基于利阿诺定受体功能增强研究低剂量联苯菊酯及溴氰菊酯神经发育毒性的新机制

Pyrethroids are a novel class of synthetic insecticides which are heavily used in the agriculture and household for insecticidal purpose. Studies have demonstrated the existence of pyrethroids and metabolites in human breast milk, blood as well as urine samples. Moreover, recent epidemiological studies suggested that low concentrations of pyrethroids may be associated with increased risk of Attention-Deficit Hyperactive Disorder (ADHD) and/or Mental Retardation. Previous researches were focusing on the acute neurotoxicity of high dose pyrethroids whose primary targets are voltage-gated sodium channels at the concentration ranges of (1 μM-100 μM). Our recent study has demonstrated that low, environmental relevant concentration of bifenthrin (10 nM) is sufficient to alter the developmental trajectory of neurite outgrowth which is independent of Na+ channel modification. Our data showed that ryanodine receptor (RyR) may represent a novel target for pyrethroid interaction. The overarching hypothesis is that low, environmental relevant concentrations of pyrethroids can affect the neuronal development through RyR gain-of function induced spontaneous Ca2+ oscillations dysregulation. In this project, we will use bifenthrin and deltamethrin as type I and II pyrthroids representatives to investigate the developmental neurotoxicity of low doses (environmental exposure relevant) of pyrethroids especially to investigate the susceptibility of neurons at different developmental stages to pyrethoids exposure. We will investigate the influence of bifenthrin and deltamethrin on spontaneous Ca2+ oscillations, neurite outgrowth, synapse formation, and how bifenthrin and deltamethrin affect neurite outgrowth, synapse formation. We will also utilize biological, pharmacological and electrophysiological approaches to investigate the physical binding of bifenthrin and deltamethrin to ryanodine receptor (RyR) and how can they modulate RyR function. Finally, we will investigate the sensitivity of mice carrying either wild type RyR or gain-of-function RyR (T4826I-RyR) to bifenthrin and deltamethrin exposure to demonstrate whether RyR is the molecular target of bifenthrin and deltamethrin-induced developmental neurotoxicity. Our goal is to demonstrate whether the evidences that pyrethroids affect neuron development from epidemiological studies can be extended to animal models. We will also demonstrate the sensitivity of neurons at distinct developmental stages to pyrethroids exposure. Molecular targets and signaling pathways profiling will help us to understand how environmental relevant concentrations of pyrethroids affect the neuron development. Our study can provide scientific evidences for how to use pyrethroids safely.

拟除虫菊酯是新一代仿生杀虫剂，在农业、居家灭虫中使用广泛。流行病学研究显示，人体内存在的微量拟除虫菊酯可能与儿童的智力发育相关。位于内质网控制细胞内Ca2+释放的利阿诺定受体（RyR）能够调节神经元的生长发育。我们提出拟除虫菊酯通过增强RyR的功能从而影响神经元钙振荡及其下游信号路径进而介导神经发育毒性的假说。本课题将以联苯菊酯和溴氰菊酯为代表研究环境暴露剂量拟除虫菊酯（<1mg/kg）对神经系统发育的影响，并探讨不同发育阶段神经系统对拟除虫菊酯的敏感性，证实环境暴露剂量拟除虫菊酯的神经发育毒性；利用生物学、药理学、电生理学的方法在分子及细胞水平研究联苯菊酯和溴氰菊酯对RyR功能的影响及其对神经突生长/突触形成的调控作用；最后探讨携带功能增强型RyR的基因插入鼠对联苯菊酯和溴氰菊酯的敏感性，阐明RyR引起的钙振荡紊乱是其神经发育毒性的新靶点及新机制，为安全使用拟除虫菊酯提供科学依据。

拟除虫菊酯具有高效、广谱、低毒和能生物降解等重要特性，广泛用于农业、家庭虫害防治。流行病学研究显示，孕妇血液、尿液、乳汁内存在的微量拟除虫菊酯可能影响子代的智力发育。位于内质网控制细胞内Ca2+释放的利阿诺定受体（RyR）能够调节神经元的生长发育。本研究以拟除虫菊酯杀虫剂代表性化合物联苯菊酯和溴氰菊酯为对象，建立了体外皮层神经元神经网络钙振荡模型，明确拟除虫菊酯对皮层神经发育毒性的新机制。研究结果：（1）建立了大脑皮层神经元钙震荡（Synchronous calcium oscillation，SCO）模型，且发现苄氯菊酯（Permethrin，PM）、七氟菊酯（Tefluthrin，TF）、氯氰菊酯（Cypermethrin，CP）、溴氰菊酯（Deltamethrin，DM）均可对神经元SCOs产生影响，其中Ⅱ型拟除虫菊酯DM对其影响最大；（2）在体外培养的皮层神经元中通过电生理学、药理学、生物学等方法证明RyR是低浓度DM的新靶点，可能参与DM调节神经元网络连接；（3）环境暴露剂量溴氰菊酯和联苯菊酯对小鼠行为和学习记忆能力产生不利影响，这可能与小鼠海马区神经元死亡及N-甲基-D-天冬氨酸受体（NMDAR）的表达变化及神经炎症的发生相关；（4）不同发育阶段的神经元对部分拟除虫菊酯存在不同的敏感性，且未成熟神经元比成熟神经元对拟除虫菊酯更敏感；（5）低浓度溴氰菊酯可能通过电压门控钠离子通道、NMDAR和RyR，及下游Erk1/2-CREB钙信号通路促进大脑皮层神经元的生长发育，包括神经突生长及神经突触形成。本研究明确了环境暴露剂量联苯菊酯和溴氰菊酯能够产生神经发育毒性，未成熟的神经元对拟除虫菊酯更加敏感，RyR是低剂量联苯菊酯和溴氰菊酯的新靶点，电压门控钠通道、NMDAR和RyR，及下游Erk1/2-CREB钙信号通路可能是其作用的信号通路。本研究将为拟除虫菊酯杀虫剂的安全合理使用提供重要科学指导意义。