靳亞茹，劉紅玲,* ，韓志華，花小雪

1. 南京大學(xué)環(huán)境學(xué)院 污染控制與資源化研究國家重點(diǎn)實(shí)驗(yàn)室，南京 210023 2. 環(huán)境保護(hù)部南京環(huán)境科學(xué)研究所，南京 210042

多溴聯(lián)苯醚(poly brominated diphenyl ethers, PBDEs)作為一種曾被廣泛應(yīng)用的溴代阻燃劑[1]，已被禁止生產(chǎn)和使用近10年[2]，但仍能在許多環(huán)境介質(zhì)包括空氣[3]、水體[4]、土壤[5]、灰塵、底泥[6]和各種生物體[7-10]甚至人體母乳、血液、頭發(fā)、胎盤以及嬰兒血液[11-15]中檢出它們的存在。其中2,4,4'-三溴聯(lián)苯醚(2,4,4'-tribromodiphenyl ether, BDE-28)和2,2',4,4',5-五溴聯(lián)苯醚(2,2',4,4',5-pentabromodiphenyl ether, BDE-99)是在長江下游底泥中檢出濃度最高的PBDE類物質(zhì)，甚至超過了2,2’,4,4’-四溴聯(lián)苯醚(2,2',4,4'-tetrabromodiphenyl ether, BDE-47)[16]。另外，在長江的水生生物如蝦類、蟹類、雜食性魚類、植食性魚類和肉食性魚類中也都有檢出BDE-28和BDE-99，其中BDE-28的檢出比例尤其高(10.9%)，超過了國外一般檢出水平[17]。因此BDE-28和BDE-99是否會(huì)對(duì)水生生物中造成負(fù)效應(yīng)引起了我們的關(guān)注。

動(dòng)物實(shí)驗(yàn)研究發(fā)現(xiàn)，研究較多的BDE-47會(huì)導(dǎo)致鳥類、魚類和哺乳動(dòng)物的神經(jīng)發(fā)育毒性、生殖毒性和內(nèi)分泌紊亂[18-23]，而且較低溴代的BDE-28和更高溴代的BDE-99也會(huì)對(duì)部分生物甚至人體健康產(chǎn)生不良影響。例如，已有毒性研究發(fā)現(xiàn)BDE-28影響斑馬魚幼魚的生長發(fā)育且對(duì)小孩的神經(jīng)發(fā)育有不良影響[24-25]，而且BDE-99也會(huì)對(duì)紅耳龜(red-eared sliders)、大鼠和鳥類的生長發(fā)育、神經(jīng)行為以及生殖行為有負(fù)面效應(yīng)[20,26-30]。由于PBDE類物質(zhì)與甲狀腺激素(thyroid hormone, TH)的結(jié)構(gòu)相似，所以它們被認(rèn)為是潛在的甲狀腺內(nèi)分泌干擾物。研究較多的BDE-47被報(bào)道作用于大鼠甲狀腺受體，改變?nèi)梭w中T3和T4濃度并對(duì)斑馬魚下丘腦-垂體-甲狀腺(hypothalamus-pituitary-thyroidal, HPT)軸基因表達(dá)有影響[31-33]。另外，也有文獻(xiàn)表明人體內(nèi)TH濃度與體內(nèi)BDE-28和BDE-99濃度相關(guān)[34]。而且有研究表明BDE-99暴露能改變青年大鱗大麻哈魚(Chinook salmon)和大鼠體內(nèi)甲狀腺激素水平，并影響大鼠小腦顆粒神經(jīng)元中甲狀腺激素受體基因的表達(dá)[35-37]。

