類(lèi)二噁英物質(zhì)及芳香烴受體(AhR)介導(dǎo)的有害結(jié)局路徑(AOP)研究進(jìn)展

魏鳳華，張俊江，夏普，張效偉，于紅霞

南京大學(xué)環(huán)境學(xué)院污染控制與資源化研究國(guó)家重點(diǎn)實(shí)驗(yàn)室，南京210023

類(lèi)二噁英物質(zhì)及芳香烴受體(AhR)介導(dǎo)的有害結(jié)局路徑(AOP)研究進(jìn)展

魏鳳華，張俊江，夏普，張效偉*，于紅霞

南京大學(xué)環(huán)境學(xué)院污染控制與資源化研究國(guó)家重點(diǎn)實(shí)驗(yàn)室，南京210023

二噁英及類(lèi)二噁英物質(zhì)(dioxin-like compounds,DLCs)是一類(lèi)高毒性化合物的統(tǒng)稱(chēng),對(duì)其毒理學(xué)的研究一直都是備受關(guān)注的焦點(diǎn)。已有證據(jù)表明,高毒二噁英及DLCs主要通過(guò)激活芳香烴受體(aryl hydrocarbon receptor,AhR),進(jìn)而導(dǎo)致一系列生物毒性。近年來(lái)越來(lái)越多的新型有機(jī)污染物被發(fā)現(xiàn)具有類(lèi)二噁英分子結(jié)構(gòu)并存在潛在生物毒性或活性。與此同時(shí),如何評(píng)估二噁英及DLCs對(duì)本土生態(tài)生物的危害及其風(fēng)險(xiǎn)也受到更多關(guān)注。本文綜述了近幾年發(fā)現(xiàn)的新型二噁英物質(zhì)、二噁英及DLCs的AhR致毒機(jī)制、相應(yīng)的有害結(jié)局路徑(adverse outcome pathway,AOP)及AOP在指導(dǎo)探索新型物質(zhì)及物種敏感性方面上的新觀(guān)點(diǎn)和發(fā)現(xiàn),同時(shí)也展望了二噁英及DLCs在生態(tài)毒理及風(fēng)險(xiǎn)評(píng)估領(lǐng)域的未來(lái)研究方向。

類(lèi)二噁英物質(zhì)；多溴聯(lián)苯醚；多氯代聯(lián)苯硫醚；新型污染物；芳香烴受體；致毒機(jī)制；物種敏感性分布

二噁英及類(lèi)二噁英物質(zhì)(dioxin-like compounds, DLCs)通常被認(rèn)為是一類(lèi)具有高毒性的物質(zhì),能引起人和野生動(dòng)物的生殖及發(fā)育毒性、免疫毒性、肝毒性及致癌、體重減少、胸腺萎縮、皮膚病變等[1-6]。二噁英及DLCs的致毒機(jī)理主要是通過(guò)激活芳香烴受體(aryl hydrocarbon receptor,AhR)通路來(lái)進(jìn)行調(diào)控的,AhR被配體激活后進(jìn)入細(xì)胞核與AhR核轉(zhuǎn)位因子(aryl hydrocarbon receptor nuclear translocator, ARNT)形成二聚體后特異性地識(shí)別結(jié)合到二噁英響應(yīng)元件(dioxin response element,DRE)上,誘導(dǎo)下游基因的表達(dá),從而產(chǎn)生相關(guān)毒性[7]。盡管一些不能激活A(yù)hR的DLCs也可能導(dǎo)致神經(jīng)毒性和免疫毒性效應(yīng),但是這一般需要很高的暴露濃度,因此在實(shí)際的化學(xué)品管理中重要性不高[8-9]。

近年來(lái)越來(lái)越多的新型有機(jī)污染物具有類(lèi)二噁英的分子結(jié)構(gòu)并存在潛在的生物毒性[10-13]。當(dāng)今新型有機(jī)污染物的種類(lèi)和數(shù)量逐年急劇增加,其生態(tài)危害和環(huán)境風(fēng)險(xiǎn)具有很高的不確定性[14]。其中一個(gè)重要的科學(xué)問(wèn)題是這些物質(zhì)中是否會(huì)有類(lèi)似二噁英的高毒物質(zhì)。然而對(duì)這些物質(zhì)開(kāi)展傳統(tǒng)的動(dòng)物測(cè)試毫無(wú)現(xiàn)實(shí)性。如何通過(guò)分子致毒機(jī)制和高通量測(cè)試技術(shù)來(lái)識(shí)別新型有毒污染物并預(yù)測(cè)其生態(tài)危害已成為當(dāng)前國(guó)際上生態(tài)毒理學(xué)研究的熱點(diǎn)。

如何評(píng)估二噁英及DLCs對(duì)本土生物的危害及其生態(tài)風(fēng)險(xiǎn)也受到更多關(guān)注。以往對(duì)二噁英及DLCs毒理學(xué)的研究主要是通過(guò)模式生物來(lái)開(kāi)展的,而證據(jù)顯示野生動(dòng)物對(duì)二噁英及DLCs的毒性反應(yīng)表現(xiàn)出顯著的差異[15-18]。從環(huán)境管理的角度,保護(hù)生態(tài)系統(tǒng)健康首先是保護(hù)敏感性的生態(tài)物種,近年來(lái)越來(lái)越多的研究開(kāi)始關(guān)注于二噁英及DLCs對(duì)野生生物的毒性敏感性差異的機(jī)制[15-16,19-20]。

隨著對(duì)二噁英及DLCs分子毒理學(xué)的深入研究,其致毒機(jī)制和模式逐漸清晰。這些研究的積累勾畫(huà)出二噁英及DLCs通過(guò)激活A(yù)hR誘導(dǎo)毒性的有害結(jié)局路徑(adverse outcome pathway,AOP)。此AOP作為連接分子啟動(dòng)事件和有害結(jié)局之間的框架[21],可為研究新型化合物的毒性及物種間敏感性提供一定指導(dǎo)。

1 二噁英及新型二噁英物質(zhì)(Dioxins and emerging dioxin-like pollutants)

二噁英及DLCs主要包括多氯二苯并對(duì)二噁英(polychlorinated dibenzo-p-dioxins,PCDDs)、多氯二苯并呋喃(polychlorinated dibenzo-furans,PCDFs)、多氯聯(lián)苯(polychorinated biphenyls,PCBs)和多環(huán)芳烴(polycyclic aromatic hydrocarbons,PAHs)等持久性有機(jī)污染物,分別有75種、135種、208種及200余種異構(gòu)體[13]。由于當(dāng)今新型有機(jī)污染物的種類(lèi)和數(shù)量每年急速增長(zhǎng),并隨著相關(guān)檢測(cè)技術(shù)的發(fā)展,越來(lái)越多的化學(xué)物質(zhì)被發(fā)現(xiàn)具有二噁英活性。最近,通過(guò)H4IIE-luc報(bào)告基因法[22]和鳥(niǎo)的AhR1-LRG報(bào)告基因法[23]發(fā)現(xiàn)了3種重要的新型二噁英物質(zhì),即甲氧基化多溴聯(lián)苯醚(methoxylated polybrominated diphenyl ethers,MeO-PBDEs)、羥基化多溴聯(lián)苯醚(hydroxylated polybrominated diphenyl ethers,OH-PBDEs)[12]和多氯代聯(lián)苯硫醚(polychlorinated diphenyl sulfides,PCDPSs)[13]。

1.1 MeO-/OH-PBDEs

MeO-PBDEs和OH-PBDEs作為最近被發(fā)現(xiàn)的具有二噁英類(lèi)活性的化合物,是多溴聯(lián)苯醚(polybrominated diphenyl ethers,PBDEs)的甲氧基化和羥基化衍生物。它們也是一種新型的環(huán)境有機(jī)類(lèi)污染物,近年來(lái)越來(lái)越受到社會(huì)各界的廣泛關(guān)注[12]。PBDEs作為常見(jiàn)的添加型溴代阻燃劑,在環(huán)境中被大量檢出[24-29]。因PBDEs與二噁英類(lèi)化合物結(jié)構(gòu)極為相似,大量的研究探索其是否為新型二噁英物質(zhì),但結(jié)果顯示這類(lèi)化合物并無(wú)二噁英類(lèi)活性,即使表現(xiàn)的微弱效應(yīng)也是由其他雜質(zhì)造成的[30-33]。

