Yuya Hayashi

Toxicity of silver nanoparticles—Nanoparticle or silver ion?

The toxicity of silver nanoparticles (AgNPs) has been shown in many publications. Here we investigated to which degree the silver ion fraction of AgNP suspensions, contribute to the toxicity of AgNPs in A549 lung cells. Cell viability assays revealed that AgNP suspensions were more toxic when the initial silver ion fraction was higher. At 1.5μg/ml total silver, A549 cells exposed to an AgNP suspension containing 39% silver ion fraction showed a cell viability of 92%, whereas cells exposed to an AgNP suspension containing 69% silver ion fraction had a cell viability of 54% as measured by the MTT assay. In addition, at initial silver ion fractions of 5.5% and above, AgNP-free supernatant had the same toxicity as AgNP suspensions. Flow-cytometric analyses of cell cycle and apoptosis confirmed that there is no significant difference between the treatment with AgNP suspension and AgNP supernatant. Only AgNP suspensions with silver ion fraction of 2.6% or less were significantly more toxic than their supernatant as measured by MTT assays. From our data we conclude that at high silver ion fractions (≥5.5%) the AgNPs did not add measurable additional toxicity to the AgNP suspension, whereas at low silver ion fractions (≤2.6%) AgNP suspensions are more toxic than their supernatant.

Global gene expression profiling of human lung epithelial cells after exposure to nanosilver

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.

Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.

Multi-platform genotoxicity analysis of silver nanoparticles in the model cell line CHO-K1

Investigation of the genotoxic potential of nanomaterials is essential to evaluate if they pose a cancer risk for exposed workers and consumers. The Chinese hamster ovary cell line CHO-K1 is recommended by the OECD for use in the micronucleus assay and is commonly used for genotoxicity testing. However, studies investigating if this cell line is suitable for the genotoxic evaluation of nanomaterials, including induction of DNA adduct and micronuclei formation, are rare and for silver nanoparticles (Ag NPs) missing. Therefore, we here systematically investigated DNA and chromosomal damage induced by BSA coated Ag NPs (15.9±7.6 nm) in CHO-K1 cells in relation to cellular uptake and intracellular localization, their effects on mitochondrial activity and production of reactive oxygen species (ROS), cell cycle, apoptosis and necrosis. Ag NPs are taken up by CHO-K1 cells and are presumably translocated into endosomes/lysosomes. Our cytotoxicity studies demonstrated a concentration-dependent decrease of mitochondrial activity and increase of intracellular reactive oxygen species (ROS) in CHO-K1 cells following exposure to Ag NPs and Ag⁺ (0-20 μg/ml) for 24h. Annexin V/propidium iodide assay showed that Ag NPs and Ag⁺ induced apoptosis and necrosis, which is in agreement with an increased fraction of cells in subG1 phase of the cell cycle. Genotoxicity studies showed that Ag NPs but also silver ions (Ag⁺) induced bulky-DNA adducts, 8-oxodG and micronuclei formation in a concentration-dependent manner, however, there were quantitative and qualitative differences between the particulate and ionic form of silver. Taken together, our multi-platform genotoxicity and cytotoxicity analysis demonstrates that CHO-K1 cells are suitable for the investigation of genotoxicity of nanoparticles like Ag NPs.

Species Differences Take Shape at Nanoparticles: Protein Corona Made of the Native Repertoire Assists Cellular Interaction

Cells recognize the biomolecular corona around a nanoparticle, but the biological identity of the complex may be considerably different among various species. This study explores the importance of protein corona composition for nanoparticle recognition by coelomocytes of the earthworm Eisenia fetida using E. fetida coelomic proteins (EfCP) as a native repertoire and fetal bovine serum (FBS) as a non-native reference. We have profiled proteins forming the long-lived corona around silver nanoparticles (75 nm OECD reference materials) and compared the responses of coelomocytes to protein coronas preformed of EfCP or FBS. We find that over time silver nanoparticles can competitively acquire a biological identity native to the cells in situ even in non-native media, and significantly greater cellular accumulation of the nanoparticles was observed with corona complexes preformed of EfCP (p < 0.05). An EfCP-nanoparticle mimicry made with a recombinant protein, lysenin, revealed its critical contribution in the observed cell-nanoparticle response. This confirms the determinant role of the recognizable biological identity during invertebrate in vitro testing of nanoparticles. Our finding shows a case of species-specific formation of biomolecular coronas, and this suggests that the use of representative species may need careful consideration in assessing the risks associated with nanoparticles.

Time-course profiling of molecular stress responses to silver nanoparticles in the earthworm Eisenia fetida.

The molecular mechanism of silver nanoparticle (AgNP) toxicity, particularly its temporal aspect, is currently limited in the literature. This study seeks to identify and profile changes in molecular response patterns over time during soil exposure of the earthworm Eisenia fetida to AgNPs (82±27 nm) with reference to dissolved silver salt (AgNO₃). Principal component analysis of selected gene and enzyme response profiles revealed dissimilar patterns between AgNO₃ and AgNP treatments and also over time. Despite the observed difference in molecular profiles, the body burdens of total Ag were within the same range (10-40 mg/kg dry weight worm) for both treatments with apparent correlation to the induction pattern of metallothionein. AgNO₃ induced the genes and enzymes related to oxidative stress at day 1, after which markers of energy metabolism were all suppressed at day 2. Exposure to AgNPs likewise led to induction of oxidative stress genes at day 2, but with a temporal pattern shift to immune genes at day 14 following metabolic upregulation at day 7. The involvement of oxidative stress and subsequent alterations in immune gene regulation were as predicted by our in vitro study reported previously, highlighting the importance of immunological endpoints in nanosilver toxicity.

Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

Abstract Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells’ protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.