INTER-INDIVIDUAL DIFFERENCES IN TRICHLOROETHYLENE TOXICOKINETICS AND TOXICODYNAMICS IN GENETICALLY-DIVERSE MICE Abhishek Venkatratnam A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Environmental Sciences and Engineering in the Gillings School of Global Public Health. Chapel Hill 2018 Approved by: Ivan Rusyn Avram Gold Wanda M. Bodnar Weihsueh Chiu Terry Wade © 2018 Abhishek Venkatratnam ALL RIGHTS RESERVED ii ABSTRACT Abhishek Venkatratnam: Inter-individual differences in trichloroethylene toxicokinetics and toxicodynamics in genetically-diverse mice. (Under the direction of Ivan Rusyn) One of the many challenging issues in toxicology is addressing human variability in chemical risk assessment. Among several intrinsic and extrinsic factors that contribute towards inter-individual differences in toxic responses, genetic differences are well known to drive variability in xenobiotic metabolism and adverse effects. Traditional toxicity studies in rodents often rely on single genotype-based models that fail to capture diverse responses similar to those observed in humans. Recent advances in mouse genetics have led to the development of large panels of recombinant lines collectively known as population-based mouse models. Although their utility in biomedical research has been sufficiently demonstrated, their application in toxicity studies needs to be investigated. My doctoral dissertation focuses on investigating inter-individual differences in toxicokinetics (TK) and toxicodynamics (TD) in population-based mouse models with trichloroethylene (TCE) as case study toxicant. The central hypothesis of my dissertation is that genetic differences in the mouse population will drive inter-individual differences in TCE metabolism and related effects. Aim 1 demonstrated sufficient statistical power and precision in the mouse population to identify genetic loci driving variability in TCE metabolism and TD. Next, extent of quantitative variability in some toxicokinetic parameters were comparable to those observed in humans suggesting the appropriateness of such model in addressing human variability in adverse outcomes. Aim 2 characterized transcriptional responses that were strongly influenced by genetic background, dose, or a combination of genetic background and dose to better iii understand individual- versus population-level responses. Aim 3 assessed the utility of different population-based models in capturing diverse responses in a standard 90-day oral toxicity study that is often conducted for safety assessments. Collectively, this doctoral dissertation serves as a comprehensive guide in incorporating population-based mouse models in toxicity studies. iv ACKNOWLEDGEMENTS I would like to thank my doctoral advisor Dr. Ivan Rusyn for the opportunity to conduct my doctoral research in his lab and for providing an environment to interact with other trainees in the lab with diverse academic backgrounds. I would like to express my sincere appreciation to my doctoral committee members Drs. Avram Gold, Wanda M. Bodnar, Weihsueh Chiu, and Terry Wade for their support and comments on my dissertation research. As an international graduate student, conducting my graduate work off-campus is a unique experience that cannot be described by words. My interaction within the lab has led to an everlasting bond of friendship with both past and current members of the Rusyn lab. I am thankful to Ms. Grace A. Chappell and Ms. Lauren Lewis for spending time with me over occasional drinks at stressful times. Scintillating conversations ranging from coffee to toxicology with Drs. Fabian A. Grimm and William (Bill) D. Klaren are indeed cherish able memories that I would carry with me after my departure from the lab. I would also like to thank both of them for their support and timely advises over the course of my doctoral research. As my doctoral work involves working with rodents, I would like to acknowledge Mrs. Oksana Kosyk (former manager of the Rusyn lab) and Dr. Shinji Furuya for training me with mouse handling and administration procedures. In a similar vein, I also recognize Dr. Joseph A. Cichocki Mr. Yu-Syuan Luo and Dr. Hisataka Fukushima for their help with experiments. My dissertation research is a collaborative effort of several individuals and labs in Texas and North Carolina. I would like to express my gratitude to Mr. Leonard Collins and Dr. Wanda v M. Bodnar for their initial support with method development for quantifying trichloroethylene metabolites. I am very grateful to Drs. Rachel Lynch and David Threadgill for their support with logistics and operations involved with the 90-day oral toxicity study. I would also like to thank Dr. Amie Perry for her histopathological observations. As one aspect of my work heavily relied on bioinformatics analysis, I acknowledge Mr. Kranti Kongati and Dr. John House for their collaborative support with differential gene expression analysis. I am very grateful to Dr. Fred Wright for making frequent visits to Texas A & M University and engaging in meetings with me to discuss the progress and scope of one of the projects in my dissertation. Similarly, I would also like to thank Dr. David Aylor and his staff for designing R-based scripts for conducting quantitative trait loci (QTL) analysis. Lastly, I thank my parents, immediate family-relatives, and friends for being supportive over the last several years. I would like to thank my significant half for providing emotional support in the midst of failures and excruciating circumstances during my dissertation and will fulfill the promises I have made to her over the coming years. Nonetheless, it has been an incredible journey and I hope to move on with my life towards a prosperous future. vi TABLE OF CONTENTS LIST OF TABLES ................................................................................................................... ix LIST OF FIGURES .................................................................................................................. x CHAPTER 1: INTRODUCTION ............................................................................................. 1 I. Risk assessment of chemicals ................................................................................ 1 II. Variability and uncertainty in components of risk assesssment ............................. 3 III. Implementation of emerging sciencs and technologies in risk assessment ........... 8 IV. Critical problems in human health assessments .................................................. 12 V. Trichloroethylene (TCE) as a case study toxicant ............................................... 13 VI. Rationale and Specific Aims ............................................................................... 15 VII. Figures and Tables............................................................................................... 18 CHAPTER 2: EVALUATION AND CHARACTERIZATION OF INTER- INDIVIDUAL DIFFERENCES OF TCE METABOLISM ................................................... 20 I. Introduction ......................................................................................................... 20 II. Materials and Methods ......................................................................................... 22 III. Results .................................................................................................................. 27 IV. Discussion ............................................................................................................ 32 V. Figures and Tables ............................................................................................... 39 CHAPTER 3: CHARACTERIZATION OF GENETIC BACKGROUND, DOSE, AND THEIR COMBINATORY EFFECTS ON LIVER GENE EXPRESSION RESPONSES WITH TCE EXPOSURE ....................................................... 54 I. Introduction ......................................................................................................... 54 vii II. Materials and Methods ......................................................................................... 56 III. Results .................................................................................................................. 61 IV. Discussion ............................................................................................................ 68 V. Figures and Tables ............................................................................................... 73 CHAPTER 4: EVALUATION OF INTER-INDIVIDUAL DIFFERENCES IN TCE METABOLISM AND TOXICITY IN A 90-DAY ORAL TOXICITY STUDY IN GENETICALLY-DIVERSE MOUSE POPULATIONS .................................... 87 I. Introduction .......................................................................................................... 87 II. Materials and Methods .......................................................................................... 90 III. Results ................................................................................................................... 93 IV. Discusssion ........................................................................................................... 97 V. Figures and Tables .............................................................................................. 101 CHAPTER 5: GENERAL DISCUSSION ............................................................................ 117 I. Conclusions………………………………………………………………………