AN ABSTRACT OF THE DISSERTATION OF Katerine Schletz Saili for the degree of Doctor of Philosophy in Toxicology presented on November 13, 2012. Title: Developmental Neurobehavioral Toxicity of Bisphenol A in Zebrafish (Danio rerio) Abstract approved: _____________________________________________________ Robert L. Tanguay Billions of pounds of bisphenol A (BPA) are produced annually around the globe for the manufacture of numerous consumer products, including polycarbonate food and water containers, the protective resin linings of food cans, thermal printing paper, and dental fillings. BPA exposure during nervous system development has been associated with learning and behavioral impairments in animal models. The mode of action for these effects is not clearly defined. While BPA is a weak estrogen receptor (ER) agonist, it is also an estrogen-related receptor gamma (ERRγ) agonist. ERRγ binds BPA with 100 times greater affinity than ERs. This study was designed to test the hypothesis that exposure to human-relevant BPA concentrations impacts nervous system development and behavior through ERRγ activation. To examine whether BPA behaves more like an ER or ERRγ ligand, two positive control compounds were used throughout the study: 17β-estradiol (E2) and GSK4716, ER and ERRγ agonists, respectively. Initial behavior testing results included the observation that neurodevelopmental exposure to 0.01 or 0.1 μM BPA led to hyperactivity in larvae, while exposure to 0.1 or 1 μM BPA led to learning deficits in adult zebrafish. Exposure to 0.1 μM E2 or GSK4716 also led to larval hyperactivity. To identify early molecular signaling events that lead to the observed neurobehavioral phenotypes, a global gene expression analysis using a 135K zebrafish microarray was conducted. The concentrations of compounds tested were anchored to the common larval hyperactivity phenotype they elicited. Gross abnormalities, in the case of higher concentrations of BPA and E2, were also anchored phenotypes included in the analysis. Functional pathway analysis of the BPA versus E2 results predicted an impact on prothrombin signaling from the two lower concentrations of BPA and E2. Both BPA and GSK4716 were also predicted to impact nervous system development, potentially involving inhibition of the upstream regulator, SIM1. Additionally, GSK4716 exposure was predicted to inhibit neuron migration. There were fewer similarities in transcriptional responses between BPA and E2 when the lower versus higher concentrations were compared, suggesting different mechanisms operated at the higher concentrations. Subsequent experiments were focused on the role of ERRγ in the larval hyperactivity phenotype. Transient ERRγ knockdown by antisense oligonucleotide morpholino during the first 24 hours of development abrogated the hyperactive phenotype induced by 0.1 µM BPA exposure. Transient ERRγ knockdown during the first 48 hours of development resulted in developmental delays, craniofacial defects, pericardial edema, and severe body axis curvature. This work is the first to identify behavioral effects in a fish from developmental BPA exposure. It is also the first study to confirm a role for ERRγ in mediating BPA’s neurobehavioral effects in any animal model. The global gene expression analysis identified similarities between BPA, E2, and GSK4716, suggesting that BPA’s mode of action may involve crosstalk between ERRγ and other ERs. These results from human-relevant BPA exposures help explain the widely documented in vivo effects of BPA, despite low binding affinity exhibited by nuclear ERs. ERRγ is an evolutionarily conserved vertebrate receptor and the developmental impacts of BPA in the zebrafish are an indication of hazard potential to vertebrates. They are also an important translational step toward knowing the hazard potential from human developmental exposure to BPA and yet unknown environmental ligands of ERRγ. © Copyright by Katerine Schletz Saili November 13, 2012 All Rights Reserved Developmental Neurobehavioral Toxicity of Bisphenol A in Zebrafish (Danio rerio) by Katerine Schletz Saili A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented November 13, 2012 Commencement June 2013 Doctor of Philosophy dissertation of Katerine Schletz Saili presented on November 13, 2012. 