Examining Mechanism of Toxicity of Copper Oxide Nanoparticles to Saccharomyces Cerevisiae and Caenorhabditis Elegans

Copper oxide nanoparticles (CuO NPs) are an up and coming technology increasingly being used in industrial and consumer applications and thus may pose risk to humans and the environment. In the present study, the toxic effects of CuO NPs were studied with two model organisms Saccharomyces cerevisiae and Caenorhabditis elegans. The role of released Cu ions during dissolution of CuO NPs in growth media were studied with freshly suspended, aged NPs, and the released Cu2+ fraction. Exposures to the different Cu treatments showed significant inhibition of S. cerevisiae cellular metabolic activity. Inhibition from the NPs was inversely proportional to size and was not fully explained by the released Cu ions. S. cerevisiae cultures grown under respiring conditions demonstrated greater metabolic sensitivity when exposed to CuO NPs compared to cultures undergoing fermentation. The cellular response to both CuO NPs and released Cu ions on gene expression was analyzed via microarray analysis after an acute exposure. It was observed that both copper exposures resulted in an increase in carbohydrate storage, a decrease in protein production, protein misfolding, increased membrane permeability, and cell cycle arrest. Cells exposed to NPs up-regulated genes related to oxidative phosphorylation but also may be inducing cell cycle arrest by a different mechanism than that observed with released Cu ions. The effect of CuO NPs on C. elegans was examined by using several toxicological endpoints. The CuO NPs displayed a more inhibitory effect, compared to copper sulfate, on nematode reproduction, feeding, and development. We investigated the effects of copper oxide nanoparticles and copper sulfate on neuronal health, a known tissue vulnerable to heavy metal toxicity. In transgenic C. elegans with neurons expressing a green fluorescent protein reporter, neuronal degeneration was observed in up to 10% of the population after copper oxide nanoparticle exposure. Additionally, nematode mutant strains containing gene knockouts in the divalent-metal transporters smf-1 and smf-2 showed increased tolerance to copper exposure. These results lend credence to the hypothesis that some toxicological effects to eukaryotic organisms from copper oxide nanoparticle exposure may be due to properties specific to the nanoparticles and not solely from the released copper ions

Decoding Cytochrome C Oxidase Biogenesis: New Insights Into Copper Trafficking

Acquisition, delivery and incorporation of metals to their respective metalloproteins are important cellular processes. These processes are tightly controlled so that cells are not exposed to free metal concentrations that would lead to harmful oxidative damages. Cytochrome c oxidases (Cox) are among these metalloproteins whose assembly and activity involves incorporation of Cu cofactor into their catalytic subunits in addition to the maturation of other subunits. In this study, we focused on the pathways of acquisition of Cu by the facultative phototroph Rhodobacter capsulatus for incorporation into the heme–Cu binuclear center of its cbb3–type Cox (cbb3–Cox). Genetic screens identified a cbb3–Cox defective mutant that requires Cu2+ supplement to produce an active cbb3–Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb3–Cox biogenesis in R. capsulatus. In the absence of CcoA, cellular content of Cu decreases, and cbb3–Cox assembly and activity becomes defective. CcoA shows pronounced homology to Major Facilitator Superfamily (MFS) type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein was implicated in cbb3–Cox biogenesis. In order to dissect the mechanism of Cu acquisition in the absence of CcoA, we isolated ΔccoA mutants that were cbb3–Cox defective after addition of Cu. Characterization of these mutants by genetic complementations revealed mutations in cytochrome c maturation (CCM) genes. These mutants were able to grow photosynthetically on the contrary to the usual phenotype of CCM genes deletion mutants. Here we show that these mutations are not directly involved in the Cu trafficking to CcoN but involved in the production of membrane bound cytochrome c subunits of cbb3–Cox. Although this study provides additional information about CCM system in R. capsulatus, the additional pathways of Cu acquisition to cbb3–Cox in the presence of exogenous Cu still remains to be identified. In the future, determination of ccoA bypass mutations will provide novel insights on the maturation and assembly of membrane-integral metalloproteins, and on hitherto unknown function(s) of MFS type transporters in bacterial Cu acquisition

Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway

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