鐘艷，汝少國(guó)

中國(guó)海洋大學(xué)海洋生命學(xué)院，青島 266003

在自然水環(huán)境中，魚類常常會(huì)遭受各種各樣的環(huán)境脅迫，如捕食者的威脅、溫度的驟變等，為了應(yīng)對(duì)外環(huán)境的脅迫，魚體通過(guò)激活腎間應(yīng)激軸(即下丘腦-垂體-腎間腺軸，hypothalamic-pituitary-interrenal, 簡(jiǎn)稱HPI軸)合成并分泌應(yīng)激激素皮質(zhì)醇，啟動(dòng)應(yīng)激反應(yīng)(stress response)，調(diào)節(jié)機(jī)體的能量分配從而維持體內(nèi)平衡。應(yīng)激反應(yīng)在動(dòng)物界中是一種普遍的、高度保守的適應(yīng)性反應(yīng)[1-2]。然而，有大量文獻(xiàn)報(bào)道了多種環(huán)境污染物的慢性或亞慢性暴露會(huì)導(dǎo)致魚類的腎間應(yīng)激軸損壞或腎間組織損傷，致使機(jī)體無(wú)法正常啟動(dòng)應(yīng)激反應(yīng)。Mishra和Mohanty[3]研究發(fā)現(xiàn)2 mg·L-1或4 mg·L-1六價(jià)鉻鹽慢性暴露一個(gè)月，可導(dǎo)致淡水鯰魚(Channapunctatus)血漿皮質(zhì)醇水平較對(duì)照組顯著降低，腎間細(xì)胞萎縮，在二次急性脅迫下HPI軸調(diào)節(jié)皮質(zhì)醇水平升高的能力缺失，表明六價(jià)鉻鹽慢性暴露損壞了鯰魚HPI軸應(yīng)激功能。環(huán)境污染物包括金屬的長(zhǎng)期暴露導(dǎo)致魚類無(wú)法正常啟動(dòng)適應(yīng)性應(yīng)激反應(yīng)，可能帶來(lái)嚴(yán)重的后果[4]，如導(dǎo)致魚類在嚴(yán)寒冬季條件下存活能力降低[5]；面對(duì)捕食者時(shí)的反應(yīng)和應(yīng)對(duì)能力減弱[6]。應(yīng)激功能的異常是污染物脅迫條件下魚體的一種早期生理反應(yīng)，腎間應(yīng)激軸的損壞還可能進(jìn)一步危害生物體的生長(zhǎng)、生殖、代謝、免疫、行為等功能，使魚類的健康水平降低。因此，利用魚類應(yīng)激功能的變化可檢測(cè)、評(píng)估和預(yù)警污染物的毒性效應(yīng)。Pankhurst[7]甚至認(rèn)為，動(dòng)物應(yīng)激功能的變化在評(píng)估環(huán)境質(zhì)量以及預(yù)測(cè)極端環(huán)境(如洪澇、暴風(fēng)雨)來(lái)臨方面也會(huì)有廣闊的應(yīng)用前景。然而在魚類應(yīng)激功能異常研究方面，以往只是利用血漿皮質(zhì)醇水平作為單一指示指標(biāo)，忽略了皮質(zhì)醇水平受到腎間應(yīng)激軸的嚴(yán)格調(diào)控。本文通過(guò)對(duì)腎間應(yīng)激軸結(jié)構(gòu)及調(diào)控機(jī)制的簡(jiǎn)要介紹，系統(tǒng)的綜述了多種環(huán)境污染物對(duì)應(yīng)激軸線的干擾效應(yīng)，初步探討污染物干擾硬骨魚應(yīng)激軸的作用機(jī)制，以期為深入研究環(huán)境污染物對(duì)魚類應(yīng)激功能的作用機(jī)理提供一定理論基礎(chǔ)。

