Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

Fibroblast growth factor-10 upregulates Na,K-ATPase via the MAPK pathway.

We studied the effects of fibroblast growth factor (FGF-10) on alveolar epithelial cell (AEC) Na,K-ATPase regulation. Within 30 min FGF-10 increased Na,K-ATPase activity and alpha1 protein abundance by 2.5-fold at the AEC plasma membrane. Pretreatment of AEC with the mitogen-activated protein kinase (MAPK) inhibitor U0126, a Grb2-SOS inhibitor (SH3-b-p peptide), or a Ras inhibitor (farnesyl transferase inhibitor (FTI 277)), as well as N17-AEC that express a Ras dominant negative protein each prevented FGF-10-mediated Na,K-ATPase recruitment to the AEC plasma membrane. Accordingly, we provide first evidence that FGF-10 upregulates (short-term) the Na,K-ATPase activity in AEC via the Grb2-SOS/Ras/MAPK pathway.

Asbestos causes apoptosis in alveolar epithelial cells: role of iron-induced free radicals.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-induced reactive oxygen species (ROS) is one important mechanism. To determine whether asbestos causes apoptosis in AECs, we exposed WI-26 (human type I-like cells), A549 (human type II-like cells), and rat alveolar type II cells to amosite asbestos and assessed apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate-biotin nick end labeling (TUNEL) staining, nuclear morphology, annexin V staining, DNA nucleosome formation, and caspase 3 activation. In contrast to control medium and TiO2, amosite asbestos and H2O2 each caused AEC apoptosis. A role for iron-catalyzed ROS was suggested by the finding that asbestos-induced AEC apoptosis and caspase 3 activation were each attenuated by either an iron chelator (phytic acid and deferoxamine) or a.OH scavenger (dimethyl-thiourea, salicylate, and sodium benzoate) but not by iron-loaded phytic acid. To determine whether asbestos causes apoptosis in vivo, rats received a single intratracheal instillation of amosite (5 mg) or normal saline solution, and apoptosis in epithelial cells in the bronchoalveolar duct regions was assessed by TUNEL staining. One week after exposure, amosite asbestos caused a 3-fold increase in the percentage of apoptotic cells in the bronchoalveolar duct regions as compared with control (control, 2.1% +/- 0.35%; asbestos, 7.61% +/- 0.15%; n = 3). However, by 4 weeks the number of apoptotic cells was similar to control. We conclude that asbestos-induced pulmonary toxicity may partly be caused by apoptosis in the lung epithelium that is mediated by iron-catalyzed ROS and caspase 3 activation.

Increased prevalence of gastroesophageal reflux symptoms in patients with COPD.

STUDY OBJECTIVES: To determine the prevalence of gastroesophageal reflux (GER) symptoms in patients with COPD and the association of GER symptoms with the severity of airways obstruction as assessed by pulmonary function tests (PFTs). DESIGN: Prospective questionnaire-based, cross-sectional analytic survey. SETTING: Outpatient pulmonary and general medicine clinics at a Veterans Administration hospital. PATIENTS: Patients with mild-to-severe COPD (n = 100) were defined based on American Thoracic Society criteria. The control group (n = 51) consisted of patients in the general medicine clinic without respiratory complaints or prior diagnosis of asthma or COPD. INTERVENTION: Both groups completed a modified version of the Mayo Clinic GER questionnaire. RESULTS: Compared to control subjects, a greater proportion of COPD patients had significant GER symptoms defined as heartburn and/or regurgitation once or more per week (19% vs 0%, respectively; p < 0.001), chronic cough (32% vs 16%; p = 0.03), and dysphagia (17% vs 4%; p = 0.02). Among patients with COPD and significant GER symptoms, 26% reported respiratory symptoms associated with reflux events, whereas control subjects denied an association. Significant GER symptoms were more prevalent in COPD patients with FEV(1) < or %, as compared with patients with FEV(1) > 50% of predicted (23% vs 9%, respectively; p = 0.08). In contrast, PFT results were similar among COPD patients with and without GER symptoms. An increased number of patients with COPD utilized antireflux medications, compared to control subjects (50% vs 27%, respectively; p = 0.008). CONCLUSIONS: The questionnaire demonstrated a higher prevalence of weekly GER symptoms in patients with COPD, as compared to control subjects. There was a trend toward higher prevalence of GER symptoms in patients with severe COPD; however, this difference did not reach statistical significance. We speculate that although GER may not worsen pulmonary function, greater expiratory airflow limitation may worsen GER symptoms in patients with COPD.

Cigarette smoke and asbestos activate poly-ADP-ribose polymerase in alveolar epithelial cells.

