Showing papers in "Particle and Fibre Toxicology in 2012"

Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

Abstract: The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells

Abstract: Carbon nanotubes (CNT) are high aspect ratio nanoparticles with diameters in the nanometre range but lengths extending up to hundreds of microns. The structural similarities between CNT and asbestos have raised concern that they may pose a similar inhalation hazard. Recently CNT have been shown to elicit a length-dependent, asbestos-like inflammatory response in the pleural cavity of mice, where long fibres caused inflammation but short fibres did not. However the cellular mechanisms governing this response have yet to be elucidated. This study examined the in vitro effects of a range of CNT for their ability to stimulate the release of the acute phase cytokines; IL-1β, TNFα, IL-6 and the chemokine, IL-8 from both Met5a mesothelial cells and THP-1 macrophages. Results showed that direct exposure to CNT resulted in significant cytokine release from the macrophages but not mesothelial cells. This pro-inflammatory response was length dependent but modest and was shown to be a result of frustrated phagocytosis. Furthermore the indirect actions of the CNT were examined by treating the mesothelial cells with conditioned media from CNT-treated macrophages. This resulted in a dramatic amplification of the cytokine release from the mesothelial cells, a response which could be attenuated by inhibition of phagocytosis during the initial macrophage CNT treatments. We therefore hypothesise that long fibres elicit an inflammatory response in the pleural cavity via frustrated phagocytosis in pleural macrophages. The activated macrophages then stimulate an amplified pro-inflammatory cytokine response from the adjacent pleural mesothelial cells. This mechanism for producing a pro-inflammatory environment in the pleural space exposed to long CNT has implications for the general understanding of fibre-related pleural disease and design of safe nanofibres.

Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver

Abstract: Background: Widespread occupational exposure to carbon black nanoparticles (CBNPs) raises concerns over their safety. CBNPs are genotoxic in vitro but less is known about their genotoxicity in various organs in vivo. Methods: We investigated inflammatory and acute phase responses, DNA strand breaks (SB) and oxidatively damaged DNA in C57BL/6 mice 1, 3 and 28 days after a single instillation of 0.018, 0.054 or 0.162 mg Printex 90 CBNPs, alongside sham controls. Bronchoalveolar lavage (BAL) fluid was analyzed for cellular composition. SB in BAL cells, whole lung and liver were assessed using the alkaline comet assay. Formamidopyrimidine DNA glycosylase (FPG) sensitive sites were assessed as an indicator of oxidatively damaged DNA. Pulmonary and hepatic acute phase response was evaluated by Saa3 mRNA real-time quantitative PCR. Results: Inflammation was strongest 1 and 3 days post-exposure, and remained elevated for the two highest doses (i.e., 0.054 and 0.162 mg) 28 days post-exposure (P < 0.001). SB were detected in lung at all doses on postexposure day 1 (P < 0.001) and remained elevated at the two highest doses until day 28 (P < 0.05). BAL cell DNA SB were elevated relative to controls at least at the highest dose on all post-exposure days (P < 0.05). The level of FPG sensitive sites in lung was increased throughout with significant increases occurring on post-exposure days 1 and 3, in comparison to controls (P < 0.001-0.05). SB in liver were detected on post-exposure days 1 (P < 0.001) and 28 (P < 0.001). Polymorphonuclear (PMN) cell counts in BAL correlated strongly with FPG sensitive sites in lung (r = 0.88, P < 0.001), whereas no such correlation was observed with SB (r = 0.52, P = 0.08). CBNP increased the expression of Saa3 mRNA in lung tissue on day 1 (all doses), 3 (all doses) and 28 (0.054 and 0.162 mg), but not in liver. Conclusions: Deposition of CBNPs in lung induces inflammatory and genotoxic effects in mouse lung that persist considerably after the initial exposure. Our results demonstrate that CBNPs may cause genotoxicity both in the primary exposed tissue, lung and BAL cells, and in a secondary tissue, the liver.

