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.     

Enzyme inactivation by metal-catalyzed oxidation of coenzyme Q1.

Ubiquinol-1 in aerated aqueous solution inactivates several enzymes--alanine aminotransferase, alkaline phosphatase, Na+/K(+)-ATPase, creatine kinase and glutamine synthetase--but not isocitrate dehydrogenase and malate dehydrogenase. Ubiquinone-1 and/or H2O2 do not affect the activity of alkaline phosphatase and glutamine synthetase chosen as model enzymes. Dioxygen and transition metal ions, even if in trace amounts, are essential for the enzyme inactivation, which indeed does not occur under argon atmosphere or in the presence of metal chelators. Supplementation with redox-active metal ions (Fe3+ or Cu2+), moreover, potentiates alkaline phosphatase inactivation. Since catalase and peroxidase protect while superoxide dismutase does not, hydrogen peroxide rather than superoxide anion seems to be involved in the inactivation mechanism through which oxygen active species (hydroxyl radical or any other equivalent species) are produced via a modified Haber-Weiss cycle, triggered by metal-catalyzed oxidation of ubiquinol-1. The lack of efficiency of radical scavengers and the almost complete protection afforded by enzyme substrates and metal cofactors indicate a 'site-specific' radical attack as responsible for the oxidative damage.     

Role of iron in adriamycin biochemistry

Adriamycin forms a chelate with Fe(III) that exhibits complex redox chemistry. The drug ligand is able to directly reduce the bound Fe(III) with the concomitant production of a one-electron oxidized drug radical. This Fe(II) can reduce oxygen to hydrogen peroxide and cleave the peroxide to yield the hydroxyl radical. In addition, the drug X Fe complex can catalyze the transfer of electrons from reduced glutathione to molecular oxygen to yield superoxide, hydrogen peroxide, and hydroxyl radicals. The adriamycin X Fe complex binds to DNA to form a ternary drug X Fe X DNA complex, which is also able to catalyze the thiol-dependent reduction of oxygen and the formation of hydroxyl radical from hydrogen peroxide. As a consequence of this chemistry, the adriamycin X Fe complex can cleave DNA on the addition of glutathione or hydrogen peroxide. Although less well defined, the adriamycin X Fe complex can bind to cell membranes and cause oxidative destruction of these membranes in the presence of thiols or hydrogen peroxide.     

Free radicals in iron-containing systems

All oxidative damage in biological systems arises ultimately from molecular oxygen. Molecular oxygen can scavenge carbon-centered free radicals to form organic peroxyl radicals and hence organic hydroperoxides. Molecular oxygen can also be reduced in two one-electron steps to hydrogen peroxide in which case superoxide anion is an intermediate; or it can be reduced enzymatically so that no superoxide is released. Organic hydroperoxides or hydrogen peroxide can diffuse through membranes whereas hydroxyl radicals or superoxide anion cannot. Chain reactions, initiated by chelated iron and peroxides, can cause tremendous damage. Chain carriers are chelated ferrous ion; hydroxyl radical .OH, or alkoxyl radical .OR, and superoxide anion O2-. or organic peroxyl radical RO2.. Of these free radicals .OH and RO2. appear to be most harmful. All of the biological molecules containing iron are potential donors of iron as a chain initiator and propagator. An attacking role for superoxide dismutase is proposed in the phagocytic process in which it may serve as an intermediate enzyme between NADPH oxidase and myeloperoxidase. The sequence of reactants is O2----O2-.----H2O2----HOCl.     

Effects of free radicals on cytosolic creatine kinase activities and protection by antioxidant enzymes and sulfhydryl compounds

The main purpose of this study was to investigate the effect of free radicals and experimental diabetes on cytosolic creatine kinase activity in rat heart, muscle and brain. Hydrogen peroxide decreased creatine kinase activity in a dose dependent manner which was reversed by catalase. Xanthine/xanthine oxidase, which produces superoxide anion, lowered the creatine kinase activity in the same manner whose effect was protected by superoxide dismutase. N-acetylcysteine and dithiothreitol also significantly ameliorated the effect of Xanthine/xanthine oxidase and hydrogen peroxide. Experimental diabetes of twenty-one days (induced by alloxan), also caused a similar decrease in the activity of creatine kinase. This led us to the conclusion that the decrease in creatine kinase activity during diabetes could be due to the production of reactive oxygen species. The free radical effect could be on the sulfhydryl groups of the enzyme at the active sites, since addition of sulfhydryl groups like N-acetylcysteine and dithiothreitol showed a significant reversal effect.     

