苯对人外周血单个核细胞S+G2/M期阻滞、细胞凋亡和DNA氧化损伤的影响

Effects of benzene on S+G2/M cell cycle arrest,apoptosis and oxidative DNA damage in human peripheral blood mononuclear cells

目的:研究苯致人外周血单个核细胞(PBMCs)周期阻滞与凋亡,探讨苯对PBMC DNA氧化损伤效应的影响。方法:离体培养24 h后加S9液,设置苯低、中、高浓度组(0.5,5,50μmol/L)和乙醇溶剂对照组,染毒处理24 h后,采用四唑盐比色法检测PBMCs相对存活率;流式细胞术检测细胞周期构成比及凋亡率;DCFH-DA荧光探针检测活性氧(ROS)含量、黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活力;硫代巴比妥酸TBA比色法测定丙二醛(MDA)含量;酶联免疫吸附法(ELISA)测定细胞中谷胱甘肽(GSH)含量;SCGE检测DNA断裂;微核实验检测染色体畸变。结果:与对照组相比,0.5μmol/L苯染毒组细胞存活率明显下降(P<0.05),G0/G1期细胞百分比明显减少(P<0.05),S期和G2/M期百分比明显增加(P<0.05),且细胞凋亡率明显上升(P<0.05),细胞内ROS、MDA、微核率、彗星率和彗星尾长呈剂量依赖性增加,差异均具有统计学意义(P<0.05),而SOD和GSH呈剂量依赖性减少(P<0.05)。结论:苯可以导致PBMCs细胞凋亡和S+G2/M期阻滞,增加细胞脂质过氧化作用,改变细胞内氧化还原状态,诱导DNA氧化损伤。

AIM：To observe and verify the benzene-induced cell cycle arrest and apoptosis in human peripheral blood mononuclear cells（PBMCs）,and explore its effects on oxidative DNA damage.METHODS：PBMCs were isolated,cultivated for 24 h and then divided into 4 groups supplemented with alcohol solvent as a control,low,middle and high concentration benzene（0.5,5,50 μmol/L）,respectively.Another 24 h later,we assayed the cell growth arrest by MTT,detected cell cycle and apoptosis rate by flow cytometry,the content of reactive oxygen species（ROS） by DCFH-DA assay,superoxide dismutase（SOD） activity by xanthine oxidase test,malondialdelhde（MDA） content by thiobarbituric acid test,glutathione（GSH） content by ELISA,DNA damage by single cell gel electrophoresis（SCGE） technology and micronucleus assay.RESULTS： Compared with the control group,benzene decreased cell viability（P〈0.05） and increased cell apoptosis（P〈0.05） in a dose-dependent manner,along with induction of S phase and G2/M phase arrest（P〈0.05）.Meanwhile,benzene induced the accumulation of intracellular ROS and MDA,micronucleus rates,comet rates and comet tail length,which were found dose-dependent and statistically significant compared to the solvent control（P〈0.05）.The activity of SOD and the contents of GSH decreased significantly（P〈0.05）.CONCLUSION： Benzene can induce cell apoptosis,S＋G2/M phase accumulation and change of oxidoreduction status in PBMCs,and strengthen the effects of lipid peroxidation as well as the DNA damage.