Ergothioneine: A Stress Vitamin with Antiaging, Vascular, and Neuroprotective Roles?

Abstract

Significance:

Ergothioneine (ET) is an unusual sulfur-containing amino acid derived from histidine, acquired predominantly from food. Its depletion is associated with deleterious consequences in response to stress stimuli in cell culture models, prompting us to classify it as a vitamin in 2010, which was later supported by in vivo studies. ET is obtained from a variety of foods and is taken up by a selective transporter. ET possesses antioxidant and anti-inflammatory properties that confer cytoprotection. ET crosses the blood–brain barrier and has been reported to have beneficial effects in the brain. In this study, we discuss the cytoprotective and neuroprotective properties of ET, which may be harnessed for combating neurodegeneration and decline during aging.

Recent Advances:

The designation of ET as a stress vitamin is gaining momentum, opening a new field of investigation involving small molecules that are essential for optimal physiological functioning and maintenance of health span.

Critical Issues:

Although ET was discovered more than a century ago, its physiological functions are still being elucidated, especially in the brain. As ET is present in most foods, toxicity associated with its deprivation has been difficult to assess.

Future Directions:

Using genetically engineered cells and mice, it may now be possible to elucidate roles of ET. This coupled with advances in genomics and metabolomics may lead to identification of ET function. As ET is a stable antioxidant with anti-inflammatory properties, whose levels decline during aging, supplementing ET in the diet or consuming an ET-rich diet may prove beneficial. Antioxid. Redox Signal. 36, 1306–1317.

Introduction

Ergothioneine (ET) is a sulfur-containing derivative of the amino acid, histidine. Discovered more than a century ago in 1909, physiological functions for this molecule are still being elucidated (126). Mammals cannot synthesize ET and acquire it predominantly from food. Its de2pletion is linked to impaired stress responses and toxicity, prompting us to designate it as a vitamin (102). This designation has gained recognition and momentum in the field, opening new avenues of investigation (4, 10). ET was first isolated from the ergot of rye, Claviceps purpurea (hence the name ergothioneine), and its structure was determined in 1911 (78). Mycobacteria, members of the genus Actinomycetales, and fungi were the first organisms reported to produce ET (37, 38). Later, ET was also discovered in cyanobacteria, methylobacteria, and other microbes (3, 75, 105). Unlike eukaryotes and Gram-negative bacteria, where glutathione (GSH) is the primary thiol, in mycobacteria, GSH is absent and small molecules such as ET and mycothiol (MSH) constitute the major thiol reserve. ET and MSH are utilized in the biosynthesis of lincomycin A, a sulfur-containing C8 sugar (lincosamide) antibiotic (137). The presence of ET in mammals was first identified in pig blood in independent studies, where it was identified as the substance that interfered with the detection of uric acid (11, 57).

Physicochemical Properties of ET

The molecular weight of ET is 229.3 Da. It is a colorless, and odorless compound that is readily soluble in water. Structurally, ET is a betaine of histidine thiol or 2-mercaptohistidine trimethylbetaine (Fig. 1). In 1911, Barger and Ewins showed that ET is the betaine of thiolhistidine. ET exhibits tautomerism and exists predominantly as a thione at physiological pH. Thus, several of its properties are different from thiol molecules. ET is relatively more stable and does not auto-oxidize at physiological pH and generates free radicals such as GSH (88). Unlike most thiols whose standard redox potential of the thiol–disulfide couple ranges from −0.2 to −0.32 V, the value for ET is −0.06 V. An important feature of ET is its thermostability (it does not decompose upon cooking), a feature desirable for its use in culinary preparations.

FIG. 1.

Thione–thiol tautomerism of ET. ET is an unusual sulfur-containing histidine derivative and is the betaine of 2-mercapto-L-histidine. Chemically, it is an N,N,N-trimethylhistidine with a sulfhydryl group linked at the C2 position of the imidazole ring. ET undergoes tautomerism and exists both in the thiol form and thione form, with the latter predominating at physiological pH. Due to this property, ET is resistant to auto-oxidation. ET, ergothioneine.

