污染場(chǎng)地土壤重金屬的生物可給性及毒性研究

馬嬌陽,田 穩(wěn),王 坤,保欣晨,汪 潔,崔道雷,向 萍

污染場(chǎng)地土壤重金屬的生物可給性及毒性研究

馬嬌陽,田 穩(wěn),王 坤,保欣晨,汪 潔,崔道雷,向 萍*

(西南林業(yè)大學(xué)生態(tài)與環(huán)境學(xué)院/環(huán)境修復(fù)與健康研究院,云南 昆明 650224)

近年來,生物可給性被用于評(píng)估場(chǎng)地土壤污染健康風(fēng)險(xiǎn),然而不同場(chǎng)地類型重金屬生物可給性差別巨大,生物可給態(tài)重金屬的人體健康危害效應(yīng)仍然鮮見報(bào)道.本研究以浙江溫嶺某電子拆解廠為研究區(qū),分析比較了5個(gè)場(chǎng)地土壤(S1-S5)中Zn、Cu、Cd、Pb的生物可給性并探究生物可給態(tài)重金屬對(duì)人小腸上皮細(xì)胞的毒性效應(yīng)機(jī)制.結(jié)果表明,場(chǎng)地土壤Cd和Cu污染較為嚴(yán)重,含量分別為4.84,438.52mg/kg. 4種重金屬在胃階段生物可給性范圍分別為2.10%～48.28%、4.84%～33.73%、16.04%～42.81%、1.81%～15.71%,小腸階段為2.05%～36.91%、13.17%～22.23%、10.19%～23.10%、0.60%～2.69%,可見胃階段的生物可給性低于小腸階段.對(duì)于腸相生物可給態(tài)重金屬暴露人體腸道上皮細(xì)胞后,除樣點(diǎn)S4外,細(xì)胞活力均顯著性下降.此外,樣點(diǎn)S3和S5土壤提取液對(duì)超氧化物歧化酶活力影響較小,但顯著抑制過氧化氫酶活力,并且該樣點(diǎn)對(duì)DNA產(chǎn)生損傷.通過研究電子拆解廠土壤生物可給性以及其毒性效應(yīng),以為我國(guó)場(chǎng)地土壤重金屬健康風(fēng)險(xiǎn)評(píng)估提供科學(xué)依據(jù).

電子拆解廠；土壤；生物可給性；Caco-2；DNA損傷

隨著人類對(duì)電子產(chǎn)品的需求增加以及其更新?lián)Q代的速度,電子垃圾的產(chǎn)生已經(jīng)成為一個(gè)嚴(yán)重的環(huán)境問題.根據(jù)全球電子垃圾檢測(cè)報(bào)告,預(yù)計(jì)2021年全球?qū)?huì)產(chǎn)生5.2×1010kg電子垃圾,但只有20%的電子垃圾以正規(guī)渠道回收.然而非正規(guī)的電子垃圾拆解和回收是不安全的、不受監(jiān)管的[1],此外,在電子垃圾拆解和回收過程中,電子產(chǎn)品中的重金屬、有機(jī)污染物等一些有毒有害物質(zhì)將會(huì)釋放到環(huán)境中[2],進(jìn)而對(duì)空氣、水以及土壤造成污染[3],甚至對(duì)人體產(chǎn)生毒害效應(yīng)[4].

土壤中的重金屬可能通過吸入、攝入和皮膚接觸途徑而暴露于人體,其中,偶然經(jīng)口攝入土壤是主要方式之一[5].當(dāng)人體攝入土壤后,達(dá)到人體消化系統(tǒng),溶解在胃腸階段的重金屬量與土壤重金屬總量之比稱之為生物可給性.近年來,因體外方法省時(shí)、簡(jiǎn)單等優(yōu)勢(shì),越來越多研究者采用體外胃腸模擬法來測(cè)定生物可給性[6-8].Li等[7]測(cè)定不同類型土壤重金屬鎘的生物可給性為60.9%～99.4%, Amponsah等[9]測(cè)定國(guó)外電子拆解廠重金屬鉛、鎘和銅生物可給性分別為70.8%、64.1%、62.3%.目前土壤重金屬對(duì)人體的健康風(fēng)險(xiǎn)評(píng)估大多是基于其總量,但重金屬并沒有100%被人體吸收,因此基于該評(píng)估方法會(huì)高估其真實(shí)風(fēng)險(xiǎn).然而,對(duì)我國(guó)電子拆解廠土壤重金屬的生物可給性鮮有報(bào)道,因此,采用SBRC體外模擬方法測(cè)定浙江溫嶺某電子拆解廠土壤鋅(Zn)、銅(Cu)、鎘(Cd)和鉛(Pb)的生物可給性.

