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B T Mossman

Publications and source records attributed to B T Mossman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Asbestos increases mammalian AP-endonuclease gene expression, protein levels, and enzyme activity in mesothelial cells.

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.

Animals↗
PubMed1998

Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers.

Erionite, a naturally occurring fibrous zeolite, is associated with the development of nonmalignant and malignant lung diseases and is more carcinogenic than asbestos fibers in man and rodent inhalation models of disease. To investigate the possible molecular mechanisms of erionite-induced toxicity and carcinogenesis and whether cationic content of erionite fibers was important, we examined c-fos and c-jun mRNA levels, activator protein-1 (AP-1) binding to DNA, and changes in cell proliferation and apoptosis in rat pleural mesothelial (RPM) cells exposed to different cation-substituted erionite fibers or crocidolite asbestos at various concentrations (1, 5, or 10 microg/cm2 dish) at time periods from 8 to 48 h after addition of minerals. c-fos mRNA levels in cells exposed to equal weight concentrations of various erionites and crocidolite fibers were increased comparably. When compared to other fibers, Na-erionite caused significantly increased levels of c-jun mRNA at lower mass concentrations (1 and 5 microg/cm2) than crocidolite asbestos, but comparable AP-1 binding to DNA. In comparison to untreated controls, numbers of RPM cells incorporating 5'-bromodeoxyuridine (BrdU) were increased dramatically after exposure to asbestos or Na-erionite at 5 and 10 microg/cm2. Significant dose-dependent increases in apoptosis were observed with asbestos at all time points, whereas erionites failed to induce apoptosis at 8 or 24 h, with minimal induction at higher concentrations than asbestos at 48 h. These data suggest that erionite increases the balance between cell proliferation (and/or abnormal DNA repair) and apoptosis, a normal mechanism of elimination of transformed or proliferating cells.

Animals↗
PubMed1998

Expression and activity of urokinase and its receptor in endothelial and pulmonary epithelial cells exposed to asbestos.

An elongated endothelial cell phenotype, which demonstrated increased ICAM-1-dependent neutrophil adherence, was induced when these cells were exposed to noncytotoxic concentrations of asbestos (Treadwell et al., Toxicol. Appl. Pharmacol. 139, 62-70, 1996). The present study examined mechanisms underlying this phenotypic change by investigating the effects of asbestos on transcription factor activation and expression of urokinase-type plasminogen activator (uPA) and its receptor uPAR. In situ zymography was used to compare the effects of these fibers on the activity of uPA. Cultures incubated with chrysotile or crocidolite asbestos, but not refractory ceramic fiber 1 (RCF-1), demonstrate localized cleavage of plasminogen, which was inhibited by amiloride. Immunocytochemistry showed that chrysotile-stimulated uPA activity was associated with a time-dependent augmentation of uPAR protein levels. RT-PCR analysis was used to investigate molecular mechanisms for these increases. Chrysotile asbestos, but not RCF-1, increased endothelial cell uPA message, relative to changes in beta-actin mRNA. This response to asbestos was not limited to endothelial cells, since both uPA and uPAR mRNA levels increase in human bronchial epithelial BEAS-2B cells exposed to chrysotile fibers. Finally, both types of asbestos, but not RCF-1, increased nuclear levels of nuclear factor-kappaB (NF-kappa B), a transcription factor common to increased expression of ICAM-1 and uPA. These data demonstrate that asbestos caused fiber-specific activation of endothelial and pulmonary epithelial cells, resulting in phenotypes capable of facilitating tissue remodeling.

Animals↗
PubMed1998

Crocidolite activates NF-kappa B and MIP-2 gene expression in rat alveolar epithelial cells. Role of mitochondrial-derived oxidants.

Nuclear factor kappa B (NF-kappa B) is a transcription factor that regulates expression of several genes coding for inflammatory and immunoregulatory proteins including the neutrophil chemotactic cytokine MIP-2. In previous studies we found that crocidolite asbestos activates the nuclear translocation of NF-kappa B as well as MIP-2 gene expression in rat alveolar type II cells. Here we report that both crocidolite-induced NF-kappa B activation of MIP-2 gene expression can be attenuated by the antioxidant tetramethylthiourea, suggesting the dependence of these responses on oxidative stress. Crocidolite exposure of RLE-TN cells also increased production of H2O2, a response that was inhibited by the mitochondrial respiratory chain inhibitor thenoyltrifluoroacetone (TTFA). TTFA treatment of RLE-6TN cells also inhibited crocidolite-induced nuclear translocation of NF-kappa B and MIP-2 gene expression. These results indicate crocidolite exposure of rat alveolar type II cells results in increased production of mitochondrial-derived hydrogen peroxide and that mitochondrial-derived oxidants contribute to crocidolite activation of NF-kappa B and increases in MIP-2 gene expression.

