Conversion of 1 nitropyrene by Brown trout Salmo trutta and turbot Scophthalamus maximus to DNA adducts detected by 32P postlabelling

Abstract

The ability of the common aquatic contaminant 1-nitropyrene to form DNA adducts in fish was investigated in vitro and in vivo using Brown trout (Salmo trutta) and turbot (Scophthalmus maximus)in comparison to the Wistar rat. In vitro studies used Brown trout (control and induced (50 mg kg-1-naphthoflavone (NF), i.p. 3 day pre-treatment single injection)) and induced rat (PB; 0 1% w/v for 7 days in drinking water, NF; 80 mg kg-1, single injection 2 days prior to sacrifice). Hepatic 9000 g supernatant (S9 fractions) were incubated for 2 hours (at 25 C for fish and 37 C for rat) with calf thymus DNA (1mg) and 1-NP (100 M). With all S9 fractions the presence of three distinct 1-NP-related DNA adducts was detected using the butanol enrichment procedure of the 32Ppostlabelling assay. A greater level of DNA adducts was observed with the uninduced compared to the induced trout S9 (37, 12 and 8 fold greaterfor adducts in chromatograph areas 1-3 respectively) suggesting the enhancement of detoxification pathways with respect to bulky adducts following NF pre-treatment. DNA adduct levels in the induced rat consistently demonstrated approximately two-fold higher levels as compared to the induced fish, reflecting the lower protein levels in the S9 fraction of Brown trout (42 and 22 mg ml-1 for rat and fish respectively). Turbot, rat and Brown trout (uninduced and induced (NF; 50 mg kg-1; i.p. single injection 3 days prior)) were dosed with 100 mg kg-1 1-NP (i.p. single injection, 24 hours). Liver DNA from both turbot and rat exhibited a 1- NP related adduct spot which was similar in position to that of area 1 in the incubations with S9 from rat and Brown trout. However, in contrast to the in vitro studies no 1-NPrelated adducts were found in liver DNA from induced and uninduced Brown trout. This study highlights the potential, in a marine and a freshwater fish, for 1-NP metabolism to reactive of binding to DNA. However, activation of 1-NP was more optimal in the S9-mediated system, possibly reflecting the influence of detoxification systems.