蔣鵬，盛南，王建設(shè)，戴家銀

中國(guó)科學(xué)院動(dòng)物研究所中國(guó)科學(xué)院動(dòng)物生態(tài)與保護(hù)生物學(xué)重點(diǎn)實(shí)驗(yàn)室，北京 100101

五氯酚(PCP)對(duì)雞肝癌細(xì)胞(LMH)毒性效應(yīng)的機(jī)制研究

蔣鵬，盛南，王建設(shè)，戴家銀*

中國(guó)科學(xué)院動(dòng)物研究所中國(guó)科學(xué)院動(dòng)物生態(tài)與保護(hù)生物學(xué)重點(diǎn)實(shí)驗(yàn)室，北京 100101

五氯酚(pentachlorophenol, PCP)是一種持久性有機(jī)污染物，廣泛用于滅釘螺、木材防腐、除草劑等方面，由于PCP在環(huán)境中的持久性和生物累積性，其對(duì)生態(tài)環(huán)境和人類健康造成潛在危害。本文以雞肝癌細(xì)胞系(chicken hepatoma cells, LMH)為受試對(duì)象，探討了PCP對(duì)細(xì)胞色素P450(CYP450)和抗氧化系統(tǒng)的影響。MTT結(jié)果顯示LMH細(xì)胞經(jīng)不同濃度PCP暴露后，呈現(xiàn)出先促進(jìn)細(xì)胞增殖后抑制的J-型曲線，PCP對(duì)LMH細(xì)胞24 h的半數(shù)效應(yīng)濃度(24 h-EC50)為427.52 μmol·L-1。LMH細(xì)胞在1.56、6.25、25、100 μmol·L-1PCP染毒條件下可增加細(xì)胞EROD、MROD、PROD和BFC活性，并可使CYP1A、1B、1C、2H及3A家族基因mRNA表達(dá)水平升高。LMH細(xì)胞在0.4～100 μmol·L-1PCP染毒下可顯著降低硫酸基轉(zhuǎn)移酶(SULT1B1和SULT 1C1)基因mRNA水平。此外，LMH細(xì)胞在6.25、25、100 μmol·L-1PCP染毒下可引起細(xì)胞內(nèi)ROS升高，同時(shí)PCP(1.56～100 μmol·L-1)可顯著增加細(xì)胞內(nèi)MDA含量和降低GSH/GSSH比值。這些結(jié)果表明細(xì)胞色素P450(CYP450)基因及酶活性的變化、細(xì)胞內(nèi)ROS和MDA含量及GSH/GSSH可作為評(píng)價(jià)LMH細(xì)胞PCP毒性效應(yīng)的敏感性生物標(biāo)志物。此研究在細(xì)胞水平上利用多個(gè)評(píng)價(jià)指標(biāo)研究PCP對(duì)細(xì)胞的毒性效應(yīng)，為PCP環(huán)境風(fēng)險(xiǎn)評(píng)價(jià)提供依據(jù)。

五氯酚；雞肝癌細(xì)胞系；細(xì)胞色素P450；硫酸基轉(zhuǎn)移酶；氧化應(yīng)激

Received12 December 2016accepted10 February 2017

Abstract: Pentachlorophenol (PCP), a persistent organic pollutant, has been used for wood preservation and as a herbicide and insecticide. Due to its persistence in the environment and bioaccumulation, PCP causes potential harm to the ecological environment and human health. In this study, chicken hepatoma cells (LMH) were used to explore the effect of PCP on antioxidant systems and cytochrome P450 (CYP450) levels. The results revealed that 1.56, 6.25, 25 and 100 μmol·L-1PCP could increase EROD, MROD, PROD and BFC levels, and remarkably upregulated the expressions of CYP1A, 1B, 1C, 2H and 3A families. Data obtained from 24 h-exposure studies demonstrated that 0.4-100 μmol·L-1PCP could decrease sulfotransferase (SULT1B1 and SULT 1C1) expression levels. When LMH cells were exposed to 6.25, 25, and 100 μmol·L-1PCP for 24 h, ROS and MDA content increased, while GSH/GSSH deceased significantly. These experimental results showed that PCP exposure could change CYP450 gene expression and activity, ROS and MDA content, and GSH/GSSH ratioin LMH cells. In this view, multiple evaluation indexes should be used to assess the cell toxicity of PCP, which will provide better understanding for PCP risk assessment and mechanism studies.

