A single exposure to hyperbaric oxygen does not cause oxidative stress in isolated platelets: no effect on superoxide dismutase, catalase, or cellular ATP

OBJECTIVES: The aim of the study was to investigate whether a single hyperbaric oxygen exposure causes oxidative stress in isolated platelets. DESIGN AND METHODS: Isolated horse platelets were exposed to 100% oxygen at 2.2 atmospheres, or 100% oxygen under normobaric conditions, or air under normobaric conditions for 90 min. RESULTS: There were no differences in platelet SOD activity between conditions, but there was a rise in SOD in all cases after 24 h (in control platelets at 24 h, SOD was 11.9 +/- 1.9 nmol/min/mg protein compared to initial background levels of 8.2 +/- 1.9 nmol/min/mg protein) (P < 0.05). Neither platelet catalase activity nor platelet GSH concentration changed over time, nor between conditions (catalase activity remained at around 12 units/mg protein, and GSH at around 1.58 nmol/mg protein). CONCLUSIONS: These data suggest that a single HBO exposure has no detrimental effect on platelet biochemistry, and does not cause overt oxidative stress in vitro.     

Reactive oxygen species during ischemia-reflow injury in isolated perfused rat liver.

The hypothesis that intracellular generation of reactive oxygen species in hepatocytes or reticuloendothelial cells may cause ischemia-reperfusion injury was tested in isolated perfused livers of male Fischer rats. GSSG was measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 30 min, the perfusion was stopped (global ischemia) for various times (30, 120 min) and the liver was reperfused for another 60 min. The bile flow (1.48 +/- 0.17 microliters/min X gram liver weight), the biliary efflux of total glutathione (6.54 +/- 0.94 nmol GSH eq/min X g), and GSSG (1.59 +/- 0.23 nmol GSH eq/min X g) recovered to 69-86% after short-term ischemia and to 36-72% after 2 h of ischemia when compared with values obtained from control livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4 +/- 2.1 nmol GSH eq/min X g) and GSSG (0.13 +/- 0.05 nmol GSH eq/min X g) did not change except for an initial 10-30-s increase during reperfusion washout. No increased GSSG secretion into bile was detectable at any time during reperfusion. The liver content of total glutathione (32.5 +/- 3.5 nmol GSH eq/mg protein) and GSSG (0.27 +/- 0.09 nmol GSH eq/mg protein) did not change significantly during any period of ischemia or reperfusion. We conclude, therefore, that at most only a minor amount of reactive oxygen species were generated during reperfusion. Thus, reactive oxygen species are unlikely to cause ischemia/reperfusion injury in rat liver by lipid peroxidation or tissue thiol oxidation.     

Glutathione, glutathione S-transferase, and transmembrane transport of glutathione conjugate in human neutrophil leukocytes

Glutathione (GSH) conjugation is important in the detoxification of carcinogens and other exogenous electrophilic drugs and chemicals. The conjugation is catalyzed by GSH S-transferase. Neutrophil GSH content was 12.3 +/- 2.5 (mean +/- SD) nmol/10(7) cells, and transferase activity of cytosol preparations was 0.187 +/- 0.035 nmol/min/mg protein with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Maximal activity was found at a pH of 7.0 and a temperature of 40 degrees C. Apparent Km of transferase was 1.25 +/- 0.18 mmol/L CDNB, and apparent Vmax was 0.621 +/- 0.22 nmol/min/mg. GSH-CDNB conjugate was quantitated by HPLC in cells and in medium after CDNB exposure. Transport of conjugate from cells to medium increased with CDNB concentration to 50 mumol/L, and kinetic data showed two saturable transport mechanisms with apparent Km of 5.90 mumol/L and 0.265 mumol/L, respectively. Cellular GSH content fell rapidly with CDNB concentration greater than 2.5 mumol/L, and was depleted in a 10-minute incubation at a concentration of greater than 50 mumol/L CDNB. Neutrophils have a significant content of GSH and a significant amount of transferase activity, and transport of GSH conjugate involves two distinct saturable pathways. GSH depletion can be accomplished with a relatively low concentration of an exogenous chemical and could impair the ability of the cells to carry out their phagocytic functions.     

