The antioxidant action of ergothioneine

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 288, No. 1, July, pp. 10-16, 1991

The Antioxidant Dola Akanmu,*

Action of Ergothioneine

Rubens Cecchini,j- Okezie I. Aruoma,*

and Barry HalliwelL#?

*Department of Biochemistry, University of London Kings College, Strand, London WC2R 2LS United Kingdom; tDepartment of General Pathology, University of Londrina, Londrina CEP 86051, Brazil; and SDivision of Pulmonary-Critical Care Medicine, University of California, Davis Medical Center, 4301 X Street, Sacramento, California 95817

Received August 8, 1990, and in revised form February

20, 1991

Ergothioneine is a product of plant origin that accumulates in animal tissues. Its suggested ability to act as an antioxidant has been evaluated. Ergothioneine is a powerful scavenger of hydroxyl radicals (‘OH) and an inhibitor of iron or copper ion-dependent generation of ‘OH from hydrogen peroxide (H,O,). It is also an inhibitor of copper ion-dependent oxidation of oxyhaemoglobin, and of arachidonic acid peroxidation promoted by mixtures of myoglobin (or haemoglobin) and H202. Ergothioneine is a powerful scavenger of hypochlorous acid, being able to protect q-antiproteinase against inactivation by this molecule. By contrast, it does not react rapidly with superoxide (0;) or hydrogen peroxide (H,O,) and it does not inhibit microsomal lipid peroxidation in the presence of iron ions. Overall, our results show that ergothioneine at the concentrations present in . . uivo could act as an antioxidant. o 1991 Academic PESS, IIIC.

Ergothioneine, which exists in two tautomeric forms (thiol and thione, Fig. l), of which the latter is favoured, was originally discovered in Clavicepspurpurea (the ergot fungus) and later identified as the betaine of 2-thiol-Lhistidine (1, 2). Early work on ergothioneine reported its presence in rat erythrocytes and liver and showed it to arise from dietary plant material (1,3), although the exact distribution of ergothioneine in the plant kingdom has not been fully investigated, probably because of the lack of an adequate assay applicable to plant material. However, the presence of ergothioneine in human, rat, and other animal tissues has been confirmed by HPLC (4-7) and spin-echo NMR (8). The function of ergothioneine in animal and plant tissues is unknown: suggestions that it is a neurotransmitter or diabetogenic agent have not been convincing (l-4). Ergothioneine has been shown to form stable complexes ’ To whom correspondence

should be addressed.

with several metal ions, including iron and copper ions (4, 9, lo), and it has been suggested that ergothioneine might function as an antioxidant in vivo (reviewed in Ref. (4)). Consistent with this view, it has been shown that ergothioneine inhibits generation of singlet oxygen by quenching the excited states of photosensitizers (4, 11, 12). Ergothioneine has additionally been reported to react with ‘OH at an almost diffusion-controlled rate (rate constant 1.2 X 10” M-’ s-l as measured by pulse radiolysis; Ref. (13)), to inhibit lipid peroxidation in liver homogenates (14) and to interact with haem proteins (4, 15, 16). Such an interaction might not always be beneficial, however. Thus ergothioneine stimulated oxidation of NADH by myoglobin or haemoglobin in the presence of Mn2+ ions, with production of superoxide radical (0,) and hydrogen peroxide (H,O,) (16). By contrast, ergothioneine diminished the mutagenicity of cumene and t-butylhydroperoxides to a Salmonella strain (17). However, many compounds have been suggested to act as antioxidants in vivo and such suggestions need to be critically evaluated. Thus it is important to know whether the rate at which the putative “antioxidant” react with biologically relevant reactive oxygen species (ROS)2 would allow them to compete with other molecules for such species in vivo (18). Biologically relevant ROS include 0,) H20z, hypochlorous acid (HOCl), hydroxyl radical (‘OH), lipid peroxides, singlet oxygen (often written as ‘02) and haem proteins in the presence of H20z (18-22). For example, exposure of myoglobin to H,O, generates a species (probably a tyrosine peroxyl radical (Ref. (23)) that can cause peroxidation of membrane lipids and fatty acids (22). Reaction of oxyhaemoglobin with H,O, generates a similar damaging species (24). The binding of copper and iron ions to chelating agents sometimes prevents these ions from participating in free radical reactions such as

’ Abbreviations used: ROS, reactive oxygen species; TBA, thiobarbituric acid; NBT, nitro-blue tetrazolium.

10 All

0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.

