Uptake Of Ergothioneine Research Articles

Does Intracellular Metabolism Render Gemcitabine Uptake Undetectable in Mass Spectrometry?

The ergothioneine transporter ETT (formerly OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). Recently, Sparreboom et al. reported that the ETT would transport nucleosides and nucleoside analogues such as cytarabine and gemcitabine with the highest efficiency. In our assay system, we could not detect any such transport. Subsequently, Sparreboom suggested that the intracellular metabolization of the nucleosides occurs so fast that the original compounds cannot be detected by LC–MS/MS after inward transport. Our current experiments with 293 cells disprove this hypothesis. Uptake of gemcitabine was easily detected by LC–MS/MS measurements when we expressed the Na+/nucleoside cotransporter CNT3 (SLC28A3). Inward transport was 1280 times faster than the intracellular production of gemcitabine triphosphate. The deoxycytidine kinase inhibitor 2-thio-2′-deoxycytidine markedly blocked the production of gemcitabine triphosphate. There was no concomitant surge in intracellular gemcitabine, however. This does not fit the rapid phosphorylation of gemcitabine. Uptake of cytarabine was very slow, but detection by MS was still possible. When the ETT was expressed and incubated with gemcitabine, there was no increase in intracellular gemcitabine triphosphate. We conclude that the ETT does not transport nucleosides.

Ergothioneine in the brain.

Ergothioneine (ERGO) is a naturally occurring food-derived antioxidant. Despite its extremely hydrophilic properties, ERGO is easily absorbed from the gastrointestinal tract and distributed to various organs, including the brain. This is primarily because its entry into brain cells is mediated by the ERGO-specific transporter OCTN1/SLC22A4. Octn1 gene knockout mice do not have ERGO in the brain, due to the absence of OCTN1 in neurons, neural stem cells, and microglia. The existence of OCTN1 and uptake of ERGO into the brain parenchymal cells may suggest that ERGO and its transporter play a pivotal role in brain function. Oral administration of ERGO has antidepressant activities in mice. Furthermore, repeated oral administration of ERGO and ERGO-containing food extract tablets enhance memory function in mice and humans, respectively. ERGO also protects against stress-induced sleep disturbance and neuronal injury induced by amyloid β in rodents. In vitro observations suggest that ERGO benefits brain function through both its antioxidative activity and by promoting neurogenesis and neuronal maturation. This review discusses the possible involvement of ERGO in brain function and its potential therapeutic properties.

The ergothioneine transporter (ETT): substrates and locations, an inventory.

In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell-surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may also transport ET. We conclude that, to date, ETT remains the only well-defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter.

Rat Kidney Slices for Evaluation of Apical Membrane Transporters in Proximal Tubular Cells

Kidney slice has been often used as a tool reflecting basolateral transport in renal tubular epithelial cells. Recently, we reported that several important apical reabsorptive transporters such as Octn1/2, Sglt1/2, and Pept1/2 were functional in mouse kidney slices as well as transporter activities in basolateral side, which have been well accepted. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. The present study investigates usefulness of kidney slices from rats for evaluation of apical membrane transporters for efflux (multidrug resistance 1a, mdr1a) as well as influx (Octn1/2, Sglt1/2, Pept1/2). Na+-dependent uptake of ergothioneine (Octn1), carnitine (Octn2), and methyl-α-D-glucopyranoside (Sglt1/2) by rat kidney slices was observed, and the uptake was decreased by selective inhibitors. In addition, uptake of glycyl-sarcosine (Pept1/2) showed H+-dependence and was decreased by selective inhibitor. Furthermore, accumulation of mdr1a substrate azasetron was increased in the presence of zosuquidar, an mdr1a inhibitor, while strain differences existed. In conclusion, rat kidney slices should be useful for evaluation of renal drug disposition regulated by transporters in apical as well as basolateral membranes of rat renal proximal tubule cells.

