北京市大气颗粒物不同有机组分毒性研究
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摘要
PM10和PM2.5对人体健康具有危害作用。大量体外毒性研究证实了颗粒物上有机物的毒性,但其作用机制至今不太清楚。研究以北京市三个采样点冬夏两季的PM10和PM2.5中多环芳烃和非烃组分为研究对象,用Ames试验检测其致突变性;以小鼠肺泡巨噬细胞、人肺泡上皮细胞为靶细胞,用MTT法测定细胞毒性;流式细胞仪检测其对细胞周期和细胞凋亡的影响;用单细胞凝胶电泳检测细胞的DNA单链断裂、双链断裂和DNA―蛋白质交联及其自发修复;对北京9个采样点冬季PM10和PM2.5及其有机物的污染变化特征进行了研究,得出主要结论如下:
(1)采样期内北京市PM10和PM2.5及其有机物污染相当严重。夜间颗粒物浓度总体上高于日间浓度。各采样点有机质族组分分布主要呈现沥青质>非烃>饱和烃>芳烃的趋势。
(2)所有的PAHs和非烃组分都具有较强的致突变性且以移码型突变为主;两个采样点的PAHs组分主要是间接致突变物。PAHs和非烃组分都可导致细胞存活率的降低,可能来自于细胞受到的氧化损伤;导致细胞阻滞于G0/G1期、G2/M期;引发细胞凋亡;导致RAW267.4细胞DNA单链断裂、双链断裂和DNA―蛋白质交联。所有毒性实验证明,与多环芳烃相比,非烃是主要的毒性物质。
(3)受污染源、降解等因素的影响致使具有不同毒性的化学组成及含量的差异,导致两种有机组分的毒性表现为冬季多环芳烃和非烃大于夏季多环芳烃和非烃;PM2.5中多环芳烃和非烃大于PM10中多环芳烃和非烃;工业区多环芳烃和非烃>商业区多环芳烃和非烃>居民区多环芳烃和非烃。
(4)PAHs和非烃组分诱导的细胞DNA单链断裂自发修复能力较强,而DNA双链断裂和DNA―蛋白质交联修复率极低。
(5)DNA单链、双链断裂和DNA―蛋白质交联是本次研究多环芳烃和非烃组分的致突变分子机制之一。DNA的损伤导致细胞周期阻滞于G0/G1期、G2/M期进行修复,无法修复的损伤细胞发生凋亡或突变,导致细胞存活率降低。而氧化性损伤可能是DNA损伤的机制之一。
PM10 and PM2.5 have adverse effects on human health. Although the results of toxicological research in vitro have shown that PM induces several types of adverse effects, the biological mechanisms behind these associations are not fully understood. The goal of this study is to investigate the mutagenicity, DNA damage and repair, cytotoxicity, apoptosis and the effect on cell cycle of PAHs and Non-hydrocarbons (NHs) extracted from the atmospheric particulates (PM10 and PM2.5) collected from different sites, seasons(summer and winter)respectively and to detect the effect of these factors on the toxicological activity of PAHs and NHs. And then determine which one is mainly responsible for the toxicity of PM. The assays used are Salmonella mutagenicity assay, using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation (S9), Comet assay, MTT assay (RAW264.7 and A549 cell lines) and flow cytometry. The characteristics of PM10 and PM2.5 from 9 sampling sites in winter is investigated either in this study. The dissertation made the following results:
1) The pollution level in Beijing is very serious. The mass concentrations of PM during daytime are higher than nighttime. In all sampling sites, the percentage of organic subfractions from big to small is asphaltene, non-hydrocarbons, aliphatic hydrocarbons and polycyclic hydrocarbons. 2) All samples are mutagenitic. The mechanism of mutagenicity of PAHs was a frame-shift mutation and PAHs from Coking plant mostly were indirect-acting mutagens. All samples have potency to make cell viability decrease (perhaps induced by oxidative stress), to retard cells at G0/G1phase and G2/M phase, to induce apoptosis and DNA single strand breakages, double strand breakages and DNA-protein crosslinks. And compared with PAHs, NHs are responsible for the toxicity from PM. 3) The sampling sites, seasons and particle size are important factors to the toxicological potency of PAHs and NHs. In general, the smaller PM size have the higher toxicity; and the toxicity of the samples in winter is higher than that in summer; and the toxicity of the samples from coking plant is the highest in 3 sampling sites. 4) DNA single strand breakages are more prone to be repaired than double strands breaks and DNA-protein crosslinks. 5) DNA damages are one of the main mechanisms of the mutagenicity induced by PAHs and NHs from 3 different sampling sites. DNA damages result in cell cycles retarding at G0/G1 phase and G2/M phase to repair. The cells in which DNA cannot be repaired, will be induced apoptosis or mutation, as a result, the cell viability decreases. Oxidative damage might be one of the main mechanisms of DNA damages.
