Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line, A549.

Nanomaterials, especially silver nanoparticles (Ag NPs), are used in a rapidly increasing number of commercial products. Accordingly, the hazards associated with human exposure to nanomaterials should be investigated to facilitate the risk assessment process. A potential route of exposure to NPs is through the respiratory system. In the present study, we investigated the effects of well-characterized PVP-coated Ag NPs and silver ions (Ag+) in the human, alveolar cell line, A549. Dose-dependent cellular toxicity caused by Ag NPs and Ag+ was demonstrated by the MTT and annexin V/propidium iodide assays, and evidence of Ag NP uptake could be measured indirectly by atomic absorption spectroscopy and flow cytometry. The cytotoxicity of both silver compounds was greatly decreased by pretreatment with the antioxidant, N-acetyl-cysteine, and a strong correlation between the levels of reactive oxygen species (ROS) and mitochondrial damage (r(s) = -0.8810; p = 0.0039) or early apoptosis (r(s) = 0.8857; p = 0.0188) was observed. DNA damage induced by ROS was detected as an increase in bulky DNA adducts by (32)P postlabeling after Ag NP exposure. The level of bulky DNA adducts was strongly correlated with the cellular ROS levels (r(s) = 0.8810, p = 0.0039) and could be inhibited by antioxidant pretreatment, suggesting Ag NPs as a mediator of ROS-induced genotoxicity.

Shell crosslinked nanoparticles carrying silver antimicrobials as therapeutics.

Amphiphilic polymer nanoparticles loaded with silver cations or/and N-heterocyclic carbene-silver complexes were assessed as antimicrobial agents against Gram-negative pathogens Escherichia coli and Pseudomonas aeruginosa.

Is Cl- protection against silver toxicity due to chemical speciation?

In freshwater teleosts, the primary mechanism of acute silver toxicity is inhibition of Na(+)/K(+) ATPase and carbonic anhydrase at the gill, leading to net Na(+) and Cl(-) loss due to the continued diffusion of these ions into the hypoosmotic external environment. External Cl(-) has been shown to protect rainbow trout (Oncorhychus mykiss) against silver toxicity presumably by complexation to form AgCl. However, Cl(-) does not appear to greatly influence silver toxicity to at least two other species, the European eel (Anguilla Anguilla) and the fathead minnow (Pimephales promelas). We hypothesized that differences in protective effects of Cl(-) at the gill were due to differing requirements or mechanisms for Cl(-) uptake among fish species. To test this hypothesis, we exposed Fundulus heteroclitus, which does not take up Cl(-) across the gills, and Danio rerio and P. promelas, which do rely on Cl(-) uptake across the gills, to Ag(+) in waters of varying Cl(-) concentration. The 96-h LC50s of F. heteroclitus exposed to Ag(+) in soft water with 10 microM Cl(-), 1mM KCl, and 0.5mM MgCl(2) were 3.88, 1.20, and 3.20 microg/L, respectively, and not significantly different. The 96-h LC50s for D. rerio exposed to Ag(+) in soft water with 10 microM Cl(-) and 1mM KCl were 10.3 and 11.3 microg/L, respectively and P. promelas exposed under the same conditions were 2.32 and 2.67 microg/L, respectively. Based on these results, increasing external Cl(-) concentration by as much as 1mM (35.5mg/L) did not offer protection against Ag(+) toxicity to any fish species tested. Although previous results in our laboratory have demonstrated that P. promelas do take up Cl(-) at the gill, a mechanism of uptake has not been identified. Additional experiments, investigating the mechanisms of Na(+) and Cl(-) influx at the gill of P. promelas and the influence of silver, demonstrated that Cl(-) uptake in P. promelas acclimated to soft water occurs through both a Na(+):K(+):2Cl(-) co-transporter and a Cl(-)/HCO(3)(-) exchanger, but is not dependent on carbonic anhydrase. Further, acclimation water chemistry was found to greatly influence subsequent branchial silver accumulation, but Cl(-) uptake was not sensitive to 10 microg/L Ag(+).

The protective effect of selenium inorganic forms against cadmium and silver toxicity in mycelia of Pleurotus ostreatus.

