Ergothioneine Prevents Copper-Induced Oxidative Damage to DNAand Protein by Forming a Redox-Inactive Ergothioneine-Copper

Complex

Ben-Zhan Zhu,*,†,‡ Li Mao,† Rui-Mei Fan,† Jun-Ge Zhu,† Ying-Nan Zhang,† Jing Wang,†

Balaraman Kalyanaraman,§ and Balz Frei*,‡

State Key Laboratory of EnVironmental Chemistry and Eco-Toxicology, Research Center for Eco-EnVironmentalSciences, Chinese Academy of Sciences, Beijing 100085, P. R. China, Linus Pauling Institute, Oregon StateUniVersity, CorVallis, Oregon 97331, United States, and Biophysics Research Institute, Medical College of

Wisconsin, Milwaukee, Wisconsin 53226-3548, United States

ReceiVed June 27, 2010

Ergothioneine (2-mercaptohistidine trimethylbetaine) is a naturally occurring amino acid analogue foundin up to millimolar concentrations in several tissues and biological fluids. However, the biological functionsof ergothioneine remain incompletely understood. In this study, we investigated the role of ergothioneinein copper-induced oxidative damage to DNA and protein, using two copper-containing systems: Cu(II)with ascorbate and Cu(II) with H2O2 [0.1 mM Cu(II), 1 mM ascorbate, and 1 mM H2O2]. Oxidativedamage to DNA and bovine serum albumin was measured as strand breakage and protein carbonylformation, respectively. Ergothioneine (0.1-1.0 mM) provided strong, dose-dependent protection againstoxidation of DNA and protein in both copper-containing systems. In contrast, only limited protectionwas observed with the purported hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, even atconcentrations as high as 100 mM. Ergothioneine also significantly inhibited copper-catalyzed oxidationof ascorbate and competed effectively with histidine and 1,10-phenanthroline for binding of cuprouscopper, but not cupric copper, as demonstrated by UV-visible and low-temperature electron spin resonancetechniques. We conclude that ergothioneine is a potent, natural sulfur-containing antioxidant that preventscopper-dependent oxidative damage to biological macromolecules by forming a redox-inactiveergothioneine-copper complex.

Introduction

L-Ergothioneine (2-mercaptohistidine trimethylbetaine) is anaturally occurring amino acid that contains an imidazole-2-thione moiety (Figure 1). The thione group exists in a tautomericthiol-thione equilibrium, with the thione being the predominantform at physiological pH, which distinguishes ergothioneinefrom other biological thiol compounds. Ergothioneine is syn-thesized by some bacteria and fungi, but not by animals (1, 2).Humans acquire ergothioneine from their diet, including mush-rooms, oats, corn, and meat (1-3). Ergothioneine is found inbrain, erythrocytes, liver, kidney, heart, seminal fluid, and oculartissues (1-4). However, the biological functions of ergothio-neine remain incompletely understood. Ergothioneine has beenshown to exert radioprotective effects (5); scavenge singletoxygen, hydroxyl radicals, hypochlorous acid, and peroxylradicals (6-8); inhibit peroxynitrite-dependent nitration ofproteins and DNA (9); protect retinal neurons from N-methyl-D-aspartate (NMDA)-induced excitotoxicity in vivo (10); andprotect against diabetic embryopathy in pregnant rats (11).Ergothioneine also inhibits peroxynitrite-induced formation of

xanthine and hypoxanthine and their metabolite, urate, whichmay have important implications for gout and other inflamma-tory disorders (9).

Copper is an essential trace element for humans (12, 13). Itis a redox-active transition metal comprising the active centerof a wide variety of metalloenzymes, such as Cu, Zn superoxidedismutase and cytochrome c oxidase (12, 13). Copper is alsoan essential component of chromatin and is closely associatedwith DNA bases, particularly near G-C sites (14-16). Althoughthe essentiality of copper in biology is well-documented, coppercan also be toxic (16-23). It is well-known that copper caninduce oxidative damage to DNA, proteins, and lipids, therebypotentially contributing to disease pathology (21-23). Coppercan accumulate in certain tissues and cells, such as hepatocytes,causing liver injury. Copper-induced injury has been hypoth-esized to result from the redox properties of copper, in particularits participation in a copper-catalyzed, Fenton-like reaction(19-23). Wilson’s disease, idiopathic copper toxicosis, andIndian childhood cirrhosis are examples of severe chronic liverdiseases that result from genetic predisposition for hepatic

* To whom correspondence should be addressed. B.-Z.Z.: State KeyLaboratory of Environmental Chemistry and Eco-Toxicology, ResearchCenter for Eco-Environmental Sciences, Chinese Academy of Sciences,Beijing 100085, P. R. China; phone, 86-10-62849030; fax, 86-10-62923563;e-mail, bzhu@rcees.ac.cn. B.F.: Linus Pauling Institute, Oregon StateUniversity, 571 Weniger Hall, Corvallis, OR 97331; phone, (541) 737-5078; fax, (541) 737-5077; e-mail, balz.frei@oregonstate.edu.

