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

Abstract

Abstract Heretofore, unscheduled DNA synthesis (UDS) induced by DNA-damaging chemicals has been measured in non-replicating human fibroblasts by autoradiographic methods that are not readily applicable to organotypic epithelial cell cultures. In order to evaluate the range of chemical sensitivity and DNA repair response of human skin epithelial cells, we have developed a novel semiquantitative in vitro method for measuring UDS induced by chemicals. Normal foreskin epithelial cells from a cryopreserved skin pool were grown from explants 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 mm) hydroxyurea treatment. [3H]Thymidine and carcinogen were simultaneously added, and UDS accumulated over a 24-hr incubation period was determined by direct scintillation counting 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 several, 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-soluble metabolites. Arginine deprivation and hydroxyurea treatment of skin epithelial cells does not impair their ability to metabolize procarcinogens to DNA-damaging metabolites. Our working hypothesis is that those procarcinogens 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-specific 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.