Cytochrome P450 and steroid hydroxylase activity in mouse olfactory and vomeronasal mucosa

Neopterin and 7,8-dihydroneopterin, two compounds which are secreted by activated macrophages, have been shown to interfere with radicals generated by cellular and certain chemical systems. Reduced pterins were reported to scavenge whereas aromatic pterins promoted or reduced radical mediated reactions or had no effect. However, recently it was found that high concentrations of 7, 8-dihydroneopterin enhanced luminol dependent chemiluminescence and T-cell apoptosis, suggesting an enhancement of free radical formation. In this study hydroxylation of salicylic acid was used for detection of hydroxyl radicals. It is shown that in solutions of 7,8-dihydroneopterin hydroxyl radicals were formed in the absence of any radical source. The presence of EDTA chelated iron enhanced hydroxyl radical formation. Whereas the addition of iron accelerated the hydroxylation reaction, 7,8-dihydroneopterin was responsible for the amount of hydroxylation products. In the presence of superoxide dismutase or catalase, as well as by helium purging, hydroxylation was inhibited. Our data suggest that in solutions of 7, 8-dihydroneopterin superoxide radicals are generated which are converted to hydroxyl radicals by Fenton or Haber-Weiss type reactions. While superoxide might be generated during autoxidation of ferrous iron, dihydroneopterin seems to be involved in regeneration of ferrous iron from the ferric form.     

Leukotriene B4, C4, D4 and E4 inactivation by hydroxyl radicals

Leukotriene B4 chemotactic activity and leukotriene C4, D4 and E4 slow reacting substance activity were rapidly decreased by hydroxyl radicals generated by two different iron-supplemented acetaldehyde-xanthine oxidase systems. At low Fe2+, leukotriene inactivation was inhibited by catalase, superoxide dismutase, mannitol and ethanol, suggesting involvement of hydroxyl radicals generated by the iron-catalyzed interaction of superoxide and H2O2 (Haber-Weiss reaction). Leukotriene inactivation increased at high Fe2+ concentrations, but was no longer inhibitable by superoxide dismutase, suggesting that inactivation resulted from a direct interaction between H2O2 and Fe2+ to form hydroxyl radicals (Fenton reaction). The inactivation of leukotrienes by hydroxyl radicals suggests that oxygen metabolites generated by phagocytes may play a role in modulating leukotriene activity.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

The metal-mediated formation of hydroxyl radical by aqueous extracts of cigarette tar

Aqueous extracts of cigarette tar produce hydroxyl radicals that are spin trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The addition of catalase almost completely inhibits and superoxide dismutase partially inhibits spin adduct formation. The addition of ethylenediamine tetraacetic acid greatly increases the amount of hydroxyl radical adduct observed; in contrast, diethylenetriamine pentaacetic acid causes complete inhibition of spin adduct formation. We suggest that the hydroxyl radical arises from the metal-mediated decomposition of hydrogen peroxide, and that hydrogen peroxide is formed from the reduction of dioxygen by the semiquinones present in the cigarette tar.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver

The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.     

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).     

Xanthine oxidase induced depolymerization of hyaluronic acid in the presence of ferritin

Oxygen free radicals generated by xanthine oxidase are able to depolymerize hyaluronic acid in the presence of ferritin-bound iron. This suggests that ferritin can catalyse the Haber-Weiss reaction, leading to the formation of highly damaging hydroxyl radicals.     

Effect of antioxidants on chemiluminescence produced by reactive oxygen species

Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.     

The involvement of biological quinones in the formation of hydroxyl radicals via the Haber-Weiss reaction

The present investigation was made to evaluate biologically relevant quinones as possible catalysts in the generation of hydroxyl radicals from hydrogen peroxide and superoxide radicals. ESR spectra demonstrated that menadione, plastoquinone, and ubiquinone derivatives could all be reduced to their semiquinone forms by electron transfer from superoxide radicals. Reductive homolytic cleavage of H₂O₂ was observed to be dependent upon the presence of semiquinones in the reaction medium. Our data strongly support the concept that quinones normally involved in physiological processes may also play a role as catalysts of the Haber-Weiss reaction in the biological generation of hydroxyl radicals.     

Hydroxyl radical formation in chondrocytes and cartilage as detected by electron paramagnetic resonance spectroscopy using spin trapping reagents

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as α-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.     

