Hydroxyl Radical Adduct Research Articles

Cataract Formation through the Polyol Pathway is associated with Free Radical Production

The relationship between sugar cataract formation and radical production was investigated. The in vivo formation of free radicals in the lenses of rats fed on a diet containing 25% and 50% galactose was studied using the electron spin resonance (ESR) spin trapping method. Effects of treatment with aldose reductase inhibitor (ARI) SNK-860 on free radical formation were determined in 25% and 50% galactose fed rats. Hydroxyl radical (·OH) adduct of the spin trap 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO) was directly detected in the superficial cortical cataract obtained from 25% and 50% galactose-fed rats.·OH production was completely inhibited by ARI SNK-860 in both galactose groups. Polyol accumulated in rat lenses given 50% galactose with a peak within the first 2 weeks, and was significantly inhibited by SNK-860. The increase in·OH production was considered with the polyol accumulation in both galactose groups. The dose of SNK-860 to inhibit·OH by 50% level was estimated at 3mby the method of kinetic competition in vitro experiment. SNK-860 is not an effective·OH scavenger compared to other·OH scavengers. The results of the present study suggest that·OH is indirectly inhibited by SNK-860 resulting from decreasing polyol and·OH formation is related to sugar cataract formation in early stages, possibly via the Fenton reaction involving H2O2produced from the activated polyol pathway. We suppose that·OH may accelerate damage to the cell membrane of lens fibers resulting from polyol accumulation.·OH may play an important role in the early stage of sugar cataract process.

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

Chromium(VI) compounds and cigarette smoke are known human carcinogens. We found that K 2Cr 2O 7 and cigarette smoke solution synergistically induced DNA single-strand breaks (0.23±0.04 breaks per DNA molecule) in pUC118 plasmid DNA. K 2Cr 2O 7 alone or cigarette smoke solution alone induced much less strand breaks (0.03±0.01 or 0.07±0.02 breaks per DNA molecule, respectively). The synergistic effect was prevented by catalase and by hydroxyl radical scavengers such as deferoxamine, dimethylsulfoxide, d-mannitol, and Tris, but not by superoxide dismutase. Ascorbic acid enhanced the synergism. Glutathione inhibited strand breakage only at high concentrations. Electron spin resonance (ESR) studies using a hydroxyl radical trap demonstrated that hydroxyl radicals were generated when DNA was incubated with K 2Cr 2O 7 and cigarette smoke solution. Hydroxyl radical adduct decreased dose-dependently when strand breakage was prevented by catalase, deferoxamine, dimethylsulfoxide, d-mannitol or Tris, but not significantly by superoxide dismutase. We also used ESR spectroscopy to study the effects of different concentration of ascorbic acid and glutathione. The results showed that hydroxyl radical, which is proposed as a main carcinogenic mechanism for both chromium(VI) compounds and cigarette smoke solution was mainly responsible for the DNA breaks they induced.

Chemical reactions of menadione with probes for superoxide: A cautionary note: [formula omitted], Lawrence J Berliner, A John Merola, Valerie P Wright, Thomas L Clanton. The Ohio State Univ, Pulm. and Crit. Care Med. and Biophysics, Columbus, OH

We have reexamined the formation and reactions of radicals formed from peroxynitrite (ONOO−)-mediated oxidation of glutathione (GSH),l-cysteine (Cys),N-acetyl-d,l-penicillamine (NAP), and sodium bisulfite (NaHSO3). Sulfur-centered and superoxide anion radicals were trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the radical adducts were analyzed by electron spin resonance (ESR) spectroscopy. The following sulfur-centered radicals were detected: glutathionyl radical (GS•) from GSH,l-cysteinyl radical (•Scys) froml-cysteine,N-acetyl-d,l-penicillamine thiyl radical (•Snap) from NAP, and sulfite anion radical[formula]from NaHSO3. Additionally the formation of the hydroxyl radical adduct of DMPO (DMPO/•OH) was observed. DMPO/•OH formation was totally inhibited by low-molecular-weight superoxide dismutase (SOD) mimics. This suggests that DMPO/•OH was formed from the decay of the superoxide radical adduct of DMPO. In the presence of SOD mimics, the DMPO-sulfur-centered adducts were more persistent, suggesting that[formula]is partially responsible for the instability of DMPO-thiyl adducts. Sulfur-centered radicals formed during oxidation of thiols and sulfite by peroxynitrite react with ammonium formate to form the carbon dioxide anion radical[formula]We conclude that sulfur-centered radicals produced from the oxidation of thiols and sulfite by peroxynitrite arise from a hydroxyl-radical-independent mechanism. Biological implications of peroxynitrite-mediated oxidation of thiols as well as the use of SOD mimics in biological spin-trapping are discussed.