(1)采样期内北京市PM10和PM2.5及其有机物污染相当严重。夜间颗粒物浓度总体上高于日间浓度。各采样点有机质族组分分布主要呈现沥青质>非烃>饱和烃>芳烃的趋势。
(2)所有的PAHs和非烃组分都具有较强的致突变性且以移码型突变为主;两个采样点的PAHs组分主要是间接致突变物。PAHs和非烃组分都可导致细胞存活率的降低,可能来自于细胞受到的氧化损伤;导致细胞阻滞于G0/G1期、G2/M期;引发细胞凋亡;导致RAW267.4细胞DNA单链断裂、双链断裂和DNA―蛋白质交联。所有毒性实验证明,与多环芳烃相比,非烃是主要的毒性物质。
(3)受污染源、降解等因素的影响致使具有不同毒性的化学组成及含量的差异,导致两种有机组分的毒性表现为冬季多环芳烃和非烃大于夏季多环芳烃和非烃;PM2.5中多环芳烃和非烃大于PM10中多环芳烃和非烃;工业区多环芳烃和非烃>商业区多环芳烃和非烃>居民区多环芳烃和非烃。
(4)PAHs和非烃组分诱导的细胞DNA单链断裂自发修复能力较强,而DNA双链断裂和DNA―蛋白质交联修复率极低。
(5)DNA单链、双链断裂和DNA―蛋白质交联是本次研究多环芳烃和非烃组分的致突变分子机制之一。DNA的损伤导致细胞周期阻滞于G0/G1期、G2/M期进行修复,无法修复的损伤细胞发生凋亡或突变,导致细胞存活率降低。而氧化性损伤可能是DNA损伤的机制之一。
PM10 and PM2.5 have adverse effects on human health. Although the results of toxicological research in vitro have shown that PM induces several types of adverse effects, the biological mechanisms behind these associations are not fully understood. The goal of this study is to investigate the mutagenicity, DNA damage and repair, cytotoxicity, apoptosis and the effect on cell cycle of PAHs and Non-hydrocarbons (NHs) extracted from the atmospheric particulates (PM10 and PM2.5) collected from different sites, seasons(summer and winter)respectively and to detect the effect of these factors on the toxicological activity of PAHs and NHs. And then determine which one is mainly responsible for the toxicity of PM. The assays used are Salmonella mutagenicity assay, using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation (S9), Comet assay, MTT assay (RAW264.7 and A549 cell lines) and flow cytometry. The characteristics of PM10 and PM2.5 from 9 sampling sites in winter is investigated either in this study. The dissertation made the following results:
1) The pollution level in Beijing is very serious. The mass concentrations of PM during daytime are higher than nighttime. In all sampling sites, the percentage of organic subfractions from big to small is asphaltene, non-hydrocarbons, aliphatic hydrocarbons and polycyclic hydrocarbons. 2) All samples are mutagenitic. The mechanism of mutagenicity of PAHs was a frame-shift mutation and PAHs from Coking plant mostly were indirect-acting mutagens. All samples have potency to make cell viability decrease (perhaps induced by oxidative stress), to retard cells at G0/G1phase and G2/M phase, to induce apoptosis and DNA single strand breakages, double strand breakages and DNA-protein crosslinks. And compared with PAHs, NHs are responsible for the toxicity from PM. 3) The sampling sites, seasons and particle size are important factors to the toxicological potency of PAHs and NHs. In general, the smaller PM size have the higher toxicity; and the toxicity of the samples in winter is higher than that in summer; and the toxicity of the samples from coking plant is the highest in 3 sampling sites. 4) DNA single strand breakages are more prone to be repaired than double strands breaks and DNA-protein crosslinks. 5) DNA damages are one of the main mechanisms of the mutagenicity induced by PAHs and NHs from 3 different sampling sites. DNA damages result in cell cycles retarding at G0/G1 phase and G2/M phase to repair. The cells in which DNA cannot be repaired, will be induced apoptosis or mutation, as a result, the cell viability decreases. Oxidative damage might be one of the main mechanisms of DNA damages.
引文
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