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Asbestos fibers are highly fibrous silicate fibers that are distinguished by having a large aspect (length to diameter) ratio and are crystallized in an asbestiform habit that causes them to separate into very thin fibers or fibrils. These fibers are distinct from nonasbestiform cleavage fragments and may appear as thick, short fibers which break along cleavage planes without the high strength and flexibility of asbestiform fibers. Since cleavage fragments of respirable dimensions have generally proven nonpathogenic in animal studies, little data exists on assessing well-characterized preparations of cleavage fragments in in vitro models. The available studies show that cleavage fragments are less bioreactive and cytotoxic than asbestiform fibers.

Exposure to asbestos is a known etiological factor in malignant mesothelioma (MM). However, in vitro cell culture studies have provided paradoxical evidence that asbestos exposure to mesothelial cells causes cytotoxicity or apoptosis rather than malignant transformation. Although it has been shown that the iron associated with asbestos participates in the cell toxicity and probably MM pathogenesis via generation of reactive oxygen species (ROS), the molecular mechanisms largely remain unknown. Here, we demonstrate that ferritin heavy chain (FHC), a core subunit of iron-binding protein ferritin, works as an anti-apoptotic protein against toxic asbestos and oxidative stress in human mesothelial cells and MM cells. We found that FHC was induced in asbestos-exposed MeT-5A human mesothelial cells. The mesothelial cell line stably expressing FHC generated less amount of hydrogen peroxide (H2O2), one of the main ROS, after asbestos exposure and was more resistant to apoptosis induced by H2O2 compared with the cells transfected with the empty vector. Next, we investigated biological roles of FHC in human MM cell. We found that NCI-H2052, a human MM cell line, had a higher expression of endogenous FHC than MeT-5A and used the cell to address FHC function in MM. NCI-H2052 showed reduced H2O2 production and an apoptosis-resistant phenotype compared with MeT-5A. Suppression of the over-expressed FHC by using FHC small interfering RNA rendered the MM cells sensitive to apoptosis, suggesting the contribution of FHC to apoptosis resistance of the MM cells. Our findings highlight the potential role of FHC in the pathogenesis of asbestos-induced mesothelioma.

We showed previously that both crocidolite and chrysotile asbestos inhalation induced a persistent macrophage inflammatory response within the pleural space of the rat. We postulated that the stimulus for pleural macrophage recruitment after asbestos exposure was the induction of monocyte chemoattractant protein-1 (MCP-1) synthesis by pleural mesothelial cells. To test this hypothesis, rat pleural mesothelial cells (RPMC) were cultured with or without chrysotile or crocidolite asbestos fibers (8 micrograms/cm2) in the presence (50 ng/mL) or absence of either tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). MCP-1 mRNA expression was assessed by RT-PCR in RPMC cultured for 2 to 24 hours, and MCP-1 protein secretion was measured by ELISA in conditioned medium from 24-hour and 48-hour cultures. Crocidolite and chrysotile fibers induced MCP-1 mRNA expression in RPMC which was maximal after 12 hours in the absence of cytokines, but which peaked after 2 hours when RPMC were challenged with asbestos + TNF-alpha or IL-1 beta. Both types of asbestos also significantly increased MCP-1 protein secretion after 24 and 48 hours (P < .0001), an effect that was potentiated by cytokine stimulation. Rats exposed by inhalation to either chrysotile or crocidolite asbestos fibers also had greater amounts of MCP-1 protein in their pleural lavage fluid than did sham-exposed rats. These findings suggest that MCP-1 secretion by RPMC may have a role in the initiation and/or potentiation of asbestos-induced pleural injury.

This study was designed to assess the effects of in vitro and in vivo asbestos exposure on the adhesion of rat pleural leukocytes (RPLs) labeled with the fluorochrome calcein AM to rat pleural mesothelial cells (RPMCs). Exposure of RPMCs for 24 h to either crocidolite or chrysotile fibers (1.25-10 microgram/cm(2)) increased the adhesion of RPLs to RPMCs in a dose-dependent fashion, an effect that was potentiated by interleukin-1beta. These findings were not observed with nonfibrogenic carbonyl iron particles. Crocidolite and chrysotile plus interleukin-1beta also upregulated vascular cell adhesion molecule-1 mRNA and protein expression in RPMCs, and the binding of RPL to asbestos-treated RPMCs was abrogated by anti-vascular cell adhesion molecule-1 antibody. PRLs exposed by intermittent inhalation to crocidolite for 2 wk manifested significantly greater binding to RPMCs than did RPLs from sham-exposed animals. The ability of asbestos fibers to upregulate RPL adhesion to RPMCs may play a role in the induction and/or potentiation of asbestos-induced pleural injury.

