Cinnamophilin as a novel antiperoxidative cytoprotectant and free radical scavenger

The antioxidant properties of cinnamophilin were evaluated by studying its ability to react with relevant reactive oxygen species, and its protective effect on cultured cells and biomacromolecules under oxidative stress. Cinnamophilin concentration-dependently suppressed non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 8.0+/-0.7 microM and iron ion/ADP/ascorbate-initiated rat liver mitochondrial lipid peroxidation with an IC50 value of 17.7+/-0.2 microM. It also exerted an inhibitory activity on NADPH-dependent microsomal lipid peroxidation with an IC50 value of 3.4+/-0.1 microM without affecting microsomal electron transport of NADPH-cytochrome P-450 reductase. Both 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azo-bis(2-amidinopropane) dihydrochloride-derived peroxyl radical tests demonstrated that cinnamophilin possessed marked free radical scavenging capacity. Cinnamophilin significantly protected cultured rat aortic smooth muscle cells (A7r5) against alloxan/iron ion/H2O2-induced damage resulting in cytoplasmic membranous disturbance and mitochondrial potential decay. By the way, cinnamophilin inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity and thiobarbituric acid-reactive substance formation in a concentration-dependent manner. On the other hand, it was reactive toward superoxide anions generated by the xanthine/xanthine oxidase system and the aortic segment from aged spontaneously hypertensive rat. Furthermore, cinnamophilin exerted a divergent effect on the respiratory burst of human neutrophil by different stimulators. Our results show that cinnamophilin acts as a novel antioxidant and cytoprotectant against oxidative damage.     

Novel thiazolo-pyrazolyl derivatives as xanthine oxidase inhibitors and free radical scavengers

Xanthine oxidase (XO) is a complex metalloflavoprotein, overproduction of which usually leads to a pathological condition called Gout. XO inhibitors may prove to be promising antigout agents. Present investigation describes synthesis, characterization and evaluation of 26 thiazolo-pyrazolyl derivatives V(a-z) for XO inhibitory and free radical scavenging activities. Derivatives Vq, Vo and Vh showed most promising XO inhibitory and free radical scavenging activities on the basis of their IC(50) values ranging from (6.5-9 μM). Significant dock scores compared with Allopurinol have been figured out using molecular docking. Evaluation of Vq, Vo and Vh for both the activities for first time may provide a new approach for antigout research.     

Effects of pretreatment with a xanthine oxidase inhibitor on free radical levels during carotid endarterectomy

Objective: Free radicals contribute to the tissue damage caused by ischaemia-reperfusion. The aim of the present study was to investigate whether preoperative antioxidant therapy (allopurinol) affects free radical levels in cerebral venous blood in connection with surgery for carotid artery stenosis.

Materials and methods: Twenty-five patients were randomised into the study. Thirteen were controls and 12 were pretreated with allopurinol the day before surgery. Before, during and after surgery, blood samples were drawn from the ipsilateral jugular vein. Radical levels were measured using the spin trap technique ex vivo using OXANOH as the spin trap. Multivariate statistics were used with Principal Component Analysis and Partial Least Square regression analysis.

Results: Radical levels increased with diabetes, high leukocyte count, high creatinine and a high degree of contralateral stenosis. Radical levels decreased with high age, blood pressure, collateral circulation as well as operation for left-side carotid artery stenosis. After pretreatment with allopurinol, several of the relationships noted in the control group were eliminated, i.e. leukocyte count, side of operation, Betapred pretreatment and collateral circulation.

Conclusions: Radical levels can be determined in connection with surgery for carotid artery stenosis using an ex vivo spin trap method. With preoperative antioxidant therapy the relationships between enhanced radical levels and clinical data, as seen in control subjects, disappeared. This might indicate a beneficial effect of preoperative pretreatment with antioxidants.     

