Investigation of the pathogenesis caused by exposure to varying doses of VX nerve agent in rat with special reference to cardiotoxicity

INVESTIGATION OF THE PATHOGENESIS CAUSED BY EXPOSURE TO VARYING DOSES OF VX NERVE AGENT IN RAT WITH SPECIAL REFERENCE TO CARDIOTOXICITY FONG XIAO JUN (B.Sc (Hons)), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ANATOMY NATIONAL UNIVERSITY OF SINGAPORE 2006 To my Family ACKNOWLEDGEMENTS I would like to express my sincere appreciation and gratitude to my supervisor, Professor P Gopalakrishnakone, Department of Anatomy, National University of Singapore, for his patient guidance, encouragement and support through the course of the whole project, as well as for his expertise in research I would like to thank Professor Ling Eng Ang (Head of Anatomy) for his kind support and concern during my study I also wish to express my gratitude to Dr Loke Weng Keong, DSO National Laboratories, Singapore, for his kind assistance and valuable expertise in the area of nerve agent research I would like to thank Dr Lee Fook Kay, DSO National Laboratories, Singapore, for his kind concern and support Special thanks shall be extended to all staff of DSO National Laboratories, Singapore, particularly Mdm Soh Poh Chiang, Emily, Mdm Chang May Ling, Joyce and Miss Tan Yong Teng for their invaluable help and assistance in my experiments and laboratory work I wish to specially thank the staff of Department of Anatomy, Mrs Ng Geok Lan, Mrs Yong Eng Siang, Mdm Manomani and Mdm Thenmozhi of the Histology Laboratory; Miss Chan Yee Gek of the Electron Microscopy Unit; Mdm Bay Song Lin, Mr Low Chun Peng and Mr Yick Tuck Yong of the Multimedia Development Unit for their technical help and patient assistance I would also like to I thank Mdm Teo Li Ching, Violet, Mdm Diljit Kour d/o Bachan Singh and Mdm Ang Lye Gek, Carolyne of the general office for their kind assistance and helpful advice in all administrative-related work I would like to thank all academic staffs and postgraduate students in the Department of Anatomy for their caring support and encouragement I wish to thank fellow research colleagues of Venom & Toxin Research Programme, Miss Hema d/o Jethanand, Dr Pachiappan Arjunan, Dr Maung Maung Thwin, Dr R Perumal Samy and Dr Ramasamy Saminathan, for their kind and friendly support Finally, I hope to take this opportunity to express my gratitude and appreciation to my family members for their endless support and encouragement during my period of postgraduate study They have been a constant inspiration and have seen me through this important and valued phase of my academic pursuit II TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY PUBLICATION LIST OF ABBREVIATIONS CHAPTER ONE I III VII IX X INTRODUCTION 1.1 Chemical warfare nerve agents 1.1.1 Introduction 1.1.2 Incidents of nerve agent use 1.1.3 Mechanism of action 1.1.4 Pathophysiology and effects on organ systems 1.1.5 Routes of exposure 1.1.5.1 Inhalation 1.1.5.2 Absorption through skin and mucous membranes 1.1.5.3 Ingestion 1.1.6 Chemical properties 1.1.7 Decontamination and treatment 2 4 9 11 12 12 15 1.2 Effects of VX (O-ethyl-S-2 diisopropylaminoethyl-methyl phosphonothiolate) and other nerve agents 18 1.2.1 Histopathological findings 18 1.2.2 Electrocardiographical studies 24 1.3 Aims and significance of present study 28 CHAPTER TWO 32 MATERIALS AND METHODS 2.1 Supply of nerve agent VX 33 2.2 Chemicals and equipments 33 2.3 Animals 35 2.4 Biochemical analysis 2.4.1 Acetylcholinesterase assay 2.4.2 Creatine Kinase – MB test 35 36 37 2.5 Injection of nerve agent VX and drug treatment 37 III 2.6 Acute and chronic VX exposure protocols and observation of clinical signs and symptoms of intoxication 38 2.7 Electrocardiographical studies 2.7.1 Telemetry system 2.7.2 Physiological parameters and calibration values 2.7.3 Implantation of electrocardiography transmitter 2.7.3.1 Surgical procedure 2.7.3.2 Post-operative regimen 2.7.4 Recording of electrocardiography signals and measurement of electrophysiologic parameters 2.7.5 ECG recording schedule for toxicity tests 40 40 41 42 42 46 2.8 Histopathological studies 2.8.1 Perfusion and fixation 2.8.1.1 Preparation of fixative 2.8.1.2 Perfusion system 2.8.1.3 Perfusion pressure 2.8.1.4 Anaesthesia of animals 2.8.1.5 Perfusion 2.8.2 Processing of tissue specimens for light microscopy 2.8.2.1 Fixation and dehydration 2.8.2.2 