The DNA cleavage induced by a chromium(V) complex and by chromate and glutathione is mediated by activated oxygen species

The number of strand breaks induced by the combination of chromate and glutathione (GSH) in PM2 DNA was effectively reduced upon addition of the hydroxyl radical scavengers dimethyl sulphoxide (DMSO), formate and benzoate. Administration of catalase also led to a depression of DNA degradation whereas superoxide dismutase (SOD) had very little influence. Essentially the same results were obtained in experiments employing a chromium(V) complex Na4(GSH)4Cr.8H20, which is an intermediate chromium species isolated from the reduction of chromate by glutathione. DNA cleavage was dependent on the presence of iron (FeCl3). When compared with the number of breaks produced by FeCl3 and GSH alone, chromate stimulated the generation of single-strand breaks. These findings suggest that hydroxyl radicals are one ultimate DNA cleaving agent in both reactions. A reaction scheme for the production of hydroxyl radicals is proposed.     

Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.     

The transport of oxidized glutathione from the erythrocytes of various species in the prescence of chromate

1. Erythrocytes from normal and glucose 6-phosphate dehydrogenase-deficient humans were subjected to hydrogen peroxide diffusion to oxidize the GSH. Studies were carried out in the presence and absence of chromate to inhibit glutathione reductase and with or without the addition of glucose. 2. The GSH content of erythrocytes from other species was oxidized by subjecting them to hydrogen peroxide diffusion in the presence of chromate and glucose. 3. Chromate (1.3mm) inhibited glutathione reductase by about 80%, whereas glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were not inhibited. 4. The GSSG formed was transported from the erythrocytes to the medium. 5. The transport rate of GSSG from glucose 6-phosphate dehydrogenase-deficient erythrocytes subjected to hydrogen peroxide diffusion in the presence of chromate was comparable with that from normal and glucose 6-phosphate dehydrogenase-deficient erythrocytes. 6. The rate of transport of GSSG from erythrocytes of various species studied could be ranked: pigeon>rabbit>rat>donkey>man>dog>horse>sheep>chicken>fish.     

Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI)

Electron spin resonance measurements provide evidence for the formation of long-lived Cr(V) intermediates in the reduction of Cr(VI) by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr(VI). Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals. Thus Cr(V) intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Thus the mechanism of Cr(VI) toxicity might involve the interaction between macromolecules and the hydroxyl radicals.     

Sulfinyl radical formation from the reaction of cysteine and glutathione thiyl radicals with molecular oxygen

Using Electron Spin Resonance spectroscopy at low temperatures, we find that thiyl radicals resulting from irradiation of frozen aqueous solutions of a variety of thiols, including cysteine, glutathione, and penicillamine react with oxygen to form sulfinyl (RSO.) radicals. The identity of the cysteine sulfinyl radical has been confirmed by the use of molecular oxygen isotopically labeled with 17O. Previous workers have suggested the reaction of thiyl radicals and molecular oxygen resulted in the formation of the potentially damaging thiol peroxyl radical, RSOO.; our work shows no evidence for this species. The sulfinyl radicals are suggested to result from a direct reaction between thiyl radicals and molecular oxygen. This reaction results in the cleavage of the dioxygen bond.     

Role of physiological antioxidants in chromium(VI)-induced cellular injury.

Chromium(VI) compounds are well known to be potent toxic and carcinogenic agents. Because chromium(VI) is easily taken up by cells and is subsequently reduced to the trivalent form, the formation of chromium(III) or other intermediate oxidation states such as chromium(V) and (IV) is believed to play a role in the adverse biological effects of chromium(VI) compounds. Recent in vitro studies have shown that this reduction process generates free radical species such as active oxygen radicals. Furthermore, physiological antioxidants are reported to modify the genotoxic and toxic effects of chromate. This article reviewed the recent in vitro and in vivo studies of the effects of antioxidants including active oxygen scavengers; glutathione; vitamins B2, E, and C, on chromate-induced injury such as DNA lesions; lipid peroxidation; enzyme inhibition; cytotoxicity; mutation; and so on. In addition, the mechanism of action of these antioxidants was discussed with respect to the formation of active oxygen radicals and paramagnetic chromium such as chromium(V) and (III). Such studies may help elucidate the mechanism of chromium(VI) toxicity as well as the mechanism of protection.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

Effect of selenium on the system of superoxide anion radical formation and detoxication in hepatocarcinogenesis

It is established that the introduction of selenium in combination with diethylnitrosamine into rat organisms has a preventive influence on the tumour formation. The intensity of superoxide radicals formation by the liver cell microsomes in this case decreases, while the activity of superoxide dismutase, glutathione peroxidase I, glutathione reductase and concentration of selenium in microsomes increases. The anticarcinogenic action of selenium is considered as a result of an increase in the activity of superoxide dismutase, glutathione peroxidase I and glutathione reductase. This increase induces detoxication of superoxide radicals forming in considerable amounts in rat liver cells under the effect of carcinogen.     