Another characteristic of ET, that contributes to its cytoprotective properties, is its capacity to absorb ultraviolet (UV) light. ET absorbs light in the UV range similar to DNA, with a molar extinction coefficient of 1.4 × 10⁻⁴ M⁻¹·cm⁻¹, λmax 257, indicating that ET acts as a physiological UV filter (16). In the caterpillar fungus, Cordyceps militaris, a mushroom harvested for its medicinal activities, irradiation with ultraviolet B (UVB) increased ET content (55). These studies also revealed that ET prevents DNA damage induced by UV irradiation in a dose-dependent manner (102). In addition to absorbing UV light, ET present in the Coprinus comatus extract protects UVB-induced DNA damage (halogenation) by inhibiting myeloperoxidase activity and scavenging halogenous species (7). The mammalian skin is particularly vulnerable to UV damage, which may induce sunburn, immunosuppression, skin aging, and carcinogenesis, in addition to other damage (26, 123). ET accumulates in skin cells and not only prevents oxidative damage but also facilitates DNA repair in UV-irradiated cells (80). For these reasons, ET has been included as an ingredient in several skin care products and cosmetics.

Biosynthesis of ET

The biosynthetic pathway of ET involves a series of reactions involving histidine and cysteine (8, 86) (Fig. 2). The use of radioactive precursors showed that histidine was first converted to hercynine by addition of three methyl groups, followed by incorporation of sulfur from cysteine, to generate ET (49–51). The conversion of hercynine to ET involved a sulfoxide intermediate (61). More recently, the gene clusters involved in ET biosynthesis were identified in Mycobacteria and Neurospora crassa (54, 60, 112). The mycobacterial pathway involves EgtA-EgtE enzymes, while fungal biosynthesis involves Egt1-Egt2 enzymes. The key steps involve a nonheme iron enzyme-catalyzed oxidative C-S bond formation (EgtB/Egt1 catalysis) and a pyridoxal 5′-phosphate (PLP)-catalyzed C-S lyase (EgtE/Egt2) reaction, which culminates in the transfer of a sulfur atom from a cysteine to a histidine side chain. Although biosynthesis of ET was believed to require oxygen, recent studies show that the anaerobic bacterium, Chlorobium limicola, might produce ET via oxygen-independent reactions, which suggests that ET may have been present on the planet in an anoxic environment (15).

FIG. 2.

Biosynthesis of ET. The biosynthesis of ET utilizes histidine as a precursor and cysteine as the sulfur donor in both Mycobacteria and Neurospora crassa. The enzymes involved in ET biosynthesis (EgtA-E) in Mycobacteria are depicted in blue and those in N. crassa (Egt1-2) are depicted in brown.

Features of ET Meriting Its Classification as a Vitamin

In this section, the features that ET shares with vitamins are described. Vitamins are essential nutrients and constituents of a healthy diet, which cannot be synthesized by humans, but if so, only in suboptimal amounts (1). The concept of vitamins, although not originally termed so, originated in the 18th and 19th centuries when it was noted that nutritional deficiencies caused disease (113). A study by Frederick Hopkins in 1912 revealed that growth of young rats was retarded on a basal diet of protein, starch, cane sugar, lard, and minerals. Normal growth resumed when a small amount of milk was added to the diet. The as yet unknown components in milk, which support life, were reported to be present in astonishingly small amounts and called accessory factors (52). Later, Casimir Funk proposed the term “vitamine” for these factors in 1912 (36). Soon these unknown factors in foods became synonymous with both “vitamine” and “accessory food factors.” Most vitamins are associated with deficiency syndromes, which led to their discovery to begin with. For instance, deficiency of vitamin A causes night blindness, vitamin C causes scurvy, and vitamin D causes rickets (84). Although ET has not been afforded the status of a classical vitamin, its importance in the well-being of humans may have gone unnoticed as it is present in a wide variety of foodstuffs. No pathological syndrome of ET deficiency has been reported. However, lack of such reports may simply reflect the relative paucity of ET research as well as the difficulty of depleting ET. ET is obtained by mammals exclusively from their diet like many vitamins. ET is concentrated in cells and tissues that are frequently exposed to oxidative stress, such as blood, liver, eye lens, and seminal fluid, and its concentration approaches high micromolar or millimolar levels in some of these tissues (97, 115, 116, 121, 124).

