Environmental chemicals such as polycyclic aromatic hydrocarbons (PAHs), which are present in cigarette smoke and charcoal-broiled meats, cause cancer in laboratory animals and are suspected human carcinogens. The stated long-term goal of this application is to prevent chemically-induced malignancies in humans. The proposed work is based on studies indicating that some PAHs cause long-term induction in rats of cytochrome P450 1A1 (P450 1A1), an enzyme that can play important roles in carcinogenesis.
Specific Aim 1 will investigate the mechanisms of 3-methylcholanthrene (3MC)-mediated persistent induction of P450 1A1 in rats. Rats will be challenged with 3MC, and nuclear A receptor levels (gel mobility shift assays), P450 1A1 gene transcription rates (run-on transcription), mRNA levels (hybridization), and P450 1A1 activities/contents will be determined at selected time points.
Specific Aim 2 will test the hypothesis that persistent MC metabolites contribute to the sustained P450 1A1 induction. Animals will be treated with [3H]-3MC, the nucle will be isolated, and it will be determined whether 3MC metabolites interact with the Ah receptor.
Specific Aim 3 will test the hypothesis that 3MC causes sequence-specific DNA adduction on the regulatory regions of the P450 1A1 promoter, leading to novel mechanisms by which DNA adducts mimic the 3MC-Ah receptor complex and upregulate P450 1A1 gene expression. Plasmids carrying P450 1A1 promoter will be used to generate in vivo or in vitro adducts with 3MC. The adducted plasmids will be transiently transfected into Hepa-1 cells t determine whether 3MC-DNA adducts on the promoter will induce P450 1A1 gene expression in the absence of the inducer.
Specific Aim 4 will measure P450 1A1 activities and DNA adduct levels (32P-postlabeling) in rat lung and mammary tissues to test the hypothesis that 3MC elicits sustained induction of P450 1A and persistent DNA damage in target organs for carcinogenesis.
In Specific Aim 5, the activities/levels of P450 1B1, glutathione S-transferase-a, NAD(P)H quinone reductase, and UDP-glucuronosyl transferases will be determined to tes whether 3MC persistently induces other enzymes of the Ah gene battery.
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