Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

Mechanism of site-specific DNA damage induced by methylhydrazines in the presence of copper(II) or manganese(III).

DNA damage induced by methylhydrazines (monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine) in the presence of metal ions was investigated by a DNA sequencing technique. 1,2-Dimethylhydrazine plus Mn(III) caused DNA cleavage at every nucleotide without marked site specificity. ESR-spin-trapping experiments showed that the hydroxyl free radical (.OH) is generated during the Mn(III)-catalyzed autoxidation of 1,2-dimethylhydrazine. DNA damage and .OH generation were inhibited by .OH scavengers and superoxide dismutase, but not by catalase. The results suggest that 1,2-dimethylhydrazine plus Mn(III) generates .OH, not via H2O2, and that .OH causes DNA damage. In the presence of Cu(II), DNA cleavage was caused by the three methylhydrazines frequently at thymine residues, especially of the GTC sequence. The order of Cu(II)-mediated DNA damage (1,2-dimethylhydrazine greater than monomethylhydrazine approximately 1,1-dimethylhydrazine) was not correlated with the order of methyl free radical (.CH3) generation during Cu(II)-catalyzed autoxidation (monomethylhydrazine greater than 1,1-dimethylhydrazine much greater than 1,2-dimethylhydrazine). Catalase and bathocuproine, a Cu(I)-specific chelating agent, inhibited DNA damage while catalase did not inhibit the .CH3 generation. The order of DNA damage was correlated with the order of ratio of H2O2 production to O2 consumption observed during Cu(II)-catalyzed autoxidation of methylhydrazines. These results suggest that the Cu(I)-peroxide complex rather than the .CH3 plays a more important role in methylhydrazine plus Cu(II)-induced DNA damage.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

The relative effectiveness of .OH, H2O2, O2-, and reducing free radicals in causing damage to biomembranes. A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (.OH, H2O2), ambivalent (O2-) and reducing free radicals (e- and CO2-) in causing damage to membranes and membrane=bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membrane-bound glyceraldehyde-3-phosphate dehydrogenase (R(enz)) were measured and the rates of damage to membranes (R(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. .OH, O2- and H2O2 were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e- and CO2- were the least effective. R(enz) values of O2- and H2O2 were 10-times and of .OH 15-times that of e-. R(mb) values were quite similar for e- and H2O2 (about twice that of O2-), while that of .OH was 3-times that of O2-. Hence, with respect to R(mb): .OH greater than e- = H2O2 greater than O2-, and with respect to R(enz): .OH greater than O2- = H2O2 much greater than e-. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H2O2 added as a chemical reagent and H2O2 formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H2O2 but markedly susceptible to the latter.     

Egg yolk phosvitin inhibits hydroxyl radical formation from the fenton reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical ((.-)OH) formation from H(2)O(2) in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited (.-)OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the (.-)OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H(2)O(2). These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings.

Leaves of maize (Zea mays L.) seedlings were supplied with different concentrations of abscisic acid (ABA). Its effects on the levels of superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and the content of catalytic Fe, the activities of several antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), the contents of several non-enzymatic antioxidants such as ascorbate (ASC), reduced glutathione (GSH), alpha-tocopherol (alpha-TOC) and carotenoid (CAR), and the degrees of the oxidative damage to the membrane lipids and proteins were examined. Treatment with 10 and 100 microM ABA significantly increased the levels of O(2)(-) and H(2)O(2), followed by an increase in activities of SOD, CAT, APX and GR, and the contents of ASC, GSH, alpha-TOC and CAR in a dose- and time-dependent pattern in leaves of maize seedlings. An oxidative damage expressed as lipid peroxidation, protein oxidation, and plasma membrane leakage did not occur except for a slight increase with 100 microM ABA treatment for 24 h. Treatment with 1,000 microM ABA led to a more abundant generation of O(2)(-) and H(2)O(2) and a significant increase in the content of catalytic Fe, which is critical for H(2)O(2)-dependent hydroxyl radical production. The activities of these antioxidative enzymes and the contents of alpha-TOC and CAR were still maintained at a higher level, but no longer further enhanced when compared with the treatment of 100 microM ABA. The contents of ASC and GSH had no changes in leaves treated with 1,000 microM ABA. These results indicate that treatment with low concentrations of ABA (10 to 100 microM) induced an antioxidative defence response against oxidative damage, but a high concentration of ABA (1,000 microM) induced an excessive generation of AOS and led to an oxidative damage in plant cells.     

