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.     

Excitatory Amino Acid Release from Rat Hippocampal Slices as a Consequence of Free-Radical Formation

The release of D-[3H]aspartate, [3H]noradrenaline, and of endogenous glutamate and aspartate from rat hippocampal slices was significantly increased when the slices were incubated with xanthine oxidase plus xanthine to produce superoxide and hydroxyl free radicals locally. Allopurinol, a specific xanthine oxidase inhibitor, the hydroxyl-radical scavenger D-mannitol, or the superoxide-radical scavenger system formed by superoxide dismutase plus catalase prevented this release. These results suggest that endogenous excitatory amino acids are released consequent to the formation of free radicals. The excess of glutamate and aspartate released by this mechanism could be one of the factors contributing to the death of neurons after anoxic or ischemic injuries.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Modification of contractile proteins by oxygen free radicals in rat heart

This study was undertaken to investigate the effects of oxygen free radicals on myofibrillar creatine kinase activity. Isolated rat heart myofibrils were incubated with xanthine+xanthine oxidase (a superoxide anion radical-generating system) or hydrogen peroxide and assayed for creatine kinase activity. To clarify the involvement of changes in sulfhydryl groups in causing alterations in myofibrillar creatine kinase activity, 1) effects of N-ethylmaleimide (sulfhydryl groups reagent) on myofibrillar creatine kinase activity, 2) effect of oxygen free radicals on myofibrillar sulfhydryl groups content, and 3) protective effects of dithiothreitol (sulfhydryl groups-reducing agent) on the changes in myofibrillar creatine kinase activity due to oxygen free radicals were also studied. Xanthine+xanthine oxidase inhibited creatine kinase activity both in a time-and a concentration-dependent manner. Superoxide dismutase (SOD) showed a protective effect on the depression in creatine kinase activity caused by xanthine+xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a concentration-dependent manner; this inhibition was prevented by the addition of catalase. N-ethylmaleimide reduced creatine kinase activity in a dose-dependent manner. The content of myofibrillar sulfhydryl groups was decreased by xanthine+xanthine oxidase; this reduction was protected by SOD. Furthermore, the depression in myofibrillar creatine kinase activity by xanthine+xanthine oxidase was protected by the addition of dithiothreitol. Oxygen free radicals may inhibit myofibrillar creatine kinase activity by modifying sulfhydryl groups in the enzyme protein. The reduction of myofibrillar creatine kinase activity may lead to a disturbance of energy utilization in the heart and may contribute to cardiac dysfunction due to oxygen free radicals.     

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 involvement in the generation of trans‐root potential

The identity of the naturally occurring compounds that accept electrons from plasma membrane-bound redox systems in vivo is obscure. We analysed the effect of ascorbate, oxygen, iron, as well as their free radical forms, and also the free radical-generating and -quenching systems on the trans-root electrical potential, which had previously been shown to be coupled to plasma membrane-bound redox systems. The material was the primary root of 8-day-old maize (Zea mays L.) seedlings. Trans-root electrical potential difference was measured across excised roots. Different ascorbate (ascorbate, dehydroascorbate and ascorbate free radical) and oxygen redox forms (superoxide and hydroxide radicals and hydrogen peroxide), as well as scavenging agents of oxygen species (superoxide dismutase, catalase, mannitol), and ferric and ferrous ions were added to the solution flowing around the root. Ascorbate free radical induced the greatest depolarization of the trans-root potential when compared to other ascorbate redox forms, which is consistent with its suggested role as a natural electron acceptor. Addition of xanthine oxidase, with or without xanthine, also produced depolarizing effects. The presence of SOD magnified this effect both with ascorbate free radical and xanthine oxidase. When ferric or ferrous chloride and ferric EDTA were applied to the bathing medium, only free ferric ion produced a very pronounced depolarization. The magnitude and kinetics of trans-root potential depolarization, induced by the ascorbate redox forms and systems for the generation and scavenging of oxygen species, argue in favour of the mutually competing electron transfer role of ascorbate free radicals and superoxide radicals in the extracellular space of the root. These results provide evidence that at least a part of the electrical potential difference occurring across plant roots arises from current flow from the symplast, via the plasma membrane-bound redox systems, to naturally occurring compounds in the apoplast, and that this transfer is achieved through the mediation of their free radical forms.     

