Macrophage-derived exosomal TNF-α promotes pulmonary surfactant protein expression in PM2.5-induced acute lung injury.

Short-term high-concentration exposure to airborne fine particulate matter (PM2.5) is strongly associated with the risk of acute lung injury (ALI). It has been recently reported that exosomes (Exos) involve in the progression of respiratory diseases. However, the molecular mechanisms by which exosome-mediated intercellular signaling exacerbate PM2.5-induced ALI remains largely unaddressed. In the present study, we firstly investigated the effect of macrophage-derived exosomal tumor necrosis factor α (TNF-α) on pulmonary surfactant proteins (SPs) expression in epithelial MLE-12 cells after PM2.5 exposure. The higher levels of exosomes in the bronchoalveolar lavage fluid (BALF) of PM2.5-induced ALI mice were found. BALF-exosomes significantly up-regulated SPs expression in MLE-12 cells. Moreover, we found that remarkably high expression of TNF-α in exosomes secreted by PM2.5-treated RAW264.7 cells. Exosomal TNF-α promoted thyroid transcription factor-1 (TTF-1) activation and SPs expression in MLE-12 cells. Furthermore, intratracheal instillation of macrophage-derived TNF-α-containing exosomes increased epithelial cell SPs expression in the lungs of mice. Taken together, these results suggest that macrophages-secreted exosomal TNF-α can trigger epithelial cell SPs expression, which provides new insight and potential target in the mechanism of epithelial cell dysfunction in PM2.5-induced ALI.

Correlation between inhibition of lung surfactant function in vitro and rapid reduction in tidal volume following exposure to plant protection products in mice.

Currently, testing of acute inhalation toxicity in animals is required for regulation of pesticide active ingredients and formulated plant protection products. The main outcome of the regulatory tests is "lethal concentration 50″ (LC50), i.e. the concentration that will kill 50% of the exposed animals. However, ongoing work aims to identify New Approach Methods (NAMs) to replace animal experiments. To this end, we studied 11 plant protection products, sold in the European Union (EU), for their ability to inhibit lung surfactant function in vitro in the constrained drop surfactometer (CDS). In vivo, inhibition of lung surfactant function can lead to alveolar collapse and reduction of tidal volume. Therefore, we also assessed changes in breathing patterns of mice during exposure to the same products. Six of the eleven products inhibited lung surfactant function, and six products reduced tidal volume in mice. In vitro inhibition of lung surfactant function predicted reduction in tidal volume in exposed mice with a sensitivity of 67% and a specificity of 60%. Two products were labelled as "harmful if inhaled", both inhibited surfactant function in vitro and reduced tidal volume in mice. Lung surfactant function inhibition in vitro predicted reduction in tidal volume for plant protection products to a lesser degree than for previously tested substances. This could owe to the requirement for rigorous testing of plant protection products prior to approval that might have selected against substances that could potentially inhibit lung surfactant, e.g. due to severe adverse effects during inhalation.

Risk assessment of consumer spray products using in vitro lung surfactant function inhibition, exposure modelling and chemical analysis.

Consumer spray products release aerosols that can potentially be inhaled and reach the deep parts of the lungs. A thin layer of liquid, containing a mixture of proteins and lipids known as lung surfactant, coats the alveoli. Inhibition of lung surfactant function can lead to acute loss of lung function. We focused on two groups of spray products; 8 cleaning and 13 impregnation products, and in the context of risk assessment, used an in vitro method for assessing inhibition of lung surfactant function. Original spray-cans were used to generate aerosols to measure aerodynamic particle size distribution. We recreated a real-life exposure scenario to estimate the alveolar deposited dose. Most impregnation products inhibited lung surfactant function at the lowest aerosolization rate, whereas only two cleaning products inhibited function at the highest rates. We used inhibitory dose and estimated alveolar deposition to calculate the margin of safety (MoS). The MoS for the inhibitory products was ≤1 for the impregnation products, while much larger for the cleaning products (>880). This risk assessment focused on the risk of lung surfactant function disruption and provides knowledge on an endpoint of lung toxicity that is not investigated by the currently available OECD test guidelines.

Interaction of industrial smelting soot particles with pulmonary surfactant: Pulmonary toxicity of heavy metal-rich particles.