Mechanism of cellular uptake of genotoxic silica nanoparticles

Abstract: Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations ≥ 1 μg/ml, but DNA damage was evident at 0.1 μg/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 μg/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4°C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal microbalance experiments on tethered bilayer lipid membrane systems show that the nanoparticles strongly bind to lipid membranes, forming an adherent monolayer on the membrane. Leakage assays on large unilamellar vesicles (400 nm diameter) indicate that binding of the silica NPs transiently disrupts the vesicles which rapidly self-seal. We suggest that an adhesive interaction between silica nanoparticles and lipid membranes could cause passive cellular uptake of the particles.

Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos

Abstract: Carbon nanotubes (CNT) and carbon nanofibers (CNF) are allotropes of carbon featuring fibrous morphology. The dimensions and high aspect ratio of CNT and CNF have prompted the comparison with naturally occurring asbestos fibers which are known to be extremely pathogenic. While the toxicity and hazardous outcomes elicited by airborne exposure to single-walled CNT or asbestos have been widely reported, very limited data are currently available describing adverse effects of respirable CNF. Here, we assessed pulmonary inflammation, fibrosis, oxidative stress markers and systemic immune responses to respirable CNF in comparison to single-walled CNT (SWCNT) and asbestos. Pulmonary inflammatory and fibrogenic responses to CNF, SWCNT and asbestos varied depending upon the agglomeration state of the particles/fibers. Foci of granulomatous lesions and collagen deposition were associated with dense particle-like SWCNT agglomerates, while no granuloma formation was found following exposure to fiber-like CNF or asbestos. The average thickness of the alveolar connective tissue - a marker of interstitial fibrosis - was increased 28 days post SWCNT, CNF or asbestos exposure. Exposure to SWCNT, CNF or asbestos resulted in oxidative stress evidenced by accumulations of 4-HNE and carbonylated proteins in the lung tissues. Additionally, local inflammatory and fibrogenic responses were accompanied by modified systemic immunity, as documented by decreased proliferation of splenic T cells ex vivo on day 28 post exposure. The accuracies of assessments of effective surface area for asbestos, SWCNT and CNF (based on geometrical analysis of their agglomeration) versus estimates of mass dose and number of particles were compared as predictors of toxicological outcomes. We provide evidence that effective surface area along with mass dose rather than specific surface area or particle number are significantly correlated with toxicological responses to carbonaceous fibrous nanoparticles. Therefore, they could be useful dose metrics for risk assessment and management.

Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles

Abstract: Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5) collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon) which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM) from ambient air particles, human embryonic lung fibroblasts (HEL12469) were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls. For this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 μg EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina). The expression of selected genes was verified by quantitative real-time PCR. Dose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goeman's global test, varied, depending on the locality, between 12 to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair), the Wnt and TGF-β signaling pathways (associated particularly with tumor promotion and progression), Steroid hormone biosynthesis (involved in the endocrine-disrupting activity of chemicals), and Glycerolipid metabolism (pathways involving the lipids with a glycerol backbone including lipid signaling molecules). The microarray data suggested a prominent role of activation of aryl hydrocarbon receptor-dependent gene expression.

Uptake and cytotoxicity of citrate-coated gold nanospheres: Comparative studies on human endothelial and epithelial cells

Abstract: Background: The use of gold nanoparticles (AuNPs) for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles) was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11 nm, 25 nm), to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier. Results: Transmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentrationdependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10 nm, 11 nm, 25 nm), the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the nanoparticles’ surface. Conclusions: In conclusion the higher amount of citrate on the particle surface resulted in a higher impairment of cell viability, but did not enhance or reduce the uptake behavior in endothelial or epithelial cells. In addition, epithelial and endothelial cells exhibited different uptake behaviors for citrate-stabilized gold nanoparticles, which might be related to different interactions occurring at the nanoparticle-cell-surface interface. The different uptake in epithelial cells might explain the higher reduction of proliferation of these cells after exposure to AuNPs treatment although more detailed investigations are necessary to determine subcellular events. Nevertheless an extrinsic effect of sodium-citrate stabilized particles could not be excluded. Thus, the amount of sodium citrate should be reduced to a level on which the stability of the particles and the safety for biomedical applications are guaranteed.

