磷酸三(2，3-二氯丙基)酯暴露誘發NRK-52E細胞纖維化的體外研究

董良佳，楊峰，王京真,2.#，劉文華,*

1.汕頭大學海洋生物研究所廣東省海洋生物技術重點實驗室，汕頭515063

2.欽州學院海洋學院廣西北部灣海洋生物多樣性養護重點實驗室，欽州535011

磷酸三(2，3-二氯丙基)酯暴露誘發NRK-52E細胞纖維化的體外研究

董良佳1，楊峰1，王京真1,2.#，劉文華1,*

1.汕頭大學海洋生物研究所廣東省海洋生物技術重點實驗室，汕頭515063

2.欽州學院海洋學院廣西北部灣海洋生物多樣性養護重點實驗室，欽州535011

自2004年禁止使用五溴聯苯醚混合物阻燃劑以來,有機磷阻燃劑(organophosphorus flame retardants,OPFR)作為替代品開始被廣泛生產和使用,因而成為當今阻燃劑研究的熱點。目前,有機磷阻燃劑在水體環境及生物體中已有較高的檢出率,而其對生物體及生態環境的潛在毒性效應還知之甚少。本研究利用磷酸三(2,3-二氯丙基)酯(tri(2,3-dichloropropyl)phosphate, TDCPP)對大鼠腎小管上皮細胞(NRK-52E)的毒性暴露實驗,探討TDCPP潛在的腎臟毒性。結果表明:一定劑量的TDCPP對NRK-52E的細胞活性有抑制作用,能誘導細胞內活性氧自由基(reactive oxide species,ROS)生成量增加,并觸發上皮-間充質轉換(epithelial-mesenchymal transition,EMT)及胞外基質沉積因子如波形蛋白(vimentin)、轉化生長因子-β1(transforming growth factor-β1,TGF-β1)、胞外基質成分纖連蛋白-1(fibronectin-1,FN-1)等基因mRNA表達的顯著上調以及上皮細胞鈣粘蛋白(E-cadherin)等基因mRNA表達的顯著下調。上述研究結果表明,TDCPP可促進NRK-52E細胞發生上皮間充質轉化及纖維化。本研究為進一步綜合評估TDCPP的生物和環境毒理效應提供了基礎實驗數據。

磷酸三(2,3-二氯丙基)酯；大鼠腎小管上皮細胞；毒理效應；有機磷阻燃劑；上皮-間充質轉換

有機磷阻燃劑逐步替代傳統的溴代阻燃劑,室內塵埃中磷酸三(2,3-二氯丙基)酯(TDCPP)的濃度和經典阻燃劑多溴聯苯醚(PBDEs)的濃度相當[1-2],TDCPP通常是以物理添加方式(而非以較穩定的共價鍵作用)被大量加入到聚氯乙烯、硬質及軟質聚氨酯泡沫塑料、環氧樹脂、聚酯纖維等材料中[3],因而很容易釋放到環境中[4]；同時由于其在水環境中很難發生轉化和降解(在沉積物中半衰期為1 600 d),因此TDCPP具有很高的環境蓄積性[5-6]。

德國、挪威、奧地利等廢水中的TDCPP的濃度可高達1 400 ng·L-1[7-8],在中國、德國、美國、澳大利亞、西班牙等地表水及飲用水中TDCPP的濃度達1 284 ng·L-1[9-12]。目前已在多種野生生物體內檢測到TDCPP的存在,在瑞典靠近一排污點的淡水鱸魚體內,TDCPP的濃度為140μg·kg-1[13]；在鱈魚肝臟、肌肉等組織中TDCPP的濃度可達到5～30μg·kg-1[7,14]。人類脂肪、精液和母乳中也相繼檢出TDCPP[13,15-16]。由于TDCPP具有相對較高的親脂性(辛醇/水分配系數Log Kow為3.65),TDCPP易進入血流系統中,因此能在各種生物體中累積和富集(BCF理論值為13.5[17]),并可隨食物鏈進行傳遞。毒理學研究顯示,多種磷系阻燃劑具有明顯的致畸性和致癌性,對生態環境和人體健康造成潛在威脅[18],因此,評估TDCPP的環境毒性風險變得尤為重要。