生物細(xì)胞活性氧(ROS)的積累會(huì)產(chǎn)生氧化損傷.有文獻(xiàn)報(bào)道,重金屬可能通過氧化損傷對(duì)人體產(chǎn)生危害效應(yīng)[10],而抗氧化酶系統(tǒng)對(duì)防御氧化應(yīng)激具有重要作用.當(dāng)體內(nèi)金屬離子平衡破壞引發(fā)氧化應(yīng)激后,并隨后可能產(chǎn)生DNA損傷等毒性效應(yīng)[10].而小腸作為人體的主要消化器官之一,當(dāng)土壤重金屬溶解于人體腸道后,是否對(duì)人體腸道細(xì)胞產(chǎn)生氧化損傷和DNA損傷鮮有報(bào)道.

本研究以浙江溫嶺某電子拆解廠為研究區(qū),通過SBRC體外模擬的方法測(cè)定土壤重金屬生物可給性,進(jìn)而基于小腸上皮細(xì)胞模型,通過細(xì)胞活力、抗氧化酶SOD和CAT酶活性以及DNA損傷等指標(biāo)探究土壤重金屬對(duì)人體的毒害效應(yīng).因此,弄清污染場(chǎng)地土壤重金屬的生物可給性及毒性效應(yīng),對(duì)于準(zhǔn)確評(píng)估其健康風(fēng)險(xiǎn)具有重要意義,對(duì)防控其人體健康危害提供重要數(shù)據(jù)支撐.

1 材料與方法

1.1 樣品采集與處理

以浙江溫嶺某電子拆解廠為研究區(qū),布設(shè)5個(gè)土壤采樣點(diǎn),并標(biāo)號(hào)S1～S5,每個(gè)樣點(diǎn)采集0～20cm的表層土,除去石塊、植物根系等雜物,將土壤在陰涼通風(fēng)處自然晾干,并過60目(250 μm)的尼龍篩以測(cè)定土壤重金屬總量以及生物可給性備用.

1.2 土壤物理化學(xué)性能表征

土壤pH值采用水提法(固液比為1:2.5),利用pH計(jì)(Starter3100)測(cè)定;使用0.5mol/L的鹽酸浸泡24h后,利用總有機(jī)碳分析儀(Vario)測(cè)定土壤總有機(jī)碳含量;粒徑分布利用激光粒度分析儀(Mastersizer 3000)測(cè)定;依據(jù)美國(guó)EPA的USEPA 3050B方法[11],土壤重金屬含量采用硝酸(HNO3)和過氧化氫(H2O2)于105℃的消解爐內(nèi)消解,利用5%硝酸定容,過0.45μm濾膜于4℃保存?zhèn)溆脺y(cè)定.重金屬Cd和Pb利用電感耦合等離子體質(zhì)譜(ICP-MS,ICAPRQ)測(cè)定,Zn和Cu利用原子吸收分光光度計(jì)(AA-6880)測(cè)定.

1.3 體外胃腸模擬

采用SBRC方法進(jìn)行體外消化,以測(cè)定胃腸階段土壤重金屬生物可給性.胃階段采用1:100的固液比加入胃模擬液,并于37℃恒溫和轉(zhuǎn)速150r/min的震蕩箱中震蕩1h,期間利用HCl使pH值穩(wěn)定控制在1.5.胃階段提取結(jié)束后,利用NaOH調(diào)節(jié)消化液pH值至7,并加入小腸模擬液,于上述相同條件下的震蕩箱中震蕩4h,期間利用HCl或者NaOH調(diào)整消化液pH值穩(wěn)定控制在7.胃腸階段分別消化完成后,離心并過0.45 μm濾膜于4℃保存?zhèn)溆脺y(cè)定.胃腸階段重金屬Cd和Pb利用ICP-MS測(cè)定,Zn和Cu利用AA測(cè)定.土壤中重金屬生物可給性由下述公式計(jì)算得出:

式中:BA為土壤中目標(biāo)重金屬的生物可給性,%;I為胃或小腸模擬液中對(duì)應(yīng)的重金屬濃度,mg/L;I為胃腸模擬液體積,L;S為土壤中重金屬總含量,mg/ kg;S為土壤質(zhì)量,kg.

