Mannitol Protects against Oxidation by Hydroxyl Radicals

Hydroxyl radicals may be responsible for oxidative damage during drought or chilling stress. We have shown that the presence of mannitol in chloroplasts can protect plants against oxidative damage by hydroxyl radicals (B. Shen, R.G. Jensen, H.J. Bohnert [1997] Plant Physiol 113: 1177-1183). Here we identify one of the target enzymes that may be protected by mannitol. Isolated thylakoids in the presence of physiological concentrations of Fe2+ generated hydroxyl radicals that were detected by the conversion of phenylalanine into tyrosine. The activity of phosphoribulokinase (PRK), a thiol-regulated enzyme of the Calvin cycle, was reduced by 65% in illuminated thylakoids producing hydroxyl radicals. Mannitol (125 mM) and sodium formate (15 mM), both hydroxyl radical scavengers, and catalase (3000 units mL-1) prevented loss of PRK activity. In contrast, superoxide dismutase (300 units mL-1) and glycine betaine (125 mM) were not effective in protecting PRK against oxidative inactivation. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity was not affected by hydroxyl radicals. We suggest that the stress-protective role of mannitol may be to shield susceptible thiol-regulated enzymes like PRK plus thioredoxin, ferredoxin, and glutathione from inactivation by hydroxyl radicals in plants.     

Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon

Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Hyaluronan degradation by copper(II) chloride and ascorbate: rotational viscometric, EPR spin-trapping, and MALDI-TOF mass spectrometric investigations

The degradation of high-molar-mass hyaluronan (HA) by copper(II) chloride and ascorbate was studied by means of rotational viscometry. It was found that even small amounts of CuCl(2) present in the oxidative system led to the pronounced degradation of HA, reflected in a rapid decrease of the dynamic viscosity of the biopolymer solution. Such degradation was induced by free radicals generated in elevated amounts in the presence of copper ions. Electron paramagnetic resonance investigations performed on a model oxidative system containing Cu(II) and ascorbic acid proved the formation of relatively stable ascorbate anion radicals resulting from the reaction of ascorbic acid with hydroxyl radicals. In this way, by scavenging the hydroxyl radicals, ascorbic acid protected HA from their degradative action. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied to analyze the degraded HA. The results showed that only regular fragmentation of hyaluronan occurred using the mentioned oxidative system that led to the formation of HA oligomers with unaffected primary chemical structure.     

Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon

Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C₃-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 × 10¹¹ M⁻¹ s⁻¹, demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions.     

Chain scission of hyaluronan by peroxynitrite

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Presence of Hydrogen Peroxide,a Source of Hydroxyl Radicals,in Acid Electrolyzed Water

Background

Acid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity.

Methodology/Principal Findings

The aim of the present study is to detect hydroxyl radicals in AEW. The hydroxyl radicals in AEW prepared under different conditions were determined using an electron spin resonance (ESR) technique. A signal from 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH, an adduct of DMPO and the hydroxyl radical, was detected in AEW prepared by double or triple electrolyses of 1% NaCl but not of 0.1% NaCl solution. Then the presence of hydrogen peroxide as a proposed source of hydroxyl radicals was examined using a combination of ESR and a Fenton reaction. The DMPO-OH signal was clearly detected, even in AEW prepared by single electrolysis of 0.1% NaCl solution, when ferrous sulfate was added to induce a Fenton reaction, indicating the presence of hydrogen peroxide in the AEW. Since sodium formate, a hydroxyl radical scavenger, did not affect the bactericidal activity of AEW, it is concluded that the radical is unlikely to contribute to the antimicrobial activity of AEW, although a small amount of the radical is produced from hydrogen peroxide. Dimethyl sulfoxide, the other hydroxyl radical scavenger used in the present study, canceled the bactericidal activity of AEW, accompanied by complete depletion of free available chlorine, suggesting that hypochlorous acid is probably a major contributor to the antimicrobial activity.

Conclusions

It is strongly suggested that although hydrogen peroxide is present in AEW as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals.     

Drought and watering-dependent oxidative stress: effect on antioxidant content in Triticum aestivum L. leaves

The purpose of the present work was to evaluate both oxidative stress and the antioxidant response system in leaves from wheat (Triticum aestivum cv. Buck Poncho) subjected sequentially to drought and watering. Drought was imposed by withholding water until soil water potential reached -2.0 MPa and maintained under those conditions for 24 h. DCFDA oxidation by wheat leaves was not significantly affected by drought, but watering led to an approximately 2-fold increase in DCFDA oxidation rate. However, no significant effect either on lipid radical content or on hydroperoxide content was measured after drought and drought followed by watering. Microsomes isolated from leaves exposed to drought, and from leaves exposed to drought followed by watering, generated a significantly higher amount of hydroxyl radical as compared to microsomes isolated for control leaves suggesting a higher production of hydroxyl radical in the cellular water soluble phase, after drought and watering as compared to control values. The content of -tocopherol in wheat leaves was increased 2.4-fold after drought and -carotene content was increased by 2.6-fold after drought. Hydration lowered lipid-soluble antioxidant content to control values. Total thiol content as increased by 70% after drought, and watering did not significantly alter the enhanced values. Drought decreased by 28.5% the content of reduced ascorbic acid. Taken as a whole, active species formed at wheat membranes after exposure to moderate water stress, are efficiently removed upon rehydration by reaction with an increased content of -tocopherol and -carotene. Moreover a co-ordinated response involving glutathione reductase activity, thiols and ascorbic acid is triggered to limit free radical dependent effects.Key words: Antioxidants, lipid radicals, oxygen radicals, water stress, wheat      