Paraffin embedding 2.8.2.3 Microtome sectioning 2.8.2.4 Haematoxylin and Eosin (H & E) staining 2.8.2.5 Observation with light microscope and histologic scoring 50 50 50 51 51 52 53 54 54 54 55 55 56 2.9 Statistical analysis 57 CHAPTER THREE OBSERVATIONS AND RESULTS 46 49 58 3.1 Acute 1.6 LD50 VX with drug treatment 3.1.1 Clinical observations after injection 3.1.2 Weight profile 3.1.3 Acetylcholinesterase activity 3.1.4 Histopathological changes 3.1.4.1 Cardiac muscle 3.1.4.2 Kidney 3.1.4.3 Skeletal muscle 3.1.4.4 Liver 3.1.4.5 Lungs 59 59 61 63 64 64 72 76 80 83 3.2 Acute LD50 VX injection 3.2.1 Clinical observations after injection 3.2.2 Weight profile 87 87 89 IV 3.2.3 Acetylcholinesterase activity 3.2.4 Time study profile of histopathological changes 3.2.4.1 Cardiac muscle 3.2.4.2 Kidney 3.2.4.3 Skeletal muscle 3.2.4.4 Lungs 3.2.4.5 Liver 3.2.5 Correlation between cardiac damage and convulsions 3.2.6 Electrocardiography 3.2.6.1 Day of exposure 3.2.6.1.1 Physiologic and electrocardiographic data 3.2.6.1.2 Cardiac rhythm 3.2.6.2 Post exposure -14 days 3.2.6.2.1 Physiologic and electrocardiographic data 3.2.6.2.2 Cardiac rhythm 91 92 94 99 102 104 106 106 107 107 107 114 124 124 132 3.3 Chronic 0.4 LD50 VX injections 3.3.1 Clinical observations after injection 3.3.2 Weight profile 3.3.3 Acetylcholinesterase activity 3.3.4 Histopathological changes 3.3.4.1 Cardiac muscle 3.3.4.2 Kidney 3.3.4.3 Skeletal muscle 3.3.4.4 Lungs 3.3.4.5 Liver 3.3.5 Correlation between cardiac damage and convulsions 3.3.6 Electrocardiography 3.3.6.1 Physiologic and electrocardiographic data 3.3.6.2 Cardiac rhythm 137 137 141 143 145 147 147 148 148 149 149 151 151 162 3.4 Creatine kinase – MB activity 165 CHAPTER FOUR 167 DISCUSSION AND CONCLUSION 4.1 Histopathological findings in acute and chronic VX poisoning 168 4.2 Electrocardiographical changes in acute high dosage and chronic low dosage VX challenges 175 4.3 Conclusions 178 4.4 Directions for further research 180 V REFERENCES 182 VI SUMMARY Cardiotoxicity was investigated in rats challenged with an organophosphorus chemical warfare nerve agent VX, S-(2-diisopropylaminoethyl)-O-ethylmethyl phosphonothiolate A paucity of literature on chronic low dose VX challenges and electrocardiographic investigations in VX exposure necessitate the need for research in the respective areas Three groups of rats followed three different dosing protocols and various organs in addition to cardiac tissues were examined for histopathological changes Cardiotoxicity was determined by electrocardiography studies as well Assays for acetylcholinesterase activity were performed to determine the level of enzyme inhibition due to VX Acute single doses of 1.6 LD50 VX were injected subcutaneously into rats which received treatment drugs namely pyridostigmine bromide, atropine methyl nitrate and pralidoxime chloride to ensure survival All animals presented tonic-clonic convulsions Light microscopic observations of the cardiac muscles revealed severe myocardial damage such as mononuclear cellular infiltration, myofibre degeneration and necrosis in all of the VX-challenged rats However, morphological changes in other organs were minimal In the group of rats that received acute dosages of LD50 VX in the absence of drug treatment, light microscopy examinations were carried out on day 1, 2, 5, and 14 following intoxication to capture the time profile of the appearance of organ VII damages The degree of myocardial degeneration and necrosis were moderate up to day and reparation of the cardiac lesions was detected as early as day postexposure Myocardial lesions were rarely observed on day 14 post-intoxication As in the 1.6 LD50 VX group, injuries in other organs were infrequently observed Electrocardiography recordings revealed QTc and PR interval prolongations in addition to abnormalities in cardiac rhythm (atrioventricular blocks, arrhythmias and ‘Torsade de pointes’ ventriclar tachycardia) in the intoxicated rats The electrocardiographic irregularities were detected up to day 14 post-injection In the third dosing regimen, chronic low doses (0.4 LD50) of VX were administered subcutaneously daily up to days Clinical symptoms of nerve agent intoxication started appearing from the fourth day of injection The rats were perfused for histologic evaluations 24 hours after designated dosing days (dosing day 1, 3, and 8) Histopathological studies