An Esr Study of Nonenzymatic Reactions of Nitroso Compounds with Biological Reducing Agents

The nonenzymatic reduction of nitrosobenzene (1), 2-nitroso-l-naphthol (II) and 2-nitroso-l-naphthol-4-sulfonic acid (III) with reducing agents such as NADPH, L-cysteine and N-acetyl-L-cysteine led to the formation of the corresponding hydronitroxide radicals, as confirmed with ESR spectroscopy. In addition to these radicals, a novel hydronitroxide radical, which was conjugated with GSH at the 4-position, was observed in the reaction of II or III with GSH. The formation of a hydronitroxide conjugated with GSH still retains the radical structure with its related redox chemistry. In this case, the formation of a GSH conjugate does not lead to the formation of chemically less reactive species.     

Biological Reduction of Aromatic Nitroso Compounds: Evidence for the Involvement of Superoxide Anions

The in vitro formation of phenylhydronitroxide and 2-methylphenylhydronitroxide free radicals from nitrosobenzene (NB) and 2-nitrosotoluene (NT), respectively, in either red blood cells (RBC) or RBC hemolysates, was confirmed by electron spin resonance spectroscopy (ESR). Free radicals were generated nonenzymatically from reaction of the respective nitroso compounds with a number of biological reducing agents as corroborated by model studies of NB or NT with NAD(P)H. Under aerobic conditions, phenylhydronitroxide and 2-methylphenylhydronitroxide underwent a subsequent one-electron transfer to oxygen, which then resulted in the formation of superoxide anion (O₂^-). The latter product was confirmed by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (cyt c). Apparently, oxygen is needed for continuous formation of the hydronitroxide radical derivatives. On the other hand, under anaerobic conditions, no phenylhydronitroxide radical was generated from NB in the presence of NADH, but the formation of phenylhydroxylamine from NB was detected by the absorption spectrometry. These results suggest that oxygen is a preferential electron acceptor for hydronitroxide radical derivatives.     

The mechanism of kinetic inhibition of Cu(II)-induced oxidation of low density lipoprotein by lanthanide ions

Oral administration of lanthanum chloride (LaCl(3)) was reported to inhibit atherosclerosis in experimental animals, but the mechanism was not clear. In the present work, the effects of La(III) and other lanthanide ions (Ln(III)) on Cu(II)-induced oxidation of isolated low-density lipoprotein (LDL) and the related mechanism were investigated. By monitoring the formation of conjugated dienes (CD), low concentrations of La(III), Gd(III) and Y(III) were found to inhibit Cu(II)-induced LDL oxidation kinetically, as characterized by the prolongation of the lag time, the decrease of the maximal accumulation of CD, and the maximal rate of CD accumulation. Using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin trapping agents, the electron spin resonance (ESR) results showed that La(III) and Gd(III) at low concentrations significantly decreased the level of free radicals, including alkoxyl radical (LO*), alkyl radical (L*), and a transient radical, alkylperoxyl radical (LOO*), generated during LDL oxidation induced by Cu(II). In addition, Fourier-transform infrared spectroscopy (FT-IR) study revealed that La(III) might cause the conformational change and the less aggregation of apolipoprotein B-100 (apoB) in LDL, as demonstrated by the decreasing contents of alpha-helix, intermolecular beta-sheet, unordered structure and beta-turns, and the increasing contents of intramolecular beta-sheet and beta-strands. The inhibitory effect of Ln(III) on Cu(II)-induced LDL oxidation was discussed on the basis of the decreased free radical level and the second structural changes of apoB in LDL.     

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

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

Hemoglobin: A mechanism for the generation of hydroxyl radicals

Oxyhemoglobin (HbO₂) reduces Fe(III) NTA aerobically to become methemoglobin (metHb) and Fe(II)NTA. These conditions are favorable for the generation via Fenton chemistry of the hydroxyl radical that was measured by HPLC using salicylate as a probe. The levels of hydroxyl radicals generated are a function of both the percent metHb formed and the chemical nature of the buffer. The rates of formation of both metHb and hydroxyl radicals were dependent upon the concentration of Fe(III)NTA. Of the buffers tested, HEPES was the most effective scavenger of hydroxyl radicals while the other buffers scavenged in the order: HEPES > Tris > MOPS > NaCl ≈ unbuffered. The addition of catalase to remove H₂0₂ or bathophenanthroline to chelate Fe(II) inhibited virtually all hydroxyl radical formation. Carbonyl formation from free radical oxidation of amino acids was found to be 0.1 mol/mol of hemoglobin. These experiments demonstrate the ability of hemoglobin to participate directly in the generation of hydroxyl radicals mediated by redox metals, and provide insight into potential oxidative damage from metals released into the blood during some pathologic disorders including iron overload.     