1 硬骨魚腎間應(yīng)激軸

當(dāng)人或動(dòng)物自身完整性受到挑戰(zhàn)并處于危險(xiǎn)狀態(tài)的時(shí)候，我們稱人或動(dòng)物受到“脅迫(stress)”[8]。動(dòng)物應(yīng)對(duì)環(huán)境脅迫的關(guān)鍵反應(yīng)，則稱為“應(yīng)激反應(yīng)(stress response)”，包括一系列的行為、生理以及細(xì)胞的變化。急性應(yīng)激反應(yīng)通常是短期的、適應(yīng)性的反應(yīng)，它能使機(jī)體應(yīng)對(duì)潛在的威脅；而慢性應(yīng)激反應(yīng)被認(rèn)為是長(zhǎng)期的、不利的反應(yīng)，它能夠?qū)ιL(zhǎng)、生殖和免疫反應(yīng)等產(chǎn)生不利的影響[9-11]。

硬骨魚的應(yīng)激反應(yīng)主要受到下丘腦-垂體-腎間腺軸的調(diào)節(jié)[12-13]。腎間應(yīng)激軸的激活首先從下丘腦開始，下丘腦接收從中樞和周邊神經(jīng)系統(tǒng)發(fā)出的“應(yīng)激”信號(hào)，“應(yīng)激”信號(hào)促進(jìn)下丘腦視前核(preoptic nucleus, NPO)神經(jīng)細(xì)胞分泌并釋放促腎上腺皮質(zhì)激素釋放因子(corticotrophin releasing factor, CRF)[14]。CRF在魚類HPI軸的調(diào)控中發(fā)揮關(guān)鍵性作用[15]，CRF通過(guò)與垂體前葉促腎上腺皮質(zhì)細(xì)胞膜上的鳥嘌呤核苷酸結(jié)合蛋白(G-蛋白)偶聯(lián)受體結(jié)合，促進(jìn)垂體分泌促腎上腺皮質(zhì)激素(adrenocorticotropic hormone, ACTH)，ACTH由前體激素阿黑皮素原(pro-opiomelanocortin, POMC)衍生而來(lái)，在脊椎動(dòng)物應(yīng)激反應(yīng)中處于中心角色[16]。垂體分泌并釋放的ACTH與頭腎腎間細(xì)胞上的黑皮質(zhì)素2型受體(melanocortin type 2 receptor, MC2R)結(jié)合后[17]，激活MC2R，經(jīng)過(guò)cAMP信號(hào)轉(zhuǎn)導(dǎo)途徑，一些調(diào)節(jié)類固醇激素生成的蛋白被磷酸化，這些蛋白質(zhì)磷酸化后由非活性變成活性，將皮質(zhì)類固醇合成原料--膽固醇由線粒體外膜轉(zhuǎn)運(yùn)至線粒體內(nèi)膜，在一系列皮質(zhì)類固醇生成酶的作用下，最終合成大量的應(yīng)激激素皮質(zhì)醇(cortisol，最主要的皮質(zhì)類固醇激素)(圖1)。皮質(zhì)醇經(jīng)血液循環(huán)到達(dá)靶組織并與靶組織上的糖皮質(zhì)激素受體(glucocorticoid receptor, GR)結(jié)合，調(diào)控一些與糖代謝、免疫功能以及行為等相關(guān)目的基因的轉(zhuǎn)錄[18-19]。GR是類固醇激素受體家族的成員之一，屬于核受體超家族[20]，主要存在于硬骨魚的鰓、肝臟、大腦和腸中[21-23]。大多數(shù)硬骨魚如虹鱒魚(Oncorhynchusmykiss)[24]、棘鰭類熱帶淡水魚伯氏樸麗魚(Haplochromisburtoni)[25]等均具有兩種GRs(GR1和GR2)，斑馬魚(Daniorerio)是迄今為止被報(bào)道的只有一種GR的硬骨魚。通過(guò)皮質(zhì)醇的合成與釋放，生物體的能量得以重新分配，從而滿足應(yīng)激引起的機(jī)體能量增加的需求[12,26]。

2 環(huán)境污染物對(duì)腎間應(yīng)激軸的干擾效應(yīng)及其潛在干擾機(jī)制

2.1 環(huán)境污染物對(duì)腎間應(yīng)激軸的干擾效應(yīng)