Effects of Enrofloxacin on Porcine Phagocytic Function

The interaction between enrofloxacin and porcine phagocytes was studied with clinically relevant concentrations of enrofloxacin. Enrofloxacin accumulated in phagocytes, with cellular concentration/extracellular concentration ratios of 9 for polymorphonuclear leukocytes (PMNs) and 5 for alveolar macrophages (AMs). Cells with accumulated enrofloxacin brought into enrofloxacin-free medium released approximately 80% (AMs) to 90% (PMNs) of their enrofloxacin within the first 10 min, after which no further release was seen. Enrofloxacin affected neither the viability of PMNs and AMs nor the chemotaxis of PMNs at concentrations ranging from 0 to 10 microg/ml. Enrofloxacin (0.5 microg/ml) did not alter the capability of PMNs and AMs to phagocytize fluorescent microparticles or Actinobacillus pleuropneumoniae, Pasteurella multocida, and Staphylococcus aureus. Significant differences in intracellular killing were seen with enrofloxacin at 5x the MIC compared with that for controls not treated with enrofloxacin. PMNs killed all S. aureus isolates in 3 h with or without enrofloxacin. Intracellular S. aureus isolates in AMs were less susceptible than extracellular S. aureus isolates to the bactericidal effect of enrofloxacin. P. multocida was not phagocytosed by PMNs. AMs did not kill P. multocida, and similar intra- and extracellular reductions of P. multocida isolates by enrofloxacin were found. Intraphagocytic killing of A. pleuropneumoniae was significantly enhanced by enrofloxacin at 5x the MIC in both PMNs and AMs. AMs are very susceptible to the A. pleuropneumoniae cytotoxin. This suggests that in serologically naive pigs the enhancing effect of enrofloxacin on the bactericidal action of PMNs may have clinical relevance.     

Mechanism of pH amelioration of 2-bromohydroquinone-induced toxicity to rabbit renal proximal tubules

The basis of extracellular acidosis amelioration of 2-bromohydroquinone (BHQ)-induced renal proximal tubular cell death was determined by comparing the metabolism, uptake and mitochondrial effects of BHQ (0.2 mM) and bromoquinone (BQ) (0.05 mM) on isolated rabbit renal proximal tubules incubated in pH 7.4 and pH 6.4 buffers. Exposure of proximal tubules in pH 7.4 buffer to [14C]BHQ resulted in a time-dependent increase in covalently bound BHQ-equivalents to tubular protein (9 +/- 1 nmol/mg of protein at 1 hr) and a decrease in nystatin-stimulated oxygen consumption (NYS-QO2). In comparison, covalently bound BHQ-equivalents were 0.7 nmol/mg of protein and NYS-QO2 was unaffected in proximal tubules incubated at pH 6.4 for 1 hr. After a 1-hr exposure, tubular content of [14C]BHQ-equivalents was 15 +/- 2 and 9 +/- 1 nmol/mg of protein in tubules incubated at pH 7.4 and 6.4, respectively. Thus, decreased covalent binding of BHQ-equivalents in proximal tubules incubated at pH 6.4 could not be accounted for by limited uptake of BHQ. The lactate dehydrogenase release induced by 0.05 mM BQ was decreased by acidic pH. Similarly, BQ induced an 85% decrease in NYS-QO2 of proximal tubules in pH 7.4 buffer, compared to a 55% inhibition when proximal tubules were incubated at pH 6.4 for 4 hr. Thus, extracellular acidosis ameliorates BHQ toxicity by altering BHQ biotransformation; that is, extracellular acidosis inhibits the oxidation of BHQ to BQ and may promote the reduction of BQ to BHQ.     

Serum from healthy pregnant women reduces oxidative stress in human umbilical vein endothelial cells

This study was conducted to compare the effects of serum from healthy pregnant women and that from pregnant women with pre-eclampsia on oxidative stress in endothelial cells in culture. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from 18 pre-eclamptic, 18 healthy pregnant and 18 healthy non-pregnant women for 24 h. The levels of reduced glutathione (GSH) and lipid peroxides (LPOs) were measured in endothelial cell lysates. Measurement of malondialdehyde in combination with 4-hydroxyalkenals has been used as an indicator of LPOs. Serum from healthy pregnant women decreased significantly the LPO content in HUVECs in comparison with serum from pre-eclamptic women and healthy non-pregnant women (30.7+/-6.6 compared with 39.3+/-10.9 and 41.0+/-12.7 pmol/mg of protein respectively; P<0.003 and P<0.01 respectively). No differences in GSH content between the three groups (18.3+/-2.1 nmol/mg of protein for healthy pregnant, 19.2+/-3.3 nmol/mg for pre-eclamptic and 18.3+/-2.0 nmol/mg for healthy non-pregnant women) were found. Thus serum from normal pregnant women contains a factor(s) that decreases oxidative stress in human endothelial cells. This mechanism might be altered in pre-eclampsia.     

