Mechanisms of flavonoid repair reactions with amino acid radicals in models of biological systems: a pulse radiolysis study in micelles and human serum albumin

Neutral tryptophan (*Trp) and tyrosine (TyrO(*)) radicals are repaired by certain flavonoids in buffer, in micelles and in human serum albumin (HSA) with corresponding formation of semioxidized flavonoid radicals. In deaerated buffer, *Trp but not TyrO(*) radicals react with catechin. In micelles, quercetin and rutin repair both *Trp and TyrO(*) radicals. In addition to amino acid reactivity, microenvironmental factors and nature of the flavonoids govern this repair. Electron transfer efficiencies from quercetin to negatively charged *Trp radicals are 100% in the micellar pseudophases of positively charged cetyltrimethylammonium bromide, (CTAB), and neutral Triton X100 (TX100), but 55% in negatively charged sodium dodecyl sulfate (SDS). In oxygen-saturated CTAB micelles, quercetin also reacts with the superoxide radical anion. When bound to domain IIA of HSA, quercetin repairs, by intra- or intermolecular encounter, less than 20% of oxidative damage to HSA. Quercetin can also repair freely circulating oxidized molecules with repair efficiencies falling to 7% for oxidized Trp, Tyr and alpha-MSH and to less than 2% for urate radical. This limited effectiveness is attributed both to the inaccessibility of bound quercetin and rutin toward radicals of circulating molecules and to the diffusion-controlled recombination of these radicals.     

Albumin-bound quercetin repairs vitamin E oxidized by apolipoprotein radicals in native HDL3 and LDL

In the minor fraction of HDL3 containing alpha-tocopherol (alphaTocOH), selective one-electron oxidation of Trp and Tyr residues of apolipoproteins A-I and A-II by *Br2- radical-anions produces the corresponding semioxidized species, TyrO* and *Trp. Repair of TyrO* by endogenous alphaTocOH generates the alpha-tocopheroxyl radical (alphaTocO*). Fast spectroscopic studies show that two populations representing 80% of alphaTocO* initially formed are repaired over several seconds with rate constants of 3.0 x 10(6) and 1.5 x 10(5) M-1 s-1 by quercetin bound to human serum albumin (HSA) at physiologically relevant concentration. Formation of HSA-bound quercetin radicals (*Qb) is observed. In the major fraction of HDL3 particles lacking alphaTocOH, TyrO* and *Trp are repaired by free and HSA-bound quercetin. In LDL particles which all contain alphaTocOH, alphaTocO* radicals are formed in the millisecond time scale by repair of TyrO* radicals produced in apolipoprotein B. Then, 75% of initial alphaTocO* are repaired over seconds by HSA-bound quercetin (rate constant: 2.0 x 10(6) M-1 s-1). HSA-bound quercetin can also repair *Trp radicals. In O2-saturated solutions, the fraction of alphaTocO* radicals (more than 50%) not repaired by superoxide radical-anions can be repaired by HSA-bound quercetin with formation of *Qb but to a much lesser extent in LDL than in HDL.     

Repair of amino acid radicals of apolipoprotein B100 of low-density lipoproteins by flavonoids. A pulse radiolysis study with quercetin and rutin

Selective oxidative damage to apolipoprotein B in LDL can be effected radiolytically by (*)Br(2)(-) radicals. Twenty-seven Trp residues constitute major primary sites of oxidation, but two-thirds of oxidized Trps ((*)Trp) that are formed are repaired by intramolecular electron transfer from Tyr residues with formation of phenoxyl radicals (TyrO(*)). Analysis of (*)Trp and TyrO(*) transient absorbance changes suggests that other apolipoprotein B residues, probably Cys, are oxidized. LDL-bound quercetin can efficiently repair this damage. Absorption studies show that a LDL particle has the capacity to bind approximately 10 quercetin molecules through interaction with apolipoprotein B. The repair occurs by intramolecular electron transfer characterized by a rate constant of 2 x 10(3) s(-)(1). In contrast, rutin, a related flavonoid which does not bind to LDL, cannot repair oxidized apolipoprotein B. Urate is a hydrophilic plasma antioxidant which displays synergistic antioxidant properties with flavonoids. Urate radicals produced by (*)Br(2)(-) can also be repaired by LDL-bound quercetin. This repair occurs with a reaction rate constant of 6.8 x 10(7) M(-)(1) s(-)(1). Comparison with previous studies conducted with human serum albumin-bound quercetin suggests that quercetin analogues tailored to be carried preferentially by lipoproteins might be more powerful plasma antioxidants than natural quercetin carried by serum albumin.     