revealed the presence of myocardial damage on the fourth and eighth days of dosing Histopathological changes in other organs were uncommon Electrocardiography measurements demonstrated cardiac arrhythmias as early as the second day of dosing, before the appearance of clinical symptoms specific for nerve agent intoxication Significant lengthening of QTc and PR intervals was evident from the first day of injection The aberrations in cardiac rhythm and QTc prolongations were reproducible up to days after the last dosing day i.e dosing day This is a novel finding in the research of cardiac toxicity of nerve agents VIII Several possible mechanisms through which increased concentrations of catecholamines could bring about cardiotoxicity have been proposed (Behonick et al., 2001) In summary, hypoxia, hemodynamic modifications, decreased coronary blood flow, metabolic changes, membrane permeability alterations resulting in electrolyte imbalances and disturbances in calcium homeostasis leading to overloading of intracellular levels of calcium ions were suggested as contributory factors to cardiotoxicity Most of the suggested mechanisms attributed cardiac myofibre necrosis to the deficit of energy supply that was essential for crucial cellular processes maintenance In addition, inadequacy of hemodynamic mechanisms such as alterations in coronary vascular resistance and coronary blood supply inefficiency might have produced ischemic conditions, paving the way to cardiac cell necrosis The decreased coronary blood flow on top of increased coronary vascular resistance could also lead to eventual coronary spasm, which consecutively triggered cardiac arrhythmias and infarction (Behonick et al., 2001) Reduction in mean arterial pressure in dogs (Robineau and Guittin, 1987b) as well as marked hypotension in rabbits (Preston and Heath, 1972) upon subcutaneous VX administration had indeed been reported Thus, the proposed catecholamine-initiated mechanism through which eventually arrhythmia develops is indeed plausible Oxidative stress elements such as free radicals and aminochromes were postulated to work in concert with catecholamines in the initiation of cardiotoxicity (Dhalla et al., 1987) Evidence supporting catecholamine- and aminochrome- induced cardiotoxicity in nerve agent poisoning was presented by Tryphonas and coworkers (1996) where myocardial 174 damage brought about by soman intoxication was shown to strongly resemble catecholamine-induced cardiac injury at the ultrastructural level 4.2 ELECTROCARDIOGRAPHICAL CHANGES IN ACUTE HIGH DOSAGE AND CHRONIC LOW DOSAGE VX CHALLENGES Electrocardiographic recordings were performed separately in two groups of rats which received different VX dosing regimens: (1) acute single exposure to LD50; (2) chronic daily 0.4 LD50 VX injections for days Acute LD50 VX exposure investigations produced results that tied in with the current literature on ECG findings in acute nerve agent and organophosphorus insecticide poisoning (Ludomirsky et al., 1982; Robineau, 1987a) Intense increase in parasympathetic tone which manifested as sinus bradycardia, AV conduction disturbances and arrhythmias was revealed in the high dosage VX-challenged animals in this study Instability in AV conduction pathways presented in the form of first- and second- degree AV blocks were detected as early as 21.7 ± 2.8 post injection Series of short-long ECG cycles that occurred in succession to extreme prolongations were detected repeatedly Malignant torsades de pointes (TdP) ventricular tachycardia followed the cardiac arrhythmias Parallel to QTc interval prolongation reported in nerve agent sarin and soman intoxication (Abraham et al., 2001; Allon et al., 2005) and organophosphorus insecticide poisoning (Saadeh et al., 1997; Karki et al., 2004), QTc interval prolongation was discernible in the VX-exposed rats up to 120 post injection Additionally, distinct lengthening of the PR segment was revealed for 120 175 following injection Impaired conduction through the AV node has been documented as a common cause for PR interval prolongation (Conover, 1996), thus justifying the occurrence of AV blocks in the animals in the present study First-degree AV blocks had been reported in LD50 VX-challenged beagle dogs which exhibited PR segment lengthening in the ECG traces (Robineau and Guittin, 1987b) It is certain that VX poisoning can produce disturbances