Hyperoxia and xanthine dehydrogenase/oxidase activities in rat lung and heart

Cell injury from hyperoxia is associated with increased formation of superoxide radicals (O2-). One potential source for O2- radicals is the reduction of molecular O2 catalyzed by xanthine oxidase (XO). Physiologically, this reaction occurs at a relatively low rate, because the native form of the enzyme is xanthine dehydrogenase (XD) which produces NADH instead of O2-. Reports of accelerated conversion of XD to XO, and increased formation of O2- formation in ischemia-reperfusion injury, led us to examine whether hyperoxia, which is known to increase O2- radical formation, is associated with increased lung XO activity, and accelerated conversion of XD to XO. We exposed 3-month-old rats either to greater than 98% O2 or room air. After 48 h, we sacrificed the rats and measured XD and XO activities and uric acid contents of the lungs. We also measured the activities of the two enzymes in the heart as a control organ. We found that the activity of XD was not altered significantly by hyperoxia in rat lungs or hearts, but XO activity was markedly lower in the lung, whether expressed per whole organ or per milligram protein, and remained unchanged in the heart. Lung uric acid content was also significantly lower with hyperoxia. The decrease in lung XO activity may reflect inactivation of the enzyme by reactive O2 metabolites, possibly as a negative feedback mechanism. The concomitant decrease in uric acid content suggests either decreased production mediated by XO due to its inactivation or greater utilization of uric acid as an antioxidant. We examined these postulates in vitro using a xanthine/xanthine oxidase system and found that H2O2, but not uric acid, has an inhibitory effect on O2- formation in the system. We therefore conclude that hyperoxia is not associated with increased conversion of XD to XO, and that the exact contribution of XO to hyperoxic lung injury in vivo remains unclear.     

Pulmonary microvascular endothelial cells constitutively release xanthine oxidase.

The generation of oxidants in reperfused ischemic tissues by xanthine oxidase (XO) may contribute to tissue damage. We exposed bovine pulmonary microvascular endothelial (BPMVE) cells to hypoxia and subsequent reoxygenation and examined alterations in intracellular and extracellular XO activities. BPMVE cells incubated 24 h under hypoxic conditions (less than 1% O2) showed a twofold increase in intracellular xanthine dehydrogenase activity and a smaller increase in intracellular XO activity compared to normoxic BPMVE. Both normoxic and hypoxic BPMVE cells constitutively released XO activity into their culture media. Incubation of hypoxic or normoxic BPMVE cells with oxygenated medium (95% O2) stimulated the release of XO activity into the extracellular medium within 5 min. The XO activity could not be detected in the oxygenated medium after 60 min incubation with 95% O2. These results indicate that endothelial cells in culture constitutively release XO and that oxygenation rapidly enhances XO release. The released XO activity may play an important role in generation of oxidants in the extracellular milieu during reperfusion.     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

Preparation of antibodies against xanthine oxidase from human milk

1. Human xanthine oxidase [XO; EC 1.2.3.2.] was isolated by a non-proteolytic method from fresh human milk. Final purification of the protein was achieved by hydroxyapatite chromatography. Most (less than 95%) of the enzyme was released in the 0.40 M phosphate fraction at pH 6.8. 2. The specific activity of this preparation was found to be 0.047 microM min-1 mg-1 with xanthine as substrate. 3. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separated two subunits, each with a mol. wt approximately 122 kDa. 4. On non-denaturing acrylamide gels both of these subunits exhibited oxidase-like activity with xanthine as substrate in the presence of nitroblue tetrazolium and molecular oxygen. 5. Immunoconjugates of XO were prepared by the keyhole limpet hemocyanin (KLH)- and glutaraldehyde-crosslinking techniques. 6. Polyclonal antibodies to XO were raised by i.m. injection of these conjugates into female New Zealand rabbits. 7. Western blot analysis using the semi-dry technique was employed to confirm the specificity of the antibody.     

