Comparison of the effects of superoxide dismutase and cytochrome c on luminol chemiluminescence produced by xanthine oxidase-catalyzed reactions

Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) (SOD) and ferricytochrome c are used to check the effects on luminol chemiluminescence induced by a xanthine or hypoxanthine/xanthine oxidase/oxygen system. Luminol chemiluminescence has been attributed to superoxide anion radical (O2.-) in this system. From kinetic studies on the light intensity vs. time curves it is demonstrated that addition of SOD into the system does not affect the mechanism of O2.- generation, whilst ferricytochrome c dramatically alters the time-course of the reaction. This is interpreted as the effect of cytochrome c redox cycling by reaction with H2O2, modifying oxy-radical generation in the reaction medium. Also, an alternative mechanism for luminol chemiexcitation is proposed under certain experimental conditions.     

Evidence for superoxide-dependent reduction of Fe3+ and its role in enzyme-generated hydroxyl radical formation.

This report describes studies yielding additional evidence that superoxide anion (O2) production by some biological oxidoreductase systems is a potential source of hydroxyl radical production. The phenomenon appears to be an intrinsic property of certain enzyme systems which produce superoxide and H2O2, and can result in extensive oxidative degradation of membrane lipids. Earlier studies had suggested that iron (chelated to maintain solubility) augmented production of the hydroxyl radical in such systems according to the following reaction sequence: O2 + Fe3+ leads to O2 + Fe2+ Fe2+ + H2O2 leads to Fe3+ + HO-+OH-. The data reported below provide additional support for the occurrence of these reactions, especially the reduction of Fe3+ by superoxide. Because the conditions for such reactions appear to exist in animal tissues, the results indicate a mechanism for the initiation and promotion of peroxidative attacks on membrane lipids and also suggest that the role of antioxidants in intracellular metabolism may be to inhibit initiation of degradative reactions by the highly reactive radicals formed extraneously during metabolic activity. This report presents the following new information: (1) Fe3+ is reduced to Fe2+ during xanthine oxidase activity and a significant part of the reduction was oxygen dependent. (2) Mn2+ appears to function as an efficient superoxide anion scavenger, and this function can be inhibited by EDTA. (3) The O2-dependent reduction of Fe3+ to Fe2+ by xanthine oxidase activity is inhibited by Mn2+, which, in view of statement 2 above, is a further indication that the reduction of the iron involves superoxide anion. (4) Free radical scavengers prevent or reverse the Fe3+ inhibiton of cytochrome c3+ reduction by xanthine oxidase. (5) The inhibition of xanthine oxidase-catalyzed reduction of cyt c3+ by Fe3+ does not affect uric acid production by the xanthine oxidase system. (6) The reoxidation of reduced cyt c in the xanthine oxidase system is markedly enhanced by Fe3+ and is apparently due to enhanced HO-RADICAL formation since the Fe3+-stimulated reoxidation is inhibited by free radical scavengers, including those with specificity for the hydroxyl radical.     

Effect of antiperoxidative drugs on gastric damage induced by ethanol in rats

Lesion formation due to oral administration of absolute ethanol could be prevented by parenteral pretreatment with antiperoxidative drugs such as butylated hydroxytoluene (BHT), quercetin and quinacrine. Also effective were allopurinol and oxypurinol, inhibitors of xanthine oxidase, but not superoxide dismutase (SOD) and hydroxyl radical scavengers, such as sodium benzoate and dimethyl sulfoxide (DMSO). BHT, quercetin, quinacrine and sulfhydryl compounds such as reduced glutathione and cysteamine which offer gastroprotection in vivo against ethanol inhibited lipid peroxidation induced in vitro by ferrous ion in porcine gastric mucosal homogenate, but SOD, sodium benzoate, DMSO, allopurinol and oxypurinol did not. These results suggest the possibility that an active species, probably derived from free iron mobilized by the xanthine oxidase system, other than oxygen radicals such as hydroxyl radicals, contributes to lipid peroxidation and lesion formation in the gastric mucosa after absolute ethanol administration.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Desulfonation of a colitis inducer 2,4,6-trinitrobenzene sulfonic acid produces sulfite radical

2,4,6-Trinitrobenzene sulfonic acid (TNBS) has been used in vivo to induce colitis. With the nitroreductase of intestinal cells, TNBS underwent redox cycling to produce TNBS-nitro and superoxide radical anions which are thought to be involved in initial oxidative reactions that lead to colonic injury. In this study, we demonstrated that the TNBS desulfonative reaction with tissue amino acids produces sulfite which is subsequently oxidized to sulfite radical. Sulfite radical was measured using a spin trapping methodology. Sulfite radical adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were detected in a mixture of TNBS and lysine, xanthine oxidase, red blood cells, colonic mucosal or submucosal muscle tissues. TNBS alone did not produce sulfite radical, indicating that its formation required the presence of amino acids. Because sulfite radical is the precursor of highly reactive sulfiteperoxyl and sulfate radicals, our data imply that these sulfite-derived free radicals may also contribute to oxidative reactions leading to colonic injury in TNBS-induced colitis.     

