GLUTATHIONE TRANSFERASE Z1-CATALYZED BIOTRANSFORMATION OF DICHLOROACETATE – ROLES OF MITOCHONDRION, SUBJECT AGE, GSTZ1 HAPLOTYPE AND CHLORIDE INTERACTION By WENJUN LI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Wenjun Li 2 To my mom and dad, and those who have loved and supported me 3 ACKNOWLEDGMENTS Five years of graduate school have helped me mature as a researcher and as an individual. First and foremost, I would like to deliver my most sincere thanks to Dr. Margaret James, my Ph.D. mentor and role model during these years. I deeply appreciate the continuous support, encouragement and enlightenment Dr. James has provided throughout my Ph.D. study. Her dedication, fairness and integrity to work and her kindness and respect to people have and will always guide me in my own research and doings. My gratitude extends to my collaborators and committee members. Dr. Peter Stacpoole has provided me with expert insights into dichloroacetate research and firsthand information from clinical studies, both of which motivated me to dig deeper into the study and reminded me of our goal of helping people. Drs. Ronald Hines and Taimour Langaee have both given me generous assistance in various aspects of the study. I am thankful to Drs. Ken Sloan, Raymond Booth and Raymond Bergeron for assisting with the dissertation and their support during graduate study. I would like to thank all of my fellow students and colleagues for their support and encouragements. In particular, Yuan Gu has made substantial contributions to our study and Sriram Ambadapadi has been so kind to hold our regular coffee breaks. Special thanks goes to my fiancé Zhuming Sun, who truly knows me and cares for me. Together, we walk through the happiness and bitterness of research and life. Finally, I am most grateful to my parents for their forever love, support and understandings. They are friends and teachers of my life. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 INTRODUCTION .................................................................................................... 14 DCA as an Environmental Agent ............................................................................ 14 DCA as a Therapeutic Agent .................................................................................. 15 DCA Biotransformation ........................................................................................... 17 GSTZ1 as the Metabolizing Enzyme ................................................................ 17 DCA-Induced Inactivation of GSTZ1 ................................................................ 19 Discrepancy in studies of GSTZ1 inactivation ............................................ 20 GSTZ1 Polymorphism ...................................................................................... 20 DCA Pharmacokinetics ........................................................................................... 22 Overview of DCA Absorption, Distribution, Metabolism and Elimination .......... 22 Subject Age and GSTZ1 Genotype in Determining DCA Pharmacokinetics .... 23 DCA Pharmacotoxicology ....................................................................................... 25 Postulated Mechanisms of DCA Toxicity .......................................................... 25 Clinical Pharmacotoxicology of DCA ................................................................ 26 Hypotheses ............................................................................................................. 27 2 MITOCHONDRION AS A NOVEL SITE OF DICHLOROACETATE BIOTRANSFORMATION BY GLUTATHIONE TRANSFERASE Z1 ....................... 35 Specific Aim ............................................................................................................ 35 Materials and Methods............................................................................................ 35 Subcellular Fractionation of Human and Rat Livers ......................................... 35 Subfractionation of Liver Mitochondria ............................................................. 36 Electrophoresis and Western Blots .................................................................. 37 GSTZ1 Activity ................................................................................................. 38 Immunoprecipitation of Mitochondrial GSTZ1 .................................................. 39 LC-MS/MS ........................................................................................................ 40 Protein Search Algorithm.................................................................................. 40 Results .................................................................................................................... 41 Expression and Activity of GSTZ1 in Hepatic Mitochondria ............................. 41 Mitochondrial GSTZ1 Is Localized in the Matrix ............................................... 42 5 LC-MS/MS Identification of the Mitochondrial GSTZ1 ...................................... 43 Kinetic Study of Cytosolic and Mitochondrial GSTZ1 ....................................... 44 Discussion .............................................................................................................. 44 3 ROLES OF SUBJECT AGE AND HAPLOTYPE ON GSTZ1 EXPRESSION AND ACTIVITY WITH DICHLOROACETATE IN HUMAN LIVER ........................... 59 Specific Aim ............................................................................................................ 59 Materials and Methods............................................................................................ 59 Human Liver Samples ...................................................................................... 59 GSTZ1 Activity with DCA .................................................................................. 60 Western Blot Analysis of GSTZ1 Expression ................................................... 60 Genotyping ....................................................................................................... 61 Data Analysis ................................................................................................... 62 Results .................................................................................................................... 62 Developmental Pattern of GSTZ1 in Human Liver Cytosol............................... 62 Impact of Z1A Variant ....................................................................................... 63 Correlation Analysis of GSTZ1 Activity and Expression ................................... 64 Role of Haplotype on GSTZ1 Expression and Activity ..................................... 64 Role of Haplotype on Mitochondrial GSTZ1 ..................................................... 65 Discussion .............................................................................................................. 66 4 CHLORIDE MODULATES GSTZ1 HAPLOTYPE-DEPENDENT INACTIVATION BY DICHLOROACETATE ............................................................. 75 Specific Aim ............................................................................................................ 75 Materials and Methods............................................................................................ 75 Human Liver Samples and GSTZ1 Genotype Information ............................... 75 Assay of DCA-Induced Inactivation of GSTZ1 in Human Liver Cytosol ............ 76 Data Analysis ................................................................................................... 76 Results .................................................................................................................... 77 Chloride Protected GSTZ1 from DCA Inactivation ........................................... 77 Time Course of GSTZ1 Inactivation by DCA .................................................... 77 Effects of Other Anions..................................................................................... 78 Discussion .............................................................................................................. 78 5 CONCLUSION ........................................................................................................ 87 LIST OF REFERENCES ............................................................................................... 89 BIOGRAPHICAL SKETCH ............................................................................................ 98 6 LIST OF TABLES Table page 1-1 Specific activities and inactivation half-lives of polymorphic variants of expressed recombinant hGSTZ1. ....................................................................... 32 1-2 Plasma kinetics of DCA in children and adults after single and repeated doses .................................................................................................................