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LD5655.V856 1991.N677.Pdf (9.869Mb) DEVELOPMENT OF A MODEL CELL CULTURE SYSTEM IN WHICH TO STUDY EARLY EFFECTS OF NEUROPATHY-INDUCING ORGANOPHOSPHATES by Amy Carol Nostrandt Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Veterinary Medical Sciences APPROVED: Marion Ehrich, Chairperson Bernard2. I,S. Jortner Teresa K. Rowles Jez lif, ty R Wheke Brian Storrie “ JéefR. Wilcke September, 1991 Blacksburg, Virginia DEVELOPMENT OF A MODEL CELL CULTURE SYSTEM IN WHICH TO STUDY EARLY EFFECTS OF NEUROPATHY-INDUCING ORGANOPHOSPHATES by Amy Carol Nostrandt Committee Chairperson: Marion F. Ehrich Veterinary Medical Sciences (ABSTRACT) Certain organophosphorus (OP) compounds produce a delayed neuropathy in man and susceptible animal species after early inhibition and aging of the enzyme, neurotoxic esterase (neuropathy target esterase, or NTE). In this study, the human neuroblastoma cell line, SY-SY, was examined for its potential to serve as a nonanimal model for the study of the early effects of neuropathy-inducing OPs. For these investigations, the time course of inhibition and aging of NTE after toxicant treatment in neuroblastoma cells was compared to that in brain tissue from the adult chicken, which is the recognized animal model for organophosphorus ester-induced delayed neuropathy (OPIDN). Concentrations of toxicants to be used for treating neuroblastoma cells were determined after observing viability of treated differentiated SY-SY cells incubated for 24 hours with a range of concentrations of an OP (mipafox) that induces neuropathy, an OP (paraoxon) that does not induce neuropathy, a carbamate (aldicarb), a neurotoxicant (8, 8’-iminodipropionitrile, or IDPN) that acts by a different mechanism than the OPs, and a cholinergic agonist (carbachol). Treatment concentrations were chosen that caused less than 30% loss of viability over the 24 hour period. The time course and extent of detrimental effects of mipafox, which induces OPIDN in hens, and the carbamate, aldicarb, on NTE were similar in the SY-S5Y cells to those observed in homogenized chicken brain tissue after the same treatments. Mipafox produced rapid inhibition and aging of NTE, with maximal effects occurring within 10 minutes of exposure. Aldicarb inhibited NTE but did not age the enzyme. Instead, spontaneous reactivation was observed both in SY-5Y cells and in brain tissue. None of the other negative control compounds (paraoxon, IDPN, carbachol) affected NTE activity in either SY-SY cells or chicken brain tissue. To determine if the neuroblastoma cells could be used to study early events that could lead to modification of OPIDN, NTE inhibition and aging were determined in the differentiated SY-SY cell line after mipafox was removed. Removal of mipafox from the cell culture medium at 5 minutes after exposure, or earlier, resulted in essentially no NTE inhibition or aging. NTE inhibition and aging were also determined after treatment of the SY-S5Y cells with the neuropathy-inducing OP, mipafox, and representatives of 2 classes of compounds (carbamates and calcium channel blockers) previously demonstrated to modify OPIDN in hens. The modifiers (aldicarb and verapamil) were used as a 5 minute pre-treatment, simultaneous treatment, and a 2 minute post-treatment. Significant prevention of most of mipafox-induced NTE inhibition and aging was observed. Effects on NTE inhibition and aging in differentiated SY-5Y cells after each mipafox-aldicarb combination and mipafox-verapamil combination of treatments were similar to those in chicken brain homogenate. These results indicated that both aldicarb and verapamil protected NTE against the early biochemical effects of mipafox that are thought to initiate OPIDN in vivo. The temporal relationship of NTE inhibition and aging to other detrimental effects on neuroblastoma cells was assessed by the capability of mipafox to cause changes in free intracellular calcium ion concentration, measured using fluorescent calcium probes, and by its capability to alter cell morphology as assessed by phase contrast microscopy. NTE inhibition and aging preceded these changes. Capability to inhibit activity of the neural enzyme, acetylcholinesterase, was also determined. The results of these studies indicate that the SY-SY model system shows promise for use in the determination of initial mechanisms contributing to the development of organophosphorus- induced delayed neuropathy. ACKNOWLEDGEMENTS I would like to express my gratitude to my advisor, Dr. Ehrich, for her patience, encouragement and support in the completion of my doctoral program. I would also like to thank her and the other members of my committee, Dr. Huber, Dr. Jortner, Dr. Rowles, Dr. Storrie, and Dr. Wilcke, for their guidance and assistance throughout my graduate studies. Thanks