A Toxicogenomic Study of the Hepatocarcinogen Furan by Anna Francina Webster (Jackson) A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Carleton University Ottawa, Ontario © 2015, Anna Francina Webster Abstract A major goal in human health risk assessment is the identification and management of chemicals that may cause cancer in human populations. The current gold- standard for assessing chemical carcinogenicity is the two-year rodent cancer bioassay, which is animal, time, and resource intensive. The use of toxicogenomics for chemical risk assessment was first proposed over 15 years ago because of its potential to produce toxicologically relevant data more quickly, using fewer animals, and at a lower cost than the two-year cancer bioassay. While the two-year cancer bioassay produces a detailed inventory of chemical-dependent lesions, toxicogenomics analyzes chemical-dependent changes to global gene expression. Moreover, toxicogenomics provides comprehensive mechanistic data that are not obtained using standard tests. In this thesis quantitative, predictive, and mechanistic approaches were applied to a toxicogenomic case study of the rodent hepatocarcinogen furan. Female B3C6F1 mice were exposed for three weeks to non-carcinogenic or carcinogenic doses of furan. The dose response of a variety of transcriptional endpoints produced benchmark doses (BMDs) similar to the furan- dependent cancer BMDs. Bioinformatic analysis of disease datasets showed strong similarity between global gene expression changes induced by furan and those associated with the appropriate hepatic pathologies. The molecular pathways that were enriched in the liver following furan exposure facilitated the development of a molecular mode of action (MoA) for furan-induced liver cancer. Finally, transcriptional changes in formalin- fixed and paraffin embedded (FFPE) samples were compared to high quality frozen samples in order to evaluate whether archival samples are a viable option for toxicogenomic studies. The advantage of using FFPE tissues is that they are very well ii characterized (phenotypically); the disadvantage is that formalin degrades biomacromolecules, including RNA. FFPE samples as old as three decades were shown to be feasible for toxicogenomics studies using a ribo-depletion RNA-seq protocol. Taken together, this case study demonstrates the utility of toxicogenomics data in human health risk assessment and the potential of archival FFPE tissue samples, and identifies viable strategies toward the reduction of animal usage in chemical testing. iii Acknowledgements I have had a wonderful 4.5 years at Carleton University and Health Canada. For this I must thank my supervisors, Iain Lambert and Carole Yauk. To Iain, for encouraging me to massively change projects (from bioremediation of contaminated soils, to toxicogenomics and FFPE genomics); and to Carole, for taking it from there. As I think is often the case, I entered this degree expecting to do one thing, and am leaving having done something completely different. Carole, my accomplishments are largely a product of your support, encouragement, and imagination. Thank you for giving me the opportunity to be a part of the Health Canada Genomics Lab, what a wonderful team of people you have built. You are an exceptional leader, scientist, (and the life of the party). In addition to learning about genomics, from you I learned a great deal about being a good collaborator, communicator, and writer. These important skills will, I’m sure, be invaluable as I move forward in my career. Iain, thank you for letting me turn your office into a thesis writing headquarters over these final few months of writing. I am sure that this process would have taken much longer had I not had you as my thesis writing chaperone. Thank you also for helping me ‘put fog in a box’ as I waded through my massive transcriptomic datasets. I sure am glad that I followed up on my email to you back in 2010! To my OCIB buds! Thank you so much Julie Cox, Marc Beal, and Ali Long for making my last year so memorable! I am so proud of all of our work on the 12th Annual OCIB Symposium: The Next Generation. What a great experience. I don’t have enough adjectives to describe you guys: creative, enthusiastic, dedicated, hardworking, persistent, iv resourceful, and wonderful; well, that’s a start. Also, thank you for being such great travel buddies over the years: King’s Canyon and Sequoia National Parks (‘Francina stop reading and look out the window!’), San Francisco (‘I’m gonna git’chu….’), Monterey (Aquarium!), Orlando (Disney!), Miami (and the Bahamas for a weekend!), Brazil (‘Aaaabacackzi’), and Philadelphia and Newark (Road trip!); so many adventures! To my other