Similar to vitamin C, ET is taken up by a very specific transporter (130). The avidity with which ET is retained in mammalian systems and toxicity associated with its depletion in response to stress and its dietary origin led us to propose that ET merits designation as a vitamin. With accumulating evidence, it appears that ET is a stress vitamin that comes into play during adverse conditions or under duress (102) (Table 1). Foods such as mushrooms are a rich source of ET, with certain species, including king oyster, enoki, and shiitake mushrooms, having higher levels. Interestingly, yellow oyster and porcini mushrooms have higher levels of ET compared with GSH, the major antioxidant in most species (65). Plants too obtain ET from the soil, presumably through fungi present in their vicinity. Other foodstuffs that have higher levels of ET include garlic and Brazilian and gingko nuts (33, 44, 65) (Fig. 3).

Table 1.

Stress Stimuli That Induce the Ergothioneine/Ergothioneine Transporter System

Stress stimuli	System	References
UV-B irradiation	Increased ET content in caterpillar mushroom, Cordyceps militaris.	(55)
Inflammatory cytokines	IL-1β and TNF-α increased the expression of the ET transporter (ETT/OCTN1/SLC22A4) in the human fibroblast-like synoviocyte cell line, MH7A, derived from RA patients.	(77)
SDS	SDS increased the population of Lactobacillus reuteri, which produced ET, in the intestine of rats exposed to SDS.	(83)
Starvation	Under starvation conditions, ET levels increase in fission yeast, Schizosaccharomyces pombe.	(106)
High-cholesterol diet	Livers of guinea pigs fed a cholesterol-rich diet accumulated higher levels of ET.	(24)
Liver fibrosis inducing stress	Injection of the hepatotoxin, DMN, which induces liver fibrosis, increased expression of ETT/SLC22A4 and ET content in wild-type mice.	(125)
Metabolic acidosis	Metabolic acidosis induced by NH4Cl caused upregulation of ETT in the mouse kidney.	(41)
AIMD	Mice treated with a mixture of antibiotics (ampicillin, vancomycin, neomycin, metronidazole, and amphotericin B) displayed upregulation of ETT.	(136)
Ni2+ ion irradiation	The human EC line (EA.hy926) irradiated with accelerated nickel ions exhibited an increase in ETT.	(9)
Vaccination using a recombinant virus	Nonhuman primates injected with vaccines directed against VSV-EBOV exhibited upregulation of ETT/SLC22A4.	(87)
RUPP rat model	In the RUPP model of preeclampsia, ET upregulates antioxidant enzymes such as Nrf2, UCP1, PGC-1α, and SOD1 and SOD2.	(133)

AIMD, antibiotic-induced microbiome depletion; DMN, dimethylnitrosamine; EC, endothelial cell; ET, ergothioneine; ETT, ergothioneine transporter; IL, interleukin; Nrf2, nuclear factor [erythroid-derived 2]-like 2; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator 1α; RA, rheumatoid arthritis; RUPP, reduced uterine perfusion pressure; SDS, social defeat stress; SOD, superoxide dismutase; TNF-α, tumor necrosis factor α; UCP1, uncoupling protein 1; UV, ultraviolet; VSV-EBOV, vesicular stomatitis virus expressing the EBOV glycoprotein.

