An uncommon redox behavior sheds light on the cellular antioxidant properties of ergothioneine

Article abstract of DOI:10.1016/j.freeradbiomed.2014.11.017

Ergothioneine (ESH), an aromatic thiol occurring in the human diet and which accumulates in particular cells, is believed to act as an antioxidant. However, its redox mechanism remains unclear and it does not seem to provide any advantage compared to other antioxidants, such as alkylthiols, which are better reducing agents and generally present in cells at higher levels. Here, we investigated by ESI-MS the products of ESH oxidation produced by neutrophils during oxidative burst and, to further elucidate ESH redox behavior, we also analyzed the oxidation products of the reaction of ESH with hypochlorite in cellfree solutions. Indeed, neutrophils are the main source of hypochlorite in humans. Furthermore, we also tested other biologically relevant oxidants, such as peroxynitrite and hydrogen peroxide. Our results indicate that treatment of human neutrophils with phorbol 12-myristate 13-acetate in the presence of ESH leads to a remarkable production of the sulfonated form (ESO₃H), a compound never described before, and hercynine (EH), the desulfurated form of ESH. Similar results were obtained when ESH was subjected to cell-free oxidation in the presence of hypochlorite, as well as hydrogen peroxide or peroxynitrite. Furthermore, when the disulfide of ESH was reacted with those oxidants, we found that it was also oxidized, with production of EH and ESO₃H, whose amount was dependent on the oxidant strength. These data reveal a unique ESH redox behavior, entirely different from that of alkylthiols, and suggest a mechanism, so far overlooked, through which ESH performs its antioxidant action in cells.

Full text of DOI:10.1016/j.freeradbiomed.2014.11.017

Author's Accepted Manuscript
An Uncommon Redox Behaviour Enlightens
the Cellular Antioxidant Properties of Er-
gothioneine
Luigi Servillo, Maria Luisa Balestrieri, Rosario
Casale, Nunzia D’Onofrio, Alfonso Giovane,
Domenico Cautela, Domenico Castaldo
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http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.017
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To appear in: Free Radical Biology and Medicine
Received date: 12 September 2014
Revised date: 18 November 2014
Accepted date: 20 November 2014
Cite this article as: Luigi Servillo, Maria Luisa Balestrieri, Rosario Casale,
Nunzia D’Onofrio, Alfonso Giovane, Domenico Cautela, Domenico Castaldo, An
Uncommon Redox Behaviour Enlightens the Cellular Antioxidant Properties of
Ergothioneine, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.
freeradbiomed.2014.11.017
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An Uncommon Redox Behaviour Enlightens the Cellular Antioxidant Properties of
Ergothioneine
Luigi Servillo a*, Maria Luisa Balestrieria, Rosario Casalea, Nunzia D’Onofrioa, Alfonso Giovanea,
Domenico Cautelab and Domenico Castaldob
a Dipartimento di Biochimica, Biofisica e Patologia Generale - Seconda Università degli Studi di
Napoli; via L. De Crecchio, 7- 80138, Napoli, Italy
b Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi (SSEA)- Azienda
Speciale della Camera di Commercio di Reggio Calabria – via Tommaso Campanella, 12 – 89125,
Reggio Calabria, Italy.
* Corresponding Author. Phone:+390815665865. Fax:+390815665863. e-mail address:
luigi.servillo@unina2.it
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ꢁ

Abstract
Ergothioneine (ESH), an aromatic thiol, assumed by diet in humans and accumulated in particular
cells, is believed to act as antioxidant. However, its redox mechanism remains unclear and it does
not seem to provide any advantage as compared to other antioxidants, such as alkylthiols, which are
better reducing agents and generally present in cells at higher levels. Here, we investigated by ESI-
MS spectrometry the products of ESH oxidation produced by neutrophils during oxidative burst
and, to further elucidate ESH redox behavior, we also analyzed the oxidation products upon
reacting ESH with hypochlorite in cell free solutions. Indeed, neutrophils are the main source of
hypochlorite in humans. Furthermore, we also tested other biologically relevant oxidants, such as
peroxynitrite, and hydrogen peroxide. Results indicate that treatment of human neutrophils with
phorbol 12-myristate 13-acetate (PMA) in the presence of ESH leads to a remarkable production of
the sulfonated form (ESO3H), a compound never described before, and hercynine (EH), the
desulfurated form of ESH. Similar results were obtained when ESH was subjected to cell free
oxidation in the presence of hypochlorite, as well as hydrogen peroxide, or peroxynitrite.
Furthermore, when the disulfide of ESH (ESSE) was reacted with those oxidants, we found that it
was also oxidized with production of EH and ESO3H whose amount was dependent on the oxidant
strength. These data enlighten a unique ESH redox behavior, entirely different from alkylthiols, and
suggest a mechanism, so far overlooked, through which ESH performs its antioxidant action in the
cells.