Cell cycle progression and apoptosis are controlled by regulatory proteins, including p53, of which functional alterations are linked to carcinogenesis. Recently, malignant mesothelioma (MM), a primary tumour related to asbestos exposure, alternatively to post therapeutic radiations, has proven to be an important problem in oncogenesis. The p53 protein does not seem mutated or deleted in MM but a possible inactivation by binding to other proteins [mdm2; SV40 large T antigen (Tag)] has been suggested. The present work investigated cell cycle regulation in normal rat pleural mesothelial cells (RPMC) and in RPMC expressing Tag (RPMC-TSV40), under exposure to asbestos and radiations. In RPMC, these agents induced activation of cell cycle checkpoints located at G1/S and G2/M and/or mitosis but a lack of control at G1/S was found in RPMC-TSV40. A loss of G2/M control may account for the formation of micronuclei observed after exposure of RPMC-TSV40 to radiations. In RPMC-TSV40 the enhancement of abnormal mitoses and apoptosis after asbestos exposure, in comparison with RPMC, suggests a loss of mitotic control and a p53-independent mechanism of apoptosis. Thus Tag expression in mesothelial cells might have both adverse and beneficial effects by impairing the control of DNA integrity and enhancing apoptosis respectively.

Stimulation of cell signaling cascades by oxidants may be important in the pathogenesis of pulmonary and pleural diseases. Here, we demonstrate in rat pleural mesothelial cells that apoptotic concentrations of crocidolite asbestos and H2O2 induce phosphorylation and activation of extracellular signal-regulated protein kinases (ERK). Activation of c-jun-NH2-terminal protein kinases (JNK)/stress-activated protein kinases was also observed in response to H2O2. In contrast, asbestos caused more protracted activation of ERK without JNK activation. Both H2O2- and asbestos-induced activation of ERK was abolished by catalase. Moreover, chelation of surface iron from crocidolite fibers or addition of N-acetyl-L-cysteine prevented ERK activation and apoptosis by crocidolite, indicating an oxidative mechanism of cell signaling. The MEK1 inhibitor PD-98059 abrogated asbestos-induced apoptosis, confirming a causal relationship between ERK activation and apoptosis. These results suggest that distinct cell-signaling cascades may be important in phenotypic responses elicited by oxidant stresses.

Mesothelial cells, the progenitor cell of the asbestos-induced tumor mesothelioma, are particularly sensitive to the toxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in mesothelial cells are not known. We asked whether asbestos induced apoptosis in mesothelial cells and whether reactive oxygen species were important. Pleural mesothelial cells (rabbit or human) were exposed to asbestos (crocidolite, amosite, or chrysotile) or control particles at moderate doses (1-10 microg/cm2) over 24 h and evaluated for oligonucleosomal DNA fragmentation, loss of membrane phospholipid asymmetry, and nuclear condensation. Asbestos fibers, not control particles, induced apoptosis in mesothelial cells by all assays and induction of apoptosis was dose dependent for all types of asbestos, with crocidolite (5 microg/cm2) inducing 15.0+/-1.1% (mean+/-SE; n = 12) apoptosis versus control particles < 4%. Apoptosis induced by asbestos, but not by actinomycin D, was inhibited by extracellular catalase, superoxide dismutase in the presence of catalase, hypoxia (8% oxygen), deferoxamine, 3-aminobenzamide [an inhibitor of poly(ADP-ribosyl) polymerase], and cytochalasin B. Only catalase and cytochalasin B decreased fiber uptake. We conclude that asbestos induces apoptosis in mesothelial cells via reactive oxygen species. Escape from this pathway could allow the abnormal survival of mesothelial cells with asbestos-induced mutations.

