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. ? ?
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