【作者】 黄晔；

【导师】 周忠良；

【作者基本信息】 华东师范大学 ， 动物学， 2008， 硕士

【摘要】 环境内分泌干扰物（EDCs）广泛存在于生活和工作的环境中,并可通过食物链进入动物和人体内,干扰机体正常内分泌系统功能。目前,对单一化合物的内分泌干扰效应已有比较详细的研究,主要造成生殖障碍、出生缺陷、发育异常、代谢紊乱以及癌症发生等不良影响。但是野外环境是非常复杂的,往往存在多种EDCs共同作用,与此相关的报道还很少,据仅有的少量资料可以发现,两种或多种化合物的联合效应并不是各个污染物作用的简单相加,值得我们深入研究。然而,在EDCs作用机理方面的研究还很有限,各实验室的结果时常出现矛盾,无法以现有的的理论来解释。近年来,许多研究者逐渐开始关注AhR-ERα交叉作用在水生动物体内EDCs效应中发挥的作用,众所周知ERα是体内介导雌激素效应的重要受体,其表达受到内源或外源化合物影响,而细胞色素P450系统中的CYP1A与大多数环境污染物的代谢有关,且受到AhR的调控。由此,我们猜想AhR-ERα途径可能在环境内分泌干扰物的联合效应中发挥着重要的调控作用。在本文的研究中,我们用壬基酚（NP）、双酚A（BPA）、苯并[a]芘和雌二醇（E2）多种内分泌干扰物对斑马鱼进行联合暴露,检测暴露后AhR-ERα相关基因的表达差异,试图通过AhR-ERα交叉作用途径来探讨EDCs的雌激素效应或抗雌激素效应的机制。为了获得一个准确、稳定、高效的AhR-ERα交叉作用检测系统,我们选择real-time RT-PCR法测定基因表达水平,并根据GenBank上已公布的序列信息设计实验所用目的基因（ERα、Vtg、AhR和CYP1A）以及内参基因（β-actin）引物。在优化的实时定量RT-PCR条件下,确保引物的特异性和灵敏度符合实验测定要求。而且,通过实验确认目的基因和内参基因的扩增效率基本一致,完全可以用2^-△△Ct相对定量方法分析real-time RT-PCR测得的数据。我们通过NP和BPA联合暴露斑马鱼来研究环境类雌激素的联合效应。将成年雄性斑马鱼置于NP（62.5和125μg/L）和BPA（250、500和1000μg/L）半静态系统中,联合暴露14d。从实验结果可以发现,NP和BPA的联合雌激素效应是通过诱导ERα来介导Vtg表达的,这种表达随NP和BPA浓度的升高而上调,有显著的浓度-效应关系。NP与低浓度（250μg/L）BPA和中浓度（500μg/L）BPA联合暴露能诱导ERαmRNA上调2.5至5.7倍,远大于单独暴露的效应之和,呈现明显的协同效应。同样,联合暴露使Vtg随ERαmRNA表达量急剧上升,协同效应也极显著。然而,NP和BPA联合暴露剂量与AhR或CYP1A表达量之间没有明显的相关性。这些结果提示,NP和BPA共同作用于ERα,在低浓度时相互促进与ERα结合,表现为相加效应。另外,从我们的数据发现,NP和BPA并不是通过CYP1A进行代谢的。为了解B[a]P的抗雌激素效应,我们将性成熟雌性斑马鱼暴露于不同浓度B[a]P中研究其与内源雌激素的联合作用,同时将成体雄性斑马鱼暴露于B[a]P和E2混合的水体中研究它与外源雌激素的联合效应机制。首先,当B[a]P暴露浓度在10～50μg/L范围内,性成熟雌性斑马鱼肝脏中ERα和Vtg表达量急剧下降;暴露浓度＞50μg/L时,ERαmRNA表达量维持在一定水平,不再随B[a]P浓度提高而下调,Vtg反而缓慢回升。同时,检测到AhR mRNA表达量显著上升,100μg/L暴露组诱导的表达量最高,然后回落。低剂量（10～20μg/L）B[a]P对CYP1A表达量影响不明显,但是高剂量组（100和200μg/L）诱导CYP1A表达量显著增加。这些结果提示,低剂量B[a]P激活AhR介导的对内源E2（诱导ERα介导的雌激素效应）的代谢作用,而高剂量B[a]P却抑制了这种作用。随后,我们在E2和B[a]P对雄性斑马鱼进行联合暴露时发现,在较低E2浓度（250ng/L）条件下,B[a]P也能诱导AhR和CYP1AmRNA上调,并抑制ERα和Vtg mRNA表达,也呈浓度-效应关系;然而,高浓度E2（500ng/L）与B[a]P联合作用于雄性斑马鱼时,E2反而对AhR有抑制作用,AhR和CYP1AmRNA急剧下降,且EROD酶活性也迅速降低。这些结果说明AhR-ERα交叉作用的机制十分复杂,通过这一途径对外源污染物进行的调控可能与所暴露物质各自的效应浓度有关,还有待今后进一步研究。更多还原