Keywords: pentachlorophenol; chicken hepatoma cells; cytochrome 450; sulfotransferase; oxidative stress

五氯酚(pentachlorophenol, PCP)作為氯代烴類殺蟲劑和滅真菌劑，具有低成本優(yōu)勢(shì)，被廣泛地用作農(nóng)業(yè)殺蟲劑、除草劑和木材防腐劑等[1]。PCP因具有穩(wěn)定的芳香環(huán)結(jié)構(gòu)和高氯含量而不容易被降解，已成為重要的環(huán)境污染物[2]，PCP污染所造成的環(huán)境問(wèn)題也日益受到全社會(huì)的高度重視[3]。PCP可以通過(guò)雨水等擴(kuò)散，從而轉(zhuǎn)移到水果、蔬菜和谷物中[4]。已有研究表明，PCP在水系統(tǒng)中半衰期長(zhǎng)達(dá)200 d[5]；PCP在人體內(nèi)的半衰期為33 h至16 d[6]。PCP對(duì)動(dòng)物體毒性研究表明慢性暴露可造成嚙齒類動(dòng)物多種不良效應(yīng)，包括免疫系統(tǒng)損傷等[7-10]。PCP是潛在的致癌物質(zhì)，在實(shí)驗(yàn)動(dòng)物中可誘發(fā)腫瘤，其氧化脫氯生成的生物大分子在PCP的毒性中具有重要作用[11]。

流行病學(xué)研究顯示，職業(yè)人群在PCP暴露后增加了患惡性淋巴瘤和白血病的風(fēng)險(xiǎn)[9]。因此開展PCP對(duì)生物體的毒理學(xué)研究，特別是在細(xì)胞水平上利用多個(gè)評(píng)價(jià)指標(biāo)研究PCP對(duì)細(xì)胞的毒性效應(yīng)，對(duì)有毒物質(zhì)的環(huán)境風(fēng)險(xiǎn)評(píng)價(jià)具有重要意義。研究發(fā)現(xiàn)PCP可通過(guò)解偶聯(lián)氧化磷酸化產(chǎn)生細(xì)胞毒性[12]。生物體細(xì)胞中CYP3A4酶承擔(dān)PCP的生物轉(zhuǎn)化作用，通過(guò)產(chǎn)生毒性更強(qiáng)的四氯代氫醌(tetrachlorohydroquinone, TCHQ)而產(chǎn)生細(xì)胞毒性[13]。TCHQ是PCP的主要毒性代謝產(chǎn)物，活性氧(ROS)參與了TCHQ的毒性作用。同時(shí)PCP的毒性也可能與生成的高反應(yīng)性代謝物——四氯-1,4-苯醌(tetrachloro-1,4-benzoquinone, TCBQ)有關(guān)[14]。作為親電子分子，TCBQ與脫氧鳥苷形成加合物，從而導(dǎo)致細(xì)胞遺傳毒性[15]。此外，TCBQ易迅速產(chǎn)生四氯醌(tetrachlorosemiquinone, TCSQ)自由基，并使細(xì)胞產(chǎn)生氧化應(yīng)激。這些研究表明PCP及代謝產(chǎn)物對(duì)人和動(dòng)物細(xì)胞具有遺傳毒性和潛在的致癌性。鳥類作為生態(tài)系統(tǒng)食物鏈中的頂級(jí)生物類群與人類處于相似的食物鏈地位，已經(jīng)成為監(jiān)測(cè)生態(tài)環(huán)境中鹵代有機(jī)污染物的指示生物[16]，因此本研究開展了PCP對(duì)雞肝癌細(xì)胞的毒性效應(yīng)，并初步探討了其毒性機(jī)制，為評(píng)價(jià)PCP環(huán)境污染水平和對(duì)人群的潛在威脅提供理論依據(jù)。

1 材料與方法(Materials and methods)

1.1 實(shí)驗(yàn)材料

PCP(CAS號(hào)87-86-5，純度97%)和Gelatin購(gòu)自Sigma-Aldrich(美國(guó))；Waymouth’s MB 752/1培養(yǎng)基(貨號(hào)：11220-035)和進(jìn)口胎牛血清(貨號(hào)：10099141)購(gòu)自Gibco(美國(guó))；H2DCF-DA(Life Technologies，美國(guó))。

1.2 細(xì)胞培養(yǎng)

雞肝癌細(xì)胞系(chicken hepatoma cells, LMH)(CRL-2117)購(gòu)自ATCC公司(美國(guó))。在無(wú)菌條件下，用0.1% gelatin溶液包被細(xì)胞培養(yǎng)瓶。LMH細(xì)胞用Waymouth’s MB 752/1培養(yǎng)基和15%進(jìn)口胎牛血清于37 ℃、5% CO2培養(yǎng)箱進(jìn)行培養(yǎng)。

1.3 細(xì)胞暴露實(shí)驗(yàn)

首先將PCP溶解在二甲基亞砜(DMSO)中，配成200 mmol·L-1PCP母液[12]。然后用含有血清的培養(yǎng)基將PCP母液稀釋成不同的濃度。對(duì)照組用含有血清的培養(yǎng)基。將培養(yǎng)瓶中的細(xì)胞消化，進(jìn)行細(xì)胞計(jì)數(shù)，然后在6或96孔板中加入一定的細(xì)胞個(gè)數(shù)。24 h后，可將原有的培養(yǎng)基倒掉，加入含不同PCP濃度的培養(yǎng)基，在培養(yǎng)箱繼續(xù)培養(yǎng)。