綜上所述，BDE-28和BDE-99與其具有相似結(jié)構(gòu)的物質(zhì)BDE-47一樣對(duì)生物體和人類有潛在的神經(jīng)發(fā)育、生殖和甲狀腺相關(guān)的內(nèi)分泌干擾作用。然而，關(guān)于BDE-28和BDE-99對(duì)水生生物的內(nèi)分泌干擾效應(yīng)和機(jī)制研究還較少。同時(shí)，有學(xué)者提出影響甲狀腺系統(tǒng)的PBDE同系物也能影響硬骨魚的生殖系統(tǒng)[38]。例如，BDE-47喂食暴露會(huì)損害黑頭呆魚(fathead minnow)的甲狀腺功能并影響其生殖發(fā)育[39]。類似地，DE-71攝入會(huì)同時(shí)影響斑馬魚的甲狀腺激素濃度和產(chǎn)卵率[40]。Yu等[41-42]報(bào)道，在胚胎到成魚階段暴露于低濃度的DE-71不僅改變了HPT軸和TH相關(guān)基因的表達(dá)，而且改變了下丘腦-垂體-性腺(hypothalamus-pituitary-gonadal, HPG)軸和性激素相關(guān)基因的表達(dá)，并減少了斑馬魚的產(chǎn)卵量。在中國稀有鮈鯽(Chinese rare minnow)中，十溴聯(lián)苯醚(decabromodiphenyl ether, BDE-209)暴露可能導(dǎo)致成魚TH相關(guān)基因表達(dá)的改變并對(duì)雄性睪丸中精子發(fā)生產(chǎn)生抑制[43]。另外，有研究表明在斑馬魚中已經(jīng)觀察到下丘腦-垂體-腎上腺(hypothalamus-pituitary-adrenal, HPA)和HPG軸之間的交互作用，表明化學(xué)物質(zhì)可以通過降低E2的濃度從而使HPA軸基因的表達(dá)下調(diào)并降低皮質(zhì)醇的濃度[44]。并且在非哺乳類脊椎動(dòng)物實(shí)驗(yàn)中也發(fā)現(xiàn)HPA軸及HPG軸之間存在交互作用[45]。除此之外也有實(shí)驗(yàn)證明，化合物對(duì)斑馬魚體內(nèi)某一內(nèi)分泌軸的影響也將間接影響斑馬魚的其他內(nèi)分泌軸[44, 46]。在脊椎動(dòng)物中，生殖、甲狀腺和腎上腺內(nèi)分泌系統(tǒng)主要由HPT、HPA和HPG軸控制，這3個(gè)系統(tǒng)通路通過協(xié)調(diào)激素的合成、分泌、運(yùn)輸和代謝來調(diào)節(jié)其動(dòng)力學(xué)過程[32,44,47]。所以本文選擇與人體基因有87%相似的斑馬魚為模式生物，展開BDE-28和BDE-99對(duì)斑馬魚HPT、HPG和HPA內(nèi)分泌軸上相關(guān)基因影響研究，從而探究它們的內(nèi)分泌干擾機(jī)制。

1　材料與方法(Materials and methods)

1.1　實(shí)驗(yàn)材料

BDE-28、BDE-99(純度>99.2%)，購自美國AccuStandard公司；二甲基亞砜(dimethyl sulfoxide, DMSO)，分析純，購自南京化學(xué)試劑有限公司，作助溶劑；RNA抽提試劑盒(RNeasy?Mini Kit)、cDNA逆轉(zhuǎn)錄試劑盒(Omniscript RT Kit)均購自德國Qiagen公司；實(shí)時(shí)定量PCR試劑盒(SYBR?Real time PCR Master Mix Plus Kits)購自日本東洋紡公司。

斑馬魚養(yǎng)殖系統(tǒng)(ZKH-BM09)購自中國青島中科海水處理有限公司；多功能連續(xù)變倍體視顯微鏡(AZ100)購自日本尼康公司；智能光照培養(yǎng)箱購自寧波海曙賽福實(shí)驗(yàn)儀器廠；分析天平(AUY120)購自日本島津公司；魚孵化器(WT-2005)購自旺通五金電器廠；全功能酶標(biāo)儀(Synergy H4)購自美國Biotek公司；q-RT-PCR儀(Applied Biosystems Stepone Plus Real-time PCR System)購自美國Applied biosystems公司。