環(huán)境污染物可通過(guò)降低魚體皮質(zhì)醇水平和糖原含量，減弱其對(duì)二次脅迫的應(yīng)答能力。Benguira等[27]利用體內(nèi)試驗(yàn)研究表明，5、20、50 mg·kg-1有機(jī)氯農(nóng)藥o,p’-DDD注射14 d可引起虹鱒魚血漿皮質(zhì)醇水平降低、高濃度處理組肝臟糖原含量顯著減少，從而導(dǎo)致魚體對(duì)二次脅迫的應(yīng)答能力減弱。某些污染物還有可能通過(guò)直接損傷腎間組織、破壞腎間細(xì)胞以及垂體促腎上腺皮質(zhì)細(xì)胞，干擾HPI軸的正常運(yùn)行。Brodeur等[28]認(rèn)為腎間組織功能異常是導(dǎo)致黃色河鱸(Percaflavescens)皮質(zhì)醇應(yīng)激反應(yīng)損壞的原因之一，受重金屬污染水域黃色河鱸的腎間組織(體外)在ACTH的刺激下皮質(zhì)醇分泌水平顯著低于對(duì)照組。因此，環(huán)境污染物對(duì)HPI軸的干擾效應(yīng)最終表現(xiàn)為血漿皮質(zhì)醇水平的降低，導(dǎo)致機(jī)體不能正常啟動(dòng)應(yīng)激反應(yīng)，從而無(wú)法提供充足能量應(yīng)對(duì)二次脅迫，使魚體處于亞健康狀態(tài)。

圖1 魚類皮質(zhì)醇合成途徑Fig. 1 Cortisol biosynthesis pathway in fish

2.2 腎間應(yīng)激軸的干擾對(duì)其它生理學(xué)功能的影響

污染物對(duì)腎間應(yīng)激軸的干擾導(dǎo)致皮質(zhì)醇水平的異常，可能會(huì)進(jìn)一步影響魚類的代謝、離子平衡的調(diào)節(jié)以及免疫等生理功能[29-30]。

皮質(zhì)醇參與調(diào)節(jié)機(jī)體的能量代謝。Aluru和Vijayan[31]研究證實(shí)皮質(zhì)醇通過(guò)與肝臟GR結(jié)合，上調(diào)肝臟葡糖異生作用的限速酶磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PEPCK)，使肝細(xì)胞葡萄糖含量升高，為恢復(fù)體內(nèi)平衡提供能量來(lái)源。污染物可能通過(guò)干擾皮質(zhì)醇合成、GR途徑等，進(jìn)一步影響魚類的代謝[32]。Gagnon等[33]研究發(fā)現(xiàn)，Cu(80 μg·L-1)暴露30 d，導(dǎo)致虹鱒魚急性皮質(zhì)醇反應(yīng)能力降低，同時(shí)伴隨血漿葡萄糖水平和肝臟糖原含量的降低。Ings[2]研究表明，100%市政污水暴露導(dǎo)致虹鱒魚在二次脅迫下皮質(zhì)醇應(yīng)答減弱，同時(shí)肝臟己糖激酶(hexokinase, HK)和葡糖激酶(glucokinase, GK)的活性顯著降低，表明虹鱒魚的能量代謝能力也受到了抑制。

皮質(zhì)醇涉及魚類離子平衡的調(diào)節(jié)。由于硬骨魚不能像哺乳動(dòng)物一樣合成鹽皮質(zhì)激素如醛固酮，皮質(zhì)醇在硬骨魚中除了發(fā)揮糖皮質(zhì)激素功能外，還具有鹽皮質(zhì)激素的作用[12,34]。污染物干擾皮質(zhì)醇應(yīng)激反應(yīng)可能會(huì)進(jìn)一步影響魚類的離子平衡。皮質(zhì)醇能夠促進(jìn)鰓氯細(xì)胞的增殖和分化[35-36]。Laflamme等[32]認(rèn)為，污染最嚴(yán)重湖泊(Osisiko)中的黃鱸血漿氯離子水平減少可能是急性應(yīng)激反應(yīng)能力降低導(dǎo)致的生理學(xué)后果。Na+,K+-ATPase涉及硬骨魚類鰓的離子攝取和鹽分泌[37]。皮質(zhì)醇能夠提高大西洋鮭魚(Salmosalar)鰓Na+,K+-ATPase的活性和mRNA水平，提高耐鹽性[38]。Levesque等[39]研究發(fā)現(xiàn)，金屬污染水域中黃鱸急性皮質(zhì)醇反應(yīng)損壞的同時(shí)伴隨鰓Na+,K+-ATPase活性的改變。Lerner等[29]認(rèn)為17β-雌二醇(2 μg·L-1)通過(guò)降低GR水平減少大西洋鮭魚鰓Na+,K+-ATPase的活性，使其離子調(diào)節(jié)能力和海水中的耐鹽性減弱。

