oxypurinol and Reperfusion-Injury

Acute respiratory distress syndrome (ARDS) remains a major cause of morbidity and mortality. Oxygen-free radicals (OFRs) produced during ischemia and reperfusion (IR) have been implicated as the final common pathway in the pathogenesis of this syndrome. Spin traps have been shown to decrease IR injury in several animal lung models. The hydroxylamine, OXANOH (2-ethyl-2,5,5-trimethyl-3-oxazolidine) has been proposed as an ideal spin trap that would trap extra- and intracellular OFRs producing the stable radical, OXANO• (2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl). Electron microscopy was used to investigate whether OXANOH would protect against IR injury in the rabbit lung.. OXANOH was obtained by hydrogenation of its stable radical, OXANO• using a safe laboratory technique. Several doses of OXANOH were tested to identify a nontoxic dose. Two quantitative methods were used based on the average surface area of the alveoli and average number of alveoli per unit surface area using scanning electron microscopy (SEM). A total of 20 animals were subjected to 2 hours of ischemia followed by 4 hours of reperfusion. On reperfusion, the 4 groups (N = 5) received no treatment, OXANOH, superoxide dismutase (SOD)/catalase, or oxypurinol.. A therapeutic dose of 250 μmol/L of OXANO• was suggested in this in vitro model. All the 3 treatments showed significantly less injury compared to the control group and that SOD/catalase was significantly different from OXANOH and oxypurinol (P < .008).. OXANOH ameliorated IR injury in the isolated rabbit lung, almost as effectively as SOD/catalase and oxypurinol.

Topics: Animals; Antioxidants; Catalase; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Lung; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Oxazoles; Oxypurinol; Rabbits; Reactive Oxygen Species; Reperfusion Injury; Spin Labels; Superoxide Dismutase; Time Factors

The detrimental role of superoxide anion (O(2)(-)) has been well documented in the pathogenesis of ischemia-reperfusion (I/R) injury. Our and other studies suggested that one critical source of O(2)(-) generation may be xanthine oxidase (XO). We thus hypothesized that I/R injury could be protected by inhibiting XO activity, which would reduce the amount of O(2)(-) and hence reduce pathogenic consequences. Among various XO inhibitors, we previously found 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) exhibited potent XO inhibitory activity. Here, we report that the covalent conjugate of AHPP with amphipathic styrene-maleic acid copolymer (SMA-AHPP) showed protective effect against I/R-induced injury in a rat hepatic I/R model. Liver ischemia was induced by occluding both the portal vein and the hepatic artery for 30 min, and followed by reperfusion. SMA-AHPP was administered via the tail vein two hours before ischemia was initiated. A remarkable increase of liver enzymes in plasma (aspartate aminotransferase, AST; alanine aminotransferase, ALT and lactate dehydrogenase, LDH) was detected three hours after reperfusion, whereas prior injection of SMA-AHPP greatly suppressed this increase of AST, ALT and LDH. Moreover, induction of inflammatory cytokines, i.e. tumor necrosis factor-alpha (TNF-alpha), interleukin-12 (IL-12) and monocyte chemotactic protein-1 (MCP-1) by I/R were significantly inhibited by SMA-AHPP treatment. Accordingly, cytotoxic effect or apoptosis in the liver caused by I/R was clearly reduced by SMA-AHPP pretreatment. Furthermore, thiobarbituric acid-reactive substance assay showed a significant decrease of lipid peroxidation in rat liver after the administration of SMA-AHPP, which is parallel with the decreased XO activity after SMA-AHPP treatment, indicating the involvement of reactive oxygen species generated by XO. In addition, SMA-AHPP was found to bind to albumin, thus to exhibit prolonged in vivo (plasma) half-life. These results suggest that SMA-AHPP exerted a potent cytoprotective effect against I/R injury in rat liver, by inhibiting XO activity and the subsequent generation of O(2)(-).

Topics: Albumins; Animals; Apoptosis; Cytokines; Enzyme Inhibitors; Heme Oxygenase-1; Liver; Male; Maleates; Molecular Weight; Oxypurinol; Polystyrenes; Rats; Rats, Wistar; Reperfusion Injury; Superoxides; Thiobarbituric Acid Reactive Substances; Tissue Distribution; Xanthine Oxidase

Ischemia-reperfusion (IR) injury is a major insult to postcapillary venules. We hypothesized that IR increases postcapillary venular hydraulic conductivity and that IR-mediated changes in hydraulic conductivity result from temporally and mechanistically separate processes. A microcannulation technique was used to determine hydraulic conductivity (Lp) in rat mesenteric postcapillary venules serially throughout ischemia (45 min) and reperfusion (5 h) induced by superior mesenteric artery occlusion and release. Mesenteric IR resulted in a biphasic increase in Lp. White blood cell (WBC) adhesion slowly increased with maximal adhesion corresponding to the second peak (P < 0.005). After IR, tissue was harvested for RT-PCR analysis of ICAM-1, E-selectin, and P-selectin mRNA. Intercellular adhesion molecule-1 (ICAM-1) mRNA in the gut showed the most significant upregulation. Quantitative real-time PCR revealed that ICAM-1 mRNA was upregulated 60-fold in the gut. An ICAM-1 antibody was therefore used to determine the effect of WBC adhesion on Lp during IR. ICAM-1 inhibition attenuated Lp during the first peak and completely blocked the second peak (P < 0.005). When rats were fed a tungsten diet to inhibit xanthine oxidase and then underwent IR, Lp was dramatically attenuated during the first peak and mildly decreased the second peak (P < 0.005). Inhibition of xanthine oxidase by oxypurinol decreased Lp during IR by over 60% (P < 0.002). Tempol, a superoxide dismutase mimetic, decreased Lp during IR by over 30% (P < 0.01). We conclude that IR induces a biphasic increase in postcapillary hydraulic conductivity. Reactive oxygen species impact both the first transient peak and the sustained second peak. However, the second peak is also dependent on WBC-endothelial cell adhesion. These serial measurements of postcapillary hydraulic conductivity may lead the way for optimal timing of pharmaceutical therapies in IR injury.

