Benzo[a]pyrene metabolism and xenobiotic-stimulated reactive oxygen species generation by subcellular fraction of larvae of turbot ( Scophthalmus maximus L.)

Abstract

NADPH-dependent 3H-benzo[a]pyrene (BaP) metabolism and basal and xenobiotic-stimulated NAD(P)H-dependent reactive oxygen species (ROS) production were investigated in 11,600 g supernatants of 4-day-old (yolk sac) larvae of turbot ( Scophthalmus maximus L.). BaP metabolites were resolved by HPLC and detected radiometrically. ROS were quantified by the iron-EDTA mediated production of hydroxyl radical (·OH) that was detected by its oxidation of 2-keto-4-methiolbutyric acid (KMBA) to yield ethylene. BaP metabolism produced phenols, dihydrodiols and diones (quinones) (respectively, 54, 32, and 14% of free metabolites) and putative protein adducts. Metabolites identified by retention time included the 7,8-dihydrodiol, the 1,6-, 3,6- and 6,12-diones and the 3- and 9-phenols. Pre-exposure of turbot larvae to 5 ppb BaP for 24 hr caused an approximate 2-fold increase in both BaP metabolism and 7-ethoxyresorufin O-deethylase activity, indicative of the induction of cytochrome P4501A and its involvement in BaP metabolism. Basal KMBA oxidation rates were similar for NADH and NADPH. Inhibition studies indicate that ·OH was formed via the production of Superoxide anion radical and hydrogen peroxide. Basal ROS production was stimulated up to 3-fold by a range of redox cycling aromatic hydrocarbon quinones and indicated to be stimulated by other xenobiotics, including nitroaromatics. The results indicate biotransformation and ROS production as potential mechanisms of toxicity in larval fish.