The harmful effects of oxidative processes in living organisms, in addition to chemical and biochemical media, can be reduced by antioxidants. The efficacy of an antioxidant depends on its reduction potential and kinetics of elimination of diverse free radicals. Redox potentials and reaction rate constants of selected gallocatechins and flavonoids were measured by pulse radiolysis and laser photolysis. The reduction potentials of the flavonoids studied were in the range of 0.33 V (quercetin) and 0.75 V (kaempferol). The rate constants of the superoxide radical with 15 flavonoids ranged from 10(5)-10(7) M-1 s-1. Singlet oxygen quenching by flavonoids was also very rapid (from 10(5) to 10(8) M-1 s-1). These studies may be crucial in optimizing health and increasing longevity by reducing oxidative stress and biological damage.

Antioxidant mechanisms of curcumin, bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, have been studied by laser flash photolysis and pulse radiolysis. The keto-enol-enolate equilibrium of the heptadienone moiety of curcumin determines its physicochemical and antioxidant properties. In neutral and acidic aqueous solutions (from pH 3 to 7), the keto form dominates, and curcumin acts as an extraordinarily potent H-atom donor. The reaction rate constant with the methyl radical (3.5 +/- 0.3) x 10(9) M-1 s(-1) is close to diffusion control in 40% aqueous DMSO at pH 5. The tert-butoxyl radical reacts with curcumin in acetonitrile solutions at a diffusion controlled rate, k = (7.5 +/- 0.8) x 10(9) M-1 s(-1). The apparent site of reaction is the central CH2 group in the heptadienone link, which has two labile hydrogens. This is supported by comparing the reaction patterns of curcumin and dehydrozingerone (DHZ) ("half-curcumin", 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one). DHZ does not react With the methyl radical, indicating that the presence of the labile hydrogens is crucial for the H-atom donating ability of curcumin. The tert-butoxyl radical reacts with DHZ at almost an order of magnitude lower rate (1.1 +/- 0.1) x 10(9) M-1 s(-1), clearly abstracting an H-atom from the phenolic OH group. The reaction mechanism of curcumin changes dramatically above pH 8, where the enolate form of the heptadienone link predominates. As a consequence, the reaction of the methyl radical diminishes completely in alkaline media, and the phenolic part of the molecule takes over as (electron donor) reaction site. The electron donating ability of curcumin is assessed from the measurements of one-electron-transfer equilibria of DHZ radicals. Reduction potential of the DHZ phenoxyl radical, E(pH = 6.5) = 0.83 +/- 0.06 V, and E(pH = 13.0) = 0.47 +/- 0.06 V vs NHE, which may be expected for an ortho-methoxy-substituted phenoxyl radical, indicate only moderate electron-donating ability. The importance of H-atom donation vs electron donation in free radical scavenging and antioxidant mechanisms of curcumin is discussed.

Iron(III) complexes of gallocatechins were studied in aqueous Solutions by UV-VIS spectrometry, HPLC, cyclic voltammetry, laser photolysis and pulse radiolysis techniques. The blue-violet colored complexes are readily formed in water. The Job plots indicate 1:1 stoichiometry for the reaction of iron(III) with gallocatechins and methyl gallate, and 1:3 for that of iron(III) and catechin. This suggests that the three phenol groups of the gallate moiety play a role in complex formation. The formation constants of the complexes are found to be pH dependent, as expected for polyhydroxybenzene derivatives. pK(a1) = 4.3 and pK(a2) = 7.4 for the polyphenols with the gallate ester moiety (epigallocatechin gallate and epicatechin gallate) are lower than those of epigallocatechin (EGC) and catechin (pK(a1) = 4.9 and pK(a2) = 8.4), very probably because of the electron-withdrawing effect of the ester. Apparent stability constants of iron(III) gallocatechin complexes are high at pH 7, log K approximate to 27, comparable to those of the catechol derivatives. Photoionization of the iron complexes by the 248 nm laser is more efficient at higher pH, phi = 0.13 at pH 7 vs. phi = 0.26 at pH 11.5. The absorption spectra, which resemble those of ligand phenoxyl radicals, indicate that photoionization yields unstable phenoxyls, t(1/2) similar to 1 ms. Similar spectra are recorded when one-electron oxidation by the azide radical, N-3(.), is used to generate the ligand radicals. The reduction potential of Fe(III)gallocatechins is -0.325 V vs. NHE, which is similar to 0.45 V less negative than the reduction potential of the Fe(II)/Fe(III) couple. In the case of the catechins with the gallate ester moiety,namely EGCG and ECG,the high pH cyclic voltammograms exhibit a quasi-reversible oxidation-reduction not seen in the other derivatives. The relevance of these findings for the physiological function and antioxidant and chemopreventive action of gallocatechins Is discussed.

