The effect of fibrous dusts on lung cells. In vitro study.
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Abstract:
The mechanism of toxicity of selected asbestos substitute mineral fibres was examined and compared to that of asbestos. Alveolar macrophages and type II cells were isolated from Fischer 344 rats and after 20 h cultivation various concentration of fibres alone (amosite, wollastonite, rockwool or glass fibres) or in combination with cigarette smoke were added to cells and the cultivation continued for another 24 h. After finishing the exposure the number of alkaline phosphatase positive type II cells was counted, the comet assay was used to detect DNA damage (strand breaks) in both cell types and ultrastructural changes were evaluated by transmission electron microscopy. The decrease of the number of alkaline positive type II cells was dose dependent in all cases. The number of DNA strand breaks (SBs) in both cell types was enhanced after exposure to all types of fiber, the enhancement was dose dependent, the highest level of SBs was observed after amosite exposure. The combined exposure to mineral fibres and cigarette smoke showed synergic effect on the level of SBs. Transmission electron microscopy showed that already 1 microg x cm(-2) amosite caused destruction of AM while other fibres were phagocytized.Keywords:
Chrysotile
Comet Assay
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The alveolar epithelium contains tight junctions and provides a barrier to passage of potentially injurious substances into the pulmonary interstitium. Alveolar epithelial injury is hypothesized to he an important early event in the pathogenesis of asbestosis. Mechanisms that may contribute to alveolar epithelial cell injury following asbestos exposure include the physicochemical interactions between asbestos fibers and cells, and the generation of reactive oxygen species such as hydrogen peroxide (H2O2). The present study examined changes in transepithelial resistance (Rt) (a measure of barrier function) and permeability of alveolar epithelium after chrysotile asbestos and H2O2 exposure. Alveolar epithelial cell monolayers, obtained from isolation of rat alveolar type II cells and grown on porous supports, were exposed to chrysotile asbestos or polystyrene beads (control) at concentrations of 5, 10, and 25 μg/cm2 for 24 h. In separate experiments, monolayers were exposed to H2O2 at concentrations of 50, 75, and 100 μM for 1 h. Rt was measured using a voltohmmeter. Prior to treatment, monolayers had a high Rt (>20()0 ohms ± cm2). Permeability was assessed by measuring flux of [3H]sucrose from apical to basolateral compartments. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) and preincorporated [14C]adenine release. The morphological integrity of the monolayers was evaluated by scanning electron microscopy. Chrysotile asbestos and H2O2 exposure resulted in dose-dependent decreases in alveolar epithelial Rt and increases in permeability under conditions that did not result in overt cytotoxicity. These results demonstrate that both chrysotile asbestos and H2O2 have effects on alveolar epithelial Rt and permeability and suggest a potential role for the alveolar epithelium in mediation of asbestos-induced pulmonary interstitial disease.
Chrysotile
Alveolar Epithelium
Asbestosis
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AIM: To assess the role of surface free radicals and electromotive voltage of fibrous mineral dusts in rabbit pulmonary alveolar macrophage injuries induced by fibrous mineral dusts. METHODS: Changes in cell death ratio, malandialdehyde (MDA) and cellur electrophoresis ratio, lactate dehydrogenate (LDH)and superoxide dismitase(SOD) activities were determined, the technique of cell culture and Scanning electron Microscopy were used to examine the change of membranous permeability, charge and cellular shape. RESULTS: Fibrous wollastonite and tabulate clinoptilolite, which had no OH-, had no cytotoxicity, while fibrous sepiolite, fibrous palygorskite, fibrous brucite and chrysolite asbestos damaged pulmonary alveolar macrophages in various degrees because of the different OH- levels. All the six fibrous mineral dusts changed the cellular electrophoresis ratio. CONCLUSION: The surface electromotive voltage of fibrous mineral dusts is not an important factor, and the cytotoxicity of them may be related to OH- levels on the mineral dust surface.
