Liposome-mediated augmentation of catalase in alveolar type II cells protects against H2O2 injury

Oxidant injury to the alveolar epithelium can be mediated by exposure to oxidant gases such as O2 at high concentrations and O3, inflammatory cell-derived reactive O2 species, and the intracellular metabolism of xenobiotics such as paraquat. An in vitro model of alveolar epithelial oxidant injury was developed based on exposure of cultured rat type II pneumocytes to superoxide and hydrogen peroxide (H2O2) enzymatically generated in the culture medium. Cytotoxicity was assessed by the release of lactate dehydrogenase (LDH) into the culture medium, which was a more reliable indicator of damage than release of 51Cr by prelabeled cells. Incubation of cells for 6-8 h with xanthine plus xanthine oxidase and glucose plus glucose oxidase induced the release of greater than 50% of total intracellular LDH. Oxidant exposure also resulted in significant detachment of cells from culture dishes. Modulation of oxidant damage was accomplished using liposomes as vectors for the delivery of catalase. Treatment of cells with catalase liposomes for 2 h resulted in augmentation of cellular catalase specific activities up to 631% of controls. Catalase was partitioned into intracellular and surface-associated compartments in catalase liposome-treated cells. Partial and complete protection against oxidant injury, induced by xanthine plus xanthine oxidase and glucose plus glucose oxidase, respectively, was achieved by pretreatment of cells with catalase liposomes. LDH release during oxidant exposure was inversely related to augmentation of cellular catalase activities. Catalase liposome-treated cells also exhibited an enhanced ability to scavenge enzymatically generated H2O2 from the culture medium. These observations suggest a useful approach to modulation of alveolar injury induced by reactive O2 species.     

NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca(2+), but whether NADPH oxidase activation directly affects Ca(2+) signaling is unknown. NADPH stimulated the production of superoxide anion and H(2)O(2) in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H(2)O(2) each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca(2+) and shifted the InsP(3)-Ca(2+) release dose-response curve to the left. Concentrations of H(2)O(2) as low as 3 microm increased the sensitivity of intracellular Ca(2+) stores to InsP(3) and decreased the InsP(3) EC(50) from 423.2 +/- 54.9 to 276.9 +/- 14. 4 nm. The effect of NADPH on InsP(3)-stimulated Ca(2+) release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H(2)O(2) increases the sensitivity of intracellular Ca(2+) stores to InsP(3) in human endothelial cells. Since Ca(2+)-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.     

Toxicity by pyruvate in HepG2 cells depleted of glutathione: role of mitochondria.

Several studies have shown that pyruvate can scavenge H(2)O(2) and protect from H(2)O(2)-mediated cell injury. Mitochondria are critical participants in the control of apoptotic and necrotic cell death. Mitochondrial GSH plays an important role in the maintenance of cell functions and viability by metabolism of oxygen free radicals generated by the respiratory chain. Since loss of GSH, especially mitochondrial GSH, is associated with increased production of reactive oxygen species and cell toxicity, the ability of pyruvate to protect against these actions was evaluated. Adding pyruvate to HepG2 cells depleted of GSH by treatment with l-buthionine sulfoximine (BSO) surprisingly caused loss of viability after 24 and 48 h of incubation. Anoxia, treatment with antioxidants, and infection with cytosolic catalase, and interestingly, catalase expressed in the mitochondrial compartment were able to rescue the HepG2 cells from this pyruvate plus BSO injury, suggesting a key role for H(2)O(2), and lipid peroxides as mediators in the cytotoxicity. This toxicity and cell death observed was linked to damage to the mitochondria as evidenced by the increased lipid peroxidation in total homogenate and mitochondrial fraction, loss of mitochondrial membrane potential, and a decrease in protein-sulfhydryl groups. The type of cell death observed under these conditions was a mixture of apoptosis and necrosis. These results suggest that the protective ability of pyruvate against oxidant damage requires a functional GSH pool, especially in the mitochondrial compartment, and that in the absence of GSH, pyruvate increases cell injury by damaging the mitochondria, presumably as a consequence of enhanced electron flow and reactive oxygen production by the respiratory chain.     

