The influence of gene-gene and gene-environment interactions on the risk of asbestosis.

This study investigated the influence of gene-gene and gene-environment interactions on the risk of developing asbestosis. The study comprised 262 cases with asbestosis and 265 controls with no asbestos-related disease previously studied for MnSOD, ECSOD, CAT, GSTT1, GSTM1, GSTP1, and iNOS polymorphisms. Data on cumulative asbestos and smoking were available for all subjects. To assess gene-gene and gene-environmental interactions, logistic regression was used. The associations between MnSOD Ala -9Val polymorphism and the risk of asbestosis and between iNOS genotypes and asbestosis were modified by CAT -262 C > T polymorphism (P = 0.038; P = 0.031). A strong interaction was found between GSTM1-null polymorphism and smoking (P = 0.007), iNOS (CCTTT) n polymorphism and smoking (P = 0.054), and between iNOS (CCTTT) n polymorphism and cumulative asbestos exposure (P = 0.037). The findings of this study suggest that the interactions between different genotypes, genotypes and smoking, and between genotypes and asbestos exposure have an important influence on the development of asbestosis and should be seriously considered in future research on occupational/environmental asbestos-related diseases.

Inducible nitric oxide synthase genetic polymorphism and risk of asbestosis.

Asbestos, a known occupational pollutant, may upregulate the activity of inducible nitric oxide synthase (iNOS) and thus the production of nitric oxide (NO). This study investigated whether iNOS (CCTTT)(n) polymorphism is associated with an increased asbestosis risk in exposed workers. The study cohort consisted of 262 cases with asbestosis and 265 controls with no asbestos-related disease. For each subject the cumulative asbestos exposure data were available. The number of CCTTT repeats was determined following PCR amplification of the iNOS promoter region. Logistic regression was performed to estimate asbestosis risk. The OR of asbestosis was 1.20 (95% CI = 0.85-1.69) for the LL genotype compared to the combined SL and SS genotypes and 1.26 (95% CI = 0.86-1.85) for the LL genotype compared to the SL genotype. The results of this study are borderline significant and suggest a possible role of iNOS (CCTTT)(n) polymorphism in the risk of asbestosis; however, further studies are needed.

Manganese and extracellular superoxide dismutase polymorphisms and risk for asbestosis.

Manganese and extracellular superoxide dismutases (SOD2 and SOD3) are part of the enzymatic defence against reactive oxygen species, which are involved in the pathogenesis of asbestosis. This study investigates whether SOD2Ala - 9Val and SOD3 Arg213Gly genetic polymorphisms represent risk factors for asbestosis in workers exposed to asbestos. The study included 262 cases with asbestosis and 265 controls with no asbestos-related disease. Cumulative asbestos exposure was calculated for each subject. A real-time PCR assay was introduced for genotyping. Logistic regression analysis was used to assess asbestosis risk. Asbestosis was associated with the homozygous SOD2 - 9Ala/Ala genotype (OR = 1.50, 95% CI 1.01-2.24), whereas the association for the SOD3 Arg/Gly genotype was not significant (OR = 1.63, 95% CI 0.62-4.27). The finding that the SOD2 - 9Ala/Ala genotype increases the risk for asbestosis indicates that, in addition to asbestos exposure, genetic factors may also have a significant influence on the development of asbestosis.

Asbestos-induced MKP-3 expression augments TNF-alpha gene expression in human monocytes.

TNF-alpha is associated with the development of interstitial fibrosis. We have demonstrated that the p38 mitogen-activated protein (MAP) kinase regulates TNF-alpha expression in monocytes exposed to asbestos. In this report, we asked if extracellular signal-regulated kinase (ERK) was also involved in TNF-alpha expression in monocytes exposed to asbestos. We found that p38 and ERK were differentially activated in alveolar macrophages obtained from patients with asbestosis compared with normal subjects. More specifically, p38 was constitutively active and ERK activation was suppressed. Since the upstream pathway leading to ERK was intact, we hypothesized that an ERK-specific phosphatase was, in part, responsible for the decreased ERK activity. We evaluated whether the dual specificity phosphatase MAP kinase phosphatase (MKP)-3, which is highly expressed in the lung and specifically dephosphorylates ERK, was increased after exposure to asbestos. We found that MKP-3 increased after exposure to asbestos, and its expression was regulated by p38. We found that p38 and ERK negatively regulated one another, and MKP-3 had a role in this differential activation. We also found that p38 was a positive regulator and ERK was a negative regulator of TNF-alpha gene expression. Cells overexpressing MKP-3 had a significant increase in TNF-alpha gene expression, suggesting than an environment favoring p38 MAP kinase activation is necessary for TNF-alpha production in monocytes exposed to asbestos. Taken together, these data demonstrate that the p38 MAP kinase down-regulates ERK via activation of MKP-3 in human monocytes exposed to asbestos to enhance TNF-alpha gene expression.