而后來(lái)研究發(fā)現(xiàn)PBDEs的衍生物MeO-PBDEs和OH-PBDEs具有二噁英類(lèi)活性,并且發(fā)現(xiàn)它們的二噁英活性對(duì)不同鳥(niǎo)類(lèi)具有敏感性差異。Su等[12]利用報(bào)告基因法將34種PBDEs衍生物(15種MeOPBDEs和19種OH-PBDEs)暴露于H4IIE-luc細(xì)胞,首次發(fā)現(xiàn)了PBDEs衍生物能夠誘導(dǎo)顯著的AhR活性,結(jié)果顯示:34種測(cè)試物質(zhì)有19種表現(xiàn)出二噁英活性,其中5-C1-6-HO-BDE-47的相對(duì)毒性效力(relative potency,ReP)最大,與八氯代二苯并二噁英(OCDD)和八氯代二苯并呋喃(OCDF)的毒性當(dāng)量因子(TEF)相當(dāng)；并且發(fā)現(xiàn)OH-官能團(tuán)和MeO-官能團(tuán)相比,可以誘導(dǎo)更大的AhR活性,這和其他文獻(xiàn)報(bào)道的MeO-和OH-官能團(tuán)的加入會(huì)大大加強(qiáng)化合物的AhR效應(yīng)的結(jié)果相一致[34]?？梢?jiàn)OH-和MeO-官能團(tuán)的加入使得PBDEs具有了極強(qiáng)的二噁英活性。張睿[35]用鳥(niǎo)的 AhR1-LRG報(bào)告基因法對(duì) 19種MeO-/OH-PBDEs二噁英活性的鳥(niǎo)類(lèi)種間敏感性進(jìn)行了研究,結(jié)果表明:1)不同鳥(niǎo)對(duì)同一MeO-/OH-PBDEs的敏感性不同；2)部分MeO-/OH-PBDEs二噁英毒性的鳥(niǎo)類(lèi)種間敏感性排序與典型二噁英化合物也不一致；3)基于H4IIE-luc試驗(yàn)推導(dǎo)的MeO-/OH-PBDEs的毒性效力比鳥(niǎo)類(lèi)毒理試驗(yàn)得到的小1～4個(gè)數(shù)量級(jí),這與典型二噁英化合物的情況一致。

1.2 PCDPSs

PCDPSs作為一種新型二噁英活性化合物,其結(jié)構(gòu)與多氯聯(lián)苯醚和多溴聯(lián)苯醚極其相似,共有209種同素異構(gòu)體。PCDPSs兩個(gè)苯環(huán)之間由硫原子連接,化學(xué)通式為C12H10-xSClx(x=1～10)。其可在冶煉廠(chǎng)的灰渣、垃圾焚燒爐的煙道廢氣、紙漿廢水、長(zhǎng)江下游水和表層底泥中被檢出[36-38]。

通過(guò)研究發(fā)現(xiàn)部分PCDPSs具有二噁英活性,并且發(fā)現(xiàn)PCDPSs的二噁英活性規(guī)律與PCDD/Fs、PCBs相似。夏潔[13]使用H4IIE-luc受體報(bào)告基因法,對(duì)19種PCDPSs的毒性進(jìn)行研究,發(fā)現(xiàn)15種PCDPSs具有二噁英類(lèi)活性,8種的二噁英活性最顯著,ReP值為5.1×10-8～3.2×10-5,AhR受體活性最高的PCDPSs的ReP值(ReP=10-5)比四氯二苯并-p-二噁英(TCDD)的ReP值(ReP=l)低105倍；且除了2,4,4 ',5-TrisCDPS和2,2',3,3',4,5,6-HeptaCDPS的ReP值與WHO公布的單臨位 PCBs相當(dāng)外,其他的PCDPSs的 ReP值均比 PCBs低；另外,19種PCDPSs的二噁英活性規(guī)律與PCDD/Fs、PCBs相似,低于四氯取代的PCDD/Fs、PCBs均不會(huì)激活芳香烴受體活性,這可能與AhR-LBD域氨基酸殘基通過(guò)疏水作用所形成的口袋有關(guān)。Zhang等[39]使用鳥(niǎo)類(lèi)AhR1-LRG試驗(yàn)對(duì)18種PCDPSs的二噁英活性進(jìn)行分析,證實(shí)了它們能夠誘導(dǎo)二噁英類(lèi)活性,且具有很大的種間敏感性差異。并發(fā)現(xiàn)PCDPSs對(duì)雞、環(huán)頸雉和日本鵪鶉的毒性效力均隨著氯代水平的提高呈現(xiàn)上升趨勢(shì)。部分PCDPSs類(lèi)二噁英毒性的鳥(niǎo)類(lèi)種間敏感性排序與典型二噁英的情況不同,這可能與鳥(niǎo)類(lèi)AhR1-LBD域氨基酸序列的差異及配體化合物結(jié)構(gòu)的差異有關(guān)。

2 二噁英及DLCs的AhR相關(guān)毒性機(jī)制(AhR toxic mechanism of dioxins and DLCs)

1976年,Poland等[40]首次發(fā)現(xiàn)了TCDD的毒性主要是由于其可與AhR特異性地結(jié)合。其后大量研究表明,具有高毒性的二噁英及DLCs的作用主要是通過(guò)激活 AhR,進(jìn)而引起各種相關(guān)毒性[41-43]。并且二噁英及DLCs在不同物種間和物種內(nèi)的毒性存在著敏感性差異。由于A(yíng)hR經(jīng)過(guò)長(zhǎng)期的復(fù)制和多樣化,產(chǎn)生了各種差異,雖然同一物種的AhR結(jié)構(gòu)特性有著廣泛的保守性,但結(jié)構(gòu)上細(xì)微的差異會(huì)導(dǎo)致功能上巨大的不同[44]。

2.1 AhR簡(jiǎn)介

AhR屬于堿性螺旋-環(huán)-螺旋(basic helix loop helix,bHLH)PER-ARNT-SIM同源域(PER-ARNT-SIM, PAS)蛋白超家族[45]。AhR是一個(gè)依賴(lài)配體激活的轉(zhuǎn)錄因子,主要由DNA結(jié)合域(DNA-binding domain,DBD)、配體結(jié)合域(ligand binding domain, LBD)和反式激活域(transactivation domain,TAD)組成[46]。

