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Background: Certain multi-walled carbon nanotubes (MWCNTs) have been shown to elicit asbestos-like toxicological effects. To reduce needs for risk assessment it has been suggested that the physicochemical characteristics or reactivity of nanomaterials could be used to predict their hazard. Fibre-shape and ability to generate reactive oxygen species (ROS) are important indicators of high hazard materials. Asbestos is a known ROS generator, while MWCNTs may either produce or scavenge ROS. However, certain biomolecules, such as albumin - used as dispersants in nanomaterial preparation for toxicological testing in vivo and in vitro - may reduce the surface reactivity of nanomaterials., Methods: Here, we investigated the effect of bovine serum albumin (BSA) and cell culture medium with and without BEAS 2B cells on radical formation/scavenging by five MWCNTs, Printex 90 carbon black, crocidolite asbestos, and glass wool, using electron spin resonance (ESR) spectroscopy and linked this to cytotoxic effects measured by trypan blue exclusion assay. In addition, the materials were characterized in the exposure medium (e.g. for hydrodynamic size-distribution and sedimentation rate)., Results: The test materials induced highly variable cytotoxic effects which could generally be related to the abundance and characteristics of agglomerates/aggregates and to the rate of sedimentation. All carbon nanomaterials were found to scavenge hydroxyl radicals (•OH) in at least one of the solutions tested. The effect of BSA was different among the materials. Two types of long, needle-like MWCNTs (average diameter >74 and 64.2 nm, average length 5.7 and 4.0 μm, respectively) induced, in addition to a scavenging effect, a dose-dependent formation of a unique, yet unidentified radical in both absence and presence of cells, which also coincided with cytotoxicity., Conclusions: Culture medium and BSA affects scavenging/production of •OH by MWCNTs, Printex 90 carbon black, asbestos and glass-wool. An unidentified radical is generated by two long, needle-like MWCNTs and these two CNTs were more cytotoxic than the other CNTs tested, suggesting that this radical could be related to the adverse effects of MWCNTs.

Purpose: Malignant mesothelioma is a devastating disease with a need for new treatment strategies. In the present study, we showed the importance of extracellular signal-regulated kinase 5 (ERK5) in malignant mesothelioma tumor growth and treatment., Experimental Design: ERK5 as a target for malignant mesothelioma therapy was verified using mesothelial and mesothelioma cell lines as well as by xenograft severe combined immunodeficient (SCID) mouse models., Results: We first showed that crocidolite asbestos activated ERK5 in LP9 cells and mesothelioma cell lines exhibit constitutive activation of ERK5. Addition of doxorubicin resulted in further activation of ERK5 in malignant mesothelioma cells. ERK5 silencing increased doxorubicin-induced cell death and doxorubicin retention in malignant mesothelioma cells. In addition, shERK5 malignant mesothelioma lines exhibited both attenuated colony formation on soft agar and invasion of malignant mesothelioma cells in vitro that could be related to modulation of gene expression linked to cell proliferation, apoptosis, migration/invasion, and drug resistance as shown by microarray analysis. Most importantly, injection of shERK5 malignant mesothelioma cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Assessment of selected human cytokine profiles in peritoneal lavage fluid from intraperitoneal shERK5 and control tumor-bearing mice showed that ERK5 was critical in regulation of various proinflammatory (RANTES/CCL5, MCP-1) and angiogenesis-related (interleukin-8, VEGF) cytokines. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the intraperitoneal model of tumor growth., Conclusion: ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in patients with malignant mesothelioma.

Human malignant mesothelioma (HMM), which is strongly related to asbestos exposure, exhibits high resistance to many anticancer drugs. Asbestos fibre deposition in the lung may cause hypoxia and iron chelation at the fibre surface. Hypoxia-inducible factor (HIF)-1alpha, which is upregulated by a decreased availability of oxygen and iron, controls the expression of membrane transporters, such as P-glycoprotein (Pgp), which actively extrude the anticancer drugs. The present study aimed to assess whether asbestos may play a role in the induction of doxorubicin resistance in HMM cells through the activation of HIF-1alpha and an increased expression of Pgp. After 24-h incubation with crocidolite asbestos or with the iron chelator dexrazoxane, or under hypoxia, HMM cells were tested for HIF-1alpha activation, Pgp expression, accumulation of doxorubicin and sensitivity to its toxic effect. Crocidolite, dexrazoxane and hypoxia caused HIF-1alpha activation, Pgp overexpression and increased resistance to doxorubicin accumulation and toxicity. These effects were prevented by the co-incubation with the cell-permeating iron salt ferric nitrilotriacetate, which caused an increase of intracellular iron bioavailability, measured as increased activity of the iron regulatory protein-1. Crocidolite, dexrazoxane and hypoxia induce doxorubicin resistance in human malignant mesothelioma cells by increasing hypoxia-inducible factor-1alpha activity, through an iron-sensitive mechanism.

