Saharan dust induces the lung disease-related cytokines granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor.

Desert dust exposure is associated with adverse respiratory health effects. Desert dust is a complex pollutant mixtures that includes respirable crystalline and amorphous particles, metals, and microbial constituents. Given the health effects of desert dust and its heterogeneity, as yet unidentified harmful biological pathways may be triggered. Therefore, we exposed human in vitro air-liquid interface co-cultures of alveolar epithelial A549 cells and THP-1 macrophages to Saharan dust (SD). For comparison, we used the known pulmonary toxicant DQ12 quartz dust. Via RNA sequencing, we identified that SD but not DQ12 increased the gene expression of granulocyte-macrophage colony-stimulating factor (GMCSF) and granulocyte colony-stimulating factor (GCSF). These findings were confirmed by quantitative reverse transcriptase PCR. SD dose-dependently upregulated GMCSF and GCSF expression with significant 7 and 9-fold changes, respectively, at the highest tested concentration of 31 µg/cm2. Furthermore, we observed that SD significantly enhanced the secretion of GM-CSF and G-CSF by 2-fold. Both cytokines have previously been associated with lung diseases such as asthma and fibrosis. Hence, we present two molecular messengers that may contribute to the adverse health effects of desert dust and might serve as drug targets for this globally relevant non-anthropogenic air pollutant.

Saharan dust induces NLRP3-dependent inflammatory cytokines in an alveolar air-liquid interface co-culture model.

BACKGROUND: Epidemiological studies have related desert dust events to increased respiratory morbidity and mortality. Although the Sahara is the largest source of desert dust, Saharan dust (SD) has been barely examined in toxicological studies. Here, we aimed to assess the NLRP3 inflammasome-caspase-1-pathway-dependent pro-inflammatory potency of SD in comparison to crystalline silica (DQ12 quartz) in an advanced air-liquid interface (ALI) co-culture model. Therefore, we exposed ALI co-cultures of alveolar epithelial A549 cells and macrophage-like differentiated THP-1 cells to 10, 21, and 31 µg/cm² SD and DQ12 for 24 h using a Vitrocell Cloud system. Additionally, we exposed ALI co-cultures containing caspase (CASP)1-/- and NLRP3-/- THP-1 cells to SD. RESULTS: Characterization of nebulized DQ12 and SD revealed that over 90% of agglomerates of both dusts were smaller than 2.5 µm. Characterization of the ALI co-culture model revealed that it produced surfactant protein C and that THP-1 cells remained viable at the ALI. Moreover, wild type, CASP1-/-, and NLRP3-/- THP-1 cells had comparable levels of the surface receptors cluster of differentiation 14 (CD14), toll-like receptor 2 (TLR2), and TLR4. Exposing ALI co-cultures to non-cytotoxic doses of DQ12 and SD did not induce oxidative stress marker gene expression. SD but not DQ12 upregulated gene expressions of interleukin 1 Beta (IL1B), IL6, and IL8 as well as releases of IL-1ß, IL-6, IL-8, and tumor necrosis factor α (TNFα). Exposing wild type, CASP1-/-, and NLRP3-/- co-cultures to SD induced IL1B gene expression in all co-cultures whereas IL-1ß release was only induced in wild type co-cultures. In CASP1-/- and NLRP3-/- co-cultures, IL-6, IL-8, and TNFα releases were also reduced. CONCLUSIONS: Since surfactants can decrease the toxicity of poorly soluble particles, the higher potency of SD than DQ12 in this surfactant-producing ALI model emphasizes the importance of readily soluble SD components such as microbial compounds. The higher potency of SD than DQ12 also renders SD a potential alternative particulate positive control for studies addressing acute inflammatory effects. The high pro-inflammatory potency depending on NLRP3, CASP-1, and IL-1ß suggests that SD causes acute lung injury which may explain desert dust event-related increased respiratory morbidity and mortality.

Investigating nanoplastics toxicity using advanced stem cell-based intestinal and lung in vitro models.