1.2　斑馬魚馴化及胚胎收集

5月齡野生AB型成年斑馬魚是由本實(shí)驗(yàn)室在斑馬魚養(yǎng)殖系統(tǒng)(ZKH-BM09)自行繁殖并養(yǎng)殖成熟。該半自動(dòng)斑馬魚養(yǎng)殖系統(tǒng)配備曝氣充氧泵、紫外殺菌燈、系統(tǒng)過濾器、溫度控制系統(tǒng)、光周期控制系統(tǒng)并可維持系統(tǒng)的水持續(xù)循環(huán)。系統(tǒng)運(yùn)行期間，維持溫度在27 ℃～29 ℃的自來水供斑馬魚生存，并維持系統(tǒng)光暗比14 h:10 h，每天喂食3次新鮮孵化的豐年蟲。

光照刺激對(duì)斑馬魚產(chǎn)卵非常重要，一般產(chǎn)卵發(fā)生在清晨開燈后30 min以內(nèi)。由于斑馬魚會(huì)自食其卵，因此我們選擇底部帶有小孔塑料板的孵化器來進(jìn)行斑馬魚產(chǎn)卵。前1天晚將3條雌魚和3條雄魚放進(jìn)一個(gè)底部帶有孔塑料板的孵化器，并用孵化器自帶的塑料隔板將雌魚與雄魚分開。第2天清晨開燈時(shí)，將孵化器中間的塑料隔板撤去，讓雄魚與雌魚在光刺激下交配產(chǎn)卵。30 min后將魚卵取出并置于培養(yǎng)皿中，使用統(tǒng)一配制充氧飽和的60 mg·L-1海鹽水清洗胚胎后，置于體視顯微鏡下觀察。剔除未發(fā)生卵裂以及卵裂不正常的胚胎，這些不正常的胚胎主要表現(xiàn)為初期無卵裂或卵裂細(xì)胞大小不均等。然后將斑馬魚胚胎培養(yǎng)于(28 ± 1) ℃恒溫光照培養(yǎng)箱中。在斑馬魚胚胎受精后4 h(4 hours post fertilization, 4 hpf)統(tǒng)一進(jìn)行暴露實(shí)驗(yàn)。

1.3　斑馬魚胚胎暴露實(shí)驗(yàn)

使用前將BDE-28和BDE-99分別配成高濃度的DMSO母液，實(shí)驗(yàn)中所用相應(yīng)濃度的暴露液由高濃度母液經(jīng)胚胎培養(yǎng)水稀釋而得，并且保證最高濃度暴露液中DMSO不超過暴露溶液體積的0.1%。暴露液現(xiàn)用現(xiàn)配，設(shè)置BDE-28和BDE-99水暴露溶液的濃度為2 μg·L-1、20 μg·L-1和200 μg·L-1，對(duì)照為0.1%的DMSO溶液。使用25 mL燒杯作為本次實(shí)驗(yàn)的暴露容器，實(shí)驗(yàn)設(shè)置3次生物學(xué)平行，每個(gè)燒杯中加入20 mL的暴露液，20枚發(fā)育正常的胚胎，放置于光照培養(yǎng)箱中。另外，為了防止溶液蒸發(fā)，覆蓋保鮮膜于暴露容器上，有實(shí)驗(yàn)表明覆蓋保鮮膜不會(huì)影響斑馬魚胚胎/幼魚的正常生長和發(fā)育[32]。暴露期間及時(shí)將停止發(fā)育或死亡的胚胎和幼魚用一次性吸管吸出，以免影響其他胚胎/幼魚的生長。在胚胎受精后120 h(120 hpf)，將各濃度對(duì)應(yīng)的斑馬魚幼魚收取到1.5 mL無RNA酶的離心管中，盡量將水分吸干，加入RNAlater試劑對(duì)樣品進(jìn)行固定(確保幼魚樣品浸沒在RNAlater溶液中)，置于-80 ℃超低溫冰箱中待后續(xù)實(shí)驗(yàn)。

