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 benzoa]anthracene(BP) and 7,12-dimethylbenza]anthracene (DMBA) was examinedin cell lines derived from bluegill fry (BF-2), rainbow trout(RTG-2) and brown bullhead (BB). All three cell lines metabolizedBP (0.5 µg/ml medium) almost completely to water-solublemetabolites within 120 h, but the maximum amount of BP boundto DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17in the BB cells and 44 in the RTG-2 cells. The major BP-DNAadduct in the BB and BF-2 cells was that formed by reactionof (+)-anti-BP-7,8-diol-9,10 epoxide (+)anti-BPDE] with deoxyguanosine.This adduct was also present in the RTG-2 cell DNA, but therewere larger amounts of unidentified polar BP-DNA adducts. Exposureof 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 inthe BF-2, BB and RTG-2 cells, respectively. More than 90% ofthe BP-7,8-diol added to the BF-2 cultures was recovered asa glucuronic acid conjugate, but the RTG-2 cells formed moreglutathione conjugates than glucuronide conjugates. The BB cellsformed both types of conjugates at a slower rate for more than75% of the 7,8-diol was recovered unchanged after 24 h. Thethree cell lines differed in the proportion of a 0.1 µg/mldose 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-2cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6pmol/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 significantamounts of DMBADE-DNA adducts were detected in the RTG-2 orBF-2 cell DNA. These results demonstrate that fish cell culturescan activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE,but the level of binding of this metabolite to DNA is much lowerthan that which occurs in rodent embryo cell cultures. In BF-2cell cultures formation of BP-7,8-diol-glucuronide effectivelyprevents the activation of this diol to (+)anti-BPDE. A substantialproportion of the BP-7,8-diol is also metabolized to glucuromdeand glutathione conjugates in BB and RTG-2 cells. DMBA alsobinds to DNA at very low levels in these fish cell cultures.Thus effective conjugation of diols and their metabolites byfish cell lines appears to greatly reduce metabolic activationof hydrocarbons through the bay-region diol epoxide pathwaythat predominates in mammalian cell cultures.