Thiyl radicals react with nitric oxide to form S-nitrosothiols with rate constants near the diffusion-controlled limit

A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.     

Effects of reactive species of oxygen and nitrogen on iron ion release from ferritin and synthesis of dinitrosyl iron complexes

Using EPR spectroscopy it was established that Fe ions released from ferritin under the action of glutathione and superoxide took part in the formation of dinitrosyl complexes of iron with glutathione (DNIC). The reaction between O2-. and NO resulted in the formation of peroxynitrite, which oxidized glutathione to the thiyl radical. In these conditions, DNIC did not inhibit the formation of thiyl radicals but effectively slowed down the oxidative destruction of beta-carotene by peroxynitrite and free radicals of lipids. In the presence of glutathione, the inversion of the antioxidant properties of DNIC into prooxidant ones took place. S-nitrosoglutathione prevented this inversion and suppressed the free-radical oxidation of beta-carotene induced by ferritin. It was proposed that the equilibrium between S-nitrosoglutathione, DNIC, "free Fe" ions and ferritin may determine the balance between prooxidant and antioxidant processes in living organisms.     

Spin trapping of glutathiyl and protein radicals produced from nitric oxide-derived oxidants

Despite the importance of protein radicals in cell homeostasis and cell injury, their formation, localization, and propagation reactions remain obscure, mainly because of the difficulties in detecting and characterizing radicals, in general, and protein radicals, in particular. New approaches based on spin trapping coupled with other methodologies are under development/testing but so far they have been applied mainly to the study of protein-tyrosyl and protein-tryptophanyl radicals. Here, our aim is to emphasize the importance of developing new methodologies for the detection of glutathyil and protein-cysteinyl radicals under physiological conditions. To this end, we summarize current EPR evidence supporting the view that glutathione and protein-cysteines are among the preferential targets of nitric oxide-derived oxidants and that they are oxidized to the glutathiyl and protein-cysteinyl radicals, respectively. The possible intermediacy of these species in the biological formation of mediators of protein-cysteine redox signaling, such as S-nitrosothiols and sulfenic acids, is also discussed.     

A bacterial flavin reductase system reduces chromate to a soluble chromium(III)-NAD(+) complex

Biological reduction of carcinogenic chromate has been extensively studied in eukaryotic cells partly because the reduction produces stable chromium(III)-DNA adducts, which are mutagenic. Microbial reduction of chromate has been studied for bioremediation purposes, but little is known about the reduction mechanism. In eukaryotic cells chromate is mainly reduced non-enzymatically by ascorbate, which is usually absent in bacterial cells. We have characterized the reduction of chromate by a flavin reductase (Fre) from Escherichia coli with flavins. The Fre-flavin system rapidly reduced chromate, whereas chemical reduction by NADH and glutathione was very slow. Thus, enzymatic chromate reduction is likely the dominant mechanism in bacterial cells. Furthermore, the end-product was a soluble and stable Cr(III)-NAD(+) complex, instead of Cr(III) precipitate. Since intracellularly generated Cr(III) forms adducts with DNA, protein, glutathione, and ascorbate in eukaryotic cells, we suggest that the produced Cr(III) is primarily complexed to NAD(+), DNA, and other cellular components inside bacteria.     

Evidence for free radical formation during horseradish peroxidase-catalyzed N-demethylation of crystal violet.

Crystal violet (gentian violet) can undergo an oxidative metabolism, catalyzed by horseradish peroxidase, resulting in formaldehyde formation. The N-demethylation reaction was strongly inhibited by reduced glutathione. Evidence for the formation of a crystal violet radical during the horseradish peroxidase catalyzed reaction was the detection of thiyl and ascorbate radicals from glutathione and ascorbate, respectively. The concentration of radicals from both compounds was significantly increased in the presence of crystal violet. Oxygen uptake was stimulated when glutathione was present in the system and this oxygen uptake was dependent on the dye and enzyme concentration. Oxygen uptake did not occur when ascorbate, instead of glutathione, was present in the system. However, when glutathione was present, ascorbate totally inhibited the glutathione-stimulated oxygen uptake in the crystal violet/horseradish peroxidase/hydrogen peroxide system. Although a weak ESR spectrum from a crystal violet-derived free radical was detected when the dye reacted with H2O2 and horseradish peroxidase, using the fast flow technique, this spectrum could not be interpreted.     

