Metabolism of benzo[a]pyrene in human epidermal keratinocytes in culture

Abstract

The metabolism of benzo[a]pyrene (BP) in cultured human epidermal keratinocytes was investigated using thin layer chromatography, high pressure liquid chromatography and cell-mediated mutagenesis assay. Epidermal keratinocytes were obtained from skin of normal subjects and all experiments were performed on primary cultures. Human epidermal keratinocytes were shown to metabolize BP. Analysis of BP metabolites by high pressure liquid chromatography indicated that epidermal keratinocytes metabolize BP preferentially at non-K-regions such as positions 7, 8, 9 and 10, forming a moderate amount of BP-7, 8-dihydrodiol, a precursor of the ultimate metabolite, BP-7, 8-dihydrodiol-9, 10-epoxide. Conjugate formation was examined by treating the medium with β-glucuronidase and arylsulfatase. No appreciable amount of conjugates was formed by epidermal keratinocytes, except in one culture which gave small peaks eluted in the phenol regions after β-glucuronidase treatment. The metabolic activity of human epidermal keratinocytes on BP was further demonstrated by a cell-mediated assay, in which V79 Chinese hamster cells were cultured on top of sheets of keratinocytes and treated with BP for 48 h. Mutation of the V79 cells, demonstrated as ouabain resistance, was induced in a dose-related fashion. The extent of induced mutation was higher than that observed using rat embryo cells as the activating layer, although the shape of the dose-response curves was different.