1.4　斑馬魚幼魚總RNA抽提、逆轉(zhuǎn)錄和q-RT-PCR實(shí)驗(yàn)

本研究使用q-RT-PCR技術(shù)對(duì)HPT、HPG及HPA軸相關(guān)基因表達(dá)進(jìn)行檢驗(yàn)，因此首先需要對(duì)所取得的樣品進(jìn)行總RNA提取和逆轉(zhuǎn)錄。各濃度組孵化出的斑馬魚樣品使用RNA抽提試劑盒進(jìn)行總RNA提取。RNA抽提結(jié)束，使用酶標(biāo)儀TAKE 3軟件測定總RNA的量和OD260/OD280的比值，比值顯示1.9～2.1之間說明純度較好。根據(jù)測得的RNA濃度，使用無RNA酶的水將所有樣品稀釋到相同的濃度后，盡快使用逆轉(zhuǎn)錄試劑盒將RNA逆轉(zhuǎn)錄為cDNA。RNA抽提與逆轉(zhuǎn)錄過程嚴(yán)格按照試劑盒所述方法。最后使用SYBR實(shí)時(shí)定量PCR試劑盒對(duì)逆轉(zhuǎn)錄后的cDNA進(jìn)行目的基因的擴(kuò)增和測定。所選擇的基因以及引物序列如之前文獻(xiàn)中所描述[48]。

該實(shí)驗(yàn)RT-PCR條件設(shè)置如下：初始變性時(shí)間10 min，擴(kuò)增40個(gè)循環(huán)，擴(kuò)增條件為首先升溫至95 ℃維持15 s，然后降至60 ℃保持1 min。目標(biāo)片段擴(kuò)增后，畫出溶解曲線確保所有cDNA樣品均僅能擴(kuò)增出唯一目標(biāo)產(chǎn)物。

圖1　斑馬魚胚胎暴露于2、20或200 μg·L-1的BDE-28至120 hpf時(shí)(A)下丘腦-垂體-甲狀腺(HPT)、(B)下丘腦-垂體-性腺(HPG)和(C)下丘腦-垂體-腎上腺(HPA)軸的基因表達(dá)變化注：結(jié)果表示為3次重復(fù)的平均值±SD，*P <0.05和**P <0.01表明暴露組與對(duì)照組相比有顯著性差異。Fig. 1　Expression of genes along the (A) HPT axis, (B) HPG axis, and (C) HPA axis of zebrafish embryo at 120 hpf following 2, 20 or 200 μg·L-1 BDE-28 exposureNote: Results were expressed as means±SD of three replicates. * P<0.05 and ** P<0.01 indicate significant differences in exposure groups compared with control group.

圖2　暴露于2、20和200 μg·L-1的BDE-28后，斑馬魚幼魚的3個(gè)軸((A)HPT軸、(B)HPG軸和(C)HPA軸)在各濃度下的效應(yīng)結(jié)果和可能的毒性途徑Fig. 2　Concentration dependent effects and proposed toxicity pathways along the three axes ((A) HPT axis, (B) HPG axis and (C) HPA axis) of zebrafish larvae exposed to 2, 20 and 200 μg·L-1 BDE-28

1.5　數(shù)據(jù)處理

使用LinRegPCR處理q-RT-PCR的原始數(shù)據(jù)，然后以持家基因18S rRNA作為mRNA表達(dá)內(nèi)參，用空白對(duì)照組進(jìn)行標(biāo)準(zhǔn)化，通過2-ΔΔCt方法計(jì)算基因的調(diào)控倍數(shù)[49]?；蛘{(diào)控?cái)?shù)據(jù)使用Graph Pad Prism 5(GraphPad，San Diego，CA，USA)進(jìn)行統(tǒng)計(jì)學(xué)分析。在GraphPad中使用單因素方差分析(ANOVA)并隨后通過多重比較Tukey檢驗(yàn)方法分析暴露組與對(duì)照組基因調(diào)控倍數(shù)之間的顯著性差異，當(dāng)P< 0.05時(shí)即表示存在顯著性差異。

