Generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate

Nitrofurantoin, misonidazole, and metronidazole were reduced to their corresponding nitro anion radicals by ascorbate in anaerobic solutions at high pH. The nitrofurantoin anion radical could be detected at neutral pH. In neutral solutions, the nitro anion radicals of misonidazole and metronidazole were too unstable to be observed by electron spin resonance spectroscopy. At neutral pH, solutions containing ascorbate, nitrofurantoin, or misonidazole consumed oxygen. The addition of superoxide dismutase, catalase, or both superoxide dismutase and catalase decreased the rate of oxygen consumption. These results show that nitro anion radicals are formed by reduction with ascorbate, and superoxide anion radical and hydrogen peroxide are produced by reactions of these radicals with oxygen.     

Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism

The EPR signal recorded in reaction medium containing L-lysine and methylglyoxal is supposed to come from the anion radical (semidione) of methylglyoxal and cation radical of methylglyoxal dialkylimine. These free radical inter-mediates might be formed as a result of electron transfer from dialkylimine to methylglyoxal. The EPR signal was observed in a nitrogen atmosphere, whereas only trace amounts of free radicals were registered under aerobic conditions. It has been established that the decay of methylglyoxal anion radical on aeration of the medium is inhibited by superoxide dismutase. Using the methods of EPR spectroscopy and lucigenin-dependent chemiluminescence, it has been shown that nonenzymatic generation of free radicals including superoxide anion radical takes place during the interaction of L-lysine with methylglyoxal — an intermediate of carbonyl stress — at different (including physiological) pH values. In the course of analogous reaction of L-lysine with malondialdehyde (the secondary product of the free radical derived oxidation of lipids), the formation of organic free radicals or superoxide radical was not observed.     

Generation of nitro radical anions of some 5-nitrofurans, 2- and 5-nitroimidazoles by norepinephrine, dopamine, and serotonin. A possible mechanism for neurotoxicity caused by nitroheterocyclic drugs

Catecholamine neurotransmitters such as norepinephrine, dopamine, and related catecholamine derivatives reduce nitroheterocyclic drugs such as nitrofurantoin, nifurtimox, nifuroxime, nitrofurazone, misonidazole, and metronidazole in slightly alkaline solutions. Drugs which contain 5-nitrofurans are reduced at lower pH than drugs which contain 2- and 5-nitroimidazoles. 5-Nitroimidazole derivatives such as metronidazole and ronidazole are known to be more difficult to reduce than 2-nitroimidazole derivatives, due to their lower redox potential. Catecholamines, when reducing nitro drugs, undergo concomitant oxidation to form semiquinone radicals. Both semiquinone radicals and nitro anion radicals formed in a reaction of nitro drug and catecholamine derivative were detected by electron spin resonance spectroscopy. Oxygen consumption studies in solutions containing nitro drug and catecholamine derivative showed that nitro anion radicals formed under aerobic conditions reduce oxygen to form the superoxide radical and hydrogen peroxide. Quinones formed in the reaction of catecholamine and nitro drug were detected by optical spectroscopy. Biosynthetic precursors and some metabolic products of catecholamines were also used in these studies, and they all exhibited reactions similar to catecholamines. Bovine chromaffin granules which synthesize and store catecholamines produced the nitrofurantoin anion radical when intact granules were treated with nitrofurantoin. These radicals formed inside the granules were observed by ESR spectroscopy. The formation of nitrofurantoin radical, semiquinone radicals of catecholamines, and oxygen-derived radicals by chromaffin granules is proposed to cause damage to adrenal medulla, and this process may lead to neurotoxicity.     

Generation of alloxan free radicals in chemical and biological systems: implication in the diabetogenic action of alloxan

Electron spin resonance (ESR) studies that on reaction with NADPH, alloxan was reduced forming labile anion radicals giving a 7-line signal with g = 2.005. These radicals were also produced on incubation of alloxan with rat liver subcellular fractions and their production was greatly enhanced by NADPH. Alloxan effectively scavenged superoxide anion generated by a xanthine-xanthine oxidase (XOD) system in association with its reduction to these anion radicals. These radicals were also formed during incubation of alloxan with rat pancreatic beta-cells. These results suggest that the cytotoxicity of alloxan is related to the formation of alloxan anion radicals.     

Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion

The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.     

Nitrobenzyl radical metabolites from microsomal reduction of nitrobenzyl chlorides

The o-, m-, and p-nitrobenzyl chlorides are reduced aerobically and anaerobically by NADPH and rat hepatic microsomes. Under aerobic conditions, these nitro anion radicals reduce oxygen to superoxide as demonstrated by oxygen consumption and spin trapping of superoxide with 5,5-dimethyl-1-pyrroline N-oxide. At low oxygen concentration, the p- and o-nitro anion radicals undergo intramolecular electron transfer and decompose to carbon-centered nitrobenzyl radicals, which can be spin-trapped with t-nitrosobutane. The p-nitrobenzyl (o-nitrobenzyl) radical adduct was characterized by a nitrogen hyperfine splitting of 16.5 G (17.1 G) and two equivalent beta-hydrogen hyperfine splittings of 10.6 G (14.4 G). The spin trap 5,5-dimethyl-1-pyrroline N-oxide also yields adducts characteristic of carbon-centered free radicals. This unimolecular decomposition is much faster than the disproportionation decay, which is characteristic of most nitro anion radicals, and the primary o- and p-nitrobenzyl chloride anion radicals never achieve detectable concentrations. The nitrobenzyl radical trapping is not inhibited by metyrapone or CO. In contrast, the m-nitrobenzyl anion radical does achieve a detectable steady-state concentration, which is increased 20% by either metyrapone or a CO atmosphere.     

Action of reumycin on the blood system in an experiment

The effect of reumycin, an antitumor antibiotic on the peripheral circulatory system and bone marrow was studied on albino rats. The drug was injected intraperitoneally in a dose of 25 mg/kg daily for 30 days. It was shown that reumycin had a comparatively low toxic effect on the peripheral circulatory system and hemopoiesis. It induced the signs of transitory anemia and did not suppress the regenerative capacity of the bone marrow. After the drug repeated use there was an increase in the platelet count and in the rate of blood coagulation. These signs vanished a month after the end of the treatment course.     

Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4

This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin-trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion, both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping are similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein.     

Effect of O-glycosilation on the antioxidant activity and free radical reactions of a plant flavonoid,chrysoeriol

Chrysoeriol and its glycoside (chrysoeriol-6-O-acetyl-4'-beta-D-glucoside) are two natural flavonoids extracted from the tropical plant Coronopus didymus. The aqueous solutions of both the flavonoids were tested for their ability to inhibit lipid peroxidation induced by gamma-radiation, Fe (III) and Fe (II). In all these assays chrysoeriol showed better protecting effect than the glycoside. The compounds were also found to inhibit enzymatically produced superoxide anion by xanthine/xanthine oxidase system; here the glycoside is more effective than the aglycone. The rate constants for the reaction of the compounds with superoxide anion determined by using stopped-flow spectrometer were found to be nearly same. Chrysoeriol glycoside reacts with DPPH radicals at millimolar concentration, but the aglycone showed no reaction. Using nanosecond pulse radiolysis technique, reactions of these compounds with hydroxyl, azide, haloperoxyl radicals and hydrated electron were studied. The bimolecular rate constants for these reactions and the transient spectra of the one-electron oxidized species indicated that the site of oxidation for the two compounds is different. Reaction of hydrated electron with the two compounds was carried out at pH 7, where similar reactivity was observed with both the compounds. Based on all these studies it is concluded that chrysoeriol exhibits potent antioxidant activity. O-glycosylation of chrysoeriol decreases its ability to inhibit lipid peroxidation and reaction with peroxyl radicals. However the glycoside is a more efficient scavenger of DPPH radicals and a better inhibitor of xanthine/xanthine oxidase than the aglycone.     

