UNIVERSITY OF CALIFORNIA RIVERSIDE Mechanisms of Selenomethionine and Hypersaline Developmental Toxicity in Japanese Medaka (Oryzias latipes) A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Environmental Toxicology by Allison Justine Kupsco August 2016 Dissertation Committee: Dr. Daniel Schlenk, Chairperson Dr. Morris Maduro Dr. Nicole zur Nieden Copyright by Allison Justine Kupsco 2016 The Dissertation of Allison Justine Kupsco is approved: Committee Chairperson University of California, Riverside Acknowledgements This dissertation would not have been possible without the support of many people. First, my advisor, Dr. Dan Schlenk, was invaluable in not only his guidance and resources for this project, but also in providing me with the opportunities to grow and develop as a scientist. Without the time and effort he put into guiding me, this would not have been possible. In addition to advice about this dissertation, he also provided me with numerous networking and presentation opportunities through attendance of many conferences. I would also like to thank my committee members, Dr. Nicole zur Nieden and Dr. Morris Maduro for their support and advice. There have been many past and present labmates that have also aided in the dissertation through training, advice and emotional support. Dr. Aileen Maldonado, Dr. Lindley Maryoung, and Dr. Jordan Crago all participated in training me in lab techniques. Rafid Sikder was always eager to help and assisted in fish care and in data collected in Chapter 2. Luisa Becker Bertotto, Scott Coffin, Marissa Giroux and Sara Vliet provided important emotional support and entertainment during long lab hours. And especially, I want to thank Graciel Diamante for her invaluable advice, commiseration and emotional support throughout our 4 years working together. Without her this would truly have not been possible. I would like to acknowledge several collaborators and funding sources in conjunction with this work. Firstly, Rodney Johnson and Frank Whiteman at the USEPA Mid-Continent Ecology Division Laboratory, Duluth, MN, provided the medaka cultures used in this research. David Lyons provided much technical assistance in chemical analysis. I would like to thank Greggory Britten, at University of California-Irvine for his iv statistical guidance. I would also like to thank Roger Phillips and Eric Kingsley at Monterey Bay Aquarium, Monterey, CA, for providing water samples and analysis for Chapter 2. This research was supported by the National Water Research Institute and Southern California Salinity Coalition Fellowship, a National Research Service Award Institutional Training Grant [2T32ES018827-06], the University of California-Riverside/ Agricultural Experiment Station Resource Allocation Program, the University of Washington Superfund Basic Research Program [NIEHS P42ES04696], and the UCR Dissertation Year Program Fellowship. Funding for attendance of numerous conferences was provided by the UCR Graduate Student Association Travel grants, Pollutant Responses in Marine Organisms Student Travel Award, Society of Toxicology Student Travel Award, Society of Environmental Toxicology and Chemistry North America Student Travel Award, and T. Roy Fukuto Environmental Toxicology travel awards. Copyright Acknowledgements The text and figures in Chapter 1: Part C in part or in full, are a reprint of the material as it appears in “Oxidative Stress, Unfolded Protein Response, and Apoptosis in Developmental Toxicity” published in International Review of Cellular and Molecular Biology, Vol. 317, pages 1-66, 2015. The co-author Dr. Daniel Schlenk provided guidance and editing for this work. The text and figures in Chapter 3 in part or in full, are a reprint of the material as it appears in “Stage Susceptibility of Japanese Medaka (Oryzias latipes) to v Selenomethionine and Hypersaline Developmental Toxicity” published in Environmental Toxicology and Chemistry, Vol. 35, pages 1247-1256, 2016. The co-author Dr. Daniel Schlenk directed and supervised this research. The text and figures in Chapter 4 in part or in full, are a reprint of the material as it appears in “Mechanisms of Selenomethionine Developmental Toxicity and the Impacts of Combined Hypersaline Conditions on Japanese Medaka (Oryzias latipes)” published in Environmental Science and Technology, Vol. 48, pages 7062-7068, 2014. The co- author Dr. Daniel Schlenk directed and supervised this research. vi Dedication This dissertation is dedicated to my fiancé Mike and my parents. Without their support this would not have been possible. vii ABSTRACT OF THE DISSERTATION Mechanisms of Selenomethionine and Hypersaline Developmental Toxicity in Japanese Medaka (Oryzias latipes) by Allison Justine Kupsco Doctor of