鯽魚(yú)對(duì)水中有機(jī)防曬劑的富集及代謝酶響應(yīng)

陸光華,李 晟,宗永臣,黃 毅(.西藏農(nóng)牧學(xué)院水利土木工程學(xué)院,西藏 林芝 860000；.河海大學(xué)環(huán)境學(xué)院淺水湖泊綜合治理與資源開(kāi)發(fā)教育部重點(diǎn)實(shí)驗(yàn)室,江蘇 南京 0098)

鯽魚(yú)對(duì)水中有機(jī)防曬劑的富集及代謝酶響應(yīng)

陸光華1*,李 晟2,宗永臣1,黃 毅1(1.西藏農(nóng)牧學(xué)院水利土木工程學(xué)院,西藏 林芝 860000；2.河海大學(xué)環(huán)境學(xué)院淺水湖泊綜合治理與資源開(kāi)發(fā)教育部重點(diǎn)實(shí)驗(yàn)室,江蘇 南京 210098)

采用半靜態(tài)水體暴露方法研究了有機(jī)防曬劑對(duì)二甲氨基苯甲酸異辛酯(OD-PABA)在鯽魚(yú)不同組織的分布、累積及生態(tài)毒理效應(yīng).結(jié)果表明,OD-PABA在不同組織中的含量隨著水體暴露濃度的升高而持續(xù)升高,并在14或28d達(dá)到最大.不同濃度的OD-PABA在不同組織的生物富集因子(BCF)存在明顯差異,基于暴露 14d和 28d計(jì)算得到的 BCF值,肝臟為 123～186,皮膚為 117～163,腎臟為90.2～147.OD-PABA對(duì)鯽魚(yú)肝臟EROD、PROD、BFCOD和GST活性都產(chǎn)生了誘導(dǎo)效應(yīng),其中EROD和GST的響應(yīng)更為顯著,最大誘導(dǎo)倍數(shù)分別為0.97和0.53倍.結(jié)果表明,水中的OD-PABA可在鯽魚(yú)體內(nèi)富集,并通過(guò)I相和II相代謝酶進(jìn)行生物轉(zhuǎn)化.

鯽魚(yú)；OD-PABA；組織分布；生物富集；代謝酶

有機(jī)防曬劑(Organic UV Filters, OUVFs)是具有羰基共軛或雜環(huán)的芳香族有機(jī)化學(xué)品,具有強(qiáng)烈地選擇性吸收紫外線的性能.OUVFs除了作為防曬成分用于個(gè)人護(hù)理品以外,還被廣泛添加到家居產(chǎn)品、紡織品、塑料、光學(xué)產(chǎn)品、農(nóng)業(yè)化學(xué)品和涂料等產(chǎn)品中,防止紫外線照射[1].近年來(lái),紫外線輻射的危害已經(jīng)引起人們高度關(guān)注, OUVFs的生產(chǎn)和使用呈現(xiàn)出激增的趨勢(shì),已經(jīng)成為一類(lèi)重要的新型污染物.

OUVFs可以通過(guò)水上娛樂(lè)活動(dòng)、涂料的雨水沖刷、生產(chǎn)廢水及生活污水排放等途徑進(jìn)入水環(huán)境[2],在水體、沉積物和生物體內(nèi)被廣泛檢出.Sang等在香港近海生物體內(nèi)檢出了7種有機(jī)防曬劑,其中甲氧基肉桂酸乙基己酯和對(duì)二甲氨基苯甲酸異辛酯(OD-PABA)是主要污染物[3]. Ma等在南京地表水中檢出8種有機(jī)防曬劑,其中二苯酮-3(BP3)具有高風(fēng)險(xiǎn)[4].研究表明,多種有機(jī)防曬劑對(duì)魚(yú)類(lèi)具有雌激素活性,并干擾其產(chǎn)卵和繁殖[5-6].中國(guó)、美國(guó)和歐盟規(guī)定化妝品中允許添加OD-PABA的最大濃度為8%.OD-PABA在自來(lái)水、地表水及污水中濃度范圍在2～110ng/L之間[4,7-9].因其疏水性強(qiáng),水環(huán)境中的 OD-PABA具有潛在的生物累積風(fēng)險(xiǎn)[10].目前,針對(duì)有機(jī)防曬劑的生物累積效應(yīng)研究很少,這些化合物進(jìn)入生物體內(nèi)可能對(duì)其生理功能產(chǎn)生負(fù)面影響,因此,這方面研究亟待加強(qiáng).