The oxidation of ubiquinol by the isolated Rieske iron-sulfur protein in solution

The pre-steady-state redox reactions of the Rieske iron-sulfur protein isolated from beef heart mitochondria have been characterized. The rates of oxidation by c-type cytochromes is much faster than the rate of reduction by ubiquinols. This enables the monitoring of the oxidation of ubiquinols by the Rieske protein through the steady-state electron transfer to cytochrome c in solution. The pH and ionic strength dependence of this reaction indicate that the ubiquinol anion is the direct reductant of the oxidized cluster of the iron-sulfur protein. The second electron from ubiquinol is diverted to oxygen by the isolated Rieske protein, and forms oxygen radicals that contribute to the steady-state reduction of cytochrome c. Under anaerobic conditions, however, the reduction of cytochrome c catalyzed by the protein becomes mechanicistically identical to the chemical reduction by ubiquinols. The present kinetic work outlines that: (i) the electron transfer between the ubiquinol anion and the Rieske cluster has a comparable rate when the protein is isolated or inserted into the parent cytochrome c reductase enzyme; (ii) the Rieske protein may be a relevant generator of oxygen radicals during mitochondrial respiration.     

Free radical scavenging reactions and antioxidant activities of silybin: Mechanistic aspects and pulse radiolytic studies

Silybin (extracted from Silybum marianum) is the major active constituent of silymarin which possesses a wide range of medicinal properties. These properties may be, in part, due to the potent scavenging capacity of oxidizing free radicals. In this context, scavenging radicals (hydroxyl, azide, dibromide anion radicals, nitrite, carbonate, etc.) of silybin have been studied to understand the mechanistic aspects of its action against free radicals. The transients produced in these reactions have been assigned and the rate constants have been measured by pulse radiolysis techniques. Reduction potential determined both by cyclic voltammetry gave a value 0.62±0.02 V vs NHE at pH 9. Quantum chemical calculations have been performed to further confirm the different activities of individual hydroxyl groups with the difference of heat of formation. Moreover, silybin also protected plasmid pUC18 DNA from soft X-ray radiation which induced strand breaks. These results are expected to be helpful for a better understanding of the anti-oxidative properties of silybin.     

The effect of oxygen on the radiolysis of tyrosine in aqueous solutions

The effect of oxygen on the radiolysis of tyrosine in aqueous solutions was investigated by using gamma and pulsed electron irradiation. Steady-state radiolysis was reexamined and extended to include the effect of pH and determination of hydrogen peroxide. The loss of tyrosine, G(-Tyr), during irradiation and yields of 3,4-dihydroxyphenylalanine, G(DOPA), and hydrogen peroxide, G(H2O2), are determined in the pH range from 1 to 9. In the whole pH range used G(-Tyr) equals G(DOPA), and a higher G(H2O2) than expected was observed. In slightly acid and neutral media, both G(-Tyr) and G(DOPA) equal the yield of hydroxyl radicals, GOH, formed in the radiolysis of water, while the excess of hydrogen peroxide equals 1/2 GOH. Hence it was concluded that all tyrosine OH-adducts react with oxygen yielding peroxy radicals. In acid and alkaline media all measured yields decrease. This is caused by formation of tyrosine phenoxyl radicals (TyrO), which react with superoxide anion (O2-) and hydroperoxy (HO2) radicals regenerating tyrosine. By using pulse radiolysis K(TyrO + O2) less than or equal to 2 X 10(5) mol-1 dm3 s-1 and k(TyrO + O2-) = (1.7 +/- 0.2) X 10(9) mol-1 dm3 s-1 were determined. On the basis of the results, a reaction mechanism is proposed.     