in atrioventricular conduction Due to technical limitations in ECG devices, ECG investigations in organophosphate compounds intoxication animal studies hitherto either used anesthetised animals or were designed as a one-time measurement where the animals were sacrificed after the procedure Constraints on follow-up recordings after exposure were present for the latter In the former case, anaesthetic procedure was speculated to have caused interference in cardiac rhythm as cardiac abnormalities had been described in anesthetised control animals (Robineau, 1987a) The use of ECG implants in this study facilitated the continual monitoring of ECG changes in conscious animals for days after VX challenge Cardiac arrhythmias including second degree AV blocks, short-long ECG cycles and immense sustained prolongations between ECG waveforms were reproducible up to 14 days post LD50 VX exposure Analysis of the ECG traces revealed evident extension of QTc duration for as long as 14 days following intoxication These results are consistent with reports of accidental human exposure showing QT segment prolongation for a period of duration of to weeks (Chuang et al., 1996; Allon et al., 2005) In addition, Abraham and coworkers 176 had reported QTc prolongation in rats for up to months post acute 0.9 LD50 sarin or soman exposures (Abraham et al., 2001) The present study investigated the presence of cardiotoxicity in chronic exposure to low levels of VX, an area of research where not much has been reported so far Cardiac rhythm disturbances emerged in 50% of the rats by dosing day after administration of the second 0.4 LD50 VX dose The proportion exhibiting cardiac arrhythmias grew to 100% by the fourth day of injection Subsequently, the rats exhibited recurrent cardiac rhythm irregularities up till the last day of injection (i.e dosing day 8) Cardiac abnormalities observed in the chronically dosed rats included sinus bradycardia, first- and second-degree AV blocks, short-long ECG cycles, extreme pauses between QRS complexes as well as significant QTc and PR interval prolongation The evidence further corroborated the conclusion drawn from histopathological and clinical observations, that toxic effects of VX are cumulative and that chronic low level VX exposure eventuates in cardiotoxicity More importantly, occurrence of the cardiac rhythm aberrations arose before appearance of specific symptoms of intoxication (i.e tonic-clonic convulsions) This is the first report demonstrating cardiac arrhythmias in animals displaying minimal signs of poisoning in the area of nerve agent research as ECG studies of nerve agent have only used convulsive doses of nerve agent i.e greater than 0.5 LD50 to date (Robineau, 1987a; Robineau, 1987b) This data verified the proposition that cardiotoxicity arising from VX intoxication is independent of convulsive activity and is a disparate entity distinct from neurotoxicity 177 Extension of QTc duration and arrhythmias were sustained in the intoxicated rats up till days after the last injection This demonstrated that akin to acute high dosage VX intoxication, cardiotoxicity arising from repeated low dose VX exposure is long-standing Consequential adverse repercussions of lengthened repolarisation phase of the ventricular action potential manifested as QTc prolongation is clinically significant as it could generate the development of polymorphic ventricular tachycardia and delayed sudden cardiac death (Ludomirsky et al., 1982; Mancuso et al., 2004) Poor prognosis such as higher respiratory failure incidence, frequency of ventricular premature contraction and mortality rate has also been reported in organophosphate-poisoned patients demonstrating QTc prolongation (Chuang et al., 1996; Grmec et al., 2004) Hence, the incessant QTc lengthening detected post acute and chronic VX challenge in this present study suggests the necessity for continual ECG monitoring in instances of nerve agent exposure in humans 4.3 CONCLUSIONS Histopathological changes in various organs of VX-challenged rats had been investigated in high level acute and low level chronic poisoning Results revealed VX to specifically instigate myocardial damage and produce distinctive myofibre necrosis and inflammatory mononuclear cellular infiltration This study also demonstrated that cardiomyopathy was exacerbated with higher doses of VX 178 By a combination of light