Topics: Animals; Antibodies; Cell Adhesion; Cricetinae; Cyclic N-Oxides; Disease Models, Animal; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Female; Intercellular Adhesion Molecule-1; Leukocytes; Male; Mesentery; Mesocricetus; Oxypurinol; P-Selectin; Permeability; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; RNA, Messenger; Spin Labels; Time Factors; Tungsten Compounds; Venules; Xanthine Oxidase

We investigated the effect of a hydroxyl radical scavenger, 1,3-dimethyl-2-thiourea (dimethylthiourea), and two xanthine oxidase inhibitors, oxypurinol and allopurinol, on the threshold shift of the compound action potential (CAP) after transient ischemia of the cochlea. Transient ischemia of 30 min duration was induced in albino guinea pigs via a skull base approach. The animals were treated with perilymphatic perfusion of dimethylthiourea, oxypurinol or allopurinol from 10 min before the onset of ischemia to 4 h after the termination of ischemia. Dimethylthiourea ameliorated the CAP threshold shifts at 4 h after the onset of reperfusion in a dose-dependent manner. However, oxypurinol and allopurinol did not affect the post-ischemic cochlear dysfunction. These results imply that the hydroxyl radical plays an important role in generation of cochlear dysfunction induced by ischemia-reperfusion and that xanthine oxidase may not be the primary source of this radical.

Topics: Action Potentials; Allopurinol; Animals; Cochlea; Enzyme Inhibitors; Free Radical Scavengers; Guinea Pigs; Hydroxyl Radical; Ischemia; Oxypurinol; Reperfusion Injury; Thiourea; Xanthine Oxidase

The relative contribution of xanthine oxidase (XO) and leukocytes to tissue injury after short-term ischemia is unknown. In this study, we subjected three groups of rat spinotrapezius muscles to 30-min ischemia and 1-h reperfusion: 1) ischemia-reperfusion (I/R) + 0.9% saline, 2) I/R + superoxide dismutase, and 3) I/R + oxypurinol. A fourth group served as nonischemic control. We quantified the increase in resistance (%DeltaR) caused by leukocyte-capillary plugging concurrently with myocyte uptake of propidium iodide (PI) [expressed as no. of PI spots per total volume of perfused tissue (N(PI)/V)] and performed assays to quantify XO activity, thiobarbituric acid-reactive substances (TBARS), and myeloperoxidase (MPO). Groups 2 and 3 exhibited significant decreases in N(PI)/V relative to group 1. MPO levels and TBARS were similar among all groups, and mean %DeltaR was significantly reduced in groups 2 and 3 relative to group 1. However, elevated XO was observed in groups 1 and 2 relative to group 3 and nonischemic controls. These data are consistent with the hypothesis that XO, rather than toxic species produced by plugging or venule-adherent leukocytes, is responsible for postischemic damage in this model.

Topics: Animals; Capillaries; Cell Adhesion; Cell Movement; Female; Ischemia; Leukocytes; Lipid Peroxides; Muscle, Skeletal; Oxidants; Oxypurinol; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Superoxide Dismutase; Time Factors; Vascular Resistance; Xanthine Oxidase

Tissue injury following reperfusion represents an essential problem of reconstructive vascular surgery. Pathogenetically toxic oxygen radicals are considered to play a pivotal role. Pharmacotherapeutical approaches are based particularly on antioxidants and vasodilators. However, a standardized regimen is not yet clinically introduced. In 48 adult Lewis-rats lower limb ischemia was induced by aortal cross-clamping. Following 3.5 hours of ischemia intravascular flushing perfusion via the distal aorta with a heparinized electrolyte solution (group B). Group C received additionally oxypurinol, group D alprostadil and group E sodium selenite into the flushing solution. At 4 hours recirculation was established. After 10 min, 30 min and 24 hours of reperfusion we determined lactate, creatine kinase, lactate dehydrogenase, urea, malondialdehyde and the laser Doppler flux. At the end of the experiments biopsies were taken from M. tibialis anterior. In comparison to control animals (group A) we observed an attenuation of reperfusion injury in the groups treated with flushing perfusion. Free oxygen radical reactions measured by malondialdehyde release were significantly reduced (30 min: A-209.1 +/- 45.4, B-127.3 +/- 36.9, C-113.2 +/- 14.1, D-99.6 +/- 24.5, E-123.6 +/- 11.2 mmol/l, p < 0.05). The laser Doppler flux measurements corresponded with the biochemical analyses (30 min: A-52.4 +/- 11.1, B-48.0 +/- 11.0, C-72.6 +/- 12.0, D-74.4 +/- 13.3, E-62.6 +/- 10.8% of baseline). Histologically, treatment with alprostadil (PGE1) and oxypurinol revealed superior results. Standardized intraarterial flushing perfusion with antioxidants and vasodilators reduces reperfusion injury. Clinical trials are urgently required to confirm the experimental findings and to optimize the therapy of extremity ischemia/reperfusion injury in humans.

