as an indicator of mancozeb, and they reported that the levels in the workers were much higher than in controls, being especially high in operators of open tractors. In contrast, infants and children may have other opportunities to be exposed to OPs and CMs. Gurunathan et al. (1998) reported that chlorpyrifos persistently accumulates on residential surfaces and toys accessible to children after household application. Exposure of children to OPs can be evaluated by measuring urinary biomarkers and their metabolites, and it may be related to their potential adverse health effects (Eskenazi et al., 1999).
CHAPTER 34
Environmental antiandrogens may affect the development of the male reproductive system (Kelce and Wilson, 1997). Because androgens initiate and maintain spermatogenesis, environmental antiandrogens may contribute to the decline in sperm content that is suspected to be occurring in humans and other animals. Furthermore, inhibition of androgen receptor-mediated actions during the embryonic stage may lead to developmental alteration of male external genitalia (Foster, 1997). In humans, although a causal relationship between exposure to endocrine-disrupting pesticides and reproductive disorders has not been established, some reports predict an effect on humans. Sherman (1996) reported birth defects of the central nervous system in children exposed in utero to chlorpyrifos. Weidner et al. (1998) noted an increased occurrence of cryptorchidism in sons of female gardeners. Abell et al. (2000) also reported reduced fecundity in female greenhouse workers. There are conflicting results concerning the association of exposure of pregnant women to pesticides with changes of fetal growth or length of gestation (Grether et al., 1987; Savitz et al., 1989; Fenster and Coye, 1990; Restrepo et al., 1990; Thomas et al., 1992; Willis et al., 1993; Kristensen et al., 1997; Xiang et al., 2000; Perera et al., 2003; Eskenazi et al., 2004). Further examination is needed to clarify the influence of OPs and CMs on humans and wildlife.
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Porter, W. R, Jaeger, J. W., and Carlson, I. H. (1999). Endocrine, immune, and behavioral effects of aldicarb (carbamate), atrazine (triazine) and nitrate (fertilizer) mixtures at groundwater concentrations. Toxicol. Ind. Health 15, 133-150. Ram, R. N. (1988). Carbofuran-induced histophysiological changes in thyroid of the teleost fish, Channa punctatus (Bloch). Ecotoxicol. Environ. Safety 16, 106-113. Ram, R. N., Joy, K. E, and Sathyanesan, A. G. (1989). Cythioninduced histophysiological changes in thyroid and thyrotrophs of the teleost fish, Channa punctatus (Bloch). Ecotoxicol. Environ. Safety 17, 272-278. Ram, R. N., Singh, I. J., and Singh, D. V. (2001). Carbofuran induced impairment in the hypothalamo-neurohypophysealgonadal complex in the teleost, Channa punctalus (Bloch). J. Environ. Biol. 22, 193-200. Rawlings, N. C., Cook, S. J., and Waldbillig, D. (1998). Effects of the pesticides carbofuran, chlorpyrifos, dimethoate, lindane, trillate, trifluran, 2,4-D and pentachlorophenol on the metabolic endocrine and reproductive endocrine system in ewes. J. Toxicol. Environ. Health A 54, 21-36. Ray, A., Chatterjee, S., Ghosh, S., Kabir, S. N., Pakrashi, A., and Deb, C. (1991). Suppressive effect of quinalphos on the activity of accessory sex glands and plasma concentrations of gonadotrophins and testosterone in rats. Arch. Environ. Contam. Toxicol. 21, 383-387. Ray, A., Chatterjee, S., Ghosh, S., Bhattacharya, K., Pakrashi, A., and Deb, C. (1992). Quinalphos-induced suppression of spermatogenesis, plasma gonadotrophins, testicular testosterone production, and secretion in adult rats. Environ. Res. 57, 181-189. Restrepo, M., Munoz, N., Day, N. E., Parra, J. E., de Romero, L., and Nguyen-Dinh, X. (1990). Prevalance of adverse reproductive outcomes in a population occupationally exposed to pesticides in Colombia. Scand. J. Work Environ. Health 16, 232-238. Rojas, M., Bustos-Obregon, E., Martinez-Garcia, E, Contreras, H., and Regadera, J. (1998). The effect of parathion on mouse testicular and epididymal development cultured in chicken allantochorion. Adv. Exp. Med. Biol. 444, 201-206. Sanghi, R., Pillai, M. K., Jayalekshmi, T. R., and Nair, A. (2003). Organochlorine and organophosphorus pesticide residues in breast milk from Bhopal, Madhya, Pradesh, India. Hum. Exp. Toxicol. 22, 73-76. Savitz, D. A., Whelan, E. A., and Kleckner, R. C. (1989). Self-reported exposure to pesticides and radiation related to pregnancy outcome m Results from National Natality and Fetal Mortality Surveys. Public Health Rep. 104, 473-477. Schein, L. G., and Thomas, J. A. (1976). Deldrin and parathion interaction in the prostate and liver of the mouse. J. Toxicol. Environ. Health 1, 829-838. Schein, L. G., Donovan, M. E, Thomas, J. A., and Felice, E R. (1979). Effects of pesticides on 3H-dihydrotestosterone binding to cytosol proteins from various tissues of the mouse. J. Environ. Pathol. Toxicol. 3, 461-470. Shain, S. A., Shaeffer, J. C., and Boesel, R. W. (1977). The effect of chronic ingestion of selected pesticides upon rat ventral prostate homeostasis. Toxicol. Appl. Pharmacol. 40, 115-130. Sherman, J. D. (1996). Chlorpyrifos (Dursban)-associated birth defects: Report of four cases. Arch. Environ. Health 51, 5-8.
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Sinha, N., Lal, B., and Singh, T. E (1991a). Carbaryl-induced thyroid dysfunction in the freshwater catfish Clarias batrachus. Ecotoxicol. Environ. Safety 21, 240-247. Sinha, N., Lal, B., and Singh, T. E (1991b). Pesticides induced changes in circulating thyroid hormones in the freshwater catfish Clarias batrachus. Comp. Biochem. Physiol. C 100, 107-110. Sinha, N., Lal, B., and Singh, T. E (1992). Thyroid physiology impairment by malathion in the freshwater catfish Clarias batrachus. Ecotoxicol. Environ. Safety 24, 17-25. Sohoni, E, Lefevre, E A., Ashby, J., and Sumpter, J. E (2001). Possible androgenic/anti-androgenic activity of the insecticide fenitrothion. J. Appl. Toxicol. 21, 173-178. Sonnenschein, C., and Soto, A. M. (1998). An update review of environmental estrogen and androgen mimics and antagonists. J. Steroid Biochem. Mol. Biol. 65, 143-150. Steenland, K., Cedillo, L., Tucker, J., Hines, C., Sorensen, K., Deddens, J., and Cruz, V. (1997). Thyroid hormones and cytogenetic outcomes in backpack sprayers using ethylenebis(dithiocarbamate) (EBDC) fungicides in Mexico. Environ. Health Perspect. 105, 1126-1130. Sunami, O., Kunimatsu, T., Yamada, T., Yabushita, S., Sukata, T., Miyata, K., Kamita, Y., Okuno, Y., Seki, T., Nakatsuka, I., and Matsuo, M. (2000). Evaluation of a 5-day Hershberger assay using young mature male rats: Methyltestosterone and p,p'-DDE, but not fenitrothion, exhibited androgenic or antiandrogenic activity in vivo. J. Toxicol. Sci. 25, 403-415. Tamura, H., Maness, S. C., Reischmann, K., Dorman, D. C., Gray, L. E., and Gaido, K. W. (2001). Androgen receptor antagonism by the organophosphate insecticide fenitrothion. Toxicol. Sci. 60, 56-62. Thomasl D. C., Petitti, D. B., Goldhaber, M., Swan, S. H., Rappaport, E. B., and Hertz-Picciotto, I. (1992). Reproductive outcomes in relation to malathion spraying in the San Francisco Bay Area, 1981-1982. Epidemiology 3, 32-39. Thomas, J. A., and Schein, L. G. (1974). Effect of parathion on the uptake and metabolism of androgens in rodent sex accessory organs. Toxicol. Appl. Pharmacol. 29, 53-58. Tilak, K. S., Veeraiah, K., and Rao, D. K. (2004). Toxicity and bioaccumulation of chlorpyrifos in Indian carp Catla catla (Hamilton), Labeo rohita (Hamilton), and Cirrhinus mrigala (Hamilton). Bull. Environ. Contam. Toxicol. 73, 933-941.
Tuomainen, A., Kangas, J. A., Meuling, W. J. A., and Glass, R. C. (2002). Monitoring of pesticide applicators for potential dermal exposure to malathion and biomarkers in urine. Toxicol. Lett. 134, 125-132. Turner, K. J., Barlow, N. J., Struve, M. E, Wallace, D. G., Gaido, K. W., Dorman, D. C., and Foster, E M. (2002). Effects of in utero exposure to the organophosphate insecticide fenitrothion on androgen-dependent reproductive development in the Crl:CD(SD)BR rat. Toxicol. Sci. 68, 174-183. Tsuda, T., Aoki, S., Kojima, M., and Harada, H. (1990). Bioconcentration and excretion of diazinon, malathion and fenitrothion by carp. Comp. Biochem. Physiol. C 96, 23-26. Tsuda, T., Kojima, M., Harada, H., Nakajima, A., and Aoki, S. (1996). Accumulation and excretion of fenthion, fenthion sulfoxide and fenthion sulfone by killifish (Oryzias latipes). Comp. Biochem. Physiol. C 113, 45-49. Usmani, K. A., Rose, R. L., and Hodgson, E. (2003). Inhibition and activation of the human liver microsomal and human cytochrome P450 3A4 metabolism of testosterone by deployment-related chemicals. Drug Metab. Disp. 31, 384-391. Vinggaard, A. M., Hnida, C., Breinholt, V., and Larsen, J. C. (2000). Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. in Vitro 14, 227-234. Vonier, E M., Crain, D. A., McLachlan, J. A., Guillette, L. J., and Arnold, S. E (1996). Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator. Environ. Health Perspect. 104, 1318-1322. Weidner, I. S., Moller, H., Jensen, T. K., and Skakkebaek, N. E. (1998). Cryptorchidism and hypospadias in sons of gardeners and farmers. Environ. Health Perspect. 106, 793-796. Willis, W. O., de Peyster, A., Molgaard, C. A., Walker, C., and MacKendrick, T. (1993). Pregnancy outcome among women exposed to pesticides through work or residence in an agricultural area. J. Occup. Med. 35, 943-949. Wong, C., Kelce, W., Sar, M., and Wilson, E. M. (1995). Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Biol. Chem. 270, 19998-20003. Xiang, H., Nuckolos, J. R., and Stallones, L. (2000). A geographic information assessment of birth weight and crop production patterns around mother's residence. Environ. Res. 82, 160-167.
CHAPTER ~ 5
Organophosphates, Carbamates, and the Immune System RAGHUBIR P. SHARMA The University of Georgia, Athens, Georgia
pesticides possess detrimental effects on various immune responses, information on the effects of these chemicals on the immune system of humans is very limited. It is controversial whether environmental or even occupational exposures to these pesticides are capable of affecting the immune system (Thomas, 1995). The level of exposure from the environment is negligible for anticholinesterase pesticides because of their short lives in the environment, including food. A greater concern of untoward effect with pesticide use is occupational exposures at the manufacturing, shipping and formulating, and application levels. In addition to pesticide exposure through environmental or occupational modes, people may also come in contact with cholinesterase inhibitors via therapeutic uses. Cholinesterase inhibitors have been employed in a variety of nervous system disorders, including Alzheimer's disease, Down syndrome, traumatic brain injury, and delirium (Giacobini, 2004). For the treatment of these disorders and cognitive enhancement, patients have to be treated for a long period (24 months or longer) with either specific acetycholinesterase inhibitors or butyrylcholinesterase inhibitors, sometime switching one with the other. A long-term treatment with such inhibitor is likely to cause various systemic effects, including those on the immune system. The possibility that OPs can be designed as selective immunosuppressors for therapeutic purposes has been considered (Becker, 1975). It was theorized that highly specific, highly active, relatively stable, and nontoxic chemicals can be prepared. Specificity against the first component of the complement system was selectively increased by adding a terminal amino group to the p-nitrophenyl ethyl pentyl phosphate. It is sometimes possible to separate the anticholinesterase activity from the protease inhibitor activity; however, this approach has had limited success. There is increasing concern regarding safety and risk assessment of chemicals on the developing immune system (Holsapple et al., 2004). The developing immune system is considered as susceptible to toxic effects; however, data supporting this assumption are limited. On the one hand,
I. I N T R O D U C T I O N Pesticides are toxic chemicals that are deliberately introduced in the environment and therefore exposure of people and animals to them is unavoidable. These chemicals are inherently toxic, especially to the organisms they are intended to eradicate. They also produce variable toxicity in all other organisms that come in contact with them. The use of pesticides is desirable because pests cause heavy losses to crops and other food commodities and they are indispensable for disease prevention in public health. Some of the original agents used as pesticides included metal compounds and chlorinated hydrocarbons. Due to the persistence and biomagnification of earlier pesticides in the food chain, their use is on the decline and they have largely been replaced by cholinesterase inhibitors and, recently, pyrethrins and analogs. Anticholinesterase agents are extensively used in agriculture because of both their effectiveness and their low cost; also, they are not persistent, so there is no concern of secondary exposures via food. Because exposure to prevalent cholinesterase-inhibiting insecticides is unavoidable, these compounds have been investigated in detail with regard to their toxic potential; however, their potential effects on the immune system are not so well characterized. Concern regarding the immunotoxic potential of chemicals in general is relatively new in the assessment of their safety. Only a few organophosphates (OPs) and carbamates (CMs) have been systematically investigated for their immunotoxic potential; studies have used different protocols and various parameters for measuring toxic outcomes. The immune system is important for defense against a variety of organic insults and is necessary for the well-being and even survival of host organisms. However, the immune system is highly complex, regulated by multistep control processes, and can be influenced by the status of other body systems and functions. There are a number of reviews on the immunotoxicity of OPs and CMs (Sharma, 1988; Sharma and Tomar, 1992; Galloway and Handy, 2003). Despite indications that these Toxicology of Organophosphate and Carbamate Compounds
495
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
496
SECTION IV. Organ Toxicity
the system undergoing development in neonates and children may be at greater risk due to differentiation and cellular proliferation occurring at this stage; on the other hand, adaptation and plasticity of developing systems suggest that the effects may not be long-lasting. However, various regulatory agencies are cautious and increasingly have been developing additional guidelines for risk assessment for developmental toxicology (Kimmel and Makris, 2001). New guidelines that involve additional information on effects in pregnant women and children are recommended in various areas of safety evaluation, including for immunotoxicity studies.
II. C O M P L E X I T I E S O F T H E I M M U N E SYSTEM AND LOCATIONS OF EFFECTS FOR TOXIC CHEMICALS The immune system is diffuse and distributed throughout the body. Various cells of this system, primarily macrophages and lymphocytes and their subpopulations, are found in the circulation and in most organs. The system possesses multistep control processes by which production and proliferation of immune-related cells are regulated depending on their need. Cells in the immune system require interaction and communication with each other or other systems to achieve their objectives. Immune functions include innate immunity, which is inherent in most tissues and is ready to neutralize invading organisms or antigens. This resident immune system in various organs employs macrophages, polymorphonuclear leukocytes, and natural killer (NK) lymphocytes. The other type is the adaptive immune system, which requires priming with specific antigens to mount an appropriate defense. The latter may be further divided into humoral immunitymthat is, production of specific antibodies [immunoglobulins (Igs)] by primed B cellsmand cell-mediated immunity carried out by T lymphocytes. Several subpopulations of T cells exist (e.g., cytotoxic T cells, helper T cells, and suppressor T cells). Some lymphocytes are NK cells, which generally require no priming or proliferation to exert their effects. The functions of immune competent cells are communicated either via cell-cell communications or through cytokines. Macrophages are derived from monocytes and are present in various body cavities, such as pulmonary alveoli and peritoneum; they are also found in the lymph nodes and liver as components of the innate immune system. Other peripheral leukocytes are often involved in various immunopathologic mechanisms. For acquired immunity, macrophages are phagocytes that concentrate antigens and confer specific immunological responses to various T or B cells and also remove cell debris. The immune system interacts with other systems and is profoundly influenced by the central nervous system, both directly via innervations of lymphatic organs and indirectly
via neuroendocrine mechanisms. The cells of the immune system produce factors that influence the nervous system. Hormones such as somatotrophin (growth hormone) and thymosin (thymic maturation factor) stimulate the immune responses, whereas steroids, including sex hormones, generally suppress the immune responses. Because the effective mounting of immune responses requires cell proliferation and production of signaling agents involving DNA and protein synthesis, chemical factors that interfere with cell cycling will modulate the r immune responses. This interference may occur at several steps in the immune functions. A schematic presentation of various locations where chemicals can interfere with the immune system is depicted in Fig. 1. This is a simplistic scheme and may not cover every aspect of either the immune system or other factors that regulate the system.
III. M E C H A N I S M S OF IMMUNOMODULATING ACTION OF ANTICHOLINESTERASES
A. Esterases and Immune Responses The immune system can be affected either by direct inhibition of esterases in effector cells or via alterations in neurotransmitters that regulate autonomic nervous system connections. Becker and Henson (1973) suggested that activated esterases may degranulate mast cells, releasing histamine, and may be involved in the process of chemotaxis by rabbit polymorphonuclear cells. Lymphocyte activation and subsequent division after antigen challenge are prerequisites for many immunological responses. It has been reported that serine esterases are activated after surface Ig cross-linking by anti-Ig. Diisopropylfluorophosphate (DFP; a potent anticholinesterase agent) inhibited the anti-Ig-induced activation of mouse B cells (Ashman, 1984). A direct role of esterases in immune responses was also suggested in the cytotoxic and NK cell-mediated cell lysis. The target cell lysis is associated with proteins stored in cytoplasmic granules of the effector cells, which are released on target cell recognition. Many of these proteins have been identified as serine esterases (Jenne and Tschopp, 1988). T cell killing was also inhibited by DFP and other serine esterase inhibitors (Ferluga et al., 1972). When mice were injected with the powerful cholinesterase inhibitor DFP, antigen processing and presentation by macrophages for immune processes requiting the cooperation of T cells were affected (Shek and Eastman, 1988). However, the response to macrophage-independent B cell antigens was not altered. DFP also interfered with the generation of memory cells that are responsible for clonal expansion when cells encounter a subsequent challenge to the antigen. Anticholinesterases may act directly on the cells possessing acetylcholine receptors located on lymphocytes.
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B. Control of the Nervous System on Immunological Functions The autonomic nervous system directly innervates thymus, spleen, lymph node, bone marrow, and other lymphoid tissues (Felton et al., 1985). Williams et al. (1981) showed that pharmacological manipulation of postganglionic noradrenergic nerve fibers affects immune functions. Increased levels of cyclic nucleotidase, which respond to neurotransmitters, may also alter the immune response (Coffey and Hadden, 1985). Accumulation of cyclic guanosine 3'5'monophosphate (cGMP) is observed after cholinergic stimulation of target tissues. Cyclic nucleotides have been reported to influence lymphocyte activation and proliferation (Strom et al., 1977).
C. Role of Hypothalamic-Pituitary-Adrenal Axis in Regulating Immunity The effect of cholinesterase inhibition on adrenocorticotrophic hormone has been demonstrated. Microinjection of neostigmine, a reversible cholinesterase inhibitor, in the hypothalamus of rats induced a stress-like syndrome characterized by c-fos expression in the hypothalamic paraventricular nucleus and increased the plasma levels of adrenocorticotrophic hormone (Zhu et al., 2001). The effects of cholinesterase inhibition were found to be gender specific; an intraperitoneal injection of physostigmine produced greater effect in male rats than in female rats (Rhodes et al., 2001). On the other hand, intravenous injection of physostigmine in patients indicated that circulating levels of cortisol increased only in women suffering major depression and in normal male subjects; normal females and depressive males were unaffected (Rubin et al., 1999). These studies point to a stress-like effect after cholinergic stimulation subsequent to cholinesterase inhibition leading to cortisol production; the latter is well known for its immunomodulatory effects.
D. Cholinesterase Inhibitors as Potential Alkylating Agents OP pesticides possess potential phosphorylating or alkylating properties. Various carcinogenic alkylating agents are immunosuppressive agents (Luebke et al., 1987); phosphorylating agents are likely to have an immunotoxic effect via this mechanism. Hilgetag and Teichmann (1965) hypothesized that dimethyl phosphates and other phosphorotriesters are good alkylating agents. Kimbrough and Gaines (1968) reported that the alkylating agents metepa, tepa, and apholate induced malformations in newborn rats perhaps via alkylating mechanism, as did other OPs such as parathion, dichlorvos, and diazinon, but the latter generally required maternally toxic doses. The relationship between phosphorylating properties of OPs and their immunotoxic effects is
a mechanistic possibility that has not been investigated in detail. The phosphorylating properties have been extensively investigated to study the mechanism of neurotoxic OPs (Johnson, 1975). A mono-substituted phosphoric acid residue was formed on proteins at specific neurotoxic esterase and other nonspecific sites. A phosphorylation of factors involved in the expression of immune responses therefore warrants consideration. Ethyl carbamate (urethane), another carcinogenic, is also an alkylating agent with anticholinesterase properties. Carcinogenic doses of urethane produced severe myelotoxicity and a markedly depressed NK cell activity but had less effect on other parameters of immune function, such as cellular and humoral immunity, including macrophage function (Luster et al., 1982). A selective phosphorylation or alkylation of various processes of the immune function by antiesterase agents is therefore possible.
IV. I M M U N O M O D U L A T I O N BY A N T I C H O L I N E S T E R A S E
PESTICIDES
There is a vast amount of information on the immunotoxic effects of OP and CM insecticides. A number of studies suggest that anticholinesterase pesticides are immunotoxic. However, these studies employed various testing protocols and obtained variable responses even with the same chemical or species. It is difficult to compare data from different OPs because of the many variations in their chemical configuration. It is not possible to describe all reported studies in detail here; selected investigations for OPs are summarized in Table 1, whereas those for CMs are indicated in Table 2. Overall evaluation of available information suggests that both suppression and enhancement of immunity can be mediated by these agents; however, effects are observed at doses that are generally systemically toxic and hence have little relevance to environmental exposure of anticholinesterase pesticides. Systemic toxicity would likely alter the immune functions as a result of general stress. Only a few studies involved long-term exposures and employed standardized protocols for immunotoxicity evaluation. Selected studies are briefly mentioned next.
A. Organophosphates and Carbamates on Human Immune Functions Studies on the environmental exposure of anticholinesterase pesticides on humans are very limited. One study involved individuals exposed to chlorpyrifos (a chlorinated OP) with respect to their lymphocyte subpopulations in blood (Thrasher et al., 2002). Chlorpyrifos-exposed individuals, whether showing clinical symptoms of anticholinesterase toxicity or not, had a decrease in the relative percentage of CD5 + (found on B 1a, a subpopulation of B cells involved in the production of autoreactive antibodies) and decreased mitogenesis of
CHAPTER 35 9OPs, CMs, and the Immune System TABLE 1.
499
Summary of Effects of Selected Organophosphate Insecticides on the Immune System
Chemical
Species, route, dose
Immunological parameters and effects
Parathion
Male mice, oral, 16 mg/kg single dose, 2 days after immunization with sheep red cells
Suppression of primary IgM plaque-forming cells
Casale et al. (1983)
Methyl parathion
Female mice, oral, 1-6 mg/kg/day, daily for 7-28 days
Methyl parathion at 1 or 3 mg/kg increased splenic natural killer cell activity In vitro response to sheep erythrocytes suppressed but in vivo response to this antigen unaltered Spleen weight decreased; germinal centers reduced Atrophy of thymic cortex Antibody-forming cells decreased in lymph node Skin reactivity to tuberculin decreased at 0.6 mg/kg/day only Increased plaque-forming cells in spleen against sheep erythrocytes Decrease in circulating white and red blood cells Decreased relative thymus weight Decreased plaque-forming cells in spleen after immunization with sheep erythrocytes Increase infectivity against Salmonella
Crittenden et al. (1998)
Male rabbits, 0.04-1.5 mg/kg/day via feed for 4 weeks
Male mice, oral, variable doses for 1-3 days or 4 weeks Male and female rats, in drinking water at 0.22-22 mg/kg/day for three generations
Mice, to provide 0.08, 0.7, and 3 mg/kg/day, in diet for 4 weeks
Male rats, orally 7, 14, and 28 mg/kg/day for 28 days
typhimurium
Decreased total or specific Ig in serum Reduced splenic blast transformation in response to mitogens Decreased spleen cellularity at the middle dose only
Reference
Street and Sharma (1975)
Institoris et al. (1992) Institoris et al. (1995)
Fan et al. (1978)
Undeger et al.
(2ooo)
Malathion
Japanese medaka (fish), malathion in water at 0.2 and 0.8 mg/liter for 7, 14, or 21 days Male rabbits, 5-100 mg/kg/day orally five times per week for 6 weeks Male mice, 720 mg/kg per os, 2 days after immunization with sheep red cells
Decreased plaque-forming cells in kidney Increased susceptibility to infection at -> 14 days Decreased S. typhi-induced immune response indicated by reduced antibody titers Suppression of primary IgM plaque-forming cells
Beaman et al. (1999) Desi et al. (1978) Casale et al. (1983)
Diazinon
Female mice, ip 0.2, 2, and 25 mg/kg/day for 28 days
Medullary atrophy of thymus with capsular and trabecular damage (all effects at the highest dose only) Decreased thymus weight and spleen cellularity Decreased number of plaque-forming cells in spleen Suppressed splenocyte proliferation in response to PHA Increased CD8 + cells at the two high doses Relative increase in circulating lymphocytes Increased splenic capsule thickness, detached from the parenchyma, hyperplasia of red and white pulp with pyknosis Thymic lesions similar to those in spleen Polysaccharides increased in spleen and lymph nodes but decreased in thymus; DNA decreased in spleen Increased IgG1 in females at high dose at 101 days but not at 400 or 800 days of age
Neishabouri et al. (2004)
Male mice, 300 ppm, sprayed on food pellets for 45 days
Pregnant mice, 0.18 or 9 mg/kg in the diet daily throughout gestation; offspring evaluated at 28 days
Handy et al. (2002)
Barnett et al. (1980) (continues)
500
SECTION IV- O r g a n
Toxicity
TABLE 1.
Chemical Dimethoate
Species, route, dose Female mice, single oral dose of 16 mg/kg, evaluated 1 4 weeks later
Male rats, orally 1.6-6.4 mg/kg for 28 days
Dichlorvos
Male rats, orally 7, 14, and 28 mg/kg/day for 28 days Male rabbits, orally 0.31, 0.62, 1.23, and 2.5 mg/kg/day, five times per week for 6 weeks Male mice, 120 mg/kg per os, 2 days after immunization with sheep
TABLE 2.
Chemical
(continued)
Immunological parameters and effects
Reference
Decrease in spleen weight; decreased rosette-forming cells in spleen lymphocytes Decreased response of splenocytes to PHA and LPS Decrease in serum total Ig and IgM Decrease in plaque-forming cells in spleen against sheep red blood cells at the highest dose Decreased delayed hypersensitivity against Keyhole limpet hemocyanin Decrease in delayed hypersensitivity (footpad swelling) at the highest dose after 24 hr only Decreased S. typhi-induced immune response indicated by reduced antibody titers
Aly and E1-Gendy (2000)
Suppression of primary IgM plaqueforming cells
Casale et al. (1983)
Institoris et al. (1999)
Undeger et al.
(2ooo) Desi et al. (1978)
Summary of Effects of Selected Carbamate Insecticides on the Immune System
Species, route, dose
Immunological parameters and effects
Reference
Aminocarb
Female mice, injected ip or gavaged (0.08-5 mg/kg), or applied to skin (up to 7.75 mg/kg in ear), single treatment
Oral and ip route (not dermal) produced immunologic effects Increased plaque-forming cells at 10 or 15 days Increased incorporation uridine or thymidine in spleen cultures stimulated with mitogens Autoimmunity potential suggested (not shown)
Bernier et al. (1995)
Carbaryl
Male rabbits, 0.23-8.38 mg/kg/day in feed for 28 days
Decreased hemagglutination titers after sheep erythrocyte inoculation; decrease in Ig: transferrin ratio Slight decrease in skin reactivity to tuberculin at the lowest dose only Dose-related atrophy of the thymic cortex Spleen cell number reduced after inhalation of the highest dose Dose-related decrease in splenic plaque-forming cells after sensitizing with sheep red cells after inhalation Peripheral leukocyte count decreased after oral gavage Dermal exposures ineffective IgG1 in male offspring elevated after 0.5 mg/kg dose but depressed in females after 0.01 mg/kg at 101 days but not at 400 or 800 days Decrease in Ig:transferrin ratio Decrease in skin reactivity to tuberculin at 0.49 mg/kg/day only Dose-related decrease in antibody-producing cells in the popliteal lymph node, spleen germinal centers, and atrophy of thymic cortex
Street and Sharma (1975)
Male rats, via inhalation to 36, 137, and 335 mg/kg aerosolized, 6 hrs/day, 5 days/ week for 2 weeks; oral gavage with 10, 25, or 50 mg/kg, 5 days/week for 2 weeks; 100, 500, or 1000 mg/kg once daily on dorsal skin, 5 days/week for 2 weeks
Carbofuran
Pregnant mice, 0.01 or 0.5 mg/kg, pups evaluated up to 800 days Male rabbits, 0.03-1.15 mg/kg/day in feed for 28 days
Ladics et al. (1994)
Barnett et al. (1980) Street and Sharma (1975)
(continues)
CHAPTER 35 9OPs, CMs, and the Immune System TABLE 2.
Chemical Propoxur
Female mice, 0.2, 2, and 10 mg/kg, ip daily for 28 days
Male rats, 0.85 or 8.5 mg/kg per os, for 4 weeks
(continued)
Immunological parameters and effects
Species, route, dose
501
Reference
Increased thymus weight Slight increase in hemagglutination titer and plaque-forming cells in spleen at 2 mg/kg only Decrease in delayed hypersensitivity reaction at 10 mg/kg/day dose Increase in splenic CD8 + cells at 10 mg/kg/day Plaque-forming cells in spleen decreased at the highest dose
Hassan et al. (2004)
Institoris et al. (2001)
Chlorpyriphos
Humans, exposure not known
Increase in CD26 ( a protease marker on cytotoxic T cells) expression Decrease in percentage of CD5 (autoantibody producing B cells) cells Decreased mitogenesis in response to PHA and Con A Increased frequency of autoantibodies
Thrasher et al. (2002)
Aldicarb
Women with 61 ppb in drinking water for 2 weeks
Increased number and percentage of peripheral T8 + lymphocytes Decrease in T4+:T8 + (helper/suppressor) cell ratio in blood Increased percentage of lymphocytes and CD2 + (dendritic) cells 2 years later Increased response of peripheral lymphocytes to candida, but no other mitogens Splenic plaque-forming cell response to sheep erythrocytes decreased at 1 ppb only; an inverse dose-response relationship observed Decreased response of spleen cells to mitogens Decreased response of T cells to Con A, splenic cells showing greater effect than peripheral cells Antigen presentation of macrophages decreased Ex vivo macrophage IL-1 production decreased A decrease in L3T4 + (CD4 +) cells after 28 days only Increased mitogenesis of splenic cells with PHA and Con A after 28 days only
Fiore et al. (1986)
Mice, drinking water with 0.1-1000 ppb for 34 days Female mice injected with 0.005-50 Ixg/kg, as single ip injection
Female mice, 0.1-10 ppb in water for 28 or 90 days
peripheral blood lymphocytes in response to phytohemagglutinin or concanavalin A. These findings were associated with an increase in CD26 + cells (adenosine deaminase binding protein found on B cells, mature T cells, NK cells, and macrophages) and the presence of autoreactive antibodies against thyroid and myelin of either the central or the peripheral nervous system. Results from this study are indicative of tissue damage by chlorpyrifos associated with a lack of tolerance to self-antigens suggesting immune dysregulation. Several other reports have studied aldicarb, a CM pesticide found in groundwater in several areas of the United States. Individuals exposed to drinking water contaminated with aldicarb showed an increase in the absolute number and the relative percentage of CD8 + (cytotoxic/suppressor) lymphocytes with a decreasing ratio of CD4 + (helper T)/CD8 + cells
Mirkin et al. (1990) Hong (1991) Olson et al. (1987) Dean et al. (1990a)
Hajoui et al. (1992)
(Fiore et al., 1986; Mirkin et al., 1990). Similar findings were also reported by another investigator (Hong, 1991), although no other immunodeficiency as a result of this change was indicated. Thomas (1995) investigated residents of the Great Lakes region who were probably exposed to a number of pesticides but found no discernible immunological impairment.
B. Experimental Studies on O r g a n o p h o s p h a t e Insecticides Rabbits fed low doses of methyl parathion were reported to have atrophy of the thymus cortex and fewer antibodyforming cells in the lymph node; splenic morphology was considerably altered (Street and Sharma, 1975). Delayed hypersensitivity response was not altered in a consistent
507.
SECTION I V .
Organ Toxicity
manner. There was no significant influence on circulating antibody levels against sheep erythrocytes. This pesticide also produced increased mortality in Swiss mice challenged with S a l m o n e l l a t y p h i m u r i u m (Fan et al., 1978); the activity was associated with an increased number of viable bacteria in blood, decreased ~/-globulin in the serum, and reduced blast formation of splenic cells in response to mitogens. In another study involving female C57B1/6 mice, methyl parathion increased NK cell activity with no other untoward effects on immune functions (Crittenden et al., 1998). Methyl parathion caused an increase in splenic plaque-forming cells in mice (Institoris et al., 1992) but a decrease in third-generation exposed rats (Institoris et al., 1995). A single oral exposure to 0.9 mg methyl parathion/ kg body weight did not affect any immunological parameters studied after 6 weeks, although a small dose of this pesticide enhanced the toxic immunological responses of propoxur, a CM insecticide (Institoris et al., 2004). Humoral immune response in rabbits exposed to malathion or dichlorvos was decreased against S. typhimurium (Desi et al., 1978). Casale et al. (1983) reported that a single high dose of malathion, parathion, or dichlorvos reduced primary IgM response to sheep erythrocytes at levels producing cholinergic symptoms. Dichlorvos inhibited the lysis of antibodysensitized sheep erythrocytes in a dose-dependent manner. The OPs had no effect when given in multiple lower doses. A cholinomimetic agent, arecoline, reduced IgM antibody response only when given in a form that would sustain prolonged cholinergic symptoms. Diazinon exposure in utero caused elevation of serum immunglobulins in male offspring of mice observed over their life span; the effect was opposite in female offspring and the changes were observed at 101 days but not at 400 or 800 days of age (Barnett et al., 1980). Immunosuppression has been reported with OPs such as dimethoate (Tiefenbach and Lange, 1980), fenitrothion fenthion, o-ethyl-o-p-nitrophenyl phenylphosphorothioate (EDN) and diazinon (Moon et al., 1986), and dichlorvos (Coffey and Hadden, 1985). Dimethoate caused a decrease in delayedtype hypersensitivity reaction measured as footpad swelling in mice (Undeger et al., 2000). Mitogen response of female mice splenic cells to phytohemagglutinin and lipopolysaccharide was reduced by this insecticide (Aly and E1-Gendy, 2000). Diazinon exposure caused pathological changes in spleen, thymus, and lymph nodes; however, these effects were dependent on dietary protein or lipid content (Handy et al., 2002). Exposure of mice to diazinon reduced splenic plaqueforming cells and delayed hypersensitivity, and the number of CD4 + cells in spleen was increased (Neishabouri et al., 2004). The OPs triphenylphosphate and triphenylphosphine oxide were reported to be immunomodulatory primarily to the innate immune functions (Fautz and Miltenburger, 1994). OPs are immunosuppressive on the function of hemopoietic stem cells. Mice given 4 mg/kg of parathion daily for 14 days showed alterations in bone marrow-derived hemopoietic stem cell colony formation for up to 2 weeks
without any cholinergic symptoms (Gallicchio et al., 1987b). Results were similar when human bone marrow cells were exposed to paraoxon or malaoxon in vitro; granulocyte-macrophage colony formation was suppressed in a dose-dependent manner (Gallicchio et al., 1987a).
C. Impurities Associated with Organophosphates Several impurities often present in OPs are potent cholinesterase inhibitors and may potentiate OP toxicity. For example, of many impurities in malathion, O, O,S-trimethyl phosphorothioate (OOS-TMP) and O,S,S-trimethyl phosphorothioate (OSS-TMP) are potent cholinesterase inhibitors, but they inhibit immune responses at doses that produce no cholinergic symptoms (Mallipudi et al., 1979). Various effects of OOS-TMP and OSS-TMP have been reviewed in detail elsewhere (Rodgers et al., 1992). Acute nontoxic doses of OOS-TMP suppressed both cellular and humoral immune responses (Devens et al., 1985; Rodgers et al., 1985a). However, subchronic treatment of mice with OOS-TMP at 0.5 mg/kg/day for 14 days elevated immune responses, characterized by generation of antibody-secreting cells against sheep red cells, generation of interleukin (IL)-2, and proliferative responses of lymphocytes concanavalin A and lipopolysaccharide (Rodgers et al., 1985b). At the higher dose of 5 mg/kg/day, antibody production and cytotoxic T lymphocyte responses were unaffected. Macrophages were suggested to be the cells most affected by this treatment (Rodgers et al., 1985c). Acute treatment of mice with OOS-TMP reduced the ability of macrophages to present antigen (Rodgers et al., 1985d). Macrophages from OOS-TMP-treated mice suppressed the proliferation of tumor cells and the supernatant from 24-hr cultures of spleen cells inhibited the proliferative response to mitogens and antibody response to sheep erythrocytes (Rodgers et al., 1986). Acute exposure to OOS-TMP increased the phagocytic activity and IL-1 production by macrophages but reduced the ability of macrophages tO present antigen (Rodgers et al., 1985d). A significant reduction in the generation of cytotoxic lymphocytes against P815 tumor cells and reduced antibody response to sheep red cells in C57BL/6 mice after a single acute dose of l0 mg OOSTMP/kg were reported (Rodgers et al., 1987). These were associated with a transient reduction in the number of thymic lymphocytes on days 3 and 5 after dosing. Nonspecific esterase activity of splenic macrophages increased after 10 mg OOS-TMP/kg in cell separation/reconstitution experiments. Thomas and Imamura (1986) suggested that the immunosuppression by OOS-TMP was via a glutathionemediated process that involved the inhibition of responder lymphocytes and macrophages. Preincubation of OOS-TMP with liver postmitochondrial supernatant blocked the generation of cytotoxic T lymphocyte response but failed to inhibit mature cytotoxic T cells (Rodgers et al., 1988). OOS-TMP
CHAPTER 3 5 9OPs, CMs, and the Immune System appears to act at the early stage of cytotoxic T cell activation or proliferation. Rodgers et al. (1988) investigated the effect of OSS-TMP on the in vivo primary and in vitro secondary cellular and humoral immune response. Single nontoxic doses of 20 or 40mg OSS-TMP/kg elevated primary immune response, but only the higher dose elevated the secondary humoral response. Addition of OSS-TMP in vitro also blocked the effector phase of the cytolytic reaction mediated by murine and human cytotoxic T lymphocytes.
D. Immunological Effects of Plasticizer Tri-o-tolyl Phosphate Some OPs cause delayed neurotoxicity by inhibition of neurotoxic esterase (Lotti and Johnson, 1978), and alterations of immunological function have been suggested (O'Brien, 1967). Watanabe and Sharma (1977) tested the latter hypothesis by administering tri-o-tolyl phosphate (TOTP; a component of a common heat-resistant lubricant tricresyl phosphate) to chickens. TOTP persistently increased diffuse lymphatic tissue in the fiver and spleen and increased total plasma protein levels (Watanabe and Sharma, 1976). However, the transfer of plasma and leukocytes from ataxic chickens to normal chickens failed to passively transfer the neurotoxicity, and immunosuppressive therapy also failed to protect chickens from TOTP-induced paralysis (Watanabe and Sharma, 1977). Foil et al. (1980) reported a failure to produce ataxic syndrome in chickens by the passive transfer of serum and/or lymphocytes from ataxic donors without subsequent TOTP treatment. However, T cellmediated graft versus host reaction and phytohemagglutinin wattle responses were significantly depressed in TOTP-treated chickens. In another study involving repeated gavage treatment of mice, neither TOTP nor its isomer, tri-m-tolyl phosphate, consistently affected immune parameters, including lymphocyte blastogenesis, splenic anti-sheep red cell plaques, or delayed hypersensitivity (Brinkerhoff et al., 1981).
E. Carbamate Insecticides Diets containing carbaryl or carbofuran fed to rabbits reduced the numbers of activated lymphocytes in lymph nodes, decreased the number of splenic germinal centers, and produced atrophy of the thymic cortex (Street and Sharma, 1975). Pipy et al. (1983)showed that carbaryl reduced the phagocytic activity of hepatic splenic macrophages, based on clearance of colloidal carbon following intravenous injection of carbaryl. They suggested that phagocytic activity was impaired due to inhibition of cell-bound serine esterases, de Maroussem et al. (1986) demonstrated that resident peritoneal macrophages stimulated with zymogen in the presence of carbaryl had reduced respiratory burst, altered phospholipid profile, and decreased prostaglandin levels. Casale et al. (1989) investigated the influence of carbaryl and carbofuran. In an in vitro assay system involving the incubation of diluted human sera with 0.5-3 mM of differ-
503
ent chemicals, carbaryl was more potent than carbofuran. The anticholinesterase properties were not related to their ability to inhibit the complement system; the OP paraoxon (also used in this study) was a potent anticholinesterase compound, whereas carbaryl was the least potent enzyme inhibitor of the group. The maximum inhibition of lysis in any serum by the highest concentration of carbaryl was <45%. Carbaryl inhibited IL-2-stimulated enhancement of NK cells in vitro (Casale et al., 1992). Decreases in thymus weight, spleen cellularity, plaque-forming cells/spleen, and serum IgM levels were noticed after inhalation of carbaryl by rats (Ladics et al., 1994). Barnett et al. (1980) exposed mice fetesus to a single maternal dose of 0.05 mg carbofuran/kg and reported a transient disturbance in Ig classes; the levels were elevated in male offspring, whereas these were depressed in females during the early periods only (101 days). No effects were observed during later periods in life (400 and 800 days). Reduced plaque-forming cells against sheep red cells were reported after subcutaneous injections of 8.5 mg/kg propoxur daily for 28 days in Wistar rats (Institoris et al., 2001). Similarly, high doses of 10 mg propoxur/kg injected intraperitoneally daily for 28 days caused histopathological changes in thymus and spleen and suppressed humoral immune responses against sheep erythrocytes in C57BL/6 mice (Hassan et al., 2004). Aminocarb, another CM insecticide, exhibited a weak immunosuppressive potential after a single intraperitoneal injection of 0.04 mg/kg in mice; inhalation of this compound had no effect on immune responses (Bernier et al., 1995). Olson et al. (1987) reported a decrease in plaqueforming cells to sheep erythrocytes in mice given aldicarb in drinking water. However, lower doses (1 ppb in water) had a greater effect than a high dose (1 ppm). In contrast, Thomas and Ratajczak (1988) reported that aldicarb did affect various parameters of immunological responsiveness in mice exposed to various concentrations (0.1-1000ppb) in drinking water for 34 days. However, in subsequent studies with mice given 0.1-1000 ppb of aldicarb in drinking water, no immunotoxicity was demonstrated (Thomas et al., 1987, 1990). When mice were injected with a single intraperitoneal dose of aldicarb (0.1 ml of 0.1-1000 ppb), a decreased macrophage function was reported; the T cell allogeneic responses were not impaired (Dean et al., 1990b). The same researchers also reported that a single intraperitoneal injection of aldicarb at doses of >-0.005 Ixg per mouse decreased the response of spleen cells to concanavalin A (Dean et al., 1990a); the effect was greater on whole spleen cells than on purified T cells. Antigenpresenting ability of macrophages from treated animals in vitro was compromised; there was decreased production of IL-1 by macrophages, the antigen presentation was restored after addition of recombinant IL-1 to cultures.
504
SECTION IV. O r g a n T o x i c i t y
V. A L L E R G I C
SENSITIZATION
BY O R G A N O P H O S P H A T E AND CARBAMATE I N S E C T I C I D E S Ercegovich (1973) proposed investigations on anticholinesterase-induced skin sensitization because of lack of evidence linking incidental exposure to allergic responses. Street (1981) reviewed the alteration in immune responses to a number of pesticides, including allergic sensitization to OPs. Of the many OPs that alter the immune response, only malathion showed a positive patch test, but the frequency of positive tests was low in people frequently handling malathion (Milby and Epstein, 1964). Matsushita and Aoyama (1981) reported sensitization and some crossreaction with OPs. Subjects from an area where several pesticides were used who tested positive to benomyl also reacted to diazinon. Similar results were obtained in a guinea pig maximization test. In a study of OP-induced contact dermatitis in 202 patients in Japan, Matsushita et al. (1985) attributed allergic reactions mainly to diazinon, fenitrothion, and supracide, with a low incidence for phosvel. However, in a controlled study no consistent allergic potential of OP insecticides (parathion and malathion) or CM (maneb, mancozeb, and zineb) could be demonstrated (Lisi et al., 1987). Cushman and Street (1983) produced IgE antibody in BALB/c mice to a metabolite of malathion conjugated to keyhole limpet hemocyanin. However, epicutaneous malathion did not elicit a delayed-type hypersensitivity response up to 1 month later. High doses of dithiocarbamates (maneb, mancozeb, and zineb) caused contact sensitization in guinea pigs; cross-sensitization among these chemicals was also shown (Matsushita et al., 1976). Although there are no reports that anticholinesterase insecticides cause autoimmune reactions, there is one in which the presence of autoreactive antibodies against thyroid and myelin of either the central or the peripheral nervous system were demonstrated, as indicated previously (Thrasher et al., 2002).
VI. F U T U R E P E R S P E C T I V E S Additional studies are needed to understand the immunotoxic potential of OPs and CMs. Since esterase activation has a role at various steps of complement cascade, release of histamine or other bioactive substances from cells, and chemotaxis of sensitized lymphocytes, the inhibition of esterases may produce opposite effects. Anticholinesterase pesticides have potent effects on various processes of the immune response in some cases. It has not been established whether these are mediated directly via inhibition of cholinesterases or via other esterases that mediate the immune reactions since potent anticholinesterase insecticides have the potential to inhibit different esterases in the body. It is not known if the anticholinesterase property can be effectively used for therapeutic modulation of the immune system. The cytotoxic
effect of many potent anticholinesterase chemicals may explain a number of in vitro effects observed; esterases also have lysosomal-induced cellular effects. Much of the available information on immunotoxic evaluation of anticholinesterases involves pesticides. However, exposure to low levels of pesticides does not appear to adversely affect the immune system. The effects observed are not always dose related and only rarely occur at exposures not producing systemic toxicity. Allergic responses and various degrees of autoimmune diseases have been reported in workers occupationally exposed to pesticides; however, this effect may be mediated by alteration of proteins rather than inhibition of cholinesterases. Modulation of immune responses, particularly the interference with host defense mechanisms, has been experimentally induced for a variety of anticholinesterase pesticides. Various anticholinesterases are biologically potent chemicals that inhibit cholinesterases via covalent interaction at the serine residue of the active site. Although enzyme inhibition may be reversible, there is the potential that other proteins may be modified and that several related enzymes may be inhibited. In many cases, it cannot be determined if the immunomodulatory effects are the result of inhibition of esterases or due to related mechanisms.
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Johnson, M. K. (1975). Organophosphorus esters causing delayed neurotoxic effects: Mechanism of action and structure-activity studies. Arch. Toxicol. 34, 259-288. Kimbrough, R. D., and Gaines, T. B. (1968). Effect of organic phosphorus compounds and alkylating agents on the rat fetus. Arch. Environ. Health 16, 805-808. Kimmel, C. A., and Makris, S. L. (2001). Recent developments in regulatory requirements for developmental toxicology. Toxicol. Lett. 120, 73-82. Ladics, G. S., Smith, C., Heaps, K., and Loveless, S. E. (1994). Evaluation of the humoral immune response of CD rats following a 2-week exposure to the pesticide carbaryl by the oral, dermal, or inhalation routes. J. Toxicol. Environ. Health 42, 143-156. Lisi, E, Caraffini, S., and Assalve, D. (1987). Irritation and sensitization potential of pesticides. Contact Dermat. 17, 212-218. Lotti, M., and Johnson, M. K. (1978). Neurotoxicity of organophosphorus pesticides: Predictions can be based on in vitro studies with hen and human enzymes. Arch. Toxicol. 41, 215-221. Luebke, R. W., Rogers, R. R., Riddle, M. M., Rowe, D. G., and Smialowicz, R. J. (1987). Alteration of immune function in mice following carcinogen exposure. Immunopharmacology 13, 1-9. Luster, M. I., Dean, J. H., Boorman, G. A., Dieter, M. E, and Hayes, H. T. (1982). Immune functions in methyl and ethyl carbamate treated mice. Clin. Exp. Immunol. 50, 223-230. Mallipudi, N. M., Umetsu, N., Toia, R. E, Talcott, R. E., and Fufuto, T. R. (1979). Toxicity of O,O,S-trimethyl and triethyl phosphorothioate to the rat. J. Agric. Food Chem. 27, 463-466. Matsushita, T., and Aoyama, K. (1981). Cross reactions between some pesticides and the fungicide benomyl in contact allergy. Ind. Health 19, 77-83. Matsushita, T., Arimatsu, Y., and Nomura, S. (1976). Experimental study on contact-dermatitis caused by dithiocarbamates maneb, mancozeb, zineb, and their related compounds. Int. Arch. Occup. Environ. Health 37, 169-178. Matsushita, T., Aoyama, K., Yoshimi, K., Fujita, Y., and Ueda, A. (1985). Allergic contact dermatitis from organophosphorus insecticides. Ind. Health 23, 145-153. Milby, T. H., and Epstein, W. L. (1964). Allergic contact sensitivity to malathion. Arch. Environ. Health 22, 434-437. Mirkin, I. R., Anderson, H. A., Hanrahan, L., Hong, R., Golubjatnikov, R., and Belluck, D. (1990). Changes in T-lymphocyte distribution associated with ingestion of aldicarb-contaminated drinking water: A follow-up study. Environ. Res. 51, 35-50. Moon, C. K., Yun, Y. E, and Lee, S. H. (1986). Effect of some organophosphate pesticide on the murine immune system following subchronic exposure II. Arch. Pharmacol. Res. (Seoul) 9, 175-182. Neishabouri, E. Z., Hassan, Z. M., Azizi, E., and Ostad, S. N. (2004). Evaluation of immunotoxicity induced by diazinon in C57BL/6 mice. Toxicology 196, 173-179. O'Brien, R. D. (1967). Insecticides: Action and Metabolism. Academic Press. New York, p. 332. Olson, L. J., Erickson, B. J., Hinsdill, R. D., Wyman, J. A., Porter, W. E, Binning, L. K., Bidgood, R. C., and Nordheim, E. V. (1987). Aldicarb immunomodulation in mice: An inverse dose-response to parts per billion levels in drinking water. Arch. Environ. Contam. Toxicol. 16, 433-439. Pipy, B., de Maroussem, D., Beraud, M., and Derache, E (1983). Evaluation of cellular and humoral mechanisms of
carbaryl-induced reticuloendothelial phagocytic depression. J. Reticuloendothel. Soc. 34, 395-412. Rhodes, M. E., O'Toole, S. M., Wright, S. L., Czambel, R. K., and Rubin, R. T. (2001). Sexual diergism in rat hypothalamicpituitary-adrenal axis responses to cholinergic stimulation and antagonism. Brain Res. Bull. 54, 101-113. Rodgers, K. E., Grayson, M. H., and Ware, C. E (1988). Inhibition of cytotoxic T lymphocyte and natural killer cell-mediated lysis by O,S,S,-trimethyl phosphorodithioate is at an early postrecognition step. J. Immunol. 140, 564-570. Rodgers, K. E., Grayson, M. H., Imamura, T., and Devens, B. H. (1985a). In vitro effects of malathion and O,O,S-trimethyl phosphorothioate on cytotoxic T-lymphocyte responses. Pestic. Biochem. Physiol. 24, 260-266. Rodgers, K. E., Imamura, T., and Devens, B. H. (1985b). Effects of subchronic treatment with O,O,S-trimethyl phosphorothioate on cellular and humoral immune response systems. Toxicol. Appl. Pharmacol. 81, 310-318. Rodgers, K. E., Imamura, T., and Devens, B. H. (1985c). Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate. I. Characterization of the immune cell population affected. Immunopharmacology 10, 171-180. Rodgers, K. E., Imamura, T., and Devens, B. H. (1985d). Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate. II. Effect on the ability of murine macrophages to present antigen. Immunopharmacology 10, 181-189. Rodgers, K. E., Imamura, T., and Devens, B. H. (1986). Organophosphorus pesticide immunotoxicity: Effects of O,O,Strimethyl phosphorothioate on cellular and humoral immune response systems. Immunopharmacology 12, 193-202. Rodgers, K. E., Imamura, T., and Devens, B. H. (1987). Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate: Generation of suppressive macrophages from treated animals. Toxicol. Appl. Pharmacol. 88, 270--181. Rodgers, K. E., Stem, M. L., and Ware, C. E (1989). Effects of subacute administration of O,S,S-trimethyl phosphorodithioate on cellular and humoral immune response systems. Toxicology 54, 183-195. Rodgers, K. E., Devens, B. H., and Imamura, T. (1992). Immunotoxic effects of anticholinesterases. In Clinical and Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 211-222. ButterworthHeinemann, Oxford. Rubin, R. T., O'Toole, S. M., Rhodes, M. E., Sekula, L. K., and Czambel, R. K. (1999). Hypothalamo-pituitary-adrenal cortical responses to low-dose physostigmine and arginine vasopressin administration: Sex differences between major depressives and matched control subjects. Psychiatr. Res. 89, 1-20. Sharma, R. E (1988). Evaluation of pesticide immunotoxicity. Toxicol. Ind. Health 4, 373-380. Sharma, R. E, and Tomar, R. S. (1992). Immunotoxicology of anticholinesterase agents. In Clinical and Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 203-210. Butterworth-Heinemann, Oxford. Shek, E N., and Eastman, T. M. (1988). Effect of diisopropylfluorophosphate on the antibody response. Immunopharmacology 15, 151-156.
CHAPTER 35 9OPs, CMs, and the Immune System Street, J. C. (1981). Pesticides and the immune system. In Immunologic Considerations in Toxicology (R. P. Sharma, Ed.), pp. 45-66. CRC Press, Boca Raton, FL. Street, J. C., and Sharma, R. P. (1975). Alteration of induced cellular and humoral immune responses by pesticides and chemicals of environmental concern: Quantitative studies of immunosuppression by DDT, aroclor 1254, carbaryl, carbofuran, and methylparathion. Toxicol. Appl. Pharmacol. 32, 587-602. Strom, T. B., Lundin, A. P., and Carpenter, C. B. (1977). The role of cyclic nucleotides in lymphocyte activation and function. Prog. Clin. Immunol. 3, 115-153. Thomas, I. K., and Imamura, T. (1986). Immunosuppressive effect of an impurity of malathion: Inhibition of murine T- and B-lymphocyte responses by O,O,S-trimethyl phosphorothioate. Toxicol. Appl. Pharmacol. 83, 456-464. Thomas, P. T. (1995). Pesticide-induced immunotoxicity: Are Great Lakes residents at risk? Environ. Health Perspect. 103(Suppl 9), 55-61. Thomas, P. T., and Ratajczak, H. V. (1988). Assessment of carbafnate pesticide immunotoxicity. Toxicol. Ind. Health 4, 381-390. Thomas, P. T., Ratajczak, H. V., Eisenberg, W. C., FurediMachacek, M., Ketels, K. V., and Barbera, P. W. (1987). Evaluation of host resistance and immunity in mice exposed to the carbamate pesticide aldicarb. Fundam. Appl. Toxicol. 9, 82-89. Thomas, P., Ratajczak, H., Demetral, D., Hagen, K., and Baron, R. (1990). Aldicarb immunotoxicity: Functional
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analysis of cell-mediated immunity and quantitation of lymphocyte subpopulations. Fundam. Appl. Toxicol. 15, 221-230. Thrasher, J. D., Heuser, G., and Broughton, A. (2002). Immunological abnormalities in humans chronically exposed to chlorpyrifos. Arch. Environ. Health 57, 181-187. Tiefenbach, B., and Lange, P. (1980). Studies on the action of dimethoate on the immune system. Arch. Toxicol. Suppl. 4, 167-170. Undeger, U., Institoris, L., Siroki, O., Nehez, M., and Desi, I. (2000). Simultaneous geno- and immunotoxicological investigations for early detection of organophosphate toxicity in rats. Ecotoxicol. Environ. Safety 45, 43-48. Watanabe, P. G., and Sharma, R. P. (1976). Lymphatic tissue response in chickens treated with tri-o-tolyl phosphate. J. ToxicoL Environ. Health 1, 777-786. Watanabe, P. G., and Sharma, R. P. (1977). Tri-o-tolyl phosphate neurotoxicity: Lack of evidence for autoimmunologic involvement. Arch. Environ. Contam. Toxicol. 6, 233-240. Williams, J. M., Peterson, R. G., Shea, P. A., Schmedtje, J. F., Bauer, D. C., and Felten, D. L. (1981). Sympathetic innervation of murine thymus and spleen: Evidence for a functional link between the nervous and immune systems. Brain Res. Bull 6, 83-94. Zhu, W., Umegaki, H., Suzuki, Y., Miura, H., and Iguchi, A. (2001). Involvement of the bed nucleus of the stria terminalis in hippocampal cholinergic system-mediated activation of the hypothalamo-pituitary-adrenocortical axis in rats. Brain Res. 916, 101-106.
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Nonspecific Toxic Effects
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CHAPTER
~ 6
Oxidative Stress in Anticholinesterase-lnduced Excitotoxicity WOLF-D. DETTBARN, 1 DEJAN MILATOVIC, z AND RAMESH C. GUPTA3 1Vanderbilt University School of Medicine, Nashville, Tennessee 2University of Washington, Seattle, Washington 3Murray State University, Hopkinsville, Kentucky
oxide synthesis and energy metabolism with the onset of increased lipid peroxidation. The common initiating mechanism of excitotoxicity is thought to be frequent stimulation of nicotinic acetylcholine receptors (nAChRs) at the mammalian neuromuscular junction (NMJ) and of muscarinic, nicotinic, and glutamatergic receptors in brain. For a more general review on pesticide-induced oxidative stress, see Banerjee et al. (2001) and Abdollahi et al. (2004).
I. I N T R O D U C T I O N Inhibitors of acetylcholinesterase (ACHE), such as organophosphates (OPs) and carbamates (CMs), when given in concentrations that cause status epilecticus, convulsions, and muscle fasciculations, can cause neuronal and muscle injury. Excessive activation of cholinergic and glutamatergic receptors is thought to be responsible for this excitotoxicity (Olney et al., 1986). Little is known about the biochemical mechanisms for producing these excitotoxic effects of AChE inhibitors. Previous studies support the suggestion that the toxicity is caused by oxidative stress and the excessive generation of free radicals. There is growing evidence supporting the involvement of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in excitotoxic injury (Dettbarn et al., 2001). During sustained seizures or muscle fasciculations, the flow of oxygen through brain and muscle is greatly increased at a time when the use of ATP is greater than the rate of its generation. This metabolic stress results in a markedly increased rate of ROS production. During normal physiological conditions, ROS are generated at a low rate and are efficiently removed by scavenger and antioxidant systems. A greatly increased rate of ROS production, however, may exceed the protective capacity of the cellular defense system and permit ROS to attack the cell membranes leading to peroxidation of lipids, inducing cell lesions and death (Sjodin et al., 1990). The research described in this chapter is based on the hypothesis that a causal relationship exists between excitotoxic injury and the generation of ROS, RNS, and lipid peroxidation. Since anticholinesterase agents primarily cause excitotoxicity in brain and skeletal muscle, this chapter describes the correlation between changes in nitric Toxicology of Organophosphate and Carbamate Compounds
II. O X I D A T I V E S T R E S S BY ACETYLCHOLINESTERASE
)
INHIBITORS
A. Cholinergic Excitotoxicity at the Neuromuscular Junction AChE at the NMJ is essential in the removal of acetylcholine (ACh) from the synaptic cleft. Inhibition of AChE profoundly modifies neuromuscular transmission, as seen in twitch potentiation, fasciculations, muscular weakness, and acute subjunctional necrosis of muscle fibers. Administration of AChE inhibitors (AChEIs) in a single sublethal dose that is sufficient to cause muscle fasciculations induces a myopathy in skeletal muscle (Preusser, 1967; Ariens et al., 1969; Wecker et al., 1978). The earliest lesions are focal areas of abnormality in the subjunctional area of the muscle fiber. Mitochondria are disrupted, as evidenced by clumping of highly reactive material of the lactate dehydrogenase (LDH) and NADH reactions. These focal changes progress to a breakdown of subjunctional fiber architecture followed by phagocytosis. Longitudinal sections reveal that the necrosis affects only a small segment of fiber lengths. During later stages, progressively greater lengths of muscle fibers are affected (Laskowski et al., 511
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1977; Gupta et al., 1986). Serial cross sections of 10-1xm thickness indicate that the number of lesions correlates with the greatest density of end-plates (Patterson et al., 1987). The longer the delay between injection and sacrifice, the greater the spatial extent of the lesions. A significant increase in serum creatine kinase (CK) and LDH activity coincides with the appearance of myonecrosis, indicating destruction of the muscle membrane (Sket et al., 1989; Gupta et al., 1991, 1994). Subjunctional changes in the muscle fibers, such as supercontraction of subjunctional sarcomeres as well as disruption of cytoarchitectural organization, are always present, The initial changes are in the mitochondria, which swell and then show lysis of the central cristae. Myelin figures beneath the end-plate are frequently observed, whereas the region more distal to the end-plate is less affected. The nucleoli of the muscle cell nuclei are enlarged and move to the periphery of the nucleus. This myopathy can be induced with several OP or CM AChE inhibitors, such as paraoxon (Ariens et al., 1969; Laskowski et al., 1975, 1977; Wecker et al., 1978), diisopropylphosphorofluoridate (DFP) (Preusser, 1967; Gupta et al., 1986; Misulis et al., 1987; Patterson et al., 1987, 1988; Sket et al., 1991), pyridostigmine (Hudson et al., 1985, 1986), aldicarb and carbofuran (Gupta et al., 1998, 1999), and the OP nerve agents soman, satin, tabun, and VX (Gupta et al., 1987a,b, 1991; Gupta and Dettbarn, 1992). Despite the diversity in structures of these AChEIs, the induced myopathic changes are the same, suggesting a common mechanism. This mechanism is an excess of ACh and its prolonged interactions with nAChRs and not a direct action of these inhibitors on the muscle. The common denominator is muscle hyperactivity, such as fasciculations (Adler et al., 1992). In vitro exposure to pyridostigmine causes the same ultrastructural changes as described in in vivo experiments, such as swelling of subjunctional mitochondria and disorganization of contractile proteins. These alterations are accompanied by continuous fasciculations. Tetrodotoxin, an inhibitor of axonal conduction, which does not affect the NMJ but prevents development of fasciculations and alterations in pre- and postjunctional morphology by blocking axonal conduction (Adler et al., 1992). In vitro experiments with the nonhydrolyzable cholinergic agonist carbamylcholine produced similar alterations as those observed with the AChEIs in vivo. In these experiments, excessive Ca 2+ influx was thought to contribute to the myopathy (Leonard and Salpeter, 1979, 1982). The increased rate of postsynaptic stimulation that accompanies fasciculations and the concomitant influx of Ca 2+ may initiate changes in the subjunctional mitochondrial morphology. The swelling of mitochondria with a parallel release of sequestered Ca 2+ would thus evoke contraction of subjunctional sarcomeres and lead to subjunctional fiber
damage. Mitochondria altered by excessive Ca 2+ may lead to a reduction of ATP synthesis causing excessive generation of oxygen radicals. The muscle injury induced by AChEIs actually depends on the events initiated by the constant interaction between ACh and the nAChRs. For a more detailed discussion of OP- and CM-induced myotoxicity, see Chapter 25.
B. Prevention of Myopathy Myopathy does not develop once hyperactivity/fasciculations are prevented by denervation of muscle, rapid reactivation of inhibited AChE by oximes, or pretreatment with d-tubocurarine, which blocks the nAChRs (Wecker et al., 1978; Patterson et al., 1988; Gupta and Dettbarn, 1992). Reduction of ACh release from nerve terminals by botulinum toxin (BTX) also prevents fasciculations and myopathy (Sket et al., 1991). By lowering the amount of ACh accumulation at the NMJ, signs of toxicity, such as antidromic nerve activity and muscle fasciculations, are prevented or reduced (Laskowski et al., 1977; Laskowski and Dettbarn, 1979). Pretreatment with BTX almost completely protects muscles from DFP-induced spontaneous activity and myopathy despite critically inhibited synaptic ACHE. These results are consistent with the conclusion that the myopathy is not mediated by direct action of the AChEIs on the muscle but through the accumulation of ACh, leading to muscle hyperactivity. The neuromuscular blocking agent d-tubocurarine in a low subparalyzing dose (40 Ixg/kg) affects the presynaptic release of ACh and prevents antidromic firing, fasciculations and muscle lesions without interfering with normal neuromuscular transmission (Gupta and Dettbarn, 1992). Under normal conditions, muscles are protected against oxidative damage through endogenous antioxidants such as reduced glutathione (GSH). To assess the importance of GSH in protecting the muscle against AChEI-induced injury, GSH levels were correlated with the muscle's susceptibility to oxidative stress. Pretreatment with the inhibitor of ~-glutamylcysteine synthetase (buthionine sulfoximine) before DFP showed a dramatic increase in the number of necrotic muscle fibers, as well as an increase in lipid peroxidation. The findings suggested that endogenous GSH is an important protectant against lesion formation by DFP treatment and supports the role of ROS in the pathogenesis of OP-induced muscle lesions (Yang and Dettbarn, 1996; Yang et al., 1996). In other studies, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, memantine (MEM), in combination with atropine sulfate, by preventing muscle hyperactivity, have been shown to attenuate OP- or CM-induced increases in ROS generation, decreases in energy metabolites, and muscle necrosis (Gupta and Dettbarn, 1992; Gupta et al., 2002; Milatovic et al., 2005).
CHAPTER 36 9OPs, CMs, and Oxidative Stress
C. Role of Glutamate at the NMJ By using quantitative electron immunocytochemistry, Waerhaug and Ottersen (1993) demonstrated glutamate (GLU)-like immunoreactivity at the rat NMJ and suggested that GLU and ACh are coreleased at the NMJ. GLU may be a mediator or modulator of neuromuscular transmission. It has also been established that GLU receptors present at the NMJ are predominantly of the NMDA subtype. Koyuncuoglu et al. (1998) demonstrated that the blockade of NMDA receptors reduced GLU release, and suppression of GLU production attenuated the contractions of the rat isolated hemidiaphragms elicited by indirect electrical stimulation. Unlike the well-understood role of cholinergic excitotoxicity, the role of GLU excitotoxicity is yet to be established in AChEI-induced muscle toxicity.
D. Oxidative Stress During highly intense muscle activity, the flow of 0 2 through the skeletal muscle cells is greatly increased and at the same time the rate of ATP utilization exceeds the rate of ATP generation. During normal conditions, ROS are generated at a low rate and subsequently taken care of by the well-developed scavenger and antioxidant systems. However, a greatly increased rate of free radical production may exceed the capacity of the cellular defense system. Consequently, a substantial attack of ROS on unsaturated fatty acids in the cell membranes leading to lipid peroxidation and loss of cell viability can initiate the process that leads to muscle necrosis. The primary reason for the increased generation of ROS appears to be the decreased rate of ATP synthesis in the mitochondria, which is related to a loss of cytochrome oxidase (COx) activity. COx is the terminal complex in the mitochondrial respiratory chain, which generates ATP by oxidative phosphorylation. During intense muscle hyperactivity, the activity of COx is reduced, leading to an increase in the electron pressure within the electron transport chain and to increased ROS production (Soussi et al., 1989; Gollnick et al., 1990; Yang and Dettbarn, 1998; Zivin et al., 1999b; Milatovic et al., 2001). More than 90% of the O2 consumption in cells is catalyzed by COx. The chance of intermediary products, such as superoxide anion, hydrogen peroxide, and the hydroxyl radical, escaping is small under conditions when COx remains active. A reduced capacity of this enzyme, however, will increase the risk for an incomplete reduction of O2 and further O2 radical formation (Sjodin et al., 1990). Another enzyme contributing to increased ROS generation is xanthine oxidase (XO). During normal conditions, 80-90% of native XO exists as xanthine dehydrogenase (XD), but during metabolic stress and increased Ca 2+, XD is converted to a reversible oxidase form. XO uses
513
molecular 0 2 instead of NAD as an electron acceptor. Molecular O2 is thereby reduced and the superoxide radical (O2) is formed. During hyperactivity of the muscle, such as induced by AChEIs, regeneration of ATP is insufficient not due to the lack of O2 but due to the greater utilization and impaired synthesis of ATP (Gupta et al., 1994, 2000a,b, 2001a,b, 2002). Unlike in ischemia, O2 is present during oxidative stress caused by prolonged contractile activity. This suggests that subsequent to the conversion of XD to XO, O2 is continuously univalently reduced to superoxide anions. This occurs during oxidative stress when ATP utilization exceeds the rate of ATP synthesis during increased muscle activity (Gollnick et al., 1990). Yang and Dettbarn (1998) provided direct evidence for the role of COx and XO in tissue injury by muscle hyperactivity, showing that during OP-induced muscle hyperactivity, a decrease in COx activity and an increase in XO activity occurred. Blockade of muscle fasciculations prevented these enzyme changes. Peroxynitrite (ONOO-) is another ROS contributing to oxidative stress and is formed by the reaction of nitric oxide (NO) with superoxide (O2) (Huie and Padmaja, 1993). Peroxynitrite has the potential to modify biomolecules through several different mechanisms, and it is a good candidate for mediation of the NO-dependent pathophysiological process (Brunelli et al., 1995). Under normal conditions, NO, a free radical gas (synthesized in a reaction catalyzed by NO synthase), is widely regarded as a multifunctional messenger/signaling molecule and is thought to have two physiological functions in skeletal muscle: to promote relaxation through the cGMP pathway (Schmidt et al., 1993) and to modulate muscle contractility that is dependent on reactive oxygen intermediates (Abramson and Salama, 1989). At the NMJ, NO appears to be a mediator of early synaptic protein clustering, synaptic receptor activity and transmitter release, and downstream signaling for transcriptional control (Blottner and Luck, 2001). NO has also been demonstrated to modulate excitationcontraction coupling in the diaphragm muscle (Reid et al., 1998). Within skeletal muscle fibers, all three known NOS isoforms [neuronal (nNOS), endothelial (eNOS), and inducible (iNOS)] are present, but nNOS, which is Ca 2+ dependent, seems to predominate. It is concentrated at the sarcolemma and postsynaptic surface of the NMJ and appears to be involved in the regulation of metabolism and muscle contractility (Stamler and Meissner, 2001). The finding of elevated NO by OP-induced muscle hyperactivity (Jeyarasasingam et al., 2000; Gupta et al., 2002) is supported by previous studies showing that increased muscle contractility generates significantly greater quantities of ROS/RNS than resting muscle (Clanton et al., 1999; Narayan et al., 1997; Yang and Dettbarn, 1996; Yang et al., 1996). A significant increase in NO is known to cause a decrease in mitochondrial function and may be the cause of the impaired synthesis of ATE
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E. I s o p r o s t a n e s as I n d i c a t o r s of L i p i d P e r o x i d a t i o n and O x i d a n t I n j u r y To explore the role of lipid peroxidation in OP- or CM-induced muscle fiber injury, it is important to use the most sensitive, specific, and reproducible assay method. A large number of studies have shown that measurement of F2isoprostanes (F2-IsoPs) and F4-neuroprostanes (F4-NeuroPs) provides an accurate measure of lipid peroxidation both in vivo and in vitro (Roberts and Morrow, 2000). In fact, quantitation of these compounds is one of the most accurate approaches for assessing oxidant injury in vivo. Isoprostanes are prostaglandin-like compounds that are formed nonenzymatically in vivo by free radical-induced peroxidation of arachidonic acid. The F4-NeuroP docosahexaenoic acid (C22:6to3; D H A ) has been the subject of considerable interest due to the fact that it is highly enriched in the brain, particularly in gray matter, where it comprises approximately 2 5 - 3 5 % of the total fatty acids in aminophospholipids. Isoprostane-like compounds are formed by free radical-induced peroxidation of DHA. Since D H A is highly enriched in the brain, these compounds are called F 4NeuroPs. Quantification of this compound should provide a unique marker o f oxidative injury in the brain. The formation of F2-IsoPs and F4-NeuroPs esterified in lipids can be expected to have significant effects on the biophysical properties of neuronal membranes and muscle fiber membranes, which might impair normal function. One of the physiological functions of D H A may be to maintain a certain state of membrane fluidity and promote interactions with membrane proteins that are optimal for neuronal function (Roberts et al., 1998; Roberts and Morrow, 2000). F2-IsoPs and F4NeuroPs are present in a readily detectable range in brain and muscle.
F. M u s c l e H y p e r a c t i v i t y A s s o c i a t e d with Enhanced Lipid Peroxidation In DFP-induced muscle hyperactivity, lipid peroxidation was originally quantified by measuring the thiobarbituric acid-malondialdehyde complex ( T B A - M D A ) using highperformance liquid chromatography (Draper et al., 1993). This technique is perhaps the most widely used method to quantify lipid peroxidation. DFP causes a dosedependent increase in A C h E inhibition, muscle fasciculations, T B A - M D A formation, and muscle necrosis (Table 1). Reducing the concentration of G S H through pretreatment with buthionine sulfoximine, an inhibitor of G S H synthesis (Table 2), potentiates the DFP-induced changes in T B A - M D A and causes an increase in the number of necrotic muscle fibers. Prevention of fasciculations by pretreatment with the nicotinic cholinergic antagonist d-tubocurarine, before DFP, prevents an increase in lipid peroxidation and significantly attenuates muscle fiber necrosis. Lazaroid (U78517F), a 21-amino acid
steroid and inhibitor of lipid peroxidation, significantly prevents the increase in lipid peroxidation and attenuates muscle necrosis without preventing the fasciculations, thus linking lipid peroxidation with the necrosis (Yang and Dettbarn, 1996; Yang et al., 1996) (Table 1).
TABLE 1. Prevention of DFP-Induced Increase in TBA-MDA and Muscle Fiber Necrosis in Rat Diaphragm by d-Tubocurarine and U-78517F a
Treatmentb Control DFP
% Difference of MDA levels from control c
96 ___ 11 d
No. of necrotic fibers/1000 muscle fibers 4 + 2 (0.4%) 479 __+45 (47.9%) d
d-Tubocurarine/DFP
-30 _ 5
73___19 (7.3%) e
U-78517F/DFP
-41 _ 6
70 _+ 31 (7.0%) e
aValues are mean _+ SEM of seven muscles. bTreatment: DFR 2.0 mg/kg; d-tubocurarine, 0.070 mg/kg; and U-78517F, 7.0mg/kg. Atropine sulfate (2.0 mg/kg) was also given to the d-tubocurarine-pretreated rats. All drugs were given ip with the exception of DFP, which was given sc. Rats were pretreated with these compounds 30 min before DFP, at the time of DFP, and 30 min later. Rats were sacrificed 1 hr after DFP treatment for TBA-MDA assay or 24 hr after DFP for histochemistry. CMDAlevel in control: 49.42 ng/mg protein. '/Significant difference between control and DFP-only treated rats (p < 0.01). eSignificant difference between DFP-only treated and pretreated rats (p < 0.01). No significant difference between the pretreatment regimens.
TABLE 2. Low Endogenous Glutathione Levels Potentiate Muscle Injury and Lipid Peroxidation Products in Response to DFP in Diaphragm Muscle a
Experimental group
GSH (Ixmol/g muscle)
Necrotic fibers/1000 2 ___2
Control
1.36 _ 0.20 (100%)
BSO
0.89 _ 0.13 (65%) b
2_ 2
DFP
1.18 _ .13 (86%) c
479 _ 34 c
DFP + BSO
0.39 _ 0.11 (28%) d
655 __+67 e
aTreatment: DFP (2.0 mg/kg, sc) was given 1 hr before decapitation. BSO (900 mg/kg, ip) was given 3.5 and 1.5 hr before DFP administration. Values are mean __+SEM for eight rats. bStatistical significance between BSO treatment and control (p < 0.01). cStatistical significance between DFP treatment and control for GSH (p < 0.05) and for necrotic fibers (p < 0.01). dStatistical significance between DFP + BSO treatment and control (p < 0.01). Statistical significance between DFP + BSO treatment and DFP-only treatment (p < 0.01) or between DFP + BSO treatment and BSO-only treatment (p < 0.01). eStatistical significance between DFP + BSO treatment and control (p < 0.01). Statistical significance between DFP + BSO treatment and BSO-only treatment (p < 0.01) or between DFP + BSO treatment and DFP-only treatment (p < 0.05).
CHAPTER 36
The values of T B A - M D A are usually higher than those of the F2-IsoPs, which may be attributable to the fact that measurements of T B A - M D A can be affected by the metabolism of the compound as well as generation of the compound in vitro. This suggests that F2-IsoP measurement appears to be a more specific and reliable indicator of lipid peroxidation than T B A - M D A (Janero, 1990).
G. F a s c i c u l a t i o n s - l n d u c e d C h a n g e s in X a n t h i n e Oxidase and Cytochrome c Oxidase Activities XO (Bratell et al., 1988; Downey et al., 1988) and COx (Soussi et al., 1989) are sources of cytotoxic ROS. XD is converted to XO through Ca2+-activated proteases, which utilizes molecular oxygen as an electron acceptor. The molecular oxygen is therefore reduced by XO and the
9OPs, CMs, and Oxidative Stress
515
superoxide radical (O2) is formed. Loss in COx activity causes incomplete oxygen reduction and increased generation of ROS. The consequences of administration of DFP on COx activity and the conversion of XD to XO were evaluated. The activities of XO, XD, and COx from fasciculating muscles were compared with activities from nonfasciculating muscles. Both a significant transient decrease in COx activity and a significant increase in XO activity occurred within 15 min of DFP treatment (Fig. 1 and Table 3). These changes were prevented by the neuromuscular blocking agent d-tubocurarine (Yang and Dettbarn, 1998), providing support for the hypothesis that the OPinduced muscle hyperactivity stimulates ROS production, leading to lipid peroxidation and muscle necrosis.
III. C H A N G E S I N B I O M A R K E R S INDICATING MUSCLE INJURY nTnmmDFP(1.7 mg/kg,sc) 65-
**
~
LDH and CK are commonly used as enzymatic biomarkers for a variety of diseases, poisonings, and myopathies. Their values are generally enhanced in serum, probably as a result of increased cell membrane permeability.
DFP(0.5 mg/kg,sc)
60
.~ 55 o g
50 45
o
40
A. CK and CK Isoenzymes
35 -~ 30 "6 25 0
g
20 15 10 5
0
e
i1, , e
e
15 min
!
30 min
o e
Illlll
,
,
,
60 min
FIG. 1. Effects of DFP (1.7 or 0.5 mg/kg, sc) on cytochrome c oxidase in rat diaphragm. Control activity of cytochrome c oxidase: 0.526 _ 0.093 ixmol/min/mg protein. Significant difference between control and DFP-treated rats: *p < 0.01; **p < 0.001.
TABLE 3.
CK catalyzes the synthesis of ATP and phosphocreatine (PCr) in a reversible Lohmann reaction and can serve as an indicator for the integrity of muscle and other cell membranes. A loss of CK in muscle followed by an increase in serum indicates cell damage. Normal distribution of CK in skeletal muscles of rats revealed that in controls, CK activity was found highest in EDL followed by diaphragm, and it was lowest in soleus. Electrophoretic separation of CK isoenzymes in all three muscles revealed the existence of only the C K - M M isoenzyme. Further separation of the CK-MM isoenzyme for subforms showed only CK-MM3 in
Conversion of Xanthine Dehydrogenase (XD) to Xanthine Oxidase (XO) in Rat Diaphragm following in Vivo DFP Treatment, with or without d-Tubocurarine Pretreatment a
Control XO XD + XO (lxmol/min/mg protein) XO/XD XO/XD increase from control (%)
DFP 30 min
DFP 60 min
DFP 30 min + d-tubocurarine
DFP 60 min + d-tubocurarine
0.54 _+ 0.13 b
0.553 _+ 0.09 b
0.264 _+ 0.07
0.236 _+ 0.07
0.242 _ 0.05 0.794 ___0.18
0.768 _+ 0.20
0.803 +_ 0.14
0.771--+0.22
0.825 + 0.021
0.44
2.39 b
2.21 b
0.52
0.40
443
402
18
0.40
aDFP was given 1.7 mg/kg sc, and d-tubocurarine was given 0.07 mg/kg ip. Rats were sacrificed after 30 or 60 min of exposure to DFP. Values are mean _ SEM of five rats. bSignificant differencebetween control and treated animals (p < 0.001).
516
SECTION V . N o n s p e c i f i c
Toxic Effects
all muscles. Compared to muscles, control serum had very little CK activity. However, the CK consisted of three distinct isoenzymes: CK-BB (15.3%), CK-MB (3.9%), and CK-MM (81.8%). Electrophoresis of control serum CK-MM isoenzyme revealed the presence of three subforms: CK-MM1 (6.3%), CK-MM2 (24%), and CK-MM3 (69.7%). Evidence shows that the CK-MM3 subform secretes from muscles into the plasma, where it converts into the MM2 and MM1 subforms by carboxypeptidase-N2 (Gupta et al., 1991). Carbofuran in concentrations that cause fasciculations and myopathy produced a significant and maximal increase (152%) in serum total CK activity that was seen as early as 0.5 hr after carbofuran injection and remained elevated for 3 hr. Under the influence of acute carbofuran intoxication, examination of the serum and diaphragm revealed several characteristic changes in CK isoenzymes. The activity of the CK-MM isoenzyme was elevated more than two-fold in the diaphragm within 0.5 hr and remained significantly higher than control at 24 hr. Such an increase in CK-MM activity was also seen in the serum at the time when fasciculations were evident (1 and 3 hr), indicating damage to the muscle fiber integrity. The increased leakage of CK subsided following restoration of normal muscle activity (Gupta et al., 1991, 1994, 2000b).
quantity. Diaphragm had predominantly LDH-5 and LDH-4 (40 and 27.7%, respectively). In control serum, isoenzyme LDH-5 comprises 87% of the total LDH activity (100%). Under normal conditions, serum contained a low quantity of LDH activity compared to diaphragm, suggesting low release of this enzyme from muscle into serum. Assay of LDH isoenzymes in serum revealed that 94% of total LDH activity consisted of LDH-5 and LDH-4 (72.6 and 21.4%, respectively). The activity of LDH-4 was 42% in the brain and 27% in diaphragm. Within 0.5 hr after carbofuran injection (1.5 mg/kg, sc), significant increases in total LDH activity were noted in the serum and diaphragm. Enzyme activity was further elevated at 1 hr, when muscle activity was at its peak. Detailed analysis of the serum for LDH isoenzymes revealed significant elevations of LDH-1, LDH2, and LDH-3 and a decrease in LDH-4. In diaphragm muscle, the activities of LDH-2 and LDH-5 were significantly elevated, whereas these of LDH-1, LDH-3, and LDH-4 were reduced (Gupta et al., 1994, 2000b).
IV. O P - A N D C M - I N D U C E D C H A N G E S
IN BIOMARKERS OF OXIDATIVE STRESS A. Carbamates 1. F2-ISoPs/LIPID PEROXIDATION A significant increase in F2-IsoPs was seen in soleus within 30 min and in EDL within 60 min following carbofuran injection (Fig. 2). The changes were more rapid in soleus than in EDL. F2-IsoPs levels in soleus returned to control levels within 2 or 3 hr and in EDL by 6 hr (EDL data not shown).
B. LDH and LDH Isoenzymes LDH catalyzes the synthesis of lactate and pyruvate in a reversible reaction, and it is commonly used as a biomarker of cell damage or death. Analysis of LDH and its isoenzymes in skeletal muscles of control rats revealed that LDH activity was highest in EDL followed by diaphragm, and it was lowest in soleus. Compared to muscles, the enzyme activity is low in serum. Diaphragm and serum contained all five electrophoretically distinct LDH isoenzymes, but with varying
2.5
2. CITRULLINE (NO/NOS) Data presented in Table 4 show the levels of citrulline, as a marker of NO/NOS, in skeletal muscles of control rats and those treated with an acute dose of carbofuran
a
2.0 0 (D
".= 1.5 [
r v
nO
1.0
m
LL
FIG. 2 Effects of carbofuran (1.5 mg/kg, sc) on F2-IsoPs in soleus muscles. Values are means _+ SEM (n = 4-6).
0.5
0.0
Ohr 0.25 hr (Control)
0.5 hr
1 hr
2 hr
3 hr
6 hr
24 hr
aSignificant difference between control and carbofuran-treatedrats (p < 0.05).
CHAPTER 36 (1.5 mg/kg, sc). Analyses of controls revealed slightly higher citrulline levels in the soleus (469.74 ___ 31.81 nmol/g) than in the EDL (417.84 ___ 18.54nmol/g). Within 15 min after exposure to carbofuran, citrulline levels were significantly elevated in both muscles (soleus, 155%; EDL, 176%). Maximum increases in citrulline levels were noted after 1 hr (267 and 304%, respectively). Levels remained significantly elevated until 6 hr and returned to control levels after 24 hr (Table 4).
3. HIGH-ENERGY PHOSPHATES The time courses of changes in HEPs (ATP and PCr) and their metabolites in diaphragm muscles of control rats and those acutely intoxicated with DFP (1.5 mg/kg, sc) are similar to those described for carbofuran in Table 5. Analysis of control muscles revealed that the levels of ATP and PCr were highest in the EDL followed by the diaphragm and the soleus. The values of adenylate energy charge in the soleus, EDL, and diaphragm were 0 . 8 6 _ <0.01, 0.91 _+ <0.01, and 0.86 _+ <0.01, respectively. At the time of maximal severity (i.e., 1 hr after DFP exposure), the levels of ATE TAN, PCr, and TCC were maximally reduced in all three muscles, and they remained reduced to the same degree at 2 hr. After 24 hr, the levels of energy metabolites were at control level in the soleus and EDL, whereas the diaphragm values remained significantly depressed. The data revealed that 6 0 m i n after carbofuran or DFP injection, when muscle hyperactivity was at its most intensive, NO levels were maximally increased (Table 4), whereas energy metabolites were maximally reduced (Table 5). However, the degree of NO elevation (272-288%) was much greater than the declines of HEPs (30-40%), suggesting that the NO/NOS system is a much
B. O r g a n o p h o s p h a t e s 1. F2-IsoPs]LIPID PEROXIDATION Similar changes in F2-IsoPs/lipid peroxidation as described in Section IV and Fig. 2 were seen following DFP injection.
Citrulline Levels in Soleus and EDL Muscles of Rats Intoxicated with an Acute Dose of Carbofuran (1.5 rng/kg, sc)
Time after carbofuran injection (hr) 0 (control) 0.25 0.5 1 2 3 6 24
Citrulline concentration (nmol/g muscle) a Soleus 469.74 729.27 992.15 1254.48 1134.72 823.62 703.53 457.81
517
2. CITRULLINE (NO/NOS) Animals treated with a fasciculation-inducing dose of DFP (1.5 ms/ks, sc) showed similar changes as those treated with carbofuran (Table 4). Within 15 rain of DFP exposure, citmlline levels were significantly elevated in all three muscles (EDL, 149%; soleus, 159%; and diaphragm, 167%). Maximum increase in citmlline levels (272-288%) was noted after 1 hr and remained significantly elevated (greater than two-fold) after 2 hr. At 24 hr post-DFP exposure, citmlline levels were at control level in all three muscles.
3. HIGH-ENERGY PHOSPHATES The time course of changes in high-energy phosphates (HEPs) (ATP and PCr) and their metabolites in skeletal muscles of control rats and those acutely intoxicated with carbofuran (1.5 mg/kg, sc) (Table 5) revealed that in controls the levels of ATP and PCr are higher in the EDL than in the soleus. At the time of maximal severity (i.e., 1 hr after carbofuran exposure), the levels of ATP, total adenine nucleotides (TAN = ATP + ADP + AMP), NAD, PCr, and total creatine compounds (TCC = PCr + Cr) were maximally reduced in both muscles. Slow recovery was seen after 2 or 3 hr, with complete recovery to control levels by 24 hr. The carbofuran-induced muscle hyperactivity increased muscle levels of Fa-IsoPs and NO and reduced levels of HEPs and their metabolites and thus seemed to produce a rapid onset of oxidative stress.
TABLE 4.
9OPs, CMs, and Oxidative Stress
+ + + + + + + +
31.81 (100) 29.92 (155) b 32.57 (211) b 39.32 (267) b 46.66 (242) b 29.87 (175) b 31.91 (150) b 10.28 (98)
EDL 417.84 733.69 1014.85 1270.95 1108.97 810.12 673.22 433.27
+_ 18.54 (100) _+ 43.27 (176) b _+ 41.74 (243) b + 58.97 (304) b _+ 30.45 (265) b +_ 26.64 (194) b _+ 12.68 (161) b + 22.34 (104)
aValues of citrulline are presented as mean ___SEM ( n = 4-6). Numbers in parentheses are percentage changes compared to controls (100%). bSignificant difference between control and carbofuran-treatedrats (p < 0.05).
51 8
TABLE 5.
SECTION V . N o n s p e c i f i c Toxic Effects
Levels of High-Energy Phosphates and Their Metabolites in Soleus Muscle of Rats Intoxicated with an Acute Dose of Carbofuran (1.5 mg/kg, sc)
Time after carbofuran injection (hr)
0 (control) 0.25 0.5 1 2 3 6 24
Soleus (ixmol/g) a ATP
3.66 2.68 2.47 1.97 2.28 2.22 3.21 3.54
_+ 0.11 __+0.07 ___0.07 _ 0.04 _+ 0.12 __+0.07 ___0.10 -+- 0.14
TAN
(100) (73) b (67) b (54) b (62) b (61) b (88) b (97)
4.66 3.34 3.16 2.66 3.08 2.96 4.09 4.56
___0.14 _+ 0.07 ___0.07 __+0.04 _+ 0.16 ___0.11 ___0.11 ___0.16
PCr
(100) (72) b (68) b (57) b (66) b (64) b (88) b (98)
7.91 5.88 5.21 4.40 4.65 5.14 5.89 7.23
___0.26 __+0.17 __+0.30 __+0.06 ___0.22 _+ 0.17 ___0.33 ___0.11
TCC
(100) (74) b (66) b (56) b (59) b (65) b (74) b (92)
26.06 18.13 17.62 14.89 16.43 16.95 22.47 24.54
___0.93 __+0.21 ___0.71 ___0.29 _ 0.76 __+0.55 ___0.31 ___0.45
NAD
(100) (70) b (68) b (57) b (63) b (65) b (86) b (94)
0.68 0.44 0.44 0.43 0.48 0.46 0.51 0.64
___0.10 ___0.01 ___0.05 ___0.02 __+0.02 + 0.04 ___0.02 ___0.02
(100) (65) b (65) b (63) b (63) b (68) b (75) b (94)
aValues are presented as mean _+ SEM (n = 4-6). Numbers in parentheses are percentage changes compared to controls (100%). ATE adenosine triphosphate; TAN, total adenine nucleotides (ATP + ADP + AMP); PCr, phosphocreatine; TCC, total creatine compounds; NAD, nicotinamide adenine dinucleotide. bSignificant difference between control and carbofuran-treated rats (p < 0.05).
more sensitive marker of oxidative injury in muscles, similar to the case reported for brain (Gupta et al., 2001b). In addition to NO elevation, other factors that contribute to the loss in energy include damage to mitochondria by AChEIs (Laskowski et al., 1975, 1977) and the higher rate of ATP utilization needed to generate NAD in the ADP ribosylation of nuclear proteins. Other ATP- and PCr-dependent processes, such as enhanced influx of sarcoplasmic Ca 2+ and an increased number of contractile protein cross-bridges (Leonard and Salpeter, 1979, 1982), in addition to the release of ATP in concert with ACh from the nerve terminals, may have lowered the ATP content. Thus, the combination of impaired synthesis of ATP and its greater utilization during muscle hyperactivity appears to result in a significant depletion of ATE The net effect is a reduced cellular energy level (Gupta et al., 2001 a). The first direct evidence that NO was involved in OP-induced myopathy derived from experiments using inhibitors of NO synthase in combination with OP (Jeyarasasingam et al., 2000). Rats injected with paraoxon showed a 90-fold increase in the number of dying muscle fibers when compared to controls. Coadministration of the nonspecific NOS inhibitor nitro-L-arginine methylester (L-NAME) or with the specific neuronal NOS inhibitor 7-nitroindazole (7-NI) dramatically reduced the number of myopathic fibers to 20% when compared to rats receiving paraoxon only. These data confirm that increased NO production is essential in causing such a myopathy. Increased NO exerts cellular toxicity primarily by depleting energy stores through multiple mechanisms: (1) by prolonging poly-(ADP-ribose) polymerase activation
(Zhang et al., 1994); (2) by inhibiting mitochondrial enzymes, such as COx (Giulivi, 1998; Brorson et al., 1999) and C K (Kaasik et al., 1999); and (3) by inhibiting glycolytic enzymes, such as phosphofructokinase (Firestein and Bredt, 1999). Studies have shown that NO, like cyanide and azide, directly and specifically inhibits mitochondrial respiration by competing with molecular O2 for binding to COx, thereby causing inhibition of ATP synthesis (Giulivi, 1998). Even nanomolar concentrations of NO can directly inhibit COx activity, and cells producing large quantities of NO can inhibit their own respiration as well as the respiration of neighboring cells (Brown and Cooper, 1994; Brown, 1995). The 50% reduction of COx activity in DFP-treated diaphragm muscle (Fig. 1) could be due to the inhibitory effect of NO, which was increased by a factor of 3 (Yang and Dettbarn, 1998). Inhibition of COx appears to be the primary mechanism for ATP depletion since COx is the terminal and ratelimiting enzyme of the mitochondrial respiratory chain, which generates ATP by oxidative phosphorylation. A reduced capacity of COx will cause an incomplete reduction of O2, leading to increased production of ROS and reduced ATP synthesis producing more oxidative damage to mitochondrial membranes (Bose et al., 1992; Yang and Dettbarn, 1998; Milatovic et al., 2001). NO appears to regulate the coupling between energy supply and demand. It increases skeletal muscle blood flow and glucose transport and inhibits glycolysis and mitochondrial respiration (Kaminski and Andrade, 2001). The findings of Fukushima et al. (1997) and Gultekin et al. (2000) further support the contention that the AChE-inhibiting OP insecticides cause lipid peroxidation and inhibition of oxidative phosphorylation.
CHAPTER 36 9OPs, CMs, and Oxidative Stress
C. Correlation of AChE Activity with Oxidative Stress Determinants The data on AChE inhibition correlate well with the changes in F2-IsoPs, citrulline, and HEPs. Maximal inhibition of AChE occurred at 1 hr postcarbofuran administration (1.5 mg/kg, sc), the time when maximal signs of toxicity were seen (Gupta and Kadel, 1989; Gupta et al., 1994; Gupta and Goad, 2000). Maximal increases in F2-IsoPs and citrulline occurred when HEPs and their metabolites decreased. One hour after DFP injection (1.5 mg/kg sc), when rats exhibited signs of maximal severity, AChE activity in skeletal muscles was reduced by 90-96% (Fig. 3), F2-IsoPs (Fig. 2) and citrulline (Table 4) levels were increased, and HEP levels were decreased (Table 5) (Gupta et al., 2002).
V. PREVENTION OF A C h E I - I N D U C E D OXIDATIVE STRESS AND M Y O P A T H Y
519
A. Behavioral Effects Pretreatment of rats with MEM and ATS 60 and 15 min, respectively, before DFP administration provided complete protection against DFP-induced behavioral changes since no muscle fasciculations were seen at any time. MEM in combination with ATS, at the same dose, did not produce any untoward effects in DFP-untreated rats (Gupta et al., 2002).
B. Acetylcholinesterase Activity One hour after DFP injection, when rats exhibited signs of maximal severity, AChE activity in skeletal muscles was reduced by 90-96%. No significant change occurred in the enzyme activity in muscles of rats (DFP-untreated) receiving MEM and ATS. However, pretreatment of animals with MEM in combination with ATS provided significant protection of AChE (soleus, 31%; EDL, 32%; and diaphragm, 44%) against DFP-induced inactivation (Fig. 3).
B Y M E M A N D ATS Previous studies have demonstrated that the noncompetitive NMDA receptor antagonist memantine (MEM) provided protection against OP- or CM-induced myopathy by multiple mechanisms. In brief, these mechanisms include blockage of nAChR-ion channel complex (Masuo et al., 1986), prevention of neural hyperexcitability (McLean et al., 1992), reduction of high-frequency repetitive activation of peripheral nerves (Wesemann and Ekenna, 1982), and reduced seizures induced by NMDA receptor blockage (Danysz et al., 1994; Carter, 1995; Parsons et al., 1999).
C. F2IsoPs (Lipid Peroxidation) As shown in Fig. 2, a significant increase in F2-IsoPs was seen in soleus within 30 min and in EDL within 60 min following carbofuran injection. The changes were more rapid in soleus than in EDL. F2-IsoP levels in soleus returned to control levels within 2 or 3 hr and in EDL by 6 hr. Pretreatment with MEM and ATS prevented the increase in carbofuran-induced F2-IsoPs, whereas MEM and ATS treatment alone did not cause changes in control levels of F2-IsoPs (Fig. 4) (EDL data not shown).
120 [] CONTROL
[] M E M + A T S T
100 >I> 1<
o DFP
o MEM+ATS+DFP T
80
W tO
< (.9 z z ,<
FIG. 3 Effects of memantine (MEM; 18 mg/kg, sc) and atropine sulfate (ATS; 16 mg/kg, sc), given prophylactically 60 and 15 min, respectively, before administration of DFP (1.5 mg/kg, sc), on AChE activity in skeletal muscles (soleus, EDL, and diaphragm) of rats. Rats were sacrificed 60 min after DFP injection. Values of AChE (expressed as percentage of remaining activity) are presented as means +__SEM (n = 5).
60
LU
n-" I-Z W O n"
[
40
a,b T
I11 EL
a,b
a
SOLEUS
EDL
DIAPHRAGM
aSignificant difference between values from control rats and DFP-treatedrats (p < 0.05). bSignificant difference between values from DFP-treated rats and MEM + ATS + DFP-treatedrats (p < 0.05).
520
SECTION
N o n s p e c i f i c Toxic Effects
V.
2.5
2.0
.....
iiiiiiE
ltfIli II!I
Or)
'~ 1.5
FIG. 4. Effects of MEM (18 mg/kg, sc) and ATS (16 mg/kg, sc), given 60 and 15 min, respectively, before carbofuran administration, on F2-IsoPs levels in soleus muscles. Rats were sacrificed 60 min after last injection. Values are means _ SEM (n = 4-6).
!itli[llitill iitilIlI~lll~
............. llllt !!!!!!!!!!!}! .........
c
!glllllltlilfl[llllllfl
(t)
O
o_ 1.0
II''l'ltIlllltt
(/)
U.
lil[lllt]lil[lllllltl[I
0.5
[!lIIllllfil[l[l[llll[I
i ' llII}lI111,i ]
Igllllill[il[llilllil[l iiltllll!lil[llllllllll
0.0
[il[l[tlli!l[l[tftIil[I Carbofuran
Control
Antidotes
Antidotes + Carbofuran
(MEM + ATS)
aSignificant difference between control and carbofuran-treatedrats (p < 0.05). bSignificant difference between values from saline + carbofuran and MEM + ATS + carbofuran-treatedrats (p < 0.05).
blocked the AChEI-induced increases in lipid peroxidation and citrulline and the decrease in HEPs and their metabolites. Other studies showed that MEM and ATS also prevented AChEI-induced leakage of biomarker enzymes (CK and LDH) and their isoenzymes from muscle into the serum (Gupta and Goad, 2000). MEM may have been particularly effective in preventing increases in citrulline and decreases in HEPs in muscles by partially protecting AChE against critical inhibition by AChEIs. MEM by itself does not cause inhibition of AChE (McLean et al., 1992; Gupta et al., 1994), unlike the CMs (neostigmine, pyridostigmine, and physostigmine), which provide protection by preventing critical inhibition of AChE activity. AChE activity must be reduced to less than 30% of control to trigger muscle hyperactivity (Wecker et al., 1978). It appears that M E M may bind to a different modulatory site,
D. C i t r u l l i n e ( N O / N O S ) Figure 5 shows the levels of citrulline as a marker of NO synthesis in skeletal muscles of control and carbofurantreated rats. MEM and ATS completely prevented the increase in citrulline levels normally seen with carbofuran or DFP.
E. High-Energy Phosphates MEM and ATS alone did not cause any significant change in energy metabolites; however, prophylactic administration of MEM and ATS prevented changes in HEPs and their metabolites 60 min after administration of an AChEI. Data presented in Figs. 4 and 5 show that combined treatment with MEM and ATS, by preventing muscle hyperactivity,
1400 272 a
1200
r7
CONTROL
[]
MEM +ATS
O
DFP
B
MEM+ATS+DFP
278 a 288 a _
1000
A
E O
800
ILl Z ,-I ,-I 600 O
100
400
101
104 100
102
2001 0
.
.....
SOLEUS
.
.
.
.
.
.
.
EDL
.
.
.
DIAPHRAGM
FIG. 5. Effects of MEM (18 mg/kg, sc) and ATS (16 mg/kg, sc), given prophylactically 60 and 15 min, respectively, before DFP (1.5 mg/kg, sc) administration, on citrulline levels in skeletal muscles (soleus, EDL, and diaphragm) of rats. Rats were sacrificed 60 min after DFP injection (1.5 mg/kg, sc). Values of citrulline are presented as means ___SEM (n = 4). Numbers above the bars are percentage changes from controls (100%). aSignificant differencebetween values from control rats and DFP-treated rats (p < 0.05). bSignificant difference between values from DFP-treated rats and MEM + ATS + DFP-treatedrats (p < 0.05).
CHAPTER 36 9OPs, CMs, and Oxidative Stress which is yet to be characterized. At the nerve terminals, MEM prevents repetitive firing and excessive Ca 2+ uptake by blocking ACh receptor-ion channels, thus preventing muscle hyperactivity and subsequent biochemical changes. Neither DFP nor CF has direct effects on muscle, and their toxic actions are therefore due to increased hyperactivity as a result of inhibition of AChE at the NMJ. No muscle lesions were found when hyperactivity was blocked by d-tubocurarine or MEM plus ATS in the presence of DFP (Patterson et al., 1988; Yang and Dettbarn, 1996; Yang et al., 1996; Gupta and Dettbarn, 1992) (Table 1).
F. Spin Trapping Agent PBN Prevents DFPInduced Excitotoxicity in Rat Skeletal Muscles Electron spin resonance (ESR) spectroscopy using spin traps allows direct identification and characterization of ROS. A synthetic spin trapping agent such as phenyl-Ntert-butylnitrone (PBN) is capable of scavenging many types of free radicals. This compound is widely used to trap ROS in a variety of physical, chemical, and biological studies using electron paramagnetic resonance spectrometry. PBN is known to be concentrated in the mitochondria, where it reacts with ROS and forms stable adducts and thereby maintains normal levels of energy metabolites. In addition, PBN has other pharmacological actions, such as reversible Ca 2+ channel blockade (Anderson et al., 1993), direct protective interaction of PBN with AChE against phosphorylation by DFP or carbamylation by carbofuran (Zivin et al., 1999a; Milatovic et al., 2000a,b), protection of COx activity (Milatovic et al., 2001), and induction of hypothermia (Pazos et al., 1999). Pretreatment with PBN (300 mg/kg, ip), using the concentration necessary for in vivo spin trapping, prevented muscle hyperactivity as well as necrosis and attenuated the DFP-induced AChE inhibition otherwise seen in DFP-only treated rats. PBN had no effect when given after fasciculations were established. Although the role of PBN as an antioxidant is well established, its prophylactic effect against excitotoxity induced by an AChEI is due to its protection of AChE from critical inhibition, an unexpected action (Milatovic et al., 2000a,b).
VI. E X C I T O T O X I C I T Y
IN B R A I N
The similarity between electroencephalographic patterns, repetitive clonic convulsions, and neuropathology in the brain following status epilepticus (SE) and seizures induced by AChEIs (Lipp, 1968; Olney et al., 1986; McDonough et al., 1987; McDonough and Shih, 1997) suggests a common mechanism of initiation and propagation of the lesions. The AChEI-induced neuronal cell death appears to be a consequence of a series of extra- and intracellular events leading to the accumulation of Ca 2+ ions in the cell and the generation of oxygen-derived free radicals, causing
521
irreversible destruction of cellular components such as plasmalemma, mitochondria, other intracellular membranes, and of the cytoskeleton. McDonough and Shih (1997) suggested that a multitransmitter system is involved in the initiation and maintenance of SE induced by AChEIs. They suggested a three-phase sequence: an early cholinergic phase lasting until 5 min after seizure onset, a mixed cholinergic and noncholinergic modulation lasting 40 min, followed by a predominantly noncholinergic phase such as the activation of NMDA receptors within the glutamatergic system. Activation of these receptors maintains seizure activity and contributes to the intracellular accumulation of Ca 2+ involved in cellular events leading to neuronal cell death (Lei et al., 1992). Seizures induced with direct- or indirectacting cholinomimetics can be prevented by pretreatment with cholinergic receptor antagonists such as atropine. Cholinergic antagonists, however, are ineffective after the SE has been fully established (Olney et al., 1986; McDonough and Shih, 1997). Pretreatment with a noncompetitive NMDA receptor antagonist, such as dizocilpine (MK-801) and ketamine, does not prevent the initiating excitatory and neurotoxic properties of cholinomimetics but is effective after a period of seizure activity (Braitman and Sparenborg, 1989; Ormandy et al., 1989). This suggests that cholinergic mechanisms trigger the onset of seizures, whereas the propagation and maintenance of SE depend primarily on the activation of excessive glutamatergic transmission involving NMDA and/or cholinergic and other non-NMDA glutamate receptors. The neurotoxic actions are thought to be mediated through Ca 2+ influx and translocation of intracellular Ca 2+ and activation of protein kinase C (PKC). Phosphorylation mediated by PKC may be essential for an oxidative burst in affected brain neurons generating ROS, which in turn initiate harmful peroxidations of membrane lipids such as arachidonic acid. ROS may thus contribute to the common cascade of events, eventually leading to seizure-induced neuronal damage (Schulz et al., 1995). Seizures, convulsions, and central nervous system (CNS) lesions are typical results of systemic application of sublethal doses of AChEI (Sparenborg et al., 1992). The most consistent pathological findings in acute experiments are degeneration and cell death in pyriform cortex, amygdala, hippocampus (where the CA1 region is preferentially destroyed), dorsal thalamus, and cerebral cortex. The early morphological changes in AChEI-induced SE may be dendritic swelling of pyramidal neurons in the CA1 region of hippocampus (Carpentier et al., 1991). Atropine applied before the AChE inhibitors prevents seizures, whereas diazepam inhibits ongoing seizure activity (Olney et al., 1986). The increase in seizure-related phosphatidylcholine hydrolysis may be the cause of the accumulation of free fatty acids and choline in brain including the hippocampus (Flynn and Wecker, 1987).
522
SECTION V .
N o n s p e c i f i c Toxic Effects
A. Seizure Activity and Oxidative Stress The brain is considered to be very sensitive to oxidative stress because of a high rate of 02 consumption, a high rate of glucose and energy consumption, large amounts of oxidizable fatty acids, and relatively low levels of antioxidants. Neurons in particular are dependent on mitochondrial energy production. OP- or CM-induced seizures cause a high rate of ATP consumption. This high utilization of energy, coupled with the inhibition of oxidative phosphorylation, compromises the cell's ability to maintain its energy and antioxidant levels, causing excessive production of ROS/RNS, which leads to neuronal damage. Two oxyradicals that play predominant roles as initiators of lipid peroxidation are the hydroxyl radical (OH-) and the peroxynitrite radical (OONO-). The superoxide anion radical (O2), which is generated during the electron transport process in mitochondria, is involved in the generation of both OH- and OONO-. Superoxide dismutase (MnSOD and Cu/ZnSOD) converts O2 to hydrogen peroxide (H202), which is then converted to OH- via the Fenton reaction, catalyzed by Fe 2+ and Cu. OONO- is generated from the interaction of NO with O2. A major stimulus for NO production is elevation of Ca 2+, which binds to calmodulin, resulting in the activation of NO synthase. Although OH- and OONO- can cause direct damage to proteins and DNA, they are also potent inducers of lipid peroxidation and mitochondrial damage. Mitochondrial dysfunction leads to reduced energy production (due to a decrease in complex I and complex IV activities); impaired cellular calcium sequestration; activation of proteases/caspases; activation of phospholipases; activation of NOS; and generation of ROS and RNS, resulting in cell death by apoptosis or necrosis, depending on the degree of ATP loss and the severity of the insult. COx is the terminal complex in the mitochondrial respiratory chain, which generates ATP by oxidative phosphorylation, involving the reduction of 02 to H20 by the sequential addition of four electrons and four H +. Electron leakage occurs from the electron transport chain, which produces O2 and H202. Under normal conditions, COx catalyzes more than 90% of the oxygen consumption in the cells. The chance of intermediate products, such as the O2 and H202 and OH- radicals, escaping is small under conditions in which COx remains active. During the hyperactivity of brain or muscle, the activity of COx is reduced (Zivin et al., 1999b; Milatovic et al., 2001), leading to increased electron pressure within the electron transport chain and thereby increasing ROS generation, oxidative damage to mitochondrial membranes, and vulnerability to excitotoxic impairment leading to secondary excitotoxicity (Soussi et al., 1989; Gollnick et al., 1990; Bose et al., 1992; Bondy and Lee, 1993; Yang and Dettbarn, 1998; Zivin et al., 1999b; Milatovic et al., 2001).
B. Role of Nitric Oxide in Seizure-Induced Stress NO is a labile RNS endowed with messenger functions (Bredt, 1999). Increases in intraneuronal Ca 2+ stimulate nNOS, which oxidizes L-arginine, with stoichiometric production of L-citrulline and NO (Knowles and Moncada, 1994). Its role in convulsive phenomena has been studied in different experimental models, but the results are far from unequivocal. For example, the proconvulsive activity of NO in seizures induced by the excitatory NMDA, as well as by the AChEIs, has been demonstrated (Dawson, 1995; Bagetta et al., 1992, 1993; DeSarro et al., 1993; Lallement et al., 1996; Kim et al., 1997, 1999; Jones et al., 1998; Jacobsson et al., 1999). Conversely, the results of other studies indicate that NO may play the role of an endogenous anticonvulsant substance (Haberny et al., 1992; Buisson et al., 1993; Rondouin et al., 1993). Part of this controversy, neurotoxic versus neuroprotective actions (Lipton et al., 1993), may be due to the redox state of NO or the nitrosonium ion (NO-). NO can cause neurotoxicity by reacting with the superoxide 02, leading to the formation of OONO- (Lipton et al., 1993). Whether the seizureinduced increase in NO has proconvulsive or anticonvulsive actions may depend also on the amount of NO generated by NOS. NO's neurotoxic effects are primarily caused by depletion of cellular energy stores through multiple mechanisms, which were discussed in Section IV, B. Many reports provide evidence that NO impairs mitochondrial/cellular respiration and other functions by inhibiting the activities of several key enzymes, particularly COx, and thereby causing ATP depletion (Yang and Dettbarn, 1998; Milatovic et al., 2001; Gupta et al., 2001a,b; Dettbarn et al., 2001). Inhibition of COx appears to be the primary mechanism for ATP depletion. This is further supported by the finding that AChE-inhibiting OP insecticides cause inhibition of oxidative phosphorylation in the rat brain (Fukushima et al., 1997).
C. Sequential Changes in Lipid Peroxidation, NO, and HEPs Induced by DFP and Carbofuran It is well established that OP and CM compounds exert brain hyperactivity, such as convulsions and seizures, because of accumulation of ACh as a consequence of AChE inhibition. The mechanisms involved in the pathogenesis of neuronal damage appear to be linked to a free radicalmediated injury. Lipid peroxidation, mitochondrial dysfunction or damage, reduced neuronal energy levels, and reduced COx activity support the contention that AChEIs, such as DFP and carbofuran (CF), cause neuronal injury by excessive formation of ROS (Yang and Dettbarn, 1998; Jeyarasasingam et al., 2000; Gupta et al., 2000b, 2001a,b;
CHAPTER 36 9OPs, CMs, and Oxidative Stress Milatovic et al., 2000a,b, 2001). These findings clearly demonstrate that both DFP and CF are neurotoxic. 1. LIPID PEROXIDATION
Rats that received DFP or CF showed typical signs of AChE toxicity, including tremors, wet dog shakes, mild to moderate seizures, and convulsions, with rearing and falling over progressing to severe seizures within 15-30min. Significant increases in F2-IsoPs and F4-NeuroPs were seen in the brain within 30 rain following DFP or CF injection (Fig. 6), indicating increased lipid peroxidation. The induced changes were rapid and returned to control levels within 2 or 3 hr. 2. INCREASES IN NO/NOS Data on citrulline levels (an indicator of NO/NOS activity) in brain regions of control rats and those intoxicated with an acute dose of DFP (1.25 mg/kg, sc) or CF (1.25 mg/kg, sc) are shown in Figs. 7 and 8, respectively. Control levels of citrulline are markedly higher in the amygdala (289.8 _+ 7.0 nmol/g), followed by the hippocampus (253.8 +_ 5.5 nmol/g) and the cortex (121.7 __ 4.3 nmol/g). Within 5 min of CF injection, the citrulline levels were elevated more than four-fold in the cortex and more than two-fold in the amygdala and hippocampus. At this time point, after DFP treatment, only the cortex levels of citrulline were elevated (224%). Within 15 min of either DFP or CF treatment, the levels of citrulline were significantly higher in all three brain regions and were maximally
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aSignificant difference between control and DFP- or carbofuran-treated rats (p < 0.05).
523
elevated at 30 min postinjection (more than six- to sevenfold in the cortex and more than three- to four-fold in the amygdala or hippocampus). They remained elevated up to 60 min. When measured 24 hr later, the citrulline levels were at control level in the amygdala and hippocampus, whereas the cortex values remained increased.
3. CHANGES IN HIGH-ENERGY PHOSPHATE LEVELS
Data on HEPs and their metabolites in the brain regions of control rats and those intoxicated with DFP (1.25 mg/kg, sc) or CF (1.25 mg/kg, sc) are shown for cortex only in Fig. 9. Control values of energy metabolites were significantly greater in the cortex than in the amygdala or hippocampus. One hour after DFP or CF treatment, the levels of ATE TAN, PCr, and TCC were significantly reduced in all three brain regions. With either DFP or CE the reductions in ATP and TAN levels were greater in the cortex than in the amygdala or hippocampus. With DFP, the levels of PCr were reduced to the same degree in all three brain regions (52-55%), whereas with CF treatment the PCr levels were reduced significantly more in the cortex (47%) than in the amygdala or hippocampus (71-72%). For each of the AChEIs examined, NO levels were significantly elevated (Figs. 7 and 8), whereas energy metabolites were reduced in all three brain regions (Fig. 9). The increase in NO was significantly greater in the cortex and correlated well with a greater depletion of HEPs in this brain region than in the amygdala or hippocampus. Other factors contribute to the energy changes, including mitochondrial and neuronal damage and a higher rate of ATP utilization needed to generate NAD in the ADP ribosylation of nuclear proteins (Gupta et al., 2000a,b), resulting in a decline in TAN and TCC. Thus, the combination of impaired synthesis of ATP with its greater utilization during brain hyperactivity appears to result in a significant depletion of ATP. From these studies, an important question that emerged was whether brain hyperactivity, such as seizure activity and the decrease in ATP, generated increases in ROS or whether hyperactivity produced ROS leading to a decrease in ATP. The findings revealed that within 5-15 min after CF or DFP injection (the time required for onset and development of clinical signs), NO levels increased more than five- to six-fold in the cortex and more than two- to threefold in the amygdala and hippocampus. For each AChEI, the maximum increase in NO occurred at 30 min postinjection in all three brain regions. The data also revealed that maximum decline in HEPs occurred 1 hr after DFP or CF injection. Therefore, the findings suggest that in the case of AChEIs, the increase in ROS preceded the decrease in ATP in the brain. All the changes in citrulline, HEPs, and lipid peroxidation (as described in Section VI, C) are prevented when pretreatment with MEM and ATS precedes DFP or CF injections.
