Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine.

The reaction of oxyhaemoglobin and acetylphenylhydrazine, which results in haemoglobin denaturation and precipitation, was found to be influenced by H202 and superoxide (O2-.) generated during the reaction. By analysing the different haemoglobin oxidation products, it was found that by influencing the rate at which oxyhaemoglobin was oxidized, H2O2 accelerated the overall haemoglobin breakdown, and O2-. inhibited it. By adding GSH (reduced glutathione) or ascorbate, it was possible to slow down the rates of both oxyhaemoglobin oxidation and O2-. production, and the overall rate of haemoglobin breakdown. These results are compatible with a mechanism involving production of the acetylphenylhydrazyl free radical, and with GSH, ascorbate and O2-. acting as radical scavengers and preventing its further reactions. The reaction produced choleglobin, as well as acetylphenyldiazine and methaemoglobin, which combined to form a haemichrome. The haemichrome was less stable and precipitated first. It was also less stable than the haemichrome formed by direct reaction of acetylphenyldiazine with methaemoglobin, and it is proposed that this is because the methaemoglobin produced from oxyhaemoglobin and acetylphenylhydrazine was modified by the free radicals and H2O2 produced in the reaction.     

Characterization of one- and two-electron oxidations of glutathione coupled with lactoperoxidase and thyroid peroxidase reactions

Glutathione (GSH) was oxidized to GSSG in the presence of H2O2, tyrosine, and peroxidase. During the GSH oxidation catalyzed by lactoperoxidase, O2 was consumed and the formation of glutathione free radical was confirmed by ESR of its 5,5'-dimethyl-1-pyrroline-N-oxide adduct. When lactoperoxidase was replaced by thyroid peroxidase in the reaction system, the consumption of O2 and the formation of the free radical became negligibly small. These results led us to conclude that, in the presence of H2O2 and tyrosine, lactoperoxidase and thyroid peroxidase caused the one-electron and two-electron oxidations of GSH, respectively. It was assumed that GSH is oxidized by primary oxidation products of tyrosine, which are phenoxyl free radicals in lactoperoxidase reactions and phenoxyl cations in thyroid peroxidase reactions. When tyrosine was replaced by diiodotyrosine or 2,6-dichlorophenol, the difference in the mechanism between lactoperoxidase and thyroid peroxidase disappeared and both caused the one-electron oxidation of GSH. Iodides also served as an effective mediator of GSH oxidation coupled with the peroxidase reactions. In this case the two peroxidases both caused the two-electron oxidation of GSH.     

Peroxidase activation of 1-naphthol to naphthoxy or naphthoxy-derived radicals and their reaction with glutathione

1-Naphthol was metabolised by horseradish peroxidase (HRP) in a H2O2-dependent reaction to methanol-soluble and covalently bound products. Spectrophotometric and electron spin resonance (ESR) studies established that HRP catalysed the one electron oxidation of 1-naphthol to naphthoxy or a naphthoxy-derived radical. Inclusion of glutathione (GSH) in the reaction caused a dose-dependent inhibition of covalent binding and an increase in the amount of unmetabolised 1-naphthol present at the end of the incubation. gamma-Radiolysis studies suggest that this is due to the reduction of naphthoxy radicals by GSH yielding 1-naphthol and GS.. In agreement with this, HRP-catalysed-oxidation of 1-naphthol in the presence of GSH, was found to stimulate oxidised glutathione (GSSG) formation.     

Reactions of glutathione and glutathione radicals with benzoquinones.

The reactions of glutathione (GSH) and glutathione radicals with a series of methyl-substituted 1,4-benzoquinones and 1,4-benzoquinone have been studied. It was found that by mixing excess benzoquinone with glutathione at pH above 6.5, the products formed were complex and unstable. All of the other experiments were carried out at pH 6.0, where the main product was stable for several hours. Stopped-flow analysis allowed the measurement of the rates of the rapid reactions between GSH and the quinones, and the products were monitored by High Performance Liquid Chromatography (HPLC). The rates of the reactions vary by five orders of magnitude and must be influenced by steric factors as well as changes in the redox states. It was observed that simple hydroquinones were not formed when the different benzoquinones were mixed with excess GSH and suggests that the initial reaction is addition/reduction rather than electron transfer. In the presence of excess quinone, the hydroquinone of the glutathione conjugate is oxidized back to its quinone. The rates of the reaction were measured. By using the technique of pulse radiolysis, it was possible to measure the reduction of the quinones by GSSG.- and the oxidation of hydroquinones by GS(.). It is proposed that the appearance of GSSG in reactions of quinones with glutathione could be due to oxidation of the hydroquinone by oxygen and the subsequent superoxide or H2O2 promoting the oxidation of GSH to GSSG.     

Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.     

Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

Myeloperoxidase, released by activated phagocytes, forms reactive oxidants by catalysing the reaction of halide and pseudo-halide ions with H(2)O(2). These oxidants have been linked to tissue damage in a range of inflammatory diseases. With physiological levels of halide and pseudo-halide ions, similar amounts of HOCl (hypochlorous acid) and HOSCN (hypothiocyanous acid) are produced by myeloperoxidase. Although the importance of HOSCN in initiating cellular damage via thiol oxidation is becoming increasingly recognized, there are limited data on the reactions of HOSCN with other targets. In the present study, the products of the reaction of HOSCN with proteins has been studied. With albumin, thiols are oxidized preferentially forming unstable sulfenyl thiocyanate derivatives, as evidenced by the reversible incorporation of (14)C from HOS(14)CN. On consumption of the HSA (human serum albumin) free thiol group, the formation of stable (14)C-containing products and oxidation of tryptophan residues are observed. Oxidation of tryptophan residues is observed on reaction of HOSCN with other proteins (including myoglobin, lysozyme and trypsin inhibitor), but not free tryptophan, or tryptophan-containing peptides. Peptide mass mapping studies with HOSCN-treated myoglobin, showed the addition of two oxygen atoms on either Trp(7) or Trp(14) with equimolar or less oxidant, and the addition of a further two oxygen atoms to the other tryptophan with higher oxidant concentrations (> or = 2-fold). Tryptophan oxidation was observed on treating myoglobin with HOSCN in the presence of glutathione and ascorbate. Thus tryptophan residues are likely to be favourable targets for the reaction in biological systems, and the oxidation products formed may be useful biomarkers of HOSCN-mediated protein oxidation.     

Cytochrome P-450-mediated oxidation of substrates by electron-transfer; role of oxygen radicals and of 1- and 2-electron oxidation of paracetamol

The mechanism by which the hepatic cytochrome P-450 (Cyt. P-450) containing mixed-function oxidase system oxidizes the analgesic drug paracetamol (PAR) to a hepatotoxic metabolite was studied. Since previous studies excluded the possibility of oxygenation of PAR, three other mechanisms, namely direct 1-electron oxidation by a Cyt. P-450-ferrous-dioxygen complex under concomitant formation of H2O2 to N-acetyl-p-semiquinone imine (NAPSQI), direct 2-electron oxidation by a Cyt. P-450-ferric-oxene complex to N-acetyl-p-benzoquinone imine (NAPQI) and indirect oxidation by active oxygen species released from Cyt. P-450, were considered. Indirect oxidation by active oxygen species was not involved, as active oxygen scavengers such as superoxide dismutase, catalase and DMSO did not affect the oxidation of PAR in hepatic microsomes. No reaction products characteristic for a direct 1-electron oxidation of PAR by Cyt. P-450 were observed: neither NAPSQI radical formation was detectable by ESR, nor PAR-dimer formation, nor stimulation of the microsomal H2O2 production was found to occur. In fact, PAR inhibited the spontaneous microsomal H2O2 formation. Studies on the reactions of NAPSQI with glutathione (GSH) revealed that NAPSQI hardly conjugated with GSH to a 3-glutathionyl-paracetamol conjugate (PAR-GSH) conjugate. The reactions of the elusive reactive metabolite formed during microsomal oxidation of PAR in the presence of GSH closely resembled those of synthetic NAPQI: both PAR-GSH and oxidized glutathione (GSSG) formation occurred. Furthermore, in agreement with a 2-electron oxidation hypothesis, iodosobenzene-dependent oxidation of PAR by cyt. P-450 in the presence of GSH resulted in the formation of the PAR-GSH conjugate. It is concluded that bioactivation of PAR by the Cyt. P-450 containing mixed-function oxidase system consists of a direct 2-electron oxidation to NAPQI.     

Glutathione oxidase activity of selenocystamine: a mechanistic study.

Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to glutathione disulfide, at neutral pH and ambient PO2. This glutathione oxidase activity required the heterolytic reduction of the diselenide bond, which produced two equivalents of the selenolate derivative selenocysteamine (RSe-), via the transient formation of a selenenylsulfide intermediate (RSe-SG). Formation of RSe- was the only reaction observed in anaerobic conditions. At ambient PO2, the kinetics and stoichiometry of GSSG production as well as that of GSH and oxygen consumptions demonstrated that RSe- performed a three-step reduction of oxygen to water. The first step was a one-electron transfer from RSe- to dioxygen, yielding superoxide and a putative selenyl radical RSe., which decayed very rapidly to RSe-SeR. In the second step, RSe- reduced superoxide to hydrogen peroxide through a much faster one-electron transfer, also associated with the decay of RSe. to RSe-SeR. The third step was a two-electron transfer from RSe- to hydrogen peroxide, again much faster than oxygen reduction, which resulted in the production of RSe-SG, presumably via a selenenic acid intermediate (RSeOH) which was trapped by excess GSH. This third step was studied on exogenous hydroperoxide in anaerobic conditions, and it could be eliminated from the glutathione oxidase cycle in the presence of excess catalase. The role of RSe- as a one- and two-electron reductant was confirmed by competitive carboxymethylation with iodoacetate. RSe- was able to rapidly reduce ferric cytochrome c to its ferrous derivative. The overall rate of catalytic glutathione oxidation was GSH concentration dependent and oxygen concentration independent. Excess glutathione reductase and NADPH increased the catalytic oxidation of GSH, probably by switching the rate-limiting step from selenylsulfide to diselenide cleavage. When GSH was substituted for dithiothreitol, it was shown to reduce RSe-SeR to RSe- in a fast and quantitative reaction, and selenocystamine behaved as a dithiothreitol oxidase, whose catalytic cycle was dependent on oxygen concentration. The oxidase cycle of glutathione was inhibited by mercaptosuccinate, while that of dithiothreitol was not affected. When mercaptosuccinate was substituted for GSH, a stable selenenylsulfide was formed. These observations suggest that electrostatic interactions affect the reductive cleavage of diselenide and selenenylsulfide linkages. This study illustrates the ease of one-electron transfers from RSe- to a variety of reducible substrates. Such free radical mechanisms may explain much of the cytotoxicity of alkylselenols, and they demonstrate that selenocystamine is a poor catalytic model of the enzyme glutathione peroxidase.     

Glutathione status in constituted physiological fluids containing albumin

1. Glutathione (GSH) and cysteine, added to the constituted incubation medium, rapidly disappeared from the medium in the presence of bovine serum albumin (BSA). The major portions of added GSH and cysteine were oxidized. Only a fraction was recovered as cysteine-GSH mixed disulfide in case of GSH. About 15-30% cysteine or GSH were not recovered in the media. 2. The rate of GSH oxidation was linear with time, however, GSH disappearance was not linear with GSH concentrations. 3. Oxidation of GSH to GSSG in the albumin supplemented media was greater under O2 atmosphere, but was significantly decreased under N2 atmosphere. 4. Catalase, a peroxy radical scavenger, but not dimethyl pyroline N-oxide (DMPO), N-tertbutyl-2(-2 sulfophenyl)-nitrone (NTBSPN), mannitol or superoxide dismutase (SOD), decreased BSA mediated GSH oxidation. 5. GSH oxidation was abolished when mono- or divalent metal ions were absent in the BSA supplemented media. 6. Alkaline pH favored and acidic pH inhibited GSH oxidation. GSH oxidation was maximum above pH 7.4. GSH oxidation was minimal in the media containing boiled BSA. 7. A reaction mechanism involving the mixed GSH-BSA disulfide formation, followed by the reduction of these disulfides by GSH and subsequent release of GSSG is proposed.     