Topics: Animals; Disease Models, Animal; Extremities; Muscle, Skeletal; Oxypurinol; Prostaglandins E; Rats; Reperfusion; Reperfusion Injury; Sodium Selenite; Vascular Surgical Procedures

Oxygen free radicals, generated by cerebral ischemia, have been widely implicated in the damage of vascular endothelium. Endothelial cells have been proposed as a significant source of oxygen free radicals. In the present study, we developed an anoxia-reoxygenation (AX/RO) model using pure cultures of cerebral endothelial cells (CECs) isolated from piglet cortex to measure CEC oxygen free radical production and determine its role in AX/RO-induced CEC injury. CEC injury, as measured by lactate dehydrogenase efflux into the culture medium, increased progressively with the duration of anoxic exposure, becoming significant after 10 h. Reoxygenation significantly increased CEC anoxic injury in a time-dependent manner. A 55% increase in oxygen free radical production, determined by fluorescence detection of dihydroethidium oxidation, was measured at the end of 4-h reoxygenation in CECs subjected to AX/RO conditions that killed 40% of the cells. Blockade of oxygen free radical production with superoxide dismutase (SOD; 250 and 1000 U/ml) or oxypurinol (50 and 200 microM), a potent xanthine oxidase inhibitor, reduced this injury by 32-36% and 30-39%, respectively. Results from our in vitro model indicate that CECs produce significant amounts of oxygen free radicals following ischemia, primarily from the xanthine oxidase pathway. These radicals ultimately have a cytotoxic effect on the very cells that produced them. Thus, reductions in oxygen free radical-mediated vascular injury may contribute to improvements in neurophysiologic outcome following treatment with oxygen free radical inhibitors and scavengers.

Topics: Animals; Brain Ischemia; Cells, Cultured; Cerebrovascular Circulation; Endothelium, Vascular; Enzyme Inhibitors; Microcirculation; Oxypurinol; Reperfusion Injury; Superoxide Dismutase; Superoxides; Swine; Xanthine Oxidase

Complement plays an important role in ischemia-reperfusion injury. We recently demonstrated that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) activated the classical complement pathway and augmented iC3b deposition. In the present study, we investigated the potential role of oxygen-derived free radicals, NF-kappaB and new protein synthesis in this model. HUVECs subjected to 12 or 24 h hypoxic stress (1% O2) and then reoxygenated (0.5, 1, 2 or 3 h; 21% O2) in 30% human serum activated complement and deposited iC3b. Addition of hydrogen peroxide (H2O2; 1-100 micromol/l) to normoxic HUVECs increased iC3b deposition in a concentration-dependent manner. H2O2 (10 micromol/l), a concentration that did not significantly increase iC3b deposition on normoxic HUVECs, augmented iC3b deposition on hypoxic/reoxygenated HUVECs. We observed a significant increase in intracellular H2O2 and hydroxyl radical (OH.) production in hypoxic/reoxygenated HUVECs using dihydrorhodamine 123. Further, treatment of HUVECs with dimethylthiourea (DMTU, 1-100 micromol/l), deferoxamine (DEF, 1-100 micromol/l), or oxypurinol (10 micromol/l), but not superoxide dismutase (SOD, 500 U/ml), catalase (300 U/ml) or iron-loaded DEF, attenuated iC3b deposition following hypoxia/reoxygenation in a concentration-dependent manner. Western analysis demonstrated hypoxia-induced nuclear NF-kappaB translocation that increased with reoxygenation. Inhibition of new protein synthesis (i.e. cycloheximide) or inhibition of NF-kappaB (ALLN or SN-50) also significantly decreased iC3b deposition on hypoxic/reoxygenated HUVECs. We conclude that (1) hypoxic/reoxygenated HUVECs generate H2O2 and OH.; (2) treatment of HUVECs with cell permeable reactive oxygen species inhibitors/scavengers (i.e. DEF, DMTU, oxypurinol) but not large molecular weight inhibitors (i.e. catalase or SOD) significantly reduces iC3b deposition and (3) inhibition of new protein synthesis or NF-kappaB activation attenuates iC3b deposition. These data suggest that iC3b deposition on the vascular endothelium may be regulated by intracellular oxygen-derived free radical-induced activation of NF-kappaB, new protein synthesis and activation of the classical complement pathway during ischemia/reperfusion.