Influence of cigarette smoking on crocidolite-induced ferritin release by human alveolar macrophages

Alveolar macrophages (AMs) mobilize iron from the surface of iron-containing minerals such as asbestos and synthesize ferritin for intracellular iron storage or secretion. Although the synthesis of iron-free ferritin (apoferritin) provides antioxidant protection, the secretion of iron-containing ferritin by AMs could increase the availability of catalytic iron in the lungs. Cigarette smoking may promote the secretion of ferritin by AMs after iron acquisition from mineral sources, because smokers' AMs are iron loaded. The first objective of this study was to determine whether ferritin secretion/release by AMs after in vitro exposure to crocidolite asbestos is enhanced by cigarette smoking. The second objective was to assess whether exogenous ferritin-bound iron could enhance the toxicity of crocidolite to lung cells in vitro. AMs recovered from nonsmokers (n = 8) or smokers (n = 8) were exposed to crocidolite or titanium dioxide (TiO2)(1 x 10(6) AMs, 50 to 200 microg/mL) for up to 18 hours. AMs exposed to crocidolite but not TiO2 showed increased cell content of iron and ferritin and increased cell supernatant ferritin concentrations. Increases in iron and ferritin content were similar for AMs recovered from smokers and those recovered from nonsmokers; however, increases in supernatant ferritin were >7-fold greater for smokers' AMs than for nonsmokers' AMs (P < .001). Exposure of A549 cells, a lung cancer-derived cell line, to crocidolite (50 to 200 microg/mL, 18 hours) caused dose-dependent cell death as indicated by lactate dehydrogenase release. The addition of ferritin (> or = 500 mg/mL) but not apoferritin to culture media enhanced crocidolite-induced LDH release (P < .01). These findings suggest that cigarette smoking and crocidolite exposure have synergistic effects that promote ferritin release by AMs, which could catalyze oxidative injury to other alveolar cells.     

Pulmonary contusion induces alveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neutrophils

Alveolar type 2 (AT-2) cell apoptosis is an important mechanism during lung inflammation, lung injury, and regeneration. Blunt chest trauma has been shown to activate inflammatory cells such as alveolar macrophages (AMs) or neutrophils (polymorphonuclear granulocytes [PMNs]), resulting in an inflammatory response. The present study was performed to determine the capacity of different components/cells of the alveolar compartment (AMs, PMNs, or bronchoalveolar lavage [BAL] fluids) to induce apoptosis in AT-2 cells following blunt chest trauma. To study this, male Sprague-Dawley rats were subjected to either sham procedure or blunt chest trauma induced by a single blast wave. Various time points after injury (6 h to 7 d), the lungs were analyzed by immunohistochemistry, for AT-2 cells, or with antibodies directed against caspase 3, caspase 8, Fas, Fas ligand (FasL), BAX, and BCL-2. Bronchoalveolar lavage concentrations of TNF-alpha, IL-1beta, and soluble FasL were determined by enzyme-linked immunosorbent assay. Furthermore, cultures of AT-2 cells isolated from healthy rats were incubated with supernatants of AMs, PMNs, or BAL fluids obtained from either trauma or sham-operated animals in the presence or absence of oxidative stress. Annexin V staining or TUNEL (terminal deoxynucleotidyl transferase) assay was used to detect apoptotic AT-2 cells. Histological evaluation revealed that the total number of AT-2 cells was significantly reduced at 48 h following trauma. Fas, FasL, active caspase 8, and active caspase 3 were markedly up-regulated in AT-2 cells after chest trauma. BAX and BCL-2 did not show any significant changes between sham and trauma. IL-1beta, but not TNF-alpha, levels were markedly increased at 24 h after the injury, and soluble FasL concentrations were significantly enhanced at 6, 12, 24, and 48 h after the insult. Apoptosis of AT-2 cells incubated with supernatants from cultured AMs, isolated at 48 h following chest trauma was markedly increased when compared with shams. In contrast, no apoptosis was induced in AT-2 cells incubated with supernatants of activated PMNs or BAL fluids of traumatized animals. In summary, blunt chest trauma induced apoptosis in AT-2 cells, possibly involving the extrinsic death receptor pathway. Furthermore, mediators released by AMs appeared to be involved in the induction of AT-2 cell apoptosis.     