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. Katerine Schletz Saili, Author ACKNOWLEDGEMENTS I extend my heartfelt appreciation to Dr. Robert Tanguay for the many opportunities he has provided me while pursuing this work under his mentorship. I thank Robert for his patience, good-humor, and sincere devotion to helping me succeed. His going to bat for me on multiple aspects of my graduate school experience has meant so much to me. I would not have made it this far without his support, and I am indebted to him in ways that cannot be repaid. Thank you to the entire Tanguay Lab, past and present, for their support and friendship. I thank Jane La Du for training me and accepting my quirks, Margaret Corvi for invaluable project support, and Dr. Siba Das for his assistance on this project during a time when it was most needed. Special thanks goes to Dr. Lisa Truong for offering me technological assistance throughout the duration of my graduate school tenure, including thesis formatting assistance. Special thanks also goes to Dr. Michael Simonich, both for teaching me lab techniques and donating his time and superb editing skills to my writing projects. I also thank Dr. Tamara Tal for her peer mentorship. I am grateful to have shared several years in the lab with Jill Franzosa, Dr. Sumitra Sengupta, Dr. Galen Miller, and Britton Goodale, who offered engaging discussions on a myriad of topics. Thank you to the SARL staff, whose efforts provided a constant supply of embryos for experiments; they make it look easy, but it is no small task. Thank you to my committee members: Drs. Jerry Heidel, Mark Leid, Nancy Kerkvliet, and Michael Kent, for valuable advice that has helped guide and define my thesis. Thank you to my collaborators and co-authors, particularly those with whom I worked closely: Jennifer Przybyla and Drs. Susan Tilton, Katrina Waters, and Kim Anderson. I would like to thank all of the collaborators who provided me opportunities and assistance on projects that did not make it into this thesis. Particularly, I thank Dr. Michael Kent, Virginia Watral, Justin Sanders, Dr. Nancy Kerkvliet, and Sam Bradford. Their time and effort is much appreciated, and the lessons and techniques I learned from them are valued components of my graduate school experience. Thank you to the Department of Environmental and Molecular Toxicology, especially the Department Head, Dr. Craig Marcus, and the unsung heroes of the front office who have helped me over the years: Sarah Haluzak, Mary Mucia, Susan Atkisson, Kerry Thomas, Tania Eng, Tania Porter, JayLene Seeley, and Anita Balleby. I also thank my training grant and fellowship administrators. Special thanks to the current Team Tox president, Steven O’Connell, for supporting me during the year I served as president. I am also grateful to Diana Rohlman and Linda Steppan for offering an outlet beyond the Tanguay Lab. To my entire family: thank you for your love and support. Thank you to my grandmother, Gertrude, and my late grandfather, Ray, for helping us get a foothold in a new place after so many years living overseas. Thank you to my grandparents, Carroll and Dorothy, for accepting my life decisions, even when it meant living far away. Thank you to my dad, Joe, for offering his perspectives on BPA, microwaves, future employers, and goats. Thank you to my brothers, Dan and Phil, for the many phone calls of support and helping me keep things in perspective. A special thank you goes to my mom, Carole, and sister, Annie, for everything they did for our family during the “Summer of the Flea” and beyond; they have been a constant source of encouragement throughout my graduate school career. Thank you to my Samoan family for their prayers of support. Malo le tapua’i, fa’afetai tatalo. To Faiilagi: thank you for standing by me, and thank you for cooking. To Niualava: you are my inspiration, even before I knew you, and especially now that you are conducting your own research on oral exposures. Finally, I would like to recognize the family members we lost while I was pursuing this degree: Mose Faiilagi, Tuimalatu Saili Vili, and Raymond Schletz. I acknowledge my funding sources, which made this work possible: the NIEHS Training grant program, WM Keck Foundation, and the EPA STAR Fellowship program. The EPA has not formally reviewed this work. The views expressed in this dissertation are solely my own and do not necessarily reflect the views of the EPA. CONTRIBUTION OF AUTHORS This dissertation is the result of a collective effort by many collaborators from the Tanguay Laboratory, Department of Environmental and Molecular Toxicology, Pacific Northwest National Laboratory, University of Wisconsin-Milwaukee, and Wenzhou Medical College. Chapter 2: Margaret Corvi provided technical support by collecting samples for tissue uptake analysis, Jennifer Przybyla conducted the HPLC analysis of the uptake