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Biomedical subjects

B T Mossman

Publications and source records attributed to B T Mossman.

At least 19 recordsLinked to original sources
PubMed2001

Modulation of cell injury and survival by high glucose and advancing age.

Old age is associated with a higher prevalence of cardiovascular disease and diabetes mellitus. Vascular smooth muscle cells (VSMC) play a role in the pathogenesis of vascular diseases, often a complication of diabetes mellitus. We examined in explanted aortic VSMC from young vs. older rats glucose-related activation of nuclear factor kappaB (NF-kappaB), a transcription factor induced by many oxidants. Data demonstrate that old age is associated with enhanced NF-kappaB activity in unstimulated VSMC that is further increased after exposure to high glucose medium. Furthermore, VSMC from old animals exhibit increased levels of protein carbonyls, an indicator of oxidative stress, and less apoptosis in response to glucose than VSMC isolated from young animals. These changes are accompanied by increased expression of NF-kappaB-related genes, gamma-glutamylcysteine synthetase, inhibitor of apoptosis protein-1 (IAP-1), and inducible nitric oxide synthase (iNOS). Results suggest that high glucose, a putative oxidative stress, causes apoptosis in VSMC from young animals and is associated with greater induction of NF-kappaB in VSMC from older animals. Increases in IAP-1 and decreased apoptosis implicate NF-kappaB as a survival factor in VSMC.

Animals↗
PubMed2001

Silica-induced activation of c-Jun-NH2-terminal amino kinases, protracted expression of the activator protein-1 proto-oncogene, fra-1, and S-phase alterations are mediated via oxidative stress.

Crystalline silica has been classified as a group 1 human carcinogen in the lung. However, its mechanisms of action on pulmonary epithelial cells which give rise to lung cancers are unclear. Using a nontransformed alveolar type II epithelial cell line (C10), we show that alpha-quartz silica causes persistent dose-related increases in phosphorylation of c-Jun-NH2-terminal amino kinases (JNKs) that are inhibited by antioxidants (P < or = 0.05). Increases in activator protein-1 (AP-1) binding to DNA and transactivation of AP-1-dependent gene expression by silica were accompanied by increases in steady-state mRNA levels of the AP-1 family members, c-jun, junB, fra-1, and c-fos at 8 h and elevated mRNA levels of fra-1 at 24 h (P < or = 0.05). Addition of tetramethylthiourea inhibited silica-associated increases infra-1 and proportions of cells in S-phase (P < or = .05). Our findings indicate that silica induces JNK activity, AP-1-dependent gene expression, ie., fra-1, and DNA synthesis via oxidative stress. Moreover, they suggest that silica may act mechanistically as a mitogen or tumor promoter, rather than a genotoxic carcinogen, in the development of lung cancers.

Animals↗
PubMed2001

Mitochondrial-derived oxidants and quartz activation of chemokine gene expression.

Macrophage inflammatory protein 2 (MIP-2) is a chemotactic cytokine which mediates neutrophil recruitment in the lung and other tissues. Pneumotoxic particles such as quartz increase MIP-2 expression in rat lung and rat alveolar type II epithelial cells. Deletion mutant analysis of the rat MIP-2 promoter demonstrated quartz-induction depended on a single NFkappaB consensus binding site. Quartz activation of NFkappaB and MIP-2 gene expression in RLE-6TN cells was inhibited by anti-oxidants suggesting the responses were dependent on oxidative stress. Consistent with anti-oxidant effects, quartz was demonstrated to increase RLE-6TN cell production of hydrogen peroxide. Rotenone treatment of RLE-6TN cells attenuated hydrogen peroxide production, NFkappaB activation and MIP-2 gene expression induced by quartz indicating that mitochondria-derived oxidants were contributing to these responses. Collectively, these findings indicate that quartz and crocidolite induction of MIP-2 gene expression in rat alveolar type II cells results from stimulation of an intracellular signaling pathway involving increased generation of hydrogen peroxide by mitochondria and subsequent activation of NFkappaB.

Animals↗
PubMed2001

Laser-based microscopic approaches: application to cell signaling in environmental lung disease.

Cell-imaging approaches using new laser-based technologies have a wide applicability to thefields of pathology and cell biology. Here, we present the application of several of these techniques, including confocal scanning laser microscopy (CSLM), laser scanning cytometry (LSC), and laser capture microdissection (LCM), to studies of cell signaling by environmental agents in lung disease. Using both cells in culture and lung tissue, we show that these technologies are powerful tools for understanding signal transduction cascades elicited by toxic agents, such as oxidants and asbestosfibers, and their relationship to the development of cell injury and proliferation, responses leading to lung disease and/or repair.

