陳欽暢，譚皓月，史薇，于紅霞

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

計(jì)算毒理學(xué)在內(nèi)分泌干擾物篩選上的應(yīng)用和展望

陳欽暢，譚皓月，史薇，于紅霞*

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

內(nèi)分泌干擾物通過干擾內(nèi)分泌系統(tǒng)導(dǎo)致多種疾病，如生殖疾病、肥胖癥甚至癌癥。然而，面對(duì)環(huán)境中大量潛在的內(nèi)分泌干擾物，傳統(tǒng)的體外、體內(nèi)評(píng)估方法由于成本高、耗時(shí)長(zhǎng)等問題，難以實(shí)現(xiàn)內(nèi)分泌干擾物的高通量篩查。計(jì)算毒理學(xué)逐漸發(fā)展成為被美國(guó)環(huán)保局(Environmental Protection Agency, EPA)、經(jīng)濟(jì)合作與發(fā)展組織(Organization for Economic Co-operation and Development, OECD)等機(jī)構(gòu)所推薦的內(nèi)分泌干擾物篩選與預(yù)測(cè)方法。本文綜述了計(jì)算毒理學(xué)在內(nèi)分泌干擾物篩選上的進(jìn)展，主要包括分子對(duì)接和分子動(dòng)力學(xué)模擬的應(yīng)用，并對(duì)有害結(jié)局路徑(adverse outcome pathway, AOP)的方法進(jìn)行介紹和展望。

計(jì)算毒理學(xué)；內(nèi)分泌干擾；分子對(duì)接；分子動(dòng)力學(xué)模擬；AOP

Received6 February 2017accepted13 March 2017

Abstract: Endocrine disrupting chemicals (EDCs) cause a variety of diseases, such as reproductive diseases, obesity and even cancer, by interfering with the endocrine system. However, in the face of a large number of potential endocrine disruptors in the environment, traditional in vitro and in vivo assays are difficult to achieve high throughput screening of endocrine disruptors due to their high cost and time consuming. Computational toxicology has been recommended as the screening and predicting method by the US Environmental Protection Agency (EPA), the Organization for Economic Co-operation and Development (OECD) and so on. Here, we discuss the application of computational toxicology methods, particularly molecular docking, molecular dynamics simulations and the developing adverse outcome pathway (AOP), in guiding the screening of EDCs.Keywords: computational toxicology; endocrine disrupting; molecular docking; molecular dynamics; AOP

1 前言(Introduction)

1.1 內(nèi)分泌干擾效應(yīng)與內(nèi)分泌干擾物

內(nèi)分泌系統(tǒng)指由一系列腺體分泌激素進(jìn)入內(nèi)循環(huán)系統(tǒng)運(yùn)輸并直接作用于目標(biāo)器官的系統(tǒng)。內(nèi)分泌系統(tǒng)的信號(hào)由激素傳遞，激素有不同的化學(xué)結(jié)構(gòu)，主要包括3種：類花生酸類(eicosanoids)、甾體類(steroids)和氨基酸衍生物(胺類、肽鏈和蛋白質(zhì))。激素通過與目標(biāo)細(xì)胞的特定受體蛋白結(jié)合，激活信號(hào)轉(zhuǎn)導(dǎo)通路，達(dá)到調(diào)節(jié)細(xì)胞功能的作用，調(diào)節(jié)著生物體幾乎所有生物學(xué)過程。已有報(bào)道表明，一些化學(xué)物質(zhì)如雙酚A(bisphenol A, BPA)、多環(huán)芳烴(polycyclic aromatic hydrocarbons, PAHs)及一系列殺蟲劑等的暴露會(huì)干擾內(nèi)分泌系統(tǒng)并產(chǎn)生有害影響，這類物質(zhì)被稱為內(nèi)分泌干擾物(endocrine disrupting chemicals, EDCs)。內(nèi)分泌干擾物的暴露會(huì)增加生殖疾病、心肺疾病、免疫系統(tǒng)疾病和神經(jīng)系統(tǒng)疾病的風(fēng)險(xiǎn)，甚至導(dǎo)致腫瘤和癌癥的發(fā)生[1]。據(jù)Attina等[2]的模型估計(jì)，2010年歐盟國(guó)家內(nèi)分泌干擾物導(dǎo)致的疾病花費(fèi)占國(guó)內(nèi)生產(chǎn)總值(GDP)的1.28%，為2 170億美元，而美國(guó)的達(dá)到3 400億美元，占GDP的2.33%，內(nèi)分泌干擾物篩選的研究迫在眉睫。

