Increase In Xanthine Oxidase Activity Research Articles

Abstract P199: Uric Acid Promotes Vascular Stiffness, Immune Inflammatory Response and Proteinuria in Western Diet Fed Mice

Increased consumption of a diet high in fructose and fat (western diet, WD) is associated with an increase in cardiovascular disease (CVD) and kidney injury. In this regard, excess hepatic production of uric acid generated from excess fructose consumption is emerging as a risk factor for vascular stiffness, which underpins CVD and kidney injury. We hypothesized that a WD would increase uric acid levels and cardiovascular and renal xanthine oxidase (XO) activity and associated increased vascular stiffness and proteinuria. Furthermore, we proposed that inhibition of XO activity would prevent arterial stiffening and reduce proteinuria in a clinically relevant model of WD-induced CVD and renal injury. Four week-old C57BL6/J male mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without allopurinol (125mg/L), a potent XO inhibitor for 16 weeks. XO inhibition significantly attenuated WD-induced increases in plasma and urine uric acid levels and aortic XO activity (WD, 0.225 + 0.031 mU/mL WD + allopurinol, 0.097+ 0.026mU/mL, P<0.05), as well as proteinuria (WD, 20.92 + 2.66 mg/ mg creatinine, WD + allopurinol, 13.48 + 1.56 mg/mg creatinine, P<0.05). XO inhibition had no effect on increases in body weight, fat mass, and HOMA-IR promoted by the WD. Blood pressure was not different between any of the groups. Stiffness of aortic endothelial cells, extracellular matrix and vascular smooth muscle cells, as determined by atomic force microscopy, was significantly increased in WD mice and this was prevented by XO inhibition. WD induced a significant macrophage pro-inflammatory response in aorta that was significantly suppressed by XO inhibition. Collectively, these findings support the notion that increased XO activity in the vasculature and kidney and increased hepatic production of uric acid secondary to consumption of a WD promotes vascular stiffness, vascular inflammation and a maladaptive immune response that lead to vascular stiffness and kidney injury.

PP58 - Oxidative Stress and Ubiquitin Ligases: their involvement in skeletal muscle atrophy

Introduction Muscle atrophy plays a relevant role in the many very prevalent diseases. Generation of reactive oxygen species (mainly by the xanthine oxidase) and inflammation are two of the main triggers of muscle atrophy. Aim The major aim of our study was to determine the mechanism by which reactive oxygen species activate E3 ubiquitin ligases (MuRF-1 and MAFbx) cause muscle atrophy. Possible prevention by allopurinol, a well-known xanthine oxidase inhibitor widely used in clinical practice; and by indomethacin, a non-steroidal antiinflamatory drug was also studied. Materials and methods Male C57BL/6J mice (3 months old) conditioned by 14 days of hindlimb unloading with or without each treatment or the combination of both of them (n=48) were compared with freely ambulating controls (n=48). Results After the experimental intervention, we found that hindlimb unloading induced a significant increase in xanthine oxidase activity and prostaglandins in plasma ( 209%, p Conclusions Our data point out the potential benefit of allopurinol and indomethacin administration for bedridden, astronauts or muscle disuse; as well as a potential benefit in other atrophy models such as pathology-related cachexia or sarcopenia. Acknowledgments This work was supported by grants SAF2010- 19498, ISCIII2006-RED13-027, PROMETEO2010/074, 35NEURO GentxGent and EU Funded COSTB35 and CM1001. The study has been co-financed by FEDER funds from the European Union.

Zingerone protects against cisplatin-induced oxidative damage in the jejunum of Wistar rats

