Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Free Radic Biol Med. 2011 Jun 1;50(11):1536-45. doi: 10.1016/j.freeradbiomed.2011.02.034. Epub 2011 Mar 5.
Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H₂O₂ oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.
氧化应激导致 DNA 大分子损伤会产生多种病变，这些病变与突变、致癌、生殖细胞死亡和衰老有关。许多这些病变已经通过各种技术进行了研究和描述。在现有的技术中，彗星试验、高效液相色谱-电化学检测（HPLC-EC）、气相色谱-质谱联用（GC-MS）、高效液相色谱-质谱联用（HPLC-MS）和特别是高效液相色谱-串联质谱联用（HPLC-MS/MS）仍然是最广泛使用的技术，为这些病变提供了非常有价值的信息。然而，DNA 损伤的准确测量一直存在争议。特别是，有报道称人工氧化会导致错误地高估损伤程度。此外，这些技术中的大多数都测量一系列事件的最终产物，因此只能提供有关初始自由基机制的有限信息。我们在这里报告了使用免疫自旋捕获（IST）与电子顺磁共振（EPR）、质谱（MS）和串联质谱（MS/MS）对 Cu(II)-H₂O₂氧化系统诱导的 DNA 损伤进行定性测量的结果。自由基一形成就立即被 DMPO 捕获。形成的 DMPO 加合物最初具有 EPR 活性，但随后被氧化为稳定的硝酮，然后可以通过 IST 检测到，并通过 MS 和 MS/MS 进一步表征。