Keywords: ergothioneine; neutrophils; oxidative burst; oxidative stress; hercynine; ergothioneine
sulfonic acid.
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Abbreviations.
EH, hercynine
EIC, extracted ion chromatogram
ES(O)2SE, ergothioneine disulfide S-dioxide
ES(O)SE, ergothioneine disulfide S-monoxide
ESH, ergothioneine
ESI, electrospray ionization
ESO2H, ergothioneine sulfinic acid
ESO3H, ergothioneine sulfonic acid
ESOH, ergothioneine sulfenic acid
ESSE, ergothioneine disulfide
FIA, flow injection analysis
HPLC, high perfomance liquid chromatography
ICC, ion charge control
LC, liquid chromatography
MetMb, ferrimyoglobin
MRM, multiple reaction monitoring
MS/MS, tandem mass
OCTN1, organic cationic transporter 1
PMA, phorbol 12-myristate 13-acetate
ROS, reactive oxygen species
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Introduction
Ergothioneine, (ESH; 2-mercaptohistidine betaine), is an ubiquitous substance occurring in fungi,
plants and animals, known to be synthesized only by few microorganisms, chiefly actinomycetales
[1]. It is supposed that some species of microorganisms occurring in the soil produce and release
ESH which is firstly absorbed by plants and, subsequently, by animals. In human body, ESH has
been found to accumulate in bone marrow, seminal fluid, eye lenses, kidneys and erythrocytes up to
millimolar concentrations [2]. A specific transporter of ESH, the organic cationic transporter 1
(OCTN1), has been discovered in the plasma membrane of various human cell types, with the
highest levels of expression in monocytes, macrophages and hematopoietic lineage cells [3,4].
Depleting HeLa cells of ESH through OCTN1 silencing leads to an increased mitochondrial
oxidative burden and damage [5]. Moreover, OCTN1 knockout mice, completely deficient in ESH,
resulted to be more susceptible to oxidative stress and with higher lethality due to intestinal
ischemia and reperfusion injury [6].
Although the prevalent opinion is that ESH acts in the cells mainly as an antioxidant (for an
exhaustive review on the ESH function and role see ref. [7]), more than a century after its
discovery, it is still obscure the mechanism through which ESH performs its antioxidant action. To
date, it has been widely demonstrated that ESH is an effective scavenger of hydrogen peroxide,
lipid peroxides, superoxide ion, singlet oxygen [8,9], hydroxyl radical [10,11] and nitric oxide
derivatives [12,13]. ESH has been shown to counteract reactive oxygen species (ROS) generated
upon interaction of hydrogen peroxide with hemoproteins, such as hemoglobin and myoglobin
[14,15]. Indeed, hemoproteins react with hydrogen peroxide turning into the high reactive ferryl
form (ferryl hemoproteins), with iron in the oxidation state of Fe+4, taking part to adverse cellular
oxidation processes. The prooxidant and proinflammatory effects of ferryl hemoglobin seem to be
involved into atherogenesis [16]. Also, it has been proposed that, in the early phases of ischemia,
deoxymyoglobin is oxidized by hydrogen peroxide to ferryl myoglobin which, during oxygenated
reperfusion, might lead to cellular injury [17]. ESH has been shown to reduce ferryl hemoprotein
(containing Fe+4) into methemoprotein (containing Fe+3), thus ameliorating the ischemia-
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reperfusion damage [18]. A similar antioxidant mechanism has been proposed for the ESH
inhibition of nitrite-induced oxyhemoglobin oxidation [19]. Another relevant ESH antioxidant
activity is exerted against hypochlorous acid. ESH, in fact, has been found to be a powerful
hypochlorous acid scavenger, being able to protect ꢀ1-antiproteinase against the inactivation by this
compound [11]. Furthermore, the current hypothesis is that the antioxidant activity of ESH is
critical in protecting circulating erythrocytes from the possible damage induced by this highly
reactive oxidant produced by activated neutrophils during inflammation or normal function. On the
whole, all these studies seem to leave no doubt on the antioxidant properties of ESH.
Chemically, ESH is a particular type of aromatic thiol, resulting from the N,N,N-trimethylhistidine
(histidine betaine or hercynine) in which a sulfhydryl group is linked to the imidazole ring at C2
position (Figure 1). At physiological pH, ESH exists in a tautomeric equilibrium between thiol-
thione forms, with the thione form predominating (Figure 1).
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The free energy of reduction of ergothioneine disulfide (ESSE), lower than those of alkyldisulfides,
has been ascribed to the thermodynamic stability of the thione form [20]. Actually, in the totality of
the proposed mechanisms of its antioxidant function, ESH is considered to oscillate between the
reduced state and the oxidized state represented by the disulfide form, similarly to alkylthiols such
as cysteine or glutathione [19, 26]. This is, perhaps, the most compelling reason of concern about
the antioxidant role of ESH in cells. Indeed, if ESH behaves like an alkylthiol, it is plausible to
wonder what particular advantage is given by its antioxidant function with respect to alkylthiols,
generally present in the cells at higher levels and also more easily oxidized than ESH.