1.4 細(xì)胞活力檢測(cè)(MTT法)

設(shè)置96孔板中間6×10陣列共60個(gè)孔為實(shí)驗(yàn)孔，取100 μL細(xì)胞懸液接種于96孔板中，每孔細(xì)胞數(shù)為3×104。細(xì)胞培養(yǎng)4 h后，棄去原有培養(yǎng)基，加入200 μL用培養(yǎng)基稀釋成不同濃度的PCP溶液(0～1 mol·L-1設(shè)定濃度梯度)，在培養(yǎng)箱繼續(xù)培養(yǎng)。每組設(shè)6個(gè)平行孔。

在PCP處理細(xì)胞24 h后，每孔中加入20 μL現(xiàn)配制的5 g·L-1MTT溶液，放入CO2細(xì)胞培養(yǎng)箱中，繼續(xù)培養(yǎng)4 h后，棄去培養(yǎng)基，每孔中加入150 μL DMSO，然后在酶標(biāo)儀中設(shè)置程序震蕩5 min，選擇光吸收模式，在570 nm讀取每孔中光吸收值。實(shí)驗(yàn)重復(fù)3次。計(jì)算細(xì)胞抑制率：細(xì)胞抑制率(%)=(1﹣實(shí)驗(yàn)組OD平均值/對(duì)照組OD平均值)×100%。

1.5 細(xì)胞內(nèi)活性氧(ROS)的測(cè)定

將H2DCF-DA配制成10 mmol·L-1的母液，再用waymouth’s MB 752/1培養(yǎng)基(不含血清)將母液稀釋1 000倍，稀釋成10 μmol·L-1的工作液。不同濃度PCP處理LMH細(xì)胞方法同前，每組設(shè)6個(gè)平行孔，重復(fù)3次。細(xì)胞染毒24 h后在96孔板中加入5 μL H2DCF-DA的工作液，37 ℃條件下反應(yīng)30 min。每孔加入200 μL不含血清的waymouth’s MB 752/1培養(yǎng)基洗滌LMH細(xì)胞3次。在每孔中加入100 μL 1×PBS，檢測(cè)反應(yīng)生成的DCF熒光。

1.6 實(shí)時(shí)熒光定量PCR

細(xì)胞樣品總RNA的提?。?孔板中每孔的LMH細(xì)胞約106個(gè)，每組設(shè)6個(gè)平行孔，重復(fù)3次。經(jīng)過(guò)不同濃度的PCP暴露后，用TRIzol法提取細(xì)胞RNA。

熒光定量PCR檢測(cè)目的基因用天根公司試劑盒。定量引物在表1中列出。

1.7 細(xì)胞P450酶活性和抗氧化指標(biāo)的檢測(cè)

細(xì)胞染毒方法同前，每組設(shè)6個(gè)平行孔，實(shí)驗(yàn)重復(fù)3次。細(xì)胞色素P450酶活性(EROD、MROD、PROD、BROD和BFC)用試劑盒(Genmed Scientifics Inc.，美國(guó))檢測(cè)。MDA含量和GSH/GSSH比值的測(cè)定采用南京建成生物工程研究所有限公司試劑盒。

1.8 數(shù)據(jù)統(tǒng)計(jì)

通過(guò)SPSS軟件(SPSS, Inc., 美國(guó))進(jìn)行數(shù)據(jù)的統(tǒng)計(jì)和分析。統(tǒng)計(jì)數(shù)據(jù)用平均值±標(biāo)準(zhǔn)誤(mean± SD)表示。使用單因素的方差及最小顯著差法(LSD)分析對(duì)照組和實(shí)驗(yàn)組間的差異，P< 0.05表示差別具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果(Results)

2.1 LMH細(xì)胞經(jīng)PCP處理后的細(xì)胞活力變化

用不同濃度的PCP處理LMH細(xì)胞24 h后，計(jì)算出各濃度PCP對(duì)細(xì)胞的抑制率，采用biphasic濃度效應(yīng)模型擬合[17]，劑量-效應(yīng)曲線見圖1。結(jié)果顯示LMH細(xì)胞經(jīng)不同濃度PCP暴露24 h后，呈現(xiàn)出先促進(jìn)細(xì)胞增殖后抑制的J-型曲線。MTT實(shí)驗(yàn)結(jié)果推導(dǎo)出PCP對(duì)LMH的24 h-EC50為427.52 μmol·L-1。

表1 雞肝癌細(xì)胞系(LMH)細(xì)胞經(jīng)PCP暴露后熒光定量PCR引物列表Table 1 The primers for genes in chicken hepatoma cells (LMH) exposed to PCP

圖1 不同濃度的PCP處理LMH細(xì)胞24 h后 細(xì)胞活力變化Fig. 1 MTT assay in LMH cells exposed to PCP for 24 h

