國(guó)曉春，盧少勇,*，謝平，陳雋，劉曉暉

1.中國(guó)環(huán)境科學(xué)研究院環(huán)境基準(zhǔn)與風(fēng)險(xiǎn)評(píng)估國(guó)家重點(diǎn)實(shí)驗(yàn)室國(guó)家環(huán)境保護(hù)湖泊污染控制重點(diǎn)實(shí)驗(yàn)室洞庭湖生態(tài)觀測(cè)研究站，北京100012

2.中國(guó)科學(xué)院水生生物研究所東湖湖泊生態(tài)系統(tǒng)試驗(yàn)站，武漢430072

3.山東師范大學(xué)地理與環(huán)境學(xué)院，濟(jì)南250014

微囊藻毒素的環(huán)境暴露、毒性和毒性作用機(jī)制研究進(jìn)展

國(guó)曉春1，盧少勇1,*，謝平2，陳雋2，劉曉暉3

1.中國(guó)環(huán)境科學(xué)研究院環(huán)境基準(zhǔn)與風(fēng)險(xiǎn)評(píng)估國(guó)家重點(diǎn)實(shí)驗(yàn)室國(guó)家環(huán)境保護(hù)湖泊污染控制重點(diǎn)實(shí)驗(yàn)室洞庭湖生態(tài)觀測(cè)研究站，北京100012

2.中國(guó)科學(xué)院水生生物研究所東湖湖泊生態(tài)系統(tǒng)試驗(yàn)站，武漢430072

3.山東師范大學(xué)地理與環(huán)境學(xué)院，濟(jì)南250014

微囊藻毒素(MCs)是富營(yíng)養(yǎng)化淡水水體中藍(lán)藻的爆發(fā)性繁殖產(chǎn)生的最常見(jiàn)的藻毒素，因其分布廣、結(jié)構(gòu)穩(wěn)定、毒性大引起了科學(xué)界的廣泛關(guān)注。本文系統(tǒng)梳理了微囊藻毒素在我國(guó)水體中的污染現(xiàn)狀和典型毒性效應(yīng)及毒性作用機(jī)制。另外，針對(duì)目前藻毒素研究的不足提出了建議，可為有效降低環(huán)境中微囊藻毒素的潛在安全風(fēng)險(xiǎn)及深入研究其生態(tài)毒性效應(yīng)提供支持。

微囊藻毒素；環(huán)境暴露；毒性效應(yīng)；致毒機(jī)制

國(guó)曉春,盧少勇,謝平,等.微囊藻毒素的環(huán)境暴露、毒性和毒性作用機(jī)制研究進(jìn)展[J].生態(tài)毒理學(xué)報(bào)，2016,11(3):61-71

Guo X C,Lu S Y,Xie P,et al.Environmental exposure,toxicity and toxic mechanism of microcystins:A review[J].Asian Journal of Ecotoxicology, 2016,11(3):61-71(in Chinese)

由淡水水體富營(yíng)養(yǎng)化引起的藍(lán)藻水華以及與之相關(guān)的藻毒素污染在全球范圍內(nèi)被廣泛報(bào)道。藍(lán)藻異常繁殖不僅破壞水體的水文化學(xué)特性以及光照，產(chǎn)生異臭異味物質(zhì)，使水質(zhì)惡化，水中溶解氧減少，水體生物的多樣性降低，嚴(yán)重破壞水生生態(tài)系統(tǒng)平衡，更為嚴(yán)重的是藍(lán)藻細(xì)胞破裂后能夠產(chǎn)生大量的藻毒素對(duì)動(dòng)物和人類的飲用水安全構(gòu)成嚴(yán)重的威脅。過(guò)去幾十年，世界各地水庫(kù)、河流、湖泊等水體因爆發(fā)藍(lán)藻水華使動(dòng)物和人因藍(lán)藻毒素中毒的事件頻繁發(fā)生[1]。

在已發(fā)現(xiàn)的藍(lán)藻毒素中，微囊藻毒素(MCs)在全球淡水水體中分布最廣、出現(xiàn)頻率最高、毒性效應(yīng)最嚴(yán)重，因而得到了普遍的研究，其對(duì)環(huán)境和人類健康的危害已成為全球關(guān)注的重大環(huán)境問(wèn)題之一。大量的研究報(bào)道MCs可以在器官中吸收、運(yùn)輸和累積，引起水生動(dòng)物和哺乳動(dòng)物的中毒和死亡[2]并對(duì)人類健康造成潛在威脅[3]。水體中的MCs可以通過(guò)水生生物的呼吸作用(鰓)、皮膚接觸和食物攝取等途徑進(jìn)入體內(nèi)，并經(jīng)過(guò)腸道吸收、血液循環(huán)以及體液循環(huán)分布于機(jī)體的各個(gè)部位。雙殼類動(dòng)物，比如蚌類，由于其濾食或者舔食特性而更容易積累懸浮或附著在藻類以及浮游動(dòng)物中的微量MCs，即通過(guò)生物濃縮的方式積累MCs。魚類對(duì)MCs的攝食主要通過(guò)2種方式，一是植食性魚類(如羅非魚)和濾食性魚類(如鳙、鰱)等直接吞食產(chǎn)MCs的藻類，經(jīng)過(guò)腸道的消化吸收使得MCs在機(jī)體內(nèi)運(yùn)輸累積[4]。另一方面，其他食性的魚類則還可通過(guò)捕食被MCs污染的水生生物而被染毒。

