Effects of tocopheryl quinone on the heart: model experiments with xanthine oxidase, heart mitochondria, and isolated perfused rat hearts

It is generally accepted that the protection effect of biological tissues by vitamin E is due to its radical scavenging potency in membranes, thereby being transformed to a vitamin E radical. A deficiency of appropriate reductants, which recycle vitamin E radicals back to its antioxidative active form, causes an irreversible degradation of vitamin E leading to tocopheryl quinone (TQ). TQ-like compounds were shown to result from both vitamin E and corresponding hydrophilic analogues of this antioxidant in vitro. In vivo elevated concentrations of tocopheryl quinones were detected after oxidative stress and TQ supplementation as well. Quinones in general are known to be efficient one-electron donors and acceptors. Therefore the question arises whether TQ-like compounds can undergo redox-cycling in conjunction with redox-active enzymes in the heart, thereby producing harmful oxygen radicals, or whether these compounds exhibit antioxidant properties. In order to elucidate this question we focused our interest on the interaction of TQ and a corresponding short-chain homologue (TQ(0)) with xanthine oxidase and heart mitochondria. Furthermore, we tested the influence of TQ on the recovery of isolated perfused rat hearts after ischemia/reperfusion. Our experiments revealed that hydrophilic TQ(0) was univalently reduced by xanthine oxidase (XOD) yielding semiquinone radicals in the absence of oxygen. However, under aerobic conditions TQ(0) enhanced the O(2)(*)(-) radical output of XOD. In the mitochondrial respiratory chain TQ was shown to interact with high potential cytochrome b in the bc(1) complex specifically. In contrast to the system XOD/TQ(0), lipophilic TQ in submitochondrial particles decreased the O(2)(*)(-) radical release during regular respiration possibly due to its interaction with b-cytochromes in the mitochondrial respiratory chain. In isolated rat hearts perfused with liposomes containing lipophilic TQ, it was efficiently accumulated in the heart tissue. When hearts were subjected to conditions of ischemia/reperfusion, infusion of TQ prior to ischemia significantly improved the recovery of hemodynamic parameters. Our results demonstrate that TQ derivatives may induce pro-oxidative and antioxidative effects depending on the distribution of TQ derivatives in the heart tissue and the interacting redox system.     

Prospects for the future: the role of free radicals in the treatment of stroke

Studies using animal models of stroke have demonstrated that free radicals are highly reactive molecules generated predominantly during cellular respiration and normal metabolism. Imbalance between cellular production of free radicals and the ability of cells to defend against them is referred to as oxidative stress. After ischemic brain damage introduced by ischemic stroke or reperfusion, production of reactive oxygen species may increase, sometimes drastically, leading to tissue damage via several different cellular molecular pathways. The damage can become more widespread due to weakened cellular antioxidant defense systems after ischemic stroke. These experimental findings have important implications for the treatment of human cerebral ischemia. Agents directed at eliminating oxygen radicals must be administered before, or in the early stages of, reperfusion after ischemia. The therapeutic window seems to be narrow and limited to, at most, a few hours. Future research may clarify the current hypothesis that the accuracy of gene expression could account for the recovery of cellular function after ischemic stroke. This may open the window to the future use of drug combinations that may be rationally administered sequentially. If the phenomenon of ischemic tolerance plays a role in this concept is still a matter of debate.     

The role of oxygen radicals in experimental acute pancreatitis

Oxygen-derived free radicals mediate an important step in the initiation of experimental acute pancreatitis. Thereby, it seems that these reactive oxygen metabolites are generated at an early stage of disease. The source of the enhanced production of oxygen radicals still remains unclear. Experimentally, the efficiency of scavenger treatment varied between different models, whereby these differences depended on the experimental model and not on the form of pancreatitis which was induced. Most studies pretreated the experimental animals before inducing acute pancreatitis. This does not mirror the clinical reality, since patients are admitted to the hospital after onset of the disease. It was shown in Cerulein pancreatitis, however, that scavenger treatment also mitigated the pancreatic tissue damages after induction of acute pancreatitis. Moreover, antioxidant treatment also attenuated the extrapancreatic complications, thus improving the final outcome of the disease. The first indirect observations also suggest that in human acute recurrent and chronic pancreatitis, oxygen free radicals are generated and add to the damages seen. Therefore, well-defined controlled clinical studies with patients suffering from acute pancreatitis are needed to validate the role of oxygen radicals in this disease.     

