Effects and uptake of gold nanoparticles deposited at the air-liquid interface of a human epithelial airway model.

The impact of nanoparticles (NPs) in medicine and biology has increased rapidly in recent years. Gold NPs have advantageous properties such as chemical stability, high electron density and affinity to biomolecules, making them very promising candidates as drug carriers and diagnostic tools. However, diverse studies on the toxicity of gold NPs have reported contradictory results. To address this issue, a triple cell co-culture model simulating the alveolar lung epithelium was used and exposed at the air-liquid interface. The cell cultures were exposed to characterized aerosols with 15 nm gold particles (61 ng Au/cm2 and 561 ng Au/cm2 deposition) and incubated for 4 h and 24 h. Experiments were repeated six times. The mRNA induction of pro-inflammatory (TNFalpha, IL-8, iNOS) and oxidative stress markers (HO-1, SOD2) was measured, as well as protein induction of pro- and anti-inflammatory cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFalpha, INFgamma). A pre-stimulation with lipopolysaccharide (LPS) was performed to further study the effects of particles under inflammatory conditions. Particle deposition and particle uptake by cells were analyzed by transmission electron microscopy and design-based stereology. A homogeneous deposition was revealed, and particles were found to enter all cell types. No mRNA induction due to particles was observed for all markers. The cell culture system was sensitive to LPS but gold particles did not cause any synergistic or suppressive effects. With this experimental setup, reflecting the physiological conditions more precisely, no adverse effects from gold NPs were observed. However, chronic studies under in vivo conditions are needed to entirely exclude adverse effects.

Long-term responses of canine lungs to acidic particles.

Sixteen beagle dogs were housed in four large chambers under minimum restraint. They were exposed for 16 months to clean air and individual baseline data of markers were obtained. For 13 months, eight dogs were further exposed to clean air and eight dogs for 6 h/d to 1-microm MMAD (mass median aerodynamic diameter) acidic sulfate particles carrying 25 micromol H(+) m(-3) into their lungs. To establish functional responses (lung function, cell and tissue integrity, redox balance, and non-specific respiratory defense capacity), each exposed animal served as its own control. To establish structural responses, the eight non-exposed animals served as controls. Acidic particles were produced by nebulization of aqueous sodium hydrogen sulfate at pH 1.5. Only subtle exposure-related changes of lung function and structure were detected. A significant increase in respiratory burst function of alveolar macrophages points to a marginal inflammatory response. This can be explained by the significant production of prostaglandin E(2), activating cyclooxygenase-dependent mechanisms in epithelia and thus inhibiting lung inflammation. The non-specific defense capacity was slightly affected, giving increased tracheal mucus velocity and reduced in vivo dissolution of moderately soluble test particles. Hypertrophy and hyperplasia of bronchial epithelia were not observed, but there was an increase in volume density of bronchial glands and a shift from neutral to acidic staining of epithelial secretory cells in distal airways. The acidic exposure had thus no pathophysiological consequences. It is therefore unlikely that long-term inhalation of acidic particles is associated with a health risk.

Influence of inflammatory mechanisms on the redox balance in interstitial lung diseases.

This study investigated the hypothesis that inflammatory, regulatory and antioxidant systems control the redox balance in interstitial lung diseases. Spontaneous mRNA expression of inflammatory cytokines and redox-active enzymes was examined in bronchoalveolar lavage (BAL) cells from patients with idiopathic pulmonary fibrosis (IPF) and sarcoidosis (SARC) using RT-PCR analysis. Pulmonary oxidative stress was characterized by carbonyl-levels in the soluble BAL-fluid protein. Protein carbonyls were normal in SARC, but 2.4-fold increased in IPF. Here, the protein carbonyls correlated inversely with glutathione peroxidase mRNA. The message for IL-8 increased 14-fold in IPF and was accompanied by a marked influx of PMN, while these parameters were not altered in SARC. Levels of IL-10 transcripts increased in both diseases, but stronger in SARC (33-fold) than in IPF (22-fold), contributing to a high IL-10/IL-8 mRNA ratio in SARC (0.86) in comparison to IPF (0.07) and controls (0.04). In SARC but not in IPF, IFN-gamma mRNA was expressed at high levels and correlated inversely with the carbonyl levels. In both diseases, IL-1beta, TNF-alpha, and IL-6 mRNA transcripts remained at baseline level. In summary, a low IL-10/IL-8 mRNA ratio was paralleled with significant oxidative stress in IPF, while a high IL-10/IL-8 ratio and enhanced IFN-gamma expression went along with a physiological redox-balance in SARC.