Comparison of non-crystalline silica nanoparticles in IL-1β release from macrophages

Abstract: Background Respirable crystalline silica (silicon dioxide; SiO2, quartz) particles are known to induce chronic inflammation and lung disease upon long-term inhalation, whereas non-crystalline (amorphous) SiO2 particles in the submicrometre range are regarded as less harmful. Several reports have demonstrated that crystalline, but also non-crystalline silica particles induce IL-1β release from macrophages via the NALP3-inflammasome complex (caspase-1, ASC and NALP3) in the presence of lipopolysaccharide (LPS) from bacteria. Our aim was to study the potential of different non-crystalline SiO2 particles from the nano- to submicro-sized range to activate IL-1β responses in LPS-primed RAW264.7 macrophages and primary rat lung macrophages. The role of the NALP3-inflammasome and up-stream mechanisms was further explored in RAW264.7 cells.

Chemical constituents of ambient particulate air pollution and biomarkers of inflammation, coagulation and homocysteine in healthy adults: a prospective panel study.

Abstract: Ambient air pollution has been associated with activation of systemic inflammation and hypercoagulability and increased plasma homocysteine, but the chemical constituents behind the association are not well understood. We examined the relations of various chemical constituents of fine particles (PM2.5) and biomarkers of inflammation, coagulation and homocysteine in the context of traffic-related air pollution. A panel of 40 healthy college students underwent biweekly blood collection for 12 times before and after their relocation from a suburban campus to an urban campus with changing air pollution contents in Beijing. Blood samples were measured for circulatory biomarkers of high-sensitivity C reactive protein (hs-CRP), tumor necrosis factor alpha (TNF-α), fibrinogen, plasminogen activator inhibitor type 1 (PAI-1), tissue-type plasminogen activator (t-PA), von Willebrand factor (vWF), soluble platelet selectin (sP-selectin), and total homocysteine (tHcy). Various air pollutants were measured in a central air-monitoring station in each campus and 32 PM2.5 chemical constituents were determined in the laboratory. We used three different mixed-effects models (single-constituent model, constituent-PM2.5 joint model and constituent residual model) controlling for potential confounders to estimate the effects of PM2.5 chemical constituents on circulatory biomarkers. We found consistent positive associations between the following biomarkers and PM2.5 chemical constituents across different models: TNF-α with secondary organic carbon, chloride, zinc, molybdenum and stannum; fibrinogen with magnesium, iron, titanium, cobalt and cadmium; PAI-1 with titanium, cobalt and manganese; t-PA with cadmium and selenium; vWF with aluminum. We also found consistent inverse associations of vWF with nitrate, chloride and sodium, and sP-selectin with manganese. Two positive associations of zinc with TNF-α and of cobalt with fibrinogen, and two inverse associations of nitrate with vWF, and of manganese with sP-selectin, were independent of the other constituents in two-constituent models using constituent residual data. We only found weak air pollution effects on hs-CRP and tHcy. Our results provide clues for the potential roles that PM2.5 chemical constituents may play in the biological mechanisms through which air pollution may influence the cardiovascular system.

An in vitro liver model - assessing oxidative stress and genotoxicity following exposure of hepatocytes to a panel of engineered nanomaterials

Abstract: Background: Following exposure via inhalation, intratracheal instillation or ingestion some nanomaterials (NM) have been shown to translocate to the liver. Since oxidative stress has been implicated as a possible mechanism for NM toxicity this study aimed to investigate the effects of various materials (five titanium dioxide (TiO2), two zinc oxide (ZnO), two multi-walled carbon nanotubes (MWCNT) and one silver (Ag) NM) on oxidative responses of C3A cell line as a model for potential detrimental properties of nanomaterials on the liver. Results: We noted a dose dependant decrease in the cellular glutathione content following exposure of the C3A cells to Ag, the ZnO and the MWCNTs. Intracellular ROS levels were also measured and shown to increase significantly following exposure of the C3A to the low toxicity NMs (MWCNT and TiO2). The antioxidant Trolox in part prevented the detrimental effect of NMs on cell viability, and decreased the NM induced IL8 production after exposure to all but the Ag particulate. Following 4 hr exposure of the C3A cells to sub-lethal levels of the NMs, the largest amount of DNA damage was induced by two of the TiO2 samples (7 nm and the positively charged 10 nm particles). Conclusions: All ten NMs exhibited effects on the hepatocyte cell line that were at least in part ROS/oxidative stress mediated. These effects included mild genotoxicity and IL8 production for all NM except the Ag possibly due to its highly cytotoxic nature.

Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint

Abstract: Background Little is known of how the toxicity of nanoparticles is affected by the incorporation in complex matrices. We compared the toxic effects of the titanium dioxide nanoparticle UV-Titan L181 (NanoTiO2), pure or embedded in a paint matrix. We also compared the effects of the same paint with and without NanoTiO2.

Effects of particulate matter on inflammatory markers in the general adult population

Abstract: Particulate air pollution is associated with increased risk of cardiovascular disease and stroke. Although the precise mechanisms underlying this association are still unclear, the induction of systemic inflammation following particle inhalation represents a plausible mechanistic pathway. We used baseline data from the CoLaus Study including 6183 adult participants residing in Lausanne, Switzerland. We analyzed the association of short-term exposure to PM10 (on the day of examination visit) with continuous circulating serum levels of high-sensitive C-reactive protein (hs-CRP), interleukin 1-beta (IL-1β), interleukin 6 (IL-6), and tumor-necrosis-factor alpha (TNF-α) by robust linear regressions, controlling for potential confounding factors and assessing effect modification. In adjusted analyses, for every 10 μg/m3 elevation in PM10, IL-1s increased by 0.034 (95 % confidence interval, 0.007-0.060) pg/mL, IL-6 by 0.036 (0.015-0.057) pg/mL, and TNF-α by 0.024 (0.013-0.035) pg/mL, whereas no significant association was found with hs-CRP levels. Short-term exposure to PM10 was positively associated with higher levels of circulating IL-1s, IL-6 and TNF-α in the adult general population. This positive association suggests a link between air pollution and cardiovascular risk, although further studies are needed to clarify the mechanistic pathway linking PM10 to cardiovascular risk.

A short history of the toxicology of inhaled particles.

Abstract: Particle toxicology arose in order to understand the mechanisms of adverse effects of 3 major particle types that had historically exerted the greatest toll of ill-health—quartz, coal and asbestos. By the middle of the last century rat inhalation studies had been carried out and the pathology documented, but true mechanistic particle toxicology did not really take off until the 1970s when cell culture techniques became available. By the 1980s glass fibres were a major focus of interest and attempts to develop a structure-toxicity paradigm centred on biopersistence. In the 1990s environmental particles dominated the particle toxicology agenda and the cardiovascular system emerged as a target for inhaled particles, raising new challenges for particle toxicologists. We are currently in the era of nanotoxicology where a large and diverse range of new nanoparticles types are under scrutiny.

Contamination of nanoparticles by endotoxin: evaluation of different test methods

Abstract: Nanomaterials can be contaminated with endotoxin (lipopolysaccharides, LPS) during production or handling. In this study, we searched for a convenient in vitro method to evaluate endotoxin contamination in nanoparticle samples. We assessed the reliability of the commonly used limulus amebocyte lysate (LAL) assay and an alternative method based on toll-like receptor (TLR) 4 reporter cells when applied with particles (TiO2, Ag, CaCO3 and SiO2), or after extraction of the endotoxin as described in the ISO norm 29701. Our results indicate that the gel clot LAL assay is easily disturbed in the presence of nanoparticles; and that the endotoxin extraction protocol is not suitable at high particle concentrations. The chromogenic-based LAL endotoxin detection systems (chromogenic LAL assay and Endosafe-PTS), and the TLR4 reporter cells were not significantly perturbed. We demonstrated that nanoparticles can interfere with endotoxin detection systems indicating that a convenient test method must be chosen before assessing endotoxin contamination in nanoparticle samples.

Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo.