Cisplatin (cis-diamminedichloroplatinum (II) (CDDP)) is a commonly used chemotherapeutic drug for the treatment of numerous forms of cancer, but it has marked adverse effects, namely nephrotoxicity, hepatotoxicity, ototoxicity, neurotoxicity, diarrhoea etc. CDDP-induced emesis and diarrhoea are also noticeable toxicities that may be due to intestinal injury. Zingerone; a phenolic alkanone, one of the active components of ginger, possesses multiple biological activities, such as antioxidant and anti-inflammatory properties. In the present study, we investigated the protective effect of zingerone against CDDP-induced jejunal toxicity. Animals were divided into five groups I-IV (n = 6). Group II, III and IV received single intraperitoneal administration of CDDP at 7.5 mg/kg body weight on day 14. Animals of group II and III received oral treatment of zingerone at doses of 25 and 50 mg/kg body weight respectively for 14 consecutive days. While groups I was given distilled water 5 ml/kg body weight for 14 days. All the animals were killed after 24 h of CDDP injection. Zingerone ameliorated CDDP-induced lipid peroxidation, increase in xanthine oxidase activity, glutathione depletion, decrease in antioxidant and phase-II detoxifying enzyme activities. Zingerone attenuated CDDP-induced nuclear factor (NF-κB) activation, enhanced levels of TNF-α and Nitrite. The results showed that zingerone had not only the antioxidant effect by suppression of ROS, but also anti-inflammatory effects by suppression of NF-κB activation. In addition, zingerone treatment suppressed gene activation of pro-inflammatory cytokine, TNF-α, which was up-regulated with CDDP administration through NF-κB activation. These experiments strongly indicate that zingerone treatment exercises a protective efficacy by suppressing both oxidative stress and inflammation through the modulation of key pro-inflammatory cytokine and transcription factors.

Normalizing dysfunctional purine metabolism accelerates diabetic wound healing.

Diabetic patients exhibit dysfunction of the normal wound healing process, leading to local ischemia by vascular occlusive disease as well as sustained increases in the proinflammatory cytokines and overproduction of reactive oxygen species (ROS). Of the many sources of ROS, the enzyme xanthine oxidase (XO) has been linked to overproduction of ROS in diabetic environment, and studies have shown that treatment with XO inhibitors decreases XO overactivity and XO-generated ROS. This study evaluates the role of XO in the diabetic wound and the impact of specifically inhibiting its activity on wound healing. Treatment of diabetic wounds with siXDH (xanthine dehydrogenase siRNA) decreased XDH mRNA expression by 51.6%, XO activity by 35.9%, ROS levels by 78.1%, pathologic wound burden by 31.5%, and accelerated wound healing by 7 days (23.3%). Polymerase chain reaction analysis showed that increased XO activity in wild-type wound may be due to XDH to XO conversion and/or XO phosphorylation, but not to gene transcription, whereas increased XO activity in diabetic wounds may also be from gene transcription. These results suggest that XO may be responsible for large proportion of elevated oxidative stress in the diabetic wound environment and that normalizing the metabolic activity of XO using targeted delivery of siXDH may decrease overproduction of ROS and accelerate wound healing in diabetic patients.

Study of serum uric acid and creatinine in hypertensive disorders of pregnancy.

BackgroundRenal dysfunction, increased xanthine oxidase activity and oxidative stress in placenta contributes to the elevated uric acid levels in preeclampsia (PE).ObjectiveTo determine serum uric acid and creatinine in hypertensive disorders of pregnancy and correlate with fetal outcome.Materials and MethodsPregnant women ≥32 weeks of gestation. Study population included 3 groups, 31 normotensive pregnant (NP) women as controls, 30 pregnant women with gestational hypertension (GH) and 30 with PE.ResultSerum uric acid and creatinine levels were significantly elevated in PE (6.26±1.19 and 0.94±0.26 mg/dL) when compared with Pregnancy induced hypertension (PIH) (4.27± 1.0 and 0.66 ±0.19 mg/dL) and NP (4.25 ± 0.8 and 0.63± 0.13 mg/dL) (P-value <0.001 and <0.001) respectively. Receiver operation characteristics curves demonstrated greater sensitivity and specificity for uric acid (86.7% and 83.9%, respectively) in PE than for creatinine (80% and 77.4%, respectively). Uric acid had strong and negative correlation with fetal birth weight in PE (r = −0.59, P = 0.006), where as creatinine had negative but weak correlation (r= −0.03, P=0.87).ConclusionSerum uric acid is a better diagnostic and predictive marker for PE and fetal outcome respectively.

