Analysis of 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) as a reliable marker of cellular oxidative DNA damage after gamma-irradiation.
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 gamma-radiation and an increase in 8-OH-Gua concentration was observed with increasing gamma-ray dose (0.3 residues per 10(7) 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 gamma-irradiation. Using an endonuclease nicking assay, we also found that gamma-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.
Mei
Nan
N
Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan.
Tamae
Kazuyoshi
K
Kunugita
Naoki
N
Hirano
Takeshi
T
Kasai
Hiroshi
H
D015415:Biomarkers / Q000032:analysis*; D002277:Carcinoma / Q000378:metabolism / Q000532:radiotherapy; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology; D004847:Epithelial Cells / Q000528:radiation effects; D005720:Gamma Rays / Q000009:adverse effects*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000532:radiotherapy; D018384:Oxidative Stress; D018675:Toxicity Tests / Q000379:methods; D014407:Tumor Cells, Cultured
D015415:Biomarkers; C063477:8-hydroxydeoxyguanosine 5'-monophosphate; C024829:8-hydroxyguanine; D006147:Guanine; D006157:Guanosine Monophosphate