Rooster frozen-thawed semen quality following sublethal xanthine oxidase treatments.

Reactive oxygen species are associated with cryodamage and may be a factor causing or exacerbating cellular cryodamage during freezing and thawing processes. Induction of sublethal oxidative stress as a new approach for preconditioning of sperm improves the cryo-resistance of sperm. The aim of this study was to investigate effects of sublethal concentrations of xanthine oxidase (XO), which induces oxidative stress before cryopreservation on values for semen quality variables of rooster sperm post-thawing. Semen samples were collected from 15 roosters and treated with different concentrations of XO [XO-0, XO-0.005, XO-0.05, XO-0.5, XO-5, and XO-50 U/ml]; then, the effects of treatments with XO as sublethal stressors, were examined. Results indicated the XO-0.5 and XO-5 treatments resulted in a greater percentage of sperm total motility, progressive motility, viability, and membrane functionality compared to other groups. There was no difference after treatments with XO-0, XO-0.005, and XO-0.05 on sperm total motility, membrane functionality, apoptosis, mitochondria activity, and viability. There was a greater percentage of mitochondria activity in sperm of the XO-0.05, XO-0.5, and XO-5 groups. Furthermore, there was the greatest concentration of malondialdehyde (MDA) in samples of the XO-50 group. Values for sperm abnormal morphology, acrosome integrity, and DNA fragmentation were not different among samples post-thawing. Sperm treated with XO-0.5 and XO-5 had a greater fertilization capacity than those of the control group. In conclusion, treatment of sperm with 0.5 and 5 U/ml XO as inducers of mild oxidative stress before cryopreservation, improved several function quality indices of sperm post-thawing.

Astaxanthin attenuated hyperuricemia and kidney inflammation by inhibiting uric acid synthesis and the NF-κ B/NLRP3 signaling pathways in potassium oxonate and hypoxanthine-induced hyperuricemia mice.

Inflammation is an important pathological feature of hyperuricemia, which in turn aggravates hyperuricemia. Astaxanthin is a carotenoid with strong antioxidant capacity and possesses many biological activities. This study was aimed to evaluate the effect of astaxanthin (ASX) on hyperuricemia and kidney inflammation in potassium oxonate (PO) and hypoxanthine (HX)-induced hyperuricemic mice. Male ICR mice were administered intragastrically with PO and HX (250 mg/kg, respectively) for 14 days. ASX was given by gavage one hour after PO and HX administration. ASX treatment significantly reversed PO and HX-induced hyperuricemia and kidney inflammation in mice as evidenced by decreased serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), and inflammatory factors (IL-1ß, IL-6, and TNF-α) and increased activities of antioxidant enzymes (CAT, SOD and GSH-Px). Furthermore, ASX administration effectively inhibited the activities of key enzymes related to UA synthesis (xanthine oxidase (XOD) and adenosine deaminase (ADA)) and modulated the protein expressions of NF-κ B p65, p-NF-κ B p65, Iκ Bα, p-Iκ Bα, NLRP3, ASC, Caspase-1, and cleavedCaspase-1 involved in inflammation pathways. Our results suggested that ASX improved hyperuricemia and kidney inflammation induced by PO and HX, probably by reducing UA synthesis and suppressing the NF-κ B and NLRP3 pathways simultaneously.

Characterization of superoxide dismutases in cardiac progenitor cells demonstrates a critical role for manganese superoxide dismutase.

Transplantation of cardiac progenitor cells (CPCs) is currently in early clinical testing as a potential therapeutic strategy. Superoxide is increased in the ischemic myocardium and poor survival of cells is one of the major limitations of cell transplantation therapy. Superoxide dismutase (SOD) levels were analyzed in c-kit-positive CPCs isolated from rat myocardium to identify their roles in protection against oxidative stress-induced apoptosis in vitro. CPCs were subjected to oxidative stress using xanthine/xanthine oxidase (XXO) and little apoptosis was detected. CPCs contained significantly higher levels of SOD1 and SOD2 as compared with adult cardiac cell types, both at the protein and activity levels. Both SOD1 and SOD2 were increased by XXO at the mRNA and protein level, suggesting compensatory adaptation. Only knockdown of SOD2 and not SOD1 with siRNA sensitized the cells to XXO-apoptosis, despite only accounting for 10% of total SOD levels. Finally, we found XXO activated Akt within 10 min, and this regulated both SOD2 gene expression and protection against apoptosis. Rat CPCs are resistant to superoxide-induced cell death, primarily through higher levels of SOD2 compared to adult cardiac-derived cells. Exposure to superoxide increases expression of SOD2 in an Akt-dependent manner and regulates CPC survival during oxidative stress.