Abstract: Background Frustrated phagocytosis has been stated as an important factor in the initiation of an inflammatory response after fibre exposure. The length of fibrous structures has been linked to the potential of fibres to induce adverse health effects for at least 40 years. However, we only recently reported for the first time the threshold length for fibre-induced inflammation in the pleural space and we implicated frustrated phagocytosis in the pro-inflammatory effects of long fibres. This study extends the examination of the threshold value for frustrated phagocytosis using well-defined length classes of silver nanowires (AgNW) ranging from 3–28 μm and describes in detail the morphology of frustrated phagocytosis using a novel technique and also describes compartmentalisation of fibres in the pleural space.

Intestinal toxicity evaluation of TiO2 degraded surface-treated nanoparticles: a combined physico-chemical and toxicogenomics approach in caco-2 cells

Abstract: Titanium dioxide (TiO2) nanoparticles (NPs) are widely used due to their specific properties, like UV filters in sunscreen. In that particular case TiO2 NPs are surface modified to avoid photocatalytic effects. These surface-treated nanoparticles (STNPs) spread in the environment and might release NPs as degradation residues. Indeed, degradation by the environment (exposure to UV, water and air contact …) will occur and could profoundly alter the physicochemical properties of STNPs such as chemistry, size, shape, surface structure and dispersion that are important parameters for toxicity. Although the toxicity of surface unmodified TiO2 NPs has been documented, nothing was done about degraded TiO2 STNPs which are the most likely to be encountered in environment. The superoxide production by aged STNPs suspensions was tested and compared to surface unmodified TiO2 NPs. We investigated the possible toxicity of commercialized STNPs, degraded by environmental conditions, on human intestinal epithelial cells. STNPs sizes and shape were characterized and viability tests were performed on Caco-2 cells exposed to STNPs. The exposed cells were imaged with SEM and STNPs internalization was researched by TEM. Gene expression microarray analyses were performed to look for potential changes in cellular functions. The production of reactive oxygen species was detected with surface unmodified TiO2 NPs but not with STNPs or their residues. Through three different toxicity assays, the STNPs tested, which have a strong tendency to aggregate in complex media, showed no toxic effect in Caco-2 cells after exposures to STNPs up to 100 μg/mL over 4 h, 24 h and 72 h. The cell morphology remained intact, attested by SEM, and internalization of STNPs was not seen by TEM. Moreover gene expression analysis using pangenomic oligomicroarrays (4x 44000 genes) did not show any change versus unexposed cells after exposure to 10 μg/ mL, which is much higher than potential environmental concentrations. TiO2 STNPs, degraded or not, are not harmful to Caco-2 cells and are unlikely to penetrate the body via oral route. It is likely that the strong persistence of the aluminium hydroxide layer surrounding these nanoparticles protects the cells from a direct contact with the potentially phototoxic TiO2 core.

Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

Abstract: Background Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation.

Wood smoke particles from different combustion phases induce similar pro-inflammatory effects in a co-culture of monocyte and pneumocyte cell lines

Abstract: Background: Exposure to particulate matter (PM) has been linked to several adverse cardiopulmonary effects, probably via biological mechanisms involving inflammation. The pro-inflammatory potential of PM depends on the particles’ physical and chemical characteristics, which again depend on the emitting source. Wood combustion is a major source of ambient air pollution in Northern countries during the winter season. The overall aim of this study was therefore to investigate cellular responses to wood smoke particles (WSPs) collected from different phases of the combustion cycle, and from combustion at different temperatures. Results: WSPs from different phases of the combustion cycle induced very similar effects on pro-inflammatory mediator release, cytotoxicity and cell number, whereas WSPs from medium-temperature combustion were more cytotoxic than WSPs from high-temperature incomplete combustion. Furthermore, comparisons of effects induced by native WSPs with the corresponding organic extracts and washed particles revealed that the organic fraction was the most important determinant for the WSP-induced effects. However, the responses induced by the organic fraction could generally not be linked to the content of the measured polycyclic aromatic hydrocarbons (PAHs), suggesting that also other organic compounds were involved. Conclusion: The toxicity of WSPs seems to a large extent to be determined by stove type and combustion conditions, rather than the phase of the combustion cycle. Notably, this toxicity seems to strongly depend on the organic fraction, and it is probably associated with organic components other than the commonly measured unsubstituted PAHs.

Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity.