† Chinese Academy of Sciences.‡ Oregon State University.§ Medical College of Wisconsin. Figure 1. Chemical structure of ergothioneine.

Chem. Res. Toxicol. 2011, 24, 30–3430

10.1021/tx100214t 2011 American Chemical SocietyPublished on Web 11/03/2010

copper accumulation (12, 18). The serum copper concentrationin these copper-overloaded patients has been shown to be 5-8times higher than in healthy individuals (12, 18). Copperaccumulating in the liver of Long Evans Cinnamon rats causesformation of hepatocellular carcinomas, suggesting that abnor-mal copper metabolism is involved in hepatic carcinogenesisin these animals (12, 18). A case-cohort study showed aU-shaped relationship between premorbid plasma copper levelsand the risk of developing breast cancer (24).

Ergothioneine has been shown to interact with metal ionsand metalloenzymes in chemical systems (6, 25, 26). However,it is not clear whether and by what mechanism(s) ergothioneinecan protect against copper-dependent oxidative damage tobiological macromolecules, such as DNA, proteins, and lipids.In this study, we investigated the role of ergothioneine in copper-dependent oxidative damage to DNA and bovine serum albumin,using two copper-containing systems: Cu(II) with ascorbate andCu(II) with H2O2. Oxidative damage to DNA and albumin wasmeasured as strand breakage and protein carbonyl formation,respectively. We found that ergothioneine strongly protectsagainst copper-induced oxidative damage of DNA and proteinby forming a redox-inactive ergothioneine-copper complex.

Materials and Methods

Materials. Ergothioneine, histidine, dimethyl sulfoxide (DMSO),mannitol, bathocuproine disulfonate, bovine serum albumin (BSA),2,4-dinitrophenylhydrazine (DNPH), R-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), cupric sulfate, ascorbate, and hydrogenperoxide were purchased from Sigma (St. Louis, MO). All buffersolutions were treated with Chelex to remove adventitious metals.

Protein Carbonyl Formation. BSA (1 mg/mL) was oxidizedwith Cu(II) and ascorbate or Cu(II) and H2O2 [0.1 mM Cu(II), 1mM ascorbate, and 1 mM H2O2] in 0.1 M phosphate buffer (pH7.4) at 37 °C for 1 h. Protein carbonyls were assayed as describedby Levine et al. (27). Briefly, 1 mL of BSA solution was mixedwith 0.5 mL of 10 mM DNPH in 2 N HCl. The mixture wasincubated at room temperature for 1 h, followed by the addition of0.5 mL of 20% trichloroacetic acid. The sample was incubated onice for 10 min and centrifuged in a benchtop centrifuge at 3000gfor 10 min. The protein pellet was washed three times with 3 mLof an ethanol/ethyl acetate mixture (1:1, v/v) and dissolved in 1mL of 6 M guanidine (pH 2.3). The peak absorbance at 370 nmwas measured to quantitate protein carbonyls. Data were expressedas nanomoles of carbonyl groups per milligram of protein, using amolar absorption coefficient of 22000 M-1 cm-1 for the DNPHderivatives (27).

DNA Damage. The conversion of covalently closed circulardouble-stranded supercoiled DNA (form I) to a relaxed open circleform (form II) and a linear form (form III) was used to investigateDNA strand breakage induced by Cu(II) and ascorbate or Cu(II)and H2O2 (28). The experiments were conducted via incubation ofplasmid pBR322 DNA (0.5 µg) at 37 °C for 1 h in Chelex-treatedsodium phosphate buffer (0.1 M, pH 7.4) with the Cu(II)-containingsystems, in the absence or presence of the indicated concentrationsof ergothioneine or other agents.

