Effects of Phenolic Compounds on Reactions Involving Various Organic Radicals

Investigation of effects produced by 26 various phenol and diphenol derivatives, including industrial and natural antioxidants (ionol, bis-phenol 2246, α-tocopherol), on final product yields of radiation-induced free-radical processes involving peroxyl, alkyl, α-hydroxyalkyl and α,β-dihydroxyalkyl radicals has been performed. Ionol and bis-phenol 2246 have been shown to be more effective than α-tocopherol or diphenol derivatives in suppressing hydrocarbon oxidation processes. At the same time, α-tocopherol and its water-soluble analogues, as well as diphenol-based substances, are more effective than phenol derivatives in regulating various homolytic processes involving carbon-centered radicals. This fact can be accounted for by taking into consideration the contribution to formation of the final product set and the respective yields made by semiquinone radicals and compounds with quinoid structure arising in the course of homolytic transformations in systems containing diphenol derivatives.     

Hydroperoxysterols as a probe for the mechanism of cytochrome P-450scc-mediated hydroxylation. Homolytic versus heterolytic oxygen-oxygen bond scission

The interaction of 20-, 23-, and 25-hydroperoxy derivatives of cholesterol with various heme proteins, including the cholesterol side-chain-cleaving enzyme, cytochrome P-450scc, was studied by means of product and spectral analyses. Quasi-Fenton homolytic decomposition via intermediate alkoxy radicals appears to prevail during nonspecific interaction. Highly stereospecific hydroperoxide-driven hydroxylations suggest the absence of free radical species and are interpreted as resulting from a heterolytic type of peroxide decomposition or, alternatively, homolytic decomposition assuming proximal base effect to stabilize a putative intermediate alkoxy radical. Spectral aberrations during the early stages of the latter interaction indicate formation of a ternary iron-peroxo-substrate complex. Decomposition of this complex results in multiple product formation suggesting that peroxide cleavage reverts to regular homolytic decomposition upon denaturation of the enzyme. The implications of these observations for the mechanism of hydroxylation and oxidative carbon-carbon bond scission during enzymic side-chain cleavage of cholesterol are discussed.     

Effects of alpha-tocopherol and related compounds on reactions involving various organic radicals

Effects of alpha-tocopherol, PMC, and a number of the respective sulfur-containing analogues on reactions involving various organic radicals were studied. The test compounds were found to interact with alkyl radicals more effectively than with peroxyl radicals. The presence of a sulfur atom in structures of the respective analogues did not produce significant effects on reactivity. Derivatives of 5-hydroxy-1,3-benzoxathiol-2-one and 6-hydroxy-1,4-benzoxathiin-2(3H)-one displayed a high reactivity toward alpha-hydroxyalkyl radicals.     

Stopped-flow investigation of antioxidant activity of tocopherols. Finding of new tocopherol derivatives having higher antioxidant activity than alpha-tocopherol

Second-order rate constants, kappa s, for H-atom abstraction by phenoxyl radicals from five tocopherol (vitamin E) derivatives have been measured spectrophotometrically at 25.0 degrees C by the stopped-flow method, as a model reaction of tocopherols with unstable free radicals (LOO., LO., and HO.) in biological systems. Three new tocopherol derivatives with a five-membered heterocyclic ring were found to be 1.9-2.1 times more active than the alpha-tocopherol which has the highest antioxidant activity among natural tocopherols. The proton hyperfine splittings for the five tocopheroxyl radicals derived from these tocopherols by the reaction with phenoxyl were also determined by ESR measurements.     

Radical-radical reactions of superoxide: a potential route to toxicity

Superoxide reacts with many radicals, such as phenoxyl radicals, at near diffusion-controlled rates. These reactions are usually considered to be repair processes and have received little biological attention. However, addition of superoxide to give hydroperoxides and secondary oxidation products can also occur. The relative contributions of addition and repair vary depending on the properties of the phenol. With tyrosine, addition to give tyrosine hydroperoxide predominates, but in peptides the efficiency of hydroperoxide formation depends on the proximity of free amine groups. Radicals from other phenolic compounds, such as alpha-tocopherol and serotonin, also undergo addition reactions with superoxide. Physiologically, these reactions are likely to be more significant than dimerization when both radicals are generated together. They warrant attention as potential contributors to superoxide toxicity.     

