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Ergothioneine, an adaptive antioxidant for the protection of injured tissues? A hypothesis
Accepted Manuscript Ergothioneine, an adaptive antioxidant for the protection of injured tissues? A hypothesis Barry Halliwell, Irwin K. Cheah, Chester L. Drum PII:
S0006-291X(15)31128-1
DOI:
10.1016/j.bbrc.2015.12.124
Reference:
YBBRC 35117
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 22 December 2015 Accepted Date: 30 December 2015
Please cite this article as: B. Halliwell, I.K. Cheah, C.L. Drum, Ergothioneine, an adaptive antioxidant for the protection of injured tissues? A hypothesis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2015.12.124. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Ergothioneine, an adaptive antioxidant for the protection of injured tissues? A hypothesis. Barry Halliwell1*, Irwin K. Cheah1, Chester L. Drum2 Department of Biochemistry, National University of Singapore, Singapore 117596
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Department of Medicine, National University of Singapore, Singapore 119228
Tel: +65-6516 3247 Fax: +65-6775 2207
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*Corresponding author email: [email protected]
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Abstract
Ergothioneine (ET) is a diet-derived, thiolated derivative of histidine with antioxidant properties, at least in vitro. Although ET is produced only by certain fungi and bacteria, it can be found at high concentrations in certain human and animal tissues and is absorbed through
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a specific, high affinity transporter (OCTN1). In liver, heart, joint and intestinal injury, elevated ET concentrations have been observed in injured tissues. The physiological role of ET remains unclear. We thus review current literature to generate a specific hypothesis: that
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the accumulation of ET in vivo is an adaptive mechanism, involving the regulated uptake and
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concentration of an exogenous natural compound to minimize oxidative damage.
Keywords
Reactive oxygen species; antioxidant; ergothioneine; adaptation; OCTN1; mushrooms
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ACCEPTED MANUSCRIPT Introduction Ergothioneine (ET), first isolated in 1909 from the ergot fungus Claviceps purpurea [1], is the trimethylbetaine of 2-thiol-L-histidine (Fig. 1). In solution, ET exists as a tautomer
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between thiol and thione forms (Fig. 1), but at physiological pH it exists predominantly as the thione [2-4]. As far as is known, ET is synthesized only by non-yeast fungi, certain bacteria and cyanobacteria [1-9], and its biosynthetic pathways in certain bacteria have been elucidated [10-16]. Despite the limited range of organisms able to make it, ET finds its way
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into a range of foodstuffs and it is widely present in human and other animal tissues
Ergothioneine as an antioxidant?
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[3,4,8,9,17-19].
Many early papers characterized the antioxidant activity of ET in vitro. It can scavenge
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certain reactive oxygen species (ROS)1 and bind transition metal ions such as Fe2+/Fe3+ and Cu2+/Cu+ in forms unable to catalyze redox reactions [2-4,20-30]. Antioxidant and related
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effects of ET on cells in culture have also been demonstrated [4,30-36]. Although several chemicals that have powerful “antioxidant” activities in vitro do not exert such actions in vivo
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[37,38], some evidence does exist that is consistent with antioxidant effects of ET in vivo in bacteria and fungi [16,39-42]), C. elegans [43], and in animals. For example, ET was shown to protect rodents against tissue damage induced by the pro-oxidant ferric-nitrilotriacetate [44], lung injury by cytokines [45], and ischaemia-reperfusion injury in liver, heart and intestine [29,46,47]. ET has also been shown to protect mice against brain damage induced by β-amyloid [48] or D-galactose [49]. However, although tissue injury in all these animal 1
‘Reactive oxygen species’ is a collective term [2] for agents more reactive than oxygen [O2] itself and includes both oxygen radicals and certain non-radicals that are oxidizing agents and/or are easily converted into radicals (such as hypochlorous acid [HOCl], peroxynitrite [ONOO-], hydrogen peroxide [H2O2] and singlet O2 1∆g [1O2]).