Induction of xanthine oxidase activity, endoplasmic reticulum stress and caspase activation by sodium metabisulfite in rat liver and their attenuation by Ghrelin

Sodium metabisulfite is used as a preservative in many food preparations but can oxidize to sulfite radicals initiating molecular oxidation. Ghrelin is a peptide hormone primarily produced in the stomach and has anti-inflammatory and anti-oxidant effects on gastrointestinal and cardiovascular systems. This study was performed to elucidate the effect of ghrelin on sulfite-induced endoplasmic reticulum (ER) stress and caspase activation in rat peripheral organs. Xanthine oxidase (XO), xanthine dehydrogenase (XDH) enzyme activities, ER stress markers [phosphorylated PKR-like ER kinase (pPERK); C/EBP-homologous protein (CHOP)], caspase-3, -8, -9 activities, nuclear factor kappa-B (NF-κB) levels were determined in liver, heart and kidney of rats treated with sodium metabisulfite and/or ghrelin for 5 weeks. Sodium metabisulfite treatment significantly elevated XO activity, induced expression of GRP78, CHOP and increased caspase-3, -8 and -9 activities in liver but had no significant effect in heart and kidney. Ghrelin treatment decreased XO activity to baseline levels and attenuated ER stress and caspase activation in liver tissue of sodium metabisulfite treated rats. In conclusion, metabolism of sodium metabisulfite in liver tissue increased XO activity, induced ER stress and caused caspase activation which was attenuated by ghrelin treatment. Ghrelin's hepatoprotective effect could be through modulation of XO activity.

P67 - Redox regulation of E3 ubiquitin ligases and their role in skeletal muscle atrophy

Muscle atrophy is linked to reactive oxygen species (ROS) production during hindlimb-unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of our study was to determine the mechanism by which ROS cause muscle atrophy and its possible prevention by allopurinol, a well-known inhibitor of XO widely used in clinical practice, and indomethacin, a nonsteroidal anti-inflammatory drug. We studied the activation of p38 MAP Kinase and NF-?B pathways, and the expression of two E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFb) and Muscle RING Finger-1 (MuRF-1).Male Wistar rats (3 mold) conditioned by 14 days of hindlimb unloading (n=18), with or without the treatment, were compared with freely ambulating controls (n=18). After the experimental intervention, soleus muscles were removed, weighted and analyzed to determine oxidative stress and inflammatory parameters. We found that hindlimb unloading induced a significant increase in XO activity in plasma (39%, p=0.001) and in the protein expression of CuZnSOD and Catalase in skeletal muscle. Inhibitionof XO partially prevented protein carbonylation, both in plasma and in soleus muscle, in the unloaded animals.The most relevant new fact reported is that allopurinol prevents soleus muscle atrophy by ~20% after hindlimb unloading. Combining allopurinol and indomethacin we found a further prevention in the atrophy process. This is mediated by the inhibition of the p38 MAPK-MAFbx and NF-?B -MuRF-1 pathways. Our data point out the potential benefit of allopurinol and indomethacin administration for bedridden, astronauts, sarcopenic and cachexic patients.

Inhibition of key markers linked with spermatogenesis and cellular ATP by subchronic exposure to leachate in a rat model.

Few or no studies have measured the toxic effects of subchronic exposure to leachate using key markers linked with spermatogenesis and cellular adenosine triphosphate (ATP) in an experimental rat model. This study was undertaken to evaluate the toxic effects of leachate obtained from the Elewi Odo municipal battery-recycling site (EOMABRL) on male reproductive function using testicular hormones and biomarker of cellular ATP. EOMABRL was administered at 0, 20, 40, 60, 80, and 100% concentrations to adult male rats for 60 days. After exposure, serum was collected for hormonal biochemistry assays, and testes were collected to determine the activity of xanthine oxidase (XO) and lactate dehydrogenase (LDH). Exposure of animals to EOMABRL resulted in a 519.7, 285.7, 569.1, 606.1, and 1,793.2% increase in XO activity with a sequential decrease in LDH activity (marker of cellular ATP) by 44.1, 55.9, 61.4, 69.3, and 89.7%, respectively, compared with the control. Furthermore, EOMABRL caused a significant inhibitory effect on serum luteinizing hormone, follicle-stimulating hormone, and testosterone levels. We conclude that some possible mechanisms by which EOMABRL elicits toxicity in male rat testes could be through inhibition of LDH activity and depletion of serum hormone levels.

