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.

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Toxicity of silver nanoparticles (Ag NPs) has been reported both in vitro and in vivo. However, the intracellular stability and chemical state of Ag NPs are still not very well studied. In this work, we systematically investigated the cellular uptake pathways, intracellular dissolution and chemical species, and cytotoxicity of Ag NPs (15.9 ± 7.6 nm) in Chinese hamster ovary cell subclone K1 cells, a cell line recommended by the OECD for genotoxicity studies. Quantification of intracellular nanoparticle uptake and ion release was performed through inductively coupled plasma mass spectrometry. X-ray absorption near-edge structure (XANES) was employed to assess the chemical state of intracellular silver. The toxic potential of Ag NPs and Ag(+) was evaluated by cell viability, reactive oxygen species (ROS) production and live-dead cell staining. The results suggest that cellular uptake of Ag NPs involves lipid-raft-mediated endocytosis and energy-independent diffusion. The degradation study shows that Ag NPs taken up into cells dissolved quickly and XANES results directly indicated that the internalized Ag was oxidized to Ag-O- species and then stabilized in silver-sulfur (Ag-S-) bonds within the cells. Subsequent cytotoxicity studies show that Ag NPs decrease cell viability and increase ROS production. Pre-incubation with N-acetyl-L-cysteine, an efficient antioxidant and Ag(+) chelator, diminished the cytotoxicity caused by Ag NPs or Ag(+) exposure. Our study suggests that the cytotoxicity mechanism of Ag NPs is related to the intracellular release of silver ions, followed by their binding to SH-groups, presumably coming from amino acids or proteins, and affecting protein functions and the antioxidant defense system of cells.

The toxic effects of silver nanoparticles (AgNPs) on cells are well established, but only limited studies on the effect of AgNPs and silver ions on the cellular transcriptome have been performed. In this study, the effect of AgNPs on the gene expression in the human lung epithelial cell line A549 exposed to 12.1 µg/ml AgNPs (EC20) for 24 and 48h was compared with the response to control and silver ion (Ag(+)) treated cells (1.3 µg/ml) using microarray analysis. Twenty-four hours to AgNP altered the regulation of more than 1000 genes (more than twofold regulation), whereas considerably fewer genes responded to Ag(+) (133 genes). The upregulated genes included members of the metallothionein, heat shock protein, and histone families. As expected from the induction of meta l lothionein and heat shock protein genes, Ag(+) and AgNP treatment resulted in intracellular production of reactive oxygen species but did not induce apoptosis or necrosis at the concentrations used in this study. In addition, the exposure to AgNPs influenced the cell cycle and led to an arrest in the G2/M phase as shown by cell cycle studies by flow cytometry and microscopy. In conclusion, although the transcriptional response to Ag(+) exposure was highly related to the response caused by AgNPs, our findings suggest that AgNPs, due to their particulate form, affect exposed cells in a more complex way.