ERGOTHIONEINE s-11+

7KH,)+,

yw3 -CH2-C-COO7

H 6

AS AN ANTIOXIDANT

IN ANIMAL

Ergothioneine globin.

11

TISSUES

alone did not cause any spectral changes in oxyhaemo-

-CH2-C-COO/-\

RESULTS Scavenging of Hydroxyl Radicals and Prevention Formation from HzOz by Ergothioneine

FIG.

1.

Structure

of the thiol and thione forms of ergothioneine.

lipid peroxidation and the generation of ‘OH from HzOz, but in other cases the reactivity of the ions can be increased by chelation (25). Since ergothioneine is known to chelate metal ions, it is important to study whether or not this chelation inhibits the ability of these metal ions to cause free radical damage. In this paper, we present a series of studies to test the feasibility of the proposal that ergothioneine exerts an antioxidant effect in vivo (4). MATERIALS

AND

METHODS

Ergothioneine, horse-heart myoglobin, and human haemoglobin were from Sigma Chemical Co., Ltd. (UK). Other reagents were of the highest quality available from either Sigma or from BDH Chemicals Ltd. (UK). Superoxide dismutase was the bovine erythrocyte copper-zinc-containing protein. a,-Antiproteinase was Sigma type A9024. Human haemoglobin and horse heart myoglobin were purified, and oxyhaemoglobin was prepared by dithionite reduction and sephadex gel filtration, as described in Refs. (24,26). Solutions of ergothioneine were made up immediately before use. Elastase and a,-antiproteinase were assayed essentially as described in Wasil et al. (Ref. (27)); full details are given in the legend to Table II. HOC1 was produced immediately before use by adjusting sodium hypochlorite (Na+OCll) to pH 6.2 with dilute H,SO, (27). Hydroxyl radical formation was measured in the presence of ascorbate, Fe& (*EDTA), and H,O, by the deoxyribose assay (28). Generation of 0; by the hypoxanthine-xanthine oxidase system was carried out essentially as described by Halliwell (29). Reaction mixtures contained, in a final volume of 3 ml, 0.1 ml of 30 mM EDTA, 100 ~1 of 3 mM cytochrome c or 3 mM nitroblue tetrazolium, 10 pl of 30 mM hypoxanthine KH*PO,-KOH buffer, in 50 mM KOH, and 50 mM (final concentration) pH 7.4. Reaction was started by adding 0.2 ml of xanthine oxidase (freshly diluted to give 1 unit, as defined in the Sigma catalogue, of enzyme activity per milliliter) and the rate of NBT or cytochrome c reduction measured at 560 or 550 nm, respectively, at 25°C. H202 was measured by a peroxidase-based assay system (30). Rat liver microsomes were prepared by differential pelleting and their peroxidation was measured by the thiobarbituric acid (TBA) test (31) except that the TBA reagents also contained 0.02% (w/v) butylated hydroxytoluene to suppress peroxidation during the test itself (32). Peroxidation of arachidonic acid by the myoglobin/H,O, system was studied in reaction mixtures containing the following reagents at the final concentrations stated: 25 mM KH,PO,-KOH buffer, pH 7.4, 0.4 mM arachidonic acid, 50 I.~M myoglobin, 0.5 mM H,O,, and 100 @M diethylenetriaminepenta-acetic acid to bind any metal ions present. Tubes were incubated at 30°C for 10 min. Peroxidation was measured by the TBA test as described above. In some experiments, metmyoglobin was replaced by oxyhaemoglobin at a final concentration of 20 +M (as haem). For studies of the protection of oxyhaemoglobin by ergothioneine against copper ion-dependent oxidation, reaction mixtures contained, in a final volume of 1 ml, 20 mM KH,PO,-KOH buffer, pH 7.4, 20 ELM oxyhaemoglobin and, where indicated, 80 FM CuSO, and/or 3 mM ergothioneine.

of their

A mixture of FeCl,-EDTA, HzOn, and ascorbic acid at pH 7.4 generates ‘OH radicals, which can be detected by their ability to degrade the sugar deoxyribose into fragments that, on heating with TBA at low pH, generate a pink chromogen (28). Fe”+-EDTA

+ ascorbate + Fe’+-EDTA

Fe’+-EDTA

+ oxidized ascorbate

+ HzOz + Fe3+-EDTA

+ ‘OH + OH-.

