An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)-linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal-regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death-related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag-immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.     

Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers.

Epidermal growth factor (EGF) is a potent mitogen for human mesothelial cells, and autophosphorylation of the EGF receptor (EGF-R) occurs in these cell types after exposure to asbestos, a carcinogen associated with the development of mesothelioma. Here, the intensity and distribution of EGF-R protein was documented by immunocytochemistry in a human mesothelial cell line (MET5A) exposed to various concentrations of crocidolite asbestos and man-made vitreous fibers (MMVF-10). Whereas cells in contact with or phagocytizing shorter asbestos fibers (<60 microm length) or MMVF-10 at a range of concentrations showed no increase in EGF-R protein as determined by immunofluorescence, elongated cells phagocytizing and surrounding longer fibers (> or =60 microm) showed intense staining for EGF-R. In contrast, human A549 lung carcinoma cells showed neither elongation nor increased accumulation of EGF-R protein in response to long fibers. Patterns of aggregation and increases in EGF-R protein in mesothelial cells phagocytizing long asbestos fibers were distinct from diffuse staining of phosphotyrosine residues observed in asbestos-exposed cultures. These studies indicate that aggregation of EGF-R by long fibers may initiate cell signaling cascades important in asbestos-induced mitogenesis and carcinogenesis.     

DNA damage in bronchial epithelial and mesothelial cells with and without associated crocidolite asbestos fibers

Mesothelioma is induced almost exclusively by exposure to asbestos fibers. We have investigated whether the induction of DNA damage in human bronchial epithelial BEAS 2B cells and human mesothelial MeT 5A cells by crocidolite asbestos (2 microg/cm2) requires the presence of asbestos fibers in the cells. DNA damage was measured microscopically by the Comet assay, and the presence of fibers in the same cells was assessed using bright-field illumination. After treatment times of 6-72 hr, damage levels were, on the average, two times higher in cells with fibers than in cells without fibers. It was further found that DNA damage decreased with time in BEAS 2B cells both with and without fibers. No decrease in damage with time was seen in MeT 5A cells, suggesting that these mesothelial cells repair the initial damage poorly, lack induction of protective systems, or constantly produce high levels of damaging species. Our results indicate that crocidolite-treated human mesothelial MeT 5A and bronchial epithelial BEAS 2B cells show an elevated level of DNA damage if they contain a fiber. In comparison with epithelial BEAS 2B cells, mesothelial MeT 5A cells have more DNA damage after the crocidolite treatment and the damage is more persistent.     

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.     

Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells

Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.     

Asbestos inhalation induces tyrosine nitration associated with extracellular signal-regulated kinase 1/2 activation in the rat lung

Nitration of proteins by peroxynitrite (ONOO-) has been shown to critically alter protein function in vitro. We have shown previously that asbestos inhalation induced nitrotyrosine formation, a marker of ONOO- production, in the rat lung. To determine whether asbestos-induced protein nitration may affect mitogen-activated protein kinase (MAPK) signaling pathways, lung lysates from crocidolite and chrysotile asbestos-exposed rats and from sham-exposed rats were immunoprecipitated with anti-nitrotyrosine antibody, and captured proteins were subjected to Western blotting with anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. Both types of asbestos inhalation induced significantly greater phosphorylation of ERK1/2 in rat lung lysates than was noted after sham exposure. Phosphorylated ERK proteins co-immunoprecipitated with nitrotyrosine. Moreover, in MAPK functional assays using Elk-1 substrate, immunoprecipitated phospho-ERK1/2 in lung lysates from both crocidolite-exposed and chrysotile-exposed rats demonstrated significantly greater phosphorylation of Elk-1 than was noted after sham exposure. Asbestos inhalation also induced ERK phosphorylation in bronchoalveolar lavage cells. Lung sections from rats exposed to crocidolite or chrysotile (but not from sham-exposed rats nor from rats exposed to "inert" carbonyl iron particles) demonstrated strong immunoreactivity for nitrotyrosine and phospho-ERK1/2 in alveolar macrophages and bronchiolar epithelium. These findings suggest that asbestos fibers may activate the ERK signaling pathway by generating ONOO- or other nitrating species that induce tyrosine nitration and phosphorylation of critical signaling molecules.     

