Silver nanoparticles induce neurotoxicity in a human embryonic stem cell-derived neuron and astrocyte network

<p>Silver nanoparticles (AgNPs) are among the most extensively used nanoparticles and are found in a variety of products. This ubiquity leads to inevitable exposure to these particles in everyday life. However, the effects of AgNPs on neuron and astrocyte networks are still largely unknown. In this study, we used neurons and astrocytes derived from human embryonic stem cells as a cellular model to study the neurotoxicity that is induced by citrate-coated AgNPs (AgSCs). Immunostaining with the astrocyte and neuron markers, glial fibrillary acidic protein and microtubule-associated protein-2 (MAP2), respectively, showed that exposure to AgSCs at the concentration of 0.1 µg/mL increased the astrocyte/neuron ratio. In contrast, a higher concentration of AgSCs (5.0 µg/ml) significantly changed the morphology of astrocytes. These results suggest that astrocytes are sensitive to AgSC exposure and that low concentrations of AgSCs promote astrogenesis. Furthermore, our results showed that AgSCs reduced neurite outgrowth, decreased the expression of postsynaptic density protein 95 and synaptophysin, and induced neurodegeneration in a concentration-dependent manner. Our findings additionally suggest that the expression and phosphorylation status of MAP2 isoforms, as modulated by the activation of the Akt/glycogen synthase kinase-3/caspase-3 signaling pathway, may play an important role in AgSC-mediated neurotoxicity. We also found that AgNO₃ exposure only slightly reduced neurite outgrowth and had little effect on MAP2 expression, suggesting that AgSCs and AgNO₃ have different neuronal toxicity mechanisms. In addition, most of these effects were reduced when the cell culture was co-treated with AgSCs and the antioxidant ascorbic acid, which implies that oxidative stress is the major cause of AgSC-mediated astrocytic/neuronal toxicity and that antioxidants may have a neuroprotective effect.</p

Upon Exposure to Cu Nanoparticles, Accumulation of Copper in the Isopod <i>Porcellio scaber</i> Is Due to the Dissolved Cu Ions Inside the Digestive Tract

The fate of nanoparticles in organisms is of significant interest. In the current work, we used a test system with terrestrial isopods (<i>Porcellio scaber</i>) fed with food spiked with Cu NPs or soluble Cu salt for 14 days. Two different doses were used for spiking to yield final concentrations of 2000 and 5000 μg Cu/g dry food. After the exposure period, part of the exposed group of animals was transferred to clean food to depurate. Cu content was analyzed in the digestive glands, gut, and the ‘rest’ of the body. Similar patterns of (i) assimilated and depurated amounts of Cu, (ii) Cu body distribution, and (iii) effect on isopods feeding behavior were observed regardless of whether the animals were fed with Cu NPs or soluble Cu salt spiked food. Thus, Cu ions and not Cu NPs were assimilated by the digestive gland cells. Solubilization of the Cu NPs applied to the leaves was also analyzed with chemical methods and recombinant Cu-sensing bacteria. The comparison of the in vitro data on solubilization of Cu NPs and in vivo data on Cu accumulation in the animal tissues showed that about 99% of accumulated copper ions was dissolved from ingested Cu NPs in the digestive system of isopods

Vesicle measurements from three experiments; the experimental parameters for each experiment (time is the duration of incubation at recording; the number of chambers is the same as the number of samples).

<p>Relative standard deviation (RSD) measures the variation between chambers of the same populations. Each population had multiple chambers; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113405#pone-0113405-t001" target="_blank">Table 1</a> contains the mean vesicle quantity per chamber mean vesicle diameter and mean vesicle isoperimetric quotient (IPQ).</p><p>Vesicle measurements from three experiments; the experimental parameters for each experiment (time is the duration of incubation at recording; the number of chambers is the same as the number of samples).</p

Histograms of vesicle diameter size distribution (a) and vesicle isoperimetric quotient (b).

<p>Data samples for both histograms were taken from one randomly selected sample and their shapes are representative of all samples.</p

Micrographs of two vesicle populations with different vesicle densities in solution.

