PROCESSES OF METAL SELECTION BY METAL ACCUMULATING CELLS

金属积累池的金属选择过程

• 批准号: 5211947
• 负责人: ANDREW Z MASON
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5211947
• 关键词: Mollusca; cadmium; chemical kinetics; copper; cytochrome c; cytotoxicity; enzyme activity; high performance liquid chromatography; ligands; mass spectrometry; metal complex; metal metabolism; metallothionein; stable isotope; thermodynamics

The proposed project aims to determine if Cd exposure causes a redistribution of intrinsic stores of Cu within the cell. It is hypothesized that metallothionein (MT), induced by exposure to Cd, acts as a thermodynamic and kinetic trap for metals such as Cu which show a higher affinity and binding constant for the protein. The sequestration of Cu from intracellular proteins and enzymes requiring its presence either allosterically or catalytically for normal functioning may result ultimately in cellular pathologies. Consequently, the toxicological effects noted with Cd may arise from cellular deficiencies in Cu rather than excesses of Cd. Preliminary data from two invertebrate phyla are provided in support of this hypothesis. Experiments are proposed to evaluate this proposition further both in vitro using cultured mammalian cells and in vivo using the invertebrate mollusc Littorina littorea as a model organism. The five specific aims of the study are to determine whether : i) MT induction in response to Cd exposure results in a concomitant increase in the cellular/organismal burden of Cu; ii) MT induction alters the subcellular distribution of Cu, Zn and other physiologically important metals; iii) the observed changes in Cu are due to a redistribution of intrinsic stores or the accumulation of extrinsic Cu from the surrounding media, iv) there is impaired functioning of Cu containing enzymes (eg. cytochrome C oxidase activity) as a result of Cd exposure: v) the cytotoxic effects of Cd can be remedied by administering Cu; Two novel applications of inductively coupled plasma mass spectroscopy (ICP-MS) have been developed to study these investigatory questions. Firstly, an ICP-MS has been coupled directly to a high performance liquid chromatography system to allow the simultaneous separation and elemental analysis of MT and other cytosolic metal binding ligands. Secondly, protocols using stable isotopic 63Cu and 65 Cu have been developed to permit long term kinetic studies of Cu metabolism to be undertaken. The continued refinement and development of techniques with these capabilities will undoubtedly facilitate the study of Menke's and Wilson's disease and other genetic disorders which result from abnormal Cu metabolism. Finally, in addition to the studies of MT, the role of intracellular, mineralized orth/pyrophosphate containing inclusions in the metabolism of Cd will also be studies. Recent evidence has shown that these deposits may be an important site for the sequestration and deposition of accumulated Cd. In combination, these studies will provide important information on the competitive interactions between essential and non-essential metals for intracellular metal-binding ligands. An appreciation of these types of interaction are important if we are to understand the mechanisms by which metals exert their toxicological action. The proposed research program is particularly suitable for both graduate and undergraduate minority student involvement and provides broad exposure to a number of modern techniques that are commonly employed in the biomedical sciences including aseptic and metal-free clean technique, cell culture, electron microscopy, plasma spectroscopy and protein separation and purification. This project therefore provides an excellent opportunity for students to be exposed to the biomedical research experience.

拟议项目旨在确定镉暴露是否会导致 细胞内铜内在储存的重新分配。 这是 假设金属硫蛋白 (MT) 在接触 Cd 后会发挥作用 作为金属（如铜）的热力学和动力学陷阱，显示出 蛋白质的亲和力和结合常数更高。 封存 来自需要其存在的细胞内蛋白质和酶的铜 正常功能的变构或催化可能会导致 最终在细胞病理学中。 因此，毒理学 Cd 引起的效应可能是由于细胞缺乏 Cu 而引起的 比 Cd 过量。 来自两个无脊椎动物门的初步数据是 提供支持这一假设。 建议进行实验以进一步评估这一命题 体外使用培养的哺乳动物细胞，体内使用无脊椎动物细胞 软体动物 Littorina littorea 作为模式生物。 该研究的五个具体目标是确定是否： i) MT 对 Cd 暴露的感应会导致相应的增加 细胞/有机体的铜负荷； ii) MT 感应改变了 Cu、Zn 和其他生理重要物质的亚细胞分布 金属； iii) 观察到的 Cu 变化是由于重新分布 内在储存或周围环境中外在铜的积累 介质，iv) 含铜酶的功能受损（例如 Cd 暴露导致的细胞色素 C 氧化酶活性：v) Cd 的细胞毒性作用可以通过给予 Cu 来补救； 电感耦合等离子体质谱法的两个新应用 (ICP-MS) 的开发就是为了研究这些调查问题。 首先，ICP-MS 已直接耦合到高性能液体 色谱系统允许同时分离和元素 MT 和其他胞质金属结合配体的分析。 第二， 使用稳定同位素 63Cu 和 65 Cu 的协议已被开发 允许进行铜代谢的长期动力学研究。 这 不断完善和发展这些技术 能力无疑将促进门克和 威尔逊氏病和其他由异常引起的遗传性疾病 铜代谢。 最后，除了MT的研究，细胞内的作用， 代谢中含有矿化正磷酸盐/焦磷酸盐的夹杂物 Cd 的含量也将被研究。 最近的证据表明，这些 矿床可能是封存和沉积的重要场所 累积的 Cd。 结合起来，这些研究将提供有关以下方面的重要信息： 必需金属和非必需金属之间的竞争性相互作用 细胞内金属结合配体。 对这些类型的欣赏 如果我们要理解相互作用的机制，相互作用就很重要 金属发挥其毒理学作用。 拟议的研究计划特别适合研究生 和本科少数族裔学生的参与，并提供广泛的 接触许多常用的现代技术 生物医学科学，包括无菌和无金属清洁技术， 细胞培养、电子显微镜、等离子体光谱和蛋白质 分离和纯化。 因此，该项目提供了一个 为学生提供接触生物医学的绝佳机会 研究经验。