An anionic impurity in preparations of cytochrome e interferes with assays of cationic catalysts of the dismutation of the superoxide anion radical

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Dipyridamole and dilazep suppress oxygen radicals in puromycin aminonucleoside nephrosis rats.

Reactive oxygen species (ROS) are involved in the pathophysiology of puromycin aminonucleoside (PAN) nephrosis. To elucidate further the role of radicals in PAN nephrosis and the to determine the particular radical species scavenged by dipyridamole (DPM) and dilazep (DZ), we applied chemiluminescence and electron spin resonance (ESR) techniques. Chemiluminescence of glomeruli, which were isolated on day 7 from rats injected with 100 mg kg-1 PAN, was measured with or without scavengers. The inhibitory effects of DPM and DZ on hydroxyl radical adduct formation in the Fenton's reaction were evaluated using ESR. Chemiluminescence was greater in glomeruli from rats with PAN nephrosis than in the the glomeruli of control rats. This increase was suppressed by superoxide dismutase, catalase, dimethylthiourea and also by DPM and DZ. ESR indicated that DPM and DZ inhibited hydroxyl radical adduct formation with a second-order rate constant of 2.9 x 10(10) and 1.6 x 10(10) (mol L(-1) s(-1) respectively, similar to that of dimethylthiourea. DPM and DZ scavenge hydroxyl radicals, thereby alleviating PAN nephrosis.

Redox Dependence of the Rate of Interaction of Hydroxyl Radical Adducts of DNA Nucleobases with Oxidants: Consequences for DNA Strand Breakage

Oxidants and oxygen enhance the sensitivity of cells to radiation. To understand this effect at the mechanistic level, the kinetics of interaction of the OH adducts of pyrimidines and 2‘-deoxynucle...

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2.

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5, 5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/.OH formation from the SOD and SOD mutants. The relative ratios of DMPO/.17OH and DMPO/.16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/.17OH and 37% DMPO/.16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/.17OH (64%); DMPO/.16OH (36%) from A4V and DMPO/.17OH (62%); and DMPO/.16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/.OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/.OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/.OH or DEPMPO/.OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Demonstration of the Production of Oxygen-Centered Free Radicals During Electrolysis Using E.S.R. Spin-Trapping Techniques: Effects on Cardiac Function in the Isolated Rat Heart

The present study was designed to identify the free radicals generated during the electrolysis of the solution used to perfuse isolated rat heart Langendorff preparations. The high reactivity and very short half-life of oxygen free radicals make their detection and identification difficult. A diamagnetic organic molecule (spin trap) can be used to react with a specific radical to produce a more stable secondary radical or “spin adduct” detected by electron spin resonance (ESR). Isovolumic left ventricular systolic pressure (LVSP) and left ventricular end diastolic pressure (LVEDP) were measured by a fluid-filled latex balloon inserted into the left ventricle. The coronary flow was measured by effluent collection. Electrolysis was performed with constant currents of 0.5, 1, 1.5, 3, 5, 7.5, and 10 mA generated by a Grass stimulator and applied to the perfusion solution for 1 min. A group of experiments was done using a 1.5 mA current and a Krebs–Henseleit (K-H) solution containing free radical scavengers (superoxide dismutase (SOD): 100 IU/ml or mannitol: 50 mM). Heart function rapidly declined in hearts perfused with K-H buffer that had been electrolyzed for 1 min. The addition of mannitol (50 mM) to the perfusion solution had no effect on baseline cardiac function before electrolysis while SOD (100 IU/ml) increased the coronary flow. However, SOD was more effective than the mannitol in protecting the heart against decreased of cardiac function, 5 min after the end of electrolysis. Samples of the K-H medium subjected to electrolysis were collected in cuvettes containing a final concentration of 125 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and analyzed by spectroscopy. The ESR spectrum consisted of a quartet signal (hyperfine couplings aN = aH = 14.9 G) originating from the hydroxyl adduct signal, DMPO–OH. The intensity of the DMPO–OH signal remained stable during the 60 s of electrolysis and the quantity of free radicals induced by electrolysis was directly proportional to the intensity of the current. The addition of mannitol and SOD to the perfusate scavenged the hydroxyl radicals present in the solution, suggesting that both hydroxyl and superoxide radicals were formed during electrolysis.