Usefulness of kidney slices for functional analysis of apical reabsorptive transporters

Kidney plays a key role in the elimination and reabsorption of drugs and nutrients, however in vitro methods to evaluate renal disposition are limited. In the present study, we investigated usefulness of isolated kidney slice, which had been used for transport only at basolateral membrane of tubular epithelial cells, for evaluation of apical membrane transporters. As transporters that are easy to discriminate between apical and basolateral transports, apical membrane specific and sodium-dependent transporters (SGLTs and OCTNs) and pH-dependent transporters (PEPTs) are selected. Uptake of ergothioneine, carnitine and methyl-α-D-glucopyranoside, which are substrates of apical Octn1, Octn2, and Sglt1/2, respectively, by mice kidney slices showed clear Na+ dependence and reduction by selective inhibitors. In addition, sodium dependence of ergothioneine uptake was negligible in the kidney slice from Octn1-gene deficient mice. Moreover, uptake of PepT1/2 substrate glycyl-sarcosine, was higher than that in the presence of glycyl-leucine, a non-specific Pept inhibitor. The Km and IC50 values for substrates and inhibitors of each transporter were mostly comparable to those obtained in transporter-transfected cells. In conclusion, it was demonstrated that kidney slices are promising tool to study transporters expressed at the apical membranes as well as basolateral membranes of kidney tubular epithelial cells.

Physiological Roles of Carnitine/Organic Cation Transporter OCTN1/SLC22A4 in Neural Cells.

Dysfunction in neurotransmission mediated by neurotransmitters causes various neurological disorders. Therefore, receptors and reuptake transporters of neurotransmitters have been focused on as a therapeutic target in neurological disorders. These membrane proteins have high affinity for a specific neurotransmitter and are highly expressed on synaptic membranes. In contrast, xenobiotic transporters have relatively lower affinity for neurotransmitters but widely recognize various organic cations and/or anions and are also expressed in brain neurons. However, it has been largely unknown why such xenobiotic transporters are expressed in neurons that play a key role in signal transduction. We have therefore attempted to clarify the physiological roles of one such xenobiotic organic cation transporter (OCT) in neural cells with the aim of obtaining new insight into the treatment of neurological disorders. Carnitine/organic cation transporter OCTN1/SLC22A4 is functionally expressed in neurons and neural stem cells. In particular, OCTN1 is expressed at much higher levels compared with other OCTs in neural stem cells and positively regulates their differentiation into neurons. OCTN1 accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a good in vivo substrate. Because ERGO is highly distributed into the brain after oral ingestion, OCTN1 may contribute to the alleviation of oxidative stress and promotion of neuronal differentiation via the uptake of ERGO in the brain, perhaps abating symptoms of neurological disorders. In this review, we introduce current topics on the physiological roles of OCTs with a focus on OCTN1 in neural cells and discuss its possible application to the treatment of neurological disorders.

Bioavailability and antioxidant effect of ergothioneine in human blood

Edible mushrooms, such as the white button mushrooms contain several biological compounds including ergothioneine (ET). The main function of ET seems to be to protect cells from oxidative stress. Although humans cannot synthesize ET, the human body does have its own ergothioneine transporter (ETT), which allows cells, for instance red blood cells (RBCs), to take up and store ET. The existence of this transporter suggests that ET might have a physiological importance in humans. The aims of this study were to examine the availability of ET in RBCs and the protective effect of ET on oxidative stress in RBCs. The uptake of ET in RBCs was studied in vitro by treating RBCs with known concentrations of ET (0-100 µM). The amount of ET in the RBCs was determined using high performance liquid chromatography (HPLC). The protective effect of ET was studied by measuring the haemolysis in oxidative stress induced RBCs, which were pre-treated with ET in concentrations ranging from 0-100 µM. In addition, a pilot study was performed where 10,6 mg ET was administered to subjects (n = 6) through a mushroom soup. Before and three hours after consumption, blood samples were collected. The blood samples were analyzed to determine the concentration ET in the RBCs using HPLC and the protective effect of ET was studied the same way as for in vitro. Main findings were that ET was taken up in RBCs after treatment with all ET concentrations, but only after 30 minutes and two hours. Furthermore, RBCs pre-treated with ET had less haemolysis than nontreated RBCs; pre-treatment with 100 µM ET was observed to significantly (p<0,016) decrease haemolysis in comparison with the non-treated RBCs. Consumption of the mushroom soup did not result in increased ET levels in the RBCs, although three out of five subjects had less haemolysis compared to consumption of the placebo soup. However, the beneficial effects after consumption of the mushroom soup cannot be assigned to ET for sure; other unobserved compounds could be involved

OCTN1‐Mediated Ergothioneine Uptake Protects Lung Epithelial Cells From Tobacco Smoke‐Induced Damage