Hyperoxia prolongs the aminoglycoside-induced postantibiotic effect in Pseudomonas aeruginosa.

The objective of this study was to determine whether hyperoxia enhances aminoglycoside activity against Pseudomonas aeruginosa. The existence of tobramycin-oxygen synergy was determined by using the in vitro postantibiotic effect (PAE). P. aeruginosa strains were incubated for 1 h in medium containing tobramycin at four times the MIC in the following gas mixtures: normoxia (21% O2), hyperoxia (100% O2, 101.3 kPa), or hyperbaric oxygen (100% O2, 274.5 kPa). Tobramycin was removed after 1 h and bacteria were incubated under normoxic conditions; growth rates were measured for 5 h. Exposure of three P. aeruginosa strains to hyperoxia prolonged the PAE of tobramycin approximately twofold compared with the PAE after exposure to normoxia (P less than 0.05). Exposure of P. aeruginosa ATCC 27853 to tobramycin and hyperbaric oxygen prolonged the time required for bacteria to increase 1 log10 CFU/ml compared with the time after exposure for this increase to occur in tobramycin-treated, normoxic or hyperoxic groups (P less than 0.02). Pulse-chase labeling of bacteria with L-[35S]methionine, immediately after removal of tobramycin, showed that protein synthesis rates were decreased compared with those in controls (P = 0.0001). Moreover, in tobramycin-treated groups, hyperoxia and hyperbaric oxygen induced 2- and 16-fold decreases, respectively, in protein synthesis rates compared with normoxia; these results did not achieve statistical significance. In the absence of tobramycin, hyperoxia increased bacterial growth (134%; P less than 0.01) and protein synthesis (24%; not significant) compared with normoxia. Hyperbaric oxygen, however, delayed the growth recovery of bacteria (P less than 0.05). We conclude that hyperoxia enhances the bacteriostatic effects of tobramycin in a synergistic manner.+     

短时间高氧对肺泡Ⅱ型上皮细胞线粒体活性氧产生及相关通路的影响

目的探讨短时间高氧对肺泡Ⅱ型上皮细胞（AECⅡ）线粒体Ca²⁺ /烟酰胺腺嘌呤二核苷酸（NAD⁺）/沉默信息调节因子3（SIRT3）/超氧化物歧化酶2（SOD2）通路及活性氧的影响。 方法将RLE-6TN细胞株细胞分为对照组、高氧组及线粒体钙通道拮抗剂组（拮抗剂组）。对照组细胞置于常规细胞培养箱中，高氧组细胞置于氧浓度为90%的培养箱中，拮抗剂组细胞加入钌红（2 μmol / L）后置于氧浓度为90%的培养箱中，各组均持续培养4 h。随后，对各组细胞线粒体内Ca²⁺、活性氧、NAD⁺、还原型烟酰胺腺嘌呤二核苷酸（NADH）含量进行检测，并计算NAD⁺ / NADH比值；同时，采用实时荧光定量PCR检测SIRT3和SOD2信使RNA（mRNA）水平。 结果各组间细胞线粒体内Ca²⁺、活性氧、NAD⁺、NADH、NAD⁺ / NADH比值及SIRT3 mRNA、SOD2 mRNA表达水平的比较，差异均有统计学意义（F = 183.500、135.900、32.140、51.520、128.300、59.970、45.020，P均< 0.001）。且与对照组及拮抗剂组比较，高氧组细胞线粒体内Ca²⁺[（19.5 ± 0.8）、（17.2 ± 0.7）、（24.3 ± 0.3）nmol / L]、活性氧[（491 ± 9）、（480 ± 5）、（530 ± 6）相对荧光单位]及NADH[（0.85 ± 0.03）、（0.87 ± 0.04）、（1.06 ± 0.06）nmol / 10⁴ cells]含量均明显升高，而NAD⁺含量[（3.30 ± 0.12）、（3.24 ± 0.14）、（2.58 ± 0.29）nmol / 10⁴ cells]、NAD⁺ / NADH比值[（3.89 ± 0.15）、（3.71 ± 0.15）、（2.44 ± 0.27）]、SIRT3 mRNA[（1.01 ± 0.11）、（0.96 ± 0.08）、（0.45 ± 0.09）]及SOD2 mRNA[（1.01 ± 0.14）、（1.05 ± 0.11）、（0.48 ± 0.10）]表达水平均显著降低（P均< 0.05）。 结论短时间高氧可通过AECⅡ线粒体内Ca²⁺ / NAD⁺ / SIRT3 / SOD2通路导致活性氧蓄积。     

