In vitro analysis and mathematical modeling of the cytotoxicity of organic contaminants in loggerhead (Caretta caretta) sea turtles



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Loggerhead (Caretta caretta) sea turtles are a threatened species, and yet little toxicological research has been conducted on them. This study entails toxicity testing of Perfluorooctanoic Acid (PFOA), Benzo[a]pyrene (B[a]P), and Polychlorinated Biphenyls 77 (PCB 77) on loggerhead sea turtle skin, ovary, and liver cells, followed by the production of a mathematical model that predicts survival based upon skin cell empirical data. These chemicals have been detected in the tissues, eggs, and prey items of sea turtles, including in loggerheads (Caretta caretta). These chemicals are known for cytotoxicity, mutagenicity, carcinogenicity, immunotoxicity, adverse reproductive and developmental effects, and negative impacts on pulmonary, renal and hepatic systems in a wide range of species. We established the primary skin cultures from biopsies collected from healthy animals reared at the NOAA Sea Turtle Facility in Galveston, Texas. Via laparoscopy, several loggerhead sea turtles had liver and ovary biopsies removed for cell culture establishment. After successful establishment, one successful SV40Tt liver line, an HPV liver cell line, and an HPV ovary cell line were developed. These are the first experimentally immortalized sea turtle cell lines and the first immortalized cell lines derived from healthy sea turtles. The establishment of these immortalized cultures provides for the expansion of sea turtle research at a more rapid pace and for organ cells that are often underrepresented. The effects of chemical exposure to all primary skin and ovary cultures and HPV liver and ovary cell lines were measured by two common viability assays – MTT and Lactate Dehydrogenase (LDH). Cells were exposed to each contaminant for toxicologically standard 24, 48, 72, and 96 hours. Concentrations of each contaminant applied are those that have been established as scientifically relevant for cell cultures. At varying time points and concentrations all cell cultures experience toxicity to some degree in MTT assays. Lethal Concentrations calculated by the General Linear Model methodology, indicate that primary cell cultures were typically more vulnerable to cytotoxicity than immortalized cell lines. Toxicological data was then used in the General Unified Threshold Model of Survival (GUTS) to provide a framework for the prediction of loggerhead sea turtle cell survivability overtime. Models produced served as a tool for conducting standard ecological risk assessments through the establishment of a fifty percent lethal concentration (LC50) prediction at various exposure time points over longer periods of time. These models provided the first GUTS models for this marine reptile and the first application of GUTS to cell populations rather than individual animals, pioneering the advent of TKTD models for endangered species that cannot undergo normal toxicity testing. Overall this project clearly advances the field of sea turtle toxicology through the introduction of new multi-organ cytotoxicity information, provision of immortalized organ cell lines, the use of laparoscopy for obtaining biopsies from live animals, and use of in vitro statistics for a model typically developed from in vivo data.



Sea Turtle, Immortalization, Cytotoxicity, MTT, LDH, General Linear Model, General Unified, GUTS (General Unified Threshold Model of Survival), Mathematical Modeling