1.2 內(nèi)分泌干擾物的實(shí)驗(yàn)檢測(cè)手段

檢測(cè)內(nèi)分泌干擾物的實(shí)驗(yàn)手段包括體外(in vitro)和體內(nèi)(in vivo)實(shí)驗(yàn)。體外實(shí)驗(yàn)包括細(xì)胞增殖實(shí)驗(yàn)(cell proliferation assays)[3]、報(bào)告基因?qū)嶒?yàn)(reporter gene assays)[4]、酵母雙雜交實(shí)驗(yàn)(yeast two-hybrid assays)[5]、結(jié)合實(shí)驗(yàn)(binding assays)[6]等。體內(nèi)實(shí)驗(yàn)多采用哺乳動(dòng)物[7-8]、鳥類[9-10]、魚類[11-12]、兩棲類[13-14]等動(dòng)物。過去十幾年來，體外實(shí)驗(yàn)被合理開發(fā)應(yīng)用到各種高通量測(cè)試篩選方法中，以應(yīng)對(duì)數(shù)量巨大的潛在危害化合物，以及動(dòng)物體內(nèi)測(cè)試的巨大開銷和倫理問題[15]。由美國(guó)環(huán)保局(Environmental Protection Agency, EPA)、國(guó)立衛(wèi)生研究院(National Institutes of Health, NIH)和食品與藥品管理局(Food and Drug Administration, FDA)等跨部門合作的21世紀(jì)毒理學(xué)(Tox21)項(xiàng)目，采用高通量篩選技術(shù)測(cè)試約10 000種環(huán)境化合物和藥物的毒性，其中內(nèi)分泌干擾是重要方面，關(guān)于雌激素干擾效應(yīng)及信號(hào)通路的研究結(jié)果已于2014年發(fā)布[16]。

現(xiàn)階段評(píng)估一個(gè)化合物的內(nèi)分泌干擾作用往往需要體內(nèi)和體外實(shí)驗(yàn)的結(jié)合。美國(guó)EPA內(nèi)分泌干擾物篩選項(xiàng)目(Endocrine Disruptor Screening Program, EDSP)于2012年開展了EDSP21項(xiàng)目，采用2個(gè)級(jí)別的篩選測(cè)試手段(Tier 1和Tier 2)評(píng)價(jià)化合物的內(nèi)分泌干擾活性。其中級(jí)別1測(cè)試包括5種體外實(shí)驗(yàn)和5種體內(nèi)實(shí)驗(yàn)，級(jí)別2則是更深層次或多代體內(nèi)測(cè)試。經(jīng)過級(jí)別1測(cè)試具有干擾活性的化合物進(jìn)入級(jí)別2測(cè)試，評(píng)估其內(nèi)分泌干擾效應(yīng)。目前，超過1 800種化合物內(nèi)分泌相關(guān)活性的高通量篩選數(shù)據(jù)(主要為包括雌激素受體ER、雄激素受體AR結(jié)合和轉(zhuǎn)錄激活的級(jí)別1體外篩查數(shù)據(jù))已可在EDSP21 Dashboard網(wǎng)站(http://actor.epa.gov/edsp21/)獲取。

1.3 內(nèi)分泌干擾物的模擬預(yù)測(cè)手段

體外和體內(nèi)實(shí)驗(yàn)手段雖然能完整評(píng)價(jià)化合物的內(nèi)分泌干擾效應(yīng)，但是其成本高、耗時(shí)長(zhǎng)，難以對(duì)全球現(xiàn)有超過126 000 000種化合物參考(http://www.cas.org)進(jìn)行逐一篩選。因此，亟需發(fā)展化學(xué)品內(nèi)分泌干擾效應(yīng)篩選的計(jì)算毒理學(xué)(computational toxicology)方法[17]。計(jì)算毒理學(xué)方法指通過綜合體內(nèi)、體外實(shí)驗(yàn)和計(jì)算機(jī)模擬等不同來源的數(shù)據(jù)，開發(fā)數(shù)學(xué)或計(jì)算機(jī)模型，以更好理解或預(yù)測(cè)化合物干擾效應(yīng)的方法[18]。近年來，計(jì)算毒理學(xué)得到越來越多的關(guān)注，美國(guó)EPA于2005年成立了國(guó)家計(jì)算毒理學(xué)中心(National Center for Computational Toxicology, NCCT)，致力于開發(fā)新的評(píng)估化合物安全性的方法，即計(jì)算毒理方法；經(jīng)濟(jì)合作與發(fā)展組織(Organization for Economic Co-operation and Development, OECD)于2008年開發(fā)的計(jì)算毒理軟件OECD QSAR Toolbox如今也進(jìn)入3.4版本，并得到各國(guó)政府、化學(xué)工業(yè)的接受和使用。