Antioxidant activities of estrogens against aqueous and lipophilic radicals; differences between phenol and catechol estrogens

Natural estrogens have much greater radical-scavenging antioxidant activity than has previously been demonstrated, with activities up to 2.5 times those of vitamin C and vitamin E. The biological significance of this finding remains to be elucidated. In this work the antioxidant activity of a range of estrogens (phenolic, catecholic and stilbene-derived) has been studied. The activity of these substances as hydrogen-donating scavengers of free radicals in an aqueous solution has been determined by monitoring their relative abilities to quench the chromogenic radical cation 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS*+). The results show that the order of reactivity in scavenging this radical in the aqueous phase is dependent on the precise estrogenic structure, with phenolic estrogens being more potent antioxidants than catecholestrogens or diethylstilbestrol. The ability of the same estrogens to scavenge lipid phase radicals has also been assessed, determined by the ex vivo enhancement of the resistance of low-density lipoprotein (LDL) to oxidation; the order of efficacy is different from that in the aqueous phase, with the phenolic estrogens estriol, estrone and 17beta-estradiol being less potent than 2-hydroxyestradiol, 4-hydroxyestradiol, or diethylstilbestrol. In this lipid-based system, phenolic estrogens were found to be unable to regenerate alpha-tocopherol from LDL subjected to oxidative stress, while at the same time 2- and 4-hydroxyestradiol significantly delayed alpha-tocopherol loss. These results indicate that the various estrogens are good scavengers of free radicals generated in both the aqueous and the lipophilic phases. The antioxidant activity of an estrogen depends not only on the hydrophilic or lipophilic nature of the scavenged radical, but also on the phenol and catechol structures of the estrogen compound.     

Critical aspects of the antioxidant function of coenzyme Q in biomembranes.

Coenzyme Q (ubiquinone, UQ) is increasingly considered as a significant natural antioxidant, which protects biomembranes in concert with alpha-tocopherol. In vitro experiments demonstrated that reduced UQ (ubiquinol) can improve the chain-breaking activities of alpha-tocopherol by recycling the antioxidant-derived reaction product, the chromanoxyl radical, to the native antioxidant. Less attention, however, was devoted to the antioxidant-derived reaction products of reduced UQ. Although both alpha-tocopherol and ubiquinol were found to be equally effective in scavenging chain-propagating lipid radicals. alpha-tocopherol protected lipid membranes from lipid peroxidation more efficiently than ubiquinol. The present study not only provides data which document this discrepancy but also contributes experimental data on the existence of ubiquinol derived pro-oxidants, which give an explanation of this phenomenon.     

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.     

Ferric bleomycin catalyzed reduction of 10-hydroperoxy-8,12-octadecadienoic acid: evidence for homolytic O-O bond scission

10-Hydroperoxy-8,12-octadecadienoic acid (1) is reduced by ferric bleomycin in aqueous and methanol solutions to yield 10-oxo-8-decenoic acid (2) as the major product (80-90%). Trace amounts of 10-oxo-8,12-octadecadienoic acid (3) (5-10%) and 10-hydroxy-8,12-octadecadienoic acid (4) (5-10%) were also detected. The reduction product ratios remained relatively constant in the presence or absence of the reducing substrate phenol, over the pH range 6.5-8.5, in incubations from 30 s to 1 h, and over a series of ferric drug concentrations. In the presence of phenol, incubations of ferric bleomycin and 1 yielded 2,2'-biphenol and 4,4'-biphenol as oxidation products. In reactions where phenol was replaced with the drug's biological substrate DNA, 1 was found to support ferric bleomycin mediated DNA degradation. Extracts from these assays also found 2 to be the major reduction product derived from the oxidant, with trace quantities of 3 and 4 present. Control experiments demonstrated the reactions to be dependent on both 1 and ferric bleomycin. The reduction products 2 and 3 have previously been shown to originate from transient alkoxyl radicals formed by homolysis of the peroxy O-O bond. Product 4 results from heterolysis of the peroxy O-O bond [Labeque, R., & Marnett, L. J. (1987) J. Am. Chem. Soc. 109, 2828-2829]. The results of this investigation indicate that ferric bleomycin catalyzes the homolytic cleavage of the O-O bond of 1 almost exclusively while supporting various oxidative reactions.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Reactions of arylamine and aminophenol derivatives, and riboflavin with organic radicals