一般認(rèn)為藻毒素進(jìn)入人體的主要途徑包括：慢性攝入被污染的飲用水；水產(chǎn)品、蔬菜等的食物攝入；游泳、洗澡等皮膚接觸污染水；靜脈途徑(例如血透析)以及通過(guò)鼻粘膜吸入；藍(lán)藻營(yíng)養(yǎng)品攝入等。有關(guān)藍(lán)藻對(duì)人類毒害作用的事件由來(lái)已久[5]，引起這些健康事件的毒素涉及很多，但以MCs最為常見(jiàn)。國(guó)內(nèi)外已有的流行病學(xué)調(diào)查顯示飲用水中的MCs可能是我國(guó)東部地區(qū)原發(fā)性肝癌高發(fā)病率的重要危險(xiǎn)因素之一，并與多種腫瘤的發(fā)病率，大腸癌發(fā)病率以及胃癌死亡率上升等均有相關(guān)性[6-7]。1996年2月，在巴西發(fā)生了最嚴(yán)重的人類中毒事件，由于血液透析用水被MCs污染，導(dǎo)致126例患者出現(xiàn)急性肝臟損傷癥狀，60例患者死亡[8]。最近的研究中，Chen等[9]和Li等[10]首次檢測(cè)出在慢性毒素暴露條件下人體血液中存在MCs，并結(jié)合血清生化學(xué)結(jié)果證明肝臟損傷的存在，是迄今為止藍(lán)藻毒素通過(guò)自然染毒途徑對(duì)人類健康產(chǎn)生影響的最直接的證據(jù)。本文綜述了微囊藻毒素典型毒性效應(yīng)及毒性機(jī)制2個(gè)方面的研究現(xiàn)狀，為進(jìn)一步研究藍(lán)藻水華的生態(tài)毒理提供信息。

1 微囊藻毒素的來(lái)源及結(jié)構(gòu)（Source and structure of microcystins）

微囊藻毒素(microcystins,MCs)是淡水水體中出現(xiàn)頻率最高、分布最廣、造成危害最嚴(yán)重的一類具有強(qiáng)烈肝毒性的環(huán)肽毒素[11]，其主要由微囊藻屬(Microcystis)、魚腥藻屬(Anabaena)、念珠藻屬(Nostoc)、束絲藻屬(Aphanizomenon)以及顫藻屬(Oscillatoria)等產(chǎn)生[12]。MCs是藍(lán)藻的次生代謝產(chǎn)物，其通過(guò)非核糖體途徑在細(xì)胞內(nèi)合成，并在細(xì)胞破裂釋放后表現(xiàn)出毒性[13]。光照、營(yíng)養(yǎng)鹽、微量元素、鹽度、溫度、pH等環(huán)境因素均能影響MCs的產(chǎn)生，其中光照強(qiáng)度和營(yíng)養(yǎng)鹽的影響作用最大[14-15]。

圖1 微囊藻毒素的結(jié)構(gòu)通式：環(huán)（-D-Ala1-L-X2-D-Masp3-L-Z4-Adda5-D-Glu6-Mdha7）

微囊藻毒素是單環(huán)七肽化合物，其結(jié)構(gòu)通式如圖1所示。MCs的特征結(jié)構(gòu)為5號(hào)位的Adda基團(tuán)，其結(jié)構(gòu)為3-氨基-9-甲氧基-2,6,8-三甲基-10-苯基-4(E),6(E)-二烯，是MCs生物活性表達(dá)所必需的基團(tuán)。此外，MCs的7個(gè)氨基酸還包含7號(hào)位Mdha(N-甲基脫氫丙氨酸，N-methyldehydroalanine)和2號(hào)、4號(hào)位的2個(gè)可變的L-氨基酸[16]以及3個(gè)D型氨基酸-1號(hào)位丙氨酸(alanine)、3號(hào)位MeAsp (也稱為Masp)(赤-β-甲基天冬氨酸，erythro-β-methylaspartic acid)和6號(hào)位谷氨酸(glutamic acid)。雖然MCs的結(jié)構(gòu)變異在各氨基酸殘基上都能夠發(fā)生，但其最為常見(jiàn)的變異主要發(fā)生在2號(hào)、4號(hào)位的2個(gè)可變的L-氨基酸X和Z，以及MeAsp和Mdha殘基的甲基化水平[17]。至今已發(fā)現(xiàn)的MCs的異構(gòu)體有100多種[18]，MC-LR、MC-RR和MC-YR是其中含量較多，毒性較大且分布較為普遍的。

2 我國(guó)微囊藻毒素的暴露水平（Environmental exposure of microcystins in China）

自然界中水華暴發(fā)時(shí)，若無(wú)外來(lái)影響使其迅速溶解，水體中的毒素含量至多只有0.1～10 μg·L-1，而細(xì)胞內(nèi)的毒素則會(huì)高出幾個(gè)數(shù)量級(jí)[19]。大型湖泊、河流中藍(lán)藻釋放的藻毒素可被大量水體稀釋，但大規(guī)模藍(lán)藻水華溶解時(shí)，就會(huì)使毒素達(dá)到較高濃度，造成潛在危害。

