Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Disulfiram (Antabuse) (DSF) has been reported to protect rats and other animals from the effects of hyperbaric hyperoxia at 4 to 6 ATA (atmospheres). In contrast, DSF and diethyldithiocarbamate (DDC), its metabolite, accelerate the toxic effects in rats of 100% oxygen at 1 to 2 ATA. We have examined the effects of DSF and DDC on glutathione (GSH) levels in bovine pulmonary artery endothelial cells and Chinese hamster ovary cells. Increases in intracellular GSH occurred 8 to 24 h after addition of DSF to the culture media. These increases in intracellular GSH were associated with increases in the rate of uptake of cystine into the cells. DDC was a less effective inducer of cystine uptake and increased intracellular GSH levels than was DSF. At the concentrations used, neither DDC nor DSF caused significant decreases in intracellular superoxide dismutase levels. Exogenous sulfhydryl compounds including GSH and cysteine partially blocked the induction of cystine transport by DSF or DDC, suggesting that the induction might be mediated through a sulfhydryl reaction between DSF and some cellular components. The increases in GSH in the cultured cells were not significant by 4 h of exposure. In contrast, other stress proteins including heme oxygenase are induced by 2 to 4 h after DSF addition. In previously reported in vivo studies, DSF treatment protected against hyperbaric oxygen damage after as little as 1 to 4 h pre-exposure. This suggests that effects of DSF exposure other than GSH augmentation may be responsible for the protective effects seen in vivo.
Am J Respir Cell Mol Biol 1997 Aug
PMID:Induction of cystine transport and other stress proteins by disulfiram: effects on glutathione levels in cultured cells. 927 11

Cells that are exposed to free radicals have increased levels of DNA strand breaks with accumulation of the tumor suppressor protein p53, which induces cell cycle arrest and/or apoptosis. Because oxidants injure pulmonary epithelial cells, it was hypothesized that exposure to hyperoxia promotes DNA strand breaks in lung epithelium, resulting in increased expression of p53 and loss of epithelial cell function. Adult male C57Bl/6J mice were exposed to > 95% oxygen for 72 h and DNA integrity was determined in their lungs by terminal transferase immunoreactivity. Both nonimmunoreactive and lightly stained nuclei were observed in cells comprising the airway and parenchyma. Exposure to hyperoxia resulted in a marked increase in the intensity of nuclear staining in distal bronchiolar epithelium and alveolar epithelial and endothelial cells. Airway epithelial cells from control lungs contained detectable levels of p53 protein, which markedly increased in both nuclei and cytoplasm of distal bronchiolar epithelial cells and to a lesser extent in alveolar epithelial cells that were morphologically consistent with type II cells. Western and Northern blot analyses revealed that hyperoxia increased total lung p53 protein expression but not levels of mRNA. Changes in terminal transferase immunoreactivity and p53 expression were not observed in large airway cells, fibroblasts underlying distal airway, or smooth muscle cells. Expression of SP-B mRNA modestly increased and Clara cell secretory protein and cytochrome P-450 2F2 mRNAs decreased, providing additional evidence that hyperoxia injured pulmonary epithelial cells. These findings support the concept that hyperoxia damages DNA of pulmonary epithelial cells, which respond by accumulating p53 and changes in epithelial cell-specific gene expression.
Am J Respir Cell Mol Biol 1998 Jan
PMID:Exposure to hyperoxia induces p53 expression in mouse lung epithelium. 944 44

p21WAF/CIP1 is an important regulator of cell cycle progression (1-4). When induced, p21WAF/CIP1 protein inhibits cell cycle progression at the G1/S interface, resulting in growth arrest of the cell. To determine if p21WAF/CIP1 is involved in growth arrest and lung injury during hyperoxia, several cell lines were exposed to high levels of hyperoxia. p21WAF/CIP1 was found to be induced by 72 h in all three cell lines. Next, using an in vivo model, p21WAF/CIP1 was found to be induced at both the mRNA and protein level in neonatal murine lung born and maintained in hyperoxia. Localization of p21WAF/CIP1 was found in the peripheral airway cells. Hyperoxia-induced p21WAF/CIP1 expression was then shown to be mediated through the p53 pathway, using adult p53 mutant mice. These studies demonstrated that p21WAF/CIP1 is induced both in cells grown in culture and in neonatal and adult lung exposed to high levels of hyperoxia. Localization of p21WAF/CIP1 expression to the peripheral airway cells suggests that p21WAF/CIP1 may act to inhibit growth of alveoli in neonatal lung and delay repopulation of alveolar cells during hyperoxic administration.
Am J Respir Cell Mol Biol 1998 Feb
PMID:Induction of p21WAF/CIP1 during hyperoxia. 947 4

