Establishment, Immortalization and Toxicological Applications of Primary Skin Fibroblasts from Three Sea Turtle Species



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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. Primary skin fibroblast cell cultures were also established and described for the green sea turtle (Chelonia mydas) and the Kemp’s ridley sea turtle (Lepidochelys kempii). Characterization of loggerhead sea turtle fibroblasts included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions using growth curve assays with a fractional factorial experimental design. Two cell viability assays, two assays assessing sublethal cytotoxic effects, and two CYP1A biomarker assays were optimized in the characterized loggerhead sea turtle cells. MTT and lactate dehydrogenase (LDH) cell cycle analysis successfully showed cytotoxicity when cells were exposed to environmentally relevant concentrations of perfluorinated compounds. Using c flow cytometry, we detected no alterations to the cell cycle as a result of exposure to any compound tested in preliminary studies. Reactive oxygen species (ROS) assays showed significant inhibition of ROS production in cells exposed to phenanthrene. Cytochrome P4501A (CYP1A) expression was analyzed by quantitative PCR which detectedinduction of CYP1A5 in cells exposed to benzo(a)pyrene for 72 hours. Enzymatic activity was quantified via AROD analysis and showed BROD and PROD activity when cells were exposed to benzo(a)pyrene, PCB77, and PCB126, but no MROD or EROD activity. This research demonstrates validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells. Additionally, immortalization of fibroblast cell lines derived from multiple, healthy sea turtles was attempted by the use of an hTERT insert transducted into the genome via a lentiviral vector. Transduction was unsuccessful for loggerhead and green sea turtle fibroblasts, but one line from a green sea turtle was successfully transduced. Immortal cells from the green sea turtle maintained normal fibroblastic morphology in culture when compared to primary cells from the same animal, but exhibited more rapid proliferation.



Toxicology, Sea turtle