除了能夠干擾能量代謝和離子平衡，皮質(zhì)醇的變化還會(huì)抑制魚類的生殖、生長(zhǎng)以及免疫等功能。如大量的文獻(xiàn)已經(jīng)證實(shí)高于生理水平的皮質(zhì)醇不僅對(duì)生殖功能具有有害影響，還能抑制魚類的生長(zhǎng)。Poursaeid等[40]研究發(fā)現(xiàn)皮質(zhì)醇長(zhǎng)期處理不僅導(dǎo)致大鱘魚Husohuso血漿睪酮和17β-雌二醇水平降低，而且最高濃度處理組身體增長(zhǎng)指數(shù)(包括體重增加率、比生長(zhǎng)速率、肥滿度)也顯著下降。過(guò)高水平的皮質(zhì)醇對(duì)免疫功能也具有抑制作用。Castillo等人[41]的研究則表明皮質(zhì)醇能夠抑制分離巨噬細(xì)胞細(xì)胞因子(TNF、TGFb、IL-6)的表達(dá)，當(dāng)皮質(zhì)醇和脂多糖(LPS)同時(shí)加入到巨噬細(xì)胞中，細(xì)胞因子的誘導(dǎo)也被皮質(zhì)醇顯著抑制。

2.3 環(huán)境污染物對(duì)腎間應(yīng)激軸的潛在干擾機(jī)制

研究表明環(huán)境污染物主要通過(guò)干擾下丘腦和垂體促皮質(zhì)類固醇激素的分泌、皮質(zhì)醇的合成與代謝、cAMP信號(hào)轉(zhuǎn)導(dǎo)以及皮質(zhì)醇負(fù)反饋途徑等，最終抑制皮質(zhì)醇的合成與分泌，減弱魚類的應(yīng)激反應(yīng)能力。通常情況下，某一種污染物往往是以HPI軸的多個(gè)位點(diǎn)為靶目標(biāo)發(fā)揮干擾效應(yīng)。就目前報(bào)道的文獻(xiàn)來(lái)看，多數(shù)污染物主要通過(guò)干擾類固醇生成和大腦糖皮質(zhì)激素信號(hào)這兩條途徑影響HPI軸的功能[42-44]。

2.3.1 對(duì)HPI軸的過(guò)度刺激

皮質(zhì)醇和ACTH等激素分泌亢進(jìn)可能是環(huán)境污染物導(dǎo)致HPI軸功能損壞的機(jī)制之一。如在野外環(huán)境下，污染物本身就作為一個(gè)“應(yīng)激源(stressor)”，長(zhǎng)期、過(guò)度激活腎間應(yīng)激軸，引起慢性應(yīng)激反應(yīng)即皮質(zhì)醇分泌亢進(jìn)，可能進(jìn)一步導(dǎo)致魚類HPI軸功能受損或衰竭，對(duì)二次脅迫的應(yīng)答能力下降[45-46]。Hontela等[47]研究表明，加拿大一條被污染河流中的黃色河鱸和白斑狗魚(Esoxlucius)在二次脅迫下應(yīng)激反應(yīng)減弱，原因可能是污染物慢性暴露引起HPI軸持續(xù)活躍、皮質(zhì)醇分泌系統(tǒng)衰竭所致。Norris等人[48]的研究進(jìn)一步證實(shí)了Hontela的推測(cè)，受金屬污染水域中的褐鱒魚(Salmotrutta)因過(guò)度分泌ACTH導(dǎo)致HPI軸功能衰竭，二次應(yīng)激的血漿皮質(zhì)醇水平顯著低于對(duì)照組。