我國(guó)是一個(gè)湖泊眾多的國(guó)家，20世紀(jì)90年代以來(lái)，藍(lán)藻水華暴發(fā)的面積、強(qiáng)度以及藻毒素含量均在大幅度增長(zhǎng)，由此帶來(lái)的環(huán)境和生物安全問(wèn)題日益引起關(guān)注。這其中，以江蘇太湖、安徽巢湖、云南滇池的藍(lán)藻水華污染最為嚴(yán)重。此外，長(zhǎng)江、黃河、松花江中下游等主要河流以及鄱陽(yáng)湖、武漢東湖、武漢蓮花湖、上海淀山湖、三峽庫(kù)區(qū)等淡水湖泊、水庫(kù)中也都相繼發(fā)生了不同程度的藍(lán)藻水華污染并檢測(cè)到了MCs的存在[20]。Song等[21]報(bào)道太湖五里湖和梅梁灣表層水最大胞外MCs含量分別為2.71和6.66 μg·L-1。Shen等[22]于太湖梅梁灣的研究顯示，微囊藻毒素隨時(shí)間和營(yíng)養(yǎng)鹽水平的不同有很大差異，胞內(nèi)毒素最高可達(dá)97.32 μg·g-1干藻。Wang等[23]在太湖貢湖灣檢測(cè)的表層水MCs組成以MCLR和MC-RR為主，胞外MC最大含量為0.391 μg· L-1，胞內(nèi)MC最大含量可達(dá)35.418 μg·L-1。徐海濱等[24]對(duì)江西鄱陽(yáng)湖的調(diào)查顯示，水體微囊藻毒素最大為1 036.9 pg·mL-1，同時(shí)發(fā)現(xiàn)魚體內(nèi)有毒素積累。王紅兵等[25]曾檢測(cè)到上海淀山湖水體中MCs濃度最高可達(dá)55.4 ng·mL-1。蔡金傍等[26]對(duì)華北地區(qū)某水庫(kù)進(jìn)行為期1年的監(jiān)測(cè)發(fā)現(xiàn)，胞內(nèi)胞外MCs的峰值出現(xiàn)在夏秋季，最高可達(dá)5.6288 μg·L-1。2005年對(duì)北京市重要飲用水水源地官?gòu)d水庫(kù)、密云水庫(kù)和懷柔水庫(kù)水源水樣進(jìn)行藻毒素調(diào)查發(fā)現(xiàn)，在藻類的高發(fā)季節(jié)，3個(gè)水庫(kù)水體中均檢出MCs，其中官?gòu)d水庫(kù)7月份MCs最高值達(dá)到20 μg·L-1[27-28]。廣東省典型供水水庫(kù)和淡水湖泊微囊藻毒素分布廣泛，毒素組成以MC-RR為主，水庫(kù)微囊藻毒素含量在0～0.919 μg·L-1[29]。楊希存等[30]對(duì)秦皇島洋河水庫(kù)的調(diào)查顯示微囊藻毒素總含量為0.13～0.93 μg·L-1。周學(xué)富等[31]在原發(fā)性肝癌高發(fā)地區(qū)江蘇泰興等地的許多溝塘中檢出大量的MCs，河水、溝塘水和淺井水內(nèi)MCs的平均含量為36 ng·L-1、29 ng·L-1和25 ng·L-1。我國(guó)廈門市同安地區(qū)水樣中MCs的陽(yáng)性檢出率高達(dá)77.5%，池塘水和水庫(kù)水中MC的最高檢出值分別為351 ng·L-1和876 ng·L-1[32]。

除天然水體及水庫(kù)源水中普遍檢測(cè)出MCs外，飲用水也存在MCs污染。世界衛(wèi)生組織(WHO)規(guī)定飲用水中MC-LR含量的安全指導(dǎo)值為1.0 μg· L-1[33]，目前我國(guó)也已經(jīng)采用此指導(dǎo)值作為飲用水標(biāo)準(zhǔn)。國(guó)內(nèi)相關(guān)研究表明，包括上海、廈門、無(wú)錫、海門、昆山及鄭州等在內(nèi)的部分城市自來(lái)水廠出水樣品中能檢測(cè)出MCs，部分樣品最大濃度接近甚至超過(guò)安全限值(1.0 μg·L-1)[34-36]。

3 微囊藻毒素的毒性效應(yīng)（Toxicity of microcystins）

3.1 肝臟毒性

目前已確定的MCs的同分異構(gòu)體已有100多種[18]，其主要作用于肝臟，有關(guān)其在生物體體內(nèi)及體外的肝毒性研究最為詳盡[37-38]。研究表明無(wú)論經(jīng)腹腔注射或者口服灌胃，MCs均能引起實(shí)驗(yàn)動(dòng)物的肝臟病變[39]。室內(nèi)實(shí)驗(yàn)結(jié)果顯示，小鼠[40]和大西洋鮭魚[41]經(jīng)染毒后，MCs均主要積累在肝臟；野外調(diào)查結(jié)果顯示，無(wú)論在無(wú)脊椎動(dòng)物如蚌、螺、蝦[42]還是在魚類[43]，MCs均在肝臟中的累積量最多。MCs的親肝臟性主要取決于其通過(guò)細(xì)胞內(nèi)的膽汁酸轉(zhuǎn)運(yùn)系統(tǒng)以及有機(jī)陰離子轉(zhuǎn)運(yùn)多肽(Oatps)轉(zhuǎn)運(yùn)機(jī)制主動(dòng)運(yùn)輸進(jìn)入細(xì)胞[44]，膽汁酸轉(zhuǎn)運(yùn)系統(tǒng)以及Oatps的器官表達(dá)特異性導(dǎo)致MCs轉(zhuǎn)運(yùn)及積累具有親器官性。Fischer等[45]用非洲爪蟾卵母細(xì)胞研究了多種Oatps對(duì)MC-LR的轉(zhuǎn)運(yùn)，結(jié)果發(fā)現(xiàn)轉(zhuǎn)運(yùn)MC-LR效能較高的主要是大鼠肝臟Oatp1b2、人體肝臟OATP1B1以及OATP1B3，這表明MCs具有親肝臟性。同時(shí)肝臟是MCs與GSH結(jié)合進(jìn)行I、II相代謝解毒的中心[46-49]，因而也是MCs毒性的靶器官[50]，受損最嚴(yán)重。此外，動(dòng)物實(shí)驗(yàn)表明，MCs不僅是潛在的肝腫瘤促進(jìn)劑，同時(shí)還可能是肝癌的啟動(dòng)劑[51]。

