asbestos--crocidolite and Pulmonary-Fibrosis

Pulmonary exposure to multiwalled carbon nanotubes (MWCNT) induces an inflammatory and rapid fibrotic response, although the long-term signaling mechanisms are unknown. The aim of this study was to examine the effects of 1, 10, 40, or 80 μg MWCNT administered by pharyngeal aspiration on bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration, lactate dehydrogenase (LDH) activity, and lung histopathology for inflammatory and fibrotic responses in mouse lungs 1 mo, 6 mo, and 1 yr postexposure. Further, a 120-μg crocidolite asbestos group was incorporated as a positive control for comparative purposes. Results showed that MWCNT increased BAL fluid LDH activity and PMN infiltration in a dose-dependent manner at all three postexposure times. Asbestos exposure elevated LDH activity at all 3 postexposure times and PMN infiltration at 1 mo and 6 mo postexposure. Pathological changes in the lung, the presence of MWCNT or asbestos, and fibrosis were noted at 40 and 80 μg MWCNT and in asbestos-exposed mice at 1 yr postexposure. To determine potential signaling pathways involved with MWCNT-associated pathological changes in comparison to asbestos, up- and down-regulated gene expression was determined in lung tissue at 1 yr postexposure. Exposure to MWCNT tended to favor those pathways involved in immune responses, specifically T-cell responses, whereas exposure to asbestos tended to favor pathways involved in oxygen species production, electron transport, and cancer. Data indicate that MWCNT are biopersistent in the lung and induce inflammatory and fibrotic pathological alterations similar to those of crocidolite asbestos, but may reach these endpoints by different mechanisms.

Topics: Air Pollutants; Animals; Asbestos, Crocidolite; Bronchoalveolar Lavage Fluid; Dose-Response Relationship, Drug; Gene Expression; Inflammation; Inhalation Exposure; L-Lactate Dehydrogenase; Lung; Male; Mice; Mice, Inbred C57BL; Nanotubes, Carbon; Neutrophil Infiltration; Neutrophils; Pulmonary Fibrosis; Time Factors

Asbestos causes asbestosis and malignancies by mechanisms that are not fully established. Alveolar epithelial cell (AEC) injury and repair are crucial determinants of the fibrogenic potential of noxious agents such as asbestos. We previously showed that mitochondrial reactive oxygen species mediate asbestos-induced AEC intrinsic apoptosis and that mitochondrial human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme, prevents oxidant-induced AEC apoptosis. We reasoned that OGG1 deficiency augments asbestos-induced pulmonary fibrosis. Compared with intratracheal instillation of PBS (50 μl) or titanium dioxide (100 μg/50 μl), crocidolite or Libby amphibole asbestos (100 μg/50 μl) each augmented pulmonary fibrosis in wild-type C57BL/6J (WT) mice after 3 weeks as assessed by histology, fibrosis score, lung collagen via Sircol, and type 1 collagen expression; these effects persisted at 2 months. Compared with WT mice, Ogg1 homozygous knockout (Ogg1(-/-)) mice exhibit increased pulmonary fibrosis after crocidolite exposure and apoptosis in cells at the bronchoalveolar duct junctions as assessed via cleaved caspase-3 immunostaining. AEC involvement was verified by colocalization studies using surfactant protein C. Asbestos increased endoplasmic reticulum stress in the lungs of WT and Ogg1(-/-) mice. Compared with WT, alveolar type 2 cells isolated from Ogg1(-/-) mice have increased mtDNA damage, reduced mitochondrial aconitase expression, and increased P53 and cleaved caspase-9 expression, and these changes were enhanced 3 weeks after crocidolite exposure. These findings suggest an important role for AEC mtDNA integrity maintained by OGG1 in the pathogenesis of pulmonary fibrosis that may represent a novel therapeutic target.

Topics: Alveolar Epithelial Cells; Animals; Asbestos, Crocidolite; DNA Damage; DNA Glycosylases; DNA, Mitochondrial; Humans; Mice; Mice, Knockout; Pulmonary Fibrosis; Time Factors

Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts.. The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF).. MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis.. In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential.

Topics: Animals; Asbestos, Crocidolite; BALB 3T3 Cells; Biological Assay; Cell Count; Cell Proliferation; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Nanotubes, Carbon; Particle Size; Predictive Value of Tests; Pulmonary Fibrosis; Reproducibility of Results; Surface Properties

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity.

