Quantitation of 8-Oxoguanine and Strand Breaks Produced by Four Oxidizing Agents

Abstract

Reactive oxygen species, produced endogenously or by exposure to environmental chemicals and ionizing radiation, induce a wide range of DNA lesions. The variety of chemistries associated with different oxidants suggests that each will produce a unique spectrum of DNA damage products. To extend our efforts to relate genotoxin chemistry to DNA damage, we measured both strand breaks and 8-oxoguanine (8-oxoG) in DNA after exposure to gamma-radiation, Fe(II)-EDTA/H2O2, Cu(II)/H2O2, and peroxynitrite at concentrations approaching physiological relevance. We found that the ratio of 8-oxoG to strand breaks varied more than 10-fold depending on the oxidizing agent: approximately 0.4 for Cu(II)/H2O2 and peroxynitrite and approximately 0.03 for Fe-(II)-EDTA/ H2O2 and gamma-radiation. In the case of Cu(II)/H2O2, the relative proportion of 8-oxoG and strand breaks was found to vary more than 2-fold (0.14-0.37) for different Cu(II) concentrations, consistent with other studies. We were able to detect 8-oxoG formation by peroxynitrite by using low peroxynitrite concentrations in conjunction with a sensitive immunoaffinity/HPLC-ECD methodology. The level of 8-oxoG relative to strand breaks produced by peroxynitrite was higher than that produced by Fe(II)-EDTA/H2O2 and gamma-radiation, which is consistent with the altered reactivity or accessibility of a non-hydroxyl radical species produced by peroxynitrite.