Environmental Health Center (EHC) and Carleton friends (especially Sarah Labib and Matt Meier), thank you so much for your help, advice, and friendship over the years. I need to thank my colleagues at the EHC. Ivy Moffat for training me when I started; Andrew Williams for processing all of my raw data and making some great figures with my data; Remi Gagne for coordinating with Genome Quebec; and Byron Kuo for running my data through BMDExpress. I feel particularly lucky to have worked with Julie Buick and Andrea Rowan-Caroll, who were always available to help me out or to have a chat. I also need to thank my American collaborators at ILS1 in NC (especially Leslie Recio and Michael Waters) and at the Weill Cornell Medical College in NY (especially Paul Zumbo and Chris Mason). Les, thank you for coordinating the animal study that produced the samples that my entire project was based on. Mike, thank you for pulling me into the EMGS community by asking me to co-chair the Toxicogenomics Symposium in Orlando, and for asking me to contribute a chapter to your book. Finally, thank you to all of the members of the ILSI HESI 2 FFPE 3 Genomics Working Group for all of your ideas and work on this project. 1 Integrated Laboratory Systems, Inc. (www.ils-inc.com) 2 International Life Sciences Institute, Health and Environmental Sciences Institute (www.hesiglobal.org) 3 Formalin-fixed and paraffin embedded v I would like to thank my committee members, Bruce McKay, Bill Willmore, and Paul White, for supporting and encouraging me. Further, I would like to acknowledge that this work was made possible by funds provided to me by NSERC 4, OGS 5, RAP 6, and Carleton University, and funds provided for the project by the Health Canada GRDI 7, ILS, HESI, and the NTP 8. Finally, I need to thank my best friend and husband, Richard Webster. Thank you for keeping me fed and on track during this final dash. I am looking forwad to getting back out onto the water with you! Thank you everyone! 4 The Natural Sciences and Engineering Research Council of Canada (www.nserc-crsng.gc.ca) 5 The Ontario Graduate Scholarship 6 The Federal Government of Canada’s Research Affiliate Program 7 Genomics Research and Development Initiative (grdi-irdg.collaboration.gc.ca) 8 National Toxicology Program (http://ntp.niehs.nih.gov/) vi Table of Contents (brief) Chapter Page 1 Introduction 2 2 Case Study on the Utility of Hepatic Global Gene Expression 31 Profiling in the Risk Assessment of the Carcinogen Furan 3 Bromodeoxyuridine treatment to measure Hepatocellular 75 Proliferation does not Mask Chemically Induced Gene Expression Changes in Mouse Liver 4 Impact of Platform and Statistical Filtering on Transcriptional 94 Benchmark Doses and Multiple Approaches for Selection of Point of Departure 5 Preparation of Archival FFPE Mouse Samples for use with the 127 Agilent Gene Expression Microarray Platform 6 Mining the Archives: A Cross-Platform Analysis of Gene 155 Expression Profiles in Archival Formalin-Fixed Paraffin- Embedded Tissue 7 Final Discussion 187 Appendix A Gene Expression Analysis of Livers from Female B6C3F1 Mice 238 Exposed to Carcinogenic and Noncarcinogenic Doses of Furan, with or without Five Days Bromodeoxyuridine Treatment B Differential Expression of Long Non-Coding RNAs int he Livers 251 of Female B6C3F1 Mice Exposed to the Carcinogen Furan C Supplemental Material 278 vii Table of Contents (full) ABSTRACT ...................................................................................................................... II ACKNOWLEDGEMENTS ........................................................................................... IV TABLE OF CONTENTS (BRIEF).............................................................................. VII TABLE OF CONTENTS (FULL) .............................................................................. VIII LIST OF TABLES ....................................................................................................... XVI LIST OF FIGURES .................................................................................................. XVIII LIST OF APPENDICES ............................................................................................. XXI LIST OF ABBREVIATIONS ................................................................................... XXII STATEMENT OF CONTRIBUTIONS .................................................................... XXV 1 CHAPTER: INTRODUCTION ................................................................................. 2 1.1 Chemical Testing and Toxicogenomics .............................................................................................. 2 1.2 Reducing Animal Use in Toxicity Testing