Toxicity Testing and Hormone Analyses for Cetacean Health Assessments

Currently, there is a severe need for health and toxicology information with regards to wild cetacean populations. They prove challenging to monitor, and data on population dynamics, health information, and relative fitness is difficult to obtain for free-ranging populations of cetaceans. 16 cetacean species are classified as endangered by the Marine Mammal Protection Act, and all marine mammals face a multitude of environmental and anthropogenic stressors. Due to their protection status as well as logistics and ethical concerns, traditional in vivo toxicity testing is not possible in cetaceans. Health information, such as reproductive rates, stress, and fitness, as well as an understanding of contaminant toxicity is critical when developing a scientific-based management and conservation plan for these populations. Here we describe the method development and optimization to conduct cetacean health assessments by analyzing blubber steroid hormones. We also describe and report on the development and optimization of an organotypic culture model to conduct cetacean toxicity testing. Two methodologies were used to measure steroid hormones in cetacean blubber samples: an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem-mass spectrometry (LC-MS/MS). ELISAs are commonly employed for steroid hormone measurement from the blubber matrix, and optimizations were needed to conduct hormone analysis in the blubber using LC-MS/MS. Analysis of blubber hormones using LC-MS/MS was possible through the incorporation of gel permeation chromatography for lipid removal from sample extract. Both methodologies were successful in determination of pregnancy from blubber progesterone analysis using minimal tissue (50 mg wet weight). Progesterone concentrations of pregnant female cetaceans were within the range of concentrations reported in the literature for other pregnant cetaceans. LC-MS/MS was also able to generate data on three additional steroid hormones from the same extract: testosterone, estradiol, and cortisol. These preliminary method optimizations for LCMS/MS provide significant advancements to the field of cetacean endocrinology and the ability to generate health assessments for wild cetaceans. Organotypic culture of cetacean skin and blubber was assessed for its use in cetacean toxicity testing. Skin and blubber were precision cut and cultured and/or exposed to common marine contaminants for up to 72 hours. Viability of non-dosed tissue was assessed through establishing fibroblast cell culture, assessing intracellular potassium of tissue, and measuring lactate dehydrogenase (LDH) activity in culture media. Non-dosed tissues were viable for up to 72 hours in culture. Intracellular potassium analysis of dosed tissue showed that tissues were viable upon exposure in culture for up to 72 hours. Expression of two biomarkers of exposure, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1), was analyzed by quantitative PCR which detected induction of CYP1A1 in tissues exposed to benzo(a)pyrene for 24 and 48 hours. CYP1A1 downregulation was detected in tissues exposed to chemical dispersant and crude oil for 24 hours. This research demonstrates the applicability of cetacean skin and blubber organotypic culture as a viable, non-lethal, minimally-invasive methodology for cetacean toxicity testing.