oa In Vivo Mutagenesis by UVB Irradiation: Detection and the Underlying Mechanisms

Human genome is continuously exposed to endogenous and exogenous genotoxic agents. To evaluate genotoxicity of the agents, we have developed gpt delta mice transgenic gene mutation assay, which allows detection of mutations in vivo in any organ of mice. The mice have been established by microinjection of lambda EG10 phage DNA into fertilized eggs of C57Bl/6J mice. A feature of the transgenic mice assay is incorporation of two selections to detect different types of mutations, i.e., point mutations such as base substitutions and frameshifts by gpt or 6-thioguanine selection and deletion mutations by Spi- (sensitive to P2 interference) selection. The gpt assay effectively detects point mutations induced by a variety of chemical agents that induce DNA adducts while Spi- assay exclusively detects deletions with the molecular sizes from 1 base pair (bp) to several kilo bps (kb). We report that UVB irradiation induces base substitutions and deletions in the epidermis of gpt delta mice and also that p53 suppresses the deletions and complex mutations in vivo. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5 and 2.0 kJ/m2. At four weeks after irradiation, mutations in the epidermis were analyzed. The UVB irradiation induced G:C to A:T mutations at dipyrimidine sites, such as 5’-TC-3’ and 5’-CC-3’. Tandem transitions such as CC to TT were also observed. In addition to point mutations, UVB induced deletions with the size of more than 1 kb. More than half of the large deletions occurred between short homologous sequences from 1 to 6 bps while others had flush ends. The results suggest that the deletions are generated by end-joining of double-strand breaks in DNA. To examine protective roles of p53 against UVB-induced mutagenesis, mutations were measured in the epidermis of UVB-induced p53+/+ and p53-/- gpt delta mice. The mice were exposed to UVB at single doses of 0.5, 1.0 and 2.0 kJ/m2, and the mutant frequencies (MF) were determined in the epidermis 4 weeks after the irradiation. Although UVB enhanced gpt MF more than 10 times over the unirradiated mice, there were no significant differences in gpt MF and the mutation spectra between p53+/+ and p53-/- mice. In contrast, the frequency of Spi- large deletions of more than 1 kb and complex mutations were significantly higher in unirradiated p53-/- mice than in unirradiated p53+/+ mice. These large deletions and complex mutations increased in a UVB-dose-dependent manner in p53+/+ mice, while no increase was observed in p53-/- mice. These results suggest that p53 does not suppress point mutations induced by UVB but suppresses large deletions and complex mutations in vivo.