Molecular toxicology studies on the quartz hazard.

Silicon makes up almost 28% of the Earth's crust and within that crust, quartz (crystalline silica) is one of the most abundant minerals. Exposure to quartz can occurin a number of occupations, including the mining and construction industries in which respirable quartz particles are generated and become airborne. Inhalation of quartz canlead to the fibrosing lung disease silicosis and cancer. Silicosis has been recognised for many decades as one of the most prevalent occupational lung diseases. In 1997, anIARC working Group classified quartz as a class 1 lung carcinogen, but only in some industries, suggesting that the quartz hazard is a variable entity. The reactivity of thequartz surface may underlie its ability to cause inflammation and treatments that ameliorate this reactivity would then reduce the quartz hazard.In the present study the effect of treating quartz with aluminium lactate, a procedure reported to decrease the quartz hazard, on the highly reactive quartz surface and on proinflammatory events in the rat lung were explored. Aluminium lactate-treated quartz showed a reduced surface reactivity as measured by electron spin resonance. Eighteenhours post-instillation of quartz into the rat lung, there was massive inflammation as indicated by the number of neutrophils in the bronchoalveolar lavage (BAL) and anincrease in BAL macrophage inflammatory protein-2 (MIP-2). However, aluminium lactate-treated quartz had no significant effect when compared to control. Epithelial damage as indicated by BAL protein and gamma glutamyl transpeptidasea lso increased with quartz instillation but not with aluminium lactate-treated quartz and furthermore,quartz induced an increase in MIP-2 mRNA content of BAL cells while aluminium lactate-treated quartz had no effect compared to controls. There was an increase innuclear binding of the transcription factor nuclear factor-kappa B (NF-xB) in the quartz exposed BAL cells and again, no effect on nuclear NF-xB binding in BAL cells fromaluminium lactate-treated quartz instilled rats.In addition, the effect of aluminium lactate and PVNO quartz treatment on DNA damage, cell cytotoxicity and particle uptake by A549 cells was assessed. DNA strandbreakage, as produced by quartz at non-toxic concentrations, could be completely prevented by both coating materials. Particle uptake by A549 cells appeared to be significantly inhibited by the PVNO coating, and to a lesser extent by the aluminium lactate coating, demonstrating that respirable quartz particles induce oxidative DNA damage in human lung epithelial cells and indicating that the surface properties of the quartz as well as particle uptake by these target cells are important in the cytotoxic and genotoxic effects of quartz in vitro.Finally, the role played by surface area and specific reactivity in the acute inflammatory response to particles was investigated. Acute inflammatory response followinginstillation of particles has been used to evaluate hazard but has been criticised because of the non-physiological delivery and the problems of local overload. Here, a numberof low toxicity dusts of various particle sizes were instilled and the neutrophil influx into the lung 18-24 hours post-instillation assessed. The extent of inflammation wasshown to be a function of the surface area instilled and ultrafine particles, which present a case of high surface area per unit mass, were inflammogenic pro rata with theirsurface area. There is no evidence that ultrafine particles of carbon black, titanium dioxide or polystyrene have any special reactivity in addition to their large surface area.We further tested whether this approach could be used to model the reactivity of highly toxic dusts. Rats were instilled with either quartz or aluminium lactate-treated quartzand, as anticipated, the high specific surface reactivity of quartz meant that it was much more inflammogenic than was predicted using the relationship described for `low toxicity'dusts. This approach represents the possibility of modelling potential toxicity for nuisance dusts based on the inflammatory response of a given instilled surface area dose

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

Neutrophil Apoptosis:Relevance to the Innate Immune Response and Inflammatory Disease

Neutrophils are the most abundant cell type involved in the innate immune response. They are rapidly recruited to sites of injury or infection where they engulf and kill invading microorganisms. Neutrophil apoptosis, the process of programmed cell death that prevents the release of neutrophil histotoxic contents, is tightly regulated and limits the destructive capacity of neutrophil products to surrounding tissue. The subsequent recognition and phagocytosis of apoptotic cells by phagocytic cells such as macrophages is central to the successful resolution of an inflammatory response and it is increasingly apparent that the dying neutrophil itself exerts an anti-inflammatory effect through modulation of surrounding cell responses, particularly macrophage inflammatory cytokine release. Apoptosis may be delayed, induced or enhanced by micro-organisms dependent on their immune evasion strategies and the health of the host they encounter. There is now an established field of research aimed at understanding the regulation of apoptosis and its potential as a target for therapeutic intervention in inflammatory and infective diseases. This review focuses on the physiological regulation of neutrophil apoptosis with respect to the innate immune system and highlights recent advances in mechanistic understanding of apoptotic pathways and their therapeutic manipulation in appropriate and excessive innate immune responses

Inhibition of cyclin-dependent kinase 9 downregulates cytokine production without detrimentally affecting human monocyte-derived macrophage viability

