Characterization of toxicological effects of complex nano-sized metal particles using in vitro human cell and whole blood model systems.

Metal oxide fumes form at high temperatures, for instance, during welding or firing ammunition. Inhalation exposure to high levels of airborne metal oxide particles can cause metal fume fever, cardiovascular effects, and lung damage in humans, but the associated underlying pathological mechanisms are still not fully understood. Using human alveolar epithelial cells, vascular endothelial cells, and whole blood model systems, we aimed to elucidate the short-term effects of well-characterized metal particles emitted while firing pistol ammunition. Human lung epithelial cells exposed to gunshot smoke particles (0.1-50 µg/ml) produced reactive oxygen species (ROS) and pro-inflammatory cytokines (interleukin 8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF)) that activate and recruit immune cells. Particles comprising high copper (Cu) and zinc (Zn) content activated human endothelial cells via a non-ROS-mediated mechanism that triggered immune activation (IL-8, GM-CSF), leukocyte adhesion to the endothelium (soluble intercellular adhesion molecule 1 (sICAM-1)), and secretion of regulators of the acute-phase protein synthesis (interleukin 6 (IL-6)). In human whole blood, metal oxides in gunshot smoke demonstrated intrinsic properties that activated platelets (release of soluble cluster of differentiation 40 ligand (sCD40L), platelet-derived growth factor B-chain homodimer(PDGF-BB), and vascular endothelial growth factor A (VEGF-A)) and blood coagulation and induced concomitant release of pro-inflammatory cytokines from blood leukocytes that further orchestrate thrombogenesis. The model systems applied provide useful tools for health risk assessment of particle exposures, but more studies are needed to further elucidate the mechanisms of metal fume fever and to evaluate the potential risk of long-term cardiovascular diseases.

In Situ Assembly of Choline Acetyltransferase Ligands by a Hydrothiolation Reaction Reveals Key Determinants for Inhibitor Design.

The potential drug target choline acetyltransferase (ChAT) catalyses the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells. Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction. This in situ synthesis yields an adduct that is the actual enzyme inhibitor. The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors. Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.

Polymorph- and size-dependent uptake and toxicity of TiO2 nanoparticles in living lung epithelial cells.

The cellular uptake and distribution of five types of well-characterized anatase and rutile TiO(2) nanoparticles (NPs) in A549 lung epithelial cells is reported. Static light scattering (SLS), in-vitro Raman microspectroscopy (µ-Raman) and transmission electron spectroscopy (TEM) reveal an intimate correlation between the intrinsic physicochemical properties of the NPs, particle agglomeration, and cellular NP uptake. It is shown that µ-Raman facilitates chemical-, polymorph-, and size-specific discrimination of endosomal-particle cell uptake and the retention of particles in the vicinity of organelles, including the cell nucleus, which quantitatively correlates with TEM and SLS data. Depth-profiling µ-Raman coupled with hyperspectral data analysis confirms the location of the NPs in the cells and shows that the NPs induce modifications of the biological matrix. NP uptake is found to be kinetically activated and strongly dependent on the hard agglomeration size-not the primary particle size-which quantitatively agrees with the measured intracellular oxidative stress. Pro-inflammatory responses are also found to be sensitive to primary particle size.

Amyloid fibrils prepared using an acetylated and methyl amidated peptide model of the α-Synuclein NAC 71-82 amino acid stretch contain an additional cross-ß structure also found in prion proteins.

The 71-82 fragment of the non-amyloid-ß component (NAC) region of the Parkinson's disease (PD) and dementia with Lewy bodies (DLB) related protein α-Synuclein, has been reported to be important during protein misfolding. Although reports have demonstrated the importance of this fragment for the aggregation properties of the full-length protein, its exact role in pre-fibrillar oligomerisation, fibrillar growth and morphology has not yet been fully elucidated. Here, we provide evidence that fibrils prepared from an acetylated and methyl amidated peptide of the NAC 71-82 amino acid stretch of α-Synuclein are amyloid and contain, in addition to the cross-ß structure detected in the full-length protein fibrils, a cross-ß structure previously observed in prion proteins. These results shed light on the aggregation propensity of the NAC 71-82 amino acid stretch of the full-length protein but also the roles of the N- and C-terminal domains of α-Synuclein in balancing this aggregation propensity. The results also suggest that early aggregated forms of the capped NAC 71-82 peptide generated structures were stabilised by an anti-parallel and twisted ß-sheet motif. Due to its expected toxicity, this ß-sheet motif may be a promising molecular target for the development of therapeutic strategies for PD and DLB.

Towards Fingermark Dating: A Raman Spectroscopy Proof-of-Concept Study.