Production of hydroxyl free radical in the xanthine oxidase system with addition of 1-methyl-3-nitro-1-nitrosoguanidine

We have examined the mechanism of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG)-induced gastric cancer with respect to the production of hydroxyl free radical (OH). Nucleophilic attack by H2O2 on the nitroso group of MNNG produces 1-methyl-3-nitroguanidine (MNG) and the intermediate peroxynitric acid (ONOOH), which splits into hydroxyl free radical (OH) and nitrogen dioxide leading to the formation of nitric and nitrate ions in water. Xanthine oxidase (XO) induces the production of O2.- or H2O2 from molecular oxygen, depending on the overall level of enzyme reduction. In this study, we examined OH production by the reaction of MNNG with H2O2 derived from the XO-HX system containing XO and the purine substrate hypoxanthine by ESR using the spin trapping reagent 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). OH was produced in the XO-HX-DMPO system with addition of MNNG (the MNNG-XO-HX-DMPO system) under aerobic conditions, but was not in the XO-HX-DMPO system, and production of OH was inhibited by catalase but not by superoxide dismutase, suggesting that OH was produced by the reaction of MNNG with H2O2 derived from the XO-HX system. The production of OH was significantly increased with increase in the reducing activity of XO, though that of O2.- was not, also suggesting the O2(.-)-independent .OH production. The productions of nitrite ion and MNG in the MNNG-XO-HX system were determined by the colorimetric method and HPLC, respectively. Based on these findings, we conclude that .OH was produced by homolytic split of the intermediate ONOOH formed by nucleophilic attack of H2O2 derived from the XO-HX system on MNNG.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Fluorescent light-induced chromosome damage in human IMR-90 fibroblasts. Role of hydrogen peroxide and related free radicals

Exposure of human fibroblasts (IMR-90) to cool-white fluorescent light causes chromatid breaks and exchanges. This chromatid damage is caused largely by the production of hydrogen peroxide (H2O2) since it can be prevented almost completely by the addition of catalase. In support of this conclusion, exogenous H2O2 is shown to induce chromatid breaks. The clastogenic amounts of H2O2 generated during light exposure are formed within the cell since cells illuminated in saline showed the same extent of damage as cells in culture medium. Addition of selenite to the cultures during light exposure significantly decreases the chromatid damage in a dose-related manner and may be necessary to maintain sufficient activity of glutathione peroxidase. The free hydroxyl radical, . OH, appears to be partially responsible for the light-induced chromatid damage. Of the free-radical scavengers tested, i.e., mannitol, vitamin E, and dimethyl sulfoxide, only mannitol, which scavenges . OH, significantly decreases the light-induced chromatid damage. Thus, both . OH and H2O2 formed within the cell during light exposure are agents that directly or indirectly cause chromatid damage.     