AhR在無(wú)脊椎動(dòng)物中并沒(méi)有結(jié)合二噁英及DLCs的能力,但在脊椎動(dòng)物中能夠結(jié)合二噁英類(lèi)物質(zhì),并且AhR基因經(jīng)過(guò)長(zhǎng)期的復(fù)制和多樣化,導(dǎo)致產(chǎn)生了至少3個(gè)AhR基因家族—AhR1、AhR2和芳香烴受體抑制因子(AhRR)[47-48]。AhR1最先在C57BL/6小鼠的肝中被發(fā)現(xiàn),后來(lái)發(fā)現(xiàn)在所有的脊椎動(dòng)物中都含有AhR1并具均有轉(zhuǎn)錄活性[49]。和哺乳動(dòng)物(包括人類(lèi))只有AhR1不同[50],其他脊椎動(dòng)物不只有AhR1,還有AhR2,只是在不同物種中兩者的表達(dá)活性不同。鳥(niǎo)的AhR1和AhR2雖然都具有轉(zhuǎn)錄活性,但AhR2轉(zhuǎn)錄活性低,即AhR1在鳥(niǎo)中占主導(dǎo)[51]。而對(duì)于魚(yú)來(lái)說(shuō),二噁英通過(guò)AhR介導(dǎo)的毒代動(dòng)力學(xué)更復(fù)雜。魚(yú)至少有3個(gè)AhR(AhR1、AhR2和AhR3),并且每個(gè) AhR又都包括多個(gè)亞型[52]。AhR1、AhR2最初在鳉魚(yú)中被確認(rèn)[44,53],大多數(shù)硬骨魚(yú)類(lèi)中,AhR2顯示是活化型,而AhR1不能被二噁英類(lèi)化合物結(jié)合和激活[54]。后來(lái)發(fā)現(xiàn)AhR1的亞型AhR1B鄰近AhR2,并且在斑馬魚(yú)胚胎中可表達(dá),而這與二噁英類(lèi)化合物無(wú)關(guān),即AhR1B在斑馬魚(yú)的胚胎發(fā)育中起著重要的生理作用[43]。另外AhR3的作用至今還沒(méi)有確認(rèn),僅僅知道在一些軟骨魚(yú)類(lèi)中可以表達(dá)[47]。AhRR是AhR作用的抑制因子,AhRR本身不結(jié)合AhR受體,但AhRR和AhR在bHLH和PAS-A域有著高度的序列一致性[48,55-56]。AhRR有些功能與AhR類(lèi)似,在鹵代和非鹵代芳香烴化合物激活A(yù)hR1或者AhR2后,其表達(dá)可被誘導(dǎo),與AhR競(jìng)爭(zhēng)可用的ARNT結(jié)合位點(diǎn),形成沒(méi)有轉(zhuǎn)錄活性的AhRR/ARNT二聚體,并可以結(jié)合DRE來(lái)抑制DRE啟動(dòng)子[48,57]。另外鳉魚(yú)的 AhRR可以抑制AhR1和AhR2的反式激活作用[48]。AhRR功能在魚(yú)類(lèi)和哺乳動(dòng)物的進(jìn)一步表征,可有助于理解在暴露于芳香烴化合物后,引起物種間及細(xì)胞類(lèi)型差異的機(jī)制。

2.2 AhR經(jīng)典作用機(jī)制

AhR活性的毒性機(jī)制研究由來(lái)已久,其毒性調(diào)控過(guò)程主要包括4個(gè)步驟:胞漿復(fù)合物形成、AhR轉(zhuǎn)運(yùn)、AhR異源二聚化及CYP1A的誘導(dǎo)表達(dá)[43]。但由于A(yíng)hR基因經(jīng)過(guò)長(zhǎng)期的復(fù)制和多樣化,導(dǎo)致其在不同物種中的形態(tài)和功能產(chǎn)生一定差異,再加上AhR的作用通路與其他通路交叉的多樣和混雜性,試圖描述清楚 AhR的機(jī)制比較困難[41]。但根據(jù)AhR結(jié)構(gòu)特性的廣泛保守性,仍存在著一個(gè)經(jīng)典的核受體機(jī)制,具體如下:

正常情況下存在于細(xì)胞質(zhì)中的AhR處于不活躍狀態(tài),因?yàn)锳hR與熱休克蛋白(Hsp90)、前列腺素E合成酶3(prostaglandin esynthase,p23)單聚體及乙型肝炎病毒X蛋白2(hepatitis B virus X-associated protein2,XAP2)形成多蛋白復(fù)合體[58-60],參與屏蔽核定位信號(hào)。當(dāng)外源性配體進(jìn)入細(xì)胞后,與AhR結(jié)合。接著進(jìn)入細(xì)胞核,AhR從Hsp90復(fù)合體上解離下來(lái),再與 ARNT形成異質(zhì)二聚體[59-61]。而由于Hsp90復(fù)合體的解離使得AhR的DNA結(jié)合位點(diǎn)暴露出來(lái),此DNA結(jié)合位點(diǎn)可特異性地識(shí)別結(jié)合DNA上的DRE,從而AhR/ARNT異質(zhì)二聚體結(jié)合在DRE上并啟動(dòng)下游靶基因,如編碼CYP1A1、醌還原酶的基因表達(dá)[43,62],由此誘導(dǎo)相應(yīng)的生物毒性。

3 AhR的有害結(jié)局路徑(Adverse outcome pathway of AhR)

隨著對(duì)AhR的分子生物學(xué)機(jī)制和二噁英及DLCs的致毒模式的深入研究,通過(guò)對(duì)這些研究數(shù)據(jù)進(jìn)行整合勾畫(huà)出二噁英及DLCs通過(guò)激活A(yù)hR受體誘導(dǎo)毒性的AOP,它是連接分子啟動(dòng)事件、細(xì)胞、器官、組織、個(gè)體和最終有害結(jié)局之間的框架[21]。這種AOP框架能為預(yù)測(cè)新型污染物和物質(zhì)的種間敏感性差異提供基礎(chǔ),進(jìn)而為生態(tài)毒理學(xué)的預(yù)測(cè)方法和生態(tài)風(fēng)險(xiǎn)評(píng)估的更廣泛使用提供保障。

圖1 AhR介導(dǎo)的有害結(jié)局路徑示意圖Fig.1 Adverse outcome pathway(AOP)mediated by AhR

AOP是一個(gè)概念性的框架,通過(guò)整合現(xiàn)有的不同生物組織水平的生態(tài)毒理信息,描述了直接分子啟動(dòng)事件和有害結(jié)局直接的聯(lián)系,從而使得評(píng)估結(jié)果更加有效[21]。即AOP描述了從一個(gè)分子啟動(dòng)事件開(kāi)始,經(jīng)過(guò)對(duì)細(xì)胞、器官、組織和個(gè)體產(chǎn)生的一系列效應(yīng),最終在群落水平上導(dǎo)致一個(gè)有害結(jié)局的過(guò)程。分子啟動(dòng)事件(MIE)是在開(kāi)始有害結(jié)局路徑時(shí),化學(xué)物和機(jī)體產(chǎn)生生化作用的一個(gè)主錨位。而關(guān)鍵事件(KE)是連接MIE和有害結(jié)局之間的多個(gè)層次生物組織上的有因果關(guān)系或者某種相關(guān)關(guān)系的事件,這些數(shù)據(jù)的獲得可能來(lái)自體外、體內(nèi)試驗(yàn)或計(jì)算模擬系統(tǒng)。

如圖1所示,AhR-AOP框架可幫助把二噁英及DLCs通過(guò)AhR介導(dǎo)的分子效應(yīng)和在細(xì)胞、器官、個(gè)體或者群體水平上觀(guān)察到的有害結(jié)局聯(lián)系起來(lái)。即二噁英及DLCs首先激活A(yù)hR這一分子啟動(dòng)事件,導(dǎo)致AhR/ARNT的二聚及相關(guān)I相和II相代謝酶的誘導(dǎo),接著引起細(xì)胞、器官、個(gè)體上的一連串效應(yīng),最終對(duì)整個(gè)種群產(chǎn)生影響。

二噁英及DLCs能夠激活A(yù)hR,并同時(shí)激活相關(guān)代謝酶,引起各種毒性[63]。直接和間接的證據(jù)表明二噁英的AOP的分子啟動(dòng)事件就是激活A(yù)hR,盡管這個(gè)事件還沒(méi)有被完全確定[21],但有研究發(fā)現(xiàn)TCDD的毒性通過(guò)AhR啟動(dòng)的直接證據(jù)是小鼠被敲除AhR后,對(duì)TCDD誘導(dǎo)的毒性具有抵抗力[64],并且對(duì)斑馬魚(yú)胚胎注射抑制AhR轉(zhuǎn)運(yùn)蛋白的嗎啉代寡核苷酸后,減少和延遲了TCDD的毒性[65]。