Spectral, acid-base, and redox properties of theaflavin radicals were studied by pulse radiolysis in aqueous solutions. Theaflavin radicals are generated by the azide radical one-electron oxidation of theaflavin, theaflavin gallates A and B, and theaflavin digallate. Being relatively strong transient oxidant, N-3 . oxidizes more than one phenolic site in the complex polyphenols. The resulting mixture of phenoxyl radicals transforms via an intramolecular electron transfer to the hydroxycycloheptenone radical, with apparently lowest reduction potential. The neutral hydroxycycloheptenone radical is more aromatic than the parent compound, which reflects in high rate constant of the formation of the radical. The rate of the reaction of theaflavin with the superoxide radical at pH 7, k = 1 x 10(7) M-1 s(-1), is an order of magnitude higher than that with epigallocatechin gallate (EGCG), k = 7.3 x 10(5) M-1 s(-1), in spite of higher reduction potential of the theaflavin radicals (E-7 = 0.53 V vs E-7 = 0.44 V for EGCG). Even the substitution in the adjacent benzene ring has relatively small effect on the electron density in the radical. Purpurogallin radical, with three hydroxy groups on the benzene ring, has pK(r1) = 4.7 and E-7 = 0.48 V, as compared to the theaflavin radical, with only two hydroxy groups on benzene, having pK(r) = 4.3 and E-7 = 0.51 V. The electron donating ability of theaflavins, which are major antioxidants in black tea, is quantitatively assessed on the basis of physicochemical characteristics of daughter radicals and their potential biological action discussed.

Model phenoxyl and more complex flavonoid radicals were generated by azide radical induced one-electron oxidation in aqueous solutions. Spectral, acid-base and redox properties of the radicals were investigated by the pulse radiolysis technique. The physicochemical characteristics of the flavonoid radicals closely match those of the ring with the lower reduction potential. In flavonoids which, have a 3,5-dihydroxyanisole (catechins), or a 2,4-dihydroxyacetophenone (hesperidin, rutin, quercetin)-like A ring and a catechol- or 2-methoxyphenol-like B ring, the antioxidant active moiety is clearly the B ring [reduction potential difference between the model phenoxyls is Delta E(A-B ring models) > 0.1 V]. In galangin, where the B ring is unsubstituted phenyl, the antioxidant active moiety is the A ring. Even though the A ring is not a good electron donor, E(7) > 0.8/NHE V, it can still scavenge alkyl peroxyl radicals, E(7) = 1.06 V, and the superoxide radical, E(7) > 1.06 V. Quercetin is the best electron donor of all investigated flavonoids (measured E(10.8) = 0.09 V, and calculated E(7) = 0.33 V). The favourable electron-donating properties originate from the electron donating O-3 hydroxy group in the C ring, which is conjugated to the catechol (B ring) radical through the 2,3-double bond. The conjugation of the A and B rings is apparently minimal, amounting to less than 2.5% of the substituent effect in either direction. Thus, neglecting the acid-base equilibria of the A ring, and using those of the B ring and the measured values of the reduction;potentials at pH 3, 7 and 13.5, the pH dependence of the reduction potentials of the flavonoid radicals can be calculated. In neutral and slightly alkaline media (pH 7-9), all investigated flavonoids are inferior electron donors to ascorbate. Quercetin, E(7) = 0.33 V, and gallocatechins, E(7) = 0.43 V, can reduce vitamin E radicals (assuming the same reduction potential as Trolox C radicals, E(7) = 0.48 V). Since all investigated flavonoid radicals have reduction potentials lower than E(7) = 1.06 V of alkyl peroxyl radicals, the parent flavonoids qualify as chain-breaking antioxidants in any oxidation process mediated by these radicals.

