Quantitative elucidation of the molecular mechanisms of hydroxyl radical quenching reactivity of phenolic compounds

Reported discrepancies have confused the understanding of the molecular mechanisms of antioxidant reactivity somewhat. The consequent problems necessitate systematic investigations on the molecular orbital features of antioxidants and their correlation with antioxidant potentials. In the present work, phenolic compounds as typical antioxidants were selected to investigate their hydroxyl radical-scavenging properties, and the related mechanisms of action were studied theoretically by computational chemistry. A good correlation was observed between antioxidant activity and theoretical parameters, such as O-H bond dissociation energy (BDE), ionization potential (IP), enthalpy of electron transfer (E(a)), chemical hardness (HOMO-LUMO gap), and spin delocalization of the phenoxyl radicals (D(s)(r)). The results demonstrate that the molecular mechanisms regulating the antioxidant action were more complex than hydrogen or electron-transfer processes and explain previous contradictions. Meanwhile, a satisfactory quantitative structure-activity relationship (QSAR) model was established which should be of predictive value in evaluating or screening hydroxyl radical-scavenging antioxidants.     

Phenolic chain-breaking antioxidants--their activity and mechanisms of action

This tutorial review is focused on some mechanistic aspects of peroxidation process and chemistry of phenolic chain-breaking antioxidants. Lipids are susceptible to oxidative degradation caused by radicals and during autoxidation (peroxidation) the chain reaction is mediated by peroxyl radicals leading to damage of integrity and the protective and organizational properties of biomembranes. Phenolic antioxidants provide active system of defence against lipid peroxidation, however, the effectiveness of their antioxidant action depends on several important parameters. Stoichiometry of the reaction with free radicals, fate of a phenoxyl radical, polarity of the microenvironment, localization of antioxidant molecules, their concentration and mobility, kinetic solvent effects, and interactions with other co-antioxidants are considered. Principal mechanisms of reaction between phenols and free radicals (Hydrogen Atom Transfer, Proton Coupled Electron Transfer and two mechanisms based on separate electron transfer and proton transfer steps) are described.     

Methemoglobin Formation from Butylated Hydroxyanisole and Oxyhemoglobin. Comparison with Butylated Hydroxytoluene and P-Hydroxyanisole

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyI-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H₂O₂ adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H₂O₂-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of the phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.     

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane,by ascorbate/dihydrolipoate in living HL-60 cells

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.     

Vitamin E analogue Trolox C. E.s.r. and pulse-radiolysis studies of free-radical reactions.

The reactions between Trolox C, a water-soluble vitamin E analogue, and several oxidizing free radicals including the hydroxyl radical and various peroxy radicals were examined by using the pulse-radiolysis technique. The results demonstrate that Trolox C may undergo rapid one-electron-transfer reactions as well as hydrogen-transfer processes; the resulting phenoxyl radical is shown to be relatively stable, in common with the phenoxyl radical derived from vitamin E. The reactions between the Trolox C phenoxyl radical and a variety of biologically relevant reducing compounds were examined by using both pulse radiolysis and e.s.r. The results demonstrate that the Trolox C phenoxyl radical is readily repaired by ascorbate (k = 8.3 x 10(6) dm3.mol-1.s-1) and certain thiols (k less than 10(5) dm3.mol-1.s-1) but not by urate, NADH or propyl gallate. Evidence from e.s.r. studies indicates that thiol-containing compounds may also enter into similar repair reactions with the alpha-tocopherol phenoxyl radical. Kinetic evidence is presented that suggests that Trolox C may 'repair' proteins that have been oxidized by free radicals.     

Conversion of Nitroxide Radicals by Phenolic and Thiol Antioxidants

Nitrone/nitroso spin traps are often used for detection of unstable hydroxyl radical giving stable nitroxide radicals with characteristic electron spin resonance (ESR) signals. This technique may be useful only when the nitroxide radicals are kept stable in the reaction system. The aim of the present study is to clarify whether the nitroxide radicals are kept stable in the presence of the hydroxyl radical scavengers. Effect of hydroxyl radical scavengers on the ESR signals of nitroxide radicals, 2,2,6,6-tetramethyI-piperi-dine-N-oxyl (TEMPO) and the spin adduct (DMPO-OH) of 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and hydroxyl radical, was examined. Although the ESR signals of TEMPO and the DMPO-OH spin adduct were unchanged on treatment with ethanol and dimethyl sulfoxide, their intensities were effectively decreased on treatment with 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid (Trolox), cysteine, glutathione, 2-mercaptoethanol and metallothionein. Hence, the results of the detection of hydroxyl radical in the presence of phenolic and thiol antioxidants by the ESR technique using nitrone/nitroso spin traps may be unreliable.     