Topics: Actins; Alveolar Epithelial Cells; Asbestos, Crocidolite; Cadherins; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; NF-kappa B; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta

Reflecting their exceptional potential to advance a range of biomedical, aeronautic, and other industrial products, carbon nanotube (CNT) production and the potential for human exposure to aerosolized CNTs are increasing. CNTs have toxicologically significant structural and chemical similarities to asbestos (AB) and have repeatedly been shown to cause pulmonary inflammation, granuloma formation, and fibrosis after inhalation/instillation/aspiration exposure in rodents, a pattern of effects similar to those observed following exposure to AB. To determine the degree to which responses to single-walled CNTs (SWCNT) and AB are similar or different, the pulmonary response of C57BL/6 mice to repeated exposures to SWCNTs, crocidolite AB, and ultrafine carbon black (UFCB) were compared using high-throughput global high performance liquid chromatography fourier transform ion cyclotron resonance mass spectrometry (HPLC-FTICR-MS) proteomics, histopathology, and bronchoalveolar lavage cytokine analyses. Mice were exposed to material suspensions (40 micrograms per mouse) twice a week for 3 weeks by pharyngeal aspiration. Histologically, the incidence and severity of inflammatory and fibrotic responses were greatest in mice treated with SWCNTs. SWCNT treatment affected the greatest changes in abundance of identified lung tissue proteins. The trend in number of proteins affected (SWCNT [376] > AB [231] > UFCB [184]) followed the potency of these materials in three biochemical assays of inflammation (cytokines). SWCNT treatment uniquely affected the abundance of 109 proteins, but these proteins largely represent cellular processes affected by AB treatment as well, further evidence of broad similarity in the tissue-level response to AB and SWCNTs. Two high-sensitivity markers of inflammation, one (S100a9) observed in humans exposed to AB, were found and may be promising biomarkers of human response to SWCNT exposure.

Topics: Animals; Asbestos, Crocidolite; Bronchoalveolar Lavage Fluid; Chromatography, High Pressure Liquid; Cytokines; Female; Instillation, Drug; Lung; Mice; Mice, Inbred C57BL; Nanotubes, Carbon; Particle Size; Peptides; Pneumonia; Proteins; Proteome; Proteomics; Pulmonary Fibrosis; Soot; Tandem Mass Spectrometry

The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use.. 8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis. Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate assay.. The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of collagen and underestimated changes between control and fibrotic lungs, conclusions: The proposed method provides accurate quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.

Topics: Analysis of Variance; Animals; Asbestos, Crocidolite; Asbestosis; Azo Compounds; Biomarkers; Collagen; Colorimetry; Coloring Agents; Disease Models, Animal; Hydroxyproline; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Reproducibility of Results; Severity of Illness Index; Spectrophotometry; Up-Regulation

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.

Topics: Animals; Antibiotics, Antineoplastic; Asbestos, Crocidolite; Bleomycin; Bronchoalveolar Lavage; Chemotaxis; Extracellular Matrix; Gene Expression Regulation, Enzymologic; Humans; Hyaluronic Acid; Inflammation; Lung; Lung Injury; Mice; Mice, Knockout; Neutrophils; Oxidation-Reduction; Pneumonia; Pulmonary Fibrosis; Superoxide Dismutase; Superoxides

Asbestos is a ubiquitous, naturally occurring fiber that has been linked to the development of malignant and fibrotic lung diseases. Asbestos exposure leads to apoptosis, followed by compensatory proliferation, yet many of the signaling cascades coupled to these outcomes are unclear. Because CREs (Ca(2+)/cAMP-response elements) are found in the promoters of many genes important for regulation of proliferation and apoptosis, CREB (CRE binding protein) is likely to play an important role in the development of asbestos-mediated lung injury. To explore this possibility, we tested the hypotheses that asbestos exposure leads to CREB phosphorylation in lung epithelial cells and that protein kinase A (PKA) and extracellular signal-regulated kinases 1/2 (ERK1/2) are central regulators of the CREB pathway. Persistent CREB phosphorylation was observed in lung sections from mice following inhalation of crocidolite asbestos. Exposure of C10 lung epithelial cells to crocidolite asbestos led to rapid CREB phosphorylation and apoptosis that was decreased by the inhibition of PKA or ERK1/2 using the specific inhibitors H89 and U0126, respectively. Furthermore, crocidolite asbestos selectively induced a sustained increase in MAP kinase phosphatase-1 mRNA and protein. Silencing CREB protein dramatically reduced asbestos-mediated ERK1/2 phosphorylation, yet significantly increased the number of cells undergoing asbestos-induced apoptosis. These data reveal a novel and selective role for CREB in asbestos-mediated signaling through pathways regulated by PKA and ERK1/2, further providing evidence that CREB is an important regulator of apoptosis in asbestos-induced responses of lung epithelial cells.

