Validation of a Simple In Vitro Comet Assay Method Using CHL Cells

抄録

The comet assay has been widely used as a genotoxicity test in vitro/vivo for detecting initial DNA damage in individual cells. One of the difficulties of the assay is slide preparation, for which agarose top and bottom layers and a cell-containing middle layer are needed to immobilize the cells. To establish a practical methodology while maintaining sensitivity and reproducibility, we assessed a simple comet assay method with a hydrophilic slide glass (MAS-coat type, Matsunami glass Ind., Ltd.) instead of an agarose bottom layer. Ethyl methanesulfonate (EMS), mitomycin C (MMC), and N-nitroso dimethylamine (DMN) as genotoxic chemicals and triton X-100 (TRX) as a non-genotoxic chemical were used for validation of this method. Chinese hamster lung (CHL) cells were used. The results showed that EMS and DMN induced a significant increase in tail intensity. However, MMC, a known interstrand cross-linker, did not increase tail intensity, and it was considered that this was because MMC-induced DNA-DNA crosslinks prevent separation of the DNA duplex. TRX did not increase tail intensity. These results are consistent with previous reports and demonstrate that the simple comet assay can clearly detect genotoxicity of chemicals other than interstrand cross-linkers.