表1 OD-PABA的結(jié)構(gòu)式、理化參數(shù)及暴露濃度Table 1 Structural formula, physicochemical parameters and exposure concentrations of OD-PABA

為了探討OD-PABA對(duì)水生生物的影響,本文以廣泛分布的鯉科鯽魚(yú)為模式生物,采用半靜態(tài)水體暴露方法,研究水中OD-PABA在魚(yú)體內(nèi)的組織分布和累積規(guī)律,分析鯽魚(yú)肝臟代謝系統(tǒng)相關(guān)酶系的脅迫響應(yīng),本文可為今后深入研究和科學(xué)評(píng)價(jià)這類(lèi)新型污染物提供重要參考.

1 材料與方法

1.1 材料

OD-PABA購(gòu)自百靈威科技有限公司(上海,中國(guó)),結(jié)構(gòu)式和理化性質(zhì)見(jiàn)表 1.一齡鯽魚(yú)(Carassius auratus)購(gòu)自南京水產(chǎn)養(yǎng)殖研究所,體長(zhǎng)10±1.5cm,體重35±2.9g.實(shí)驗(yàn)開(kāi)始前將鯽魚(yú)馴化兩周,每天定時(shí)喂食搖蚊幼蟲(chóng).

1.2 水體暴露

根據(jù)OECD測(cè)試指南(OECD 305)進(jìn)行鯽魚(yú)暴露實(shí)驗(yàn).采用曝氣自來(lái)水稀釋OD-PABA的甲醇儲(chǔ)備液,制成OD-PABA試驗(yàn)溶液,加入50L的玻璃魚(yú)缸中,最后暴露溶液體積為 30L,甲醇濃度為0.01%(V/V).每個(gè)魚(yú)缸放入隨機(jī)選取的12條鯽魚(yú),采用半靜態(tài)的方式暴露 28d,每天更換一半試驗(yàn)溶液,暴露過(guò)程中喂食占魚(yú)重 3%的搖蚊幼蟲(chóng).根據(jù)文獻(xiàn)報(bào)道的 OD-PABA的半數(shù)效應(yīng)濃度(EC50=3000μg/L)[12],為了避免在富集實(shí)驗(yàn)過(guò)程中對(duì)魚(yú)產(chǎn)生毒性效應(yīng),OD-PABA的暴露濃度設(shè)置為 20,100,500μg/L,同時(shí)設(shè)置空白和溶劑對(duì)照組,每組設(shè)置3個(gè)平行.暴露期間,每天采集水樣分析OD-PABA的濃度.在第3,7,14d每個(gè)平行采集2條魚(yú),解剖并取肝臟,用于生物標(biāo)志物測(cè)定;在第7,14,28d每個(gè)平行采集2條魚(yú),解剖并取肝、腦、肌肉、皮、鰓、腎臟等組織,用于OD-PABA含量測(cè)定.

1.3 樣品預(yù)處理

水樣首先過(guò) 0.45μm的玻璃纖維濾膜濾,再采用Oasis HLB 固相萃取柱(200mg,6mL,Waters, Milford, USA)進(jìn)行萃取.萃取前,依次用 5mL二氯甲烷、5mL甲醇和5mL超純水對(duì)萃取柱進(jìn)行活化處理.隨后,將水樣以3～5mL/min流速通過(guò)萃取柱,再用10mL超純水淋洗后,繼續(xù)抽濾30min,以去除柱子中的水分.然后用 10mL二氯甲烷與甲醇等體積比混合液洗脫,洗脫液經(jīng)氮吹濃縮后,定容至1mL,在-20℃條件下儲(chǔ)存待測(cè).