1.4 Caco-2細(xì)胞毒性效應(yīng)

1.4.1 細(xì)胞培養(yǎng)、細(xì)胞形態(tài)和活力檢測(cè) 結(jié)腸上皮細(xì)胞Caco-2來自美國(guó)模式培養(yǎng)物寄存庫(ATCC),細(xì)胞生長(zhǎng)于含MEM(含NEAA)基礎(chǔ)培養(yǎng)基、10%血清和1%PS的完全培養(yǎng)基中,于37℃、5%CO2的二氧化碳培養(yǎng)箱中培養(yǎng).待細(xì)胞于培養(yǎng)皿生長(zhǎng)匯合到80%時(shí),可利用胰蛋白酶溶液消化傳代.

將1.3中不同土壤樣點(diǎn)提取的小腸模擬液于95℃的水浴鍋中加熱5min,以使相關(guān)消化酶變性,并過0.22μm濾膜以備用,細(xì)胞暴露液以1:3的比例將小腸模擬液和MEM基礎(chǔ)培養(yǎng)基配置[12].為研究小腸模擬液中生物可給態(tài)重金屬對(duì)Caco-2細(xì)胞形態(tài)以及活力的變化,將細(xì)胞以1í104個(gè)cells/孔/100μL的密度接種于96孔板中,待細(xì)胞生長(zhǎng)24h后,將孔內(nèi)完全培養(yǎng)基吸棄,加入不同土壤樣點(diǎn)的細(xì)胞暴露液并在CO2培養(yǎng)箱中孵育24h,之后,利用倒置顯微鏡(尼康TS-100)觀察細(xì)胞形態(tài)并拍照.隨后加入10μLCCK-8溶液并于CO2培養(yǎng)箱中孵育2h,用酶標(biāo)儀(SpectraMax Plus 384)在波長(zhǎng)450nm的條件下檢測(cè)每孔的OD值,以研究Caco-2細(xì)胞活力變化.

1.4.2 抗氧化酶活力測(cè)定 為了測(cè)定細(xì)胞超氧化物歧化酶(SOD)和過氧化氫酶(CAT)酶活力,將Caco-2細(xì)胞以5í105個(gè)cells/孔/1.5mL的密度接種于12孔板中,細(xì)胞過夜培養(yǎng)后,加入不同土壤樣點(diǎn)細(xì)胞暴露液孵育24h后,吸棄細(xì)胞暴露液,用預(yù)冷的PBS緩沖液洗滌Caco-2細(xì)胞,隨后利用細(xì)胞裂解液裂解細(xì)胞并于4℃,12000的條件下離心5min,取上清液作為待測(cè)樣品.按總SOD活性檢測(cè)試劑盒(WST-8法)和過氧化氫酶檢測(cè)試劑盒(碧云天生物技術(shù))說明書進(jìn)行酶活力測(cè)定.

1.4.3 DNA損傷研究 采用免疫熒光方法研究土壤重金屬對(duì)人腸道細(xì)胞的DNA損傷情況,將Caco-2細(xì)胞以5í104個(gè)cells/孔/1mL的密度接種于24孔板中,并將土壤樣點(diǎn)S3和S5的暴露液暴露于Caco-2細(xì)胞24h,之后,在室溫的條件下,用4%的多聚甲醛固定細(xì)胞30min、10%的Triton X-100透化15min和1%BSA封閉60min,并加入γH2A.X單克隆抗體,在4℃孵育過夜后,在室溫復(fù)溫1h,并加入山羊抗兔FITC-IgG二抗(南京翼飛雪生物科技有限公司)在室溫避光孵育1h.滴加DAPI染色液避光孵育10min以將細(xì)胞核染色,使用倒置熒光顯微鏡(OLYMPUS IX37)對(duì)細(xì)胞進(jìn)行觀察,其中綠色熒光為DNA損傷陽性,藍(lán)色為細(xì)胞核.

1.5 數(shù)據(jù)統(tǒng)計(jì)分析

實(shí)驗(yàn)數(shù)據(jù)以平均值±標(biāo)準(zhǔn)差表示,采用Microsoft Office Excel 2010進(jìn)行數(shù)據(jù)計(jì)算,GraphPad Prism 8進(jìn)行作圖以及顯著性差異分析,認(rèn)為<0.05有顯著性差異.