Saccharomyces cerevisiae was supported on chrysotile, crocidolite and lixiviated chrysotile. Samples of the supported cells and free cells were observed by confocal laser scanning microscopy. After 30 days, the free cells showed no viability when stored at 30 degrees C, and a viability of 40% when stored at 4 degrees C. Supported cells stored at 30 degrees C were more viable than the free cells at early times, but showed no viability after 30 days. Samples stored at 4 degrees C showed that the adhered cells are more viable than the free cells, up to 30 days. Cells supported on chrysotile and lixiviated chrysotile had 80% viability, and on crocidolite 70% viability. Scanning electron microscopy showed that cells supported on lixiviated chrysotile are fully covered by the support, but crocidolite fibers adhere less, since they are stiffer. Fermentation experiments performed after 3 years storage showed that four from the five lixiviated chrysotile samples and one of the three crocidolite samples were active. In all cases, a delay time for the onset of fermentation was observed indicating a state of latency.

Asbestos fibers are biopersistent particles that are capable of stimulating chronic inflammatory responses in the pleura of exposed individuals. Exposure of pleural mesothelial cells, the progenitor cell of malignant mesothelioma, to asbestos induces an array of cellular responses. The present studies investigated whether the p38 mitogen-activated protein kinase cascade was induced under asbestos-exposed conditions. p38 plays a vital role in the response to stressful stimuli and enables the cell to enter an inflammatory state characterized by cytokine production. Western blot and in vitro kinase assays showed increases in dual phosphorylation and actual activity of p38 after exposure to fibrous and nonfibrous (milled) crocidolite; in contrast, polystyrene beads and iron (III) oxide had no such effects. In common with other asbestos-induced events, this was shown to be an oxidative stress-sensitive effect, inasmuch as preincubation with N-acetyl-L-cysteine or -tocopherol (vitamin E) ameliorated the effect. The present studies show that p38 activity is important for crocidolite-induced activator protein-1 DNA binding, inasmuch as an inhibitor of p38, SB-203580, reduced this activity. Crocidolite-induced cytotoxicity was also reduced with SB-203580, indicating a role for p38 in asbestos-mediated cell death. Our studies suggest that p38 activity could be a crucial factor in the chronic immune response elicited by asbestos and may represent a target for future pharmacological intervention.

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.

Apoptosis of mesothelial cells has been demonstrated in vitro but not in vivo. To identify apoptotic pleural cells as mesothelial, we used cytokeratin as a marker and found a striking spheroid, aggregated appearance of cytokeratin in apparently apoptotic mesothelial cells. In in vitro studies, we found that the aggregated cytokeratin pattern correlated with apoptosis in primary mesothelial cells from mice, rabbits, and humans and was not seen with necrosis. In in vivo studies in mice, we then used this cytokeratin pattern to identify and quantitate apoptotic mesothelial cells. Apoptotic mesothelial cells were best harvested by pleural lavage, indicating that they were loosely adherent or nonadherent. Instillation of RPMI 1640 medium or wollastonite for 24 h induced apoptosis in 0.1 +/- 0. 1 (SE) and 1.0 +/- 0.7%, respectively, of all mesothelial cells recovered, whereas instillation of known apoptotic stimuli, crocidolite asbestos (25 microg) for 24 h or actinomycin D plus murine tumor necrosis factor-alpha for 12 h, induced apoptosis in 5. 1 +/- 0.5 and 22.4 +/- 4.5%, respectively (significantly greater than in control experiments, P < 0.05). By analysis of cytokeratin staining, mesothelial cell apoptosis has been confirmed in vivo.

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.