目前,國內外已開展了部分TDCPP的毒性研究,以往研究顯示TDCPP具有內分泌干擾、神經發育以及致畸形和致癌等毒性[19-26]。研究顯示,TDCPP可通過皮膚滲透和食道系統進入生物體,而隨血液迅速擴散至肺、肝臟和腎臟等器官[27]。慢性毒理學實驗證實喂食一定濃度的TDCPP可誘發小鼠腎臟、睪丸和肝臟等組織腫瘤的產生[28]。Ren等[29]在兔腎臟近曲小管細胞中發現磷酸三氯乙酯(TCEP)可以降低細胞活性及DNA合成,TCEP還可以促進細胞周期調控蛋白p21WAF/Cip1和p27Kip1的表達；另外TCEP可以影響近曲小管細胞中離子、非離子物質的轉運,并抑制相應轉運載體的表達[30]；后續研究發現TCEP可以通過促進c-Jun氨基末端激酶(JNK)磷酸化程度及半胱氨酸蛋白酶-8(Caspase-8)的表達誘導腎臟近曲小管細胞凋亡[31]。腎臟是人體重要的代謝器官。腎小管上皮細胞的上皮-間充質轉分化以及胞外基質的過度積累與腎臟功能的缺失有密切的關系[32],而關于OPFR對腎小管間質纖維化是否具有促進作用的研究尚未見報道。

本研究以TDCPP為暴露化合物,以腎小管上皮細胞為實驗對象,通過對高濃度TDCPP(50μmol·L-1)作用下NRK-52E細胞中上皮-間充質轉換標識分子、纖維化誘導因子、胞外基質成分等的mRNA表達量的檢測,初步探討TDCPP暴露是否具有引起腎小管上皮細胞發生上皮-間充質轉換及胞外基質過度積累的毒性,進而評估TDCPP可能存在的環境毒性。TDCPP廣泛存在于水體壞境及某些海洋生物體內[7,14,33-34],本研究對評估TDCPP對海洋生物及生態系統的危害提供了基礎的實驗數據及理論支持。

1 材料與方法(Materials and methods)

1.1 試劑及儀器設備

試劑:TDCPP(購于美國 Sigma公司,純度98.5%)；2′,7′-二氯雙乙酸鹽熒光探針(DCFH-DA) (購于Sigma公司,純度≥97%)；二甲基亞砜(購于Sigma公司,純度≥99.7%)；低糖無酚紅細胞培養基(購于Sigma公司)；胎牛血清(購于美國Thermo公司)；青霉素-鏈霉素-谷氨酰胺溶液(購于美國Gibco公司)；1%的雙抗、胰蛋白酶-乙二胺四乙酸、磷酸緩沖鹽溶液(購于美國Invitrogen公司)等。

儀器:Eppendorf 170R二氧化碳培養箱(購于德國Eppendorf公司)；Infinite 200Pro全波段掃描酶標儀(購于瑞士Tecan公司)；超凈工作臺(購于蘇州安泰公司)。

1.2 細胞培養及毒物暴露處理

細胞培養:NRK-52E細胞株購于中國科學院細胞庫；細胞培養基為低糖無酚紅的細胞培養基,加入5%(v/v)的胎牛血清,青霉素-鏈霉素-谷氨酰胺溶液(100 U·mL-1的青霉素,100μg·mL-1的鏈霉素, 29.2mg·mL-1的L-谷氨酰胺)；培養條件為37℃, 5%CO2,飽和濕度培養。

毒物暴露處理:以二甲基亞砜作為助溶劑,溶解TDCPP,用完全培養基稀釋TDCPP配制成不同工作濃度(0.5、5、15、25、50μmol·L-1)[23,35]；待 NRK-52E細胞貼壁(24 h)后進行不同濃度組的毒物暴露。

1.3 WST-8法檢測細胞活性

將NRK-52E細胞均勻接種于96孔細胞培養板中,進行不同濃度組TDCPP的暴露處理,每組設置6個復孔,分別于毒物暴露12、24、36 h后加入碧云天細胞活性檢測試劑盒中的細胞計數試劑,37℃避光孵育2～4 h,用酶標儀測定450 nm波長下的吸光值。

1.4 DCFH-DA法檢測活性氧水平

將NRK-52E細胞均勻接種于96孔全黑細胞培養板中進行培養,待細胞完全貼壁(24 h)后加入濃度為5μmol·L-1的DCFH-DA染液,37℃避光孵育30 min；棄掉含有探針染液的細胞培養基,磷酸緩沖鹽溶液清洗細胞2次,加入含有不同濃度TDCPP的細胞培養基,毒物暴露處理1 h；用酶標儀測定激發光波長為485 nm、發射光波長為538 nm下的熒光值。

