3-Methylcholanthrene Induces Chylous Ascites and Lethality in Tiparp Knockout Mice by Tiffany Elizabeth Cho A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Pharmacology and Toxicology University of Toronto © Copyright by Tiffany Elizabeth Cho (2015) 3-Methylcholanthrene Induces Chylous Ascites and Lethality in Tiparp Knockout Mice Tiffany Elizabeth Cho Master of Science Department of Pharmacology and Toxicology University of Toronto 2015 Abstract The aryl hydrocarbon receptor (AHR) is a ligand-regulated transcription factor that is activated upon binding to various ligands. The activated AHR modulates the expression of many genes including cytochrome P450s (CYPs) such as Cyp1a1, Cyp1b1, and 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (Tiparp). We recently reported that TIPARP is a transcriptional repressor of AHR and Tiparp knockout mice show increased sensitivity to dioxin-induced toxicities. Because of these findings, we examined the sensitivity of Tiparp knockout mice to 3-methylcholanthrene (3-MC), another potent AHR ligand. Tiparp knockout mice treated with 100mg/kg of 3-MC exhibited increased hepatotoxicity, increased lipolysis, and developed chylous ascites compared with treated wildtype mice. No treated Tiparp knockout mice survived beyond day 16; all wildtype mice survived the 30 day treatment. Collectively, this thesis shows that Tiparp knockout mice exhibit increased sensitivity to 3-MC- induced toxicity and lethality supporting our previous findings that TIPARP is an important negative regulator of AHR activity. ii Acknowledgements My M.Sc. has been a great learning experience and a fulfilling journey. I am forever grateful for the support and help that my supervisor, Dr. Jason Matthews, has provided me. I thank him the most for the amazing opportunity he has given me to learn and excel in research. Overall, it has been a pleasure to work under his guidance and mentorship; no words can describe how thankful I am for his supervision and generosity throughout. Dr. Peter McPherson has been invaluable as my advisor, as he always makes himself available to his students and is always willing to lend an ear. I would also like to extend my sincere thanks to the members of our lab: Alvin Gomez, Debbie Bott, Laura Tamblyn, Susanna Tan, Sunny Yang, and David Hutin. I am grateful for their help and support; the laughter and fun moments that we indulged in; and for sharing their knowledge with me. I have learned so much from everyone and I am always appreciative of the assistance and support that I have received from our lab members throughout the years. A special thank you to Susanna, my partner-in-crime, whom I will dearly miss “holding hands” with. I would also like to acknowledge all the applicable funding agencies for their financial support that made this research possible: the Canadian Institutes of Health Research, the DOW Chemical Company, the government of Ontario, and the University of Toronto. As always, I would like to extend my most sincere thanks and appreciation to my family: my parents, my aunt and uncle, and my grandmother for supporting me with anything and everything. iii Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii List of Abbreviations .................................................................................................................... x Chapter 1: Introduction ............................................................................................................... 1 1.1 Aryl Hydrocarbon Receptor ...................................................................................................... 1 1.1.1 Structure ......................................................................................................................... 1 1.1.2 AHR Signalling Pathway ............................................................................................... 3 1.1.3 AHR Responsive Genes ................................................................................................. 4 1.2 AHR Ligands ............................................................................................................................ 5 1.2.1 Natural Ligands .............................................................................................................. 5 1.2.2 Synthetic Ligands........................................................................................................... 7 1.2.3 3-Methylcholanthrene (3-MC) ....................................................................................... 9 1.3 Functional role of AHR in Toxicology and Physiology ......................................................... 11 1.3.1 AHR-mediated Toxicity............................................................................................... 11 1.3.2 Physiological Role of AHR ......................................................................................... 13 1.4 ADP-Ribosylation ................................................................................................................... 14 1.4.1 Poly(ADP-ribose) Polymerase (PARP) ....................................................................... 16 1.4.2 Mono(ADP-ribosyl)transferase .................................................................................... 16 1.5 Macrodomains......................................................................................................................... 18 1.6 TCDD-inducible poly(ADP-ribose) polymerase (TIPARP) ................................................... 19 1.6.1 Structure ....................................................................................................................... 19 1.6.2 Function ....................................................................................................................... 20 1.7 AHRR and TIPARP: Similarities and Differences ................................................................. 23 1.8 Characterization of the TIPARP Knockout Mouse Model ..................................................... 24 Rationale and Research Objectives ........................................................................................... 26 Research Aims .............................................................................................................................. 26 iv Chapter 2: Materials and Methods ........................................................................................... 27 2.1 Materials ................................................................................................................................. 27 2.1.1 Chemicals and Biological Reagents............................................................................. 27 2.1.2 Plasticware and Other Equipment ................................................................................ 29 2.1.3 Instruments ................................................................................................................... 30 2.2 Methods................................................................................................................................... 31 2.2.1 Animal Facility ............................................................................................................ 31 2.2.2 Maintenance of Animal Colony ................................................................................... 31 +/+ -/- 2.2.3 Genotyping of Tiparp and Tiparp Mice ................................................................ 32 2.2.4 Animals and Treatments .............................................................................................. 33 2.2.5 Blood and Tissue Collection ........................................................................................ 34 2.2.6 RNA Extraction and Isolation ...................................................................................... 35 2.2.7 cDNA Synthesis and Gene Expression ........................................................................ 36 2.2.8 Tissue Processing and Sectioning for Histology ......................................................... 38 2.2.9 Haematoxylin and Eosin (H&E) Stain ......................................................................... 39 2.2.10 Oil Red O Stain .......................................................................................................... 39 2.2.11 Wright-Giemsa Stain ................................................................................................. 40 2.2.12 Serum ALT Activity .................................................................................................. 40 2.2.13 Triglyceride Level Determination.............................................................................. 41 2.2.14 Statistical Analysis ....................................................................................................