Role of different mechanisms in pro-inflammatory responses triggered by traffic-derived particulate matter in human bronchiolar epithelial cells.

BACKGROUND: Traffic-derived particles are important contributors to the adverse health effects of ambient particulate matter (PM). In Nordic countries, mineral particles from road pavement and diesel exhaust particles (DEP) are important constituents of traffic-derived PM. In the present study we compared the pro-inflammatory responses of mineral particles and DEP to PM from two road tunnels, and examined the mechanisms involved. METHODS: The pro-inflammatory potential of 100 µg/mL coarse (PM10-2.5), fine (PM2.5-0.18) and ultrafine PM (PM0.18) sampled in two road tunnels paved with different stone materials was assessed in human bronchial epithelial cells (HBEC3-KT), and compared to DEP and particles derived from the respective stone materials. Release of pro-inflammatory cytokines (CXCL8, IL-1α, IL-1ß) was measured by ELISA, while the expression of genes related to inflammation (COX2, CXCL8, IL-1α, IL-1ß, TNF-α), redox responses (HO-1) and metabolism (CYP1A1, CYP1B1, PAI-2) was determined by qPCR. The roles of the aryl hydrocarbon receptor (AhR) and reactive oxygen species (ROS) were examined by treatment with the AhR-inhibitor CH223191 and the anti-oxidant N-acetyl cysteine (NAC). RESULTS: Road tunnel PM caused time-dependent increases in expression of CXCL8, COX2, IL-1α, IL-1ß, TNF-α, COX2, PAI-2, CYP1A1, CYP1B1 and HO-1, with fine PM as more potent than coarse PM at early time-points. The stone particle samples and DEP induced lower cytokine release than all size-fractionated PM samples for one tunnel, and versus fine PM for the other tunnel. CH223191 partially reduced release and expression of IL-1α and CXCL8, and expression of COX2, for fine and coarse PM, depending on tunnel, response and time-point. Whereas expression of CYP1A1 was markedly reduced by CH223191, HO-1 expression was not affected. NAC reduced the release and expression of IL-1α and CXCL8, and COX2 expression, but augmented expression of CYP1A1 and HO-1. CONCLUSIONS: The results indicate that the pro-inflammatory responses of road tunnel PM in HBEC3-KT cells are not attributed to the mineral particles or DEP alone. The pro-inflammatory responses seem to involve AhR-dependent mechanisms, suggesting a role for organic constituents. ROS-mediated mechanisms were also involved, probably through AhR-independent pathways. DEP may be a contributor to the AhR-dependent responses, although other sources may be of importance.

The pro-inflammatory effects of combined exposure to diesel exhaust particles and mineral particles in human bronchial epithelial cells.

BACKGROUND: People are exposed to ambient particulate matter (PM) from multiple sources simultaneously in both environmental and occupational settings. However, combinatory effects of particles from different sources have received little attention in experimental studies. In the present study, the pro-inflammatory effects of combined exposure to diesel exhaust particles (DEP) and mineral particles, two common PM constituents, were explored in human lung epithelial cells. METHODS: Particle-induced secretion of pro-inflammatory cytokines (CXCL8 and IL-1ß) and changes in expression of genes related to inflammation (CXCL8, IL-1α, IL-1ß and COX-2), redox responses (HO-1) and xenobiotic metabolism (CYP1A1 and CYP1B1) were assessed in human bronchial epithelial cells (HBEC3-KT) after combined exposure to different samples of DEP and mineral particles. Combined exposure was also conducted using lipophilic organic extracts of DEP to assess the contribution of soluble organic chemicals. Moreover, the role of the aryl hydrocarbon receptor (AhR) pathway was assessed using an AhR-specific inhibitor (CH223191). RESULTS: Combined exposure to DEP and mineral particles induced increases in pro-inflammatory cytokines and expression of genes related to inflammation and redox responses in HBEC3-KT cells that were greater than either particle sample alone. Moreover, robust increases in the expression of CYP1A1 and CYP1B1 were observed. The effects were most pronounced after combined exposure to α-quartz and DEP from an older fossil diesel, but enhanced responses were also observed using DEP generated from a modern biodiesel blend and several stone particle samples of mixed mineral composition. Moreover, the effect of combined exposure on cytokine secretion could also be induced by lipophilic organic extracts of DEP. Pre-incubation with an AhR-specific inhibitor reduced the particle-induced cytokine responses, suggesting that the effects were at least partially dependent on AhR. CONCLUSIONS: Exposure to DEP and mineral particles in combination induces enhanced pro-inflammatory responses in human bronchial epithelial cells compared with exposure to the individual particle samples. The effects are partly mediated through an AhR-dependent pathway and lipophilic organic chemicals in DEP appear to play a central role. These possible combinatory effects between different sources and components of PM warrant further attention and should also be considered when assessing measures to reduce PM-induced health effects.

