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Secretory leukocyte peptidase inhibitor (SLPI) is a biomarker present in the respiratory tract that protects against tissue destruction and aids in wound healing. We examined whether SLPI in pleural effusion can be used to distinguish benign asbestos pleural effusion (BAPE) from early-stage malignant pleural mesothelioma (MPM) and other diseases. We measured the levels of SLPI, hyaluronic acid (HA), soluble mesothelin-related peptides (SMRP), CCL2, galectin-3, and CYFRA21-1 in 51 patients with BAPE, 37 patients with early-stage MPM, 77 patients with pleural effusions due to non-small-cell lung cancer (LCa), and 74 patients with other pleural effusions. SLPI levels in the pleural fluid of patients with BAPE were significantly lower than those in patients with MPM, LCa, and other pleural effusions (p < 0.0001). The area under the curve (AUC) for SLPI's ability to distinguish BAPE from MPM was 0.902, with a sensitivity of 82.4% and a specificity of 86.5%. This AUC was not only favourable but was better than the AUC for the ability of CYFRA21-1 to distinguish BAPE (0.853). The combination of SLPI and CYFRA21-1 achieved an AUC of 0.965 for the differentiation between BAPE and MPM. Pleural fluid SLPI as well as CYFRA21-1 and HA is useful as a biomarker to diagnose BAPE, which needs to be distinguished from early-stage MPM.

Alveolar epithelial cell (AEC) apoptosis, arising from mitochondrial dysfunction and mitophagy defects, is important in mediating idiopathic pulmonary fibrosis (IPF). Our group established a role for the mitochondrial (mt) DNA base excision repair enzyme, 8-oxoguanine-DNA glycosylase 1 (mtOGG1), in preventing oxidant-induced AEC mtDNA damage and apoptosis and showed that OGG1-deficient mice have increased lung fibrosis. Herein, we determined whether mice overexpressing the mtOGG1 transgene ( mtOgg1 tg ) are protected against lung fibrosis and whether AEC mtOGG1 preservation of mtDNA integrity mitigates phosphatase and tensin homolog-induced putative kinase 1 (PINK1) deficiency and apoptosis. Compared with wild type (WT), mtOgg1 tg mice have diminished asbestos- and bleomycin-induced pulmonary fibrosis that was accompanied by reduced lung and AEC mtDNA damage and apoptosis. Asbestos and H 2 O 2 promote the MLE-12 cell PINK1 deficiency, as assessed by reductions in the expression of PINK1 mRNA and mitochondrial protein expression. Compared with WT, Pink1 -knockout ( Pink1-KO ) mice are more susceptible to asbestos-induced lung fibrosis and have increased lung and alveolar type II (AT2) cell mtDNA damage and apoptosis. AT2 cells from Pink1-KO mice and PINK1-silenced (siRNA) MLE-12 cells have increased mtDNA damage that is augmented by oxidative stress. Interestingly, mtOGG1 overexpression attenuates oxidant-induced MLE-12 cell mtDNA damage and apoptosis despite PINK1 silencing. mtDNA damage is increased in the lungs of patients with IPF as compared with controls. Collectively, these findings suggest that mtOGG1 maintenance of AEC mtDNA is crucial for preventing PINK1 deficiency that promotes apoptosis and lung fibrosis. Given the key role of AEC apoptosis in pulmonary fibrosis, strategies aimed at preserving AT2 cell mtDNA integrity may be an innovative target.

Macrophage activation is implicated in the development of pulmonary fibrosis by generation of profibrotic molecules. Although NADPH oxidase 4 (NOX4) is known to contribute to pulmonary fibrosis, its effects on macrophage activation and mitochondrial redox signaling are unclear. Here, we show that NOX4 is crucial for lung macrophage profibrotic polarization and fibrotic repair after asbestos exposure. NOX4 was elevated in lung macrophages from subjects with asbestosis, and mice harboring a deletion of NOX4 in lung macrophages were protected from asbestos-induced fibrosis. NOX4 promoted lung macrophage profibrotic polarization and increased production of profibrotic molecules that induce collagen deposition. Mechanistically, NOX4 further augmented mitochondrial ROS production and induced mitochondrial biogenesis. Targeting redox signaling and mitochondrial biogenesis prevented the profibrotic polarization of lung macrophages by reducing the production of profibrotic molecules. These observations provide evidence that macrophage NOX4 is a potentially novel therapeutic target to halt the development of asbestos-induced pulmonary fibrosis.

Fibrosis in multiple organs, including the liver, kidney, and lung, often occurs secondary to environmental exposure. Asbestos exposure is one important environmental cause of lung fibrosis. The mechanisms that mediate fibrosis is not fully understood, although mitochondrial oxidative stress in alveolar macrophages is critical for fibrosis development. Mitochondrial Ca 2+ levels can be associated with production of reactive oxygen species. Here, we show that patients with asbestosis have higher levels of mitochondrial Ca 2+ compared with normal patients. The mitochondrial calcium uniporter (MCU) is a highly selective ion channel that transports Ca 2+ into the mitochondrial matrix to modulate metabolism. Asbestos exposure increased mitochondrial Ca 2+ influx in alveolar macrophages from wild-type, but not MCU +/- , mice. MCU expression polarized macrophages to a profibrotic phenotype after exposure to asbestos, and the profibrotic polarization was regulated by MCU-mediated ATP production. Profibrotic polarization was abrogated when MCU was absent or its activity was blocked. Of more importance, mice that were deficient in MCU were protected from pulmonary fibrosis. Regulation of mitochondrial Ca 2+ suggests that MCU may play a pivotal role in the development of fibrosis and could potentially be a therapeutic target for pulmonary fibrosis.-Gu, L., Larson-Casey, J. L., Carter, A. B. Macrophages utilize the mitochondrial calcium uniporter for profibrotic polarization., (© FASEB.)

Background: Myofibroblasts play a major role in the synthesis of extracellular matrix (ECM) and the stimulation of these cells is thought to play an important role in the development of silicosis. The present study was undertaken to investigate the anti-fibrotic effects of dibutyryl-cAMP (db-cAMP) on rats induced by silica., Methods: A HOPE MED 8050 exposure control apparatus was used to create the silicosis model. Rats were randomly divided into 4 groups: 1)controls for 16 w; 2)silicosis for 16 w; 3)db-cAMP pre-treatment; 4) db-cAMP post-treatment. Rat pulmonary fibroblasts were cultured in vitro and divided into 4 groups as follows: 1) controls; 2) 10 -7 mol/L angiotensin II (Ang II); 3) Ang II +10 -4 mol/L db-cAMP; and 4) Ang II + db-cAMP+ 10 -6 mol/L H89. Hematoxylin-eosin (HE), Van Gieson staining and immunohistochemistry (IHC) were performed to observe the histomorphology of lung tissue. The levels of cAMP were detected by enzyme immunoassay. Double-labeling for α-SMA with Gαi3, protein kinase A (PKA), phosphorylated cAMP-response element-binding protein (p-CREB), and p-Smad2/3 was identified by immunofluorescence staining. Protein levels were detected by Western blot analysis. The interaction between CREB-binding protein (CBP) and Smad2/3 and p-CREB were measured by co-immunoprecipitation (Co-IP)., Results: Db-cAMP treatment reduced the number and size of silicosis nodules, inhibited myofibroblast differentiation, and extracellular matrix deposition in vitro and in vivo. In addition, db-cAMP regulated Gαs protein and inhibited expression of Gαi protein, which increased endogenous cAMP. Db-cAMP increased phosphorylated cAMP-response element-binding protein (p-CREB) via protein kinase A (PKA) signaling, and decreased nuclear p-Smad2/3 binding with CREB binding protein (CBP), which reduced activation of p-Smads in fibroblasts induced by Ang II., Conclusions: This study showed an anti-silicotic effect of db-cAMP that was mediated via PKA/p-CREB/CBP signaling. Furthermore, the findings offer novel insight into the potential use of cAMP signaling for therapeutic strategies to treat silicosis.