Inhibition of cardiac phosphatidylethanolamine N-methylation by oxygen free radicals

This study was undertaken to examine the effects of oxygen free radicals on phosphatidylethanolamine (PE) N-methylation in rat heart sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes. Three catalytic sites involved in the sequential methyl transfer reaction were studied by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (0.055, 10, and 150 microM) into SL or SR PE molecules under optimal conditions. In the presence of xanthine + xanthine oxidase (superoxide anion radicals generating system), PE N-methylation was inhibited at site I and III in the heavy SL fraction isolated by the hypotonic shock-LiBr treatment method. In the light SL fraction isolated by sucrose-density gradient, a significant inhibition of PE N-methylation was seen at all three sites. These inhibitory effects of xanthine + xanthine oxidase on PE N-methylation were prevented by the addition of superoxide dismutase. Hydrogen peroxide showed a significant inhibition of PE N-methylation at site I in the heavy SL fraction, and at site I and II in the light SL fraction. Catalase blocked the inhibitory effects of hydrogen peroxide. The effects of both xanthine + xanthine oxidase and hydrogen peroxide on the SR membranes were similar to those seen for the heavy SL fraction. These results suggest that, in addition to lipid peroxidation, the oxygen free radicals may affect the function of cardiac membranes by decreasing the phospholipid N-methylation activity.     

Free radicals and Dupuytren's contracture.

The concentration of substrate expressed as hypoxanthine capable of reacting with xanthine oxidase to release superoxide free radicals (O2-) was measured in control and Dupuytren''s contracture palmar fascia. In Dupuytren''s contracture palmar fascia the concentration of hypoxanthine was six times that of control and was greatest in "nodular" areas. Xanthine oxidase activity was also detected in Dupuytren''s contracture palmar fascia. These results suggest a greater potential for hypoxanthine-xanthine oxidase generated oxygen free radical formation in Dupuytren''s contracture than in control palmar fascia. Production of free radicals may be an important factor in the pathogenesis of Dupuytren''s contracture. The benefit of allopurinol in the management of Dupuytren''s contracture and other fibrotic conditions may thus be explained, as allopurinol binds to xanthine oxidase and prevents release of free radicals.     

Free radical and freezing injury to cell membranes of winter wheat

The symptoms of injury in microsomal membranes isolated from crowns of seedlings of Triticum aestivum , L. cultivar Fredrick after a lethal freeze-thaw stress included an increased lipid phase transition temperature, loss of lipid phosphate (lipid-P), and increased free fatty acid levels. However, minimal changes in fatty acid saturation were observed, suggesting minimal amounts of lipid peroxidation. All of these injury symptoms, including the lack of lipid peroxidation, were simulated in vitro by treatment of isolated membranes with oxygen free radicals, generated from either xanthine oxidase (EC 1.1.3.22) or paraquat (l,r-dimethyl-4,4'-bipyridinium dichloride). Further evidence indicating a relationship between free radicals and freezing injury comes from the observation that both protoplasts and microsomal membranes isolated from wheat seedlings, that had been acclimated to induce freezing tolerance, also had increased tolerance of oxygen free radicals, and contained higher lipid-soluble antioxidant levels, than those from non-acclimated seedlings. Lipid-soluble antioxidants accumulated in the crown tissue of the wheat seedling during the acclimation period. Freezing stress accelerated the formation of oxygen free radicals. Membranes isolated from crowns after a freeze–thaw stress tended to produce higher levels of superoxide as shown by the reduction of Tiron (1,2-dihydroxy-l,3-benzenedisulfonic acid). In protoplasts, increased superoxide production coincided with lethal freezing injury. These results are discussed in terms of the possible involvement of oxygen free radicals in mediating aspects of freezing injury to cell membranes.     

Effect of zinc on superoxide-dependent hydroxyl radical production in vitro

Trace elements play an important role in oxygen metabolism and therefore in the formation of free radicals. Whereas iron and copper are usually the main enhancers of free radical formation, other trace elements, such as zinc and selenium, protect against the harmful effects of these radicals. To investigate the different protective mechanisms of zinc on radical formation, we examined the effects of added zinc and copper on superoxide dismutase activity. We also studied the effects of copper and iron on xanthine oxidase activity and on the Haber-Weiss cycle (iron, superoxide, and hydrogen peroxide), which generates hydroxyl radicals in vitro. The hypoxanthine/xanthine oxidase radical generating system contained a variety of different physiological ligands for binding the iron. This study confirmed the inhibitory effect of copper on xanthine oxidase activity. Moreover, it demonstrated that zinc inhibited hydroxyl radical formation when this formation was catalyzed by a citrate-iron complex in the hypoxanthine/xanthine oxidase reaction. Finally, human blood plasma inhibited citrate-iron-dependent hydroxyl radical formation under the same conditions. Although trace elements seemed responsible for this antioxidant activity of plasma, it is likely that zinc played no role as a plasma antioxidant. Indeed, calcium appeared to be responsible for most of this effect under our experimental conditions.     