Spectral, acid-base, and redox properties of 4-mercaptoimidazoles were investigated by pulse radiolysis in aqueous solutions. Thiyl radicals of 1-methyl-5-ethyl-4-mercaptoimidazole (MEMI) have weak absorption band at 330 nm, ϵ = 300 ± 60 M−1 cm−1. Because the ionic strength variation from 0.01 to 0.1 M in the pH range from 3 to 14 does not influence the rate constant of the radical decay, it is concluded that the MEMI thiyl radical is neutral. At pH 7, the reduction potential of the MEMI radical, E7 = 0.45 V, is lower than E7 = 0.48 V of the Trolox C radical, which means that MEMI may restitute vitamin E under physiological conditions (assuming similar reduction potentials of Trolox C and vitamin E radicals). Because pKa = 10.3 of the SH group in MEMI is lower than pKa = 11.9 of the OH group of Trolox C, the redox equilibrium with Trolox C is reversed at pH 13, and E13(MEMI-radical) = 0.29 ± 0.04 V is determined against E13(Trolox C - radical) = 0.19 V. In contrast to extraordinary electron donating properties, MEMI is only a moderately good H-atom donor. k(·CH3 + MEMI) = (1.5 ± 0.3) × 105 M−1 s−1 in neutral media is considerably lower than k(·CH3 + GSH) = 5 × 107 M−1 s−1, which is explained by the zwitterionic structure of MEMI. The ability of MEMI to act as antioxidant in biological systems is further demonstrated by its ability to efficiently scavenge superoxide and linoleate peroxyl radicals. On the basis of the two-fold increase in the rate of inhibition of radiation-induced oxidation of linoleic acid by MEMI and vitamin E (1:1) as compared with that by MEMI alone, it is concluded that MEMI shows synergism with vitamin E.

Gallocatechins and catechins, which are constituents of green tea, and related, simpler single-ring model compounds undergo one-electron oxidation by the azidyl radical (k = (1.4-4.8) x 10(9) M(-1) s(-1)), which was used as a model one-electron, rapid oxidant. The initial oxidation leads to the formation of a mixture of A- and B- (or C-) ring phenoxyl radicals. This finding was confirmed by comparison with the spectra of 3,5-dihydroxyanisole (the model for A ring) and methyl gallate (the model for B or C ring) radicals and by photoionization experiments in which only the B-ring radical of epigallocatechin was generated, as expected from its lower ionization potential. The A-ring phenoxyl radical is converted to the B- (or C-) ring phenoxyl radical by inter- and intramolecular electron and proton transfer. The activation parameters clearly indicate solvent-assisted intermolecular electron and proton transfer, whereas intramolecular transfer in epigallocatechin gallate radicals is suggested to proceed through an intermediate molecular complex formation. Acid-base equilibria of parent gallocatechins (pK(al) > 8.0) are significantly altered in the corresponding phenoxyl radicals (pK(rl) = 4.4-5.5). The low reduction potentials of gallocatechin radicals, E(7) = 0.42 V (which is lower than that of vitamin E radicals, E(7) = 0.48 V), are responsible for their antioxidant efficacy, which may include the repair of vitamin E radicals. These low reduction potentials also imply high susceptibility of parent gallocatechins to rapid oxidation in aerated aqueous media. The reactivity of epigallocatechin gallate with superoxide radical at pH 7, k = 7.3 x 10(5) M(-1) s(-1) is one of the highest measured rates of reduction of superoxide radical by any chemical antioxidant. In this reaction, superoxide is converted to hydrogen peroxide, thus eliminating the redox cycling that may be involved in the corresponding oxidation reaction. The high rates of quenching of singlet oxygen by gallocatechins in acetonitrile, k = (1.1-2.2) x 10(8) M(-1) s(-1), are comparable to quenching by vitamin E, k = 5 x 10(8) M(-1) s(-1).