Background: The identification of diagnostic/prognostic biomarkers for asbestos-related diseases is relevant for early diagnosis and patient survival and may contribute to understanding the molecular mechanisms underlying the disease development and progression., Aims: To identify a pattern of miRNAs as possible diagnostic biomarkers for patients with malignant pleural mesothelioma (MPM) and asbestosis (ASB) and as prognostic biomarkers for MPM patients., Methods: miRNA-16, miRNA-17, miRNA-126, and miRNA-486 were quantified in plasma and formalin-fixed paraffin-embedded samples to evaluate their diagnostic and prognostic roles compared to patients with other noncancerous pulmonary diseases (controls). Results. The expression of all the miRNAs was significantly lower in patients with MPM and ASB than that in controls. miRNA-16, miRNA-17, and miRNA-486 in plasma and tissue of MPM patients were significantly correlated. Furthermore, the expression of miRNA-16 in plasma and tissue, and miRNA-486 only in tissue, was positively related with cumulative survival in MPM patients., Conclusions: All the miRNA levels were decreased in patients with MPM or ASB, supporting the role of circulating miRNAs as a potential tool for diseases associated with exposure to asbestos fibers. miRNA-16 was directly related to MPM patient prognosis, suggesting its possible use as a prognostic marker in MPM patients.

Libby amphibole (LA) causes a unique progressive lamellar pleural fibrosis (LPF) that is associated with pulmonary function decline. Pleural fibrosis among the LA-exposed population of Libby, MT, has been associated with the production of anti-mesothelial cell autoantibodies (MCAA), which induce collagen production from cultured human mesothelial cells. We hypothesized that the progressive nature of LPF could be at least partially attributed to an autoimmune process and sought to demonstrate that LA-induced MCAA trigger collagen deposition in vivo. C57BL/6 mice were exposed to LA for 7 mo, and serum was tested for MCAA by cell-based ELISA on primary mouse mesothelial cells. When treated in vitro with serum from mice exposed to LA, mesothelial cells upregulated collagen matrix production. This effect was lost when the serum was cleared of IgG using protein G beads, implicating IgG autoantibodies. Using the peritoneal cavity as a surrogate for the pleural cavity, groups of naïve (non-asbestos-exposed) mice were injected intraperitoneally with 1) control serum, 2) one dose of serum from LA-exposed mice (LA serum), 3) two doses of LA serum, or 4) two doses of LA serum cleared of IgG. After 1 mo, analysis of collagen in peritoneal walls using two-photon confocal microscopy (SHG analysis) and a hydroxyproline assay demonstrated significant increases in collagen by LA serum but not control or cleared serum. These data support the hypothesis that MCAA in LA-exposed mice induce fibrotic responses in vivo, demonstrating that an autoimmune component may be contributing to the progressive pleural fibrosis seen in LA-exposed patients., (Copyright © 2016 the American Physiological Society.)

We tested the postulate that iron homeostasis is altered among patients diagnosed to have asbestosis. Lung tissue from six individuals diagnosed to have had asbestosis at autopsy was stained for iron, ferritin, divalent metal transporter 1 (DMT1), and ferroportin 1 (FPN1). Slides from six individuals having pneumonectomy for lung cancer were employed as controls. Lung tissue from those patients with asbestosis demonstrated stainable iron, whereas control lung tissue did not. Staining for this metal was observed predominantly in airway and alveolar macrophages. Expression of the iron-related proteins ferritin, DMT1, and FPN1 was elevated in lung tissue from the six asbestosis patients relative to controls. This increased expression of iron-transport and iron-storage proteins was evident in both airway and alveolar epithelial cells. Asbestos bodies were abundant in lung tissue from patients diagnosed to have had asbestosis. While staining for iron, ferruginous bodies did not demonstrate uptake of antibodies for ferritin, DMT1, and FPN1. We conclude that iron homeostasis is altered in lung disease among those diagnosed to have asbestosis with an accumulation of the metal and a modified expression of iron-related proteins being evident.

Although the mechanisms for fibrosis development remain largely unknown, recent evidence indicates that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) may act as an important fibrotic stimulus in diseased lungs. ER stress is observed in lungs of patients with idiopathic pulmonary fibrosis. In this study we evaluated if ER stress and the UPR was present in macrophages exposed to chrysotile asbestos and if ER stress in macrophages was associated with asbestos-induced pulmonary fibrosis. Macrophages exposed to chrysotile had elevated transcript levels of several ER stress genes. Macrophages loaded with the Ca(2+)-sensitive dye Fura2-AM showed that cytosolic Ca(2+) increased significantly within minutes after chrysotile exposure and remained elevated for a prolonged time. Chrysotile-induced increases in cytosolic Ca(2+) were partially inhibited by either anisomycin, an inhibitor of passive Ca(2+) leak from the ER, or 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator known to deplete ER Ca(2+) stores. Anisomycin inhibited X-box-binding protein 1 (XBP1) mRNA splicing and reduced immunoglobulin-binding protein (BiP) levels, whereas BAPTA-AM increased XBP1 splicing and BiP expression, suggesting that ER calcium depletion may be one factor contributing to ER stress in cells exposed to chrysotile. To evaluate ER stress in vivo, asbestos-exposed mice showed fibrosis development, and alveolar macrophages from fibrotic mice showed increased expression of BiP. Bronchoalveolar macrophages from asbestosis patients showed increased expression of several ER stress genes compared with normal subjects. These findings suggest that alveolar macrophages undergo ER stress, which is associated with fibrosis development., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Chrysotile has been frequently used in the past in manufacturing brakes and continues to be used in brakes in many countries. This study was designed to provide an understanding of the biokinetics and potential toxicology following inhalation of brake dust following short term exposure in rats. The deposition, translocation and pathological response of brake dust derived from brake pads manufactured with chrysotile were evaluated in comparison to the amphibole, crocidolite asbestos. Rats were exposed by inhalation 6 h/day for 5 days to either brake dust obtained by sanding of brake-drums manufactured with chrysotile, a mixture of chrysotile and the brake dust or crocidolite asbestos. No significant pathological response was observed at any time point in either the brake dust or chrysotile/brake dust exposure groups. The long chrysotile fibers (>20 μm) cleared quickly with T(½) estimated as 30 and 33 days, respectively in the brake dust and the chrysotile/brake dust exposure groups. In contrast, the long crocidolite fibers had a T(½)>1000 days and initiated a rapid inflammatory response in the lung following exposure resulting in a 5-fold increase in fibrotic response within 91 days. These results provide support that brake dust derived from chrysotile containing brake drums would not initiate a pathological response in the lung following short term inhalation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