Decreased catalase activity is the underlying mechanism of oxidant susceptibility in glucose-6-phosphate dehydrogenase-deficient erythrocytes

Historically, it has been theorized that the oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes arises as a direct consequence of an inability to maintain cellular gluthione (GSH) levels. This study alternatively hypothesizes that decreased NADPH concentration leads to impaired to catalase activity which, in turn, underlies the observed oxidant susceptibility. To investigate this hypothesis, normal and G6PD-deficient erythrocytes and hemolysates were challenged with a H₂O₂-generating agent. The results of this study demonstrated that catalase activity was severely impaired upon H₂O₂ challenge in the G6PD-deficient cell whiel only decrease was observed in normal cells. Supplmentation of either normal or G6PD-deficient hemolysates with purified NADPH was found to significantly (P < 0.001) inhibit catalase inactivation upon oxidant challenge while addition of NADP⁺ had no effect. Analysis of these results demonstrated direct correlation between NADPH concentration and catalase activity (r = 0.881) and an inverse correlation between catalase activity and erythrocyte oxidant sensitivity (r = 0.906). In contrast, no correlation was found to exist between glutathione concentration (r = 0.170) and oxidant sensitivity. Analysis of NADPH/NADPt ration in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required. This study supports the hypothesis that impaired catalase activity underlies the enhanced oxidant sensitivity of G6PD-deficient erythrocytes and elucidates the importance of NADPH in the maintenance of normal catalase activity.     

Adenosine deaminase enzyme activity is increased and negatively correlates with catalase,superoxide dismutase and glutathione peroxidase in patients with Behçet's disease: original contributions/clinical and laboratory investigations

AIM: Behçet''s disease (BD) is an inflammatory vasculitis with immunologic, endothelial and neutrophil alterations. Adenosine deaminase (AD) is a marker of T-cell activation and is related to the production of reactive oxygen species by neutrophils with the production of NO(*), O(2)(*-), H(2)O(2) and OH(*). We reported increased tumour necrosis factor-alpha, soluble interleukin-2 receptor, interleukin-6, interleukin-8 and NO(*) in active BD. As there is a relation between cytokines, T cells and oxidative stress in inflammatory diseases, this study further evaluated: (1) plasma AD activity and its correlation with acute phase reactants; (2) thiobarbituric acid-reactive substances (TBARS) as an indicator for lipid peroxidation; and (3) antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase in patients with BD. The effect of disease activity and correlations between the measured parameters were explored. METHODS: A total of 35 active (n=17) or inactive (n=18) patients with BD (16 men, 19 women) satisfying International Study Group criteria, and 20 age-matched and sex-matched controls (nine men, 11 women) were included in this cross-sectional case-control study. AD and TBARS were measured in plasma, catalase in red blood cells (RBC), and SOD and GSHPx in both plasma and RBC in both groups. Acute phase reactants (alpha(1)-antitrypsin, alpha(2)-macroglobulin, neutrophils, erythrocyte sedimentation rate) were used to classify patients as active or inactive. RESULTS: Plasma AD (mean+/-standard error of the mean, 36.1+/-0.7 U/l) and TBARS (4.2+/-0.1 nmol/ml) levels were significantly (for each, p<0.001) higher in BD than in controls (24.1+/-0.8 U/l and 1.6+/-0.1 nmol/ml, respectively). RBC catalase activity was significantly (p<0.001) lower in BD than in controls (120.9+/-3.8 versus 160.3+/-4.1 k/g haemoglobin). SOD and GSHPx activities were significantly lower in both plasma and erythrocytes of patients with BD than in controls (plasma SOD, 442.4+/-8.6 versus 636.4+/-9.2 U/ml, p<0.001; RBC SOD, 3719.2+/-66.0 versus 4849.7+/-49.0 U/g haemoglobin, p<0.001; plasma GSHPx, 73.1+/-1.5 versus 90.6+/-2.9 U/ml, p<0.001; RBC GSHPx, 600.7+/-8.0 versus 670.6+/-10.1 U/g haemoglobin, p<0.001). Active BD patients had significantly lower antioxidant enzymes (except RBC catalase) and higher AD and TBARS levels than inactive subjects (for each, p<0.01). When considering all BD patients, a significant positive correlation was present between AD and TBARS (p<0.001) whereas both AD and TBARS were negatively correlated with antioxidant enzymes (for each, p<0.05). CONCLUSIONS: AD and lipid peroxidation are increased and associated with defective antioxidants in BD, suggesting interactions between activated T cells and neutrophil hyperfunction. Measures of pro-oxidative stress and antioxidative defence with AD activity as an indicator of T-cell activation can be considered as significant supportive diagnostic indicators, especially in active disease. In addition, strengthening the antioxidant defence may contribute to treatment modalities.     

Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: implication of NADPH oxidase

Modified low-density lipoprotein (LDL) induces reactive oxygen species (ROS) production by vascular cells. It is unknown if specific oxidized components in these LDL particles such as oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) can stimulate ROS production. Bovine aortic endothelial cells (BAEC) were incubated with ox-PAPC (50 microg/ml). At 4 h, ox-PAPC significantly enhanced the rate of O2- production. Pretreatment of BAEC in glucose-free Dulbecco's modified Eagle's medium plus 10 mM 2-deoxyglucose (2-DOG), the latter being an antimetabolite that blocks NADPH production by the pentose shunt, significantly reduced the rate of O2- production. The intensity of NAD(P)H autofluorescence decreased by 28 +/- 12% in BAEC incubated with ox-PAPC compared to untreated cells, with a further decrease in the presence of 2-DOG. Ox-PAPC also increased Nox4 mRNA expression by 2.4-fold +/- 0.1 while pretreatment of BAEC with the small interfering RNA (siNox4) attenuated Nox4 RNA expression. Ox-PAPC further reduced the level of glutathione while pretreatment with apocynin (100 microM) restored the GSH level (control = 22.54 +/- 0.23, GSH = 18.06 +/- 0.98, apocynin = 22.55 +/- 0.60, ox-PAPC + apocynin = 21.17 +/- 0.36 nmol/10(6) cells). Treatment with ox-PAPC also increased MMP-2 mRNA expression accompanied by a 1.5-fold increase in MMP-2 activity. Ox-PAPC induced vascular endothelial OO2-(.) production that appears to be mediated largely by NADPH oxidase activity.     

Role of vitamin E in glutathione-induced oxidant stress: methemoglobin, lipid peroxidation, and hemolysis.

Red blood cells (RBC) from normal and vitamin E-deficient rats were incubated in a hypertonic solution of reduced glutathione adjusted to pH 8. Methemoglobin formation occurred in intact RBC from both normal and vitamin E-deficient rats. Hemolysis was significantly greater in RBC from vitamin E-deficient rats. Experiments with catalase, superoxide dismutase, and methional showed that H(2)O(2) was the primary extracellular source of oxidant stress. Extracellular superoxide and hydroxyl radical were not involved in oxidant stress. Experiments with dimethyl sulfoxide showed that intracellular hydroxyl radical, generated from H(2)O(2), was the hemolytic agent. Neither methemoglobin formation nor lipid peroxidation involved hydroxyl radical. Indeed, lipid peroxidation and hemolysis in RBC from vitamin E-deficient rats were concurrent rather than consecutive events. Phase contrast microscopy showed that rigid, crenated RBC with a precipitate around the interior periphery formed during glutathione-induced oxidant stress. The precipitate dissolved slowly as the crenated RBC were converted to smooth ghosts. It appeared that protein precipitates involving mixed disulfide bonds were reduced and solubilized when extracellular glutathione penetrated the ruptured cell. Comparisons between normal RBC and vitamin E-deficient RBC suggest that vitamin E has little effect on the inward diffusion of extra-cellular H(2)O(2). Vitamin E apparently interacts with different oxidant species derived from intracellular H(2)O(2) in preventing lipid peroxidation and the sulfhydryl group oxidation leading to hemolysis.     