不同組織樣品先用0.15mol/L的KCl沖洗,再用濾紙吸干,然后采用加速溶劑萃取儀(ASE 350,美國(guó) DIONEX公司)提取生物組織中的OD-PABA.首先取0.5～1g組織樣品,與硅藻土混勻后,裝入萃取池.萃取條件為 160℃,壓力1500psi,萃取溶劑為乙酸乙酯與正己烷混合液(4/1,V/V).萃取液定量濃縮至1mL以下,再用甲醇定容.然后在0℃以下以12000×g離心15min除去脂質(zhì),取上清液 0.5mL,保存于-20℃待測(cè).組織脂質(zhì)含量采用減差法測(cè)定[13],稱(chēng)取生物樣 5g,加速溶劑萃取后,定量濃縮至恒重,根據(jù)濃縮器皿加樣前及濃縮后的重量差,計(jì)算脂肪含量.

1.4 OD-PABA定量分析

采用正離子電噴霧電離(ESI+)為離子源的高效液相色譜與-質(zhì)譜聯(lián)用儀(LC/MS/MS, Agilent 1290/6460),色譜柱型號(hào)為 Eclipse Plus C18(2.1mm×30mm×1.7μm),柱溫 30℃,進(jìn)樣量10μL.采用多反應(yīng)離子監(jiān)測(cè)模式分析目標(biāo)化合物.水樣的檢出限(LOD)和定量限(LOQ)分別為 0.9和 4ng/L,方法回收率 88%～102%.生物樣品的LOD為0.2～0.4ng/L,LOQ為0.8～2ng/L,回收率為81%～107%.

1.5 生物標(biāo)志物測(cè)定

肝臟樣品用KCl溶液洗滌后,放入5mL離心管,按照1:9的質(zhì)量體積比加入Tris-HCl緩沖液(0.1mol/L Tris-HCl溶液,pH值7.4,0.15mol/L KCl)勻漿,勻漿液在9000×g下冷凍離心30min,取上清液作為粗酶液.EROD、PROD和BFCOD活性采用類(lèi)似的熒光法測(cè)定[14-15].在96孔板中加入Tris緩沖液、底物和粗酶液,加入 NADPH啟動(dòng)反應(yīng),10min后加入冰甲醇終止反應(yīng),測(cè)定產(chǎn)物的濃度.底物分別為7-乙氧基異吩唑酮、7-羥基吩噁唑嗪酮二戊醚和 7-羥基-4-三氧甲基香豆素,產(chǎn)物分別為試鹵靈、試鹵靈和7-羥基香豆素.采用酶標(biāo)儀(TECAN Infinite 200)測(cè)定產(chǎn)物的熒光強(qiáng)度(試鹵靈:激發(fā)波長(zhǎng) 530nm,發(fā)射波長(zhǎng) 590nm;7-羥基香豆素:激發(fā)波長(zhǎng) 410nm,發(fā)射波 538nm),酶活性單位為pmol/mg protein/min.

GST活性采用Frasco和Guilhermino的方法測(cè)定[16].將 100μL pH 為 6.5的磷酸緩沖溶液(0.1mM),10μL CDNB(1.0mM),10μL GSH(1.0mM)和880μL超純水混勻后,組成反應(yīng)體系.在96孔板中加入 170μL反應(yīng)體系和 10μL粗酶液.在340nm測(cè)定反應(yīng)產(chǎn)物2,4-二硝基苯-谷胱甘肽的生成速率,活性單位為nmol/mg protein/min.

肝組織蛋白質(zhì)含量測(cè)定采用改進(jìn)的Bradford方法進(jìn)行,以牛血清蛋白為標(biāo)準(zhǔn)物質(zhì)[17].

1.6 統(tǒng)計(jì)分析

采用Shapiro-Wilk和Levene方法對(duì)數(shù)據(jù)進(jìn)行正態(tài)性檢驗(yàn)和同方差檢驗(yàn).采用ANOVA分析方法中的 Dunnet’s檢驗(yàn)來(lái)比較不同組別數(shù)據(jù)之間的差異性,當(dāng)P＜0.05時(shí)表示兩組數(shù)據(jù)之間有顯著性差異.數(shù)據(jù)的統(tǒng)計(jì)分析均采用 SPSS 17.0軟件進(jìn)行處理.