Chrysotile
Alveolar macrophage
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Introduction. The exposure to dust, including chrysotile asbestos, is known to lead to the mobilization of alveolar macrophages, accompanied by the activation of free radical oxidation and the release of mediators stimulating fibroblast proliferation and collagen synthesis. Material and methods. Thirty outbred male rats were divided into two groups: 1 - control with a period of 4 months (n = 15), the 2-experienced group subjected to 4-month seed with chrysotile asbestos dust (n = 15). Under ether anesthesia, animals of the experimental group once were installed intratracheally in the respiratory tract using a syringe 1.0 ml of the sterile saline solution containing a suspension (50 mg) of chrysotile dust - asbestos. Then, the animals were killed, their bronchial washes, centrifuged, smears from the sediment, were subsequently visualized with a microscope. Fat metabolism was assessed by the content of phospholipids in the cell, according to G.A. Merkulov. Determination of hydroxyproline in the pulmonary homogenate. The statistical differences between the two groups were assessed with the Student’s t-test. Data were expressed as mean ± SE. Probability values of p <0.05 were considered significant. Results. The chronic exposure to chrysotile asbestos dust with a period of 4 months was found to causes a decrease in the activity of phagocytic cells and an increase in the destructive forms of alveolar macrophages in bronchoalveolar washes, excessive accumulation of phospholipids and an increase in oxyproline. Pneumofibrosis develops due to the cytotoxic and membrane-damaging effect of chrysotile asbestos dust. Conclusion. Thus, chrysotile asbestos dust from the Zhitikarinsky site, attributed to nanoparticles and multicomponent in chemical composition, has a cytotoxic effect, accompanied by activation of phagocytic pulmonary membrane and membrane-destructive changes in cells with accumulation of phospholipids.
Chrysotile
Alveolar macrophage
Respiratory tract
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Inhalation of asbestos fibres causes a progressive interstitial pulmonary fibrosis. To understand the basic cellular mechanisms which lead to this disease, we have studied the earliest proliferative events at the bronchiolar-alveolar regions of rats and mice exposed to chrysotile asbestos for 5 h. Animals were injected with tritiated thymidine 4 h prior to sacrifice at varying times ranging from immediately after cessation of exposure to one month post exposure. Light microscopic autoradiography showed that air-exposed control animals never had more than 1% of cells labelled. Rats and mice studied immediately after exposure also had normal numbers of labelled cells. However, between 12 and 48 h post exposure, asbestos-exposed animals exhibited up to 4-fold increases in the percentages of labelled epithelial and interstitial cells. Normal labelling returned by 8 days after exposure and was maintained through the one-month period studied. We conclude that inhalation of chrysotile asbestos induces rapid and highly significant increases in proliferation of epithelial and interstitial cells of the bronchiolar-alveolar regions where asbestos fibres were initially deposited.
Chrysotile
Inhalation exposure
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Asbestos resembles the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), in its ability to elicit release of superoxide (O2-.) from rodent alveolar macrophages (AM) in vitro. In addition, superoxide dismutase (SOD), the antioxidant enzyme scavenging O2-, is increased in cultures of tracheobronchial epithelial cells and lung fibroblasts after exposure to either crocidolite or chrysotile asbestos. Our objectives here were to determine: (1) the chemical and physical properties of asbestos important in the generation of O2- from rat AM; and (2) the effects of O2- in comparison with asbestos on biosyntheses of collagen and non-collagen protein in rat lung fibroblasts in vitro. We were also interested in whether increased production of SOD occurred in the lungs of rats after inhalation of crocidolite asbestos. To determine whether O2- was elicited in response to a variety of asbestiform fibres, AM lavaged from Fischer 344 rat lungs were exposed in vitro to equivalent non-toxic amounts of crocidolite asbestos, erionite, Code 100 fibreglass, sepiolite, and their non-fibrous analogues, riebeckite, mordenite and glass particles. In addition, sized preparations of long (greater than 10 microns) and short (less than 2 microns) asbestos were introduced at identical concentrations to determine whether length of fibres is critical in O2- release. The amount of O2- released from AM in response to dusts was then determined by measuring SOD-inhibitable reduction of cytochrome C. All asbestiform fibres caused a significant (p less than 0.05) increase in generation of O2- from epithelial cells, whereas non-fibrous particles were less active at comparable concentrations. Experiments with long (greater than 10 microns) versus short (less than 2 microns) chrysotile showed that long fibres caused a more striking, dosage-dependent release of O2-. To determine whether O2- plays a role in the causation of fibrotic lung disease, rat lung fibroblasts were exposed to a biochemical generation system (xanthine-xanthine oxidase) for O2- before quantitation of cell-associated collagen and non-collagen protein at 24, 48 and 72 h thereafter. At the latter time periods, significant increases in total collagen per ng DNA were observed. In comparison with controls, the generation system for O2- also caused an initial decrease in synthesis of non-collagen protein followed by increases in synthesis of non-collagen protein at 48 and 72 h.(ABSTRACT TRUNCATED AT 400 WORDS)
