AN ABSTRACT OF THE DISSERTATION OF Derik Haggard for the degree of Doctor of Philosophy in Toxicology present on December 2, 2016. Title: Classifying Chemical Bioactivity by Coupling High-throughput Phenotypic Anchoring and Transcriptome Profiling in Zebrafish. Abstract approved: ______________________________________________________________ Robert L. Tanguay There are more than 87,000 chemicals in current use with little to no toxicity information available. Assessing such a large number of chemicals using traditional methods would take an unreasonable amount of time and money, and require the use a large number of animals. The incorporation of high- throughput in vivo model systems that can overcome limitations of in vitro screens while providing a rapid genome-wide view of chemical bioactivity are needed to help fully realize the vision of 21st century toxicity testing. In this dissertation, I employed transcriptomics using the high-throughput in vivo developmental zebrafish model to generate hypotheses regarding the mechanism of action of specific chemicals and to identify unique transcriptional signatures for a set of endocrine disrupting chemicals that can be used to classify the activity of known and unknown chemicals. We employed comparative transcriptomics in wild-type and ahr2-null zebrafish exposed to mono-substituted isopropylated triaryl phosphate (mITP), a component of the Firemaster 550 flame retardant mixture, and demonstrated that the cardiotoxic effects of mITP are likely mediated through inhibition of retinoic acid receptor signaling and not the aryl hydrocarbon receptor (AhR). Although mITP induced a transcriptional profile indicative of AhR activation in wild-type zebrafish, these signatures were not related to cardiotoxicity. I found that mITP exposure, independent of ahr2 status, decreased the expression of many Hox genes as well as enzymes responsible for retinoic acid metabolism, which are known to be regulated by retinoic acid receptors. Several of the dysregulated Hox genes are involved in cardiac cell lineage determination in early heart development, and in heart tube elongation and looping via cell recruitment in the second heart field. I also employed transcriptomics to investigate the mechanism of developmental toxicity of the antimicrobial agent, triclosan (TCS). Exposure to TCS resulted in robust transcriptome changes with a large number of transcripts being significantly decreased. Downstream functional analyses showed that many of the transcripts significantly affected by TCS are involved in liver functioning and development, suggesting that TCS is hepatotoxic in embryonic zebrafish. I also compared our transcriptomic analysis with the comprehensive in vitro bioactivity profile of TCS from the Environmental Protection Agency’s Toxicity Forecaster (ToxCast) program, and observed low concordance between these two screening strategies. Lastly, I conducted transcriptome profiling of 25 endocrine disrupting chemicals in order to identify discriminatory transcriptional signatures that would help classify chemicals with estrogen, androgen, or thyroid hormone activity. Clustered correlation analysis of the top 1000 significantly differentially expressed transcripts revealed four chemicals with highly similar and unique transcriptional profiles compared to the other 21 chemicals. These four chemicals included three known thyroid hormone receptor agonists, and one unknown chemical, which was later identified as a failed pharmaceutical with thyroid receptor agonist activity. I identified a panel of 27 transcripts as well as a unique pigmentation phenotype for these four chemicals which can be used in future chemical screens to detect other thyroid receptor agonist chemicals in zebrafish. Overall, the work presented here demonstrates the utility of phenotypically anchored whole genome transcriptomics in zebrafish to identify putative mechanisms of action for many chemicals. Furthermore, the information obtained from these types of analyses can be used to develop predictive models to classify the bioactivity of known and unknown chemicals, or can identify biomarkers that can be used in future high-throughput screens. Copyrighted by Derik Haggard December 2, 2016 All Rights Reserved Classifying Chemical Bioactivity by Coupling High-throughput Phenotypic Anchoring and Transcriptome Profiling in Zebrafish. by Derik Haggard A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented December 2, 2016 Commencement June 2017 Doctor of Philosophy dissertation of Derik Haggard presented on December 2, 2016. APPROVED: Major Professor, representing Toxicology Head of the