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ACCEPTED MANUSCRIPT systems involves ROS, it is often the case in vivo that the protective effects of “antioxidants” are not mediated by antioxidant mechanisms [50]. Nevertheless, a few studies have used biomarkers of oxidative damage to directly demonstrate antioxidant effects of ET in animals. For example, lack of a putative ET transporter in C. elegans [43] raised the levels of protein
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carbonyls (a biomarker of oxidative protein damage [51]), and in zebrafish lack of ET increased levels of 8-oxo-7, 8-dihydroguanine [52], a biomarker of oxidative damage to
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nucleic acids [51].
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Discovery of the ET transporter
Fig. S1 shows the number of papers published mentioning ET since 1909, based on a Web of Science search on 16 November 2015. It can be seen that after the initial limited research in the early to mid 1900s, interest waned, with only a handful of papers appearing every year.
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This began to change in 2005 when Grundemann et al [53] identified a Na+-dependent transporter for ET, the organic cation transporter novel, type 1 (i.e. OCTN1) in a wide range of animal tissues, especially bone marrow and intestine but also kidney, trachea and brain
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(Fig. 2), and more recently in zebrafish [52]. The specificity of OCTN1 was demonstrated by
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the lack of uptake of other organic cations, including a much lower transport rate, relative to ET, of the structurally similar compounds, methimazole, carnitine and hercynine, and the low Km for ET (~ 21 µM) [53-56]. Silencing the gene (SLC22A4) encoding OCTN1 in cell cultures inhibits uptake of ET [30]. Similarly, metabolomic analysis of OCTN1 knockout (octn1−/−) mice revealed that tissues were almost completely deficient in ET, indicating an absence of alternative mechanisms of uptake [56,57]. The same was seen in zebrafish [52]. The presence of a specific transporter suggests that animals (including humans) may gain benefit from obtaining ET and retaining it in their tissues. It is an interesting contrast to 3
ACCEPTED MANUSCRIPT polyphenols, also powerful antioxidants in vitro, which are rapidly metabolized and excreted from the body [37,38], suggesting that they are not required by animals and thus promptly disposed of. As another example, four tocopherols and four tocotrienols from the diet can exert antioxidant effects [38,58], but only RRR-α-tocopherol is retained by the body [58]; the
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others are discarded, implying they are less useful. Like ET, ascorbate, an essential dietderived enzyme cofactor and possible in vivo antioxidant [38,59], also has selective
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transporters for intestinal uptake and delivery to tissues [59].
Hence, the data indicate that ET is useful to the body, perhaps even essential [30],
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although exactly why is still uncertain. Polymorphisms in the SLC22A4 gene encoding OCTN1 in humans that may affect the transport of ET by OCTN1 and thus might influence ET uptake by the intestine and its distribution to tissues have been described [54,61,62].
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Is ergothioneine harmful?
ET is widespread in the diet and some people, especially mushroom lovers, consume substantial amounts [8,19,63]. Toxicology studies on ET to date have revealed no adverse Nevertheless, there are
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effects (references [64-66] and Cheah et al. in preparation).
suggestions in the literature that ET might be harmful. In one study, patients with rheumatoid
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arthritis (RA) were found to have higher erythrocyte and monocyte levels of ET, which were closely correlated to expression of OCTN1 mRNA in CD14+ cells [67]. Mice with collageninduced arthritis also highly expressed OCTN1 in inflamed joints [68]. Analysis of the inflamed intestinal mucosa of patients with Crohn’s disease (CD) revealed elevated levels of ET, which correlated with increases in OCTN1 mRNA [69]. The 503F variant of OCTN1 suggested to be associated with CD was found to have a 50% greater transport efficiency compared to the normal variant (503L) [70]. Hence in these chronic inflammatory conditions,
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ACCEPTED MANUSCRIPT accumulation of ET was suggested to be an aggravating factor [68,69]. It was even suggested that consumption of mushrooms rich in ET could be a bad thing [71], although the bulk of available evidence suggests the opposite [72-75]. Some studies have indicated that the elevated levels of ET can be due to the cytokine-
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mediated increased expression of the gene encoding OCTN1. Thus, incubation of Caco-2 cells with TNF-α increased OCTN1 gene expression and tissue ET levels through activation of the TNF-α receptor-1 [69]. Similarly OCTN1 gene expression was found to be increased
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by RA-associated inflammatory cytokines such as interleukin-1 (IL-1β), and by the transcription factor RUNX1 [76]. The presence of specific SLC22A4 polymorphisms
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allegedly associated with CD or RA in certain populations, suggests the existence of ethnic differences in OCTN1 levels and resultant tissue ET levels [54,77-83]. There may also be species differences [56]. However, it would seem odd that inflamed and damaged tissues
Thinking out of the box
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would increase their ET levels to do themselves further harm.