Redox Activity of Copper. The redox activity of copper wasdetermined by measuring the rate of copper-catalyzed oxidation ofascorbate. Ergothioneine and Cu(II) (1 µM) were added to a solutionof 0.1 mM ascorbate in 0.1 M phosphate buffer (pH 7.4), and thesample was incubated at room temperature. The oxidation ofascorbate was monitored spectrophotometrically at 265 nm. Theextinction coefficient for ascorbate at 265 nm is 14500 M-1 cm-1

(29).Electron Spin Resonance (ESR) Spin-Trapping Studies. The

basic system used in this study was composed of Cu(II) (0.1 mM),H2O2 (1 mM), ascorbate (0.1 mM), DMSO (5%), and the spin-trapping agent, R-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN,100 mM), in Chelex-treated phosphate buffer (100 mM, pH 7.4),

with or without ergothioneine. Ergothioneine was added to thereaction mixture 1 min before H2O2 was added. ESR spectra wererecorded at room temperature in a Bruker ER 200 D-SRCspectrometer operating at 9.8 GHz in a cavity equipped with aBruker Aquax liquid sample cell. Typical spectrometer parameterswere as follows: scan range, 100 G; field set, 3470 G; time constant,200 ms; scan time, 100 s; modulation amplitude, 1.0 G; modulationfrequency, 100 kHz; receiver gain, 1.25 × 104; and microwavepower, 9.8 mW.

Low-Temperature ESR Studies. ESR spectra were recordedusing a Varian E109 Century Series spectrometer at a lowtemperature (77 K). The sample was placed in a standard quartzESR tube prior to being frozen. The sides of the tube were thenwarmed slightly to allow the frozen sample to slide into a fingerDewar filled with liquid nitrogen for the acquisition of spectra.Spectral measurements of the Cu(II)(histidine)2 complex wereconducted in 0.1 M phosphate buffer (pH 7.4) with or withoutergothioneine.

UV-Visible Spectral Analysis. Competition between ergoth-ioneine and histidine or 1,10-phenanthroline to bind copper ionswas assessed in a Beckman DU-640 spectrophotometer. Cu(II)(his-tidine)2 and Cu(II)(1,10-phenanthroline)2 complexes were preparedby mixing Cu(II) with either histidine or 1,10-phenanthroline at a1:2 molar ratio. Reduction of Cu(II) to Cu(I) was accomplished byadding an excess amount of ascorbate, and the spectra were recordedbetween 400 and 800 nm at room temperature.

Results

Oxidation of proteins results in the formation of carbonylgroups in quantities that reflect the extent of the oxidativedamage. Thus, in this study, copper-mediated oxidative damageto BSA was measured by protein carbonyl formation. Incubationof BSA (1 mg/mL) for 1 h at 37 °C with Cu(II) and ascorbateor Cu(II) and H2O2 [0.1 mM Cu(II), 1 mM ascorbate, and 1mM H2O2] led to the formation of 44.7 or 25.5 nmol of proteincarbonyls/mg of protein, respectively (Table 1). Ergothioneinemarkedly inhibited protein carbonyl formation in a concentra-tion-dependent manner in both copper-containing systems. Incontrast, only limited inhibition was observed with DMSO andmannitol, even at concentrations as high as 100 mM (Table 1).For example, protein carbonyl formation induced by Cu(II) withascorbate was inhibited by ∼80 and ∼20% by 0.1 mMergothioneine and 100 mM DMSO, respectively, and wascompletely inhibited by 0.2 mM ergothioneine (Table 1).Ergothioneine also markedly inhibited DNA damage in aconcentration-dependent manner (0.1-1.0 mM) in both copper-containing systems, Cu(II) with ascorbate (Figure 2A) and Cu(II)with H2O2 (Figure 2B). Histidine was less effective thanergothioneine in inhibiting DNA damage, especially in theCu(II)/ascorbate system (Figure 2), suggesting that the thiol

Table 1. Effect of Ergothioneine, DMSO, Mannitol, and theCopper Chelating Agent, Bathocuproine Disulfonate, on

Protein Carbonyl Formation Induced by Cu(II) withAscorbate or Cu(II) with H2O2

a

Cu(II) with ascorbate Cu(II) with H2O2

compoundcarbonyls(nmol/mg)

inhibition(%)

carbonyls(nmol/mg)

inhibition(%)

Control 44.7 25.5Ergothioneine (0.1 mM) 7.6 83.0 15.2 40.0Ergothioneine (0.2 mM) 0.1 99.7 7.9 69.0DMSO (100 mM) 34.8 22.1 18.3 28.2Mannitol (100 mM) 39.3 12.1 18.1 29.1Bathocuproine disulfonate (1 mM) 0.1 99.7 0.1 99.6

a Reactions were conducted in 0.1 M phosphate buffer (pH 7.4) at 37°C for 1 h. Reaction mixtures contained 1 mg/mL BSA, 0.1 mM Cu(II),1 mM ascorbate, and 1 mM H2O2. Each value represents the mean ofthree separate experiments.

