Effect of vitamin E deficiency and supercritical fluid aerosolized vitamin E supplementation on interleukin-1-induced oxidative lung injury in rats.

We hypothesized that alterations in lung vitamin E levels would impact the development of acute oxidative lung injury. We found that dietary induced deficiency of vitamin E diminished lung tissue levels of vitamin E and increased lung leak following intratracheal administration of interleukin-1 (IL-1) to rats. Conversely, rats administered vitamin E directly to the lungs as an inhaled aerosol (0.3-3 microns particles) formed by supercritical fluid aerosolization (SFA) had increased lung tissue vitamin E levels and decreased IL-1 induced lung leak compared to control rats. Lung myeloperoxidase (MPO) activities, reflecting neutrophil concentrations, were increased in rats given IL-1 intratracheally compared to rats given saline intratracheally but were not different for control or vitamin E depleted rats. Lung MPO activities in rats given IL-1 intratracheally were slightly higher in SFA vitamin E treated rats than in control rats. Our results suggest that vitamin E levels affect susceptibility to IL-1 induced, neutrophil-dependent lung injury. We speculate that supercritical fluid aerosol (SFA) delivery of vitamin E can rapidly increase lung vitamin E levels and decrease acute oxidative lung injury.

Xanthine oxidase promotes neutrophil sequestration but not injury in hyperoxic lungs.

Neutrophil accumulation in alveolar spaces is a conspicuous finding in hyperoxia-exposed lungs. We hypothesized that xanthine oxidase (XO)-derived oxidants contribute to retention of neutrophils in hyperoxic lungs. Rats were subjected to normobaric hyperoxia (100% O2) for 48 h, and lungs were assessed for neutrophil sequestration (morphometry and lavage cell counts) and injury (lavage albumin levels and lung weights). In rats exposed to hyperoxia, we found increased (P < 0.05) lung neutrophil retention, lavage albumin levels, and lung weights compared with normoxia-exposed control rats. Suppression of XO activity by pretreatment with allopurinol decreased (P < 0.05) lung neutrophil retention but increased (P < 0.05) lavage albumin concentrations and lung weights in hyperoxic rats. Allopurinol treatment had no effect (P > 0.05) on the numbers of macrophages or lymphocytes recoverable by lung lavage. Depletion of XO activity by an independent method, tungsten feeding, also decreased (P < 0.05) lung lavage neutrophil counts and increased (P < 0.05) lavage albumin concentrations. We conclude that XO may be involved in lung neutrophil retention but not lung injury during exposure to hyperoxia.

Interleukin-1-induced lung neutrophil accumulation and oxygen metabolite-mediated lung leak in rats.

We found that intratracheal administration of recombinant interleukin-1 alpha (IL-1) into rats rapidly (< 5 h) increased neutrophils in lung lavages and caused an acute edematous lung injury which was reflected by lung albumin accumulation (lung leak) and histological abnormalities (perivascular cuffing). These IL-1-dependent processes were inhibited by prior administration of recombinant IL-1 receptor antagonist and did not occur following administration of heated IL-1. Several lines of evidence suggested that neutrophil-derived oxygen metabolites contributed to lung leak. First, lung leak did not occur in rats rendered neutropenic by vinblastine treatment 4 days before IL-1 administration but did occur in neutrophil-replete rats given vinblastine 1 day before IL-1 administration and control rats given IL-1. Second, treatment with a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO) or a superoxide anion scavenger, manganese superoxide dismutase, decreased lung leak, lung lavage neutrophils, and histological abnormalities in rats given IL-1 intratracheally. Third, intratracheal IL-1 administration increased lung oxidized glutathione (GSSG) levels and expired H2O2 concentrations, and these two indices of oxidative stress were decreased by dimethyl sulfoxide or manganese superoxide dismutase treatment. We conclude that intratracheal administration of IL-1 increases neutrophils in the lung and causes a neutrophil and oxygen metabolite-dependent acute edematous lung injury.

Toxic effects of dimethylthiourea in rats.

Dimethylthiourea (DMTU) is a small, highly diffusible molecule that effectively scavenges toxic oxygen metabolites in vitro and reduces oxidative injury in many biologic systems. Nonetheless, for unknown reasons, DMTU has occasionally failed to decrease damage in some systems where injury is presumed to be mediated by oxygen metabolites. We hypothesized that the inconsistent pattern of protection might partially reflect a direct toxicity of DMTU. Our results supported this premise. We found that rats treated with commonly used doses of highly purified DMTU had increased lung accumulation of intravenously injected iodine 125-labeled albumin (4 hours after DMTU treatment) and decreased blood glutathione levels (24 hours after DMTU treatment) when compared with saline-injected control rats. In contrast, rats treated with dimethylurea, a analog of DMTU, did not develop increased accumulation of labeled albumin in the lungs or decreased blood glutathione levels. We conclude that DMTU has intrinsically toxic effects in rats and that DMTU toxicity may at times obscure its protective action.