The importance of mineralogical composition for the cytotoxic and pro-inflammatory effects of mineral dust.

BACKGROUND: Respirable mineral particles represent a potential health hazard in occupational settings and ambient air. Previous studies show that mineral particles may induce cytotoxicity and inflammatory reactions in vitro and in vivo and that the potency varies between samples of different composition. However, the reason for these differences is largely unknown and the impact of mineralogical composition on the biological effects of mineral dust remains to be determined. METHODS: We have assessed the cytotoxic and pro-inflammatory effects of ten mineral particle samples of different composition in human bronchial epithelial cells (HBEC3-KT) and THP-1-derived macrophages, as well as their membranolytic properties in erythrocytes. Moreover, the results were compiled with the results of recently published experiments on the effects of stone particle exposure and analysed using linear regression models to elucidate which mineral components contribute most to the toxicity of mineral dust. RESULTS: While all mineral particle samples were more cytotoxic to HBEC3-KT cells than THP-1 macrophages, biotite and quartz were among the most cytotoxic in both cell models. In HBEC3-KT cells, biotite and quartz also appeared to be the most potent inducers of pro-inflammatory cytokines, while the quartz, Ca-feldspar, Na-feldspar and biotite samples were the most potent in THP-1 macrophages. All particle samples except quartz induced low levels of membranolysis. The regression analyses revealed associations between particle bioactivity and the content of quartz, muscovite, plagioclase, biotite, anorthite, albite, microcline, calcite, chlorite, orthopyroxene, actinolite and epidote, depending on the cell model and endpoint. However, muscovite was the only mineral consistently associated with increased cytotoxicity and cytokine release in both cell models. CONCLUSIONS: The present study provides further evidence that mineral particles may induce cytotoxicity and inflammation in cells of the human airways and that particle samples of different mineralogical composition differ in potency. The results show that quartz, while being among the most potent samples, does not fully predict the toxicity of mineral dust, highlighting the importance of other particle constituents. Moreover, the results indicate that the phyllosilicates muscovite and biotite may be more potent than other minerals assessed in the study, suggesting that this group of sheet-like minerals may warrant further attention.

Road tunnel-derived coarse, fine and ultrafine particulate matter: physical and chemical characterization and pro-inflammatory responses in human bronchial epithelial cells.

BACKGROUND: Traffic particulate matter (PM) comprises a mixture of particles from fuel combustion and wear of road pavement, tires and brakes. In countries with low winter temperatures the relative contribution of mineral-rich PM from road abrasion may be especially high due to use of studded tires during winter season. The aim of the present study was to sample and characterize size-fractioned PM from two road tunnels paved with different stone materials in the asphalt, and to compare the pro-inflammatory potential of these fractions in human bronchial epithelial cells (HBEC3-KT) in relation to physicochemical characteristics. METHODS: The road tunnel PM was collected with a vacuum pump and a high-volume cascade impactor sampler. PM was sampled during winter, both during humid and dry road surface conditions, and before and after cleaning the tunnels. Samples were analysed for hydrodynamic size distribution, content of elemental carbon (EC), organic carbon (OC) and endotoxin, and the capacity for acellular generation of reactive oxygen species. Cytotoxicity and pro-inflammatory responses were assessed in HBEC3-KT cells after exposure to coarse (2.5-10 µm), fine (0.18-2.5 µm) and ultrafine PM (≤ 0.18 µm), as well as particles from the respective stone materials used in the pavement. RESULTS: The pro-inflammatory potency of the PM samples varied between road tunnels and size fractions, but showed more marked responses than for the stone materials used in asphalt of the respective tunnels. In particular, fine samples showed significant increases as low as 25 µg/mL (2.6 µg/cm2) and were more potent than coarse samples, while ultrafine samples showed more variable responses between tunnels, sampling conditions and endpoints. The most marked responses were observed for fine PM sampled during humid road surface conditions. Linear correlation analysis showed that particle-induced cytokine responses were correlated to OC levels, while no correlations were observed for other PM characteristics. CONCLUSIONS: The pro-inflammatory potential of fine road tunnel PM sampled during winter season was high compared to coarse PM. The differences between the PM-induced cytokine responses were not related to stone materials in the asphalt. However, the ratio of OC to total PM mass was associated with the pro-inflammatory potential.

