Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Increased levels of superoxide in brains from old female rats

Hypertension, aging and a range of neurodegenerative diseases are associated with increased oxidative damage. The present study examined whether superoxide (O₂^•-) levels in brain are increased during aging in female rats, and the role of superoxide dismutase (SOD) and oestrogen in regulating O₂^•- levels.

Young adult (3 month) and old (11 month) female spontaneously hypertensive stroke prone rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied. O₂^•- levels were measured in brain homogenates by lucigenin chemiluminescence and SOD expression by Western blotting. Ageing significantly increased brain O₂^•- levels in WKY (cortex +216%, hippocampus +320%, striatum +225%) and to a greater extent in SHRSP (cortex +540%, hippocampus +580%, striatum +533%). Older SHRSP showed a decline in cortical Cu/Zn SOD expression compared to young adult SHRSP. Oestrogen did not attenuate O₂^•- levels.

The results show a significant age-dependent increase in brain O₂^•- levels which is exaggerated in SHRSP. The excess cortical O₂^•- levels in the SHRSP may be associated with a down-regulation of Cu/Zn SOD but are not related to a decrease in oestrogen.     

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.     

Optimization of the enzymatic hydrolysis and analysis of plasma conjugated γ-CEHC and sulfated long-chain carboxychromanols, metabolites of vitamin E

Natural forms of vitamin E are metabolized by ω-hydroxylation and β-oxidation of the hydrophobic side chain to generate urinary-excreted 2-(β-carboxyethyl)-6-hydroxychroman (CEHC) and CEHC conjugates (sulfate, glucuronide, or glucoside). We recently showed that sulfated long-chain carboxychromanols, the conjugated intermediate β-oxidation products, are formed from tocopherols and tocotrienols in human cells and in rats. CEHC conjugates have been quantified after being converted to its unconjugated counterpart by sulfatase/glucuronidase. Although the enzymatic hydrolysis is critical for appropriate quantification of conjugated CEHC, it is not clear whether brief incubation of the plasma with sulfatases/glucuronidases results in complete deconjugation of conjugated CEHC. Here we show that quantitative hydrolysis of the conjugated vitamin E metabolites in the plasma requires an extraction procedure using methanol/hexane (2 ml/5 ml) and an overnight sulfatase/glucuronidase hydrolysis. Using this procedure, we demonstrate that conjugated γ-CEHC and some sulfated long-chain carboxychromanols are fully deconjugated. In contrast, direct enzymatic hydrolysis of the whole plasma underestimates the conjugated metabolites by at least threefold. This protocol may be also useful for the analysis of other conjugated phenolic compounds in complicated biological matrices such as plasma.     

Preeclampsia inactivates glucose-6-phosphate dehydrogenase and impairs the redox status of erythrocytes and fetal endothelial cells

Inactivation of glucose-6-phosphate dehydrogenase (G6PD) may contribute to vascular dysfunction in preeclampsia, and oxidative stress has been implicated in the pathogenesis of this disease. We have compared the susceptibility of erythrocytes and human umbilical vein endothelial cells (HUVEC) to oxidative stress in women with normotensive or preeclamptic pregnancies. The redox status of erythrocytes was also correlated with neutrophil-mediated superoxide (O₂⁻) production in women recruited to the “Vitamins in Preeclampsia” (VIP) trial. Erythrocytes and HUVEC from women with preeclampsia demonstrated impaired redox regulation and diminished response to glucose, detectable at 14–20 weeks gestation prior to onset of the clinical disease. Hexokinase and G6PD activities were decreased in erythrocytes and G6PD activity was decreased in HUVEC from preeclamptic pregnancies. Phorbol-ester-stimulated O₂⁻ was enhanced in preeclamptic neutrophils. Impaired redox regulation in erythrocytes and HUVEC in preeclampsia may be due to diminished hexokinase and G6PD activities resulting from increased release of reactive oxygen species from activated neutrophils. Our findings provide the first evidence that decreased G6PD activity in preeclampsia is associated with impaired redox regulation in erythrocytes and fetal endothelial cells. The deficiency in G6PD in preeclampsia potentially accounts for the lack of protection against oxidative stress afforded by antioxidant vitamin C/E supplementation in the VIP trial.     

Examination of the Inhibitory Effect of Norathyriol in Formylmethionyl-Leucyl-Phenylalanine-Induced Respiratory Burst in Rat Neutrophils

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O₂^˙−) generation and O₂ consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O₂^˙− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca²⁺]_i and the formation of inositol trisphosphate (IP₃) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O₂^˙−, however, the inhibition of O₂^˙− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.     