Targeting the MEK1 cascade in lung epithelium inhibits proliferation and fibrogenesis by asbestos.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated after inhalation of asbestos. The effect of blocking this signaling pathway in lung epithelium is unclear. Asbestos-exposed transgenic mice expressing a dominant-negative mitogen-activated protein kinase kinase-1 (dnMEK1) (i.e., the upstream kinase necessary for phosphorylation of ERK1/2) targeted to lung epithelium exhibited morphologic and molecular changes in lung. Transgene-positive (Tg+) (i.e., dnMEK1) and transgene-negative (Tg-) littermates were exposed to crocidolite asbestos for 2, 4, 9, and 32 days or maintained in clean air (sham controls). Distal bronchiolar epithelium was isolated using laser capture microdissection and mRNA analyzed for molecular markers of proliferation and Clara cell secretory protein (CCSP). Lungs and bronchoalveolar lavage fluids were analyzed for inflammatory and proliferative changes and molecular markers of fibrogenesis. Distal bronchiolar epithelium of asbestos-exposed wild-type mice showed increased expression of c-fos at 2 days. Elevated mRNA levels of histone H3 and numbers of Ki-67-labeled proliferating bronchiolar epithelial cells were decreased at 4 days in asbestos-exposed Tg+ mice. At 32 days, distal bronchioles normally composed of Clara cells in asbestos-exposed Tg+ mouse lungs exhibited nonreplicating ciliated and mucin-secreting cells as well as decreased mRNA levels of CCSP. Gene expression (procollagen 3-a-1, procollagen 1-a-1, and IL-6) linked to fibrogenesis was also increased in lung homogenates of asbestos-exposed Tg- mice, but reduced in asbestos-exposed Tg+ mice. These results suggest a critical role of MEK1 signaling in epithelial cell proliferation and lung remodeling after toxic injury.

COX-2 is widely expressed in metaplastic epithelium in pulmonary fibrous disorders.

Idiopathic usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF) and asbestosis represent progressive and often fatal pulmonary fibrous disorders, whereas cryptogenic organizing pneumonia (COP), desquamative interstitial pneumonia (DIP), and respiratory bronchiolitis-interstitial lung disease (RB-ILD) usually are reversible or nonprogressive conditions. Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and myofibroblast transition, its production depending on cyclooxygenase-2 (COX-2). In patients with UIP/IPF, levels of PGE2 and COX-2 are reduced in fibroblasts, and levels of PGE2 in bronchioalveolar lavage fluid may be lowered. We analyzed the immunohistochemical expression of COX-2 in UIP/IPF, asbestosis, COP, DIP, and RB-ILD. Our results show that the metaplastic epithelium in UIP/IPF, asbestosis, and COP is widely COX-2+, whereas COX-2 positivity is scant in DIP and RB-ILD. The mesenchymal cells remained negative. Our results suggest that irrespective of the underlying disease, lung injury that causes extensive fibrosis induces wide expression of COX-2 in the regenerating metaplastic epithelium.

Persistent localization of activated extracellular signal-regulated kinases (ERK1/2) is epithelial cell-specific in an inhalation model of asbestosis.

Asbestos fibers up-regulate the extracellular signal-regulated kinase (ERK1/2) pathway in mesothelial and pulmonary epithelial cells in vitro, but the cell-type expression patterns and intracellular localization of activated, ie, phosphorylated, ERK in the lung after inhalation of asbestos are unclear. C57/BL6 mice were exposed to 7-mg/m(3) air of crocidolite asbestos for 5 and 30 days, the times required for the development of epithelial cell hyperplasia and fibrotic lesions, respectively. Exposure to asbestos caused striking increases in both unphosphorylated and phosphorylated ERK (p-ERK), which were most marked at 30 days and co-localized in bronchiolar and alveolar epithelial cells using an antibody to cytokeratin. Alveolar macrophages, detected with an anti-macrophage antibody, did not express p-ERK. p-ERK was localized at the apical cell surface of bronchiolar and alveolar type II epithelial cells exposed to asbestos fibers, and was most marked in areas of epithelial hyperplasia in association with fibrotic lesions. Because translocation of p-ERK to the nucleus is associated with activation of early response genes and transcription factors, laser scanning cytometry was used to determine the kinetics of activation and nuclear translocation of p-ERK in an alveolar type II epithelial cell line in vitro after exposure to asbestos or the ERK stimuli, epidermal growth factor, or H(2)O(2). Results showed that cytoplasmic to nuclear translocation of p-ERK occurred in a protracted manner in cells exposed to asbestos. The immunolocalization of p-ERK at the membrane surface, a site of initial exposure to asbestos fibers, and the chronic activation of p-ERK in epithelial cells at sites of fibrogenesis are consistent with the concept that epithelial cell signaling through the ERK pathway contributes to remodeling of the lung during the development of pulmonary fibrosis.