microscopic study and ECG monitoring, chronic low dose VX exposure was demonstrated to precipitate cardiotoxicity in the form of cardiomyopathy and cardiac arrhythmias Toxic effects of VX can therefore be deduced as cumulative Appearance of myocardial injury surfaced after dosing day and became more evident after dosing day However, arrhythmias manifested as early as the second day of injection Through systematic comparison between severe symptoms of intoxication (i.e tonic-clonic convulsions with histopathological changes in the cardiac muscle), both 0.4 LD50 and LD50 VX experiments showed that cardiotoxicity is not related to convulsive activity and therefore not neurogenic in origin This proposition is further supported electrophysiologically with the observation that cardiac arrhythmia and QTc prolongation in the chronic 0.4 LD50 poisoned animals arose prior to manifestation of convulsions Both high dose acute and low dose repeated VX exposure produced longlasting irregularities in cardiac rhythm such as short-long ECG cycles and QTc prolongations The rhythm aberrations were reproducible up to 14 days post LD50 VX injection Anticholinergic drug atropine was administrated with 1.6 LD50 VX in acute intoxication and the severe myocardial injury observed in the rats invalidates the hypothesis that VX-induced cardiomyopathy is a result of overstimulation by acetylcholine accumulation and that anticholinergic treatment in VX exposure protects from myocardial damage Moreover, acetylcholinesterase 179 assays showed that non-treated LD50 VX-challenged animals presented with less severe myocardial injury had more inhibited acetylcholinesterase activity compared to atropine-treated 1.6 LD50 VX-exposed rats which possessed less inhibited acetylcholinesterase activity 4.4 DIRECTIONS FOR FUTURE RESEARCH Mechanisms by which organophosphates induce cardiotoxicity deserve further investigations The present study has shown that the genesis of cardiac lesions and ECG complications from VX exposure not originate from neurogenic cause nor are due to acetylcholine accumulation Catecholamines and its oxidative derivatives (aminochromes) remain very feasible candidates as genuine biochemical initiators of cardiotoxicity Catecholamine assays could be incorporated in toxicology tests to detect the level of plasma noradrenaline and adrenaline as well as aminochromes in the circulation to determine if a correlation between cardiotoxicity and elevated catecholamine and aminochrome levels exists Chronic low level 0.4 LD50 VX exposure giving rise to cardiotoxicity has been demonstrated in this study ECG and histological studies involving chronic challenges to lower concentrations of nerve agent can be designed to establish the maximally tolerated or no-adverse-effects-level dose where toxic effects on the heart will not be observed The information will be valuable to incident responders of chemical warfare agent attacks for the demarcation of 180 safety zones around a nerve agent incident site and for subsequent reopening of the area to public 181 REFERENCES 182 REFERENCES Aaron CK and Howland MA (1990) Insecticides: Organophosphates and carbamates In: Goldfrank's Toxicologic Emergencies 6th edition, pp 1429-1448, Goldfrank LR, Flomenbaum NE, Lewin NA, Weisman RS, Howland MA and Hoffman RS (Eds) Stamford, Ct: Appleton & Lange Abraham S, Oz N, Sahar R and Kadar T (2001) QTc 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Thomas RG (2000) In: Advanced hazmat life support provider manual, 2nd Edition, pp 279–304, Tucson, AZ, Arizona Board of Regents Yates JC and Dhalla NS (1975) Induction of necrosis and failure in the isolated perfused rat heart with oxidized isoproterenol J Mol Cell Cardiol 7: 807-816 Zorpette G and Frank SJ (1998) Patent blunder Sci Am 279: 42 188 ... injection of VX was performed by pinching a bit of skin between the fingers to form a fold and the needle was inserted into the skin at the bottom of the fold where the loose skin meets the body... study aims to investigate possible cardiac toxicity in continual exposure of low doses of nerve agent VX Similar to other nerve agents, the effects of VX are cumulative and chronic exposure to low... abnormalities in rats following challenges to single high doses of VX (Robineau, 1987a) Rats anaesthetised with pentobarbital were subcutaneously injected with 12µg/ml or 0.76 LD50 of VX The LD50 in the