Ergothioneine Prevents Copper-Induced Oxidative Damage to DNA

Nov 3, 2010 - B.F.: Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR 97331; phone, (541) 737-5078; fax, (541) 737-507...
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Chem. Res. Toxicol. 2011, 24, 30–34

Ergothioneine Prevents Copper-Induced Oxidative Damage to DNA and 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-EnVironmental Sciences, Chinese Academy of Sciences, Beijing 100085, P. R. China, Linus Pauling Institute, Oregon State UniVersity, 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 found in up to millimolar concentrations in several tissues and biological fluids. However, the biological functions of ergothioneine remain incompletely understood. In this study, we investigated the role of ergothioneine in 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]. Oxidative damage to DNA and bovine serum albumin was measured as strand breakage and protein carbonyl formation, respectively. Ergothioneine (0.1-1.0 mM) provided strong, dose-dependent protection against oxidation of DNA and protein in both copper-containing systems. In contrast, only limited protection was observed with the purported hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Ergothioneine also significantly inhibited copper-catalyzed oxidation of ascorbate and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous copper, but not cupric copper, as demonstrated by UV-visible and low-temperature electron spin resonance techniques. We conclude that ergothioneine is a potent, natural sulfur-containing antioxidant that prevents copper-dependent oxidative damage to biological macromolecules by forming a redox-inactive ergothioneine-copper complex. Introduction L-Ergothioneine (2-mercaptohistidine trimethylbetaine) is a naturally occurring amino acid that contains an imidazole-2thione moiety (Figure 1). The thione group exists in a tautomeric thiol-thione equilibrium, with the thione being the predominant form at physiological pH, which distinguishes ergothioneine from other biological thiol compounds. Ergothioneine is synthesized by some bacteria and fungi, but not by animals (1, 2). Humans acquire ergothioneine from their diet, including mushrooms, oats, corn, and meat (1-3). Ergothioneine is found in brain, erythrocytes, liver, kidney, heart, seminal fluid, and ocular tissues (1-4). However, the biological functions of ergothioneine remain incompletely understood. Ergothioneine has been shown to exert radioprotective effects (5); scavenge singlet oxygen, hydroxyl radicals, hypochlorous acid, and peroxyl radicals (6-8); inhibit peroxynitrite-dependent nitration of proteins and DNA (9); protect retinal neurons from N-methylD-aspartate (NMDA)-induced excitotoxicity in vivo (10); and protect against diabetic embryopathy in pregnant rats (11). Ergothioneine also inhibits peroxynitrite-induced formation of

* To whom correspondence should be addressed. B.-Z.Z.: State Key Laboratory of Environmental Chemistry and Eco-Toxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, P. R. China; phone, 86-10-62849030; fax, 86-10-62923563; e-mail, [email protected]. B.F.: Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR 97331; phone, (541) 7375078; fax, (541) 737-5077; e-mail, [email protected]. † Chinese Academy of Sciences. ‡ Oregon State University. § Medical College of Wisconsin.

xanthine and hypoxanthine and their metabolite, urate, which may have important implications for gout and other inflammatory disorders (9). Copper is an essential trace element for humans (12, 13). It is a redox-active transition metal comprising the active center of a wide variety of metalloenzymes, such as Cu, Zn superoxide dismutase and cytochrome c oxidase (12, 13). Copper is also an essential component of chromatin and is closely associated with DNA bases, particularly near G-C sites (14-16). Although the essentiality of copper in biology is well-documented, copper can also be toxic (16-23). It is well-known that copper can induce oxidative damage to DNA, proteins, and lipids, thereby potentially contributing to disease pathology (21-23). Copper can accumulate in certain tissues and cells, such as hepatocytes, causing liver injury. Copper-induced injury has been hypothesized to result from the redox properties of copper, in particular its participation in a copper-catalyzed, Fenton-like reaction (19-23). Wilson’s disease, idiopathic copper toxicosis, and Indian childhood cirrhosis are examples of severe chronic liver diseases that result from genetic predisposition for hepatic

Figure 1. Chemical structure of ergothioneine.