Plastic particles in the nanometer range-called nanoplastics-are environmental contaminants with growing public health concern. As plastic particles are present in water, soil, air and food, human exposure via intestine and lung is unavoidable, but possible health effects are still to be elucidated. To better understand the Mode of Action of plastic particles, it is key to use experimental models that best reflect human physiology. Novel assessment methods like advanced cell models and several alternative approaches are currently used and developed in the scientific community. So far, the use of cancer cell line-based models is the standard approach regarding in vitro nanotoxicology. However, among the many advantages of the use of cancer cell lines, there are also disadvantages that might favor other approaches. In this review, we compare cell line-based models with stem cell-based in vitro models of the human intestine and lung. In the context of nanoplastics research, we highlight the advantages that come with the use of stem cells. Further, the specific challenges of testing nanoplastics in vitro are discussed. Although the use of stem cell-based models can be demanding, we conclude that, depending on the research question, stem cells in combination with advanced exposure strategies might be a more suitable approach than cancer cell lines when it comes to toxicological investigation of nanoplastics.

Carbon nanoparticles adversely affect CFTR expression and toxicologically relevant pathways.

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that can lead to terminal respiratory failure. Ultrafine carbonaceous particles, which are ubiquitous in ambient urban and indoor air, are increasingly considered as major contributors to the global health burden of air pollution. However, their effects on the expression of CFTR and associated genes in lung epithelial cells have not yet been investigated. We therefore evaluated the effects of carbon nanoparticles (CNP), generated by spark-ablation, on the human bronchial epithelial cell line 16HBE14o- at air-liquid interface (ALI) culture conditions. The ALI-cultured cells exhibited epithelial barrier integrity and increased CFTR expression. Following a 4-h exposure to CNP, the cells exhibited a decreased barrier integrity, as well as decreased expression of CFTR transcript and protein levels. Furthermore, transcriptomic analysis revealed that the CNP-exposed cells showed signs of oxidative stress, apoptosis and DNA damage. In conclusion, this study describes spark-ablated carbon nanoparticles in a realistic exposure of aerosols to decrease CFTR expression accompanied by transcriptomic signs of oxidative stress, apoptosis and DNA damage.

Mechanistic Insights into the Role of Iron, Copper, and Carbonaceous Component on the Oxidative Potential of Ultrafine Particulate Matter.

Transition metals play a key role in the pathogenic potential of urban particulate matter (PM). However, air quality regulations include exposure limits only for metals having a known toxic potential like Pb, As, Cd, and Ni, neglecting other transition metals like Fe and Cu. Fe and Cu are mainly found in the water-soluble fraction of PM. However, a fraction of the ions may persist strongly bound to the particles, thus potentially acting as surface reactive sites. The contribution of surface ions to the oxidative potential (OP) of PM is likely different from that of free ions since the redox activity of metals is modulated by their local chemical environment. The aim of this study was to investigate how Fe and Cu bound to carbonaceous particles affect the OP and associated toxicity of PM toward epithelial cells and macrophages. Carbonaceous nanoparticles (CNPs) having well-defined size were loaded with controlled amounts of Cu and Fe. The effect of Cu and Fe on the OP of CNPs was evaluated by electronic paramagnetic resonance (EPR) spectroscopy associated with the spin-trapping technique and correlated with the ability to induce cytotoxicity (LDH, WST-1), oxidative stress (Nrf2 translocation), and DNA damage (comet assay) on lung macrophages (NR8383) and/or epithelial cells (RLE-6TN). The release of pro-inflammatory cytokines (TNF-α, MCP-1, and CXCL2) by macrophages and epithelial cells was also investigated. The results indicate a major contribution of surface Cu to the surface reactivity of CNPs, while Fe has a minor role. At the same time, Cu increases the cytotoxicity of CNPs and their ability to induce oxidative stress and DNA damage. In contrast, surface Fe increases the release of pro-inflammatory cytokines by macrophages. Overall, these results confirm the role of Cu and Fe in PM toxicity and suggest that the total metals content in PM might be a better indicator of pathogenicity than water-soluble metals.