SECTION V.
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N o n s p e c i f i c Toxic Effects
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CHAPTER
36
9 OPs, CMs, and Oxidative Stress
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FIG. 9. Protection by PBN of DFP- or CF-induced changes in ATE total adenine nucleotides (TAN), PCr, and total creatine compounds (TCC) in rat brain cortex. Values of energy metabolites are presented as means + SEM (n = 4-5). Rats were sacrifices 60 min after DFP or CF Treatment aSignificant difference between values from control rats and DFP- or CF-treated rats (p < 0.05). bSignificant difference between values from DFP-treated rats and PBN + DFP-treated rats (p < 0.05). cSignificant difference between values from CF-treated rats and PBN + CF-treated rats (p < 0.05).
D. P r e v e n t i o n of C h a n g e s in N O a n d E n e r g y M e t a b o l i t e s by P B N or V i t a m i n E NO is a free radical that has been widely regarded as a messenger molecule or neurotransmitter in the CNS and has also been considered to cause toxicity (Bredt, 1999). Rats receiving DFP or CF showed typical signs of anticholinesterase toxicity, including tremors, convulsions, wet dog shakes, and mild to moderate seizures with rearing and falling over, progressing to severe seizures within 7-15 min. PBN or vitamin E treatment alone produced no effects, but when given as pretreatment PBN prevented the development of seizures, whereas vitamin E had no effect on seizures. 1. C I T R U L L I N E , PBN, AND VITAMIN E When given alone, PBN or vitamin E did not alter the levels of NO (citrulline) in any of the three brain regions examined, but when given as pretreatment to DFP or CE they provided significant protection against DFP- or CF-induced increases in citrulline (Figs. 10 and 11, respectively). For each of the antioxidants, protection was greater against DFP than against CF in the cortex and was equally effective in amygdala and hippocampus.
2. H I G H - E N E R G Y P H O S P H A T E S ,
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AND V I T A M I N E
PBN treatment alone had no effect on the levels of HEPs or their major metabolites, but when given as pretreatment it provided significant protection against DFP- or CF-induced depletion of energy metabolites. (Fig. 9). In contrast to pretreatment with vitamin E, the spin trapping agent PBN prevented DFP- or CF-induced convulsions and seizures. This was primarily due to a protective interaction of PBN with ACHE, sufficient to protect a critical fraction of AChE against phosphorylation by DFP or carbamylation by CF (Zivin et al., 1999a; Milatovic et al., 2000a,b). Figures 10 and 11 show that AChE inhibitor-induced increases in NO (citrulline) were significantly prevented by PBN as well as by vitamin E. There is evidence that PBN inhibits the induction of iNOS by reducing the expression of iNOS protein (decrease in mRNA expression) and thus prevents the overproduction of NO (Miyajima and Kotake, 1995, 1997). These findings demonstrate that AChEI-induced depletion of energy metabolites is, in part, also prevented by antioxidants (PBN or vitamin E), supporting the suggestion that increased generation of ROS/RNS contributes to depletion of energy phosphates (Gupta et al., 200 lb)
526
SECTION V.
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Although the preventive effects of PBN and vitamin E against DFP- or CF-induced elevation of NO and depletion of energy-rich phosphates in rat brain are of the same degree, the two antioxidants may act by different mechanisms (Gupta et al., 2001a,b). The main difference is that PBN prevents seizure activity and thus inhibits the cascade of ROS generation and loss of energy metabolites (Zivin et al., 1999a). Vitamin E pretreatment partially prevented the depletion of HEPs and their metabolites without preventing seizures. Previous studies have shown that mitochondria contain the highest concentration of vitamin E (Bjorneboe et al., 1991) which accelerates ATP resynthesis in tissues subjected to ischemia/reperfusion (Punz et al., 1998). Vitamin E also prevented metasystox (OP insecticide)-induced changes in lipase activity and lipid peroxidation in the brain and spinal cord of rats (Tayyaba and Hasan, 1985). Vitamin E mainly acts as a chain-breaking
antioxidant and radical scavenger, protecting cell membranes against oxidative damage (van Acker et al., 1993). In addition, vitamin E regulates ROS production (Chow et al., 1999), maintains oxidative phosphorylation in mitochondria, and accelerates restitution of high-energy metabolites (Kotegawa et al., 1993; Punz et al., 1998). The protection provided by PBN and vitamin E against DFP- or CF-induced changes in energy metabolites was of varying degrees in different brain regions and could partly be due to pharmacokinetic variables involved in attaining different levels of PBN or vitamin E in specific brain regions. Spin trapping agents and antioxidants such as vitamin E, by preventing seizure activity and/or by scavenging ROS, provide partial protection against depletion of energy metabolites, maintain adequate cellular energy status, and thus diminish neuronal and muscle injury (Zivin et al., 1999b; Milatovic et al., 2001; Dettbarn et al., 2001).
CHAPTER
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9OPs, CMs, a n d O x i d a t i v e Stress
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E. Changes in Biomarkers during and after Seizure Activity There are additional useful markers of pathological changes in response to methyl parathion (MPTH) and CF toxicity, such as LDH and CK and their isoenzymes. Their changes in serum are related to the brain damage caused by MPTHor CF-induced SE. Both insecticides produced characteristic signs of anticholinesterase toxocity within 5-7 min after injection. In controls, analyses of the brain regions revealed a wide variability in the values of noncholinergic biomarkers (CK and LDH and their isoenzymes). The highest activities of LDH were found in the striatum and the lowest in the cerebellum. However, the activity of CK was highest in the cerebellum. Each brain region showed a characteristic profile of CK and LDH isoenzymes. Among the CK isoen-
zymes, the activity of CK-BB was highest (77.5-89.3%), followed by CKIMM (6.7-15.6%) and CK-MB (0-6.9%). The cerebellum had no CK-MB activity. In all brain regions, CK-MM isoenzyme had only the CK-MM3 subform. Among the LDH isoenzymes, the activity of LDH-4 was highest in all brain regions (23-40%) except the cerebellum, in which LDH-1 was highest (29%). Compared to the brain, control serum contained very little CK and LDH activity, but serum had three distinct CK and five distinct LDH isoenzymes. Unlike brain regions, serum had three CK-MM subforms. Each insecticide induced characteristic alterations in brain biomarkers. MPTH induced characteristic alterations in CK, LDH, and their isoenzymes in the brain, which were also reflected in serum as a result of their leakage from the brain (Gupta et al., 2000b).
528
SECTION V .
N o n s p e c i f i c Toxic Effects
VII. R O L E O F N O IN K A I N I C A C I D AND AChEI EXCITOTOXICITY The close relationship between NO and excitotoxicity has been well established with a variety of experimental models, such as kainic acid (KA) and AChEI-induced seizures in brain (Lothman and Collins, 1981; Jones et al., 1998; Lallement et al., 1996; Jacobsson et al., 1999; Kim et al., 1997; Milatovic et al., 2002; Gupta and Dettbarn, 2003). Other investigations have shown that NO is involved in AChEI-induced myotoxicity (Gupta et al., 2001b, 2002; Dettbarn et aL, 2001; Jeyarasasingam et al., 2000; Milatovic et al., 2005). There is sufficient evidence to support the role of NO in NMDA receptor-mediated excitotoxicity in the brain during SE. Studies have shown protection of rat hippocampal neurons against KA-induced excitotoxicity by pretreatment with NOS-Inhibitors L-NAME or 7-NI. 7-NI, a selective inhibitor of nNOS, does not inhibit A eNOS NOS (Moore et al., 1993), suggesting that nNOS contributes to neuronal damage in the KA model of SE (Jones et al., 1998; Montecot et al., 1998). Other studies established that pretreatment of rats with the chain-breaking antioxidant vitamin E or the spin trapping agent PBN prevented the KAinduced increase in NO, the decrease in HEPs, and attenuated neuronal damage without preventing seizures (Milatovic et al., 2001; 2002). This suggests that PBN and vitamin E protected neurons from oxidative damage by trapping oxygen radicals such as O2. 7-NI significantly prevented KA-initiated increases in citrulline (determinant of NO) and decreases in HEPs, consistent with results obtained using PBN and vitamin E, two well-known antioxidants (Milatovic et al., 2002). In vivo studies have shown that KA as well as AChEIs cause significant increases in NO as well as significant decreases in energy metabolites in brain and muscle within 30 min of injection. The decrease in levels of HEPs may be due to a combination of increased consumption as well as inhibition of synthesis, including uncoupling of oxidative phosphorylation. The inhibition of NOS by 7-NI reduces the production of NO and subsequent peroxynitrite formation, thus protecting neurons and muscle from KA and AChEIinduced toxicity. These in vivo findings are consistent with the hypothesis that excitotoxicity is mediated in part by activation of nNOS resulting in generation of OONO-, which initiates mitochondrial dysfunction and injury.
of which there are approximately 220,000 deaths. In the United States, the incidence of pesticide-caused illnesses is estimated to be between 150,000 and 300,000 (Coye, 1985). The toxic symptoms in humans are similar to those seen in animal experiments. Death results from overstimulation of the cholinergic system leading to paralysis and respiratory arrest. There are few reports about humans exposed to OPs and CMs regarding oxidative stress and lipid peroxidation (Banerjee et aL, 2001; Abdollahi et al., 2004). Oxidative stress is seen as an imbalance between free radical production and antioxidant activity. Measurements were limited in most reports to TBA-MDA formation and changes in the total antioxidant capacity of the blood. The few reports of acute poisonings showed a significant correlation between increased levels of TBA-MDA and a decrease in antioxidant capacity. In studies on severely poisoned patients, a significant increase in blood levels of TBA-MDA was seen. These levels were twice as high in patients who did not survive, suggesting severe lipid peroxidation and cell damage (Vidyasagar et al., 2004). Because the TBA-MDA technology is not very specific, problems concerning the significance of these values arise. Better and more significant data may be obtained with the determination of specific indicators of lipid peroxidation (Janero, 1990). The measurement of F2-IsoPs levels in blood is far more superior to the measurement of TBA-MDA as an index of lipid peroxidation in vivo (Roberts et al., 1998). The discovery of F2-IsoPs is important for two reasons. The first relates to the fact that compared to other methods, measurement of F2-IsoPs appears to provide a far superior approach to assess oxidative stress status in vivo. With the general availability of a reliable method for the measurement of F2-IsoPs in blood and tissues, it is expected that new and exciting insights into the role of free radicals in human disease will be forthcoming in the near future. The other area of importance is related to the use of measurements of F2-IsoPs to define the clinical pharmacology of antioxidant agents. Because of the shortcomings of methods previously available to quantify the level of oxidative stress in vivo, little information is available regarding the optimal doses and combinations of antioxidants that effectively suppress oxidant injury in humans. This applies not only to the endogenous antioxidants but also to antioxidant drugs.
IX. C O N C L U S I O N S VIII. H U M A N S T U D I E S O F O X I D A T I V E S T R E S S IN A C U T E O P A N D C M P O I S O N I N G OPs and CMs are used worldwide for pest control. Unfortunately, their use is not always specific target oriented. In 1962, a report by the World Health Organization noted that 3 million cases of acute poisonings occur annually,
AChEIs such as OPs and CMs have neuro- and myotoxic effects that destroy neurons and muscle fibers by excitotoxic actions, mediated by ACh receptor overstimulation following AChEI application. A rapid and significant increase in NO precedes increases in lipid peroxidation, mitochondrial dysfunction, loss of energy metabolites, as well as a reduction of COX activity, and an increase in xanthine oxidase.
CHAPTER 3 6 Inhibitors of NOS prevent the previously mentioned changes and protect neurons and muscle fibers from injury. These findings are supported by similar actions of the spin trapping agent PBN and the antioxidant Vitamin E and by agents that prevent convulsions and fasciculations, such as M E M and ATS or d-tubocurarine. Understanding the relationship between excitotoxicity and oxidative stress is important since this offers opportunities for developing pharmacological approaches that interfere with mechanisms significantly involved in neuro- and myotoxicity without compromising normal neurotransmitter function.
Acknowledgments This work was supported in part by NIH grants ES04957 and DAMD 17-83C-3244. The authors thank Susan Taylor, Lynda Shaver, and Debra Britton for their assistance in the preparation of the manuscript.
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CHAPTER
~ 7
DNA Damage, Gene Expression, and Carcinogenesis by Organophosphates and Carbamates MANASHI BAGCHI, 1,2 SHIRLEY ZAFRA, 1 AND DEBASIS BAGCHI 1,2 llnterHealth Research Center, Benicia, California 2Creighton University Medical Center, Omaha, Nebraska
I. I N T R O D U C T I O N
et al., 1995, 1997). Experimental evidence also demonstrated OP potential for neurotoxicity. Kaplan et al. (1993)
Residential and industrial use of organophosphate (OP) and carbamate (CM) insecticides is widespread in the United States. According to 1997 findings of the U.S. Environ' mental Protection Agency (EPA), more than 40 OP pesticides and 22 CM pesticides are among the 900 pesticides registered for use in the United States that pose the highest risks to human health (Davies, 1990; Pound and Lawson, 1976; Sandoz et al., 1998). Numerous studies have demonstrated the acute toxicity potential of OPs and CMs that are associated with developmental birth defects, DNA damage, abnormal spermocytes and oocytes, and fetal death (Botham, 1990; Eskenazi et al., 1999; Pope, 1999; Pound and Lawson, 1976; Sandoz et al., 1998; Vidair, 2004). Due to their high level of pest control and relatively low environmental toxicity, OP insecticides, such as chlorpyrifos and fenthion, are widely used in residential and industrial environments (Pope, 1999). OPs have been used extensively to control a wide range of sucking and chewing pests of field crops, fruits, and vegetables. They have many structural similarities with naturally occurring compounds, and their primary target of action in insects is the nervous system via inhibiting the release of the enzyme acetylcholinesterase (ACHE) at the synaptic junction (Pope, 1999). Eserine, parathion, and malathion are further examples of cholinesterase inhibitors responsible for the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses (Cabello et al., 2001). Experimental exposure of animals to these xenobiotics elicits a number of effects, including lipid mobilization, porphyria, hypothyroidism, an increased liver-to-body weight ratio, testicular atrophy, a progressive weight loss with hypophagia, and depletion of adipose tissues (Bagchi Toxicology of Organophosphate and Carbamate Compounds
presented case reports of residential exposure to chlorpyrifos resulting in cognitive slowing, cognitive problems, and sensory neuropathy weeks to months after application. Studies indicate that toxic manifestations induced by these pesticides are associated with the enhanced production of reactive oxygen species (ROS), which explains the multiple types of toxic responses as well as the characteristic wasting syndrome (Bagchi et al., 1995, 1996, 1997). In response to many toxicants and pathological conditions, sufficient evidence supports the hypothesis that ROS mediate cell injury, and most of these xenobiotics may also serve as common mediators in the activation of protein kinase C, oncogene expression, as well as programmed cell death (apoptosis) and tumor formation. The ability to produce ROS in vivo with successive tissue damage was examined by hepatic and brain lipid peroxidation and DNA single-strand breaks (SSBs). Chemiluminescence, lactate dehydrogenase (LDH) leakage, and DNA SSBs were also assessed to determine the in vitro production of ROS. OPs have also shown the mechanism involved in the induction of oxidative tissue damaging effects, including lipid peroxidation and nuclear DNA SSBs by the expression of specific heat shock/stress protein (Bagchi et al., 1995, 1996, 1997). Cellular exposure to the OP pesticide chlorpyrifos has been shown to elicit an immediate decrement in DNA synthesis, with a significantly greater effect on the gliotypic C6 cells than on neuronotypic PC-12 cells (Bagchi et al., 1995, 1997). It was demonstrated that chlorpyrifos, a suspected neuroteratogen, exerts antimitotic actions on developing neural cells independently of cholinesterase inhibition. Adverse effects of chlorpyrifos on glial cell replication are of critical importance in defining the sensitive period for 533
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
534
SECTION V .
N o n s p e c i f i c Toxic Effects
effects on central nervous system development. Glia provide nutritional, structural, and homeostatic support essential to the architectural modeling of the brain. Glial development continues well into the postnatal period, glial targeting implies a prolonged vulnerability extending into childhood (Aschner et al., 1999; Barone et al., 2000; Guerri and Renau-Piqueras, 1997; Morita et al., 1999; Tacconi, 1998). In keeping with this interpretation, chlorpyrifos administration in vivo inhibits DNA synthesis and causes loss of brain cells during gliogenesis, with maximal effects on neural function appearing during peaks of glial development (Whitney et al., 1995; Campbell et al., 1997; Dam et al., 1998). Other OPs such as diazinon have immediate, direct inhibitory actions on DNA synthesis and hence on neural cell replication, with preferential targeting of gliotypic cells. CM insecticides are derivatives of carbamic acid. They vary in their spectrum of activity, mammalian toxicity, and persistence. CMs are relatively unstable compounds that break down in the environment within weeks or months. CM insecticides act by a similar mechanism as OP pesticides but have a shorter duration of action (Botham, 1990; Davies, 1990; Pesticides Information Profiles, 1996; Vidair, 2004). Like OPs, CMs are widely used and have varying degrees of toxicity. CMs interfere with the conduction of signals in the nervous system of insects in which cholinergic reactions are thought to take place. However, the insect neuromuscular junction is not cholinergic, as it is in mammals. The immediate toxic effect of CMs is very similar to that of OPs, but the recovery is comparatively rapid. Spontaneous recovery without medical treatment occurs generally within 4 hr of exposure that produces symptoms and signs of headache, dizziness, weakness, excessive salivation, nausea, or vomiting (Botham, 1990; Davies, 1990). More severe signs of exposure include visual disturbances, profuse sweating, abdominal pain, incoordination, muscle fasciculations, breathing difficulties, or changes in heart rate (Pesticides Information Profiles, 1996). This chapter focuses on DNA damage, oxidative stress, gene expression, and carcinogenesis induced by OPs and CMs.
II. I N V I T R O A N D I N V I V O G E N E R A T I O N O F R O S BY O P s Reactive Oxygen Species (ROS) may serve as common mediators in programmed cell death or apoptosis in response to many toxicants and pathological conditions. Studies have demonstrated that ROS may be involved in the cytotoxicity of widely used OP pesticides, such as chlorpyrifos and fenthion. Highly toxic chlorpyrifos and fenthion are organic triesters of phosphoric acid and phosphorothioic acid and are also lipophilic (Murphy, 1986). Neurotransmitter systems play a key role in behavioral function and disturbances. Other OPs, such as phosphamidon, trichlorfon, and dichlorvos, have also been reported to induce oxidative stress, both
in vivo and in hepatocytes, as shown by inhibition of superoxide dismutase (SOD) activity, enhanced malondialdehyde production, LDH leakage, and a decrease in glutathione peroxidase activity (Julka et al., 1992). OP intoxication has also been shown to induce oxidative stress at the tubular level and may play a role in the pathogenesis of acute tubular necrosis (Poovala et al., 1999). In relevant studies, the comparative in vivo and in vitro effects of OPs such as chlorpyrifos and fenthion were assessed in cultured neuroactive PC-12 cells and in treated animals (Bagchi, et al., 1995, 1997). Although dissimilar polyhalogenated cyclic hydrocarbons, such as endrin and chlordane, and chlorinated acetamide herbicides, such as alachlor, were also compared to the OP insecticides, the in vivo and in vitro effects of OPs, chlorpyrifos, and fenthion were evaluated (Bagchi et al., 1995, 1997). The ability to produce ROS in vivo with successive tissue damage was examined by hepatic and brain lipid peroxidation and DNA SSBs. Chemiluminescence, LDH leakage, and DNA SSBs were also assessed to determine the in vitro production of ROS. The results clearly demonstrated that administration of OPs results in the in vivo and in vitro induction of hepatic and brain lipid peroxidation, chemiluminescence response, LDH leakage, and DNA SSBs, suggesting that the ROS and/or free radicals may be involved in the toxic manifestations of OPs (Bagchi et al., 1995, 1996, 1997). Furthermore, results indicate the tissue specificity of OPs with respect to the responses.
A. In Vivo Generation of ROS and Lipid
Peroxidation by OPs OP-induced in vivo production of ROS and membrane damage were assessed by hepatic and brain lipid peroxidation. Chlorpyrifos and fenthion were administered E O. to female Sprague-Dawley rats in two 0.25 LDs0 doses at 0 and 21 hr and killed at 24 hr. Lipid peroxidation assay was determined on the hepatic and brain whole homogenates from control and treated animals according to the method of Buege and Aust (1972) based on the formation of thiobarbituric acid reactive substances (TBARS). Malondialdehyde was used as the standard and prepared according to the method of Largilliere and Melancon (1988). Following treatment of the rats with chlorpyrifos and fenthion, 4.3- and 4.8-fold increases, respectively, were observed in hepatic lipid peroxidation, whereas at the same doses, increases of 4.6- and 5.3-fold, respectively, were observed in brain homogenates. The effects of the pesticides on lipid peroxidation in hepatic and brain homogenates based on the TBARS assay are summarized in Table 1 (Bagchi et al., 1995). The effect of dichlorvos exposure on lipid peroxidation and the antioxidant defense system in different regions of the rat central nervous system was also studied. Inhibition of AChE activity was used as an index of dichlorvos
CHAPTER 37
TABLE 1. Effects of OP Pesticides on Lipid Peroxidation (TBARS Content) in Liver and Brain Homogenates a
9OPs, CMs, DNA Damage, and Gene Expression
535
dichlorvos in the brain (Barron, 1991; Chan, 1989; Julka et al., 1992).
Lipid peroxidation (nmol/mg protein) Treatment
Liver
Brain
Control Chlorpyrifos Fenthion
3.8 +__0.6 a 15.9 +- 1.4c 18.0 +_ 1.7c
5.4 + 0.6 a 25.0 _+ 2.1 d 28.6 ~ 2.5 d
aFemale Sprague-Dawley rats were given two 0.25 LDs0 doses of the pesticides orally at 0 and 21 hr in corn oil and killed at 24 hr. Control animals received the vehicle. Lipid peroxidation was determined as the content of (TBARS) using malondialdehyde as the standard. Each value is the mean + SD of at least four to six animals in each group. Values with nonidentical superscripts are significantly different (p < 0.05).
neurotoxicity. Significant increases in the activities of the antioxidant enzymes SOD and catalase were accompanied by a decrease in the values of lipid peroxidation. Dichlorvos exposure also resulted in a significant decrease in glutathione peroxidase activity. The decreased levels of both reduced and oxidized glutathione as observed on dichlorvos exposure affected the glutathione-toglutathione disulfide ratio. These results indicate that the enzymes SOD and catalase may enhance the disposal of potentially toxic radicals. Furthermore, the decrease in G S H levels may be a mechanism for the detoxification of
B. C h e m i l u m i n e s c e n c e R e s p o n s e to O P s for G e n e r a t i o n of R O S The chemiluminescence assay is a nonspecific test for the identification of ROS. The sustained chemiluminescence produced by liver and brain tissues following treatment with chlorpyrifos and fenthion is presumably due to the continued production of ROS. The chemiluminescence responses produced following i n vivo administration of these pesticides on liver and brain tissues are shown in Fig. 1. The greatest chemiluminescence responses in hepatic tissues were induced by fenthion. The fenthion resulted in a moderate chemiluminescence response in the brain tissues (Bagchi et al., 1995). The results of chemiluminescence assay for the production of ROS by liver and brain homogenates from control and pesticide-treated rats are presented in Figs. 2 and 3, respectively. The chemiluminescence response produced by liver and brain homogenates from pesticide-treated rats rapidly increased, reaching a maximum between 6 and 8 min of incubation, whereas liver and brain homogenates from control animals reached a peak rate of chemiluminescence (CPM) at 6 min. The chemiluminescence persisted for more than 10 min. Increases of 2.9- and 3.4-fold were observed in the chemiluminescence responses in the liver homogenates of the animals treated with chlorpyrifos
FIG. 1. (A) Westem blot analysis of protein samples derived from the organs of control and pesticide-treated rats. 1, Control brain; 2, alachlor-treated brain; 3, endrin-treated brain; 4, chlordane-treated brain; 5, chlorpyrifos-treated brain; 6, fenthion-treated brain; 7, control liver; 8, alachlor-treated liver; 9, endrin-treated liver; 10, chlordane-treated liver; 11, chlorpyrifos-treated liver; 12, fenthion-treated liver; 13, control lung; 14, alachlor-treated lung; and 15, endrin-treated lung. (B) Western blot analysis of protein samples derived from control and pesticide-treated cultured PC-12 cells. 1, Control DMSO (50 nM); 2, control DMSO (100 nM); 3, control DMSO (200 nM); 4, alachlor (50 nM); 5, alachlor (100nM); 6, alachlor (200 nM); 7, endrin (50nM); 8, endrin (100 nM); 9, endrin (200 nM); 10, chlordane (50 nM); 11, chlordane (100 nM); 12, chlordane (200 nM); 13, chlorpyrifos (50 nM); 14, chlorpyrifos (100 nM); 15, chlorpyrifos (200 nM); 16, fenthion (50 nM); 17, fenthion (100 nM); 18 fenthion (200 nM).
536
SECTION V . N o n s p e c i f i c Toxic Effects
FIG. 2. Production of chemiluminescence by liver homogenate (1 mg protein/ml) following treatment with pesticides in two equal doses (0.25 LDs0) at 0 and 21 hr. All animals treated at 24 hr posttreatment. Each value is the mean of four to six experiments.
FIG. 3. Production of chemiluminescence by brain homogenate (1 mg protein/ml) following treatment with pesticides in two equal doses (0.25 LDs0) at 0 and 21 hr. All animals treated at 24 hr posttreatment. Each value is the mean of four to six experiments.
and fenthion, respectively, whereas increases of 2.9- and 2.4-fold, respectively, were observed in brain homogenates (Bagchi et al., 1995).
C. Lactate Dehydrogenase (LDH) Leakage by OPs OPs cause toxicological problems due to their high environmental persistence and ability to accumulate in adipose tissues. OPs exhibit similar abilities as inducers of hepatic drug-metabolizing enzymes (Viviani et al., 1978) and act as potent competitive and stereospecific inhibitors of ligand binding to specific types of brain receptors (Botham, 1990; Campbell et al., 1997; Davies, 1990). As an index of membrane and cellular damage, the release of LDH from cultured PC-12 cells was measured. Increased release of LDH into the media of cultured cells is indicative of cellular and membrane damage. Cultured PC-12 cells were incubated in the presence of 0, 50, 100, or
200 nM of chlorpyrifos and fenthion, and the release of LDH by the cells was measured after 24 hr of incubation as an index of cytotoxicity. The maximum LDH release was observed with a 100 nM concentration of the pesticides with chlorpyrifos and fenthion, producing immense leakage of LDH. LDH enzyme activities in the media of the cultured neuroactive PC-12 cells were determined as a function of concentration of the pesticides. The data are presented in Table 2 (Bagchi et al., 1995). The amount of LDH released by the pesticides was concentration dependent. However, the differences in the release of LDH into the media were not significantly different at concentrations of 100 and 200 nM. Following 24-hr incubation, increases in LDH leakage of 1.9- and 2.1-fold were observed with 50 nM concentrations of chlorpyrifos and fenthion, respectively, whereas at 100 nM concentration of the pesticides, increases of 3.1- and 3.4-fold, respectively, in LDH leakage were observed from the cultured PC-12 cells (Bagchi et al., 1995).
CHAPTER 37
TABLE 2.
9OPs, CMs, DNA D a m a g e , and G e n e Expression
537
Concentration-Dependent Effects of OP Pesticides on the Release of LDH from Cultured PC- 12 Cells a
Concentration of LDH in the media (U LDH/liter) Treatment Control
50 nM
100 nM
200 nM
77.4 + 9.2 a
76.9 + 10.3 a
79.2 _ 8.6 a
Chlorpyrifos
147.1 + 15.3 c,d
239.9 + 20.8 c,d
241.0 + 25.7 c,d
Fenthion
162.5 _+ 13.7 d
263.2 ___24.6 d
266.5 _+ 20.4 d
apc-12 cells (25 • 104 cells/35-mm petri dish) in 2 ml of RPMI 1640 were incubated for at least 3 hr to allow cell adherence, and 50, 100, or 200 nM concentrations of pesticides were added to the cultures in two equally divided portions at 0 and 24 hr. The incubation was continued at 37 ~ in an atmosphere of 5% CO2 for 24 hr. Media were collected from the cultures and assayed for LDH activity. Data are expressed as the mean value of six experiments + SD. Values with nonidentical superscripts are significantly different (p < 0.05).
D. O P - I n d u c e d
DNA Damage
D N A SSBs are another index of oxidative stress and cellular d a m a g e (Bagchi et al., 1995; Poovala et al., 1999; P o u n d and L a w s o n , 1976). In a parallel study, the effects of the pesticides on D N A SSBs were d e t e r m i n e d in cultured neuroactive P C - 1 2 cells and in the hepatic and brain tissues of OP-treated rats (Bagchi et al., 1995). D N A SSBs were d e t e r m i n e d in cultured neuroactive P C - 1 2 cells following incubation with a 100 n M concentration of chlorpyrifos and fenthion for 24 hr. Rats were orally administered two 0.25 LDs0 doses at 0 and 21 hr and killed at 24 hr. Nuclei were isolated f r o m both hepatic and brain tissues. F o l l o w i n g treatment of the rats with chlorpyrifos and fenthion, increases of 3.0- and 3.5-fold were observed in hepatic D N A SSBs, respectively, whereas increases in D N A SSBs of 1.4- and 1.4-fold, respectively, were observed in the brain nuclei c o m p a r e d to control animals. T h e D N A SSB data f r o m control and pesticide-treated cultured P C - 1 2
TABLE 3.
cells were also obtained (Table 3). F o l l o w i n g incubation, i m m e n s e D N A SSBs were observed following treatment of the cultured PC-12 cells with chlorpyrifos and fenthion. Increases in D N A SSBs of 2.4- and 2.5-fold, respectively, were observed c o m p a r e d to control cultures (Bagchi et al., 1995). T h e detection of genotoxicity caused by OP pesticides has also been d e t e r m i n e d using the single-cell gel electrophoresis assay or c o m e t assay. In a s e p a r a t e study, the OP pesticides chlorpyrifos and acephate were tested for their ability to induce in v i v o genotoxic effects in leukocytes of Swiss albino mice ( R a h m a n et al., 2002). T h e m i c e were a d m i n i s t e r e d oral doses of 0 . 2 8 - 8 . 9 6 m g / k g b o d y weight of chlorpyrifos and 1 2 . 2 5 - 3 9 2 . 0 0 m g / k g b o d y w e i g h t of acephate. T h e c o m e t assay was p e r f o r m e d on w h o l e blood at 24, 48, 72, and 96 hr. A significant increase in m e a n c o m e t tail length indicating D N A d a m a g e was observed at 24 hr p o s t t r e a t m e n t (p < 0.05) with both
Effect of Pesticides on Nuclear DNA SSBs in Sprague-Dawley Rats and in Cultured PC- 12 Cells a
DNA elution constants ( X 10 3) Sprague-Dawley rats Pesticides Control
Liver 5.0 _+ 0.12 a
Cultured PC-12 cells
Brain 7.9 + 2.1 a
3.4
-+
Fenthion
17.3
___ 3 . 3 b
11.0 _+ 0.8 c
8.6
+ 0.3 b
0.4 a
Chlorpyrifos
15.0 + 1.7 b
11.0 ___ 1.6 c
8.3 + 0.5 b
aFemale Sprague-Dawley rats were given two 0.25 LDs0 doses of the pesticides orally at 0 and 21 hr in corn oil and killed at 24 hr. Control animals received the vehicle. Effects of 100 nM concentrations of the pesticides on DNA SSBs of cultured PC-12 cells at 24 hr posttreatment were determined. DNA-SSBs were determined by the alkaline elution method and are expressed as DNA elution constants. Each value is the mean _ SD of at least four to six animals in each group. Values with nonidentical superscripts are significantly different (p < 0.05).
538
SECTION V . N o n s p e c i f i c
Toxic Effects
pesticides in comparison to cyclophosphamide (positive control), demonstrating that DNA damage was dose related. The mean comet tail length revealed a clear dose-dependent increase. From 48 hr posttreatment, a gradual decrease in mean tail length was noted. By 96 hr posttreatment, the mean comet tail length had reached control levels, indicating repair of the damaged DNA (Rahman et al., 2002). OP-induced oxidative stress at the tubular level has been hypothesized to play a role in the pathogenesis of acute tubular necrosis. B idrin, an OP insecticide formulation with dicrotophos as the active ingredient, has been associated with renal tubular epithelial cell (LLC-PK1) toxicity. Poovala et al. (1999) assessed LDH release, H202 levels (~mol/mg protein/hr), and malondialdehyde formation (nmol/mg protein). Results showed that LDH significantly increased with concentration and time after exposure of the cells to 1000, 1250, 1500, 1750, and 2000 ppm of Bidrin for 6, 12, 24, and 48 hr. Antioxidants 2-methylaminochroman (2-MAC) and desferrioxamine reduced cell damage induced by 1250 ppm of Bidrin over a 24-hr incubation period in a concentration-related manner. The greatest reductions in the percentage of LDH were produced by 2 mM desferrioxamine and 2.5 p~M, 2-MAC both significantly lower than Bidrin alone. H20 2 levels were significantly elevated after exposure to 1250ppm of Bidrin. Significantly increased malondialdehyde formation compared to control was also found in Bidrin-exposed cells, indicating enhanced lipid peroxidation. Malondialdehyde generation was significantly suppressed by 2-MAC and desferrioxamine. These results demonstrate that the OP Bidrin can cause direct tubular cytotoxicity and implicate, at least in part, a role for ROS and accompanying lipid peroxidation in cytotoxicity (Poovala et al., 1999).
III. GENE EXPRESSION OF CHLORPYRIFOS AND FENTHION Living organisms respond at the cellular level to unfavorable conditions, such as heat or other stressful situations of different origins, by a rapid, vigorous, and transient acceleration in the rate of expression of a small number of specific heat shock/stress genes, resulting in the production of heat shock proteins (HSPs) (Harboe and Quayle, 1991; Pratt, 1993; Schlesinger et al., 1982). HSPs are believed to assist cells to adapt or survive by a rapid but transient reprogramming of cellular metabolic activity to protect cells from further oxidative and thermal stress in responsive tissues (Harboe and Quayle, 1991; Pratt, 1993). Potential mechanisms of protection from oxygen free radicals by HSPs include prevention of protein degradation; inhibition of membrane lipid peroxidation or calcium intrusion; maintenance of ATP levels; and induction of classical scavengers such as SOD or GSH, which also plays a role in the induction of HSPs (Freeman and Meredith, 1989). Based on the
study by Bagchi et al. (1996), the mechanism involved in the induction of oxidative stress by these xenobiotics is the expression of Hsp89oL and Hsp8913 genes in hepatic and brain tissues of rats. The comparative in vivo and in vitro effects of the OPs chlorpyrifos and fenthion were determined in cultured neuroactive PC-12 cells and in treated animals. "Other dissimilar" polyhalogenated cyclic hydrocarbons, such as endrin and chlordane, and chlorinated acetamide herbicides, such as alachlor, were also compared (Bagchi et al., 1996). Hsp89ot and Hsp8913 genes may be mechanistically involved in protecting tissues against oxidative stress induced by structurally diverse pesticides. Dramatically induced expression of Hsp89ot and overexpression of Hsp8913 in both hepatic and brain tissues were observed. Chlorpyrifos and fenthion also induced overexpression of Hsp89ot and Hsp8913 in cultured PC-12 cells. Concentration-dependent overexpression of Hsp89ot and Hsp8913 was also observed (Bagchi et al., 1996). OPs were administered E O. individually to female Sprague-Dawley rats in two 0.25 LDs0 doses at 0 and 21 hr. The animals were killed at 24 hr, and liver, brain, heart, and lung tissues were removed to examine induction of HSPs by Westem blot analysis and Northern blot analysis. In a separate series of experiments, cultured neuroactive PC-12 cells were treated for 24 hr with 50, 100, or 200 nM concentrations of these pesticides. Chlorpyrifos and fenthion induced Hsp89et and Hsp8913 in hepatic and brain tissues as well as in cultured PC-12 cells. Oxidative stress inducible proteins by OPs were detected in cultured neuroactive PC-12 cells as well as in liver, brain, heart, and lung by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using antibody to Hsp90. The expression of "Pesticide-induced" genes was further monitored by Northern blot analysis using cDNA probes for Hsp89oL (Bagchi et al., 1996). Chlorpyrifos and fenthion were assayed by SDS-PAGE and Western blot analysis and thus induced overexpression of 90- and 60-kDa proteins in hepatic, brain, and lung tissues (Figs. 4A-4C). Enhanced expression of the 90-kDa protein was also observed in cultured PC-12 cells treated with OPs (Fig. 4D). The 90-kDa protein was further recognized by Hsp90 polyclonal mouse antibody by Western blot analysis in all pesticide-treated hepatic tissues (Fig. 4A). Expression of Hsp90 was also observed in OP-treated brain tissues by Western blot analyses (Fig. 4A) (Bagchi et al., 1996). Expression of Hsp90 in OP-treated culture PC-12 cells was in agreement with the in vivo results in rats. Cultured neuroactive PC-12 cells were treated with 50, 100, or 200 nM concentrations of chlorpyrifos and fenthion for 24 hr. The 90-kDa protein was recognized by Hsp90 polyclonal mouse antibody by Westem blot analysis in both OP-treated cultured PC-12 cells. Concentration-dependent enhanced expression of Hsp90 was observed (Bagchi et al., 1996).
CHAPTER 37 9OPs, CMs, DNA Damage, and Gene Expression
539
FIG. 4. SDS-PAGE of the hepatic (A), brain (B), and lung (C) tissues from control and pesticidetreated rats and cell lysates (D) from control and pesticide-treated cultured PC-12 cells. 1, Control sample; 2, alachlor-treated sample; 3, endrintreated sample; 4, chlordane-treated sample; 5, chlorpyrifos-treated sample; and 6, fenthiontreated sample.
The quantitation of Hsp gene expression results in rats and cell cultures as obtained from counts of the radioactivity (32p counts/min) is shown in Tables 4 and 5, respectively. Table 4 demonstrates the radioactivity of Hsp89oL and Hsp8913 mRNA expression in brain and liver tissues. Chlorpyrifos and fenthion induced increases of 3.4- and 3.3-fold in brain Hsp89oL, respectively, whereas in liver this same protein increased by 3.3- and 2.7-fold, respectively. With respect to both pesticides in the brain, Hsp8913 increased 2.0- and 2.2-fold, respectively, whereas in the liver there were increases of 2.7- and 2.6-fold, respectively (Bagchi et al., 1996). Table 5 depicts the radioactivity associated with Hsp89oL and Hsp89~ mRNA expression in control and pesticidetreated cultured PC-12 cells. The cultured PC-12 cells were incubated with 50, 100, or 200 nM concentrations of the pesticides and the concentration-dependent effects of the pesticides were determined. At 50 nM concentration of chlorpyrifos and fenthion, the expression of Hsp89oL increased by approximately 3.6- and 4.1-fold, respectively, whereas the expression of Hsp8913 increased approximately
4.9' and 5.3-fold, respectively, compared to control values. The expression of Hsp89ot increased 5.1- and 5.2-fold following incubation of cultured PC-12 cells with 100 nM concentration of chlorpyrifos and fenthion, respectively, whereas at the same concentration the expression of Hsp8913 increased 6.1- and 6.8-fold, respectively, compared to control values. At 200 nM concentration of chlorpyrifos and fenthion, the expression of Hsp89oL increased by approximately 6.1- and 5.8-fold, respectively, whereas the expression of Hsp8913 increased by approximately 6.0- and 5.6-fold, respectively, compared to the control values. The production of the Hsp90 proteins in response to the pesticides may serve as a protective mechanism against further damage (Bagchi et al., 1996). The results indicate striking regional and cell type differences in the patterns of induction of the Hsp mRNAs by different classes of pesticides, suggesting that different organs and cell types respond differently to OPs. This study demonstrates that OPs can induce the expression of Hsp89oL and Hsp8913 genes in various target organs in rats as well as cultured PC-12, and the results support the
SECTION V . N o n s p e c i f i c Toxic Effects
540
TABLE 4.
Radioactivity (3Zp Counts/min) Associated with Hsp89c~ and Hsp8913 mRNA Expression in Brain and Liver Tissues of Rats a
Hsp89t~ Treatment
Brain
Control
Hsp8913 Liver
Brain
Liver
610 -+- 80 a
570 + 90 a
607 + 65 a
Chlorpyrifos
2072 + 175 d
1896 + 158 e
1216 -+- 69 e
1491 + 133 b,d
550 + 46 a
Fenthion
2006 + 197 d
1564 _+ 133 c
1313 ___71 e
1428 _+ 125 d
aFemale Sprague-Dawley rats were treated orally with 0.25 LDs0 doses of chlorpyrifos or fenthion in corn oil at 0 and 21 hr. Liver and brain tissues were obtained at 24 hr. RNAs were isolated and subjected to Northern blot analyses using 32p-labeled Hsp89oL and Hsp8913 probes. Values are expressed as mean counts/min _ SD of at least four experiments. Values with nonidentical superscripts in each column are significantly different (p < 0.05).
TABLE 5.
Radioactivity
(32p Counts/min)
Associated with H s p 8 9 a and Hsp8913 mRNA Expression in Control and Pesticide-Treated, Cultured PC-12 Cells a
Hsp89t~ Treatment
Hsp8913
50 nM
100 nM
200 nM
50 nM
100 nM
200 nM
Control Chlorpyrifos
637 __. 52 a 2287 _ 218 b,c
716 ___73 a 3626 _ 275 b
754 +__66 a 4573 +_ 422 b
596 +__34 a 2938 _ 232 e
634 -+- 39 a 3885 +__367 b,d
787 ___55 a 4744 _ 411 b
Fenthion
2642 _ 223 b
3711 _ 307 b
4388 __+414 b
3182 +__274 e
4312 _ 323 d
4418 _+ 409 b
aCultured PC-12 (1 • 10 7) cells were incubated individually with 50, 100, or 200 nM of chlorpyrifos or fenthion, and total RNA was isolated after 24 hr using the guanidium isothiocynate method. The RNAs were run on 1% agarose gels and transferred to nylon membranes, and Northern blot analyses were performed using 32p-labeled Hsp89c~ and Hsp8913 probes. Values are expressed as mean counts/min _+ SD of at least four experiments. Values with nonidentical superscripts in each column are significantly different (p < 0.05).
hypothesis that expression of Hsp89c~ and Hsp8913 genes m a y be involved in modulating the oxidative stress and toxicity induced by these pesticides (Bagchi et al., 1996). The effect of chlorpyrifos and its metabolite, chlorpyrifos-oxon, on multidrug resistance- 1 (MDR- 1) gene expression and efflux transporter function in Caco-2 cells was also studied (Agarwala et al., 2004). The effect of chlorpyrifos and chlorpyrifos-oxon on gene expression in Caco-2 cells was tested as a function of time using reversetranscriptase p o l y m e r a s e chain reaction (RT-PCR) and competitive P C R ( c o m p P C R ) techniques. The RT-PCR results depicted a m a x i m a l effect of chlorpyrifos exposure on M D R 1 expression at 8 hr, which decreased at 24 hr. Studies on chlorpyrifos-oxon displayed an initial increase in expression at 4 hr only. The c o m p P C R assays were conducted with the chlorpyrifos-treated group to quantify the changes in gene expression levels. The c o m p P C R data confirmed and quantified the results from the time course study using semiquantitative RT-PCR. In addition to the gene expression studies, changes in effiux transporter function were investigated using Caco-2 cells grown on s e m i p e r m e a b l e m e m b r a n e s in Transwell plates. The permeability of verapamil was determined in cells treated for 8 hr
with chlorpyrifos. Effiux ratios demonstrated that verapamil was effiuxed at a higher rate from the chlorpyrifos-treated cells compared to the control group, confirming the inductive action of chlorpyrifos on transporter function. These results suggest that chlorpyrifos has the potential to modulate the bioavailability of drugs via changes in expression and function of m e m b r a n e effiux transporters (Agarwala et al., 2004).
IV. I N V I T R O A N D I N V I V O P R O T E I N KINASE C ACTIVATION BY OPs Various pesticides and transition metals induce oxidative deterioration of biological m a c r o m o l e c u l e s in which protein kinase C (PKC) m a y mediate these effects. PKC is a family of isoenymes with distinct roles in normal and pathogenic activities within cells. PKC is involved in signaling pathways mediating the regulation of m a n y cell processes, including cell differentiation, cell survival, gene expression, secretion, cytoskeletal function, and c e l l - c e l l interactions (Lord and Pongracz, 1995). Thus, P K C is involved in a cascade of events associated with cell regulation that is
CHAPTER 37 9OPs, CMs, DNA Damage, and Gene Expression subject to both internal and external factors. A growing body of evidence indicates that free radicals and ROS may be involved in mediating signal transduction through interaction with PKC (Brawn et al., 1995; Gundimeda et al., 1993; Lander et al., 1995; Pronzato et al., 1993). Studies have demonstrated that PKC is rapidly activated in cells following oxidative exposure (Brawn et al., 1995). For example, modulation of PKC activity by oxidant tumor promoters and phorbol esters is well-known (Gundimeda et al., 1993; Lord and Pongracz, 1995; Prasad and Jones, 1992). Modulation of PKC by carbon tetrachloride has been shown to depend on the degree of oxidative unbalance provoked by various concentrations of this haloalkane (Brawn et al., 1995; Pronzato et al., 1993). In a related study, the comparative in vitro and in vivo abilities of OPs to modulate PKC activity were assessed in the brain and hepatic tissues of control and chlorpyrifosand fenthion-treated animals and in cultured neuroactive PC-12 cells (Bagchi et al., 1997). Following treatment of the animals with chlorpyrifos and fenthion, approximately 2.0- and 1.7-fold increases in PKC activity were observed in the hepatic tissues, respectively, whereas under the same conditions, approximately 3.5- and 3.3-fold increases were observed in the brain tissues, respectively, compared to control tissues (Table 6) (Bagchi et al., 1997). Concentration-dependent effects of chlorpyrifos and fenthion on cultured neuroactive PC-12 cells were studied (Table 7). Increases in PKC activity were induced by chlorpyrifos and fenthion. Significant changes in PKC activity were observed only in the cytosol fractions. In these in vitro experiments, maximum activation of PKC was also observed primarily with 100 nM concentrations of the pesticides. Following treatment of cultured PC-12 cells with 5 0 n M concentrations of chlorpyrifos and fenthion, approximately 2.9- and 2.4-fold increases in PKC activity were observed, respectively. Under the same conditions, approximately 4.3- and 4.2-fold increases in
TABLE 6. Comparative Protein Kinase C Activity in Female Sprague-Dawley Rats following Treatment with OP Pesticides a Protein kinase C activity/ixg of protein/min Treatment
Liver
Brain
Control
27.2 __ 2.4a
10.7 __ 1.1a
Chlorpyrifos
53.8 + 7.8 b
37.4 +__ 4.2 d
Fenthion
46.2 ___ 6.1 b,c
35.4 ___ 2.9 d
aFemale Sprague-Dawley rats were treated orally with two 0.25 LDs0 doses at 0 and 21 hr with pesticides and killed at the 24-hr time point. Protein kinase C activity was monitored using a kit from Upstate Biotechnology. Values with nonidentical superscripts are significantly different (p < 0.05).
541
TABLE 7. Comparative Protein Kinase C Activity in Cultured Neuroactive PC-12 Cells following Treatment with OP Pesticides a Protein kinase C activity/l~g of protein/min Treatment
Control Chlorpyrifos Fenthion
50 nM
100 nM
200 nM
0.78 ___0.08a 2.26 ___ 0.20 b 1.88 _+ 0.14c
0.77 + 0.10 a 3.31 + 0.38c 3.26 _+ 0.43c
0.95 + 0.13a 3.22 _+ 0.40c 3.12 _+ 0.35c
aCultured cells were treated individually with 50, 100, or 200 nM concentrations of pesticides and incubated at 37 ~ The protein kinase C inhibitor H-7 was added at 20 IxM. Protein kinase C activity was monitored using a kit from Upstate Biotechnology at the 24-hr time point. Values with nonidentical superscripts are significantly different (p < 0.05).
PKC activity were observed, respectively, following treatment with 100nM concentrations of these pesticides compared to control cells. No further increases in PKC activity were observed following treatment with 200 nM concentrations of these pesticides (Bagchi et al., 1997). The results of these experiments clearly indicate PKC activation, following exposure to these OPs, which are also known to enhance the production of oxygen free radicals and induce oxidative stress (Bagchi et al., 1995, 1997). These results demonstrate that PKC activation occurs in hepatic and brain tissues of rats as well as in cultured cells following treatment with OPs, which are known to induce oxidative stress. Thus, OP pesticides can modulate a vital component in the cell signaling pathway.
V. D E V E L O P M E N T A L N E U R O T O X I C I T Y OF CHLORPYRIFOS IN PC-12 AND C6 CELLS The widespread use of chlorpyrifos has raised concerns about its potential to cause fetal or neonatal neurobehavioral damage, even at doses that do not evoke acute toxicity (Aschner et al., 1999). Chlorpyrifos has been shown to inhibit replication of brain cells, to elicit alterations in neurotrophic signaling governing cell differentiation and apoptosis, as well as to evoke oxidative stress. Studies have demonstrated that glial-type cells have been targeted by chlorpyrifos through the same multiple mechanisms that have been demonstrated for the effects of chlorpyrifos on brain development in vivo (Campbell et al., 1997; Dam et al., 1998; Garcia et al., 2001; Whitney et al., 1995). Postneurogenesis, glial development continues, and given that chlorpyrifos targets events in both glial cell replication and the later stages of differentiation, the vulnerable period for developmental neurotoxicity of chlorpyrifos is likely to
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extend well into childhood or even early adolescence. Early in vitro studies also demonstrated the methods causing neurotoxicity by chlorpyrifos, by means of assessment of cultures o f immature brain tissue (Cosenza and Bidanet, 1995; Roy et al., 1998; Monnet-Tschudi et al., 2000) or transformed neural cell lines, such as neuronotypic PC-12 cell lines. PC-12 cell lines are less receptive to neurotoxins and have been shown to effectively establish cell replication as a major target because they maintain a fixed pattern of mitosis until differentiation is triggered by addition of trophic factors and deletion of serum (Ehrich et al., 1997; Li and Casida, 1998; Song et al., 1998; Das and Barone, 1999; Crumpton et al., 2000; Garcia et al., 2001). In two in vitro models, PC-12 cells and gliotypic C6 cells were compared to demonstrate the antimitotic effects of chlorpyrifos (Campbell et al., 1997; Dam et al., 1998; Garcia et al., 2001; Monnet-Tschudi et al., 2000; Qiao et al., 2001). In the first set of experiments, PC-12 and C6 cells were exposed to chlorpyrifos or chlorpyrifos metabolites for 1 hr in the absence of serum to obviate any potential protective effect of serum proteins selecting a chlorpyrifos concentration (30 IxM) that was previously found to cause robust but submaximal inhibition of DNA synthesis in vitro (Braeckman et al., 1983; Das and Barone, 1999; Garcia et al., 2001; Pond et al., 1996; Song et al., 1998). Equimolar concentrations of chlorpyrifos-oxon also produced significant inhibition of DNA synthesis, again with C6 cells showing a greater effect than PC-12 cells; however, chlorpyrifos-oxon was also significantly less effective than chlorpyrifos (Monnet-Tschudi et al., 2000). The effects of chlorpyrifos were also compared with other cholinesterase inhibitors; using equivalent concentrations (30 IxM) of each compound demonstrated that both diazinon (an OP) and physostigmine (a CM) caused significant inhibition of DNA synthesis in C6 cells. In PC-12 cells, diazinon caused a significant decrement, although it was smaller than the effect of chlorpyrifos while physostigmine was ineffective. These results are consistent with those of previous studies in which it was demonstrated that chlorpyrifos exerts antimitotic actions on developing neural cells independently of cholinesterase inhibition (Das and Barone, 1999; Pope, 1999; Slotkin, 1999). It was also observed that chlorpyrifos was more effective than chlorpyrifos-oxon, despite the fact that the latter is a far more potent cholinesterase inhibitor (Slotkin, 1999). The effects of chlorpyrifos and its major metabolites in two in vitro models, PC-12 cells and gliotypic C6 cells, showed that chlorpyrifos inhibited DNA synthesis in both cell lines but had a greater effect on gliotypic cells. Chlorpyrifos-oxon, the active metabolite that inhibits cholinesterase, also decreased DNA synthesis in PC-12 and C6 cells, with a preferential effect on the latter. However, diazinon also inhibited DNA synthesis with predilection toward C6 cells, and it was less effective than chlorpyrifos. It was also found that the addition of sera protected the
cells from the adverse effects of chlorpyrifos and that the effect could be reproduced by addition of albumin. These results indicate that OPs such as chlorpyrifos and diazinon have immediate and direct effects on neural cell replication, preferentially for gliotypic cells. In light of the protective effect of serum proteins, the fact that the fetus and newborn possess lower concentrations of these proteins suggests that greater neurotoxic effects on them may occur at blood levels of chlorpyrifos that are nontoxic to adults (Slotkin, 1999; Song et al., 1997, 1998). Adverse effects of chlorpyrifos on glial cell replication are of critical importance in defining the sensitive period for effects on central nervous system development. Glia cells provide nutritional, structural, and homeostatic support essential to architectural modeling of the brain (Guerri and Renau-Piqueras, 1997; Tacconi, 1998; Aschner et al., 1999; Morita et al., 1999; Barone et al., 2000), and because glial development continues well into the postnatal period, glial targeting implies a prolonged vulnerability, extending into childhood. Chlorpyrifos administration in vivo inhibits DNA synthesis and causes loss of brain cells during gliogenesis (Campbell et al., 1997; Dam et al., 1998; Whitney et al., 1995), with maximal effects on neural function appearing during peaks of glial development (Campbell et al., 1997; Dam et al., 1999; Monnet-Tschudi et al., 2000; Song et al., 1997; Slotkin, 1999). In aggregating brain cell cultures, chlorpyrifos affects glial markers, again unrelated to cholinesterase inhibition (Monnet-Tschudi et al., 2000). The previous results thus confirm conclusively that chlorpyrifos, rather than its active metabolite chlorpyrifos-oxon, is the primary agent responsible for these effects.
A. Carcinogenic and Genotoxic Potential of OPs OP pesticides have been associated with pathology and chromosomal damage in humans as well as epidemiologic links with cancer. However, despite overwhelming experimental evidence of OP toxicity, studies have also demonstrated modifying effects of OP pesticide mixtures on tumorigenesis with medium-term carcinogenesis protocols for rapid detection of carcinogenic agents using male F344 rats. In an 8-week study, 19 OP pesticides and 1 organochlorine were added to the diet, each at acceptable daily intake (ADI) levels (Ito et al., 1996). Results indicated that exposure to the pesticides did not enhance rat liver preneoplastic lesion development initiated by diethylnitrosamine. In contrast, a mixture of these 20 pesticides at 100 times the ADI significantly increased the number and area of liver lesions. In a parallel experiment using a multiorgan carcinogenicity protocol of 28 weeks, mixtures of 40 high-production pesticides or 20 suspected carcinogenic pesticides were added to the diet at their respective ADI levels. Results revealed that the pesticides did not modulate carcinogenesis in any organ treated by 5 known potent
CH A PTE R 3 7 9OPs, CMs, DNA Damage, and Gene Expression carcinogens in combination. These results thus provide direct support for the safety factor approach using ADI values for the quantitative risk evaluation of pesticides (Ito et al., 1996). Several screening tests for low-level occupational exposure to OPs are of doubtful sensitivity and require further investigation. Blood samples were studied from 10 farmers before and after occupational exposure to OP pesticides and five unexposed controls. The standard cholinesterase test was insensitive to the exposure. However, a significant increase in Howell-Jolly bodies within erythrocytes was observed. Cytogenetic studies on routine and aphidicolininduced blood cultures revealed that following OP exposure the total number of gaps and breaks on human chromosomes was significantly increased, and they concluded that Howell-Jolly body and fragile site analyses were sensitive indicators of nuclear damage resulting from low-level occupational exposure to an OP. Such nuclear damage could be implicated in carcinogenesis. The development of bladder cancer is one such example (Webster et al., 2002). As a result of the controversy surrounding pesticide use and animal and human health concems, many municipalities in Canada have restricted the use of pesticides for cosmetic purposes. In some cases, pesticide use on golf courses is also being phased out at the municipal level. One of the dominant health effects of concem in relation to pesticide exposure is the occurrence of cancer. With more than 1600 golf courses in Canada and between 400 and 600 new courses developed each year in Canada and the United States, there appears to be increasing potential for unintentional human and animal exposure to turf pesticides. The debate regarding the extent of pesticide exposure and the onset of cancer has led to controversial Canadian municipal bylaws regulating pesticide use, and a biomonitoring study has shown genotoxicity in a rodent species living on golf courses (Knopper and Lean, 2004). Diazinon, the active ingredient in products such as Diazinon, Basudin, and Knox Out, is a nonsystemic OP insecticide. Diazinon has a half-life in soil of 2-4 weeks (Pesticide Information Profiles, 1996). In water, diazinon's half-life is related to pH, with faster breakdown related to increasing pH, whereas in neutral conditions, half-life can be as long as 6 months (Pesticide Information Profiles, 1996). Although use of diazinon on golf courses and sod farms in the United States was banned in 1988, primarily due to its high toxicity and lethality to birds (Pesticide Information Profiles, 1996), diazinon is still widely used in Canada, although several bird deaths have been reported (Frank et al., 1991). According to the EPA (2000), diazinon has been shown to be nonmutagenic in both in vivo and in vitro mutagenicity assays. Bianchi-Santamaria et al. (1997) demonstrated that diazinon produced a statistically significant increase in micronucleated human lymphocytes in vivo, but not in a dose-dependent manner. In CD-1 mice, no significant
543
change in micronucleated erythrocytes were observed when males and females were given either 60 or 120 mg/kg diazinon by gavage. Also, diazinon did not induce sister chromatid exchange in vitro in Chinese hamster V79 cells at 0.05-0.4 txg/ml, nor did it change the mitotic and second mitotic index more than in controls. Based on the available negative evidence, the EPA classifies diazinon as a group E not likely human carcinogen (EPA, 2000; Kuroda et al., 1992). Experimental data have also linked OP exposure during the gestation period to adverse neurocognitive sequalae in offspring (Eskenazi et al., 1999; Landrigan et al., 1999). Due to their lipophilic characteristic, OPs readily cross the placenta (Richardson, 1995). Research on biologic markers, important for understanding the role of environmental toxicants in fetal development (Perera et al., 1998; Whyatt and Perera, 1995; Whyatt et al., 1998), and the effects of OPs on prenatal exposure has been limited by the lack of biologic markers reflecting cumulative exposures. Furthermore, biologic markers such as urine and blood measurements provide only short-term exposure estimates. Validation research by Whyatt et al. (2001) determined that measurements of OPs in postpartum meconium yielded a longer term dosimeter of prenatal exposure. Background levels, detection limits, and the stability of six OP metabolites in meconium (diethyl phosphate, diethyl thiophosphate, diethyl dithiophosphate, dimethyl phosphate, dimethyl thiophosphate, and dimethyl dithiophosphate) were determined. Results indicate that OP metabolites in meconium show promise as biomarkers of prenatal exposure (Whyatt et al., 1998; Whyatt and Perera, 1995; Whyatt and Barr, 2001).
B. Carcinogenesis Induced by OPs in a Rat Mammary Tumor Model OPs have many structural similarities with naturally occurring compounds in the body, and their primary target of action is inhibition of ACHE. Eserine, parathion, and malathion are cholinesterase inhibitors (Klaassen, 1990; Taylor, 1990). OPs such as parathion and malathion are extensively used to control a wide range of pests of field crops, fruits, and vegetables. Malathion is also present in lotions and shampoos marketed for the treatment of head lice and mites in humans. Dermal exposure to these two pesticides has been shown to result in a small amount of systemic absorption (Taylor, 1990). The CM compound physostigmine (eserine) is a miotic drug and also used for atony of the gastrointestinal tract, but it is very toxic if inhaled or swallowed (Klaassen, 1990; Taylor, 1990). Eserine, parathion, and malathion are administered dermally, orally, or via inhalation (Klaassen, 1990; Taylor, 1990). In the liver, parathion and malathion are activated by enzymatic processes producing paraoxon and malaoxon, respectively. Eserine, parathion, and malathion are AChE inhibitors that interfere with the hydrolysis of body choline
544
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N o n s p e c i f i c Toxic Effects
esters, including ACh at cholinergic synapses (Silman and Futerman, 1987; Taylor, 1990). As a result, there is an increase in ACh, which in turn can stimulate cholinergic receptors, producing both nicotinic and muscarinic effects in the organism such as muscle contractions and glandular secretions (Taylor, 1990). Due to the high level of cell proliferation and differentiation that occurs during mammary gland development, Cabello et al. (2001) determined the susceptibility of rat mammary glands to different carcinogenic actions. Thirty-five 16-day-old virgin female Sprague-Dawley rats received injections twice a day for 5 days subcutaneously or intraperitoneally in the inguinal region of the body. Animals were injected with one of the following substances: saline solution, eserine, parathion, malathion, atropine, a combination of eserine and atropine, or a combination of malathion and atropine. The results showed that eserine, parathion, and malathion increased cell proliferation of terminal end buds of the 44-day-old mammary gland of rats, followed by formation of 8.6, 14.3, and 24.3% of mammary carcinomas, respectively, after approximately 28 months. At the same time, AChE activity decreased in the serum of these animals from 9.78 _+ 0.78 U/ml in the control animals to 3.05 _+ 0.06, 2.57 _+ 0.15, and 3.88 _+ 0.44 U/ml in the eserine-, parathion-, and malathion-treated groups, respectively. These results indicate that the malathion- and parathion-induced tumor formation changes in the epithelium of mammary gland influence the process of carcinogenesis, and such changes appear to occur due to increasing cholinergic stimulation.
VI. C A R C I N O G E N E S I S I N D U C E D BY C M s Experimental evidence indicates that CMs alter gene expression and downregulation of the antioxidant defense system, cause adverse effects on the dopamine system, and alter neurotoxicity and carcinogenesis (Barlow et al., 2005; Sandoz et al., 1998). In a study conducted by Pound and Lawson (1976), CMs were shown to induce carcinogenesis in mice. Cheng and Conner (1982) showed that six different CMs had a striking similarity in relative potencies for sister chromatid exchange induction and known tumorigenic potencies. The mutagenic potential of propoxur was demonstrated to significantly induce the formation of micronuclei in bone marrow cells of mice at different dose levels at 24 and 48 hr by the intraperitoneal and oral routes (Agrawal and Mehrotra, 1997). In a study by Pound and Lawson (1976), the tumorinitiating potency of three simple alkyl CMs and mono-Nsubstituted ethyl CMs was examined in Hall strain mice. The binding of 14C-labeled CMs to DNA was measured in Crackenbush mice. Ethyl CM was the most potent carcinogen for the epidermis, liver, and lung, followed by its N-alkyl derivatives. Methyl CM was without effect, but n-propyl and n-butyl were possible carcinogens. The ethyl
esters bound to a greater extent to DNA in liver and skin than did the methyl, n-propyl, and n-butyl esters, and only this binding persisted. A preliminary application of croton oil increased the yield of skin tumors but not liver or lung tumors. It also increased the binding of the alkyl CMs to DNA in skin, with the increase being greatest for ethyl CM. Ethyl CM binding persisted longer in ]4C-labeled CM-treated mice than in croton oil-treated mice. In a study by Cheng and Conner (1982), in vivo sister chromatid exchange induction by vinyl and allyl CMs was examined in alveolar macrophage, bone marrow, and regenerating liver cells of C57BL/6J • DBA/2J F1 mice. Allyl CM was effective in producing increases in sister chromatid exchange frequencies (relative to baseline sister chromatid exchange) over a dose range of 220 ixmol/kg (approximately 2 times baseline) to 2.2 mmol/kg (3 times baseline). In general, alveolar macrophage and regenerating liver cells had higher responses, although not significantly, than did bone marrow. Vinyl CM produced significant increases in sister chromatid exchange frequencies over a dose range of 10 txmol/kg (2 times baseline) to 75 ixmol/kg (8-10 times baseline). At the highest dose, sister chromatid exchange frequencies in extrahepatic tissues of hepatectomized mice were significantly higher than those in intact mice, and in hepatectomized mice, alveolar macrophage and regenerating liver cell responses were greater than bone marrow responses. Vinyl CM was approximately 30 times as potent a sister chromatid exchange inducer as reported previously for ethyl CM (Cheng and Conner, 1982). The mutagenic potential of propoxur, a widely used dithiocarbamate pesticide, has also been studied. Since the restriction of the use of DDT in most industrialized countries, propoxur has been proposed as a replacement for DDT (World Health Organization, 1989), resulting in a major increase in the use of propoxur during the past 10 years. However, studies indicate that nitrosopropoxur, the nitroso derivative of propoxur, is highly mutagenic in Salmonella typhimurium (Seiler, 1977), Escherichia coli (Lijinsky and Elespuru, 1976), Saccharomyces cerevisiae (Siebert and Eisenbrand, 1974), and human lymphocytes in vitro. Propoxur has an effect on induced dominant lethal mutations in mice (Tyrkiel, 1977) and produces sister chromatid exchange and micronuclei formation in human lymphocytes (Gonzalez et al., 1990). A study by Agrawal and Mehrotra (1997) further demonstrated the mutagenic potential of propoxur to significantly induce the formation of micronuclei in bone marrow cells of mice at different dose levels at 24 and 48 hr by the intraperitoneal and oral routes. A maximum tolerated dose 25 mg/kg, and 12.5 and 6.25 mg/kg doses were administered to male Swiss albino mice. Fifty and 25 mg/kg of the maximum tolerated doses were found ineffective in inducing micronuclei formation after 24 and 48 hr. However, the polychromatic erythrocyte-to-normochromatic erythrocyte ratio was inhibited significantly at all dose levels at both time periods. Oral administration of propoxur at different dose levels was also
CHAPTER 3 7 9OPs, CMs, DNA Damage, and Gene Expression shown to induce micronuclei formation. A single application of 50 and 25 mg/kg dose levels of propoxur, which are the maximum tolerated dose and 50% of maximum tolerated dose, respectively, also significantly induced micronuclei formation after 24 and 48 hr in bone marrow cells of Swiss mice compared to the solvent control group, whereas a 12.5 mg/kg dose of propoxur was ineffective in inducing micronuclei formation. Single application of indole-3carbinol, a glucobrassicin derivative present in cruciferous vegetables, significantly inhibited propoxur-induced micronuclei formation when it was given at a dose level of 500 mg/kg body weight 48 hr before the single application of propoxur. Therefore, it seems that propoxur is mutagenic in these test systems and indole-3-carbinol significantly inhibits the mutagenicity of propoxur (Agrawal and Mehrotra, 1997).
VII. CM AND GENE EXPRESSION The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a variety of hydrophobic natural synthetic chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Induction of CYP1A1 is one such response known to be regulated by the AhR complex. Although carbaryl, a CM insecticide, can induce AhR-dependent expression of CYP1A1, it is not an AhR ligand (Ledirac et al., 1997). Denison et al. (1998) examined the ability of carbaryl to stimulate the AhR signaling pathway. Not only was dioxin responsive but also luciferase gene expression was induced by carbaryl in stably transfected mouse, rat, guinea pig, and human cells. Gel retardation analysis revealed that carbaryl stimulated AhR transformation and DNA binding in vitro and in cells in culture. Carbaryl was also suggested as an inducing agent from dose-response experiments demonstrating that carbaryl was 300,000-fold less potent than the prototype inducer, TCDD, in both inducing luciferase gene expression and stimulating AhR transformation and DNA binding in vitro. The identification of carbaryl as an AhR ligand was demonstrated by its ability to completely inhibit [3H]TCDD to the guinea pighepatic cytosolic AhR. Denison et al. (1998) confirmed that carbaryl is both a weak AhR ligand and an inducer of AhR-dependent gene expression.
VIII. CONCLUSIONS OPs can produce a'variety of toxicological effects: These compounds are inducers of hepatic drug-metabolizing enzymes and thereby act as potent competitive and stereospecific inhibitors of ligand binding to specific brain receptors. A significant number of studies demonstrate that contact with OPs, such as chlorpyrifos and
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fenthion, can induce oxidative stress resulting in tissue damage, including lipid peroxidation, nuclear DNA damage, PKC activation and altered gene expression, as well as sensory neuropathy, central nervous system dysfunction, and teratogenesis. OPs such as diazinon have been demonstrated to cause immediate and direct inhibitory actions on DNA synthesis, and hence on neural cell replication, with preferential targeting of gliotypic cells. Thus, OPs may cause human toxicity by several mechanisms as well as through the cell signaling pathway. CMs have also been shown to produce similar genetic effects. Much experimental evidence demonstrates that CMs induce oxidative stress, have adverse effects on dopamine and neurotoxicity, alter gene expression, downregulate the antioxidant defense system, and induce carcinogenesis.
References Agarwala, S., Chen, W., and Cook, T. J. (2004). Effect of chlorpyrifos on effiux transporter gene expression and function in Caco-2 cells. Toxicol. in Vitro 18, 403-409. Agrawal, R. C., and Mehrotra N. (1997). Assessment of mutagenic potential of propoxur and its modulation by indole-3carbinol. Food Chem. Toxicol. 35, 1081-1084. Aschner, M., Allen, J. W., Kimelberg, H. K., LoPachin, R. M., and Streit, W. J. (1999). Glial cells in neurotoxicity development. Annu. Rev. Pharmacol. Toxicol. 39, 151-173. Bagchi, D., Bagchi, M., Hassoun, E. A., and Stohs, S. J. (1995). In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides. Toxicology 104, 129-140. Bagchi, D., Bhattacharya, G., and Stohs, S. J. (1996). In vitro and in vivo induction of heat shock (stress) protein (Hsp) gene expression by selected pesticides. Toxicology 112, 57-68. Bagchi, D., Bagchi, M., Tang, L., and Stohs, S. J. (1997). Comparative in vitro and in vivo protein kinase C activation by selected pesticides and transition metal salts. Toxicol. Lett. 91, 31-37. Barlow, B. K., Lee, D. W., Cory-Slechta, D. A., and Opanashuk, L. A. (2005). Modulation of antioxidant defense systems by the environmental pesticide maneb in dopaminergic cells. NeuroToxicology 26, 63-75. Barone, S., Das, K. P., Lassiter, T. L., and White, L. D. (2000). Vulnerable processes of nervous system development: A review of markers and methods. NeuroToxicology 21, 15-36. Barron, T. (1991). Discussion of the Carcinogenicity of Dichlorovos. Pesticides Control Service, Department of Agriculture, Castleknock, Dublin, Ireland. Bianchi-Santamaria, A., Gobbi, M., Cembran, M., and Arnaboldi, A. (1997). Human lymphocyte micronucleus gen0toxicity test With mixtures of phytochemicals in environmental concentrations. Mutat. Res. 388, 27-32. Botham, E A. (1990). Are pesticides immunotoxic. Adverse Drug React. Acute Poisoning Rev. 9, 91-101. Braeckman, R. A., Audenaert, E, Willems, J. L., Belpaire, F. M., and Bogaert, M. G. (1983). Toxicokinetics of methyl parathion and parathion in the dog after intravenous and oral administration. Arch. Toxicol. 54, 71-82.
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SECTION V .
N o n s p e c i f i c Toxic Effects
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Freeman, M. L., and Meredith, M. J. (1989). Glutathione conjugation and induction of a 32,000 dalton stress protein. Biochem. Pharmacol. 38, 299-304. Garcia, S. J., Seidler, E J., Crumpton, T. L., and Slotkin, T. A. (2001). Does the developmental neurotoxicity of chlorpyrifos involve glial targets? Macromolecule synthesis, adenylyl cyclase signaling, nuclear transcription factors, and formation of reactive oxygen in C6 glioma cells. Brain Res. 891, 54-68. Gonzalez, C. M., Loria, D., and Matos, E (1990). Genotoxicity of the pesticide propoxur and its nitroso derivative, no-propoxur on human lymphocyte in vitro. Mutat. Res. 232, 45-48. Guerri, C., and Renau-Piqueras, J. (1997). Alcohol, astroglia, and brain development. Mol. Neurobiol. lg, 65-81. Gundimeda, U., Hara, S. K., Anderson, W. B., and Gopalakrishna, R. (1993). Retinoids inhibit the oxidative modification of protein kinase C induced by oxidant tumor promoters. Arch. Biochem. Biophys. 300, 527-530. Harboe, M., and Quayle, A. J. (1991). Heat shock proteins: Friend and foe? Clin. Exp. Immunol. 86, 2-5. Ito, N., Hagiwara, A., Tamano, S., Futacuchi, M., Imaida, K., and Shirai, T. (1996). Effects of pesticide mixtures at the acceptable daily intake levels on rat carcinogenesis. Food Chem. Toxicol. 34, 1091-1096. Julka, D., Pal, R., and Gill, K. D. (1992). Neurotoxicity of dichlorvos: Effect of antioxidant defense system in rat central nervous system. Exp. Mol. Pathol. 56, 144-152. Kaplan, J. G., Kessler, J., Rosenberg, N., Pack, D., and Schaumburg, H. H. (1993). Sensory neuropathy associated with Dursban (chlorpyrifos) exposure. Neurology 43, 2193-2196. Klaassen, C. (1990). Nonmetallic environmental toxicants: Air pollutants, solvents and vapors, and pesticides. In The Pharmacological Basis of Therapeutics (A. Goodman Gilman, T. W. Rall, A. S. Nies, and E Taylor, Eds.), pp. 1615-1635. Pergamon, New York. Knopper, L. D., and Lean, D. R. S. (2004). Carcinogenic and genotoxic potential of turf pesticides commonly used on golf courses. J. Toxicol. Environ. Health B 7, 267-279. Kuroda, K., Yamaguchi, Y., and Endo, G. (1992). Mitotic toxicity, sister chromatid exchange and rec assay of pesticides. Arch. Environ. Contam. Toxicol. 23, 13-18. Lander, H. M., Ogiste, J. S., Teng, K. K., and Novogrodsky, A. (1995). p2 lras as a common signaling target of reactive free radicals and cellular redox stress. J. Biol. Chem. 270, 21195-21198. Landrigan, P. J., Claudio, L., Markowitz, S. B., Berkowitz, G. S., Brenner, B. L., Romero, H., Wetmur, J. G., Matte, T. D., Gore, A. C., Godbold, J. H., and Wolff, M. S. (1999). Pesticides and inner-city children: Exposures, risks, and prevention. Environ. Health Perspect. 107, 431-437. Largilliere, C., and Melancon, S. B. (1988). Free malondialdehyde determination in human plasma by high performance liquid chromatography. Anal. Biochem. 170, 123-126. Ledirac, N., Delescluse, C., de Sousa, G., Pralavorio, M., Lesca, E, Amichot, M., Berge, J. B., and Rahmani, R. (1997). Carbaryl induces CYP1A1 gene expression in HepG2 and HaCaT cells but is not a ligand of the human hepatic Ah receptor. Toxicol. Appl. Pharmacol. 144, 177-182. Li, W. W., and Casida, J. E. (1998). Organophosphorus neuropathy target esterase inhibitors selectively block outgrowth of neurite-like and cell processes in cultured cells. Toxicol. Lett. 98, 139-146.
CHAPTER 37 Lijinsky, W., and Elespuru, R. K. (1976). Mutagenicity and carcinogenicity of N-nitroso derivatives of carbamate insecticides. In Environmental-N-Nitroso Compound Analysis and Formation (E. A. Walker, P. Bogowsky, and L. Griciute, Eds.), IARC scientific publication No.14, pp. 425-428. International Agency for Research on Cancer, Lyon, France. Lord, J. M., and Pongracz, J. (1995). Protein kinase C: A family of isoenzymes with distinct roles in pathogenesis. Clin. Mol. Pathol. 48, M57-M64. Monnet-Tschudi, E, Zurich, M. G., Schilter, B., Costa, L. G., and Honegger, P. (2000). Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures. Toxicol. Appl. Pharmacol. 165, 175-183. Morita, K., Ishimura, K., Tsuruo, Y., and Wong, D. L. (1999). Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors. Brain Res. 816, 309-316. Murphy, S. D. (1986). Toxic effects of pesticides. In Toxicology: The Basic Science of Poisons (C. D. Klassen, M. V. Amdur, and J. Doull, Eds.), 3rd ed., pp. 519-581. Macmillan, New York. Perera, E P., Whyatt, R. M., Jedrychowski, W., Rauh, V., Manchester, D., Santella, R. M., and Ottman, R. (1998). Recent developments in molecular epidemiology: A study of the effects of environmental polycyclic aromatic hydrocarbons on birth outcomes in Poland. Am. J. Epidemiol. 147, 309-314. Pesticide Information Profiles (1996). Diazinon. http://ace.orst.edu. Pond, A. L., Coyne, C. P., Chambers, H. W., and Chambers, J. E. (1996). Identification and isolation of two rat serum proteins with A-esterase activity toward paraoxon and chlorpyrifosoxon. Biochem. Pharmacol. 52, 363-369. Poovala, V. S., Huang, H., and Salahudeen, A. K. (1999). Role of reactive oxygen metabolites in organophosphate-bidrin-induced renal tubular cytotoxicity. J. Am. Soc. Nephrol. 10, 1746-1752. Pope, C. N. (1999). Organophosphorus pesticides: Do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health 2, 161-181. Pound, A. W., and Lawson, T. A. (1976). Carcinogenesis by carbamic acid esters and their binding to DNA. Cancer Res. 36, 1101-1107. Prasad, M. R., and Jones, R. M. (1992). Enhanced membrane protein kinase C activity in myocardial ischemia. Basic Res. Cardiol. 87, 19-26. Pratt, W. B. (1993). The role of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J. Biol. Chem. 268, 21455-21458. Pronzato, M. A., Domenicotti, C., Rosso, E., Bellocchio, A., Patrone, M., Marinari, U. M., Melloni, E., and Poli, G. (1993). Modulation of rat liver protein kinase C during "in vivo" CC14induced oxidative stress. Biochem. Biophys. Res. Commun. 194, 635-641. Qiao, D., Seidler, F. J., and Slotkin, T. A. (2001). Developmental neurotoxicity of chlorpyrifos modeled in vitro: Comparative effects of metabolites and other cholinesterase inhibitors on DNA synthesis in PC12 and C6 cells. Environ. Health Perspect. 109, 909-913. Rahman, M. F., Mahboob, M., Danadevi, K., Saleha Banu, B., and Grover, R (2002). Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes. Murat. Res. 516, 139-147.
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Richardson, R. J. (1995). Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: A critical review of the literature. J. Toxicol. Environ. Health 44, 135-165. Roy, T. S., Andrews, J. E., Seidler, F. J., and Slotkin, T. A. (1998). Chlorpyrifos elicits mitotic abnormalities and apoptosis in neuroepithelium of cultured rat embryos. Teratology 58, 62-68. Sandoz, C., Lesca, P., Carpy, A., Laguionie, M., and Narbonne, J. (1998). Effects of carbaryl and naphthalene on rat hepatic CYP1A1/2: Potential binding to Ah receptor and 4S benzo(a)pyrene-binding protein. Int. J. Mol. Med. 2, 615-623. Schlesinger, M. J., Aliperti, G., and Kelley, P. M. (1982). The response of cells to heat shock. Trends Biochem. Sci. 7, 222-225. Seiler, J. P. (1977). Nitrosation in vitro and in vivo by sodium nitrite, and mutagenicity of nitrogenous pesticides. Mutat. Res. 48, 225-236. Siebert, D., and Eisenbrand, G. (1974). Induction of mitotic gene conversion in Saccharomyces cerevisiae by N-nitrosated pesticides. Murat. Res. 22, 121-126. Silman, I., and Futerman, A. (1987). Modes of attachment of acetylcholinesterase to the surface membrane. Eur. J. Biochem. 170, 11-20. Slotkin, T. A. (1999). Developmental cholinotoxicants: Nicotine and chlorpyrifos. Environ. Health Perspect. 107, 71-80. Song, X., Seidler, F. J., Saleh, J. L., Zhang, J., Padilla, S., and Slotkin, T. A. (1997). Cellular mechanisms for developmental toxicity of chlorpyrifos: Targeting the adenylyl cyclase signaling cascade. Toxicol. Appl. Pharmacol. 145, 158-174. Song, X., Violin, J. D., Seidler, F. J., and Slotkin, T. A. (1998). Modeling the developmental neurotoxicity of chlorpyrifos in vitro: Macromolecule synthesis in PC12 cells. Toxicol. Appl. Pharmacol. 151, 182-191. Tacconi, M. T. (1998). Neuronal death: Is there a role for astrocytes? Neurochem. Res. 23, 759-765. Taylor, P. (1990). Anticholinesterase agents. In The Pharmacological Basis of Therapeutics (A. Goodman Gilman, T. W. Rail, A. S. Nies, and P. Taylor, Eds.), pp. 131-147. Pergamon, New York. Tyrkiel, E. (1977). Mutagenic action of O-isopropoxyphenyl-Ncarbamate (propoksur) on mouse gamete. Rocz. Panstw. Zakl. Hig. 28, 601-613. U.S. Environmental Protection Agency (2000). Diazinon. Revised HED preliminary human health risk assessment for the reregistration eligibility decision (RED) D262343. PC Code 057801, List A Case No. 0238. U.S. Environmental Protection Agency, Washington, DC. Vidair, C. A. (2004). Age dependence of organophosphate and carbamate neurotoxicity in the postnatal rat: Extrapolation to the human. Toxicol. Appl. Pharmacol. 15, 287-302. Viviani, A., Lutz, W. K., and Schlatter, C. (1978). Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclei and microsomes by phenobarbital, 3-methyl cholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, dieldrin and other inducers. Biochem. Pharmacol. 27, 2103-2108. Webster, L. R., McKenzie, G. H., and Moriarty, H. T. (2002). Organophosphate-based pesticides and genetic damage implicated in bladder cancer. Cancer Genet. Cytogenet. 133, 112-117.
548
SECTION V . N o n s p e c i f i c
Toxic Effects
Whitney, K. D., Seidler, E J., and Slotkin, T. A. (1995). Developmental neurotoxicity of chlorpyrifos: Cellular mechanisms. Toxicol. Appl. Pharmacot. 134, 53-62. Whyatt, R. M., and Barr, D. B. (2001). Measurement of organophosphate metabolites in postpartum meconium as a potential biomarker of prenatal exposure: A validation study. Environ. Health Perspect. 109, 417-420. Whyatt, R. M., and Perera, E E (1995). Application of biologic markers to studies of environmental risks in children and the developing fetus. Environ. Health Perspect. 103, 105-110.
Whyatt, R. M., Santella, R. M., Jedrychowski, W., Garte, S. J., Bell, D. A., Ottman, R., Gladek-Yarborough, A., Cosma, G., Young, T. L., Cooper, T. B., Randall, M. C., Manchester, D. K., and Perera, E E (1998). Relationship between ambient air pollution and DNA damage in Polish mothers and newborns. Environ. Health Perspect. 106, 821-826. World Health Organization (1989). Pesticide Residues in Food. Evaluation Part I I - - Toxicology of Propoxur. pp. 183-214. World Health Organization, International Programme on Chemical Safety, Geneva.
CHAPTER
~8
Temperature Regulation in Experimental Mammals and Humans Exposed to Organophosphate and Carbamate Agents* CHRISTOPHER I. GORDON', 1 CINA M. MACK, 1 AND PAMELA I. ROWSEYz 1U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 2University of North Carolina, Chapel Hill, North Carolina
The control of body temperature is an autonomic process that is a hallmark of physiological homeostasis. Considering the multitude of parameters of the internal milieu that are regulated by homeostatic processes, thermoregulation is one with a high degree of stability. That is, in the evolution of eutherian mammals that range in body mass by over seven orders of magnitude, internal body temperature ranges from 36 to 40 ~ On the other hand, other autonomic processes, such as blood pressure, cardiac output, and respiration, vary considerably within and between species. It is the nature of these processes to vary because the autonomic nervous system responds to the ever-changing demands of the organism to deliver oxygen and nutrients and extract carbon dioxide and other waste products. These demands are intimately related to the organism's body size, age, environment, and many other factors. The stability of the thermoregulatory system should make it an ideal focal point in the study of the toxicology of anti-ChE agents and other toxicants. If a homeostatic process that is normally stable sustains marked deviations in its regulator patterns as a result of a toxic insult, then this suggests profound dysfunction in regulatory mechanisms. There is a wealth of data on the effects of OP and CM insecticides and related agents on thermoregulation in test mammals and birds as well as humans. Moreover, the advent of computerized, multianimal radiotelemetry systems in the past 25 years has been a boon to pharmacologists and toxicologists who use body temperature as a critical end point. To this end, this chapter reviews the effects of
I. I N T R O D U C T I O N Many classes of pesticides and other environmental toxicants affect the regulation of body temperature in mammals, birds, and other species (Gordon et al., 1988; Gordon, 1993a, 2004). The anticholinesterase (anti-ChE) agents, including organophosphate (OP)- and carbamate (CM)-based insecticides, were one of the first class of toxicants to be studied systematically for their effects on thermoregulation in experimental mammals (Baetjer and Smith, 1956; Meeter and Wolthuis, 1968). This field emerged in the 1960s as it became clear that the cholinergic pathways in the central nervous system (CNS) played a critical role in the control of body temperature. Moreover, toxicologists had firmly established that the primary mechanism of toxicity of the OP and CM insecticides was the inhibition of acetylcholinesterase (ACHE) activity, which resulted in stimulation of peripheral and central cholinergic pathways, With hypothermia being one of the principal symptoms of anti-ChE poisoning in laboratory mammals, it followed that thermoregulatory mechanisms would be evaluated by toxicologists studying the anti-ChE agents.
*This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Toxicology of Organophosphate and Carbamate Compounds
549
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
550
SECTION
V . N o n s p e c i f i c Toxic Effects
anti-ChE agents on the thermoregulatory system mammals and humans, including studies on the CNS.
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300 II. F U N D A M E N T A L S O F T E M P E R A T U R E REGULATION This section provides a brief overview of thermoregulation in mammals that will allow the reader to interpret the studies on anti-ChE agents. The reader is referred to a variety of review articles for detailed explanation of temperature regulation (Gordon, 1993a,b, 2 0 0 4 ; Wang and Lee, 1989). Body temperature in mammals and birds is regulated via a balance between the sum of the sources for heat production and heat loss. Birds and mammals are termed tachymetabolic species, meaning that they have a relatively high basal metabolic rate compared to the so-called lower vertebrates (i.e., bradymetabolic species). They are able to maintain a stable internal or core body temperature over a wide range of ambient temperatures by using physiological and behavioral mechanisms to balance the internal heat production and heat loss to the environment. The animal exchanges heat with the environment through four avenues: radiation, convection, conduction, and evaporation. These avenues of heat loss are more or less important depending on the species and ambient temperature. Measuring metabolic rate, evaporative water loss, and skin temperature (or skin blood flow) over a range of ambient temperatures reveals a general pattern of thermoeffector activity that is typical for most mammals (Fig. 1). There is a range of ambient temperatures termed the t h e r m o n e u t r a l z o n e in which metabolic rate is at its minimal or basal level. The thermoneutral zone is bound by the lower and upper critical temperatures. Temperature regulation in the thermoneutral zone is achieved by control of sensible heat loss - - that is, without regulatory changes in metabolic rate or evaporative. As ambient temperature decreases below the thermoneutral zone, the blood flow to the skin is minimal as a result of peripheral vasoconstriction. With further cooling, metabolism must increase above basal levels by shivering and nonshivering thermogenesis in order for heat production to match heat loss to the environment. There is a minimal ambient temperature at which metabolic rate cannot maintain the pace of high rate of heat loss and the animal becomes hypothermic. At temperatures above the thermoneutral zone, evaporative heat loss mechanisms (i.e., panting, sweating, salivation, and grooming) are activated to maintain thermal balance. This is identified as the upper critical temperature, which is also the temperature at which core temperature and metabolism usually increase above their basal levels. At approximately the point of the upper critical temperature, skin temperature has increased to a level that is just below core temperature, reflecting
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maximal redistribution of warm blood from core to the periphery. With further increase in ambient temperature, skin and core temperature parallel each other until the point of thermoregulatory failure. At this point, evaporative heat loss is ineffective and core temperature spirals upward, leading to hyperthermic death. Mice and rats have a lower critical temperature ranging from 28 to 31 ~ (depending on strain and species) that is notably much warmer than the standard temperature for housing in most laboratory settings. That is, under standard testing conditions for most toxicological and pharmacological studies, rodents experience mild cold stress and thermoregulate by maintaining a metabolic rate above basal levels. The thermoneutral zone varies widely among species of mammals and birds. In general, a large body size and greater insulation result in a lowering of the lower critical temperature (Folk et al., 1998). The lower critical temperature of the rabbit is relatively low, reflecting this species' adaptation to cold environments. Rabbits are more susceptible to ambient heat stress compared to rodents. It is interesting to note that the
CHAPTER 38 9Anticholinesterase Agents and Thermoregulation adult rat and nude human have approximately the same lower critical temperature of 28 ~
Warmreceptors
A. CNS Control of Body Temperature Temperature regulation, like other autonomic systems, is mediated by a complex interaction of neurotransmitters, modulators, and hormones in the central and peripheral nervous systems. The work of Feldberg and Myers in the 1960s demonstrating specific thermoregulatory responses when adrenergic and serotonergic neurotransmitters were microinjected into the CNS was a landmark study that spurred innumerable studies on the neurochemical control of body temperature (Myers, 1980). Reviews by W. G. Clark and others bear witness to the hundreds of studies on the responses of mammals, birds, and other species to neurotransmitters, peptides, drug agonists and antagonists, and other agents administered peripherally or directly into the CNS (Clark and Lipton, 1985a,b; Lipton and Clark, 1986; Wang and Lee, 1989). Although there was a tremendous effort to use neurochemical techniques to understand thermoregulation, there remains today a considerable controversy. Indeed, one will find opposite thermoregulatory responses for the same neurochemical given by the same route of exposure to the same species (e.g., Clark reviews). It is likely that much of the variability in the rodent studies was a result of using restrained or otherwise stressed animals. In most of the early work dating back to the 1960s, radiotelemetry was unavailable and core temperatures were measured using rectal probes or with implanted probes that were tethered to the subject. Overall, neurochemical studies of temperature regulation have generally shown distinct patterns of how neurotransmitters operate in the CNS thermoregulator centers, but the studies are occasionally found to be contradictory and should be viewed with the aforementioned caveats in mind. One relatively simple working model for the rat and mouse comprises a heat dissipatory pathway that is stimulated by serotonin and a heat producing/conserving pathway stimulated by low levels of cholinergic stimulation but suppressed when synaptic levels of acetylcholine (ACh) are excessive (Fig. 2). Norepinephrine may either stimulate or suppress these pathways. This model is useful for explaining the hypothermic effects of anti-ChE insecticides that lead to stimulation of central and peripheral cholinergic pathways. Microinjection of muscarinic agonists such as oxotremorine leads to a marked hypothermic response. Systemic injection of oxotremorine along with a peripheral acting muscarinic antagonist such as methyl scopolamine will also lead to a prolonged hypothermic response (Gordon, 1994a). Likewise, the hypothermic response resulting from central or peripheral administration of antiChE agents is thought to be a result of the cholinergic stimulation of heat loss pathways in the CNS and/or
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FIG. 2. A simple version of a neurochemical model to explain how neurotransmitters interact with warm- and cold-sensitive neurons in the preoptic area and anterior hypothalamus (POAH) to effect a regulated core temperature. ACh, acetylcholine; 5-HT, 5-hydroxytryptamine; DA, dopamine; WS, warm-sensitive neuron; CS, cold-sensitive neuron; W-INT, warm-sensitive integrating neuron; C-INT, cold-sensitive integrating neuron. Modified from Bligh (2001) and Gordon (2004). PVMT-Peripheral Vasometer tone.
suppression of heat production/conserving pathways. Small doses of ACh injected into the POAH have been shown to induce hyperthermia. 5-Hydroxytryptamine (5-HT) microinjected into the CNS generally elicits a heat dissipatory response (Gordon, 1994a). Dopaminergic pathways are also thought to be critical in activating heat loss pathways and evoking a regulated hypothermic response (Barros et al., 2004).
III. A C U T E E F F E C T S O F A N T I - C h E s O N
BODY T E M P E R A T U R E Hypothermia is the most frequently observed thermoregulatory response of mice, rats, and other relatively small mammals when they are administered acute doses of antiChE insecticides while being maintained at an ambient temperature below their thermoneutral zone (Gordon et al., 1988; Gordon, 2004). Many of the studies that have
SECTION V . N o n s p e c i f i c Toxic Effects
552
documented the thermorgulatory effects of anti-ChEs discussed in this chapter relied on colonic probe methods to measure body temperature. The advent of radiotelemetry has revolutionized the ways in which toxicologists can study thermoregulation. With telemetry, relatively small radiotransmitters with thermosensors implanted into the abdominal cavity provide accurate (i.e., _0.1 ~ and continuous monitoring of the core body temperature in unrestrained and undisturbed mice, rats, and other species. For example, the core temperature and motor activity of unrestrained Long-Evans rats monitored for 1 day before and several days after oral administration of two doses of the OP insecticide chlorpyrifos are presented in Fig. 3. Monitoring core temperature by telemetry allows one to clearly observe the acute hypothermic response that is characteristic of OP exposure as well as the subtle elevation in daytime temperature that can persist for several days after dosing. Telemetry also shows how motor activity is suppressed primarily during the first night after dosing. Past studies using colonic probes have documented the hypothermic response in mice and rats (Table 1), but telemetry provides a continuous record of the magnitude and time to recovery. The daytime hyperthermic response to OPs was unknown in rodents until the application of telemetry (Gordon 1993a,b, 1994a,b, 1996a; Gordon et al., 1997). The subtle hyperthermia was not detected with colonic probe techniques
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FIG. 3. Recording of body temperature and motor activity from unrestrained and awake Long-Evans rats (males) before and after dosing with the corn oil vehicle and two doses of the OP insecticide chlorpyrifos. Data from Gordon and Mack (2001).
because the repeated handling of the rodents to insert a colonic probe resulted in a stress-induced hyperthermia that obviated any difference in temperature between treated and control animals. The acute hypothermic response and delayed hyperthermic response to this insecticide and other anti-ChE's are discussed in more detail next.
IV. A N T I - C h E - I N D U C E D H Y P O T H E R M I A AS A B I O M A R K E R OF T O X I C I T Y IN R O D E N T S In a summary of studies on the thermoregulatory effects of anti-ChE agents (Gordon, 1994a), 22 of 25 studies on OPs listed a hypothermic response within 24 hr after exposure. Likewise, among the studies of CMs, hypothermia was reported in 15 of 16 studies. The hypothermic efficacy of the anti-ChEs in rodents is dependent on dose, route of exposure, species, and especially ambient temperature (Table 1). The absolute change in core temperature is generally proportional to the magnitude of the dose of a toxicant, but the temperature change between doses at a given time point may be indiscernible. On the other hand, the integration of the change in temperature with time, termed the temperature index, is an ideal means of quantifying the thermoregulatory effects of anti-ChE and other toxic agents (Clement, 1991; Gordon and Mack, 2001). Although hypothermia is referred to as a symptom in this discussion, the hypothermic response to anti-ChEs may actually be an integrated physiological response to minimize toxicity. Due to their small size, rodents are prone to become hypothermic following a toxic insult. Thermoregulatory stability of small rodents at ambient temperatures below the thermoneutral zone is dependent in large part on the maintenance of a high metabolic rate. Healthy rodents are generally capable of maintaining a constant body temperature at relatively cold ambient temperatures for long periods provided they are given an adequate supply of food. Thermal homeostasis in the cold is contingent on a steady rate of heat production; this process is critical in small rodents that have a relatively high rate of heat loss. A drug or toxicant that impairs the ability to maintain a normal metabolic rate will result in a rapid reduction in core temperature of rats and mice. Note that the same would be true for larger mammals such as humans, but the effect on core temperature is much slower due to the large thermal inertia and better capacity to control heat loss through peripheral vascular mechanisms. When considering the common symptoms of OP poisoning, such as miosis, diarrhea, urination, lacrimation, and salivation, hypothermia is an ideal parameter to study because it can be quantified in terms of a magnitude of change as well as duration of effect. The other symptoms of the cholinergic crisis listed are not as easy to measure in a quantified manner in the undisturbed animal.
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SECTION V . N o n s p e c i f i c
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A. Effects of Ambient Temperature
but this may reflect the physiological response to mediate a reduction in temperature regardless of ambient temperature (Gordon et al., 1991). That is, the rat is capable of activating appropriate thermoeffector mechanisms to mediate a hypothermic response at warm and cold ambient temperatures. This is discussed in more detail next.
Most of the data in Table 1 show the thermoregulatory response of rodents to anti-ChEs when exposed at room temperature (22 ~ well below the thermoneutral zone. The relatively few studies dealing with the influence of ambient temperature have shown that the hypothermia induced by anti-ChE exposure in rodents is attenuated or reversed by housing the test animal in its thermoneutral zone (Fig. 4). For example, the hypothermic response of the OP sarin is undetectable at an ambient temperature of 31 ~ but increases in a near linear manner with decreasing temperature (Wheeler, 1989). In fact, when housed at 31 ~ there was a hyperthermic effect of soman as the dose was increased from 50 to 150 txg/kg. The delayed hyperthermic and febrile effects of these agents are discussed in more detail later. The acute hypothermic response to 0.5 mg/kg physostigmine in the rat maintained at an ambient temperature of 3-5 ~ was nearly three times greater than that observed in animals housed at 22-25 ~ (Maickel et al., 1988). The hypothermic response to DFP in the unrestrained rat is slightly less at 30 ~ compared to 20 ~
V. CNS MECHANISMS The stimulation of CNS muscarinic pathways appears to be a primary cause of the acute hypothermic response elicited by anti-ChE insecticides. Coadministration of muscarinic antagonists such as scopolamine and atropine blocks much of the hypothermic response elicited by anti-ChE agents (Gordon and Grantham, 1999; Maickel et al., 1991; Meeter and Wolthuis, 1968). For example, in rats monitored by radiotelemetry, intraperitoneal administration of 1.0 mg/kg scopolamine given 3 hr after oral dosing with chlorpyrifos causes an abrupt recovery in core temperature (Fig. 5). Note that this dose of scopolamine in control rats elicited
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CHAPTER 38 9Anticholinesterase Agents and Thermoregulation
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for activation of heat loss and heat production pathways to mediate a decrease in core temperature (Fig. 2). OP exposure led to a significant turnover of norepinephrine in the hypothalamus, suggesting that a noradrenergic pathway is also involved in mediating the hypothermic and/or hyperthermic effects (Coudray-Lucas et al., 1983). Acute exposure to CMs and OPs led to marked increases in forebrain levels of DOPAC and 5-HIAA in the rat. This response is indicative of increases in metabolism of DA and 5-HT (Soininen et al., 1990). Chronic treatment with physostigmine for 9 days led to significant elevations in forebrain levels of 5-HT but no changes in other monamines or their metabolites. Administration of prazocin, a peripheral oL-adrenergic antagonist, was shown to exacerbate the hypothermic response of soman and physostigmine in mice (Clement, 1993). It is interesting to note that the OP DEF (S,S,S,-phosphorotrithioate) induces a profound hypothermic response but is a weak inhibitor of AChE activity (Ray, 1980). DEF-induced hypothermia is not affected by administration of cholinergic antagonists. Thus, although the cholinergic pathways are very important in the acute hypothermic response to anti-ChEs, it is clear that other neurochemical pathways are also operative in the mediation of anti-ChE-induced hypothermia (Gordon, 1994a).
24
FIG. 5. Time course of core temperature monitored by radiotelemetry in rats dosed initially with chlorpyrifos or corn oil vehicle by oral gavage and then injected intraperitoneally with saline or 1.0 mg/kg scopolamine. Modified from Gordon and Grantham (1999).
rapid hyperthermia, a response markedly greater than that of the rats treated with chlorpyrifos (Gordon and Grantham, 1999). On the other hand, scopolamine does not appear to be nearly as effective at reversing the hypothermic effects of the OP DFP compared to chlorpyrifos (Gordon, 1996a). It appears that ambient temperature will determine which neurotransmitter systems are operative in the thermoregulatory responses of anti-ChEs. For example, Maickel et al. (1991) found that the efficacy of atropine and scopolamine to block physostigmine-induced hypothermia was attenuated with decreasing ambient temperature. When temperature was shifted above or below thermoneutrality, specific neurotransmitter systems were activated or suppressed, and this modulation may well alter the relative activity of cholinergic systems in animals exposed to anti-ChEs. The stimulation of muscarinic and nicotinic cholinergic pathways in the CNS is well documented. In addition to the cholinergic pathways, acute exposure to anti-ChEs elicits changes in metabolism of biogenic amines suggesting activation of dopaminergic, serotonergic, and noradrenergic pathways (Coudray-Lucas et al., 1983; Soininen et al., 1990). Selective stimulation of these pathways may allow
VI. RELATIONSHIP B E T W E E N CORE TEMPERATURE AND C H O L I N E S T E R A S E INHIBITION The human health risk assessment of anti-ChE insecticides has utilized the inhibition in plasma or serum AChE activity as a threshold to limit exposure to these insecticides. To this end, it is important to characterize the relationship between the threshold inhibition in AChE activity in the brain and/or peripheral tissues that is associated with hypothermia or other physiological response. In a survey of five studies of rats exposed to various OP agents, an inhibition in brain AChE activity of 82.5% was associated with a - 3 . 0 ~ reduction in core temperature (Gordon and Fogelson, 1993). Clement (1991) showed that hypothermia in mice treated with satin occurred when AChE inhibition in the hypothalamus exceeded 52%. In fact, it appears that for rodents tested at room temperature, an inhibition in brain AChE of 50% was the approximate threshold for eliciting a hypothermic effect (Gordon, 1994a). In rats exposed to the OP soman, the degree of hypothermia over a wide range of ambient temperatures was highly correlated with the inhibition in brain ChE but not with plasma or red blood cell (RBC) ChE activity (Maickel et al., 1991). It is also interesting to note a clear negative correlation between the hypothermic response to sarin and the level of blood glucose. CMs lower core temperature with less inhibition in brain AChE activity compared to OPs. For example, a 22% inhibition in brain AChE activity is associated with
556
SECTION V .
N o n s p e c i f i c Toxic Effects
a 3.5 ~ reduction in core temperature measured 60 min after administration of physostigmine (Maickel et al., 1991). However, in nearly all of these studies, core temperature was measured with colonic probes at standard room temperature. These correlation studies are performed within relatively brief periods after dosing with the anti-ChE agent because the core temperature will recover despite continued inhibition in ChE activity. For example, the hypothermic response of the rat dosed intramuscularly with 1.0 mg/kg DFP reaches a peak response at 6 hr postdosing and then recovers over the next 24 hr despite persistent inhibition in ChE activity in the anterior preoptic area (Kozar et al., 1976). The inhibition in brain AChE activity is the crucial facet of the neurotoxic effects of anti-ChEs in rodents and other test species, but such a measurement is obviously impossible to make in human subjects and indirect methods must be utilized. The inhibition in serum or plasma ChE activity is often used as a index of potential inhibition in brain AChE activity in humans who may be exposed to anti-ChE. In the rat exposed to DFP, a significant hypothermic response was associated with an inhibition in serum ChE activity of 54% (Gordon and Fogelson, 1993). With other OPs, plasma or serum ChE activity can plummet to below 25% of normal before there is a significant hypothermic effect. A 5-min exposure to vapors of DFP in the mouse causes core temperature to decrease by more than 4 ~ within 30 min and remains depressed for approximately 4 hr (Scimeca et al., 1985). This was associated with a 66% inhibition in brain AChE activity that remained depressed despite a full recovery in core temperature and motor coordination. One endeavor of risk assessment is to identify the threshold for a toxicological effect such as hypothermia. It seems that the threshold AChE inhibition for induction of hypothermia would be lowered if the animals were subjected to colder temperatures and more sensitive methods were used to monitor core temperature (i.e., telemetry). Most rodent studies are performed at ambient temperatures that are comfortable for humans (--22 ~ but are approximately 6 ~ below the rat's lower critical temperature. Our database on threshold doses to achieve physiological responses to anti-ChEs as well as other toxicants could change significantly if the choice of temperatures to house and test rodents was reconsidered.
VII. ANTI-ChE EFFECTS O N
THERMOEFFECTORS A. Autonomic Responses A hypothermic response in a rodent exposed to an anti-ChE as well as many other toxicants has often been concluded to represent a dysfunction or failure of the thermoregulatory
system. This interpretation derives from the observation that there is an approximate linear relationship between the decrease in core temperature and ambient temperature in rodents dosed with an anti-ChE agent (Wheeler, 1989; Meeter, 1969; Fig. 4). Investigators not trained in thermal physiology have often interpreted the hypothermia to essentially represent the response of a "sick" animal, suffering from many symptoms including thermoregulatory failure. In reality, little is known about what happens to body temperature when there is "thermoregulatory failure." Failure could mean an abnormal increase or decrease in temperature, and the response would be affected by ambient temperature and other environmental factors. Electrolytic lesioning of the CNS thermoregulatory centers can lead to hypothermia or hyperthermia, depending on the location of the lesion, environmental temperature, species, and time of recovery. Hence, one should not presume that an ambient temperature-dependent hypothermia in the rodent exposed to a toxicant is thermoregulatory failure until thermoeffectors (i.e., motor outputs) are monitored. As explained later, studies on the activity of behavioral and autonomic thermoeffectors show that rodents exposed to anti-ChEs elicit an integrated response to lower body temperature in a regulated manner. Heat loss thermoeffector pathways in rodents are activated with muscarinic and other neurochemical pathways located in CNS thermoregulatory centers. Stimulating the cholinergic pathways, either by CNS or systemic injections of cholinomimetic agents or by administering anti-ChE agents, leads to a stimulation of the heat loss pathways and a hypothermic response. Meeter and colleagues first determined that the control of skin blood flow in rats played a critical role in the development of hypothermia following exposure to OPs (Meeter, 1969). The tail of the rat is a critical site for the regulation of dry heat loss (Gordon, 1993a,b). The decrease in body temperature following exposure to OPs such as sarin, DFP, and chlorpyrifos occurred concomitantly with an increase in tail skin temperature of the rat (Meeter, 1969; Gordon and Fogelson, 1993; Gordon et al., 2002). The effects of OPs on vasomotor control in the tail are integrated with other thermoeffector systems, including heat production, evaporation, and behavior. The interplay between these systems in the rat can be seen by varying ambient temperature (Gordon et al., 1991). At a standard laboratory temperature of 22-24 ~ DFP elicited a marked elevation in tail ~skin temperature and a moderate decrease in metabolic rate. However, at a colder temperature of 10 ~ DFP injection resulted in a reduction in tail skin temperature along with a reduction in metabolic rate and hypothermia. In other words, the hypothermic response in the cold was mediated primarily by a decrease in heat production and the rat appeared to restrict heat loss from the tail to prevent an excessive hypothermic response. At a thermoneutral
CHAPTER 38
9Anticholinesterase Agents and Thermoregulation
temperature of 30 ~ tail vasodilation was ineffective to dissipate much additional heat and metabolic rate could not be lowered below basal levels. Hence, the rat was unable to lower body temperature as much as in the thermoneutral environment, and DFP elicited an increase in evaporative water loss, presumably as an additional measure to increase heat loss and lower body temperature. This illustrates the balance between three thermoeffectors to achieve a hypothermic response under a wide range of ambient temperatures (Gordon et al., 1991).
B. Behavioral Responses With few exceptions, all toxicological and pharmacological studies on rodents are performed at a constant ambient temperature (usually 22 ~ at which the animal has little, if any, option of altering its thermal environment with behavioral mechanisms. A constant environmental temperature is essential for consistent and replicable laboratory studies, but it represents a challenge to rodents, which use their behavioral thermoregulatory reflexes to seek a comfortable thermal environment. In other words, rodents hou~sed at a standard room temperature of 22 ~ which is approximately 6 ~ below their thermoneutral zone, have to use metabolically costly autonomic thermoeffectors to thermoregulate. They are unable to use their behavior to maintain a comfortable thermal environment that minimizes metabolic requirements for thermoregulation. A temperature gradient, composed of a long tube with a range of ambient temperatures, provides an ideal means of studying the effects of toxicants on thermoregulatory behavior. When left undisturbed in a temperature gradient, rats and mice select a temperature of approximately 30 ~ during the daytime and cooler temperatures at night as their motor activity increases (Gordon, 1993a). The daytime selected temperature is considerably warmer than the typical environment used for housing and testing rodents in toxicological and pharmacological studies. The physiological response to an anti-ChE agent in a rodent allowed to select its thermal environment compared to one maintained at a constant temperature of 22 ~ is likely to differ considerably. The time course of selected ambient temperature and core temperature in the rat monitored by telemetry exemplifies the regulated hypothermic response induced by J administration of chlorpyrifos (Fig. 6). When dosed with a control vehicle (corn oil), there was a transient decrease in selected temperature that reflects a heat dissipatory response from the stress of handling and injection. When dosed with chlorpyrifos, selected ambient temperature decreased from 30 to 25 ~ The behavioral response to select a cooler temperature preceded a 2.5 ~ decrease in core temperature. At the nadir of the decrease in core temperature, selected temperature increased rapidly,
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a response that presumably facilitated the recovery of core temperature. Administration of the OP DFP also induced an abrupt selection for cooler temperatures that occurred concomitantly with a decrease in core temperature (Gordon, 1994b, 1997). It is important to note that in the temperature gradient the rat has the option of selecting ambient temperatures as warm as 36 ~ The rat can simply select a warm temperature that would attenuate or block the hypothermic effects of the anti-ChE agent. Overall, the mechanism of action of anti-ChEs and other toxicants on temperature regulation can be better understood if their behavioral thermoregulatory responses can be monitored. Based on measurements of tail skin temperature and blood flow, Meeter and colleagues (1971) proposed that OPs such as soman elicited a reduction in the set point for the regulation of core temperature in the rat. The behavioral thermoregulatory responses showing a preference for cooler ambient temperature provide further evidence of a reduction in set point as proposed by Meeter. The acute response of the rat when dosed with a variety of OPs appears to be an integrated autonomic and behavioral response to effect a regulated decrease in core temperature.
558
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SECTION V- N o n s p e c i f i c
Toxic Effects
VIII. BENEFITS OF HYPOTHERMIC RESPONSE With few exceptions, a hypothermic body temperature affords protection to a variety of toxicants (Gordon et al., 1988; Gordon, 2004). A mild hypothermic body temperature has been shown to increase the lethal dose of a variety of toxicants, including ethanol, pesticides, and heavy metals. To this end, one must wonder if the hypothermic response to an anti-ChE agent represents an integrated physiological response to improve the survival and recovery from the toxic agent. Relatively little is known about the mechanisms of the protective action of hypothermia. Based on the Arrhenius principles of thermal kinetics, it is reasonable to assume that hypothermia is most likely protecting tissues by slowing the rate at which the toxicant exerts its damaging effects at the cellular and intracellular levels (Gordon, 1996a, 2004). This is a rather simplistic explanation and there is a need for a better understanding of how toxicant damage is ameliorated with hypothermia. Does the regulated reduction in body temperature following exposure to anti-ChE agents afford protection? Lower air and skin temperatures will likely reduce the transcutaneous absorption of OPs applied to the skin. Once the toxicants have entered the circulation, a lower body temperature will also affect the toxicity of this class of compounds. Baetjer and Smith (1956) performed a systematic series of studies on the effects of environmental temperature on the toxicity of anti-ChEs in mice. Maintaining mice at an environmental temperature of 35.5 ~ accelerated the rate of mortality from intravenously injected parathion compared to mice maintained at 22.7 ~ Interestingly, the lethality of intraperitoneal injections of ACh in mice (Baetjer and Smith, 1956) and methacholine in rats (Keplinger et al., 1959) was similarly affected by ambient temperature. That is, the toxicity of these cholinergic agonists increases dramatically when ambient temperature is raised to above the species' thermoneutral zone. Overall, the toxicity of anti-ChE agents appears to be reduced in rodents when they select a cooler environment allowing body temperature to decrease by 2 or 3 ~ The mechanisms of this protective effect can be complex. That toxicity of cholinergic agonists such as methacholine and ACh is ameliorated with cooler temperatures suggests that the lower body temperature operates similarly with the anti-ChE agents. One would expect that the magnitude of the "cholinergic crisis" on inhibition of AChE activity is proportional to body temperature. Cooler temperatures would be expected to attenuate the symptoms of the classic signs of the cholinergic crisis. This is a simplistic explanation and should be viewed with caution because there are a variety of mitigating circumstances of hypothermia that could augment or
attenuate chemical toxicity. For example, the metabolic conversion of many OP insecticides to their oxon in the liver is a temperature-dependent process. The metabolic deactivation of the parent compounds and oxons is also temperature dependent. Hence, a lower temperature may attenuate toxicity, but it also slows the excretion of the toxicant from the body. In fact, in a review of the effects of physical factors on drug toxicity, Doull (1972) stated that temperature is directly correlated with the magnitude and inversely correlated with the duration of drug response in biological systems. In other words, although the concentration of a drug or toxicant will persist longer during hypothermia, the toxicity of the agent is reduced.
IX. D E V E L O P M E N T A L
EFFECTS
The rat pup is generally more sensitive to OP insecticides based on parameters such as lethal dose and the efficacy to inhibit AChE activity (Pope and Chakraborti, 1992). One would also expect that the thermoregulatory effects of antiChEs would be induced at lower doses in the young rodent with underdeveloped thermoregulatory control. The ability to maintain a stable core temperature in the mouse and rat is developed at approximately 15 days of age (Gordon, 1993a). Thermoregulatory stability during heat and cold stress and a distinct circadian temperature rhythm are developed during the next month. Studying thermoregulation in the preweaned rat pup using conventional colonic probes is fraught with problems in addition to those discussed for adult rodents. Body temperature should be monitored in undisturbed pups that are housed with their dam and littermates if one is interested in assessing the long-term effects of a toxicant under natural conditions. Radiotelemetry implants are available that are small enough to be surgically implanted into rat pups at an early age (--12 days) and left in place such that body temperature and other physiological parameters can be monitored in the pups while housed with dam and littermates. Our laboratory measured core temperature by telemetry in preweaned rats dosed with an OP insecticide (Fig. 7). Comparing these responses to the adult responses discussed previously (see Fig. 3), it can be seen that the hypothermic response of the 17-day-old rat pup to chlorpyrifos is qualitatively similar to that of adults. On the other hand, the rat pup was found to be markedly sensitive to chlorpyrifos, with the hypothermic response to 10 mg/kg chlorpyrifos in the rat pup equivalent to a dose of approximately 50 mg/kg in the adult. It is also interesting that the rat pup regulates a stable core temperature of approximately 37.5 ~ but has no discernable circadian rhythm until approximately day 35. The lack of a circadian rhythm in the pup presents a different time course of recovery of body temperature, including lack of a delayed fever as is seen in the adult rat.