二噁英及DLCs在啟動(dòng)AhR關(guān)鍵分子事件后,最關(guān)鍵的一條通路是能誘導(dǎo)持續(xù)的AhR/ARNT二聚,從而導(dǎo)致細(xì)胞形態(tài)發(fā)育期間的ARNT和原來(lái)的結(jié)合對(duì)象分離,最終干擾依賴(lài) ARNT的細(xì)胞功能[66-67]。比如,HIF-1α作為ARNT的二聚對(duì)象,在供氧不足時(shí),與ARNT能形成一個(gè)轉(zhuǎn)錄因子復(fù)合體,來(lái)結(jié)合DNA上的缺氧反應(yīng)增強(qiáng)子,激活相關(guān)基因的表達(dá),如激活與血管生成有關(guān)的血管內(nèi)皮生長(zhǎng)因子,而二噁英及DLCs通過(guò)干擾ARNT和HIF-1α的二聚,從而改變心血管的發(fā)育和相關(guān)功能[68-71]。

二噁英及DLCs還和轉(zhuǎn)錄輔助因子作用,改變大量基因的轉(zhuǎn)錄,包括增加細(xì)胞色素基因(CYP1A)的轉(zhuǎn)錄[72]。雞的CYP1A有2個(gè)亞型:CYP1A4和CYP1A5[73]。其中CYP1A4具有芳香烴酶(aromatic enzyme,AE)和脫乙基酶(ethoxy-resorufin-o-deethylase,EROD)的催化特異性。而CYP1A5主要是負(fù)責(zé)內(nèi)源性脂肪酸花生四烯酸代謝和特異性催化尿卟啉原的氧化。尿卟啉原氧化會(huì)帶來(lái)羧酸鹽卟啉的積累,其對(duì)肝臟、腎臟、骨骼和血液等都會(huì)帶來(lái)影響,最終導(dǎo)致尿卟啉癥?；ㄉ南┧崮軌虍a(chǎn)生大量活性氧,誘導(dǎo)氧化應(yīng)激,使得氧化系統(tǒng)和抗氧化系統(tǒng)失衡,導(dǎo)致組織損傷。

AhR的激活還能導(dǎo)致II相代謝酶的誘導(dǎo),如谷胱苷肽硫轉(zhuǎn)移酶(glutathione S-transferases,GSTs)和半乳糖基轉(zhuǎn)移酶(uridine diphosphate-galactosyl transfer-ase,UDP-GT)。因羥基化多氯聯(lián)苯(OH-PCBs)與甲狀腺激素(T4)比,和甲狀腺素運(yùn)載蛋白(TTR)的親和力更大,能與T4競(jìng)爭(zhēng)結(jié)合TTR,故PCBs可取代T4,導(dǎo)致 T4代謝和排泄,從而使 T4的含量降低[72,74-75]。同時(shí),PCBs可能會(huì)加速新陳代謝和在沒(méi)有AhR介導(dǎo)下,與一些載體作用(如維生素結(jié)合蛋白和TTR),導(dǎo)致維生素A的降低[76-77]。而T4和類(lèi)維生素A的減少會(huì)直接導(dǎo)致器官、個(gè)體發(fā)育的異常。

二噁英及DLCs的的毒性效應(yīng)在個(gè)體上表現(xiàn)為引起發(fā)育異常[78]、胚胎致死[79]、增加不孕癥及改變父母行為[80]等,進(jìn)而降低繁殖率[81],引起種群下降[82]。Kim等[83]研究了TCDD、PCB77和PCB126對(duì)日本青鳉胚胎發(fā)育毒性,發(fā)現(xiàn)心血管發(fā)育和功能異常,并且引起幼魚(yú)顱面畸形,心包及卵黃囊水腫并抑制魚(yú)鰾的形成,并且孵化后3 d的半數(shù)致死濃度(LC50)分別是8.1 pg·mL-1、0.25 ng·mL-1和0.6μg·mL-1。黃莉等[84]研究了TCDD暴露對(duì)小鼠胚胎毒性的影響,發(fā)現(xiàn)其可造成著床前胚胎丟失并導(dǎo)致雌性生殖器官的多種生殖激素的紊亂。并且20世紀(jì)60年代,人們發(fā)現(xiàn)美國(guó)的一個(gè)水紹人工養(yǎng)殖農(nóng)場(chǎng)里,水紹不孕,種群下降,研究發(fā)現(xiàn)喂食水貂的飼料里含有TCDD和PCBs[85]。Giesy等[86]研究了二噁英類(lèi)物質(zhì)對(duì)北美五大湖區(qū)域鳥(niǎo)的繁殖率的影響,發(fā)現(xiàn)禿鷹和污染相對(duì)較輕地區(qū)的鷹比,繁殖率要低,并且隨后在此地區(qū)發(fā)現(xiàn)了小鷹的畸形[87]。Henny等[88]對(duì)北美魚(yú)鷹的數(shù)量從歷史和現(xiàn)代的角度進(jìn)行了綜述,發(fā)現(xiàn)在20世紀(jì)50到70年內(nèi)期間,DDT及PCDD/Fs、PCB等環(huán)境持久性有機(jī)污染物極大降低了魚(yú)鷹的數(shù)量,而由于禁止和減少了此類(lèi)化合物的生產(chǎn)和使用,目前魚(yú)鷹的數(shù)量趨于穩(wěn)定。二噁英及DLCs對(duì)哺乳動(dòng)物的影響也很?chē)?yán)重。大量調(diào)查發(fā)現(xiàn)世界各大海洋哺乳動(dòng)物體內(nèi),如鯨、海豚、海豹等的體內(nèi)PCBs濃度比較高,都面臨著種群減少的風(fēng)險(xiǎn)[89]。

雖然從20世紀(jì)70年代末,全世界范圍內(nèi)開(kāi)始對(duì)二噁英及DLCs的生產(chǎn)和使用進(jìn)行明令禁止,但由于其具有環(huán)境持久性、親脂性及高度的生物蓄積性,生態(tài)危害仍持續(xù)存在。并且在中國(guó),PCDD/Fs的污染仍較為嚴(yán)重,并主要存在于沉積物中,其他介質(zhì)中則相對(duì)較少。張烴等[90]對(duì)中國(guó)蘇南25個(gè)城市群河流沉積物樣品中的二噁英類(lèi)物質(zhì)進(jìn)行了調(diào)查, 96%和88%的樣點(diǎn)總毒性當(dāng)量濃度(TEQs)分別超過(guò)加拿大和美國(guó)環(huán)境保護(hù)局(EPA)沉積物質(zhì)量指導(dǎo)值,表明蘇南城市群沉積物二噁英及DLCs具有一定生態(tài)風(fēng)險(xiǎn)。夏潔[13]對(duì)長(zhǎng)江和太湖流域環(huán)境介質(zhì)中二噁英類(lèi)活性進(jìn)行篩查,發(fā)現(xiàn)水樣在濃度設(shè)置范圍內(nèi)(濃縮50倍)均無(wú)二噁英活性,而所有沉積物釆樣點(diǎn)均表現(xiàn)出顯著的二噁英活性,且高于美國(guó)沉積物的二噁英類(lèi)物質(zhì)的風(fēng)險(xiǎn)閾值(30 pg·g-1,干重),表明太湖和長(zhǎng)江沉積物具有一定的生態(tài)和健康風(fēng)險(xiǎn)。Zhang等[91]對(duì)中國(guó)珠江三角地區(qū)的土壤進(jìn)行二噁英類(lèi)物質(zhì)調(diào)查發(fā)現(xiàn),62個(gè)樣品中有27.4%的樣品超過(guò)了加拿大的背景濃度。

