Effects of subchronic dietary exposure to the engineered nanomaterials SiO2 and CeO2 in C57BL/6J and 5xFAD Alzheimer model mice.

BACKGROUND: There is an increasing concern about the neurotoxicity of engineered nanomaterials (NMs). To investigate the effects of subchronic oral exposures to SiO2 and CeO2 NMs on Alzheimer's disease (AD)-like pathology, 5xFAD transgenic mice and their C57BL/6J littermates were fed ad libitum for 3 or 14 weeks with control food pellets, or pellets dosed with these respective NMs at 0.1% or 1% (w/w). Behaviour effects were evaluated by X-maze, string suspension, balance beam and open field tests. Brains were analysed for plaque load, beta-amyloid peptide levels, markers of oxidative stress and neuroinflammation. RESULTS: No marked behavioural impairments were observed in the mice exposed to SiO2 or CeO2 and neither treatment resulted in accelerated plaque formation, increased oxidative stress or inflammation. In contrast, the 5xFAD mice exposed to 1% CeO2 for 14 weeks showed significantly lower hippocampal Aß plaque load and improved locomotor activity compared to the corresponding controls. CONCLUSIONS: The findings from the present study suggest that long-term oral exposure to SiO2 or CeO2 NMs has no neurotoxic and AD-promoting effects. The reduced plaque burden observed in the mice following dietary CeO2 exposure warrants further investigation to establish the underlying mechanism, given the easy applicability of this administration method.

Ingested Engineered Nanomaterials Affect the Expression of Mucin Genes-An In Vitro-In Vivo Comparison.

The increasing use of engineered nanomaterials (ENM) in food has fueled the development of intestinal in vitro models for toxicity testing. However, ENM effects on intestinal mucus have barely been addressed, although its crucial role for intestinal health is evident. We investigated the effects of ENM on mucin expression and aimed to evaluate the suitability of four in vitro models of increasing complexity compared to a mouse model exposed through feed pellets. We assessed the gene expression of the mucins MUC1, MUC2, MUC5AC, MUC13 and MUC20 and the chemokine interleukin-8 in pre-confluent and confluent HT29-MTX-E12 cells, in stable and inflamed triple cultures of Caco-2, HT29-MTX-E12 and THP-1 cells, and in the ileum of mice following exposure to TiO2, Ag, CeO2 or SiO2. All ENM had shared and specific effects. CeO2 downregulated MUC1 in confluent E12 cells and in mice. Ag induced downregulation of Muc2 in mice. Overall, the in vivo data were consistent with the findings in the stable triple cultures and the confluent HT29-MTX-E12 cells but not in pre-confluent cells, indicating the higher relevance of advanced models for hazard assessment. The effects on MUC1 and MUC2 suggest that specific ENM may lead to an elevated susceptibility towards intestinal infections and inflammations.

Effects of dietary exposure to the engineered nanomaterials CeO2, SiO2, Ag, and TiO2 on the murine gut microbiome.

Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent ad libitum feeding studies were performed. In study 1, female mice from the local breeding facility received feed pellets containing 1% CeO2 or 1% SiO2 for three weeks. In study 2, both female and male mice were purchased and exposed to 0.2% Ag-PVP or 1% TiO2 for four weeks. A next generation 16S rDNA sequencing-based approach was applied to assess impacts on the gut microbiome. None of the ENM had an effect on the α- or ß-diversity. A decreased relative abundance of the phylum Actinobacteria was observed in SiO2 exposed mice. In female mice, the relative abundance of the genus Roseburia was increased with Ag exposure. Furthermore, in study 2, a sex-related difference in the ß-diversity was observed. A difference in the ß-diversity was also shown between the female control mice of the two studies. We did not find major effects on the gut microbiome. This contrast to other studies may be due to variations in the study design. Our investigation underlined the important role of the sex of test animals and their microbiome composition prior to ENM exposure initiation. Hence, standardization of microbiome studies is strongly required to increase comparability. The ENM-specific effects on Actinobacteria and Roseburia, two taxa pivotal for the human gut homeostasis, warrant further research on their relevance for health.