The pleuropulmonary response to inhaled asbestos frequently involves inflammation and release of various cytokines from lung cells. Among these, interleukin-8 (IL-8) released from the mesothelium could augment inflammation of the pleura by attracting neutrophils to the pleural space. We used cultures of human pleural mesothelial cells (HPMC) to examine the mechanism of IL-8 production by asbestos and cytokines. Suspensions of amosite, chrysotile, or crocidolite asbestos in concentrations as low as 5 micrograms/ml enhanced release of IL-8 from HPMC during 6 h of incubation at 37 degrees C. Electron microscopy of asbestos-treated HPMC showed that the cells avidly engulfed each of the different types of asbestos fibers. Two proinflammatory cytokines, interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha, enhanced IL-8 release within 2 h and had an even greater effect after 6 h. Release of IL-8 was measured by an enzyme-linked immunosorbent assay, and functional activity of the cytokine was assessed by chemotaxis of human neutrophils. Identity of IL-8 in HPMC supernatants was established by absorption with an antibody to IL-8. Preincubation of HPMC with IL-1 receptor antagonist (IL-1ra) significantly decreased release of IL-8 after stimulation with amosite or crocidolite asbestos. We conclude that HPMC release IL-8 in response to asbestos stimulation and that the response is, in part, mediated by IL-1, mainly in the form of IL-1 alpha.

The cytogenetic effects of asbestos fibers on rat pleural mesothelial cells were studied in vitro. Crocidolite UICC significantly enhanced aneuploidy and produced few structural chromosome aberrations, whereas anatase, an isomorphic particle, induced no numerical or structural changes. Mitomycin C (300 nM) produced a tenfold increase in abnormal anaphases compared with controls. Asbestos produced anaphase/telophase abnormalities in a concentration-dependent manner. The majority of the abnormalities involved lagging chromosomes. Crocidolite UICC induced abnormalities at a dose of 7.0 micrograms/cm2, whereas Canadian chrysotile did so at 1.0 to 2.0 micrograms/cm2. When the response was assessed by the number of long and thin fibers per cm2 (length > 8 microns; diameter < or = 0.25 microns), crocidolite UICC produced more abnormalities than Canadian chrysotile at all concentrations. On a per-weight basis, these findings differ from those obtained after intrapleural inoculation, as crocidolite induced more mesotheliomas than chrysotile; however, on a per-fiber basis, the in vitro and in vivo effects were similar. These results show that anaphase/telophase analysis is sensitive and complementary to metaphase analysis, and suggest that asbestos might produce cell transformation by inducing chromosome missegregation and aneuploidy.

To investigate the mechanisms of asbestos-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in rat pleural mesothelial cells and hamster tracheal epithelial cells after exposure to crocidolite or chrysotile asbestos. In contrast to phorbol 12-myristate 13-acetate, which induces rapid and transient increases in c-fos and c-jun mRNA, asbestos causes 2- to 5-fold increases in c-fos and c-jun mRNA that persist for at least 24 hr in mesothelial cells. The induction of c-fos and c-jun mRNA by asbestos in mesothelial cells is dose-dependent and is most pronounced with crocidolite, the type of asbestos most pathogenic in the causation of pleural mesothelioma. Induction of c-jun gene expression by asbestos occurs in tracheal epithelial cells but is not accompanied by a corresponding induction of c-fos gene expression. In both cell types, asbestos induces increases in protein factors that bind specifically to the DNA sites that mediate gene expression by the AP-1 family of transcription factors. The persistent induction of AP-1 transcription factors by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of the early response gene pathway that includes c-jun and/or c-fos.

Tumor necrosis factor (TNF) is a cytokine released predominantly by monocytes/macrophages that has been shown to modulate a variety of different immune and metabolic functions. To understand the regulatory mechanisms of TNF in governing responses in the pleural cavity following deposition of fibrous dust in the airspace of the lung, we studied the capability of leukocytes, lavaged from the pleural cavity, to release TNF in culture. TNF production by lavaged pleural leukocytes was measured using the L-929 TNF-sensitive cell line, after intratracheal instillation of crocidolite asbestos. A high level of TNF activity was found in the supernatants of normal, unstimulated pleural leukocytes; the addition of 100 ng/ml lipopolysaccharide to the culture increased the activity up to threefold. Following intratracheal instillation of 5 mg crocidolite asbestos, the pleural leukocytes secreted less TNF than the control. With increasing mass of intratracheally instilled asbestos, there was a dose-dependent reduction in TNF release. Changes in the population of the pleural leukocytes or their number could not be related to variation in TNF activity. These results suggest that exposure of rat lungs to crocidolite asbestos by intratracheal instillation affects the response of pleural leukocytes without causing changes in the population. Such changes in the bronchoalveolar space may be related to the pleural pathology found in asbestos-exposed individuals.