Allopurinol acutely increases adenosine triphospate energy delivery in failing human hearts.

OBJECTIVES: This study tested the hypothesis that acute administration of the xanthine oxidase (XO) inhibitor allopurinol improves cardiac high-energy phosphate concentrations in human heart failure (HF) and increases the rate of adenosine triphosphate (ATP) synthesis through creatine kinase (CK), the primary myocardial energy reserve. BACKGROUND: Studies of patients and animal models implicate impaired myocardial high-energy phosphate availability in HF. The XO reaction is a critical terminal step in ATP and purine degradation and an important source of reactive oxygen species. Thus, XO inhibition is a potentially attractive means to improve energy metabolism in the failing human heart. METHODS: We randomized 16 patients with nonischemic cardiomyopathy in a double-blind fashion to allopurinol (300 mg intravenously) or placebo infusion, 4-to-1, the latter for purposes of blinding only. The myocardial concentrations of ATP and creatine phosphate (PCr) and the rate of ATP synthesis through CK (CK flux) were determined by (31)P magnetic resonance spectroscopy. RESULTS: Allopurinol infusion increased mean cardiac PCr/ATP and PCr concentration by ∼11% (p < 0.02), and mean CK flux by 39% (2.07 ± 1.27 µmol/g/s to 2.87 ± 1.82 µmol/g/s, p < 0.007). Calculated cytosolic adenosine diphosphate concentration decreased, whereas the free energy of ATP hydrolysis (ΔG(∼ATP)) increased with allopurinol. The increased CK flux was disproportionate to substrate changes, indicating increased CK enzyme activity. CONCLUSIONS: Intravenous administration of the XO inhibitor allopurinol acutely improves the relative and absolute concentrations of myocardial high-energy phosphates and ATP flux through CK in the failing human heart, offering direct evidence that myofibrillar CK energy delivery can be pharmaceutically augmented in the failing human heart. (Intravenous Allopurinol in Heart Failure; NCT00181155).

Addition of erythrocytes to in vitro culture medium attenuates the detrimental effects of reactive oxygen species on bovine preimplantation embryo development.

Erythrocytes were recently found to improve the early development of mice embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine in vitro fertilized (IVF) embryos in medium supplemented with reactive oxygen species (ROS). IVF embryos were cultured in CR1aa medium supplemented with oxidizing agents, 0.5mmol/L hypoxanthine and 0.01U/mL xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5×10(4) , 5×10(5) , 5×10(6) and 5×10(7) erythrocytes/mL). After 8 days, blastocysts were examined with a stereomicroscope. HX/XOD blocked development to the blastocyst stage (HX/XOD: 0%, control: 33%), but in the presence of both erythrocytes and HX/XOD, blastocyst development was restored to about one-third to two-thirds the normal rate (5×10(5) to 5×10(7) erythrocytes/mL: 12 to 23%). Furthermore, adding erythrocytes or erythrocyte hemolysate to medium without HX/XOD increased the blastocyst rate. These results suggest that the addition of erythrocytes can attenuate the detrimental effects of ROS on embryo development in bovine species as well as in mice.

Potential contribution of oxidative stress and inflammation to anxiety and hypertension.

Previously, we have published that pharmacological induction of oxidative stress causes anxiety-like behavior in rats and also is associated with hypertension in these animals. Here, we report that sub-chronic induction of oxidative stress via pharmacological induction leads to i) reduction in glyoxalase (GLO)-1 and glutathione reductase (GSR)-1 expression; ii) calpain mediated reduction of brain derived neurotrophic factor (BDNF) levels; iii) NFκB mediated upregulation of proinflammatory factors interleukin (IL)-6 and tumor necrosis factor (TNF)-α and elevated angiotensin (AT)-1 receptor levels in hippocampus, amygdala and locus coeruleus regions of the brain. Acute oxidative stress has opposite effects. We speculate that regulation of GLO1, GSR1, BDNF, NFκB and AT-1 receptor may contribute to anxiety-like behavior and hypertension in rats.