Abstract 379: Modulation of Inflammatory Signaling by PPARα and PPARγ in Acrolein-Treated gp91phox Knockout Mice

Acrolein, a copious environmental pollutant plays a role in development of inflammation in vasculature. Oxidative stress &amp; inflammation are important in acrolein-induced toxicity. Peroxisome Proliferator Activated receptor alpha and gamma (PPARα and PPARγ) and their ability to influence oxidative stress (OS) has been the focus of inflammatory signaling. NAD(P)H oxidase is a source of free radical generation. This study investigated the influence of PPARα and γ ligands (clofibrate &amp; GW1929) in attenuating oxidative stress in acrolein-induced insult. gp91phox knockout (KO) mice and age/weight matched wild type (WT) were treated with acrolein (0.5 μg/kg; i.p.; 7 days) with/without clofibrate (Clof: 250 mg/kg; i.p; 10 days) or GW1929 (5 mg/kg, gavage, 10 days). KO mice (1.44±0.14 pg/μg prt) had higher (97%) 8-Isoprostane in response to acrolein than WT (2.19±0.11 pg/μg prt). Clof had no effect on 8-Isoprostane but GW1929 reduced it in both WT and KO mice by 35% and 43% (p&lt;0.05) respectively. There was a significant reduction in Total Antioxidant Status (TAS) which was more prominent in WT (69%) than KO (53%). Clof and GW1929 improved TAS in WT (21% and 19%). Xanthine Oxidase (XO) enzyme activity was higher in KO (0.53±0.05) than WT (0.35+0.02). Acrolein and clof increased XO activity in WT while GW1929 had no effect. Acrolein increased cycloxygenase (COX) in WT (4.58±0.3) and remained at basal level in KO. Clof and GW1929 reduced COX in WT (54% and 48% respectively). Clof reduced Cox only in KO mice (31%). NFκB activity was twice in acrolein treated WT and remained unchanged in KO. Both GW1929 and clof blunted this increase. Acrolein increased CD36 activity, an inflammatory marker, in KO (56%). Clof attenuated this increase in KO but increased it in acrolein treated WT. GW1929 blunted CD36 increase in KO mice but did not affect WT. These results suggest that in absence of a functioning NAD(P)H oxidase other oxygenase contributes in acrolein-induced oxidative stress and PPARα ligand, clofibrate, is beneficial via attenuating inflammatory signaling of NFκB and COX. We also suggest that beneficial effect of PPARγ ligand involves inhibition of NAD(P)H oxidase as in absence of NAD(P)H this effects are minimal.

Sleeve gastrectomy reduces xanthine oxidase and uric acid in a rat model of morbid obesity

BackgroundSerum uric acid (sUA) plays a major role in the development of morbidities associated with obesity, especially cardiovascular diseases. Within the purine pathway, xanthine oxidase (XOD) represents the key enzyme. The aim of this study was to investigate the dynamics of sUA and XOD following sleeve gastrectomy (SG) in a rat model of high-fat-diet (HFD) induced obesity. PatientsOver a period of 11 weeks, 30 rats received a HFD, and 10 rats received a low fat diet (LFD). Thereafter, 10 randomly selected HFD rats and 10 LFD rats were sacrificed. The remaining 20 HFD rats were randomly assigned to either SG or sham operation (SH) and studied 14 days postoperatively. MethodsThe white adipose tissues (WAT) from visceral (intestinal and retroperitoneal) and inguinal (subcutaneous) depots were collected. sUA and urine UA (uUA) were measured by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Abundance and activity of XOD was investigated in the liver, colon, adipose tissue, and skeletal muscle by enzyme-linked immunosorbent assay (ELISA). ResultsHFD led to significant weight gain, elevated sUA levels, increased WAT and increase of XOD activity. Fourteen days postoperatively, SG rats showed a significant decrease of weight and adipose tissue, improved glucose metabolism, and changes of gut hormones. The sUA and uUA levels were significantly decreased following SG. Furthermore, XOD activity was significantly down-regulated in WAT. ConclusionHFD induces elevated sUA levels by gain of WAT and increase of XOD activity. Following SG, the reduction of WAT as the major source of XOD and the lowering of XOD activity are the basis for the decrease of sUA.