Animals↗
PubMed1998

Modulation of mitochondrial gene expression in pulmonary epithelial cells exposed to oxidants.

Oxidants are important in the regulation of signal transduction and gene expression. Multiple classes of genes are transcriptionally activated by oxidants and are implicated in different phenotypic responses. In the present study, we performed differential mRNA display to elucidate genes that are induced or repressed after exposure of rat lung epithelial (RLE) cells to H2O2 or crocidolite asbestos, a pathogenic mineral that generates oxidants. After 8 or 24 hr of exposure, RNA was extracted, reverse transcribed, and amplified by polymerase chain reaction with degenerate primers to visualize alterations in gene expression. The seven clones obtained were sequenced and encoded the mitochondrial genes, NADH dehydrogenase subunits ND5 and ND6, and 16S ribosomal RNA. Evaluation of their expression by Northern blot analysis revealed increased expression of 16S rRNA after 1 or 2 hr of exposure to H2O2. At later time periods (4 and 24 hr), mRNA levels of 16S rRNA and NADH dehydrogenase were decreased in H2O2-treated RLE cells when compared to sham controls. Crocidolite asbestos caused increases in 16S rRNA levels after 8 hr of exposure, whereas after 24 hr of exposure to asbestos, 16S rRNA levels were decreased in comparison to sham controls. In addition to these oxidants, the nitric oxide generator spermine NONOate caused similar decreases in NADH dehydrogenase mRNA levels after 4 hr of exposure. The present data and previous studies demonstrated that all oxidants examined resulted in apoptosis in RLE cells during the time frame where alterations of mitochondrial gene expression were observed. As the mitochondrion is a major organelle that controls apoptosis, alterations in expression of mitochondrial genes may be involved in the regulation of apoptosis.

Animals↗
PubMed1998

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.

Asbestos↗
PubMed1997

Inhibition of protein kinase C prevents asbestos-induced c-fos and c-jun proto-oncogene expression in mesothelial cells.

Asbestos and the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), increase c-fos and c-jun mRNA levels and AP-1 DNA binding activity in rat pleural mesothelial (RPM) cells, a target cell of asbestos-induced mesotheliomas (N. H. Heintz et al., Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Because protein kinase C (PKC) is the intracellular receptor of phorbol ester tumor promoters and asbestos is a putative tumor promoter in the respiratory tract, we hypothesized that PKC might play a critical role in asbestos-induced cell signaling pathways associated with regulation of proto-oncogenes. Using a panel of PKC antibodies, we identified PKC alpha as the major PKC isozyme in RPM cells. We then pretreated cells with phorbol ester dibutyrate to down-modulate PKC or with calphostin C, a specific PKC inhibitor, to determine if depletion of PKC alpha could block asbestos-induced c-fos/c-jun expression. Quantitation of Northern blots showed that fiber-associated c-fos/c-jun mRNA levels were significantly lower either after PKC alpha down-modulation or pretreatment with calphostin C. In addition, to determine whether tyrosine kinases also were involved in proto-oncogene activation by asbestos, tyrphostin AG82 or herbimycin A was added to RPM cells before exposure to asbestos. These inhibitors decreased crocidolite-induced c-fos but not c-jun levels, suggesting that tyrosine kinases have different regulatory roles in asbestos-induced c-fos versus c-jun signaling pathways. The ability to block induction of asbestos-induced proto-oncogene expression using pharmacological intervention may be important in prevention and treatment of asbestos-induced proliferative diseases including lung cancers, mesothelioma, and pulmonary fibrosis.

Animals↗
PubMed1997

Mineralogical features associated with cytotoxic and proliferative effects of fibrous talc and asbestos on rodent tracheal epithelial and pleural mesothelial cells.