2 結(jié)果與討論

2.1 鯽魚(yú)對(duì)OD-PABA的累積效應(yīng)

在28d的暴露期內(nèi),未發(fā)現(xiàn)鯽魚(yú)死亡或畸形的情況.空白和溶劑對(duì)照組的樣品均未檢出OD-PABA.不同處理組OD-PABA的實(shí)測(cè)濃度介于設(shè)定濃度的86.5%～92.4%之間(表1).鯽魚(yú)各組織脂肪含量分別為肝5.1%、腦9.8%、鰓1.8%、腎4.4%、皮6.5%、肌肉3.8%.鯽魚(yú)不同組織中OD-PABA的濃度隨暴露時(shí)間的變化見(jiàn)圖1.

從圖1可見(jiàn),OD-PABA在不同組織中的含量隨著暴露濃度的升高而持續(xù)升高.在低濃度暴露組(W1),OD-PABA在肝和皮中的累積量最高,鰓和腎累積量相對(duì)較低.不同組織累積量隨時(shí)間的變化趨勢(shì)不同,所有組織在前 14d的累積量都不斷增加,到第28d,腦的累積量繼續(xù)升高,肝、腎、鰓有所下降,而肌肉、皮膚變化不大.在W2組暴露28d后,腎的累積量最高,肝、腦和皮的累積量次之,鰓和肌肉的累積量相對(duì)較低.暴露14d各組織濃度較第7d都有明顯升高,暴露28d腦和腎組織的累積量繼續(xù)升高,其余組織累積量變化不大.在最高濃度組(W3)組,整個(gè)暴露期肝和腦組織中的OD-PABA含量持續(xù)升高,在第28d達(dá)到最大,而鰓、腎、皮和肌肉的累積在第 14d達(dá)到最大,之后變化不顯著.

對(duì)比不同暴露濃度,組織分布規(guī)律存在差異.但是,對(duì)于所有暴露濃度,肝臟都是重要的累積組織,這與雙氯芬酸、羅紅霉素等藥物化合物在鯽魚(yú)體內(nèi)的累積分布規(guī)律類(lèi)似[18-19].肝臟是進(jìn)行有機(jī)物代謝清除的重要場(chǎng)所,當(dāng)代謝速率低于吸收速率就容易發(fā)生累積.此外,兩個(gè)高濃度組在皮膚和腎臟的累積量也比較大.由于皮膚可直接從水中吸收親脂性的防曬劑,因此,對(duì)于水體暴露而言,皮膚也是主要的累積部位.腎臟是生物的重要排泄組織,當(dāng)肝臟不能有效地代謝轉(zhuǎn)化攝入的 ODPABA時(shí),可導(dǎo)致腎臟中的OD-PABA濃度升高[20].OD-PABA在肝臟、鰓、肌肉組織中的累積量隨時(shí)間變化表現(xiàn)為先升高再下降的趨勢(shì),這可能是由于污染物在組織中的濃度達(dá)到一定水平后(也可以認(rèn)為是一個(gè)閾值),鯽魚(yú)會(huì)啟動(dòng)或者在已有基礎(chǔ)上增強(qiáng)機(jī)體代謝功能,加快體內(nèi)有毒成分的清除,以降低外源性物質(zhì)對(duì)自身的傷害[21-22].

圖1 OD-PABA在鯽魚(yú)組織中的濃度及暴露28d的BCF值(平均值±標(biāo)準(zhǔn)偏差,n=3)Fig.1 Concentrations of OD-PABA in fish tissues and BCF values at day 28, and values are expressed as mean±standard deviation (n=3)

環(huán)境中的污染物在生物體內(nèi)的累積是引起亞慢性和慢性毒性作用的基礎(chǔ),生物富集因子(BCF)是生態(tài)毒理效應(yīng)的重要評(píng)價(jià)指標(biāo)之一.本文利用暴露14d和28d后,鯽魚(yú)各組織中污染物的濃度與水體實(shí)際暴露濃度的比值得到BCF值,結(jié)果見(jiàn)圖1.不同暴露濃度的OD-PABA在不同組織的BCF值存在一定差異,主要的累積部位是肝臟、皮膚和腎臟,BCF值范圍分別為123～186、117～163和 90.2～147.在以前的斑馬魚(yú)暴露研究中,得到BP3的BCF值為19～94[23],奧克立林為41～136[24].由于這幾種防曬劑的親脂性都比較強(qiáng),實(shí)驗(yàn)得到的BCFs明顯低于基于Kow的理論預(yù)測(cè)值.這些物質(zhì)可能在魚(yú)體內(nèi)發(fā)生了代謝轉(zhuǎn)化和排除,導(dǎo)致生物富集水平比較低.比如,BP3在魚(yú)體內(nèi)能夠代謝生成二苯酮-1(BP1),并排出體外[23].此外,親脂性強(qiáng)的 OUVFs可能被魚(yú)的排泄物吸收,使其生物可利用性降低,導(dǎo)致魚(yú)的生物富集減少[25].