Topics: Animals; Apoptosis; Asbestos, Crocidolite; Bronchi; Cell Cycle Proteins; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dual Specificity Phosphatase 1; Immediate-Early Proteins; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Pulmonary Fibrosis; Respiratory Mucosa; RNA, Small Interfering; Up-Regulation

Profibrogeneic cytokines contribute to the accumulation of myofibroblasts in the lung interstitium in idiopathic pulmonary fibrosis (IPF). Imatinib mesylate, a tyrosine kinase inhibitor specific for Abl, platelet-derived growth factor receptor (PDGFR) and c-Kit tyrosine kinases, has been shown to inhibit fibrosis and profibrotic signaling in mouse models of inflammation-mediated lung reactions. The authors tested imatinib mesylate in vivo in a mouse model of crocidolite asbestos-induced progressive fibrosis. The ability of imatinib mesylate to inhibit profibrogeneic cytokine-induced human pulmonary fibroblast migration was tested in vitro and the expression of its target protein tyrosine kinases was assessed with immunofluorescence. In vivo, 10 mg/kg/day imatinib mesylate inhibited histological parenchymal fibrosis and led to a decrease in collagen deposition, but had no significant effect on asbestos-induced neutrophilia. However, 50 mg/kg/day imatinib mesylate did not inhibit collagen deposition. In vitro, IPF fibroblasts expressed Abl, PDGFR-alpha, PDGF-beta, but not c-Kit, and 1 microM imatinib mesylate inhibited profibrogeneic cytokine-induced IPF fibroblast migration. These results suggest that imatinib mesylate is a potential and specific inhibitor of fibroblast accumulation in asbestos-induced pulmonary fibrosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Asbestos, Crocidolite; Benzamides; Cell Culture Techniques; Cell Movement; Cells, Cultured; Fibroblasts; Humans; Imatinib Mesylate; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred Strains; Piperazines; Pneumonia; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pulmonary Fibrosis; Pyrimidines

Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7-28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.

Topics: Animals; Asbestos, Crocidolite; Dipeptides; Lung; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Pneumonia; Protease Inhibitors; Pulmonary Fibrosis

Oxidative stress is thought to be the pathogenesis of pulmonary fibrosis induced by asbestos, and heme oxygenase-1 (HO-1) protects lung tissue against oxidative stress. We hypothesized that HO-1 is associated with oxidative lung injury caused by exposure to asbestos. This study was conducted to investigate the time course of HO-1 expression of lungs exposed to crocidolite asbestos in vivo. Male Wistar rats were administered 1 mg or 2 mg crocidolite asbestos suspended in saline by a single intratracheal instillation and were sacrificed at 3 d, 1 wk, 1 mo, 3 mo, and 6 mo of recovery time. The expression of HO-1 was observed by Western blot analysis and immunostaining. Protein levels of HO-1 increased at from 3 d to 6 mo following intratracheal instillation of 2 mg crocidolite asbestos. The levels of HO-1 increased at 1 wk and 1 mo following intratracheal instillation of 1 mg crocidolite asbestos. Many HO-1-positive cells were found, particularly in the alveolar macrophages, during immunostaining. These findings suggest that HO-1 may be related to lung disorder induced by dust and therefore can act as a biomarker of lung injury due to dust exposure.