Oxidizing Effects of Exogenous Stressors in Huntington’s Disease Knock-in Striatal Cells—Protective Effect of Cystamine and Creatine

Huntington's disease (HD) is a polyglutamine-expansion disease associated to degeneration of striatal and cortical neurons. Previously, we showed that oxidative stress occurs in HD knock-in striatal cells, but little is known regarding cell antioxidant response against exogenous stimuli. Therefore, in the present study we analyzed cellular antioxidant profile following hydrogen peroxide (H2O2) and staurosporine (STS) exposure and tested the protective effect of cystamine and creatine in striatal cells expressing mutant huntingtin with 111 glutamines (STHdh (Q111/Q111); mutant cells) versus wild-type cells (STHdh (Q7/Q7)). Mutant cells displayed increased mitochondrial reactive oxygen species (ROS) and decreased NADPH oxidase and xanthine oxidase (XO) activities, reflecting lower superoxide cytosolic generation, along with increased superoxide dismutases (SODs) and components of glutathione redox cycle. Exposure to H2O2 and STS enhanced ROS in mutant cells and largely increased XO activity; STS further boosted the generation of mitochondrial ROS and caspase-3 activity. Both stimuli slightly increased SOD1 activity, without affecting SOD2 activity, and decreased glutathione reductase with a consequent rise in oxidized glutathione or glutathione disulfide in mutant cells, whereas H2O2 only increased glutathione peroxidase activity. Additionally, creatine and cystamine increased mutant cells viability and prevented ROS formation in HD cells subjected to H2O2 and STS. These results indicate that elevation of the antioxidant systems accompanies mitochondrial-driven ROS generation in mutant striatal cells and that exposure to noxious stimuli induces a higher susceptibility to oxidative stress by increasing XO activity and lowering the antioxidant response. Furthermore, creatine and cystamine are efficient in preventing H2O2- and STS-evoked ROS formation in HD striatal cells.

English

&nbsp; The aim of this study was to quantify xanthine oxidase (XO) levels during the transition period in Murrah buffalo (Bubalus bubalis) and determine its association with certain postpartum infections. For this, six healthy buffaloes were selected from the National Dairy Research Institute (NDRI) herd and managed under standard managemental practices as followed at the institute. Blood samples were drawn weekly from each buffalo from day -21 to +21 relative to parturition by jugular vein puncture. Additional blood samples were collected from buffaloes suffering from metritis (n=5), endometritis (n=6) and mastitis (n=8) on alternate days. XO activity followed a defined pattern with values gradually declining from day -21 up to the day of calving followed by an increase to day +21, but the difference was statistically not significant between pre- and post-partum stages. The activity significantly declined on the day of calving when compared to the pre-partum mean value (p&lt;0.05). The activity was significantly enhanced in buffaloes with bacterial infections, endometritis, and mastitis compared to healthy controls (p&lt;0.05). However, the levels were not significantly altered among buffalo with metritis. The results indicate that increased XO activity during the postpartum period was associated with some bacterial infections in buffaloes, which could be due to increased phagocytic activity as a part of the innate defense system. &nbsp; Key words:&nbsp;Xanthine oxidase, transition period.

Hyperuricemia induces endothelial dysfunction by increasing reactive oxygen species

Background: Hyperuricemia is frequently observed in patients with obesity, insulin resistance, and hyperlipidemia, and is linked to endothelial dysfunction. Elevated levels of circulating uric acid (UA) resulted in a decreased level of bioavailable nitric oxide (NO), but the molecular mechanism for this NO reduction is unclear. Therefore, the aim of the present study was to evaluate molecular changes elicited by increasing concentration of UA in endothelial cells. Methods: Human aortic endothelial cells (HAEC) were cultured in EGM-2 cell culture media and incubated with increasing concentrations of UA (0–500 μmol/l) for 24 hours. Acethylcholine (Ach)-induced NO production was measured in HAEC by quantification of DAF fluorescence at the single cell level. A proteome analysis of the UA incubated HAEC was performed by LC-MS/MS. Protein carbonylation and protein nitrosylation was assessed by western blot analysis. Enzyme actives of radical scavenging enzymes (gluthatione peroxidase, catalase) and xanthine oxidase were measured by commercial available assays. Results: Incubation of HAEC with increasing UA concentrations for 24h blunted the acethylcholine-induced NO production (0 μM Ach: 20±1.5%, 300 μM Ach: 9.5±1.3%, 500 μM Ach 5.0±4.8% stimulation in NO production vs. baseline). By LC-MS/MS 771 proteins were identified, and major changes were observed in cells stimulated with 300μM UA when compared to unstimulated cells. Ingenuity pathway analysis revealed that mainly the ubiquitin-proteasome pathway and proteins regulating oxidative stress were affected. Western blot analysis demonstrated a significant increase in protein carbonylation (0μM UA: 0.21±0.01 vs. 300μM UA: 0.28±0.02 arb units; p<0.05) and nitrostyrosinylation (0μM UA: 2.5±0.3 vs. 300μM UA: 9.8±0.6 arb. Units; p<0.01). This was accompanied by a reduction in catalase activity (0μM UA: 0.75±0.45 vs. 300μM UA: 0.25±0.2 arb units; p<0.01) and an increase in xanthine oxidase activity (0μM UA: 6.5±0.1 vs. 300μM UA: 9.1±0.3 arb. Units; p<0.01). The proteasome activity was significantly increased at the highest UA concentration (0μM UA: 0.42±0.08 vs. 500μM UA: 0.61±0.09 arb. units). Conclusion: Exposure of endothelial cells to UA, that is elevated in patients with hyperuricemia and associated endothelial dysfunction, resulted in increase in reactive oxygen species (ROS) due to a downregulation of radical scavenging enzymes and an upregulation of ROS producing enzymes. In addition, this leads to an activation of the ubiquitin proteasome system, which culminates in less bioavaible NO and subsequently endothelial dysfunction.