Cytoprotective effect of reduced glutathione in hydrogen peroxide-induced endothelial cell injury

The kinetic effects of hydrogen peroxide (H2O2) on cultured endothelial cells isolated from bovine carotid artery were studied. The cytoprotective effects of glutathione (GSH) on H2O2-induced cell injury were also investigated. H2O2-induced a dose- and time-dependent cell injury in cultured endothelial cells. H2O2-induced cell injury was blocked by simultaneous treatment by catalase, but not by superoxide dismutase. H2O2 also induced endogenous PGI2 biosynthesis, and the maximum PGI2 production was reached after 1 h treatment. Stimulation of PGI2 production was parallel with arachidonate release from H2O2-treated cells. However the prostaglandin biosynthesis enzyme activity in cells was inhibited by H2O2 treatment. When the cells were treated with GSH, the intracellular GSH reached a plateau after 3 h treatment. Both H2O2-induced cell injury and PGI2 production were significantly inhibited by the 3 h pretreatment with GSH. The cytoprotective effect of GSH was completely inhibited by buthionine sulfoximine which is a specific inhibitor of gamma-glutamylcysteine synthetase. The results indicate that the cytoprotective effect of GSH on H2O2-induced cell injury in cultured bovine carotid artery endothelial cells depends on the increase in intracellular GSH content.     

Progressive increases in serum catalase activity in advancing human immunodeficiency virus infection.

We found that serum from individuals with Acquired Immunodeficiency Syndrome (AIDS) had more (p less than .05) catalase activity (31.5 +/- 5.2 U/mL) than serum from healthy control subjects (7.3 +/- 0.8 U/mL). Moreover, serum catalase (but not glutathione peroxidase) activity increased progressively with advancing human immunodeficiency virus (HIV) infection (i.e., AIDS greater than symptomatic infection greater than asymptomatic infection greater than controls). Increases in serum catalase activity correlated with increases in serum hydrogen peroxide (H2O2) scavenging ability and reached levels which decreased exogenous H2O2-mediated injury to cultured endothelial cells without altering neutrophil bactericidal activity or mononuclear cell cytotoxicity in vitro. Serum catalase activity correlated with serum lactate dehydrogenase (LDH) activity but did not appear to be a consequence of erythrocyte (RBC) hemolysis since RBC fragility and serum haptoglobin levels were comparable in HIV-infected and control subjects. Increases in serum catalase activity may reflect and/or compensate for systemic glutathione and other antioxidant deficiencies in HIV-infected individuals.     

Effect of renal medullary H2O2 on salt-induced hypertension and renal injury

Dahl salt-sensitive (SS) and consomic, salt-resistant SS-13(BN) rats possess substantial differences in blood pressure salt-sensitivity even with highly similar genetic backgrounds. The present study examined whether increased oxidative stress, particularly H2O2, in the renal medulla of SS rats contributes to these differences. Blood pressure was measured using femoral arterial catheters in three groups of rats: 1) 12-wk-old SS and consomic SS-13(BN) rats fed a 0.4% NaCl diet, 2) SS rats fed a 4% NaCl diet and chronically infused with saline or catalase (6.9 microg x kg(-1) x min(-1)) directly into the renal medulla, and 3) SS-13(BN) fed high salt (4%) and infused with saline or H2O2 (347 nmol x kg(-1) x min(-1)) into the renal medullary interstitium. After chronic blood pressure measurements, renal medullary interstitial H2O2 concentration ([H2O2]) was collected by microdialysis and analyzed with Amplex red. Blood pressure and [H2O2] were both significantly higher in SS (126 +/- 3 mmHg and 145 +/- 17 nM, respectively) vs. SS-13(BN) rats (116 +/- 2 mmHg and 56 +/- 14 nM) fed a 0.4% diet. Renal interstitial catalase infusion significantly decreased [H2O2] (96 +/- 41 vs. 297 +/- 52 nM) and attenuated the hypertension (146 +/- 2 mmHg catalase vs. 163 +/- 4 mmHg saline) in SS rats after 5 days of high salt (4%). H2O2 infused into the renal medulla of consomic SS-13(BN) fed high salt (4%) for 7 days accentuated the salt sensitivity (145 +/- 2 mmHg H2O2 vs. 134 +/- 1 mmHg saline). [H2O2] was also increased in the treated group (83 +/- 1 nM H2O2 vs. 44 +/- 9 nM saline). These data show that medullary production of H2O2 may contribute to salt-induced hypertension in SS rats and that chromosome 13 of the Brown Norway contains gene(s) that protect against renal medullary oxidant stress.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

Protection of human endothelial cells from oxidant injury by adenovirus-mediated transfer of the human catalase cDNA.