[l]

PI

Provided that an excess of EDTA is used, any ‘OH generated by reaction [2] that escapes direct scavenging by EDTA enters “free solution” and is equally accessible to deoxyribose (the detector molecule) and to any other scavenger of ‘OH added (28). Thus the ability of a substance to inhibit competitively deoxyribose degradation under these reaction conditions is a measure of its ability to scavenge ‘OH and can be used to calculate a rate constant for reaction of ‘OH with the scavenger (28,33). Pulse radiolysis studies have shown that ergothioneine is a powerful scavenger of ‘OH (13). Figure 2 shows that the deoxyribose assay confirms this ability in a biologically

FIG. 2. Action of ergothioneine on degradation of deoxyribose by hydroxyl radicals generated by reaction of HzOz with iron ions. Reactions mixtures contained, in a final volume of 1.0 ml, the following reagents at the final concentrations stated: 10 mM KH,PO,-KOH buffer, pH 7.4, 1.42 mM H,O*, 2.8 mM deoxyribose, 20 pM FeCl, (premixed with 100 fiM EDTA before addition to the reaction mixture, where stated). Ascorbate (0.1 mM) was added to start the reaction and the tubes were incubated at 37°C for 1 h. Products of ‘OH attack upon deoxyribose were measured as in Ref. (28). (Line A) EDTA present. A rate constant of 1.59 X 10”’ Mm’ s-i can be calculated from this data using the method described in Ref. (29). (Line B) EDTA absent.

12

AKANMU

relevant ‘OH-generating system. Scavenging of ‘OH by ergothioneine was competitive with deoxyribose. A rate constant of (1.6 + 0.2) X 10” M-’ so’ was calculated by the method described in Ref. (28) from the results of four such experiments, in good agreement with the value of 1.2 X lOlo M-r s-l measured by pulse radiolysis (13). Control experiments showed that ergothioneine did not interfere with the measurement of deoxyribose degradation (it had no effect when added with the TBA reagents) or itself react with ‘OH to give TBA-reactive material (omission of deoxyribose from the reaction mixture completely abolished colour formation). However, many molecules present in uiuo can scavenge ‘OH with equally high rate constants (18). The deoxyribose assay was therefore used to examine the ability of ergothioneine to inhibit transition metal ion-dependent ‘OH generation. When iron ions are added to the reaction mixture as FeC& instead of as FeCl,,-EDTA, some of the iron ions form a complex with deoxyribose (34). This complex can be reduced by ascorbate to give Fe’+, which remains attached to deoxyribose (35) and can subsequently react with Hz02. This reaction appears to give ‘OH, which immediately attacks the deoxyribose in a sitespecific manner (18, 33-35), and cannot be intercepted by most ‘OH scavengers. The only molecules that can inhibit deoxyribose degradation under these reaction conditions are those that can bind the iron ions in the reaction mixture and render them inactive or poorly active in Fenton reactions (33, 34). Figure 2 (line B) shows that ergothioneine at low concentrations was able to inhibit iron ion-dependent deoxyribose degradation under these reaction conditions. The kinetics of the inhibition were complex and a maximum inhibition of 48-56% (three experiments) was obtained with 1.5-2.0 mM of ergothioneine. These data suggest that ergothioneine has some ability to bind iron ions into chelates poorly active in generating ‘OH. When Cu2+ ions are incubated with H202 and ascorbic acid, a number of reactive species are produced, one of which is ‘OH (18,25,36). Table I shows that ergothioneine was a powerful inhibitor of copper ion-dependent deoxyribose degradation: concentration ratios of 0.5:1 ergothioneine to copper ion inhibited-OH generation by 83 t 6% (in four experiments) and a 1:l ratio inhibited ‘OH formation completely, in five experiments. Action of Ergothioneine on Copper Ion-Dependent Oxidation of Haemoglobin Copper ions cause a rapid oxidation of oxyhaemoglobin (37,38). Incubation of 20 pM oxyhaemoglobin with 80 pM CuSO., caused a drop in oxyhaemoglobin concentration to 11 pM within 5 min (as calculated from absorbance measurements at 577, 560, and 630 nm; Refs. (37, 38)). Addition of ergothioneine to the reaction mixture (at

ET AL. TABLE Inhibition

[Ergothioneine] (mM) 0.00 0.10 0.20 0.50 1.00 1.25 1.50 2.00

of Copper Degradation

I

Ion-Dependent Deoxyribose by Ergothioneine Extent of deoxyribose degradation AS,,

