ANTICHOLINESTERASE PESTICIDES

Metabolism, Neurotoxicity, and Epidemiology

Edited by

TETSUO SATOHChiba University

RAMESH C. GUPTAMurray State University

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ANTICHOLINESTERASE PESTICIDES

ANTICHOLINESTERASE PESTICIDES

Metabolism, Neurotoxicity, and Epidemiology

Edited by

TETSUO SATOHChiba University

RAMESH C. GUPTAMurray State University

Copyright # 2010 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Anticholinesterase pesticides : metabolism, neurotoxicity, and epidemiology / edited by Tetsuo Satoh, Ramesh C. Gupta.p.; cm.

Includes bibliographical references.ISBN 978-0-470-41030-1 (hardback)1. Cholinesterase-inhibiting insecticides—Toxicology. I. Satoh, Tetsuo, Ph. D. II. Gupta, Ramesh C. (Ramesh Chandra), 1949–[DNLM: 1. Pesticides—toxicity. 2. Cholinesterase Inhibitors—metabolism. 3. Cholinesterase Inhibitors—toxicity.

4. Occupational Diseases—epidemiology. 5. Pesticides—metabolism. WA 240 A629 2010]RA1270.I5.A58 2010363.7380498—dc22

2010003432

Printed in the United States of America10 9 8 7 6 5 4 3 2 1

http://www.copyright.comhttp://www.wiley.com/go/permissionhttp://www.wiley.com

Dedicated to the late Professor Kenneth P. DuBois at the University of Chicagofor his great mentorship and inspiration

Tetsuo Satoh

Dedicated to my daughter Rekha, wife Denise, andmy parents, the late Chandra and Triveni Gupta

Ramesh C. Gupta

CONTENTS

PREFACE xi

FOREWORD xiii

CONTRIBUTORS xv

PART I METABOLISM AND MECHANISMS 1

1 Acetylcholinesterase and Acetylcholine Receptors: Brain Regional Heterogeneity 3Haruo Kobayashi, Tadahiko Suzuki, Fumiaki Akahori and Tetsuo Satoh

2 Genomic Implications of Anticholinesterase Sensitivities 19Jonathan E. Cohen, Alon Friedman, Gabrial Zimmermann and Hermona Soreq

3 Butyrylcholinesterase: Overview, Structure, and Function 25Oksana Lockridge, Ellen G. Duysen and Patrick Masson

4 Carboxylesterases: Overview, Structure, Function, and Polymorphism 43Masakiyo Hosokawa and Tetsuo Satoh

5 Carboxylesterases in the Metabolism and Toxicity of Pesticides 57Colin J. Jackson, John G. Oakeshott, Juan Sanchez-Hernandez and Craig E. Wheelock

6 The Metabolic Activation and Detoxication of Anticholinesterase Insecticides 77Janice E. Chambers, Edward C. Meek and Matthew Ross

7 Paraoxonase 1: Structure, Function, and Polymorphisms 85Lucio G. Costa, Clement E. Furlong

8 Long-Term Neurotoxicological Effects of Anticholinesterases After either Acute orChronic Exposure 97Angelo Moretto, Manuela Tiramani and Claudio Colosio

9 Molecular Toxicology of Neuropathy Target Esterase 109Yi-Jun Wu and Ping-An Chang

10 Detoxication of Anticholinesterase Pesticides 121Miguel A. Sogorb and Eugenio Vilanova

vii

PART II TOXICITY AND BIOMONITORING 133

11 Involvement of Oxidative Stress in Anticholinesterase Pesticide Toxicity 135Dejan Milatovic, Michael Aschner, Ramesh C. Gupta, Snjezana Zaja-Milatovic and Gregory Barnes

12 Central Mechanisms of Seizures and Lethality Following Anticholinesterase Pesticide Exposure 149Andrzej Dekundy and Rafal M. Kaminski

13 Apoptosis Induced by Anticholinesterase Pesticides 165Qing Li

14 Gene Expression 175Shirin Pournourmohammadi and Mohammad Abdollahi

15 Organophosphates as Endocrine Disruptors 189Shigeyuki Kitamura, Kazumi Sugihara, Nariaki Fujimoto and Takeshi Yamazaki

16 Developmental Neurotoxicity of Anticholinesterase Pesticides 203John Flaskos and Magdalini Sachana

17 Toxicity of Anticholinesterase Pesticides in Neonates and Children 225Diane Rohlman and Linda McCauley

18 Neurotoxicity of Organophosphates and Carbamates 237Kiran Dip Gill, Govinder Flora, Vidhu Pachauri and Swaran J.S. Flora

19 Biomonitoring of Pesticides: Pharmacokinetics of Organophosphorus and Carbamate Insecticides 267Charles Timchalk

20 Novel Biomarkers of Organophosphate Exposure 289Tetsuo Satoh, Salmaan H. Inayat-Hussain, Michihiro Kamijima and Jun Ueyama

21 Biomarkers of Carcinogenesis in Relation to Pesticide Poisoning 303Manashi Bagchi, Shirley Zafra-Stone, Francis C. Lau and Debasis Bagchi

22 Anticholinesterase Pesticides Interactions 315Ramesh C. Gupta and Dejan Milatovic

23 Interaction of Anticholinesterase Pesticides with Metals 329Jitendra K. Malik, Avinash G. Telang, Ashok Kumar and Ramesh C. Gupta

PART III EPIDEMIOLOGICAL STUDIES 341

24 Epidemiological Studies of Anticholinesterase Pesticide Poisoning: Global Impact 343Claudio Colosio, Francesca Vellere and Angelo Moretto

25 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Chile 357Floria Pancetti, Muriel Ramı́rez and Mauricio Castillo

26 Epidemiological Studies of Anticholinesterase Pesticide Poisoning In China 365Yueming Jiang

27 Epidemiological Studies of Anticholinesterase Pesticide Poisoning In Egypt 379Sameeh A. Mansour

viii CONTENTS

28 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Greece 403M. Stefanidou, S. Athanaselis, C. Spiliopoulou and C. Maravelias

29 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in India 417P.K. Gupta

30 Poisoning with Anticholinesterase Insecticides in Iran 433Mohammad Abdollahi

31 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Israel 447Yoram Finkelstein, Elihu D. Richiter and Michael Aschner

32 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Japan 457Takemi Yoshida and Yumiko Kuroki

33 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Korea 463Hyung-Keun Roh, Bum Jin Oh, Mi-Jin Lee and Joo-Hyun Suh

34 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Mexico 471Betzabet Quintanilla-Vega, Norma Pérez-Herrera and Elizabeth Rojas-Garcı́a

35 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Serbia 481Milan Jokanović, Biljana Antonijević and Slavica Vuc̆inić

36 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Spain 495Antonio F. Hernández, Tesifón Parrón, José L. Serrano and Porfirio Marı́n

37 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Taiwan 509Tzeng Jih Lin, Dong Zong Hung, Jin Lian Tsai, Sheng Chuan Hu and Jou-Fang Deng

38 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Thailand 523Winai Wananukul