Inactivation of alcohol dehydrogenase by piroxicam-derived radicals

Alcohol dehydrogenase (ADH) was used as a marker molecule to clarify the mechanism of gastric mucosal damage as a side effect of using piroxicam. Piroxicam inactivated ADH during interaction of ADH with horseradish peroxidase and H₂O₂ (HRP-H₂O₂). The ADH was more easily inactivated under aerobic than anaerobic conditions, indicating participation by oxygen. Superoxide dismutase, but not hydroxyl radical scavengers, inhibited inactivation of ADH, indicating participation by superoxide. Sulfhydryl (SH) groups in ADH were lost during incubation of piroxicam with HRP-H₂O₂. Adding reduced glutathione (GSH) efficiently blocked ADH inactivation. Other SH enzymes, including creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, were also inactivated by piroxicam with HRP-H₂O₂. Thus SH groups in the enzymes seem vulnerable to piroxicam activated by HRP-H₂O₂. Spectral change in piroxicam was caused by HRP-H₂O₂. ESR signals of glutathionyl radicals occurred during incubation of piroxicam with HRP-H₂O₂ in the presence of GSH. Under anaerobic conditions, glutathionyl radical formation increased. Thus piroxicam free radicals interact with GSH to produce glutathionyl radicals. Piroxicam peroxyl radicals or superoxide, or both, seem to inactivate ADH. Superoxide may be produced through interaction of peroxyl radicals with H₂O₂. Thus superoxide dismutase may inhibit inactivation of ADH through reducing piroxicam peroxyl radicals or blocking interaction of SH groups with O₂^-, or both. Other oxicam derivatives, including isoxicam, tenoxicam and meloxicam, induced ADH inactivation in the presence of HRP-H₂O₂.     

Maintenance of left ventricular function (90%) after twenty-four-hour heart preservation with deferoxamine.

During 24-h in vitro heart preservation and reperfusion, irreversible tissue damage occurs caused by reactive oxygen intermediates, such as superoxide radicals, singlet oxygen, hydrogen peroxide, hydroperoxyl, hydroxyl radicals, as well as the peroxynitrite radical. Reduction of the related oxidative damage of reperfused ischemic tissue by free radical scavengers and metal chelators is of primary importance in maintaining heart function. We assessed whether deferoxamine (DFR) added to a cardioplegia solution decreased free radical formation during 24-h cold (5 degrees C) heart preservation and normothermic reperfusion (37 degrees C) in the Langendorff isolated perfused rat heart. The deferoxamine treated hearts were significantly (p less than .001) better preserved than the control hearts after 24 h of preservation with regard to recovery of left ventricular diastolic pressure, contractility (+dP/dt), relaxation (-dP/dt), creatine kinase release, and lipid peroxidation. DFR preserved cell membrane integrity and maintained 93% of left ventricular contractility. The evidence suggests that DFR reduces lipid peroxidation damage by reducing free radical formation and thereby maintaining normal coronary perfusion flow and myocardial function.     