LMH細(xì)胞在0.4～25 μmol·L-1PCP染毒劑量下，細(xì)胞活性未受到影響。100 μmol·L-1PCP使細(xì)胞活力增加17%(圖2)。為了研究在不引起LMH細(xì)胞活性受到抑制的情況下，低濃度PCP對(duì)細(xì)胞內(nèi)酶活性和抗氧化反應(yīng)的影響，LMH細(xì)胞經(jīng)0.4、1.56、6.25、25和100 μmol·L-1PCP處理后測(cè)定細(xì)

胞內(nèi)CYP450酶活性等指標(biāo)。

2.2 PCP處理LMH細(xì)胞CYP450及SULT1s基因表達(dá)的變化

經(jīng)不同濃度PCP暴露24 h的LMH細(xì)胞，細(xì)胞CYP1A4、CYP1A5、CYP1B1、CYP1C1、CYP3A4和CYP3A37基因表達(dá)變化見圖3。其中，25和100 μmol·L-1PCP可引起LMH細(xì)胞內(nèi)CYP1A4表達(dá)量升高，CYP1A5在6.5～100 μmol·L-1PCP濃度下較對(duì)照組顯著升高，在100 μmol·L-1組升高6.27倍。而CYP1B1基因只在6.5 μmol·L-1PCP暴露組升高，CYP1C1只在25 μmol·L-1PCP暴露組升高。芳香烴受體(aryl hydrocarbon receptor, AHR)和芳香烴受體核轉(zhuǎn)運(yùn)蛋白(aryl hydrocarbon receptor nuclear translocator, ARNT)在1.56 μmol·L-1PCP濃度暴露下可被誘導(dǎo)。PCP濃度為0.4～100 μmol·L-1可引起細(xì)胞中SULT1B1和SULT1B1表達(dá)明顯下降(圖3)。

圖2 不同濃度PCP處理LMH細(xì)胞24 h后細(xì)胞活力變化Fig. 2 The viability of LMH cells treated with different concentration of PCP for 24 h

圖3 PCP處理LMH細(xì)胞24 h后，CYP450及SULT1B基因表達(dá)的變化Fig. 3 Effects of PCP on CYP450 and SULT1B mRNA expression in LMH cells

圖4 PCP處理LMH細(xì)胞24 h后，CYP450酶活性變化注：乙氧基異酚惡唑脫乙基酶(EROD)，甲氧基異酚惡唑脫甲基酶(MROD)，芐氧基試鹵靈-O-脫烷基化酶(BROD)， 戊氧基異酚惡唑脫甲基酶(PROD)，芐氧基三氟甲基香豆素脫烷基化酶(BFC)。Fig. 4 Effects of PCP on the CYP450 enzyme activities of the LMH cellsNote: ethoxyresorufin O-deethylation (EROD), methoxyresorufin O-demethylation (MROD), benzyloxyresorufin O-debenzylation (BROD), pentoxyresorufin O-depentylation (PROD), benzyloxy-trifluoromethyl-coumarin (BFC).

2.3 PCP處理的LMH細(xì)胞CYP450酶活性

LMH細(xì)胞暴露于PCP 24 h后，發(fā)現(xiàn)1.56、6.25、25、100 μmol·L-1PCP可增加細(xì)胞EROD、MROD、PROD和BFC活性，而并未引起LMH細(xì)胞BROD酶活性的變化(圖4)。其中，100 μmol·L-1PCP對(duì)LMH細(xì)胞中的EROD、MROD和PROD酶活性的促進(jìn)作用最顯著。

2.4 LMH細(xì)胞對(duì)PCP處理的氧化應(yīng)激響應(yīng)

LMH細(xì)胞暴露于PCP 24 h后，發(fā)現(xiàn)6.25、25、100 μmol·L-1PCP可誘導(dǎo)細(xì)胞ROS升高，在100 μmol·L-1PCP作用下，ROS可升高23%。GSH/GSSH在1.56～100 μmol·L-1PCP作用下顯著降低，其中100 μmol·L-1PCP可使GSH/GSSH比值下降45%，同時(shí)，1.56～100 μmol·L-1PCP可以增加LMH細(xì)胞內(nèi)MDA的含量(圖5)。

3 討論(Discussion)

本研究中PCP對(duì)雞肝癌細(xì)胞(LMH)活性影響表現(xiàn)為低濃度0.4～25 μmol·L-1PCP對(duì)細(xì)胞活性無(wú)影響，100 μmol·L-1PCP使LMH細(xì)胞增殖，高濃度PCP明顯抑制LMH生長(zhǎng)。AML12小鼠正常肝細(xì)胞經(jīng)PCP染毒后，MTT結(jié)果也顯示0～3.87 μg·mL-1PCP促進(jìn)AML12細(xì)胞增殖，7.75～31.0 μg·mL-1PCP對(duì)AML12細(xì)胞出現(xiàn)明顯抑制效應(yīng)[18]。已有研究結(jié)果表明PCP暴露對(duì)不同類型細(xì)胞的生長(zhǎng)影響表現(xiàn)出差異性[19]。本研究中PCP對(duì)LMH細(xì)胞24 h的半數(shù)效應(yīng)濃度24 h-EC50為427.52 μmol·L-1，而PCP對(duì)人肝癌細(xì)胞系HepG2(human liver carcinoma cell)細(xì)胞24 h-EC50為(23.0 ± 5.6) μg·L-1[20]，PCP對(duì)人宮頸癌細(xì)胞的24 h-EC50為66.59 mmol·L-1[21]，說(shuō)明與雞肝癌細(xì)胞相比，人肝癌細(xì)胞對(duì)PCP更敏感。