Topics: Animals; Asbestos, Crocidolite; Disease Models, Animal; Gene Expression; Heme Oxygenase-1; Inhalation Exposure; Lung; Male; Pulmonary Fibrosis; Rats; Rats, Wistar

To further study the pathogenic mechanism of crocidolite, the imperceptible changes of crocidolite surface in rat were observed.. The animal model was established and the changes in the rat infected with dust were observed by use of microscopy, SEM, differential thermal analysis and IR spectroscopy.. In the course of interaction between organism protein and crocidolite, the protein symmetry decreased and structure loosened. The silicon of crocidolite was bonded with the alkyl, amido- of protein. New absorption bands of Si-O-C(N), Si-R clearly appeared. The organism cleared the dust by means of dissolution, enwrapping, winding or in the way of biochemical dissolution, and the fibre became shortened, broken, bifurcated, ends-rounded, and also it could dissolve, transfer and chemically react on surface.. The results showed that the surface radicals of asbestos fibre reacted with some albumen in tissue and hence formed new surface mediator. It is a new form of dissolution and reaction of fibre in vivo that fibres in alveoli transform to carbonate. The residual substances of crocidolite are mainly Si-O. Tissue membrane is the retardation cingulum of dust transference in vivo.

Topics: Animals; Asbestos, Crocidolite; Asbestosis; Dust; Microscopy, Electron, Scanning; Mineral Fibers; Pleural Diseases; Pulmonary Fibrosis; Rats; Silicon

The present study was undertaken to further define the role of alveolar macrophages (AM) in the pulmonary response to crocidolite fibers. Briefly, groups of 4 male F344 rats were intratracheally instilled with saline or saline suspensions of crocidolite at 2 or 20 mg/kg body weight. Animals were sacrificed 3, 7, 14, and 28 d after exposure and the lung response was characterized by analysis of bronchoalveolar lavage fluid (BALF) for markers of lung injury and inflammation. AM obtained in BALF were cultured and their production of the pro-inflammatory cytokines, tumor necrosis factor alpha (TNF alpha), and interleukin-1 (IL-1) were characterized along with fibronectin, a protein known to stimulate fibroblast migration and proliferation. Lung hydroxyproline content was determined 28 d after exposure and lung histopathology was characterized on d 28 and 90 after exposure. Crocidolite instillation resulted in transient dose-related pulmonary inflammation as evidenced by increased numbers of BALF neutrophils at the low dose and neutrophils, macrophages, and lymphocytes at the high dose. Cytotoxicity and increased permeability were demonstrated by increased levels of BALF lactate dehydrogenase (LDH) and total protein, respectively. AM TNF alpha and IL-1 production were increased only at the high crocidolite dose. This cytokine response was greatest at d 3 and decreased thereafter. AM TNF alpha and IL-1 release were positively correlated with the increased BALF neutrophils. In contrast to TNF alpha and IL-1, AM fibronectin release was increased at both the low and high doses, with the magnitude of response increasing over time. Consistent with previous acute asbestos inhalation studies, histopathology revealed inflammation localized at the level of the terminal bronchioles and alveolar ducts. Fibrosis was demonstrated at both doses by increased trichrome staining of lung tissue sections. Only the high dose resulted in a detectable increase in lung hydroxyproline. Given the bioactivities of TNF alpha, IL-1, and fibronectin, their increased production after crocidolite exposure indicates they contribute to the pulmonary inflammation and fibrosis occurring with this mineral fiber. In addition, the correlation of increased AM TNF alpha and IL-1 production with increased BALF neutrophils supports a role for these cytokines in crocidolite-induced inflammatory cell recruitment. Lastly, association of a persistent increase in AM fibronectin production with an eventual i

Topics: Analysis of Variance; Animals; Asbestos, Crocidolite; Asbestosis; Bronchoalveolar Lavage Fluid; Cells, Cultured; Cytokines; Fibronectins; Growth Substances; Hydroxyproline; Interleukin-1; Intubation, Intratracheal; Macrophages, Alveolar; Male; Pulmonary Fibrosis; Rats; Rats, Inbred F344; Specific Pathogen-Free Organisms; Tumor Necrosis Factor-alpha

It has been suggested that glass fibers in the respirable size range may pose a health hazard similar to asbestos because of the similarities in physical characteristics. To compare the pulmonary cell response with that described earlier with crocidolite asbestos, we administered a milled fiberglass sample to mice by intratracheal instillation. Little effect was seen at a dose of 0.1 mg, but at 1 mg there was epithelial injury and an inflammatory cell response concentrated at bronchiolar-alveolar duct regions. Cellular incorporation of tritiated thymidine showed that repair of both bronchiolar and alveolar epithelium occurred rapidly. This was followed by an extended increase in cell labeling, particularly in peribronchiolar fibroblasts, from 2 to 8 wk after fiber instillation. Granulomas formed at this site and later there was morphologic evidence of fibrosis, which was confirmed biochemically by a significant increase in lung collagen at 4-16 wk. Although 10 times higher dose is required, the results show that the lung response to fiberglass in this experimental system is similar to that described previously for crocidolite asbestos; the sites of cell injury and repair are the same, and the subsequent fibrotic response produces small airway disease.

