OXIDATIVE MECHANISMS AND THE TOXICITY OF METALS

氧化机制和金属的毒性

基本信息

批准号：
6181007
负责人：
DONALD AHEARN
金额：
$ 13.59万
依托单位：
GEORGIA STATE UNIVERSITY
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 2004-04-30
项目状态：
已结题

项目摘要

Heavy metals have been implicated in a range of degenerative conditions and they pose a serious threat to human health. Mounting evidence suggests that reactive oxygen species (ROS) play a key role in mediating metal-induced damage. However, to date the evidence is inconclusive and the key cellular mechanism(s) underlying metal toxicology remains undefined. The limitations imposed on studies of this nature by the use of animal models, will be circumvented in this proposal by investigating cellular metal-effects in the yeast Saccharomyces cerevisiae. The completion of the yeast genome sequence has underscored this organism as the eukaryotic model of choice, and our preliminary data using S. cerevisiae have highlighted its applicability to the studies proposed here. The objective of this research proposal is to test the hypothesis that cellular heavy metal toxicity is primarily attributable to oxidative mechanisms. To achieve this, we will employ two principal strategies, each of which is distinct from previous approaches to this problem. The work will focus mainly on copper and cadmium, as examples of redox-active and -inactive toxic metals, respectively. Both metals have the potential to promote oxidative damage. First, we will use a flow cytometric procedure to answer a novel question: do proteins involved in protection against oxidative stress determine differential metal sensitivity among cells within isogenic populations? The strategy proposed diverges markedly from previous approaches in which the role of various gene-products has been estimated by comparison of non-isogenic manipulated strains. For the first time, our approach will provide an answer that pertains to a truly in vivo situation, i.e. using intact cells in which the expression of genes-of-interest has not been manipulated artificially. Second, we will directly test the dependence of metal-induced cellular damage on metal-induced oxidation. We will use non-metallic oxidants to determine the gross macromolecular damage incurred by specific levels of oxidative damage, and test whether the derived relationships correspond to those evident during cellular metal-exposure. We will also construct strains mutated for and overexpressing oxidative-damage repair systems. The specificity of the selected systems for particular macromolecules and for oxidative damage has not been exploited previously in this context. We will determine the degree to which alterations in the strains' susceptibilities to oxidative damage are matched by alterations in their susceptibilities to metal-induced damage. By testing the hypothesis in the manners described, the proposed studies will significantly advance our understanding of metal toxicology at the cellular level, and will concurrently provide greater insight into the impact of ROS on biological systems.

重金属与一系列退化性疾病有关，对人类健康构成严重威胁。越来越多的证据表明，活性氧 (ROS) 在介导金属引起的损伤中发挥着关键作用。然而，迄今为止，证据尚无定论，金属毒理学的关键细胞机制仍未明确。本提案将通过研究酿酒酵母中的细胞金属效应来规避使用动物模型对这种性质的研究施加的限制。酵母基因组序列的完成强调了这种生物体作为选择的真核模型，并且我们使用酿酒酵母的初步数据强调了它对此处提出的研究的适用性。本研究计划的目的是检验细胞重金属毒性主要归因于氧化机制的假设。为了实现这一目标，我们将采用两种主要策略，每种策略都与以前解决此问题的方法不同。这项工作将主要集中在铜和镉上，分别作为氧化还原活性和非活性有毒金属的例子。这两种金属都有可能促进氧化损伤。首先，我们将使用流式细胞术来回答一个新问题：参与氧化应激保护的蛋白质是否决定同基因群体内细胞之间金属敏感性的差异？所提出的策略与以前的方法明显不同，在以前的方法中，通过比较非同基因操作菌株来估计各种基因产物的作用。我们的方法将首次提供与真正的体内情况有关的答案，即使用完整的细胞，其中感兴趣的基因的表达未被人为操纵。其次，我们将直接测试金属诱导的细胞损伤对金属诱导的氧化的依赖性。我们将使用非金属氧化剂来确定特定水平的氧化损伤所引起的总体大分子损伤，并测试推导的关系是否与细胞金属暴露期间明显的关系相对应。我们还将构建针对氧化损伤修复系统进行突变并过度表达的菌株。所选系统对特定大分子和氧化损伤的特异性此前尚未在这方面得到利用。我们将确定菌株对氧化损伤的敏感性的变化与它们对金属引起的损伤的敏感性的变化相匹配的程度。通过以所描述的方式检验假设，拟议的研究将显着增进我们对细胞水平金属毒理学的理解，同时将更深入地了解 ROS 对生物系统的影响。