Improvement of the method to estimate the relative reaction rate constants of hydroxyl radical with polyphenols using ESR spin trap: X-ray irradiation of water with a flowing system

UV-photolysis of hydrogen peroxide is a useful technique to produce hydroxyl radical. However, it is not an appropriate method to estimate the reactivity of polyphenols with hydroxyl radicals because many of the polyphenol derivatives also absorb the UV-light to generate hydroxyl radicals. In this study, X-ray irradiation of water with a flowing system was applied to estimate the reactivity of hydroxyl radicals with polyphenols using electron spin resonance (ESR) spin trap. The obtained relative reaction rates reasonably agreed with previous data by pulse radiolysis. This method will be a useful technique to estimate the reactivity of antioxidants including polyphenols with hydroxyl radicals.     

Hierarchy of ozone scavenging reactions in the plant cell wall

To estimate protection of the plasmalemma against ozone by the cell apoplast, the decomposition network of ozone in the mesophyll cell wall is analysed in consideration of data on published bimolecular reaction rate constants and concentrations of the reactants involved. The effect of dimerization of ascorbate free radicals (AFR) on the stoichiometric ratio of ozone reduction by ascorbate is quantified over the range of cell wall acidity, pH = 5.0‐6.5. As the disproportionation of AFR decreases sharply towards higher pH, the flow of AFR through dimerization is low over the pH range 5.5‐6.5, allowing abstraction of the second electron from AFR and formation of dehydroascorbate with a nearly 1:1 stoichiometric ratio in relation to ozone. The direct reaction between ozone and ascorbate (AA) in cell walls 0.3‐0.5 µm thick and at an AA concentration of 0.5 m M is able to detoxify 50‐70% of the O₃ that impinges on the wall surface. Generation of singlet oxygen and the hydroxyl radical, which are more reactive to AA than O₃, decreases markedly the O₃ flow to the plasmalemma. The question is raised whether cell wall alkalinization under ozone may hasten the decomposition of the pollutant due to the more rapid generation of hydroxyl radical by phenolic compounds.     

Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

A Photochemical System for Generating Free Radicals: Superoxide, Phenoxyl, Ferryl and Methyl

The photosensitizer flavin mononucleotide (FMN), in conjunction with the reducing agents diethylenetria-minepentaacetic acid (DTPA), hydrazine and hydroxylamines derived from nitroxides, generates superoxide radicals in a strictly light-dependent reaction in aerobic solution. Addition of superoxide dismutase (SOD) converts this system to a hydrogen peroxide generator. In the presence of horseradish peroxidase the latter system becomes a phenoxyl radical generator with appropriate phenolic substrates. Under anaerobic conditions FMN, hydrogen peroxide and an iron chelate generate ferryl and when this system is combined with dimethylsulfoxide, methyl radicals are produced. All the radicals can be generated with little contamination from other radicals, in high yields and the reaction can be terminated immediately upon cessation of illumination. Useful applications of this photochemical system include ESR studies of transient free radical species.     

Scavenging of reactive oxygen species by the plant phenols genistein and oleuropein.

The plant-derived phenolic compounds genistein and oleuropein are known to exhibit several biological properties, many of which may result from their antioxidant and free radical scavenger activity. In this paper we report the results of a complex study of antioxidant activity of genistein and oleuropein, using electron spin resonance (ESR), chemiluminescence, fluorescence and spectrophotometric techniques. Different reaction systems were applied to study the inhibitory effect of the phenolic compounds studied: (a) the potassium superoxide/18-crown-6 dissolved in DMSO system, which generates superoxide radical (O(2).(-)) and hydrogen peroxide (H(2)O(2)); (b) the Co(II)-EDTA-H(2)O(2) system (the Fenton-like reaction), which generates hydroxyl radical (HO.); (c) 2,2'-azobis(2-amidino-propane)dichloride (AAPH) as the peroxyl radical (ROO.) generator, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical test. Results showed that genistein and oleuropein decreased the chemiluminescence sum from the O(2).(-) generating system, an inhibitory effect that was dependent on their concentration. These compounds also reacted with ROO radicals and they showed activity about two-fold greater than the standard Trolox. The antioxidant effects were studied at different concentrations and reflected in protection against the fluorescence decay of beta-phycoerythrin (beta-PE), due to ROO. attack on this protein. Using the Fenton-like reaction and the spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the phenolic compounds examined were found to inhibit DMPO-.OH radical formation in the range 10-90% at concentrations of 0.1 mmol/L to 2 mmol/L. Furthermore, these compounds also inhibited HO.-dependent deoxyribose degradation; about 20% and 60% inhibitions were observed in the presence of 0.5 mmol/L genistein and oleuropein, respectively. It was also demonstrated that genistein had a weaker DPPH radical scavenging activity than oleuropein. Our results confirm good scavenging activity towards O(2).(-), HO. and ROO. and the antioxidant effect of genistein and oleuropein.     

