INTEGRATING EXPERIMENTAL AND PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELING APPROACHES TO EVALUATE NEUROTOXICITY OF THE HERBICIDE ATRAZINE ACROSS THE LIFESPAN by ZHOUMENG LIN (Under the Direction of Nikolay M. Filipov) ABSTRACT Atrazine (ATR) is a widely used chlorotriazine herbicide. Available experimental evidence and computational tools are insufficient for proper assessment of ATR’s risk to human health. This dissertation project aimed to determine neurotoxicity of ATR overexposure during adulthood, development, or in vitro, and to create physiologically based pharmacokinetic (PBPK) models for ATR across the lifespan in rodents. In adult male C57BL/6 mice, short-term oral ATR exposure (5-250 mg/kg) caused dose- dependent reduced performance in a novel object recognition test (NOR), open field hypoactivity and increased swimming time in a forced swim test at higher doses; the latter effects were accompanied with altered dopamine and serotonin homeostasis in the striatum and prefrontal cortex. Low-level drinking water (DW) ATR exposure (3 mg/L) during gestation and lactation resulted in hyperactivity and decreased NOR performance in mouse dams, hyperactivity in male and female juvenile offspring, decreased swimming time in male juveniles, increased marble burying in female juveniles, and decreased NOR performance in female adults. Neurochemically, DW ATR exposure increased striatal dopamine in dams and juvenile offspring. In vitro exposure (24-48 h; 12-300 µM) to ATR or its main metabolite didealkylatrazine (DACT) affected morphological differentiation of N27 dopaminergic cells with ATR mainly targeting soma enlargement (dose-dependent effect) and DACT decreasing neurite outgrowth (high-dose effect). A PBPK model for ATR and its metabolites desethylatrazine, desisopropylatrazine and DACT was developed in adult male mice and then extrapolated to rats. This adult rodent model and recent experimental data were the foundation for a subsequent developmental PBPK model that accurately described ATR’s kinetic behavior during fetal, neonatal, pregnant and lactating stages in rats. Model simulations aligned well with experimental data, including with a new pharmacokinetic study conducted with pregnant mice orally exposed to ATR, validating the cross-species extrapolation of the gestational model. In conclusion, ATR overexposure affects multiple behavioral domains and perturbs brain dopamine and serotonin homeostasis, with some effects on the offspring being sex-specific. ATR- and/or DACT-induced neuronal differentiation disruption may contribute to the observed developmental neurotoxicity. The newly developed PBPK models can be used in brain dosimetry predictions and, together with the experimental data, may improve ATR’s risk assessment. INDEX WORDS: Atrazine, Pesticides, Toxicity, Brain, Behavior, Dopamine, Serotonin, PBPK modeling, Developmental exposure, Neuronal differentiation INTEGRATING EXPERIMENTAL AND PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELING APPROACHES TO EVALUATE NEUROTOXICITY OF THE HERBICIDE ATRAZINE ACROSS THE LIFESPAN by ZHOUMENG LIN B.Med., Southern Medical University, China, 2009 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2013 © 2013 Zhoumeng Lin All Rights Reserved INTEGRATING EXPERIMENTAL AND PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELING APPROACHES TO EVALUATE NEUROTOXICITY OF THE HERBICIDE ATRAZINE ACROSS THE LIFESPAN by ZHOUMENG LIN Major Professor: Nikolay M. Filipov Committee: Jia-Sheng Wang Mary Alice Smith Julie A. Coffield Xiaoqin Ye Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2013 ACKNOWLEDGEMENTS Firstly, I would like to thank my advisor Dr. Nikolay M. Filipov for proper guidance, continuous support and readily-available help throughout my PhD training. I really appreciate him and will always remember what he has taught or given me, such as (1) a variety of techniques and knowledge, (2) the opportunities to receive comprehensive training in the field of toxicology by learning in vitro, in vivo, and computational techniques, (3) the ways of experimental design, scientific thinking and effective writing, (4) the opportunities to receive professional training (e.g., grant writing, public presentation, and leadership), (5) the suggestions on my career development, and (6) many others. I am also grateful for his confidence in my ability to become a successful toxicologist in the future. I enjoy the time working with him, especially during the most impressive weekly mentor meetings. To me, he is not only an excellent advisor, but also a great friend. Secondly, I