Hyperoxia- and hypoxia-mediated activation of polymorphonuclear leukocytes: a comparison of cord and adult venous blood.

BACKGROUND: Among the most prominent changes occurring in newborn infants is the exposure of tissues and blood cells to increased oxygen tension. This increase is even more pronounced in neonatal resuscitation using 100% oxygen, currently recommended in the published guidelines. OBJECTIVE: To analyse the response of neonatal and adult polymorphonuclear neutrophils (PMN) to high or low oxygen tension in vitro. MATERIALS: Neonatal cord blood and adult venous blood without previous contact to ambient air was exposed to 0, 21, or 100% oxygen for 30 min followed by incubation for up to 24 h. METHODS: Flow cytometry was used to assess PMN activation as indicated by downregulation of L-selectin expression. Cell viability was quantified by the amount of propidium iodide uptake. RESULTS: In adult PMN, L-selectin downregulation was greatly accelerated by hypoxia (PO2=27.2+/-3.4 mmHg) compared with both normoxia (PO2=71.0+/-11.0 mmHg) or hyperoxia (PO2=653.2+/-9.4) (P<0.05). In contrast, hyperoxia was the most potent stimulus for cord blood PMN, compared with both normoxia and hypoxia (P<0.05). Evidence of necrosis as indicated by positive staining for propidium iodide was similar in cord blood (10 h: 5.83% in oxygen) and in adult blood (10 h: 6.45% in oxygen). No differences were found between exposure to hypoxia, normoxia, or hyperoxia. CONCLUSION: Oxygen exposure of neonatal PMN leads to a more pronounced activation as compared with adult cells. Exposure towards high concentrations of oxygen may contribute to inflammatory processes during early neonatal life.     

Effects of hypoxia and nitric oxide on ferritin content of alveolar cells

Concentrations of ferritin in alveolar cells and on the alveolar surface are increased in patients with a variety of respiratory disorders. Ferritin synthesis by cells is modulated by iron content but is also influenced by stimuli other than iron. In this study we sought to determine whether in vitro exposure to hypoxia- or nitric oxide (NO)-induced ferritin accumulation or release by human alveolar macrophages (AMs) or a lung cancer-derived epithelial cell line (A549). Changes in cell content of iron and ferritin (L- and H-types), as well as ferritin content of cell supernatants, were determined after in vitro exposure to hypoxia (1% or 10% O(2), 18 hours) or the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.01-1.0 mmol/L, 18 hours). Exposure to 1% O(2) increased ferritin content in both cell types (>fourfold increase; P <.005) without changing iron content. Treatment with SNAP increased ferritin content of A549 cells in a dose-dependent manner, whereas treatment of AMs decreased cellular iron and ferritin content and increased supernate ferritin content. Pretreatment of cells with N-acetylcysteine (500 micromol/L) reduced hypoxia-induced ferritin accumulation in alveolar cells and completely inhibited NO-induced ferritin accumulation in A549 cells. These findings indicate that exposure to 1% O(2)can increase ferritin content in alveolar cells, whereas NO can increase ferritin content (A549 cells) or decrease ferritin content (AMs).     