自由基和基质金属蛋白酶介导脑缺血血脑屏障损伤的研究进展

血脑屏障的破坏是引起脑缺血损伤及继发水肿、出血、炎症的微观原因。缺血缺氧和再灌注过程产生的自由基,以及后续基质金属蛋白酶的激活,是破坏血脑屏障结构和功能的重要分子机制。因而,在脑缺血早期及时抑制自由基产生并清除自由基,抑制基质金属蛋白酶的活性,是降低脑缺血血脑屏障损伤及其并发症的关键环节。本文将从血脑屏障损伤的角度,概述自由基与基质金属蛋白酶在脑缺血损伤过程中的作用。     

Pathophysiology of ischemic skin flaps: differences in xanthine oxidase levels among rats, pigs, and humans

Oxygen-derived free radicals have been implicated in a variety of diseases and pathologic processes, including ischemia reperfusion injury (IRI). Based on experimental work with rat skin-flap models, the enzyme xanthine oxidase (XO) has been proposed as a major source of free radicals responsible for tissue injury and flap necrosis. The presence of this enzyme is variable within different tissues of a specific species and between species. Xanthine oxidase levels in pig and human skin have not previously been reported. The activity of xanthine oxidase in the skin of rats (N = 16), pigs (N = 7), and humans (N = 8) was measured after varying intervals of ischemia and in the rat also following reperfusion. Control pig and human skin were found to contain minimal enzyme activity, almost 40 times less than that of the rat. In the rat, xanthine oxidase activity was stable throughout a prolonged period of ischemia, and a significant decrease in activity was found after 12 hours of reperfusion (p less than 0.05). In humans, xanthine oxidase activity was unaffected by ischemia time, and in the pig, it did not increase until 24 hours of ischemia (p less than 0.05). The potential sources of free radicals and the mechanism of action of xanthine oxidase and its inhibitor allopurinol in improving flap survival in different species are reviewed.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Free radical scavengers in myocardial ischemia

Reperfusion of ischemic myocardium is recognized as potentially beneficial because mortality is directly related to infarct size, and the latter is related to the severity and duration of ischemia. However, reperfusion is associated with extension of the injury that is additive to that produced by ischemia alone. The phenomenon of reperfusion injury is caused in large part by oxygen-derived free radicals from both extracellular and intracellular sources. The loci of oxygen-free radical formation include: myocardial sources (mitochondria), vascular endothelial sources (xanthine oxidase and other oxidases), or the inflammatory cellular infiltrate (neutrophils). Experimental studies have shown that free radical scavengers and agents that prevent free radical production can reduce myocardial infarct size in dogs subjected to temporary regional ischemia followed by reperfusion. Superoxide dismutase and catalase, which catalyze the breakdown of superoxide anion and hydrogen peroxide, respectively, limit experimental myocardial infarct size. The free radical scavenging agent N-(2-mercaptopropionyl)glycine (MPG) is reported to be effective in limiting infarct size. The ischemic-reperfused myocardium derives significant protection when experimental animals are pretreated with the xanthine oxidase inhibitor allopurinol. Neutrophils also serve as a significant source of oxygen-derived free radicals at the site of tissue injury. A number of agents have been shown to directly inhibit neutrophil-derived oxygen free radical formation and neutrophil accumulation within the reperfused myocardium. These agents include ibuprofen, nafazatrom, BW755C, prostacyclin, and iloprost. Thus, free radical scavengers and agents that prevent free radical formation can provide significant protection to the ischemic-reperfused myocardium.     

