Acute Inhalation Toxicity After Inhalation of ZnO Nanoparticles: Lung Surfactant Function Inhibition In Vitro Correlates With Reduced Tidal Volume in Mice.

People can be exposed to zinc oxide (ZnO) by inhalation of consumer products or during industrial processes. Zinc oxide nanoparticle (NP) exposure can induce acute inhalation toxicity. The toxicological mechanisms underlying the acute effects on the lungs have long focused on the phagolysosomal dissolution of ZnO NPs in macrophages followed by the release of free Zn2+ ions. However, we postulate an alternative mechanism based on the direct interaction of ZnO NPs with the lung surfactant (LS) layer covering the inside of the alveoli. Therefore, we tested the effect of ZnO NPs and Zn2+ ions on the function of LS in vitro using the constrained drop surfactometer. We found that the ZnO NPs inhibited the LS function, whereas Zn2+ ions did not. To examine the role of lung macrophages in the acute toxicity of inhaled ZnO NPs, mice were treated with Clodrosome, a drug that depletes alveolar macrophages, or Encapsome, the empty carrier of the drug. After macrophage depletion, the mice were exposed to an aerosol of ZnO NPs in whole body plethysmographs recording breathing patterns continuously. Mice in both groups developed shallow breathing (reduced tidal volume) shortly after the onset of exposure to ZnO NPs. This suggests a macrophage-independent mechanism of induction. This study shows that acute inhalation toxicity is caused by ZnO NP interaction with LS, independently of NP dissolution in macrophages.

Pre-conceptional exposure to multiwalled carbon nanotubes suppresses antibody production in mouse offspring.

Prenatal particle exposure has been shown to increase allergic responses in offspring. Carbon nanotubes (CNTs) possess immunomodulatory properties, but it is unknown whether maternal exposure to CNTs interferes with offspring immune development. Here, C57Bl/6J female mice were intratracheally instilled with 67 of µg multiwalled CNTs on the day prior to mating. After weaning, tolerance and allergy responses were assessed in the offspring. Offspring of CNT-exposed (CNT offspring) and of sham-exposed dams (CTRL offspring) were intranasally exposed to ovalbumin (OVA) once weekly for 5 weeks to induce airway mucosal tolerance. Subsequent OVA sensitization and aerosol inhalation caused low or no OVA-specific IgE production and no inflammation. However, the CNT offspring presented with significantly lower OVA-specific IgG1 levels than CTRL offspring. In other groups of 5-week-old offspring, low-dose sensitization with OVA and subsequent OVA aerosol inhalation led to significantly lower OVA-specific IgG1 production in CNT compared to CTRL offspring. OVA-specific IgE and airway inflammation were non-significantly reduced in CNT offspring. The immunomodulatory effects of pre-gestational exposure to multiwalled CNTs were unexpected, but very consistent. The observations of suppressed antigen-specific IgG1 production may be of importance for infection or vaccination responses and warrant further investigation.

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition.

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia.

Focal cerebral ischaemia has an initial phase of inflammation and tissue injury followed by a later phase of resolution and repair. Mass spectrometry imaging (desorption electrospray ionization and matrix assisted laser desorption ionization) was applied on brain sections from mice 2 h, 24 h, 5d, 7d, and 20d after permanent focal cerebral ischaemia. Within 24 h, N-acyl-phosphatidylethanolamines, lysophosphatidylcholine, and ceramide accumulated, while sphingomyelin disappeared. At the later resolution stages, bis(monoacylglycero)phosphate (BMP(22:6/22:6)), 2-arachidonoyl-glycerol, ceramide-phosphate, sphingosine-1-phosphate, lysophosphatidylserine, and cholesteryl ester appeared. At day 5 to 7, dihydroxy derivates of docosahexaenoic and docosapentaenoic acid, some of which may be pro-resolving mediators, e.g. resolvins, were found in the injured area, and BMP(22:6/22:6) co-localized with the macrophage biomarker CD11b, and probably with cholesteryl ester. Mass spectrometry imaging can visualize spatiotemporal changes in the lipidome during the progression and resolution of focal cerebral inflammation and suggests that BMP(22:6/22:6) and N-acyl-phosphatidylethanolamines can be used as biomarkers for phagocytizing macrophages/microglia cells and dead neurones, respectively.

