Study On The Mechanism And QSAR Of Triazole Fungicides In Sex Differentiation Of Zebrafish (Danio Rerio)

1,2,4-triazole fungicides are widely used for crop disease control,but their impact on non-target organisms and aquatic ecosystem is becoming increasingly concerning.Triazole fungicides are known to cause fungicidal effects mainly by inhibiting 14α-lanosterol demethylase（CYP51）which blocks ergosterol synthesis in fungal cell membranes,but CYP51 also acts in eukaryotes.Studies have found their reproductive endocrine disrupting effects in non-target organisms.However,there is currently insufficient research on the toxicity of triazole fungicides to aquatic organisms,particularly their effects on fish sex differentiation.Although computational toxicology methods based on compound structures show promise,toxicological data still have gaps to be filled.To address these issues,the toxicity mechanism of a triazole fungicide tebuconazole was investigated through multiple techniques,including kinase profiling,reporter genes,quantitative real-time PCR,molecular docking,and enzyme activity detection.Additionally,aromatase（CYP19）inhibitory activities of 23 commonly used triazole fungicides were analyzed using radioactive isotope tracing technology,with the related Quantitative Structure-Activity Relationship（QSAR）model constructed based on structural features.The study contributes to closing gaps in reproductive endocrine disruption-related toxicity data of triazole fungicides and provides valuable insights for developing environmentally friendly drugs while fulfilling government requirements for monitoring and evaluating related toxicities.The main results are as follows.1.The study on the mechanism of zebrafish sex differentiation with tebuconazole and inhibition of aromatase activity by triazole fungicides.The kinase profiling assay was performed,which showed that tebuconazole did not exhibit antagonistic effects on 113 kinases that commonly regulate homeostasis in vivo,such as PIK3C2A,AKT and PKA.For the CALUX bioassays,tebuconazole induced androgen receptor antagonism,suggesting that tebuconazole could cause disruption of sex hormone-mediated signalling pathways.Aromatase has been used as an important biomarker as the rate-limiting enzyme that converts androgens to estrogens.The effects of tebuconazole on zebrafish aromatase related genes（cyp19a1a,cyp19a1b,ar,esr2a,gata4,nr5a2 etc.）were investigated.Zebrafish was exposed at embryo stage（0 dpf-5 dpf）and pre-sexual differentiation（19 dpf-30 dpf）.The results showed that 2 mg/L tebuconazole caused significant down-regulation of cyp19a1b（0.64±0.04-fold）,esr2a（0.49±0.01-fold）,esr2b（0.12±0.02-fold）,ar（0.42±0.09-fold）,gata4（0.39±0.08-fold）,cebpa（0.27±0.09-fold）and ahr2（0.68±0.03-fold）during zebrafish pre-sexual differentiation,while cyp19a1b（2.03±0.46-fold）,esr2b（0.22±0.02-fold）and cebpa（0.45±0.07-fold）showed significantly compensatory effects with addition of 50 ng/L estradiol,and it can be speculated that tebuconazole is involved in the regulatory pathway of aromatase.When tebuconazole was molecularly docked to the aromatase by CDOCKER algorithm,the drug could form non-covalent forces such as van der Waals forces and alkyl interactions with the protein and formπ-πstacking interactions at the Heme in active site,indicating the potential of tebuconazole to competitively bind to the aromatase catalytic site.Further comparison of the protein structures of human-sourced（CYP19A1）and zebrafish-sourced aromatases（cyp19a1a and cyp19a1b）using sequence alignment and homology modelling techniques revealed that the structural similarities,especially in the active site,which could be analyzed as substitutes for each other.In vitro inhibition of aromatase by tebuconazole was verified using a fluorescent assay kit.Based on the release of ³H-H₂O analysis,varying degrees of inhibition of 23triazole fungicides were revealed,ranging from the strongest flusilazole(IC₅₀=44 n M)to the lowest bitertanol(IC₅₀=0.33 m M).The 200μM tebuconazole,100μM myclobutanil and 100μM epoxiconazole could cause complete inhibition of enzyme activity.2.QSAR model construction for the inhibitory effect of triazole fungicides on aromatase activity.The zebrafish embryo acute toxicity of the triazole fungicides ranged from the highest for difenoconazole(LC₅₀=1.96 mg/L)to the lowest for triadimenol(LC₅₀=59.15 mg/L).For the ZF4 cytotoxicity,the IC₅₀ ranged from 71.05μM difenoconazole to 728.4μM cyproconazole,and above 1 m M for simeconazole,paclobutrazol,triticonazole and triadimefon.The relation between embryonic toxicity,cytotoxicity and enzyme inhibition of triazole fungicides was analyzed using Pearson correlation analysis and linear regression,which showed that there was a positive correlation between zebrafish embryo toxicity and cytotoxicity,but enzyme inhibition had no significant correlation with embryonic or cell toxicity,and could not be directly predicted.The acute toxicity of zebrafish embryos with triazole fungicides was selected to evaluate QSAR modeling methods,including 1D,2D and 3D QSAR.The R_adj² of the parabolic and bilinear models in 1D QSAR by using log P with bioactivity were 0.51and 0.48,respectively.In contrast,the 3D QSAR models constructed under Co MFA and Co MSIA analysis using spatial information of compounds in the molecular force field,the cross-validated Q² of the models were all much less than 0.50.The 2D QSAR performed most consistently,combining physicochemical,electrical and topological parameters of compounds to stepwise regression multiple linear analysis with biological activity.The optimal model,as p LC₅₀=-7.24-0.30×X^VPC₄+0.76×log D-26.15×Q_N1-0.08×μ,shown R² and R_adj² of 0.91 and 0.88.The study further performed the same procedure to cytotoxicity and obtained the model p IC₅₀=-4.20-0.20×N_Acceptor+0.51×log P+0.017×PSA,which also had good predictability.The same modelling method was applied to inhibition of aromatase activity and the model was defined as p IC₅₀=-22.94-2.67×E_HOMO+0.94×log D-0.72×N_Donor evaluated by internal validation,external validation,residual assessment within acceptable limits and the Williams Plot method defined for the application domain.3.Validation of triazole fungicides on sex differentiation in zebrafish.Given the predictability of QSAR,diniconazole,flutriafol and myclobutanil were selected for exposure during the gonadal development and sex differentiation stages（2 hpf-100 dpf）of zebrafish,and the anatomical analysis revealed that drug exposure caused inhibition of development,reduction in the gonadosomatic index and increase in the proportion of males in zebrafish（VC:50.95%±12.39%,diniconazole:74.52%±5.73%,flutriafol:83.33%±3.33%and myclobutanil:70.00%±17.32%）,further verifying the efficiency of the QSAR model employed to forecast the likelihood of reproductive disruption in zebrafish.The thesis revealed the reproductive toxicity mechanism of tebuconazole which was further extended to triazole fungicides.It uncovers the compounds’inhibitory effects on the activity of aromatase,and measures their acute toxicity in zebrafish embryos and cells.The research also constructs QSAR models incorporating various toxicity endpoints.The findings offer data support for the potential risks associated with triazole fungicides in aquatic environment and provide theoretical guidance to expedite drug development and risk assessment.