Radical chemistry of epigallocatechin gallate and its relevance to protein damage

The radical chemistry of the plant polyphenolics epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were investigated using electron paramagnetic resonance spectroscopy. Radical species formed spontaneously in aqueous solutions at low pH without external oxidant and were spin stabilized with Zn(II). The spectra were assigned to the gallyl radical and the anion gallyl radical, with only 10% of the signal assigned to a radical from the galloyl ester. Spectral simulations were used to establish a pK(a) of 4.8 for the EGCG radical and a pK(a) of 4.4 for the EGC radical. The electrochemical redox potentials of EGCG and EGC varied from 1000 mV at pH 3 to 400 mV at pH 8. The polyphenolics did not produce hydroxyl radicals unless reduced metal ions such as iron(II) were added to the system. Zinc(II)-stabilized EGCG radicals were more effective protein-precipitating agents than unoxidized EGCG and produced irreversibly complexed protein. EGCG and other naturally occurring polyphenolics are effective radical scavengers but their radical products have the potential to damage biological molecules such as proteins.     

Experimental study of the action of the antitumor antibiotic reumycin on the macroorganism]

The side effect of reumycin, an antitumor antibiotic was studied experimentally. The average lethal dose for mice and rats on intravenous administration of the drug was 45.5 and 42.2 mg/kg respectively. When reumycin was administered to rats and dogs for prolong periods of time, an increase in the levels of total protein, urea nitrogen and inorganic phosphorus in the blood was observed, while the levels of hemoglobin and thrombocyte counts decreased, the process of the blood coagulation being slower. The changes in the ECG were similar to those observed in the myocardium dystrophy. The morphological changes in the organs were characterized by perivascular edema, swelling of the vessel walls and edema of the interstitial tissues.     

Immobilized radicals. IV. Biological semiquinone anions and neutral semiquinones

The semiquinone anion and neutral semiquinone radicals of benzoquinone, vitamin K-1, ubiquinone and plastoquinone were generated in both protic and aprotic solvents and frozen to produce immobilized spectra. The linewidths of the neutral semiquinones were always much larger than those of the corresponding anion radicals. Furthermore, the spectra of the neutral radicals often exhibit fine structure. When compared with in vivo spectra of semiquinones, these model systems suggest that the ubisemiquinone anion radical observed in photosynthetic bacteria can exist in either a protic or aprotic environment. There is also the implication that Signal II in chloroplasts may be a plastosemiquinone radical with a spin distribution similar to that of the neutral radical.     

Generation of Oxygen Radicals from Iron Complex of Orellanine, A Mushroom Nephrotoxin; Preliminary ESR and Spin-Trapping Stukes

Orellanine, [2,2'-bipyridine]-3,3',4,4'-tetrol-l,I'-dioxide, is the toxin responsible for the lethal nephrotoxicity of some Cortinarius mushrooms. Our present ESR and spin-trapping studies of the redox properties of the system of non-illuminated orellanine, ferrous iron and dioxygen contribute to understanding the molecular mechanism of its toxicity. UV-visible spectrophoto-metry, cyclic voltammetry and ESR in frozen medium showed the formation of a wine-red tris complex, Fe(III)Or₃. This ferric complex is easily reducible (E_P =-565 mV vs Ag/AgCl/3M KCl at pH7), involving a one-electron reversible process. Spin-trapping using DMPO is employed to detect the generation of super-oxide anion and hydroxyl radicals. The instantaneous one-electron oxidation of ferrous ions in the presence of the toxin under air is concomitant with dioxygen consumption as supported by dioxygen consumption. GSH involves the toxin and ferrous ions under air in a redox cycling process resulting in the production of glutathionyl and oxygen free radicals, observed for the first time with an iron complex of a mushroom toxin. In most cases, EDTA is not able to prevent the Fe(III)Or₃ and radical formation. The ortho-dihydroxylated groups borne by the di-N-oxidized bipyridine structure and not the bipyridine structure itself, are responsible for the formation of a stable ferric complex at pH 7, as they are for the generation of an apparently stable ortho-semiquinone anion radical. These one-electron mechanisms may play a major role in some of the known toxic effects of orellanine.     