also to Dr. Ludeman Eng, Linda Correll, Karen diLorenzo, Joan Kalnitsky-Aliff, and Haywood Pyle for their assistance and support. Finally, I would like to thank my friends and family for being there when I needed them. ACKNOWLEDGEMENTS iV TABLE OF CONTENTS Page ABSTRACT. ou. cssssssecesessesecsccessssessssscecescesscsssesecnseasecesssssensees ii ACKNOWLEDGEMENTS ..........ccessssssssssecessssecessensscsesessesecscocsesesssaessesansoseeees ves . iv TABLE OF CONTENTS au... csesssssessssecssseccscsessssesscssceessesecsacscseceasscsaceecsesesecessesseasseseesnssees Vv LIST OF TABLES. o.oo ccseececcescnsessesscsecsenscssccesensoessescesssesecsacsesesserecseseosssvasseseateesossesoees vill LIST OF FIGURES oo esssececsssseseccecensessaseccecsescessnsssceacssserenssseoes ix ABBREVIATIONS ou... cssssssssssserssrescsecessecscesescsssscateccsesseccacsccsesesseseacensacecsneseesesseseacanseseseenass x PART I. INTRODUCTION TIYPOTHESIS ooo eccccscssecssescsssesssecescesecescessceessesceseceeseccessccensscessaseseesscscesssscasscecsscesossssoosass 2 LITERATURE REVIEW 0... esessscscsscsscsccecssssccssencscsrsscessstescsecsscscecenseacsacecesseaesnsacsesensensasens 4 Historical background ...........cssssssssssssssssssesesenescecsenecsesccecssssessesees 4 Organophosphate toxicity ........cccssssssescseesecesecsetenessensessesetseseeseeees 4 Clinical signs Of OPIDN ..........sccssssssecssssssesereensecseceesececececeecseeneeeoes 5 Pathological aspects Of OPIDN uu... esesecessscecsetcssesesescsceseesesecees 6 The chicken as an animal model for OPIDN ...........csssssssseeseeeeee 7 NTE and the initiation of OPIDN uc eseesscesscessssssssneneeseeesers 7 OP effects determined using in vitro systems ............scsceceeeeeeeees 10 Development of OPIDN and the role of Ca** in neurotoxicity. 13 References ......ccsssesessscecsssesscessesecsscecscseessscssssesecsucseseceessscensneseenessesenes 18 PART II. MATERIALS AND METHODS MATERIALS AND METHODS ..... ce ecssssssessessessscsecessssssessssescsssssecsesscsseasatensareneneseeersees 23 References .........ccccccsccccssscsscccscccosssccescsscscccsssccsesscssscsssssscssccesesssessesesces 32 TABLE OF CONTENTS PART II. RESULTS INTRODUCTION TO RESULTS ou. cescssesesseesseresseecsesseees 34 CHAPTER I. DEVELOPMENT OF A MODEL CELL CULTURE SYSTEM IN WHICH TO STUDY EARLY EFFECTS OF ORGANOPHOSPHORUS ESTERS ........essssscecesssssseseeseecececscacssseseseees 36 SUMMALY ........sscesscscssssccsscscsasersecsseseessoessenscssesssssessesssssesessessesessessesaees 37 Trntroduction ...........cssssscsssssssssssceceessececesecesessssensnssssessssessssssseesssssenenees 37 Materials and Methods ..............sc0e00 39 Results o......cesscesecscesssecssesssecscsseseeseecscecsescecsesencsssensnesensececeasceceesseseeseses 41 DISCUSSION. .......ccsssssessesseesscstecscecscacsrecerscscersencccesscersescavatseenesseseensnseseees 43 References ........csssssssssssssssesssscssssssecscececeeeseccncececscecenecessersesessseseseenese® 46 CHAPTER II. MODIFICATION OF MIPAFOX-INDUCED INHIBITION AND AGING OF NEUROTOXIC ESTERASE IN NEUROBLASTOMA CELLS oun eesssescssscecesenesesceesececesesscseecscececeeececscesecscesecsesseseneseseseceseseeesees 55 SUMIMALY ....cseeecscscssssscsecssecesssssnesssesscessesseeseessssasssesesseseessceseseesseeseeees 56 Tintroduction ..........ccccsssssssssessssssssessecesesseececseseccsececnesesesecececesecsesessneseees 56 Materials and Methods ............cscssscssscessssecesececesecsesssseeeecncerscesnceeees 58 ReSUlts oo... eseseescesccscesescsecececscncsesstcessesesececscaesseesesecssessesessseseenenseeees 60 DiSCUSSION .........ssscssssescscesscsssesssessesecesesscnesesenecncecsessaeseseseesceeseacotensseces 62 References 0... .ececssssssssssssssssencsesscecscscsesscseseessssesssecscseacsenesesessenesenenses 67 CHAPTER III. CYTOTOXIC EFFECTS OF ORGANOPHOSPHORUS ESTERS AND OTHER NEUROTOXIC CHEMICALS ON CULTURED CELLS wove cccsscccscssssesssecsesssccsccsccssscsesssssssccsssscssssscccesssssosssesscesscscessesseeses 73 SUMIMALY 000... eeeeceecssssssscnsececcscscsccacecececesscsenessessscensnseneesssseesessescanensanees 74 Introduction co.ccccccccccccsccscecccscssscsscssscsssesscesseccssscssessessccsesssscsscssecceas 74 TABLE OF CONTENTS Materials and Methods ...........ccccccssssssscsssscesssscssscsesscessecesscsscscssoseee 75 ReSUullts 0... ecsssscscscececessesseeccececssssssessssssessnsocscececessscscscesceanacssecerseees 78 DISCUSSION .........sssscscsssecesceesssseceesesesseseeneas . 80 REfEreNCES ........cecssesesssesscnsesscscecscscscasecscsescacsesencecs 86 APPCNGix o.0.......escsscssscscssessesscssesssesecenssensnssesesesesesecesssesesccesecnscaeasesases 96 DISCUSSION uu. tecccsssccsscssscsscccsscnssscescscsssssscsscsssessccsscccsscsesssssssecscsssssessoesecssssosssscssscsscossesases
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