Metabolism and DNA adduct formation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in fish cell lines in culture

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Abstract
The metabolic activation of the carcinogens benzo[a]anthracene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was examined in cell lines derived from bluegill fry (BF-2), rainbow trout (RTG-2) and brown bullhead (BB). All three cell lines metabolized BP (0.5 μg/ml medium) almost completely to water-soluble metabolites within 120 h, but the maximum amount of BP bound to DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17 in the BB cells and 44 in the RTG-2 cells. The major BP-DNA adduct in the BB and BF-2 cells was that formed by reaction of (+)-anti-BP-7,8-diol-9,10 epoxide [(+)anti-BPDE] with deoxyguanosine. This adduct was also present in the RTG-2 cell DNA, but there were larger amounts of unidentified polar BP-DNA adducts. Exposure of the cells to [3H]BP-7,8-diol, a metabolic precursor of (+)anti-BPDE, resulted in binding of 1.5, 12 and 35 pmol BP per mg DNA in the BF-2, BB and RTG-2 cells, respectively. More than 90% of the BP-7,8-diol added to the BF-2 cultures was recovered as a glucuronic acid conjugate, but the RTG-2 cells formed more glutathione conjugates than glucuronide conjugates. The BB cells formed both types of conjugates at a slower rate for more than 75% of the 7,8-diol was recovered unchanged after 24 h. The three cell lines differed in the proportion of a 0.1 μg/ml dose of DMBA metabolized in 48 h: the values ranged from 47% in the BF-2 cells to 78% in the BB cells and 97% in the RTG-2 cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6 pmol/mg DNA in the three cell lines: DMBA-3,4-diol-1,2-epoxide (DMBADE) adducts were present in the BB cell DNA, but no significant amounts of DMBADE-DNA adducts were detected in the RTG-2 or BF-2 cell DNA. These results demonstrate that fish cell cultures can activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE, but the level of binding of this metabolite to DNA is much lower than that which occurs in rodent embryo cell cultures. In BF-2 cell cultures formation of BP-7,8-diol-glucuronide effectively prevents the activation of this diol to (+)anti-BPDE. A substantial proportion of the BP-7,8-diol is also metabolized to glucuromde and glutathione conjugates in BB and RTG-2 cells. DMBA also binds to DNA at very low levels in these fish cell cultures. Thus effective conjugation of diols and their metabolites by fish cell lines appears to greatly reduce metabolic activation of hydrocarbons through the bay-region diol epoxide pathway that predominates in mammalian cell cultures.