Chemical induction of unscheduled DNA synthesis in human skin epithelial cell cultures.

Heretofore, unscheduled DMA synthesis (UDS) induced by DMA-damaging chemicals has been measured in nonreplicating human fibroblasts by autoradiographic meth ods that are not readily applicable to organotypic epithe lial cell cultures. In order to evaluate the range of chemi cal sensitivity and DNA repair response of human skin epithelial cells, we have developed a novel semiquantitative in vitro method for measuring UDS induced by chemi cals. Normal foreskin epithelial cells from a cryopreserved skin pool were grown from expiants and replanted in replicate culture wells. Cultures were then treated for 3 days in arginine-deficient medium and further inhibited in S-phase DNA synthesis by a 2-hr (10 ITIM) hydroxyurea treatment. [3H]Thymidine and carcinogen were simulta neously added, and UDS accumulated over a 24-hr incu bation period was determined by direct scintillation count ing of acid-precipitable whole-cell radioactivity. With this rapid assay, UDS is detected in dose-related fashion with an array of ultimate and proximate carcinogens and sev eral, but not all, classes of procarcinogens. Upon serial subculture of organotypic primary skin cultures, the UDS response elicited by the procarcinogen 3-methylcholanthrene decreased in parallel with a decreased ability of cells to metabolize polycyclic hydrocarbons to water-solu ble metabolites. Arginine deprivation and hydroxyurea treatment of skin epithelial cells does not impair their ability to metabolize procarcinogens to DMA-damaging metabolites. Our working hypothesis is that those procar cinogens efficiently activated by human skin-specific schemes of metabolism will be detected with UDS as an end point. Because no single human cell strain or microsomal enzyme preparation possesses all the organ-spe cific schemes of carcinogen metabolism, similar UDS measurement in a panel of organotypic epithelial cell cultures may provide a relevant technique for detecting specific human carcinogens.