He等[52]以大鼠為研究模型，進(jìn)行了長(zhǎng)期低劑量暴露于MC-LR的代謝組學(xué)研究，結(jié)果表明MC-LR對(duì)肝臟代謝的干擾與營(yíng)養(yǎng)物質(zhì)的吸收抑制有關(guān)，因?yàn)楦闻K中多達(dá)12種氨基酸含量顯著下降，而這些氨基酸在回腸中含量相應(yīng)上升。MC-LR顯著干擾了肝臟中酪氨酸的合成和分解代謝，干擾了膽堿3條主要代謝通路，通過(guò)抑制谷胱甘肽合成以及促進(jìn)谷胱甘肽與MC-LR結(jié)合導(dǎo)致肝臟谷胱甘肽耗竭，并阻礙了肝臟核苷酸的從頭合成。

流行病學(xué)研究發(fā)現(xiàn)MCs長(zhǎng)期暴露嚴(yán)重危害肝臟功能。Ueno等[53]推測(cè)長(zhǎng)期飲用MCs污染的水與我國(guó)原發(fā)性肝癌發(fā)病率的上升有關(guān)。1996年巴西發(fā)生了腎透析用水被MCs污染，導(dǎo)致126例患者出現(xiàn)亞急性肝毒性癥狀，約60例患者死亡的嚴(yán)重事故，免疫分析表明患者的肝臟和血清中均存在高濃度的MCs[54]，這也是世界上首次關(guān)于MCs直接引起人群肝損傷并致死的報(bào)道。此外，關(guān)于通過(guò)飲用水和食用水產(chǎn)品方式長(zhǎng)期慢性暴露于MCs的巢湖漁民[9]的相關(guān)研究顯示，指示肝臟損傷的生化指標(biāo)出現(xiàn)顯著升高，并且在受試者血漿樣品中檢測(cè)到的MCs與這些指標(biāo)具有顯著相關(guān)性，以上結(jié)果暗示了MCs對(duì)人體的肝臟毒性。

3.2 腎臟毒性

動(dòng)物體的腎臟是MCs作用的另一重要靶器官，其在MCs的代謝和排泄中起著重要的作用，MCs通過(guò)與肝細(xì)胞類似的轉(zhuǎn)運(yùn)機(jī)制被轉(zhuǎn)運(yùn)至腎小管細(xì)胞內(nèi)，從而對(duì)腎臟造成損傷[55]。野外及室內(nèi)急性毒性實(shí)驗(yàn)顯示，生活于微囊藻爆發(fā)水體的鳙、鰱；腹腔染毒的鰱、虹鱒；以及口腔飼喂的鯉，均表現(xiàn)出MCs引起的腎小管和腎小球細(xì)胞壞死、溶酶體增生、細(xì)胞核固縮以及退行性病變等，這些現(xiàn)象都顯示在微囊藻毒素的毒性作用下，細(xì)胞開(kāi)始出現(xiàn)了自噬、凋亡甚至壞死的現(xiàn)象[56]。放射自顯影研究顯示，動(dòng)物體經(jīng)MC-LR染毒后，MC-LR在腎臟中有較高的含量，并且主要位于腎皮質(zhì)區(qū)域內(nèi)的腎細(xì)胞核內(nèi)[57]。MCs可以引起哺乳動(dòng)物的腎臟細(xì)胞損傷。組織學(xué)研究發(fā)現(xiàn)，小鼠經(jīng)注射染毒后，腎小球的管腔增大并伴有大量的紅細(xì)胞，毛細(xì)血管簇被破壞，紅細(xì)胞減少，遠(yuǎn)曲小管和近曲小管的管腔也表現(xiàn)出增大的現(xiàn)象，其上皮細(xì)胞脫落或消失，細(xì)胞質(zhì)中出現(xiàn)液泡并且細(xì)胞間隙中浸潤(rùn)有淋巴細(xì)胞，說(shuō)明在該組織中有壞死現(xiàn)象產(chǎn)生；而凋亡小體的產(chǎn)生、肌動(dòng)蛋白絲的破壞以及其他細(xì)胞形態(tài)學(xué)上的改變說(shuō)明微囊藻毒素引發(fā)了腎臟細(xì)胞的凋亡[58]。此外，離體灌注實(shí)驗(yàn)表明，MCs可以改變腎臟的血管阻力、腎小球率過(guò)濾和灌注壓等一系列功能指標(biāo)[59]。