Spectral, acid-base, and redox properties of the phenoxyl radicals derived from 3,4-dihydroxybenzene derivatives and selected flavonoids were studied by pulse radiolysis of aqueous solutions. From the pH-dependent changes in the phenoxyl spectra, the dissociation constants were derived. The pK(a), values for the deprotonation of the 3'-OH group in the catechin (pK(a) = 4.6) and rutin (pK(a) = 4.3) radicals are similar to the pK(a) value of the 3,4-dihydroxybenzoate radicals, pK(a) = 4.2, which is expected from their similar electronic structures. Deprotonation of 5- and 7-OH in the catechin and rutin and of 5-OH in the hesperidin radicals has no effect on the radical spectra, which is explained by the inefficient coupling of the A-ring of the flavonoid radicals with the unpaired electron. Because of favorable reduction potentials of the phenoxyl radicals, E(7) = 0.56-0.7 V vs NHE, flavonoids may act as efficient antioxidants of alkylperoxyl and superoxide/hydroperoxyl radicals. The ac kinetic conductivity method was developed for the measurements of the low reaction rate constants of the superoxide radical reactions with flavonoids and phenols in aqueous solutions at pH 10. The rates of the superoxide radical reactions with flavonoids, k = 3 X 10(2)-5.1 X 10(4) M(-1) s(-1), depend on the redox properties and the charge of the flavonoids. The highest rates are measured for the oxidation of quercetin and rutin, whereas the lowest are those for the B-ring monosubstituted derivatives, with substantially higher redox potentials. Uncharged catechin at pH 7 reacts at k = 6.6 X 10(4) M(-1) s(-1), whereas the rate at pH 10, where catechin is doubly negatively charged, is approximately 4 times lower, k = 1.8 x 10(4) M(-1) s(-1). The activation parameters of the oxidation of rutin and trolox at pH 10 and methyl gallate at pH 7 were determined in an attempt to understand why the rates of the superoxide reactions are low despite high driving forces of Delta E greater than or equal to 0.4 V. Low activation enthalpies, Delta H-double dagger = 2.3-3.6 kcal/mol, and negative activation entropies, Delta S-double dagger = -25-28 cal/(mol K), point to an inner-sphere electron-transfer mechanism.

Understanding endogenous mechanisms of carcinogenesis through measuring oxidative markers has advanced greatly in the past decade, paralleling similar achievements in exogenous carcinogenesis through measurements of DNA-adduct markers. Understanding the mechanisms of genesis, metabolism, and physiological properties of the products of oxidative stress is essential in determining products that are specific molecular markers. Measurement technology allows sensitive detection, monitoring, and quantitation of oxidative DNA markers both locally in tissue and systemically in body fluids. Both approaches can be used to assess oxidative stress. Although measurement of markers of oxidative stress relevant to carcinogenesis is at an early stage of development, this approach will probably become an integral part of early diagnostics and the assessment of tumor metabolism. For comprehensive understanding of endogenous carcinogenesis, oxidative markers of protein and lipid damage are also necessary. A larger and perhaps more important application of oxidative markers is in anticarcinogenesis, particularly chemoprevention. Because urinary markers are a noninvasive methodology, they are especially appropriate for assessing and indexing the anticarcinogenic potential of diets and foods from modulation of the rate of DNA damage, which may be correlated with mutagenic and, ultimately, carcinogenic potential.

Hydroxylated and polyhydroxylated aromatic and heterocyclic compounds may have antioxidant and anticarcinogenic properties. Whether an antioxidant is an anticarcinogen may depend on its efficacy as an oxygen radical (peroxyl, alkoxyl, superoxide, hydroxyl) inactivator and inhibitor. Location, concentration in situ, reaction kinetics (rate constants), energetics (redox potentials), and products (intermediates and final) contribute to the efficacy of an antioxidant. The kinetics and energetics of an antioxidant are governed by the type and position of the substituents. The rate constant for the reaction of an antioxidant and an oxy radical depends on the type of radical. In general, the reactivity of a radical decreases in the following order: hydroxyl > alkoxyl > peroxyl > superoxide. One-electron oxidation potential of an antioxidant at pH 7, E7 , can be calculated from the Hammett correlation using Brown substituent constants.

A concise overview of the kinetics, energetics, and mechanisms of oxidative processes involving fatty acids in simple model systems relevant to autoxidation of foods is presented. Endogenous and exogenous factors that initiate oxidative processes in biochemical systems are reviewed and elements of and conditions for propagation of chain peroxidation processes are defined. Mechanistic aspects of chain-breaking antioxidants, including redox potentials, are presented. Biomarkers of fatty acid peroxidation are briefly reviewed and their relevance and specificity assessed.

