Michael G. Simic

Mechanisms of inhibition of free-radical processes in mutagenesis and carcinogenesis.

Mechanisms of the defense system against genotoxic damage, with emphasis on free-radical processes, are presented. Components of the defense system, interrelationships between components, and the stages in defense are discussed. Examples of free-radical mechanisms of protective agents (mannitol, DMSO) and repair agents (thiols and antioxidants), and the kinetics of their reactions are reviewed. Future trends in the development of novel protective agents are invoked.

Antioxidants in Nutrition

Abstract: 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 105‐107 M−1 s−1. Singlet oxygen quenching by flavonoids was also very rapid (from 105 to 108 M−1 s−1). These studies may be crucial in optimizing health and increasing longevity by reducing oxidative stress and biological damage.

Reduction potentials of flavonoid and model phenoxyl radicals. Which ring in flavonoids is responsible for antioxidant activity

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 ΔE(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, E7 > 0.8/NHE V, it can still scavenge alkyl peroxyl radicals, E7= 1.06 V, and the superoxide radical, E7 > 1.06 V. Quercetin is the best electron donor of all investigated flavonoids (measured E10.8= 0.09 V, and calculated E7= 0.33 V). The favourable electron-donating properties originate from the electron donating 0-3 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, E7= 0.33 V, and gallocatechins, E7= 0.43 V, can reduce vitamin E radicals (assuming the same reduction potential as Trolox C radicals, E7= 0.48 V). Since all investigated flavonoid radicals have reduction potentials lower than E, =1.06 V of alkyl peroxyl radicals, the parent flavonoids qualify as chain-breaking antioxidants in any oxidation process mediated by these radicals.

Generation of oxy radicals in biosystems.

Many recent lines of evidence indicate that endogenous free radicals contribute to spontaneous mutagenesis through the direct induction of DNA damage. However, the mechanisms underlying this process are not yet fully understood. A brief overview of the knowledge that is currently available is provided here, with emphasis on the generation of oxy radicals in biosystems, the reactions of those radicals with biomolecules, and the induction of oxidative DNA base damage that might lead to mutation.

Dietary modulation of DNA damage in human

Manipulation of human diet can modulate urinary biomarkers of oxidative DNA base damage (UBODBD), reflecting changes in levels of DNA damage. When dietary composition is maintained but caloric intake is decreased (caloric restriction), UBODBD excretion is suppressed. At isocaloric dietary intake the level of damage depends on diet composition. For diets consisting of foods containing carbohydrates, proteins, and fats but lacking fruits and vegetables, the level of damage is higher than for diets including fruits and vegetables, which are rich in natural antioxidants. Assay of urinary biomarkers is suggested as a potential test for quantitative assessment of the carcinogenic or anticarcinogenic properties of foods, food components, and diets and for individual responses to nutritional regimens.

OH Radical-induced Products of Tyrosine Peptides

Reactions of radiation-generated OH radicals with tyrosine and its homopeptides, i.e. L-Tyr-L-Tyr and L-Tyr-L-Tyr-L-Tyr, in N2O saturated solutions were shown to give crosslinks between the peptide chains with high yields. High-performance liquid chromatography, capillary gas chromatography and mass spectrometry were used for isolation and identification of the monomeric and dimeric products. Evidence is presented for the crosslinking to occur through C-C and C-O-C bonds. To the best of our knowledge, the formation of the ether type of crosslink is demonstrated for the first time. Mechanisms of product formation are also discussed, which involve radicals that were described in previous pulse radiolysis studies.

Biomarkers of oh Radical Damage In Vivo

Mechanisms of formation of o-tyrosine (o-Tyr) and thymine glycol (TG), the two possible markers of OH radical generation in biosystems and in vivo are described. The o-Tyr measurements require invasive approaches, while TG detection may be accomplished by noninvasive analysis in the urine.

