Mutagenic effect of BUdR in diploid human fibroblasts

Abstract

It has only recently been possible to demonstrate the expected mutagenic effect of 5-bromodeoxyuridine (BUdR) in heteroploid hamster cells in culture. We have now extended this observation to diploid human fibroblasts utilizing techniques adapted from the work of Albertini and DeMars on X-tay mutagenesis at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in these cells. In four separate experiments, fibroblasts from a female donor were exposed to 500 μg/ml ethylmethane sulfonate (EMS) or 3 μg/ml BUdR yielding survivals of 9% and 5%, respectively. After a 6-day expression period, survivors were plated in selection medium containing 0.3 μg/ml 8-azaguanine (8-AG). After 3–5 weeks, azaguanine-resistant colonies were isolated for characterization or stained for counting. The average spontaneous mutation rate/cell/generation was 0.6·10 −6. The average induced mutation rates for EMS and BUdR were 7.8·10 −6 and 6.3·10 −6/cell/generation, respectively. Similar results were obtained in two experiments with an additional fibroblast line. Mutant colonies isolated following BUdR treatment demonstrated from 1.4 to 61.5% of the HGPRT activity of the parental line and showed at least 8% Barr bodies, excluding the possibility of contamination by Lesch-Nyhan cells. This demonstration of a BUdR effect comparable to that of an alkylating agent or X-irradiation opens the study of mutation due to base-analog substitution in diploid human cells.