In a variety of disorders, endothelial cells are exposed to high levels of oxidants, generated within the cells and/or consequent to local inflammation. In the context of the sensitivity of endothelial cells to oxidant stress, particularly related to H2O2, we have designed a replication deficient recombinant adenovirus containing the human catalase cDNA (AdCL) to transfer the catalase cDNA to the endothelial cells, in order to augment intracellular anti-H2O2 protection. Human umbilical vein endothelial cells that were not infected or infected with control adenovirus maintained low levels of catalase mRNA. Endothelial cells infected with AdCL expressed AdCL-driven exogenous catalase mRNA, as early as 24 hr and at least for 7 days. Catalase protein levels were increased significantly over controls in cells infected with AdCL, as were catalase activity levels, with catalase activity correlated closely with levels of catalase protein. Importantly, when the endothelial cells were exposed to 500 microM H2O2, all the AdCL infected endothelial cells survived, compared to only 37% of the control cells. Thus, a recombinant adenovirus containing the human catalase cDNA is able to infect human endothelial cells in vitro and express high levels of functional intracellular catalase, protecting the cells against H2O2-mediated oxidant stress. These observations support the feasibility of the transfer of catalase cDNA to human endothelium to protect against oxidant injury.     

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.     

Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals.

Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.     

Apoptosis in arsenic trioxide-treated Calu-6 lung cells is correlated with the depletion of GSH levels rather than the changes of ROS levels

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We investigated an involvement of ROS such as H(2)O(2) and O(2)(*-), and GSH in ATO-treated Calu-6 cell death. The levels of intracellular H(2)O(2) were decreased in ATO-treated Calu-6 cells at 72 h. However, the levels of O(2)(*-) were significantly increased. ATO reduced the intracellular GSH content. Many of the cells having depleted GSH contents were dead, as evidenced by the propidium iodine staining. The activity of CuZn-SOD was strongly down-regulated by ATO at 72 h while the activity of Mn-SOD was weakly up-regulated. The activity of catalase was decreased by ATO. ROS scavengers, Tiron and Trimetazidine did not reduce levels of apoptosis and intracellular O(2)(*-) in ATO-treated Calu-6 cells. Tempol showing a decrease in intracellular O(2)(*-) levels reduced the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment with NAC showing the recovery of GSH depletion and the decreased effect on O(2)(*-) levels in ATO-treated cells significantly inhibited apoptosis. In addition, BSO significantly increased the depletion of GSH content and apoptosis in ATO-treated cells. Treatment with SOD and catalase significantly reduced the levels of O(2)(*-) levels in ATO-treated cells, but did not inhibit apoptosis along with non-effect on the recovery of GSH depletion. Taken together, our results suggest that ATO induces apoptosis in Calu-6 cells via the depletion of the intracellular GSH contents rather than the changes of ROS levels.     

Hypoxia increases glutathione redox cycle and protects rat lungs against oxidants

Preexposure to hypoxia increased survival and lung reduced glutathione-to-oxidized glutathione ratios (GSH/GSSG) and decreased pleural effusions in rats subsequently exposed to continuous hyperoxia. In addition, lungs from hypoxia-preexposed rats developed less acute edematous injury (decreased lung weight gains and lung lavage albumin concentrations) than lungs from normoxia-preexposed rats when isolated and perfused with hydrogen peroxide (H2O2) generated by xanthine oxidase (XO) or glucose oxidase (GO). In contrast, when perfused with elastase or exposed to a hydrostatic left atrial pressure challenge, lungs isolated from hypoxia-preexposed rats developed the same acute edematous injury as lungs from normoxia-preexposed rats. The mechanism by which hypoxia preexposure conferred protection against H2O2 appeared to depend on hexose monophosphate shunt (HMPS)-dependent increases in lung glutathione redox cycle activity. First, before perfusion with GO, lungs from hypoxia-preexposed rats had increased glutathione peroxidase and glucose 6-phosphate dehydrogenase (but not catalase or glutathione reductase) activities compared with lungs from normoxia-preexposed rats. Second, after perfusion with GO, lungs from hypoxia-preexposed rats had increased H2O2 reducing equivalents, as reflected by increased GSH/GSSG and NADPH/NADPH+, compared with lungs from normoxia-preexposed rats. Third, pretreatment of rats with an HMPS inhibitor, (6-aminonicotinamide) or a glutathione reductase inhibitor, [1,3-bis(2-chloroethyl)-1-nitrosourea] prevented hypoxia-conferred protection against H2O2-mediated acute edematous injury in isolated lungs. These findings suggest that increased detoxification of H2O2 by glutathione redox cycle and HMPS-dependent mechanisms contributes to tolerance to hyperoxia and resistance to H2O2 of lungs from hypoxia-preexposed rats.     