Ovothiols as free-radical scavengers and the mechanism of ovothiol-promoted NAD(P)H-O2 oxidoreductase activity

Racemic ovothiol A [(+/-)-1a] and the ovothiol model compound 1,5-dimethyl-4-mercaptoimidazole (DMI, 2) were found to scavange the free radicals Fremy's salt (4) and Banfield' radical (5) much more rapidly than did the thiol antioxidant glutathione. Ovothiol A also scavenges the tyrosyl radical, with efficiency comparable to that of ascorbic acid and the tocopherol analogue trolox (3). The ovothiol model compound DMI was found to scavenge superoxide with a rate constant comparable to that of the reaction between superoxide and glutathione. These results suggest both a free-radical scavenging role for the ovothiols and a mechanism by which the ovothiols confer NAD(P)H-O2 oxidoreductase activity upon the enzyme ovoperoxidase. Investigation of this mechanism implicates the ovothiol thiyl radical and the NAD radical as key intermediates. The ovothiyl radical appears to be unreactive toward oxygen but highly reactive toward NADH. An estimate of the one-electron oxidation potential of the ovothiol anion is presented. The physical basis for the stability of the ovothiol free radical is discussed.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation.

Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H2O2. When low levels of thiocyanate (SCN-) were used in the GO system, H2O2 was detected and lactoperoxidase (LP) was inactivated when SCN- was depleted. With 10-fold higher SCN-, LP remained active and H2O2 was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H2O2 in a system employing low SCN-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H2O2 was not detected during the course of the experiment. At high SCN- levels, relatively high concentrations of oxidation product were produced immediately, with H2O2 undetectable. The results suggest that the final product of the LP reaction depends on the method of H2O2 generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37 degrees C.     

In vivo protection of synaptosomes from oxidative stress mediated by Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride by the glutathione mimetic tricyclodecan-9-yl-xanthogenate

D609 (tricyclodecan-9-yl-xanthogenate) is a phosphatidylcholine-specific phospholipase C inhibitor that also has been reported to protect rodents against oxidative damage caused by lethal doses of ionizing radiation. We previously showed that D609 mimics glutathione. D609 has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies from our laboratory have also shown that D609 reduces the Alzheimer amyloid beta-peptide (1-42)-induced oxidative stress and cytotoxicity in neuronal cell culture. The present study was undertaken to test the hypothesis that D609 would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes isolated from gerbils, previously injected intraperitoneally (ip) with D609, were treated with the oxidants Fe2+/H2O2 or 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH), which produce free radicals. Synaptosomes isolated from the gerbils ip injected with D609 and treated with Fe2+/H2O2 or AAPH showed significant reduction in reactive oxygen species, levels of protein carbonyl, protein-bound hydroxynonenal (a lipid peroxidation product), and 3-nitrotyrosine (another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to oxidative stress in synaptosomes isolated from gerbils that were injected with saline, but treated with Fe2+/H2O2 or AAPH. These results are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders.     

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.     

Reversal by nickel(II) of inhibitory effects of some scavengers of active oxygen species upon hydroxylation of 2'-deoxyguanosine in vitro.

Effects of ethanol (EtOH), mannitol (Man), L-histidine (His) and glutathione (GSH) on the oxidation of 2'-deoxyguanosine (dG) to its 8-hydroxy derivative (8-OH-dG) with H2O2 plus L-ascorbic acid (Ascb) in the absence and presence of Ni(II) were investigated in order to unveil the nature of active oxygen species involved in that oxidation. In the absence of Ni(II), production of 8-OH-dG was inhibited by His much greater than GSH greater than or equal to GSSG (oxidized glutathione) much greater than EtOH, but not by Man. The latter tended to enhance the production of 8-OH-dG. In the presence of Ni(II), the inhibition by His, GSH and GSSG, but not EtOH, was prevented. The results indicate involvement of a 'crypto-hydroxyl' radical as the dG oxidizing species in both the absence and presence of Ni(II). Also, the results provide evidence that Ni(II) complexes with His, GSH and GSSG may lack antioxidant capacity. Moreover, the Ni(II) complex with His was found capable of enhancing 8-OH-dG production by the Ascb+H2O2 system to a greater extent than Ni(II) alone. Likewise, although to a lesser extent, the formation of 8-OH-dG was enhanced by the combination of Ni(II) and Man which do not form complexes at pH 7.4. Since His is a major Ni(II) carrier in animal tissues, the dG oxidation enhancing capacity of the Ni(II) complex with His may contribute to the toxic and carcinogenic effects of Ni(II).     

