Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries, Inc, 10-1, Tebiro 6-chome, Kamakura, Kanagawa 248-8555, Japan.
Toxicol In Vitro. 2010 Apr;24(3):1039-44. doi: 10.1016/j.tiv.2009.11.014. Epub 2009 Nov 16.
The comet assay, also known as the single cell gel electrophoresis (SCGE) assay, is a rapid, simple, visual and sensitive technique for measuring DNA damage in mammalian cells. In the present study, Methyl methanesulfonate (MMS), 4-Nitrosoquinoline-Oxide (4NQO), Cyclophosphamide (CPA), and Benzo(a)pyrene (BP)-induced DNA damage was assayed in vitro in a murine bone-marrow cell line (FDC-P2), with or without an activation mixture (rat liver S9). All compounds caused significant DNA damage. With MMS and 4NQO, the frequency of comet tails, scored manually under a fluorescence microscope, increased dose-dependently, and reached a maximum of 53.2 and 74.8% respectively. Three parameters indicating DNA damage in the comet assay with the two-layer method, tail length, %DNA in tail, and tail moment, calculated using the automated image analysis software "Comet Analyzer v1.5" increased with all compounds. With MMS and 4NQO, all parameters increased at concentrations over 40 and 0.25 micromol/L, respectively. The in vitro comet assay with rat liver S9 could detect DNA damage caused by the metabolites of CPA and BP. The comet assay using the two-layer method is easy and efficient, and so can be conducted on a routine as basis. The assay with FDC-P2 cells was highly sensitive in detecting DNA damage with the frequency of comet tails, tail moment, %DNA in tail and tail length as indicators of the damage. Metabolism-mediated DNA damage could be detected with the addition of a rat S9 mixture at a final concentration of 6% for 6h exposure.
彗星试验，又称单细胞凝胶电泳（SCGE）试验，是一种快速、简便、直观和灵敏的技术，用于测量哺乳动物细胞中的 DNA 损伤。在本研究中，使用鼠骨髓细胞系（FDC-P2），在体外检测了甲磺酸甲酯（MMS）、4-亚硝基喹啉-1-氧化物（4NQO）、环磷酰胺（CPA）和苯并（a）芘（BP）诱导的 DNA 损伤，有无激活混合物（大鼠肝 S9）。所有化合物均引起明显的 DNA 损伤。对于 MMS 和 4NQO，手动在荧光显微镜下评分的彗星尾的频率呈剂量依赖性增加，分别达到 53.2%和 74.8%的最大值。使用双层法的彗星试验的三个参数（彗星尾长度、尾部 DNA 百分比和尾部矩），使用自动图像分析软件“Comet Analyzer v1.5”计算，随着所有化合物的浓度增加而增加。对于 MMS 和 4NQO，所有参数均在浓度超过 40 和 0.25 μmol/L 时增加。使用大鼠肝 S9 的体外彗星试验可以检测 CPA 和 BP 代谢物引起的 DNA 损伤。使用双层法的彗星试验简单高效，可以常规进行。使用 FDC-P2 细胞的试验，以彗星尾的频率、尾部矩、尾部 DNA 百分比和尾部长度作为损伤的指标，对 DNA 损伤具有高度的敏感性。通过添加终浓度为 6%的大鼠 S9 混合物，在 6 小时暴露时，可以检测到代谢介导的 DNA 损伤。