Enhanced production of hydroxyl radicals by the xanthine-xanthine oxidase reaction in the presence of lactoferrin

The generation of hydroxyl radicals by the xanthine-xanthine oxidase reaction (C. Beauchamp and I. Fridovich (1970) J. Biol. Chem. 245, 4641-1616) has been shown to be increased by iron-saturated lactoferrin isolated from pig neutrophils. Hydroxyl radical production, measured by EPR spin trapping and by ethylene production from alpha-keto-gamma-methiol butyric acid, has been demonstrated to be produced by a Fenton-type Haber-Weiss reaction catalysed by lactoferrin. The possibility that lactoferrin catalyses such a reaction in vivo is considered.     

Inhibition of peroxidase-catalyzed reactions by deferoxamine

Phagocytes generate superoxide (O2-.) and hydrogen peroxide (H2O2) and their interaction in an iron-catalyzed reaction to form hydroxyl radicals (OH.) (Haber-Weiss reaction) has been proposed. Deferoxamine chelates iron in a catalytically inactive form, and thus inhibition by deferoxamine has been employed as evidence for the involvement of OH. generated by the Haber-Weiss reaction. We report here that deferoxamine also inhibits reactions catalyzed by the peroxidases of phagocytes, i.e., myeloperoxidase (MPO) and eosinophil peroxidase (EPO). The reactions inhibited include iodination in the presence and absence of chloride and the oxidation of guaiacol. Iodination by MPO and H2O2 is stimulated by chloride due to the intermediate formation of hypochlorous acid (HOCl). Iodination by reagent HOCl also is inhibited by deferoxamine with the associated consumption of HOCl. Iron saturation of deferoxamine significantly decreased but did not abolish its inhibitory effect on iodination by MPO + H2O2 or HOCl. Deferoxamine did not affect the absorption spectrum of MPO, suggesting that it does not react with or remove the heme iron. The conversion of MPO to Compound II by H2O2 was not seen when H2O2 was added to MPO in the presence of deferoxamine, suggesting either that deferoxamine inhibited the formation of Compound II by acting as an electron donor for MPO Compound I or that deferoxamine immediately reduced the Compound II formed. Iodination by stimulated neutrophils also was inhibited by deferoxamine, suggesting an effect on peroxidase-catalyzed reactions in intact cells. Thus deferoxamine has multiple effects on the formation and activity of phagocyte-derived oxidants and therefore its inhibitory effect on oxidant-dependent damage needs to be interpreted with caution.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation

The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.     

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.     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Characteristics of luminol chemiluminescence induced by the catalytic action of myeloperoxidase

The effects of pH, luminol myeloperoxidase and hydrogen peroxide concentrations on the intensity of luminol chemiluminescence induced by myeloperoxidase catalysis were investigated. It was found that the intensity of luminescence is proportional to the enzyme concentration (up to 8.10(-8) M) and reaches the saturation level at higher enzyme concentrations. The dependence of chemiluminescence intensity on [H2O2] is bell-shaped: at H2O2 concentrations above 1.10(-4) M the luminescence is inhibited with a maximum at neutral values of pH. Luminol at concentrations above 5.10(-5) M inhibits this process. It was demonstrated that the effects of singlet oxygen, superoxide and hydroxyl radicals on the chemiluminescence reaction are insignificant. Luminol oxidation in the course of the myeloperoxidase reaction is induced by hypochlorite.     

The Haber-Weiss cycle--70 years later

The chain reactions HO* + H2O2 --> H2O + O2*- + H+ and O2*- + H+ + H2O2 --> O2 + HO* + H2O, commonly known as the Haber-Weiss cycle, were first mentioned by Haber and Willst?tter in 1931. George showed in 1947 that the second reaction is insignificant in comparison to the fast dismutation of superoxide, and this finding appears to have been accepted by Weiss in 1949. In 1970, the Haber-Weiss reaction was revived by Beauchamp and Fridovich to explain the toxicity of superoxide. During the 1970s various groups determined that the rate constant for this reaction is of the order of 1 M(-1) s(-1) or less, which confirmed George's conclusion. The reaction of superoxide with hydrogen peroxide was dropped from the scheme of oxygen toxicity, and superoxide became the source of hydrogen peroxide, which yields hydroxyl radicals via the Fenton reaction, Fe2+ + H2O2 --> Fe3+ + HO- + HO*. In 1994, Kahn and Kasha resurrected the Haber-Weiss reaction again, but this time the oxygen was believed to be in the singlet (1delta(g)) state. As toxicity arises not from a Fenton-catalysed Haber-Weiss reaction, but from the Fenton reaction, the Haber-Weiss reaction should not be mentioned anymore.     

The production of hydroxyl radicals by adriamycin in red blood cells

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.