Department of Environmental and Molecular Toxicology Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Derik Haggard, Author ACKNOWLEDGEMENTS There are many people that have aided, provided guidance, and given me the confidence to pursue a career in toxicology. First and foremost, I want to express my most sincere appreciation to Dr. Robert Tanguay for being my research advisor and mentor throughout my time at Oregon State University. Not only did he allow me to join the amazing research environment his lab represents, but he has provided continuous support and opportunities to better myself as a scientist and as a person. Without his guidance and encouragement, I would not be who I am today, nor would I have discovered the challenge, excitement, and rewards the field of computational toxicology has to offer. I would also like to extend my deepest gratitude to my undergraduate mentor, Dr. Sara Heggland, who was kind enough to allow a lowly freshman the opportunity to experience a research lab, and had the patience to keep me there for four years. I would never have been inspired to continue on to graduate school, nor would I have found a love of researching endocrine disrupting chemicals without her. Dr. Pamela Noyes also deserves particular thanks for her help and constructive discussions on the experimental design of my dissertation research described in Chapter 4. From surviving the challenging summer of 2014, to her assistance with screening and helping in the mornings of RNA collections, her help was paramount to the success of that project, and I am so very thankful. There have been so many great friendships and thoughtful discussions with so many people over my 6 years here that acknowledging everyone would likely double the length of this dissertation. I want to thank all of the members of the Tanguay lab that I have worked with: Jane LaDu for training me when I first joined that lab; Siba Das for being my primary mentor during my rotation in the lab; Jill Franzosa, Britton Goodale, Galen Miller, Margaret Corvi, Kate Saili, Kitae Kim, Andrea Knecht, Joe Fisher, Leah Wehmas, Yasmeen Nkrumah-Elie, and Sean Bugel for being the original members of the lab when I joined, who had profound effects on my initial research directions, and gave thoughtful advice and encouragement as I was finding my place in the lab; Cory Gerlach, Amber Roegner, Mitra Geier, Anna Chlebowski, Mike Garland, Lindsay St. Mary, Paroma Chatterjee, Courtney Roper, and Gloria Garcia for our conversations and assistance during my later stages in the lab; Chris Sullivan for help and support at the command line throughout my time here. I would also like to thank all of my committee members (both past, present, and temporary) who have helped guide my research and evaluate my ability to be a successful scientist: Nancy Kerkvliet, Michael Freitag, Siva Kolluri, David Hendrix, Kim Anderson, and Anna Harding. I would like to thank the staff at the Sinnhuber Aquatic Research Laboratory for all of their help and support: Chapell Miller, Greg Gonnerman, Eric Johnson, Michael Simonich, and Carrie Barton. Lisa Truong for all of her help with manuscript organization and career advice and encouragement. I would also like to thank Katrina Waters at the Pacific Northwest National Laboratory, for guidance during the chemical selection process and microarray analysis advice for the research presented in Chapter 3 and 4. I would also like to thank the many people in the Department of Environmental and Molecular Toxicology for their support, conversations, administrative assistance, and for allowing me the opportunity to even apply as a graduate student to this esteemed research program. I also thank the National Institutes of Health and the Environmental Protection Agency who provided the funding necessary to conduct my studies. Throughout my time here, I have made a many great friendships from people affiliated and unaffiliated with the research department for which I am deeply grateful. To those who helped keep my sanity by climbing, exploring, going to Timbers games, and showing me the joys of surfing on the Oregon Coast. I am so thankful to TJ Newton, for his love and support through the struggles, anxieties, and trials of being with a graduate student/mad scientist. Lastly, I would like to thank my family. To my brother, Mason: Thank you for always being there and letting me enjoy our mutual love of fly fishing with you whenever I visited. To my father: Thank you for always encouraging my pursuits in higher education and for teaching me to be independent. To my mother: I would have never made it this far without your love