MECHANISMS OF PREDICTION AND POTENTIAL CAUSATION OF ORGANOPHOSPHATE INDUCED DELAYED NEUROTOXICITY by Nichole DeEtta Hein A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Toxicology) in The University of Michigan 2009 Doctoral Committee: Professor Rudy J. Richardson, Chair Professor Paul F. Hollenberg Associate Professor Peter Mancuso Research Assistant Professor Jeanne Stuckey To my sunshine - Daphne Louise ii ACKNOWLEDGEMENTS I am absolutely appreciative of the opportunity to study and grow in the Toxicology Program of the Department of Environmental Health Sciences in the University of Michigan. The work that has been completed herein could not have been accomplished without the invaluable teachings of the faculty and support from the staff. I would like to thank Dr. Rudy Richardson for inviting me into his laboratory to work and learn, and for his guidance, patience, and support as I completed this program. I am grateful for the advice and direction provided by my committee members Dr. Rudy Richardson, Dr. Jeanne Stuckey, Dr. Paul Hollenberg, and Dr. Peter Mancuso. I also extend a thank you to Jen Pierce and Dr. Tim Kropp who introduced me to the laboratory and helped me initiate the work that I would continue. In addition, I would like to thank Dr. Rob Christner for his guidance on the SELDI TOF-MS. I need to thank Dr. Jeanne Stuckey and the members (former and current) of her laboratory, Pat Gee, Ann Kendall, Dr. Jen Meagher, and Dr. Ron Rubin at the Life Sciences Institute for their assistance in the production of NEST and the mutations, without whom much of this work would not have been completed. I would like to thank Dr. Steve Bursian and Angelo Napolitano who provided the source of hen brain enzymes from the poultry research and training center at Michigan State University. In addition I would like to thank Dr. John Fink and Dr. Shirley Rainier iii for providing the source of mouse brain enzymes and for discovering the mutations that led to the work in Chapter 4 of the dissertation. I would like to thank the researchers from the Institute of Physiologically Active Compounds in Chernogolovka, Russia, particularly Dr. Galina Makhaeva, who provided the fluorinated aminophosphonate compounds. I want to include a word of appreciation to all of the other students, my friends, and my family that have supported me through this journey. Finally, I acknowledge the support of the U.S. Army Research Office grant number DAAD19-02-1-0388, Pharmaceutical Sciences Training Program, Environmental Toxicology Training Program Grant NIH ES07062, Dow Chemical Company, Dow Agrosciences, AstraZeneca Pharmaceuticals, and Dr. Martin Philbert and Dr. Rita Loch- Caruso for providing the funding or materials that allowed me to complete my research. iv TABLE OF CONTENTS DEDICATION_________________________________________________________ ii ACKNOWLEDGEMENTS ______________________________________________iii LIST OF TABLES ____________________________________________________viii LIST OF FIGURES ____________________________________________________ x LIST OF ABBREVIATIONS ____________________________________________ xi ABSTRACT__________________________________________________________ xvi CHAPTER 1 BACKGROUND AND INTRODUCTION__________________________________ 1 History of Organophosphorus Compounds_______________________________ 1 Fluorinated Aminophosphonates _____________________________________ 2 Mechanism of OP Toxicity ____________________________________________ 3 Acute OP Toxicity _________________________________________________ 6 Chronic OP Toxicity ________________________________________________ 8 Modeling of OPIDN _________________________________________________ 10 Hypothesis_________________________________________________________ 13 Specific Aims ______________________________________________________ 13 References_________________________________________________________ 20 CHAPTER 2 MASS SPECTROMETRY REVEALS THAT BUTYRYLCHOLINESTERASE IS PHOPSPHORYLATED BY FLUORINATED AMINOPHOSPHONATE COMPOUNDS THROUGH A SCISSION IN THE CARBON-PHOSPHORUS BOND_______________________________________________________________ 26 Abstract___________________________________________________________ 26 Introduction _______________________________________________________ 27 Materials and Methods ______________________________________________ 28 Materials________________________________________________________ 28 v Measurement of Cholinesterases Activity and Inhibition ______________________ 29 Calculation of ki __________________________________________________ 29 Mass Spectrometry ________________________________________________ 30 Results ____________________________________________________________ 31 Discussion _________________________________________________________ 33 References_________________________________________________________ 40 CHAPTER 3 ASSESSMENT OF DELAYED NEUROTOXICITY POTENTIAL OF ORGANOPHOSPHORUS COMPOUNDS IN VITRO _______________________ 43 Abstract___________________________________________________________ 43 Introduction _______________________________________________________ 44 Experimental Protocol_______________________________________________ 46 Suppliers________________________________________________________ 46 Preparation of Brain Tissue ________________________________________ 46 Production and Purification of Human Recombinant NEST ______________ 47 Transformation for plasmid and protein expression ___________________ 47 DNA isolation__________________________________________________ 48 Protein expression ______________________________________________ 49 Protein purification _____________________________________________ 49 Measurement of AChE Activity and Inhibition _________________________ 50 Measurement of AChE Aging _______________________________________ 51 Measurement of NTE/NEST Activity and Inhibition_____________________ 52 Measurement of NEST Aging _______________________________________ 53 Calculation of Bimolecular Rate Constant of Inhibition, ki _______________ 53 Calculation of First-Order Rate Constant of Aging, k4 ___________________ 54 Results ____________________________________________________________ 55 Discussion _________________________________________________________ 55 References_________________________________________________________ 70 CHAPTER 4 vi HUMAN RECOMBINANT NEUROPATHY TARGET ESTERASE DOMAIN CONTAINING MUTATIONS RELATED TO MOTOR NEURON DISEASE HAVE ALTERED ENZYMATIC PROPERTIES __________________________ 73 Abstract___________________________________________________________ 73 Introduction _______________________________________________________ 74 Materials and Methods ______________________________________________ 76 Chemicals _______________________________________________________ 76 Protein Expression and Purification__________________________________ 76 Site-Directed Mutagenesis __________________________________________ 77 Determination of NEST activity _____________________________________ 78 Calculation of ki __________________________________________________ 78 Determination of NEST aging_______________________________________ 79 Calculation of k4__________________________________________________ 79 Statistical Analysis ________________________________________________ 79 Results ____________________________________________________________ 80 Discussion _________________________________________________________ 81 References_________________________________________________________ 92 CHAPTER 5 SUMMARY AND CONCLUSIONS ______________________________________ 95 Conclustions _______________________________________________________ 95 Summary of Data Chapters __________________________________________ 97 The Phosphorylation of BChE by FAP Compounds Revealed Through MS __ 97 Assessment of Delayed Neurotoxic Potential of OP Compounds ___________ 99 Altered Kinetic Properties of Mutations Discovered in NTE-MND ________ 100 Significance and Further Investigations _______________________________ 101 Phosphorylation by FAP Compounds _______________________________ 101 Assay development for prediction of neuropathic potential_______________ 102 NTE-related motor neuron disease mutations _________________________ 103 References________________________________________________________ 105 vii LIST OF TABLES Table 2.1 Bimolecular rate constants of inhibition (ki) for FAP compounds. ________ 35 Table 2.2 Theoretical and calculated mass shifts of inhibited and aged adducted serine peptides. _____________________________________________________________ 36 Table 3.1 Bimolecular rate constants of inhibition (ki) and corresponding fixed-time IC50 values for organophosphrus inhibitors against acetylcholinesterase (AChE) from different species. ______________________________________________________________ 60 Table 3.2 Bimolecular rate constants of inhibition (ki) and corresponding fixed-time IC50 values for organophosphrus inhibitors against neuropathy target esterase (NTE) or NTE catalytic domain (NEST) from different species. ______________________________ 61 Table 3.3 Relative inhibitory potential (RIP) for organophosphorus inhibitors against acetylcholinesterase (AChE) versus neuropathy target esterase (NTE) or NTE catalytic domain (NEST) from different species. _____________________________________ 62 Table 3.4 Rate constant of aging (k4) and calculated half-time (t1/2)for neuropathy target esterase catalytic domain (NEST). _________________________________________