Ergothioneine PreVents Damage to DNA and Protein Chem. Res. Toxicol., Vol. 24, No. 1, 2011 31

group of ergothioneine is critical for its inhibitory effect onCu(II)/ascorbate system-induced DNA damage. In contrast, noinhibition was observed with DMSO and mannitol, even atconcentrations as high as 100 mM (data not shown).

The observations that protein carbonyl formation and DNAdamage were strongly inhibited by ergothioneine, but not byDMSO or mannitol, indicate that the effect of ergothioneine isnot due to scavenging of hydroxyl radicals. In contrast, theinhibition of copper-induced protein oxidation and DNA damageby the known copper chelating agent, bathocuproine disulfonate(Table 1 and Figure 2), suggests that the effect of ergothioneinemight be due to binding of copper and inhibition of its redoxactivity. It should be noted that BSA has a specific high-affinitycopper binding site, rendering the copper redox-inactive.However, this does not change the conclusion reached abovebecause the molar concentration of BSA is ∼0.015 mM whilethe Cu(II) concentration employed in this study is 0.1 mM.

Although it has been shown that ergothioneine can bindcopper and form ergothioneine-copper complexes under non-physiological conditions (25, 26), little is known about the effectof ergothioneine on the redox activity of copper and whetherergothioneine combines preferentially with Cu(II) or Cu(I) atphysiological pH. Hence, we investigated the effect of ergot-hioneine on the redox activity of copper by measuring Cu(II)-catalyzed oxidation of ascorbate. We found that this reactionwas significantly inhibited in a dose-dependent manner by

ergothioneine (Figure 3), but not by histidine (data not shown).These results suggest that ergothioneine forms a redox-inactivecomplex with copper.

To further support this notion, we studied the effect ofergothioneine on free radical production by a copper-catalyzed,Fenton-like reaction. The most direct technique for detectingfree radicals is ESR spectroscopy using spin-trapping agents(30). Hydroxyl radicals can be detected by ESR spectroscopyusing POBN as a spin-trapping agent. As shown in Figure 4,the interaction of ascorbate with H2O2 and Cu(II) producedPOBN-CH3 adducts in the presence of DMSO and POBN,suggesting that hydroxyl radicals or equivalent reactive inter-mediates were generated from the ascorbate/H2O2/Cu(II) system.Ergothioneine progressively decreased the level of formationof the POBN-CH3 adducts as the ergothioneine concentration,and hence its ratio with Cu(II), increased. At an ergothioneine:Cu(II) ratio of g3:1, little or no POBN-CH3 adduct formationwas observed (Figure 4). Because the concentrations of ergot-hioneine (0.1-1 mM) used in these experiments were muchlower than the concentrations of POBN (100 mM) and DMSO(700 mM), the inhibition of POBN-CH3 adduct formation byergothioneine cannot be due to its scavenging of hydroxylradicals or equivalent reactive intermediates produced by theascorbate/H2O2/Cu(II) system, but most likely is due to its abilityto form a redox-inactive ergothioneine-copper complex.

To investigate whether ergothioneine combines with Cu(II)or Cu(I), we used the two model copper complexes, Cu(II)(his-

Figure 2. Inhibition by ergothioneine, histidine, and bathocuproinedisulfonate of DNA strand break formation induced by Cu(II) withascorbate (A) and Cu(II) with H2O2 (B). Reactions were conducted in0.1 M phosphate buffer (pH 7.4) at 37 °C for 1 h. Reaction mixturescontained 5 µg/mL plasmid DNA, 0.1 mM Cu(II), 0.1 mM ascorbate,and the indicated concentrations of ergothioneine, histidine, andbathocuproine disulfonate (BCS). Different DNA forms: form I, closedcircular double-stranded supercoiled DNA; form II, relaxed open circleDNA; and form III, linear DNA.