MCI-186 (edaravone), a novel free radical scavenger, protects against acute autoimmune myocarditis in rats

In this study, we tested the hypothesis that MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one; edaravone), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats by the radical scavenging action associated with the suppression of cytotoxic myocardial injury. Recent evidence suggests that oxidative stress may play a role in myocarditis. We administered MCI-186 intraperitoneally at 1, 3, and 10 mg.kg(-1).day(-1) to rats with EAM for 3 wk. The results were compared with untreated rats with EAM. MCI-186 treatment did not affect hemodynamics. MCI-186 treatment (3 and 10 mg.kg(-1).day(-1)) reduced the severity of myocarditis as assessed by comparing the heart-to-body weight ratio and pathological scores. Myocardial interleukin-1beta (IL-1beta)-positive cells and myocardial oxidative stress overload with DNA damage in rats with EAM given MCI-186 treatment were significantly less compared with those of the untreated rats with EAM. In addition, MCI-186 treatment decreased not only the myocardial protein carbonyl contents but also the myocardial thiobarbituric acid reactive substance products in rats with EAM. The formation of hydroxyl radicals in MCI-186-treated heart homogenates was decreased compared with untreated heart homogenates. Furthermore, cytotoxic activities of lymphocytes of rats with EAM treated with MCI-186 were significantly lower compared with those of the untreated rats with EAM. Hydroxyl radicals may be involved in the development of myocarditis. MCI-186 protects against acute EAM in rats associated with scavenging hydroxyl free radicals, resulting in the suppression of autoimmune-mediated myocardial damage associated with reduced oxidative stress state.     

A novel strategy of natural plant ferritin to protect DNA from oxidative damage during iron oxidation

Plant ferritin is a naturally occurring heteropolymer in plastids, where Fe(2+) is oxidatively deposited into the protein. However, the effect of this process on the coexistence of DNA and plant ferritin in the plastids is unknown. To investigate this effect, we built a system in which various plant ferritins and DNA coexist, followed by treatment with ferrous ions under aerobic conditions. Interestingly, naturally occurring soybean seed ferritin (SSF), a heteropolymer with an H-1/H-2 ratio of 1 to 1 in the apo form, completely protected DNA from oxidative damage during iron oxidative deposition into protein, and a similar result was obtained with its recombinant form, but not with its homopolymeric counterparts, apo rH-1 and apo rH-2. We demonstrate that the difference in DNA protection between heteropolymeric and homopolymeric plant ferritins stems from their different strategies to control iron chemistry during the above oxidative process. For example, the detoxification reaction occurs only in the presence of apo heteropolymeric SSF (hSSF), thereby preventing the production of hydroxyl radicals. In contrast, hydroxyl radicals are apparently generated via the Fenton reaction when apo rH-1 or rH-2 is used instead of apo hSSF. Thus, a combination of H-1 and H-2 subunits in hSSF seems to impart a unique DNA-protective function to the protein, which was previously unrecognized. This new finding advances our understanding of the structure and function of ferritin and of the widespread occurrence of heteropolymeric plant ferritin in nature.     

Quantitative measurement of hydroxyl radical induced DNA double-strand breaks and the effect of N-acetyl-L-cysteine

Reactive oxygen species, such as hydroxyl or superoxide radicals, can be generated by exogenous agents as well as from normal cellular metabolism. Those radicals are known to induce various lesions in DNA, including strand breaks and base modifications. These lesions have been implicated in a variety of diseases such as cancer, arteriosclerosis, arthritis, neurodegenerative disorders and others. To assess these oxidative DNA damages and to evaluate the effects of the antioxidant N-acetyl-L-cysteine (NAC), atomic force microscopy (AFM) was used to image DNA molecules exposed to hydroxyl radicals generated via Fenton chemistry. AFM images showed that the circular DNA molecules became linear after incubation with hydroxyl radicals, indicating the development of double-strand breaks. The occurrence of the double-strand breaks was found to depend on the concentration of the hydroxyl radicals and the duration of the reaction. Under the conditions of the experiments, NAC was found to exacerbate the free radical-induced DNA damage.     

Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow trout erythrocytes

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K(+)-Cl(-) cotransport). 5 mM hydrogen peroxide caused an increase in K(+)-Cl(-) cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K(+)-Cl(-) cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K(+)-Cl(-) cotransport significantly at hypoxic conditions (1% O(2)). These data suggest that hydroxyl radicals, formed from O(2) in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K(+)-Cl(-) cotransporter.     

