Ascertainment of the effect of differential growth rates of mutants on observed mutant frequencies in X-irradiated mammalian cells An indication for the occurrence of X-ray-induced untargeted mutagenesis

Abstract

As it is not known to what extent differential growth rates of induced mutants lead to over- and under-representation of mutants in treated populations and thereby affect the determination of mutant frequencies, the mutation induction in X-irradiated L5178Y mouse lymphoma cells was determined via two methods. The first method involves the standard protocol which may suffer from the effect of differential growth rates, while the second method is based upon the fluctuation test in which the differential growth rates can be actually measured. It appeared that the standard protocol led to a mutant frequency that was similar to the mutant frequency determined in the fluctuation test. Therefore, the standard protocol appears to lead to only a minor under-estimation if any. Substantial heterogeneity in growth rates of induced mutants was observed, but the mutants with a selective advantage appear largely to compensate for the mutants that are lost because of selective disadvantage. It was calculated that the chance for isolating the same mutant twice from a treated population had been increased 2.2-fold because of the observed differential growth rates. Therefore, our data indicate that the standard protocol does not lead to serious errors in the determination of mutant frequencies and in the sampling of mutants. The fluctuation tests were also used to determine the spontaneous mutation frequency per cell per generation. The mutation rate appeared more than 10-fold enhanced in X-irradiated cells which may be attributed to the induction of a process of untargeted mutagenesis in mammalian cells.