39 Epidemiological Studies of Anticholinesterase Pesticide Poisoning in Turkey 533İsmet Çok

40 Epidemiology of Anticholinesterase Pesticide Poisoning in the United States 541Anna M. Fan

PART IV REGULATORY ASPECTS 567

41 Regulatory Aspects of Anticholinesterase Pesticides 569Kai Savolainen

PART V MEDICAL TREATMENT OF POISONING WITH ORGANOPHOSPHATESAND CARBAMATES 581

42 Medical Treatment of Poisoning with Organophosphates and Carbamates 583Milan Jokanović

INDEX 599

CONTENTS ix

PREFACE

Anticholinesterase pesticides are primarily comprised oforganophosphate (OP) and carbamate (CM) compoundsthat constitute a large number of synthetic chemicals. Thefirst OP compound, tetraethyl phosphate, was synthesizedby Philippe de Clermont in France in 1854. After a lapse ofalmost 80 years, a series of OP compounds were synthesizedin Germany by Gerhard Schrader in the quest for pesticides.But prior to and during World War II, priority shiftedfor these compounds to be used as nerve agents/nervegases in chemical warfare. OP nerve agents of the G series,including tabun (GA), sarin (GB), and soman (GD) were syn-thesized and stockpiled on a mass scale. During and soonafter World War II, deadly toxic OP nerve agents of the Vseries (such as VX) were also synthesized and produced.Fortunately, none of these nerve agents were used in thewar because of the fear of retaliation by the Allied Forces.Soon after the war, thousands of OP compounds were syn-thesized for pesticidal use in many countries around theworld. During the same time period, hundreds of CM com-pounds were synthesized and thoroughly investigated fortheir pesticidal activity. During the second half of the twenti-eth century, OPs and CMs became very popular around theglobe because the organochlorine pesticides were found tobe persistent in the environment posing serious health risksto humans, animals, and the environment as a whole. In1962, the book Silent Spring by Rachel Carson was aneye-opener regarding the need to protect our environmentfrom the use of DDT-related organochlorine pesticides. Inaddition, many of the organochlorine pesticides were foundto be carcinogenic. In the recent past, there has been a conti-nuing trend for preference of CMs over OPs, because CMsproduce reversible toxic effects, while OPs can elicit acuteas well as delayed polyneuropathy in mammalian and avianspecies. Throughout this book, OPs and CMs are collectivelyreferred to as “anticholinesterase pesticides.”

Currently, about 250 OPs and 24 CMs are commonlymarketed worldwide for many purposes as pesticides. Eachyear, an estimated one million people are poisoned from pes-ticides, including several hundred thousand fatalities from

inadvertent, accidental, and occupational exposures, as wellas suicidal intent. In the case of animals and birds, poisoningsare often due to accidental exposure or malicious activity.

Anticholinesterase pesticides produce a variety of toxico-logical manifestations in nontarget species, including mam-malian, avian, and aquatic. Although OPs and CMs bothexert initial toxicity by virtue of acetylcholinesterase (AChE)inactivation at the nerve synapses and neuromuscularjunctions, several other cholinergic and noncholinergicmechanisms are involved, with a variety of adverse effectscontributing to the overall toxicity of these compounds.Anticholinesterase pesticides primarily affect the central ner-vous system, skeletal muscles, respiratory and cardiovascularsystems, but many other vital and nonvital organs and systemsare affected as well. Furthermore, great variations exist amongOPs and CMs in the context of toxic effects. Therefore, antic-holinesterase pesticides should not be generalized for exertingtheir toxicity based on a single underlying common mechan-ism. Many of these OPs and CMs are used in different combi-nations to deal with the problem of development of resistancein insects, but as a result of additive or synergistic interactions,potentiation of toxicity is observed in mammalian systems.This complexicity makes the risk assessment of these pesti-cides rather difficult. During the past decade, many novel bio-markers (enzymatic and nonenzymatic) of exposure and effectsof OPs and CMs have been developed, which are commonlyused for surveillance and risk assessment. In this regard, federalregulatory agencies (the Centers for Disease Control andPrevention [CDC], U.S. Environmental Protection Agency[EPA], and National Institute for Occupational Safety andHealth [NIOSH]) and international regulatory agencies(the World Health Organization [WHO] and Food andAgriculture Organization [FAO]) play pivotal roles in develop-ing strategies to minimize or prevent workers from pesticideexposure and putting plans for therapeutic measures in place.

This book entitled Anticholinesterase Pesticides: Meta-bolism, Neurotoxicity and Epidemiology is published withtwo major objectives: (1) thorough characterization of targetand nontarget enzymes and proteins involved in toxicity and

xi

metabolism of OPs and CMs; and (2) epidemiology ofpoisonings and fatalities in people from short- and long-termexposures to these pesticides in different occupational settings,on an individual country basis as well as on a global basis.Metabolism and neurotoxicity studies presented in this bookare primarily from experimental studies, while the epidemiolo-gical studies are from humans. The book has 42 chaptersorganized into five sections. The early portion of the bookdeals with metabolism, mechanisms, and biomonitoring ofanticholinesterase pesticides, while the later part deals withepidemiological studies, regulatory issues, and therapeuticintervention. It needs to be emphasized that this book is heavilyfocused on epidemiology, which is a novelty.

The editors of this book would like to offer sincere thanksand deepest appreciation to all the authors for their hardwork in contributing the chapters. The editors also thankDr. Satoshi Suzuki, Ms. Shinobu Yui, Ms. Yuko Fukasawa,Mrs. Robin Doss, and Mrs. Kristie Rohde for their technicalassistance. Sincere thanks also go to Mr. Jonathan Rose, whois the acquiring editor at John Wiley & Sons.

TETSUO SATOHChiba University Chiba, Japan

RAMESH C. GUPTAMurray State University Hopkinsville, Kentucky

xii PREFACE

FOREWORD

We are all part of globalization, the global economy, or theglobal village, to use only three of the many descriptiveterms, each with different meanings. The emerging nationsare searching for ways and means to improve their economies,high food prices encouraging agricultural expansion andniche markets for the international trade of fruits andvegetables shipped from tropical or subtropical regions tocountries of temperate or cold climates during part of theyear. Such endeavors will require significant changes inagricultural practices, with attendant problems of pollution.

My experiences in Southeast Asia and South Americahave demonstrated the demographic shifts of people tocities seeking employment, leaving those less educationallyadvantaged on the farms, faced with the problems of notonly growing food for themselves but for those living inthe cities, and being encouraged by governments to increasefarm incomes by participating in the developing exportmarkets for agricultural products. This has become morecomplicated by the specter of biofuels being manufacturedfrom essential food grains, increasing costs as well as creatingfood shortages.

Some of the needs for increased food production have beenmet by using high-yield varieties or genetically modifiedorganisms (rice, corn, wheat, etc.). Other approaches haverequired techniques to reduce significant pre- and post-harvestloss of produce through predation by fungi, insects, andmam-mals. Monocultures of various crops will lead to dynamicincreases in crop-specific pests—the brown hopper plaguein Southeast Asian rice-growing regions being one recentexample. Ultimately, such problems will require chemicalcontrol (pesticides), thereby placing these unfamiliar agentsin the hands of naı̈ve farmers. The use of such agents oncrops is being encouraged and even subsidized by govern-ments. The switch from traditional mixed farming to cropmonoculture results in elevated crop infestations and escalat-ing use of pesticides, a sure recipe for disaster, both environ-mentally and in terms of effects on human health.

One of the most important classes of chemicals is theanticholinesterase insecticides, the organophosphorus and

carbamate esters, the topic of this timely book. Reviews ofthe published literature and government reports reveal thatthis class of agents is responsible for the increased incidencesof accidental and intentional poisonings, particularly intropical countries in Southeast Asia, South America, andAfrica. Frequently, the agents involved in such poisoningsare older, beyond-patent, and more potent agents now bannedin North America and Europe but which can be made cheaplyanywhere in the world as components of global trade.Frequently, these agents contain toxic impurities that areformed during manufacture and storage and are not removed.

It is time to revisit the subject of anticholinesterase-typeinsecticide toxicology in depth. This book will examinemechanisms of biotransformation (metabolism) particularlythe roles of various types of esterases: as “sinks” to bindcirculating toxicants, as target sites in nervous systems, andas indices for monitoring the severity of poisoning.

In the past decade, significant advances have been madein knowledge regarding mechanisms of neurotoxicity ofthese chemicals indirectly at muscarinic and nicotinic recep-tors either as a consequence of adversely high levels of unme-tabolized, circulating acetylcholine or by direct action onnerve cell membranes. In addition to effects at specific,known target sites, the more subtle adverse effects on thewhole nervous organism and particularly developmentalneurotoxicity are of special concern, given the many studiesnow demonstrating subtle, subclinical, persistent, adverseeffects in infants and young children.