Inhalable particles can influence the interfacial behavior of pulmonary surfactant (PS) resulting in various pulmonary diseases. However, the effects of actually airborne particles on the interfacial behavior of PS and its role in the alteration for soluble metal fraction in particles are entirely unexplored. Herein, we investigated the interaction of PS extracted from porcine lungs with smelting soot fine particles as a model of inhaled heavy metal-rich particles. Our results showed that the phase behavior and foamability of PS were obviously altered in the presence of smelting soot fine particles. In addition, the soluble heavy metals in smelting soot fine particles notably increased in the presence of PS as compared to that of saline solution. Further experiments conducted by adding PS's major components (dipalmitoylphosphatidylcholine, DPPC; bovine serum albumin, BSA) demonstrated that comparison of DPPC, adsorbed BSA is beneficial for the dissolution of heavy metals in smelting soot fine particles. Dynamic light scattering experiments verified that the well dispersion of smelting soot fine particles in the presence of BSA may be responsible for the higher solubility of heavy metals. These findings indicate that PS's interfacial behavior change and PS-enhanced solubilization release of metal components may increase the potentially pulmonary risk in the exposure of airborne fine particles enriched with heavy metals.

Response surface methodological (RSM) approach for optimizing the removal of trihalomethanes (THMs) and its precursor's by surfactant modified magnetic nanoadsorbents (sMNP) - An endeavor to diminish probable cancer risk.

Response surface methodology (RSM) approach was used for optimization of the process parameters and identifying the optimal conditions for the removal of both trihalomethanes (THMs) and natural organic matter (NOM) in drinking water supplies. Co-precipitation process was employed for the synthesis of magnetic nano-adsorbent (sMNP), and were characterized by field emission scanning electron microscopy (SEM), trans-emission electron microscopy (TEM), BET (Brunauer-Emmett-Teller), energy dispersive X-ray (EDX) and zeta potential. Box-Behnken experimental design combined with response surface and optimization was used to predict THM and NOM in drinking water supplies. Variables were concentration of sMNP (0.1 g to 5 g), pH (4-10) and reaction time (5 min to 90 min). Statistical analysis of variance (ANOVA) was carried out to identify the adequacy of the developed model, and revealed good agreement between the experimental data and proposed model. The experimentally derived RSM model was validated using t-test and a range of statistical parameters. The observed R2 value, adj. R2, pred. R2 and "F-values" indicates that the developed THM and NOM models are significant. Risk analysis study revealed that under the RSM optimized conditions, a marked reduction in the cancer risk of THMs was observed for both the groups studied. Therefore, the study observed that the developed process and models can be efficiently applied for the removal of both THM and NOM from drinking water supplies.

Polymer-Lipid Microparticles for Pulmonary Delivery.

Toward engineering approaches that are designed to optimize the particle size, morphology, and mucoadhesion behavior of the particulate component of inhaler formulations, this paper presents the preparation, physicochemical characterization, and preliminary in vitro evaluation of multicomponent polymer-lipid systems that are based on "spray-drying engineered" α-lactose monohydrate microparticles. The formulations combine an active (budesonide) with a lung surfactant (dipalmitoylphosphatidylcholine) and with materials that are known for their desirable effects on morphology (polyvinyl alcohol), aerosolization (l-leucine), and mucoadhesion (chitosan). The effect of the composition of formulations on the morphology, distribution, and in vitro mucoadhesion profiles is presented along with "Calu-3 cell monolayers" data that indicate good cytocompatibility and also with simulated-lung-fluid data that are consistent with the therapeutically useful release of budesonide.

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.

Pulmonary surfactant and nanocarriers: Toxicity versus combined nanomedical applications.

Pulmonary surfactant is a membrane-based lipid-protein system essential for the process of breathing, which coats and stabilizes the whole respiratory surface and possesses exceptional biophysical properties. It constitutes the first barrier against the entry of pathogens and harmful particles in the alveolar region, extended through the lungs, but on the other hand, it can offer novel possibilities as a shuttle for the delivery of drugs and nanocarriers. The advances in nanotechnology are opening the doors to new diagnostic and therapeutic avenues, which are not accessible by means of the current approaches. In this context, the pulmonary route is called to become a powerful way of entry for innovative treatments based on nanotechnology. In this review, the anatomy of the respiratory system and its properties for drug entry are first revisited, as well as some current strategies that use the respiratory route for both local and peripheral action. Then, a brief overview is presented on what pulmonary surfactant is, how it works and why it could be used as a drug delivery vehicle. Finally, the review is closed with a description of the development of nanocarriers in the lung context and their interaction with endogenous and clinical pulmonary surfactants. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Dynamics of Structural Parameters and Accumulation of Collagen Fibrils in Rat Lung after Inhalations of Surfactant-BL at Various Terms of Bleomycin-Induced Alveolitis.

