The nature and origin of DNA single-strand breaks determined with the comet assay

Abstract

After treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and hydrogen peroxide, the level of alkali-labile sites and single-strand breaks (ssb) in DNA was investigated, using the comet assay. The ability of antioxidant pre-treatment to decrease DNA damage was assessed. Results showed the following. (a) All single-strand (ss) DNA breaks detected immediately after MNNG- and MMS-treatment in hamster V79 cells had the character of alkali-labile sites while true ssb of DNA were represented only as a minor statistically significant (p<0.01) fraction at the highest MMS concentration. (b) Most ss DNA breaks detected immediately after H2O2-treatment had the character of true breaks in DNA and alkali-labile sites represented only a minor fraction. (c) Pre-treatment of hamster V79 and human CaCo2 cells with vitamin E significantly reduced the number of breaks induced by hydrogen peroxide, but has no effect on the level of breaks induced by MNNG or MMS. We suggest that MNNG and MMS do not induce significant oxidative damage of DNA. Most of breaks induced by hydrogen peroxide have the nature of oxidative lesions of DNA. (d) In contrast to the effect of vitamin E, stobadine (STB) decreased not only the breaks induced by hydrogen peroxide but also those induced by MNNG and MMS. The reduced level of DNA damage in STB pre-treated samples could be due to inactivation of these alkylating agents by STB.