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

The metabolic activation of the carcinogens benzo[a]pyrene (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 microgram/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 microgram/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 glucuronide 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.

Benz[a]anthracene-induced alterations in the metabolic activation of benzo[a]pyrene by hamster embryo cell cultures.

Co-administration of benz[a]anthracene (BA) with benzo[a]pyrene (B[a]P) to hamster embryo cell cultures for 24 h resulted in a decrease in the metabolism of benzo[a]pyrene by 40%, a decrease in the level of binding of B[a]P to DNA by 70% and a 10-fold reduction in mutation induction in a hamster embryo cell-mediated V79 cell mutation assay. This data indicates that the biological effects of co-administration of BA with B[a]P result from inhibition of the metabolic activation of B[a]P rather than induction of enzymes that detoxify the B[a]P.

Benzo[a]pyrene-DNA adduct formation in target and activator cells in a Wistar rat embryo cell-mediated V79 cell mutation assay.

To determine the relationship of the benzo[a]pyrene (BaP)-DNA adducts formed in the activator cells of a cell-mediated mutation assay to the adducts formed in the target cells and to mutation induction, irradiated second passage Wistar rat embryo (WRE) cells and V79 Chinese hamster lung cells were exposed to [3H]BaP for 5, 24 and 48 h under the conditions of a cell-mediated mutation assay. The V79 target cells were separated from the WRE activator cells by an immunoseparation procedure; the resulting V79 cell pellet contained less than 7% WRE cells. The percentage of the BaP-DNA adducts containing cis vicinal hydroxyl groups and the h.p.l.c profile of individual adducts in the V79 target cells were similar to those of the WRE activator cells for each time point. The transfer of reactive BaP metabolites from the activator cells to the target cell DNA was detectable after only 5 h of exposure to BaP, however, exposure for this length of time did not result in significant mutation induction. The (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE)-deoxyguanosine (dGuo) adduct was essentially absent after 5 h of exposure, but the amount of this adduct increased at longer times of exposure as did the mutation frequency. The complex mixture of BaP-DNA adducts formed in the WRE cells was also present in the V79 cells after all three times of exposure to BaP, a result which demonstrates the value of this cell-mediated mutation assay for investigating the role of species-specific differences in the activation of BaP. The correlation between the increase in mutation induction and the relative amount of the (+)-anti-BaPDE-dGuo adduct present in V79 cell DNA suggests the importance of this adduct in mutation induction by BaP.