<p>Vesicle abundance is higher in (a) than in (b). The lower abundance of vesicles (b) is preferred for better performance of the computer vision algorithms.</p

Harmful at non-cytotoxic concentrations: SiO₂-SPIONs affect surfactant metabolism and lamellar body biogenesis in A549 human alveolar epithelial cells

<p>The pulmonary delivery of nanoparticles (NPs) is a promising approach in nanomedicine. For the efficient and safe use of inhalable NPs, understanding of NP interference with lung surfactant metabolism is needed. Lung surfactant is predominantly a phospholipid substance, synthesized in alveolar type II cells (ATII), where it is packed in special organelles, lamellar bodies (LBs). <i>In vitro</i> and <i>in vivo</i> studies have reported NPs impact on surfactant homeostasis, but this phenomenon has not yet been sufficiently examined. We showed that in ATII-like A549 human lung cancer cells, silica-coated superparamagnetic iron oxide NPs (SiO₂-SPIONs), which have a high potential in medicine, caused an increased cellular amount of acid organelles and phospholipids. In SiO₂-SPION treated cells, we observed elevated cellular quantity of multivesicular bodies (MVBs), organelles involved in LB biogenesis. In spite of the results indicating increased surfactant production, the cellular quantity of LBs was surprisingly diminished and the majority of the remaining LBs were filled with SiO₂-SPIONs. Additionally, LBs were detected inside abundant autophagic vacuoles (AVs) and obviously destined for degradation. We also observed time- and dose-dependent changes in mRNA expression for proteins involved in lipid metabolism. Our results demonstrate that non-cytotoxic concentrations of SiO₂-SPIONs interfere with surfactant metabolism and LB biogenesis, leading to disturbed ability to reduce hypophase surface tension. To ensure the safe use of NPs for pulmonary delivery, we propose that potential NP interference with LB biogenesis is obligatorily taken into account.</p

Changes in samples from different vesicle populations over time (from 3 to 60 minutes).

<p>Each experiment was conducted using a new initial vesicle population and each sample point represents a single chamber. Vesicle quantities (a), mean projected diameter sizes (b), and mean isoperimetric quotients (c). The box plot consists of mean minimal and maximal values 25th 50th and 75th percentile.</p

Light micrograph of vesicles in a micrograph (a), vesicles with overlaid segmentation (b), mask with segmented vesicles separated from the background in which each vesicle is marked with a number (c).

<p>Light micrograph of vesicles in a micrograph (a), vesicles with overlaid segmentation (b), mask with segmented vesicles separated from the background in which each vesicle is marked with a number (c).</p

Scheme of the perfusion chambers (a).

<p>Silicone model with four perfusion chambers named C1–C4 (b). The side view is given in the figure and the depth of the chambers is 0.5 mm. A single chamber with two tracks (P1 and P2) locations of two tracks recorded in each chamber (c). The two tracks together cover approximately 3% of the chamber area.</p

Effect of Ingested Tungsten Oxide (WO_<i>x</i>) Nanofibers on Digestive Gland Tissue of Porcellio scaber (Isopoda, Crustacea): Fourier Transform Infrared (FTIR) Imaging

Tungsten nanofibers are recognized as biologically potent. We study deviations in molecular composition between normal and digestive gland tissue of WO_<i>x</i> nanofibers (nano-WO_<i>x</i>) fed invertebrate Porcellio scaber (Iosopda, Crustacea) and revealed mechanisms of nano-WO_<i>x</i> effect <i>in vivo</i>. Fourier Transform Infrared (FTIR) imaging performed on digestive gland epithelium was supplemented by toxicity and cytotoxicity analyses as well as scanning electron microscopy (SEM) of the surface of the epithelium. The difference in the spectra of the Nano-WO_<i>x</i> treated and control cells showed up in the central region of the cells and were related to lipid peroxidation, and structural changes of nucleic acids. The conventional toxicity parameters failed to show toxic effects of nano-WO_<i>x</i>, whereas the cytotoxicity biomarkers and SEM investigation of digestive gland epithelium indicated sporadic effects of nanofibers. Since toxicological and cytological measurements did not highlight severe effects, the biochemical alterations evidenced by FTIR imaging have been explained as the result of cell protection (acclimation) mechanisms to unfavorable conditions and indication of a nonhomeostatic state, which can lead to toxic effects