FIG. 3.

Dietary sources of ET. ET (depicted as a ball and stick model) is present in a variety of foodstuffs. It is enriched in mushrooms and fungi in the soil, which is taken up by plants. ET is also enriched in red meat, black beans, nuts, milk, and oats.

Similar to several vitamins, which have specific transporters for their uptake, ET is imported into cells by a specific transporter, the ergothioneine transporter (ETT/SLC22A4), in a sodium- and pH-dependent manner (42, 130). Knockdown of ETT decreases ET uptake in cell lines and mice, indicating that ETT is the major transporter for ET (67, 102). Additionally, a general evolutionary ancient genomic approach, which identifies genetic variants with frequency changes that are significantly greater over a given time period than expected under genetic drift alone, revealed ETT or SLC22A4 as one of the genes positively selected over evolutionary time, attesting to its importance (79). Differences in abundance of fungi in soil may also give rise to variations in the ET content of crops. It has been reported that excessive tillage of the soil can deplete ET levels in crops by disruption of mycelia of mycorrhizal fungi in symbiotic association with plants (10, 14, 100). A functional variant in the ETT is proposed to have provided protection against ET deficiency through increased absorption of this unusual amino acid in European agriculturalists (56). Although this allele was present at low frequencies in the early Neolithic populations, its enrichment only occurred within the last 4000 years (82). Functional variants of ETT such as L503F, which increase absorption of ET, have been linked to Crohn's disease and believed to be protective in nature (103).

Interestingly, ET levels increase during periods of starvation in both yeast and humans (106). Metabolomic studies have also confirmed these findings in the blood of humans, where ET is enriched (70). Additionally, levels of ET decrease as a function of aging in blood (17, 72, 121). In a study measuring age-related decline in gait speed, ET content was positively correlated with gait speed in middle-aged adults (96). ET levels also diminished twofold in the blood of sickle cell anemia patients and were associated with increased markers of oxidative stress (18). In a longitudinal study analyzing mortality and coronary artery disease (CAD), ET was identified as the metabolite most significantly associated with lower morbidity and mortality, being associated with a lower risk of CAD (117). This study also proposed ET as a biomarker for a healthy diet and low cardiometabolic risk. Consumption of an ET-based nutritional supplement has also been reported to improve joint range of motion and reduction of chronic pain (12). More recently, decrease in ET in the whole blood of human subjects has identified it as a potential marker of frailty (66). In the subsequent sections, the properties of ET that set it apart from other known antioxidant cytoprotectants and its role in neuroprotection are discussed.

ET as an Antioxidant

The fact that mammals do not synthesize ET, but import it via a specific evolutionarily conserved transporter, and retain it with high avidity suggests important physiological functions. ET accumulates in cells and tissues frequently exposed to oxidative stress, with concentrations approaching the millimolar range in blood, lens of the eye, liver, bone marrow, and seminal fluid (97, 115, 116, 120). One of the principal functions of ET is its antioxidant–cytoprotectant function (2, 44, 46, 102). High levels of ET are present in red blood cells, which also express the transporter. At the cellular level, ET has been reported to be present in the mitochondria, which produce reactive oxygen species during respiration (68). Thus, it is not surprising that its transporter has been localized to the mitochondria (73). ET mitigates deleterious effects of several free radicals, including reactive oxygen and reactive nitrogen species (Table 2). ET protects against the deleterious effects of hydroxyl radicals (^•OH), peroxynitrite (ONOO⁻), hypochlorous acid (HOCl), and singlet oxygen ¹O₂ (2, 47). ET is a better scavenger of ¹O₂ than GSH (122). ¹O₂ is generated by photosensitizers activated by sunlight in the eye and skin, which can also affect red blood cells, where ET is enriched. Sunlight exposure causes generation of ¹O₂ from protoporphyrin IX, the iron-free precursor of heme, while the iron-bound form does not produce ¹O₂. The peroxidase activity of hemoglobin can also lead to ¹O₂ production (32, 92). Cell culture studies have revealed that ET protects against ONOO⁻-induced DNA damage (5). Knocking down ETT causes increased levels of oxidative damage, as reflected by increase in protein carbonylation, lipid peroxidation, and DNA damage (102). Knocking out this transporter in Caenorhabditis elegans leads to increased oxidative stress and decrease in longevity (23). Similarly, knocking out the transporter in zebrafish, Danio rerio, results in increased oxidation of DNA, as revealed by accumulation of 8-oxoguanine (104). ET scavenges ¹O₂ more efficiently than GSH or ascorbate (99). In addition to these properties, ET can chelate divalent cations such as Cu²⁺, Zn²⁺, Ni²⁺, and Zn²⁺, and chelation of Cu²⁺ accounts for its ability to counteract Cu²⁺-mediated DNA damage (45, 91, 138).