In this paper, we aim at elucidating aspects of ESH redox behaviour, gone completely unnoticed so
far, which unveil that the chemical antioxidant mechanism of ESH is absolutely different from
alkylthiols.
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Materials and Methods
Materials
Ergothioneine, myoglobin from equine heart, histopaque-1077, histopaque-1119, iodomethane,
iodomethane-d3, hydrogen peroxide 30 wt. % in H2O, calcium hypochlorite, sodium nitrite were
purchase from Sigma-Aldrich, Milan, Italy. SPE-C18 columns for flash chromatography were
obtained from Phenomenex (Anzola Emilia, Italy). All other solvents and reagents used were of
analytical grade.
The concentration of the hypochlorite stock solution in 0.1 M NaOH was determined, after
appropriate dilution, by the absorbance at 292 nm (ꢁ292 =350 M-1 cm-1) [21].
Preparations of neutrophils from human peripheral blood.
Neutrophils were isolated from six ml of heparinized peripheral blood from human healthy
volunteers after centrifugation (400 x g for 40 min at 4°C) on discontinuous density gradient on
Histopaque-1077 and Histopaque-1119, following manufacturer’ instructions. After centrifugation,
the interface cells between the Histopaque-1119 and the Histopaque-1077 were carefully removed
and transferred to a new conical tube. Cells were washed twice with PBS, centrifuged at 300 x g for
10 min at 4°C, seeded on gelatin-coated culture dishes (2 x 106 cells/ml) and immediately used for
the experiment.
Neutrophil oxidative burst assay.
The purified neutrophils were stimulated in the presence (100 ng/ml) or absence of phorbol 12-
myristate 13-acetate (PMA) in a final volume of 3 ml of HBSS containing ESH (0.4 mM). After
PMA addition, aliquots of the incubation media (200 µl) were taken from time zero up to 30 min
and centrifuged at 16.000 x g for 10 min. The supernatants were then removed and subjected to
mass spectrometric analysis.
Preparation of peroxynitrite
Peroxynitrite was synthesized as described [22]. Briefly, 50 ml of ice-cold 1.2 M HCl and 50 ml of
1.4 M H2O2 were mixed and maintained under continuous stirring in an ice cold container. Then
100 ml of ice cold 0.6 M sodium nitrite solution was added, followed immediately by addition of
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100 ml of 1.5 M NaOH solution. The excess of hydrogen peroxide in the reacting solution was
decomposed by passing the solution through a column containing manganese dioxide. The eluate
was frozen overnight in a glass cylinder at -20 °C. The top layer which separated out, containing
enriched peroxynitrite, was collected in aliquots and used in the successive experiments. The
peroxynitrite concentration in the stock solution, after appropriate dilution in 0.4 M NaOH, was
determined by its absorbance at 302 nm (ꢁ302= 1670 M-1cm-1) [22].
Preparation of ergothioneine disulfide
ESH disulfide was prepared essentially as described [23]. Briefly, 3.9 mg of ESH dissolved in 200
µl of 5 M HCl were reacted with 8.5 µl of a standardized 2.0 M H2O2 solution in 1.5 ml conical
tube. After standing at room temperature for 10 min, the solution was divided in aliquots of 20 µl
in conical tubes, evaporated to dryness under vacuum overnight, and stored in dry nitrogen
atmosphere at -20 °C. At the time of use, an aliquot was dissolved again in 100 µl of the due
solvent and immediately employed.
Preparation of hercynine and deuterated hercynine.
As hercynine was not commercially available, it was prepared by a modified heterogeneous phase
reaction employing iodomethane as the methylation agent in the presence of KHCO3 [24,25]. Also
deuterated hercynine (EH-d9) was prepared in the same way by substituting iodomethane with
deuterated iodomethane (methyl-d3 iodide). In both cases, histidine (50 mg) was dissolved in 5 ml
of methanol, then 200 mg of KHCO3 and, subsequently, 2 ml of the methylating agent was added.
The mixture was stirred at room temperature overnight. The reaction completeness was monitored
by LC-ESI-MS. Then, the mixture was centrifuged and the supernatant collected and evaporated to
dryness at 40°C in a rotavapor. The residue was dissolved in 10 ml of Milli Q grade water and
purified by flash chromatography on SepPac C18 cartridge (Phenomenex, Anzola Emilia, Italy).
The sample-loaded column was washed with 100 ml of Milli Q water and eluted by applying 50
ml of H2O/acetonitrile solution (80:20 v:v). The eluate containing the purified product was
evaporated to dryness under a stream of air and dried overnight in vacuum over P2O5.
Mass spectrometric determinations.
