Analysis of 8-Hydroxyguanine (8-OH-Gua) Released from DNA by the Formamidopyrimidine DNA Glycosylase (Fpg) Protein: A Reliable Method to Estimate Cellular Oxidative Stress

Abstract

To improve the analyses of a form of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), we treated isolated DNA with formamidopyrimidine DNA glycosylase (Fpg) and analyzed the released 8-OH-Gua by using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The human lung carcinoma cells (A549) and human keratinocyte (HaCaT) were irradiated with γ-rays. After the isolated DNA was treated with the Fpg protein, we analyzed the released 8-OH-Gua by using an HPLC-ECD. With this method, the background level of 8-OH-Gua in DNA from human lung carcinoma cells was determined to be 3.4 residues per 10⁷ guanine (Gua). A similar background level of 8-OH-Gua (3.1 residues per 10⁷ Gua) was also detected in human keratinocyte DNA with this method. These background 8-OH-Gua levels in cellular DNA are comparable to that obtained previously by an analysis of 8-OH-dGMP after nuclease P1 digestion of cellular DNA (4.3 residues per 10⁷ dCMP). A dose-dependent increase of 8-OH-Gua (0.17/10⁷ Gua/Gy) was observed after cells were irradiated with γ-rays. Twenty hours after γ-irradiation with 60 Gy, 75% of the 8-OH-Gua produced in keratinocyte DNA was repaired. With our new analysis method, it is possible to detect the small changes in the 8-OH-Gua levels in cellular DNA induced by various environmental factors.