A new type of O(2)(-)-generating tool for oxidative stress studies by remodeling neutrophil NADPH oxidase

The effects of reactive oxygen species on cells have attracted much attention in relation to redox regulation and oxidative stress-related diseases. Superoxide (O₂⁻) is the reactive oxygen species primarily formed in biological systems. However, no convenient O₂⁻-generating device has been available for use in cell or tissue culture. The neutrophil NADPH oxidase, a professional enzyme for killing bacteria, has a high ability to produce O₂⁻. However, the cell-free activation process requires several protein factors and an anionic amphiphile, and moreover, the activation is transient. To utilize the enzyme as an O₂⁻ generator, we improved the cell-free activation method by remodeling regulatory components, optimizing lipid composition, and modifying the mixing conditions. We established a new method to produce an active enzyme that is stable, efficient, and preservable. As an application, we examined the effect of the device on cultured HEK293 cells and observed that it caused cell death. This system has several advantages over the xanthine oxidase system often used. The new device will be useful for studies of oxidative stress and related diseases.     

Direct measurement of oscillatory generation of superoxide anions by single phagocytes

Phagocytic cells such as neutrophils generate superoxide anions (O₂⁻) within phagocytic vacuoles for killing and digesting microorganisms. Here we report the simultaneous observation of morphological changes and O₂⁻ generation in single phagocytic cells during phagocytosis. Point stimulation of a cell by contact with an opsonized microelectrode at the cell surface induced significant deformation to engulf the electrode, and also induced the O₂⁻ generation which was measured by the electrode. Periodic fluctuations in the magnitude of the O₂⁻ generation were observed in the time course. These oscillations may be caused by metabolic regulation of the formation of NADPH, which is the substrate for the O₂⁻ generation.     

A new method for the analysis of urinary vitamin E metabolites and the tentative identification of a novel group of compounds

There is currently interest in measuring urinary metabolites of vitamin E. It has been suggested that alpha-to-copheronolactone (alphaTL), with an oxidized chroman ring, may be an indicator of in vivo oxidative stress and 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), with a shortened side chain but intact hydroxychroman ring, may provide a measure of adequate or excess vitamin E status. To date, methods in the literature have tended to concentrate on the estimation of single metabolites. We describe the establishment and validation of a relatively simple and reproducible method to extract and quantitate a range of vitamin E metabolites using 0.5 ml of human urine. The vitamin E metabolites were extracted from urine using solid phase extraction cartridges, deconjugated enzymatically, and analyzed using gas chromatography-mass spectrometry. Using this method we have identified alphaTL and the CEHC metabolites derived from alpha-, delta-, and gamma-tocopherol. In addition we have tentatively identified a novel group of vitamin E metabolites, which are related to the CEHCs but have three extra carbons in the side chain. The possibility of the artifactual oxidation of alpha-CEHC to alphaTL during the assay procedure is also discussed.     

Kinetics of the reactions of nitrogen dioxide with glutathione,cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO₂^•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO₂^• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO₂^• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO₂^• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO₂^• with glutathione, cysteine, and urate were estimated as 2 × 10⁷, 5 × 10⁷, and 2 × 10⁷ M⁻¹ s⁻¹, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO₂^• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10⁷ M⁻¹ s⁻¹. The implications of these values are: (i) the lifetime of NO₂^• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO₂^• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO₂^• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO₂^•; and (v) reactions between NO₂^• and thiols/urate severely limit the likelihood of reaction of NO₂^• with NO• to form N₂O₃ in the cytoplasm.     

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

Solid Phase Synthesis of Mitochondrial Triphenylphosphonium-Vitamin E Metabolite Using a Lysine Linker for Reversal of Oxidative Stress

Mitochondrial targeting of antioxidants has been an area of interest due to the mitochondria''s role in producing and metabolizing reactive oxygen species. Antioxidants, especially vitamin E (α-tocopherol), have been conjugated to lipophilic cations to increase their mitochondrial targeting. Synthetic vitamin E analogues have also been produced as an alternative to α-tocopherol. In this paper, we investigated the mitochondrial targeting of a vitamin E metabolite, 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC), which is similar in structure to vitamin E analogues. We report a fast and efficient method to conjugate the water-soluble metabolite, α-CEHC, to triphenylphosphonium cation via a lysine linker using solid phase synthesis. The efficacy of the final product (MitoCEHC) to lower oxidative stress was tested in bovine aortic endothelial cells. In addition the ability of MitoCEHC to target the mitochondria was examined in type 2 diabetes db/db mice. The results showed mitochondrial accumulation in vivo and oxidative stress decrease in vitro.     

Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats

The metabolism of vitamin E involves oxidation of the phytyl chain to generate the terminal metabolite 7,8-dimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (CEHC) via intermediate formation of 13'-hydroxychromanol and long-chain carboxychromanols. Conjugated (including sulfated) metabolites were reported previously but were limited to CEHCs. Here, using electrospray and inductively coupled plasma mass spectrometry, we discovered that gamma-tocopherol (gamma-T) and delta-T were metabolized to sulfated 9'-, 11'-, and 13'-carboxychromanol (9'S, 11'S, and 13'S) in human A549 cells. To further study the metabolites, we developed a HPLC assay with fluorescence detection that simultaneously analyzes sulfated and nonconjugated intermediate metabolites. Using this assay, we found that sulfated metabolites were converted to nonconjugated carboxychromanols by sulfatase digestion. In cultured cells, approximately 45% long-chain carboxychromanols from gamma-T but only 10% from delta-T were sulfated. Upon supplementation with gamma-T, rats had increased tissue levels of 9'S, 11'S, and 13'S, 13'-hydroxychromanol, 13'-carboxychromanol, and gamma-CEHC. The plasma concentrations of combined sulfated long-chain metabolites were comparable to or exceeded those of CEHCs and increased proportionally with the supplement dosages of gamma-T. Our study identifies sulfated long-chain carboxychromanols as novel vitamin E metabolites and provides evidence that sulfation may occur parallel with beta-oxidation. In addition, the HPLC fluorescence assay is a useful tool for the investigation of vitamin E metabolism.     

