Analysis of 8‐hydroxydeoxyguanosine 5′‐monophosphate (8‐OH‐dGMP) as a reliable marker of cellular oxidative DNA damage after γ‐irradiation-猫眼课题宝

分析 8-羟基脱氧鸟苷 5′-单磷酸 (8-OH-dGMP) 作为 γ 辐射后细胞氧化 DNA 损伤的可靠标记物

DOI：

发表时间：

2003

期刊：

Environmental and Molecular Mutagenesis

影响因子：

2.8

通讯作者：

中科院分区：

环境科学与生态学3区

文献类型：

作者：

N. Mei;K. Tamae;N. Kunugita;T. Hirano;H. Kasai

为了提高8 -羟基鸟嘌呤（8 - OH - Gua）在DNA中的检测，我们用核酸酶P1消化分离的DNA，并使用配备电化学检测器（HPLC - ECD）的高效液相色谱系统分析8 -羟基脱氧鸟嘌呤5 ' -单磷酸（8 - OH - dGMP）。DNA中8‐OH‐Gua的量以8‐OH‐dGMP与脱氧胞苷单磷酸（dCMP）的比例表示。通过该分析，人肺癌细胞（A549） DNA中8‐OH‐Gua的背景水平比以前的方法低几倍。A549细胞暴露于20-60 Gy的γ射线照射下，8 - OH - Gua浓度随γ射线剂量的增加而增加（每107 dCMP / Gy 0.3个残基）。此外，通过使用商业FITC试剂盒的免疫组织化学程序，可以在A549细胞中清楚地检测到8‐OH‐Gua，并且氧化DNA损伤细胞的荧光强度随着γ‐照射剂量的增加而增加。通过核酸内切酶切痕实验，我们还发现γ射线降低了8‐OH‐Gua的修复活性。结果表明，8 - OH - dGMP是一种有用且敏感的DNA氧化损伤标志物。环绕。生物工程学报，2003,31(1):332 - 338。©2003 Wiley‐Liss, Inc。

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 γ‐radiation and an increase in 8‐OH‐Gua concentration was observed with increasing γ‐ray dose (0.3 residues per 107 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 γ‐irradiation. Using an endonuclease nicking assay, we also found that γ‐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. Environ. Mol. Mutagen. 41:332–338, 2003. © 2003 Wiley‐Liss, Inc.