基于A(yíng)hR介導(dǎo)的AOP的構(gòu)建為研究新型化合物的作用模式、生態(tài)毒性預(yù)測(cè)及物種間敏感性研究提供了指導(dǎo)性的框架。面對(duì)種類(lèi)和數(shù)量急劇增長(zhǎng)的各種新型化學(xué)物質(zhì),探索其中是否存在新AhR配體及物種敏感性是二噁英及DLCs毒理學(xué)研究的重要方向之一。而AOP正好為此提供了支撐,可以實(shí)現(xiàn)從作用機(jī)制到最終生態(tài)毒性的反推。我們可以設(shè)想:根據(jù)新型化合物的關(guān)鍵作用模式,即分子啟動(dòng)事件,和現(xiàn)有已知化合物的AOP模式的進(jìn)行比對(duì),并輔以相應(yīng)的細(xì)胞、器官、個(gè)體及種群水平上的比較,從而能為識(shí)別此新型化合物和推斷相應(yīng)的物種敏感性提供可能。進(jìn)而也許無(wú)需傳統(tǒng)的生態(tài)毒理學(xué)實(shí)驗(yàn)即可進(jìn)一步推斷此新型化合物可能導(dǎo)致的最終結(jié)局效應(yīng),最終可為實(shí)現(xiàn)此類(lèi)化合物的生態(tài)毒性預(yù)測(cè)和風(fēng)險(xiǎn)評(píng)估提供一定指導(dǎo)。

然而要預(yù)測(cè)一個(gè)化合物是否能夠誘導(dǎo)類(lèi)二噁英毒性,存在以下幾種假設(shè):

(1)分子啟動(dòng)事件:化合物能夠結(jié)合和激活A(yù)hR；

(2)需要有顯著的效能；

(3)分子啟動(dòng)事件激活是高度專(zhuān)一的,且是最敏感的內(nèi)源性分子事件；

(4)由于A(yíng)hR蛋白(序列和構(gòu)象)基因多樣性,生態(tài)物種間的對(duì)二噁英的毒性敏感性是不同的。

4 AhR-AOP相關(guān)的檢測(cè)方法(Testing method of AhR-AOP)

要實(shí)現(xiàn)上述假設(shè),必須有相應(yīng)的檢測(cè)方法來(lái)支撐。主要包括生物檢測(cè)方法和化學(xué)分析方法兩大類(lèi)。生物檢測(cè)方法又包括報(bào)告基因法和酶活力誘導(dǎo)法(ethoxyresorufin-O-deethylase,EROD)、免疫法等。

目前基于細(xì)胞的的體外實(shí)驗(yàn)生物測(cè)試方法,能夠?yàn)闇y(cè)試新型二噁英物質(zhì)和物種敏感性提供有效的技術(shù)支持。而傳統(tǒng)上是通過(guò)大量的活體模式動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)的生態(tài)毒性類(lèi)型和大小來(lái)判斷其是否為類(lèi)二噁英物質(zhì),這種做法不僅違背動(dòng)物保護(hù)理論,工作量巨大[35],而且對(duì)當(dāng)今數(shù)量巨大并一直在增加的新型化合物來(lái)說(shuō),也是不現(xiàn)實(shí)的。這種利用體外實(shí)驗(yàn)的動(dòng)物替代性試驗(yàn)技術(shù),高效價(jià)廉,很好地減少了對(duì)活體動(dòng)物長(zhǎng)期測(cè)試的需要[40]。EROD法作為一種體外實(shí)驗(yàn)生物方法,是基于二噁英與AhR結(jié)合活化后,經(jīng)過(guò)一系列過(guò)程,通過(guò)測(cè)定激活7-乙氧基-異吩惡唑酮-脫乙基(7-ethoxyresorufin-O-deethylase,EROD)酶的活性,來(lái)了解二噁英激活A(yù)hR的能力。而由于EROD酶不能由肝細(xì)胞內(nèi)源表達(dá),故其表達(dá)量與二噁英的暴露量成存在定量關(guān)系。報(bào)告基因法是近年來(lái)根據(jù)AhR受體作用模式發(fā)展起來(lái)的體外細(xì)胞測(cè)試法(如H4IIE-luc報(bào)告基因法和鳥(niǎo)的AhR1-LRG報(bào)告基因法),與EROD法比,靈敏度更高,檢測(cè)周期更短,更適合大量樣品的篩選和半定量測(cè)定[92],能為快速發(fā)現(xiàn)這些新型二噁英物質(zhì)提供有效的技術(shù)支撐,故目前得到了更廣泛的應(yīng)用。報(bào)告基因法是應(yīng)用基因重組技術(shù),把哺乳動(dòng)物細(xì)胞的細(xì)胞色素P450基因(CYP1A1)和螢火蟲(chóng)熒光酶合成,作為報(bào)告基因重組到真核細(xì)胞內(nèi),當(dāng)二噁英及DLCs進(jìn)入細(xì)胞和AhR結(jié)合后,經(jīng)過(guò)一系列過(guò)程激活下游的熒光合成酶基因表達(dá)[22]。該測(cè)定系統(tǒng)合成的熒光素酶表達(dá)量及熒光強(qiáng)度與加入的二噁英及DLCs的量成正比,最終測(cè)定結(jié)果以ReP表示。鳥(niǎo)的AhR1-LRG報(bào)告基因法已經(jīng)在雞、環(huán)頸雉、日本鵪鶉、鸕鶿、黑足信天翁和游隼中應(yīng)用,并表現(xiàn)出顯著的敏感性差異。報(bào)告基因法與高通量測(cè)試技術(shù)結(jié)合,為快速探索環(huán)境介質(zhì)中大量存在的潛在二噁英活性物質(zhì)提供了更加有效的支撐[12,93]。報(bào)告基因法還可以和化學(xué)方法聯(lián)合,構(gòu)成用于識(shí)別關(guān)鍵致毒物的技術(shù),即基于毒性測(cè)試的污染物鑒別技術(shù)(effect directed analysis,EDA)[94]。Shi等[95]和Hu等[96]用EDA的方法分別對(duì)長(zhǎng)江水源水和中國(guó)東部不同地區(qū)的自來(lái)水廠(chǎng)出水、自來(lái)水、煮沸的自來(lái)水進(jìn)行了篩查分析,首先用生物測(cè)試方法進(jìn)行了生物活性測(cè)試,對(duì)高活性的樣品再進(jìn)行進(jìn)一步的分級(jí)分離,最后用化學(xué)儀器定量檢測(cè),最終識(shí)別出有機(jī)氯農(nóng)藥、鄰苯二甲酸二丁酯、鄰苯二甲酸二異辛酯、雙酚A、壬基酚、辛基酚等關(guān)鍵致毒物。細(xì)胞水平的相關(guān)指標(biāo),如T4的檢測(cè)可使用放射免疫法(RIA)和酶聯(lián)免疫法(ELISA)。傳統(tǒng)上,廣泛使用RIA研究鋸齒動(dòng)物的內(nèi)分泌和毒性,而目前,ELISA已經(jīng)成為鋸齒動(dòng)物研究常用的方法,對(duì)其分析測(cè)定則采用高效液相色譜(HLPC)和質(zhì)譜(MS)[97]。氧化應(yīng)激的測(cè)定主要通過(guò)檢測(cè)活性氧(ROS)、還原型谷胱甘肽(GSH)、脂質(zhì)過(guò)氧化、過(guò)氧化物等指標(biāo),ROS主要是用熒光進(jìn)行檢測(cè),不過(guò)目前出現(xiàn)了基于發(fā)光進(jìn)行檢測(cè)的技術(shù)。GSH水平可由HLPC、毛細(xì)管電泳(CE)檢測(cè)。另外通過(guò)熒光或吸收光檢測(cè)技術(shù)可檢測(cè)脂質(zhì)過(guò)氧化反應(yīng)。