Free Radical Generation at the Solid/Liquid Interface in Iron Containing Minerals

The potential for free radical release has been measured by means of the spin trapping technique on three kinds of iron containing particulate: two asbestos fibers (chrysotile and crocidolite); an iron-exchanged zeolite and two iron oxides (magnetite and haematite). DMPO (5,5'-dimethyl-1 -pirroline-N-oxide), used as spin trap in aqueous suspensions of the solids, reveals the presence of the hydroxyl and carboxylate radicals giving rise respectively to the two adducts [DMPO-OH] and [DMPO-CO2], each characterized by a well-defined EPR spectrum. Two target molecules have been considered: the formate ion to evidence potential for hydrogen abstraction in any biological compartment and hydrogen peroxide, always present in the phagosome during phagocytosis. The kinetics of decomposition of hydrogen peroxide has also been measured on all solids. Ferrozine and desferrioxamine, specific chelators of Fe(II) and Fe(III) respectively, have been used to remove selectively iron ions. Iron is implicated in free radical release but the amount of iron at the surface is unrelated to the amount of radicals formed. Only few surface ions in a particular redox and coordination state are active. Three different kinds of sites have been evidenced: one acting as H abstractor, the other as a heterogeneous catalyst for hydroxyl radical release, the third one related to catalysis of hydrogen peroxide disproportionation. In both mechanisms of free radical release, the Fe-exchanged zeolite mimics the behaviour of asbestos whereas the two oxides are mostly inert. Conversely magnetite turns out to be an excellent catalyst for hydrogen peroxide disproportionation while haematite is inactive also in this reaction. The results agree with the implication of a radicalic mechanism in the in vitro DNA damage and in the in vivo toxicity of asbestos.     

Enhancement of adriamycin toxicity by iron chelates is not a free radical mechanism

The possible involvement of metal ions and free radicals in the cytotoxic mechanism of Adriamycin (ADR) was investigated, using a model system ofEscherichia coli cells. It is shown thatE. coli mediated the production of free radicals under anaerobic (ADR-semiquinone) and aerobic (superoxide) conditions. ADR-induced loss of colony-forming ability was enhanced by the addition of iron (Fe) chelates. These observations suggested that a Fenton-type free radical mechanism was responsible for ADR toxicity. However, the mortality rate was essentially unchanged by the exclusion of oxygen. It was also unaffected by the addition of H₂O₂, catalase, or chelating agents. Cu(II), Zn(II) or Mg(II) had no effect on ADR toxicity. ADR and iron chelates did not induce measurable amounts of DNA strand-breaks. These observations suggest a mechanism of ADR-induced cell killing that is enhanced by Fe chelates, but does not directly involve oxygen-derived free radicals.     

Interaction between albumin-bound dinitrosyl iron complexes and reactive oxygen species

Dinitrosyl iron complexes (DNIC) bound to BSA are shown to be destroyed by superoxide radicals generated in the xanthine oxidase-xanthine system. Peroxynitrite is also efficient in this respect. By contrast, neither hydrogen peroxide nor tert-butyl hydroperoxide appreciably destroy BSA-DNIC even at a tenfold molar excess. Evidence is obtained for the vasodilatory properties of BSA-DNIC. It is suggested that in this way peroxynitrite and superoxide radical can affect the physiological functions of nitric oxide.     

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.     

The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger

Free radicals are by-products of metabolism and exist in a homeostasis between generation and scavenging in vivo. Excessive free radicals cause various diseases, including nervous system diseases. Neuroglobin (Ngb), a nervous system-specific oxygen-binding protein, has been suggested to be a potential free radical scavenger in the nervous system in vivo; however, its underlying mechanism remains unclear. In this study, we investigated the antioxidant potential and free radical scavenging properties of recombinant human Ngb (rhNgb) in vitro. Interestingly, we found that the rhNgb protein itself has a direct and distinct antioxidant capacity and can efficiently scavenge a variety of free radicals, including the [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid)] (ABTS) cation, superoxide anion, hydrogen peroxide, and hydroxyl radical. The capacity of rhNgb to scavenge the superoxide anion and hydrogen peroxide was even comparable to that of vitamin C. In addition, rhNgb had Fe(2+) chelating activity but hemoglobin did not. In conclusion, our results indicated that the rhNgb protein itself has antioxidant and free radical scavenging activities, providing fundamental evidence for the neuroprotective function of Ngb. These data provide key information for the origin of the neuroprotective and physiological role of Ngb and will promote the treatment of reactive oxygen species (ROS)-related diseases using this novel oxygen-binding globin.     

Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+-ATPase activity of cardiac sarcoplasmic reticulum

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe3+-ADP, we studied lipid peroxidation and Ca2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15 micrograms/ml) or iron chelator, deferoxamine (1.25-1000 microM) protected Ca2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.     

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.     

Lipid peroxidation induced by phenylbutazone radicals

Lipid peroxidation was investigated to evaluate the deleterious effect on tissues by phenylbutazone (PB). PB induced lipid peroxidation of microsomes in the presence of horseradish peroxidase and hydrogen peroxide (HRP-H2O2). The lipid peroxidation was completely inhibited by catalase but not by superoxide dismutase. Mannitol and dimethylsulfoxide had no effect. These results indicated no paticipation of superoxide and hydroxyl radical in the lipid peroxidation. Reduced glutathione (GSH) efficiently inhibited the lipid peroxidation. PB radicals emitted electron spin resonance (ESR) signals during the reaction of PB with HRP-H2O2. Microsomes and arachidonic acid strongly diminished the ESR signals, indicating that PB radicals directly react with unsaturated lipids of microsomes to cause thiobarbituric acid reactive substances. GSH sharply diminished the ESR signals of PB radicals, suggesting that GSH scavenges PB radicals to inhibit lipid peroxidation. Also, 2-methyl-2-nitrosopropan strongly inhibited lipid peroxidation. R-Phycoerythrin, a peroxyl radical detector substance, was decomposed by PB with HRP-H2O2. These results suggest that lipid peroxidation of microsomes is induced by PB radicals or peroxyl radicals, or both.     

Ascorbate/iron mediation of hydroxyl free radical damage to PBR322 plasmid DNA

Plasmid PBR322 DNA has been exposed to hydroxyl free radicals generated from an ascorbate/Fe system. Hydroxyl free radical scavengers as well as the iron chelator desferroxamine and catalase inhibit the DNA nicking which occurs, but superoxide dismutase had no effect. The DNA nicking was temperature dependent, occuring more rapidly at higher temperatures. The rate of DNA nicking was accelerated by the addition of hydrogen peroxide. There was an early lag phase in DNA nicking, even though the rate of hydroxyl free radical generation, as assessed by salicylate hydroxylation, showed no lag phase. It is considered that the early hydroxyl free radical damage to DNA may be biologically very important in mutagenic and carcinogenic processes.     

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.     

Mechanisms for survival of bacteria in oxidative stress and iron ions deficiency

The influence of culture medium Fe2+ content on the resistance of Escherichia coli to hydroxyl radicals formed in the presence of Fe2+ and hydrogen peroxide in Fenton reaction was investigated. It was founded that a lack of Fe2+ in a culture medium increased resistance of bacteria to hydroxyl radicals but not to hydrogen peroxide. The suggestion was made that the lack of Fe2+ starts up synthesis of metabolites which inactivate hydroxyl radical or block Fe2+ ions participating in Fenton reaction. The phenomenon under study is considered to be a possible mechanism for survival of bacteria in oxidative stress and iron ions deficiency.     

Ascorbate peroxidase–thioredoxin interaction

Proteomics data have suggested ascorbate peroxidase (APX) to be a potential thioredoxin-interacting protein. Using recombinant enzymes, we observed that incubation of pea cytosolic APX with reduced poplar thioredoxins h drastically inactivated the peroxidase. A similar inactivation is induced by reduced glutathione and dithiothreitol, whereas diamide and oxidized glutathione have no effect. Oxygen consumption measurements, modifications of the APX visible spectrum and protection by hydrogen peroxide scavenging enzymes suggest that APX oxidizes thiols leading to the generation of thiyl radicals. These radicals can in turn react with thiyl anions to produce the disulfide radical anions, which are responsible for oxygen reduction and subsequent hydrogen peroxide production. The APX inactivation is not due solely to hydrogen peroxide since fluorimetry indicates that the environment of the APX tryptophan residues is dramatically modified only in the presence of thiol groups. The physiological implications of this interaction are discussed.     

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.