Oxidative injury of isolated cardiomyocytes: dependence on free radical species

The contribution of lipid peroxidation to myocardial injury by free radicals (FR) is still unclear. Consequently, we examined the functional damages inflicted on cultured rat cardiomyocytes (CM) during FR stress provoked by the xanthine/xanthine oxidase system (X/XO) or by a hydroperoxidized fatty acid ((9 Z, 11 E, 13 (S), 15 Z)-13-hydroperoxyocta-decatrienoic acid; 13-HpOTrE), in order to simulate in vitro the initial phase and the propagation phase of the FR attack, respectively. Transmembrane potentials were recorded with glass microelectrodes and contractions were monitored photometrically. The EPR spectroscopy showed that X/XO produced superoxide and hydroxyl radicals during 10 min. The X/XO system altered sharply and irreversibly the spontaneous electrical and mechanical activities of the CM. However, the gas chromatographic analysis showed that these drastic functional damages were associated with comparatively moderate membrane PUFA degradation. Moreover, the EPR analysis did not reveal the production of lipid-derived FR. 13-HpOTrE induced a moderate and reversible decrease in electrical parameters, with no change in CM contractions. These results indicate that the functional consequences of FR attack are dependent on the radical species present and do not support the idea that the membrane lipid breakdown is a major factor of myocardial oxidant dysfunction.     

Effects of oxygen radicals on substrate oxidation by cardiac myocytes

Freshly isolated adult rat heart cells were used to study the effects of oxygen-free radicals on the myocardial oxidation of different substrates. The calcium-tolerant quiescent cells were incubated with xanthine plus xanthine oxidase as the source of free radicals. The oxidation of exogenous glucose, lactate and octanoate was severely inhibited (approx. 70%) by products of xanthine oxidase activity. Superoxide dismutase plus catalase effectively prevented the inhibition of oxidation. Cellular high energy phosphate levels were decreased in the presence of the oxygen free radical generating system although cell viability determined by Trypan blue exclusion and light microscopic assessment of normal morphology was not affected. These data suggest that oxygen free radicals decrease myocardial substrate oxidation which may contribute to the functional and ultrastructural changes in the myocardium under conditions such as reoxygenation after hypoxia and reperfusion after ischemia.     

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.     

Tourniquet shock in rats: effects of allopurinol on biochemical changes of the gastrocnemius muscle subjected to ischemia followed by reperfusion

Recent data suggest that oxygen free radicals are implicated in the pathogenesis of ischemic injury. This study evaluates the effects of allopurinol, a xanthine oxidase (XO) inhibitor, on malonaldehyde generation, free sulfhydryl levels, oxygen consumption, and water contents of rat gastrocnemius muscles of female Sprague-Dawley rats subjected to tourniquet shock and after hind-limb reperfusion. Serum lactic dehydrogenase isozyme patterns after ligature release were also examined. Our results show that the four muscle parameters were not altered during 5 hr of ischemia, but that on hind-limb reperfusion, malonaldehyde production, SH levels, O2 consumption, and water contents were significantly altered in the control animals, but not in those pretreated with allopurinol. LDH serum patterns of the untreated animals showed the presence of all five isoforms; these were much less evident in the drug-protected rats. Our data suggest that following ischemia, the affected muscles are unable to recover their normal function when reperfusion is resumed. The subsequent damage is probably due to the generation of cytotoxic superoxide radicals formed during the XO-catalyzed transformation of hypoxanthine to uric acid on tissue reoxygenation. The severity of tissue damage is related to the duration of the ischemic episode possibly due to hypoxanthine accumulation during ischemia.     

Free radicals and myocardial ischemia: overview and outlook

Much evidence suggests that free radicals and active oxygen species derived from molecular oxygen (superoxide, hydrogen peroxide, and hydroxyl radical) contribute to the tissue injury which accompanies myocardial ischemia and reperfusion. Three possible sources have been identified for the production of active oxygen species: the enzyme xanthine oxidase; the activated polymorphonuclear leukocyte; the disrupted mitochondrial electron transport system. These sources may be mutually interactive. Once triggered, they may lead to the loss of antioxidant enzymes and to the release of iron, both of which are exacerbatory events.     

