China-Japanese Joint Institute for Medical and Pharmaceutical Science, Dalian Medical University, West Segment of South Lvshun Road, Dalian, Liaoning, China.
Toxicol Ind Health. 2011 Feb;27(1):87-95. doi: 10.1177/0748233710387001. Epub 2010 Oct 14.
Ambient particulate matter (PM) has been reported to be associated with increased respiratory, cardiovascular, and malignant lung diseases. The aim of the present study was to investigate the variability of the DNA-damage induced by thoracic particles (PM( 10)) sampled in different locations and seasons (2006) in Dalian, China, in human hepatoma G2 (HepG2) cells. Significant differences in percentage of tail DNA induced by the extractable organic matter of PM(10) were revealed between summer and winter seasons and among monitoring sites in single cell gel electrophoresis (SCGE) assay. The percentage of tail DNA in HepG2 cells significantly increased in a dose-dependent manner after exposure to 7.5 and 30 μg/mL extractable organic matter of PM(10) for 1 hour. In order to clarify the underlying mechanisms, we evaluated the level of reactive oxygen species (ROS) production with the 2, 7-dichloro-fluorescein diacetate (DCFH-DA) assay. Significantly increased level of ROS was observed in HepG2 cells at higher concentrations (15 and 30 μg/mL). Significantly increased levels of 8-hydroxydeoxyguanosine (8-OHdG) were also shown in HepG2 cells. In this study, the accumulation of nuclear factor kappa B (NF-κB) p65 protein induced by the extractable organic matter of PM(10) was detected by western blotting in HepG2 cells, and the protein expression of NF-κB p65 significantly increased after the treatment with 30 μg/mL extractable organic matter of PM(10) for 24 hours. These results indicate that the extractable organic matter of PM(10) causes DNA strand breaks in HepG2 cells, and significant differences in percentage of tail DNA in dependence on locality and season are revealed. The extractable organic matter of PM(10) exerts DNA damage effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS, increase of 8-OHdG formation, and protein expression of NF-κB p65.
据报道，环境颗粒物（PM）与呼吸系统疾病、心血管疾病及肺部恶性疾病的增加有关。本研究的目的是调查2006年在中国大连不同地点和季节采集的空气中颗粒物（PM₁₀）诱导人肝癌G2（HepG2）细胞DNA损伤的变异性。在单细胞凝胶电泳（SCGE）试验中，夏季和冬季以及各监测点之间，PM₁₀可提取有机物诱导的尾DNA百分比存在显著差异。暴露于7.5和30 μg/mL的PM₁₀可提取有机物1小时后，HepG2细胞中的尾DNA百分比呈剂量依赖性显著增加。为了阐明潜在机制，我们用2,7-二氯荧光素二乙酸酯（DCFH-DA）试验评估了活性氧（ROS）的产生水平。在较高浓度（15和30 μg/mL）下，HepG2细胞中观察到ROS水平显著升高。HepG2细胞中8-羟基脱氧鸟苷（8-OHdG）水平也显著升高。在本研究中，通过蛋白质印迹法检测了PM₁₀可提取有机物诱导的HepG2细胞中核因子κB（NF-κB）p65蛋白的积累，用30 μg/mL的PM₁₀可提取有机物处理24小时后，NF-κB p65的蛋白表达显著增加。这些结果表明，PM₁₀的可提取有机物会导致HepG2细胞中的DNA链断裂，且尾DNA百分比在不同地点和季节存在显著差异。PM₁₀的可提取有机物可能通过细胞内ROS诱导的氧化性DNA损伤、8-OHdG形成增加以及NF-κB p65的蛋白表达，对HepG2细胞产生DNA损伤作用。