Mechanism of free radical production in exhaustive exercise in humans and rats; role of xanthine oxidase and protection by allopurinol

Exhaustive exercise generates free radicals. However, the source of this oxidative damage remains controversial. The aim of this paper was to study further the mechanism of exercise-induced production of free radicals. Testing the hypothesis that xanthine oxidase contributes to the production of free radicals during exercise, we found not only that exercise caused an increase in blood xanthine oxidase activity in rats but also that inhibiting xanthine oxidase with allopurinol prevented exercise-induced oxidation of glutathione in both rats and in humans. Furthermore, inhibiting xanthine oxidase prevented the increases in the plasma activity of cytosolic enzymes (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase) seen after exhaustive exercise. Our results provide evidence that xanthine oxidase is responsible for the free radical production and tissue damage during exhaustive exercise. These findings also suggest that mitochondria play a minor role as a source of free radicals during exhaustive physical exercise.     

Lithospermic acid as a novel xanthine oxidase inhibitor has anti-inflammatory and hypouricemic effects in rats

Lithospermic acid (LSA) was originally isolated from the roots of Salvia mitiorrhiza, a common herb of oriental medicine. Previous studies demonstrated that LSA has antioxidant effects. In this study, we investigated the in vitro xanthine oxidase (XO) inhibitory activity, and in vivo hypouricemic and anti-inflammatory effects of rats. XO activity was detected by measuring the formation of uric acid or superoxide radicals in the xanthine/xanthine oxidase system. The results showed that LSA inhibited the formation of uric acid and superoxide radicals significantly with an IC50 5.2 and 1.08 microg/ml, respectively, and exhibited competitive inhibition. It was also found that LSA scavenged superoxide radicals directly in the system beta-NADH/PMS and inhibited the production of superoxide in human neutrophils stimulated by PMA and fMLP. LSA was also found to have hypouricemic activity on oxonate-pretreated rats in vivo and have anti-inflammatory effects in a model of gouty arthritis. These results suggested that LSA is a competitive inhibitor of XO, able to directly scavenge superoxide and inhibit superoxide production in vitro, and presents hypouricemic and anti-inflammatory actions in vivo.     

6-(N-benzoylamino)purine as a novel and potent inhibitor of xanthine oxidase: inhibition mechanism and molecular modeling studies

The inhibition of xanthine oxidase (XO) activity by the purine analogue 6-(N-benzoylamino)purine was evaluated and compared with the standard inhibitor, allopurinol and the parent compound adenine. 6-(N-benzoylamino)purine is a highly potent inhibitor of XO (IC50 = 0.45 microM) and comparable to allopurinol (IC50 = 0.80 microM). Furthermore, 6-(N-benzoylamino)purine neither produced any enzymatic superoxide nor reduced XO by an electron transfer reaction unlike allopurinol. 6-(N-benzoylamino)purine (Ki = 0.0475 microM) is about 10000-fold more potent as a XO inhibitor compared to the only known purine analogue 8-bromoxanthine (Ki = 400 microM). 6-(N-Benzoylamino)purine is a competitive inhibitor of XO and the inhibition was not completely reversed even at 100 microM xanthine concentration. The calculated interaction energy [Ecomplex - (Eligand + Eprotein)] of -30.5, -22.6, and -17.2 kcal/mol, respectively, of 6-(N-benzoylamino)purine, 8-bromoxanthine and the parent compound adenine provided the rationale for the better enzyme inhibitory activity of 6-(N-benzoylamino)purine. To understand the role of the benzamido group in the inhibition process, molecular docking studies were carried out and it was revealed that the hydrogen bonding interactions involving N-7 of the purine ring and the N-H of Arg880, N-H of the purine ring and OH of Thr1010, as well as non-bonded interactions of the benzamido group of 6-(N-benzoylamino)purine with amino acid residues Gly799, Glu802, Phe914, Ala1078, Ala1079 and Glu1261 in the active site of XO play an important role in the stabilization of the E-I complex.     