Abstract: Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded. CPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced. Despite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs.

Controlled human wood smoke exposure: oxidative stress, inflammation and microvascular function.

Abstract: Exposure to wood smoke is associated with respiratory symptoms, whereas knowledge on systemic effects is limited. We investigated effects on systemic inflammation, oxidative stress and microvascular function (MVF) after controlled wood smoke exposure. In a randomised, double-blinded, cross-over study 20 non-smoking atopic subjects were exposed at rest to 14, 220, or 354 μg/m3 of particles from a well-burning modern wood stove for 3 h in a climate controlled chamber with 2 week intervals. We investigated the level of oxidatively damaged DNA, inflammatory markers and adhesion molecules before and 0, 6 and 20 h after exposure. Six h after exposure we measured MVF non-invasively by digital peripheral artery tonometry following arm ischemia. The MVF score was unaltered after inhalation of clean air (1.58 ± 0.07; mean ± SEM), low (1.51 ± 0.07) or high (1.61 ± 0.09) concentrations of wood smoke particles in atopic subjects, whereas unexposed non-atopic subjects had higher score (1.91 ± 0.09). The level of oxidatively damaged DNA, mRNA of ITGAL, CCL2, TNF, IL6, IL8, HMOX1, and OGG1 and surface marker molecules ICAM1, ITGAL and L-selectin in peripheral blood mononuclear cells were not affected by inhalation of wood smoke particles. Exposure to wood smoke had no effect on markers of oxidative stress, DNA damage, cell adhesion, cytokines or MVF in atopic subjects.

Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP Kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages

Abstract: Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2). RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E2 (PGE2) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively. MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE2 and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction. This study identifies COX-2 and subsequent PGE2 production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.

Critical role of surface chemical modifications induced by length shortening on multi-walled carbon nanotubes-induced toxicity.

Abstract: Given the increasing use of carbon nanotubes (CNT) in composite materials and their possible expansion to new areas such as nanomedicine which will both lead to higher human exposure, a better understanding of their potential to cause adverse effects on human health is needed. Like other nanomaterials, the biological reactivity and toxicity of CNT were shown to depend on various physicochemical characteristics, and length has been suggested to play a critical role. We therefore designed a comprehensive study that aimed at comparing the effects on murine macrophages of two samples of multi-walled CNT (MWCNT) specifically synthesized following a similar production process (aerosol-assisted CVD), and used a soft ultrasonic treatment in water to modify the length of one of them. We showed that modification of the length of MWCNT leads, unavoidably, to accompanying structural (i.e. defects) and chemical (i.e. oxidation) modifications that affect both surface and residual catalyst iron nanoparticle content of CNT. The biological response of murine macrophages to the two different MWCNT samples was evaluated in terms of cell viability, pro-inflammatory cytokines secretion and oxidative stress. We showed that structural defects and oxidation both induced by the length reduction process are at least as responsible as the length reduction itself for the enhanced pro-inflammatory and pro-oxidative response observed with short (oxidized) compared to long (pristine) MWCNT. In conclusion, our results stress that surface properties should be considered, alongside the length, as essential parameters in CNT-induced inflammation, especially when dealing with a safe design of CNT, for application in nanomedicine for example.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro

Abstract: Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (−NH2) and carboxyl (−COOH) surface modifications. Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies.

Oxidative stress-induced telomeric erosion as a mechanism underlying airborne particulate matter-related cardiovascular disease.