3.3 生殖毒性

MCs具有顯著的生殖及胚胎發(fā)育毒性。雌、雄青鳉魚30 d低劑量暴露實(shí)驗(yàn)顯示，MC-LR能夠造成雄魚的睪丸損傷，在細(xì)精管出現(xiàn)大面積的溶解性區(qū)域，指示趨向凋亡的異常細(xì)胞增殖過(guò)程有所增加。MC-LR同時(shí)對(duì)卵巢有明顯的影響，特別是使性腺組織萎縮，降低卵黃含量[60]。斑馬魚慢性染毒實(shí)驗(yàn)發(fā)現(xiàn)，肝臟卵巢、睪丸均有顯著的組織學(xué)損傷，孵化率和性腺的17β-雌二醇含量均顯著降低并且發(fā)現(xiàn)Bcl-2轉(zhuǎn)錄水平的顯著下調(diào)[61]。急性毒性試驗(yàn)顯示，MC-LR染毒后雌性斑馬魚卵巢出現(xiàn)顯著的組織學(xué)損傷并且呈現(xiàn)出時(shí)間劑量依賴關(guān)系，同時(shí)檢測(cè)到MDA含量，抗氧化酶CAT、SOD和GPX酶活性和轉(zhuǎn)錄水平的升高[62]。雄性大鼠腹腔注射染毒實(shí)驗(yàn)發(fā)現(xiàn)，藻毒素粗提物能夠造成雄鼠的睪丸損傷，精子的運(yùn)動(dòng)能力和生存發(fā)育能力下降，生精小管內(nèi)精子的質(zhì)量降低[63]。針對(duì)銅銹環(huán)棱螺的野外調(diào)查研究發(fā)現(xiàn)，性腺是MCs除肝臟外的第二個(gè)靶器官[64]，并且可以從母代傳遞給子代，對(duì)子代的發(fā)育造成危害。妊娠孕鼠腹腔注射染毒實(shí)驗(yàn)顯示不同劑量的MCLR(4～62 μg·kg-1)都能夠造成胎盤屏障的損傷，導(dǎo)致胎盤細(xì)胞水腫、變性以及間質(zhì)疏松[65]。MCs除了對(duì)睪丸和卵巢有直接的毒性作用外，還可以通過(guò)損傷下丘腦-垂體-性腺(HPG)軸和肝臟來(lái)間接地作用于性激素。Chen等[66-67]研究發(fā)現(xiàn)，MC-LR處理后睪酮含量減少而LH和FSH的水平升高，LH和FSH的分泌受到了睪酮的負(fù)反饋調(diào)控。在雌性斑馬魚，MCs同樣干擾類固醇合成基因和HPG軸上性激素的含量，導(dǎo)致卵泡發(fā)育、卵母細(xì)胞成熟和排卵受到抑制[68]。

斑馬魚胚胎染毒實(shí)驗(yàn)發(fā)現(xiàn)，經(jīng)不同濃度的MCLR處理后斑馬魚胚后發(fā)育受到影響，并且幼魚的存活率下降[69]。此外，Bu等[70]研究發(fā)現(xiàn)藻毒素提取物會(huì)導(dǎo)致孕鼠的體重下降，胎兒的重量、體長(zhǎng)以及尾巴長(zhǎng)度均下降，還會(huì)出現(xiàn)胎鼠尾巴卷曲的情況。

3.4 神經(jīng)毒性

1996年，巴西血液透析MCs素中毒事件的大部分病人都伴有頭暈、頭痛、惡心、嘔吐、昏睡、視覺(jué)障礙、眼盲、耳鳴、耳聾、癲癇等一系列的神經(jīng)毒性癥狀。進(jìn)一步的研究顯示，MCs可經(jīng)由有機(jī)陰離子轉(zhuǎn)運(yùn)多肽(Oatps)通過(guò)血腦屏障轉(zhuǎn)運(yùn)至腦組織中引起神經(jīng)毒性病癥[71]。懷孕SD大鼠10 μg·kg-1體重MCLR的暴露實(shí)驗(yàn)顯示，前腦超微結(jié)構(gòu)稀疏，內(nèi)質(zhì)網(wǎng)腫脹，且線粒體腫脹[72]。MC-LR暴露后可以引起海馬區(qū)顯著的組織和結(jié)構(gòu)損傷以及嚴(yán)重的氧化性損傷[73]。MCs能夠引起神經(jīng)性生理功能的改變，比如魚類的游泳能力(平均速度、活動(dòng)百分率)在低MCRR的暴露條件下增加，而在高的暴露條件下降低[74]。此外，MCs還可以引起生物體行為的變化。0.1 μg·L-1MC-LR暴露24 h后，線蟲的身體彎曲頻率和頭部鞭打頻率顯著降低[75]。水迷宮測(cè)試實(shí)驗(yàn)顯示，MC-LR暴露3 d后，大鼠出現(xiàn)顯著的長(zhǎng)期逃避延遲并且發(fā)現(xiàn)其進(jìn)入月臺(tái)區(qū)域的頻率較低[73,76]。

MCs可能是通過(guò)影響腦組織中與細(xì)胞骨架[72,77]、能量代謝、信號(hào)轉(zhuǎn)導(dǎo)和氧化應(yīng)激等功能相關(guān)的蛋白的表達(dá)而對(duì)神經(jīng)系統(tǒng)發(fā)育與功能造成損害，并可能進(jìn)一步引發(fā)相關(guān)的神經(jīng)退行性疾病[73,78-79]。

3.5 其他毒性

MCs除了具有強(qiáng)烈的肝臟和腎臟毒性，還表現(xiàn)出一定的遺傳毒性和免疫毒性[80]。

大量的研究表明MCs可以通過(guò)損傷DNA、染色體和基因等遺傳物質(zhì)，對(duì)機(jī)體產(chǎn)生危害。MCs可誘導(dǎo)小鼠肝臟、倉(cāng)鼠幼體腎臟細(xì)胞、小鼠胚胎纖維原細(xì)胞、原代培養(yǎng)的大鼠肝細(xì)胞、人肝癌細(xì)胞以及人外周血淋巴細(xì)胞的DNA損傷。細(xì)胞體外微核試驗(yàn)顯示MCs能明顯引起染色體損傷，并且呈現(xiàn)良好的劑量-反應(yīng)關(guān)系[81]。Zhan等[82]應(yīng)用人類淋巴母細(xì)胞TK6研究MC-LR的體外遺傳毒性的分子機(jī)理中顯示MC-LR可導(dǎo)致TK6細(xì)胞tk位點(diǎn)雜合性丟失。