We have previously reported that normal pleural leukocytes secrete a urokinase-type plasminogen activator inhibitor (PAI) in culture. In view of the pathogenic effects of asbestos on the pleura, in particular pleural fibrosis, we have extended these observations to crocidolite asbestos-exposed rats. Pleural leukocytes from rats exposed to crocidolite asbestos were found to secrete more PAI in culture than controls. The activity of PAI in pleural leukocyte-conditioned medium increased in a dose-dependent manner in relation to the quantity of asbestos injected into the lung. However, with increasing time post asbestos instillation, there was no significant change in the secretion of PAI by pleural leukocytes in culture compared with earlier time points of crocidolite-exposed rats. Plasminogen activator was not detectable in the conditioned medium at any time point. The data derived from this study may help to elucidate the pathogenesis of some pleural disorders caused by exposure to fibrous dusts in the lungs.

Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.

Cultures of rat pleural mesothelial cells (PMC) were exposed to nonlethal doses of UICC chrysotile A. The morphology was studied by optical and electron microscopy. The consequences of chrysotile ingestion on the rate of pinocytosis of horseradish peroxidase (HPR) metabolism and benzo-3-4-pyrene (BP) were studied. Nonlethal doses of chrysotile (5 micrograms/mL) induced a time-dependent vacuolation of PMC; a dose-dependent inhibition of the vacuolation was observed when PMC were pretreated with DMSO. The origin of the vacuoles is not clear, but some features of autophagy and lysosomal storage were observed. Chrysotile fibers did not modify the rate of pinocytosis of HRP. Similarly, the metabolism of BP was unchanged when BP and chrysotile were both added to the culture medium or when PMC were preincubated with the fibers 24 hr prior to the addition of BP.

The modification of the chemistry of asbestos chrysotile fibres (Mg3(Si2O5)(OH)4) after their ingestion by cultured cells has been studied. Two types of cells involved in asbestos related pulmonary disease were used, rabbit alveolar macrophages (AM), recovered by bronchoalveolar lavage, and pleural mesothelial cells (PMC) obtained from the rat parietal pleura. Chemical characterisation of intracellular fibres was performed on unstained ultrathin sections by electron probe microanalysis. The results showed a progressive leaching of Mg, characterised by a time dependent decrease of Mg/Si. AM were more efficient than PMC at leaching intracellular chrysotile fibres since it took longer to obtain the same proportion of leached fibres with PMC than with AM. As in vitro Mg-leaching can be obtained by acid treatment, chrysotile fibres were incubated, either untreated or pretreated with cell membranes, at pH 4 or 7 for various times. The data show that the kinetic of leaching by AM was comparable with leaching at pH 4. The leaching by PMC was of the same order as leaching at pH 7. When membranes were adsorbed on to the fibres, a delayed leaching was observed. The results indicate that the solubilisation of chrysotile by AM could be an intraphagolysosomal event due to a pH effect. With PMC, however, it is not possible to draw this conclusion since nothing is known about the intracellular pH.

The effect of conditioned media obtained from in vivo or in vitro asbestos-transformed rat pleural mesothelial cells on growth of normal mesothelial cells was studied. It was found that both conditioned media led to a reduction of cell proliferation as shown by a decrease in the following parameters: cell number, tritiated thymidine incorporation and percentages of S cells. Similar effects were obtained with EGF (Epidermal Growth Factor).

Rats were instilled intrabronchially with 1 mg UICC amosite suspended in 0.2 mL of filtered saline; control animals received the saline instillation only. Five animals from each group were killed on various days after instillation, up to day 128/129. Total retrieved cell counts and differential cell analysis were performed from lung and pleural lavages. In particular, the appearance of peroxidase-positive macrophages (PPM) as indicators of newly arrived macrophages was investigated. Polymorphonuclear cells (PMN) and PPMs in lung lavages increased in number 24 hr after amosite instillation and remained at increased levels until day 62. Alveolar macrophage numbers were significantly decreased after amosite instillation. There was only a very transient increase of PPMs and PMNs in the saline group. The number of PPMs in pleural lavage fluid was already increased 24 hr after amosite instillation. The pleural PPM increase was sustained throughout the study. No pleural reaction was seen in the saline instilled group. The inflammatory reactions indicated by the composition of the lavaged cells of the lung represent the in vivo toxicity of intrabronchially instilled amosite. The stimulus for recruitment of PMNs and PPMs is different, since no PMN response was detected in the pleural space. It is suggested that the response of the pleural PPMs is caused by the early arrival of fibers at the pleural sites, which results in the recruitment of PPMs to this space by an unknown mechanism.