In vivo bioassays of acute asbestosis and its correlation with ESR spectroscopy and imaging in redox status.

In vivo electron spin resonance (ESR) spectroscopy and whole body imaging were used to investigate the toxicity of biological reactions and organ specific oxidative changes associated with the development of acute asbestosis. Pathogen-free mice were exposed to 100 microg of crocidolite asbestos suspended in 50 microL of a 0.9% NaCl solution by aspiration. The bio-assay group had broncho-alveolar lavage (BAL) and serum draws performed on control and treated mice at 1, 3, and 7 days post-instillation. The ESR spectroscopic measurements and whole body imaging were performed with a separate group of mice at the same time points. Bio-assays included measurements of albumin, lactate dehydrogenase (LDH), N-acetyl-beta-D-glucoaminidase (NAG), and catalase in acellular lavage fluids, and total antioxidants status in blood serum. ESR spectroscopic and imaging measurements were performed after intraperitoneal injection of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-15N-1-oxyl (TEMPOL) or 3-carbamoylproxyl (3-CP) nitroxides at a final concentration of 344 mg/kg body weight. Albumin showed a significant increase in BAL fluid at the 3 day exposure time point. The presence of this protein in lavage fluid indicates that the gas/blood barrier has been damaged in the lung. LDH in BAL fluid also exhibited a significant increase at 3 days post-exposure, an indication of enhanced cell membrane damage in the lung. Similar results were observed for NAG, a lysosomal enzyme, implying activation of phagocytic cells. Contemporaneously with the development of acute asbestosis at day 3 post-exposure, there were significant increases in the levels of total antioxidants in the serum and catalase in the BAL fluid. Significant impairment in the ability of asbestos exposed animals to clear TEMPOL radical during acute disease progression was evident at days 1 and 3 post exposure. ESR image measurements provided information on the location and distribution of the 3-CP label within the lungs and heart of the mouse and its clearance over time. Bioassays in concert with ESR spectroscopy and imaging presented in this study provide congruent data on the early acute phase of pulmonary injury and oxidant generation in response to asbestos exposure and their decline after 7 days. The increased levels of total antioxidants in the serum and catalase in BAL fluid correlated with the reduction in the clearance rate for TEMPOL, suggesting that a change in the redox status of the lung is associated with lung injury induced by asbestos.

Dual role of inducible nitric oxide synthase in acute asbestos-induced lung injury.

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of asbestos fibers-associated pulmonary diseases. By comparing the responses of inducible nitric oxide synthase (iNOS) knockout and wild-type mice we investigated the consequences of iNOS expression for the development of the inflammatory response and tissue injury upon intratracheal instillation of asbestos fibers. Exposure to asbestos fibers resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. Moreover, iNOS knockout mice exhibited an exceeded pulmonary expression and production of TNF-alpha as well as a higher influx of neutrophils into the alveolar space than wild-type mice. In contrast, iNOS knockout animals displayed an attenuated oxidant-related tissue injury reflected in a decrease in protein leakage and LDH release into the alveolar space as well as weaker nitrotyrosine staining of lung tissue compared to wild-type mice. Data presented here indicate that iNOS-derived NO exerts a dichotomous role in acute asbestos-induced lung injury in that iNOS deficiency resulted in an exacerbated inflammatory response but improved oxidant-promoted lung tissue damage.

Early inflammatory response to asbestos exposure in rat and hamster lungs: role of inducible nitric oxide synthase.

