DNA oxidation matters: The HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine
DNA oxidation matters: The HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine
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DOI：
10.1073/pnas.95.1.288
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发表时间：
1998-01-06
影响因子：
11.1
通讯作者：
Ames, BN
中科院分区：
文献类型：
--
作者：
Helbock, HJ;Beckman, KB;Ames, BN
氧化DNA损伤在衰老和诸如癌症等衰老的退行性疾病中是重要的，估计通常依赖于8-氧代-2‘-脱氧鸟苷(oxo(8)dg)的测量，8-oxo-2’-脱氧鸟苷(oxo(8)dG)是DNA中的一种加合物，也是DNA修复后在尿液中排泄的一种加合物。在这里，我们检查oxo(8)dG分析中固有的困难，识别伪影的来源，并为一些常见的方法学问题提供解决方案。经常有人批评DNA提取溶液中的苯酚人为地增加了oxo(8)dG的测量水平，我们发现，酚提取DNA有助于真正但轻微的增加oxo(8)DG水平当在同等条件下，与成功的非酚方法相比，通过对最近引入的杂化NaI方法的修改，基线水平实现了更显著的降低，将我们对稳态氧化加合物水平的估计降低了一个数量级，在年轻大鼠的每个细胞中为24,000个加合物，在老年大鼠中为每个细胞66,000个加合物，在几种替代测试方法中，使用这种通过使用NaI分离DNA的杂化技术产生了最低和变化最小的oxo(8)DG值。在进一步的研究中，我们发现人类尿中8-氧鸟嘌呤(oxo(8)gua)的排泄不受给药别嘌醇的影响，这表明oxo(8)gua不同于某些甲基化加合物，它不是从黄嘌呤氧化酶酶促而来的，最后，我们讨论了在稳态oxo(8)DG测量和基于oxo(8)dg和oxo(8)gua排泄的内源性氧化(“命中率”)估计中所固有的剩余不确定性。
Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer, Estimates commonly rely on measurements of 8-oxo-2'-deoxyguanosine (oxo(8)dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair, Here we examine difficulties inherent in the analysis of oxo(8)dG, identify sources of artifacts, and provide solutions to some of the common methodological problems, A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo(8)dG, We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo(8)dG when compared, under equivalent conditions, with a successful nonphenol method, A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats, Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo(8)dG values. In further studies we show that human urinary 8-oxo-guanine (oxo(8)Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo(8)Gua is not derived enzymatically from xanthine oxidase, Lastly, we discuss remaining uncertainties inherent both in steady-state oxo(8)dG measurements and in estimates of endogenous oxidation ("hit rates") based on urinary excretion of oxo(8)dG and oxo(8)Gua.