Electron paramagnetic resonance spin trapping investigation into the kinetics of glutathione oxidation by the superoxide radical: re-evaluation of the rate constant

The ability of glutathione to scavenge the superoxide radical is a matter of serious contention in the literature: reported values for the second-order rate constant range from 10(2) to greater than 10(5) M(-1) s(-1). The physiological implications of this discrepancy will determine, for example, whether or not glutathione can compete with Mn-superoxide dismutase for reaction with the radical in the mitochondrial matrix, leading to formation of the potentially harmful glutathionyl radical. Several authors have investigated the kinetics of glutathione oxidation by superoxide using spectrophotometric assays, based on competition between either ferricytochrome c or epinephrine for reaction with the radical. However, these approaches have received criticism because the contributions of various secondary reactions to the overall kinetics have been largely overlooked (e.g., the reduction of ferricytochrome c by glutathione). In the present investigation, we have used electron paramagnetic resonance spectroscopy to monitor competition between GSH and the spin trap 5,5-dimethyl-1-pyrroline N-oxide for reaction with superoxide. This method has been used previously and a rate constant of 1.8 x 10(5) M(-1) s(-1) obtained (Dikalov, S.; Khramtsov, V.; Zimmer, G. Arch. Biochem. Biophys. 326:207-218; 1996). However, we demonstrate that this value is a gross overestimation because the spectrum of the hydroxyl radical adduct of the spin trap was incorrectly assigned to the glutathionyl radical adduct. The relatively high yield of the DMPO hydroxyl radical adduct is shown to be due to the two-electron reduction of the corresponding superoxide radical adduct by glutathione. Taking these factors into consideration, we estimate the second order rate constant for the oxidation of glutathione by superoxide to be approximately 200 M(-1) s(-1).     

Dopamine-derived dopaminochrome promotes H(2)O(2) release at mitochondrial complex I: stimulation by rotenone, control by Ca(2+), and relevance to Parkinson disease

Inhibitors of Complex I of the mitochondrial respiratory chain, such as rotenone, promote Parkinson disease-like symptoms and signs of oxidative stress. Dopamine (DA) oxidation products may be implicated in such a process. We show here that the o-quinone dopaminochrome (DACHR), a relatively stable DA oxidation product, promotes concentration (0.1-0.2 mum)- and respiration-dependent generation of H(2)O(2) at Complex I in brain mitochondria, with further stimulation by low concentrations of rotenone (5-30 nm). The rotenone effect required that contaminating Ca(2+) (8-10 mum) was not removed. DACHR apparently extracts an electron from the constitutively autoxidizable site in Complex I, producing a semiquinone, which then transfers an electron to O(2), generating O(2)(.) and then H(2)O(2). Mitochondrial removal of H(2)O(2) monoamine, formed by either oxidase activity or DACHR, was performed largely by glutathione peroxidase and glutathione reductase, which were negatively regulated by low intramitochondrial Ca(2+) levels. Thus, the H(2)O(2) formed accumulated in the medium if contaminating Ca(2+) was present; in the absence of Ca(2+), H(2)O(2) was completely removed if it originated from monoamine oxidase, but was less completely removed if it originated from DACHR. We propose that the primary action of rotenone is to promote extracellular O(2)(.) release via activation of NADPH oxidase in the microglia. In turn, O(2)(.) oxidizes DA to DACHR extracellularly. (The reaction is favored by the lack of GSH, which would otherwise preferably produce GSH adducts of dopaminoquinone.) Once formed, DACHR (which is resistant to GSH) enters neurons to activate the rotenone-stimulated redox cycle described.     

Oxygen therapy at low flow causes oxidative stress in chronic obstructive pulmonary disease: Prevention by N-acetyl cysteine

Exposure to high oxygen concentration produces toxicity by free radical release. We aimed to study: whether stable chronic obstructive pulmonary disease (COPD) patients present an unbalance in the blood redox status; the effect of oxygen administration on blood redox balance; the efficacy of N-acetyl-cysteine (NAC) treatment against the oxidative stress-induced by oxygen administration and whether it is dose-related. To this, 45 stable state III COPD patients were recruited and reduced glutathione (GSH) and oxidised glutathione (GSSG) in erythrocytes and thiol proteins (P-SH) and carbonyl proteins (PC) in both erythrocytes and plasma were evaluated. All COPD patients underwent 2 l/m oxygen for 18 h and NAC at 1200 or 1800 mg/day or placebo for 48 h starting with oxygen administration. Blood samples were collected at basal conditions, after 8 and 18 h of oxygen administration and 24 h after oxygen withdrawal. Results: COPD patients present an unstable redox equilibrium mainly due to plasma sulphydryl protein depletion. Oxygen administration oxidize erythrocyte GSH, decrease P-SH and increase PC levels in both plasma and erythrocytes. NAC administration counteract the oxidative stress and at the highest dose completely prevent protein oxidation. In conclusion, stable state III COPD patients present an unstable redox balance; long term low flow oxygen administration induces systemic oxidative stress, which is prevented by NAC treatment.     