Smoking is a major contributing factor for chronic obstructive pulmonary disease (COPD). Tobacco smoke exposure causes oxidative stress which results in alveolar wall destruction, mucus hyper secretion, inflammation and defective tissue repair. Ergothioneine (ET), an amino acid transported by OCTN1, has been shown to exhibit antioxidant and cytoprotective capacities in vitro. Hence, the objective of this study is to understand the role of OCTN1 and the effect of ET on human alveolar cells when subjected to tobacco smoke‐induced oxidative stress.Confocal microscopy and Western blot was used to determine the presence of OCTN1 in A549 cells and freshly isolated human alveolar epithelial type II (ATII) and type I‐like (ATI‐like) cells in primary culture. A549 cells were cultured in medium containing 1 mM of ET and its intracellular accumulation was measured by LC‐MS/MS over a 3‐week period. A549 cells were also was exposed to various concentrations of cigarette smoke extract (CSE). Prior to the experiment, cells were either cultured for 1 week in the presence or absence of 1 mM ET. An MTT assay was used to assess cell viability for the difference exposures to CSE.Immunoblot and CLSM showed OCTN1 expression in A549, ATII and ATI‐like cells. The uptake of ET by A549 epithelial cells was saturated after approximately 7 days of culture. Western blot data supported the CLSM results and also indicated that OCTN1 levels were increased in the presence of ET. The MTT assay showed that cells pre‐treated with ET were less affected by the cytotoxic effects of CSE.These preliminary data indicate that OCTN1 might play a role in the pathogenesis of tobacco smoke‐induced COPD by regulating ergothioneine transport.

Knockout of the ergothioneine transporter ETT in zebrafish results in increased 8-oxoguanine levels

Ergothioneine (ET) is a natural compound that humans and other vertebrates must absorb from dietary sources. In general, ET is considered an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. The ergothioneine transporter ETT (human gene symbol SLC22A4) is a highly specific transporter for the uptake of ET. Here, we sought to identify and knock out ETT from zebrafish (Danio rerio) to determine the function of ET. We cloned and assayed three related proteins from zebrafish, only one of which catalyzed the uptake of ET. RT-PCR analysis revealed that the protein is strongly expressed in the skin, brain, kidney, intestine, and eye. In ETT-knockout animals generated by retroviral insertion into exon 1, ET content was reduced by more than 1000-fold compared to the wild type. Thus, ETT is the sole transporter responsible for uptake of ET into zebrafish. ETT-knockout fish did not exhibit obvious differences in morphology or behavior. In whole-fish homogenates, an increase in 4-hydroxy-2,3-trans-nonenal and malondialdehyde was observed, but only after stress caused by incubation with Pb2+ or Cu2+. Comparison of unstressed fish at the level of small molecules by LC–MS difference shading revealed a 3.8-fold increase in 8-oxoguanine (8-oxo-7,8-dihydroguanine) in the skin of ETT-knockout animals. Our knockout represents a new model for examining the consequences of complete absence of ET. Based on the phenotype observed here, we hypothesize that the specific purpose of ET could be to eliminate singlet oxygen.

Organic cation transporter Octn1-mediated uptake of food-derived antioxidant ergothioneine into infiltrating macrophages during intestinal inflammation in mice

OCTN1/SLC22A4 is expressed on apical membranes of small intestine, and is involved in gastrointestinal absorption of its substrates, including the food-derived antioxidant ergothioneine (ERGO). ERGO concentration in circulating blood of patients with inflammatory bowel disease (Crohn's disease) is lower than that in healthy volunteers; thus, circulating ERGO is a potential diagnostic marker, although the mechanisms underlying low ERGO concentration in patients are unknown. Here, we focused on intestinal macrophages, which infiltrate sites of inflammation, and examined possible first-pass uptake of ERGO by macrophages. ERGO concentration in blood was lower in mice with dextran sodium sulfate (DSS)-induced colitis than in controls. On the other hand, expression of octn1 gene product and ERGO concentration in intestinal tissues of DSS-treated mice were higher than in controls. Interestingly, lamina propria mononuclear cells (LPMCs) isolated from DSS-treated mice contained ERGO and showed [3H]ERGO uptake and Octn1 expression, whereas ERGO was undetectable in LPMCs of control mice. Functional expression of OCTN1 was also confirmed in LPS-stimulated human macrophage-like cell line, THP-1. In conclusion, OCTN1 is functionally expressed on activated intestinal macrophages, and ERGO uptake into these immune cells could contribute at least in part to the altered disposition of ERGO in intestinal inflammation.