2 結(jié)果與討論

2.1 土壤性狀及重金屬污染特征

浙江溫嶺某電子拆解廠土壤基本性狀以及重金屬含量如表1所示.該場(chǎng)地土壤pH值平均值為6.02,除樣點(diǎn)S2呈堿性外,其他土壤樣點(diǎn)均呈酸性.有機(jī)碳范圍為1.18%～1.39%,粒徑分布除S2外都以粉粒(51.78%～73.01%)為主.重金屬Zn、Cu、Cd和Pb的含量平均值分別為491.43,438.52,4.84,133.93mg/ kg,該場(chǎng)地土壤4種重金都已超出浙江省土壤背景值[13],且重金屬含量平均值分別超出背景值6.96倍、24.9倍、69.2倍、5.65倍,其中Cd的超標(biāo)最為嚴(yán)重,其次為Cu.依據(jù)土壤環(huán)境質(zhì)量農(nóng)用地土壤污染風(fēng)險(xiǎn)管控標(biāo)準(zhǔn)(試行)[14],樣點(diǎn)S3、S4的Zn以及S1、S2的Pb未超標(biāo),Cu和Cd五個(gè)樣點(diǎn)均超出該風(fēng)險(xiǎn)篩選值.同樣,趙等[15]發(fā)現(xiàn)該地電子拆解區(qū)土壤重金屬對(duì)當(dāng)?shù)厣鷳B(tài)以及人類可能存在健康風(fēng)險(xiǎn),其中Cu和Cd污染較嚴(yán)重,與本研究結(jié)果較為一致.因此,該場(chǎng)地土壤存在的重金屬污染風(fēng)險(xiǎn)可能受到電子拆解活動(dòng)的影響.

表1 土壤基本性質(zhì)及重金屬含量

2.2 土壤重金屬生物可給性

土壤重金屬在胃腸階段的生物可給性如圖1,不同土壤樣點(diǎn)胃腸階段4種重金屬生物可給性差異明顯,Zn、Cu、Cd、Pb在胃階段生物可給性范圍分別為2.10%～48.28%、4.84%～33.73%、16.04%～42.81%、1.81%～15.71%,小腸階段為2.05%～36.91%、13.17%～ 22.23%、10.19%～23.10%、0.60%～2.69%.除樣點(diǎn)S5的Zn和S3的Cu,胃階段生物可給性均高于小腸階段,反應(yīng)出土壤重金屬從胃相到腸相是一個(gè)逐步消化吸收的過程[16].從胃階段進(jìn)入小腸階段,pH值從1.5調(diào)至7.0,重金屬在酸性條件下,其酶活性增加而使得易從土壤中釋放出,從而胃階段生物可給性較高[17],而進(jìn)入小腸階段,隨pH值的升高,重金屬易發(fā)生沉淀使得釋放出的重金屬再次被固定鈍化[18],使其小腸階段的生物可給性降低.而樣點(diǎn)S5的Zn胃階段生物可給性高于小腸階段的,但兩階段生物可給性并無顯著性差異;樣點(diǎn)S3的Cu胃階段生物可給性明顯低于小腸階段,可能與該樣點(diǎn)土壤粒徑分布有關(guān),其黏粒與粉粒所占比重最大(74.2%),有研究表明在土壤顆粒較細(xì)的情況下,Cu的生物可給性較高[19],因此在小腸階段較細(xì)的土壤顆粒中的Cu可能易被溶解,從而增加生物可給性.該結(jié)果與Cao等[20]研究類似,他們發(fā)現(xiàn)利用體外模擬方法研究電子拆解廠土壤Cu的生物可給性,胃階段進(jìn)入小腸階段后,生物可給性從1.3%～57.5%提高到7%～100%.

以上4種重金屬的生物可給性均小于100%,由此說明并不是土壤全部重金屬可溶解在人體消化系統(tǒng)中,基于重金屬總量進(jìn)行的評(píng)估可能會(huì)高估其健康風(fēng)險(xiǎn)[21].在胃階段,土壤重金屬生物可給性平均值呈現(xiàn)出Cd(32.09%) > Zn(22.76%) > Cu(19.93%) > Pb(7.36%)的趨勢(shì);在小腸階段,呈現(xiàn)出Cu(17.05%) > Cd(16.88%) > Zn(11.88) > Pb(1.35%)的趨勢(shì).因此,該場(chǎng)地土壤Cd和Cu的生物可給性相對(duì)較高,與其總量污染較為嚴(yán)重相吻合,而Pb的生物可給性最低,與國(guó)外電子拆解廠相反,其胃階段Pb生物可給性可達(dá)49.0%～90.2%[9],可能與其場(chǎng)地所釋放的Pb氧化物較多有關(guān),而使得其生物可給性較高.此外,與其他研究區(qū)相比,不同污染場(chǎng)地土壤重金屬生物可給性差異較大,林等[22]所測(cè)定的農(nóng)田土壤的生物可給性均比本研究高,這也說明不同類型土壤中重金屬賦存的形態(tài)不同.