The effect of two inorganic selenium forms has been investigated in the mycelia of Pleurotus ostreatus exposed to cadmium and silver salts in the shaken cultures. The degree of toxicity was assessed by the determination of malondialdehyde (MDA; a common biomarker of lipid peroxidation). The mycelia were exposed to one element form (up to 5 mg l(-1)) and also to the following combinations: cadmium(II) + selenium(IV); cadmium(II) + selenium(VI); silver(I) + selenium(IV); silver(I) + selenium(VI). The concentrations of cadmium, silver, selenium, and MDA were assessed in the mixed cytosol and cell membrane fractions (CCM). A positive correlation between MDA and cadmium was found in the CCM (beta=0.7775, P=0.0001), whereas the effect of silver was less significant (beta=0.4642, P=0.039). These results indicate that silver(I) and cadmium(II) have different capacities to induce lipid peroxidation in P. ostreatus. The protective role of selenium against metal-induced oxidative damage was found to be dependent on the oxidation state of the element form in the growth medium. The strongest beneficial effect was observed in mycelia exposed to cadmium(II) + selenium(IV) (inverse correlation between MDA and selenium in the CCM: beta=-0.7129, P=0.009) and it has been ascribed to a lower incorporation of the toxic metal and/or to possible intracellular interaction between selenium and cadmium. Under exposure to silver(I), the protective effect of selenium(IV) was less noticeable (correlation between MDA and selenium in the CCM; beta=-0.6068, P=0.036); in the presence of selenium(VI), no beneficial effect was observed.

Heterogeneity of natural organic matter amelioration of silver toxicity to Daphnia magna: effect of source and equilibration time.

Despite the heterogeneity of natural organic matter (NOM) in the aquatic environment, current models that predict metal toxicity to aquatic biota treat these important metal-complexing agents in a homogeneous manner. In this investigation, the ability of 11 commercial and naturally isolated NOM samples to ameliorate silver toxicity to the freshwater crustacean Daphnia magna was examined. The commercially available Aldrich humic acid (AHA) increased the 48-h median lethal concentration for daphnid neonates from nominally NOM-free levels of 0.29 to 3.80 microg/L (at 6.9 mg C/L) in a concentration-dependent manner. Three of the tested samples exhibited similar protective effects, but the additional seven NOM samples displayed significantly stronger ameliorative actions. In fact, four samples of both commercial and naturally isolated origin demonstrated greater than fourfold increases in protection compared to that of AHA. Additional investigations showed that increased silver-AHA equilibration time resulted in decreased toxicity. Increased equilibration time also decreased whole-body silver accumulation at NOM levels less than 1 mg C/L. The present results suggest that heterogeneity of NOM and silver-NOM equilibration time will have to be accounted for in future models of silver toxicity to D. magna and that laboratory toxicity testing using NOM and metals should account for the effects of metal-NOM equilibration time.

Heterogeneity in physicochemical properties explains differences in silver toxicity amelioration by natural organic matter to Daphnia magna.

Recently collected data have shown that natural organic matter (NOM) source is an important parameter influencing the toxicity of silver to the freshwater crustacean Daphnia magna. The present study attempted to correlate the physicochemical properties of 11 naturally isolated and commercially available NOM sources with their ameliorative effects. The protection offered by these samples was standardized to the protective effect of Aldrich humic acid using geochemical modeling approaches that accounted for associated changes in water chemistry and, consequently, silver speciation. The protective ability of NOM was not correlated with reactive sulfide or nitrogen content, which are considered to be strong silver-binding ligands. Color (specific absorbance coefficient) was positively correlated with protection but narrowly eluded statistical significance. The peak wavelength of emission fluorescence following excitation at 370 nm and the fluorescence index values of NOM samples were significantly correlated with protective effects, suggesting that aromatic carbon content may govern the ameliorative actions of NOM. Simple optical properties may therefore act as a suitable indicator for the ability of a given NOM to protect against waterborne silver toxicity to D. magna as long as changes in water chemistry and, thus, silver speciation, are considered.

Evaluation of the protective effects of reactive sulfide on the acute toxicity of silver to rainbow trout (Oncorhynchus mykiss).

Acute 96-h toxicity tests were performed with juvenile rainbow trout (Oncorhynchus mykiss) exposed to AgNO3 in either the absence or the presence of 100 nM reactive sulfide to evaluate the protective effect of aqueous sulfides against ionic Ag toxicity. The sulfide was presented in the form of zinc sulfide (ZnS) clusters under oxic conditions. Silver was lost from the water column during the course of the experiment, so mean measured Ag concentrations were used to generate all median lethal concentration (LC50) data. The system was complicated in that Ag2S precipitated because of the need for large amounts of Ag to obtain lethal effects in the presence of ZnS. Some of the losses of Ag could be explained by complexation with ZnS and formation of solid Ag2S. Other losses were probably the result of partial adsorption to exposure-chamber walls or to complexation with ligands or functional groups within organic material produced by the fish. The LC50 (95% confidence interval) values generated using measured concentrations for total Ag were 139 (122-162) nM in the absence of sulfide and 377 (340-455) nM in the presence of 100 nM sulfide. The LC50 values generated using measured concentrations from filtered (pore size, 0.45 microm) water samples were 122 (105-145) nM in the absence of sulfide and 225 (192-239) nM in the presence of 100 nM sulfide. These results suggest a stoichiometric protection of sulfides up to a 2:1 ratio of Ag:sulfide. Greater accumulation of Ag at the gills was measured in fish exposed to AgNO3 in the presence of sulfide.