Superoxide-dependent and -independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Implications for oxygen-free-radical-induced tissue destruction during ischaemia and inflammation.

Xanthine oxidase is able to mobilize iron from ferritin. This mobilization can be blocked by 70% by superoxide dismutase, indicating that part of its action is mediated by superoxide (O2-). Uric acid induced the release of ferritin iron at concentrations normally found in serum. The O2(-)-independent mobilization of ferritin iron by xanthine oxidase cannot be attributed to uric acid, because uricase did not influence the O2(-)-independent part and acetaldehyde, a substrate for xanthine oxidase, also revealed an O2(-)-independent part, although no uric acid was produced. Presumably the amount of uric acid produced by xanthine oxidase and xanthine is insufficient to release a measurable amount of iron from ferritin. The liberation of iron from ferritin by xanthine oxidase has important consequences in ischaemia and inflammation. In these circumstances xanthine oxidase, formed from xanthine dehydrogenase, will stimulate the formation of a non-protein-bound iron pool, and the O2(-)-produced by xanthine oxidase, or granulocytes, will be converted by 'free' iron into much more highly toxic oxygen species such as hydroxyl radicals (OH.), exacerbating the tissue damage.     

慢性肾衰病人血清和红细胞抗氧化能力的ESR研究

用电子自旋共振(ESR)自旋捕集技术研究了正常人和肾衰病人血清和红细胞对黄嘌呤 黄嘌呤氧化酶体系产生的氧自由基的作用.结果发现:(1)正常人血清和红细胞能够有效地清除超氧阴离子自由基(O_2~-),而肾衰病人血清和红细胞清除O_2~-的能力明显比正常人血清和红细胞低;(2)正常人血清能有效地把黄嘌呤 黄嘌呤氧化酶体系产生的O_2~-转化为·OH,病人血清在这方面与正常人血清有显著性差异.     

Enzymatic and Non-Enzymatic Oxygenation of Tyrosine

Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. l -Tyrosine and d -tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detection as the product of oxygenation. Incubations were performed in the presence or absence of dopamine as co-substrate. Oxygenation of l -tyrosine occurred only in the presence of dopamine as co-substrate. No oxygenation of d -tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the l -isomer of tyrosine in its oxygenase function. It has recently been suggested that superoxide anion is a preferential oxygen substrate for human tyrosinase. Incubations were therefore performed with l - and d -tyrosine, human tyrosinase, and xanthine/xanthine oxidase in the system, generating superoxide anion and hydrogen peroxide. Considerable formation of dopa was observed, but the quantity was the same irrespective of whether d -tyrosine or l -tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosinase. In the incubate containing xanthine/xanthine oxidase, catalase completely inhibited dopa formation, and superoxide dismutase and mannitol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, since such radicals may be secondarily formed in the presence of superoxide anion and hydrogen peroxide.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

How relevant is the reoxidation of ferrocytochrome c by hydrogen peroxide when determining superoxide anion production?

In a recent publication [(1987) FEBS Lett. 210, 195-198] the authors claim the use of cytochrome c to detect superoxide anion underestimates the real rate of superoxide anion formation on the basis that: (i) the rate of uric acid formation by xanthine oxidase is about 4-fold faster than the rate of cytochrome c reduction and (ii) hydrogen peroxide formed upon dismutation of the superoxide anion generated by xanthine oxidase is capable of reoxidizing ferrocytochrome c. That paper may have been misleading for readers not very familiar with the field of oxygen radicals, since both assumptions are, in fact, incorrect. In this report we demonstrate that the build up in concentration of H2O2 during most reactions in which superoxide anion is being produced is not enough to affect the rate of cytochrome c reduction. Our results suggest that the authors may have been misled by an artifact due to exposure of the samples containing H2O2 to UV light, which generates hydroxyl radicals by photolysis.     

