Christiane Beer

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.

Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

ABSTRACT Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t ≈5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.

Fast intracellular dissolution and persistent cellular uptake of silver nanoparticles in CHO-K1 cells: implication for cytotoxicity

Abstract 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.

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.

Silver nanoparticles – wolves in sheep's clothing?

Silver nanoparticles (Ag NPs) are one of the most widely utilized engineered nanomaterials (ENMs) in commercial products due to their effective antibacterial activity, high electrical conductivity, and optical properties. Therefore, they have been one of the most intensively investigated nanomaterials in terms of their toxic effects on humans and the environment. It has become clear during recent years that nanomaterials can behave unexpectedly due to new and unique characteristics when their particle size reaches the nanoscale (1–100 nm). Consequently, their effect on human health and the environment has been hard to predict. Widespread applications increase the chances of public and environmental exposure to Ag NPs and have thereby increased concerns regarding the potential adverse effects of Ag NPs on human health and environmental safety. To fully understand and predict possible health effects following exposure to Ag NPs, information about the mechanisms for their cytotoxicity and genotoxicity is necessary. The present paper attempts to review the cellular and molecular mechanisms behind Ag NP toxicity. In addition, the role of silver ions in the toxicity of Ag NPs is discussed.

The temperature stability of mouse retroviruses depends on the cholesterol levels of viral lipid shell and cellular plasma membrane

To delineate parameters contributing to the extracellular lifetime of retroviral vectors, we carried out stability tests of retroviruses derived from cell lines of different origin and kept under different cultivation conditions. Results show that amphotropic mouse retroviruses (MLV-A) derived from human and hamster cells exhibit 2- to 3-fold higher half-lives compared to retroviruses from mouse cells. Cultivation at 32 degrees C has been reported to yield high virus titers. However, the benefit of virus production in mouse cells at 32 degrees C is controversial. In our hands the cultivation temperature affected, hitherto not noticed, the half-life time of MLV-A. The 37/32 degrees C shift resulted in a 3-fold decrease of viral half-lifes compared to MLV-A released from mouse cells at 37 degrees C. Thus, MLV-A released at 37 degrees C is phenotypically different from MLV-A synthesized at 32 degrees C. Increased virus stability was inversely correlated with the level of cholesterol in the viral membrane. Finally, depletion of viral cholesterol in vitro resulted in intact virus with increased thermal stability. Thus, retrovirus lability depends on the host cell and parallels the cholesterol amount in the viral lipid shell.

Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro

Proteins adsorbing at nanoparticles have been proposed as critical toxicity mediators and are included in ongoing efforts to develop predictive tools for safety assessment. Strongly attached proteins can be isolated, identified and correlated to changes in nanoparticle state, cellular association or toxicity. Weakly attached, rapidly exchanging proteins are also present at nanoparticles, but are difficult to isolate and have hardly been examined. Here we study rapidly exchanging proteins and show for the first time that they have a strong modulatory effect on the biotransformation of silver nanoparticles. Released silver ions, known for their role in particle toxicity, are found to be trapped as silver sulphide nanocrystals within the protein corona at silver nanoparticles in serum-containing cell culture media. The strongly attached corona acts as a site for sulphidation, while the weakly attached proteins reduce nanocrystal formation in a serum-concentration-dependent manner. Sulphidation results in decreased toxicity of Ag NPs.

Amphotropic murine leukaemia virus envelope protein is associated with cholesterol-rich microdomains

BackgroundCholesterol-rich microdomains like lipid rafts were recently identified as regions within the plasma membrane, which play an important role in the assembly and budding of different viruses, e.g., measles virus and human immunodeficiency virus. For these viruses association of newly synthesized viral proteins with lipid rafts has been shown.ResultsHere we provide evidence for the association of the envelope protein (Env) of the 4070A isolate of amphotropic murine leukaemia virus (A-MLV) with lipid rafts. Using density gradient centrifugation and immunocytochemical analyses, we show that Env co-localizes with cholesterol, ganglioside GM1 and caveolin-1 in these specific regions of the plasma membrane.ConclusionsThese results show that a large amount of A-MLV Env is associated with lipid rafts and suggest that cholesterol-rich microdomains are used as portals for the exit of A-MLV.

Subchronic toxicity and cardiovascular responses in spontaneously hypertensive rats after exposure to multiwalled carbon nanotubes by intratracheal instillation.

The tremendous demand of the market for carbon nanotubes has led to their massive production that presents an increasing risk through occupational exposure. Lung deposition of carbon nanotubes is known to cause acute localized pulmonary adverse effects. However, systemic cardiovascular damages associated with acute pulmonary lesion have not been thoroughly addressed. Four kinds of multiwalled carbon nanotubes (MWCNTs) with different lengths and/or iron contents were used to explore the potential subchronic toxicological effects in spontaneously hypertensive (SH) rats and normotensive control Wistar-Kyoto (WKY) rats after intratracheal instillation. MWCNTs penetrated the lung blood-gas barrier and accumulated in the liver, kidneys, and spleen but not in the heart and aorta of SH rats. The pulmonary toxicity and cardiovascular effects were assessed at 7 and 30 days postexposure. Compared to the WKY rats, transient influences on blood pressure and up to 30 days persistent decrease in the heart rate of SH rats were found by electrocardiogram monitoring. The subchronic toxicity, especially the sustained inflammation of the pulmonary and cardiovascular system, was revealed at days 7 and 30 in both SH and WKY rat models. Histopathological results showed obvious morphological lesions in abdominal arteries of SH rats 30 days after exposure. Our results suggest that more attention should be paid to the long-term toxic effects of MWCNTs, and particularly, occupationally exposed workers with preexisting cardiovascular diseases should be monitored more thoroughly.