Inhibition of deoxyribose degradation (%I

0.906 0.614 0.405 0.157 0.000 0.000 0.000 0.000

0 32 55 83 100 100 100 100

Note. Results of a representative experiment are shown. Reaction mixtures contained, in a final volume of 1.2 ml, 10 mM KH,PO,-KOH buffer, pH 7.4, 1.42 mM H,02, 1 mM CuS04, and 1 mM ascorbate, added to start the reaction. Tubes were incubated at 37OC for 1 h and formation of products of ‘OH attack upon deoxyribose measured as in Ref. (28). Results have been corrected for the rate of deoxyribose degradation observed in the absence of added Cu”, which is due to trace iron ion contamination in the reagents (34). Ergothioneine, when added, was present in the reaction mixture at the final concentrations stated, before adding copper ions.

concentrations equimolar to or greater than, those of Cu*+) before adding Cu2+ ions prevented the haemoglobin oxidation. Figure 3 depicts a representative experiment (in which 3 mM ergothioneine was used). Ergothioneine itself produced no spectral changes in oxyhaemoglobin. Protection by Ergothioneine of a,-Antiproteinase Inactivation by Hypochlorous Acid

against

One of the most important targets attacked by HOC1 in uivo is cr,-antiproteinase (aI-AP), the major inhibitor in body fluids of serine proteinases such as elastase (21, 39). ai-AP is rapidly inactivated by HOCl, losing its ability to inhibit elastase (39). Although many compounds can react with HOCl, few do so sufficiently rapidly to protect ol,-AP against inactivation. A good test of whether a compound might be capable of scavenging HOC1 at a biologically significant rate is, therefore, to examine its ability, at the concentrations present in uiuo, to protect (r,-AP against inactivation by HOC1 (18, 27, 40, 41). Table II shows that (rl-AP inhibited elastase: a concentration just sufficient to inhibit completely was used (this concentration is comparable to that of (Y~-AP present in human plasma; Ref. (27)). Treatment of the cul-AP with 60 PM HOC1 almost completely abolished its elastase-inhibitory capacity (Table II, third line). However, if ergothioneine was included in the reaction mixture, it protected oll-AP against inactivation by HOCl. A concentration of 500 PM protected almost completely (82 + 10% protection in eight

ERGOTHIONEINE

AS AN ANTIOXIDANT

IN ANIMAL

TABLE Action

13

TISSUES II

on the Ability

of Ergothioneine

the Elastase-Inhibitory

Capacity

of HOC1 to Inactivate

of cY,-Antiproteinase

Elastase activity in final reaction mixture L+L,ol~~c)

Addition to first reaction mixture

(cri-AP)

Activity of a,-AP in inhibiting elastase (100. % of elastase activity detected)

Buffer only

0.042

Buffer,n,-AP

0.000

100

Buffer, RM + RM + RM + RM +

0.036 0.017 0.011 0.005 0.008

14 60 14 88 81

a,-AP, HOC1 (RM) ERT (0.05 mM) ERT (0.10 mM) ERT (0.50 mM) ERT (2.00 mM)

Note. cu,-Antiproteinase (1.2 mg/ml), HOC1 (60 PM), and ergothioneine (if present) were incubated in a final volume of 1.0 ml in phosphatebuffered saline at pH 7.4 (full details in Ref. (27)) for 20 min at 25°C to allow HOC1 to inactivate a,-AP. Then 2 ml of buffer and 0.05 ml of porcine pancreatic elastase solution (27) were added, followed by incubation at 25°C for a further 20 min to allow any active ni-AP remaining to inhibit elastase. (Any HOC1 remaining is diluted to a point at which it cannot affect elastase itself.) The uninhibited elastase is then measured with a rise in A,,,. by adding elastase substrate, which is hydrolysed Concentrations of ergothioneine quoted were those present in the first (1.0 ml) reaction mixture. Ergothionine (ERT) was mixed with (u,-AP and buffer immediately before adding HOCl.