Fingermarks have, for a long time, been vital in the forensic community for the identification of individuals, and a possibility to non-destructively date the fingermarks would of course be beneficial. Raman spectroscopy is, herein, evaluated for the purpose of estimating the age of fingermarks deposits. Well-resolved spectra were non-destructively acquired to reveal spectral uniqueness, resembling those of epidermis, and several molecular markers were identified that showed different decay kinetics: carotenoids > squalene > unsaturated fatty acids > proteins. The degradation rates were accelerated, less pronounced for proteins, when samples were stored under ambient light conditions, likely owing to photo-oxidation. It is hypothesized that fibrous proteins are present and that oxidation of amino acid side chains can be observed both through Raman and fluorescence spectroscopy. Clearly, Raman spectroscopy is a useful technique to non-destructively study the aging processes of fingermarks.

Field evaluation and calibration of a small axial passive air sampler for gaseous and particle bound polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs.

Polycyclic aromatic hydrocarbons (PAHs) and their oxygenated analogues (OPAHs) are ubiquitous air pollutants known to cause adverse health effects. PAH air levels are commonly monitored by active sampling but passive sampling has become popular because of its lower cost and simplicity, which facilitate long-term sampling and increased spatial coverage. However, passive samplers are less suitable for short-term sampling and are in general less accurate than active samplers because they require reliable sampling rate (Rs) measurements for individual analytes under diverse environmental conditions. In this study a small passive sampler designed to sample both particle-bound and gaseous compounds was evaluated and calibrated for PAHs and OPAHs in a traffic environment by co-deployment with active samplers for two weeks. Despite the relatively low average air concentrations of PM10 (20 µg/m(3)), PM2.5 (5 µg/m(3)), total PAHs (4.2 ng/m(3)), and OPAHs (2.3 ng/m(3)) at the site, detectable quantities (on average 24 times above blank values) of the full range of PAHs and OPAHs were captured, with low variability (average RSD of 16%). This was accomplished by using a Tenax(®) TA-modified glass fiber substrate that is compatible with highly sensitive thermal desorption GC-MS analysis, which made it possible to achieve detection limits per sample in the pg range. Experiments with inverted samplers revealed that the relative contribution of gravitational settling to the sampling of particles carrying PAHs and OPAHs was around 3.5 times larger than other deposition mechanisms. Average Rs values for individual OPAHs and PAHs were 0.046 ± 0.03 m(3)/day and 0.12 ± 0.07 m(3)/day, respectively, with no appreciable difference between the values for particle-associated and gaseous compounds. Furthermore, the Rs values were competitive with other currently used passive samplers if normalized for substrate area. Overall, the new sampler's performance, simplicity and ability to generate relatively time-resolved data make it a promising candidate for diverse SVOC monitoring studies.

Noise Removal with Maintained Spatial Resolution in Raman Images of Cells Exposed to Submicron Polystyrene Particles.

The biodistribution of 300 nm polystyrene particles in A549 lung epithelial cells has been studied with confocal Raman spectroscopy. This is a label-free method in which particles and cells can be imaged without using dyes or fluorescent labels. The main drawback with Raman imaging is the comparatively low spatial resolution, which is aggravated in heterogeneous systems such as biological samples, which in addition often require long measurement times because of their weak Raman signal. Long measurement times may however induce laser-induced damage. In this study we use a super-resolution algorithm with Tikhonov regularization, intended to improve the image quality without demanding an increased number of collected pixels. Images of cells exposed to polystyrene particles have been acquired with two different step lengths, i.e., the distance between pixels, and compared to each other and to corresponding images treated with the super-resolution algorithm. It is shown that the resolution after application of super-resolution algorithms is not significantly improved compared to the theoretical limit for optical microscopy. However, to reduce noise and artefacts in the hyperspectral Raman images while maintaining the spatial resolution, we show that it is advantageous to use short mapping step lengths and super-resolution algorithms with appropriate regularization. The proposed methodology should be generally applicable for Raman imaging of biological samples and other photo-sensitive samples.

Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B.

We have compared the cellular uptake and responses of five preparations of nanocrystalline titanium dioxide (TiO(2)) between normal human bronchial epithelial (NHBE) cells and epithelial cell lines (A549 and BEAS-2B). The P25 nanoparticles, containing both anatase and rutile modifications, induced reactive oxygen species (ROS) and secretion of the neutrophil chemoattractant IL-8 in all three cell types used. Pure anatase and rutile particles provoked differential IL-8 response in A549 and no response in BEAS-2B cells despite similar formation of ROS. The pure TiO(2) modifications also provoked release of the inflammatory mediators: IL-6, G-CSF and VEGF, in NHBE cells but not in the two cell lines. We conclude that the responsiveness of lung epithelial cells is strongly dependent on both the physicochemical properties of TiO(2) nanoparticles and the type of responder cells. The differential pro-inflammatory responsiveness of primary lung epithelial cells compared with immortalized cell lines should be considered in the assessment of adverse reactions to inhaled nanoparticles.