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E F F E C T S O F ANTI-CHES In a study on the thermoregulatory responses to a single dose of DFP in the rat monitored by radiotelemetry, a sustained elevation in core temperature was observed following recovery from the acute period of hypothermia (Gordon, 1993b). The magnitude of the hyperthermia was relatively small compared to the hypothermic effect. However, although the hypothermic effect of DFP recovered 5 hr after exposure, the hyperthermic response persisted for 3 days after dosing. Acute oral dosing with OP insecticides such as chlorpyrifos and diazinon (Gordon et al., 1997; Gordon, 1997; Gordon and Mack, 2003) and carbaryl (Gordon and Mack, 2001) elicited a similar pattern of hypothermia followed by a delayed elevation in core temperature that persisted for at least 1 day (see Fig. 3). These acute studies demonstrate a prolonged hyperthermic response following a single exposure to an OP. The hyperthermic response has been shown to persist with repeated or chronic exposure to OPs. For example, Haque et al. (1987) dosed rats repeatedly with the OP malathion for 7 days and found a 1.0 ~ increase in core temperature on the day after the last dose. Female rats showed a hyperthermic response the day after injection for 4 consecutive days of oral dosing with chlorpyrifos (Rowsey and Gordon, 1997). Rats allowed to feed on a diet containing chlorpyrifos and monitored by radiotelemetry underwent an -0.2 ~ elevation in core temperature during the daytime but not during the night during a 15-day treatment period (Gordon and Padnos, 2002). The dosage of chlorpyrifos to elicit this response was only 7 mg/kg/day but nonetheless resulted in an 87% inhibition in serum cholinesterase activity. This is the first demonstration using radiotelemetry to show a relatively small increase in core temperature following subchronic, dietary treatment with an anti-ChE insecticide. Although the hypothermic effects of anti-ChE agents abate with repeated dosing as a result of
FIG. 7. Time course of core temperature in male rat pups dosed by oral gavage with corn oil or 10 mg/kg chlorpyrifos. Radiotelemetry units implanted at 15 days of age and pups dosed on day 17 while housed with dam and 5 littermates at ambient temperature of 22 ~ Data from Mack and Gordon (unpublished observations).
the development of tolerance, the few studies on the hyperthermic effects of OPs suggest little development of tolerance. Overall, the hyperthermic effect of these agents has largely gone undetected in previous rodent studies because of a limitation in technology. It was thought that rodents simply became hypothermic in response to acute exposure to anti-ChE insecticides because the response can be marked and is easy to detect with conventional colonic temperature probes. On the other hand, a hyperthermic or febrile response is relatively small, generally less than 1.0 ~ and can only be detected in undisturbed and unstressed rodents using telemetry. A fever appears to be a predominant thermoregulatory response of humans exposed acutely to OP and CM insecticides. Hence, it behooves one to understand the mechanism of the delayed fever in rodents. The delayed fever following exposure to anti-ChE agents in rodents may represent an important biological end point and mechanism of neurotoxicity that can be extrapolated from experimental animals to humans. Autonomic and behavioral thermoregulatory responses during anti-ChE-induced hyperthermia suggest that the increase in temperature is regulated, akin to that of a fever. A hyperthermic response following acute hypothermia may suggest that the response is essentially a compensatory rebound, That is; as the rat recovers from being hypothermic for 10 or more hours, heat gain and conserving mechanisms overwhelm heat loss processes and there is a transient overshoot in body temperature. However, rebound hyperthermia does not seem to explain the anti-ChEinduced hyperthermia. Blocking the chlorpyrifos-induced hypothermia by increasing ambient temperature from 22 to 31 ~ during the period in which the rat would be hypothermic led to an increase in the magnitude of the delayed hyperthermic response (Gordon, 1997). The response of thermoeffectors during the period of the hyperthermic response also suggests the development of a regulated hyperthermia. Rats allowed to behaviorally
560
SECTION
V.
N o n s p e c i f i c Toxic Effects
thermoregulate in a temperature gradient prefer normal ambient temperatures during the period of the chlorpyrifosand DFP-induced fever (Gordon, 1994b, 1997). If the elevation in temperature was a forced hyperthermic response as a result of peripheral vasoconstriction and/or an increase in metabolism, then one would expect the rat to prefer cooler temperatures to dissipate the excess heat load. In other words, the rats behave in a manner suggesting that they do not feel hot despite being hyperthermic. Chronic measurements of tail skin temperature during the period of chlorpyrifos-induced fever show no indication of vasodilation of tail skin blood flow to dissipate the excess heat (Gordon and Padnos, 2002). Finally, the hyperthermia persists during the day, disappears at night, but then may return the following day. Overall, a prolonged and reoccurring elevation in temperature suggests a resetting of the set point for temperature regulation, meaning that the delayed hyperthermia is a fever. Pharmacological data also reveal unique aspects of the anti-ChE fever. The fever is not blocked with the muscarinic antagonist scopolamine (Gordon and Grantham, 1999). In fact, although scopolamine and atropine are very effective at blocking the hypothermic effects of anti-ChEinduced hypothermia, scopolamine exacerbates the increase in temperature when given during the febrile period. This suggests that the elevated temperature is being maintained by mechanisms other than the stimulation of cholinergic pathways. The delayed hyperthermia from chlorpyrifos, DFR and diazinon (Gordon, 1996b; Gordon et al., 1997; Gordon and Mack, 2003) has been found to be effectively blocked with the antipyretic sodium salicylate (Fig. 8). In view of the similarity of the anti-ChE-induced fever to that of infectious fever, it was reasonable to assume that activation of the immune-neural pathways involved in infectious fever is also operative in anti-ChE fever. Tumor necrosis factor-cx (TNF-ot) and interlevtin-6 (IL-6) are two of several cytokines involved in the mediation of infectionmediated fevers. Gordon and Rowsey (1999) measured plasma levels of these cytokines at several time points following exposure to chlorpyrifos in male and female rats. The only significant finding was an increase in plasma levels of TNF-oL but no change in IL-6 when measured 48 hr after dosing with chlorpyrifos (i.e., during the time when a fever develops). An 89% increase in TNF-cx levels in female rats 48 hr after chlorpyrifos was associated with a febrile state. However, this increase in TNF-oL was relatively small compared to the greater than 10-fold increase in blood levels of this cytokine in animals subjected to an lipopolysaccharide (LPS) fever (Gordon and Rowsey, 1999; Kluger et al., 1995). Moreover, IL-6 increased by 1000-fold in the blood of rats subjected to an LPS fever but changed little following chlorpyrifos administration. In summary, data do not show the robust elevations in circulating cytokines as seen with an infectious fever. However, this
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FIG. 8. (A) Time course of core temperature in the rat following oral dosing with corn oil or 200 mg/kg diazinon. (B) Antipyretic effect of 200 mg/kg sodium salicylate injected intraperitoneally 48 hr after rats were dosed with corn oil or 200 mg/kg diazinon. Modified from Gordon and Mack (2003).
does not rule out participation of the neuroimmune fever response in the mediation of the anti-ChE fever. Overall, the anti-ChE agent-induced fever appears to be a unique thermoregulatory response that may involve the participation of multiple phy~siological systems.
CHAPTER 38 9Anticholinesterase Agents and Thermoregulation
A. Fever and Hyperthermic Responses in Humans Exposed to Anti-ChEs Humans are acutely exposed to anti-ChE agents as a result of accidental exposures in the home and work place and from their use as suicidal or homicidal agents. Information collected during treatment of victims from these poisonings accounts for much of our understanding of the thermoregulatory effects of anti-ChEs in humans. Analysis of these data is hampered by the innumerable uncontrolled variables in these studies, such as variations in time to treatment in the emergency room, age, gender, ambient conditions, pharmacological treatments, lack of information on the patient's normal body temperature, and dose of the antiChE agent. The effects of these poisoning incidents are often recorded during emergency room treatment, a period associated with marked stress on the thermoregulatory system. Despite these drawbacks, these clinical reports provide insight into the thermoregulatory responses of humans exposed to anti-ChEs as well as other toxicants. Thermoregulation is one of many autonomic processes affected by exposure to OP and CM insecticides (Table 2). There are also incidences of hypothermia, especially when subjects are first admitted to the emergency room, when their symptoms of cholinesterase inhibition are severe. A hyperthermic or febrile response is often seen as a complication during the recovery from exposure to the antiChE agent. Namba et al. ( 1 9 7 1 ) performed one of the first studies to document the occurrence of fever in humans acutely exposed to OP insecticides. They noted an association between the degree of inhibition in serum ChE activity and incidence of fever in patients exposed to the OP insecticides parathion and methyl parathion. It was noted that mild fevers could persist for more than 1 week in some cases. Others have also reviewed case reports of exposure to anti-ChE insecticides and found a high incidence of
fever. Hirshberg and Lerman (1984) performed a retrospective analysis of 236 cases of OP or CM poisoning in Israel between 1958 and 1979. They found that a fever was a prevalent symptom in 25% of cases labeled as a "complication or delayed effect" that usually appeared more than 24 hr after exposure. In this study, fever was defined as a condition in which core temperature was >37.5 ~ Saadeh et al. (1996) described the clinical and sociodemographic data of 70 adults exposed acutely to OP or CM insecticides and found that 49% had a low-grade fever of 37.5-38.5 ~ with no evidence of infection. The onset and recovery from fever occurred between 1 and several days after exposure, with nearly half of the fevers persisting for 3 days. They noted that patients with fevers had received significantly more atropine than those without fevers. One might consider if the administration of atropine is in some way responsible for the fever since muscarinic antagonists have well-known hyperthermic effects in humans (Christoph, 1989). On the other hand, the authors noted that the fever persisted in 27% of the patients for 4 days despite discontinuing atropine therapy. A presumed terrorist attack with the nerve gas sarin in Matsumoto, Japan, led to the poisoning of approximately 600 residents (Morita et al., 1995). A mild, low-grade fever was reported in some subjects for up to 1 month after exposure, and one man had a low-grade fever 6 months after exposure. It is interesting to note that the aforementioned studies rarely mention whether standard antipyretics such as aspirin were prescribed for alleviating the fever. Overall, it appears that fever is the most frequent thermoregulatory response in humans acutely exposed to anti-ChEs. On the other hand, an acute hypothermic effect of OP and CM insecticides has occasionally been reported in humans poisoned with anti-ChEs. In one instance, a 16-year-old male suffering from OP poisoning
TABLE 2. Summary of Studies That Have Shown a Febrile or Hyperthermic Response in Humans Exposed to OP and/or CM Insecticides
Study Saadeh et al. (1996) Hirshberg and Lerman (1984)
Namba et ai.~(1971)
Morita et al. (1995)
561
Findings Fever was a symptom in 49% of 70 subjects exposed acutely to OP or CM insecticides Fever was a symptom in 25% of cases labeled as "complicated or delayed" in humans exposed to OP insecticides from 1958 to 1979 One of first studies to document delayed fevers in humans exposed to parathion or methylparthion Mild or low-grade fever reported in a number of subjects lasting up to 1 month following terrorist attack with satin nerve gas in Matsumoto, Japan
562
SECTION V .
N o n s p e c i f i c Toxic Effects
was admitted with a high blood pressure (152/102) but a rectal temperature of only 34.5 ~ (Cupp et al., 1975). One hour after a 17-year-old male was admitted to the hospital following ingestion of a large amount of malathion, his blood pressure was also high (170/80), and he was mildly hypothermic with a core temperature of 36.2 ~ (Hassan et al., 1981). A 58-year-old woman who ingested a large amount of diazinon had a low blood pressure and was markedly hypothermic with a core temperature of 34.4 ~ 1 hr after poisoning (Hassan et al., 1981). In another series of case reports on diazinon poisoning, there were incidences of transient hypothermia (34.4 ~ and delayed fever (38.9 ~ (Klemmer et al., 1978). A 39-year-old man was admitted following malathion poisoning with high blood pressure (140/90), profuse sweating, and a core temperature of 34 ~ (Meller et al., 1981). By 15 hr after admission, core temperature had recovered to normal. In the same study, an 81-year-old woman was also admitted with high blood pressure (180/100) and a core temperature of 35 ~ It is possible that the very young and old may be more susceptible to the hypothermic effects of these pesticides, but this has not been studied. The hypothermic effects of the anti-ChEs in humans are most likely mediated by the stimulation of muscarinic cholinergic pathways that lead to profuse sweating and peripheral vasodilation. However, it is difficult to determine whether the hypothermic response is regulated as it appears to be in rodents because whenever the hypothermia is reported during the course of treatment in the emergency room, measures are usually taken to raise the subject's core temperature to normal. Information on the subject's state of thermal comfort during the onset of poisoning would be useful to determine if the thermal set point is reduced in the same manner as in rodents subjected to acute antiChE exposure. That is, if the subjects felt warm during the period of hypothermia, then it would suggest, as is evident in the behavioral thermoregulatory response of rodents, that the anti-ChEs induce a regulated hypothermia. In addition, because of the large body mass and small surface area: body mass ratio of adult humans, the ability to lower core temperature in response to a toxicant is markedly attenuated with increasing body mass (Gordon, 2004). As the muscarinic effects of anti-ChE poisoning abate, either naturally or due to prophylactic intervention, it appears that thermoeffectors for increasing heat gain and/or reduction in heat loss take over, leading to a prolonged albeit low-grade fever.
XI. EXERCISE AND HEAT STRESS The factors that affect the biological dosage of an antiChE agent are also linked to thermal homeostatic mechanisms. Toxic agents can enter the body by three principal routes: respiratory surfaces, gastrointestinal tract, and
transcutaneously (Casarett and Doull, 1975). These routes of entry into the body are also intimately connected to ambient temperature and the organism's thermoregulatory state. That is, under ideal environmental conditions (e.g., thermoneutral environment), a stable core temperature is maintained with minimal strain on physiological systems. However, in the face of marked changes in ambient temperature, relative humidity, and/or workload, a constant core temperature is maintained but at the expense of activation of thermoeffectors and physiological stress. The surface of the respiratory tract and skin are integral for the operation of thermoeffectors for evaporative and dry heat loss. Hence, when a homeotherm is in an environment in which it must actively dissipate heat, it is likely to be more susceptible to lower doses and/or concentrations of certain types of toxicants. On the other hand, the increased demand for heat production in a cold environment results in an elevation in respiratory rate, thus increasing the intake of airborne toxicants and also raising susceptibility. The thermoregulatory system responds to heat stress and exercise by activating three key systems to dissipate excess heat: cardiovascular, respiratory, and sudomotor (sweating). The combination of peripheral vasodilation to increase skin blood flow and raise skin temperature along with sweating results in an effective mechanism to dissipate a heat load (Folk et al., 1998; Blatteis, 1998). True panting animals exhibit marked increases in breathing frequency during heat stress. Nonpanting homeotherms, including humans and rodents, also exhibit increases in breathing frequency and minute volume that contribute to a modest increase in evaporative water loss when heat stressed (Ingram and Mount, 1975). The added heat load of exercise will further increase ventilation and augment the total intake of airborne pollutants (Mautz, 2003). Sweating is the principal thermoeffector response in heat-stressed humans and some other mammals. Eccrine sweat glands in humans are activated by cholinergic pathways, and stimulation of these pathways generally occurs concurrently with an increase in skin blood flow. The flow of warm blood from the core to the surface combined with evaporative cooling from sweating is an effective mechanism to dissipate excess body heat. On the other hand, the combination of moisture, warm temperatures on bare skin, and increased skin blood flow also provides an ideal environment to accelerate the transcutaneous absorption of many types of pesticides (Chang et al., 1994; Wester et al., 1996). Both in vitro and in vivo studies suggest that activation of thermoeffectors during heat stress and/or exercise to dissipate heat will accelerate pesticide absorption in humans. An in vitro model of cutaneous absorption of parathion has been used to show how temperature, blood flow, and relative humidity affect the absorption of parathion. A small section of porcine skin positioned over a flow-through diffusion cell provides an ideal means to control air temperature, relative humidity, perfusate
CHAPTER 3 8 .
temperature (i.e., an indication of body temperature), and flow of the perfusate (i.e., an indication of the potential effects of blood flow) while studying the percutaneous absorption of a pesticide (Chang and Riviere, 1991). The absorption of radiolabeled parathion across porcine skin increases dramatically with an elevation in air and/or perfusate temperature. For example, a 5 ~ increase in air and perfusate temperature leads to a more than two-fold increase in parathion absorption. It is possible that skin warming can raise lipid fluidity and permeability of the dermal tissues, leading to increased penetration of the pesticide. The cutaneous absorption of parathion is directly affected by relative humidity and perfusate flow. The effects of humidity are profound, suggesting that increased moisture on the skin raises the permeability to parathion. Parathion is a lipophillic molecule, and it is thus not clear why percutaneous absorption would increase with additional moisture on the skin. Studies on humans performed decades ago showed how perspiration can accelerate the cutaneous absorption of OPs. Human volunteers were exposed to ambient temperatures of 14, 21, 28, and 40.5 ~ while their hand and arm were exposed for 2 hr to a 2% parathion dust (Funckes et al., 1963). The absorption of the insecticide was estimated by the quantity of paranitrophenol, a metabolite of parathion, that was excreted in the urine. The dermal absorption of parathion was mildly affected by skin warming at low temperatures and markedly affected at warm temperatures. Parathion absorption increased by 25% when the temperature of exposure was raised from 14 to 21 ~ however, from 21 to 28 ~ parathion absorption increased by only 17%. Raising ambient temperature from 28 to 40.5 ~ led to a 180% increase in absorption (Funckes et al., 1963). Although the rate of sweating was not measured, it was clear that subjects perspired profusely at the warmest ambient temperature. It is also interesting to note the increase in parathion absorption at the lower ambient temperatures despite a lack of sweating. The warmer skin temperature is likely to be a critical factor affecting parathion absorption even without sweating. The dose of parathion used in this study was relatively low because RBC and plasma cholinesterase activity was unaffected by the treatment. The dosage from exposure to anti-ChE pesticides should be exacerbated in a warm and humid environment because of cholil~ergic stimulation of sweating combined with greater transcutaneous absorption across moist skin. In another human study, volunteers had small amounts of the nerve gas VX [S-(2-diisopropylaminoethyl) o-ethyl methylphosphonothioate] applied topically to their cheek and forearm at ambient temperatures of - 1 8 , 2, 18, or 46 ~ (Fig. 9). The VX was left on the skin for 3 hr and its penetration into the body was estimated by measuring the inhibition of RBC ChE activity (Craig et al., 1977). The absorption of VX in humans was directly dependent on ambient temperature~ The decimal fraction of penetration
Anticholinesterase Agents and Thermoregulation
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of VX on the cheek was 0.04 at an extremely cold ambient temperature of - 1 8 ~ and 0.32 under conditions of extreme heat stress with an ambient temperature of 46 ~ For the skin on the forearm, the VX penetration was 0.004 at 18 ~ and increased to 0.029 at 46 ~ Overall, penetration across the cheek was much more effective than that on the forearm, with greater than 50% inhibition in ChE activity occurring at a dose of less than 10 Ixg/kg. It was postulated that after exposure to VX or a comparable agent, cooling the skin would delay absorption, thus allowing for safer decontamination of an exposed subject.
XII. S U M M A R Y The regulation of body temperature is an ideal benchmark to study the toxicity of the anti-ChE insecticides in experimental mammals and humans. The inhibition in AChE activity and subsequent stimulation of cholinergic pathways in the central and peripheral nervous systems is a key mechanism responsible for driving changes in body temperature and activation of thermoregulatory effectors. Body size and ambient temperature are perhaps the most critical factors governing the thermoregulatory effects of these toxicants. Small mammals with a relatively large surface area: body mass ratio undergo a marked increase in heat loss and become hypothermic when exposed to anti-ChEs. After recovery from the acute hypothermic effects, rats have been shown to exhibit a small but prolonged elevation in temperature manifested during the daytime that is similar in many ways to a fever. A fever predominates in large mammals such as humans following an acute exposure. The hypothermic response in large mammals is relatively small and transient compared to that seen in small rodents. Small mammals exposed to these agents lower their body temperature by selective activation of heat loss thermoeffectors. This hypothermic response is often protective
564
SECTION V . N o n s p e c i f i c Toxic Effects
and increases the likelihood of surviving the toxic insult. Large mammals such as humans are unable to take advantage of this hypothermic response, and it is not clear if a fever from these toxicants will affect the health effects of these agents. It does appear that exercise and/or heat stress will exacerbate the toxic effects of anti-ChE insecticides and nerve gas agents. The redistribution of blood to the skin combined with sweating will increase the cutaneous absorption of OP agents that are applied to the skin. The increase in pulmonary ventilation during exercise combined with higher tissue temperature is bound to increase the intake and toxicity of airborne anti-ChE agents.
XIII. FINAL PERSPECTIVE All life processes depend on chemical reactions that, in turn, are directly dependent on temperature, as based on the principles of the Arrhenius equation. The toxicology of the anti-ChE agents, like all other toxicants, is intimately related to temperature and the thermoregulatory system. We have shown that the intake through the lungs and skin, metabolic activation and deactivation, and physiological effects of anti-ChE agents can be affected by ambient, skin, and core temperature. The message of this chapter is that toxicologists working at the subcellular, cellular, in vitro, and/or in vivo level should always consider how temperature can influence the toxicological efficacy of anti-ChE agents.
Acknowledgments We thank Dr. Amir Rezvani and Andrew Geller for reviewing the manuscript.
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CHAPTER 38
9Anticholinesterase Agents and Thermoregulation
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Meeter, E. (1969). The mode of action of cholinesterase inhibitors on the temperature regulation of the rat. Arch. Int. Pharmacodyn. 182, 416-419. Meeter, E., and Wolthuis, O. L. (1968). The effects of cholinesterase inhibitors on the body temperature of the rat. Eur. J. Pharmacol. 4, 18-24. Meeter, E., Woithuis, O. L., and Van Benthem, R. M. (1971). The anticholinesterase hypothermia in the rat: Its practical application in the study of the central effectiveness of oximes. Bull. World Health Organization 44, 251-257. Meller, D., Fraser, I., and Kryger, M. (1981). Hyperglycemia in anticholiesterase poisoning. Can. Med. Assoc. 124, 763-748. Morita, H., Yanagisawa, N., Nakajima, T., Shimizu, M., Hirabayshi, H., Okudera, H. O., Hohara, M., Midorikawa, Y., and Mimura, S. (1995). Sarin poisoning in Matsumoto, Japan. Lancet 346, 290-293. Moser, V. C. (1995). Comparisons of the acute effects of cholinesterase inhibitors using a neurobehavioral screening battery in rats. Neurotoxicol. Teratol. 17, 617-625. Myers, R. D. (1980). Hypothalamic control of thermoregulation, neurochemical mechanisms. In Handbook of the Hypothalamus (E J. Morgane and J. Panskeep, Eds.), Vol. 3, Part A, pp. 83-210. Dekker, New York. Namba, T., Nolte, C. T., Jackrel, J., and Grob, G. (1971). Poisoning due to organophosphate insecticides. Am. J. Med. 50, 475-492. Pope, C. N., and Chakraborti, T. K. (1992). Dose-related inhibition of brain and plasma cholinesterase in neonatal and adult rats following sublethal organophosphate exposures. Toxicology 73, 35-43. Ray, D. E. (1980). Selective inhibition of thermogenesis by tributyl S,S,S,-phosphorotrithioate (DEF). Br. J. Pharmacol. 69, 257-264.
Rowsey, R J., and Gordon, C. J. (1997). Tolerance to the hypothermic and hyperthermic effects of chlorpyrifos. Toxicology 121, 215-221. Rupniak, N. M., Tye, S. J., Brazell, C., Heald, A., Iversen, S. D., and Pagella, E G. (1992). Reversal of cognitive impairment by heptyl physostigmine, a long-lasting cholinesterase inhibitor, in primates. J. Neurol. Sci. 107, 246-249. Saadeh, A. M., A1-Ali, M. K., Farsakh, N. A., and Ghani, M. A. (1996). Clinical and sociodemographic features of acute carbamate and organophosphate poisoning: A study of 70 adult patients in North Jordan. Clin. Toxicol. 34, 45-51. Scimeca, J. A., Little, E J., and Martin, B. R. (1985). Relationship between the pharmacological effects and the biodisposition of [3H]diisopropylfluorophosphate in mice after inhalation. Toxicol. Appl. Pharmacol. 79, 502-510. Soininen, H., Unni, L., and Shillcutt, S. (1990). Effect of acute and chronic cholinesterase inhibition on biogenic amines in rat brain. Neurochem. Res. 15, 1185-1190. Stemler, E W., Corcoran, K. D., Parrish, J. H., Hurt, H. H., TezakReid, T., Kaminskis, A., and Jaeger, J. J. (1990). Effects of physostigmine on the cardiopulmonary system of conscious pigs. Fundam. Appl. Toxicol. 14, 96-103. Wang, L. C. H., and Lee, T. E (1989). Temperature regulation. In Psychoendocrinology pp. 437-539. Academic Press, New York. Brush, E R. and Levine, S. (eds.). Wester, R. C., Quan, D., and Maibach, H. I. (1996). In vitro percutaneous absorption of model compounds glyphosate and malathion from cotton fabric into and through human skin. Food Chem. Toxicol. 34, 731-735. Wheeler, T. G. (1989). Soman toxicity during and after exposure to different environmental temperatures. J. Toxicol. Environ. Health 26, 349-360.
CHAPTER ~ 9
Occupational Toxicology and Occupational Hygiene Aspects of Organophosphate and Carbamate Anticholinesterases with Particular Reference to Pesticides* BRYAN BALLANTYNE 1 AND HARRY SALEM z 1Charleston, West Virginia 2U.S. Army Chemical and Biological Center, Aberdeen Proving Ground, Maryland
hence the necessary protective and precautionary measures dictated by occupational hygiene considerations may be extensive and vary for differing chemical groups. The most geographically extensive and quantitatively greatest application of OPs and CMs is as pesticides used in agriculture, horticulture, public health protection, and domestically. Most of this chapter concerns these uses.
I. I N T R O D U C T I O N Organophosphate (OP) and carbamate (CM) anticholinesterases (anti-ChEs) have a wide spectrum of applications that includes pesticides in agriculture and horticulture, insecticides in domestic and public health applications, use in general commerce, use in therapeutic medicine, and as chemical warfare and terrorist agents. During normal civilian use, and when handled correctly with the appropriate recommended protective and precautionary measures, they can be used safely. However, because of their widespread and sometimes uncontrolled usage, there is a potential for misuse. Also, because of the high biological activity of many OPs and CMs, the likelihood for adverse effects occurring from accidental, and sometimes deliberate, exposure is high. Although the principal mechanism of action that underlies the practical use of OPs and CMs, namely inhibition of the cholinesterase group of enzymes, is also responsible for some of their known human toxicity, because of the wide range of chemical structures involved, the potential for numerous and differing toxic effects that are mediated by other mechanisms also exists (e.g., inflammation, immunotoxicity, myopathy, genetic toxicity, oncogenicity, and developmental and reproductive toxicity) (Ballantyne and Marrs, 1992). Thus, OPs and CMs may have a wide spectrum of potential occupational toxicity, and
II. S O U R C E S , P A T T E R N S , A N D R O U T E S OF E X P O S U R E Human adverse health effects from overexposure tO pesticides are, or should be, documented from carefully prepared clinical case notes of single and/or group poisonings, results from the findings of forensic toxicologists and pathologists in fatal cases of poisoning, the records and published works of poison control centers, and formal epidemiological studies. To the latter can be added the newer techniques of geographic processes for the capture, storage, retrieval, analysis, and display of spatial data (Clarke et al., 1996; Gunier et al., 2001; Ward et al., 2000). These information systems, which are automated, can be effectively utilized to study regional and temporal variations in the incidence of human symptomatic pesticide exposures (Sudakin et al., 2002). Occupational exposure to OP and CM pesticides occurs at different locations that include the workplace at production and formulation facilities, during transportation and distribution, during warehouse storage, and at in-use sites. The resultant potential health hazards and consequent published safety information
*The opinions and interpretations expressed in the chapter are those of the authors and do not necessarily represent the position of the U.S. Army and U.S. Department of Defense.
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sources, recommended safe handling practices, protective practices, and precautionary measures differ significantly for these different locations.
ing an accident, when there may be exposure of local inhabitants and others in the vicinity of the incident with a potential for adverse health effects and possible environmental effects on soil, rivers, and groundwater.
A. Production Facility Exposures can be to raw starting materials for the synthesis of pesticides, to intermediates in the manufacturing process, and to the OP or CM pesticides. Exposure to these various materials is generally limited to the workers at the facility ~ who produce and formulate the pesticide. However, the possibility for the escape of materials into the immediate area around a production plant needs to be considered in respect to possible local and general environmental and human adverse effects. This was most dramatically demonstrated in an incident involving the escape of the highly irritant material methyl isocyanate, used in the manufacture of the CM aldicarb, at a production facility in Bhopal, India. This resulted in the deaths of more than 2000 people (Lewinsohn, 1992) and more than 4000 animals (Gupta, 2004) in the surrounding area. Lethal adverse effects included pulmonary edema, bronchiolitis, and secondary pulmonary infections (Jain and Dave, 1986; Pandey, 1986). However, in general, losses of materials into the area around a production facility, where the general population could be exposed, are small in quantity and are controlled to "safe" levels by use of appropriate acute inhalation reference exposure levels and guidance levels (Collins et al., 2003). Formulators convert the pesticide into a desired physical state for use, such as a solution, emulsifiable concentrate, dust, powder, granule, and wettable powder. Thus, they may be exposed to various physical forms of the pesticide together with additional formulating materials of widely differing toxicity, which may significantly influence exposure considerations. Thus, these employees may represent a special health risk group requiring additional protective and precautionary measures. Exposure of workers in the production facility to starting materials, intermediates, and final product can be limited by good engineering controls and industrial hygiene practices, including the use of closed reactors, personal protective equipment (clothing and respirators), and precautionary measures that include medical surveillance, atmosphere and personnel monitoring, and ventilation for collective protection. Worker education, training sessions, the availability of appropriate first aid measures and emergency antidotes, and the provision for random audits of the health and safety program are all key elements for maintainence of a healthy working environment.
B. Transport Transportation does not usually result in direct exposure, although this may occur during loading and unloading of the road or rail tanker, for which monitoring techniques are available. Most problems with transportation occur follow-
C. In Use The in-use occupational applications of pesticides can be comparatively uncontrolled or difficult to control since they may occur in various and differing enclosed spaces (e.g., greenhouses) or open spaces (fields). Als0, the modes of applications may differ qualitatively and quantitatively, varying from hand application to land-based spray and powder applications and aerial applications. Since the atmospheric concentration of a pesticide cannot be controlled in these situations, particularly in open conditions, as it can by a plant workplace exposure guideline [e.g., Threshold Limit Value (TLV)], direct worker protection must be mainly by protective clothing and respirators. Also, there is a potential not only for those directly involved in applications to be contaminated but also for workers subsequently entering the treated areas before expiration of the reentry time to be contaminated. This may result in a significant degree of exposure. For example, the Centers for Disease Control (1999) reported an incident in California involving 34 farm workers who became ill after entry into a cotton field 2 hr after it had been aerially sprayed with a solution containing 0.26% carbofuran (N-methyl carbamate); the restricted entry interval for carbofuran is 48 hr. Approximately 4 or 5 hr after entering the sprayed field, they began to feel ill. The symptoms reported included nausea (7%), headache (94%), eye irritation (5%), muscle weakness (2%), tearing (68%), vomiting (79%), and salivation (56%); the most commonly observed signs were bradycardia (21%), diaphoresis (15%), and miosis (12%). Foliage samples showed carbofuran levels up to 0.77 ixgcm -2, and workers' clothing contained carbofuran residue up to 1 mg per item. Carbofuran metabolites were detected in urine samples obtained up to 11 days following exposure. Clearly, those who live in close proximity to the area being treated may also become contaminated from spray or dust drift ("farm proximity pathway"). For example, a study in Washington State (Fenske et al., 2002a) demonstrated that children of nonagricultural workers living close to farms (up to 200 ft), and who had been exposed to drift of azinphos-methyl, phosmet, chlorpyrifos, and ethyl parathion, had detectable amounts of urinary dimethyl OP metabolites similar to those in children of agricultural workers. Transient passers-by can also be subject to acute overexposure. Drift of pesticides after aerial application has been well documented (Ames and Stratton, 1991; Chester and Ward, 1984; Cone et al., 1994; Draper and Street, 1981; Draper et al., 1981). These considerations indicate the importance of various health and safety measures required with respect to the in-use application of pesticides; notably, monitoring of workers for exposure, medical surveillance,
C H APT ER 3 9 9Occupational Toxicology and Hygiene the use of physical protective measures, careful planning and timing of operations, and adherence to reentry times (Kahn, 1980; Lewinsohn, 1992; Spear, 1980). Drift also indicates the need to consider environmental protective measures, including adherence to derived exposure guidelines to protect the public (e.g., inhalation reference exposure levels, acute emergency guidance levels, short-term public emergency guidance levels, and emergency response planning guidelines) (American Industrial Hygiene Association, 1991; Collins et al., 2003; National Research Council, 2001). Considerable interest has been shown in the "take-home" (paraoccupational) exposure pattern, in which pesticide operatives contaminate members of their family with the material that is carried back to the home on worker clothing or skin (Baker et al., 2004). Thus, family members may face an increased risk of pesticide exposure (Fenske et al., 2002b; Quandt et al., 2004; Renner, 2002). A typical example is given by Fenske et al. (2002a), who studied children in the state of Washington and demonstrated that those from agricultural families compared to children from reference (control, nonexposed) families had a greater exposure to OPs as indicated by higher levels of urinary OP metabolites. In addition to the potential for occupational and paraoccupational exposure to pesticides, there may be significant and uncontrolled in-use exposures in nonoccupational settings, notably pesticide-treated general workplaces and residences. Also, OP and CM anti-ChEs have found a not insignificant use in deliberate (intentional) poisoning, both self-administered and for homicide (Ballantyne, 1992). Self-poisoning with anti-ChEs, notably OPs, is particularly prevalent in underdeveloped and developing countries. For example, in Turkey OPs are the most common agent employed for suicidal poisoning (Akgtir et al., 2003).
D. Routes of Exposure
The principal route(s) of potential exposure will depend on a variety of factors, including the chemistry and physical properties of the pesticide (e.g., solid, liquid, vapor pressure, and viscosity), its formulation and formulation constituents, and the mode of use and application (e.g., as liquid, emulsil-/ fiable concentrate, dust, aerosol, and spray). The route(s) of exposure together with the nature of the toxicity of the pesticide and its formulation constituents, the mode of exposure, and the degree of exposure will be major determinants of the potential hazards and the necessary protective and precautionary measures. The exposure routes discussed next may be encountered to varying degrees. 1. SKIN CONTACTAND PERCuTANEOUS ABSORPTION Clearly, unprotected skin may become contaminated during accidental spills and with the in-use application of pesticides during transfer of formulations, mixing and loading of applicators, and applications involving dusts and spray dispersions from land-based equipments or airplanes. This route
569
also presents a problem for entry of unprotected agricultural workers into recently treated areas before the expiration of a recommended reentry interval. Contact with the skin may result in local irritancy and/or sensitization reactions; in California, 15-25% of adverse pesticide reports are due to skin reactions (O'Malley, 1997). More important with regard to OPs and CMs is the possibility for systemic toxicity following percutaneous absorption. The likelihood for absorption of pesticide across the skin, and its magnitude, will be determined by a variety of interacting factors, principal among which are the following: 1. Physicochemical properties, including molecular weight, charge, and hydrophilic/lipophilic characteristics. 2. The surface area of the skin contaminated (Maibach and Feldman, 1974); clearly, the larger the area contaminated, the greater the amount of material absorbed, i 3. The concentration of the material or amount of applied material; the higher the concentration, the greater the gradient to facilitate percutaneous absorption. 4. The time of contact will clearly influence the amount of pesticide absorbed, increasing with increasing duration of contact; hence the importance of early decontamination of skin. Related to this is the degree of occlusion i for example, material becoming trapped between skin and clothing (Semple, 2004). Clothing saturated with pesticide residue may promote continuing skin exposure and also percutaneous absorption (Freed et al., 1980; Webster and Maibach, 1985a,b). 5. The presence of formulation surfactants and solvents, which will facilitate skin penetration of the pesticide. Absorption rate is more effective for lipophilic materials. Some UV-absorbing chemicals can act as skin penetration enhancers, which may increase the percutaneous absorption of pesticides and other formulation chemicals (Morgan et al., 1998; Nakai and Chu, 1997). Thus, it has been shown that the application of sunscreens to the skin can increase the penetration of pesticides (Brand et al., 2002). 6. Anatomical site contamination. With many materials, including pesticides, the absorption rate through the skin of a specific material varies with the regional location of the skin area contaminated. For example, with parathion the absorption rate is faster through skin of the scrotum, axillae, and face than through skin of the hands and arms (Maibach et al., 1971). Also, Maibach and Feldman (1974) applied 4 txg cm -2 of parathion to the forearm, abdomen, and forehead of volunteers and found that the absorbed doses were 8.6, 18.5, and 36.3%, respectively. 7. The integrity of the absorption surface may be an important practical consideration. Thus, materials may be absorbed more effectively through diseased and abraded skin, particularly with more recently injured skin (Ballantyne, 1989; Ballantyne et al., 1999; Grissom and Shah, 1992). For example, parathion was absorbed 8.5 times more through damaged than through normal skin (Maibach and Feldman, 1975).
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8. Environmental factors, such as increased temperature and relative humidity, may facilitate skin penetration of a chemical (Ballantyne et al., 1999). All those factors emphasize the need, with the more toxic materials, for physical protection of skin (gloves, clothing, and face shields/respirators) and for decontamination of accidentally contaminated skin. Although cleansing of contaminated skin is advised to remove residual pesticide, a simple soap and water wash may not be sufficient since it has been shown that a considerable residuum may remain on or in the area of washed skin; most of the residual material remains in the stratum corneum (Fredriksson, 1961; Webster and Maibach, 1983; Zendzian, 1994, 2003). Although there is constant exfoliation of the stratum corneum, the turnover time is on the order of 14 days (Halpran, 1973). Thus, there is a potential for the residual pesticide in skin to contribute to potential local and/or systemic toxicity. This may be compounded by the fact that washing the contaminated site can lead to a transient increase in the absorption flux of the pesticide (Webster and Maibach, 1985a,b, 1999). The timing and the magnitude of any increase in absorption will vary with the specific pesticide and its physicochemical properties and the rate/magnitude of absorption in comparison with the rate/magnitude of excretion of the absorbed pesticide. Zendzian (2003) found that of 19 pesticides studied postwash in the rat, absorption continued for 15 at all dosages tested, in 2 it continued but not at all dosages, and in 2 absorption did not continue postwash. Of the 15 pesticides in which absorption continued postwash, only 9 had an increase in systemic concentration indicating a potential for increased toxicity.
2. RESPIRATORYTRACT EXPOSURES Inhalation of vapor is the more likely hazard with exposure to employees in plant production facilities, but with in-use applications there may inhalation exposure to vapor, dusts, mists, and aerosols. Vapor will be transported to the pulmonary alveoli, but the degree of penetration of dusts and aerosols into the respiratory tract will depend primarily on particle size (Dodd, 1992). Depending on the toxicity of the inhaled material, general effects that may be caused are irritation and inflammation within the respiratory tract, and possibly induction of an asthmatic response. OP and CM anti-ChEs will produce local respiratory tract toxicity particularly affecting the bronchiolar smooth muscle, resulting in bronchoconstriction and increased tracheobronchial secretions (Dodd, 1992). Systemic toxicity may result from absorption of OPs and CMs through the respiratory mucosa or, if the alveoli are reached, absorption into the pulmonary alveolar capillaries. The specific occupation of the worker may influence the degree of inhalation exposure. For example, Wolfe (1973) found that exposure of an operator of an air blast sprayer directing the spray upward into trees was 12 times higher than that of a boom operator directing
a comparable formulation down into rows of crops. Hayes (1975) reported that during an aerial application of tetraethyl pyrophosphate dust, the pesticide loader received approximately three times more exposure to the concentrate than did the airplane pilot and 4.5 times that received by the flagman. 3. GASTROINTESTINALTRACT Ingestion of OPs and CMs is not usually a major problem in the production facility, unless there is poor personal hygiene and unwrapped food or drink is kept in close proximity to anti-ChEs. Peroral exposure may also result from transfer of pesticide from contaminated hands (e.g., during eating or smoking). Worker ingestion of OPs and CMs is more likely to occur from swallowing saliva contaminated with pesticide inhaled during a dust or aerosol application session or from coughed mucus. Clearly, ingestion of foods from crops with pesticide residues is a general public health possibility and consideration. In some cases, this may present a significant acute hazard. Toxic illnesses caused by the CM aldicarb are typical; one case involved consumption of contaminated watermelons in Oregon (Hall and Rumack, 1992), and another was due to consumption of hydroponically grown cucumbers (Lewinsohn, !992). 4. EYE CONTACT Contamination of the eye with anti-ChE OPs and CMs may occur accidentally during pesticide manufacture and use and deliberately with therapeutic agents applied to the eye. This may result in both local and systemic toxicity.
III. G L O B A L A N D R E G I O N A L S C A L E S OF H U M A N H E A L T H P R O B L E M S D U E TO PESTICIDE POISONING Accidental and intentional poisoning of humans with CMs and particularly OPs is a worldwide occupational, public health, environmental, and forensic problem. Thus, acute pesticide poisoning represents a significant cause of morbidity and mortality in underdeveloped and developing counties. The World Health Organization (WHO) estimated that there are approximately 3 million cases of acute pesticide poisoning annually, with 220,000 deaths (Bryant et al., 2003; Jeyaratnam, 1990). The majority of these incidents occur in developing counties, notably Africa, Asia, and Central and South America (He, 2000; Van der Hook et al., 1998). A study in Zimbabwe from January 1998 to December 1999 showed that OPs and rodenticides were the leading cause of acute pesticide poisoning admissions to major hospitals; thus, of a total of 914 acute pesticide exposures, 42.2% were to anti-ChEs, mainly OPs (Tagwireyi et al., 2004). In Central America between 1992 and 2000, there was a significant increase in the importation of pesticides and this was associated with an increase in the incidence rate for acute
CHAPTER 39 9Occupational Toxicology and Hygiene poisoning from 6.3 per 100,000 to 19.5 per 100,000 population, and the mortality rate increased from 0.3 per 10,000 to 2.1 per 100,000 population (Hena and Arbelaez, 2000). A systematic randomized 10% retrospective screen was conducted of all hospital-referred poisoned patients from March 1993 to March 2000 in Imam Reza University Hospital of Mashhad, Iran; this country has no center for poison control and surveillance to gather information and analyze data (Afshari et al., 2004). There were 71,589 total cases, of which 7158 were selected for analysis. The annual crude rate of referral due to poisonings was 3.9 per 1000 population per year, with an annual crude rate of mortality of 2.3 per 100,000 population. Main poisoning groups in the analysis were pharmaceuticals (61.4%), chemicals (22.8%), and natural toxins (16.6%). For chemicals (industrial, domestic, and environmental), there were 1486 cases analyzed, of which OP and CM insecticides accounted for 250 (16.8% of group and 3.5% of the total poisonings analyzed). In Mexico, among 200 seasonal workers, 20% experienced acute pesticide poisoning (De Jesus et al., 1998), and in Costa Rica, an overall rate of pesticide poisoning of 5.3 per 100 workers per year was calculated based on data from the National Insurance Institute (Verga and Fuotes, 1998). Several major pesticide poisoning incidents have been reported from India. For example, more than 100 people died in Kerala in 1958 after consuming wheat flour contaminated with ethyl parathion (Karunakaran, 1958). In the same year in Kerala, 102 people died due to the careless handling and storage of wheat. Thirty-five cases of malathion poisoning were reported from Indore, of which there were 5 deaths (Sethuraman, 1977). In a report from Madhya Pradesh, 12 people who consumed wheat for 6 months that had been contaminated with aldrin and gammexane developed neurological symptoms that included myoclonic jerks, generalized clonic convulsions, and limb weakness (Gupta, 1975). In general terms, and with respect to the general population, in India there are currently approximately 145 pesticides registered for use, with an annual production of 85,000 metric tons (Gupta, 2004). The Poisons InformationCenter of the National Institute of Occupational Health in Ahmedabad has noted that OPs have been responsible for the largest number of poisonings (73%) among agricultural workers (Dewan and Sayed, 1998). Despite the fact that the consumption of pesticides is very low in India at 0.5 kg ha -1, there has been widespread contamination of foods with pesticide residues, basically due to the ill-informed use of pesticides. Thus, in India approximately 51% of food commodities are contaminated with pesticides, which the levels of 21% are higher than world accepted maximum residue levels (Gupta, 2004). Hence, in India there appear to be deficiencies in the understanding of pesticide health problems, safety in use, intelligent approaches to registration, and monitoring for overexposure. There is thus a need for local education at all strata in government, industry, and worker populations on the monitoring, correct handling, and protective measures neces-
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sary for the agricultural use of pesticides. Severe OP self-poisoning is a major clinical problem in the Asia Pacific region, where some hospitals treat 500-1000 patients annually, with a case fatality of more than 20% (Eddleston et al., 2004). Although the potential for adverse effects from working with agrochemicals in developed counties is generally well appreciated, there is still concern in such countries about the occurrence of pesticide-related illnesses from acute and/or repeated overexposure. It has been noted that working conditions in agriculture are among the most hazardous in the United States, and seasonal farm workers in particular are at a significant risk for occupational illness and injury (Arcury and Quandt, 1998; Bureau of Labor Statistics, 1999; Das et al., 2001; Griffith and Duncan, 1992; Mobed et al., 1992; National Institute for Occupational Safety and Health, 1996; Wilk, 1986). This applies notably for injury and illness resulting from pesticide exposure (Coye and Fenske, 1988; Moses, 1989). The greatest source of exposure is from dislodgeable residues (Fenske, 1997). Several authorities have noted that OPs continue to pose a risk to human health in the United States, and there is a clear need for continuous biological monitoring to regulate exposure (Jaga and Dharmani, 2003). An indication of the scale of the problem of exposure to pesticides in the United States is provide by the following examples. In 1999, more than 13,000 cases of OP poisoning were reported to poison control centers, there were more than 3000 cases seen in hospital emergency departments, and 83 fatalities occurred (Riegel, 2002). Data provided by the Toxic Exposure Surveillance System of the American Association of Poison Control Centers recorded there were 10,073 exposures to OP insecticides in adults in 2000, of which 9609 were unintentional and 276 were intentional (Litovitz et al., 2001). For 2003, the U.S. Bureau of Labor Statistics recorded 707 fatal work injuries under the heading of "agriculture, forestry, fishing, and hunting" out of a total of 5559 workplace fatalities for that year. In agriculture, the fatalities were crop production, 333 (of which 17 were classified as being due to exposure to harmful substances); vegetable and melon farming, 17; fruit and tree nut farming, 13; greenhouse, 18; and other crop farming (tobacco, cotton, sugarcane, hay, and miscellaneous), 77 (American College of Occupational and Environmental Medicine, 2004). Among a cohort of more than 50,000 pesticide applicators in the United States, 16% reported having a high pesticide exposure event (which did not always result in toxic effects) (Kiem and Alavanga, 2001); within the same cohort, and on the basis of nested case-control analysis, it was estimated that approximately 0.6% of the applicators had developed symptoms (Alavanja et al., 2001). In a study of acute occupational pesticide-related illnesses among adolescents in the United States, Calvert et al. (2003) concluded that there is still a need for greater effort to prevent such illnesses. They Suggested the need to improve surveillance in order to overcome the limitations of underreporting. Also, they advised that because of problems in
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relating signs and symptoms to acute pesticide overexposure, there is a need for vigilance on the part of health care professionals to consider the roles of environmental and occupational exposures. In an interview survey of Latino farm workers by Arcury et al. (2000), it was reported that farmers do not necessarily adhere to regulations mandating training and basic sanitation facilities. It was concluded that additional regulation by itself is not an advantageous starting point and the emphasis for intervention should include educating farmers as well as farm workers. A particular area of concern in the United States is morbidity from pesticide overexposure in hired migrant farm workers, many of whom are undocumented, have no legal authorization to work, are underpaid, have little or no formal education, may not be able to speak the language and thus have significant communication problems, and have barriers to health care access. It is therefore not surprising that they have a high incidence of systemic effects notably from OP and CM antiChEs, inorganic compounds, and pyrethroids (Das et al., 2001; Jaga and Dharmani, 2003).
TABLE 1. Clinical Signs and Symptoms of Anticholinesterase Intoxication a Muscarinic
Nicotinic
IV. M E D I C A L S U R V E I L L A N C E OF W O R K E R S P O T E N T I A L L Y EXPOSED TO OP AND CM ANTICHOLINESTERASES Details of the mechanism of action and clinical consequences of overexposure to OP and CM anti-ChEs are given in other chapters of this book. However, the possible adverse effects that may result from acute and repeated overexposure are briefly summarized here as a preliminary to the needs and requirements for surveillance of workers potentially exposed to OPs and CMs. Although the objectives of medical, and indeed any, surveillance programs are to detect the early onset of adverse effects and to confirm that health and safety measures are proceeding in a satisfactory, effective, objective, and planned manner, the ultimate goal is prevention of work-related morbidity and mortality (Schnitzer and Shannon, 1999).
A. Clinical Concerns Forming the Basis for Medical Surveillance 1. CHOLINERGIC EFFECTS The immediate adverse effects (type 1 syndrome) resulting from an acute overexposure to OP and CM anti-ChEs are summarized in Table 1. With OPs, these effects are the result of the OP binding with the catalytic center of ACHE, inhibiting its enzyme activity, and causing an accumulation of ACh at muscarinic receptors in skeletal neuromuscular sites, at nicotinic receptors in autonomic ganglia, and at central nervous system (CNS) cholinergic receptors (mainly muscarinic). The resultant increases in synaptic ACh at muscarinic, nicotinic, and CNS sites form the basis for the cholinergic signs and symptoms and the acute lethal
CNS
Excess lacrimation Rhinorrhea Hypersalivation Bronchorrhea Bronchoconstriction Hyperhidrosis Miosis Decreased visual acuity Urination Defecation Bradycardia Hypotension Mydriasis Tachycardia Hypertension Ataxia Weakness Skeletal muscle twitching Skeletal muscle fasciculations Skeletal muscle weakness Skeletal muscle paralysis Pallor Ataxia Lethargy Slurred speech Confusion Depression Tremors Convulsions Respiratory failure Hypothermia Coma
aAfter Ballantyneand Marrs (1992). toxicity of anti-ChEs. In general, because the carbamylation of AChE results in a more rapidly reversible inhibition complex with AChE than does phosphorylation, the clinical effects produced by OPs are generally more severe and more sustained than those caused by CMs. Also, since CMs penetrate the blood-brain barrier less effectively than do OPs, the CNS component is usually smaller with CMs (Ballantyne and Marrs, 1992).
2. ACUTE DELAYED-ONSET NEUROLOGICAL EFFECTS Following recovery from the cholinergic toxicity ("cholinergic crisis") of an acute overexposure to OPs, a proportion of patients may develop, after approximately 2 4 weeks, a delayed-onset peripheral neurotoxicity of the central peripheral-distal axonopathy type with secondary demyelination
CHAPTER 39 9Occupational Toxicology and Hygiene (Cavanagh, 1963; Davis et al., 1985; Genel et al., 2003). This is often referred to as organophosphorus-induced delayed-onset polyneuropathy (OPIDP). The onset of OPIDP is characterized by cramping muscle pains in the legs, rapidly followed by distal paresthesia and leg weakness with reduced deep tendon reflexes. In severe cases, the upper limbs can become affected and quadriplegia may develop; in these cases, pyramidal signs may occur because of spinal cord involvement (Lotti, 2003). Electrophysiological studies show reduced amplitude of compound muscle action potentials and delayed terminal latencies after supramaximal stimulation of motor nerves; maximal conduction velocity is generally normal or slightly reduced (Lotti et al., 1984). Electromyography reveals a denervation of affected muscles (fibrillation potentials and positive sharp waves) and reduced interference pattern (Lotti, 2001). Some functional recovery of peripheral nerves can occur. Mechanistically, OPIDP is a result of an inhibition of neurotoxic esterase (Johnson, 1992). Delayed-onset polyneuropathy in humans has been described as occurring with the following OP anti-ChEs: dichlorvos (Vasilescu and Florescu, 1980; Wadia et al., 1985), mipafox (Bidstrup et al., 1953), isofenphos (Moretto and Lotti, 2002), leptofos (Xintaras et al., 1978), methamidophos (Senanayake and Johnson, 1982), trichlorofon (Heirons and Johnson, 1978; Johnson, 1981), trichloronat (Jedrzejowska et al., 1980), Oethyl O-4-nitrophenyl phenylphosphonothioate (EPN; Xintaras and Burg, 1980), and chlorpyrifos (Lotti and Morreto, 1986; Lotti et al., 1986). Another delayed-onset neurological/myopathic condition is the "intermediate syndrome," identified by Senanayake and Karalliedde (1987). This appears after the cholinergic crisis but before the time anticipated for the development of delayed-onset peripheral neuropathy. It is characterized by weakness or paralysis of proximal limb muscles, neck flexors, respiratory muscles, and motor cranial nerves (Ballantyne and Marrs, 1992; Senanayake and Karalliedde, 1992). 3. NEUROPSYCHOBEHAVIORALEFFECTS
Following symptomatic acute overexposures to OP anti-ChEs particularly, there may be clinical evidence of neurobehavioral, psychological, and psychiatric effects, which may take months to regress. For example, Le~,in and Rodnitzky (1976) showed that when serum butyrylcholinesterase (BChE) or red blood cell (RBC) AChE activity is inhibited, some or all of the following may be impaired: (i) cognition m vigilance, information processing and psychomotor speed, and memory; (ii) speech, both performance and perception; (iii) psychic state m increased tendencies to depression, anxiety, and irritability; and (iv) electroencephalographic (EEG) records m tendency to faster frequencies and higher voltages. They concluded that EEG abnormalities were positively related to the degree of AChE activity inhibition in the early stages. Several authors have noted that neuropsychobehavioral effects may occur as a consequence of
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occupational overexposure to OPs. The effects that have been recorded include impaired cognition, memory deficits, impaired mental function, retrograde amnesia, increased vibrotactile threshold, depression, dizziness, irritability, anxiety, schizoid reactions, and exacerbation of preexisting psychiatric problems (Levin and Rodnitzky, 1976; Namba et al., 1971; Savage et al., 1988; Stephens et al., 1995). These abnormalities following acute intoxication were grouped together in a syndrome called COPIND 1 (chronic organophosphate-induced neuropsychiatric disorders) by Jamal (1997). However, Lotti (2001) and Ray (1998a) did not regard it as appropriate to include all these possible different effects together into a single syndrome such as COPIND 1. Also, they concluded that there is little evidence, in the absence of hypoxia and/or convulsions in the early cholinergic phase of OP intoxication, that acute OP poisoning results in late permanent neurological or psychiatric effects other than OPIDR Available evidence indicates that asymptomatic acute exposure to OPs is generally not a precursor to neuropsychiatric sequelae, but repetitive acute episodes of OP intoxication with clear signs of cholinergic symptoms are associated with long-term neuropsychobehavioral effects (Eyer, 1995). A long-term low-concentration/low-dosage exposure to OPs is associated with a number of psychiatric, neurobehavioral, and neurological effects, which may not be correlated with symptoms of acute exposure (Brown and Brix, 1998; European Center for Ecotoxicology and Toxicology, 1998; Lotti, 2001; Ray, 1998a,b; Salvi et al., 2003; Steenland, 1996). These were grouped under a syndrome called COPIDN phenomenon 2 by Jamal (1997), an approach that also has been subject to criticism by Lotti (2001). Most studies in which neurobehavioral tests were performed have given negative results, except when subjects were still exposed and had evidence of AChE inhibition (Gomes et al., 1998). Studies have been conducted to determine if there is an association between OP exposures and suicide rates or psychiatric disorders. The results were either negative (Pickett et al., 1998; Stoller et al., 1965) or inconclusive due to lack of information on either the actual exposure or confounding factors (Amr et al., 1997; Levin and Rodnitsky, 1976; Parr6n et al., 1996). However, there is a general consensus that exposure to low-level doses of OPs, as well as acute poisoning, can lead to persistent neurological and neurobehavioral effects, which cannot be explained by AChE inhibition alone (Smulders et al., 2004). It has been suggested that other, more sensitive brain proteins may be involved (Ray and Richards, 2001). Studies of the effects of several OPs (parathion-ethyl, chlorpyrifos, and disulfoton) on rat neuronal Ot.4[~2 nicotinic ACh receptors expressed in X e n o p u s laevis oocytes, using the two-electrode voltage clamp technique, showed that these OPs inhibited the ACh-induced ion current with potencies in the micromolar range (Smulders et al., 2004). The potency of inhibition increased with increasing concentrations of the agonist ACh. Comparison of the potency of inhibition of the nicotinic ACh
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Toxic
Effects
receptor with that of AChE activity inhibition demonstrated that some OPs inhibit nicotinic ACh receptors more potently than AChE activity. The inhibitory effects of OPs on nicotinic ACh receptors can be described and explained by a sequential two-step mechanism, in which rapidly reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It was concluded that OPs interact directly with neuronal ot4~ 2 nicotinic ACh receptors to inhibit the agonist-induced response, and this implicates neuronal OL4~2 nicotinic ACh receptors as additional targets for some OPs (Smulders et al., 2004). These findings may have implications for explaining the mechanism of production of the neurobehavioral effects of OPs by repeated low-concentration exposure. Thus, mice lacking the oL4 nicotinic ACh receptor subunit exhibit a reduced antinociceptive effect of nicotine (Marubio et al., 1999) and elevated anxiety (Ross et al., 2000). Knockout of the ~2 nicotinic ACh receptor subunit showed that it is involved in the reinforcing properties of nicotine (Picciotto et al., 1998) and i n passive avoidance learning (CorderoErausquin et aL, 2000). It follows that repeated desensitization of Ot.4~2 nicotinic ACh receptors may result in some of the neurobehavioral deficits of OP poisoning. Related to this is the clinical observation that one of the effects resulting from long-term repeated exposure of workers to OPs is anxiety (Salvi et al., 2003). Kamel et al (2005) analyzed crosssectional data from licensed pesticide operators enrolled in an agricultural health study under the auspices of the National Institute of Health and Environmental Protection Agency. They found that prevelance of neurologic symptoms was associated with cumulative life time exposure to pesticides, particularly OPs and organochlorines. These associations were present in individuals with no history of pesticide poisoing or high exposure events, and were independent of recent exposures. The investigators consider them due to chronic moderate exposure, and that more attention should be paid to risks associated with moderate exposure. 4. ASTHMA There are differing reports on possible adverse respiratory effects of exposure to airborne pesticides. Some investigators have associated pesticide exposure with effects that include cough, bronchospasm, hypersensitivity pneumonitis, and pulmonary fibrosis (Do Pica, 1992; Schenker et al., 1991; Weisenburger, 1993). Others, however, have not found evidence for occupational asthma among workers with occupational exposure to pesticides, including serial pesticide applicators (Jones et al., 2003). However, since inhaled anti-ChEs have local pharmacological effects on the airways, including bronchospasm as discussed previously, there is reason to suspect that this group of pesticides could cause adverse airflow problems, including asthma, by aerial dispersion. In a cross-sectional study of Canadian farmers, Senthilselvan et al. (1992) found an association between self-reported asthma and CM insecticide use.
Several clinical and epidemiological studies have linked OP exposure to airways hyperreactivity and other symptoms of asthma (Bryant, 1985; Deschamps et al., 1994; Hoppin et al., 2002; O'Malley, 1997). In the respiratory tract, vagal cholinergic nerves mediate airway tone and reactivity by the release of ACh with stimulation of M3 muscarinic receptors resulting in bronchoconstriction. This vagally induced bronchoconstriction is limited by autoinhibitory M2 muscarinic receptors, and loss of M2 function leads to increased release of ACh from the parasympathetic nerves, resulting in potentiation of vagally induced bronchoconstriction. It is generally believed that inhibition of respiratory tract AChE is responsible for OP-induced bronchoconstriction by increasing ACh availability, and in this way contributing to the development of asthma (Ernst, 2002). However, experimental studies by Fryer et al. (2004) have shown that chlorpyrifos altered M2 receptor function in the lung at concentrations that did not inhibit ACHE. It was therefore postulated that OP-induced bronchoconstriction may be a consequence of blockade of M2 receptors resulting in increased ACh release from M3 parasympathetic fibers, and thus vagally induced bronchospasm is potentiated by disruption of the control of airway responsiveness. 5. MISCELLANEOUS Other less frequently described adverse effects that have been described as a consequence of overexposure to anti-ChEs include cardiomyopathy (Singer et al., 1981), rhabdomyolysis (Bright et al., 1991; Dettbarn, 1992; Futagami et al., 2001; Vanneste and Lison, 1993; Yeh et al., 1993; Young and Koplovitz, 1995), nephrotoxicity (Kaedtisuke et al., 1989; Wedin, 1992), and pancreatic injury (Hayes et al., 1978; 'Ma, 1983). 6. ANTI-ChE POTENTIATION Agricultural formulators and field workers may be exposed to more than one OP during a given time period. This is relevant because some OPs (e.g., carbophenothion, fenthion, and dioxathion) inhibit aliesterases, such as diethylsuccinase and tributyrinase, that detoxify other OPs and at doses lower than those inhibiting ChEs. Thus, such OPs are likely to enhance the toxicity of OPs that are normally detoxified by aliesterases and potentiate their activity. OPs that inhibit ChEs and aliesterases at the same rate are likely to cause additive rather than synergistic effects. A health and safety program designed to ensure safe working conditions for employees who may potentially be exposed to OPs and CMs should have a medically supervised surveillance system that involves an initial preemployment screen for suitability to work with the antiChEs, and for those who are actively employed, there should be provision for periodic medical examinations to exclude clinical evidence of short- or long-term adverse health effects from the OPs and CMs to which they may be potentially overexposed. Also, the medical surveillance program should
CHAPTER 39 9Occupational Toxicology and Hygiene be integrated into the general monitoring and industrial hygiene elements of the health and safety program.
B. Preemployment Medical Examination Before being employed in situations in which they may be exposed to OPs and CMs, and to ensure that they are medically, psychologically, and intellectually suitable for such employment, potential employees should have the following documented: 1. Detailed history and physical examination to exclude the presence of medical factors that may indicate a susceptibility of the individual to adverse effects, including precipitation of incipient disease(s), resulting from pesticide overexposure. This should pay particular attention to hepatorenal, respiratory, peripheral and central nervous, visual, cutaneous, and cardiovascular systems. This should include a determination of a history of allergies or asthma that could be aggravated by the anti-ChE or common formulation ingredients to which they may be exposed. Where there is reason to suspect a susceptibility, appropriate biochemical, physiological, and/or radiological investigations should be conducted. Potential employees with hepatorenal disease, glaucoma, cardiovascular disease, and CNS dysfunctions and those using anticholinergic drugs should be warned of the aggravation that could be caused by exposure to OPs and CMs. 2. Preemployment (preexposure) measurement of baseline (control) RBC AChE and serum BChE activities. 3. Exclusion of BChE (pseudo-ChE) deficiency or the presence of a genetically determined atypical pseudocholinesterase (BChE) variant (Bonderman and Bonderman, 1971; Ostergaard et al., 1992). 4. Confirm suitability to wear a respirator. Factors that may be considered as contraindicating respirator use include obstructive pulmonary disease, moderate cardiovascular disease, physical features that may result in poor fit (e.g., facial scars), and psychological problems such as claustrophobia and anxiety (Ballantyne, 1981).
C. Employee Periodic Medical Examination The periodic medical examination should ideally be conducted annually for full-time operatives (e.g., in pesticide manufacture) or at midseason and the end of the work season for part-time employees (e.g., field workers). However, in determining the periodicity of medical examinations and special studies for individual cases, the advising physician should take into account factors that include age, gender, frequency and duration of exposures, and potential exposure dosages. Additionally, employees should be advised, ideally at the preemployment medical examination, that if they believe that they are developing pesticide exposure-
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related symptoms and/or signs, they should immediately seek medical attention. Examinations should include the following, the results of which should be documented: 1. History to include questions concerning, but not limited to, headache, dizziness, chest tightness, muscle weakness, neurobehavioral effects, and disturbance of visual function, including difficulty with focusing. 2. Physical examination to exclude, particularly, hepatorenal, cardiorespiratory, neurological, and visual dysfunction. Where there is reason to suspect adverse effects, appropriate special investigations may need to be undertaken (biochemical, physiological, and/or radiological). Occasionally, there may be difficulty in clinically differentiating neurobehavioral effects from exposure to anti-ChE and those resulting as a consequence of chronic alcoholism. In such cases, the measurement of ~/-glutamyl transferase and erythrocyte mean corpuscular volume, with the use of the CAGE questionnaire, can assist in the differential diagnosis (Lewinsohn, 1992; WHO, 1987). 3. Measurement of RBC AChE and serum BChE to detect any overexposure to anti-ChE.
D. Monitoring for Exposure to OP and CM Anticholinesterases A number of methods are available to monitor workers for exposure to anti-ChE pesticides that differ in approach, sophistication, technical basis, sensitivity, specificity, and cost. Choice of the method(s) will depend on several factors, including the chemical(s) applied, size and nature of the operation, facilities available, reason for monitoring, and regulatory needs. Exposure of the skin to pesticides may estimated by the following methods: R e m o v a l methods: This involves measuring residues on skin and clothing that remain after a specified exposure time. It can be carried out by measuring residues on swabs taken by skin wipes, by hand rinses, by skin stripping techniques, and determining residues on clothing. For rinses, the hand is placed in a plastic container holding approximately 200 ml of 95% ethanol and the fingers are briskly rubbed against the thumb and palm to remove particles. Two hand rinses were found to remove 96% of parathion from one hand soon after exposure (Durham and Wolfe, 1962). Wipe sampling and wash techniques may show a high degree of variability in recovery and recovery efficiency. They may also be of limited use when the pesticide or formulation used is highly volatile or has a rapid percutaneous absorption (Brouwe et al., 2000). It has been stressed by Durham and Wolfe (1962) that the hands of the worker should be clean before entering the field or trial area. Tape stripping involves the
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physical removal of the outer layers of the epidermis and can be used to estimate the quantity of material that has been absorbed into the skin (Nylander-French, 2000). With removal methods, the skin wipe and hand washing approaches have been successfully employed in studies on premature reentry into sprayed lands, paraoccupational exposure, and contamination from the domestic use of pesticides (Ballantyne and Marrs, 2004; Quandt et al., 2004; Thompson et al., 2003). Surrogate skin techniques: These involve placing a collection medium against the skin or clothing and subsequently analyzing it for pesticide. Common approaches are the use of whole body suits or the "patch technique," which involves attaching sorbent pads to the skin or clothing and subsequently measuring the pesticide load of the patches. Although a simple technique with limitations, it does allow a semiqauntitative estimate of surface contamination. Bias may be introduced because of the fact that patches measure pesticide deposition over a selected area. Also, differences in the type of material used for patch sampling, the body location sampled, and patch substance interaction with pesticide will all increase the variability of measurements. Highly absorbent knit white cotton garments that cover the entire body area of interest may provide a more accurate estimate of exposure (e.g., gloves to estimate hand exposure and short-sleeved undershirts for upper torso). The patch technique may be employed in predictive studies for contamination during in-use applications of pesticide. Visualization methods: For practical qualitative assessment of skin exposure, fluorescent tracer methods can be used. Fluorescent tracer can be added to the material being handled or processed to assess the area of contamination (Cherrie et al., 2000). This approach can be combined with video imaging analysis to allow some degree of quantitation (Fenske, 1997). This is achieved by determining the amount of surface area exposed and correlated with image intensity (Semple, 2004). Biomonitoring: This can be employed as an indirect method to provide an estimate of actual exposure dose (or at least the cutaneously absorbed dose). It involves quantitative analysis of parent material and/or metabolites in expired air, blood, and urine; from these analyses it can be calculated how much material has been percutaneously absorbed (Honeycutt et al., 2001). Cutaneous exposure modeling" This approach utilizes statistical and deterministic methods to aid in estimating the amount of pesticide deposited on the skin. One conceptual model of cutaneous exposure (Schneider et al., 1999) divides the worker's environment into six compartments" the source, air, surface contaminant layer, outer clothing layer, inner clothing layer, and skin. The following transport processes then characterize movement of the chemical within the environment: emission, deposition, resuspension/evaporation, transfer,
removal, decontamination, penetration/permeation, and redistribution. Inhalation exposures to pesticides in the work area can be estimated from measurements of concentrations of vapor, aerosol, or dust in environmental air (Ballantyne and Marrs, 2004). This can be done using passive samplers in the general work area or personal samplers in the employees breathing zone (Griffith and Duncan, 1992). However, in field situations in which pesticide samples are not of uniform size, and there may be moving variable concentrations in the air, the estimates may be misleading.
V. BLOOD CHOLINESTERASE MEASUREMENTS Mechanistically, OPs act by covalently binding with ACHE, which involves a chemical reaction between the phosphoryl ( ~ O ) moiety of the OP and the active site serine hydroxyl group of the catalytic center of AChE to form a phosphorylated enzyme. The phosphorylation of the catalytic center occurs with varying affinity constants, depending on the OP involved. Phosphorothionates ( ~ S ) must first be converted to the more reactive oxon ( ~ O ) form via oxidative desulfuration or, less commonly, by isomerization (Gallo and Lawryk, 1991; Thompson, 1992). The phosphorylated enzyme is stable and the rate of spontaneous reactivation depends on the chemistry and chirality of the attached phosphoryl residue. In the process of aging, there is loss of an alkyl group leading to stabilization of phosphorylated ACHE. This inhibition of AChE underlies the toxicity of OPs, leading to an excess accumulation of ACh and producing a cholinergic crisis. It follows that the measurement of AChE is a mechanistic basis for monitoring exposure to OP anti-ChEs and is a biomarker of effect. A similar consideration applies to CMs, except that since carbamylated cholinesterase is spontaneously and rapidly reactivated, ChE measurements may be unreliable and misleading in diagnosing or confirming CM poisoning. The mechanism of inhibition of AChE by CMs is similar to the catalytic hydrolysis of ACh (Wilson et al., 1966). Like the reaction with substrate, the inhibitor first forms a Michaelis-type complex with the enzyme. The activated serine hydroxyl group in the catalytic triad of the active sites then reacts with the carbonyl group of the CM to form a carbamylated ACHE. The overall inactivation of the enzyme is characterized kinetically by the bimolecular rate constant of inhibition (ki), which provides a measure of the inhibitory potency of the compound. When first-order kinetics are observed, the 150 may be calculated from the ki by the relationship 150 = 0.693/(ki • t), where t is the time of preincubation of inhibitor and enzyme before the addition of substrate, and 150 is the concentration of inhibitor resulting in a 50% loss of the enzymatic activity after preincubating the enzyme for a fixed time (Thompson and
CHAPTER 39 9Occupational Toxicology and Hygiene Richardson, 2004). Kinetic determinations of inhibitory potency are preferable to fixed-time measurements because they yield a more complete understanding of the inhibitory process (Aldridge and Reiner, 1972; Richardson, 1992; Taylor and Radid, 1994). Anti-ChEs inhibit both erythrocyte (RBC) AChE [Enzyme Commission Number (E.C.) 3.1.1.7] and plasma/ serum BChE (pseudocholinesterase; E.C. 3.1.1.8). Serum BChE is inhibited more rapidly than RBC AChE and restored to normal (control) activity within 3 months. RBC AChE is inhibited less rapidly by OPs and takes several weeks to several months to retum to normal activity (Jaga and Dharmani, 2003). RBC AChE is the most frequently measured enzyme activity for assessing chronic OP exposure since activity may not be restored to control values for approximately 2 or 3 months, but in acute cases it is preferable to measure both RBC AChE and serum BChE since inhibition of RBC AChE activity may take a few hours to occur. Serum BChE is the more reliable enzyme determination for acute OP poisoning, but with decreased serum BChE activity there is a need to exclude acute/chronic inflammation, liver disease, malnutrition, and atypical pseudocholinesterase variant. An individual with symptoms of acute cholinergic poisoning may not necessarily have low RBC AChE activity during this phase, and the degree-of inhibition may not correlate with acute cholinergic symptoms. Several different analytical methodologies are available for measuring serum and RBC ChE, including visible spectrophotomettic, gasometric, fluorimetric, radiometric, electrometric (pH stat), and gas chromatography; they have been reviewed in detail by St. Omer and Rottinghaus (1992). A portable battery-operated test kit, based on a colorimetric approach, is available for field use (Higgins et al., 2001). A variety of factors may influence measured normal ChE activities and should be taken into consideration before the practical significance in relation to working conditions is interpreted. These include the methodology (technique) used for the measurement, intra- and interperson variation, age, race, gender, disease, and genetic factors: Methodology: Laboratory methods may account for 40% of the variability in RBC AChE activity and 24% in plasma BChE (Hartvig et al., 1980; Yager et al., 1976). Using the same methodology in the same laboratory can reduce this variability. Intraperson variation: Although there is less variability between samples taken serially from the same individual than when samples are compared between individuals (Sawitsky et al., 1948), a significant intraperson variation does exist. This variation is less for RBC AChE than for plasma BChE. For example, Callaway et al. (1951) found that the percentage standard deviation [coefficient of variation (C~V)] for an individual around his own RBC AChE was 10.7% and for plasma BChE was 22.8%. Similarly, Sawitsky et al. (1948) determined the CV for
577
RBC AChE to be 10.2%, and for plasma BChE it was 30.2%. Davies and Rutland (1950) determined the CV for RBC AChE to be 15.4% and for plasma BChE to be 25.8%. It was proposed by Hayes (1982) that in normal workers unexposed to OPs, the expected variations in RBC AChE would be in the range of 13-25% and in plasma BChE 20-23%. Interperson variation: A review of 10 studies comparing OP-exposed groups to nonexposed controls showed a CV of interperson plasma BChE ranging from 14.9 to 30.7% among unexposed controls (Duncan et al., 1986). For a group of unexposed individuals, Kilgore et al. (1977) suggested that the CV for plasma BChE values was 19-33% (mean, 25.9%), with a standard deviation of 5.9%; the RBC AChE CV for the group was 5.97-27.5% (mean, 16.2%), with a standard deviation of 1.06%. These data indicate that interperson variations in ChE activities are greater than intraperson variations. Genetic factors: The presence of an atypical ChE leading to a reduced plasma BChE is relatively rare but should be screened for at the preemployment medical examination of potential employees. Disease factors: Plasma BChE activity may be reduced in liver cirrhosis, hepatic parenchymal disease, protein malnutrition, low serum albumin, myocardial infarction, and dermatomyositis (Balistreri and Rej, 1994; Duncan and Griffith, 1992; Vorhaus and Kark, 1953). Age, sex, and race: RBC AChE decreases with increasing age in adults (Gage, 1967), but serum BChE is not so affected. In general, gender has little influence on ChE activity, although plasma BChE is somewhat lower in females and fluctuates during the menstrual cycle (Gage, 1967), and during the first trimester of pregnancy and days 2-7 postpartum plasma BChE is reduced (Evans and Wroe, 1980). Although race is not considered to be a factor in determining normal ChE activity, Reinhold et al. (1953) reported lower plasma BChE activities in black compared to white people of the same sex. The major objective of ChE screening programs is to detect potential overexposure to anti-ChEs before the onset of definitive poisoning. To this end, it is necessary for a "cutoff" point to be decided on that dictates the need for action to be taken to avoid further exposure of the affected individual(s) and to undertake reviews to determine the cause for the overexposure and institute corrective measures. Ideally, the most appropriate time for measurements to be made is as soon as possible after exposure; this is particularly important with CMs because of their relatively rapid reactivation, and thus prompt analysis is also needed. Clearly, there is a need to avoid contamination of blood with anti-ChEs from the environment or the skin during sampling. Interpretation of the significance of ChE measurement in relation to working conditions may differ somewhat between individual experts and authorities.
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However, a typical discussion would indicate the following for serum BChE: 1. When the BChE activity is 75-100% of baseline, the individual is asymptomatic and should not be restricted from working. 2. When activity is approximately 50% of normal, symptoms may be present. 3. Values in the range of 20-50% of baseline activity indicate there has only been mild overexposure, and minimal symptoms may be present. 4. In the range of 10-20%, there has been moderate exposure, and more marked effects may be present, including muscle fasciculation and miosis. 5. Less than 10% activity indicates there is severe poisoning, with life-threatening effects from an acute cholinergic crisis. A decrease in serum BChE activity may result from a variety of disease and other processes, which need to be considered in the differential diagnosis. These include hepatitis, hepatic cirrhosis, and various drugs. Similar considerations apply to inhibition of RBC AChE. Inhibition of RBC AChE to <50% of baseline is usually regarded as diagnostic of OP intoxication but is not directly related to the severity of poisoning. This could be regarded as somewhat surprising since symptomatic poisoning by OPs is caused by inhibition of neural ACHE, the same gene product as the RBC ACHE, but the nervous system is usually less accessible than the erythrocyte to inhibitor. RBC AChE is associated with the plasma membrane and thus readily accessible for inhibition [International Programme on Chemical Safety (IPCS), 1986a; Jeyaratnam and Maroni, 1994; Ple~tina, 1984; Wilson et al., 1992, 1997]. Thus, the ratio of AChE inhibition in nervous tissue compared to the RBC is less than unity. However, there are significant differences in the penetration of the CNS by different OPs, and although OPs will have better access to RBC ACHE, the differences in AChE inhibition between the CNS and the RBC may be trivial for those OPs that readily cross the blood-brain barrier. Hence, measurement of RBC AChE inhibition may overestimate that in CNS tissue. Also to be taken into consideration when interpreting the significance of RBC AChE inhibition are the high interindividual and somewhat lower intraindividual variability. Thus, it is generally believed that only reductions of >20% on normal average values and of 10-20% on individual preexposure values can be considered significant (Lotti, 2001). The following surveillance protocol recommendations have been suggested: (i) If AChE activity is reduced by 30% or more of the preexposure level, repeat the test, (ii) decreases of 20-25% are diagnostic of exposure but not of hazard, and (iii) decreases of 30-50% are an indication for removal from further exposure with anfi-ChE until AChE returns to control levels (Lewinsohn, 1992). Inhibition of RBC AChE by dimethyl phosphates can be partially reversible; thus, this needs to be taken into account if a meas-
urement is not carried out immediately postexposure. Additionally, the rate of regain of AChE activity due to resynthesis of the enzyme occurs more rapidly in nervous tissue than in the RBC; animal data demonstrate a tl/2 in nervous issue of 5-7 days (Lotti, 1992) compared with reappearance of enzyme activity due to erythropoiesis at 1% per day (Lotti, 2001; Mason, 2000). Severe symptoms and mortality are generally believed to occur with RBC AChE inhibition of > 8 0 and >90%, respectively, in the absence of treatment (Moretto, 2004). The specific decreases in ChE activity that demand active intervention in employee work status vary among different authorities, but typical of the thresholds for removal of workers from exposure are those given by the California Health Services Department, which recommends removal of workers from further exposure when the RBC AChE activity reduces to 60% of baseline and serum BChE reduces to 50%; they are permitted to return to work when ChE activities increase to 75% of baseline (Ames et al., 1989; Jaga and Dharmani, 2003). For inhibition of ACHE, other values for intervention range from 30-60% against preexposure values to 50-70% against normal reference values (Jeyaratnam and Maroni, 1994). Ple~tina (1984) suggested waiting until RBC AChE returns to normal before allowing readmission to work. The World Health Organization (WHO, 1987) commented on the suggestion that a decrease of 30% in AChE activity from the baseline is a biological threshold for withdrawal of workers from anti-ChE exposure; they noted that this threshold has not been substantiated by existing medical and epidemiological information and may have been proposed based on the accuracy of the methods available at the time of its proposal. In the context of individuals with established OP poisoning who were admitted to the hospital within 6 hr, Aygun et al. (2002) found that low serum AChE levels (>50% of minimal normal value) supported a diagnosis of OP poisoning, but there was no significant association between serum ChE and the severity of poisoning on day 1, with the serum ChE activities of patients with severe poisoning not being statistically significantly different from those with mild poisoning. There was no significant difference in AChE levels between patients with delayed-onset neuropathy and those without, indicating that serum ChE is not useful as a predictor for delayed-onset neuropathy. However, patients who died did not show an increase in serum AChE between day 1 and time of death, in contrast with patients who survived, who showed a significant increase from day 1, indicating that serum AChE may be a useful predictor of prognosis of acute OP intoxication. Therefore, the investigators concluded that although a decrease in serum AChE activity during the cholinergic crisis of acute OP poisoning supports a diagnosis, it does not reflect the severity of poisoning, and although it is a useful indicator for following the acute prognosis, it is not a predictor for the development of
CHAPTER 39 9OccupationalToxicology and Hygiene delayed-onset polyneuropathy. CM anti-ChEs inhibit ChE activity by a reversible spontaneous hydrolysis and carbamylation of ACHE, with a rapid onset. ChE activity in workers exposed to CMs is as low as that with OPs, making it difficult to differentiate OP and CM poisoning on the basis of enzyme inhibition alone. Measurement of CM-inhibited AChE is limited by the rapid reversibility of the complex, and the time interval between sampling and analysis should be as short as possible. Blood samples should be kept cold and sample dilution during measurement should be kept to a minimum (Lotti, 1991).
VI. URINE/BLOOD METABOLITE MEASUREMENTS Monitoring anti-ChEs or their metabolites in urine and blood can be employed in two ways. The first is to give an accurate estimate of potential risk and requires a determination of the systemic dose received by the worker that can be equated to doses used in laboratory studies. This requires knowledge of the metabolism and toxicokinetics of the material and obtaining specific timed samples of blood and urine for analysis and accurate calculation of absorbed doses of the parent material. Second, for screening workers for exposure to an anti-ChE, there is a need to measure metabolite to qualitatively determine that there has or has not been exposure to the anti-ChE, or else to quantitatively measure the concentration of metabolite to ensure that it has not exceeded a specific level known to be associated with the development of pesticide-induced adverse health effects. Measurement of metabolites of anti-ChEs in urine or blood samples is an established method of monitoring for OP exposure and is a biomarker of exposure. For example, alkyl phosphates have been used to monitor exposure to OPs Carrier et al., (1999). However, the metabolism of OP compounds is variable, with some being extensively altered and others excreted mostly unchanged. The most common hydrolytic pathway is fission of the P-ester bond giving the alcoholic moiety ("leaving group") and the acidic moiety [alkyl(thio)phosphates]. Dimethylated OPs give dimethyl phosphates, dimethyl thiophosphates, and dimethyl phosphorothioates, whereas diethylated OPs give the corresponding diethylated metabolites (Coye et al., 1986; IPCS, 1986a). Since they can be derived from a large number of compounds, alkyl phosphates are nonspecific metabolites. Therefore, the compounds to which the employee is exposed must be known if a toxicological significance is to be attached to the analytical data. For example, dimethylated OPs have a wide range of acute toxicity and yet may give the same amount of dimethyl phosphates; the same consideration applies to diethylated OPs. Alkyl phosphate metabolites of OPs that have been extensively used as markers of occupational exposure to OPs include O,O-dimethyl phosphate (DMP), O,O-diethyl
579
phosphate (DEP), O,O-diethyl thiophosphate (DTP), O,O-dimethyl dithiophosphate (DMDTP), O,O-diethyl dithiophosphate (DEDTP), and O,O-dimethyl thiophosphate (Bravo et al., 2002; Hardt and Angerer, 2000; Linet al., 2002; Oglobline et al., 2001). Measurement of the urinary excretion of the alcoholic moiety may be more specific but is less frequently employed (Moretto, 2004). These include 3,5,6trichloro-2-pyridinol after exposure to chlorpyrifos-methyl and chlorpyrifos-ethyl (Nolan et al., 1984), malathion monoand dicarboxylic acids after exposure to malathion (Bradway and Shafik, 1977), and p-nitrophenol after exposure to parathion (Michalke and Daldrup, 1982; Morgan et al., 1977). Differences in the toxicokinetics of individual OPs and the route of exposure may further complicate the interpretation of data. Therefore, the timing of urine sampling is critical and has to be chosen according to the characteristics of the compound and the exposure route. In most field studies using urinary metabolites to assess OP exposure, there is insufficient data to allow a determination of any correlation between the amount of urinary metabolite and the degree of inhibition of RBC AChE and/or plasma BChE. In most published field studies, RBC AChE was found to be not or only minimally inhibited (Aprea et aL, 1997; Griffin et al., 1999; Jauhiainen et aL, 1991; Kraus et aL, 1977; Krieger and Thongsinthusak, 1993; Maroni et aL, 1990; Popendorf et aL, 1979; Spear et al., 1977). The only good correlation found has been between urinary p-nitrophenol and RBC AChE in workers exposed to parathion (Arterberry et al., 1961), which supports the biological exposure index (BEI) established by the American Conference of Governmental Hygienists (ACGIH, 2004) of 0.5 mg p-nitrophenol g-1 creatinine. Measurement of urinary p-nitrophenol is a relatively specific monitor for parathion exposure, and it is also a metabolite of parathion-ethyl and EPN. An approximate time interval of 8 or 9 hr is required from first exposure to parathion to peak p-nitrophenol excretion. Concentrations of urinary p-nitrophenol in the general population are 0.01-0.03 mg liter -1 (Baselt, 1980), and little or no decrease in ChE activity occurs when the urinary pnitrophenol concentration does not exceed 2.0 mg m1-1. For the rapid and sensitive quantitation of unchanged OP pesticide in urine and other biological samples, Tarbah et al. (2001) used a gas chromatograph with nitrogen/phosphorussensitive detection and electron impact gas chromatographymass spectrometry (GC-MS). A r a p i d and sensitive quantitative analysis for alkyl phosphate metabolites in urine, using GC-MS, has been developed by Kupfermann et al. (2004); they examined DMP, DEP, DMDTP, and DEDTP. A GC-MS method with deuterated DMP-d6 as an internal standard was developed and validated by Tarbah et al. (2004) for measurement of blood and urinary DMP. This is a metabolite of, for example, phosphamidon, mevinphos, monocrotophos, dicrotophos, dichlorvos, and trichlorofon. From the previous considerations, it follows that the measurement of plasma or urinary parent OP or its metabolite(s) can be used to monitor occupational exposure, providing the appropriate analytical
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Toxic Effects
techniques are available, but the resulting data can only be used to give a qualitative index of exposure rather than a toxicologically significant value (Moretto, 2004). However, this approach can be used to widely and noninvasively examine anti-ChE exposure in occupational situations, paraoccupational conditions, domestic applications, and exposure from food residues (Fenske, 1997; Hendorf et al., 2004). Many OPs are unstable in aqueous solution and, because of the presence of esterases, are not stable in blood. OPs are degraded more rapidly by esterases than by other biochemical mechanisms (Bouaid et al., 2001; Bowman and Sans, 1980; Drevenkar et al., 1983; O'Brien, 1960; Singh et al., 1985). Organophosphothioates, such as chlorpyrifos-methyl, are unstable in aqueous media but stable in blood for at least 2 hr, possibly due to stabilization of chlorpyrifos-methyl by proteins and lipids (Moriya et al., 1999). Stability of OPs in serum samples during storage was measured for 23 different OPs, which were mixed with serum containing 10 mg m1-1 EDTA and stored up to 10 days at 4 and - 2 0 ~ (Tarbah et al., 2001). Recovery rates of the OPs ranged from 50% (dimethoate) to 133% (dialifos). Stability studies on DMP have shown it to be stable over 2 weeks at 4 and - 2 0 ~ and additional studies showed no evidence of degradation when spiked in fresh blood and stored at 4 ~ for 1 week and stored in water for 10 months (Tarbah et al., 2004). In a case of selfpoisoning with phosphamidon by a 19-year-old female, the concentration of phosphamidon in serum decreased from 10 to 4.4 mg liter-1 after storage at - 2 0 ~ for 6 months; nearly complete degradation was found after 3 years (Tarbah et al., 2004). DMP was found in the body fluids stored at - 2 0 ~ for up to 3 years (respective values at 20 and 36 months of storage were blood, 3.9 and 4.9 mg liter-l; urine, 33.5 and 50.4mg liter-l; gastric fluid, 8.1 mg liter -1 and not detected). It is recommended that an examination for stable metabolites in suspected cases of OP poisoning should be performed. Unlike the situation with blood ChE inhibition measurements, the determination of CM metabolites may be useful to confirm and follow suspected CM-induced toxicity. CMs usually undergo extensive metabolism, particularly by carboxylesterases forming an aryl alcohol and a methylor dimethyl-amine. However, the rate of metabolism is dependent on the structure of the individual CM. Side chains may also undergo oxidation (e.g., hydroxymethylation), N-demethylation of secondary amines attached to the aryl moiety, or ring hydroxylation via the formation of an epoxide intermediate (Moretto, 2004). Thiocarbamates (e.g., aldicarb) may undergo S-oxidation forming the corresponding sulfane and sulfoxide. Urinary metabolites are mainly in the form of glucuronide or sulfate derivatives of the aryl groups. The parent compound may be found in small amounts in urine (IPCS, 1986b). Specific measurements for assessing worker exposure to CMs have included free and conjugated 1-naphthol from carbaryl (IPCS, 1986b, Lauwerys, 1982), 3-hydroxycarbofuran and
3-ketocarbofuran from carbofuran (Huang et al., 1989), 2- (di)methylamino-4-hydroxy-5,6-dimethylpyrimidime and other hydroxypyrimidimes from pirimicarb (Hardt et al., 1999; Verberk et al., 1990), and 2-isopropoxyphenol from propoxur (Brouwer et al., 1991, 1993). With carbaryl, in healthy nonexposed individuals the urinary 1-naphthol concentration is <0.3 mg liter -1 and in carbaryl-exposed subjects it is >4.0 mg liter -1 (Lauwerys, 1982). However, correlations of the chemical analyses of CMs or CM metabolites in urine have not been obtained with blood ChE measurements. Although sensitive GC and GC-MS methods are available for OP and CM measurements, they can be timeconsuming, require cleanup procedures, and involve the use of expensive equipment. Immunochemical assays may overcome some of these problems. Assays have been developed for parathion (Ercegovich et al., 1981) and paraoxon (Brimfield et al., 1985; Hunter and Lenz, 1982), as well as for certain CMs, including aldicarb (Mumma and Brady, 1987), benomyl, and carbendazim (Lukens et al., 1977; Newsome and Shields, 1981). These immunochemical assays have the advantages over traditional chemical analyses in that they require only minimal sample cleanup and permit high sample throughput, but they have the limitation that any one antibody allows only a very restricted set of structurally similar cross-reacting residues. Genetic engineering approaches for the development of monoclonal antibodies should enhance the potential for immunochemical techniques in qualitative and quantitative screening for anti-ChEs and metabolites.
VII. OTHER BIOCHEMICAL AND PHYSIOLOGICAL MONITORS OF OP/CM EXPOSURE Studies of greenhouse workers chronically exposed to antiChEs showed significant increases in acid phosphatase and [3-glucuronidase activities with decreased paraoxanase; the paraoxanase B allele was associated with a higher susceptibility to anti-ChE pesticides (Hermindez et al., 2004). Increases in creatine phosphokinase (CPK) may be an indication of myopathy resulting from exposures to anti-ChEs years previously. Friedman et al. (2003) described two patients who had elevations in CPK a decade after exposure to anti-ChEs. Both patients suffered from progressive generalized muscle weakness, chronic fatigue, myopathy, neuropathy, and marked neurobehavioral impairments. Measurement of lymphocytic neuropathy target esterase (Bertoncin et al., 1985) has been suggested as a monitor for exposure to neuropathic OPs, but its large interindividual variability, the different access of OPs to the nervous system, and the different rates of resynthesis between the nervous system and lymphocytes limit, its usefulness (Moretto, 2004).
C H APT ER 3 9 9Occupational Toxicology and Hygiene Neurophysiological monitors for peripheral effects include electromyography (EMG), nerve conduction studies, neuromuscular conduction techniques, and microelectrode studies. For central effects, techniques available are EEG and evoked potentials. Monitoring for delayed-onset neurotoxicity can be undertaken using EMG; with conventional EMG the motor units recorded are summated potentials of muscle fibers in a motor unit within a few millimeters of the recording electrode. EMG signals are analyzed for insertion activity, spontaneous activity, motor unit potential analysis, and recruitment pattern on voluntary muscle contraction. Some authorities regard EMG as a useful and sensitive technique (Jager et al., 1970; Roberts, 1976), but its sensitivity has been questioned by others (Le Quesne and Maxwell, 1981; WHO, 1986). EMG changes were found in 50% of workers exposed to dimethyl phosphate esters and consisted of low-voltage and repetitive activity (Jager et al., 1970). Motor nerve conductiqn involves stimulating the nerve, usually with a supramaximal square wave pulse, and recording the compound motor action potential using surface or needle electrodes. The evoked compound motor action potential is described by latency, amplitude, duration, and configuration. AChEs produce twitch potentiation, fasciculations, and tetanic fade. In twitch potentiation, there is potentiation of the tension developed by skeletal muscle in response to submaximal or maximal indirect stimulation at low frequency (Koelle and Gillman, 1949; Werner and Kuperman, 1963). Fasciculations are produced by OPs (Meer and Van der Meter, 1956) and CMs (Blaber and Goode, 1968) and result from intermittent synchronized contraction of muscle fibers of a motor unit, which is probably due to the initiation of an axon reflex allowing released ACh to have access to the first node of Ranvier and to depolarize it in the absence of nerve stimulation (Misra, 1992). Fasciculations occur within a limited range of AChE inhibition, disappearing when inhibition is >95% (Barnes and Duff, 1953). Indirect stimulation of skeletal muscle at >20 Hz for a brief period results in a sustained increase in tension, but following exposure to anti-ChEs the response to such stimulation consists of a rapid increase in tension followed by partial or complete relaxation while the stimulation is maintained; this is called tetanic fade (Blaber and Bowman, 1963). Tetanic fade is associated with inhibition of AChE (Misra, 1992) and caused mainly by postsynaptic block. Singlefiber EMG records from individual fibers within 300 lxm of the electrode, and the potential is biphasic, with a rise time of the negative phase of 200 lxsec, duration of 1 msec, and amplitude usually of 5-10 mV. Motor nerve stimulation propagates distally (orthodromically) and also proximally (antidromically) toward the anterior horn neuron cell bodies, which can be activated. The recurrent discharge produces small potentials after a delay of approximately 20-50 msec, which is called the F-response (Magladrey and McDougal, 1950). They provide information about
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conduction in the proximal segment of the motor fibers and may be a more sensitive index of peripheral nerve dysfunction than standard nerve conduction studies (Latchman et al., 1980). Sensory nerve conduction can be evaluated by stimulating and recording from a cutaneous nerve using surface or needle electrodes. Repetitive nerve stimulation techniques involve stimulating a muscle by repetitive supramaximal stimuli through the motor nerve. A decline in the amplitude of the evoked potential provides a measure of the degree of neuromuscular block. EEG changes in acute OP exposure have been reported to cause marked desynchronization and a triad of changes consisting of increased high-frequency activity, decreased low-frequency activity, and lowered background voltage. High-concentration exposures cause slowing of the EEG followed by the development of spike wave discharges that accompany convulsions (Burchfeil et al., 1976). The EEG is regarded by many as not being sufficiently sensitive or specific to discriminate between normal (control) subjects and neurobehaviorally affected OP-exposed individuals (Savage et al., 1988). Details on the use of neurophysiological monitors for exposure to anti-ChEs have been discussed in detail by Misra (1992) and WHO (1986).
VIII. WORKPLACE BIOMONITORING In addition to the control of workplace exposure to pesticides by the use of airborne exposure guidelines, BEIs may be recommended based on appropriate urine or blood analyses (Helath and Safety Execution, 1987). These BEIs can be applied not only to plant exposures but also to accident sites and where there are formulation and in-use applications. BEIs are guidance values for assessing biological monitoring results and represent the levels of determinants that are most likely to be observed in specimens collected from healthy workers who have been exposed to the chemical under examination to the same extent as workers with inhalation exposure at the TLV. Exceptions may be chemicals whose TLV is based on nonsystemic effects (e.g., irritation). The BEI in general indicates a concentration below which nearly all workers should not experience adverse health effects. They apply to 8-hr exposures for 5 days a week. Biological monitoring can assist the occupational health professional to determine absorbed dose of chemical and can assist in determining the efficacy of personal protective equipment, engineering controls, and general work practices. For example, the OP parathion has BEIs based on a urinary metabolite and on RBC ACHE. In urine, the determinant is total p-nitrophenol, with a BEI of 0.5 mg g-1 creatinine, and for RBC ACHE the BEI is 70% of the individual's baseline (ACGIH, 2004). Gosselin et al. (2005) developed a multicompartment model to describe the kinetics of parathion and its metabolites, p-nitrophenol and alkyl phosphates, in order to assess worker exposure and health risks. For percutaneous
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Toxic Effects
absorption, except for the cutaneous absorption fraction and \ absorption rate, which are known to show wide intraperson and interperson variability, a single set of parameter values for the internal body kinetics enabled the model to simulate accurately the available kinetic data. For dermal absorption to parathion, with a typical absorption rate of 0.085 hr-1, model simulations showed that it takes 20 hr to recover half of the total amounts of p-nitrophenol eventually excreted in urine and 30 hr for the alkyl phosphates. The model can be used to estimate the dose of parathion absorbed under different exposure routes and temporal scenarios, based on measurements of the amounts of metabolites accumulated in the urine over given time periods. Using the dose-excreta links and the human no observed effect level for parathion inhibition of cholinesterase activities, biological reference values can be established to prevent adverse health effects in exposed workers in the form of specific amounts of urinary metabolites excreted over selected periods of time.
IX. O C C U P A T I O N A L H Y G I E N E CONSIDERATIONS FOR RESTRICTION OF ANTICHOLINESTERASE EXPOSURES In view of the fact that exposure to pesticides occurs in a variety of circumstances and conditions, although there are factors common to all these, it is necessary to define occupational hygiene measures for each individual situation. For example, although workplace exposure guidelines such as TLVs are appropriate to use for worker protection in enclosed environments such as production plants, these cannot be applied to open-air situations. Occupational hygiene measures are developed and designed in order to give reliable protective and precautionary approaches against the development of morbidity and/or mortality in circumstances of potential overexposure.
X. G E N E R A L I Z A T I O N S A N D A C T I O N S COMMON TO ALL POTENTIAL OCCUPATIONAL OVEREXPOSURE CIRCUMSTANCES The following considerations apply to all situations in which OP and CM anti-ChEs may be manufactured, formulated, transported, and used in occupational environments.
A. Advance Planning This covers items such as ensuring that the least toxic material and formulation to achieve the desired pesticidal effect are used; ensuring that all necessary inspection and repairs have been carried out; and, where possible, determining the best time from a meterological perspective to undertake application (such as low wind speed to limit drift).
B. Education and Training It has been estimated that in the United States approximately two-thirds of farm workers have not received any information or training on pesticide safety (Arcury et al., 1999). Also, as noted previously, in a survey of Latino farm workers, Arcury et al. (2000) found that it was believed that not all farmers necessarily adhere to regulations mandating training and basic sanitation. They noted that regulation by itself is not an advantageous starting point, and intervention must include educating farmers as well as farm workers. For a developed country, it should be mandatory that everyone, at all levels of employment status, who may involved in work in which there is a potential for exposure to anti-ChEs should be adequately informed and trained about general aspects of anti-ChEs and specific aspects relevant to their occupation. Education and training sessions should at least cover the following: 1. The names, chemistry, and physicochemical properties of the anti-ChEs that may be encountered during work activities. 2. Routes of exposure and how overexposures to pesticides may occur. 3. The potential health hazards of pesticides and other chemicals that may be handled or encountered, and the signs and symptoms of intoxication. 4. Protective and precautionary measures needed to ensure safe working conditions, including protective clothing and equipment. There is a clear need for a respirator training program. 5. Emergency first aid measures (including controlled decontamination) and procedures to be adopted locally in the event of an accident leading to overexposure, including those who are to be immediately informed and how to obtain emergency medical information and care. The latter should include ensuring ready availability to telephone communications to a local hospital accident/ emergency center and the nearest poison control center. 6. Environmental and health hazards from contaminated clothing, spills, drift, and take-home path. 7. Warnings necessary, including oral and written reentry and warning notices, informing local inhabitants about applications. 8. Any relevant written information should be given to employees, who should be instructed to read manufacturers literature, material safety data sheets (MSDS), and container labels. Compound-specific MSDSs should be written by the pesticide manufacturer giving all necessary physicochemical, toxicology, medical, protective and precautionary, and regulatory information. These MSDSs should be made available for wide distribution to employees, users, poison control centers, and those who request them. Details on the intent, structure, and contents of the MSDS have been discussed elsewhere (Tyler and
CHAPTER 39 9Occupational Toxicology and Hygiene Ballantyne, 1988). Product labeling is a very important component in ensuring the safe use of an anti-ChE pesticide (Laughlin and Gold, 1988). Registration of a pesticide requires that the manufacturer provide an understandable and informative label for the user that must have the approval of the appropriate registration authority [e.g., the Environmental Protection Agency (EPA)]. The label should provide information on the following: active ingredient and concentration; approved uses; specific methods of handling the chemical; methods on preparation and application; guidance on storage and disposal of unused chemical; safe use, including protective and precautionary measures; statement on significant adverse health effects and of medical management and antidotes; first aid measures; and a manufacturer's emergency telephone number (ideally manned for 24 hr) from which expert advice on dealing with accidental situations or emergency health issues can be obtained (Ballantyne, 1975; Griffith and Duncan, 1992).
C. Audit of the Health and Safety Program There should be provision to undertake frequent, but random, audits of the following: 1. Educational and training sessions. 2. That collective and personal protective and precautionary measures are being strictly followed, including adherence to workplace exposure guidelines (e.g., TLVs). 3. Protective equipment and clothing are being kept clean and are readily available. 4. Respirators are being maintained, and respirator training and fitting sessions are adequate. 5. Engineering controls are being adhered to. This includes confirmation of the following: (i) Raw materials are delivered in bulk by railcar, pipeline, barge, or tanker truck and are mechanically unloaded, thus ensuring minimal handling by employees; (ii) raw materials are packaged in sealed containers; (iii) where possible, weighing and dispensing of raw materials are automated; (iv) mixing equipment, milling machines, and baggers should be enclosed, and emissions should be dealt with by other engineering controls; (v) a plant manufacturing potent anti-ChEs should not be sited ina heavily populated area; and (vi) there should be closed-transfer systems for use by employees mixing and loading liquid pesticide. 6. There is provision for atmosphere monitoring in situations in which a workplace exposure guideline is applicable (e.g., plant facility); the sampling times, sampling methods, and analysis should all be examined and judged to be adequate and appropriate. 7. Periodic medical surveillance is carried out, with all necessary health monitoring requirements for general health and to exclude anti-ChE overexposure.
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D. Supervision There should be one or more senior operatives who are knowledgeable and well trained in precautionary and protective measures and also compliance with pesticide regulations. They should be responsible for the day-to-day supervision of plant operations, applications in the field, etc. No person should work alone while handling or likely to be exposed to anti-ChEs. There should always be an additional person nearby, designated as safety officer, who can view the operation at a distance sufficient, if necessary, to raise an alarm, to allow protective clothing and equipment to be donned, and to undertake the initial rescue and possible first aid and resuscitation measures. Thus, ideally this person should be trained in the emergency measures for an anti-ChE leak, spill, and exposure, and it is desirable for him or her to be trained in first aid measures to treat OP and CM poisoning.
E. Protective Clothing Where there is likely to be direct skin contact with antiChEs, protective clothing should be available, and when the circumstances indicate the need, it should be donned. This includes loss of containment in a production facility; an accident during transportation; handling of the pesticide during formulation, mixing, or loading; and during in-use application. In such circumstances, there is a need for eye protection (goggles) and clothing that should cover the trunk, arms, hands, legs, feet, and face. Although face shields give protection of facial skin, they may provide only partial protection of the eyes. Thus, for full protection of the eyes, goggles are needed. Clothing should not have external cuffs or open pockets in which hazardous materials can accumulate, and sleeves should be of adequate length. Gloves should be chosen on the basis of impermeable characteristics and be carefully inspected for integrity before use. Boots or shoes should be mechanically sound and made of appropriate impermeable material. It is of absolute necessity to ensure that the recommended protective practices are followed; for example, agricultural workers frequently do not wear protective gloves even though they have been advised to do so (Webster and Maibach, 1985a,b).
F. Respirator Use Where there is likely to be inhalation overexposure to anti-ChE vapor, aerosols, or dusts, appropriate respiratory equipment should be available. There are, descriptively, two main types of respirators (Ballantyne and Schwabe, 1981). First are air-purifying respirators in which environmental air is drawn through a nonreturn valve and an appropriate absorbent filter material that removes atmospheric contaminants (particulates, vapor, or gas), and
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N o n s p e c i f i c Toxic Effects
expired air is eliminated through a nonreturn exhalation valve. This type of respirator is clearly of use for working in an atmosphere containing a toxic material for which an absorbent is available, but clearly not of use if there is also an oxygen-deficient atmosphere. Second are atmosphere-supplied respirators (breathing apparatus) in which an independent source of uncontaminated air or cylinder gas is introduced into the respiratory airspace. They can be used for prolonged periods and in many types of atmospheres. A decision regarding the type of respirator to be used needs to take into consideration various issues, including the type, toxicity, and physicochemical properties of the pesticide used; whether required for indoor or outdoor use; and whether they can be adequately stored and serviced at the user facility. A respirator-servicing program needs to be introduced to ensure that the respirators are periodically cleaned, inspected for damage, and checked for adequacy of function. A periodic check of individual respirator fit is needed to ensure continued adequacy of function with fit. As part of the preemployment medical, it should be determined if the potential employee can psychologically tolerate the wearing of a respirator and if there are any medical contraindications, such as respiratory disease. An employee training program should be introduced to ensure the continued appropriate maintenance and use of the equipment.
G. Good Personal Hygiene Good personal hygiene should be ensured to prevent contamination of the employee and avoid a take-home path. This includes frequent change of clothing, adequate washing facilities, and no eating, drinking, or smoking while there is a potential for exposure. In one survey, Griffith and Duncan (1983) found that 56% of field workers did not wash their hands within 15 min of leaving the work area. The provision of washing facilities, ideally with showers, is an important occupational hygiene necessity for agricultural workers.
H. Treatment of Established Poisoning If an on-site physician is not employed, there should be appropriately trained and qualified nursing or first aid staff who are promptly available and can undertake the initial elements in the management of acute anti-ChE poisoning. First responders should be familiar with the clinical presentation of acute anti-ChE poisoning, such that they are confident in the recognition of an individual with intoxication who requires treatment. The management of anti-ChE poisoning involves the following: 1. Decontamination. The person(s) undertaking decontamination should be suitably protected to avoid becoming contaminated from the victim (Abraham and Weinbrom,
2003). Generally, decontamination measures are removal of clothing and washing affected areas. 2. Maintenance of an adequate airway and breathing is necessary if there is obstruction resulting from bronchial secretions and bronchospasm. This may require the use of an oral airway (or insertion of an endotracheal tube if medical assistance is available) and assisted ventilation (e.g., mask with manual inflator). Oxygen is valuable because of the potential for hypoxia resulting from airway obstruction secondary to increased tracheobronchial secretions and bronchospasm (Munidasa et al., 2004). When hospitalized, endotracheal intubation and assisted ventilation may be required (Proudfoot and Vale, 1996). 3. Antidotes. Atropine, a muscarinic receptor antagonist, is particularly effective and important in the acute management of OP poisoning (Leenders et al., 2003). Full and early atropinization is essential to reverse cholinergic excess and improve respiratory function, heart rate, and blood pressure (Eddleston et al., 2004). Atropine should be given intramuscularly at the accident site and may be given by intravenous infusion in the hospital. Atropine is important in reducing tracheobronchial secretions, but it does not have a significant effect on nicotinic ACh receptors and therefore ventilatory muscle weakness persists. Early deaths result from respiratory failure and cardiovascular collapse (Eddleston et al., 2004; Munidasa et al., 2004). However, atropine does cross the blood-brain barrier and counteracts the convulsive effects of OPs. Because of the possibility for ventricular arrhythmias when atropine is used in the presence of cyanosis and/or an ischemic myocardium, oxygen should be administered in these situations. Dosage is best titrated on the basis of clinical signs, notably resolution/reduction of bronchospasm and bronchosecretion and a sinus tachycardia to approximately 80-100 beats/min (Leenders et al., 2003). Oximes are of importance for reactivation of phosphorylated AChE and used in combination with atropine (Eyer et al., 2003; Vucinic et al., 2003; Zilker, 2004; Zilker et al., 2004). Oximes are therapeutic by removing the phosphoryl group from AChE and thus restoring its catalytic sites (ChE reactivators). This antidotal action only occurs when the phosphorylated AChE has not undergone the intramolecular rearrangement of aging. Ideally, for the immediate treatment of OP poisoning, they would best be given intramuscularly by an autoinjector, but these are not readily available in a civilian first aid situation. In the context of occupational OP exposure, there are no clinically important differences between pralidoxime, obidoxime, and HI-6 (Marrs et al., 2003). Therapeutic benefit requires early and sustained treatment with high (effective) oxime doses (Lotti, 2003; Proudfoot and Vale, 1966; Thermann et al., 2003). Although the administration of oximes should occur at the earliest possible
CHAPTER 39 9Occupational Toxicology and Hygiene time after intoxication, their late administration can be beneficial in cases in which there is a possibility of prolonged absorption and/or high lipid solubility, such as may occur with fenitrothion, fenthion, and clofenthion (Burillo-Putze et al., 2004). Anti-ChE-induced centrally mediated seizures may develop into a clinical condition resembling status epilepticus. The administration of an anticonvulsant early in the management of intoxication is recommended (Ballantyne and Marrs, 1992). Diazepam and phenytoin have been recommended, although these may not provide optimal protection. Fosphenytoin, alone or in conjunction with diazepam, has little or no therapeutic anticonvulsant effectiveness for OP-induced convulsions (McDonough et al., 2004).
I. Environmental Issues In order to ensure that there is no harm to the environment from accidental discharges from plants or from drainage as a consequence of in-use applications, and for compliance with state and federal regulations, there should be frequent measurements of anti-ChEs in soil, water (rivers and groundwater), and surrounding air.
XI. O C C U P A T I O N A L HYGIENE CONSIDERATIONS IN CLOSED FACILITIES In the manufacture of pesticides, exposure of factory workers is probably negligible in well-organized plants with good industrial hygiene practices, but during formulation there may be additional factors to be considered, Overall, in closed facilities, the general considerations discussed previously need to be followed, and in production sites for anti-ChE pesticides, special attention is needed as follows.
A. Atmosphere Dust particles in the air of a closed environment can be inhaled or trapped in saliva or mucus and swallowed. Also, the volatility of liquids has to be taken into consideration during the design of engineering controls using local exhaust methods, which are the most widely used methods of atmosphere control in manufacturing facilities. They are designed to capture or contain emissions at their source before they can reach the workplace atmosphere. General ventilation systems provide clean air to, and remove air from, the workplace to provide frequent and adequate "tumover" of air and hence maintain contaminants at concentrations below those considered to represent a health hazard; however, this method is not generally considered appropriate to control exposures to antiChEs. Exhausted air may need to be treated to ensure that anti-ChE concentrations do not cause any local environmental or human population risks. In the atmosphere of a production
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plant, there may be accumulation of anti-ChEs if there is a breakage of containment, if the engineering controls for ventilation fail, or if a spill occurs. Ideally, there should be an automatic warning system to indicate if this situation develops, and this should be maintained and tested periodically. A senior person at the site (ideally an industrial hygienist) should ensure that sampling is taken for routine measurements to confirm adherence to the recommended workplace exposure guidelines, such as the TLVs, with any qualifications proposed by ACGIH (2004); examples of these are given in Table 2. Govemment-mandated permitted exposure limits may apply in many countries. For example, in the United States, the Occupational Safety and Health Administration sets permissible exposure limits, and in the United Kingdom maximum exposure limits and occupational exposure standards are set under the Control of Substances Hazardous to Health regulations of the Advisory Committee on Toxic Substances and Working Group on the Assessment of Toxic Chemicals of the Health and Safety Executive. Clearly, these airbome workplace exposure guidelines are relevant to enclosed areas (factories, greenhouses, etc.) but cannot be applied to outdoor work situations (Ballantyne and Marrs, 2004). The acceptable occupational exposure level (AOEL) is defined by the Directive of the European Union 91/414 concerning the placing of plant protection products on the market as "the maximum amount of active substance to which the operator may be exposed without any adverse health effect" and it is expressed as mg kg -1 (body weight) day -1 of absorbed (by any route of exposure) dose available for systemic distribution (Council of Europe, 1991). AOELs for agricultural pesticides are derived from the toxicological database of the active substance involved. These levels are considered to be safe for workers exposed to the formulated product used as recommended. Therefore, worker exposure, either measured or estimated, must be compared to the established AOEL. In the European Union, an estimated exposure above the AOEL prevents the registration of the active ingredient or some of its formulations and/or uses. AOELs are intended for preregistration risk assessment purposes and not as a tool to control worker exposure. In this respect, AOELs differ from OELs used in the industrial setting, which are typically recommended for an 8-hr time-weighted average, based on a working life time of 40 hr per week, or 15-rain short-term exposure levels, which are used to qualify occupational exposure (Ballantyne and Marrs, 2004).
B. Respiratory Protection If a break in containment or a spill occurs in the plant manufacturing situation, then vapor may accumulate in the air at concentrations above the workplace exposure guideline and may reach toxic concentrations. Therefore, appropriate respiratory protective equipment should be available for emergency use. As a support for this, there should be a respirator training and fitting program.
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TABLE 2. Examples of Workplace Exposure Guidelines (Threshold Limit Values) Assigned by ACGIH (2004) for OP and CM anti-ChEs a
Pesticide
Adopted value
Notation
Effects noted
Organophosphates Azinphos-methyl Chlorpyrifos
TWA8 0.2 mg m -3 (IV) TWA 8 0.1 mg m -3 (IV)
Skin: SENS: A4: BEI A Skin: BEIA: A4
Cholinergic Cholinergic
Demeton Demeton-S-methyl Diazinon Dichlorvos Dicrotophos Disulfoton EPN Ethion Malathion Methyl demeton
TWA8 0.05 g m -3 (IV) TWA80.05 mg m -3 (IV) TWA8 0.01 mg m -3 (IV) TWA8 0.1 g m -3 (IV) TWA8 0.05 mg m -3 (IV) TWA8 0.05 mg m -3 (IV) TWA8 0.1 mg m -3 (I) TWA8 0.05 mg m -3 (IV) TWA8 1 mg m -3 (IV) TWA8 0.5 mg m -3
Skin: BEIA Skin: SENS: A4: BEI A Skin: BEIA: A4 Skin: SENS: BEIA: A4 Skin: A4: BEIA Skin: A4: BEIA Skin: A4: BEIA Skin: A4; BEIA Skin: A4: BEIA Skin: BEIA
TWA8 0.2 mg m -3 TWA8 0.01 mg m -3 (IV) TWA8 0.0 mg m -3 TWA8 1 mg m -3 (I)
Skin: A4: BEIA Skin: A4: BEIA Skin: BEIA A4: BEIA
Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic, irritation Cholinergic Cholinergic Cholinergic Cholinergic
Carbaryl
TWA8 5 mg m -3
A4
Carbofuran Methomyl Propoxur
TWA8 0.1 mg m -3 (IV) TWA8 2.5 mg m -3 TWA 8 0.5 mg m -3
A4: BEIA A4: BEIA A3: BEIA
Methyl parathion Mevinphos TEPP Trichlorphon Carbamates
Cholinergic, reproductive Cholinergic Cholinergic Cholinergic
aAbbreviations used: EPN, O-ethyl-O-4-nitrophenylphenylphosphonothioate; TEPP, tetraethyl pyrophosphate; TWA8, time-weightedaverageconcentrationover 8 hr; I, inhalable fraction;V, vapor and aerosol; BEIA, Biological Exposure Index for cholinesterase-inhibitingpesticides (RBC AChE 70% of individual's baseline);A3, confirmed animal carcinogen with no known relevance to relevance to humans; A4, not classifiable as a human carcinogen; SENS, sensitizer(cutaneous or respiratory; animal and/or human data); skin, possibilityfor percutaneousabsorption and resultant systemictoxicity;TLV basis, critical effect(s).
C. Medical and Emergency Considerations The plant should have access to a full- or part-time occupational health physician supported by an occupational health nurse. They should be responsible for the conduct and recording of pre- and periodic medical surveillance and be available for any emergency overexposures and poisoning. Equipment and antidotes should be readily available in the event of an accidental poisoning (e.g., airways, oxygen, mask with manual inflator, and atropine and oxime).
XII. ADDITIONAL OCCUPATIONAL HYGIENE CONSIDERATIONS FOR IN-USE OPEN-AIR CONDITIONS The following discussion applies to the application of pesticides in open-air situations, such as fields and orchards.
A. Supervision During loading and application of pesticide, there should be supervision of employees by experienced people. The employees should be carefully instructed, before the working day starts, about the intended applications and where pesticide handling and exposure may occur. As a reminder, there should be posters and notices drawing attention to, and clearly defining the health hazards and risks from, overexposure to anti-ChEs. Provision should be made for preemployment medicals for new workers and for regular periodic medical examinations for established workers.
B. Protective Clothing Protective clothing, including gloves and a face shield, should be readily available where overexposure is likely. Although impermeable clothing provides the greatest
CHAPTER 39 9Occupational Toxicology and Hygiene protection against anti-ChEs (Griffith and Duncan, 1983), 100% cotton fabric reduces skin exposure more effectively than synthetic fibers (Griffith and Duncan, 1992). Clothing saturated with pesticide residue may promote continuing exposure of the skin (Freed et al., 1980; Maibach and Feldman, 1974; Wicker et al., 1979), which may enhance percutaneous absorption and the potential for systemic toxicity. Clearly, workers should wash and/or change clothing at least daily to prevent accumulation of potentially harmful residue (Finley et al., 1978). Additionally, washing facilities should be made available to employees.
C. Health Care Facilities When anti-ChEs are to be used, there should be provision for a trained medical or nursing professional to be on site during the application phase. They should have access to necessary emergency first aid and treatment needs (e.g., airways, oxygen, and antidotes). When there are no local health facilities nearby, there should be arrangements for transporting overexposed and poisoned workers to the nearest capable hospital.