Rats were subjected to surfactant-BL inhalations at the early and late phases of bleomycininduced alveolitis. In both regimens, the drug reduced the severity of inflammation. In the acute phase of alveolitis, the therapeutic effect of inhalation was accompanied by activation of the synthesis of fine lose collagen fibrils. In the late phase of alveolitis, inhalation of surfactant-BL thickened the fibrils and diminished their population in alveolar walls.

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.

Phospholipid surfactant adsorption by respirable quartz and in vitro expression of cytotoxicity and DNA damage.

Respirable-sized quartz was treated with a saline dispersion of dipalmitoyl phosphatidylcholine (DPPC), a primary component of pulmonary surfactant, to model the adsorption of phospholipid surfactant onto quartz dust following particle deposition in the bronchoalveolar region of the lung. Control and surfactant-treated dusts were used to challenge lavaged rat pulmonary macrophages in vitro over a 1-week period, to determine the effects of adsorbed surfactant on the expression of quartz cytotoxicity and genotoxicity. DNA damage was determined by the single cell gel electrophoresis 'comet' assay. Untreated quartz induced DNA damage, increasing with dose and with time of incubation of dust with macrophages over a 5 day period. DPPC treatment of quartz suppressed DNA damage through 1 day of macrophage challenge. DNA damage then increased over a 5 day period, to approximately half the positive control (untreated quartz) values. Cytotoxicity was measured by trypan blue dye exclusion and by the Live-Dead fluorescence assay for cell viability. Cytotoxicity of surfactant-treated quartz measured one day after challenge of lavaged macrophages was suppressed to values near those of the negative controls, and then increased over a 1 week incubation period to levels near those expressed by native quartz positive controls. Quartz similarly treated with dioleoyl phosphatidylcholine mixed with DPPC substituted in one acyl group with a boron-containing fluorescent chromophore was used with confocal microscopy to measure particle-associated fluorescent surfactant in cells. Approximately half of the fluorescence intensity was lost over a 1 week period following challenge of lavaged macrophage. Results are discussed in terms of a model of restoration of quartz particle surface toxicity as prophylactic surfactant is removed from particle surface by cellular enzymatic digestion processes.

Lysis of red blood cells and alveolar epithelial toxicity by therapeutic pulmonary surfactants.

The risk of pulmonary hemorrhage is increased in extremely low birth weight infants treated with surfactant. The pathogenesis of this increased risk is far from clear. We tested whether exposure of cell membranes to surfactant may lead to increased membrane permeability, hypothesizing that this process may contribute to the occurrence of alveolar hemorrhage after surfactant treatment. Aliquots of washed packed red blood cells (used as membrane model) were suspended in 0.9% NaCl with various concentrations of Survanta or Exosurf for either 2 or 24 h at 37 degrees C. Cytolysis was measured by spectrophotometric determination of free Hb after centrifugation. Red cells suspended in 0.9% NaCl alone, distilled water, or various concentrations of melittin were used as negative and positive controls. Both surfactants were associated with increased hemolysis to 35% of maximum at concentrations of 1.25 mg/2 mL. Above these concentrations, Survanta was associated with no increase in hemolysis, whereas Exosurf increased hemolysis to 60% of maximum at concentrations of 12.5 mg/2 mL. In additional experiments, primary cultures of alveolar type II cells from adult rats were treated with Survanta, Exosurf, the Exosurf components tyloxapol and hexadecanol, melittin, or culture medium alone. After 24 h of incubation, lactate dehydrogenase release into the media was measured as a percent of total lactate dehydrogenase activity to indicate cytotoxicity. Lactate dehydrogenase release was < 10% for control experiments but increased sharply with Exosurf and its components tyloxapol and hexadecanol. These results indicate that surfactant may be associated with in vitro cytotoxicity and that this property differs for different surfactants and different dosages.