Topics: Cell Hypoxia; Cells, Cultured; Complement Activation; Complement C3b; Deferoxamine; Dose-Response Relationship, Drug; Endothelium, Vascular; Free Radical Scavengers; Humans; Hydrogen Peroxide; Hydroxyl Radical; NF-kappa B; Oxypurinol; Protein Biosynthesis; Reactive Oxygen Species; Reperfusion Injury; Thiourea; Umbilical Veins

It has been shown that hypercholesterolemia (HCh) exaggerates the microvascular dysfunction that is elicited by ischemia and reperfusion (I/R). The objective of this study was to determine whether oxidants contribute to the exaggerated inflammatory responses and enhanced albumin leakage observed in HCh rat mesenteric venules exposed to I/R (10 minutes of ischemia and 30 minutes of reperfusion). Intravital videomicroscopy was used to quantify the number of adherent and emigrated leukocytes, albumin extravasation, platelet-leukocyte aggregation in postcapillary venules, and the degranulation of adjacent mast cells. Oxidation of the fluorochrome dihydrorhodamine 123 (DHR) was used to monitor oxidant production by venular endothelium. I/R was shown to elicit an increased DHR oxidation in venules of both control and HCh rats, with the latter group exhibiting a significantly larger response. Treatment with either oxypurinol or superoxide dismutase largely prevented the leukocyte recruitment, platelet-leukocyte aggregation, mast cell degranulation, and enhanced DHR oxidation elicited by I/R in HCh rats. The enhanced albumin leakage was reduced by superoxide dismutase but not by oxypurinol. These results indicate that HCh amplifies the oxidant stress elicited by I/R and that interventions that blunt the oxidant stress effectively attenuate the leukocyte, platelet, and mast cell activation that result from I/R.

Topics: Albumins; Animals; Cell Adhesion; Enzyme Inhibitors; Hypercholesterolemia; Leukocytes; Male; Mesenteric Arteries; Oxidants; Oxypurinol; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rhodamines; Superoxide Dismutase

Little is known about the nature of biochemical disturbances during reperfusion after retinal ischemia. Previous studies have suggested that adenosine is responsible for regulation of retinal blood flow soon after ischemia has ended. Therefore, in this study we measured concentrations of adenosine and its metabolites in the rat retina/choroid after brief (10 min) or prolonged (60 min) periods of ischemia, and the functional consequences of inhibiting adenosine metabolism.. Ischemia was produced in anesthetized rats by ligation of the central retinal artery. The eyes were frozen in situ and purine nucleoside concentration was determined by high performance liquid chromatography. The functional effects of pre-ischemic inhibition of xanthine dehydrogenase/xanthine oxidase were assessed by measurement of the electroretinogram before, during, and up to 7 days following 60 min ischemia.. Changes in the concentrations of adenosine and its metabolites were significant early in the reperfusion period, and were greater in magnitude and occurred earlier in prolonged, compared to brief, ischemic periods. Concentrations of adenosine, inosine, and hypoxanthine remained elevated for 30 min following the end of 60 min ischemia, and xanthine concentration was significantly elevated until 60 min after the end of either 10 or 60 min of ischemia. The onset of its peak value after ischemia was delayed in comparison to that of adenosine. Ischemia-evoked increases in xanthine concentration were attenuated by inhibition of adenosine deaminase or xanthine oxidase/xanthine dehydrogenase. Pre-ischemic inhibition of xanthine oxidase/xanthine dehydrogenase by oxypurinol (40 or 80 mg/kg intraperitoneally [IP]) resulted in a significant improvement in recovery of the a and b waves of the electroretinogram in comparison to a saline-treated control group.. These results indicate that adenosine is a major component of the biochemical changes that occur after retinal ischemia. Long-lasting increases in xanthine concentration during reperfusion after ischemia could be a source of oxygen free radicals that may contribute to delayed injury of the retina, attempts to decrease xanthine concentration would ideally be initiated within one hour after the end of ischemia.

Topics: Adenosine; Animals; Choroid; Electroretinography; Enzyme Inhibitors; Ischemia; Osmolar Concentration; Oxypurinol; Pentostatin; Purines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Retina; Retinal Diseases

We evaluated experimentally (80 Lewis-rats) possible pharmacological strategies in the treatment of intestinal reperfusion injury in hypo- and normothermia. We used a specific perfusion solution containing PGI2 or radical scavengers (superoxide dismutase, oxypurinol, tocopherol, ascorbate). Decreased malondialdehyde (MDA) plasma release after reperfusion proved the antioxidative efficiency of the administered radical scavengers (normothermia-control group: MDA increase after 15 min of reperfusion to 160 +/- 30% compared to level at the end of ischemia, oxypurinol: 110 +/- 23%, tocopherol: 112 +/- 12%, ascorbate: 104 +/- 20%; p < 0.05). The ATP/ADP-ratio of the therapy groups was stable in contrast to the control group. Alkaline phosphatase release was significantly diminished under radical scavenger administration (normothermia/15 min reperfusion-control group: 7.7 +/- 0.9 mumol/ls, oxypurinol: 4.4 +/- 0.4 mumol/ls, tocopherol: 3.5 +/- 0.1 mumol/ls, ascorbate: 5.9 +/- 0.3 mumol/ls; p < 0.05). Histologically we observed a mucosa protective effect particularly in the ascorbate group. Other pharmacological strategies are discussed.