Effects of vanadate on the oxidation of NADH by xanthine oxidase

Vanadate (V(V)) stimulates the oxidation of NADH by xanthine oxidase and superoxide dismutase eliminates the effect of V(V). Paraquat stimulates both the oxidation of NADH by xanthine oxidase and the V(V) enhancement of that oxidation. Xanthine, which is a better substrate for xanthine oxidase than is NADH, causes a V(V)-dependent co-oxidation of NADH which is transient and eliminated by SOD. Urate inhibits the V(V)-stimulated oxidation of NADH by xanthine oxidase or by Rose Bengal plus light. Measurement of rates of both O2- production and V(V)-stimulated NADH oxidation showed that many molecules of NADH were oxidized per O2-. These chain lengths were an inverse function of overall reaction rate. Minimum chain lengths, calculated on the basis of 100% univalent reduction of O2 to O2-, were smaller than measured average chain lengths by a factor of five. All of these results are in accord with the view that V(V) does not directly affect the activity of the enzyme, but rather catalyzes the free radical chain oxidation of NADH by O2-. It was further shown that phosphate was not involved and that the active form of V(V) was orthovanadate, rather than decavanadate.     

Cytotoxicity of the Hypoxanthine-Xanthine Oxidase System on V79 Cells: Comparison of the Effects of Sod and Cudips

The hypoxanthine — xanthine oxidase system generates an extracellular flux of superoxide anion radical (O₂^?) and hydrogen peroxide (H₂O₂). Catalase but not superoxide dismutase (SOD) protects V79 cells exposed to the hypoxanthine — xanthine oxidase system, showing that H₂O₂ is the major reactive oxygen species involved in the cytotoxicity of such a system. In contrast to SOD, the lipophilic SOD like compound CuII (diisopropylsalicylate)₂ (CuDIPS) exhibits some protection at non cytotoxic concentration. It is also found that methanol partially protects cells exposed to the hypoxanthine-xanthine oxidase system. It appears that in our experimental conditions (temperature, ionic strength and pH) the protective effect afforded by methanol and CuDIPS is due to the inhibition of the xanthine oxidase activity.     

Oxygen radicals alter the cell membrane potential in a renal cell line (LLC-PK1) with differentiated characteristics of proximal tubular cells

We employed a carbocyanine dye (1,1',3,3,3',3'-hexamethylindocarbocyanine iodide) to measure the plasma membrane potential of LLC-PK1 renal epithelial cells exposed to either xanthine oxidase-generated oxygen radicals or to hydrogen peroxide. Measurements were performed using a fluorescent-activated cell sorter to record fluorescence on a cell by cell basis. Initial exposure of cells to low concentrations of either H2O2 or xanthine oxidase resulted in a transient increase in membrane potential relative to control cells (P less than 0.001), followed by an exponential decline in potential (P less than 0.001). The addition of extracellular catalase diminished the H2O2-related decline in potential, consistent with a role for hydrogen peroxide in producing this effect. Pretreatment of cells with inhibitors of intracellular catalase and superoxide dismutase prior to exposure to xanthine oxidase caused an even larger decline in potential (P less than 0.001). Cells could be partially protected from the radical-mediated loss of potential by incubating them in a hypertonic (400 mosmolal) environment during radical exposure. Similarly, the loss of membrane potential was increased after incubation of cells in a hypotonic (200 mosmolal) environment during radical exposure. These observations are consistent with a reduction in membrane potential effected by exposure to oxygen radicals (including superoxide anion and hydrogen peroxide). This reduction may be prevented, in part, by radical scavenging enzymes and by reducing the degree of cellular swelling in response to oxygen radical exposure.     

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

Coronary reperfusion in dogs inhibits endothelium-dependent relaxation: role of superoxide radicals

