Abstract
Ergothioneine is a thiol/thione molecule synthesised only by some fungi and bacteria. Nonetheless, it is avidly taken up from the diet by humans and other animals through a transporter, OCTN 1, and accumulates to high levels in certain tissues. Ergothioneine is not rapidly metabolised, or excreted in urine and is present in many, if not all, human tissues and body fluids. Ergothioneine has powerful antioxidant and cytoprotective properties in vitro and there is evidence that the body may concentrate it at sites of tissue injury by raising OCTN 1 levels. Decreased blood and/or plasma levels of ergothioneine have been observed in some diseases, suggesting that a deficiency could be relevant to the disease onset or progression. This brief Review explores the possible roles of ergothioneine in human health and disease.
Abbreviations
Aβ, amyloid beta peptide
CKD, chronic kidney disease
ET, ergothioneine
GSH, reduced glutathione
OCTN1, organic cation transporter (novel type‐1)
RNS, reactive nitrogen species
ROS, reactive oxygen species
The antioxidant paradox
The generation of free radicals and related reactive oxygen (ROS),11 For detailed explanations of these terms please see reference [1].
nitrogen (RNS) and other reactive species11 For detailed explanations of these terms please see reference [1].
is an inevitable consequence of aerobic life and serves many useful purposes [1-4]. Indeed, these species were key drivers of the evolution of the huge range of aerobic organisms that we find in the world today, including ourselves [1, 3, 4]. Without them, we would die early from infectious disease and other disorders [1, 3, 5]. However, there is a downside. Certain reactive species can damage biological molecules (this is called “oxidative damage”) and this oxidative damage contributes to the development of certain age‐related human diseases. The evidence for a role of ROS/RNS in the origin and progression of human disease is probably strongest for certain cancers [1, 6, 7] and for neurodegenerative diseases such as Parkinson disease and the dementias (especially Alzheimer disease) [1, 8-10], but they play roles in many other diseases as well (reviewed in [1, 11]). There has, therefore, been considerable interest in developing antioxidant agents with the aim to slow the onset of, and/or to treat such diseases. Interest originally focussed (and to some extent still does) on the established diet‐derived antioxidants (such as vitamins E and C) or putative ones including carotenoids and polyphenols such as the flavonoids (we say “putative” because the evidence for their antioxidant roles in vivo is not as strong as for vitamins E and C, as reviewed in reference [1]). However in general, the effects of diet‐derived antioxidants in intervention trials on human subjects have been modest at best, and sometimes, they appear to have caused harm [1, 11-15]. Multiple reasons can account for this failure, one of which is that many of these agents, administered at the high doses used in the trials, are not very effective at decreasing levels of oxidative damage in the human body [1, 11, 16]. There is, therefore, an ongoing search for better antioxidant agents, including synthetic ones such as NADPH oxidase inhibitors [3, 17] and metalloporphyrins that can scavenge a range of ROS/RNS: some of each type are presently in clinical trials [17, 18]. Another approach is to use reagents that activate the Nrf2 system, which leads to increases in the in vivo levels of several antioxidant enzymes and of reduced glutathione (GSH), a key cellular antioxidant [19]. Indeed, the beneficial effects (if any, the jury is still out) of polyphenols against certain diseases, such as cancer, have been suggested to relate more to their pro‐oxidant abilities (raising endogenous antioxidant levels in a hormetic fashion [1, 20]) than to direct antioxidant effects. However, too much Nrf2 activation may not be good [16, 19, 21], especially if it disturbs the normal essential cellular function of ROS/RNS.
Ergothioneine, a natural “antioxidant”?