圖1 土壤重金屬胃腸階段生物可給性

2.3 土壤腸相提取液對(duì)小腸上皮細(xì)胞活力和形態(tài)的影響

腸上皮細(xì)胞可維持人體腸上皮功能,并保持其持續(xù)更新能力以維持組織穩(wěn)態(tài),但是腸上皮細(xì)胞死亡會(huì)導(dǎo)致腸通透性增加和腸屏障功能障礙[23].因此,利用小腸上皮細(xì)胞以研究該場(chǎng)地土壤重金屬對(duì)其毒性效應(yīng).

細(xì)胞活力是評(píng)價(jià)細(xì)胞對(duì)污染物所反映出的毒性效應(yīng)的一個(gè)重要指標(biāo)[24],將制備的不同土壤樣點(diǎn)的暴露液暴露于Caco-2細(xì)胞以測(cè)定其細(xì)胞活力變化,暴露液的重金屬濃度如表2.Caco-2細(xì)胞暴露于不同土壤樣點(diǎn)的細(xì)胞活力差異性明顯(圖2A).樣點(diǎn)S4細(xì)胞存活率下降<5%,與空白對(duì)照組無顯著性差異,其余土壤樣點(diǎn)都可抑制Caco-2細(xì)胞活力,其中樣點(diǎn)S5抑制細(xì)胞活力最為顯著,細(xì)胞活力下降至74.48%.可見,腸相生物可給態(tài)重金屬對(duì)細(xì)胞活力有顯著性影響.不同重金屬對(duì)細(xì)胞活力的影響有所差別,重金屬Zn在低濃度對(duì)人骨肉瘤細(xì)胞U-2OS有促進(jìn)作用,但濃度>60μmol后,Zn對(duì)細(xì)胞活力有顯著性抑制作用[25],同樣,Zn濃度達(dá)到50μmol后,使人乳腺癌細(xì)胞MDAMB231的細(xì)胞活力下降到～80%[26]. Caco-2細(xì)胞對(duì)Cu的半抑制濃度為300μmol,而人肝細(xì)胞Hep-G2和胃細(xì)胞SGC-7901對(duì)Cu的IL50分別為700μmol和168μmol[27-28],表明Caco-2細(xì)胞比人肝細(xì)胞對(duì)Cu更為敏感,而比人胃細(xì)胞更有耐性.重金屬Cd暴露于人肺細(xì)胞A594 24h后,濃度為20nmol～2μmol時(shí),細(xì)胞活力降低<10%,與對(duì)照組顯著性差異較小(<0.05),但濃度達(dá)到10μmol時(shí),細(xì)胞活力差異性顯著增強(qiáng)(<0.001)[29].相似地,重金屬Pb隨著濃度的增加對(duì)細(xì)胞活力有所影響,當(dāng)Pb濃度<25μmol時(shí),對(duì)小鼠海馬神經(jīng)元細(xì)胞HT-22無顯著性影響,而當(dāng)濃度提高到50～100μmol后,細(xì)胞活力明顯下降[30].可見,不同重金屬對(duì)不同類型的細(xì)胞活力影響有所差別,也表明不同細(xì)胞類型可能通過不同機(jī)制途徑抵御重金屬的外部脅迫.然而,土壤成分復(fù)雜,盡管本研究細(xì)胞暴露液中的重金屬均低于以上研究的濃度,但土壤中多種重金屬共存往往產(chǎn)生協(xié)同效應(yīng),導(dǎo)致毒性顯著增強(qiáng),研究[27]證實(shí)Cd和Cu復(fù)合產(chǎn)生的毒性效應(yīng)較Cd或Cu暴露強(qiáng)數(shù)倍.可見,當(dāng)該場(chǎng)地土壤進(jìn)入人體腸道后,可能會(huì)損壞腸上皮細(xì)胞進(jìn)而產(chǎn)生毒害效應(yīng).

