含半胱氨酸残基蛋白介导1-溴丙烷中枢神经毒性的机制研究

1-Bromopropane(1-BP),an alternative to ozone-depleting solvents,was largely produced and wildly used for adhesives,aerosol products,metal,electronics, and precision cleaning. It is well documented that exposure to 1-BP causes neurotoxic effects in both experimental animals and humans.Several lines of evidences suggest the hippocampus as a potential target of 1-BP neurotoxicity in the central nerve system (CNS).However,the molecular basis and mode of action of 1-BP-induced neurotoxicity in the CNS remain to be elucidated.Considering that 1-BP is an electrophilic haloalkane and its primary macromolecular targets are nucleophilic sulfhydryl thiolate groups on cysteine residues of protein,detection of alteration in expression and function of these cysteine-containing proteins will contribute to the knowledge of mechanism underlying 1-BP-induced neurotoxicity in the CNS.We have hypothesized that 1-BP intoxication causes cumulative hippocampal damage by forming adducts with nucleophilic cysteine sulfhydryl groups on critical hippocampal proteins. To determine the cumulative effects of 1-BP on the cysteine-containing proteome of hippocampus and to understand their roles in the 1-BP-induced neurotoxicity in the CNS,we exposed male F344 rats to 1-BP at 0, 400, or 1000 ppm for 8 h/day for 1 week or 4 weeks by inhalation and quantitated changes in cysteine-containing proteins using cleavable isotope-coded affinity tagging (ICAT) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry(MALDI-TOF-TOF/MS).ICAT analysis uses a sulfhydryl-specific tag to identify and quantitate cysteine-containing proteins.The hippocampal proteins of each experimental group were labeled with either light (control) or heavy (1-BP exposure) ICAT reagent.Morris water maze (MWM)was employed to investigate the change of hippocampal function in spatial learning and memory after 1-BP exposure.Statistical analysis was ran to identify hippocampal cysteine-containing proteins which showed dose-dependent and/or time-course alteration with 1-BP exposure.Western blotting and immunohistochemistry were conducted to confirm the alteration of hippocampal cysteine-containing proteins after 1-BP exposure using rat hippocampal samples.On the other hand,neuron cells were cultured and SiRNA was used to explore the function of candidate cysteine-containing proteins selected by statistical analysis.Finally,candidate biomarkers will be validated in 1-BP-exposed workers in epidemiological investigation.This study will uncover the primary alteration of hippocampal cysteine-containing proteins after 1-BP exposure and understand their roles in 1-BP-induced neurotoxicity in the CNS, as well as their relationships with secondary adverse effects in 1-BP neurotoxicity. Furthermore,this study also provides the possibility to develop cysteine-containing proteins as sensitive biomarkers for 1-BP neurotoxicity in the CNS,and to provide new targets for early prevention and clinical treatment.

1-溴丙烷(1-BP)作为臭氧层破坏溶剂的替代溶剂在国内外被大量生产和使用，职业接触人数众多，神经中毒病例层出不穷，对其神经毒性分子机理的深入研究迫在眉睫。含半胱氨酸残基蛋白是1-BP的初始效应分子，其表达和功能的改变与1-BP的中枢神经毒性密切关联。为明确含半胱氨酸残基蛋白介导1-BP中枢神经毒性的机制，本课题通过吸入染毒制作1-BP中枢神经毒性动物模型,Morris水迷宫检测大鼠海马区功能变化，ICAT定量分析1-BP的初始靶向含半胱氨酸残基蛋白，筛选与1-BP暴露呈剂量和时间依赖关系的关键蛋白，蛋白印迹和免疫组化确认，SiRNA技术在细胞水平研究蛋白功能，最终在1-BP暴露人群中进行验证。本研究将揭示1-BP的初始靶向含半胱氨酸残基蛋白，阐明其表达和功能的改变与1-BP中枢神经毒性的关系，探讨关键靶蛋白作为1-BP神经毒性早期损害的敏感生物标志物，为早期预防和临床药物治疗提供新靶点。

1-溴丙烷(1-BP)作为臭氧层破坏溶剂的替代溶剂其神经毒性机制尚待阐明。本课题吸入染毒制作1-BP神经毒性动物模型，ICAT和磷酸化蛋白质组学分析1-BP靶向含半胱氨酸残基和磷酸化蛋白，筛选与1-BP呈剂量和时间依赖关系的关键蛋白，最终在1-BP暴露人群中验证。发现（1）动物实验：通过Pro-Q Diamond染色法和2D-DIGE辨认26个差异表达的磷酸化蛋白；质谱鉴定18个上调和8个下调蛋白。参与生物过程包括刺激反应、代谢、凋亡信号等。9个蛋白的表达与1-BP呈剂量-反应和剂量-时间依赖关系。Mn2+-Phos-tag SDS-PAGE确认14-3-3 θ的差异表达。蛋白印迹发现1-BP引起Bax蛋白在线粒体内高表达，但在细胞浆中低表达；1-BP引起cytochrome C蛋白在细胞浆中高表达而在线粒体中低表达。TUNEL发现1-BP在1000 ppm引起海马区TUNEL阳性细胞数量增加。（2）细胞实验：1-BP引起HA细胞凋亡率的显著性提高。（3）人群研究：对深圳市某企业发生的群体性1-BP职业性中毒事故进行调查发现该企业使用含1-BP的清洗剂进行超声波作业，导致4名工人在接触1-BP后均出现周围神经系统损害。检测结果显示清洗房空气中1-BP的短时间接触浓度为1 038.8~1 466.1 mg/m3，时间加权平均浓度为371.5~654.1 mg/m3；1-BP清洗剂顶空气中1-BP相对百分比为97.1%。清洗房作业工人班后尿中1-BP水平为25.50~155.64 μg/L。4例病例均为男性，均接触高水平1-BP，接触时间为9~11个月。临床表现主要有不同程度的感觉、运动障碍，主要症状为渐进性加重的肢体麻木、乏力，以下肢多见，可伴有步态不稳。体格检查可见双下肢肌力减弱；双下肢或四肢痛觉和触觉减退，位置觉和振动觉减退；双侧跟腱反射多呈减弱或消失；1例病例出现感觉性共济失调。神经-肌电图检查均存在不同程度的周围神经损害，以运动和感觉神经传导速度减慢多见，且轴索和髓鞘的损害均可见。依据GBZ/T 247—2013均诊断为职业性慢性1-溴丙烷中毒性周围神经病。本研究阐明1-BP通过抑制海马区抗凋亡分子作用而引起凋亡相关的神经毒性；筛选到的与1-BP呈现剂量和时间依赖关系的磷酸化蛋白质可作为1-BP神经毒性的分子标志物，并作为早期预防和临床药物治疗提供新靶点。