We recently examined ET levels in a guinea pig model of diet-induced liver damage [84]. If
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guinea pigs are fed a cholesterol-rich diet, they rapidly develop liver damage [84,85]. We
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observed that, despite the fact that the diet of the animals contained little ET (~2-3 ng/mg), the damaged liver accumulated high levels of ET (Fig. 3) [86], much higher than in the healthy livers of control animals. Indeed, an early paper [9] pointed out that even very low levels of ET in the diet can lead to significant amounts in animal tissues. The rise in ET appeared to result from increased OCTN1 levels [86]. We noted that, although animal models of fatty liver disease often show increased oxidative damage [87-89], this was not the case in our model. We put forward the hypothesis, summarized in Fig. 4, that ET accumulation is done deliberately as cytoprotective event helping to protect tissues against further injury by
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ACCEPTED MANUSCRIPT oxidative damage. Thus the observed accumulation of ET in tissues in some human diseases is not pathological, but cytoprotective. Such accumulation of ET in at risk tissues could function in parallel to the known adaptive antioxidant systems, such as activation of the Nrf2 system [90-92]. We examined the literature to search for other examples of ET accumulation
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in injured tissues. A metabolomics study identified, but did not comment on, a marked increase in ET levels in both pressure overloaded and infarcted mouse hearts [93]. There is some suggestion that ET levels in blood cells are elevated in diabetes [94,95]. Pre-eclampsia,
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which can involve oxidative stress [96,97], is also associated with ET accumulation [98]. In a mouse model of intestinal inflammation, expression of the OCTN1 gene and levels of ET
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were elevated in intestinal macrophages [99]. All these observations are consistent with our hypothesis (Fig. 4). It would also be interesting to examine if other diet-derived antioxidants are accumulated similarly.
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Effects of age?
Blood ET levels decline with age, which could of course be due to several mechanisms (references [100,101], Cheah et al. in preparation). Hence the adaptive mechanism (Fig. 4),
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like some other antioxidant defence mechanisms [50], may be less efficient in the elderly.
Some caveats
The studies reviewed above highlight the importance of considering dietary ET levels and possible accelerated tissue ET uptake when undertaking studies in animal models of human disease. This dietary variable could have a significant impact on the outcomes. Our study [86], like reference [9], revealed that ET can accumulate in tissues despite very low dietary concentrations, e.g. as typically found in normal animal feeds. We also measured a variety of other normal laboratory animal feeds and observed similarly low but detectable levels of ET
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ACCEPTED MANUSCRIPT (data not shown). Few, if any, studies have monitored the presence of ET in the diets of laboratory animals, but it could be a very important variable and confounding factor. ET has been shown to accumulate in a wide range of tissues, including the brain [4]. Hence dietary levels of it may also be relevant to animal models of neurodegeneration, and indeed of many
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other diseases.
Our laboratory has previously identified numerous artefacts caused by oxidative stress in interpreting studies on cells in culture [102,103]. Cells possessing the ET transporter will
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be able to take up ET and accumulate it to exert antioxidant effects, whereas cells without the transporter cannot [30]. We therefore measured the ET content in cell culture media and
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found it to be present in several batches of foetal calf serum, at levels varying between supplier and batch (Cheah et al. in preparation). This presence of ET and the variable expression of the transporter in cells are important factors that also need to be considered
Acknowledgements
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when interpreting cell culture data, but have not been to date.