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OBJECTIVES--Mounting evidence suggests that asbestos fibres can stimulate alveolar macrophages to generate the potent inflammatory and fibrogenic mediator, tumour necrosis factor-alpha (TNF-alpha), and that this may play an important part in the onset and development of airway inflammation and lung fibrosis due to asbestos fibre inhalation. Little is known, however, about the ability of other mineral fibres to initiate formation and release of TNF-alpha by alveolar macrophages. Therefore the effects of different fibres (crocidolite, chrysotile A, chrysotile B, two man made mineral fibres (MMVF 21 and MMVF 22), a ceramic fibre (RCF 1), and a silicon carbide whisker fibre (SiCwh)) on formation and release of TNF-alpha by rat alveolar macrophages were examined. METHODS--Cells were isolated and incubated at 37 degrees C with the different fibres, or with culture medium alone (controls), and the amounts of TNF-alpha messenger RNA (mRNA) in the cells and TNF-alpha bioactivity released into the culture medium were measured at different time points. RESULTS--Significantly (P < 0.05 v control) increased amounts of TNF-alpha mRNA were found in cells exposed to crocidolite, chrysotile A, chrysotile B, MMVF 21, RCF 1, or SiCwh for 90 minutes, and significantly (P < 0.05 v control) increased activities of TNF-alpha were found in the medium of macrophages exposed to crocidolite, chrysotile A, chrysotile B, or MMVF 21 for four hours. CONCLUSION--These observations suggest that not only natural mineral fibres but also certain man made mineral fibres are able to induce the formation and release of TNF-alpha by alveolar macrophages in vitro.
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Alpha (finance)
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Alveolar macrophages are considered to play a major role in the pathophysiology of lung diseases caused by exposure to various kinds of pathogens and particles. In this study, the cytotoxic effect of different shapes of titanium dioxide (TiO 2 ) was evaluated on macrophages using a unique magnetometry method and was compared with conventional methods of lactate dehydrogenase (LDH) release, apoptosis measurement, and morphological observations. Alveolar macrophages obtained from Fischer rats (F344) by bronchoalveolar lavage were incubated in vitro for 18 h with Fe 3 O 4 as a magnetometric indicator and fibrous and particulate forms of TiO 2 as test materials. In the control and particulate exposed group, rapid attenuation of the residual magnetic field, so-called "relaxation," was observed immediately after cessation of the external magnetic field. In comparison, a delay of relaxation was observed in alveolar macrophages exposed to fibrous TiO 2 . LDH released into serum-free medium induced by exposure to TiO 2 increased significantly in a concentration-dependent manner in macrophages exposed to fibrous TiO 2 , while negligible LDH release was observed in macrophages exposed to particulate TiO 2 . The DNA ladder detection method and morphological examination detected no apoptosis in macrophages exposed to 60 µg/ml of fibrous or particulate TiO 2 . Electron microscopic examination revealed vacuolar changes and cell surface damage in macrophages exposed to fibrous TiO 2 , but no significant changes in macrophages exposed to particulate TiO 2 . The results of magnetometry, LDH release, and electron microscopy suggest that cytotoxicity of TiO 2 depends on the shape of the material.
Alveolar macrophage
Titanium Dioxide
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Chrysotile
Alveolar Epithelium
Mesothelium
Asbestosis
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The objective of this study was to evaluate the combined effects of mineral fibres and cigarette smoke on the production of tumour necrosis factor (TNF) by alveolar macrophages. Rats were exposed to cigarette smoke in vivo, and production of TNF by alveolar macrophages was measured in the presence of mineral fibres in vitro. For smoke exposure, rats were divided into two groups. Five were exposed to a daily concentration of 10 mg/m3 of cigarette smoke for an eight hour period, and five rats (controls) were not exposed to smoke. Bronchoalveolar lavage was performed after exposure to smoke and the recovered alveolar macrophages were incubated with either chrysotile or ceramic fibres on a microplate for 24 hours. Activity of TNF in the supernatant was determined by the L-929 fibroblast cell bioassay. When alveolar macrophages were not stimulated by mineral fibres, production of TNF by rats exposed to smoke and unexposed rats was essentially the same. When alveolar macrophages were stimulated in vitro by chrysotile or ceramic fibres, production of TNF by alveolar macrophages from rats exposed to smoke was higher than that by alveolar macrophages from unexposed rats. The findings suggest that cigarette smoke and mineral fibres have a synergistic effect on TNF production by alveolar macrophages.
Chrysotile
Pulmonary alveolus
Alveolar macrophage
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