It has been proposed that an epithelial injury may be one of the multiple primary events in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The aim of this study was to characterize the tight junction and adherens junction proteins in normal human lung, IPF, cryptogenic organizing pneumonia, and asbestosis. We determined the immunohistochemical cell-specific expression of tight junction proteins claudin-1, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-7, as well as 3 adherens junction proteins, E-cadherin, N-cadherin, and β-catenin. We further analyzed the expression of claudin-1, claudin-3, and claudin-4 and E-cadherin, N-cadherin, and β-catenin at the transcriptional level by quantitative real-time reverse transcriptase polymerase chain reaction. The expression levels of both tight junction and adherens junction proteins were elevated in regenerative alveolar epithelium in pulmonary fibrosis as compared with the expression of these proteins in normal alveolar epithelium. In particular, the expression levels of claudins-1 and claudin-3 were clearly elevated in all diseases. Furthermore, the amounts of adherens junction proteins messenger RNAs (mRNAs) were also all increased in pulmonary fibroses in comparison with healthy controls, with N-cadherin showing the greatest increase in mRNA levels in all diseases. However, the amounts of claudin-1, claudin-3, and claudin-4 mRNAs in fibrotic lung were similar to or even lower than those measured in the healthy controls. It is possible that the diminished capacity to produce claudin mRNAs may be one explanation for poor repair capacity of alveolar epithelial cells in IPF., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This review provides a basis for substantiating both kinetically and pathologically the differences between chrysotile and amphibole asbestos. Chrysotile, which is rapidly attacked by the acid environment of the macrophage, falls apart in the lung into short fibers and particles, while the amphibole asbestos persist creating a response to the fibrous structure of this mineral. Inhalation toxicity studies of chrysotile at non-lung overload conditions demonstrate that the long (>20 µm) fibers are rapidly cleared from the lung, are not translocated to the pleural cavity and do not initiate fibrogenic response. In contrast, long amphibole asbestos fibers persist, are quickly (within 7 d) translocated to the pleural cavity and result in interstitial fibrosis and pleural inflammation. Quantitative reviews of epidemiological studies of mineral fibers have determined the potency of chrysotile and amphibole asbestos for causing lung cancer and mesothelioma in relation to fiber type and have also differentiated between these two minerals. These studies have been reviewed in light of the frequent use of amphibole asbestos. As with other respirable particulates, there is evidence that heavy and prolonged exposure to chrysotile can produce lung cancer. The importance of the present and other similar reviews is that the studies they report show that low exposures to chrysotile do not present a detectable risk to health. Since total dose over time decides the likelihood of disease occurrence and progression, they also suggest that the risk of an adverse outcome may be low with even high exposures experienced over a short duration.

Asbestos causes asbestosis and malignancies by molecular mechanisms that are not fully understood. The modes of action underlying asbestosis, lung cancer, and mesothelioma appear to differ depending on the fiber type, lung clearance, and genetics. After reviewing the key pathologic changes following asbestos exposure, we examine recently identified pathogenic pathways, with a focus on oxidative stress. Alveolar epithelial cell apoptosis, which is an important early event in asbestosis, is mediated by mitochondria- and p53-regulated death pathways and may be modulated by the endoplasmic reticulum. We review mitochondrial DNA (mtDNA)-damage and -repair mechanisms, focusing on 8-oxoguanine DNA glycosylase, as well as cross talk between reactive oxygen species production, mtDNA damage, p53, OGG1, and mitochondrial aconitase. These new insights into the molecular basis of asbestos-induced lung diseases may foster the development of novel therapeutic targets for managing degenerative diseases (e.g., asbestosis and idiopathic pulmonary fibrosis), tumors, and aging, for which effective management is lacking.

Asbestos is a potent carcinogen associated with malignant mesothelioma and lung cancer but its carcinogenic mechanisms are still poorly understood. Asbestos toxicity is ascribed to its particular physico-chemical characteristics, and one of them is the presence of and ability to adsorb iron, which may cause an alteration of iron homeostasis in the tissue. This observational study reports a combination of advanced synchrotron-based X-ray imaging and micro-spectroscopic methods that provide correlative morphological and chemical information for shedding light on iron mobilization features during asbestos permanence in lung tissue. The results show that the processes responsible for the unusual distribution of iron at different stages of interaction with the fibres also involve calcium, phosphorus and magnesium. It has been confirmed that the dominant iron form present in asbestos bodies is ferritin, while the concurrent presence of haematite suggests alteration of iron chemistry during asbestos body permanence.

Leukotrienes (LTs) are involved in the pathogenesis of lung fibrosis and were increased in exhaled breath condensate (EBC) of the patients with pneumoconiosis. However the possible influence of extra-pulmonary disorders on the EBC markers is not known. Therefore in parallel with EBC, LTs' levels in the plasma and urine were measured in patients with pneumoconiosis (45 × asbestos exposure, 37 × silica exposure) and in 27 controls. Individual LTs B4, C4, D4 and E4 were measured by liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). In EBC, LT D4 and LT E4 were increased in both groups of patients (p<0.001 and p<0.05), comparing with the controls. Both LT B4 and cysteinyl LTs were elevated in asbestos-exposed subjects (p<0.05). Asbestosis with more severe radiological signs (s1/s2-t3/u2) and lung functions impairment has shown higher cysteinyl LTs and LT C4 in the EBC (p<0.05) than mild asbestosis (s1/s0-s1/s1). In addition, in the subjects with asbestosis, cysteinyl LTs in EBC correlated with TLC (-0.313, p<0.05) and TLCO/Hb (-0.307, p<0.05), and LT C4 with TLC (-0.358, p<0.05). In pneumoconioses, EBC appears the most useful from the 3 fluids studied.

Inflammation and lung remodeling are hallmarks of asbestos-induced fibrosis, but the molecular mechanisms that control these events are unclear. Using laser capture microdissection (LCM) of distal bronchioles in a murine asbestos inhalation model, we show that osteopontin (OPN) is up-regulated by bronchiolar epithelial cells after chrysotile asbestos exposures. In contrast to OPN wild-type mice (OPN(+/+)) inhaling asbestos, OPN null mice (OPN(-/-)) exposed to asbestos showed less eosinophilia in bronchoalveolar lavage fluids, diminished lung inflammation, and decreased mucin production. Bronchoalveolar lavage fluid concentrations of inflammatory cytokines (IL-1β, IL-4, IL-6, IL-12 subunit p40, MIP1α, MIP1β, and eotaxin) also were significantly less in asbestos-exposed OPN(-/-) mice. Microarrays performed on lung tissues from asbestos-exposed OPN(+/+) and OPN(-/-) mice showed that OPN modulated the expression of a number of genes (Col1a2, Timp1, Tnc, Eln, and Col3a1) linked to fibrosis via initiation and cross talk between IL-1β and epidermal growth factor receptor-related signaling pathways. Novel targets of OPN identified include genes involved in cell signaling, immune system/defense, extracellular matrix remodeling, and cell cycle regulation. Although it is unclear whether the present findings are specific to chrysotile asbestos or would be observed after inhalation of other fibers in general, these results highlight new potential mechanisms and therapeutic targets for asbestosis and other diseases (asthma, smoking-related interstitial lung diseases) linked to OPN overexpression., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aim: The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use., Methods: 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., Results: 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.

Pulmonary toxicity induced by asbestos is thought to be mediated through redox-cycling of fiber-bound and bioavailable iron (Fe). We hypothesized that Libby amphibole (LA)-induced cute lung injury will be exacerbated in rat models of cardiovascular disease (CVD)-associated Fe-overload and oxidative stress. Healthy male Wistar Kyoto (WKY), spontaneously hypertensive (SH) and SH heart failure (SHHF) rats were intratracheally instilled with 0.0, 0.25 or 1.0  mg/rat LA and examined at 1 day, 1 week or 1 month. Although histologically it was not possible to distinguish severity differences between strains in LA-induced initial inflammation and later fibrosis, quantitative assessment of biomarkers showed strain-related differences. LA-induced neutrophilic inflammation was reversible in WKY but persisted more in SH and SHHF. Lung MIP-2 mRNA increased only in WKY at 1 day in response to LA but not in SH and SHHF. Bronchoalveolar lavage fluid (BALF) protein increased in SH but not WKY at 1 week and 1 month, while γ-glutamyltransferase and N-acetyl-β-D-glucosaminidase activities increased in all strains (WKY>SH=SHHF). BALF ferritin levels were high at baseline and increased following LA exposure only in SH and SHHF. Ferritin heavy chain mRNA increased only in SHHF at 1 day. At 1 month ferritin light chain mRNA declined from already high baseline levels in SHHF but increased in WKY and SH suggesting its differential involvement in LA-induced injury in Fe-overload. Unlike WKY, both SHHF and SH failed to increase the lung lining antioxidant, ascorbate, in response to LA. We conclude that underlying CVD-associated Fe-overload is likely linked to persistent lung injury, inflammation and antioxidant decompensation following LA exposure in rats.