Intra and intermolecular charge effects on the reaction of the superoxide radical anion with semi-oxidized tryptophan in peptides and N-acetyl tryptophan

The reaction of the superoxide radical anion (O^-·₂), with the semi-oxidized tryptophan neutral radical (Trp^·) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4-5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O^-·₂ with the Trp^· radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp^· radical formed from NAT with O^-·₂ is reduced to half that of the free Trp^· in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp^· and O^-·₂ may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.     

Intra and intermolecular charge effects on the reaction of the superoxide radical anion with semi-oxidized tryptophan in peptides and N-acetyl tryptophan

The reaction of the superoxide radical anion (O^-·₂), with the semi-oxidized tryptophan neutral radical (Trp^·) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4–5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O^-·₂ with the Trp^· radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp^· radical formed from NAT with O^-·₂ is reduced to half that of the free Trp^· in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp^· and O^-·₂ may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.     

Antioxidant activity of some polyphenol constituents of the medicinal plant Phyllanthus amarus Linn

Phyllanthus amarus Linn is a widely distributed tropical medicinal plant highly valued for its therapeutic properties. The antioxidant activity of some of its principal constituents, namely amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D, rutin and quercetin 3-O-glucoside were examined for their ability to scavenge free radicals in a range of systems including 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)/ferrylmyoglobin, ferric reducing antioxidant power (FRAP) and pulse radiolysis. In addition, their ability to protect rat liver mitochondria against oxidative damage was determined by measuring the ROO* radical induced damage to proteins and lipids and *OH radical induced damage to plasmid DNA. The compounds showed significant antioxidant activities with differing efficacy depending on the assays employed. Amariin, repandusinic acid and phyllanthusiin D showed higher antioxidant activity among the ellagitannins and were comparable to the flavonoids, rutin and quercetin 3-O-glucoside.     

Inhibitions of the autoxidation of linoleic acid by flavonoids in micelles.

The activities of five flavonoids as chain-breaking antioxidants have been studied for the autoxidation of linoleic acid in cetyl trimethylammonium bromide (CTAB) micelles at 37 degrees C. Flavonols such as quercetin, rutin and morin exhibited antioxidant activities, while two flavanones, naringin and hesperidin, did not suppress the oxidation appreciably. The ratio of rate constants for inhibition and propagation kinh/kp and stoichiometric factor n were determined.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a "constant *OH radical scavenging environment", k of 1.5 x 10(7) s(-1) by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0+/-0.2 at [F] < 100 microM by a mechanism other than through direct scavenging of *OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H* atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from *OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

On the repair of oxidative damage to apoferritin: a model study with the flavonoids quercetin and rutin in aerated and deaerated solutions

Abstract

Ferritin (Ft) impairment through ^?O₂^–, H₂O₂, and ^?OH production occurs in the cases of ketoses, diabetes mellitus, acute intermittent porphyria and tyrosinemia. In addition to ^?Trp and TyrO^? radical production, ferrous iron liberation and Ft synthesis stimulation, site-specific oxidation reactions are induced leading to toxic iron accumulation in organs with high Ft content, for example, liver and brain. To elucidate the potential pathways to Ft recovery, repair of oxidative damage to horse spleen apoferritin (apoFt) and Ft by quercetin (QH) or rutin (RH) was studied in the presence and absence of oxygen. ^?Trp and TyrO^? radicals were produced in pulse radiolysis through apoFt oxidation by ^?Br₂^– radicals. QH and RH bind to apoFt on eight sites with binding constants of ?80,000 and ?32,000 M^?1, respectively. In deaerated solutions, a repair of apoFt radicals is observed involving both bound and free flavonoids. This repair occurs by a fast intra- and a slow inter-molecular electron transfer from bound and free flavonoids, respectively. With QH, the rate constants are 10⁴ s^–1 and 3.5 × 10⁷ M^–1 s^–1 for the intra- and intermolecular repair reactions, respectively. Oxygen does not interfere with repair of apoFt or Ft by bound QH but inhibits 90% of Ft repair by RH. These results taken together indicate that flavonoid antioxidants may help alleviate Ft impairment in diseases involving an oxidative stress.     