THE EFFECT OF PROPOFOL, EMULSIFIED PROPOFOL (DIPRIVAN[registered sign]) AND EMULSIFIER (INTRALIPID) ON HYDROXYL RADICAL GENERATED BY FENTON REACTION

S493 INTRODUCTION: Recent studies have established that propofol (2, 6-Diisopropylphenol) is an important free radical scavenger which can inhibit radical-induced lipid peroxidation that produces tissue damage [1-4]. Especially, hydroxyl radical ([center dot]OH), a strong oxygen related radical, is the most reactive in lipid peroxidation and its scavenging activity may explain some properties of propofol such as the anti-emetic effect. The purpose of this study is to compare the scavenging activity of propofol, emulsified propofol (Diprivan[registered sign]) and the emulsifier of propofol (10% intralipid) by using electron spin resonance (ESR, JES-RE1X, JEOL, Akisima, Japan) spectrometry. METHODS: The ESR spectrum was measured to assess the magnitude of hydroxyl radical adduct trapped by 5,5-dimethyl-1-pyrroline-n-oxide (DMPO). In the hydroxyl radical formation mixture, 1% propofol, 1% Diprivan or 10% intralipid was added to make final concentrations of 2.8, 5.6, and 8.4 mM for both propofol and Diprivan[registered sign] and 0.5, 1.0, and 1.5 % for intralipid, respectively. The amount of the hydroxyl radical was quantified by the intensity of the DMPO-OH adducts measured directly from the field scan. RESULTS: Typical signals of the DMPO-OH adducts in control (without Diprivan[registered sign]) and experimental mixtures (with Diprivan[registered sign]) are shown in Figure 1, where Diprivan[registered sign] appeared to decrease OH-adduct. Propofol significantly decreased OH-adduct in a dose dependent manner. Diprivan[registered sign] and intralipid also caused a decrease of [center dot]OH-adduct. The 8.4 mM propofol, 8.4 mM Diprivan[registered sign] and 1.5% intralipid decreased [center dot]OH-adduct by 34.7%, 63.4% and 53.5%, respectively compared with the control.Figure 1DISCUSSION: Our results show that both propofol and the emulsifier scavenge hydroxyl radical adduct, and the Diprivan[registered sign] mixture also has a scavenging property. The magnitude of scavenging activity of Diprivan[registered sign] might be greater than propofol itself.

Synthesis of alpha-aryl N-adamant-1-yl nitrones and using them for spin trapping of hydroxyl radicals.

alpha-Aryl N-adamant-1-yl nitrones were synthesized and evaluated with respect to the stability of the hydroxyl radical adduct. The polarity and water solubility of nitrones were altered with changing the alpha-aryl groups. Introduction of adamantane ring instead of tert-butyl group resulted in a reasonable good stability of hydroxyl radical adduct for biological measurements.

Use of a phosphorylated spin trap to discriminate between the hydroxyl radical and other oxidising species

The spin-trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) is shown to provide a simple method to distinguish free-radical reactions which lead to the production of ˙OH from those which proceed via one-electron transfer followed by hydration or nucleophilic substitution of another adduct. In the first case, only an ˙OH adduct is detected; in the latter, signals from the hydroxyl adduct (labelled with 17O when H217O is employed) are accompanied by a second signal believed to result via internal trapping of a radical formed from an intermediate radical cation. This provides a potentially useful mechanistic probe, for example for use in biochemical systems.

