Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

Reptiles are an underrepresented taxon in ecotoxicological literature, and the means by which toxicants play a role in population declines are only partially understood. Among the contaminants of interest for reptiles are polycyclic aromatic hydrocarbons (PAHs), a class of organic compounds that is already a concern for numerous other taxa. The objectives of the present review are to summarize the existing literature on reptilian exposure to PAHs and synthesize general conclusions, to identify knowledge gaps within this niche of research, and to suggest future directions for research. Results confirm a relative scarcity of information on reptilian exposure to PAHs, although research continues to grow, particularly after significant contamination events. The orders Testudines and Squamata are better represented than the orders Crocodilia and Rhynchocephalia. For the taxonomic orders with relevant literature (all but Rhynchocephalia), some species are more frequently represented than others. Few studies establish solid cause-effect relationships after reptilian exposure to PAHs, and many more studies are suggestive of effect or increased risk of effect. Despite the scarcity of information in this area, researchers have already employed a wide variety of approaches to address PAH-related questions for reptiles, including molecular techniques, modeling, and field surveys. As more research is completed, a thoughtful interpretation of available and emerging data is necessary to make the most effective use of this information. Environ Toxicol Chem 2017;36:25-35. #