Of particular importance today in developing countries isthe fourth section discussing epidemiological studies essen-tial to an understanding of acute and delayed toxicities,including neural, developmental, and endocrine effects.While acute intoxications are monitored to some extent indeveloping countries, the health infrastructure does notextend to studying chronic or delayed toxicity, sometimesappearing years after exposure. The latter aspect is extremelyimportant in countries where control of pesticide use is mini-mal. Information, however anecdotal or detailed, is crucial todemonstrate to health providers and investigators in various

xiii

countries that they are not working on unique scenarios butare encountering the same problems. The “small” picture ofcountry-related pesticide toxicities must be amalgamatedinto the “large,” global viewpoint. Some major steps in thisdirection will be achieved with Section III of this book.

Given the worldwide problems of pesticide use andmisuse, this book dealing with the anticholinesterase class

of insecticides is important. Developing countries shouldfind it valuable, being faced with the challenge of buildingagricultural infrastructures to control pesticide use througheducation and training in addition to monitoring agriculturalworker and general population health and safety.

DONALD J. ECOBICHON

xiv FOREWORD

CONTRIBUTORS

Mohammad Abdollahi, Laboratory of Toxicology andPharmacology, Faculty of Pharmacy, and PharmaceuticalSciences Research Center, Tehran University of MedicalSciences, IRAN

Fumiaki Akahori, Department of Veterinary Medicine,Azabu University, JAPAN

Biljana Antonijević, Faculty of Pharmacy, University ofBelgrade, Vojvode Stepe, Belgrade, SERBIA

Michael Aschner, Departments of Pediatrics and Pharma-cology, and the Kennedy Center for Research on HumanDevelopment, Vanderbilt University Medical Center,Nashville, TN, USA

S. Athanaselis, Department of Forensic Medicine andToxicology, University of Athens, School of Medicine,Athens, GREECE

Debasis Bagchi, Department of Pharmacological andPharmaceutical Sciences, College of Pharmacy, Univer-sity of Houston, Houston, TX, USA

Manashi Bagchi, PhD, Research and Development, Inter-Health Research Center, Benicia, CA, USA

Gregory Barnes, Vanderbilt University School ofMedicine, Department of Neurology, Nashville, TN, USA

Mauricio Castillo, Department of Public Health, Faculty ofMedicine, Universidad Catolica del Norte, Coquimbo,CHILE

Janice E. Chambers, Center for Environmental HealthSciences, College of Veterinary Medicine, MississippiState University, MS, USA

Ping-An Chang, Key Laboratory of Molecular Biology,College of Bio-information, Chongqing University ofPosts and Telecommunications, Chongqing, CHINA

Jonathan E. Cohen, Department of Physiology and Neuro-surgery, Zlotowski Center for Neuroscience, Ben-GurionUniversity of the Negev, Beer-Sheva, ISRAEL

Ismet COK, Gazi University, Faculty of Pharmacy,Department of Toxicology, Hipodrom, Ankara, TURKEY

Claudio Colosio, Department of Occupational and Environ-mental Health, University of Milano, S. Paolo HospitalUnit, and International Centre for Rural Health (ICRH),Milano, ITALY

Lucio G. Costa, Department of Environmental Healthand Occupational Health Sciences, University ofWashington, Seattle, WA, USA, and Department ofHuman Anatomy Pharmacology and Forensic Sciences,University of Parma Medical School, Parma, Italy

Andrzej Dekundy, InVivo Pharmacology, RþDCNS,MerzPharmaceuticals GmbH, Frankfurt am Main, GERMANY

Ellen G. Duysen, University of Nebraska Medical Center,Eppley Institute, Omaha, NE, USA

Donald J. Ecobichon, RR1, Elgin, ON KOG lE0,CANADA

Anna M. Fan, Office of Environmental Health HazardAssessment, California Environmental ProtectionAgency, CA, USA

Yoram Finkelstein, Service and Unit of Neurology andToxicology, Shaare Zedek Medical Center, Jerusalem,ISRAEL

John Flaskos, Laboratory of Biochemistry and Toxicology,School of Veterinary Medicine, Aristotle University ofThessaloniki, GREECE

Govinder Flora, Spherix Inc., 6430 Rockledge Dr. West-moreland Bldg. #503, Bethesda, MD, USA

Swaran J. S. Flora, Division of Pharmacology & Toxi-cology, Defence Research & Development EstablishmentJhansi Road, Gwalior, INDIA

Alon Friedman, Department of Physiology, Faculty forHealth Sciences, Ben-Gurion University, Beer-Sheva,ISRAEL

xv

Nariaki Fujimoto, Research Institute for RadiationBiology and Medicine, Hiroshima University, Hiroshima,JAPAN

Clement E. Furlong, Departments of Genome Sciences andMedicine (Medical Genomics), University ofWashington,WA, USA

Kiran Dip Gill, Department of Biochemistry, PostgraduateInstitute of Medical Education and Research Chandigarh,INDIA

Pawan K. Gupta, Division of Pharmacology & ToxicologyC-44, Rajendra Nagar, Bareilly INDIA, and Advisor toWHO, Geneva, SWITZERLAND

Ramesh C. Gupta, Toxicology Department, BreathittVeterinary Center, Murray State University, Hopkinsville,KY, USA

Antonio F. Hernández-Jerez, Department of LegalMedicine and Toxicology, University of Granada Schoolof Medical, on behalf of the ESPAPP group, SPAIN

Masakiyo Hosokawa, Laboratory of Drug Metabolismand Biopharmaceutics, Faculty of PharmaceuticalSciences, Chiba Institute of Science, Choshi, Chiba,JAPAN

Salmaan H. Inayat-Hussain, Faculty of Allied HealthSciences, Universiti Kebangsaan Malaysia, JaranRajaMuda Abdul Aziz, Kuala Lumpur, MALAYSIA

Cohn J. Jackson, CSIRO Entomology, Black Mountain,Canberra, AUSTRALIA

Yueming Jiang, Department of Toxicology, School ofPublic Health, Guangxi Medical University, Nanning,CHINA

Milan Jokanović, Experta Consulting, Belgrade, SERBIA,and Academy of Sciences and Arts of Republic Srpska,Banja Luka, Republic Srpska, BOSNIA ANDHERZEGOVINA

Rafal M. Kaminski, UCB Pharma S.A., CNS Research,Epilepsy Pharmacology, Chemin du Foriest, Alleud,BELGIUM

Michihiro Kamijima, Department of Occupational andEnvironmental Health, Nagoya City University GraduateSchool of medical Sciences, Nagoya, JAPAN

Shigeyuki Kitamura, Department of EnvironmentalScience, Nihon Pharmaceutical University Saitama,JAPAN

Haruo Kobayashi, Department of Veterinary Medicine,Faculty of Agriculture, Iwate University, Morioka, and7-272 Aza-Mukaishinden, Ukai, Takizawa-mura, Iwate,JAPAN

AshokKumar, Division of Biochemistry, Indian VeterinaryResearch Institute, Izatnagar, INDIA

Yumiko Kuroki, Japan Poison Information Center,Tsukuba, Ibaraki, JAPAN

Francis C. Lau, Research and Development, InterHealthResearch Center, Benicia, CA, USA

Mi-Jin Lee, Department of Emergency Medicine, KonyangUniversity Hospital, Daejeon, KOREA

Qing Li, Department of Hygeine and Public Health, NipponMedical School Tokyo, JAPAN

Tzeng Jih Lin, Department of Emergency, KaohsiungMedical University Hospital, and Department ofEmergency Medicine, Faculty of Medicine, College ofMedicine, Kaohsiung Medical University, Kaohsiung,TAIWAN

Oksana Lockridge, University of Nebraska MedicalCenter, Eppley Institute, Omaha, NE, USA

Jitendra K. Malik, National Referral Laboratory (ChemicalResidues), Division of Pharmacology and Toxicology,Indian Veterinary Research Institute, Izatnagar, INDIA

Sameeh A. Mansour, Pesticides and EnvironmentalToxicology, Environmental Toxicology Research Unit(ETRU), Pesticide Chemistry Department, NationalResearch Centre, Cairo, EGYPT