Philosophy, Graduate Program in Environmental Toxicology University of California, Riverside, August 2016 Dr. Daniel Schlenk, Chairperson Selenium toxicity to oviparous vertebrates is often attributed to selenomethionine (SeMet), which can maternally transfer to developing embryos. The mechanism of SeMet toxicity is unclear. Furthermore, salinity of fresh waterways is increasing due to climate change and anthropogenic disturbance. Hypersalinity can potentiate SeMet toxicity to Japanese medaka (Oryzias latipes). The current study aimed to characterize the molecular mechanisms of SeMet and hypersalinity at sensitive developmental stages. Developmental toxicity of seawater was compared to desalination brine (DSB) and artificial water based on the San Joaquin River (SJR), CA. DSB toxicity was equal to seawater, while SJR water was the most toxic to embryos and larvae. Flavin-containing monooxygenases (FMOs) initiate SeMet toxicity and are induced by hypersalinity. However, developmental expression and regulation of FMOs in fish are unknown. Five putative medaka FMOs were identified with differential developmental mRNA expression patterns: two FMOs increased during mid-organogenesis; two FMOs decreased beginning at early organogenesis; and one FMO remained constant. Promoter viii analysis indicated regulation by developmental factors and the UPR. Treatments with UPR-inducer tunicamycin increased expression of two FMOs. In contrast, dithiothreitol inhibited the UPR and three FMOs, suggesting that FMOs are differentially regulated by the UPR. The developmental stage sensitivity of medaka embryos to SeMet was investigated in freshwater and SJR water. Stages 9-25 were most sensitive to SeMet; and hypersalinity potentiated SeMet toxicity during the onset of liver organogenesis, osmoregulation, and chondrogenesis. The mechanisms behind the potentiation of SeMet toxicity by hypersalinity indicated no involvement of oxidative stress or apoptosis; however, results suggested a role for the unfolded protein response (UPR) when animals were treated with 50µM SeMet for 12hrs. Mechanisms of SeMet-induced spinal deformities (5µM and 2.5µM for 24hrs) were further elucidated using imaging methods and showed increased oxidative stress and apoptosis in tails of embryos with spinal malformations. Gene expression analysis demonstrated a UPR activation pattern unique from UPR positive controls. Furthermore, these effects prematurely repressed chondrogenesis and induced osteogenesis. Overall, results will be useful for the risk assessment of hypersalinity and Se under hypersaline conditions; and inform studies on developmental mechanisms of toxicity. ix Table of Contents Chapter 1: Introduction 1 Selenium 1 Selenium Overview 1 Selenium in the Environment 4 Toxicity of Selenium to Fish 9 Selenium in the SJRV 18 Hypersalinity 21 Salinity Toxicity 21 Hypersalinity in Multiple Stressor Situations 25 Mechanisms of Developmental Toxicity 34 Oxidative Stress 35 The Unfolded Protein Response 52 Apoptosis 65 Integration of Oxidative Stress, the UPR and Apoptosis 72 Proposed Mechanism of Selenium and Hypersaline Toxicity 83 References 85 Chapter 2: Comparative Developmental Toxicity of Desalination Brine and 115 Sulfate Dominated Saltwater in a Euryhaline Fish Abstract 115 Introduction 116 Methods 117 Results 121 Discussion 127 References 134 Supplemental Information 136 x Chapter 3: Flavin-containing Monooxygenase Developmental Expression 138 and Regulation by the Unfolded Protein Response in Japanese Medaka (Oryzias latipes) Abstract 138 Introduction 139 Methods 141 Results 145 Discussion 152 References 158 Supplemental Information 162 Chapter 4: Stage Susceptibility of Japanese Medaka (Oryzias latipes) to 172 Selenomethionine and Hypersaline Developmental Toxicity Abstract 172 Introduction 173 Methods 175 Results 181 Discussion 195 References 203 Chapter 5: Mechanisms of Selenomethionine Developmental Toxicity and 208 the Impacts of Combined Hypersaline Conditions on Japanese Medaka (Oryzias latipes) Abstract 208 Introduction 209 Methods 212 Results 216 Discussion 220 References 227 xi Chapter 6: Molecular Mechanisms of Selenium-Induced Spinal 232 Deformities in Fish Abstract 232 Introduction 233 Methods 235 Results 240 Discussion 248 References 255 Chapter 7: Conclusions 259 References 266 xii List of Figures Figure 1.1. Selenium Metabolism. 3 Figure 1.2. Mosquito fish from Belews lake, NC. 10 Figure 1.3. Proposed mechanism of SeMet induced oxidative stress in the 15 presence of methioninase. Figure 1.4. Oxidation of SeMet by FMO and reduction by GSH. 15 Figure 1.5. Mean salinity in May in A. Fort Point, CA, and B. Pittsburg, CA. 23 Figure 1.6. Endogenous formation of reactive oxygen species/reactive nitrogen 37 species.