定量結(jié)構(gòu)-效應(yīng)關(guān)系(quantitative structure-activity relationship, QSAR)是最早開發(fā)和發(fā)展的計(jì)算毒理學(xué)方法，將代表化合物結(jié)構(gòu)、物理、化學(xué)性質(zhì)的分子描述符(molecular descriptors)與特定效應(yīng)終點(diǎn)或有害結(jié)局(adverse outcome, AO)建立聯(lián)系，達(dá)到預(yù)測(cè)目的，在不同尺度的內(nèi)分泌干擾效應(yīng)，如核受體結(jié)合[19-20]、轉(zhuǎn)錄激活[21-22]、器官和個(gè)體有害結(jié)局[23-24]等，都得到廣泛運(yùn)用。我國(guó)陳景文教授、張愛茜教授、高士祥教授、王連生教授和于紅霞教授等的團(tuán)隊(duì)在內(nèi)分泌干擾物的QSAR研究上都做了大量工作[25-29]，比如Li等[25]計(jì)算了517種有機(jī)化合物的705個(gè)分子描述符，并選取其中的13個(gè)分子描述符建立雌激素效應(yīng)的QSAR模型，發(fā)現(xiàn)有機(jī)分子的雌激素活性主要與分子尺寸、形狀特征、電負(fù)性和范德華體積等相關(guān)。如今，QSAR已經(jīng)發(fā)展成較為成熟的計(jì)算毒理學(xué)方法，得到OECD等組織的認(rèn)可，基于QSAR開發(fā)的毒性預(yù)測(cè)軟件，如TEST、ECOSAR、OncoLogic等，也得到廣泛應(yīng)用。然而，QSAR往往忽略干擾物的效應(yīng)機(jī)制，采用的分子描述符往往也沒有直接或明確的藥理學(xué)或生物學(xué)意義[30]。

激素分子與體內(nèi)調(diào)節(jié)相關(guān)生理功能的大分子，如受體蛋白等之間的相互關(guān)系在內(nèi)分泌系統(tǒng)信號(hào)傳遞中具有重要作用。因此對(duì)干擾物與受體作用關(guān)系的研究是內(nèi)分泌干擾物篩選的重要研究手段，很多體外實(shí)驗(yàn)都是以干擾物與受體作用關(guān)系為對(duì)象研究化合物的內(nèi)分泌干擾效應(yīng)的[31-32]，而分子對(duì)接(molecular docking)和分子動(dòng)力學(xué)(molecular dynamics, MD)模擬方法作為基于干擾物與受體作用關(guān)系的計(jì)算毒理學(xué)研究方法也得到越來越多的應(yīng)用。在此基礎(chǔ)上，為了更加深入地理解內(nèi)分泌干擾效應(yīng)作用機(jī)制，OECD、美國(guó)EPA等組織開展了開發(fā)有害結(jié)局路徑(adverse outcome pathway, AOP)的項(xiàng)目，將極大促進(jìn)基于效應(yīng)機(jī)制的計(jì)算毒理學(xué)的發(fā)展。因此，本文將對(duì)分子對(duì)接、MD模擬和AOP等基于效應(yīng)機(jī)制的計(jì)算毒理學(xué)方法在內(nèi)分泌干擾物篩選上的應(yīng)用進(jìn)行綜述。

2 分子對(duì)接及其應(yīng)用(Molecular docking and its applications)

分子對(duì)接是預(yù)測(cè)配體與受體結(jié)合成穩(wěn)定復(fù)合體時(shí)配體所處的最佳位置和方向的方法[33]。內(nèi)分泌系統(tǒng)中主要的受體蛋白(圖1)是包括雌激素受體(estrogen receptor, ER)、雄激素受體(androgen receptor, AR)、甲狀腺激素受體(thyroid hormone receptor, TR)、糖皮質(zhì)激素受體(glucocorticoid receptor, GR)等在內(nèi)的核受體(nuclear receptor, NR)，它們都受激素調(diào)節(jié)并控制大量基因的表達(dá)[34]。隨著晶體學(xué)和生物化學(xué)技術(shù)的發(fā)展，越來越多核受體的晶體結(jié)構(gòu)被解析出來(圖1)[35-39]，這些晶體結(jié)構(gòu)都可以通過Protein Data Bank網(wǎng)站(http://www.rcsb.org/pdb/home/home.do)獲得，使采用對(duì)接和MD模擬等方法篩選內(nèi)分泌干擾物成為可能[40]。但已有的多為人類的受體，對(duì)于其他物種，往往需要通過同源建模(homology modeling)構(gòu)建受體結(jié)構(gòu)[41]。