C. Maravelias, Department of Forensic Medicine andToxicology, University of Athens, School of Medicine,Athens, GREECE

Porfirio Marı́n, Delegacion Provincial de Salud, Almeria,SPAIN

Patrick Masson, University of Nebraska Medical Center,Eppley Institute, Omaha, NE, USA

Linda A. McCauley, Nell Hodgson Woodruff School ofNursing, Emory University, Atlanta, GA, USA

Edward C. Meek, Center for Environmental HealthSciences, College of Veterinary Medicine, MississippiState University, MS, USA

Dejan M.Milatovic, Vanderbilt University Medical Center,Department of Pediatrics/Pediatric Toxicology Nashville,TN, USA

Angelo Moretto, Department of Occupational and Environ-mental Health of the University of Milano, InternationalCentre for Pesticides and Health Risk Prevention,Ospedale Luigi Sacco. Via Stephenson, Milano, ITALY

Elizabeth de Souza Nascimento, University of Salo Paulo,School of Pharmaceutical Sciences, Department ofClinical Chemistry and Toxicology, Salo Paulo, BRAZIL

xvi CONTRIBUTORS

John G. Oakeshott, CSIRO Entomology, Black Mountain,Canberra, AUSTRALIA

Bum Jin Oh, Department of Emergency Medicine,Seoul Asan Hospital, Ulsan University, Seoul,KOREA

Floria Pancetti, Laboratory of Environmental Neurotoxi-cology, Department of Biomedical Sciences, UniversiddCatólica del Norte, Coquimbo, CHILE

Tesifón Parrón, Delegacion Provincial de Salud, Almeria,SPAIN

Norma Perez-Herrera, Seccion Externa de Toxicologia,Mexico City, MEXICO

Shirin Pournourmohammadi, Laboratory of Toxicology,Institute of Medicinal Plants, ACECR, Teheran, andFaculty of Pharmacy, Kerman University of MedicalSciences, Kerman, IRAN

Betzabet Quintanilla-Vega, Sección Externa deToxicologia, CINVESTAV-IPN, Mexico City,MEXICO

Muriel Ramirez, Department of Public Health, Faculty ofMedicine, Universidad Catolica del Norte, Coquimbo,CHILE

Elihu D. Richter, Occupational and EnvironmentalMedicine Hebrew University-Hadassah School ofPublic Health and Community Medicine, Jerusalem,ISRAEL

Hyung-Keun Roh, Internal Medicine, Division of ClinicalPharmacology Gachon University Gil Hospital, Incheon,KOREA

Diane Rohlman, Center for Research on Occupational andEnvironmental Toxicology, Oregon Health & ScienceUniversity, Portland, OR, USA

Elizabeth Rojas-Garcia, Dirección de Fortalecimiento dela Investigación, Universidad Autónoma de Nayarit,Tepic, Nayarit, MEXICO

Matthew Ross, Center for Environmental Health Sciences,College of Veterinary Medicine, Mississippi StateUniversity, MS, USA

Magda Sachana, Laboratory of Biochemistry and Toxi-cology, School of Veterinary Medicine, Aristotle Univer-sity of Thessaloniki, Thessaloniki, GREECE

Juan Sanchez-Hernadez, Laboratory of Ecotoxicology,Faculty of Environmental Science, University ofCastilla-La Mancha, Teledo, SPAIN

Tetsuo Satoh, Department of Pharmacology and Toxi-cology; Graduate School of Pharmaceutical Sciences,

Chiba University, Chiba, and HAB Research Institute,Ichikawa, Chiba, JAPAN

Jose L. Serrano, Delegacion Provincial de Salud, Almeria,SPAIN

Miguel A. Sogorb, Unidad de Toxicologı́a y SeguridadQuı́mica, Instituto de Bioingenı́eria UniversidadMiguel Hernández de Elche. Avenida de Ia Universidad,SPAIN

Hermana Soreq, Department of Biological Chemistry,The Institute of Life Sciences, The Hebrew University ofJerusalem, ISRAEL

C. Spiliopoulou, Department of Forensic Medicine andToxicology, University of Athens, School of Medicine,Athens, GREECE

Maria Stefanidou, Department of Forensic Medicine andToxicology, University of Athens, School of Medicine,Athens, GREECE

Kazumi Sugihara, Graduate School of BiomedicalSciences, Hiroshima University, Hiroshima, JAPAN

Joo-Hyun Suh, Department of Emergency Medicine,Konyang University Hospital, Daejeon, KOREA

Tadahiko Suzuki, Department of Veterinary Medicine,Faculty of Agriculture, Iwate University, Morioka,Iwate, Japan

Avinash G. Telang, National Referral Laboratory(Chemical Residues), Division of Pharmacology andToxicology, Indian Veterinary Research Institute,Izatnagar, INDIA

Manuela Tiramani, European Food Safety Authority,Parma, Italy

Charles Timchalk, MSIN P7-59, Biological Monitoringand Modeling, Pacific Northwest National Laboratory,Richland, WA, USA

Jun Ueyama, Ph.D, Department of Medicinal Technology,Nagoya University School of Health Sciences, Nagoya,JAPAN

Francesca Vellere, Department of Occupational andEnvironmental Health of the University of Milan, andInternational Centre for Rural Health (ICRH), San PaoloHospital, Milano, ITALY

Eugenio Vilanova, Unidad de Toxicologı́a y SeguridadQuı́mica, Instituto de Bioingenı́eria, UniversidadMiguel Hernández de Elche. Avenida de la Universidad,SPAIN

Slavica Vučinić, National Poison Control Center, MilitaryMedical Academy, Belgrade, SERBIA

CONTRIBUTORS xvii

Winai Wananukul, Ramathibodi Poison Center, Depart-ment of Medicine, Faculty of Medicine Ramathibodi Hos-pital, Mahidol University, Bangkok, THAILAND

Craig E. Wheelock, Department of Medical Biochemistryand Biophysics, Division of Physiological Chemistry II,Scheeles, väg 2, Karolinska Institut, Stockholm,SWEDEN

Yi JunWu, Laboratory of Molecular Toxicology, State KeyLaboratory of Integrated Management of Pest Insects andRodents, Institute of Zoology, Chinese Academy ofSciences, Beijing, CHINA

Takesh Yamazaki, Graduate School of Integrated Artsand Sciences, Hiroshima University, Higashihiroshima,JAPAN

Takemi Yoshida, Department of Biochemical Toxicology,School of Pharmacy, Showa University, Shinagawa,Tokyo, JAPAN

Jiang Yueming, Department of Occupational Health andToxicology, School of Public Health, Guangxi MedicalUniversity, Nanning, CHINA

Shirley Zafra-Stone, Research and Development, Inter-Health Research Center, Benicia, CA, USA

Snjezana Zaja-Milatovic, Vanderbilt University Schoolof Medicine, Department of Cancer Biology, TN, USA

Gabriel Zimmermann, Department of Biological Chem-istry, The Institute of Life Sciences, The Hebrew Univer-sity of Jerusalem, ISRAEL

xviii CONTRIBUTORS

PART I

METABOLISM AND MECHANISMS

1ACETYLCHOLINESTERASE AND ACETYLCHOLINERECEPTORS: BRAIN REGIONAL HETEROGENEITY

HARUO KOBAYASHI AND TADAHIKO SUZUKIFaculty of Agriculture, Department of Veterinary Medicine, Iwate University, Morioka, Japan

FUMIAKI AKAHORIFaculty of Veterinary Medicine, Azabu University, Japan

TETSUO SATOHDepartment of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, andHAB Research Institute Cornea Center, Ichikawa, Chiba, Japan

1.1 Introduction 3

1.2 Role of Acetylcholinesterase and Mechanism ofCholinergic Neurotransmission 4

1.3 Effects of Organophosphates and Carbamates onAcetylcholinesterase 51.3.1 Determination of Acetylcholinesterase Activity 51.3.2 Effects of Organophosphates on Regional

Brain Acetylcholinesterase Activity 51.3.3 Effects of Carbamates on Brain Regional

Acetylcholinesterase Activity 7

1.4 Effects on Release, Synthesis, and Storage ofAcetylcholine 9

1.5 Effects on Acetylcholine Receptors 111.5.1 Effects on Muscarinic Receptors 111.5.2 Effects on Nicotinic Acetylcholine Receptors 13

1.6 Effects on Neuroactivities Other than the CholinergicSystem 13

1.7 Conclusions 14

Acknowledgments 14

References 14

1.1 INTRODUCTION

Acetylcholinesterase (AChE) inhibitors are used throughoutthe world for many purposes. Probably the best known arethe pesticides that are used to control the insects that affectpublic health (e.g., mosquitoes, flies, cockroaches, ticks,fleas, and bedbugs) as well as those that affect agricultureand gardening (e.g., grasshoppers, aphids, caterpillars, riceinsects, and stinkbugs). Although compounds with compara-tively low toxicity, such as pyrethroids and novel insecticidesincluding fipronil and neonicotinoids, have been developed

and are widely used, carbamates and organophosphorus com-pounds (organophosphates) are still commonly used through-out the world for the control of these various insects.