10.1021/tx100214t  2011 American Chemical Society Published on Web 11/03/2010

Ergothioneine PreVents Damage to DNA and Protein

copper accumulation (12, 18). The serum copper concentration in these copper-overloaded patients has been shown to be 5-8 times higher than in healthy individuals (12, 18). Copper accumulating in the liver of Long Evans Cinnamon rats causes formation of hepatocellular carcinomas, suggesting that abnormal copper metabolism is involved in hepatic carcinogenesis in these animals (12, 18). A case-cohort study showed a U-shaped relationship between premorbid plasma copper levels and the risk of developing breast cancer (24). Ergothioneine has been shown to interact with metal ions and metalloenzymes in chemical systems (6, 25, 26). However, it is not clear whether and by what mechanism(s) ergothioneine can protect against copper-dependent oxidative damage to biological macromolecules, such as DNA, proteins, and lipids. In this study, we investigated the role of ergothioneine in copperdependent oxidative damage to DNA and bovine serum albumin, using two copper-containing systems: Cu(II) with ascorbate and Cu(II) with H2O2. Oxidative damage to DNA and albumin was measured as strand breakage and protein carbonyl formation, respectively. We found that ergothioneine strongly protects against copper-induced oxidative damage of DNA and protein by 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-tertbutylnitrone (POBN), cupric sulfate, ascorbate, and hydrogen peroxide were purchased from Sigma (St. Louis, MO). All buffer solutions were treated with Chelex to remove adventitious metals. Protein Carbonyl Formation. BSA (1 mg/mL) was oxidized with Cu(II) and ascorbate or Cu(II) and H2O2 [0.1 mM Cu(II), 1 mM ascorbate, and 1 mM H2O2] in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 1 h. Protein carbonyls were assayed as described by Levine et al. (27). Briefly, 1 mL of BSA solution was mixed with 0.5 mL of 10 mM DNPH in 2 N HCl. The mixture was incubated at room temperature for 1 h, followed by the addition of 0.5 mL of 20% trichloroacetic acid. The sample was incubated on ice for 10 min and centrifuged in a benchtop centrifuge at 3000g for 10 min. The protein pellet was washed three times with 3 mL of an ethanol/ethyl acetate mixture (1:1, v/v) and dissolved in 1 mL of 6 M guanidine (pH 2.3). The peak absorbance at 370 nm was measured to quantitate protein carbonyls. Data were expressed as nanomoles of carbonyl groups per milligram of protein, using a molar absorption coefficient of 22000 M-1 cm-1 for the DNPH derivatives (27). DNA Damage. The conversion of covalently closed circular double-stranded supercoiled DNA (form I) to a relaxed open circle form (form II) and a linear form (form III) was used to investigate DNA strand breakage induced by Cu(II) and ascorbate or Cu(II) and H2O2 (28). The experiments were conducted via incubation of plasmid pBR322 DNA (0.5 µg) at 37 °C for 1 h in Chelex-treated sodium phosphate buffer (0.1 M, pH 7.4) with the Cu(II)-containing systems, in the absence or presence of the indicated concentrations of ergothioneine or other agents. Redox Activity of Copper. The redox activity of copper was determined by measuring the rate of copper-catalyzed oxidation of ascorbate. Ergothioneine and Cu(II) (1 µM) were added to a solution of 0.1 mM ascorbate in 0.1 M phosphate buffer (pH 7.4), and the sample was incubated at room temperature. The oxidation of ascorbate was monitored spectrophotometrically at 265 nm. The extinction 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 spintrapping agent, R-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN, 100 mM), in Chelex-treated phosphate buffer (100 mM, pH 7.4),

Chem. Res. Toxicol., Vol. 24, No. 1, 2011 31 Table 1. Effect of Ergothioneine, DMSO, Mannitol, and the Copper Chelating Agent, Bathocuproine Disulfonate, on Protein Carbonyl Formation Induced by Cu(II) with Ascorbate or Cu(II) with H2O2a Cu(II) with ascorbate compound Control Ergothioneine (0.1 mM) Ergothioneine (0.2 mM) DMSO (100 mM) Mannitol (100 mM) Bathocuproine disulfonate (1 mM)

Cu(II) with H2O2

carbonyls inhibition carbonyls inhibition (nmol/mg) (%) (nmol/mg) (%) 44.7 7.6 0.1 34.8 39.3 0.1

83.0 99.7 22.1 12.1 99.7

25.5 15.2 7.9 18.3 18.1 0.1

40.0 69.0 28.2 29.1 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 of three separate experiments.

