COMPARISON OF MUTATION SPECTRA INDUCED BY N-ETHYL-N-NITROSOUREA IN THE HPRT GENE OF MER(+) AND MER(-) DIPLOID HUMAN FIBROBLASTS

N-Ethyl-N-nitrosourea (ENU) forms several major adducts upon reaction with DNA, of which ethylation at the O-6 position of guanine and the O-1, O-2 and N-3 positions Of thymine have been implicated to be mutagenic lesions. To investigate what specific kinds of ENU-induced mutations were affected by the repair ability of O-6-alliylguanine-DNA alkyltransferase (AGT), we examined the mutations in the hypoxanthine (guanine) phosphoribosyltransferase gene (hprt) in 87 independent mutants derived from ENU-treated AGT proficient (Mer(+)) or deficient (Mer(-)) diploid human fibroblasts. Of the characterized mutations, 97% were single base substitutions. The major difference in the mutation spectra was that the frequency of G C to A T transitions was significantly higher in Mer(-) mutants (16/38) than in Mer(+) mutants (4/33). The results indicate that AGT removes O-6-ethylguanine, thus protecting human cells from parts of the cytotoxic and mutagenic effects of ENU. A high frequency of T A to A T transversions induced by ENU was observed in both Mer(+) (52%) and Mer(-) (34%) mutants. This type of mutation was less frequently observed (10%) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutants derived from the same Mer(+) cells in our previous report (J. Mol. Biol., 221, 421, 1991). Comparison of alkylating lesions formed by MNNG and ENU indicates that O-2-ethylthymine and N-3-ethylthymine are potent mutational adducts for T to A transversions. The occurrence of ENU-induced T A base pair transversions showed a strong strand bias; 35/37 were located on the non-transcribed strand, assuming thymine is the mutagenic lesion. The result suggests a difference in repair capacity of ethylthymine on the two strands. In addition, this type of mutation preferentially occurred at 5'-Pu-T sequences.