Postinsult treatment with N-acetyl-L-cysteine decreases IL-1-induced neutrophil influx and lung leak in rats.

We found that intratracheal administration of interleukin-1 alpha (IL-1) rapidly (5 h) increased leak of 125I-labeled albumin from the blood into the lung (lung leak), influx of neutrophils into lung lavages, lung oxidized glutathione (GSSG) levels, breath hydrogen peroxide (H2O2) concentrations, and lung histological abnormalities in intact rats. Since N-acetyl-L-cysteine (NAC) increases glutathione (GSH) levels in vivo and scavenges oxygen radicals in vitro, we tested the effect of NAC given intravenously on lung changes following intratracheal IL-1 administration. We found that administration of NAC immediately before or 2.5 h after intratracheal administration of IL-1 decreased lung leak, neutrophil influx into lung lavages, and defects in lung histology. NAC treatment also increased blood acid soluble sulfhydryl levels, reduced lung GSSG increases, and decreased breath H2O2 levels in rats given IL-1 intratracheally. The latter findings are consistent with the possibility that NAC is enhancing GSH or other sulfhydryls and, as a result, reducing oxidative stress due to H2O2 or H2O2-derived products. Since postinsult treatment with NAC is effective in this relevant intact animal model of acute lung injury, we speculate that NAC may have promise in the treatment of patients with the adult respiratory distress syndrome.

Effects of dimethylthiourea in hyperoxic injury.

Pretreatment with a single dose of the oxygen metabolite scavenger 1,3-dimethyl-2-thiourea (DMTU) decreased hyperoxia-induced injury (as assessed by measurement of pleural effusions and increases in hematocrits and blood acid-soluble sulfhydryl levels) in rats that were exposed to hyperoxia for 48 hours. However, the degree of protection was not proportional to DMTU dose. An intermediate dose of DMTU (250 mg/kg) reduced injury more than a lower dose of 125 mg/kg and at least as effectively as the higher, widely used dose of 500 mg/kg DMTU. In contrast to its protective action with respect to hyperoxic injury, none of the doses of DMTU that were tested decreased the elevations in lung oxidized glutathione levels or oxidized glutathione/reduced glutathione ratios associated with hyperoxia exposure. These findings indicate that maximal protection from hyperoxic injury may be achieved with doses of DMTU that are lower than the doses used routinely. The failure of DMTU to decrease lung oxidized glutathione and lung oxidized glutathione/reduced glutathione ratio increases after hyperoxia exposure suggests that the mechanism by which DMTU confers protection requires careful evaluation.

Human phagocytic cells as oxygen metabolite scavengers.

Human neutrophils or monocytes decreased hydrogen peroxide (H2O2) concentrations in vitro. Neutrophils or monocytes decreased H2O2 concentrations as well as human erythrocytes. Treatment with aminotriazole or azide decreased both phagocyte and erythrocyte catalase activity and the ability of each cell to decrease H2O2 concentrations in vitro. Prestimulation of phagocytic cells with phorbol myristate acetate (PMA) or opsonized zymosan decreased neither their catalase activity nor their ability to decrease H2O2 concentrations. The results suggest that unstimulated or stimulated phagocytic cells can scavenge H2O2 and may potentially decrease H2O2-mediated tissue injury. The H2O2 scavenging potential of phagocytic cells is due at least partially to their catalase activity.

Xanthine oxidase is increased and contributes to paraquat-induced acute lung injury.

Two lines of investigation suggested that xanthine oxidase- (XO) derived O2 metabolites contribute to paraquat- (PQ) induced acute lung injury. First, PQ treatment increased lung XO activity and decreased lung xanthine dehydrogenase activity. Second, lung albumin uptake increased compared with control values in untreated XO-replete but not tungsten-treated XO-depleted lungs in rats treated with PQ.

Albumin decreases hydrogen peroxide and reperfusion injury in isolated rat hearts.

Perfusion with human serum albumin decreased myocardial hydrogen peroxide (H2O2) levels (as assessed by inactivation of myocardial catalase activities following aminotriazole pretreatment) and increased myocardial ventricular developed pressures (DP), contractility (+dP/dt) but not relaxation rate (-dP/dt) in isolated crystalloid perfused rat hearts subjected to normothermic global ischemia (20 min) and then reperfusion (40 min). Albumin also decreased H2O2 concentrations in vitro. The findings support the possibility that albumin may act as a protective O2 metabolite scavenger in vivo.

Blood sulfhydryl level increases during hyperoxia: a marker of oxidant lung injury.