分子對(duì)接的使用有助于加深對(duì)配體受體相互作用機(jī)制的理解。Nose等[42]用對(duì)接的方法從14個(gè)酚類物質(zhì)中篩選出4-(1-adamantyl)phenol為擬雌激素物質(zhì)，經(jīng)驗(yàn)證確實(shí)具有很強(qiáng)的雌激素活性。對(duì)對(duì)接結(jié)果的分析發(fā)現(xiàn)，與雌激素類似，4-(1-adamantyl)phenol的羥基也能與ERα中Glu353和Arg394氨基酸形成氫鍵。D'Ursi等[43]采用柔性對(duì)接的方法探索了內(nèi)分泌干擾物與ER、孕酮受體(progesterone receptor, PR)和AR的相互作用，發(fā)現(xiàn)這些內(nèi)分泌干擾物與受體的相互作用主要取決于化合物與配體結(jié)合腔(ligand binding cavity, LBC)中多個(gè)氨基酸殘基之間的疏水性作用，對(duì)于親脂性內(nèi)分泌干擾物，它們有能力適應(yīng)甾體受體的疏水性LBC，并呈現(xiàn)非特異性結(jié)合模式。

圖1 部分已解析的核受體結(jié)構(gòu)[35-39]Fig. 1 Some of the structures of nuclear receptors (NRs) that have been refined[35-39]

由于內(nèi)分泌干擾物往往能同時(shí)作用于不同受體，研究干擾物與不同受體的相互作用能預(yù)測(cè)潛在的效應(yīng)終點(diǎn)。Yuriev等[45]針對(duì)人體14種NR，選取了18個(gè)完整、可靠的晶體結(jié)構(gòu)建立對(duì)接模型，通過對(duì)接得到化合物與各個(gè)NR的結(jié)合能，判斷對(duì)哪些受體更敏感，從而判斷化合物潛在的內(nèi)分泌干擾效應(yīng)[44]，這種方法被稱為反向?qū)臃āＳ诩t霞教授團(tuán)隊(duì)最近也采用反向?qū)娱_發(fā)了一個(gè)針對(duì)39個(gè)人類NR的程序SPEN，該程序經(jīng)過10種化合物的驗(yàn)證具有良好的表現(xiàn)[46]。

分子對(duì)接可以與QSAR結(jié)合，構(gòu)建多維QSAR模型。Vedani等[47]采用柔性對(duì)接和QSAR結(jié)合的方法建立了預(yù)測(cè)ER結(jié)合能力的6維QSAR模型，并用于對(duì)106種內(nèi)分泌干擾物的篩選，結(jié)果r2達(dá)到0.885，表明具有很好的篩選能力。Vedani團(tuán)隊(duì)將這種方法拓展到AR、TR、GR等11個(gè)核受體，并建立了基于網(wǎng)站的預(yù)測(cè)平臺(tái)VirtualToxLab[48]。分子對(duì)接在藥物開發(fā)領(lǐng)域也產(chǎn)生了一些新方法，如基于分子碎片的對(duì)接技術(shù)正在快速發(fā)展，雖然在內(nèi)分泌干擾物篩選上還沒有得到應(yīng)用，但是不失為提高對(duì)接結(jié)果精確度的選擇[49]。然而分子對(duì)接在很大程度上仍受受體柔性的制約，而對(duì)受體賦予過多的柔性會(huì)導(dǎo)致對(duì)計(jì)算的要求呈指數(shù)增加并且變得不切實(shí)際[50]。