Glutamate-cysteine ligase modulatory subunit in BAL alveolar macrophages of healthy smokers.

The antioxidant glutathione (GSH) is increased in the epithelial lining fluid (ELF) of chronic smokers. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), also known as gamma-glutamylcysteine synthetase, consisting of a heavy, catalytic (GCLC) and a light, modulatory (GCLM) subunit. To determine the contribution of bronchoalveolar lavage (BAL) cells to GSH levels in ELF, BAL was performed in eight smokers and eight never-smokers. Intra- and extracellular total glutathione (GSHt) levels and GCL subunit expression were assessed. GSHt was increased in ELF from smokers (1,090.1 +/- 163.0 microM versus 559.2 +/- 48.2 microM). GSHt content of BAL cells (nmol x mg protein(-1)) was decreased in smokers without differences reaching statistical significance (8.0 +/- 1.4 versus 12.4 +/- 2.6). GCLM expression was also reduced in smokers (0.6 +/- 0.1 versus 2.8 +/- 0.4) and correlated with intracellular GSHt content. There was no significant difference in GCLC expression and in differential cell counts in BAL fluid. The authors conclude that smoking does increase glutathione levels in the epithelial lining fluid but not intracellular levels in bronchoalveolar lavage cells. The data suggest that the intracellular glutathione concentration of bronchoalveolar lavage cells (predominately alveolar macrophages) is regulated by the modulatory glutamate-cysteine ligase subunit rather than the catalytic subunit.

Health effects of sulfur-related environmental air pollution. I. Executive summary.

The motivation of simulating real-world environmental exposure in a number of long-term studies with dogs was to address the question of whether or not perpetual inhalation of air pollutants can initiate diseases in healthy lungs and can thus contribute to the increasing prevalence of respiratory diseases in industrialized countries. The major conclusion of this article is that this question has to be answered in the negative for the simultaneous inhalation of the major constituents of combustion-related air pollution, particle-associated sulfur(IV), and particle-associated hydrogen ions. Over 13 mo, 8 healthy beagle dogs were exposed in 2 whole-body chambers daily for 16.5 h to 1 microm neutral sulfite [sulfur(IV)] particles at a mass concentration of 1.5 mg m-3 and for 6 h to 1.1 microm acidic sulfate particles carrying 15 micromol m-3 hydrogen ions into the canine lungs. This longitudinal study was characterized by repeated observations of individual respiratory response patterns. To establish baseline data the dogs were repeatedly examined preexposure while the chambers were ventilated over 16 mo with clean air. Each individual served thus as its own control. Another eight dogs served as additional controls. They were housed in 2 chambers ventilated with clean air over the entire study period of 29 mo. To assess response patterns, respiratory lung function tests were performed pre- and postexposure, segmental lung lavages were repeatedly performed to obtain epithelial lining fluid from the lungs for analysis of cell content, cell function, and biochemical indicators of lung injury, and radiolabeled test particles were used to study pathways of intrapulmonary particle elimination. At the end of the study, the lungs of all animals were morphologically and morphometrically examined. Functional and structural responses were finally compared to those observed previously as a result of a sole exposure of canine lungs to neutral sulfite particles over 10 mo (Heyder et al., 1992). Interactions between responses induced by neutral sulfite and acidic sulfate particles occurred, but antagonism rather than synergism was observed. The responses induced by sulfur(IV) were less pronounced, not detectable, or even reversed when hydrogen ions were also delivered to the lungs. On the other hand, responses not induced by the sole exposure to sulfur(IV) were observed: The activity of alkaline phosphatase was elevated and type II pneumocytes proliferated. It can, however, be concluded that long-term exposure of healthy lungs to particle-associated neutral sulfur(IV) and hydrogen ions at concentration close to ambient levels causes subtle respiratory responses but does not initiate pathological processes in the lungs. In other words, the perpetual inhalation of sulfur(IV) and hydrogen ions from the atmospheric environment presents no health risk to the healthy lungs. It is thus also very unlikely that respiratory diseases can be initiated by the inhalation of these pollutants.

