Differentially transcriptional regulation on cell cycle pathway by silver nanoparticles from ionic silver in larval zebrafish (Danio rerio).

Silver nanoparticles (AgNPs) have a strong antibacterial activity and the relevant modes of actions have regarded as direct or indirect causes of toxicity observed in the environment. In this study, the transcriptomic profiles in larval zebrafish (Danio rerio) exposed to AgNPs (about 50 nm in size) and AgNO3 as a comparative ionic silver were investigated and analyzed using differential expressed gene (DEG), Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses. Results indicated that underlying molecular mechanisms are different each other. Interestingly, the global gene expression profiling showed that cell cycle pathway is affected by both AgNPs and dissolved Ag+, however its regulation pattern was opposite each other. To the best of our knowledge, the up-regulation of cell cycle pathway by AgNPs and down-regulation by Ag+ is the first reporting and suggests the distinguished toxicological perspective from a well-known hypothesis that Ag+ mainly regulates the cell cycle. This study provides novel insights onto the genotoxicological mechanisms of AgNPs.

Quantitatively profiling the dissolution and redistribution of silver nanoparticles in living rats using a knotted reactor-based differentiation scheme.

Whether silver nanoparticles (AgNPs) degrade and release silver ions (Ag(+)) in vivo has remained an unresolved issue. To evaluate the biodistribution and dissolution behavior of intravenously administered AgNPs in living rats, we employed a knotted reactor (KR) device to construct a differentiation scheme for quantitative assessment of residual AgNPs and their released Ag(+) ions in complicated animal tissues; to do so, we adjusted the operating parameters of the KR, namely, the presence/absence of a rinse solution and the sample acidity. After optimization, our proposed differentiation system was confirmed to be tolerant to rat tissue and organ matrix and provide superior reliability of differentiating AgNPs/Ag(+) than the conventional centrifugal filtration method. We then applied this differentiation strategy to investigate the biodistribution and dissolution of AgNPs in rats 1, 3, and 5 days postadministration, and it was found that the administered AgNPs accumulated predominantly in the liver and spleen, then dissolved and released Ag(+) ions that were gradually excreted, resulting in almost all of the Ag(+) ions becoming deposited in the kidney, lung, and brain. Histopathological data also indicated that toxic responses were specifically located in the AgNP-rich liver, not in the Ag(+)-dominated tissues and organs. Thus, the full-scale chemical fate of AgNPs in vivo should be integrated into future assessments of the environmental health effects and utilization of AgNP-containing products.

Evaluation of the effect of silver nanoparticles and silver ions using stress responsive gene expression in Chironomus riparius.

Silver nanoparticles (AgNPs) are extensively used in many commercial products because of their antimicrobial properties and they are therefore released into the environment from various products. A number of genes, especially those representing antioxidant and detoxification pathways, have potential application for studying mechanism of action of environmental pollutants at molecular level. In the present study, the stress responsive transcription of antioxidant and detoxification genes in response to AgNPs and Ag(+) ions exposure is studied in the ecotoxicologically important model species Chironomus riparius. The selected genes were superoxide dismutases (CuZnSOD and MnSOD), catalase (CAT), phospholipid hydroperoxide glutathione peroxidase 1 (PHGPx1), thioredoxin reductase 1 (TrxR1), and delta-3, sigma-4 and epsilon-1 classes of glutathione S-transferases (GSTs). The mRNA expression levels of each gene were determined after exposure of animals for 24h to three different AgNP and Ag(+) ion concentrations using Real-Time PCR method. Significant up-regulation of CuZnSOD and MnSOD was found after exposure to Ag(+) ions and AgNPs, respectively. The transcript levels of CAT, PHGPx1 and TrxR1 were significantly up-regulated only after exposure to AgNPs and no significant change was observed after exposure to Ag(+) ions. The expression levels of all the GSTs were more pronounced after exposure to AgNPs as compared to Ag(+) ions. The overall results suggest that AgNPs led to pronounced induction of genes related to oxidative stress and detoxification than Ag(+) ions.

Silver nanoparticles compromise neurodevelopment in PC12 cells: critical contributions of silver ion, particle size, coating, and composition.