1.5 實時熒光定量PCR

待NRK-52E細胞貼壁(24 h),用 RNAisolate Plus溶液(Takara)收集細胞并于-80℃低溫儲存,用于細胞總RNA的提取。用Takara RNA反轉試劑盒(Takara)將20μg總RNA逆轉錄為cDNA。

反應體系為20μL,擴增程序為:預加熱95℃, 30 s,1個循環；擴增95℃,5 s；60℃,31 s,40個循環。溶解曲線:95℃,5 s；60℃,1 min；95℃,15 s。各基因的特異性引物序列見表1。

利用LightCycler 480實時熒光定量PCR儀檢測經50μmol·L-1TDCPP暴露處理細胞24 h后上述基因的表達情況。本實驗以β-肌動蛋白(β-actin)為內參基因,以2-△△Ct法計算目的基因mRNA的相對表達水平。

表1 RT-PCR各基因引物序列Table 1 The sequence of primers for the RT-PCR

1.6 數據統計與分析

每組數據至少經過3次重復實驗得到,且每組實驗設置6個平行實驗孔,描述性統計分析以6個平行實驗孔的平均數±標準差SD表示。運用SPSS 21.0軟件ANOVA程序進行組間的數據顯著性差異水平的分析(單因素方差分析,One-way ANOVA),若P＜0.05則認為毒物暴露實驗組與空白對照組的數據具有統計學意義上的顯著性差異,以*P表示；若P＜0.01則認為毒物暴露實驗組與空白對照組的數據具有統計學意義上的極顯著性差異,以**P表示。

2 結果與分析(Results and analysis)

2.1 TDCPP對NRK-52E細胞增殖的影響

暴露實驗組的細胞活性相對于空白對照組細胞活性降低,一定濃度的TDCPP可以抑制NRK-52E細胞的增殖,且TDCPP對細胞的抑制效應與TDCPP存在時間--劑量依賴關系(見圖1)。TDCPP暴露處理12 h時,較高濃度的實驗組(25μmol·L-1、50μmol·L-1)的細胞活性被顯著性抑制(*P＜0.05)；暴露處理24 h時, 5μmol·L-1以上濃度的TDCPP對細胞活性都具有顯著性抑制作用(*P＜0.05),且50μmol·L-1的TDCPP的抑制效應極顯著性大于空白對照組(**P＜0.01)；暴露處理36 h時,5μmol·L-1以上濃度的TDCPP對細活性都具有極顯著性抑制作用(**P＜0.01)。

圖1 TDCPP對NRK-52E細胞活力的抑制Fig.1 Inhibitory effect of TDCPP on viability of NRK-52E cells

2.2 TDCPP對NRK-52E細胞內活性氧含量的影響

25μmol·L-1、50μmol·L-1暴露實驗組與空白對照組的細胞內活性氧含量有統計學意義上的顯著性差異(*P＜0.05、**P＜0.01)。經不同濃度TDCPP暴露處理1 h后,5、25、50μmol·L-1暴露實驗組的細胞內活性氧含量分別為空白對照組的113%、130%、152%；25、50μmol·L-1暴露實驗組細胞內活性氧含量顯著高于空白對照組(*P＜0.05、**P＜0.01)(見圖2)。TDCPP與NRK-52E細胞內活性氧水平的升高存在劑量依賴關系。

圖2 TDCPP對NRK-52E細胞內活性氧含量的影響Fig.2 Effect of TDCPP on content of reactive oxygen species in NRK-52E cells

2.3 TDCPP對NRK-52E細胞上皮基質轉換相關基因表達的影響

50μmol·L-1TDCPP可以使NRK-52E細胞內的E-cadherin mRNA表達量極顯著下調(**P＜0.01),毒物暴露實驗組的E-cadherin mRNA表達量為空白對照組的16.83%；而vimentin mRNA表達量顯著性上調(*P＜0.05),毒物暴露實驗組的vimentin mRNA表達量為空白對照組的126.43%(見圖3)。

圖3 TDCPP影響E-cadherin和vimentin mRNA的表達Fig.3 Effect of TDCPP on mRNA expression of E-cadherin and vimentin

