Actin polymerization plays a significant role in asbestos-induced inflammasome activation in mesothelial cells in vitro.

Asbestos exposure leads to malignant mesothelioma (MM), a deadly neoplasm of mesothelial cells of various locations. Although there is no doubt about the role of asbestos in MM tumorigenesis, mechanisms are still not well explored. Recently, our group demonstrated that asbestos causes inflammasome priming and activation in mesothelial cells, which in part is dependent on oxidative stress. Our current study sheds light on yet another mechanism of inflammasome activation by asbestos. Here we show the role of actin polymerization in asbestos-induced activation of the nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome. Using human mesothelial cells, we first demonstrate that asbestos and carbon nanotubes induced caspase-1 activation and high-mobility group box 1, interleukin 1 beta and interleukin 18 secretion was blocked by Cytochalasin D (Cyto D) an actin polymerization inhibitor. Next, to understand the mechanism, we assessed whether phagocytosis of fibers by mesothelial cells is affected by actin polymerization inhibition. Transmission electron microscopy showed the inhibition of fiber uptake by mesothelial cells in the presence of Cyto D. Furthermore, localization of components of the inflammasome, apoptotic speck-like protein containing a CARD domain (ASC) and NLRP3, to the perinuclear space in mitochondria or endoplasmic reticulum in response to fiber exposure was also interrupted in the presence of Cyto D. Taken together, our studies suggest that actin polymerization plays important roles in inflammasome activation by fibers via regulation of phagocytosis and/or spatial localization of inflammasome components.

Diallylsulfide attenuates asbestos-induced genotoxicity.

It is well known that asbestos fibers induced genotoxicity is mediated by reactive oxygen species (ROS) and insufficient endogenous antioxidant protection. Asbestos exposure can result in ROS generation in two different ways: (a) by catalyzation of Fe(2+), which is present in asbestos fibers, and (b) via oxidative bursts during phagocytosis of the fibers. On the other hand, it has been discussed that the physical presence of the fibres may mechanically influence the normal segregation of chromosomes during mitosis resulting in the induction of micronuclei in late ana/telophase, and aneuploidy. Glutathione and the glutathione redox system protect cells from the oxidative damage after exposure to xenobiotics. Glutathione depletion occurs during oxidative stress conditions and it is observed that supplementation of a precursor of glutathione and other sulphur containing compounds results in reduced toxicity. In the present study, diallyl sulfide (DAS), a lipid soluble allyl sulphur compound present in fresh extracts of garlic was evaluated for its protective effects. The micronucleus (MN) assay was performed in human mesothelial cells (HMC) exposed to 1 microg/cm(2) of crocidolite and chrysotile. Simultaneously, the cells were treated with 5 and 10 microM DAS, incubated for 48 and 66 h. Asbestos fibers induced significant genetic damage in HMC. The assay revealed a significant reduction in MN induction after treatment of cells with 5 microM but not with 10 microM DAS in mesothelial cells. The study revealed that at appropriate concentrations DAS protects mesothlelial cells against asbestos induced genotoxicity.

Asbestos-induced alveolar epithelial cell apoptosis: role of mitochondrial dysfunction caused by iron-derived free radicals.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-inducedAEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (delta(psi)m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces delta(psi)m after a 4 h exposure period. Further, the delta(psi)m after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell delta(psi)m and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell delta(psi)m after 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos.

Protective effect of natural flavonoids on rat peritoneal macrophages injury caused by asbestos fibers.

Exposure of macrophages to asbestos fibers resulted in enhancement of the production of oxygen radicals, determined by a lucigenin enhanced chemiluminescence (LEC) assay, a formation of thiobarbituric acid reactive substances (TBARS), a LDH release into the incubation mixture, and a rapid lysis of the cells. Rutin (Rut) and quercetin (Qr) were effective in inhibiting LEC, TBARS formation, and reducing peritoneal macrophages injury caused by asbestos. The concentrations pre-treatment of antioxidants that were required to prevent the injury of peritoneal macrophages caused by asbestos by 50% (IC50) were 90 microM and 290 microM for Qr and Rut, respectively. Both flavonoids were found to be oxidized during exposure of peritoneal macrophages to asbestos and the oxidation was SOD sensitive. The efficacy of flavonoids as antioxidant agents as well as superoxide ion scavengers was also evaluated using appropriate model systems, and both quercetin and rutin were found to be effective in scavenging O2.-. These findings indicate that flavonoids are able to prevent the respiratory burst in rat peritoneal macrophages exposed to asbestos at the stage of activated oxygen species generation, mainly as superoxide scavengers. On the basis of this study it was concluded that natural flavonoids quercetin and rutin would be promising drug candidates for a prophylactic asbestos-induced disease.

Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers.

The mutagenic effect of chrysotile asbestos fibers and zeolite and latex particles on human lymphocytes in whole blood has been studied. It was concluded that their mutagenic activities were mediated by oxygen radicals because they were inhibited by antioxidant enzymes (SOD and catalase) and oxygen radical scavengers (rutin, ascorbic acid, and bemitil). It was proposed that oxygen radicals were released by phagocytes activated upon exposure to mineral dusts and fibers. The study of lucigenin- and luminol-amplified chemiluminescence of peritoneal macrophages stimulated by chrysotile fibers and zeolite and latex particles has shown that their mutagenic action is probably mediated by different oxygen species, namely, by the iron-oxygen complexes (perferryl ions) plus hydrogen peroxide, hydrogen peroxide, and superoxide ion, respectively. From the oxygen radical scavengers studied, rutin was the most effective inhibitor of the mutagenic effect of mineral fibers and dusts.

Asbestos fibers induce release of collagenase by human polymorphonuclear leukocytes.

The asbestos fibers chrysotile and crocidolite cause a dose-dependent release of specific granule collagenase by human polymorphonuclear leukocytes (PMNL). Release of azurophil granule elastase was induced by the asbestos fibers at higher concentrations, suggesting that asbestos fibers primarily cause the release of specific granule contents of human PMNL. Wollastonite, a fibrous silicate mineral, causes a weaker collagenase release and no elastase release. The collagenase was released in inactive, latent form. Carboxymethyl cellulose (CMC), an agent known to blunt chrysotile-induced hemolysis and production of reactive oxygen metabolites by human PMNL, specifically inhibits chrysotile-induced release of collagenase. Chrysotile asbestos was found to bind the PMNL serine proteinase cathepsin G. A role of collagenase release, production of reactive oxygen metabolites and cathepsin G binding by chrysotile for the perpetuation of the asbestos-induced alveolitis is suggested.

Evidence supporting a role for active oxygen species in asbestos-induced toxicity and lung disease.

Asbestos is an important occupational and environmental toxicant that affects several cell types in the respiratory tract. In an effort to understand how asbestos causes cell injury and/or altered proliferation and differentiation of cells, this laboratory has focused on reactive oxygen species as mediators of asbestos-induced biological effects. A compendium of experimental results reported by this laboratory and others supports this hypothesis. For example, scavengers of reactive oxygen metabolites and iron chelators (i.e., desferroxamine) prevent cytotoxicity after addition of asbestos to a variety of cell lines and macrophages in vitro. DNA strand breakage associated with toxicity of crocidolite asbestos in C3H10T 1/2 cells also is ameliorated with use of desferroxamine. All types of asbestos cause lipid peroxidation in mammalian cells and artificial membranes, a phenomenon that can be prevented by removal of catalytic iron. Last, asbestos causes generation of active oxygen species after interaction with leukocytes or by reduction of oxygen on the surface of the fibers.

Prevention of asbestos-induced cell death in rat lung fibroblasts and alveolar macrophages by scavengers of active oxygen species.

Cell injury and inflammation caused by asbestos are critical to the pathogenesis of pulmonary fibrosis (asbestosis). Our goal in studies here was to investigate the possible modulation of asbestos-related cell death using antioxidants in both target and effector cells of asbestosis. After exposure to crocidolite asbestos at a range of concentrations (2.5-25 micrograms/cm2 dish), the viability of a normal rat lung fibroblast line (RL-82) and freshly isolated alveolar macrophages (AM) was determined by exclusion of trypan blue and nigrosin, respectively. In comparison to fibroblasts, AM were more resistant to the cytotoxic effects of asbestos. Cytotoxic concentrations of asbestos then were added to both cell types in combination with the antioxidants, superoxide dismutase (SOD), a scavenger of superoxide (O2-.), and catalase, an enzyme scavenging H2O2. Dimethylthiourea (DMTU), a scavenger of the hydroxyl radical (OH.) and deferoxamine, an iron chelator, also were evaluated in similar studies. Results showed significant dosage-dependent reduction (P less than 0.001) of asbestos-associated cell death with all agents. In contrast, asbestos-induced toxicity was not ameliorated after addition of chemically inactivated SOD and catalase or bovine serum albumin. Results above suggest asbestos-induced cell damage is mediated by active oxygen species. In this regard, the iron associated with the fiber and/or its interaction with cell membranes might be critical in driving a modified Haber-Weiss (Fenton-type) reaction resulting in production of OH(.).

