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Development and Application of Methods for Extraction and LC/MS/MS Analysis of Sex Steroids and Conjugates from Fish Feces

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Development and Application of Methods for Extraction and LC/MS/MS Analysis of Sex Steroids and Conjugates from Fish Feces Development and Application of Methods for Extraction and LC/MS/MS Analysis of Sex Steroids and Conjugates from Fish Feces By Lisa E. Peters A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of Doctor of Philosophy Department of Environment and Geography University of Manitoba Winnipeg Copyright © 2014 Lisa E. Peters 1 Acknowledgments I would like to start by thanking my supervisor, Dr. Gregg Tomy, for his support, patience and resources during my Ph.D. program. He took a chance when I tried to convince him that my biology background would be a great addition to his chemistry lab. My husband, Vince Palace, and I also appreciated his support and genuine happiness for us when I announced we were expecting a baby in the middle of my studies. A sincere thanks also goes to my co-supervisor, Dr. Mark Hanson, for his support on so many levels (I wouldn’t even know where to start), and for doing his best to keep me on track, which was no small task. I would also like to thank my other thesis committee members, Drs. Gary Anderson and Feiyue Wang, for their encouragement and insightful comments. Gary gave a lot of extra time to review various thesis chapters and data, and to lend his expertise during the fish surgeries. I also had the pleasure of taking his Endocrinology course, which was probably the most informative and enjoyable class of my entire university student career. I would like to thank the technical staff and students from DFO, Suzanne Mittermuller, Kerry Wautier, Alea Goodmanson, Danielle Godard, Lisa Friedrich and Kirstin Dangerfield, and my lab mates Bruno Rosenberg, Kerri Pleskach, Colin Darling, Bonnie Gemmill and Lianna Bestvater for their technical input and help during my experiments. A special thanks to those ladies that assisted during the much needed wine, ice cream and coffee breaks. I am not sure what I would have done without the support of my “non-science, personal cheerleading team” Patty Fisher, Jo-Anne Harrison and Jodi Bachynski. They i were always available when I needed a “meeting”, and provided the best therapy ever; lots of laughs and great food. Last, but not least, I would like to thank my family; Mom, Dad, Lori, Vince, Danny, Leah and Brooke for their love and support while we road this crazy rollercoaster together (even when they didn’t buy a ticket). To my Vince, thank you for being a wonderful husband, friend, mentor and punching bag. I could not have done this without your patience and encouragement; I love you with all my heart. To my sweet, beautiful Brookins, you danced into our lives when we thought it wasn’t possible. You have brought pure joy, love and laughter to my life. Mommy loves you. ii Abstract Non-lethal monitoring of animals using fecal steroid analysis is frequently employed to assess the health/reproductive status of individuals or populations, and may be applied to environmental monitoring of fish. Fecal steroid analysis requires both the non-polar parent and the polar glucuronide/sulfate conjugated forms to be considered. To address this challenge in fish, a reproductive steroid extraction method with HPLC/MS/MS analysis was first developed for Cortland’s in vitro bioassay medium. A 2-step liquid:liquid extraction method successfully captured parent sex steroids and their conjugates. This method was then applied to investigate routes of endocrine disruption in trout gonads exposed to environmentally relevant concentrations of polybrominated diphenyl ether (PBDE) flame retardants, and selected metabolites. Of the two classes, hydroxylated metabolites of PBDE-47 had the greatest effect on steroidogenesis in both male and female trout gonadal tissues. The extraction and LC/MS/MS analysis techniques were then applied to fish feces. The most effective matrix treatment for feces was a lipid removal agent, CleanasciteTM. It removed lipid and pigmented compounds left by acid washing, and prolonged column life without affecting hormone recoveries. Using paired plasma and feces samples from rainbow trout it was determined that fecal estrogens could predict plasma concentrations. Changes in plasma estrogens were typically not reflected in feces, which varied less, until the subsequent week. Fecal concentrations of E2-17 glucuronide best predicted plasma E2 concentrations in female rainbow trout, while the strongest relationship was between plasma E2-3 sulfate and fecal E2-17 glucuronide. iii Plasma clearance time and partitioning of estrogens into feces, urine and bile was then monitored in two experiments. The first involved in vivo administration of radiolabeled E2 into the blood of rainbow trout, and in the second radiolabelled E2 was introduced into the fish gut. Overall, E2 was cleared from plasma in 72 hours, and estrogens in feces can be a composite of hormones metabolized over > 4 days. 3 Approximately 68% of E2-H injected into the gut entered enterohepatic circulation. Understanding hormone metabolism and clearance, and the roles of dietary uptake and enterophepatic circulation, will facilitate further development of fecal steroid analysis as a non-lethal assessment tool for monitoring fish reproduction. iv Table of Contents Acknowledgments .............................................................................................................. i Abstract ............................................................................................................................ iii Table of Contents .............................................................................................................. v List of Tables .................................................................................................................... xi List of Figures ................................................................................................................. xiv Chapter 1 - General Introduction ................................................................................... 1 Traditional Environmental Monitoring of Fish ....................................................... 1 Non-Lethal Sampling of Fish.................................................................................. 3 Reproductive Hormones in Feces ........................................................................... 6 Problems with current hormone analysis .............................................................. 11 Focus and Highlights of This Thesis .................................................................... 12 Research Objectives .............................................................................................. 15 Hypotheses ............................................................................................................ 16 References ............................................................................................................. 17 Chapter 2 - Extraction of multiple sex steroids and their conjugates from steroidogenesis assay medium followed by simultaneous HPLC/MS/MS analysis .............................................................................................................. 28 Abstract ................................................................................................................. 28 Introduction ........................................................................................................... 30 Materials & Methods ............................................................................................ 32 v Hormones, Solutions and Buffers ....................................................................... 32 LC/MS/MS Optimization .................................................................................... 34 Solvent Tests ....................................................................................................... 35 pH Tests ............................................................................................................... 37 Addition of Ion Pair ............................................................................................. 37 Fraction Recoveries and Solvent Volume ........................................................... 37 Final Protocol Tests and Validation of Method .................................................. 38 Results & Discussion ............................................................................................ 40 Validation of Method .......................................................................................... 54 References ............................................................................................................. 59 Chapter 3: Investigating effects of polybominated diphenyl ethers (PBDEs) and their hydroxylated metabolites on in vitro gonadal steroidogenesis in rainbow and brown trout using LC/MS/MS analysis. .................................. 66 Abstract ................................................................................................................. 66 Introduction ........................................................................................................... 68 Methods................................................................................................................. 69 Chemicals: PBDEs, Hormones and in vitro Medium .......................................... 69 Fish ...................................................................................................................... 70 In vitro Incubations ............................................................................................
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