BACKGROUND: Cigarette smoke augments asbestos-induced bronchogenic carcinoma in a synergistic manner by mechanisms that are not established. One important mechanism may involve alveolar epithelial cell (AEC) injury resulting from oxidant-induced DNA damage that subsequently activates poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair that can deplete cellular energy stores. We previously showed that whole aqueous cigarette smoke extracts (CSE) augment amosite asbestos-induced DNA damage and cytotoxicity to cultured AEC in part by generating iron-induced free radicals. We hypothesized that CSE increase asbestos-induced AEC injury by triggering PARP activation resulting from DNA damage caused by iron-induced free radicals. METHODS: Aqueous CSE were prepared fresh on the day of each experiment. PARP activity in WI-26 (a type I-like cell line) and A549 (a type II-like cell line) cells was assessed by the uptake of labeled NAD over 4 hours and confirmed on the basis of the reduction of PARP levels in the presence of a PARP inhibitor, 3-aminobenzamide (3-ABA). Cell survival was assessed by trypan blue dye exclusion. RESULTS: Hydrogen peroxide (H2O2; 1-250 microM), CSE (0.4-10 vol%), and amosite asbestos (5-250 micrograms/cm2) each caused PARP activation in WI-26 and A549 cells. The combination of asbestos (5 micrograms/cm2) and CSE (0.04-10%) induced WI-26 and A549 cell PARP activation without evidence of synergism. 3-ABA significantly attenuated WI-26 and A549 cell PARP activity and cell death after exposure to H2O2, CSE, and asbestos. Phytic acid, an iron chelator, catalase, and superoxide dismutase each decreased WI-26 cell PARP activation caused by asbestos and CSE. CONCLUSIONS: CSE and asbestos induced PARP activation in cultured AEC in a nonsynergistic manner. These data provide further support that asbestos and cigarette smoke are genotoxic to relevant lung target cells and that iron-induced free radicals in part cause these effects.

Glutathione redox system in oxidative lung injury.

Glutathione (GSH) is a ubiquitous intracellular thiol present in all tissues, including lung. Besides maintaining cellular integrity by creating a reduced environment, GSH has multiple functions, including detoxification of xenobiotics, synthesis of proteins, nucleic acids, and leukotrienes. Present in high concentrations in bronchoalveolar lavage fluid (BALF), GSH provides protection to the lung from oxidative injury induced by different endogenous or exogenous pulmonary toxicants. Its depletion in the lung has been associated with the increased risk of lung damage and disease. The redox system of GSH consists of primary and secondary antioxidants, including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PD). Alterations in the activities of these enzymes may reflect reduced cellular defense and may serve as surrogate markers of many lung diseases. As GSH is also involved in the regulation of expression of protooncogenes and apoptosis (programmed cell death), the development of diseases such as cancer and human immune deficiency may be affected by depleting or elevating cellular GSH levels. Exogenous delivery of GSH or its precursor N-acetyl cysteine (NAC) is being used as chemotherapeutic approach.

Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: role of free radicals.

Cigarette smoke augments asbestos-induced bronchogenic carcinoma by mechanisms that are not established. Alveolar epithelial cell (AEC) injury due to oxidant-induced DNA damage and depletion of glutathione (GSH) and adenosine triphosphate (ATP) may be one important mechanism. We previously showed that amosite asbestos-induces hydroxyl radical production and DNA damage to cultured AEC and that phytic acid, an iron chelator, is protective. We hypothesized that whole cigarette smoke extracts (CSE) augment amosite asbestos-induced AEC injury by generating iron-induced free radicals that damage DNA and reduce cellular GSH and ATP levels. Asbestos or CSE each caused dose-dependent toxicity to AEC (WI-26 and rat alveolar type I-like cells) as assessed by 51chromium release. The combination of asbestos (5 microg/cm2) and CSE (0.O1-0.1%) caused synergistic injury whereas higher doses of each agent primarily had an additive toxic effect. Asbestos (5 microg/cm2) augmented CSE-induced (0.01-1.0%) AEC DNA damage over a 4 h exposure period as assessed by an alkaline unwinding, ethidium bromide fluorometric technique. These effects were synergistic in A549 cells and additive in WI-26 cells. Asbestos (5 microg/cm2) and CSE (0.5-1.0%) reduced A549 and WI-26 cell GSH levels as assessed spectrophotometrically and ATP levels as assessed by luciferin/luciferase chemiluminescence but a synergistic interaction was not detected. Phytic acid (500 microM) and catalase (100 microg/ml) each attenuated A549 cell DNA damage and depletion of ATP caused by asbestos and CSE. However, neither agent attenuated WI-26 cell DNA damage nor the reductions in GSH levels in WI-26 and A549 cells exposed to asbestos and CSE. We conclude that CSE enhance asbestos-induced DNA damage in cultured alveolar epithelial cells. These data provide additional support that asbestos and cigarette smoke are genotoxic to relevant target cells in the lung and that iron-induced free radicals may in part cause these effects.

