The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions

Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.     

Formation of long-lived hydroxyl free radical adducts of proline and hydroxyproline in a Fenton reaction

Proline and hydroxyproline when exposed to the hydroxyl free radical generating system of ADP-Fe(II)-H2O2 yielded long-lived free radicals. An analysis of the electron paramagnetic resonance spectra of the long-lived hydroxyl free radical adducts of proline and hydroxyproline is consistent with a free electron on a nitroxyl group interacting with the nitrogen atom as well as with three separate protons. In the case of proline, nitroxide formation was observed under the influence of tert-butyl-hydroperoxide, giving a similar EPR spectrum (Lin, J.S., Tom, T.C. and Olcott, H.S. (1974) J. Agr. Food Chem. 22, 526-528); however, the hydroxyl free radical adduct of hydroxyproline has not been described yet. In the case of the proline nitroxide radical, two of the three protons involved interact with the free electron equivalently. The coupling constants for the hydroxyl free radical adduct of proline are AN = 1.58 mT, AH1 beta = AH2 beta = 2.13 mT, AH3 beta = 1.77 mT and for hydroxyproline are AN = 1.54 mT, AH1 beta = 2.56 mT, AH2 beta = 2.03 and AH3 beta = 1.51. The data are consistent with the amine nitrogen of proline and hydroxyproline being oxidized to a nitroxyl group and the free electron of the nitroxyl interacting with the beta-protons of these amino acid hydroxyl free radical adducts.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Quantitation of the hydroxyl radical by reaction with dimethyl sulfoxide

This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO.) in aqueous systems. Reaction of HO. with DMSO produces methane sulfinic acid as a primary product, which can be detected by a simple colorimetric assay. To evaluate this method for estimating total HO. production, we studied three model systems, including the Fenton reaction, gamma irradiation of water, and ultraviolet photolysis of hydrogen peroxide, for which the theoretical maximum yield of HO. could be calculated and compared to measured DMSO oxidation. The results confirm that 0.05 to 1 M DMSO may be used to capture nearly all of the expected HO. radicals formed. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO. formation in aqueous systems pretreated with DMSO.     

Enhanced hydroxyl radical production by dihydroxybenzene-driven Fenton reactions: implications for wood biodegradation

Brown rot fungi degrade wood, in initial stages, mainly through hydroxyl radicals (•OH) produced by Fenton reactions. These Fenton reactions can be promoted by dihydroxybenzenes (DHBs), which can chelate and reduce Fe(III), increasing the reactivity for different substrates. This mechanism allows the extensive degradation of carbohydrates and the oxidation of lignin during wood biodegradation by brown rot fungi. To understand the enhanced reactivity in these systems, kinetics experiments were carried out, measuring •OH formation by the spin-trapping technique of electron paramagnetic resonance spectroscopy. As models of the fungal DHBs, 1,2-dihydroxybenzene (catechol), 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid were utilized as well as 1,2-dihydroxy-3,5-benzenedisulfonate as a non-Fe(III)-reducing substance for comparison. Higher amounts and maintained concentrations of •OH were observed in the driven Fenton reactions versus the unmodified Fenton process. A linear correlation between the logarithms of complex stability constants and the •OH production was observed, suggesting participation of such complexes in the radical production.     

Evidence against transition metal-independent hydroxyl radical generation by xanthine oxidase

It is shown by the use of EPR spectroscopy that formation of the hydroxyl radical adduct with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the xanthine-xanthine oxidase system is hydrogen peroxide-independent. Production of the DMPO-hydroxyl radical adduct is inhibited by superoxide dismutase but is unaffected by purified grades of catalase. Hydroxyl radicals are a secondary product of the decomposition of the DMPO-superoxide radical adduct and are also formed as a result of trace metals such as iron present in the buffer. These results are in contrast with a recent report (Kuppusamy, P., and Zweier, J. W. (1989) J. Biol. Chem. 264, 9880-9884) in which the assertion is made that the hydroxyl radical adduct arises from the trapping of hydroxyl radicals generated via the direct reduction of hydrogen peroxide by xanthine oxidase. It is demonstrated here that treatment of phosphate buffer with the chelator deferoxamine mesylate is not in itself sufficient to suppress the effect of contaminating adventitious metal ions in xanthine-xanthine oxidase incubations.     

Primaquine can mediate hydroxyl radical generation by Trypanosoma cruzi extracts

Primaquine increases the NAD(P)H dependent oxygen consumption and hydroxyl radical generation by extracts of Trypanosoma cruzi, the causative agent of Chagas' disease. Spin-trapping studies show that hydroxyl radical yield is completely inhibited by catalase and slightly increased by SOD, indicating that radical generation is dependent on the pair primaquine-NAD(P)H and their interaction product, H2O2. Primaquine effects upon Trypanosoma cruzi extracts are compared with those obtained with nifurtimox, a compound effective in the treatment of Chagas' disease. The observed similarity suggests that hydroxyl radical may be involved in the antiprotozoan activity of primaquine.     

Quinolinic acid-iron(ii) complexes: slow autoxidation, but enhanced hydroxyl radical production in the Fenton reaction

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti³⁺/hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.     

Noninvasive detection of hydroxyl radical generation in lung by diesel exhaust particles

Diesel exhaust particles (DEP) induce pulmonary tumors, asthma-like symptoms, and the like in experimental animals. The involvement of reactive oxygen species (ROS) is suggested in the injuries induced by DEP, though the generation of ROS has not been proven. The present study provided the first direct evidence of *OH generation in the lungs of living mice after intratracheal instillation of DEP, using noninvasive L-band ESR spectroscopy and a membrane-impermeable nitroxyl probe. *OH generation is confirmed with the enhancement of in vivo ESR signal decay rate of the probe. The decay rate at mid-thorax was significantly enhanced in DEP-treated mice compared to that in vehicle-treated mice. The enhancement was completely suppressed by the administration of either *OH scavengers, catalase, or desferrioxamine, while the administration of SOD further increased the rate. The administration of Fenton's reagents into the lung also enhanced the decay rate of the probe at mid-thorax of mice. These results clearly provided evidence that the intratracheal exposure to DEP in mice produced *OH in the lung through an iron-catalyzed reaction of superoxide/H(2)O(2). This first direct evidence of *OH generation in DEP-treated mice lung may be utilized to determine treatments for DEP-induced lung injury.     