Figure 3. Inhibition by ergothioneine of Cu(II)-catalyzed oxidation ofascorbate. Reactions were conducted in 0.1 M phosphate buffer (pH7.4) at room temperature for 15 min. Reaction mixtures contained 0.1mM ascorbate and 1 µM Cu(II): (A) Cu(II) and ascorbate, (B) Cu(II),ascorbate, and 1 µM ergothioneine, (C) Cu(II), ascorbate, and 3 µMergothioneine, (D) Cu(II), ascorbate, and 10 µM ergothioneine, and(E) ascorbate only. The oxidation of ascorbate was monitored spec-trophotometrically at 265 nm.

Figure 4. Inhibition by ergothioneine of copper-catalyzed POBN-CH3

adduct formation. Reactions were conducted at room temperature inChelex-treated phosphate buffer (100 mM, pH 7.4). All reactionmixtures contained POBN (100 mM), Cu(II) (0.1 mM), H2O2 (1 mM),ascorbate (0.1 mM), and DMSO (5%). Where indicated, ergothioneinewas added to the reaction mixture 1 min before the addition of H2O2.The hyperfine splitting constants for the POBN-CH3 adduct were asfollows: aN ) 15.96 G, and a�

H ) 2.74 G. The overlapping signal inthe center of the spectra was identified as ascorbate radical.

32 Chem. Res. Toxicol., Vol. 24, No. 1, 2011 Zhu et al.

tidine)2 and Cu(I)(1,10-phenanthroline)2. It is known thathistidine binds Cu(II) in a redox-active manner to form anintensely blue Cu(II)(histidine)2 complex with a λmax of 640 nm.Addition of ergothioneine (2 mM) to the Cu(II)(histidine)2

complex (2 mM) led to an only slight decrease in absorbanceat 640 nm (Figure 5), suggesting that ergothioneine cannotcompete effectively with histidine for Cu(II). In contrast, in thepresence of ascorbate (10 mM), addition of ergothioneine (2mM) led to a dramatic decrease in the absorbance at 640 nm(Figure 5), suggesting that ergothioneine can compete effectivelywith histidine for Cu(I). Similar experiments with the Cu(I)(1,10-phenanthroline)2 complex, which was prepared by mixing theCu(II)(1,10-phenanthroline)2 complex with ascorbate, showedthat ergothioneine effectively competes with 1,10-phenanthrolinefor Cu(I) (Figure 6). These data indicate that in the presence ofascorbate, ergothioneine reacts with Cu(I) to form a redox-inactive ergothioneine-Cu(I) complex.

To further confirm this notion, we employed low-temperatureESR. The Cu(II)(histidine)2 complex has a characteristic ESRspectrum at a low temperature (77 K), showing the typicalpattern of a square-planar copper complex (31). The Cu(II)(his-tidine)2 ESR signal completely disappeared when both ergot-hioneine and ascorbate were added (data not shown). These ESRresults indicate that in the presence of ascorbate, ergothioneinecan compete effectively with histidine to bind copper and

stabilize it in the Cu(I) state. All the results described abovestrongly suggest that ergothioneine may combine with Cu(I) toform a redox-inactive ergothioneine-Cu(I) complex, mostprobably with a molar ratio of 2:1.

Discussion

Ergothioneine is synthesized exclusively by fungi and my-cobacteria (1, 2) and is present in human food items in highlyvariable amounts. By far the highest levels of ergothioneine arefound in mushrooms (0.1-1.0 mg/g of dried material) (2).Mammals ingest ergothioneine and, interestingly, conserve itwith minimal metabolism (1). Ergothioneine in its free form isstored in tissues that may be exposed to oxidative stress, suchas erythrocytes, seminal fluid, liver, kidney, heart, and oculartissues, where ergothioneine can reach up to millimolar con-centrations (1). In addition, micromolar concentrations ofergothioneine are found in the brain, another tissue potentiallyexposed to oxidative stress (1).

Since its discovery in 1909, the physiological role ofergothioneine, if any, has remained elusive (1). Most authorsconsider it an intracellular antioxidant. However, intracellularconcentrations of ergothioneine (∼0.1-1 mM) are considerablylower than those of the ubiquitous hydrophilic antioxidants,glutathione (GSH) and ascorbate, which are present intracellu-larly at ∼3-7 and ∼1-4 mM, respectively (32). Therefore,the question of whether ergothioneine, when compared to othercommon antioxidants and thiol compounds, including GSH andcysteine, plays a unique biological role arises. Interestingly, ithas been shown that ergothioneine, unlike GSH and ascorbate,does not autoxidize at physiological pH in the presence oftransition metal ions, such as copper or iron (1). In general,ergothioneine has been suggested to serve as a powerful catalyticscavenger of oxidizing species that are not free radicals (32).By contrast, GSH and ascorbate are regarded as free radicalscavengers.