Superoxide removal and radiation protection in bacteria

Previous work with procaryotic cells has identified one kind of lethal damage from ionizing radiation which occurs only within a specific range of low O2 concentrations, about 10(-6) to 10(-4) M. Within this range, protection can occur in three ways: through the enzymatic decomposition of hydrogen peroxide (H2O2) by added catalase, through the enzymatic degradation of superoxide anion radicals (.O2-) by added superoxide dismutase (SOD), and through scavenging hydroxyl radicals (.OH) by various additives. These results indicate that three radiolytic products, H2O2, .OH, and .O2- (and/or the conjugate acid, the perhydroxyl radical, .HO2) are involved in this single kind of radiation-induced damage. Although the radiolytic productions of H2O2 and .O2- are strongly enhanced in higher O2 concentrations, neither enzyme protects when these air-equilibrated bacteria are irradiated. These experiments address this apparent contradiction and focus on the specific issue of why the addition of SOD protects at low but not at high O2 concentrations. We propose that, at a given O2 concentration, .O2- (and/or .HO2) may either react (with some cellular component?) to cause damage or react (with itself) to form hydrogen peroxide (H2O2). The specific O2 concentration during irradiation would determine the relative rates of these competing reactions and therefore the O2 concentration itself would establish whether or not we will observe damage from .O2-.     

Hyperoside prevents oxidative damage induced by hydrogen peroxide in lung fibroblast cells via an antioxidant effect

We elucidated the cytoprotective effects of hyperoside (quercetin-3-O-galactoside) against hydrogen peroxide (H2O2)-induced cell damage. We found that hyperoside scavenged the intracellular reactive oxygen species (ROS) detected by fluorescence spectrometry, flow cytometry, and confocal microscopy. In addition, we found that hyperoside scavenged the hydroxyl radicals generated by the Fenton reaction (FeSO4)+H2O2) in a cell-free system, which was detected by electron spin resonance (ESR) spectrometry. Hyperoside was found to inhibit H2O2-induced apoptosis in Chinese hamster lung fibroblast (V79-4) cells, as shown by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population, and decreased DNA fragmentation. In addition, hyperoside pretreatment inhibited the H2O2-induced activation of caspase-3 measured in terms of levels of cleaved caspase-3. Hyperoside prevented H2O2-induced lipid peroxidation as well as protein carbonyl. In addition, hyperoside prevented the H2O2-induced cellular DNA damage, which was established by comet tail, and phospho histone H2A.X expression. Furthermore, hyperoside increased the catalase and glutathione peroxidase activities. Conversely, the catalase inhibitor abolished the cytoprotective effect of hyperoside from H2O2-induced cell damage. In conclusion, hyperoside was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing antioxidant enzyme activity.     

Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation

We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway.     

Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related hydrazines: non-Fenton-type hydroxyl radical formation.

The mechanism by which hydrazines induce damage to cellular and isolated DNA in the presence of metal ions has been investigated by pulsed-field gel electrophoresis (PFGE), DNA sequencing methods, and the ESR spin-trapping technique. For the detection of single-strand breaks by PFGE, an experimental procedure with alkali treatment has been designed. Isoniazid, hydrazine, and phenylhydrazine induced DNA single- and double-strand breaks in cells pretreated with Mn(II), whereas iproniazid did not. With isolated 32P-DNA, isoniazid produced DNA damage in the presence of Cu(II), Mn(II), or Mn(III). Iproniazid damage isolated DNA only in the presence of Cu(II). The Cu(II)-mediated DNA damage by isoniazid or iproniazid is due to active oxygen species other than hydroxyl free radical (.OH), presumably the Cu(I)-peroxide complex. Cleavage of isolated DNA by isoniazid plus Mn(II) occurred without marked site specificity. The DNA damage was inhibited by .OH scavengers and superoxide dismutase (SOD) but not by catalase, suggesting the involvement of .OH formed via O2- but not via H2O2. Consistently, in ESR experiments .OH formation was observed during Mn(II)-catalyzed autoxidation of isoniazid, and the .OH formation was inhibited by SOD, but not by catalase. Iproniazid plus Mn(II) produced no or little .OH. We propose a reaction mechanism for the .OH formation without a H2O2 intermediate during manganese-catalyzed autoxidation of hydrazine. The present and previous data raise the possibility that hydrazines plus Mn(II)-induced cellular DNA damage may occur, at least in part, through the non-Fenton-type reaction.     

Progressive increases in serum catalase activity in advancing human immunodeficiency virus infection.