The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells.

Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.

Effect of oxidative stress on glial cell volume.

Cytotoxic brain edema is a major contributor of tissue damage following cerebral ischemia and traumatic brain injury. The pathophysiology of cytotoxic edema formation is still not well understood. Although it is widely believed that oxidative stress causes cytotoxic brain edema, experimental proof is lacking. The aim of the present study was therefore to examine the effect of oxidative stress on cell volume of glial cells. C6 glial cells were exposed to hydrogen peroxide and the superoxide forming complex hypoxanthine/xanthine oxidase (HX/XO). Exposure to hydrogen peroxide (0.5-5 mM) resulted in initial cell shrinkage by 5.7 +/- 1.5% (mean +/- SEM; p < 0.05) and was followed by a dose-dependent recovery to baseline. Exposure to superoxide anions generated by HX/XO provoked a delayed, but sustained decrease of cell volume by 11.8 +/- 0.9% (p < 0.05). Cell volume showed no tendency to recover upon sustained exposure to superoxide. Neither hydrogen peroxide nor HX/XO exposure was associated with a decrease of cell viability. Thereby, the present study demonstrates that oxidative stress by hydrogen peroxide and superoxide anions does not induce cytotoxic cell swelling and suggests that free radicals are not directly involved in the formation of cytotoxic brain edema.

Direct vascular effects of HR780, a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor.

1. We have examined the effects of HR780, a novel 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on porcine endothelial cell (EC) injury induced by xanthine (X)/xanthine oxidase (XO), a source of superoxide anion. Furthermore, the effects of HR780 on platelet-derived growth factor (PDGF)-induced migration and fetal calf serum (FCS)-induced proliferation of rabbit smooth muscle cells (SMC) were investigated. 2. Probucol, at 10 micro mol/L, significantly (P < 0.001) and completely suppressed lactate dehydrogenase leakage induced by X/XO. At 10 micro mol/L, HR780 significantly (P = 0.010) inhibited X/XO-induced EC injury. 3. HR780 dose-dependently inhibited PDGF-induced SMC migration and FCS-induced SMC proliferation. The addition of mevalonate completely abolished the inhibitory effect of HR780 on SMC proliferation. Another HMG-CoA reductase inhibitor, simvastatin (0.1-100 micro mol/L), also inhibited the migration and proliferation responses as potently as HR780. In contrast, pravastatin (0.1-100 micro mol/L) did not show any effects. 4. This in vitro study provides the first evidence that HR780 protects the vascular endothelium from oxidant stress and inhibits the migration and proliferation of SMC.

Neutrophil elastase and acute lung injury: prospects for sivelestat and other neutrophil elastase inhibitors as therapeutics.

OBJECTIVES: To review the evidence and rationale that suggest that neutrophil elastase (NE) may contribute to the development of acute lung injury (ALI) and the acute respiratory distress syndrome. To review selected preliminary data regarding the effectiveness of NE inhibition in animals, in in vitro models, and in patients with ALI. DATA SOURCES: The published literature and observations provided by Ono Pharmaceutical and Eli Lilly investigators and their colleagues. DATA SUMMARY: Taken en toto, the data suggest that NE could contribute to ALI and endothelial cell injury that is relevant to ALI. Moreover, the toxic effects of NE are greatly enhanced by increased oxidative stress, which commonly occurs in patients with ALI. In addition to neutrophils, xanthine oxidase, a constituent of endothelial cells, is a potential source of oxidative stress in ALI; xanthine oxidase-derived oxidants enhance NE toxicity in in vivo, isolated lung, and in vitro endothelial cell test systems. Not surprisingly, endogenous nonoxidatively sensitive NE inhibitors (e.g., eglin C) are more effective in combating the detrimental effects of NE than oxidatively sensitive NE inhibitors (e.g., alpha-1-proteinase inhibitor). In addition, a synthetic NE inhibitor, sivelestat (ONO-5046 and LY544349), is effective in reducing measures of inflammation and injury in multiple animal models of ALI. In a trial of ALI patients with systemic inflammatory response syndrome, conducted in Japan by Ono Pharmaceutical scientists, sivelestat treatment improved the investigator assessment of global improvement and the percentages of patients who were removed from ventilators and transferred out of the intensive care unit. CONCLUSIONS: Further study of the role of NE inhibition as a treatment for ALI is warranted. Additional clinical and preclinical studies with sivelestat and various other NE inhibitors should not only clarify the clinical potential of this intervention strategy, but also better define the activities of NE in inflammatory disorders such as ALI and multiple organ failure.