Submolecular adventures of brain tyrosine: what are we searching for now?

This overview summarizes recent findings on the role of tyrosyl radical (TyrO(*)) in the multitudinous neurochemical systems of brain, and theorizes on the putative role of TyrO(*) in neurological disorders [Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)]. TyrO(*) and tyrosine per se can interact with reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radical mechanisms and chain propagating reactions. The concentration of TyrO(*), ROS and RNS can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive as well, and this can induce damage directly (by lipid peroxidation) or indirectly (by proteins oxidation and/or nitration), potentially causing apoptotic neuronal cell death or autoschizis.Evidence of lesion-induced neuronal oxidative stress includes the presence of protein peroxides (TyrOOH), DT (o,o'-dityrosine) and 3-NT (3-nitrotyrosine). Mechanistic details of protein- and enzymatic oxidation/nitration in vivo remain unresolved, although recent in vitro data strongly implicate free radical pathways via TyrO(*). Nitration/denitration processes can be pathological, but they also may play: 1). a signal transduction role, because nitration of tyrosine residues through TyrO(*) formation can modulate, as well the phosphorylation (tyrosine kinases activity) and/or tyrosine hydroxylation (tyrosine hydroxylase inactivation), leading to consequent dopamine synthesis failure and increased degradation of target proteins, respectively; 2). a role of "blocker" for radical-radical reactions (scavenging of NO(*), NO(*)(2) and CO(3)(*-) by TyrO(*)); 3). a role of limiting factors for peroxynitrite formation, by lowering O(2)(*-) formation, which is strongly linked to the pathogenesis of neural diseases.It is still not known if tyrosine oxidation/nitration via TyrO(*) formation is 1). a footprint of generalized stress and neuronal disorders, or 2). an important part of O(2)(*-) and NO(*) metabolism, or 3). merely a part of integral processes for maintaining of neuronal homeostasis. The full answer to these questions should be of top research priority, as the problem of increased free radical formation in brain and/or imbalance of the ratios ROS/RNS/TyrO(*) may be all important in defining whether oxidative stress is the critical determinant of tissue and neural cell injury that leads to pathological end-points.     

Fast repair of purine deoxynucleotide radical cations by rutin and quercetin

Repair effects of rutin and quercetin on purine deoxynucleotide radical cations were studied using pulse radiolysis technique. On electron pulse irradiation of N₂ saturated deoxynucleotide aqueous solution containing 20 mmol/L K₂S₂O₈, 200 mmol/Lt-BuOH and rutin or quercetin, the transient absorption spectra of the deoxynucleotide radical cations decayed quickly. At the same time, the spectra of flavonoid phenoxyl radicals formed within several dozen microseconds. The results indicated that deoxynucleotide radical cations can be repaired by flavonoids. The rate constants of the repair reactions were 3.8 ×10⁸-4.4 ×10⁸ mol⁻¹ · L · s⁻¹ and 1.3×10⁸-1.8×10⁸ mol⁻¹ · L · s⁻¹ for dAMP and dGMP radical cations, respectively.     

Fast repair of purine deoxynucleotide radical cations by rutin and quercetin

Repair effects of rutin and quercetin on purine deoxynucleotide radical cations were studied using pulse radiolysis technique. On electron pulse irradiation of N2 saturated deoxynucleotide aqueous solution containing 20 mmol/L K2S2O8, 200 mmol/L f-BuOH and rutin or quercetin, the transient absorption spectra of the deoxynucleotide radical cations decayed quickly. At the same time, the spectra of flavonoid phenoxyl radicals formed within several dozen microseconds. The results indicated that deoxynucleotide radical cations can be repaired by flavonoids. The rate constants of the repair reactions were 3.8 × 108-4.4×108 mol-1 · L · s-1 and 1.3×108-1.8×108 mol-1 · L · s-1 for dAMP and dGMP radical cations, respectively.     