The dietary antioxidant quercetin reduces hallmarks of bleomycin-induced lung fibrogenesis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, lethal disease of which the etiology is still not fully understood. Current treatment comprises two FDA-approved drugs that can slow down yet not stop or reverse the disease. As IPF pathology is associated with an altered redox balance, adding a redox modulating component to current therapy might exert beneficial effects. Quercetin is a dietary antioxidant with strong redox modulating capacities that is suggested to exert part of its antioxidative effects via activation of the redox-sensitive transcription factor Nrf2 that regulates endogenous antioxidant levels. Therefore, the aim of the present study was to investigate if the dietary antioxidant quercetin can exert anti-fibrotic effects in a mouse model of bleomycin-induced pulmonary fibrogenesis through Nrf2-dependent restoration of redox imbalance. METHODS: Homozygous Nrf2 deficient mice and their wildtype littermates were fed a control diet without or with 800 mg quercetin per kg diet from 7 days prior to a single 1 µg/2 µl per g BW bleomycin challenge until they were sacrificed 14 days afterwards. Lung tissue and plasma were collected to determine markers of fibrosis (expression of extracellular matrix genes and histopathology), inflammation (pulmonary gene expression and plasma levels of tumor necrosis factor-α (TNFα) and keratinocyte chemoattrachtant (KC)), and redox balance (pulmonary gene expression of antioxidants and malondialdehyde-dG (MDA)- DNA adducts). RESULTS: Mice fed the enriched diet for 7 days prior to the bleomycin challenge had significantly enhanced plasma and pulmonary quercetin levels (11.08 ± 0.73 µM versus 7.05 ± 0.2 µM) combined with increased expression of Nrf2 and Nrf2-responsive genes compared to mice fed the control diet in lung tissue. Upon bleomycin treatment, quercetin-fed mice displayed reduced expression of collagen (COL1A2) and fibronectin (FN1) and a tendency of reduced inflammatory lesions (2.8 ± 0.7 versus 1.9 ± 0.8). These beneficial effects were accompanied by reduced pulmonary gene expression of TNFα and KC, but not their plasma levels, and enhanced Nrf2-induced pulmonary antioxidant defences. In Nrf2 deficient mice, no effect of the dietary antioxidant on either histology or inflammatory lesions was observed. CONCLUSION: Quercetin exerts anti-fibrogenic and anti-inflammatory effects on bleomycin-induced pulmonary damage in mice possibly through modulation of the redox balance by inducing Nrf2. However, quercetin could not rescue the bleomycin-induced pulmonary damage indicating that quercetin alone cannot ameliorate the progression of IPF.

Crystalline silica alters Sulfatase-1 expression in rat lungs which influences hyper-proliferative and fibrogenic effects in human lung epithelial cells.

Lung epithelial cells are the first cell-type to come in contact with hazardous dust materials. Upon deposition, they invoke complex reactions in attempt to eradicate particles from the airways, and repair damage. The cell surface is composed of a heterogeneous network of matrix proteins and proteoglycans, which act as scaffold and control cell-signaling networks. These functions are controlled, in part, by the sulfation patterns of heparin-sulfate proteoglycans (HSPGs), which are enzymatically regulated. Although there is evidence of altered HSPG-sulfation in idiopathic pulmonary fibrosis (IPF), this is not investigated in silicosis. Our previous studies revealed down-regulation of Sulfatase-1 (SULF1) in human bronchial epithelial cells (BECs) by crystalline silica (CS). In this study, CS-induced down-regulation of SULF1, and increases in Sulfated-HSPGs, were determined in human BECs, and in rat lungs. By siRNA and plasmid transfection techniques the effects of SULF1 expression on silica-induced fibrogenic and proliferative gene expression were determined. These studies confirmed down-regulation of SULF1 and subsequent increases in sulfated-HSPGs in vitro. Moreover, short-term exposure of rats to CS resulted in similar changes in vivo. Conversely, effects were reversed after long term CS exposure of rats. SULF1 knockdown, and overexpression alleviated and exacerbated silica-induced decrease in cell viability, respectively. Furthermore, overexpression of SULF1 promoted silica-induced proliferative and fibrogenic gene expression, and collagen production. These findings demonstrate that the HSPG modification enzyme SULF1 and HSPG sulfation are altered by CS in vitro and in vivo. Furthermore, these changes may contribute to CS-induced lung pathogenicity by affecting injury tolerance, hyperproliferation, and fibrotic effects.