Separable detection of lipophilic- and hydrophilic-phase free radicals from the ESR spectrum of nitroxyl radical in transient MCAO mice

Free radicals are believed to be key factors that promote ischemia reperfusion injury in the brain. This study used the characteristic spectrum of methoxycarbonyl-PROXYL to detect free radical reactions in hydrophilic and lipophilic compartments in a transient middle cerebral artery occlusion (MCAO) mouse model. Methoxycarbonyl-PROXYL, which has a high water/octanol partition coefficient, allows the detection of nitroxyl radical in both compartments simultaneously. Free radicals generation was analysed from the enhanced ESR signal decay rate of methoxycarbonyl-PROXYL. The signal decay rate in the lipidic compartment was significantly enhanced 1 h after reperfusion following MCAO. The enhanced signal decay rate was significantly suppressed by Trolox. The accumulation of lipid peroxidation products increased by 6 h post-reperfusion and was suppressed by methoxycarbonyl-PROXYL or Trolox. These results demonstrate that information pertaining to different sites of free radical generation in vivo can be obtained simultaneously and that lipid-derived radicals are generated in transient MCAO mice.     

Mechanism of Kainate Toxicity to Cerebellar Neurons In Vitro Is Analogous to Reperfusion Tissue Injury

The neuroexcitotoxin kainate has been used as a selective lesioning agent to model the etiology of a number of neurodegenerative disorders. Although excitotoxins cause susceptible neurons to undergo prolonged or repeated depolarization, the proximate metabolic pathology responsible for neuronal necrosis has remained elusive. We report here that kainate-induced death of cerebellar neurons in culture is prevented by inhibiting the enzyme xanthine oxidase, a cellular source of cytotoxic superoxide radicals (O2-.). Moreover, neurons are also protected from excitotoxin-induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenge superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalls formation of xanthine oxidase. These findings indicate that excitotoxin-induced neuronal degeneration is mediated by superoxide radicals generated by xanthine oxidase, a mechanism partially analogous to that proposed for tissue damage seen upon reperfusion of ischemic tissues.     

Cell therapy in critical limb ischemia: current developments and future progress

Critical limb ischemia (CLI) is a syndrome manifested by ischemic rest pain, non-healing ulcers and tissue loss. CLI patients are at very high risk of amputation and experience poor physical function, leading to severe morbidity and mortality. The fundamental goal for CLI treatment is to relieve ischemic rest pain, heal ulcers, prevent limb loss and improve the quality of life, thereby extending the survival of the patient. Surgical or endovascular revascularization aimed at increasing blood flow is currently available for limb salvage in CLI. However, up to 30% of CLI patients are not suitable for such interventions because of high operative risk or unfavorable vascular anatomy. Therefore exploring new and more effective strategies for revascularization of ischemic limbs is imperative for the establishment of a viable therapeutic alternative. With the emergence of new approaches, this review describes up-to-date progress and developments in cell-based therapy as a novel and promising alternative for CLI treatment. Preliminary clinical data have established the safety, feasibility and efficacy of stem cells, and numerous studies are underway to consolidate this evidence further. However, significant hurdles remain to be addressed before this research can be responsibly translated to the bedside. In particular, we need better understanding of the behavior of cells post-transplantation and to learn how to control their survival and migration proliferation/differentiation in the hostile pathologic environment. Future research should focus on methods of isolation, optimal dosage, appropriate cell type, route of administration, role of tissue-derived factors and supportive endogenous stimulation.     