Topics: alpha-Tocopherol; Animals; Ascorbic Acid; Energy Metabolism; Epoprostenol; Free Radical Scavengers; Hypothermia, Induced; Intestinal Mucosa; Intestines; Male; Malondialdehyde; Oxypurinol; Rats; Rats, Inbred Lew; Reperfusion Injury; Superoxide Dismutase; Tocopherols; Vasodilator Agents; Vitamin E

To apply an electron spin resonance (ESR) spectroscopic technique as a means of determining the oxidising capacity of reactive oxygen species produced during hypoxia and reoxygenation of diseased human synovial tissue.. Twenty four specimens of fresh synovial tissue were obtained from patients undergoing primary total knee joint replacement and graded according to the degree of inflammation present. Tissue samples were subjected to an ex vivo hypoxia-reoxygenation cycle in the presence of the nitroso based spin trap, 3,5-dibromo-4-nitrosobenzene sulphonate. The degree of oxidation of the spin trap to a stable free radical was determined and followed with time. Control samples were subjected to hypoxia only.. The results indicate that the oxidising capacity of reactive oxygen species produced by human synovial tissue varies with the degree of inflammation present. Only the more inflamed specimens, from both rheumatoid arthritis and osteoarthritis patients, demonstrated increased production of reactive oxygen species when subjected to a hypoxia-reoxygenation cycle. This change was reduced by both competitive and non-competitive inhibitors of the endothelial based enzyme xanthine oxidase. The relative concentration of reactive oxygen species generated by the synovial tissue samples correlated with the mean capillary density of the specimens.. This study supports the hypothesis of movement induced hypoxicreperfusion injury of the chronically inflamed joint by demonstrating the generation of reactive oxygen species within inflamed human synovium following an ex vivo hypoxia-reoxygenation cycle. Evidence is presented that the microvascular endothelial based enzyme xanthine oxidase is the predominant source of ESR detectable oxidising species in inflamed synovial specimens exposed to hypoxia-reoxygenation.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Culture Techniques; Electron Spin Resonance Spectroscopy; Humans; Knee Joint; Middle Aged; Osteoarthritis; Oxypurinol; Reactive Oxygen Species; Reperfusion Injury; Synovial Membrane; Thiazoles; Xanthine Oxidase

The present study investigated the effect of the administration of oxypurinol (40 mg/kg), an inhibitor of xanthine oxidase, on adenosine and adenine nucleotide levels in the rat brain during ischemia and reperfusion. The brains of the animals were microwaved before, at the end of a 20-min period of cerebral ischemia, and after 5, 10, 45, and 90 min of reperfusion. Cerebral ischemia was elicited by four-vessel occlusion with arterial hypotension to 45-50 mm Hg. Adenosine and adenine nucleotide levels in the oxypurinol-pretreated (administered intravenously 20 min before ischemia) rats were compared with those in nontreated animals exposed to the same periods of ischemia and reperfusion. Oxypurinol administration resulted in significantly elevated ATP levels at the end of ischemia and 5 min after ischemia, but not at 10 min after ischemia. ADP levels were also elevated, in comparison with those in the control rats, at the end of the ischemic period. Conversely, AMP levels were significantly reduced at the end of ischemia and during the initial (5 min) period of reperfusion. Adenosine levels were lower in oxypurinol-treated rats, during ischemia, and in the initial reperfusion phase. Oxypurinol administration resulted in a significant increase in the energy charge both during ischemia and after 5 min of reperfusion. Physiological indices, namely, time to recovery of mean arterial blood pressure and time to onset of respiration, were also shortened in the oxypurinol-treated animals. These beneficial effects of oxypurinol may have been a result of its purine-sparing (salvage) effects and of its ability to inhibit free radical formation by the enzyme xanthine oxidase. Preservation of high-energy phosphates during ischemia likely contributes to the cerebroprotective potency of oxypurinol.

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Blood Pressure; Electrochemistry; Hydrogen-Ion Concentration; Ischemic Attack, Transient; Kinetics; Male; Oxypurinol; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Respiration; Xanthine Oxidase

Xanthine oxidase (XO) activity and hydroxyl radical (.OH) formation are widely proposed mediators of renal reperfusion injury, potentially altering the severity of, and recovery from, postischemic acute renal failure. The goal of this study was to ascertain whether combination XO inhibitor (oxypurinol) and .OH scavenger (Na benzoate) therapy, given at the time of renal ischemia, alters the extent of: (1) tubular necrosis and filtration failure; (2) DNA fragmentation/apoptosis (assessed in situ by terminal deoxynucleotidyl transferase reactivity); (3) early tubular regenerative responses (proliferating cell nuclear antigen expression; (3H)thymidine incorporation); and (4) the rate and/or degree of functional and morphologic repair. The effects of XO inhibition, .OH scavengers, and "catalytic" iron (FeSO4) on human proximal tubular cell proliferation in vitro were also assessed with a newly established cell line (HK-2). Male Sprague-Dawley rats were subjected to 35 min of bilateral renal arterial occlusion with or without oxypurinol/benzoate therapy. These agents did not alter the extent of tubular necrosis or filtration failure, proliferating cell nuclear antigen expression or thymidine incorporation, or the rate/extent of renal functional/morphologic repair. DNA fragmentation did not precede tubular necrosis, and it was unaffected by antioxidant therapy. By 5 days postischemia, both treatment groups demonstrated regenerating epithelial fronds that protruded into the lumina. These structures contained terminal deoxynucleotidyl transferase-reactive, but morphologically intact, cells, suggesting the presence of apoptosis. Oxypurinol and .OH scavengers (benzoate; dimethylthiourea) suppressed in vitro tubular cell proliferation; conversely, catalytic Fe had a growth-stimulatory effect. These results suggest that: (1) XO inhibition/.OH scavenger therapy has no discernible net effect on postischemic acute renal failure; (2) DNA fragmentation does not precede tubular necrosis, suggesting that it is not a primary mediator of ischemic cell death; and (3) antioxidants can be antiproliferative for human tubular cells, possibly mitigating their potential beneficial effects.