2　結(jié)果 (Results)

本研究使用q-RT-PCR技術(shù)研究了BDE-28和BDE-99對(duì)斑馬魚胚胎/幼魚HPT、HPG和HPA軸上相關(guān)基因轉(zhuǎn)錄表達(dá)的影響。結(jié)果表明斑馬魚胚胎暴露于BDE-28和BDE-99在不同程度上引起3個(gè)內(nèi)分泌軸上相關(guān)基因的表達(dá)變化，總的來說，BDE-28主要引起相關(guān)基因發(fā)生顯著性下調(diào)而BDE-99引起相關(guān)基因表達(dá)發(fā)生顯著性上調(diào)。

圖1和圖2展示了BDE-28暴露后HPT、HPG和HPA軸相關(guān)基因的表達(dá)情況。從圖1(A)和圖2(A)可以看出，與下丘腦相關(guān)的促甲狀腺激素釋放激素基因(trh)表達(dá)在2、20和200 μg·L-1分別顯著下調(diào)3.53、3.89和2.43倍。與HPT軸相關(guān)的垂體基因的表達(dá)在BDE-28作用下都有下調(diào)趨勢。促甲狀腺激素β基因(tshb)和促甲狀腺激素釋放激素受體2基因(trhr2)在暴露于2 μg·L-1的BDE-28之后分別顯著下調(diào)2.17和2.44倍，而在暴露于200 μg·L-1的BDE-28之后分別顯著下調(diào)2.99和2.70倍。甲狀腺激素受體α和β(trα and trβ)在3個(gè)暴露濃度下都發(fā)生了顯著下調(diào)，在由低到高暴露濃度下trα和trβ分別下調(diào)2.20、2.16、2.31倍和3.64、2.30和2.42倍。同時(shí)，在3個(gè)暴露濃度下促甲狀腺激素受體基因(tshr)分別顯著下調(diào)了4.14、3.00和3.93倍。暴露于2和20 μg·L-1的BDE-28后，Na+/I同向載體(nis)的轉(zhuǎn)錄顯著下調(diào)了2.79倍和2.54倍，而甲狀腺球蛋白(tg)顯著下調(diào)了2.53倍和2.32倍。脫碘酶1(dio1)在2 μg·L-1的BDE-28暴露下顯著上調(diào)5.04倍。

BDE-28還對(duì)HPG軸相關(guān)基因的表達(dá)產(chǎn)生了影響，具體如圖1(B)和圖2(B)。下丘腦相關(guān)基因促性腺激素釋放激素基因2和3(gnrh2和gnrh3)在2、20和200 μg·L-1的BDE-28暴露后分別顯著下調(diào)4.59、3.22、2.34倍和2.43、2.18、2.60倍。垂體中相關(guān)基因基本都呈下調(diào)趨勢，其中促性腺激素釋放激素類似物1和4(gnrhr1和gnrhr4)在2、20和200 μg·L-1分別下調(diào)2.79、2.19、2.92倍和3.23、2.35、2.65倍，而gnrhr2只有在最低暴露濃度顯著下調(diào)2.10倍。斑馬魚幼魚體內(nèi)雌激素受體基因a(era)在最高暴露濃度下顯著下調(diào)2.84倍，而雄激素受體(ar)在低和中暴露濃度下分別下調(diào)2.44和2.26倍。BDE-28也使性腺中相關(guān)基因表現(xiàn)出不同程度地變化，如促濾泡激素受體(fshr)和雌激素合成的細(xì)胞色素P450(cyp17)在2、200 μg·L-1的暴露濃度下分別顯著下調(diào)2.65、4.18倍和2.90、4.38倍，而芳香化酶a(cyp19a)在高濃度暴露下顯著下調(diào)4.07倍。3β-羥類固醇脫氫酶(3βhsd)在中濃度下顯著下調(diào)2.08倍，而在高濃度下顯著下調(diào)4.75倍。羥甲基戊二酰輔酶a還原酶(hmgra)和類固醇合成急性調(diào)節(jié)蛋白基因(star)在低、中暴露濃度下分別顯著下調(diào)3.41、2.59倍和4.08、2.65倍。

