Immunoreactive levels of UCP2.

<p>Immunoblotting was performed on A549 cell homogenates following treatment with vehicle or nano-SiO₂ and developed with antibodies against (A) UCP2 or (B) actin. The relative intensity of the immunoreactive bands was determined by densitometry as described in the Materials and Methods.</p

Elucidation of Toxicity Pathways in Lung Epithelial Cells Induced by Silicon Dioxide Nanoparticles

<div><p>A study into the effects of amorphous nano-SiO₂ particles on A549 lung epithelial cells was undertaken using proteomics to understand the interactions that occur and the biological consequences of exposure of lung to nanoparticles. Suitable conditions for treatment, where A549 cells remained viable for the exposure period, were established by following changes in cell morphology, flow cytometry, and MTT reduction. Label-free proteomics was used to estimate the relative level of proteins from their component tryptic peptides detected by mass spectrometry. It was found that A549 cells tolerated treatment with 100 µg/ml nano-SiO₂ in the presence of 1.25% serum for at least 4 h. After this time detrimental changes in cell morphology, flow cytometry, and MTT reduction were evident. Proteomics performed after 4 h indicated changes in the expression of 47 proteins. Most of the proteins affected fell into four functional groups, indicating that the most prominent cellular changes were those that affected apoptosis regulation (<i>e.g.</i> UCP2 and calpain-12), structural reorganisation and regulation of actin cytoskeleton (<i>e.g.</i> PHACTR1), the unfolded protein response (<i>e.g.</i> HSP 90), and proteins involved in protein synthesis (<i>e.g.</i> ribosomal proteins). Treatment with just 10 µg/ml nano-SiO₂ particles in serum-free medium resulted in a rapid deterioration of the cells and in medium containing 10% serum the cells were resistant to up to 1000 µg/ml nano-SiO₂ particles, suggesting interaction of serum components with the nanoparticles. A variety of serum proteins were found which bound to nano-SiO₂ particles, the most prominent of which were albumin, apolipoprotein A-I, hemoglobin, vitronectin and fibronectin. The use of a proteomics platform, with appropriately designed experimental conditions, enabled the early biological perturbations induced by nano-SiO₂ in a model target cell system to be identified. The approach facilitates the design of more focused test systems for use in tiered evaluations of nanomaterials.</p></div

Volcano plot analysis showing the effect of nano-SiO₂ treatment on protein expression in A549 cells.

<p>For each protein detected the relative level of protein expression following treatment is depicted on the basis of both fold change and statistical difference. The main proteins of interest are those furthest from the origin, and these are indicated as open triangles.</p

Analysis of proteins that bind to nano-SiO₂ particles.

<p>Nano-SiO₂ particles were incubated with 1.25% serum only or 1.25% serum and A549 cells. The nanoparticles were recovered and washed by centrifugation and then bound proteins separated by SDS-PAGE which were stained with InstantBlue. For proteomic analysis the gel was cut into 11 horizontal slices based on the migration of proteins markers and the bands in the material eluted from the nanoparticles. Each slice was further divided between each of the protein lanes for analysis by proteomics as detailed in the Materials and Methods.</p

Typical morphology and flow cytometry of A549 cells.

<p>(A) Cells with a normal morphology as observed by light microscopy (magnification X250). Flow cytometry showed a relatively discrete distribution of fluorescent signals within the indicated area of interest. (B) Following treatment at effective doses a fraction of the cells took on a rounded appearance and became detached from the surface. Flow cytometry indicated a spread in the distribution of fluorescent signals outside the region of interest. (C). Later the cells became largely detached from the surface, started to clump together and lose their integrity; under such conditions very few cells were detected by flow cytometry.</p

Serum proteins that bind to nano-SiO₂ particles.

<p>Nano-SiO₂ particles were incubated with fetal bovine serum under the same conditions as those used to treat A549 cells. The nanoparticles were recovered and bound proteins identified by proteomics as described in Materials and Methods. The table lists the main proteins identified in each row (the 4 with the highest Sf scores; minimum score of 3.0) and the rows in which they were present. Underlined symbols indicate the row where the highest protein coverage was found. The MW equivalent to the centre of each row is indicated. The experiment was performed twice and also repeated in the presence of A549 cells. A similar result was found on each occasion, i.e. all proteins identified were bovine in origin and none could be attributed to a human source (i.e. A549 cells). MS details supporting the identification of the proteins are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072363#pone.0072363.s003" target="_blank">Table S2</a>.</p

Physico-chemical characteristics of the nano-SiO₂ preparation.

<p>(A) XRD analysis confirming the sample is amorphous silica. (B) FTIR spectrum indicating the presence of non-functionalised surfaces in the sample. (C) TEM image showing monodispersed particles with an average size of 25±2 nm. (D) DLS intensity weighed particle size distributions of nano-SiO₂ particles in water, cell culture medium containing 1.25% serum (in the absence of nano-SiO₂ particles), nano-SiO₂ particles in cell culture medium containing 1.25% serum immediately after dispersion (0 h) and then after incubation at 37°C for 4 h and 24 h. (E) Changes in the average particle size (estimated from DLS) of nano-SiO₂ dispersed in culture medium containing 1.25% serum at 37°C for up to 24 h.</p

Flow cytometric analysis of A549 cells following treatment with nano-SiO₂.

<p>A549 cells were treated for up to 24% serum (filled circles), 100 µg/ml nano-SiO₂ in medium containing 1.25% serum (solid squares), and 10 µg/ml nano-SiO₂ in serum-free medium (solid diamonds). At each time point the relative number of cells with fluorescent measurements in the region of interest (ROI) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072363#pone-0072363-g002" target="_blank">figure 2</a>) are shown.</p