Chemical properties of water-soluble porphyrins. 4. The reaction of a 'picket-fence-like' iron (III) complex with the superoxide oxygen couple

Solution properties of the iron-(III) 'picket-fence-like' porphyrin, Fe(III)-alpha,alpha,alpha, beta-tetra-ortho (N-methyl-isonicotinamidophenyl) porphyrin, (Fe(III)PFP) were investigated. These were acid/base properties of the aquo complex with pKa of 3.9 and its aggregation (formation of dimer with K = 1 X 10(-10) dm3 mol-1), complex formation with cyanide ions and 1-methyl imidazole (1-MeIm), spectral properties of the three iron complexes in their ferric and ferrous form and the one-electron reduction potential of these complexes. Knowing these properties, the reaction of the ferric complexes, aquo, dicyano and bis (1-MeIm), with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the iron (III) aquo complex which governs the catalytic efficiency of the metalloporphyrin upon the disproportionation of the superoxide radical was 7.6 X 10(7) dm3 mol-1 s-1, two orders of magnitude faster when compared to the reaction of each of the other complexes. The reduction by other radicals with all iron (III) complexes had similar second-order rate constants (10(9) to 10(10) dm3 mol-1 s-1). The reduction reaction in all cases produced Fe(II)PEP and no intermediate was found. The oxidation reaction of Fe(II)PEP by O2- was one order of magnitude faster when compared to the reduction of Fe(III)PFP by the same radical. Since the reactivity of O2- toward the three iron (III) porphyrin complexes follows their reduction potentials, it is suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The reactions of the Fe(II)PFP complexes with dioxygen were also studied. The aquo complex was found to be first order in O2 and second order in Fe(II)PFP, suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The intermediate formation was corroborated by evidence of the rapid CO binding reaction to the aquo complex of Fe(II)PFP. The two other complexes reacted very slowly with O2 as well as with CO.     

Protein radical formation during lactoperoxidase-mediated oxidation of the suicide substrate glutathione: immunochemical detection of a lactoperoxidase radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct

A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H(2)O(2). The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.     

Glutathione propagates oxidative stress triggered by myeloperoxidase in HL-60 cells. Evidence for glutathionyl radical-induced peroxidation of phospholipids and cytotoxicity

Glutathione acts as a universal scavenger of free radicals at the expense of the formation of the glutathionyl radicals (GS*). Here we demonstrated that GS* radicals specifically interact with a reporter molecule, paramagnetic and non-fluorescent 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-Tempo), and convert it into a non-paramagnetic fluorescent product, identified as 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidine (Ac-piperidine). Horseradish peroxidase-, myeloperoxidase-, and cyclooxygenasecatalyzed oxidation of phenol in the presence of H2O2 and GSH caused the generation of phenoxyl radicals and GS* radicals, of which only the latter reacted with Ac-Tempo. Oxidation of several other phenolic compounds (e.g. etoposide and tyrosine) was accompanied by the formation of GS* radicals along with a characteristic fluorescence response from Ac-Tempo. In myeloperoxidase-rich HL-60 cells treated with H2O2 and phenol, fluorescence microscopic imaging of Ac-Tempo revealed the production of GS* radicals. A thiol-blocking reagent, N-ethylmaleimide, as well as myeloperoxidase inhibitors (succinyl acetone and azide), blocked formation of fluorescent acridine-piperidine. H2O2/phenolinduced peroxidation of major classes of phospholipids in HL-60 cells was completely inhibited by Ac-Tempo, indicating that GS* radicals were responsible for phospholipid peroxidation. Thus, GSH, commonly viewed as a universal free radical scavenger and major intracellular antioxidant, acts as a pro-oxidant during myeloperoxidase-catalyzed metabolism of phenol in HL-60 cells.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Detection of 2-deoxy-D-ribose radicals generated by the reaction with the hydroxyl radical using a rapid flow-ESR method

ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO*) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO* at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

OH radical formation by photolysis of aqueous porphyrin solutions. A spin trapping and e.s.r. study

