Analysis of 8‐hydroxydeoxyguanosine 5′‐monophosphate (8‐OH‐dGMP) as a reliable marker of cellular oxidative DNA damage after γ‐irradiation

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Abstract
In order to improve 8-hydroxyguanine (8-OH-Gua) detection in DNA, we digested isolated DNA with nuclease P1 and analyzed for 8-hydroxydeoxyguanosine 5′-monophosphate (8-OH-dGMP) using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The amount of 8-OH-Gua in the DNA was expressed as the ratio of 8-OH-dGMP to deoxycytidine monophosphate (dCMP). Using this analysis, the background level of 8-OH-Gua in DNA from human lung carcinoma cells (A549) was several-fold lower than that obtained by a previous method. A549 cells were exposed to 20–60 Gy of γ-radiation and an increase in 8-OH-Gua concentration was observed with increasing γ-ray dose (0.3 residues per 107 dCMP per Gy). Moreover, by an immunohistochemical procedure using a commercial FITC-kit, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of γ-irradiation. Using an endonuclease nicking assay, we also found that γ-rays decreased 8-OH-Gua repair activity. The results indicate that 8-OH-dGMP is a useful and sensitive marker for estimating oxidative damage in DNA. Environ. Mol. Mutagen. 41:332–338, 2003.