Hyperoxia exposure impairs surfactant function and metabolism

OBJECTIVE: To evaluate the effects of hyperoxia exposure on lung function and the endogenous surfactant system in spontaneously breathing adult rats. DESIGN: Analysis of the pulmonary surfactant system isolated from adult rats following exposure to > 90% oxygen or room air for 48 or 72 hrs. SETTING: A basic science research laboratory in a university setting. SUBJECTS: Sixty pathogen-free Sprague-Dawley rats. INTERVENTIONS: Exposure to > 90% oxygen for 72 hrs. MEASUREMENTS AND MAIN RESULTS: Exposure to > 90% oxygen for 72 hrs resulted in significant lung dysfunction and an increase in neutrophils and total protein concentrations within the airspace compared with animals exposed to room air or 48 hrs of 90% oxygen exposure. Total alveolar surfactant and large aggregate pool sizes were increased after 72 hrs of hyperoxia compared with the other groups, and there was evidence of lipid peroxidation within these large aggregate subtractions. The biophysical function of large aggregate isolated from 72-hr hyperoxia-exposed animals was also impaired and converted into small aggregate forms faster than large aggregate from normoxia-exposed animals when assessed using in vitro surface area cycling techniques. CONCLUSIONS: Oxidative stress on the endogenous surfactant system may represent an important mechanism contributing to the surfactant dysfunction and abnormal surfactant metabolism associated with hyperoxia-induced lung injury.     

Oxidative tissue damage following regional intestinal ischemia and reperfusion in the cat

Regional intestinal ischemia in cats resulted in an accumulation of hypoxanthine within 2 h, the concentration of which rose from 0.062 to 1.131 nmol/mg protein. A similar rise in AMP content (from 0.5 to 3.2 nmol/mg protein) was observed, but not in the ADP level. In parallel, ATP content decreased from 7.5 to 2.8 nmol/mg protein. Reperfusion of the ischemic tissue was followed by rapid metabolism of the purine metabolites; after 1 h of reperfusion the tissue level of hypoxanthine was 0.186 nmol/mg protein, of AMP 0.7 nmol/mg protein, and of ATP 4.3 nmol/mg protein. The oxidation of hypoxanthine, mediated by xanthine oxidase, is accompanied by the release of superoxide ions. Consequently, the concentration of oxidized glutathione was doubled upon reperfusion, while marked lipid peroxidation took place, as evidenced by the rise in conjugated diene content from 2.8 mumol/g tissue before reperfusion to 5.6 mumol/g tissue after 10 min of reoxygenation. In line with these findings is the fact that histologically observable damage occurred mainly in the presence of oxygen. These data indicate that, at least in our model, rapid reoxygenation is a major cause of "ischemic" tissue damage.     

Leptin attenuates oxidative stress and neuronal apoptosis in hyperglycemic condition

One of the main pathological mechanisms of neurotoxicity in diabetic situation is oxidative stress promoted by hyperglycemia. It has been shown that leptin has neuroprotective effects and may provide neuronal survival signals. This study was designed to reveal the possible neuroprotective effects of leptin in hyperglycemic conditions. Pheochromocytoma (PC12) cell viability was assessed via the MTT test. Cellular reactive oxygen species (ROS) generation was determined by DCFH‐DA analysis. The malondialdehyde (MDA) and glutathione (GSH) levels were measured in high‐glucose‐treated PC12 cells with and without leptin cotreatment. Western blotting was performed to measure apoptosis markers (Cleaved caspase‐3 and Bax/Bcl2 ratio). Elevation of glucose levels (100 mmol/L) consecutively increased intracellular ROS and MDA level, and apoptosis in PC12 cells after 24 h leptin administration (12 and 24 nmol/L) decreased the high‐glucose‐induced cell toxicity, caspase‐3 activation, and Bax/Bcl‐2 ratio. Also, cotreatment with leptin (12 and 24 nmol/L) significantly reduced oxidative damage to PC12 cells in high‐glucose condition, as reflected by the diminution in MDA and ROS levels and the increase in GSH content. Our finding demonstrates that leptin has protective effects against hyperglycemia‐induced neural damage. This could be related to the attenuation of oxidative stress and neural apoptosis.     

Lung Cell Oxidant Injury: ENHANCEMENT OF POLYMORPHONUCLEAR LEUKOCYTE-MEDIATED CYTOTOXICITY IN LUNG CELLS EXPOSED TO SUSTAINED IN VITRO HYPEROXIA

