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 | Zendy

Hiroshi Orimo | Zendy; Nan Mei | Zendy; Serge Boiteux | Zendy; Y. Tokura | Zendy; Hiroshi Kasai | Zendy

Open Access

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

Author(s) -

Hiroshi Orimo,

Nan Mei,

Serge Boiteux,

Y. Tokura,

Hiroshi Kasai

Publication year - 2004

Publication title -

journal of radiation research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.643

H-Index - 60

eISSN - 1349-9157

pISSN - 0449-3060

DOI - 10.1269/jrr.45.455

Subject(s) - dna , dna glycosylase , hacat , chemistry , microbiology and biotechnology , dna damage , guanine , dna repair , oxidative stress , biochemistry , gene , biology , in vitro , nucleotide

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.

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