Airway irritation, inflammation, and toxicity in mice following inhalation of metal oxide nanoparticles.

Metal oxide nanoparticles are used in a broad range of industrial processes and workers may be exposed to aerosols of the particles both during production and handling. Despite the widespread use of these particles, relatively few studies have been performed to investigate the toxicological effects in the airways following inhalation. In the present study, the acute (24 h) and persistent (13 weeks) effects in the airways after a single exposure to metal oxide nanoparticles were studied using a murine inhalation model. Mice were exposed 60 min to aerosols of either ZnO, TiO2, Al2O3 or CeO2 and the deposited doses in the upper and lower respiratory tracts were calculated. Endpoints were acute airway irritation, pulmonary inflammation based on analyses of bronchoalveolar lavage (BAL) cell composition, DNA damage assessed by the comet assay and pulmonary toxicity assessed by protein level in BAL fluid and histology. All studied particles reduced the tidal volume in a concentration-dependent manner accompanied with an increase in the respiratory rate. In addition, ZnO and TiO2 induced nasal irritation. BAL cell analyses revealed both neutrophilic and lymphocytic inflammation 24-h post-exposure to all particles except TiO2. The ranking of potency regarding induction of acute lung inflammation was Al2O3 = TiO2 < CeO2 ≪ ZnO. Exposure to CeO2 gave rise to a more persistent inflammation; both neutrophilic and lymphocytic inflammation was seen 13 weeks after exposure. As the only particles, ZnO caused a significant toxic effect in the airways while TiO2 gave rise to DNA-strand break as shown by the comet assay.

Generation and Characterization of Indoor Fungal Aerosols for Inhalation Studies.

In the indoor environment, people are exposed to several fungal species. Evident dampness is associated with increased respiratory symptoms. To examine the immune responses associated with fungal exposure, mice are often exposed to a single species grown on an agar medium. The aim of this study was to develop an inhalation exposure system to be able to examine responses in mice exposed to mixed fungal species aerosolized from fungus-infested building materials. Indoor airborne fungi were sampled and cultivated on gypsum boards. Aerosols were characterized and compared with aerosols in homes. Aerosols containing 10(7)CFU of fungi/m(3)air were generated repeatedly from fungus-infested gypsum boards in a mouse exposure chamber. Aerosols contained Aspergillus nidulans,Aspergillus niger, Aspergillus ustus, Aspergillus versicolor,Chaetomium globosum,Cladosporium herbarum,Penicillium brevicompactum,Penicillium camemberti,Penicillium chrysogenum,Penicillium commune,Penicillium glabrum,Penicillium olsonii,Penicillium rugulosum,Stachybotrys chartarum, and Wallemia sebi They were all among the most abundant airborne species identified in 28 homes. Nine species from gypsum boards and 11 species in the homes are associated with water damage. Most fungi were present as single spores, but chains and clusters of different species and fragments were also present. The variation in exposure level during the 60 min of aerosol generation was similar to the variation measured in homes. Through aerosolization of fungi from the indoor environment, cultured on gypsum boards, it was possible to generate realistic aerosols in terms of species composition, concentration, and particle sizes. The inhalation-exposure system can be used to study responses to indoor fungi associated with water damage and the importance of fungal species composition.

A Proposed In Vitro Method to Assess Effects of Inhaled Particles on Lung Surfactant Function.