We found that serum from individuals with Acquired Immunodeficiency Syndrome (AIDS) had more (p less than .05) catalase activity (31.5 +/- 5.2 U/mL) than serum from healthy control subjects (7.3 +/- 0.8 U/mL). Moreover, serum catalase (but not glutathione peroxidase) activity increased progressively with advancing human immunodeficiency virus (HIV) infection (i.e., AIDS greater than symptomatic infection greater than asymptomatic infection greater than controls). Increases in serum catalase activity correlated with increases in serum hydrogen peroxide (H2O2) scavenging ability and reached levels which decreased exogenous H2O2-mediated injury to cultured endothelial cells without altering neutrophil bactericidal activity or mononuclear cell cytotoxicity in vitro. Serum catalase activity correlated with serum lactate dehydrogenase (LDH) activity but did not appear to be a consequence of erythrocyte (RBC) hemolysis since RBC fragility and serum haptoglobin levels were comparable in HIV-infected and control subjects. Increases in serum catalase activity may reflect and/or compensate for systemic glutathione and other antioxidant deficiencies in HIV-infected individuals.     

On the formation of oxygenated radicals by fredericamycin A and implications to its anticancer activity: an ESR investigation

It has been recently suggested that the exceptionally high antitumor and antibacterial activity of natural fredericamycin A (FMA) is related to its ability to spontaneously generate the superoxide anion (O2-) and hydroxyl (.OH) radicals in aerobic solutions [Hilton, B. D., Misra, R., & Zweier, J. L. (1986) Biochemistry 25, 5533]. With a view to understand the mechanistic details, attempts were made to reproduce earlier electron spin resonance (ESR) evidence for the oxygenated free radical formation in well-aerated solutions of natural FMA in dimethyl sulfoxide and dilute H2O2. Little or no evidence was obtained for the formation of the O2- and methoxy (.OCH3) radicals, while the detected formation of the .OH and methyl (.CH3) radicals was attributable largely to mechanisms not involving FMA. These results thus reopen the question regarding the mechanism of its exceptionally high tumoricidal-bacteriocidal activity.     

Free radical-mediated transgene inactivation of macrophages by endotoxin

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process.     

The oxidative inactivation of mitochondrial electron transport chain components and ATPase

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

One-electron reduction of chromate by NADPH-dependent glutathione reductase

Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl (.OH) radical formation during the GSSG-R catalyzed reduction of Cr(VI) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the .OH radical and Cr(V) formation, indicating that the O2- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the .OH radical formation, indicating the involvement of hydrogen peroxide (H2O2) in the reaction mechanism. Addition of H2O2 suppresses Cr(V) and enhances the .OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(VI) by GSSG-R, react with H2O2 to generate .OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.     

DNA damage and lethal effects of hydrogen peroxide and menadione in Chinese hamster cells: distinct mechanisms are involved

Menadione (Md)-resistant variants of V79 Chinese hamster cells were derived by culturing cells in progressively higher concentrations of this drug. Along with Md resistance these cells acquired cross-resistance to H2O2, which was retained when cells were cultured back in the absence of Md for 18 cell passages. This indicates that some stable alteration is responsible for the modification and may suggest that the toxicity of Md is mediated by oxygen activation. In agreement with the latter a 2.8-fold increase in catalase activity and a 1.5-fold increase in glutathione content were observed in Md-resistant cells as compared to parental cells, whereas superoxide dismutase and glutathione peroxidase remained unaltered. The use of inhibitors of Fenton reaction, inhibitors of enzymatic and nonenzymatic lipid peroxidation and OH radical scavengers, indicated that both DNA damage and cytotoxic effects of H2O2 and Md are mediated by OH radical, without intervention of lipid peroxides. However whereas the catalysis by iron was required for toxicity of extracellular H2O2, it was not involved in Md-induced toxicity. Possible explanations for this difference have been considered, one of them assuming that an excess of the semiquinone form of Md in the cell might replace iron II as a H2O2 reductant, producing OH radical by an organic Fenton reaction.