D. Reentry Restrictions Entering a pesticide-treated zone before the material has decreased to nonhazardous levels can result in significant acute illness (Centers for Disease Control, 1999). After pesticide application, workers should be prevented from entering the treated zone until it is safe to do so; this is normally controlled by a restricted entry interval (REI). The duration of the REI depends on the specific pesticide used. However, a reentry interval established for intact residues of selected OPs may be of little value for those compounds that degrade to their more toxic analogues, such as parathion. The establishment of reentry intervals requires the following: (i) examination of dose-response data, (ii) an estimate of the relationship between surface residue and total body exposure, and (iii) examination of time versus residue data. These data should permit an estimate of the dose at which there is no effect or minimum risk (Zweig et al., 1984). California has set 48-hr worker safety reentry intervals and 96-hr harvest intervals based on, but not limited to, toxicity, rate of persistence and degradation curves, human exposure practices, usage patterns, frequency of documented poisoning cases, rate of pesticide application, formulation, concentrated or dilute application, and the possibility for potentiation (Knaak, 1980; Maddy, 1996). Reentry standards for field workers were first proposed in the United States by the EPA in 1974. The reentry interval or standard has been defined as the period of time, in hours or days, following pesticide application after which a worker may legally enter a treated field to engage in normal field activity resulting in prolonged contact with foliage (Kraus et al., 1981). Workers without appropriate personal protective
587
equipment and clothing should not enter the pesticidetreated area until expiration of the REI. Posted and oral warnings, based on the REI, should be made available immediately after pesticide application. Where reentry standards have been appropriately set and applied, there has been a reduction in systemic illness related to residues (Maddy, 1996).
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CHAPTER 40
Public Health Impacts of Organophosphates and Carbamates DAPHNE B. MOFFETT U.S. Department of Health and Human Services, Atlanta, Georgia
some OPs were developed as potential chemical warfare agents. The first OP to be used commercially was tetraethylpyrophosphate (TEPP); although effective, it proved chemically unstable and extremely toxic to all forms of life (Ecobichon, 2001). During the same time period, the first pesticidal carbamic acid esters were synthesized and marketed as fungicides. Because of TEPP's undesirable ubiquitous toxicity and due to poor insecticidal activity of the existing carbamic esters, insecticide development was directed toward the synthesis of more stable chemicals with moderate environmental persistence, which gave rise to parathion in 1944 and paraoxon at a later date (Ecobichon, 2001). In the mid-1950s, there was renewed interest in insecticides with anticholinesterase (anti-ChE) activity but reduced mammalian toxicity, thus leading to the synthesis of several potent aryl esters of methyl carbamic acid. The insecticidal CMs were synthesized as analogs of the drug physostigmine, a toxic anti-ChE alkaloid extracted from the seeds of Physostigma venenosum, the calabar bean (Ecobichon, 2001).
I. I N T R O D U C T I O N The public health impacts from the use of organophosphorus (OP) and carbamate (CM) pesticides are broad, ranging from benefits seen through control of vector-borne diseases (Le., diseases transmitted by insects or other arthropods) and increased survival rates, particularly in subtropical and tropical countries, to severe illness and death associated with acute OP poisoning. People of all ages are exposed to pesticides. Whether it is through diet (including breast milk and water), indoor spraying, aerial spraying, or outdoor activity, kids and adults are exposed. Occupational exposures are common, particularly among applicators and farm workers. The widespread use of pesticides feeds into the exposure cycle. In the United States, more than 18,000 products are licensed for use, and each year more than 2 billion pounds of pesticides are applied to crops, homes, schools, parks, and forests [U.S. Environmental Protection Agency (EPA), 2002]. Discussions of pesticide use and exposure, including the OP and CM classes, are often marked by ominous tones and monopolized emphasis on the negative contributions made to the environment and public health. The public health importance of the continued application of pesticides in integrated pest management and the control of outbreaks and vector-borne disease is overshadowed by the concern for human exposure and health effects, particularly in small children. In an effort to portray the positive and negative impacts of OPs and CMs in public health, two subtopics are reviewed in this chapter: uses of OPs and CMs in the control of vector-borne diseases and children's exposures to OPs and CMs.
A. Eradication of Vector-Borne Diseases The development of effective OPs and CMs historically played a critical role in the control of vector-borne diseases. Malaria, dengue, yellow fever, plague, filariasis, louse-borne typhus, trypanosomiasis, leishmaniasis, and other vector-borne diseases were responsible for more human disease and death in the 17th through the early 20th centuries than all other causes combined (Gubler, 1991). During the 19th and 20th centuries, vector-borne diseases prevented the development of large areas of the tropics, especially in Africa; it was not until these diseases were controlled that wider spread habitation and travel could occur. Once the disease transmission cycles were known, prevention and control programs became focused on vector control. These public health programs included the use of pesticides, initially organochlorine pesticides such as DDT and later OPs and CMs (Table 1).
II. C O N T R O L OF V E C T O R - B O R N E DISEASES OP insecticides were first synthesized in 1937 by a group of German chemists led by Gerhard Schrader (Ecobichon, 2001). Unfortunately, due to Nazi oversight in World War II, 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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SECTION VI 9Risk Assessment & Regulations TABLE 1. Selected Commonly Used OPs and CMs and Targeted Vector-Borne Diseases Chemical classification
Selected chemical
Organophosphates
Chlorpyrifos Fenitrothion Fenthion Malathion
Carbamates
Vector-borne disease
Naled Pirimiphos-methyl Temephos
Malaria, West Nile virus (WNV) Malaria, dengue, yellow fever, leishmaniasis WNV, dengue, yellow fever Malaria, WNV, dengue, yellow fever, leishmaniasis, head lice WNV, dengue, yellow fever Malaria, dengue, yellow fever Malaria, WNV, dengue, yellow fever, onchocerciasis
Bendiocarb Carbosulfan Propoxur
Malaria Malaria Malaria, leishmaniasis
In 1900, yellow fever in Cuba was the first vector-borne disease to be effectively controlled, followed quickly by yellow fever and malaria in Panama (Gubler, 1998). By the 1960s, vector-borne diseases were no longer considered major public health problems outside of Africa. Urban yellow fever and dengue, both transmitted by the mosquito Aedes aegypti, were effectively controlled in Central and South America and eliminated from North America; malaria was nearly eradicated in the Americas, the Pacific Islands, and Asia (Gubler, 1998). The effective use of residual insecticides from the 1940s through the 1960s contributed greatly to these successes. Major mosquito vectors responsible for some of the most widespread diseases include Culex genus, which is the vector for West Nile virus (WNV), filariasis, and Japanese encephalitis; Aedes of dengue, dengue hemorrhagic fever, and yellow fever; and Anopheles of malaria. DDT was first introduced for mosquito control in 1946. In 1955, the World Health Organization (WHO) assembly proposed the global eradication of malaria with DDT (Hemingway, 2003). DDT was the insecticide of choice globally for many years. It remains in use in developing nations because of its affordability and continued effectiveness in controlling diseases such as malaria. However, public concern over the environmental persistence of DDT and other organochlorine pesticides and their banning from manufacture and use in the United States in the 1970s led to the development and use of alternative insecticides, including OPs (malathion, pirimiphos-methyl, and fenitrothion) and CMs (bendiocarb, carbosulfan, and propoxur). The WHO Pesticide Evaluation Scheme identified and recommended these insecticides for indoor residual spraying (Chavasse and Vap, 1997). Since 1964, ultralow-volume (ULV) aerial applications of the OPs (malathion and naled) have been used many times in the United States and its territories for controlling mosquitoes in disaster areas and for controlling epidemics of mosquito-borne disease. The ULV method was used to
kill infected Culex pipiens quinquefaseiqtus during the Dallas, Texas, epidemic of St. Louis encephalitis; in 1967 to kill species of Aedes, Psorophora, Culex, and Anopheles in a 3-million acre flooded area in Texas; in 1969 in Ohio to kill Aedes vectors of LaCross encephalitis during an epidemic; in 1972 and 1974 in New England to kill species of Aedes, Coquillettidia, and Culiseta during an outbreak of eastern equine encephalitis; in 1975 in North Dakota and Minnesota to kill infected Culex tarsalis during an outbreak of western equine encephalitis; and in 1975 in Guam and Puerto Rico to control the vectors of dengue [U.S. Centers for Disease Control and Prevention (CDC), 2000]. With the dramatic appearance of epidemic WNV in the New York City area in 1999 and its subsequent spread to Canada and the eastern United States, ULV spraying of malathion was a frontline public health response to control the epidemic. B. R e e m e r g e n c e and R e s i s t a n c e of Vector-Borne Disease
The 1999 WNV emergence in the United States demonstrated that without sustained vector mosquito control in urban areas, even the world's most affluent cities are at risk for vectorborne disease. WNV, an Old World flavivirus related to St. Louis encephalitis virus and transmitted through mosquitoes, caused a serious outbreak (62 cases, with 7 deaths) and signaled the potential for similar outbreaks in the Western Hemisphere (Rose, 2001). This emergence or reemergence of vector-borne diseases thought to be under control was first observed in the 1970s in Asia and the Americas as rates of malaria and dengue began to climb (Gubler, 1998). The ease of travel and the increase in migrant laborers are two elements for the increased frequency in cases of dengue and malaria in the United States. Epidemic dengue was effectively controlled in the American region when the principal mosquito vector, Ae. aegypti, was controlled in the 1950s and 1960s. Unfortunately, the program was disbanded in the early 1970s
CHAPTER 40 9Public Health Impacts of OPs and CMs and the mosquito reinfested most countries of the region during the next 30 years (Gubler, 2001). According to Gubler (2001), global reports of dengue or dengue hemorrhagic fever were practically nonexistent in 1955. Data provided by decade show a dramatic increase during a 45-year span such that by 2000, more than 500,000 cases were reported. Each year, depending on the epidemic activity in the world, there are an estimated 50-100 million cases of dengue fever and several hundred thousand cases of the severe form of the disease, dengue hemorrhagic fever (Gubler, 2001). The resurgence of these diseases has been linked to demographic changes, societal changes, agricultural changes, and changes in public health practices. The use of insecticides has played a very important role in genetic changes in pathogens leading to increased epidemic potential. OP resistance has been recorded in all the major Culex vectors, which has ramifications for the control of WNV, Japanese encephalitis, and filariasis. OP and CM resistance occurs in the Aedes vector, which is the vector for dengue, dengue hemorrhagic fever, and yellow fever. The Simulium damnosum complex, vectors of onchocerciasis or "fiver blindness" have been under insecticide-based control in West Africa since 1974. Temephos (an OP insecticide) resistance prompted a switch to chlorphoxim, but resistance to this insecticide occurred within 1 year. Resistance in Simulium is currently being managed by a rotation of temephos, Bacillus thuringiensis, and permethrin (Hemingway, 2003). Investigations are under way in countries, provinces, and states to evaluate vector resistance to insecticides. A study in Brazil examined the resistance of the primary mosquito vector of dengue and yellow fever. Since 1967, public health programs have used mostly OPs in the control of Ae. aegypti. The mosquito vector was first eradicated from the country in 1955 following 8 years of a DDT application program. It was reintroduced into Brazil in 1967 as a DDT-resistant species, so temephos was used to eradicate it for a second time in 1973. The vector was reintroduced in 1976, and since then malathion and fenitrothion have been used for controls of adults concomitantly with the larvicide temephos (Lima et al., 2003). With the extensive use of temephos and malathion, dengue epidemics continue to emerge. The increasing insecticide resistance points to the need for new vector control strategies. Studies examining the resistance of Culex pipiens quinquefasciatus to propoxur, temephos, and chlorpyrifos in Martinique (Yebakima et al., 2004) and studies examining resistance of the same vector to malathion, chlorpyrifos, and other non-OP/CM insecticides in Alabama and Florida (Liu et al., 2004) have revealed important but not unanticipated results. In all cases, resistance and even cross-resistance to OP and CM insecticides and nonOP/CM insecticides were recorded in all species. Control of the reemergence of vector-borne disease and reversing the trend have become public health priorities. Controlling and preventing these diseases require strategies
601
including the application of larvicides and adult insecticides for mosquito control. For example, control of the main mosquito vector, Ae. aegypti, is currently the only option available for prevention and control of dengue, and it is carried out mainly by applying insecticides (generally OPs and CMs) to larval habitats, destroying unwanted containers, and educating the public. During epidemics, this is complemented by insecticide space spraying against adult mosquitoes (Corbel et al., 2004). Simultaneously, however, society is concerned with the effects of these chemicals on untargeted beneficial arthropods and vertebrates as well as their effects on mammals. Regulatory actions including the Food Quality and Protection Act of 1996 (FQPA) are intended to ensure that existing public health pesticide uses are not lost without economically effective alternatives. However, if FQPA results in cancellation of major agricultural uses of a pesticide that is also used in public health, it may become no longer profitable for the manufacturer to produce small quantifies for mosquito control, thus ending production of the pesticide (Rose, 2001). Reversing the trend of emergent/resurgent vector-borne disease is a major challenge. Vaccines are available for only a few vector-borne diseases (yellow fever, Japanese encephalitis, tick-borne encephalitis, tularemia, and plague) and are not widely used. Vaccine prospects for major vector-borne disease are not good. With the exception of malaria, few other vector-borne diseases have funding for vaccine research (Gubler, 1998). In the next decade, vector control will be required to interrupt transmission of most emergent/resurgent vector-borne diseases. Therefore, we must maintain the public health arsenal of effective, "safe" pesticides. This arsenal is likely to include a suite of traditional OP- and CM-based insecticides as well as newer pesticides and alternative approaches (e.g., biological control). Because adulticides used for mosquito control were registered decades ago, the data supporting their registrations may be insufficient to meet current requirements. None of the mosquito adulticides commonly used in the United States was developed recently; their registrations are up to 44 years old (Rose, 2001). Shrinking availability of insecticides as a result of resistance is exacerbated by removal from the market of insecticides no longer registered for public health use, especially in the past decade; the cost to keep certain compounds on the market is higher than can be recouped from such use. Local mosquito control programs would use an integrated program if resources were available, but often funding is limited to the extent that adulticiding trucks are the only means of mosquito intervention. Malathion, naled, and fenthion are examples of OP insecticides commonly used for adult mosquito control in the United States and for control of WNV (CDC, 2003a). Implementation of the public health pesticide provisions of FQPA must include comparative risk-benefit analyses of the sig~ficance of vector-borne disease impacts versus potential human and environmental toxic effects for pesticides used to control public health pests both
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SECTION Vl 9Risk A s s e s s m e n t & Regulations
in the United States and in other countries affected by EPA pesticide regulatory decisions (Rose, 2001).
III. CHILDREN'S EXPOSURES As discussed previously, pesticides are a part of the frontline public health defense :against vector-borne diseases such as malaria, dengue, WNV, and other major causes of infectious disease. OPs, CMs, and the broader group of pesticides assist in the control of food-borne and vector-borne diseases, which affect millions of children and adults and kill thousands annually in the United States (CDC, 2002; Gubler, 1998). Additionally, pesticide usage, including OPs and CMs, improves human nutrition through greater availability, greater crop diversity, and lower costs of food. The OP and CM insecticides are used to control insect pests that otherwise would lower crop yields significantly. In addition, OP/CM insecticides are inexpensive relative to many other pest control options. It has been argued that the benefits of OP/CM insecticide usage for crop pest control outweigh the countervailing risks to human health in large part due to the availability and affordability of nutritious fruits, vegetables, and grains that have been linked to chronic disease prevention (Taylor and Smith, 1999). Although there are benefits to the usage of OPs and CMs, there are also many public health dangers. Children reap the benefits from the use of OPs and CMs, but they also reap the negative consequences, often disproportionately compared to adults. Children's exposures to pesticides may be particularly increased because they have a tendency to explore their environments with their mouths and because their breathing zones are close to the ground where OP and CM residues accumulate, increasing inhalation to heavier-than-air toxicants and low-lying particulates. Children are susceptible populations in that they are still growing and developing (e.g., the brain grows rapidly during infancy and myelination is not completed until the second year of life). Children's vulnerabilities have fueled the public's escalating concerns about the large-scale use of pesticides (particularly AChE inhibitors, including OPs and CMs), resulting in the passage into law of FQPA, which requires the EPA to address risks to infants and children and requires pesticide tolerances to be safe, defined as "a reasonable certainty that no harm will result from aggregate exposure," including all exposure through the diet and other nonoccupational exposures, including drinking water, for which there is reliable information. As elements of their aggregate pesticide exposure, children's exposures will be explored by examining accidental poisonings, dietary exposures, indoor spraying, and agricultural exposures in the United States.
A. Accidental Poisonings In the United States, a total of 4.5 billion pounds of chemicals are applied annually as pesticides (Aspelin and Grube, 1999).
This total consists of 1.2 billion pounds of conventional pesticides (975 million pounds) and other pesticide chemicals, such as sulfur, wood preservatives, specialty biocides, and chlorine/hypochlorite compounds (Weiss et aL, 2004). OPs account for approximately half of the insecticides used in the United States, and CMs are widely used in homes and gardens (Weiss et al., 2004). The amount of OP insecticides used has declined nearly 45% since 1980, from an estimated 131 million pounds in 1980 to 73 million pounds in 2001. Since 1980, however, OP use as a percentage of total insecticide use has increased, from 58% in 1980 to 70% in 2001. The increase in use in 1999 was due mainly to the increased amount of malathion used as part of the U.S. Department of Agriculture-sponsored Boll Weevil Eradication Program. Malathion use in this program decreased during 2003 and 2004, resulting in a decline in total OP use. The estimates of OP insecticide use rely on the estimated amount used and changes in the amount of OPs used from public and proprietary EPA databases (Table 2). Since nearly 74 million households (or three-fourths of all U.S. households) use an estimated 76 million pounds of pesticides annually (Aspelin and Grube, 1999), the potential for children's exposure is high. Pesticides are often stored in relatively large quantities in garages, sheds, basements, barns, and other areas accessible to children. A study estimated that nearly half of all households with a child younger than 5 years old stored pesticide in an unlocked cabinet within reach of the child (Bass et al., 2001). Although these exposures are unintentional, they have significant immediate public health consequences. Accidental exposure to pesticides is a common cause of acute poisoning, particularly among young children. More than half (57%) of all reported pesticide poisonings in the United States occur in children younger than 6 years of age; this translates into approximately 50,000 children per year (Litovitz et al., 2002). Of these poisonings, at least 2000 are attributed to exposure to OPs and CMs (Watson et al., 2004). The number is potentially much greater due to cases reported in which the pesticide agent was not known. OPs are generally much more toxic than CMs. Unlike OPs, CMs do not irreversibly inhibit ACHE. Thus, their activity is quickly reversed after excretion of the pesticide. Acute, high-level exposure can result in immediate health effects potentially seen within minutes of exposure. The most frequent acute symptoms of OP poisoning in children include miosis, excessive salivation, nausea and vomiting, lethargy, muscle weakness, tachycardia, hyporeflexia and hypertonia, respiratory distress, and, in severe cases, death (Zwiener and Ginsburg, 1988). No studies have examined the long-term sequelae of acute pesticide poisoning in children.
B. Dietary Exposures Dietary exposures to OPs and CMs in children are a major pathway and main contributor to pesticide intake and body
CHAPTER 40 9Public Health Impacts of OPs and CMs TABLE 2.
603
Most Commonly Used OP Insecticide Active Ingredients, All Market Sectors J 2001 and 1999 Estimates a 2001
1999
Active ingredient
Rank
Range
Rank
Range
Malathion Chlorpyrifos Diazinon Terbufos Acephate Phorate Methyl parathion Phosmet Azinphos-methyl Dimethoate
1 2 3 4 5 6 7 8 9 10
23-32 11-16 4-7 3-5 2-3 2-3 1-3 1-2 1-2 1-2
1 2 4 3 7 6 5 9 8 10
30-38 13-19 4-7 5-7 2-3 2-3 2-4 1-2 1-2 1-2
Source: EPA estimates based on CroplifeAmerica annual surveys, USDA/NASS, and EPA proprietarydata aRanked by range in millions of pounds of active ingredient (www.epa.gov).
burden. In particular, dietary exposures to CMs are the primary route of exposure for children to this class of pesticide. Physiological differences between children and adults contribute to their increased risk of exposure to pesticides in food. As infants are weaned and progress to solid foods, they consume, per unit body weight, proportionally more fruit and more fruit juice than adults. Lu et al. (2005) determined the prevalence of dialkylphosphates (DAPs), which are metabolic products of most OP pesticides, in fruit juices. DAPs were found in both conventional and organic juices purchased from local grocery stores, and the original levels were higher, for both apple and orange juices, in conventional than in organic juices. The typical 1-year-old child drinks, per unit of body weight, 16 times as much apple juice as an adult (Wargo, 1998). The National Research Council (1993) reported that children's dietary exposures to pesticides differed from those of adults both quantitatively and qualitatively. The report noted that children experienced higher exposure to pesticides from agricultural crop residues because of their higher intake of fruits and vegetables, per body weight, relative to adults. The report estimated that 50% of lifetime pesticide exposure occurs during the first 5 years of fife. Fenske et al. (2002) conducted an assessment of OP exposures in the diets of children 2-5 years old in Washington State. Twenty-four-hour duplicate diet sampling was employed to examine the dietary OP exposures. Samples were collected from children living in the Seattle metropolitan area and from children living in counties in central Washington. A total of 88 food category samples were collected and analyzed for 15 OPs. Of the 15 targeted OPs, 6 were detected: azinphos-methyl, chlorpyfifos, malathion, methidathion, methyl parathion, and phosmet (Fenske et al., 2002). The fresh fruits and vegetables category had the most frequent pesticide determinations, followed by
beverages. No detections were above the legal tolerances for residues on produce; however, the acute population-adjusted reference dose for chlorpyrifos exposure of 1.7 ixg/kg/day was exceeded by one subject during one sampling event. This subject's cumulative daily dose of chlorpyrifos equivalents was estimated to be 2.5 lxg/kg/day. Since the family did not spray inside or outside the home, it was determined that his exposure was attributable to agricultural residues on food (Fenske et al., 2002). In 2003, the CDC published a national survey of human exposure to environmental chemicals based on laboratory analysis of blood and urine specimens obtained in 1999 and 2000 (CDC, 2003b). The report provides reference ranges for 116 chemicals, including selected pesticides, measured in a randomly selected subsample of participants in the National Health and Nutrition Examination Survey (NHANES), 6-59 years of age. Urine levels of dimethyl thiophosphate (a major metabolite of many OP pesticides) were approximately twice as high in children 6-11 years of age as in adults 20-59 years of age. Measurement of these metabolites in urine reflects exposure to OP pesticides that has occurred primarily in the past few days. The increased levels seen in children suggest that children in the United States have had higher levels of exposure to OPs than adults (Weiss et al., 2004). The long-term human health and children's health implications of this finding are not known, but further study is warranted. Exposure of the general population to CM pesticides occurs primarily from ingestion of food products or from residential use. In examining the CM metabolites also published in the CDC report, the levels of the carbaryl metabolite 1-naphthol seen in the 6- to 11-year age group were similar to those reported for Minnesota schoolchildren age 3-13 years (CDC, 2003b).
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FQPA explicitly requires the EPA to address risks to infants and children and to publish a specific safety finding before a pesticide tolerance can be established. It also provides for an additional safety factor (10-fold, unless reliable data show that a different factor will be safe) to ensure that tolerances are safe for infants and children and requires collection of better data on food consumption patterns, pesticide residue levels, and pesticide use. Diet is considered a significant source of aggregate pesticide exposure for children, and one aim of FQPA is to monitor pesticide exposure levels for children that are a result of agricultural residue from pesticide use and to adjust recommendations for pesticide use and/or registration in a manner that is protective of children's health. Although increased levels of OP metabolites were seen in children 6-11 years of age relative to levels in adults in the CDC cross-sectional study of the U.S. population, the meaning of this finding is not well understood, particularly as it relates to potential long-term health effects.
C. Indoor Spraying Exposures Recurrent broadcast spraying of pesticides in homes is another exposure pathway for children to OPs and CMs. Children's breathing zones are closer to the ground, where pesticide residues accumulate, increasing inhalation exposure to heavier-than-air toxicants and low-lying particulates. For example, higher chlorpyrifos concentrations have been demonstrated closer to the floor (25 vs 100 cm above the carpet) after the indoor broadcasting of Dursban (chlorpyrifos) (Fenske, 1997). Children are more likely to crawl around on the carpet and floors and to place their fingers in their mouths. Deposition of pesticides on toys and other objects has a greater impact on children's exposures because children frequently place objects in their mouths. Children are also less likely to wash their hands before eating, and they are less likely to use utensils when eating. Gurunathan et al. (1998) conducted a study examining residential treatment with broadcast spraying of chlorpyrifos. Peak levels on surfaces such as toys and furniture were measured 36 hr after the original application. The semivolatile pesticide accumulated on and in toys and other sorbant surfaces in a home via a two-phase physical process that continues for at least 2 weeks postapplication. The total dose of chlorpyrifos for a 3- to 6-year-old child whose home was treated depended on the frequency of that child's hand-to-mouth activity, but it was estimated to be 208 Ixg/ kg/day, which far exceeded the EPA's reference dose of 3 txg/ kg/day. The Gurunathan study examined the exposures from an indoor broadcast spray, but similar attention was given to a crack-and-crevice application of chlorpyrifos with very different results (Hore et al., 2005). The study was conducted to examine the distribution of chlorpyrifos within a home environment for 2 weeks after a routine professional crackand-crevice application and to determine the amount of the
chlorpyrifos that is absorbed by a 2- to 5-year-old child living within the home. The daily amount of chlorpyrifos estimated to be absorbed by the children postapplication ranged from 0.04 to 4.8 Ixg/kg/day. Comparison of results from the Hore et al. study and the Gurunathan et al. study suggests that selection of the application method will greatly influence the children's exposure and dose received from pesticides applied indoors. Another study assessed the exposure to OP pesticides by biomonitoring in epidemiologic studies of birth outcomes (Needham, 2005). The author evaluated three different exposure studies that had been conducted in California and New York. The New York studies (Berkowitz et al., 2004; Whyatt et al., 2004) involved recurrent indoor broadcast sprayings, primarily of chlorpyrifos. In the California study (Eskenazi et al., 2004), the primary exposure focus was agricultural applications of several pesticides, pesticide drift from the spraying, and the "bringing home" of the pesticides on workers' clothing; however, pesticides were also potentially used indoors, for landscape maintenance, for insect control, and for prevention of structural damage. The studies examined the relationship between pregnant women's exposures to OPs, particularly chlorpyrifos, and birth outcomes. Chlorpyrifos or its metabolite, 3,5,6-trichloro-2pyrinidol, were detected and measured in umbilical cord and maternal blood samples and in urine. Although the birth outcomes from these exposures were not identical across the studies, significant relations reported between their exposure assessments and birth outcomes included decreased birth size (Whyatt e t a l . , 2004), decreased head circumference with levels of paraoxonase-1 activity as a modifier (Berkowitz et al., 2004), and decreased gestational age at birth (Eskenazi et al., 2004). Although the birth outcomes may not be identical across the studies and may be difficult to interpret, clearly the application of indoor pesticides contributes to fetal and children's exposures to pesticides (including OPs), such that pregnant women should also be considered an at-risk population.
D. Exposures of Children of Agricultural Workers Children of agricultural families are very likely to be exposed to pesticides, even if they are not actively involved in farm activities. This same exposure risk applies to children of migrant farmworkers who are at increased risk of exposure to OPs because of "carry-home" transport processes and residential location (Lu et al., 2000). Simcox et al. (1995) studied 59 families in the Yakima Valley and compared levels of four OP pesticides in the homes of hired farmworkers, families residing on farms, and nonagricultural families. Chlorpyrifos was detected in 95% of the homes. House dust concentrations were consistently higher for agricultural families than for nonagricultural families, and pesticide applicators tended to have higher house dust concentrations compared to
CHAPTER 40
nonapplicators. There was a threefold difference in median chlorpyrifos house dust concentration between farmworkers who did not directly handle pesticides and reference families of non-farmworkers living in agricultural communities (median, 172 ng/g for farmworkers vs 53 ng/g for nonagricultural families). Bradman et al. (1997) conducted a pilot study of pesticide exposures to children of migrant farmworkers and nonfarmworkers living in California. Floor dust samples and child hand wipes were collected from the homes of 10 families, 5 of which had at least one resident farmworker. Higher levels of the OPs diazinon, chlorpyrifos, and malathion were found in house dust in farmworker homes. Residues of diazinon and chlorpyrifos were detected on the hands of two and three farmworker toddlers, respectively, who also lived in the homes with the highest dust concentrations. Overall findings from studies suggest that inadvertent carry-homes of occupational pesticides are occurring and that contamination in the homes of farm families is likely to be higher than in other homes. Furthermore, a significant source of exposure to farmworker families may derive from their residential proximity to fields (Eskenazi et al., 1999). As of 1999, studies of the effects of pesticide exposure on children's health were limited to those of birth defects, childhood cancer, and acute poisoning following~ingestion. Some case-control studies have associated parental exposure to pesticides or pesticide use in the home with childhood brain tumors, leukemia and lymphomas, testicular cancers, and other cancers. Other studies have reported that parental exposure to pesticides or application of pesticides in the home are associated with certain birth defects, including neural tube and other defects (Eskenazi et al., 1999). Evaluations of OP and CM exposures, levels of exposures, and health impacts to children are needed.
IV. C O N C L U S I O N S The public health impacts of OPs and CMs are historic and of current and future importance. They embody the frontline public health response for prevention and control of outbreaks of vector-borne diseases. Their applications have afforded society to enjoy a wide range of foods in large quantities at low costs. The nutritional benefits received from crop diversity are numerous. However, their use is somewhat of a "double-edged sword." At the same time the use of these chemicals is preventing disease, it is also promoting the mutation of vectors such that new diseases and reemergences of diseases once nearly eradicated are at epidemic levels in many tropical and subtropical regions of the world. At the same time they are used to protect public health from disease, they have been associated with undesirable chronic health effects in adult populations and developmental effects in children. At the same time their use provides diverse, low-cost food, it also taints breast milk and
9Public Health Impacts of OPs and CMs
605
foods commonly consumed by children. With the implementation of FQPA, regulatory and public health agencies will be charged with maintaining a delicate balance for use of OPs and CMs in the broader pesticide category. It will be necessary to address aggregate exposures and children's health and simultaneously maintain a cache of pesticides that are effective in the prevention and control of disease.
References Aspelin, A. L., and Grube, A. H. (1999). Pesticides Industry Sales and Usage: 1996 and 1997 Market Estimates. U.S. Environmental Protection Agency, Office of Pesticide Programs, Washington, DC. Bass, J. K., Ortega, L., Rosales, C., Petersen, N. J., and Philen, R. M. (2001). What's being used at home: A household pesticide survey. Pan Am. J. Public Health 9, 138-144. Bradman, M. A., Hamly, M. E., Draper, W., Seidel, S., Teran, S., Wakeham, D., and Neutra, R. (1997). Pesticide exposures to children from California's Central Valley: Results of pilot study. J. Exposure Anal. Environ. Epidemiol. 7, 217-234. 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. Chavasse, D. C., and Vap, H. H. (Eds.). (1997). Chemical Methods for the Control of Vectors and Pests of Public Health Importance, Document WHO/CTD/WHOPES/97.2. World
Health Organization, Geneva. Corbel, V., Duchon, S., Zaim, M., and Hougard, J.-M. (2004). Dinotefuran: A potential neonicotinoid insecticide against resistant mosquitoes. J. Med. Entomol. 41, 712-717. Ecobichon, D. J. (2001). Toxic effects of pesticides. In Casarett and Doull's Toxicology: The Basic Science of Poisons (C. D. Klassen, Ed.), 6th ed., pp. 763-810. McGraw-Hill, New York. Eskenazi, B., Bradman, A., and Castorina, R. (1999). Exposure of children to organophosphate pesticides and their potential adverse health effects. Environ. Health Perspect. 107, 409-419. Eskenazi, B., Harley, K., Bradman, A., Weltzien, E., Jewell, N. E, Barr, D. B., Furlong, C. E., and Holland, N. T. (2004). Association of in utero organophophate pesticide exposure and fetal growth and length of gestation in an agricultural population. Environ. Health Perspect. 112, 1116-1124. Fenske, R. (1997). Pesticide exposure assessment of workers and families. Occup. Med. 12, 221-237. Fenske, R. A., Kedan, G., Lu, C., Fisker-Andersen, J. A., and Curl, C. L. (2002). Asssessment of organophosphorous pesticide exposures in the diets of preschool children in Washington State. J. Exposure Anal. Environ. Epidemiol. 12, 21-28. Gubler, D. J. (1991). Insects in disease transmission. In Hunter Tropical Medicine (G. T. Strickland, Ed.), 7th ed., pp. 981-1000. Saunders, Philadelphia. Gubler, D. J. (1998). Resurgent vector-borne diseases as a global health problem. Emerging Infect. Dis. 4, 442-450. Gubler, D. J. (2001). Human arbovirus infections worldwide. Ann. N. Y. Acad. Sci. 951, 13-24. Gurunathan, S., Robson, M., Freeman, N., Buckley, B., Roy, A., Meyer, R., Bukowski, J., and Lioy, E J. (1998). Accumulation
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of chlorpyrifos on residential surfaces and toys accessible to children. Environ. Health Perspect. 106, 9-16. Hemingway, J. (2003). Insecticide resistance in insect vectors of human disease. In The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health Strategies for ContainmentmWorkshop Summary, pp. 79-106. National Academies Press, Washington, DC. Hore, P., Robson, M., Freeman, N., Zhang, J., Wartenberg, D., Ozkaynak, H., Tulve, N., Sheldon, L., Needham, L., Barr, D., and Lioy, P. J. (2005). Chlorpyrifos accumulation patterns for child-accessible surfaces and objects and urinary metabolite excretion by children for 2 weeks after crack-and-crevice application. Environ. Health Perspect. 113, 211-219. Lima, J. B. P., Da-Cunha, M. P., Da Silva, R. C., Jr., Galardo, A. K. R., Stares, S. D. S., Braga, I. A., Ramos, R. P., and Valle, D. (2003). Resistance of Aedes aegypti to organophosphates in several municipalities in the state of Rio de Janeiro and Espirito Santo, Brazil. Am. J. Trop. Med. Hyg. 68, 329-333. Litovitz, T. L., Klein-Schwartz, W., Rodgers, G. C., Jr., Cobaugh, D. J., Youniss, J., Omslaer, J. C., May, M. E., Woolf, A. D., and Benson, B. E. (2002). 2001 annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am. J. Emerg. Med. 20, 391-401. Liu, H., Cupp, E. W., Micher, K. M., Gut, A., and Liu, N. (2004). Insecticide resistance and cross-resistance in Alabama and Florida strains of Culex quinquefaciatus. J. Med. Entol. 41, 408-4 13. Lu, C., Fenske, R. A., Simcox, N. J., and Kalman, D. (2000). Pesticide exposure of children in an agricultural community: Evidence of household proximity to farmland and take home exposure pathways. Environ. Res. A 84, 290-302. Lu, C., Bravo, R., Caltabiano, L. M., Irish, R. M., Weerasekera, G., and Barr, D. B. (2005). The presence of dialkylphosphates in fresh fruit juices: Implication for organophosphorus pesticide exposure and risk assessments. J. Toxicol. Environ. Health A 68, 209-227. National Research Council (1993). Pesticides in the Diets of Infants and Children. National Academy Press, Washington, DC. Needham, L. (2005). Assessing exposure to organophosphorous pesticides by biomonitoring in epidemiologic studies of birth outcomes. Environ. Health Perspecr 113, 494-498. Rose, R. I. (2001). Pesticides in public health: Integrated methods of mosquito management. Emerging Infect. Dis. 7, 17-23. Simcox, N. J., Fenske, R. A., Wolz, S. A., Lee, I. C., and Kalman, D. A. (1995). Pesticides in household dust and soil: Exposure pathways for children of agricultural families. Environ. Health Perspecr 103, 1126-1134.
Taylor, C. R., and Smith, H. A. (1999). Aggregate economic evaluation of banning organophosphate and carbamate pesticides, AFPC Policy Research Report 99-15. Agricultural and Food Policy Center, Texas A&M University, College Station, TX. U.S. Centers for Disease Control and Prevention (2000). Arboviral Encephalitis Cases Reported in Humans, by Type, United States, 1964-2000. U.S. Centers for Disease Control and Prevention, Atlanta. Available at www.cdc.gov/ncidod/dvbid/arbor/arbocase.htm. (accessed April 2, 2005). U.S. Centers for Disease Control and Prevention (2002). Disease Information: Food-Borne Illness Technical Information. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National center for Infectious Diseases, Division of Bacterial and Mycotic Diseases, Atlanta. Available at www.cdc.gov (accessed April 2, 2005). U.S. Centers for Disease Control and Prevention (2003a). Epidemic/Epizootic West Nile Virus in the United States: Revised Guidelines for Surveillance, Prevention, and Control U.S. Centers for Disease Control and Prevention, Atlanta. Available at www.cdc.gov (accessed April 2, 2005). U.S. Centers for Disease Control and Prevention (2003b). Second National Report on Human Exposure to Environmental Chemicals. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta. Available at www.cdc.gov (accessed April 2, 2005). U.S. Environmental Protection Agency, Office of Pesticide Programs (2002). FY 2002 Annual Report. U.S. Environmental Protection Agency, Washington, DC. Available at www.epa.gov (accessed April 2, 2005). Wargo, J. (1998). Our Children's Toxic Legacy: How Science and Law Fail to Protect Us from Pesticides, 2nd ed. Yale Univ. Press, New Haven, CT. Watson, W. A., Litovitz, T. L., Klein-Schwartz, W., Rodgers, G. C., Jr., Reid, N., Rouse, W. G., Rembert, R. S., and Borys, D. (2004). 2003 annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am. J. Emerg. Med. 22, 335-404. Weiss, B., Amler, S., and Amler, R. W. (2004). Pesticides. Pediatrics 113, 1030-1036. Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. E, Garfinkel, R., Hoepner, L. A., Diaz, D., Dietrich, J., Reyes, A., Tang, D., Kinney, P. L., and Perera, E P. (2004). Prenatal insecticide exposures, birth weight and length among an urban minority cohort. Environ. Health Perspecr 112, 1125-1132. Yebakima, A., Marquine, M., Rosine, J., Yp-Tcha, M. M., and Pasteur, N. (2004). Evolution of resistance under insecticide selection pressure in Culex pipiens quinquefasciatus (Diptera, Culcidae) from Martinique. J. Med. Entomol. 41, 718-725. Zwiener, R. J., and Ginsburg, C. M. (1988). Organophosphate and carbamate poisoning in infants and children. Pediatrics 81, 121-126.
CHAPTER 4
|
Cumulative Effects of O r g a n o p h o s p h o r u s or Carbamate qr
Pesticides
STEPHANIE PADILLA U.S. Environmental Protection Agenc); Research Triangle Park, North Carolina
pesticide mixtures, and mixtures of OP and CM pesticides; and (iii)address future research priorities. Also, this chapter considers the N-methyl CMs or the cholinesteraseinhibiting CMs and not the thio- or dithiocarbamates, which have other mechanisms of toxicity. Therefore, only OP and CM pesticides that inhibit cholinesterase are considered. Very few investigators have ventured into the realm of mixture experiments during the past 40 years. These types of studies can be complicated and time-consuming. The overall design of these studies is usually one of two types: those that determine the toxicity of a given mixture or those that determine if the interaction of two or more OP or CM pesticides is synergistic (i.e., if the toxicity of a mixture is more toxic than the sum of the toxicity of the individual pesticides that make up the mixture).
I. I N T R O D U C T I O N Although there is no doubt that humans are exposed to low doses of mixtures of pesticides (Castorina et al., 2003; Duggan et al., 2003; Fenske et aL, 2002; S~inchez-Pefia et al., 2004), the question is whether mixtures are more toxic than the sum of the toxicity for each pesticide alone. In the literature describing the toxicity of mixtures of organophosphorus (OP) or carbamate (CM) pesticides, concern is expressed that exposure to mixtures of pesticides may be much more toxic than the toxicity of the individual pesticides. This concern about potentiated toxicity is all the more worrisome because data are lacking on the toxicity of pesticide mixtures, whether OP or CM, at less than lethal dosages. There is such scarcity of data, and the data are so disparate, that a science-based conclusion on the toxicity of OP or CM pesticide mixtures is not possible. There are some excellent review articles on various aspects of the toxicology of mixtures of chemicals (Calabrese, 1995; Krishnan and Brodeur, 1991), pesticides (Iyaniwura, 1990; Murphy, 1980), or OP compounds (Cohen, 1984; DuBois, 1961; Murphy, 1969, 1980). Many of these reviews, although not recent, provide a comprehensive overview of the possible mechanisms underlying interactions of chemicals in a mixture and, specifically, interactions of OP compounds in a mixture. This chapter does not provide an in-depth discussion of the mechanisms underlying interactions of OP or CM pesticides in a mixture. Rather, this chapter is designed to (i) address issues of experiment design of mixture studies; (ii) summarize the available literature on OP pesticide mixtures, CM
II. D E F I N E D M I X T U R E S Some investigators have chosen to study defined mixtures. The intention was not to determine the pattern of interaction of the OP or CM compounds in the mixture but to characterize the toxicity of a given mixture. For example, a basic adult rat neurotoxicity study was conducted using a 1:1 ratio of two cholinesterase-inhibiting metabolites of the N-methyl CM pesticide aldicarb: aldicarb sulfoxide and aldicarb sulfone (DePass et al., 1985). This study defined the dosage level that precipitated plasma, erythrocyte, or brain cholinesterase inhibition. The authors made no effort to assess the toxicity of either metabolite alone but were solely interested in the toxicity of the mixture. Another study used a mixture of the 15 most commonly used pesticides in Italy (not all were O P or CM compounds) to determine if that mixture was capable of precipitating adverse genetic effects in human lymphocytes (Dolara et al., 1994). It was found that the inclusion of benomyl in
*The views expressed are those of the author and do not necessarily reflect the views and policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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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the mixture increased its toxicity markedly, leading the authors to suggest that removal of benomyl could reduce the potential for human toxicity due to pesticide contaminants in food. In related studies of mixtures based on probable human exposure (Ito et al., 1995a,b), rats were fed a mixture of either lx or 10x the Acceptable Daily Intake (ADI) of 10 OPs plus 1 organochlorine pesticide in an effort to determine if the mixture showed increased toxicity beyond the regulated levels. The authors were concerned with possible carcinogenic effects of a mixture of commonly used pesticides and therefore toxicity was defined as liver preneoplastic lesions. No lesions were found in the animals fed the pesticide mixture at lx the ADI, but the mixture at 100x the ADI did precipitate lesions. The authors concluded that humans were protected from possible carcinogenicity by current regulated pesticide exposure levels. In another study, the concentration of OP and CM pesticides in rainwater correlated well with the cholinesterase-inhibiting potency of the rainwater (Hamers et al., 2001). Because the in vitro anticholinesterase potency of the rainwater sample was directly related to its pesticide profile, it is very unlikely that the interaction of the pesticides in the mixture was more than additive. Although this is not a whole animal study, this study is especially relevant because the pesticide levels in the mixture were low and the end point, cholinesterase inhibition, was more sensitive than the commonly used lethal end points. Another important aspect of studies on defined mixtures involves interaction of the impurities with the pesticide in a formulation. One of the most well-known and wellstudied examples is the malathion epidemic poisoning of malaria pesticide appliers in Pakistan (Baker et al., 1978; Krishnan and Brodeur, 1994). Ordinarily, malathion has very low mammalian toxicity, but in this instance, many workers applying the pesticide became gravely ill and some died. The culprit was assumed to be higher than normal levels of a breakdown product of malathion, isomalathion, in the formulations used by the sprayers. In subsequent years, many investigators studied the toxicity of "contaminated" formulations (Aldridge et al., 1979; Toia et al., 1980; Umetsu et al., 1977, 1981). Two laboratories (Aldridge et al., 1979; Umetsu et al., 1977) confirmed potentiation of malathion toxicity by isomalathion and identified other contaminants that may also potentiate malathion toxicity. Although malathion tends to become more toxic if it is stored in a manner that promotes the production of isomalathion or trimethyl phosphorothioates, this is not the case for impurities found in other OP compounds, such as acephate (Umetsu et al., 1977) or fenthion (Toia et al., 1980). Some investigators have also studied the interaction of the pesticide with the other compounds found in pesticide formulations. For example, in a study comparing the
toxicity of a fenitrothion formulation (fenitrothion + emulsifier + cosolvent) with fenitrothion alone, Durham and coworkers (1982) found that the formulation did not appear to affect the toxicity of fenitrothion. In a more recent study using depressed neurite outgrowth in neuroblastoma cells as a measure of toxicity, some solvents found in commercial formulations appeared to potentiate the toxicity of chlorpyrifos (Axelrad et al., 2002). Obviously, the interaction potential for the formulation with a pesticide depends on the formulation and the pesticide because no consistent pattern can be ascertained from the scarce data.
lII. D E S I G N O F I N T E R A C T I O N S T U D I E S OP and N-methyl CM pesticides inhibit cholinesterase activity. It is assumed that the primary mechanism of toxicity is inhibition of cholinesterase activity, and this represents a common mechanism for grouping these pesticides for risk assessment (Mileson et al., 1998). There is no doubt, however, that these pesticides, especially the OP pesticides, have other mechanisms of toxicity (Reviewed in Pope, 1999). Nevertheless, for the purpose of assessing mixtures of these pesticides, the most reasonable, initial approach is to consider them as a "homergic" mixture (Wessinger, 1986; Woolverton, 1987) that is, having the same mechanism of toxicity. The appropriate manner to test for interactions of mixtures of homergic chemicals is to use a dose-additive (similar joint action or concentration addition) experimental design (Bliss, 1939; Plaa and V6zina, 1990" Wessinger, 1986; Wessinger and Evans, 1988; Woolverton, 1987). A dose-additive design assumes that the chemicals in the mixture have the same mode of toxicity and contribute to the same toxic response. An effect-additive model, on the other hand, assumes that the chemicals have different modes of toxicity and produce different physiological responses (Bliss, 1939; Plaa and V6zina, 1990; Wessinger, 1986; Wessinger and Evans, 1988; Woolverton, 1987). Unfortunately, only a small proportion of studies on mixtures of OP or CM pesticides appear to have used the appropriate, dose-additive experimental design. Most studies have instead used an effect-additive design (independent joint action and response-additive design). To illustrate the difference, consider the following example of a binary mixture of two chemicals, A and B" The LD25 for A is 25 mg/kg and that for B is 2.5 mg/kg. A doseadditive model would combine one-half of the LD25 dose of each chemical (i.e., 12.5 mg/kg of A + 1.25 mg/kg of B) and ask if the response of the animals to the mixture was an LDe5 (additive interaction), less than an LD25 (less-thanadditive interaction), or more than an LDe5 (more-thanadditive interaction). On the other hand, an effect-additive model would combine the LD25 dose of each (i.e.,
CHAPTER 41 25 mg/kg of A and 2.5 mg of B) and ask if the response of the mixture produced an LDs0. Of course, this is a simple example because compounds can be combined using either model in different ratios depending on their toxicity. An illustration of a dose-addition design for a binary mixture of OP compounds is shown in Fig. 1. Seume and O'Brien (1960b) characterized the interaction of O-ethyl O-p-nitrophenyl phenylphosphonothionate (EPN) and O,O-dimethyl S-(N-ethylcarbamoylmethyl) phosphorodithioate (CL 18706). After determining the LD10 in mice for the individual compounds, they created five mixtures of the two compounds using different proportions of the LD10 for each compound. If the interaction had been additive across all the mixtures, then each of the bars in Fig. 1 would have hovered around the 10% mark. Instead, most of the mixtures produced a marked morethan-additive interaction, with 80-100% of the animals dying. Only the 5% EPN + 95% CL 18706 showed the expected 10% lethality. These results illustrate two important points: There is a marked toxic interaction between these two OP compounds, and the toxicity profile is directly related to the proportion of each in the mixture. Other investigators working with either pesticides or nonpesticide chemicals have also noted that the ratio of the components in the mixture can influence the type of interaction (DuBois, 1961; Gessner, 1974; Gessner and Cabana, 1970; Wessinger, 1986). Unfortunately, remarkably few investigations have altered the pesticide proportions in mixtures. In summary, the study of the interaction of OP or CM pesticides should employ a dose-additiye experimental
9Cumulative Effects of OP or CM Pesticides
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design varying ratios of the components to fully characterize the interaction profile.
IV. MIXTURES O F O R G A N O P H O S P H O R U S PESTICIDES One of the defining historical moments in pesticide mixture research occurred in 1957 when Frawley and coworkers showed the marked potentiation of malathion toxicity by EPN. When nonlethal dosages of both compounds were given to rats or dogs, 100% mortality was noted. These observations spurred a flurry of studies. The first group of studies identified one of the primary mechanisms underlying the potentiation of the malathion toxicity by EPN (Murphy and DuBois, 1957; Seume and O'Brien, 1960a): EPN inhibited the hydrolysis of malathion by carboxylesterases (CarbEs). Other investigators showed a potentiation between EPN and other OP compounds (Rosenberg and Coon, 1958; Seume and O'Brien, 1960b). Soon, these studies were followed by studies showing a more-than-additive interaction between other OP compounds that were potent inhibitors of CarbE activity, such as tri-o-cresylphosphate combined with malathion (Cohen and Murphy, 1971b; Lauwerys and Murphy, 1969; Murphy et aL, 1959; Polak and Cohen, 1969). In 1963, 112 OP compounds were assessed for their ability to lower the LDs0 of malathion in mice (Casida et al., 1963). Wide variation was obtained in the degree to which the compounds potentiated malathion toxicity; potentiation
EPN: % of Total Mixture CL18706: % of Total Mixture 100 90 80 -9 1= o
70 60 5O
o~ 4o 3o 20 10 0
Ep.oo.: 1 20.000 0o 1 23, i 33.000 000 1,2,01 0 00 1 156.750 0,2, t 165.000 0000 t 8.250 82.500 132.000
13L18706 Dose:
FIG. 1. Dose-additiveexperimental design exploring the interaction of two OP pesticides in female mice. Initially, an individual LD10 (ip) was determined for EPN and CL18706. The LD10 for EPN was 2.5 Ixg/g body weight and that for CL18706 was 165 ~g/g. The graph shows the results of the various mixtures of the two OP compounds, each as a percentage of its respective LD10. If the chemicals showed a purely additive interaction, then each of the mixtures should have shown lethality of 10%, no matter what the mixing ratio. The two compounds alone (solid black bar on left and solid gray bar on fight) produced only 10% lethality, but when the two were mixed, no matter what the ratio, 80-100% lethality was noted--a more-than-additive interaction. The only exception to this was the 5% EPN + 95% CL 18706, for which 10% lethality was noted. (Adapted from Table 4 of Seume and O'Brien, 1960b).
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varied considerably not only by class of compounds but also within a class of compounds. Further research has shown that CarbE inhibition does not totally explain the potentiation of OP toxicity by either EPN (Cohen and Murphy, 1971a) or tri-o-cresylphosphate (Lauwerys and Murphy, 1969; Polak and Cohen, 1969). It is thought that the potentiating chemical may interfere with the binding of the OP compound to nonspecific binding sites in nonvital tissues (Lauwerys and Murphy, 1969; Polak and Cohen, 1969) or that the potentiating compound may have other mechanisms for potentiation in addition to CarbE inhibition (Cohen and Murphy, 1971a). In addition, it has also been demonstrated that preexposure of rats to a CarbE inhibitor (iso-OMPA) also potentiated the toxicity of some CM pesticides (carbofuran, propoxur, or aldicarb) but not the toxicity of other CM compounds (physostigmine) (Gupta and Dettbam, 1993). Beyond the studies of CarbE inhibitors and potentiation of OP compounds, there are basic studies of the interaction of OP pesticides. Only a few of these studies used a dose-additive experimental design. One of the earliest and most extensive studies was described in DuBois's (1961) review that investigated 43 pairs of OP pesticides using a dose-additive design and lethality in female rats as an end point. Of the 43 pairs, 21 showed the expected additive interaction, 18 showed a less-than-additive interaction, and only 4 pairs (3 of the pairs contained malathion) showed a more-than-additive interaction [malathion + EPN, malathion + dipterex (Dylox; trichlorfon; O,O-dimethyl 1-hydroxyl-2,2,2-trichloroethylphosphonate), dipterex + Guthion (O, O-dimethyl S- [4-oxo- 1,2,3-benzotriazin3(4H)-ylmethyl] phosphorodithioate), and malathion + Co-Ral (O,O-dimethylO-[(1-methyl-2-methoxycarbonyl) vinyl] phosphate (oL-2-carbomethoxy-1-methylvinyl dimethyl phosphate)]. This study was followed by one of similar experimental design, except the end point was lethality in birds rather than rats (Kreitzer and Spann, 1973). Again, a more-than-additive interaction was found for the malathion + EPN and the malathion + trichlorfon mixtures; however, the malathion + Co-Ral and the trichlorfon + Guthion mixtures did not show a markedly more-thanadditive interaction as they had in rats. The phosphamidon + fenthion and malathion + parathion mixtures did not appear to show a substantial more-than-additive interaction. In another study using a dose-additive experimental design exploring the effects of pesticide mixtures on rainbowtrout lethality, the trichlorfon + Volaton [oL-([(diethyloxyphosphinothioyl)-ox]-imino) benzeneacetonitrile] produced only an additive interaction, whereas the trichlorfon + Guthion mixture may have produced a slightly more-than-additive interaction (Marking and Mauck, 1975). A dose-additive study of the mutagenic effects of mixtures of trichlorfon + methyl parathion or malathion or methyl azinphos in mice revealed that most end points were affected in a purely additive manner, with no potentiation noted (Degraeve et al., 1985).
In the studies that assessed the interaction of two OP compounds using a dose-additive design, the majority used lethality as an end point. Even when assessing that artificial, high-dose situation, many combinations did not produce a more-than-additive interaction. In fact, the majority of the binary mixtures of OP compounds produced a less-than-additive or additive interaction (DuBois, 1961). Only a few pairs of OP compounds definitively potentiated one another, such as malathion + EPN (Cohen and Murphy, 1971a; DuBois, 1961; Frawley et al., 1957; Kreitzer and Spann, 1973; Murphy and DuBois, 1957; Seume and O'Brien, 1960a)or malathion + trichlorfon (DuBois, 1961; Kreitzer and Spann, 1973), and the majority of other binary mixtures did not show definitive potentiation in these high-dose situations. A recent study of Moser & coworkers (2005) Moser et al. (2005), using a dose-additive design with mixtures of five commonly used OP pesticides (chlorpyrifos, diazinon, dimethoate, acephate, and malathion), showed a more-thanadditive interaction on multiple end points: blood and brain cholinesterase inhibition, motor activity, and gait score (tailpinch response did not show a more-than-additive interaction). This study is noteworthy because (i) relatively sensitive end points were used to test the toxic interaction of the OP pesticides, such as cholinesterase inhibition or depression of motor activity; (ii) more than two OP compounds were used in the mixture; and (iii) comprehensive statistical analyses of the data were performed. The pharmacokinetic interaction of two of the compounds in the mixture, chlorpyrifos and diazinon, has been studied in rats (Timchalk et al., 2004). The authors found that one compound did not affect the pharmacokinetics of the other unless high doses were given, concluding that a more-than-additive interaction is unlikely at environmentally relevant concentrations. In summary, there is some evidence that high doses of some but not all binary combinations of OP compounds show a more-than-additive interaction. In the one study using lower dosages of multiple OP compounds (Moser et al., 2005), a more-than-additive interaction was noted for multiple end points at low dosages.
V. M I X T U R E S O F C A R B A M A T E P E S T I C I D E S A perusal of the literature reveals virtually no studies of mixtures of CM pesticides in mammals. An in vitro study investigated the interaction of CM pesticides using an effectadditive model (Kok and Hasirci, 2004). In this investigation, an acetylcholinesterase biosensor was used to measure the cholinesterase-inhibiting potency of three single CM pesticides (aldicarb, carbaryl, and carbofuran) as well as two binary mixtures (aldicarb + carbofuran and aldicarb + carbaryl). The mixtures showed less cholinesterase inhibition than was predicted from the single compounds; it was unclear, however, what statistical comparison was used to reach this conclusion.
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VI. MIXTURES OF O R G A N O P H O S P H O R U S COMPOUNDS AND N-METHYL CARBAMATES Perhaps the most well-known CM/OP interaction studied in both laboratory animals and humans is CM protection against subsequent challenge with a nerve agent (Koster, 1946; reviewed in Bajar 2004; Gunderson et al., 1992; Lee, 1997). Normally, the prophylactic CM is the peripherally acting pyridostigmine; however, in an interesting study comparing the protective efficacy of pyridostigmine, mobam, physostigmine, and carbofuran, all four were able to protect guinea pigs against poisoning by sarin, tabun, or VX (Gordon et al., 1978). Pyridostigmine was however preferred because its protective effects were longer lasting. The generally accepted mechanism of this protection is based on the assumption that the CM reaches the cholinesterase molecule first and carbamylates the active site. This carbamylation does inhibit enzyme activity, but only for a matter of minutes to hours. Most important, this temporary occupation of the active site of the cholinesterase by the CM prevents the irreversible binding of the nerve agent to the active site. In time, the bond between the CM and the cholinesterase breaks and the cholinesterase activity is regenerated. If however, the CM had not occupied the active site of the cholinesterase, the nerve agent, which is much more reactive and potent than the CM, would have phosphorylated the active site of the cholinesterase, permanently inhibiting the enzyme activity for days. The temporary inhibition of cholinesterase by the CM is preferable to the permanent inhibition by the OP nerve agent. Beyond the nerve agent literature, there are few studies on the interaction of OP and CM compounds and these have produced mixed results. In one series of studies, pretreatment with fenthion (Miyaoka et al., 1984) or fenitrothion (Takahashi et al., 1984; Tsuda et al., 1984) markedly lowered the LD50 of a subsequent challenge with an N-methyl CM (BPMC; 2-sec-butylphenyl methylcarbamate) in mice. The increased lethality precipitated by pretreatment with the OP pesticides was correlated with an increase in the plasma concentration of BPMC (Miyaoka et al., 1984; Takahashi et al., 1984; Tsuda et al., 1984). A subsequent study (Takahashi et al., 1987), extending the work to various OP compounds (cyanophos, fenitrothion, malathion, and dichlorvos) and other structurally similar N-methyl CMs, found that cyanophos, fenitrothion, and malathion, but not dichlorvos, potentiated the toxicity of the N-methyl CMs and that the increase in plasma concentration of the N-methyl CM did not fully explain the potentiation by the OP compounds. An early investigation using a dose-additive design compared the expected to the actual LDs0 of a series of binary combinations of various pesticides in rats. (Keplinger and Deichmann, 1967). In the study, the expected:actual ratio for the carbaryl + malathion combination was 1.82, for the carbaryl + parathion combination it was 1.58, and for
9Cumulative Effects of OP or CM Pesticides
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the carbaryl + diazinon combination it was 1.30. Because the actual LDs0 was less than expected, the results indicate a more-than-additive interaction between the N-methyl CM and OP pesticide. Two other studies investigated the carbaryl + malathion mixture with different results. Using lethality in female houseflies, the carbaryl + malathion mixture was reported to show a slight antagonism (less-than-additive interaction) (Kulkarni, 1976). A carbaryl + malathion mixture was also tested using teratological end points in rats (Lechner and Abdel-Rahman, 1984). The authors used an effectadditive design, so it is difficult to attribute interaction, but the effects of the mixture did not appear to differ markedly from the high dose of either compound alone. Thus, it appears that carbaryl + malathion mixtures produce various types of interactions, depending on the end point and the species. In an early in vitro investigation using human plasma or erythrocyte cholinesterase inhibition, the carbaryl + dichlorvos or physostigmine + dichlorvos combination produced slightly less cholinesterase inhibition than was expected from the individual IC50 curves (Carter and Maddux, 1974). Using fish mortality as an end point, carbaryl + methyl or ethyl parathion both exhibited an additive interaction pattern (Macek, 1975). Unfortunately, in both of the latter studies, it is difficult to determine whether the experimental design was dose- or effect-additive. In a study that did use a dose-additive design, two OP/CM mixtures exhibited lessthan-additive interaction when tested using a 96-hr LCs0 in rainbow trout (Marking and Mauck, 1975). In an interesting study that may shed some light on the disparate results described in this section, mixtures of carbaryl and an OP pesticide [phenthoate; ethyl-(dimethoxyphosphinothioyl)thio benzeneacetate] were tested using LC50 determinations for a fish (Channa punctatus) (Sambasiva Rao et al., 1985). The type of interaction varied according to the ratio of the chemicals in the mixture, with 3:1 (carbaryl:phenthoate) showing more-than-additive interaction and 1:3 showing moderate less-than-additive interaction. In summary, CM/OP interactions are not necessarily less than additive, as is the general expectation given the wellknown antagonism between CMs and nerve agents. In fact, there are quite a few indications that in some systems there is a more-than-additive interaction between the two. Moreover, the type of interaction likely depends on the proportion of each pesticide in the mixture.
VII. T I M I N G O F A D M I N I S T R A T I O N AND SEQUENCE OF ADMINISTRATION The timing of the administration of the pesticides also influences the nature of the interaction. For example, in a pioneering study in cats, Koster (1946) showed that physosfigmine protection against diisopropylfluorophosphate (DFP) toxicity was related to the time interval between the administration of physostigmine and DFP. Mortality increased as the time between the administration
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of physostigmine and DFP increased. Specifically, when physostigmine was administered minutes before DFP, mortality was only 12.5% (one of eight cats), but when physostigmine was given 5 or 6 hr before DFP, the mortality was 100%. Likewise, a detailed time course study of the interaction of malathion with tri-o-tolyl phosphate showed maximal potentiation at shorter intervals separating the two OP compounds, waning as the interval between administration of the two increased (Cohen and Murphy, 1971b). In this case, however, the time intervals were days rather than hours, as in the Koster study, and, of course, Koster was assessing protection, whereas Cohen and Murphy were assessing potentiation. Subsequently, other authors have delineated very detailed time courses for the interaction of two OP compounds, showing that the time course of the interaction depends on the compounds. In their investigation of the interaction of an N-methyl CM with various OP pesticides, Takahashi and coworkers (1987) mapped out the influence of the time between the two doses on the subsequent lethality (LDs0). In general, although the degree of potentiation of N-methyl CM lethality differed according to the OP compound, the shorter the time interval between the doses, the higher the lethality. There were substantial differences in how long the potentiation lasted, with cyanophos and fenitrothion showing potentiation for 1 or 2 days, whereas malathion showed potentiation for only 2 hr and dichlorvos did not show any potentiation. In all the previous examples, it appears that the shorter the time interval between the two anticholinesterases, the more potent the interaction; however, in an extensive study of the interaction of numerous OP compounds with malathion, Casida and coworkers (1963) demonstrated that this was not always the case. Specifically, in two of four compounds for which the interval between the two doses was varied between 0 and 120 hr, it was found that the maximal increase in malathion lethality was not achieved at the shorter interval but, rather, at an interval of 6-24 hr. In summary, one anticholinesterase pesticide can influence the toxicity of another without being administered to the animal at the same time. Therefore, they can be considered a mixture without necessarily physically occurring together. This can occur due to a pharmacodynamic interaction, as in the case of physostigmine and DFP (Koster, 1946), or a pharmacokinetic interaction, as with triorthotoyl phosphate and malathion (Cohen and Murphy, 1971a,b; Murphy et al., 1959). A caveat is that all of the previously mentioned time course studies used high doses and lethality as an end point, which makes one wonder if these same types of interactions would still be seen at lower, sublethal doses. In an interesting study relevant to the previous discussion, an interaction was found between first and second dosages of the same compound. When chlorfenvinphos was administered to rats in two separate dosages 24 hr apart, it was found that the toxicity of the second dosage was dampened by the previous administration of the same compound (Ikeda et al.,
1990). Both lethality and brain cholinesterase inhibition were lessened in the rats that had received the first dose of chlorfenvinphos 24 hr before the second dose. Exploring the mechanisms for this apparent protection, Ikeda and coworkers found that the first dose increased the hepatic clearance of the second dose of chlorfenvinphos (Ikeda et aL, 1992), possibly by the induction of the hepatic cytochrome P450s (Ikeda et al., 1991). This series of studies raises the interesting possibility that even the same compound may produce different toxic profiles depending on the time between successive exposures. Another consideration is the sequence of administration of two compounds. Some early studies explored how the order of administration of two different cholinesterase-inhibiting compounds affected their toxicity. Physostigmine given before DFP protected cats from lethality, but if DFP was given before physostigmine, there was lethality (Koster, 1946). In another study, tri-o-cresylphosphate given 24 hr before malathion killed all the dosed rats, but when the same dosages were given in reverse order (i.e., malathion given 24 hr before tri-o-cresylphosphate), none of the rats died or even showed toxic signs (Murphy et al., 1959). Karanth and coworkers explored the sequence of administration of chlorpyrifos and parathion (Karanth et al., 2001) or methyl parathion (Karanth et al., 2004). Chlorpyrifos given before either parathion or methyl parathion produced more lethality, toxic signs, and brain cholinesterase inhibition than when either parathion or methyl parathion was given before chlorpyrifos. As with the tri-o-cresylphosphate/malathion (Murphy et al., 1959), Karanth and coworkers interpreted these findings to be due to the first compound (either tri-o-cresylphosphate or chlorpyrifos) affecting the detoxification of the second compound (malathion, parathion, or methyl parathion), but not vice versa. In other words, tri-o-cresylphosphate was likely inhibiting the CarbEs that detoxify malathion (Murphy et al., 1959), and chlorpyrifos was likely inhibiting the carboxylesterases that detoxify parathion or methyl parathion (Karanth et al., 2001, 2004). Significant detoxification of chlorpyrifos is mediated via A-esterases (Li et al., 1995; Padilla et al., 2000, 2004), which cannot be inhibited by either parathion or methyl parathion. Obviously, at least at high doses, the order or sequence of administration of two OP compounds markedly affects toxicity.
VIII. FUTURE DIRECTIONS Many obvious research needs are apparent after a perusal of the anticholinesterase mixture literature. Most important is the need to conduct mixture testing in the low range of the dose-response relationship; researchers need to use sensitive end points such as cholinesterase inhibition :rather than lethality. Experimental design and statistical analyses are also very important to the interpretation of results. In their paper on the use of mixture data in risk assessment,
CHAPTER 41
Teuschler and Hertzberg (1995) suggest directions for future mixture research, emphasizing both experimental design and statistical analyses, with suggestions for each. Because the ratio of the OP or CM pesticides in the mixture, the sequence of administration, and the timing of the administration can all have a marked influence on the toxicity profile of a mixture, more research investigating these relationships is needed to define comprehensively the interaction profile of a group of pesticides. Only a very small percentage of the mixture studies in the literature used more than two anticholinesterase pesticides, whereas in the real world humans are routinely exposed to many more than two chemicals. More studies need to be conducted using multiple chemicals. There have been advances in experiment design and analysis so that this type of multiple chemical mixture study is much easier and more approachable (Gennings, 1995) than the traditional full factorial design. Another issue that has not been mentioned is the quality of the effects of a mixture. With anticholinesterase compounds, one always expects cholinesterase inhibition, but when investigating the effects of a mixture of anticholinesterase pesticides, one must always be watchful for novel effects that may not be manifested by the individual pesticides.
Acknowledgments The author thanks Michael Cummings of the U.S. EPA library for all his excellent and timely help finding copies of the cited articles and Dr. Robert MacPhail for his careful review of earlier versions of this chapter.
References Aldridge, W. N., Miles, J. W., Mount, D. L., and Verschoyle, R. D. (1979). The toxicological properties of impurities in malathion. Arch. Toxicol. 42, 95-106. Axelrad, J. C., Howard, C. V., and McLean, W. G. (2002). Interactions between pesticides and components of pesticide formulations in an in vitro neurotoxicity test. Toxicology 173, 259-268. Bajar, J. (2004). Organophosphates/nerve agent poisoning: Mechanism of action, diagnosis, prophylaxis, and treatment. Adv. Clin. Chem. 38, 151-216. Baker, E. L., Jr., Zack, M., Miles, J. W., Alderman, L., Warren, M., Dobbin, R. D., Miller, S., and Teeters, W. R. (1978). Epidemic malathion poisoning in Pakistan malaria workers. Lancet 1, 31-34. Bliss, C. I. (1939). The toxicity of poisons applied jointly. Ann. Appl. Biol. 26, 585-615. Calabrese, E. J. (1995). Toxicological consequences of multiple chemical interactions: A primer. Toxicology 105, 121-135. Carter, K. M., and Maddux, B. (1974). Interaction of dichlorvos and anticholinesterases on the in vitro inhibition of human blood cholinesterases. Toxicol. Appl. Pharmacol. 27, 456-463.
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Casida, J. E., Baron, R. L., Eto, M., and Engel, J. L. (1963). Potentiation and neurotoxicity induced by certain organophosphates. Biochem. Pharmacol. 12, 73-83. Castorina, R., Bradman, A., McKone, T. E., Barr, D. B., Harnly, M. E., and Eskenazi, B. (2003). Cumulative organophosphate pesticide exposure and risk assessment among pregnant woman living in an agricultural community: A case study from the CHAMACOS cohort. Environm. Health Perspect. 111, 1640-1648. Cohen, S. D. (1984). Mechanisms of toxicological interactions involving organophosphate insecticides. Fundam. Appl. Toxicol. 4, 315-324. Cohen, S. D., and Murphy, S. D. (1971a). Carboxylesterase inhibition as an indicator of malathion potentiation in mice. J. Pharmacol. Exp. Ther. 176, 733-742. Cohen, S. D., and Murphy, S. D. (1971b). Malathion potentiation and inhibition of hydrolysis of various carboxylic esters by triorthotolyl phosphate (TOTP) in mice. Biochem. PharmacoL 20, 575-587. Degraeve, N., Chollet, M.-C., and Moutschen, J. (1985). Mutagenic efficiency of organophosphorus insecticides used in combined treatments. Environ. Health Perspect. 60, 395-398. DePass, L. R., Weaver, E. V., and Mirro, E. J. (1985). Aldicarb sulfoxide/aldicarb sulfone mixture in drinking water of rats: Effects on growth and acetylcholinesterase activity. J. Toxicol. Environ. Health 16, 163-172. Dolara, P., Torricelli, E, and Antonelli, N. (1994). Cytogenetic effects on human lymphocytes of a mixture of fifteen pesticides commonly used in Italy. Murat. Res. 325, 47-51. DuBois, K. P. (1961). Potentiation of the toxicity of organophosphorus compounds. Adv. Pestic. Cont. Res. 4, 117-151. Duggan, A., Charnley, G., Chen, W., Chukwudebe, A., Hawk, R., Krieger, R. I., Ross, J., and Yarborough, C. (2003). Di-alkyl phosphate biomonitoring data: Assessing cumulative exposure to organophosphate pesticides. Regul. Toxicol. Pharmacol. 37, 382-395. Durham, H. D., Comeau, A. M., Cameron, P. H., and Ecohichon, D. J. (1982). Subacute toxicity in rats of orally administered fenitrothion alone and in a selected formulation. Toxicol. Appl. Pharmacol. 62, 455--464. Fenske, R. A., Kedan, G., Lu, C., Fisker-Andersen, J. A., and Cuff, C. L. (2002). Assessment of organophosphorous pesticide exposures in the diets of preschool children in Washington State. J. Exp. Anal. Environ. Epidemiol. 12, 21-28. Frawley, J. P., Fuyat, H. N., Hagan, E. C., Blake, J. R., and Fitzhugh, O. G. (1957). Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinesterase compounds. J. Pharmacol. Exp. Ther. 121, 96-106. Gennings, C. (1995). An efficient experimental design for detecting departure from additivity in mixtures of many chemicals. Toxicology 105, 189-197. Gessner, P. K. (1974). The isobolographic method applied to drug interactions. In Drug Interactions (P. L. Morselli, S. Garattini, and S. N. Cohen, Eds.), pp. 349-362~ Raven Press, New York. Gessner, P. K., and Cabana, B. E. (1970). A study of the interaction of the hypnotic effects and of the toxic effects of chloral hydrate and ethanol. J. Pharmacol. Exp. Ther. 174, 247-259. Gordon, J. J., Leadbeater, L., and Maidment, M. P. (1978). The protection of animals against organophosphate poisoning by
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pretreatment with a carbamate. Toxicol. Appl. Pharmacol. 43, 207-216. Gunderson, C. H., Lehmann, C. R., Sidell, E R., and Jabbari, B. (1992). Nerve agents: A review. Neurology 42, 946-950. Gupta, R. C., and Dettbarn, W.-D. (1993). Role of carboxylesterases in the prevention and potentiation of N-methylcarbamate toxicity. Chem.-Biol. Interact. 87, 295-303. Hamers, T., Smit, M. G. D., Murk, A. J., and Koeman, J. H. (2001). Biological and chemical analysis of the toxic potency of pesticides in rainwater. Chemosphere 45, 609-624. Ikeda, T., Kojima, T., Yoshida, M., Takahashi, H., Tsuda, S., and Shirasu, Y. (1990). Pretreatment of rats wtih an organophosphorus insecticide, chlorfenvinphos, protects against subsequent challenge with the same compound. Fundam. Appl. Toxicol. 14, 560-567. Ikeda, T., Tsuda, S., and Shirasu, Y. (1991). Metabolic induction of the hepatic cytochrome P-450 system by chlorfenvinphos in rats. Fundam. Appl. Toxicol. 17, 361-367. Ikeda, T., Tsuda, S., and Shirasu, Y. (1992). Pharmacokinetic analysis of protection by an organophosphorus insecticide, chlorfenvinphos, against the toxicity of its succeeding dosage in rats. Fundam. Appl. Toxicol. 18, 299-306. Ito, N., Hagiwara, A., Tamano, S., Hasegawa, R., Imaida, K., Hirose, M., and Shirai, T. (1995a). Lack of carcinogenicity of pesticide mixtures administered in the diet at acceptable daily intake (ADI) dose levels in rats. Toxicol. Lett. 82/83, 513-520. Ito, N., Hasegawa, R., Imaida, K., Kurata, Y., Hagiwara, A., and Shirai, T. (1995b). Effect of ingestion of 20 pesticides in combination at acceptable daily intake levels on rat liver carcinogenesis. Food Chem. Toxicol. 33, 159-163. Iyaniwura, T. T. (1990). Mammalian toxicity and combined exposure to pesticides. Vet. Hum. Toxicol. 32, 58-62. Karanth, S., Olivier, K., Jr., Liu, J., and Pope, C. (2001). In vivo interaction between chlorpyrifos and parathion in adult rats: Sequence of administration can markedly influence toxic outcome. Toxicol. Appl. Pharmacol. 177, 247-255. Karanth, S., Liu, J., Olivier, K., Jr., and Pope, C. (2004). Interactive toxicity of the organophosphorus insecticides chlorpyrifos and methyl parathion in adult rats. Toxicol. Appl. Pharmacol. 196, 183-190. Keplinger, M. L., and Deichmann, W. B. (1967). Acute toxicity of combinations of pesticides. Toxicol. Appl. Pharmacol. 10, 586-595. Kok, E N., and Hasirci, V. (2004). Determination of binary pesticide mixtures by an acetylcholinesterase-choline oxidase biosensor. Biosens. Bioelectron. 19, 661-665. Koster, R. (1946). Synergisms and antagonisms between physostigmine and di-isopropyl fluorophosphate in cats. J. Pharmacol. Exp. Ther. 88, 39-46. Kreitzer, J. E, and Spann, J. W. (1973). Tests of pesticidal synergism with young pheasants and Japanese quail. Bull. Environ. Contam. Toxicol. 9, 250-256. Krishnan, K., and Brodeur, J. (1991). Toxicological consequences of combined exposure to environmental pollutants. Arch. Complex Environ. Studies 3, 1-106. Krishnan, K., and Brodeur, J. (1994). Toxic interactions among environmental pollutants: Corroborating laboratory observations with human exposure. Environ. Health Perspecr 102, 11-17. Kulkarni, A. E (1976). Joint action of insecticides against houseflies. J. Toxicol. Environ. Health 1, 521-530.
Lauwerys, R. R., and Murphy, S. D. (1969). Interaction between paraoxon and tri-o-tolyl phosphate in rats. Toxicol. Appl. Pharmacol. 14, 348-357. Lechner, D. M. W., and Abdel-Rahman, M. S. (1984). A teratology study of carbaryl and malathion mixtures in rat. J. Toxicol. Environ. Health 14, 267-278. Lee, E. J. (1997). Pharmacology and toxicology of chemical warfare agents. Ann. Acad. Med. Singapore 26, 104-107. Li, W. E, Furlong, C. E., and Costa, L. G. (1995). Paraoxonase protects against chlorpyrifos toxicity in mice. Toxicol. Lett. 76, 219-226. Macek, K. J. (1975). Acute toxicity of pesticide mixtures to bluegills. Bull. Environ. Contam. Toxicol. 14, 648-652. Marking, L. L., and Mauck, W. L. (1975). Toxicity of paired mixtures of candidate forest insecticides to rainbow trout. Bull. Environ. Contam. Toxicol. 13, 518-522. Mileson, B. E., Chambers, J. E., Chen, W. L., Dettbarn, W., Ehrich, M,, Eldefrawi, A. T., Gaylor, D. W., Hamernik, K., Hodgson, E., Karczmar, A. G., Padilla, S., Pope, C. N., Richardson, R. J., Saunders, D. R., Sheets, L. E, Sultatos, L. G., and Wallace, K. B. (1998). Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41, 8-20. Miyaoka, T., Takahashi, H., Tsuda, S., and Shirasu, Y. (1984). Potentition of acute toxicity of 2-sec-butylphenyl N-methylcarbamate (BPMC) by fenthion in mice. Fundam. Appl. Toxicol. 4, 802-807. Moser, V. C., Casey, M., Hamm, A., Carter, J., W. H., Simmons, J. E., and Gennings, C. (2005). Neurotoxicological and statistical analyses of a mixture of five organophosphorus pesticides using a ray design. Toxicol. Sci., 86, 101-115. Murphy, S. D. (1969). Mechanisms of pesticide interaction in vertebrates. Residue Rev. 25, 201-222. Murphy, S. D. (1980). Assessment of the potential for toxic interactions among environmental pollutants. In The Principles and Methods in Modem Toxicology (C. L. Galli, S. D. Murphy, and R. Paoletti, Eds.), pp. 277-294. Elsevier/North-Holland, Amsterdam. Murphy, S. D., and DuBois, K. P. (1957). Quantitative measurement of inhibition of the enzymatic detoxification of malathion by EPN (ethyl p-nitrophenyl thionobenzenephosphonate). Proc. Soc. Exp. Biol. Med. 96, 813-818. Murphy, S. D., Anderson, R. L., and DuBois, K. E (1959). Potentiation of toxicity of malathion by triortotolyl phosphate. Proc. Soc. Exp. Biol. Med. 100, 483-487. Padilla, S., Buzzard, J., and Moser, V. C. (2000). Comparison of the role of esterases in the differential age-related sensitivity to chlorpyrifos and methamidophos. NeuroToxicology 21, 49-56. Padilla, S., Sung, H. J., and Moser, V. C. (2004). Further assessment of an in vitro screen that may help identify organophosphorus pesticides that are more acutely toxic to the young. J. Toxicol. Environ. Health A 67, 1477-1489. Plaa, G. L., and V6zina, M. (1990). Factors to consider in the design and evaluation of chemical interactions studies in laboratory animals. In Toxic interactions (R. S. Goldstein, W. R. Hewitt, and J. B. Hook, Eds.), pp. 3-30. Academic Press, New York. Polak, R. L., and Cohen, M. E. (1969). The influence of triorthocresylphosphate on the distribution of 32p in the body of the rat after the injection of 32p-sarin. Biochem. Pharmacol. 18, 813-820.
CHAPTER 41 Pope, C. N. (1999). Organophosphorus pesticides: Do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health B 2, 161-181. Rosenberg, E, and Coon, J. M. (1958). Potentiation between cholinesterase inhibitors. Proc. Soc. Exp. Biol. Med 97, 836-839. Sambasiva Rao, K. R. S., Prasada Rao, K. S., Ahammad Sahib, I. K., and Ramana Rao, K. V. (1985). Combined action of carbaryl and phenthoate on a freshwater fish (Channa punctatus Bloch). Ecotoxicol. Environ. Safety 10, 209-217. Sfinchez-Pefia, L. C., Reyes, B. E., L6pez-Carrillo, L., Recio, R., Mor~in-Martinez, J., Cebrifin, M. E., and Quintanilla-Vega, B. (2004). Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers. Toxicol. Appl. Pharmacol. 196, 108-113. Seume, E W., and O'Brien, R. D. (1960a). Metabolism of malathion by rat tissue preparations and its modification by EPN. Agric. Food Chem. 8, 36-41. Seume, E W., and O'Brien, R. D. (1960b). Potentiation of the toxicity to insects and mice of phosphorothionates containing carboxylester and carboxyamide groups. Toxicol. Appl. Pharmacol. 2, 495-503. Takahashi, H., Miyaoka, T., Tsuda, S., and Shirasu, Y. (1984). Potentiated toxicity of 2-sec-butylphenyl methylcarbamate (BPMC) by O,O,-dimethyl O-(3-methyl-4-nitrophenyl)phosphorothioate (fenitrothion) in mice; Relationship between actue toxicity and metabolism of BPMC. Fundam. Appl. Toxicol. 4, 718-723. Takahashi, H., Kato, A., Yanashita, E., Naito, Y., Tsuda, S., and Shirasu, Y. (1987). Potentiations of N-methylcarbamate toxicities by organophosphorus insecticides in male mice. Fundam. Appl. Toxicol. 8, 139-146.
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Teuschler, L. K., and Hertzberg, R. C. (1995). Current and future risk assessment guidelines, policy, and methods development for chemical mixtures. Toxicology 105, 137-144. Timchalk, C., Poet, T. S., Hinman, M. N., Busby, A. L., and Kousba, A. A. (2004). Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat. Toxicol. Appl. Pharmacol. 205, 31-42. Toia, R. E, March, R. B., Umetsu, N., Mallipudi, N. M., Allahyari, R., and Fukuto, T. R. (1980). Identification and toxicological evaluation of impurities in technical malathion and fenthion. J. Agric. Food Chem. 28, 599-604. Tsuda, S., Miyaoka, T., Iwasaki, M., and Shirasu, Y. (1984). Pharmacokinetic analysis of increased toxicity of 2-secbutylphenyl methylcarbamate (BPMC) by fenitrothion pretreatment in mice. Fundam. Appl. Toxicol. 4, 724-730. Umetsu, N., Grose, E H., Allahyari, R., Abu-E1-Haj, S., and Fukuto, T. R. (1977). Effect of impurities on the mammalian toxicity of technical malathion and acephate. J. Agric. Food Chem. 25, 946-953. Umetsu, N., Mallipudi, N. M., Toia, R. E, March, R. B., and Fukuto, T. R. (1981). Toxicological properties of phosphorothioate and related esters present as impurities in technical organophosphorus insecticides. J. Toxicol. Environ. Health 7, 481-497. Wessinger, W. D. (1986). Approaches to the study of drug interactions in behavioral pharmacology. Neurosci. Biobehav. Rev. 10, 103-113. Wessinger, W. D., and Evans, E. B. (1988). Modeling multiple agent interactions in behavioral pharmacology. J. Am. Coll. Toxicol. 7, 953-962. Woolverton, W. L. (1987). Analysis of drug interactions in behavioral pharmacology. Adv. Behav. Pharmacol. 6, 275-302.
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CHAPTER 4
Federal Regulations and Risk A s s e s s m e n t of Organophosphate and Carbamate Pesticides A N N A B. LOWIT U.S. Environmental Protection Agency, Washington, DC
and technical analyses for specific risk assessments, can be found at www.epa.gov/pesticides/reregistration/status.htm and the EPA's pesticide docket and electronic EPA dockets Web site, www.epa.gov/edocket. Because this chapter focuses primarily on regulations, activities, and efforts in the United States, particularly those at the EPA, the reader may want to search other sources of information regarding risk assessment activities for the OPs and N-methyl CMs by other organizations, such as the World Health Organization's Joint FAO/WHO Meeting on Pesticide Residues, Canada's Pest Management Regulatory Agency, the Organization for Economic Co-operation and Development, and the California Department of Pesticide Regulation. People are exposed to OPs and N-methyl CMs through various pathways. OPs and N-methyl CMs are applied to various agricultural crops and therefore can be present in food. OPs and N-methyl CMs can be present in water. Some OPs and N-methyl CMs can be purchased by consumers for use in or around the home; thus, residential exposure to children and adults is possible. Agricultural workers and professional pesticide applicators are exposed to these pesticides through occupational activities. EPA-OPP develops risk assessments for many exposure pathways and scenarios, including exposures to food and water, residential use, and occupational exposure. Risk is composed of two major components: hazard assessment and exposure assessment. Aspects of chemical toxicity and actual environmental exposure impact potential risk to a particular agent(s). This chapter reviews the toxicology of OPs and N-methyl CMs; thus, hazard assessment of the OPs and N-methyl CMs is discussed in detail. There are many publicly available sources of information regarding exposure to OPs and N-methyl CMs, including the U.S. Department of Agriculture's pesticide data program, the Food and Drug Administration's food monitoring program, U.S. Geological Survey water monitoring programs, and the National Home and Garden Pesticide Use Survey. Specific
I. I N T R O D U C T I O N In the United States, pesticide chemicals are registered for use by the U.S. Environmental Protection Agency (EPA). Although some are no longer registered in the United States, there are more than 30 organophosphates (OPs) and more than 15 N-methyl carbamates (CMs). A complete, exhaustive review of the history and risk assessments for each of these chemicals is beyond the scope of this chapter. Instead, this chapter provides an overview of the federal regulations governing pesticide chemicals in the United States and the risk assessment practices and approaches used by the EPA's Office of Pesticide Programs (EPA-OPP) to evaluate the OP and N-methyl CM pesticides. The text focuses on aspects of the OP and N-methyl CM risk assessments that provide the most robust overview, particularly noncancer effects related to the inhibition of acetylcholinesterase (ACHE). This chapter highlights key milestones and accomplishments regarding risk assessments of the OP and N-methyl CM pesticides, particularly after the passage of the Food Quality Protection Act (FQPA) of 1996. It is important to note that while this chapter was being written the EPA was actively working on risk assessments for some OPs and N-methyl CMs and the cumulative risk assessments for both common mechanism groups. Reference doses and benchmark dose estimates provided in this chapter are subject to change as risk assessments develop or are refined and/or as new data and information become available for evaluation. The values listed reflect the best information as of January 1, 2005. For the most recent status of individual chemicals of interest and/or the cumulative risk assessments, the reader is referred to the EPA's Web site (www.epa.gov/ pesticides), which contains a wealth of information about many areas of pesticide regulation and risk assessment. Additional information, including supporting documentation
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exposure assessments for each pesticide can also be found at the EPA's Web site and/or at the EPA's e-docket.
II. F E D E R A L R E G U L A T I O N S GOVERNING P E S T I C I D E S IN T H E U N I T E D S T A T E S The first modern form of today's pesticide laws, the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), was passed in 1947, and it has undergone significant revisions since that time. Under FIFRA, EPA registration is required prior to manufacture, transport, or sale in the United States. FIFRA also provides EPA with the authority to require data to be provided prior to registration. In 1984, extensive data requirements were published for toxicity testing in mammal and nonmammalian species, physical-chemical properties, environmental fate, and field trial studies (to evaluate potential food exposure). These requirements are listed in 40 CFR part 158 (www.access.gpo.gov/nara/cfr/index/html). Numerous toxicity studies in laboratory animals are required to evaluate metabolism and pharmacokinetics in addition to potential subchronic and chronic toxicity, neurotoxicity, developmental toxicity, carcinogenicity, and genotoxicity. Typically, toxicology studies are performed via oral administration; however, dermal and/or inhalation toxicology studies may be required in some cases, depending on the exposure pattern(s) of the particular pesticide, physical-chemical properties, and/or potential route-specific hazard concerns. EPA also regulates pesticides under the Federal, Food, Drug, and Cosmetic Act (FFDCA). The FFDCA provides EPA with the authority to set pesticide tolerances (maximum allowable pesticide residue levels) in food and feed. Food and feed are considered adulterated if they contain a pesticide residue greater than the tolerance or if they contain a pesticide not covered by an existing tolerance. The most recent revision to both FFDCA and FIFRA occurred in 1996 with the passage of the Food Quality Protection Act (FQPA). Among other things, FQPA required that EPA reevaluate all existing food tolerances by August 2006. This process is called "tolerance reassessment." By early 2005, EPA had reassessed more than two-thirds of all food tolerances. FQPA also imposed key requirements on EPA that substantially changed some approaches used by EPA-OPP in many of its pesticide risk assessments. Specifically, FQPA mandated that EPA (i) perform aggregate risk assessment in its single chemical risk assessment, (ii) perform cumulative risk assessment for groups of pesticides with a common mechanism of action, and (iii) apply an additional FQPA 10x safety factor specifically for infants and children. Aspects of each of these provisions are discussed in this chapter. As part of tolerance reassessment, Congress required EPA to prioritize tolerance reassessment by evaluating the "worst first." As such, many of the single chemical assessments for the OPs and N-methyl CMs were identified for development early in the tolerance reassessment process; many of the risk
assessments for the individual OPs and N-methyl CMs were complete or close to completion at the time of writing of this chapter. However, several chemicals, namely malathion, dimethoate, dichlorvos, formetanate HC1, aldicarb, and carbofuran, were not yet complete or finalized. Although available for OPs in a well-developed form, the cumulative risk assessments for neither common mechanism were finalized.
III. S I N G L E - C H E M I C A L , AGGREGATE
RISK ASSESSMENTS FOR OPs AND N-METHYL CMs Historically, EPA has focused on single pathways of exposure (e.g., pesticide residues in food, water, or residential/ nonoccupational uses) for individual chemicals and not on the potential for individuals to be exposed to multiple pathways or to multiple pesticides concurrently. The requirement by FQPA to perform aggregate and cumulative risk assessments substantially changed some approaches used by EPA-OPP. "Aggregate risk" is defined by EPA-OPP as the sum total of all exposure to pesticides through inhalation or dermal, oral, or optic contact (EPA, 2001a). Aggregate risk is the risk to a single pesticide chemical from multiple routes and pathways of exposure (e.g., food + water + residential).
A. Hazard Assessment and Hazard Identification Procedures When performing hazard assessment and hazard identification for aggregate risk assessment, dose-response aspects of all the potential toxicities for a single pesticide are considered. EPA's guidance documents for developing reference doses (RfDs) and reference concentrations (RfCs) (EPA, 1994, 2002a) provide a comprehensive discussion of performing dose-response analysis for risk assessment purposes. EPA-OPP uses two different risk metrics in its pesticide noncancer risk assessments: the RfD and the margin of exposure. In both methods, a point of departure (POD) is first established, followed by the identification of appropriate uncertainty and extrapolation factors. The information contained in EPA's RfD guidance document, the review of RfD procedures, and the benchmark dose (BMD) guidance (EPA, 1994, 2000, 2002a) together provide a thorough description of the history, purpose, and procedures for developing PoDs. The PoD is selected to be protective of the critical effect for a particular pesticide. This approach is based, in part, on the assumption that if the critical toxic effect is prevented, then all toxic effects are prevented. A PoD can be a BMD estimate (EPA, 2000), no-observed-adverse-effect-level (NOAEL), or a lowest-observed-adverse-effect-level (LOAEL). The types of effects that have been identified as critical effects for the single chemical assessments for OPs and N-methyl
CHAPTER 42 9Federal Regulations and Risk Assessment CMs include AChE inhibition, particularly in the blood and brain; behavioral and/or motor activity changes; and developmental toxicity or developmental neurotoxicity. When EPA evaluates AChE data for risk assessment purposes, measurements of AChE from the target tissue m the central or peripheral nervous system m are preferred. Toxicology studies submitted to EPA for pesticide registration typically measure plasma, red blood cell (RBC), and brain cholinesterase (ChE) inhibition and most often do not measure peripheral tissues. In the absence of such data, EPA considers blood measures as surrogates of peripheral tissues. The next step is to identify the appropriate uncertainty and extrapolation factors. Historically, EPA-OPP has used a 10X factor for interspecies extrapolation (e.g., extrapolation from animal to human) and a 10X factor for intraspecies extrapolation to account for human variability. In some cases, studies on human subjects are available and contain information regarding human pharmacokinetic (PK) or pharmacodynamic (PD) effects. In these cases, it may be appropriate to reduce or remove the interspecies extrapolation factor. The LOAEL to NOAEL uncertainty factor may be applied when a NOAEL is not identified in a toxicology study and the LOAEL must be used as the PoD. B MD modeling offers a preferred alternative to using NOAELs or LOAELs since the entire dose-response curve along with uncertainty are considered in the PoD development. When using B MD modeling, the LOAEL to NOAEL factor may not be necessary. A subchronic to chronic duration extrapolation factor may be applied when a chronic study is not available. In cases in which a key study (e.g., developmental toxicity) is not available in the database of toxicology studies, the database uncertainty factor may be used. When applying each of these factors, particular attention is paid to the mode or mechanism of action for a particular chemical. For example, as discussed later for the N-methyl CMs, the mechanism of action for the N-methyl CMs involves rapid recovery of AChE inhibition. For most N-methyl CMs, recovery occurs in minutes to hours. The toxic effects observed after exposure to N-methyl CMs generally do not increase with repeated days of exposure. Acute effects are the primary concern; the subchronic to chronic uncertainty factor may not be relevant for this class of pesticides. FQPA requires that EPA apply a 10X factor to account for added sensitivity of infants and children unless there are sufficient data to reduce this factor. This factor is called the FQPA 10X factor for the protection of infants and children. In single-chemical risk assessments, EPA-OPP evaluates the adequacy of the toxicology database for evaluating potential sensitivity in infants or children on a chemical-by-chemical basis. EPA published a data-call in notice (DCI) requiring developmental neurotoxicity (DNT) studies for each of the OPs with active pesticide registrations. In addition to the DNT study, a companion
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study to evaluate the comparative sensitivity for AChE inhibition in adult and juvenile animals was required. Chapter 42 describes aspects of the DNT and comparative AChE studies along with the DCI. Many of the DNT and comparative AChE studies for the OPs are under review by EPA. Several comparative AChE studies or DNT in several N-methyl CMs were planned or ongoing. As such, FQPA factors for some OPs and N-methyl CMs may be revised in the future; chemical-specific FQPA 10X factors are not provided in this chapter. Particularly through EPA's Integrated Risk Information System program, the agency typically develops RfDs and RfCs only for long-term or chronic exposures. However, because of the requirements of FQPA, EPA-OPP also develops risk assessments for exposure scenarios of varying duration and pathways of exposure. These include dietary exposure from food and water, residential exposure, and occupational activities. EPA-OPP also considers specific population groups (e.g., adults and children) in its risk assessments. In dietary risk assessment, EPA-OPP considers both acute (single exposures) and chronic exposures to food and drinking water. In residential and occupational risk assessments, EPA-OPP evaluates potential toxic effects that could result following dermal and/or inhalation exposure from applying pesticides in or around the home or from various activities such as playing on the lawn. Potential oral exposure to young children and toddlers from mouthing behaviors is also evaluated. When selecting PoDs, to the degree possible, EPA-OPP attempts to select end points from available toxicology studies that match the duration and route of exposure with specific exposure scenarios. For example, for acute dietary risk assessment, EPA prefers to use a single oral dosing study or a multiple dosing study in which clinical observations were made after an oral single exposure. Regarding the acute RfDs, AChE inhibition following single oral exposures is the critical effect for most OPs and N-methyl CMs. In cases in which developmental effects are observed in a developmental toxicity or developmental neurotoxicity study, EPA first considers the mechanistic information available that could characterize the progression of the noted teratogenic effect. In the absence of such mechanistic information, EPA may assume the observed developmental effect could potentially occur following a single exposure and may select the noted developmental effect for estimation of acute dietary risk. When estimating residential risk to a pesticide used to treat lawns, EPA evaluates potential dermal exposures of various durations. EPA prefers to use toxicity studies performed via the dermal route but again may consider developmental end points from oral studies in specific cases in which developmental toxicity provides the critical effect(s). When effects from an oral study are selected as the PoD, a dermal absorption factor is also identified to adjust for differential absorption between the oral and dermal absorption routes.
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B. Acute and Chronic Dietary Risk Assessment for OPs and N-Methyl CMs The oral route of administration provides the most robust database of toxicology studies and also provides the most extensive set of regulatory end points that can be used for comparison purposes. Regulatory end points used to develop dermal and inhalation risk assessments can be found for specific chemicals at EPA's Web site or e-docket. Tables 1 and 2 provide the PoDs identified for acute and chronic dietary exposure assessment, the selected uncertainty and/or extrapolation factors (FQPA 10• factors are not provided), and the derived acute and chronic RfDs for the OPs and N-methyl CMs. The mechanism of action for the N-methyl CMs involves rapid recovery of AChE inhibition, and the effects observed after exposure to N-methyl CMs generally do not increase with repeated exposure. Thus, for some N-methyl CMs, a chronic PoD has not been established. The acute PoD, along with uncertainty/extrapolation factors for these pesticides, is protective of effects observed following acute, subchronic, or chronic exposures. ChE inhibition provides the critical effect for acute and chronic RfDs for almost all the OPs and N-methyl CMs. Regarding acute exposures, the rat is most commonly used in acute neurotoxicity studies; thus, rat studies were selected most often for derivation of acute RfDs. Studies performed in rat and dog are commonly used for chronic RfD derivation.
IV. CUMULATIVE RISK ASSESSMENT FOR OPs AND N-METHYL CMs As mentioned previously, in addition to aggregate risk assessment, FQPA required EPA to consider "available evidence concerning the cumulative effects on infants and children of such residues and other substances that have a common mechanism of toxicity." Mechanism of toxicity is defined by EPA-OPP (EPA, 1999a) as the major steps leading to an adverse health effect following interaction of a pesticide with biological targets. All steps do not need to be specifically understood. Rather, it is the identification of the crucial events following chemical interaction that are required in being able to describe a mechanism of toxicity. Common mechanism of toxicity pertains to cases where two or more chemicals produce or may be expected to produce adverse effects by the same crucial steps(s). Thus, "cumulative risk" is defined as the risk that may result from dietary, residential, or other nonoccupational exposure to multiple chemicals that have a common mechanism of toxicity (i.e., cumulative risk). Cumulative risk assessments include multiple pathways and multiple chemicals. EPA-OPP has developed a guidance document for developing cumulative risks assessments under FQPA (EPA, 2002b). Single-chemical, aggregate assessments differ from cumulative risk assessments in focus and intent. Regarding hazard
assessment, single-chemical, aggregate assessments consider all potential toxicities; cumulative risk assessments focus on the common toxic effect for the common mechanism group. The first step in producing a cumulative risk assessment is to identify a group of chemicals that produce a common toxic effect(s) by a common mechanism of toxicity. EPA-OPP has developed a general framework for identifying the chemicals that belong to that group (EPA, 1999a). The cumulative guidance states that in determining this common mechanism group (CMG), careful attention should be given to a variety of factors, including the mechanism of toxicity, the time dimensions of the toxic effects and exposure, and the pesticide exposure patterns and treatment scenarios. Thus, assessing the potential for two or more pesticides to act by the same mechanism involves the consideration of the following principles: (i) They cause the same critical effect(s), (ii) they act on the same molecular target at the same target tissue, and (iii) they act by the same biochemical mechanism of action perhaps because they share a common toxic intermediate (Miteson et al., 1998). The OPs and N-methyl CMs have been identified as CMGs (EPA, 1999b, 2001b). The OP pesticides were grouped by a common mechanism based on their shared ability to inhibit AChE by phosphorylation of the active site of the enzyme. Regarding the CM pesticides, EPA distinguishes pesticides with the CM chemical structure that have the potential for inhibiting AChE and those CM pesticides that do not inhibit AChE (EPA, 1999b). Specifically, EPA separates CM pesticides into three distinct subgroups: N-methyl CMs, thiocarbamates, and dithiocarbamates. The following discussion focuses on the ChE-inhibiting N-methyl CMs; thiocarbamates and dithiocarbamates are not discussed further. The subgroup of N-methyl CMs share similar structural characteristics and share the ability to inhibit AChE by carbamylation of the serine hydroxyl group located in the active site of the enzyme (EPA, 2001c), leading to rapid recovery of ChE inhibition. Based on these similarities, the N-methyl CMs have been grouped into a CMG. Because the OPs and N-methyl CMs have been grouped into CMGs, these two classes of pesticides are subject to cumulative risk assessment.
A. OP Cumulative Risk Assessment Pesticides are determined to have a "common mechanism of toxicity" if they act the same way in the body; that is, if scientifically reliable data demonstrate that upon exposure to these chemicals, the same toxic effect occurs in or at the same organ or tissue by essentially the same sequence of major biochemical events. The OPs were the first common mechanism of toxicity group identified by EPA and are the first pesticides to undergo a full cumulative risk assessment. More than 30 OPs are included in the CMG. However, not all are included in the cumulative assessment group (CAG). After the CMG is established, the next step involves selecting a subset of these chemicals as the CAG. This subset
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of C M G chemicals is selected because not all chemicals grouped by common mechanism of toxicity should necessarily be included in a quantitative cumulative risk assessment. For example, initial cumulative assessments should not attempt to quantify risk resulting from chemicals with low hazard potential or from minor exposure scenarios but should instead focus on those chemicals that are likely to be risk contributors. Specifically, the CAG, and consequently the cumulative risk assessment, should exclude those chemicals, chemical uses, and exposure scenarios/routes/pathways for
TABLE 3.
9Federal Regulations and Risk Assessment
which risk and exposure do not contribute in any meaningful or substantive way to the total cumulative risk (EPA, 2002b). The OPs included in the cumulative risk assessment are provided in Table 3. These chemicals were selected based on registered agricultural and/or residential uses in the United States. OPs, such as parathion, that have been canceled or are under phase-out, were excluded. Since the passage of FQPA in 1996, EPA has held more than 25 public technical briefings and external scientific peer reviews with the FIFRA Scientific Advisory Panel to discuss methods, approaches,
Oral BMD10s and BMDLs for the OPs (mg/kg/Day) Estimated for Brain ChE Activity for the OP Cumulative Risk Assessment a Female rat
Chemical
Acephate Azinphos-methyl Bensulide Chlorethoxyfos Chlorpyrifo s Chlorpyrifos-methyl Diazinon Dichlorvos
BMDlo
Male rat BMDL
BMDlo
BMDL
0.99 0.86
0.53 0.79
0.77 1.14
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31.91
30.44
40.88
37.11
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0.61
0.69
0.62
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1.50 14.26
1.27 4.21
6.24 2.35
2.89 1.61
9.62 1.71
5.39 0.08
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0.04
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0.25
0.22
0.35
0.31
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0.07
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Ethoprop
1.37
0.70
1.35
0.69
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1.96
0.69
1.73
0.63
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0.24 1.28
0.21 0.32
0.18 1.48
0.15 0.38
Malathion
313.91
221.12
212.02
119.31
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0.08 0.25
0.07 0.17
0.07 0.24
0.06 0.16
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0.67 0.11
0.50 0.10
0.70 0.15
0.51 0.13
Naled Omethoate
1.00 0.09
0.82 0.07
1.00 0.14
0.82 0.12
Oxydemeton-methyl Phorate
0.09 0.21
0.09 0.20
0.07 0.29
0.07 0.26
Phosalone
6.93
6.27
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3.56 0.37 2.25
2.03 0.24 1.61
4.15 0.40 1.58
2.25 0.26 0.93
20.58
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21.86
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0.10
0.08
0.18
0.17
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60.69 4.27
20.97 3.31
369.27 4.52
102.31 3.47
Trichlorfon
31.74
28.62
58.49
45.39
aData from EPA (2002c).
627
SECTION VI 9Risk Assessment & Regulations
628
data, and parameters that should be considered or included when estimating cumulative risk associated with common mechanism pesticides by multiple pathways of exposure. EPA used the relative potency factor (RPF) method in its cumulative hazard assessment of the OPs. The RPF method is based on the assumption of dose additivity. Dose additivity is the agency's assumption when evaluating the joint risk of chemicals that are toxicologically similar and act at the same target site (EPA, 2001c). Briefly, with the RPF approach, the toxic potency of each chemical is first determined. One chemical, called the index chemical, is then selected. The index chemical provides the basis for comparison. Relative potency is determined by converting the toxic potency of each chemical into toxic equivalents of the index chemical. According to the cumulative guidance (EPA, 2002b), relative potency estimates should be made, to the extent possible, based on a common species and sex to provide a uniform estimate. For the OPs, although toxicology studies in human, mouse, rabbit, and dog are available, the toxicology studies in the rat provided the most extensive and complete database of ChE inhibition data for all routes of
BMDlo's Male
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interest (oral, dermal, and inhalation), compartments (blood and brain), and both sexes. EPA used rabbit studies for five chemicals with residential/nonoccupational exposure potential because dermal toxicity data in rats were not available. Toxic potency for the OPs was determined using the BMD technique (EPA, 2002c). The BMD predicted to result in 10% ChE inhibition (BMD~0) was used as the response level for determining toxic potency. EPA collected plasma, RBC, and brain ChE inhibition data from more than 30 OPs, more than 100 subchronic and chronic studies, and more than 1100 measurements o f C h E inhibition. EPA's OP ChE data set is the largest compilation of ChE data ever collected; this data set is available for public download on EPA's Web site. BMD estimates for each OP represent a whole brain ChE measured at 21 days of study or longer for each chemical. Some OPs have only one data set; however, two or more data sets are available for most. Table 3 provides BMD10 and BMDL]0 (lower 95% confidence interval on the BMD]0) estimates for female and male rat brain ChE inhibition. Figures 1 and 2 provide a plot of the BMD]0 estimates with their confidence intervals. Potency of OPs spans five orders of magnitude.
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CHAPTER
42
9 F e d e r a l R e g u l a t i o n s a n d Risk A s s e s s m e n t
629
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EPA critically evaluated the quality and quantity of the blood and brain ChE data and determined that the female brain ChE inhibition was the most appropriate data for developing RPFs. The brain ChE data typically have tighter confidence limits compared to RBC ChE data and therefore confer less uncertainty on cumulative risk estimates. Brain ChE inhibition also represents a direct measure of the common mechanism of toxicity as opposed to using surrogate measures. The toxic potencies and PoDs for brain cholinesterase inhibition for these OPs are generally similar to the RBC data for the oral, inhalation, and dermal exposures (EPA, 2002c). For some OPs, female rats are more sensitive than male rats. The cumulative risk assessment guidance document (EPA, 2002) states that the index chemical should be selected based on the availability of high-quality
FIG. 2. Plot of benchmark dose estimates for female rat brain ChE inhibition. Data from EPA (2002c).
dose-response data for the common mechanism end point and that it acts toxicologically similar to other members of the CMG. The index chemical selected for the OP cumulative risk assessment is methamidophos. Methamidophos was selected because this chemical exhibited high-quality dose-response data in plasma, RBC, and brain for both sexes of a single species for all exposure routes of interest (oral, dermal, and inhalation). Figure 3 provides dose-response curves representing brain ChE data from male and female rats for three different oral studies with methamidophos. There is remarkable similarity between male and female rats particularly as the three studies plotted were performed by three different laboratories across a 20-year span of time. Table 4 provides RPFs for each OP; values represent methamidophos-adjusted equivalents.
630
SECTION Vl
9
Risk A s s e s s m e n t & Regulations
U/G
V. C O N C L U S I O N S A N D F U T U R E CONSIDERATIONS
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EPA-OPP released the revised cumulative risk assessment for the OPs in June 2002 (EPA, 2002c); this assessment represents the first-ever cumulative risk assessment developed by EPA.
B. N-Methyl CM Cumulative Risk Assessment The N-methyl CMs were grouped by a common mechanism of action based on their similar structural characteristics and shared ability to inhibit AChE by carbamylation of the serine hydroxyl group located in the active site of the enzyme (EPA, 2001b). This carbamylation leads to rapid recovery of ChE inhibition, typically within minutes to hours. At the time of writing of this book, the N-methyl CM cumulative risk assessment was in the early stages of development. The CAG was identified to include aldicarb (and its aldoxycarb metabolite), carbaryl, carbofuran, formetanate HC1, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, and thiodicarb (Carbamate Cumulative Assessment Group, 2004). This identification was based on similar criteria used to establish the CAG for the OP cumulative risk assessment. Only those pesticides with active agricultural and/or residential uses are included. N-methyl CMs undergoing cancellation or phase-out are not included. EPA expects to release the preliminary cumulative risk assessment for this group in 2005 and a revised assessment prior to the August 2006 FQPA deadline for tolerance reassessment. The N-methyl CM cumulative hazard assessment will focus on the ChE inhibition following acute (single) exposures. EPA is also analyzing the available time course data in order to characterize recovery (EPA, 2005).
Since the passage of FQPA, EPA has worked diligently to develop risk assessments for the OPs and N-methyl CMs. These efforts have led to substantial reduction in exposures to these chemicals in food and in or around the home. For example, several pesticides have been canceled, including parathion and ethion. Numerous uses of agricultural chemicals, such as methyl parathion, phosmet, and dimethoate, have been canceled or are being phased out. Regarding the residential uses of these pesticides, most indoor use of chlorpyrifos has been canceled or is being phased out; home owner uses of diazinon and acephate are also being phased out. The OP cumulative risk assessment was the first cumulative risk assessment ever developed by EPA. EPA's groundbreaking efforts to develop the OP cumulative risk assessment have received accolades throughout the world. This assessment is one of the largest, most complex risk assessments ever developed. The N-methyl CM cumulative risk assessment will likely be more refined and sophisticated. This chapter described EPA's typical methods for evaluating pesticide chemicals. EPA is committed to incorporating the most robust and up-to-date scientific information in its risk assessments and is actively pursuing improvements in risk assessment approaches and methods. Specifically, improved characterization of PK and PD factors that impact species differences, age-related sensitivities, and the understanding of mechanism of action are important scientific areas that will improve EPA's risk assessments. The development and utilization of physiologically based pharmacokinetic/dynamic models (PBPK/PD) has expanded significantly. Regarding OPs and N-methyl CMs, there are several ongoing efforts to develop PK or mechanistic-based risk assessment approaches. Although many of these efforts may not be incorporated into EPA's risk assessments for the 2006 FQPA deadline, these efforts demonstrate EPA's commitment to improving risk assessment approaches. The knowledge gained during the development of these methods is likely to improve risk assessments in the future. Timchalk et al. (2002) and Poet et al. (2004) have developed PBPK models for chlorpyrifos and diazinon, respectively. Bayer CropSciences, pesticide registrant for aldicarb, is also developing a simple pharmacokinetic approach for considering rapid recovery explicitly in risk estimates. EPA is developing a simple PK approach for estimating AChE inhibition for exposure to the N-methyl CMs as part of research being performed for the cumulative risk assessment (EPA, 2005). EPA is developing PBPK/PD models for malathion and carbaryl. The Chemical Industry Institute for Toxicology Centers for Health Research is also developing such a model for carbaryl. The ongoing efforts to develop PBPK/PD models
CHAPTER 4 2
9Federal Regulations and Risk Assessment
631
TABLE 4. OP Relative Potency Factors (Methamidophos-Equivalents) Estimated for EPA's Cumulative Risk Assessment for the Oral, Dermal, and Inhalation Routes of Exposure a Chemical
Oral
Acephate
0.08
Azinphos-methyl B ensulide Chlorethoxyfos Chlorpyrifos Chlorpyrifos-methyl Diazinon Dichlorvos Dicrotophos Dimethoate Disulfoton
0.10 0.003 0.13 0.06 0.005 0.01 0.03 1.91 0.32 1.26
Ethoprop Fenamiphos Fenthion Fosthiazate Malathion Methamidophos Methidathion Methyl parathion Mevinphos Naled
0.06 0.04 0.33 0.07 0.0003 1.00 0.32 0.12 0.76 0.08
Omethoate Oxydemeton-methyl Phorate Phosalone Phosmet Phostebupirim Pirimiphos-methyl Profenofos Terbufos Tetrachlorvinphos Tribufos Trichlorfon
0.93 0.86 0.39 0.01 0.02 0.22 0.04 0.004 0.85 0.001 0.02 0.003
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Inhalation
0.0025
0.208
0.0015
0.677
0.47
6.596
1.5 0.015
0.315
0.015 1.00
0.003 1.00
0.075
0.82
0.00075 0.0075
0.087
aData from EPA (2002c).
for carbaryl are only possible because of PK studies developed by Bayer CropScience targeted to evaluate the time course of internal exposures to carbaryl. One critical limitation to improving the methods used in risk assessments is the current database of toxicology studies, particularly the current protocol(s) for metabolism and PK studies. PK studies typically submitted to EPA-OPP for purposes of pesticide registration were designed to evaluate absorption, distribution in tissues and organs, metabolism, and elimination in fluids and excreta. The study protocols,
however, were not specifically designed to obtain parameter values needed for developing robust PBPK/PD models. For example, sample collection is typically not targeted or specified to obtain blood/tissue partition coefficients or kinetic rates of metabolism or AChE inhibition for particular chemicals that may be identified with the critical metabolic pathways or mechanisms of action. Radiometric measurements of tissue concentrations are not sufficient to identify the specific metabolites that would constrain the parameter values associated with chemical absorption, distribution,
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S E CTI O N V I 9Risk A s s e s s m e n t & Regulations
metabolism, and excretion. Therefore, mass balance of parent chemical and metabolites in tissues must be inferred from excretion data as mass equivalents remaining. The current database of toxicology studies includes a standard battery of animal toxicology studies and does not emphasize mechanism or mode of action. Improved understanding of mechanism or mode of action for pesticide chemicals, not just ChE-inhibiting chemicals, will improve risk characterization. For example, specifically for ChE inhibitors, the time course for toxicity following acute or multiple exposures (e.g., time to peak effect and time to recovery) is often lacking. In addition, in most acute and subchronic neurotoxicity studies available to EPA, the time of peak effect is determined based on the timing of onset of clinical signs following large doses, not the time for peak AChE inhibition. Because ChE inhibition is expected to be the critical precursor event leading to more serious effects, understanding the time course of these events is important. Better characterization of sensitive subpopulations and the biological basis for the sensitivity is also an important future research area. Understanding population variability in toxicological response(s) and enzyme activities and levels, particularly those related to activation and detoxification pathways and age-related ontogeny, will be critical before the agency can consistently implement probabilistic methods into variability and uncertainty evaluations.
References Carbamate Cumulative Assessment Group (2004, February 4). Notices, OPP-2003-0360; FRL-7334-4. Fed. Reg. 69(23). Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (2001). Transmittal of the final report of the FIFRA Scientific Advisory Panel (SAP) meeting held September 7, 2001. Available at www.epa.gov. Mileson, B., Chambers, J. E., Chen, W. L., Dettbarn, W., Ehrich, M., Eldefrawi, A. T., Gaylor, D. W., Hammernik, K., Hodgson, E., Karczmar, A. G., Padilla, S., Pope, C. N., Richardson, R. J., Saunders, D. R., Sheets, L. R, Sultatos, L. G., and Wallace, K. B. (1998). Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41, 8-20. Poet, T. S., Kousba, A. A., Dennison, S. L., and Timchalk, C. (2004). Physiologically based pharmacokinetic/pharmacodynamic model for the organophosphorus pesticide diazinon. NeuroToxicology 25(6), 1013-1030. Timchalk C., Nolan, R. J., Mendrala, A. L., Dittenber, D. A., Brzak, K. A., and Mattsson, J. L. (2002). A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD)
model for the organophosphate insecticide chlorpyrifos in rats and humans. Toxicol. Sci. 66(1), 34-53. U.S. Environmental Protection Agency (1994). Methods for derivation of inhalation reference concentrations and applications of inhalation dosimetry, EPA/600/8-90/066F. Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1999a, February). Guidance for identifying pesticide chemicals and other substances that have a common mechanism of toxicity. Available at www.epa.gov. U.S. Environmental Protection Agency (1999b, August). A science policy on a common mechanism of toxicity: The carbamate pesticides and the grouping of carbamate with the organophosphorus pesticides, draft document. Available at www.epa.gov/ scipoly/sap/1999/september/carbam.pdf U.S. Environmental Protection Agency (2000). Benchmark dose technical guidance document, draft report. Risk Assessment Forum, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2001a, December 2). General principles for performing aggregate exposure and risk assessments. Office of Pesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. Available at www.epa.gov. U.S. Environmental Protection Agency (2001b, July 12). Memorandum from Marcia Mulkey to Lois Rossi. "Implementation of the determinations of a common mechanism of toxicity for N-methyl carbamate pesticides and for certain chloroacetanilide pesticides." Available at www.epa.gov. U.S. Environmental Protection Agency (2001c). Supplementary guidance for conducting health risk assessment of chemical mixtures, NCEA C 0148. Risk Assessment Forum, Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC. Available at www.epa.gov. U.S. Environmental Protection Agency (2002a). A review of the reference dose and reference concentration processes, EPA/630/P-02/002E Risk Assessment Forum, U.S. EPA, Washington, DC. U.S. Environmental Protection Agency (2002b, January 14). Guidance on cumulative risk assessment of pesticide chemicals that have a common mechanism of toxicity, 67 FR 2210. Available at www.epa.gov. U.S. Environmental Protection Agency (2002c, June 10). Revised organophosphorus pesticide cumulative risk assessment. Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC. Available at www.epa.gov./pesticides/ U.S. Environmental Protection Agency (2005, January 19). Cumulative hazard assessment: Issues for the FIFRA SAR Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC. Available at www.epa.gov.