2.3.2 干擾下丘腦以及垂體主要促激素對(duì)皮質(zhì)醇的調(diào)節(jié)

污染物對(duì)CRF和ACTH的干擾可能直接影響HPI軸功能的正常發(fā)揮。在哺乳動(dòng)物中研究發(fā)現(xiàn)，Cd可通過(guò)干擾Y-1型鼠腎上腺腫瘤細(xì)胞ACTH的分泌影響皮質(zhì)類固醇的分泌[49]。因魚體CRF、ACTH等激素水平較難測(cè)定，相關(guān)研究主要是在基因水平上開展的，如Fuzzen等[50]研究表明，100 ng·L-1E2暴露48 h導(dǎo)致雄性斑馬魚皮質(zhì)醇應(yīng)答減弱、crfmRNA的表達(dá)水平降低了三倍。然而，基因水平的變化與激素水平的變化有時(shí)并不成正相關(guān)關(guān)系。因此，未來(lái)還需要從蛋白水平進(jìn)一步研究污染物暴露下CRF和ACTH對(duì)皮質(zhì)醇的調(diào)節(jié)作用。

2.3.3 干擾cAMP信號(hào)途徑

普遍認(rèn)為cAMP信號(hào)級(jí)聯(lián)反應(yīng)的過(guò)程主要為：ACTH與受體MC2R結(jié)合后，激活G蛋白，G蛋白激活后促進(jìn)腺苷酸環(huán)化酶生成大量的cAMP[51]。cAMP通過(guò)激活多個(gè)靶目標(biāo)如cAMP-依賴性蛋白激酶A(protein kinase A, PKA)等促進(jìn)類固醇激素的生成[52-53]。cAMP信號(hào)途徑的各個(gè)因子都有可能是環(huán)境污染物干擾皮質(zhì)類固醇合成的關(guān)鍵目標(biāo)。研究發(fā)現(xiàn)，有機(jī)氯農(nóng)藥o,p’-DDD能顯著抑制G蛋白激動(dòng)劑NaF、腺苷酸環(huán)化酶激動(dòng)劑毛喉素以及cAMP類似物對(duì)皮質(zhì)醇生成的促進(jìn)效應(yīng)，表明cAMP信號(hào)途徑的多個(gè)因子(G蛋白、腺苷酸環(huán)化酶等)均是o,p’-DDD干擾虹鱒魚腎間細(xì)胞皮質(zhì)醇生成的靶位點(diǎn)(體外)[54]。Sandhu和Vijayan[55]研究認(rèn)為鎘(10、100、1 000 nmol)干擾虹鱒魚頭腎細(xì)胞皮質(zhì)醇合成的靶目標(biāo)位于cAMP生成的上游，并推測(cè)MC2R信號(hào)途徑(ACTH誘導(dǎo)皮質(zhì)醇生成首要的一步)可能是鎘干擾虹鱒魚頭腎細(xì)胞皮質(zhì)醇生成的關(guān)鍵目標(biāo)。

2.3.4 干擾皮質(zhì)醇的合成與代謝

皮質(zhì)類固醇生物合成途徑的關(guān)鍵限速步驟主要包括：類固醇急性調(diào)節(jié)蛋白(steroidogenic acute regulatory protein, StAR)和外周型苯二氮受體(peripheral-type benzodiazepine receptor,PBR)共同將膽固醇從線粒體外膜轉(zhuǎn)移到細(xì)胞色素P450側(cè)鏈裂解酶(cytochrome P450 side chain cleavage enzyme, P450scc)的活性部位[56-58]；P450scc將膽固醇轉(zhuǎn)化成孕烯醇酮[59]。皮質(zhì)類固醇合成途徑中關(guān)鍵酶或蛋白轉(zhuǎn)錄水平的微小變化可能會(huì)對(duì)皮質(zhì)醇激素水平產(chǎn)生顯著的影響[44]。多篇文獻(xiàn)研究結(jié)果表明皮質(zhì)醇合成限速步驟中的關(guān)鍵酶或蛋白是環(huán)境污染物干擾腎間應(yīng)激軸的關(guān)鍵靶目標(biāo)，環(huán)境污染物可通過(guò)降低該限速步驟中關(guān)鍵因子的轉(zhuǎn)錄水平，進(jìn)而干擾皮質(zhì)醇的合成。Aluru等[60]研究表明，芳香烴受體(aryl hydrocarbon receptor, AhR)激動(dòng)劑(β-萘黃酮,BNF)和拮抗劑(α-萘黃酮,ANF)可通過(guò)降低StAR和P450scc的mRNA含量使腎間類固醇生成能力減弱，顯著抑制了ACTH對(duì)虹鱒魚頭腎細(xì)胞皮質(zhì)醇分泌的促進(jìn)效應(yīng)(體外)。100 mg·kg-1水楊酸鹽喂食虹鱒魚3 d，可導(dǎo)致頭腎StAR和PBR的mRNA水平顯著降低[43]。