Topics: Animals; Asbestos, Crocidolite; Bronchoalveolar Lavage Fluid; DNA; Environmental Exposure; Glass; Granuloma, Foreign-Body; Lung; Male; Mice; Microscopy, Electron; Pulmonary Fibrosis

An early proliferative response of mesothelial and subpleural cells has been reported in animals after inhalation or intratracheal (I.T.) instillation to the lung of long asbestos fibers, which also induce pulmonary fibrosis. To determine whether this cell proliferation is directly related to asbestos exposure or is a nonspecific response to injury, we examined [3H]thymidine (3HT) uptake by cells at the pleura after exposing mice to 5 days of hyperoxia, to intravenous (I.V.) (3 mg) or I.T. (0.15 mg) bleomycin, to I.T. (1 mg) silica, and to I.T. (0.1 mg) crocidolite asbestos of mixed length. All exposures induced acute lung injury, as shown by high levels of protein in lavage fluid. After hyperoxia, the percentage of total lung cells labeled by 3HT in autoradiographs was high for only a few days, as repair took place with no increase in fibroblast growth and no subsequent development of fibrosis. Particle or bleomycin exposure induced a prolonged increase in 3HT uptake with enhanced fibroblast labeling over a 4- to 6-wk period. In each case, labeled subpleural cells, mainly fibroblasts, increased up to 10-fold in the first 2 to 4 wk. At the same time, 3HT uptake by mesothelial cells ranged from 1.4 to 3% compared with almost zero in controls and in oxygen-exposed mice after a few days upon return to air. These results indicate that mesothelial and subpleural cell proliferation occurs after various types of injury to the lung. The close temporal association between 3HT uptake by mesothelial cells and fibroblasts during the reparative phase suggests that mesothelial cells may respond to the same cytokines that trigger interstitial fibrosis.

Topics: Animals; Asbestos, Crocidolite; Autoradiography; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Division; Epithelial Cells; Fibroblasts; Hydroxyproline; Injections, Intravenous; Lung; Male; Mice; Oxygen; Pleura; Pulmonary Fibrosis; Silicon Dioxide; Thymidine

We focused here on steady-state mRNA levels of genes involved in antioxidant defense, i.e., manganese superoxide dismutase and copper-zinc superoxide dismutase, and in cell proliferation, i.e., ornithine decarboxylase, c-jun, and glyceraldehyde-3-phosphate-dehydrogenase in whole-lung homogenates from Fischer 344 rats at 3 h to 20 d after exposure to crocridolite asbestos. Changes in gene expression were correlated with histopathologic findings, total and differential cell counts in bronchoalveolar lavage, and levels of hydroxyproline in lung. Dosage-dependent increases in mRNA levels of antioxidant enzymes and proliferation-related genes were observed. Differential cell counts revealed a dose-related infiltration of neutrophils that preceded elevations in gene expression. Neutrophil infiltration into lung and focal lesions of fibrosis as well as increased levels of hydroxyproline were observed only at high concentrations of asbestos. These results indicate that high airborne concentrations of asbestos cause molecular changes in lung that may be related to antioxidant defense and the triggering of cell proliferation, a feature of asbestosis and lung cancer.