DNA single strand breaks induced by asbestos fibers in human pleural mesothelial cells in vitro

The mechanisms of the cellular effects and DNA damage caused by asbestos fibers in human mesothelial cells are not well understood. We exposed transformed human pleural mesothelial cells to 1-4 microg/cm2 crocidolite and to 10-100 ng/ml tumor necrosis factor alpha for up to 48 hr and studied the induction of DNA damage using the Comet assay. As a positive control, 100 microM H2O2 was used. The DNA single strand breaks were assessed as the mean tail moments and as distributions of the tail DNA in the cell. The Comet assay showed significant but reversible increases in the mean tail moments, but not in the distribution of Comet tails in the histograms in cells exposed to 1 microg/cm2 crocidolite for 6 hr. At higher concentrations of asbestos fibers all the indices in the Comet assay showed significant and irreversible change. All the doses of TNF-alpha caused marginal increase in the mean tail moments. The mean tail moments were highest in the cells with concurrent treatment to TNF-alpha and crocidolite. In the cells pretreated with inhibitors of antioxidant enzymes (aminotriazole for catalase and buthionine sulfoximine for gamma-glutamylcysteine synthetase) asbestos fibers slightly increased oxidant-related fluorescence of dichlorofluorescein (DCFH) but did not cause any further increases in the mean tail moments. This study shows that asbestos fibers cause DNA single strand breaks in human mesothelial cells. Since the inhibition of antioxidant enzymes did not have an effect on the DNA damage caused by the fibers, other mechanisms than free radicals seem to be involved in the induction of DNA damage by mineral fibers.     

Asbestos upregulates expression of the urokinase-type plasminogen activator receptor on mesothelial cells.

Inhalation of asbestos is associated with pathologic changes in the pleural space, including pleural thickening, pleural plaques, and mesothelioma. These processes are characterized by altered local proteolysis, cellular proliferation, and cell migration, suggesting that the urokinase-type plasminogen activator receptor (uPAR) could be involved in the pathogenesis of asbestos-induced pleural disease. We hypothesized that mesothelial cell uPAR expression is induced by exposure to asbestos. To test this hypothesis, we used complementary techniques in rabbit and human mesothelial cells to determine whether uPAR expression is altered by exposure to asbestos. uPAR expression was induced by chrysotile and crocidolite asbestos, but not by wollastonite, as indicated by binding of radiolabeled urokinase-type plasminogen activator (uPA) to rabbit or human mesothelial cells. uPA was not induced by fiber exposure. Exposure to exogenous uPA increased uPA activity of cells exposed to wollastonite but not asbestos-treated MeT5A cells. uPAR expression increased further when asbestos was preincubated with vitronectin (VN) or serum. Increases in uPAR expression were confirmed by binding of uPA to uPAR in cell membrane preparations and immunofluorescent staining of uPAR at the cell surface, and were associated with increases in steady-state uPAR messenger RNA. Mesothelial cell uPAR expression was also induced by media from monocytes cultured with asbestos incubated with VN and serum. By antibody neutralization, the latter effect appeared to be in part mediated by transforming growth factor-beta. We found that asbestos increases uPAR at the surface of rabbit and human mesothelial cells, suggesting that altered expression of this receptor could be involved in asbestos-induced remodeling of the pleural mesothelium.     