Post-irradiation free radical generation: evidence from the conversion of xanthine dehydrogenase into xanthine oxidase

The xanthine oxidoreductase (XOR) system which consists of xanthine dehydrogenase (XDH) and xathine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOR system is pre-dominantly present as XDH in normal tissues and converts into the free radical generating XO-form in the damaged tissue. Therefore, the XO-form of the XOR system is expected to be mainly found in radiolytically damaged tissues. In such an event, XO may catalyze the generation of free radicals and potentiate radiation effects in the post-irradiation period. Recent findings on the effect of ionizing radiation on the XOR system in the liver of mice, peroxidative damage and lactate dehydrogenase support this possibility. From these results it has been hypothesized that free radical generating systems could be activated in the radiolytically damaged cell and in turn contribute to the cause and complications of late effects and their persistence in post-irradiation period. This aspect may have great significance in the understanding of radiation-induced damages. It may also have serious implication in various fields like radiation therapy, health physics, carcinogenesis, space travelling radiation exposures and post nuclear accident care. Further, it is suggested that efforts need to be made to search more system(s) which could be activated particularly at lower doses of radiation to generate free radicals in the post-exposure period.     

Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki' values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 muM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.     

Free-radical mechanism of antimicrobial action of xanthine oxidase and lactoperoxidase]

The interaction between milk xanthine oxidase (XO) and lactoperoxidase (LP) in model system and antimicrobial action of these enzymes on Escherichia coli 0-111 were studied. It was shown, that bacterial superoxide dismutase (SOD), which transforms O2-. (XO-reaction product) into H2O2 (substrate of LP), is necessary for binding of the reaction sequence: XO-->LP-->antimicrobial products. It is suggested, that these enzymes unite in the protective system in intestinal infections of newborns. Bacterial SOD in this case acts as the key factor, creating the system.     

Effect of normothermic ischemic cardiac arrest and of reperfusion on the free oxygen radical scavenger enzymes and xanthine oxidase (a generator of superoxide anions)

Recent evidence suggests that free oxygen radicals are produced by ischaemic tissues, accounting for at least part of the damage that results. These free oxygen radicals are produced by xanthine oxidase, amongst others, and removed by scavenger enzymes (catalase, superoxide dismutase and glutathione peroxidase) and anti-oxidants. As mitochondria are oxygen-utilising organelles, they are capable of producing free oxygen radicals. Our results indicate that the removal of free oxygen radicals are not diminished during ischaemia, but the activity of the free oxygen radical generator, xanthine oxidase, is increased. This could lead to an increased superoxide anion concentration.     

Discrepancy between biochemical normalization and morphological recovery of jejunal mucosa during postischemic reperfusion in presence of the xanthine oxidase inhibitor oxypurinol

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.     

Inhibition of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol

Allopurinol (1H-pyrazolo-[3,4-d]pyrimidine-4-ol), an inhibitor of xanthine oxidation in ureide-producing nodulated legumes, was taken up from the rooting medium, translocated in xylem, and transferred to nodules of both the ureide-forming cowpea (Vigna unguiculata L. Walp.) and the amide-forming white lupin (Lupinus albus L.). Cowpea suffered severe nitrogen deficiency, extreme chlorosis, and reduced growth, whereas lupin was unaffected by the inhibitor. Similar results were obtained with oxypurinol (1H-pyrazolo-[3,4-d]pyrimidine-4,6-diol). Xylem composition of symbiotic cowpea was markedly changed by allopurinol. Ureides fell to a very low level, but xanthine and, to a lesser extent, hypoxanthine increased markedly. Xylem glutamine was also reduced, but there was little change in other amino acids. Nitrogenase (EC 1.7.99.2) activity of intact nodulated plants or nodulated root segments of plants treated with allopurinol or oxypurinol for 24 hours or more was severely inhibited in cowpea but unaffected in lupin for periods of exposure up to 9 days. Nitrogenase activity of slices of nodules prepared from allopurinol-treated cowpea showed inhibition comparable to that of intact plants. Breis prepared from nodules of treated plants showed no reduction in nitrogenase, nor was there reduction in activity of breis following addition of allopurinol, xanthine, or a range of purine pathway intermediates. Increasing the O₂ concentration in assays above 20% (volume/volume) reversed inhibition of nitrogenase by allopurinol in intact nodulated roots. It was concluded for cowpea that allopurinol not only inhibited ureide synthesis but also caused inhibition of nitrogenase activity, thereby leading to progressive dysfunction and eventual senescence of nodules. The mechanistic relationships between inhibition of ureide biosynthesis, changes in gaseous diffusion resistance, and reduced nitrogenase activity remain obscure.     

Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence

Multiple reactive oxygen/nitrogen species induce oxidative stress. Mammals have evolved with an elaborate defense network against oxidative stress, in which multiple antioxidant compounds and enzymes with different functions exert their respective roles. Radical scavenging is one of the essential roles of antioxidants and vitamin E is the most abundant and important lipophilic radical-scavenging antioxidant in vivo. The kinetic data and physiological molar ratio of vitamin E to substrates show that the peroxyl radicals are the only radicals that vitamin E can scavenge to break chain propagation efficiently and that vitamin E is unable to act as a potent scavenger of hydroxyl, alkoxyl, nitrogen dioxide, and thiyl radicals in vivo. The preventive effect of vitamin E against the oxidation mediated by nonradical oxidants such as hypochlorite, singlet oxygen, ozone, and enzymes may be limited in vivo. The synergistic interaction of vitamin E and vitamin C is effective for enhancing the antioxidant capacity of vitamin E. The in vitro and in vivo evidence of the function of vitamin E as a peroxyl radical-scavenging antioxidant and inhibitor of lipid peroxidation is presented.     

Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages

As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.     

Pharmacokinetics/pharmacodynamics of Y-700, a novel xanthine oxidase inhibitor, in rats and man

The pharmacokinetics and pharmacodynamics of a novel xanthine oxidase (XO) inhibitor, Y-700, were evaluated in rats and healthy male volunteers. In a rat model of hyperuricemia, oral Y-700 (0.3-10 mg/kg) showed a more potent and a longer-lasting hypouricemic action than allopurinol. A single oral dosing of Y-700 (5, 20 or 80 mg) to volunteers caused a dose-dependent reduction of serum uric acid levels indicating close relationship to plasma concentrations of the compound. In addition, Y-700 was hardly excreted in urine but mainly excreted in feces in rats and volunteers. These results suggested that Y-700 is a new effective inhibitor of XO in rats and humans with high oral bioavailability being predominantly eliminated via the liver unlikely to allopurinol.     

Oxygen free radical induced damage during intestinal ischemia/reperfusion in normal and xanthine oxidase deficient rats

This study looks at the role of xanthine oxidase (XO) in ischemia/reperfusion (I/R) induced intestinal mucosal damage using normal and xanthine oxidase deficient rats. Tungstate feeding for 3 days depleted the intestinal mucosal XO by 80%. A ligated loop of the rat small intestine (both normal and XO-deficient) was subjected to 1 h of total ischemia followed by 5 min revascularisation. The ensuing mucosal damage was assessed by biochemical and histological studies. Ischemia or I/R increased the XO levels in normal rats without any change in XO-deficient rats. Myeloperoxidase (a neutrophil marker) level was increased in both group of rats but it was comparatively higher in the XO-deficient rats. Accumulation of peroxidation products such as malondialdehyde, conjugated diene and increased production of hydroxyl radicals by microsomes were seen after ischemia and I/R and were similar in normal and XO-deficient rats. Studies on other parameters of peroxidation showed a decrease in polyunsaturated fatty acids and alpha-tocopherol, an increase in cysteine and cystine levels after I/R and were similar in both normal and XO-deficient rats. Histological results indicated gross morphological changes in the intestinal mucosa due to ischemia and I/R, and the damage was more severe in XO-deficient rats. These observations suggest that oxygen-derived free radicals are involved in the intestinal mucosal damage during I/R and infiltrated neutrophils rather than XO may be the primary source of free radicals under these conditions.