Topics: Administration, Inhalation; Animals; Asbestos, Crocidolite; Asbestosis; Blotting, Northern; Blotting, Western; Bronchoalveolar Lavage Fluid; Cell Count; Cell Division; Dust; Female; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Hydroxyproline; Lung; Ornithine Decarboxylase; Proto-Oncogene Proteins c-jun; Pulmonary Fibrosis; Rats; Rats, Inbred F344; RNA, Messenger; Superoxide Dismutase

Several in vitro studies suggest the involvement of active oxygen metabolites in cell damage caused by asbestos. To determine if lung injury, inflammation, and asbestosis could be inhibited in vivo in a rapid-onset, inhalation model of disease, a novel method of chronic administration of antioxidant enzymes was developed. In brief, Fischer 344 rats were treated with polyethylene glycol-conjugated (PEG-) superoxide dismutase or catalase in osmotic pumps over a 10-day (5 days/wk for 2 wk) or 20-day (5 days/wk for 2 wk) period of exposure to crocidolite asbestos. Control rats included sham-exposed animals and those exposed to asbestos but receiving chemically inactivated enzymes. After 10 days of exposure to asbestos, lactic dehydrogenase (LDH), alkaline phosphatase, and total protein in bronchoalveolar lavage (BAL) were measured in one group of rats. Total and differnetial cell counts in BAL also were assessed. After 20 days of exposure, lungs of an additional group of rats were evaluated by histopathology and by measurement of hydroxyproline. Asbestos-associated elevations in LDH, protein, and total cell numbers in BAL were reduced in rats receiving PEG-catalase. Decreases in numbers of alveolar macrophages, polymorphonuclear leukocytes, and lymphocytes occurred in these animals. Exposure to asbestos for 20 days caused significant increases in both the amount of hydroxyproline in lung and the severity and extent of fibrotic lesions as determined by histopathology. These indicators of asbestosis were inhibited in a dosage-dependent fashion in rats receiving PEG-catalase. Use of inactivated PEG-catalase failed to boost serum levels of catalase and did not inhibit asbestos-induced elevation of hydroxyproline in lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Asbestos; Asbestos, Crocidolite; Asbestosis; Bronchoalveolar Lavage Fluid; Catalase; Disease Models, Animal; Hydroxyproline; Lung; Male; Polyethylene Glycols; Pulmonary Fibrosis; Rats; Rats, Inbred F344; Superoxide Dismutase

The interaction of UICC crocidolite asbestos with biological membranes in vivo was studied in rats after a single intratracheal dose of a suspension of 20 mg of fibres per rat. Development of lung fibrosis (increased level of hydroxyproline, a collagen index together with corresponding pathomorphological alteration) confirmed the penetration of crocidolite fibres into the lungs in the course of seven months exposure. The pulmonary deposition of crocidolite affected the lysosomal membranes of lung cells as manifested by (1) enhanced lipid peroxidation with (2) stimulation (release) of activity of beta-glucuronidase and cathepsin D. Enhanced lipid peroxidation and activity of beta-glucuronidase may contribute to the delayed, carcinogenic effects of crocidolite asbestos.

Topics: Animals; Asbestos; Asbestos, Crocidolite; Cathepsin D; Glucuronidase; Lipid Metabolism; Lung; Lysosomes; Male; Membrane Proteins; Pulmonary Fibrosis; Rats; Rats, Inbred Strains

To determine the cellular and fibrogenic responses of the lung to long asbestos fibres, mice were instilled intratracheally with 0.1 mg of a sample of long crocidolite fibres. Animals were killed at intervals to 20 weeks with 3H thymidine injected one h before death. Following bronchoalveolar lavage, an increase in polymorph neutrophils (PMN) and alveolar macrophages (AM) was found during the first week, accompanied by elevated glucosaminidase and alveolar protein levels. Although the PMN number dropped, some were always recovered by lavage to 20 weeks. Early multifocal necrosis of bronchiolar epithelium was followed by a large increase in labelling of epithelial cells and underlying fibroblasts. Epithelial overgrowth of luminal long fibres and inflammatory exudates was followed by giant cell and granuloma formation in the interstitium. After four weeks collagen levels were significantly increased and fibrosis was seen in these peribronchiolar locations. A few small fibres were observed in AM but no evidence of fibrosis was seen in alveolar walls. These findings suggest that injury to bronchial and bronchiolar epithelium allows long fibres to reach the interstitium where subsequent macrophage-fibroblast interactions result in a severe fibrotic reaction that resembles the bronchiolar component of human asbestosis.