Keratinocyte growth factor promotes alveolar epithelial cell DNA repair after H2O2 exposure.

Alveolar epithelial cell (AEC) injury and repair are important in the pathogenesis of oxidant-induced lung damage. Keratinocyte growth factor (KGF) prevents lung damage and mortality in animals exposed to various forms of oxidant stress, but the protective mechanisms are not yet established. Because DNA strand break (DNA-SB) formation is one of the earliest cellular changes that occurs after cells are exposed to an oxidant stress, we determined whether KGF reduces H2O2-induced pulmonary toxicity by attenuating AEC DNA damage. KGF (10-100 ng/ml) decreased H2O2 (0.05-0.5 mM)-induced DNA-SB formation in cultured A549 and rat alveolar type II cells measured by an alkaline unwinding, ethidium bromide fluorometric technique. The protective effects of KGF were independent of alterations in catalase, glutathione (GSH), or the expression of bcl-2 and bax, two protooncogenes known to regulate oxidant-induced apoptosis. Actinomycin D and cycloheximide abrogated protective effects of KGF. Furthermore, protection by KGF was completely blocked by 1) genistein, a tyrosine kinase inhibitor; 2) staurosporine and calphostin C, protein kinase C (PKC) inhibitors; and 3) aphidicolin, butylphenyl dGTP, and 2',3'-dideoxythymidine 5'-triphosphate, inhibitors of DNA polymerase. We conclude that KGF attenuates H2O2-induced DNA-SB formation in cultured AECs by mechanisms that involve tyrosine kinase, PKC, and DNA polymerases. These data suggest that the ability of KGF to protect against oxidant-induced lung injury is partly due to enhanced AEC DNA repair.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells.

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

Asbestosis: clinical spectrum and pathogenic mechanisms.

Asbestosis is a diffuse pulmonary fibrotic process caused by the inhalation of asbestos fibers. Despite extensive investigations, the precise mechanisms regulating asbestos-induced lung damage are not fully understood. This review summarizes the important clinical manifestations and pathogenic mechanisms of asbestosis. We focus on the relatively new information that has emerged over the last several years. The diagnosis of asbestosis is often easily established by well-characterized criteria. Pulmonary physiologic testing and high-resolution computed tomography can detect clinically occult disease. The finding of asbestos bodies in the bronchoalveolar lavage fluid confirms that an individual has been exposed to asbestos but is of unclear significance in diagnosing asbestosis. Evidence reviewed herein suggests that asbestos pulmonary toxicity is due in part to the physical properties of the fibers, iron-catalyzed reactive oxygen species (ROS), and macrophage-derived cytokines and growth factors. Special emphasis is given to the hypothesis that iron-catalyzed hydroxyl radicals (HO.-) have a pivotal role in causing asbestosis. Definitive proof of this hypothesis is difficult to obtain since HO.- are highly reactive and their deleterious effects to cells may have occurred years prior to disease presentation. Despite these limitations, considerable data firmly support the notion that ROS have an important role in causing asbestos toxicity. Further, the iron content of asbestos or the redoxactive iron associated with or mobilized from the surface of the fibers is important in generating HO.- as well as in activating inflammatory cells. There also appears to be a close association between asbestos-induced ROS production and cellular toxicity and DNA damage. The full expression of asbestos-induced diseases likely involves the contribution of cytokines, growth factors, proteases, and other inflammatory cell products. Many of the mechanisms by which asbestos- and inflammation-induced ROS activate specific genes in pulmonary cells remain to be elucidated.

Mechanisms of carcinogenesis and clinical features of asbestos-associated cancers.

Exposure to asbestos, particularly members of the amphibole subgroup (crocidolite, amosite), is associated with the development of malignant mesothelioma and lung cancer. Although management of asbestos in buildings and increased regulation of asbestos in workplace settings are viable approaches to the prevention of disease, the prognosis of asbestos-associated tumors is generally dismal. Moreover, although a vast amount of information is available on the responses of cells and tissues to fibers, understanding the pathogenesis of asbestos-associated malignancies is hampered by the complexity of and differences between various fiber types. Multiple interactions between components of cigarette smoke and asbestos may be important in the development of lung cancer. In this article, the general properties of asbestos fibers will be discussed with an emphasis on chemical and physical features implicated in tumorigenesis. We will then provide a brief overview of the clinical features and treatment of cancers associated with exposure to asbestos. Finally, we will review recent experimental data providing some insight into the cellular and molecular mechanisms of carcinogenesis by asbestos.