The lung surfactant (LS) lining is a thin liquid film covering the air-liquid interface of the respiratory tract. LS reduces surface tension, enabling lung surface expansion and contraction with minimal work during respiration. Disruption of surface tension is believed to play a key role in severe lung conditions. Inhalation of aerosols that interfere with the LS may induce a toxic response and, as a part of the safety assessment of chemicals and inhaled medicines, it may be relevant to study their impact on LS function. Here, we present a novel in vitro method, based on the constrained drop surfactometer, to study LS functionality after aerosol exposure. The applicability of the method was investigated using three inhaled asthma medicines, micronized lactose, a pharmaceutical excipient used in inhaled medication, and micronized albumin, a known inhibitor of surfactant function. The surfactometer was modified to allow particles mixed in air to flow through the chamber holding the surfactant drop. The deposited dose was measured with a custom-built quartz crystal microbalance. The alterations allowed the study of continuously increasing quantified doses of particles, allowing determination of the dose of particles that affects the LS function. The tested pharmaceuticals did not inhibit the function of a model LS even at extreme doses--neither did lactose. Micronized albumin, however, impaired surfactant function. The method can discriminate between safe inhaled aerosols--as exemplified by the approved inhaled medicines and the pharmaceutical excipient lactose--and albumin known to impair lung functionality by inhibiting LS function.

Interactions between nanoparticles and lung surfactant investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: Inhaled nanoparticles may cause adverse effects due to inactivation of lung surfactants. We have studied how three different nanoparticles interact with dipalmitoyl-phosphatidylcholine (DPPC), the main component in lung surfactant. METHODS: DPPC in solution was mixed with a suspension of nanoparticles, both in organic solvent, and allowed to interact for 40 min under conditions partly resembling the alveolar lining. Nanoparticles were isolated by centrifugation, washed, and re-suspended in ethanol/water 1:1 (v/v). The resulting solution was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) using dihydroxybenzoic acid as matrix. RESULTS: The developed methodology was successfully applied for quantitative detection of phospholipid lung surfactant bound to three different types of nanoparticles. Titanium dioxide nanoparticles had a strong affinity for binding of lipid lung surfactant in contrast to pristine and methylated silica nanoparticles. When the concentration of lipid surfactant was raised in the reaction mixture, the titanium dioxide nanoparticles showed an apparently non-linear binding process. CONCLUSIONS: This work demonstrates that MALDI-TOFMS can be used for direct determination of the binding of surfactant lipids to nanoparticles and represents an important initial step towards a simple and quantitative in vitro method for assessment of interactions of nanoparticles with lung surfactants.

An in vitro method for predicting inhalation toxicity of impregnation spray products.

Impregnation spray products are used for making surfaces water and dirt repellent. The products are composed of one or more active film-forming components dissolved or suspended in an appropriate solvent mixture. Exposure to impregnation spray products may cause respiratory distress and new cases are reported frequently. The toxicity appears to be driven by a disruption of the pulmonary surfactant film, which coats the inside of the lungs. Due to the complex chemistry of impregnation spray products, it is impossible to predict if inhalation of an aerosolized product is toxic in vivo. The aim of this study was to evaluate whether disruption of the pulmonary surfactant film can be used as a predictor of the toxic effects in vivo. Nine impregnation products with various chemical compositions were selected for testing and the main constituents of each product, e.g., solvents, co-solvents and film-forming compounds, were identified by mass spectrometry. We used a capillary surfactometry method to assess disruption of pulmonary surfactant function in vitro and a mouse model to evaluate acute respiratory toxicity during inhalation. Concentration-response relationships were successfully determined both in vitro and in vivo. The true positive rate of the in vitro method was 100%, i.e. the test could correctly identify all products with toxic effects in vivo, the true negative rate was 40%. Investigation of inhibition of the pulmonary surfactant system, e.g. by capillary surfactometry, was found useful for evaluation of the inhalation toxicity of impregnation spray products and thus may reduce the need for animal testing.

Mechanism of action of lung damage caused by a nanofilm spray product.