Inhaled nitric oxide (NO) is an important new therapeutic agent used to treat pulmonary arterial hypertension in a variety of disease states. However, the effects of NO on cells in the lung are uncertain. Previously, we have shown that NO gas depresses neutrophil oxidative cell function and increases neutrophil cell death. The purpose of this in vitro study was to determine the mechanism of neutrophil death. We hypothesized that NO hastened cell death by inducing apoptosis. To mimic the clinical environment of patients with respiratory failure, we also studied the effects of hyperoxia on neutrophil cell viability and apoptosis. Isolated human neutrophils were exposed to 80% O2 (O2), NO at 20 ppm in room air (NO/RA), 20 ppm NO blended with 80% O2 (NO/O2), or RA alone (control) for 2 to 24 h. Experiments were repeated with NO concentrations of 5 and 50 ppm and with 20 ppm in the presence of superoxide dismutase (SOD). Neutrophils were also incubated in the absence or presence of neutrophil stimulant fMLP (10 nM). Neutrophil cell viability was measured by fluorescence viability/cytotoxicity assay. Neutrophil apoptosis was assessed by cell death detection ELISA for histone-associated DNA fragments, TdT transferase-mediated fluorescence-labeled dUTP nick end labeling (TUNEL) assay, and DNA fragmentation gel electrophoresis. NO/O2-exposed neutrophils showed decreased viability at 2 h (31.7 +/- 3.7%, mean % viability +/- SD) compared with control (94.7 +/- 4.7%), O2 (75.6 +/- 9.3%), and NO/RA (62.8 +/- 14.9%; P < 0.05 by ANOVA; n = 9). Although control neutrophils demonstrated marked apoptosis at 24 h, there was no significant apoptosis at 2, 4, or 6 h (P < 0.001 by Kruskal-Wallis, n = 20) as assessed by ELISA and TUNEL assays. When compared with RA controls at 2 h, neutrophils exposed to NO/O2 showed significantly more apoptosis (292% of control, range: 106 to 2,488%, P < 0.001 by ANOVA and Kruskal-Wallis) but not with exposure to NO/RA or O2 alone. These findings were confirmed by TUNEL assay (n = 4, P < 0.05). NO/ RA and NO/O2-exposed neutrophils demonstrated both evidence of necrosis and enhanced DNA fragmentation at 2 h by gel electrophoresis (n = 2). Fifty parts per million NO produced similar findings, but exposure to 5 ppm NO did not induce significant DNA fragmentation. Coincubation with SOD inhibited NO/ O2-associated apoptosis, suggesting peroxynitrite contributed to cell death. Stimulation with fMLP did not alter apoptosis induced in neutrophils exposed to NO/RA or NO/O2. We conclude that exogenous NO gas, at clinically relevant concentrations under hyperoxic conditions, induces cell death in neutrophils in part by enhancing DNA fragmentation.
Am J Respir Cell Mol Biol 1998 Mar
PMID:Exogenous nitric oxide enhances neutrophil cell death and DNA fragmentation. 949 Jun 60

Pulmonary oxygen toxicity occurs after prolonged administration of increased fractions of inspired oxygen. Lung damage in this setting manifests as diffuse alveolar damage. In animals exposed to hyperoxia, increased numbers of alveolar macrophages are noted 72 h after initiation of high concentrations of oxygen. Monocyte chemotactic protein-1 (MCP-1) is a cytokine released by a number of cell types that has potent chemotactic activity for monocytes, precursor cells for alveolar macrophages. In the current study, we examined whether MCP-1 production was increased in response to hyperoxia. We used the monocyte/ histiocytic U937 cell line and exposed these cells to hyperoxia for variable amounts of time, then determined MCP-1 concentrations by enzyme-linked immunosorbent assay and MCP-1 mRNA levels by Northern blot analysis. We also examined the effects of dexamethasone on the response of U937 cells to hyperoxia. Finally, as a potential mechanism for regulation of U937 MCP-1 production, we examined effects of hyperoxia on MCP-1 mRNA stability. The results demonstrate that hyperoxia stimulates MCP-1 production after 6 and 24 h of exposure. MCP-1 mRNA levels are also increased after initiation of hyperoxia in part through effects on MCP-1 transcript stability. Dexamethasone significantly reduces MCP-1 production and mRNA levels also in part through effects on transcript stability. These studies suggest monocytes may be attracted to hyperoxia-exposed lungs through enhanced MCP-1 production. MCP-1 production appears to be upregulated in part through post-transcriptional processes in this setting.
Am J Respir Cell Mol Biol 1998 Apr
PMID:Modulation of monocyte chemotactic protein-1 production by hyperoxia: importance of RNA stability in control of cytokine production. 953 39

