Micronuclei, hemoglobin adducts and respiratory tract irritation in mice after inhalation of toluene diisocyanate (TDI) and 4,4'-methylenediphenyl diisocyanate (MDI).

Toluene diisocyanate (TDI) and 4,4'-methylenediphenyl diisocyanate (MDI), used in the production of polyurethane foam, are well known for their irritating and sensitizing properties. Contradictory results have been obtained on their genotoxicity. We investigated the genotoxicity and protein binding of inhaled TDI and MDI in mice by examining micronucleated polychromatic erythrocytes (PCEs) in bone marrow and peripheral blood and TDI- and MDI-derived adducts in hemoglobin. Male C57Bl/6J mice (8 per group) were exposed head-only to TDI vapour (mean concentrations 1.1, 1.5, and 2.4mg/m(3); the mixture of isomers contained, on the average, 63% 2,4-TDI and 37% 2,6-TDI) or MDI aerosol (mean concentrations 10.7, 20.9 and 23.3mg/m(3)), during 1h/day for 5 consecutive days. Bone marrow and peripheral blood were collected 24h after the last exposure. Inhalation of TDI caused sensory irritation (SI) in the upper respiratory tract, and cumulative effects were observed at the highest exposure level. Inhalation of MDI produced SI and airflow limitation, and influx of inflammatory cells into the lungs. Hemoglobin adducts detected in the exposed mice resulted from direct binding to globin of 2,4- and 2,6-TDI and MDI, and dose-dependent increases were observed especially for 2,4-TDI-derived adducts. Adducts originating from the diamines of TDI (toluene diamine) or MDI (methylene dianiline) were not observed. No significant increase in the frequency of micronucleated PCEs was detected in the bone marrow or peripheral blood of the mice exposed to TDI or MDI. The ratio of PCEs and normochromatic erythrocytes (NCEs) was reduced at the highest concentration of MDI, and a slight reduction of the PCE/NCE ratio, dependent on cumulative inhaled dose, was also seen with TDI. Our results indicate that inhalation of TDI or MDI (1h/day for 5 days), at levels that induce toxic effects and formation of TDI- or MDI-specific adducts in hemoglobin, does not have detectable genotoxic effects in mice, as studied with the micronucleus assay.

Level of Fatty Acid Binding Protein 5 (FABP5) Is Increased in Sputum of Allergic Asthmatics and Links to Airway Remodeling and Inflammation.

BACKGROUND: The inflammatory processes in the upper and lower airways in allergic rhinitis and asthma are similar. Induced sputum and nasal lavage fluid provide a non-invasive way to examine proteins involved in airway inflammation in these conditions. OBJECTIVES: We conducted proteomic analyses of sputum and nasal lavage fluid samples to reveal differences in protein abundances and compositions between the asthma and rhinitis patients and to investigate potential underlying mechanisms. METHODS: Induced sputum and nasal lavage fluid samples were collected from 172 subjects with 1) allergic rhinitis, 2) asthma combined with allergic rhinitis, 3) nonallergic rhinitis and 4) healthy controls. Proteome changes in 21 sputum samples were analysed with two-dimensional difference gel electrophoresis (2D-DIGE), and the found differentially regulated proteins identified with mass spectrometry. Immunological validation of identified proteins in the sputum and nasal lavage fluid samples was performed with Western blot and ELISA. RESULTS: Altogether 31 different proteins were identified in the sputum proteome analysis, most of these were found also in the nasal lavage fluid. Fatty acid binding protein 5 (FABP5) was up-regulated in the sputum of asthmatics. Immunological validation in the whole study population confirmed the higher abundance levels of FABP5 in asthmatic subjects in both the sputum and nasal lavage fluid samples. In addition, the vascular endothelial growth factor (VEGF) level was increased in the nasal lavage fluid of asthmatics and there were positive correlations between FABP5 and VEGF levels (r=0.660, p<0.001) and concentrations of FABP5 and cysteinyl leukotriene (CysLT) (r=0.535, p<0.001) in the nasal lavage fluid. CONCLUSIONS: FABP5 may contribute to the airway remodeling and inflammation in asthma by fine-tuning the levels of CysLTs, which induce VEGF production.

Invariant Natural Killer T Cells Play a Role in Chemotaxis, Complement Activation and Mucus Production in a Mouse Model of Airway Hyperreactivity and Inflammation.