Which brings us to ergothioneine (ET) a natural product with considerable in vitro antioxidant properties (reviewed in [22]). Figure 1 shows its structure; the tautomeric equilibrium favours the thione form. Studies into this thiol/thione derivative of histidine date back to the early 1900s when ET was first identified in the ergot fungus [23], hence the name. ET is synthesised by certain fungi and bacteria only, not by animals or higher plants [22, 24-26]. Nevertheless, ET can be found in a wide range of foods (Table 1 shows some data from our laboratory). However, mushrooms, which are capable of ET biosynthesis along with several other fungi, are a major source in the human diet [22, 25, 27-29]. Interestingly, some foods have large variations in the levels of ET, for example, asparagus, which we believe to be attributed to possible symbiotic relationships of these plants with soil fungi or bacteria, or pre‐ or postharvest fungal contamination (Table 1). Dietary ET in animals (including humans) is absorbed by means of an intestinal transporter, OCTN1, that has a high degree of specificity [30]. The same transporter then distributes ET to most or all body tissues: excretion from the body is slow and administered ET is highly retained in human and other animal body tissues and red blood cells [22, 31-33]. This implies that ET has a useful function, otherwise why absorb and retain it? By contrast, many polyphenols (which have frequently been suggested to act as important antioxidants in vivo ) are rapidly metabolised or excreted from the body, which instead suggests that they are unwanted xenobiotics [1]. Indeed, numerous in vitro studies have demonstrated the abilities of ET to scavenge ROS and RNS (such as hydroxyl radicals [34], hypochlorous acid [34], singlet oxygen [35, 36], and peroxynitrite [37]), modulate inflammation [38], chelate divalent metal cations such as iron and copper (thereby decreasing the ability of these metal ions to stimulate oxidative damage) [34, 39, 40], and protect against UV radiation‐induced damage [41, 42], amongst other cytoprotective activities [43]. Conversely, OCTN1 knockout animal models appear to be predisposed to oxidative stress [44, 45], although more studies in rodents to characterise the phenotype in detail and verify this are needed. 
| Mushrooms varieties | Ergothioneine (mg per 100 g dry weight) | Fruits and vegetables | Ergothioneine (mg per kg dry weight) |
|---|---|---|---|
| Boletus edulis (cepes) | 181.24 | Garlic | 34.60 |
| King Oyster | 54.17 | Japanese Seaweed | 2.34 |
| Buna Shimeji | 43.26 | Parsnip | 2.23 |
| Shiitake | 35.35 | Kiwi fruit | 1.99 |
| Enoki | 34.64 | Onion | 1.13 |
| Willow | 29.68 | Persimmon | 1.52 |
| Abalone | 32.47 | Pomegranate | 1.3 |
| White Shimeji | 19.75 | Passion fruit | 1.22 |
| Portobello | 19.09 | Durian | 1.09 |
| White button | 15.44 | Broccoli | 0.38 |
| Brown button | 10.41 | Kale | 0.22 |
| Black fungus | 9.42 | Tomato | 0.20 |
| Maitake | 2.02 | Ginger | 0.17 |
| Wood ear | 0.64 | Rice | LOQ |
| White fungus | 0.58 |
| Nuts, beans and spices | Ergothioneine (mg per kg dry weight) | Milk and soy products | Ergothioneine (mg per kg dry weight) |
|---|---|---|---|
| Basil leaf | 4.92 | Tempeh | 201.13 |
| Brazilian nut | 4.45 | Soy beancurd | 3.71 |
| Gingko nut | 3.98 | Soy milk | 2.31 |
| Cumin | 2.60 | Fresh milk (average of 4 varieties) | 0.25 |
| Pepper | 2.57 | Greek yogurt | LOQ |
| Kidney beans | 2.09 | ||
| Pistachio nut | 1.90 | *Asparagus varieties | |
| Almond | 1.87 | Asparagus (Malaysia) | 0.57 |
| Oats | 1.84 | Asparagus (Thailand) | 10.24 |
| Macadamia nut | 1.65 | Asparagus (Mexico) | 163.25 |
| Sweet bean | 1.33 | White asparagus | 18.20 |
| Ginseng root | 0.69 | ||
An “adaptive” antioxidant?
Some studies have presented evidence that ET may be accumulated at sites of tissue injury, in particular in fatty liver disease, liver fibrosis, pressure overloaded and infarcted hearts and pre‐eclampsia. This accumulation seems to relate to an increased expression of the gene encoding OCTN1, resulting in increased transporter activity [46-49]. We have suggested [46, 50] that this is a deliberate cytoprotective mechanism (Fig. 2). Arguably in these situations of tissue injury, supplementation of humans with extra ET might be of therapeutic benefit [50, 51]. Animal studies have suggested that ET may play a limited antioxidant role in healthy animals but may come into play when levels of reactive species rise due to tissue injury, in the presence of ROS/RNS‐generating toxins, or diseases that involve increased levels of oxidative damage (reviewed in [22, 50]). This concept is consistent with in vivo studies of the effects of dietary ET supplementation on oxidative damage in young healthy adults [32]; there was a trend to a decrease in oxidative damage, as detected in plasma and urine using several established biomarkers of oxidative damage, but no major decreases. However, this could be argued to be a useful property of ET: not interfering with the important roles of ROS/RNS in healthy tissues [1, 3-5] but coming into play when oxidative damage becomes excessive due to tissue injury, toxin exposure or disease [50] and ET is then accumulated, as summarised in Fig. 2. Some cells where the risk of oxidative stress is high and constant, such as erythrocytes [1], may always keep ET levels high [32, 33, 49, 52]. 