Topics: Acute Kidney Injury; Animals; Antioxidants; Apoptosis; Benzoates; Benzoic Acid; Cell Division; Cells, Cultured; DNA Damage; DNA Nucleotidylexotransferase; Free Radical Scavengers; Humans; Ischemia; Kidney; Kidney Tubular Necrosis, Acute; Male; Nuclear Proteins; Oxypurinol; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Regeneration; Reperfusion Injury; Xanthine Oxidase

Isolated rat hepatocytes were used for the evaluation of nucleotide depletion and oxidative stress as two causal components of postischemic injury following oxygen deficiency. The ATP and GTP loss during anoxia was accompanied by temporary increases of nucleotide degradation products. The critical duration of anoxia for a complete ATP restoration during reoxygenation was between 30 and 60 min. The oxidative stress during reoxygenation was demonstrated by decrease of GSH concentration and increase of TBA-RS level. The tremendous GSH loss could not be balanced by the slight GSSG increase during reoxygenation. Prevention of GSH decrease and TBA-RS increase in parallel to prevention of viability loss in presence of oxipurinol in contrast to lacking improvement of ATP and GTP restoration by this drug speak in favor for the oxidative stress as major causal component for postischemic injury of hepatocytes in comparison with depletion of energy-rich purine nucleotides. The inhibition of formation of reactive oxygen species via xanthine oxidase reactions was found to be the dominant protective effect of oxipurinol against postischemic injury of hepatocytes in comparison with lacking influence on nucleotide salvage and ATP/GTP regeneration and with radical scavenging.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Cell Hypoxia; Cell Survival; Cells, Cultured; Energy Metabolism; Glutathione; Guanosine Triphosphate; Liver; Male; Nucleotides; Oxidation-Reduction; Oxygen; Oxygen Consumption; Oxypurinol; Rats; Rats, Wistar; Reperfusion Injury; Stress, Physiological; Thiobarbituric Acid Reactive Substances

Free radical generation and release from the cerebral hemispheres of rats subjected to four vessel occlusion followed by reperfusion was monitored using a cortical cup technique in conjunction with the spin-trapping agent alpha(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Electron spin resonance (ESR) was used to detect the presence of free radical adducts of POBN in the cortical superfusates. 30 min of ischemia plus reperfusion resulted in the release of .OH radical adducts during the period of ischemia and, especially, during the initial phases of reperfusion. No radical adducts were detectable 90 min after the onset of reperfusion. Pretreatment with the xanthine oxidase inhibitor, oxypurinol (40 mg/kg i.p.), virtually abolished free radical formation and release. The results of this study are consistent with earlier evidence of free radical formation during ischemia/reperfusion and suggest that the cerebroprotective actions of oxypurinol may be related to its ability to prevent the cascade of free radical generation.

Topics: Animals; Cerebral Cortex; Electron Spin Resonance Spectroscopy; Hydroxyl Radical; Male; Nitrogen Oxides; Oxypurinol; Pyridines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spin Labels

We investigated whether xanthine oxidase (XO) is a major source of oxygen-derived free radicals (oxy-radicals) in the pig and human skeletal muscles. It was observed that xanthine dehydrogenase and XO activities in nonischemic pig latissimus dorsi (LD) and gracilis muscles and human LD and rectus abdominis (RA) muscles were < 0.5 mU/g wet wt. The pig LD muscle hypoxanthine content increased significantly from 0.33 +/- 0.02 to 2.33 +/- 0.44 mumol/g dry wt after 5 h of warm ischemia, but the muscle uric acid content remained unchanged up to 2 h of reperfusion. Similarly, the hypoxanthine content in the human LD and RA muscles increased from 0.33 +/- 0.03 to 0.84 +/- 0.23 mumol/g dry wt after 2.0-3.5 h of warm ischemia, and the muscle uric acid content remained unchanged at the end of 15-90 min of reperfusion. Furthermore, 5 days of allopurinol treatment (25 mg/kg iv twice daily) starting 2 days before ischemia or 3 days of oxypurinol treatment (25 mg/kg iv twice daily) starting 15 min before reperfusion did not attenuate the extent of skeletal muscle necrosis in pig LD muscles subjected to 5 h of ischemia and 48 h of reperfusion. However, deferoxamine treatment (250 mg/kg iv twice daily) starting before or after ischemia, as described above, significantly reduced the extent of pig LD muscle necrosis. Finally, at 2 and 48 h of reperfusion significantly higher muscle neutrophil contents were seen in ischemic than in nonischemic control pig LD muscles. Neutrophil depletion with mechlorethamine (0.75 mg/kg iv) significantly reduced the extent of necrosis in pig LD muscles. These observations indicate that XO is not a major source of oxy-radicals in ischemia/reperfusion injury in the pig gracilis and LD muscles and human RA and LD muscles.

