Keratinocyte cell-mediated mutagenesis assay: correlation with in vivo tumor studies

A murine keratinocyte cell-mediated mutagenesis assay was characterized and examined as an in vitro model system for studying the biotransformation of promutagens/procarcinogens by mouse skin. The assay used living cultured newborn SENCAR keratinocytes for the metabolic activation of promutagens and Chinese hamster lung V-79 fibroblasts for detection of resulting mutagens. Mutations at, or affecting, the hypoxanthine-guanine phosphoribosyltransferase locus were scored by resistance to 6-thioguanine. The relative mutagenicities of several polycyclic aromatic hydrocarbons (PAHs) in the cell-mediated assay correlated with the in vivo skin tumorigenicity of the PAHs determined in a two-stage carcinogenesis protocol. Metabolic activation of the promutagenic PAHs to ultimate mutagens was dependent upon the presence of the cultured keratinocyte feeder layer. 7,8-Benzoflavone, a potent inhibitor of 7,12-dimethylbenz[a]anthracene (DMBA)-dependent initiation in mouse skin, inhibited DMBA-dependent mutagenesis in the cell-mediated assay in a concentration responsive manner. The non-PAH promutagens, dimethylnitrosamine (DMN) and sterigmatocystin (STC) were both activated by cultured keratinocytes to cytotoxic derivatives. DMN was neither mutagenic in the cell-mediated assay nor tumorigenic in mouse skin when tested in a two-stage carcinogenesis protocol. STC was weakly mutagenic and tumorigenic in mouse skin.