三唑酮干扰斑马鱼致内分泌-生殖毒性分子机制的研究

Triadimefon inhibit fungal growth by specifically inhibiting the cytochrome P450-mediated 14 alpha-demethylase step in the biosynthesis of ergosterol, a biomolecule unique in fungi. The ability of triazoles to bind to the heme protein and inhibit CYP-dependent enzymes raises concerns over triazole effects on gonadal steroidogenesis and reproductive function. Previous study indicated that triadimefon has the estrogen-like effect to zebrafish, which can disrupt the normal function of reproductive system. However, mechanistic understanding of the key biological pathways perturbed by triadimefon that lead to toxicity is still unknown. For this reason, combined with high throughput screening assay (such as UPLC-MS/MS and Rt-PCR), developmental stages of zebrafish will be exposed to triadimefon at environmentally relevant concentrations to display the expression alternation of related steroids and genes in biotransformation processes and to discover the key biological pathways triggered by CYPs and main life-stage where triadimefon effects on. Moreover, the present study will be undertaken to investigate the modulatory potential of triadimefon for CYPs-involved biotransformation metabolism including induction of defensome in response to chemicals. Furthermore, in order to better explain the mode of action for the reproductive toxicity of triadimefon, the cross-talk between AhR and ER signaling pathway will be selected as target, and in vivo effects of triadimefon will be studied on hepatic expression of genes involved in the AhR pathway and in steroidogenesis, and of ER genes in zebrafish. The prospective result will provide the scientific evidence of reasonable application of triadimefon and triazole fungicides, ecological risk assessment and environment protection, and so on.

三唑酮通过影响甾醇脱甲基酶（CYP51）抑制麦角甾醇合成，且P450依赖型单加氧酶含有可被三唑类化合物结合的血红素，因此三唑酮可以对生物转化过程中涉及的CYP450产生干扰，影响生物体内类固醇激素的合成，从而影响生殖发育。前期研究发现三唑酮对斑马鱼具有拟雌激素活性，对斑马鱼生殖内分泌系统具有干扰效应，但这种干扰效应发生的关键环节及其分子机制等仍然不明。为此，本课题拟采用不同发育阶段的斑马鱼，结合高通量测定技术，研究鱼体内类固醇/激素含量、参与类固醇/激素生物转化过程相关基因表达量在药剂暴露后的变化，力图在生物转化途径探究三唑酮对斑马鱼类雌激素作用产生的关键环节和敏感期；同时研究生物转化对三唑酮富集、转化、分布的影响，阐释斑马鱼的防御机制；进一步结合AhR-ER信号通路，以期更好地解释三唑酮致斑马鱼内分泌-生殖毒性分子通路机制。研究结果为探讨三唑酮农药生态风险评估、环境保护等提供科学依据。

三唑酮是目前应用最广泛的广谱杀菌剂之一，在环境和目标农产品中时有检出，其可以对生物转化过程中涉及的CYP450产生干扰，影响生物体内类固醇激素的合成、代谢，从而诱导生殖损伤。然而，由三唑酮介导受体信号途径触发的鱼类生殖毒性的关键环节和敏感发育期仍然未知。故此，申请人利用模式生物斑马鱼，开展环境剂量三唑酮生殖毒性效应及作用机制的研究。研究发现：1）肝脏是三唑酮进入生物体后的主要富集器官，并伴随着肝脏受体信号通路及其下游相关靶基因的表达变化。三唑酮激活了肝脏中生物转化途径，诱导外源物感知受体PXR和AhR2，加速内源类固醇激素代谢作用和三唑酮转化（形成毒性代谢物三唑醇）、外排作用；2）成熟期斑马鱼暴露于环境相关浓度三唑酮中，其成熟生殖细胞发育延迟，产卵量降低；3）通过串联质谱确证技术靶向类固醇合成途径结合RT-PCR，Western-Blot方法的研究，发现由于不同的代谢需求，斑马鱼雌雄之间存在着性别差异表达的基因，且主要富集在芳香烃﹣雌激素受体途径。三唑酮在0.069 mg/L及以上浓度成熟期暴露激活了肝脏中AhR受体信号通路，显著抑制了雄鱼体内cyp19a1a的表达，并伴随着E2生物合成速率的降低，进而导致肝脏中esr1和vtg1表达的下调，显现抗雌激素效应；而雌鱼中，E2水平的增加伴随着由三唑酮引起的Esr1蛋白表达的增加，表明三唑酮可能通过作用AhR受体—ER受体途径诱导雌激素活性；4）性腺转变期（43-71 dpf）和性腺发育期（72-99 dpf）是三唑酮诱导生殖毒性的发育关键期。三唑酮发育关键期暴露导致斑马鱼性腺损伤是不可逆的。在性腺转变期，三唑酮影响斑马鱼性别分化的关键环节是通过抑制雌激素合成并加速雌激素代谢途径，性别趋向表现为雄性偏向性分化；而性腺发育期，三唑酮通过抑制雄激素激素合成并加速雄激素激素代谢途径，性别趋向于雌性偏向性分化。项目研究结果为三唑酮生态安全性评价提供了科学依据，同时促进了对三唑类农药对鱼类造成的内分泌干扰效应机制的认识，具有重要的学术价值和社会意义。