Lung carcinomas and pulmonary fibrosis (asbestosis) occur in asbestos workers. Understanding the pathogenesis of these diseases is complicated because of potential confounding factors, such as smoking, which is not a risk factor in mesothelioma. The modes of action (MOA) of various types of asbestos in the development of lung cancers, asbestosis, and mesotheliomas appear to be different. Moreover, asbestos fibers may act differentially at various stages of these diseases, and have different potencies as compared to other naturally occurring and synthetic fibers. This literature review describes patterns of deposition and retention of various types of asbestos and other fibers after inhalation, methods of translocation within the lung, and dissolution of various fiber types in lung compartments and cells in vitro. Comprehensive dose-response studies at fiber concentrations inhaled by humans as well as bivariate size distributions (lengths and widths), types, and sources of fibers are rarely defined in published studies and are needed. Species-specific responses may occur. Mechanistic studies have some of these limitations, but have suggested that changes in gene expression (either fiber-catalyzed directly or by cell elaboration of oxidants), epigenetic changes, and receptor-mediated or other intracellular signaling cascades may play roles in various stages of the development of lung cancers or asbestosis.

Increased incidences of asbestosis have been reported in workers from Libby, MT, associated with exposures to amphibole-contaminated vermiculite. In this study pulmonary and histopathological changes were investigated following Libby amphibole (LA) exposure in a rat model. Rat respirable fractions of LA and amosite (aerodynamic diameter <2.5 μm) were prepared by water elutriation. Male F344 rats were exposed to single doses of either saline (SAL), amosite (0.65 mg/rat), or LA (0.65 or 6.5 mg/rat) by intratracheal instillation. At times from 1 d to 3 mo after exposure, bronchoalveolar lavage (BAL) was performed and right and left lungs were removed for reverse-transcription polymerase chain reaction (RT-PCR) and histopathological analysis, respectively. Data indicated that 0.65 mg amosite resulted in a higher degree of pulmonary injury, inflammation, and fibrotic events than LA at the same mass dose. Exposure to either amosite or high dose LA resulted in higher levels of cellular permeability and injury, inflammatory enzymes, and iron binding proteins in both BAL fluid and lung tissue at most time points when compared to SAL controls. However, mRNA expression for some growth factors (e.g., platelet-derived growth factor [PDGF]-A and transforming growth factor [TGF]-1β), which contribute to fibrosis, were downregulated at several time points. Furthermore, histopathological examination showed notable thickening of interstitial areas surrounding the alveolar ducts and terminal bronchioles. On a mass dose basis, amosite produced a greater acute and persistent lung injury for at least 3 mo after exposure. However, further testing and analysis of LA are needed with regard to the dose metric to fully evaluate its potential fibrogenicity and carcinogenicity.

The cellular and molecular mechanisms of how asbestos fibers induce cancers and other diseases are not well understood. Both serpentine and amphibole asbestos fibers have been shown to induce oxidative stress, inflammatory responses, cellular toxicity and tissue injuries, genetic changes, and epigenetic alterations in target cells in vitro and tissues in vivo. Most of these mechanisms are believe to be shared by both fiber-induced cancers and noncancerous diseases. This article summarizes the findings from existing literature with a focus on genetic changes, specifically, mutagenicity of asbestos fibers. Thus far, experimental evidence suggesting the involvement of mutagenesis in asbestos carcinogenicity is more convincing than asbestos-induced fibrotic diseases. The potential contributions of mutagenicity to asbestos-induced diseases, with an emphasis on carcinogenicity, are reviewed from five aspects: (1) whether there is a mutagenic mode of action (MOA) in fiber-induced carcinogenesis; (2) mutagenicity/carcinogenicity at low dose; (3) biological activities that contribute to mutagenicity and impact of target tissue/cell type; (4) health endpoints with or without mutagenicity as a key event; and finally, (5) determinant factors of toxicity in mutagenicity. At the end of this review, a consensus statement of what is known, what is believed to be factual but requires confirmation, and existing data gaps, as well as future research needs and directions, is provided.

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumour associated with asbestos exposure that has only a limited response to conventional therapy; therefore, diagnosing MPM early is very important. We have previously reported that angiopoietin (Ang)-1 was correlated with bleomycin-induced pulmonary fibrosis. Here, we investigated the association of Ang-1 with the development of MPM cells, which originate from mesenchymal cells similar to lung fibroblasts, and demonstrated that Ang-1 stimulated the growth and migration of MPM cells in vitro. We also demonstrated that patients with MPM had significantly higher serum levels of Ang-1 in comparison to a population who had been exposed to asbestos but had not developed MPM. The patients with advanced-stage MPM showed higher levels of Ang-1 than the early-stage MPM patients and the Kaplan-Meier method revealed a significant correlation between serum Ang-1 levels and survival. We propose the possibility that Ang-1 plays an important role in MPM tumour growth and our data suggest that the serum concentration of Ang-1 could be useful as prognostic factor.

Many asbestos-exposed subjects have minor parenchymal changes on high resolution computed tomography (HRCT) that do not fulfil the diagnostic criteria for pulmonary fibrosis and asbestosis. We investigated if these borderline parenchymal changes in asbestos-exposed subjects are related to pulmonary inflammatory activity. Exhaled nitric oxide was measured, exhaled breath condensate collected and HRCT scanned in 104 subjects with moderate to high occupational asbestos exposure. Forty-one healthy unexposed subjects served as a comparison group. After excluding other pulmonary diseases, 35 asbestos-exposed subjects had normal parenchymal findings and 31 subjects had borderline parenchymal changes on HRCT. Lung function was poorer in the latter group, but there was no difference in the degree of asbestos exposure between these groups. As compared with the unexposed comparison group, asbestos-exposed subjects with borderline parenchymal changes had increased alveolar NO concentration (3.0 + or - 0.2 vs. 2.3 + or - 0.1 ppb, p = 0.008) and increased levels of leukotriene B(4) (12.2 + or - 1.1 vs. 3.3 + or - 0.8 pg/ml, p < 0.001) and 8-isoprostane (9.4 + or - 0.7 vs. 7.3 + or - 0.6 pg/ml, p = 0.021) in breath condensate. Asbestos-exposed subjects with normal parenchymal findings had only increased breath condensate levels of leukotriene B(4) (11.4 + or - 0.9, p < 0.001). Borderline parenchymal changes on HRCT in asbestos-exposed subjects are associated with increased markers of pulmonary inflammation. Such borderline parenchymal changes are likely a mild or early form of the same pathological process that leads to asbestosis., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The immunohistochemical expression of retinoblastoma protein (Rb) and its phosphorylated form (pRb) was investigated in an in vivo sheep model, i.e., lung tissue exposed to asbestos-like fibers. Rb is a phosphoprotein with a central role in controlling the cell cycle, cell growth regulation, differentiation, and apoptosis. pRb functions as a checkpoint in the G(1) phase, favoring cell growth, while the hypophosphorylated form (Rb) not only promotes cell-cycle arrest and a return to the G(1) phase, but also appears to be a key player in most instances of apoptosis. pRb overexpression was detected in alveolar epithelium and the interstitium, especially close to the fluoro-edenite fibers, whereas Rb immunostaining was faint or absent. The data suggest that the altered balance between pRb and Rb expression may be a programmed response to protect the organism against uncontrolled cell proliferation.