BACKGROUND: Silver exposures are rising because of the increased use of silver nanoparticles (AgNPs) in consumer products. The monovalent silver ion (Ag+) impairs neurodevelopment in PC12 cells and zebrafish. OBJECTIVES AND METHODS: We compared the effects of AgNPs with Ag+ in PC12 cells for neurodevelopmental end points including cell replication, oxidative stress, cell viability, and differentiation. First, we compared citrate-coated AgNPs (AgNP-Cs) with Ag+, and then we assessed the roles of particle size, coating, and composition by comparing AgNP-C with two different sizes of polyvinylpyrrolidone-coated AgNPs (AgNP-PVPs) or silica nanoparticles. RESULTS: In undifferentiated cells, AgNP-C impaired DNA synthesis, but to a lesser extent than an equivalent nominal concentration of Ag+, whereas AgNP-C and Ag+ were equally effective against protein synthesis; there was little or no oxidative stress or loss of viability due to AgNP-C. In contrast, in differentiating cells, AgNP-C evoked robust oxidative stress and impaired differentiation into the acetylcholine phenotype. Although the effects of AgNP-PVP showed similarities to those of AgNP-C, we also found significant differences in potencies and differentiation outcomes that depended both on particle size and coating. None of the effects reflected simple physical attributes of nanoparticles, separate from composition or coating, as equivalent concentrations of silica nanoparticles had no detectable effects. CONCLUSIONS: AgNP exposure impairs neurodevelopment in PC12 cells. Further, AgNP effects are distinct from those of Ag+ alone and depend on size and coating, indicating that AgNP effects are not due simply to the release of Ag+ into the surrounding environment.

Biochemical and genetic analyses of the role of yeast casein kinase 2 in salt tolerance.

Saccharomyces cerevisiae cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the gene CKB1, display a phenotype of hypersensitivity to Na(+) and Li(+) cations. The sensitivity of a strain lacking ckb1 is higher than that of a calcineurin mutant and similar to that of a strain lacking HAL3, the regulatory subunit of the Ppz1 protein phosphatase. Genetic analysis indicated that Ckb1 participates in regulatory pathways different from that of Ppz1 or calcineurin. Deletion of CKB1 increased the salt sensitivity of a strain lacking Ena1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is not mediated by Ena1. Consistently, ckb1 mutants did not show an altered cation efflux. The function of Ckb1 was independent of the TRK system, which is responsible for discrimination of potassium and sodium entry, and in the absence of the kinase regulatory subunit, the influx of sodium was essentially normal. Therefore, the salt sensitivity of a ckb1 mutant cannot be attributed to defects in the fluxes of sodium. In fact, in these cells, both the intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-type cells. The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the light of these findings.

Activated calcineurin confers high tolerance to ion stress and alters the budding pattern and cell morphology of yeast cells.

The PP2B protein phosphatase, also known as calcineurin, is a regulator of ion homeostasis in yeast cells. We have investigated the physiological consequences of constitutive expression of a recombinant form of calcineurin in which the Ca2+/calmodulin-binding and autoinhibitory domains of the catalytic subunit were deleted. The concomitant expression of the regulatory subunit along with the truncated catalytic subunit resulted in high tolerance to toxic levels of Na+ and Li+. This activated form of calcineurin substituted for the Na+ stress signal to promote the expression of the ENA1 gene, encoding a P-ATPase pump, and to induce the transition of the K+ uptake system to the high affinity mode that restricts influx of Na+ and Li+. In addition, the transcriptional responsiveness of ENA1 to Na+ stress was enhanced. These results demonstrate that calcineurin has a pivotal role in a signaling cascade activated by ion stress in yeast. Moreover, we found that changes in the level of calcineurin activity affected budding pattern and cell morphology. Cells expressing the truncated calcineurin were elongated and budded in an unipolar pattern, whereas calcineurin-deficient mutants budded randomly. These results suggest that calcineurin may also act in the establishment of cell polarity.

Hydroxyl radical formation from cuprous ion and hydrogen peroxide: a spin-trapping study.

Copper toxicity has been presumed to involve catalytic hydroxyl radical (.OH) formation from hydrogen peroxide. Addition of Cu1+ to a solution containing ethanol or dimethylsulfoxide (Me2SO) and the spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) results in formation of the alpha-hydroxyethyl radical or methyl radical adduct of 4-POBN, respectively. Adduct formation was prevented by inclusion of catalase, but not by superoxide dismutase. Inclusion of exogenous H2O2 in the reaction mixture increased the yield of ethanol- or Me2SO-derived radical adduct and also enhanced the formation of secondary radical adducts, including 4-POBN/.H and the methyl radical adduct of 2-methyl-2-nitrosopropane. The alpha-hydroxyethyl adduct of 4-POBN is rapidly decomposed in the presence of copper, but not iron salts, whereas the methyl radical adduct is relatively stable in the presence of inorganic copper. The total concentration of radical adduct detected from the reaction between Cu1+ and H2O2, determined by comparison of the integrated spectral intensity with that of the stable 2,2,6,6-tetramethyl-1-piperidinyloxy free radical, was only 1-5% of the maximum amount predicted assuming radical adduct formation from all of the added copper. A variety of copper chelators inhibit formation of carbon-centered radical adducts of 4-POBN, including penicillamine and triethylenetetramine, which are the primary drugs used to treat the copper metabolism disorder Wilson's disease. The results provide clear evidence for hydroxyl radical formation from Cu1+ and H2O2 (either added or formed during the autoxidation of reduced copper.