端正花,王勛功,王華,李寧濤,朱琳
1. 天津理工大學(xué)環(huán)境科學(xué)與安全工程學(xué)院,天津300384 2. 天津出入境檢驗(yàn)檢疫局工業(yè)產(chǎn)品安全技術(shù)中心,天津300191 3. 南開大學(xué)環(huán)境科學(xué)與工程學(xué)院,天津300071
全氟辛烷羧酸(PFOA)與全氟辛烷磺酸(PFOS)的細(xì)胞毒性效應(yīng)
端正花1,王勛功1,王華2,李寧濤2,朱琳3,*
1. 天津理工大學(xué)環(huán)境科學(xué)與安全工程學(xué)院,天津300384 2. 天津出入境檢驗(yàn)檢疫局工業(yè)產(chǎn)品安全技術(shù)中心,天津300191 3. 南開大學(xué)環(huán)境科學(xué)與工程學(xué)院,天津300071
新型污染物全氟辛烷羧酸(PFOA)與全氟辛烷磺酸(PFOS)在全球范圍內(nèi)的各種環(huán)境介質(zhì)中被廣泛檢出,對(duì)生態(tài)安全和人體健康造成威脅。利用MTT(噻唑藍(lán))法研究了PFOA/PFOS對(duì)人體細(xì)胞的毒性效應(yīng)。結(jié)果表明,低劑量PFOA和PFOS對(duì)人體正常肝細(xì)胞增殖具有顯著毒性,其3 d半數(shù)抑制濃度(IC50值)分別約為5和0.5 μg·L-1。此外,PFOA和PFOS均對(duì)MCF-7細(xì)胞增殖表現(xiàn)出顯著的非單調(diào)劑量-毒性效應(yīng),并且在環(huán)境濃度范圍(< 0.6 μg·L-1)內(nèi)均可促進(jìn)MCF-7細(xì)胞增殖,表現(xiàn)出潛在的類雌激素作用風(fēng)險(xiǎn)。
全氟辛烷羧酸(PFOA); 全氟辛烷磺酸(PFOS);肝細(xì)胞;MCF-7;非單調(diào)劑量效應(yīng)關(guān)系
近年來,新型污染物全氟化合物(perfluorinated compounds, PFCs)在生產(chǎn)領(lǐng)域被大規(guī)模使用,導(dǎo)致這類物質(zhì)廣泛進(jìn)入全球環(huán)境。由于很難被降解且具有生物蓄積性,PFCs被認(rèn)為是一類新型的持久性有機(jī)污染物[1],在水體[2]、生物體[3]、人體組織[4]和食物[5]中被廣泛檢出,甚至在北極地區(qū)的生物體及環(huán)境介質(zhì)中均有PFCs檢出[6]。PFCs類化合物最終會(huì)部分轉(zhuǎn)化為全氟辛烷羧酸(PFOA)和全氟辛烷磺酸(PFOS),這2種物質(zhì)是PFCs中應(yīng)用最為廣泛的,尤其是PFOS,它在生物體內(nèi)的蓄積水平是二惡英等的數(shù)百至數(shù)千倍[1]。因此,PFOA和PFOS的毒性研究已成為生態(tài)環(huán)境污染領(lǐng)域的熱點(diǎn)問題[7-9]。
PFOA與PFOS作為持久性新型環(huán)境污染物,已發(fā)現(xiàn)其在生物的生殖、發(fā)育、神經(jīng)、免疫、遺傳等方面具有毒性[10]。但是目前PFCs的毒性研究多數(shù)集中于哺乳動(dòng)物的急性毒性研究,且使用的暴露劑量明顯高于環(huán)境中的實(shí)際含量[11]。PFCs對(duì)人類的毒性效應(yīng)僅限于小范圍人群的流行病學(xué)研究[12]。許多種類的外源性物質(zhì)都有可能對(duì)不同的人體細(xì)胞產(chǎn)生毒性作用,同時(shí)研究發(fā)現(xiàn)肝臟比其他內(nèi)臟更容易富集長鏈的PFCs[13],因此本實(shí)驗(yàn)首先探討了PFOA與PFOS對(duì)人體正常肝細(xì)胞株的細(xì)胞增殖毒性。另外,據(jù)動(dòng)物體內(nèi)實(shí)驗(yàn)證明,PFCs具有類雌激素作用[14-15]。因此,本實(shí)驗(yàn)進(jìn)一步通過研究PFOA與PFOS對(duì)雌激素受體陽性乳腺癌細(xì)胞株MCF-7的毒性作用,初步分析其類雌激素作用。從而為PFCs的多介質(zhì)環(huán)境行為、健康風(fēng)險(xiǎn)和控制原理的研究提供依據(jù)。
