氟咯草酮诱导的睾丸毒性分子机制研究

Fluorochloridone （FLC）is a ketopyrrolidine herbicide widely used in the world, and it is believed that more and more people will have chance to be exposed to this chemical. European Food Safety Authority (EFSA) and European Commission Health & Consumers Directorate-general (ECHCD) considered this chemical as a potential endocrine disruptor for its male reproductive toxicity. ..Our studies supported by NSFC showed that FLC was quickly absorbed, widely distributed in body tissues and slowly eliminated through urine. It was able to pass the blood-testis barrier. The metabolites become more polar and are distributed in testis, epididymis, heart and muscle with Tmax at 24h after multiple biotransformation. Our 28-day and 90-day rat oral administration studies found that the functional changes occurred though no obvious pathological changes of testis at dose level of 3mg/kg/day were observed. Urinary metabolomics study revealed that 16 metabolic pathways were potentially associated, including steroid hormone. The liver metabolomics study showed that metabolic differences were statistically associated with 30 lipid biomarkers, linked with cellar membrane components and signaling molecules which control cellar differentiation and proliferation. The studies in primary cultured rat testicular Sertoli cells verified FLC-induced ROS generation disrupted intracellular calcium homeostasis by attacking ER, and the elevated intracellular calcium levels resulted in ERK1/2 over-phosphorylation, which consequently promoted apoptosis. FLC perturbed the expression of blood-testis barrier associated proteins and increased germ cell detachment. ..According to such findings, to understand high sensitivity to testis of FLC, the proposals is aimed to focus on the Nrf2 pathway-oxidative stress, the Immuno-inflammation and metabolomics of testis based on the experimental design of a 28-day orally administrated mice (both Nrf2-WT and Nrf2-KO mice) toxicity study plus 14-day recovery period. Consequently, we can understand the internal linking among Nrf2 function and change of Immuno-inflammation and metabolomics of testis, know fully the molecular mechanism of FLC –induced testicular toxicity, predict its potential toxicity on human and ecological system, and further provide scientific evidence for the pesticide management.

氟咯草酮（FLC）是一种广泛应用的吡咯烷酮类除草剂，人群接触机会越来越多。它具有睾丸毒性，已被欧盟食品安全局和欧盟健康与消费品理事会归为可能的环境内分泌干扰物。刚结题的自然科学基金研究显示，FLC在大鼠体内吸收快、分布广、消除慢，可破坏血睾屏障。经过多次代谢-分布-再代谢过程后的高极性代谢物能进入睾丸、附睾组织。肝脏及尿液代谢组学初步研究示它能诱导代谢扰动。FLC能诱导睾丸支持细胞胞内ROS水平上升，进而攻击内质网致胞内钙离子水平升高并激活ERK1/2导致细胞凋亡。项目拟在这些研究的基础上应用野生型小鼠和Nrf2敲除型小鼠模型，以28天给药以及14天恢复实验设计，针对Nrf2信号通路参与的氧化应激在FLC睾丸毒性中的作用以及睾丸免疫炎性反应和睾丸的代谢组学三个环节开展深入研究，观察氧化应激改变与氟咯草酮染毒后小鼠免疫炎性变化及代谢组学变化的内在联系，以期全面认识FLC睾丸毒作用分子机制。

氟咯草酮作为20世纪80年代中期投产使用的吡咯烷酮类芽前施用选择性除草剂在欧盟和北美国家中广泛应用。近年来，对其慢性毒性的研究日渐增多。氟咯草酮在土壤和地下水中具有中等持久性，其环境命运与哺乳动物和人类健康高度相关。欧洲食品安全局于 2010年发表了 氟咯草酮危险度评估报告，对氟咯草酮的生态毒性、哺乳动物毒性以及代谢特点做出报告提示 氟咯草酮的靶器官为睾丸和附睾，并将其归类为R62（可能损害生育能力）和R61（可能对胎仔造成伤害）的化学物。我们前期实验发现，氟咯草酮诱导支持细胞凋亡和BTB完整性的丧失，从而导致大鼠睾丸异常并影响生育能力。氟咯草酮诱导大鼠睾丸损伤，但是否造成小鼠雄性生殖损伤以及潜在的分子机制仍存在数据匮乏。本研究通过构建C57BL/6小鼠体内染毒模型和 TM4小鼠睾丸支持细胞系体外染毒模型，探究了氟咯草酮染毒后小鼠睾丸发育、附睾精子参数、氧化应激状态、线粒体损伤、自噬及其相关通路的改变等。此外，我们还通过TM4细胞模型及其相关干预实验模型建立研究氟咯草酮体外染毒诱导细胞自噬的分子机制，探讨活性氧（ROS）及AKT/mTOR信号通路在该过程中的作用；深入探讨了线粒体损伤、未折叠蛋白反应三条信号通路在氟咯草酮诱导 TM4细胞凋亡中的作用；探究 Nrf2、NFκB信号通路以及睾丸代谢改变及代谢相关通路AMPK、SIRT在氟咯草酮所致雄性生殖毒性中的作用。本研究为氟咯草酮睾丸毒性机制研究提供了方向，为全面理解氟咯草酮睾丸毒性提供数据支持。