The effect of EDTA and iron on the oxidation of hydroxyl radical scavenging agents and ethanol by rat liver microsomes

Rat liver microsomes catalyzed an NADPH-dependent oxidation of dimethylsulfoxide, 2-keto-4-thiomethylbutyrate and ethanol. The addition of EDTA and iron (ferric)-EDTA increased the oxidation of the hydroxyl radical scavenging agents and ethanol. Unchelated iron had no effect; therefore, appropriately chelated iron is required to stimulate microsomal production of hydroxyl radicals. Catalase strongly inhibited control rates as well as EDTA or iron-EDTA stimulated rates of hydroxyl radical production whereas superoxide dismutase had no effect. The rate of ethanol oxidation was ten- to twenty-fold greater than the rate of oxidation of hydroxyl radical scavengers in the absence of EDTA or iron-EDTA, suggesting little contribution by hydroxyl radicals in the pathway of ethanol oxidation. In the presence of EDTA or iron-EDTA, the rate of ethanol oxidation increased, and under these conditions, hydroxyl radicals appear to play a more significant role in contributing toward the overall oxidation of ethanol.     

Adaptational effect of dimethylsulfoxide during chronic emotional-painful stress in rats

Intraperitoneal injection of hydroxyl radical scavenger dimethyl sulfoxide (DMSO) to rats (1 g/kg body weight, daily for 3 weeks) did not change their behaviour in the open field and had no effect on blood pressure and heart and respiratory rates. DMSO injection before stress prevented changes usually induced by chronic (three-week) emotional-painful stress. DMSO increased superoxide dismutase activity in brain homogenates and serum. It is suggested that hydroxyl radicals (OH) play the key role in the realization of stress-induced effects and that molecular mechanisms of anti-stress effects of DMSO include both scavenging of hydroxyl radicals and activation of superoxide dismutase.     

Oxyradicals under UV-b stress and their quenching by antioxidants

Formation of oxyradicals under UV-B stress was investigated using cucumber cotyledons. UV-B radiation induced production of free radicals which were analyzed by ESR spectroscopy. Evidence was obtained for the formation of superoxide and hydroxyl radicals in the tissues by comparing PBN-adducts formed with radicals obtained by chemical autooxidation of KO2 and Fenton's reaction. Addition of superoxide dismutase (SOD) to the reaction mixture partially reduced the intensity of signals confirming the production of superoxide radical as well as hydroxyl radicals. These radicals were quenched in vitro by the natural antioxidants alpha-tocopherol, ascorbic acid and benzoquinone. Changes in the level of antioxidants were also monitored under UV-B stress. The endogenous level of ascorbic acid was enhanced and alpha-tocopherol level was reduced in the tissue after exposure to UV-B radiation. The present report happens to be the first direct evidence obtained for the formation of superoxide and hydroxyl radicals in plant tissues exposed to UV-B radiation.     

Synergistic induction of hydroxyl radical-induced DNA single-strand breaks by chromium(VI) compound and cigarette smoke solution

Chromium(VI) compounds and cigarette smoke are known human carcinogens. We found that K2Cr2O7 and cigarette smoke solution synergistically induced DNA single-strand breaks (0.23+/-0.04 breaks per DNA molecule) in pUC118 plasmid DNA. K2Cr2O7 alone or cigarette smoke solution alone induced much less strand breaks (0.03+/-0.01 or 0.07+/-0.02 breaks per DNA molecule, respectively). The synergistic effect was prevented by catalase and by hydroxyl radical scavengers such as deferoxamine, dimethylsulfoxide, d-mannitol, and Tris, but not by superoxide dismutase. Ascorbic acid enhanced the synergism. Glutathione inhibited strand breakage only at high concentrations. Electron spin resonance (ESR) studies using a hydroxyl radical trap demonstrated that hydroxyl radicals were generated when DNA was incubated with K2Cr2O7 and cigarette smoke solution. Hydroxyl radical adduct decreased dose-dependently when strand breakage was prevented by catalase, deferoxamine, dimethylsulfoxide, d-mannitol or Tris, but not significantly by superoxide dismutase. We also used ESR spectroscopy to study the effects of different concentration of ascorbic acid and glutathione. The results showed that hydroxyl radical, which is proposed as a main carcinogenic mechanism for both chromium(VI) compounds and cigarette smoke solution was mainly responsible for the DNA breaks they induced.     

The catalytic effect of the carcinogen "4-nitroquinoline-N-oxide" on the oxidation of vitamin C.

