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Establishment, Characterization, and Toxicological Application of Loggerhead Sea Turtle (Caretta caretta) Primary Skin Fibroblast Cell Cultures

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† The Institute of Environmental and Human Health, Department of Environmental Toxicology, Texas Tech University, 1207 Gilbert Drive, Lubbock, Texas 79409, United States
‡ National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 4700 Avenue U, Galveston, Texas 77551, United States
§ Department of Veterinary Integrative Biosciences, Texas A&M University, 4458 TAMU, College Station, Texas 77843, United States
∥ Department of Biological Sciences, 2901 Main, Texas Tech University, Lubbock, Texas 79409, United States
*E-mail: [email protected]; telephone: (806) 885-0337; fax: (806) 885-2132.
Cite this: Environ. Sci. Technol. 2014, 48, 24, 14728–14737
Publication Date (Web):November 10, 2014
https://doi.org/10.1021/es504182e
Copyright © 2014 American Chemical Society
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    Abstract

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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. 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.

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    Additional text with description of alternative methods for establishing fibroblast growth from tissue explants, explanation of cell count and population doubling level calculations, and methods for analyzing dosing stocks of B[a]P and PFOA via chromatography; two tables comparing cell number yields from different explant techniques and showing fractional factorial design. This material is available free of charge via the Internet at http://pubs.acs.org.

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