Silver nanoparticles induce hormesis in A549 human epithelial cells.

Despite the gaps in our knowledge on the toxicity of silver nanoparticles (AgNPs), the application of these materials is fast expanding, from medicine, to food as well as the use in consumer products. It has been reported that prolonged exposure might make cells more resistant to AgNPs. This prompted us to investigate if AgNPs may give rise to a hormetic response. Two types of AgNPs were used, i.e. colloidal AgNPs and an AgNP powder. For both types of nanosilver it was found that a low dose pretreatment of A549 human epithelial cells with AgNPs induced protection against a toxic dose of AgNPs and acrolein. This protection was more pronounced after pretreatment with the colloidal AgNPs. Interestingly, the mechanism of the hormetic response appeared to differ from that of acrolein. Adaptation to acrolein is related to Nrf2 translocation, increased mRNA expression of γGCS, HO-1 and increased GSH levels and the increased GSH levels can explain the hormetic effect. The adaptive response to AgNPs was not related to an increase in mRNA expression of γGCS and GSH levels. Yet, HO-1 mRNA expression and Nrf2 immunoreactivity were enhanced, indicating that these processes might be involved. So, AgNPs induce adaptation, but in contrast to acrolein GSH plays no role.

Oxidant generation, DNA damage and cytotoxicity by a panel of engineered nanomaterials in three different human epithelial cell lines.

Due to the steeply increased use of nanomaterials (NMs) for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. In this study, we compared the DNA-damaging properties and concurrent cytotoxicity of a panel of 10 engineered NMs in three different cell lines in relation to their intrinsic oxidant generating properties. The human epithelial cell lines A549, HK-2 and HepG2 were chosen to represent relevant target organs for NMs in the lung, kidney and liver. Cytotoxicity, evaluated by WST-1 assay in the treatment concentration range of 0.3-80 µg/cm2, was shown for Ag and ZnO NM in all three cell lines. Cytotoxicity was absent for all other NMs, i.e. five types of TiO2 and two types of multiwalled carbon nanotubes. DNA damage, evaluated by the alkaline comet assay, was observed with Ag and ZnO, albeit only at cytotoxic concentrations. DNA damage varied considerably with the cell line. The oxidant generating properties of the NMs, evaluated by electron spin resonance spectroscopy in cell free conditions, did not correlate with their cytotoxic or DNA-damaging properties. DNA damage by the nanosilver could be partly attributed to its surfactant-containing dispersant. The coating of a TiO2 sample with the commercial surfactant Curosurf augmented its DNA-damaging properties in A549 cells, while surface modification with serum tended to reduce damage. Our findings indicate that measurement of the intrinsic oxidant-generating capacity of NMs is a poor predictor of DNA damage and that the cytotoxic and DNA-damaging properties of NMs can vary substantially with experimental conditions. Our study also underlines the critical importance of selecting appropriate cell systems and aligned testing protocols. Selection of a cell line on the mere basis of its origin may provide only poor insight on organ-specific hazards of NMs.

Air pollution and cytokine responsiveness in asthmatic and non-asthmatic children.