The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis

The mechanisms underlying asbestos-induced pulmonary toxicity are not fully understood. Alveolar epithelial cell (AEC) apoptosis by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. The two major pathways regulating apoptosis include (i) the mitochondrial death (intrinsic) pathway caused by DNA damage, and (ii) the plasma-membrane death receptor (extrinsic) pathway. However, it is unknown whether asbestos activates either death pathway in AEC. We determined whether asbestos triggers AEC mitochondrial dysfunction by exposing cells (A549 and rat alveolar type II) to amosite asbestos and assessing mitochondrial membrane potential changes (deltapsi(m)) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. Unlike inert particulates (titanium dioxide and glass beads), amosite asbestos caused dose- and time-dependent reductions in deltapsi(m). Asbestos-induced deltapsi(m) was associated with the release of cytochrome c from the mitochondria to the cytoplasm as well as activation of caspase 9, a mitochondrial-activated caspase. In contrast, a lower level of caspase 8, the death receptor-activated caspase, was detected in asbestos-exposed AEC. An iron chelator (phytic acid or deferoxamine) or a hydroxyl radical scavenger (sodium benzoate) each blocked asbestos-induced reductions in deltapsi(m) and caspase 9 activation, suggesting a role for iron-derived ROS. Finally, Bcl-X(L), a mitochondrial antiapoptotic protein that prevents cell death by preserving the outer mitochondrial membrane integrity, blocked asbestos-induced decreases in A549 cell deltapsi(m) and reduced apoptosis as assessed by DNA fragmentation. We conclude that asbestos-induced AEC apoptosis results from mitochondrial dysfunction, in part due to iron-derived ROS, which is followed by the release of cytochrome c and caspase 9 activation. Our findings suggest an important role for the mitochondria-regulated death pathway in the pathogenesis of asbestos-associated pulmonary toxicity.     

Relationship of fiber surface iron and active oxygen species to expression of procollagen, PDGF-A, and TGF-beta(1) in tracheal explants exposed to amosite asbestos

To investigate the role of iron and active oxygen species (AOS) in asbestos-induced fibrosis, we loaded increasing amounts of Fe(II)/Fe(III) onto the surface of amosite asbestos fibers and then applied the fibers to rat tracheal explants. Explants were harvested after 7 d in air organ culture. Asbestos by itself doubled procollagen gene expression, and a further increase was seen with increasing iron loading; actual collagen content measured as hydroxyproline was increased in a similar pattern. Iron loading also increased gene expression of platelet-derived growth factor (PDGF)-A and transforming growth factor (TGF)-beta(1). Neither asbestos alone nor iron-loaded asbestos affected gene expression of PDGF-B, tumor necrosis factor-alpha, or TGF-alpha. The AOS scavenger tetramethylthiourea or treatment of fibers with the iron chelator deferoxamine prevented asbestos-induced increases in procollagen, PDGF-A, and TGF-beta gene expression, whereas glutathione had no effect. The proteasome inhibitor MG-132 abolished asbestos-induced increases in procollagen gene expression but did not affect increases in PDGF-A or TGF-beta(1) expression, whereas the extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 had exactly the opposite effect. We conclude that surface iron as well as the iron-catalyzed generation of AOS play a role in asbestos-induced matrix (procollagen) production and that this process is driven in part through oxidant-induced nuclear factor kappa B activation. Surface iron and AOS also play a role in PDGF-A and TGF-beta gene expression, but through an ERK-dependent mechanism.     

Effects of asbestos and man-made vitreous fibers on cell division in cultured human mesothelial cells in comparison to rodent cells

We report the effects of chrysotile and crocidol-ite asbestos, and glass and rock wool fibers (man-made vitreous fibers, MMVF) on the induction of binucleate cells in vitro. The response of human mesothelial cells (target cells in fiber carcinogenesis) and rodent cells was compared. Human primary mesothelial cells, MeT-5A cells (an immortalized human mesothelial cell line), and rat liver epithelial (RLE) cells were exposed to asbestos and MMVF samples of similar size range. Milled glass wool, milled rock wool, and titanium dioxide were used as non-fibrous particle controls. All four fiber types caused statistically significant increases in the amount of binucleate cells in human primary mesothelial cells and MeT-5A cells (in the dose range 0.5–5.0 ug/ cm²). Chrysotile and crocidolite asbestos were more effective (1.3–3.0-fold increases) than thin glass wool and thin rock wool fibers (1.3–2.2–fold increases). However, when the fiber doses were expressed as the number of fibers per culture area, the asbestos and MMVF appeared equally effective in human mesothelial cells. In RLE cells, chrysotile was the most potent inducer of binucleation (2.9–5.0-fold increases), but the response of the RLE cells to crocidolite, thin glass wool, and thin rock wool fibers was similar to the response of the human mesothelial cells. No statistically significant increases in the number of bi- or multinucleate cells were observed in human primary mesothelial cells or RLE cells exposed to the non-fibrous dusts. In MeT-5A cells exposed to 5 ug/cm² of milled glass wool and milled rock wool, as well as in cultures exposed to 2 and 5 ug/cm² of TiO₂, significant increases were, however, observed. Our results show that rodent cells respond differently to mineral fibers than human cells. The results also add evidence to the suggested importance of disturbed cell division in fiber carcinogenesis. © 1995 Wiley-Liss, Inc.     

