Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.
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.
The Institute of Environmental and Human Health, Department of Environmental Toxicology, Texas Tech University , 1207 Gilbert Drive, Lubbock, Texas 79409, United States.
Sarah J
SJ
Webb
Gregory V
GV
Zychowski
Sandy W
SW
Bauman
Benjamin M
BM
Higgins
Terje
T
Raudsepp
Lauren S
LS
Gollahon
Kimberly J
KJ
Wooten
Jennifer M
JM
Cole
Céline
C
Godard-Codding
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D001564:Benzo(a)pyrene / Q000633:toxicity; D002210:Caprylates / Q000633:toxicity; D002470:Cell Survival; D002478:Cells, Cultured; D054750:Ecotoxicology / Q000379:methods; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005466:Fluorocarbons / Q000633:toxicity; D007621:Karyotyping; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012867:Skin / Q000166:cytology*; D018675:Toxicity Tests / Q000379:methods*; D014426:Turtles
D002210:Caprylates; D005466:Fluorocarbons; D001564:Benzo(a)pyrene; C023036:perfluorooctanoic acid; D007770:L-Lactate Dehydrogenase; D001189:Aryl Hydrocarbon Hydroxylases; C108015:cytochrome P-450 CYP1A5