城市不同源雾霾颗粒物健康风险差异评估比较
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摘要
通过研究不同来源霾颗粒物对大鼠气管上皮细胞(RTEcells)电阻抗变化和细胞自噬因子的影响,评价不同来源霾颗粒物对人体健康风险的差异性.分别将RTE暴露于从居民区(I),高架交通源(II)和化工园区(III)采集的3种雾霾颗粒物中,统一暴露浓度和时间分别为100mg/L和24h.通过电子细胞基质阻抗检测(ECIS)细胞增长引起的阻抗变化和细胞电损伤恢复时间;通过蛋白免疫印迹测定p62, Atg5, Atg7, Beclin1, LC3B和m TOR蛋白表达量来分析比较不同来源雾霾颗粒物对RTE细胞自噬的影响.结果表明,与空白对照组相比,不同雾霾颗粒物处理组细胞电损伤恢复时间分别延长了34.6%,63.2%和78.0%; p62蛋白表达量差异显著性下降, Atg5, Atg7, Beclin1, LC3B蛋白表达量差异显著性上升.此外, mTOR相关蛋白表达量差异显著性下降,分别下降了4.38%, 3.34%和2.36%; p-m TOR蛋白表达量与空白组相比,实验组I下降24.2%,实验组II下降37.0%,实验组III下降60.9%.由以上结果可知,不同来源雾霾颗粒物对RTE细胞均有一定的毒性损伤作用,能够减小细胞增长速度和削弱细胞修复能力,增强细胞自噬因子蛋白的表达,且化工园区采集的雾霾颗粒物毒性强于居民区和高架交通源.不同来源雾霾颗粒物的细胞毒性存在明显差异,基于细胞电损伤恢复时间的测定以及自噬相关蛋白的检测方法能够为雾霾颗粒物健康风险评价提供一种快速的生物学手段.
To evaluate the health risks induced by haze particles sampled from three different areas. The present study assessed the effects of haze particles on electrical impedance and autophagy factors of the tracheal epithelial cells. Rat tracheal epithelial(RTE) cells were exposed to haze particles collected from three different areas, including residential area(I), traffic area(II), and chemical industry park(III) in the megacity Hangzhou, China. The particle concentration was treated and designed as 100mg/L and then a period of 24 h exposure was given to cells in treatment groups. The electronic impedance and the time needed for the recovery of electricity damage were determined by ECIS(Electric Cell-substrate Impedance Sensing). Western blot was used to analyze the protein expressions of p62, Atg5, Atg7, Beclin1, LC3 B and mTOR to compare the effects of different haze particles on RTE cells autophagy. The results showed that the recovery time of cell injury was prolonged by 34.6%, 63.2% and 78.0%, respectively, when compared with the control group. The expression of Atg5, Atg7, Beclin1 and LC3B proteins increased, whereas that of p62 protein significantly decreased. The expression of mTOR related proteins decreased significantly by 4.38%, 3.34% and 2.36%, respectively. Compared with the control group, the expression of p-mTOR decreased by 24.2% in group I, 37.0% in group II and 60.9% in group III. All the results suggested that haze particles collected from different functional areas could induce various toxic damages on RTE cells such as reduction of growth speed, impairment of revovery ability, and increased authophagy protein expression. The haze particles from chemical industrial areas showed more toxic effects than those from residential areas and traffic areas. The cytotoxicity of haze particles from different sources is obviously different The determination of cell electrical injury recovery time and the detection of autophagy-related proteins can provide a rapid biological method for health risk assessment of haze particles.
To evaluate the health risks induced by haze particles sampled from three different areas. The present study assessed the effects of haze particles on electrical impedance and autophagy factors of the tracheal epithelial cells. Rat tracheal epithelial(RTE) cells were exposed to haze particles collected from three different areas, including residential area(I), traffic area(II), and chemical industry park(III) in the megacity Hangzhou, China. The particle concentration was treated and designed as 100mg/L and then a period of 24 h exposure was given to cells in treatment groups. The electronic impedance and the time needed for the recovery of electricity damage were determined by ECIS(Electric Cell-substrate Impedance Sensing). Western blot was used to analyze the protein expressions of p62, Atg5, Atg7, Beclin1, LC3 B and mTOR to compare the effects of different haze particles on RTE cells autophagy. The results showed that the recovery time of cell injury was prolonged by 34.6%, 63.2% and 78.0%, respectively, when compared with the control group. The expression of Atg5, Atg7, Beclin1 and LC3B proteins increased, whereas that of p62 protein significantly decreased. The expression of mTOR related proteins decreased significantly by 4.38%, 3.34% and 2.36%, respectively. Compared with the control group, the expression of p-mTOR decreased by 24.2% in group I, 37.0% in group II and 60.9% in group III. All the results suggested that haze particles collected from different functional areas could induce various toxic damages on RTE cells such as reduction of growth speed, impairment of revovery ability, and increased authophagy protein expression. The haze particles from chemical industrial areas showed more toxic effects than those from residential areas and traffic areas. The cytotoxicity of haze particles from different sources is obviously different The determination of cell electrical injury recovery time and the detection of autophagy-related proteins can provide a rapid biological method for health risk assessment of haze particles.