2.2 OD-PABA暴露下鯽魚(yú)肝臟代謝酶系的響應(yīng)

OD-PABA對(duì)EROD、PROD、BFCOD和GST活性的影響見(jiàn)圖 2.由于空白對(duì)照與溶劑對(duì)照無(wú)顯著差異,本文采用溶劑對(duì)照與處理組進(jìn)行對(duì)比分析.

OD-PABA暴露增加了鯽魚(yú)肝臟EROD的活性,兩個(gè)低濃度組在第3d顯著誘導(dǎo)了EROD的活性(P＜0.05),誘導(dǎo)倍數(shù)分別為0.58和0.65倍.隨著暴露時(shí)間的延長(zhǎng),各濃度組的EROD活性逐漸降低至對(duì)照水平.從濃度效應(yīng)來(lái)看,不同暴露濃度組之間EROD活性無(wú)顯著差異.水體OD-PABA暴露條件下,不同處理組鯽魚(yú)肝臟PROD的活性都有所增加,但是只有W2組在第3d和第14d顯著誘導(dǎo)了鯽魚(yú)肝臟 PROD的活性(P＜0.05),其余暴露組 PROD活性與對(duì)照組并無(wú)顯著差異. BFCOD的活性變化與PROD相似,不同濃度和不同時(shí)間OD-PABA暴露都增加了BFCOD的活性,但是除了W2組在第14d的顯著誘導(dǎo)效應(yīng)外,其余處理組與對(duì)照組無(wú)顯著差異.OD-PABA暴露增加了鯽魚(yú)肝臟 GST的活性,最高濃度的OD-PABA顯著誘導(dǎo)了 GST活性(P＜0.05),誘導(dǎo)倍數(shù)在 0.40～0.53之間,隨暴露時(shí)間的延長(zhǎng),誘導(dǎo)倍數(shù)逐漸降低,但是并無(wú)顯著差異.

生物體在自身的生長(zhǎng)過(guò)程中,需要不斷地代謝清除生物體自身合成的(內(nèi)源)以及從外部環(huán)境中累積的(外源)各種有機(jī)化合物,以達(dá)到代謝清除或?qū)τ卸净衔镞M(jìn)行解毒的目的.P450酶系作為生物體 I相代謝酶的重要組成部分,在環(huán)境污染物的生物監(jiān)測(cè)和生態(tài)效應(yīng)評(píng)價(jià)領(lǐng)域有著廣闊的應(yīng)用前景.其中,魚(yú)類(lèi) CYP1A家族在致癌物的代謝和活化中發(fā)揮重要作用,也被稱(chēng)為藥物代謝酶[26].研究發(fā)現(xiàn)多種藥物如羅紅霉素、普萘洛爾、磺胺甲惡唑、咖啡因等,可以誘導(dǎo)鯽魚(yú)肝臟與CYP1A關(guān)聯(lián)的EROD活性[27-29],而EROD的誘導(dǎo)效應(yīng)與藥物在魚(yú)體內(nèi)的生物富集和生物轉(zhuǎn)化有關(guān)[27].目前,有關(guān)魚(yú)類(lèi) I相代謝酶對(duì) OUVFs的響應(yīng)研究還比較少.Grabicova等[30]研究發(fā)現(xiàn)2-苯基苯并咪唑-5-磺酸在 1～1000μg/L濃度范圍內(nèi)暴露21d,對(duì)虹鱒魚(yú)(Oncorhynchus mykiss)的EROD、PROD和BFCOD活性都產(chǎn)生了顯著誘導(dǎo),這與本文結(jié)果相似.