圖4 TDCPP影響TGF-β1 mRNA的表達Fig.4 Effect of TDCPP on mRNA expression of TGF-β1

50μmol·L-1TDCPP可以使NRK-52E細胞內的TGF-β1 mRNA表達量極顯著上調(**P＜0.01),毒物暴露實驗組的TGF-β1 mRNA表達量為空白對照組的128.63%(見圖4)。

50μmol·L-1TDCPP可以使NRK-52E細胞fibronectin-1的mRNA表達量極顯著性上調(**P＜0.01),毒物暴露實驗組的fibronectin-1的mRNA表達量為空白對照組的185.32%(見圖5)。

圖5 TDCPP影響fibronectin-1的表達Fig.5 Effect of TDCPP on mRNA expression of fibronectin-1

3 討論(Discussion)

腎臟是藥物在體內代謝與排泄主要器官[38],也是生態毒理學研究中的一種模式器官,如Albina等[39]利用腎臟發現2,2',4,4',5-pentabromodiphenyl ether(BDE-99)有引起氧化應激的作用,Gobe和Crane[40]研究了金屬鉻對腎臟產生的毒性等。以往研究顯示,TDCPP可通過皮膚滲透和進食等進入生物體,腎臟是其中的一個靶標器官[27]；同時慢性毒理學的實驗進一步證實,喂食一定濃度(20mg·kg-1·d-1)的TDCPP可誘發小鼠腎臟腫瘤的產生[28]。然而,TDCPP腎毒性的分子機制尚不清楚。在腎臟中最易受到外源物影響的是近曲小管上皮細胞[41],在正常生理情況下,活性氧參與細胞信號轉導過程,但是當腎細胞受到機械損傷或有害刺激時,體內氧化還原平衡被破壞,會產生過多的活性氧,增加腎細胞的氧耗量[42-43]。這些過多的活性氧可以促進炎性細胞在腎聚集,并促進趨化因子、血管緊張素II、TGF-β1、表皮生長因子等致纖維化關鍵性因子表達量增加[44-45],進一步介導其下游的致纖維化效應,導致腎臟功能的損害[46],而近曲小管上皮細胞較易受到氧化應激的損害[47]。由于動物機體的富集作用,本研究設置了高濃度的TDCPP作用于NRK-52E腎細胞,發現TDCPP可以降低NRK-52E的細胞活性；使上皮-間充質轉換的誘導因子TGF-β1以及間充質標識vimentin和細胞外基質成分fibronectin-1表達上調,而降低E-cadherin的表達。表明TDCPP具有潛在的促腎臟纖維化作用,對腎臟具有一定的毒性效應。

在腎臟間質纖維化過程中,腎小管上皮細胞參與纖維母細胞的形成[48],上皮細胞通過上皮-間充質轉分化轉換成具有分泌胞外基質功能的纖維母細胞[49]。腎小管上皮細胞的上皮-間充質轉換可誘導腎小管間質纖維化,進而與間質中的成纖維細胞、淋巴細胞等相互作用,促進間質發生纖維化,最終誘發腎臟病變[50]。上皮-間充質轉分換是一個比較復雜的過程,其中TGF-β1是最主要的上皮-間充質轉分化誘導因子[51]。上皮細胞發生上皮-間充質轉分換時,細胞間的粘附性降低(如E-cadherin的表達減少),細胞之間的緊密連接性減弱甚至丟失,基底膜被破壞,從而使上皮細胞中維持上皮形態的基因表達受到抑制,而間質細胞形態的基因得以表達,具有遷移特性,繼而會影響到器官的正常生理功能[52],同時,胞外基質中fibronectin-1的分泌量也會增大,增大纖維化程度[48]。

本研究發現TDCPP可以使NRK-52E細胞內活性氧含量增加,并使TGF-β1 mRNA的表達上調；分析了TDCPP對上皮細胞的起粘附作用的標志分子E-cadherin[53]和間充質標識分子 vimentin[54]的 mRNA表達的影響,發現NRK-52E細胞經50μmol·L-1TDCPP處理24 h后,細胞內的E-cadherin mRNA表達量極顯著性下調,而vimentin mRNA表達量顯著性上調,說明TDCPP可以誘導NRK-52E上皮細胞發生間質轉換；纖維化的主要特點是胞外基質的過度累積[29,47],TDCPP可以誘導NRK-52E細胞里的fibronectin-1的表達,由于fibronectin-1是胞外基質的組成成分之一[37,55],所以可以說明TDCPP可促進胞外基質累積,對腎臟纖維化有促進作用。