Phytic acid, an iron chelator, attenuates pulmonary inflammation and fibrosis in rats after intratracheal instillation of asbestos.

Reactive oxygen species, especially iron-catalyzed hydroxyl radicals (.OH) are implicated in the pathogenesis of asbestos-induced pulmonary toxicity. We previously demonstrated that phytic acid, an iron chelator, reduces amosite asbestos-induced .OH generation, DNA strand break formation, and injury to cultured pulmonary epithelial cells (268[1995, Am. J. Physiol.(Lung Cell. Mol. Physiol.) 12:L471-480]). To determine whether phytic acid diminishes pulmonary inflammation and fibrosis in rats after a single intratracheal (it) instillation of amosite asbestos, Sprague-Dawley rats were given either saline (1 ml), amosite asbestos (5 mg; 1 ml saline), or amosite treated with phytic acid (500 microM) for 24 hr and then instilled. At various times after asbestos exposure, the rats were euthanized and the lungs were lavaged and examined histologically. A fibrosis score was determined from trichrome-stained specimens. As compared to controls, asbestos elicited a significant pulmonary inflammatory response, as evidence by an increase (approximately 2-fold) in bronchoalveolar lavage (BAL) cell counts at 1 wk and the percentage of BAL neutrophils (PMNs) and giant cells at 2 wk (0.1 vs 6.5% and 1.3 vs 6.1%, respectively; p < 0.05). Asbestos significantly increased the fibrosis score at 2 wk (0 +/- 0 vs 5 +/- 1; p < 0.05). The inflammatory and fibrotic changes were, as expected, observed in the respiratory bronchioles and terminal alveolar duct bifurcations. The increased percentage of BAl PMNs and giant cells persisted at 4 wk, as did the fibrotic changes. Compared to asbestos alone, phytic acid-treated asbestos elicited significantly less BAL PMNs (6.5 vs 1.0%; p < 0.05) and giant cells (6.1 vs 0.2%; p < 0.05) and caused significantly less fibrosis (5 vs 0.8; p < 0.05) 2 wk after exposure. We conclude that asbestos causes pulmonary inflammation and fibrosis in rats after it instillation and that phytic acid reduces these effects. These data support the role of iron-catalyzed free radicals in causing pulmonary toxicity from asbestos in vivo.

Infectivity of Legionella pneumophila mip mutant for alveolar epithelial cells.

Legionella pneumophila can invade and grow within explanted alveolar epithelial cells. Given its potential clinical significance, an examination of the molecular basis of epithelial cell infection was initiated. The mip gene encodes a 24-kilodalton surface protein that promotes macrophage infection and virulence. To determine whether this gene is required for pneumocyte infection, we tested a strain bearing a mip null mutation for its ability to infect both explanted type II cells and type I-like cell lines. For infection of type II cells, the infective dose 50% for the Mip-strain was 25-fold higher than an isogenic Mip+ strain. Type I cell monolayers infected with the mutant for 3 days yielded approximately 50-fold fewer bacteria than did monolayers infected with the parental strain. These data indicate that Mip enhances infection of pneumocytes and that L. pneumophila employs some of the same genes (mechanisms) to infect epithelial cells and macrophages.

Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals.

