Transcriptional Regulation Of Key Genes On Important Signaling Pathways In Zebrafish Associated With Climbazole Exposure

In recent years,azole fungicides were received increasing attention due to their ubiquitous occurrence in the environment and potential eco-toxicological effects on aquatic organisms.Azole fungicides are chemicals used to inhibit or destroy fungi.They are widely applied as antifungal ingredients in pharmaceuticals and personal care products.After application,azole fungicides will directly or indirectly enter the aquatic environment via incomplete removal in wastewater treatment plants.The concentrations of azole fungicides were several ng/L or?g/L in the aquatic environment.The azole fungicide residues will pose potential risk to aquatic organisms.Previous reports have indicated azole fungicides can bind to cytochrome P450 enzymes(CYP450),leading to inhibit the activities of CYP450 and further disrupt the metabolic balance of organisms.However,the research about is limited.Based on the zebrafish(Danio rerio)model,the aims of this study were to investigate the influence on the transcriptional expression of important signaling pathways of embryonic or adult zebrafish associated with exposure to a typical azole fungicide climbazole,to assess the relationships among various climbazole-induced signaling pathways,and to screen the potential biomarkers in zebrafish eleutheroembryos following climbazole exposure.The main findings from this study are given as follows:(1)The influence on the transcriptional expression of 73 genes involved in important signaling pathways of embryonic zebrafish(2,7,and 14 dpf)following climbazole exposure(100 and 10000 ng/L)was investigated.The results showed significant down-regulation of steroidogenesis-(cyp17a1,cyp19a1b,ar,and hsd20b)and circadian rhythm-related genes(cry1a,cry1b,cry2a,nr1d1,nr1d2a,and nr1d2b)in zebrafish embryos and larvae after exposure to environmentally relevant concentrations of climbazole.Significant down-regulation of steroidogenesis-(cyp17a1,cyp19a1a,cyp19a1b,ar,and esr2a),circadian rhythm-(cry1a,cry1b,clock1a,arntl2,nr1d1,nr1d2a,nr1d2b,and per1a),and oocyte maturation-related genes(mpr?and igf3),and significant up-regulation of inflammation-related genes(il-1?,il-8,cxcl-clc,and cc-chemokine)in embryonic zebrafish after exposure to high concentrations of climbazole.Climbazole at environmental relevant concentrations may inhibit the synthesis of steroid,lengthen the periods of biological rhythms of zebrafish,and cause a phase delay of circadian rhythms.In addition to steroidogenesis and circadian rhythm signaling pathways,high-dose climbazole could have the ability to facilitate the inflammation and suppress the oogenesis.(2)The correlation between different climbazole-induced signaling pathways was assessed by the linear regression analysis based on transcript indices.Cytochrome P450enzyme-related steroidogenesis,gonadotropin-releasing hormone/follicle-stimulating hormone/luteinizing hormone,and oocyte maturation signaling pathways displayed significant correlations(R~2=0.74-0.94;P<0.05).These three pathways were involved in the secretion of steroid hormones.The transcript index of hydroxysteroid dehydrogenase-related steroidogenesis,but not other steroidogenesis-related signaling pathways,was significantly correlated to that of circadian rhythm(R~2=0.92;P<0.05).So that the circadian clock and its modulation by climbazole may directly regulate the production of steroid hormones via hydroxysteroid dehydrogenases.(3)The potential biomarkers in zebrafish eleutheroembryos following climbazole exposure were screened by the supervised partial least squares discriminant analysis.One(mpr?)and 5 genes(igf3,nr1d1,nr1d2b,cyp19a1a,and vtg1)were extracted as differential genes(VIP>1)in 6 and 5 climbazole-treated groups,respectively.The VIP values of the inflammation-related genes(il-1?and il-8)were greater than 1 in all high-dose climbazole-treated groups.Considered together with the transcriptional analysis,mpr?,igf3,nr1d1,nr1d2b,cyp19a1a,vtg1,il-1?,and il-8 were selected as the potential biomarkers for climbazole exposure to zebrafish eleutheroembryos.(4)The influence on the transcriptional expression of genes involved in steroidogenesis,circadian rhythm,inflammation,oxidative stress,and oocyte maturation signaling pathways of adult zebrafish(28 d)following climbazole exposure(100,1000 and 10000 ng/L)was preliminary investigated.Significant up-regulation of steroidogenesis-related genes(ar,cyp17a1,cyp19a1a,hsd17b3,and hsd20b)was found in the testis of male zebrafish,whereas significant down-regulation of those genes was observed in the ovary of female zebrafish.In addition,the transcriptional level of vtg1 was significantly reduced in the testis and ovary tissues after exposure to climbazole.These findings showed that climbazole could induce the masculinization of female zebrafish and facilitate the spermatogenesis in male zebrafish.Taken together,the above results show that climbazole could disrupt the signaling pathways of steroid biosynthesis,circadian rhythm,inflammation,oxidative stress and oocyte maturation in embryonic or adult zebrafish at transcriptional levels,leading to potential risks to the endocrine system of zebrafish.Interfere on the steroidogenesis implies that climbazole is a potential endocrine disruptor.