would like to express my sincere gratitude to Dr. Jeffrey W. Fisher. I was fortunate to take the last course of PBPK modeling offered by Dr. Fisher before he moved to FDA. Even though he works far away from Athens and is not in my Advisory Committee, he is so nice that he keeps mentoring me various computational modeling techniques throughout my graduate study and providing me with excellent professional suggestions to help me find a job. To my respected Advisory Committee members, Dr. Jia-Sheng Wang, Dr. Mary Alice Smith, Dr. Xiaoqin Ye and Dr. Julie A. Coffield, I would like to say Thank You so much for helping me review my research proposal and dissertation, attend annual Committee Meetings, provide a number of constructive suggestions and write multiple reference letters. Special thanks iv are also given to my Society of Toxicology Mentor Match Program mentor, Dr. Joshua A. Harrill, for excellent professional advice and helping me revise my CV and Cover Letter. Additionally, I would like to give my warm appreciation to my undergraduate advisor and friend, Dr. Lang Yao in the Department of Toxicology at the Southern Medical University in Guangzhou, China, who brought me into this exciting field of Toxicology and keeps encouraging me to pursue my dream. Many thanks are given to my lab members, Dr. Celia A. Dodd, Irina I. Georgieva, and Saritha Krishna. Celia and Irina showed me many techniques and Saritha taught me a lot of oral presentation skills. I really enjoyed the time working with them. Additionally, to my good friends, Dr. Sheppard A. Martin, Dr. Shuang Li, Dr. Shuo Xiao, Fei Zhao, Rong Li, and Xia Guo, thank you for helping me a lot in my dissertation research. I sincerely appreciate the Interdisciplinary Toxicology Program, Graduate School, and Department of Physiology and Pharmacology for offering me research assistantships. My home department, the Department of Physiology and Pharmacology is really an excellent environment that allows me to focus on research. Dr. Gaylen L. Edwards, Kali King and Misty Patterson in my home department, and Dr. Brain S. Cummings, Dr. Mary Alice Smith, and Joanne Mauro in the Interdisciplinary Toxicology Program always provide me with a variety of timely assistance. Thanks are given to them. Last but not least, I would like to thank my family. My parents have been doing their best to raise, educate, support and encourage me. While I was not able to be with them most of the time during the last four years, they had never complained. Instead, they always understand, support, and encourage me. I am in particularly indebted to my wife Chunla He, who is also a PhD student at the University of Georgia. We have been studying together for nine years. She v has been constantly supporting and helping me in my research and in my life without any complaints, regardless of success or failure. Even endless thanks cannot express my gratitude. For those many other friends at the University of Georgia, thank you for helping me in my daily life. Once again, I would like sincerely thank my advisor, committee members, mentors, friends, and my family. Without your support and help, I would not have completed this dissertation project. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ..................................................................................................................... xiii ABBREVIATIONS ................................................................................................................... xviii CHAPTER 1 INTRODUCTION .........................................................................................................1 Problem statement ....................................................................................................1 Objectives, hypotheses, and specific aims ...............................................................3 2 LITERATURE REVIEW ..............................................................................................6 Pesticides..................................................................................................................6 Atrazine ..................................................................................................................12 3 SHORT-TERM ATRAZINE EXPOSURE CAUSES BEHAVIORAL DEFICITS AND DISRUPTS MONOAMINERGIC SYSTEMS IN MALE C57BL/6 MICE ......31 Abstract ..................................................................................................................32 Introduction ............................................................................................................33