5 AhR-AOP的物種敏感性(Species sensitivity of AhR-AOP)

由上述可知,AOP反映的是關(guān)鍵分子啟動(dòng)事件及隨后產(chǎn)生的細(xì)胞、器官、組織、個(gè)體和種群水平上效應(yīng)的框架,可為化合物的物種敏感性差異研究提供指導(dǎo),進(jìn)而據(jù)此或許可進(jìn)行跨物種外推的生態(tài)風(fēng)險(xiǎn)評(píng)估。二噁英及DLCs化合物對(duì)大多數(shù)脊椎動(dòng)物具有高毒性,但是不管在物種內(nèi)還是物種間敏感性上存在很大的不同[51,98-101]。主要是由于在長(zhǎng)期的進(jìn)化過(guò)程中,生物體的遺傳變異產(chǎn)生了多種AhR的變形體,雖然脊椎動(dòng)物的AhR結(jié)構(gòu)特性有著廣泛的保守性,但結(jié)構(gòu)上細(xì)微的差異會(huì)導(dǎo)致功能上巨大的不同[42],即導(dǎo)致了對(duì)二噁英及DLCs的生化和毒性效應(yīng)的敏感性差異[50,102]。而這種差異是由AhR配體結(jié)合區(qū)域的氨基酸序列和構(gòu)象的不同導(dǎo)致的。另外不同配體化合物由于不同的分子結(jié)構(gòu),導(dǎo)致其和AhR的結(jié)合力也不同,進(jìn)而產(chǎn)生不同的物種敏感性。

5.1 敏感性差異

已有大量研究表明,TCDD、PCB77和PCB126等其他二噁英類(lèi)物質(zhì)對(duì)鳥(niǎo)類(lèi)的毒效應(yīng)存在種間敏感性差異[103-106]。對(duì)于TCDD來(lái)說(shuō),發(fā)現(xiàn)雞在鳥(niǎo)類(lèi)中最敏感,日本鵪鶉的敏感性比雞低1 000倍[103]。而對(duì)于哺乳動(dòng)物,大鼠對(duì)TCDD的敏感性比雞高1個(gè)數(shù)量級(jí)左右[93,107]。而TCDD對(duì)人類(lèi)AhR的親和力通常比大多數(shù)標(biāo)準(zhǔn)的實(shí)驗(yàn)室嚙齒類(lèi)動(dòng)物低,研究發(fā)現(xiàn),人類(lèi)細(xì)胞的CYP1A1誘導(dǎo)的劑量效應(yīng)曲線(xiàn)在這些嚙齒類(lèi)動(dòng)物的右側(cè),大約10倍左右[104,108]。由TCDD引起的鮭魚(yú)和斑馬魚(yú)早期階段致死率敏感性相差40倍[109]。

圖2 二噁英及DLCs對(duì)各個(gè)物種的EC50值注:1.數(shù)據(jù)來(lái)自不同文獻(xiàn)[35,39,23,110],2.圖2-b是圖2-a的部分物質(zhì)數(shù)據(jù)圖。Fig.2 EC50value of dioxins and DLCs to different speciesNote:1.Dates are from different references[35,39,23,110], 2.Fig.2-b is a part of Fig.2-a.

圖2 是二噁英及DLCs對(duì)大鼠和3種鳥(niǎo)類(lèi)(雞、環(huán)頸雉、日本鵪鶉)的通過(guò)報(bào)告基因法得到的半效應(yīng)濃度(EC50)比較圖。顯示:

(1)對(duì)各個(gè)物種間敏感性比較,發(fā)現(xiàn)對(duì)大多數(shù)物質(zhì)來(lái)說(shuō),4種物種的敏感性具有一定的差異,濃度相差1～2個(gè)數(shù)量級(jí)左右,特別是對(duì)TCDD來(lái)說(shuō),在4個(gè)物種的EC50值相差一個(gè)數(shù)量級(jí)左右。對(duì)于大多數(shù)物質(zhì),3種鳥(niǎo)類(lèi)中,雞最敏感,環(huán)頸雉次之,日本鵪鶉最弱,分別相差一個(gè)數(shù)量級(jí)左右。而大鼠比雞的敏感性又高0～1個(gè)數(shù)量級(jí)左右。

(2)對(duì)于單個(gè)物種,不同二噁英及DLCs的物種敏感性也存在差異。除對(duì) 2,3,3',4,4',5,6-Hepta-CDPS、2,3,3',4,4'-PentaCB和2,3',4,4',5-PentaCB物種敏感性在一個(gè)數(shù)量級(jí)內(nèi),相差不大外,其他物質(zhì)的敏感性差別最大可達(dá)一個(gè)多數(shù)量級(jí)。

(3)TCDD、2,3,4,7,8-PentaCDF和2,3,7,8-TetraCDF的毒性最強(qiáng),2,4',5-TrisCDPS的毒性最弱。

通過(guò)圖2我們可推想,對(duì)于某未知新型化合物,若通過(guò)報(bào)告基因法進(jìn)行毒性測(cè)試,得到的EC50值在某個(gè)區(qū)間內(nèi),我們或許可以通過(guò)此EC50的所落區(qū)間范圍來(lái)判斷其所屬物質(zhì)類(lèi)型,實(shí)現(xiàn)物質(zhì)的半定性判斷,對(duì)化學(xué)分析方法起到輔助補(bǔ)充作用。另外,對(duì)某已知其結(jié)構(gòu)的化合物,通過(guò)其結(jié)構(gòu)上與其他化合物結(jié)構(gòu)的類(lèi)似性,可以推斷此化合物的相應(yīng)二噁英毒性。并且可以據(jù)此進(jìn)一步對(duì)此物質(zhì)的敏感性和毒性進(jìn)行跨物種外推。而要實(shí)現(xiàn)上述設(shè)想,最重要的前提是進(jìn)行更多化合物和更多物種的二噁英類(lèi)毒性測(cè)試,圖中的物質(zhì)數(shù)量太少,判斷結(jié)果存在很大不確定性。只有建立大量物質(zhì)和大量物種的毒性數(shù)據(jù)庫(kù),才能為更好的物質(zhì)毒性識(shí)別和判斷提供可靠的數(shù)據(jù)支撐。

5.2 敏感性差異原因

不同的二噁英及DLCs在不同物種間存在很大敏感性差異,導(dǎo)致這種差異的主要原因有兩點(diǎn):(1)化學(xué)物質(zhì)由于其分子結(jié)構(gòu)不同,導(dǎo)致其在同一物種的活性不同；(2)不同物種由于其AhR蛋白序列和構(gòu)象差異,導(dǎo)致同一化學(xué)物質(zhì)在不同物種中的活性不同。而后者是敏感性差異研究的主要關(guān)注點(diǎn)。

對(duì)于生態(tài)系統(tǒng),鳥(niǎo)類(lèi)、哺乳動(dòng)物及魚(yú)類(lèi)通常作為主要的研究對(duì)象,對(duì)于鳥(niǎo)和哺乳動(dòng)物的敏感性差異機(jī)制已經(jīng)比較清楚,而對(duì)于魚(yú)類(lèi),由于基因經(jīng)過(guò)長(zhǎng)期的進(jìn)化和多樣化,產(chǎn)生了多種AhR變異體,故其敏感性機(jī)制比較復(fù)雜,目前還不是很清楚。

5.2.1 鳥(niǎo)類(lèi)

對(duì)于鳥(niǎo)類(lèi)來(lái)說(shuō),AhR1配體LBD區(qū)域的氨基酸序列的差異是引起二噁英及DLCs效應(yīng)產(chǎn)生鳥(niǎo)類(lèi)種間敏感性差異的原因[103-106]。