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The analyses were performed with an HPLC Agilent 1100 series coupled on line with an Agilent
LC-MSD SL quadrupole ion trap. All MS acquisitions were performed by using ESI in positive ion
mode with nitrogen as the nebulizing and drying gas. Nitrogen was used at a flow rate of 7 L/min
and a pressure of 30 psi both as a drying and a nebulising gas. The nebulizer temperature was set at
350°C. The ion charge control (ICC) was applied with a target set at 30000 and maximum
accumulation time at 20 ms. The measurements were performed from the peak area of the extracted
ion chromatogram (EIC). The quantitation was achieved either by comparison with the calibration
curves obtained with standard solutions or by internal standard addition. The optimization of the
instrumental parameters for the analyses of ESH, ESSE and EH was performed by continuous
infusion (FIA-ESI-MS/MS measurements) of 5 )M standard solution in 0.1% formic acid. The
mass cutoff and amplitude were optimized to obtain the most efficient MS/MS transitions from the
positively charged precursor ion [M+H]+ to the fragment ions. Multiple reaction monitoring (MRM)
was used for the detection of analytes in HPLC-ESI-MS/MS analyses. The most intense transitions
utilized for MS/MS detection were 230.1ꢂ186 for ESH, 198.1ꢂ154 for EH, 207.1ꢂ163 for EH-
d9, 229.1ꢂ185 for ESSE (as bipositive ion) or 457.2ꢂ310 (as unipositive ion), 278.1ꢂ154 for
ESO3H. The chromatographic analyses with ESI-MS detection were performed on a 250 mm x 3.0
mm i.d., 5 )m, Discovery-C8 analytical column (Supelco, PA, USA). The mobile phase was
composed with 0.1% formic acid in water and pumped at 0.1 mL/min in isocratic conditions at
room temperature. Sample volumes of 5-10 )L were injected.
Cell free oxidation of ESH
All the oxidation reactions of ESH were conducted in 1.0 mM sodium phosphate buffer at pH 7.4.
The final composition of the reaction mixtures was 100 )M ESH and 100 )M of the oxidant,
according to the employed ESH and oxidant concentrations in similar in vitro studies (11). The
reactions were initiated by adding into the reaction mixture the due amount of the oxidant stock
solution. 5 )l aliquots of the reaction mixture were analyzed over time by flow injection analysis
(FIA) with MS detection. When hydrogen peroxide was employed as oxidant, the reactions were
conducted both in the presence or absence of ferrimyoglobin (MetMb). MetMb was prepared by
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ꢇ

myoglobin oxidation with ferricyanide and purified as reported [14]. In all the experiments, the final
concentration of MetMb was 50 )M.
Cell free oxidation of ESSE
All the oxidation reactions of ESSE were conducted in 1.0 mM sodium phosphate buffer at pH 7.4.
The final composition of the reaction mixtures was 93 )M ESSE and 93 )M of the oxidant. The
reactions were initiated by adding into the reaction mixture the due amount of the oxidant stock
solution. 5 )l aliquots of the reaction mixture were analyzed over time by FIA with MS detection.
Hydrolytic decomposition of ESSE
Assay conditions: Solutions of ESSE (93 )M) in 1.0 mM sodium phosphate buffer, pH 7.4. 5 )l
aliquots of the solutions were analyzed over time both by FIA with MS detection and by HPLC-
ESI-MS/MS.
Oxidative formation of sulfate from ergothioneine.
The experiments were conducted using only water as a solvent, in order to avoid interference from
phosphate buffer. 1 ml of ESH solution (1.75 mM) in water was reacted with hydrogen peroxide
(10 mM) for 18 h. Then, 200 )l of barium chloride solution, 22 mM in water, was added to the
reaction mixture. The white precipitate formed was collected by centrifugation in a conical tube and
treated with 5 M HCl. The formation of barium sulfate was demonstrated by the insolubility of the
white precipitate in 5 M HCl solution. As blanks, two solutions of ESH and H2O2 in water, at the
same concentrations as in the reaction mixture, were treated, after 18 h, in the same manner with the
barium chloride solution.
Statistical analysis
The results of the FIA analyses reported in the figures are the mean S.E.M of 4 determinations.
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Results
Stimulation of human neutrophils in the presence of ergothioneine
ESH has been proven to be a powerful scavenger of hypochlorous acid (11). As the main cellular
source of this strong oxidant in human body is represented activated neutrophils, we first analyzed
the products of ESH oxidation in whole cell experiments. To this end, human neutrophils were
subjected to oxidative burst by stimulation with PMA in the presence of ESH. The time-dependent
HPLC-ESI-MS analyses of the incubation medium revealed the increases over time of two
unknown peaks at m/z 198.1 and m/z 278.1, with retention times of 12.1 min and 16.2 min,
respectively. Both MS/MS fragmentation patterns of the peaks at m/z 198.1 and at m/z 278.1
showed the more intense fragment at m/z 154. Therefore, the transitions 198.1ꢂ154 and
278.1ꢂ154 were utilized for the analytical determinations (Figure 2). In order to ascertain whether
the unknown compounds derived from ESH oxidation or were released in the medium by the cells,
we performed a set of cell free ESH oxidation experiments in the presence of hypochlorous acid
and, as a comparison, also in presence of other important cellular oxidants, such as hydrogen
peroxide and peroxynitrite.