Inhalation of asbestos fibers causes cell damage and increases in cell proliferation in various cell types of the lung and pleura in vivo. By using a colony-forming efficiency (CFE) assay, the cytotoxicity and proliferative potential of three mineral samples containing various proportions of fibrous talc were compared to NIEHS samples of crocidolite and chrysotile asbestos in cell types giving rise to tracheobronchial carcinomas, i.e., hamster tracheal epithelial (HTE) cells, and mesotheliomas, i.e., rat pleural mesothelial (RPM) cells. Characterization of mineralogical composition, surface area, and size distributions as well as proportions of fibers in all mineral samples allowed examination of data by various dose parameters including equal weight concentrations, numbers of fibers >5 micron in length, and equivalent surface areas. Exposure to samples of asbestos caused increased numbers of colonies of HTE cells, an indication of proliferative potential, but fibrous talc did not. RPMs did not exhibit increased CFE in response to either asbestos or talc samples. Decreased numbers of colonies, an indication of cytotoxicity, were observed in both cell types and were more striking at lower weight concentrations of asbestos in comparison to talc samples. However, all samples of fibrous minerals produced comparable dose-response effects when dose was measured as numbers of fibers greater than 5 micron or surface area. The unique proliferative response of HTE cells to asbestos could not be explained by differences in fiber dimensions or surface areas, indicating an important role of mineralogical composition rather than size of fibers.

Animals↗
PubMed1997

Patterns of 8-hydroxydeoxyguanosine formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos.

Oxidative damage is a proposed mechanism of asbestos-induced carcinogenesis, but the detection of oxidative DNA lesions in target cells of asbestos-induced mesothelioma has not been examined. In studies here, DNA was isolated from both rat pleural mesothelial (RPM) cells and a human mesothelial cell line (MET5A) after exposure in vitro to crocidolite asbestos at various concentrations. DNA was then examined for formation of 8-hydroxydeoxyguanosine (8-OHdG) at 24, 48 and 72 h using HPLC with electrochemical detection. In addition, steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) were assessed as an indication of oxidative stress. Whereas RPM cells showed dose-dependent and significant increases in 8-OHdG formation in response to crocidolite asbestos or iron-chelated crocidolite fibers (but not after exposure to glass beads), MET5A cells showed decreases in 8-OHdG. Both cell types exhibited elevations in message levels of MnSOD. In comparison with human MET5A cells, RPM cells exhibited increased cytotoxicity and apoptosis in response to asbestos, as documented by cell viability assays and flow cytometry analysis using propidium iodide. Results in RPM cells indicate that asbestos causes oxidative damage that may result in potentially mutagenic lesions in DNA and/or apoptosis, despite compensatory increases in expression of an antioxidant enzyme.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Differential induction of c-fos, c-jun, and apoptosis in lung epithelial cells exposed to ROS or RNS.

Reactive oxygen (ROS) or nitrogen (RNS) species can affect epithelial cells to cause acute damage and an array of pulmonary diseases. The goal of this study was to determine patterns of early response gene expression and functional end points of exposure to nitric oxide (NO.), H2O2, or peroxynitrite (ONOO-) in a line of rat lung epithelial (RLE) cells. Our focus was on c-fos and c-jun protooncogenes, as these genes play an important role in proliferation or apoptosis, possible end points of exposure to reactive metabolites in lung. Our data demonstrate that NO. generated by spermine 1,3-propanediamine N-14-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl] or S-nitroso-N-acetylpenicillamine as well as H2O2 cause increased c-fos and c-jun mRNA levels, nuclear proteins, and complexes binding the activator protein-1 recognition sequence in RLE cells. These agents also lead to apoptosis and increased membrane permeability. In contrast, exogenously administered ONOO- or 3-morpholinosydnonimine do not induce protooncogenes or apoptosis in RLE cells despite nitration oftyrosines. We conclude that ROS and RNS can elicit distinct molecular and phenotypic responses in a target cell of pulmonary disease.

Animals↗
PubMed1997

Role of extracellular signal-regulated protein kinases in apoptosis by asbestos and H2O2.

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.

Acetylcysteine↗
PubMed1997

Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos.

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.

Animals↗
PubMed1997

Cell signaling pathways elicited by asbestos.

In recent years, it has become apparent that minerals can trigger alterations in gene expression by initiating signaling events upstream of gene transactivation. These cascades may be initiated at the cell surface after interaction of minerals with the plasma membrane either through receptorlike mechanisms or integrins. Alternatively, signaling pathways may be stimulated by active oxygen species generated both during phagocytosis of minerals and by redox reactions on the mineral surface. At least two signaling cascades linked to activation of transcription factors, i.e., DNA-binding proteins involved in modulating gene expression and DNA replication, are stimulated after exposure of lung cells to asbestos fibers in vitro. These include nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) cascade important in regulation of the transcription factor, activator protein-1 (AP-1). Both NF kappa B and AP-1 bind to specific DNA sequences within the regulatory or promoter regions of genes that are critical to cell proliferation and inflammation. Unraveling the cell signaling cascades initiated by mineral dusts and pharmacologic inhibition of these events may be important for the control and treatment of mineral-associated occupational diseases.