Asbestos-induced apoptosis is protein kinase C delta-dependent

Alveolar epithelial and mesothelial cells undergo apoptosis in response to asbestos, a phenomenon that may be important in injury and/or initiation of compensatory proliferation. Here, we report a functional role of protein kinase (PKC)delta in apoptosis by crocidolite asbestos. We first show that asbestos increases the kinase activity of PKC delta in alveolar type II epithelial cells (C10 line) and causes its translocation to mitochondria, events associated with caspase-9 cleavage and apoptosis as detected by the Apostain technique. Pretreatment of C10 cells with rottlerin (Rot), a PKC delta-selective inhibitor, before addition of asbestos prevented cleavage of caspase-9 and blocked the appearance of apoptotic cells. Asbestos-induced apoptosis also was inhibited in cells stably expressing a dominant-negative kinase-deficient mutant of PKC delta (dnPKC delta), but not dnPKC alpha. Activities of PKC alpha and PKC zeta increased after exposure to asbestos, but neither isoform migrated to mitochondria. A general inhibitor of PKCs, bisindolylmaleimide I, had no effect on asbestos-induced apoptosis. Hydrogen peroxide (H2O2) induced activation of PKCs delta, alpha, zeta, and theta, translocation of PKC delta to mitochondria, and caspase-9 cleavage. However, H2O2-induced apoptosis was not inhibited by cell lines stably expressing either dnPKC delta or dnPKC alpha, suggesting that activation of PKC delta has a distinct role in the development of asbestos-induced apoptosis.     

The formation of asbestos bodies by mouse peritoneal macrophages. An in vitro study.

For studies on the mechanism of asbestos body formation, Union Internationale Contre Cancer (UICC) crocidolite asbestos fibers were added to a culture of mouse peritoneal macrophages. Small asbestos fibers were totally ingested by the macrophages, but fibers too long to be taken up completely remained as a consequence extracellular. These long asbestos fibers became the basis for asbestos body formation. The basic mechanism underlying asbestos body formation was found to be the exocytotic activity of macrophages. The number of iron-rich inclusion bodies was dependent on the availability of iron in the culture media, and the same holds for the amount of iron in the asbestos body coat. This means that asbestos body formation is a phenomenon that occurs accidentally when macrophages come into contact with long fibers in an iron-rich environment. A time-dependent increase in the number, average size, and rate of segmentation of the asbestos bodies was observed. The present report is the first to describe asbestos body formation in vitro.     

Differential in vitro cellular response induced by exposure to synthetic vitreous fibers (SVFs) and asbestos crocidolite fibers

In this study, we analyzed the effects of synthetic vitreous fibers (SVFs) on a mesothelial (MeT5A) and a fibroblast cell line (NIH3T3), compared to those exerted by crocidolite asbestos fibers. SVFs (glass wool, rock wools) do not induce significant changes in cell mortality, whereas crocidolite asbestos fibers caused a dose-dependent cytotoxicity. We investigated the correlation between the fiber-induced cytotoxicity and the extent and type of interaction of the fibers with the cell surface, and we observed that SVFs, unlike crocidolite asbestos fibers, establish few and weak interactions. Moreover, after internalization, crocidolite asbestos fibers are often found free in the cytoplasm, whereas glass wool fibers are mainly localized inside cytoplasmic vacuoles. After treatments, we also detected signs of oxidative stress, revealed by an increased reactive oxygen species (ROS) production and by an induction of superoxide dismutase (SOD) activity. The lipoperoxidative damage was characterized by a decrease in polyunsaturated fatty acids (PUFA), an increase in the content of thiobarbituric reactive species (TBARS) and a consumption of vitamin E, as a lipophilic antioxidant. Furthermore, we investigated the effect of fiber exposure on cell proliferation. and it was found that, unlike crocidolite asbestos fibers, SVFs did not induce a significant increase in DNA synthesis.     