為了進(jìn)一步說(shuō)明氨基酸差異在物種敏感性上所起的重要作用,研究發(fā)現(xiàn),鳥(niǎo)AhR1的LBD域的異亮氨酸324(Ile324)位點(diǎn)和絲氨酸380(Ser380)位點(diǎn)的差異決定著二噁英及DLCs誘導(dǎo)的鳥(niǎo)種間敏感性不同[16,106,110-111]。Karchner等[111]研究發(fā)現(xiàn)雞對(duì)二噁英類(lèi)物質(zhì)的敏感性明顯大于燕鷗(約250倍),為評(píng)估雞和歐燕的AhR1-LBD域的3個(gè)氨基酸殘基(雞的丙氨酸Ala 257、異亮氨酸Ile324和絲氨酸Ser380,分別對(duì)應(yīng)于歐燕的蘇氨酸Thr258、纈氨酸Val325和丙氨酸Ala381)對(duì)AhR特性的貢獻(xiàn),通過(guò)嵌合AhR蛋白和定點(diǎn)誘變,把歐燕AhR的每個(gè)相應(yīng)的殘基變?yōu)橄鄬?duì)應(yīng)的雞的殘基,來(lái)檢測(cè)其結(jié)合TCDD的能力和反式激活熒光報(bào)告基因的能力,結(jié)果顯示:野生型的歐燕結(jié)合TCDD的能力是雞的20%,歐燕AhR誘變型Thr258Ala(258位點(diǎn)的Thr誘變?yōu)锳la)結(jié)合TCDD的能力和野生型的相似,而歐燕的誘變型Val325Ile和Ala381Ser結(jié)合TCDD的能力趨于雞；并且反式激活熒光報(bào)告基因的能力的結(jié)果顯示, Thr258Ala誘變型不能激活熒光素酶的轉(zhuǎn)錄表達(dá),而誘變型Val325Ile和Ala381Ser能夠激活其轉(zhuǎn)錄表達(dá),所以雞的Ile324和Ser380兩個(gè)氨基酸位點(diǎn)決定此2種鳥(niǎo)對(duì)二噁英及DLCs的敏感性差異。Head等[106]為了進(jìn)一步證明這2個(gè)關(guān)鍵氨基酸差異在鳥(niǎo)敏感性上所起的關(guān)鍵作用,在Karchner研究的基礎(chǔ)上,選取了更多的鳥(niǎo)類(lèi)(12種)進(jìn)行研究,進(jìn)一步顯示了AhR1-LBD的Ile324和Ser380兩個(gè)關(guān)鍵氨基酸位點(diǎn)能夠預(yù)測(cè)更多鳥(niǎo)的敏感性差異。隨后Farmahin等[18]和 Manning等[110]對(duì) 86種鳥(niǎo)類(lèi)對(duì)二噁英及DLCs的敏感性和AhR1-LBD域的氨基酸位點(diǎn)進(jìn)行分析,并從256、257、297、324、337和380六個(gè)氨基酸位點(diǎn)中,又一次確認(rèn)了氨基酸位點(diǎn) Ile324和Ser380能夠影響86種鳥(niǎo)AhR1表達(dá)的敏感性。并據(jù)此將鳥(niǎo)類(lèi)分為三大類(lèi):高敏感型(Ⅰ型,Ile324_ Ser380)、中敏感型(Ⅱ型,Ile324_Ala380)和低敏感型(Ⅲ型,Val324_Ala380),各個(gè)敏感型的代表物種分別為雞、環(huán)形雉、日本鵪鶉。張睿[35]采用Farmahin[23]建立的鳥(niǎo)類(lèi)AhR1-LRG實(shí)驗(yàn)對(duì)MeO-/OH-PBDEs鳥(niǎo)類(lèi)特異的類(lèi)二噁英毒性及其類(lèi)二噁英毒性的鳥(niǎo)類(lèi)種間敏感性差異進(jìn)行了研究,結(jié)果表明,同一MeO-/OHPBDEs對(duì)不同鳥(niǎo)類(lèi)的二噁英毒性存在差異；且由分子動(dòng)力模擬可知,鳥(niǎo)類(lèi)AhR1的380和324氨基酸點(diǎn)位會(huì)影響4號(hào)螺旋的運(yùn)動(dòng)和構(gòu)象的變化,進(jìn)而影響AhR1的激活,從而導(dǎo)致MeO-/OH-PBDEs在鳥(niǎo)類(lèi)間的二噁英活性敏感性差異；且Ile324Val誘變型和Ser380Ala誘變型分別增加了雞AhR1-LBD的空腔體積[111],故二噁英及DLCs的敏感性差異可能與AhR蛋白空腔體積大小有關(guān),另外殘基側(cè)鏈上羥基的氫鍵等也可能影響二噁英及DLCs的敏感性差異。Farmahin等[18]也研究發(fā)現(xiàn),日本鵪鶉AhR1的氨基酸324位點(diǎn)從Val到Ile(Val324Ile)的誘變可以提高AhR的熒光素酶活性(約12倍)。通過(guò)對(duì)日本鵪鶉AhR1的氨基酸380位點(diǎn)從Ala到Ser的誘變可以分別提高TCDD和PeCDF的敏感性約25和3.5倍。并同時(shí)會(huì)導(dǎo)致AhR空腔體積的減少,引起殘基側(cè)鏈上羥基的氫鍵和雙氧橋或配體的氯原子進(jìn)行相互作用,這些變化可能使得配體-受體的相互作用更穩(wěn)定[18],從而導(dǎo)致二噁英及DLCs的種間敏感性產(chǎn)生差異。

5.2.2 哺乳動(dòng)物

研究發(fā)現(xiàn)人類(lèi)的AhR-LBD的苯丙氨酸318 (Phe318)位點(diǎn)和纈氨酸381(Val381)位點(diǎn)在二噁英的敏感性差異上起著關(guān)鍵作用。Goryo等[112]研究了人的AhR的5個(gè)氨基酸在敏感性差異上可能起到的作用,發(fā)現(xiàn)這些氨基酸通過(guò)誘變方法改變?yōu)锳la后,通過(guò)檢測(cè)它們的反激活效應(yīng)發(fā)現(xiàn)Phe318Ala完全丟失了活性,而其他點(diǎn)位的活性?xún)H稍微被損壞。并對(duì) 318鄰近位點(diǎn)進(jìn)行誘變發(fā)現(xiàn) Ile319Ala和His320Ala誘變型(小鼠的319位點(diǎn)和鳥(niǎo)的324位點(diǎn)是對(duì)等的)的活性也完全喪失,顯示這兩個(gè)氨基酸也在配體結(jié)合上起著重要作用[112-113]。Ema等[114]發(fā)現(xiàn)了人類(lèi)AhR的381位點(diǎn)(和小鼠AhR的375位點(diǎn)對(duì)等)在配體結(jié)合上的重要性,由381位點(diǎn)的Val到天門(mén)冬氨酸(Asp)的誘變完全喪失了人類(lèi)AhR的配體結(jié)合活性。故二噁英及DLCs對(duì)人類(lèi)敏感性差異的原因是 AhR-LBD的2個(gè)氨基酸位點(diǎn) Phe318和Val381的不同。

5.2.3 魚(yú)類(lèi)

和鳥(niǎo)類(lèi)和哺乳動(dòng)物不同的是,在長(zhǎng)期的進(jìn)化和演變中,魚(yú)類(lèi)的AhR產(chǎn)生了更多變異體,所以對(duì)魚(yú)類(lèi)來(lái)說(shuō),二噁英的種間敏感性和AhR氨基酸序列的類(lèi)似關(guān)系還沒(méi)有確定[52]。斑馬魚(yú)是已知的最不敏感的魚(yú)類(lèi),和已知的最敏感的魚(yú)類(lèi)虹鱒魚(yú)比,斑馬魚(yú)的胚胎對(duì)TCDD的敏感性低40倍[109,115]。在鳥(niǎo)類(lèi)中, AhR配體結(jié)合域的關(guān)鍵氨基酸殘基能預(yù)測(cè)二噁英類(lèi)化合物的物種敏感性,但不清楚是否類(lèi)似的預(yù)測(cè)關(guān)系存在于魚(yú)類(lèi)中[106]。因?yàn)殡m然二噁英效應(yīng)在魚(yú)類(lèi)中也是通過(guò)AhR介導(dǎo),但對(duì)于魚(yú)類(lèi)來(lái)說(shuō)動(dòng)力學(xué)更復(fù)雜。魚(yú)至少有3個(gè)AhR(AhR1、AhR2和AhR3),并且每個(gè)AhR又都包括多個(gè)亞型[52]。在大西洋鮭魚(yú)中,共有6種不同的AhR亞型被發(fā)現(xiàn),包括2個(gè)AhR1s(α、β)和4個(gè)AhR2s(α、β、γ、ζ)[116]。目前主要推測(cè)脊椎動(dòng)物,如一些魚(yú),包括大馬哈魚(yú),經(jīng)歷了古老基因復(fù)制,并且經(jīng)歷了二次復(fù)制,從而導(dǎo)致了魚(yú)類(lèi)的多種AhR分支和多種AhR亞型[117]。且基因復(fù)制后的完整冗余功能是不穩(wěn)定的,隨著時(shí)間推移會(huì)導(dǎo)致復(fù)制的基因的失活或功能差異[118]。另外魚(yú)類(lèi)間的AhR序列和鳥(niǎo)相比,保守性更低,保守性的缺乏使得識(shí)別結(jié)合配體的關(guān)鍵氨基酸更加困難[52]。

