The etiology of human breast cancer is currently undefined. However, it has been hypothesized that exposure to chemical carcinogens may be an important factor. Extrapolation from rodent models for chemically-induced mammary cancer suggests the possibility that human mammary epithelial cells in situ might contain DNA adducts due to exposure to environmental chemicals. We have therefore screened breast epithelial cells from 10 donors for the existence of DNA adducts using the 32P-postlabeling assay. In order to validate this analysis technique, we also examined the DNA adducts formed in human mammary cells exposed to benzo[a]pyrene (B[a]P) in vitro, and adducts formed in rat mammary epithelial cells exposed to B[a]P in vitro and in vivo. Confirming previous results using HPLC analysis of [3H]B[a]P-DNA adducts, the major B[a]P-DNA adduct formed by human mammary epithelial cells in vitro was (+)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE):deoxyguanosine. This adduct did not appear to be formed by rat mammary cells exposed to B[a]P in vitro. However, 32P-postlabeling analysis of mammary epithelial cell DNA from rats exposed to B[a]P in vivo indicated that (+)-anti-BPDE-deoxyguanosine was a major B[a]P-DNA adduct under these exposure conditions. When the mammary epithelial cells from 10 human donors were screened for DNA adducts formed in situ, cells from three donors exhibited distinct adduct patterns. None of these adducts appeared to be (+)-anti-BPDE-deoxyguanosine. The existence of DNA adducts in human mammary epithelial cells in situ, coupled with the data indicating that rat mammary cells form different B[a]P adducts in vitro and in situ, suggests the need for further study of human breast cell adducts.

The capacity of polycyclic aromatic hydrocarbons such as 7,12-dimethylbenz[a]anthracene (DMBA) to induce mammary carcinomas has been studied in three rat strains. Wistar/Furth (WF) rats are highly susceptible to DMBA-induced mammary carcinogenesis, Copenhagen (Cop) rats are completely resistant, and Fischer 344 (F344) rats have an intermediate susceptibility. We have previously shown that WF rats possess 'enhancer genes', which enhance susceptibility to induced mammary cancer. Cop rats, however, possess a single 'suppressor' gene which confers complete resistance to mammary cancer. Both gene types are apparently absent in F344 rats. In order to determine possible mechanisms of action of these enhancer and suppressor genes, we have examined DMBA metabolism and DNA binding in mammary epithelial cells isolated from each rat strain. Quantitative analyses of both metabolism and DNA binding indicate no significant differences among the strains. In addition, HPLC analyses of DMBA metabolites and DMBA-DNA adducts were essentially identical. These data suggest that the genes controlling susceptibility and resistance to mammary carcinogenesis in these rat strains are likely to be active at later stages of the carcinogenic process.

Rat mammary epithelial cells (RMEC) in culture have been shown to activate polycyclic aromatic hydrocarbon (PAH) carcinogens. This study investigates the role of mammary cytochrome P-450 monooxygenases in these metabolic processes. Monooxygenation of 7,12-dimethylbenz[a]anthracene (DMBA) by RMEC in culture exhibited a 6-h lag period before reaching a constant rate. The mechanism for this time-dependent expression of DMBA monooxygenase activity was investigated in lysed cells, where both conjugation and in situ induction of P-450 are prevented. Although metabolism of DMBA by untreated RMEC lysates was undetectable (less than 1 pmol/mg cell protein/h), prior exposure of cultured cells to benz[a]anthracene (BA) induced DMBA metabolism, (approximately 100 pmol/mg cell protein/h). BA pretreatment also eliminated the lag period for metabolism of DMBA by cultured RMEC but did not prevent additional induction of DMBA monoxygenase activity by the substrate. The distribution of monooxygenated DMBA metabolites formed by BA-induced cell lysates was clearly different from that obtained with purified P-450c, the predominant PAH-inducible isozyme in rat liver. For example, the carcinogen precursor DMBA 3,4-dihydrodiol, which is not formed by P-450c, was a clearly detectable product in RMEC. The low epoxide hydratase activity of BA-induced lysate (approximately 400-fold lower compared to that in the liver) limited formation of all DMBA dihydrodiols. The formation of DMBA 3,4-dihydrodiol increased by 5-fold following addition of exogenous purified epoxide hydratase. The DMBA monooxygenase activity of BA-induced RMEC lysates was completely inhibited by alpha-naphthoflavone but was only partially inhibited (50%) by a polyclonal antibody raised against cytochrome P-450c. Anti P-450c completely inhibited formation of some of the metabolites, partially inhibited formation of others and notably stimulated formation of DMBA 3,4-dihydrodiol by 60%. A polyclonal antibody that recognized both rat hepatic P-450a and a group of P-450 isozymes related to P-450h, and which totally inhibited DMBA 3,4-dihydrodiol formation by rat liver microsomes, did not inhibit formation of any DMBA metabolite in RMEC, including DMBA 3,4-dihydrodiol. Western blot analyses of RMEC homogenates demonstrated that BA pretreatment induces P-450c, but not P-450a or any of the P-450h-related isozymes. We conclude that metabolism of DMBA by RMEC depends on induction of P-450c and at least one additional form of cytochrome P-450 which is immunochemically distinct from rat hepatic P-450a and P-450h related isozymes, but is sensitive to alpha-naphthoflavone.

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Primary hepatocyte cultures from six human donors were established and their abilities to metabolize the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) were examined. Cells from each donor were plated at similar densities (1 X 10(7) cells/100 mm dish). All cultures metabolized B[a]P to a significant extent (24-35 nmol in 24 h) and h.p.l.c. profiles of the organic solvent-soluble and glucuronidated B[a]P metabolites were obtained for all donors. The predominant extracellular organic solvent-soluble B[a]P metabolites were the 9,10- and 7,8-dihydrodiols, 9-hydroxy-B[a]P, and a mixture of tetrols, but the general ratios of these metabolites varied widely among the cells from different donors. In contrast, profiles were highly reproducible in cells from the same donor treated with B[a]P at either 8 or 24 h after initial plating. There was less variability in the amounts of specific B[a]P metabolites conjugated to glucuronic acid by cells from various donors. This variability could not be correlated with cell viability or overall levels of B[a]P metabolism. In addition, B[a]P metabolism by fresh and cryopreserved hepatocytes from the same donor was compared. While there was only a small reduction in the level of total B[a]P metabolism after cell freezing, there was a 3- to 5-fold increase in production of B[a]P-7,8-dihydrodiol, found both in the extracellular medium and as glucuronic acid conjugates, by the cryopreserved cells tested.