Gold ion inhibits silver ion induced contracture and activates ryanodine receptors in skeletal muscle.

Effects of Au3+ on Ag(+)-induced contractures and Ca2+ release channel activity in the sarcoplasmic reticulum were studied in frog skeletal muscles. Single fibres spontaneously produced phasic and tonic contractures upon addition of 5-20 microM Ag+ or more than 50 microM Au3+. Simultaneous application of 5 microM Ag+ and 20 microM Au3+ inhibited contractures induced by Ag+. Au3+ applied immediately after development of Ag(+)-induced contractures shortened the duration of the phasic contracture and markedly decreased the subsequent tonic contracture. Pretreatment of fibres with Au3+ inhibited the Ag(+)-induced phasic contracture. Ca2+ release channels incorporated into planar lipid bilayers were activated in response to Au3+ at 20 to 200 microM. A close relationship was observed between Ca2+ release channel open probability and amplitude of the Au(3+)-induced tonic contracture. Channel activity was inhibited by 5 microM ruthenium red. We conclude that extracellular Au3+ at low concentrations modifies the interaction of Ag+ with voltage sensors in the transverse tubules to inhibit the Ag(+)-induced contracture and, if it enters the cell, Au3+ may directly activate the sarcoplasmic reticulum Ca2+ release channel to partially contribute to the tonic contracture.

Glutathione inhibition of silver-enhanced sodium transport across toad skin.

Silver stimulated short-circuit current and transepithelial potential difference. Glutathione inhibited the silver-induced short-circuit current. There was a dose-response inhibition of silver-induced short-circuit current by glutathione. The silver-induced short-circuit current is carried by a net active sodium transfer from the outside to the inside bathing solution.

N-ethylmaleimide inhibition of silver-enhanced sodium transport across toad (Bufo macinus) skin.

1. Silver stimulated short-circuit current and transepithelial potential difference. 2. N-Ethylmaleimide inhibited the silver-induced short-circuit current. 3. There was a biphasic inhibition of silver-induced short-circuit current by N-ethylmaleimide. 4. There is a specific, maximal number of sulfhydryl groups associated with active sodium absorption. 5. Stimulation of active sodium transport by antidiuretic hormone was blunted by the presence of silver. 6. The silver-induced short-circuit current is carried by a net active sodium transfer from the outside to the inside bathing solution.

Effects of selenium on toxicity and ultrastructural localization of silver in cultured macrophages.

The effects of selenium on cellular toxicity and histochemical distribution of silver were examined in a cell culture system of mouse peritoneal macrophages. Selenium caused a significant delay in the appearance of coagulation necrosis induced by high silver concentrations and reduced the cytostatic effect of lower doses of silver when long-term toxicity was examined. Furthermore, selenium increased the amount of silver that could be visualized by autometallography. The additional silver made available for this histochemical demonstration was located in the cytosol as well as in lysosomes, the sole localization of silver when selenium was not administered.

A local anesthetic, tetracaine, similarly inhibits Ag+ and K+ contractures in frog skeletal muscle.

To evaluate usefulness of Ag+ contracture as a tool for elucidating the mechanism underlying the excitation-contraction coupling, the effects of tetracaine on Ag+ contracture were compared with those on K+ and caffeine contractures in frog skeletal muscle. Tetracaine less than 100 microM shortened the duration of 120 mM K+ contracture, without affecting tension amplitude. At higher concentrations of tetracaine, K+ contracture was inhibited dose-dependently and the duration shortened. Treatment of the fibers with 20-500 microM tetracaine for 3 min did not block the contracture induced by 25 mM caffeine. Effects of tetracaine on Ag+ contracture were similar to those on K+ contracture. In the presence of 200 microM tetracaine, 41% inhibition was observed in 120 mM K+ contracture, while 43% in 100 microM Ag+ contracture. Also, 200 microM tetracaine completely inhibited the contractures induced by 40 mM K+ or 5 microM Ag+. These findings suggest that the Ag+ may induce contractures via its action on the T/SR junction, not a direct action on the SR. Therefore, understanding the mechanism involved in the development of Ag+ contracture would be helpful to elucidate the mechanism of excitation-contraction coupling.