細(xì)胞色素P450(CYPs)是含有血紅素結(jié)合位點(diǎn)的蛋白家族。由于它們參與多種內(nèi)源和外源物質(zhì)的代謝，在生物體內(nèi)發(fā)揮重要的作用[22]。不同的藥物和其他外源化合物對(duì)生物體CYPs的表達(dá)和酶活性影響不同[23]。在眾多CYPs中，CYP1A家族是有機(jī)污染物轉(zhuǎn)化中發(fā)揮主要作用的亞族[24]。PCP可引起HepG2細(xì)胞CYP1A1表達(dá)升高[20]，在LMH細(xì)胞中也發(fā)現(xiàn)25 μmol·L-1和6.5 μmol·L-1PCP可分別誘導(dǎo)CYP1A4和CYP1A5的表達(dá)。CYP1A基因通過(guò)外源物質(zhì)與芳香烴受體(AHR)結(jié)合進(jìn)而使芳香烴受體核轉(zhuǎn)運(yùn)蛋白(ARNT)與特定的DNA識(shí)別位點(diǎn)結(jié)合而被誘導(dǎo)表達(dá)[25]。激活的AHR/ARNT復(fù)合物識(shí)別位位于基因啟動(dòng)子區(qū)的外源性反應(yīng)元件序列(XRE)，進(jìn)而調(diào)控下游基因表達(dá)，如CYP1A1和UGTA1酶等[26]。本研究中PCP可顯著誘導(dǎo)LMH細(xì)胞中AHR和ARNT的表達(dá)，進(jìn)而通過(guò)AHR通路誘導(dǎo)其調(diào)控基因CYP1A的表達(dá)。已有文獻(xiàn)表明PCP在生物體內(nèi)通過(guò)形成二聚物導(dǎo)致四氯二苯-p-二噁英(TCDD)生成，而TCDD可以結(jié)合AHR誘導(dǎo)CYP1A[27]。本研究中LMH細(xì)胞中CYP1B1和CYP1C1分別在6.25和25 μmol·L-1PCP作用下表達(dá)水平升高，說(shuō)明PCP是CYP1B1和CYP1C1的誘導(dǎo)劑。細(xì)胞色素P450單加氧酶3A4(CYP3A4)可代謝多種外源物質(zhì)，體外暴露實(shí)驗(yàn)表明PCP可誘導(dǎo)小鼠肝細(xì)胞CYP3A4表達(dá)[28]，在LMH細(xì)胞中也發(fā)現(xiàn)6.25～100 μmol·L-1PCP可誘導(dǎo)CYP3A4表達(dá)。由于CYP450對(duì)外源物質(zhì)有解毒作用，CYP450基因表達(dá)的改變可減輕有毒物質(zhì)發(fā)揮毒性作用[29]。

圖5 PCP處理LMH細(xì)胞24 h后其氧化應(yīng)激反應(yīng)的測(cè)定Fig. 5 Effects of PCP on the ROS of the LMH cells

EROD、MROD、BROD和PROD是重要的CYP450酶，在外源化學(xué)品解毒中發(fā)揮重要作用[30-31]。目前，對(duì)外源性物質(zhì)的代謝研究中，CYP450基因表達(dá)的誘導(dǎo)或抑制已經(jīng)成為生態(tài)毒理學(xué)和環(huán)境污染生物監(jiān)測(cè)的一個(gè)有效手段[32-34]。本研究中1.25 μmol·L-1PCP可使細(xì)胞EROD、MROD、PROD和BFC活性增加。這表明PCP可改變不同CYP450的酶活性，CYP450酶活性可成為PCP污染監(jiān)測(cè)的指標(biāo)之一。這些結(jié)果說(shuō)明雖然低濃度PCP未對(duì)細(xì)胞活性抑制，但細(xì)胞內(nèi)CYP450活性增加，低濃度PCP可通過(guò)激活CYP450酶活性而使細(xì)胞免受損傷。

