Møller Peter, Jantzen Kim, Løhr Mille, Andersen Maria Helena, Jensen Ditte Marie, Roursgaard Martin, Danielsen Pernille Høgh, Jensen Annie, Loft Steffen
Department of Public Health, Section of Environmental Health, University of Copenhagen, Øster Farimagsgade, Copenhagen K, Denmark.
Mutagenesis. 2018 Feb 24;33(1):9-19. doi: 10.1093/mutage/gex015.
The formamidopyrimidine DNA glycosylase (Fpg) and human 8-oxoguanine DNA glycosylase (hOGG1)-modified comet assays have been widely used in human biomonitoring studies. The purpose of this article is to assess differences in reported levels of Fpg- and hOGG1-sensitive sites in leukocytes and suggest suitable assay controls for the measurement of oxidatively damaged DNA. An assessment of the literature showed a large variation in the reported levels of Fpg-sensitive sites (range 0.05-1.31 lesions/106 bp). The levels of Fpg-sensitive sites are lower in studies where Fpg has been obtained from commercial suppliers or unknown sources as compared to Fpg from one particular non-commercial source (χ2 = 7.14, P = 0.028). The levels of hOGG1-sensitive sites are lower (range: 0.04-0.18 lesions/106 bp in leukocytes) compared to the Fpg-sensitive sites. Surprisingly, few publications have reported the use of oxidising agents as assay controls, with the exception of hydrogen peroxide. This may be due to a lack of consensus about suitable controls for the Fpg- and hOGG1-modified comet assay. A major challenge is to find an oxidising agent that only oxidises nucleobases and does not generate DNA strand breaks because this reduces the dynamic range of Fpg- and hOGG1-sensitive sites in the comet assay. Based on a literature search we selected the photosensitiser Ro19-8022 plus light, KBrO3, 4-nitroquinoline-1-oxide, Na2Cr2O7 and ferric nitrilotriacetate as possible assay controls. A subsequent assessment of these compounds for generating cryopreserved assay controls in mononuclear blood cells showed that Ro19-8022 plus light, KBrO3 and 4-nitroquinoline-1-oxide provided suitable assay controls. We recommend these compounds as comet assay controls for oxidatively damaged DNA.
甲酰胺嘧啶DNA糖基化酶（Fpg）和人8-氧代鸟嘌呤DNA糖基化酶（hOGG1）修饰的彗星试验已广泛应用于人体生物监测研究。本文旨在评估白细胞中Fpg和hOGG1敏感位点报告水平的差异，并为氧化损伤DNA的测量提出合适的试验对照。对文献的评估表明，报告的Fpg敏感位点水平存在很大差异（范围为0.05 - 1.31个损伤/106碱基对）。与来自一个特定非商业来源的Fpg相比，从商业供应商或未知来源获得Fpg的研究中Fpg敏感位点水平较低（χ2 = 7.14，P = 0.028）。与Fpg敏感位点相比，hOGG1敏感位点水平较低（白细胞中范围为0.04 - 0.18个损伤/106碱基对）。令人惊讶的是，除过氧化氢外，很少有出版物报道使用氧化剂作为试验对照。这可能是由于对于Fpg和hOGG1修饰的彗星试验的合适对照缺乏共识。一个主要挑战是找到一种仅氧化核碱基而不产生DNA链断裂的氧化剂，因为这会降低彗星试验中Fpg和hOGG1敏感位点的动态范围。基于文献检索，我们选择了光敏剂Ro19 - 8022加光、KBrO3、4-硝基喹啉-1-氧化物、Na2Cr2O7和次氮基三乙酸铁作为可能的试验对照。随后对这些化合物在冷冻保存的单核血细胞中生成试验对照的评估表明，Ro19 - 8022加光、KBrO3和4-硝基喹啉-1-氧化物提供了合适的试验对照。我们推荐这些化合物作为氧化损伤DNA的彗星试验对照。