在實(shí)際應(yīng)用中分子對(duì)接受受體結(jié)構(gòu)和配體種類的影響較大，針對(duì)相同種類、數(shù)量較少的化合物時(shí)，分子對(duì)接能得到較好的預(yù)測(cè)效果，對(duì)接的結(jié)合能可以很好預(yù)測(cè)化合物與受體的結(jié)合效力[51]；而針對(duì)大量具有不同結(jié)構(gòu)的化合物時(shí)，預(yù)測(cè)效果一般，甚至比普通的QSAR模型差[52]。因此，選擇合適、可靠的受體模型是分子對(duì)接模型構(gòu)建的重點(diǎn)。針對(duì)不同種類的化合物提供不同對(duì)接模型是可行的解決方法；也可以借助分子動(dòng)力學(xué)模擬，產(chǎn)生多個(gè)受體模型進(jìn)行對(duì)接[53]。

3 分子動(dòng)力學(xué)模擬及其應(yīng)用(Molecular dynamics simulations and the applications)

隨著計(jì)算機(jī)技術(shù)的發(fā)展和計(jì)算能力的提升，分子動(dòng)力學(xué)模擬逐漸成為研究生物大分子作用的標(biāo)準(zhǔn)方法[54]。分子動(dòng)力學(xué)模擬是研究原子和分子物理運(yùn)動(dòng)的計(jì)算機(jī)模擬方法，所有分子和原子在給定的時(shí)間范圍內(nèi)相互作用，形成一個(gè)動(dòng)態(tài)變化的系統(tǒng)，以此研究生物分子之間的相互作用[55]。

MD模擬有助于探索干擾物作用下受體蛋白及配體本身的構(gòu)象變化。Li等[56]用MD模擬研究了內(nèi)分泌干擾物的雌激素干擾效應(yīng)，發(fā)現(xiàn)干擾物與ER在2 ns的模擬下都能達(dá)到穩(wěn)定狀態(tài)，并且干擾效力更強(qiáng)的化合物能與ER的His524氨基酸穩(wěn)定形成氫鍵。采用MD模擬還能發(fā)現(xiàn)蛋白質(zhì)的關(guān)鍵結(jié)構(gòu)及活性產(chǎn)生的關(guān)鍵變化[57]。Wang等[58]通過對(duì)AR骨架變構(gòu)情況的比較發(fā)現(xiàn)12號(hào)螺旋(Helix 12, H12)在MD模擬過程中具有最顯著的位置變化，認(rèn)為H12的位置變化是抗雄激素活性產(chǎn)生的關(guān)鍵。Wang等[58]還發(fā)現(xiàn)H12在10 ns模擬時(shí)間內(nèi)達(dá)到平衡是抗雄性活性產(chǎn)生的重要特征，且穩(wěn)定時(shí)間與活性強(qiáng)弱呈負(fù)相關(guān)。

干擾物與受體蛋白LBC的結(jié)合情況仍然是MD研究的重點(diǎn)。Martínez等[59]通過MD模擬發(fā)現(xiàn)了配體逃離TR-LBC的3種可能途徑。有學(xué)者進(jìn)一步用操縱分子動(dòng)力學(xué)(steered molecular dynamics, SMD)模擬探究配體從各逃離途徑逃離配體結(jié)合腔的難易程度[60-62]。Martínez等[61]發(fā)現(xiàn)配體逃離TR的最佳途徑是位于H1、H2和H3處的通道3，而且當(dāng)配體親水部分能與受體外部的水分子接觸時(shí)逃離過程會(huì)變得更輕松。另一方面，有研究表明，干擾物的誘導(dǎo)能使受體H12的位置發(fā)生變化，而其穩(wěn)定位置正好擋住配體，使配體無法從LBC中逃離，受體形成的這種結(jié)構(gòu)稱為“老鼠夾(mousetrap)”結(jié)構(gòu)[56, 63]。

熱力學(xué)計(jì)算也是MD模擬的常用分析方法。采用MM/PBSA或MM/GBSA(molecular mechanics with Poisson-Boltzmann or generalized Born and surface area)方法計(jì)算配體-受體結(jié)合自由能ΔGbinding可用于預(yù)測(cè)配體與受體間的結(jié)合效力。van Lipzig等[64]將計(jì)算得到的雌激素干擾物與ER的結(jié)合自由能和實(shí)驗(yàn)測(cè)得的結(jié)合效力比較，發(fā)現(xiàn)兩者的相關(guān)系數(shù)達(dá)到0.94。結(jié)合自由能還能區(qū)分干擾物對(duì)受體不同亞型的選擇性[65-66]。Martínez等[65]分別計(jì)算了配體Triac與TRα和TRβ相互作用的結(jié)合自由能，發(fā)現(xiàn)Triac與TRα的結(jié)合自由能顯著低于TRβ，導(dǎo)致其對(duì)TRα具有高度選擇性。