Based on product yield data on radiolysis of hexane, ethanol and 3 M aqueous ethylene glycol solutions, the ability of a number of arylamine, aminophenol and quinonimine derivatives to affect processes involving peroxyl, alkyl or alpha-hydroxyalkyl radicals was assessed. It has been shown that the introduction of a hydroxyl group into aromatic amine structure enhances its antioxidant performance and makes it significantly more reactive with respect to carbon-centered organic radicals. Replacement of the hydrogen atom of a hydroxyl group by a methyl group decreases the anti-radical activity of aminophenols drastically. Compounds containing (or capable of forming) a quinonimine moiety interact with alkyl or alpha-hydroxyalkyl radicals most effectively, suppressing recombination and fragmentation reactions of the latter. In the sequence: aromatic amines--aminophenols--quinonimines, a trend towards enhancement of the ability of the compounds studied to react with carbon-centered radicals was noted. Also, this study presents for the first time evidence of riboflavin reactivity with respect to organic radicals.     

Inhibition of peroxyl radical-mediated lipid oxidation by plasmalogen phospholipids and alpha-tocopherol

The recently discovered peroxyl radical scavenging properties of plasmalogen phospholipids led us to evaluate their potential interactions with alpha-tocopherol. The oxidative decay of plasmalogen phospholipids and of polyunsaturated fatty acids as induced by peroxyl radicals (generated from 2,2'-azobis-2-amidinopropane hydrochloride; AAPH) was studied in micelles using 1H-NMR and chemical analyses. In comparison with alpha-tocopherol, a 20- to 25-fold higher concentration of plasmalogen phospholipids was needed to induce a similar inhibition of peroxyl radical-mediated oxidation of polyunsaturated fatty acids. Plasmalogen phospholipids and alpha-tocopherol protected each other from oxidative degradation. In low-density lipoproteins (LDL) and micelles supplemented with plasmalogen phospholipids plus alpha-tocopherol, the peroxyl radical-promoted oxidation was additively diminished. The differences in the capacities to inhibit oxidation processes induced by peroxyl radicals between the plasmalogen phospholipids and alpha-tocopherol were less pronounced in the LDL particles than in the micelles. In conclusion, plasmalogen phospholipids and alpha-tocopherol apparently compete for the interaction with the peroxyl radicals. Oxidation processes induced by peroxyl radicals are inhibited in an additive manner in the presence of the two radical scavengers. The contribution of the plasmalogen phospholipids to the protection against peroxyl radical promoted oxidation in vivo is expected to be at least as important as that of alpha-tocopherol.     

Reactions of Arylamine and Aminophenol Derivatives,and Riboflavin with Organic Radicals

Based on product yield data on radiolysis of hexane, ethanol and 3 M aqueous ethylene glycol solutions, the ability of a number of arylamine, aminophenol and quinonimine derivatives to affect processes involving peroxyl, alkyl or α-hydroxyalkyl radicals was assessed. It has been shown that the introduction of a hydroxyl group into aromatic amine structure enhances its antioxidant performance and makes it significantly more reactive with respect to carbon-centered organic radicals. Replacement of the hydrogen atom of a hydroxyl group by a methyl group decreases the anti-radical activity of aminophenols drastically. Compounds containing (or capable of forming) a quinonimine moiety interact with alkyl or α-hydroxyalkyl radicals most effectively, suppressing recombination and fragmentation reactions of the latter. In the sequence: aromatic amines--aminophenols--quinonimines, a trend towards enhancement of the ability of the compounds studied to react with carbon-centered radicals was noted. Also, this study presents for the first time evidence of riboflavin reactivity with respect to organic radicals.     

