HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2′-deoxyguanosine, in Cultured Cells and Animal Tissues
HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2′-deoxyguanosine, in Cultured Cells and Animal Tissues
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DOI：
10.3791/52697
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发表时间：
2015-08-01
影响因子：
1.2
通讯作者：
White, Paul A.
中科院分区：
文献类型：
--
作者：
Chepelev, Nikolai L.;Kennedy, Dean A.;White, Paul A.
氧化应激与许多生理和病理过程以及异生物质代谢有关，导致生物大分子（包括DNA）的氧化。因此，DNA氧化的有效检测对于包括医学和毒理学在内的各种研究学科都很重要。氧化损伤DNA的常见生物标志物是8-氧代-7，8-二氢-2'-脱氧鸟苷（8-氧代-dGuo;通常被错误地称为8-羟基-2'-脱氧鸟苷（8-OH-dGuo或8-氧代-dG））。本文介绍了几种用高压液相色谱电化学检测器（HPLC-ED）测定8-氧代-dGuo的方法。然而，这些主要应用于用促氧化剂处理的纯化的DNA。此外，由于实验室之间的方法差异，主要是由于分析设备的差异，采用已发表的HPLC-ED检测8-oxo-dGuo的方法需要每个实验室进行仔细优化。一个全面的协议，描述这样的优化过程，是缺乏的。在这里，描述了通过HPLC-ED检测来自培养细胞或动物组织的DNA中的8-氧代-dGuo的详细方案。它说明了如何DNA样品制备可以很容易地和快速优化，以尽量减少不必要的DNA氧化，可以在样品制备过程中发生。该方案显示了如何检测培养的人肺泡腺癌细胞中的8-氧代-dGuo（即，A549细胞），以及暴露于多环芳烃二苯并（def，p）芘（DBC，以前称为二苯并（a，l）芘，DalP）的小鼠脾脏。总体而言，这项工作说明了HPLC-ED方法如何容易地优化用于检测生物样品中的8-oxo-dGuo。
Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.