Topics: Adult; Allopurinol; Animals; Blood Cell Count; Chromatography, High Pressure Liquid; Female; Humans; Hypoxanthines; Male; Mechlorethamine; Muscles; Necrosis; Neutrophils; Orchiectomy; Oxypurinol; Peroxidase; Reactive Oxygen Species; Reperfusion Injury; Swine; Uric Acid; Xanthine Dehydrogenase; Xanthine Oxidase; Xanthines

Oxygen-based free radicals produced by the enzyme xanthine oxidase may be involved in postischemic reperfusion injury. To determine whether oxypurinol, a xanthine oxidase inhibitor and the major metabolite of allopurinol, attenuates renal ischemic reperfusion injury, and, if so, to determine its most effective dose, oxypurinol 2.5, 5, 10 or 20 mg/kg BW was infused 20 min prior to 20 min of complete renal ischemia in uniephrectomized rats. Animals treated with 5 mg/kg BW oxypurinol had significantly higher creatinine clearances on the first and second days postischemia than did untreated animals. In other animals given either buffered saline or oxypurinol at 5 mg/kg BW i.v. 20 min before ischemia, the inulin clearance (CIn) returned to near-control values within 1 h after ischemia. At 24 h there was a secondary decline in the CIn in animals receiving buffered saline, whereas in the animals treated with oxypurinol, this decline was less evident. In animals given oxypurinol at 5 mg/kg BW 40 min after ischemia, the CIn was significantly greater than in those receiving buffered saline. No changes in renal blood flow or renal vascular resistance were observed, suggesting that the effect of oxypurinol was not hemodynamically mediated. Analysis of plasma hypoxanthine, xanthine, uric acid and oxypurinol levels by high-pressure liquid chromatography revealed that in the absence of oxypurinol, a significant increase in uric acid production occurred between 20 and 170 min after the period of ischemia. In the presence of oxypurinol, there was a marked reduction in the rate of production of uric acid for the first 3 h postischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Free Radical Scavengers; Kidney; Kidney Function Tests; Male; Oxypurinol; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Uric Acid

The objective of this study was to determine whether agents that either scavenge or inhibit the production of oxygen radicals can alter the adhesive interactions between leukocytes and venular endothelium elicited by ischemia-reperfusion. Cat mesenteric and intestinal blood flows were reduced to 20% of baseline for 1 hr, followed by 1 hr of reperfusion. Sixty minutes after reperfusion, red blood cell velocity (Vr), leukocyte rolling velocity (Vw), and the number of adherent leukocytes were measured in mesenteric venules. Then, either manganese-superoxide dismutase (Mn-SOD), catalase, desferrioxamine, or oxypurinol was administered intravascularly. Ten minutes later, repeat measurements were obtained and compared with pretreatment values. Catalase, Mn-SOD, and oxypurinol significantly attenuated neutrophil adherence while neither inactivated-catalase nor desferrioxamine altered the reperfusion-induced leukocyte adhesion. The ratio of Vw to erythrocyte velocity, an index of the fracture stress between rolling leukocytes and venular endothelium, was not altered by any of the agents studied. These results and data in the literature indicate that many of the agents that are commonly used to either scavenge or inhibit the production of oxygen radicals in postischemic tissues exert a significant inhibitory influence on leukocyte adhesion to microvascular endothelium in vivo. Our results are also consistent with the view that xanthine oxidase-derived oxidants contribute to the leukocyte-endothelial cell adhesive interactions associated with reperfusion of ischemic tissues.

Topics: Animals; Catalase; Cats; Cell Adhesion; Cell Communication; Deferoxamine; Dose-Response Relationship, Drug; Endothelium, Vascular; Leukocytes; Manganese; Oxypurinol; Reperfusion Injury; Superoxide Dismutase

An increased formation of oxygen free radicals in the reperfused rat small intestine is concluded from accumulations of oxidized glutathione, of thiobarbituric acid-reactive substances and of 4-hydroxynonenal. Xanthine oxidase inhibition prevented these biochemical changes. The histological and electronmicroscopic studies of intestinal sucosa showed significant structural deteriorations already at the end of the ischemic period obviously due to disturbances of cellular energy metabolism. The extent of dosage was increased during the reperfusion without qualitative changes of the pattern of structural dosage. The beneficial effects of oxypurinol on biochemical criteria which occurred already in the early phase of reperfusion were not reflected in significant morphological differences within the first hour of reperfusion. Differences of morphological findings between oxypurinol-treated and untreated animals could be observed after longer periods of reperfusion--during the regeneration of the tissue.

Topics: Animals; Free Radicals; Glutathione; Intestinal Mucosa; Ischemia; Jejunum; Male; Microscopy, Electron; Oxypurinol; Rats; Rats, Inbred Strains; Reperfusion Injury

An in vitro canine tibia model was used to assess the effects of 48 h of hypothermic (4 degrees C) ischemia on bone vascular resistance and on responsiveness of intraosseous blood vessels to circulating norepinephrine. Three groups of bones were studied: Group I (n = 11), 48 h hypothermic ischemia; Group II (n = 11), 48 h hypothermic ischemia with pretreatment with allopurinol and oxypurinol; and Group III (n = 10), no ischemia. Resting vascular resistance in both ischemic groups (79 and 74 mmHg/ml/min) was significantly higher (p less than 0.0001) than in the nonischemic group (22 mmHg/ml/min). Effects of norepinephrine on vascular resistance were significantly greater in both ischemic groups (p less than 0.004). In all three groups, acetylcholine infusion attenuated the increases in perfusion pressure caused by norepinephrine. This demonstrates secretion of endothelial-mediated relaxing factors (EDRF) and prostaglandin for up to 48 h of hypothermic ischemia. As no significant differences were detected between the two ischemic groups, this study failed to demonstrate any protective effect of xanthine oxidase inhibitors.