Epidemiological studies indicate that asthmatic children are more susceptible to traffic-related air pollution exposure than non-asthmatic children. Local and systemic inflammation in combination with oxidative stress have been suggested as a possible susceptibility factor. We investigated effect modification by asthma status for the association between air pollution exposure and systemic effects using whole blood cytokine responsiveness as an inflammatory marker. The study was nested within the two German birth cohort studies GINIplus and LISAplus and initially designed as a random sub-sample enriched with asthmatic children. Using data from 27 asthmatic and 59 non-asthmatic six-year-old children we measured the production of Interleukin-6 (IL)-6, IL-8, IL-10, monocyte chemotactic protein-1 (MCP-1), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in whole blood after ex-vivo stimulation with urban particulate matter (EHC-93). Air pollution exposure (nitrogen dioxide (NO2), nitrogen oxides (NOx), particulate matter with an aerodynamic diameter <10µm (PM10), particulate matter with an aerodynamic diameter <2.5µm (PM2.5mass), coarse particulate matter (PMcoarse) and PM2.5absorbance (PM2.5abs)) was modelled for children´s home addresses applying land-use regression. To assess effect modification by asthma status linear regression models with multiplicative interaction terms were used. In asthmatics exposure to NO2 was associated with higher production of pro-inflammatory cytokines: adjusted means ratio (MR) 2.22 (95% confidence interval 1.22-4.04) for IL-6 per 2.68µg/m³ NO2. The interaction term between asthma status and NO2 exposure was significant. Results for NOx, PM10, PM2.5mass and PM2.5abs were in the same direction. No association between air pollution and cytokine responsiveness was found in the group of non-asthmatic children and in the overall group. Traffic-related air pollution exposure is associated with higher pro-inflammatory cytokine responsiveness in whole blood of asthmatic children.

Silica-induced NLRP3 inflammasome activation in vitro and in rat lungs.

RATIONALE: Mineral particles in the lung cause inflammation and silicosis. In myeloid and bronchial epithelial cells the inflammasome plays a role in responses to crystalline silica. Thioredoxin (TRX) and its inhibitory protein TRX-interacting protein link oxidative stress with inflammasome activation. We investigated inflammasome activation by crystalline silica polymorphs and modulation by TRX in vitro, as well as its localization and the importance of silica surface reactivity in rats. METHODS: We exposed bronchial epithelial cells and differentiated macrophages to silica polymorphs quartz and cristobalite and measured caspase-1 activity as well as the release of IL-1ß, bFGF and HMGB1; including after TRX overexpression or treatment with recombinant TRX. Rats were intratracheally instilled with vehicle control, Dörentruper quartz (DQ12) or DQ12 coated with polyvinylpyridine N-oxide. At days 3, 7, 28, 90, 180 and 360 five animals per treatment group were sacrificed. Hallmarks of silicosis were assessed with Haematoxylin-eosin and Sirius Red stainings. Caspase-1 activity in the bronchoalveolar lavage and caspase-1 and IL-1ß localization in lung tissue were determined using Western blot and immunohistochemistry (IHC). RESULTS: Silica polymorphs triggered secretion of IL-1ß, bFGF and HMGB1 in a surface reactivity dependent manner. Inflammasome readouts linked with caspase-1 enzymatic activity were attenuated by TRX overexpression or treatment. At day 3 and 7 increased caspase-1 activity was detected in BALF of the DQ12 group and increased levels of caspase-1 and IL-1ß were observed with IHC in the DQ12 group compared to controls. DQ12 exposure revealed silicotic nodules at 180 and 360 days. Particle surface modification markedly attenuated the grade of inflammation and lymphocyte influx and attenuated the level of inflammasome activation, indicating that the development of silicosis and inflammasome activation is determined by crystalline silica surface reactivity. CONCLUSION: Our novel data indicate the pivotal role of surface reactivity of crystalline silica to activate the inflammasome in cultures of both epithelial cells and macrophages. Inhibitory capacity of the antioxidant TRX to inflammasome activation was evidenced. DQ12 quartz exposure induced acute and chronic functional activation of the inflammasome in the heterogeneous cell populations of the lung in associated with its crystalline surface reactivity.

A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes.