The role of oxidant injury in the pathophysiology of human thalassemias

The anemia in beta-thalassemia major is caused by a combination of hemolysis and ineffective erythropoiesis, with the latter being more important. Studies of the underlying cause of the hemolysis have indicated that oxidant injury to circulating red blood cells (RBCs) was of critical importance, with evidence of oxidant damage to RBC membrane proteins 4.1 and band 3. Therefore, it seemed reasonable that oxidant damage to thalassemic erythroid precursors would cause their accelerated apoptosis and ineffective erythropoiesis. However, direct analysis showed that the apoptotic programs turned on in thalassemics were not those triggered by oxidative damage but were dependent on activation of FAS/FAS-Ligand interaction. Thus, destruction of thalassemic erythroid precursors may involve different mechanisms from those that cause RBC hemolysis.     

nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis

We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)), induces apoptosis of human airway epithelial (HAE) cells. Under oxidant exposure, a lung sphingomyelinase (SMase) is activated and displays continued ceramide generation and pro-apoptotic signaling, thus leading to the pathological apoptosis that causes lung injury. In a search for a specific SMase that is modulated by oxidative stress, we recently cloned nSMase2 from monkey lung tissue and HAE cells. Here, we show that this nSMase2 is up-regulated by an oxidant (H(2)O(2)) and is inhibited by an antioxidant (glutathione (GSH)). Moreover, nSMase2 subcellular localization is governed by oxidant exposure, which leads to its preferential trafficking to the plasma membrane, where it generates ceramide and induces apoptosis. On the other hand, exposure to GSH results in nSMase2 trafficking to the nucleus, where it neither generates ceramide nor induces apoptosis.     

A superoxide anion generator, pyrogallol induces apoptosis in As4.1 cells through the depletion of intracellular GSH content

We investigated the involvement of ROS such as H2O2 and O2*-, and GSH in As4.1 cell death induced by pyrogallol. The intracellular H2O2 levels were decreased or increased depending on the concentration and incubation time of pyrogallol. The levels of O2*- were significantly increased. Pyrogallol reduced the intracellular GSH content. And ROS scavengers, Tempol, Tiron, Trimetazidine and NAC could not significantly down-regulate the production of H2O2 and O2*-. However, these ROS scavengers slightly inhibited apoptosis. Interestingly, Tempol showing the recovery of GSH depletion induced by pyrogallol significantly decreased apoptosis without the significant reduction of intracellular O2*- levels. SOD and catalase did not change the level of H2O2 but decreased the level of O2*-. The inhibition of GSH depletion by these was accompanied with the decrease of apoptosis, as evidenced by sub-G1 DNA content, annexin V staining, mitochondria membrane potential (DeltaPsi(m)) and Western data. In addition, ROS scavengers and SOD did not alter a G2 phase accumulation of the cell cycle induced by pyrogallol. However, catalase changed the cell cycle distributions of pyrogallol-treated cells to those of pyrogallol-untreated cells. In summary, we have demonstrated that pyrogallol potently generates ROS, especially O2*-, in As4.1 JG cells, and Tempol, SOD and catalase could rescue to a lesser or greater extent cells from pyrogallol-induced apoptosis through the up-regulation of intracellular GSH content.