2,4-Dichlorophenol Induces Feminization In Zebrafish By Affecting Levels Of Sex Hormones

2,4-dichlorophenol(2,4-DCP),as a common industrial material,has been discharged into the aquatic environment in the process of industrial and agricultural production,and has become one of the most common chlorophenol pollutants.Studies have shown that 2,4-DCP is an endocrine disruptor with potential estrogenic effects and causes change in the levels of sex hormones and the expression of steroidogenic genes in organisms.However,the effects of 2,4-DCP on sex differentiation of fish remain poorly reported.In this study,zebrafish were exposed to 2,4-DCP at different concentrations from 20 to 40 pdf(a critical period for sex differentiation).The effects of 2,4-DCP on sex ratio,sex hormone levels,gene expression and DNA methylation were detected by tissue section,enzyme-linked immunosorbent assay(ELISA),real-time fluorescence quantitative analysis and bisulfite sequencing PCR.On the basis of this study,zebrafish were exposed to combined treatment of 2,4-DCP and fadrozole(an aromatase inhibitor,inhibiting synthesis of E2),to explore whether 2,4-DCP could induce the feminization of zebrafish by affecting the synthesis of estradiol(E2),and to further explore the mechanism of 2,4-DCP altering the sex ratio of zebrafish.This study will provide us new information for understanding the effect of chlorophenol pollutants on the sex differentiation of fish.The following results were obtained in this study:1.2,4-DCP at different concentrations(80 and 160 ?g/L)and 17?-E2(5 ng/L)could significantly increase the proportion of female zebrafish,leading to the feminization of zebrafish.2.Both 80 ug/L 2,4-DCP and 17?-E2 up-regulated the expression of vitellogenin gene(vtg)and levels of vitellogenin(VTG),suggesting that 2,4-DCP have potential estrogenic effect.3.2,4-DCP at different concentrations resulted in the decrease of 11-ketotestosterone(11-KT)levels in whole fish while 80 ug/L 2,4-DCP increased the levels of E2.Meanwhile,2,4-DCP could increase the ratio of E2/11-KT in whole fish.These results indicated that 2,4-DCP could destroy the balance between estrogen and androgen in zebrafish.It is noteworthy that 80 ug/L 2,4-DCP can increase the levels of estrone(E1)and estriol(E3),and decrease the levels of testosterone(T),suggesting that the increase of E2 level may be due to the promotion of E2 synthesis by 2,4-DCP.4.Compared with 2,4-DCP treatment alone,the combined treatment of fadrozole and 2,4-DCP could significantly decrease the proportion of female zebrafish,resulting in a male-biased sex ratio in zebrafish.Compared with 2,4-DCP treatment alone,the combined treatment of fabazole and 80 ug/L 2,4-DCP could significantly decrease the levels of E2.In addition,combined treatment of fadrozole and 17?-E2 caused the masculinization of zebrafish,and female proportion of zebrafish in combined treatment of fadrozole and 17?-E2 was higher than that of the combined treatment of fadrozole and 2,4-DCP.These results indicated that estrogen E2 plays an important role in the feminization of zebrafish induced by 2,4-DCP.5.2,4-DCP resulted in changes in the expression patterns of steroidogenic genes(cyp19a1a,cyp19a1 b,11?-hsd and 17?-hsd)in zebrafish.Both concentrations of 2,4-DCP up-regulated the expression of cyp19a1 a,suggesting that cyp19a1 a may play an important regulatory role in the promotion process of E2 synthesis by 2,4-DCP exposure.In addition,the expressions of 11?-hsd and 17?-hsd were significantly increased by 160 ug/L 2,4-DCP while the expression of cyp11b2 was unchanged at different concentrations of 2,4-DCP,suggesting that there are other mechanisms involved in the regulation of T and 11-KT levels by 2,4-DCP,which need to be further explored.In addition,compared with 2,4-DCP alone,fadrozole combined with 2,4-DCP decreased the expression of cyp19a1 b.However,there were no obvious regularity in the expression of other genes in these groups.6.2,4-DCP could cause demethylation of CpG-61(61 bp upstream of cyp19a1 a promoter)in the promoter region of cyp19a1 a with concentration-dependent effects.In addition,there is a cis-acting element binding to estrogen-related receptors(ESRR)near CpG-61 locus.The results suggest 2,4-DCP up-regulate the expression of cyp19a1 a by inducing demethylation at CpG-61 site.In addition,2,4-DCP did not cause significant changes in the methylation levels of promoter region of cyp19a1 b,suggesting that the up-regulation of cyp19a1 b expression induced by 2,4-DCP may not be related to DNA methylation or demethylation.In conclusion,2,4-DCP can promote the synthesis of estrogen by up-regulating the expression of cyp19a1 a in zebrafish,destroy the balance of estrogen and androgen,and then cause a female-biased sex ratio in zebrafish.DNA demethylation involved in the regulation of cyp19a1 a expression by 2,4-DCP.However,it needs to be further explored whether 2,4-DCP induces DNA demethylation by affecting expression of demethyltransferase TETs(ten-eleven translocation proteins).This study explored the toxic effect and mechanisms of 2,4-DCP on sex differentiation of zebrafish,provided us new evidence and data for understanding the effect of chlorophenols on sex differentiation of fish,and also provided theoretical basis for human to recognize the harm of chlorophenols to aquatic organisms.