AhR表達(dá)的自動(dòng)調(diào)節(jié)和AhR蛋白的穩(wěn)定性已經(jīng)被證實(shí)和魚(yú)類(lèi)的敏感性有關(guān)。然而,目前還不清楚AhR2的上調(diào)是否對(duì)物種間的AhR激動(dòng)劑的效應(yīng)差異存在影響[119]。Wirgin等[120]推測(cè)了AhR蛋白構(gòu)象的不同是大西洋小鱈對(duì)二噁英及DLCs產(chǎn)生種間敏感性差異的決定因素。但研究中沒(méi)有發(fā)現(xiàn)大西洋小鱈的LBD氨基酸序列的不同和配體結(jié)合的親和力有關(guān),然而,LBD以外的氨基酸的檢測(cè)顯示影響蛋白的穩(wěn)定性,因此導(dǎo)致親和力的降低。

另外關(guān)于A(yíng)hRR、ARNT和Hsp蛋白的表達(dá)和功能在決定魚(yú)類(lèi)對(duì)二噁英及DLCs的敏感性差異方面的信息還不完善[120-121]。而魚(yú)類(lèi)鐘間還有AhR動(dòng)力學(xué)的其他未知差異,致使得魚(yú)種間對(duì)二噁英及DLCs敏感性差異的原因更加復(fù)雜。

6 展望(Prospect)

(1)雖然目前對(duì)二噁英及DLCs的毒理學(xué)的研究由來(lái)已久,包括一個(gè)經(jīng)典的核受體機(jī)制。但由于在物種漫長(zhǎng)的進(jìn)化過(guò)程中,AhR經(jīng)過(guò)了各種變異,雖然AhR結(jié)構(gòu)特性有著廣泛的保守性,但是將其具體致毒機(jī)制試圖描述清楚還比較困難,并且二噁英及DLCs的各種毒性數(shù)據(jù)還不齊全,故基于此建立的AOP還需要進(jìn)一步完善,從而為更好的風(fēng)險(xiǎn)評(píng)估提供有效的支持。

(2)二噁英及DLCs在不同的物種間存在顯著的敏感性差異,找到最敏感性物種是生態(tài)毒理的一個(gè)重要任務(wù),而要在我國(guó)進(jìn)行二噁英及DLCs對(duì)環(huán)境污染物的生態(tài)評(píng)估,必須加強(qiáng)本土物種的研究,建立基于本土物種的二噁英及DLCs毒性數(shù)據(jù),從而為風(fēng)險(xiǎn)評(píng)價(jià)及基準(zhǔn)和標(biāo)準(zhǔn)的制定提供更加有效的數(shù)據(jù)支持。

(3)本文介紹了最近幾年所發(fā)現(xiàn)的環(huán)境中的新型二噁英物質(zhì)。而目前環(huán)境中的污染物數(shù)量巨大,并且一小部分物質(zhì)具有相關(guān)的毒性數(shù)據(jù),而大部分物質(zhì)的毒性數(shù)據(jù)非常缺乏。故應(yīng)該進(jìn)一步開(kāi)展新型物質(zhì)的毒性篩查工作,探索新型二噁英物質(zhì)。

(4)環(huán)境中有大量的有機(jī)污染具有潛在二噁英類(lèi)毒性和生態(tài)風(fēng)險(xiǎn)。基于物種特異性的報(bào)告基因技術(shù),不僅可以用于檢測(cè)復(fù)合暴露條件下有機(jī)污染物的二噁英類(lèi)物質(zhì)的TEQs,并以EDA方法來(lái)鑒別關(guān)鍵有毒物質(zhì)；此外,采用本土敏感性物種的報(bào)告基因法的檢測(cè)策略,還可以預(yù)測(cè)二噁英類(lèi)毒性物質(zhì)的生態(tài)風(fēng)險(xiǎn)。

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Research Progress on Dioxin-like Compounds and AhR-Mediated Adverse Outcome Pathway(AOP)

Wei Fenghua,Zhang Junjiang,Xia Pu,Zhang Xiaowei*,Yu Hongxia
State Key Laboratory of Pollution Control and Resource Reuse,School of the Environment,Nanjing University,Nanjing 210023,China

12 April 2015 accepted 6 July 2015

Dioxins and DLCs(dioxin-like compounds,DLCs)are normally considered as a class of highly toxic compounds and their toxicological mechanisms have been extensively studied in the past decades.It is well accepted that all highly toxic dioxins and DLCs exert their toxic and biological interference effects through the binding and activation of Aryl hydrocarbon receptor(AhR).Recently increasing evidences showed that many emerging environmental pollutants had similar structure as dioxins and could potentially induce high toxicity.There is widely regulatory concern that how to efficiently and effectively evaluate the hazard and ecological risk to local species by this large amount of untested chemicals.Here this paper reviewed 1)discovery of emerging dioxin-like pollutants, 2)recent development on the understanding of AhR related toxicological mechanism,3)development of AhR-mediated adverse outcome pathway(AOP),4)development of predictive method to assess the AhR mediated toxicity in local species.A perspective was provided on future research direction in the ecotoxicological and risk assessment of dioxins and DLCs.

DLCs;emerging pollutants;PBDEs;PCDPSs;AhR;toxicological mechanism;species sensitivity distribution

2015-04-12 錄用日期:2015-07-06

1673-5897(2016)1-037-15

X171.5

A

10.7524/AJE.1673-5897.20150412001

魏鳳華,張俊江,夏普,等.類(lèi)二噁英物質(zhì)及芳香烴受體(AhR)介導(dǎo)的有害結(jié)局路徑(AOP)研究進(jìn)展[J].生態(tài)毒理學(xué)報(bào),2016,11(1):37-51

Wei F H,Zhang J J,Xia P,et al.Research progress on dioxin-like compounds and AhR-mediated adverse outcome pathway(AOP)[J].Asian Journal of Ecotoxicology,2016,11(1):37-51(in Chinese)

國(guó)家重大“水專(zhuān)項(xiàng)”(2012ZX07506)；環(huán)保公益性行業(yè)科研專(zhuān)項(xiàng)經(jīng)費(fèi)項(xiàng)目(201209016)；高校博士學(xué)科點(diǎn)專(zhuān)項(xiàng)科研基金(20120091110034)

魏鳳華(1986-),女,碩士,研究方向?yàn)樯鷳B(tài)毒理及風(fēng)險(xiǎn)評(píng)價(jià)研究,E-mail:fenghua0722@163.com；

),E-mail:zhangxw@nju.edu.cn

簡(jiǎn)介:張效偉(1978—)，男，動(dòng)物學(xué)和環(huán)境毒理學(xué)博士、教授、博士生導(dǎo)師，主要從事生態(tài)毒理學(xué)和健康風(fēng)險(xiǎn)評(píng)估方面的研究，發(fā)表英文SCI論文80多篇，包括以一作或通訊作者在環(huán)境領(lǐng)域高影響刊物Environmental Science& Technology和Toxicological Science等上發(fā)表論文20多篇。