Comparative Transcriptome Analysis On Immunotoxic Effect And Mechanisms Under Different Exposure Time Of EE2 On Oryzias Melastigma

17a-ethinylestradiol(EE2)is an endocrine disrupting chemical(EDC)that exists extensively in marine environment.It possesses various toxic effects which can impact on the reproduction and immunity health of fish.There are some studies mainly focus on the reproductive and immune toxicity effects of EE2 on fish,however,few researches on the immunotoxicity mechanism of EE2 are reported at present.In our study,a typical endocrine disrupting chemical EE2 was chosen as tested substance,and marine model animal Oryzias melastigma was chosen as study objects.The liver transcriptome library of male O.melastigma was constructed using RNA-seq.The exposure time(12 h and 48 h)and concentration of EE2(500 ng/L)were set referring to the preliminary experiment result of which the gene expression of hepcidin,an important immune factor,were significantly effected.We compared and analyzed genes expression patterns related to endocrine,stress response and immune in liver of male O.melastigma at time point 12 h and 48 h post exposure.The result offers overall evaluation of toxic effect of EE2 and the time sequence of the process.Then we set a experimental group of fish exposed to EE2 after injection of ICI 182780,an inhibitor of estrogen receptor(ER),screened out target genes regulated by ER and its signal pathway,then further analyzed and surmised its potential ER-mediated immunotoxication mechanism on transcriptome level.This study contributes to intensive understanding on the potential regulatory role of ER in the process of EDCs'immunotoxic effect on marine fish.The main results for this study are as follows:1.The liver transcriptome library of male O.melastigma was constructed using RNA-seq.High-throughput sequencing resulted in 122,987 unigenes,with a mean length of 686 bp,distribute in the range of 201 bp to 16884 bp.In total,67.70%(83267)of these unigenes have the length between 200 bp to 500 bp,and 38,233(20.8%)unigenes were annotated from NR database and 72,662 unigenes(4.5%)were annotated from NT database.2.Illustrated the gene expression patterns related to endocrine,endoplasmic reticulum stress and immunity in liver of male O.melastigma at different time point post EE2 exposure.We screened 839(at 12 h)and 1361(at 48 h)differential expression genes after EE2 exposure.The expression of endocrine related genes(such as Esr1,Vtg1,Vtg21,CHG-H and CHG-L)and genes participate in estrogen receptor signaling pathway(such as Hsp90b1,Hspa4,Creb,Adcy6 and Shcl/2)was induced significantly by EE2 exposure,indicating that 12 h-exposure could induce endocrine disrupting activity.The expression of genes related to endoplasmic reticulum stress(such as Eif2ak3,Ire1,atf4,Xbp1 and so on)was significantly up-regulated after 12 h,suggesting 12h-exposure could cause ER response.Antimicrobial peptide gene OM-hep2 was activated by EE2 exposure,while signaling pathways related to innate immunity(TNF,JAK-STAT,complement system)and genes involved in antigen-presenting process were significantly surppressed,indicating that 12h-exposure of EE2 had a certain toxic effect on innate immunity.After 48 h exposure of EE2,some of genes related to endoplasmic reticulum stress(Hspa5,Eif2ak3,atf6,atf4 and so on)and genes related to protein processing and trafficking were still induced,indicating the continuously activation of ER response.Innate immunity signaling pathways(TNF,JAK-STAT,complement system,etc.),antimicrobial peptide gene OM-hep1 and genes invloved in TLRs and apoptosis were down-regulated,suggesting EE2 exposure exerted persistently impact on innate immunity which could further cause certain toxic effect on adaptive immunity.Above all,EE2 exposure might pose its immunotoxic effect on fish through multiple ways.3.Preliminarily revealed immunotoxic effect and mechanism of EE2 mediated by ERa.Compared with group T,the genes related to endoplasmic reticulum stress(such as Hspa5,Dnajc3,Hsp90bl and so on)and genes function for processing and transportation of protein(such as Ergic53,Sec61,Osts and so on)were significantly down-regulated by EE2 exposure in group Ti after 48 h.The expression of OM-hepl increased in group Ti.Meanwhile,EE2 exposure effected on the expressions of the genes involved in TLRs-signaling pathway(Tlr5,Map3k8,Junb),JAK-STAT-signaling pathway(Socs3,Pik3r1,Spry4)and complement system(C4,Cfh,Masp3b)in group T were eliminated by ICI in group Ti.These results indicated that EE2 mediated the expressions of immune related genes through ERa and its signaling pathways and caused immunotoxic effect on fish.In summary,RNA-seq accompanied with intraperitoneal injection technique were applied to evaluate the toxic effect of EE2 on O.melastigmain this study.Moreover,we illustrated the function mechanism of the effect on the target genes from EE2 mediated by ER and its signaling pathway using estrogen inhibitor.It will contribute to the further research on the relationship between immunotoxicity of marine environmental endocrine disruptors.In addition,it will lay foundation for the mechanism study of the relationship between endocrine system and immune system of marine fish.