Exposure of liver cell culture from the orange-spotted grouper, Epinephelus coioides, to benzo[a]pyrene and light results in oxidative damage as measured by antioxidant enzymes

Abstract

Among the various toxicants discharged into aquatic environments, benzo (a) pyrene (BaP) has been shown to effect on the antioxidant system of fish and the evaluation of its impact on biota is of considerable concern. The aim of the present study was to use the primary hepatocyte culture obtained from the orange-spotted grouper, Epinephelus coioides, to evaluate the adverse effects of benzo (a) pyrene (BaP) on cell viability and liver antioxidant system. BaP was selected for its high ability to produce reactive oxygen species (ROS) and oxidative stress. The liver was minced by a scalpel and digested in the PBS solution with 0.1% collagenase IV at room temperature for 20 min. Then, the cell suspension was transferred to a plate contained an equal amount of Leibovitz's L-15 medium with 20% fetal bovine serum (FBS), 100 IU mL−1 of penicillin and 100 μg mL−1 streptomycin. 5 mL of cell suspension were plated into sterile 25 cm2 tissue culture flasks at the density of 1.5 × 106 cell/ml L-15 and incubated at 30 °C for two weeks. The medium was renewed after 24–48 h. The number of the liver cells was adjusted to 4 × 106 after two weeks. 10−4 mol l−1 was verified by MTT assay as the IC50 of BaP. Then, hepatocytes were exposed to three concentrations of BaP (10−5, 2 × 10−5, 3 × 10−5 mol L−1) and incubated for 24 h. Samples were collected after 6, 12 and 24 h and the amounts of SOD, CAT, GPx, LPO, LDH, AST, ALT, ALP and total protein were analyzed. The results showed that, 10−5 mol L−1 of BaP was not significantly toxic to cultivated hepatocytes, however, the sensitivity of cells to BaP increased in a dose-related pattern. The activity of the antioxidant enzymes (SOD, CAT and GPx) and liver enzymes (ALT, AST, ALP, LDH) significantly increased, though the amount of LPO, total antioxidant power and total protein decreased dose-dependently in BaP-exposed cells. In conclusion, according to the finding of the present study, BaP has a high potential to induce the oxidative stress in primary liver cell culture of E. coioides.