Air Pollutants↗
PubMed2000

Asbestos and cigarette smoke cause increased DNA strand breaks and necrosis in bronchiolar epithelial cells in vivo.

Coexposures to asbestos and cigarette smoke cause increased risks of lung cancer in asbestos workers. Although these carcinogens cause DNA damage to epithelial cells in vitro via generation of reactive oxygen species (ROS), it is unclear whether they cause injury to bronchiolar epithelial cells (i.e., the target cells of lung cancers in vivo). We exposed rats to amosite asbestos, cigarette smoke, and the two agents in combination for 1, 2, and 14 d. Numbers of cells exhibiting DNA strand breaks in comparison to sham rats were then evaluated in lungs using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method and by transmission electron microscopy (TEM). Increases in TUNEL-positive, necrotic epithelial cells occurred after exposure to asbestos alone and in an additive fashion after smoke and asbestos in combination. These results indicate that DNA strand breakage and necrosis are prominent mechanisms of injury by asbestos fibers and cigarette smoke in vivo to epithelial cells of the respiratory tract, thus validating in vitro observations from a number of laboratories.

Animals↗
PubMed2000

Expression of GPC3, an X-linked recessive overgrowth gene, is silenced in malignant mesothelioma.

Gene expression changes in rat asbestos-induced malignant mesothelioma (MM) cells were investigated by differential mRNA display. A mRNA transcript identified by this approach was abundant in normal rat mesothelial cells but not expressed in rat MM cell lines. Northern blot analysis confirmed that this transcript is uniformly silenced in rat MM cell lines and primary tumors. Nucleotide sequence analysis revealed that this transcript is encoded by the rat glypican 3 gene (GPC3), whose human homolog is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. Allelic loss at the GPC3 locus was infrequent (6.9%) in MM cell lines, and no mutations were found. GPC3 transcript levels were markedly decreased in 16 of 18 primary tumors and 17 of 22 human MM cell lines. Most of the cell lines were shown to have aberrant methylation of the GPC3 promoter region. In two of four human MM cell lines tested, GPC3 expression was restored after 2-deoxy 5-azacytidine (DAC)-mediated demethylation of its promoter region. Ectopic expression of GPC3 inhibited in vitro colony formation of human MM cells. Collectively, these data suggest that down-regulation of GPC3 is a common occurrence in MM and that GPC3, an X-linked recessive overgrowth gene, may encode a negative regulator of mesothelial cell growth.

Alleles↗
PubMed2000

Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.

Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers.

Administration, Inhalation↗
PubMed2000

Strategies for evaluation of signaling pathways and transcription factors altered in aging.

Aging is characterized by an accumulation of oxidative injury to DNA, RNA, proteins, lipids, and carbohydrates. In addition to damage, oxidative stress can initiate cell signaling cascades that modulate cell function, growth, and death. Aging and two common age-related diseases, diabetes mellitus and atherosclerosis, may share common oxidant-related signaling pathways that lead to abnormal transcription factor activation and ultimately to cellular dysfunction, degeneration, or death. This review will focus on approaches to evaluate key redox-sensitive signaling pathways and the transcription factors altered by diabetes, atherosclerosis, and aging.

Aging↗
PubMed2000

Silica, silicosis, and lung cancer: a response to a recent working group report.

The relationship between crystalline silica and lung cancer has been the subject of many recent publications, conferences, and regulatory considerations. An influential, international body has determined that there was sufficient evidence to conclude that quartz and cristobalite are carcinogenic in humans. The present authors believe that the results of these studies are inconsistent and, when positive, only weakly positive. Other, methodologically strong, negative studies have not been considered, and several studies viewed as providing evidence supporting the carcinogenicity of silica have significant methodological weaknesses. Silica is not directly genotoxic and is a pulmonary carcinogen only in the rat, a species that seems to be inappropriate for assessing particulate carcinogenesis in humans. Data on humans demonstrate a lack of association between lung cancer and exposure to crystalline silica. Exposure-response relationships have generally not been found. Studies in which silicotic patients were not identified from compensation registries and in which enumeration was complete did not support a causal association between silicosis and lung cancer, which further argues against the carcinogenicity of crystalline silica.

