Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories:: an approach to consensus
Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories:: an approach to consensus
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DOI：
10.1093/carcin/23.12.2129
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发表时间：
2002-12-01
期刊：
影响因子：
4.7
通讯作者：
Smith, E
中科院分区：
文献类型：
--
作者：
Collins, A;Gedik, C;Smith, E
欧洲氧化性DNA损伤标准委员会（ESCODD）的成立是为了解决与测量人类细胞中氧化性DNA损伤（特别是8-氧代-7，8-二氢鸟嘌呤或8-氧代Gua）的背景水平相关的问题。在层析之前的样品制备期间发生DNA氧化的趋势已被认为是非常重要的伪影的来源。为了评估消除人工制品的努力是否成功，ESCODD向其成员分发了猪肝和HeLa细胞的标准样品进行分析。使用色谱技术，估计猪肝中的8-oxoGua为2.23 - 441/10（6）鸟嘌呤，中位数为10.47/10（6）鸟嘌呤。HeLa细胞DNA的色谱分析给出了每10（6）个鸟嘌呤1.84至214的范围，中位数为每10（6）个鸟嘌呤5.23。HeLa细胞DNA也进行了分析，通过酶的方法，其中整个细胞的DNA处理与formamidopyrimidine DNA糖基化酶，切口DNA的网站8-oxoGua，和断裂测量彗星试验，碱性解旋或碱性洗脱。这些方法的值范围为0.06 - 4.98 8-oxoGua/10（6）鸟嘌呤，中位数为0.79/10（6）鸟嘌呤。虽然方法和实验室之间显然仍然存在严重的差异，但色谱法的最低估计值（可以说是人工制品得到最好控制的那些）仅比酶法获得的中值高2.5倍。
The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve the problems associated with the measurement of background levels of oxidative DNA damage (in particular 8-oxo-7,8-dihydroguanine, or 8-oxoGua) in human cells. A tendency for DNA oxidation to occur during sample preparation prior to chromatography has been recognized as the source of a very substantial artefact. To assess the success of attempts to eliminate the artefact, ESCODD has distributed to its members standard samples of pig liver and HeLa cells for analysis. Estimates of 8-oxoGua in pig liver, using chromatographic techniques, ranged from 2.23 to 441 per 10(6) guanines, with a median of 10.47 per 10(6) guanines. Chromatographic analysis of HeLa cell DNA gave a range of 1.84 to 214 per 10(6) guanines with a median of 5.23 per 10(6) guanines. HeLa cell DNA was also analysed by an enzymic approach, in which whole cell DNA was treated with formamidopyrimidine DNA glycosylase, which nicks DNA at sites of 8-oxoGua, and the breaks measured with the comet assay, alkaline unwinding or alkaline elution. Values with these methods ranged from 0.06 to 4.98 8-oxoGua per 10(6) guanines, with a median of 0.79 per 10(6) guanines. Although there are clearly still serious discrepancies between methods and laboratories, the lowest estimates by chromatography (arguably those in which the artefact was best controlled) are only 2.5 times higher than the median value obtained with the enzymic approach.