生物體內(nèi)發(fā)生的硫酸化反應(yīng)與環(huán)境污染物、致癌物質(zhì)、激素等各種內(nèi)源性和外源性化合物的活化及解毒作用有關(guān)[35]。硫酸基轉(zhuǎn)移酶(SULTs)可催化從3′-磷酸腺苷-5′-磷酰硫酸(PAPS)轉(zhuǎn)移SO3-至親核受體底物而發(fā)揮生物轉(zhuǎn)化作用[36]。大量研究表明PCP是SULTs的抑制劑[37-39]，如小鼠SULT1C1活性可被PCP抑制[40]。SULT1B1與脂質(zhì)代謝有關(guān)，芯片的數(shù)據(jù)顯示，SULT1B1在生長(zhǎng)快速的雞肝組織中比生長(zhǎng)慢的雞肝臟組織中表達(dá)量高7倍[41]。已有數(shù)據(jù)顯示雞SULT1C1酶與小鼠SULT1C1結(jié)構(gòu)類似，表明鳥類及哺乳類硫酸基轉(zhuǎn)移酶在結(jié)構(gòu)和功能上是相似的[42]。本研究中發(fā)現(xiàn)0.4～100 μmol·L-1PCP暴露可引起LMH細(xì)胞SULT1B1和SULT1C1的表達(dá)降低，但在此濃度下細(xì)胞活性有增強(qiáng)，說(shuō)明PCP在細(xì)胞內(nèi)通過(guò)硫酸基轉(zhuǎn)移酶發(fā)生生物轉(zhuǎn)化，減少了PCP對(duì)細(xì)胞的毒性作用。

活性氧(ROS)通常是線粒體能量代謝的產(chǎn)物，與細(xì)胞生長(zhǎng)、細(xì)胞信號(hào)傳導(dǎo)和內(nèi)環(huán)境穩(wěn)定有關(guān)[43]。持續(xù)升高的ROS水平是腫瘤發(fā)生、腫瘤生長(zhǎng)和腫瘤轉(zhuǎn)移的特征現(xiàn)象[44]。LMH細(xì)胞經(jīng)低濃度PCP暴露后可引起細(xì)胞內(nèi)ROS升高，同時(shí)引起GSH/GSSG比值下降和MDA含量增加，說(shuō)明PCP也可引起細(xì)胞內(nèi)氧化應(yīng)激反應(yīng)，這與白腰文鳥體內(nèi)暴露實(shí)驗(yàn)的結(jié)果一致[45]。

本論文中雞肝癌細(xì)胞(LMH)經(jīng)6.25～100 μmol·L-1PCP染毒后細(xì)胞內(nèi)ROS及MDA含量升高，進(jìn)而造成機(jī)體發(fā)生氧化應(yīng)激反應(yīng)，同時(shí)PCP影響CYP450基因及激活A(yù)HR1表達(dá)，表明PCP可通過(guò)AHR1與ARNT1通路作用于CYP1A基因。此外，PCP在LMH細(xì)胞內(nèi)還參與硫酸基轉(zhuǎn)移酶的生物轉(zhuǎn)化作用。用離體培養(yǎng)的細(xì)胞來(lái)評(píng)價(jià)環(huán)境中化學(xué)物質(zhì)的毒性作用，是近年來(lái)生物和環(huán)境相關(guān)領(lǐng)域研究的熱點(diǎn)之一。本文從細(xì)胞、蛋白質(zhì)和基因水平上分析了PCP對(duì)細(xì)胞的毒性作用，對(duì)PCP環(huán)境風(fēng)險(xiǎn)評(píng)價(jià)具有重要意義。

[1] Crosby D G. IUPAC reports on pesticides. 14. Environmental chemistry of pentachlorophenol [J]. Pure and Applied Chemistry, 1981, 53: 1051-1080

[2] Okeke B C, Paterson A, Smith J E, et al. Comparative biotransformation of pentachlorophenol in soils by solid substrate cultures of Lentinula edodes [J]. Applied Microbiology and Biotechnology, 1997, 48: 563-569

[3] Zheng W W, Yu H, Wang X, et al. Systematic review of pentachlorophenol occurrence in the environment and in humans in China: Not a negligible health risk due to the re-emergence of schistosomiasis [J]. Environment International, 2012, 42: 105-116

[4] Jorens P G, Schepens P J C. Human pentachlorophenol poisoning [J]. Human & Experimental Toxicology, 1993, 12:479-495

[5] Law W M, Lau W N, Lo K L, et al. Removal of biocide pentachlorophenol in water system by the spent mushroom compost of Pleurotus pulmonarius [J]. Chemosphere, 2003, 52: 1531-1537

[6] Reigner B G, Bois F Y, Tozer T N. Assessment of pentachlorophenol exposure in humans using the clearance concept [J]. Human & Experimental Toxicology, 1992, 11: 17-26

[7] Blakley B R, Yole M J, Brousseau P, et al. Effect of pentachlorophenol on immune function [J]. Toxicology,1998, 125: 141-148

[8] Chhabra R S, Maronpot R M, Bucher J R, et al. Toxicology and carcinogenesis studies of pentachlorophenol in rats [J]. Toxicological Sciences, 1999, 48: 14-20

[9] Roberts H J. Pentachlorophenol-associated aplastic anemia, red cell aplasia, leukemia and other blood disorders [J]. The Journal of the Florida Medical Association, 1990, 77:86-90

[10] Umemura T, Kai S, Hasegawa R, et al. Pentachlorophenol (PCP) produces liver oxidative stress and promotes but does not initiate hepatocarcinogenesis in B6C3F1 mice [J]. Carcinogenesis, 1999, 20: 1115-1120