In addition to their use as pesticides, AChE inhibitors suchas sarin, tabun, and VX, highly toxic organophosphates, havealso been used as nerve gases. These compounds were usedin the Iran–IraqWar (1980–1988), and in the terrorist attackson the Tokyo subway (1995) and in Matsumoto (1994),Japan (Okumura et al., 2003). There continues to be a realthreat that these types of nerve agents can be misused in thefuture.

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

3

On the other hand, since carbamates and organophos-phates are highly effective inhibitors of AChE, they canalso be used for the treatment of diseases where cholinergicfunction is inadequate (Giacobini, 2000). By the inhibitionof AChE, acetylcholine concentrations are increased withinthe synapses and clinical improvement can be produced inperipheral and central disorders such as myasthenia gravisand Alzheimer’s disease, respectively. The AChE inhibitorsthat have been used either clinically for therapeutic purposesor experimentally for research purposes include organophos-phates such as diisopropylfluorophosphate (DFP) and tetra-isopropyl pyrophosphoramide (iso-OMPA), carbamates suchas neostigmine, physostigmine, and rivastigmine, and antide-mentia drugs or candidates, such as 2-[(1-benzyl-4-piperi-dyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one (done-pezil), tetrahydroamino acridine (THA), galantamine, andmethanesulfonyl fluoride.

In spite of the toxicological or therapeutic importance ofAChE inhibitors, the regional effects of these compoundson various cholinergic systems of the brain are not adequatelyunderstood. Many of the prior studies have focused ongrossly defined changes in whole brain or major subdivisionssuch as the forebrain or hindbrain. Since higher brain func-tions involve the integration of information from severaldifferent regions, neurochemical, biochemical, physiological,pharmacological, immunochemical, and electrophysiologicalinvestigation of brain activities can probably be best under-stood by detailed study of specific structures or areas. Forexample, very small changes that might be observed indetailed studies of specific areas such as the striatum andhippocampus may be lost when analyzed as a part of thewhole brain, forebrain, and hindbrain.

The study of specific areas of the brain is particularlyimportant because the cholinergic system has major functionsin the brain, especially in the cerebral cortex (cortex), limbicsystem, and hippocampus. In addition, the striatum is denselyinnervated by cholinergic interneurons that are crucial formotor behavior (Pisani et al., 2001), and this structure ishighly enriched in cholinergic markers such as AChEactivity, choline acetyltransferase (ChAT) activity, andacetylcholine (ACh) content.

Carbamates and organophosphates produce toxicitythrough the inhibition of AChE whether by acute, repetitive,or chronic exposure. Changes in cholinergic neurons pro-duced by AChE inhibition also have secondary effects ondopaminergic, g-aminobutyric acid (GABAergic), and gluta-matergic neurons, especially in the central nervous system(CNS). The two cholinergic receptors, muscarinic (mAChR)and nicotinic (nAChR), are located postsynaptically orpresynaptically on cholinergic neurons and these noncholi-nergic neurons, such as dopaminergic, GABAergic andglutamatergic neurons, and are involved in mediating theeffects of AChE inhibitors. Therefore, the cholinergic regu-lation and modulation of the brain can be determined by the

density of innervation in brain regions, cholinergic activities,such as AChE activity, ChAT activity, and ACh level, and thedistribution or sensitivity of AChRs.

In this chapter, the toxicological effects of cholinesteraseinhibitors, mainly organophosphates and carbamate insec-ticides, on brain cholinergic mechanisms are described. Thefocus of this chapter is on the effects of AChE inhibitorson ACh dynamics such as the synthesis, storage, and releaseof ACh, changes in ACh receptor density and function, andAChE activity in discrete brain regions of experimental ani-mals. For further reading on brain regional heterogeneity inrelation to the cholinergic system, readers are referred torecent publications elsewhere (Gupta, 2004, 2006b).

1.2 ROLE OF ACETYLCHOLINESTERASEAND MECHANISM OF CHOLINERGICNEUROTRANSMISSION

ACh is stored, in part, in synaptic vesicles (about 50 nm indiameter) in the cytoplasm of cholinergic nerve terminals(Bloom, 2002; Dani and Bertrand, 2007; Deutch and Roth,2004). When impulses arrive at the terminal membrane (8to 10 nm thick) causing depolarization, a portion of theACh-containing vesicles fuse with membrane and undergoexocytosis and release ACh into the fluid of the synapticcleft. Since the clearance of the cleft between the pre- andpostsynaptic membranes is about 20 nm, the extremelyhydrophilic ACh molecules diffuse and bind to AChRs onthe postsynaptic and/or presynaptic membranes almostinstantly (0.1 to 0.2 msec). Immediately after binding onthe AChRs, the ACh is hydrolyzed by AChE into cholineand acetic acid, destroying it within a few milliseconds afterexocytosis (Taylor, 2002). This rapid destruction of AChis required for normal cholinergic function. Any delayin the hydrolysis of ACh causes the accumulation of ACharound AChRs and prolongs excitation or transmission.There are several different mechanisms for terminating theactions of other neurotransmitters, such as dopamine,noradrenaline, GABA, histamine, and 5-hydroxytriptamine(serotonin). The biggest difference in inactivation of releasedneurotransmitter between ACh and other neurotransmittersis that most noncholinergic neurotransmitters are inactivatedby the reuptake into presynaptic nerve terminals, enzymaticcatabolism, and diffusion away from the receptor sites fol-lowed by dilution in extracellular fluid or plasma to subthres-hold concentration (Bloom, 2002; Deutch and Roth, 2004).Therefore, inactivation of noncholinergic transmitters maytake much longer than the hydrolysis of ACh by AChE.

Because the inactivation of ACh depends critically on theaction of AChE, increases in the synaptic effects of ACh canbe produced through the action of AChE inhibitors. The pop-ular AChE inhibitors used to affect cholinergic activity areorganophosphates and carbamates (Gupta, 2006a, 2006b).

4 ACETYLCHOLINESTERASE AND ACETYLCHOLINE RECEPTORS: BRAIN REGIONAL HETEROGENEITY

1.3 EFFECTS OF ORGANOPHOSPHATES ANDCARBAMATES ON ACETYLCHOLINESTERASE

The primary mechanism of action and the most acutelylife-threatening effect of exposure to carbamates and organo-phosphates result from the inhibition of AChE. Organophos-phates are grossly classified into oxon-type compounds,which directly inhibit AChE, such as dichlorvos, DFP, tri-chlorphon, and sarin, and thion-type compounds, such aschlorpyrifos, parathion, fenitrothion, and malathion. Thelatter organophosphates do not inhibit AChE directly butrequire the replacement of a sulfur atom with oxygen tobecome oxon-type compounds. It is well known that the inhi-bition of AChE by organophosphates is persistent, lastinghours to days, and potentially may not be reversible if anonenzymatically mediated dealkylation, termed “aging,”occurs. The phosphorylated aged AChE is refractory toreactivation.

