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By Ivy D. Moffat a Thesis Submitted in Conformity with the Requirements For MECHANISMS OF GENETIC RESISTANCE TO DIOXIN-INDUCED LETHALITY by Ivy D. Moffat A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmacology and Toxicology University of Toronto © Copyright by Ivy D. Moffat, 2008 Mechanisms of genetic resistance to dioxin induced lethality Ivy D. Moffat Doctor of Philosophy, 2008 Graduate Department of Pharmacology University of Toronto THESIS ABSTRACT Dioxins are environmental contaminants that raise concern because they are potent and persistent. The most potent dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a wide variety of biochemical and toxic effects in laboratory animals and in humans. Major toxicities of TCDD are initiated by their binding to the AH receptor (AHR), a ligand-activated transcription factor that regulates expression of numerous genes. However, the specific genes whose dysregulation leads to major toxicities such as wasting, hepatotoxicity, and lethality are unknown. The objective of this thesis research was to identify the molecular mechanisms by which dioxins cause lethality. To this end, a powerful genetic rat model was utilized – the Han/Wistar (Kuopio) rat which is highly resistant to dioxin toxicity due to a major deletion in the AHR’s transactivation domain (TAD) leading to 3 potential AHR variant transcripts. We found that insertion-variant transcripts (IVs) are the dominant forms of AHR expressed in H/W rats, constitutively and after TCDD treatment. Gene expression array analysis revealed that the total number of TCDD-responsive genes in liver was significantly lower in H/W rats (that carry the TAD deletion) than in dioxin-sensitive rats (that carry wildtype AHR). Genes that are well-known to be AHR-regulated and dioxin-inducible − such as CYP1 transcripts − remained responsive to TCDD in H/W rats; thus the TAD deletion selectively interferes with expression of a subset of hepatic genes rather than abolishing global AHR-mediated ii responses. Genes that differed in response to TCDD between dioxin-sensitive rats and dioxin-resistant rats are integral parts of pathways known to be disrupted by dioxin treatment such as protein synthesis/degradation, fatty acid transport/metabolism, and apoptosis. These genes are worthy candidates for further mechanistic studies to test their role in major dioxin toxicities. Numerous differentially-regulated genes were downregulated; however, microRNAs, which downregulate mRNA levels in other systems, likely play no role in downregulation of mRNAs by dioxins in adult liver and are unlikely to be involved in hepatotoxicity. Findings in this research support the hypothesis that H/W rats are resistant to TCDD lethality because the TAD deletion prevents the AHR from dysregulating specific mRNA transcripts but not hepatic miRNAs. iii ACKNOWLEDGEMENTS The foundation and support for these research projects was laid by many amazing people to whom I owe a world of gratitude. A very special “thank you” to: [ Dr. Allan Okey, there are not enough words to express my gratitude. Though opportunity, advice, and independence you have trained many high caliber scientists; I am privilege to be among these fortunate scientists. [ Raimo Pohjanvirta for a highly productive collaboration, exhaustive knowledge of molecular and biochemical physiology, and fruitful scientific discussions. [ PhD advisory committee: Dr. Riddick, Dr. Harper, and Dr. Grant for their genuine interest and guidance since the beginning of this research. Thank you for challenging this research and polishing this scientist. [ Amazing friends who are more like family. Michael Dorr, Lucy Reed, Howard Kim, Heather Runions, Jenny & Katherine Clark, Martha Weber, and Jane Macaulay thank you for sharing in my best times and making me laugh in the worst. [ Paul Boutros, a valued colleague, for numerous discussions and statistical advice. [ Trine Celius for valuable conversations both scientific and other, you made the thesis writing experience more enjoyable. [ The chocolate manufacturing industry and to Paracelsus who stated “it is the dose that makes the poison”, a concept to guide research and life. [ The Canadian Institutes of Health Research, the Academy of Finland, the Natural Sciences & Engineering Council, and the University of Toronto for financial support of this research. While this research focused on the genetics of the Han/Wistar (Kuopio) rat, it was powered by the genetics of my parents. In particular, the ‘curiosity’ gene and the ‘persistence’ gene to which I owe my father a great deal. I am also grateful to all my family members, who allowed me the independence to follow my dreams in the ‘big city’, but were always there when times got tough. iv TABLE OF CONTENTS THESIS ABSTRACT ....................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................ iv TABLE OF CONTENTS ................................................................................................. v LIST OF PUBLICATIONS ..........................................................................................viii ABBREVIATIONS.......................................................................................................... ix LIST OF TABLES ........................................................................................................... xi LIST OF FIGURES ........................................................................................................ xii CHAPTER 1: General Introduction ............................................................................... 1 Dioxins and dioxin-like compounds ............................................................................... 1 Effects of dioxins in humans........................................................................................... 3 Acute effects of dioxins in laboratory animals ............................................................... 4 Adaptive response................................................................................................................... 4 Wasting syndrome .................................................................................................................. 5 Hepatotoxicity......................................................................................................................... 5 Tumor promotion.................................................................................................................... 6 Acute lethality......................................................................................................................... 6 The AH Receptor: Transcription factor and essential mediator of dioxins toxicities..... 7 Repertoire of ligands which bind to the AHR ...................................................................... 13 Structure of the AHR............................................................................................................ 14 The AHR mediates dioxin toxicities..................................................................................... 16 Expanding the AHR-mediated transcriptome in relation to TCDD lethality and associated toxicities ................................................................................................. 30 Potential model systems to identify genes relevant to dioxin lethality and associated toxicities................................................................................................................... 20 Humans................................................................................................................................. 20 Animal models...................................................................................................................... 21 The TCDD-resistant H/W rat model..................................................................................... 23 AIMS OF MY THESIS RESEARCH ....................................................................... 28 SPECIFIC AIMS OF EACH THESIS PROJECT .................................................. 29 CHAPTER 2: Aryl Hydrocarbon Receptor (AHR) Splice Variants in the Dioxin- Resistant Rat: Tissue Expression and Transactivational Activity .. 36 ABSTRACT.................................................................................................................. 37 INTRODUCTION ........................................................................................................ 38 MATERIALS & METHODS ....................................................................................... 41 RESULTS ..................................................................................................................... 48 v Insertion variants were the predominant AHR splice-variant transcripts constitutively expressed in dioxin-resistant rats...................................................................................... 48 Dioxin treatment increases expression of AHR splice-variant transcripts. .......................... 49 Functional differences in intrinsic transactivation activity among the splice variants ......... 51 AHR structures predicted in silico: comparison between species and within species.......... 54 DISCUSSION............................................................................................................... 59 Constitutive expression of splice variants ...........................................................................
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