To determine the potential inhalatory risk posed by carbon nanotubes (CNTs), a tier-based approach beginning with an in vitro assessment must be adopted. The purpose of this study therefore was to compare 4 commonly used in vitro systems of the human lung (human blood monocyte-derived macrophages [MDM] and monocyte-derived dendritic cells [MDDC], 16HBE14o- epithelial cells, and a sophisticated triple cell co-culture model [TCC-C]) via assessment of the biological impact of different CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) over 24h. No significant cytotoxicity was observed with any of the cell types tested, although a significant (p < .05), dose-dependent increase in tumor necrosis factor (TNF)-α following SWCNT and MWCNT exposure at concentrations up to 0.02mg/ml to MDM, MDDC, and the TCC-C was found. The concentration of TNF-α released by the MDM and MDDC was significantly higher (p < .05) than the TCC-C. Significant increases (p < .05) in interleukin (IL)-8 were also found for both 16HBE14o- epithelial cells and the TCC-C after SWCNTs and MWCNTs exposure up to 0.02mg/ml. The TCC-C, however, elicited a significantly (p < .05) higher IL-8 release than the epithelial cells. The oxidative potential of both SWCNTs and MWCNTs (0.005-0.02mg/ml) measured by reduced glutathione (GSH) content showed a significant difference (p < .05) between each monoculture and the TCC-C. It was concluded that because only the co-culture system could assess each endpoint adequately, that, in comparison with monoculture systems, multicellular systems that take into consideration important cell type-to-cell type interactions could be used as predictive in vitro screening tools for determining the potential deleterious effects associated with CNTs.

Zinc oxide nanoparticles induce necrosis and apoptosis in macrophages in a p47phox- and Nrf2-independent manner.

In view of the steadily increasing use of zinc oxide nanoparticles in various industrial and consumer applications, toxicological investigations to evaluate their safety are highly justified. We have investigated mechanisms of ZnO nanoparticle-induced apoptosis and necrosis in macrophages in relation to their important role in the clearance of inhaled particulates and the regulation of immune responses during inflammation. In the murine macrophage RAW 264.7 cell line, ZnO treatment caused a rapid induction of nuclear condensation, DNA fragmentation, and the formation of hypodiploid DNA nuclei and apoptotic bodies. The involvement of the essential effector caspase-3 in ZnO-mediated apoptosis could be demonstrated by immunocytochemical detection of activated caspase-3 in RAW 264.7 cells. ZnO specifically triggered the intrinsic apoptotic pathway, because Jurkat T lymphocytes deficient in the key mediator caspase-9 were protected against ZnO-mediated toxicity whereas reconstituted cells were not. ZnO also caused DNA strand breakage and oxidative DNA damage in the RAW 264.7 cells as well as p47(phox) NADPH oxidase-dependent superoxide generation in bone marrow-derived macrophages. However, ZnO-induced cell death was not affected in bone marrow-derived macrophages of mice deficient in p47(phox) or the oxidant responsive transcription factor Nrf2. Taken together, our data demonstrate that ZnO nanoparticles trigger p47(phox) NADPH oxidase-mediated ROS formation in macrophages, but that this is dispensable for caspase-9/3-mediated apoptosis. Execution of apoptotic cell death by ZnO nanoparticles appears to be NADPH oxidase and Nrf2-independent but rather triggered by alternative routes.

Toxicology of ambient particulate matter.

It is becoming increasingly clear that inhalation exposure to particulate matter (PM) can lead to or exacerbate various diseases, which are not limited to the lung but extend to the cardiovascular system and possibly other organs and tissues. Epidemiological studies have provided strong evidence for associations with chronic obstructive pulmonary disease (COPD), asthma, bronchitis and cardiovascular disease, while the evidence for a link with lung cancer is less strong. Novel research has provided first hints that exposure to PM might lead to diabetes and central nervous system (CNS) pathology. In the current review, an overview is presented of the toxicological basis for adverse health effects that have been linked to PM inhalation. Oxidative stress and inflammation are discussed as central processes driving adverse effects; in addition, profibrotic and allergic processes are implicated in PM-related diseases. Effects of PM on key cell types considered as regulators of inflammatory, fibrotic and allergic mechanisms are described.

Neutrophils augment LPS-mediated pro-inflammatory signaling in human lung epithelial cells.

