THE MOLECULAR MECHANISMS of ORGANOPHOSPHORUS COMPOUND-INDUCED CYTOTOXICITY Kent Richard Carlson
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THE MOLECULAR MECHANISMS OF ORGANOPHOSPHORUS COMPOUND-INDUCED CYTOTOXICITY Kent Richard Carlson 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 Marion Ehrich, Co-Chairperson Bernard S. Jortner, Co-Chairperson Brad Klein Jeff Bloomquist Mitzi Nagarkatti May 19th, 2000 Blacksburg, Virginia Keywords: Organophosphorus, SH-SY5Y, cytotoxicity, apoptosis Copyright 2000, Kent R. Carlson THE MOLECULAR MECHANISMS OF ORGANOPHOSPHORUS COMPOUND-INDUCED CYTOTOXICITY Kent Richard Carlson (ABSTRACT) Certain organophosphorus compounds have the ability to induce a delayed neuropathic condition in sensitive species termed organophosphorus compound-induced delayed neurotoxicity (OPIDN). Esteratic changes associated with OPIDN have been successfully modeled in vitro. The physical characteristics of lesion development in OPIDN including the mode of nerve cell death, cytotoxic initiator and effector molecules, and cytoskeletal involvement have received little in vitro investigation. This dissertation evaluated the mode of cell death (apoptosis versus oncotic-necrosis), and cell cycle, cytoskeletal, nuclear, and mitochondrial alterations induced by OP compounds in SH-SY5Y cultures, an in vitro human neuroblastoma model. The distribution of in vivo neural degeneration in white Leghorn hen models was also assessed as a prelude to validating the mode of OP compound- induced in vivo neural cell death. These endpoints were evaluated by utilizing flow cytometry, spectrophotometry, gel electrophoresis, immunohistochemistry, light, and electron microscopy. Initial data gathered on culture parameters revealed that the mitotic status, basal rates of cell death, and total culture density were dependent on the condition of the media and the initial seeding density. Subsequent in vitro investigations used standardized culture conditions with OP compounds (diisopropylphosphorofluoridate (DFP), paraoxon, parathion, phenyl saligenin phosphate (PSP), tri- ortho-tolyl phosphate (TOTP), and triphenyl phosphite (TPPi); 1mM - 1mM). These studies revealed that OP compounds altered the cell cycle phase, decreased the amount of intracellular f-actin, altered the mitochondrial membrane potential, and induced caspase-3 activation and nuclear partitioning characteristic of apoptosis. The amount of change in these parameters was strongly dependent on the OP compound, the length of incubation time, and the presence of modulators of cytotoxicity such as phenylmethylsulfonyl fluoride (PMSF), carbachol, Ac-DEVD-CHO, Ac-IETD-CHO, and cyclosporin A. Preliminary in vivo experiments designed to validate in vitro results revealed neural degeneration involving fibers, terminals, and cell soma in spinal cord and brain tissue of PSP- and TPPi- exposed hens. The distribution and magnitude of these changes were contingent on the OP compound and length of time post-exposure. Subsequent experiments designed to evaluate the mode of cell death in these tissues revealed little evidence of either necrosis or apoptosis. These results, therefore, did not support or refute in vitro observations. Many OP compound-induced subcellular alterations have been demonstrated in our in vitro SH-SY5Y neuroblastoma model. Even though the mode of cell death observed in SH-SY5Y cells was not validated in in vivo experiments, in vitro observations nonetheless provide stimulating areas to further research the mechanisms of OPIDN and OP compound-induced cell death. Funding for the research involved in this dissertation was provided by a Novartis - Society of Toxicology graduate student fellowship, US EPA grant R825356, and the Virginia-Maryland Regional College of Veterinary Medicine. ACKNOWLEDGMENTS I would like to express my sincerest gratitude to my advisors, Dr. Marion Ehrich and Dr. Bernard Jortner. Both have exhibited extreme patience, provided economic support, and taken the time to review critically all manuscripts and ideas associated with this project. I would also like to thank other members of my committee, Dr. Jeff Bloomquist, Dr. Brad Klein, and Dr. Mitzi Nagarkatti for their guidance during proposal writing and for continual encouragement during my Ph.D. candidacy. Finally, I would like to thank the Virginia-Maryland Regional College of Veterinary Medicine for funding (Departmental and US EPA R825356), technical support, and the liberty to work when and how I chose. iii DECLARATION OF WORK PERFORMED I declare that I, Kent Richard Carlson, performed all of the work reported in this dissertation except that which is reported below. Cells for flow cytometry were collected and prepared by me and then given to Joan Kalnitsky for processing through the flow cytometer (Flow Cytometry Laboratory). Cells destined for ultrastructural observations were collected and fixed by me and then given to Kathy Lowe and Virginia Viers for embedding and sectioning (Ultrastructure Laboratory). Hen nervous system tissue destined for immunohistopathology was harvested by me, Linda Correll, and Jason Hunt (Toxicology Laboratory) and embedded and sectioned by Jill Songer (Histopathology Laboratory). iv TABLE OF CONTENTS i. ABSTRACT................................................................................................... ii ii. ACKNOWLEDGMENTS..............................................................................iii iii. DECLARATION OF WORK PERFORMED................................................ iv iv. TABLE OF CONTENTS............................................................................... v v. LIST OF FIGURES........................................................................................ ix vi. LIST OF TABLES..........................................................................................xiv vii. ABBREVIATIONS........................................................................................ xvi PART I HYPOTHESIS HYPOTHESIS Hypothesis...................................................................................................... 2 PART II LITERATURE REVIEW Chapter 1 LITERATURE REVIEW A. History of Organophosphorus (OP) Compounds......................................... 9 B. Importance of OP Compounds in Our Daily Lives ................................... 10 C. Proposed Mechanisms of Toxicity Associated with OP Compounds 1. Cellular Toxicity Studies Putative Serine Substrates........................................................ 11 Viability................................................................................... 12 Nuclear Structure and Function................................................ 12 Mitochondrial Structure and Function....................................... 13 Enzyme Activity....................................................................... 14 Cytostructural Components......................................................15 Summary............................................................................................. 16 2. Whole Animal Studies Acutely Toxic Effects............................................................... 17 Delayed Neuropathic Effects....................................................18 Chickens (Gallus domesticus).................................... 19 v Embryonic and Juvenile Chicks (Gallus domesticus)................................................................ 22 Japanese Quail (Coturnix coturnix), Bobwhites (Colinus virginianus), Mallard Ducks (Anas platyrhychos), or Ring-necked Pheasants (Phasianus colchicus)............ 23 Summary............................................................................................. 25 D. In Vitro SH-SY5Y Human Neuroblastoma Model 1. Morphology and Background.......................................................... 25 2. Receptors and Cell Signaling............................................................ 26 3. Enzymes and Neurotransmitters....................................................... 26 4. Nuclear Considerations....................................................................27 5. Cytoskeleton................................................................................... 27 6. Growth and Death........................................................................... 27 7. Differentiation.................................................................................. 29 Summary............................................................................................. 30 PART III MATERIALS AND METHODS Chapter 2 Experimental Methods and Protocols............................................. 32 PART IV RESULTS Chapter 3 Human Neuroblastoma Cell Viability and Growth are Affected by Altered Culture Conditions Abstract.............................................................................................. 37 Introduction......................................................................................... 38 Experimental Methods......................................................................... 40 Results ............................................................................................... 42 Discussion........................................................................................... 46 References.......................................................................................... 50 Chapter 4 Organophosphorus Compounds Alter the Cell Cycle Phase of SH-SY5Y Human Neuroblastoma Cells Abstract.............................................................................................