除了干擾物與核受體的結(jié)合，核受體與其他蛋白質(zhì)的相互作用，如與共調(diào)節(jié)因子作用、二聚現(xiàn)象等[67-69]，也是影響內(nèi)分泌干擾效應(yīng)產(chǎn)生的重要過程。研究表明ER的二聚作用大大抑制了E2逃離LBC[69]。于紅霞教授團(tuán)隊(duì)[67]最近的MD研究也表明，共調(diào)節(jié)因子在化合物甲狀腺激素干擾活性產(chǎn)生過程具有重要作用，抗甲狀腺激素干擾物與TR結(jié)合能促進(jìn)共抑制因子而不是共激活因子與TR結(jié)合，從而導(dǎo)致抗性的產(chǎn)生。因此，考慮蛋白質(zhì)受體與其他調(diào)劑因子的作用過程，對(duì)生物大分子間作用，如共調(diào)節(jié)因子結(jié)合、二聚作用和與DNA的結(jié)合等進(jìn)行模擬，是MD模擬在內(nèi)分泌干擾物篩選上的重要發(fā)展方向。另外，在MD模擬中采用量子力學(xué)/分子力學(xué)(QM/MM)耦合的方法，將配體部分用QM計(jì)算，其他部分用MM模擬的方法，有助于提高模擬的精確度，并有利于更深入探索配體受體之間的相互作用。陳景文教授團(tuán)隊(duì)[70]采用QM/MM的方法，探索了電中性和陰離子形態(tài)下酚類內(nèi)分泌干擾物與甲狀腺素運(yùn)載蛋白(transthyretin, TTR)的結(jié)合，發(fā)現(xiàn)陰離子形態(tài)比電中性的酚類物質(zhì)與TTR結(jié)合更強(qiáng)，認(rèn)為離子形態(tài)的考慮是內(nèi)分泌干擾物虛擬篩選過程不可忽視的機(jī)制。

4 AOP的發(fā)展和展望(Development and prospect of AOP)

隨著對(duì)效應(yīng)機(jī)制理解的不斷深化，AOP概念逐漸發(fā)展起來。AOP就是描繪從分子啟動(dòng)事件(MIE)的開始，由一系列關(guān)鍵事件(KE)和之間關(guān)系(KER)連接，到有害結(jié)局(AO)之間關(guān)系的框架[71]，與AOP相關(guān)的各種概念如表1所示。內(nèi)分泌干擾效應(yīng)的產(chǎn)生不只是干擾物與靶標(biāo)相互作用，還包括生物大分子間、細(xì)胞層次、器官層次的變化(如圖2)，因此，對(duì)AOP的研究有助于獲得更加精確和透徹的預(yù)測(cè)效果。然而這種預(yù)測(cè)方法是建立在對(duì)干擾效應(yīng)作用通路足夠明晰的基礎(chǔ)上，這也是目前面臨的最大挑戰(zhàn)[72]。隨著有害結(jié)局路徑知識(shí)庫(Adverse Outcome Pathway Knowledge Base, AOP-KB: http://aopkb.org/)的建立，越來越多AOP被開發(fā)并在AOP-KB平臺(tái)上共享，這將大大促進(jìn)AOP在計(jì)算毒理學(xué)預(yù)測(cè)上的應(yīng)用。

表1 與有害結(jié)局路徑(AOP)相關(guān)概念的定義[71]Table 1 Definition of concepts relevant to adverse outcome pathway (AOP)[71]

圖3 雄激素受體激動(dòng)效應(yīng)導(dǎo)致生殖紊亂的AOP[73]Fig. 3 AOP: Androgen receptor agonism leading to reproductive dysfunction[73]

目前已有的AOP為20個(gè)，其中包括與雌激素、雄激素、甲狀腺激素受體等相關(guān)的5個(gè)內(nèi)分泌干擾AOP。Villeneuve[73]開發(fā)并發(fā)表了有關(guān)雄激素受體激動(dòng)效應(yīng)導(dǎo)致生殖功能紊亂的AOP(AOP23，如圖3所示)，干擾物激活雄激素受體(MIE)，導(dǎo)致睪酮、雌激素合成下降，血液雌激素濃度降低，進(jìn)而肝臟卵黃原蛋白合成下降，血液中卵黃原蛋白濃度降低，卵母細(xì)胞吸收量減少，進(jìn)而使產(chǎn)卵下降，最終導(dǎo)致生殖功能紊亂的AO產(chǎn)生。Villeneuve等[74]還總結(jié)了AOP開發(fā)的五大原則：(1)AOP不具有化合物特異性；(2)AOP是模塊化的，且構(gòu)成要素可重復(fù)利用；(3)每個(gè)獨(dú)立的AOP都由單一系列的KE和KER構(gòu)成；(4)由具有相同KE和KER的AOP構(gòu)成的網(wǎng)絡(luò)(圖2)是預(yù)測(cè)真實(shí)世界情況的基礎(chǔ)單元；(5)AOP是可以隨著新認(rèn)識(shí)的形成而不斷演化的。這些原則的考慮有助于對(duì)AOP的理解和應(yīng)用。