細(xì)胞形態(tài)也是評(píng)價(jià)外部污染物對(duì)細(xì)胞毒性的有效指標(biāo).其中3個(gè)土壤樣點(diǎn)細(xì)胞暴露液對(duì)Caco-2細(xì)胞形態(tài)的影響如圖2C、D、E,其變化與細(xì)胞活力變化相一致.空白對(duì)照組(圖2B: CK)的Caco-2細(xì)胞形態(tài)大多呈鵝卵石不規(guī)則的圓形,樣點(diǎn)S4(圖2D)所暴露的細(xì)胞形態(tài)與CK組相似,而樣點(diǎn)S3(圖2C)和S5(圖2E)暴露的細(xì)胞形態(tài)發(fā)生改變,由圓形形狀變成不規(guī)則多邊形、松散的形狀,并且部分細(xì)胞發(fā)亮變圓以導(dǎo)致細(xì)胞死亡,進(jìn)而使得細(xì)胞活力也顯著降低,樣點(diǎn)S1和S2與S3無顯著性差異.

表2 不同土壤樣點(diǎn)細(xì)胞暴露液重金屬濃度

圖2 土壤小腸提取液對(duì)Caco-2細(xì)胞形態(tài)和活力的影響

圖B～圖E為細(xì)胞形態(tài)圖,放大倍數(shù)為200′;CK為空白腸液的對(duì)照組;不同字母表示具有顯著性差異(<0.05)

可見,腸相中生物可給態(tài)重金屬暴露使得細(xì)胞活力和形態(tài)發(fā)生明顯改變.我們對(duì)細(xì)胞活力改變程度與生物可給態(tài)重金屬含量進(jìn)行相關(guān)性分析(圖3),發(fā)現(xiàn)細(xì)胞活力降低與重金屬Zn無相關(guān)性(2=0.03) (圖3A),與Cu和Pb含量有相關(guān)性(2=0.13、0.54),但細(xì)胞活力隨Cu濃度的增加而增加,與細(xì)胞活力的增加呈正相關(guān)(圖3B),可見低濃度Cu對(duì)Caco-2細(xì)胞增殖有促進(jìn)作用,而重金屬Pb與細(xì)胞活力的增加呈負(fù)相關(guān)(圖3D),Pb濃度的增加對(duì)細(xì)胞起到抑制作用.重金屬Cd與細(xì)胞活力有較強(qiáng)的相關(guān)性(2=0.80),并呈負(fù)相關(guān)關(guān)系(圖3C).與Husejnovic等[31]研究相似,人皮膚細(xì)胞HaCaT細(xì)胞活力與重金屬Cd毒性呈正相關(guān)(2=0.56).此外,生物有效態(tài)的重金屬能夠改變小鼠腸道的形態(tài),并且可能破壞腸道屏障以紊亂消化系統(tǒng)[32].因此,通過重金屬濃度與Caco-2細(xì)胞活力相關(guān)性也可說明樣點(diǎn)S5所暴露的細(xì)胞活力最低可能是由于其小腸提取液中的生物可給態(tài)Cd和Pb濃度較高導(dǎo)致的.

圖3 不同土壤暴露液重金屬濃度與Caco-2細(xì)胞活力相關(guān)性

2.4 土壤腸相提取液改變小腸上皮細(xì)胞抗氧化酶活力

外界污染物重金屬的脅迫下,可能會(huì)增加細(xì)胞內(nèi)活性氧產(chǎn)生,進(jìn)而觸發(fā)抗氧化酶系統(tǒng)的作用以避免生物機(jī)體氧化損傷[33].其中,抗氧化酶SOD和CAT對(duì)維持細(xì)胞內(nèi)的穩(wěn)態(tài)至關(guān)重要[34].為了進(jìn)一步研究腸相生物可給態(tài)重金屬對(duì)人腸道細(xì)胞的毒性效應(yīng)機(jī)制,分析了暴露后Caco-2細(xì)胞內(nèi)抗氧化酶改變情況.

