FMO3) As a Novel Genetic Determinant of Acetaminophen (APAP) Induced Hepatotoxicity Swetha Rudraiah University of Connecticut - Storrs, [email protected]

University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 8-4-2014 Characterization of Flavin-containing Monooxygenase-3 (FMO3) as a Novel Genetic Determinant of Acetaminophen (APAP) Induced Hepatotoxicity Swetha Rudraiah University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Rudraiah, Swetha, "Characterization of Flavin-containing Monooxygenase-3 (FMO3) as a Novel Genetic Determinant of Acetaminophen (APAP) Induced Hepatotoxicity" (2014). Doctoral Dissertations. 506. https://opencommons.uconn.edu/dissertations/506 Characterization of Flavin-containing Monooxygenase-3 (FMO3) as a Novel Genetic Determinant of Acetaminophen (APAP) Induced Hepatotoxicity Swetha Rudraiah, Ph.D. University of Connecticut, 2014 Mice pretreated with a mild toxic dose of acetaminophen (APAP) acquire resistance to a second, higher APAP dose. This phenomenon is termed APAP autoprotection and the exact mechanism by which such resistance develops is not clearly known. Given the prevalence of APAP- hepatotoxicity and the human health impact of this potentially hepatotoxic agent, a further understanding of the mechanism(s) involved in such protection are of considerable significance and could lead to new modalities of treatment of acute drug-induced liver injury. The work presented in this thesis investigates FMO3 gene expression during APAP-induced liver injury as well as the functional significance of FMO3 over-expression during APAP- induced liver injury. Furthermore, FMO3 gene regulation during oxidative stress conditions is also examined. Acetaminophen treatment resulted in up-regulation of liver Fmo3 protein in male mice. Female mice express higher liver Fmo3 than males and are highly resistant to APAP hepatotoxicity. Inhibition of Fmo3, by methimazole, renders female mice susceptible to APAP-induced liver injury. These findings are suggestive of a protective function for Fmo3. In addition to Swetha Rudraiah – University of Connecticut, 2014 APAP, ANIT and BDL also increase Fmo3 gene expression in mice. Because these hepatotoxicants also induce oxidative stress, we also investigated the potential role of the oxidative stress sensor and transcription factor, NRF2, in FMO3 gene regulation. Both in vivo and in vitro results show that transcriptional regulation of FMO3 might not involve the NRF2-KEAP1 regulatory pathway. Human liver can adapt to APAP-induced hepatotoxicity similar to our APAP autoprotection mouse model. The last part of this dissertation examined FMO3 gene induction in a human hepatoma cell line, HepaRG. APAP induced FMO3 gene expression in HepaRG cells, and over-expression of FMO3 protects cells against APAP-induced cytotoxicity. The unexpected observation of a faster differentiation phenotype in HepaRG cells overexpressing FMO3 suggests that FMO3 may play an important role in cellular differentiation. Collectively, data presented in this thesis provide evidence for Fmo3 as a novel genetic determinant of APAP-induced liver injury. Furthermore, this thesis describes a novel protective function for FMO3. Findings from HepaRG cells suggest that FMO3 over-expression in response to APAP may be a driving force for differentiation in regenerating hepatocytes. Characterization of Flavin-containing Monooxygenase-3 (FMO3) as a Novel Genetic Determinant of Acetaminophen (APAP) Induced Hepatotoxicity Swetha Rudraiah B.V.Sc. & A.H., University of Agricultural Sciences, India, 2004 M.V.Sc., Karnataka Veterinary, Animal and Fisheries Sciences University, India, 2006 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2014 i COPYRIGHT BY Swetha Rudraiah 2014 ii APPROVAL PAGE Doctor of Philosophy Dissertation Characterization of Flavin-containing Monooxygenase-3 (FMO3) as a Novel Genetic Determinant of Acetaminophen (APAP) Induced Hepatotoxicity Presented by Swetha Rudraiah Major Advisor: ___________________________________________ José E. Manautou, Ph.D. Associate Advisor: ________________________________________ Ronald N. Hines, Ph.D. Associate Advisor: ________________________________________ Urs A. Boelsterli, Ph.D. Associate Advisor: ________________________________________ Richard S. Bruno, Ph.D. University of Connecticut 2014 iii DEDICATION To everyone who taught me, guided me, and inspired me. iv ACKNOWLEDGEMENTS I would first and foremost like to thank my major advisor, Dr. José Manautou. Dr. Manautou has been an excellent mentor and has provided me invaluable guidance, continued advice and critical discussions that have allowed me to grow both professionally and personally. I would also like to thank my committee members Drs. Ronald