1.1 實(shí)驗(yàn)材料
人張氏肝細(xì)胞株(Chang liver),購自武漢博士德生物工程有限公司;人乳腺癌細(xì)胞株(MCF-7),南開大學(xué)生命科學(xué)學(xué)院惠贈(zèng)。PFOS和PFOA(純度≥99.9%)購自Sigma-Aldrich公司,其他試劑購自武漢博士德生物工程有限公司。
配制PFOS和PFOA母液5 mg·L-1:稱取一定量的PFOS和PFOA粉末,加入少量二甲基亞砜(DMSO)助溶(實(shí)際暴露溶液中DMSO的體積分?jǐn)?shù)不超過0.1%,經(jīng)溶劑對(duì)照實(shí)驗(yàn),此濃度對(duì)實(shí)驗(yàn)結(jié)果無影響),再溶于含有10%(質(zhì)量分?jǐn)?shù))胎牛血清的1640完全培養(yǎng)基中。
1.2 細(xì)胞的傳代和培養(yǎng)
將人體肝細(xì)胞和MCF-7細(xì)胞分別置于含有10%(質(zhì)量分?jǐn)?shù))胎牛血清的1640完全培養(yǎng)基中,在條件為37 ℃、5% CO2的細(xì)胞培養(yǎng)箱(美國Thermo, 3110)內(nèi)培養(yǎng),每隔2 d更換1次培養(yǎng)基。
吸去培養(yǎng)皿中的完全培養(yǎng)基,用磷酸鹽緩沖液(PBS)清洗培養(yǎng)皿,洗去細(xì)胞表面上的完全培養(yǎng)基;吸去PBS,加入1 mL胰蛋白酶放入培養(yǎng)箱中消化細(xì)胞2 min;取出培養(yǎng)皿加入1 mL完全培養(yǎng)基終止消化;吹散貼在壁上的細(xì)胞后,將細(xì)胞懸液置于離心管中,以1 000 r·min-1離心(德國Hettich, Rotina 420)3 min;棄去上清液,加入完全培養(yǎng)基用移液槍反復(fù)吹散至單細(xì)胞狀態(tài),以1/2比例傳代培養(yǎng)。
1.3 MTT法檢測(cè)
取對(duì)數(shù)生長期的人體肝細(xì)胞和MCF-7細(xì)胞制成單細(xì)胞懸液,將細(xì)胞懸液接種于96孔板(美國Thermo),調(diào)整細(xì)胞懸液的濃度,使每孔加入100 μL細(xì)胞懸液后每孔約有1×104個(gè)細(xì)胞。在37 ℃、5% CO2條件下置于細(xì)胞培養(yǎng)箱中培養(yǎng)24 h待細(xì)胞貼壁。24 h后吸去每孔中的完全培養(yǎng)基,空白對(duì)照組加入含有10%(質(zhì)量分?jǐn)?shù))胎牛血清的1640完全培養(yǎng)基100 μL,實(shí)驗(yàn)組分別在每孔中加入含有不同濃度(0、0.5、5、500和5 000 μg·L-1)的PFOS或PFOA的完全培養(yǎng)基100 μL,每組設(shè)置個(gè)10復(fù)孔。將細(xì)胞分別培養(yǎng)24 h、48 h和72 h。培養(yǎng)結(jié)束后,吸去完全培養(yǎng)基,每孔加入20 μL濃度為5 mg·mL-1的MTT溶液(噻唑藍(lán)溶液),繼續(xù)在培養(yǎng)箱中培養(yǎng)4 h。而后吸去MTT溶液,每孔加入100 μL DMSO溶液,震蕩10 min,用酶標(biāo)儀(美國Thermo, FC)在492 nm波長處測(cè)定各孔的吸光度(A)值。
1.4 數(shù)據(jù)統(tǒng)計(jì)