Copper redox-dependent activation of 2-tert-butyl(1,4)hydroquinone: formation of reactive oxygen species and induction of oxidative DNA damage in isolated DNA and cultured rat hepatocytes

The biotransformation of butylated hydroxyanisole (BHA), a possible carcinogenic food antioxidant, includes o-demethylation to 2-tert-butyl(1,4)hydroquinone (TBHQ) which can subsequently be oxidized to 2-tert-butyl(1,4)paraquinone (TBQ). In this study, we have examined the capacity of Cu, a nuclei- and DNA-associated transition metal, to mediate the oxidation of TBHQ. In phosphate buffered saline (PBS), autooxidation of TBHQ to TBQ was not detectable, while Cu(II) at micromolar concentrations strongly catalyzed the oxidation of TBHQ to TBQ. Oxidation of TBHQ by Cu(II) was accompanied by the utilization of O(2) and the concomitant generation of H(2)O(2). Using electron spin resonance spectroscopy, it was observed that Cu(II) mediated the one electron oxidation of TBHQ to a semiquinone anion radical. The formation of a semiquinone anion radical, the utilization of O(2) and the generation of H(2)O(2) and TBQ could be completely blocked by bathocuproinedisulfonic acid (BCS) and reduced glutathione (GSH), two Cu(I)-chelators. 4-Pyridyl-1-oxide-N-tert-butylnitrone (POBN)-spin trapping experiments showed that the reaction of TBHQ with Cu(II) resulted in the generation of POBN-CH(3) and POBN-CH(OH)CH(3) adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting the formation of hydroxyl radical or a similar reactive intermediate. The formation of POBN-CH(3) adduct from the TBHQ/Cu(II)+DMSO could be completely inhibited by catalase, GSH or BCS, indicating that the hydroxyl radical or its equivalent is generated from the interaction of H(2)O(2) with Cu(I). Incubation of supercoiled phiX-174 plasmid DNA with the TBHQ/Cu(II) resulted in extensive DNA strand breaks, which could be prevented by catalase or BCS. Incubation of rat hepatocytes with TBHQ in PBS led to increased formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in nuclear DNA. The TBHQ-induced formation of 8-OHdG was markedly reduced in the presence of cell permeable Cu(I)-specific chelator, bathocuproine or neocuproine, suggesting that a Cu(II)/Cu(I) redox mechanism may also be involved in the induction of oxidative DNA damage by TBHQ in hepatocytes. Taken together, the above results conclusively demonstrate that the activation of TBHQ by Cu(II) results in the formation of TBQ, semiquinone anion radical and reactive oxygen species (ROS), and that the ROS formed may participate in oxidative DNA damage in both isolated DNA and intact cells. These reactions may contribute to the carcinogenicity as well as other biochemical activities observed with BHA in animals. To our knowledge this study provides the first evidence that endogenous cellular Cu may be capable of bioactivating TBHQ, leading to oxidative DNA damage in cultured cells.     

Salivary thiocyanate/nitrite inhibits hydroxylation of 2-hydroxybenzoic acid induced by hydrogen peroxide/Fe(II) systems under acidic conditions: possibility of thiocyanate/nitrite-dependent scavenging of hydroxyl radical in the stomach

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.     

Oxidative degradation of thymine with O2 promoted by L-ascorbic acid and Cu(II) ion

Oxidation of thymine with O2 was promoted by copper(I) ion generated from reaction of L-ascorbic acid (AA) with copper (II) ion. The main oxidation products were thymine glycol (TG) and N-formyl-N'-pyruvylurea (FPU). At higher concentration of O2, formation of FPU was favored, while TG was dominant at higher Cu(II) ion and lower O2 concentrations. Reaction mechanism involving hydroxy thyminyl radical was proposed.