Study On Mechanisms Of Genistein On Vitellogenin Induction In Zebra Fish (Danio Rerio) Hepatocyte

High concentration of Genistein in water bodies and fish food could significantly induce vitellogenin (VTG) synthesis in fish, while the mechanism is still unknown. In this work, the primary cultured hepatocyte of zebrafish (Danio Rerio) was used for the study on mechanisms of genistein on VTG induction. The ERs or the ERs subtypes were blocked by antagonists to study the function of estrogen reporters (ERs) on VTG synthesis induced by genistein. The expression of vtg mRNA in zebrafish hepatocyte was quantified by real-time RT-PCR .The ERαmRNA and ERβmRNA expression level was detected to further study whether the transactivation of vtg was mediated by different ER subtypes. The competitive inhibition of peroxisome proliferator-activated receptors (PPARs) on ER mediated vitellogenin synthesis was preliminary investigated.The results as follows:(1) 1~1000μmol/L genistein induce vtg-1 mRNA synthesis in zebra fish hepatocyte with dose-dependent manner. The 1000μmol/L genistein-treated group has similar expression lever with 1μmol/L E2 on induction of vtg-1 mRNA. This result shows the weak estrogenic effects of genistein, which is about 10-3 of E2.(2) Following direct co-exposure to 1000μmol/L genistein and ICI 182780 for 48hr, the expression level of vtg-1 mRNA was significantly decreased which indicated that the introduction of vtg-1 mRNA by genistein was blocked. This result unequivocally demonstrate that genistein induce zebrafish hepatocyte VTG synthesis via ERs-mediated transcriptional pathway.(3) Following direct co-exposure to 1000μmol/L genistein and ERβantagonist (Mifepristone) for 48 hr, the expression level of vtg-1 mRNA was almost completely inhabited, while Following direct co-exposure to 1000μmol/L genistein and ERαantagonist (Raloxifene) for 48 hr, the expression level of vtg-1 mRNA was partly inhabited. These results show that genistein selectively trigger ERβ-mediated transcriptional pathway, ERαis also partly involved.(4) further study of ERs expression in zebrafish hepatocyte show that 1000μmol/L genistein remarkably increased the transcription of both ERαand ERβ,meanwhile the expression of ERβmRNA was 1.9-fold. higher than ERα.(5) PPARs is also the molecular target for genistein. for the purpose of investigate whether PPARs participate in the induction of VTG by genistein, three PPARs antagonists (MK-886,GSK0660,GW9662) were adopted. The result show that the PPARs antagonists had no effect on the zebrafish hepatocyte vtg-1 mRNA transcription induced by genistein, preliminary demonstrate that PPARs don't participate in the synthesis of VTG and have no competitive inhibition on ER mediated vitellogenin synthesis.In conclusion, genistein is of weak estrogen activity and induce zebrafish hepatocyte VTG synthesis via ERs-mediated transcriptional pathway, mainly mediated by ERβ. Genistein can also raise the level of ERβto promote the synthesis of VTG. In addition, PPARs have no the competitive inhibition on ER mediated vitellogenin synthesis. This paper used vitro tests studied the mechanisms of genistein on induction of VTG, and provide basis for the use of VTG as a biomarker in phytoestrogen screen. It also laid a theoretical foundation which is meaningful for assessing the effect of phytoestrogen on fish.