Table 2.

Free Radical Scavenging/Neutralizing Activity of Ergothioneine

Free radical and oxidants	System	References
ONOO−	Prevented peroxynitrite-dependent nitration of tyrosine and inactivation of α1-antiproteinase.	(6)
1O2	Prevented oxidation of BHMF. Scavenges singlet oxygen in vitro.	(31, 108)
O2•−	Prevented cell death induced by pyrogallol, a superoxide generator in HeLa cells. Scavenged superoxide and singlet oxygen in UV-irradiated human dermal fibroblasts. Reduced cytotoxicity of paraquat, a superoxide generating agent in ECs, and formed the hercynine and sulfonic acid derivative (ESO3H) in both cell-free systems when reacted with superoxide and also in ECs exposed to high glucose.	(98, 102, 114)
HOCl	ET protected α1-antiproteinase against inactivation by HOCl.	(132)
•OH	ET is a powerful scavenger of hydroxyl radicals and an inhibitor of iron or copper ion-dependent generation of •OH from hydrogen peroxide.	(2)

¹ O₂, singlet oxygen; BHMF, 2,5-bis(hydroxymethyl) furan; HOCl, hypochlorous acid; O₂^•−, superoxide; ^•OH, hydroxyl radical; ONOO⁻, peroxynitrite.

ET and Inflammation

A link between ET and inflammation was observed in rheumatoid arthritis, where an SNP was found to be associated with the disease (129). In addition to these observations, expression of ETT was increased in response to the proinflammatory cytokine, tumor necrosis factor α (TNF-α), in inflamed joints. Moreover, mice lacking ETT exhibit increased susceptibility to inflammation after ischemia–reperfusion injury (67). The anti-inflammatory property of ET was evident in studies where both H₂O₂ and TNF-α mediated activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and release of interleukin 8 (IL-8) was prevented by ET (107). Furthermore, NF-κB has binding sites in the promoter of human ETT and regulates its expression, further supporting a role for ETT in modulating inflammatory processes (77).

ET Functions in the Nervous System

The presence of ET in the brain was observed as early as the sixties and was initially believed to be a neurotransmitter and identified as the cerebellar factor (28, 29). Later, it was shown that ET did not support neurotransmission and the cerebellar factor and ET had distinct properties, although they shared several similar features (13, 71). It is not surprising that ET is enriched in the cerebellum as its transporter is abundant in this tissue (134). ET is also present in other brain regions. The basal concentration of ET in the cortex of the brain has been reported to be ∼3.73 ± 0.59 ng/mg (124). Exogenous administration of ET revealed that ET is widely distributed in the brain in regions such as the cerebellum, striatum, medulla and pons, midbrain, hippocampus, hypothalamus, and cortex and the concentration correlates with the expression of its transporter, indicating its ability to cross the blood–brain barrier (94, 95).