Formation of Spin Trap Adducts during the Decomposition of Peroxynitrite

Peroxynitrite-mediated one-electron oxidations may be an important event in its cytotoxic mechanisms, and yet, free radical formation in the presence of peroxynitrite is difficult to study by EPR–spin trapping because adducts from most spin traps are destroyed by the oxidant. This led to some controversy with regard to the interpretation of experiments in the presence of 5,5-dimethyl-1-pyrrolineN-oxide (DMPO), an adequate spin trap to study most of free radicals. In this report we reexamined peroxynitrite-mediated formation of spin-trap adducts. Kinetic studies and EPR experiments with water labeled with17O are in agreement with the reaction of DMPO with a highly reactive intermediate derived from peroxynitrite to produce the DMPO–hydroxyl radical adduct by a mechanism not involving the oxidation of DMPO to a cation radical followed by water addition. The results cannot discriminate between two mechanisms of DMPO–hydroxyl radical formation, either spontaneous peroxynitrite homolysis to the hydroxyl radical or DMPO-assisted peroxynitrite homolysis. The formation of DMPO adducts during peroxynitrite-mediated oxidation of dimethyl sulfoxide, ethanol, and formate occurs through free radical mechanisms as confirmed by studies of oxygen consumption and product formation. Accordingly, spin-trapping experiments in the presence of 3,5-dibromo-4-nitrosobenzenesulfonic acid, a spin trap that is more resistant to nitrogen dioxide, led to the detection of the methyl and the β-hydroxyethyl radical during peroxynitrite-mediated oxidation of dimethyl sulfoxide and ethanol, respectively. Oxidation of these hydroxyl radical scavengers to detectable radicals favors the hypothesis that the hydroxyl radical is produced during peroxynitrite homolysis. Bicarbonate was able to modulate peroxynitrite-mediated one-electron oxidations.

Synthesis of creatol, a hydroxyl radical adduct of creatinine and its increase by puromycin aminonucleoside in isolated rat hepatocytes

Creatol is a hydroxyl radical adduct of creatinine and the precursor of methylguanidine (MG), a uremic toxin. We investigate the synthesis of creatol and MG from creatinine and the effect of substances that affect the hydroxyl radical in isolated rat hepatocytes. In the presence of increasing concentrations of creatinine, rising level of creatol were found after 2 h incubation in Krebs-Henseleit bicarbonate buffer. However, further increase of creatol was not observed after 4 and 6 h incubations. On the other hand, MG after 2 h incubation achieved a level of about 50% that of creatol and increased depending on both the creatinine concentration and the incubation period. DMSO, a hydroxyl radical scavenger decreased the generation of creatol and MG by about 50% at 2.5 mM and the inhibition depended on DMSO concentration. Puromycin aminonucleoside (PAN) increased both by about 170%. These findings demonstrated that hepatocytes synthesize creatol prior to MG and are inhibited by a hydroxyl radical scavenger. They also show that PAN increased hydroxyl radical generation in tissue cells.

Correlation Between Direct ESR Spectroscopic Measurements and Electromechanical and Biochemical Assessments of Exogenous Free Radical Injury in Isolated Rat Cardiac Myocytes

