Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells?
Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells?
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DOI：
10.1016/j.abb.2003.12.022
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发表时间：
2004-03-01
影响因子：
3.9
通讯作者：
Viña, J
中科院分区：
文献类型：
--
作者：
Collins, AR;Cadet, J;Viña, J
氧化性DNA损伤的最常测量的标志物是8-氧代-7，8-二氢鸟嘌呤（8-oxoGua）或其脱氧核糖核苷（8-oxodGuo）。已发表的正常人细胞DNA中8-oxoGua/8-oxodGuo浓度的估计值在三个数量级的范围内变化。通过色谱方法（GC-MS、具有电化学检测器（ECD）的HPLC或HPLC-MS/MS）进行的分析受到样品制备期间鸟嘌呤的外源氧化问题的困扰。另一种方法，基于使用DNA修复酶甲酰氨基嘧啶DNA N-糖基化酶（FPG）在氧化碱基的位点处使DNA断裂，得到的值要低得多。ESCODD，欧洲标准委员会对氧化性DNA损伤，已经测试了不同的实验室使用各种方法来测量8-oxodGuo，小牛胸腺DNA，猪肝，寡核苷酸和HeLa细胞的标准样品中的8-oxoGua的能力，以及从志愿者血液中分离的淋巴细胞。HPLC-ECD能够以高准确度测量小牛胸腺DNA或HeLa细胞中实验诱导的8-oxodGuo。然而，没有迹象表明这种损害的背景水平的共识，这表明，即使使用标准提取程序，变量氧化Gua仍在发生。GGMS未能检测到诱导的8-oxoGua的剂量响应，不能被视为一个可靠的方法，用于测量低水平的损伤。HPLC-MS/MS尚未被证明能够测量低水平的氧化DNA损伤。基于FPGA的方法似乎不太容易出现额外氧化的假象。虽然它们可以定量使用，但如果要用于人体生物监测，它们需要仔细校准和标准化。正常人体细胞中DNA氧化的背景水平可能约为0.3-4.2 8-oxoGua/10（6）Gua。应努力开发替代的、有效的方法来估计氧化DNA损伤。(C)2003年爱思唯尔公司All rights reserved.
The most commonly measured marker of oxidative DNA damage is 8-oxo-7,8-dihydroguanine (8-oxoGua) or its deoxyribonucleoside (8-oxodGuo). Published estimates of the concentration of 8-oxoGua/8-oxodGuo in DNA of normal human cells vary over a range of three orders of magnitude. Analysis by chromatographic methods (GC-MS, HPLC with electrochemical detection (ECD) or HPLC-MS/MS) is beset by the problem of adventitious oxidation of guanine during sample preparation. An alternative approach, based on the use of the DNA repair enzyme formamidopyrimidine DNA N-glycosylase (FPG) to make breaks in the DNA at sites of the oxidised base, gives much lower values. ESCODD, the European Standards Committee on Oxidative DNA Damage, has been testing the ability of different laboratories using a variety of methods to measure 8-oxoGua in standard samples of 8-oxodGuo, calf thymus DNA, pig liver, oligonucleotides, and HeLa cells, and in lymphocytes isolated from blood of volunteers.HPLC-ECD is capable of measuring 8-oxodGuo induced experimentally in calf thymus DNA or HeLa cells with high accuracy. However, there is no sign of consensus over the background level of this damage, suggesting that, even though standard extraction procedures were used, variable oxidation of Gua is still occurring.GGMS failed to detect a dose response of induced 8-oxoGua and cannot be regarded as a reliable method for measuring low levels of damage.HPLC-MS/MS as yet has not proved capable of measuring low levels of oxidative DNA damage. FPG-based methods seem to be less prone to the artefact of additional oxidation. Although they can be used quantitatively, they require careful calibration and standardisation if they are to be used in human biomonitoring.The background level of DNA oxidation in normal human cells is likely to be around 0.3-4.2 8-oxoGua per 10(6) Gua.An effort should be made to develop alternative, validated methods for estimating oxidative DNA damage. (C) 2003 Elsevier Inc. All rights reserved.