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.

Evaluation of the neurotoxic effects of engineered nanomaterials in C57BL/6J mice in 28-day oral exposure studies.

In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays. Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO2. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO2 and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.

Model Complexity as Determining Factor for In Vitro Nanosafety Studies: Effects of Silver and Titanium Dioxide Nanomaterials in Intestinal Models.

With the rising interest in the effects of orally ingested engineered nanomaterials (ENMs), much effort is undertaken to develop and advance intestinal in vitro models. The cytotoxic, proinflammatory, and DNA damaging properties of polyvinylpyrrolidone-capped silver (Ag-PVP) and titanium dioxide (TiO2 , P25) ENM in four in vitro models of increasing complexity-from proliferating Caco-2 and HT29-MTX-E12 monocultures to long-term transwell triple cultures including THP-1 macrophages to reproduce the human intestine in healthy versus inflamed-like state-are studied. Results are compared against in vivo effects of the same ENM through intestinal tissue analysis from 28-day oral exposure studies in mice. Adverse responses are only observed in monocultures and suggest toxic potential for both ENM, typically showing stronger effects for Ag-PVP than for TiO2 . By contrast, no adverse effects are observed in either the transwell cultures or the analyzed murine tissues. The data provide further support that monoculture models represent a cost and time efficient tool for early-phase hazard assessment. However, the observed similarities in morphology and ENM effects in murine intestinal tissue and the in vitro triple culture model suggest that advanced multifacetted research questions concerning oral ENM exposure are more adequately addressed by the more complex and time intensive models.

Evaluation of the NLRP3 Inflammasome Activating Effects of a Large Panel of TiO2 Nanomaterials in Macrophages.

TiO2 nanomaterials are among the most commonly produced and used engineered nanomaterials (NMs) in the world. There is controversy regarding their ability to induce inflammation-mediated lung injuries following inhalation exposure. Activation of the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome and subsequent release of the cytokine interleukin (IL)-1ß in pulmonary macrophages has been postulated as an essential pathway for the inflammatory and associated tissue-remodeling effects of toxic particles. Our study aim was to determine and rank the IL-1ß activating properties of TiO2 NMs by comparing a large panel of different samples against each other as well as against fine TiO2, synthetic amorphous silica and crystalline silica (DQ12 quartz). Effects were evaluated in primary bone marrow derived macrophages (BMDMs) from NALP3-deficient and proficient mice as well as in the rat alveolar macrophage cell line NR8383. Our results show that specific TiO2 NMs can activate the inflammasome in macrophages albeit with a markedly lower potency than amorphous SiO2 and quartz. The heterogeneity in IL-1ß release observed in our study among 19 different TiO2 NMs underscores the relevance of case-by-case evaluation of nanomaterials of similar chemical composition. Our findings also further promote the NR8383 cell line as a promising in vitro tool for the assessment of the inflammatory and inflammasome activating properties of NMs.

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.

Evaluation of neurological effects of cerium dioxide nanoparticles doped with different amounts of zirconium following inhalation exposure in mouse models of Alzheimer's and vascular disease.