Ergothioneine in tissues, extracellular fluids and cell culture
Ergothioneine can accumulate to high levels in some human and animal tissues, including red blood cells (with basal levels of ~ 125 μm and ~ 220 μm in human and mouse whole blood, respectively, and millimolar levels reported in red blood cells [52, 53]), liver and spleen (with basal levels of ~ 350 μmol·g−1 tissue and ~ 100 μmol·g−1 tissue in mouse liver and spleen, respectively) [22, 31-33]. Our recent study [33] demonstrated that when ET is orally administered to mice, it accumulates rapidly in the liver and blood cells but also enters most (or perhaps all) other tissues, including brain, heart, lung, kidney, spleen and eye. Since animal sera are commonly used in cell culture media, ET is even present in cell cultures. The variable ET levels in different batches and sources of sera may be a possible confounder in cell culture studies [50], given the propensity of cell culture to cause oxidative stress [20, 54, 55].
When ET is administered to humans, ET levels in plasma and whole blood are significantly elevated and, interestingly, continued to increase in whole blood for up to 4 weeks after administration ceased [32]. Moreover, excreted levels (in urine) were extremely low, indicating the avid retention of ET by the body [32]. We (paper in preparation) have found ET to be present in a range of other human extracellular fluids and secretions such as cerebrospinal fluid (with levels around 250 nm ) and aqueous humour of the eye (with levels in the high μm range). Animal seminal fluids are reported to contain especially high levels of ET, with early studies showing that ET levels in boar seminal plasma were around six times greater than blood ET levels, and in some animals, ET is the predominant thiol/thione in seminal plasma, far exceeding levels of GSH [56-58]. Data on humans are awaited with interest.
Ergothioneine and the mother–baby axis
As a further illustration of the widespread distribution of ET and possibly the essentiality of this compound to humans (as suggested by the presence of a transporter that appears largely specific for ET [30]), we have recently identified the presence of ET in human breast milk (with ET concentrations ranging from 5 to 150 nm ; paper in preparation). ET can also be found in cow and goat's milk (with mean concentrations ~ 13 nm and ~ 9 nm , respectively) and a range of infant formulas (with a mean concentration ~ 9 nm ). The latter is not unexpected, since baby formulas are typically based on milk powders. Indeed, the urine and brains of newborn babies have been reported to contain ET [59, 60]. This implies that ET can cross the placenta into the baby (presumably via OCTN1, which is known to be present in placenta [61, 62]) and/or that ET is absorbed from breast milk through OCTN1 in the intestines of the baby. Additionally, ET has been detected in amniotic fluid, in sheep [63]. Studies revealed that the OCTN1 mRNA expression levels in cultured human mammary epithelial cells are elevated more than sixfold during lactation, relative to nonlactating mammary epithelial cells [64]. If this happens in the intact breast, it suggests that this is a mechanism to deliver ET to the baby, from which it would follow that ET is important to the baby. However, more work is needed to establish this in vivo .
Ergothioneine as a potential treatment for diseases
Several studies have identified decreased levels of ET in certain tissues relative to controls, in subjects with various diseases (reviewed in [22, 50], also see the sections below). This suggests potential interventions with administered ET to raise the levels as both a therapeutic and possibly a preventative agent. Studies in animals and humans have found no toxicity or adverse effects to be associated with ET administration, even at high doses, and recently, ET (Tetrahedron, Paris, France) has attained European Food Safety Authority approval in the European Union and is generally recognised as safe by the Food and Drug Administration in the US (GRAS notice 734) [65] as a supplement. Hence, the possible beneficial effects of supplementation with ET to correct these low levels are worth further investigation.