Blood acid-soluble sulfhydryl, but not glutathione (GSH), levels increased during the development of acute edematous lung injury in rats exposed to normobaric hyperoxia for 48 h or more. A relationship between increases in blood sulfhydryl levels, lung injury, and O2 metabolite generation during exposure to hyperoxia was suggested by two observations. First, increases in blood sulfhydryl levels occurred simultaneously with increases in lung oxidized glutathione (GSSG) levels and lung GSSG-to-GSH ratios (GSSG/GSH). Second, hyperoxia-induced increases in blood sulfhydryl levels, blood hematocrits, pleural effusion volumes, lung GSSG levels, and lung GSSG/GSH were decreased by pretreating rats with dimethylthiourea (DMTU), an O2 metabolite scavenger. Our findings indicate that exposure of rats to hyperoxia increases blood acid-soluble sulfhydryl levels in vivo and that increases in blood sulfhydryl levels may provide an accessible marker of increased oxidant exposure and/or oxidant-mediated lung injury.

Erythrocytes decrease myocardial hydrogen peroxide levels and reperfusion injury.

Reperfusion with untreated, carbon monoxide-treated, or glutaraldehyde-fixed human erythrocytes (RBC) increased ventricular function and decreased myocardial hydrogen peroxide (H2O2) levels [assessed by H2O2-dependent aminotriazole (AMT) inactivation of myocardial catalase activities] of ischemic, isolated rat hearts. In contrast, reperfusion with RBC that lacked catalase (AMT treated) and/or glutathione (N-ethylmaleimide treated) did not increase ventricular function or decrease myocardial H2O2 levels as much as reperfusion with untreated RBC. By comparison, reperfusion with superoxide dismutase-depleted (diethyldithiocarbamate-treated) or anion channel-inhibited (diisothiocyanodisulfonic acid stilbene-treated) RBC increased ventricular function and decreased myocardial H2O2 levels the same as untreated RBC. The results suggest that catalase and/or glutathione in intact RBC can decrease endogenously generated H2O2 and related reperfusion injury in ischemic, isolated perfused hearts.

Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process.

Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro. DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro. By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro. Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury. DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes. Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells. In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO. Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions. In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers. Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.

Hyperoxia and self- or neutrophil-generated O2 metabolites inactivate xanthine oxidase.

Xanthine oxidase (XO) and xanthine dehydrogenase (XD) activities decreased in lungs isolated from rats and cultured lung endothelial cells that had been exposed to hyperoxia. Purified XO activity also decreased after addition of a variety of chemically generated O2 metabolite species (superoxide anion, hydrogen peroxide, hydroxyl radical, or hypochlorous acid), hypoxanthine, or stimulated neutrophils in vitro. XO inactivation by chemically, self-, or neutrophil-generated O2 metabolites was decreased by simultaneous addition of various O2 metabolite scavengers but not their inactive analogues. Since XO appears to contribute to a variety of biological processes and diseases, hyperoxia- or O2 metabolite-mediated decreases in XO activity may be an important cellular control mechanism.

Persistent bactericidal defect in neutrophils from a young woman who recovered from toxic shock syndrome.

We have previously found transient menstruation-associated abnormalities in the in vitro bactericidal function of neutrophils from females who have recovered from toxic shock syndrome (TSS). We now report the case of a young woman who has also recovered from TSS, but who has a persistent, non-menstruation-associated defect in the ability of her neutrophils to kill Staphylococcus aureus in vitro.

Erythrocytes from cigarette smokers contain more glutathione and catalase and protect endothelial cells from hydrogen peroxide better than do erythrocytes from nonsmokers.

Recent observations regarding the ability of intracellular erythrocyte (RBC) antioxidants to decrease O2 metabolite-mediated injury to lung tissues has prompted interest in the RBC antioxidants of patients with lung disease. We found that RBC from 14 healthy, age- and gender-matched cigarette smokers contained more (p less than 0.05) glutathione (6.3 +/- 0.4 microM/g Hgb versus 5.0 +/- 0.3 microM/g Hgb) and catalase (249,533 +/- 8,307 units/g Hgb versus 222,617 +/- 7,180 units/g Hgb) than did RBC from nonsmokers. In contrast, RBC from cigarette smokers and nonsmokers contained the same activities of glutathione peroxidase (21.4 +/- 1.2 units/g Hgb versus 20.4 +/- 5.5 units/g Hgb). RBC from cigarette smokers also protected bovine pulmonary artery endothelial cells in culture from hydrogen peroxide (H2O2) better (p less than 0.005) than did RBC from nonsmokers (52.1 +/- 6.1% protection versus 31.9 +/- 5.7% protection). The results suggest that alterations in RBC antioxidants may reflect exposure and/or affect susceptibility to oxidant-induced injury.