Cell free ESH oxidation
Several reaction mixtures were prepared each containing 100 )M ESH and hydrogen peroxide or
peroxynitrite or hypochlorite all at the same concentrations (100 )M) in sodium phosphate buffer
1.0 mM, pH 7.4. Furthermore, being reported a more intense oxidative action of H2O2 in the
presence of hemoproteins (14, 15), ESH oxidation by hydrogen peroxide was also performed in
solutions containing 50 )M MetMb.
Each reaction started with the addition of the due amount of the oxidant stock solution. The time
courses of ESH consumption during oxidation is reported in Figure 3, panel A. As expected, the
formation of ESSE was always detected when analyzing over time the reaction mixtures with each
oxidant (Figure 3, panel B). However, surprisingly, the same unknown peaks at m/z 198.1 and m/z
278.1, found during neutrophil oxidative burst, were always found among the products of the ESH
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oxidation by all the oxidants employed (Figure 3, panels C and D). The compound at m/z 278.1
(Figure 3, panel D), formed at different amounts depending on the strength of the oxidant
employed, was found to be ESO3H by successive HPLC-ESI-MS/MS analyses. Also, the identity of
the compound at m/z 198.1 was revealed to be hercynine (EH), confirmed by its mass spectrometric
and chromatographic behaviour, and substantiated by comparison with authentic standard which
was synthesized labelled with deuterium at the methyl groups (N,N,N-trimethylhistidine-d9, EH-d9).
As an example, a typical HPLC-ESI-MS/MS analysis of the reaction mixture in which ESH was
reacted with hypochlorite for 10 min, is reported in Figure 4 (panel A).
The chromatographic analysis allowed, in less than 30 min, the complete chromatographic
separation of ESH and its oxidation products, that is, EH, ESSE and ESO3H. In particular, EH
eluted at a retention time (TR) of 12.1 min, followed by ESO3H at TR 16.2 min, ESH at TR 17.2
min, and ESSE at TR 25.8 min. The MS/MS fragmentation patterns confirmed the identity of each
chromatographic peak (Figure 4, panel B, C, D, E).
Indeed, after certain reaction times, HPLC-ESI-MS/MS analyses of the reaction mixtures were
performed to identify and characterize the oxidation products. However, to monitor over time these
rather fast reactions, the analyses were conducted by FIA, which allowed the determination of the
components in the reaction mixtures with higher frequency and in much shorter times. The rate of
ESH oxidation follow the order hypochlorite > peroxynitrite > H2O2 + myoglobin > H2O2 (Figure
3, panel A). When ESH oxidation was conducted with hypochlorite, a more relevant formation of
ESO3H was observed (Figure 3, panel D). Indeed, some formation of ESO3H was also observed
when ESH was oxidized by peroxynitrite and H2O2 but at significantly lower levels than oxidation
by hypochlorite. The mass spectrometric characterization of ESO3H is reported in Figure 4, panel
D and E.
Oxidation of ESSE
In the light of the above results showing that ESSE was always formed during ESH oxidation, we
sought to evaluate oxidative behaviour of ESSE in the presence of the same oxidants.
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Each ESSE oxidation reaction started with the addition of the due amount of ESSE to the oxidant
solution at physiological pH. At certain reaction times, identification and characterization of the
oxidation products were performed by HPLC-ESI-MS/MS analyses of each reaction mixture.
In order to follow over time the oxidation reactions of ESSE, which is oxidized more rapidly than
ESH, the analyses of the reaction mixtures were performed by FIA. Results show that the rates of
ESSE oxidation follow the order hypochlorite > peroxynitrite > H2O2 (Figure 3, panel E). The
rates of the product formations in the reaction mixtures are reported in Figure 3 (panels F, G, H).
Similarly to ESH, the main product of all ESSE oxidation reactions was EH. Levels of ESO3H were
more consistent only when hypochlorite was employed as an oxidant.
Decomposition of ESSE in the absence of oxidants
ESSE was reported to have an uncommon behaviour compared to alkylthioldisulfides (23).
Specifically, ESSE, which is stable in very acidic aqueous solution, slowly decomposes at
increasing pH, partially reforming ESH along with an unidentified compound (23). In this respect,
we studied over time the decomposition of ESSE dissolved in 1 mM sodium phosphate buffer at pH
7.4 (Figure 5). After 36 h, ESSE was no longer detectable in the solution and the concentrations of
its decomposition products were identified and quantified by HPLC-ESI-MS/MS. We found that the
products of the hydrolytic decomposition of ESSE are ESH and EH, the latter very likely
corresponding to the unknown compound reported by Heath and Tonnies (23). The quantitative
analyses, reported as mean STD of four determinations, each in duplicate, on four identical ESSE
solutions (93 )M) were the following: [ESH] = 137.7+/-12.5 ()M) and [EH] = 45.2+/-6.3 ()M).