Radical production during the photolysis of deaerated aqueous alkaline solutions (pH 11) of some water-soluble porphyrins was investigated. Metal-free and metallo complexes of tetrakis (4-N-methylpyridyl)porphyrin (TMPyP) and tetra (4-sulphonatophenyl)porphyrin (TPPS4) were studied. Evidence for the formation of OH radicals during photolysis at 615, 545, 435, 408 and 335 nm of Fe(III) TPPS4 is presented. Fe(III) TMPyP, Mn(III) TPPS4 and Mn(III) TMPyP also gave OH radicals but only during photolysis at 335 nm. The method of spin trapping with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 4-pyridyl-1-oxide-N-tert-butylnitrone (POBN) combined with e.s.r. was used for the detection of OH, H and hydrated electrons. With the spin trap DMPO, photolysis generated DMPO-OH adducts under certain conditions but no DMPO-H adducts could be observed. With POBN, no POBN-H adducts were found. The formation of OH was confirmed by studying competition reactions for OH between the spin traps and OH scavengers (formate, isopropanol) and the concomitant formation of the CO-2 adduct and the (CH3)2COH adduct with both DMPO and POBN. The photochemical generation of OH radicals was pH dependent; at pH 7.5 no OH radicals could be detected. Photolysis (615-335 nm) of dicyanocomplexes of the Fe(III) porphyrins did not produce OH radicals. When corresponding Cu(II), Ni(II), Zn(II) and metal-free porphyrins were photolysed at 615 and 335 nm, no OH radicals could be spin trapped. These results tend to associate the well-known phenomenon of photoreduction of Fe(III) and Mn(III) porphyrins with the formation of OH radicals. This process is described mainly as the photoreduction of the metal ion by the ligand-bound hydroxyl ion via an intramolecular process.     

Modulation of oxidative DNA damage and DNA-crosslink formation induced by cis-diammine-tetrachloro-platinum(IV) in the presence of endogenous reductants

Platinum(IV) [Pt(IV)] complex, satraplatin, is currently in clinical trials for the treatment of various cancers. As a key step of the anti-cancer effect exertion, satraplatin is supposed to be reduced by endogenous reductants to platinum(II) [Pt(II)] complex. In this study, we investigated the interaction of DNA, Pt(IV), and the endogenous reductants such as ascorbic acid (AsA) and glutathione (GSH). As a model Pt(IV) compound, cis-diammine-tetrachloro-Pt(IV) [cis-Pt(IV)], which is a prodrug of cisplatin [cis-diammine-dichloro-Pt(II), cis-Pt(II)], was incubated with calf thymus DNA in the presence of AsA or GSH. In the presence of AsA, cis-Pt(IV) induced oxidative DNA damage. Hydroxyl radical scavengers suppressed the AsA-associated oxidative damage, thereby suggesting that hydroxyl radicals are involved in the DNA oxidation. cis-Pt(II)-like CD spectral change and crosslink formation in calf thymus DNA were also observed during this DNA oxidation, suggesting cis-Pt(IV) reduction by AsA and DNA conformational change induced by the newly formed cis-Pt(II) binding to DNA. GSH did not induce oxidative DNA damage likely due to its own hydroxyl radical scavenging ability. Further, GSH suppressed the Pt(II)-mediated DNA conformational change and crosslink formation, suggesting that GSH sequesters the cis-Pt(II) away from DNA by GSH-cis-Pt(II) complex formation.     

Hydroxyl radical production during oxidative deposition of iron in ferritin

The chemistry of oxidative deposition of iron(III) in ferritin and apoferritin is poorly understood. This study was undertaken to look for radicals formed as the hydrous ferric oxide core is developed from Fe(II) and O2. Radicals were observed indirectly by using the spin-trapping reagent N-tert-butyl-alpha-phenylnitrone (PBN) at room temperature and directly by measuring ESR spectra of frozen solutions at 77 K. In both instances, radical production was inhibited by the hydroxyl radical scavenging agents dimethyl sulfoxide, thiourea, and mannitol and enhanced by the addition of hydrogen peroxide. These findings strongly suggest that hydroxyl radical, produced from the iron-catalyzed Haber-Weiss reaction, is a by-product of core formation in ferritin and is a precursor to the observed radicals. The yield of ESR-observable and spin-trapped radicals is quite low, being at the micromolar level when millimolar concentrations of ferrous ion are employed. Furthermore, radical production appears to be confined to the interior of the ferritin molecule, where cellular components would be protected from the oxygen-derived toxic effects of iron. It is postulated that hydroxyl radical-medicated oxidative damage to the protein, a process that may contribute to the formation of hemosiderin from ferritin, leads to the observed radicals. By serving as a sink for hydroxyl radical, the protein shell may therefore efficiently minimize damage to other biomolecules in the cell.     