Cinnamate and cinnamate derivatives in plants

Cinnamic acid, an ubiquitous alpha beta unsaturated acid, upon hydroxylation yields p-hydroxy cinnamic acid or p-coumarate, a plant mono phenol. Being, precursor for the production of various di (lignans), polyphenols (lignins) and also substituted derivatives, it seems to be an important aromatic chemical in growth and development of plants. This aromatic chemical substance synthesized primarily by almost all forms of plants, seemingly involves in the regulation of various physiological processes. The presence of this ubiquitous plant alpha beta unsaturated acid and its derivatives have been adopted by plants for various mechanisms. An effort towards the consolidation of these is made here.     

The effects of ascorbic acid on homolytic processes involving alpha-hydroxyl-containing carbon-centered radicals

Effects of ascorbic acid and 5,6-O-isopropylidene-2,3-O-dimethylascorbic acid on final product formation in radiolysis of ethanol, aqueous solutions of ethanol, ethylene glycol, alpha-methylglycoside, maltose, alpha-glycerophosphate, and alpha-glucose phosphate were studied. It was found that ascorbic acid is able to suppress reactions involving various alpha-hydroxyl-containing carbon-centered radicals and depending on the experimental conditions can either oxidize or reduce alpha-hydroxyethyl radicals.     

Quinones as free-radical fragmentation inhibitors in biologically important molecules

Effects of a number of quinones and diphenols of various structures on free-radical fragmentation processes taking place in alpha-diols, glycerol, 2-aminoethanol, glycero-1-phosphate, ethylene glycol monobutyrate, maltose, and some lipids were investigated. Quinone additions have been found to change the direction of free-radical transformations of the compounds cited above by inhibiting formation of the respective fragmentation products owing to oxidation of radicals of the starting compounds. The results obtained and literature data available allow a suggestion to be made that the system quinone/diphenol is able to not only deactivate or generate such active species as O2.- but also control the realization probability of free-radical processes of peroxidation and fragmentation in biologically important molecules.     

Reaction of substituted pyrimidines with photochemically generated t-BuO* radicals

Humans are exposed to various organic peroxides through chemical, pharmaceutical and cosmetic products. On photolysis, these peroxides produce alkoxyl radicals and hydroxyl radicals. The reaction of *OH radicals with DNA and its constituents have been extensively studied, but very little is known about the reactions of alkoxyl radicals with DNA and its constituents. In view of this, the oxidation of pyrimidine bases viz., thymine, uracil, cytosine, 5-bromouracil, 6-methyluracil and 1,3-dimethyluracil by t-BuO* radicals in aqueous solution at pH 7.5 has been carried out. The reaction between pyrimidine and t-BuO* is followed by measuring the absorbance of pyrimidine at the respective lambdamax. The rates of oxidation of pyrimidines are calculated from the plot of absorbance vs time. The rates of oxidation of pyrimidines have been found to increase with increase in [t-BuOOH], [pyrimidine] and light intensity. The quantum yields are calculated from the initial rates of oxidation of pyrimidine and the measured light intensity at 254 nm the wavelength at which t-BuOOH is activated to give radicals. The quantum yields are found to depend on [pyrimidine] as well as on [t-BuOOH] while they are independent of light intensity. The product analysis was carried out on HPLC with UV-visible detector. The corresponding 5,6-dihydroxypyrimidine and isobarbituric acid have been identified by comparing the retention times of the authentic samples. On the basis of experimental results and product analysis, it is suggested that t-BuOOH on photolysis gives t-BuO* radical, which initiates the reaction by adding to C (5) or C (6) position of pyrimidine base, leading to the formation of pyrimidine base radical via hydrolysis. The pyrimidine radical further reacts with t-BuO* radical to give the final product. This study predicts the probable transient pyrimidine radicals.     