Inhibition occurs as a result of phosphorylation of theserine (S200) included in the catalytic triad of the activecenter by the organophosphate (Aldridge, 1950; Fukuto,1990). Although this inhibition is usually considered irrevers-ible, some reactivation of phosphorylated acetylcholinester-ase can occur slowly as a result of hydrolytic cleavage, ifthe process of aging is not complete (Sultatos, 1994, 2006).Aging is a poorly understood mechanism in which onealkoxy group is hydrolyzed leaving the monoalkoxy phos-phate bound essentially irreversibly to the active site ofAChE (Sultatos, 1994, 2006).

The inhibition of AChE by an organophosphate is a func-tion of both binding affinity at the active site and the rate ofphosphorylation (Main, 1964). As a result, the bimolecularrate constant that can be determined experimentally continuesto be considered the single best approach to compare theinhibitory power of various organophosphates (Fukuto,1990; Mortensen et al., 1998). This strategy, however,depends upon the assumption that these in vitro reactionsapproximate first-order conditions because the oxon concen-trations are much higher than the uninhibited enzyme.However, a new approach based on continuous systems mod-eling to determine the apparent inhibition rate constant ofparaoxon and methyl paraoxon towards mouse brain AChEhas challenged the validity of that assumption. Theseexperiments have shown that the bimolecular rate constantsfor organophosphate-induced inhibition appear to change asa function of oxon concentrations, indicating that theefficiency of phosphorylation appeared to decrease as theparaoxon concentration increased (Kardos and Sultatos,2000).

In addition, in studies of the direct effects of AChE inhibi-tors, AChE knockout animals may be useful as a model forinvestigating the effects of selective, complete, and chroni-cally diminished AChE activity on AChRs, other cholinergicactivities and functions. AChE knockout mice were

developed recently and have provided a valuable tool forexamining the effects of long-term complete and selectiveabolition of AChE activity in brain regions (Duysen et al.,2002; Volpicelli-Daley et al., 2003; Xie et al., 2000). AChEknockout mice showed dramatic and selective reduction inmAChR, a marked redistribution of mAChRs to intracellularcompartments, upregulation of the high-affinity cholinetransporter, and altered behavior induced by mAChR antag-onists. In contrast, there was no change in the activity ofChAT, the levels of vesicular ACh transporter, and the b2subunit of nAChRs.

1.3.1 Determination of Acetylcholinesterase Activity

Excessive inhibition of AChE, a critical enzyme involved inboth peripheral and central cholinergic functions, by organo-phosphates or carbamates produces a cholinergic crisis that isthe mechanism of both acute and chronic toxicity. Themeasurement of AChE inhibition produced by these com-pounds is, therefore, important for evaluating and predictingmammalian toxicity. Studies of brain AChE are traditionallybased on biochemical assays, immunoreactivity, and histo-chemistry. A variety of methods have been developed forquantifying AChE activity, including detection of the thiolgroup formed by hydrolysis of acetylthiocholine as a sub-strate (Ellman et al., 1961) or another colorimetric methodof Hestrin (1949), or determination of radiolabeled acetatefollowing hydrolysis of radiolabeled ACh (Johnson andRussell, 1975). Several additional histochemical methodsdeveloped in recent years, including microdensitometry,microphotometry, and video-based histochemistry, are effec-tive in quantitative and detailed study of AChE in tissuesections (Ma et al., 2001; Sun et al., 2003).

1.3.2 Effects of Organophosphates on RegionalBrain Acetylcholinesterase Activity

Many studies of the effects of organophosphates on the brainhave focused on the effects of chlorpyrifos, dichlorvos,malathion, ethyl parathion (parathion), and methyl parathion.These pesticides have been used extensively as agriculturaland commercial insecticides throughout the world.

Most studies of the effects of AChE inhibitors on the CNShave reported AChE activity in gross neuroanatomical areassuch as the whole brain or forebrain and they have focusedon the consequences of high levels of organophosphateswith either acute or repeated exposures in either animals orpostmortem human victims. Relatively little attention hasbeen given to the effects of low-level organophosphateexposure that is not associated with acute cholinergic symp-toms (Ray and Richards, 2001). There are only a few reportsfocused on persistent effects of repeated and low-levelexposures to organophosphates on brain regional AChEactivity and other neurochemical and behavioral parameters

1.3 EFFECTS OF ORGANOPHOSPHATES AND CARBAMATES ON ACETYLCHOLINESTERASE 5

(Karanth et al., 2007; Kobayashi et al., 2007; Ma et al., 2001;Sun et al., 2003; Terry et al., 2007).

Terry et al. (2007) injected rats with chlorpyrifos at dosesof 2.5, 10.0, and 18.0 mg/kg, subcutaneously (s.c.) everyother day over the course of 30 days and then gave a two-week washout period. The activity of acetylcholinesterasewas measured in six brain regions, including prefrontalcortex, cortex, anterior hippocampus, posterior hippocampus,basal forebrain, and striatum at the end of the washout period(Fig. 1.1). For five of the six brain regions, AChE activity wassignificantly decreased even14days after the10and18 mg/kgregimens. For the striatum, AChE inhibition was significant14 days after all three dose regimens. Across the six brainregions, 14 days after the final 18 mg/kg dose of chlorpyri-fos, AChE activity was still inhibited by 55%. The authorsalso reported that the brain/plasma ratio of cholinesteraseactivity varied from a low of 0.67 in the striatum to a highof 1.04 in the anterior hippocampus and averaged 0.82across all three dose regimens and all six brain regions.They also determined the levels of chlorpyrifos and its metab-olite 3,5,6-trichloro-2-pyridinol in brain tissue. Although thelevels of chlorpyrifos and its metabolite were low or nearlyundetectable 14 days after the 10 and 18 mg/kg regimens,AChE activity continued to be inhibited by at least 50%.

In another study, Kobayashi et al. (2007) measured AChEactivity in the striatum, hippocampus, and cerebral cortex ofrats after they were treated with dichlorvos (DDVP) at 3 mg/kg/day, s.c., for 7 and 14 days. AChE activity was assayed 1,6, and 11 days after the last treatment with DDVP (Kobayashi

et al., 2007). AChE activity was markedly decreased inthe three brain regions 1 day after treatment over both 7 and14 days. AChE activity showed gradual recovery at 6 and11 days (Fig. 1.2a). The depression of AChE activity in thethree brain regions appears to be more severe in groupsadministered for 14 days than for 7 days. Although theactivity increased depending on days after withdrawal, therecovery was about 20% for 10 days in all brain regions.The irreversibility and slow recovery from DDVP treatmentare considered to be due to an aging and a new synthesis ofAChE (Taylor, 2002).

Sun et al. (2003) treated rats repeatedly with either vehicleor methyl parathion at a dose of 3 mg/kg/day, s.c. for oneweek or three weeks. The animals were sacrificed 24 h afterthe last treatment and AChE activity was measured histo-chemically in different brain regions, including striatum, hip-pocampus, frontal cortex, thalamus, and midbrain (Fig. 1.3,Table 1.1). The activity of AChE in the striatum, cortex, thala-mus, and midbrain was reduced to about 40%, 45%, 35%,33%, and 25% of respective controls after 1 week of treatmentand 20%, 20%, 13%, 13%, and 15% of respective controlsafter three weeks of treatment. As shown in Figure 1.3 andTable 1.1, AChE is distributed heterogeneously throughoutthe brain, and the relative regional preponderance is in thestriatum and thalamus. This study demonstrated that repeatedexposures to methyl parathion inhibit AChE to a similardegree in different brain regions.