The oxidant damage of lung tissue during in vivo hyperoxic exposure appears to be amplified by neutrophils that release toxic amounts of oxygen metabolites. In our studies cloned lung epithelial cells (L2 cells), lung fibroblasts, and pulmonary artery endothelial cells were cultured under either ambient (Po(2) approximately 140 torr) or hyperoxic (Po(2) approximately 630 torr) conditions for 48 h (24 h for endothelial cells). After cultivation, phorbol myristate acetate- or opsonized zymosan-stimulated neutrophils were added to the cultivated monolayers for 4 h, and lung cell damage was quantitated using (51)Cr release as an index. The data show that stimulated neutrophils are able to injure the three lung cell lines tested, with endothelial cells being highly susceptible to this injury and L2 cells being slightly more susceptible than lung fibroblasts. The studies also demonstrate that all three lung cell lines exposed to sustained hyperoxia are more susceptible to neutrophil-mediated cytotoxicity than their time-matched air controls. Hydrogen peroxide was the main toxic oxygen metabolite because catalase (2,500 U/ml) completely protected the target cells. Equivalent quantities of hydrogen peroxide generated by glucose oxidase instead of by neutrophils gave a similar degree of target cell injury. Superoxide dismutase at high concentrations (250 mug/ml) provided some protection. Other systems that detoxify oxygen metabolites were without protective effect. These findings indicate that the increase in susceptibility of lung cells to neutrophil-mediated oxidant damage is a toxic effect of hyperoxia on lung cells. This specific manifestation of oxygen damage provides insight into the integration between primary mechanisms (oxygen exposure) and secondary mechanisms (release of oxygen metabolites by neutrophils) with respect to the cellular basis for pulmonary oxygen toxicity.     

高氧所致氧化应激状态下肺泡上皮细胞Toll样受体的表达及其功能研究

目的 观察高氧暴露后肺泡上皮细胞内活性氧(ROS)水平与Toll样受体(TLRs)基因、蛋白表达变化及其与信号通路功能之间的关系,探讨高氧所致肺损伤炎症反应的可能机制.方法 将体外培养的人肺腺癌A549细胞株分为空气对照组、高氧组、N-乙酰半胱氨酸(NAC)预处理组;高氧组细胞在＞90％O2的高氧环境中暴露2、6、12、24及48 h,NAC预处理组细胞予以ROS清除剂NAC预处理后高氧暴露6h.于相应时间点用流式细胞术检测细胞内ROS含量以及TLR2/4蛋白在细胞中的分布和表达;用逆转录-聚合酶链反应(RT-PCR)法检测TLR2/4 mRNA表达;用酶联免疫吸附法(ELISA)检测细胞上清液中白细胞介素(IL-6、IL-8)的含量.结果 A549细胞有TLR2/4表达,并且以细胞质内表达为主.与空气对照组比较,高氧组暴露2h细胞内ROS含量(荧光强度)即明显增高(11.820±3.123比7.223±1.170,P＜0.01),随高氧暴露时间延长呈进行性增高,于48 h达高峰(113.837±5.247,P＜0.01);高氧暴露2 h TLR2/4 mRNA表达至高峰(TLR2 mRNA:1.820±0.056比1.263±0.023;TLR4 mRNA:2.080±0.220比1.317±0.107,均P＜0.01),随高氧暴露时间延长,TLR2/4 mRNA表达虽仍有增多,但无显著变化;高氧暴露后TLR2/4蛋白表达显著增高,6h表达至高峰(TLR2蛋白:8.370±1.548比3.930±0.277;TLR4蛋白:25.803±5.783比8.867±2.230,均P＜0.01),以细胞质内表达为主;随高氧暴露时间延长,细胞上清液中IL-6(ng/L)、IL-8(ng/L)水平呈进行性增高,于48 h达高峰(IL-6:2 213.41±69.99比9.76±1.47;IL-8:11 520.38±429.93比159.56±20.80,均P＜0.01).在NAC预处理情况下,高氧刺激后,细胞内ROS水平明显降低(14.050±1.257比31.180±2.336,P＜0.01),细胞TLR2/4 mRNA和蛋白表达显著降低(TLR2 mRNA:1.270±0.061比1.683±0.025; TLR4 mRNA:1.507±0.058比1.650±0.139;TLR2蛋白:3.458±0.258比8.370±1.548;TLR4蛋白:11.611±3.403比25.803±5.783,均P＜0.05),细胞上清液中IL-6、IL-8水平显著下降(IL-6:8.42±0.70比73.51±16.70;IL-8:134.94±5.19比772.82±96.05,均P＜0.05),与空气对照组比较差异均无统计学意义.结论 A549细胞在高氧暴露后,细胞内ROS能够激活人肺泡上皮细胞TLR2/4的表达,导致前炎症细胞因子IL-6和IL-8的大量释放.     

