戊唑醇对斑马鱼HPG轴内分泌干扰作用的分子机制

Triazole fungicides are currently the most important class of fungicides, and the application amount of tebuconazole is the biggest one in all triazole fungicides in recent years. Due to its widely application, the potantial harm to environmental organisms is paid more and more attention. Previous study indicated that tebuconazole has the estrogen-like effect to zebrafish, which can disrupt the normal fuction of HPG axis of endocrine systerm. However, the key location where tebuconazole effects on and its molecular disrupting mechanism are still unknown. For this reason, combined with high throughput screening assay (such as UPLC-MS/MS and microarray screening of genes), developmental stages of zebrafish will be exposed to tebuconazole at environmental dosages to display the expression alternation of HPG axis related steroids and genes which participate in the synthesis, transportation and metabolism of these steroids and to discover the he key location and main lifestage where and when tebuconazole effects on. Furthermore, in order to better explain the molecular mechanism of tebuconazole disrupting effect on the HPG axis of zebrafish, steroidogenic factor 1 (SF-1) will be selected as target which tebuconazole effects on. The SF-1lack of phenotype mutant zebrafish by CRISPR/Cas9 knockdown assay will be cultured and used for exposure to tebuconazole and then the expression alternation of HPG axis related steroids and genes mentioned above will be also test. The prospective result will be benefit to explain the molecular mechanism of EEDs, provide the scientific evidence of reasonable application of tebuconzole and even triazole fungicides, policy making of environment protection and food safty, and so on.

三唑类杀菌剂是目前最重要的一类杀菌剂，而戊唑醇是其中用量最大的一种杀菌剂。因其广泛使用造成环境生物的潜在危害逐渐受到关注。前期研究发现戊唑醇对斑马鱼具有类雌激素样作用，对斑马鱼内分泌系统的HPG轴具有干扰效应，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，研究鱼体内类固醇含量在戊唑曝露前后的变化，以及参与类固醇合成、转运、代谢的相关酶及其基因在药剂曝露前后表达量的变化，力图在类固醇/激素代谢途径上探究该药对斑马鱼类雌激素样干扰作用产生的关键环节和敏感期。进一步结合CRISPR/Cas9基因敲除技术，探讨类固醇生成因子SF-1在戊唑醇内分泌干扰中的作用，以期更好地解释戊唑醇对斑马鱼HPG轴干扰效应的分子通路机制。研究结果为揭示环境内分泌干扰的作用机制，探讨戊唑醇等三唑类农药的合理使用、环境保护和食品安全政策制定，提供理论依据。

近年来，农药大量频繁使用后的生态毒理学效应日益受到关注。作为农田常用药剂，三唑类杀菌剂在田间与水生生态系统中被广泛检出，且在环境中稳定。作为单个品种年销售超过5亿美金的代表性三唑类杀菌剂戊唑醇，其生态毒性尚未知。故此，申请人利用模式生物斑马鱼，开展环境剂量戊唑醇对环境生物长期毒性效应及作用机制研究。研究发现，1）戊唑醇可在成年斑马鱼性腺中蓄积，表明其存在潜在的内分泌干扰风险。幼鱼期斑马鱼暴露于环境相关浓度戊唑醇中，斑马鱼性别趋向于雄性偏向性分化；2）青春期和性成熟期斑马鱼暴露于环境相关浓度戊唑醇中，其成熟生殖细胞发育延迟，产卵量降低；3）通过检测性激素合成通路上的类固醇含量以及RNA-Seq研究，发现戊唑醇影响斑马鱼性别分化和繁殖力的关键环节是戊唑醇抑制了芳香化酶Cyp19a的表达，从而抑制睾酮或雄烯二酮向雌二醇的转化。RNAi试验表明，将不同发育阶段的斑马鱼胚胎中cyp19a基因沉没，斑马鱼性别分化仍趋向雄性化，而通过同期补偿雌二醇，可使的雌雄性别比趋向正常化。此外，检测了戊唑醇暴露后斑马鱼体内PKA的活性及其调控产物磷酸化蛋白量的变化。发现PKA活性受到抑制，且抑制率与暴露浓度正相关，总磷酸化蛋白含量也降低。采用CRISP/CAS9技术，构建了yak1基因缺失型H295R细胞株，使得PKA过表达。戊唑醇暴露后发现PKA活性及总磷酸化蛋白含量依然下降。最后，采用SPR技术，研究发现戊唑醇可与PKAɑ催化亚基结合，抑制其磷酸化催化活性。上述研究结果表明戊唑醇可能通过与PKA的ɑ催化亚基结合，抑制cAMP/PKA/SF-1通路介导的信号传导（即抑制了PKA的磷酸化功能，从而抑制了SF-1的表达，导致了Cyp19a下调，进而阻碍了雌激素E2的合成），引发抗雌激素样生殖内分泌干扰效应。研究结果对戊唑醇的使用提出了安全预警，为三唑类农药生态安全性评价提供了科学依据，同时促进了对农药乃至环境污染物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。