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The authors wish to thank Tetrahedron (Parc Technologique Biocitech 102 avenue Gaston
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Roussel, Romainville, F 93230, France) for providing the L-ergothioneine used in our studies. The authors are also grateful to Mr. Richard Tang for analysis of data. We thank the National Medical Research Council (grant number: NMRC/1264/2010/082/12) of Singapore for support.
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Figure
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Figure 1. Structure of thione-thiol tautomers of ET (2-mercaptohistidine trimethylbetaine). In solution at physiological pH, ET exists predominantly in the thione (a) rather the thiol (b),
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hence conferring greater stability over other thiols such as GSH.
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Figure 2. Expression of the gene encoding the ET transporter, OCTN1, in human tissues, analyzed by real-time PCR, adapted from 2 independent sources [53,69]. Data are relative to mRNA level of the ileum for each set (Grundemann et al, 2005, National Academy of Sciences and Taubert et al, 2009, BJ Journals).
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Figure 3. ET levels in guinea pig liver. Liver ET levels were measured using LC-MS/MS and found to be significantly elevated with increasing dietary cholesterol and for the moderate cholesterol diet also with duration of feeding (A). n=7; ***p<0.001, ****p<0.0001 versus respective controls for feeding duration, ###p<0.001 versus 2-month moderate cholesterol diet; one-way ANOVA (Bonferroni’s post-test). (C). There was no difference in ET
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concentrations (average of 3 measurements) between the three diets. A level of 40-50 ng/mg tissue corresponds approximately to a concentration of 0.19 mM ET. Figure taken with
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permission from [86] by courtesy of Taylor & Francis publishers.
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Figure 4. Hypothesis: ET as an adaptive antioxidant. Tissue injury by any mechanim leads to
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increased oxidative damage that can worsen the injury [50]. ET may help protect against this.
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Highlights •
Ergothioneine (ET) is a powerful antioxidant.
•
It originates from fungi and bacteria but can accumulate at high levels in human
•
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tissues. We propose that it is deliberately accumulated at sites of tissue injury in vivo as a cytoprotective mechanism.
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This may be achieved by increasing the levels of OCTN1, the ergothioneine
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transporter.
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•
S0006-291X(15)31128-1
DOI:
10.1016/j.bbrc.2015.12.124
Reference:
YBBRC 35117
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 22 December 2015 Accepted Date: 30 December 2015
Please cite this article as: B. Halliwell, I.K. Cheah, C.L. Drum, Ergothioneine, an adaptive antioxidant for the protection of injured tissues? A hypothesis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2015.12.124. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Ergothioneine, an adaptive antioxidant for the protection of injured tissues? A hypothesis. Barry Halliwell1*, Irwin K. Cheah1, Chester L. Drum2 Department of Biochemistry, National University of Singapore, Singapore 117596
2
Department of Medicine, National University of Singapore, Singapore 119228
Tel: +65-6516 3247 Fax: +65-6775 2207
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*Corresponding author email: [email protected]
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1
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Abstract
Ergothioneine (ET) is a diet-derived, thiolated derivative of histidine with antioxidant properties, at least in vitro. Although ET is produced only by certain fungi and bacteria, it can be found at high concentrations in certain human and animal tissues and is absorbed through
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a specific, high affinity transporter (OCTN1). In liver, heart, joint and intestinal injury, elevated ET concentrations have been observed in injured tissues. The physiological role of ET remains unclear. We thus review current literature to generate a specific hypothesis: that
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the accumulation of ET in vivo is an adaptive mechanism, involving the regulated uptake and
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concentration of an exogenous natural compound to minimize oxidative damage.
Keywords
Reactive oxygen species; antioxidant; ergothioneine; adaptation; OCTN1; mushrooms
1
ACCEPTED MANUSCRIPT Introduction Ergothioneine (ET), first isolated in 1909 from the ergot fungus Claviceps purpurea [1], is the trimethylbetaine of 2-thiol-L-histidine (Fig. 1). In solution, ET exists as a tautomer
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between thiol and thione forms (Fig. 1), but at physiological pH it exists predominantly as the thione [2-4]. As far as is known, ET is synthesized only by non-yeast fungi, certain bacteria and cyanobacteria [1-9], and its biosynthetic pathways in certain bacteria have been elucidated [10-16]. Despite the limited range of organisms able to make it, ET finds its way
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into a range of foodstuffs and it is widely present in human and other animal tissues
Ergothioneine as an antioxidant?