實(shí)驗(yàn)所得數(shù)據(jù)為96孔板各孔的吸光度(A)值,用Excel中的常用函數(shù)AVERAGE計(jì)算每組10個(gè)復(fù)孔吸光度(A)值的平均值,用常用函數(shù)STDEV計(jì)算每組的標(biāo)準(zhǔn)偏差,用統(tǒng)計(jì)函數(shù)TTEST比較每個(gè)濃度組與空白對(duì)照組的差異,如p<0.05,則認(rèn)為有顯著的差異。
2.1 PFOA和PFOS對(duì)人體肝細(xì)胞的細(xì)胞增殖毒性
如圖1所示,3 d內(nèi)空白對(duì)照組人體正常肝細(xì)胞都呈對(duì)數(shù)增長趨勢(shì),說明本實(shí)驗(yàn)細(xì)胞體系穩(wěn)定可靠。
經(jīng)PFOA和PFOS暴露1 d后,從5 μg·L-1濃度組開始,細(xì)胞增殖受到顯著的抑制作用(p(PFOA)=0.006;p(PFOS)=0.005)。暴露2 d后,最低濃度組(0.5 μg·L-1)也都表現(xiàn)為抑制肝細(xì)胞增殖(p(PFOA)=0.005;p(PFOS)=0.0005)。PFOA的3 d半數(shù)抑制濃度(3 d-IC50)約為5 μg·L-1,而PFOS的3 d-IC50約為0.5 μg·L-1,從這個(gè)指標(biāo)來看,PFOS對(duì)肝細(xì)胞增殖抑制的毒性更大。
2.2 全氟辛烷羧酸(PFOA)與全氟辛烷磺酸(PFOS)對(duì)MCF-7的細(xì)胞增殖毒性
如圖2所示,5 d內(nèi)空白對(duì)照組MCF-7都呈對(duì)數(shù)增長趨勢(shì),說明本實(shí)驗(yàn)細(xì)胞體系穩(wěn)定可靠。
由圖2可見,經(jīng)PFOA暴露1~3 d后,各濃度組均促進(jìn)MCF-7細(xì)胞增殖(p<0.05);暴露4 d后低濃度組(0.5 μg·L-1和5 μg·L-1)繼續(xù)促進(jìn)細(xì)胞增殖,表現(xiàn)為類雌激素效應(yīng),500 μg·L-1組無顯著細(xì)胞毒性(p=0.112),最高濃度組(5 000 μg·L-1)表現(xiàn)出抑制細(xì)胞增殖作用(p=0.023);暴露5 d后低濃度組(0.5 μg·L-1和5 μg·L-1)繼續(xù)促進(jìn)細(xì)胞增殖(p<0.05),500 μg·L-1組無細(xì)胞毒性(p=0.272),最高濃度組(5 000 μg·L-1)抑制細(xì)胞增殖(p=0.039)。由此可見,PFOA對(duì)MCF-7的細(xì)胞增殖表現(xiàn)出非單調(diào)劑量-效應(yīng)毒性。 PFOS暴露1 d后,各濃度組均無毒性效應(yīng),而PFOA暴露1 d后甚至最低濃度組(0.5 μg·L-1)都表現(xiàn)出刺激MCF-7細(xì)胞增殖作用(p=0.01),因此從這個(gè)指標(biāo)看PFOA的類雌激素作用更強(qiáng);暴露2 d后PFOS各濃度組均促進(jìn)細(xì)胞增殖(p<0.05);暴露3 d后則表現(xiàn)出非單調(diào)劑量-效應(yīng)毒性,即低濃度(0.5、5和500 μg·L-1組)繼續(xù)促進(jìn)細(xì)胞增殖(p<0.05),最高濃度組(5 000 μg·L-1)表現(xiàn)為抑制細(xì)胞增殖。可見,PFOA和PFOS對(duì)MCF-7細(xì)胞增殖的毒性作用總體規(guī)律相同。
圖1 PFOA/PFOS對(duì)人體肝細(xì)胞增殖的毒性作用注:* p<0.05,** p<0.01,與空白對(duì)照組相比,下同。Fig. 1 Toxicity of PFOA/PFOS to the proliferation of human liver cellsNote: *p<0.05, ** p<0.01, compared with the control group, the same below.