Activation of activator protein (AP-1) by crocidolite asbestos was examined in vitro in a JB6 P+ cell line stably transfected with AP-1-luciferase reporter plasmid and in vivo using AP-1-luciferase reporter transgenic mice. In in vitro studies, crocidolite asbestos caused a dose- and time-dependent induction of AP-1 activation in cultured JB6 cells. The elevated AP-1 activity persisted for at least 48 h. Crocidolite asbestos also induced AP-1 transactivation in the pulmonary and bronchial tissues of transgenic mice. AP-1 activation was observed at 2 days after intratracheal instillation of the mice with asbestos. At 3 days postexposure, AP-1 activation was elevated 10-fold in the lung tissue and 22-fold in bronchiolar tissue as compared with their controls. The induction of AP-1 activity by asbestos appeared to be mediated through the activation of mitogen-activated protein kinase family members, including extracellular signal-regulating protein kinase, Erk1 and Erk2. Aspirin inhibited asbestos-induced AP-1 activity in JB6 cells. Pretreatment of the mice with aspirin also inhibited asbestos-induced AP-1 activation in bronchiolar tissue. The data suggest that further investigation of the role of AP-1 activation in asbestos-induced cell proliferation and carcinogenesis is warranted. In addition, investigation of the potential therapeutic benefits of aspirin in the prevention/amelioration of asbestos-induced cancer is justified.

Only two DNA repair enzymes, DNA polymerase beta and O6-methylguanine-DNA methyltransferase, have been shown to be inducible in mammalian cells by genotoxic agents. We show here that crocidolite asbestos induces the DNA repair enzyme, apurinic/apyrimidinic (AP)-endonuclease, in isolated mesothelial cells, the progenitor cells of malignant mesothelioma. Asbestos at nontoxic concentrations of 1.25 and 2.5 microg/cm2 significantly increased AP-endonuclease mRNA and protein levels as well as enzyme activity (P < 0.05) in a dose-dependent manner in rat pleural mesothelial cells. These increases were persistent from 24 to 72 h after initial exposure to fibers. Changes were not observed with glass beads, a noncarcinogenic particle. Confocal scanning laser microscopy showed that AP-endonuclease was primarily localized in the nucleus but also in mitochondria. Our data are the first to demonstrate the inducibility of AP-endonuclease by a human class I carcinogen associated with oxidant stress in normal cells of the lung.

The control of DNA integrity in mammalian cells is important to maintain the cell homeostasis and prevent neoplastic transformation. Control of cell division and cell death permits repair or elimination of damaged cells. Since asbestos fibers can produce DNA damage, chromosome alterations and apoptosis in several sorts of cells, including mesothelial cells, it was interesting to investigate cell cycle disturbances in rat pleural mesothelial cells (RPMC) treated with asbestos fibers. Cell cycle analyses were performed in RPMC exposed to crocidolite (10 and 20 microg/cm2) and chrysotile (5 and 10 microg/cm2) for different times (4 to 48 h). Both fiber types entailed a G2/M accumulation in agreement with a delay in the mitosis course. Chrysotile fibers produced a G0/G1 accumulation associated with a time-dependent p53 and p21 expression. Crocidolite exposure resulted in a delay in the G1/S transition paralleling a low rate of p53 expression. These results are in agreement with a DNA damaging potential of asbestos fibers since similar results were found following RPMC exposure to gamma rays. In asbestos-treated RPMC, a low rate of apoptosis was found suggesting that RPMC may follow a DNA repair pathway that could contribute to the formation of DNA lesions. In addition, the cell cycle disturbances at the G2/M checkpoint suggest that genetically altered cells have progressed through the cycle and support the already published findings on the ability of asbestos fibers to impair cell division.

We developed in situ dual-fluorescence detection techniques for measuring apoptosis and proliferation simultaneously in single dishes of cells. The deoxyribonucleic acid (DNA)-specific labeling method, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), first was used in conjunction with a 4',6-diamidino-2-phenylindole (DAPI) counterstain to detect and measure morphologic characteristics of apoptotic rat pleural mesothelial (RPM) cells isolated from Fischer 344 rats and exposed to 300 microM hydrogen peroxide (H2O2). For this purpose, 100 TUNEL-positive nuclei were measured while being viewed with DAPI counterstaining for area, perimeter, longest diameter, and average diameter, using imaging software and an image-collection apparatus. We then exposed cells to a range of concentrations of crocidolite asbestos and putative apoptotic and mitogenic agents. Exposure to crocidolite asbestos (5 microg/cm2) caused a striking dose-dependent apoptotic response at 24 h, 48 h, and 72 h. The nonfibrous crocidolite analogue riebeckite failed to induce apoptosis. At 24 h, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) caused an increase in apoptotic nuclei. A second method, utilizing an antibody to 5'-bromodeoxyridine (BrdU) and oxazole yellow homodimer (YOYO), showed a dose-dependent increase in proliferation occurring in cells exposed to asbestos (5 microg/cm2) at 48 h and 72 h. In addition, increased numbers of rat pleural mesothelial (RPM) cells exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA), TNF-alpha, and epidermal growth factor (EGF) exhibited incorporation of BrdU at these time points, although total numbers of cells per unit area were unchanged. Results indicate a dynamic balance between apoptosis and increased DNA synthesis after exposure of mesothelial cells to asbestos.

