| With the rapid development of the agro-industry,the broad-spectrum antibacterial agent triclosan(TCS)has been widely added to agricultural products,food packaging and toiletries,so a large amount of triclosan has been detected in the environment.This also makes its ecological safety and toxicological effects on organisms become a research hotspot.In this paper,the laboratory model animal zebrafish was used as the test object.The interference effect and the molecular mechanism of triclosan on the sex differentiation and reproductive toxicity of zebrafish were systematically studied.The following conclusions were obtained:1.To verify the effect of TCS on the sex differentiation of zebrafish,24 hpf zebrafish embryos were exposed to 50 ?g/L,100 ?g/L and 200 ?g/L TCS and exposed to 60 dpf,respectively,and the results showed that at 200 ?g/L In the TCS treatment group,the number of female zebrafish individuals increased significantly,and the proportion of females reached 84.67%.In order to further study the mechanism of TCS affecting the sex differentiation of zebrafish,the expression of sex-related genes and the changes of sex hormone content were detected in the critical stages of juvenile sex differentiation(7dpf,30 dpf and 45dpf).The results showed that 7dpf zebrafish In vivo,the dmrt1 gene associated with development of males was significantly down-regulated in the 200 ?g/L treatment group,while the expression of sox9 a and dmrt1 was significantly down-regulated at 200 ?g/L at 30 dpf and 45 dpf.The genes related to the development of females,such as brac2,aromatase genes(cyp19a and cyp19b),sox9 b,were up-regulated in different degrees,and the effect was up-regulated in the 200 ?g/L TCS-treated group.The levels of sex hormones(estradiol and testosterone)in 45 dpf zebrafish juveniles were determined by gender differentiation.The results showed that the estradiol content and testosterone content of zebrafish in 100 ?g/L and 200 ?g/L treatment groups were significant.Increased and significantly reduced,suggesting that it may induce the synthesis and secretion of estrogen in zebrafish,suggesting that TCS may induce zebrafish to develop into females,as evidenced by sex ratio statistics.2.To verify the effect of TCS on the reproductive system of adult zebrafish,female and male zebrafish were exposed to 50 ?g/L,100 ?g/L and 200 ?g/L TCS for 15 and 30 days,respectively,blank control and solvent control were set up.The results showed that the gonad tissue structure has pathological change.After 15 days of treatment with 200 ?g/L,the sperm density in the testes of male zebrafish was significantly reduced.After 30 days of continuous exposure,severe testicular vacuolation was observed in the 100 ?g/L and 200 ?g/L treatment groups.In the female ovary,the proportion of mature oocytes decreased and the plasma membrane separated after 15 days of TCS treatment.After 30 days,a large number of oocyte membranes were damaged in the 200 ?g/L treatment group.The results of tests for sex steroid hormones and vitellogenin showed that estradiol and vitellogen levels increased significantly at higher concentrations of TCS(100 ?g/L and 200 ?g/L),testosterone and 11-Ketotestosterone levels were down-regulated;these results indicate that TCS has some interference with the zebrafish reproductive system.To further study its mechanism of action,real-time quantitative PCR was used to detect the expression of hypothalamic-pituitary-gonadal axis-related genes after TCS exposure.The results showed that the expression of gonadotropin-releasing hormone and its receptor gene was up-regulated under the action of TCS.For example,on the effect of TCS under 200 ?g/L,gnrh3 were significantly up-regulated expression in the brains of both female and male zebrafish at 15 and 30 days.The steroid transporter and aromatase genes such as star and cyp19 a were significantly up-regulated in each treatment group.The brain aromatase gene cyp19 b was up-regulated after TCS exposure for 15 days.After 30 days of continuous TCS exposure,the upregulation of cyp19 b in the male fish brain was inhibited,probably due to the negative feedback mechanism of estradiol.The genes such as 3?hsd and 17?hsd in the pathway of catalyzing cholesterol-synthesizing steroids also showed significant up-regulation after 15 days of TCS exposure and insignificant up-regulation after 30 days,which may also be due to the feedback effect of estrogen on the starting point of cholesterol synthesis.3.According to previous studies,the estrogen effect of TCS on zebrafish can be preliminarily determined.To further study the transduction pathway of estrogen in zebrafish gonadal target cells,zebrafish were exposed to 50,100,200 ?g/L TCS for 30 days,the gene expression associated with estrogen signaling in the gonads was examined.The results showed that there was no significant change in the expression of estrogen receptors er? and er? in the gonads of females,but genes closely related to mER-mediated cAMP pathway and MAPK pathway,such as gper1,prkacaa,adcyap1 a,efgra,etc.Significant up-regulation in group 200 ?g/L indicates that high concentration of TCS may induce estrogen to transduce into target cells in female fish through these two pathways;in male testis,er? expression is up-regulated and akt1 expression is down-regulated,indicating that TCS may be Inhibition of PI3 K pathway makes nER mediate more obvious,while cAMP pathway and MAPK pathway are less affected by TCS,indicating that mER-mediated estrogen rapid signal transduction pathway is affected by TCS after male fish.Inhibition.In conclusion,TCS has a certain effect on the transduction of estrogen in the gonads of zebrafish. |
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