Previous studies indicate that release of superoxide radicals during coronary reperfusion following occlusion may relate to the loss of endothelium-dependent coronary arterial relaxation. We examined coronary arterial ring relaxation in dogs subjected to temporary circumflex (Cx) coronary artery occlusion and treated with saline or the superoxide radical scavenger superoxide dismutase (SOD). In dogs treated with saline, Cx coronary ring relaxation in response to leukotriene D4 (LTD4) and acetylcholine (ACh) was attenuated (p less than 0.01), but coronary relaxation in response to nitroglycerin was preserved, suggesting loss of endothelium-dependent relaxation following coronary reperfusion. In contrast, Cx coronary relaxation in response to LTD4 and ACh was preserved in the SOD-treated dogs (p less than 0.01 compared to saline-treated dogs). To further examine the role of superoxide radicals in the loss of endothelium-dependent relaxation, normal nonischemic canine coronary artery and rat aortic rings were exposed to a superoxide radical generating system of xanthine and xanthine oxidase in vitro. Xanthine plus xanthine oxidase treatment caused a significant (p less than 0.01) decrease in the relaxant effects of ACh. Pretreatment of rat aortic rings with SOD protected against the loss of ACh-induced relaxation. These observations suggest that release of superoxide radicals during reperfusion is the basis of loss of endothelium-dependent coronary arterial relaxation. Treatment with superoxide radical scavengers prior to coronary reperfusion protects against this loss.     

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.     

Superoxide-induced bleaching of streptocyanine dyes: Application to assay the enzymatic activity of superoxide dismutases

With the aim of developing a novel superoxide dismutase (SOD) activity assay, a series of polymethinium salts (streptocyanines) were prepared and studied for their ability to be reduced by superoxide radical anion generated either from the pyrogallol autoxidation or by the xanthine oxidase-catalyzed oxidation of xanthine. The nonacarbon chain streptocyanine 9Cl(NEt₂)₂ was found to be relatively stable in neutral buffered aqueous solutions, to be reduced at a significant rate by superoxide, and addition of iron-dependent superoxide dismutase (Fe-SOD) prevented its bleaching, thus constituting a good candidate as a possible superoxide indicator in a spectrophotometric SOD assay. The values found to be optimal for a SOD assay were defined as pH 7.4, wavelength 728 nm, xanthine and xanthine oxidase as superoxide source, and a reaction time of 5 min. Based on the color change caused by the superoxide-induced bleaching of the streptocyanine, a qualitative colorimetric method for the SOD activity detection is proposed, enabling visual detection within a short time without any instrument.     

Biosensors for determination of total and natural antioxidant capacity of red and white wines: comparison with other spectrophotometric and fluorimetric methods

Research was carried out to experimentally evaluate the antioxidant capacity of several red and white wines using a superoxide dismutase (SOD) biosensor recently developed by the present authors. Measurements were performed by comparing the biosensor response to increasing concentration of the superoxide radical produced in solution by the xanthine/xanthine oxidase system, both in the presence and absence of the test sample.The results were compared with those of two traditional spectrophotometric methods and of a spectrofluorimetric method described in literature.Lastly, also the polyphenol, sulfite and ascorbic acid contents of the different wine samples examined were measured using a tyrosinase biosensor, a sulfite oxidase biosensor and an ascorbate oxidase biosensor, respectively.     

A role for xanthine oxidase in the loss of cytochrome P-450 evoked by interferon.

It has been suggested that the loss of cytochrome P-450, which is mediated by interferon and its inducers, can result from the generation of free radical species by the enzyme xanthine oxidase. Cytochrome P-450, aminopyrine N-demethylase, and ethoxyresorufin deethylase were depressed by 35, 36, 38%, respectively, in the livers of hamsters 24 h following the administration of a synthetic interferon (IFN-alpha-Con1) which contains the most frequent amino acid sequences of the human subtypes. Interferon increased the activities of the D and O forms of xanthine oxidase by 65 and 74%, respectively, in the same animals. The induction of the D form of xanthine oxidase, which is the precursor of the O form, preceded the loss in cytochrome P-450. The protein synthesis inhibitor, actinomycin D, prevented the interferon-induced loss of drug biotransformation and the increase in xanthine oxidase. The free radical scavenger, alpha-tocopherol, and the xanthine oxidase inhibitor, allopurinol, also prevented the loss of cytochrome P-450 mediated by the interferon inducer poly rI.rC. In chickens in which xanthine oxidase cannot be formed, poly rI.rC had no effect on cytochrome P-450 levels. These results suggest that xanthine oxidase induction may play some role in the interferon-mediated loss of cytochrome P-450.     

The effect of some tannins on trout erythrocytes exposed to oxidative stress

In order to gain more knowledge on the role of tannins as antioxidants, their ability to protect (Salmo irideus) erythrocytes against oxidative stress was investigated. Antioxidant activity of different tannins (tannic, gallic and ellagic acid) was evaluated by chemiluminescence (CL) techniques using lucigenin and luminol as chemiluminogenic probes for the superoxide radical generated by the xanthine/xanthine oxidase system and hydrogen peroxide, respectively. The superoxide-scavenging activity of these tannins was shown for all the compounds; however, it is not clear if this is due to their ability of scavenging the superoxide radical or to their inhibitory activity on xanthine oxidase. Tannic and ellagic acid showed a marked effect on the reduction of H2O2-luminol chemiluminescence. The influence of these tannins on the rate of hemolysis in stressed trout erythrocytes was investigated and the results indicate that tannic acid accelerates the hemolytic event while gallic and ellagic acid have no significant effect. The possible protective action of these compounds against oxidative DNA damage was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique, used to detect primary DNA damage in individual cells. The results here reported show that tannins under study are capable at low concentrations of protecting DNA breakage, while at high concentrations they can be genotoxic.     