除了限速步驟中的StAR、PBR和P450scc，皮質(zhì)類固醇合成途徑中的主要蛋白還包括下游的17α-羥化酶(CYP17)、3β-羥化類固醇脫氫酶(3β-HSD)、21-羥化酶(CYP21)和11β-羥化酶(CYP11B)[59]。其中11β-羥化酶(CYP11B)是一個(gè)重要的多功能類固醇合成酶，它參與皮質(zhì)醇生物合成的最后一步，將11-脫氧皮質(zhì)醇轉(zhuǎn)化為皮質(zhì)醇[61]。Kortner等[62]研究發(fā)現(xiàn)，0.1、1、10 mg·kg-1三丁基錫(TBT)灌食幼年大馬哈魚72 h，可顯著抑制腎間11β-羥化酶的mRNA水平。但Walsh等[63]認(rèn)為，與限速步驟因子StAR、P450scc等相比，類固醇合成途徑下游的一些酶對(duì)污染物的暴露可能表現(xiàn)較低的敏感性。如芳香烴受體(AhR)激動(dòng)劑(β-萘黃酮,BNF)和拮抗劑(α-萘黃酮,ANF)顯著降低了虹鱒魚頭腎細(xì)胞StAR和P450scc的轉(zhuǎn)錄水平，但對(duì)CYP11B表達(dá)無(wú)影響[60]。Gravel和Vijayan[43]研究表明，水楊酸鹽暴露可顯著降低虹鱒魚頭腎StAR和PBR的mRNA水平，而11β-羥化酶的mRNA水平無(wú)明顯變化。

皮質(zhì)類固醇的代謝對(duì)血漿皮質(zhì)醇水平的維持也起關(guān)鍵性的作用，環(huán)境污染物可能通過(guò)干擾皮質(zhì)醇的代謝，促進(jìn)或抑制皮質(zhì)醇轉(zhuǎn)化為失活代謝物，進(jìn)而影響其發(fā)揮正常生理功能。Ings等[2]研究發(fā)現(xiàn)，城市污水暴露虹鱒魚14 d，在急性脅迫后24 h，100%未稀釋污水處理組皮質(zhì)醇合成酶(StAR、P450scc、11β-羥化酶)mRNA水平均顯著降低，而血漿皮質(zhì)醇含量較對(duì)照組顯著升高，由此推測(cè)城市污水的暴露可能干擾了皮質(zhì)醇的代謝。Wiseman等[64]研究表明，用含硒代蛋氨酸8.47 mg·(kg·dm)-1的食物喂養(yǎng)雌性虹鱒魚126 d后，給予空氣暴露脅迫，血漿皮質(zhì)醇水平較對(duì)照組降低，而其失活代謝物--皮質(zhì)酮水平顯著升高，進(jìn)而導(dǎo)致虹鱒魚對(duì)二次脅迫的應(yīng)激應(yīng)答能力減弱。雖然現(xiàn)有文獻(xiàn)已經(jīng)證明皮質(zhì)醇的代謝是污染物干擾魚類應(yīng)激反應(yīng)的靶位點(diǎn)，但是污染物是如何干擾皮質(zhì)醇的代謝途徑尚不清楚。Wiseman等[64]研究中缺少對(duì)11β-羥化類固醇脫氫酶(將皮質(zhì)醇轉(zhuǎn)化成皮質(zhì)酮的直接指標(biāo))水平的測(cè)定。