with or without ergothioneine. Ergothioneine was added to the reaction mixture 1 min before H2O2 was added. ESR spectra were recorded at room temperature in a Bruker ER 200 D-SRC spectrometer operating at 9.8 GHz in a cavity equipped with a Bruker Aquax liquid sample cell. Typical spectrometer parameters were as follows: scan range, 100 G; field set, 3470 G; time constant, 200 ms; scan time, 100 s; modulation amplitude, 1.0 G; modulation frequency, 100 kHz; receiver gain, 1.25 × 104; and microwave power, 9.8 mW. Low-Temperature ESR Studies. ESR spectra were recorded using a Varian E109 Century Series spectrometer at a low temperature (77 K). The sample was placed in a standard quartz ESR tube prior to being frozen. The sides of the tube were then warmed slightly to allow the frozen sample to slide into a finger Dewar filled with liquid nitrogen for the acquisition of spectra. Spectral measurements of the Cu(II)(histidine)2 complex were conducted in 0.1 M phosphate buffer (pH 7.4) with or without ergothioneine. UV-Visible Spectral Analysis. Competition between ergothioneine and histidine or 1,10-phenanthroline to bind copper ions was assessed in a Beckman DU-640 spectrophotometer. Cu(II)(histidine)2 and Cu(II)(1,10-phenanthroline)2 complexes were prepared by mixing Cu(II) with either histidine or 1,10-phenanthroline at a 1:2 molar ratio. Reduction of Cu(II) to Cu(I) was accomplished by adding an excess amount of ascorbate, and the spectra were recorded between 400 and 800 nm at room temperature.

Results Oxidation of proteins results in the formation of carbonyl groups in quantities that reflect the extent of the oxidative damage. Thus, in this study, copper-mediated oxidative damage to BSA was measured by protein carbonyl formation. Incubation of BSA (1 mg/mL) for 1 h at 37 °C with Cu(II) and ascorbate or Cu(II) and H2O2 [0.1 mM Cu(II), 1 mM ascorbate, and 1 mM H2O2] led to the formation of 44.7 or 25.5 nmol of protein carbonyls/mg of protein, respectively (Table 1). Ergothioneine markedly inhibited protein carbonyl formation in a concentration-dependent manner in both copper-containing systems. In contrast, only limited inhibition was observed with DMSO and mannitol, even at concentrations as high as 100 mM (Table 1). For example, protein carbonyl formation induced by Cu(II) with ascorbate was inhibited by ∼80 and ∼20% by 0.1 mM ergothioneine and 100 mM DMSO, respectively, and was completely inhibited by 0.2 mM ergothioneine (Table 1). Ergothioneine also markedly inhibited DNA damage in a concentration-dependent manner (0.1-1.0 mM) in both coppercontaining systems, Cu(II) with ascorbate (Figure 2A) and Cu(II) with H2O2 (Figure 2B). Histidine was less effective than ergothioneine in inhibiting DNA damage, especially in the Cu(II)/ascorbate system (Figure 2), suggesting that the thiol

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Figure 3. Inhibition by ergothioneine of Cu(II)-catalyzed oxidation of ascorbate. Reactions were conducted in 0.1 M phosphate buffer (pH 7.4) at room temperature for 15 min. Reaction mixtures contained 0.1 mM 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 µM ergothioneine, (D) Cu(II), ascorbate, and 10 µM ergothioneine, and (E) ascorbate only. The oxidation of ascorbate was monitored spectrophotometrically at 265 nm.

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

group of ergothioneine is critical for its inhibitory effect on Cu(II)/ascorbate system-induced DNA damage. In contrast, no inhibition was observed with DMSO and mannitol, even at concentrations as high as 100 mM (data not shown). The observations that protein carbonyl formation and DNA damage were strongly inhibited by ergothioneine, but not by DMSO or mannitol, indicate that the effect of ergothioneine is not due to scavenging of hydroxyl radicals. In contrast, the inhibition of copper-induced protein oxidation and DNA damage by the known copper chelating agent, bathocuproine disulfonate (Table 1 and Figure 2), suggests that the effect of ergothioneine might be due to binding of copper and inhibition of its redox activity. It should be noted that BSA has a specific high-affinity copper binding site, rendering the copper redox-inactive. However, this does not change the conclusion reached above because the molar concentration of BSA is ∼0.015 mM while the Cu(II) concentration employed in this study is 0.1 mM. Although it has been shown that ergothioneine can bind copper and form ergothioneine-copper complexes under nonphysiological conditions (25, 26), little is known about the effect of ergothioneine on the redox activity of copper and whether ergothioneine combines preferentially with Cu(II) or Cu(I) at physiological pH. Hence, we investigated the effect of ergothioneine on the redox activity of copper by measuring Cu(II)catalyzed oxidation of ascorbate. We found that this reaction was significantly inhibited in a dose-dependent manner by