引文
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[20]霍婷婷,董发勤,邓建军,等.几种高硅质矿物细颗粒的A549细胞毒性对比[J].环境科学,2016,37(11):4410-4418.Huo Tingting,Dong Faqin,Deng Jianjun,et al.Comparation of toxic effect of silicious mineral dusts on lung epithelial A549 Cells[J].Environmental Science,2016,37(11):4410-4418.
[21]苏凯麒.基于ECIS细胞传感器和图像检测的海洋毒素分析系统设计[D].浙江大学,2014.Su Kaiqi.Design of marine toxin analysis system based on ECIS and image detection[D].Hangzhou:Zhejiang University,2014.
[22]Marco Diociaiuti,Maria Balduzzi,Barbara De Berardis,et al.The two PM2.5(fine)and PM2.5-10(coarse)fractions:evidence of different biological activity[J].Environmental Research,2001,86(3):254-262.
[23]Hetland R B,Cassee F R,Refsnes M,et al.Release of inflammatory cytokines,cell toxicity and apoptosis in epithelial lung cells after exposure to ambient air particles of different size fractions[J].Toxicology.in Vitro,2004,18(2):203-212.
[24]吕子峰,郝吉明,段菁春,等.北京市夏季二次有机气溶胶生成潜势的估算[J].环境科学,2009,30(4):969-975.Lv Zifeng,Hao Jiming,Duan Jingchun,et al.Estimate of the formation potential of secondary organic aerosol in Beijing summertime[J].Environmental Science,2009,30(4):969-975.
[25]Ghio A J,Silbajoris R,Carson J L,et al.Biological effects of oil fly ash[J].Environmental Health Perspectives,2002,110(6):88-94.
[26]Zhang Jin,Liu Jianhui,Ren Lihua,et al.PM2.5 induces male reproductive toxicity via mitochondrial dysfunction,DNA damage and RIPK1mediated apoptotic signaling pathway[J].Science of the Total Environment,2018,634(1):1435-1444.
[27]Yao Cui,Yu An,Jin Tongyu,et al.Real-time monitoring of skin wound healing on nano-grooves atopography using electric cellsubstrate impedance sensing(ECIS)[J].Sensors and Actuators BChemical,2017,250:461-468.
[28]Levine B,Kroemer G.Autophagy in the pathogenesis of disease[J].Cell,2008,132(1):27-42.
[29]Chen Zhihua,Lam Hilaire C,Jin Yang,et al.Autophagy protein microtubule-associated protein 1light chain-3B(LC3B)activates extrinsic apoptosis during cigarette smoke-induced emphysema[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(44):18880-18885.
[30]Kiyono K,Suzuki H I,Matsuyama H,et al.Autophagy is activated by TGF-beta and potentiates TGF-beta-mediated growth inhibition in human hepatocellular carcinoma cells[J].Cancer Research,2009,69(23):8844-8852.
[31]Kong H,Xia K,Pan L,et al.Autophagy and lysosomal dysfunction:Anew insight into mechanism of synergistic pulmonary toxicity of carbon black-metal ions co-exposure[J].Carbon,2017,111:322-333.
[32]Fujita N,Itoh T,Omori H,et al.The Atg16L complex specifies the site of LC3lipidation for membrane biogenesis in autophagy[J].Molecular biology cell,2008,19(5):2092-2100.
[33]Nezis I P,Stenmark H.p62 at the interface ofautophagy,oxidative stress signaling,and cancer[J].Antioxidants&Redox Signaling,2012:17(5):786-793.