Inhibition of the peroxidation of linoleic acid by the flavonoid quercetin within their complex with human serum albumin

This work provides a quantitative kinetic analysis of oxidative pathways involving linoleic acid and the common dietary antioxidant quercetin (flavonoid), both bound to human serum albumin (HSA). In particular, it is shown that quercetin, although embedded in drug site I, is oxidized as quickly as free quercetin under a flux of hydrophilic peroxyl radicals. This observation suggests that efficient charge relays are established between the periphery of HSA and bound quercetin. Moreover, the peroxidation of HSA-bound linoleic acid is shown to take place at some specific fatty acid binding sites once one to two critical HSA residues are themselves oxidized. Quercetin efficiently delays the onset of lipid peroxidation. The inhibition persists long after the total consumption of quercetin, in agreement with some quercetin oxidation products exerting a residual antioxidant activity. Consistently, HSA markedly increases the maximal concentration of a two-electron oxidation product of quercetin that is accumulated and then consumed in the course of the peroxidation. The additional observation of the faster consumption of the single Trp residue in the presence of quercetin suggests that HSA enhances the antioxidant activity of quercetin by regenerating some of its oxidation products retaining a H-donating activity.     

Antioxidant activity of some polyphenol constituents of the medicinal plant Phyllanthus amarus Linn

Abstract

Phyllanthus amarus Linn is a widely distributed tropical medicinal plant highly valued for its therapeutic properties. The antioxidant activity of some of its principal constituents, namely amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D, rutin and quercetin 3-O-glucoside were examined for their ability to scavenge free radicals in a range of systems including 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)/ferrylmyoglobin, ferric reducing antioxidant power (FRAP) and pulse radiolysis. In addition, their ability to protect rat liver mitochondria against oxidative damage was determined by measuring the ROO? radical induced damage to proteins and lipids and ?OH radical induced damage to plasmid DNA. The compounds showed significant antioxidant activities with differing efficacy depending on the assays employed. Amariin, repandusinic acid and phyllanthusiin D showed higher antioxidant activity among the ellagitannins and were comparable to the flavonoids, rutin and quercetin 3-O-glucoside.     

The interaction of dietary carotenoids with radical species

Dietary carotenoids react with a wide range of radicals such as CCl3O2*, RSO2*, NO2*, and various arylperoxyl radicals via electron transfer producing the radical cation of the carotenoid. Less strongly oxidizing radicals, such as alkylperoxyl radicals, can lead to hydrogen atom transfer generating the neutral carotene radical. Other processes can also arise such as adduct formation with sulphur-centered radicals. The oxidation potentials have been established, showing that, in Triton X-100 micelles, lycopene is the easiest carotenoid to oxidize to its radical cation and astaxanthin is the most difficult. The interaction of carotenoids and carotenoid radicals with other antioxidants is of importance with respect to anti- and possibly pro-oxidative reactions of carotenoids. In polar environments the vitamin E (alpha-tocopherol) radical cation is deprotonated (TOH*+ --> TO* + H+) and TO* does not react with carotenoids, whereas in nonpolar environments such as hexane, TOH*+ is converted to TOH by hydrocarbon carotenoids. However, the nature of the reaction between the tocopherol and various carotenoids shows a marked variation depending on the specific tocopherol homologue. The radical cations of the carotenoids all react with vitamin C so as to "repair" the carotenoid.     

Different effects of the constituents of EGb761 on apoptosis in rat cerebellar granule cells induced by hydroxyl radicals

The present study was conducted to evaluate the different effects of the constituents of EGb761 (Ginkgo biloba Extract) on apoptosis in cerebellar granule cells induced by hydroxyl radicals. The total flavonoid component of EGb761, two pure EGb761 components (rutin and quercetin), and a mixture of flavonoids and terpenes protected cerebellar granule cells from oxidative damage and apoptosis induced by hydroxyl radicals. ESR(electron spin resonance) results showed that the IC50 of the flavonoids for scavenging hydroxyl radicals was almost the same as that of EGb761, even though flavonoids make up only 24% of EGb761, implying that other constituents of EGb761 besides flavonoids can scavenge hydroxyl radicals. Total terpenes of EGb761 did not protect against apoptosis. Flavonoids and terpenes did not show a synergistic effect in this regard. Terpenes did not scavenge hydroxyl radicals directly, which might be related to their "cage-like" structures.     

Redox reactions of the urate radical/urate couple with the superoxide radical anion, the tryptophan neutral radical and selected flavonoids in neutral aqueous solutions

The kinetics of several processes involving the potential antioxidant role of urate in physiological systems have been investigated by pulse radiolysis. While the monoanionic urate radical, ^·UH^-, can be produced directly by oxidation with ^·Br^-₂ or ^·OH, it can also be generated by oxidation with the neutral tryptophan radical, ^·Trp, with a rate constant of 2 × 10⁷ M^-1s^-1. This radical, ^·UH^-, reacts with ^·O^-₂ with a rate constant of 8 × 10⁸ M^-1s^-1. Also, ^·UH^- is reduced by flavonoids, quercetin and rutin in CTAB micelles at rate constants of 6 × 10⁶ M^-1s^-1 and 1 × 10⁶ M^-1s^-1, respectively. These results can be of value by providing reference data useful in further investigation of the antioxidant character of urate in more complex biological systems.     