【Abstract】 Endocrine disrupting chemicals（EDCs）are widely distributed in living environment, which can be absorbed by animals and human beings through the food chain.At present, scientists have pay more attention to the endocrine interferential effect induced by single chemical,which result in reproductive disfunction,birth defect,abnomaly development, metabolic disorder,carcinogenesis and so on.However,there is often co-effect of two or more kinds of EDCs on the wild lives because of the complicated environment.Only a few articals have reported this,and they revealed that the co-effect of the chemicals is not the simple addition of each one.The mechanisms of the co-response are also not clear so that it is difficult to understand the contradictive experimental results from various laboratories.Fortunately,recent researches have focused on the role of AhR-ERαcross-talk in endocrine disrupting regulation in aquatic animals.It has been widely recognized that ERαis one of nuclear receptors which take important roles in mediating the effect of estrogen,and the level of ERαis affected by endogenetic and ectogenous EDCs.In addition,most EDCs can be metabolized by CYP1A,one member of cytochrome P450 family,which is regulated by AhR.We supposed that the AhR-ERαpathway might take a great part in the regulation of co-response.In the study,we investigated the expression differences of AhR-ERαrelevant genes in zebrafish（Danio rerio）after exposure to 4-nonylphenol（NP）,Bisphenol A（BPA）,Benzo[a]Pyrene（B[a]P） and 17β-estradiol（E2）in combination.We tried to understand the estrogen or anti-estrogen activity mechanism of these EDCs via the AhR-ERαcross-talk pathway.To establish an accurate,stable and efficient examine system of AhR-ERαcross-talk, we detected the gene expression levesl by real-time RT-PCR.The primers of target genes （ERα,Vtg,AhR and CYP1A）and reference gene（β-actin）were designed by Primer Express 2.0 software,and the sequences information came from GenBank.Each primer was specific and sensitive after optimizing the condition of real-time RT-PCR.Moreover, because we ensured that the amplification efficiencies of target genes and reference were approximately equal,the 2^-△△Ctmethod was valid in the analysis of real-time quantitative PCR data.In the study of estrogen co-effects,adult male zebrafish were exposed to a nominal concentration of NP（62.5 and 125μg/L）and BPA（250,500 and 1000μg/L）in combination in a semi-static exposure system for 14 days.When exposed to NP and BPA at low（250μg/L）and middle concentration（500μg/L）in combination,the expression of ERαobservably increased to 2.5- to 5.7-fold of solvent control.It showed a dose-response relationship between the concentration of NP-BPA and the expression of ERα.On the other hand,co-treatment dramatically elevated the expression of Vtg mRNA with the up-regulation of ERα,and it revealed a significant synergetic effect.However, there was no remarkable relationship between the dose of co-exposed NP and BPA and expression of AhR or CYP1A.These results suggested that NP allied with BPA for the binding to ERα.In addition,NP and BPA did not be degraded through CYP1A pathway.To understand the anti-estrogen of B[a]P,female adult zebrafish were exposed to nominal concentrations of 10,20,50,100 and 200μg/L B[a]P for 14 days.The expressions of ERαand Vtg were obviously descent when exposed to 10 to 50μg/L B[a]P. But the higher dose（＞50μg/L）didn’t induce the highest anti-estrogen activity. Meanwhile,it was observed a significant increase of the expression of AhR mRNA,the highest induced concentration was 100μg/L.There is no obviously alteration of CYP1A expression at lower dose（10～20μg/L）,but the higher concentration of B[a]P（100 and 200μg/L）evidently ascent expression of CYP1A.The results indicate that the AhR mediated metabolism of endogenous E2 which induce the ERαmediated estrogen-responsiveness may be activated at lower concentration of B[a]P,but inhibited at higher dose.Furthermore,we treated the adult male with E2（250,500ng/L）and B[a]P （20,50,100μg/L）in combination.It showed that B[a]P elevated the expression of AhR and CYP1A mRNA when co-exposed with lower dose of E2（250ng/L）.In contrast,E2 inhibited the up-regulation of AhR mRNA which induced by B[a]P when co-treated with higher dose of E2（500ng/L）.Furthermore,the expression of CYP1A mRNA and the EROD activity dramaticly decreased.This implies that the mechanism of AhR- ERαcrosstalk is complex,the regulation by this way may be relevant to the effective concentration of each chemical.更多还原