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

<p>Abstract</p> <p>Background</p> <p>The objective of this study was to examine the threshold fibre length for the onset of pulmonary inflammation after aspiration exposure in mice to four different lengths of silver nanowires (AgNW). We further examined the effect of fibre length on macrophage locomotion in an <it>in vitro</it> wound healing assay. We hypothesised that exposure to longer fibres causes both increased inflammation and restricted mobility leading to impaired clearance of long fibres from the lower respiratory tract to the mucociliary escalator <it>in vivo</it>.</p> <p>Methods</p> <p>Nine week old female C57BL/6 strain mice were exposed to AgNW and controls via pharyngeal aspiration. The dose used in this study was equalised to fibre number and based on 50 μg/ mouse for AgNW₁₄. To examine macrophage migration <it>in vitro</it> a wound healing assay was used. An artificial wound was created in a confluent layer of bone marrow derived macrophages by scraping with a pipette tip and the number of cells migrating into the wound was monitored microscopically. The dose was equalised for fibre number and based on 2.5 μg/cm² for AgNW_14.</p> <p>Results</p> <p>Aspiration of AgNW resulted in a length dependent inflammatory response in the lungs with threshold at a fibre length of 14 μm. Shorter fibres including 3, 5 and 10 μm elicited no significant inflammation. Macrophage locomotion was also restricted in a length dependent manner whereby AgNW in the length of ≥5 μm resulted in impaired motility in the wound closure assay.</p> <p>Conclusion</p> <p>We demonstrated a 14 μm cut-off length for fibre-induced pulmonary inflammation after aspiration exposure and an <it>in vitro</it> threshold for inhibition of macrophage locomotion of 5 μm. We previously reported a threshold length of 5 μm for fibre-induced pleural inflammation. This difference in pulmonary and pleural fibre- induced inflammation may be explained by differences in clearance mechanism of deposited fibres from the airspaces compared to the pleural space. Inhibition of macrophage migration at long fibre lengths could account for their well-documented long term retention in the lungs compared to short fibres. Knowledge of the threshold length for acute pulmonary inflammation contributes to hazard identification of nanofibres.</p

Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma

The unique hazard posed to the pleural mesothelium by asbestos has engendered concern in potential for a similar risk from high aspect ratio nanoparticles (HARN) such as carbon nanotubes. In the course of studying the potential impact of HARN on the pleura we have utilised the existing hypothesis regarding the role of the parietal pleura in the response to long fibres. This review seeks to synthesise our new data with multi-walled carbon nanotubes (CNT) with that hypothesis for the behaviour of long fibres in the lung and their retention in the parietal pleura leading to the initiation of inflammation and pleural pathology such as mesothelioma. We describe evidence that a fraction of all deposited particles reach the pleura and that a mechanism of particle clearance from the pleura exits, through stomata in the parietal pleura. We suggest that these stomata are the site of retention of long fibres which cannot negotiate them leading to inflammation and pleural pathology including mesothelioma. We cite thoracoscopic data to support the contention, as would be anticipated from the preceding, that the parietal pleura is the site of origin of pleural mesothelioma. This mechanism, if it finds support, has important implications for future research into the mesothelioma hazard from HARN and also for our current view of the origins of asbestos-initiated pleural mesothelioma and the common use of lung parenchymal asbestos fibre burden as a correlate of this tumour, which actually arises in the parietal pleura

Combustion-derived nanoparticles: A review of their toxicology following inhalation exposure

This review considers the molecular toxicology of combustion-derived nanoparticles (CDNP) following inhalation exposure. CDNP originate from a number of sources and in this review we consider diesel soot, welding fume, carbon black and coal fly ash. A substantial literature demonstrates that these pose a hazard to the lungs through their potential to cause oxidative stress, inflammation and cancer; they also have the potential to redistribute to other organs following pulmonary deposition. These different CDNP show considerable heterogeneity in composition and solubility, meaning that oxidative stress may originate from different components depending on the particle under consideration. Key CDNP-associated properties of large surface area and the presence of metals and organics all have the potential to produce oxidative stress. CDNP may also exert genotoxic effects, depending on their composition. CDNP and their components also have the potential to translocate to the brain and also the blood, and thereby reach other targets such as the cardiovascular system, spleen and liver. CDNP therefore can be seen as a group of particulate toxins unified by a common mechanism of injury and properties of translocation which have the potential to mediate a range of adverse effects in the lungs and other organs and warrant further research

Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes

Background: Large production volumes of zinc oxide nanoparticles (ZnONP) might be anticipated to pose risks, of accidental inhalation in occupational and even in consumer settings. Herein, we further investigated the pathological changes induced by ZnONP and their possible mechanism of action.Methods: Two doses of ZnONP (50 and 150 cm(2)/rat) were intratracheally instilled into the lungs of rats with assessments made at 24 h, 1 wk, and 4 wks after instillation to evaluate dose- and time-course responses. Assessments included bronchoalveolar lavage (BAL) fluid analysis, histological analysis, transmission electron microscopy, and IgE and IgA measurement in the serum and BAL fluid. To evaluate the mechanism, alternative ZnONP, ZnONP-free bronchoalveolar lavage exudate, and dissolved Zn2+ (92.5 mu g/rat) were also instilled to rats. Acridine orange staining was utilized in macrophages in culture to evaluate the lysosomal membrane destabilization by NP.Results: ZnONP induced eosinophilia, proliferation of airway epithelial cells, goblet cell hyperplasia, and pulmonary fibrosis. Bronchocentric interstitial pulmonary fibrosis at the chronic phase was associated with increased myofibroblast accumulation and transforming growth factor-beta positivity. Serum IgE levels were up-regulated by ZnONP along with the eosinophilia whilst serum IgA levels were down-regulated by ZnONP. ZnONP are rapidly dissolved under acidic conditions (pH 4.5) whilst they remained intact around neutrality (pH 7.4). The instillation of dissolved Zn2+ into rat lungs showed similar pathologies (eg., eosinophilia, bronchocentric interstitial fibrosis) as were elicited by ZnONP. Lysosomal stability was decreased and cell death resulted following treatment of macrophages with ZnONP in vitro.Conclusions: We hypothesise that rapid, pH-dependent dissolution of ZnONP inside of phagosomes is the main cause of ZnONP-induced diverse progressive severe lung injuries

Efficacy of Simple Short-Term in Vitro Assays for Predicting the Potential of Metal Oxide Nanoparticles to Cause Pulmonary Inflammation

Background: There has been concern regarding risks from inhalation exposure to nanoparticles (NPs). The large number of particles requiring testing means that alternative approaches to animal testing are needed. Objectives: We set out to determine whether short-term in vitro assays that assess intrinsic oxidative stress potential and membrane-damaging potency of a panel of metal oxide NPs can be used to predict their inflammogenic potency. Methods: For a panel of metal oxide NPs, we investigated intrinsic free radical generation, oxidative activity in an extracellular environment, cytotoxicity to lung epithelial cells, hemolysis, and inflammation potency in rat lungs. All exposures were carried out at equal surface area doses. Results: Only nickel oxide (NiO) and alumina 2 caused significant lung inflammation when instilled into rat lungs at equal surface area, suggesting that these two had extra surface reactivity. We observed significant free radical generation with 4 of 13 metal oxides, only one of which was inflammogenic. Only 3 of 13 were significantly hemolytic, two of which were inflammogenic. Conclusions: Potency in generating free radicals in vitro did not predict inflammation, whereas alumina 2 had no free radical activity but was inflammogenic. The hemolysis assay was correct in predicting the proinflammatory potential of 12 of 13 of the particles examined. Using a battery of simple in vitro tests, it is possible to predict the inflammogenicity of metal oxide NPs, although some false-positive results are likely. More research using a larger panel is needed to confirm the efficacy and generality of this approach for metal oxide NPs

Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles

BACKGROUND: Hazard identification for risk assessment of nanoparticles (NPs) is mainly composed of in vitro cell-based assays and in vivo animal experimentation. The rapidly increasing number and functionalizations of NPs makes in vivo toxicity tests undesirable on both ethical and financial grounds, creating an urgent need for development of in vitro cell-based assays that accurately predict in vivo toxicity and facilitate safe nanotechnology. METHODS: In this study, we used 9 different NPs (CeO(2), TiO(2), carbon black, SiO(2), NiO, Co(3)O(4), Cr(2)O(3), CuO, and ZnO). As an in vivo toxicity endpoint, the acute lung inflammogenicity in a rat instillation model was compared with the in vitro toxicity endpoints comprising cytotoxicity, pro-inflammatory cytokine expression, or haemolytic potential. For in vitro assays, 8 different cell-based assays were used including epithelial cells, monocytic/macrophage cells, human erythrocytes, and combined culture. RESULTS: ZnO and CuO NPs acting via soluble toxic ions showed positive results in most of assays and were consistent with the lung inflammation data. When compared in in vitro assays at the same surface area dose (30 cm(2)/mL), NPs that were low solubility and therefore acting via surface reactivity had no convincing activity, except for CeO(2) NP. Cytotoxicity in differentiated peripheral blood mononuclear cells was the most accurate showing 89% accuracy and 11% false negativity in predicting acute lung inflammogenicity. However, the haemolysis assay showed 100% consistency with the lung inflammation if any dose, having statistical significance was considered positivity. Other cell-based in vitro assays showed a poorer correlation with in vivo inflammogenicity. CONCLUSIONS: Based on the toxicity mechanisms of NPs, two different approaches can be applied for prediction of in vivo lung inflammogenicity. Most in vitro assays were good at detecting NPs that act via soluble ions (i.e., ZnO and CuO NP). However, in vitro assays were limited in detecting NPs acting via surface reactivity as their mechanism of toxicity, except for the haemolysis assay