Effect of oxidants on small intestinal brush border membranes and colonic apical membranes--a comparative study

This study compares composition of the rat small intestinal brush border membranes (BBM) and colonic apical membranes (CAM) and their susceptibility to in vitro exposure to various oxidants. Differences were observed between BBM and CAM in their lipid composition, sugar content, alkaline phosphatase (ALP) activity and cholesterol/phospholipid ratio. BBM and CAM were exposed to superoxide generated by xanthine+xanthine oxidase (X-XO) or peroxides such as tertiary butyl hydroperoxide (tBuOOH) and hydrogen peroxide (H(2)O(2)) and alterations in ALP activity, peroxidation parameters and membrane lipids were analyzed. Exposure of BBM and CAM to superoxide resulted in decrease in ALP activity and increase in peroxidation parameters such as protein carbonyl content, malondialdehyde and conjugated diene. Superoxide exposure also resulted in lipid alterations specifically in certain phospholipids. These alterations were prevented either by superoxide dismutase or by allopurinol. Peroxides did not have any significant effect. These results suggest that both BBM and CAM are susceptible to superoxide, which can bring about peroxidation and degradation of membrane lipids specifically, certain phospholipids.     

Mechanism of Kainate Toxicity to Cerebellar Neurons In Vitro Is Analogous to Reperfusion Tissue Injury

The neuroexcitotoxin kainate has been used as a selective lesioning agent to model the etiology of a number of neurodegenerative disorders. Although excitotoxins cause susceptible neurons to undergo prolonged or repeated depolarization, the proximate metabolic pathology responsible for neuronal necrosis has remained elusive. We report here that kainate-induced death of cerebellar neurons in culture is prevented by inhibiting the enzyme xanthine oxidase, a cellular source of cytotoxic superoxide radicals (O2-.). Moreover, neurons are also protected from excitotoxin-induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenge superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalls formation of xanthine oxidase. These findings indicate that excitotoxin-induced neuronal degeneration is mediated by superoxide radicals generated by xanthine oxidase, a mechanism partially analogous to that proposed for tissue damage seen upon reperfusion of ischemic tissues.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Permeation of the erythrocyte stroma by superoxide radical.

Superoxide anion, generated by xanthine oxidase within vesicles formed from washed erythrocyte ghosts, crosses the vesicle membrane to reduce cytochrome c in the medium (Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem, 253, 1838-1845). To determine whether O2- could travel through the membrane in the "channel" for the exchange of stable anions, the effects of two specific inhibitors of anion exchange, 4-acetamido-4'-isothiocyano-2,2' disulfonic acid stilbene (SITS) and 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS), on the escape of O2- from vesicles were studied. The reduction of external cytochrome c, caused by O2- produced by the enzymic turnover of internal xanthine oxidase, was 85 to 90% inhibited by SITS and DIDS. If SITS impeded the egress of O2- from vesicles, it should enhance the internal effects of O2- and antagonize the inhibition of these effects by external superoxide dismutase. External superoxide dismutase inhibited the lysis of vesicles containing xanthine oxidase. SITS (60 micron) partially reversed this inhibition. It appears that O2- can cross the membrane of the erythrocyte in the anion channel.     

Human platelets attenuate oxidant injury in isolated rabbit lungs

Because platelets contain active antioxidant systems, the capacity of platelets to attenuate oxidant lung injury was investigated. Purine and xanthine oxidase were infused into isolated perfused rabbit lungs (IPL) to generate H2O2, thereby causing increased membrane permeability edema. The coinfusion of washed human platelets (1.20 +/- 0.07 x 10(10) cells) attenuated the degree of edema formation as measured by lung weight gain and lung lavage albumin concentration. Electron microscopy of lung preparations demonstrated platelet adherence to capillary endothelial luminal surfaces of oxidant-injured lungs, but there was no evidence of vascular plugging with platelet macroaggregates. The platelet glutathione redox cycle or platelet catalase were inhibited before infusion of platelets into the IPL with purine and xanthine oxidase. Inhibition of the glutathione redox cycle with 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-chloro-2,4-dinitrobenzene, or buthionine sulfoximine prevented platelet attenuation of lung injury. Inactivation of platelet catalase with 3-amino-1,2,4-triazole, however, did not significantly reduce the platelet-induced lung protection. We conclude that the platelet glutathione redox cycle plays a major role in reducing enzymatically generated toxic O2 metabolites and attenuating lung injury.