[34]Su Ruijun,Jin Xiaoting,Zhang Weifeng,et al.Particulate matter exposure induces the autophagy of macrophages via oxidative stress-mediated PI3K/AKT/m TOR pathway[J].Chemosphere,2017,167:444-453.
[35]Wang Yu,Nie Hao,Zhao Xin,et al.Bicyclol induces cell cycle arrest and autophagy in HepG2 human hepatocellular carcinoma cells through the PI3K/AKTand Ras/Raf/MEK/ERK pathways[J].BMCCancer,2016,16(1):742.
[2]王朔,赵卫雄,徐学哲,等.北京一次严重雾霾过程气溶胶光学特性与气象条件[J].中国环境科学,2016,36(5):1305-1312.WANG Shuo,ZHAO Weixiong,XU Xuezhe,et al.Analysis of aerosol optical properties and meteorological parameters in a severe haze-fog episode in Beijing[J].China Environmental Science,2016,36(5):1305-1312.
[3]Huang Y C,Ghio A J.Vascular effects of ambient pollutant particles and metals[J].Current Vascular Pharmacology,2006,4(3):199-203.
[4]Künzli N,Tager I B.Air pollution:from lung to heart[J].Swiss Medical Weekly,2005,135(47/48):697-702.
[5]Sharma R K,Agrawal M.Biological effects of heavy metals:an overview[J].Journal of Environmental Biology,2005,26(2):301.
[6]Cohen A J,Ross A H,Ostro B,et al.The global burden of disease due to outdoor air pollution[J].Journal of Toxicology&Environmental Health,2005,68(14):1301-1307.
[7]Strasser A,Cory S,Adams J M.Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases[J].EMBOJournal,2011,30(18):3667-3683.
[8]Bravo L.Polyphenols:Chemistry,dietary sources,metabolism,and nutritional significance[J].Nutrition Reviews,1998,56(11):317-333.
[9]蒋燕,熊好琴,鲁绍伟,等.2015年北京采暖季城市森林内外SO2浓度的时空变化特征[J].环境科学研究,2017,30(11):1689-1696.Jiang Yan,Xiong Haoqin,Lu Shaowei,et al.Spatial-temporal variation of SO2 concentration in Beijing's Urban forest in heating season,2015[J].Research of Environmental Sciences,2017,30(11):1689-1696.
[10]Zou Yajuan,Wu Yizhao,Li Yinsheng,et al.Physicochemical properties,in vitro cytotoxic and genotoxic effects of PM1.0,and PM2.5,from Shanghai,China[J].Environmental Science&Pollution Research,2017,24(24):19508-19516.
[11]Alfaro-Moreno E,Martínez L,García-Cuellar C,et al.Biologic effects induced in Vitro by PM10 from three different zones of Mexico City[J].Environmental Health Perspectives,2002,110(7):715-720.
[12]Liu Ying,Chen Yanyan,Cao Jiyu,et al.Oxidative stress,apoptosis,and cell cycle arrest are induced in primary fetal alveolar type II epithelial cells exposed to fine particulate matter from cooking oil fumes[J].Environmental Science&Pollution Research,2015,22(13):9728-9741.
[13]胡睿,尹燕,陈奎,等.南京雾霾天气中碳质颗粒特征的研究[J].中国环境科学,2017,37(6):2007-2015.Hu Rui,Yin Yan,Chen kui,et al.Insights into characteristics of carbonaceous particles during haze and fog events in Nanjing[J].China Environmental Science,2017,37(6):2007-2015.
[14]Feng Yuchen,Yao Zhiyuan,Daniel J.Klionsky.How to control selfdigestion:transcriptional,post-transcriptional,and post-translational regulation of autophagy[J].Trends in Cell Biology,2015,25(6):354-363.
[15]Noda N N,Inagaki F.Mechanisms of autophagy[J].Annual Review of Biophysics,2015,44(1):101-122.
[16]Sabrina Di Bartolomeo,Marco,Corazzari,Francesca,Nazio,et al.The dynamic interaction of AMBRA1with the dynein motor complex regulates mammalian autophagy[J].Journal of Cell Biology,2010,191(1):155-168.
[17]Mizushima N,Yoshimori T,Levine B.Methods inmammalian autophagy research[J].Cell,2010,140(3):313-326.
[18]Kang R,Zeh H J,Lotze M T,et al.The Beclin 1 network regulates autophagy and apoptosis[J].Cell Death&Differentiation,2011,18(4):571-580.