Inhalation of waterproofing spray products has on several occasions caused lung damage, which in some cases was fatal. The present study aims to elucidate the mechanism of action of a nanofilm spray product, which has been shown to possess unusual toxic effects, including an extremely steep concentration-effect curve. The nanofilm product is intended for application on non-absorbing flooring materials and contains perfluorosiloxane as the active film-forming component. The toxicological effects and their underlying mechanisms of this product were studied using a mouse inhalation model, by in vitro techniques and by identification of the binding interaction. Inhalation of the aerosolized product gave rise to increased airway resistance in the mice, as evident from the decreased expiratory flow rate. The toxic effect of the waterproofing spray product included interaction with the pulmonary surfactants. More specifically, the active film-forming components in the spray product, perfluorinated siloxanes, inhibited the function of the lung surfactant due to non-covalent interaction with surfactant protein B, a component which is crucial for the stability and persistence of the lung surfactant film during respiration. The active film-forming component used in the present spray product is also found in several other products on the market. Hence, it may be expected that these products may have a toxicity similar to the waterproofing product studied here. Elucidation of the toxicological mechanism and identification of toxicological targets are important to perform rational and cost-effective toxicological studies. Thus, because the pulmonary surfactant system appears to be an important toxicological target for waterproofing spray products, study of surfactant inhibition could be included in toxicological assessment of this group of consumer products.

Pulmonary toxicity of perfluorinated silane-based nanofilm spray products: solvent dependency.

A number of cases of pulmonary injury by use of aerosolized surface coating products have been reported worldwide. The aerosol from a commercial alcohol-based nanofilm product (NFP) for coating of nonabsorbing surfaces was found to induce severe lung damage in a recent mouse bioassay. The NFP contained a 1H,1H,2H,2H-perfluorooctyl trialkoxysilane (POTS) and the effects were associated with the hydrolyzed forms of the silane; increase in hydrolyzation resulted in faster induction of compromised breathing and induction of lung damage. In this study, the impact of the solvent on the toxicity of POTS has been investigated. BALB/cA mice were exposed to aerosolized water-based NFPs containing POTS, and solutions of hydrolyzed POTS in methanol, ethanol, and 2-propanol, respectively. No acute respiratory effect was observed at exposure concentrations up to 110 mg/m³ with an aqueous solution of POTS. However, exposure to POTS in methanol resulted in a decrease of the tidal volume--an effect that did not resolve within the recovery period. After 27 min of exposure, the tidal volume had decreased by 25%, indicating partial alveolar collapse. For POTS in ethanol and 2-propanol, a 25% reduction of the tidal volume was observed after 13 and 9 min, respectively; thus, the tidal volume was affected by increase of the chain length. This was confirmed in vitro by investigating lung surfactant function after addition of POTS in different solvents. The addition of vaporized methanol, 2-propanol, or acetone to aerosolized POTS in methanol further exacerbated the tidal volume reduction, demonstrating that the concentration of vaporized solvent participated in the toxicity of POTS.

Human reference values for acute airway effects of five common ozone-initiated terpene reaction products in indoor air.

Ozone-initiated monoterpene reaction products have been hypothesized to cause eye and airway complaints in office environments and some have been proposed to cause skin irritation and sensitization. The respiratory effects of 60 min exposures to five common oxidation products from abundant terpenoids (e.g. limonene), used as solvent and fragrance in common household products or present in skin lipids (e.g. squalene), were studied in a head out mouse bioassay. This allowed determination of acute upper airway (sensory) irritation, airflow limitation in the conducting airways, and pulmonary irritation in the alveolar region. Derived human reference values (RFs) for sensory irritation were 1.3, 0.16 and 0.3 ppm, respectively, for 4-acetyl-1-methylcyclohexene ( 0.2 ppm) [corrected], 3-isopropenyl-6-oxo-heptanal (IPOH), and 6-methyl-5-heptene-2-one (6-MHO). Derived RFs for airflow limitation were 0.8, 0.45, 0.03, and 0.5 ppm, respectively, for dihydrocarvone (DHC), 0.2 ppm [corrected], 4-oxo-pentanal (0.3 ppm) [corrected], and 6-MHO. Pulmonary irritation was unobserved as a critical effect. The RFs indicate that the oxidation products would not contribute substantially to sensory irritation in eyes and upper airways in office environments. Reported concentrations in offices of 6-MHO and 0.3 ppm [corrected]would not result in airflow limitation. However, based upon the RFs for IPOH and 0.3 ppm [corrected], precautionary actions should be considered that disfavor their formation in excess.

