Establishment And Application Of The Comet Assay Of Primary Mouse Hepatocytes

The comet assay, also called single cell gel electrophoresis assay( SCGE), is a method of detecting DNA damage and repair of single cell. Because of it's convenience, quickness, sensitivity, little sample, repetition, and no radioactive marker, is applied all sorts of vivo and vitro tests, it has been most widely used to detect DNA damage. With the development of this method, it has been induced into the field of environmental sanitation, it has now played an important role in detecting and assessing the genotoxicity of environmental factors, biomonitoring for the people who engage in some occupation, entironment monitoring, investigating of environmental epidemiology , et al.The cell line is mostly used in the comet assay; it can be obtained from cell culture. But the protocols of cell culture are comparatively complex, long-range and expensive. In hence, we established a simpler, quicker and more economical comet assay based on primary cells. In this research, we firstly contrasted primary mouse cells with traditional target cell line V79 to analyze the effects in comet assay respectively and established a new protocol of comet assay using the former cells. Then we detected the genotoxicity of the drinking water in Luzhou City with the mew protocol to evaluate the practicability and reliability of this protocol.Primary hepatocytes, spleen cells and renal cells of mouse are selected as target cells in comet assay. K2Cr2O67 is selected as genotoxic substance to detect the damage of DNA. Our data show that the three kinds of primary cells are all sensitive in detection. Hepatocytes show most high sensitivity and even show auto-activated capability. So hepatocytes are selected again in comet assay in contrast with cell line V79. The data show that primary mouse hepatocytes are more sensitive than V79 cell lines.Luzhou City is located in upper reache of Yangtse Rive and lower reaches of Tuojiang River, which is heavily polluted by industry, agriculture sewage and wastewater. There has been no report on the genotoxicity of drinking water of this area till now. source water ,Out-factory water, and tip water from Luzhou City were tested with the comet assay of primary mouse hepatocytes. In contrast, the same samples of water were tested with Ames test. The data show that Ames test can only detect the mutagenicity of the chlorinated disinfection drinking water, while the comet assay of primary mouse hepatocytes can detect the mutagenicity of source water and chlorinated disinfection drinking water. The detection liminal value of the comet assay is much lower than that of Ames test. The conclusion is that the comet assay of primary mouse hepatocytes is more sensitive, more convenient, quicker and more economical.Draw a conclusion in the protocols of the comet assay of primary mouse hepatocytes we established, material is easy to obtain and cells is easily prepared ,no need to cell or bacterial culture, no need to activate cell in vitro. This kind of comet assay also has the merit of sensitivity, quickness and economy. There will be extensiveapplication of primary mouse hepatocytes comet assay in detection of genotoxicity and entironment monitoring.