Animals↗
PubMed1997

Asbestos causes translocation of p65 protein and increases NF-kappa B DNA binding activity in rat lung epithelial and pleural mesothelial cells.

The mechanisms of cell signaling and altered gene expression by asbestos, a potent inflammatory, fibrogenic, and carcinogenic agent, are unclear. Activation of the transcription factor, nuclear factor (NF)-kappa B, is critical in up-regulating the expression of many genes linked to inflammation and proliferation. Inhalation models of crocidolite- and chrysotile-induced inflammation and asbestosis were used to study the localization of p65, a protein subunit of the NF-kappa B transcription factor, in sham control rats and those exposed to asbestos. In addition, we investigated, using electrophoretic mobility shift analysis, whether in vitro exposure of rat lung epithelial cells and rat pleural mesothelial cells to asbestos increased binding of nuclear proteins, including p65, to the NF-kappa B DNA response element. Furthermore, translocation of p65 into the nucleus was determined by confocal microscopy. In comparison with sham animals, striking increases in p65 immunofluorescence were observed in airway epithelial cells of rats at 5 days after inhalation of asbestos. These increases were diminished by 20 days, the time period necessary for development of fibrotic lesions. In contrast, although inter-animal variability was observed, immunoreactivity for p65 was more dramatic in the interstitial compartment of asbestos-exposed rat lungs at both 5 and 20 days. Changes in p65 expression in pleural mesothelial cells exposed to asbestos in inhalation experiments were unremarkable. Exposure to asbestos also caused significant increases in nuclear protein complexes that bind the NF-kappa B consensus DNA sequence in both rat lung epithelial and rat pleural mesothelial cells. Using confocal microscopy, we observed partial nuclear translocation of p65 in rat pleural mesothelial cells exposed to asbestos. This partial response contrasted with the effects of lipopolysaccharide, which caused rapid and complete translocation of p65 from cytoplasm to nucleus. Our studies are the first to show the presence of the NF-kappa B system in lung tissue and evidence of activation in vitro and in vivo after exposure to a potent inflammatory, fibrinogenic, and carcinogenic environmental agent.

Animals↗
PubMed1996

Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor.

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.

Animals↗
PubMed1996

Comparative proliferative and histopathologic changes in rat lungs after inhalation of chrysotile or crocidolite asbestos.

Patterns of cell proliferation in lung and pleura and development of histopathologic lesions were studied in lungs from Fischer 344 rats after inhalation exposure to chrysotile or crocidolite asbestos at average airborne concentrations of approximately 8 mg/m3 air for 5 and 20 days and after 20 days of exposure followed by an additional 20 days in room air (20 + 20 days). To assess cell proliferation rats were injected with 5-bromo-2'-deoxyuridine (BrdU) at various time points after initiation of exposure to asbestos. Image analysis was used to quantitate the effects of chrysotile and crocidolite on BrdU labeling indices in the following lung compartments: (1) interstitium, (2) alveolar duct region, (3) bronchial epithelium, and (4) visceral mesothelium. With the exception of mesothelium, which exhibited significant increases in BrdU incorporation in rats exposed to crocidolite at 20 + 20 days, asbestos-induced elevations in BrdU uptake in other compartments were transient with labeling comparable to sham controls at later time points. Histopathology of rat lungs revealed fibrotic lesions of a greater extent and severity at 20 days in rats exposed to crocidolite, but fibrosis occurred in both asbestos-exposed groups after an additional 20 days in clean air (20 + 20). Quantification of fiber burden in rat lung after inhalation of comparable airborne concentrations of either fiber type demonstrated that inhalation of crocidolite asbestos led to a higher fiber retention when compared to chrysotile asbestos. Our results indicate that chrysotile and crocidolite asbestos induce different patterns of cell proliferation in lung and pleural cells. The protracted increases in BrdU labeling of mesothelial cells by crocidolite may reflect increased retention of fibers and/or inherent differences between types of asbestos.

Administration, Inhalation↗