2.3.5 干擾大腦GR信號(hào)途徑

皮質(zhì)醇可通過(guò)負(fù)反饋途徑作用于下丘腦和垂體，調(diào)節(jié)CRF和ACTH的分泌，從而維持血漿中皮質(zhì)醇的水平。其中大腦GR信號(hào)途徑是皮質(zhì)醇負(fù)反饋調(diào)節(jié)環(huán)的一個(gè)關(guān)鍵組成部分，Alderman等[65]研究表明，GR涉及調(diào)節(jié)虹鱒魚下丘腦視前區(qū)(preoptic area, POA)CRF轉(zhuǎn)錄水平，進(jìn)而影響血漿皮質(zhì)醇的水平。大腦GR信號(hào)途徑是污染干擾腎間應(yīng)激軸的靶位點(diǎn)之一。在分子水平上，Aluru等[42]研究表明，高濃度PCBs 100 mg·(kg·bm)-1暴露北極紅點(diǎn)鮭(Salvelinusalpinus)引起神經(jīng)毒性，使含GR的神經(jīng)細(xì)胞減少，大腦GR基因表達(dá)下降，從而干擾了皮質(zhì)醇的負(fù)反饋調(diào)節(jié)，導(dǎo)致血漿皮質(zhì)醇水平降低。在蛋白水平上，Gravel和Vijayan[43]的研究也獲得了類似結(jié)果，100 mg·(kg·bw)-1水楊酸鹽處理虹鱒魚3 d導(dǎo)致大腦GR蛋白含量顯著降低約50%，從而干擾了皮質(zhì)醇負(fù)反饋調(diào)節(jié)。

3 研究展望

3.1 開發(fā)環(huán)境腎間腺干擾物篩選的分子標(biāo)志物

三大內(nèi)分泌軸線下丘腦-垂體-性腺軸(hypothalamic-pituitary-gonadal, HPG軸)、下丘腦-垂體-甲狀腺軸(hypothalamic-pituitary-thyroid axis, HPT軸)以及HPI軸共同調(diào)節(jié)魚體的內(nèi)分泌平衡。污染物干擾任一內(nèi)分泌軸線均可導(dǎo)致魚體內(nèi)分泌紊亂，甚至威脅個(gè)體的存活。利用指示內(nèi)分泌軸線干擾的分子標(biāo)志物可以準(zhǔn)確、敏感地評(píng)價(jià)污染物對(duì)魚類早期、低水平的內(nèi)分泌擾亂作用，例如卵黃原蛋白(vitellogenin, VTG)是指示HPG軸是否受到環(huán)境雌激素干擾的理想分子標(biāo)志物。目前，對(duì)HPI軸分子標(biāo)志物的研究工作還未見開展。而開發(fā)指示腎間腺軸干擾的分子標(biāo)志物，對(duì)鑒別和篩選“環(huán)境腎間腺干擾物”具有重要意義。早期關(guān)于哺乳動(dòng)物的研究發(fā)現(xiàn)，具有皮質(zhì)類固醇受體啟動(dòng)元件的基因可能是研究污染物干擾皮質(zhì)醇應(yīng)答的有效標(biāo)志物，這可能同樣適用于魚類。Quabius等[66]研究表明，PCBs暴露能引起虹鱒魚前腎細(xì)胞中糖皮質(zhì)激素受體和白細(xì)胞介素-1-β的升高。因此，今后應(yīng)該著重研究含有皮質(zhì)醇受體啟動(dòng)元件的基因，在基因表達(dá)水平上篩選“環(huán)境腎間腺干擾物”的分子標(biāo)志物。