The development of the pleural inflammatory response to asbestos remains poorly defined. Importantly, the role of the pleural mesothelial cell in recruitment of neutrophils to the pleural space is not known. We hypothesized that rabbit pleural mesothelial cells stimulated by asbestos fibers release chemotactic factor(s) for neutrophils. Primary cultures of rabbit pleural mesothelial cells were established, and their purity verified by the presence of keratin and hyaluronic acid mucin. Mesothelial cells in serum-free media, in the presence of 30 micrograms/ml of crocidolite asbestos, released chemotaxins for neutrophils. This activity was not dependent on the type of asbestos fiber or fiber length. It was dose-dependent until 30 micrograms/ml of asbestos. The chemotactic fractions had the ability to increase both directed and random migration of neutrophils. The chemotactic activity was not present in sonicated fractions of unstimulated mesothelial cells, nor in supernates of asbestos fibers alone. Characterization of the chemotactic activity showed that it was heat stable (56 degrees C per 30 min) and sensitive to digestion with trypsin and papain. On Sephadex G-50 chromatography, it had a molecular weight between 6,000 and 9,000. Production of the chemotactic activity was inhibitable by cycloheximide. These results demonstrate that pleural mesothelial cells can actively synthesize a protein fraction with chemotactic activity for neutrophils. Production of this mesothelial cell-derived chemotactic activity for neutrophils may play an important role in the initiation of the inflammatory response of the pleura to asbestos.

The effects of the UICC asbestos fibers chrysotile A and crocidolite on the morphology and growth of rat pleural mesothelial cells were examined. The response was different according to the fiber used. Within 4 hr of treatment with 5, 10, 20, or 50 micrograms/ml of chrysotile fibers, cell morphology showed intense vacuolization, in both logarithmic and confluent cells. Following treatment of growing cells with 20 or 50 micrograms/ml of chrysotile fibers, cell spreading was also observed. Four hours after treatment of cells with 5, 10, or 20 micrograms/ml of crocidolite fibers, no such changes were seen. Vacuolization appeared later and was much less marked than for chrysotile fibers. The mesothelial cell population-doubling time of about 30 hr was not significantly altered by addition of 5 to 20 micrograms/ml of crocidolite fibers, but a concentration of 50 micrograms/ml lengthened doubling time to about 90 hr. Treatment with 5 or 10 micrograms/ml chrysotile fibers usually prolonged this time, but 20 or 50 micrograms/ml caused cell lysis. After 48 hr of incubation with both types of fiber, the proportion of mitosis was the same as in control cultures, whatever the fiber concentration, and asbestos fibers were often seen inside dividing cells.

Pleural mesothelial cells (PMC) from the parietal pleura of rats were incubated in culture with UICC A chrysotile fibers. The sequence of events in phagocytosis was studied by electron microscopy: phases of attachment sequestration, and degranulation of lysosomal content into the phagocytic vacuole were observed. This demonstrates that PMC can engage in phagocytosis of chrysotile fibers.

The effects of UICC crocidolite and chrysotile A, either oxalic acid-leached or unleached, on the viability, morphology and growth characteristics of rat pleural mesothelial cells (PMC) were examined; DQ12 quartz particles were also used. When asbestos fibers were added for 48 hr at the beginning of exponential growth, 20 or 50 micrograms/mL of chrysotile fibers were cytotoxic and no growth occurred; with 5 or 10 micrograms/mL a latent period was observed, and the mean population doubling time was increased. Chrysotile ingestion was associated with morphological changes (spreading, intense vacuolation); moreover, a large proportion of the cells was binucleated (more than 30% with 10 micrograms/mL). The oxalic acid-leached chrysotile inhibited growth at a concentration of 50 micrograms/mL; with 5 or 10 micrograms/mL, no spreading occurred, but a shrinkage of some cells was observed. A few large vacuoles were seen in the cytoplasm of the cells; there were fewer binucleated cells. Addition of 5 or 10 micrograms/mL of crocidolite, either leached or unleached, did not significantly change the growth rate, in spite of the presence of a large number of fibers inside the cells which persisted when the cells reached confluency. With 20 or 50 micrograms/mL, the mean population doubling time was increased in a dose-dependent manner. A slight vacuolation of the cells occurred. The sample of quartz did not modify the parameters studied in this report. The results confirm the different in vitro reactivities of the two kinds of unleached asbestos fibers. Leaching of chrysotile fibers decreased their reactivity; alternatively, leaching of crocidolite increased the effects on PMC.

When studying total pleural mesothelium films in rats after intrapleural injection of chrysotile the pathological regeneration of the mesothelium is observed. During 12 months of the experiment rare mitoses, multinuclear cells and symplasts were found.