Optimization of the concurrent assay for gene mutations and chromosomal aberrations in vivo: expression time in rats

Ethylnitrosourea was used as a model mutagen to determine the time at which the most mutants and chromosomal aberrations could be detected. Primary fibroblasts derived from Fischer (F344) rats that had been mutagenized by ethylnitrosourea in vivo, were grown in culture and sampled at increasing intervals to analyze, quantitatively, gene mutations and chromosomal aberrations. Since ethylnitrosourea reacts very quickly and the cells were isolated five hours after treatment, expression time represents the intracellular processes involved, not the pharmacokinetics of the mutagen. A thioguanine selection procedure was used to quantify gene mutations. A cytochalasin B technique for micronuclei was used to quantify chromosomal aberrations. A significant increase in the number of mutations was observed on the ninth day in an experiment wherein sampling was done at increasing intervals of three days. One of two experiments wherein sampling was done at increasing intervals of five days showed maximum number of mutations in dishes plated with cells, exposed to maximum dose (200 mg/kg), on the tenth day for selection. Cells that received dose 100 mg/kg on the contrary produced maximum number of mutant colonies in dishes that were plated on the 20th day for selection. A repeat of the 5-day interval experiment showed maximum number of mutant colonies in dishes selected on the tenth day, irrespective of the dose used. The number of micronuclei reached a maximum on the third day.