The effects of organophosphate exposure on the brain alsoseems to depend to some degree on age. For example,Karanth et al. (2007) compared the effect of acute exposureto ethyl parathion on striatal AChE levels in adult (3-month-old) and aged (18-month-old) male Sprague-Dawleyrats. The activity of AChE was determined 3 and 7 daysafter a single subcutaneous treatment with a range of dosagesof ethyl parathion (adult: 1.8, 3.4, 6.0, 9.0, 18, and 27 mg/kg;aged: 1.8, 3.4, 6.0, and 9.0 mg/kg). It is interesting to notethat in this experiment, striatal AChE activity was signifi-cantly lower in control aged rats compared to control adultrats. Comparison of identical dosages in adult and aged rats(i.e., 9 mg/kg versus 9 mg/kg) showed that AChE inhibitionwas significantly higher in aged rats than in adults. Similarresults were reported by Scali et al. (1997) who foundhigher levels of brain AChE inhibition in aged rats than inadults following acute exposure to metrifonate (trichlorfon).Karanth et al. (2007), however, found that similar maximaldegrees of inhibition of 92% to 94% were noted in adultsand aged rats treated at the highest dose used for each agegroup, 27 and 9 mg/kg, respectively (Karanth et al., 2007).AChE inhibition in the striatum peaked at 3 days afterthe single injection and there was little evidence of recoveryby 7 days in either age group. Although it remains unclearwhy striatal AChE is more sensitive to ethyl parathion-induced inhibition in aged rats than adults, the authors pro-posed that the higher sensitivity in aged rats could be due

Figure 1.1 Brain acetylcholinesterase (AChE) activities measuredin six brain regions 14 days after the last chlorpyrifos administration.Each value represents the mean+SEM derived from five to six rats.�, p , 0.05 with respect to the vehicle control mean. Adapted fromTerry et al. (2007).

6 ACETYLCHOLINESTERASE AND ACETYLCHOLINE RECEPTORS: BRAIN REGIONAL HETEROGENEITY

to lower tissue AChE (about 14% significantly lowercompared to adult rats).

It seems that the inhibition of AChE activity and itsirreversibility after repeated administrations of the respectiveorganophosphates described above is not specific to variousbrain areas and is not selective for specific regions.

1.3.3 Effects of Carbamates on Brain RegionalAcetylcholinesterase Activity

Although carbamates react with the same serine moiety in thecatalytic site of AChE as do the organophosphates, the effectsof carbamates may be very different because of the duration

Figure 1.2 The activity of acetylcholinesterase in the striatum, hippocampus, and cortex of rats 1, 6, and 11 days after withdrawal of repeatedadministration of DDVP (a), propoxur (b), or oxotremorine (c) for 7 and 14 days. The activity of brain regional AChE was determined 1, 6, and11 days after withdrawal of repeated administration of DDVP, propoxur, or oxotremorine for 7 days (†) or 14 days (O) and expressed aspercent of control (W). Data are expressed as mean+SEM (n ¼ 4–6). Asterisks indicate values that are significantly different from controlvalue (�p , 0.05, ���P , 0.01). Adapted from Kobayashi et al. (2007).

1.3 EFFECTS OF ORGANOPHOSPHATES AND CARBAMATES ON ACETYLCHOLINESTERASE 7

of the inhibition. In contrast to the organophosphates, recov-ery of AChE activity after a carbamate (e.g., carbofuran)-induced inhibition is quite rapid since recovery simplyrequires the spontaneous hydrolysis of the covalent bondbetween the methyl carbamyl moiety and the enzyme(Ferguson et al., 1984; Gupta and Kadel, 1989). This obser-vation is also true for several other carbamate pesticides,including aldicarb, methomyl, and propoxur (Gupta, 1994,2004; Gupta and Kadel, 1990, 1991a, 1991b).

In an experiment conducted by Kamboj et al. (2006),carbofuran was administered to rats orally (in corn oil) at adose of 1 mg/kg/day for 28 days. The activity of acetylchol-inesterase was measured in three brain regions (cerebral

cortex, cerebellum, brain stem) one day after the finaladministration (Table 1.2). Carbofuran treatment resulted ina significant decrease in AChE activity in cortex (66.9%),cerebellum (71.7%), and brain stem (66.6%) compared tothe control animals.

Kobayashi et al. (2007) measured AChE activity in thestriatum, hippocampus, and cortex of rats 1, 6, and 11 daysafter the last treatment with propoxur at a dose of 10 mg/kg/day, s.c. for 7 and 14 days. As shown in Figure 1.2b,repeated injections of propoxur did not produce a uniformchange in the activity of AChE in every brain region 1, 6,and 11 days after withdrawal from repeated treatments withpropoxur across 7 or 14 days. The administration of propoxur

Figure 1.3 Typical images of AChE histochemistry staining from control and treated rats. Rats were treated with either vehicle or methylparathion (3 mg/kg) daily for 1 week or 3 weeks. Adapted from Sun et al. (2003).

8 ACETYLCHOLINESTERASE AND ACETYLCHOLINE RECEPTORS: BRAIN REGIONAL HETEROGENEITY

for 7 days suppressed the activity in the three brain regions 1,6, and 11 days after withdrawal. Surprisingly, the activity ofAChE in brain regions was generally higher in rats treatedwith propoxur for 14 days than for 7 days.

Other experiments have shown the complexity of theeffects of repeated exposures to carbamates. For example,Costa et al. (1981a) found that administering propoxur indrinking water decreased the activity of forebrain AChEonly in rats treated for 5 weeks but not for 2, 3, 4, and 6weeks. Kobayashi et al. (1988) also noted that a single injec-tion of propoxur (10 mg/kg, s.c.) resulted in a significantdecrease in the activity of AChE in the forebrain of micefor 180 min, but the repeated administration (5 mg/kg/day,s.c.) did not produce a significant change in activity. It hasalso been shown that chronic administration of rivastigmine, acarbamate that is used to ameliorate dementia in Alzheimer’sdisease, did not decrease the activity of AChE in the ratbrain regions studied (frontal cortex, hippocampus, striatum,and thalamus þ midbrain) (Tanaka et al., 1994). However,rivastigmine with an acute dose (0.35 mg/kg, intraperitone-ally, i.p.) reduced 40 to 50% AChE activity in the cortex andhippocampus of rats. Furthermore, rivastigmine at double thedose (0.7 mg/kg, i.p.), produced about 80% AChE inhibition

(Gupta and Dekundy, 2005, 2007). It is possible, therefore,that chronic administration of a carbamate may not producea uniform and predictable effect on brain regional AChEactivity even after recurring episodes of inhibition causedby repeated administrations. Chronic exposure to carbamatescauses reversible but recurring inhibition of AChE and mayinduce alterations in metabolic kinetics of the compounds(Tang et al., 2006), which may result in different activity ofAChE independent of the dose and frequency of exposure.These factors may explain the reason for inconsistent effectsof some carbamates on brain regional AChE activity follow-ing chronic exposure, while a carbamate compound like aldi-carb produces consistent effect on AChE activity (Gupta andKadel, 1991b).

1.4 EFFECTS ON RELEASE, SYNTHESIS, ANDSTORAGE OF ACETYLCHOLINE

As described previously, ACh released from the presynapticcholinergic nerve terminal is hydrolyzed into choline andacetic acid very rapidly. Since choline is a quaternaryammonium compound, its membrane permeability through

TABLE 1.2 Effect of Carbofuran Administration on the Activity of Acetylcholinesterase in Rat Brain

Acetylcholinesterase (nmol AcetylthiocholineHydrolyzed/min/mg Protein)

Lipid Peroxidation(nmol MDA/mg Protein)

Cerebral Cortex Cerebellum Brainstem Cerebral Cortex Cerebellum Brainstem

Control 148.97+ 6.62 125.66+ 10.94 107.46+10.48 2.06+0.22 2.59+0.22 2.02+0.21CF treated 49.26+ 4.10a 35.62+ 2.43a 35.88+2.86a 3.40+0.20a 3.45+0.35a 2.68+0.42a

NAC treated 154.83+ 8.43 131.10+ 4.44 116.75+3.84 2.11+0.09 2.70+0.12 2.13+0.10CF þ NAC treated 101.29+ 5.09a,b 75.83+ 10.06a,b 66.29+2.72a,b 2.16+0.23a,b 2.55+0.21b 2.16+0.12b

Note: CF, carbofuran; NAC, N-acetylcysteine; Values are expressed as mean+S.D., n ¼ 6.aSignificantly different from control group ( p , 0.05).bSignificantly different from carbofuran treated group ( p, 0.05).