目前關(guān)于MCs的免疫毒性研究主要集中于其對(duì)免疫細(xì)胞和免疫分子的影響兩方面。研究表明低劑量MCs暴露可導(dǎo)致小鼠免疫抑制[83]。另外，體外和體內(nèi)動(dòng)物實(shí)驗(yàn)結(jié)果顯示，MC-LR染毒能夠降低小鼠NK細(xì)胞對(duì)YAC-1細(xì)胞的殺傷活性。Chen等[84]的體外研究結(jié)果表明不同劑量MC-LR處理后，巨噬細(xì)胞的NO產(chǎn)生量減少并且IL-1?、GM-CSF、iNOS、IFN-r和TNF-n的表達(dá)降低。動(dòng)物實(shí)驗(yàn)證明MCs對(duì)細(xì)胞因子的表達(dá)具有調(diào)節(jié)作用[85]。此外，Yea等[86]的研究結(jié)果也表明MCs對(duì)小鼠淋巴細(xì)胞功能的抑制作用是通過(guò)降低IL-2mRNA的穩(wěn)定性，MCs對(duì)免疫系統(tǒng)多個(gè)方面、多個(gè)層次的功能顯示出明顯的抑制作用[87]。

4 微囊藻毒素的毒性作用機(jī)制（Toxic mechnism of microcystins）

4.1 有機(jī)陰離子轉(zhuǎn)運(yùn)多肽轉(zhuǎn)運(yùn)機(jī)制

微囊藻毒素不易通過(guò)被動(dòng)運(yùn)輸方式通過(guò)細(xì)胞膜，只能依靠相應(yīng)載體的運(yùn)輸進(jìn)入細(xì)胞。有機(jī)陰離子轉(zhuǎn)運(yùn)多肽類(organic anion transporting polypeptides,簡(jiǎn)稱OATP)的蛋白家族能夠介導(dǎo)對(duì)于不依賴鈉離子的兩性有機(jī)化合物的吸收，如膽汁鹽類、有機(jī)陰離子染料、甾類和甾類軛合物、藥物、不同的多肽類以及毒素等[88]。OATP家族有許多成員，到目前為止研究發(fā)現(xiàn)OATP家族成員有：OATP1、OATP2、OATP3、OATP4、OATP5以及OATP6。不同的OATP在人或哺乳動(dòng)物的組織和器官中具有不同的分布，而不同的OATP對(duì)于不同種類的微囊藻毒素的吸收也具有特殊性。研究顯示，OATP在肝臟、腦、腎臟、腸道、心臟以及生殖腺中均有不同的表達(dá)，這也被認(rèn)為是MCs之所以具有較為明顯的器官選擇性毒性的重要原因[89-94]。

4.2 抑制蛋白磷酸酶活性

微囊藻毒素在經(jīng)過(guò)相應(yīng)載體的運(yùn)輸作用進(jìn)入細(xì)胞后，能夠引起細(xì)胞產(chǎn)生一系列的反應(yīng)，從而改變細(xì)胞正常的內(nèi)部環(huán)境，進(jìn)而發(fā)揮對(duì)細(xì)胞的毒性作用。MCs代表性的致毒機(jī)理為蛋白磷酸酶抑制。MCs進(jìn)入細(xì)胞后，MeAsp殘基首先通過(guò)非共價(jià)結(jié)合作用抑制絲氨酸/蘇氨酸蛋白磷酸酶1和2A(簡(jiǎn)稱PP1/ PP2A)的活性，隨后，MCs的Mdha基團(tuán)與PP1和PP2A的半胱氨酸殘基共價(jià)結(jié)合，進(jìn)而導(dǎo)致PP1和PP2A不可逆的改變[95]。就是通過(guò)這種抑制作用造成了細(xì)胞內(nèi)多種蛋白的磷酸化和去磷酸化失衡，細(xì)胞內(nèi)一系列的生化過(guò)程發(fā)生紊亂，最終導(dǎo)致細(xì)胞的損傷。

研究表明，MCs通過(guò)抑制PP1和PP2A的活性，從而抑制單鏈核苷酸剪切修復(fù)(nucleotide excision repair,即NER)和DNA雙鏈斷裂修復(fù)(double-strand breaks,即DSB)這2種通過(guò)非同源末端連接(non-homologous end joining,即NHEJ)方式來(lái)修復(fù)DNA的通路[96-97]。有研究發(fā)現(xiàn)微囊藻毒素能夠促進(jìn)腫瘤的生成，而這可能與其抑制PP2A進(jìn)而對(duì)細(xì)胞周期產(chǎn)生影響有關(guān)。Takumi等[98]的研究發(fā)現(xiàn)MC-LR可能通過(guò)抑制PP2A來(lái)激活A(yù)kt，并造成Akt下游靶蛋白GSK-3βS激酶的磷酸化而失活。GSK-3β激酶的失活能介導(dǎo)β-catenin的磷酸化并能引起該蛋白向核內(nèi)的轉(zhuǎn)運(yùn)，β-catenin在核內(nèi)的聚集可以通過(guò)促進(jìn)一系列基因的表達(dá)而引起細(xì)胞增殖和分化。此外，MCs還可以通過(guò)影響微管相關(guān)蛋白tau和小熱休克蛋白27(heat shock protein27，即HSP27)兩類蛋白的磷酸化而對(duì)細(xì)胞骨架造成破壞[99-100]。同時(shí)，PP2A被MCs抑制與p53、Bcl-2家族蛋白的表達(dá)以及鈣調(diào)蛋白激酶II(簡(jiǎn)稱CaMKII)的調(diào)控誘導(dǎo)的細(xì)胞凋亡密切相關(guān)[101-103]。