The carcinogen 4-nitroquinoline-N-oxide was found to mediate the reaction between ascorbate and oxygen. The oxidation of ascorbate was initiated by the production of the nitro radical anion which reacted with oxygen to produce the oxygen superoxide radical anion, peroxide and hydroxyl radical. The production of partially reduced oxygen intermediates resulted in additional reactions with ascorbate. The consumption of oxygen could be either completely blocked by reacting the nitro radical with ferricytochrome c or partially blocked by the combined effects of superoxide dismutase and catalase. The consumption of oxygen could be enhanced by reducing the hydroxyl radicals with dimethylsulfoxide.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

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.     

Leukotriene B4, C4, D4 and E4 inactivation by hydroxyl radicals

Leukotriene B4 chemotactic activity and leukotriene C4, D4 and E4 slow reacting substance activity were rapidly decreased by hydroxyl radicals generated by two different iron-supplemented acetaldehyde-xanthine oxidase systems. At low Fe2+, leukotriene inactivation was inhibited by catalase, superoxide dismutase, mannitol and ethanol, suggesting involvement of hydroxyl radicals generated by the iron-catalyzed interaction of superoxide and H2O2 (Haber-Weiss reaction). Leukotriene inactivation increased at high Fe2+ concentrations, but was no longer inhibitable by superoxide dismutase, suggesting that inactivation resulted from a direct interaction between H2O2 and Fe2+ to form hydroxyl radicals (Fenton reaction). The inactivation of leukotrienes by hydroxyl radicals suggests that oxygen metabolites generated by phagocytes may play a role in modulating leukotriene activity.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes.

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

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.     

Deoxyribose breakdown by the adriamycin semiquinone and H2O2: evidence for hydroxyl radical participation

We report our finding that the reaction between the adriamycin semiquinone (produced by reduction of the drug by xanthine oxidase) and H2O2 in N2 causes deoxyribose degradation to a thiobarbituric acid-reactive chromogen. Deoxyribose breakdown was inhibited by scavengers of hydroxyl radicals, providing evidence for the participation of hydroxyl radicals. The reaction was detected in air, but was less efficient in air than in N2. Deoxyribose degradation did not require a metal catalyst, and was inhibited by superoxide dismutase in air, but not N2. A similar reaction with deoxyribose in DNA may be of major importance in the antitumour action of adriamycin.     

The role of oxygen radicals in dye-mediated photodynamic effects in Escherichia coli B

Photosensitive dyes representative of the thiazines, xanthenes, acridines, and phenazines mediated phototoxicity in Escherichia coli B. The observed phototoxicity was sensitizer-, light-, and oxygen-dependent and is therefore a photodynamic effect. Hydroxyl radical scavengers conferred protection against the photodynamic action of all of the representative dyes. The extent of protection was dependent on the concentration of scavenger and on the in vitro reactivity of the scavenger with the hydroxyl radical. Exogenous superoxide dismutase and catalase partially protected the cells against the dye-mediated phototoxicity, and prior induction of intracellular superoxide dismutase and catalase by growth in glucose minimal medium containing manganese and paraquat substantially protected E. coli B against the photodynamic action of all of the dyes examined. Combinations of protective treatments against the phototoxicity of all four classes of dyes, including superoxide dismutase and catalase preinduction and addition of extracellular superoxide dismutase and catalase or the addition of hydroxyl radical scavengers, provided nearly complete protection against the oxygen-dependent component of dye-mediated lethality. E. coli B grown in glucose minimal medium containing manganese and photosensitive dyes induced manganese superoxide dismutase. The extent of induction was correlated with the dyes' ability to photooxidize NADH in vitro. Thus, oxygen radicals are primarily responsible for the oxygen-dependent toxicity of the photosensitive dyes examined, and one adaptive response of E. coli B to a dye-mediated oxidative threat is to induce superoxide dismutase.     

Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat

Thiourea and superoxide dismutase were effective antidotes to paraquat toxicity in an HL60 cell culture system, whereas other hydroxyl scavengers were ineffective. The efficacy of thioureas was not due to blockage of intracellular paraquat uptake, inhibition of NADPH-P-450 reductase, or reaction with the paraquat radical. Thiourea also competitively inhibited the reduction of cytochrome c by the xanthine/xanthine oxidase superoxide-generating system, and the release of iron from ferritin by superoxide radicals. The reaction of superoxide with thiourea produced a sulfhydryl compound distinct from products formed by hydrogen peroxide or hydroxyl radicals. Spectrophotometric and chromatographic studies indicated the carbon-sulfide double bond was converted to a sulfhydryl group which reacted with Ellman's reagent. Additional confirmatory evidence for the sulfhydryl compound was obtained with carbon-13 NMR and mass spectroscopies. Thus, thioureas are direct scavengers of superoxide radicals as well as hydroxyl radicals and hydrogen peroxide. The rate constant for the reduction of thiourea by superoxide was estimated at 1.1 x 10(3) M-1 s-1. The implication of this finding on free radical studies, the mechanism of paraquat toxicity, and the metabolism of thioureas is discussed.     

