Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.
A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, standard reference material (SRM) 1597, has been shown to initiate tumor formation in mouse initiation-promotion assays in our laboratory [(2001) Carcinogenesis 22 (7), 1077-1086]. To determine the effects of SRM 1597 on PAH activation in human cells, we investigated the PAH-DNA adduct formation in the human mammary carcinoma-derived cell line MCF-7. We examined the effects of SRM 1597 on the metabolic activation to DNA binding derivatives of two carcinogenic PAHs, the bay region containing benzo[a]pyrene (B[a]P) and the more carcinogenic fjord region containing dibenzo[a,l]pyrene (DB[a,l]P). PAH-DNA adduct analysis by 33P-postlabeling and reversed phase high-performance liquid chromatography revealed a significant decrease in the levels of both B[a]P and DB[a,l]P DNA adduct formation on cotreatment with SRM 1597 in comparison to cells exposed to B[a]P or DB[a,l]P alone. However, the inhibition of PAH-DNA adduct formation only occurred within the first 48 h of exposure in cells cotreated with SRM 1597 and B[a]P. In contrast, SRM 1597 significantly inhibited the level of DB[a,l]P DNA adducts throughout the 120 h of exposure. Induction of human cytochrome P450 (P450) enzymes 1A1 and P4501B1 on treatment with SRM 1597 was observed by immunoblots. These results suggest that the important factors in determining the carcinogenic activity of PAH within a complex mixture would depend on the ability of other components of the mixture to promote or inhibit the activation of carcinogenic PAH by the induction of P450 enzymes followed by the formation of DNA adducts.
A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, the Standard Reference Material (SRM) 1597, was recently shown to decrease the levels of DNA binding of the 2 strong carcinogens benzo[a]pyrene (BP) and dibenzo [a,l]pyrene (DBP) in the human mammary carcinoma-derived cell line MCF-7 (Mahadevan et al., Chem Res Toxicol 2005;18:224-231). The present study was designed to further elucidate the biochemical mechanisms involved in this inhibition process. We examined the effects of SRM 1597 on the metabolic activation of BP and DBP toward DNA-binding derivatives in Chinese hamster cells expressing either human cytochrome P450 (CYP) 1A1 or CYP1B1. SRM 1597 inhibited BP-DNA adduct formation through the entire exposure time in cells expressing human CYP1A1, while it significantly inhibited adduct formation only up to 48 hr when co-treated with DBP. Conversely, human CYP1B1-expressing cells were unable to catalyze PAH-DNA adduct formation on treatment with SRM 1597 alone, and on co-treatment with BP or DBP. The data obtained from biochemical experiments revealed that SRM 1597 competitively inhibited the activity of both human enzymes as analyzed by 7-ethoxyresorufin O-deethylation assays. While the Michaelis-Menten constant (K M ) was <0.4 lM in the absence of SRM 1597, this value increased up to 1.12 (CYP1A1) or 4.45 lM (CYP1B1) in the presence of 0.1 lg/ml SRM 1597. Hence the inhibitory effects of the complex mixture on human CYP1B1 were much stronger when compared to human CYP1A1. Taken together, the decreases in PAH-DNA adduct formation on co-treatment with SRM 1597 revealed inhibitory effects on the CYP enzymes that convert carcinogenic PAH into DNA-binding metabolites. The implications for the tumorigenicity of complex environmental PAH mixtures are discussed. ' 2006 Wiley-Liss, Inc.
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