Animals↗
PubMed2000

Inhaled particulate matter causes expression of nuclear factor (NF)-kappaB-related genes and oxidant-dependent NF-kappaB activation in vitro.

High levels of ambient air pollution are associated with exacerbation of asthma and respiratory morbidity, yet little is known concerning the mechanisms of inflammation and toxicity by components of inhaled particulate matter (PM). Brief inhalation of PM(2.5) (particles of an aerodynamic diameter of < 2.5 microns) (300 microg/m(3) air for 6 h followed by a period of 24 h in clean air) by either C3H/HeJ or C57/BL6 mice caused significant (P </= 0.05) increases in steady-state messenger RNA (mRNA) levels of a number of nuclear factor (NF)-kappaB-associated and/ or -regulated genes, including tumor necrosis factor-alpha and -beta, interleukin-6, interferon-gamma, and transforming growth factor-beta. Lung mRNA levels of lymphotoxin-beta and macrophage migration inhibitory factor were unchanged. In murine C10 alveolar cells and an NF-kappaB-luciferase reporter cell line, exposure to PM(2.5) at noncytotoxic concentrations resulted in increases in transcriptional activation of NF-kappaB-dependent gene expression which were inhibited in the presence of catalase. Early and persistent increases in intracellular oxidants, as measured by flow cytometry and cell imaging using the oxidant probe 2'-7'-dichlorofluoroscin diacetate, were observed in epithelial cells exposed to PM(2.5) and ultrafine carbon black particles. Studies here are the first to show NF-kappaB-related inflammatory and cytokine gene expression after inhalation of PM(2.5) and oxidant-dependent induction of NF-kappaB activity by PM(2.5) in pulmonary epithelial cells.

Air Pollutants↗
PubMed1999

Measurement of oxidant-induced signal transduction proteins using cell imaging.

In addition to their capacity to damage macromolecules, oxidants play important roles in initiation of a number of signal transduction pathways. These include phosphorylation and dephosphorylation of members of the extracellular-regulated kinase (ERK) family of the mitogen-activated protein kinase (MAPK) cascade and events leading to activation of the transcription factor nuclear factor-kappaB (NF-kappaB). These cascades are key to transcriptional upregulation of genes important for cell survival, apoptosis, proliferation, transformation, and inflammation. To complement biochemical assays, cell-imaging approaches are necessary to detect the phosphorylated proteins of these cascades and their nuclear translocation, i.e., activation in cells. Protocols for these studies are presented, and the advantages of in situ microscopy-based techniques to detect oxidant-induced signaling pathways are discussed.

Animals↗
PubMed1999

Glutathione redox system in oxidative lung injury.

Glutathione (GSH) is a ubiquitous intracellular thiol present in all tissues, including lung. Besides maintaining cellular integrity by creating a reduced environment, GSH has multiple functions, including detoxification of xenobiotics, synthesis of proteins, nucleic acids, and leukotrienes. Present in high concentrations in bronchoalveolar lavage fluid (BALF), GSH provides protection to the lung from oxidative injury induced by different endogenous or exogenous pulmonary toxicants. Its depletion in the lung has been associated with the increased risk of lung damage and disease. The redox system of GSH consists of primary and secondary antioxidants, including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PD). Alterations in the activities of these enzymes may reflect reduced cellular defense and may serve as surrogate markers of many lung diseases. As GSH is also involved in the regulation of expression of protooncogenes and apoptosis (programmed cell death), the development of diseases such as cancer and human immune deficiency may be affected by depleting or elevating cellular GSH levels. Exogenous delivery of GSH or its precursor N-acetyl cysteine (NAC) is being used as chemotherapeutic approach.

Antioxidants↗
PubMed1999

Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis.

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c-fos/c-jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR (P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c-fos but not of c-jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c-fos and the development of apoptosis. The ability to block asbestos-induced elevations in c-fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.

Animals↗
PubMed1999

Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappaB: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappaB by oxidants.