3.2 環(huán)境污染物對(duì)腎間應(yīng)激軸作用機(jī)制的深入探討

目前，環(huán)境污染物干擾腎間應(yīng)激軸的機(jī)制研究遠(yuǎn)不夠深入。Ings等[2]研究發(fā)現(xiàn)，城市污水暴露導(dǎo)致虹鱒魚皮質(zhì)醇合成酶基因表達(dá)下降而血漿皮質(zhì)醇水平顯著升高，推測(cè)原因是皮質(zhì)醇的代謝也受到干擾，但是該作者并沒(méi)有進(jìn)一步證實(shí)。多篇文獻(xiàn)表明大腦GR信號(hào)途徑是污染物干擾HPI軸的靶位點(diǎn)，但垂體水平的負(fù)反饋調(diào)節(jié)卻未見報(bào)道。其次，環(huán)境污染物因其種類、結(jié)構(gòu)和性質(zhì)的不同，其干擾機(jī)制也存在較大差異，有些是對(duì)魚類腎間應(yīng)激軸直接的作用，還有些可能是污染物對(duì)其它內(nèi)分泌軸線的作用間接導(dǎo)致腎間軸線的變化。腎間應(yīng)激軸與生殖軸[67-68]、甲狀腺軸均存在交互作用[69]。三大軸線之間也可以進(jìn)行復(fù)雜的交叉反應(yīng)，Liu等[70]研究表明咪鮮胺(prochloraz, PCZ)可通過(guò)降低雌性斑馬魚血漿E2濃度下調(diào)HPI軸CRF的基因表達(dá)，并進(jìn)一步導(dǎo)致血漿皮質(zhì)醇水平的降低；而CRF的降低又能夠下調(diào)HPT軸促甲狀腺激素(thyroid-stimulating hormone, TSH)水平，最終導(dǎo)致甲狀腺素(thyroxine, T4)含量下降。因此，作用機(jī)制的進(jìn)一步探討首先要從軸線外角度明確污染物干擾腎間應(yīng)激軸是直接作用還是間接作用，其次要深入研究軸線內(nèi)的靶位點(diǎn)，皮質(zhì)醇代謝途徑的酶和因子以及垂體水平的正負(fù)反饋調(diào)節(jié)等應(yīng)是今后深入探討污染物干擾HPI軸作用機(jī)制的研究重點(diǎn)，而污染物對(duì)魚類完整內(nèi)分泌系統(tǒng)的影響研究應(yīng)是未來(lái)的研究熱點(diǎn)和難點(diǎn)。

3.3 大腦神經(jīng)遞質(zhì)系統(tǒng)與應(yīng)激軸的聯(lián)合研究

目前已知大腦單胺能神經(jīng)遞質(zhì)系統(tǒng)涉及脊椎動(dòng)物的完整應(yīng)激反應(yīng)，其中大腦5-羥色胺(5-hydroxytryptamine, 5-HT)系統(tǒng)在復(fù)雜的神經(jīng)內(nèi)分泌環(huán)中發(fā)揮關(guān)鍵作用，在鼠中已經(jīng)證實(shí)5-HT神經(jīng)末端與室旁核內(nèi)包含CRH的神經(jīng)元之間存在直接的突觸聯(lián)系[71]，5-HT能夠促進(jìn)哺乳動(dòng)物下丘腦CRF[72]和垂體ACTH的釋放[73]；同時(shí)，5-HT活性也受到應(yīng)激反應(yīng)中的因子如皮質(zhì)醇、CRF等影響[74]。早期文獻(xiàn)就已表明大腦5-HT能系統(tǒng)在魚類HPI軸的調(diào)節(jié)中也發(fā)揮關(guān)鍵作用[75]，但具體機(jī)制還不清楚。近來(lái)，Gesto等[76]研究表明，急性應(yīng)激能夠誘導(dǎo)虹鱒魚前腦血清素活性和終腦多巴胺能活性的增加。因此，環(huán)境污染物對(duì)魚類完整應(yīng)激反應(yīng)的影響除了與應(yīng)激軸有關(guān)，應(yīng)該還要考慮大腦神經(jīng)遞質(zhì)系統(tǒng)是否也受到干擾，上游神經(jīng)遞質(zhì)系統(tǒng)受到干擾可能也會(huì)影響下游軸線的調(diào)節(jié)。由于單胺能系統(tǒng)與應(yīng)激軸之間存在復(fù)雜的交互作用，在哺乳動(dòng)物中相關(guān)研究很有限，魚類中的文獻(xiàn)更是非常罕見，魚類大腦神經(jīng)遞質(zhì)系統(tǒng)與應(yīng)激軸的聯(lián)系還有待于進(jìn)一步揭示。

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