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: An EPR spectroscopy study

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of ^.OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were a_N = 15.3 G, and a_H = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with ^.OH radicals were close to a diffusion-controlled rate (≈ 2 × 10¹⁰ M⁻¹s⁻¹). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H₂O₂, and CAP, or EPICAP, ^.OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no ^.OH or other Fenton oxidants were observed in the presence of H₂O₂. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of ^.OH radicals. The direct ^.OH radical scavenging, together with the reductive “repair” of other sites of ^.OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with ^.OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.     

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.     

Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells

We have compared the abilities of ascorbate and reduced glutathione (GSH) to act as intracellular free radical scavengers and protect cells against radical-mediated lipid peroxidation. Phenoxyl radicals were generated in HL60 cells, through the action of their myeloperoxidase, by adding H2O2 and phenol. Normally cultured cells, which contain no ascorbate; cells that had been preloaded with ascorbate; and those that had been depleted of GSH with buthionine sulfoximine were investigated. Generation of phenoxyl radicals resulted in the oxidation of ascorbate and GSH. Ascorbate loss was much greater in the absence of GSH, and adding glucose gave GSH-dependent protection against ascorbate loss. Ascorbate, or glucose metabolism, had little effect on the GSH loss. Glutathionyl radical formation was detected by spin trapping with DMPO in cells lacking ascorbate, and the signal was suppressed by ascorbate loading. Addition of phenol plus H2O2 to the cells caused lipid peroxidation, as measured with C11-BODIPY. Peroxidation was greatest in cells that lacked both ascorbate and GSH. Either scavenger alone gave substantial inhibition but optimal protection was seen with both present. These results indicate that GSH and ascorbate can each act as an intracellular radical scavenger and protect against lipid peroxidation. With both present, ascorbate is preferred and acts as the ultimate radical sink for phenoxyl or glutathionyl radicals. However, GSH is still consumed by metabolically recycling dehydroascorbate. Thus, recycling scavenging by ascorbate does not spare GSH, but it does enable the two antioxidants to provide more protection against lipid peroxidation than either alone.     

Reaction between protein radicals and other biomolecules

The present study investigates the reactivity of bovine serum albumin (BSA) radicals towards different biomolecules (urate, linoleic acid, and a polypeptide, poly(Glu-Ala-Tyr)). The BSA radical was formed at room temperature through a direct protein-to-protein radical transfer from H(2)O(2)-activated immobilized horseradish peroxidase (im-HRP). Subsequently, each of the three different biomolecules was separately added to the BSA radicals, after removal of im-HRP by centrifugation. Electron spin resonance (ESR) spectroscopy showed that all three biomolecules quenched the BSA radicals. Subsequent analysis showed a decrease in the concentration of urate upon reaction with the BSA radical, while the BSA radical in the presence of poly(Glu-Ala-Tyr) resulted in increased formation of the characteristic protein oxidation product, dityrosine. Reaction between the BSA radical and a linoleic acid oil-in-water emulsion resulted in additional formation of lipid hydroperoxides and conjugated dienes. The results clearly show that protein radicals have to be considered as dynamic species during oxidative processes in biological systems and that protein radicals should not be considered as end-products, but rather as reactive intermediates during oxidative processes in biological systems hereby supporting recent data.     

One-electron oxidation of Trolox C (a vitamin E analogue) by peroxidases

The oxidation mechanism of Trolox C (a vitamin E analogue) by peroxidases was examined by stopped flow and ESR techniques. The results revealed that during the oxidation of Trolox C, peroxidase Compound II was the catalytic intermediate. The rate constants for the reaction of Compound II with Trolox C, which should be the rate-determining step, were estimated to be 2.1 X 10(4) and 7.2 X 10(3) M-1.s-1 for horseradish peroxidase and lactoperoxidase, respectively, at pH 6.0. The formation of the Trolox C radical was followed by ESR. The time course of the signal was similar to that of the optical absorbance changes at 440 nm, assigned as the peak of the Trolox C radical. The signal exhibited a hyperfine structure characteristic of phenoxyl radicals. From an estimation of the radical concentration in the steady state and the velocity of the radical formation, the dismutation constant was calculated to be 5 X 10(5) M-1.s-1. The concentration of the signal in the steady state was reduced by the addition of GSH. The spectrum changed from that of the Trolox C radical to that of the ascorbate radical when the reaction was carried out in the presence of ascorbate.     

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.