Asbestos causes pulmonary fibrosis and various malignancies by mechanisms that remain uncertain. Reactive oxygen species in part cause asbestos toxicity. However, it is not known whether asbestos-induced free radical production causes alveolar epithelial cell (AEC) cytotoxicity by inducing DNA strand breaks (DNA-SB). We tested the hypothesis that asbestos-induced AEC injury in vitro is due to iron-catalyzed free radical generation, which in turn causes DNA-SB. We found that amosite asbestos damages cultured human pulmonary epithelial-like cells (WI-26 cells) as assessed by 51Cr release and that an iron chelator, phytic acid (500 microM), attenuates these effects. A role for iron causing these effects was supported by the observation that ferric chloride-treated phytic acid did not diminish WI-26 cell injury. Production of hydroxyl radical-like species (.OH) was assessed based upon the .OH-dependent formation of formaldehyde (HCHO) in the presence of dimethyl sulfoxide. A variety of mineral dusts induced significant levels of .OH formation (nmol HCHO at 30 min: carbonyl iron, 85 +/- 21; amosite asbestos, 14 +/- 2; chrysotile asbestos, 7 +/- 1; titanium dioxide, 2.5 +/- 0.5). Phytic acid significantly diminished the asbestos-induced .OH production. DNA damage to AEC was assessed by the alkaline unwinding, ethidium bromide fluorometric technique. Hydrogen peroxide caused dose-dependent DNA-SB in WI-26 cells after a 30-min exposure period [50% effective dose (ED50): 5 microM] that was similar to other cell lines. Amosite asbestos induced dose-dependent DNA-SB in WI-26, A549, and primary isolated rat alveolar type II cells maintained in culture for 7-10 days (alveolar type I-like). Lower doses of amosite (0.5-5 micrograms/ml or 0.25-2.5 micrograms/cm2) caused significant WI-26 cell DNA-SB after prolonged exposure periods (> or = 2 days). Phytic acid ameliorated DNA damage in all three cultured AEC. There was a direct correlation between mineral dust-induced .OH production at 30 min and DNA-SB in WI-26 cells at 4 h (P < 0.0005). These data suggest that mineral dusts can be directly genotoxic to relevant target cells of asbestos, AEC. Furthermore, these results provide additional support for the premise that iron-catalyzed free radicals mediate asbestos-induced pulmonary toxicity.

Dibutyryl cAMP attenuates asbestos-induced pulmonary epithelial cell cytotoxicity and decline in ATP levels.

Adenosine 3',5'-cyclic monophosphate (cAMP) analogues prevent lung injury in various models by mechanisms that remain unknown. We speculated that cAMP attenuates asbestos-induced pulmonary epithelial cell injury by limiting the effects of an oxidant stress. Agents that increase intracellular cAMP [dibutyryl cAMP (DBcAMP), terbutaline, or aminophylline] but not guanosine 3',5'-cyclic monophosphate (cGMP) attenuated WI-26 cell-specific 51Cr release caused by asbestos. The protective effects of DBcAMP were associated with negligible alterations in asbestos-induced .OH formation or decline in WI-26 cell glutathione levels. Cycloheximide, an inhibitor of protein synthesis, failed to diminish the effects of DBcAMP. ATP levels were measured to determine whether the effects of DBcAMP are due to preservation of cellular ATP. Asbestos caused dose-dependent reductions in cellular ATP and DB-cAMP attenuated these effects. To determine whether the protective effects of DBcAMP related to alterations in WI-26 cell growth, we assessed the effects of DBcAMP on WI-26 cell number over time. DBcAMP diminished WI-26 cell replication and increased the doubling time. These results demonstrate that DBcAMP diminishes asbestos-induced cytotoxicity to cultured WI-26 cells in part by maintaining intracellular ATP levels and inhibiting cellular replication. The reduction in asbestos-induced WI-26 cell injury occurs despite a persistent oxidant stress. The data suggest a novel strategy to limit pulmonary toxicity from asbestos that warrants further investigation.

Contrasting effects of alveolar macrophages and neutrophils on asbestos-induced pulmonary epithelial cell injury.

Pulmonary toxicity from asbestos may be due in part to oxidant-mediated mechanisms. The purpose of this study was to determine whether alveolar macrophages (AM) contribute to asbestos-induced alveolar epithelial cell injury by oxidant-dependent mechanisms similar to that previously described for polymorphonuclear leukocytes (PMN). We assessed 51Cr release from cultured rat alveolar epithelial cells (RAEC) and transformed human pulmonary epithelial-like cell lines (rat L2 and human WI-26: HPEC). Amosite asbestos caused dose-dependent injury to both RAEC and L2 cells after an 18-h incubation period. Rat PMN increased asbestos-induced injury to RAEC (11 vs. 20% 51Cr release). In contrast, rat AM diminished asbestos-induced injury to RAEC and L2 cells by 60-80%. Human monocytes cultured for 72 h also attenuated asbestos-induced HPEC damage. Asbestos stimulated more H2O2 release from PMN than from AM isolated from the same rats (5.3 +/- 0.6 vs. 0.3 +/- 0.1 nmol x 10(6) cells-1 x 2h-1). The protective effect of rat AM, as opposed to PMN, was not due to differences in asbestos-induced toxicity to each cell type, since > 90% of AM and PMN were nonviable after 18 h. Transmission electron microscopy demonstrated comparable uptake of asbestos by AM and PMN after a 2-h incubation period. However, after an 18-h exposure period, the PMN were completely lysed, whereas over 90% of the AM contained fibers, despite morphologic evidence of cytotoxicity. These results demonstrate that AM, unlike PMN, can reduce alveolar epithelial cell injury in this model.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The role of free radicals in asbestos-induced diseases.

Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral fibers have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.