Hypercapnic acidosis may attenuate acute lung injury by inhibition of endogenous xanthine oxidase.

Relative hypoventilation, involving passively-or "permissively"-generated hypercapnic acidosis (HCA), may improve outcome by reducing ventilator-induced lung injury. However, the effects of HCA per se on pulmonary microvascular permeability (Kf,c) in noninjured or injured lungs are unknown. We investigated the effects of HCA in the isolated buffer-perfused rabbit lung, under conditions of: (1) no injury; (2) injury induced by warm ischemia-reperfusion; and (3) injury induced by addition of purine and xanthine oxidase. HCA (fraction of inspired carbon dioxide [FICO2] 12%, 25% versus 5%) had no adverse microvascular effects in uninjured lungs, and prevented (FICO2 25% versus 5%) the increase in Kf,c following warm ischemia-reperfusion. HCA (FICO2 25% versus 5%) reduced the elevation in Kf,c, capillary (Pcap), and pulmonary artery (Ppa) pressures in lung injury induced by exogenous purine/xanthine oxidase; inhibition of endogenous NO synthase in the presence of 25% FICO2 had no effect on Kf,c, but attenuated the reduction of Pcap and Ppa. HCA inhibited the in vitro generation of uric acid from addition of xanthine oxidase to purine. We conclude that in the current models, HCA is not harmful in uninjured lungs, and attenuates injury in free-radical-mediated lung injury, possibly via inhibition of endogenous xanthine oxidase.

Cardiac depressant effects of oxygen free radicals.

In many clinical situations, including cardiac ischemia/reperfusion, elective cardiac arrest, and renal dialysis, the chances of increased production of oxygen free radicals (OFR) exist. OFR have been implicated as a causative factor of cell damage in several pathologic conditions. The effects of exogenous OFR, generated by xanthine plus xanthine oxidase, in the absence and in the presence of OFR scavenger (superoxide dismutase [SOD]) on the contractility of isolated perfused heart of rabbit were studied. OFR produced concentration-dependent decreases in the contractility of perfused heart. SOD prevented the OFR-induced decreases in the left ventricular contractility. Xanthine produced an increase in the contractility of isolated perfused rabbit's heart. Xanthine oxidase produced a marked decrease in the left ventricular contractility. Repeated administration of xanthine oxidase produced accelerated and greater decreases in the contractility of perfused heart when compared with that of the initial administration of the drug. Effects of xanthine or xanthine oxidase on the cardiac function and contractility were also studied in anesthetized dogs. Xanthine alone had no significant effect on the cardiac function and indices of myocardial contractility. However, xanthine oxidase produced a marked decrease in the mean aortic pressure, left ventricular work index, heart rate, cardiac index, left ventricular systolic pressure, left ventricular end-diastolic pressure, (+) and (-) dp/dt of left ventricular pressure, and other indices of myocardial contractility [(dp/dt)/PAW (pulmonary arterial wedge pressure)]; and an increase in the total systemic and pulmonary vascular resistance. Repeated administration of xanthine oxidase in anesthetized dogs had lesser effects on the cardiovascular system when compared with those from the initial dose of the drug. These results suggest that OFR are cardiac depressant. Clinical situations wherein there is an increased production of OFR or increased formation of xanthine and xanthine oxidase may be associated with decreased cardiac function and contractility. Scavengers of OFR may protect the heart from the deleterious effects of OFR in such clinical conditions.

Protective effect of anisodamine on cultured bovine pulmonary endothelial cell injury induced by oxygen-free radicals.