Respirable stone particles differ in their ability to induce cytotoxicity and pro-inflammatory responses in cell models of the human airways.

BACKGROUND: Respirable stone- and mineral particles may be a major constituent in occupational and ambient air pollution and represent a possible health hazard. However, with exception of quartz and asbestos, little is known about the toxic properties of mineral particles. In the present study, the pro-inflammatory and cytotoxic responses to six stone particle samples of different composition and with diameter below 10 µm were assessed in human bronchial epithelial cells (HBEC3-KT), THP-1 macrophages and a HBEC3-KT/THP-1 co-culture. Moreover, particle-induced lysis of human erythrocytes was assessed to determine the ability of the particles to lyse biological membranes. Finally, the role of the NLRP3 inflammasome was assessed using a NLRP3-specific inhibitor and detection of ASC oligomers and cleaved caspase-1 and IL-1ß. A reference sample of pure α-quartz was included for comparison. RESULTS: Several stone particle samples induced a concentration-dependent increase in cytotoxicity and secretion of the pro-inflammatory cytokines CXCL8, IL-1α, IL-1ß and TNFα. In HBEC3-KT, quartzite and anorthosite were the most cytotoxic stone particle samples and induced the highest levels of cytokines. Quartzite and anorthosite were also the most cytotoxic samples in THP-1 macrophages, while anorthosite and hornfels induced the highest cytokine responses. In comparison, few significant differences between particle samples were detected in the co-culture. Adjusting responses for differences in surface area concentrations did not fully account for the differences between particle samples. Moreover, the stone particles had low hemolytic potential, indicating that the effects were not driven by membrane lysis. Pre-incubation with a NLRP3-specific inhibitor reduced stone particle-induced cytokine responses in THP-1 macrophages, but not in HBEC3-KT cells, suggesting that the effects are mediated through different mechanisms in epithelial cells and macrophages. Particle exposure also induced an increase in ASC oligomers and cleaved caspase-1 and IL-1ß in THP-1 macrophages, confirming the involvement of the NLRP3 inflammasome. CONCLUSIONS: The present study indicates that stone particles induce cytotoxicity and pro-inflammatory responses in human bronchial epithelial cells and macrophages, acting through NLRP3-independent and -dependent mechanisms, respectively. Moreover, some particle samples induced cytotoxicity and cytokine release to a similar or greater extent than α-quartz. Thus, these minerals warrant further attention in future research.

Potential role of polycyclic aromatic hydrocarbons in air pollution-induced non-malignant respiratory diseases.