關于TDCPP的毒理研究主要集中在其內分泌干擾性或神經毒性等方面[19,23],本實驗首次以腎小管上皮細胞為實驗對象,探討了TDCPP對腎臟可能產生的危害,結果表明高濃度TDCPP對NRK-52E細胞上皮-間質轉換及纖維化具有促進作用,說明TDCPP對生物體具有一定的毒性,這對評估TD-CPP的生態毒理效應提供基礎的實驗數據；但細胞形態學上并無明顯變化,造成這個結果的機制正在深入研究。本研究所用的TDCPP暴露劑量較大,還需研究低劑量TDCPP對腎小管上皮細胞的影響,以對TDCPP的環境毒理效應進行更嚴謹的評估。由于在水體環境及一些海洋生物體內檢測到TDCPP的存在,所以研究TDCPP對海洋生物尤其是具有海洋生態環境指示作用的大型海洋哺乳動物的危害具有重要的生態意義。

致謝:感謝李平老師、吳應林師兄、金瑛師姐、孫亞菁師妹等在實驗及文章修改中給予的幫助。

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*共同通訊作者(Co-),E-mail:wangjingzhen-1@163.com

Tris(1,3-dichloro-2-propyl)Phosphate Induces NRK-52E Cells Fibrosis in vitro

Dong Liangjia1,Yang Feng1,Wang Jingzhen1,2,#,Liu Wenhua1,*
1.Shantou University Marine Biology Institute,Guangdong Province Marine Biotechnology Key Laboratory,Shantou 515063,China
2.Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation,Qinzhou University,Qinzhou,China

14 May 2015 accepted 10 October 2015

Since the prohibition of the commercialized pentabromodiphenyl ether compound fire retardants in 2004, organic phosphorus flame retardants(OPFRs)have been produced and applied comprehensively as the substitute. Recently,the detectable rate of OPFRs in water environment and organisms is quite high,but studies about the potential adverse effect of OPFRs on organisms and thus ecosystems are scarce.The aim of the present study was toexamine the possible toxic effect of tris(1,3-dichloro-2-propyl)phosphate(TDCPP)as a model OPFR substance. NRK-52E cells were exposed to TDCPP,cell viability and extracellular matrix gene expression levels were measured.The results showed that the viability of NRK-52E cells was significantly inhibited by TDCPP in both a dosedependent and time-dependent manner.TDCPP augmented the reactive oxygen species production in NRK-52E cells.The expression of vimentin,transforming growth factor-β1(TGF-β1)and fibronectin-1 was significantly upregulated,while E-cadherin was significantly down-regulated by TDCPP.In conclusion,TDCPP may have the ability to promote epithelial-mesenchymal transition and fibrosis in NRK-52E cells.The result forms the foundation for future comprehensive evaluation of TDCPP toxic effects on the environment.

tris(1,3-dichloro-2-propyl)phosphate;NRK-52E cells;toxicological effect;OPFR;epithelial-mesenchymal transition

2015-05-14 錄用日期:2015-10-10

1673-5897(2016)1-329-08

X171.5

A

10.7524/AJE.1673-5897.20150514003

董良佳,楊峰,王京真,等.磷酸三(2,3-二氯丙基)酯暴露誘發NRK-52E細胞纖維化的體外研究[J].生態毒理學報,2016,11(1):329-336

Dong L J,Yang F,Wang J Z,et al.Tris(1,3-dichloro-2-propyl)phosphate induces NRK-52E cells fibrosis in vitro[J].Asian Journal of Ecotoxicology, 2016,11(1):329-336(in Chinese)

國家自然科學基金項目(41306180)；國家自然科學基金項目(41176155)；教育部高等學校博士點科研基金項目(20134402110005)；海洋公益性行業科研專項經費項目(201405007)

董良佳(1991-),女,碩士研究生,研究方向為環境毒理學,E-mail:13ljdong@stu.edu.cn

),E-mail:whliu@stu.edu.cn

簡介:劉文華(1970—)，男，博士，研究員，主要研究方向生態毒理學、海洋哺乳動物保護生物學。

共同通訊作者簡介:王京真(1982—)，男，博士，主要研究方向體外毒理學，保護生物學，生態和演化生物學。