Effects of selenite on O2 consumption, glutathione oxidation and NADPH levels in isolated hepatocytes and the role of redox changes in selenite toxicity

Isolated hepatocytes incubated with selenite (30–100 μM) exhibited changes in the glutathione redox system as shown by an increase in O₂ consumption, oxidation of glutathione and loss of NADPH. Selenite (50 μM) raised O₂ consumption within the 1 h and induced an partial depletion of thiols with a concomitant increase in oxidized glutathione, as well as a decrease in NADPH levels within 2 h. With 100 μM selenite more pronounced effects were obtained such as a total depletion of thiols. This concentration of selenite also lysed cells within 3 h. Arsenite, HgCl₂ and KCN prevented the increase in O₂ uptake, counteracted loss of thiols and delayed selenite induced lysis. p-Tert-butylbenzoic acid, an inhibitor of gluconeogenesis, decreased selenite dependent O₂ consumption and potentiated the effect on NADPH levels as well as the toxic effect. Finally, methionine further enhanced O₂ consumption by selenite and also delayed loss of thiols and potentiated selenite toxicity. These results indicated that selenite catalyzed a reduction of O₂ in glutathione dependent redox cycles with NADPH as an electron donor. With subtoxic concentrations of selenite (50 μM) there were indications that O₂ reduction was terminated by selenite biotransformation to methylated metabolites. With toxic concentrations of selenite (100 μM) it appeared that O₂ reduction was eventually limited by the capacity of the cell to regenerate NADPH. It is suggested that a depletion of NADPH mediated the observed cytotoxicity of selenite.     

Clarification of the Relationship Between Free Radical Spin Trapping and Carbon Tetrachloride Metabolism in Microsomal Systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/^•CCl₃) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/^•CO₂⁻) or the glutathione (GSH) and CCl₄-dependent PBN radical adduct (PBN/[GSH-^•CCl₃]). Inclusion of PBN/^•CCl₃ in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ radical adducts. Microsomes alone or with GSH had no effect on the PBN/^•CCl₃ radical adduct. Addition of NADPH to a microsomal system containing PBN/^•CCl₃ presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/^•CCl₃. We conclude that PBN/^•CCl₃ is not metabolized into either PBN/[GSH-^•CCl₃] or PBN/^•CO₂⁻ in microsomal systems.     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding

The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O₂⁻) scavenging activity. The O₂⁻ scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O₂⁻ scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O₂⁻ flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O₂⁻ but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O₂⁻ dismutation capacity than the apo-protein, ranging from moderate (Zn²⁺-OTf and Fe²⁺-OTf) to high (Mn²⁺-OTf and Cu²⁺-OTf) activity with the Cu²⁺-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H₂O₂ production in the X/XOD reaction than the apo-OTf, providing evidence that Zn²⁺-, Mn²⁺- and Cu²⁺-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O₂⁻ scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug.     

Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity

The free radical scavenging properties and possible antioxidant activity of folic acid are reported. Pulse radiolysis technique is employed to study the one-electron oxidation of folic acid in homogeneous aqueous solution. The radicals used for this study are CCl₃O₂^•, N₃^•, SO₄^•−, Br₂^•−, √OH, and O^•−. All these radicals react with folic acid under ambient condition at an almost diffusion-controlled rate producing two types of transients. The first transient absorption maximum is around 430 nm, which decays, and a simultaneous growth at around 390 nm is observed. Considering the chemical structure of folic acid, the absorption maximum at 430 nm has been assigned to a phenoxyl radical. The latter one is proposed to be a delocalized molecular radical. A permanent product has been observed in the oxidation of folic acid with CCl₃O₂^• and N₃^• radicals, with a broad absorption band around 370–400 nm. The bimolecular rate constants for all the radical-induced oxidation reactions of folic acid have been measured. Folic acid is seen to scavenge these radicals very efficiently. In the reaction of thiyl radicals with folic acid, it has been observed that folic acid can not only scavenge thiyl radicals but can also repair these thiols at physiological pH. While carrying out the lipid peroxidation study, in spite of the fact that folic acid is considerably soluble in water, we observed a significant inhibition property in microsomal lipid peroxidation. A suitable mechanism for oxidation of folic acid and repair of thiyl radicals by folic acid has been proposed.     

Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2/CO2 studied by fast pulse radiolysis

The reduction of ferricytochrome c by O₂⁻ and CO₂⁻ was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O₂⁻ and correspondingly with O₂ of ferrocytochrome c) is by a factor of approx. 10³ slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O₂⁻/O₂ and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.