Anisodamine, a Chinese traditional medicine herb, has been used for treatment of adult respiratory distress syndrome effectively, but little is known about its mechanism. We attempted to investigate if anisodamine could protect bovine pulmonary endothelial cell injury induced by exogenous oxygen-free radicals that were generated by xanthine/xanthine oxidase or opsonized zymosan-stimulated polymorphonuclear leukocytes. Results showed that with the addition of xanthine/xanthine oxidase into cultured bovine pulmonary endothelial cells, production of malondialdehyde and release of lactate dehydrogenase in supernatant increased, and synthesis of prostacyclin decreased. Damaged cellular membranes were revealed by scanning electron microscopy. The same was true for the addition of opsonized zymosan-stimulated polymorphonuclear leukocytes. While treatment with anisodamine greatly attenuated all of the above-mentioned parameters, results showed that (1) cultured bovine pulmonary endothelial cells could be damaged by oxygen-free radicals, (2) anisodamine had a protective effect on this injury as effective as that of superoxide dismutase and catalase, and (3) the membrane-stable action might contribute to the mechanism of protective effect against this injury.

[Effect of oxygen free radicals on the rat pancreas in vivo].

Many reports concerning the involvement of active oxygen free radicals in the pathogenesis and progression of acute pancreatitis have been published. In this study, the direct toxic effect of active oxygen free radicals on the rat pancreas was evaluated in vivo. Superoxide anions, generated via the xanthine/xanthine oxidase (X/XO) system, and hydrogen peroxide (H2O2) were used. After continuous arterial injection of X/XO into the celiac artery hemorrhage and extensive edema developed. However, additional continuous injection of superoxide dismutase (SOD) into the external jugular vein completely suppressed the hemorrhage and relieved the edema. When hydrogen peroxide (100 microM/Kg/hour) was injected continuously through the celiac artery made hemorrhage and edema were recognized in the pancreas, both of which were suppressed by continuous injection of catalase (10 mg/Kg/hour) or gabexate mesilate (10 mg/Kg/hour) into the external jugular vein. The amylase and lipase levels in the intraperitoneal fluid rose to more than 10 times the preoperative values 5 hours after drug administration. These levels were lowered to 2 times the preoperative values by the continuous venous injection of SOD or catalase (which are specific scavengers of superoxide anions or hydrogen peroxide, respectively) or by gabexate mesilate. On the other hand, serum amylase and lipase levels remained almost constant throughout the entire experiment. Thus, the administration of active oxygen free radicals caused acute pancreatitis, which was suppressed by the systemic administration of specific scavengers for each free radical. Active oxygen free radicals were shown to have a direct, toxic effect on the pancreas.

Homogenized bovine milk xanthine oxidase: a critique of the hypothesis relating to plasmalogen depletion and cardiovascular disease.

A hypothesis has repeatedly been promoted that xanthine oxidase from homogenized bovine milk is absorbed intact, damaging cardiovascular tissue by depleting plasmalogens and initiating atherosclerotic changes that culminate in heart disease. In the light of recent experimental evidence, the present paper examines the validity of this hypothesis and associated claims. The evidence leads to the conclusion that 1) absorption of dietary xanthine oxidase has not been demonstrated; 2) a relationship between intakes of homogenized milk and levels of serum xanthine oxidase activity have not been established; 3) a direct role for xanthine oxidase in plasmalogen depletion has not been established; 4) neither liposome formation during homogenization of milk nor absorption of intact liposomes from the gastrointestinal tract has been demonstrated; and 5) data are lacking to support the claim that large doses of folic acid inhibit xanthine oxidase in vivo and/or are therapeutic in heart disease. Experimental evidence has failed to substantiate, and in many cases has refuted, the xanthine oxidase/plasmalogen depletion hypothesis.

Homogenized milk and atherosclerotic disease: a review.

The theory that consumption of homogenized milk containing active xanthine oxidase is a causative factor in development of atherosclerosis is reviewed. Biologically available xanthine oxidase in consumed milk products may be absorbed in the small intestine and enter the blood stream. However, there appears to be no unequivocal evidence that the absorbed enzyme has any pathological effects that may contribute to development of atherosclerotic heart disease.