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：
7090001
负责人：
SIMON V AVERY
金额：
$ 21.09万
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）：金属与一系列退化条件有关，它们对人类健康构成了严重威胁。大量证据表明，活性氧（ROS）可能在引起金属的有害生物学作用方面起关键作用。但是，该证据仍然尚无定论，主要的细胞和分子机制（S）基础金属毒理学尚未清楚地解决。这项工作利用了酿酒酵母酵母模型的实验优势来阐明细胞金属毒性，因此避免了动物模型中对这种性质的研究的局限性。酵母独有的功能基因组技术的最新进展以及导致我们实验室的提案的结果，强调了酿酒酵母作为选择的真核模型。目的是检验氧化机制是细胞金属毒性的主要原因的假设。这将使用我们的实验室中开发的两种主要策略来实现，每种主要策略与以前的该问题的方法高度不同。这项工作将集中于铜，铬和镉，作为模型氧化还原活性和 - 非活性金属的示例。首先，我们将更深入地探索一个完全新颖的问题，它为我们提供了对金属毒性的主要新见解：防止氧化损伤的蛋白质是否决定了等生群体中各个细胞的不同金属抗性？我们已经发现，这种异质金属抗性的基础机制与引起培养平均抗药性的机制不同（过去研究的重点）。此外，特定的抗氧化功能似乎是异质性的基础，并且该关键问题将直接在该项目中解决。此外，将进行创新的全基因组搜索，以找到异质性的新决定因素。这些异质性研究至关重要，因为它们首次允许对原位基因的作用进行真实评估，即与完整的细胞有关，其中表达没有人为地进行操纵。其次，我们将基于最近成功使用用于金属毒性问题的特定氧化损伤修复酶。除了这种方法外，该项目还将引入强大的新功能基因组学工具，以识别细胞中金属作用的基本分子目标。我们将直接确定氧化机制是否引起这些靶标失活和全细胞毒性。通过测试所描述的举止中的假设，提出的研究应显着提高我们对细胞水平金属毒理学的理解，并同时对ROS对生物系统的影响有更深入的了解。