Cyclin-dependent kinase (CDK) inhibitor drugs (CDKi), such as R-roscovitine and AT7519, induce neutrophil apoptosis in vitro and enhance the resolution of inflammation in a number of in vivo models. This class of compounds are potential novel therapeutic agents that could promote the resolution of acute and chronic inflammatory conditions where neutrophil activation contributes to tissue damage and aberrant tissue repair. In this study we investigated CDKi effects on macrophage pro-inflammatory mediator production and viability. Treatment of human monocyte-derived macrophages (MDMs) with the CDKi AT7519 and R-roscovitine at concentrations that induce neutrophil apoptosis had no significant effect on control or LPS-activated MDM apoptosis and viability, and did not detrimentally affect MDM efferocytosis of apoptotic cells. In addition, enhanced efferocytosis, induced by the glucocorticoid dexamethasone, was also unaffected after a short time treatment with R-roscovitine. Macrophage cytokine responses to inflammatory stimuli are also of importance during inflammation and resolution. As a key target of CDKi, CDK9, is involved in protein transcription via the RNA polymerase II complex, we investigated the effect of CDKi drugs on cytokine production. Our data show that treatment with AT7519 significantly downregulated expression and release of key MDM cytokines IL-6, TNF, IL-10 and IL-1β, as well as markers of pro-inflammatory macrophage polarisation. R-Roscovitine was also able to downregulate inflammatory cytokine protein secretion from MDMs. Using siRNA transfection, we demonstrate that genetic knock-down of CDK9 replicates these findings, reducing expression and release of pro-inflammatory cytokines. Furthermore, overexpression of CDK9 in THP-1 cells can promote a pro-inflammatory phenotype in these cells, suggesting that CDK9 plays an important role in the inflammatory phenotype of macrophages. Overall, this study demonstrates that pharmacological and genetic targeting of CDK9 inhibits an inflammatory phenotype in human MDMs. As such these data indicate that CDK9 may be key to therapeutically targeting pro-inflammatory macrophage functions during chronic inflammation

Diesel exhaust particulate induces pulmonary and systemic inflammation in rats without impairing endothelial function ex vivo or in vivo

<p>Abstract</p> <p>Background</p> <p>Inhalation of diesel exhaust impairs vascular function in man, by a mechanism that has yet to be fully established. We hypothesised that pulmonary exposure to diesel exhaust particles (DEP) would cause endothelial dysfunction in rats as a consequence of pulmonary and systemic inflammation.</p> <p>Methods</p> <p>Wistar rats were exposed to DEP (0.5 mg) or saline vehicle by intratracheal instillation and hind-limb blood flow, blood pressure and heart rate were monitored <it>in situ </it>6 or 24 h after exposure. Vascular function was tested by administration of the endothelium-dependent vasodilator acetylcholine (ACh) and the endothelium-independent vasodilator sodium nitroprusside (SNP) <it>in vivo </it>and <it>ex vivo </it>in isolated rings of thoracic aorta, femoral and mesenteric artery from DEP exposed rats. Bronchoalveolar lavage fluid (BALF) and blood plasma were collected to assess pulmonary (cell differentials, protein levels & interleukin-6 (IL-6)) and systemic (IL-6), tumour necrosis factor alpha (TNFα) and C-reactive protein (CRP)) inflammation, respectively.</p> <p>Results</p> <p>DEP instillation increased cell counts, total protein and IL-6 in BALF 6 h after exposure, while levels of IL-6 and TNFα were only raised in blood 24 h after DEP exposure. DEP had no effect on the increased hind-limb blood flow induced by ACh <it>in vivo </it>at 6 or 24 h. However, responses to SNP were impaired at both time points. In contrast, <it>ex vivo </it>responses to ACh and SNP were unaltered in arteries isolated from rats exposed to DEP.</p> <p>Conclusions</p> <p>Exposure of rats to DEP induces both pulmonary and systemic inflammation, but does not modify endothelium-dependent vasodilatation. Other mechanisms <it>in vivo </it>limit dilator responses to SNP and these require further investigation.</p

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

Characterisation of fume from hyperbaric welding operations

We report preliminary work characterising dust from hyperbaric welding trials carried out at increased pressure in a helium and oxygen atmosphere. Particle size and concentration were measured during welding. Samples for quartz and metal analysis and toxicity assessment were taken from a filter in the local fume extraction system. The residue of dust after metal extraction by nitric acid in hydrogen peroxide predominantly a non-metallic white powder assumed to be dust from welding rod coatings and thermal insulation material. Metallic analysis showed predominantly calcium, from the welding rod coating, and period 4 transition metals such as iron, manganese, magnesium and titanium (inductively coupled mass spectrometry, Agilent 7500c). The presence of zirconium indicated a contribution from grinding. The fume was nanoparticulate in nature with a mean particle diameter of 20-30 nm (MSI Inc WPS 1000XP). It showed an intermediate level of oxidative potential regarding the low-molecular weight respiratory tract lining fluid antioxidants ascorbate and glutathione and caused release of the inflammatory marker IL-8 in a human lung A 549 epithelial cell culture with no indication of cytotoxicity. The study findings have strong implications for the measurement techniques needed to assess fume exposure in hyperbaric welding and the provision of respiratory protection