The molar ratio [ESH]/[EH] resulted 3.05, thus indicating that 3 moles of ESH and 1 mole of EH
are produced from decomposition of 2 moles of ESSE.
Detection of sulfate ion as a reaction product of ESH with hydrogen peroxide
The desulphurization of ESH with the consequent formation of EH was observed in the course of
all the oxidation reactions performed. Therefore, we undertook additional experiments, using only
hydrogen peroxide as an oxidant, in order to ascertain the fate of sulphur atoms. The experiments,
in order to avoid interference from phosphate buffer, were conducted in water as a solvent by
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mixing ESH and hydrogen peroxide in the molar ratio of about 1:6 and reacting for 18 h. After the
addition to the reaction mixture of a barium chloride solution, a white precipitate soon formed,
whose identity as barium sulfate was confirmed by its insolubility in 5 M HCl. Conversely, two
blank solutions of ESH and H2O2 in water, at the same concentrations as in the reaction mixture,
did not give any precipitate when treated in the same manner with the barium chloride solution.
Discussion
In this study we reveal the unique redox behaviour, so far overlooked, that makes ESH different
from alkylthiols, shedding a new light on the chemical antioxidant mechanism of ESH in the cell.
To date, the antioxidant action of ESH has been described with reaction mechanisms considered,
more or less explicitly, to function in cyclic manner: ESH is believed to be first oxidized to
disulfide [19], or to a mixed disulfide with another thiol [26], and subsequently regenerated by the
disulfide reduction, similarly to what is known for alkylthiols. As demonstrated in this study, in the
presence of oxidants, ESH can form the disulfide according to the usual pattern, but this disulfide
(ESSE) can behave very differently from alkylthiol disulfides, in that, being unstable at
physiological pH, it undergoes a progressive decomposition by disproportion. Our data show that,
from two moles of ESSE, three moles of ESH and one mole of EH are formed. Notably, the partial
ESH regeneration from ESSE does not require reducing substances, being the process a
disproportion. The possible mechanism accounting for that stoichiometry is reported in Figure 6.
The pathway starts with the hydrolysis of ESSE (Figure 6, reaction a) into ESH and the highly
reactive sulfenic acid (ESOH) which then disproportionates into ESO2H and EH [27] (Figure 6,
reaction b). Then, ESO2H decomposes into EH + H2SO3 (Figure 6, reaction c). The global reaction
accounts for the observation that from the decomposition of two moles of ESSE, three moles of
ESH and one mole of EH are formed (Figure 6, reaction d). The whole process is driven toward the
end products by the irreversible decomposition of the sulfinic acid, ESO2H (Figure 6, reaction c).
It is well known, in fact, that the oxidative desulfurization of 2-imidazolethiones is to be ascribed to
the formation of 2-imidazolesulfinc acids, highly instable intermediates, which rapidly and
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ꢁꢃ

irreversibly decompose into the corresponding imidazoles and sulfurous acid [28]. On the contrary,
it is important to point out that 2-imidazole sulfonic acids are stable toward hydrolysis [28].
From the overall mechanism, it clearly emerges the great advantage of ESH as compared to
alkylthiols from a biological point of view. In fact, ESH can be partially reformed from the disulfide
ESSE, without the need of reducing agents. The reaction produces also sulfurous acid, an other
effective reducing agent, which could counteract the cell oxidative stress by further conversion into
sulfate. This oxidative behaviour also makes ESH a rich source of reducing equivalents as the
oxidation of ESH to EH and sulfate involves the irreversible transfer to intracellular oxidants of six
electrons per each ESH molecule. This is at variance with the classical oxidation of alkylthiols into
their respective alkyldisulfides, where only one electron per thiol molecule is transferred to the
oxidant and cellular reducing equivalents are required to regenerate alkylthiols.
As for the pathway(s) of ESH oxidation in the presence of oxidants, in our experimental conditions,
ESSE was always observed to form, more or less rapidly, and then gradually disappear. The main
product of the oxidation reactions was EH in all cases (Figure 3, panel C). When hypochlorite was
used, ESO3H was observed at the highest levels (Figure 3, panel D).
It is likely that ESH oxidation entails the preliminary formation of ESOH, as for other alkyl and aryl
thiols [27]. Successively, ESOH could form disulfide by reacting with ESH according to the
reversible reaction [27]: ESOH + ESH = ESSE + H2O. When the oxidant is hypochlorite, the
disulfide is likely formed through the preliminary formation of the sulfenyl chloride intermediate
ESCl [27, 29], according to the reaction ESCl + ESH = ESSE + H+ + Cl-. In the presence of excess
of oxidants, ESSE could not only disproportionate but be further transformed into more oxidized
species, such as disulfide S-monoxide (ES(O)SE) and disulfide S-dioxide (ES(O)2SE) (Figure 7,
left side), which can be seen as the thiosulfinate and the thiosulfonate derivatives of ESH.
