Mutational spectra in human B-cells: Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene

Abstract

To test the hypothesis that reactive species in the oxygen cascade are responsible for spontaneous mutation, we examined the spectra of oxygen and hydrogen peroxide-induced mutations at the hprt locus in a human B-lymphoblastoid cell line. We compared these spectra with the spontaneous mutational spectrum. Large gene alterations were studied by Southern analysis of individual TG R clones. A combination of high fidelity polymerase chain reaction, denaturing gradient gel electrophoresis and direct DNA sequencing were used to detect and identify point mutations in exon 3 of hprt. With regard to spontaneous mutations, a previous study showed that 39% of the spontaneous TG R clones had large gene alterations. In the present study, the analysis of spontaneous point mutations within exon 3 revealed two hotspots. A one base-pair deletion (−A) at base-pair 256 or 257 and a two base-pair deletion (−GG) at base-pair 237 and 238, were detected in triplicate cultures. Each of the hotspots comprised about 1% of the TG R mutants. The analysis of individual oxygen-induced TG R clones (48 h, 910 μ m-O 2) showed 43% had large gene alterations similar to the spontaneous TG R clones. However, none of the spontaneous point mutation hotspots was found among triplicate oxygen-treated cultures. Two point mutations in common with H 2O 2-treated cultures were found in one of the three oxygen-treated cultures. Hydrogen peroxide-induced mutations (1 h, 20 μ m) also differed from spontaneous mutations. Only 24% of the hydrogen peroxide-induced TG R clones had large gene alterations. The analysis of point mutations showed three hotspots within exon 3 of hprt. An AT to TA transversion at base-pair 259 had an average frequency of 3 % of all TG R mutants (present in all of 3 H 2O 2-treated cultures). Two GC to CG transversions at base-pairs 243 and 202 were present at a frequency of 0.6% and 0.4%, respectively. A five base-pair deletion (base-pair 274 to 278) was present at an average frequency of 0.3%. The latter three mutations were detected in two of three H 2O 2-treated cultures. Thus, the point mutation spectra of both oxygen and hydrogen peroxide were significantly different from the spontaneous spectrum. The oxygen and hydrogen peroxide-induced spectra shared some features, suggesting that oxygen and hydrogen peroxide share some but not all pathways for induction of mutations within the DNA sequence studied here. Subject to the caveat that the mutation rates differed among the three conditions characterized, and that these differences may well affect mutation mechanisms, we conclude that spontaneous point mutations at the hprt gene in human cells in vitro do not arise primarily from reactions of the DNA with elements of the active oxygen cascade. However, it is possible that the pathway(s) of spontaneous large deletions do involve active oxygen species.