Sarcomatoid mesothelioma is the least common, but most aggressive of the three major histological types of mesotheliomas. This study comprises 326 cases of sarcomatoid mesotheliomas among 2000 consecutive malignant mesothelioma cases received in consultation (16%). Patients included 312 men (96%) and 14 women (4%), with a median age of 70 years (range 41-94 years). Most tumors were pleural (319; 98%), and 7 were peritoneal (2%). Some desmoplastic features were identified in 110 cases (34%), and 70 (21%) were classified as desmoplastic. Rare subtypes included two cases with a lymphohistiocytoid pattern (<1%) and eight heterologous mesotheliomas (2%). Labeling for cytokeratins (CKs) was observed in 261/280 cases (93%), and for calretinin and vimentin in 31 and 91%, respectively. Pleural plaques were present in 79% of cases for which information was available, and asbestosis was diagnosed in 34/127 cases (27%). Median survival was 3.5 months. Fiber analysis was performed in 61 cases. The median asbestos body count was 1640/g wet lung tissue (by light microscopy). Amosite fibers were the most commonly identified fibers using energy-dispersive X-ray analysis and were significantly higher in the sarcomatoid cases, as were uncoated fibers using scanning electron microscopy. This study represents the largest series of sarcomatoid and desmoplastic malignant mesotheliomas to date and confirms the diagnostic usefulness of CK immunohistochemistry. The relationship with asbestos exposure--particularly amosite--and an association with pleural plaques and less often asbestosis is confirmed.

The sensitive assay method was developed for a parallel, rapid and precise determination of the most prominent oxidative stress biomarkers: 8-iso-prostaglandin F(2α) a lipid oxidation biomarker, o-tyrosine an amino acid oxidation biomarker and 8-hydroxy-2'-deoxy-guanosine a nucleic acid oxidation biomarker. The method consisted of a pre-treatment part, freeze drying (lyophilization), serving the purpose of biomarkers concentration from the exhaled breath condensate and detection method LC-ESI-MS/MS, where the selected reaction-monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method is characterized by the following parameters: the precision was higher than 84.3% and the mean accuracy (relative error) was determined lower than 11.6%. The method was tested on samples obtained from patients diagnosed with asbestosis and silicosis, occupational diseases induced by oxidative stress, and then compared with samples from healthy subjects. The difference in biomarkers' concentration levels found between the two groups was statistically significant.

To prevent and control disease caused by exposure to various agents, it is necessary to determine the harmful level of intervention and to establish a method for measuring that level. In-air microparticle-induced X-ray emission (in-air micro-PIXE) analysis is based on irradiation of specimens with a proton ion microbeam, and has been modified for biological application. Two-dimensional analysis and quantitative analysis using the system confirmed that asbestos induced apoptosis by upregulating Fas expression and also revealed the accumulation of CD163-expressing macrophages in the lungs of patients with asbestosis. By quantitative comparison of the area of Fas or CD163 expression and the Fas- or CD163-negative area in asbestos lung tissue, the harmful levels which caused the expression of Fas or CD163 could be estimated on Silica, Ferrous iron, and Magnesium (the components of asbestos) deposition. These results indicate that the system could be useful for investigating the pathogenesis of inhaled particle-induced immune reactions and for determining harmful levels of exogenous agents.

Asbestosis has long been defined as a diffuse interstitial "fibrotic" process, in similarity to other chronic interstitial pulmonary diseases. To address the hypothesis (which was based on morphological nuances) that the interstitial connective tissue response in asbestosis may be fibroelastotic rather than fibrotic, a comparative characterization of the connective response in cases of asbestosis and other forms of interstitial lung disease was performed. Archival open lung biopsies or autopsy specimens of pulmonary diseases featuring interstitial connective tissue abnormalities (15 of asbestosis, 21 of organizing pneumonia, 15 usual interstitial pneumonitis/idiopathic pulmonary fibrosis [IPF], 9 organizing diffuse alveolar damage, 9 "nonspecific" interstitial pneumonitis, 4 sarcoidosis, 3 each of desquamative interstitial pneumonia and chronic amiodarone toxicity, 2 cryptogenic organizing pneumonias, and 1 each of chronic hypersensitivity pneumonitis and chronic eosinophilic pneumonitis [85 total]) were stained histochemically with hematoxylin and eosin, Perl's method, Gomori's trichrome procedure, and the Verhoeff-van Gieson technique. Representative subsets of the cases (n = 20) were also studied immunohistologically using an antibody to elastin. Fibroelastosis in each of the samples was assessed for the degree of response and its location using a 3-tiered scale. The degree of fibroelastosis in the 15 cases of asbestosis was variable, with the pattern being peribronchial and perivascular in all instances; at least 2 asbestos bodies were identified in fibroelastotic foci in each of the 15 cases as highlighted with Perl's stain. Forty-seven cases of nonasbestotic lung disease (71%) showed interstitial fibrosis with a variable (usually modest) amount of admixed elastic tissue; when present, elastic fibers were distributed in a diffuse interstitial pattern, with or without perivascular accentuation. All cases of IPF also showed areas of fibroelastosis, but those foci were confined to regions of overt "honeycomb" change. No asbestos bodies were seen in any disease except asbestosis, and a predominantly peribronchial pattern of fibroelastosis was not identified in any nonasbestotic interstitial lung disease in this study. The authors conclude that the types and patterns of pulmonary connective tissue response in interstitial lung diseases may provide additional diagnostic clues to the presence of asbestosis.

Objectives: Asbestos induces generation of reactive oxygen and nitrogen species in laboratory studies. Several such species can be measured non-invasively in humans in exhaled breath condensate (EBC) but few have been evaluated. This study aimed to assess oxidative stress and lung inflammation in vivo., Methods: Eighty six men were studied: sixty subjects with asbestos-related disorders (asbestosis: 18, diffuse pleural thickening (DPT): 16, pleural plaques (PPs): 26) and twenty six age- and gender-matched normal individuals., Results: Subjects with asbestosis had raised EBC markers of oxidative stress compared with normal controls [8-isoprostane (geometric mean (95% CI) 0.51 (0.17-1.51) vs 0.07 (0.04-0.13) ng/ml, p<0.01); hydrogen peroxide (13.68 (8.63-21.68) vs 5.89 (3.99-8.69) microM, p<0.05), as well as increased EBC total protein (17.27 (10.57-28.23) vs 7.62 (5.13-11.34) microg/ml, p<0.05), and fractional exhaled nitric oxide (mean+/-SD) (9.67+/-3.26 vs 7.57+/-1.89ppb; p<0.05). EBC pH was lower in subjects with asbestosis compared with subjects with DPT (7.26+/-0.31 vs 7.53+/-0.24; p<0.05). There were no significant differences in exhaled carbon monoxide, EBC total nitrogen oxides and 3-nitrotyrosine between any of the asbestos-related disorders, or between these and controls., Conclusion: In asbestos-related disorders, markers of inflammation and oxidative stress are significantly elevated in subjects with asbestosis compared with healthy individuals but not in pleural diseases.

Background: Asbestos fibers are known to accumulate in lung parenchyma and thoracic lymph nodes, but their presence and translocation into the extrapulmonary tissues need clarification. We assessed the presence of asbestos in the para-aortic (PA) and mesenteric (ME) lymph nodes., Methods: PA and ME lymph nodes and lung tissue from 17 persons who underwent medicolegal autopsy for suspicion of asbestos-related disease and from five controls were analyzed for asbestos fibers using transmission electron microscopy., Results: High concentrations of amphibole asbestos fibers were detected in several lung tissue samples and in the respective PA and ME lymph nodes. The mean concentration for the 10 persons with a lung asbestos content of >/=1 million fibers/g of dry tissue (f/g) was 0.85 (<0.05-4.36) million f/g in the PA lymph nodes and 0.55 (<0.02-2.86) million f/g in the ME lymph nodes. The respective mean values for the 12 persons with a lung asbestos concentration of <1 million f/g were 0.07 for the PA lymph nodes and 0.03 million f/g for the ME nodes. The lung asbestos burden that predicted the detection of asbestos in abdominal lymph nodes was 0.45 million f/g., Conclusions: In addition to their accumulation in lung tissue, asbestos fibers also collect in the retroperitoneal and the mesenteric lymph nodes. Even low-level occupational exposure results in the presence of crocidolite, amosite, anthophyllite, tremolite, or chrysotile in these abdominal lymph nodes. Our results support the hypothesis of lymph drainage as an important translocation mechanism for asbestos in the human body.