Asbestos fibers are human carcinogens with undefined mechanisms of action. In studies here, we examined signal transduction events induced by asbestos in target cells of mesothelioma and potential cell surface origins for these cascades. Asbestos fibers, but not their nonfibrous analogues, induced protracted phosphorylation of the mitogen-activated protein (MAP) kinases and extracellular signal-regulated kinases (ERK) 1 and 2, and increased kinase activity of ERK2. ERK1 and ERK2 phosphorylation and activity were initiated by addition of exogenous epidermal growth factor (EGF) and transforming growth factor-alpha, but not by isoforms of platelet-derived growth factor or insulin-like growth factor-1 in mesothelial cells. MAP kinase activation by asbestos was attenuated by suramin, which inhibits growth factor receptor interactions, or tyrphostin AG 1478, a specific inhibitor of EGF receptor tyrosine kinase activity (IC50 = 3 nM). Moreover, asbestos caused autophosphorylation of the EGF receptor, an event triggering the ERK cascade. These studies are the first to establish that a MAP kinase signal transduction pathway is initiated after phosphorylation of a peptide growth factor receptor following exposure to asbestos fibers.

Asbestos fibers cause dose-dependent, persistent increases in mRNA levels of c-jun and c-fos proto-oncogenes in rat pleural mesothelial (RPM) cells, the progenitor cells of asbestos-induced mesothelioma (N. Heintz, Y. M. W. Janssen, and B. T. Mossman. Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Here we report that addition of N-acetyl-L-cysteine decreases asbestos-mediated induction of c-fos and c-jun mRNA levels in a dose-dependent fashion. Exposure of RPM cells to asbestos causes depletion of total cellular glutathione, a response that can be abolished by pretreatment with N-acetyl-L-cysteine. Pretreatment of cells with buthionine sulfoximine, an agent which diminishes glutathione pools, increases the magnitude of induction of c-fos and c-jun mRNA by asbestos. To determine whether asbestos-induced effects on proto-oncogene expression could be attributed to extracellular generation of active oxygen species (AOS), RPM cells were exposed to H2O2 or xanthine and xanthine oxidase, a generating system of AOS. These oxidant stresses did not decrease cellular glutathione levels nor alter mRNA levels of c-fos or c-jun. However, increased mRNA levels of manganese-containing superoxide dismutase and heme oxygenase were observed, indicating that RPM cells respond to AOS by increased expression of genes encoding antioxidant enzymes. These data indicate that the signaling pathways leading to c-fos/c-jun proto-oncogene induction by asbestos are not triggered directly by formation of extracellular AOS. However, intracellular thiol levels appear to influence the expression of c-fos and c-jun, suggesting a redox-sensitive component in the signaling cascade which modulates gene expression of c-fos and c-jun by asbestos.

We have used a rat intratracheal instillation model to study the effect of crocidolite asbestos exposure on cytokine production by bronchoalveolar lavage (BAL) leucocytes. In unexposed controls, the normal BAL leucocytes were mostly macrophages which spontaneously produced interleukin 1 (IL-1)-like activity and tumour necrosis factor (TNF-alpha) in culture; these levels were enhanced by stimulation with LPS. In animals exposed to crocidolite asbestos, two new types of cell, neutrophils and eosinophils, were recruited into the bronchoalveolar space by 1-3 days after instillation. However, the BAL profile had returned to normal by 14 days. The production of IL-1-like activity was decreased considerably compared to control from 1 to 14 days after asbestos instillation, but was increased at 30 days. However, the leucocytes produced increased TNF-alpha as early as 3 days after asbestos instillation and maintained this elevated level throughout the experimental period. Crocidolite asbestos in vitro also stimulated normal BAL leucocytes to release significantly increased amounts of IL-1-like activity and TNF-alpha. We conclude that the deposition of crocidolite asbestos fibre in the lung by instillation causes reduced production of IL-1-like activity in the acute phase, but elevated production of both IL-1-like activity and TNF in the chronic phase, suggesting that enhanced activities of these cytokines may contribute to the development of lung pathological changes in the long term.