Recent studies have suggested that inducible nitric oxide synthase (iNOS) plays a role in the development of asbestos-related pulmonary disorders. The pulmonary reactions of rats and hamsters upon exposure to asbestos fibers are well known to be disparate. In addition, in vitro experiments have indicated that mononuclear phagocytes from hamsters, in contrast to those from rats, lack the iNOS pathway. Therefore, the purpose of this study was to investigate whether rats and hamsters differ in lung iNOS expression in vivo upon exposure to asbestos fibers and whether differences in iNOS induction are associated with differences in the acute pulmonary inflammatory reaction. Body weight, alveolar-arterial oxygen difference, differential cell count in bronchoalveolar lavage fluid, total protein leakage, lung myeloperoxidase activity and lipidperoxidation, wet/dry ratio, iNOS mRNA and protein expression, and nitrotyrosine staining of lung tissue were determined 1 and 7 days after intratracheal instillation of asbestos fibers in CD rats and Syrian golden hamsters. Exposure of rats to asbestos fibers resulted in enhanced pulmonary iNOS expression and nitrotyrosine staining together with an acute inflammation that was characterized by an influx of neutrophils, enhanced myeloperoxidase activity and lipid peroxidation, damage of the alveolar-capillary membrane, edema formation, and impairment of gas exchange. In comparison, instillation of asbestos fibers in hamsters resulted in a significantly milder inflammatory reaction of the lung with no induction of iNOS in pulmonary cells. The data obtained provide important information to understand the underlying mechanisms of species differences in the pulmonary response upon exposure to asbestos fibers.

Manganese superoxide dismutase genotypes and asbestos-associated pulmonary disorders.

Manganese superoxide dismutase (MnSOD) activity is highly elevated in the biopsies of human asbestos-associated malignant mesothelioma. We therefore examined if polymorphism in the mitochondrial targeting sequence of the MnSOD gene modified individual susceptibility to this malignancy or related asbestos-associated pulmonary disorders. The study population consisted of 124 male Finnish asbestos insulators who were all classified as having been exposed to high levels of asbestos; 63 of the workers had no pulmonary disorders and 61 either had malignant mesothelioma or the non-malignant pulmonary disorders asbestosis and/or pleural plaques. No significant associations were found between the MnSOD genotypes and these ill-health. This study therefore suggest no major modifying role for the MnSOD polymorphism in development of asbestos-associated pulmonary disorders.

Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.

Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers.

Pulmonary expression of glutathione S-transferase M3 in lung cancer patients: association with GSTM1 polymorphism, smoking, and asbestos exposure.

To characterize the relative roles of glutathione S-transferases (GST) M1 and M3 in the susceptibility to lung cancer, the pulmonary expression of GSTM3 was quantified immunochemically and related to the GSTM1 genotype in 100 lung cancer patients. Among active smokers and recent ex-smokers (for 6 years or less), parenchymal GSTM3 expression was lower in patients with a homozygous GSTM1 null genotype than in those who were GSTM1 positive and had similar smoking habits (P < 0.001 and P = 0.004, respectively). However, in long-term ex-smokers (for 15 years or longer) GSTM3 was not affected by the GSTM1 genotype. Among active smokers and recent ex-smokers who were homozygous GSTM1 null, those with a definite or probable exposure to asbestos expressed GSTM3 at significantly higher levels than those for whom it was unlikely (P = 0.04). A similar effect of the homozygous GSTM1 null genotype on GSTM3 expression was not detected in the bronchial epithelium when GSTM3 was visualized immunohistochemically. Different mechanisms may result in an increased risk of either squamous cell or adenocarcinomas in patients with the homozygous GSTM1 null genotype. Low expression of GSTM3 due to smoking in the parenchymal lung of GSTM1 null individuals can theoretically favor the development of adenocarcinoma. Our data indicated a predominance of this tumor type in patients with low expression of GSTM3.

Radiographic changes and lung function in relation to activity of the glutathione transferases theta and mu among asbestos cement workers.

Experimental data indicate that active oxygen species may be casually involved in the development of asbestos-related disease. Thus, it was hypothesized that individual differences in glutathione transferase activity, which may affect the ability to inactivate molecules formed in relation to oxidative stress, could influence the biological response to asbestos exposure. We could, however, not demonstrate an increased risk for radiographic changes or reduced lung function among asbestos cement workers deficient for glutathione transferase theta (GSTT1), glutathione transferase mu (GSTM1), or having a combined deficiency of enzyme activity.

Asbestos-induced injury to cultured human pulmonary epithelial-like cells: role of neutrophil elastase.