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[3,4,8,9,17-19].
Many early papers characterized the antioxidant activity of ET in vitro. It can scavenge
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certain reactive oxygen species (ROS)1 and bind transition metal ions such as Fe2+/Fe3+ and Cu2+/Cu+ in forms unable to catalyze redox reactions [2-4,20-30]. Antioxidant and related
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effects of ET on cells in culture have also been demonstrated [4,30-36]. Although several chemicals that have powerful “antioxidant” activities in vitro do not exert such actions in vivo
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[37,38], some evidence does exist that is consistent with antioxidant effects of ET in vivo in bacteria and fungi [16,39-42]), C. elegans [43], and in animals. For example, ET was shown to protect rodents against tissue damage induced by the pro-oxidant ferric-nitrilotriacetate [44], lung injury by cytokines [45], and ischaemia-reperfusion injury in liver, heart and intestine [29,46,47]. ET has also been shown to protect mice against brain damage induced by β-amyloid [48] or D-galactose [49]. However, although tissue injury in all these animal 1
‘Reactive oxygen species’ is a collective term [2] for agents more reactive than oxygen [O2] itself and includes both oxygen radicals and certain non-radicals that are oxidizing agents and/or are easily converted into radicals (such as hypochlorous acid [HOCl], peroxynitrite [ONOO-], hydrogen peroxide [H2O2] and singlet O2 1∆g [1O2]).
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ACCEPTED MANUSCRIPT systems involves ROS, it is often the case in vivo that the protective effects of “antioxidants” are not mediated by antioxidant mechanisms [50]. Nevertheless, a few studies have used biomarkers of oxidative damage to directly demonstrate antioxidant effects of ET in animals. For example, lack of a putative ET transporter in C. elegans [43] raised the levels of protein
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carbonyls (a biomarker of oxidative protein damage [51]), and in zebrafish lack of ET increased levels of 8-oxo-7, 8-dihydroguanine [52], a biomarker of oxidative damage to
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nucleic acids [51].
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Discovery of the ET transporter
Fig. S1 shows the number of papers published mentioning ET since 1909, based on a Web of Science search on 16 November 2015. It can be seen that after the initial limited research in the early to mid 1900s, interest waned, with only a handful of papers appearing every year.
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This began to change in 2005 when Grundemann et al [53] identified a Na+-dependent transporter for ET, the organic cation transporter novel, type 1 (i.e. OCTN1) in a wide range of animal tissues, especially bone marrow and intestine but also kidney, trachea and brain
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(Fig. 2), and more recently in zebrafish [52]. The specificity of OCTN1 was demonstrated by
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the lack of uptake of other organic cations, including a much lower transport rate, relative to ET, of the structurally similar compounds, methimazole, carnitine and hercynine, and the low Km for ET (~ 21 µM) [53-56]. Silencing the gene (SLC22A4) encoding OCTN1 in cell cultures inhibits uptake of ET [30]. Similarly, metabolomic analysis of OCTN1 knockout (octn1−/−) mice revealed that tissues were almost completely deficient in ET, indicating an absence of alternative mechanisms of uptake [56,57]. The same was seen in zebrafish [52]. The presence of a specific transporter suggests that animals (including humans) may gain benefit from obtaining ET and retaining it in their tissues. It is an interesting contrast to 3
ACCEPTED MANUSCRIPT polyphenols, also powerful antioxidants in vitro, which are rapidly metabolized and excreted from the body [37,38], suggesting that they are not required by animals and thus promptly disposed of. As another example, four tocopherols and four tocotrienols from the diet can exert antioxidant effects [38,58], but only RRR-α-tocopherol is retained by the body [58]; the
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others are discarded, implying they are less useful. Like ET, ascorbate, an essential dietderived enzyme cofactor and possible in vivo antioxidant [38,59], also has selective
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transporters for intestinal uptake and delivery to tissues [59].