Figure 4. Inhibition by ergothioneine of copper-catalyzed POBN-CH3 adduct formation. Reactions were conducted at room temperature in Chelex-treated phosphate buffer (100 mM, pH 7.4). All reaction mixtures contained POBN (100 mM), Cu(II) (0.1 mM), H2O2 (1 mM), ascorbate (0.1 mM), and DMSO (5%). Where indicated, ergothioneine was added to the reaction mixture 1 min before the addition of H2O2. The hyperfine splitting constants for the POBN-CH3 adduct were as follows: aN ) 15.96 G, and aβH ) 2.74 G. The overlapping signal in the center of the spectra was identified as ascorbate radical.

ergothioneine (Figure 3), but not by histidine (data not shown). These results suggest that ergothioneine forms a redox-inactive complex with copper. To further support this notion, we studied the effect of ergothioneine on free radical production by a copper-catalyzed, Fenton-like reaction. The most direct technique for detecting free radicals is ESR spectroscopy using spin-trapping agents (30). Hydroxyl radicals can be detected by ESR spectroscopy using POBN as a spin-trapping agent. As shown in Figure 4, the interaction of ascorbate with H2O2 and Cu(II) produced POBN-CH3 adducts in the presence of DMSO and POBN, suggesting that hydroxyl radicals or equivalent reactive intermediates were generated from the ascorbate/H2O2/Cu(II) system. Ergothioneine progressively decreased the level of formation of 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 formation was observed (Figure 4). Because the concentrations of ergothioneine (0.1-1 mM) used in these experiments were much lower than the concentrations of POBN (100 mM) and DMSO (700 mM), the inhibition of POBN-CH3 adduct formation by ergothioneine cannot be due to its scavenging of hydroxyl radicals or equivalent reactive intermediates produced by the ascorbate/H2O2/Cu(II) system, but most likely is due to its ability to 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-

Ergothioneine PreVents Damage to DNA and Protein

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stabilize it in the Cu(I) state. All the results described above strongly suggest that ergothioneine may combine with Cu(I) to form a redox-inactive ergothioneine-Cu(I) complex, most probably with a molar ratio of 2:1.

Discussion

Figure 5. Competition between ergothioneine and histidine for binding of copper ions. The Cu(II)(histidine)2 complex was prepared by mixing Cu(II) with histidine at a 1:2 molar ratio. Reaction mixtures contained 2 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-phenanthroline for binding of Cu(I). The Cu(II)(1,10-phenanthroline)2 complex was prepared 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 ascorbate. Reaction mixtures contained 0.5 mM Cu(II)(1,10-phenanthroline)2, 10 mM ergothioneine, and 10 mM ascorbate: (A) Cu(II)(1,10phenanthroline)2 and ascorbate and (B) Cu(II)(1,10-phenanthroline)2, ascorbate, and ergothioneine.

tidine)2 and Cu(I)(1,10-phenanthroline)2. It is known that histidine binds Cu(II) in a redox-active manner to form an intensely 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 absorbance at 640 nm (Figure 5), suggesting that ergothioneine cannot compete effectively with histidine for Cu(II). In contrast, in the presence of ascorbate (10 mM), addition of ergothioneine (2 mM) led to a dramatic decrease in the absorbance at 640 nm (Figure 5), suggesting that ergothioneine can compete effectively with histidine for Cu(I). Similar experiments with the Cu(I)(1,10phenanthroline)2 complex, which was prepared by mixing the Cu(II)(1,10-phenanthroline)2 complex with ascorbate, showed that ergothioneine effectively competes with 1,10-phenanthroline for Cu(I) (Figure 6). These data indicate that in the presence of ascorbate, ergothioneine reacts with Cu(I) to form a redoxinactive ergothioneine-Cu(I) complex. To further confirm this notion, we employed low-temperature ESR. The Cu(II)(histidine)2 complex has a characteristic ESR spectrum at a low temperature (77 K), showing the typical pattern of a square-planar copper complex (31). The Cu(II)(histidine)2 ESR signal completely disappeared when both ergothioneine and ascorbate were added (data not shown). These ESR results indicate that in the presence of ascorbate, ergothioneine can compete effectively with histidine to bind copper and

