Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35,000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3-9 J/m2; the D0 of the survival curve was 4.2 J/m2. Delaying subculture to low density for 1.5-24h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6h as compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels. (orig./AJ)