不同土壤樣點(diǎn)小腸提取液暴露于Caco-2細(xì)胞后,SOD和CAT酶活水平(U/mg 蛋白)如圖4.對(duì)照組的SOD酶活力為35.47U/mg 蛋白,與空白對(duì)照組相比,暴露組5個(gè)土壤樣點(diǎn)的酶活水平與其都無顯著性差異,為對(duì)照組的0.89～1.22倍;不同土壤樣點(diǎn)相比,S2和S5的SOD酶活力相對(duì)較高,且無顯著性差異,其余3個(gè)樣點(diǎn)酶活力相對(duì)較低.對(duì)照組與樣點(diǎn)S1、S2和S4的CAT酶活力無顯著性差異,但樣點(diǎn)S3和S5酶活力顯著降低.SOD可通過催化歧化反應(yīng)生成氧氣和過氧化氫,進(jìn)而通過CAT將過氧化氫轉(zhuǎn)化為水[35],從而達(dá)到清除活性氧的效果以防御機(jī)體受到氧化損傷.盡管土壤樣點(diǎn)S3和S5所暴露的Caco-2細(xì)胞SOD酶活力無明顯變化,但其CAT的酶活力顯著降低,因此細(xì)胞內(nèi)可能會(huì)有部分過氧化氫未被分解,進(jìn)而對(duì)人腸道產(chǎn)生氧化損傷.

圖4 土壤小腸提取液對(duì)Caco-2細(xì)胞SOD和CAT酶活力的影響

CK為空白腸液的對(duì)照組;不同字母表示具有顯著性差異(<0.05)

不同重金屬對(duì)SOD和CAT酶活力的影響有所差異,重金屬Cd在5μmol時(shí),對(duì)人胃細(xì)胞SGC-7901的SOD和CAT酶活力無顯著性差異,而Cu在10μmol和20μmol時(shí),SOD酶活力顯著增加,但CAT酶活力顯著降低[27].此外,重金屬Cd(0.5mg/L)也會(huì)使水生動(dòng)物腸道SOD和CAT酶活力發(fā)生改變[36],且Zn也可使水生動(dòng)物腸道中CAT酶活力下降[37],進(jìn)而引發(fā)氧化損傷.因此,樣點(diǎn)S3和S5所暴露的Caco-2細(xì)胞CAT酶活力顯著下降可能與其細(xì)胞暴露液中重金屬濃度有關(guān),多種金屬的聯(lián)合作用比單獨(dú)重金屬的毒性更大,進(jìn)而引發(fā)酶活力下降并誘導(dǎo)氧化損傷.

2.5 土壤腸相提取液誘導(dǎo)腸道細(xì)胞DNA損傷

基于上述細(xì)胞活力以及抗氧化酶活力結(jié)果,利用免疫熒光技術(shù)進(jìn)一步從分子層面分析了土壤樣點(diǎn)S3和S5暴露Caco-2后,其DNA損傷情況.DNA雙鏈斷裂是DNA損傷最嚴(yán)重的形式[38].H2AX 組蛋白是染色質(zhì)核小體組蛋白核心的成員之一,其在DNA加工、修復(fù)中起到重要作用[39],但當(dāng)DNA雙鏈斷裂時(shí),會(huì)促使DNA周邊的H2AX 組蛋白磷酸化,從而形成γH2A.X[40].因此,γH2A.X是DNA雙鏈斷裂的標(biāo)志物.利用倒置顯微鏡觀察Caco-2細(xì)胞暴露土壤S3和S5腸提取液后DNA損傷如圖5.圖5A、B、C為γH2A.X抗體熒光染色(綠色),圖5A′、B′、C′為DAPI細(xì)胞核熒光染色(藍(lán)色),圖5A″、B″、C″為兩種染色所疊加圖像.與空白對(duì)照CK組相比,土壤樣點(diǎn)S3和S5所暴露的Caco-2細(xì)胞中,γH2A.X陽性細(xì)胞(綠色)百分比有所增加,表明該場(chǎng)地土壤腸相生物可給態(tài)重金屬引發(fā)了明顯的DNA損傷.土壤腸相生物可給態(tài)重金屬中存在多種,但已有文獻(xiàn)表明重金屬Cu、Cd和Pb均能夠使人細(xì)胞引發(fā)DNA損傷[31,41],其是因?yàn)橥寥乐械闹亟饘倏赡苣軌蛞种艱NA合成或干擾DNA修復(fù)[42].因此,土壤樣點(diǎn)S3和S5的小腸提取液中的Cd和Pb的濃度較高,因而引發(fā)DNA損傷.相似地,Husejnovic等[31]所研究的Cd和Hg含量較高的土壤樣點(diǎn)對(duì)Caco-2細(xì)胞和人皮膚細(xì)胞HaCaT誘發(fā)DNA損傷.此外,這些數(shù)據(jù)也可表明,土壤中的多種重金屬的聯(lián)合作用產(chǎn)生的毒害效應(yīng)可能比單獨(dú)重金屬更強(qiáng).