Ergothioneine is synthesized exclusively by fungi and mycobacteria (1, 2) and is present in human food items in highly variable amounts. By far the highest levels of ergothioneine are found in mushrooms (0.1-1.0 mg/g of dried material) (2). Mammals ingest ergothioneine and, interestingly, conserve it with minimal metabolism (1). Ergothioneine in its free form is stored in tissues that may be exposed to oxidative stress, such as erythrocytes, seminal fluid, liver, kidney, heart, and ocular tissues, where ergothioneine can reach up to millimolar concentrations (1). In addition, micromolar concentrations of ergothioneine are found in the brain, another tissue potentially exposed to oxidative stress (1). Since its discovery in 1909, the physiological role of ergothioneine, if any, has remained elusive (1). Most authors consider it an intracellular antioxidant. However, intracellular concentrations of ergothioneine (∼0.1-1 mM) are considerably lower than those of the ubiquitous hydrophilic antioxidants, glutathione (GSH) and ascorbate, which are present intracellularly at ∼3-7 and ∼1-4 mM, respectively (32). Therefore, the question of whether ergothioneine, when compared to other common antioxidants and thiol compounds, including GSH and cysteine, plays a unique biological role arises. Interestingly, it has been shown that ergothioneine, unlike GSH and ascorbate, does not autoxidize at physiological pH in the presence of transition metal ions, such as copper or iron (1). In general, ergothioneine has been suggested to serve as a powerful catalytic scavenger of oxidizing species that are not free radicals (32). By contrast, GSH and ascorbate are regarded as free radical scavengers. Ergothioneine has been shown to scavenge hydroxyl radicals. However, most biological molecules react with hydroxyl radicals at diffusion-controlled rates. Perhaps more important than direct scavenging of hydroxyl radicals is our finding that ergothioneine binds copper ions in a way that prevents them from generating reactive oxygen species and free radicals from hydrogen peroxide. Our data show that ergothioneine is especially effective in inhibiting copper-mediated oxidation of protein and DNA, and that the complex of ergothioneine with Cu(I) does not decompose to generate reactive oxygen species. It should be 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 while H2O2 tends to maintain copper in its Cu(II) state, ascorbate can reduce Cu(II) to Cu(I), which can be oxidized back to Cu(II) by oxygen. The redox cycling between Cu(I) and Cu(II) then will produce a large amount of reactive oxygen species. This indicates that in the presence of “ubiquitous antioxidants” such as ascorbate, loosely bound copper may use reactions with those antioxidants to produce oxidants unless the reactivity of copper is constrained. This may also explain why ergothioneine provided 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 by copper ions with production of toxic radical species. Copper ions also readily promote the oxidation of NAD(P)H, hemoglobin, erythrocyte membranes, and low-density lipoproteins (6). Hence, chelation of copper ions in a redox-inactive form might

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be a major biological function of ergothioneine in erythrocytes and other human tissues. In summary, our data show that ergothioneine effectively protects against copper-dependent oxidative damage to DNA and protein by forming a redox-inactive complex with Cu(I). Therefore, ergothioneine is a natural thiol compound with a strong copper chelating ability that is present in human tissues at concentrations of up to 1 mM. Our data suggest that ergothioneine may be useful in the treatment of conditions where excess, redox-active copper plays a detrimental role, such as Wilson’s disease, idiopathic copper toxicosis, and Indian childhood cirrhosis, as well as ischemia-reperfusion injury and Alzheimer’s disease (12, 18, 33, 34). Acknowledgment. The work in this paper was supported by Project 973 (2008CB418106); Hundred-Talent Project, CAS; NSFC Grants (20925724, 20777080, 20877081, 20890112 and 20921063); National Institutes of Health Grants ES11497, RR01008, and ES00210 (B.-Z.Z.), and National Center for Complementary and Alternative Medicine Center of Excellence Grant AT002034 (B.F.). We also acknowledge the excellent technical assistance provided by Shantibhushan Jha, Jack Zhang, and Drs. Christopher Felix and William E. Antholine.

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