Source: Adapted from Kamboj et al. (2006).

TABLE 1.1 Intensity of AChE Staining in Rat Brain Regions After 1 Week and 3 Weeks of Methyl ParathionRepeated Treatment

Brain Region

AChE Staining (O.D.)

1 Week Treatment 3 Week Treatment

Control MP3 (mg/kg) Control MP3 (mg/kg)

Striatum 0.55+0.03 0.21+0.02 (263%) 0.53+0.03 0.11+0.01 (280%)Frontal cortex 0.05+0.01 0.02+0.00 (266%) 0.02+0.00 0.00+0.00 (287%)Hippocampus 0.08+0.01 0.04+0.01 (254%) 0.05+0.00 0.01+0.00 (280%)Thalamus 0.14+0.01 0.05+0.01 (267%) 0.11+0.01 0.01+0.00 (287%)Midbrain 0.15+0.01 0.04+0.01 (274%) 0.11+0.01 0.02+0.00 (285%)

Note: Images of AChE histochemical staining (Figure 1.3) were analyzed with a digital scanning densitometer. Values are the Mean O.D.+SEM (n ¼ 7 or 8).Values in parentheses represent difference to control value in percentage at same region.

Source: Adapted from Sun et al. (2003).

1.4 EFFECTS ON RELEASE, SYNTHESIS, AND STORAGE OF ACETYLCHOLINE 9

the blood–brain barrier is very low, indicating that bloodis not the main source of choline for ACh synthesis.Therefore, choline derived from the hydrolysis of ACh inthe synaptic cleft is transported into the presynaptic nerveterminal by high-affinity choline uptake (or transport)(HACU or HACT). This high-affinity choline transporter(Apparsundaram et al., 2001; Okuda et al., 2000) is asodium ion- and chloride ion-dependent process with a Kmvalue of 1 to 2mM for choline and it is the rate-limitingstep in the biosynthesis of ACh (Kuhar and Zarbin, 1978).Following reuptake, choline and acetylcoenzyme A (pro-vided by mitochondria) are transformed into ACh by cholineacetyltransferase (ChAT) in the cytoplasm of the nerve term-inal. Although ChAT and HACU are specific markers ofcholinergic innervation in the CNS, the enzyme is notaccepted to be a rate-limiting step in the synthesis of ACh(Apparsundaram et al., 2001; Kuhar and Zarbin, 1978).

The newly synthesized ACh is subsequently incorporatedinto synaptic vesicles via the vesicular ACh transporter(VAChT) that is located in the vesicular membrane (Prioret al., 1992). VAChT exchanges luminal protons for cyto-plasmic ACh and concentrates ACh inside the synapticvesicles. It is well known that this action of VAChT is specifi-cally blocked by 2-(4-phenyl piperidino)-cyclohexanol(vesamicol) (Prior et al., 1992; Schuldiner et al., 1995).

Organophosphates and carbamates exert acute toxicityprimarily through persistent inhibition of AChE at cholin-ergic junctions, resulting in prolonged residence time ofACh within the synaptic cleft. Because of this persistentAChE inhibition, certain compensatory mechanisms arise tocombat excess ACh present in the synaptic cleft. Thesehomeostatic mechanisms involve modulation of both presyn-aptic and/or postsynaptic components of the cholinergicsynapse. These may involve changes in HACU, ChAT, andthe vesicular VAChT in the presynaptic terminal and modifi-cations of mAChRs and nAChRs for both the postsynapticand presynaptic components (Costa et al., 1981a, 1981b,1982a, 1982b; Kobayashi et al., 1986, 1997; Padilla, 1995;Richardson and Chambers, 2003, 2004, 2005; Russell andOverstreet, 1987; Schwab et al., 1981, 1983; Whalley andShih, 1988).

Several studies have reported consistent inhibition ofAChE by organophosphates. However, the effects of organo-phosphates on ChAT are shown to be variable, ranging fromno effects (Kobayashi et al., 1986; Sivam et al., 1984), someinhibition in PC cells (Jameson et al., 2006), to activation invitro (Brooks and Goldberg, 1979) or in vivo (Gupta et al.,1985; Khan et al., 2000). Furthermore, the effects of organo-phosphates on the cholinergic elements, such as AChE, ACh,ChAT, HACU, and VAChT, depend, at least in part, on devel-opmental level. Rats exposed in utero to methyl parathion(1.5 mg/kg, perorally, p.o. daily from day 6 through day 20of gestation) showed significant reduction in AChE activityand increase in ChAT activity in brain regions (cortex,

brainstem, striatum, and hippocampus) that persisted throughpostnatal day 28. Some studies have also shown that repeatedpostnatal exposure of rats to chlorpyrifos resulted in persistentbrain AChE inhibition and a decrease in ChAT activity in ratsexposed to the organophosphate on postnatal day 1 through 4.These changes persisted through postnatal day 30 and wereaccompanied by decreases in HACU levels (Dam et al.,2000; Slotkin et al., 2001). It was also reported that gesta-tional exposure to chlorpyrifos results in persistent reductionsin ChAT activity, HACU levels, and also vesicular AChtransporter (VAChT) levels (Richardson and Chambers,2003, 2004). Therefore, it is suggested that presynapticcholinergic neurons may be especially vulnerable to earlypostnatal and gestational exposure to organophosphates likechlorpyrifos.

Avariety of acute neurotoxic effects of sarin, in addition tothe AChE inhibition, have also been studied in rats (Khanet al., 2000). The animals were treated with a single intramus-cular injection of sarin at 0.01, 0.1, 0.5, or 1 � LD50 andsacrificed 0.5, 1, 3, 6, 15, or 20 h later. Brain regionalAChE activity was inhibited (44% to 55% of control)30 min after the LD50 dose and it remained inhibited for upto 20 h. ChAT activity was increased in the cortex, brainstem,and midbrain 6 h after the LD50 dose and the elevated activitypersisted up to 20 h after treatment. Midbrain and brainstemseemed to be most responsive to sarin treatment at lowerdoses as these regions exhibited inhibition (�49% and10%, respectively) in AChE activity 20 h following 0.1 �LD50 treatment. Cortical ChAT activity was significantlyincreased following a 0.1 � LD50 dose, whereas the activityin the brainstem and midbrain did not show any effect atthis lower dose. The authors speculate from various reportsand their previous findings that increased ChAT activity fol-lowing sarin exposure may be a consequence of protease-mediated activation of the enzyme (Khan et al., 2000).

Microdialysis is a useful tool with which to investigatethe extracellular release of neurotransmitters successivelyevery 10 min or so in the brains of awake freely moving ani-mals (Bradberry, 2000; Chang et al., 2006; Mas et al., 1996;Westerink, 1995; Young, 1993). Microdialysis methods havebeen used frequently to investigate the effect of drugs onACh, especially those that have been proposed for the treat-ment of Alzheimer’s disease. However, there is a paucity ofstudies that have utilized this method to evaluate the effectsof toxicological exposure to organophosphates and carba-mates on the release or extracellular level of ACh (Bueterset al., 2002; Karanth et al., 2007). Intracerebral microdialysisstudies in rats have shown that sarin (0.144 mg/kg, intramus-cularly, i.m.) and VX (0.024 mg/kg, i.m.) produced an11-fold and 18-fold increase in ACh levels, respectively, 10minutes after exposure (Bueters et al., 2002). Parathion atdoses of 6, 9, 18, and 27 mg/kg, s.c. dose-dependently elev-ated extracellular ACh levels in the striatum of adult (three-month-old) and aged (18- to 19-month-old) rats 3 and 7 days

10 ACETYLCHOLINESTERASE AND ACETYLCHOLINE RECEPTORS: BRAIN REGIONAL HETEROGENEITY