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.
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.
Orimo
Hiroshi
H
Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan.
Mei
Nan
N
Boiteux
Serge
S
Tokura
Yoshiki
Y
Kasai
Hiroshi
H
D002460:Cell Line; D002477:Cells / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D043382:DNA-Formamidopyrimidine Glycosylase / Q000494:pharmacology*; D004563:Electrochemistry; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism; D018384:Oxidative Stress
C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D043382:DNA-Formamidopyrimidine Glycosylase