Western University Scholarship@Western Electronic Thesis and Dissertation Repository 12-12-2017 2:00 PM Effects of Prenatal Bisphenol A Exposure on Adrenal Gland Development and Steroidogenic Function Samantha Medwid The University of Western Ontario Supervisor Yang, Kaiping The University of Western Ontario Graduate Program in Physiology and Pharmacology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Samantha Medwid 2017 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Hormones, Hormone Substitutes, and Hormone Antagonists Commons Recommended Citation Medwid, Samantha, "Effects of Prenatal Bisphenol A Exposure on Adrenal Gland Development and Steroidogenic Function" (2017). Electronic Thesis and Dissertation Repository. 5071. https://ir.lib.uwo.ca/etd/5071 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Developmental exposure to bisphenol A (BPA), a ubiquitous endocrine disrupting chemical, is associated with organ dysfunction and diseases in adulthood. However, little is known about its effects on the adrenal glands. Therefore, this thesis addresses this important question using both in vivo and in vitro approaches. BPA at environmentally relevant doses was administrated via diet to pregnant mice from embryonic day 7.5 to birth, following which mice were switched to a standard chow. At two months postnatally, adrenal glands and blood samples were collected from adult mouse offspring for structural and functional analysis. I found that: (a) BPA increased adrenal gland weight as well as plasma corticosterone levels; (b) BPA did not alter plasma levels of ACTH; and (c) BPA stimulated expression of the two key steroidogenic factors, steroidogenic acute regulatory protein (StAR) and cyp11A1 in female but not male offspring. To determine the molecular mechanisms underlying the BPA-induced StAR expression, I used human fetal adrenal cortical H295A cells as an in vitro model system, and showed that BPA increased StAR protein expression likely through an estrogen receptor (ER)-mediated mechanism independent of StAR gene transcription, translation and protein half-life. I then investigated the molecular mechanisms underlying the BPA- induced increase in adrenal gland weight using the same in vitro model system. I demonstrated that (a) BPA increased cell number and protein levels of the three universal markers of proliferation (proliferating cell nuclear antigen (PCNA), cyclin D1 and D2, as well as sonic hedgehog (shh) and its key transcriptional regulator Gli1; (b) cyclopamine, a shh pathway inhibitor, blocked these stimulatory effects of BPA on cell proliferation; (c) BPA increased the nuclear translocation of ERβ; and (d) the ERb-specific agonist DPN mimicked while the ERb antagonist PHTPP abrogated the stimulatory effects of BPA on cell proliferation, and prevented BPA-induced activation of the shh signaling. Taken together, these findings demonstrate that developmental exposure to BPA adversely affects adrenal gland development and steroidogenic function in adult mouse offspring. Furthermore, they reveal novel molecular signaling mechanisms of BPA actions in regulating adrenal steroidogenic function and adrenal cortical cell proliferation. ii Keywords Bisphenol A, endocrine disruptor, adrenal gland, steroidogenesis, steroidogenic acute regulatory protein, fetal development, estrogen receptor, sonic hedgehog iii Co-Authorship Statement Chapter 3: Medwid S, Guan H, and Yang K (2016) Prenatal exposure to bisphenol A disrupts adrenal steroidogenesis in adult mouse offspring. Environ Toxicol Pharmacol. 43: 203-8 SM, GH, and KY designed the experiments. SM was responsible for animal care, blood and tissue collection and conducted the experiments. SM, GH and KY analyzed and interpreted the data. SM and KY wrote the manuscript. All authors approved the final version of the manuscript. Chapter 4: Medwid S, Guan H, Yang K (2017) Bisphenol A induces steroidogenic acute regulatory protein (StAR) expression via an unknown mechanism independent of transcription, translation and protein half-life in human adrenal cortical cells. Submitted Steroids. SM, GH and KY designed experiments. SM conducted all experiments. SM, GH and KY analyzed and interpreted the data. SM and KY wrote the manuscript. All authors approved the final version of the manuscript. Chapter 5: Medwid S, Guan H, Yang K (2017) Bisphenol A stimulates adrenal cortical cell proliferation via ERβ-mediated activation of the sonic hedgehog signaling pathway. Submitted Journal of Steroid Biochemistry and Molecular Biology. SM, GH and KY designed experiments. SM conducted all experiments. SM, GH and KY analyzed and interpreted the data. SM and KY wrote the manuscript. All authors approved the final version of the manuscript. iv Acknowledgments I would first like to express my deepest appreciation to my advisor, Dr. Kaiping Yang, whose expertise, leadership, and encouragement has been invaluable to me throughout this whole experience. I appreciate all your guidance as it has helped me to mature as a researcher and an individual. It is under your direction that I have discovered the confidence in my capacity to grow, both academically and personally. I will always remember the lessons I have learned from you as I begin the next chapter of my life. I would like to thank my advisory committee members, Dr. Dean Betts, Dr. Andy Babwah, Dr. Dan Hardy, and Dr. Rommel Tirona. Your guidance, advice and support through my graduate studies is deeply appreciated. I would like to express my heartfelt appreciation of Dr. Haiyan Guan. Thank you for sharing all your technical expertise and support with me over the years but above all, thank you for your amazing friendship. Special thanks to all the members of the Yang lab, including Dr. Maria Cernea, Dr. Ayten Hijazi, Bianca DeBenedictis, Colleen Westerman and Maria Abou Taka. Thank you, to all the other CHRI trainees for all the many coffee breaks, especially, Amanda Oakie, Jason Peart, Kurt Berger, and Phyo Win. Throughout the years, each has had an impact in my life in some way or another. The friendships we have created over the years will always remain in my life and I will remember all the great times we shared together. Lastly, I would like to thank my parents and my partner. Thank you to my mom and dad for their constant encouragement and endless love, which kept me motivated during difficult times. You have taught me that I can achieve anything I put my mind too. Lastly, my partner and best friend, Martin, thank you for always supporting and believing in me. Without the support and encouragement of my family, this process would have been much more difficult. Thank you! v Table of Contents Abstract ................................................................................................................................ i Co-Authorship Statement ................................................................................................... iii Acknowledgments .............................................................................................................. iv Table of Contents ................................................................................................................ v List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi List of Appendices ........................................................................................................... xiii List of Abbreviations ....................................................................................................... xiv 1 INTRODUCTION ......................................................................................................... 1 1.1 Bisphenol A ............................................................................................................ 2 1.1.1 Mechanism of Action .................................................................................. 3 1.1.2 Dose Response and Low Dose Effects ....................................................... 6 1.1.3 Pharmacokinetics ........................................................................................ 7 1.1.4 Human Exposure to BPA .......................................................................... 11 1.2 The Adrenal Gland ................................................................................................ 13 1.2.1 The HPA Axis ........................................................................................... 16 1.2.2 Physiological Function.............................................................................. 18 1.2.3 Adrenal Development ............................................................................... 21 1.2.4 Adrenal Remodeling and Growth ............................................................. 26 1.2.5 Sex Specificity in Adrenal Glands ............................................................ 34 1.3 Adrenal Steroidogenesis ....................................................................................... 34 1.3.1 Steroidogenic Pathway .............................................................................