Original Contribution An uncommon redox behavior sheds light on the cellular antioxidant properties of ergothioneine Luigi Servillo a,n , Domenico Castaldo b , Rosario Casale a , Nunzia D’Onofrio a , Alfonso Giovane a , Domenico Cautela b , Maria Luisa Balestrieri a a Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli, 7-80138 Napoli, Italy b Stazione Sperimentale per le Industrie delle Essenze e dei Derivati dagli Agrumi, Azienda Speciale della Camera di Commercio di Reggio Calabria, 12-89125 Reggio Calabria, Italy article info Article history: Received 12 September 2014 Received in revised form 18 November 2014 Accepted 20 November 2014 Available online 4 December 2014 Keywords: Ergothioneine Neutrophils Oxidative burst Oxidative stress Hercynine Ergothioneine sulfonic acid Free radicals abstract 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 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. 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 (ESO 3 H), 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 ESO 3 H, 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. & 2015 Published by Elsevier Inc. Ergothioneine (ESH 1 ; 2-mercaptohistidine betaine) is a ubiqui- tous substance occurring in fungi, plants, and animals, known to be synthesized by only a few microorganisms, chiefly actinomy- cetales [1]. It is supposed that some species of microorganisms occurring in the soil produce and release ESH, which is absorbed first by plants and subsequently by animals. In the human body, ESH has been found to accumulate in bone marrow, seminal fluid, eye lenses, kidneys, and erythrocytes in up to millimolar concen- trations [2]. A specific transporter of ESH, the organic cationic transporter 1 (OCTN1), has been discovered in the plasma mem- brane 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, were more susceptible to oxidative stress and experienced 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, the mechanism through which ESH performs its anti- oxidant action is still obscure. To date, it has been widely demon- strated 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 generated upon interaction of hydrogen peroxide with hemoproteins, such as hemoglobin and myoglobin [14,15]. Indeed, hemoproteins react with hydrogen peroxide, turning into the highly reactive ferryl form (ferryl hemoproteins), with iron in the oxidation state of Fe 4 þ , and taking part in adverse cellular oxidation processes. The pro-oxidant and proinflammatory effects of ferryl hemoglobin Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine http://dx.doi.org/10.1016/j.freeradbiomed.2014.11.017 0891-5849/& 2015 Published by Elsevier Inc. Abbreviations: EH, hercynine; EIC, extracted ion chromatogram; ES(O) 2 SE, ergothioneine disulfide S-dioxide; ES(O)SE, ergothioneine disulfide S-monoxide; ESH, ergothioneine; ESI, electrospray ionization; ESO 2 H, ergothioneine sulfinic acid; ESO 3 H, ergothioneine sulfonic acid; ESOH, ergothioneine sulfenic acid; ESSE, ergothioneine disulfide; FIA, flow-injection analysis; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MetMb, ferrimyoglobin; MS/ MS, tandem mass; OCTN1, organic cationic transporter 1; PMA, phorbol 12- myristate 13-acetate n Corresponding author. Fax: þ390815665863. E-mail address: luigi.servillo@unina2.it (L. Servillo). Free Radical Biology and Medicine 79 (2015) 228–236