Our inability to observe these derivatives directly could be due to their instability inasmuch as the
higher electrophylicity of the sulphur atom bound to oxygen makes their decomposition in water
much faster than that of ESSE. It is well known, in fact, that thiosulfinates and thiosulfonates are
easily hydrolyzed to sulfenic and sulfonic acid [27]. These reactions closely resembles the proposed
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ꢁꢄ

hydrolytic decomposition of ESSE. Obviously, only one molecule of ESH and one of EH and
sulfurous acid are formed from the hydrolysis of one molecule of ES(O)SE. While one molecule of
ESH and one of ESO3H are formed when one molecule of ES(O)2SE is hydrolyzed (Figure 7, left
side).
However, we cannot rule out that, depending on the strength and concentration of the oxidants, ESH
could be, at least in part, also sequentially oxidized to ESOH, ESO2H and ESO3H (Figure 7, right
side), without passing through the formation of disulfide and its oxidation products. Anyway, no
matter how ESO2H, the key intermediate of both pathways, is produced, its irreversible
decomposition leads to the formation of EH and sulfurous acid in both cases (Figure 7).
Our findings that ESH can be oxidized through the proposed oxidative route give reason of the
observation, so far unexplained, that, while being not metabolized to any notable extent in
mammalian tissues [30, 31], ESH is excreted by humans as EH and free sulfate [32,33], which are
the ultimate products of the oxidative pathway described for the first time in this paper. While these
studies [30-33] give no explanation of how EH and sulfate are formed, their conclusions strongly
reinforce our hypothesis that excreted sulphate and EH derive from the proposed oxidative
degradation of ESH.
In this regard, the remarkable lower affinity of the ESH transporter for EH [34] could suggest an
evolutionary purpose according to which cells possessing the transporter can avidly accumulate
ESH, while its oxidative by-product EH can be easily excreted.
Conclusions
Our studies show peculiar antioxidant properties of ESH which make this compound a cell adjuvant
of noticeable importance, mainly because of its capacity to be irreversibly oxidized by relevant
oxidants occurring in cells. In particular, we report for the first time that the oxidation of ESH by
those oxidants produces mainly EH and variable amounts, depending on the oxidant strength, of
ESO3H, a compound never described before. It is worth to point out that, being available in the diet,
ESH represents a free and rich source of reducing equivalents that, through the herein described
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redox mechanism, could be slowly and continuously utilized by cells in basal conditions and ensure
additional protection during extreme oxidative stress.
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Competing financial interests
The authors declare no competing financial interests.
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Legends to figures
Figure 1. Chemical structures. Ergothioneine (ESH), hercynine (EH), ergothioneine sulfenic
acid (ESOH), ergothioneine sulfinic acid (ESO2H), ergothioneine sulfonic acid (ESO3H),
ergothioneine disulfide (ESSE), ergothioneine disulfide S-monoxide (ES(O)SE), ergothioneine
disulfide S-dioxide ES(O)2SE. The equilibrium between the thiol-thione forms of ergothioneine is
indicated.
Figure 2. HPLC-ESI-MS/MS analyses of ESH oxidation by stimulated neutrophils. The
chromatographic conditions and the stimulation of human neutrophil by phorbol-myristate-acetate
(PMA) in the presence of ergothioneine are described in the experimental section. 10 µl of
incubation medium was analyzed immediately after PMA addition (t = 0 min), and then at 15 min
and 30 min. The extracted ion chromatograms (EIC) at the indicated MS/MS transitions are
reported. The fragmentation patterns of the peaks corresponding to m/z 198.1 and m/z 278.1 are the
same as those reported in panel B and panel D of Figure 4.
Figure 3. Time course oxidations of ESH and ESSE. Upper panel. FIA analyses of ESH
oxidation with the oxidants hypochlorite (ꢃ); peroxynitrite (P); hydrogen peroxide in the presence
of myoglobin (L); hydrogen peroxide (D). All the reaction mixtures contained 100 )M
ergothioneine and 100 )M of the oxidant, in phosphate buffer 1 mM pH 7.4. The concentration of
myoglobin, when present, was 50 )M. The reactions started with the addition of the oxidant
solution. At set time intervals, 5 )l of the reaction mixture was injected in a stream of 0.1% formic
acid in water at flow rate of 100 )l/min. FIA analyses were performed in full scan in the range 190-
290 amu. The data are reported as the peak area by extracting at m/z 198.1, for EH; at m/z 229.1 for
ESSE; at m/z 230.1 for ESH; at m/z 278.1 for ESO3H. Panel A. Time courses of ESH consumption.
Panel B. Time courses of ESSE production. Panel C. Time courses of EH production. Panel D.
