NEUROTOXIC MECHANISMS IN PRIMARY CNS CELL CULTURES

原代中枢神经系统细胞培养中的神经毒性机制

• 批准号: 3251658
• 负责人: NEVILLE BROOKES
• 金额: $ 9.9万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-15 至 1989-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3251658
• 关键词: aminoacid metabolism; aminoacid transport; astrocytes; brain metabolism; central nervous system; cryoscience; cytotoxicity; electron microscopy; electron probe spectrometry; environmental toxicology; fluorimetry; freeze etching; glucose metabolism; glutamates; immunofluorescence technique; leucine; mercury poisoning; methylmercury; mixed tissue /cell culture; neurotoxins; organ culture; paper chromatography; phase contrast microscopy; scintillation counter

Mercury is a pervasive occupational and environmental neurotoxicant of unknown cellular mechanism. A preliminary study by the P.I. showed that mercury in low concentration selectively inhibits glutamate uptake into primary cell cultures of mouse cerebral astrocytes. Glutamate and related endogenous neuroexcitants are themselves suspected of involvement in adult-onset primary neurodegenerative disorders. It is hypothesized that selective blockade of glutamate inactivation may be a contributory mechanism in mercury neurotoxicity. The proposed research will determine whether a cellular basis exists for this hypothesis. The aim of the research is to explore the selective impairment of characterized amino acid transport systems by the toxic action of mercuric chloride and methylmercury(II) chloride. Anionic (Systems X-AG, X-A), neutral (Systems A, L, ASC, N, beta, GABA), and cationic (System y+) amino acid transport systems will be probed. This analysis will be correlated with cell mercury content and subcellular distribution, and with functional and morphologic status in mouse cerebral astrocytes. Radioactive tracers will be used to measure amino acid transport, leucine incorporation into protein, glucose utilization and mercury content of the astrocytes. Membrane integrity will be probed by vital dye exclusion and tracer efflux. Ultrastructural analysis of the cells and scanning X-ray microanalysis of mercury distribution will be carried out using quick-freeze fixation and cryopreparative techniques. A consultative and collaborative arrangement has been made for fluorometric assay of intermediates and metabolites of glycolytic and oxidative pathways. The proposed research will provide answers to fundamental questions concerning the cellular neurotoxic mechanism of mercury. These answers will be in a form that can be quantitatively, and therefore meaningfully, related to in vivo studies. The results will have a direct bearing upon (a) the need to provide a sound basis for defining tolerable tissue burdens of mercury in humans, (b) alternative therapeutic approaches in management of mercury intoxication, and (c) exploration of possible linkages between environmental neurotoxicants and primary neurodegenerative disorders.

汞是一种普遍存在的职业和环境神经毒物 未知的细胞机制。 P.I. 的初步研究表明 低浓度的汞选择性地抑制谷氨酸的吸收 小鼠脑星形胶质细胞的原代细胞培养物。 谷氨酸及相关 内源性神经兴奋剂本身被怀疑参与 成人发病的原发性神经退行性疾病。 假设 选择性阻断谷氨酸失活可能是一个促成因素 汞神经毒性机制。 拟议的研究将确定 该假设是否存在细胞基础。 该组织的目标是 研究目的是探索特征氨基酸的选择性损伤 氯化汞的毒性作用对运输系统的影响 氯化甲基汞(II)。 阴离子（系统 X-AG、X-A）、中性（系统 A、L、ASC、N、β、GABA) 和阳离子（系统 y+）氨基酸转运 系统将受到调查。 该分析将与电池汞相关 含量和亚细胞分布，以及功能和形态 小鼠大脑星形胶质细胞的状态。 放射性示踪剂将用于 测量氨基酸转运、亮氨酸掺入蛋白质、葡萄糖 星形胶质细胞的利用率和汞含量。 膜的完整性将 通过活体染料排除和示踪剂流出来探测。 超微结构 细胞分析和汞的扫描 X 射线微量分析 分配将使用快速冷冻固定和 冷冻制备技术。 协商和合作安排 已用于荧光测定中间体和代谢物 糖酵解和氧化途径。 拟议的研究将提供 有关细胞神经毒性的基本问题的答案 汞的作用机制。 这些答案将采用以下形式： 定量地，因此与体内研究有意义。 结果将直接影响 (a) 是否需要提供健全的 确定人类可耐受的组织汞负荷的基础，(b) 管理汞中毒的替代治疗方法， (c) 探索环境之间可能的联系 神经毒物和原发性神经退行性疾病。