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.     

Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase-contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of O*- and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating O*- and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.     

Effect of radiation on the xanthine oxidoreductase system in the liver of mice.

The xanthine oxidoreductase system is one of the major sources of free radicals in many pathophysiological conditions. Since ionizing radiations cause cell damage and death, the xanthine oxidoreductase system may contribute to the detrimental effects in irradiated systems. Therefore, modulation of the xanthine oxidoreductase system by radiation has been examined in the present study. Female Swiss albino mice (7-8 weeks old) were irradiated with gamma rays (1-9 Gy) at a dose rate of 0.023 Gy s(-1) and the specific activities of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were determined in the liver of the animals. The mode and magnitude of change in the specific activities of XO and XDH were found to depend on radiation dose. At doses above 3 Gy, the specific activity of XO increased rapidly and continued to increase with increasing dose. However, the specific activity of XDH was decreased. These findings are suggestive of an inverse relationship between the activity of XO and XDH. The ratio of the activity of XDH to that of XO decreased with radiation dose. However, the total activity (XDH + XO) remained constant at all doses. These results indicate that XDH may be converted into XO. An intermediate form, D/O, appears to be transient in the process of conversion. The enhanced specific activity of XO may cause oxidative stress that contributes to the radiation damage and its persistence in the postirradiation period. Radiation-induced peroxidative damage determined in terms of the formation of TBARS and the change in the specific activity of lactate dehydrogenase support this possibility.     

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.     

N-ethylmaleimide inhibits xanthine oxidase activity with no detectable change in xanthine dehydrogenase activity in rabbit liver

The effect of an alkylating agent, N-ethylmaleimide (NEM), on the activities of xanthine oxidase (XO) and xanthine dehydrogenase (XD) in the presence and absence of Cu2+ or trypsin in the cytosolic fraction from rabbit liver was examined. At concentrations ranging from 0.25 to 2.0 microM, allopurinol, which is generally considered to be a XO inhibitor, suppressed the XD activity (41.5-93.4% inhibition) in addition to the XO activity (28.6-88.4% inhibition) under basal conditions, without the addition of Cu2+ or trypsin. In contrast, NEM (100-400 microM) inhibited the XO activity (35.7-85.7% inhibition) without affecting the XD activity. Also, NEM inhibited the Cu2+- and trypsin-induced XO activities, but did not affect the XD activity at the same concentration range. These results demonstrate that NEM can be a selective inhibitor of XO activity in rabbit liver.     

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.     

Binding of xanthine oxidase to glycosaminoglycans limits inhibition by oxypurinol

Although the binding of xanthine oxidase (XO) to glycosaminoglycans (GAGs) results in significant alterations in its catalytic properties, the consequence of XO/GAG immobilization on interactions with clinically relevant inhibitors is unknown. Thus, the inhibition kinetics of oxypurinol for XO was determined using saturating concentrations of xanthine. When XO was bound to a prototypical GAG, heparin-Sepharose 6B (HS6B-XO), the rate of inactivation for uric acid formation from xanthine was less than that for XO in solution (k(inact) = 0.24 versus 0.39 min(-1)). Additionally, the overall inhibition constant (K(i)) of oxypurinol for HS6B-XO was 2-5-fold greater than for free XO (451 versus 85 nm). Univalent electron flux (O(2)(.) formation) was diminished by the binding of XO to heparin from 28.5% for free XO to 18.7% for GAG-immobilized XO. Similar to the results obtained with HS6B-XO, the binding of XO to bovine aortic endothelial cells rendered the enzyme resistant to inhibition by oxypurinol, achieving approximately 50% inhibition. These results reveal that GAG immobilization of XO in both HS6B and cell models substantially limits oxypurinol inhibition of XO, an event that has important relevance for the use of pyrazolo inhibitors of XO in clinical situations where XO and its products may play a pathogenic role.     