Structural effects on the reactivity 1,4-dihydropyridines with alkylperoxyl radicals and ABTS radical cation

A series of eight commercial C-4 substituted 1,4-dihydropyridines and other synthesized related compounds were tested for direct potential scavenger effect towards alkylperoxyl radicals and ABTS radical cation in aqueous Britton-Robinson buffer pH7.4. A direct quenching radical species was established. The tested 1,4-dihydropyridines were 8.3-fold more reactive towards alkylperoxyl radicals than ABTS cation radical, expressed by their corresponding kinetic rate constants. Furthermore, NPD a photolyte of nifedipine and the C-4 unsubstituted 1,4-DHP were the most reactive derivatives towards alkylperoxyl radicals. The pyridine derivative was confirmed by GC/MS technique as the final product of reaction. In consequence, the reduction of alkylperoxyl and ABTS radicals by 1,4-dihydropyridines involved an electron transfer process. Also, the participation of the hydrogen of the 1-position appears as relevant on the reactivity. Results of reactivity were compared with Trolox.     

Scavenging effects of phenolic compounds on reactive oxygen species

Phenolic compounds are widely present in plants and they have received considerable attention due to their antioxidant property. In this article we report the results of a study of the reactivity of 10 selected phenolics (sesamol, three phenolic acids, three flavonols, one flavone, and two flavanones) with superoxide anion radical (O(2) (*)), hydroxyl radical (HO(*)) and singlet oxygen ((1)O(2)). The following generators of reactive oxygen species were used: 18-crown-6/KO(2)/dimethylsulfoxide (DMSO) or hypoxanthine/xanthine oxidase as sources of O(2) (*), the Fenton reaction carried out in a sodium trifluoroacetate (pH 6.15) for HO(*), and a mixture of alkaline aqueous H(2)O(2) and cobalt ions for (1)O(2). We have employed chemiluminescence, electron spin resonance spin trapping, and spectrophotometry techniques to examine an antioxidative property. All tested compounds acted as scavengers of various reactive oxygen species. The reactivity indexes (beta) for the reaction of the phenolic compounds with HO(*) were calculated.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6 ± 1 × 10(7) M(-1) s(-1) at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7 ± 3 × 10(7) M(-1) s(-1) at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK(a) of phenolic hydroxyl dissociation in tyrosine is ≈ 10.3, this infers a much lower rate constant, about 3 × 10(5) M(-1) s(-1), for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Fast repair of hydroxy radical purine deoxynucleotide adducts by phenylpropanoid glycosides and their derivatives from Chinese herbs

DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very significant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells' inadequate repair capacity. The repair activities and reaction mechanism of phenylpropanoid glycosides (PPGs) and their derivatives, isolated from Chinese folk medicinal herbs, towards both dGMP-OH* adducts and dAMP-OH* adducts were studied with the pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mM dGMP or dAMP aqueous solution containing one of the PPGs or their derivatives, the transient absorption spectra of the hydroxyl adduct of dGMP or dAMP decayed with the formation of that of phenoxyl radicals of PPGs or their derivatives within several decades of microseconds after electron pulse irradiation. The result indicated that dGMP or dAMP hydroxyl adducts can be repaired by PPGs or their derivatives. The rate constants of the repair reactions were deduced to be 0.641-1.28 x 10(9) M(-1) s(-1) for dGMP-OH* and 0.2-0.491 x 10(9) M(-1) s(-1) for dAMP-OH*, which positively correlated to the number of phenolic hydroxyl groups in the glycoside structure. A deeper understanding of this new repair mechanism may help researchers to design strategies to prevent and/or intervene more effectively in free radical related diseases.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

Kinetic Study of the Inhibition of Linoleic Acid Oxidation in Aqueous Media by Phenolic Compounds

The antioxidant activity of several phenolic compounds has been evaluated in terms of the inhibition of the lipid oxidation. The extent of linoleic acid oxidation was monitored by the absorption of the conjugated diene hydroperoxydes formed as a result of oxidation. The antioxidant activity of phenolic compounds was evaluated in aqueous media consisting of negatively-charged micelles of sodium dodecyl sulfate (SDS) or positively-charged micelles of hexadecyl trimethylammonium bromide (HDTBr). 2,2′-azobis-(2-amidinopropane)-dihydrochloride (ABAP) was employed as the oxidation initiator. The assayed phenolic compounds showed good antioxidant efficiency, closely related to the presence of hydroxyl groups and the electron delocalization within the structure, which may stabilize the formed phenoxyl radicals. The kinetic analysis revealed that the oxidizability of linoleic acid was 10-fold higher in SDS than in HDTBr media, which could indicate that oxidation is favoured in negatively charged SDS micelles regarding to positive HDTBr medium. Furthermore, a greater antioxidant efficiency of phenolic compounds was found in HDTBr than in SDS micelles.