TOXICOGENOMIC BIOMARKER DISCOVERY OF AHR- MEDIATED TCDD-INDUCED HEPATOTOXICITY By Edward Dere A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Biochemistry and Molecular Biology 2010 ABSTRACT TOXICOGENOMIC BIOMARKER DISCOVERY OF AHR- MEDIATED TCDD-INDUCED HEPATOTOXICITY By Edward Dere 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that causes a wide array of species-specific adverse biochemical and physiological responses, including increased tumor promotion, lethality and hepatotoxicity. Most, if not all of the effects elicited by TCDD are due to inappropriate changes in gene expression that are mediated through activation of the aryl hydrocarbon receptor (AhR). Although the mechanism of AhR gene regulation is well known, the full spectrum of targeted genes leading to the subsequent toxicological responses remains poorly understood. The objective of this research was to integrate disparate and complementary toxicogenomic approaches to identify putative biomarkers of TCDD-induced hepatotoxicity that would aide in reducing the uncertainties involved in cross-species and cross-model extrapolations. In vitro microarray investigation of a mouse hepatoma cell line treated with TCDD identified complex temporal and dose-dependent gene expression responses. Comparative analysis with in vivo hepatic gene expression responses in mice identified a small subset of conserved genes with biological functions related to xenobiotic metabolism, consistent with the known responses observed in vivo. Furthermore, in vitro cross-species comparison using human, mouse, and rat hepatoma cell lines identified relatively few species-conserved gene expression and is corroborates prior reports of species-specific TCDD-induced toxicities. Genome-wide computational identification and characterization of dioxin response elements (DREs) using a position weight matrix identified species-specific regulons in the promoter regions of targeted genes that may account for the observed species-divergent and -specific responses. In order to better understand the molecular mechanisms responsible for regulating the transcriptional responses and downstream hepatotoxicity, ChIP-chip analysis was performed to globally identify TCDD-induced AhR/DNA interactions in mouse hepatic tissue. Interestingly, integration of the DRE, ChIP-chip and gene expression analyses found that only ~32% of all TCDD-elicited hepatic gene expression responses are mediated by a DRE-dependent mechanism. These direct targets of AhR regulation have biological functions related to xenobiotic and lipid metabolism, which correspond with the physiological responses observed in vivo. The remaining transcriptional responses that are mediated through a DRE-independent mechanism illustrate the diverse regulatory role of the AhR. Collectively, these results have expanded our knowledge of the hepatic AhR regulatory network and provide insight into the species-conserved responses elicited by TCDD. ACKNOWLEDGEMENTS Graduate school has been a tremendous learning experience of not only science and research, but also of life in general. This memorable experience has defined me scientifically and personally in ways that I never thought were possible. I would never have been able to accomplish this without the guidance, support and love from all those around me during my time at Michigan State University and I wish to thank you all. First, I give many thanks to my advisor, Dr. Timothy Zacharewski. He has provided me with abundant support both in and outside of the lab. Without his initial faith in hiring me as a co-operative education student while as an undergraduate student, I would never have had the opportunity to be at Michigan State University. Second, I would like to thank my Graduate Committee members: Drs. Kristina Chan, David DeWitt, Gregg Howe and Jack Watson for their availability, insight and unique perspectives in contributing to the success of my project during my committee meetings. I would also like to acknowledge all my fellow lab members, both past and present, Darrell Boverhof, Josh Kwekel, Lyle Burgoon, Ania Kopec, Suntae Kim, Michelle Angrish, and many others, including numerous co-operative education students. Thank you all for putting up with me over the years. I’ve learned so much from each and every single one of you and never would have made it through graduate school without your support. I also would like to thank iv members of the LaPres lab, especially Dr. John LaPres and Ajith Vengellur for all their help and insightful conversations about everything. Finally, I could never have endured this experience without the encouragement, support and love from those closest to me. To my father and mother, my brother, sister-in-law, and even my little nephew Riley, I owe you all a tremendous debt for all that you have given me. And to May, you are the highlight of my graduate experience. You’ve always been there for me when I needed you most and I hope that I can somehow return the favor. v TABLE OF CONTENTS LIST OF TABLES .....................................................................................................................viii LIST OF FIGURES ...................................................................................................................... x! LIST OF ABBREVIATIONS ...................................................................................................xiii! CHAPTER 1 ! Review of the Literature: Toxicogenomics and TCDD-Induced Toxicity Mediated by the Aryl Hydrocarbon! Receptor.............................................................................................. 1 Introduction............................................................................................................................. 2 Toxicogenomics...................................................................................................................... 2! TCDD and its Elicited Effects ................................................................................................ 3! The Aryl Hydrocarbon Receptor ............................................................................................ 5! Conclusions............................................................................................................................. 9! References............................................................................................................................. 10 ! ! CHAPTER 2 Rationale, Hypothesis and Specific Aims ............................................................................. 16 Rationale! ............................................................................................................................... 17 Hypothesis............................................................................................................................. 17! Specific Aims........................................................................................................................ 17 ! ! CHAPTER 3 In Vivo – In Vitro Toxicogenomic Comparison of TCDD-Elicited Gene Expression in Hepa1c1c7! Mouse Hepatoma Cells and C57BL/6 Hepatic Tissue................................. 19 Abstract................................................................................................................................. 20 Introduction........................................................................................................................... 21! Materials and Methods.......................................................................................................... 23! Results................................................................................................................................... 27! Discussion............................................................................................................................. 48! References............................................................................................................................. 53 ! ! CHAPTER 4 Comparison of TCDD-Elicited Gene Expression in Human HepG2, Mouse Hepa1c1c7! and Rat H4IIE Hepatoma Cells ......................................................................... 59 Abstract................................................................................................................................. 60 Introduction........................................................................................................................... 60! Materials and Methods.......................................................................................................... 62! Results................................................................................................................................... 63! Discussion............................................................................................................................. 81! ! ! vi References............................................................................................................................. 84 CHAPTER 5 Genome-Wide Computational Analysis of Dioxin Response Element Location and Distribution! in the Human, Mouse and Rat Genomes................................................. 88 Abstract................................................................................................................................. 89 Introduction..........................................................................................................................