CHAPTER 4
Regulatory Considerations in Developmental Neurotoxicity of Organophosphorus and Carbamate Pesticides qr
SUSAN L. MAKRIS U.S. Environmental Protection Agency, Washington, DC
and Cosmetic Act. These regulations were both amended in 1996 with the promulgation of the Food Quality Protection Act (FQPA). The FQPA mandated that "an additional tenfold margin of safety for the pesticide chemical residue and other sources of exposure shall be applied for infants and children to take into account potential pre- and postnatal toxicity and completeness of data with respect to exposure and toxicity to infants and children." It further stated that "the administrator may use a different margin of safety for the pesticide chemical residue only if, on the basis of reliable data, such margin will b e safe for infants and children." In Executive Order No. 13045, signed by President Clinton in 1997, U.S. federal agencies were required to identify and assess environmental health risks and safety risks that may disproportionately affect children and to ensure that agency policies, programs, activities, and standards address disproportionate risks to children. The evaluation of the potential for developmental neurotoxicity falls directly within these mandates, and extensive consideration of the most appropriate implementation of these principles was conducted by the EPA Office of Pesticide Programs (OPP) through public comment and peer review and through the publication of guidance on the application of the FQPA 10X factor (EPA, 2002a,b). Health effect test guidelines, which are standardized protocols that describe the conduct of defined toxicology studies, have been published by the EPA and by a number of other regulatory agencies throughout the world. They include guidelines that are specific for the evaluation of effects on development (Kimmel and Makris, 2001), among them the developmental neurotoxicity guideline OPPTS 870.6300 (EPA, 1998a,b). The determination of which toxicology studies are necessary for pesticide registration is primarily
I. I N T R O D U C T I O N The assessment of chemical pollutants, including pesticides, for developmental neurotoxicity potential has been, and continues to be, an issue of intense scientific and public concern. It has been estimated that 5-10% of the 4 million infants born annually in the United States are affected by neurobehavioral disorders, including learning disabilities, dyslexia, mental retardation, attention deficit disorder, and autism (Trasande and Landrigan, 2004). It is believed that exposure to environmental agents contributes to these adverse developmental outcomes [National Academy of Sciences (NAS), 2000]. Many pesticides and classes of pesticides, including the organophosphorus (OP) and carbamate (CM) pesticides, are known to affect the nervous system, and there are concerns regarding the potential for developmental neurotoxicity following early life exposures to these substances (NAS, 1993). This is particularly important since the unique behaviors and activities of children place them at greater risk for increased exposure to pesticides by multiple routes (Weiss et al., 2004).
II. R E G U L A T O R Y C O N T E X T Pesticide registration in the United States is conducted by the U.S. Environmental Protection Agency (EPA) under the authority of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Federal Food Drug
*The views expressed are those of the author and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.
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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defined in the Code of Federal Regulations, 40 CFR Part 158, which specifies data requirements for pesticides that will be used in or on products that may enter the food supply ("food use") and for those that are used for other ("non-food use") purposes. The need for developmental neurotoxicity testing for pesticides has been historically based on evidence of neurotoxic potential in the chemical database or by inference of chemical class (Levine and Butcher, 1990; EPA, 2002a).
III. THE D E V E L O P M E N T A L NEUROTOXICITY GUIDELINE The developmental neurotoxicity (DNT) guideline was developed in the late 1980s, firmly based on experience gained from many decades of extensive experimentation in the fields of neurobehavioral and developmental toxicology. The study design relied on the use of end points that represented a broad sampling across recognized physical and functional neurological domains that may be affected by exposures, including developmental exposures, to neurotoxicants. Additionally, interlaboratory studies had been conducted to assess the ability of several behavioral test methods to detect effects of neurotoxic insult (i.e., from methylmercury and amphetamine exposure) and to determine the reliability and sensitivity of results within and among laboratories. One example of such a study was the Collaborative Behavioral Teratology Study (Buelke-Sam et al., 1985). The results of this study showed that replicability of data among six participating laboratories using a standardized protocol was excellent, and that both positive effects (e.g., with methylmercury exposure) and the lack of effects (e.g., after low-level amphetamine exposure) were replicable. Even for behavioral measures that exhibited some degree of variability, no more than a 5-20% change from control values was required to detect an effect. Another interlaboratory evaluation of neurobehavioral screening methodologies that are used in both adult and developmental neurotoxicity studies was initiated by the International Programme on Chemical Safety (MacPhail et al., 1997; Moser et al., 1997). A total of eight laboratories participated in the study, evaluating seven neurotoxic positive control chemicals: triethyl tin, acrylamide, parathion, p,p'-DDT, toluene, N,N'-methylene bis-acrylamide, and lead acetate. The overall conclusion was that there was general "agreement across laboratories in terms of their ability to detect dose-related changes in behavioral end points with prototypic neurotoxic agents" (Tilson et al., 1997). Further examination of the validity of the protocol described in the DNT guideline was addressed by the Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity (Kimmel et al., 1990). In this extensive effort, the measures of developmental neurotoxicity in humans and laboratory animals were evaluated for several known human
developmental neurotoxicants drugs of abuse (cannabis, cocaine, methadone, and phencycline), methylmercury, phenytoin, lead, ethanol, polychlorinated biphenyls, and ionizing radiation (respectively, Hutchings, 1990; Burbacher et al., 1990; Adams et al., 1990; Davis et al., 1990; Driscoll et al., 1990; Tilson et al., 1990; Schull et al., 1990). The overall conclusion of this workshop (as reported by BuelkeSam and Mactutus, 1990; Stanton and Spear, 1990; Tyl and Sette, 1990; Levine and Butcher, 1990; and summarized by Francis et al., 1990) was that the DNT protocol would have identified each of the agents presented at the workshop as a potential developmental neurotoxicant, although the critical effects, and the dose at which the effects were observed, could vary across species. These studies supported the finalization of the EPA DNT guideline in 1991 (as Health Effects Test Guideline w EPA, 1991a); it underwent minor revisions during a guideline harmonization effort and was reissued in 1998 as OPPTS 870.6300 (EPA, 1998a). The guideline specifies that the test can be used as a stand-alone study, as a follow-up to a standard developmental toxicity (OPPTS 870.3700; EPA, 1998b) and/or adult neurotoxicity study (OPPTS 870.6200; EPA 1998c), or in combination with a two-generation reproduction study (870.3800; EPA, 1998d), with assessment of the offspring conducted on the second (F2) generation. The DNT study guideline presents a study design that is intended to develop data on the potential functional and morphological hazards to the nervous system that may arise in the offspring from exposure of the mother during pregnancy and lactation (EPA, 1998a). It includes measurements that are not assessed in any other available guideline studies, particularly in relationship to the developing organism, and has been shown to be a sensitive indicator of developmental insult (Makris et al., 1998; EPA, 2002c). In a standard guideline DNT study, pregnant rats are administered the chemical orally from gestation day 6 to postnatal day 10; extension of treatment to postnatal day 21 is preferentially recommended by OPP regulatory scientists. These testing days are defined in relation to the day of mating and the day of birth, designated as gestation and postnatal (lactation) days 0, respectively. The offspring are therefore potentially exposed to the chemical, via the maternal circulation and/or milk, during in utero and postnatal development for approximately 25-36 days. The dams are examined grossly at least once daily before treatment, and detailed clinical observations are conducted on approximately half of the dams in each group twice during gestation and twice during lactation. Maternal body weight is recorded at least weekly. In the guideline DNT study, the offspring are assessed for evidence of deficits in functional development. The typical study schedule is illustrated in Fig. 1. Litters are randomly standardized on postnatal day 4 to yield 4 pups per sex per litter, and the pups are assigned for testing. End points that are evaluated between birth and day 60 of age include measures of physical development, reflex ontogeny, motor
CHAPTER 43 9Regulatory Considerations in Developmental Neurotoxicity Gestation Day: 0 Postnatal Day:
[
I
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1No Treatment
635
11 L ~ \ \ ~ Treatment
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21
t:':':':':l Preferred Extension of Treatment
Maternal Observations (Gestation Day 0-Lactation Day 21) Clinical Observations & Mortality Body weight & Food Consumption Functional Observations
Offspring Observations (Postnatal Days 0-60)
Sexual Maturation
Growth & Survival Functional Observations Motor Activity Auditory Startle Learning & Memory Brain Wt & Neuropathology Assessment time point
[,r
activity, motor function, sensory function, and learning and memory. Daily cage-side observations are conducted, and a group of 10 pups per sex per group is examined outside the cage on days 4, 11, 21, 35, 45, and 60. Pups are counted and weighed individually at birth; on postnatal days 4, 11, 17, and 21; and at least once every 2 weeks thereafter. The ages of vaginal opening and preputial separation are recorded. Motor activity is monitored by an automated activity recording apparatus on postnatal days 13, 17, 21, and 60 (_+2). Tests of auditory startle habituation (preferably using prepulse inhibition) and associative learning and memory are performed on the offspring at approximately the time of weaning (day 21) and on approximately day 60. Flexibility is allowed in the choice of tests for learning and memory, although the guideline provides criteria for selection and some examples of tests that could be used. On postnatal day 11 (and/or postnatal day 21, if dosing is extended to weaning) and at study termination, the offspring are subjected to extensive neuropathological examination including simple morphometric analysis. One pup per sex per litter is killed on day 11 (and/or day 21). Of these, at least 6 pups per sex per group are assigned to neuropathological evaluation (10 pups/ sex/group are preferently recommended by OPP scientists); their brains are removed and immersed in an aldehyde fixative. At study termination, all remaining offspring are killed; 6 (or 10) rats per sex per group are prepared for neuropathological evaluation with in situ transcardial perfusion of appropriate fixatives (paraformaldehyde and gluteraldehyde). Brain weight is recorded at both a preweaning time point (postnatal day 11 and/or 21) and at study termination (postnatal day 60). Qualitative neuropathological examina-
Optional (Instead of PND 11)
FIG. 1. Developmental neurotoxicity study design (OPPTS 870.6300; EPA, 1998).
tion is conducted for the control and high-dose groups, and if a treatment-related finding is evident, the low- and middose groups are also examined. The guideline provides guidance on the regions of the brain to be examined and the types of alterations on which to focus, particularly emphasizing structural changes indicative of developmental insult. Simple morphometric analysis, performed on offspring killed on postnatal day 11 and/or 21 and at termination, is defined as consisting, at a minimum, of a reliable estimate of the thickness of major layers at representative locations within the neocortex, hippocampus, and cerebellum.
IV. D N T T E S T I N G R E Q U I R E M E N T S FOR OP AND C M P E S T I C I D E S Under the mandate of 1988 revisions to FIFRA, all registered pesticides are subjected to a cyclical reregistration process, which includes a reevaluation of the hazard database. The promulgation of FQPA in 1996, with its focus on adequate assessment of potential risk to infants and children, focused a significant amount of attention on the need for developmental neurotoxicity testing for the OP and CM pesticides. Although an extensive number of toxicology studies had been submitted historically to EPA for these pesticides, generally including acute and subchronic neurotoxicity studies in adult rats, few standard DNT studies were available. A review of the nine DNT studies that had been submitted to the OPP by December 1998 identified only one OP and three CM pesticides (Makris et al., 1998). Following Scientific Advisory Panel (SAP) recommendations
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SECTION VI 9Risk A s s e s s m e n t & Regulations
for DNT testing for all neurotoxic pesticides (FIFRA SAE 1999a,b), the OPP issued a data call-in (DCI) for the submission of a DNT study for all registered OP pesticides with established tolerances (Kimmel and Makris, 2001). The DCI, which was issued to applicable registrants for a total of 34 OPs (Table 1), included a preliminary list of chemical classes with neurotoxic potential, including cholinesterase-inhibiting CM, for which adult and developmental neurotoxicity testing would be required at some unspecified future time. However, since the OP class DNT DCI was issued, the EPA has issued only chemical-specific DCIs based on testing decisions made during the process of reviewing the toxicology and exposure databases for registration and reregistration actions. In addition to requiting the submission of adult and DNT studies, the OP DNT DCI specified the following modifications to the guideline DNT study: 9 Increase the duration of dosing to postnatal day 21. 9 Increase the number of animals examined for neuropathology (from 6/sex/dose to 10/sex/dose). 9 Demonstrate the adequacy of postnatal dosing for substances that are not present to any significant extent in the milk of the dams (including an assessment of the need for direct exposure of pups to the test substance). 9 Conduct a comparative evaluation of cholinesterase inhibition (or other biomarkers) and behavior in adults and young organisms. These criteria were intended to (i) encourage the development of customized DNT protocols that use other known information on the chemical and the chemical class to optimize the ability of the study to detect treatment-related effects; (ii) optimize exposure to ensure as broad as possible a test of potential developmental neurotoxic effects under relevant conditions; and (iii) provide data to analyze the comparative sensitivity of the young, their dams, and other adults, in accordance with the intent of the FQPA.
TABLE 1. Acephate Azinphos-methyl Bensulide Chlorethoxyfos Chlorpyrifos Chlorpyrifos-methyl Coumaphos Diazinon Dichlorvos (DDVP)
Whereas the first three of these requirements are applicable to any substance undergoing DNT testing, the last requirement is only applicable to cholinesterase-inhibiting pesticides. DCI notices issued by OPP for cholinesteraseinhibiting CM pesticides since the OP DNT DCI was issued in 1999 have typically specified all four of these modification criteria. Table 2 presents a list of CM pesticides for which a determination was made by OPP to require DNT and/or comparative cholinesterase testing. Without a structured chemical class DCI effort, as was implemented for the OP pesticides, it appears that data needs for the CM have not been systematically or consistently articulated. A large subset of the CM chemicals in this list consists of N-methyl CMs with active registrations, which will be included in a cumulative CM risk assessment based on their common mechanism of action [i.e., cholinesterase inhibition (EPA, 2005)]. OPP separately issued guidance for conducting cholinesterase measures (in DNT and comparative cholinesterase studies) to registrants that were required to conduct such assessments. The rationale for conducting these studies was that they could provide reliable data for determining an alternative to the 10-fold margin of safety factor applied under FQPA and be used in selection of end points and doses for specific risk assessments (i.e., acute or repeated exposures). Age- or life stage-related differences in the response to exposures with cholinesterase-inhibiting chemicals have been observed in humans and in laboratory animal models. These differences are not unidirectional; that is, for some chemicals under a specific treatment paradigm or for a particular life stage, immature individuals are more sensitive than adults, in some cases there is no differential sensitivity, and in some cases the young are less sensitive than adults (EPA, 2002a). They appear to be generally attributable to toxicokinetic (e.g., the interplay of metabolic activation and detoxification processes, particularly in early life; Ginsberg et al., 2002) and
OP Pesticides Included in the 1999 DNT Data Call-Ina Dicrotophos Dimethoate Disulfoton Ethion Ethoprop Ethyl parathion Fenamiphos Fenitrothion Fenthion
Malathion Methamidophos Methidathion Methyl parathion Naled Oxydemeton-methyl Phorate Phosmet Phostebupirim
Pirimiphos-methyl Profenophos Propetamphos Terbufos Tetrachlorvinphos Tribufos Trichlorfon
!
aFinal determination of whether a DNT study will be required is based on both toxicology and exposure considerations (i.e., potential for aggregate exposure to fetuses, infants, and children). In some cases, registrants elected to cancel a pesticide registration rather than conduct additional neurotoxicity testing. Chemicals for which a DNT study was submittedto EPA (as of 12/31/04) are indicated with a check mark.
CHAPTER 4 3 TABLE 2.
9Regulatory Considerations in Developmental Neurotoxicity
637
OPP Decisions Regarding the Need for DNT or Comparative Cholinesterase Testing for CM Pesticides a
Chemical name
DNT required
Aldicarb Bendiocarb Carbaryl Carbendazim Carbofuran Fenoxycarb Formetanate HC1 Methiocarb Methomyl
X c X
Oxamyl Pirimicarb Propoxur Thiodicarb Thiophanate-methyl
ChE required
Cumulative assessment group b
X X Xd
X X X X X X X
c
c c
aFinal determination of whether a DNT study will be required is based on both toxicology and exposure considerations (i.e., potential for aggregate exposure to fetuses, infants, and children). Chemicals for which a DNT study was submitted to EPA (as of 12/31/04) are indicated with a check mark. bproposal to FIFRA Scientific Advisory Panel (EPA, 2005). CDNTtesting was "reserved" pending receipt and review of adult neurotoxicity data. dComparative cholinesterase study required in lieu of a DNT study.
toxicodynamic (e.g., receptor downregulation; Pope et al., 1991) considerations. The D N T study design offers two reasonable opportunities for collection of offspring blood and tissue samples for cholinesterase measuresmthat is, when standardizing litter sizes on postnatal day 4 (when extra pups are killed and discarded) and when weaning litters on postnatal day 2 1 (when extra weanlings and their dams are killed). Samples collected at these time points can be utilized to help determine qualitatively whether some exposure to the offspring had occurred. (Such exposures of pups during the lactation period could occur by three pathways, dependent on the study protocol: maternal transfer via milk, consumption of treated diet by pups, and/or direct dosing of pups.) However, in order to fully characterize differences in the response of adult and immature rats to discrete quantified acute (i.e., single dose) or repeated doses of the test substance, separate studies need to be conducted. An example of a design for such testing is shown in Fig. 2. In the acute exposure study, evaluation of comparative sensitivity is assessed at two time points during lactation and at one adult time point. The first lactation time point, would be no later than postnatal day 11, and the second would be 7-10 days later (corresponding to the final day measured in the repeated dose study). The evaluation of data for these two time points is expected to provide specific information
regarding differences in sensitivity between pups and adults for two different ages of pups as well as some information about the direction and magnitude of the change over time. In the repeated dose exposure study, multiple daily exposures are administered to pups or young adult rats, where the time point assessed at the end of the repeated exposure corresponds to one of the time points assessed following acute exposures. The comparison of results from these two studies should enable specific determination of whether repeated Acute Exposure ~.
ChE M easur es
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FIG. 2. Timing of cholinesterase measures in comparative cholinesterase study.
638
SECTION Vl 9Risk A s s e s s m e n t
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exposure leads to cumulative cholinesterase inhibition in pups. Despite the extensive information collected in the standard DNT study and the comparative cholinesterase studies, data on maternal versus fetal exposures are not easily assayed without the evaluation of maternal versus fetal tissues; maternal and fetal test subjects can be included as a satellite group in the DNT study or, alternatively, included in another separate study. In this protocol, dams would be dosed daily from approximately gestation day 6 through gestation day 20, at which point they would be killed and samples extracted from maternal and fetal tissues. Important criteria established for the conduct of all these studies include the need to (i) assay both sexes; (ii) consider the litter of origin in assignment of pups to test groups or in conducting data analyses; (iii) base the adequacy of group sizes on the power of the data to detect statistically significant changes on the order of 5-10% in brain, 10% in erythrocytes, and 20% in plasma; (iv) establish a dose response compared to an untreated control group for all compartments (i.e., plasma, erythrocyte, and brain cholinesterase); (v) collect analytical samples at the time of peak cholinesterase inhibition, which would need to be established by gender for each age of rat tested; and (vi) utilize appropriate methodologies in the measurement of cholinesterase activity (EPA, 1992) for OP (Wilson et al., 1996) or CM (Hunter et al., 1997). Measurements of cholinesterase activity in peripheral system tissues (e.g., the atria of the heart, skeletal muscle, lung, diaphragm, and/or salivary glands) were highly recommended by OPP but not required; guidance for such assays can be found in Mileson et al. (1999).
V. THE USE OF DNT AND COMPARATIVE CHOLINESTERASE DATA IN RISK ASSESSMENT Evaluation and interpretation of the DNT and comparative cholinesterase study data are conducted in accordance with agency risk assessment guidelines for developmental toxicity, reproductive toxicity, and neurotoxicity (EPA, 1991b, 1996, 1998e). Risk assessments, including determination of reference values and application of traditional uncertainty factors in the calculations, are conducted based on historically established principles (NAS, 1983). The DNT study assesses a number of parameters in offspring, and to a limited extent in their dams, that could be utilized to provide a point of departure for noncancer risk assessment calculations (Makris et al., 1998). The study is not designed to identify the exact timing or duration of the exposure that results in an adverse developmental event. However, conservative assumptions are utilized in the absence of specific pharmacokinetic or experimental data that utilize timed dosing to examine specific critical windows of developmental susceptibility. These assumptions are that adverse outcomes in the offspring could have
resulted from developmental exposures and that developmental outcomes could have resulted from either acute or repeated doses of the test substance. Evidence abounds in the peer-reviewed literature to support the contention that developmental neurotoxicity could result from a single maternal or perinatal exposure. Neurobehavioral or neuropathological alterations of the offspring have been observed in studies of a number of chemicals in various chemical classes, including halothane or nitrous oxide (Rodier and Koeter, 1986), methyl mercury (Sager et al., 1984), 5-azacytidine (Rodier et al., 1979), valproic acid (Rodier et al., 1996), ethanol (Goodlett et al., 1989; Gavin et al., 1994), methylazoxymethanol (Rodier et al., 1991; Gavin et al., 1994), acetylsalicylic acid (Voorhees et al., 1982), and alkyltins (Reiter et al., 1981; Cook et al., 1984; Stine et al., 1988). Since the offspring in a DNT study are treated for a period of approximately 28-43 days (based on initiation of treatment as early as gestation day 0 and termination of treatment as late as postnatal day 21), there is also the possibility that any adverse outcomes are the result of multiple exposures; therefore, it is also considered appropriate to utilize offspring end points from DNT studies in risk assessments of greater than acute duration. Although the dosing duration in a DNT study does not approximate a lifetime (chronic) exposure to a chemical, it may still be appropriate in some instances to select maternal or offspring end points from this study for longer term or chronic reference values. This could occur, for example, if no other appropriate end point were identified in long-term studies, or if the point of departure derived from the DNT study were more conservative than that derived from any other study in the chemical database. In summary, EPA has determined that the offspring results from DNT studies can be used (as appropriate) in reference dose calculations for risk assessments of any duration (Table 3)mthat is, acute, short term, longer term (intermediate term), or chronic (EPA, 2002c). Maternal data from the DNT study generally have much less impact due to the limited number and scope of end points assessed. The comparative cholinesterase studies, as described, provide information on adverse outcomes (cholinesterase inhibition) following either acute or repeated exposures in populations at various life stages. In a review of cumulative risk assessment procedures for the OP pesticides (EPA, 2002b), acute and/or repeated dose comparative cholinesterase data in rats were available for a limited number of OP pesticides. These data raised uncertainties regarding the adequacy of adult risk potency factors to be protective of potential age-dependent sensitivity to cholinesterase inhibition and of potential adverse neurodevelopmental outcomes that are a result of the inhibition of cholinesterase. As a result, a three-fold database uncertainty factor was applied in the cumulative risk calculation for those chemicals that did not have comparative cholinesterase data available for evaluation. This
CHAPTER 43 TABLE 3.
9Regulatory Considerations in Developmental Neurotoxicity
639
Standardized Definitions for Various Duration Reference Values a
Reference value duration
Acute Short term Longer term Chronic
Definition
24 hr or less >24 hr up to 30 days >30 days up to approximately 10% of the life span in humans b Approximately 10% of the life span in humans
aFrom EPA (2002c). bMore than 30-90 days in typically used laboratory animal species.
approach was consistent with general OPP guidance on the application of the 10X FQPA factor (EPA, 2002a), as well as with the conclusions of a review of the reference dose (RfD) and reference concentration (RfC) setting process at EPA. This RfD/RfC report, conducted under the auspices of the Risk Assessment Forum, concluded that the absence of data or information that is reasonably expected to have the potential for lowering the reference value may indicate the need for the use of an additional uncertainty factor in the risk calculation (EPA, 2002c). For individual chemicals, such determinations are made following careful consideration of the entire toxicology database. EPA has committed resources to providing an updated peer-reviewed evaluation of the results of submitted DNT and comparative cholinesterase studies and their use and impact on individual chemical risk assessments. Although such reviews have been conducted in the past (Makris et al., 1998; Makris, 2004), the universe of submitted studies examined at the time of these earlier analyses did not include a broad sampling across chemical classes. In the 1998 analysis, DNT studies for only one OP (chlorpyrifos) and three CM (aldicarb, carbaryl, and carbofuran) pesticides were available. At the time of the 2004 review, only two additional OP DNT studies were available for inclusion (dimethoate and malathion). Although these analyses showed, in general, that the DNT study is a sensitive study compared to other studies in the toxicology database, and demonstrated the value of the DNT in risk assessment, the impact of the DNT study on any specific chemical class has not been determined. However, the database of DNT studies available for consideration and analysis increasingly includes a substantive number of OP and CM pesticides, in part as a result of the 1999 OP DNT DCI. Therefore, any future reviews should be able to expand on previous retrospective analyses of submitted DNT studies and examine the impact of the studies, including the comparative cholinesterase data, on risk assessment for the OP and CM pesticides. Additionally, these future reviews will be able to incorporate findings from cross-laboratory comparisons of DNT study methodologies and results previously conducted
by EPA scientists in an ongoing surveillance of agency study submissions. These cross-laboratory comparisons have focused on various neurobehavioral (Crofton et al., 2004; Raffaele et al., 2003, 2004; Sette et al., 2004) and neuropathological (Crofton et al., 2001; Raffaele et al., 2005) end points as well as general methodological considerations (Makris et al., 2005). Although they included information and data from all available OP and CM studies, they did not focus on specific chemical class issues. It is anticipated that information and conclusions derived from the evaluation and analysis of the OP and CM DNT and comparative cholinesterase studies will prove to be quite useful from a much broader chemical-class perspective.
References
Adams, J., Voorhees, C. V., and Middaugh, L. D. (1990). Developmental neurotoxicity of anticonvulsants: Humans and animal evidence on phenytoin. Neurotoxicol. Teratol. 12(3), 203-214. Buelke-Sam, J., and Mactutus, C. E (1990). Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity. Work Group II report: Testing methods in developmental neurotoxicity for use in human risk assessment. Neurotoxicol. Teratol. 12(3), 269-274. Buelke-Sam, J., Kimmel, C. A., Adams, J., Nelson, C. J., Vorhees, C. V., Wright, D. C., St. Omer, V., Korol, B. A., Butcher, R. E., Geyer, M. A., Holson, J. E, Kutscher, C. L., and Wayner, M. J. (1985). Collaborative behavioral teratology study: Results. Neurobehav. Toxicol. Teratol. 7, 591-624. Burbacher, T. M., Rodier, E M., and Weiss, B. (1990). Methylmercury developmental neurotoxicity: A comparison of effects in humans and animals. Neurotoxicol. Teratol. 12(3), 191-202. Cook, L. L., Jacobs, K. S., and Reiter, L. W. (1984). Tin distribution in adult and neonatal rat brain following exposure to triethyltin. Toxicol. Appl. Pharmacol. 72(1), 75-81. Crofton, K. M., Sutton, J. L., Makris, S. L., Raffaele, K., and Sette, W. E (2001). Developmental neurotoxicity testing guidelines: Variability in morphometric assessments of neuropathology, poster presentation No. 539, 40th annual meeting of the Society of Toxicology, San Francisco, CA. Toxicologist 60(1), 113.
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MacPhail, R. C., Tilson, H. A., Moser, V. C., Becking, G. C., Cuomo, V., Frantik, E., Kulig, B. M., and Winneke, G. (1997). The IPCS collaborative study on neurobehavioral screening methods: I. Background and genesis. NeuroToxicology 18(4), 925-928. Makris, S. L. (2004). A retrospective analysis of developmental neurotoxicity studies submitted to the U.S. EPA, presentation abstract No. 630, 43rd annual meeting of the Society of Toxicology, Baltimore, MD. Toxicologist 78(S- 1), 130. Makris, S., Raffaele, K., Sette, W., and Seed, J. (1998). A Retrospective Analysis of Twelve Developmental Neurotoxicity Studies Submitted to the U.S. EPA Office of Prevention, Pesticides, and Toxic Substances (OPPTS). U. S. Environmental Protection Agency, Washington, DC. Makris, S. L., Raffaele, K. C., and Mendez, E. (2005). A retrospective review of studies utilizing gavage dosing of pre-weaning rats demonstrates no adverse consequences of dosing procedures, poster presentation No. 380, 44th annual meeting of the Society of Toxicology, New Orleans, LA. Toxicologist 84(S- 1), 77. Mileson, B. E., Brimijoin, S., Chambers, J. E., Dass, E D., Padilla, S., Sheets, L. E, Tayler, E W., Van Pelt, C., and Wallace, K. B. (1999). Guidance for the design and interpretation of studies intended to characterize acetylcholinesterase activity in the peripheral nervous system. Unpublished report of the International Life Sciences Institute, Risk Sciences Institute, Washington, DC. Moser, V. C., Tilson, H. A., MacPhail, R. C., Becking, G. C., Cuomo, V., Frantik, E., Kulig, B. M., and Winneke, G. (1997). The IPCS collaborative study on neurobehavioral screening methods: II. Protocol design and testing procedures. NeuroToxicology 18(4), 929-938. National Academy of Sciences, National Research Council (1983). Risk Assessment in the Federal Government: Managing the Process. National Academy Press, Washington, DC. National Academy of Sciences, National Research Council (1993). Pesticides in the Diets of Infants and Children. National Academy Press, Washington, DC. National Academy of Sciences, Committee on Developmental Toxicology. (2000). Scientific Frontiers in Developmental Toxicology and Risk Assessment. National Academy Press, Washington, DC. Pope, C. N., Chakrabourti, T. K., Chapman, M. L., Farrar, J. D., and Arthun, D. (1991). Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorous pesticides. Environ. Toxicol. Pharmacol. 4, 309-314. Raffaele, K., Gilbert, M., Crofton, K., Makris, S., and Sette, W. (2004). Learning and memory tests in developmental neurotoxicity testing: A cross-laboratory comparison of control data, poster presentation No. 1342, 43rd annual meeting of the Society of Toxicology, Baltimore, MD. Toxicologist 78(S-1), 276. Raffaele, K. C., Sette, W. E, Makris, S. L., Moser, V. C., and Crofton, K. M. (2003). Motor activity in developmental neurotoxicity testing: A cross-laboratory comparison of control data, poster presentation No. 598, 42nd annual meeting of the Society of Toxicology, Salt Lake City, UT. Toxicologist 72(S 1), 123. Raffaele, K. C., Sette, W., Doherty, J. D., Makris, S., and Crofton, K. M. (2005). Neuropathological findings in developmental neurotoxicity testing: Comparison of qualitative and quantitative evaluations, poster presentation No. 977, 44th
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annual meeting of the Society of Toxicology, New Orleans, LA. Toxicologist 84(S- 1), 200. Reiter, L. W., Heavner, G. B., Dean, K. E, and Ruppert, P. H. (1981). Developmental and behavioral effects of early postnatal exposure to triethyltin in rats. Neurobehav. Toxicol. Teratol. 3(3), 285-293. Rodier, P. M., and Koeter, H. B. (1986). General activity from weaning to maturity in mice exposed to halothane or nitrous oxide. Neurobehav. Toxicol. Teratol. 8(2), 195-199. Rodier, P. M., Reynolds, S. S., and Roberts, W. N. (1979). Behavioral consequences of interference with CNS development in the early fetal period. Teratology 19(3), 327-336. Rodier, P. M., Kates, B., White, W. A., and White, A. L. (1991). The relationship of rat brain weight and pituitary weight to postnatal growth after prenatal exposure to methylazoxymethanol. Neurotoxicol. Teratol. 13(6), 583-590. Rodier, P. M., Ingram, J. L., Tinsdale, B., Nelson, S., and Romano, J. (1996). Embryological origin for autism: Developmental anomalies of the cranial nerve motor nuclei. J. Comp. Neurol. 370(2), 247-261. Sager, P. R., Aschner, M., and Rodier, P. M. (1984). Persistent, differential alterations in developing cerebellar cortex of male and female mice after methylmercury exposure. Brain Res. 314(1), 1-11. Schull, W. L., Norton, S., and Jensh, R. P. (1990). Ionizing radiation and the developing brain. Neurotoxicol. Teratol. 12(3), 249-260. Sette, W., Crofton, K., Makris, S., Doherty, J., and Raffaele, K. (2004). Auditory startle reflex habituation in developmental neurotoxicity testing: A cross-laboratory comparison of control data, poster presentation No. 1341, 43rd annual meeting of the Society of Toxicology, Baltimore, MD. Toxicologist 78(S 1), 275. Stanton, M. E., and Spear, L. P. (1990). Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: Comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol. Teratol. 12(3), 261-268. Stine, K. E., Reiter, L. W., and Lemasters, J. J. (1988). Alkyltin inhibition of ATPase activites in tissue homogenates and subcellular fractions from adult and neonatal rats. Toxicol. Appl. Pharmacol. 94(3), 3394-406. Tilson, H. A., Jacobson, J. L., and Rogan, W. J. (1990). Polychlorinated biphenyls and the developing nervous system: Cross-species comparisons. Neurotoxicol. Teratol. 12(3), 239-248. Tilson, H. A., MacPhail, R. C., Moser, V. C., Becking, G. C., Cuomo, V., Frantik, E., Kulig, B. M., and Winneke, G. (1997). The IPCS collaborative study on neurobehavioral screening methods: VII. Summary and conclusions. NeuroToxicology 18(4), 1065-1070. Trasande, L., and Landrigan, P. J. (2004). Editorial. The National Children's Study: A critical national investment. Environ. Health Perspect. 112(14), A789-A790. Tyl, R. W., and Sette, W. (1990). Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group III report: Weight of evidence and quantitative evaluation of developmental neurotoxicity data. Neurotoxicol. Teratol. 12(3), 275-280. U.S. Environmental Protection Agency (1991a). Developmental neurotoxicity study, series 83-6, Addendum 10 (neurotoxicity), subdivision F: Hazard evaluation: Human and domestic animals, EPA Publication No. 540/09-91-123; NTIS Publication No. PB
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91-154617. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1991b). Guidelines for developmental toxicity risk assessment. Fed. Reg. 56, 63798-63826. U.S. Environmental Protection Agency (1992). Proceedings of the U.S. EPA Workshop on Cholinesterase Methodologies. March 1, 1992, Arlington, VA. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1996). Reproductive toxicity risk assessment guidelines. Fed. Reg. 61, 56274-56322. U.S. Environmental Protection Agency (1998a). Health effects test guidelines, OPPTS 870.6300, Developmental Neurotoxicity Study, EPA 712-C-98-239. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998b). Health effects test guidelines, OPPTS 870.3700, Prenatal Developmental Toxicity Study, EPA 712-C-98-207. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998c). Health effects test guidelines, OPPTS 870.6200, Neurotoxicity Screening Battery, EPA 712-C-98-238. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998d). Health effects test guidelines, OPPTS 870.3800, Reproduction and Fertility Effects, EPA 712-C-98-208. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998e). Guidelines for neurotoxicity risk assessment. Fed. Reg. 63, 26926-26954. U.S. Environmental Protection Agency (2002a), (February 28). Determination of the appropriate FQPA safety factor(s) for use in the tolerance-setting process. Office of Pesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2002b), June 10. Determination of the appropriate FQPA safety factor(s) in the organophosphorus pesticide cumulative risk assessment Evaluation of sensitivity and susceptibility to the common mechanism of toxicity, acetylcholinesterase inhibition. Office of Pesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2002c). A review of the reference dose and reference concentration processes, risk assessment forum, EPA/630/P-02/002E U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2005). The N-methyl carbamate cumulative risk assessment: Pilot analysis, Background document for FIFRA Scientific Advisory Panel, February 15-18. U.S. Environmental Protection Agency, Washington, DC. Voorhees, C. V., Klein, K. L., and Scott, W. J. (1982). Aspirinindflced psychoteratogenesis in rats as a function of embryonic age. Teratogen. Carcinogen. Mutagen. 2(1), 77-84. Weiss, B., Amler, S., and Amler, R. W. (2004). The vulnerability, sensitivity, and resiliency of the developing embryo, infant, child, and adolescent to the effects of environmental chemicals, drugs, and physical agents as compared to the adult pesticides. Pediatrics 113(4, Pt 2), 1030-1036. Wilson, B. W., Padilla, S., Henderson, J. D., Brimijoin, S., Dass, E D., Elliot, G., Jaeger, B., Lanz, D., Pearson, R., and Spies, R. (1996). Factors in standardizing automated cholinesterase assays. J. Toxicol. Environ. Health 48, 187-195.
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CHAPTER 44
WHO/FAO Guidelines for Cholinesterase-lnhibiting Pesticide Residues in Food P. K. GUPTA Toxicology Consulting Services, Bareilly, India
Agency (EPA) in 1970. The reorganization did not significantly alter the process of pesticide registration, but it did condense the functions of the previous two agencies (FDA and FDA) into a single governing body. Likewise, OECD guidelines for testing of chemicals and risks associated with pesticides have been issued (OECD, 1987). In general, when making recommendations international agencies consider the efforts and progress made in the area of international harmonization. For example, the conduct of toxicity studies [good laboratory practices (GLPs)] and agricultural practices [good agricultural practices (GAPs)] are governed by regulations and monitored by specific quality assurance functions. Such regulations specify the need for clear and consistent procedures (i.e., standard operating procedures) and documentation of studies such that all aspects can be re-created and verified. The precision and discipline are the characteristics frequently associated with safety assessment procedure; the interpretation and extrapolation process in toxicology is far from an exacting science. This chapter highlights guidelines and recommendations issued by various expert panels of the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) with particular reference to guidelines issued for OP and CM pesticide residues in food.
I. I N T R O D U C T I O N The banning of DDT and other commonly used organochlorine (OC) pesticides led to increased use of other, more acutely toxic synthetic compounds that were also effective in pest control but were much less persistent in the environment. Subsequently, several cases of pesticide poisoning, including the Bhopal disaster in India, have been reported (Gupta, 2004). Research efforts led to the development of different types of organic compounds, such as organophosphates (OPs) and carbamates (CMs). OP pesticides are typically much less persistent in the environment than the early OCs. In fact, broad-spectrum OP and CM pesticides were the foundation of insect control chemicals in the 1980s. OPs are toxic due to their inhibition of acetylcholinesterase (ACHE) activity of nervous tissue. Like the OPs, the mode of action of CMs is inhibition of ACHE. However, CMs are generally less toxic than OPs because inhibition is more rapidly reversed (Sandhu and Brar, 2000). For judicious use, the regulation of pesticides is covered by several legislative acts and enforced by several international, federal, and state agencies. The risks associated with the use of pesticides are evaluated by assessing the toxicity and by estimating the magnitude of exposure from sources in the workplace, the environment, food, and water. The need to balance the risk to man and the environment against benefits associated with using pesticides is an important issue. Various agencies, such as the U.S. Food and Drug Administration (FDA) and Organization for Economic Co-operation and Development (OECD), provide guidelines and maximum residue limits (MRLs) of pesticides. The FDA guidelines tend to coincide with those o f the OECD. Public concern of environmental and health issues as well as an interest in greater efficiency in regulation led to the formation of the U.S. Environmental Protection Toxicology of Organophosphate and Carbamate Compounds
II. W H O C L A S S I F I C A T I O N O F PESTICIDES BY HAZARDS In 1973, the WHO executive board took the initiative to develop a tentative classification of pesticides that would distinguish between the more and less hazardous forms of each pesticide. Taking into account the views of members of the WHO Expert Advisory Panel on Insecticides and other expert advisory panels with special competence and interest in pesticide technology, as well as the comments of WHO 643
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&
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member states and international agencies, a proposal for a WHO-recommended classification of pesticides by hazard was prepared. The WHO Recommended Classification of Pesticides by Hazard was approved by the 28th World Health Assembly in 1975 and has since gained wide acceptance. Guidelines for this classification were first issued in 1978 and have since been revised and reissued at 2-year intervals [International Programme on Chemical Safety (IPCS), 2002]. In brief, part I of the document includes the classification as recommended by WHO. This part is not subject to periodic review, and the classification table and the text can only be changed by resolution of the WHO assembly. Part II includes guidelines to the classification of individual products in a series of tables, according to the oral or dermal toxicity of the technical product and its physical state. The tables are subject to review periodically. The hazard referred to in this recommendation is the acute risk to health (i.e., the risk of single or multiple exposures over a relatively short period of time) that may be encountered accidentally by any person handling the product in accordance with the directions for handling by manufacturers or in accordance with the rules for storage and transportation developed by competent international bodies. The classification is based primarily on the acute oral and dermal toxicity to the rat since these determinations are standard procedures in toxicology. The recommended classification is given in Table 1.
III. W H O / F A O ACTIVITIES
PESTICIDE
Additives (JECFA) and the Joint WHO/FAO Meeting on Pesticide Residues in Food (JMPR). The first meeting of JECFA was held in 1956. The WHO expert panel of JMPR provides information on toxicological evaluations. This has been accomplished through the establishment of acceptable daily intakes (ADIs). The FAO expert panel of JMPR is an authoritative voice on the levels of pesticides that can be ingested daily by humans without appreciable risk; this has been accomplished through the establishment of MRLs. The first meeting on pesticide residues was held in 1963. Since then, JMPR has been establishing ADIs and MRLs of pesticides in food commodities. In 1980, WHO established IPCS, which is a joint venture with the United Nations Environment Programme and the International Labor Organization. IPCS devotes significant resources to pesticides, primarily for their assessment but also for management support. The importance that IPCS places on these activities is based on the widespread use of pesticides, both in agriculture and in public health, and on a high level of public concern in both developed and developing countries because of the intrinsic toxicity of and potential exposure to the chemicals. The primary responsibilities of IPCS within WHO are risk assessment, provision of support for risk assessment, and holding the Joint Meeting on Pesticides. The detailed activities of WHO/IPCS are beyond the scope of this chapter. Since 1980, WHO activities concerned with the safety assessment of food chemicals have been incorporated into IPCS. Members invited by WHO review toxicological and related data and estimate, when possible, ADIs of the pesticides for humans. Members invited by FAO review pesticide use patterns (GAPs), data on chemistry composition of pesticides and methods of analysis for pesticide residues, and estimate MRLs that may occur as a result of the use of the pesticides according to GAPs. These MRLs serve as the basis for international standards established by the Codex Alimentarius Commission, which is administered
RESIDUE
In 1953, the World Health Assembly noted that the increasing use of various chemicals in the food industry had created a new public health problem. In response, WHO, in conjunction with FAO, initiated two series of annual meetings, the Joint FAO/WHO Expert Committee on Food
TABLE 1. WHO Classification of Pesticides by Hazard LD5o for the rat (mg/kg body weight) b
Class
Oral
Ia, extremely hazardous Ib, highly hazardous II, moderately hazardous III, slightly hazardous
Dermal
Solids a
Liquids a
Solids a
Liquids a
--<5 5-50 50-500 >500
-<20 20-200 200-2000 >2000
-<10 10-100 100-1000 > 1000 ~
-<40 40-400 400-4000 >4000
aAdapted with permission from IPCS (2002). bThe terms solids and liquids refer to the physical state of the active ingredients being classified.
CHAPTER 44 9OP and CM Pesticide Residues in Food by the Joint FAOAVHO Food Standards Program. JMPR advises the Codex committee of the Codex that is responsible for recommending Codex MRLs, which is hosted by the government of The Netherlands. The objectives of this expert group also include the formulation of guiding principles for exposure limits, such as ADIs for food additives and pesticide residues, and tolerances for toxic substances in food, air, water, soil, and the working environment. Since 1963, JMPR has evaluated approximately 100 OP and CM pesticides.
IV. APPROACH FOR T O X I C O L O G I C A L EVALUATIONS This chapter reflects the broad guidelines of the panel of experts of JMPR that is involved with the toxicological assessment of pesticides. The WHO expert panel of J M P R is responsible for reviewing toxicological and related data and for estimating (where possible) an ADI for humans (WHO, 2000). Routine standard procedures are used for the assessment of the toxicity of pesticide residues and are briefly summarized here.
A. Biological Data The biological data are subgrouped under three headings: biochemical aspects, toxicological studies, and observations in humans. 1. BIOCHEMICALASPECTS These studies are required to measure the concentration/time profiles of the ingested substance and its metabolites in the various organs and tissues of the body. Human biochemical studies that fall into these categories are also included in this section, whereas other human studies are included under observations in humans. The evaluation of metabolism studies in plants is the responsibility of the FAO expert panel of JMPR:
Absorption, distribution, and excretion: These studies generally focus on hydrolysis/digestion of the parent compound and its products in the mammalian gastrointestinal tract. These studies also include bioavailability of the unchanged compound and hydrolysis or digestion products, the pattern and rate of distribution of absorbed substances, and the mode and excretion/elimination of parent as well as identified metabolites. Biotransformation: These studies include metabolism of the parent compound, jf absorbed as such, and of its products if they are not normal dietary or body constituents. Effects on enzymes and other biochemical parameters: The effects of absorbed substances and/or their metabolites on cellular and tissue enzyme production and morphology, chemical constitution, enzyme activity, and physiochemical
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state are also evaluated while performing a toxicological evaluation of the compound. 2. TOXICOLOGICALSTUDIES These studies generally comprise routine acute studies, short-term feeding studies, long-term feeding studies, and studies on specific effects [e.g., carcinogenicity, genotoxicity, and reproduction toxicity (development toxicity and teratogenicity)]. Sometimes, these routine studies indicate the need to examine particular target organs or tissues; such studies are classified as special studies. Special studies are designed to test specific effects, such as neurotoxicity, immunotoxicity, allergenicity, cardiovascular effects, cataractogenicity, delayed neuropathy/neurotoxicity, acute neurotoxicity, development neurotoxicity, immune responses, macromolecular binding, photosensitization, and thyroid functions. Several parameters are of special consideration for OP and CM pesticides.
a. Delayed Neurotoxicity/Neuropathy Since 1974, delayed neurotoxicity has been discussed in a series of JMPR meetings. In 1987, JMPR recommended that in view of the presence of optical isomers of OP esters, two types of studies should be conducted on chemicals suspected of being neurotoxic (WHO, 1990). The first is the use of a suitable sensitive species (usually the adult hen), in which test substance is administered at two acute exposures (separated by 21 days) to atropineprotected animals at a level at or above the LDs0 of the compound. Observations on body weight, ataxia, and signs of delayed neurotoxicity are made while the animals are alive. At termination, usually 42 days after the first dose, histopathological examination of the brain, spinal cord, and proximal and distal sections of (usually) the sciatic nerve is performed. Data from this type of test suffer from two major drawbacks: The evaluation is often subjective, and a negative result cannot be graded. The second type of test is the determination of neurotoxic esterase activity (NTE). In its simplest form, this involves treatment of the adult hen with a single maximum tolerated dose of the test substance and subsequent assay of the brain enzyme after the time of peak inhibition but before substantial resynthesis of new enzyme has occurred (Johnson, 1982). The time of peak inhibition, which can be from 3 to 48 hr postdosing (and is determined by the pharmacokinetics of the compound), can often be assessed by observation of the time of onset of cholinergic signs. The threshold level of NTE inhibition at this early stage, which correlates with delayed neurotoxicity, is approximately 80%. No clinical signs are associated with an inhibition of 60% or less. When multiple determinations of NTE are made during chronic exposures, plateau levels are observed after 2 or 3 weeks. If inhibition of NTE in the brain and spinal cord is less than 50%, delayed neuropathy does not occur.
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b. Acute Neurotoxicity (ACHE Inhibition) It is established that the correlation between AChE inhibition (AChEI) in erythrocytes and that in the nervous system is usually unknown, and data on brain AChEI are considered to be of greater value than those on erythrocytes in assessing the cholinergic effects of cholinesterases (ChEs). However, in the absence of measurements of brain AChE, those of erythrocyte AChE serve as a better indicator of toxicity than those of plasma ChE activity. In vitro kinetic studies may be necessary for pesticides with antiesterase activity. Results of these studies in different species may be combined with in vivo study findings to establish ADIs for these compounds. Guidelines issued by JMPR have drawn attention to the currently used methods for the determination of ChE activity that may lead to erroneous conclusions when applied to rapidly reversible ChE inhibitions (e.g., N-methyl and N,Ndimethyl CMs). It has been suggested that in vitro kinetic studies should be made to elucidate the nature of reversible inhibition reactions and the results obtained from in vivo studies should be interpreted cautiously. The method for the determination of ChE inhibition of CMs is inadequate. Occasionally, data are inconsistent with respect to dose and the degree of ChE inhibition because CMs are reversible inhibitors of ChE with a short duration of action. Because of the reversible inhibition of the enzyme by dilution, as would occur during the preparation of the assay, inhibition cannot be accurately measured. JMPR has stressed that in order to permit evaluation of ChE inhibition by CMs in vivo, special care is required in reporting all details of such studies. CM-induced ChE inhibition studies should utilize minimal dilution during the preparation of the assay, minimal incubation times, and minimal times between blood sampling and assay (Ellman et al., 1967). It is recommended that 1. The information on delayed neurotoxicity appears to follow a dose-response relationship. Thus, with an adequate margin of safety, an ADI can be allocated. 2. Delayed neurotoxicity testing should be conducted routinely for OPs. However, it need not be done for monomethyl CMs, phosphinates, or sulfonates. 3. Tri-O-cresyl phosphate is recommended as a positive control substance only for OPs. 4. The NTE assay should be included in the database for OP evaluations. 5. While making assessment of effects on peripheral nervous tissues, comparison of the dose-response curves for erythrocyte AChE and brain AChEI and the occurrence of clinical signs may aid in the establishment of no-observable-adverse-effect levels (NOAELs). 6. For the purpose of establishing ADIs, the inhibition of plasma and brain butyrylcholinesterase (BuChE) is not a toxicologically significant effect because there is no evidence that BuChE inhibition has any adverse effect. It can be used as an indicator of absorption of the inhibitor
and, as such, is still a useful tool for monitoring occupational exposure. Data on statistically significant inhibition of BuChE activity should therefore always be induced. 7. For brain and erythrocyte ACHE, statistically significant inhibition by 20% or more represents a clear toxicological effect. Less than 20% or statistically insignificant inhibition indicates that a more detailed analysis of the data should be undertaken. The toxicological significance of these findings should be determined on a case-by-case basis. Considerations affecting such determinations include inter alia the shape of the dose-response curve, assay variability, and correlation with clinical signs. For neurotoxic compounds, an acute neurotoxicity study is required, unless short-term studies include a detailed neurotoxicological assessment.
c. Developmental Neurotoxicity In recent years, developmental neurotoxicity tests for assessing the safety of OP and CM pesticides to developing fetuses, infants, and children have been performed. The objective of these tests is to examine the use of such studies in the establishment of ADIs and acute reference dose (RfD). JMPR has recommended that if the toxicological profile of a chemical indicates developmental neurotoxicity end points, appropriate testing parameters should be incorporated into a multigeneration study of reproductive toxicity. While assessing such effects, the chances of introduction of artifacts due to stress resulting from directly dosing the pups and bolus (gavage) administration should be kept in mind (JMPR, 2002a).
3. OBSERVATIONSIN HUMANS Observations in humans are useful for assessing the relevance of the results of animal studies and for confirming ADIs. Studies dealing with epidemiological surveys, clinical experience, anecdotal observations, health effect studies relating to occupational exposure, reports of abuse, and volunteer studies measuring intolerance provide important observations.
V. A S S E S S M E N T OF E N D POINTS The overall objective of toxicological evaluation is to determine a NOAEL, based on consideration of the total toxicology database, that is utilized in conjunction with an appropriate safety factor to determine the ADI. The ADI has been defined as follows (JMPR, 1975): "ADI" of a chemical is the daily intake which, during an entire lifetime, appears to be without appreciable risk to the health of the consumer on the basis of all the known facts at the time of the evaluation of the chemical by the JMPR. It is expressed in milligrams of the chemical per kilogram of body weight. The initial stage of the evaluation is a critical examination of individual studies. In some cases, a study initially
CHAPTER 44 considered to be of marginal value in fact is useful when considered in the context of the entire database. Integration of the results from all studies permits an appraisal of the toxicity of the compound. Data from acute oral studies are rarely considered to be relevant to the establishment of the ADI. However, such data may provide information that permits a ranking of the sensitivity of different species and assist in the selection of dose levels in subsequent studies. Studies also indicate types of pharmacological activities, degree of absorption, or potential target organs. JMPR has occasionally required additional acute data to determine the relative toxicity of salts of a pesticide or required further metabolic studies to determine species differences in acute toxicity. Short-term feeding studies were considered to be the basis for the determination of ADIs for a number of compounds evaluated by JMPR before long-term toxicity studies were indicated to be an essential part of the toxicological database for evaluating the safety of pesticides. Currently long-term feeding studies have become an essential part of the toxicological database, and the major use of the data from short-term toxicity studies has been to determine suitable dose levels to be utilized in long-term and reproduction studies. However, multigeneration reproduction and teratogenicity studies have also been used for establishing ADIs for certain compounds (WHO, 1990). For carcinogenicity studies, a statistically significant difference between experimental and control groups is considered in light of its biological relevance. Thus, an increased incidence of a rare tumor type in treated animals may be of concern even if the incidence is not significantly different statistically from that in the concurrent control animals. Conversely, a statistically significant change in an isolated parameter (e.g., erythrocyte count) is usually not considered to be biologically relevant unless supported by changes in other parameters (e.g., bone marrow or spleen histopathology or methemoglobin formation). In interpreting carcinogenicity data, JMPR bases its evaluations on the threshold concept, which is the basis for evaluating most other toxicological effects (WHO, 1974). In assessing tumor incidence, benign and malignant tumors have been considered as separate entities in the majority of cases (WHO, 1987). Sometimes, the use of more than one species for the same type of toxicity study may complicate interpretation in those cases in which an effect occurs in one species but not in a second species or in which one species is much more sensitive to the agent than the other species. In such cases, it is often difficult to determine the most appropriate species for extrapolation to man. Generally, unless adequate data are available to indicate the most appropriate species (usually comparative pharmacokinetic or pharmacodynamic data), the most sensitive species (i.e., the species in which the adverse effect occurred at the lower dose) is used in determining the NOAEL and allocating the ADI.
9OP and CM Pesticide Residues in Food VI. E X T R A P O L A T I O N
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O F DATA
AND DETERMINATION OF ADIs The objective of the safety evaluation of pesticide residues in food is to determine the ADIs of pesticides that will not result in adverse effects at any stage in the human life span. Since in the majority of cases data on humans are inadequate to permit such a determination, effects observed in other species must be extrapolated to man. This approach is used to determine the NOAEL from the experimental animal data or, preferably, from data in humans, if available. Generally, the 100-fold safety factor is used as the starting point for extrapolating animal data to humans and may be modified depending on the data that are available and the various concerns that arise when considering these data. The safety factors generally involved in establishing an ADI also serve to provide assurance that exposure exceeding the ADI for short time periods is unlikely to result in any deleterious effects on health. However, consideration should be given to the potentially acute toxic effects that are not normally considered in the assessment of an ADI. When relevant human data are available, the 10-fold factor for interspecies variability may not be necessary. However, relatively few parameters are studied in humans in the assessment of pesticide safety, and data on carcinogenicity, reproduction, and chronic effects are rarely available. Consequently, JMPR rarely utilizes safety factors as low as 10-fold. ADIs of selected OP and CM pesticides are summarized in Table 2. It is evident that certain factors may serve to increase and others to decrease the choice of the final safety factor. The total weight of evidence has to be considered when determining the appropriate safety factor to be used and the determination of safety factors must be considered on a case-by-case basis. If the metabolites in food commodities are qualitatively and quantitatively the same as those observed in laboratory test species, the ADI would apply to the parent compound as well as the metabolite(s). If the metabolite(s) is not identical or not present at the same order of magnitude, separate studies on the metabolite(s) may be necessary. When one or several pesticides are degradation products of another pesticide, a single ADI may be appropriate for the pesticide and its metabolites (e.g., oxydemeton-methyl, demeton-S-methyl sulfone, and demeton-S-methyl) (IPCS, 2003). In 1988, JMPR recommended that temporary acceptable daily intakes should not be allocated for new compounds and that an ADI should not be allocated in the absence of an adequate database (FAO/WHO, 1988). The monographs are published for all chemicals that are reviewed, regardless of whether an ADI is allocated. The data requirements are clearly specified for those chemicals with an inadequate database. The concept of the "conditional acceptable daily intake" adopted by the 1969 JMPR, was limited to those compounds for which the use was at that time considered essential but for which the toxicological database was
648
SECTION Vl
9 RiskAssessment &
TABLE 2.
Regulations
WHO/FAO Recommended Acceptable Daily Intakes (ADIs) of OP and CM Pesticides a
Compound
Year
ADI (mg/kg body weight)
Acephate
2002
0-0.01
N-acetyl glufosinate/NAGb
1999
0-0.02
Aldicarb
1995
0-0.003
Aminocarb
1979
No ADI
AMPA (metabolite of glyphosate)
1997
0-0.3
Azinphos-ethyl
1973
No ADI
Azinphos-methyl
1991
0-0.005
Bendiocarb
1984
0-0.004
Benomyl
1995
0-0.1
Bromophos
1977
0-0.04
Bromophos-ethyl
1975
0-0.003
Butacarboxim
1985
No ADI
Cadusaphos
1991
0-0.0003
Carbaryl
2001
0-0.008
Carbendazim
1995
0-0.03
Carbofuran
2002
0-0.002
Carbophenothion
1980
0-0.0005
Carbosulfan
1986
0-0.01
Chlorfenvinphos
1994
0-0.0005
Chlorpropham
2000
0-0.03
Chlorpyrifos
1999
0-0.01
Chlorpyrifos-methyl
2001
0-0.01
Chlorthion
1965
No ADI
Coumaphos
1990
No ADI
Crufomate
1968
0-0.1
Demeton
1984
No ADI
Demeton-S-methyl and related compounds c
1989
0-0.0003
Dialifos
1982
ADI withdrawn
Diazinon
2001
0-0.002
Dichlorvos
1993
0-0.004
Dimethoatea
1996
0-0.002
Dioxathion
1968
0-0.0015
Disulfoton
1996
0-0.0003
Edifenphos
1981
0-0.003
Ethephon
2002
0-0.05
Ethiofencarb
1982
0-0.1
Ethion
1990
0-0.002
Ethoprophos
1999
0-0.0004
Etrimfos
1986
0-0.003
Fenamiphos
2002
0-0.0008
Fenchlorphos
1968
0-0.01
Fenitrothion
2000
0-0.005
Fensulfothion
1982
0-0.0003
Fenthion
1997
0-0.007
Formothion
1996
ADI withdrawn
Glufosinate ammoniume
1999
0-0.02
(continues)
CHAPTER 44
TABLE 2.
9OP and CM Pesticide Residues in Food
(continued) ADI (mg/kg body weight)
Compound
Year
Glyphosate b
2004
0-1
Isofenphos
1986
0-0.001
Leptophos
1978
Temporary ADI withdrawn
Malathion
1997
0-0.3
Mecarbam
1986
0-0.002
Methacriphos
1990
0-0.006
Methamidophos
2002
0-0.004
Methidathion
1997
0-0.001
Methiocarb
1998
0-0.02
Methomyl
2001
0-0.02
Mevinphos
1997
0-0.0008
Monocrotophos
1995
0-0.0006
Omethoate
1996
ADI withdrawn
Oxamyl
2002
0-0.009
Oxydemeton-methyl/demeton-S-methyl
2002
0-0.0003
Parathion
1995
0-0.004
Parathion-methyl
1995
0-0.003
Phenthoate
1984
0-0.003
Phorate
2004
0-0.0007
Phosalone
2001
0-0.02
Phosmet
1998
0-0.01
Phosphamidon
1986
0-0.0005
Phoxim
1999
0-0.004
Primicarb
1983
0-0.02
Pirimiphos-methyl
1992
0-0.03
Profenofos
1990
0-0.01
Propamocarb
1986
0-0.1
Propham
1992
No ADI
Propoxur
1989
0-0.02
Pyrazophos
1992
0-0.004
Terbufos
1989
0-0.0002
Thiodicarb
2000
0-0.03
Thiometon
1979
0-0.003
Thiophanate-methyl
1998
0-0.08
Tolcfos-methyl
1994
O-0.07
Triazophos
2002
0-0.001
Trichlorfon (metrifonate)
2000
0-0.02
Trichlornate
1971
No ADI
Vamidothion
1988
0-0.008
sulfoxide c
aData from IPCS (2003) and JMPR (2004). bMetabolite of glufosinate ammonium. CGroup ADI, alone or in combination, evaluated under oxydemeton-methyl/demetonS-methyl sulfoxide/sulfone. dSum of dimethoate and omethoate as dimethoate. eADI applies to glufosinate, N-acetyl glufosinate, and 3-[hydroxy (methyl)phosphinoyl] propionic acid, alone or in combination.
649
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SECTION Vl 9Risk A s s e s s m e n t & Regulations
incomplete (FAO/WHO, 1970). This concept, which is unacceptable, has been abandoned. Long-term dietary intakes are expressed as percentage of the ADI for a 60-kg person, with the exception of the intake calculated for the Far East, for which a body weight of 55 kg is used (Codex, 1997b). The detailed procedure for calculations of long-term dietary intake can be found in Annexure 3 of JMPR (2002a).
VII. PREDICTING DIETARY INTAKE O F PESTICIDE RESIDUES The FAO expert panel of JMPR (WHO, 1997a) holds the responsibility for reviewing pesticide use pattems, data on the chemistry and composition of pesticides, and methods of analysis of pesticide residues and for recommending MRLs that may occur in food commodities following the use of pesticides according to GAPs. The MRL has been defined as follows: "MRL" is the maximum concentration of a pesticide residue (expressed as mg/kg), recommended by the Codex Alimentarius Commission (Codex) to be legally permitted in or on food commodities and animal feeds. MRLs are based on GAP data and foods derived from commodities that comply with the respective MRLs are intended to be toxicologically acceptable. Extraneous maximum residue limit (EMRL) refers to a pesticide residue or a contaminant arising from environmental sources (including former agricultural uses) other than the use of a pesticide or contaminant substance directly or indirectly on the commodity. It is the maximum concentration of a pesticide residue or contaminant that is recommended by the Codex (1997a) to be legally permitted or recognized as acceptable in or on a food, agricultural commodity, or animal feed. The concentration is expressed in milligrams of pesticide residue or contaminant per kilogram of the commodity. "GAP" includes the nationally authorized safe uses of pesticides under actual conditions necessary for effective and reliable pest control. It encompasses a range of levels of pesticide applications up to the highest authorized use, applied in a manner which leaves a residue, and is the smallest amount practicable. Authorized safe uses are determined at the national level and include nationally registered or recommended uses, which take into account public and occupational health and environmental safety considerations. Actual conditions include any stage in the production, storage, transport, distribution, and processing of food commodities and animal feed. MRLs which are primarily intended to apply in international trade are derived from estimations made by the JMPR following: (a) toxicological assessment of the pesticide and its residue; and (b) review of residue data from supervised trials and supervised uses including those reflecting national food agricultural practices. Data from supervised trials conducted at the highest nationally recommended, authorized, or registered uses are included. In order to
accommodate variations in national pest control requirements, Codex MRLs take into account the higher levels shown to arise in such supervised trials, which are considered to represent effective pest control practices. Estimates of dietary intake play an important role in ensuring safe food for consumers throughout the world. Consideration is given to the identity, purity, stability, quality of data, structure-activity relationship, and GAPs used during the application of pesticides. Generally, most treated crops contain residues well below the MRL at the harvest: Residues are usually reduced during storage, preparation, commercial processing, and cooking, and it is unlikely that every food for which MRL is proposed will have been treated with the pesticide over the lifetime of the consumer. Therefore, MRL represents the maximum residue level that is expected to occur in a commodity following the application of pesticide according to GAP. As per international standards, five regional diets (Middle Eastern, Far Eastern, African, Latin American, and European) are considered, and the average daily food consumption values are used in predicting pesticide residue intake for long-term hazard. Consideration of the various dietary residue estimates and determinations at both the national and international level in comparison with the ADI should indicate that foods complying with Codex MRLs are safe for human consumption.
A. MRLs for Milk and Milk Products For a "milk product" with a fat content less than 2%, the MRL applied should be half that specified for milk. The MRL for "milk products" with a fat content of 2% or more should be 25 times the MRL specified for milk, expressed on a fat basis. The residues in whole milk are usually expressed on a fat basis (4% fat).
B. MRLs for Meat and Meat Products Where adequate data are not available, MRLs are specified for the fat portion of animal products. Consumption data for mammalian and poultry meat are corrected on the assumption that mammalian meat contains 20% fat and poultry meat (with adhering skin) contains 10% fat. Dietary intake calculations are based on MRLs (fat) for fat-soluble pesticides and for non-fat-soluble pesticides. For mammalian animals, 20% of the meat consumption is used for fat-soluble pesticides and 80% of meat consumption is used for non-fat-soluble pesticides. For poultry, the corresponding values are 10 and 90% (JMPR, 2002a).
C. MRLs for Fruits and Vegetables For residues in whole commodities, including the edible portion, there is no universal distribution ratio because the amount of residue in the edible portion varies from pesticide
CHAPTER 44 to pesticide. To calculate the international estimated daily intake for assessing exposure to pesticide residues, the supervised trials median residue level (SMTR) is used. In practice, the STMR is estimated directly from residue data on the edible portion. Residue data on the edible portion are obtained to refine estimates of dietary intake for commodities such as citrus fruits, bananas, pineapples, kiwifruits, and other fruits with inedible peel; cereals and oilseeds; and cucurbits with inedible peel, such as melons. The consumption of the edible portion of food commodities should be used in estimating intakes rather than that of entire commodities. Correction factors currently used for the weight of the edible portion include 30% reduction for both citrus and bananas. In some countries, edible portions of some food commodities may vary, and their national governments may consider local dietary habits when using such data (WHO, 1997a). Risks associated with long-term dietary intake are calculated by multiplying the concentrations of residues (STMRs or STMR-P values or recommended MRLs) by the average daily per capita concentration estimated for each commodity on the basis of the GEMS/Food diet (WHO, 1997b, 1998). For risk assessment of long-term hazards, STMRs should be used together with mean reduction or mean concentration factors. However, data on the effects of processing on residues are needed.
D. MRLs and EMRLs in and on Spices There is a proposal to develop guidance for the submission of monitoring data for setting MRLs and EMRLs for spices. Since most of the spices moving in international trade are produced by millions of small farmers of less than 10 ha, and usually by intercropping, the presence of residues is frequently associated with products used for pest control on the main crop rather than on the spices. For estimation of MRLs in or on spices, it will be more meaningful if the commodities are subgrouped on the basis of parts of plants from which they are obtained (JMPR, 2004).
E. MRLs for Metabolite(s) of Parent Compounds Information is often available on the conversion of a parent compound to a metabolite or degradation product of toxicological concern that is produced or persists in processed food (e.g., oxygen analogs of phosphorothiolates). Estimates of dietary intake should be made on the basis of information on the consumption of processed commodities, provided an ADI or other assessment of toxicological properties of the metabolite products has been made.
9OP and CM Pesticide Residues in Food
651
VIII. EVALUATIONS O F M I X T U R E S The possibility of pesticide interaction was recognized as early as 1961 when the FAO/WHO Meeting on Principles Governing Consumer Safety in Relation to Pesticide Residues recognized that "different pesticides and other chemicals are often absorbed simultaneously during occupational use, or in food, by man or animals" (FAO/WHO, 1962). A survey of data indicates that residues of more than one pesticide may be detected in food. This gives rise to concern over the possibility of unanticipated interactions between such residues leading to adverse toxicological effects. There are, of course, a virtually unlimited number of combinations of pesticides on various crops. There are also a very large number of combinations of foods containing pesticide residues. Therefore, man could be exposed to unlimited chemicals leading to unlimited possibilities, and there is no special reason why the interactions of pesticide residues (which are at very low levels) should be highlighted as being of particular concern. Where isomeric mixtures exist, the ratio of isomers in the test material must be clearly specified since it has been amply documented that different isomers frequently have different toxicological activities. Second, very little data on these interactions are available, and the data obtained from acute potentiation studies are of little value in assessing ADIs for man. However, there is a need for further data on interactions of pesticides with each other and with other common contaminants (e.g., metals and mycotoxins) of food (FAO/WHO, 1988).
IX. ACUTE DIETARY RISK ASSESSMENT The establishment of an acute RfD forms the basis of shortterm intake and a short-term risk assessment. The acute RfD is defined as an estimate of the amount of a substance in food and/or drinking water, normally expressed on a body weight basis, that can be ingested in a period of 24 hr or less without appreciable health risk to the consumer on the basis of all known facts at the time of the evaluation. In other words, it is a short-term dietary intake assessment of pesticide residue intake over a single day (JMPR, 2002a). Since 1995, JMPR has established an acute RfD for a number of pesticides (Table 3). The meeting has followed the basic principle and an acute RfD has been considered on a case-by-case basis for all compounds that are evaluated. For this estimate, two population groups have been modeled: the general population and children (1-6 years of age). There may be certain use patterns that are unlikely to give rise to residues, and in such cases a full intake assessment may not be necessary. Most of the scientific concepts used for the establishment of ADIs apply equally to acute RfDs. The decision to establish an acute RfD is normally
652
S E CTI O N VI 9 Risk A s s e s s m e n t & Regulations
TABLE 3. WHO/FAO Recommended Acute Reference Dose (RE)) of OP and CM Pesticides a
Compound
Year
Acute RtI) (mg/kg body weight)
Acephate Aldicarb Carbaryl Carbofuran Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Demeton-S-methyl and related compounds Diazinon Disulfoton Ethephon Ethoprophos Fenitrothion Fenthion Glufosinate ammonium b Glyphosate Methamidophos Methidathion Methiocarb Methomyl Mevinphos Monocrotophos Oxamyl Oxydemeton-methyl/ demeton-S-methyl sulfoxide c Parathion Parathion-methyl Phorate Phosalone Phosmet Primicarb Thiodicarb Thiophanate-methyl Triazophos
2002 1995 2001 2002 2000 1999 2001
0.05 0.003 0.2 0.009 0.03 0.1 Unnecessary
2002 2001 1996 2002 1999 2000 1997 1999 2004 2002 1997 1998 2001 1997 1995 2002
0.002 0.03 0.003 0.05 0.05 0.04 0.01 Unnecessary Unnecessary 0.01 0.01 0.02 0.02 0.003 0.002 0.009
2002 1995 1995 2004 2001 1998 2004 2000 1998 2002
0.002 0.01 0.03 0.003 0.3 0.02 0.1 0.04 Unnecessary 0.001
aData from IPCS (2003) and JMPR (2004). bAcute RfD applies to glufosinate, N-acetyl glufosinate, and 3-[hydroxy (methyl)phosphinoyl]propionic acid, alone or in combination. CGroup acute RfD, alone or in combination, evaluated under demetonS-methyl and related compounds. based on toxicological grounds because an acute RfD is a toxicological reference value. Therefore, the establishment of an acute RfD is considered for all substances. In considering whether an acute RfD for a pesticide is necessary, it is not advisable to take into account current agricultural prac-
tice and related residues for existing crop use because with different application rates or new applications on other crops (or other crop groups), higher residue values and/or higher dietary intakes may occur.
A. Toxicological Effects Relevant for Derivation of the Acute RfD A number of effects could be due to a single exposure to a compound. The following list is not necessarily comprehensive and the omission of a toxicological effect does not mean that it should be discounted when considering the establishment of an acute RfD: The effects could be clinical signs, behavioral signs, pharmacological effects, or effects on target organs observed in single-dose studies (e.g., miosis) or early in studies with repeated doses such as acute neurotoxicity (e.g., acute delayed polyneuropathy or inhibition of AChE activity); clinical chemical or hematological effects (e.g., methemoglobin formation, hemolysis, or anemia); immunotoxicity, kidney toxicity, liver toxicity, and reproductive or development effects (e.g., teratogenicity or development neurotoxicity); or endocrine effects or other biochemical alterations observed in studies with repeated doses that may conceivably be elicited by a single exposure. Selection of the most relevant end point providing the lowest NOAEL is the most appropriate method (JMPR, 2004). Direct effects on the gastrointestinal tract should be assessed carefully to determine their relevance to human exposure. Are they due to irritation or a pharmacological action? Are they relating to the method of administration (present with bolus dosing but not by dietary admixture)? The relevance of these effects should be considered on a case-by-case basis. The route of administration should be considered carefully to minimize influences that are not relevant to the intake of residues (e.g., effects induced by gavage or the vehicle).
B. Determination of Acute RfD As discussed previously, toxicological effects will help to determine NOAELs. To determine acute RfD, safety factors higher or lower than the default values of 100 and 10 depending on animal or human data are used by JMPR (2000). It is considered inappropriate to have a higher ADI value than the acute RfD. If the previously derived ADI is higher than the acute RfD, then the complete database has to be reevaluated and the reference values need to be reconciled. Such a situation can occur for a number of reasons, such as the availability of additional studies or when compounds produce more severe effects when given by gavage than in the diet. Because short-term consumption data are for a 24-hr period, this is a precautionary approach for rapidly reversible effects (e.g., inhibitors of ChE activity by CM) for which the acute RfD is applicable to a shorter period.
CHAPTER 4 4
X. CONCLUSIONS The hazards associated with the use of OP and CM pesticides are evaluated by assessing the toxicity and by estimating the magnitude of exposure from sources in the workplace, the environment, food, and water. The need to balance the risk to man and the environment against benefits associated with pesticide use is an important issue. The conduct of toxicity studies (GLP) and agricultural practices (GAP) are important considerations for consistent procedures and documentation of studies so that all aspects can be recorded and verified. A joint panel of WHO/FAO (JMPR) is responsible for reviewing toxicological and residue data and estimating (where possible) an ADI and acute RfD for humans and MRLs for residues in foods. The overall objective of toxicological evaluation is to determine a NOAEL, based on consideration of the total toxicology database, that is utilized in conjunction with an appropriate safety factor to determine the ADI. The use of GAP helps to determine MRL values for pesticide residues in food. Integration of the results from all studies permits an appraisal of toxicity of the compound. The establishment of an acute RfD forms the basis of short-term intake and short-term risk assessment. Approximately 100 OP and CM pesticides have been evaluated to derive their MRL values in foods and ADIs for humans. Innumerable factors, such as crop pattern, food habits, processing conditions, species variations, and multiple uses of pesticides and toxic metabolites, complicate the determination; however, human data on a pesticide, whether from volunteer studies or from other investigations of human exposures in the workplace or environment, can be extremely valuable in placing the animal data in context and, when available, should always be evaluated even when they are not used to derive ADIs.
References Codex (1997a, June). Codex maximum residue limit for pesticides and extraneous residue limits. Twenty-second Session, Codex Alimentarius Commission report. World Health Organization, Geneva. Codex (1997b). Codex Alimentarius Commission report, CX/PR 98/5. World Health Organization, Geneva. Ellman, G. I., Courtney, K. D., Andres, V., Jr. and Featherstone, R. M. (1967). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Food and Agriculture Organization/World Health Organization (1962). Principles governing consumer safety in relation to pesticide residues, FAO Plant Production and Protection Division Report, No. PL/1961/ll; WHO Technical Report Series No. 240. World Health Organization, Geneva. Food and Agriculture Organization/World Health Organization (1970). Pesticide residues in food. Report of the 1969 joint meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Group on Pesticide Residues,
9OP and CM Pesticide Residues in Food
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FAO Agricultural Series No. 84; WHO Technical Report Series No. 458. World Health Organization, Geneva. Food and Agriculture Organization/World Health Organization (1988). Pesticide residues in f o o d - 1988. Report of the joint meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues, FAO Plant Production and Protection Paper 92. Food and Agriculture Organization, Rome. Gupta, E K. (2004). Pesticide exposure u Indian scene. Toxicology 198, 83-90. International Programme on Chemical Safety (2002). The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification. World Health Organization, Geneva. International Programme on Chemical Safety (2003). Inventory of IPCS and other WHO pesticide evaluations and summary of toxicological evaluations performed by the JMPR, WHO/PCS/02.3. World Health Organization, Geneva. Johnson, M. K. (1982). The target for initiation of delayed neurotoxicity by organophosphorus esters: Biochemical studies and toxicological applications. Rev. Biochem. Toxicol. 4, 141-212. Joint FAO/WHO Meeting on Pesticide Residues (1975). Report of the 1975 Joint FAO/WHO Meeting on Pesticide Residues, FAO Plant Production and Protection Series No. 1; WHO Technical Report Series No. 592. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2000). Proposed guidance for interpretation of data generated in studies with single oral doses (for use in establishing acute RfDs for chemical residues in food and drinking water), FAO/WHO Pesticide Residues in Food, Report 2000, Annex 5, pp. 197-198. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2002a). Pesticide residues in food. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticides Residues, Rome, September 16-25. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2002b). Pesticide residues in food. Evaluations (Part II-Toxicological) of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticides Residues, Rome, September 16-25. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2004). Pesticide residues in food. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticides Residues, Rome, September 19-29. World Health Organization, Geneva. Organization for Economic Co-operation and Development (1987). Guidelines for Testing of Chemicals by Organization for Economic Co-operation and Development. Organization for Economic Co-operation and Development, Paris. Sandhu, H. S., and Brar, R. S. (2000). Textbook of Veterinary Toxicology. Kalyani, New Delhi. World Health Organization (1974). Assessment of the carcinogenicity and mutagenicity of chemicals, Report of a WHO Scientific Group, WHO Technical Report Series No. 546. World Health Organization, Geneva.
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World Health Organization (1987). IPCS Environmental Health Criteria 70: Principles for the safety assessment of food additives and contaminants in food. World Health Organization, Geneva. World Health Organization (1990). Principles for the toxicological assessment of pesticide residues in food, Environmental Health Criteria No. 104. World Health Organization, Geneva. World Health Organization (1997a). Guidelines for predicting dietary intake of pesticide residues, 2nd rev. edn., GEMS/Food document WHO/FSF/FOS/97.7. World Health Organization, Geneva.
World Health Organization (1997b). Food consumption and exposure assessment of chemicals, report of a FAO/WHO consultation, February 10-14. World Health Organization, Geneva. World Health Organization (1998). GEMS/Food regional diets. Food Safety Issues, WHO/FSF/98.3. World Health Organization, Geneva. World Health Organization (2000). Guidelines for the Preparation
of Toxicological Working Papers for the WHO Core Group of the JMPR. World Health Organization, Geneva.
Aquatic Life & Wildlife
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CHAPTER 4
5
Aquatic Toxicity of Carbamates and O rgano p hosp hates ARUN K. RAY AND MANIK C. GHOSH Bose Institute, Calcutta, India
environment cannot be ignored. The health hazards of pesticides are well recognized. It is a global problem for agricultural workers and rural communities. For the safeguard of human health, the potential adverse effects of different pesticides were studied in different mammalian models. There a~'e many routes by which pesticides can reach the aquatic environment Foy and Bingham, 1969; Nicholson, 1969; Edwards, 1973a,b):
I. I N T R O D U C T I O N Use of pesticides in the control of agricultural pests was a turning point in human civilization that ushered in the new horizon in production of food grain necessary for survival of humankind. Pest coevolution with the host plant sometimes seems to be more advanced and complex, interfering with and disrupting human civilization from time immemorial. The discovery of pesticides resulted in millions of tons of global food grains being saved every year. A wide spectrum of pesticides have contributed to the control of a variety of pests in the agricultural field. In modern times, toxicologists have been designing pesticides based on the physiological or anatomical characteristics of a particular pest so that use of a particular pesticide in the field kills a particular pest without harming the other fauna (O'Brein, 1967; Winteringham, 1969). The deciphering of the pesticidal properties of dichlorodiphenyltrichloroethane (DDT) by Paul Muller in 1939 marks the birth of modem insecticide chemistry. This served as the cornerstone for its subsequent development. Humans have faced difficulties in cohabitation with insects since the beginning of agriculture in 8000 BC. Sumerians first used insecticide as sulfur compounds in 2500 BC to combat insects and mites. In 1936, the German chemist Gerhard Schrader synthesized a variety of organophosphorus compounds with potential use as insecticides. After organochlorines were phased out, the organophosphate (OP) and carbamate (CM) pesticides became the preferred choice. This chapter focuses on the toxicity of OPs and CMs on aquatic system.
1. Surface runoff and sediment transported from treated soil 2. Industrial waste discharged from factory effluents 3. Direct application as aerial spray or granules to control pests inhabiting water 4. Spray drift from normal agricultural operation 5. Municipal waste discharge Runoff is generally considered to represent the major movement of pesticides in the aquatic environment. During runoff, pesticides remain suspended in the runoff water and are transported to the aquatic ecosystem (Malins and Ostrander 1991). Nicholson and coworkers conducted an extended study on the routes of pesticide movements into the water. During runoff, pesticides may be adsorbed on the eroding soil particles suspended in runoff water. Heavy rainfall immediately after application of pesticides has a higher potential for pesticide transport. The effects of pesticides on aquatic life may be acute, resulting in mass mortality of fishes, chronic changes in their behavior, or reductions in survival, growth, and reproduction (Malins and Ostrander, 1991). The problem of pesticide contamination is a unique environmental problem because pesticides are deliberately introduced into the environment for beneficial purposes. One of the most disastrous effects of runoff was reported in the Punarbhaba River in West Bengal, India, where several tons of commercially important fishes were killed. Pesticides in general are also toxic to many nontarget organisms and cause ecological imbalance by indiscriminate killing of
II. P E S T I C I D E S IN T H E A Q U A T I C ECOSYSTEM: ACCUMULATION AND BIOMAGNIFICATION Although the use of pesticides has become indispensable due to their beneficial effect on crop yield, forest protection, and domestic living, their potential threat to the Toxicology of Organophosphate and Carbamate Compounds
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aquatic insects, worms, and mollusks by contaminating the soil and water, thereby disturbing the general ecosystem. The distribution of pesticides in water influences the pathway of biological uptake. The quantity accumulated by each biological entity is dependent on the chemical properties of the pesticides and the physiology and behavior of the organisms. Laboratory studies have shown that 14C-labeled DDT can be absorbed from water quickly by Atlantic salmon and is distributed to the major organs in approximately 5 min (Premdas and Anderson, 1963). When pesticides are present for a short time in water, adsorption onto water and food organisms may play an important contributory role in determining the amount of residue found in fishes (Chadwick and Brocksen, 1969). Pesticides in the aquatic environment constitute both direct and indirect hazards to aquatic organisms and humans. The organism at the end of the food chain may be exposed to a higher concentration of the pesticide than originally found in the environment due to biomagnification. This sort of magnification mechanism is of greater importance in aquatic than in terrestrial organisms. All pesticides can be categorized as organochlorines, pyrethrins/pyrethroids, OPs, and CMs according to their chemical nature. Also, the mechanism of action varies depending on their chemical nature. Among them, CMs are the most widely used pesticides throughout the world since organochlorines have longlasting residue persistence (Parmar and Dureja, 1990; Hayes, 1982) and OPs are extremely toxic and pose a delayed neurotoxicity problem (Gupta, 1994). CM pesticides are preferred in agriculture more than OPs due to their rapid biodegradation, less bioaccumulation, and also due to their quick action compared to OPs and organochlorine (Corbett et al., 1984). Among the CMs, carbofuran is widely used in agriculture and forestry as a broad-spectrum systemic insecticide, nematicide, and acaricide that kills insects, nematodes, and mites on contact or after ingestion (Gupta, 1994). It is used for soil and foliar pests of field, fruits, vegetables, and forest crops. Carbofuran is available in liquid and granular form (Worthing, 1987; U.S. Environmental Protection Agency, 1991).
III. D E T E R M I N A T I O N O F P E S T I C I D E R E S I D U E IN W A T E R The determination of pesticide contents in water, organic substrates, sediments, and animal tissues depends on the chemical methods. The solid materials of plant and animal tissues are homogenized and extracted with acetone or hexane, evaporated to a small volume for microdetermination by various chromatographic methods. The concept of in situ bioassays is mainly based on exposure of test animals in the contaminated water and determination of
percentage survival. Most simple is the process of exposing fish to test the toxicity of water. Cages containing the experimental fish are hung in the contaminated water column or anchored at the bottom. Mortality is measured after exposure of 96 hr or longer.
IV. M E T H O D S T O D E T E C T A C U T E A N D DELAYED TOXICITY Several invertebrate organisms, among approximately 1 million species, have been selected to test the adverse effects of toxicants. These invertebrates include snail (Helisoma trivolvis), the clams (Spanerium, Corbicula, and Musculium), the amphipods (Hyalella azteca, Gammarus lacustris, and G. pulex fossarum), the burrowing mayfly larvae (Hexagenia rigida and H. bilineta), and larvae of the midge (Chironomus tentans). Rarely, marine amphipods or freshwater benthic oligochaetes are used (Swartz et al., 1982; Bailey and Liu, 1980). Approximately 25 fish species are also used to test waterborne pesticides. The most frequently used fishes are common carp (Cyprinus carpio), rainbow trout (Salmo gairdnari), brown trout (Salmo trutta), goldfish (Carassius auretus), zebra fish (Brachydanio rerio), and bluegill (Lepomis macrochirus). Nevertheless, any type of fish may be used for pesticide toxicity. Acute toxicity is generally expressed as LC50/48 hr or LC5/48 hr (Mayer and Ellersiek, 1986). In fish, most of the toxic effects are expressed through inhibition of some hydrolytic enzymes, mainly acetylcholinesterase. Acute or delayed toxic effects of pesticides are measured in bioassays in aquarium organisms as follows: 1. The hatch rate (cumulative percentage hatch per treatment) and initiation of hatching from eggs of live female mayfly, Hexagenia, at 20~ are tested with different doses of particular pesticide and LCs0s are reported. 2. In sediments, tests for pesticides are performed using the Chironomus adult emergence test. The test is performed with 10-day-old second-instar larva at 20~ and the end point is the number of emerged adults collected in an Erlenmayer flask trap (Nebecker et al., 1984). 3. In the Daphnia magma life cycle test, 5-day old Daphnia are exposed for 10 days through maturation and release of young (three broods). Experimental specimen is fed at the rate of 2 mg/liter of algae every other day. For water or sediment toxicity, the total number of survivors is reported. 4. Ramshorn snail (H. trivolvis) is used to measure mortality, growth, and fecundity in successive generations. The snails are cultured according to standard conditions and exposed to various concentrations of a required pesticide (Flannagan, 1974).
CHAPTER 45 9Aquatic Toxicity of CMs and OPs 5. Crayfish (Orconectes virilis) exposed to different concentrations of pesticides are used for measuring intermolt phase and hemolymph calcium. The absence of elevation of calcium concentration in specimens exposed to pesticides in laboratory defines failure to molt. Hemolymph samples are taken directly from the heart and analyzed calcium biochemically (Leonhard, 1974). 6. Most common effects of pesticides in fish are monitored through changes in respiratory rate, fin and body movements, heartbeat, and response to light and touch. Chronic or sublethal effects are monitored through long-term toxicity tests, such as growth and development, timing and duration of hatch, or even ovarian failure. Fish eggs are also used for toxicity tests (McKim, 1977; McDonald, 1979). 7. Fish serum and brain cholinesterase activity, serum glucose, serum protein, or total serum lipid contents are measured biochemically to monitor the strength of pesticide exposure in fish (Lockhart et al., 1951). Since most OP pesticides are known to inhibit cholinesterase, monitoring of this enzyme activity is of prime importance. 8. Zebra fish (B. rerio) can be used for its small size, egg-laying performance, and short life cycle of approximately 75 days to monitor chronic toxicity and bioaccummulation (Lillie et al., 1979).
V. A C U T E T O X I C I T Y ( M A M M A L S VS F I S H ) Carbofuran is highly toxic by inhalation and ingestion and moderately toxic by dermal absorption (Hayes, 1982) in mammalian species. The LDs0 in experimental rat by oral exposure was reported to be 5.0 mg/kg (Ben-Dyke et al., 1970) and by inhalation 85 mg/m 3 (Tobin, 1970), whereas intraperitoneal exposure of 2 mg/kg was found to be lethal (Gupta and Kadel, 1989a). Following oral and parenteral exposure to carbofuran, experimental laboratory animals exhibit the onset of toxic signs such as salivation, chewing, and fine tremors within 5-15 min with frequent propensity; signs of maximal severity, including muscle fasciculation and convulsion, are evident from 15 min to 1 hr and persist for approximatey 2 hr. In general, toxic manifestations are characteristics of acetylcholinesterase inhibition, with parasympathetic preponderance exhibiting profuse salivation, lacrimation, miosis, hypothermia, muscle twitch and fasciculations, body tremors, and convulsion. Overall, carbofuran elicits toxic manifestation of central and peripheral nervous systems origin by overstimulation of muscarinic and nicotinic acetylcholine (ACh) receptors (Cambon et al., 1979; Gupta and Kadel, 1989a,b). In humans, symptoms of carbofuran toxicosis include salivation, diaphoresis, abdominal pain, chest tightness, drowsiness, dizziness, vomiting, muscular twitching, convulsions, and coma
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(Tobin et al., 1970). Similar clinical signs are observed in other species due to acute carbofuran poisoning. Although carbofuran is a rapidly degrading pesticide in soil and water, its residue may persist in soil and water for several weeks, months, or up to a 1 year, depending on the environmental conditions (Finlayson et al., 1991). Results from a study conducted by the Environmental Hazards Assessment Group of California, Department of Food and Agriculture, revealed the presence of carbofuran in the runoff water from rice and sugar beet fields in agricultural drain and in the Sacramento River (Nicosia et al., 1990). Thus, carbofuran reaches the aquatic ecosystem from the agricultural field and aquatic organisms face the toxicity when adsorbed. During the past 30 years, extensive studies have been conducted on various species, regarding carbofuran inhibition of AChE but most studies were targeted on mammalian species rather than fish or other invertebrates. This chapter focuses on the toxicity of carbofuran on aquatic organisms in retrospect to the effect in comparison to the mammalian system. Second, aquatic organisms encounter the toxic effect of this pesticide as the first consumers of the ecological food chain rather the second consumer such as mammals. Bhattacharyya (1985) reported carbofuran-induced acetylcholinesterase (ACHE) inhibition in the fish (Channa punctatus and Anabas testudineus). Brain ACHE activity decreased significantly when these fish were exposed to carbofuran in laboratory conditions. Carbofuran-induced inhibition of AChE in juvenile goldfish (Carassius auratus) was reported by Bretaud et al. (2000). Brain and muscle AChE were inhibited significantly by 50 txg/liter (19-28%) or 500 txg/liter (85-87%) of carbofuran. Carbofuran inhibits AChE significantly in C. carpio exposed to a sublethal dose (Dembele et al., 2000). This inhibited enzyme activity can be recovered in time, indicating the reversibility of binding of this pesticide with the enzyme (Dembele et al., 1999). Toxic and teratogenic effects of carbofuran to Cyprinus carpio communis were reported by Pawar (1994) when the pesticide was administered to the ambient water. The percentage of hatching and survival of hatching decreased with the increasing concentration of pesticide. Abnormalities observed included deformed body curvature, eye pigmentation, enlargement of pericardial sacs, circulatory failure, loss of balance, and abnormal behavior. Carbofuran causes a significant decrease in blood hemoglobin and remarkable changes in erythrocytes in fish (Bhakthavathasalam, 1991). Carbofuran-induced histopathological and biochemical changes in the liver of the teleost fish Channa punctatus (Bloch) were reported by Ram and Singh (1988). Channa punctatus exposed to a safe dose (4.5 ppm) of the commercial pesticide carbofuran for 6 months exhibited a varying degree of histopathological changes, including cytoplasmolysis, nuclear pyknosis, and necrosis in the liver. In some regions of the liver, extensive degeneration of proliferative hepatocytes and induction of the tumor were indicative
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of carcinogenic action of this pesticide (Gopalkrisna and Ram, 1994). Heath et al. (1992) simulated the environment of the Sacramento River, which is constantly exposed to rice pesticide originating as surface runoff from the agricultural field. They showed inhibition of AChE activity of striped bass (Morone saxatilis) larvae exposed to water mixed with parathion, carbofuran, and molinate. Carbofuran is well known for extensive field killing of fish, wildlife, and invertebrates, and therefore protection of such sensitive species is recommended. Anton et al. (1993) reported the ecotoxicity of carbofuran to freshwater fish and algae, which demonstrated heavy mortality of Carassius auratus L. (Cyprinidae) and Chlorella pyrenoidosa, the important species in the tropic chain of the Spanish freshwater ecosystem. Singh and Garg (1992) showed the localization of the carbofuran in the body of Clarius batracus after its exposure to the pesticide. They demonstrated that carbofuran accumulates in the gills, liver, and kidney of the fish, with a residue range of 0.01-0.095%. Heath et al. (1993) reported toxic effects of carbofuran on Japanese medaka (Oryzias latipes) larvae exposed for 4 days in the rice field. Pesticide concentration was half of the 96-hr LCs0 dose level simulating a condition of rice field water runoff. This sublethal dose resulted in impairment of swimming speed and inhibited AChE activity. Newly hatched fathead minnow (Pimephales promelas) larvae were exposed for 4 days to two pesticides in water in a simulated condition of the Sacramento River during the striped bass spawning season, which coincided with real pesticide use in adjacent rice culture. Carbofuran and molinate were tested at two concentrations a higher level approximating one-half of the LCs0 and a much lower level of LCs0 as found in the Colusa basin drain. The higher concentration of carbofuran and molinate caused a reduction in swimming capacity and inhibited AChE activity, whereas the lower concentration showed no significant changes. The study indicates that regulated use of pesticide in the agriculture field may restrict the deleterious effects on the fauna through surface runoff (Heath et al., 1997). Rice field surface runoff containing carbofuran caused a decline in the population of striped bass in the Sacramento River by either direct killing or reducing food items such as mycids (Finlayson etal., 1993). Since 1980, when molinate was demonstrated to kill carp in agricultural drain, an intensive research effort has been under way to assess the impact of rice pesticide on the aquatic system in the Sacramento River and its delta. Laboratory bioassay indicated the presence of carbofuran or methyl parathion and also bufencarb in the fiver water, which were responsible for the deaths of aquatic organisms and early life stages of striped bass. After 1981, management protocol for pesticide use was executed, which resulted in a dramatic decrease in rice pesticide levels in the fiver and delta water. The nontoxic level of pesticide in the fiver was commensurate with the recovery
of striped bass and Chinuk salmon (Byard, 1999). The effect of carbofuran on the fish reproductive system has been elaborately studied in our laboratory (Chatterjee et al., 1997). The study showed impairment of the hypothalamohypophysial-gonadal axis in Heteropneustes fossilis after treatment with carbofuran. The effects included retarded ovarian maturity, reduced gonadosomatic index, an increase in stage-1 oocyte and decrease in stage-3 oocytes numbers, atrophy of gonadotrophs in the pituitary glands, and increased total lipid in liver but its loss in ovary along with essential fatty acids. Carbofuran-induced atresia in freshwater fish Colisa lalia has been reported by Sukumar and Karpagaganapathy (1992). Saxena and Mani (1987) reported testicular recrudescence in freshwater murrel Channa punctatus by carbofuran treatment. Neuroendocrine dysfunction by carbofuran treatment in freshwater murrel Channa punctatus has been reported by Ram and Singh (1988). With prolong carbofuran exposure (4 months at 4.5 ppm), the thyroid gland showed abnormalities including hypertrophy, hyperplasia, degeneration of follicular cells, and reduction in colloidal material. Carbofuran, which is washed into the water system in small quantities, induces adverse histopathological alterations in thyroid, depending on the age and body weight of the animal, possibly by acting directly on thyroid or through the hypothalamuo-pituitary-thyroid axis. This pesticide may influence the growth and protein conversion efficiency in freshwater fish such as Oreochromis mossambicus, Mystus vittatus, and Channa striatus, as reported by Vasanthi et al., (1990). Carbofuran at sublethal concentration alters the adenylate parameters in the blue gill sunfish (Lepomis macrochirus) (Horneiter etal., 1991). Adenylate parameters (ATE total adenylate nucleotides, and adenylate energy charge) decreased in gill, liver, muscle, and stomach. The adenylate parameter in the stomach is very much susceptible to the concentration of the carbofuran (0.2-30 mg/liter). An important study was performed by Zayed et al. (1988) in which they showed the distribution of 14C-labeled carbofuran residue and the parent compound in fish exposed to paddy pisiculture for 90 days. Carbofuran residue was present in the rice plant after 30 days of application. Fish showed the presence of carbofuran in the body even after 30 days of exposure without any sign of accumulation of an appreciable amount. In water, the parent compound was decreased by 10% after 30 days. Analysis of residue in fish, plant, and soil showed the presence of several metabolites (60% of residue) apart from the parent compound. Among the degradation products, 3-hydroxycarbofuran and carbofuran phenol were identified as major metabolites. An excellent study was conducted on the transient carbofuran runoff from farmland and its effect on the bioassay organism, Gammarus pulex, by Matehiessen et al. (1995). One month after an application of carbofuran of 3 kg/ha as
CHAPTER 45 9Aquatic Toxicity of CMs and OPs broadcast granules or as broad granules to oilseed rape crop, a carbofuran concentration up to 26 mg/liter was measured in a nearby water reservoir after heavy rainfall. The majority of carbofuran translocates via field drain at a concentration up to 264 mg/liter. Maximum concentration persisted only for approximately 24 hr after the rainfall event, although measurable amounts could be detected for the next 4 days. An in situ bioassay of stream water that monitors the feeding rate of the bioassay species G. pulex showed that feeding stopped completely during the rainfall events, leading to the death of the organisms. Subsequently, laboratory studies demonstrated the LCs0 of carbofuran to be 21 mg/liter for G. pulex at 24 hr, which could reduce the feeding rate. Persistence of carbofuran in the fish of the Oconee River in central Georgia that receives surface runoff from adjacent agricultural orchard fields has been reported by Bush et al. (1986).
VI. M E C H A N I S M O F A C T I O N Carbofuran elicits acute intoxication by virtue of reversible inhibition (carbamylation) of AChE (Casida, 1963; O'Brien, 1967; Yu et al., 1972; Kuhr and Dorough, 1976; Gupta and Kadel, 1989a,b), which hydrolyzes ACh, a neurotransmitter. The inhibition of AChE consequently leads to excessive ACh accumulation at the synapses and neuromuscular junctions, resulting in overstimulation of ACh receptors (muscarinic and nicotinic), which could ultimately lead to death due to respiratory failure. When carbofuran mimics an alternate substrate, the alcohol moiety is cleaved, giving rise to the carbamylated ACHE. In contrast to the acetylated ACHE, carbamylated AChE is more stable (Wilson and Harrison, 1961). Sequestration of the AChE in carbamylated form thus precludes the hydrolysis of ACh, leading to ACh accumulation. Potent AChE inhibition results from rapid carbamylation (Yu et al., 1972; Kuhr and Dorough, 1976) because the carbamylation rate constant (kl) is directly correlated with toxicity (Fergusen et al., 1984). The IC50 value for AChE inhibition by carbofuran was reported to be 3.3 • 10 -8 (Dorough, 1968). In general, IC50 values for CMs decrease as the side chain becomes longer and bulkier (Soreq and Zakut, 1989). It should be noted that IC50 values vary in different tissues depending on experimental conditions, such as incubation time, temperature, and pH. Skeletal muscle, brain, heart, etc. are the main target organs for toxicity of carbofuran and other AChE-inhibiting pesticides (Petras, 1981). Recovery of AChE from carbofuran-induced inhibition (i.e., decarbamylation) is quite rapid since recovery simply requires dissociation of the methylcarbamyl moiety from the enzyme (O'Brien, 1967). The short enzyme recovery period is explained in part by kinetics of the reversible AChE-carbofuran complex. It has been reported that the
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in vivo carbofuran hydrolysis rate constant (krn 1 = 0.028 min -1, tl/2 = 25 min), representing all esterases active in carbofuran (Fergusen et al., 1984), is similar to that of the in vitro value (tl/2 = 20-63 min), suggesting rapid enzyme recovery (O'Brien et al., 1967; Aldridge, 1971). Enzyme recovery is faster in skeletal muscle than in the brain region (Gupta and Kadel, 1989a,b). Besides the inhibition of ACHE, carbofuran has a severe impact on other enzymes and compounds, such as butyrylcholinesterase (Gupta and Kadel, 1989a,b), lactate dehydrogenase (Gupta et al., 1991a,b; Gupta and Kadel, 1989b), neurotransmitter-like epinephrine, ~/-aminobutyric acid, protein, lipid and lipoprotein, and high-energy phosphates (Gupta et al., 1994) in rat or mice. It may also upset the immune system in laboratory rats (Burnett, 1980). The mechanism involved in toxicity of OPs is similar to that described for CMs (as described previously), except that OPs cause irreversible inhibition of AChE by phosphorylation, whereas CMs cause reversible AChE inhibition by carbamylation. Once OPs enter the body, they are mainly metabolized in the liver, gastrointestinal tract, malpighian tubules, and body fat by mixed function oxidases, which further increase their toxicity. Oxidative desulfuration of OPs leads to maximum anticholinesterase activity. To consider the possible toxic effect of OPs, it is necessary to take into account the type of chemical and the species in question because there are wide differences. For example, in vertebrates, after one single exposure to OP pesticides the recovery time from the anticholinesterase effects ranges from 1 to 3 weeks, whereas only approximately 1 day is necessary to recover from exposure to a CM pesticide. Birds and mammals have similar metabolic responses to anticholinesterase pesticides. However, birds tend to be more sensitive to their toxic action, probably due to lower levels of metabolizing enzymes. Based on their low LDs0s, most OPs and CMs are classified as "extremely toxic" to birds and mammals after a single dose. However, by virtue of their short half-life, they ~are quickly metabolized by the body and excreted, allowing for a fast recovery from the toxicity. A different outcome is observed when exposure occurs by means of continuous smaller doses. In this case, an accumulative anticholinesterase effect may be observed, even resulting in death after a few days. For aquatic animals, discrete studies have obtained no substantial information regarding the molecular mechanism of action of OPs.
VII. N E U R O T O X I C I T Y
OF ORGANOPHOSPHATES Several subacute and chronic syndromes can result from OP exposure, and these have been summarized by Jamal (1997) as the intermediate syndrome (IMS), the
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organophosphate-induced delayed neuropathy (OPIDN), and the chronic organophosphate-induced neuropsychiatric disorder (COPIND). Some OPs are known to produce IMS and it can occur within 3 or 4 days after poisoning. It is not responsive to atropine or oxime therapy. The patient usually recovers within 2 or 3 weeks, but this is usually an intermediary stage occurring prior to the delayed effects. OPIDN occurs usually 10 days to 3 weeks after exposure and is serious and irreversible. It consists of a distal symmetrical sensorimotor mixed peripheral neuropathy mainly affecting the lower limbs. It is unrelated to the effect of OPs on ACHE. It is thought to act by causing an aging (dealkylation) of an enzyme in nerve cells called neuropathy target esterase (NTE). This is only induced in certain species, including humans and chickens. Other induced abnormalities include axon transport and physicochemical changes of proteins and axon membrane integrity. The exact mechanism(s) is not fully understood. Evidence indicates that OPs can induce chronic effects on both the peripheral and central nervous systems following acute intoxication (Jamal, 1997). The mechanisms for this condition are not related to the inhibition of AChE or NTE. Two types of such disorders have been documented: type I, representing COPIND following an acute poisoning episode(s), and type II, representing COPIND following a chronic long-term exposure to subclinical doses. There is no distinction in the chemical nature of OPs for their apparent ability to cause COPIND. Several components of COPIND have been described, including neurobehavioral and cognitive changes, psychiatric and mental manifestations, chronic fatigue, peripheral neuropathy, neuromuscular junctional dysfunction, electroencephalographical changes, autonomic nervous system disturbances, frontal lobe syndrome, and abnormalities of cognitive evoked potentials (Jamal, 1997). Some patients have all markers, whereas others show a variety of combinations. However, no such studies at the neurophysiological level have been performed in aquatic animals. Toxicants, including OPs, often completely eliminate the performance behaviors that are essential to fitness and survival in the natural ecosystem (Scott and Sloman, 2004). The behavioral toxicity of many xenobiotics is unknown and warrants future study. Physiological effects of toxicants available in the literature have shed light on the disruption of sensory, hormonal, neurobiological, and metabolic systems that are likely to have profound effects on behavioral responses in fish. The environmental toxicants may possibly be linked with the disruption of behavioral responses in fish through cholinesterase inhibition, altered brain neurotransmitter levels, sensory deprivation, and impaired gonadal and thyroid hormone levels (Scott and Sloman, 2004).
VIII. M U T A G E N I C I T Y ,
CARCINOGENICITY, AND OXIDATIVE STRESS Nelson et al. (1981) reported the mutagenicity of N-nitroso derivatives of carbofuran and its toxic metabolites (3hydroxycarbofuran and 3-ketocarbofuran) by the Ames assay method with S. typhimurium strains TA98 and TA100. The nitroso derivatives of all three compounds were similar, giving a mutation ratio of 45 at 5 ixg/plate on TA100. In addition, all three compounds produced chromosomal aberration in Chinese hamster ovary cells. Nitrosocarbofuran and 3-hydroxynitrosocarbofuran were also found capable of inducing a large number of sister chromatid exchanges in the same cells. OPs are also known to induce tumorigenic risks (Brown etal., 1991; Soreq and Zakut, 1993). In a chronic study, isofenphos (OP) was shown to cause myeloid leukemia blast crisis and altered function of lymphocyte DNA. Several studies have demonstrated oxidative stress induced by OPs in rat brain and human erythrocytes (Abdollahi et al., 2004). OP-induced seizures have been reported to be associated with oxidative stress (Gupta et al., 2001).
IX. METABOLISM OF CARBOFURAN Metabolism of carbofuran has been studied in rats (Dorough, 1968; Lucier et al., 1972; Fergusen et al., 1984; Marshall and Dorough, 1977), mice (Metcalf et al., 1968), hens (Hicks et al., 1970), fish (Bruce, 1972; Chatterjee and Ghosh, 1995), and worms and houseflies (Metcalf et al., 1968; Dorough, 1968). Generally, in the body, carbofuran undergoes hydroxylation and oxidation reaction, producing 3-hydroxycarbofuran, 3-hydroxycarbofuran-7-phenol, 3-ketocarbofuran, and 3-ketofuran-7-phenol (Dorough, 1968; Metcalf et al., 1968). The key metabolites are produced by hydroxylation at the benzylic carbon to give 3-hydroxycarbofuran, which is oxidized to the 3-ketocarbofuran when not blocked by formation of conjugates. Dorough and Metcalf et al. first identified the metabolites with certainty: 3-hydroxycarbofuran, 3-ketocarbofuran, and their respective 7-hydroxy hydrolysis product. There is also evidence of the formation of N-CH2OH derivatives in mammals and insects produced from carbofuran or 3-keto derivatives. In an extensive investigation, Marshall and Dorough (1977) revealed that oral exposure of rats to 14C-labeled carbofuran resulted in enterohepatic circulation of some metabolites. Radiocarbon in the urine of carbofuran-treated rats consisted largely of sulfate and glucuronide conjugates of carbofuran hydrolytic derivatives. This enterohepatic cycling of glucuronides involves cleavage of conjugates in the gut and biliary
CHAPTER 45 9Aquatic Toxicity of CMs and OPs excretion, which may lead to increased systemic activity of toxic CM metabolites. 3-Hydroxycarbofuran and 3-ketocarbofuran as the main metabolites of carbofuran were identified in catfish (H. fossilis) liver by high-performance liquid chromatography (Chatterjee and Ghosh, 1995).
A. Cytochrome P450 and Pesticide Metabolism Among the CMs, carbofuran is one of the main pesticidal contaminants in the aquatic ecosystem and threatens the aquatic fauna and flora due to its detrimental effects associated with multipotent toxicity. The half-life of this pesticide is sufficient to exert its harmful effects on the living organism. The aquatic environment covers approximately 71% of the total earth surface (Goksoyr and Forlin, 1992), and 28,000 fish species inhabit this environment (Moyle and Cech, 1988), along with other innumerable living organisms. Deposition of large amounts of toxicants into the water occurs from a variety of anthropogenic sources, particularly in urban and industrial areas (Malins and Ostrander, 1991). Fish constitute the largest group of vertebrates in the aquatic ecosystem. They first come into contact with anthropogenic, industrial, or agricultural contaminants that enter the aquatic ecosystem through surface runoff. So fish face the challenge of pesticide-like carbofuran intoxication in water from agricultural fields. The integuments and the gills are the major sites of uptake of the contaminants into the body. These contaminants are rapidly distributed in the body through their efficient circulatory system. This poses a threat not only to the health of the fish but also to the consumers, including humans. Several attempts have been made to demonstrate the role of the cytochrome P450 monooxygenase system in metabolism of carbofuran in liver of catfish. Lee etal. (1998) reported the metabolism of ethyl carbamate by cytochrome P450 2El (CYP 2El) and carboxylesterase (hydrolase A) enzymes in murine liver microsomes. Binding of ethyl carbamate (ethyl 14C) to liver microsomes, followed by its metabolism in the microsomal system, has been found (Lee et al., 1998). Such involvement of cytochrome P450 2El in metabolism of ethyl carbamate was shown by Forkert and Lee (1997) in lung microsomes. Jeong et al. (1996) reported the role of P450 enzymes in the detoxification of ethyl carbamates in female BALB/c mice splenocytes in culture. Scientific interest in the cytochrome P450 system has increased dramatically since the unheralded discovery in 1958 of a hepatic microsomal carbon monoxide binding pigment with a maximum absorption at 450nm (Klingenberg, 1958; Garfinkel, 1957). Omura and Sato (1964) subsequently showed that the pigment had biochemical properties of a cytochrome and designated it P450 because of its absorption maxima in the CO-bound condi-
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tion at 450 nm (Omura and Sato, 1964). The cytochrome P450 superfamily is ancient and has expanded via divergent evolution. It is found from prokaryotes to human beings. The ancestral P450 gene probably appeared 1.5 billion years ago among the prokaryotes. The evolution of this gene among the eukaryotes is considered to be due to a defensive mechanism against the phytotoxin that accumulate during the ingestion of plants by herbivores. This gene later evolved in response to detoxification of drugs and lipophilic pollutants (Nebert and Gonzalez, 1987). Cytochrome P450 exists in bacteria, plants, and animals, and these hemoproteins catalyze the monooxygenation of a broad spectrum of lipophilic substrates (Guengerich and Shimada, 1991; Nelson etal., 1996; Mansuy, 1998). Biochemical analysis of cytochrome P450 has revealed the presence of multiple isozymes that often have broad and overlapping substrate specificity. Cytochrome P450 belongs to a superfamily of structurally and functionally related hemoproteins. The numerous CYP isozymes are classified on the basis of proposed evolutionary relationship as inferred from the degree of identity among amino acid sequences between the isozymes. Cytochrome P4501A has attracted attention because it is involved in the biotransformation of many foreign compounds, including polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons (PAHs) (Stegeman and Hahn, 1994; Parkinson, 1995). CYP1A converts the lipophilic xenobiotics by monooxygenation to more water-soluble metabolites, which is the first step toward excretion and detoxification. However, some intermediates are highly reactive and may interact with biological macromolecules, thereby appearing to be more toxic than the parent compounds (Parkinson, 1995). Purification and characterization of the P450 proteins in fish have progressed faster than the study of teleost CYP gene. Only a limited number of freshwater and marine fish have been studied. Multiple P450 forms have been purified from the freshwater fish rainbow trout (Oncorhynchus mykiss) and perch (Perca fluviatilis) and from the marine fish scup (Stenotomus chrysops and Gadus morhua). Many of these forms have been discussed in reviews (Buhler and Williams, 1988, 1989; Stegeman, 1989; Stegeman and Klopper-Sams, 1987; Stegeman et al., 1990). In some cases, the isoenzymes were purified from fish treated with a PAHtype inducer, such as [3-napthoflavone or 3-methylcholanthrene. The fish were taken directly from the environment. In mammals, the members of the CYP1A subfamily and their response to exogenous inducer, such as [3-napthoflavone, PAH, or 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), are among the best characterized and well studied of the P450 superfamily (Nebert and Gonzalez, 1987; Nebert et al., 1989) because of their role in metabolism and activation by aromatic hydrocarbons, carcinogens, and other toxic chemicals. Two genes, CYP1A1 and-1A2, being the two distinct protein products, are known to exist in all mammals studied to date.
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Observations reveal that typical CYP1A1 activities, such as aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin-odeethylase (EROD), and ethoxycoumarin-o-deethylase, may be differentially induced by different inducers, such as diesel oil and isosafarol (Leaver et al., 1993; Celander and Foflin, 1991). The characteristic feature of both mammalian and teleostean CYP1A forms is that they are inducible in nature (Nebert and Gonzalez, 1987; Nebert et al., 1989). The rate of gene transcription, affecting messenger RNA formation, new synthesis of cytochrome P450 protein, and subsequent processing, heme insertion, and folding lead to the availability of catalytically active enzyme. The previously mentioned process can be analyzed with suitable probes to detect their induction in fish (Goksoyr and Husoy, 1998; Stegeman and Hahn, 1994; Bucheli and Fent, 1995). The induction response is not fully characterized in all CYP families; the best studied family is the CYP1A subfamily. The induction response in this subfamily is known to occur via the high-affinity binding of aromatic hydrocarbons to an intercellular receptors complex (the Ah receptor), which remains associated with the 90-kDa heat shock protein (HSP90). Binding of ligand with the Ah receptor results in the dissociation of HSP90. Then, the ligandreceptor complex translocates to the nucleus with the help of aromatic hydrocarbon nuclear translocator protein (Arnt), where the heterodimer complex interacts with the xenobiotic responsive element of nucleus (XRE) to initiate the transcription of the CYP1A gene. Ligand for the Ah receptor includes the hydrophobic planar molecules that fit into the rectangular receptor binding site. The prototype ligand for the Ah receptor is TCDD, which is one of the potent CYP1A inducers (Safe, 1988; Hahn et al., 1994; Nebert et al., 1989; Stegeman and Hahn, 1994; Hankinson, 1995; Segner and Braunbeck, 1998). The inducibility of CYP1A by xenobiotic exposure led to its use as a biomarker in a pollution monitoring study of the aquatic environment (Whyte et al., 2000). EROD and AHH activities appear to be the most sensitive catalytic probes for determining response in fish. In most fish, they are highly inducible by a PAH-type inducer. Pollutants of major concern in the aquatic environment are PAH, polychlorinated dibenzo-p-dioxin and dibenzofuran, polyhalogenated biphenyls (PCB/PBB), and halogenated organic compounds and herbicides. The responsiveness of the CYP1A1 gene varies depending on the gradient of CYP1Ainducing chemicals, such as PAHs and PCBs. For instance, a complex mixture of inducing and noninducing xenobiotics as they are normally present in the environment can modify the CYP1A induction response (Croce et al., 1995; Beyer etal., 1997). Likewise, nonchemical exogenous parameters, such as temperature and season, and endogenous factors, such as hormone and physiological condition of the fish, can modulate the responsiveness of the CYP1A system (Achazi et al., 1994; Machala et al., 1997; Kopeonen et al., 1998; Lange et al., 1998.
X. A N A L Y T I C A L D E T E C T I O N O F C Y P EXPRESSION IN FISH For the assessment of CYP1A in fish, protein and catalytic activity can be used (Goksoyr and Husoy, 1998; Stegeman and Hahn, 1994; Brucheli and Fent, 1995). Mainly two enzymes translated from the CYP1A gene are used for assay: ethoxyresorufin-o-deethylase and AHH (Nebert and Gelboin, 1968; Burke and Mayer, 1974; Eggens and Galgani, 1992). CYP1A-associated catalytic activity in the fish tissue is conventionally analyzed on the microsomes prepared by ultracentrifugation from homogenized tissue. However, methods have been developed to study the activity directly from the cell without preparation of microsomes (Kennedy et al., 1995; Clemons et al., 1996; Hahn et al., 1996; Behrens et al., 1998). Assessment of CYP1A activity at the protein level was done as protein purification protocol for teleostean P450 protein was developed. CYP1A homologous protein has been purified from rainbow trout (Oncorhynchus mykiss; Williams and Buhler, 1984), and P450 LM4b/4a (Scholz et al., 1997) has been purified from scup (Stenotomus chrysops; Klotz etal., 1983), cod (Gadus morhua; Goksoyr, 1985), perch (Perca fluviatilis; Zhang etal., 1991), and tilapia (Oreochromis niloticus; Uang and Uang, 1995). For each of these proteins, polyclonal and monoclonal anti-CYP1A antibodies have been prepared (Williams and Buhler, 1984; Goksoyr, 1985; Park et al., 1986; Kloepper-Sams et al., 1987; Celander and Forlin, 1991; Husoy et al., 1996; Scholz et al., 1997). The available antibody toward fish CYP1A has generally shown broad interspecies cross-reactivity (Kloepper-Sams et al., 1987; Goksoyr, 1991; Goksoyr and Forlin, 1992). For immunochemical detection of teleostean CYP1A, the available antibody has been used in Western blotting, enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry. Immunochemical assay may be particularly advantageous in environmental monitoring programs because it does not rely on adequate conversion of the enzymatic activity of the samples (Goksoyr etal., 1991; Collier etal., 1995). Xenobiotics may inhibit CYP1A enzymatic activity in any assay in which such a compound may be present. Disadvantages of immunochemical CYP1A detection are that it is less sensitive compared to enzymatic assay (Scoltz et al., 1997) and problems in quantification (Goksoyr, 1991; Van Veld et al., 1997).
