Surprisingly little is known about the activities responsible for polycyclic aromatic hydrocarbon (PAH) metabolism in the epidermis. Our recent studies suggest that proliferating and differentiating keratinocytes have different capacities for PAH metabolism. The overall goal of this proposal is to determine whether epidermal PAH metabolism and tumorigenesis are influenced by the differentiation status of the tissue. Specifically, we will a) characterize the capacities of basal, proliferating and differentiating keratinocytes for PAH metabolism, b) characterize the enzymatic activities responsible for metabolism, and c) relate these activities to PAH-dependent epidermal tumorigenesis. Analyses will be made in primary cultures of proliferating and differentiating murine keratinocytes, and freshly isolated suspensions of keratinocytes separated by density gradient centrifugation into basal and differentiating cell populations. Capacities for oxidation of the PAHs, DMBA, BP and BP-7,8-diol will be measured by monitoring their metabolism or comparing mutant frequencies obtained in a keratinocyte- mediated mutation assay. Metabolite profiles will provide an indication of whether there are quantitative or qualitative differences in metabolizing activities, and studies with (+)BP-7,8- diol will provide estimates of mixed-function monooxygenase (MFO) and non-MFO (peroxidase-like) mediated metabolism. Antibodies to specific cytochrome P-450 species will be used in immunoblot studies to quantify these components of the MFO system. In vivo skin tumor studies in conjunction with PAH-DNA adduct analyses, using (+)BP-7,8-diol as the initiator, will be used to assess the in vivo relevance of MFO and non-MFO activities in the various keratinocyte cell types to epidermal carcinogenesis. Lastly, the differentiation status of murine epidermis will be manipulated to determine its influence on in vivo PAH metabolism (measured as PAH-DNA adducts) and carcinogenesis.
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