ET exerts potent neuroprotective effects in the brain (Fig. 4). In the brain, the transporter, ETT, is functionally present in neurons, but not in astrocytes (58, 95). ET protects neuronal cells against oxidative stress (5). ET also protected against neurotoxicity induced by the excitotoxin, N-methyl-d-aspartate (NMDA) and cisplatin in vivo (90, 118). The protective effect of ET was also observed against β-amyloid toxicity. Mice injected with β-amyloid developed learning and memory deficits, while those pretreated with ET were spared (135). In addition, ET prevented oxidative stress, as revealed by decreased lipid peroxidation and maintenance of the GSH/GSH disulfide ratio and superoxide dismutase (SOD). ET has also been shown to be protective against learning and memory impairment induced by d-galactose in mice (119). Other studies have reported a role for ET in neuronal differentiation (63, 95). The effects on neuronal differentiation are partly attributed to phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1), a component of the mTOR signaling pathway, at Thr389 and by activation of the neurotrophin receptor, Tropomyosin receptor kinase B (TrkB) signaling, by upregulation of the neurotrophin, NT5 (62). Recently, it has been reported that ET activates human carbonic anhydrase VII at nanomolar levels (89). Carbonic anhydrases have been linked to modulation of redox homeostasis in cells and thus activation of these enzymes could be beneficial in the treatment of conditions involving redox imbalance. Neurodegenerative diseases have been associated with elevated oxidative and nitrosative stress, and ET may be beneficial in decreasing damage caused by reactive oxygen and nitrogen species in these diseases. ET prevented cisplatin-induced neuronal injury in neuronal cultures as well as mice and enhanced cognition, likely through inhibition of oxidative stress and restoration of acetylcholinesterase (AChE) activity in neuronal cells (118). Metabolomic analysis of Parkinson's disease (PD), the second most common neurodegenerative disease after Alzheimer's disease (AD), revealed a significant decline in ET levels, suggesting a decreased ability in antioxidant defenses (48). In addition, decrease in ET levels has also been observed in vascular dementia and dementia (21, 127). A study of subjects who consumed mushrooms, a rich source of ET, revealed an inverse correlation between mild cognitive impairment and mushroom intake, which was independent of age, gender, or lifestyle habits such as smoking or alcohol consumption (35). Oral administration of ET also promoted memory in rodents, as measured by the novel object recognition test (93). In another study, ET was reported to mitigate the deleterious effects of 7-ketocholesterol (7KC), an oxidation product of cholesterol, in the human brain endothelial cell line (69). 7KC induced elevation in messenger RNA (mRNA) levels of proinflammatory cytokines, IL-1β, IL-6, IL-8, TNF-α, and cyclooxygenase-2 (COX2), and COX2 activity was decreased by ET. Increased 7KC levels have been observed in the AD brain and administering ET may afford neuroprotection (128). Indeed, ET counters neurotoxicity induced in the cell line, C. elegans, and mouse models of AD (22, 64, 135).

FIG. 4.

Effects of ET on brain function. ET (depicted as a ball and stick model) is a neuroprotective molecule affecting multiple aspects of brain function. ET promotes neuronal differentiation and increases neurotrophin levels in the brain. ET prevents neurotoxicity induced by the excitotoxin, NMDA, and cisplatin in vivo. ET also ameliorates learning and memory deficits induced by amyloid β in mice. ET produced by the gut microbiota such as Lactobacillus reuteri protects against stress-induced sleep disturbances and social defeat stress. NMDA, N-methyl-d-aspartate.

ET has also been implicated in behavioral responses to social stress. Oral delivery of ET significantly prevented major depressive disorder (MDD)-like social avoidance and sleep abnormalities in a social defeat stress (SDS) model in rats (83). Symptoms of MDD include lack of interest or pleasure and depressed mood in addition to sleep abnormalities, a psychiatric disorder affecting millions worldwide (25). SDS had effects on the gut microbiota as well. The study reported increases in fecal Lactobacillus reuteri in correlation with ET levels at around day 11, which continued for at least 1 month following SDS administration. Thus, ET may participate in the gut–brain axis via the microbiota that produce it.