此外，BDE-28還影響HPA軸相關(guān)基因的表達(dá)(如圖1(C)和圖2(C))。在2 μg·L-1的BDE-28作用下下丘腦中促腎上腺皮質(zhì)激素釋放激素(crh)和垂體中促腎上腺皮質(zhì)激素釋放激素結(jié)合蛋白(crhbp)分別顯著上調(diào)5.80倍和4.53倍。促腎上腺皮質(zhì)激素釋放激素受體2(crhr2)在低、中濃度下分別下調(diào)2.52和2.09倍。另外，垂體中阿片黑素促皮質(zhì)素原(pomc)在中、高濃度下分別顯著下調(diào)3.08和3.54倍。鹽皮質(zhì)激素受體(mr)和糖皮質(zhì)激素受體(gr)在低、中、高暴露濃度下分別顯著下調(diào)4.04、2.33、2.69倍和3.69、2.30、2.34倍。在腎上腺中，hmgra、hmgrb、3βhsd、star、cyp17和cyp19a表達(dá)情況跟上述HPG軸中一樣，其余基因都沒有顯著性變化。

BDE-99與BDE-28的作用方式不同，BDE-99暴露后HPT、HPG和HPA軸相關(guān)的基因大部分呈下調(diào)狀態(tài)(如圖2)，且大多基因效應(yīng)強(qiáng)度與BDE-99濃度成反比，在較低濃度下具有較大的倍數(shù)變化。在HPT軸中(如圖3(A)和圖4(A))，下丘腦相關(guān)的基因trh在2 μg·L-1的BDE-99暴露下顯著上調(diào)2.77倍，而在20或200 μg·L-1下沒有顯著變化。在垂體中，trhr2和tra在最低暴露濃度下分別顯著上調(diào)4.20和3.91倍，其他基因與空白組相比沒有顯著性變化。在甲狀腺中，nis、tshr、tg和dio1在最低暴露濃度下分別上調(diào)5.35、3.43、3.74和5.04倍。

BDE-99暴露后，HPG軸上基因也發(fā)生了變化(如圖3(B)和圖4(B))。下丘腦相關(guān)基因gnrh2和gnrh3在2 μg·L-1的BDE-99暴露下分別下調(diào)了3.00倍和4.83倍，而在較高濃度下沒有顯著變化。同樣地，垂體相關(guān)基因gnrhr1、gnrhr2、erb、ar和fshb也在最低暴露濃度下分別顯著上調(diào)2.50、3.46、2.18、2.78和3.93倍，而在其他濃度沒有顯著變化。性腺相關(guān)的基因cyp19a、lhr、hmgra、hmgrb、3βhsd3和cyp17在最低暴露濃度下分別顯著上調(diào)3.81、4.06、2.69、4.02、4.04和3.01倍，其中3βhsd3 和cyp17在最高濃度下顯著下調(diào)2.44和2.29倍。

圖3　斑馬魚胚胎暴露于2、20或200 μg·L-1的BDE-99至120 hpf時(shí)(A)下丘腦-垂體-甲狀腺(HPT)、(B)下丘腦-垂體-性腺(HPG)和(C)下丘腦-垂體-腎上腺(HPA)軸的基因表達(dá)變化注：結(jié)果表示為3次重復(fù)的平均值±SD，* P <0.05和** P <0.01表明暴露組與對(duì)照組相比有顯著性差異。Fig. 3　Expression of genes along the (A) HPT axis, (B) HPG axis, and (C) HPA axis of zebrafish embryo at 120 hpf following 2, 20 or 200 μg·L-1 BDE-99 exposureNote: Results were expressed as means±SD of three replicates. * P<0.05 and ** P<0.01 indicate significant differences in exposure groups compared with control group.