Reactive free radical species appear to be involved in the ischemic injury of cardiac muscle, although the mechanisms by which oxygen-derived free radicals affect the heart cell function are not known. In the present study, cultured ventricular myocytes were exposed to an exogenous oxygen radical generating system. The myocyte-enriched, primary cultures were prepared from ventricles of new-born rat heart and exposed to a xanthine/xanthine oxidase (X+XO) system. The transmembrane potentials were recorded with glass microelectrodes. Cell contractions were monitored photometrically. The release of lactate dehydrogenase (LDH) in the medium was analysed. Quantitative measurement and the time course of the radical generation were performed by the electron paramagnetic resonance (EPR) spin trapping technique with the spin trap 5,5-dimethyl-1-pyroline- N-oxide (DMPO). We verified that X and XO alone had no significant functional and biochemical effects. The X+XO system produced a rapid decrease in the action potential amplitude. This effect was accompanied by a strong decrease in contractility and spontaneous rate. The time course of these functional defects were correlated with a progressive efflux of LDH from the cardiomyocytes. Prolonging the exposure to the X+XO system provoked the cessation of the spontaneous beatings and the progressive loss of the resting diastolic potential, together with a near total release of the cellular LDH. The LDH release and the functional depression were both efficiently prevented by catalase. On the contrary, superoxide dismutase (SOD) slowed down but did not protect against the functional and biochemical effects of the free radicals. In comparison, the EPR spectra obtained indicated that the X+XO system was associated with an important generation of superoxide anions but also with a small hydroxyl production. SOD scavenged the superoxide but a small ·OH production persisted. Catalase (CAT) did not modify the superoxide generation but decreased the hydroxyl adduct formation. These results suggest that, although the generation of superoxide anions by the X+XO system was higher than the hydroxyl production, the functional injury and enzyme leakage seemed mainly mediated through a hydrogen peroxide-hydroxyl radical pathway. Cultured ventricular myocytes can be thus used as a valuable model to investigate the cellular mechanism of oxidant-induced damage in the heart.

ESR Identification of Free Radicals Formed from the Oxidation of Catechol Estrogens by Cu2+

Catechol estrogens are genotoxic, indirectly through redox cycling mechanisms leading to oxidative DNA damage and directly by formation of quinone–DNA adducts. Previously, we demonstrated that Cu2+can oxidize estradiol (E2) catechols, establishing a copper redox cycle leading to the formation of DNA strand breaks. The goal of this study was to use electron spin resonance techniques to identify the free radical intermediates formed. The 2- and 4-OH catechols of E2 and ethinyl estradiol (EE) were oxidized to semiquinone intermediates, stabilized by Mg2+, when incubated with Cu2+. The 4-OH-EE semiquinone decayed more slowly than the 2-OH-EE semiquinone. Using the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, 4-OH-E2 plus Cu2+generated hydroxyl radicals at a greater rate than 2-OH-E2 plus Cu2+. Formation of hydroxyl and methyl radical adducts was detected, using 5,5-dimethyl-1-pyrroline-N-oxide as the spin trap, when 2-OH-E2 was incubated with Cu2+and 1% dimethyl sulfoxide. This was inhibited by the Cu1+chelator bathocuproinedisulfonic acid and catalase. These data demonstrate that the oxidation of estrogen catechols by Cu2+leads to a Cu-dependent mechanism of hydroxyl radical production via a hydrogen peroxide intermediate and suggest a mechanism for estrogen-associated site-specific DNA damage and mutagenesis.

Free radical activity of PM10: iron-mediated generation of hydroxyl radicals.

The purpose of this study was to test the hypothesis that particulate matter < or = 10 microns in aerodynamic diameter (PM10) particles have the ability to generate free radical activity at their surface. We collected PM10 filters from the Edinburgh, United Kingdom, Enhanced Urban Network sampling site, removed particles from the filter, and tested their ability to cause free radical damage to supercoiled plasmid DNA. We found that the PM10 particles did cause damage to the DNA that was mediated by hydroxyl radicals, as shown by inhibition of the injury with mannitol. The PM10-associated hydroxyl radical activity was confirmed using a high-performance liquid chromatography-based assay to measure the hydroxyl radical adduct of salicylic acid. Desferrioxamine abolished the hydroxyl radical-mediated injury, which suggests that iron was involved. Analysis of PM10 filters confirmed the presence of large amounts of iron and leaching studies confirmed that the PM10 samples could release substantial amounts of Fe(III) and lesser amounts of Fe(II). To investigate the size of the particles involved in the hydroxyl radical injury, we centrifuged the suspension of PM10 to clarity, tested the clear supernatant, and found that it had all of the suspension activity. We conclude, therefore, that the free radical activity is derived either from a fraction that is not centrifugeable on a bench centrifuge, or that the radical generating system is released into solution.