Respiratory exposure to asbestos fibers has been associated with diffuse malignant mesothelioma (DMM) in humans. Despite advancements in the molecular analyses of human DMM and the development of animal models, the carcinogenic mechanisms of the disease remain unclear. There are basically three hypotheses regarding the pathogenesis of asbestos-induced DMM, which may be summarized as follows: (1) the "oxidative stress theory" is based on the fact that phagocytic cells that engulf asbestos fibers produce large amounts of free radicals due to their inability to digest the fibers, and epidemiological studies indicating that iron-containing asbestos fibers appear more carcinogenic; (2) the "chromosome tangling theory" postulates that asbestos fibers damage chromosomes when cells divide; and (3) the "theory of adsorption of many specific proteins as well as carcinogenic molecules" states that asbestos fibers in vivo concentrate proteins or chemicals including the components of cigarette smoke. Elucidation of the major mechanisms underlying DMM would be helpful for the development of novel strategies to prevent DMM induction in people who have already been exposed to asbestos.

Background: In many previous studies, the asbestos fibres retained in the lung were regarded as a good index of cumulative occupational asbestos exposure. Twelve workers suffering from asbestos-related diseases and had been employed in an asbestos-cement factory operating from 1961 to 1994 underwent post mortem investigations in the course of a criminal law suit., Objectives: Samples of lung tissues were collected for electron microscopy analysis to measure the asbestos fibre burden of the lungs in workers with high exposure, and assess the possible correlation between asbestos fibre lung burden and the estimated levels of cumulative exposure., Methods: Samples of lung parenchyma obtained from a consecutive series of 12 post-mortem examinations that were performed between 1994 and 2007and included 5 cases of malignant pleural mesothelioma, 4 lung cancers, 1 case of asbestosis and2 ofpleuralplagues, were collected, stored and analysed by SEM electron microscopy, according to the methods suggested by the current scientific literature. For each worker, all males, a detailed occupational history was reconstructed by means ofpersonal interviews; both the measurements of airborne asbestos fibresperformed by the factory in the 1970's and the duration of each single job in the plant were taken into account to estimate an individual cumulative exposure index., Results: A wide variation of total asbestos fibre concentrations in the lung (1,320-118 million) was observed; in all 12 workers, the lung amphibole fibre burden exceeded 1,000,000 fibres per g/dry tissue, The highest values were detected in the mesothelioma cases, in which the mean fibre concentrations differed statistically (t=2.29, p=0.045) from the mean calculated for the other asbestos-related diseases; in 9 subjects only amphibole fibres were detected. There was a good correlation between total asbestos fibre concentration and cumulative exposure index (r=0.91, p<0.0001)., Conclusion: This study, which was numerically the biggest ever performed in Italy for this category of workers, confirms a wide range of total asbestos fibre burden in heavily occupationally exposed workers and showed that of the asbestos-related diseases, the highest lung concentrations of asbestos fibres were reached in cases of mesothelioma. It was also observed that almost the entire lung burden consists of only amphibole fibres, all exceeding 1 million per gramme of dry tissue. This study tested a synthetic cumulative occupational exposure index, which appears to be well correlated to the level of exposure established by biological analysis.

Asbestosis and pleural plaques exhibit unpredictable but progressive development, and there are no markers routinely available to measure their prognosis. Asbestos exposure induces the generation of reactive oxygen species, and 8-isoprostane is involved in experimental asbestos-related lung toxicity. This oxidative stress marker was measured in exhaled breath condensate (EBC) in 92 former asbestos workers with mean age 68.8+/-1.7 yr and mean duration of asbestos exposure 24.1+/-2.0 yr. The control group had 46 subjects with mean age 65.2+/-3.3 yr. The mean level of 8-isoprostane, analyzed by liquid chromatography-electrospray ionization-mass spectrometry, was higher in asbestos-exposed subjects (69.5+/-6.6 pg/ml, p=0.0001) compared with the control group, where the concentration was 47.0+/-7.8 pg/ml. The results presented support the hypothesis that oxidative stress due to asbestos is the main cause of increased 8-isoprostane in EBC. Measurement of 8-isoprostane in EBC is a promising non-invasive means for assessing the activity of asbestos-induced diseases.

More than 1 asbestos body (AB) per ml of bronchoalveolar lavage fluid (BALF) under light microscopy was defined as AB positive (ABP) and suggests an occupational asbestos exposure. We microscopically evaluated the AB number per one ml of BALF, which we defined as the AB concentration (ABC), using bronchoalveolar lavage (BAL) cytocentrifuge slides obtained from 35 patients having pulmonary nodular lesions (20 carcinoma and 15 nonneoplastic disease) and examined the correlation between ABC and clinicopathological data including findings on Helical computed tomography scan (HCTS) and occupational history of asbestos exposure (OHAE). BAL was performed by the standard technique without removing mucous with a gauze filter. AB was microscopically defined as a structure consisting of a core of transparent asbestos surrounded by an iron-protein coat. Twenty of 35 patients were ABP (ABP rate; 57%) and ABC ranged from 0 to 207.98/ml (mean ABC; 11.33/ml). Mean ABC was significantly higher in patients with OHAE (15.04/ml) compared to that in patients without OHAE (3.23/ml). Twenty-two of 35 patients (63%) lacked abnormality on HCTS and among these, 12 patients (55%) were ABP. In 20 pulmonary carcinoma patients, the ABP rate was 85% and ABC ranged from 0 to 31.1/ml (Mean ABC; 2.99/ml). The ABP rate of pulmonary carcinoma patients was 40% (8 patients) and among these, 5 patients (63%) did not show any abnormality on HCTS. In conclusion, our method was simple and useful and should be applied to patients with pulmonary nodular lesions and OHAE, even if there are no abnormalities on HCTS.

Atelectatic induration (AI) and honeycombing (HNCB) are two distinct macroscopic features of asbestosis. The current study was undertaken to clarify whether or not there might be a relation between the macroscopic type of asbestosis and burden of asbestos fibers in the lung. The material (N = 63) derived from 58 autopsies and 5 surgically removed lungs with lung cancers, which comprised 22 cases of AI (mean age: 67 years: 20 males and 2 females) and 41 cases of HNCB (mean age: 66 years: 38 men and 3 women) types of asbestosis, respectively. The quantification of asbestos bodies (AB) and fibers (AF) was carried out using Kohyama's method. The AI type showed 260,000 +/- 460,000 AB per 1g of wet lung and 920,000 +/- 1,360,000 AF per 1g of dry lung, whereas HNCB showed 44,000 +/- 93,000 AB and 200,000 +/- 490,000 AF, respectively. These differences were statistically significant between the types of asbestosis (p < 0.01). Diffuse pleural thickening and upper lobe involvement were more advanced in the AI type and the differences were statistically significant between subtypes (p < 0.05). In conclusion, asbestos load is considered to be a factor related to the macroscopic subtype of asbestosis.

