Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Silent Spring Institute, Newton, MA 02460, USA.
Int J Mol Sci. 2022 Oct 4;23(19):11776. doi: 10.3390/ijms231911776.
The comet assay is a versatile assay for detecting DNA damage in eukaryotic cells. The assay can measure the levels of various types of damage, including DNA strand breaks, abasic sites and alkali-sensitive sites. Furthermore, the assay can also be modified to include purified DNA glycosylases so that alkylated and oxidized bases can be detected. The CometChip is a higher throughput version of the traditional comet assay and has been used to study cultured cells. Here, we have tested its utility for studies of DNA damage present in vivo. We show that the CometChip is effective in detecting DNA damage in multiple tissues of mice exposed to the direct-acting methylating agent methylmethane sulfonate (MMS) and to the metabolically activated methylating agent -nitrosodimethylamine (NDMA), which has been found to contaminate food, water, and drugs. Specifically, results from MMS-exposed mice demonstrate that DNA damage can be detected in cells from liver, lung, kidney, pancreas, brain and spleen. Results with NDMA show that DNA damage is detectable in metabolically competent tissues (liver, lung, and kidney), and that DNA repair in vivo can be monitored over time. Additionally, it was found that DNA damage persists for many days after exposure. Furthermore, glycosylases were successfully incorporated into the assay to reveal the presence of damaged bases. Overall, this work demonstrates the efficacy of the in vivo CometChip and reveals new insights into the formation and repair of DNA damage caused by MMS and NDMA.
彗星实验是一种用于检测真核细胞 DNA 损伤的多功能检测方法。该检测方法可以测量各种类型的损伤水平，包括 DNA 链断裂、脱碱基位点和碱敏性位点。此外，该检测方法还可以进行修改，以包括纯化的 DNA 糖苷酶，从而可以检测烷基化和氧化碱基。彗星芯片是传统彗星检测的高通量版本，已被用于研究培养细胞。在这里，我们测试了它在体内 DNA 损伤研究中的实用性。我们表明，彗星芯片可有效检测暴露于直接作用的甲基化试剂甲磺酸甲酯（MMS）和代谢激活的甲基化试剂亚硝二甲胺（NDMA）的小鼠的多种组织中的 DNA 损伤，已发现 NDMA 会污染食物、水和药物。具体而言，MMS 暴露小鼠的结果表明，可以在来自肝脏、肺、肾、胰腺、脑和脾的细胞中检测到 DNA 损伤。NDMA 的结果表明，可以在代谢活跃的组织（肝、肺和肾）中检测到 DNA 损伤，并且可以随时间监测体内 DNA 修复。此外，发现暴露后 DNA 损伤会持续数天。此外，成功地将糖苷酶纳入该检测方法，以揭示受损碱基的存在。总的来说，这项工作证明了体内彗星芯片的功效，并揭示了关于 MMS 和 NDMA 引起的 DNA 损伤的形成和修复的新见解。