Xanthine Oxidase and Paraoxonase – 1 as a new markers in the diagnosis and prognosis of organophosphorus pesticide poisoning

Background: Acetylcholinesterase is already well known marker for organophosphorus poisoning in human beings. But, our interest was to known for another new markers. There is high limited study on xanthine oxidase and paraoxonase 1 in human beings having organophosphorus poisoning. Thus, aim of our this research was to estimate and correlate activities of xanthine oxidase and paraoxonase 1 in different stages of organophosphorus poisoning in a way to find out potential of these enzymes as marker of organophosphorus poisoning in human beings. Method: Xanthine oxidase (XO) activity was determined by Roussos method while paraoxonase (PON1) activity was determined by spectrophotometric method. Result: As compared to healthy controls, the activities of paraoxonase 1 were continuously and significantly decreased (p < 0.01) from group I to group V, while, the activities of xanthine oxidase were continuously and significantly increased (p < 0.01) from group I to group V in organophosphorus poisoned patients. Conclusion: We found a proportional increase in xanthine oxidase (XO) and a proportional decrease in paraoxonase 1 activity with severity of organophosphorus poisoning. Increase in xanthine oxidase activity is less significant compared to decrease in activity of paraoxonase 1. However, xanthine oxidase activity is a marker of oxidative stress developed in organophosphorus poisoning. Thus, paraoxonase 1 (PON1) can be used as a potent biochemical marker in the diagnosis and prognosis of organophosphorus poisoning along with xanthine oxidase (XO).

P4: Nitrite-mediated, xanthine oxidase-dependent diminution of obesity-related hyperglycemia and cardiopulmonary dysfunction