The effect of lung irradiation on subsequent inflammatory or fibrotic lung injuries remains poorly understood. We postulated that irradiation and bone marrow transplantation might impact the development and progression of lung remodeling resulting from asbestos inhalation. Our objective was to determine whether irradiation and bone marrow transplantation affected inflammation and fibrosis associated with inhaled asbestos exposure. Inflammation, cytokine production, and fibrosis were assessed in lungs of naïve and sex-mismatched chimeric mice exposed to asbestos for 3, 9, or 40 days. Potential engraftment of donor-derived cells in recipient lungs was examined by fluorescence in situ hybridization and immunohistochemistry. Compared with asbestos-exposed naïve (nonchimeric) mice, chimeric mice exposed to asbestos for 3, 9, or 40 days demonstrated significant abrogation of acute increases in asbestos-associated inflammatory mediators and fibrosis. Donor-derived cells trafficked to lung but did not significantly engraft as phenotypic lung cells. Irradiation and bone marrow transplantation alters inflammatory and fibrotic responses to asbestos, likely through modulation of soluble inflammatory mediators.

Experiments on white outbred rats helped to establish placental transmission of chrysotile asbestos fibers from mother to fetus with reliable increased incidence of neoplasma in first generation, mainly of respiratory malignancies. Chronic exposure to chrysotile asbestos damaged peripheral lymphocytes and did not affect red bone marrow. Extrapolating the experimental data on placental transmission of chrysotile asbestos to children requires epidemiologic research.

The deceased was a 75-year-old male, found dead in his home. He had a history of occupational asbestos exposure for 13 years. At autopsy, there was diffuse fibrosis of the lung, with diffuse pleural thickening. Large amounts of asbestos bodies were detected in the lung tissue. The findings of transmission electron microscopy with energy-dispersive X-ray microanalysis (TEM-EDX) also showed asbestos fibers deposited in the lung tissue. From the macroscopic and histological findings of the lung, the number of asbestos bodies in the lung tissue and the TEM-EDX findings, we concluded that the cause of his death was chronic respiratory failure due to asbestosis. We recognize the importance of a detailed occupational history, which provides useful information for determining the cause of death.

Background: Inhaled asbestos fibres can cause inflammation and fibrosis in the lungs called asbestosis. However, there are no non-invasive means to assess and follow the severity of the inflammation. Exhaled nitric oxide (NO) measured at multiple exhalation flow rates can be used to assess the alveolar NO concentration and bronchial NO flux, which reflect inflammation in the lung parenchyma and airways, respectively. The aim of the present study was to investigate whether exhaled NO or markers in exhaled breath condensate could be used to assess inflammation in asbestosis., Methods: Exhaled NO and inflammatory markers (leukotriene B(4) and 8-isoprostane) in exhaled breath condensate were measured in 15 non-smoking patients with asbestosis and in 15 healthy controls. Exhaled NO concentrations were measured at four constant exhalation flow rates (50, 100, 200 and 300 ml/s) and alveolar NO concentration and bronchial NO flux were calculated according to the linear model of pulmonary NO dynamics., Results: The mean (SE) alveolar NO concentration was significantly higher in patients with asbestosis than in controls (3.2 (0.4) vs 2.0 (0.2) ppb, p = 0.008). There was no difference in bronchial NO flux (0.9 (0.1) vs 0.9 (0.1) nl/s, p = 0.93) or NO concentration measured at ATS standard flow rate of 50 ml/s (20.0 (2.0) vs 19.7 (1.8) ppb, p = 0.89). Patients with asbestosis had increased levels of leukotriene B4 (39.5 (6.0) vs 15.4 (2.9) pg/ml, p = 0.002) and 8-isoprostane (33.5 (9.6) vs 11.9 (2.8) pg/ml, p = 0.048) in exhaled breath condensate and raised serum levels of C-reactive protein (2.3 (0.3) vs 1.1 (0.2) mug/ml, p = 0.003), interleukin-6 (3.5 (0.5) vs 1.7 (0.4) pg/ml, p = 0.007) and myeloperoxidase (356 (48) vs 240 (20) ng/ml, p = 0.034) compared with healthy controls., Conclusions: Patients with asbestosis have an increased alveolar NO concentration and high levels of leukotriene B4 and 8-isoprostane in exhaled breath. Measurement of exhaled NO at multiple exhalation flow rates and analysis of inflammatory markers in exhaled breath condensate are promising non-invasive means for assessing inflammation in patients with asbestosis.

Inhaled dust particulates are able to relocate to the extrapulmonary compartments, particularly the lymph nodes that drain the lung. There is little information about the concentration and type of asbestos in the lymphatics and lymph nodes. Quantitative analysis of asbestos lymph node burden conducted by light and analytical transmission electron microscopy has shown ferruginous bodies in lymph nodes that drain the lung and appreciable numbers of short asbestos fibers accumulate in lymph nodes in occupationally exposed individuals. The location of lymph nodes in the thoracic cavity was categorized according to the Naruke anatomical map. Tissue from eleven individuals with a history of asbestos exposure were selected for a comparative study of the asbestos content of lung with that found in the thoracic lymph nodes. The study used a digestion technique for tissue preparation and evaluated ferruginous body burden and concentration of asbestos fibers (> 0.5 microm in length). Comparison was made between sites and analysis was made as to the population of fibers detectable by light microscopy and defined as "Stanton fibers." The findings indicated the vast majority of all asbestos fiber types in all sites were shorter than 5 microm and would not have been counted in a light microscopy count scheme that included only those fibers > 5 microm. There were reproducible patterns of asbestos types of found in various lymph nodes, although there were variations in the amount of asbestos found in the sites sampled. In summary, asbestos fibers found in thoracic lymph nodes have predominately short fibers and, in this study group, consisted of a mixture of commercial and noncommercial amphiboles. When a long/thin fiber was found in the lung or lymph tissue, its detection required the use of analytical transmission electron microscopy for identification.

Background: Diagnosis of malignant pleural mesothelioma is a challenging issue. Potential markers in mesothelioma diagnosis include soluble mesothelin-related peptides (SMRPs) and osteopontin, but no subsequent validation has been published yet., Methods: We prospectively evaluated SMRPs in serum and pleural effusion from patients with mesothelioma (n = 74), pleural metastasis of carcinomas (n = 35), or benign pleural lesions associated with asbestos exposure (n = 28), recruited when first suspected for mesothelioma., Findings: Mean serum SMRP level was higher in patients with mesothelioma (2.05 +/- 2.57 nM/L [median +/- interquartile range]) than in patients with metastasis (1.02 +/- 1.79 nM/L) or benign lesions (0.55 +/- 0.59 nM/L). The area under the receiver operating characteristic curve (AUC) for serum SMRP was 0.872 for differentiating mesothelioma and benign lesions, cut-off = 0.93 nM/L (sensitivity = 80%, specificity = 82.6%). The AUC for serum SMRP differentiating metastasis and mesothelioma was 0.693, cut-off = 1.85 nM/L (sensitivity = 58.3%, specificity = 73.3%). SMRP values in pleural fluid were higher than in serum in all groups (mesothelioma: 46.1 +/- 83.2 nM/L; benign lesions: 6.4 +/- 11.1 nM/L; metastasis: 6.36 +/- 21.73 nM/L). The AUC for pleural SMRP-differentiating benign lesions and mesothelioma was 0.831, cut-off = 10.4 nM/L (sensitivity = 76.7%, specificity = 76.2%). The AUC for pleural SMRP-differentiating metastasis and mesothelioma was 0.793., Interpretation: We show that SMRPs may be a promising marker for mesothelioma diagnosis when measured either in serum or pleural fluid. The diagnostic value of SMRPs was similar in both types of samples, but pleural fluid SMRPs may better discriminate mesothelioma from pleural metastasis.

