Establishment of a sensitive assay system for detecting mutations induced by oxidative DNA damage

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Oxygen radicals are produced through normal cellular metabolism, and formation of such radicals is enhanced further by ionizing radiation and by various chemicals. The oxygen radicals attack nucleic acids and generate various modified bases in DNA. Among them, 8-oxoguanine (8-oxoG) is the most abundant, and seems to play a critical role in mutagenesis as well as carcinogenesis and aging. 8-OxoG can pair with both cytosine and adenine during DNA synthesis, and as a result, G : C to T : A transversions are induced. Oxidation of guanine also occurs in the cellular nucleotide pool. 8-Oxo-dGTP, when formed, is a potent mutagenic substrate for DNA synthesis ; it is equally incorporated opposite both adenine and cytosine in DNA, resulting in both A : T to C : G and G : C to T : A transversions. In order to establish a sensitive assay system for detecting mutagenesis caused by oxidative DNA damage, we used the rpsL transgene as a reporter for the mutation assay, a suitable system for identifying a spectrum of base substitutions and single-base frameshifts. Using the resulting DNA repair-deficient mice with the rpsL transgene (rpsL hemizygotes), we examined oxidative stress-induced mutations occurring in the tissues of different DNA repair-deficient mouse lines. The frequency of G : C to T : A transversions was significantly increased both in Ogg1^<-/-> and Mutyh^<-/-> mice when administered with KBrO_3 in drinking water. We observed an increased incidence of G : C to A : T transitions in Ogg1^<-/-> mice after exposure to low dose X-ray irradiation (4 Gy). We further detected a characteristic spectra of A : T to G : C transitions in the spleens of X-ray-irradiated Mth1^<-/-> mice. In this study, we have shown a characteristic difference in the mutational spectra of wild-type mice and several DNA repair-deficient mice, such as Mth1,Ogg1,and Mutyh.We also examined the interaction between mammalian enzymes and oxidatively damaged ribonucleotides/RNA.