Mechanisms of action of malondialdehyde and 4-hydroxynonenal on xanthine oxidoreductase

Studies have been made on the possible involvement of malondialdehyde (MDA) and (E)-4-hydroxynon-2-enal (HNE), two terminal compounds of lipid peroxidation, in modifying xanthine oxidoreductase activity through interaction with the oxidase (XO) and/or dehydrogenase (XDH) forms. The effect of the two aldehydes on XO (reversible, XO(rev), and irreversible, XO(irr)) and XDH was studied using xanthine oxidase from milk and xanthine oxidoreductase partially purified from rat liver. The incubation of milk xanthine oxidase with these aldehydes resulted in the inactivation of the enzyme following pseudo-first-order kinetics: enzyme activity was completely abolished by MDA (0.5-4 mM), while residual activity (5% of the starting value) associated with an XO(irr) form was always observed when the enzyme was incubated in the presence of HNE (0.5-4 mM). The addition of glutathione to the incubation mixtures prevented enzyme inactivation by HNE. The study on the xanthine oxidoreductase partially purified from rat liver showed that MDA decreases the total enzyme activity, acting only with the XO forms. On the contrary HNE leaves the same level of total activity but causes the conversion of XDH into an XO(irr) form.     

The screening and characterization of 6-aminopurine-based xanthine oxidase inhibitors

Xanthine oxidase (XO) is a key enzyme which can catalyze xanthine to uric acid causing hyperuricemia in humans. By using the fractionation technique and inhibitory activity assay, an active compound that prevents XO from reacting with xanthine was isolated from wheat leaf. It was identified by the Mass and NMR as 6-aminopurine (adenine). A structure-activity study based on 6-aminopurine was conducted. The inhibition of XO activity by 6-aminopurine (IC(50)=10.89+/-0.13 microM) and its analogues was compared with that by allopurinol (IC(50)=7.82+/-0.12 microM). Among these analogues, 2-chloro-6(methylamino)purine (IC(50)=10.19+/-0.10 microM) and 4-aminopyrazolo[3,4-d] pyrimidine (IC(50)=30.26+/-0.23 microM) were found to be potent inhibitors of XO. Kinetics study showed that 2-chloro-6(methylamino)purine is non-competitive, while 4-aminopyrazolo[3,4-d]pyrimidine is competitive against XO.     

Disordered functioning of the superoxide radical-superoxide dismutase system in rat liver ischemia

The rate of O2 radical generation in microsomal membranes (VO2), the activity of cytosol superoxide dismutase (Cu, ZnSOD) and mitochondrial superoxide dismutase (MnSOD), and the activity of xanthine oxidizing system (XO) after a two-hour ischemia following a 24-hour reoxygenation of the rat liver were investigated. The high value of VO2, as compared to Cu, ZnSOD activity, may result in regulation disorders in O2-SOD system during ischemia. During reoxygenation, xanthine oxidizing system in combination with lowered Cu, ZnSOD activity may substantially contribute to the disturbance.     

Mechanisms of extracellular reactive oxygen species injury to the pulmonary microvasculature.

We investigated the effect of xanthine (X) plus xanthine oxidase (XO) on pulmonary microvascular endothelial permeability in isolated rabbit lungs perfused with Krebs buffer containing bovine serum albumin (5 g/100 ml). Addition of five mU/ml XO and 500 microM X to the perfusate caused a twofold increase in the pulmonary capillary filtration coefficient (Kf,c) 30 min later without increasing the pulmonary capillary pressure. This increase was prevented by allopurinol or catalase but not by superoxide dismutase or dimethyl sulfoxide. Because these data implicated hydrogen peroxide (H2O2) as the injurious agent, we measured its concentration in the perfusate after the addition of X and XO for a 60-min interval. In the absence of lung tissue and albumin, H2O2 increased with time, reaching a concentration of approximately 250 microM by 60 min. If albumin (5 g/100 ml) was added to the perfusate, or in the presence of lung tissue, the corresponding values were 100 microM and less than 10 microM, respectively. To understand the mechanisms of H2O2 scavenging by lung tissue, we added a 250 microM bolus of H2O2 to the lung perfusate. We found that H2O2 was removed rapidly, with a half-life of 0.31 +/- 0.04 (SE) min. This variable was not increased significantly by inhibition of lung catalase activity with sodium azide or inhibition of the lung glutathione redox cycle with 1-chloro-2,4-dinitrobenzene. However, inhibition of both enzymatic systems increased the half-life of H2O2 removal to 0.71 +/- 0.09 (SE) min (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairment of thioredoxin reductase activity by oxidative stress in human rheumatoid synoviocytes

