Polycyclic aromatic hydrocarbons (PAHs), which are present in tobacco smoke and in charcoal broiled meats, cause cancers in laboratory animals and are suspected human carcinogens. The central hypothesis of this application is that a cytochrome P4501A2 (CYP1A2)-dependent) metabolite of 3-methylcholanthrene (MC) plays an important role in the sustained induction of CYP1A1, a phenomenon that may have important implications for carcinogenesis. We propose the following specific aims. 1. To investigate the molecular mechanisms of sustained induction of CYP1A1 by MC, and test the hypothesis that a CYP1A2-dependent metabolite of MC contributes to the sustained CYP1 Al induction by MC. Wild-type (WT) and CYP1A2- knockout (KO) mice will be treated with MC (100 fmol/kg), once daily for 4 days, or a single dose of MC (100 fmol/kg) and at selected time points, levels of ligand-bound Ah receptor (AHR) in hepatic or pulmonary nuclei will be determined by electrophoretic mobility shift assay (EMSA)/Western blotting. 2.To test the hypothesis that CYP1A2-dependent metabolism of MC in hepa-1 cells or in mice leads to formation of sequence-specific DNA adduction, as determined by ligation-mediated polymerase chain reaction (LM-PCR), on the regulatory regions of the CYP1 Al promoter [e.g., AHR response elements (AhREs)], leading to a novel mechanism by which MC-DNA adducts will upregulate CYP1A1 gene. 3. Using a transgenic mouse line carrying the human CYP1A1 promoter, the hypothesis, that persistent induction of CYP1A1 expression by MC occurs in a tissue-specific manner, and is preceded by sustained activation of the CYP1A1 promoter, will be tested. 4. To test the hypothesis that CYP1A2-null mice will be more susceptible to MC-induced pulmonary carcinogenesis than similarly exposed WT mice. WT or KO mice will be exposed to MC, and CYP1A1 expression, DNA adducts, alteration in the expression of other cancer-related genes, as determined by cDNA microarray analyses, and tumor histology will be studied. The long-term goals are to: (i) define the molecular mechanisms of regulation of CYP1A1 gene expression by PAHs and (ii) develop rational strategies for the prevention/treatment of humans cancers caused by environmental chemicals.
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