An interesting aspect of neuroprotection mediated by ET has been observed in the parasitic interaction between a fungus, Ophiocordyceps kimflemingiae, and the carpenter ant, Camponotus castaneus (76). The fungal infection triggers neurobehavioral alterations in behavior of these ants, which then invade plants and bite into them, before being killed by the fungus. The metabolic profile of the ant's brain revealed an elevation of ET levels, which presumably prevents neurodegeneration and preserves brain function.

ET and Antiaging Effects

Blood ET levels have been found to decrease significantly beyond 60 years of age. The serum concentrations of ET showed an inverse correlation with age (121). Moreover, a subset of the population exhibiting mild cognitive impairment had significantly lower plasma ET levels compared with age-matched controls, indicating that ET deficiency could contribute to aging (19). ET was found to delay endothelial cell senescence caused by high glucose through a mechanism involving the histone deacetylases, sirtuin 1 (SIRT1) and sirtuin 6 (SIRT6) (30). Due to its cytoprotective properties and UV filtering capability, ET is one of the top ingredients used in antiaging creams (27). ET protects UV-irradiated human dermal fibroblasts by scavenging ¹O₂ and O₂^•− and reduces levels of inflammation (98). ETT is present on skin cells, allowing them to import ET and reduce levels of reactive oxygen species and DNA, protein, and lipid damage in keratinocytes subjected to solar-simulating UV oxidative stress (80). ET has been reported to protect ultaviolet A (UVA)-irradiated human dermal fibroblasts via inhibition of the activator protein-1 (AP-1) pathway and activation of nuclear factor [erythroid-derived 2]-like 2 (Nrf2)-mediated antioxidant genes (53). ET is also protective in the eye, and formation of cataract is associated with a decline in ET levels (116). Interestingly, levels of ET in the eye lens exceed that of GSH, unlike the scenario in other tissues.

Cardiovascular Benefits

Cardiovascular disease is responsible for a vast majority of deaths worldwide and there is a constant search for drugs that can improve cardiovascular function. Endothelial dysfunction is a major cause of cardiovascular disease with links to oxidative and nitrosative stress (34, 59). ET, with its proven in vitro antioxidant functions, has also been reported to be imported by endothelial cells and reduce markers of oxidative damage (74). ET prevents toxicity induced by mercury chloride and preserves acetylcholine-mediated relaxation, improves the ratio of reduced GSH to oxidized GSH and catalase levels, and reduces overall oxidative stress (40). ET elicits a concentration-dependent relaxation in endothelium-intact aortic rings, which is abrogated endothelial denudation or NO synthase inhibition (39). The study also describes protection against the Cu/Zn SOD inhibitor, diethyldithiocarbamate (DETCA), and hypoxanthine/xanthine oxidase-induced impairment in vasorelaxation, all of which involved decreases in superoxide production. In addition to these effects, ET also prevents the binding of monocytes to endothelial cells, an early event in cardiovascular dysfunction (81).