圖2 水中OD-PABA暴露對(duì)鯽魚(yú)肝臟酶活性的影響(n=3,不同字母表示存在顯著性差異,P＜0.05)Fig.2 Effects of OD-PABA in water on enzymatic activities in fish liver (n=3, values that do not share the same superscript letters are significantly different, P＜0.05)

GST是生物體進(jìn)行II相代謝反應(yīng)的一個(gè)重要酶系,不僅參與外源污染物的II相代謝,還能夠消除生物體內(nèi)的過(guò)氧化物,抵御DNA損傷和脂質(zhì)氧化[31-32].一些藥物化合物誘導(dǎo)魚(yú)類(lèi)肝臟 EROD的同時(shí),GST活性也顯著增加[28,33].Liu等[34]將鯽魚(yú)暴露于0.5和5mg/L的二苯酮類(lèi)防曬劑(BP-1和BP-3)7d和14d,肝臟GST活性顯著增加.GST的誘導(dǎo)效應(yīng)表明魚(yú)類(lèi)產(chǎn)生了氧化應(yīng)激的適應(yīng)性反應(yīng),或者該物質(zhì)發(fā)生了代謝和排泄[35].

3 結(jié)論

3.1 水體暴露的OD-PABA能夠在鯽魚(yú)腦、肝、腎、鰓、肌肉、皮膚等組織中分布和累積,暴露28d生物富集因子在71～184之間,其中肝臟、皮膚和腎臟是主要的累積組織.

3.2 OD-PABA對(duì)鯽魚(yú)肝臟P450酶系EROD、PROD和BFCOD產(chǎn)生了誘導(dǎo)效應(yīng),其中,EROD的活性增加最為顯著,誘導(dǎo)倍數(shù)最高達(dá)到0.97倍.高濃度的OD-PABA對(duì)鯽魚(yú)肝臟GST活性產(chǎn)生了顯著的誘導(dǎo)效應(yīng),誘導(dǎo)倍數(shù)最高達(dá)到0.53倍.

3.3 結(jié)果表明,鯽魚(yú)可以從水環(huán)境中吸收和累積OD-PABA,并通過(guò)I相和II相代謝酶進(jìn)行生物轉(zhuǎn)化.

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Bioconcentration of organic UV filters in Carassius auratus and metabolic enzyme response.

LU Guang-hua1*, LI Sheng2, ZONG Yong-chen1, HUANG Yi1(1.College of Hydraulic and Civil Engineering, Xizang Agriculture and Animal Husbandry College, Linzhi 860000, China；2.Key Laboratory of Integrated Regulation and Resources Development of Shallow Lakes, Ministry of Education, College of Environment, Hohai University, Nanjing 210098, China). China Environmental Science, 2017,37(9)：3576～3582

The semi static exposure test was used to investigate the distribution and accumulation of organic UV filter 2-ethylhexyl 4-dimethylaminobenzoate (OD-PABA) in different tissues in Carassius auratus and to determine its ecotoxicological effects. The contents of OD-PABA in different tissues increased corresponding to the increasing exposure concentrations, and reached the maximum at day 14 or 28. The bioconcentration factor (BCF) of OD-PABA in different tissues obviously depended on the different exposure concentrations, and the BCF values calculated at day 14 and 28 were 123～186 in liver, 117～163 in skin and 90.2～147 in kidney, respectively. OD-PABA significantly induced EROD, PROD, BFCOD and GST activities in fish liver, EROD and GST activities showed more remarkable responses, and the maximum induction folds were 0.97 and 0.53, respectively. Our results suggest that OD-PABA in water can concentrate in Carassius auratus and be biotransformed by I phase and II phase metabolic enzymes.

Carassius auratus；OD-PABA；tissue distribution；bioconcentration；metabolic enzyme

X171.5

A

1000-6923(2017)09-3576-07

2017-02-15

西藏農(nóng)牧學(xué)院雅江學(xué)者計(jì)劃(2015XYA01);國(guó)家自然科學(xué)基金創(chuàng)新群體項(xiàng)目(51769034)

* 責(zé)任作者, 教授, ghlu@hhu.edu.cn

陸光華(1969-),女,吉林乾安人,教授,博士,主要從事水環(huán)境保護(hù)研究.發(fā)表論文170余篇.