Abstract: Particulate matter (PM) pollution is responsible for hundreds of thousands of deaths worldwide, the majority due to cardiovascular disease (CVD). While many potential pathophysiological mechanisms have been proposed, there is not yet a consensus as to which are most important in causing pollution-related morbidity/mortality. Nor is there consensus regarding which specific types of PM are most likely to affect public health in this regard. One toxicological mechanism linking exposure to airborne PM with CVD outcomes is oxidative stress, a contributor to the development of CVD risk factors including atherosclerosis. Recent work suggests that accelerated shortening of telomeres and, thus, early senescence of cells may be an important pathway by which oxidative stress may accelerate biological aging and the resultant development of age-related morbidity. This pathway may explain a significant proportion of PM-related adverse health outcomes, since shortened telomeres accelerate the progression of many diseases. There is limited but consistent evidence that vehicular emissions produce oxidative stress in humans. Given that oxidative stress is associated with accelerated erosion of telomeres, and that shortened telomeres are linked with acceleration of biological ageing and greater incidence of various age-related pathology, including CVD, it is hypothesized that associations noted between certain pollution types and sources and oxidative stress may reflect a mechanism by which these pollutants result in CVD-related morbidity and mortality, namely accelerated aging via enhanced erosion of telomeres. This paper reviews the literature providing links among oxidative stress, accelerated erosion of telomeres, CVD, and specific sources and types of air pollutants. If certain PM species/sources might be responsible for adverse health outcomes via the proposed mechanism, perhaps the pathway to reducing mortality/morbidity from PM would become clearer. Not only would pollution reduction imperatives be more focused, but interventions which could reduce oxidative stress would become all the more important.

Cytotoxicity and cellular uptake of tri-block copolymer nanoparticles with different size and surface characteristics.

Abstract: Polymer nanoparticles (PNP) are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce. Fluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm) and surface charges (positive and negative) were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP45 (45 nm) showed a higher cytotoxicity compared to the positive bigger PNP90 (90 nm) particles including reduction in mitochondrial membrane potential (ΔΨm), induction of reactive oxygen species (ROS) production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨm), uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM). Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP), caveolin (for negative PNP) and mannose receptors (for hydroxylated PNP) were found with smaller PNP45 showing stronger interactions with the receptors than bigger PNP90. The size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges) play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose) leading to cellular internalization were observed to depend on size and surface properties of the different PNP. These properties of the nanoparticles also dominate their cytotoxicity, which was analyzed for many factors. The effective reduction in the mitochondrial membrane potential (ΔΨm), uncoupling of the electron transfer chain in mitochondria and resulting ATP depletion, induction of ROS and oxidative stress likely all play a role in the mechanisms behind the cytotoxicity of these PNP.

Use of silver nanowires to determine thresholds for fibre length-dependent pulmonary inflammation and inhibition of macrophage migration in vitro

Abstract: Background The objective of this study was to examine the threshold fibre length for the onset of pulmonary inflammation after aspiration exposure in mice to four different lengths of silver nanowires (AgNW). We further examined the effect of fibre length on macrophage locomotion in an in vitro wound healing assay. We hypothesised that exposure to longer fibres causes both increased inflammation and restricted mobility leading to impaired clearance of long fibres from the lower respiratory tract to the mucociliary escalator in vivo.

Amorphous silica nanoparticles size-dependently aggravate atopic dermatitis-like skin lesions following an intradermal injection

Abstract: Due to the rising use of nanomaterials (NMs), there is concern that NMs induce undesirable biological effects because of their unique physicochemical properties. Recently, we reported that amorphous silica nanoparticles (nSPs), which are one of the most widely used NMs, can penetrate the skin barrier and induce various biological effects, including an immune-modulating effect. Thus, it should be clarified whether nSPs can be a risk factor for the aggravation of skin immune diseases. Thus, in this study, we investigated the relationship between the size of SPs and adjuvant activity using a model for atopic dermatitis. We investigated the effects of nSPs on the AD induced by intradermaly injected-mite antigen Dermatophagoides pteronyssinus (Dp) in NC/Nga mice. Ear thickness measurements and histopathological analysis revealed that a combined injection of amorphous silica particles (SPs) and Dp induced aggravation of AD in an SP size-dependent manner compared to that of Dp alone. In particular, aggravation was observed remarkably in nSP-injected groups. Furthermore, these effects were correlated with the excessive induction of total IgE and a stronger systemic Th2 response. We demonstrated that these results are associated with the induction of IL-18 and thymic stromal lymphopoietin (TSLP) in the skin lesions. A particle size reduction in silica particles enhanced IL-18 and TSLP production, which leads to systemic Th2 response and aggravation of AD-like skin lesions as induced by Dp antigen treatment. We believe that appropriate regulation of nanoparticle physicochemical properties, including sizes, is a critical determinant for the design of safer forms of NMs.