XI. C E L L AND T I S S U E D I S T R I B U T I O N O F C Y P 1 A IN T E L E O S T F I S H Intracellular immunochemical localization of CYP1A appears to be a potential target for the assessment of the degree of toxic action of xenobiotics and carcinogens. CYP1A is commonly expressed in many epithelial cells of many organs. The highest CYP1A activity is found in
CHAPTER 45 9Aquatic Toxicity of CMs and OPs liver, but it also occurs in extrahepatic organs, such as gallbladder, gonads, nervous tissue, and endocrine cells (Sarasquete and Segner, 2000). Although the biochemically detectable CYP1A level in extrahepatic organs is usually lower than that in liver, the prominent expression and induction of CYP1A in fish liver are consistent with the involvement of the organ in xenobiotic metabolism and excretion. Several studies have demonstrated the expression of CYP1A in hepatocytes of fish by means of immunohistochemistry (Reinecke and Segner, 1998; Sarasquete et al., 1999). In contrast to mammals, CYP1A shows heterogeneous distribution throughout the liver parenchyma and no zonation is observed in fish liver (Smolowitz et al., 1991). Subcellularly, in fish hepatocytes CYP1A is localized at the rough endoplasmic reticulum, the nuclear envelop, and in the plasma membrane of the microvillae of the bile canaliculi (Lester et al., 1993). Although the role of cytochrome P450 in the metabolism of carbofuran has not been explored in any system, the metabolism of ethyl carbamate by P450 2El has been reported by Lee et al. (1998). The study implicated cytochrome-P450 2El and carboxylesterase enzyme in the metabolism of carcinogens such as ethyl carbamate in the murine liver microsomal system. The fish taken from a lake in Newfoundland with a history of hydrocarbon pollution showed increased AHH activity (Payne and Penrose, 1975). Thus, many workers have studied the EROD activity of fish in laboratory conditions or naturally exposed samples as a molecular biomarker for aquatic pollution. These studies are reviewed by Whyte et al. (2000) and Goldfarb et al. (1998). In addition, EROD activity in carbofuran-treated fish has been measured in laboratory conditions. Carbofuran is a lipophilic PAH; therefore, it always accumulates in the lipid-lipid bilayer of the liver cell membrane (Gupta, 1994). Biophysical study of another pesticide, lindane, in relation to membrane partition has been performed by Madeira-Antues and Madeira (1985). This study suggests that lindane localizes in the hydrophobic part of the membrane phospholipid and decreases the midpoint temperature of the phase transition. It appeared that lindane tends to be excluded from the gel phase. The finding is reinforced by the fact that membrane cholesterol withdraws the lindane effectively from the membrane, and lindane thus takes a position in the region of the bilayer. The effects of the membrane fluidity and geometry have also drawn attention in the interpretation the cholesterol-related lindane withdrawal from the membrane. Such observations were made previously for parathion (Madeira-Antues and Madeira, 1984) and several other drugs. By virtue of its lipophilic nature, carbofuran accumulates in membrane lipid moieties en route to their target sites. Chatterjee and Ghosh (1995) showed that total cholesterol and phospholipid increased in the carbofuraninduced liver of fish compared to controls. In mice, treatment of Furadan (commercial name for carbofuran)
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increased total lipid, cholesterol (free and esterified), triglyceride, and phospholipid and its fraction (lecithin, lysolecithin, phosphatidylethanolamine, and lysophosphatidylethanolamine) in liver, kidney, and serum. The elevation of cholesterol by carbofuran treatment can be justified by the inhibition of Ca2+-ATPase activity. Warren e t a l . (1975) stated that phospholipid annulus surrounding the Ca-2+ATPase enzyme is necessary for its catalytic activity. Many workers, such as Hunter et al. (1990, 1999), supported this finding. Ghiasuddin et al. (1982) reported that Ca2+-ATPase activity could be restored in the nerve by the addition of phosphatidylcholine. Ghiasuddin et al. (1982) showed inhibition of membrane Ca2+-ATPase activity by the pesticide treatment. Thus, changes in cholesterol and phospholipid spectrum due to the involvement of pesticide in the membrane induced an impetus for the measurement of cholesterol and phospholipid in the carbofuran-treated hepatocyte membrane that can be correlated with Ca2+-ATPase activity of the membrane. This study may provide information about the retention and persistence of the pesticide in the lipid bilayer. Currently, heat shock protein (HSP) is used as a biomarker in various ecotoxicological studies. HSP is the family of proteins with varying molecular weights, such as Hsp28, Hsp70, and Hsp90, which are induced during thermal stress. HSP functions as armor, which safeguards the native structure of another protein during temperature stress on the cell. However, it is established that HSP may be induced by any other cellular stress and act as a molecular chaperone, which plays a critical role during the stress, and the native structure of the cellular protein is threatened. Pyza et al. (1997) studied Hsp70 in centipede L i t h o b i u s m u t a b i l e s exposed to the insecticide dimethoate. Bagchi et al. (1996) demonstrated the induction of HSP in vivo and in vitro by selected pesticides. Regulation of P4501A1 (CYP1A1) through protein kinase C (PKC) has been reported by Stephen et al. (1997) in lymphoid tissue. Reiners (1993) studied the regulation of CYP1A1 in lclc7 murine hepatoma cells through PKC, in which treatment of cathepsin, staurosporine, or H7, the PKC blockers, suppressed the induction of EROD in that cell line. Lee and Dasmahapatra (1993) performed a similar type of study in the hepatocyte culture of rainbow trout (0. mykiss) in which they showed the regulation of EROD activity by PKC. This study suggests that pretreatment of 12-O-tetradecanoynl phorbol 13-acetate, a PKC activator, to the culture hepatocytes inhibits EROD activity with a decreased CYP1A1 protein level. Pretreatment of the cell with the PKC inhibitor staurosporine effectively blocked the EROD activity. The study indicated the role of PKC in the modulation of the CYP1A1 gene in fish liver by [3-napthoflavone. All these results indicate a change in the phosphorylation state of the cell during induction of the CYP1A1 gene. The role of PKC in regulating CYP1A1
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gene induction in the cultured human hepatoma cell 101L derived from the HEPG2 cell line has been studied. It was clearly shown that PKC was essential for the cellular and molecular events that controlled the induction of CYP1A1 gene transcription. Formation of PKC is an essential step in the signaling cascade initiated by the change of minor lipid spectrum, such as phosphatidyl-inositol diphosphate (PIP2), followed by the formation of diacylglycerol to activate the PKC (Hannun et al., 1985; Bonser et al., 1988). This study of PKC, which is a ubiquitous part of membrane signaling during stress, indicates the requirement to study prior cascade phenomenon, such as diacylglycerol or PIP2 and membrane phospholipid status. All these studies can examine signaling initiated in the membrane and culminating in the cytosol modulating the expression of HSP as well as aromatic hydrocarbon receptor protein. This may identify the details of the response of a cell under stress by the pesticide or related compound. Ding etal. (1998) studied the role of PKC in the expression of Hsp70 and showed that overexpression of Hsp70 inhibits the phosphorylation of heat shock protein-1 (HSF1) by activating protein phosphatase and inhibiting protein kinase activity. Sakai et al. (1997) and Holmberg et al. (1997) studied the effect of inhibitor and activator of the PKC in response to HSP in cultured mammalian cell. There are no reports in the fish system in this regard. HSP has been linked to CYP1A1 gene regulation. The involvement of PKC in regulation of the CYP 1A1 gene, which has been the subject of study by many workers in the past few years, has been analyzed by Ding et al. (1998). The study indicated the downregulation of PKC by HSF1, which is a transcription factor for production of HSP70. The transcription factor (HSF1) requires phosphorylation for initiation of transcription, in which PKC plays an indispensable role. However, overexpression of HSF1 downregulates PKC, which in turn can modulate CYP1A1 expression (Ding et al., 1998; Holmberg et al., 1997). It is well demonstrated that worldwide discharge of metal into the aquatic ecosystem has an impact on cytochrome P450, which must be considered in the role of regulation of this important protein. Considerable attention has been given to glutathione, which plays an important role in modulating the catalytic activity of P450 enzymes such as EROD (Otto et al., 1996). Copper is one of the main components among the discharges of metal into the aquatic ecosystem (Gumgum et al., 1994).
XII. BIOMARKERS AND TOXICITY ASSESSMENT The presence of carbofuran and its major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) in food, water, and air may be considered as the source of contamination. The presence of carbofuran and its metabolites in the urine,
feces, and bile or any other body tissues can be considered as the most specific biomarker of recent or ongoing exposure. The excretion of 3-hydroxycarbofuran has been found to be more associated with carbofuran exposure than 3-ketocarbofuran in urine. Huang et al. (1989) reported that the level of 3-hydroxycarbofuran in patients with acute carbofuran poisoning may increase to 80.64 ppb. Monitoring of AChE in erythrocytes gives a good measure of the effect of carbofuran exposure (He, 1993). Erythrocyte membrane AChE is more sensitive than plasma AChE (Vandekar etal., 1971; Wills, 1972). Hussain et al. (1990) demonstrated significant inhibition of red blood cell AChE in farmers exposed to carbofuran. Plasma AChE was not affected. The recovery of CM AChE inhibition is much faster than that of OP exposure because carbamylation of the enzyme is easily and rapidly reversed (Coye et al., 1986). AChE should be determined when assessing CM exposure. Blood samples in such cases should be drawn within 4 hr of exposure to CM (World Health Organization Task Group, 1986). This critical inhibition of AChE in blood, brain, and muscle in humans, animals, birds, or fish is indicative of poisoning due to either CM or OP. Cholinergic symptoms are usually associated in CM-exposed workers with a blood AChE lower than 70% of the individual's baseline level (Huang etal., 1989; He, 1993). Analysis of serum enzymes (creatine kinase, lactate dehydrogenase, and others) and their isozymes and subforms provides additional information on tissue-specific damage (Gupta etal., 1991 a,b, 1994), and these can be considered as markers of the effects.
XIII. EXPERIMENTS USING FISH HEPATOCYTES IN C U L T U R E Some interesting observations were noticed in the author's laboratory. Although carbofuran was present in all cellular compartments, it was highest in the membrane and lowest in the nucleus of the singi fish (Hetropneustes fossilis Bloch) hepatocytes in culture. Metabolites of carbofuran, such as 3-ketocarbofuran and 3-hydroxycarbofuran, also showed a qualitatively similar distribution as carbofuran. Hepatocytes incubated in the carbofuran-containing medium at different concentrations caused dose-dependent inhibition of Ca2+-ATPase activity in comparison to the untreated control. During carbofuran treatment, a higher amount of cholesterol was found in hepatocyte membranes. Indirectly, insertion of cholesterol to normal hepatocyte membranes also showed inhibition in Ca2+-ATPase activity depending on the amount of cholesterol inserted. Such effect has been studied by measuring EROD activity, a marker enzyme of CYP1A1, in different conditions of carbofuran treatment. Incubation of hepatocytes in different doses of carbofuran for 24 hr showed a dose-dependent increase in EROD
CHAPTER 45
activity, with the highest activity observed with 1 ~zM concentration of carbofuran in the medium. [3-Napthoflavone (BNF), a known inducer of CYP1A, also demonstrated a dose-dependent increase in EROD activity, ot-Napthoflavone (ANF), an effective blocker of the Ah receptor, demonstrated a dose-dependent inhibition of EROD activity in the hepatocytes maintained for 24 hr in ANF-containing medium. The carbofuran- and BNF-induced EROD activity in the cultured hepatocytes was counteracted by actinomycin-D and cycloheximide when either of the compounds was administered simultaneously with carbofuran or BNF in the medium. Interestingly, the same compounds showed a superinduction of EROD activity when added to hepatocytes pretreated with carbofuran or BNF for 12 hr. Phosphatidyl-inositol phosphate and PIP2 showed significant increases in carbofuran-treated hepatocytes in comparison to DMSO-treated control. Equimolar amounts of carbofuran or BNF used in the separate culture significantly increased the hepatocyte membrane PKC activity compared to the control. Use of staurosporine, a known inhibitor of PKC, counteracted the carbofuran- or BNF-induced increase in PKC activity. Interestingly, phorbol ester demonstrated an attenuation of the carbofuran- or BNF-induced increase in PKC activity, but it remained higher than the control value. Because PKC activity is related to the formation of HSP, this relationship has been examined in a hepatocyte culture system. Carbofuran or BNF increased the HSP70 content 8- to 11-fold higher than the control. Addition of Staurosporine (ST) or 12-Phorbol,13-myristate acetate (PE) to carbofuran or B NF attenuated the carbofuran- or B NFinduced increase in Hsp70 but maintained its level higher than that of the DMSO-treated control. Similar results were obtained for the EROD activity of the hepatocytes in the previously mentioned treatment conditions (Ghosh et al., 2000). Furthermore, it was demonstrated that carbofuran and BNF elevated the glutathione (GSH) level and EROD activity in hepatocytes in comparison to the control. Equimolar concentrations of carbofuran + BNF demonstrated a synergistic elevation of GSH level in the culture hepatocytes. Addition of Cu 2+ with carbofuran or BNF caused a decrease in GSH level and EROD activity (Ghosh et al., 2001). The catfish hepatic microsomal protein that contained EROD activity was purified up to approximately 82-fold and its authenticity was checked by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, Western blot, dot blot, capillary electrophoresis, and ELISA. Analysis of circular dichroism (CD) indicated functional aspects of oL and [3 helices in relation to the EROD activity of the purified protein (Ghosh et al., 2001). The study of the Indian catfish, Singhi, clearly indicated the impact of carbofuran in fish and evolutionary conservation of the detoxification system involving cytochrome P450. Further studies using OPs should be performed, which may show the major and minor differences in the
9Aquatic Toxicity of CMs and OPs
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molecular pathways of metabolism and detoxification in different organs in fish of the tropical and temperate zones.
References Abdollahi, M., Ranjbar, A., Shadnia, S., Nikfar, S., and Rezaie, A. (2004). Pesticide and oxidative stress. Med. Sci. Monit. 10, 141-147. Achazi, R. K., Chroscz, G., Heimig, E., Neunaber, R., and Steudel, I. (1994). A high molecular regulator of 7-ethoxyresorufino-deethylase activity in fish. Comp. Biochem. Physiol. C 108, 243-256. Aldridge, W. N. (1971). The nature and reaction of organophosphorus compounds and carbamates with esterases. Bull. WHO 44, 25-30. Anton, E A., Laborda, E., Laborda, E, and Ramos, E. (1993). Carbofuran acute toxicity to fresh water algae and fish. Bull. Environ. Contam. Toxicol. 30(3), 400-406. Bagchi, D., Bhattacharya, G., and Stohs, S. J. (1996). In vitro and in vivo induction of heat shock (stress) protein (HSP) gene expression by selected pesticides. Toxicology 112(1), 57-68. Bailey, H. C., and Liu, D. H. E. (1980). Lumbriculus veriegatus, a benthic oligochaete, as bioassay organism. In Aquatic Toxicology (J. C. Eaton, E R. Parrish, and A. C. Hendrick, Eds.), Special Technical Publication No. 707. pp. 205-215. ASTM, Philadelphia. Behrens, A., Sehirmer, K., Bols, N. C., and Segner, H. (1998). Microassay for rapid measurement of 7-ethoxyresrufin-odeethylase activity in intact fish hepatocytes. Mar. Environ. Res. 46, 369-373. Ben-Dyke, R., Sanderson, D. M., and Noakes D. N. (1970). Acute toxicity data for pesticides. World. Rev. Pest Control 9, 119-127. Beyer, J., Sandvik, M., Skare, J. U., et al. (1997). Time and dose dependent biomarker responses in flounder (Platichthys flesus L) exposed to benzo[a]pyren, 2,3,3,4,4,5-hexachlorobiphenyl (PCB-256) and cadmium. Biomarkers 2, 35-44. Bhakthavathasalam, R. (1991). Hematology of the fish Anabas testiduneus exposed to lindane and carbofuran at submerged condition and on exposure to air. Environ. Ecol. 9(1), 124-127. Bhattacharyya, S. (1985). Toxicity of carbofuran and phenthoate in Channa punctatus and Anabus testudineus. J. Environ. Biol. 6(2), 129-137. Bonser, R. W., Thompson, N. T., Hodson, H. E, Beams, R. M., and Garland, L. G. (1988). Evidence that a second stereochemical centre in diacylglycerol defines interaction at the recognition site on protein kinase C. FEBS Lett. 234, 341-344. Bretaud, S., Toutant, J. E, and Saglio, E (2000). Effects of carbofuran, diuron and nicosulfuron on acetylcholinestarase activity in goldfish (Carassius auratus). Ecotoxicol. Environ. Safety. 47(2), 117-124. Brown, L. M., Dosemeci, M., Blair, A., and Burnmeister, L. (1991). Comparability of data obtained from farmers and surrogate respondents on use of agricultural pesticides. Am. J. Epidemiol. 134, 348-355. Bruce, W. (1972). Separation and identification of carbofuran, its metabolites and conjugates found in fish exposed to ring 14C-labeled carbofuran using TLC silica gel strips. J. Econ. Entomol. 65, 1003-1009.
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Sakai, K., Suzuki, N., Itoh, H., and Kubodera, A. (1997). Effects of an inhibitor of protein kinase on the response to heat treatment in cultured mammalian cells. Int. J. Hyperthermia 13(5), 535-545. Sarasquete, C., and Segner, H. (2000). Cytochrome P4501A (CYP1A) in teleostean fishes. A review of immunohistochemical studies. Sci. Total Environ. 247, 313-332. Sarasquete, C., Munoz-Cueto, J.A., Ortiz, J. B., RodriguezGomez, E J., Dinis, M. T., and Segner, H. (1999). Immunocytochemical distribution of cytochrome P4501A (CYP1A) in developing gilthead seabream, Sparus aurata. Histol. Histopathol. 14, 407-415. Saxena, P. K., and Mani, K. (1987). Effect of safe concentrations of some pesticides on testicular recrudescence in the fresh water murrel, Channa punctatus (B1). A morphological study. Ecotoxicol. Environ. Safety 14(1), 56-63. Scholz, S., Behin, I., Honeck, H., Hauck, C., Braunbeck, T., and Segner, H. (1997). Development of a monoclonal antibody for ELISA of CYP1A in primary cultures of rainbow trout (Onchorynchus mykiss) hepatocytes. Biomarkers 2, 287-294. Scott, G. R., and Sloman, K. A. (2004). The effects of environmental pollutants on complex fish behaviour: Integrating behavioural and physiological indicators of toxicity. Aquat. Toxicol. 68, 369-392. Segner, H., and Braunbeck, T. (1998). Cellular response profile to chemical stress. In Ecotoxicology (G. Sehuuramanna and B. Markert, Eds.), pp. 521-569. Wiley/Spektrums, New York Heidelberg. Singh, B., and Garg, A. K. (1992). Observation on the accumulation of carbon 14-labeled carbofuran in different organs of fish. Natl. Acad. Sci. Lett. (India) 15(10), 343-354. Smolowitz, R. M., Hahn, M. E., and Stegeman, J. J. (1991). Immunohistochemical histochemical localization of cytochrome P450 1A1 by 3,3,4,4-tetrachloro biphenyl and by 2,3,7,8-tetrachlorodibenzofuran in liver and extrahepatic tissues of the teleost Stenotomus chrysops (scup). Drug Metab. Disp. 19, 113-123. Soreq, H., and Zakut, M. (1989). Human Cholinesterase and Anticholinesterase, pp. 26-33. Academic Press, San Diego. Soreq, H., and Zakut, H. (1993). Human Cholinesterases and Anticholinesterases. Academic Press, San Diego. Stegeman, J. J. (1989). Cytochrome P450 forms in fish: Catalytic, immunological and sequence similarities. Xenobiotica 19(10), 1093-1110. Stegeman, J. J., and Hahn, M. E. (1994). Biochemistry and molecular biology of monooxygenases" Current perspectives on forms, functions and regulation of cytochrome P450 in aquatic species. In Aquatic Toxicology (D. C. Malins and G. K. Ostrander, Eds.), pp. 87-203. Lewis, Boca Raton, FL. Stegeman, J. J., and Kloepper-Sams, P. J. (1987). Cytochrome P450 isoenzymes and mooxygenase activity in aquatic animals. Environ. Health Perspect. 71, 87-95. Stegeman, J. J., Woodin, B. R., and Smolowitz, R. M. (1990). Structure, function and regulation of cytochrome P450 forms in fish. Biochem. Soc. Trans. 18, 19-21. Stephen, E D., Drahushuk, A. T., and Olson, J. R. (1997). Cytochrome P4501A induction in rat lymphoid tissues following in vivo and in vitro exposure to 2,3,7,8-tetrachlorodibenzop-dioxin requires protein kinase C. Toxicology 124(1), 39-51.
672
SECTION V l l . A q u a t i c
Life & Wildlife
Sukumar, A., and Karpagaganapathy, R R. (1992). Pesticide induced atresia in ovary of fresh water fish, Colisa lalia (HamiltonBuchanoa). Bull Environ. Contam. Toxicol. 88(3), 457-462. Swartz, R. C., DeBeb, W. A., Sercu, K. A., and Lamberson, J. O. (1982). Sediment toxicity and the distribution of amphipods in Commencement Bay. Pollut. Bull. 13, 359-364. Tobin, J. E (1970). Carbofuran. A new carbamate insecticide. J. Occup. Med. 12, 16-19. Uang, Y. E, and Uang, T. H. (1995). Induction and purification of cytochrome P4501A from 3-methylcholanthrene-treated tilapia, Oreochromis niloticus and O. aureus. Arch. Biochem. Biophys. 322, 347-356. U.S. Environmental Protection Agency (1991). Environmental FactSheets for Carbofuran. U.S. Environmental Protection Agency, Washington, DC. Vandekar, M., Pleastina, R., and Wilhem, K. (1971). Toxicity of carbamate for mammals. WHO Bull. 44, 241-249. Van Veld, E A., Vogelbein, W. K., Cochran, M. K., Goksoyr, A., and Stegeman, J. J. (1997). Route specific cellular expression of cytochrome P4501A (CYPIA) in fish (Fundulus heteroclirus) following exposure of aqueous and dietary benzo[a]pyrene. Toxicol. Appl. Pharmacol. 142, 348-359. Vasanthi, R., Baskaran, E, and Palanichamy, S. (1990). Influence of carbofuran on growth and protein conversion efficiency in some fresh water fish. J. Ecobiol. 2(1), 85-88. Warren, G. B., Houslay, M. D., and Metcalfe, J. C. (1975). Cholesterol is excluded from phospholipid annulus surrounding an active calcium transport protein. Nature 255, 684-687. Whyte, J. J., Jung, R. E., Schmitt, C. J., and Tillitt, D. E. (2000). Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit. Rev. Toxicol. 30(4), 347-350.
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46
CHAPTER
Toxicity of O r g a n o p h o s p h o r u s and Carbamate Insecticides Using Birds as Sentinels for Terrestrial Vertebrate Wildlife SPENCER R. MORTENSEN Syngenta Crop Protection, Greensboro, North Carolina
I. I N T R O D U C T I O N an emphasis on those that have a U.S. registration. Two methods of estimating the relative toxicity of each compound to birds are also given: the HD5 method (Mineau et al., 2001), which is based on the avian acute oral toxicity test, and the U.S. Environmental Protection Agency's (EPA) category of toxicity to birds, which is also based on the avian acute oral toxicity test (EPA, 1994). Additionally, the number of avian incidents in the United States from 1968 to 2004 is provided where available, and a ranking of the top 10 OPs used in the United States in 2001 is given.
Although the use and number of registered organophosphorus (OP) and likely carbamate (CM) insecticides has steadily decreased during the past two decades (Fig. 1), the importance of these broad-spectrum classes of chemistry for pest control and integrated pest management programs remains unchanged. There are currently more than 40 OP and CM active ingredients and hundreds of formulations registered in the United States (Table 1). The purpose of this chapter is to summarize the OP and CM insecticides that are registered in the world, with
250
* u
A
y =-5.9138x + 11910 R2=0.8
OP Use (MM Ibs ai)
m Total Insecticide Use (MM Ibs ai)
200
. n
t~ o
~~ o
150
*-.....
~
--- 100
-
Z
y =-3.4248x + 6908.4 R2 = 0.9385 50-
0
1978
19'80
19'82
19'84
19'86
19'88
19'90
19'92
19'94
19'96
19'98
20'00
Year
FIG. 1. Decline in OP and total insecticide use in the United States from 1978 to 1998. Data from Kiely et al., (2004).
Toxicology of Organophosphate and Carbamate Compounds
673
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
674
SECTION V l l .
II. S U M M A R Y TABLE 1.
A q u a t i c Life & W i l d l i f e
DATA
Avian Toxicity and Incident Data for OP and CM Insecticides Registered in the United States and Other Areas of the World*
HD5 a
U.S. EPA category of toxicity b
Current U.S. registration? c
OP
18.52
3
Yes
OP
2.28
2
Yes
1
2921-88-2
OP
3.76
2
Yes
70
Chlorpyrifos-methyl
5598-13-0
OP
25.32
4
Yes
NRf
Coumaphos
56-72-4
OP
0.69
1
Yes
1
Diazinon
333-41-5
OP
0.59
1
Yes
390
Dichlorvos (DDVP)
62-73-7
OP
0.61
2
Yes
2
Dicrotophos
141-66-2
OP
0.42
1
Yes
1
Dimethoate
60-51-5
OP
5.78
2
Yes
8
Disulfoton
298-04-4
OP
0.81
2
Yes
NR
Ethion
563-12- 2
OP
1.06
3
Yes
1
Ethoprop
13194-48-4
OP
2.41
1
Yes
2
Fenamiphos
22224-92-6
OP
0.43
1
Yes
8
Fenitrothion
122-14-5
OP
3.37
3
Yes
NR
Fenthion
55-38-9
OP
0.87
1
Yes
54
Isofenphos
25311-71-1
OP
0.44
2
Yes
10
Malathion
121-75-5
OP
139.10
3
Yes
NR
Methamidophos
10265-92-6
OP
1.70
2
Yes
3
Methidathion
950-37-8
OP
3.53
2
Yes
NR
Methyl parathion
298-00-0
OP
2.13
2
Yes
2
Naled
300-76-5
OP
1.72
3
Yes
1
Oxydemeton-methyl
301 - 12-2
OP
13.96
3
Yes
1
Parathion
56-38-2
OP
0.40
1
Yes
46
Compound
CAS No.
OP or CM
Acephate
30560-19-1
Azinphos-methyl
86-50-0
Chlorpyrifos
No. of U.S. incidents d
Use rank, 2001 e
1
3
10
1
7
Phorate
298-02-2
OP
0.34
1
Yes
21
6
Phosmet
732-11-6
OP
1.24
3
Yes
NR
8
Pirimiphos-methyl
29232-93-7
OP
13.51
3
Yes
NR
Profenofos
41198-08-7
OP
NDg
3
Yes
NR
Propetamphos
31218-83-4
OP
7.09
3
Yes
NR
Tebupirimfos
96182-53-3
OP
2.36
2
Yes
NR
Temephos
3383-96-8
OP
8.68
3
Yes
NR
Terbufos
13071-79-9
OP
0.16
1
Yes
5
Tetrachlorvinphos
961-11-5
OP
25.32
5
Yes
1
Trichlorfon
52-68-6
OP
13.36
3
Yes
2
Aldicarb
116-06-3
CM
0.43
1
Yes
12
Aldoxycarb
1646-88-4
CM
ND
2
Yes
NR
Carbaryl
63-25-2
CM
30.05
4
Yes
6
Carbofuran
1563-66-2
CM
0.21
1
Yes
345
Methiocarb
2032-65- 7
CM
1.06
1
Yes
NR
Methomyl
16752-77-5
CM
8.46
2
Yes
2
Oxamyl
23135-22-0
CM
0.78
1
Yes
NR
Pirimicarb
23103-98-2
CM
6.78
2
Yes
NR
Propoxur
114-26-1
CM
1.31
2
Yes
3
Thiodicarb
59669-26-0
CM
234.96
5
Yes
NR
4
(continues)
CHAPTER 46
TABLE 1.
Compound
CAS No.
9Wildlife Toxicity of OPs and CMs
(continued)
OP or CM
HD5 a
U.S. EPA category of toxicity b
Current U.S. registration? c
No. of U.S. incidents d
Amidithion
919-76-6
OP
ND
ND
No
NR
Anilofos
64249-01-0
OP
ND
4
No
NR
Azamethiphos
35575-96-3
OP
3.98
2
No
NR
Azinphos-ethyl
2642-71-9
OP
1.53
1
No
NR
122-10-1
OP
0.25
1
No
NR
Bromophos
2104-96-3
OP
491.14
5
No
NR
Bromophos-ethyl
4824-78-6
OP
12.88
3
No
NR
Butonate
126-22-7
OP
40.00
3
No
NR
Bomyl
Cadusafos
95465-99-9
OP
6.33
3
No
NR
Carbophenothion
786-19-6
OP
2.00
3
No
NR
Chlorethoxyfos (Fortress)
54593-83-8
OP
3.25
2
No
NR
Chlorfenvinphos
470-9O-6
OP
2.73
2
No
NR
Chlormephos
24934-91-6
OP
25.10
3
No
NR
Chlorphoxim
14816-20-7
OP
11.61
3
No
NR
Chlorthiophos
60238-56-4
OP
ND
ND
No
NR
Crotoxyphos
7700-17-6
OP
14.23
3
No
NR
Cyanophos
2636-26-2
OP
0.83
ND
No
NR
Cyanthoate
3734-95-0
OP
ND
ND
No
NR
DAEP
13265-60-6
OP
ND
ND
No
NR
Demephion
8065-62-1
OP
ND
ND
No
NR
1
No
NR
Demeton
8065-48-3
OP
Demeton-methyl
867-27-6
OP
Dialifos
10311-84-9
OP
Diamidafos
1754-58-1
OP
1.04
7.24
2
No
NR
4
No
NR
3.37
2
No
NR
4.13
ND
No
NR
ND
Dicapthon
2463-84-5
OP
Dichlofenthion
97.17-6
OP
3
No
NR
Dioxabenzofos
3811-49-2
OP
ND
ND
No
NR
Dioxathion
78-34-2
OP
25.50
3
No
NR
ND
No
NR
7.54
DMCP
3309-87-3
OP
ND
Endothion
2778-04-3
OP
ND
ND
No
NR
EPBP
3792-59-4
OP
ND
ND
No
NR
EPN
2104-64-5
OP
1
No
NR
ND
No
NR NR
0.53
Ethoate-methyl
116-01-8
OP
ND
Etrimfos
38260-54-7
OP
23.65
ND
No
Famphur
52-85-7
OP
0.45
1
No
33
Fenchlorphos (Ronnel)
299-84-3
OP
12.23
3
No
NR
Fensulfothion
115-90-2
OP
0.13
1
No
5
3.86
2
No
NR
'
Fonofos
944-22-9
OP
Formothion
2540-82-1
OP
ND
3
No
NR
Fosmethilan
83733-82-8
OP
ND
ND
No
NR
Fosthiazate
98886-44-3
OP
1.47
2
No
NR
3.04
3
No
NR
ND
No
NR
2
No
1
23560-59-0
OP
IPSP
5827-05-4
OP
Isazofos
42509-80-8
OP
Heptenophos
ND 0.51
675
Use rank, 2001 e
(continues)
676
S E C T I O N V l l . A q u a t i c Life & W i l d l i f e
TABLE 1.
Compound
CAS No.
OP or CM
Isocarbophos
24353-61-5
OP
Isothioate
36614-38-7
OP
HD5 a
0.26
(continued) U.S. EPA category of toxicity b
Current U.S. registration? c
No. of U.S. incidents d
1
No
NR
ND
ND
No
NR
Isoxathion
18854-01-8
OP
ND
ND
No
NR
Jodfenphos
18181-70-9
OP
ND
ND
No
NR
Leptophos
21609-90-5
OP
3
No
NR
Kayaphos
7292-16-2
OP
ND
ND
No
NR
Mazidox
7219-78-5
OP
ND
ND
No
NR
Mecarbam
2595-54-2
OP
ND
ND
No
NR
Mephosfolan (Cytrolane)
950-10-7
OP
1
No
NR
Mevinphos
7786-34-7
OP
0.70
1
No
Monocrotophos
6923-22-4
OP
0.42
1
No
3 4
ND
No
NR
2
0.09
0.14
Morphothion
144-41-2
OP
Omethoate
1113-02-6
OP
No
NR
Oxydeprofos
2674-91-1
OP
ND
ND
No
NR
Oxydisulfoton
2497-07-6
OP
ND
ND
No
NR
Phenkapton
2275-14-1
OP
ND
ND
No
NR
Phenthoate
2597-7-3
OP
23.17
3
No
NR
Phosalone
2310-17-0
OP
106.27
5
No
NR
Phosfolan
947-02-4
OP
0.69
1
No
NR
Phosphamidon
297-99-4
OP
1.08
1
No
1
Phoxim
14816-18-3
OP
1.71
2
No
NR
Pirimiphos-ethyl
23505-41-1
OP
1.90
1
No
NR
Propaphos
7292-16-2
OP
0.18
1
No
NR
Prothidathion
20276-83-9
OP
ND
ND
No
NR NR
ND 4.14
Prothiofos
34643-46-4
OP
13.65
3
No
Prothoate
2275-18-5
OP
5.52
3
No
NR
Pyraclofos
77458-01-6
OP
ND
No
NR
No
NR
ND
Pyridaphenthion
119-12-0
OP
7.94
3
Quinalphos
13593-03-8
OP
0.42
2
No
NR
Schradan
152-16-9
OP
2.02
2
No
NR
Sulfotep
3689-24-5 ~
OP
50.63
3
No
NR
Sulprofos
35400-43-2
OP
6.85
2
No
NR
TEPP
107-49-3
OP
Thiometon
640-15-3
OP
ND 5.07
1
No
NR
3
No
NR
1
No
NR
ND
No
NR
Thionazin
297-97-2
OP
Triamiphos
1031-47-6
OP
Triazophos
24017-47-8
OP
1.68
1
No
NR
Trichloronat
327-98-0
OP
0.73
2
No
NR
2
No
NR
ND
No
NR
1.02 ND
Vamidothion
2275-23-2
OP
Alanycarb
83130-01-2
CM
Allyxycarb
6392-46-7
CM
3.37
2
No
NR
Aminocarb
2032-59-9
CM
6.59
2
No
NR
3.72 ND
Bendiocarb
22781-23-3
CM
0.72
2
No
11
Benfuracarb
82560-54-1
CM
4.23
3
No
NR
Bufencarb
8065-36-9
CM
3.09
2
No
NR
Butacarb
2655-19-8
CM
ND
No
NR
ND
Use rank, 2001 e
(continues)
CHAPTER 4 6
TABLE 1.
Compound
9Wildlife Toxicity of OPs and CMs
(continued)
U.S. EPA category of toxicity b
Current U.S. registration? c
No. of U.S. incidents d
6.17
3
No
NR
18.58
3
No
NR
9.52
3
No
NR
CM
0.43
ND
No
NR
CM
25.32
3
No
NR
87130-20-9
CM
234.13
5
No
NR
644-64-4
CM
0.92
2
No
NR NR
CAS No.
OP or CM
Butocarboxim
34681-10-2
CM
Butoxycarboxim
34681-23-7
CM
Carbosulfan
55285-14-8
CM
Cloethocarb
51487-69-5
Crufomate
299-86- 5
Diethofencarb Dimetilan
HD5 a
677
Dioxicarb
6988-21-2
CM
3.36
3
No
Ethiofencarb
29973-13-5
CM
14.96
1
No
NR
Fenobucarb
3766-81-2
CM
31.12
3
No
NR
Formetanate
22259- 30-9
CM
8.77
2
No
NR
Furathiocarb
65907-30-4
CM
2.41
2
No
NR
Isoprocarb
2631-40-5
CM
14.23
ND
No
NR
Metolcarb
1129-41-5
CM
ND
ND
No
NR
Mexacarbate
315-18-4
CM
1.39
1
No
NR
Promecarb
2631-37-0
CM
0.94
2
No
NR
Thiofanox
39196-18-4
CM
0.12
1
No
NR
Triazamate
112143-82-5
CM
0.93
1
No
NR
Trimethacarb XMC
12407-86-2 2655-14-3
CM CM
16.28 ND
3 ND
No No
NR NR
Xylylcarb
2425-10-7
CM
6.20
ND
No
NR
Use rank, 2001 e
*The views expressed are those of the author and do not necessarily reflect the views of syngenta Crop Protection, Inc. aSee Mineau et al. (2001) for details. bCategories 1-5 represent a median LDs0 value (from Mineau et al., 2001) that is < 10, 10-50, >50-500, > 500-2000, or >2000 mg ai/kg body weight, respectively (see EPA, 1994). CThe following sources were used to determine the current U.S. registration status of each OP and CM: EPA/OPP Pesticide-Related Database Queries (www.cdpr.ca.gov/docs/epa/epamenu.htm), Tomlin (2000), and the Crop Protection Handbook (2004). aNumber of U.S. incidents in the AIMS database (www.abcbirds.org/aims) as of April 2005. eTop 10 most commonlyused OP insecticide active ingredients in all market sectors of the United States in 2001 (Kiely et al., 2004). fNR, not reported. gND, not determined.
III. DATA EVALUATION Several interesting observations can be made from Table 1. First, there are an estimated 116 OP and 38 CM insecticides used worldwide. There are also an estimated 33 OPs and 10 CMs registered in the United States. Second, not surprisingly, there appears to be a negative relationship between HD5 or categorization of toxicity and avian incidents, which suggests that the more toxic OPs and CMs are responsible for the majority of the avian incidents. This is in agreement with an earlier review of bird-kill incidents (Grue et al., 1983) that indicated that most incidents could be explained by pesticide toxicity and use. Third, approximately 60% of the avian incidents involving OPs and > 9 0 % of the avian incidents involving CMs in the United States (reported in the AIMS database by April 2005) were
associated with diazinon and carbofuran, respectively. A word of caution is in order here. It would be easy to jump to the conclusion that simply removing diazinon and carbofuran from the market would necessarily reduce the number of avian incidents. However, the records in the AIMS database are a compilation of incidents from 1968 to 2004. Many of the incidents occurred prior to current restrictions for these products (i.e., restrictions to limit application rates, the types of crops on which the products can be applied, and market sectors). It will be interesting to determine if the same trend continues after all of the restrictions are in force. Also, it is important to perform a risk-risk analysis (i.e., weigh the risk of using a particular product against the risk of not having a particular product available) prior to making decisions regarding the cancellation of a pesticide. In some cases, the risk of having a more toxic
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TABLE 2. Comparison of the Toxicity of Methyl and Ethyl Analogs of the OP Insecticides
Compound Azinphos-methyl Azinphos-ethyl Bromophos Bromophos-ethyl Methyl chlorpyrifos Chlorpyrifos Demeton-methyl Demeton Pirimiphos-methyl Pirimiphos
HD5
Toxicity category
2.28 1.53 491.14 12.88 25.32 3.76 7.24 1.04 13.51 1.90
2 1 5 3 4 2 2 1 3 1
alternative outweighs the risk of not having it. This risk-risk analysis could vary from country to country and even state to state. Fourth, it is interesting to note that the methyl analogs of the OPs are less toxic to birds than the ethyl analogs, as shown in Table 2. The data in Table 2 agree well with results from an experiment by Clothier et al. (1981), who showed that bovine red blood cell AChE inhibited with dimethoxysubstituted compounds spontaneously reactivated and aged more rapidly than AChE inhibited by the diethoxysubstituted compounds. Importantly, however, the rate of spontaneous reactivation of the dimethoxy-substituted compounds was approximately an order of magnitude more rapid than the rate of aging.
IV. F I N A L T H O U G H T S Perhaps Ecobichon (1991) described the paradox of pesticide use best: "All pesticides possess an inherent degree of toxicity to some living organism; otherwise they would be of no practical use" and "There is no such thing as a 'completely safe' pesticide." Pesticides are unique among chemicals in that they are registered specifically to kill their intended target. The question is how can we most effectively reduce nontarget incidents? There are times when the correct choice is to discontinue the use of a pesticide, as was the case with the use of monocrotophos in Argentina (Hooper et al., 2003). This was a very focused and collaborative effort that involved a number of government agencies, university personnel, industry, nongovernmental
organizations, and many others. Perhaps the easiest and most effective first step that we as individuals can take is to read and follow pesticide labels because many of the avian incidents are a direct result of product misuse.
Acknowledgments The author wishes to thanks Dr. Joh Akins and Jeff Stabnau for their help finding referenced materials and Alan Hosmer for his review of an earlier version of this chapter.
References Clothier, B., Johnson, M. K., and Reiner, E. (1981). Interaction of some trialkyl phosphorothiolates with acetylcholinesterase: Charaterization of inhibition, aging, and reactivation. Biochem. Biophys. Acta 660, 306-316. Crop Protection Handbook, Vol. 90. (2004). Meister Media Worldwide, Willoughby, OH. Ecobichon, D. J. (1991). Toxic effects of pesticides. In Casarett and Doull's Toxicolgy: The Basic Science of Poisons (M. O. Amdur, J. Doull, and C. D. Klaassen, Eds.), 4th ed., pp. 565-622. Pergamon, New York. Grue, C. E., Flemming, W. J., Busby, D. G., and Hill, E. E (1983). Assessing hazards of organophosphate pesticides to wildlife. Trans. N. Am. Wildl. Nat. Res. Conf. 48, 200-220. Hooper, M. J., Mineau, P., Zaccagnini, M. E., and Woodbridge, B. (2003). Pesticides in international migratory bird conservation. In Handbook of Ecotoxicology (D. J. Hoffman, B. A. Rattner, G. A. Burton, Jr., and J. Cairns, Jr., Eds.), 2nd ed., pp. 737-754. CRC Press, Boca Raton, FL. Kiely, T., Donaldson, D., and Grube, A. (2004). Pesticides Industry Sales and Usage: 2000 and 2001 Market Estimates. Biological and Economic Analysis Division, Office of Pesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. Mineau, P., Jobin, B., and Baril, A. (1994). A critique of the avian 5-day dietary test (LCs0) as the basis of the avian risk assessment, Technical Report No. 215. Canadian Wildlife Service Headquarters, Hull, Quebec, Canada. Mineau, P., Baril, A., Collins, B. T., Duffe, J., Joerman, G., and Luttik, R. (2001). Pesticide acute toxicity reference values for birds. Rev. Environ. Contam. Toxicol. 170, 13-74. Smith, G. J. (1987). Pesticide Use and Toxicology in Relation to Wildlife: Organophosphorus and Carbamate Compounds, Resource Publication 170. U.S. Department of the Interior, Fish and Wildlife Service, Washington, DC. Tomlin, C. D. S. (2000). The Pesticide Manual 12th ed. British Crop Protection Council, Farnham, Surrey, UK. U.S. Environmental Protection Agency (1994). Pesticide reregistration rejection rate analysis: Ecological effects, EPA 738-R-94-035. U.S. Environmental Protection Agency, Washington, DC.
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CHAPTER 4
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Analysis of Organophosphate and Carbamate Pesticides and Anticholinesterase Therapeutic Agents ANANT V. lAIN University of Georgia, Athens, Georgia
of the sample extract, (iii) concentration of purified extract, (iv) separation from impurities and detection, and (v) confirmation. In this chapter, the various steps are discussed briefly, and recent trends for each step are discussed.
I. I N T R O D U C T I O N Organophosphate (OP) and carbamate (CM) pesticides are an integral part of modern agriculture. The publication of Silent Spring by Rachel Carson led to the withdrawal of many organochlorine (OC) pesticides due to their persistence in the environment. At this time, the use of OPs and CMs soared due to their availability and quick degradation in the environment. Although OPs and CMs degrade quickly and are much less persistent in the environment, they are much more toxic to mammals than OC pesticides. Because of their adverse health affects, various governmental agencies set limits on allowable levels of pesticide residues in foods, animal feeds, and the environment. In order to enforce these allowable levels, pesticides are monitored in various types of samples. Also, humans and animals may be poisoned accidentally or maliciously. Thus, there is a need for analytical methods to determine OPs and CMs in food and feedstuffs as well as biological specimens. By nature, analytical results are variable. The science of trace analysis (analysis at parts per million or below levels) is not as precise as most layman and many scientists view it to be (Rogers, 1986). Once devised, analytical methods are like life-forms, subject to evolution. Natural selection is mediated by analytical chemists, which ensures that only the fittest analytical methods survive. Therefore, an analytical method must be fit for purpose. In order to be able to determine trace levels of OPs and CMs in the environment and biological, food, and feed samples, it is necessary to follow a series of operations. Note that most of the advancements and improvements in the analytical methods have occurred by making necessary changes and improvements in various steps used in analytical methods. The papers by Sawyer (1988) and Seiber (1988) clearly demonstrate this point. The various steps in an analytical method are (i) extraction of the sample, (ii) cleanup and purification Toxicology of Organophosphate and Carbamate Compounds
II. S A M P L E E X T R A C T I O N It is essential to separate the target pesticide(s) from the sample matrix. Traditionally, this has been done by blending a homogeneous ground sample with an organic solvent. Since OPs and CMs are nonpolar to polar compounds, the polarity of extraction solvents varies accordingly. There are three general methods for extracting pesticides from solids with organic solvents: soxhlet extraction, homogenization with a solvent, and ultrasonication of the ground sample with an organic solvent. Homogenization is the preferred method for the extraction of pesticides with solvents in the methods described in the Pesticide Analytical Manual (PAM) of the U.S. Food and Drug Administration (FDA, 1994). Handling of the dry samples usually requires the addition of water to moisten the sample, along with the organic solvents. Sonication of the blended mixture expedites the extraction of target compounds. However, it is not recommended for OPs [SW-846 method 8141A; U.S. Environmental Protection Agency (EPA, 1994)]. Acetone, methylene chloride, hexane, petroleum ether, acetonitrile, and methanol are some of the solvents used for extraction of pesticides depending on the sample matrix and the nature of the pesticide(s). Liquid samples are extracted directly from the sample with organic solvents. Water samples are extracted with organic solvents; these methods use solvents that are immiscible with the sample phase. Ideally, one selectively extracts the target compounds by using a solvent whose polarity is similar to that of the target compound. Volatile 681
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solvents, such as ether, ethyl acetate, and methylene chloride, are usually used for the extractions of OPs and CMs. For air samples, a certain amount of air is pumped through a trap tube (OSV-2) that contains an adsorbent such as XAD-2, which efficiently traps OPs and CMs, and then the trap is eluted with organic solvents in the case of OPs and buffer in the case of CMs [National Institute for Occupational Safety and Health (NIOSH), 1994].
A. Current Trends in Sample Extraction Current trends have been to accelerate the extraction of pesticides from the sample matrix by accelerated solvent extraction (ASE) or pressurized solvent extraction (PSE), microwave-assisted solvent extraction (MASE), and super critical fluid extraction (SFE). 1. ACCELERATEDSOLVENT EXTRACTION OR
PRESSURIZEDSOLVENTEXTRACTION ASE or PSE performs extractions at elevated solvent temperatures and pressures to achieve higher extraction efficacy. The process may be compared to pressure cooking. After loading a sample into the extraction cell, the cell is filled with solvent, heated and pressurized, and held at the pressure and temperature for a predetermined time. The clean solvent is pumped into the sample cell, and the sample cell is purged with nitrogen gas. This extract is collected into a collection vial. ASE has been used for the determination of OP and N-methyl CM pesticides in foods (Obana et al., 1997; Okihashi et al., 1997; Richter et al., 2001). ASE has also been used for the extraction of OPs and other pesticides from soils, clays, sediments, sludge, and waste solids (SW-846, method 3545A; EPA, 1994). 2. MICROWAVE-ASSISTEDSOLVENT EXTRACTION This method is similar to ASE, except that heating power is supplied by microwaves. MASE has been used for the extraction of pesticides from soil (de Andrea. et al., 2001). Sun and Lee (2003) report a method for the optimization of MASE as well as supercritical fluid extraction of CM pesticides from soil. Lopez-Avila et al. (1998) studied the stability of OCs and OPs when extracted from solid matrixes with microwave energy. 3. SUPERCRITICALFLUID EXTRACTION In this method, CO2 gas at the critical temperature and pressure is used to extract pesticides and other chemicals from solid samples or liquid samples soaked in an inert powder. CO2 and other gases become fluid (neither gas nor liquid) when temperature and pressure reach a critical point called the supercritical phase. In SFE, this fluid is used to extract target chemicals (OPs and CMs) from solid sampies. Instruments connecting the SFE apparatus with online gas chromatography (GC) have been developed in which the series of steps from extraction to analysis have been
automated. The sample is placed in an extraction chamber through which the supercritical fluid is forced, and the target substances are extracted from the samples and trapped in vials by a small amount of methanol or adsorbent such as florisil or ODS resin. The most common gas used in SFE is CO2. However, supercritical CO2 is very nonpolar, so modifiers are added to improve extraction efficiency for polar compounds. Modifiers for CO2 include methanol, dichloromethane, acetonitrile, and water. Modifiers are mixed with the CO2 using a pump or added directly into the samples. The application and effectiveness of SFE for the extraction of pesticides from various types of samples for pesticide analysis have been demonstrated (Hopper, 1997; Hopper et al., 1995; Kim et al., 1998; Lehotay, 2002; Lehotay and Eller, 1995; Lehotay and Valverdegarcia, 1997; Lehotay et al., 1995). A method for the SFE extraction of methamidophos residue from vegetables has been described (Valverdegarcia et al., 1995). A limitation of SFE is that extraction of fatty foods and animal tissues with SFE extracts the fat along with pesticides. A systemic approach to optimization of SFE of pesticides is presented by Juhler (1997). A review of the usefulness of SFE for pesticide determination is presented by Camel (1998). ASE, MASE, and SFC, and their uses and potential pitfalls, have been discussed in a review (Camel, 2001).
III. C L E A N U P A N D P U R I F I C A T I O N Sample extracts for pesticide analysis from food, feed, biological, and environmental samples are usually complicated mixtures. The other chemical components in the sample are extracted along with the pesticide residue. Usually, the residues are present at the parts per million (ppm) level in the sample, whereas other components are present in very large amounts (Rogers, 1986). The other component chemicals in the sample extract can interfere with GC and highperformance liquid chromatography (HPLC) analysis due to coelution of the impurities with target compounds, and affecting the separation capacity of the analytical column due to overloading by impurities. Furthermore, large amounts of nonvolatile or polar compounds can contaminate GC injection ports and columns. It is therefore necessary to clean up or remove nontarget compounds as much as possible. For OPs and CMs, the usual techniques for purification are liquid-liquid partitioning and column chromatography. For cleanup of OPs and CMs, the adsorption columns used are Florisil, charcoal/Celite, and ODS, in which an octadecyl function is bonded to silica particles (FDA, 1994).
A. Current Trends in Cleanup and Purification 1. SOLIDPHASE EXTRACTION This technique came in vogue with the development of HPLC techniques. This technique is a form of partition
CHAPTER 47 9Analysis of OPs and CMs chromatography and works according to the partition equilibrium between a solid phase and a solvent. If the solid phase is polar, the solvent used is nonpolar. On the other hand, if the solid phase is nonpolar, such as ODS, the solvent used is polar. The advantage of this system is that it is much faster than the traditional column chromatography and uses much less solvent. Solid phases are even available in the form of disks, which can be used to separate OPs and CMs from water (Chiron and Barcelo, 1993; Mersie et al., 2002). The application of SPE has been described for the extraction of pesticides from vegetables (Casanova, 1996), oranges (Yamazaki and Ninomiya, 1999), water (Van-Hoof et al., 2002), and beef muscle (Kuivinen and Bengtsson, 2002). In the case of water, the samples are percolated through the column or filtered through the disk; the pesticides are retained on the column or disk. The pesticides are eluted with appropriate solvents to be used for determination. In the method of Kuivinen and Bengtsson, the bovine muscle is homogenized with ethyl acetate (EtOAc); the homogenate is centrifuged and filtered through anhydrous sodium sulfate. The fat in the filtered extract is precipitated in methanol by cooling, and the extract is diluted with water and passed through an SPE column (isoelute ENV +). After elution with EtOAc, evaporation, and redissolution, the sample is injected into a GC fitted with a capillary column (DB-1701) and detected with a flame photometric detector. In a variation of SPE, honey samples were mixed with Florisil and anhydrous sodium sulfate and loaded in a column, and the column was eluted with hexane-ethyl acetate (90/10 V/V) (Sanchez-Brunete et al., 2002). The column eluate was analyzed by GC and nitrogen phosphorus detector (NPD), as well as GC and mass spectrometry (MS). 2. GEL PERMEATION CHROMATOGRAPHY Gel permeation chromatography (GPC) separates molecules by size. Compounds are separated when sample mixtures are passed through a column packed with material of known pore size. Larger molecules that cannot penetrate the pore size of the packed material (usually known as a gel) elute faster. GPC is used as a general separation method for semivolatile compounds. The separation ability is poorer than that of other forms of chromatography. Thus, GPC is generally used to remove lipids, proteins, and natural resins from samples. It is a very good technique for removing fat in the analysis of fatty samples for pesticides. The GPC technique for pesticide residue determination has been used since 1972. An automated GPC instrument was introduced by Ault et al. (1979) for chromatographic preparation of vegetable, fruits, and crops for OP residue determination utilizing GC with flame photometric detection (FPD). Sannino et al. (1995) described a multiresidue method for the quantitative determination of 39 OPs in seven fatty foods using GPC as a cleanup technique. A report of a cooperative trial to validate the use of GPC
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cleanup for the isolation of pesticide residues from fats and oils was published by the Committee for Analytical Methods (Anonymous, 1992). A multiresidue screen for quantitative determination of 43 OPs, 17 OCs, and 11 CMs in plant and animal tissues, including ingesta, was reported by Holstege et al. (1994). Ali (1989) applied GPC cleanup and HPLC (with postcolumn derivatization) for the determination of CMs in animal tissues. 3. IMMUNOEXTRACTIONS Immunoaffinity sorbents (ISs) are more selective compared to ODS sorbents. The first commercial ISs were introduced for the cleanup of samples for the determination of aflatoxins (Groopman and Donahue, 1988). ISs have been synthesized for a limited number of pesticides and pesticide classes. Immunosorbents are formed by covalently bonding antibodies to an appropriate sorbent. Immunosorbent is packed into a solid phase extraction cartridge or precolumn as a classical extraction sorbent (Bouzige and Pichon, 1998). There is much interest in developing ISs for single analytes, which are particularly difficult to analyze at the trace level because of a lack of available extraction methods, such as acephate and methamidophs. 4. SOLID PHASE MICROEXTRACTION The solid phase microextraction (SPME) technique is used in many areas. SPME combines the use of solid phase extraction and concentration of the eluate from SPE in one step. SPME probes are silica fibers coated with organic polymers (DB 1, DB 1701, etc.). These fibers capture and concentrate pesticides and, of course, some other chemicals. The fibers are sealed in a syringe. The sample can be injected directly into a GC by using an accessory. The needle of the syringe is lowered into the injection port and then fiber is introduced in the injection port. Pesticides are thermally desorbed from the fiber and then separated on the analytical column and detected. A review of the application of SPME to the analysis of pesticides has been published (Boyd Boland et al., 1996). Methods for the analysis of pesticides using the SPME technique coupled with GC or HPLC have been described for water (Sng et al., 1997; Beltran et al., 1998; Aguilar et al., 1998) and in a food plant (Chen et al., 1998), biological samples (Musshoff et al., 1999), and soil samples (Ng etal., 1999; Moder etal., 1999). SPME fibers are also finding use in the analysis of chemical warfare agents (CWAs) in water (Lasko and Ng, 1997).
IV. C O N C E N T R A T I O N The cleaned up sample extract has to be concentrated in order to inject microliter amounts of the sample extract in GC or HPLC systems connected to high-sensitivity detectors used in the determinative step. Large volumes of solvents in the sample extracts overload the analytical column, particularly in GC. Several types
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of concentrators, such as Kuderna-Danish (K-D), Rotory Evaporator (RE), Turbovap, and N-EVAP, are available for sample concentration. The use of K-D, RE, and Turbovap is described in the Pesticide A n a l y t i c a l M a n u a l (FDA, 1994). Information about the use of N-EVAP nitrogen evaporators for sample concentration is available from Organomation Associates.
V. SEPARATION FROM IMPURITIES, D E T E C T I O N , AND D E T E R M I N A T I O N The cleaned up sample extract still contains many impurities; thus, these impurities have to be separated from the analyte before it is determined. The detection and determination of OPs is usually with GC coupled with selective detectors, such as electron capture detector, NPD, FPD, and mass spectrometric detector (MSD or MS). Large varieties of packed as well as megabore and capillary columns are used with these detectors. CMs are determined with GC coupled to NPD or FPD (in S mode). CMs are effectively determined with HPLC/photo diode array or HPLC/fluorescence. For a trace amount, it is essential to do a postcolumn derivatization (Ali, 1989).
A. Chromatography Chromatographic methods in general are the most common methods used at the determinative step in pesticide analysis. Thus, basic understanding of chromatographic procedures and the detection devices is important for pesticide analysis. According to the International Union of Pure and Applied Chemistry, chromatography is the method used primarily for the separation of components of a sample in which the components are distributed between two phases, one of which is stationary while the other moves. The stationary phase may be a solid, a liquid supported on a solid, or a gel. The stationary phase may be packed in a column, spread as a layer, or distributed as a film, etc. The mobile phase may be gaseous or liquid. Thus, there are two movements in the chromatographic system: the mobile phase movement, which is usually at a constant rate, and the movement of the components of the sample. The movement of the components depends on their relative distribution between the stationary phase and the mobile phase. This results in the separation of the components. This separation process coupled with the detection and measuring device completes the chromatographic system.
1. GAS CHROMATOGRAPHY In GC, the basic instrument consists of a sample introduction port (injector), an analytical column in an oven (to separate the components of the mixture), a detector (which can detect the presence of chemicals eluting from the analytical column), and a recording or data handling device. The injector,
oven, and detectors are heated zones; the temperature regulation of these zones depends on the application. Initially, packed columns were used in GC, but these columns had only limited resolving power. With the advent of capillary and megabore columns in which the liquid phase is crosslinked to pure silica capillary tubes, the resolving power of the analytical column increased severalfold. Lawrence (1987) showed the separation of 38 OPs on a DB-17 capillary column used for GC with FPD.
2. HIGH-PERFORMANCELIQUID CHROMATOGRAPHY HPLC is a variant of traditional liquid chromatography. The resolution and separation are improved by using small, uniform particle size columns. These columns require the use of a high-pressure pump that can force the liquid mobile phase through the columns, presumably at a constant rate. The basic HPLC instrument consists of an injector, an analytical column (which may or may not be temperature controlled), and a detector. With the development of hardware for pumps, the performance of liquid chromatography has increased manyfolds. The greatest impediment to the use of HPLC in residue analysis has been the absence of selective detectors, as are available for GC. However, the use of HPLC coupled with the MSD in trace analysis of pesticides is increasing, particularly for polar and thermally labile compounds. The technique of postcolumn derivatization (attaching a fluorophore) and fluorometric analysis has proved very useful for the determination of N-methyl CMs in foods (Holstege et al., 1994) and tissues (Ali, 1989; Ali et al., 1993a,b).
B. Detectors 1. ELECTRONCAPTURE DETECTOR The modem ECD contains a 63Ni source. This source emits high-energy beta particles. When these particles collide with the carrier gas molecules to produce low-energy electrons, the electrons continually collect at the cell anode by applying voltage pulses to the cell electrode. Cell current thus produced is measured and the pulse interval (frequency) adjusted to maintain constant cell current. A standing pulse frequency describes the equilibrium condition that exists when only carrier gas is passing through the cell. When molecules of an electrophilic substance enter the detector, electrons are "captured" to a degree depending on the electron affinity of the substance. The electron supply in the cell decreases, and pulse frequency increases to generate constant current. Change in the frequency required to keep the cell current constant is converted to voltage and is sent to the recording device as the detectors response to the analyte.
2. FLAME PHOTOMETRICDETECTOR When GC column effluent is burned in a hydrogen/air flame, compounds containing phosphorus and/or sulfur
CHAPTER 47 9Analysis of OPs and CMs produce characteristic emissions. A narrowband pass (interference) filter of appropriate wavelength isolates emissions produced by either phosphorus or sulfur. These emissions can be viewed by a conventional photomultiplier tube; a filter with maximum transmittance at 526 nm permits detection of phosphorus compounds, whereas one with maximum transmittance of 304 nm detects sulfur compounds. A single optical filter and photomultiplier tube may be used, or two filters and photomultiplier tubes can be assembled to permit response to both phosphorus and sulfur compounds simultaneously. A variation of FPD known as the pulsed flame photometric (PFPD) has been introduced. The PFPD uses the time-delayed chemiluminescence produced by the heteroatom in a molecule. It is claimed that PFPD is much more sensitive than FPD. Moreover, several other heteroatoms, such as S, P, N, and Se, can be detected by PFPD (Jing and Amirav, 1998). PFPD has been used simultaneously with MS to enhance pesticide detection capabilities (Amirav and Jing, 1998) 3. NITROGEN PHOSPHORUS DETECTOR The NPD is selective to residues containing nitrogen and/or phosphorus atoms. Modem NPD detectors evolved from the potassium chloride thermionic detector. GC effluent impinges onto the surface of an electrically heated and polarized alkali metal salt source (usually rubidium) in the presence of air/hydrogen plasma; ionization occurs and the flow of ions between plasma and an ion collector is amplified and recorded. Detector response to analytes results from the increased ionization that occurs when compounds containing nitrogen or phosphorus elute from the column. At gas flow rates used for NPD operation, the degree of ionization of compounds containing nitrogen or phosphorus is 10,000 times greater than for hydrocarbons. The mechanisms of enhanced response to nitrogen and phosphorus are not completely clear. However, both gas phase and surface ionization processes have been proposed. ECD, FPD, and NPD are discussed in detail in Chapter 5 of PAM (FDA, 1994).
4. MASS SPECTROMETRIC DETECTOR A mass spectrometer is an instrument that can separate charged atoms or molecules according to their massto-charge ratio. Relative molecular masses of organic compounds and biopolymers can be measured in this way, and the instrument is also capable of generating structural information. The sample is introduced into the mass spectrometer, which is generally kept under high vacuum (5-10 mbar). Compounds are converted into gas phase molecules either before or during the charging or ionization process, which takes place in the ion source. Many types of ionization mode are available: The type of compound to be analyzed and the specific information required determine which ionization mode is the most suitable. Once ionized, the molecule ion may fragment, producing ions of lower mass than the original precursor molecule. These fragment ions are dependent
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on the structure of the original molecule. The ions produced are repelled out of the ion source and accelerated toward the analyzer region. Although both positive and negative ions may be generated at the same time, one polarity is chosen and either positive or negative ions are analyzed and recorded. Molecules that do not ionize (i.e., remain neutral) are pumped away and will not be detected. There are various types of ionization techniques, such as electron impact ionization (EI), chemical ionization, fast atom bombardment/ liquid secondary ionization, matrix-assisted laser desorption ionization, and electrospray ionization. Recently, tandem mass spectrometers (MS-MS) have been introduced. In a tandem mass spectrometer, two mass spectrometers are coupled together separated by a collision cell. By coupling together two analyzers, separated by a collision cell, additional information can be obtained. The first analyzer is used to select the ion of interest. This ion is then passed into the collision cell, which usually will have been pressurized with an inert gas such as argon. Collision of the ion with the atoms in the cell can induce dissociation of the ion. This is known as collision-induced dissociation: The original ion is referred to as the "precursor" ion and the dissociated ions are known as "product" ions. Product ions are then analyzed in the second mass spectrometer, thus generating a product ion mass spectrum of the original precursor ion. The tandem mass spectrometer provides much more structural information about the analyte molecule and thus is an excellent confirmation technique. The greatest impediment for the use of mass spectrometers in common laboratories has been their high price. A direct sample introduction device has been used for the GC/MS-MS determination and confirmation of pesticides (Lehotay, 2002). It has been claimed that the sample extracts need not be cleaned up before determination. A method for the deterruination of 37 OPs in human tissue using GC/MS has been described (Russo et al., 2002). A HPLC/MS method for the determination of eight different types of CM pesticides in serum has been reported (Kawasaki et al., 1993). In this method, 1.5 ml of serum is mixed with 1.5 ml of 0.2 M phosphate buffer. The mixture is applied to an Extrelute column and then eluted with 15 ml of methylene chloride. The eluate is evaporated and the residue is dissolved in mobile phase. Carbofuran has been determined in stomach/rumen contents by GC/MS. Stomach/rumen contents are extracted with methylene chloride and cleaned up with GPC, and then the sample is analyzed by GC/MS (Osheim et al., 1985). Detection of pesticides in various types of samples without any cleanup has also been reported (Smith and Lewis, 1988). In this method, the samples are shaken with a suitable solvent and then analyzed by GC/MS. This method is qualitative in nature. 5. ION MOBILITY SPECTROMETRY Ion mobility spectrometry (IMS) has been known for more than 25 years. In IMS, the analyte and impurity molecules
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are ionized by a 63Ni source, which is housed in a shielded chamber. As discussed previously, the 63Ni source gives off high-energy electrons, which collide with sample molecules to produce ions. The aspirated air or any other gas used to introduce the sample in the reaction chamber also produces ions. The ions move through an electric field in the drift tube. Smaller ions move faster compared to larger ions. Thus, this technique is based on the size-to-charge ratio of ions. This technique differs from mass spectrometry, in which the separation is on the basis of mass-to-charge ratio of ions. Also, in IMS the ions are formed at atmospheric pressure, whereas in mass spectrometry ions are formed at very low pressures (5-10mbar). A walk-through portal based on IMS has already been installed at the Statue of Liberty for detecting traces of explosives and other illegal chemicals. Compact units are in use at many airports for the detection of traces of explosives and illicit drugs. The application of IMS for monitoring mevinphos (phosdrin), an OP, has already been demonstrated (Tuovinen et al., 2001). Fematoscan (1999), a company involved in the manufacture of IMS-based detectors, has demonstrated the application of a handheld detector, which is a combination of GC and IMS for pesticide detection, including some OPs.
VI. CONFIRMATION Mass spectrometry is the technique of choice for confirmation of pesticides. In the case of OPs, the sample extract can be run on different columns. Relative retention times (RRt) of a given peak can be compared with a table of RR t for different columns (FDA, 1994). For CMs, the alkylated CMs can be chromatographed using GC with NPD detector. The retention time of peaks can be compared with the retention time of the standard compound. If enough of the residue compound is present, then thin-layer chromatography can be used to confirm the presence of the pesticide (FDA, 1982).
VII. SOURCES OF A N A L Y T I C A L METHODS Several excellent sources of analytical methods for OPs and CMs are available. These sources also contain methods for other classes of pesticides:
1. Pesticide Analytical Manual, Vol. 1 (FDA, 1994) 2. Pesticide Analytical Manual, Vol. 2 (FDA, 1991) 3. Manual of Analytical Methods for the Analysis of Pesticides in Humans and Environmental Samples (EPA, 1980)
4. NIOSH Manual of Analytical Methods (NIOSH, 1994) 5. Test Methods for Evaluating Solid Wastes (EPA, 1994) 6. Manual of Pesticide Residue Analysis, Vol. 1 [Deutsche Forschungsgemeinschaft (DFG), 1987]
7. Manual of Pesticide Residue Analysis, Vol. 2 (DFG, 1992)
8. A World Compendium. The Pesticide Manual (Tomlin, 1997)
9. Official Methods of Analysis (OMA) of AOAC International [Association of Analytical Communities (AOAC), 2003a]
10. ATSDR Pesticide Profiles [Agency for Toxic Substances and Disease Registry (ATSDR), 1994]
11. Residue Analytical Methods (RAM) (EPA, 2003) 12. Environmental Chemistry Methods (ECM) (EPA, 2004) 13. Scientific reviews
A. Pesticide Analytical Manual, Vol. 1 (FDA, 1994) PAM, Vol. 1, is the most exhaustive and complete compendium of analytical methods for the multiresidue method (MRM) analysis of pesticides in food stuffs. There are six chapters in PAM. The first chapter deals with regulatory operations, which describes what pesticide tolerance is and under which laws and statutes the pesticide tolerances are regulated. It also describes preparation of analytical sample and method application. Chapter 2 provides information about general analytical operations and discusses basic analytical techniques such as column chromatography; solvent selection and evaporation; equipment and procedures for comminuting samples; and procedures for specific commodities, such as crabs, eggs, fish, hay, straw, food, and feed ingredients. Also discussed in this chapter are special reagent preparation, tests and purification for applicable reagents, reference standards for pesticides, quality assurance and quality control, preparation of standard operating procedures, hazardous waste disposal, and safety issues. Chapter 3 discusses the need for MRMs of analysis. When the target analyte (residue compound) is not known, it is essential to use a method that can detect the maximum number of residues. For this purpose, multiclass MRMs are used. This chapter describes multiclass MRMs, their capabilities, and their limitations. Three MRMs are described in detail for nonfatty foods as well as fatty foods. Methods 302 and 303 are for nonfatty foods, and method 304 is for fatty foods. Each method is composed of various modules, such as for extraction, cleanup, identification, and determination. The extraction, cleanup, and detection modules are selected based on the nature of the sample. For example, for method 302 for nonfatty foods, there are 7 extraction modules, 6 cleanup modules, and 19 determinative step modules. Thus, method 302 is not a single method but a combination of various analyrical methods. The validation references for each module are also provided. The methods described in PAM are all validated methods. The extraction modules are described as El, E2, E3, etc.; the cleanup modules are described as C1, C2, etc.; and those for the determinative step are described with a prefix D. The complete description of the determinative step
CHAPTER 4 7
is DG or DL followed by a number. The DG modules refer to GC methods and DLs refer to HPLC methods. It should be noted that the extraction module E1 and cleanup module C1 of method 302 are not same as described for method 303 or any other method. Thus, in order to define a method of analysis, the method number, such as 302, 303, or 304, as well as the extraction module and cleanup module should be specified. The determinative step modules, such as DG1, DG2, or DL, are independent of method number and can be used with sample extract prepared by method 302, 303, or 304. Chapter 4 of PAM discusses methods 401-404. Method 401 is for N-methylCMs, method 402 is for acids and phe-
TABLE 1.
Method
PAM 302
PAM 303
Matrix
Nonfatty, high-moisture (>75%) commodities
Fruits and vegetables, moisture >75 %, sugar <5%, fat <2%
Whole eggs
Dried egg whites, grains, and other foods with low moisture (<75%); fat < 2%. Fruits and other foods with 5-15% sugar. Fruits and other foods with > 15% sugar
9Analysis of OPs and CMs
687
nols, method 403 is for phenylurea herbicides, and method 404 is for benzimidazoles. Again, each method contains various extractions and cleanup step modules. Chapters 3 and 4 also contain the table for recovery data and notes for special situations. For example, the recovery of acephate is complete (>80%) using 302, E l - E 3 extractions, and GC/FPD. For the determinative step, it is recommended that wide-bore capillary DEGS column be used instead of packed column; on the other hand, for azinophos-methyl, although recovery is complete, DEGS column is unsuitable. The applicable methods for various modules applicable to OPs and CMs are shown in Table 1.
Multi Residue Methods for OPs and CMs a Extraction module b
El. Extraction with acetone, liquid-liquid partitioning with petroleum ether/ methylene chloride E5. Extraction with acetone, liquid-liquid partitioning with acetone/methylene chloride; alternative to E1 for relatively polar residues E4. Extraction with water/ acetone, liquid-liquid partitioning with petroleum ether/methylene chloride E6. Extraction with acetone, liquid-liquid partitioning with acetone/methylene chloride; alternative to E4 for relatively polar residues El. Extraction with acetonitrile, partition into petroleum ether with high moisture E2. Extraction with acetonitrile, partition into petroleum ether E3. Extraction with water/ acetonitrile, partition into petroleum ether E4. Extraction with acetonitrile and water, partition into petroleum ether Extraction with heated acetonitrile and water, acetonitrile and water, partition into petroleum ether
Cleanup modul@
Determinative module c
None
DG2/14, DG3/DG 16, DG 4/5/17 DG15 DG12 Residues detectable with element selective detectors
C3. Charcoal/silanized celite column cleanup C4. C- 18 cartridge cleanup
DL1 for N-methyl CMs
C1. Florisil column cleanup, with three ethyl ether/petroleum ether eluants; applicable to all extraction modules
DG2/DG14 for residues with phosphorus; DG4/5/17 for residues with nitrogen
C4. Florisil columns cleanup, with three methylene chloride eluants; alternative to C3; some additional residues are recovered
(continues)
688
SECTION Vlll 9Analytical & Biomarkers
TABLE 1. Method
PAM 304
PAM 401
Matrix
(continued)
Extraction moduleb
Cleanup moduleb
Fatty foods, animal tissue, fatty fish
El. Extraction of fat with sodium sulfate, petroleum ether
Butter, oils, cheese, milk, egg yolks, dried egg whites, oil seeds, high-fat feed, nuts
E3. Extraction of fat by filtering E4. Extraction of fat with solvents from denatured product E5. Extraction of fat with solvents from feed materials, grains, nuts
Fatty, nonfatty, and variable moisture
E 1. Extraction with methanol E2. Extraction with methanol, reduced sample size for low products
C 1. Acetonitrilepetroleum ether partitioning, Florisil column cleanup with three mixed ether eluants C2. Acetonitrilepetroleum ether partitioning, Florisil column cleanup with three methylene chloride eluants C3. Acetonitrilepetroleum ether partitioning, Florisil column cleanup with petroleum ether and three mixed eluants C4. Acetonitrilepetroleum ether partitioning, Florisil column cleanup, petroleum ether and three methylene chloride eluants C5. Gel permeation chromatography m the preferred technique due to automation C 1. Two-stage liquidliquid partitioning, and charcoal/Celite column cleanup
Determinative modulec DG12/14 for residues with phosphorus
DG4/5/17 for residues with nitrogen
DL 1 for N-methyl CMS; method includes postcolumn hydrolysis and derevatization
aFrom FDA (1994). bExtraction and cleanup modules are dependent on method number, such as 302 and 303. CDeterminativemodules are independent of method number; a given determinativemodule is applicable to all methods. Chapters 5 and 6 of PAM discuss the techniques of GC and HPLC, respectively. The descriptions of GC and HPLC as well as the detectors used with each technique are explained in detail. These descriptions keep the residue analyst in mind and provide many helpful points for the analyst.
B. P A M , Vol. 2 (FDA, 1991) PAM, Vol. 2, contains methods designed for the analysis of commodities for residues of only a single compound (although some methods are capable of determining several related compounds). These methods are most often used when the likely residue is known and/or when the residue
of interest cannot be determined by common MRMs. An updated index for Vol. 2 is available online (http:// vm.c fs an. fda. gov/-- frf/pami 1.html).
C. Manual of Analytical Methods for the Analysis o f Pesticides in Humans and Environmental Samples (EPA, 1980) This publication contains methods for the determination of pesticides in humans and environmental samples. The manual describes the collection, preservation, and storage of samples, as well as cleaning of glassware and preparation, storage, and use of pesticide standards. Section 6 of the
C H A PT ER 4 7 9Analysis of OPs and CMs manual describes methods for OPs and metabolites in tissues and excreta and metabolites in urine. The manual is somewhat outdated because all the GC methodology is based on packed column. The capillary and megabore column could be used after method validation.
D. NIOSH Manual of Analytical Methods
(NIOSH, 1994) The NIOSH Manual of Analytical Methods (NMAM) is a collection of methods for sampling and analysis of contaminants in workplace air and in the blood and urine of workers who are occupationally exposed. These methods have been developed or adapted by NIOSH or its partners and have been evaluated according to established experimental protocols and performance criteria. NMAM also includes chapters on quality assurance, sampling, portable instrumentation, etc. Methods 5600 and 5601 are applicable to OPs and CMs, respectively. In these methods, air is pulled through a sampler tube (OVS-2), which contains XAD-2 adsorbent, for a prescribed time at a given flow rate. The pesticides are trapped in the sampler tube. The pesticides are extracted from the sampler tube by elution with 2 ml of 90% toluene/10% acetone for OPs and by 2 ml extraction solution (0.2% V/V, 0.1 M aqueous triethylamine phosphate buffer in acetonitrile) for CMs. OPs are determined by GC/FPD, and CMs are determined by HPLC with detection at 200 and 225 nm. Method 5006 deals with the determination of carbaryl (which can also be determined by method 5601). Methods 5012 and 5514 describe the determination of the OPs demeton and EPN, respectively.
E. Test Methods for Evaluating Solid Wastes,
(EPA, 1994) This manual deals with the analysis of many contaminants, including OPs and CMs, in wastes. Method 8414 A is applicable to OPs and method 8318 covers CMs.
F. Manual of Pesticide Residue Analysis, Vol. 1
(DFG, 1987) Part 1 of this manual discusses the preparation of reagents and samples, micromethods and equipment for sample processing, and limits of detection determination. Part 2 deals with cleanup methods. Part 3 includes methods for single residue determination. It includes methods for OPs and CMs such as acephate, methamidophos, aldicarb, chlorthiophos, heptenophos, methomyl, pirimiphos-methyl, pyrazophos, tetrachlorvinphos, and triazophos. Part 4 describes MRMs for OCs and OPs (methods S 10 and S 11); OP insecticides (method S13); dithiocarbamates and thiram fungicides (method S15); OPs with thioester group (method S 16); and OPs, OCs, and nitrogen-containing pesticides.
689
G. Manual of Pesticide Residue Analysis, Vol. 2
(DGF, 1992) Part 1 contains mass spectrometric EI data for confirmation of results. Part 2 deals with the updated cleanup methods, including SPE with various types of sorbents. Part 3 describes some single residue methods (SRMs). The pertinent methods for OPs and CMs are method 261-378-370 for methomyl, carbendazim, and thiophenate-methyl; method 378 for carbendazim; method 658-344 for cabosulfan and carbofuran; method 522 for fonofos; method 405 for glyphosate; and method 441 for oxamyl. Part 4 deals with MRMs, and a pertinent method for OPs and CMs is method $8, an updated version of method S 19. Method $25 is for methyl CMs. Thinlayer chromatographic methods using the automated multiple development technique are described in part 6.
H. A World Compendium. The Pesticide Manual
(Tomlin, 1997) This publication is a reservoir of references for analytical methods for the determination of SRMs for pesticides and insecticides. This also includes references for the analysis of commercial pesticide products. Pesticides and insecticides used worldwide are listed in alphabetical order. Each entry in the manual deals with a single pesticide and lists the properties, types of formulations in which the product is used, and toxicological properties, as well as reference(s) for analysis.
I. Official Methods of Analysis (OMA) of AOAC International (AOAC, 2003a) This is a compilation of fully validated analytical methods. Every method in OMA has been tested for ruggedness and validated through a multilaboratory collaborative study (a minimum of 8 labs for quantitative methods and 10 labs for qualitative methods), undergone rigorous scrutiny by recognized experts, and met AOAC criteria. These methods are preferred by regulators and are cited in the U.S. Code of Federal Regulations, the Codex Alimentarius, and other regulatory codes throughout the world; they are also routinely accepted in compliance actions and in courts. Chapter 7 of OMA deals with pesticide formulation analysis, and Chapter 10 deals with residue analysis in various types of samples.
J. ATSDR Pesticide Profiles (ATSDR, 1994) ATSDR publishes toxicological profiles for hazardous substances. The toxicological profiles for the pesticides chlorpyrifos, chlorfenvinphos, diazinon, dichlorvos, disulfoton, ethion, malathion, and methyl parathion have been published. Each of these profiles contains a section on analytical methods for the pesticide and its metabolites. The references for various analytical methods are given.
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S E CT I 0 N V I I I 9 A n a l y t i c a l & B i o m a r k e r s
Many of the analytical methods used for environmental samples are approved by federal agencies such as EPA and NIOSH. Other methods included are those approved by groups such as AOAC International and the American Public Health Association. Additionally, analytical methods that modify previously used methods to obtain lower detection limits and/or to improve accuracy and precision are referenced.
K. Residue Analytical Methods (EPA, 2003) This is a compilation of residue analytical methods for food, feed, and animal commodities to identify and quantify the pesticide residue of interest, determining the total toxic residue of the pesticide regulated by the tolerance (maximum legal residue level), including significant metabolites and breakdown products. EPA's laboratory has tested most of the methods in the RAM index. When some of these methods were tested, EPA's laboratory clarified certain sections of the methods to improve performance or remove ambiguity. In most of these cases, an addendum was added to the method to explain the necessary clarifications. A few of the methods in the RAM index have not been tested in the laboratory but have undergone extensive review regarding their suitability for collection of pesticide residue monitoring data and for tolerance enforcement. Although most of the methods perform satisfactorily, some may have deficiencies. These methods are very similar to those included in PAM, Vol. 2 (FDA, 1991).
L. Environmental Chemistry Methods (EPA, 2004) Environmental chemistry methods for soil and water are used to determine the fate of pesticides in the environment. The methods identify and quantify the pesticide residue of interest, determining the total concentration of pesticides, including the extractable parent compound and significant metabolites and breakdown products. Although the EPA reviews all analytical methods submitted in support of pesticide registration, only approximately 25% of the currently available environmental chemistry methods have been evaluated in EPA's laboratory. Most of the methods perform satisfactorily, but some have deficiencies, particularly some of the older methods. The sites for ECM as well as RAM are continually updated; thus, the date of reference will change with time.
M. Reviews Two reviews on pesticide residue analysis have been published (Sherma, 1999, 2001). These reviews cover various technological developments and methods published during 1997-1998 and 1999-2000, respectively. Sherma (2003) has also written a review on recent advances in the thinlayer chromatography of pesticides.
VIII. QuEChERS METHOD This method for multiclass multiresidue determination of pesticides was introduced by Anastassiades et al. (2002). QuEChERS stands for "quick, easy, cheap, effective, rugged, and safe." The method is applicable to a wide variety of sample types. The method involves the extraction of the sample with acetonitrile, and then MgSO4 and NaC1 are added to the sample and solvent mixture. The mixture is centrifuged and an aliquot of the upper layer is transferred to another tube, in which primary-secondary amine SPE sorbent and MgSO4 are added, mixed, and centrifuged. The upper layer is used for analysis by GC/FPD, GC/MS, GC/MS-MS, or HPLC (Lehotay et al., 2003). This method has also been applied to fatty foods, such as milk and eggs, and an interlaboratory study for the applicability of the method has been conducted (Lehotay and Mastovaska, 2004). The method has been evaluated by Schenck and Hobbs (2004), and it is also applicable to CMs.
IX. I M M U N O A S S A Y S FOR OPs A N D C M s Several immunoassay test kits are commercially available for the determination of OPs and CMs by the enzymelinked immunosorbent assay (ELISA) technique. A complete description of ELISA is outside the scope of this chapter. Briefly, these test kits are based on the use of antibodies, which bind both pesticide and a pesticide-enzyme conjugate for a limited number of antibody binding sites. Antibodies for pesticide are immobilized to the inside of a small plastic tube (well). Since there are the same number of antibody sites in the test well, and the same amount of pesticide-enzyme conjugate is added to each test well, a low concentration of pesticide in the test sample allows more antibody sites to bind with more pesticide--enzyme conjugate molecules. Thus, a low concentration of pesticide will produce a dark blue color on the addition of enzyme substrate. Conversely, a high concentration of pesticide will allow fewer pesticide-enzyme conjugate molecules to bind with antibody binding sites, resulting in a lighter blue color on the addition of the enzyme substrate. In a variation of the previously discussed scheme, the antibodies are attached to paramagnetic particles. The sample to be tested and the pesticide-enzyme conjugate are added to a disposable test tube followed by the antibody-attached paramagnetic particles. Both the pesticide and pesticide-enzyme molecules compete for the available antibody binding sites. At the end of the incubation period, a magnetic field is applied to the test tube to hold the magnetic particles. The contents of the test tube are decanted holding the magnetic particles. The magnetic particles in the test tube are washed, and a substrate and a chromogen are added to the tube. The color produced is inversely proportional to the concentration of pesticide in the sample.
CHAPTER 47 9Analysis of OPs and CMs During the past 15 years, there has been a consolidation of vendors producing these test kits. Currently, there is only one vendor, Strategic Diagnostics. These kits are available for several OPs and CMs. Note that none of these kits have been validated to receive the PTM status from AOAC International.
X. OP AND CM SCREENS A qualitative colorimetric test based on the inhibition of AChE can screen the presence of OPs and CMs. The thioesters of OPs have to be activated by a dilute bromine solution. Commercial test kits are available for the colorimetric test from Strategic Diagnostic.
A. Pesticide Detector Ticket for OPs and CMs Detection ticket is based on the inhibition of AChE by OPs and CMs. The ticket consists of two parts, one with AChE immobilized on paper disk and the other part is a paper disk impregnated with a substrate. The package is opened, and the enzyme paper is soaked in an aqueous sample extract. Subsequently, the two parts are put together and held between a thumb and a finger (to provide heat) for 2 or 3 min. If the enzyme part of the ticket turns a blue color, CMs and some of the OPs are not present in the sample. OPs have to be treated with dilute bromine water to activate them for inhibition of ACHE. If the enzyme part of the ticket turns blue, then organothiophosphates are also not present in the sample. On the other hand, if the enzyme part of ticket remains white, then OPs and/or CMs are present in the sample. The pesticide detector ticket is marketed by Neogen. The samples can be extracted with organic solvent(s), and methods for different procedures are available from the company online (www.neogen.com). The ticket can also be used for the detection of nerve agents.
XI. M E T A B O L I T E A N A L Y S I S
691
phosphate (DMP), dimethyl thiophosphate (DMTP), and dimethyl dithiophosphate (DMDTP). Alkyl phosphates are excreted in urine as sodium or potassium salts. Alky phosphates are excreted rapidly. More than 80% of the total dose of parent pesticide is excreted as alkyl phosphate in urine within 48 hr (Aprea et al., 2002). Traditionally, alkyl phosphates have been analyzed by GC coupled with element selective detectors such as FPD and NPD. The first method for the determination of alkyl phosphates was described by St. John and Lisk (1968). This method used methylation of alkyl phosphates due to their polar nature. The alkali thermionic detector used in the method has been replaced with NPD. Other derivatizing agents, such as diazopentane, triazines, and pentafluobenzylbromide (PFBBr), have also been used. The use of PFBBr is advantageous since DMTP and DETP each produce a single product on derivatization with PFBBr, whereas other derivatizing agents produce two isomers for either DMTP or DETE Medlin (1996) introduced the concept of confirming DETE DEDTP, DMTP, and DMDTP by converting them to DEP and DMP, respectively, by reacting thio and/or dithio analogs with 1% bromine water. The conversion of DETP and DEDTP to DEE and that of DMTP and DMDTP to DMP, is complete when urine is buffered with K2CO 3. Figure 1 shows the complete conversion of
ml FI
m.
(a)
[-i
A_
The analysis of OP and CM metabolites for monitoring pesticide exposure has been extensively discussed by Murray and Franklin (1992). Thus, in the following sections, a brief history of analytical methods for OP and CM metabolites is given, and advances in their analysis are discussed.
%_
(b)
A. OPs Metabolites One of the advantages of OPs is that they do not persist very long in the environment; they degrade and/or metabolize rapidly. Nearly 70% of all OPs produce one or more of the six dialkyl phosphates (DAPs) on degradation or due to metabolism: diethyl phosphate (DEP), diethyl thiophosphate (DETP), diethyl dithiophosphate (DEDTP), dimethyl
Time/minutes
FIG. 1. Chromatograms of rat urine (a) before and (b) after oxidative desulfuration. *Unidentified peaks.
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SECTION VIII 9A n a l y t i c a l & B i o m a r k e r s
DMTP and DETP to DMP and DEE respectively. These chromatograms are from urine samples for which the rats were dosed with a mixture of chlorpyrifos, parathion, and coumaphos. The reaction of organothiophosphates with a dilute solution of bromine is called "oxidative desulfuration." We also found that when reference samples for calibration curve are prepared by adding known amounts of the metabolites (DAPs) to the urine of nonexposed people or animals of the same species, the recovery of incurred metabolites improves drastically. In our experiments with rat urine, the recovery of various alkyl phosphates ranged from 85% for DMDTP to 112% for DEE Drevenkar et al. (1991) used the same approach for the preparation of reference samples for calibration. A thin-layer chromatography procedure for the determination of DETE DEDTP, DMTE and DEDTP has been described (Sherma et al., 1999). These compounds were separated on C-18 chemically bonded silica gel plates and detected by TCQ chromogenic reagent. This method in conjunction with GC/FPD method could be used for further confirmation of these compounds. A method for the determination of DAPs in water has been described (Chang et al., 2000). This method uses a strong anion exchange disk to isolate DAPs from water and DAPs are derivatized with methyl iodide in acetonitrile. Capillary GC with FPD was used for determination. A residue analytical method for the determination of DEP
and DETP in fecal samples has been described (Schenke, 2000). The fecal samples were homogenized in water, and DEP and DETP were subsequently alkylated to pentafluorobenzyl esters by a phase transfer reaction. The determination was carried out with GC/MS (Schenke, 2000). An excellent review of the analytical methods for biological monitoring of pesticide exposure is presented by Aprea et al. (2002). This review provides information regarding sample preparation and analytical procedures. MS or MS-MS coupled with GC or HPLC has been used for the determination of DAPs in urine (Bravo et al., 2002; Hardt and Angerer, 2000; Hernandez et al., 2004; Oglobine et al., 2001). The use of GC/MS or GC/MS-MS requires alkylation of DAPs in order to render them volatile for GC separation. HPLC coupled with MS or MS-MS has the advantage that DAPs need not be alkylated. Furthermore, the use of labeled (isotopic) internal standards compensates for the losses in recovery and improves precision. However, the use of labeled internal standards is very expensive since these compounds have to be custom synthesized. B. S p e c i f i c M e t a b o l i t e s ~
OPs
When OPs degrade or metabolize, nonspecific (e.g., DAPs) as well as specific degradation products are formed. The specific metabolites that have been analyzed are listed in Table 2.
TABLE 2. Specific Metabolites m OPs Parent compound
Specific metabolite
Azinphos-methyl/-ethyl Chlorpyrifos-methyl/-ethyl
1,2,3-Benzotriazin-4-one 3,5,6-Trichloro-2-pyridinol
BTA TCPY
Coumaphos
3-Chloro-4-methyl-7hydroxycoumarin 2-Isopropyl-6-methylpyrimidin-4-ol 3-Methyl-4-nitrophenol 5-Chloro- 1,2-dihydro- 1isopropyl- [3H]- 1,2,4triazol-3-one 2-[(Dimethoxyphos phorothi oyl)sulfanyl] succinic acid Malathion monocarboxylic acid Malathion dicarboxylic acid O,S-dimethyl hydrogen phosphorthioate 4-Nitrophenol, also known as p-nitrophenol 2-Diethylamino-6-methyl pyrimidin-4-ol
CMHC
Olsson et al. (2003) Olsson et al. (2003), Aprea et al. (1999) Olsson et al. (2003)
IMPY
Olsson et al. (2003)
MNP CIT
Ameno et al. (1995) Olsson et al. (2003)
MDA
Olsson et al. (2003)
MCA
Aprea et al. (2002)
DCA O,S-DMPT PNP
Aprea et al. (2002) Tomaszewska and Hebert (2003) Olsson et al. (2003), Aprea
DEAMPY
Olsson et al. (2003)
Diazinon Fenitrothion Isazofos, methyl/ethyl
Malathion
Methamidaphos/acephate Parathion-methyl/-ethyl Primiphos-methyl
Abbreviation
References
et al. (2002)
CHAPTER 47 9Analysis of OPs and CMs The methods of analysis for many of the specific metabolites have been reviewed by Aprea et al. (2002). Most of the specific metabolites are conjugate glucurornides or sulfates; thus, enzyme or acid hydrolysis is required to liberate the metabolites. Most of the methods for the analysis of specific metabolites use GC/ECD or GC/MS. The urine sample (after hydrolysis) is extracted with a solvent, such as ether or toluene. The filtration of urine is performed through a Sep-Pak C18 cartridge followed by extraction of the eluate with solvents. Several derivatizing reagents such as BSA [N, O-bis(trimethylsilyl)acetamide], 1-chloro-3-iodopentane, or MTBSTFA [N-(tertbutyldimethylsilyl)-N-methyl trifluoroacetamide] have been used to convert the metabolites into volatile compounds. Tomaszewaska and Hebert (2003) reported a method for the analysis of O,S-dimethyl hydrogen phosphorothioate (O,S-DMPT) in urine. O,S-DMPT is a specific metabolite of methamidophos. The urine sample was extracted with a C18 column, and the sample was lyophilized at low temperature to prevent loss of highly volatile and thermally unstable metabolite (O,S-DMPT). The lyophilized residue was derivatized using MTBSTFA and 1% tert-butyldimethylchlorosilane in acetonitrile. After filtration, the derivatized product was analyzed with GC/FPD (pulse FPD) in the phosphorus mode. The limit of detection for the method is reported as 0.004 ppm, with a mean recovery of 108%. A liquid chromatography/electrospray ionization-tandem mass spectrometry for the analysis of specific metabolites, such as BTA, TCPY, CHMC, IMPY, CIT, MDA, PNE and DEAMPY, has been reported (Olsson et al., 2003). The urine sample (2 ml) was hydrolyzed with glucurornides and loaded on an SPE cartridge (Oasis, HLB, or Waters). The cartridge was washed with 0.8 ml of 5% MeOH in 1% acetic acid and then eluted with 2 ml of MeOH. The wash and eluate were collected in separate tubes. The wash fraction was further cleaned by applying it tO a Chem Elute cartridge and eluted with 14 ml of CHC13. It was evaporated to dryness and reconstituted in 100 lxl of 1% acetic acid. The SPE cartridge eluate was also evaporated to dryness and reconstituted in 100 txl of 1% acetic acid. The reconstituted samples were analyzed by LC/MS-MS. The data for the analysis of acephate and methamidophos were also provided. Acephate and methamidophos are two OPs that do not produce DAPs. Acephate produces methamidophos on degradation; thus, it is considered a metabolite of acephate. Olsson et al. (2003) included both acephate and methamidophos in their analytical scheme for specific metabolites. Chang and Lin (1995) reported a HPLC method for the determination of 3-methyl-4 nitrophenol in urine of rats orally dosed with fenitrothion. Although some of these specific metabolites may be derived from several analogs of the same OP or from other non-OP sources, these are termed as specific metabolites. In combination with the DAPs, these metabolites can pro-
693
vide information regarding the parent compound. For example, p-nitrophenol may be derived from ethyl/methyl parathion or p-aminophenol. The DAP metabolites will be produced only by parathion. If DMP is found in the same urine sample, it clearly indicates exposure to methyl parathion. C. Specific M e t a b o l i t e s - - C M s
Most of the CMs are phenyl N-methyl esters of carbamic acid. On hydrolysis or metabolism, CMs produce phenols. Phenols are polar compounds and thus have to be alkylated before GC determination with either ECD or NPD. HPLC analysis combined with the appropriate detector appears to be the better choice. Aprea et al. (2002) reviewed various methods for unchanged CMs in blood and urine, as well methods for specific CM metabolites, such as benomyl metabolites carbendazim and methyl-5-hydroxy-2-benzaimidazolecarbamate; 1-napthol; 2-isopropoxyphenol (metabolite of propoxur); and carbofuran phenol, which is a metabolite of several pesticides (carbofuran, benfuracarb, carbosulfan, and furathiocarb).
XII. N E R V E A G E N T A N A L Y S I S Nerve agents such as tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and venom toxin (VX) are extremely toxic OP compounds. Although these agents have been controlled by international treatises, some of them have been used in terrorist attacks, such as the satin attacks in Japan. All these compounds are liquids with high vapor pressure except VX, which has a low vapor pressure and is thus more persistent in the environment. All these compounds are unstable in the environment and degrade to methyl phosphonate alkyl esters, which are stable (Elashvili, 2004). The methyl phosphonate alkyl esters further degrade over time, and in the presence of phosphonate ester hydrolase they degrade to methyl phosphonic acid. Different methyl phosphonic alkyl esters are produced based on the other substituents present in the original nerve agents. For example, satin gives isopropyl methyl phosphonic acid, whereas VX degradation results in ethyl methyl phosphonic acid. Since methyl phosphonic acid and alkyl esters, as well as the parent nerve agents, contain phosphorus, the most logical analytical procedure is GC/FPD-P (FPD in phosphorus mode). The same author used GC/FPD-P for the detection of nerve agents. The nerve agents, their intermediate degradation products (alkyl esters of nerve agents) and the final degradation product, methyl phosphonic acid, and their tri-methyl silylated products were determined with GC/FPD-E This publication provided the retention times of the parent, intermediate methyl phosphonic acid alkyl esters, and methyl phosphonic acid. However, the GC column specifications and other GC conditions were not
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specified. Harper (2002) described a method for nerve agents in air. The air is pulled through a bed of Hayesep D (known as the PCT tube) for a few minutes at 300-700 ml/min. After collection, the PCT tube is heated and the effluent is introduced onto a GC column coupled with FPD detector. The GC/MS-MS methods for the quantitative determination of nerve agents have been reported (Driskell et al., 2002; Barr et al., 2004). The difference between the two methods is that in the study by Driskell et al., the urine samples were concentrated by forming an azeotrope with acetonitrile, whereas in the study by Barr et al., the acidified urine samples were extracted into ether acetonitrile. The samples were derivatized by methylation with diazomethane and analyzed by GC/MS-MS. A microcolumn LC capillary electrophoresis (CE) with FPD for the screening of degradation products of chemical warfare agents, including nerve agents in water and soil, has been described (Hooijschuur et al., 2001). This study was a proficiency study to test the ability of participating laboratories to unambiguously identify chemical warfare agents and their degradation products. This study contains several references to the LC and CE method with FPD of various nerve agents. A method for the detection of nerve agent metabolites based on GC coupled with an atomic emission detector (AED) has been reported (Creasy et al., 1995). The nerve agent degradation products were extracted from spiked water, wipes, and soil samples. The extracted samples were derivatized with 1% trimethylchlorosilane in bis-(trimethylsilyl) trifluoroacetamide. The GC/AED technique was used for separation, detection, and determination of OP nerve agent metabolites. A miniaturized analytical system for separating and detecting toxic nerve agent compounds based on the coupling of a micromachined capillary electrophoresis chip with thick-film amperometric detection has been described (Wang et al., 2001). "Lab on a chip" technology is utilized in the measurement of CWA degradation products using an electrophoresis microchip with a contactless conductivity detector (Wang et al., 2002a). Wang et al. (2002b) also described a single-channel microchip for fast screening and detailed identification of nitroaromatic explosives or OP nerve agents.
A. Retrospective Detection of Nerve Agents Nerve agents strongly bind to acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE); however, the measurement of either AChE or BuChE does not identify the nerve agent or any other compound that may inhibit any of these esterases. It has been shown that both AChE and BuChE inhibited by sarin can be reactivated by high concentrations of fluoride ions (Polhuijs et al., 1997). Van der Schans et al. (2004) reported a method for the retrospective detection of nerve agents by reactivating BuChE in human plasma. The
plasma samples were treated with KF and passed through a Sep-Pak C18 cartridge. The generated phosphonofluoridates were isolated and analyzed by GC/NPD. They also used GC/MS to confirm the nerve agent(s). Besides the laboratory procedures, many field detectors are available for the detection of nerve agents as well as other CWAs.
B. Detection Paper The detection paper is based on certain dyes being soluble in CW agents. Normally, two dyes and one pH indicator are used, which are mixed with cellulose fibers in an unbleached paper. When a drop of CW agent is absorbed by the paper, it dissolves one of the pigments. Mustard agent dissolves a red dye, and nerve agent dissolves a yellow dye. In addition, VX causes the indicator to turn blue, which together with yellow will become green/green black.
C. Detection Tubes Detection tubes, such as civil defense kit, contain special reagents impregnated on inert supports. Air is sucked through the tube with a special pump. Reaction between the reagents in the tube and CWA takes place, and different colors are produced based on the CWA.
D. Detection Ticket This was discussed in Section X,A.
E. Other Detectors Hill and Martin (2002) presented a review of conventional analytical methods for CWAs. They discussed various sensors, such as surface acoustic wave sensors, electrochemical sensors, spectrophotometric sensors, immunochemical sensors, and IMS detector. For OP nerve agents, FPD and MS are the detectors of choice when coupled with GC or LC. Miniature ion trap mass spectrometer has been described for the detection of nerve agents in the field (Patterson et al., 2002; Riter et al., 2002). A book published by the Institute of Medicine and the National Research Council explains the use of various types of detectors for nerve agents as well as CWAs (IOM, 1999).
XIII. ANALYSIS OF AChE INHIBITOR
THERAPEUTIC AGENTS Some AChE inhibitors, such as donepezil, rivastigmine, galantamine, tacrine, eptastigmine, neostigmine, pyridostigmine, and ambenonium, are used as therapeutic agents. Table 3 shows the compound analyzed, methods Used, and reference for the method.
C H APT E R 4 7 9Analysis of OPs and CMs
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TABLE 3. Methods for AChE Inhibitor Therapeutic Agents
Compound
Method
References
Ambenonium Donepezil
HPLC HPLC HPLC/MS-MS
Epastigmine
HPLC
Galantamine
HPLC Capillary electrophoresis HPLC GC HPLC
Neostigmine
Pyridostigmine
HPLC GC
Rivastigmine
Tacrine
GC/MS HPLC/MS HPLC/MS-MS HPLC Capillary electrophoresis
XIV. M E T H O D V A L I D A T I O N There are several types of validation for analytical methods. AOAC is the leading organization that conducts methods validation under its auspices. AOAC conducts the following types o f method validation: performance tested method (PTM), peer verified method (PVM), and official methods of analysis (OMA). AOAC OMAs are referenced in the U.S. Code of Federal Register and are used worldwide by regulated industry, product testing laboratories, and academic institutions. AOAC (formed in 1884 as the Association of Official Agricultural Chemists) is an independent association devoted to promoting methods validation and quality measurements in analytical sciences. It does so by reviewing and validating approved standards methods of analysis, promoting uniformity and reliability in statements of results, and developing and promoting criteria useful for laboratory accreditation and analyst certification.
A. Performance-Tested Methods The AOAC Research Institute maintains a frequently updated list of PTMs. PTMs have been independently tested, rigorously evaluated, and thoroughly reviewed by the AOAC Research Institute and its expert reviewers. None of
Yamamoto et al. (1998) Yasui-Furukori et al. (2002), Lu et al. (2004), Matsui et al. (1999) Zecca et al. (1993), Unni and Becker (1992), Herold et al. (1992) Claessens et al. (1983, 1998) Pokorna et al. (1999) Tencheva et al. (1987) Chan et al. (1980) Ellin et al. (1982), Chan and Dehghan (1978) Abu-Qare and Abou-Donia (2001a, g) Chan et al. (1980), Chan and Dehghan (1978) Sha et al. (2004) Enz et al. (2004) Pommeir and Frigola (2003) Hansen et al. (1998) Vargas et al. (1998)
the pesticide detecting kits have PTM status. The reason appears to be that the kit manufacturer(s) has not yet sought this status.
B. Peer-Verified Programs The AOAC PVM program is intended to provide a class of tested methods that have not been the subject of a full collaborative study. Through a less intensive process, the program provides a rapid entry point for methods that are recognized by the AOAC at a level of validation for methods not otherwise evaluated. The distinguishing aspect of an AOAC PVM is that its performance has been checked in at least one other independent laboratory. It is expected that eventually most PVMs will undergo full interlaboratory collaborative studies and obtain OMA status.
C. Single Laboratory Validation In order for a method to be validated as PTM, PVM, and, finally, OMA, it must be validated in a single laboratory. In international trade and regulatory affairs, only validated methods are acceptable. The method need not be validated under the auspices of AOAC, but evidence is required that the method is suitable for the intended purpose and that similar results are expected from other competent laboratories
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and analysts. In order to gather this information, single laboratory validation is essential. In the pesticide arena, new pesticides are introduced regularly. In the United States, the registrant of a pesticide is required to submit a method for the analysis of the active ingredient as well as its residues on the intended commodity. Often, a situation arises in which a given pesticide is used on another commodity, or it may have been used in the environment illegally. The question then arises whether the original method is fit for a new matrix(s). The other scenario is that an old pesticide no longer in use or withdrawn from use is to be analyzed. It may be that the instrumentation and reagents are no longer available, instruments must be modified, and other unanticipated problems may require the method to return to the development phase. Frequently, a method works satisfactorily in one laboratory but fails to operate in another laboratory in the same manner. Validation is a process and not a result. AOAC (2003a) defines validation as the process of demonstrating or confirming the performance characteristics of a method of analysis, and the performance characteristics of a method of analysis are the functional qualities and the statistical measures of the degree of reliability exhibited by the method under specified operating conditions. The performance characteristics are specificity, the ability to distinguish the analyte from other substances, applicability (the matrices and the concentration range), and reliability. Reliability is the most important characteristic of an analytical method and is expressed in terms of percentage recovery, repeatability, and reproducibility. Repeatability and reproducibility are expressed in terms of relative standard deviation within laboratory (RSDr) and between laboratories (RSDR), respectively. Why worry about between-laboratory relative standard deviation (RSDR) when the method is validated only in a single laboratory? It is true that RSDR cannot be determined in a single laboratory; however, it can be predicted based on the concentration of the analyte (Horwitz, 1982; Horwitz et al., 1980). Based on the data from 100 years' worth of interlaboratory method validation studies conducted under the auspices of AOAC, Horwitz determined that relative standard deviation between laboratories and within laboratory is dependent on the analyte concentration. The relationship is expressed by the equation PRSDR % = 2C -~ where C is expressed as the mass fraction. This equation can be expressed in spread sheet notation as PRSDR % = 2 * C^(-0.15). For example, a concentration of 1 ppm is expressed as 1.000E-6 (1 p,g/g). Thus, for an analyte concentration of 1 ppm, PRSDR % = 2 * (1.00E-07) ^ ( - 0 . 1 5 ) = 16%
Another observation of Horwitz, confirmed by Thompson and Lowthian (1997), is that the precision of analytical methods at any given concentration does not improve with time, despite advances in analytical technology.
D. H O R R A T Value The concept of HORRAT values was introduced by Horwitz et al. (1989). The HORRAT value is the ratio of RSDR obtained from the actual experimental data from interlaboratory data to the PRSDR calculated from the Horwitz formula: HORRATR = RSD R / PRSD R The acceptable values for a good interlaboratory validated method are 0.5-2. Similarly, for intralaboratory work, HORRATr = RSDr/PRSDR Horwitz also noted that within-laboratory variation was in general one-half to two-thirds of the variation between laboratories. Thus, the acceptable values for HORRATr are 0.3-1.3 (1/2 of 1/2 = 0.25 rounded to 0.3; 2/3 of 2 = 1.3). Note that the equation for HORRATr differs from the equation given in the AOAC guidelines (AOAC, 2003a). The error stems from the formula used to calculate these values in the AOAC guidelines. HORRATr values should always be calculated using the formula HORRATr = RSDr/PRSDR, rather than the formula HORRATr = RSDr /PRSDr, since RSDr is more variable than RSDR. (W. Horwitz, 2004, personal communication). This relationship is widely used to predict a reasonable relative standard deviation for a given analyte concentration within a laboratory. AOAC (2003a) guidelines for single-laboratory validation of chemical methods also provide guidelines for acceptable recovery values, as shown in Table 4. Thus, armed with these guidelines it is possible to discern whether the experimental recovery rate, as well as the precision of the method, is within acceptable boundaries. TABLE 4.
Acceptable Recovery Values for Various Analyte Concentrations
Concentration 100% 10% 1% 0.1% 0.01% 0.001% (10 ppm) 0.0001% (1 ppm) 1 ppb
Recovery limits (%) 98-101 95-102 92-105 90-108 85-110 80-115 75-120 70-125
CHAPTER 47
XV. C O N C L U S I O N S A mosquito was heard to complain That a chemist has poisoned his brain The cause of his sorrow Was para-Dichloro-Diphenyl-Trichloroethane (DDT). mAuthor unknown (appeared in "lighter elements" in Today's Chemist at Work) Chemists are always developing new pesticides, and analytical chemists are busy developing methods for the analysis of pesticides. This will continue in the future. With the technological advancements in gas and liquid chromatography as well as detection devices, faster and more reliable analytical methods will be possible. With advancements in the technology of mass spectrometry and data handling units, which are coupled with libraries (NIST, Wiley, TOX, etc.) of a large number of chemical compounds (mass spectral data), it has become easier to identify and confirm pesticides as well as other compounds. Miniature mass spectrometers are being developed and, in time, their size and price shall shrink. The future of analytical methods for OPs and CMs is bright. Analytical methods for the analysis of new specific metabolites of OPs will be highly useful for the retrospective evaluation of OP exposure. Because CMs are the phenyl N-methyl esters of carbamic, they produce mainly phenols as the metabolites. Although phenols are ubiquitous in the environment, a multiphenol method that possibly covers all CM metabolites will be useful. The history of the person or the animal may give an indication if the person or animal was exposed to phenols or CMs. The development of M R M methods for both specific and nonspecific DAPs would considerably aid in the diagnosis of OP and CM exposure.
Acknowledgments My sincere thanks to my wife, Gagan, whose encouragement made this chapter possible. I also thank Anu, Shephali, Ritu, and Amit for helping me with computer problems and collating the references.
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CHAPTER 48
Biomarkers of Organophosphate Exposure OKSANA LOCKRIDGE AND LAWRENCE M. SCHOPFER University of Nebraska Medical Center, Omaha, Nebraska
nicotinic receptors leads to desensitization and paralysis of the breathing muscles. In the lung, overstimulation of receptors causes vast amounts of fluid to be secreted so that a person drowns in his or her own fluids. Heart rate is increased by sympathetic stimulation and decreased by parasympathetic stimulation. Depending on the relative effects on the two branches, OP compounds may produce tachycardia, bradycardia, fibrillation, or cardiac arrest.
I. I N T R O D U C T I O N Most of the U.S. population has been exposed to organophosphorus (OP) pesticides in their homes, workplaces, outdoors, or through trace contaminants in food (Barr et al., 2004; Casida and Quistad, 2004). OPs are used most heavily in agriculture. They are also used in the urban setting, for example, to protect home foundations from termites and to prevent the spread of West Nile virus carried by mosquitoes. OPs for use in the home and garden can be purchased at hardware stores. The Environmental Protection Agency has approved 38 different OPs for pesticide use (Pope, 1999). At nonlethal doses, the signs of toxicity caused by each OP are distinct (Moser, 1995; Pope, 1999; Lockridge et al., 2005). This suggests that each OP reacts not only with acetylcholinesterase but also with additional targets. A more complete understanding of the proteins modified by exposure to OPs will aid in understanding why some people cannot tolerate doses of OPs that are harmless to the majority. Furthermore, identification of new biomarkers of OP exposure will aid in diagnosis of exposure. Potential new bioscavengers that could be used for protection against OP toxicity may be discovered.
III. E V I D E N C E F O R S I T E S O F A C T I O N I N ADDITION TO ACETYLCHOLINESTERASE Not all the toxic effects of OP compounds can be attributed to inhibition of ACHE. Evidence that additional sites of action contribute to toxicity includes the following: 1. Organophosphate-induced delayed neuropathy is unrelated to the anticholinesterase (anti-ChE) effects of OP agents because many highly potent cholinergic OPs do not cause neuropathy, and other OP compounds such as tri-o-cresyl phosphate (which is not used as a pesticide) have only weak anti-ChE activity but are powerful inducers of neuropathy. The enzyme involved in organophosphate-induced delayed neuropathy is neuropathy target esterase-lysophospholipase (Lush et al., 1998; Glynn, 2003). When neuropathy target esterase is inhibited above a threshold of 70%, the axons in the periphery degenerate, leading to crippling paralysis of the legs (Glynn et al., 1999; Glynn, 2000; Kropp et al., 2004). Only a select few OP compounds inhibit neuropathy target esterase, and none of these are sold for use as pesticides in the United States. 2. The AChE knockout mouse has no AChE activity in any tissue. Nevertheless, this mouse is supersensitive to OP toxicity, thus demonstrating the existence of non-AChE protein targets (Xie et al., 2000; Duysen et al., 2001; Lockridge et al., 2005). It has been suggested that the AChE knockout mouse owes its life to butyrylcholinesterase
II. M E C H A N I S M O F O P T O X I C I T Y The function of acetylcholinesterase (ACHE) is to degrade the neurotransmitter acetylcholine (ACh). There is general agreement that the acute toxicity of OP is explained by irreversible inhibition of AChE activity at cholinergic synapses (Chambers and Levi, 1992; McDonough and Shih, 1997; Mileson et al., 1998; Pope, 1999; Taylor, 2001; Casida and Quistad, 2004). Inhibition of AChE (>70%) leads to accumulation of ACh at central and peripheral sites. In the brain, overstimulation of ACh receptors can lead to seizures. Inhibition of the breathing center in the brain results in asphyxiation. In the diaphragm muscle, overstimulation of Toxicology of Organophosphate and Carbamate Compounds
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Copyright 2006, Elsevier, Inc. All fights of reproduction in any form reserved.
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(BuChE) because the mouse has normal levels of BuChE and BuChE hydrolyzes ACh (Mesulam et al., 2002; Casida and Quistad, 2004; Cousin et al., 2005). In this view, the important target for OP in the AChE knockout mouse would be BuChE, although this has not been proven. 3. The specificity of AChE inhibitors was tested in zebra fish by comparing the phenotype of inhibitor-treated animals with the phenotype of a zebra fish mutant lacking AChE activity. It was concluded that three out of four inhibitors demonstrated secondary target effects and were not specific AChE inhibitors (Behra et al., 2004). 4. Different OP pesticides cause different degrees of toxicity despite similar levels of AChE inhibition. For example, when AChE in rat brain is inhibited to a level that leaves 20% residual activity, the rat shows signs of toxicity. When the OP that produced this inhibition is chlorpyrifos, there are more signs of toxicity than when the OP is parathion (Chaudhuri et al., 1993). To explain these observations, toxicologists have postulated the existence of toxicologically relevant sites of action in addition to AChE (Moser, 1995; Pope, 1999; Richards et al., 1999). 5. There is no correlation between AChE inhibition and the disposition of [3H]-soman, [3H]-DFP, and [3H]-sarin in brain (Kadar et al., 1985; Little et al., 1988). For example, the hypothalamus binds two to five times more OP compared to the striatum, but the striatum has more AChE activity than the hypothalamus. This suggests that in the hypothalamus, targets other than cholinesterase bind OP (Little et al., 1988). In a related experiment, binding of [3H]-DFP to whole brain homogenate was decreased only 15% by preincubation with 1 p~M eserine (a specific inhibitor of AChE and BChE), suggesting that only 15% of the DFP-labeled proteins are cholinesterases (Richards et al., 1999). 6. Low doses of cholinesterase inhibitors produce distinct effects that depend on the identity of the OP. For example, a low dose of fenthion decreased motor activity in rats by 86% but did not alter the tail-pinch response, whereas a low dose of parathion did not affect motor activity but did decrease the tail-pinch response (Moser, 1995). This observation supports the conclusion that there are other toxicologically important targets of OP, besides ACHE. 7. Low levels of chlorpyrifos administered to weanling rats impaired cognitive function without significantly inhibiting AChE activity and without downregulating cholinergic receptors. The rats showed no overt signs of cholinergic intoxication, but they performed poorly in the Morris swim task (Jett et al., 2001). Chlorpyrifos is a developmental neurotoxicant, targeting the immature brain at doses below the threshold for systemic toxicity. It has been proposed that chlorpyrifos has a noncholinergic mechanism of toxicity, acting on the pathway that controls expression and function of adenylyl cyclase. Adenylyl cyclase controls cell signaling through synthesis of the second messenger, cyclic AMP. Thus, chlorpyrifos may
indirectly affect the phosphorylation and function of nuclear transcription factors that control cell differentiation (Aldridge et al., 2003). 8. The biotinylated OP called FP-biotin, when given to mice at a dose that caused no cholinergic signs of toxicity, labeled at least 12 proteins in mouse plasma. Four of these proteins have been identified as ACHE, BuChE, carboxylesterase, and albumin (Peeples et al., 2005). This experiment demonstrates that this particular OP binds to at least 12 proteins. ~
IV. HYPOTHESIS The proteins that have been identified to date as proteins that react covalently with OP in living animals are just a small fraction of the actual number. We hypothesize that many additional proteins react in vivo at doses that cause no acute signs of cholinergic toxicity. The set of OP-reactive proteins is unique for each OP, although almost all OPs react with AChE and BuChE.