Increasing evidence from toxicological and epidemiological studies indicates that the brain is an important target for ambient (ultrafine) particles. Disturbance of redox-homeostasis and inflammation in the brain are proposed as possible mechanisms that can contribute to neurotoxic and neurodegenerative effects. Whether and how engineered nanoparticles (NPs) may cause neurotoxicity and promote neurodegenerative diseases such as Alzheimer's disease (AD) is largely unstudied. We have assessed the neurological effects of subacute inhalation exposures (4 mg/m3 for 3 h/day, 5 days/week for 4 weeks) to cerium dioxide (CeO2) NPs doped with different amounts of zirconium (Zr, 0%, 27% and 78%), to address the influence of particle redox-activity in the 5xFAD transgenic mouse model of AD. Four weeks post-exposure, effects on behaviour were evaluated and brain tissues were analysed for amyloid-ß plaque formation and reactive microglia (Iba-1 staining). Behaviour was also evaluated in concurrently exposed non-transgenic C57BL/6J littermates, as well as in Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice as a model of vascular disease. Markers of inflammation and oxidative stress were evaluated in brain cortex. The brains of the NP-exposed 5xFAD mice revealed no accelerated amyloid-ß plaque formation. No significant treatment-related behaviour impairments were observed in the healthy C57BL/6J mice. In the 5xFAD and ApoE-/- models, the NP inhalation exposures did not affect the alternation score in the X-maze indicating absence of spatial working memory deficits. However, following inhalation exposure to the 78% Zr-doped CeO2 NPs changes in forced motor performance (string suspension) and exploratory motor activity (X-maze) were observed in ApoE-/- and 5xFAD mice, respectively. Exposure to the 78% doped NPs also caused increased cortical expression of glial fibrillary acidic protein (GFAP) in the C57BL/6J mice. No significant treatment-related changes neuroinflammation and oxidative stress were observed in the 5xFAD and ApoE-/- mice. Our study findings reveal that subacute inhalation exposure to CeO2 NPs does not accelerate the AD-like phenotype of the 5xFAD model. Further investigation is warranted to unravel whether the redox-activity dependent effects on motor activity as observed in the mouse models of AD and vascular disease result from specific neurotoxic effects of these NPs.

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.

Diesel engine exhaust accelerates plaque formation in a mouse model of Alzheimer's disease.

BACKGROUND: Increasing evidence from toxicological and epidemiological studies indicates that the central nervous system is an important target for ambient air pollutants. We have investigated whether long-term inhalation exposure to diesel engine exhaust (DEE), a dominant contributor to particulate air pollution in urban environments, can aggravate Alzheimer's Disease (AD)-like effects in female 5X Familial AD (5XFAD) mice and their wild-type female littermates. Following 3 and 13 weeks exposures to diluted DEE (0.95 mg/m3, 6 h/day, 5 days/week) or clean air (controls) behaviour tests were performed and amyloid-ß (Aß) plaque formation, pulmonary histopathology and systemic inflammation were evaluated. RESULTS: In a string suspension task, assessing for grip strength and motor coordination, 13 weeks exposed 5XFAD mice performed significantly less than the 5XFAD controls. Spatial working memory deficits, assessed by Y-maze and X-maze tasks, were not observed in association with the DEE exposures. Brains of the 3 weeks DEE-exposed 5XFAD mice showed significantly higher cortical Aß plaque load and higher whole brain homogenate Aß42 levels than the clean air-exposed 5XFAD littermate controls. After the 13 weeks exposures, with increasing age and progression of the AD-phenotype of the 5XFAD mice, DEE-related differences in amyloid pathology were no longer present. Immunohistochemical evaluation of lungs of the mice revealed no obvious genetic background-related differences in tissue structure, and the DEE exposure did not cause histopathological changes in the mice of both backgrounds. Luminex analysis of plasma cytokines demonstrated absence of sustained systemic inflammation upon DEE exposure. CONCLUSIONS: Inhalation exposure to DEE causes accelerated plaque formation and motor function impairment in 5XFAD transgenic mice. Our study provides further support that the brain is a relevant target for the effects of inhaled DEE and suggests that long-term exposure to this ubiquitous air pollution mixture may promote the development of Alzheimer's disease.

The effect of zirconium doping of cerium dioxide nanoparticles on pulmonary and cardiovascular toxicity and biodistribution in mice after inhalation.