BACKGROUND: The role of polymorphonuclear neutrophils in pulmonary host defense is well recognized. The influence of a pre-existing inflammation driven by neutrophils (neutrophilic inflammation) on the airway epithelial response toward pro-inflammatory exogenous triggers, however, is still poorly addressed. Therefore, the aim of the present study is to investigate the effect of neutrophils on lipopolysaccharide (LPS)-induced pro-inflammatory signaling in lung epithelial cells. Additionally, underlying signaling pathways are examined. METHODS: Human bronchial epithelial cells (BEAS-2B) were co-incubated with human peripheral blood neutrophils or bone-marrow derived neutrophils from either C57BL/6J wild type or nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase deficient (p47(phox-/-)) mice. Upon stimulation with LPS, interleukin (IL)-8 production and reactive oxygen species (ROS) generation were measured. Additionally, activation of the extracellular signal-regulated kinases (ERK) 1/2 and nuclear factor (NF)-κB signaling pathways was analyzed. RESULTS: Our studies show that the presence of neutrophils synergistically increases LPS-induced IL-8 and ROS production by BEAS-2B cells without inducing cytotoxicity. The observed IL-8 response to endotoxin increases in proportion to time, LPS-concentration and the number of neutrophils present. Moreover, this synergistic IL-8 production strongly correlated with the chemotactic properties of the co-incubations and significantly depended on a functional neutrophilic NADPH oxidase. The presence of neutrophils also augments LPS-induced phosphorylation of ERK1/2 and IκBα as well as NF-κB RelA DNA binding activity in BEAS-2B cells. CONCLUSIONS: Our results indicate that the pro-inflammatory effects of LPS toward lung epithelial cells are amplified during a pre-existing neutrophilic inflammation. These findings support the concept that patients suffering from pulmonary neutrophilic inflammation are more susceptible toward exogenous pro-inflammatory triggers.

Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells.

Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO(2) particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO(2)-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO(2)-toxicity toward intestinal cells.

Evaluation of apoptosis induced by nanoparticles and fine particles in RAW 264.7 macrophages: facts and artefacts.

Current hazard characterisation of nanoparticles (NP) is predominantly based on in vitro test systems, being established for small molecules of drugs and chemicals. However, specific physicochemical properties of NP may result in interference with assay components, biomarkers, or detection systems. In the present study, six types of (nano)particles were screened in RAW 264.7 macrophages by common cytotoxicity methods (WST-1, LDH). Our specific focus was on the investigation of apoptosis (analysis of hypodiploid DNA, phosphatidylserine exposure, caspase 3/7 activation, and Cell Death Detection ELISA). Assays were validated by the well-known apoptosis inducer staurosporine. Our results show that ZnO, DQ12 quartz and amorphous silica are cytotoxic with strong indications for apoptotic effects in RAW 264.7 macrophages, whereas toxicity was absent for MgO. For fine as well as ultrafine TiO(2), no apoptotic effects could be detected except for induction of DNA fragmentation. The results of our study demonstrate the necessity to control on a case-by-case basis for assay interference to avoid misinterpretation of specific in vitro test findings. To obtain valid statements on the potential induction of apoptosis by specific NP the measurement of multiple endpoints is a prerequisite.

Contrasting macrophage activation by fine and ultrafine titanium dioxide particles is associated with different uptake mechanisms.

Inhalation of (nano)particles may lead to pulmonary inflammation. However, the precise mechanisms of particle uptake and generation of inflammatory mediators by alveolar macrophages (AM) are still poorly understood. The aim of this study was to investigate the interactions between particles and AM and their associated pro-inflammatory effects in relation to particle size and physico-chemical properties.NR8383 rat lung AM were treated with ultrafine (uf), fine (f) TiO2 or fine crystalline silica (DQ12 quartz). Physico-chemical particle properties were investigated by transmission electron microscopy, elemental analysis and thermogravimetry. Aggregation and agglomeration tendency of the particles were determined in assay-specific suspensions by means of dynamic light scattering.All three particle types were rapidly taken up by AM. DQ12 and ufTiO2 , but not fTiO2 , caused increased extracellular reactive oxygen species (ROS), heme oxygenase 1 (HO-1) mRNA expression and tumor necrosis factor (TNF)-α release. Inducible nitric oxide synthase (iNOS) mRNA expression was increased most strongly by ufTiO2 , while DQ12 exclusively triggered interleukin (IL) 1ß release. However, oscillations of intracellular calcium concentration and increased intracellular ROS were observed with all three samples. Uptake inhibition experiments with cytochalasin D, chlorpromazine and a Fcγ receptor II (FcγRII) antibody revealed that the endocytosis of fTiO2 by the macrophages involves actin-dependent phagocytosis and macropinocytosis as well as clathrin-coated pit formation, whereas the uptake of ufTiO2 was dominated by FcγIIR. The uptake of DQ12 was found to be significantly reduced by all three inhibitors. Our findings suggest that the contrasting AM responses to fTiO2 , ufTiO2 and DQ12 relate to differences in the involvement of specific uptake mechanisms.