Experiences from occupational exposure limits set on aerosols containing allergenic proteins.

Occupational exposure limits (OELs) together with determined airborne exposures are used in risk assessment based managements of occupational exposures to prevent occupational diseases. In most countries, OELs have only been set for few protein-containing aerosols causing IgE-mediated allergies. They comprise aerosols of flour dust, grain dust, wood dust, natural rubber latex, and the subtilisins, which are proteolytic enzymes. These aerosols show dose-dependent effects and levels have been established, where nearly all workers may be exposed without adverse health effects, which are required for setting OELs. Our aim is to analyse prerequisites for setting OELs for the allergenic protein-containing aerosols. Opposite to the key effect of toxicological reactions, two thresholds, one for the sensitization phase and one for elicitation of IgE-mediated symptoms in sensitized individuals, are used in the OEL settings. For example, this was the case for flour dust, where OELs were based on dust levels due to linearity between flour dust and its allergen levels. The critical effects for flour and grain dust OELs were different, which indicates that conclusion by analogy (read-across) must be scientifically well founded. Except for subtilisins, no OEL have been set for other industrial enzymes, where many of which are high volume chemicals. For several of these, OELs have been proposed in the scientific literature during the last two decades. It is apparent that the scientific methodology is available for setting OELs for proteins and protein-containing aerosols where the critical effect is IgE sensitization and IgE-mediated airway diseases.

Airway effects of inhaled quaternary ammonium compounds in mice.

Quaternary ammonium compounds (QAC) constitute a family of widely used chemical substances. The QAC benzalkonium chloride (BAC) has caused bronchoconstriction in human beings by poorly understood mechanisms and lung damage at high concentration as shown in a single rat study. This study evaluates acute airway effects in mice after inhalation of aerosols of the QACs, BAC, hexadecyl trimethyl ammonium bromide (HTA), cetyl pyridinium chloride (CPC) and dimethyl dioctadecyl ammonium bromide (DDA). The QACs gave rise to concentration-dependent decreases in the tidal volume (VT) and a concomitant increase in respiratory rate indicating pulmonary irritation. The potencies of the QAC to induce these effects were in the order: BAC > HTA = CPC > DDA. Furthermore, inhalation of BAC and CPC aerosols gave rise to pulmonary inflammation as apparent from bronchoalveolar lavage. Stimulation of nasal trigeminal nerve endings by QAC, which may serve as a warning signal, was absent.

Acute airway irritation of methyl formate in mice.

Methyl formate (MF) is a volatile solvent with several industrial applications. The acute airway effects of MF were evaluated in a mouse bioassay, allowing the assessment of sensory irritation of the upper airways, airflow limitation of the conducting airways and deep lung (pulmonary) irritation. MF was studied at vapour concentrations of 202-1,168 ppm. Sensory irritation was the only effect observed, which developed slowly over the 30-min exposure period. The potency at steady state was at least 10-fold higher than expected from a hypothetically similar, but non-reactive compound. Methyl formate may be hydrolysed in vivo to formic acid, a potent sensory irritant, and methanol, a low-potent sensory irritant. Hydrolysis may be catalysed by carboxyesterases, and therefore, the role of the esterases was studied using the esterase inhibitor tri-ortho-cresyl phosphate (TOCP). TOCP pre-treatment reduced the irritation response of MF, suggesting that carboxyesterase-mediated hydrolysis plays a role in the irritative effect. However, even after administration of TOCP, MF was considerably more irritating than expected from a quantitative structure-activity relationship (QSAR) model. The slope of the concentration-effect relationship for formic acid was lower than that for the MF in the low-dose range, suggesting that different receptor activation mechanisms may occur, which may include an effect of MF itself, in addition to an effect of formic acid and potentially an effect from formaldehyde.

Acute and subchronic airway inflammation after intratracheal instillation of quartz and titanium dioxide agglomerates in mice.