Efficacy of glutathione in ameliorating sulfur mustard analog-induced toxicity in cultured skin epidermal cells and in SKH-1 mouse skin in vivo

Exposure to chemical warfare agent sulfur mustard (HD) is reported to cause GSH depletion, which plays an important role in HD-linked oxidative stress and skin injury. Using the HD analog 2-chloroethyl ethyl sulfide (CEES), we evaluated the role of GSH and its efficacy in ameliorating CEES-caused skin injury. Using mouse JB6 and human HaCaT epidermal keratinocytes, we observed both protective and therapeutic effects of exogenous GSH (1 or 10 mM) in attenuating a CEES-caused decrease in cell viability and DNA synthesis, as well as S and G(2)M phase arrest in cell cycle progression. However, the protective effect of GSH was stronger than its ability to reverse CEES-induced cytotoxic effect. The observed effect of GSH could be associated with an increase in intracellular GSH levels after its treatment before or after CEES exposure, which strongly depleted cellular GSH levels. N-Acetyl cysteine, a GSH precursor, also showed both protective and therapeutic effects against CEES-caused cytotoxicity. Buthionine sulfoximine, which reduces cellular GSH levels, caused an increased CEES cytotoxicity in both JB6 and HaCaT cells. In further studies translating GSH effects in cell culture, pretreatment of mice with 300 mg/kg GSH via oral gavage 1 h before topical application of CEES resulted in significant protection against CEES-caused increase in skin bifold and epidermal thickness, apoptotic cell death, and myeloperoxidase activity, which could be associated with increased skin GSH levels. Together, these results highlight GSH efficacy in ameliorating CEES-caused skin injury and further support the need for effective antioxidant countermeasures against skin injury by HD exposure.     

Hyperoxia prolongs tumor necrosis factor-alpha-mediated activation of NF-kappaB: role of IkappaB kinase

Hyperoxia and tumor necrosis factor-alpha (TNFalpha) are two canonical signals centrally involved in the pathophysiology of acute lung injury. We have attempted to elucidate the effects of these two stimuli on the signal transduction pathways of lung parenchymal cells. In cultured human lung epithelial cells, exposure to hyperoxia alone (95% oxygen) did not affect NF-kappaB activation or degradation of the NF-kappaB inhibitory protein, IkappaB alpha. Stimulation with TNFalpha alone increased NF-kappaB activation within 1 h and induced IkappaB alpha degradation within 0.5 h. After TNFalpha alone, NF-kappaB activation returned to baseline within 2 h and this corresponded with near complete IkappaB alpha resynthesis within 1 h of stimulation. In contrast, simultaneous exposure to hyperoxia and TNFalpha prolonged NF-kappaB activation up to 4 h, and IkappaB alpha degradation up to 2 h after stimulation. Hyperoxia did not affect TNFalpha-mediated resynthesis of IkappaB alpha mRNA. Hyperoxia alone did not induce IkappaB kinase (IKK) activity, but significantly prolonged TNFalpha-mediated activation of IKK activity. Hyperoxia alone did not activate the intercellular adhesion molecule-1 (ICAM-1) promoter, but augmented TNFalpha-mediated activation of the ICAM-1 promoter. These data demonstrate that while hyperoxia alone does not affect activation of NF-kappaB, hyperoxia prolongs TNFalpha-mediated activation of NF-kappaB. The mechanism of this effect involves, in part, prolonged degradation of IkappaB alpha resulting from prolonged activation of IKK.     

Detection of organic hydroperoxides in rabbit lung lavage fluid, but not in lung tissue homogenate, using GSH peroxidase and GSH reductase

A specific method for the detection of organic hydroperoxides in lung lavage fluid (lung surfactant system) and lung tissue homogenate is described. After the inactivation of endogenous GSH peroxidase and GSH reductase and preincubation with catalase, organic hydroperoxides are consumed by addition of GSH and GSH peroxidase. The increase of GSSG, compared to a blank without addition of GSH peroxidase, is measured in a second enzymatic step with GSH reductase. The recoveries of t-butyl hydroperoxide and of peroxidized, free fatty acids added to lavage fluid or to lung homogenate are higher than 85% in each case. The detection limits of this assay for organic hydroperoxides are 0.9 nmol/mg surfactant phospholipid (molar ratio of 0.00066) and 40 nmol/g wet lung weight. The assay detects organic hydroperoxides in the surfactant system of normal rabbit lungs, but not in lung tissue homogenate.