Topics: Acetylcholine; Allopurinol; Animals; Cold Temperature; Disease Models, Animal; Dogs; Drug Hypersensitivity; Ischemia; Nitric Oxide; Norepinephrine; Oxypurinol; Regional Blood Flow; Reperfusion Injury; Tibia; Vascular Resistance; Xanthine Oxidase

Reoxygenation injury that occurs when blood circulation is restored to previously ischemic tissues is currently discussed as a pathophysiological entity distinct from the primary anoxic injury that develops during ischemia per se. To test the hypothesis that reoxygenation injury in hepatocytes is caused by a postischemic burst of reactive oxygen species (ROS), including superoxide radicals, O2-., and hydrogen peroxide, H2O2, we performed a cytochemical study exploiting the peroxidase activity within peroxisomes as a sensitive ultrastructural detector of intracellular H2O2 generation. The osmiophilic polymer formed when tissue peroxidase is incubated with 3,3'-diaminobenzidine (DAB) and H2O2 was used as a marker for endogenous H2O2 in rat liver slices in short-term organ culture subjected to a cycle of 60-min ischemic anoxia and 30-min reoxygenation in the presence of DAB without exogenous H2O2. Peroxisomal reaction product was quantitatively evaluated in transmission electron micrographs of systematically sampled hepatocytes. Mean densities of positive peroxisomes per 1,000 micron2 (+/- SE) in liver slices subjected to various treatments were as follows: continuous anoxia (negative control) 0 +/- 0; normoxia + exogenous H2O2 (positive control) 45 +/- 12; normoxia only 26 +/- 2; ischemia-reoxygenation 13 +/- 6; ischemia-reoxygenation + xanthine oxidase inhibitor, oxypurinol 5 +/- 3; ischemia-reoxygenation + peroxidase inhibitor, aminotriazole 7 +/- 3. Endogenous H2O2 can be detected in hepatocytes by electron microscopic cytochemistry and may in part derive from xanthine oxidase, but it is not substantially increased in the postischemic state. We conclude that hepatocytes do not exhibit a postischemic burst of reactive oxygen species that could cause reoxygenation injury.

Topics: Amitrole; Animals; Histocytochemistry; Hydrogen Peroxide; Ischemia; Liver; Liver Diseases; Male; Microbodies; Microscopy, Electron; Oxygen; Oxypurinol; Rats; Rats, Inbred Strains; Reperfusion Injury

Skeletal muscle edema secondary to an increase in capillary permeability after reflow is an important cause of the compartment syndrome after acute arterial revascularization. The purpose of this study was to investigate the possible role of oxygen free radicals, generated at reperfusion, in the pathogenesis of the compartment syndrome secondary to acute arterial ischemia/reperfusion. A reproducible model of this syndrome was produced in anesthetized rabbits by femoral artery occlusion after surgical devascularization of collateral branches from the aorta to the popliteal artery. Increasing periods of ischemia from 6 to 12 hours, followed by 2 hours of reperfusion, were associated with corresponding increases in the anterior muscle compartment hydrostatic pressure and inversely proportional decreases in tibialis anterior muscle blood flow within that compartment as assessed by xenon 133 washout (n = 46) (r = -0.62, p less than 0.001). Anterior compartment pressure increased from 5 +/- 1 to 48 +/- 5 mm Hg (n = 46) (p less than 0.001) after 7 hours of total arterial ischemia and 2 hours of reperfusion. Ablation of free radicals generated from xanthine oxidase with either allopurinol (n = 8) or oxypurinol (n = 8), by scavenging the superoxide radical at reperfusion with superoxide dismutase (n = 8), or by blocking secondary hydroxyl radical formation with deferoxamine (n = 8) significantly ameliorated the rise in compartment pressure (p less than 0.05) in each case; it also significantly improved muscle perfusion in the superoxide dismutase-, allopurinol-, and deferoxamine-treated animals (p less than 0.05). These findings indicate that development of the compartment syndrome after acute arterial revascularization may be due, at least in part, to microvascular injury mediated by oxygen-derived free radicals generated from xanthine oxidase at reperfusion.

Topics: Allopurinol; Animals; Blood Pressure; Compartment Syndromes; Deferoxamine; Free Radicals; Ischemia; Oxygen; Oxypurinol; Rabbits; Regional Blood Flow; Reperfusion Injury; Superoxide Dismutase

During the last few years, the scientific field has focused its attention on the pathogenic role of free radicals in the process of ischemia-revascularization. It is a well-known fact that xanthine oxidase is an important source of tissular free radicals. Bearing this in mind, we designed an experimental protocol to analyse the effect of allopurinol (a xanthine oxidase inhibitor) in the survival of rats after the occlusion of the superior mesenteric artery during a period of 90 minutes and its action on the superoxide radical liberation. The concentration of oxipurinol and allopurinol in the ischemic area (intestine), liver and blood were measured. We concluded that the administration of allopurinol increased the survival rate, which is correlated to higher concentrations of allopurinol and oxipurinol in the inner part of the intestinal cells. A correlation between the survival rate and superoxide radicals was not found.

Topics: Acute Disease; Allopurinol; Animals; Female; Free Radicals; Intestines; Ischemia; Male; Oxypurinol; Prognosis; Purines; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxides; Xanthine Oxidase