Life span extension is a fascinating goal that has captured human imagination throughout history, prompting much speculation and numerous investigations. Since the early experiments of McCay and coworkers (1935, 1943) with rats, the mechanisms of dietary restriction (DR) and its effects on extension of maximum life span (MLS) and reduction of degenerative diseases has been of great interest (Weindruch and Walford 1988). More recently, reducing the incidence of diseases such as cancer and cardiovascular disorders through nutritional interventions (Ames 1983) has become an economic issue and a driving force in investigations of DR. In contrast to gross observations, however, the progress in mechanistic understanding has been quite modest. Although research on free radical and oxidative processes in biology and medicine (Simic et al. 1988) appears to have growing relevance to our understanding of DR, there has been relatively little interaction between the two areas of study. Nevertheless, novel ideas bridging these fields have been introduced by Harman (1981).

Reversible one-electron-transfer reactions involving the tryptophan cation and neutral radicals were investigated byh pulse radiolysis. The one-electron-reduction potential fo the neutral tryptophan radical at pH 7 was determined to be E7 = 1.01 +/- 0.03 V by using the bis(1,4,7-triazacyclononane)nickeld (III/II) redox couple with E = 0.95 V as a standard. The value obtained with promethazine (E = 0.98 V) as a standard at pH 6 was E6 = 1.11 V. From these measurements, a mean value E7 = 1.03 V results, in agreement with some earlier determinations (1.05 and 1.08 V) obtained in experiments with inorganic redox standards. The kietics and energetics of the reactions of the tryptophan neutral and cation radicals with selected electron donors were also investigated. The reactivity of the typtophan radical cation was found to be 1-2 orders of magnitude higher than that of the neutral radical. The lower reactivity of the neutral tryptophan radical is explained by the entropy loss due to protonation of the tryptophan anion, resulting from the changes in the solvation shell.

Spectral, acid-base, and redox properties of methoxyphenoxyl radicals were determined in aqueous solutions by pulse radiolysis. Except for 2,6-dimethoxyphenoxyl, with lambda(max) < 300 nm, methoxyphenoxyl radicals absorb in the visible spectrum, with high molar absorptivities. From the effect of ionic strength on the rate of radical decay, a pK(a) < 2 was estimated for the deprotonation of phenolic radical cations. The oxidation potentials of methoxyphenols were determined from the electron-transfer equilibria with inorganic redox standards. The largest effect of methoxy groups on oxidation potential was observed for 2,6-dimethoxyphenol, E7 = 0.58 V vs NHE, whereas the effect of meta substitution was minimal for 3,5-dimethoxyphenol, E7 = 0.85 V, yet this was 0.12 V lower than the measured E7 of 0.97 V for unsubstituted phenol. The reduction potential E7 of methoxyphenoxyl radicals satisfies the Hammett correlation using Brown sigma+ substituent constants, as do other monosubstituted phenoxyls if the additivity of sigma+ values is assumed and the state of protonation of the substituents is taken into consideration. The derived sigma+(CH3O)m value of -0.14, in contrast to reported positive values, indicates the contribution of the methoxy group, even in the meta position, to charge delocalization and resonant stabilization of the phenoxyl radical. The contribution of the ring structure to more negative sigma+ values was observed, as expected. Use of the Hammett correlation is suggested for rapid estimation and accurate calculation of the redox potentials of polysubstituted phenolic antioxidants.

Fresh chicken breast and beef incubated in water were found to contain no o-Tyr at the current levels of detection (0.1 ppm) by capillary gas chromatography/mass spectrometry and selective ion monitoring. In contrast, samples incubated at 37 degrees C in the presence of ethanol, benzene, or carbon tetrachloride (used in fat extraction) contained large quantities (2.5-5.1 ppm) of o-Tyr. No o-Tyr was detected in the water-insoluble fraction of meat treated with carbon tetrachloride after triple extraction by water. However, reaction of radiation generated .OH in gamma-irradiated fresh chicken tissue with endogenous phenylalanine yields o-Tyr with a linear yield-dose response in both water-soluble and -insoluble tissue fractions. Nonradiolytically generated .OH is suggested to be formed through a mitochondrion-mediated Haber-Weiss reaction in association with water-soluble proteins since the yields of o-Tyr in beef, a tissue with a higher mitochondrial content, are four times greater than in the chicken breast tissue.

The ability to monitor noninvasively the biological effect of a radiation dose in humans is potentially beneficial for screening cancer patients during the course of radiotherapy. Yet no universal approach exists for this purpose other than routine examination of patients and visible tumors, although in limited situations specific parameters are measured after treatment is completed (e.g., determinations of antibody levels after breast cancer therapy). In all cases, no appreciable indicators of the effects of the irradiation are apparent during the early part of the therapy regimen.