Mechanism Of Tebuconazole On Reproductive Endocrine Disruption In Zebrafish Based On CAMP/PKA/SF-1 Signaling Pathway

Various chemical pesticides play an important role in protecting agricultural production and increasing production.However,in the last half century,more and more pesticides have been confirmed to have environmental endocrine disrupting effects.They disrupt the regulation of normal hormones in the organism,causing so many problems in nervrous system,secretory system,immune system.These problems include male degeneration,frequent hermaphrodite,and population degradation.Tebuconazole is a broad-spectrum,highly efficient systemic triazole fungicide that is widely used worldwide because of its low toxicity and high efficiency.With the widespread use of tebuconazole fungicides,the impact on China's ecological environment should be highly concerned.Related studies have shown that tebuconazole can cause endocrine disrupting effects on zebrafish,but the specific interference mechanism is still not clear.So,this study is to develop a study on the mechanism of cAMP/PKA/SF-1 signal transduction disruption by tebuconazole.(1)In order to investigate the effect of Tebuconazole on cAMP/PKA/SF-1 signaling pathway,in vitro and in vivo experiments were mainly used.First,11 time points between 0 and 72 h were selected.The determined tebuconazole exposure time was 48 h through the method of cell counting.At the same time,the activity of H295R cells was detected by MTS kit and ATP kit after exposure to tebuconazole in the range of 0.1 mg/L-50 mg/L for 48 h.MTS and ATP results showed that at 20 mg/L,the cell xviability was about 80%of the control.The final selection was 48 h,and the tebuconazole exposure concentration was 20 ma/L as the subsequent in vitro experimental exposure standard.(2)Before investigating the effect of tebuconazole on cAMP/PKA/SF-1 signal transduction pathway,tebuconazole 20 mg/L,H295R cells exposed for 48 h and unexposed H295R cells were transcribed and sequenced.Sequencing results showed that there were significant differences in 165 genes in the tebuconazole-treated group compared xwith the blank control,of xwhich 102 genes were significantly up-regulated and 63 genes were significantly down-regulated.KEGG enrichment analysis revealed that the Ras signaling pathway?PI3K-AKT signaling pathway and Rapl signaling path"way were associated with the cAMP/PKA/SF-1 pathway.(3)In order to further explore the effect of tebuconazole on cAMP/PKA/SF-1 signal transduction pathway,in vitro experiments combined with in vivo experiments were carried out by RT-PCR,PKA Kit assay,Western blotting and other experimental methods.Results of PKA kit assay showed that 20 mg/L and 0.5 mg/L tebuconazole resulted in dose-dependent significant decreases of PKA activities in H295R cells(20mg/L,P=0.0001)and zebrafish embryos(0.5mg/L,P=0.016).Western Blotting indicated that exposure to tebuconazole resulted in decreased expression of Phosphoserine,Phosphothreonine and Phosphotyrosine proteins in zebrafish embryos and expression of Phosphothreonine protein in H295R cells,playing as the similar.Effects of H-89(A PKA inhibitor to be set as positive control in the present study)Addtionally,it was found that forskolin,a PKA agonist,could alleviate the adverse effects caused by tebuconazole.RT-PCR was used to investigate the effect of tebuconazole on SF-1 pathway-related genes.After exposure to tebuconazole for 48 h,the expression of ERu,,SF-1,GPR30,ER? and CYP17 genes in H295R cells decreased significantly,and expoursed H-89 led to a similar effect that of tebuconazole.However,forskolin exposure could alleviate the expression reductions of SF-1 pathway genes caused by tebucinazole(4)The experiment successfully constructed the ?YAK1 H295R cell line by CRISPR-Cas9 method,and over-expressed PKA,which was used to further verify the effect of tebuconazole on cAMP/PKA/SF-1 signaling pathway.The PKA Kit,Western blot,and RT-PCR were also used for verification.The results of PKA Kit and Western blotting were consistent with the exposure of H295R cells in vitro and the exposure of zebrafish embryos in vivo,further validating the effect of tebuconazole on this pathway.The RT-PCR results of AYAK1 H295R showed that the expression of ER?,GPR30 and CYP17 genes was significantly lower in H295R cells than in AYAK1 H295R cells.In addition,exposure to 20 mg/L tebuconazole resulted in ?YAK1 H295R.The expression of ER?,SF-1,CYP450,ER?,GPR30,CYP17 and GPER genes was significantly down-regulated,which was consistent with the RT-PCR results of H295R.(5)To investigate whether tebuconazole affects the cAMP/PKA/SF-1 signal transduction pathway by binding to PKA,the surface plasmon resonance(SPR)method was used to investigate the binding of tebuconazole to hPKA-Ca.In the binding experiments of tebuconazole and hPKA-Ca,the concentrations of tebuconazole at different concentrations after 180s injection were 27.6 Ru(0.2?M),34.1 Ru(0.4?M),37.7Ru(0.8 pM),43.6 Ru(1.6 pM),54.4 Ru(3.2?M),66.2 Ru(6.4?M),75.8Ru(12.5?M),106.2 Ru(25?M),the binding oftebuconazole to hPKA-C?(Ru)increases with increasing concentration of tebuconazole,kinetic constant KD=2.72x10-7;in the positive control H-89 and hPKA-Ca binding experiments,the different concentrations of H-89 after 180s injection were 93.6 Ru(0.32?M),110.1 Ru(0.64?M),121.5 Ru(1.25?M),132.6 Ru(2.5?M),144.1 Ru(5?M),showed a good concentration gradient effect with the increase of injection concentration,the kinetic constant KD=3.21×<10-8;the negative control chlorpyrifos combined with hPKA-Ca protein The concentrations are superimposed and there is no significant concentration gradient effect.The results indicate that tebuconazole may bind to hPKA-Ca in a manner similar to H-89.