[11] Zhu B Z,Shan G Q. Potential mechanism for pentachlorophenol-induced carcinogenicity: A novel mechanism for metal-independent production of hydroxyl radicals [J]. Chemical Research in Toxicology, 2009, 22: 969-977

[12] Shan G Q, Ye M Q, Zhu B Z, et al. Enhanced cytotoxicity of pentachlorophenol by perfluorooctane sulfonate or perfluorooctanoic acid in HepG2 cells [J]. Chemosphere, 2013, 93: 2101-2107

[13] Westerink W M A, Schoonen W G E J. Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells [J]. Toxicology in Vitro, 2007, 21: 1581-1591

[14] Dong H, Xu D M, Hu L H, et al. Evaluation of N-acetyl-cysteine against tetrachlorobenzoquinone-induced genotoxicity and oxidative stress in HepG2 cells [J]. Food and Chemical Toxicology, 2014, 64: 291-297

[15] Nguyen T N T, Bertagnolli A D, Villalta P W, et al. Characterization of a deoxyguanosine adduct of tetrachlorobenzoquinone: Dichlorobenzoquinone-1,N-2-etheno-2'-deoxyguanosine [J]. Chemical Research in Toxicology,2005, 18: 1770-1776

[16] 孫毓鑫. 鳥類作為指示生物監(jiān)測(cè)陸生環(huán)境中鹵代有機(jī)污染物的研究[D]. 北京: 中國(guó)科學(xué)院研究生院, 2012

[17] Hu J Y, Li J, Wang J S, et al. Synergistic effects of perfluoroalkyl acids mixtures with J-shaped concentration-responses on viability of a human liver cell line [J]. Chemosphere, 2014, 96: 81-88

[18] Dorsey W C, Tchounwou P B, Ford B D. Neuregulin 1-beta cytoprotective role in AML 12 mouse hepatocytes exposed to pentachlorophenol [J]. International Journal of Environmental Research and Public Health, 2006, 3: 11-22

[19] Ling B, Gao B, Yang J. Evaluating the effects of tetrachloro-1,4-benzoquinone, an active metabolite of pentachlorophenol, on the growth of human breast cancer cells [J]. Journal of Toxicology, 2016(5): 1-8

[20] Dorsey W C,Tchounwou P B. CYP1a1, HSP70, P53, and c-fos expression in human liver carcinoma cells (HepG2) exposed to pentachlorophenol [J]. Biomedical Sciences Instrumentation, 2003, 39: 389-396

[21] 金幫明, 王輔明, 熊力, 等. 五氯酚對(duì)Hela細(xì)胞毒性及DNA損傷的研究[J]. 環(huán)境科學(xué), 2012, 33(2): 658-664

Jin B M, Wang F M, Xiong L, et al. Effects of pentachlorophenol on DNA damage and cytotoxicity of Hela cells [J]. Environmental Science, 2012, 33(2): 658-664 (in Chinese)

[22] Ibrahim Z S. Chenodeoxycholic acid increases the induction of CYP1A1 in HepG2 and H4IIE cells [J]. Experimental and Therapeutic Medicine, 2015, 10(5): 1976-1982

[23] Pelkonen O, Turpeinen M, Hakkola J, et al. Inhibition and induction of human cytochrome P450 enzymes: Current status [J]. Archives of Toxicology, 2008, 82: 667-715

[24] He X T, Nie X P, Wang Z H, et al. Assessment of typical pollutants in waterborne by combining active biomonitoring and integrated biomarkers response [J]. Chemosphere,2011, 84: 1422-1431

[25] Marshall N B, Kerkvliet N I. Dioxin and immune regulation: Emerging role of aryl hydrocarbon receptor in the generation of regulatory T cells [J]. Annals of the New York Academy of Sciences, 2010, 1183: 25-37

[26] Sorg O. AhR signalling and dioxin toxicity [J]. Toxicology Letters, 2014, 230: 225-233

[27] Jung D K, Klaus T, Fent K. Cytochrome P450 induction by nitrated polycyclic aromatic hydrocarbons, azaarenes, and binary mixtures in fish hepatoma cell line PLHC-1 [J]. Environmental Toxicology and Chemistry, 2001, 20: 149-159

[28] Thummel K E,Wilkinson G R. In vitro and in vivo drug interactions involving human CYP3A [J]. Annual Review of Pharmacology and Toxicology, 1998, 38: 389-430

[29] Mortensen A S, Arukwe A. Modulation of xenobiotic biotransformation system and hormonal responses in Atlantic salmon (Salmo salar) after exposure to tributyltin (TBT) [J]. Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP, 2007, 145: 431-441

[30] Hirakawa S, Iwata H, Takeshita Y, et al. Molecular characterization of cytochrome P450 1A1, 1A2, and 1B1, and effects of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners on their hepatic expression in Baikal seal (Pusa sibirica) [J]. Toxicological Sciences, 2007, 97: 318-335