Topics: Animals; Asbestos; Asbestos, Crocidolite; Bronchi; Cell Division; Lung; Macrophages; Mice; Mice, Inbred Strains; Microscopy, Electron; Phagocytosis; Pulmonary Fibrosis

To determine the relationship between the development of pulmonary fibrosis and the size of deposited asbestos, we prepared a pure sample of short crocidolite fibres and instilled 0.5 mg of 0.1 mg to the lungs of mice. Animals were killed up to 20 weeks later with 3H thymidine injected 1 h before death. By bronchoalveolar lavage, there was a rapid transient increase in polymorph neutrophils (PMN) and in glucosaminidase levels; alveolar macrophage (AM) numbers were elevated in the 0.5 mg group for eight weeks. Most fibres were phagocytized by AM, many of which were heavily laden and cleared from the lung over the 20 week period. Some fibres were seen in type 1 epithelial cells, frequently associated with cell injury. From cell kinetic studies, a very brief proliferative response was seen in bronchiolar epithelial and Type 2 alveolar epithelial cells. A greater response was seen in interstitial fibroblasts which showed increased labelling up to two weeks after 0.5 mg asbestos. However no granulomas were seen and very little fibrosis was found by morphology or by biochemistry at any time after 0.5 mg; no fibrosis was seen after instilling 0.1 mg. The results show that a high dose of exclusively short asbestos fibres produces minimal lung injury and fibrosis in spite of long standing macrophage-fibre interaction in the alveoli.

Topics: Animals; Asbestos; Asbestos, Crocidolite; Cell Division; Lung; Mice; Mice, Inbred Strains; Microscopy, Electron; Phagocytosis; Pulmonary Alveoli; Pulmonary Fibrosis

The responses of pulmonary alveolar and bronchial cells to asbestos exposure were studied by relating the cytokinetic changes of injury and repair to the inflammatory process and subsequent fibroblastic activity. The lesions were induced by intratracheal instillation of 1 mg crocidolite asbestos in mice, which were killed up to 20 weeks thereafter; 3H-thymidine was injected 1 hour before death. A rapid inflammatory response with elevated polymorphonuclear leukocytes and lysosomal enzyme release was largely over by 2 weeks, but the increase in alveolar macrophages was maintained. Focal necrosis of bronchial epithelial cells was repaired by cell regeneration, whereby new epithelial cells overgrew luminal exudates to incorporate long asbestos fibers into the peribronchial interstitium, where macrophagic granulomas formed. Increased collagen levels were largely due to stimulation of peribronchial fibroblasts. A lesser reaction of epithelial damage, Type 2 cell proliferation, and fibroblast stimulation also occurred in the alveolar walls. The results suggest that macrophage-fibroblast interactions associated with enhanced fibrosis occur readily in the peribronchial interstitium following injury and repair of epithelial cells by long asbestos fibers.

Topics: Animals; Asbestos; Asbestos, Crocidolite; Autoradiography; Bronchi; Cell Division; Collagen; DNA; Epithelium; Hydroxyproline; Kinetics; Macrophages; Male; Mice; Neutrophils; Protein Biosynthesis; Pulmonary Alveoli; Pulmonary Fibrosis

Asbestosis is generally considered to result in restrictive pulmonary disease associated with interstitial fibrosis. Recently, however, attention has focused upon bronchiolar lesions and concomitant obstruction to air flow. The responses of conducting airways and alveoli were studied over a 20 week period following instillation of crocidolite to mice. The location of the lesions and the sequential inflammatory changes were studied by bronchoalveolar lavage, light and electron microscopy; regenerative activity was monitored by autoradiographs. Within 48 h there was multifocal necrosis of bronchiolar epithelium, maximal at bifurcations where longer fibres tend to adhere. Subsequently, intralumenal exudates were overgrown by epithelium and incorporated into the bronchiolar connective tissue where active peribronchiolar granulomas persisted until 20 weeks. Alveolar lesions were located predominantly in peribronchiolar air sacs and at the junctions of bronchioles and alveolar ducts. Focal acute injury of type 1 cells and transepithelial passage facilitated transport of short asbestos fibres to the interstitium where they were phagocytozed by macrophages. Regenerative activity was prompt with active division of type 2 epithelial cells. Biochemically, collagen increased after 4 weeks, when fibrosis was predominantly located in the bronchiolar lumens and in peribronchiolar connective tissue with lesser amounts in the centrilobular alveolar interstitium. The results suggest that the longer fibres induce bronchiolar injury and a more severe fibrotic pattern similar to the recently described changes seen in human asbestosis.

