Waterborne Tebuconazole Exposure Induces Male-Biased Sex Differentiation in Zebrafish (Danio rerio) Larvae via Aromatase Inhibition

Tebuconazole is a widely used fungicide for various crops
that
targets sterol 14-α-demethylase (CYP51) in fungi. However, attention
has shifted to aromatase (CYP19) due to limited research indicating
its reproductive impact on aquatic organisms. Herein, zebrafish were
exposed to 0.5 mg/L tebuconazole at different developmental stages.
The proportion of males increased significantly after long-term exposure
during the sex differentiation phase (0–60, 5–60, and
19–60 days postfertilization (dpf)). Testosterone levels increased
and 17β-estradiol and cyp19a1a expression levels
decreased during the 5–60 dpf exposure, while the sex ratio
was equally distributed on coexposure with 50 ng/L 17β-estradiol.
Chemically activated luciferase gene expression bioassays determined
that the male-biased sex differentiation was not caused by tebuconazole
directly binding to sex hormone receptors. Protein expression and
phosphorylation levels were specifically altered in the vascular endothelial
growth factor signaling pathway despite excluding the possibility
of tebuconazole directly interacting with kinases. Aromatase was selected
for potential target analysis. Molecular docking and aromatase activity
assays demonstrated the interactions between tebuconazole and aromatase,
highlighting that tebuconazole poses a threat to fish populations
by inducing a gender imbalance.

特布萘唑是一种广泛应用于各类作物的广谱杀菌剂，其主要作用靶点是真菌中的甾醇14-α-去甲基酶（CYP51）。然而，鉴于有限的研究表明其对水生生物繁殖的影响，研究焦点已转向芳香化酶（CYP19）。本研究中，将斑马鱼暴露于0.5 mg/L特布萘唑中，观察其在不同发育阶段的反应。长期暴露于性别分化期（受精后0-60天、5-60天和19-60天）后，雄性比例显著增加。在受精后5-60天的暴露期间，睾酮水平升高，而17β-雌二醇和cyp19a1a表达水平下降。在同时暴露于50 ng/L 17β-雌二醇的情况下，性别比保持均衡。通过化学激活的荧光素酶基因表达生物分析，确定特布萘唑并非通过直接与性激素受体结合而导致雄性倾向的性别分化。尽管排除了特布萘唑直接与激酶相互作用的可能，但血管内皮生长因子信号通路中的蛋白质表达和磷酸化水平发生了特异性改变。芳香化酶被选为潜在靶点进行分析。分子对接和芳香化酶活性分析揭示了特布萘唑与芳香化酶之间的相互作用，凸显了特布萘唑通过诱导性别失衡对鱼类种群构成的威胁。