Alteration of superoxide dismutase activity in tracheal epithelial cells by asbestos and inhibition of cytotoxicity by antioxidants.

We report here the inhibition of asbestos-induced cytotoxicity in a hamster tracheal epithelial cell line by superoxide dismutase, a scavenger of superoxide (O2-.), and by mannitol and dimethylthiourea, scavengers of the hydroxyl radical (OH.). By using these agents, cell damage was ameliorated in cultures exposed to long (greater than 10 microns in length) fibers of chrysotile and crocidolite asbestos. In contrast, injury to epithelial cells by short (less than or equal to 2 microns) chrysotile or glass fibers was not prevented by scavengers of O2-., OH., H2O2 or 1O2 (singlet oxygen). These results implicate active oxygen species as mediators of injury by long asbestos fibers to cells of the respiratory tract. By using immunocytochemical and biochemical techniques, we detected appreciable amounts of copper-zinc superoxide dismutase in hamster tracheobronchial epithelial cells and alveolar macrophages in vitro and in histologic sections of rat and human respiratory tract. Activity of total endogenous superoxide dismutase (copper-zinc and manganese forms) increased in tracheal epithelial cells exposed for several days in vitro to either crocidolite or chrysotile asbestos but was unchanged in untreated cells and those exposed to comparable amounts of glass fibers. After inhalation of asbestos by rats, or exposure of cells in culture to asbestos, long fibers were observed protruding from both epithelial cells and alveolar macrophages. The unsuccessful phagocytosis of long fibers of asbestos coupled with generation of oxygen free radicals might explain the increased pathogenic potential of long fibers in asbestos-associated diseases of the respiratory tract.

Asbestos-induced epithelial changes in organ cultures of hamster trachea: inhibition by retinyl methyl ether.

The epithelium of the hamster trachea in organ culture undergoes hyperplasia and squamous metaplasia after exposure to the amphibole types of asbestos, crocidolite and amosite. These changes are inhibited when the synthetic vitamin A analog, retinyl methyl ether, is incorporated into the culture medium. These findings suggest a possible use for retinoids in the prevention and treatment of respiratory tract disease associated with environmental exposure to asbestos.

Inhibition by phospholipids of haemolytic action of asbestos.

Haemolysis by asbestos fibres results from an increase in membrane permeability and not from rupture of red blood cells (RBC). The effect of chrysotile asbestos on RBC is at least partly, if not completely, attributable to lipid extraction and adsorption on to the fibres. This was suggested by the hyperbolic relationship between the haemolytic activity of chrysotile and the relative concentration of both chrysotile and RBC. Moreover, it was shown that pre-incubation of chrysotile with lipids, either as RBC membranes or with pure lipids in the form of liposomes, prevents haemolysis.

Macrophage plasminogen activator: induction by asbestos is blocked by anti-inflammatory steroids.

Intraperitoneal injection of asbestos fibres into mice induces the formation of exudates containing macrophages that produce plasminogen activator. Like-wise, in vitro addition of asbestos to macrophage cultures stimulates plasminogen activator secretion; the synthesis and secretion of lysozyme and lysosomal enzymes are not changed under these conditions. The enhanced secretion of plasminogen activator by macrophages exposed to asbestos is suppressed by low concentrations of anti-inflammatory steroids.

The biological effects of mineral fibres, especially asbestos, as seen from in vitro and in vivo studies.

Two in vitro models have been extensively used to compare the biological action of different types of asbestos fibres: the haemolytic effect and the cytotoxic one on macrophages grown in cell culture. The use of both techniques as led towards a better understanding of the chemical reactions which occur between fibres and the biological membranes of cells or intracellular organelles. Studies on the prevention of haemolysis and cytotoxicity have also been of use in explaining how asbestos acts a the cellular and molecular levels. Regarding in vivo studies, useful comparisons have been made of the fibrogenic and carcinogenic effects of different types of fibres in man and experimental animals. Both the in vitro and the in vivo aspects of the problem are discussed in some detail and an attemps is made to provide a reasonably unified concept for both.