Objective and Background: Asbestos exposure induces generation of reactive oxygen and nitrogen species. Nitric oxide is involved in asbestos-related lung toxicity in vitro and can be measured non-invasively in humans in exhaled breath. The authors hypothesized that fractional exhaled nitric oxide concentration (FENO) would be increased in subjects with asbestos-related lung disorders., Methods: FENO was measured in 56 subjects with asbestos-related disorders (asbestosis: 12; pleural plaques: 32; asbestos-related diffuse pleural thickening: 12) and in 35 normal subjects. The authors also measured exhaled carbon monoxide, another marker of lung inflammation., Results: Median (25-75 percentile) FENO was increased in subjects with asbestosis (7.9 (6.6-15.7) p.p.b.; P=0.001) and pleural plaques (6.3 (5.3-9) p.p.b.; P=0.03) compared with normal controls (4.6 (3.5-6) p.p.b.). Subjects with DPT had a median FENO of 5.6 p.p.b., similar to controls. No significant differences in exhaled carbon monoxide were observed between controls (1.0+/-0.3 p.p.m.) and subjects with asbestosis (1.3+/-0.3 p.p.m.), pleural plaques (1.2+/-0.3 p.p.m.) or diffuse pleural thickening (1.1+/-0.3 p.p.m.)., Conclusions: FENO is raised in asbestosis consistent with lung inflammation, and also in pleural plaques.

Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA damage. We previously showed that the mitochondria-regulated (intrinsic) death pathway mediates alveolar epithelial cell (AEC) DNA damage and apoptosis. Because p53 regulates the DNA damage response in part by inducing intrinsic cell death, we determined whether p53-dependent transcriptional activity mediates asbestos-induced AEC mitochondrial dysfunction and apoptosis. We show that inhibitors of p53-dependent transcriptional activation (pifithrin and type 16-E6 protein) block asbestos-induced AEC mitochondrial membrane potential change (DeltaPsim), caspase 9 activation, and apoptosis. We demonstrate that asbestos activates p53 promoter activity, mRNA levels, protein expression, and Bax and p53 mitochondrial translocation. Further, pifithrin, E6, phytic acid, or rho(0)-A549 cells (cells incapable of mitochondrial reactive oxygen species production) block asbestos-induced p53 activation. Finally, we show that asbestos augments p53 expression in cells at the bronchoalveolar duct junctions of rat lungs and that phytic acid prevents this. These data suggest that p53-dependent transcription pathways mediate asbestos-induced AEC mitochondria-regulated apoptosis. This suggests an important interactive effect between p53 and the mitochondria in the pathogenesis of asbestos-induced pulmonary toxicity that may have broader implications for our understanding of pulmonary fibrosis and lung cancer.

To elucidate genes important in development or repair of asbestos-induced lung diseases, gene expression was examined in mice after inhalation of chrysotile asbestos for 3, 9, and 40 days. We identified changes in the expression of genes linked to proliferation (cyclin B2, CDC20, and CDC28 protein kinase regulatory subunit 2), inflammation (CCL9, CCL6, complement component 1, chitinase3-like 3, TNF superfamily member 10, and IL-1B), and matrix remodeling (MMP12, MMP3, integrin alphaX, and cathepsins K, Z, B, and S). The most highly induced gene at all time points was mclca3 (gob5), a putative calcium-activated chloride channel involved in the regulation of mucus production and/or secretion. Using histochemistry, we demonstrated accumulation of mucus and increased mClca3 protein in the bronchiolar epithelium of asbestos-exposed mice at all time points but peaking at 9 days. Cytokine levels (interleukin-1beta, interleukin-4, interleukin-6) in bronchoalveolar lavage fluid also increased at 9 days, suggesting Th2-mediated immunity may play a role in asbestos-induced mucus production. In contrast, levels of cathepsin K, a potent elastase, increased between 3 and 40 days at both the mRNA and protein levels, localizing primarily in CD45-positive leukocytes and interstitial cells. Identification of genes involved in lung injury and remodeling after asbestos exposure could aid in defining mechanisms of airborne particulate-induced disease and in developing therapeutic strategies.

Unlabelled: Antisense techniques that inhibit intracellular expression of insulin-like growth factor-I (IGF-I) were efficient in gene therapy of some tumor diseases. IGF-I in the airways is considered to induce lung fibrosis in interstitial lung diseases, inhibit apoptosis of epithelial cells and participate in local carcinogenesis., Aim of the Study: The aim of the study was--by examining the IGF-I expression in the lower airways--to evaluate preliminary the efficacy of anti-IGF-I antisense technicques in the treatment of airways diseases., Material and Methods: The IGF-I expression was examined in the reverse transcriptase--polimerase chain reaction (RT-PCR) applied to A549 human cell line, that is representative for lower airway epithelium and exemplifies the model of bronchioloalveolar adenocarcinoma. IGF-I expression in non-neoplastic lower airways cells was assessed by immunocytochemical staining (anti-IGF-I) in cytological materials originating from bronchoalveolar lavage (BAL) of sarcoidosis and asbestosis patients and in individuals free of lung pathology., Results: The IGF-I expression was detected in A549 cells with use of RT-PCR method (3 independent probes). In BAL cytological specimens the appearance of IGF-I was found, mainly in alveolar macrophages (62 +/- 6, 5 in sarcoidosis vs. 36 +/- 6 in controls, p=0,09), as well as in BAL lymphocytes., Conclusions: Summing up, the antisense technicques, blocking the intracellular IGF-I expression may be potentially useful in treatment of selected lower airways, both tumor (non-small cell lung carcinoma) and non-tumor (ILD complicated with lung fibrosis) conditions.

In a rural community in South Africa historically exposed to asbestos environmentally and occupationally, 200 women who had worked with asbestos and applied for medical examination to determine compensable asbestos disease were evaluated. Clinical and radiologic evaluation, sputum collection, and microscopic analysis were done. A questionnaire elicited type of exposure, duration, decade of first work exposure, and environmental exposure. Crackles were present in the lungs of 166 women and asbestos fibers and ferruginous bodies were present in 122. Asbestosis was identified in 26 and plural plaques in 62. Auscultation for crackles (rales) is useful in the initial examination of former asbestos workers in rural communities of developing countries.

Platelet-derived growth factor (PDGF) isoforms and PDGF receptor-alpha are upregulated in fibroproliferative lesions in response to asbestos exposure. To examine the functional role of PDGF in asbestos-induced lung disease, we have evaluated the impact of PDGF-B overexpression in the lung on the development of pulmonary fibrosis induced by asbestos inhalation. Transgenic mice expressing PDGF-B from the surfactant protein C promoter and wild-type C57BL/6 mice were exposed to aerosolized chrysotile asbestos fibers via three different exposure regimens: 3 consecutive days to 9 mg/m(3), once a week for 5 weeks to 12 mg/m(3), or once a week for 8 weeks to 11 mg/m(3). The 3-day exposure did not produce fibroproliferative lesions in SPC-PDGFB or wild-type mice, indicating that PDGF expression did not increase susceptibility to a subthreshold dose of asbestos. Transgenic and wild-type mice subjected to the 5-week exposure protocol exhibited similar fibrogenic lesions histologically 48 hours and 8 weeks postexposure, but lungs from transgenic mice had elevated lung hydroxyproline content 8 weeks postexposure relative to wild-type mice. In addition, SPC-PDGFB transgenic mice developed pronounced thickening of arterioles following the 5-week exposure regimen. Mice exposed to asbestos for 8 weeks and examined 10 months later showed pronounced, diffuse fibrotic lesions of terminal bronchioles and alveolar ducts, but no histological differences between transgenic and nontransgenic mice were observed. These results indicated that PDGF-B overexpression can stimulate increased collagen deposition and vascular smooth muscle hyperplasia following asbestos inhalation and that a limited exposure (8 times) to chrysotile aerosol can produce long-lasting fibrotic lesions. The 8-week exposure regimen provides an animal model that encompasses an important aspect of human asbestosis-i.e., persistence of fibrosis for long periods after cessation of asbestos exposure.