This study investigated the acute and subchronic inflammatory effects of micrometer-size (micro-size) and nanometer-size (nano-size) particles after intratracheal (i.t.) installation in mice. The role of the type of compound, polymorphism, and size of the particles was investigated. Studied compounds were the two micro-size reference quartzes, SRM1878a and DQ12, a micro- and nano-size rutile titanium dioxide (TiO2), a nano-size anatase, and an amorphous TiO2. Particles were administered by a single i.t. instillation in mice at a fixed dose of 5, 50, and 500 micrograms, respectively. Inflammation was evaluated from the bronchoalveolar lavage fluid (BALF) content of inflammatory cells, the cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), as well as from lung histology. Evaluations were at 24 h (acute effects) and 3 months (subchronic effects) after instillations. Both types of quartz induced a dose-dependent acute increase of neutrophils, IL-6, and total protein in BALF. Limited subchronic inflammation was observed. All types of TiO2 induced a dose-dependent acute increase of neutrophils in BALF. In the acute phase, micro- and nano-size rutile and nano-size amorphous TiO2 induced elevated levels of IL-6 and total protein in BALF at the highest dose. At the nano-size rutile and amorphous TiO2, subchronic lung inflammation was apparent from a dose-dependent increase in BALF macrophages. Histology showed little inflammation overall. The two types of quartz showed virtually similar inflammatory effects. Nearly similar effects were observed for two sizes of rutile TiO2. Differences were seen between the different polymorphs of nano-size TiO2, with rutile being the most inflammogenic and amorphous being the most potent in regard to acute tissue damage.

Sub-chronic lung inflammation after airway exposures to Bacillus thuringiensis biopesticides in mice.

BACKGROUND: The aim of the present study was to assess possible health effects of airway exposures to Bacillus thuringiensis (Bt) based biopesticides in mice. Endpoints were lung inflammation evaluated by presence of inflammatory cells in bronchoalveolar lavage fluid (BALF), clearance of bacteria from the lung lumen and histological alterations of the lungs. Hazard identifications of the biopesticides were carried out using intratracheal (i.t.) instillation, followed by an inhalation study. The two commercial biopesticides used were based on the Bt. subspecies kurstaki and israelensis, respectively. Groups of BALB/c mice were i.t instilled with one bolus (3.5 × 105 or 3.4 × 106 colony forming units (CFU) per mouse) of either biopesticide. Control mice were instilled with sterile water. BALFs were collected and the inflammatory cells were counted and differentiated. The BALFs were also subjected to CFU counts. RESULTS: BALF cytology showed an acute inflammatory response dominated by neutrophils 24 hours after instillation of biopesticide. Four days after instillation, the neutrophil number was normalised and inflammation was dominated by lymphocytes and eosinophils, whereas 70 days after instillation, the inflammation was interstitially located with few inflammatory cells present in the lung lumen.Half of the instilled mice had remaining CFU recovered from BALF 70 days after exposure. To gain further knowledge with relevance for risk assessment, mice were exposed to aerosols of biopesticide one hour per day for 2 × 5 days. Each mouse received 1.9 × 104 CFU Bt israelensis or 2.3 × 103 CFU Bt kurstaki per exposure. Seventy days after end of the aerosol exposures, 3 out of 17 mice had interstitial lung inflammation. CFU could be recovered from 1 out of 10 mice 70 days after exposure to aerosolised Bt kurstaki. Plethysmography showed that inhalation of Bt aerosol did not induce airway irritation. CONCLUSIONS: Repeated low dose aerosol exposures to commercial Bt based biopesticides can induce sub-chronic lung inflammation in mice, which may be the first step in the development of chronic lung diseases. Inhalation of Bt aerosols does not induce airway irritation, which could explain why workers may be less inclined to use a filter mask during the application process, and are thereby less protected from exposure to Bt spores.

Ozone increases airway hyperreactivity and mucus hyperproduction in mice previously exposed to allergen.

