Development of Tester Strains Deficient in Nth/Nei DNA Glycosylases to Selectively Detect the Mutagenicity of Oxidized DNA Pyrimidines

Abstract

Oxidative DNA damage is a major cause of mutation and cell death in aerobic organisms. In addition to 8-hydroxyguanine, oxidized DNA pyrimidines play important roles in mutagenesis. Salmonella typhimurium TA1535, widely used in mutagenicity assays, carries a hisG46 missense mutation and efficiently detects mutations at G:C base pairs. To detect oxidative mutagens that selectively modify pyrimidines, we constructed a derivative of strain TA1535, termed YG3206, which lacks the Nei and Nth DNA glycosylases that excise oxidized pyrimidines from DNA. This novel strain easily detected the mutagenicity of L-cysteine, L-penicillamine, dopamine-HCl, and phenazine methosulfate, which are non-mutagenic or only weakly mutagenic in the TA1535 parent strain. A second strain that is equivalent to YG3206 but harbors the plasmid pKM101 which carries mucAB encoding DNA polymerase R1, termed YG3216, was significantly sensitive to phenazine ethosulfate. The compound was not mutagenic in either YG3206 or the Fapy-glycosylase-defective strain YG3001. Potassium bromate and methylene blue plus visible light with metabolic activation induced mutations in YG3001 but not YG3206 or YG3216. The number of spontaneous His+ revertants per plate was 82 ± 16 (YG3206, ΔnthΔnei), 19 ± 4 (YG3001, Δfpg), and 6 ± 2 (TA1535), suggesting a significant contribution to spontaneous mutagenesis by endogenous pyrimidine oxidation. In the absence of exogenous chemical treatment, exposure to fluorescent light enhanced the spontaneous mutation frequency by approximately two-fold (YG3206), 13-fold (YG3001), and 10-fold (TA1535). These results suggest that certain environmental chemicals may selectively introduce mutagenic damage at DNA pyrimidines, and that these changes can be monitored by the use of YG3206.