Surface iron inhibits quartz-induced cytotoxic and inflammatory responses in alveolar macrophages.

The mechanism of enhancement/inhibition of quartz toxicity induced by iron is still unclear. Here the amount of iron on a fibrogenic quartz (Qz) was increased by wet impregnation (Fe(NO(3))(3) 0.67 and 6.7 wt %). X-ray diffraction (XRD), XRF diffuse reflectance, UV-vis, and infrared (IR) spectroscopies revealed dispersed ferric ions, and hematite aggregates at the higher loading. Surface features relevant to pathogenicity and cell responses were compared not only to the original quartz but also to reference quartz DQ12. Surface charge (ζ-potential) was more negative on the original and low-loaded specimen than on the high-loaded one. DQ12 had a less negative ζ-potential than Qz, ascribed to the absence of aluminium present in Qz (1.7 wt %). All quartz specimens were able to generate HO(•) radicals, iron-loaded samples being more reactive than original quartz. Iron deposition inhibited the rupture of a C-H bond. All quartzes were phagocytized by alveolar macrophages (AMΦ cell line NR8383) to the same extent, irrespective of their surface state. Conversely, iron loading increased AMΦ viability (evaluated by cytotoxicity and induction of apoptosis). Qz was found to be much less cytotoxic than DQ12. The induction of oxidative stress and inflammatory responses (evaluated by HO-1 mRNA expression and TNF-α mRNA and protein expression) revealed a reduction in inflammogenicity upon iron loading and a more inflammogenic potency of DQ12 ascribed to undissociated SiOH interacting via H-bonding with cell membrane components. The results suggest that besides aluminium also iron at the quartz surface may have an inhibitory effect on adverse health responses.

Neutrophil-derived ROS contribute to oxidative DNA damage induction by quartz particles.

The carcinogenicity of respirable quartz is considered to be driven by reactive oxygen species (ROS) generation in association with chronic inflammation. The contribution of phagocyte-derived ROS to inflammation, oxidative stress, and DNA damage responses was investigated in the lungs of C57BL/6J wild-type and p47(phox-/-) mice, 24h after pharyngeal aspiration of DQ12 quartz (100 mg/kg bw). Bone-marrow-derived neutrophils from wild-type and p47(phox-/-) mice were used for parallel in vitro investigations in coculture with A549 human alveolar epithelial cells. Quartz induced a marked neutrophil influx in both wild-type and p47(phox-/-) mouse lungs. Significant increases in mRNA expression of the oxidative stress markers HO-1 and γ-GCS were observed only in quartz-treated wild-type animals. Oxidative DNA damage in lung tissue was not affected by quartz exposure and did not differ between p47(phox-/-) and WT mice. Differences in mRNA expression of the DNA repair genes OGG1, APE-1, DNA Polß, and XRCC1 were also absent. Quartz treatment of cocultures containing wild-type neutrophils, but not p47(phox-/-) neutrophils, caused increased oxidative DNA damage in epithelial cells. Our study demonstrates that neutrophil-derived ROS significantly contribute to pulmonary oxidative stress responses after acute quartz exposure, yet their role in the associated induction of oxidative DNA damage could be shown only in vitro.