CD1d-restricted invariant natural killer T (iNKT) cells play a critical role in the induction of airway hyperreactivity (AHR). After intranasal alpha-galactosylceramide (α-GalCer) administration, bronchoalveolar lavage fluid (BALF) proteins from mouse lung were resolved by two-dimensional differential gel electrophoresis (2D-DIGE), and identified by tandem mass spectroscopy. A lack of iNKT cells prevented the development of airway responses including AHR, neutrophilia and the production of the proinflammatory cytokines in lungs. Differentially abundant proteins in the BALF proteome of α-GalCer-treated wild type mice included lungkine (CXCL15), pulmonary surfactant-associated protein D (SFTPD), calcium-activated chloride channel regulator 1 (CLCA1), fragments of complement 3, chitinase 3-like proteins 1 (CH3LI) and 3 (CH3L3) and neutrophil gelatinase-associated lipocalin (NGAL). These proteins may contribute to iNKT regulated AHR via several mechanisms: altering leukocyte chemotaxis, increasing airway mucus production and possibly via complement activation.

Phagocytosis of nano-sized titanium dioxide triggers changes in protein acetylation.

Nano-sized titanium dioxide (nTiO2) is one of the most produced engineered nanomaterials and therefore carries a high risk for workplace exposure. In several nanosafety studies, exposure to nTiO2 has been shown to trigger inflammation in mice lung and to cause oxidative stress. Here, cytoplasmic proteome changes in human monocyte derived macrophages were investigated with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry to evaluate the adverse cellular effects after exposure to different types of TiO2 nanoparticles (NPs). Both studied TiO2 NPs (rutile TiO2 with or without silica coating) evoked similar proteome alterations. The identified proteins were linked to metabolic homeostasis, cytoskeleton remodeling and oxidative stress. The abundances of chloride intracellular channel protein 1 and cathepsin D changed only after exposure to nTiO2 as compared to a coarse particle analog. Enrichment analysis revealed that 70% of the proteins with changed intensities contained known acetylation sites, and it was possible to confirm a significant induction of cytoplasmic protein acetylation after nTiO2 exposure. The course of the events during phagocytosis could account for the observed membrane maintenance, metabolic and cytoskeletal protein expression changes. Lysine acetylation of cytoplasmic proteins in macrophages is emerging as a major cell regulation mechanism after nTiO2 exposure. BIOLOGICAL SIGNIFICANCE: While the amount of nanosafety research conducted in recent years has been constantly increasing, proteomics has not yet been utilized widely in this field. In addition, reversible protein post-translational modifications (PTMs) such as acetylation and phosphorylation have not been investigated in-depth in nanomaterial exposed cells. Proteome changes observed in nanomaterial exposed macrophages revealed active phagocytosis of the particles and provided new insights into underlying mechanisms of biological responses to nTiO2 exposures. Moreover, reversible protein acetylation might be a major cellular regulation event occurring in nanomaterial exposed cells.

Proteomic changes of alveolar lining fluid in illnesses associated with exposure to inhaled non-infectious microbial particles.

BACKGROUND: Hyperresponsiveness to inhaled non-infectious microbial particles (NIMPs) has been associated with illnesses in the airways. Hypersensitivity pneumonitis (HP) is considered to be the prototype for these NIMPs-related diseases; however, there is no consensus on the definitions or diagnostic criteria for HP and the spectrum of related illnesses. METHODS AND FINDINGS: In order to identify the possible diagnostic markers for illnesses associated with NIMPs in alveolar lining fluid, we performed a proteomic analysis using a two-dimensional difference gel electrophoresis on bronchoalveolar lavage (BAL) fluid from patients with exposure to NIMPs in the context of damp building-related illness (DBRI) or conditions on the borderline to acute HP, designated here as agricultural type of microbial exposure (AME). Samples from patients with HP and sarcoidosis (SARC) were included for reference. Results were compared to results of healthy subjects (CTR). Western blot was used for validation of potential marker proteins from BAL fluid and plasma. Protein expression patterns suggest a close similarity between AME and HP, while DBRI was similar to CTR. However, in DBRI the levels of the inflammation associated molecules galectin-3 and alpha-1-antitrypsin were increased. A novel finding emerging from this study was the increases of semenogelin levels in BAL fluid from patients with AME, HP and SARC. Histone 4 levels were increased in AME, HP and SARC. Elevated plasma levels of histone 2B were detected in HP and SARC, suggesting it to be a potential blood indicator for inflammatory diseases of the lungs. CONCLUSIONS: In this study, the proteomic changes in bronchoalveolar lavage of DBRI patients were distinct from other NIMP exposure associated lung diseases, while changes in AME overlapped those observed for HP patient samples. Some of the proteins identified in this study, semenogelin and histone 4, could function as diagnostic markers for differential diagnosis between DBRI and HP-like conditions.