Oxidatively modified proteins in bronchoalveolar lavage fluid of patients with ARDS and patients at-risk for ARDS.

Oxidative stress in acute respiratory distress syndrome (ARDS) is considered as an important pathophysiological mechanism in acute impairment of lung function. The present study investigated whether a pulmonary oxidant-antioxidant imbalance is indicated by substantial oxidative modification of proteins in bronchoalveolar lavage (BAL) fluid. Oxidatively modified proteins in BAL fluid, as measured by the reduction of protein carbonyl groups with tritiated borohydride, were studied in control subjects, patients with clinically established ARDS, and patients considered at-risk for ARDS because they had had coronary bypass surgery. Subsets of these at-risk patients were pretreated either with methylprednisolone or N-acetylcysteine. The carbonyl content of BAL fluid proteins was greatly increased in ARDS patients (5.0+/-13 nmol carbonyl x mL(-1) BAL fluid; mean+/-SEM; p=0.0004; n=10) and moderately increased in the untreated patients at-risk for ARDS (1.3+/-0.2 nmol x mL(-1); p=0.027; n=19) compared with controls (0.8+/-0.2 nmol x mL(-1); n=12). The two other at-risk groups pretreated either with methylprednisolone or N-acetylcysteine showed carbonyl values that were statistically not different from the controls (1.2+/-0.2 nmol x mL(-1); p=0.13; n=13, and 1.1+/-0.3 nmol x mL(-1); p=0.40; n=8, respectively). These results show that oxidatively modified proteins clearly accumulated in bronchoalveolar lavage fluid of acute respiratory distress syndrome patients, and to a minor extent in untreated at-risk patients. These data suggest a severe oxidant-antioxidant imbalance in acute respiratory distress syndrome.

Tracheobronchial surface active material in cystic fibrosis.

Surface active material potentially present in the airway is difficult to analyse due to the tight binding of surfactant components to mucins. A surface active sol-fraction was obtained from sputum of patients with cystic fibrosis (CF), analysed and compared with the sol-fraction from sputum of tracheomized, non-CF patients. The release of phospholipids from CF sputum was relatively fast being completed within minutes, temperature dependent and averaged 5.6 +/- 2.2% of total phospholipid mass. In comparison to sputum, the phospholipid composition of the sol fraction was the same except for a lower percentage of phosphatidylethanolamine, which is usually found primarily cell membrane associated. The sol-fraction from the CF patient group had a lower percentage of phosphatidylcholine and about 3 times more surfactant protein A than that from the non-CF patients. Surface activity did not differ between CF and non-CF samples. Of interest, the adsorption rate (gamma ads, about 30-35 mN/m) and the minimal surface tension (gamma min, about 20-25 mN/m) were relatively low. These data support the hypothesis that surface active material can be released from sputum and that it might support its transport by reducing mucus adhesiveness to the airways.

Metal-catalyzed inactivation of bovine glucose-6-phosphate dehydrogenase--role of thiols.

The role of thiols as oxidant scavengers during inactivation of bovine glucose-6-phosphate dehydrogenase by metal-catalyzed oxidation systems has been studied in vitro. Partial inactivation of the enzyme was achieved by the metal-catalyzed oxidation systems Fe(II)/H202/EDTA or Fe(II)/H202/ADP under specific conditions. When EDTA as chelator was present in the oxidation system, both cysteine and N-acetylcysteine at low concentrations (0.1-1 mM) drastically enhanced inactivation, while cysteinyl-glycine and glutathione did not. The thiol-mediated inactivation was inhibitable by superoxide dismutase. Depletion of enzyme activity by cysteine was paralleled by an increase of the carbonyl content, which indicates oxidative injury. However, when EDTA as chelator was replaced by the natural chelator ADP, all thiols studied acted as antioxidants. It is therefore concluded that the nature of the chelator as a constituent of the metal-catalyzed oxidation systems determines whether the antioxidative function of some thiols is shifted to a prooxidative function against glucose-6-phosphate dehydrogenase.

