Stability of Epiderm-200 XME expression profiles in culture.

<p>Epiderm-200 cultures were derived from either donor 254 or 1188 and were maintained for up to 3 days. The relative amount of each protein is indicated by the number of different tryptic peptides specific to each protein or protein family that were detected with adjacent bars representing results from 0, 1, 2 and 3 days, respectively. Details of the protein accession numbers and their subcellular location are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041721#pone.0041721.s003" target="_blank">Table S2</a>. Shading indicates different enzyme classes: oxidoreductase (black), transferase (red), antioxidant (green), and other (blue).</p

Detection of CYP proteins in skin and liver microsomal fraction by LC-MS/MS.

<p>Samples of skin microsomal fraction were spiked with a range of quantities of either recombinant CYP1A1 (expressed in lymphoblast cells) or with human liver microsomal fraction that contains known amounts of CYP1A2, CYP2E1, CYP3A4, and CYP3A5. The normal proteomics workflow was followed to identify peptides corresponding to the CYP proteins. Limits of detection based on the use of at least 2 tryptic peptides were established and based on these values the minimum level detectable by this technique was calculated for skin and compared with the mean level measured in liver. From these values the minimum comparative level in skin was calculated. CYP1A1 was not detected in either skin or liver making any comparison redundant (n/a; not applicable).</p

Analysis of CYP expression in skin by immunoblotting.

<p>Samples of human whole skin microsomal fraction (75 µg) prepared from 5 donors were separated by SDS-PAGE, transferred to nitrocellulose filters and the presence of CYP1A1, CYP1A2, CYP2E1 and CYP3A4 detected using antibodies specific to each form. The lane on the left hand side contained either 25 µg lymphoblast cell microsomes containing recombinant human CYP1A1 (∼2 pmol), or a sample of human liver loaded with 25, 35 or 5 µg microsomal fraction for detection of CYP1A2, CYP2E1, or CYP3A4, respectively. Immunoreactive bands were developed using goat anti-rabbit-horseradish peroxidase and ECL detection.</p

Flow diagram summarizing the methodology.

<p>Flow diagram summarizing the methodology.</p

Comparison of XME profiles from <i>in vitro</i> skin models and whole skin.

<p>The relative amount of each protein is represented by the number of different tryptic peptides specific to each protein or protein family that were detected. Details of the protein accession numbers and their subcellular location are shown in Table S1. Shading indicates different enzyme classes: oxidoreductase (black), hydrolase (magenta), transferase (red), antioxidant (green), and other (blue).</p

XMEs detected in whole skin. Protein identification was based on the presence of ≥2 different tryptic peptides in at least two donors.

<p>The proteins identified have been classified into functional groups as indicated. The corresponding NCBI numbers are indicated for each protein and for all members of groups of related proteins. The sub-cellular fraction in which each protein was principally detected is shown. The proportion of donor samples (skin n = 10, liver n = 5) in which each protein was identified is indicated. Fold difference was calculated by summing the intensity values of all detected peptides for a protein and comparing the values obtained for skin and liver. Where no peptides were detected, an intensity value equivalent to the limit of detection was used. Statistical significance was assessed using the Mann-Whitney U test.</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

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

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