The mechanisms responsible for asbestos-induced pulmonary epithelial cell cytotoxicity, especially oxidant-independent mechanisms, are not established. We determined whether human polymorphonuclear leukocyte (PMN) proteases contribute to asbestos-induced damage to human pulmonary epithelial-like cells (PECs) assessed using an in vitro chromium-51 release assay. Serine antiproteases, phenylmethylsulfonyl fluoride and alpha 1-antitrypsin, each ameliorated PEC injury induced by amosite asbestos and PMNs. A role for a specific proteinase, human neutrophil elastase (HNE), is supported by the facts that (1) asbestos increased HNE release assessed by an enzyme-linked immunosorbent assay technique (1.7 +/- 0.5 vs. 2.8 +/- 0.5 micrograms/ml; P < .025), (2) purified HNE or porcine pancreatic elastase (PPE) each alone caused PEC detachment, (3) asbestos plus either HNE or PPE caused PEC lysis similar to that mediated by asbestos and PMNs, and (4) cationic agents released from PMNs were unlikely to be involved because polyanions did not ameliorate injury resulting from asbestos and PMNs. Compared to elastase, cathepsin G caused less PEC detachment and negligible augmentation in asbestos-induced PEC lysis. Asbestos increased the association of 125I-labeled elastase with PECs nearly 50-fold compared with PPE alone (14.4% vs. 0.3%, respectively; P < .01) and nearly 10-fold compared with another particle, opsonized zymosan. We conclude that PMN-derived proteases, especially elastase, may contribute to asbestos-induced lung damage by augmenting pulmonary epithelial cell injury.

Development and characterization of a rapid-onset rodent inhalation model of asbestosis for disease prevention.

A short-term inhalation model of asbestosis was developed in rodents to examine possible preventive approaches to lung disease. Fischer 344 (F344) rats were exposed for 10 and 20 days to National Institute of Environmental Health Sciences (NIEHS) crocidolite asbestos while sham controls were exposed to air only. To determine quantitative biochemical indicators of asbestos-induced lung disease, bronchoalveolar lavage (BAL) fluids were analyzed for lactic dehydrogenase (LDH), alkaline phosphatase, angiotensin-converting enzyme (ACE), and protein. Total and differential cell counts were performed on cell pellets from BAL. Lungs from additional rats were processed for histopathology, measurement of hydroxyproline, and autoradiography after injection of rats with 3H-thymidine. Exposure to asbestos for 10 and 20 days caused increases in LDH, alkaline phosphatase, and protein in BAL. In contrast, ACE was undetectable in BAL fluids from sham or asbestos-exposed rats. At both time periods, the percentages of polymorphonuclear leukocytes (PMNs) and lymphocytes in BAL were increased in asbestos-exposed rats. Total cell numbers in BAL were increased significantly at 20 days in animals inhaling asbestos. Exposure to asbestos for 10 and 20 days caused elevated amounts of hydroxyproline in lung and the development of fibrotic lesions. Asbestos-exposed rats exhibited increased numbers of interstitial cells and airspace epithelial cells incorporating 3H-thymidine, whereas labeled bronchiolar epithelial cells were not elevated significantly. The quantitative changes in asbestos-associated enzyme levels, cell types and protein in BAL, as well as increases in hydroxyproline and morphologic evidence of fibrosis, are useful indices of asbestos-related lung injury which enable preventive and therapeutic approaches to disease.

Serum peptide III-procollagen and ACE in asbestos workers.

Type-III-procollagen peptide (P-III-P) concentration and angiotensin-converting-enzyme (ACE) activity were measured in the serum of asbestos workers. The subjects were classified into four groups (A, B, C, D) according to the duration of exposure (in years). A gradual increase of ACE activity in group B and P-III-P concentration in group C was found. The increase in ACE activity can be regarded as the response to the progressing inflammatory state, and the increase in P-III-P concentration may be related to the fibrosis of the lung tissue.

Increases in endogenous antioxidant enzymes during asbestos inhalation in rats.

Although the pathogenesis of asbestos-induced pulmonary damage is still not completely understood, an important role has been attributed to active oxygen species. In the present paper we present results of a study investigating the effect of crocidolite asbestos inhalation on different lung antioxidant enzymes in rats. During the development of pulmonary fibrosis induced by crocidolite asbestos, lung superoxide dismutase, catalase and selenium-dependent glutathione peroxidase activities increased, indicating an adaptive response to increased pulmonary oxidant stress. However, this adaptive response obviously is not sufficient to protect the lung from asbestos-induced pulmonary damage. Considering the role of active oxygen species in both the fibrotic process and tumor promotion, it is hypothesized that antioxidants may also protect the lung from chronic asbestos-induced pulmonary damage such as bronchogenic carcinoma.