Organic Cation Transporter-Mediated Ergothioneine Uptake in Mouse Neural Progenitor Cells Suppresses Proliferation and Promotes Differentiation into Neurons

The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [3H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [3H]ERGO uptake. On the other hand, exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin, but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP), with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly, edaravone and ascorbic acid did not affect such differentiation of NPCs, in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP, but decreased the number immunoreactive for βIII-tubulin, with concomitant down-regulation of Math1 in P19-NPCs. Thus, OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress, and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action.

Oxaliplatin Transport Mediated by Organic Cation/Carnitine Transporters OCTN1 and OCTN2 in Overexpressing Human Embryonic Kidney 293 Cells and Rat Dorsal Root Ganglion Neurons

The organic cation/carnitine transporters OCTN1 and OCTN2 are related to other organic cation transporters (OCT1, OCT2, and OCT3) known for transporting oxaliplatin, an anticancer drug with dose-limiting neurotoxicity. In this study, we sought to determine whether OCTN1 and OCTN2 also transported oxaliplatin and to characterize their functional expression and contributions to its neuronal accumulation and neurotoxicity in dorsal root ganglion (DRG) neurons relative to those of OCTs. [(14)C]Oxaliplatin uptake, platinum accumulation, and cytotoxicity were determined in OCTN-overexpressing human embryonic kidney (HEK) 293 cells and primary cultures of rat DRG neurons. Levels of mRNA and functional activities of rat (r)Octns and rOcts in rat DRG tissue and primary cultures were characterized using reverse transcription-polymerase chain reaction and uptake of model OCT/OCTN substrates, including [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) (OCT1-3), [(14)C]tetraethylammonium bromide (TEA(+)) (OCT1-3 and OCTN1/2), [(3)H]ergothioneine (OCTN1), and [(3)H]l-carnitine (OCTN2). HEK293 cells overexpressing rOctn1, rOctn2, human OCTN1, and human OCTN2 showed increased uptake and cytotoxicity of oxaliplatin compared with mock-transfected HEK293 controls; in addition, both uptake and cytotoxicity were inhibited by ergothioneine and L-carnitine. The uptake of ergothioneine mediated by OCTN1 and of L-carnitine mediated by OCTN2 was decreased during oxaliplatin exposure. rOctn1 and rOctn2 mRNA was readily detected in rat DRG tissue, and they were functionally active in cultured rat DRG neurons, more so than rOct1, rOct2, or rOct3. DRG neuronal accumulation of [(14)C]oxaliplatin and platinum during oxaliplatin exposure depended on time, concentration, temperature, and sodium and was inhibited by ergothioneine and to a lesser extent by L-carnitine but not by MPP(+). Loss of DRG neuronal viability during oxaliplatin exposure was inhibited by ergothioneine but not by L-carnitine or MPP(+). OCTN1 and OCTN2 both transport oxaliplatin and are functionally expressed by DRG neurons. OCTN1-mediated transport of oxaliplatin appears to contribute to its neuronal accumulation and treatment-limiting neurotoxicity more so than OCTN2 or OCTs.

Abstract 4391: OCTN1- and OCTN2-mediated uptake of oxaliplatin in rat dorsal root ganglion neurons and HEK293 cells over-expressing OCTN1 or OCTN2