Platelet activation independent of pulmonary inflammation contributes to diesel exhaust particulate-induced promotion of arterial thrombosis

BACKGROUND: Accelerated thrombus formation induced by exposure to combustion-derived air pollution has been linked to alterations in endogenous fibrinolysis and platelet activation in response to pulmonary and systemic inflammation. We hypothesised that mechanisms independent of inflammation contribute to accelerated thrombus formation following exposure to diesel exhaust particles (DEP). METHODS: Thrombosis in rats was assessed 2, 6 and 24 h after administration of DEP, carbon black (CB; control carbon nanoparticle), DQ12 quartz microparticles (to induce pulmonary inflammation) or saline (vehicle) by either intra-tracheal instillation (0.5 mg, except Quartz; 0.125 mg) or intravenous injection (0.5 mg/kg). Thrombogenicity was assessed by carotid artery occlusion, fibrinolytic variables and platelet-monocyte aggregates. Measures of inflammation were determined in plasma and bronchoalveolar lavage fluid. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI)-1 were measured following direct in vitro exposure of human umbilical vein endothelial cells (HUVECs) to DEP (10–150 μg/mL). RESULTS: Instillation of DEP reduced the time to thrombotic occlusion in vivo, coinciding with the peak of DEP-induced pulmonary inflammation (6 h). CB and DQ12 produced greater inflammation than DEP but did not alter time to thrombotic occlusion. Intravenous DEP produced an earlier (2 h) acceleration of thrombosis (as did CB) without pulmonary or systemic inflammation. DEP inhibited t-PA and PAI-1 release from HUVECs, and reduced the t-PA/PAI-1 ratio in vivo; similar effects in vivo were seen with CB and DQ12. DEP, but not CB or DQ12, increased platelet-monocyte aggregates. CONCLUSION: DEP accelerates arterial thrombus formation through increased platelet activation. This effect is dissociated from pulmonary and systemic inflammation and from impaired fibrinolytic function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-016-0116-x) contains supplementary material, which is available to authorized users

Inhaled Nanoparticles Accumulate at Sites of Vascular Disease

The development of engineered nanomaterials is growing exponentially, despite concerns over their potential similarities to environmental nanoparticles that are associated with significant cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental unanswered question remains: Do inhaled nanoparticles translocate from the lung in man and directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting detection by high-resolution inductively coupled mass spectrometry and Raman microscopy. Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated sampling of blood and urine. Gold was detected in the blood and urine within 15 min to 24 h after exposure, and was still present 3 months after exposure. Levels were greater following inhalation of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated the accumulation in the blood and liver following pulmonary exposure to a broader size range of gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles <10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of vascular inflammation provides a direct mechanism that can explain the link between environmental nanoparticles and cardiovascular disease and has major implications for risk management in the use of engineered nanomaterials

Metal Oxide Nanoparticles Induce Unique Inflammatory Footprints in the Lung: Important Implications for Nanoparticle Testing

BACKGROUND: Metal oxide nanoparticles (NPs) have been widely used in industry, cosmetics, and biomedicine. OBJECTIVES: We examined hazards of several well-characterized high production volume NPs because of increasing concern about occupational exposure via inhalation. METHODS: A panel of well-characterized NPs [cerium oxide (CeO(2)NP), titanium dioxide (TiO(2)NP), carbon black (CBNP), silicon dioxide (SiO(2)NP), nickel oxide (NiONP), zinc oxide (ZnONP), copper oxide (CuONP), and amine-modified polystyrene beads] was instilled into lungs of rats. We evaluated the inflammation potencies of these NPs 24 hr and 4 weeks postinstillation. For NPs that caused significant inflammation at 24 hr, we then investigated the characteristics of the inflammation. All exposures were carried out at equal-surface-area doses. RESULTS: Only CeO(2)NP, NiONP, ZnONP, and CuONP were inflammogenic to the lungs of rats at the high doses used. Strikingly, each of these induced a unique inflammatory footprint both acutely (24 hr) and chronically (4 weeks). Acutely, patterns of neutrophil and eosinophil infiltrates differed after CeO(2)NP, NiONP, ZnONP, and CuONP treatment. Chronic inflammatory responses also differed after 4 weeks, with neutrophilic, neutrophilic/lymphocytic, eosinophilic/fibrotic/granulomatous, and fibrotic/granulomatous inflammation being caused respectively by CeO(2)NP, NiONP, ZnONP, and CuONP. CONCLUSION: Different types of inflammation imply different hazards in terms of pathology, risks, and risk severity. In vitro testing could not have differentiated these complex hazard outcomes, and this has important implications for the global strategy for NP hazard assessment. Our results demonstrate that NPs cannot be viewed as a single hazard entity and that risk assessment should be performed separately and with caution for different NPs