A Diphenol Oxidase Gene Is Part of a Cluster of Genes Involved in Catecholamine Metabolism and Sclerotization in Drosophila. I. Identification of the Biochemical Defect in Dox-A2 [l(2)37Bf] Mutants

Phenol oxidase, a complex enzyme, plays a major role in the processes of sclerotization and melanization of cuticle in insects. Several loci have been reported to affect levels of phenol oxidase activity, but to date only one structural locus has been identified [Dox-3F (2-53.1+)]. Recently isolated Dox-A2 mutations (2-53.9) are recessive, early larval lethals, which as heterozygotes reduce phenol oxidase activity. A homozygous mutant escaper had weak, completely unpigmented cuticle and unpigmented bristles. Enzyme assays show that Dox-A2 heterozygotes have diphenol oxidase activity reduced to 47-79% of wild type, whereas monophenol oxidase activity, at 94-106% of wild type, is normal. Elevated pool sizes of the diphenol oxidase substrates DOPA, dopamine, and N-acetyldopamine are observed in the mutant, confirming the enzyme assay results. Separation of the three phenol oxidase A component activities on polyacrylamide gels shows that Dox-A2 mutations reduce the activity of only the A2 component. Dox-A2 may identify a structural locus for the A2 component of the diphenol oxidase enzyme system. The Dox-A2 locus is one of 18 loci in the dopa decarboxylase, Df (2L)TW130 region of the second chromosome, at least 14 of which affect the formation, melanization or sclerotization of cuticle in some way. These loci form an apparent cluster of functionally related genes.     

Dynamics of antioxidant action of ubiquinol: a reappraisal.

The dynamics of action of ubiquinol as an antioxidant against lipid peroxidation was reinvestigated and compared with that of alpha-tocopherol. It was found that ubiquinol was 2.5 and 1.9 times more reactive than alpha-tocopherol toward phenoxyl and peroxyl radicals, respectively, at 25 degrees C in ethanol and that it was capable of donating two hydrogen atoms toward oxygen radicals but that the apparent stoichiometric number decreased in the inhibition of lipid peroxidation, to even smaller than 1, due to its autoxidation. The autoxidation of ubiquinol proceeded even in the micelles and liposomal membranes in aqueous dispersions as well as in organic homogeneous solution. The apparent antioxidant activity of ubiquinol was smaller than that of alpha-tocopherol against lipid peroxidation in organic solution as judged from either rate of oxidation or duration of inhibition period. They exerted similar antioxidant potency against lipid peroxidation in the membranes and micelles in aqueous dispersions. The combination of ubiquinol and alpha-tocopherol was suggested to be effective.     

Oxidation of C4-hydroxyphenyl 1,4-dihydropyridines in dimethylsulfoxide and its reactivity towards alkylperoxyl radicals in aqueous medium

This work reports the electrochemical oxidation of three newly synthesized C4-hydroxyphenyl-substituted 1,4-dihydropyridine derivatives in dimethylsulfoxide. The reactivity of the compounds with ABAP-derived alkylperoxyl radicals in aqueous buffer pH 7.4, was also studied. The oxidation mechanism involves the formation of the unstable dihydropyridyl radical, which was confirmed by controlled-potential electrolysis (CPE) and ESR experiments. The final product of the CPE, that is, pyridine derivative, was identified by GC-MS technique for the three derivatives. A direct reactivity of the synthesized compounds toward ABAP-derived alkylperoxyl radicals was found. The pyridine derivative was identified by GC-MS as the final product of the reaction. Results reveal that this type of 1,4-DHPs significantly reacts with the radicals, even compared with commercial 1,4-DHP drugs with a well-known antioxidant ability.