Alveolar cell stretching in the presence of fibrous particles induces interleukin-8 responses.

Inhalation of fibrous particulates is strongly associated with lung injury, but the molecular and cellular mechanisms that could explain the fiber-induced pathogenesis are not fully understood. We hypothesized that the physical stress exerted on the alveolar epithelium by the deposited fibers is greatly enhanced by the tidal cyclic motion of the epithelial cells that is associated with breathing, and that this initial mechanical interaction triggers a subsequent cell response. To test this hypothesis, we developed a dynamic model of fiber-induced cell injury using a cell-stretcher device. We exposed a cyclically stretched monolayer of the human alveolar epithelial cell line A549 to glass or crocidolite asbestos fibers for 8 h and then measured the production of the proinflammatory cytokine interleukin (IL)-8 as a readout of fiber-induced cell injury. Cyclic stretching significantly increased IL-8 production in the fiber-treated cultures, suggesting that the physical stress on the cells caused by the fibers was indeed enhanced by the motion. Coating of the asbestos fibers with fibronectin, a glycoprotein abundant in the alveolar lining fluid, further increased the fiber-induced cell response when the cells were cyclically stretched. This response was, however, significantly reduced by introducing into the culture medium, before fiber treatment, soluble RGD (Arg-Gly-Asp)-containing peptides, which specifically block binding to integrin receptors upon RGD attachment. These results suggested that adhesive interactions between protein-coated fibers and cell surface molecules are involved in the fiber-induced pathogenic process. Our novel findings indicate the importance of physical insults in fiber-induced cell stress, and bring to the forefront the need to study the mechanisms involved in this process.     

Oxalate deposition on asbestos bodies

We report on a deposition of oxalate crystals on ferruginous bodies after occupational exposure to asbestos demonstrated in 3 patients. We investigated the mechanism and possible significance of this deposition by testing the hypothesis that oxalate generated through nonenzymatic oxidation of ascorbate by asbestos-associated iron accounts for the deposition of the crystal on a ferruginous body. Crocidolite asbestos (1000 microg/mL) was incubated with 500 micromol H(2)O(2) and 500 micromol ascorbate for 24 hours at 22 degrees C. The dependence of oxalate generation on iron-catalyzed oxidant production was tested with the both the metal chelator deferoxamine and the radical scavenger dimethylthiourea. Incubation of crocidolite, H(2)O(2), and ascorbate in vitro generated approximately 42 nmol of oxalate in 24 hours. Oxalate generation was diminished significantly by the inclusion of either deferoxamine or dimethylthiourea in the reaction mixture. Incubation of asbestos bodies and uncoated fibers isolated from human lung with 500 micromol H(2)O(2) and 500 micromol ascorbate for 24 hours at 22 degrees C resulted in the generation of numerous oxalate crystals. We conclude that iron-catalyzed production of oxalate from ascorbate can account for the deposition of this crystal on ferruginous bodies.     

Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos

The protein kinase C (PKC) family consists of several isozymes whose substrates may be necessary for the regulation of key cellular events important in the pathogenesis of proliferative diseases. Asbestos is a carcinogen and fibroproliferative agent in lung that may cause cell signaling events through activation of PKC. Here we used a murine inhalation model of asbestos-induced inflammation and fibrosis to examine immunoreactivity of PKC delta and its substrate, phosphorylated-adducin (p-adducin), in cells of the lung. Moreover, we characterized PKC delta and p-adducin expression in a pulmonary epithelial cell line (C10) in both log versus confluent cells and in cells after mechanical wounding or crocidolite asbestos exposure. Both PKC delta and p-adducin were almost exclusively expressed in bronchiolar and alveolar type II (ATII) epithelial cells in lung sections and increased in these cell types after inhalation of asbestos by mice. Increases in membrane and nuclear localization of PKC delta were seen in log phase as compared to confluent C10 cells. Moreover, enhanced immunoreactivity of PKC delta was observed in epithelial cells expressing proliferating cell nuclear antigen (PCNA) after mechanical wounding or exposure to asbestos fibers. These studies show that activated PKC delta in pulmonary epithelial cells is a consequence of inhalation of asbestos and may be linked to the activation of cell proliferation.     

Fiber size and number in amphibole asbestos-induced mesothelioma

Numbers and sizes of fibers from the lungs of 10 patients who had an amphibole asbestos-induced malignant pleural mesothelioma were analyzed. Amosite was found in 10 lungs and crocidolite in 9; the average ratio of amosite to crocidolite was approximately 14:1. In the 8 patients who were not long-time asbestos insulators , the mean number of amosite fibers was 2.3 X 10(6) fibers/g dry lung, and of crocidolite fibers, 0.2 X 10(6)/g; these values represent an approximately 250-fold increase over those found in the general population. Crocidolite fibers were significantly narrower than amosite fibers (mean width, 0.13 versus 0.23 mu), were significantly shorter (mean length, 4.0 versus 5.8 mu), and had a significantly higher mean aspect (length to width) ratio (48 versus 34). Aspect ratios in general increased with increasing fiber length and decreasing fiber width, but the highest values were found for thin amosite fibers at about 13 mu in length, and thin crocidolite fibers at 8 or 15-17 mu in length. Comparison with data from other asbestos-exposed populations indicates that mesothelioma can be induced by relatively small numbers of amphibole fibers and also indicates that amosite is an effective mesothelial carcinogen in humans. Comparison of these data with epidemiologic and experimental predictions of carcinogenic size ranges for mesothelioma induction implies that either the carcinogenic size range is much broader than has been claimed (in particular, fibers considerably shorter than 8 mu and broader than 0.05 mu can produce mesothelioma), or, alternately, that extraordinarily small absolute numbers of fibers in certain size ranges can induce tumors in humans.     

Asbestos fibers and pleural plaques in a general autopsy population.

It has been claimed that symmetric lower zone pleural or diaphargmatic plaques are markers of asbestos exposure both in asbestos workers and the general population. In this study, total pulmonary asbestos burden was analyzed for 29 patients selected because pleural plaques were found at autopsy, and the results were compared with values obtained for 25 patients who had no occupational asbestos exposure. The average number of asbestos bodies in the plaque groups was 1732/g wet lung, and in the control group, 42/g wet lung. Uncoated asbestos fibers were extracted from lung and counted, measured, and identified by morphologic examination, electron diffraction, and energy-dispersive x-ray spectroscopy. The total number of fibers/per gram wet lung in the plaque group (114 x 10(3)) was similar to that in the control group (99 x 10(3), as was the number of chrysotile fibers (51 x 10(3) versus 29 x 10(3)). However, the plaque patients had a marked increase in the number of the commercially used high aspect ratio amphiboles, amosite and crocidolite (50 x 10(3) versus 1 x 10(3). A retrospective history of fairly certain asbestos exposure was obtained for 16 of the plaque patients, and such a history correlated strongly with increased numbers of commercial amphiboles in lung. It is concluded that 1) in this general autopsy population, two subgroups of patients are present. About one half of the patients appear to have developed pleural plaques as a result of asbestos exposure, while the etiology of the plaques in the other half is unclear; 2) the presence of pleural plaques correlates with a modest (50-fold) increase in numbers of long high-aspect ratio commercial amphiboles in lung tissue but does not correlate with numbers of chrysotile fibers, noncommercial amphiboles, or the total number of asbestos fibers; 3) asbestos-induced lesions are related to a complex set of mineralogic parameters and not to mere numbers of fibers in lung.