Effects Of Eugenol On Zebrafish Embryonic Development And Its Mechanism

Eugenol,as a plant-derived natural compound,has many advantages such as good anesthetic effect,low toxicity,lower price,and low residue.As a new generation of fish anesthetic,it has received more and more attention and application.There are also many reports on the application of eugenol as a fish anesthetic,but most of them discussed the anesthesia effect of eugenol on fish,such as anesthesia concentration and anesthesia time.There are few studies on the safety of eugenol to the water environment,and the potential risks of water pollution and aquatic organisms caused by its improper or excessive use still need to be studied.Therefore,this paper studies the effects of eugenol on fish embryo development,in order to provide a reference for the potential risks of eugenol on fish breeding.In this study,zebrafish(Danio rerio,AB line)embryos,a model experimental organism,were used as the experimental object.Under a gradient concentration 0,10,15,20,25,30mg/L of eugenol,96 h exposure experiment was performed with 24 hpf zebrafish embryos.To investigate the effects of eugenol exposure on zebrafish embryo development and its mechanism through analysis in morphological observation,wholeembryo transcriptomics,and real-time PCR were performed.The main findings are as follows:The results of morphological studies show that exposure of 10～30mg/L eugenol will delay the embryo hatching of zebrafish,the absence rate of swim bladder increased significantly,and the growth rate of body lengthdecreased significantly.Compared with the control group,there was no significant difference in the mortality rate of the exposure groups,and the larvae which the swim bladder absence during the mouth-opening period were difficult to swim and feed normally,and eventually died.The cumulative deaths of each concentration group increased significantly compared with the control group.These morphological abnormalities are positively correlated with the concentration of eugenol exposure.Whole-embryo RNA-seq transcriptomic analyzed the gene expression profile of zebrafish embryos in the control group and the lower concentration of eugenol(15mg/L)during the hatching stage(72hpf)and mouth-opening stage(120hpf).Compared with the control group,275 genes were significantly different in the eugenol-exposed group,among them 109 genes were up-regulated and 166 genes were down-regulated;and 297 genes were significantly different in the exposed group compared with the control group,among them 66 genes were up-regulated and 231 genes were down-regulated.The hierarchical clustering tree analysis showed,that the differences between biological replicates are small,but the differences between groups are large.These results indicate that eugenol exposure has significant effects on the gene expression level of zebrafish embryo development.Compared with the control group,the 15mg/L exposure group had37 genes that had significant differences in expression during the hatching stage and the mouth-opening stage.These differential genes were mainly focus on the drug catabolic pathway and the antioxidant system.The genes regulating the drug catabolic pathway are cyp1c2,cyp2r1,cox5b2,hbae5,and zgc:101040,and the genes regulating the antioxidant system are cyp1c2,cyp2r1,cox5b2,dao.1,xdh,fads2,nfkbiaa,and gadd45 ba.In addition,the expression of genes that regulate digestive-related enzymes trypsin and zinc carboxypeptidase activitiesdecreased significantly.Among them,the genes that regulate trypsin activity were bfb,CELA1(1 of many),prss1,prss59.1,prss59.2,and ctrb1.Genes that regulate zinc carboxypeptidase activity included cpa4,cpa5,and cpb1.These results indicate that the tissues and organs of larvae may have suffered oxidative damage after exposure to eugenol,and normal digestive function may be impacted.Real-time PCR verified the expression levels of drug catabolism pathways and the antioxidant system some related genes(cyp1c2,cyp2r1,cox5b2,hbae5,dao.1,xdh)in the RNA-seq results during the hatching and mouth-opening stages.The verification results are consistent with the transcriptomics results.The expression levels of several genes(wif1,dkk1 b,fzd3b,fzd6,ctnnb1,lef1)in the Wnt-β-catenin signaling pathway,which involved in the development of fish swim bladder,during hatching and mouth-opening stageswere also analyzed with real-time PCR,and the results showed that this signaling pathway transduction was supressed.Among them,the expression of the Wnt signaling pathway inhibitor gene wif1 was significantly up-regulated,and the genes in the pathway fzd3 b,fzd6,ctnnb1 and lef1 were significantly down-regulated.There was a certain concentration dependencebetween the expression level of these genes and the exposure concentrations of eugenol at 10～30mg/L.These results indicated that eugenol certainly exerted embryonic toxicity in zebrafish,which caused oxidative damage in some tissues and organs.The development of swim bladder may be inhibited by down-regulating Wnt-β-catenin signaling pathway.Above results suggestedthat Eugenol has a dose-dependent toxicity on zebrafish embryos.Eugenol exposure delay the development of zebrafish embryos,cause oxidative damage of tissues and decrease the activity of digestive enzymes.The absence of swim bladder development in the embryonic mouth-opening stage induced by eugenol exposure may be due to the inhibition of Wnt-β-catenin signaling pathway.Lack of swim bladder,larvae eventually died.The results of this study will enrich our knowledge of eugenol embryo toxicology and provide reference data for its environmental toxicology.