Significance of Thiol-Disulfide Exchange in Resting Stages of Plant Development

Abstract: Desiccation tolerance is a fundamental principle for resting stages of plant development which include the dormancy of seeds and the quiescent stages of resurrection plants. To prevent the deleterious effects of cellular desiccation, a complex interplay of several adaption mechanisms is required. The ability to cope with free radicals, the formation of which is well documented in desiccated tissues, is one of these basic requirements. Detoxification of free radicals by several antioxidants and scavenging enzymes include reactions of reduced glutathione (GSH) resulting in the formation of glutathione disulfide (GSSG). In free radical processing pathways GSSG is considered to be immediately reduced back to GSH by the action of glutathione reductase (EC 1.6.4.2.). However, in desiccated tissues GSSG accumulates. Protein-glutathione mixed disulfides (PSSG) are also reported to increase in plants under drought leading to the hypothesis that glutathione protects protein thiol groups from auto-oxidation. The irreversible formation of intramolecular disulfides resulting in denaturation of proteins would be one of the primary sites of desiccation injury. We suggest that PSSG is formed by the reaction of GSSG with high molecular weight thiols and introduce a thiol-disulfide cycle that involves reduction/oxidation processes of glutathione and protein thiol groups during the dehydration/rehydration processes in desiccation tolerant tissues.     

Interaction of Oxoferrylmyoglobin and Dinitrosyl-Iron Complexes

It is shown that dinitrosyl-iron complexes (DNIC) with glutathione can reduce oxoferrylmyoglobin forming on interaction of tert-butyl hydroperoxide and metmyoglobin. A rapid decrease in the DNIC concentration was observed under the conditions of production of tert-butyl free radicals; however, destruction of DNIC in the presence of oxoferrylmyoglobin alone was negligible. It is demonstrated that DNIC reduces oxoferrylmyoglobin more than an order more efficiently than S-nitrosoglutathione and glutathione. DNIC also inhibits formation of the thiyl radicals of glutathione in a medium containing metmyoglobin and tert-butyl hydroperoxide. A mechanism of the antioxidant action of DNIC based on regeneration of the nitrosyl complexes from the products of their interaction with oxoferrylheme is proposed.     

Semiquinone anion radicals formed by the reaction of quinones with glutathione or amino acids

Using ESR spectroscopy, we show that benzoquinone, 1,4-naphthoquinone and 5-hydroxy-1,4-naphthoquinone react readily with thiol containing compounds, such as glutathione, to form their respective semiquinone anion radicals. These quinones react similarly, but less readily, with the amino group of amino acids. The therapeutic or toxicological significance of the formation of semiquinone anion radicals from the reaction of quinones with nucleophiles, such as thiols and amines, remains to be assessed.     

Peroxidase-mediated formation of glutathione conjugates from polycyclic aromatic dihydrodiols and insecticides

Using two peroxidative systems (prostaglandin H synthase/arachidonic acid and horseradish peroxidase/H2O2) we observed GSH conjugate formation with a number of compounds including polycyclic aromatic hydrocarbon-diols (PAH-diols), insecticides, and steroids. Several of the conjugates were characterized by chromatography, uv-vis spectrophotometry, and FAB mass spectroscopy. Conjugate formation is dependent upon a functioning peroxidase, GSH, and is markedly enhanced (3- to 10-fold) by the inclusion of a number of reducing cosubstrates including phenol, uric acid, phenylbutazone, and acetaminophen. The mechanism of conjugate formation appears to involve addition of thiyl radical to alkene bonds conjugated to an electron releasing group probably by resonance stabilization of the carbon-centered radical intermediate. Thiyl radicals are formed either directly by GSH reduction of the peroxidase or indirectly by GSH reduction of radicals formed from reducing cosubstrates. The nitrone spin trap, 5,5-dimethyl-1-pyrroline N-oxide, which traps thiyl radicals, totally inhibits production of GSH conjugates in both peroxidative systems. Conjugation of PAH-diols, some of which are penultimate carcinogens, would prevent their metabolism to the diol-epoxides, an ultimate carcinogenic species of PAH. Conjugation by peroxidases appears to be a general pathway for glutathione conjugate formation that may lead to potential detoxification of chemicals.     