Hence, the data indicate that ET is useful to the body, perhaps even essential [30],
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although exactly why is still uncertain. Polymorphisms in the SLC22A4 gene encoding OCTN1 in humans that may affect the transport of ET by OCTN1 and thus might influence ET uptake by the intestine and its distribution to tissues have been described [54,61,62].
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Is ergothioneine harmful?
ET is widespread in the diet and some people, especially mushroom lovers, consume substantial amounts [8,19,63]. Toxicology studies on ET to date have revealed no adverse Nevertheless, there are
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effects (references [64-66] and Cheah et al. in preparation).
suggestions in the literature that ET might be harmful. In one study, patients with rheumatoid
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arthritis (RA) were found to have higher erythrocyte and monocyte levels of ET, which were closely correlated to expression of OCTN1 mRNA in CD14+ cells [67]. Mice with collageninduced arthritis also highly expressed OCTN1 in inflamed joints [68]. Analysis of the inflamed intestinal mucosa of patients with Crohn’s disease (CD) revealed elevated levels of ET, which correlated with increases in OCTN1 mRNA [69]. The 503F variant of OCTN1 suggested to be associated with CD was found to have a 50% greater transport efficiency compared to the normal variant (503L) [70]. Hence in these chronic inflammatory conditions,
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ACCEPTED MANUSCRIPT accumulation of ET was suggested to be an aggravating factor [68,69]. It was even suggested that consumption of mushrooms rich in ET could be a bad thing [71], although the bulk of available evidence suggests the opposite [72-75]. Some studies have indicated that the elevated levels of ET can be due to the cytokine-
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mediated increased expression of the gene encoding OCTN1. Thus, incubation of Caco-2 cells with TNF-α increased OCTN1 gene expression and tissue ET levels through activation of the TNF-α receptor-1 [69]. Similarly OCTN1 gene expression was found to be increased
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by RA-associated inflammatory cytokines such as interleukin-1 (IL-1β), and by the transcription factor RUNX1 [76]. The presence of specific SLC22A4 polymorphisms
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allegedly associated with CD or RA in certain populations, suggests the existence of ethnic differences in OCTN1 levels and resultant tissue ET levels [54,77-83]. There may also be species differences [56]. However, it would seem odd that inflamed and damaged tissues
Thinking out of the box
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would increase their ET levels to do themselves further harm.
We recently examined ET levels in a guinea pig model of diet-induced liver damage [84]. If
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guinea pigs are fed a cholesterol-rich diet, they rapidly develop liver damage [84,85]. We
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observed that, despite the fact that the diet of the animals contained little ET (~2-3 ng/mg), the damaged liver accumulated high levels of ET (Fig. 3) [86], much higher than in the healthy livers of control animals. Indeed, an early paper [9] pointed out that even very low levels of ET in the diet can lead to significant amounts in animal tissues. The rise in ET appeared to result from increased OCTN1 levels [86]. We noted that, although animal models of fatty liver disease often show increased oxidative damage [87-89], this was not the case in our model. We put forward the hypothesis, summarized in Fig. 4, that ET accumulation is done deliberately as cytoprotective event helping to protect tissues against further injury by
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ACCEPTED MANUSCRIPT oxidative damage. Thus the observed accumulation of ET in tissues in some human diseases is not pathological, but cytoprotective. Such accumulation of ET in at risk tissues could function in parallel to the known adaptive antioxidant systems, such as activation of the Nrf2 system [90-92]. We examined the literature to search for other examples of ET accumulation
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in injured tissues. A metabolomics study identified, but did not comment on, a marked increase in ET levels in both pressure overloaded and infarcted mouse hearts [93]. There is some suggestion that ET levels in blood cells are elevated in diabetes [94,95]. Pre-eclampsia,
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which can involve oxidative stress [96,97], is also associated with ET accumulation [98]. In a mouse model of intestinal inflammation, expression of the OCTN1 gene and levels of ET
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were elevated in intestinal macrophages [99]. All these observations are consistent with our hypothesis (Fig. 4). It would also be interesting to examine if other diet-derived antioxidants are accumulated similarly.