We hypothesized that manganese superoxide dismutase (MnSOD), known to be induced in rat mesothelial cells by asbestos fibers, cytokines, and hyperoxia, may also be induced in asbestos-related pleural diseases such as mesothelioma. MnSOD was assessed in healthy human pleural mesothelium (n = 6), in biopsy samples of human pleural mesothelioma (n = 7), in transformed nonmalignant human mesothelial cells (Met5A), and in two human mesothelioma cell lines (M14K and M38K) established from the tumor tissue of mesothelioma patients. There was no MnSOD immunoreactivity in five of the six samples of healthy pleural mesothelium, whereas MnSOD immunoreactivity was high in the tumor cells in all the mesothelioma samples. Northern blotting, immunohistochemistry, Western blotting, and specific activity measurements showed lower MnSOD in the nonmalignant Met5A mesothelial cells than in the M14K and M38K mesothelioma cells. In additional experiments the mesothelial and mesothelioma cells were exposed to menadione, which generates superoxide intracellularly, and to epirubicin, a cytotoxic drug commonly used to treat mesothelioma. The M38K mesothelioma cells were most resistant to menadione and epirubicin when assessed by LDH release or by adenine nucleotide (ATP, ADP, and AMP) depletion. These same cells showed not only the highest MnSOD levels, but also the highest mRNA levels and activities of catalase, whereas glutathione peroxidase and glutathione reductase levels did not differ significantly. We conclude that MnSOD expression is low in healthy human pleural mesothelium and high in human malignant mesothelioma. The most resistant mesothelioma cells contained coordinated induction of MnSOD and catalase.
Am J Respir Cell Mol Biol 1998 Apr
PMID:Manganese superoxide dismutase in healthy human pleural mesothelium and in malignant pleural mesothelioma. 953 46

Hyperoxia has deleterious effects on lung form and function; however, the molecular events initiated by oxygen exposure remain unclear. We hypothesized that macrophages function as important intermediaries in the protective response of lung tissues after exposure to hyperoxia. This hypothesis was tested by exposing cultured macrophages (RAW 264.7 cells) to hyperoxia for 24 h and then applying the conditioned medium from these cells to cultured pulmonary epithelial cells or to pulmonary microvascular endothelial cells. We observed that the expression of manganese superoxide dismutase mRNA increased in both target cell lines. Therefore, we next hypothesized that exposure of these macrophages to hyperoxia results in a change in gene expression which could be detected by differential display PCR (ddPCR). This hypothesis was tested by exposing RAW 264.7 cells to > or = 95% oxygen (or normoxia) for 24 h, harvesting RNA, and performing ddPCR. A cDNA fragment upregulated by hyperoxia was identified and reamplified. Verification of differential expression of mRNA was done by Northern analysis. A mRNA which was reproducibly upregulated by hyperoxia, as well as by lipopolysaccharide and interferon gamma, was identified. The differentially expressed PCR product was cloned and sequenced, revealing a product with 99% identity to mouse urokinase mRNA. We speculate that one function of pulmonary macrophages following a hyperoxic exposure is to secrete urokinase.
Mol Genet Metab 1998 Apr
PMID:Identification of urokinase as a hyperoxia-inducible gene. 963 98