Carbon dioxide stimulates the production of thiyl, sulfinyl, and disulfide radical anion from thiol oxidation by peroxynitrite

Reaction of peroxynitrite with the biological ubiquitous CO(2) produces about 35% yields of two relatively strong one-electron oxidants, CO(3) and ( small middle dot)NO(2), but the remaining of peroxynitrite is isomerized to the innocuous nitrate. Partial oxidant deactivation may confound interpretation of the effects of HCO3-/CO(2) on the oxidation of targets that react with peroxynitrite by both one- and two-electron mechanisms. Thiols are example of such targets, and previous studies have reported that HCO3-/CO(2) partially inhibits GSH oxidation by peroxynitrite at pH 7.4. To differentiate the effects of HCO3-/CO(2) on two- and one-electron thiol oxidation, we monitored GSH, cysteine, and albumin oxidation by peroxynitrite at pH 5.4 and 7.4 by thiol disappearance, oxygen consumption, fast flow EPR, and EPR spin trapping. Our results demonstrate that HCO3-/CO(2) diverts thiol oxidation by peroxynitrite from two- to one-electron mechanisms particularly at neutral pH. At acid pH values, thiol oxidation to free radicals predominates even in the absence of HCO3-/CO(2). In addition to the previously characterized thiyl radicals (RS.), we also characterized radicals derived from them such as the corresponding sulfinyl (RSO.) and disulfide anion radical (RSSR.-) of both GSH and cysteine. Thiyl, RSO. and RSSR.- are reactive radicals that may contribute to the biodamaging and bioregulatory actions of peroxynitrite.     

Generation of free radicals from metronidazole and other nitroimidazoles by Tritrichomonas foetus

Metronidazole, ronidazole, secnidazole, benznidazole, and misonidazole are reduced by intact Tritrichomonas foetus cells to nitro anion radicals that can be detected by electron spin resonance spectroscopy. This activity appears to be related to the cellular content of reducing substrates, since nitro anion radical formation is stimulated in the presence of glucose and pyruvate. The nitro anion radicals could not be detected under aerobic conditions. Anaerobic homogenates of T. foetus also reduce metronidazole to the nitro anion radical when pyruvate, NADH, or NADPH is added as the ultimate source of reducing equivalents. Free radical formation may be the basic cause of nitroimidazole toxicity in trichomonads.     

The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates.

Electron spin resonance spectra are observed during the enzymatic reduction of many nitrophenyl derivatives by rat hepatic microsomes or mitochondria. The spectra indicate that nitroaromatic anion radicals are present and are freely rotating in aqueous solution at a steady-state concentration of 0.1-6 muM. The rate of formation of p-nitrobenzoate (NBZO) dianion radical in microsomal incubates is consistent with the radical being an obligate intermediate in the reduction of NBZO to p-aminobenzoic acid. A model system consisting of NBZO, NADPH, and FMN, but no heme-containing compounds, also reduced NBZO to the NBZO dianion free radical. The steady-state concentration of the anion radicals in microsomal systems is not altered by CO. This observation, together with the results from the model system, suggests that the formation of nitroaromatic anion radicals is mediated through a flavine and not cytochrome P-450. The oxidation of the anion radical intermediate by O2 to the parent nitro compound is proposed to account for the well-known O2 inhibition of microsomal nitroreductase.     

Evaluation of Superoxide Scavenging Activities of Hamamelis Extract and Hamamelitannin

Hamamelitannin, which is a component of bark extract of hamamelis (Hamamelis virginior L.), was found to be a potent scavenger of superoxide anion radicals. Superoxide anion scavenging activity of the compound was evaluated by ESR-spin trap method using DMPO (5,5'-dimethyl-1-pyrroline-N-oxide) as a spin trapping agent. The IC₅₀ value (the concentration producing 50% inhibition of superoxide anion radicals) of hamamelitannin was found to be 1.38 ± 0.06 μM much lower than that of ascorbic acid (23.31 ± 2.23 μM). Supporting the superoxide scavenging activity of hamamelitannin, the compound showed both suppresive ability against depolymelization of hyaluronic acid and protective ability against cytotoxicity induced by superoxide anion radicals. Hamamelitannin increased the survival rate of fibroblast to 85.5 ± 3.3%, compared with that of control (27.2 ± 4.3%).