Damage to hemoglobin by radiation-generated serum albumin radicals.

We have studied the effects of the interaction of radiation generated human serum albumin radicals (HSA*) with human hemoglobin molecules (Hb). Diluted Hb aqueous solutions were irradiated under N2O or argon without HSA and in the presence of HSA. Analysis of Hb absorbance spectra in the visible range, cross-linking of HSA* radicals with Hb molecules and functional properties of Hb were investigated. The degree of Hb destruction estimated on the basis of changes in the absorption spectra indicated that the effectiveness of HSA* radicals generated under N2O for Hb destruction was approximately equal to that of *OH radicals. In this case mainly *OH radicals formed the secondary HSA* radicals. However, during the irradiation Hb + HSA under argon the presence of equivalent amounts of oxidizing and reducing products of water radiolysis lowers the degree of Hb destruction. Some reactions of HSA* radicals with Hb molecules lead to the formation of covalent bonds between the molecules of both proteins. The following types of hybrids could be distinguished: Hb monomer-HSA, Hb dimer-HSA and higher aggregates. Structural changes of Hb by HSA* radicals were reflected by alterations in the oxygen affinity (increase) and cooperativity (decrease) of Hb. The results obtained indicate that in the experimental systems studied, the HSA* radical reactions with Hb molecules are favoured over recombination reactions of HSA* radicals. On this basis one can suggest that in the studied systems Hb plays the role of an acceptor of radical energy located on HSA.     

Site-specific induction of lipid peroxidation by iron in charged micelles

Generation of hydroxyl radicals by the Fenton reaction resulted in lipid peroxidation of linoleic acid (LA) (H2O2-Fe2+-induced lipid peroxidation) in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles, but not in negatively charged sodium dodecyl sulfate (SDS) micelles. However, more OH radicals formed via the Fenton reaction were trapped by N-t-butyl-alpha-phenylnitrone (PBN) in SDS micelles than in TTAB micelles. When detergent-dispersed LA was contaminated with linoleic acid hydroperoxide (LOOH), lipid peroxidation was catalyzed by Fe2+ via reductive cleavage of LOOH (LOOH-Fe2+-induced lipid peroxidation), and Fe2+ was oxidized simultaneously in SDS micelles, even when H2O2 was not present. In contrast, LOOH-Fe2+-induced lipid peroxidation and simultaneous oxidation of Fe2+ were not observed in TTAB micelles. An ESR spectrum presumed to be due to an alkoxy radical trapped by PBN was also detected in SDS micelles, but not in TTAB micelles in the LOOH-Fe2+-induced lipid peroxidation system. The results are discussed in the light of the localization of iron, the unsaturated bonding moiety of LA, the OOH-group of LOOH, and the trapping site of PBN in different charged micelles.     

Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco-2 cells

Protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide (tert-BOOH)- and menadione-induced DNA single strand breaks was investigated in Caco-2 cells. Both tert-BOOH and menadione induced DNA single strand breaks in a concentration-dependent manner. Pre-incubation of Caco-2 cells with either quercetin or rutin for 24 h significantly decreased the formation of DNA single strand breaks evoked by tert-BOOH (P <.05). Iron chelators, 1,10-phenanthroline (o-Phen) and deferoxamine mesylate (DFO), also protected against tert-BOOH-induced DNA damage, whereas butylated hydroxytoluene (BHT) had no effect. Quercetin, and not rutin, decreased the extent of menadione-induced DNA single strand breaks. DFO and BHT, and not o-Phen, protected against menadione-induced DNA strand break formation (P <.05). From the results of this study, iron ions were involved in tert-BOOH-induced DNA single strand break formation in Caco-2 cells, whereas DNA damage evoked by menadione was far more complex. We demonstrated that the flavonoids, quercetin and rutin, protected against tert-BOOH-induced DNA strand breaks by way of their metal ion chelating mechanism. However, quercetin, and not rutin, protected against menadione-induced DNA single strand breaks by acting as both a metal chelator and radical scavenger.