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 gamma-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 10(7)
guanine (Gua). A similar background level of 8-OH-Gua (3.1 residues per 10(7) 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 10(7)
dCMP). A dose-dependent increase of 8-OH-Gua (0.17/10(7) Gua/Gy) was observed after cells were irradiated with gamma-rays. Twenty hours after gamma-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.