圖4　暴露于2、20和200 μg·L-1的BDE-99后，斑馬魚幼魚的3個(gè)軸((A)HPT軸、(B)HPG軸和(C)HPA軸)在各濃度下的效應(yīng)結(jié)果和可能的毒性途徑Fig. 4　Concentration dependent effects and proposed toxicity pathways along the three axes ((A) HPT axis, (B) HPG axis and (C) HPA axis) of zebrafish larvae exposed to 2, 20 and 200 μg·L-1 BDE-99

除此之外，BDE-99也對(duì)HPA軸相關(guān)的基因產(chǎn)生了影響(如圖3(C)和圖4(C))。與下丘腦相關(guān)基因crh只在最低暴露濃度下顯著上調(diào)3.80倍，并且與垂體相關(guān)基因pomc、crhbp、crhr2和gr也只在最低暴露濃度下顯著上調(diào)2.46、2.53、3.87和3.87倍，而下丘腦和垂體中其他基因在任何暴露濃度下都沒有顯著變化。與腎上腺相關(guān)基因mc2r、hmgra、hmgrb和cyp19a在2 μg·L-1的BDE-99暴露下分別顯著上調(diào)4.40、2.69、4.02和3.81倍，而在其他暴露濃度無顯著變化。3βhsd和cyp17在2 μg·L-1的BDE-99暴露下分別顯著上調(diào)4.04和3.01倍，而在200 μg·L-1時(shí)分別顯著下調(diào)2.44和2.29倍。

圖5　BDE-28和BDE-99暴露后(A)HPT軸(B)HPG軸及(C)HPA軸相關(guān)基因表達(dá)情況的主成分分析注：每個(gè)點(diǎn)都代表相應(yīng)物質(zhì)暴露后基因的表達(dá)情況，而橢圓區(qū)域代表95%的置信區(qū)間。Fig. 5　Principal component analysis (PCA) of genes expression associated with (A) HPT (B) HPG (C) HPA axes after BDE-28/BDE-99 exposureNote: Each point represents the genes expression after exposure to the corresponding substance and the elliptical area represents the 95% confidence interval.

3　討論 (Discussion)

本文研究了BDE-28和BDE-99對(duì)斑馬魚體內(nèi)HPT軸的影響，結(jié)果發(fā)現(xiàn)BDE-28暴露后與甲狀腺激素合成通路相關(guān)的基因nis、tg、tshr、tra和trb顯著下調(diào)，這可能會(huì)使斑馬魚體內(nèi)TH的合成量改變。脫碘酶1(dio1)顯著上調(diào)導(dǎo)致T4更多地轉(zhuǎn)化為有活性的T3，從而可能導(dǎo)致體內(nèi)有活性的甲狀腺激素水平失衡。這一結(jié)果與之前相關(guān)研究一致，之前研究表明人體妊娠期間體內(nèi)BDE-28的存在與T4和T3濃度變化有關(guān)[34]。trh涉及下丘腦向垂體的信號(hào)傳遞過程，所以trh下調(diào)也導(dǎo)致垂體中相關(guān)基因trhr2和tshb顯著下調(diào)，這些基因的調(diào)控變化也會(huì)最終影響TH的合成。BDE-99暴露使斑馬魚體內(nèi)甲狀腺合成通路相關(guān)的基因nis、tg、tshr、dio1、tra和trb顯著上調(diào)，從而有可能使斑馬魚體內(nèi)TH濃度升高。而下丘腦和垂體中相關(guān)基因trh與trhr2的上調(diào)傳遞給甲狀腺相應(yīng)的上調(diào)信號(hào)，從而也導(dǎo)致TH合成增加。之前有研究也表明人體內(nèi)BDE-99含量與T3含量成正比關(guān)系[50]。但是，最近有研究報(bào)道BDE-99暴露降低青年大鱗大麻哈魚和大鼠體內(nèi)T3、T4濃度[35, 37]。本研究中BDE-99對(duì)斑馬魚HPT軸的作用與大鱗大麻哈魚和大鼠相關(guān)研究結(jié)果的不一致可能是由生物種類差異導(dǎo)致的。