Epidemiological studies have found strong associations between air pollution and respiratory effects including development and/or exacerbation of asthma and chronic obstructive pulmonary disease (COPD) as well as increased occurrence of respiratory infections and lung cancer. It has become increasingly clear that also polycyclic aromatic hydrocarbons (PAHs) may affect processes linked to non-malignant diseases in the airways. The aim of the present paper was to review epidemiological studies on associations between gas phase and particle-bound PAHs in ambient air and non-malignant respiratory diseases or closely related physiological processes, to assess whether PAH-exposure may explain some of the effects associated with air pollution. Based on experimental in vivo and in vitro studies, we also explore possible mechanisms for how different PAHs may contribute to such events. Epidemiological studies show strongest evidence for an association between PAHs and asthma development and respiratory function in children. This is supported by studies on prenatal and postnatal exposure. Exposure to PAHs in adults seems to be linked to respiratory functions, exacerbation of asthma and increased morbidity/mortality of obstructive lung diseases. However, available studies are few and weak. Notably, the PAHs measured in plasma/urine also represent other exposure routes than inhalation. Furthermore, the role of PAHs measured in air is difficult to disentangle from that of other air pollution components originating from combustion processes. Experimental studies show that PAHs may trigger various processes linked to non-malignant respiratory diseases. Physiological- and pathological responses include redox imbalance, oxidative stress, inflammation both from the innate and adaptive immune systems, smooth muscle constriction, epithelial- and endothelial dysfunction and dysregulated lung development. Such biological responses may at the molecular level be initiated by PAH-binding to the aryl hydrocarbon receptor (AhR), but possibly also through interactions with beta-adrenergic receptors. In addition, reactive PAH metabolites or reactive oxygen species (ROS) may interfere directly with ion transporters and enzymes involved in signal transduction. Overall, the reviewed literature shows that respiratory effects of PAH-exposure in ambient air may extend beyond lung cancer. The relative importance of the specific PAHs ability to induce disease may differ between the biological endpoint in question.

Pro-inflammatory effects of crystalline- and nano-sized non-crystalline silica particles in a 3D alveolar model.

BACKGROUND: Silica nanoparticles (SiNPs) are among the most widely manufactured and used nanoparticles. Concerns about potential health effects of SiNPs have therefore risen. Using a 3D tri-culture model of the alveolar lung barrier we examined effects of exposure to SiNPs (Si10) and crystalline silica (quartz; Min-U-Sil) in the apical compartment consisting of human alveolar epithelial A549 cells and THP-1-derived macrophages, as well as in the basolateral compartment with Ea.hy926 endothelial cells. Inflammation-related responses were measured by ELISA and gene expression. RESULTS: Exposure to both Si10 and Min-U-Sil induced gene expression and release of CXCL8, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-1ß (IL-1ß) in a concentration-dependent manner. Cytokine/chemokine expression and protein levels were highest in the apical compartment. Si10 and Min-U-Sil also induced expression of adhesion molecules ICAM-1 and E-selectin in the apical compartment. In the basolateral endothelial compartment we observed marked, but postponed effects on expression of all these genes, but only at the highest particle concentrations. Geneexpressions of heme oxygenase-1 (HO-1) and the metalloproteases (MMP-1 and MMP-9) were less affected. The IL-1 receptor antagonist (IL-1RA), markedly reduced effects of Si10 and Min-U-Sil exposures on gene expression of cytokines and adhesion molecules, as well as cytokine-release in both compartments. CONCLUSIONS: Si10 and Min-U-Sil induced gene expression and release of pro-inflammatory cytokines/adhesion molecules at both the epithelial/macrophage and endothelial side of a 3D tri-culture. Responses in the basolateral endothelial cells were only induced at high concentrations, and seemed to be mediated by IL-1α/ß released from the apical epithelial cells and macrophages.

Potential role of polycyclic aromatic hydrocarbons as mediators of cardiovascular effects from combustion particles.

Air pollution is the most important environmental risk factor for disease and premature death, and exposure to combustion particles from vehicles is a major contributor. Human epidemiological studies combined with experimental studies strongly suggest that exposure to combustion particles may enhance the risk of cardiovascular disease (CVD), including atherosclerosis, hypertension, thrombosis and myocardial infarction.In this review we hypothesize that adhered organic chemicals like polycyclic aromatic hydrocarbons (PAHs), contribute to development or exacerbation of CVD from combustion particles exposure. We summarize present knowledge from existing human epidemiological and clinical studies as well as experimental studies in animals and relevant in vitro studies. The available evidence suggests that organic compounds attached to these particles are significant triggers of CVD. Furthermore, their effects seem to be mediated at least in part by the aryl hydrocarbon receptor (AhR). The mechanisms include AhR-induced changes in gene expression as well as formation of reactive oxygen species (ROS) and/or reactive electrophilic metabolites. This is in accordance with a role of PAHs, as they seem to be the major chemical group on combustion particles, which bind AhR and/or is metabolically activated by CYP-enzymes. In some experimental models however, it seems as PAHs may induce an inflammatory atherosclerotic plaque phenotype irrespective of DNA- and/or AhR-ligand binding properties. Thus, various components and several signalling mechanisms/pathways are likely involved in CVD induced by combustion particles.We still need to expand our knowledge about the role of PAHs in CVD and in particular the relative importance of the different PAH species. This warrants further studies as enhanced knowledge on this issue may amend risk assessment of CVD caused by combustion particles and selection of efficient measures to reduce the health effects of particular matters (PM).

Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s).

BACKGROUND: Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis. RESULTS: Exposure-relevant concentrations of DEP (0.12 µg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 µg/cm2) induced low to moderate increases in IL-1α, IL-1ß, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects. CONCLUSION: Lipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.

Effect of silver nanoparticles on mitogen-activated protein kinases activation: role of reactive oxygen species and implication in DNA damage.

Large quantities of engineered nanoparticles (NP), such as nanosilver (AgNP), have been widely applied, leading to an increased exposure and potential health concerns. Herein, we have examined the ability of AgNP to induce reactive oxygen species (ROS), their role in genotoxic effects and the involvement of mitogen-activated protein kinases (MAPK). AgNP exposure induced ROS production in human epithelial embryonic cells which could be decreased by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation, induced by AgNP, was an early response but not sustained in time. Furthermore, JNK and ERK activation could be inhibited by both DPI and a free radicals scavenger N-acetyl cysteine. We also investigated the role of MAPK in the DNA damage. Using a modified comet assay for the specific detection of hOGG1 sensitive sites, we showed that AgNP induced DNA oxidation after 30-min treatment, whereas no response was observed after 2h. In conclusion, AgNP seem to induce DNA damage via a mechanism involving ROS formation. The oxidative DNA damage observed was transient, likely due to DNA repair; furthermore, higher damage was achieved upon inhibition of ERK activation by pre-treatment with U0126, suggesting a role for ERK in DNA damage repair. Activation of different MAPK might play an important role in the NP toxicity outcomes; understanding this process may be helpful for the identification of NP toxicity.

Silica nanoparticles induce cytokine responses in lung epithelial cells through activation of a p38/TACE/TGF-α/EGFR-pathway and NF-κΒ signalling.

Amorphous silica nanoparticles (SiNPs) have previously been shown to induce marked cytokine (interleukin-6; IL-6 and interleukin-8; CXCL8/IL-8) responses independently of particle uptake in human bronchial epithelial BEAS-2B cells. In this study the involvement of the mitogen-activated protein kinases (MAP-kinases), nuclear factor-kappa Β (NF-κΒ) and in particular tumour necrosis factor-α converting enzyme (TACE) and-epidermal growth factor receptor (EGFR) signalling pathways were examined in triggering of IL-6 and CXCL8 release after exposure to a 50nm silica nanoparticle (Si50). Exposure to Si50 increased phosphorylation of NF-κΒ p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK), but not extracellular signal regulated kinases (ERK). Inhibition of NF-κΒ and p38 reduced the cytokine responses to Si50, whereas neither JNK- nor ERK-inhibition exerted any significant effect on the responses to Si50. Increases in membrane-bound transforming growth factor-α (TGF-α) release and EGFR phosphorylation were also observed after Si50 exposure, and pre-treatment with inhibitors of these pathways reduced the release of IL-6 and CXCL8, but did not affect the Si50-induced phosphorylation of p38 and p65. In contrast, p38-inhibition partially reduced Si50-induced TGF-α release, while the p65-inhibition was without effect. Overall, our results indicate that Si50-induced IL-6 and CXCL8 responses in BEAS-2B cells were regulated through combined activation of several pathways, including NF-κΒ and p38/TACE/TGF-α/EGFR signalling. The study identifies critical, initial events in the triggering of pro-inflammatory responses by nanoparticles.

AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells.

BACKGROUND: The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B). RESULTS: Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C. CONCLUSION: AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

The effects of fine particulate matter (SRM 2786) on three different 3D lung models exposed at the air-liquid interface - A comparative study.