Time courses of ESO3H production. Lower panel. FIA analyses of ESSE oxidation with the
oxidants hypochlorite (ꢃ); peroxynitrite (P); hydrogen peroxide (D). All the reaction mixtures
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ꢂꢁ

contained 93 )M ESSE and 93 )M of the oxidant, in phosphate buffer 1 mM pH 7.4. The reactions
started with the addition of the oxidant solution. At set time intervals, 5 )l of the reaction mixture
was injected in a stream of 0.1% formic acid in water at flow rate of 100 )l/min. FIA analyses were
performed in full scan in the range 190-290 amu. The data are reported as the peak area by
extracting at m/z 198.1, for EH; at m/z 229.1 for ESSE; at m/z 230.1 for ESH; at m/z 278.1 for
ESO3H. Panel E. Time courses of ESSE decrease. Panel F. Time courses of ESH production.
Panel G. Time courses of EH. Panel H. Time courses of ESO3H production.
Figure 4. LC-ESI-MS/MS chromatogram of a reacting mixture containing ESH and
hypochlorite. Panel A. Chromatography of an aliquot of the reaction mixture containing initially
ESH and hypochlorite at the same concentrations, after 10 min of reaction. The initial composition
of the reaction mixture and the chromatographic conditions are reported in the experimental section.
Synthesized EH-d9 was added to the reaction mixture as an internal standard. The extracted ion
chromatograms (EIC) at the indicated MS/MS transitions are reported. Panel B. The upper part
shows the MS/MS fragmentation pattern of EH, produced by the oxidation reaction, by isolating at
m/z 198.1. The lower part shows the MS/MS fragmentation pattern of EH-d9, added as an internal
standard to the reaction mixture, by isolating at m/z 207.1. Panel C. The upper part shows the
MS/MS fragmentation pattern of ergothioneine disulfide (ESSE) produced by the oxidation
reaction, isolated as mono-charged positive ion at m/z 457.2. The lower part shows the MS/MS
fragmentation pattern of ESSE isolated as doubly charged positive ion at m/z 229.1. Panel D.
MS/MS fragmentation pattern of the oxidation product of ESH isolated at m/z 278.1. The MS/MS
fragments clearly indicate the identity of this compounds as the sulfonate derivative of ESH. Panel
E. MS/MS fragmentation pattern of the oxidation product of ESH isolated at m/z 280.1,
corresponding to the sulfonate containing 34S. It can be observed that, excepting the fragments
containing the sulphur atom, all other fragments are the same as those of the compound isolated at
m/z 278.1.
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Figure 5. Time course of ESSE decomposition at neutral pH. The HPLC-ESI-MS/MS analyses
of a solution of ESSE in 1 mM buffer phosphate at pH 7.4. The initial concentration of ESSE was
93 )M. At the times of 0, 5, 10, and 15 h are reported, 10 µL of the solution were injected. The
extracted ion chromatograms (EIC) at the indicated MS/MS transitions are reported.
Figure 6. Pathway of ESSE decomposition at neutral pH. The pathway starts with the hydrolysis
of ESSE (reaction a) into ESH and the highly reactive sulfenic acid (ESOH) which then
disproportionates into ESO2H and EH (reaction b). Then, ESO2H decomposes into EH + H2SO3
(reaction c). The global reaction accounts for the observation that from the decomposition of two
moles of ESSE, three moles of ESH and one mole of EH are formed (reaction d).
Figure 7. Oxidative patterns of ESH. Left side. ESH is first oxidized into the disulfide (ESSE);
ESSE is oxidized into S-monoxide (ES(O)SE) and, then, ES(O)SE into S-dioxide (ES(O)2SE). The
hydrolytic decompositions of ESSE, ES(O)SE and ES(O)2SE have in common the partial formation
of ESH which can restart a new oxidative cycle. Moreover, the hydrolytic decomposition of
ES(O)2SE generates the stable ESO3H, whereas the decomposition of ES(O)SE generates the
highly instable thiosulfinate, ESO2H, which rapidly and irreversibly decomposes into hercynine
(EH) and sulfurous acid. Instead, the hydrolytic decomposition of ESSE produces ESOH which
disproportionate into an additional ESH molecule and ESO2H which, as seen above, decomposes
into hercynine (EH) and sulfurous acid. Right side. ESH is first oxidized into the sulfinic acid,
ESOH, which is successively oxidized into the sulfenic acid, ESO2H and, then, this into the sulfonic
acid ESO3H. The fate of these three compounds is the same as described above.
Highlights
ꢀ
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1) Ergothioneine (ESH) is oxidized by cell oxidants differently from alkylthiols
2) Upon oxidation, ESH mainly produces hercynine and sulfurous acid.
3) Upon oxidation with hypochlorite, ESH forms its sulfonated derivative
4) The sulfonated derivative of ESH is produced by stimulated human neutrophils
5) Its peculiar oxidative behavior makes ESH a rich source of reducing equivalents
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Figure 1

Figure 3

Figure 4