Spin-Trapping of Free Radicals Formed during the Oxidation of Glutathione by Tetramethylammonium Peroxynitrite

Glutathionyl radical (GS•) formed during the oxidation of glutathione by tetramethylammonium peroxynitrite ([NMe4][ONOO]) was spin-trapped with 5,5′-dimethyl-1-pyrrolineN-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrrolineN-oxide (DEPMPO). This radical reacted with ammonium formate to form the carbon dioxide anion radical (CO•−2). The superoxide anion formed during oxidation of GSH by peroxynitrite salt was trapped with DMPO and detected as the DMPO-hydroxyl adduct. Addition of SOD mimic completely abolished the spectrum of the hydroxyl adduct but not the spectrum of the DMPO-glutathionyl radical adduct. Addition of seleno-dl-cystine or its reduced form caused a dramatic inhibition in the formation of spin adducts, suggesting that seleno-dl-cysteine is a more effective scavenger of peroxynitrite. The oxygen uptake observed during oxidation of GSH by peroxynitrite salt was inhibited by spin traps. In the presence of catalase, approximately 50% of the oxygen consumed was restored, indicating stoichiometric conversion of O2to H2O2during oxidation of GSH by peroxynitrite salt. Results indicate that nitrite and glutathione disulfide are formed as the major products during oxidation of GSH by peroxynitrite.

A Comparative Study of the Redox-Cycling of a Quinone (Rifamycin S) and A Quinonimine (Rifabutin) Antibiotic by Rat Liver Microsomes

Rifamycin S and rifabutin are clinical drugs used to treat tuberculosis and leprosy. The formation of reactive oxygen species during the redox-cycling of rifamycin S (quinone) and rifabutin (quinonimine) was evaluated. The semiquinone (or semiquinonimine) and hydroquinone (or hydroquinonimine) formed during the reduction of the parent molecules by microsomal electron transfer in the presence of nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide-adenine dinucleotide, reduced (NADH) reoxidizes in air to generate superoxide radical and hydrogen peroxide. In the presence of added iron, hydroxyl radicals, formed by the Fenton reaction, were detected using 5,5′-dimethyl-1-pyrroline-N-oxide as the spin-trap. Rifamycin S, a quinone, redox cycles more efficiently than rifabutin, a quinonimine, as approximately five times the concentration of hydroxyl radical adduct of 5,5′-dimethyl-1-pyroline-N-oxide (DMPO) was detected, when compared with rifabutin. The NADPH-dependent microsomal production of hydroxyl radical in the presence of rifamycin S was somewhat higher than the NADH-rifamycin S system with most iron chelators. However, with rifabutin, NADH-dependent microsomal production of hydroxyl radical was higher than that found with the NADPH-rifabutin system. An exception was the iron chelator, diethylene-triamine-pentacetic acid (DTPA), in which NADPH-dependent rates exceeded the rates with NADH with both antibiotics. Rat liver sub-mitochondrial particles also generated hydroxyl radical in the presence of NADH and either rifamycin S or rifabutin. The electron transport chain inhibitors such as rotenone and antimycin A enhanced the signal intensity of DMPO-OH, suggesting NADH dehydrogenase (complex I) as the major component involved in the reduction of rifamycin S. Rifamycin S was shown to be readily reduced to rifamycin SV, the corresponding hydroquinone by Fe(II); under similar conditions Fe(II) did not reduce rifabutin. Using optical spectroscopy, we determined that rifamycin S forms a complex with Fe(II). The stoichiometry of the complex was Fe(rifamycin S) 3 in phosphate buffer at pH 7.4. Rifabutin did not form a detectable complex with Fe(II). The redox cycling of rifamycin S and rifabutin did not cause microsomal lipid peroxidation. In fact, the Fe:ATP induced lipid peroxidation was completely inhibited by these two molecules. These results indicate that rifamycin S and rifabutin can interact with rat liver microsomes to undergo redox-cycling, with the subsequent production of hydroxyl radicals when iron complexes are present. Compared to NADPH, NADH is almost as effective (rifamycin S) or even more effective (rifabutin) in promoting these interactions. These interactions may play a role in the hepatotoxicity associated with the use of these antibiotics. Copyright © 1996 Elsevier Science Inc.

