In vitro toxicology and hormone assessment in cetaceans



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Endocrinological and toxicological data is imperative to the conservation of marine mammals. Because of their protected status, it is necessary to conduct both these analyses using minimally invasive biopsies and in vitro techniques. Therefore, it is critical that tissue be used to gather the largest amount of data possible. Steroid hormone analysis has been used to assess reproduction and stress in multiple species. Minimally invasive blubber biopsies can be obtained from free ranging animals and split for numerous investigations. We developed methodology and analyzed several sex and stress steroid hormones via liquid chromatography mass spectrometry (LC-MS). The hormone panel consisted of, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 17ß-estradiol, estrone, 17α-hydroxyprogesterone, progesterone, and testosterone. We verified the optimal, yet minimal tissue mass required, by comparing results between paired 50 mg and 150 mg samples and then paired 150 mg and 400 mg samples. In addition, we identified the optimal method of homogenization by comparing lysing (via Fast Prep) and shredding (via polytron) techniques. Our results indicated that 50 mg of tissue was enough for hormone analysis. We also identified significant differences in the blubber matrices between species. These differences have possible implications for not only homogenization technique but also matrix match calibrations. Although hormone data is imperative to conservation, identifying the toxicants of concern is just as vital. Currently there is very little toxicological data in gray whales. Therefore, we tested several toxicants that animals are potentially exposed to. Those included polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), per/poly-fluorinated compounds (PFASs), crude oil, and crude oil dispersant (COREXIT™). Despite the presence of these xenobiotics in the marine environment very little is understood about their effects in the species. Therefore, we tested these xenobiotics on gray whale skin cell fibroblasts. Our aim was to determine whether these xenobiotics were of concern. Contaminants selected for analysis included benzo[a]pyrene (B[a]P), PCB 126, perfluorooctanoic acid (PFOA), or media accommodated fractions of crude oil and/or Corexit™ 9500A.We measured cytotoxicity via both MTT (methylthiazolyldiphenyl-tetrazolium bromide) and LDH (lactate dehydrogenase) assays. We found that all toxicants significantly reduced cellular viability. In addition, we pinpointed the toxicants that may pose the most risk. In this dissertation, we present and describe a methodology for the simultaneous analysis of multiple sex and stress steroid hormones from small blubber masses using liquid chromatography mass spectrometry. We also describe the establishment of skin fibroblasts, propagated from skin biopsies, and the results from subsequent toxicity testing. Our results will help aid future science-based conservation efforts as well as expand our knowledge regarding gray whale endocrinology and toxicology.

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Marine mammal, Hormone, Cell culture, Toxicology, Gray whale, Fin whale, Liquid chromatography mass spectrometry (LC-MS), MTT (methylthiazolyldiphenyl-tetrazolium bromide), LDH (lactate dehydrogenase)