圖5 Caco-2細(xì)胞暴露土壤S3和S5腸提取液后DNA損傷

圖放大倍數(shù)為400X

3 結(jié)論

3.1 浙江溫嶺某電子拆解廠土壤4種重金屬含量呈現(xiàn)出不同程度的超標(biāo)情況,Zn、Cu、Cd和Pb分別超出浙江省土壤背景值6.96倍、24.9倍、69.2倍、5.65倍.此外,Cu和Cd的含量均超出國(guó)家農(nóng)業(yè)土壤所規(guī)定的限值.

3.2 土壤中Cd和Cu生物可給性相對(duì)較高,胃腸階段的生物可給性分別為32.09%、16.88%(Cd)和19.93%、17.05%(Cu);而Pb的生物可給性較低(7.36%、1.35%).總體上,4種重金屬小腸階段的生物可給性低于胃階段.

3.3 不同土壤樣點(diǎn)提取的小腸提取液對(duì)Caco-2細(xì)胞活力變化有所差異,其中樣點(diǎn)S5使細(xì)胞活力下降最為顯著,此外,土壤樣點(diǎn)S3和S5所暴露的Caco-2細(xì)胞CAT酶活力顯著下降并產(chǎn)生DNA損傷,該樣點(diǎn)對(duì)人體腸道細(xì)胞產(chǎn)生毒性效應(yīng)可能與其含有較高的生物可給態(tài)重金屬有關(guān),為此該結(jié)果可為土壤重金屬對(duì)人體健康的影響提供數(shù)據(jù)支撐.

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Bioaccessibility and their toxic effects of heavy metal in field soils from an electronic disassembly plant.

MA Jiao-yang, TIAN Wen, WANG Kun, BAO Xin-chen, WANG Jie, CUI Dao-lei, XIANG Ping*

(Institute of Environment Remediation and Human Health, School of Ecology and Environment, Southwest Forestry University, Kunming 650224, China)., 2021,41(10)：4885～4893

Recently, bioaccessibility of metals has been applied for assessing the health risk of field soils, however the bioaccessibility of metals is varied and there are few studies on the toxic effects of bioaccessible metals on human body. In this study, fives soil samples (S1-S5) from an electronic disassembly plant in Wenling, Zhejiang Province were sampled. The total concentrations and bioaccessibility of Zn, Cu, Cd and Pb in the soils were determined. Moreover, the toxic effects of bioaccessible heavy metals on human intestinal epithelial cells were also evaluated. The results demonstrated that all samples were polluted by Cd, Pb, Zn, and Cu, with the greatest concentrations of Cd (4.84mg/kg) and Cu (438.52mg/kg). The bioaccessibility of the four heavy metals in the gastric and intestinal phase was 2.10%～48.28%, 4.84%～33.73%, 16.04%～42.81%, 1.81%～15.71%, and 2.05%～36.91%, 13.17%～22.23%, 10.19%～23.10%, 0.60%～2.69%, respectively. In general, the bioaccessibility of four heavy metals in the gastric phase was lower than that in the intestine phase. After exposure to intestinal bioaccessible extracts of different soils for 24h, cell viability significantly decreased except for sample S4. In addition, there was no significant change in SOD activity after treatment with S3 and S5samples, but CAT activity was decreased. Furthermore, DNA damage was trigged by those samples. To investigate the bioaccessibility and its toxic effects of fields soils, this study provides a scientific basis for the health risk assessment of soil in China.

electronic dismantling；field soils；bioaccessibility；Caco-2；DNA damage

X53

A

1000-6923(2021)10-4885-09

馬嬌陽(1998-),女,河北石家莊人,西南林業(yè)大學(xué)碩士研究生,主要從事環(huán)境健康研究.發(fā)表論文7篇.

2021-02-18

國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2018YFC1800504);云南省創(chuàng)新團(tuán)隊(duì)項(xiàng)目(202005AE160017);國(guó)家自然科學(xué)基金項(xiàng)目(41967026);國(guó)家林業(yè)和草原局林草科技創(chuàng)新青年拔尖人才項(xiàng)目(2020132613);云南省高層次人才引進(jìn)計(jì)劃青年人才項(xiàng)目(YNQR-QNRC-2018-049);云南省教育廳科學(xué)研究基金項(xiàng)目資助(2021Y237)

* 責(zé)任作者, 副研究員, ping_xiang@126.com