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Effects of age?
Blood ET levels decline with age, which could of course be due to several mechanisms (references [100,101], Cheah et al. in preparation). Hence the adaptive mechanism (Fig. 4),
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like some other antioxidant defence mechanisms [50], may be less efficient in the elderly.
Some caveats
The studies reviewed above highlight the importance of considering dietary ET levels and possible accelerated tissue ET uptake when undertaking studies in animal models of human disease. This dietary variable could have a significant impact on the outcomes. Our study [86], like reference [9], revealed that ET can accumulate in tissues despite very low dietary concentrations, e.g. as typically found in normal animal feeds. We also measured a variety of other normal laboratory animal feeds and observed similarly low but detectable levels of ET
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ACCEPTED MANUSCRIPT (data not shown). Few, if any, studies have monitored the presence of ET in the diets of laboratory animals, but it could be a very important variable and confounding factor. ET has been shown to accumulate in a wide range of tissues, including the brain [4]. Hence dietary levels of it may also be relevant to animal models of neurodegeneration, and indeed of many
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other diseases.
Our laboratory has previously identified numerous artefacts caused by oxidative stress in interpreting studies on cells in culture [102,103]. Cells possessing the ET transporter will
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be able to take up ET and accumulate it to exert antioxidant effects, whereas cells without the transporter cannot [30]. We therefore measured the ET content in cell culture media and
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found it to be present in several batches of foetal calf serum, at levels varying between supplier and batch (Cheah et al. in preparation). This presence of ET and the variable expression of the transporter in cells are important factors that also need to be considered
Acknowledgements
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when interpreting cell culture data, but have not been to date.
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The authors wish to thank Tetrahedron (Parc Technologique Biocitech 102 avenue Gaston
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Roussel, Romainville, F 93230, France) for providing the L-ergothioneine used in our studies. The authors are also grateful to Mr. Richard Tang for analysis of data. We thank the National Medical Research Council (grant number: NMRC/1264/2010/082/12) of Singapore for support.
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Figure
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Figure 1. Structure of thione-thiol tautomers of ET (2-mercaptohistidine trimethylbetaine). In solution at physiological pH, ET exists predominantly in the thione (a) rather the thiol (b),
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hence conferring greater stability over other thiols such as GSH.
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Figure 2. Expression of the gene encoding the ET transporter, OCTN1, in human tissues, analyzed by real-time PCR, adapted from 2 independent sources [53,69]. Data are relative to mRNA level of the ileum for each set (Grundemann et al, 2005, National Academy of Sciences and Taubert et al, 2009, BJ Journals).
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Figure 3. ET levels in guinea pig liver. Liver ET levels were measured using LC-MS/MS and found to be significantly elevated with increasing dietary cholesterol and for the moderate cholesterol diet also with duration of feeding (A). n=7; ***p<0.001, ****p<0.0001 versus respective controls for feeding duration, ###p<0.001 versus 2-month moderate cholesterol diet; one-way ANOVA (Bonferroni’s post-test). (C). There was no difference in ET
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concentrations (average of 3 measurements) between the three diets. A level of 40-50 ng/mg tissue corresponds approximately to a concentration of 0.19 mM ET. Figure taken with
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permission from [86] by courtesy of Taylor & Francis publishers.
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Figure 4. Hypothesis: ET as an adaptive antioxidant. Tissue injury by any mechanim leads to
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increased oxidative damage that can worsen the injury [50]. ET may help protect against this.
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Highlights •
Ergothioneine (ET) is a powerful antioxidant.
•
It originates from fungi and bacteria but can accumulate at high levels in human
•
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tissues. We propose that it is deliberately accumulated at sites of tissue injury in vivo as a cytoprotective mechanism.
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This may be achieved by increasing the levels of OCTN1, the ergothioneine
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transporter.
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•