Neurodegenerative diseases
A range of in vitro and in vivo studies have demonstrated the neuroprotective capabilities of ET. Supplementation with ET dose‐dependently protected rat phaeochromocytoma cells against β‐amyloid (Aβ)‐induced apoptotic death [66] and decreased neuronal injury caused by direct injection of Aβ into the hippocampus of mice [67]. Our own studies (paper in preparation) have demonstrated that ET can dose‐dependently extend lifespan of a transgenic Caenorhabditis elegans model of Alzheimer disease [68], through reduction in Aβ‐oligomer load. Other in vivo studies have also demonstrated that ET attenuates oxidative stress and prevents cognitive deficits in a D‐galactose‐induced mouse model of dementia [69], protects against N ‐methyl‐D‐aspartate‐induced cytotoxicity in rat retinal neurons [70] and cisplatin‐induced neuronal damage in mice [71].
In humans, plasma ET levels were found to be significantly decreased in subjects with mild cognitive impairment [72] and Parkinson disease [73] when compared to age‐matched controls. This could happen by multiple mechanisms of course, such as changes in diet and/or OCTN1 transporter activity. Dietary ET does cross the blood–brain barrier, since it can be measured in human cerebrospinal fluid and postmortem brain samples (our unpublished data; also see [59]) and readily enters the brain when administered to mice [33]. A Japanese study [74] has found a correlation between increased intake of mushrooms (one of the most important dietary sources of ET [27-29]; Table 1) and lower incidence of dementia. However, mushrooms are known to contain a wide range of possibly bioactive compounds that could account for this observation. Hence, direct studies (placebo‐controlled, double‐blinded intervention trials) are needed to investigate whether ET administration will have beneficial effects in neurodegenerative disorders.
Eye disorders
Early studies demonstrated that substantial levels of ET are found in the eye [75], where it was suggested to protect against chronic exposure to ROS due to high oxygen tension (the cornea is exposed to 21% oxygen), UV exposure and high metabolic activity [76]. The ocular surface, consisting of a layer of tear fluid, the cornea and the aqueous humour, forms the first line of defence against oxidative damage [1, 77]. We recently identified significant levels of ET in human tear and aqueous humour samples (~ 0.35 μm and ~ 28 μm , respectively; paper in preparation), and we have demonstrated that ET readily accumulates within the eye when mice are fed with ET [33]. It is, therefore, of interest that some earlier studies revealed that significantly lower levels of ET in the lens and cornea are found in individuals with cataracts; the levels continued to decrease with increasing severity of the cataract formation [78, 79]. Another study found that treatment with ET afforded modest protection against glucocorticoid‐induced cataract formation in chicks [80]. No further studies have since been undertaken to follow up on this. However, if ET levels in tear fluids correlate with levels in the lens and aqueous humour, this could provide a noninvasive means of measuring eye ET levels and thereby establish the risk of developing eye disorders. Since the pathology of the major eye diseases involves oxidative damage [1, 77, 81], the possible protective effects of ET seem to be worthy of further investigation in various human ocular diseases.
Cardiovascular diseases
Myocardial ischaemia–reperfusion injuries are known to generate, and be exacerbated by, excessive formation of ROS/RNS, leading to oxidative myocyte damage [1, 82, 83]. This suggests potential benefits of antioxidants as cardioprotectants [1, 82, 83]. Early studies by Arduini et al . [84] suggested that one of the mechanisms by which ET might protect the heart is by countering the oxidation of myoglobin by ROS/RNS to the cytotoxic ferryl myoglobin, which can be a critical event in myocyte damage during cardiac ischaemia–reperfusion. The ability of ET to scavenge ROS/RNS and chelate transition metal ions may also be relevant [1, 82, 83]. Since administered ET readily accumulates in the blood [32] and enters the heart (at least in mice [33]), it could be useful therapeutically.
Ischaemia–reperfusion injury is not a phenomenon confined to the heart; it can occur in almost all organs including brain, liver, skin, gut, muscle and kidney [1, 82]. Indeed, animal studies have shown that ET supplementation can protect several tissues, including liver [85], intestine [86] and lung (following intestinal ischaemia–reperfusion [87]), from ischaemia–reperfusion injury. Conversely, removing ET by knocking out the gene encoding the transporter in mice appears to predispose them to more injury following ischaemia–reperfusion injury [44]. Hence, ET could conceivably have therapeutic potential in these various ischaemic injuries.