[31] Kubota A, Watanabe M X, Kim E Y, et al. Accumulation of dioxins and induction of cytochrome P450 1A4/1A5 enzyme activities in common cormorants from Lake Biwa, Japan: Temporal trends and validation of national regulation on dioxins emission [J]. Environmental Pollution, 2012, 168: 131-137

[32] Esler D, Ballachey B E, Trust K A,et al. Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow's goldeneyes to residual Exxon Valdez oil [J]. Marine Pollution Bulletin, 2011, 62: 609-614

[33] Bigorgne E, Custer T W, Dummer P M, et al. Chromosomal damage and EROD induction in tree swallows (Tachycineta bicolor) along the Upper Mississippi River, Minnesota, USA [J]. Ecotoxicology, 2015, 24: 1028-1039

[34] Iwata H, Nagahama N, Kim E Y, et al. Effects of in ovo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic AHR/ARNT-CYP1A signaling pathways in common cormorants (Phalacrocorax carbo) [J]. Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP, 2010, 152: 224-231

[35] Honma W, Kamiyama Y, Yoshinari K, et al. Enzymatic characterization and interspecies difference of phenol sulfotransferases, ST1A forms [J]. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 2001, 29:274-281

[36] Meloche C A, Falany C N. Expression and characterization of the human 3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b) [J]. The Journal of Steroid Biochemistry and Molecular Biology, 2001, 77: 261-269

[37] Mulder G J, Scholtens E. Phenol sulphotransferase and uridine diphosphate glucuronyltransferase from rat liver in vivo and vitro. 2,6-Dichloro-4-nitrophenol as selective inhibitor of sulphation [J]. The Biochemical Journal, 1977, 165: 553-559

[38] Koster H, Scholtens E, Mulder G J. Inhibition of sulfation of phenols in vivo by 2,6-dichloro-4-nitrophenol: Selectivity of its action in relation to other conjugations in the rat in vivo [J]. Medical Biology, 1979, 57: 340-344

[39] Wang L Q, James M O. Inhibition of sulfotransferases by xenobiotics [J]. Current Drug Metabolism, 2006, 7:83-104

[40] Nagata K, Ozawa S, Miyata M, et al. Isolation and expression of a cDNA-encoding a male-specific rat sulfotransferase that catalyzes activation of N-hydroxy-2-acetylaminofluorene [J]. Journal of Biological Chemistry,1993, 268: 24720-24725

[41] D'Andre H C, Paul W, Shen X, et al. Identification and characterization of genes that control fat deposition in chickens [J]. Journal of Animal Science and Biotechnology, 2013, 4(1): 1-16

[42] Wilson L A, Reyns G E, Darras V M, et al. cDNA cloning, functional expression, and characterization of chicken sulfotransferases belonging to the SULT1B and SULT1C families [J]. Archives of Biochemistry and Biophysics, 2004, 428: 64-72

[43] Landry W D, Cotter T G. ROS signalling, NADPH oxidases and cancer [J]. Biochemical Society Transactions, 2014, 42: 934-938

[44] Chen E I. Mitochondrial dysfunction and cancer metastasis [J]. Journal of Bioenergetics and Biomembranes, 2012, 44: 619-622

[45] Jiang P, Wang J, Zhang J, et al. Effects of pentachlorophenol on the detoxification system in white-rumped munia (Lonchura striata) [J]. Journal of Environmental Sciences,2016, 44: 224-234

◆

TheToxicityandMechanismofPentachlorophenolExposureonChickenHepatomaCells

Jiang Peng, Sheng Nan, Wang Jianshe, Dai Jiayin*

Institute of Zoology, Chinese Academy of Sciences, Key Laboratory of Animal Ecology and Conservation Biology of Chinese Academy of Sciences, Beijing 100101, China

2016-12-12錄用日期2017-2-10

1673-5897(2017)3-373-09

X171.5

A

戴家銀(1965—)，男，博士，研究員，主要從事持久性污染物的生態(tài)毒理學(xué)研究工作，發(fā)表學(xué)術(shù)論文100余篇。

廣東省自然科學(xué)基金(2015A030312005)

蔣鵬(1981-)，女，博士，研究方向?yàn)樯鷳B(tài)毒理學(xué)，E-mail: jiangpeng169@163.com；

*通訊作者(Corresponding author)， E-mail: daijy@ioz.ac.cn

10.7524/AJE.1673-5897.20161212001

蔣鵬, 盛南, 王建設(shè), 等. 五氯酚(PCP)對(duì)雞肝癌細(xì)胞(LMH)毒性效應(yīng)的機(jī)制研究[J]. 生態(tài)毒理學(xué)報(bào)，2017, 12(3): 373-381

Jiang P, Sheng N, Wang J S, et al. The toxicity and mechanism of pentachlorophenol exposure on chicken hepatoma cells [J]. Asian Journal of Ecotoxicology, 2017, 12(3): 373-381 (in Chinese)