Responses of hepatic cytochrome P450 1A and formation of DNA-adducts in juveniles of turbot ( Scophthalmus maximus L.) exposed to water-borne benzo[a]pyrene

Abstract

The time-course of the induction of hepatic cytochrome P450 1A (CYP1A) and the formation of DNA-adducts in liver and surrounding tissues were 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 CYPlA-immunopositive protein (Western blotting using polyclonal antibody to hepatic CYP1A of perch, Perca fluviatilis). The formation of BaP-DNA-adducts was determined by 32P-postlabelling analysis of extracted DNA. Hepatic EROD 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, indicating induction of CYP1A at the higher BaP exposure concentration. Western blot analysis identified major immunopositive bands of apparent molecular weight 58 kDa (consistent with the presence of a CYP1A protein) and 48.5 kDa. Although levels of the 58kDa CYPlA-immunopositive protein were higher at 25 ppb than 1 ppb BaP 48 h after exposure, no overall consistent meaningful correlation between EROD activity and CYPlA-immunopositive protein could be discerned, probably owing to the relatively low levels of CYP1A induction and the sensitivity of the Western blot analysis. Consistent with the results for EROD activity, formation 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 remained high at 24 h but decreased by 50% after 48 h. The marked formation of 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. maximum injected intraperitoneally with 20 mg kg −1 BaP, respectively, 583 ± 131 and 650 ± 106 attomoles adducts μg −1 DNA. Co-chromatography of extracted adducted-bases with (+) and (−)- anti-benzo[a]pyrene diol epoxide- N 2-guanine and N6-adenine nucleic acid standards failed to identify any specific adducts.