Background Obesity is associated with elevated tissue and plasma xanthine oxidase (XO) levels and allied enhancement of reactive species formation contributory to systemic inflammation. Despite a long standing association between increased XO activity and negative clinical outcomes, recent reports describe a paradigm shift where XO may mediate beneficial actions by reducing NO 2 - to NO. While provocative, these observations contradict both reports of improved outcomes in similar models when XO is inhibited and reports revealing anoxia as a requisite for XO-mediated NO formation. Herein, we identify a vascular microenvironment where NO 2 - reductase activity of XO is operative in the presence of O2 as well as examine effects of XO inhibition vs. NO 2 - supplementation in a high-fat diet (HFD) model of obesity. Methods Purified XO, XO bound to heparin-Sepharose 6B and XO sequestered by endothelial cell glycosaminoglycans (GAGs) was used to examine NO 2 - reductase activity as assessed by several detection platforms including enhanced chemiluminescence (NO), EPR spin trapping O 2 - and amperometric O2 detection. Male C57Blk/j6 mice were subjected to a HFD (60% calories derived from fat) for 20 weeks and treated (drinking water) with the XO-specific inhibitor febuxostat (0.5 mg/L) or NaNO2 (100 mg/L) or both for the final 6 weeks. Age-matched controls were maintained on a standard rodent chow diet consisting of 15% of adjusted calories from fat. After 20 weeks on diets mice were analyzed for right ventricular (RV) pressure volume (PV) relationship using a PV conductance catheter under steady state conditions. Results Whereas NO 2 - reductase activity of XO free in solution is only operative under anoxia, sequestration of XO on endothelial cell GAGs confers the capacity for substantive NO formation in the presence of O2 (1–2% or ∼13–26 μM) with concomitant diminution of XO-derived ROS production (↓40%). Treatment with febuxostat: reduced fasting blood glucose (223 vs. 181 mg/dL), improved (39%) impaired glucose tolerance and diminished oxidative stress in lung, liver, heart and skeletal muscle. Febuxostat also improved indices related to obesity-mediated right ventricular (RV) dysfunction and the onset of pulmonary arterial hypertension including RV end systolic pressure (RVESP) (41.2 ± 7.3 vs. 27.2 ± 5.2 mmHg), RV contractility index, pulmonary vascular resistance (PVR) (2.51 ± 0.47 vs. 1.96 ± 0.24 mmHg/mL/min), mean pulmonary artery pressure (mPAP) (25.2 ± 3.7 vs. 18.5 ± 3.0 mmHg) and Tau (diastolic function) (6.8 ± 1.3 vs. 4.5 ± 0.9). Febuxostat significantly reduced RV hypertrophy (Fulton Index), pulmonary arteriole smooth muscle (SM) remodeling (α-SM actin staining) and pulmonary tissue macrophage infiltration. Treatment of HFD mice with NO 2 - produced similar yet more pronounced beneficial effects than febuxostat in all the parameters listed above. However, combined treatment with NO 2 - + febuxostat completely abolished these protective effects, suggesting the salutary actions of NO 2 - were mediated by XO. Conclusion These data demonstrate that under hypoxic/inflammatory conditions where vascular XO levels and subsequent XO-GAG interactions are enhanced, XO-catalyzed NO 2 - reduction increases NO generation, decreases ROS production and serves to diminish indices of obesity-mediated pathology. Disclosure Supported by AHA National Scientist Development-10SDG3560005 (E.E.K.).

PPARα Modulates Acrolein‐Induced Inflammatory Signaling in gp91phox Knock‐Out Mice

Objective of this study was to identify the effect of Peroxisome Proliferator Activated Receptor (PPAR) α induction on acrolein‐induced oxidative stress (OS) in gp91phox knockout mice. gp91phox knockout (KO) mice were treated with acrolein (Acr: 0.5μg/kg; i.p) with/without PPAR α ligand Clofibrate (clof: 250mg/kg; i.p). Age/weight matched wild type (WT) were used as control. KO mice had a higher (97%) 8‐Isoprostane in response to Acr than WT, and clof had no effect. Total Antioxidant Status (TAS) was significantly reduced and this was more prominent in WT (69%) than KO (53%). Xanthine Oxidase (XO) activity was higher in KO (0.53±0.05) than WT (0.35+0.02). Acr and clof increased XO activity in WT. Acr increased cycloxygenase (COX) in WT (4.58±0.3) and was unchanged in KO. Clof reduced COX in WT (54%) and KO mice (31%). NFκB activity was twice in Acr‐treated WT and stayed unchanged in KO. Clof blunted this increase in WT. Acr increased CD36 activity in KO (56%) which was attenuated by Clof. We propose that in absence of a NAD(P)H oxidase other oxygenase contributes in Acr‐induced OS and PPARα ligand clof is beneficial in attenuating inflammatory signaling.

Melatonin protects rat thymus against oxidative stress caused by exposure to microwaves and modulates proliferation/apoptosis of thymocytes