The thioredoxin/thioredoxin reductase system is strongly induced in patients with rheumatoid arthritis (RA). We have investigated the impact on TR activity of doses of superoxide anion generated by the hypoxanthine (HX)/xanthine oxidase (XO) system and by hydrogen peroxide, H(2)O(2), for various times and compared the findings with synoviocytes obtained from osteoarthritis (OA) patients. At baseline, TR activity in RA cells was significantly higher than in OA cells (2.31 +/- 0.65 versus 0.74 +/- 0.43 mUnit/mg protein, p < 0.01). HX/XO and H(2)O(2) in RA cells decreased TR activity, which was found to be unchanged in OA cells. H(2)O(2) and superoxide anion caused a time-dependent accumulation of oxidized TR and induced the formation of carbonyl groups in TR protein in RA cells rather than OA cells, and oxidized the selenocysteine of the active site. The oxidation in TR protein was irreversible in RA cells but not in OA cells. In conclusion, we report that the oxidative aggression generates modifications in the redox status of the active site of the TR and induces an alteration of the Trx/TR system, concomitant with those of the other antioxidant systems that could explain the causes of oxidative stress related to RA disease.     

Radiomodfication of xanthine oxidoreductase system in the liver of mice by phenylmethylsulfonyl fluoride and dithiothreitol

The widely distributed xanthine oxidoreductase (XOR) system has been shown to be modulated upon exposure of animals to ionizing radiation through the conversion of xanthine dehydrogenase (XDH) into xanthine oxidase (XO). In the present work, radiomodification of the XOR system by phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) was examined using female Swiss albino mice which were irradiated with gamma rays at a dose rate 0.023 Gy s(-1). PMSF, a serine protease inhibitor, and DTT, the sulfhydryl reagent, were administered intraperitoneally prior to irradiation. The specific activities of XDH and XO as well as the XDH/XO ratio and the total activity (XDH+XO) were determined in the liver of the mice. The inhibition of XO activity, restoration of XDH activity, and increase in the XDH/XO ratio upon administration of PMSF were suggestive of irreversible conversion of XDH into XO mediated through serine proteases. The biochemical events required for the conversion were probably initiated during the early phase of irradiation, as the treatment with PMSF immediately after irradiation did not have a modulatory effect. Interestingly, DTT was not effective in modulating radiation-induced changes in the XOR system or oxidative damage in the liver of mice. The DTT treatment resulted in inhibition of the release of lactate dehydrogenase. However, the protection appears to be unrelated to the formation of TBARS. On the other hand, the presence of PMSF during irradiation inhibited radiation-induced oxidative damage and radiation-induced increases in the specific activity of lactate dehydrogenase. These findings suggest that a major effect of ionizing radiation is irreversible conversion of xanthine to xanthine oxidase.     

Xanthine Oxidase Catalyzes the Synthesis of Retinoic Acid

Milk xanthine oxidase (xanthine: oxygen oxidore-ductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min^?1, determined at pH 7.0 with 1nM XO and all trans-retinaldehyde varying between 0.05 to 2μM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4μM and 2μM allopurinol respectively and inhibited 48% by 10 μM xanthine in enzyme assays performed at 2μM all trans-retinaldehyde. The K_i value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 μM.