Acute exacerbations of asthma represent a common clinical problem with major economic impact. Air pollutants including ozone have been shown to contribute to asthma exacerbation, but the mechanisms underlying ozone-induced asthma exacerbation are only partially understood. The present study aimed to develop a mouse model to gain insight into the development of airway hyperresponsiveness (AHR) to methacholine (MCh) in mice after exposure to both allergen and ozone. Mice were exposed for 20 min per day for 10 consecutive days to an aerosol of 1% ovalbumin (OVA) or saline followed by a single 3-h exposure to clean air or 100, 250, or 500 ppb ozone. Ozone induced AHR in mice previously exposed to OVA when compared to non-exposed (saline) control mice. After a 10-d exposure to OVA, a single exposure to a low (100 ppb) ozone concentration was sufficient to induce AHR. The AHR response was associated with goblet-cell metaplasia. Even the lowest concentration of ozone tested, 100 ppb, which may be exceeded in urban environments and in the workplace, resulted in a significant increase in AHR, most prominent 24 h after exposure in the OVA-exposed mice.

Lung damage in mice after inhalation of nanofilm spray products: the role of perfluorination and free hydroxyl groups.

Exposures to two commercial nanofilm spray products (NFPs), a floor sealant (NFP 1) and a coating product for tiles (NFP 2), were investigated for airway irritation, airway inflammation, and lung damage in a mouse inhalation model. The particle exposure was characterized by particle number, particle size distribution, and gravimetric analysis. BALB/cJ mice were exposed for 60 min to the aerosolized products at 3.3-60 mg/m(3) (10(5)-10(6) fine particles/cm(3)) measured in the breathing zone of the mice. Lung inflammation and lung damage were assessed by study of bronchoalveolar lavage fluid (BALF) cytology, protein in BALF, and histology. Mass spectral analysis showed that NFP 1 and NFP 2 contained hydrolysates and condensates of a perfluorosilane and alkylsilane, respectively. NFP 1 induced a concentration-dependent decrease of the tidal volume lasting for at least 1 day. Exposure concentrations above 16.1 mg/m(3) (2.1 x 10(6) fine particles/cm(3)) gave rise to significant increases of protein level in BALF and reduced body weight, and histological examination showed atelectasis, emphysema, and hemorrhages. A narrow interval between the no-effect level (16.1 mg/m(3)) and the lethal concentrations (18.4 mg/m(3)) was observed. The alkylsilane-based product (NFP 2) had no effect at the concentrations studied. Experiments with different types of perfluorinated silanes and alkylsiloxanes showed that the toxic effects did not arise solely from the perfluorination. The number of free hydroxyl groups in the silanes/alkylsiloxanes was also critical for the toxicity.

Time-response relationship of nano and micro particle induced lung inflammation. Quartz as reference compound.

An increasing number of engineered particles, including nanoparticles, are being manufactured, increasing the need for simple low-dose toxicological screening methods. This study aimed to investigate the kinetics of biomarkers related to acute and sub-chronic particle-induced lung inflammation of quartz. Mice were intratracheal instilled with 50 µg of microsized or nanosized quartz. Acute inflammation was assessed 1, 2, 4, 8, 16 or 48 hours post exposure, whereas sub-chronic inflammation was investigated 3 months after exposure. Markers of acute inflammation in the bronchoalveolar lavage fluid (BALF) were neutrophils (PMN), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, macrophage inflammatory protein-2 (MIP-2), keratinocyte derived chemokine (KC) and total protein, which were all close to maximum 16 hours post instillation. No major differences were seen in the time-response profiles of nano- and micro-sized particles. The potency of the two samples cannot be compared; during the milling process, a substantial part of the quartz was converted to amorphous silica and contaminated with corundum. For screening, BALF PMN, either TNF-α or IL-1ß at 16 hours post instillation may be useful. At 3 months post instillation, KC, PMN and macrophages were elevated. Histology showed no interstitial inflammation three months post instillation. For screening of sub-chronic effects, KC, PMN, macrophages and histopathology is considered sufficient.