Aromatic Substitution Reaction of Ergothioneine - a Novel Mechanism of Xenobiotic Deactivation

The xenobiotic amino acid ergothioneine (ET) has been described to have antioxidant properties, similar to glutathione (GSH). However, many of ET's physiological functions remain unclear to date. GSH is involved in xenobiotic deactivation via several mechanisms, e.g. nucleophilic substitution‐like reactions, which are catalysed by enzymes of the glutathione sulphur transferase (GST) family. 1‐Chloro‐2,4‐dinitrobenzene (CDNB) is a prototypical GST substrate. The molecule is conjugated to a single molecule of reduced glutathione. Once covalently bound, 2,4‐dinitrophenyl‐S‐glutathione is effluxed by MRP transporters, to fully detoxify the cell. The aim of this research was to determine whether ET can also undergo nucleophilic substitution‐type reactions with CDNB to effectively counteract toxins in vitro. Ergothioneine was reacted with CDNB at 50°C for 24 hours in methanol. The formation of ET‐CDNB product was monitored by UV spectroscopy at 405 nm. The formed product was purified by solid phase extraction and its structure confirmed by proton and carbon NMR as well as infrared spectroscopy. Quantification was achieved by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). From the product ion spectra, the following molecules were chosen for selected reaction monitoring (molecule of interest, m/z parent, m/z fragment and the magnitude of voltage): ET, 395, 352 and 16 V. Moreover, product formation was carried out in NCI‐H441 human distal lung epithelium cells. Cytotoxicity of the product was investigated by Alamar blue assay, in the presence or absence of the MRP inhibitor, MK‐571 (50 μm, 1 hour). An orange solid with melting point of 178–181°C was successfully produced and its structure confirmed by NMR and infrared spectroscopy. LC‐MS/MS could successfully detect formation of the product in vitro and in cell culture. Upon co‐incubation with ET, the toxicity of CDNB in NCI‐H441 cells decreased, illustrating the cytoprotective potential of ET. In addition, a decrease in cell viability was observed after MRP inhibition, suggesting a role of MRP transporters in CDNB and ET‐CDNB extrusion. These data confirm, for the first time, that ET is able to undergo nucleophilic substitution reactions which could point at a new and important role for the compound in xenobiotic deactivation. ET‐conjugates appear to be effluxed from the cell by a transporter belonging to the MRP family. Further studies are required to elucidate the enzyme(s), involved in the formation of ET‐CDNB and also to investigate the molecular identity of the ET efflux transporter. Moreover, it needs to be determined which other GSH‐like reactions ET can perform. Support or Funding Information This work has been financially supported by a Clinical Investigator Awards from the Flight Attendant Medical Research Institute (FAMRI CIA 130016). M.A.S. is the recipient of a PhD bursary from the Iraqi Ministry of Higher Education and Scientific Research (MOHESR) and C.G.C. is supported by a Trinity College Research Studentship Award. The authors would like to acknowledge the contribution of the COST Actions BM1201 and MP1404.