The role of free radicals in cold injuries

Cold injury is a tissue trauma produced by exposure to freezing temperatures and even brief exposure to a severely cold and windy environment. Rewarming of frozen tissue is associated with blood reperfusion and the simultaneous generation of free oxygen radicals. In this review is discussed the current understanding of the mechanism of action of free oxygen radicals as related to cold injury during rewarming. Decreased energy stores during ischaemia lead to the accumulation of adenine nucleotides and liberation of free fatty acids due to the breakdown of lipid membranes. On rewarming, free fatty acids are metabolized via cyclo-oxygenase and adenine nucleotides are metabolized via the xanthine oxidase pathway. These may be the source of free oxygen radicals. Leukocytes may also play a major role in the pathogenesis of cold injury. Oxygen radical scavengers, such as superoxide dismutase and catalase, may help to reduce the cold induced injury but their action is limited due to the inability readily to cross the plasma membrane. Lipid soluble antioxidants are likely to be more effective scavengers because of their presence in membranes where peroxidative reactions can be arrested.     

Hydroxyl radicals mediate injury to endothelium-dependent relaxation in diabetic rat

The purpose of this study was to determine the radical species which mediates the toxic effects of exogenous oxygenderived free radicals on endothelial function of chronic diabetic rat aorta. Endothelium-dependent relaxation to acetylcholine was impaired in diabetic vessels. Exposure to the exogenous free radical generating system of xanthine plus xanthine oxidase selectively impaired endothelium-dependent relaxation to acetylcholine in control and diabetic aorta with relaxations essentially abolished in diabetic aorta. The loss of relaxation to acetylcholine in diabetic aorta was prevented or attenuated by pretreatment with catalase, dimethylthiourea or desferrioxamine, but not by mannitol or superoxide dismutase. These results suggest that hydroxyl radicals play an important role in the endothelial injury produced by oxygen-derived free radicals in chronic diabetic rat aorta. Furthermore, the site of the injury is likely due to intracellular generation of hydroxyl radicals.     

Cytokinin interaction with free radical metabolism and senescence: Effects on endogenous lipoxygenase and purine oxidation

A typical system comprising xanthine-xanthine oxidase, which produces superoxide free radicals, significantly increased endogenous levels of the senescence-associated lipoxygenase enzyme while cytokinin reversed this effect. It is suggested that in its interaction with free radicals cytokinin may have a dual effect: a) it may inhibit purine oxidation by the formation of a 2,8 dihydroxy purine which lowers the substrate affinity of xanthine oxidase; b) it may act as a direct free radical scavenger by virtue of H abstraction from the α-carbon atom in the amine bond.     

Free radical scavenging actions of hippocampal metallothionein isoforms and of antimetallothioneins: an electron spin resonance spectroscopic study.

The high concentration of zinc in the hippocampal mossy fiber axon boutons is localized in the vesicles and is mobilized by exocytosis of the zinc-laden vesicles. Furthermore, the mammalian hippocampi contain metallothionein (MT) isoforms which regulate the steady state concentration of zinc, an important antioxidant. Indeed, zinc deprivation leads to an increased lipid peroxidation, reduces the activity of Cu++-Zn++ superoxide dismutase, and protect against oxidative stress such as exposure to ultraviolet A irradiation. By employing electron spin resonance (ESR) spectroscopy, we have demonstrated that rat hippocampal MT isoforms 1 and 2 were able to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), hydroxyl radicals (*OH) generated in a Fenton reaction, and superoxide anions (O2*-) generated by the hypoxanthine and xanthine oxidase system. In addition, MT-1 isoform protected the isolated hepatocytes from lipid peroxidation as determined by thiobarbituric acid bound malondialdehyde. MT antibodies scavenged DPPH radicals, hydroxyl radicals and reactive oxygen species but not superoxide anions. The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-1 appears to be a superior scavenger of superoxide anions and 1,1-diphenyl-2-picrylhydrazyl radicals. Moreover, antibodies formed against MT isoform retain some, but not all, free radical scavenging actions exhibited by MT-1 and MT-2.     

Superoxide radical as electron donor for oxidative phosphorylation of ADP

When isolated rat heart mitochondria are subject to xanthine/xanthine oxidase generated free radicals, nmol quantities of ADP are phosphorylated to ATP. This effect is proportional to xanthine oxidase concentration, and is relatively independent of ADP concentration. Exogenous superoxide dismutase partially suppresses the phosphorylation. Micromolar concentrations of iron salts completely eliminate the phosphorylation. Catalase has no effect. The likely electron source, then, is superoxide radicals. The reduced minus oxidised spectra of superoxide-bombarded mitochondria show that superoxide enters the electron transport chain by reducing cytochrome c and complex IV. Mitochondria retain their ability to phosphorylate ADP in more traditional ways under the experimental conditions described. Superoxide under physiological conditions in vivo may be a source of electrons for the oxidative phosphorylation of ADP.