Measurement of unscheduled DNA synthesis in primary cultures of adult mouse epidermal keratinocytes.

Skin is a major target tissue for many environmental carcinogens. In order to provide a tool to study the role of DNA-repair processes in relation to DNA damage and carcinogenesis in this tissue, we have developed an assay that measures chemically induced DNA repair as unscheduled DNA synthesis (UDS) using cultures of adult mouse epidermal keratinocytes (MEKs) from SENCAR mice. Primary MEKs were prepared and incubated for 24 h in the presence of the test chemical and 10 microCi/ml [3H]thymidine. UDS was quantitated autoradiographically as net grains per nucleus. This assay detected DNA damage caused by the direct-acting agents N-methyl-N'-nitro-N-nitrosoguanidine, N-methylnitrosourea, methyl methanesulfonate, ethyl methanesulfonate and (+/-)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene and the indirect-acting carcinogens 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, adriamycin and 4-nitroquinoline-1-oxide. The growth conditions used allowed the epidermal cells to retain the tissue specificity of carcinogen activation of mouse epidermis in vivo in that 2-acetylaminofluorene was inactive in this assay. This assay system should be useful for determining genotoxic and potential carcinogenic agents for skin as well as for mechanistic studies involving DNA repair and chemical carcinogenesis in this tissue.