圖2 PFOA/PFOS對(duì)MCF-7細(xì)胞增殖的毒性作用Fig. 2 Toxicity of PFOA/PFOS to the proliferation of MCF-7 cells
城市工業(yè)污水中PFCs的含量較高,比如泰國工業(yè)區(qū)污水處理廠出水中檢測(cè)出的PFCs總濃度高于0.6 μg·L-1[16]。人體血清中PFOA和PFOS的平均濃度約為10 μg·L-1[17]。Liu等[18]以原代培養(yǎng)的羅非魚肝臟細(xì)胞為實(shí)驗(yàn)對(duì)象,證明高濃度(>1 000 μg·L-1)的PFOA和PFOS具有細(xì)胞毒性效應(yīng),并且認(rèn)為目前的污染程度對(duì)魚類生殖發(fā)育帶來的風(fēng)險(xiǎn)很小。本實(shí)驗(yàn)所選取的最低濃度0.5 μg·L-1低于環(huán)境暴露濃度。PFOA和PFOS在該濃度下抑制人正常肝細(xì)胞的增殖,并且隨著濃度的增大,抑制作用增強(qiáng),證明了環(huán)境中PFOA和PFOS對(duì)于人體正常細(xì)胞存在毒性。究其原因,PFOA和PFOS都能誘導(dǎo)氧化自由基(ROS)的產(chǎn)生[19-20],從而導(dǎo)致肝細(xì)胞凋亡。但是本實(shí)驗(yàn)中PFOS對(duì)正常肝細(xì)胞增殖抑制的IC50值顯著低于PFOA,即PFOS對(duì)正常肝細(xì)胞增殖抑制的相對(duì)毒性較大。這與前人的研究結(jié)論是一致的[21-22]。研究發(fā)現(xiàn)PFOS和PFOA都能抑制細(xì)胞間歇連接通訊這一細(xì)胞膜功能[23],但是PFOS還能提高細(xì)胞膜對(duì)疏水性配體的滲透性能,從而使PFOS在生物個(gè)體中具有較高的生物富集速度,而PFOA的滲透及富集速度則比較緩慢[13]。因此可推測(cè)PFOS在同濃度下比PFOA進(jìn)入生物體速度快,富集速度也快,對(duì)生物影響更大。
本實(shí)驗(yàn)以應(yīng)用最為廣泛的具有環(huán)境雌激素受體陽性特征的MCF-7細(xì)胞為研究對(duì)象,探討了環(huán)境濃度的PFOA和PFOS的內(nèi)分泌干擾風(fēng)險(xiǎn)。Henry等[24]研究發(fā)現(xiàn)PFOA在高濃度(3 000 μg·L-1)對(duì)MCF-7細(xì)胞具有細(xì)胞毒性,而在30 μg·L-1則無任何毒性表征。本實(shí)驗(yàn)在高濃度組(5 000 μg·L-1)也發(fā)現(xiàn)PFOA和PFOS對(duì)MCF-7細(xì)胞具有細(xì)胞毒性。但是在低濃度范圍(0.5~5 μg·L-1)下,PFOA和PFOS對(duì)MCF-7細(xì)胞增殖還有具有顯著的促進(jìn)作用,提示其在環(huán)境濃度下具有類雌激素作用。PFOA和PFOS對(duì)MCF-7的細(xì)胞毒性存在非單調(diào)劑量-毒性效應(yīng)。暴露1 d后,PFOS各濃度組均無毒性效應(yīng),而PFOA甚至在最低濃度組(0.5 μg·L-1)表現(xiàn)出刺激MCF-7細(xì)胞增殖,從這個(gè)指標(biāo)看PFOA的類雌激素作用更為明顯。據(jù)報(bào)道PFOA能抑制G-蛋白偶聯(lián)的受體蛋白信號(hào)通路[25]。G-蛋白偶聯(lián)的雌激素受體(G protein-coupled estrogen receptor1, GPER)是迄今發(fā)現(xiàn)的最重要的雌激素膜性受體[26]。但是,是否由于這些特性的異同造成PFOA和PFOS類雌激素效應(yīng)的差距,還有待于進(jìn)一步分析。
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TheCytotoxicEffectofPFOAandPFOS
Duan Zhenghua1, Wang Xungong1, Wang Hua2, Li Ningtao2, Zhu Lin3,*