4.3 誘導(dǎo)細(xì)胞內(nèi)氧化應(yīng)激

相關(guān)的研究顯示，氧化損傷是MCs毒性作用的另一重要機(jī)制。在正常生理狀態(tài)下，機(jī)體活性氧(ROS)的產(chǎn)生和抗氧化系統(tǒng)對(duì)活性氧的消除存在著動(dòng)態(tài)的平衡。超氧化物歧化酶(SOD)和過(guò)氧化氫酶(CAT)分別作用于過(guò)氧根離子(O-2)和過(guò)氧化氫(H2O2)；谷胱甘肽過(guò)氧化物酶(GPx)則主要消除過(guò)氧化氫和脂質(zhì)過(guò)氧化產(chǎn)物；氧化型谷胱甘肽(GSSG)在谷胱甘肽還原酶(GR)作用下NADPH供氫還原為還原型谷胱甘肽(GSH)，以維持細(xì)胞內(nèi)充足的GSH水平。同時(shí)，抗氧化系統(tǒng)還包括許多其他的抗氧化物質(zhì)，如GSH，維生素A、C、E以及胡蘿卜素等。GSH的合成又通過(guò)其自身對(duì)r-谷氨酰半胱氨酸合成酶(γ-GCS)的反饋抑制來(lái)調(diào)控。細(xì)胞的功能狀態(tài)取決于ROS與抗氧化能力的平衡[104]，當(dāng)抗氧化系統(tǒng)活性下降或者活性氧產(chǎn)物增加時(shí)，即發(fā)生氧化損傷。大量的體外和原位系統(tǒng)實(shí)驗(yàn)表明MCs能夠引起機(jī)體產(chǎn)生高濃度的活性氧，并產(chǎn)生由脂質(zhì)過(guò)氧化(LPO)所帶來(lái)的氧化損傷，破壞細(xì)胞膜的結(jié)構(gòu)與功能，導(dǎo)致過(guò)氧化產(chǎn)物丙二醛(MDA)的產(chǎn)生[105]，引起機(jī)體內(nèi)抗氧化防御系統(tǒng)的變化。另外，MCs通過(guò)誘導(dǎo)細(xì)胞活性氧產(chǎn)生和氧化應(yīng)激，進(jìn)而導(dǎo)致細(xì)胞凋亡。MCs誘發(fā)細(xì)胞內(nèi)ROS含量過(guò)量上升，導(dǎo)致肝細(xì)胞脂質(zhì)過(guò)氧化、巰基狀態(tài)改變以及酶活性抑制。加上細(xì)胞磷酸化平衡和細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)功能失調(diào)，從而改變了肝細(xì)胞膜的結(jié)構(gòu)和通透性并損傷內(nèi)質(zhì)網(wǎng)等細(xì)胞內(nèi)膜系統(tǒng)，造成細(xì)胞骨架結(jié)構(gòu)重排和細(xì)胞核損傷，引發(fā)細(xì)胞膜發(fā)泡，并由此導(dǎo)致了細(xì)胞凋亡。McDermott等[106]率先報(bào)道了MC-LR能誘導(dǎo)各種類型哺乳動(dòng)物細(xì)胞凋亡，Chen等[107]則系統(tǒng)研究了MCs誘導(dǎo)小鼠肝細(xì)胞凋亡的分子基礎(chǔ)，提出了MCs誘導(dǎo)的細(xì)胞凋亡由線粒體介導(dǎo)的觀點(diǎn)。近年來(lái)的研究也發(fā)現(xiàn)，與細(xì)胞增殖和癌變有關(guān)的原癌基因、抑癌基因參與了MCs誘導(dǎo)的細(xì)胞凋亡過(guò)程。

4.4 誘發(fā)DNA損傷

微囊藻毒素侵入機(jī)體后，基于抑制PP2A和產(chǎn)生ROS這2種生理過(guò)程，通過(guò)誘導(dǎo)DNA突變、損傷DNA結(jié)構(gòu)、抑制DNA修復(fù)這3種方式誘發(fā)DNA損傷。Zegura等[108]的研究發(fā)現(xiàn)，細(xì)胞暴露于微囊藻毒素-LR 8 h后，氧化嘧啶開(kāi)始修復(fù)，但氧化嘌呤并未同時(shí)得到修復(fù)，如果這種嘌呤的氧化損傷沒(méi)有在DNA復(fù)制之前得到修復(fù)，就會(huì)導(dǎo)致DNA中的GCA得到轉(zhuǎn)化突變。此外，大量實(shí)驗(yàn)證明，微囊藻毒素不僅直接影響細(xì)胞核的形態(tài)，其誘導(dǎo)產(chǎn)生的ROS，同樣作用于DNA結(jié)構(gòu)。Repavich等[109]發(fā)現(xiàn)，MCs可以誘導(dǎo)人體淋巴細(xì)胞染色體斷裂，并呈劑量相關(guān)性。另有研究則表明MC-LR導(dǎo)致人體血淋巴細(xì)胞的凋亡和DNA鏈斷裂現(xiàn)象[110]。除了直接損傷DNA，微囊藻毒素還會(huì)抑制DNA修復(fù)過(guò)程，進(jìn)一步增強(qiáng)促突變、促癌效果。Lankoff等[111]發(fā)現(xiàn)，用MC-LR處理后的人類成膠質(zhì)瘤細(xì)胞MO59K及其非同源的MO59J細(xì)胞并未得到同時(shí)修復(fù)，并且雙鏈修復(fù)系統(tǒng)的關(guān)鍵酶DNA-PK的活性被抑制。