Bronchial epithelial cells are the first cells to encounter high concentrations of inspired oxygen, and their damage is a typical feature in many airway diseases. The direct effect of oxygen on the expression of the main antioxidant enzymes (AOEs) in human bronchial epithelial cells is unknown. We investigated the messenger RNA (mRNA) levels of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), catalase (CAT), and glutathione peroxidase (GPx), as well as the specific activities of MnSOD, CuZnSOD, CAT, GPx, and glutathione reductase, in BEAS-2B bronchial epithelial cells exposed to hyperoxia (95% O2, 5% CO2) for 16 to 48 h. We also assessed the resistance of cells preexposed to hyperoxia to subsequent oxidant stress. Significant cell injury was observed after 72 h exposure to hyperoxia; release of lactate dehydrogenase (LDH) from control cells and cells exposed to hyperoxia for 72 h was 7.0 +/- 1.0% and 22.0 +/- 1.0%, respectively. Hyperoxia for 16 h, 24 h, or 48 h had no effect on the mRNA levels or specific activities of any of these enzymes. Despite their unchanged AOE levels, cells exposed to hyperoxia for 48 h showed increased resistance to H2O2 and menadione. Total glutathione content of the cells increased by 55% and 58% after 24 h and 48 h, respectively, compared with normoxic controls. However, glutathione depletion with buthionine sulfoximine (BSO) did not diminish the oxidant resistance of hyperoxia-exposed cells. We conclude that AOEs in human bronchial epithelial cells are not directly upregulated by high oxygen tension, and that increases in AOE-specific activities or glutathione are not necessary for the development of increased oxidant resistance in these cells.
Am J Respir Cell Mol Biol 1998 Aug
PMID:Antioxidant enzyme regulation and resistance to oxidants of human bronchial epithelial cells cultured under hyperoxic conditions. 969 1

Previously it was reported that hyperoxia induced death of the human lung adenocarcinoma cell line (A549 cells) by necrosis, not by apoptosis. This study examined proliferation and death of untransformed human small airway epithelial (SAE) cells in normoxia or hyperoxia in comparison with A549 cells. We tested the hypothesis that SAE cells respond differently to hyperoxic injury than do A549 cells. We measured total cell number and viability, thymidine incorporation (SAE cells only), lactate dehydrogenase (LDH) release, and apoptotic changes as markers for cell proliferation and death. Protective effects of antioxidant vitamins also were examined in SAE cells. In normoxia, subconfluent SAE cells had less apoptosis and fewer detached cells, but higher thymidine incorporation than did near-confluent cells. Hyperoxia suppressed thymidine incorporation and augmented apoptosis in both subconfluent and near-confluent SAE cells. Hyperoxia decreased the total cell number only in subconfluence, whereas SAE cell viability declined with hyperoxia in near confluence, but not in subconfluence. For SAE cells, necrosis assessed by LDH release was minimal in all conditions and was not augmented by hyperoxia in SAE cells. In contrast, normoxic A549 cells proliferated more rapidly than did SAE cells with a large number of cells detached during the culture. A549 cells underwent necrotic cell death under confluent or in hyperoxic conditions, but had much less apoptotic cell death. In SAE cells, vitamin E partially prevented the decline of thymidine incorporation with hyperoxia in subconfluence and protected against apoptotic changes with hyperoxia in both subconfluent and near-confluent conditions. Vitamin C prevented apoptosis with hyperoxia only in near-confluent SAE cells. Thus, SAE cells maintained balanced apoptosis and cell proliferation that were altered by cell density and hyperoxia and demonstrated very little necrosis with hyperoxia. Although A549 cells underwent cell death mainly by necrosis, they also were influenced by cell density and hyperoxia. Cell density also determined specific antioxidant vitamin protection in SAE cells.
Am J Respir Cell Mol Biol 1998 Sep
PMID:The effects of hyperoxic injury and antioxidant vitamins on death and proliferation of human small airway epithelial cells. 973 Aug 70

Lung injury is a frequent consequence of oxygen (O2) therapy administered to newborns and adults with respiratory distress. Acute exposure to hyperoxia results in a well-described pathophysiologic response in the lungs. Because inflammation is an important component of pulmonary O2 toxicity, we have an interest in identifying the inflammatory mediators that increase during hyperoxia. Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a member of the immunoglobulin superfamily that is expressed at the junctions between endothelial cells, is essential to the transendothelial migration of leukocytes. We hypothesized that increased expression of PECAM-1 occurs in pulmonary endothelial cells during hyperoxic lung injury. Adult mice were exposed to 100% O2 for up to 96 h. We analyzed PECAM-1 expression by RNA blot hybridization, in situ hybridization, and immunohistochemistry. A increase in PECAM-1 mRNA was seen as soon as 2 d of hyperoxia relative to unexposed control mice. PECAM-1 mRNA and protein were found in endothelial cells of both large and small arteries. The expression of PECAM-1 in capillary vessels was further confirmed using in situ hybridization at the electron microscope level. This increase in PECAM-1 expression coincided with the appearance of leukocytes in lung tissue. These observations suggest that PECAM-1 expression is a relatively early step in the inflammation cascade, and intervention at this phase may be critical to the prevention of further damage.
Am J Respir Cell Mol Biol 1998 Oct
PMID:Increased endothelial cell expression of platelet-endothelial cell adhesion molecule-1 during hyperoxic lung injury. 976 50


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