Mechanism of reaction of a suggested superoxide-dismutase mimic, Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The interaction of a recently developed intracellular superoxide dismutase analogue, Fe(II)-N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (Fe(II)-TPEN), with reactive oxygen species was investigated under in vitro conditions. The complex catalyzed the dismutation of enzyme- or radiolysis-generated superoxide with the production of H2O2; under steady-state conditions the equilibrium was strongly shifted toward Fe(III)-TPEN. Fe(II)-TPEN reacted with H2O2 to generate hydroxyl radicals in a Fenton reaction. The oxidized Fe(III)-TPEN was readily reduced by ascorbate or glutathione. Given the capacity to produce hydroxyl radicals and the reaction with cellular reductants it seems unlikely that Fe-TPEN may find widespread use as an intracellular superoxide dismutase substitute.     

In vivo and in vitro oxidative biotransformation of dimethylformamide in rat

In rats and in humans, dimethylformamide (DMF) is mainly metabolized into N-hydroxymethyl-N-methylformamide (DMF-OH). The in vitro oxidation of DMF by rat liver microsomes is decreased in the presence of catalase and superoxide dismutase. The radical scavengers, dimethylsulfoxide (DMSO), tertiary butyl alcohol (t-butanol), aminopyrine, hydroquinone and trichloroacetonitrile reduce the oxidation of DMF to DMF-OH in vitro and in vivo. Conversely, DMF inhibits the demethylation of DMSO, t-butanol and aminopyrine. The addition of iron-EDTA to the incubation system induces the production of N-methylformamide (NMF) from DMF. These results support the hypothesis that the metabolic pathway leading from DMF to DMF-OH and NMF involves hydroxyl radicals. Superoxide radical and hydrogen peroxide take part in the metabolic process. DMF is preferentially metabolized into DMF-OH. NMF appears mainly when the production of hydroxyl radicals is stimulated, the methyl group being recovered as formic acid.     

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reaction mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both .O2- and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H2O2 and .O2- are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (less than 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal chelating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (less than 5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (less than 10%) inhibition, but inhibited by 30% in the presence of catalase + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalase + superoxide dismutase + DETAPAC.     

Possible cytotoxic mechanisms of TNF in vitro

TNF is a protein originally isolated by its ability to induce hemorrhagic necrosis of some tumors in vivo, and cytotoxic effects against certain tumor cells, but not against normal cells in vitro. Though mechanisms of the cytotoxicity and selectivity of TNF action in vitro are not clear, there is much evidence that free radicals such as superoxide and hydroxyl radicals, mediated by TNF, are correlated with TNF cytotoxicity. Recently, manganous superoxide dismutase (MnSOD) in mitochondria has been demonstrated to be a rescue protein against TNF cytotoxicity and to be induced when the cells are exposed to TNF in the resistant cells. Two steroid hormones, glucocorticoid and 1,25-dihydroxyvitamin D3, block the cytotoxicity of TNF. Glucocorticoid may reduce TNF cytotoxicity by inhibiting phospholipase A2 and 1,25-dihydroxyvitamin D3 by inducing MnSOD in combination with TNF.     

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.     

The production of free radicals during the autoxidation of cysteine and their effect on isolated rat hepatocytes

Autoxidizing cysteine has been shown to produce thiyl and hydroxyl radicals. Hydrogen peroxide increased the yield of both radicals which was inhibited by catalase but stimulated by copper / zinc superoxide dismutase. This effect is due to increased bydrogen peroxide production by copper / zinc superoxide dismutase as a result of superoxide dismutation. The production of superoxide radicals could not be detected probably because of its low reactivity, however, measurement of oxygen uptake and reduction of ferricytochrome c by autoxidizing cysteine clearly implicate the involvement of super oxide radicals. The production of hydroxyl radicals is postulated to proceed through a Fenton reaction, however, this may not necessarily be metal ion controlled. Autoxidizing cysteine disrupts the integrity of heptocytes causing release of glutathione, adenosine triphosphate and lactate dehydrogenase indicating that it is of little use as a therapeutic agent.