Ergothioneine has been shown to scavenge hydroxyl radicals.However, most biological molecules react with hydroxyl radicalsat diffusion-controlled rates. Perhaps more important than directscavenging of hydroxyl radicals is our finding that ergothioneinebinds copper ions in a way that prevents them from generatingreactive oxygen species and free radicals from hydrogenperoxide. Our data show that ergothioneine is especiallyeffective in inhibiting copper-mediated oxidation of protein andDNA, and that the complex of ergothioneine with Cu(I) doesnot decompose to generate reactive oxygen species. It shouldbe noted that the level of carbonyl formation by the Cu(II)/ascorbate system is higher than that caused by the Cu(II)/H2O2

system. One possible reason for this might be the fact that whileH2O2 tends to maintain copper in its Cu(II) state, ascorbate canreduce Cu(II) to Cu(I), which can be oxidized back to Cu(II)by oxygen. The redox cycling between Cu(I) and Cu(II) thenwill produce a large amount of reactive oxygen species. Thisindicates that in the presence of “ubiquitous antioxidants” suchas ascorbate, loosely bound copper may use reactions with thoseantioxidants to produce oxidants unless the reactivity of copperis constrained. This may also explain why ergothioneineprovided better protection in the Cu(II)/ascorbate system,because ergothioneine could stabilize copper in its Cu(I) state.By contrast, other thiols, such as GSH, are rapidly oxidized bycopper ions with production of toxic radical species. Copperions also readily promote the oxidation of NAD(P)H, hemo-globin, erythrocyte membranes, and low-density lipoproteins (6).Hence, chelation of copper ions in a redox-inactive form might

Figure 5. Competition between ergothioneine and histidine for bindingof copper ions. The Cu(II)(histidine)2 complex was prepared by mixingCu(II) with histidine at a 1:2 molar ratio. Reaction mixtures contained2 mM Cu(II)(histidine)2, 2 mM ergothioneine, and 4 mM ascorbate:(A) Cu(II)(histidine)2, (B) Cu(II)(histidine)2 and ergothioneine, (C)Cu(II)(histidine)2 and ascorbate, and (D) Cu(II)(histidine)2, ascorbate,and ergothioneine.

Figure 6. Competition between ergothioneine and 1,10-phenanthrolinefor binding of Cu(I). The Cu(II)(1,10-phenanthroline)2 complex wasprepared by mixing Cu(II) with 1,10-phenanthroline at a 1:2 molar ratio,and Cu(I)(1,10-phenanthroline)2 was prepared by adding excess ascor-bate. Reaction mixtures contained 0.5 mM Cu(II)(1,10-phenanthroline)2,10 mM ergothioneine, and 10 mM ascorbate: (A) Cu(II)(1,10-phenanthroline)2 and ascorbate and (B) Cu(II)(1,10-phenanthroline)2,ascorbate, and ergothioneine.

Ergothioneine PreVents Damage to DNA and Protein Chem. Res. Toxicol., Vol. 24, No. 1, 2011 33

be a major biological function of ergothioneine in erythrocytesand other human tissues.

In summary, our data show that ergothioneine effectivelyprotects against copper-dependent oxidative damage to DNAand protein by forming a redox-inactive complex with Cu(I).Therefore, ergothioneine is a natural thiol compound with astrong copper chelating ability that is present in human tissuesat concentrations of up to 1 mM. Our data suggest thatergothioneine may be useful in the treatment of conditions whereexcess, redox-active copper plays a detrimental role, such asWilson’s disease, idiopathic copper toxicosis, and Indianchildhood cirrhosis, as well as ischemia-reperfusion injury andAlzheimer’s disease (12, 18, 33, 34).

Acknowledgment. The work in this paper was supported byProject 973 (2008CB418106); Hundred-Talent Project, CAS;NSFC Grants (20925724, 20777080, 20877081, 20890112 and20921063); National Institutes of Health Grants ES11497,RR01008, and ES00210 (B.-Z.Z.), and National Center forComplementary and Alternative Medicine Center of ExcellenceGrant AT002034 (B.F.). We also acknowledge the excellenttechnical assistance provided by Shantibhushan Jha, Jack Zhang,and Drs. Christopher Felix and William E. Antholine.

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