Role of Oxidative Mechanisms in the Toxicity of Metals

氧化机制在金属毒性中的作用

基本信息

批准号：
6823696
负责人：
SIMON V AVERY
金额：
$ 21.6万
依托单位：
UNIVERSITY OF NOTTINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-05-01 至 2008-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Metals are associated with a range of degenerative conditions and they pose a serious threat to human health. Considerable evidence indicates that reactive oxygen species (ROS) may play a key role in causing the deleterious biological effects of metals. However, this evidence remains inconclusive and the principal cellular and molecular mechanism(s) underlying metal toxicology have yet to be clearly resolved. This work exploits the experimental advantages of the yeast model Saccharomyces cerevisiae to elucidate cellular metal toxicity, so avoiding the limitations imposed on studies of this nature in animal models. Recent advances in functional genomics technologies that are unique to yeast, in conjunction with results leading up to this proposal from our laboratory, underscore S. cerevisiae as the eukaryotic model of choice here. The objective is to test the hypothesis that oxidative mechanisms are the primary cause of metal toxicity in cells. This will be achieved using two principal strategies developed in our laboratory, each highly distinct from previous approaches to this problem. The work will focus on copper, chromium and cadmium as examples of model redox-active and - inactive toxic metals. First, we will explore in greater depth an entirely novel question that is giving us major new insight to metal toxicity: do proteins that protect against oxidative damage determine the differing metal resistances of individual cells within isogenic populations? We have already discovered that the mechanisms underlying this heterogeneous metal resistance are distinct from those giving rise to culture-averaged resistance (the focus of past studies). Moreover, specific antioxidant functions appear to underpin the heterogeneity, and this key issue will be resolved directly in this project. In addition, an innovative genome-wide search will be carried out to find new determinants of heterogeneity. These heterogeneity studies are vital because, for the first time, they allow true evaluation of the roles of genes-of-interest in situ, i.e., pertaining to intact cells in which expression is not manipulated artificially. Second, we will build on our recent successful use of specific oxidative-damage repair enzymes applied to the problem of metal toxicity. Alongside such approaches, this project will introduce powerful new functional-genomics tools to identify the essential molecular-target(s) of metal action in cells. We will determine directly whether oxidative mechanisms cause the inactivation of these targets, and whole-cell toxicity. By testing the hypothesis in the manners described, the proposed studies should advance significantly our understanding of metal toxicology at the cellular level, and concurrently provide greater insight into the impact of ROS on biological systems.

描述（由申请人提供）：金属与一系列退化状况有关，对人类健康构成严重威胁。大量证据表明，活性氧（ROS）可能在引起金属的有害生物效应中发挥关键作用。然而，这一证据仍然没有结论，并且金属毒理学的主要细胞和分子机制尚未得到明确解决。这项工作利用酵母模型酿酒酵母的实验优势来阐明细胞金属毒性，从而避免了动物模型中这种性质的研究所受到的限制。酵母独有的功能基因组学技术的最新进展，与我们实验室提出这一建议的结果相结合，强调了酿酒酵母作为此处选择的真核模型。目的是检验氧化机制是细胞中金属毒性的主要原因的假设。这将通过我们实验室开发的两种主要策略来实现，每种策略都与以前解决此问题的方法截然不同。这项工作将重点关注铜、铬和镉，作为氧化还原活性和非活性有毒金属模型的例子。首先，我们将更深入地探索一个全新的问题，该问题为我们提供了对金属毒性的重大新见解：防止氧化损伤的蛋白质是否决定了同基因群体中单个细胞的不同金属抵抗力？我们已经发现，这种异质金属耐药性的机制与引起文化平均耐药性（过去研究的焦点）的机制不同。此外，特定的抗氧化功能似乎支撑了异质性，这个关键问题将在本项目中直接解决。此外，还将进行创新的全基因组搜索，以寻找异质性的新决定因素。这些异质性研究至关重要，因为它们第一次允许对感兴趣的基因的原位作用进行真正的评估，即涉及不人为操纵表达的完整细胞。其次，我们将基于最近成功使用的特定氧化损伤修复酶来解决金属毒性问题。除了这些方法之外，该项目还将引入强大的新功能基因组学工具来识别细胞中金属作用的基本分子靶标。我们将直接确定氧化机制是否导致这些靶标失活以及全细胞毒性。通过以所描述的方式检验假设，拟议的研究将显着促进我们对细胞水平金属毒理学的理解，同时更深入地了解 ROS 对生物系统的影响。