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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057945-05
Application #
6636254
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Preusch, Peter C
Project Start
1999-05-01
Project End
2004-06-30
Budget Start
2003-05-01
Budget End
2004-06-30
Support Year
5
Fiscal Year
2003
Total Cost
$148,339
Indirect Cost
Name
Georgia State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
837322494
City
Atlanta
State
GA
Country
United States
Zip Code
30302
Alhebshi, Alawiah; Sideri, Theodora C; Holland, Sara L et al. (2012) The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species. Mol Biol Cell 23:3582-90
Holland, Sara L; Ghosh, Ekalabya; Avery, Simon V (2010) Chromate-induced sulfur starvation and mRNA mistranslation in yeast are linked in a common mechanism of Cr toxicity. Toxicol In Vitro 24:1764-7
Holland, Sara L; Avery, Simon V (2009) Actin-mediated endocytosis limits intracellular Cr accumulation and Cr toxicity during chromate stress. Toxicol Sci 111:437-46
Sideri, Theodora C; Willetts, Sylvia A; Avery, Simon V (2009) Methionine sulphoxide reductases protect iron-sulphur clusters from oxidative inactivation in yeast. Microbiology 155:612-23
Nargund, Amrita M; Avery, Simon V; Houghton, John E (2008) Cadmium induces a heterogeneous and caspase-dependent apoptotic response in Saccharomyces cerevisiae. Apoptosis 13:811-21
Payne, Tom; Hanfrey, Colin; Bishop, Amy L et al. (2008) Transcript-specific translational regulation in the unfolded protein response of Saccharomyces cerevisiae. FEBS Lett 582:503-9
Smith, Matthew C A; Sumner, Edward R; Avery, Simon V (2007) Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells. Mol Microbiol 66:699-712
Holland, Sara; Lodwig, Emma; Sideri, Theodora et al. (2007) Application of the comprehensive set of heterozygous yeast deletion mutants to elucidate the molecular basis of cellular chromium toxicity. Genome Biol 8:R268
Bishop, Amy L; Rab, Faiza A; Sumner, Edward R et al. (2007) Phenotypic heterogeneity can enhance rare-cell survival in 'stress-sensitive'yeast populations. Mol Microbiol 63:507-20
Sumner, Edward R; Shanmuganathan, Anupama; Sideri, Theodora C et al. (2005) Oxidative protein damage causes chromium toxicity in yeast. Microbiology 151:1939-48

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