Jun-ichi Ueda

Kinetic study on ESR signal decay of nitroxyl radicals, potent redox probes for in vivo ESR spectroscopy, caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.

Production of hydroxyl radicals by copper-containing metallothionein: roles as prooxidant.

Production of hydroxyl radicals by copper (Cu)-containing metallothionein (MT) and its relation to zinc (Zn) bound to MT were studied in vitro with reference to the mechanism of the Cu toxicity in the liver of LEC rats. Zn-MT prepared from the liver of Zn-injected rats was reacted with cupric ions at various Cu/Zn ratios, and the concentrations of the two metals bound to MT and in the solution, valence states of Cu in the solution, production of hydroxyl radicals were determined. Cupric ions replaced Zn in MT after being reduced by thiol groups, and MT, worked as an antioxidant. Cupric ions added to MT that did not contain Zn were reduced to cuprous ions by thiol groups in Cu-MT, and the Cu bound to MT was liberated in a form of cuprous ions. Hydroxyl radicals were produced in the presence of hydrogen peroxide in proportion to the amount of cuprous ions liberated from MT. Cu-containing MT was proposed to work as a prooxidant until all thiol groups in MT were oxidized when Zn was not present in MT. The results indicate that MT works as an antioxidant as long as Zn is present in Cu-containing MT, while it works as a prooxidant when Zn is not present by liberating 1.5 M equivalents of cuprous ions relative to cupric ions added, and hydroxyl radicals are produced in the presence of hydrogen peroxide. On the other hand, MT not bound by Cu does not work as a prooxidant throughout.

Efficient radical scavenging ability of artepillin C, a major component of Brazilian propolis, and the mechanism

Hydrogen transfer from artepillin C to cumylperoxyl radical proceeds via one-step hydrogen atom transfer rather than via electron transfer, the rate constant of which is comparable to that of (+)-catechin, indicating that artepillin C can act as an efficient antioxidant.

Reaction of para-hydroxybenzoic acid esters with singlet oxygen in the presence of glutathione produces glutathione conjugates of hydroquinone, potent inducers of oxidative stress.

The determination and toxicological characterization of products of the reaction between p-hydroxybenzoic acid esters (parabens) and singlet oxygen (1O2) are very important because of the frequent use of parabens in cosmetics and possible generation of 1O2 in the skin. We observed 1O2-dependent production of mono-, di-, and tri-substituted glutathione (GSH) conjugates of hydroquinone (HQ) during visible light-irradiation of a mixture of methyl or ethyl paraben and GSH in the presence of rose bengal (RB). 1,4-Benzoquinone (BQ) and HQ were produced during the irradiation in the absence of GSH. While a mixture of BQ and GSH produced only mono-substituted conjugate, irradiation of the mixture with RB produced mono-, di-, and tri-substituted conjugates. These observations indicate that 1O2 is involved both in the production of BQ and HQ from parabens and in the formation of multi-substituted GSH conjugates from mono-substituted conjugate. Tri-substituted conjugate generated larger amounts of hydrogen peroxide in an aqueous solution than mono-substituted conjugates or HQ did. Detection of semiquinone radical suggests that the autoxidation of conjugates is related to the generation of hydrogen peroxide. The results obtained in this study indicate that parabens may induce oxidative stress in the skin after conversion to GSH conjugates of HQ by reacting with 1O2 and GSH.

Singlet Oxygen–mediated Hydroxyl Radical Production in the Presence of Phenols: Whether DMPO–·OH Formation Really Indicates Production of ·OH?¶

The reaction of singlet oxygen (1O2) generated by ultraviolet‐A (UVA)–visible light (λ > 330 nm) irradiation of air‐saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO–hydroxyl radical spin adduct (DMPO–·OH). In contrast, the ESR signal of 5,5‐dimethyl‐2‐pyrrolidone‐N‐oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO–·OH decreased in the presence of either a ·OH scavenger or a quencher of 1O2 and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both 1O2‐ and DMPO‐mediated formation of free ·OH during the reaction. When DMPO was replaced with 5‐(diethoxyphosphoryl)‐5‐methyl‐1‐pyrroline‐N‐oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that 1O2, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron‐withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO–·OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron‐donating properties of phenolic compounds contribute to the appearance of ·OH. These observations indicate that 1O2 reacts first with DMPO, and the resulting DMPO–1O2 intermediate is immediately decomposed/reduced to give ·OH. Phenolic compounds would participate in this reaction as electron donors but would not contribute to the direct conversion of 1O2 to ·OH. Furthermore, DEPMPO did not cause the spin‐trapping agent–mediated generation of ·OH like DMPO did.

Oxidation of spin-traps by chlorine dioxide (ClO2) radical in aqueous solutions: first ESR evidence of formation of new nitroxide radicals.