Abstract Purpose: Organic cation transporters (OCTs) have been recently implicated in the uptake and toxicity of oxaliplatin. Peripheral neuropathy is the dose-limiting toxicity of oxaliplatin due to accumulation of platinum in the dorsal root ganglia (DRG) and damage to sensory neurons. This study sought to determine the role of OCTs in the DRG neuronal uptake of oxaliplatin. Experimental Design: [14C] Oxaliplatin uptake and cytotoxicity in HEK/rOctn1, HEK/rOctn2, HEK/hOCTN1, HEK/hOCTN2, and primary culture of DRG neurons was determined to evaluate the role of Octn1 and Octn2 in oxaliplatin transport and neurotoxicity. mRNA, protein and functional expression of organic cation transporters in rat lumbar DRG were characterised using RT-PCR, Western blotting and uptake of model OCT substrates [3H] MPP, [14C] TEA, [3H] ergothioneine and [3H] L-carnitine. Results: HEK293 cells over-expressing rOctn1, rOctn2, hOCTN1 and hOCTN2 showed increased uptake and cytotoxicity of oxaliplatin compared to Mock-transfected HEK293 controls, and Octn2-mediated uptake of oxaliplatin was significantly higher than Octn1 in both human and rat. Uptake of ergothioneine mediated by Octn1, and L-carnitine mediated by Octn2, was decreased during oxaliplatin exposure. Rat DRG showed high Octn1 and Octn2 mRNA levels, and uptake of ergothioneine and L-carnitine that was temperature- and sodium-dependent and significantly inhibited by model substrates of Octn1 and Octn2. In contrast, DRG had negligible functional activity and low or undetectable mRNA levels of Oct1, Oct2, and Oct3. [14C] Oxaliplatin uptake by DRG neurons was temperature- and sodium-dependent and inhibited by ergothioneine and L-carnitine. Ergothioneine and L-carnitine uptake by DRG neurons was inhibited in the presence of oxaliplatin. Conclusions: In conclusion, Octn1 and Octn2, but not Oct1, Oct2 or Oct3, are functionally expressed in rat DRG neurons. Oxaliplatin uptake by rat DRG neurons and HEK293 cells was mediated by Octn2 and to lesser extent by Octn1. Supported by Grant-in-Aid for Scientific Research (B) (I. T.), Research Activity Start-up (T. N.), Auckland Medical Research Foundation (Doctoral Scholarship) and Cancer Society of New Zealand (Research Grant). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2011-4391

Probing the substrate specificity of the ergothioneine transporter with methimazole, hercynine, and organic cations

Recently, we have identified the ergothioneine (ET) transporter ETT (gene symbol SLC22A4). Much interest in human ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic inflammatory diseases. ETT was originally designated a multispecific novel organic cation transporter (OCTN1). Here we reinvestigated, based on stably transfected 293 cells and with ET as reference substrate, uptake of quinidine, verapamil, and pyrilamine. ETT from human robustly catalyzed transport of ET (68 μl/(min mg protein)), but no transport of organic cations was discernible. With ET as substrate, ETT was relatively resistant to inhibition by selected drugs; the most potent inhibitor was verapamil ( K i = 11 μmol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, efficiency of ETT-mediated transport of methimazole ( K i = 7.5 mmol/l) was 130-fold lower, and transport of hercynine ( K i = 1.4 mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high affinity, sodium-driven uptake of ET very similar to ETT from human. Additional real-time PCR experiments based on 16 human tissues revealed ETT mRNA levels considerably lower than in bone marrow. Our experiments establish that ETT is highly specific for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high affinity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific molecular marker of intracellular ET activity.

Decreased Proliferation and Erythroid Differentiation of K562 Cells by siRNA-induced Depression of OCTN1 (SLC22A4) Transporter Gene

Recently, it was reported that OCTN1 transporter (SLC22A4) is associated with rheumatoid arthritis (RA) and Crohn's disease. Additionally, we reported that OCTN1 is expressed in hematopoietic cells, preferentially in erythroid cells. Accordingly, we assessed the physiological role of OCTN1 by examining the effect of knockdown of OCTN1 in blood cells using siRNA method. Vector-based short hairpin RNA (shRNA) was used to establish K562 cell line which shows stably decreased expression of OCTN1. The characteristic of knockdown of OCTN1 in K562 cells was investigated by cell proliferation, cell differentiation, and uptake of ergothioneine that is a good substrate of OCTN1. Several clones of K562 cells exhibited significantly reduced expression of OCTN1 mRNA and protein. They also showed a decreased growth rate and butyrate-dependent differentiation to erythrocytes compared with control-vector transfected cells. In addition, uptake of [(3)H]ergothioneine by K562 cells suggested that Na(+)-dependent and high-affinity transporter which is similar to the characteristics of OCTN1 is functional. Moreover, uptake of ergothioneine by K562 cells which exhibit decreased-expression of OCTN1 was decreased in comparison with wild type K562 cells. It was suggested that OCTN1 is involved in the transport of physiological compounds that are important for cell proliferation and erythroid differentiation.

The uptake of ergothioneine from the soil into the latex of Hevea brasiliensis

The uptake of ergothioneine from the soil into the latex of Hevea brasiliensis