1. School of Environmental Science and Safety Engineering, Tianjin University of Technology, Tianjin 300384, China 2. Technical Center for Safety of Industrial Products, Tianjin Entry-Exit Inspection Quarantine Bureau, Tianjin 300191, China 3. College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China
6 December 2013accepted3 January 2014
The new pollutants perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) is widely detected in various media within global scope, and thus posing a hazard to environment and human health. The cytotoxic effect of PFOA and PFOS was studied with the method of MTT in this paper. The results showed that low-dose PFOA/PFOS exposure had significant toxicities to the proliferation of normal human liver cells, and the values of their 3 d half maximal inhibitory concentration (IC50) were 5 and 0.5 μg·L-1. Meanwhile, both of PFOA and PFOS had significant non monotonic dose-effect relationship in the toxicity to the proliferation of MCF-7 cells, and they could promote the proliferation of MCF-7 cells within the scope of environmental concentrations (< 0.6 μg·L-1), which showed their potential estrogen actions.
PFOA;PFOS; liver cell; MCF-7; non monotonic dose-effect
國家質(zhì)檢總局科技計(jì)劃項(xiàng)目(2012IK213)
端正花(1981-),女,博士,講師,主要研究方向?yàn)榄h(huán)境生物技術(shù),E-mail: duanzhenghua@mail.nankai.edu.cn;
*通訊作者(Corresponding author),E-mail: zhulin@nankai.edu.cn
10.7524/AJE.1673-5897.20131206001
端正花, 王勛功, 王華,等. 全氟辛烷羧酸PFOA與全氟辛烷磺酸PFOS的細(xì)胞毒性效應(yīng)研究[J]. 生態(tài)毒理學(xué)報(bào), 2014, 9(2): 353-357
Duan Z H, Wang X G, Wang H, et al. The cytotoxic effect of PFOA and PFOS [J]. Asian Journal of Ecotoxicology, 2014, 9(2): 353-357 (in Chinese)
2013-12-06錄用日期2014-01-03
1673-5897(2014)2-353-05
X171.5
A
朱琳(1957—),男,環(huán)境科學(xué)博士,教授,主要研究方向?yàn)樯鷳B(tài)毒理學(xué)和環(huán)境生物學(xué),發(fā)表學(xué)術(shù)論文30余篇。