Concluding Remarks: Future Perspectives and Therapeutic Avenues

The body has evolved multiple mechanisms to counteract stress. Some of these defenses act constitutively, while others are inducible and act during stress. No single antioxidant can scavenge or neutralize the wide variety of reactive oxygen and nitrogen species single-handedly. Thus, the search is on for molecules that counter a wide variety of reactive oxygen and nitrogen species. Additionally, molecules that possess anti-inflammatory effects in addition to antioxidant scavenging roles would provide improved neuroprotection. ET is an unusual antioxidant, in that it is exceptionally stable and does not auto-oxidize at physiological pH and is not destroyed upon heating. ET is water soluble and neutralizes several reactive oxygen and nitrogen species, including ^•OH, O₂^•−, ONOO⁻, HOCl, and ¹O₂. Accumulating evidence suggests that ET is endowed with cytoprotective signaling functions in addition to its antioxidant and anti-inflammatory role in cells. It has also been posited that ET is an adaptive antioxidant, with cells deliberately accumulating ET in times of stress (43). Thus, ET is a stress metabolite that is obtained via specific transport, implying that regulation of the transporter is part of an adaptive stress response. Accumulation of ET has been observed in infarcted mouse hearts, diabetes, and preeclampsia (109, 110, 131). Similarly, metabolomic analysis of mice repeatedly injected with metamphetamine, a drug of abuse, led to increase in ET levels in the brain (85). These observations, in conjunction with the fact that ET has not been associated with any toxic or adverse effects, support its use in therapies against a wide range of diseases and conditions, ranging from cardiovascular diseases to aging and neurodegeneration. ET is a rare antioxidant–cytoprotectant capable of crossing the blood–brain barrier, a feature that is necessary to treat neurodegenerative disorders where oxidative stress plays a central role in disease progression (111). It is present in mitochondria, which is a feature that can be harnessed in therapies for disorders involving mitochondrial dysfunction such as PD, where this molecule is significantly depleted (48). Another avenue of exploration could be its anti-inflammatory potential to develop a new series of nonsteroidal anti-inflammatory drugs. Because of its antioxidant and anti-inflammatory properties, the use of ET as a therapeutic in the treatment of COVID-19 patients has been proposed (20). A feature of COVID-19 is dysregulated redox balance, which is also observed in patients exhibiting chronic fatigue (COVID-19 long haulers) long after the infection was cleared. Presumably, ET may be beneficial in this aspect of the disease as well (101). Future studies that elucidate its precise mechanism of action in signal transduction cascades could pave the way for development of novel strategies to combat aging and disease. In summary, ET may afford a more stable mode of cytoprotection. It is not metabolized to any significant extent in mammalian tissues, the half-life of dietary ET being ∼1 month. Its existence as a tautomer of thiol and thione forms confers resistance to auto-oxidation, distinguishing it from other common thiols. These properties suggest a role for ET as a bulwark, a final defense for cells against oxidative damage. Evidence that ET is a physiological antioxidant raises the question of its status in biology. Despite its high concentration and ubiquitous presence, mammalian ET is mostly derived from dietary sources. The existence of ETT establishes ET as an important normal body constituent, and in this regard, ET fits the definition of a vitamin.

Abbreviations Used

¹O₂

singlet oxygen

7KC

7-ketocholesterol

AD

Alzheimer's disease

AIMD

antibiotic-induced microbiome depletion

BHMF

2,5-bis(hydroxymethyl) furan

CAD

coronary artery disease

COX2

cyclooxygenase-2

DMN

dimethylnitrosamine

EC

endothelial cell

ET

ergothioneine

ETT

ergothioneine transporter

GSH

glutathione

HOCl

hypochlorous acid

IL

interleukin

MDD

major depressive disorder

MSH

mycothiol

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NMDA

N-methyl-D-aspartate

Nrf2

nuclear factor [erythroid-derived 2]-like 2

O₂^•−

superoxide

^•OH

hydroxyl radical

ONOO⁻

peroxynitrite

PD

Parkinson's disease

PGC-1α

peroxisome proliferator-activated receptor-γ coactivator 1α

RA

rheumatoid arthritis

RUPP

reduced uterine perfusion pressure

SDS

social defeat stress

SIRT

sirtuin

SOD

superoxide dismutase

TNF-α

tumor necrosis factor α

UCP1

uncoupling protein 1

UV

ultraviolet

VSV-EBOV

vesicular stomatitis virus expressing the EBOV glycoprotein

Author Disclosure Statement

The author declares no conflicts of interest.

Funding Information

This work was supported by the American Heart Association/Paul Allen Frontiers Group Project 19PABH134580006 (to B.D.P. and associates) and NIH 1R21AG073684-01 (to B.D.P).