Topics: Animals; Asbestos; Asbestos, Crocidolite; Bronchi; Connective Tissue; Epithelium; Hydroxyproline; Lung; Male; Mice; Mice, Inbred Strains; Microscopy, Electron; Phagocytosis; Pulmonary Alveoli; Pulmonary Fibrosis; Time Factors; Vacuoles

Although all commercial types of asbestos can cause pulmonary fibrosis, little is known about ultrastructural differences in the evolution of pulmonary lesions induced by amphiboles and serpentines. The present study was designed to compare the histological and ultrastructural effects produced by chronic inhalation of either crocidolite (amphibole) or chrysotile (serpentine) asbestos in the rat. Animals, exposed by intermittent inhalation for 3 months, were killed after 2 to 16 months. When inhaled, both types of asbestos caused thickened alveolar duct bifurcations associated with macrophage aggregates. Crocidolite inhalation also produced subpleural collections of alveolar macrophages and lymphocytes. Electron microscopy revealed some similarities, but also distinct differences, in the pulmonary distribution of inhaled chrysotile and crocidolite. Whereas both asbestos varieties were identified within the pulmonary interstitium, only crocidolite was detected inside alveolar macrophages. Chrysotile fibres were seen infrequently within the vascular compartment. Microcalcifications were noted after chrysotile inhalation, but were never observed following crocidolite exposure. Both asbestos types induced slight pulmonary fibrosis. These findings indicate that crocidolite and chrysotile produce different pathogenetic features, although both are fibrogenic.

Topics: Animals; Asbestos; Asbestos, Crocidolite; Asbestos, Serpentine; Lung; Macrophages; Male; Microscopy, Electron; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Inbred F344

This account reviews the clinical and pathological features of asbestosis, with a brief comment on the incidence of asbestosis in workers exposed occupationally to Western Australian crocidolite at Wittenoom Gorge. In keeping with common usage, the term "asbestosis" is restricted to pulmonary parenchymal fibrosis--with or without associated visceral pleural fibrosis--related to inhalation of asbestos fibres, thereby excluding parietal pleural fibrous plaques. The mechanisms underlying the development of the interstitial fibrosis are incompletely understood, but they appear to involve complex interactions of asbestos with alveolar macrophages, fibroblasts, lymphocytes and the complement system.

Topics: Asbestos; Asbestos, Crocidolite; Asbestosis; Australia; Biological Transport; Biomechanical Phenomena; Disease Susceptibility; Dose-Response Relationship, Drug; Humans; Lung; Pulmonary Fibrosis; Radiography

The relative in vitro and in vivo toxicity of several types of manufactured fibrous glass insulation and crocidolite asbestos was investigated to aid in selection of a suitable glass fiber for subsequent use in inhalation exposures. The in vitro cytotoxicity to pulmonary alveolar macrophages of small glass fibers from microfiber insulation (count median diameter (CMD) approximately 0.1-0.2 micrometer) was greater than that of the larger fibers from household insulation (CMD approximately 2.4 micrometers). To screen for in vivo pulmonary toxicity, 2-21 mg of glass or asbestos fibers were administered in divided doses to male Syrian hamsters by intratracheal instillation. Animals were sacrificed at 1, 3.5 and 11 months following initial administration of material. One type of glass microfiber [count median diameter (CMD) approximately 0.1 micrometer] caused deaths from pulmonary edema at early times after instillation. High levels of asbestos, a second glass microfiber (CMD approximately 0.2 micrometer) and one type of household insulation fiber (CMD 2.3 micrometers) all resulted in increase in total collagen and mild pulmonary fibrosis at later times after instillation, although microfiber insulation produced a greater response than household insulation. Asbestos insulation produced the greatest response. A five-day inhalation exposure to a high level of glass microfibers deposited in lung less than 10 percent of the lowest instilled amount which elicited indications of lung injury. This amount did not produce significant biological changes at 1 to 12 months after exposure.

Topics: Animals; Asbestos; Asbestos, Crocidolite; Construction Materials; Cricetinae; Environmental Exposure; Glass; Lung Diseases; Macrophages; Male; Mesocricetus; Pulmonary Alveoli; Pulmonary Edema; Pulmonary Fibrosis