Antioxidant Activity of 5-Hydroxytryptophan, 5-Hydroxyindole, and Dopa Against Microsomal Lipid Peroxidation and Its Dependence on Vitamin E

The antioxidant capacity of 5-hydroxy-tryptophan, 5-hydroxy-indole, and DOPA (3,4-dihydroxy-phenylalanine) was tested in the Fe-induced lipid peroxidation of liver microsomes of normal- and vitamin E-deficient rats, using ascorbate as a reductant. Lipid peroxidation was monitored as low-level chemiluminescence, indicative of generation of electronically-excited states arising from the recombination of secondary lipid peroxyl radicals. The lag phase (tau 0) preceding the rise in chemiluminescence intensity was used as indicator of antioxidant efficiency, in the absence (tau 0) and the presence (tau) of these polar compounds. The increase in duration of the lag phase exerted by these hydroxy compounds was expressed and quantified as the relationship: tau-tau 0. The tau-tau 0 values were considerably higher in the presence of vitamin E and almost negligible in the absence of tocopherol. It is postulated that the observed increased protection against lipid peroxidation by the above 5-hydroxy derivatives is displayed in a fashion dependent on the presence of vitamin E and probably involving recovery of the chromanoxyl radical by means of an electron-transfer process.

URINE BIOMARKERS FOR OXIDATIVE DNA DAMAGE

The maintenance of macromolecular integrity and function, particularly in DNA, appears to be a major determinant of the longevity and functional capacity of biological systems.1 DNA damage induced by active oxygen species therefore may be of primary importance in cancer and aging2–4. This suggests the need for specific biomarkers (a) to elucidate the biochemical nature of DNA lesions and their repair and (b) to monitor noninvasively the generation and removal of DNA damage in biological systems. Several types of damage are known to result from the interactions of free radicals with chromatin, including single- and double-strand breaks, base and sugar alterations, and DNA-protein crosslinks.5 Each of these types of damage seems to be amenable to chemical analysis by the techniques of high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS), alkaline and neutral elution, and other chromatographic and filter techniques. Of particular interest have been the measurements of specific products of oxidative damage to DNA and proteins, such as thymine glycol, thymidine glycol, base-amino acid crosslinks, and altered amino acids. Recently, the measurement of thymine glycol (TG), thymidine glycol (dR-TG), and 5-hydroxymethyluracil (HMU) in urine has been suggested to be a suitable approach for the in situ assessment of oxidative DNA damage caused by every day metabolic processes.References

Intramolecular H atom abstraction from the sugar moiety by thymine radicals in oligo- and polydeoxynucleotides.

Hydroxyl radical addition to uracil (U) has been suggested to lead to strand breaks in polyuridylic acid, an occurrence attributed in part to H atom abstraction by .U-OH radicals from the ribose moiety [D.G.E. Lemaire et al., Int. J. Radiat. Biol. 45, 351-358 (1984)]. We have investigated this particular reaction by means of the hydroxyl radical-induced products of thymine (T), pT, TpT, TpTpT, polythymidylic acid (poly-T), (T + dR) poly-dA.poly-T, and a mixture of T and 2-deoxyribose (dR). The major monomeric product of .T-OH in TpT, TpTpT, poly-T, and poly-dA.poly-T was found to be 5-hydroxy-6-hydrothymine (H-T-OH), while that in T, pT, and T plus dR was thymine glycol (HO-T-OH). These results indicated that the intramolecular H atom abstraction from a nearby sugar (in this case, deoxyribose) moiety by base radicals, i.e., .T-OH, occurs in oligo- and polydeoxynucleotides of T. In poly-T, the yield of H-T-OH is not much greater than in TpT or TpTpT, indicating that the abstraction of an H atom from the sugar moiety of a nucleotide subunit further than two nucleotides along the chain may not be significant. Additionally, a corresponding decrease in the yield of HO-T-OH with an increase in the yield of H-T-OH suggests that the formations of these two types of thymine products are competitive.