圖2 微囊藻毒素生物毒性作用典型機(jī)制

5 結(jié)論與展望（Conclusions and prospects）

日趨嚴(yán)重的富營(yíng)養(yǎng)化污染已經(jīng)導(dǎo)致我國(guó)成為藍(lán)藻水華頻發(fā)的地區(qū)，藍(lán)藻水華衍生污染物微囊藻毒素的污染已成為全球性環(huán)境問(wèn)題，其對(duì)自然界中的多種生物有著嚴(yán)重的毒性作用，目前對(duì)于其環(huán)境暴露水平和毒性效應(yīng)的研究已取得階段性的成果，但是以下幾個(gè)方面以后應(yīng)加深研究：(1)加強(qiáng)對(duì)全國(guó)各區(qū)域水體MCs含量的詳細(xì)調(diào)查及其在食物鏈各營(yíng)養(yǎng)級(jí)水平生物上的積累和傳遞作用的研究，探索其光化學(xué)降解、化學(xué)氧化及生物降解的機(jī)制和途徑，為相關(guān)生態(tài)風(fēng)險(xiǎn)評(píng)價(jià)提供基礎(chǔ)數(shù)據(jù)；(2)針對(duì)MCs轉(zhuǎn)運(yùn)機(jī)理的解析，目前基于基因表達(dá)模式以及藥物實(shí)驗(yàn)鑒定了4個(gè)有機(jī)陰離子轉(zhuǎn)運(yùn)肽，但是到目前一直沒(méi)有直接的分子與遺傳證據(jù)。尤其是這些有機(jī)陰離子轉(zhuǎn)運(yùn)肽究竟具體經(jīng)由怎樣的分子機(jī)制調(diào)控著MCs在生物體內(nèi)的轉(zhuǎn)運(yùn)尚不清楚；(3)環(huán)境污染物在自然環(huán)境中并非單一存在，生物體常常暴露于多種環(huán)境污染物的綜合毒性影響之下，因而關(guān)于不同類型藻毒素以及其與其他有機(jī)、無(wú)機(jī)環(huán)境污染物的復(fù)合毒性研究對(duì)于準(zhǔn)確預(yù)測(cè)環(huán)境風(fēng)險(xiǎn)，評(píng)價(jià)和診斷環(huán)境安全，具有十分重要的理論和現(xiàn)實(shí)意義；(4)不同物種及性別生物對(duì)MCs的抗性差異很大，開(kāi)展不同物種及性別間的比較毒理學(xué)研究可以為該類毒素的毒性作用模式和風(fēng)險(xiǎn)評(píng)估提供依據(jù)；(5)關(guān)于MCs的毒性效應(yīng)研究相對(duì)全面，但是其致毒機(jī)理全貌尚不清楚，亟需基于如代謝組學(xué)、蛋白組學(xué)、轉(zhuǎn)錄組學(xué)等更為敏感的新興系統(tǒng)毒理學(xué)研究手段，尋找生物標(biāo)志物，深入剖析其毒性機(jī)制；(6)在深入研究MCs微觀致毒機(jī)理的基礎(chǔ)上，加強(qiáng)指示藍(lán)藻水華污染的相關(guān)分子標(biāo)志物研究，以期篩選出可用于湖泊生態(tài)安全的早期診斷指標(biāo)體系和分子水平上的安全閾值。

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Environmental Exposure，Toxicity and Toxic Mechanism of Microcystins：A Review

Guo Xiaochun1,Lu Shaoyong1,*,Xie Ping2,Chen Jun2,Liu Xiaohui3

1.Observation and Research Station for Lake Dongtinghu(SEPSORSLD),Research Centre of Lake Environment,State Environmental Protection Key Laboratory for Lake Pollution Control,State Key Laboratory of Environmental Criteria and Risk Assessment,Chinese Research Academy of Environmental Sciences,Beijing 100012,China
2.Donghu Experimental Station of Lake Ecosystems,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China
3.Institute of Geography and Environment,Shandong Normal University,Jinan 250014,China

13 October 2015 accepted 5 February 2016

Microcystins(MCs),which is the most popular algae toxins generated by the explosive propagation of Cyanobacteriain the eutrophic fresh water,have caused worldwide concerns for their great toxicity,widespread distribution and structural stability.This review summarized the recent development of pollution status of MCs and their representative toxicity and toxic mechanism.In addition,the suggestions were put forward for present studies, in order to provide the basis for effectively reducing the potential security risks and studying deeply the ecotoxicological effects of microcystins.

microcystins;environmental exposure;toxic effects;toxic mechanism

2015-10-13 錄用日期：2016-02-05

1673-5897（2016）3-061-11

X171.5

A

10.7524/AJE.1673-5897.20151013002

簡(jiǎn)介：盧少勇(1976-)，男，環(huán)境科學(xué)博士，研究員，主要研究方向湖泊生態(tài)修復(fù)與污染防治，發(fā)表學(xué)術(shù)論文140余篇。

科技基礎(chǔ)性工作專項(xiàng)重點(diǎn)項(xiàng)目(2015FY110900)；國(guó)家水體污染控制與治理重大專項(xiàng)(2013ZX07101-014,2012ZX07105-002)

國(guó)曉春(1987-)，女，理學(xué)博士，研究方向?yàn)榈鷳B(tài)毒理學(xué)，E-mail:guoxiaochun419@163.com

*通訊作者（Corresponding author），E-mail:lusy@craes.org.cn