Drugs to cure avian influenza infection – multiple ways to prevent cell death

New treatments and new drugs for avian influenza virus (AIV) infection are developed continually, but there are still high mortality rates. The main reason may be that not all cell death pathways induced by AIV were blocked by the current therapies. In this review, drugs for AIV and associated acute respiratory distress syndrome (ARDS) are summarized. The roles of antioxidant (vitamin C) and multiple immunomodulators (such as Celecoxib, Mesalazine and Eritoran) are discussed. The clinical care of ARDS may result in ischemia reperfusion injury to poorly ventilated alveolar cells. Cyclosporin A should effectively inhibit this kind of damages and, therefore, may be the key drug for the survival of patients with virus-induced ARDS. Treatment with protease inhibitor Ulinastatin could also protect lysosome integrity after the infection. Through these analyses, a large drug combination is proposed, which may hypothetically greatly reduce the mortality rate.     

Repair of DNA damage induced by oxygen radicals in human non-proliferating and proliferating lymphocytes.

Repair of DNA lesions induced by oxygen radicals, generated by xanthine/xanthine oxidase (X/XO), was studied in human peripheral blood lymphocytes and in PHA-stimulated proliferating lymphocytes from 4 healthy subjects. The lesions included DNA-strand breaks (SSB) and other lesions that are converted to SSB under alkaline conditions. The frequencies of SSB were estimated by fluorometric analysis of DNA unwinding. Maximum production of SSB occurred within 10 min of incubation with X/XO at 22 degrees C; with 0.5 mM or higher concentrations of xanthine; and with 0.1-0.5 units/ml of xanthine oxidase. Proliferating lymphocytes repaired X/XO-induced SSB about 4 times more rapidly than lymphocytes. Lymphocytes repaired X/XO-induced SSB more slowly than SSB caused by gamma-radiation. These findings are consistent with the evidence that a number of DNA-repair enzymes have greater activity in proliferating cells than in resting cells. These findings also support the view that there are differences between the DNA damage due to oxygen radicals and that due to ionizing radiation.     

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.     

Altered levels of tissue and urinary prostacyclin in rats subjected to pancreas transplantation

Significant increases of TXB2 and PGE2 are reported to occur in pancreas transplantation. These increases are prevented with scavengers of oxygen-free radicals. In this communication, we report on changes of prostacyclin metabolites such as tissue 6-keto prostaglandin F1 alpha and urinary 2,3-dinor 6-keto prostaglandin F1 alpha in rats subjected to pancreas transplantation after different periods of organ cold preservation ischemia as well as the effect of superoxide dismutase (SOD) on these changes. For this purpose, male Lewis rats were classified as follows: Group I, Control; Group II, syngenic pancreas transplantation after 15 min of organ preservation in Collins solution at 4 degrees C; Group III, same as II but with 12 hours of organ preservation; Group IV, same as III, but with SOD pretreatment. Results have shown significant posttransplantation increases of both tissue 6-keto PGF1 alpha and urinary 2, 3 dinor 6-keto PGF1 alpha, the latter being a useful marker to evaluate systemic prostacyclin (PGI2) production by rat pancreas. This effect was prevented when the organ had been exposed to SOD during the period of cold preservation ischemia. These results confirm the implication of oxygen-free radicals (OFR) in the ischemia-reperfusion process associated to rat pancreas transplantation leading to enhanced arachidonic acid metabolism.     

Contracture and cell damage in calcium paradox is not caused by lipid peroxidation

The role of oxygen radicals and lipid peroxidation in calcium-paradox injury in isolated perfused rat hearts was studied by examining the effects of mannitol and (or) allopurinol on this phenomenon. Myocardial changes due to calcium paradox were characterized by contractile failure, a rise in resting tension, and cell damage. These changes were also accompanied by increased lipid peroxidation, as indicated by an increase in malondialdehyde content. Mannitol (an effective quencher of hydroxyl radicals) treatment resulted in a dose-dependent decrease in lipid peroxidation but did not affect other changes due to calcium paradox. Allopurinol (an inhibitor of xanthine oxidase) neither affected lipid peroxidation nor modified any of the structure-function changes due to calcium paradox. These data demonstrate the occurrence of lipid peroxidation which, however, may not be involved in the observed structure-function changes due to calcium paradox. It is also suggested that in this experimental model, xanthine oxidase may not be the inducer of oxygen radicals or of lipid peroxidation.