3D cell culture models exposed at the air-liquid interface (ALI) represent a potential alternative to animal experiments for hazard and risk assessment of inhaled compounds. This study compares cocultures composed of either Calu-3, A549 or HBEC3-KT lung epithelial cells, cultured together with THP-1-derived macrophages and EA.hy926 endothelial cells, in terms of barrier capacity and responses to a standard reference sample of fine particulate matter (SRM 2786). High-content imaging analysis revealed a similar cellular composition between the different cell models. The 3D cell cultures with Calu-3 cells showed the greatest barrier capacity, as measured by transepithelial electrical resistance and permeability to Na-fluorescein. Mucus production was detected in 3D cell cultures based on Calu-3 and A549 cells. Exposure to SRM 2786 at ALI increased cytokine release and expression of genes associated with inflammation and xenobiotic metabolism. Moreover, the presence of THP-1-derived macrophages was central to the cytokine responses in all cell models. While the different 3D cell culture models produced qualitatively similar responses, more pronounced pro-inflammatory responses were observed in the basolateral compartment of the A549 and HBEC3-KT models compared to the Calu-3 model, likely due to their reduced barrier capacity and lower retention of secreted mediators in the apical compartment.

Characterization of elements, PAHs, AhR-activity and pro-inflammatory responses of road tunnel-derived particulate matter in human hepatocyte-like and bronchial epithelial cells.

The aims were to characterize the content of elements and polycyclic aromatic hydrocarbons (PAHs) in size-separated particulate matter (PM) sampled in a road tunnel, estimate the contribution of PAHs to the toxic potential, and measure the pro-inflammatory potential of PM samples and extracts with increasing polarity. Several elements/metals previously associated with cytokine responses were found. Based on PAHs levels and published PAHs potency, the calculated mutagenic and carcinogenic activities of size-separated samples were somewhat lower for coarse than fine and ultrafine PM. The AhR-activity of the corresponding PM extracts measured in an AhR-luciferase reporter model (human hepatocytes) were more similar. The highest AhR-activity was found in the neutral (parent and alkylated PAHs) and polar (oxy-PAHs) fractions, while the semi-polar fractions (mono-nitrated-PAHs) had only weak activity. The neutral and polar aromatic fractions from coarse and fine PM were also found to induce higher pro-inflammatory responses and CYP1A1 expression in human bronchial epithelial cells (HBEC3-KT) than the semi-polar fractions. Fine PM induced higher pro-inflammatory responses than coarse PM. AhR-inhibition reduced cytokine responses induced by parent PM and extracts of both size fractions. Contributors to the toxic potentials include PAHs and oxy-PAHs, but substantial contributions from other organic compounds and/or metals are likely.

Silver nanoparticles induce premutagenic DNA oxidation that can be prevented by phytochemicals from Gentiana asclepiadea.

Among nanomaterials, silver nanoparticles (AgNPs) have the broadest and most commercial applications due to their antibacterial properties, highlighting the need for exploring their potential toxicity and underlying mechanisms of action. Our main aim was to investigate whether AgNPs exert toxicity by inducing oxidative damage to DNA in human kidney HEK 293 cells. In addition, we tested whether this damage could be counteracted by plant extracts containing phytochemicals such as swertiamarin, mangiferin and homoorientin with high antioxidant abilities. We show that AgNPs (20 nm) are taken up by cells and localised in vacuoles and cytoplasm. Exposure to 1, 25 or 100 µg/ml AgNPs leads to a significant dose-dependent increase in oxidised DNA base lesions (8-oxo-7,8-dihydroguanine or 8-oxoG) detected by the comet assay after incubation of nucleoids with 8-oxoG DNA glycosylase. Oxidised DNA base lesions and strand breaks caused by AgNPs were diminished by aqueous and methanolic extracts from both haulm and flower of Gentiana asclepiadea.