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.     

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.     

Effect of gamma radiation on E. coli ribosomes, tRNA and aminoacyl-tRNA synthetases

Gamma-irradiated E coli ribosomes and tRNA, in aerated solutions, were inactivated with D37 doses of 144 and 77 Gy, respectively. Aminoacyl-tRNA-synthetases were only slightly inactivated under comparable conditions. Effects of additives to ribosome and tRNA solutions suggest that hydroxyl radicals were the major damaging species, that superoxide anions were not damaging and that radiolytically-formed hydrogen peroxide was also unimportant. Part of the damage by hydroxyl radicals is expressed through secondary radicals produced from additives and buffers. Results obtained with three different buffers suggest that (1) acetate ions provide protection by competing for hydroxyl radicals, (2) chloride ions are without effect and (3) inactivation of ribosomes and aminoacyl-tRNA-synthetases in Tris-HCl/MgCl2 and phosphate/MgCl2 buffered solutions was similar but the tRNA inactivation was lower in Tris-HCl/MgCl2 buffer.     

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.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6 ± 1 × 10(7) M(-1) s(-1) at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7 ± 3 × 10(7) M(-1) s(-1) at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK(a) of phenolic hydroxyl dissociation in tyrosine is ≈ 10.3, this infers a much lower rate constant, about 3 × 10(5) M(-1) s(-1), for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Role of hydroxyl radicals in the iron-ethylenediaminetetraacetic acid mediated stimulation of microsomal oxidation of ethanol

The microsomal oxidation of ethanol or 1-butanol was increased by ferrous ammonium sulfate-ethylenediaminetetraacetic acid (1:2) (Fe-EDTA) (3.4-50 microM). The increase was blocked by hydroxyl radical scavenging agents such as dimethyl sulfoxide or mannitol. The activities of aminopyrine demethylase or aniline hydroxylase were not affected by Fe-EDTA. The accumulation of H2O2 was decreased in the presence of Fe-EDTA, consistent with an increased utilization of H2O2. Other investigators have shown that Fe-EDTA increases the formation of hydroxyl radicals in systems where superoxide radicals are generated. The stimulation by Fe-EDTA appears to represent a pathway involving hydroxyl radicals rather than catalase because (1) stimulation occurred in the presence of azide, which inhibits catalase, (2) stimulation occurred in the presence of 1-butanol, which is not an effective substrate for catalase, and (3) stimulation was blocked by hydroxyl radical scavenging agents, which do not affect catalase-mediated oxidation of ethanol. A possible role for contaminating iron in the H2O or buffers could be ruled out since similar results were obtained with or without chelex-100 treatment of these solutions. The stimulatory effect by Fe-EDTA required microsomal electron transfer with NADPH, and H2O2 could not replace the NADPH-generating system. In the absence of microsomes or catalase, Fe-EDTA also stimulated the coupled oxidation of ethanol during the oxidation of xanthine by xanthine oxidase. These results suggest that during microsomal electrom transfer, conditions may be appropriate for a Fenton type or a modified Haber-Weiss type of reaction to occur, leading to the production of hydroxyl radicals.     

Production of 4-hydroxypyrazole from the interaction of the alcohol dehydrogenase inhibitor pyrazole with hydroxyl radical

Pyrazole, an effective inhibitor of alcohol dehydrogenase, was previously shown to be a scavenger of the hydroxyl radical. 4-Hydroxypyrazole is a major metabolite in the urine of animals administered pyrazole in vivo. Experiments were conducted to show that 4-hydroxypyrazole was a product of the interaction of pyrazole with hydroxyl radical generated from three different systems. The systems utilized were the iron-catalyzed oxidation of ascorbate, the coupled oxidation of hypoxanthine by xanthine oxidase, and NADPH-dependent microsomal electron transfer. Ferric-EDTA was added to all the systems to catalyze the production of hydroxyl radicals. A HPLC procedure employing either uv detection or electrochemical detection was utilized to assay for the production of 4-hydroxypyrazole. The three systems all supported the oxidation of pyrazole to 4-hydroxypyrazole by a reaction which was sensitive to inhibition by competitive hydroxyl radical scavengers such as ethanol, mannitol, or dimethyl sulfoxide and to catalase. The sensitivity to catalase implicates H2O2 as the precursor of the hydroxyl radical by all three systems. Superoxide dismutase inhibited production of 4-hydroxypyrazole only in the xanthine oxidase reaction system. In the absence of ferric-EDTA (and azide), microsomes catalyzed the oxidation of pyrazole to 4-hydroxypyrazole by a cytochrome P-450-dependent reaction which was independent of hydroxyl radicals. This latter pathway may be primarily responsible for the in vivo metabolism of pyrazole to 4-hydroxypyrazole. The production of 4-hydroxypyrazole from the interaction of pyrazole with hydroxyl radicals may be a sensitive, rapid technique for the detection of these radicals in certain tissues or under certain conditions, e.g., increasing oxidative stress.