The transcription factor nuclear factor (NF)-kappaB is activated by oxidative stress or cytokines and is critical to the activation of inflammatory genes. Here, we report that hydrogen peroxide or 3-morpholinosydnonimine, which simultaneously releases nitric oxide and superoxide, synergize with the cytokine tumor necrosis factor (TNF)-alpha to activate NF-kappaB in rat lung epithelial cells, suggesting that signaling pathways elicited by reactive oxygen species (ROS)/reactive nitrogen species (RNS) are different from TNF-induced signaling. These findings were substantiated by observations that levels of IkappaB-alpha did not change after exposure to ROS/RNS, whereas a rapid depletion of IkappaB-alpha was observed in cells exposed to TNF. In addition, the proteosome inhibitor MG132 did not affect activation of NF-kappaB by ROS/RNS, whereas it abolished the TNF response. Transfection of a dominant negative Ras construct prevented the activation of NF-kappaB by ROS/RNS, demonstrating the requirement for Ras in the activation of NF-kappaB by oxidants. In contrast, TNF activated NF-kappaB in a Ras-independent fashion. Evaluation of members of the mitogen-activated protein kinase (MAPK) family as downstream effectors of Ras revealed the requirement of MAPK/ extracellular-regulated kinase (ERK) kinase kinase (MEKK)1 and c-Jun N-terminal kinases in the induction of NF-kappaB by both oxidants and TNF, whereas the MEK-ERK pathway negatively regulates NF-kappaB. Our findings demonstrate that cytokines and oxidants cooperate in the activation of transcription factors through distinct pathways, and suggest that anti-inflammatory and antioxidant therapies may be required in concert to prevent the activation of NF-kappaB-regulated genes important in the development of inflammatory diseases.

Animals↗
PubMed1998

Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models.

To evaluate the contribution of reactive nitrogen species to inflammation by asbestos, Fischer 344 rats were exposed to crocidolite or chrysotile asbestos by inhalation to determine whether increases occurred in nitric oxide (NO.) metabolites from alveolar macrophages (AMs). AMs from animals inhaling asbestos showed significant elevations (p < .05) in nitrite/nitrate levels which were ameliorated by NG-monomethyl-L-arginine (NMMA), an inhibitor of inducible nitric oxide synthase (iNOS) activity. Temporal patterns of NO. generation from AMs correlated with neutrophil influx in bronchoalveolar lavage samples after asbestos inhalation or bleomycin instillation, another model of pulmonary fibrosis. To determine the molecular mechanisms and specificity of iNOS promoter activation by asbestos, RAW 264.7 cells, a murine macrophage-like line, and AMs isolated from control rats were exposed to crocidolite asbestos in vitro. These cells showed increases in steady-state levels of iNOS mRNA in response to asbestos and more dramatic increases in both iNOS mRNA and immunoreactive protein after addition of lipopolysaccharide (LPS). After transfection of an iNOS promoter/luciferase reporter construct, RAW 264.7 cells exposed to LPS, crocidolite asbestos and its nonfibrous analog, riebeckite, revealed increases in luciferase activity whereas cristobalite silica had no effects. Studies suggest that NO. generation may be important in cell injury and inflammation by asbestos.

Administration, Inhalation↗
PubMed1998

GRP78, HSP72/73, and cJun stress protein levels in lung epithelial cells exposed to asbestos, cadmium, or H2O2.

Occupational exposure to crocidolite asbestos is associated with the development of nonmalignant and malignant pulmonary disease. Considerable evidence indicates that the mechanisms of asbestos-induced toxicity involve the production of active oxygen species (AOS). Production of AOS in excess of cellular defenses creates an environment of oxidative stress and stimulates the expression of a number of different genes whose products may be involved in mediating responses from oxidant injury. To further investigate the mechanisms of asbestos-induced pathogenicity, we have examined by Western blot analyses the induction of the stress response proteins GRP78 and HSP72/73 in rat lung epithelial cells (RLE) exposed to crocidolite asbestos. In comparative studies, we also examined GRP78, HSP72/73, and cJun expression in RLE cells exposed to equitoxic concentrations of cadmium chloride (CdCl2) and hydrogen peroxide (H2O2). Our results demonstrate that asbestos and H2O2 do not alter GRP78 or HSP72/73 protein levels in RLE cells, but do increase levels of cJun protein. Increases by asbestos and H2O2 were not accompanied by alterations in cellular glutathione levels in this cell type, but asbestos caused elevations in protein levels of manganese-containing superoxide dismutase (MnSOD), an indirect indicator of oxidant stress. In contrast, exposure of cells to CdCl2 led to no changes in MnSOD protein levels, but increases in GRP78, HSP72/73, and cJun proteins as well as significant increases in oxidized and reduced thiol pools. Results suggest that environmental agents causing oxidative injury to lung epithelium elicit different patterns of stress responses.

Animals↗