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

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-1ß 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-1ß 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. RESULTS: In the present study, we have shown that 6 h exposure to non-crystalline SiO2 particles in nano- (SiNPs, 5-20 nm, 50 nm) and submicro-sizes induced strong IL-1ß responses in LPS-primed mouse macrophages (RAW264.7) and primary rat lung macrophages. The primary lung macrophages were more sensitive to Si-exposure than the RAW-macrophages, and responded more strongly. In the lung macrophages, crystalline silica (MinUsil 5) induced IL-1ß release more potently than the non-crystalline Si50 and Si500, when adjusted to surface area. This difference was much less pronounced versus fumed SiNPs. The caspase-1 inhibitor zYVAD and RNA silencing of the NALP3 receptor reduced the particle-induced IL-1ß release in the RAW264.7 macrophages. Furthermore, inhibitors of phagocytosis, endosomal acidification, and cathepsin B activity reduced the IL-1ß responses to the different particles to a similar extent. CONCLUSIONS: In conclusion, non-crystalline silica particles in the nano- and submicro-size ranges seemed to induce IL-1ß release from LPS-primed RAW264.7 macrophages via similar mechanisms as crystalline silica, involving particle uptake, phagosomal leakage and activation of the NALP3 inflammasome. Notably, rat primary lung macrophages were more sensitive with respect to silica-induced IL-1ß release. The differential response patterns obtained suggest that silica-induced IL-1ß responses not only depend on the particle surface area, but on factors and/or mechanisms such as particle reactivity or particle uptake. These findings may suggest that bacterial infection via LPS may augment acute inflammatory effects of non-crystalline as well as crystalline silica particles.

Short-term exposure to stone minerals used in asphalt affect lung function and promote pulmonary inflammation among healthy adults.

OBJECTIVE: Stone minerals are a partially ignored environmental challenge but a significant contributor to urban air pollution. We examined if short-term exposure to two stone minerals - quartz diorite and rhomb porphyry - commonly used in asphalt pavement would affect lung function, promote pulmonary inflammation, and affect bronchial reactivity differently. METHODS: Our randomized crossover study included 24 healthy, non-smoking young adults exposed to the stone minerals quartz diorite, rhomb porphyry, and control dust (lactose). Exposure occurred in an exposure chamber, in three separate 4-hour exposure sessions. Fractional exhaled nitric oxide (FeNO) and lung function were monitored before exposure, then immediately following exposure, and 4 and 24 hours after exposure. In addition, methacholine was administered 4 hours following exposure, and exhaled breath condensate (EBC) was collected before exposure, then immediately and 4 hours after exposure. EBC was analyzed for pH, thiobarbituric acid reactive substances (TBARS), intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), IL-10, P-Selectin, surfactant protein D (SP-D), and tumor necrosis factor-α (TNF-α). RESULTS: Our results showed significantly elevated concentrations of FeNO after exposure to quartz diorite compared to rhomb porphyry, suggesting that quartz diorite is more likely to trigger pulmonary inflammation after short-term exposure. Moreover, short-term exposure to rhomb porphyry was associated with a modest but statistically significant decline in forced vital capacity (FVC) compared to quartz diorite. CONCLUSION: These results emphasize that using stone material in asphalt road construction should be reconsidered as it may affect lung inflammation and lung function in exposed subjects.

Role of scavenger receptors in silica nanoparticle-induced cytokine responses in bronchial epithelial cells.

A major challenge in nanoparticle (NP) research is to elucidate how NPs activate initial targets in cells, leading to cytotoxicity and inflammation. We have previously shown that silica (Si)NPs induce pro-inflammatory responses in bronchial epithelial cells (BEAS-2B) via mechanisms involving transforming growth factor (TGF)-α release, and activation of MAP-kinase p38 and JNK besides NF-κB (p65). In the present study, the roles of scavenger receptors (SRs) in SiNP-induced cytokine responses in BEAS-2B cells were examined by siRNA silencing. Cells exposed to Si10 and Si50 (nominal sizes 10 and 50 nm) showed marked interleukin (IL)-6, CXCL8, IL-1α, IL-1ß responses. Transient knockdown of SR-B1, LOX-1 and CXCL16 reduced the Si10- and Si50-induced cytokine responses, to a different magnitude dependent on the particle size, SR and cytokine. Si10-induced TGF-α responses were also markedly reduced by knockdown of SR-B1 and CXCL16. Furthermore, the role of SR-B1 in Si10-induced phosphorylations of p65 and MAP-kinases p38 and JNK were examined, and no significant reductions were observed upon knockdown of SR-B1. In conclusion, LOX-1 and CXCL16 and especially SR-B1 seem to have important roles in mediating cytokine responses and TGF-α release due to SiNP exposure in BEAS-2B cells, without a down-stream role of MAP-kinase and NF-κB.