V. WHICH PROTEINS HAVE BEEN IDENTIFIED TO BIND OPs COVALENTLY? Humans were exposed to the OPs listed in Table 1 as research subjects (Mazur and Bodansky, 1946; Grob et al., 1947; Grob and Harvey, 1958) by accident while applying insecticide (Grob et al., 1950), through chemical attack by religious fanatics (Fidder etal., 2002), by suicide attempt (Eddleston and Phillips, 2004), or by ingestion of cooking oil and alcoholic beverages contaminated with OPs (Davis and Richardson, 1980). Only three proteins have been shown to react with OPs as a consequence of these in vivo exposures. In the first four instances of exposure, both AChE in red blood cells (RBCs) and BuChE in plasma were shown to be irreversibly inhibited. With these enzymes, the OP attaches covalently to the active site serine (Jansz et al., 1959), which is located at the bottom of the active site gorge (Millard etal., 1999; Nachon et al., 2005). The fifth instance of exposure involved food contaminated with tri-o-cresyl phosphate. Toxicity due to exposure to tri-o-cresyl phosphate is associated with inhibition of neuropathy target esterase (Johnson, 1990). Decades of work were required before neuropathy target esterase was identified, isolated, and sequenced and its function in lipid metabolism established (Lush et al., 1998; Glynn et al., 1999; van Tienhoven et al., 2002). A direct connection between inhibition of neuropathy target esterase in human lymphocytes and organophosphate-induced delayed neuropathy in a person has been demonstrated (Lotti et al., 1986). No other enzymes or proteins in OP-exposed humans have been shown to bind OPs covalently.
CHAPTER 48 TABLE 1.
OP
Acetylcholinesterase (EC 3.1.1.7)
Nerve agents, pesticides, DFP
Butyrylcholinesterase (EC 3.1.1.8)
DFR nerve agents, pesticides
Neuropathy target esterase-lysophospholipase (EC 3.1.1.5)
Metabolite of tri-o-cresyl phosphate
VI. ENZYMES AND PROTEINS THAT BIND OPs IN RODENTS EXPOSED TO OPS Table 2 lists the enzymes and proteins that have been shown to bind OPs after rats, mice, and guinea pigs were treated with OPs in vivo. All the enzymes and proteins in
TABLE 2.
Cannabinoid CB 1 receptor in brain Arylformamidase in liver M2 muscarinic receptor in lung Albumin in plasma
References
Grob et al. (1947), Grob and Harvey (1958), Chambers and Levi (1992), Somani and Romano (2001) Mazur and Bodansky (1946), Grob et al. (1947), Fidder et al. (2002) Casida et al. (1961), Lotti et al. (1986), Glynn (1999), van Tienhoven et al. (2002)
this table, with the exception of albumin, were detected because the investigator was searching for effects on specific, preselected targets. Electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry allow identification of OP-labeled proteins without any prior knowledge of the identity of the protein.
Proteins Found to Bind OP in Rodents Exposed to OP
OP
Protein
705
Proteins That Bind OP in Living Humans
Protein
Acetylcholinesterase in plasma and brain Butyrylcholinesterase in plasma Carboxylesterase in plasma Acylpeptide hydrolase in brain and RBC Fatty acid amide hydrolase in brain Neuropathy target esterase in brain
9Biomarkers of Organophosphate Exposure
Method of detection
References
Pesticides, nerve agents, DFP, echothiophate, FP-biotin Pesticides, nerve agents, DFP, echothiophate, FP-biotin Pesticides, nerve agents, DFP, FP-biotin DFP
Activity assay, MALDI-TOF Activity assay Activity assay, mass spectrometry Activity assay
Jennings et al. (2003), Casida and Quistad (2004), Peeples et al. (2005) Mazur and Bodansky (1946), Casida and Quistad (2004), Peeples et al. (2005) Gupta and Dettbarn (1987), Maxwell et al. (1987), Peeples et al. (2005) Richards et al. (2000), Quistad et al. (2005)
Profenofos, tribufos, ethyl octylphosphonofluoridate Octylbenzodioxaphosphorin oxide, ethyloctylphosphono fluoridate, tribufos Chlorpyrifos oxon
Activity assay
Quistad et al. (2001)
Activity assay
Quistad et al. (2002b)
Binding assay
Quistad et al. (2002a)
Diazinon
Activity assay
Chlorpyrifos, parathion, diazinon FP-biotin
Bronchoconstriction
Seifert and Pewnim (1992), Pewnim and Seifert (1993) Lein and Fryer (2005)
Mass spectrometry
Peeples et al. (2005)
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VII. E N Z Y M E S A N D P R O T E I N S T H A T B I N D OPS I N V I T R O I n vitro, hundreds of proteins have been shown to bind OPs
covalently. Representative examples are listed in Table 3. All serine esterases, serine peptidases, and serine proteases bind OPs covalently, resulting in enzyme inhibition. The site of OP binding is the serine residue in the catalytic site. There is at least one example of a serine esterase from rabbit liver in which DFP was found to bind not only to the active site serine but also to the histidine in the catalytic triad (Korza and Ozols, 1988). The serine proteases are orders of magnitude less sensitive to OPs than are the serine esterases. Most OP pesticides react preferentially with BuChE. In contrast, most OP nerve agents react preferentially with ACHE. Carboxylesterase is also highly reactive with most OPs. This complicates the comparison of studies on rodents with studies on monkeys and humans because rodents have very high concentrations of carboxylesterase in plasma, whereas monkeys and humans have no carboxylesterase in plasma. On the other hand, albumin has esterase activity (Means and Wu, 1979), and this is sometimes mistaken for carboxylesterase activity in human plasma.
TABLE 3.
Protein class Serine esterases
Serine peptidase
Serine proteases
Cysteine proteases Serine amidase Receptors
Phospholipase
Other
Albumin binds OPs on tyrosine 411 of human albumin or tyrosine 410 of bovine albumin (Sakurai et al., 2004). The adjacent arginine residue activates this tyrosine for reaction with esters as well as with OPs. Other non-serine hydrolases that react with OPs include papain and bromelain, which are cysteine proteases that stochiometrically bind OPs on tyrosine, without loss of protease activity (Murachi, 1963; Chaiken and Smith, 1969).
VIII. P O S S I B L E A D D I T I O N A L MECHANISMS OF TOXICITY In addition to inhibition of AChE and the consequent disruption in the function of cholinergic nerve transmission, a number of other scenarios have been suggested that could lead to toxicity upon exposure to OPs. In some of these scenarios, exposure to OPs may actually provide beneficial effects. 1. OP binding to a subset of muscarinic receptors can be either beneficial or toxic depending on the location of the target tissue (central or peripheral), the agonist or antagonist action of the OP, and the location and function
A Selection of Proteins That Covalently Bind OP in Isolated Systems a
Examples
AAb
References
ACHE, butyrylcholinesterase, carboxylesterase, long-chain acyl-CoA hydrolase, fatty acid amide hydrolase Acylpeptide hydrolase, prolyloligopeptidase, dipeptidyl-peptidase IV Trypsin, chymotrypsin, elastase, thrombin, plasminogen, tissue plasminogen activator Papain, bromelain Arylformamidase, fatty acid amide hydrolase Muscarinic acetylcholine receptor M2, nicotinic acetylcholine receptor, cannabinoid CB 1 receptor
Ser, His
Jansz et al. (1959), Korza and Ozols (1988), Kidd et al. (2001), Quistad et al., (2002b)
Ser
Liu et al. (1999), Richards et al. (2000), Kidd et al. (2001)
Ser
www.biochem.wustl.edu/~protease
Tyr Ser
Murachi (1963), Chaiken and Smith (1969) Cravatt et al. (2001), Omeir et al. (1999)
9
Ward et al. (1993), Huff et al. (1994), Katz et al. (1997), Chebabo et al. (1999), Bomser and Casida (2001), Howard and Pope, (2002), Quistad et al. (2002a), Smulders et al. (2004) Sato et al. (1997), Kidd et al. (2001), Glynn (2003)
Platelet-activating factor acetylhydrolase (phospholipase A2), neuropathy target esterase-lysophospholipase Albumin
Ser
Tyr
Murachi (1963), Means and Wu (1979), Black et al. (1999)
aThis table lists only a representative sampling of the large number of OP reactive proteins that have been identified in vitro. bThe amino acid to which the OP binds covalently.
CHAPTER 48 9Biomarkers of Organophosphate Exposure of the receptor (presynaptic vs postsynaptic, inhibitory vs excitatory) (Ward et al., 1993). In the central nervous system, the predominant location of the M2/M4 receptor subtypes is thought to be presynaptic, where they act as autoreceptors to regulate the release of ACh. If OPs act as agonists at these autoreceptors, the release of ACh would be reduced and the toxicity resulting from AChE inhibition would be modulated. This was found to be the case for paraoxon and methyl paraoxon in a study of rat striatum slices (Liu et al., 2002). On the other hand, in cardiac tissue, OPs have a high affinity for postsynaptic M2 receptors. Agonist activity at these receptors would contribute to the toxicity resulting from the accumulation of ACh. In lung tissue, chlorpyrifos, parathion, and diazinon potentiated vagally induced bronchoconstriction in the absence of AChE inhibition (Lein and Fryer, 2005). OP binding to M2 muscarinic receptors decreased the function of these receptors. Vagally induced bronchoconstriction is normally limited by autoinhibitory M2 muscarinic receptors. Loss of M2 receptor function leads to increased release of ACh from the parasympathetic nerves, resulting in potentiation of vagally mediated bronchoconstriction, which contributes to airway hyperreactivity (asthma). 2. Fatty acid amide hydrolase regulates the action of endocannabinoids and hydrolyzes a sleep-inducing factor, oleamide. Large, hydrophobic OPs react selectively with fatty acid amide hydrolase rather than with ACHE. Up to 99% inhibition of mouse brain fatty acid amide hydrolase does not lead to any overt neurotoxicity or change in behavior (Quistad et al., 2002b). This led Quistad et al. to conclude that inhibition of fatty acid amide hydrolase in mouse brain does not appear to be a primary target for OP pesticide-induced neurotoxic action. However, mice lacking fatty acid amide hydrolase display reduced pain sensation. When these mice are treated with the endogenous substrate for fatty acid amide hydrolase, anandamide, they exhibit hypomotility, analgesia, catalepsy, hypothermia, and seizure susceptibility (Cravatt et al., 2001; Clement et al., 2003). These symptoms are frequently associated with OP toxicity and therefore support the possibility that OP binding to fatty acid amide hydrolase contributes to OP toxicity. 3. OPs bind directly to nicotinic ACh receptors (Katz et al., 1997; Smulders et al., 2004). This desensitizes the receptors and contributes to paralysis of skeletal muscles, thereby enhancing respiratory failure and asphyxia.' Desensitization of neuronal OL4[~2 nicotinic ACh receptors may result in increased anxiety. 4. Liver arylformamidase (previously called kynurenine formamidase), the enzyme that catalyzes N-formyl-L-kynurenine hydrolysis in the L-kynurenine pathway of L-tryptophan metabolism, is susceptible to OP inhibition. Diazinon given to mice at doses that were one-sixth of the LDs0 completely inhibited
707
arylformamidase activity in liver and altered the formation of several L-tryptophan metabolites. The level of N-formylL-kynurenine increased 100-fold. Such high increases in metabolites could disrupt intracellular homeostasis and inhibit formation of essential tryptophan metabolites. The increased plasma kynurenine pool and the induced xanthurenic acid urinary excretion have several implications in the assessment of diazinon noncholinergic toxicity. An increase in xanthurenic acid formation may alter glucose metabolism. Xanthurenic acid has been reported to form a complex with insulin and damage pancreatic [3 cells. Elevated plasma kynurenin may alter kynurenin transport into the brain. Since more than 40% of brain kynurenin originates from the systemic circulation, cerebral biosynthesis of neuroactive kynurenin metabolites such as quinolinic acid and kynurenic acid may change. Finally, the availability of L-tryptophan for other L-tryptophan-dependent processes may be reduced. Tryptophan is the metabolic precursor for serotonin and nicotinic adenine dinucleotide. Diabetes, bladder cancer, and neurological disorders may be the toxic consequences of diazinon-altered L-tryptophan metabolism (Seifert and Pewnim, 1992; Pewnim and Seifert, 1993). 5. Acylpeptide hydrolase is present in RBCs, brain, and liver. Acylpeptide hydrolase hydrolyzes the N-terminal acetylated amino acid residue on peptides. It also selectively degrades proteins that have been modified by oxidation or glycation (Fujino et al., 2000). Inhibition of this function may lead to cell death since intracellular accumulation of denatured proteins impairs cellular function (Shimizu et al., 2004). Failure to degrade misfolded and aggregated proteins is a dominant contributing factor to neuronal cell death in many neurodegenerative diseases. 6. Platelet-activating factor acetylhydrolase (also called phospholipase A2) is found both in the cytoplasm and in plasma. This enzyme inactivates the proinflammatory phospholipid, platelet-activating factor by hydrolyzing an ester bond. Inhibition of the acetylhydrolase could be a risk factor for coronary artery disease (Chen, 2004) or cause impairment of spermatogenesis (Koizumi et al., 2003). 7. Binding of OPs to BuChE, carboxylesterase, and albumin is not thought to result in toxicity. These proteins stoichiometrically detoxify OP compounds and are therefore considered a protective buffer for AChE (Mileson et al., 1998). However, the idea that BuChE has no physiological function other than to bind poisons is being undermined by detailed histochemical studies of brain and heart (Darvesh et al., 2003) showing that certain neurons contain only BuChE and no ACHE. ACHE- and BuChE-positive neurons together influence overall tonic activity of intrinsic cardiac ganglia (Darvesh et al., 2003). Therefore, inhibition of BuChE could disrupt tonic balance. 8. Acute toxicity levels are influenced by the hepatic cytochrome P450 enzymes that activate phosphorothionate
708
SECTION V l l l . A n a l y t i c a l
& Biomarkers
insecticides to oxons, as well as by enzymes that inactivate the OPs, including paraoxonase, glutathione transferase, and flavin-containing oxygenases (Chambers and Levi, 1992; Chambers and Carr, 1995; Costa et al., 2005). Species differences in response to OPs are explained partly by differences in the activity of OP-metabolizing enzymes.
AChE and BuChE. The signs of toxicity are characteristic of a particular OP and reflect the set of proteins that have reacted with that particular OE Proof of this hypothesis is expected to come through the use of new mass spectrometry tools. This chapter presents the evidence for this hypothesis.
Acknowledgment IX. RELEVANCE TO HUMAN HEALTH Identification of the set of proteins that bind a particular OP will be useful for understanding the toxicity of low-dose exposure. Why do only some people have neurologic and cognitive deficits after low-dose exposure to OPs? Do they have mutations in proteins involved in OP metabolism? For example, paraoxonase mutations may explain OP susceptibility in some individuals by causing a reduction in catabolism of OPs, thereby enhancing the effective dose (Costa et al., 2005). Alternatively, it is likely that mutations in other enzymes that bind OPs may also play a role. For example, people with no BuChE activity have a diminished OP-scavenging ability and therefore may be more susceptible to OP toxicity, although this has not been demonstrated. Another value of knowing other proteins that bind OPs is for the discovery of new bioscavengers. Human BuChE is being developed as a new therapeutic for treatment of OP toxicity (Broomfield etal., 1991; Doctor etal., 1991; Ashani, 2000; Lenz et al., 2004). Less expensive alternatives may be identified as potential bioscavengers. For example, the finding that albumin binds OPs covalently in mice (Peeples et al., 2005) suggests that albumin should be evaluated for in vivo protection against OP toxicity. Knowledge of the set of proteins that bind a particular OP could also be useful in diagnosis of exposure. Some of the OP-binding proteins will have a longer residence time in the circulation than others, giving a longer window for analysis. Furthermore, the set of OP-bound proteins could be a fingerprint for identifying the OP to which the person was exposed.
X. C O N C L U S I O N S In this chapter, biomarkers of (OP) agent exposure are defined as proteins that covalently bind OPs. In OP-exposed humans, only three proteins m ACHE, BuChE, and neuropathy target esterase have been shown to covalently bind OPs. From in vivo-treated rodents, six additional OP-binding proteins have been identified: acylpeptide hydrolase in brain and RBCs, carboxylesterase in plasma, arylformamidase in liver, M2 muscarinic receptor in lung, fatty acid amide hydrolase in brain, and albumin in plasma. It is our hypothesis that a given OP reacts with a set of proteins, and that the set of OP-reactive proteins is unique to each OP, although almost all OPs also covalently bind
This work was supported by U.S. Army Medical Research and Material Command grant DAMD17-01-1-0776, UNMC Eppley Cancer Center Support grant P30CA36727, and U.S. Army Research, Development & Engineering Command grant W911 SR04-C-0019 from the Edgewood Biological Chemical Center. The information does not necessarily reflect the position or the policy of the U.S. government, and no official endorsement should be inferred.
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Therapeutic Measures
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CHAPTER
49
Management of Organophosphorus Pesticide Poisoning TIMOTHY C:~MARR.S, 1,2 AND J. ALLISTERVALEz 1Food Standards Agency, London, UK 2National Poison Information Service (Birmingham Center) and West Midland Poison unit, City Hospital, Birmingham, UK
Phosphates are biologically active, whereas phosphorothioates need bioactivation to the corresponding metabolite (oxon) to become biologically active. As a consequence, the features of intoxication may be delayed unless aerial oxidation of the phosphorothioate has occurred already to generate traces of oxon. OP compounds are metabolically activated to the corresponding oxon by oxidative desulfuration mediated by P450 isoforms, ravincontaining monooxygenase enzymes, N-oxidation, and S-oxidation. The oxons that inhibit acetylcholinesterase (ACHE) can be deactivated by hydrolases such as the carboxylesterases and by A-esterases such as paraoxonase. OP compounds undergo other transformations mediated by cytochrome P450 that do not result in the production of an active metabolite, including oxidative dealkylation and dearylation. OP compounds may also be transformed by enzymatic action on the side chains, including ring hydroxylation, thioether oxidation, deamination, alkyl and N-hydroxylation, N-oxide formation, and N-dealkylation. The products of biotransformation may be conjugated with glucuronide, sulfate, or glycine. Elimination of metabolites occurs mostly in urine, with lesser amounts in feces and expired air. Some OPs, such as dichlorvos, which is not stored in fat to any great extent, may be eliminated in hours, whereas the inhibitory oxon of chlorpyrifos or demeton-S-methyl may persist for days because of their extensive storage in fat. Inhibition of cholinesterases by OPs is by a reaction in which the OP reacts with a serine residue at the active site of the enzyme, in which the leaving group of the OP is lost. The efficacy of oxime reactivators is attributable to the quaternary nitrogen that binds to the anionic site of the enzyme and the nucleophilic oxime moiety, to which the dialkoxyphosphate, at the active site of the enzyme, is transferred. The enzyme is thus again able to hydrolyze ACh and cholinergic neurotransmission capability is restored. When OP inhibitors bind to ACHE, the leaving group is lost (Burgen and Hobbiger, 1951), and in the case
I. I N T R O D U C T I O N The successful management of organophosphorus (OP) pesticide poisoning depends on: 1. The clinician possessing an understanding of the mechanisms of OP pesticide toxicity and applying them to the treatment options; 2. Accurate diagnosis and assessment of the severity of intoxication; 3. Maintenance of vital body functions and adequate clinical monitoring; 4. Further absorption of the OP pesticide being minimized; 5. Appropriate use of atropine, oxime; and diazepam.
II. M E C H A N I S M S O F O P P E S T I C I D E TOXICITY AND IMPLICATIONS FOR MANAGEMENT
Because the majority of OP insecticides are lipophilic and not ionized, they are absorbed rapidly following inhalation or ingestion. Although dermal absorption of OP compounds tends to be slow, severe poisoning may still ensue if exposure is prolonged. The degree of absorption depends on the contact time with the skin, the lipophilicity of the agent involved, and the presence of solvents (e.g., xylene) and emulsifiers in the formulation that can facilitate absorption. Following absorption, OP compounds accumulate rapidly in fat, liver, kidneys, and salivary glands. The phosphorothioates ( P = S ) , such as diazinon, parathion, and bromophos, are more lipophilic than phosphates (P--O), such as dichlorvos, and are therefore stored extensively in fat, which may account for the prolonged intoxication and clinical relapse after apparent recovery that has been observed in poisoning from these OP insecticides. OP compounds vary in the ease with which they cross the blood-brain barrier. Toxicology of Organophosphate and Carbamate Compounds
715
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71 6
SECTION IX 9T h e r a p e u t i c
Measures
of nearly all pesticides because most are dialkylphosphates or dialkylphosphorothioates, a dialkoxyphosphoryl derivative of AChE is formed. The rates of spontaneous reactivation, aging, and oxime-induced reactivation of AChE are dependent solely on the nature of the alkoxy groups and independent of the nature of the leaving group. Because OP pesticides are mostly dimethyl (e.g., demeton-S-methyl, dichlorvos, dimethoate, and malathion) or diethyl (e.g., chlorpyrifos, diazinon, and parathion) compounds, the vast majority of pesticides give rise to either dimethoxyphosphoryl AChE or diethoxyphosphoryl AChE (Table 1). The rate of spontaneous reactivation is greater for dimethoxyphosphoryl than for the diethoxyphosphoryl enzyme (International Programme on Chemical Safety, 1986; Wilson et al., 1992), and this also appears to be true of oximeinduced reactivation (Hansen and Wilson, 1999). Spontaneous reactivation of dimethoxy phosphorylated AChE proceeds quite rapidly so that the patient's condition should improve even without oxime therapy. Unless oximes are employed, however, there is no such expectation of rapid recovery for patients intoxicated with diethoxy phosphoryl insecticides. There are exceptions to the typical pesticidal OP structures described previously, and in the case of AChEs inhibited by these pesticides, less is known about reactivation rates, both spontaneous and oxime induced. These pesticides include EPN, leptophos, and cyanophenfos, which have one ethoxy and one phenyl group, and methamidophos, which is a methoxy S-methyl compound. Trichloronat, a phosphonate, has one ethoxy substituent and an ethyl group attached directly to the phosphorus atom. For some pesticides, it is not clear which group is
TABLE 1. Main OP Pesticides by Alkoxy Group a Dimethoxy pesticides Azinphos-methyl Chlorpyrifos-methyl Demeton-S-methyl Dichlorvos Fenitrothion Malathion Methidathion Mevinphos Oxydemeton-methyl Parathion-methyl Pirimiphos-methyl Temephos Trichlorfon aAdapted with permissionfromMarrs (2001). bAn OP fungicide.
Diethoxy pesticides Chlorfenvinphos Chlorpyrifos Diazinon Disulfoton Ethion Mephospholan Parathion Phorate Phosalone Pyrazophosb Quinalphos Terbufos Triazophos
acting as the leaving group in particular circumstances, and therefore the nature of the inhibited complex is not clear. Moreover, metabolism can change the leaving group, an example being profenofos (Edwards, 2001; Glickman et al., 1984). The efficacy of oximes against the structures produced by AChE inhibition by compounds such as diisopropylphosphorofluoridate (DFP) and the nerve agent soman (pinacolyl methylphosphonoflouridate) is poor, but this is not relevant to pesticide poisoning. In summary, the chemical structure of the OP pesticide and the presence of other ingredients in the formulation may have an impact on the speed of onset of features of intoxication. In addition, the fact that many OP insecticides are lipophilic means that they are distributed to and stored in body fat, and therefore elimination takes place slowly. Thus, the severity of intoxication may increase for 12-36 hr after exposure, intoxication may be prolonged, or relapse may occur after apparent clinical recovery. An understanding of the toxicodynamic aspects also explains why oximes may be of particular value in intoxication due to diethyl phosphates.
III. A C C U R A T E D I A G N O S I S A N D A S S E S S M E N T OF T H E S E V E R I T Y OF INTOXICATION The diagnosis of OP pesticide poisoning is based on the patient's history, clinical presentation, and laboratory tests. In a patient with a positive history, a typical odor on the breath, characteristic symptoms, and depressed erythrocyte and plasma cholinesterase activities, diagnosis is not difficult to make. Unfortunately, the history is often unobtainable. Moreover, the clinical features of OP poisoning may not be recognized as such if the patient presents, for example, with heart block, gastroenteritis, convulsions, or ketoacidosis. An awareness of this diversity of presentation is the first step to accurate diagnosis. The activity of two enzymes may be estimated to diagnose and/or monitor the progress of OP poisoning: red blood cell AChE and plasma butyrylcholinesterase (plasma cholinesterase, plasma pseudocholinesterase). Both are surrogates for activity of AChE in the central and peripheral nervous systems, the inhibition of which causes the cholinergic syndrome, probably the intermediate syndrome, but not organophosphorus-induced delayed polyneuropathy (OPIDP). A number of factors need to be borne in mind when interpreting cholinesterase activity measurements. AChE and butyrylcholinesterase (BuChE) are different gene products, although there are similarities in their structures (Darvesh et al., 2003). There are differences in the kinetics of the inhibition of the two enzymes by OPs and in the kinetics of reactivation and aging of the resulting inhibited enzymes (Wilson et al., 1992). Thus, on enzyme kinetic grounds, measurement of the activity of plasma BuChE would be expected to be a poorer surrogate than
CHAPTER 49 measurement of the activity of red blood cell AChE for the all-important enzyme in nervous tissue. However, even erythrocyte AChE measurements cannot be expected to be a perfect surrogate for the nervous tissue enzyme: This is because pharmacokinetic factors may result in differential access of the inhibitor to the red blood cells and to neural structures. A further consideration is that whereas OPs react with the enzyme to produce a phosphorylated structure that does not spontaneously reactivate, red blood cells of mammals lack the protein synthetic capability to synthesize new ACHE. In contrast, in nervous tissue, after inhibition by OPs whose e n z y m e inhibitor complex with AChE does not readily reactivate, activity reappears surprisingly quickly. Thus, Wehner et al. (1985) observed approximately 30% recovery after 24 hr in DFP-treated mouse central nervous system (CNS) reaggregates, which was clearly due to synthesis de novo of ACHE. Another consideration in the interpretation of BuChE activity measurements is that the normal range is relatively wide, rendering interpretation in individual patients difficult unless the results of previous estimations in the patient are available (Swaminathan and Widdop, 1992). Although erythrocyte cholinesterase is invariably more specific than BuChE activity as a marker of OP insecticide exposure, some OP insecticides (e.g., chlorpyrifos, demeton, and malathion) depress plasma BuChE activity to a greater degree. Few laboratories can quantitatively determine the insecticide responsible for the intoxication and measurement of the parent compound or metabolite in body fluids. Furthermore, such measurement has little place in the immediate diagnosis or early management of poisoning. In many cases, rapid hydrolysis prevents the detection of the parent compound, although urinary metabolites may persist for several days. The measurement of metabolites is most helpful as a measure of low-level chronic exposure. It is generally true that the presence of certain clinical features is more helpful in determining the severity of intoxication and prognosis than measurement of erythrocyte AChE activity alone soon after presentation. Patients who are moderately or severely poisoned, as indicated by drowsiness, hypotension, severe bronchorrhea, and marked muscle fasciculation, require treatment in a critical care unit because further deterioration may occur and mechanical ventilation may be required.
IV. MANAGEMENT A. Maintenance of Vital Body Functions and Adequate Clinical Monitoring Bronchorrhea requires prompt relief with intravenous atropine, and supplemental oxygen should be given to maintain PaO2 > 10 kPa (75 mmHg). If these measures fail,
9Management of OP Pesticide Poisoning
717
the patient should be intubated and mechanical ventilation (with positive end expiratory pressure) should be instituted. In severely poisoned patients who are hypotensive, it may be necessary not only to expand plasma volume but also to use a vasopressor (e.g., dopamine titrated to a systolic pressure >90 mmHg) or an inotrope (e.g., dobutamine 2.5-10 txg/kg/min to maintain cardiac output). Cardiac arrhythmias should be treated conventionally, and hypoxia must be considered as a possible etiology. The management of convulsions and muscle fasciculation with diazepam is discussed later. Careful attention must be given to fluid and electrolyte balance and adjustments to infusion fluids made as necessary. Heart rate, blood pressure, electrocardiogram, and arterial blood gases should be monitored routinely.
B. Minimizing Further Absorption of the OP Pesticide If exposure is dermal, thorough skin decontamination should be carried out, without caregivers themselves being contaminated, after resuscitation and stabilization. All contaminated clothing should be removed and affected skin should be washed thoroughly with soap and cold water, including exposed areas (e.g., hands, arms, face, neck, and hair). Gastric lavage may be considered in all potentially serious cases if ingestion has occurred less than 1 hr previously, although its value is unproven. Lavage should be performed with care and with an endotracheal tube in situ if the level of consciousness is depressed because hydrocarbons are present in many OP insecticide formulations. Syrup of ipecacuanha should be avoided because emesis is dangerous in a patient whose level of consciousness may deteriorate because of either the OP or the solvent in the insecticide formulation; aspiration pneumonia is a wellrecognized complication in these circumstances. Moreover, aspiration can also occur in conscious patients given syrup of ipecacuanha and is more likely to happen if hydrocarbons are present in the pesticide mixture. The capacity of activated charcoal to adsorb most OP compounds has not been demonstrated. On theoretical grounds, a single dose of activated charcoal (50-100 g) may be beneficial if administered less than 1 hr after OP insecticide ingestion. No cathartic should be administered with the charcoal because this may exacerbate OP-induced gastroenteritis.
V. APPROPRIATE USE OF ATROPINE, OXIMES, AND DIAZEPAM The development of specific therapies effective in poisoning by OP pesticides was undertaken in a number of institutions, including chemical defense establishments and academic laboratories. The first development was the discovery of the usefulness of anticholinergic drugs such as atropine, which was soon followed in the 1950s by the
718
SECTION IX 9T h e r a p e u t i c
Measures
demonstration of oxime-induced cholinesterase reactivation (Wilson and Ginsburg, 1955). A later development was the addition of anticonvulsants to the atropine/oxime combination. Atropine competes with ACh and other muscarinic agonists for a common binding site on the muscarinic receptor, thus effectively antagonizing the actions of ACh at muscarinic receptor sites, which leads to increased tracheobronchial and salivary secretions, bronchoconstriction, and bradycardia. The usefulness of atropine is virtually undisputed. Clinically, the main benefit of oximes is to reverse cholinergic effects at peripheral nicotinic sites so that, for example, muscle strength may improve. Oximes are much less effective than atropine at peripheral muscarinic sites, and their effects on CNS-mediated symptoms and signs may not be clinically significant. Although the therapeutic combination of oxime and atropine is well established in the treatment of OP pesticide poisoning, there is still no international consensus on the choice of oxime and on aspects of dosing; indeed, some have doubted the worth of oxime therapy altogether. For these reasons, the discussion on the oximes is relatively long. Benzodiazepines (most often diazepam and sometimes midazolam) may also be of benefit by reducing anxiety and restlessness, reducing muscle fasciculation, arresting seizures, and possibly reducing morbidity and mortality when used in conjunction with atropine and pralidoxime.
A. Atropine Atropine competes with ACh and other muscarinic agonists for a common binding site on the muscarinic receptor, thus effectively antagonizing the actions of ACh at muscarinic receptor sites. However, the peripheral antimuscarinic effects of atropine may not be the only antidotal property of the drug in OP poisoning. Atropine may also be of value in treating acute dystonic reactions occasionally observed in acute OP poisoning (Joubert et al., 1984; Joubert and Joubert, 1988; Smith, 1977; Wedin, 1988). |. PHARMACOKINETICS After intravenous dosing, atropine distributes rapidly, with only 5% remaining in the blood compartment after 5 min (Berghem et al., 1980). The apparent volume of distribution (Vd) is 1-1.7 liters/kg. Atropine is partly metabolized in the liver by microsomal monooxygenases to noratropine, tropine, atropine-N-oxide, and tropic acid (Van der Meer et al., 1983) and partly excreted unchanged in the urine. Elimination kinetics can be fitted to a two-compartment model with a clearance of 5.9-6.8 ml&g/min and a half-life of 2.6-4.3 hr in the elimination phase (Aaltonen et al., 1984; Kanto et al., 1981; Virtanen et al., 1982). Since the renal plasma clearance ( 6 5 6 _ 18 ml/min) was found to approach the renal plasma flow (712 _+ 38 ml/min), tubular
excretion may occur. Thus, both liver and renal disease can be expected to influence the kinetics of atropine (Hinderling etal., 1985). The elimination half-life of atropine is longer in children younger than 2 years of age and in the elderly. In the neonate, the half-life is between 5 and 10 hr due to an increased volume of distribution. In the elderly (70 years or older), the half-life may be prolonged to 30 hr due to reduced clearance. 2. EFFICACY Sanderson (1961) studied the effect of intraperitoneally administered atropine 17.4 mg/kg given alone or combined with oximes on the survival of rats poisoned orally by 10 different OPs, excluding dimethoate. Atropine alone prevented the development of toxicity. Although the numbers of rats in each group were small (n = 6) and statistical analysis was not performed, this study demonstrated that atropine treatment alone did reduce mortality. In calves poisoned with intravenous dichlorvos, atropine was shown to reverse the respiratory effects of the OP (Lekeux et al., 1986). The OP-induced reduction in dynamic lung compliance, arterial oxygen tension, increase in total pulmonary resistance, work of breathing, and alveolar arterial oxygen gradient were reversed by atropine. Atropine may therefore reverse changes in ventilation-perfusion inequalities resulting from uneven distribution of ventilation caused by ACh-mediated airway constriction (Slocombe and Robinson, 1981). Although the clinical efficacy of atropine in OP poisoning is well established (Bardin et al., 1987; DuToit et al., 1981 ; Namba et al., 1971; Senanayake and Karalliedde, 1987; Zilker and Hibler, 1996), no controlled studies have been published. Finkelstein et al. (1989) performed a noncontrolled prospective study of severe OP poisoning. In this study of 53 adult patients, atropine 2 mg by intravenous bolus, followed by the same dose at intervals of 10 min or more, was administered and the dose was adjusted as necessary to control tracheobronchial secretions and bronchospasm. All 53 patients were mechanically ventilated and obidoxime was also given. Although it is not possible to quantify any beneficial effect from atropine administration alone, atropine appeared to counteract the muscarinic features.
3. DOSAGEREGIMEN Atropine sulfate 2 mg (0.02-0.1 mg/kg in a child) intravenously should be given as soon as possible to patients who have increased secretions or bronchorrhea. Repeated injections of atropine, every few minutes if necessary, may be required during the first few hours of therapy; the dose should be titrated to control peripheral muscarinic signs, notably bronchorrhea and bronchospasm. In very severe cases, 100 mg or more per day may be required to control symptoms.
CHAPTER 49 The degree of atropinization may be assessed by dryness of the mouth and the magnitude of the tracheobroncheal secretions, pupil size, and heart rate. Tachycardia and mydriasis can be unreliable indicators, however, since both may also result from nicotinic stimulation in severely poisoned patients or, in the case of mydriasis, be a direct local effect of exposure. Tachycardia may also reflect hypoxia. The most sensitive and useful measure of adequate atropinization is the repeated evaluation of the quantity of secretions produced (Bardin et al., 1987; DuToit et al., 1981). B. Oximes
Early work on cholinesterase reactivators has been reviewed (Hobbiger, 1963). The earliest reactivators of AChE studied were hydroxylamine, choline, and hydroxamic acids. Pralidoxime (PAM), a much faster reactivator than hydroxylamine, was introduced by Wilson and Ginsburg (1955). The next major advance was the introduction of obidoxime by Ltittringhaus and Hagedorn (1964) and von Erdmann and Engelhart (1964). Large numbers of pyridinium oximes, both mono- and bis-pyridinium compounds, have been studied in OP poisoning, but only PAM salts and obidoxime have achieved widespread use (Table 2). Other oximes, particularly the Hagedorn oximes, have been studied for use in nerve agent poisoning, especially poisoningby soman (Dawson, 1994), and one (HI-6) has been the subject of a large case series in former Yugoslavia (Kusic et al., 1991), where it was employed to treat OP pesticide poisoning. This discussion focuses predominantly on PAM because this oxime is clinically used most frequently. 1. ACTION OF OXIMES The fundamental action o f the pyridinium oximes is to reactivate AChE inhibited by OPs. The pyridinium oximes also reactivate BuChE (Hobbiger, 1963; Worek etal.,
TABLE 2. Oxime
Pralidoxime
Obidoxime Asoxime
9Management of OP Pesticide Poisoning
1999), but dimethyl BuChE is reactivated more slowly than dimethyl ACHE. The action on BuChE is probably not clinically significant in the action of the pyridinium oximes, although it has been suggested that BuChE acts as a sink for anticholinesterases, in which case reactivation of the enzyme would presumably restore the capacity of the sink. The reactivation of AChE allows ACh to be hydrolyzed in the normal way, and therefore normal cholinergic neurotransmission will resume. It is usually considered that the beneficial effects of oximes in OP poisoning are confined to peripheral nicotinic sites and that CNS effects are clinically insignificant (Bismuth et al., 1992), although there is evidence that PAM can cross the blood-brain barrier (Sakurada etal., 2003). This means that the beneficial effects will mainly be on neuromuscular transmission, and that there will be little action on parasympathetic effects, such as bronchorrhea, bronchoconstriction, and rhinorrhea, or on CNS effects. 2. FACTORS LIMITING OXIME EFFICACY Three main factors limit the success of oxime therapy: the continuing presence of high concentrations of inhibitor in the plasma (Willems et al., 1993; Worek et al., 1997), aging, and the formation of phosphoryl oximes. The first depends on the dose and pharmacokinetics of the inhibitor: Fat-soluble OPs may form a deep compartment. Aging results from monodealkylation of the dialkoxyphosphorylated AChE (Karalliedde and Szinicz, 2001). Aged enzyme reactivates neither spontaneously nor under the influence of oximes so that recovery of enzymic activity depends on synthesis de novo of more enzyme. Although the tl/2 for aging of inhibited human AChE by soman is on the order of a few minutes, it is clear that for most OP pesticides the tl/2's for aging are much longer; for dimethoxyphosphoryl ACHE, it is 2-32 hr and for diethoxyphosphoryl AChE > 3 6 hr (Mason etal., 2000; Wilson etal., 1992; Worek et al., 1999). Similar differences exist in aging rates of dimethoxy- and diethoxy-inhibited BuChE (Mason et al.,
Oximes That Have Been Used in Human OP Pesticide Poisoning Salt
Iodide Chloride Mesilate, methanesulfonate Methylsulfate Chloride Chloride
719
Abbreviation a
2-PAMI 2-PAMC1 P2S 2-PAM methylsulfate
Proprietary name
Protopam
Contrathion Toxogonin
HI-6
aThere is some discrepancy in the use of the abbreviation2-PAM, so PAM is used for pralidoximein general in this chapter and the abbreviations as above for the salts. Many papers fail to state which salt was used. Note that the mesilate and methylsulfate are not the same salt.
720
SECTION I X . Therapeutic M e a s u r e s
1993), and these differences are important when considering BuChE activity after exposure to OPs and during recovery from their effects. With the typical dimethoxy and diethoxy structure of pesticides, aging rates are such that the phenomenon only becomes a problem when treatment is delayed and/or exposure prolonged. It is commonly, but erroneously, believed that 1 day after intoxication with an OP insecticide virtually all the phosphorylated enzyme will be in the aged form so that oxime therapy will be ineffective after this time. However, this interpretation derives from in vitro studies in which AChE is rapidly inhibited and is maintained fully inhibited thereafter by the presence of an excess of inhibitor and in the absence of oxime throughout the experiment. Such experiments do not represent the case in vivo and should not be used as a reason to abandon the use of oxime therapy after 24 hr. There is evidence from case reports and series that with dimethoxy pesticides, the efficacy of oximes may be reduced by aging. Because of concerns that dimethylphosphoryl enzyme might age sufficiently fast to interfere with the efficacy of oximes, studies have been carried out in vitro to investigate this (Ganendran and Balabaskaran, 1976; Skrinjaric-Spoljar et al., 1973). Less is known about structures other than the typical dimethyl and dethylphosphoryl enzyme and the effects of oximes. With AChE inhibited by profenofos (Glickman et al., 1984; Wing et al., 1984), an O-ethyl, S-propyl structure, aging occurs rapidly and oximes would be expected to be ineffective. With phosphoramidothioates, such as methamidophos, spontaneous and oxime-induced reactivation occurs and aging does not seem to be a problem: These data should not be extrapolated to all phosphoramidothioates, however, because fairly rapid aging occurs with the dithio analog of methamidophos (de Jong etal., 1982; Langenberg etal., 1988). Indeed, human plasma cholinesterase inhibited by propetamphos and crufomate, the former a phosphoramidothioate and the latter a phosphoramidate, appears not to undergo spontaneous reactivation (Mason et al., 1993). A third factor that may interfere with the reactivating capability of oximes is the formation of phosphoryl oximes in the AChE reactivating reaction because phosphylated oximes can act as AChE inhibitors (de Jong and Ceulen, 1978; Nenner, 1974). Fortunately, those produced by reactivation of AChE inhibited by pesticides appear not to be very stable in the plasma because they are hydrolyzed rapidly (Kiderlen et al., 2000; Leader et al., 1999). 3. ACTIONS OF OXIMES OTHER THAN REACTIVATION OF CHOLINEsTERASES
In a study on guinea pigs performed by Inns and Leadbeater (1983) involving pretreatment with pyridostgmine in both treatment regimens, the administration of bis-pyridinium compounds that are not oximes with atropine and diazepam was more effective than atropine and diazepam alone in
poisoning by certain OP nerve agents. This suggested that bis-pyridinium compounds did not depend entirely on the oxime moiety for their antidotal effects. There is evidence that oximes may react directly with OPs and such effects have been reviewed (Van Helden et al., 1996). Oximes may have direct activating and inhibitory effects on AChE (Alkondon et al., 1988; Kuhnen, 1971). It has been shown that both 2-PAMI and HI-6 interact with the nicotinic receptor-ion channel complex (Alkondon etal., 1988). It is unclear how much these actions contribute to the efficacy of oximes. Kusic et al. (1991) suggested that a general improvement in the clinical condition of poisoned patients, which was more rapid than the rise of AChE activity with HI-6, indicated direct beneficial pharmacological effects. In a study on the rat to evaluate noncholinesterase reactivating effects of the Hagedorn oximes, Van Helden et al. (1994) treated atropinized rats iv with three times the LD50 of crotylsarin, O-(2-butenyl) methylphosphonofluoridate. This compound produces an inhibited AChE that ages instantly, thus precluding any beneficial reactivating effects of the oxime. After 5 min, the poisoned rats were treated with saline or an oxime (obidoxime, HI-6, HL6-7, HGG-12, or HGG-42). The oximes significantly prolonged survival time compared to the saline-treated rats and the authors suggested that this effect must be by action other than cholinesterase reactivation. However, because the prolongation of survival time did not correlate with differences in pharmacokinetics of the oximes, potency in restoring neuromuscular transmission, and affinity for muscarinic receptors, the authors also concluded that the effect must be mediated through an unknown mechanism.
C. Pralidoxime 1. PHARMACOKINETICS
The majority of kinetic studies on PAM have been performed in healthy, nonpoisoned subjects. There is evidence from animal (Green et al., 1985, 1986) and human (Jovanovic, 1989) studies that OP compounds can alter PAM kinetics in a complex manner; this may be due to the cardiovascular changes and reduced blood flow seen in OP pesticide poisoning. Therefore, it may be inappropriate, to extrapolate the results of volunteer studies to severely poisoned patients. Because they are quaternary amines, PAM salts and obidoxime are not well absorbed after oral administration, although PAM chloride tablets remain commercially available. Ethanol does not appear to interfere with the absorption of PAM (Calesnick et al., 1967). The distribution of PAM is largely determined by its small molecular size and quaternary amine structure. It is thus widely distributed in most body fluids and is not significantly bound to plasma proteins (Sidell etal., 1972b). PAM penetrates the erythrocyte membrane by
CHAPTER 49 9Management of OP Pesticide Poisoning simple diffusion and does not bind to either red blood cell stroma or hemoglobin (Ellin et al., 1974). PAM does not pass readily into the CNS because of its quaternary nitrogen structure (Uehara et al., 1993). The in vivo rat brain microdialysis technique with high-performance liquid chromatography/ultraviolet was used by Sakurada et al., (2003) to determine whether 2-PAMI penetrated the blood-brain barrier. After intravenous injection of 2-PAMI at doses of 10, 50, or 100 mg/kg, PAM appeared in the dialysate and concentrations were dose dependent; the striatal extracellular:blood concentration ratio at 1 hr after a 50 mg/kg dose was 0.093 _+ 0.053 (mean _+ SEM). Neural uptake of PAM was sodium dependent. These results suggest that PAM may have some capability in reactivating CNS ACHE. Furthermore, Ligtenstein and Kossen (1983) found some entry of HI-6 into the CNS in the rat, as did Klimmek and Eyer (1986) in the dog. The values for the apparent volume of distribution in the central compartment (V1), peripheral compartment (V2), and at steady state [(Vd)ss] in volunteer studies are given in Table 3; the values at steady state are 0.6-0.8 liters/kg. However, in poisoned patients treated with methylsupfate and chloride salts, respectively, the mean (_+SD) volumes of distribution were found to be 2.77 +_ 1.45 liters/kg (Willems and Belpaire, 1992) and 2.8 + 2.21iters/kg (Jovanovic, 1989). For children treated for OP poisoning with a loading dose and then continuous infusion of PAM, the volume of distribution was higher in those severely poisoned (Schexnayder et al., 1998). PAM is metabolized only to a minor extent in humans (Gibbon et al., 1979). PAM is excreted rapidly in urine (Calesnick et al., 1967; Jager and Stagg, 1958; Loomis, 1963; Rivero Gonz~ilez et al., 2001; Sidell et al., 1972a,b; Sidell and Groff, 1971; Swartz and Sidell, 1973; Vojvodic and Maksimovic, 1972). Most studies, whether performed on humans or animals, appear to demonstrate first-order disappearance of PAM from the plasma (Jager and Stagg, 1958; Kondritzer et al., 1968; Sidell e t a l . , 1972b). Because urine clearance of PAM exceeds simultaneously measured creatinine clear-
TABLE 3.
ance, it is probable that PAM is secreted by the renal tubules, at least in part, by secreting mechanisms shared by several other strong bases (Swartz and Sidell, 1973). In several volunteer studies, the PAM half-life varied from 67 to 84 min (Table 3) after intravenous dosing of PAM 5-10mg/kg body weight. In poisoned patients treated with the methylsulpfate and chloride salts, respectively, the mean (_+ SD) elimination half-lives were found to be 3.44 +_ 0.9 hr (Willems and Belpaire, 1992) and 2.9 +_ 1.18 hr (Jovanovic, 1989). Josselson and Sidell (1978) investigated the effect of intravenous thiamine hydrochloride on the elimination of pralidoxime chloride 5 mg/kg body weight administered intravenously. The addition of thiamine lengthened the elimination half-life and the oxime concentration increased, whereas the intercompartment clearances and rate constant for elimination of oxime declined. The authors suggested that either thiamine and PAM compete for a common secretory mechanism or thiamine alters the membrane transport of PAM. PAM is excreted preferentially in acid urine (Berglund et al., 1962) and intravenous sodium bicarbonate markedly reduces PAM excretion. Swartz and Sidell (1973) observed that in six volunteers given 2-PAMC1 5 mg/kg body weight (but not atropine), exercise alone and exercise and heat stress significantly (p < 0.05) increased the PAM elimination half-life [71.2 _+ 7.4 min (control) vs 87.7 + 14.7 and 86.2 +_ 13.5 min, respectively). This suggests that exercise and heat reduce the renal elimination of PAM. Because oximes are quaternary ammonium compounds, they will be fully ionized in aqueous solution. Unless the accompanying anion has pharmacological properties of its own, there would thus be no expectation that the PAM salts would differ in activity, if used on molar equivalent bases, either qualitatively or quantitatively. Sidell e t a l . (1972b) compared the pharmacokinetics of 2-PAMC1 and P2S after intravenous administration to human volunteers. The two PAM salts at the same (mass) dose (5 mg/kg) produced virtually identical plasma concentration-time curves. The
Pralidoxime Kinetics after Intravenous Dosing in Volunteers Vd (liters/kg) a
Dose and salt
5 mg/kg chloride 5 mg/kg chloride 5 mg/kg chloride 5 mg/kg P2S 10 mg/kg chloride
721
tl/2 [~ (min)
V1
V2
(Vd)ss
Reference
78 71 67 84 79
0.27 0.37 0.18 0.20 0.30
0.54 0.39 0.42 0.58 0.46
0.82 0.76 0.60 0.78 0.76
Sidell et al. (1972b) Swartz and Sidell (1973) Josselson and Sidell (1978) Sidell et al. (1972b) Sidell and Groff (1971)
agl, central compartment; V2,peripheral compartment;(Vd)ss,volumeof distribution at steady state.
722
SECTION IX. T h e r a p e u t i c
Measures
tl/2's were similar, being 1.31 and 1.41 for 2-PAMC1 and P2S, respectively. The major difference was in Vd, which at steady state was 815 ___ 105 ml/kg for 2-PAMC1 and 775 ___204 ml/kg for P2S (mean ___SD).
VI. E F F I C A C Y O F O X I M E S A N D A T R O P I N E De Silva etal. (1992) concluded that nothing is to be gained in cases of severe acute OP insecticide poisoning by the addition of oximes to the standard regimen of atropine plus mechanical ventilation. This conclusion was based on a study in which 21 patients received atropine alone and 24 patients received atropine plus 2-PAMC1 (median doses, 4 g in the first 24 hr and 1 g daily thereafter). The mortality in both groups was 29%, which is not dissimilar to that reported from other centers managing severe cases of OP insecticide poisoning. Thus, the need for more effective treatment for OP poisoning is undeniable. However, in all probability the supposed failure of oxime therapy in this study does not indicate ineffectiveness of the drug employed nor, necessarily, does it indicate delay in administration but, rather, inadequate oxime dosing. The value of oximes has also been challenged on the basis of a systematic review of clinical trials (Eddleston et al., 2002). What, then, is the role of oximes?
A. Pharmacodynamic Studies in Animals Many of the relevant pharmacodynamic studies on pesticides are in the proprietary literature supporting registration of pesticides. These data can be accessed through the Joint Expert Meeting on Pesticide Residues monographs or through national pesticide registration authorities. Note that in these studies, the oxime therapy is usually given a few minutes after the OP challenge. This is not particularly representative of the clinical situation, in which the patient reaches the hospital some hours after poisoning: in such circumstances, these studies may give an unrealistic idea of the likely efficacy of oxime therapy, particularly with dimethoxy OPs, where aging may occur if oxime therapy is delayed. Examples of such studies are those with mevinphos, a dimethoxy OP (Kassa and Fusek, 1997), and methidathion, also a dimethoxy OP. In a rat study, atropine sulfate, PAM, obidoxime, and a combination of atropine sulfate and obidoxime were all effective when given at the first sign of poisoning with methidathion [Food and Agriculture Organization/World Health Organization (FAO/WHO), 1993]. A number of diethoxy OPs have been studied in relation to oximes, including diazinon, phosalone, pyrazophos, triazophos, and terbufos. In the study with diazinon, antidotal therapy comprised atropine and 2-PAMC1 and studies were undertaken in both rats and rabbits (FAO/WHO, 1994). In the latter, although there was enzyme reactivation, recrudescence of
poisoning was seen at 2 hr and the authors suggested that this indicated the need for repeated oxime dosing. In three studies reviewed by FAO/WHO (1994) on phosalone and 2-PAM methylsulfate in mice, P2S in rats, and obidoxime in mice, all appeared effective, although various aspects of the design of the studies were not optimal. Oximes (PAM or obidoxime) in combination with atropine were successful in rats experimentally poisoned with pyrazophos, and there was some indication that repeated dosing was required for optimal antidotal efficacy (FAO/WHO, 1993). In a rat study of experimental terbufos poisoning, little benefit was observed from PAM and atropine (FAO/WHO, 1991). In rat studies on triazophos, combinations of atropine sulfate and 2-PAMI or atropine sulfate and obidoxime were successful as experimental therapies (FAOAVHO, 1994). The effects of oximes in profenofospoisoned chicks and mice were reported to be limited, as expected, although atropine was effective (FAOAVHO, 1991). An interesting study was undertaken in rats by Jokanovic and Maksimovic (1995) in which the efficacy of PAM, obidoxime, HI-6, and trimedoxime, given with atropine and diazepam 1 min after poisoning, was tested in the treatment of poisoning with two LDs0's of 25 different OP insecticides and one OP fungicide (pyrazophos). It was shown that the oximes were potent antidotes in poisoning with phosphate insecticides. Obidoxime, PAM, and HI-6 had low effectiveness in the treatment of poisoning with the phosphonate trichlorfon. However, none of the oximes were effective antidotes in poisoning with dimethoate and pyridaphenthion. The reasons for some of these differences are unclear. Trimedoxime was the most effective oxime in the treatment of insecticide poisoning, being especially efficacious at the lowest tested doses. In animal toxicology and pharmacodynamic studies, "CNS AChE activity" is often reported, usually measured on brain homogenate. Studying the effect of the nerve agents tabun, satin, and VX on ACHE, Gupta et al. (1987, 1991) found that there were major differences in the degree and time course of AChE inhibition between different brain regions (and between brain and muscles). The use of whole brain homogenates could clearly obscure regional changes in AChE activity (see review by Gupta, 2004). In interpreting brain and erythrocyte AChE and plasma BuChE data in animal studies, the same considerations should be borne in mind as would be for erythrocyte AChE and plasma BuChE data in humans. That is, red blood cells cannot synthesize AChE de novo; BuChE is a different gene product from ACHE, with its own kinetics of inhibition, reactivation, and aging; and the enzyme one is really interested in is AChE in the central and peripheral nervous systems. It is important not to overinterpret such data, which are influenced by numerous pharmacokinetic factors. Additionally, it is sometimes necessary to examine the methodology of enzyme activity measurement in detail because BuChE
CHAPTER 49 9Management of OP Pesticide Poisoning can hydrolyze acetylcholine and therefore some "ACHE" activity may in fact be due to BuChE: Specific inhibitors are available for use in assays (St. Omer and Rottinghaus, 1992). A further consideration is that recovery of activity of brain enzyme cannot be assumed to be due to reactivation of the enzyme because synthesis de novo of AChE in brain may be quite rapid. The literature on atropine, oximes, and nerve agents is enormous and some of it is of relevance to the management of pesticide poisoning. However, some caution is necessary because pharmacodynamic studies on nerve agents and oximes frequently employ pretreatment with carbamate drugs. Moreover, much of the literature is primarily concerned with treatment of soman poisoning, in which the inhibited enzyme rapidly ages; here, beneficial actions of oximes are presumably due to their noncholinesterase reactivating properties. Rapidly aging pesticide-inhibited AChE only occurs with one or two pesticides, such as profenofos, and is therefore not a widespread problem. The lessons of the pharmacodynamic studies on OPs, with relevance to pesticides, can be (cautiously) summarized as follows: 1. Typical inhibited cholinesterases produced by dimethoxy and diethoxy OPs can be reactivated by oximes. 2. Delay in the treatment of OP poisoning produced by dimethoxy OPs may result in a significant proportion of the inhibited cholinesterase undergoing the aging reaction and becoming refractory to oxime-induced reactivation. 3. Prolonged therapy may be of value in poisoning by diethoxy OPs. 4. Nonreactivating effects of oximes may produce therapeutic benefit in poisoning by those few pesticides for which aging is a problem.
B. Efficacy: Case Reports and Case Series in Humans In a case series, Willems et al. (1993) reported that ethyl parathion and methyl parathion poisoning could be treated effectively with 2-PAM methylsulfate (plasma concentrations approximately 4 mg/liter) and atropine provided that the pesticide plasma concentrations were low. In more severe cases, when the plasma pesticide concentrations were >30 ixg/liter, even plasma PAM concentrations of 14.6 mg/liter did not produce improvement. In dimethoate poisoning, omethoate-inhibited enzyme could not be reactivated at PAM concentrations of 6.3 mg/liter (Willems et al., 1993). In a case report, continuous PAM infusion (with atropine sulfate) was successful in treating chlorpyrifos poisoning, and the nicotinic signs and symptoms were controlled (Tush and Anstead, 1997). In a case series, Thiermann et al. (1997) reported that in parathion poisoning, obidoxime
723
(250 mg iv as a bolus followed by 750 mg/day by infusion) was effective, but that in severe poisoning, reactivation did not occur until the concentration of inhibitor in the plasma had declined. Obidoxime (250 mg iv as a bolus followed by 750 mg/day by infusion) was ineffective with oxydemeton methyl when oxime therapy was delayed more than 1 day after poisoning. In another case series, Thiermann et al. (1999) reported that in parathion poisoning, reactivation was possible 7 days after poisoning, whereas with oxydemeton methyl, response was only seen when obidoxime therapy was instituted soon after poisoning. Similarly, Zilker et al. (1997) reported that obidoxime (750 mg/day by infusion) drastically reduced the need for atropine in parathion poisoning, but that demeton-S-methyl poisoning only responded to obidoxime if therapy was instituted soon after intoxication. Human experience of oximes, other than PAM salts and obidoxime, is scanty. An exception is a case series reported by Kusic et al. (1991). The oxime HI-6, administered four times daily as a single intramuscular injection of 500 mg, accompanied by atropine and diazepam therapy, was studied in OP pesticide poisoning. Oxime treatment was started on admission and continued for a minimum of 48 hr and a maximum of 7 days. Most patients were treated with HI-6, but nine patients severely poisoned with quinalphos were treated with 2-PAMC1 (1000 mg four times daily). HI-6 rapidly reactivated human red blood cell AChE inhibited by diethoxy OPs (phorate, pyridaphenthion, and quinalphos) as well as that inhibited by dichlorvos. With the exception of dichlorvos, enzyme inhibited by dimethoxy OPs (dimethoate and phosphamidon) was reported to be generally resistant to treatment by HI-6, whereas reactivation with malathion was poor. Both HI-6 and 2-PAMC1 reactivated erythrocyte AChE in quinalphos-poisoned subjects, but reactivation was much more rapid following the use of HI-6. The general improvement of poisoned patients, which was sometimes more rapid than the increase in AChE activity with HI-6, suggested that direct pharmacological effects were occurring. However, this may be an overinterpretation of the data because the clinical status of patients is determined by the degree of inhibition of neuronal ACHE, which may not be mirrored by the inhibition of red blood cell cholinesterase. No adverse side effects were noted when plasma concentrations of HI-6 were maintained above therapeutic concentrations for up to 7 days.
C. The Choice of Oxime Possibly for historical reasons, as discussed previously, in English-speaking countries together with France, the use of PAM salts is standard, whereas in German-speaking countries, obidoxime is used. Thus, it is of interest to consider whether one or the other oxime is better. Based on studies using OP pesticide-inhibited human AChE in vitro, Worek
724
SECTION I X .
Therapeutic Measures
et al. (1996) concluded that obidoxime was superior to 2-PAMI in reactivation capability. The pesticides and pesticide oxons used were chlorfenvinphos, dichlorvos, dicrotophos, heptenophos, mevinphos, monocrotophos, paraoxon, phosphamidon, trichlorfon, malaoxon, omethoate, oxydemeton-methyl, and methamidophos, and the inhibition time was 30 min. The Hagedorn oxime HI-6 appears to work in pesticides poisoning, although there are data in vitro using human AChE suggesting that HI-6 would be less effective in poisoning by dimethoxy pesticides than either PAM or obidoxime (Worek et al., 1999). Another Hagedorn oxime, HL6-7, has a broad spectrum of activity in nerve agent poisoning, being capable of reactivating human erythrocytic AChE inhibited by the nerve agent tabun (de Jong et al., 1989). However, there is no evidence that the use of HL6-7 would produce any benefit compared to existing oximes in pesticide poisoning.
D. O x i m e D o s i n g R e g i m e n Oxime administration must produce clear, irreversible, clinical improvement and achieve rapid reactivation of ACHE. This must be achieved without producing major adverse effects and the need to monitor oxime concentrations. Reactivation of inhibited AChE and clinical improvement depend on the chemical form of inhibited ACHE, the plasma oxime concentration, the duration of oxime therapy, and the plasma OP concentration. Early experiments on anesthetized cats (possibly seven animals) given lethal doses of intravenous sarin and P2S 10 mg/kg intramuscularly, but not atropine, established that plasma PAM concentrations higher than 4 mg/liter were required to counteract neuromuscular block in vitro and bradycardia, hypotension, and respiratory failure in vivo (Sundwall, 1961). Crook e t a l . (1962) gave dogs oral P2S and 2-PAM lactate, 30-115 mg/kg body weight, 1-5 hr before exposure to sarin vapor. Atropine 5 mg/kg body weight was administered 1 min after the dogs were exposed
TABLE 4.
to sarin. The authors extrapolated from this study of dogs to humans and concluded that a plasma PAM concentration of at least 3 mg/liter would be required "for reasonably protective attenuation of the toxic effects of OP anfi-ChEs." The relationship between plasma oxime concentrations after dosing with PAM and obidoxime and protection against sarin poisoning has been investigated by Shiloff and Clement (1987) in rats (Table 4) and by Bokonjic et al. (1987) in quinalphos-poisoned rats (Table 5). A study on poisoned patients showed that reactivation of dimethoateinhibited enzyme was not achieved with a plasma PAM concentration of 6.37 mg/liter (Willems et al., 1993). In the same study, it was shown that reactivation of inhibited enzyme did not occur, even in the presence of a plasma PAM concentration of 14.6 mg/liter, when plasma ethyl and methyl parathion concentrations were >30 t~g/liter. Thus, the modest doses of PAM that have often been recommended in the past (to achieve plasma oxime concentrations of approximately 4 mg/liter) will be insufficient to produce not only reactivation of phosphorylated enzyme but also a lasting clinical improvement, unless the patient is only mildly poisoned. Moreover, clinically effective PAM concentrations need to be maintained as long as inhibitory oxons (active OP metabolites) are circulating. Data from case reports suggest that PAM concentrations of 40 rag/liter may be required to produce reactivation of inhibited AChE in some cases (Casey et al., 1995a,b). How may such plasma PAM concentrations be achieved and maintained if reactivation of inhibited AChE is to occur and clinical improvement is to ensue in severe cases? Sidell and Groff (1971) administered 2-PAMC1 at a dose of 10.0 mg/kg intravenously to volunteers: PAM concentrations > 10 mg/liter were maintained for approximately 30 rain. Medicis et al. (1996) administered 16 mg/kg 2-PAMC1 over 30 min or 4 mg/kg over 15 min, followed by 3.2 mg/kg/hr for 3 hr 45 min to human volunteers, using a randomized crossover design. Concentrations in plasma > 10 mg/liter were maintained for approximately 60 min after bolus infusion, but were not achieved following
Relationship between Plasma Oxime Concentrations and Mortality in Sarin-Poisoned Rats Also Given Atropine 17.4 mg/kg a
Oxime
n
Mean (_+ SD) oxime concentration (mg/liter)
Pralidoxime Pralidoxime Pralidoxime Obidoxime Obidoxime Obidoxime
5 4 5 10 8 5
0.7 _+ 0.1 2.0 _+ 0.4 3.3 _+ 2.3 3.6 + 0.2 9.2 _+ 0.6 19.7 _+ 3.7
aAfter Shiloff and Clement(1987).
% Mortality 100 80 20 90 62.5 0
CHAPTER 49
9Management of OP Pesticide Poisoning
725
TABLE 5. Relationship between Plasma Pralidoxime Concentrations and LD50 in Quinalphos-Poisoned Rats Also Given Atropine 10 mg/kg and Diazepam 2.5 mg/kg a Mean (--. SEM) plasma PAM concentration (mg/liter) 0 0.8 1.5 + 0.6 2.9 _+ 0.7
Mean (-4--SEM) LDs0 (mg/kg)
Protective index
10.5 _+ 3.8 353.6 + 33.6 457.9 _+ 121.6 498.7 +_ 137.9
33.7 43.6 47.5
aAfter Bokonjic et al. (1987).
the continuous infusion, when concentrations of approximately 6 mg/liter were observed. After a single intramuscular injection of 2-PAMC1 30 mg/kg, PAM concentrations >10 mg/liter were maintained for approximately 2 hr in volunteers (Calesnick et al., 1967). Also, after an intravenous infusion of 2-PAMC1 30 mg/kg, PAM concentrations > 10 mg/liter were maintained for approximately 30 min (Calesnick et al., 1967). In children with OP poisoning, Schexnayder et al. (1998) found that mean (_+SD) PAM steady-state concentrations were 22.2 mg/liter after a loading dose of 2-PAM C1 25-50mg/kg, followed by a continuous infusion of 10-20 mg/kg/hr. Green et al. (1985) investigated the impact of poisoning on PAM concentrations. Guinea pigs were administered 2-PAMC1 25 mg/kg and atropine 16 mg/kg intramuscularly 1 min after subcutaneous sarin and soman at various multiples of the LDs0. The PAM concentrations remained above 25 mg/liter for the duration of observation (10 min). Generally, the higher the LDs0, the higher the PAM concentration (>30 mg/liter). The plasma PAM concentrations were measured both in volunteers and in poisoned patients after the administration of 2-PAMC1 1000 mg intramuscularly (Jovanovic, 1989). Mean plasma PAM concentrations were almost one and half times higher in patients compared to volunteers. Based on these data (Casey et al., 1995a,b; Willems et al., 1993; Willems and Belpaire, 1992), we propose that 2-PAMC1 or P2S 30 mg/kg should be administered by intravenous injection as soon as possible after exposure. Repeat doses at 4- to 6-hr intervals or, preferably, an intravenous infusion of 8-10 mg/kg/hr should then be given. Administration of PAM should continue for as long as atropine is r e q u i r e d - that is, until clear, irreversible clinical improvement is achieved, which may take many days while residual insecticide is cleared from the body stores. E. A d v e r s e Effects of O x i m e s The toxicology of oximes has been reviewed (Dawson, 1994; Marrs, 1991). With the first oximes used in OP poisoning, such as diacetylmonoxime, it was suggested that
some toxic effects could be due to cyanogenesis. In the case of P2S, this seems unlikely to be important in toxicity (Ballantyne et al., 1975; Enander et al., 1961). In the context of the treatment of acute OP pesticide poisoning, the acute toxicity is the only consideration of importance. The acute toxicity of PAM salts and of obidoxime is of the same order of magnitude. Impaired liver function has been observed after treatment of human parathion poisoning with obidoxime (Barckow et al., 1969; Wirth, 1968), and reversible fatty change has been reported in the liver of rats treated with that oxime (Bisa et al., 1964). However, Boelcke and Gaaz (1970) did not find evidence of hepatotoxicity in mice in terms of enzyme elevation, and bilirubin clearance and bromsulfthalein retention were unaffected in the rat (Boelcke and Kamphenkel, 1970). Therefore, some or all of the hepatotoxicity of obidoxime may be attributable to other factors, such as the OP or solvents. In human volunteer studies, side effects have been seen with oximes. Thus, Jager and Stagg (1958) found that medical students given 2-PAMI developed dizziness, blurred vision, diplopia, impairment of accommodation, and headache. Dizziness, blurred vision, and, occasionally, diplopia have been reported in human volunteer studies after intravenous or intramuscular administration of 2-PAMC1 (Sidell and Groff, 1971). In a study on obidoxime, male volunteers were given tablets in quantifies ranging from 1.84 to 3.58 g as a single dose or 7.36 g divided into four equal doses. More than half the subjects complained of one or more side effects: pallor, nausea, burning sensation, headache, generalized weakness, sore throat, and paresthesia of the face. Activities of blood cholinesterase, alanine, and aspartate aminotranferases, as well as hematocrit values, heart rate, and blood pressure, were not affected (Simon and Picketing, 1976).
VII. A N T I C O N V U L s A N T S A large number of anticonvulsants have been studied in animals or used in OP poisoning (Sellstr6m, 1992). Many candidate anticonvulsants have been investigated in attempts
726
SECTION IX- T h e r a p e u t i c
Measures
to improve the treatment of OP nerve agent poisoning, such as the water-soluble diazepam prodrug avizafone (Lallement et al., 2000); other benzodiazepines, such as clonazepam (Lipp, 1974) and midazolam (Pieri etal., 1981); and anticonvulsants of other types, such as barbiturates and phenytoin. Other drugs that have been studied include tiagabine (a GABA uptake inhibitor) and glutamate receptor antagonists (Shih and McDonough, 1999). Only diazepam and midazolam have achieved widespread use in the treatment of OP pesticide poisoning. Anticonvulsants including benzodiazepines, especially diazepam, were originally studied in OP poisoning for the symptomatic relief of OP-induced convulsions. Benzodiazepines are CNS depressants, anxiolytics, and muscle relaxants (Diamantis and Kletzkin, 1966). The main site of action of benzodiazepines is the 7-aminobutyric acid A (GABAA) receptor. The GABAA receptor is a ligand-gated chloride ion channel (Ortells and Lunt, 1995), the GABAergic system being the major inhibitory neurotransmission system in the mammalian CNS. Benzodiazepines including diazepam alter GABA binding at the GABAA receptor in an allosteric manner, but these drugs do not activate the GABAA receptor by direct action (Charney et al., 2001). Nevertheless, the overall effect is to increase the inhibitory action of the GABAergic system. Data from experimental nerve agent poisoning (Anderson et al., 1997; Hayward et al., 1990) suggest that benzodiazepines, such as diazepam and midazolam, ameliorate or prevent the development of pathological changes in the CNS.
A. Diazepam Diazepam is the anticonvulsant that has been most studied for use in OP pesticide poisoning. In this context, the most likely mode of administration is by intravenous injection, but other modes of administration have been considered for self-administration or when administration by those not trained in intravenous injection is required. In OP-induced convulsions, diazepam would be given intravenously. 1. PHARMACOKINETICS In healthy human subjects, a peak mean serum concentration of 1607 ~g/liter was found 15 rain after single bolus intravenous injections of 20 mg (Hillestad et al., 1974). A two-compartment open model has been used to describe elimination kinetics of diazepam in humans after single intravenous injections were reported (Andreasen etal., 1976; Klotz et al., 1975, 1976). A two-compartment open model has also been used to describe elimination kinetics of diazepam in experimental animals; however, there were major interspecies differences in parameters such as tl/2 and Vd (Klotz et al., 1976), which indicated caution in the interpretation of animal studies. In human volunteers, the plasma protein binding of diazepam was greater than 95% (Klotz et al., 1976). The tl/2 of diazepam appears
to increase when liver damage is present and with age (Andreasen etal., 1976; Herman and Wilkinson, 1996; Klotz et al., 1975). It appears that intramuscularly administered diazepam produces lower peak blood levels in humans than does either intravenous injection or oral administration (Hillestad et al., 1974), whereas absorption following oral administration is almost complete with bioavailability close to 1 (Mandelli et al., 1978). 2. PHARMACODYNAMICS In a variety of pharmacodynamic studies on experimental animals, it can be concluded that diazepam adds to the effects of atropine and of the classical combination of atropine and pyridinium oximes (Bokonjic etal., 1987; Gupta, 1984; Kassa and Bajgar, 1994; Kleinrok and Jagiello-Wojtowicz, 1977; Krutak-Krol and Domino, 1985; Rump and Grudzinska, 1974). There seems to be little doubt that in experimental animal models, diazepam can prevent, stop, and/or ameliorate convulsions due to OP pesticides and render less severe or even prevent structural changes in the brain, but the effects on lethality are less clear (Marrs, 2003).
3. EFFICACY: CASE REPORTSAND CASE SERIES In the scientific literature, there are many case reports and series of the apparently successful treatment of OP insecticide poisoning with diazepam as an adjunctive but widely accepted therapy (Karalliedde and Szinicz, 2001). Examples include Barckow et al. (1969), Vale and Scott (1974), Yacoub etal. (1981), Merrill and Mihm (1982), Martf etal. (1985), LeBlanc etal. (1986), de Kort etal. (1988), Jovanovic etal. (1990), Kusic etal. (1991), and Weissmann-Brenner et al. (2002). In most cases, the indication for the use of diazepam was convulsions, but diazepam has also been used to control muscle fasciculation and agitation. In the case of convulsions, the adult dose is 10-20 mg iv, whereas that for children is 0.2 or 0.3 mg/kg iv. The elderly should receive half the adult dose. In the absence of convulsions, diazepam administration at doses of 5-10 mg intravenously has also been recommended in cases of OP poisoning accompanied by anxiety and restlessness (Johnson and Vale, 1992). If large doses of diazepam are required to suppress seizure activity, phenytoin should be considered as an alternative (Johnson and Vale, 1992).
VIII. O T H E R M E T H O D S OF A N T I D O T A L TREATMENT A number of novel approaches to the antidotal treatment of OPs have been studied, often using prophylactic protocols and mostly in relation to nerve agent poisoning, but some may be applicable, at least in principle, to OP pesticide poisoning.
CHAPTER 49 9Management of OP Pesticide Poisoning
A. Enzymes 1. CHOLINESTERASE Both AChE (Maxwell e t a l . , 1999; Wolfe, 1992; Wolfe et al., 1987) and BuChE (Broomfield etal., 1999) have been studied as scavengers for nerve agents.
2. PHOSPHOTRIESTERASE McGuinn et al. (1993) described a study in which squid DFP-hydrolyzing enzyme (DFPase) was entrapped within mouse erythrocytes. These red blood cells were shown to be capable of hydrolyzing DFP. This approach would presumably be effective against other OP esters that are hydrolyzed by DFPase. In a similar approach, Pei et al. (1995) reported that resealed murine erythrocyte cells containing recombinant phosphotriesterase protected against the lethal effect of paraoxon in mice. Also, when these carrier cells were administered in combination with 2-PAM and/or atropine, synergy was reported.
B. Calcium Channel Blockers Calcium channel blockers have been studied in OP poisoning (e.g., nimodipine) (Dretchen et al., 1992; Karlsson et al., 1994).
C. Adenosine Receptor Agonists Adenosine receptor agonists were reported to prevent clinical signs and increase survival in soman, satin, and DFP poisoning (Harrison et al., 2003; Tuovinen, 2004; Van Helden et al., 1998).
D. N-Methyl-D-Aspartate Receptor Antagonists Although the initial stimulus for seizures appears to be cholinergic overactivity, as the seizures develop, other excitatory neurotransmission systems become involved, including the glutamatergic system. The N-methyl-Daspartate (NMDA) receptor is a subtype of glutamatergic receptor: antagonists at this receptor, dizocilpine and 3-((R, S)-2-carboxypiperazin-4-yl)-propyl- 1-phosphonic avid, were found in mice experimentally poisoned with chlorfenvinphos to block seizures (Dekundy et al., 2001). It has been suggested that the beneficial activity of caramiphen, an anticholinergic drug, in soman poisoning may be modulated through activity at the NMDA receptor (Raveh et al., 1999, 2003).
E. Blockade of Acetylcholine Synthesis or Uptake An obvious therapeutic measure in AChE decrease the synthesis of ACh. Sterling et al. that administration of acetylsecocholinium to soman enhanced the protective effects of
would be to (1988) found 30 min prior atropine and
727
2-PAMC1 in the rat, whereas administration of N-hydroxyethylnaphthylvinylpyridine a few minutes before soman reduced mortality due to soman. Acetylsecocholinium is an inhibitor of high-affinity choline uptake and a choline acetyltransferase (CHAT) inhibitor, and N-hydroxyethylnaphthylvinylpyridine is a ChAT inhibitor. N-allylquinuclinidol, another inhibitor of high-affinity choline uptake, reduced mortality. Gray et al. (1988)studied naphthylvinylpyridine derivatives as antidotes for nerve agent poisoning and concluded that their beneficial experimental action in mice and guinea pigs was not related to ChAT inhibition.
IX. PREVENTION AND TREATMENT OF INTERMEDIATE SYNDROME Assuming that intermediate syndrome (IMS) is a consequence of ACh accumulation at the neuromuscular junction, oxime cholinesterase reactivators should protect against the development of IMS. It is noteworthy, and possibly relevant, that 2-PAM methylsulfate was reported to protect against the development of muscle fiber necrosis consequent on exposure to OP pesticides (Cavaliere et al., 1998). Johnson et al. (1996) reported experience with two treatment regimes of PAM in the treatment of patients with OP poisoning in a prospective trial. Seventy-two adults presenting with a history of consumption of OP compounds and requiting intensive care were entered into the trial. Patients were randomized using a block randomization to receive either a single bolus dose of 1 g PAM at admission followed by placebo infusion during the next 4 days or a single placebo bolus at admission followed by 12 g PAM as a continuous infusion during the next 4 days. A higher prevalence of IMS was observed in the latter group. Analysis of patients who received at least 1 g PAM within 12 hr of ingestion of the OP poison and those who received PAM after 12 hr suggested that the time of administration of PAM was important because the first group was less likely to develop IMS. Other case reports have suggested that a high dose of oxime may not avert IMS. Thus, in a case of severe malathion self-poisoning, 1 g PAM was administered iv approximately 7 hr after poisoning and at 12 hr an infusion of 400 mg/hr PAM was administered iv. The patient developed IMS on hospital day 3 (Sudakin et al., 2000).
X. PREVENTION AND TREATMENT OF ORGANOPHOSPHATE-INDUCED DELAYED POLYNEUROPATHY There is no recognized antidotal treatment for OPIDR Numerous substances, including certain carbamates, phenyl methane sulfonyl fluoride, n-butane-sulfonyl fluoride, and some phosphinates, have been shown to prevent the development of OPIDP when given before neuropathic OPs to
728
SECTION I X .
Therapeutic Measures
sensitive species such as hens (Johnson and Vale, 1992). Furthermore, Petrovic et al. (2000) found that elements of the conventional treatment used in humans (atropine, trimedoxime, and midazolam), if injected into hens prophylactically, ameliorated the subsequent development of OPIDP induced by D F E Jokanovic et al. (2001) found that a combination of trimedoxime, atropine, and methylprednisolone given 20 min before experimental poisoning of hens with DFP reduced the severity of the OPIDP that developed subsequently. The effect was less if the treatment was given after the D F E None of the previously mentioned studies provide any support for the efficacy of any postexposure antidotal therapy, so the treatment of OPIDP is essentially symptomatic.
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SECTION I X . T h e r a p e u t i c
Measures
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9M a n a g e m e n t of OP Pesticide Poisoning
733
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Index
Page references in bold type refer to tables.
A (atypical) variant, butyrylcholinesterase, 189-194, 204, 216 Abortions, spontaneous, 474 Absorption anticholinesterases, interspecies variation, 148 body temperature and, 558 dermal, s e e Dermal absorption/toxicity PBPK models, 108-114 percutaneous, 569-570; s e e a l s o Dermal absorption/toxicity sweating and, 562-563 transappendageal, 411 Accelerated solvent extraction, 682 Acceptable Daily Intakes pesticide mixtures, 608 in risk assessment, 543 WHO/FAO guidelines, 644-645,646-647, 648-649
Accidental poisonings, in children, 602 Acephate degradation products, 693 homeowner use, cancellation/phase out, 630 Acetylation, 130 carbamates metabolism, 137-138 xenobiotics metabolism, 130 Acetylcholine, 3 accumulation, 703 and adenosine receptor agonists, 260 and muscarinic receptor downregulation, 236 and myopathy, 511-512 active site, 90 Alzheimer's disease, levels, 26, 30, 42 autoregulation of, 259 cholinergic crisis, 451 cholinergic receptor binding, 145, 209 cholinesterase hydrolysis of, 199, 211-213 developmental chlorpyrifos exposure, 298-300 glutamate and, 513 hyperthermic response, 551 regulation, 271,275-276, 282
retinal, 435 role in placenta, 472-473 in senile plaques, 29 synthesis, 275 tissue distribution, 451 vascular smooth muscle effects, 29, 381-382 Acetylcholine receptors carbamates and, 5 overstimulation, 703 Acetylcholinesterase active site, 104, 213 activity measurements, 199-205 aged, 104, 152, 215-216, 716 and oxime efficacy, 719 binding sites, 175-179 active center gorge, 174-176, 210 acyl pocket, 177-179, 209, 210, 214 choline, 176, 177-178, 179-180 oxyanion hole, 162, 211 peripheral, 212 serine residue, 165-168, 213 stereoselectivity, 210, 214 as biomarker, carbofuran exposure, 666 brain, and core temperature, 555-556 carbamylation of, 151-152 catalytic turnover rates, 104, 175, 182, 213 control of cholinergic neurotransmission, 276 distribution in eye, 423-424 erythrocyte activity measurements, 577-579, 716-717 organophosphate-binding, 704 genes, 168, 172-173, 194, 206 genetic variants, 204-206, 216 knockout mice, 260-261,703 morphogenic activity, 240-241 nerve agent antidote, 727 organophosphate deactivation, 150 pharmacogenetics, 194-195 phosphorylation, 152, 214-216 placental, 472-473 prophylaxis, soman, 195
735
reactivation, s e e Acetylcholinesterase inhibition, reactivation reactivators, 719-722; s e e a l s o Oximes role of, 209 structure interspecies variation, 152-153 primary, 161-162, 163-167, 168, 169-171 quaternary, 181 secondary/tertiary, 168, 174-181 variants, 181-182 substrate specificity, 209 tissue distribution, 161, 187 tolerance development and, 263 Acetylcholinesterase inhibition carbamates, 90 structure-activity relationships, 214 carbofuran-induced, 660 interspecies variation, 151-152 irreversible, 703 kinetic modeling, 211-213 and muscarinic receptor down-regulation, 236 organophosphates, 90 neurotoxicity, 234-235 structure-activity relationships, 213-214 and oxidative stress biomarkers, 519 reactivation, 152 and behavioral recovery, 350-351 carbamates, 103, 104 d e n o v o synthesis, 263 organophosphates, 103, 104 oxime-induced, 214-215, 716 spontaneous, 716 time course, 199-200 tolerance, s e e Tolerance development WHO/FAO activity guidelines, 646 Acetylcholinesterase inhibitors for activity measurements, 201 in Alzheimer's disease, 17, 22, 27-30 analytical methods, 694-695 and dementia with Lewy bodies, 29-30
736
Index
Acetylcholinesterase inhibitors (continued) effects on amyloid/3 peptide, 28-29 on cerebrovascular parameters, 29 excitotoxic, 511 on tau protein, 29 first generation, 17 glaucoma and, 22 history of, 3-4 immune system and~ 495 indications, 389 irreversible, 4; see also Organophosphates memantine and, 39-42 myasthenia gravis and, 22 in organophosphate nerve agent poisoning, 23 oxidative stress, 511-515 and Parkinson's disease dementia, 29-30 reversible, 4; see also Carbamates second generation, 17 urine voiding dysfunction and, 22 uses, 3 in veterinary medicine, 23 Acetylcholine synthesis, blocking, 727 Acetyl coenzyme A, 275 Acetylesterases, 129 Acetylpeptide hydrolase, 707 Acetylsalycylic acid, developmental neurotoxicity, 638 Acetylsecocholinium, 727 Acetylthiocholine, 112-113, 182, 199 Acetyltransferases, role of, 130 ACHE, 194-195,206 Acid phosphatase, 453-454 Acrylamide, developmental neurotoxicity, 634 Actin-myosin cross-bridges, 382 Action potential effect of pesticides, 339-340 organophosphate-induced cardiotoxicity, 383-386 Activated charcoal, 717 Active transport, placental toxicity, 465 Acute akinetic rigidity syndrome, 280 Acute Emergency Guideline Levels, 399 Acute Exposure Guideline Levels, 48, 57-62 Acyl peptide hydrolase, 281 Acyl pocket binding site acetylcholinesterase, 177-179, 209, 210,214 butyrylcholinesterase, 176, 214 carboxylesterases, 224 cholinesterases, 175-176, 177-179, 209, 210,214 AD-DX 384, 258-259 Addictive behaviors, cholinergic modulation of, 272 Adenosine, 260 Adenosine receptor, tolerance development, 260
Adenosine receptor agonists, and acetylcholine accumulation, 260 Adenosine receptor antagonists, 727 Adenylyl cyclase signaling, 704 chlorpyrifos-induced alterations, 239, 295, 296-297, 301 inhibition, 236, 237, 240 Adipose tissue, accumulation of organophosphates, 536 Adolescents, acute poisoning incidences, 572 Adrenocorticotrophic hormone, 498 Adulticides, 601 Adverse drug reactions, pharmacogenetics and, 187 Adverse-observed effect level, agricultural pesticides, 585 Aedes, pesticide resistance, 601 Aedes aegypti, 600, 601 Aerodynamic diameter, inhaled particles, 401 Aerosols inhalation exposure, 401-402, 570 respiratory absorption of, 404 A-esterases, 128-129 activity, interspecies variation, 150 detoxification potential, 52 in organophosphate metabolism, 106, 109-110, 133 modeling, 109-118 role of, 247 Affective function cholinergic modulation of, 274 and chronic pesticide exposure, 351,352 Africa, vector-b0rne disease, 599-600 Age acute pesticide exposure, neurobehavioral effects, 354-356 and anticholinesterase sensitivity, 154-155 and butyrylcholinesterase activity, 204 and dermal absorption, 416 OPIDN and, 362 organophosphate sensitivity and, 91, 118-119 and paraoxonase activity, 251 pesticide sensitivity and, 448 Aggregate risk, defined, 618 Aggregate risk assessment, 618-620 Aggression, cholinergic modulation of, 274 Aging acetylcholinesterase, 104, 152, 215-216, 716 and oxime efficacy, 719 cholinesterases, 215-216 nerve agents, 51, 83 neuropathy target esterase, 361,662 of organophosphates, 183 push-pull mechanism, 215 Agitation, and Alzheimer's disease, 26 Agriculture workers children of, 604-605 neurobehavioral effects in, 351,352
Agrochemicals and male reproduction, 451 and Parkinson's disease, 281 AIDS, dementia associated with, 39 Airborne exposure guidelines, 581-582, 585 Air samples extraction methods, 682 nerve agent analysis, 694 Airway hyperreactivity, 237-239, 574, 707 Airway resistance, 391 Albumin, organophosphate binding, 148, 706, 707, 708 Aldicarb age-related sensitivity, 355 developmental neurotoxicity, 639 endocrine disruption, 485 history of, 3, 134 immune system effects, 501,503 -induced myopathy, 512 interaction with memantine, 41 metabolism, 137 metabolites, 607 mixtures, 610 poisoning incidences, 4 sulfone, 607 toxicity, 4 Aldicarb sulfoxide, 607 Algae, carbofuran toxicity, 659 Aliesterases, 148, 150, 574 Alimentary tract, and ocular absorption, 431-432 Alkali thermionic detector, 691 Alkylating agents, cholinesterase inhibitors as, 498 Alkylphenols, estrogen receptor binding, 452, 458 Alkyl phosphates analysis, 691 excretion, 691 occupational exposure, 490 Alkyl pyrophosphates, lethal inhalation toxicity, 405 Alkyltins, developmental neurotoxicity, 638 Allergic sensitization, pesticides, 504 a-fl hydrolase fold carboxylesterases, 224 cholinesterases, 174 Alveoli, pulmonary toxicity, 394 Alzheimer's disease acetylcholine levels in, 42 amyloid hypothesis, 28-29, 30 cholinergic hypothesis, 25-26 cognitive reserve, 28 dementia, 26, 36-38 functional decline in, 26 nicotinic agonists and, 260 pathogenesis, 25, 28-29, 30, 35 prevalence, 25, 42 risk factors, 28 seizures and, 279
Index
symptoms, 26, 42 treatment acetylcholinesterase inhibitors, 3, 4, 17, 22, 27-30 acetylcholinesterase variants and, 194 approaches to, 41 cholinesterase inhibitors, 283 galantamine, 28 memantine, 26, 36-38 memantine/acetylcholinesterase inhibitor combination therapy, 41-42 donepezil, 27 rivastigmine, 27-28 tacrine, 27 "use it or lose it" hypothesis, 28 visual symptoms, 437-438 Alzheimer's Disease Assessment Scale-Cognitive Subscale, 26, 37 Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory, 36 Amantadine, 35, 36-38, 280 Ambenonium analysis, 693,694 in myasthenia gravis, 22 Ambient temperature, and anticholinesteraseinduced hypothermia, 554, 555-557 American Association of Poison Control Centers, 94, 571 Ameridine, 17 Amerindians, butyrylcholinesterase genotypes, 191 Ames assay method, 662 Aminocarb, immune system effects, 503 Aminopyrine, induction of carboxylesterases, 227 Ammonium polyphosphate, 406 Amnesia, scopolamine-induced, 27 Amphibians, anticholinesterases sensitivity, 148, 150, 152 Amygdala, cholinergic innervation, 271 Amyloid/3 peptide, effects of acetylcholinesterase inhibitors, 28-29 Amyloid precursor protein, 28-29 Amyotrophic lateral sclerosis, in Gulf War veterans, 74-75 Anabas testudineus, carbofuran toxicity, 659 Analgesia, cholinergic regulation of, 275 Analytical methods acetylcholinesterase inhibitor therapeutic agents, 694-695 cleanup/purification, 682-683 detectors, 684-686 HORRAT value, 696 immunoassays, 690-691 impurities separation, 684 metabolites, 691-693 nerve agents, 693-694 pesticide confirmation, 686 pesticide screens, 691 QuEChERS method, 690
sample concentration, 683-684 sample extraction, 681-682 sources for, 686-690 Androgen production, site of, 449 Androgen receptor, pesticide binding, 452-453,481,485-487 Angiotensin-converting enzyme inhibitors, 219 Anhedonia, 300 Anopheles, 600 Anthelmintics, 23 Anthrax vaccine, 69 Antiandrogenic compounds, 481, 491 Anticholinesterase eye drops, and accomodative capacity, 433 Anticholinesterase poisoning/intoxication biomarkers, 91-93 blood-brain barrier, 278-279, 283 chronic exposure, 281-282 clinical aspects, 91-93 CNS symptoms, 145 diazepam for, 93,277, 386, 394 extrapyramidal effects, 280-281 fatality rates, 91 global epidemiology, 93-98 Gulf War illnesses, 279-280 hypothermia and, 549, 551-554 interspecies variation age and, 154-155 gender and, 154-155 LDs0, 145-148 pharmacodynamics, 151-155 pharmacokinetics, 148-151 mechanism of, 90 muscarinic symptoms, 145 neuropathy, 278-279, 283 nicotinic symptoms, 145 respiratory effects, 277 seizures, 278-279, 283 signs/symptoms, 89, 276-277, 283 treatment, 93 Anticholinesterases. see Cholinesterase inhibitors Anticonvulsants, 726-727 Antidotes anticonvulsants, 725-726 atropine, 584, 718-719 atropine/oxime, 722-725 diazepam, 93,277, 386, 394 HI-6, 584-585 obidoxime, 584-585 oximes, 200, 584-585, 719-722 pralidoxime, 584-585 Antioxidants, see also specific antioxidant endogenous, 512 pesticide-induced alterations, 456 Antitumor drugs, carboxylesterase metabolism of, 220 Anxiety, 260 and acute pesticide exposure, 350 Bhopal accident-associated, 84
737
chlorpyrifos-associated, 354 and organophosphate exposure, 574 AOAC, validation methods, 695-606 AP5, 341 Apathy, and Alzheimer's disease, 26 Aphids, acetylcholinesterase genes, 168, 172-173 Apnea, scoline, 188, 189 Apoptosis neuronal stem cells, 318 organophosphate-induced, 235,454-456 AP prolongation, organophosphate-induced, 383-386 Aquatic ecosystem biomagnification of pesticides, 658 metals in, 666 pesticide accumulation, 657 pollutants, 664 Aquatic toxicity, methods, 658-659 Aqueous humor fluorescein flare, 425 and intraocular pressure, 426-427 ophthalmic toxicity, 434 Arachidonic acid, 514 Arecoline, 502 Areflexia, 376 Aricept, 27 Arousal, behavioral, 273-274 Arsenicals, Lewisite, 49 Arylalkylphosphatase/paraoxonase, 263 Arylesterases, 128-129, 150; see also A-esterases Arylformamidase, 707 Aryl hydrocarbon hydroxylase, 664 Aryl hydrocarbon receptor, 545 Asphyxia, organophosphate-induced, 394-395 Aspiration pneumonia, 717 Asthma irritant receptor activation, 391 organophosphate exposure and, 237, 395,574 Astrocyte-endothelial interactions, organophosphate effects, 331-332 Astrocytes, 315-316 and blood-brain barrier integrity, 331-332 function of, 330 Astroglia, 330 Asulam, acetylation of, 138 Atlantic salmon, DDT absorption, 658 Atmosphere closed facilities, 585 inhalable fraction, 401 respirable fraction, 401 sampling, 402 ATP carbamate-induced changes, 517 organophosphate-induced changes, 517-518 and oxidative stress, 513 Atresia, carbofuran-induced, 659
738
Index
Atropine blocking of hypothermic response, 554-555 clinical efficacy, 718 dosage regimen, 718-719 hyperthermic effect, 561 mechanism of action, 718 and methyl isocyanate toxicity, 82 muscarinic receptor blocking, 281 organophosphate poisoning/intoxication, 93,584 cardiotoxicity, 386 ocular exposure, 433 pulmonary toxicity, 394, 395 oxidative stress prevention, 519-521 and oximes, efficacy of, 722-725 pharmacokinetics, 718 ATSDR Pesticide Profiles, 689-690 Attention deficit disorder, 633 Attention/vigilance, 274, 350 Australia, pesticide poisoning in, 93-94 Autism, 633 Autoimmune myasthenia gravis, 376 Autonomic nervous system disorders, in Gulf War veterans, 75 Autoradiography, cholinergic pathways, 271 Avian acute oral toxicity test, EPA, 673 Avian toxicity, 673,674-677, 677-678 Avizafone, 726 Axonopathies, promotion of, 365-366 Axons degeneration, 234, 362-363,703 myelination, 330 outgrowth, effects of organophosphates on, 241 5-Azacytidine, developmental neurotoxicity, 638 Bacillus thuringiensis, 601
Balance, chronic pesticide exposure and, 353 Bambuterol, butyrylcholinesterase binding, 161 Barbiturates, 726 Basal forebrain, 26, 35, 271-272, 274 Basal ganglia, cholinergic neurons, 272-273 Basudin, 543 Bayer Crop Sciences, 630-631 Baygon, endocrine disruption, 485 BCHE, 190-194, 204-206 de novo amplification of, 195 variants, 204 Behavioral toxicity, 573 acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 Benchmark dose, 618, 619, 627, 628 Bendiocarb, endocrine disruption, 485 Benfuracarb, metabolites, 693 Benomyl, 134 inhalation toxicity, 403 metabolites, 693
in mixtures, 607-608 reproductive toxicity, 454 Benzodiazepines, 726 Benzoylcholine, 176, 201,202 Bergmann glia, 330 B-esterases, 129 activity, interspecies variation, 150 in organophosphate metabolism, 106, 109-110 modeling, 109-118 role of, 247 Beta-adrenoreceptor agonists, and developmental neurotoxicity, 302 BeWo cells, 469 Bhopal accident, 4, 79-80, 568 clinical toxicity of methyl isocyanate, 83-85 methyl isocyanate toxicity, 81-83 Bicyclophosphorus esters, neurotoxicity, 407 Bidrin, 538 Binding globulins, 452 Biochemical studies, WHO/FAO guidelines, 645 Bioconcentration pesticides, 481 placental, 471-472 Biological exposure index, 579, 581 Biomagnification, of pesticides in aquatic ecosystems, 658 Biomarkers muscle injury, 512, 515-516 organophosphate intoxication cholinesterase, 91-92 egasyn, 92-93 oxidative stress carbamate-induced changes, 516-517 organophosphate-induced changes, 517-519 parathion poisoning, 92 Biomonitoring pesticide exposure, 576 workplace, 581-582 Bioscavengers, 708 Biotransformation carbamates, 106 organophosphates, 104-106, 715 in vitro models, 332-333 Birds anticholinesterases sensitivity, 147-148, 150, 152 insecticide toxicity, 673, 674--677, 677-678 paraoxonase activity, 249 pesticide metabolism, 661 thermoregulation, 550 Birth defects, 84, 605 Bispyridinium SAD-128, 341 Bitot spots, 83 Blepharitis, eye irritation tests, 425 Blepharospasm, 431
Blood human, measuring acetylcholinesterase activity, 199-204 measuring anticholinesterase metabolites, 679-680 sample collection/storage, 200 Blood-brain barrier and anticholinesterase intoxication, 278-279, 283 artificial, 332 cell culture models, 331-332 factors effecting permeability, 279-280 and organophosphates neurotoxicity, 149 pralidoxime and, 721 role of, 278 Blue gill sunfish, carbofuran toxicity, 659 Body temperature core, 551 effects of cholinesterase inhibitors on, 551-554 Boll Weevil Eradication Program, 602 Botulinum toxin, pretreatment, and myopathy prevention, 512 Botulinum toxin poisoning, 376 Botulinum toxoid vaccine, 69 Bovine adrenal chromaffin cells, 329 Bovine microvascular endothelial cells, 332 Bradycardia, 703 Brain cholinergic pathways, 271,273,282-282 developmental neurotoxicity long-term outcomes, 298-301 mechanisms, 294-298 vulnerable subpopulations, 301-303 excitotoxicity in, 521-527 muscarinic receptors, and tolerance development, 258-259 sex differentiation, 299 Brain morphometry, effects of developmental organophosphate exposure, 241 Brain slice model, soman, 341 Brain stem, upper, cholinergic neurons, 273 Breathing pattern, and properties of inhaled materials, 404 Bromophos, metabolism of, 715 Bronchi, pulmonary toxicity, 393-394 Bronchioles, pulmonary toxicity, 394 Bronchoconstriction methacholine-induced, 237 organophosphate-induced, 393, 574 vagally-induced, 237, 574, 707 Bronchospasm, 574 Brownian diffusion, inhaled particles, 401 BSA, 693 n-Butane-sulfonyl fluoride, OPIDN prevention, 727 Buthionine sulfoximine, 514 Butylate, 17
Index Butyrylcholinesterase A (atypical) variant, 189-194, 204, 216 activity decreases in, 188 measurements, 199-204 aged, 215-216 in Alzheimer's disease, 17, 25 binding sites, 175-179 active gorge, 174-175 acyl pocket, 176, 209, 214 choline, 176 oxyanion hole, 211 as biomarker of organophosphate exposure, 187 C5 variant, 190 catalytic turnover rates, 175, 182 deficiencies, 187 mode of inheritance, 188-193 dibucaine-resistant variant, 192 distribution in eye, 423-424 fluoride-resistant variant, 192, 204 function, 703-704 gene for, 190-194, 204-206 genetic variants, 204-206, 216 H (Hammersmith) variant, 189-194, 204 J (James) variant, 189-194 kinetic modeling, 211-213 K (Kalow) variant, 189-194, 204, 216 nerve agent antidote, 727 in organophosphate metabolism, 106, 109-110, 150, 707, 708 modeling, 109-118 oxime reactivation of, 719 pharmacogenetics clinical aspects, 187-188 genetic variants, 189-194, 204, 216 inhibition studies, 188-189 phenotypes, 188-189, 204 phenotyping and succinyldicholine sensitivity, 201,202, 216 placental, 472-473 plasma activity measurements, 577-579, 716-717 organophosphate-binding, 704 Sc (scoline) variant, 194 S (silent) variant, 189-194, 204, 216 structure primary, 161-168 quaternary, 181 secondary/tertiary, 168, 174-181 variants, 181-182 substrate specificity, 209 tissue distribution, 161, 187 tolerance development and, 263 U (usual) variant, 189-194, 204, 216 Butyrylcholinesterase inhibitors for activity measurements, 201 immune system and, 495
Butyrylthiocholine, 112-113 butyrylcholinesterase binding sites, 176 BW286c51, cholinesterase binding site, 176, 179 BW2845c51, acetylcholinesterase inhibition, activity measurements, 201 C5 variant, butyrylcholinesterase, 190, 204 C6 rat glioma cell line, 330-331, 541-542 Ca2+-ATPase, pesticide inhibition of, 665 Ca2+/cAMP-response element binding protein, 240, 331,363 Ca2+/cAMP response element binding protein, organophosphate activation of, 239-240 Calabar bean, 4, 48, 599 Calcium/calmodulin-dependent protein kinase II, organophosphate activation of, 234, 239 Calcium channel blockers, 727 Calcium ions homeostasis, in OPIDN pathogenesis, 363 myopathy and, 512 and organophosphate-induced cardiotoxicity, 383-386 pesticide disruption of, 453 and smooth muscle regulation, 382 California Health Services Department, 578 Canal of Schlemm, 434 Cancer, childhood, 605 Cannabis, developmental neurotoxicity, 634 Capecitabine, 220 Capillary electrophoresis, 694 Caramiphen, 727 Carbachol, and down-regulation of muscarinic receptors, 258 Carbamate compounds acetylcholinesterase inhibition, 90, 214 allergic sensitization, 504 androgen receptor binding, 481 behavioral toxicity, 347-356 bioaccumulation, 658 biodegradation, 658 biotransformation, 106 body temperature and, 553, 554 carcinogenesis, 544-545 chemistry, 17, 18--21, 103 CNS effects, 451 crop pest control, 602 cumulative effects, 607-613 dermal absorption/toxicity, 411-4 19 ectoparasiticides, 23 embryocidal/fetocidal effects, 474-475 endocrine disruption, 481-491 EPA subgroups, 620 estrogenic effects, 447, 485-487 extraction solvents, 682 and gene expression, 545 and Gulf War syndrome, 69-75 history of, 3-4
739
in human medicine, 17, 22-23 immunomodulation by, 498-503 inhalation pharmacology/toxicology, 399-407 mechanism of action, 3, 103-104, 211, 339-344 memantine interactions, 40-41 metabolism in v i t r o studies using human tissues, 138-140 phase I reactions, 134-137 phase II reactions, 137-138 mixtures, 610 mosquito resistance, 601 neuropathy target esterase inhibition, 362 neurotoxicity, 276-283, 339-344 occupational exposure, s e e Occupational toxicology ophthalmic toxicity, 423-438 OPIDN prevention, 727 peripheral neuropathy, 364 pharmacokinetics, 104 placental toxicity, 463-475 pulmonary toxicity, 393 reproductive toxicity, 447-459 residue persistence, 4 resistance, 4 routes of exposure, 104 selectivity, 4 specific metabolites, analysis, 693 thermolabile, 406 thermoregulatory effects, 549-564 tolerance, s e e Tolerance development toxicity interspecies variation, 145-155 potentiation in mixtures, 610 uses, 4, 5, 17, 22-23,567 U.S. registered, 673,674-677 U.S. use of, 602 vector-borne disease control, 599-602 Carbamate poisoning/intoxication behavioral toxicity acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 blood cholinesterase measurements, 576-579 children's exposures, 602-605 interspecies variation age and, 154-155 gender and, 154-155 LDs0s, 145-148 pharmacodynamics, 151-155 pharmacokinetics, 148-151 medical surveillance, 572-576 neurotoxicity, 276-283, 339-344 signs/symptoms, 276-277, 348 treatment, 584-585 urine/blood metabolite measurement, 579-580
740
Index
Carbamic acid, 134 Carbamylcholine, 341, 512 Carbaryl absorption/elimination of, 148 aryl hydrocarbon receptor and, 545 cholinesterase inhibition, interspecies variation, 152 cognitive effects, 350 dermal absorption, 416-417 developmental neurotoxicity, 639 ectoparasiticide, 23 endocrine disruption, 485,489-490 history of, 4 immune system effects, 503 metabolism, 106, 135 CYP isoforms in, 139 interspecies variation, 139 metabolites, 580 mixtures, 610, 611 nitric oxide synthase inhibition, 455 PBPK/PD model for, 631 peripheral neuropathy, 364 placental toxicity, 475 Carbendazim, 693 Carbofuran and cholesterol, 665 CNS effects, 451 and creatine kinase activity, 516 developmental neurotoxicity, 639 effect on blood-brain barrier permeability, 279 endocrine disruption, 489-490 half-life, 663 immune system effects, 503 -induced myopathy, 512 interaction with memantine, 41 mechanism of action, 661 metabolism, 135, 662-664 CYP isoforms in, 139 interspecies variation, 139 metabolites, 580, 662, 693 biomarkers, 666 mixtures, 610 nerve agent protective effects, 611 peripheral neuropathy, 364 and phospholipids, 665 reactive oxygen species formation, 455 reproductive toxicity, 454 residue persistence, 659 restricted entry level, 568 toxicity, 4 in fish, 659-661 and route of exposure, 659 signs/symptoms, 659 uses, 658 Carbofuran phenol, 693 Carbon dioxide, 682 Carbon tetrachloride, protein kinase C modulation, 541 Carbosulfan, metabolites, 693
Carboxylesterase inhibitors, and tolerance development, 261-262 Carboxylesterases activity, interspecies variation, 150 acyl pocket binding site, 224 catalytic triad, 224 CES 1 isozyme family, 221-222 genes, 228 induction of, 227-228 structure-activity relationships, 222 CES2 isozyme family, 221-222 genes, 228-229 induction of, 227-228 structure-activity relationships, 222 CES3 isozyme family, 221-222 CES4 isozyme family, 221-222 detoxification potential, 52 gene structure, 228-229 human gene for, 224 induction of, 227-228 inhibition, and organophosphate potentiation, 610 isozymes as biomarkers, 92-93 structure and catalytic mechanism, 223-227 in organophosphate metabolism, 106, 109-110, 150, 706, 707 modeling, 109-118 oxyanion hole, 224 regulation, 228-229 role of, 133 scavenger function, 263 structure, 3-D, 224 substrate specificity, 219, 227, 229 tissue distribution, 219 tolerance development, 261-263 -UGT interaction, 220 Carboxylic ester hydrolases, 129 Carcinogenicity carbamates, 544-545 methyl isocyanate, 84 organophosphates, 542-544, 662 rat mammary tumor model, 543-544 WHO/FAO guidelines, 647 Cardiac arrest, 703 Cardiolipin, 455 Cardiomyopathy, anticholinesterase exposure and, 574 Cardiotoxicity organophosphate-induced effect on contractile tissue, 381-382 in silico approaches, 382-386 treatment, 386 Cardiovascular disease, role of paraoxonase, 247, 248-249 Carson, Rachel, 681 Caspase-3,455-456 Cat, as OPIDN model, 153 Catalase, 456
Catalytic triad carboxylesterases, 224 cholinesterases, 162, 165-168 organophosphate binding, 706 Cataractogenesis, 434-435 Cathepsin, 665 Caucasians butyrylcholinesterase genotypes, 191,204 paraoxonase polymorphisms, 247 Caudate putamen, cholinergic innervation, 273 CBDP, 262 Cell cultures, see also specific cell line fish hepatocytes, 666-667 gonadotoxicity, 457 neurotoxicant screening, 303-304 neurotoxicity testing, 317-318, 319-327, 328-329 placental, 469 Cell damage, see Cytotoxicity Cell signaling, adenylyl cyclase, 704 developmental chlorpyrifos exposure and, 239, 295,296-297, 301 inhibition, 236, 237,240 Cell swelling, organophosphate-induced, 383-386 Centers for Disease Control and Prevention, National Health and Nutrition Examination Survey, 603 Centers for Disease Control and Prevention, no adverse effects limit, 47 Central America, acute pesticide poisonings, 570-571 Central nervous system anticholinesterase intoxication blood-brain barrier, alterations, 278-279, 283 chronic exposure, 281-282 extrapyramidal effects, 280-281 Gulf War illnesses, 279-280 neuropathy, 277-278, 283 respiratory effects, 277 seizures, 277-278, 283 signs/symptoms, 276-277, 283 sites of action, 145 brain excitotoxicity, 521-527 cell types, 315-316 cholinergic failure, 26, 35 cholinergic neurons, 271-273,282-283 cholinergic neurotransmission, 275-276 functions associated with, 273-275 and thermoregulation, 549 cholinergic nuclei, 271 cholinesterase inhibitors, effects, 451 GABAergic transmission, 51-52 inhaled cholinesterase inhibitors, systemic effects, 404-405 muscarinic pathways, and hypothermic response, 554-555 muscarinic receptors, 236, 707 nerve agents and, 51-52
Index