Oxidized BAL fluid proteins in patients with interstitial lung diseases.

Oxygen-derived free radicals, released by phagocytic cells, have been postulated to contribute to lung tissue damage. We therefore investigated oxidative damage to proteins from bronchoalveolar lavage fluid (BALF) as an indicator of oxidative stress and to assess antioxidant defences in the lungs. We examined BAL fluids from patients with interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF, nonsmokers (NS) and smokers (S)), sarcoidosis (SARC, nonsmokers), and asbestosis (ASB, ex-smokers (EXS)). The oxidation of BALF proteins is accompanied by the introduction of carbonyl groups into their amino acid side-chains and can be quantitated by labeling these groups with tritiated borohydride. The total lung content of oxidized proteins recovered by bronchoalveolar lavage (BAL) was 0.3 +/- 0.07 nmol carbonyl.mL-1 BALF (mean +/- SEM) in the NS control group (n = 9) and tended to be increased, in the asymptomatic S group (n = 8; 0.59 +/- 0.14 nmol.mL-1). This parameter was significantly elevated both in IPF-NS (n = 14; 0.84 +/- 0.2 nmol carbonyl.mL-1 BALF) and SARC-NS (n = 15; 0.73 +/- 0.16 nmol.mL-1) as compared with the NS control. On the contrary, in smoking patients with IPF (n = 6; 0.41 +/- 0.1 nmol carbonyl.mL-1 BALF) and also in ASB-EXS (n = 6; 0.37 +/- 0.06 nmol.mL-1) it was not different from NS controls. The total amount of oxidized proteins correlated positively with the absolute number of eosinophils (EOS) in IPF-NS, IPF-S and SARC, and also with absolute polymorphonuclear neutrophil (PMN) numbers in IPF-NS and IPF-S. In conclusion, oxidative damage of BALF proteins occurred in nonsmoking patients with IPF and SARC. The amount of oxidized bronchoalveolar lavage fluid protein may provide a quantitative assessment of oxygen burden, a balance between oxidant stress and antioxidant defences.

Responses of human neutrophils to sulfite.

Exposure to sulfur dioxide or sulfite aerosols induce inflammatory reactions in the respiratory tract characterized by an influx of neutrophils into the airways. To determine direct intracellular effects of sulfite on human neutrophils, these cells were evaluated ultrastructurally by electron microscopy and analyzed for their extracellular and intracellular respiratory burst activity after incubation with sulfite (0.01-10 mM) in vitro. The respiratory burst was quantitated by measuring both the extracellular release of superoxide anions (O2-) by superoxide dismutase-inhibitable lucigenin-dependent chemiluminescence (CL) and the intracellular generation of hydrogen peroxide (H2O2) by flow cytometry using the reagent dichlorofluorescein diacetate. The addition of sulfite in concentrations of 0.01-1 mM resulted in sixfold increases in CL of resting neutrophils. Neutrophils stimulated with zymosan, phorbol myristate acetate (PMA), or N-formyl-methionine-leucine-phenylalanine further increased CL when sulfite was added. Higher sulfite concentrations (2-10 mM) decreased CL of resting, zymosan-stimulated, and PMA-stimulated cells. When sulfate was added, no changes in CL of resting and zymosan-stimulated neutrophils were seen, indicating that the effect is specific for sulfite. The intracellular generation of H2O2 in resting and PMA-stimulated neutrophils incubated with sulfite (0.1-2 mM) was increased twofold. These findings suggest that sulfite in low concentrations stimulates neutrophils by activating the respiratory burst to produce O2- and H2O2. Ultrastructural studies confirm the stimulating effect of sulfite on neutrophils with sulfite-treated cells exhibiting increased ruffled surface membranes, degranulation changes, and vesiculation similar to those seen in PMA-stimulated cells.

Bronchoalveolar lavage fluid proteins in human lung disease: analysis by two-dimensional electrophoresis.