Development and manufacture of nanomaterials is growing at an exponential rate, despite an incomplete understanding of how their physicochemical characteristics affect their potential toxicity. Redox activity has been suggested to be an important physicochemical property of nanomaterials to predict their biological activity. This study assessed the influence of redox activity by modification of cerium dioxide nanoparticles (CeO2 NPs) via zirconium (Zr) doping on the biodistribution, pulmonary and cardiovascular effects in mice following inhalation. Healthy mice (C57BL/6 J), mice prone to cardiovascular disease (ApoE-/-, western-diet fed) and a mouse model of neurological disease (5 × FAD) were exposed via nose-only inhalation to CeO2 NPs with varying amounts of Zr-doping (0%, 27% or 78% Zr), or clean air, over a four-week period (4 mg/m3 for 3 h/day, 5 days/week). Effects were assessed four weeks post-exposure. In all three mouse models CeO2 NP exposure had no major toxicological effects apart from some modest inflammatory histopathology in the lung, which was not related to the amount of Zr-doping. In ApoE-/- mice CeO2 did not change the size of atherosclerotic plaques, but there was a trend towards increased inflammatory cell content in relation to the Zr content of the CeO2 NPs. These findings show that subacute inhalation of CeO2 NPs causes minimal pulmonary and cardiovascular effect four weeks post-exposure and that Zr-doping of CeO2 NPs has limited effect on these responses. Further studies with nanomaterials with a higher inherent toxicity or a broader range of redox activities are needed to fully assess the influence of redox activity on the toxicity of nanomaterials.

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.

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.

Signalling-dependent adverse health effects of carbon nanoparticles are prevented by the compatible solute mannosylglycerate (firoin) in vitro and in vivo.

The inhalation of combustion-derived nanoparticles leads to adverse health effects in the airways. In this context the induction of membrane-coupled signalling is considered as causative for changes in tissue homeostasis and pro-inflammatory reactions. The identification of these molecular cell reactions allowed to seek for strategies which interfere with these adverse effects. In the current study, we investigated the structurally different compatible solutes mannosylglycerate (firoin) from thermophilic bacteria and ectoine from halophilic bacteria for their capability to reduce signalling pathways triggered by carbon nanoparticles in target cells in the lung. The pre-treatment of lung epithelial cells with both substances decreased the particle-specific activation of mitogen-activated protein kinases and also the endpoints proliferation and apoptosis. Firoin applied into the lungs of animals, like ectoine, led to a significant reduction of the neutrophilic lung inflammation induced by particle exposure. The pro-inflammatory effect of carbon nanoparticles on human neutrophil granulocytes ex vivo was significantly reduced by both substances via the reduction of the anti-apoptotic membrane-dependent signalling. The data of this study together with earlier studies demonstrate that two structurally non-related compatible solutes are able to prevent pathogenic reactions of the airways to carbon nanoparticles by interfering with signalling events. The findings highlight the preventive or therapeutic potential of compatible solutes for adverse health effects caused by particle exposure of the airways.

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.

Influence of simulated gastrointestinal conditions on particle-induced cytotoxicity and interleukin-8 regulation in differentiated and undifferentiated Caco-2 cells.

Novel aspects of engineered nanoparticles offer many advantages for optimising food products and packaging. However, their potential hazards in the gastrointestinal tract require further investigation. We evaluated the toxic and inflammatory potential of two types of particles that might become increasingly relevant to the food industry, namely SiO2 and ZnO. The materials were characterised for their morphology, oxidant generation and hydrodynamic behaviour. Cytotoxicity and interleukin-8 mRNA and protein expression were evaluated in human intestinal Caco-2 cells. Particle pretreatment under simulated gastric and intestinal pH conditions resulted in reduced acellular ROS formation but did not influence cytotoxicity (WST-1 assay) or IL-8 expression. However, the differentiation status of the cells markedly determined the cytotoxic potency of the particles. Further research is needed to determine the in vivo relevance of our current observations regarding the role of particle aggregation and the stage of intestinal epithelial cell differentiation in determining the hazards of ingested particles.

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.