Toxicological properties of emission particles from heavy duty engines powered by conventional and bio-based diesel fuels and compressed natural gas.

Abstract: Background: One of the major areas for increasing the use of renewable energy is in traffic fuels e.g. bio-based fuels in diesel engines especially in commuter traffic. Exhaust emissions from fossil diesel fuelled engines are known to cause adverse effects on human health, but there is very limited information available on how the new renewable fuels may change the harmfulness of the emissions, especially particles (PM). We evaluated the PM emissions from a heavy-duty EURO IV diesel engine powered by three different fuels; the toxicological properties of the emitted PM were investigated. Conventional diesel fuel (EN590) and two biodiesels were usedrapeseed methyl ester (RME, EN14214) and hydrotreated vegetable oil (HVO) either as such or as 30% blends with EN590. EN590 and 100% HVO were also operated with or without an oxidative catalyst (DOC+POC). A bus powered by compressed natural gas (CNG) was included for comparison with the liquid fuels. However, the results from CNG powered bus cannot be directly compared to the other situations in this study. Results: High volume PM samples were collected on PTFE filters from a constant volume dilution tunnel. The PM mass emission with HVO was smaller and with RME larger than that with EN590, but both biofuels produced lower PAH contents in emission PM. The DOC+POC catalyst greatly reduced the PM emission and PAH content in PM with both HVO and EN590. Dose-dependent TNFα and MIP-2 responses to all PM samples were mostly at the low or moderate level after 24-hour exposure in a mouse macrophage cell line RAW 264.7. Emission PM from situations with the smallest mass emissions (HVO+cat and CNG) displayed the strongest potency in MIP-2 production. The catalyst slightly decreased the PM-induced TNFα responses and somewhat increased the MIP-2 responses with HVO fuel. Emission PM with EN590 and with 30% HVO blended in EN590 induced the strongest genotoxic responses, which were significantly greater than those with EN590+cat or 100% HVO. The emission PM sample from the CNG bus possessed the weakest genotoxic potency but had the strongest oxidative potency of all the fuel and catalyst combinations. The use of 100% HVO fuel had slightly weaker and 100% RME somewhat stronger emission PM induced ROS production, when compared to EN590. (Continued on next page)

Experimental determination of the respiratory tract deposition of diesel combustion particles in patients with chronic obstructive pulmonary disease

Abstract: Background Air pollution, mainly from combustion, is one of the leading global health risk factors. A susceptible group is the more than 200 million people worldwide suffering from chronic obstructive pulmonary disease (COPD). There are few data on lung deposition of airborne particles in patients with COPD and none for combustion particles.

A mixture of anatase and rutile TiO2 nanoparticles induces histamine secretion in mast cells

Abstract: Histamine released from mast cells, through complex interactions involving the binding of IgE to FceRI receptors and the subsequent intracellular Ca2+ signaling, can mediate many allergic/inflammatory responses. The possibility of titanium dioxide nanoparticles (TiO2 NPs), a nanomaterial pervasively used in nanotechnology and pharmaceutical industries, to directly induce histamine secretion without prior allergen sensitization has remained uncertain. TiO2 NP exposure increased both histamine secretion and cytosolic Ca2+ concentration ([Ca2+]C) in a dose dependent manner in rat RBL-2H3 mast cells. The increase in intracellular Ca2+ levels resulted primarily from an extracellular Ca2+ influx via membrane L-type Ca2+ channels. Unspecific Ca2+ entry via TiO2 NP-instigated membrane disruption was demonstrated with the intracellular leakage of a fluorescent calcein dye. Oxidative stress induced by TiO2 NPs also contributed to cytosolic Ca2+ signaling. The PLC-IP3-IP3 receptor pathways and endoplasmic reticulum (ER) were responsible for the sustained elevation of [Ca2+]C and histamine secretion. Our data suggests that systemic circulation of NPs may prompt histamine release at different locales causing abnormal inflammatory diseases. This study provides a novel mechanistic link between environmental TiO2 NP exposure and allergen-independent histamine release that can exacerbate manifestations of multiple allergic responses.