RESPONSES OF HEPATIC CYTOCHROME-P450 1A AND FORMATION OF DNA-ADDUCTS IN JUVENILES OF TURBOT (SCOPHTHALMUS-MAXIMUS L) EXPOSED TO WATER-BORNEBENZO[A]PYRENE

The time-course of the induction of hepatic cytochrome P450 1A (CYP1A) and the formation of DNA-adducts in liver and surrounding tissues wer e studied in juvenile turbot (Scophthalmus maximus) (size range 6.0 +/ - 0.5 cm) exposed to a single water-column dosage of 1 or 25 ppb benzo [a]pyrene (BaP) for up to 48 h. CYP1A induction was measured in terms of 7-ethoxyresorufin O-deethylase (EROD) activity and CYP1A-immunoposi tive protein (Western blotting using polyclonal antibody to hepatic CY P1A of perch, Perca fluviatilis). The formation of BaP-DNA-adducts was determined by P-32-postlabelling analysis of extracted DNA. Hepatic E ROD activity was not elevated by exposure to 1 ppb BaP at any time but increased 2- to 3-fold, 24 and 48 h after exposure to 25 ppb BaP, ind icating induction of CYP1A at the higher BaP exposure concentration. W estern blot analysis identified major immunopositive bands of apparent molecular weight 58 kDa (consistent with the presence of a CYP1A prot ein) and 48.5 kDa. Although levels of the 58kDa CYP1A-immunopositive p rotein were higher at 25 ppb than 1 ppb BaP 48 h after exposure, no ov erall consistent meaningful correlation between EROD activity and CYP1 A-immunopositive protein could be discerned, probably owing to the rel atively low levels of CYP1A induction and the sensitivity of the Weste rn blot analysis. Consistent with the results for EROD activity, forma tion of DNA-adducts was indicated at 1 ppb BaP, but a 6-fold increase after 16 h was seen at 25 ppb. The latter level of DNA-adducts remaine d high at 24 h but decreased by 50% after 48 h. The marked formation o f DNA-adducts at 16 h; before the increase in EROD activity, indicates a constitutive capacity for the metabolism of BaP to DNA-adducts. The maximal levels of DNA-adducts observed in BaP-exposed juveniles were similar to those in adult S. maximus injected intraperitoneally with 2 0 mg kg(-1) BaP, respectively, 583 +/- 131 and 650 +/- 106 atto-moles adducts mu g(-1) DNA. Co-chromatography of extracted adducted-bases wi th (+) and (-)-anti-benzo[a]pyrene diol epoxide-N-2-guanine and N6-ade nine nucleic acid standards failed to identify any specific adducts.