Biological effects of combustion-derived particles from different biomass sources on human bronchial epithelial cells.

Combustion-derived particles (CDPs), in particular from traffic, are regarded as a central contributor for adverse health effects linked to air pollution. Recently, also biomass burning has been recognized as an important source for CDPs. Here, the effects of CDPs (PM10) originating from burning of pellet, charcoal and wood on key processes associated to lung carcinogenesis were explored. Human bronchial epithelial cells (HBEC3-KT) were exposed to 2.5 µg/cm2 of CDPs for 24 h and biological effects were examined in terms of cytotoxicity, inflammation, epithelial to mesenchymal transition (EMT)-related effects, DNA damage and genotoxicity. Reduced cell migration, inflammation and modulation of various PM-associated genes were observed mainly after exposure to wood and pellet. In contrast, only particles from pellet burning induced alteration in cell proliferation and DNA damage, which resulted in cell cycle alterations. Charcoal instead, appeared in general less effective in inducing pro-carcinogenic effects. These results illustrate differences in the toxicological profile due to the CDPs source. The different chemical compounds adsorbed on CDPs seemed to be central for particle properties, leading to an activation of various cellular signaling pathways involved in early steps of cancer progression.

Diesel exhaust particles induce CYP1A1 and pro-inflammatory responses via differential pathways in human bronchial epithelial cells.

BACKGROUND: Exposure to diesel engine exhaust particles (DEPs) has been associated with several adverse health outcomes in which inflammation seems to play a key role. DEPs contain a range of different inorganic and organic compounds, including polycyclic aromatic hydrocarbons (PAHs). During the metabolic activation of PAHs, CYP1A1 enzymes are known to play a critical role. In the present study we investigated the potential of a characterised sample of DEPs to induce cytotoxicity, to influence the expression of CYP1A1 and inflammation-related genes, and to activate intracellular signalling pathways, in human bronchial epithelial cells. We specifically investigated to what extent DEP-induced expression of interleukin (IL)-6, IL-8 and cyclooxygenase (COX)-2 was regulated differentially from DEP-induced expression of CYP1A1. RESULTS: The cytotoxicity of the DEPs was characterised by a marked time- and concentration-dependent increase in necrotic cells at 4 h and above 200 µg/ml (~ 30 µg/cm2). DEP-induced DNA-damage was only apparent at high concentrations (≥ 200 µg/ml). IL-6, IL-8 and COX-2 were the three most up-regulated genes by the DEPs in a screening of 20 selected inflammation-related genes. DEP-induced expression of CYP1A1 was detected at very low concentrations (0.025 µg/ml), compared to the expression of IL-6, IL-8 and COX-2 (50-100 µg/ml). A CYP1A1 inhibitor (α-naphthoflavone), nearly abolished the DEP-induced expression of IL-8 and COX-2. Of the investigated mitogen-activated protein kinases (MAPKs), the DEPs induced activation of p38. A p38 inhibitor (SB202190) strongly reduced DEP-induced expression of IL-6, IL-8 and COX-2, but only moderately affected the expression of CYP1A1. The DEPs also activated the nuclear factor-κB (NF-κB) pathway, and suppression by siRNA tended to reduce the DEP-induced expression of IL-8 and COX-2, but not CYP1A1. CONCLUSION: The present study indicates that DEPs induce both CYP1A1 and pro-inflammatory responses in vitro, but via differential intracellular pathways. DEP-induced pro-inflammatory responses seem to occur via activation of NF-κB and p38 and are facilitated by CYP1A1. However, the DEP-induced CYP1A1 response does not seem to involve NF-κB and p38 activation. Notably, the present study also indicates that expression of CYP1A1 may represent a particular sensitive biomarker of DEP-exposure.