MIE和KE都是AOP必不可少的組成部分。由于MIE直接與干擾物相互作用，由此開始整條通路的調(diào)節(jié)，并最終到達(dá)AO終點(diǎn)，干擾物的結(jié)構(gòu)和性質(zhì)與MIE之間的聯(lián)系比其他任一節(jié)點(diǎn)和效應(yīng)終點(diǎn)都強(qiáng)[75]。因此，正如QSAR和分子模擬所做的，大多數(shù)計(jì)算機(jī)預(yù)測(cè)模型都以MIE為研究對(duì)象。然而，MIE與AO并不是直接的相互關(guān)系，它們之間至少存在一個(gè)KE，并且不同的MIE都有可能導(dǎo)致相同的AO產(chǎn)生，形成AOP網(wǎng)絡(luò)(圖2)[76]。以前面Villeneuve[73]開發(fā)的雄激素受體激動(dòng)效應(yīng)導(dǎo)致生殖功能紊亂的AOP為例，血液雌激素濃度降低同時(shí)會(huì)與芳香酶活性降低導(dǎo)致生殖毒性的AOP(AOP7)產(chǎn)生交聯(lián)，通過另一條AOP造成生殖問題，多條通路的交聯(lián)從而形成AOP網(wǎng)絡(luò)(圖3)。干擾物與最終AO之間的網(wǎng)絡(luò)關(guān)系，使針對(duì)單一靶標(biāo)受體的預(yù)測(cè)方法具有更大的不確定性。

將MIE、KE、AO之間用一系列的數(shù)學(xué)模型聯(lián)系起來[77]，可建立定量AOP(qAOP)模型。學(xué)者們相信，通過建立qAOP可以將體外實(shí)驗(yàn)得到的數(shù)據(jù)，如配體受體結(jié)合效力，作為qAOP的輸入信息，通過系列數(shù)學(xué)模型的模擬計(jì)算預(yù)測(cè)潛在的內(nèi)分泌干擾活性，甚至模擬劑量-效應(yīng)關(guān)系和時(shí)間進(jìn)程行為[71]。AOP-KB平臺(tái)上推出了Effectopedia模塊，通過量化KE之間的關(guān)系，建立qAOP。目前，Effectopedia模塊還處于發(fā)展階段，但Beta版本的軟件已發(fā)布(http://www.effectopedia.org/)。此外，還有其他系統(tǒng)生物學(xué)計(jì)算模擬軟件，如PK-Sim和MoBi都具有很好的qAOP模型構(gòu)建和模擬功能[78]。然而，qAOP需建立于明確的作用機(jī)制之下，目前還處于發(fā)展階段，其定量預(yù)測(cè)能力還有待實(shí)驗(yàn)驗(yàn)證。

既然可以利用體外實(shí)驗(yàn)的數(shù)據(jù)，通過AOP預(yù)測(cè)最終的干擾效應(yīng)，那么計(jì)算預(yù)測(cè)方法與AOP的結(jié)合也將成為可能。事實(shí)上，AOP概念正是來源于利用QSAR、生物標(biāo)記物(biomarker)和其他機(jī)制數(shù)據(jù)提高對(duì)毒理學(xué)的認(rèn)識(shí)和預(yù)測(cè)化學(xué)品暴露的潛在有害影響的想法[64]。由于對(duì)接和MD模擬的靶標(biāo)往往與AOP中的MIE或KE相對(duì)應(yīng)，分子模擬與AOP結(jié)合進(jìn)行模擬預(yù)測(cè)將成為AOP發(fā)展中的重要研究方向。然而，限于目前AOP仍處于起步階段，還沒有較為成功的AOP與模擬預(yù)測(cè)方法結(jié)合的案例。

5 總結(jié)和展望(Conclusions and prospect)