OPIDN effects, 234 interspecies variation, 153-154 organophosphate toxicity, 91 chronic, 234-235 delayed, 662; see also Organophosphate-induced delayed neuropathy respiratory centers organophosphate-induced effects, 391,394 pulmonary toxicity and, 394 thermoregulation, 549, 551,554-555 Cerebral cortex alterations in Alzheimer's disease, 26 cholinergic innervation, 271 Cerebrovascular disease, dementia and, 29 CES1, 228 CES2, 228-229 C-esterases, 129 Channa punctatus, carbofuran toxicity, 659 Chemical Industry Institute for Toxicology Centers for Health Research, 630 Chemical Materials Agency, 47-48, 62 Chemical/physical properties, nerve agents, 50 Chemical Stockpile Emergency Preparedness Program, 47, 62 Chemical warfare agents, see also specific agents; Matsumoto incident; Nerve agents; Tokyo subway attack analytical methods, 693-694 at Khamisiyah, 73-74 decontamination/cleanup, 48 emergency preparedness plans, 47-48, 62 exposure guidelines, estimated reference doses, 48, 55-57 history of, 48-49 non-stockpile material, 48 organophosphate-induced delayed neuropathy, 54-55 properties, 49-52 response to release of, 48, 55 stockpile destruction, 332-333 stress and, 71, 73 toxicity, 52-55 mechanisms of, 51-52 Chemical weapons of mass destruction, 3 Chemiluminescence, time-delayed, 685 Chemiluminescence assay, 535-536 Chemotaxis, 496 Chest tightness, organophosphate exposure and, 392 Chick eggs, developmental neurotoxicity screening, 305 Chickens, as OPIDN model, 153 Children anticholinesterase poisoning/intoxication, signs of, 276-277 breathing zones, 604 and chlorpyrifos exposure, 293
developmental neurotoxicity, 633 FQPA 10x factor, 618, 619, 633,635,639 organophosphates sensitivity, 118-119 pesticide exposure, 490, 602-605 sensitivity to, 354-356 Chironomus adult emergence test, 658 Chlorfenvinphos, 23 chronic exposure, neurobehavioral effects, 353 CNS effects on respiration, 277 metabolism, interspecies variation, 139 pharmacokinetics, interspecies variation, 148-149 retinal effects, 436 seizure induction, 278 successive exposures, 612 Chlorothalonil, endocrine effects, 474 Chlorphoxim, mosquito resistance, 601 Chlorpyrifos, 23 adenylyl cyclase signaling and, 239, 295, 296-297, 301 age-related sensitivity, 354-355 airway hyperreactivity, 237-239, 395 antidotal treatment, 723 anxiogenic effects, 354 biotransformation, 104-106 cognition effects, 350 developmental neurotoxicity, 293-294, 639, 704 glial targeting, 541-542 long-term outcomes, 298-301 mechanisms, 294-298 vulnerable subpopulations, 301-303 and dioxalon binding, 259 DNA synthesis inhibition, 331,533 effects on Ca2+/cAMP response element binding protein, 240, 331 estrogenic activity, 485 gene expression of, 538-540 and Gulf War, 69 hyperthermia, delayed, 559, 560 hypothermic response to, 555 immune system effects, 498, 501 indoor use, 604, 630 induction of heat shock proteins, 538-540 inhibition of cAMP synthesis, 239 metabolism CYP isoforms in, 139, 140 interspecies variation, 139 metabolites, 579 mixtures, 612 neonate vs. adult toxicity, 293 neurobehavioral effects, chronic exposure, 352,353 neurotoxicity testing cell culture systems, 330 whole rat embryo, 329 nicotinic receptor inhibition, 259 oxidative stress and, 534 partitioning coefficient, 111
741
PBPK/PD model for, 108-121 potency, 131 prenatal exposure, 253,604 protein kinase C modulation, 541 and tolerance to acetylcholinesterase inhibition, 258-259 toxicity and paraoxon status, 249-250, 264 transendothelial permeability, 332 uses, 293, 533 Chlorpyrifos-methyl adrenal gland toxicity, 454 endocrine disruption, 488 Chlorpyrifos-oxon activation, 104-106 developmental neurotoxicity, 542 effects on Ca2+/cAMP response element binding protein, 240 hydrolysis of, 133 multidrug resistance-1 gene and, 540 neurotoxicity testing, cell culture systems, 330 noncompetitive muscarinic receptor binding, 237 protective effects of paraoxonase, 249 Choice behavior, effects of anticholinesterase poisoning, 350 Cholesterol, and carbofuran, 665 Choline, 275, 719 Choline acetyltransferase, 271 in Alzheimer's disease, 25-26 developmental chlorpyrifos exposure, 298 levels in mild cognitive impairment, 26 Choline binding site, cholinesterases, 175-176, 177-179, 179-180 Cholinergic agonists chronic exposure and muscarinic receptor decrease, 258 neurobehavioral teratology, 294 Cholinergic crisis, 145,209, 233 acetylcholine accumulation, 451 signs/symptoms, 552, 572-574 Cholinergic fibers, arborization, 272-273 Cholinergic hypothesis, Alzheimer's disease, 25-26 Cholinergic neurons, 271-273, 282-283 Cholinergic neurotransmission, 275-276 and cholinesterase inhibitors, 276-283 functions associated with, 273-275 Cholinergic nuclei, central nervous system, 271 Cholinergic receptors acetylcholine binding, 209 age-related pesticide sensitivity, 355 postsynaptic, 275 presynaptic, 276 tolerance and, 236 Cholinergic system distribution, 103 impairment of, 35 placental, 472-474
742
Index
Cholinesterase inhibitors as alkylating agents, 498 allergic sensitization, 504 autonomic thermoeffectors and, 556-557 behavioral thermoeffectors and, 557 behavioral toxicity acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 cardiotoxicity, 381-386 and cholinergic neurotransmission, 276-283 combustion toxicology, 406-407 and core temperature, 555-556 dermal absorption/toxicity, 411-4 19 endocrine disruption, 481-491 eye accommadative capacity and, 433 glaucoma treatment, 434 history of, 3-4 hypersensitivity to, 194-195 immunomodulation, 498-503 mechanism, 496-498 inhalation pharmacology/toxicology, 399-407 LDs0, interspecies variation, 145-148 lens opacities, 435 low doses, 704 mechanism of action, 51-52 apoptosis, 454-456 carbamates, 211 central nervous system effects, 451 endocrine -related effects, 452-453 hypothalamic-pituitary-gonadal axis, 452,457 metabolic effects, 453-454 organophosphates, 209-211 oxidative stress, 454-456 neurotoxicity, electrophysiological mechanisms, 339-344 occupational exposure, s e e Occupational toxicology ophthalmic toxicity, 423-438 placental toxicity, 463-475 potentiation, 574 progressive, irreversible, 183-184, 199-200 and pulmonary toxicity, 389-395 reproductive toxicity, 447-459 retinal effects, 436-438 reversible, 182-183 therapeutic uses, 283,495 thermoregulation, 551-564 tolerance, s e e Tolerance development uses, 567 vapor pressure, 399 Cholinesterases activity measurements, 716-717 expressing, 200-201 methods, 201-204 quality control, 204 sample collection, 200
standards, 204 substrates, 201 aged, 215-216 Alzheimer's disease and, 25-26 binding sites, 175-179, 199 active center gorge, 174-176, 179-180 acyl pocket, 175-176, 177-179, 209, 210,214 amino acid sequence, 165-168 catalytic triad, 162, 165-168 choline, 175-176, 177-179, 179-180 oxyanion hole, 162, 165-168, 175,211 peripheral, 175-176, 177-179, 209-211, 212,214 serine residue, 162, 165-168, 213 catalytic turnover rates, 104, 175, 213 characteristics of, 161 cysteine residues, 168, 169-171 disulfide loops, 168, 169-171 erythrocyte, 52; s e e a l s o Acetylcholinesterase as biomarker of organophosphate exposure, 91-92 eye, distribution in, 423-424 genetic variants, 204-206 inhibition as biomarker of organophosphate exposure, 91-92 and neurotoxicity, 233-236 oxime reactivation of, 21 4-215 pharmacodynamic models, 111-118 spontaneous reactivation, 200, 213 placental, 472-473 plasma, 52; s e e a l s o Butyrylcholinesterase activity measurements, 576-579 as biomarker of organophosphate exposure, 91-92 human, 112 rat, 112 structure primary, 161-168, 169-171,177-179 quaternary, 181 secondary/tertiary, 168, 174-181 variants, 181-182 substrate activation, 182 substrate hydrolysis, 182, 199-200 substrate inhibition, 182 tissue distribution, 161, 187, 423-424, 451 WHO/FAO activity guidelines, 646 Cholinomimetic agents, effect on motor activity, 274-275 Choreiform movements, 280 Choreoathetosis, 280 Chorioallantoic membrane, in eye irritation tests, 427-428 Choriocarcinoma cell line, 469 Chorionic gonadotropin, placental, 473-474 Chorionic somatomammotrophin, 474
Chromatography, 683,684 Chronic organophosphate-induced neuropsychiatric disorder, 573,662 Cilazapril, 219 Ciliary body, ophthalmic toxicity, 433-434 Citrulline and anticholinesterase-induced seizures, 278 carbamate-induced changes, 516-517 correlation with acetylcholinesterase inhibition, 519 organophosphate-induced changes, 517 pesticide-induced changes memantine/atropine pretreatment, 520 and PBN pretreatment, 525-527 and vitamin E pretreatment, 525-527 CL 18706, in mixtures, 609 Clara cells, 394, 403 Clinical Global Impression of Change, 36 Clinician's Interview-Based Impression of Change Plus Caregiver Input, 36 Clonazepam, 726 CNQX, 341 Cocaine butyrylcholinesterase hydrolysis of, 187 carboxylesterase metabolism of, 220, 222 developmental neurotoxicity, 634 Cockroaches, synaptic transmission, 343-344 Code of Federal Regulations, 40 CFR Part 158, 618, 634 Codex Alimentarius Commission, 644-645 Cognex, 27 Cognition cholinergic regulation of, 274 impaired in Alzheimer's disease, 26 Bhopal accident-associated, 84 measures of, 26-27, 36 organophosphate-induced deficits, 234-235, 282 pesticide exposure acute, 573 chronic, 351,353 Coho salmon, carbamates sensitivity, 342 Collaborative Behavioral Teratology Study, 634 Collision cell, 685 Collision-induced dissociation, 685 Colorimetric test, pesticides, 691 ColQ, 181 Combat stress, 70 Combustion toxicology, 406-407 Comet assay, 458, 537-538 Common mechanism group, 620, 627, 629 Compartmental pharmacokinetic models, 107-108 Compound muscle action potential, 371 Conditioned behaviors, 350-351 Confocal microscopy, corneal thickness, 426
Index
Conjunctivae absorption from, 431 inflammation of, 425 Contact dermatitis, organophosphateinduced, 504 Contractile tissue, effect of cholinesterase inhibitors, 381-382 Convulsions and anticholinesterase intoxication, 278-279, 283 role of nitric oxide, 522 treatment, 725-726 Cooperative Threat Reduction Program, 49 Copper, in aquatic ecosystems, 666 Coquillettidia, 600 Co-Ral, mixtures, 610 Comea cholinergic activity, 424-425 eye irritation tests, 425-428 isolated, cell preparations, 427 Coronary arterial disease, and butyrylcholinesterase activity, 204 Cortex, cholinergic innervation, 273,274 Corticotrophin-releasing factor, 474 Cortisol, immunomodulatory effects, 498 Coughing, 392, 393-394, 574 Coumaphos, 23 CPT- 11,220 Crack-and-crevice application, 604 Cranial nerve palsies, intermediate syndrome, 371 Crayfish, 659 Creatine kinase as biomarkers of muscle injury, 512, 515-516 isoenzymes, 512, 515-516 myonecrosis and, 512 pesticide-induced changes in, 527 Creatine phosphokinase, as biomarker, 580 o-Cresyl saligenin phosphate, and OPIDN syndrome, 7 Critical toxic effect, risk assessment, 618, 621-626
Crop yield, and pesticide usage, 602 Cross-resistance, insecticides, 601 Croton oil, 544 Crotylsarin, 720 Crufomate, 23 Crystal violet staining, eye irritation tests, 428 Cule tarsalis, 600 Culex, 600, 601 Culex pipiens quinquefasciatus, 600, 601 Culiseta, 600 Cumulative assessment group, 620, 627, 630 Cutaneous exposure modeling, 576 Cyanide poisoning, 80 Cyanophos, 716 carbamates interaction, 611 mixtures, 612
cyclicAMP, 704 chlorpyrifos interactions, 297, 301 organophosphate inhibition of, 239 cyclicAMP/PKA signaling pathway, organophosphate modulation of, 239 Cyclic nucleotidase, 498 Cycloate, 17 Cyclopentolate, 432, 433 N6-Cyclopentyladenosine, 260 Cycloplegia, 432 Cyclosarin, 62 chemistry, 7 history, 389 neuropathy induced by, 278 properties, 49-52 CYP 19-aromatase, 487 Cyprinus carpio communis, carbofuran toxicity, 659 Cysteine, cholinesterases, amino acid sequence, 168-171 Cythioate, 23 Cytochrome c oxidase, 455 fasciculations-induced changes, 515 loss of activity, 513 Cytochrome P450 activity, interspecies variation, 150 ancestral gene, 663 and inhalation toxicity, 403 inhibitory effects of pesticides, 140 isozymes CYP1A2, 663-664 CYP1A EROD activity, 666-667 in fish, 664-666 function, 663-664 induction of, 664 placenta and, 465,470, 474 polymorphisms, 139-140 selectivity, 139-140 organophosphate metabolism, 104-106, 111,707-708, 715 oxon activation, 211 pesticide metabolism, 663-664 role of, 128 Cytokines, 560 Cytoskeletal proteins, in OPIDN, 363 Cytotoxicity biomarkers creatine kinase, 512, 515-516 lactate dehydrogenase, 512, 516 nitric oxide and, 518 organophosphate-induced DNA single strand breaks, 537-538 reactive oxygen species-mediated, 533 tubular 538 i ' Cytotoxicity assays, eye irritation tests, 428 Cytotrophoblasts, 463,474 2,4-D, dermal absorption, 416
Daphnia magma life cycle test, 658
743
Data-call in notice, 619, 636 Data extrapolation, WHO/FAO guidelines, 650, 7647 DBCP, and male reproduction, 451 DDT developmental neurotoxicity, 634 endocrine disruption, 481 environmental persistence, 600 estrogenic effects, 447, 452 history of, 657 and male reproduction, 451 mosquito control, 600-602 Dealkylation carbamate metabolism, 136 organophosphates metabolism, 131 Dearylation, organophosphates metabolism, 131 Decamethonium, 168, 176, 179 de Clermont, Phillipe, 3 Decontamination, 584 DEET dermal absorption, regional variability, 416 and Gulf War, 69 interaction with pyridostigmine bromide, 71 Delayed neuropathy, nerve agents, 53-55 Delayed neurotoxicity, WHO/FAO guidelines, 645-646 Dementia and Alzheimer's disease, 26 and cerebrovascular disease, 29 Lewy bodies with, 29-30 memantine for, 36-38 mixed, 29 Parkinson's disease, 29-30 vascular, 37 Demeton, tolerance development and carboxylesterase, 261 Demyelination, organophosphate-induced, 234 Dengue, 599-601 Dengue hemorrhagic fever, 600, 601 Department of Veterans Affairs, Gulf War illness, 71 Depression and Alzheimer's disease, 26 Bhopal accident-associated, 84 Derivatizing agents, 691,693 Dermal absorption factor, 619 Dermal absorption/toxicity additives and, 417 dose and, 413 factors affecting, 416-418 formulation and, 417 mechanism, 411-413 models, 413-415 occlusion and, 418 organophosphates, 715 species differences, 415-416 vehicle effects, 417-418 Dermis, 411 Desferrioxamine, 538
744
Index
Desulfuration, organophosphates metabolism, 131 Detection paper, nerve agents, 694 Detection tickets, pesticides, 691 Detection tubes, nerve agents, 694 Detectors analytical, 684-686 nerve agents, 694 Detoxification age-related differences, 355 organophosphates, in vitro models, 332-333 Developmental neurotoxicity children, 633 chlorpyrifos, 704 glial targeting, 541-542 long-term outcomes, 298-301 vulnerable subpopulations, 301-303 chlorpyrifos-oxon, 542 comparative cholinesterase data, 638-639 diazinon, 542 EPA guidelines, 619 guideline, 634-635 mechanisms, 294-298 neurotoxicants screening, 303-305 organophosphate-induced, 235-236 pituitary-gonadal axis, 448 testing requirements, 635-638 WHO/FAO guidelines, 646 Dexamethasone, induction of carboxylesterases, 228 Di(2-ethylhexyl) phthalate, induction of carboxylesterases, 227 Diacetyl monoxime, 262, 263, 725 Diacylglycerol, 239, 666 Diacylglycerol lipase, 239 Dialkyl phosphates analysis, 691-692 in fruit juices, 603 in water, 692 Diazepam, 726 for anticholinesterases poisoning, 93,277, 386, 394 for convulsions, 585 Diazinon, 23 avian toxicity, 677 chronic exposure, central effects, 281-282 delayed hyperthermia, 560 developmental neurotoxicity, 303,542 DNA synthesis inhibition, 534 endocrine disruption, 487, 489 environmental persistence, 543 EPA classification, 543 homeowner use, cancellation/phase out, 630 immune system effects, 502 intermediate syndrome, 371-372, 377 metabolism of, 715 mixtures, 611 partitioning coefficient, 111 PBPK/PD model for, 108-118
retinal effects, 436 Saku disease, 430 Diazopentane, 691 Diazoxon, 133,248, 249-250 Dibromochloropropane, reproductive toxicity, 448 Dibucaine and butyrylcholinesterase phenotyping, 204 cholinesterase inhibition, 188,204 Dibucaine number, 189 Dibucaine-resistant variant, butyrylcholinesterase, 192 Dibutyryl cyclic AMP, 328 Dichlorodiphenyltrichloroethane. see DDT Dichlorvos, 23 absorption/elimination of, 148 blood-brain barrier permeability and, 279 CNS effects, 277, 281 extrapyramidal effects, 280, 281 immune system effects, 502 intermediate syndrome, 376 lipid peroxidation and, 534-535 memantine and, 41 mixtures, 611 noncompetitive muscarinic receptor binding, 236 Saku disease, 430 seizure induction, 278 tolerance development and carboxylesterase, 261 Dieldrin, estrogenic activity, 485 Diesel oil, 664 Diet, pesticide exposure, in children, 602-604 Diethiofencarb, 134 O,O-Diethyl dithiophosphate, 579, 691 O, O-Diethyl phosphate, 579, 691 Diethyl phosphorofluoridate, 3 Diethylsuccinase, 574 O,O-Diethyl thiophosphate, 106, 579, 691 Diethylumbelliferyl phosphate, 239 Diffusion cell model, dermal absorption/toxicity, 413-414 Diisopropyl fluorophosphatase, 727 Diisopropyl fluorophosphatase/somanase, 263 Diisopropyl fluorophosphate acetylcholinesterase knockout mice and, 234 central respiratory depression, 394 cholinesterase inhibition, interspecies variation, 152 chronic exposure central effects, 281 neurobehavioral effects, 353, 354 cognitive effects, 350 delayed hyperthermia, 560 hypothermic response to, 555 immune system effects, 496 -induced myopathy, 512 inhalation toxicity, 404 memantine and, 39-40 neuropathy target esterase inhibition, 362
neurotoxicity, 277-278 electrophysiological, 340, 341 nicotinic receptor effects, 260 PBPK model for, 108 reactive oxygen species formation, 455 structure of, 390 and tolerance to acetylcholinesterase inhibition, 258 toxicity, physostigmine protection, 611-612 N,N'-Diisopropylphosphorodiamidic fluoride, 7 Diisopropyl phosphorofluoridate, 3 and OPIDN syndrome, 7 Dimethoate birth defects and, 475 cancellation/phase out, 630 immune system effects, 502 intermediate syndrome, 371,372, 376 reproductive toxicity, 454 Dimethyl-4-phenylpiperazinium, 260 O, O-Dimethyl dithiophosphate, 579, 691 O, S-Dimethyl hydrogen phosphorothioate, 693 O, O-Dimethyl phosphate, 579, 580, 691 Dimethyl phosphorofluoridate, 3 O, O-Dimethyl S-(N-ethylcarbamoylmethyl) phosphorodithioate (CL 18706), 609 O, O-Dimethyl thiophosphate, 579, 691 Dioxalan binding, effect of organophosphates on, 259 Dioxins estrogenic effects, 447 and male reproduction, 451 Diplopia, oxime-related, 725 Dipterex, mixtures, 610 Diquat, 415 Disposition, PBPK models, 108-114 Distigmine bromide, in urine voiding dysfunction, 22 Distribution, anticholinesterases, interspecies variation, 148-149 Disulfide loops, cholinesterases, amino acid sequence, 168-171 Disulfiram, methylation of, 138 Disulfoton intermediate syndrome, 377 nicotinic receptor inhibition, 259 tolerance development and carboxylesterase, 261 and tolerance to acetylcholinesterase inhibition, 258 5,5'-Dithiobis-2-nitrobenzoic acid, 202 Diuron, endocrine disruption, 487 Dizziness, oxime-related, 725 DNA adducts, carbamates and, 137 organophosphate-induced damage, 537-538 single strand breaks, 537-538 DNA flow cytometry, 458 DNA stability assay, 458
Index
DNA synthesis inhibition cell cultures, 303-304 and developmental chlorpyrifos exposure, 297 DNQX, 341 Docosahexaenoic acid, 514 Donepezil for Alzheimer's disease, 27,283 analysis, 693,694 combination therapy with memantine, 40, 41-42 mechanism of action, 27, 28 Dopamine, 272-273 effects of developmental chlorpyrifos exposure, 300 retinal, 435 Dopaminergic fibers, arborization, 272-273 Dose-additive design, mixture studies, 608-609 Dosimetry, and PBPK/PD models, 108-118 Doxorubicin, 470 Draize injury scores, 427 Drift, sprays/dusts, 568 Drosophila melanogaster, cholinesterases 3-D structure, 168, 174-181 amino acid sequence, 162, 163-164 Drug metabolism, see also Xenobiotics metabolism butyrylcholinesterase, 187 carboxylesterases induction of, 227-228 isozymes, 219-220, 229 organophosphate enzyme-induction, 536 role of paraoxonase, 247 Drugs of abuse, developmental neurotoxicity, 634 Dugway Proving Grounds, 3 Dusts in closed environments, 585 dermal absorption, 411 drift, 568 inhalation exposure, 570 Dyskinesia, 280 Dyslexia, 633 Dyspnea nerve agent exposure and, 52, 59-61 organophosphate exposure and, 392, 393-394 Dystonia, 280 Eastern equine encephalitis, 600 Echothiophate effect on nicotinic receptors, 260 for glaucoma, 22, 283,432, 433 noncompetitive muscarinic receptor binding, 237 Ectoparasiticides, organophosphates, 23 Eel, freshwater, 168 Effect-additive design, mixture studies, 608-609
Egasyn, 92-93,223-224 Electrocardiogram, organophosphate-induced changes, 92, 383-386 Electroencephalogram, pesticide-related abnormalities, 573 Electromyography in intermediate syndrome, 373, 374, 375-376 for monitoring exposure, 581 OPIDN, 365,573 single-fiber, 376 Electron capture detector, 684 Electron impact ionization, 685 Electron spin resonance spectroscopy, 521 Electroolfactogram, 342 Electrophoresis microchips, 694 Electrophus electricus, 168 Electrophysiological mechanisms, insecticide neurotoxicity action potential conductance, 339-340 neuromuscular transmission, 340-341 synaptic transmission, 341-344 Electroretinography, 430, 435-438 Electrospray ionization, 685 Electrospray ionization mass spectrometry, 705 Elimination anticholinesterases, interspecies variation, 148 PBPK models, 108-114 ELISA, pesticide analysis, 690 Ellman method, cholinesterase activity measurements, 202-203 Embryolethality, pesticide-induced, 474-475 Embryotoxicity, pesticide-induced, 474-475 Emergency preparedness plans, chemical warfare agents, 47-48 Emotion, cholinergic modulation of, 274 Employee periodic medical examination, 574 Encephalitis, 600 Endocannabinoids, 707 Endocrine disruption, 448, 452-453 carbamates in vitro, 485-487 in vivo, 489-490 in humans, 490-491 organophosphates in vitro, 481-485 in vivo, 488-489 Endoplasmic reticulum KDEL receptor, 223,229 localization of carboxylesterase, 219, 220, 223-227 " retention tetrapeptide, 223,229 Endosulfan, estrogenic activity, 485 Endothelium-derived relaxing factor, 473 Endplate potential, 340 Endpoint assessment, WHO/FAO guidelines, 646-647 Endpoint methods, cholinesterase activities, 201-202
745
Enucleated eyes, eye irritation tests, 427 Environmental Chemistry Methods, 690
Environmental Genome Project, 248 Environmental Protection Agency avian acute oral toxicity test, 673 benchmark dose guidance document, 618 canceled/phased out pesticides, 630 carbamates subgroups, 620 chlorpyrifos restrictions, 293, 306 cumulative risk, defined, 620 developmental neurotoxicity guidelines, 633-639 Integrated Risk Information System, 619 mechanism of toxicity, defined, 620 ocular toxicity testing, 430-431 Office of Pesticide Programs, 617-632, 633 anticholinesterase toxicity guidelines, 52-53 data call-in, 619, 636 pesticide additives regulations, 417 pesticide docket, 617 pesticide exposures, 94 pesticide regulations, 618 pesticide reregistration, 635 pesticide use, 523 reference doses, 55-57, 618 registered pesticides, 617, 633, 643 residue guidelines, 690 website, 617 Enzyme activities, measuring, 201 Epichlorhydrin, and male reproduction, 451 Epidemics, mosquito-borne diseases, 600 Epidemiology, organophosphate intoxication, 93-98 Epidermis, 411-412 EPN, 609-610, 716 Eptastigmine in Alzheimer's disease, 17 analysis, 693,694 toxicity, 27 EPTC, methylation of, 138 Equilibrium dialysis, partitioning coefficient determination, 110-111 Erectile dysfunction, 447 Eserine. see Physostigmine Esotropia, 433 Esterases activity, interspecies variation, 150 and immune response, 496 role of, 128-129 Ester drugs/prodrugs, carboxylesterases metabolism of, 219-220, 227-229 Estimated reference doses, nerve agent exposure, 48, 55, 57 Estrogen hypothesis, reproductive toxicity, 447, 448, 452-453,458-459 Estrogen proliferative screening assay, 481 Estrogen receptor carbamate binding, 485-487 pesticide binding, 452-453,481-485
746
Index
Estrogen receptor competitive binding assay, 481 Estrogens, environmental, 447, 448, 452-453,458-459 Ethanol developmental neurotoxicity, 634, 638 hypothermic response, 558 Ethenoadenosine, 137 Ethion, 23,630 Ethopropazine, 161, 176, 201 Ethoxycoumarin-o-deethylase, 664 Ethoxyresorufin-o-deethylase, 664, 665-667 Ethyl carbamate carcinogenicity, 137, 544 immunotoxicity, 498 Ethyldichlorvos, aging of, 183 S-Ethyl dipropylthiocarbamate, 17 Ethylene dibromide, and male reproduction, 451 Ethyl methyl phosphonic acid, 693 O-Ethyl O-p-nitrophenyl phenylphosphonothionate, potentiation of malathion, 609-610 Ethyl parathion antidotal treatment, 723 intermediate syndrome, 376 retinal effects, 436 Ethylthiometon, 433-434, 438 Europeans, butyrylcholinesterase genotypes, 191 European Union, airborne exposure guidelines, 585 Excitotoxicity, 42, 521-527 glutamate, 513 mechanism, 511 neuromuscular junction, 511-512 and nitric oxide, 528 Executive Order No. 13045, 633 Exelon, 28 Exercise effect on pralidoxime, 721 and heat stress, 562-563 Exhaust methods, closed facilities, 585 Explant cultures, placental, 469 Explosives, IMS analysis, 686 Extracellular signal-regulated kinase signaling pathway, 239 Extraction methods, 681-684 Extraneous maximum residue limit, 650, 651 Extrapolation factors, risk assessment, 619 Extrapyramidal motor effects, 280, 281 Eye(s) absorption pathways, 431-432 accomodative capacity, 433-434 cholinergic activity, 429-430 distribution of cholinesterases, 423-424 enucleated, 427 methyl isocyanate toxicity, 83 occupational pesticide exposure protection, 583
as route of absorption, anticholinesterases, 423-430 as target organ, 423,430-431 Eye irritation tests, 424-426 crystal violet staining, 428 cytotoxicity assays, 428 fluorescein staining, 425 in vitro alternatives, 427-429 in vivo alternatives, 426-427 neutral red uptake, 428 ocular/periocular lesions, 425 photobacterium phosphoreum luminescence, 428 plasminogen activation assays, 428 slit-lamp biomicroscopy, 425,426 structure-activity relationships, 429 tiered scheme, 429 Eyelid twitching, 438 F2-isoprostanes, 514 carbamate-induced changes, 516 correlation with acetylcholinesterase inhibition, 519 pesticide-induced changes in, memantine/atropine pretreatment, 519 F4-neuroprostanes, 514 Facial nerve paralysis, 438 Famphur, 23 Farm workers children of, pesticide exposure, 604-605 occupational pesticide exposure, 571 neurobehavioral effects, 352 Fasciculations, evaluating, 581 Fasciculins, 168, 176, 182-183 Fas ligand, 464 Fast atom bombardment/liquid secondary ionization, 685 Fatality rates, organophosphate intoxication, 91 Fatty acid amide hydrolase, 707 Febrile response, anticholinesterases, 559-562 Fecal samples, metabolite analysis, 692 Federal Emergency Management Agency, 47, 62 Federal, Food, Drug, and Cosmetic Act, 618, 633 Federal Insecticide, Fungicide, and Rodenticide Act, 633,635 Feeding studies, WHO/FAO guidelines, 647 Fematoscan, 686 Fenarimol, 474, 485 Fenitrothion antiandrogenic activity, 485 carbamates interaction, 611 endocrine disruption, 488 formulations, 608 immune system effects, 502 mixtures, 612 partitioning coefficient, 110-111 retinal effects, 438
Saku disease, 430 steroid receptor binding, 453 Fenthion, 23 antiandrogenic activity, 485 carbamates interaction, 611 cholinergic crisis, clinical course, 372 endocrine disruption, 488 gene expression of, 538-540 induction of heat shock proteins, 538-540 intermediate syndrome, 371,372 mixtures, 610 nicotinic receptor inhibition, 259 oxidative stress and, 534 protein kinase C modulation, 541 retinal effects, 436-437 Saku disease, 430 uses, 533 Ferbam, 17 Fetotoxicity, pesticide-induced, 474-475 Fetus, pesticide sensitivity, 448 Fever, anticholinesterase-induced, 559-562 Fick's law of diffusion, 412 Filariasis, 599-600 Fire retardants, thermal decomposition products, 406-407 Fires, thermal decomposition products, 406 Fish anticholinesterases sensitivity, 148, 150, 152 carbofuran toxicity, 659-661 cholinesterases, amino acid sequence, 162, 163-164
CYP isozymes, 663-666 malathion toxicity, 489 organophosphate metabolism, 661 pesticides residues, 481 species used in toxicity testing, 658 xenobiotics metabolism, 664-666 Fish eggs, toxicity testing, 659 Flame photometric detector, 683,684-685 " Flame retardants chemistry, 7, 17 organophosphate, 5, 406 Flavin-containing monooxygenase, role of, 128 Flavin-containing oxygenases, 708 Flea collars, 70 Florisil, 682 Fluorescein staining, eye irritation tests, 425 Fluorescein uptake, blood-brain barrier integrity, 279 Fluorescent tracer methods, pesticide exposure, 576 Fluoride number, 189 Fluoride-resistant variant, butyrylcholinesterase, 192, 204 Fluorochromes, corneal permeability, 425,427 Flutamide, 488 Follicle-stimulating hormone, pesticideinduced alterations, 452
Index Food pesticide residues, 603 pesticide tolerances, 618 Food and Drug Administration acetylcholinesterase inhibitors approved for Alzheimer's disease, 27-28 food monitoring program, 617 Pesticide Analytical Manual, 681 pesticide guidelines, 643 Food Quality and Protection Act, 617, 618 public health pesticide uses, 601, 602, 604 10x factor, 618,619, 633,635,639 Formulations, pesticide, interaction of compounds, 608 Formulators, pesticide, 568 FP-biotin, 704 Free radicals and heat shock proteins, 538 and male infertility, 454-456 and pesticide-induced oxidative stress, 90 protein kinase C interaction, 541 Free radical scavenging system, 456 Fruit juices, dialkylphosphates in, 603 Fruits, maximum residue limits, 650-651 Fungicides, thiocarbamates, 17 Furathiocarb, metabolites, 693 Galantamine for Alzheimer's disease, 28,283 analysis, 693,694 mechanism of action, 28 in myasthenia gravis, 22 Gallamine, cholinesterase binding site, 176 Gamma-aminobutyric acid retinal, 435 VX-induction of, 51 Gamma aminobutyric acid receptor benzodiazepines and, 726 TMPP blocking of, 407 Gamma-glutamylcysteine synthetase, 512 Gamma-glutamyl transpeptidase, 456 Gammarus plexus, 660-661 Ganstigmine, in Alzheimer's disease, 17, 22 GAPs. see Good agricultural practices Gas chromatography, 684 Gastrointestinal tract, occupational pesticide exposure, 570 Gel permeation chromatography, 683 Gender and anticholinesterase sensitivity, 154-155 and butyrylcholinesterase activity, 204 Gene expression carbamates and, 545 organophosphates and, 538-540 Genotoxicity, organophosphates, 537-538, 542-544 Germany, nerve agent production, 49, 389
Gestation chlorpyrifos exposure during, 296 and placental choline acetyltransferase activity, 473 Gills, 663 Glaucoma cholinesterase inhibitors for, 3, 4, 22, 434 echothiophate for, 22, 283,432, 433 physostigmine for, 4, 283 Glia chlorpyrifos targeting, 297-298, 533-534, 541-542 organophosphate neurotoxic effects, 330-332 types of, 330 Globus pallidus, cholinergic innervation, 272,273 GLPs. see Good laboratory practices Glucagon, ocular absorption, 431 Glucocorticoids, pretreatment and methyl isocyanate toxicity, 82 Glucose-6-phosphate dehydrogenase, 456 Glucosidation, 129 Glucuronic acid, 129 /3-Glucuronidase, 92-93 Glucuronidases, 129-130 Glucuronidation, 129-130 Gluphosinate, toxicity, 5 Glutamate dementia and, 42 memantine and, 36 role at neuromuscular junction, 513 Glutamate receptor, nerve agents and, 51 Glutamate receptor antagonists, 726 Glutathione carbofuran plus/3-napthoflavone, 667 composition, 129 conjugation reactions, 137 protective effects, 512 Glutathione peroxidase, 456 Glutathione reductase, 456 Glutathione transferases, 129, 456, 708 Glyceraldehyde-3-phosphate dehydrogenase, 363 Glycerophosphocholine, 362 Glycine, retinal, 435 Glycogen synthase kinase-3, 29 Glycopyrrolate, 93, 377 Glycosolation, 129 Glyphosate, toxicity, 5 Goldfish, carbofuran toxicity, 659 Gonadotoxicity, assessing, 456-458 Gonads, male, 449-450 Good agricultural practices, 643,650, 653 Good laboratory practices, 643,653 gpl20, inhibition by memantine, 39 G protein/cAMP-dependent protein kinase A, pesticide disruption of, 453 G protein-coupled receptors, 259, 275-276 down-regulation, 258
747
G proteins, 258 chlorpyrifos interactions, 295,297, 301 Gravitational settlement, inhaled particles, 401 Greenhouse workers, pesticide exposure central effects, 282 endocrine disruption, 490 Growth, methyl isocyanate exposure and, 84 G series agents, see also Sarin; Soman; Tabun delayed neuropathy, 54-55 history, 3, 48-49, 389-390 properties, 49-52 Guinea pigs plasma carboxylesterase, 263 tolerance development to carboxylesterase inhibitors, 261-262 Gulf War environmental exposures, 69, 252 nerve gas warning systems, 73 products used during, 70 Gulf War syndrome animal studies, 72-73 anticholinesterase intoxication, long-term effects of, 281-282 carbamates and, 69-75 and cholinesterases variants, 195 cognitive dysfunction, 234 neurological diseases, 74-75 organophosphates and, 69-75 and paraoxonase status of veterans, 252 pyridostigmine and, 279-280 self-reported exposures, 70-71 "symptom belief", 283 symptoms, 69, 252 synergistic effects of multiple exposures, 71-73 Guthion, mixtures, 610 H7,665 Haber's rule, 392 Hagedorn oximes, 719, 720 efficacy, 722, 723,724 Half-maximal effective concentrations, 318 Half-maximal inhibitory concentrations, 318 Halothane, developmental neurotoxicity, 638 Haloxon anthelmintic use, 23 and OPIDN syndrome, 7 Hand rinses, 575 Hatch rate, 658 Hazard assessment, 618-619 Hazard identification, 618-619 HD5 method, 673, 674-677 Health care facilities, workplace, 587 Health and Safety Program, auditing, 583 Heart, organophosphate-induced toxicity, 381-386, 703 Heat, dissipation methods, 562 Heat loss pathways cholinergic stimulation, 551 rodents, 556-557
748
Index
Heat shock proteins overexpression of, 666 pesticide induction of, 538-540, 665 and protein kinase C, 667 Heat shock/stress genes, 538-540 Heat stress effect on pralidoxime, 721 and exercise, 562-563 and toxicity, 562-563 Heavy metals, hypothermic response, 558 Hematocrit, effects of organophosphate intoxication, 92 Hematopoietic system, organophosphate suppression of, 502 Hemicholinium-3,274, 298 Hemolymph calcium, 659 Hen, as OPIDN model, 361 Hen egg, chorioallantoic membrane, in eye irritation tests, 427-428 Hepatocytes, fish, 666-667 Herbicides, thiocarbamates, 17 Heroin butyrylcholinesterase metabolism of, 187 carboxylesterase metabolism of, 220, 222 Hestrin method, cholinesterase activities, 201-202 H (Hammersmith) variant, butyrylcholinesterase, 189-194, 204 HI-6, 215, 719, 724 efficacy, 722, 723 organophosphate poisoning/intoxication, 485-486 and pulmonary toxicity, 393-394 High-affinity choline uptake, 271,276 High-energy phosphates and anticholinesterase-induced seizures, 278 carbamate-induced changes, 517 correlation with acetylcholinesterase inhibition, 519 organophosphate-induced changes, 517-518 pesticide-induced changes, 522-523 memantine/atropine pretreatment, 520 and PBN pretreatment, 525-527 and vitamin E pretreatment, 525-527 High-performance liquid chromatography, 684 High-stepping gait, 365 Hippocampal slices, insecticide mechanism of action studies, 341 Hippocampus alterations in Alzheimer's disease, 26 cholinergic innervation, 272 developmental chlorpyrifos exposure, 298-299 HIV, and neuronal damage, 39 HLr-7, 724 Homogenization, 681 Hormones, pesticide-induced alterations, 452 HORRAT value, 696 Households, pesticide use, 602, 604 HPTE, steroid receptor binding, 452
Human chorionic gonadotropin, 458 Human placental endothelial cells, 469 Human placental perfusion method, 466-469 Humans anticholinesterase poisoning behavioral effects, 351-352 neurobehavioral effects, 348-351 sensitivity to, 148, 150, 152 signs of, 276-277 carbofuran toxicosis, 659 carboxylesterases genes, 228 plasma, 263 cholinesterases 3-D structure, 174-181 amino acid sequence, 162, 163-164 variants, 181-182 endocrine disruption, pesticide-induced, 490-491 fever/hyperthermia, anticholinesteraseinduced, 561-562 immunotoxicity and cholinesterase inhibitors, 498, 501 observations in, WHO/FAO guidelines, 646 OPIDN, clinical aspects, 364-365 organophosphate-binding proteins, 704-705 placenta, cholinergic system, 472-473 spermatogenesis, 450 Huperzines, 176, 201,386 Hydraulic fluids, 364 Hydrogen cyanide and Bhopal accident, 79-80 inhalation hazard, 400 Hydrogen peroxide, detoxification, 456 Hydroxamic acids, 3-4, 719 3-Hydroxycarbofuran, 666 Hydroxylamine, 3-4, 719 Hydroxylation, carbamate metabolism, 135-136 Hydroxyl radical, lipid peroxidation initiation, 522 3-Hydroxynitrosocarbofuran, 662 5-Hydroxytryptamine, 551 Hygiene occupational, 582-587 personal, 584 Hyperactive airway, 395 Hypersensitivity pneumonitis, 574 Hyperthermia, 559-562 Hypoglycemia, 92 Hypogonadism, 452 Hypokalemia, 92, 384 Hypomagnesaemia, 92 Hyporeflexia, 376 Hypothalamic-hypophysial-gonadal axis, 659 Hypothalamic-pituitary-adrenal axis, role in regulating immunity, 498 Hypothalamic-pituitary-gonadal axis, effects of pesticides on, 452, 457
Hypothalamic-pituitary-thyroid axis, 660 Hypothalamus, acetylcholine activity, 704 Hypothermia and anticholinesterase poisoning, 549, 551-554 benefits of, 558 development of, 550 Idolamine, retinal, 435 Imidapril, 219 Immune system adaptive, 496 allergic sensitization, 504 anticholinesterase pesticides and, 496-503 developing, 496 developmental chlorpyrifos exposure and, 301 innate, 496 Immunity cell-mediated, 496 humoral, 496 Immunoaffinity sorbents, 683 Immunochemical assays, metabolites, 580 Immunoextractions, 683 Immunoglobulins, 496 Immunomodulation, cholinesterase inhibitors, 496-498 Immunosuppression, organophosphates, 495,502 Immunotoxicity methyl isocyanate, 84 and phosphorylating properties of organophosphates, 498 Impaction, inhaled particles, 401 Indentation tonometry, 426 India acute pesticide poisonings, 571 Bhopal accident, 79-85,568 butyrylcholinesterase genotypes, 191 pesticide poisoning in, 93-94 Indian Council of Medical Research, 79-80 Industrial chemicals, and male reproduction, 451 Infants dietary pesticide exposure, 603 FQPA 10x factor, 618, 619, 633,635, 639 neurobehavioral disorders, 633 organophosphate sensitivity, 118-119 Ingestion, pesticides, 570 Inhalable fraction, 401 Inhalation toxicity, 392; see a l s o Pulmonary toxicity acute lethal toxicity, 399-401 exposure route, 399 exposure studies, 402 lethality, 405 material distribution, 401-402 material formulation, 399 respiratory tract effects, 402-403 study design, 399
Index
systemic effects, 403-406 vapor inhalation hazards, 399 Inhibition rate constant, 113 Inositol triphosphate, 237 Inotrope, 717 Insecticides, s e e a l s o Pesticides biotransformation, 104-106 carbamates, 339 cross-resistance, 601 mechanism of action, 339-344 organochlorine, 130 organophosphates, 339 vector resistance, 601-602 Insects acetylcholinesterase genes, 168, 172-173 cholinesterases, amino acid sequence, 162, 163-164 resistance development, 176, 183 Insulin, ocular absorption, 431 Integuments, 663 Interception, inhaled particles, 401 Interleukin-6, 560 Intermediate syndrome, 7 clinical aspects, 91,373-375 diagnostic criteria, 377 experimental animal studies, 372-373 future studies, 377-378 inhalation toxicity, cholinesterase inhibitors, 404 pathogenesis, 376-377 prevention, 727 signs/symptoms, 371-372, 373-374, 573, 661-662 treatment, 371,372, 377, 727 Intermolt phase, 659 International Programme on Chemical Safety, 634 International Union of Pure and Applied Chemistry, 684 Interspecies variation, anticholinesterase intoxication LDs0s, 145-150 pharmacodynamics, 151-155 pharmacokinetics, 148-151 Intracellular neurofibrillary tangle, 26 Intracellular signaling pathways, 239 Intracytoplasmic sperm injection, 447 Intraocular pressure, 426-427, 434 Inuits, butyrylcholinesterase genotypes, 191 Invertebrates, aquatic toxicity testing, 658 Ionization techniques, 685 Ion mobility spectrometry, 685-686 Iran acute pesticide poisonings, 571 butyrylcholinesterase genotypes, 191 Iris eye irritation tests, 425 ocular toxicity, anticholinesterases, 432-433 Iritis, 433 Irritant receptors, 391
Islands of Calleja, cholinergic innervation, 273 Isocyanates, 80 Isomalathion, 608 Isoniazid, 187 iso-OMPA butyrylcholinesterase inhibition, 161,176, 214 activity measurements, 201 lethal inhalation toxicity, 405 and tolerance development, 262 toxicity potentiation, 610 2-Iso-propoxyphenol, 693 Isopropyl methyl phosphonic acid, 693 Isoprostanes as biomarkers of oxidant injury, 514,516,517 correlation with acetylcholinesterase inhibition, 519 Isosafrol, 664 Japan butyrylcholinesterase genotypes, 192 nerve agent poisoning incidents, 49 pesticide poisoning in, 94-95 Japanese encephalitis, 600 Japanese medaka, carbofuran toxicity, 659 Jar cells, 469 Jeg3 cells, 469 J (James) variant, butyrylcholinesterase, 189-194
Joint FAO/WHO Expert Committee on Food Additives, 644 Joint WHO/FAO Meeting on Pesticide Residues in Food, 644-653 Juxtacapillary (J) receptors, 391 Kainic acid, 528 KDEL receptor, endoplasmic reticulum, 223,229 Kepone and male reproduction, 451 nitric oxide synthase inhibition, 455 Kerala (India), acute pesticide poisonings, 571 Keratinocytes, 411 Ketamine, 39 Ketanserin, 300 3-Ketocarbofuran, 666 Ketoconazole, 139 Khamisiyah, and chemical warfare agents exposure, 73-74 Kinetic disorders, anticholinergic agents for, 274-275 Kinetic modeling, acetylcholinesterase metabolism/inhibition, 211-213 K (Kalow) variant, butyrylcholinesterase, 189-194, 204, 216 Knox Out, 543 Kuderna-Danish, 684
749
Kuhn, Richard, 389 Kynurenic acid, 707 Kynurenine, plasma, 707 "Lab on a chip", 694 Lacrimation due to methyl isocyanate toxicity, 83 excess, ocular absorption and, 431 eye irritation tests, 425 LaCross encephalitis, 600 Lactate dehydrogenase as biomarker of muscle injury, 512, 516 isozymes, 512, 516 leakage by organophosphates, 536 myonecrosis and, 511-512 pesticide-induced changes in, 527 Lactation, developmental neurotoxicity guideline, 634, 637 Lambert-Eaton myasthenic syndrome, 376 Lange, Willy, 390 Lannate, reproductive toxicity, 453-454 Larvicides, 601 Laskin nebulizer, 402 Laterodorsal tegmental nucleus (Ch6) cholinergic innervation, 273 role in respiratory depression, 277 Lavage, gastric, 717 Lazaroid (U78517F), 514 Lead acetate, developmental neurotoxicity, 634 Learning, 36 cholinergic modulation of, 273,274 chronic pesticide exposure effects, 354 conditioned, 273 developmental chlorpyrifos exposure and, 299, 300 disabilities, 633 effects of memantine on, 39 measuring deterioration, 39 reward-based, 272 Leishmaniasis, 599 Lens isolated, 427 ophthalmic toxicity, 434-435 Leptophos, 330, 716 Leukocytes, 496 Leukocytosis, organophosphate intoxicationassociated, 92 Leutinizing hormone, 449, 450, 452 Lewisite, 49 Lewy bodies, 29-30 Leydig cells, 449-450 Libido, development of, 450 Ligand binding methods, insecticide mechanism of action, 339 Lindane dermal absorption, 416, 417 hormone alterations, 452 metabolism of, 665 Linear free energy relationships, 412-413 Linuron, 452, 487
750
Index
Lipid peroxidation biomarkers, carbamate-induced changes, 516 dichlorvos and, 534-535 initiators, 522 isoprostanes as indicators, 514, 516, 517 and muscle hyperactivity, 514-515 organophosphate-induced, 534-535 pesticide-induced changes, 522-523 pesticide-induced oxidative stress, 90 Lipids intercellular, and dermal absorption, 412 metabolism, role of paraoxonase, 247 Liquid chromatography/electrospray ionization-tandem mass spectrometry, 693 Liver disease, and butyrylcholinesterase activity, 204 Lou Gehrig's disease, in Gulf War veterans, 74-75 Lowest-observed-adverse-effect level anticholinesterase exposure, interspecies variation, 145-147 estimated reference doses, 55-57 risk assessment, 618-619, 621--626 Lowest-observed-effect level, anticholinesterase exposure, interspecies variation, 145-147 Lubricants, 364, 406 LtiH6, 215 Lungs control of ventilation, 391 methyl isocyanate toxicity, 82, 83 muscarinic receptors, 237-239 organophosphates effects, 237-239, 703 receptors, 391 respiratory physiology, 390-391 Lymphocyte activation, 496 Macrophages, 496 Madhya Pradesh (India), acute pesticide poisonings, 571 Malaoxon, inhibition of cAMP synthesis, 239 Malaria, 599-600 malathion epidemic poisoning, 608 reemergence, 600-601 vaccine research, 601 Malathion, 23 absorption/elimination of, 148 allergic response to, 504 boil weevil eradication, 602 carbamates interaction, 611 carboxylesterase hydrolysis of, 133 cholinesterase inhibition interspecies variation, 152 specificity, 183 decomposition products, 406 delayed hyperthermia, 559, 560 history of, 3 immune system effects, 502 intermediate syndrome, 372, 376
metabolites, 579 mixtures, 610, 611, 612 nitric oxide synthase inhibition, 455 oxidative stress induction, 454 poisoning incidences, 3 potentiation, 608 by EPN, 609-610 by tri-o-cresylphosphate, 609-610 rat mammary tumor model, 543-544 retinal effects, 436 Saku disease, 430 and thyroid activity, 489 toxicity to fish, 489 ultra-low volume spraying, 600 uses, 543 Male reproduction and antiandrogen compounds, 491 assessing gonadotoxicity, 456-458 pesticides and, 450-451 reproductive tract, 449-450 sperm quality and, 448 Malondialdehyde, 538 Mammals anticholinesterases sensitivity, 148, 152 paraoxonase activity, 249 pesticide metabolism, 661 thermoregulation, 549, 550-551 Mancozeb, 17 endocrine disruption, 487, 490 reproductive toxicity, 454 Maneb, 17 Manual of Analytical Methods for the Analysis of Pesticides in Humans and Environmental Samples, 688-689 Manual of Pesticide Residue Analysis, 689 Mass balance studies, dermal absorption/toxicity, 414 Mass spectrometer, 658 Matching accuracy, effects of anticholinesterase poisoning, 350 Material safety data sheets, 582 Maternal-placental-fetal unit, 471-472 Matrix-assisted laser desorption ionization, 685 Matrix-assisted laser desorption time-offlight, 705 Matsumoto incident, delayed hyperthermia, 561 Maximum residue limits, WHO/FAO guidelines, 644, 650-651 Meat, maximum residue limits, 650 Mecamylamine, 259, 278 Meconium, organophosphate metabolites in, 543 Medical surveillance, occupational pesticide exposure, 572-576 Medulla, respiratory centers, 391,394 Memantine adverse effects, 38 clinical use, 36-38 interaction with acetylcholinesterase inhibitors, 39-42
in vitro profile, 38-39 mechanism of action, 42-43 and pesticide-induced reactive oxygen species, 512 postmarketing surveillance studies, 38 prevention of acetylcholinesterase inhibitorinduced oxidative stress, 519-521 properties, 35-36 safety, 38 Membrane signaling, and stress, 665-666 Memory, 36 acute pesticide poisoning effects, 573 Ca2+/cAMP response element binding protein and, 240 cholinergic modulation of, 273, 274 chronic pesticide exposure and, 353 developmental chlorpyrifos exposure and, 299, 300 impairment, 236 loss of, in Alzheimer's disease, 26 Mental retardation, 633 Meperidine, carboxylesterase metabolism of, 220 Merphos, 5 Metabolism anticholinesterases, interspecies variation, 149-151 carbamates in vitro studies, 138-140 phase I reactions, 134-137 phase II reactions, 137-138 effects of pesticides on, 453--454 organophosphates in vitro studies, 138-140 phase I reactions, 131-133 phase II reactions, 134 PBPK models, 108-114 Metabolites carbamates, analytical methods, 693 maximum residue limits, 651 organophosphates, analytical methods, 691-693 Metals, in aquatic ecosystems, 666 Metam, methylation of, 138 Methadone, developmental neurotoxicity, 634 Metham, 17 Methamidophos, 716 age-related sensitivity to, 355 birth defects and, 475 cumulative risk assessment, 629 extraction of, 682 extrapyramidal effects, 280 metabolites, 693 neurotoxicity, 340, 353, 354 Methidathion, 722 Methiocarb, 485,487 Methomyl endocrine disruption, 474, 485 interaction with memantine, 41 seizure induction, 278
Index
Methoxychlor, 452, 481 Methyl-5-hydroxy-2benzaimidazolecarbamate, 693 Methyl amines, toxicity, animal models, 82-83 2-Methylaminochroman, 538 Methylation, carbamates metabolism, 137-138 Methyl azinphos, 610 Methylazomethanol, developmental neurotoxicity, 638 Methylcarbamates, anticholinesterase activity, 214 Methyl-carbamic acid, 106 Methyl chlorpyrifos, neurotoxicity, 234 Methyl isocyanate Bhopal accident, 4, 79-80, 568 conjugates, 81 degradation products, 81 physicochemical characteristics, 80-81 toxicity animal models, 81-83 clinical, 83-85 Methyl mercury, developmental neurotoxicity, 634, 638 Methyl parathion activation of, 150 antiandrogenic activity, 485 antidotal treatment, 723 cancellation/phase out, 630 demethylation of, 131 dermal absorption, 417 immune system effects, 501-502 memantine and, 41 mixtures, 610, 612 neurobehavioral effects, chronic exposure, 353 nicotinic receptor and, 259 protein synthesis and, 474 Saku disease, 430 and tolerance to acetylcholinesterase inhibition, 259 transplacental transfer of, 470 Methyl phosphonate alkyl esters, 693 Methyl phosphonic acid, 693 Methylprednisolone hemisuccinate, carboxylesterase metabolism of, 222, 228 Metolcarb, peripheral neuropathy, 364 Metrifonate in Alzheimer's disease, 17 chronic exposure, central effects, 281 interaction with memantine, 40 Mevinphos birth defects and, 475 IMS analysis, 686 retinal effects, 436 Mexico, acute pesticide poisonings, 571 Michaelis complexes, 210-214 Microarray technology, neurotoxicant screening, 304
Microchips electrophoresis, 694 single-channel, 694 Microelectrode studies, 581 Microglia, 330 Microwave-assisted solvent extraction, 682 Midazolam, 726 Mild cognitive impairment, 26 Milk, maximum residue limits, 650 Mimivirus, cholinesterases, amino acid sequence, 162-164 Miniature endplate currents, 340 Miniature endplate potential, 260, 340 Mini-Mental Status Examination, 26 Miosis nerve agent poisoning, 52-53, 58-61 organophosphate-induced, 392, 432 and sarin exposure, 58-59 Mipafox neurotoxicity testing, cell culture systems, 329, 330 OPIDN, 7, 316 Miscarriages, methyl isocyanate-associated, 83-84 Mission Oriented Protective Posture protection, 73 Mists, inhalation exposure, 570 Mitochondria acetylcholinesterase inhibitor-induced changes, 512 dysfunction, 522 nitric oxide and, 518 Mivacurium, butyrylcholinesterase hydrolysis of, 187 Mixtures administration sequence, 612 carbamates, 610 defined, 607-608 homergic, 608 more-than additive interactions, 610 organophosphates, 609-610 organophosphates and N-methyl carbamates, 611-612 study design, 608-609 time course of interactions, 611-612 WHO/FAO guidelines, 651 MK-801, 39, 341 Mobam, nerve agent protection, 611 Modifying factors, estimating reference doses, 56-57 Molinate, 17 endocrine disruption, 490 metabolism, interspecies variation, 139 toxicity in fish, 659 Monitoring, occupational, 575-576 Monkey, placental cholinergic system, 472 Monoamine systems, effects of developmental chlorpyrifos exposure, 300
751
Monocrotophos, 678 extrapyramidal effects, 280 intermediate syndrome, 371 Monoethylamine, 106 Mood, cholinergic modulation of, 274 Morbidity, organophosphate intoxication, 91 Morphine, carboxylesterase metabolism of, 222 Morris swim tank, 704 Mortality, organophosphate intoxication, 91 Mosquitoes acetylcholinesterase genes, 168, 172-173 disease vectors, 600-601 resistance, 600-602 Motivation, cholinergic modulation of, 274 Motor function, cholinergic modulation of, 273,274-275 Motor nerve conduction, evaluating, 581 Mouse acetylcholinesterase knockout, 234, 260-261 carboxylesterases genes, 228 cholinesterases 3-D structure, 174-181 amino acid sequence, 162, 163-164 neurotoxic target esterase knockout, 234 paraoxonase knockout, 249-250, 264 paraoxonase transgenic, 250 Mouse neuroblastoma cell lines, 328 MRLs. s e e Maximum residue limits MTBSTFA, 693 Muller, Paul, 657 Multidrug resistance- 1 gene, 540 Multidrug resistance-associated protein, 2, 220 Multiresidue method analysis, 686 Munitions binary, 48 unitary, 48, 49 Muscarinic effects nerve agents, 52 organophosphate intoxication, 91 Muscarinic receptors activation, 273 antagonists, 237, 259 binding assays, 258 chlorpyrifos effects, 294 cholinergic hyperstimulation, 145 cholinesterase-independent effects, 236-237 down-regulation, 236 inhibition of acetylcholine, 271 lung, direct effects of organophosphates, 237-239 nerve agents and, 51 organophosphate binding, 706-707 overstimulation of, 233,236 postsynaptic, 707 presynaptic, 707 respiration and, 274 subtypes, 236-239, 257-258 tolerance development and, 236, 257-259, 261
752
Index
Muscle fasciculations, acetylcholinesterase inhibitor-induced, 511-512 Muscle hyperactivity, and lipid peroxidation, 514-515 Muscle injury, biomarkers, 512, 515-516 Muscle weakness intermediate syndrome, 371,374 in OPIDN, 364-365 Mutagenicity, organophosphates, 662 Myasthenia gravis characteristics, 375 treatment acetylcholinesterase inhibitors, 3, 4, 22, 194 cholinesterase inhibitors, 283 galantamine, 28 genetic variability in, 194 neostigmine, 4 Mydriasis, 432 Myelin, organophosphate-induced degradation, 234 Myocardium, organophosphate-induced toxicity, 382-386 Myonecrosis, in intermediate syndrome, 372, 374 Myopathy acetylcholinesterase inhibitor-induced, 511-512 memantine/atropine and, 519-521 organophosphate-induced, role of nitric oxide, 518 prevention of, 512 Myopia, ethylthiometon-induced, 433-434 Myotoxicity, acetylcholinesterase inhibitorinduced, memantine and, 41 NADPH cytochrome P450 reductase, 128 Naled, 600 N-allylquinuclinidol, 727 Naphthalophos, 23 a-Naphthol, 106 a-Napthoflavone, 667 /3-Napthoflavone, 665,667 1-Napthol, 693 Narcotics, carboxylesterase metabolism of, 220 Nasal mucosa, absorption and, 431 Nasal passages, inhalation toxicity, 392-393 Nasolacrimal drainage system, 431 National Health and Nutrition Examination Survey, 603 National Home and Garden Pesticide Use Survey, 617 National Research Institute of Police Sciences, 94-95 Necrotizing myopathy, in intermediate syndrome, 372, 374 Neonates and anticholinesterase sensitivity, 154 chlorpyrifos neurotoxicity, 293-294, 296
paraoxonase activity, 252-253 pesticide sensitivity, 448 Neostigmine analysis, 693,694 in myasthenia gravis, 22 neurotoxicity, electrophysiological mechanisms, 340 respiratory effects, 393 in urine voiding dysfunction, 22 uses, 4 Neostriatum, cholinergic innervation, 272 Nephrotoxicity, anticholinesterase exposure and, 574 Nerve agents, see also specific agent; Chemical warfare agents aging of, 51, 183 analytical methods, 693-694 antidotes acetylcholine synthesis blockade, 727 adenosine receptor antagonist, 727 anticonvulsants, 726 cholinesterase, 727 blood flow alterations, 382 carbamate protection against, 611 cardiotoxicity, 382 chemical/physical properties, 50 CNS effects, 52, 277 degradation products, 693-694 delayed neuropathy, 53--55 destruction of, in vitro models, 332-333 dissemination, 389 exposure guidelines, estimated reference doses, 48, 55-57, 62 history of, 389-390 hypothermic response to, 555 -induced myopathy, 512 muscarinic effects, 52 nicotinic effects, 52 ocular toxicity, 430 OPIDN, 54-55 organophosphate, 5 history of, 3 prophylactic agents, 3, 4 protective effects of memantine, 41 properties of, 49-52 pulmonary toxicity, 277, 392-394, 395 pyridostigmine prophylaxis, 3, 23, 69, 71, 279-280, 365, 611 relative potency, 60-61 retrospective detection, 694 Russian stockpiles, 49 toxicity, 52-55 mechanisms of, 51-52 U.S. stock piles, 47--48, 49 Nerve conduction studies, 581 Nerve excitability, neurotoxicity and, 340 Neural cell cultures, neurotoxicant screening, 303 Neural tracts, interspecies variation, 154 Neural tube defects, 296, 605
Neurite growth, 241,297 Neurobehavioral disorders, infants, 633 Neuroblastoma cell lines, human (SY5Y), 328-329, 332-333 Neuroeffector junctions, acetylcholine accumulation, 209 Neuroendocrine dysfunction, in fish, carbofuran-induced, 659 Neurofibrillary tangles, tau and, 29 Neuromotor function, chronic pesticide exposure and, 353 Neuromuscular blockade, reversal of, galantamine, 28 Neuromuscular conduction techniques, 581 Neuromuscular junction cholinergic excitotoxicity, 511-512 nitric oxide at, 513 role of glutamate, 513 Neuromuscular transmission and acetylcholinesterase inhibition, 511-512 effect of pesticides, 340-341 glutamate and, 513 Neuromuscular transmission disorders, intermediate syndrome comparisons, 375-376 Neuronal stem cells, apoptosis, 318 Neurons, 315-316 cholinergic, see Cholinergic neurons effects of organophosphate neurotoxicants, 318, 328-330, 333 Neuropathology, and anticholinesterase intoxication, 278-279, 283 Neuropathy, delayed, see Delayed neuropathy; Organophosphate-induced delayed neuropathy Neuropathy target esterase, 7, 54, 316 aging of, 361,662 as biomarker, 580 placental, 473 tissue distribution, 362 WHO/FAO guidelines, 645-646 Neuropathy target esterase inhibition, 234, 703 DFP, 362 interspecies variation, 153-154 and neurotoxicity testing, 329-330 and OPIDN, 704 phenylmethane sulfonyl fluoride, 363 phenylpentyl phosphinate, 363 Neuropsychiatric disorder, organophosphateinduced, 89; see also Organophosphateinduced delayed neuropathy Neuropsychiatric Inventory, 26 Neurospora crassa, cholinesterases, amino acid sequence, 162-164 Neurotoxicity bicyclophosphorus esters, 407 carbamate-induced acetylcholinesterase inhibition, 276-283 electrophysiological mechanisms, 339-344
Index
and cholinesterase inhibition, 233-236 organophosphate-induced, 661-662 acetylcholinesterase inhibition, 276-283 acetylcholinesterase knockout mice and, 234 blood-brain barrier, 149 chronic, 234-235 developmental, see Developmental neurotoxicity effects, 233-234 electrophysiological mechanisms, 339-344 intracellular targets, 239-241 molecular targets, 239-241 muscarinic receptor effects, 236-239 neurotoxic target esterase knockout mice and, 234 and paraoxonase status, 251-253 pathways, 316 studying, 315 symptoms, 234 trimethylolpropane phosphate, 406, 407 tri-o-cresyl phosphate, 406 WHO/FAO guidelines, 646 Neurotoxicity testing bioavailability and, 318 cell/tissue culture models, 317-318, 319-327, 328-329 developmental cell culture models, 303-304 nonmammalian models, 305 effects on glia, 330-332 effects on neurons, 318, 328-330, 333 exposure regimens, 318, 333 extrapolation of results, 333 future directions, 333-334 goals of, 315 in vitro vs. in vivo systems, 316-317 neural cell types, 315-316 neuronal morphology and differentiation, 330 neuron-like cell lines, 328-329 noncholinergic/nonantiesterase mechanisms, 329-330 primary cultures, 329 prophylactic organophosphate detoxification, 332-333 whole embryo cultures, 329 Neurotransmission, cholinergic, 161,275-276 Neurotransmitters developing brain, 294 retinal, 435-436 Neutral red uptake, eye irritation tests, 428 NEvap, 684 N-formyl-L-kynurenine, 707 NGF, 328 NGF receptor, 271 N-hydroxyethylnaphthylvinylpyridine, 727 Nicotinamide adenine dinucleotide, 474 Nicotine, 259, 302-303
Nicotinic acetylcholine receptors organophosphate binding, 707 prolonged stimulation, 511, 512 Nicotinic agonists, and Alzheimer's disease, 260 Nicotinic effects nerve agents, 52 organophosphate intoxication, 91 Nicotinic receptors acetylcholine and, 271 activation, 273 antagonists, 259 chlorpyrifos effects, 294 cholinergic hyperstimulation, 145,233, 236 direct effects of insecticides, 340, 343-344 nerve agents and, 51 organophosphate inhibition of, 574 respiration and, 274 and sodium influx, 276 tolerance development, 259-260 Nictitating membrane, inflammation of, 425 Nimodipine, 727 N I O S H Manual o f Analytical Methods, 689 Nitric oxide acetylcholine and, 29 at neuromuscular junctions, 513 biomarkers carbamate-induced changes, 516-517 organophosphate-induced changes, 517-518 developmental neurotoxicity, 638 and excitotoxicity, 528 male infertility and, 454--455 neurotoxic effects, 522 and organophosphate-induced cardiotoxicity, 381 pesticide-induced changes, 522-523 respiration regulation, 274 role in organophosphate-induced myopathy, 518 sleep regulation, 274 Nitric oxide synthase activation of, 235 biomarkers carbamate-induced changes, 516-517 organophosphate-induced changes, 517-518 effects of memantine pretreatment, 41 inhibition, 235, 274, 455 isoforms, 513 N~-Nitro-L-arginine, 274 Nitro-L-arginine methyl ester, 518 Nitrogen phosphorus detector, 685 7-Nitroindazole, 518 p-Nitrophenol, urinary, 92, 579 p-Nitrophenylacetate, carboxylesterases and, 228 Nitrosocarbofuran, 662 Nitrosopropoxur, mutagenic potential, 544 NK cells, 496
753
NMDA. see N-methyl-D aspartate N-methyl-D aspartate antagonists, analgesic properties, 39 N-methyl-D aspartate receptors, 35, 36 activation, 235 antagonists, 39, 727 blocking, 513 memantine inhibition of, 38-39 N-methyl carbamates, cumulative risk assessment, 620, 630 N-methyl hydroxylamine, 214 N-methyl scopolamine, 258 N,N-diethyl m-toluamide, see DEET N,N'-methylene bi-acrylamide, developmental neurotoxicity, 634 No-observed-adverse-effect level estimated reference doses, 55-57 risk assessment, 618-619, 621-626 WHO/FAO guidelines, 646, 647, 652-653 No-observed-effect level, anticholinesterase exposure, interspecies variation, 145-147 Noradrenergic pathway, thermoregulation, 555 Norepinephrine, 300 North Americans, butyrylcholinesterase genotypes, 191 Northern Europeans, paraoxonase polymorphisms, 247 Nucleic acid synthesis, 474 Nucleophilic agents, 3 Nucleus accumbens, cholinergic innervation, 272,273 Nucleus basalis of Meynert, 26, 272 Nurse cells, 450 Nutrition, and pesticide usage, 602 Obesity, and butyrylcholinesterase activity, 204 Obidoxime, 719, 720 adverse effects, 725 efficacy of, 722-723 organophosphate poisoning/intoxication, 584-585 Occlusion, and dermal absorption, 418 Occupational hygiene, 582-585 closed facilities, 585-586 open air, 586-587 Occupational Safety and Health Administration, exposure limits, 585 Occupational toxicology biomarkers, 580 blood cholinesterase measurements, 576-579 exposure, 423,424, 448, 567-570 behavioral deficits, 351-354 "carry home", 605 dermal absorption, 411 and endocrine disruption, 490-491 NIOSH guidelines, 689 routes of, 569-570 sources of, 567-569
754
Index
Occupational toxicology (continued) medical surveillance, 572-576 neurophysiological monitors, 581 urine/blood metabolite measurement, 579-580 workplace biomonitoring, 581-582 Oconne River, 661 Octamethyl pyrophosphortetramide, lethal inhalation toxicity, 405 ODS resin, 682 ODS sorbents, 683 Office of the Army Surgeon General, AEGLs, 48, 57-62 Office of Pesticide Programs, 617-632, 633 developmental neurotoxicity guidelines, 633-639 Official Methods o f Analysis (OMA) o f AOAC International, 689 Oikopleura dioica, cholinesterases, amino
acid sequence, 162, 163--164 Oleamide, 707 Olfactory bulb, cholinergic innervation, 271 Olfactory tubercle, cholinergic innervation, 273 Oligodendrocytes, 316 Oligodendroglia, 330 Onchocerciasis, 601 ONT (Osterrieder-Noma-Trautwein) formulation, 386 O, O,S-trimethyl phosphorothioate, immunotoxic effects, 502-503 Operation Desert Shield/Storm, 69 pyridostigmine bromide, prophylactic use, 23 Ophthalmic toxicity anticholinesterases intraocular effects, 430 ocular effects, 424-428 aqueous humor, 434 ciliary body, 433-434 exposure accidental, 423,424 deliberate (medicinal), 423,424 occupational, 423,424 extraocular muscles, 438 eye, cholinergic activity, 429-430 eye irritation tests, 424-426 glaucoma and, 434 intraocular pressure, 434 iris, 432-433 lens, 434-435 retina, 435-438 systemic effects extraocular absorption, 430-431 transocular absorption, 431-432 Ophthalmoscopy, retinal lesions, 435 OPIDN. see Organophosphate-induced delayed neuropathy Optic neuritis, 438 Oral studies, WHO/FAO guidelines, 647 Ordered Uni Bi Bi kinetic scheme, 212-213
Organization for Economic Co-operation and Development, pesticide guidelines, 643 Organochlorine pesticides, 130 environmental persistence, 600 estrogenic activity, 481 withdrawal of, 681 Organomation Associates, 684 Organophosphate compounds acetylcholinesterase inhibition, 90 structure-activity relationships, 213-214 activation, interspecies variation, 150 aging, 183 allergic sensitization, 504 androgen receptor binding, 481 anthelmintics, 23 asthma and, 395 behavioral toxicity, 347-356 biotransformation, 104-106 body temperature and, 553, 554 carcinogenicity, 542-544, 662 rat mammary tumor model, 543-544 cardiotoxicity, 381-386 chemistry/structure, 5-7, 8-16, 103, 130-131,390 children, sensitivity to, 118-119 classes of, 104 crop pest control, 602 cumulative effects, 607-613 deactivation, interspecies variation, 150-151 decline in use, 673 delayed neurotoxicity/neuropathyassociated, 7; see also Organophosphate-induced delayed neuropathy dermal absorption/toxicity, 411-419 developmental neurotoxicity chlorpyrifos, 293-306 and paraoxonase, 251 DNA damage, 537-538 ectoparasiticides, 23 embryocidal/fetocidal effects, 474-475 endocrine disruption, 481-491 estrogenic effects, 447, 485 extraction solvents, 682 flame retardants chemistry, 7, 17 neurotoxic, 406 gene expression, 538-540 genotoxicity, 537-538, 542-544 and Gulf War syndrome, 69-75 history of, 3, 89, 130, 599 in human medicine, 17, 22-23 immune system and, 495-504 impurities associated with, 502-503 inhalation pharmacology/toxicology, 399-407 intermediate syndrome-associated, 7 lung muscarinic receptors and, 237
mechanism of action, 3, 90, 103-104, 209-211,715-716 electrophysiological, 339-344 non-acetylcholinesterase inhibition, 706-708 memantine interactions, 40-41 metabolism, 130-131 in fish, 661 and genetic polymorphisms, 119-120 in vitro studies using human tissues, 138-140 phase I reactions, 131-133 phase II reactions, 134 metabolite analysis, 691-693 metabolites, blood/urinary, 579-580 mixtures, 609-610 more-than additive interactions, 610 mosquito resistance, 601 mutagenicity, 662 nerve agents chemistry, 7 prophylactic agents, 23 neurotoxicity electrophysiological mechanisms, 339-344 noncholinesterase mechanisms, 233-241 noncholinesterase interactions, 236 occupational exposure, see Occupational toxicology ophthalmic toxicity, 423-438 OPIDN associated, 573 paraoxonase activity, and chronic exposure, 252-253 pesticides, chemistry, 5-7, 8-16 pharmacokinetics, 104 compartmental models, 107-108 physiologically based models, 108-114 principles, 107 placental toxicity, 463-475 prenatal exposure, biomarkers, 543 prophylactic detoxification, in vitro testing, 332-333 protein binding in vitro, 706 in vivo, 704-705 protein kinase C modulation, 540-541 pulmonary toxicity, 389-395 reproductive toxicity, 447-459 residue persistence, see Pesticide residues resistance, 4 risk assessment, see Risk assessment routes of exposure, 104 selectivity, 4 sensitivity to, 118-119, 708 sites of action, 703-704 specific metabolites, analysis, 692-693 thermoregulatory effects, 549-564 tolerance, see Tolerance development
Index
toxicity biomarkers, 91-93 and butyrylcholinesterase variants, 195 characteristics of, 89 clinical aspects, 91-93 fatality rates, 91 global epidemiology, 93-98 interspecies variation, 145-155 mechanism of, 3, 90, 103-104, 706-708, 715-716 and paraoxonase status, 249-250, 251 tolerance, s e e Tolerance development types of, 5-7, 17, 89 uses, 4, 5, 7, 17, 567 U.S. registered, 673,674-677 U.S. use of, 602 vector-borne disease control, 599-602 Organophosphate-induced delayed neuropathy, 7, 316, 703 animal models, 365-366 characterization of, 234, 361 clinical aspects, 91,364-365 DFP-induced, 728 early events, 363 inhalation toxicity, cholinesterase inhibitors, 404 intermediate events, 363 interspecies variation, 153-154 late events, 363 mechanism, 361-362, 573 nerve agents, 54-55 nerve conduction, 340 organophosphates associated with, 573 prevention/treatment, 727-728 signs/symptoms, 573,662 Organophosphate poisoning/intoxication, 717 behavioral toxicity acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 biomarkers, 91-93 blood cholinesterase measurements, 576-579 and butyrylcholinesterase variants, 195 characteristics of, 89 children's exposures, 602-605 chronic, low level, 281-282 clinical aspects, 91-93 CNS effects, 91 diagnosis/assessment of, 716-717 fatality rates, 91 global epidemiology, 93-98 intermediate syndrome, 371-378 interspecies variation age and, 154-155 gender and, 154-155 LDs0s, 145-148 pharmacodynamics, 151-155 pharmacokinetics, 148-151 management of, 715
mechanism of, 90 medical surveillance, 572-576 muscarinic effects, 91 neurotoxicity, 276-283 nicotinic effects, 91 signs/symptoms, 90, 276-277, 348, 552 treatment, 93,584-585 acetylcholine synthesis blockade, 727 adenosine receptor agonists, 727 anticonvulsants, 726-727 atropine, 718-719 calcium channel blockers, 727 cholinesterases, 727 NMDA receptor antagonists, 727 oxime/atropine, 722-725 oximes, 200, 719-722 phosphotriesterase, 727 urine/blood metabolite measurement, 579-580 Organophosphate-specific PBPK/PD model, 108-114 O , S , S - t r i m e t h y l phosphorothioate immunotoxic effects, 502-503 and inhalation toxicity, 403 Oxamyl, 41,485 Oxidant injury, isoprostanes as indicators, 514,516,517 Oxidant tumor promotors, protein kinase C modulation, 541 Oxidation carbamates, 135-137 organophosphates metabolism, 131-132 Oxidative destruction, 692 Oxidative phosphorylation, inhibition of, 518 Oxidative stress acetylcholinesterase inhibitor-induced, 511-515 memantine/atropine, 519-521 antioxidant protection, 512 and ATP, 513 biomarkers carbamate-induced changes, 516-517 organophosphate-induced changes, 517-519 chlorpyrifos and, 534 delayed, 278 and developmental neurotoxicity, 302 fenthion and, 534 and heat shock proteins, 538-540 isoprostanes as indicators of, 514, 516,517 and memantine pretreatment, 41 organophosphate-induced, 662 DNA single strand breaks, 537-538 and male infertility, 454-456 protein kinase C activation, 540-541 pesticide-induced, 90 seizures and, 278, 522 skeletal muscle, 513
755
Oximes adverse effects, 725 antidotal effect, 340 choice of, 723-724 clinical efficacy, 719-720 dosing regimen, 724-725 efficacy as reactivators, 200, 715, 716 history of, 3-4 intermediate syndrome treatment, 377 mechanism of action, 719-720 electrophysiological, 341 myopathy prevention, 512 organophosphate poisoning/intoxication, 584-585 plus atropine, efficacy of, 722-725 reactivation of inhibited cholinesterases, 214-215 Oxonases, organophosphate deactivation, 150-151 Oxons anticholinesterase activity, 211 carboxylesterase detoxification of, 133,715 Oxotremorine, hypothermic response to, 551 Oxyanion hole carboxylesterases, 224 cholinesterases, 162, 165-168, 175, 211 Oxydemeton-methyl, birth defects and, 475 Pachymetry, 425,426 Pakistan, malathion epidemic poisoning, 608 2-PAM. s e e Pralidoxime Pancreas, anticholinesterase exposure and, 574 Paralysis, due to organophosphate intoxication, 91 Paraoxon, 247 aging of, 183 anticholinesterase activity, 213 axon growth and, 241 blood-brain barrier permeability and, 279 central effects, chronic exposure, 281 cholinergic crisis, clinical course, 372 -induced myopathy, 512 inhibition of cAMP synthesis, 239 neurobehavioral effects, 354 neurotoxicity, 278 electrophysiological, 340, 341 neurotoxicity testing cell culture systems, 329 exposure regimens, 318 noncompetitive muscarinic receptor binding, 236 and OPIDN, 7 potency, 131 respiratory effects, 393 sensitivity in paraoxonase knockout mice, 249-250 tolerance development and carboxylesterase, 261
756
Index
Paraoxonase, 133, 708; see also A-esterases activity, interspecies variation, 150-151 clinical relevance of paraoxonase status, 251-253 genetic polymorphisms, 119-120 genotype/phenotype, 248-249 levels in newborns, 154 mutations, 708 in organophosphate metabolism, 106, 109-110, 249-250, 251 modeling, 109-118 polymorphisms, 247-248 structure, 247 substrates, 247 type Q, 75 Paraquat, 95, 97, 415 Parasiticides carbamate, 5 organophosphate, 5 Parathion activation of, 150 antiandrogenic activity, 485 antidotal treatment, 723 biological exposure index, 581 cancellation/phase out, 630 cholinesterase inhibition specificity, 183 dermal absorption, 563,582 regional variability, 416 developmental neurotoxicity, 303, 634 and dioxalon binding, 259 endocrine disruption, 488-489 history of, 3 immune system effects, 502 inhibition of CYE 140 intermediate syndrome, 376 metabolism of, 715 metabolites, 579 mixtures, 610, 611, 612 nicotinic receptor inhibition, 259 and OPIDN, 7 poisoning, biomarkers of, 92 rat mammary tumor model, 543-544 retinal effects, 436 Saku disease, 430 steroid receptor binding, 453 tolerance development, 236, 264 and tolerance to acetylcholinesterase inhibition, 258 transplacental transfer of, 470 uses, 543 Parkinson's disease and agrochemical exposure, 281 dementia and acetylcholinesterase inhibitors, 29-30 Partition coefficient, 110-111,412 Passive diffusion and dermal absorption, 412 placental toxicity, 465 Patch clamp analyses, 342
Patch technique, pesticide exposure monitoring, 576 PC-12 cell line, 297,328 chlorpyrifos developmental neurotoxicity, 541-542 PCBs, estrogen receptor and, 452 Pebulate, 17 Pedunculopontine nucleus (Ch5), 274 cholinergic innervation, 273 innervation, 273 role in respiratory depression, 277 Peer-reviewed programs, AOAC, 695 Pentaerythritol, 406 Pentafluobenzylbromide, 691 Pentylenetetrazol, 277-278, 350 Performance-tested methods, AOAC, 695 Perfused skin preparations, dermal absorption/toxicity, 414 Peripheral binding site, cholinesterases, 175-176, 177-179, 209-211,212, 214 Peripheral nervous system anticholinesterases, sites of action, 145 cell types, 316 cholinergic neurotransmission, 275-276 chronic organophosphate neurotoxicity, 234-235 compensation/repair mechanisms, 365 muscarinic receptors, distribution, 236 OPIDN effects, 234, 662 Permeability coefficient, dermal absorption, 412 Permethrin, 70, 71 Peroxisome proliferators, induction of carboxylesterases, 227 Peroxynitrite radicals, 455, 513,522 Persian Gulf War and paraoxonase status of veterans, 252 pyridostigmine bromide, prophylactic use, 23 Perspiration, and toxicant absorption, 562-563 Pesticide Analytical Manual, 681 Pesticide Analytical Manual, Vol. 1,686-688 Pesticide Analytical Manual, Vol. 2, 688 Pesticide applicators acute poisoning, 571-572 chronic poisoning, central effects, 282, 35 l, 352 Pesticide exposure assessment, 448 Pesticide poisoning blood cholinesterase measurements, 576-579 in children, 602-605 diagnosis/assessment of, 716-717 exposure accidental, 423,424 deliberate (medicinal), 423,424 dermal, 411 inhalation, 570 occupational, 423,424 paraoccupational "take-home", 569
routes of, 567-569 sources of, 567-569 global incidence of, 4, 570-572 in homicides, 569 management of, 715, 717 medical surveillance, 572-576 self, 569 toxicological effects of, 4 treatment, 584-585 acetylcholine synthesis blockade, 727 adenosine receptor agonists, 727 anticonvulsants, 726-727 atropine, 718-719 calcium channel blockers, 727 cholinesterases, 727 NMDA receptor antagonists, 727 oxime/atropine, 722-725 oximes, 719-722 phosphotriesterase, 727 urine/blood metabolite measurement, 579-580 Pesticide residues analysis, sources for, 686-690 carbamates, 4 carbofuran, 659 fish, 481 in food/beverages, 603 organophosphates, 4 persistence, 448 water, 657-658 WHO/FAO guidelines, 644-645, 650-651 Pesticides, see also Insecticides additives, classification, 417 aerial drift, 568 age-related sensitivity to, 354-356 aging of, 183 allergic sensitization, 504 androgen receptor binding, 485-487 bioaccumulation, 481 bioconcentration, 481 carbamates, chemistry, 17, 18-21 detection tickets, 691 estrogenic effects, 447, 481-485 exposure pathways, 617 extraction methods, 681-682 formulators, 568 household use, 602 impurities, toxicity and, 608 inhibition of CYP, 140 labeling, 583 and male reproduction, 450-451 mechanism of action, 451-456 apoptosis, 454-456 central nervous system effects, 451 endocrine -related effects, 452-453 hypothalamic-pituitary-gonadal axis, 452, 457 metabolic effects, 453-454 oxidative stress, 454-456
Index
metabolism of, and cytochrome P450, 139, 663-664 mixtures, see Mixtures organophosphates, chemistry, 5-7, 8-17 progesterone receptor binding, 484-485 registration, 583 reentry restrictions, 587 reregistration, 635 resistance, see Resistance development storage, 602 transplacental transfer, 465-474 turf, 543 U.S. registered, 673,674-677 Pesticides in the Diets of infants and Children, 354
P-glycoproteins mammalian, 149 placental transfer, 470 Phagocytes, 496 Pharmacodynamic models cholinesterase inhibitors, 111-118 validation, 115-118 Pharmacodynamics, anticholinesterases, interspecies variation, 151-155 Pharmacogenetics acetylcholinesterase, 194-195 butyrylcholinesterase BCHE, 190-194, 204-206 clinical aspects, 187-188 genetic variants, 189-194, 204, 216 inhibition studies, 188-189 phenotypes, 188-189, 204 role of, 187 Pharmacokinetic models compartmental, 107-108 physiologically based, 108-121 Pharmacokinetics anticholinesterases, interspecies variation, 148-151 applications, 103, 107 role of, 103 Phase I metabolism, 219, 220 carbamates hydrolysis reactions, 134-135 oxidation reactions, 135-137 enzymes, 127-129 age and, 154 carboxylesterases, see Carboxylesterases placental, 465-466 organophosphates hydrolysis reactions, 132-133 oxidation reactions, 131-132 Phase II metabolism, 219, 220 carbamates, 137-138 enzymes, 128, 129-130 age and, 154 placental, 466 organophosphates, 134 Phencyclidine, developmental neurotoxicity, 634
Phenobarbital, induction of carboxylesterases, 227 Phenols, 693 Phenserine, in Alzheimer's disease, 17 Phenylmethane sulfonyl fluoride, 363 Phenyl methane sulfonyl fluoride, OPIDN prevention, 727 Phenyl-N-tert-butylnitrone
action of, 521 pretreatment with, 525-527 Phenylpentyl phosphinate, 363 Phenytoin, 726 for convulsions, 585 developmental neurotoxicity, 634 Pheochromocytoma cell line, 328 Phorbol esters, protein kinase C modulation, 541 Phosdrin, IMS analysis, 686 Phosmet, 23, 630 Phosphamidon, mixtures, 610 Phosphatidylcholine, deacylation, 362 Phosphatidyl inositol, pesticide disruption of, 453 Phosphatidyl inositol diphosphate, 666, 667 Phosphatidyl-inositol-phosphate, 667 Phosphinates, OPIDN prevention, 727 Phosphine, 406 3'-Phosphoadenosine 5'-phosphosulfate, 130 Phosphocreatine carbamate-induced changes, 517 organophosphate-induced changes, 517-518 Phosphoinositide-specific phospholipase C, 237 Phospholine, in glaucoma, 22 Phospholine iodide, 278-279 Phospholipase 2, 707 Phospholipase C, activation, 236 Phospholipids, and carbofuran, 665 Phosphonofluoridates, 7 lethal inhalation toxicity, 405 Phosphonothioates, 7 Phosphoric triester hydrolases, 129 and tolerance development, 263-264 Phosphoroamidothiolates, biotransformation, 104 Phosphorodithioates, biotransformation, 104 Phosphorofluoridates, lethal inhalation toxicity, 405 Phosphorothioates bioactivation, 715 and inhalation toxicity, 403 Phosphorothionates biotransformation, 104 desulfuration, 131 oxons, 131 S,S,S,-Phosphorotrithioate, hypothermic response to, 555 Phosphotriesterase, 727 Photobacterium phosphoreum luminescence, eye irritation tests, 428
757
Photophobia, due to methyl isocyanate toxicity, 83 Physiologically-based pharmacokinetic/dynamic models, and risk assessment, 630-631 Physiologically based pharmacokinetic models, 108-121 organophosphate-specific, 108-114 validation, 115-118 Physostigma venenosum, 599 Physostigmine central effects, chronic exposure, 281 cholinesterase inhibition, interspecies variation, 152 cognition and, 27 DFP toxicity and, 611-612 for glaucoma, 22, 283 history of, 48, 599 hypothermic response to, 555 inhalation toxicity, 403 mixtures, 611, 612 in myasthenia gravis, 22 nerve agent protection, 611 neuropathy induced by, 278 neurotoxicity, electrophysiological, 340, 341 rat mammary tumor model, 543-544 respiratory effects, 393 in urine voiding dysfunction, 22 uses, 4, 17, 35 Pig, as dermal absorption/toxicity model, 415 Pigeon chest, 84 Pilocarpine, 237 Ping Pong Bi Bi kinetic scheme, 211-213 Pirimicarb endocrine disruption, 474, 485, 487 metabolites, 580 Pirimiphos-methyl, 23 Pituitary-gonadal axis, developmental toxicity, 448 Pituitary hormone, pesticide-induced alterations, 452 Placenta as barrier, 470 bidiscoid, 463 bioconcentration of toxicants, 471-472 cholinergic system, 472-474 endotheliochorial, 463 function of, 463 hemochorial, 463 human, 463-465 perfusion method, 466-469 multicotyledonary, 463 pesticide metabolism, 470 syndesmochorial, 463 term, 464-465 xenobiotic-metabolizing enzymes, 465-466, 467 zonary, 463 Placental choline acetyltransferase, gestation age and, 473
758
Index
Placental toxicity abnormal pregnancy outcomes, 474-475 experimental methods, 466-469 placenta structure/function, 463-465 species differences, 463-464 toxicodynamics, cholinesterase inhibitors, 470-474 toxicokinetics, 465-466 cholinesterase inhibitors, 469-470 Plague, 599 Plaques, senile, 26, 29 Plasminogen activation assays, eye irritation tests, 428 Plasticizers, 364 Platelet-activating factor acetylhydrolase, 707 Point of departure, 618-620, 629 Poiseuille's law, 391 Poisonings, pesticide, see Pesticide poisoning Polychlorinated biphenyls developmental neurotoxicity, 634 estrogenic effects, 447 Polydactyly, 475 Polyurethane foams, thermal decomposition products, 406 Pons, respiratory centers, organophosphateinduced effects, 391,394 Pons medulla, cholinergic innervation, 273,274 Pontomesencephalic tegmentum, innervation, 273 Porton Down, 389 Posttraumatic stress disorder, 70 Potassium ions, and organophosphateinduced cardiotoxicity, 383-386 Potentiation anticholinesterases, 574 pesticide mixtures, 607-613 Potentiometric methods, cholinesterase activities, 202 Pralidoxime, 719 adverse effects, 725 history of, 3-4 intermediate syndrome treatment, 727 mechanism of action, electrophysiological, 341 organophosphate poisoning/intoxication, 93, 395, 584-585 pharmacokinetics, 720-722 and pulmonary toxicity, 393, 394 reactivation of inhibited cholinesterases, 214,215 Prazocin, 555 Precinorm S, 204 Precinorm U, 204 Precursor ions, 685 Preemployment medical examination, 575 Pregnancy abnormal outcomes, 474-475 and butyrylcholinesterase activity, 204 chlorpyrifos exposure and, 293,604
developmental neurotoxicity guideline, 634 nicotine, developmental neurotoxicity, 302-303 and paraoxonase activity, 252-253 Presenilin 1 and 2, 29 Pressurized solvent extraction, 682 Preterm labor, pharmacotherapy and developmental neurotoxicity, 302 PRIMA, 181 Primary cultures containing glia, 331 neurotoxicity testing, 329 Primates cholinergic neurons, 271-272 plasma carboxylesterase, 263 Procaine, 187 Prochloraz, estrogenic activity, 474, 485,487 Procymidone, steroid receptor binding, 452 Produce, pesticide residues, 603 Production facility, occupational exposure, 568 Product ions, 685 Profenofos, 716 Progesterone, toxic effects of methyl isocyanate, 82 Progesterone receptor, pesticide binding, 453,484--485 Propamocarb, estrogenic activity, 485, 487 Propanil, endocrine disruption, 487 Propham, 134, 135 Propidium, cholinesterase binding site, 176 Propoxur cognition effects, 350 ectoparasiticide, 23 immune system effects, 503 metabolites, 580, 693 mutagenic potential, 544-545 neurobehavioral effects, chronic exposure, 353 Protamine sulfhydryl, 458 Protective clothing, 583,586-587 Protein kinase C and developmental chlorpyrifos exposure, 297 and heat shock proteins, 667 organophosphates and, 239, 540-541 pesticide disruption of, 453 regulation of CYP1A, 665-666 role of, 540-541 Protein kinases, OPIDN and, 363 Protein phosphatase, 39 Protein synthesis, effects of anticholinesterases on, 474 Proxyfur, intermediate syndrome, 376 pS2 expression assay, 481 Pseudocholinesterase. see Butyrylcholinesterase Psorophora, 600 Psychic state, acute pesticide poisoning effects, 573
Psychomotor function, and chronic pesticide exposure, 351,353 Psychosis, and Alzheimer's disease, 26 Public health control of vector-borne disease, 599-602 pesticide exposure, children, 602-605 Pulmonary fibrosis, 574 Pulmonary stretch receptors, 391 Pulmonary toxicity alveoli, 394 bronchi, 393-394 bronchioles, 394 exposure Ct, 392 nasal airway, 392-393 organophosphate-induced, 395 and respiratory center, 394 signs/symptoms, 392 trachea, 393-394 Pulsed flame photometric detector, 685 Punarbhaba River, 657 Pupils, soman-induced constriction, 257 Putamen, cholinergic innervation, 272 Pyrazophos, antidotal treatment, 722 Pyrethrin, dermal absorption, regional variability, 416 Pyridinium oximes, 719 Pyridostigmine analysis, 693,694 blood-brain barrier permeability and, 279 blood flow alterations, 382 and Gulf War, 69, 71 hypersensitivity to, 194-195 -induced myopathy, 512 in myasthenia gravis, 22 neurotoxicity, electrophysiological, 341 potentiation by stress, 280 prophylactic use, 3, 23, 69, 71,279-280, 395, 611 respiratory effects, 393 QNB, 258-259 QT prolongation, organophosphate-induced, 383-386 Quadriplegia, in OPIDN, 365 Quantitative structure permeability relationships, 412-413 QuEChERS method, 690 Quinalphos antidotal treatment, 723 endocrine disruption, 488, 489 male infertility and, 457 steroid receptor binding, 453 Quinapril, 219 Quinidine, butyrylcholinesterase inhibition, activity measurements, 201 Quinolinic acid, 707 Rabbits, paraoxonase activity, 249 Radial glia, 330 Radiation, developmental neurotoxicity, 634
Index
Radiometric methods, cholinesterase activities, 201 Radiotelemetry, 551 Rainwater, pesticides in, 608 Ramathibodi Poison Center, 96 Ramshorn snail, 658 Rapid-eye movement sleep, 274, 277 Rat adrenal pheochromocytoma cell line, 328 Rat brain endothelium-4 cell line, 332 Rat embryos, neurotoxicity testing, 329 Rating Scale for Geriatric Patients, 36 Rat mammary tumor model, 543-544 Rats anticholinesterases sensitivity, 148, 150, 152 inhalation toxicology, 401 neurobehavioral effects, anticholinesterase poisoning, 348-351,352-354 neuropathic response to organophosphates, 153 paraoxonase activity, 249 plasma carboxylesterase, 263 spermatogenesis, 450 toxicity testing, 457 tolerance development to carboxylesterase inhibitors, 261-262 Reactivation acetylcholinesterase, 152 and behavioral recovery, 350-351 nucleophilic agents, 3-4 cholinesterases by oximes, 214-215 time course, 200, 213 oxime-induced, 21 4-215, 716 resistance to, 216 spontaneous, 716 Reactivators, 214-215 Reactive airways dysfunction syndrome, 83 Reactive nitrogen species and excitotoxic injury, 511 male infertility and, 454-455 and pesticide-induced oxidative stress, 90 Reactive oxygen species chemiluminescence assay, 535-536 and cytochrome oxidase, 513, 515 and excitotoxic injury, 511 male fertility and, 454 organophosphate-induced, 534-535 peroxynitrite, 513 pesticide-induced, 90, 533 protein kinase C interaction, 541 role in organophosphate-induced myopathy, 512 spin trapping agents, 521 and xanthine dehydrogenase, 513, 515 Red nucleus, cholinergic innervation, 272 Reentry restrictions, pesticides, 587 Reference concentration, risk assessment, 618, 639
Reference dose nerve agents, 48, 55-57, 62 risk assessment, 618-620, 621--626, 639 WHO/FAO guidelines, 651-652 Reflexes, alterations in intermediate syndrome, 371,374 Registry of Toxic Effects of Chemical Substances, database, 147 Relative potency factor, 628-629 Relative retention times, 686 Relative standard deviation, 696 Remediation, organophosphates, in vitro models, 332-333 Reminyl, 28 Repeatability, 696 Reproducibility, 696 Reproductive toxicity assessing gonadotoxicity, 456-458 carbofuran, in fish, 659 estrogen hypothesis, 447, 448, 452-453, 458-459 male reproductive tract, 449-450 mechanisms, 451-456 methyl isocyanate, 83-84 Residue Analytical Methods, 690 Residues. see Pesticide residues Resistance development carbamates, 4 insects, acetylcholinesterase structural changes, 183 mosquito vectors, 600-602 organophosphates, 4 pesticides, cholinesterase binding sites and, 176 Resource Utilization in Dementia Scale, 38 Respirable fraction, 401 Respiration anticholinesterase intoxication, central effects, 277 cholinergic regulation of, 274 control of, 391,394 muscles of, 391 Respirators air-purifying, 583-584 atmospheric, 584 Respiratory system control of ventilation, 391 distress/paralysis, and intermediate syndrome, 371,372, 373-374 methyl isocyanate toxicity, 82, 83 organophosphate-induced failure, 394-395 physiology, 390-391 pulmonary toxicity, 392-394 Respiratory tract distribution of inhaled materials, 401-402 local pharmacological effects, 402-403 local toxicity, 403 occupational pesticide exposure, 570
759
Restricted entry interval, 587 Retention tetrapeptide, endoplasmic reticulum, 223,229 Rete testis, 449 Reticular activating system, cholinergic innervation, 273 Retina detachment of, 436 ophthalmic toxicity, 435438 Retinoic acid, 328 Reward, cholinergic modulation of, 272, 273,274 Rhabdomyolysis, anticholinesterase exposure and, 574 Rhinorrhea, 392-393 nerve agent exposure, 52-53, 58 Rice fields, pesticide-contaminated runoff, 660 Rigidity, 280 Risk assessment, see also World Health Organization/Food and Agriculture Organization acceptable daily intake, 543 cumulative common mechanism of toxicity, 620, 627, 629 cumulative assessment group, 620, 627 N-methyl carbamates, 620, 630 organophosphates, 620, 627-630 relative potency factor, 628-629 dermal absorption/toxicity, 414, 620 developmental neurotoxicity, 633-635 comparative cholinesterase data, 638-639 testing requirements, 635-638 dietary, 619, 620 gonadotoxicity, 456-458 inhalation, 620 occupational, 619 and paraoxonase status, 248 and PBPK/PD models, 630-631 pesticides, 448 pharmacokinetic approach, 630-632 pharmacokinetic modeling, 107-121 reference concentration, 639 reference dose, 639 residential, 619 single chemical, aggregate, 618-620 Risk-risk analysis, pesticide cancellation, 677-678 Rivastigmine for Alzheimer's disease, 17, 27-28, 283 analysis, 693,694 effects on tau levels, 29 mechanism of action, 28 memantine and, 40, 41-42 River blindness, 601 Ro 02-0683, and butyrylcholinesterase phenotyping, 204
760
Index
Rodents anticholinesterase-induced hypothermia, 551-559 anticholinesterase sensitivity, 148, 150, 152 anticholinesterases-induced hyperthermia, 559-560 heat loss pathways, 556-557 organophosphate binding proteins, 705 tolerance development to carboxylesterase inhibitors, 261-262 Ronnel, 23 RO number, 189 Root mean square, 174-175 Rotory Evaporator, 684 Runoff, 657, 660-661 Russia, nerve agent stockpiles, 389 Sacramento River, 660 Safety factors, data extrapolation, 647, 650 St. Louis encephalitis, 600 Saku disease, 430 Samples cleanup/purification, 682-683 concentration, 683-684 impurities separation, 684 Sampling atmosphere, 402 skin exposure, 575-576 Sarcomeres, 512 Sarin AEGLs, 58-62 analytical methods, 693-694 chemistry, 7 Ct, 392 estimated reference dose, 56-57 history, 3, 48, 389 hypothermic response to, 555 -induced myopathy, 512 inhalation toxicity, 405 long-term effects, 73-74 neuropathy induced by, 278 noncompetitive muscarinic receptor binding, 237 ocular toxicity, 433 paraoxonase hydrolysis of, 248 properties, 49-52 respiratory effects, 277, 393, 394-395 retinal effects, 436 structure of, 390 tolerance development and carboxylesterase, 261-263 toxicity and adenosine receptors, 260 delayed neuropathy, 54-55 effects, 52-55 mechanism of, 51-52 and paraoxonase status, 251 use in warfare, 3
Satin poisoning delayed hyperthermia, 561 Matsumoto incident, 49 oximes for, 724 Tokyo subway attacks, 3, 47, 49 paraoxonase status and, 251 postmortem findings, 97 Schradan, tolerance development and carboxylesterase, 261 Schrader, Gerhard, 3,389, 599, 657 Schwann cells, 316, 330 Scoline apnea, 188, 189 Scoline (Sc) variant, butyrylcholinesterase, 194 Scopolamine, blocking of hypothermic response, 554-555 Sea urchins, developmental neurotoxicity screening, 305 Seizures and anticholinesterase intoxication, 278-279, 283 anticonvulsants for, 585 organophosphate-induced, 235,662 and oxidative stress, 522 role of nitric oxide, 235,522 Semen, decreased quality, and environmental toxicants, 448 Seminiferous tubules, 449 Senile plaques, 26, 29 Sensory nerve conduction, evaluating, 581 September 11,2001 attacks, 3 Septohippocampal (Chl) pathway, 274 Serine residue acetylcholinesterase active site, 165-168, 213 cholinesterases, 162, 165-168, 213 organophosphate binding, 706 Serotonin chlorpyrifos interactions, 294, 300-301 heat loss pathways, 551 Sertoli cells, 450 Serum, organophosphate stability in, 580 Severe Impairment Battery, 36 Sexdifferentiation, developmental chlorpyrifos exposure and, 299 Sex steroid hormone-binding globulin, 452 Sheep dippers chronic pesticide exposure, neurobehavioral effects, 351-352 paraoxonase status and organophosphates exposure, 252 Signaling pathways and developmental chlorpyrifos exposure, 295,296-297, 301 pesticide disruption of, 453 Silent Spring, 681 Silicon microphysiometer, 428 Simulium damnosum, 601 Single-channel microchips, 694 Single fiber electromyography, 59 .Single laboratory validation, 695-696
Single nucleotide polymorphisms acetylcholinesterases, 181-182 butyrylcholinesterase variants, 191 paraoxonase, 248 Skeletal muscle, oxidative stress, 513 Skin acidity, 412 composition, 411 decontaminating, 717 monitoring pesticide exposure, 575-576 permeability humidity effects on, 418, 562-563 regional variation, 416 species differences, 415-416 pesticide absorption, 569-570 protection, 570, 583 sensitization, anticholinesterase-induced, 504 Sleep, cholinergic regulation of, 274 Slit-lamp biomicroscopy, eye irritation tests, 425,426 Slow-wave sleep, 274 SLUD, 354 Smoking, and developmental neurotoxicity, 302-303 Smooth muscle, vascular, effect of cholinesterase inhibitors, 381-382 SN-38, 220 Snake venom toxins, 168, 176, 182-183 S-(N-methylcarbamoyl) glutathione, 81, 83 Sodium ion, and organophosphate-induced cardiotoxicity, 383-386 Sodium salicylate, 560 Sodium thiosulfate, and Bhopal accident, 79-80, 82 Solid phase extraction, 682-683 Solid phase microextraction, 683 Solvents, extraction, 681 Soman acetylcholinesterase prophylaxis, 195 AEGLs, 59-62 aging of, 183 analytical methods, 693-694 blood-brain barrier permeability and, 279 brain slice model, 341 cardiotoxicity, 382, 383 chemistry, 7 estimated reference dose, 57 history of, 3, 48--49, 389 hypothermic response to, 555 -induced myopathy, 512 inhalation exposure, 403,404 memantine and, 40-41 miotic effect, 257 neuropathy induced by, 54-55,277-278 noncompetitive muscarinic receptor binding, 237 paraoxonase hydrolysis of, 248 pharmacokinetics, interspecies variation, 149 poisoning, oximes for, 719
Index
properties, 49-52 pyridostigmine pretreatment, 395 respiratory arrest, 394-395 respiratory effects, 277, 393 structure of, 390 tolerance development and carboxylesterase, 261-263 toxicity delayed neuropathy, 54-55 effects, 52-55 mechanism of, 51-52 Somanase activity, 263-264 Somatotrophin, 496 Sonication, 681 Soviet Union, nerve agent stockpile, 49 Spastic ataxia, 365 Spectrophotometric methods, cholinesterase activities, 201 Speech, acute pesticide poisoning effects, 573 Sperm, function, and oxidative stress, 454-456 Spermatogenesis, 450 and environmental antiandrogens, 491 parathion exposure and, 489 site of, 449 Sperm chromatin structure assay, 458 Spermination, 450 Sperm nuclear integrity assessment, 458 Spices, maximum residue limits, 651 Spinal cord, OPIDN-associated lesions, 365 Spin trapping agents, reactive oxygen species, 521 Spraying, indoor, children and, 604 Sprays drift, 568 pesticide, dermal absorption, 411 S (silent) variant, butyrylcholinesterase, 189-194, 204, 216 Status epilepticus, 585 Staurosporine, 665 Steroid hormones pesticide-induced alterations, 452 placental, 473-474 Steroidogenesis, disruption of, in vitro, 487-488 Steroid receptor, pesticide binding, 452 Stillbirths, 474 Strategic Diagnostics, 691 Stratum corneum, 411-4 13 species differences, 415-4 16 tape stripping, 414 Stress blood-brain barrier permeability and, 279-280 and membrane signaling, 665-666 and susceptibility to chemical warfare agents, 71, 73 Striatum acetylcholine, 704 cholinergic innervation, 272, 273 dopaminergic transmission, 272-273
Structure-activity relationships carbamates, 214 organophosphates, 213-214 Substantia nigra, cholinergic innervation, 272, 273 Substantia pars compacta, 272 Subthalamic nucleus, cholinergic innervation, 272, 273 Succinylcholine, 188 Succinylcholine sensitivity, and butyrylcholinesterase phenotyping, 181-182, 201,202, 216 Succinyldithiocholine, 202 Suicide by pesticide poisoning, 93,569 chronic pesticide exposure and, 352 with organophosphates, 91 Sulfation reaction, 130 Sulfones, 136 Sulfonyl fluorides, neuropathy target esterase inhibition, 362 Sulfotransferases, role of, 130 Sulfoxidation carbamate metabolism, 136-137 organophosphates metabolism, 132 Sulfoxides, 136 Sulfur compounds, as insecticides, 657 Sulfur mustard agent, 49 Sulphanilamide, acetylation of, 138 Sulphorhodamine B, 425 Supercritical fluid extraction, 682 Superoxide anion radical, 455,522 Superoxide dismutase, 456, 522 Supervised trials median residue level, 651 Surrogate skin techniques, 576 Suxamethonium, butyrylcholinesterase hydrolysis of, 187, 188 Sweating, 562-563 SY5Y human neuroblastoma cell line, 328-329, 332-333 Synapses acetylcholine accumulation, 145,209, 233 cholinergic, 275-276 transmission, effects of pesticides on, 341-344 Synaptogenesis, impaired, 328 Syncytiotrophoblasts, 463,474 Syndactyly, 84 Syrup of Ipecacuanha, 717 Tabun AEGLs, 59-62 analytical methods, 693-694 chemistry, 7 delayed neuropathy, 54-55 estimated reference dose, 57 history of, 3, 48-49, 389 -induced myopathy, 512 noncompetitive muscarinic receptor binding, 237
761
properties, 49-52 pulmonary toxicity, 277 respiratory arrest, 394-395 structure of, 390 toxicity, 278 and adenosine receptors, 260 effects, 52-55 electrophysiological mechanisms, 340 mechanism of, 51-52 Tachycardia, 703 Tacrine for Alzheimer's disease, 27, 35,283 analysis, 693,694 cholinesterase binding site, 176 combination therapy with memantine, 41-42 retinal effects, 437-438 toxicity, 17, 27 Tail, rat, skin temperature, 556-557 Taiwan, pesticide poisoning in, 95, 97 TAK-802, 22 Tamoxifen, 453 Tandem mass spectrometers, 685 Tape stripping, 414, 575 Tau protein effects of acetylcholinesterase inhibitors, 29 memantine and, 39 and OPIDN, 363 T cells, 496 Teleost fish carbofuran toxicity, 659 CYP1A in, 664-666 Temephos, mosquito resistance, 601 Temocapril, 219, 222, 227 Temperature gradient tube, 557 Temperature regulation, see Thermoregulation Teratogenesis, pesticide-induced, 474-475 Terbutaline, and developmental neurotoxicity, 302 Termiticide applicators, chronic pesticide exposure, neurobehavioral effects, 352 Terrorism, chemical, 47--48; see also Chemical warfare agents; Nerve agents Testicular cancer, 448 Testis composition, 449-450 dysfunction, and oxidative stress, 454-456 Test kits, cholinesterase activity measurements, 203 Test-mate field kit, 203 Test Methods for Evaluating Solid Wastes, 689 Testosterone effects, 449-450 effects of organophosphates on, 488-489 metabolism, pesticide inhibition of, 140 production, 449 Testosterone-estrogen-binding globulin, 452 Tetanic fade, 581 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 545
762
Index
Tetrachlorfenvinphos, 23 Tetraethyl pyrophosphate, 3 central respiratory depression, 394 extraocular effects, 438 noncompetitive muscarinic receptor binding, 236 and OPIDN syndrome, 7 structure of, 390 toxicity, 599 Tetrodotoxin, 512 Thailand, pesticide poisoning in, 95-98 Thalamus, cholinergic innervation, 273 Thermogenesis, 550 Thermoneutral zone, 550 Thermoregulation autonomic response, 556-557 behavioral response, 557 cholinergic pathways, 551 cholinesterase inhibitors and core temperature, 555-556 CNS control of, 551,554-555 developmental effects, 558 dopaminergic pathways, 551,555 exercise and heat stress, 562-563 fundamentals of, 550-551 hyperthermia, 559-562 hypothermia, 551-559 neurochemical studies, 551 noradrenergic pathways, 555 serotonergic pathways, 555 Thiamine, effect on pralidoxime, 721 Thin-layer chromatography, 692 5-Thio-2-nitrobenzoic acid, 202 Thiobarbituric acid-malondialdehyde complex, 514 Thiobarbituric acid reactive substances, 534 Thiocarbamates, 17, 138 Thiocholine, 202 Thionophosphorus organophosphate insecticides, PBPK/PD model for, 108-110 Thiram, 17 Threshold Limit Values, 399 Thymosin, 496 Thyroid gland, fish, carbofuran-induced abnormalities, 660 Thyroid hormone carbamate-related effects, 490 organophosphate-related effects, 489 Tiagabine, 726 Timolol, ocular absorption, 431 Titration methods, cholinesterase activities, 202 T-maze test, 39 TMB4, 215 Tobacco workers, organophosphate exposure, extrapyramidal effects, 280 Tokyo subway attack, 3, 47, 49 paraoxonase status and, 251 postmortem findings, 97
Tolclofosmethyl, estrogenic activity, 485 Tolerance development in acetylcholinesterase knockout mice, 260-261 and acetylcholinesterases, 263 adenosine receptors, 260 and butyrylcholinesterases, 263 carboxylesterases, 261-263 cholinesterase inhibitors, 257-264 muscarinic receptors and, 235, 257-259, 261 nicotinic receptors and, 259-260 parathion, 236, 258, 264 pathways, 257 phosphoric triester hydrolases and, 263-264 presynaptic changes, 260 Tolerance reassessment, 618 Toluene, developmental neurotoxicity, 634 Tonometry, indentation, 426 Torpedo californica cholinesterases, 3-D structure, 168, 174-181 cholinesterases, amino acid sequence, 162, 163-164 Torsade de Pointes, and hypokalemia, 384 Toxic Exposure Surveillance System, 571 Toxicity body temperature and, 558 heat stress and, 562-563 pesticide mixtures, 607-613 Toxicity studies LOAEL, 55-56 NOAEL, 55-56 WHO/FAO guidelines, 645,647 Trace analysis, 681 Trachea, 393-394 Transappendageal pathways, absorption, 411 Transcription factors, 239-240 Transport, and occupational exposures, 568 Transthyretin, 489 Trialkyl phosphates, thermal decomposition products, 406 Trialkyl phosphorothioates, and inhalation toxicity, 403 Triarylphosphates, 362, 364 Triazines, 691 Triazoles, cholinesterase inhibition, 176, 183 Triazophos, antidotal treatment, 722 S,S,S-Tributyl phosphorotrithioate, 5 S,S,S-Tributyl phosphorotrithioite, 5 Tributyrinase, 574 Trichlorfon, 23 in Alzheimer's disease, 17 anthelmintic use, 23 effect on action potential conductance, 340 intermediate syndrome, 376 mixtures, 610 Saku disease, 430 3,5,6-Trichloro-2-pyridinol, 106 in fetal/maternal samples, 604 Trichloroethane. see DDT
Trichloronat, 716 Trichloropyridinol, 117, 240 Triethyl tin, developmental neurotoxicity, 634 Trifluoroacetophenones, cholinesterase inhibition, 183 Trimedoxime, efficacy, 722 Trimethylopropane phosphate, 406, 407 Trimethyl phosphorodithioate, pulmonary toxicity, 394 Tri-o-cresyl phosphate, 7 neurotoxicity, 406, 703 neurotoxicity testing, cell culture systems, 330 OPIDN and, 316, 364-365 potentiation of malathion, 609-610, 612 potentiation of paraoxon toxicity, 148 pretreatment and tolerance development to carboxylesterase inhibitors, 262 Tri-o-tolyl phosphate, 503 Triphenyl phosphate antiandrogenic activity, 485 neurobehavioral effects, 353 thermal decomposition products, 406 Tris (1,3-dichloroisopropyl) phosphate, 7, 17 Tris (2-chloroethyl) phosphate, 7, 17 "Iris (2-chloropropyl) phosphate, 7, 17 Trophoblasts, in cell cultures, 469 Tropicamide, eye drops, 432 Trypsanomiasis, 599 Tryptophan, 707 d-Tubocurarine, 512, 514, 515 alpha-Tubulin, 363 Tumorigenesis, organophosphate modifying effects, 542-543 Tumor necrosis factor-a, 560 Turbovap, 684 TV3326, 17 T-wave abnormalities, 383-386 Twitch potentiation, 581 Typhus, louse-borne, 599 Tyrosine kinase, pesticide disruption of, 453 syn-TZ2PA6, 179 Ultrafiltration, partitioning coefficient determination, 110-111 Ultralow-volume aerial application, 600 Uncertainty factors, risk assessment, 56-57, 59, 62, 619, 621-626 Unconditioned behaviors, acute effects of anticholinesterases, 348-350 Union Carbide plant, 4, 79-85 United Kingdom, airborne exposure guidelines, 585 United States federal pesticide regulations, 617; see also Environmental Protection Agency nerve agent production, 49 pesticide poisoning in, 94, 528, 571 pesticide use, 599, 602 registered pesticides, 673,674-677
Index
U.S. Department of Agriculture, pesticide data program, 617 U.S. Environmental Protection Agency. see Environmental Protection Agency U.S. Federal Emergency Management Agency, 47, 62 U.S. Food and Drug Administration. see Food and Drug Administration U.S. Geological Survey, water monitoring, 617 Urethane carcinogenicity, 137 immunotoxicity, 498 Uridine diphosphate glucuronic acid, 129 Uridine diphosphate glucuronosyltransferase, 129, 220 Urine children, metabolites in, 603 collection, dermal absorption/toxicity, 414 metabolite analysis, 679-680, 692, 693 nerve agents analysis, 694 Urine voiding dysfunction, and acetylcholinesterase inhibitors, 22 U (usual) variant, butyrylcholinesterase, 189-194, 204, 216 Vaccines, vector-borne diseases, 601 Valproic acid, developmental neurotoxicity, 639 Vapor diffusion rate, 404 Vapor pressure cholinesterase inhibitors, 399 nerve agents, 693 Vapors, inhalation exposure, 401,404, 570 Vascular smooth muscle, effect of cholinesterase inhibitors, 381-382 Vasopressors, 717 Vector-borne disease, eradication of, 599-600; see also Public health Vegetables maximum residue limits, 650-651 pesticide residues, 603 Velnacrine, toxicity, 27 Ventilation assisted, 584, 717 control of, 391 Ventilation systems, closed facilities, 585 Ventral striatum, cholinergic innervation, 272 Ventral tegmental area, 272 Ventricular fibrillations, organophosphateinduced, 384 Vernolate, 17 Vertebrates, pesticide metabolism, 661 Vesicant (blister) agents, in unitary munitions, 49 Vestibular function, chronic pesticide exposure and, 353 Veterinary medicine, acetylcholinesterase inhibitors in, 23
Vial equilibrium method, partitioning coefficient determination, 110-111 Vinclozolin, androgen receptor binding, 452 Vineyard workers, endocrine disruption, 490 Vinyl carbamate epoxide, carcinogenicity, 137 Vision, blurred organophosphate-induced, 433 oxime-related, 725 Visual evoked response, 62 Vitamin E, pretreatment with, 525-527 Vitellogenin assay, 484 Volaton, 610 Voltage-dependent calcium channel, 329 Volume transmission, 272-273 von Krueger, Gerde, 390 VR, neuropathy induced by, 278 V series compounds, 3 VX acetylcholinesterase knockout mice and, 234 AEGLs, 59-62 analytical methods, 693-694 chemistry, 7 CNS effects on respiration, 277 dermal absorption, 563 estimated reference dose, 57 history of, 3,389 -induced myopathy, 512 muscarinic receptors, noncompetitive binding, 237 neurotoxicity testing, cell culture systems, 329 nicotinic receptors and, 260 properties, 49-52 respiratory arrest, 394-395 stereoselectivity of acetylcholinesterase toward, 210 structure of, 390 supralethal doses, 55 toxicity, 278 effects, 52-55 electrophysiological, 341,342 mechanism of, 51-52 Wasting syndrome, 533 Water dialkyl phosphates in, 692 pesticide residues, 657-658 Water samples, extraction of, 681 Western equine encephalitis, 600 West Nile virus, 600 organophosphate insecticides used for, 601 U.S emergence, 600 Wipe sampling, 575 Workers occupationally-exposed, withdrawal of, 578 protective clothing, 583, 586-587 supervision of, 583, 586
763
Workplace biomonitoring, 581-582 health care facilities, 587 NIOSH guidelines, 689 Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity, 634 A World Compendium. The Pesticide Manual, 689 World Health Organization acute pesticide poisonings, 570 eradication of malaria, 600 IPCS, 644 Neurobehavioral Core Test Battery, 282 pesticide classification, 643-644 pesticide poisoning, incidence of, 93,528 World Health Organization/Food and Agriculture Organization acceptable daily intakes, 644-645, 646-647,648-649 acute reference dose, 651-652 assessment of endpoints, 646-647 data extrapolation, 647, 650 maximum residue limit, 644, 650-651 mixtures guidelines, 651 pesticide residues, 644-645,650-651 toxicity assessment guidelines, 645-646 World War II, nerve agent stockpiles, 389 Worms, acetylcholinesterase genes, 168
Xanthine dehydrogenase, and reactive oxygen species, 513, 515 Xanthine oxidase, fasciculations-induced changes, 515 Xanthurenic acid, 707 Xenobiotic-metabolizing enzymes phase I, 127-129 phase II, 127, 129-130 placental, 465-466, 467 Xenobiotic responsive element of nucleus, 664 Xenobiotics estrogenic effects, 447 metabolism, see also Drug metabolism carboxylesterases, 219-220, 227-228, 229 in fish, 664-666 transplacental transfer, 465-474 Xenopus oocytes, 343 Y chromosome deletions, 447 Yellow fever, 599-600 YT blood group, 194 Zebra fish, 704 developmental neurotoxicity screening, 305 toxicity testing, 659 Zimbabwe, acute pesticide poisonings, 570
Index U.S. Page Department of Agriculture, pesticide This Intentionally Left Blank data program, 617 U.S. Environmental Protection Agency. see Environmental Protection Agency U.S. Federal Emergency Management Agency, 47, 62 U.S. Food and Drug Administration. see Food and Drug Administration U.S. Geological Survey, water monitoring, 617 Urethane carcinogenicity, 137 immunotoxicity, 498 Uridine diphosphate glucuronic acid, 129 Uridine diphosphate glucuronosyltransferase, 129, 220 Urine children, metabolites in, 603 collection, dermal absorption/toxicity, 414 metabolite analysis, 679-680, 692, 693 nerve agents analysis, 694 Urine voiding dysfunction, and acetylcholinesterase inhibitors, 22 U (usual) variant, butyrylcholinesterase, 189-194, 204, 216 Vaccines, vector-borne diseases, 601 Valproic acid, developmental neurotoxicity, 639 Vapor diffusion rate, 404 Vapor pressure cholinesterase inhibitors, 399 nerve agents, 693 Vapors, inhalation exposure, 401,404, 570 Vascular smooth muscle, effect of cholinesterase inhibitors, 381-382 Vasopressors, 717 Vector-borne disease, eradication of, 599-600; see also Public health Vegetables maximum residue limits, 650-651 pesticide residues, 603 Velnacrine, toxicity, 27 Ventilation assisted, 584, 717 control of, 391 Ventilation systems, closed facilities, 585 Ventral striatum, cholinergic innervation, 272 Ventral tegmental area, 272 Ventricular fibrillations, organophosphateinduced, 384 Vernolate, 17 Vertebrates, pesticide metabolism, 661 Vesicant (blister) agents, in unitary munitions, 49 Vestibular function, chronic pesticide exposure and, 353 Veterinary medicine, acetylcholinesterase inhibitors in, 23
Vial equilibrium method, partitioning coefficient determination, 110-111 Vinclozolin, androgen receptor binding, 452 Vineyard workers, endocrine disruption, 490 Vinyl carbamate epoxide, carcinogenicity, 137 Vision, blurred organophosphate-induced, 433 oxime-related, 725 Visual evoked response, 62 Vitamin E, pretreatment with, 525-527 Vitellogenin assay, 484 Volaton, 610 Voltage-dependent calcium channel, 329 Volume transmission, 272-273 von Krueger, Gerde, 390 VR, neuropathy induced by, 278 V series compounds, 3 VX acetylcholinesterase knockout mice and, 234 AEGLs, 59-62 analytical methods, 693-694 chemistry, 7 CNS effects on respiration, 277 dermal absorption, 563 estimated reference dose, 57 history of, 3,389 -induced myopathy, 512 muscarinic receptors, noncompetitive binding, 237 neurotoxicity testing, cell culture systems, 329 nicotinic receptors and, 260 properties, 49-52 respiratory arrest, 394-395 stereoselectivity of acetylcholinesterase toward, 210 structure of, 390 supralethal doses, 55 toxicity, 278 effects, 52-55 electrophysiological, 341,342 mechanism of, 51-52 Wasting syndrome, 533 Water dialkyl phosphates in, 692 pesticide residues, 657-658 Water samples, extraction of, 681 Western equine encephalitis, 600 West Nile virus, 600 organophosphate insecticides used for, 601 U.S emergence, 600 Wipe sampling, 575 Workers occupationally-exposed, withdrawal of, 578 protective clothing, 583, 586-587 supervision of, 583, 586
763
Workplace biomonitoring, 581-582 health care facilities, 587 NIOSH guidelines, 689 Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity, 634 A World Compendium. The Pesticide Manual, 689 World Health Organization acute pesticide poisonings, 570 eradication of malaria, 600 IPCS, 644 Neurobehavioral Core Test Battery, 282 pesticide classification, 643-644 pesticide poisoning, incidence of, 93,528 World Health Organization/Food and Agriculture Organization acceptable daily intakes, 644-645, 646-647,648-649 acute reference dose, 651-652 assessment of endpoints, 646-647 data extrapolation, 647, 650 maximum residue limit, 644, 650-651 mixtures guidelines, 651 pesticide residues, 644-645,650-651 toxicity assessment guidelines, 645-646 World War II, nerve agent stockpiles, 389 Worms, acetylcholinesterase genes, 168
Xanthine dehydrogenase, and reactive oxygen species, 513, 515 Xanthine oxidase, fasciculations-induced changes, 515 Xanthurenic acid, 707 Xenobiotic-metabolizing enzymes phase I, 127-129 phase II, 127, 129-130 placental, 465-466, 467 Xenobiotic responsive element of nucleus, 664 Xenobiotics estrogenic effects, 447 metabolism, see also Drug metabolism carboxylesterases, 219-220, 227-228, 229 in fish, 664-666 transplacental transfer, 465-474 Xenopus oocytes, 343 Y chromosome deletions, 447 Yellow fever, 599-600 YT blood group, 194 Zebra fish, 704 developmental neurotoxicity screening, 305 toxicity testing, 659 Zimbabwe, acute pesticide poisonings, 570