WAVELENGTH nm

FIG. 3. The inhibition of the effect of Cue+ on oxyhaemoglobin by ergothioneine. (Line A) Oxyhaemoglobin (20 GM). (Line B) A plus 80 fiM CuSO+ (Line C) Ergothioneine (3 mM) was premixed with oxyhaemoglobin prior to the addition of copper ions as in B. Reagents were contained in 20 mM KH,PO,-KOH buffer, pH 7.4.

experiments). Raising the ergothioneine concentration to 2 mM did not increase the protection observed. Control experiments showed that ergothioneine did not inhibit elastase directly, nor did it interfere with the ability of (rl-AP to inhibit elastase. It is concluded that ergothioneine is a good scavenger of HOCL Action of Ergothioneine on Lipid Per-oxidation Stimulated by FeC&/Ascorbate and Myoglobin/H202 or Haemoglobin/H202 The rate of peroxidation of rat liver microsomes in the presence of FeC13 plus ascorbate (both at 100 PM), or of ferrous ammonium sulphate (100 PM) was measured by the thiobarbituric acid test. In none of the eight experiments did ergothioneine, tested up to a final concentration of 2 mM, have any effect on the rate of lipid peroxidation. In incubation mixtures containing FeCl, (100 PM) only, ergothioneine (2 mM) did not increase the low rate of peroxidation observed, i.e., the ergothioneine/FeCl, system did not stimulate peroxidation.

Peroxidation of certain lipids, such as arachidonic acid, can also be induced by a mixture of metmyoglobin and H202. Ergothioneine inhibited myoglobin/H,O,-dependent peroxidation of arachidonic acid: Table III shows some representative results. Addition of HzOz to myoglobin produced the expected (22,24) spectrum of myoglobin

TABLE

III

Action of Ergothioneine on Peroxidation of Arachidonic by the Myoglobin/HzOz System Extent of peroxidation A 632

[Ergothioneine] (mM)

0.000

myoglobin) H,O,)

0.244 0.515 0.459 0.346 0.333 0.289 0.280 0.275 0.244

0.15 0.30 0.60 1.20 1.80 2.40 3.00 3.00 (omit H,O,) Note. Reaction Methods. stated.

% Inhibition

0.540

0 0 (omit 0 (omit

Acid

mixtures

Ergothioneine

was

8 27 66 70 85 88 90 100

were as described added

to give

the

under Materials final

concentrations

and

14

AKANMU

ET AL.

(IV). However, if ergothioneine was present the spectrum was very similar to that of myoglobin (III), as shown in Fig. 4. Similarly, ergothioneine inhibited peroxidation of arachidonic acid by the oxyhaemoglobin/H202 system to a comparable extent. Table IV shows some representative data. Reaction of Ergothioneine

with 0, and H,O,

A mixture of hypoxanthine and xanthine oxidase at pH 7.4 generates 02, which can be detected by its ability to reduce ferricytochrome c or nitro-blue tetrazolium, NBT (42,43). Any added compound that is itself able to react with 0; should decrease the rate of reduction of these substances. Ergothioneine, tested at concentrations up to 3 mM (which did not themselves reduce cytochrome c or NBT directly under our reaction conditions) had no significant effect on the rate of reduction of 100 PM ferricytochrome c, whereas superoxide dismutase inhibited its reduction by >90%. 3 mM ergothioneine inhibited reduction of 100 PM NBT by <25%, although NBT reduction was inhibited >90% by superoxide dismutase. At pH 7.4 cytochrome c reacts with 0; with a rate constant of about 2.6 X lo5 Mm’ s-l, whereas NBT reacts with a rate constant of about 6 X lo4 Mm1 s-l (44). The inability of ergothioneine, at concentrations 30 times greater than that of NBT or cytochrome c, to significantly inhibit their reduction by 0, suggest that its reaction with 0, , if any,

O-50

TABLE

IV

Inhibition by Ergothioneine of the Peroxidation of Arachidonic Acid by the Oxyhaemoglobin/H,02 System

Additions to reaction mixture AA, RM RM RM RM RM RM

Oxyhaem, HZ02 (RM) + ERT (0.3 mM) t ERT (0.6 mM) + ERT (1.2 mM) + ERT (1.8 mM) + ERT (2.0 mM) + ERT (3.0 mM)

Extent of AA peroxidation A,,*

% Inhibition

0.178 0.105

0.092 0.091 0.085 0.078 0.080

41 48

49 52 56 55

Note. Reaction mixtures contained (where appropriate), in a final volume of 1.0 ml, the following reagents at the final concentrations tested: arachidonic acid (AA) (0.4 mM), H,O, (200 PM), oxyhaemoglobin (prepared as described under Materials and Methods, 20 FM [as oxyhaem]), 25 mM KH2P0,-KOH buffer, pH 7.4, ergothioneine (ERT) as acid to bind any indicated, and 100 pM diethylenetriaminepentaacetic released metal ions. Representative data are given, but the results were reproducible in three experiments. Results have been corrected for the background level of peroxidation observed when haemoglobin alone was added to the arachidonic acid.