The roles of thiol-derived radicals in the use of 2',7'-dichlorodihydrofluorescein as a probe for oxidative stress

2',7'-Dichlorodihydrofluorescein (DCFH2) is one of the most widely used probes for detecting intracellular oxidative stress, but requires a catalyst to be oxidized by hydrogen peroxide or superoxide and reacts nonspecifically with oxidizing radicals. Thiyl radicals are produced when many radicals are "repaired" by thiols, but are oxidizing agents and thus potentially capable of oxidizing DCFH2. The aim of this study was to investigate the reactivity of thiol-derived radicals toward DCFH2 and its oxidized, fluorescent form 2',7'-dichlorofluorescein (DCF). Thiyl radicals derived from oxidation of glutathione (GSH) or cysteine (CysSH) oxidized DCFH2 with rate constants at pH 7.4 of approximately 4 or approximately 2x10(7) M(-1) s(-1), respectively. Both the rates of oxidation and the yields of DCF were pH-dependent. Glutathione-derived radicals interacted with DCF, resulting in the formation of DCFH* absorbing at 390 nm and loss of fluorescence; in contrast, cysteine-derived radicals did not cause any depletion of DCF fluorescence. We postulate that the observed apparent difference in reactivity between GS* and CysS* toward DCF is related to the formation of carbon-centered, reducing radicals from base-catalyzed isomerization of GS*. DCF formation from interaction of DCFH2 with GS* was inhibited by oxygen in a concentration-dependent manner over the physiological range. These data indicate that in applying DCFH2 to measure oxidizing radicals in biological systems, we have to consider not only the initial competition between thiols and DCFH2 for the oxidizing radicals, but also subsequent reactions of thiol-derived radicals, together with variables--including pH and oxygen concentration--which control thiyl radical chemistry.     

An ESR investigation of the reactions of glutathione, cysteine and penicillamine thiyl radicals: competitive formation of RSO., R., RSSR-., and RSS(.)

The reactions of the cysteine, glutathione and penicillamine thiyl radicals with oxygen and their parent thiols in frozen aqueous solutions have been elucidated through electron spin resonance spectroscopy. The major sulfur radicals observed are: (1) thiyl radicals, RS.; (2) disulfide radical anions. RSSR-.; (3) perthiyl radicals, RSS. and upon introduction of oxygen; (4) sulfinyl radicals, RSO., where R represents the remainder of the cysteine, glutathione or penicillamine moiety. The radical product observed depends on the pH, concentration of thiol, and presence or absence of molecular oxygen. We find that the sulfinyl radical is a ubiquitous intermediate in the free radical chemistry of these important biological compounds, and also show that peroxyl radical attack on thiols may lead to sulfinyl radicals. We elaborate the observed reaction sequences that lead to sulfinyl radicals, and, using 17O isotopic substitution studies, demonstrate that the oxygen atom in sulfinyl radicals originates from dissolved molecular oxygen. In addition, the glutathione thiyl radical is found to abstract hydrogen from the alpha-carbon position on the cysteine residue of glutathione to form a carbon-centered radical.     

Ultrasound-Induced Formation of S-Nitrosoglutathione and S-Nitrosocysteine in Aerobic Aqueous Solutions of Glutathione and Cysteine

S-Nitrosocompounds are formed when aqueous solutions of cysteine or glutathione are exposed to ultrasound (880 kHz) in air. The yield of the S-nitrosocompounds was as high as 10% for glutathione and 4% for cysteine of the initial thiol concentrations (from 0.1 to 10 mM) in the aqueous solutions. In addition to the formation of S-nitrosocompounds, thiol oxidation to disulfide forms was observed. After the oxidation of over 70% of the sulfhydryl groups, formation of peroxide compounds as well as cysteic acid derivatives was recorded. The formation of the peroxide compounds and peroxide radicals in the ultrasound field reduced the yield of S-nitrosocompounds. S-Nitrosocompounds were not formed when exposing low-molecular-weight thiols to ultrasound in atmospheres of N₂ or CO. In neutral solutions, ultrasound-exposed cysteine or glutathione released NO due to spontaneous degradation of the S-nitrosocompounds. N₂O₃, produced due to the spontaneous degradation of the S-nitrosocompounds in air, nitrosylated sulfhydryl groups of glutathione manifested in the appearance of new absorption bands at 330 and 540 nm. The nitrogen compounds formed in an ultrasound field modified the sulfhydryl groups of apohemoglobin and serum albumin. The main target for ultrasound-generated oxygen free radicals were cystine residues oxidized to cysteic acid residues.