64. Study on the DNA damage induced by coal tar pitch fume extracts in rat alveolar macrophage and it”s mechanism
The carcinogenic mechanism of coal tar pitch (CTP) as a recognized carcinogen has been studying. It is widely believed that the carcinogenicity of CTP is based on the genotoxicity of CTP. In the process of carcinogenesis caused by extrinsic chemical substance, the DNA damage mainly occurred in the initiation phase. UP to now, the most sensitive detecting endpoint for DNA damage is to detect DNA single strand breaks. The single cell gel electrophoresis has been rapidly becoming a widely used analytical procedure during the last few years, which can detect DNA strand breaks. The method is a fast, relatively inexpensive, easy to perform, non-radioactive, and very sensitive method. This method suits to different tests in vitro or in vivo. Virtually any eukaryotic cell, which could be made into single cell suspensions, can be processed for analysis of DNA damage using the single cell gel electrophoresis. The aim of the study is to investigate the role of DNA damage induced by CTP fume in rat AM, to examine the changes of ROS, MDA and SOD, and to explore the mechanism of DNA damage by CTP fume. The present study is in favor of studying the mechanism of mutagenesis and carcinogenesis induced by CTP. Method: The healthy male Wistar rats were anesthetized intraperitoneally with 40 mg pentobarbital sodium per kilogram of body weight. The animals were exanguinated by excising femoral, and collected the rat alveolar macrophage by Joseph”s method. The concentration of AM had been regulated to 1.5×106 cell/ml. AMs, which had been cultured in 24-well culture plate, were divided into 4 groups. These cells were exposured to 5.0 μg/ml extracts of coal tar pitch fume, and contacted with 500 μM, 1 000 μM, and 2 000 μM of GSH respectively. These cells were divided into 4 groups. After incubation 24 hours, the indexes that had been used above were measured. Results: ①The DNA strand breaks induced by coal tar pitch fume extracts: After undergoing electrophoresis, the ‘comet’ cells are seldom in control group, but the ‘comet’ cells increased significantly in experimental groups. When the concentration of CTP fume extracts increased from 0 to 10.0 μg/ml, the incidences of ‘comet’ cell increased from 6.0% to 92.0%. There were significant differences in all groups (P<0.001). There was a dose-response relationship between the concentrations of CTP fume extracts and the incidences of ‘comet’ cell. There were correlation relationship between the grade of DNA damage and the concentration of CTP fume extracts (P<0.001). ②The level of ROS induced by CTP fume extracts in AM: After incubation 24 h, the concentration of ROS increased. The difference between control group and experimental groups were significant (P<0.001). ③ The changes of MDA and SOD in AM induced by CTP fume extracts: As the concentration of coal tar pitch fume extracts increased, the concentration of MDA increased too. The differences between experimental groups and control group were significant. But the difference of SOD activity in all groups weren’t significant. ④The role of GSH interfere: GSH could protect the DNA of AM from DNA-damage induced by CTP fume extracts. With the increasing of the concentration of GSH, the incidences of ‘comet’ cell decreased from 77.0% to 16.0%. The differences of incidences of ‘comet’ cell in all groups were different significantly. There was a dose-response relationship between the concentrations of GSH and the incidences of ‘comet’ cell. There was correlation relationship between the grade of DNA damage and the concentration of GSH (P<0.001). GSH could inhibit the increasing of ROS and MDA. The differences of SOD activity in all groups weren”t significant (P>0.05). Conclusion: ①Coal tar pitch fume extracts can induce DNA single strand breaks in rat alveolar macrophage; the single cell gel electrophoresis appears to be suitable to detect DNA strand breaks in rat alveolar macrophage. ②Coal tar pitch fume extracts can induce the rat alveolar macrophage to produce reactive oxygen species (ROS), and can induce lipid peroxidation: the DNA strand breaks induced by coal tar pitch fume extracts in AM are associated with the producing of ROS. ③GSH can inhibit coal tar pitch fume extracts-induced DNA damage in rat alveola r macrophage.
single cell gel electrophoresis、DNA damage、alveolar macrophage、coal tar pitch
13
R73(肿瘤学)
2004-03-26（万方平台首次上网日期，不代表论文的发表时间）
共2页
248-249