cannot proceed with M-

a rate constant

much above lo3

1 s-1

No evidence for a reaction of ergothioneine (tested up to 2 mM) with HzOz was obtained using a peroxidasebased assay system (30). DISCUSSION

A

0.25

WAVELENGTH

nm

FIG. 4. The effect of ergothioneine upon the myoglobin system. (Line 1) Myoglobin (50 pM) in 10 mM KH2P04-KOH buffer. (Line 2) As line 1 plus 100 pM H202. (Line 3) As line 1 plus 1 mM ergothioneine. (Line 4) As line 2 plus 1 mM ergothioneine. Reagents were at the final concentrations quoted, in the reaction mixtures.

Ergothioneine has been suggested to act as an antioxidant in viuo (4) and has been reported to be present in human and other mammalian tissues at concentrations up to l-2 mM (l-8). For this reason, the maximum concentration tested in any of our experiments was 3 mM. At this concentration, ergothioneine had no inhibitory effect on peroxidation of liver microsomes stimulated by addition of Fe’+ ions or of Fe3+ ions and ascorbate. Previous reports (14) that ergothioneine inhibited such peroxidation used unphysiologically high concentrations (>lO mM). However ergothioneine, unlike some other thiols (45,46), did not stimulate peroxidation in the presence of ferric ions. Perhaps this is because the equilibrium (Fig. 1) favours the thione form, rather than the morereducing thiol form. By contrast, ergothioneine inhibited the peroxidation of arachidonic acid induced by mixtures of H202 and haem proteins. The identity of the species that initiates lipid peroxidation in the microsomal system is unknown: it is known not to be hydroxyl radical, ‘OH (reviewed in Refs. (18, 25)). The oxidizing species that is produced by reaction of H,Oz with the haem ring (22) may be a tyrosine peroxyl radical, with which ergothioneine can probably

ERGOTHIONEINE

AS AN ANTIOXIDANT

react, since this radical has already been shown to react with thiol compounds (23). The ability of ergothioneine to inhibit peroxidation induced by mixtures of haem compounds and H,Oz may be very important in vivo. Thus oxyhaemoglobin plays an important role in the peroxidation induced when erythrocytes are exposed to excess H,Oz (47), and myoglobin may contribute to reoxygenation injury when previously ischaemic heart and muscle are reperfused (22). Thus ergothioneine could conceivably have a protective role in vivo against peroxidation induced by haem proteins plus H,O,. Indeed, since this paper was submitted, Hochstein and co-workers (48,49) have demonstrated an interaction of ergothioneine with ferryl-myoglobin in isolated tissues and have shown that ergothioneine can protect against reperfusion injury in isolated rat hearts (48). Ergothioneine did not react with 0; or HzOz at high rates (if at all), but we confirmed its previously reported ability to react with ‘OH radical at a diffusion-controlled rate (rate constant approximately lOlo Mm’ s-l). However, most molecules present in uivo react with ‘OH at equally fast rates. Perhaps of greater importance than the direct scavenging of ‘OH is our demonstration of the ability of ergothioneine to bind transition metal ions in a way that inhibits generation of ‘OH by reaction of H202 with these ions. Ergothioneine has some effect against iron ion-dependent ‘OH-mediated damage to deoxyribose, but was especially effective in inhibiting copper ion-dependent damage (Table II). The complex of ergothioneine with copper ions is stable (9), i.e., ergothioneine-copper ion complexes do not decompose to generate oxygen radicals. By contrast, other thiols such as GSH are rapidly oxidized by copper ions with production of toxic radical species (51). Copper ions also readily promote the oxidation of NAD(P)H (52), erythrocyte membranes (53) and lowdensity lipoproteins (54). Copper ions also oxidize haemoglobin (38, 39). Hence chelation of copper ions in non-redox active forms might be a major function of ergothioneine in the erythrocyte and in other tissues of the human body. This molecule, at the concentrations present in vivo, might also help protect tissue against toxic effects of HOCl. Thus 500 PM ergothioneine almost completely prevented damage to ol,-antiproteinase induced by 60 PM HOCl. a,-Antiproteinase is an especially sensitive target of damage, so ergothioneine’s protective action might be even greater in uiuo depending, of course, on the precise location of the ergothioneine in relation to the site of HOC1 generation. ACKNOWLEDGMENTS We are grateful to the Arthritis and Rheumatism Council, and to the National Council of Research (CNP,) of Brazil, for research support.

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