Proteins of human bronchoalveolar lavage fluids, obtained by washing the epithelial lining fluid of the lungs with phosphate-buffered saline, were analyzed by two-dimensional electrophoresis under denaturating and reducing conditions. The two-dimensional pattern of bronchoalveolar lavage fluid proteins of healthy volunteers (controls) were compared with those of patients with idiopathic pulmonary fibrosis, sarcoidosis, and asbestosis. Particular interest was paid to the proteins present in minor amounts mainly in the low molecular weight region of the gels. Marked changes in single protein spots were observed. In idiopathic pulmonary fibrosis the spot intensity of the surfactant-associated protein, SP-A, showing isomeric forms both in charge and in molecular weight, was markedly decreased. In sarcoidosis, the immunoglobulins (IgG, IgA) and a group of protein spots at an isoelectric point of 4.5-5.0 and a molecular mass of 55 kDa were increased. An additional spot appeared at an isoelectric point of 4.5 and a molecular mass of 12 kDa. In particular in asbestosis, but also in some cases of idiopathic pulmonary fibrosis and sarcoidosis, the number and intensity of low molecular weight proteins were increased strongly.

Oxidative stress in vivo and in vitro: modulation by quartz dust and hyperbaric atmosphere.

Changes in the oxidative status in the soluble proteins of bronchoalveolar lavage (BAL) fluid from monkeys were examined during 26 months of individual or combined exposure to quartz dust (5 mg/m3 of DQ12) and a hyperbaric atmosphere (2.5 bar). The oxidation of BAL proteins, assumed to be an indicator for oxidative stress in the lungs, was determined by measuring the amount of carbonyl groups in their amino acid side chains. The carbonyl content of BAL proteins (nmol carbonyl/mg protein) increased steadily to a maximum value of 156% of the control after 6 months exposure to hyperbaric atmosphere, and decreased below 50% of control levels in both the quartz alone exposed group and the group exposed to quartz in combination with a hyperbaric atmosphere. The effect of quartz on the production of reactive oxygen species by BAL cells was investigated in vitro. BAL cells from healthy monkeys preincubated with quartz and stimulated with phorbol-myristate acetate (PMA) produced reduced levels of extracellular superoxide anion and intracellular hydrogen peroxide compared with PMA-only stimulated cells. Thus the lowered carbonyl content of BAL proteins in the quartz exposed groups may have resulted from reduced production of the hydrogen peroxide which is essential for carbonyl formation by phagocytes. Changes in carbonyl content of BAL protein in vivo may be a new indicator for potential subsequent lung damage.

Two-dimensional electrophoresis of dog bronchoalveolar lavage fluid proteins.

Proteins of dog bronchoalveolar lavage fluid, obtained by washing the epithelial lining layer of lungs with phosphate-buffered saline, were separated by two-dimensional electrophoresis. Due to the low protein and high salt content of the bronchoalveolar lavage fluid, samples had to be concentrated and desalted. Following electrophoresis the protein spots were visualized by silver staining. Comparing the two-dimensional protein patterns of bronchoalveolar lavage fluid with that from serum, several lung-specific proteins were detected. The most prominent protein, most probably a surfactant-associated protein, showed isoforms with isoelectric points in the range of pH 4.2-4.8, and a molecular mass of 32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol.

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride.

Oxidatively modified proteins have been implicated in a variety of physiologic and pathologic processes. Oxidative modification typically causes inactivation of enzymes and also the introduction of carbonyl groups into amino acid side chains of the protein. We describe a method to quantify oxidatively modified proteins through reduction of these carbonyl groups with tritiated borohydride. The technique was applied to purified, oxidatively modified glutamine synthetase and to bronchoalveolar lavage fluid from dogs and from humans. Since the protein content of lung lavage fluid is low, a very sensitive method was required to measure the oxidized residues. Reduction of the carbonyl group generated during oxidation of proteins with tritiated borohydride provided excellent sensitivity. Incorporation of tritium was directly proportional to the amount of protein with a range from 10 to 1000 micrograms. Should moieties other than amino acids be labeled, they are easily removed by rapid benchtop hydrolysis of the protein followed by chromatography on Dowex 50.