[19]张建英,张杭君,陈英旭.微囊藻毒素导致鲫鱼淋巴细胞凋亡的研究[J].环境科学学报,2005,25(8):1101-1104.Zhang Jianying,Zhang Hangjun,Chen Yingxu.Induction of apoptosis in the lymphocytes of the crucian carp(Garassius auratus)by microcystins[J].Acta Scientiae Circumstantiae,2005,25(8):1101-1104.
[20]霍婷婷,董发勤,邓建军,等.几种高硅质矿物细颗粒的A549细胞毒性对比[J].环境科学,2016,37(11):4410-4418.Huo Tingting,Dong Faqin,Deng Jianjun,et al.Comparation of toxic effect of silicious mineral dusts on lung epithelial A549 Cells[J].Environmental Science,2016,37(11):4410-4418.
[21]苏凯麒.基于ECIS细胞传感器和图像检测的海洋毒素分析系统设计[D].浙江大学,2014.Su Kaiqi.Design of marine toxin analysis system based on ECIS and image detection[D].Hangzhou:Zhejiang University,2014.
[22]Marco Diociaiuti,Maria Balduzzi,Barbara De Berardis,et al.The two PM2.5(fine)and PM2.5-10(coarse)fractions:evidence of different biological activity[J].Environmental Research,2001,86(3):254-262.
[23]Hetland R B,Cassee F R,Refsnes M,et al.Release of inflammatory cytokines,cell toxicity and apoptosis in epithelial lung cells after exposure to ambient air particles of different size fractions[J].Toxicology.in Vitro,2004,18(2):203-212.
[24]吕子峰,郝吉明,段菁春,等.北京市夏季二次有机气溶胶生成潜势的估算[J].环境科学,2009,30(4):969-975.Lv Zifeng,Hao Jiming,Duan Jingchun,et al.Estimate of the formation potential of secondary organic aerosol in Beijing summertime[J].Environmental Science,2009,30(4):969-975.
[25]Ghio A J,Silbajoris R,Carson J L,et al.Biological effects of oil fly ash[J].Environmental Health Perspectives,2002,110(6):88-94.
[26]Zhang Jin,Liu Jianhui,Ren Lihua,et al.PM2.5 induces male reproductive toxicity via mitochondrial dysfunction,DNA damage and RIPK1mediated apoptotic signaling pathway[J].Science of the Total Environment,2018,634(1):1435-1444.
[27]Yao Cui,Yu An,Jin Tongyu,et al.Real-time monitoring of skin wound healing on nano-grooves atopography using electric cellsubstrate impedance sensing(ECIS)[J].Sensors and Actuators BChemical,2017,250:461-468.
[28]Levine B,Kroemer G.Autophagy in the pathogenesis of disease[J].Cell,2008,132(1):27-42.
[29]Chen Zhihua,Lam Hilaire C,Jin Yang,et al.Autophagy protein microtubule-associated protein 1light chain-3B(LC3B)activates extrinsic apoptosis during cigarette smoke-induced emphysema[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(44):18880-18885.
[30]Kiyono K,Suzuki H I,Matsuyama H,et al.Autophagy is activated by TGF-beta and potentiates TGF-beta-mediated growth inhibition in human hepatocellular carcinoma cells[J].Cancer Research,2009,69(23):8844-8852.
[31]Kong H,Xia K,Pan L,et al.Autophagy and lysosomal dysfunction:Anew insight into mechanism of synergistic pulmonary toxicity of carbon black-metal ions co-exposure[J].Carbon,2017,111:322-333.
[32]Fujita N,Itoh T,Omori H,et al.The Atg16L complex specifies the site of LC3lipidation for membrane biogenesis in autophagy[J].Molecular biology cell,2008,19(5):2092-2100.
[33]Nezis I P,Stenmark H.p62 at the interface ofautophagy,oxidative stress signaling,and cancer[J].Antioxidants&Redox Signaling,2012:17(5):786-793.
[34]Su Ruijun,Jin Xiaoting,Zhang Weifeng,et al.Particulate matter exposure induces the autophagy of macrophages via oxidative stress-mediated PI3K/AKT/m TOR pathway[J].Chemosphere,2017,167:444-453.
[35]Wang Yu,Nie Hao,Zhao Xin,et al.Bicyclol induces cell cycle arrest and autophagy in HepG2 human hepatocellular carcinoma cells through the PI3K/AKTand Ras/Raf/MEK/ERK pathways[J].BMCCancer,2016,16(1):742.