| 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.
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