內(nèi)分泌干擾物是導(dǎo)致多種疾病，如生殖疾病、肥胖癥和與激素相關(guān)的癌癥等的重要誘因，眾多化合物都具有潛在的內(nèi)分泌干擾效應(yīng)，使內(nèi)分泌干擾問題在化合物風(fēng)險(xiǎn)評(píng)估上顯得尤為突出。然而，評(píng)估一個(gè)化合物的內(nèi)分泌干擾效應(yīng)需要耗費(fèi)大量的成本，無法對(duì)成千上萬種化學(xué)品進(jìn)行逐一測(cè)試。計(jì)算毒理學(xué)大大簡(jiǎn)化了這一過程，其使用也逐漸受到認(rèn)可。本文基于內(nèi)分泌干擾的效應(yīng)機(jī)制，介紹了分子對(duì)接、MD模擬和AOP這3種計(jì)算毒理學(xué)方法及其在內(nèi)分泌干擾物篩選上的應(yīng)用。

分子對(duì)接與QSAR相比更有助于效應(yīng)機(jī)制的理解，通過反向?qū)幽茴A(yù)測(cè)化合物可能的內(nèi)分泌干擾活性終點(diǎn)，還能與QSAR結(jié)合構(gòu)建多維QSAR模型。MD模擬有助于探索配體-受體相互作用關(guān)系及兩者的變化，探索重要的結(jié)構(gòu)變化，并借助熱力學(xué)計(jì)算預(yù)測(cè)結(jié)合效力。AOP將MIE與最終AO用一系列KE和KER連接，形成完整的、明晰的通路甚至網(wǎng)絡(luò)，借助AOP和AOP網(wǎng)絡(luò)將推動(dòng)計(jì)算毒理學(xué)進(jìn)入新的階段。

內(nèi)分泌干擾物主要通過與內(nèi)分泌系統(tǒng)相關(guān)靶標(biāo)的相互作用，正如AOP中的MIE和KE的激活，因此，分子對(duì)接、MD模擬和AOP的結(jié)合將使計(jì)算毒理學(xué)更加面向效應(yīng)機(jī)制。在內(nèi)分泌干擾物的篩選中，將反向?qū)蛹夹g(shù)與AOP模擬結(jié)合，不僅能預(yù)測(cè)干擾物潛在的內(nèi)分泌干擾敏感靶標(biāo)，還能進(jìn)一步通過AOP網(wǎng)絡(luò)預(yù)測(cè)可能造成的有害結(jié)局。MD模擬的發(fā)展使得通過模擬區(qū)分促進(jìn)和抑制作用逐漸成為可能，甚至可以模擬生物大分子之間的相互作用，研究MIE、KE和KE、KE之間的關(guān)系。因此，將對(duì)接和分子動(dòng)力學(xué)模擬技術(shù)運(yùn)用到AOP和AOP網(wǎng)絡(luò)中進(jìn)行預(yù)測(cè)和篩選，將有助于計(jì)算毒理學(xué)在內(nèi)分泌干擾物篩選上的發(fā)展與應(yīng)用。

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◆

ApplicationandProspectofComputationalToxicologyinScreeningofEndocrineDisruptingChemicals

Chen Qinchang, Tan Haoyue, Shi Wei, Yu Hongxia*

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China

10.7524/AJE.1673-5897.20170206001

2017-02-06錄用日期2017-03-13

1673-5897(2017)3-038-11

X171.5

A

于紅霞(1963-)，女，博士，教授，主要從事有機(jī)污染化學(xué)、環(huán)境監(jiān)測(cè)和毒理分析等領(lǐng)域的研究。

國(guó)家自然科學(xué)基金(21577058)；國(guó)家環(huán)保部公益性行業(yè)科研專項(xiàng)(201409040)

陳欽暢(1991-)，男，博士研究生，研究方向?yàn)橛?jì)算毒理學(xué)，E-mail:cqchang@outlook.com；

*通訊作者(Corresponding author)， E-mail: yuhx@nju.edu.cn

陳欽暢, 譚皓月, 史薇, 等. 計(jì)算毒理學(xué)在內(nèi)分泌干擾物篩選上的應(yīng)用和展望[J]. 生態(tài)毒理學(xué)報(bào)，2017, 12(3): 38-48

Chen Q C, Tan H Y, Shi W, et al. Application and prospect of computational toxicology in screening of endocrine disrupting chemicals [J]. Asian Journal of Ecotoxicology, 2017, 12(3): 38-48 (in Chinese)