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Early-Life Exposure to the Antidepressant Fluoxetine Induces A Early-life exposure to the antidepressant fluoxetine induces a male-specific transgenerational disruption of the stress axis and exploratory behavior in adult zebrafish, Danio rerio Marilyn Nohely Vera Chang Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate in Philosophy degree in Biology with specialization in Chemical and Environmental Toxicology Department of Biology Faculty of Science University of Ottawa © Marilyn Nohely Vera Chang, Ottawa, Canada, 2018 ACKNOWLEDGMENTS Finishing this thesis has been both a challenging and gratifying experience. There are many people whom I must express my sincere gratitude toward as the work presented in this thesis would not have been possible without their contributions and never-ending support. First and foremost, I would like to acknowledge my supervisors, Dr. Vance Trudeau and Dr. Thomas Moon. I want to say how grateful I am for providing me with the opportunity to purse my academic and professional interests. I was first accepted by Tom and I will be forever thankful that he took a shot on me. Also, thanks to you Vance for adopting me when I was lab-less and for believing in my abilities as a scientist. Thank you to both of you for your outstanding guidance, patience and friendship which have given me an amazing experience in these past 6 years, you have been excellent mentors. Your concerns for your student’s well-being and mental health were bonus that I will not soon forget. I don’t think there are enough words to describe how thankful and fortunate I am to have benefit from your supervision not only at the academic and scientific level but also at the personal level, for all my accomplishments during this doctorate, I will forever be indebted to both of you. To my Ph.D. advisory committee, Dr. Marie-Andrée Akimenko, Dr. Michael Jonz and Dr. William Willmore, I would also like to express my sincerest gratitude for your time, and insightful and valuable suggestions. To my dear friend and mentor, Dr. James Fenwick, I express my heartfelt gratitude, and deep regards for all your time invested on me and foremost for your friendship and exceptional guidance. You will always be a very special person in my life. To Dr. Katie Gilmour, even if you were not my supervisor, I consider you a great mentor. Thank you from the bottom of my heart for having always kept your door open and for being there ii when I needed it either on the professional / academic level as well as on the personal level. Thanks again! To my fellow colleagues: the Moon and TeamENDO research group, both past and present members and to the numerous colleagues and friends I met throughout my 6 years of Ph.D., I want to express my gratitude to all of you, for your support and for providing a fun and friendly work environment. I will not forget the words of encouragement when an experiment did not work or our celebrations/parties…what good times we had!!!, they will not be forgotten. I would also like to thank each of my family members, those ones close and the ones back in Peru, whose encouragement and support during the course of my studies was greatly appreciated. To my brother and sister, Martin and Kattia, my brother-in-law Andrei and my lovely nephew Gabriel, thanks for all your love and constant encouragement. Particularly to my parents, Carolina Chang and Saul Vera for having always looked out for us and for giving me this amazing opportunity to thrive in Canada even if it meant a lot of sacrifices for both of you, gracias Papis, los amo! Lastly, I would like to especially thank and acknowledge my thesis writing buddy and companion for life, Antony D. St-Jacques, whose patience, care, support and understanding have given me the confidence to keep pushing through and reached the final line. Tony, I know one day, we will be looking back at these times and even though we will have probably suppressed these memories of sleepless nights, horrible headaches, and all the down-sides that we went through, I know for sure that we will be happy to have accomplished this together. It was an amazing experience and a great encouragement to know that I was not alone in the science building late at night. iii ABSTRACT Selective serotonin (5-HT) reuptake inhibitors (SSRIs) particularly fluoxetine (FLX, Prozac®), are often the first-line of pharmacological treatment for affective disorders in pregnant women. Given that SSRIs readily cross the placenta, a fetus from a SSRI-treated pregnant woman is potentially at risk from the disruptive effects of the SSRIs-induced 5-HT actions during this highly plastic stage of development. One of the prominent roles of 5-HT that we will explore here is its involvement in the development and programming of the stress axis. Pharmaceuticals including FLX and other SSRIs reach aquatic ecosystems through sewage release, so fish may also be inadvertently exposed. We investigated the premise that early-life exposure to FLX induces a transgenerational disruption of the stress axis using the zebrafish (ZF) Danio rerio, as a research model to encompass both the environmental and human health concerns. The FLX concentrations studied were environmentally relevant (0.54 µg·L-1) or comparable to concentrations detected in the cord blood of FLX-treated pregnant women (54 µg·L-1). Exposure to FLX during the first 6 days of life induced a reduction of whole-body cortisol levels in adult ZF (filial generation 0; F0), an effect that persisted across 4 consecutive generations without diminution, even though the descendants (F1 to F4) were not directly exposed to FLX. This effect was more pronounced and persistent in males than females. The in vivo cortisol response of the interrenal cells (the fish ‘adrenal’) to an intraperitoneal injection of adrenocorticotropic hormone was also reduced in the F0 and F3 FLX-exposed males. RNA sequencing of the F0 and F3 male whole kidney containing the interrenal cells detected an array of differentially expressed genes (>500) altered by FLX treatment. Enrichment analysis of these genes revealed that early FLX exposure significantly modified numerous canonical pathways (>40) including key pathways related to steroidogenesis. These findings provide further insights on the underlying mechanism iv of the transgenerational disruption induced by FLX. We also showed that altered cortisol levels were linked to reduced exploratory behavior in adult males from the F0 to F2 FLX lineage. In contrast, females were susceptible to the effects of FLX-induced hypocortisolism during a different window of development. Exposure to FLX in the sex differentiation period (15 to 42 days post- fertilization) increased exploratory behavior in the adult females. Transcriptional profile of selected steroidogenic genes in the whole-larvae from the F0 varied in magnitude and direction in both treatments, despite the same low cortisol phenotype induced by both concentrations. We also found an up-regulation in the transcript levels of steroidogenic-related genes and a down- regulation of a gene involved in the inactivation of cortisol in the F3 larvae ancestrally exposed to the human-relevant concentration. These findings on the transcript levels of the selected genes in the larvae from F0 and F3 suggest that the larvae adopted specific coping mechanism(s) to the disruptive effects of FLX depending on the exposure concentration and the filial generation. The pigmentation patterns in some of the descendants of the exposed fish (F1 to F3) were reduced by the 6-day early-FLX treatment. In response to a 6-day embryonic exposure to a second antidepressant, venlafaxine, the F4 adult females that were ancestrally exposed (in the F0) to the human-relevant FLX concentration displayed an intensified reduction of cortisol levels. Therefore, FLX exposure of the great-great-grandparents (F0) permanently and most likely epigenetically shaped the response of future generations to other antidepressants. Collectively, our data are cause for concern, given the high-prescription rates of FLX to pregnant women and the potential long- term negative impacts on humans and aquatic organisms exposed to ever rising levels of SSRIs. v TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................ ii ABSTRACT .................................................................................................................................. iv LIST OF FIGURES ...................................................................................................................... x LIST OF TABLES ..................................................................................................................... xiv LIST OF ABBREVIATIONS ................................................................................................... xvi General Introduction ........................................................................................... 1 1.1. Rationale for the study ......................................................................................................... 1 1.2. Depression ............................................................................................................................ 3 1.3. The Selective Serotonin Reuptake Inhibitor (SSRI) family ................................................ 6 1.3.1. SSRI mechanism of action ........................................................................................ 8 1.3.2. Maternal transfer
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