Effects Of Natural Estrogen And Paraben On The Transcriptome Of Oryzias Sinensis Primary Hepatocytes

Natural estrogens are steroid hormones,including estrone(E1),estradiol(E2),and estriol(E3).Natural estrogens have high fat solubility,low water solubility and strong estrogen effect.They have been widely detected in the environment and have received wide attention.Natural estrogen affect the endocrine system and reproductive system of aquatic organisms,causing hermaphrodite and abnormal gonads in fish.Parabens,including Methylparaben(MP),Ethylparaben(EP),Propylparaben(PP),Butylparaben(BP),Benzoparaben(Bz P),Isopropylparaben(IPP)and Isobutylparaben(IBP),are among the most widely used preservatives and are commonly used in foods,medicine and cosmetics.In recent years,they have been reported to have potential endocrine disrupting activity,which mimics estrogen function and have been regarded as an emerging pollutant.It is important to study the toxic effects of natural estrogens and parabens on aquatic organisms.In this paper,the transcriptome analysis of Oryzias sinensis hepatocytes exposed to different concentrations of E1(1,10,100 n M),E2(0.01,0.1,1,10 n M),E3(1,10,100 n M)and parabens(50 μM),was carried out based on the method of establishing the primary hepatocyte culture of O.sinensis,based on next-generation sequencing technology(NGS).After exposure to natural estrogen,some genes with good dose-response relationships were obtained by transcriptome sequencing analysis.GO and KEGG analysis found that estrogen mainly affects oxidation-reduction process,phosphorylation,lipid transport,neural retina development,ion transport and Notch signaling pathway,as well as oxidative phosphorylation,Insulin signaling pathway,Calcium signaling pathway,lipid transport and other signaling pathways.By analyzing the results of RT-q PCR of estrogen-responsive genes,it was found that both vitellogenin VTG and choriogenin CHG were significantly increased,and the estrogen response of VTG2 is most pronounced.After exposure to parabens,GO and KEGG enrichment analysis revealed that the major differentially expressed genes(DEG)were mainly enriched in estrogen response,lipid transport,ion transport,transmembrane transport,cell differentiation,G protein-coupled receptor signaling pathway,signal transduction and transcript ional regulation,neural retina development,regulation of insulin secretion,these biological processes have an important impact on the reproductive development of O.sinensis.The main signaling pathways are calcium signaling pathway,endocytosis,RNA transport,MAPK signaling pathway,and m RNA surveillance pathway.The transcriptome data and RT-q PCR results showed that the exposure of parabens significantly increased the expression of estrogen-related genes.Parabens have estrogenic activity,and their order of estrogenic activity is IPP>IBP>PP>EP>BP>BZP>MP.This study established a method for detecting environmental estrogen using culture of Oryzias sinensis primary hepatocytes,and obtained a large number of estrogen response genes,which can be used as new biomarkers.The toxic effects of natural estrogens and parabens were studied at the cellular level.