Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and some are believed to be causal in human cancer. This proposal has two objectives. The first objective is to elucidate the mechanism(s) of metabolic activation of 3-methylcholanthrene (3MC), which is a potent carcinogen and a cytochrome P450 isozyme inducer widely used as a model compound in cancer and drug research. The second objective is to examine the formation of proximate carcinogenic metabolites and subsequent oxidation to ultimate carcinogenic product in the metabolism of benzo[a]pyrene (BaP) and 3MC respectively in an enzyme system containing one of thirteen forms of human liver P450s. Specific forms of human P450s (1A2, 2A3, 2B7, 2C8, 2C9, 2D6, 2E1, 2F1, 3A3, 3A4, 3A5, and 4B1) are each contained in human hepatoma Hep G2 cells infected with recombinant vaccinia virus containing a human P450 cDNA or in AHH-1 lymphoblastoid cells in which the cDNA encoding for human P450 1A1 is incorporated in a herpes-like vector. BaP is known to be metabolically activated along the BaP->BaP 7,8-epoxide->BaP 7,8-dihydrodiol->BaP 7,8-diol-9,10-epoxide pathway. However, pathway(s) of metabolic activation of 3MC is less clear. We propose to employ in vitro and in vivo methods to elucidate the activation pathways of 3MC. Individual cDNA-expressed human P450s will be used to determine P450-specific activation/detoxification metabolic pathways. 3MC is metabolized by mammalian drug metabolizing enzyme system to form a complex mixture of metabolites. Among synthetic 3MC derivatives, 2-hydroxy-3MC (2-OH-3MC) and 3MC-2-one are known to be potent carcinogens. Based on recent findings in our laboratory, we have developed a hypothesis that three highly optically active 9, 10-diol-7,8-epoxides derived from further metabolism of 2S-hydroxy- 3MC (2S-OH-3MC), 3MC-2-one, and 3-hydroxymethylcholanthrene (3-OHMC) may be responsible for the carcinogenic activities of 3MC. Each activation pathway differs in detail from that of BaP and involves four or five enantioselective enzymatic steps catalyzed by cytochrome P450s and epoxide hydrolase in the microsomal enzyme complex. Experiments including high-performance liquid chromatographic isolation and physicochemical characterization of metabolites, DNA binding, and tumor-initiating activity test on mouse skin are designed to determine activation/detoxification pathways of 3MC. The well-established species differences in P450 expression indicate that it is unlikely to reliably extrapolate rodent carcinogen activation data to humans. Direct analysis of human P450-based carcinogen metabolite formations is therefore essential to identify the form(s) responsible for carcinogen activation and detoxification. The results may eventually allow us to specifically induce certain P450s engaged in detoxification processes relative to those involved in carcinogen activation and thus reduce toxicity. This research will contribute to the much needed human enzyme-based risk assessment of environmental carcinogens.
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