Pyrimido[1,2-a]-purin-10(3H)-one, M1G, is less prone to artifact than base oxidation

Pyrimido[1,2-a]-purin-10(3H)-one (M(1)G) is a secondary DNA damage product arising from primary reactive oxygen species (ROS) damage to membrane lipids or deoxyribose. The present study investigated conditions that might lead to artifactual formation or loss of M(1)G during DNA isolation. The addition of antioxidants, DNA isolation at low temperature or non-phenol extraction methods had no statistically significant effect on the number of M(1)G adducts measured in either control or positive control tissue samples. The number of M(1)G adducts in nuclear DNA isolated from brain, liver, kidney, pancreas, lung and heart of control male rats were 0.8, 1.1, 1.1, 1.1, 1.8 and 4.2 M(1)G/10(8) nt, respectively. In rat liver tissue, the mitochondrial DNA contained a 2-fold greater number of M(1)G adducts compared with nuclear DNA. Overall, the results from this study demonstrated that measuring M(1)G is a reliable way to assess oxidative DNA damage because the number of M(1)G adducts is significantly affected by the amount of ROS production, but not by DNA isolation procedures. In addition, this study confirmed that the background number of M(1)G adducts reported in genomic DNA could have been overestimated by one to three orders of magnitude in previous reports.