已有大量研究表明HPG軸與HPT軸有交互作用[38,44]，所以BDE-28和BDE-99也可能直接或間接地作用于HPG。因此，本研究展開了2種物質(zhì)對(duì)HPG軸基因表達(dá)影響的研究，結(jié)果顯示暴露于BDE-28和BDE-99影響了HPG軸上的基因表達(dá)，從而可能影響了斑馬魚體內(nèi)11KT和E2的合成。在暴露于BDE-28之后，下丘腦和垂體中基因gnrh2、gnrh3、gnrhr1、gnrhr2和gnrhr4顯著下調(diào)，這可能導(dǎo)致性腺激素合成發(fā)生改變。且與性腺激素合成過程相關(guān)的基因fshr、hmgra、star、3βhsd、cyp17、cyp19a、era和ar都顯著下調(diào)，這可能導(dǎo)致性腺激素合成減少。關(guān)于BDE-99的研究結(jié)果表明，在2 μg·L-1的BDE-99暴露后，lhr、hmgra、hmgrb、3βhsd、cyp17、cyp19a、erb和ar都顯著上調(diào)，這可能導(dǎo)致E2和11KT合成和釋放增加。而且另一項(xiàng)研究也表明BDE-99能刺激雄激素生成[51]。另外，之前關(guān)于斑馬魚的研究表明PBDE不僅改變了HPT軸和TH濃度中的基因的表達(dá)，而且改變了HPG軸基因的表達(dá)和性激素濃度并降低了成年魚的產(chǎn)卵量[41-42]。本研究中BDE-28和BDE-99的研究結(jié)果也驗(yàn)證了這一結(jié)果，它們不僅影響斑馬魚HPT軸基因的表達(dá)，而且也可能直接或間接地影響HPG軸基因表達(dá)從而可能導(dǎo)致魚類繁殖性能的破壞。

到目前為止，關(guān)于PBDE對(duì)HPA軸的影響基本上尚未被研究。而從本研究結(jié)果可以看出斑馬魚經(jīng)過BDE-28和BDE-99暴露后，腎上腺中相關(guān)基因cyp17、3βhsd、cyp19a、hmgra、hmgrb以及mr和gr都有顯著變化，這說明這2種物質(zhì)可能導(dǎo)致腎上腺激素合成發(fā)生改變。有研究表明化學(xué)物質(zhì)可以通過降低斑馬魚體內(nèi)E2的濃度從而使HPA軸基因的表達(dá)下調(diào)并降低皮質(zhì)激素的濃度[44]。所以本研究結(jié)果表明，BDE-28和BDE-99也可能通過HPG軸間接地作用于HPA軸。另外，已有學(xué)者證明在非哺乳類脊椎動(dòng)物中的促腎上腺皮質(zhì)激素釋放激素(corticotropin releasing hormone, CRH)(激活HPA軸的主要神經(jīng)激素)也是促甲狀腺激素(thyroid stimulating hormone, TSH)的刺激因子[45]。在魚類中，CRH比促甲狀腺激素釋放激素(thyrotropin releasing hormone, TRH)更能有效地刺激TSH的釋放[45]。所以本研究中BDE-28和BDE-99對(duì)HPT軸的影響也可能是由于其對(duì)HPA軸的作用間接導(dǎo)致的。

通過BDE-28與BDE-99暴露后3個(gè)內(nèi)分泌軸的基因表達(dá)情況的主成分分析(principal component analysis, PCA)發(fā)現(xiàn)，BDE-28和BDE-99暴露后，斑馬魚HPT(如圖5(A))、HPG(如圖5(B))和HPA(如圖5(C))軸相關(guān)基因表達(dá)情況的橢圓區(qū)域都沒有重疊部分。這一結(jié)果表明BDE-28和BDE-99可能通過不同的毒性通路影響斑馬魚的HPT、HPG和HPA軸。

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