The aim of the study was to evaluate the effect of melatonin on oxidative stress, DNA fragmentation, apoptsis and proliferation in thymus tissue of rats exposed to microwaves. Wistar rats were divided in four groups: I - treated with saline; II - treated with melatonin; III - microwaves exposed; IV - microwaves exposed and melatonin treated. Melatonin (2 mg/kg i.p.) was administered daily. Animals were sacrificed after 20, 40 and 60 days. A significant increase in malondialdehyde and carbonyl group content, as well as decrease in catalase and increase in xanthine oxidase activity were registered under microwave exposure. Melatonin prevented the increase in malondialdehyde and carbonyl group content, and reversed the effect on catalase and xanthine oxidase activity. Both, alkaline and acid DNase activity were increased due to microwave exposure. Furthermore, microwaves caused increase in apoptosis rate (detected using Annexin V-FITC/PI kit) and reduced proliferative capacity of thymocytes (induced by ConA). However, melatonin caused decrease in alkaline and acid DNase activity, decrease in apoptotic rate and increase in proliferation rate of thymocytes. Melatonin exerts protective effects on rat thymocytes by modulating processes of apoptosis and proliferation, and causes decrease in DNA fragmentation and oxidative stress intensity under exposure to microwaves.

Acute creatine administration improves mitochondrial membrane potential and protects against pentylenetetrazol-induced seizures

A growing body of evidence indicates that creatine (Cr) exerts beneficial effects on a variety of pathologies where energy metabolism and oxidative stress play an etiological role. However, the benefits of Cr treatment for epileptics are still shrouded in controversy. In the present study, we found that acute Cr treatment (300 mg/kg, p.o.) prevented the increase in electroencephalographic wave amplitude typically elicited by PTZ (30, 45 or 60 mg/kg, i.p.). Cr treatment also increased the latency periods of first myoclonic jerks, lengthened the latency periods of the generalized tonic-clonic seizures and reduced the time spent in the generalized tonic-clonic seizures induced by PTZ (60 mg/kg). Administration of PTZ (all doses) decreased Na(+), K(+)-ATPase activity as well as adenosine triphosphate (ATP) and adenosine diphosphate levels in the cerebral cortex, but Cr treatment prevented these effects. Cr administration also prevented increases in xanthine oxidase activity, adenosine monophosphate levels, adenosine levels, inosine levels and uric acid levels that normally occur after PTZ treatment (60 mg/kg, i.p.). We also showed that Cr treatment increased the total Cr (Cr + PCr) content, creatine kinase activity and the mitochondrial membrane potential (ΔΨ) in the cerebral cortex. In addition, Cr prevented PTZ-induced mitochondrial dysfunction characterized by decreasing ΔΨ, increasing thiobarbituric acid-reactive substance levels and increasing protein carbonylation. These experimental findings reinforce the idea that mitochondrial dysfunction plays a critical role in models of epileptic seizures and suggest that buffering brain energy levels through Cr treatment may be a promising therapeutic approach for the treatment of this neurological disease.

Inhibition of Xanthine Oxidase by Allopurinol Prevents Skeletal Muscle Atrophy: Role of p38 MAPKinase and E3 Ubiquitin Ligases

Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ∼20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.

Effects of Oxidative Stress on Immunosuppression Induced by Selenium Deficiency in Chickens

Selenium (Se) is an important nutritional trace element possessing immune-stimulatory properties. The aim of this 75-day study was to investigate effect of oxidative stress on immunosuppression induced by selenium deficiency by determining antioxidative function, morphological changes, DNA damage, and immune function in immune organ of chickens. One hundred sixty 1-day-old chickens (egg-type birds) were randomly assigned to two groups of 80 each and were fed on a low-Se diet (0.032 mg/kg Se) or a control diet (0.282 mg/kg Se, sodium selenite), respectively. Se contents in blood and immune organ (thymus, spleen, bursa of Fabricius) were determined on days 30, 45, 60, and 75, respectively. Antioxidative function was examined by total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and xanthine oxidase (XOD), and oxidative damage was examined by malondialdehyde (MDA) detection. DNA damage was measured by comet assay, and immune function was examined by determining serum interleukin-1β (IL-1β), interleukin-2 (IL-2), and tumor necrosis factor (TNF) contents. The results showed that Se concentrations in the low-Se group were significantly lower (P < 0.05) than in the control group. Low-Se diet caused a decrease in the activities of T-AOC, SOD, GSH-Px, and an increase in XOD activity and MDA content. Pathological lesions and DNA damage of immune tissues were observed in low-Se group, while the serum IL-1β and IL-2 contents decreased, and TNF content increased. The present study demonstrated that chickens fed deficient in Se diets exhibited lesions in immune organs, decreased serum IL-1β, IL-2 content, and serum TNF content, indicating that oxidative stress inhibited the development of immune organs and finally impaired the immune function of chickens.