EPR spin-trapping studies of the reaction of hydroxyl and other electrophilic radicals with uridine and related compounds. Isotopic substitution to refine analyses and aid quantification †

EPR spin-trapping experiments using MNP (2-methyl-2-nitrosopropane) have been employed to study radicals formed by reaction of HO˙ (generated from reaction of H2O2 with Fe2+) with uridine as a model nucleoside; studies with [5-2H]uridine, [5,6-2H2]uridine and [1,3-15N2] have allowed us to obtain a more detailed analysis of the spectra following HO˙ attack on uridine, for which EPR spectra cannot at present be unambiguously assigned. These studies provide detailed information concerning the analysis of hyperfine splittings and hence the quantification of the different amounts of the C(5)- and C(6)-hydroxyl-radical adducts formed following HO˙ attack, which are comparable to results obtained from pulse radiolysis studies. Studies have also been extended to investigate the reactions of both SO4˙– and ButO˙ with uridine.

The Metabolism of Tyramine by Monoamine Oxidase A/B Causes Oxidative Damage to Mitochondrial DNA

Monoamine oxidases A/B (EC 1.4.3.4, MAO), flavoenzymes located on the outer mitochondrial membrane, catalyze the oxidative deamination of biogenic amines, such as dopamine, serotonin, and norepinephrine. In this study, we examined whether the H2O2formed during the two-electron oxidation of tyramine [4-(2-aminoethyl)phenol] (a substrate for monoamine oxidases A/B) may contribute to the intramitochondrial steady-state concentration of H2O2([H2O2]ss) and, thus, be involved in the oxidative impairment of mitochondrial matrix components. Supplementation of intact, coupled rat brain mitochondria with benzylamine, β-phenylethylamine, or tyramine showed initial rates of H2O2production ranging from 0.4- to 1.6 nmol H2O2/min/mg protein. ESR analysis of the oxidative deamination of tyramine by intact rat brain mitochondria revealed the formation of hydroxyl (HO) and carbon-centered radical adducts—the latter probably originating by the HO-mediated oxidation of mannitol. The signals were substantially enhanced upon addition of FeSO4and were abolished by catalase. The intramitochondrial [H2O2]sscalculated in terms of glutathione peroxidase activity during the metabolism of tyramine was 48-fold higher (7.71 ± 0.25 × 10−7M) than that obtained during the oxidation of succinate via complex II in the presence of antimycin A (1.64 ± 0.2 × 10−8M). Oxidative damage to the brain mtDNA was assessed by single strand breakage. The ratio of nicked DNA for the preparations treated with tyramine and those without the amine was 1.5 ± 0.29 (n= 4), 2.12 ± 0.28 (n= 8,P≤ 0.05), and 3.12 ± 0.69 (n= 3,P≤ 0.05) at 15, 30, and 60 min, respectively. Preincubation of mitochondria with tranylcypromine (trans-2-phenylcyclopropylamine), an inhibitor to MAO A/B, abolished mtDNA oxidative damage. Catalase inhibited mtDNA strand breakage by approximately 60%. Incubation of intact, coupled rat brain mitochondria with chlorodinitrobenzene (CDNB) depleted mitochondrial GSH by 72%. Tyramine-dependent damage of mtDNA was decreased by 68% in CDNB-treated mitochondria (with 28% remaining GSH). The [H2O2]sswas slightly increased in CDNB-treated mitochondria: 1.38- and 1.28-fold increase during the oxidation of succinate in the presence of antimycin A and during the oxidation of tyramine, respectively. These results suggest that the H2O2generated during the MAO-catalyzed oxidation of biogenic amines and possibly certain neurotransmitters at the outer mitochondrial membrane contributes to the intramitochondrial [H2O2]ssand may cause oxidative damage to mtDNA. This is effected by the intramitochondrial concentration of GSH and might have potential implications for aging and neurodegenerative processes.