The reactivities of the chlorine dioxide (ClO2), which is a stable free radical towards some water-soluble spin-traps were investigated in aqueous solutions by an electron spin resonance (ESR) spectroscopy. The ClO2 radical was generated from the redox reaction of Ti3+ with potassium chlorate (KClO3) in aqueous solutions. When one of the spin-traps, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was included in the Ti3+-KClO3 reaction system, ESR spectrum due to the ClO2 radical completely disappeared and a new ESR spectrum [aN(1) = 0.72 mT, aH(2) = 0.41 mT], which is different from that of DMPO-ClO2 adduct, was observed. The ESR parameters of this new ESR signal was identical to those of 5,5-dimethylpyrrolidone-(2)-oxyl-(1) (DMPOX), suggesting the radical species giving the new ESR spectrum is assignable to DMPOX. The similar ESR spectrum consisting of a triplet [aN(1) = 0.69 mT] was observed when the derivative of DMPO, 3,3,5,5-tetramethyl-1-pyrroline N-oxide (M4PO) was included in the Ti3+-KClO3 reaction system. This radical species is attributed to the oxidation product of M4PO, 3,3,5,5-tetramethylpyrrolidone-(2)-oxyl-(1) (M4POX). When another nitrone spin-trap, alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) was used as a spin-trap, the ESR signal intensity due to the ClO2 radical decreased and a new ESR signal consisting of a triplet [aN(1) = 0.76 mT] was observed. The similar ESR spectrum was observed when N-t-butyl-alpha- nitrone (PBN) was used as a spin-trap. This ESR parameter [a(N)(1) = 0.85 mT] was identical to the oxidation product of PBN, PBNX. Thus, the new ESR signal observed from POBN may be assigned to the oxidation product of POBN, POBNX. These results suggest that the ClO2, radical does not form the stable spin adducts with nitrone spin-traps, but oxidizes these spin-traps to give the corresponding nitroxyl radicals. On the other hand, nitroso spin-traps, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS), and 2-methyl-2-nitrosopropane (MNP) did not trap the ClO2 radical. This result indicates that an unpaired electron of the ClO2 radical is localized on oxygen atom, because nitroso spin-traps cannot form the stable spin adduct with oxygen-centered radical.

Activation of hydrogen peroxide by copper(II) complexes with some histidine-containing peptides and their SOD-like activities

The reactivities of copper(II) complexes with histidine-containing oligopeptides towards active oxygen species such as hydrogen peroxide (H2O2) and superoxide ion (O2-) were investigated by electron spin resonance (ESR)-spin trapping and thiobarbituric acid (TBA) methods. At physiological pH values, Cu(II) complexes with oligopeptides containing histidyl residue at the N-terminal could more easily activate H2O2 to yield hydroxyl radical (.OH) than other Cu(II)-oligopeptide complexes containing histidyl residue in the second or third position. Further, it was suggested that since all Cu(II)-oligopeptide complexes examined could scavenge O2-, these complexes have SOD-like activities.

Reactions of copper(II)-oligopeptide complexes with hydrogen peroxide: Effects of biological reductants

Cu(II) complexes with oligopeptides containing histidyl residue in the second or third position could scarcely activate hydrogen peroxide (H2O2), but they could activate H2O2 to yield hydroxyl radical (.OH) in the presence of biological reductants such as cysteine and ascorbic acid. Further, DNA single strand breakage was also observed during the reactions of Cu(II)-glycylglycylhistidylglycine (GGHG) with H2O2 in the presence of same biological reductants.

Copper(II) complexes of l-histidylglycyl-l-histidylglycine and l-histidyl-l-histidylglycylglycine: Coordination mode of histidyl residues

Abstract The complex formations of L-histidylglycyl-L- histidylglycine (His-Gly-His-Gly) and L-histidyl-L- histidylglycylglycine (His-His-Gly-Gly) with copper- (II) ion were studied in a slightly alkaline medium by visible absorption, circular dichroism and electron spin resonance spectroscopies. His-Gly-His-Gly coordinates to copper(II) ion via a terminal amino nitrogen, two deprotonated peptide nitrogens and an imidazole nitrogen of the histidyl residue in the third position. The copper(II) ion in the His-His- Gly-Gly complex is coordinated by three nitrogen donors; i.e. , a terminal amino nitrogen, an adjacent deprotonated peptide nitrogen and an imidazole nitrogen of the histidyl residue in the second position. The imidazole nitrogen at the N-terminal does not participate in the chelate formation with the copper(II) ion, but it bridges between the two monomeric complexes so that a broad ESR spectrum without the hyperfine structure is observed.

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants.

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (1O2) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of 1O2 with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from 1O2-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from 1O2, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.