Further insights into the reaction of melatonin with hydroxyl radical

Recent interest has focused on the use of exogenous melatonin as an antioxidant, particularly to scavenge the highly cytotoxic hydroxyl radical (HO(z.rad;)) which may be generated in many pathological conditions. However, in vitro and in vivo studies aimed at assessing the antioxidant properties of melatonin have produced conflicting results. While it is known that HO(z.rad;) reacts with melatonin at a diffusion limited rate, very little is known about the products of this reaction. In this investigation it is shown that incubation of melatonin with a Fenton-type HO(z.rad;)-generating system at pH 7.4 forms a complex mixture of primary products. These include 2-hydroxymelatonin, which was isolated as its more stable oxindole tautomer, 4- and 6-hydroxymelatonin, N-acetyl-N(2)-formyl-5-methoxykynurenine and 7,7(')-bi-(5-methoxy-N-acetyltryptamine-4-one). Reaction pathways that might lead to these products are described. The differing biological effects of these products, while currently incompletely understood, might account for the controversy concerning the antioxidant properties of melatonin.     

Detection of hydroxyl radicals by D-phenylalanine hydroxylation: a specific assay for hydroxyl radical generation in biological systems

Hydroxylation of l-phenylalanine (Phe) by hydroxyl radical (*OH) yields 4-, 3-, and 2-hydroxyl-Phe (para-, meta-, and ortho-tyrosine, respectively). Phe derivative measurements have been employed to detect *OH formation in cells and tissues, however, the specificity of this assay is limited since Phe derivatives also arise from intracellular Phe hydroxylase. d-Phe, the d-type enantiomer, is not hydroxylated by Phe hydroxylase. We evaluate whether d-Phe reacts with *OH as well as l-Phe, providing a more reliable probe for *OH generation in biological systems. With *OH generated by a Fenton reaction or xanthine oxidase, d- and l-Phe equally gave rise to p, m, o-tyr and this could be prevented by *OH scavengers. Resting human neutrophils (PMNs) markedly converted l-Phe to p-tyr, through non-oxidant-mediated reactions, whereas d-Phe was unaffected. In contrast, when PMNs were stimulated in the presence of redox cycling iron the *OH formed resulted in more significant rise of p-tyr from d-Phe (9.4-fold) than l-Phe (3.6-fold) due to the significant background formation of p-tyr from l-Phe. Together, these data indicated that d- and l-Phe were equally hydroxylated by *OH. Using d-Phe instead of l-Phe can eliminate the formation of Phe derivatives from Phe hydroxylase and achieve more specific, sensitive measurement of *OH in biological systems.     

The centennial of the Fenton reaction

A short account is given of Fenton's life and research, with special emphasis on the Fenton reactions.     

Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide

To understand chemical characteristics of the asbestos minerals which might contribute to tissue damage, the catalytic properties of three different varieties were studied. Using spin trapping techniques it was determined that crocidolite, chrysotile, and amosite asbestos were all able to catalyze the generation of toxic hydroxyl radicals from a normal byproduct of tissue metabolism, hydrogen peroxide. The iron chelator desferroxamine inhibits this reaction, indicating a major role for iron in the catalytic process, and suggesting a possible mechanism by which asbestos toxicity might be reduced.     

Scavenging of hydroxyl radical by catecholamines

The direct effects of the four catecholamines (CATs), adrenaline (A), noradrenaline (NA), dopamine (D) and isoproterenol (I), on free radicals were investigated using the free radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) and hydroxyl radial (HO^?). The CATs examined were found to inhibit the ESR signal intensity of DPPH^? in a dose‐dependent manner over the range 0.1–2.5 mmol/L in the following order: NA > A > I > D, with IC₅₀ = 0.30 ± 0.03 for noradrenaline and IC₅₀ = 0.86 ± 0.02 for dopamine. Hydroxyl radicals were produced using a Fenton reaction in the presence of the spin trap 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO), and ESR technique was applied to detect the CATs reactivity toward the radicals. The reaction rates constant (k_r) of CATs with HO^? were found to be in the order of 10⁹ L/mol/s, and the k_r value for noradrenaline was the highest (k_r = 8.4 × 10⁹ L/mol/s). The CATs examined exhibited also a strong decrease in the light emission (62–73% at 1 mmol/L concentration and 79–89% at 2 mmol/L concentration) from a Fenton‐like reaction. These reactions may be relevant to the biological action of these important polyphenolic compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

The reaction of methionine with hydroxyl radical: reactive intermediates and methanethiol production

The mechanisms of reaction of methionine with hydroxyl radical are not fully understood. Here, we unequivocally show using electron paramagnetic resonance spin-trapping spectroscopy and GC-FID and GC-MS, the presence of specific carbon-, nitrogen- and sulfur-centered radicals as intermediates of this reaction, as well as the liberation of methanethiol as a gaseous end product. Taking into account the many roles that methionine has in eco- and biosystems, our results may elucidate redox chemistry of this amino acid and processes that methionine is involved in.     

Egg yolk phosvitin inhibits hydroxyl radical formation from the fenton reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical ((.-)OH) formation from H(2)O(2) in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited (.-)OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the (.-)OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H(2)O(2). These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.