Hines, Urs Boelsterli and Richard Bruno for their scientific inputs during the completion of my graduate degree. My special thanks to Dr. Hines for his willingness to help and answer my questions even before being a part of my thesis committee. Thank you for your guidance throughout. Many thanks to other faculty at the University of Connecticut School of Pharmacy for their assistance and advise during this research. Thanks to our collaborators Drs. Angela Slitt, Lauren Aleksunes, and Sarah Campion for providing samples and for insightful scientific discussions. My special thanks to Dr. Carolina Ghanem for the inspiration and friendship. I am very grateful for the support and friendship of the other members of our lab, both past and present, Xinsheng Gu, Amy Bataille, Ron Scialis, Dan Ferreira, Phil Rohrer, and Meeghan O’Connor. Also, thanks to fellow graduate students and postdocs including Bindu, Igor, Supriya, Joe, Greg, Kyle, Vince, Janine, Ritvik, Chad, Stephanie, Lai and Erica for making my stay here at UCONN memorable. I am grateful for the assistance provided by staff at the Department of Pharmaceutical Sciences especially Mark Armati and Leslie v Lebel, Denise Long at the histology laboratory, and all the hardworking staff in the animal care facility. I would also like to acknowledge the financial support of the National Institutes of Health Grant (DK069557) and the UCONN School of Pharmacy Department of Pharmaceutical Sciences without which none of this would have been possible. Thanks to the Society of Toxicology Mechanisms Specialty Section, for recognizing this work through Carl C. Smith Graduate Student Award. Lastly, I would like to extend my sincere thanks to my parents for instilling in me the values of hard work, perseverance, and good education. Their unconditional love, sacrifices, continued support, encouragement and constant pride in my accomplishments have led me so far. Thanks to my sister Shylaja and brother Omprakash, for always being there for me and for believing in me. Thanks to my extended family for all the motivation. To my husband, Sangamesh, thanks for the daily support and love and for this I will always be grateful. Finally, to my daughter, Gauri, your smile and cuteness have immensely helped me cheer, especially on some of those gloomy days. I am very fortunate to have you all in my life. vi TABLE OF CONTENTS APPROVAL PAGE iii DEDICATION iv ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii LIST OF FIGURES ix LIST OF TABLES xii LIST OF ABBREVIATIONS xiii CHAPTER 1. Review of Literature 1 1.1 Liver Function and Structure 1 1.2 Hepatic Metabolizing Systems 4 1.3 APAP Hepatotoxicity 6 1.4 Liver Injury Models and Mechanisms of Hepatotoxicity 12 1.5 Nrf2 Signaling Pathway 17 1.6 APAP Autoprotection 24 1.7 Flavin-containing Monooxygenase-3 27 1.8 Summary 40 CHAPTER 2. Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection is Associated With Induction of Flavin-containing Monooxygenase-3 (FMO3) in Hepatocytes 48 2.1 Abstract 48 2.2 Introduction 50 2.3 Materials and Methods 55 2.4 Results 63 2.5 Discussion 98 CHAPTER 3. Differential FMO3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice 107 3.1 Abstract 107 3.2 Introduction 109 3.3 Materials and Methods 114 3.4 Results 118 3.5 Discussion 138 vii CHAPTER 4. Establishment and Characterization of HepaRG Cell Line Overexpressing Human Flavin-containing Monooxygenase-3 (FMO3) 145 4.1 Abstract 145 4.2 Introduction 147 4.3 Materials and Methods 150 4.4 Results 156 4.5 Discussion 186 CHAPTER 5. Human Hepatic Flavin-containing Monooxygenase-3 (FMO3) Gene Expression is not Regulated by Nuclear Factor Erythroid 2-Like 2 (NRF2/NFE2L2) Under Oxidative Stress Conditions 193 5.1 Abstract 193 5.2 Introduction 195 5.3 Materials and Methods 196 5.4 Results 202 5.5 Discussion 212 CHAPTER 6. Summary 215 REFERENCES 223 viii LIST OF FIGURES Page Figure 1.1 Scheme showing structural organization of the liver lobule 42 Figure 1.2 Schematic drawing of various domains of Nrf (Nrf1, Nrf2, Nrf3) and Keap1/INrf2 43 Figure 1.3 A schematic representation of Nrf2-mediated gene transcription 44 Figure 1.4 A schematic representation of catalytic cycle of FMO 45 Figure 2.1 Plasma ALT activity and quantitative RT-PCR analysis of liver Fmo3 transcripts following a single dose APAP treatment 74 Figure 2.2 Analysis of liver Fmo3 protein expression following a single dose APAP treatment by western blotting, enzyme activity assay as well as by immunohistochemistry 76 Figure 2.3 Plasma ALT activity in the mouse model of APAP autoprotection 78 Figure 2.4 Analysis of liver Fmo3 protein expression in the mouse model of APAP autoprotection by western blotting and enzyme activity

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