Chemical Demethylation of Methylmercury by Selenoamino Acids Mohammad A. K. Khan † and Feiyue Wang* ,†,‡ Department of Chemistry, and Department of EnVironment and Geography, UniVersity of Manitoba, Winnipeg, MB R3T 2N2, Canada ReceiVed March 2, 2010 A new chemical demethylation pathway for methylmercury under physiologically and environmentally relevant conditions is reported. The pathway involves the reaction between methylmercury and a selenoamino acid (L-selenocysteine, L-selenoglutathione, D,L-selenopenicillamine, or L-selenomethionine) via the formation of bis(methylmercuric)selenide and dimethylmercury as intermediates. The final degradation product is HgSe(s). Introduction Methylmercury (CH 3 Hg + and its complexes; MeHg hereafter) is a known developmental neurotoxin (1), capable of crossing the placental and blood-brain barriers (2, 3) causing neurologi- cal damage in humans and death in extreme cases (4, 5). Produced mainly in the aquatic environment by microbial- mediated methylation of inorganic Hg (6), MeHg biomagnifies along food chains, resulting in elevated concentrations in high trophic level animals such as predatory fish and marine mammals and in humans via dietary consumption of these animals (1, 5). Much less is, however, known about the demethylation process once MeHg is formed (7). Because of the kinetic stability of the C-Hg bond (8), MeHg demethylation in the aquatic environment is thought to occur primarily via photolysis or microbial processes (7). The only established mechanism for chemical demethylation of MeHg in nature is the reaction between MeHg and H 2 S via a bis(methylmercu- ric)sulfide ((CH 3 Hg) 2 S) intermediate, ultimately forming HgS(s) (9, 10). MeHg demethylation, however, is known to occur in vivo in animals, particularly in the liver of marine mammals, and Se has long been postulated to be involved in this demethylation process (11). Indirect evidence has suggested that the Se-aided demethylation may have involved the formation of bis(meth- ylmercuric)selenide ((CH 3 Hg) 2 Se) (12, 13), similar to (CH 3 Hg) 2 S in the H 2 S case, but the presence of (CH 3 Hg) 2 Se has never been analytically proven due to its in vivo instability (13), and the pathway leading to the formation of (CH 3 Hg) 2 Se remains unknown. When synthesizing MeHg complexes with several sele- noamino acids (14), we noticed the formation of a black precipitate from their aqueous solutions after various storage times. NMR, X-ray diffraction (XRD), and mass spectrometry studies were subsequently carried out to characterize the reactions, which revealed a new chemical demethylation pathway of MeHg in the presence of selenoamino acids. Four selenoamino acids were studied, including L-selenocysteine (CSeH), L-selenoglutathione (GSeH), D,L-selenopenicillamine (SePen), and L-selenomethionine (SeMet). All the four sele- noamino acids were shown to be capable of demethylating MeHg under physiologically and environmentally relevant conditions. Materials and Methods Reagents and Apparatus. Methylmercury hydroxide, D,L- penicillamine, and Na 2 SeO 3 were purchased from Alfa Aesar, methylmercury chloride, L-selenocystine, L-glutathione (reduced form), and ethanol (anhydrous, 99.8%) from Sigma-Aldrich, L-selenomethionine from CalBioChem, and benzene from Fluka. All of the chemicals were of ACS grade or greater and were used as received. L-Selenocysteine was prepared from L-selenocystine following the procedure of Carty et al. (15). L-Selenoglutathione and D,L-selenopenicillamine were prepared from L-glutathione and D,L-penicillamine, respectively, following the procedure of Khan et al. (14). Ultrapure deionized water (Milli-Q Element; Millipore) was used as the laboratory water in all of the experiments. All reactions were carried out under an inert atmosphere of Ar. Real-Time NMR Spectra. Real-time 1 H and 199 Hg NMR spectra were collected at 37 °C. For the MeHg-SeMet study, a 0.1 M L-selenomethionine solution was added to 0.1 M CH 3 HgOH solution at pH 9.0 in a standard NMR tube. 1 H NMR spectra were then recorded in D 2 O on a Bruker AMX 500 MHz NMR spectrometer equipped with a 5-mm broadband probe at various time intervals after mixing. Chemical shifts δ were reported in ppm relative to TMS and coupling constants J in Hz. 1 H NMR spectra were referenced to the residue of D 2 O at δ ) 4.75 ppm. 199 Hg NMR spectra were recorded in D 2 O on a Bruker AMX 600 MHz NMR spectrometer equipped with a 5 mm broadband probe, with referencing 107.4043151 Hz as 0 ppm frequency for 199 Hg. Real- time 1 H and 199 Hg NMR spectra of CH 3 HgOH in the presence of CSeH, GSeH, or SePen were studied following a similar procedure except that L-selenomethionine was replaced with L-selenocysteine, L-selenoglutathione, and D,L-selenopenicillamine, respectively, and that the pH was adjusted to 10.5 when working with D,L- selenopenicillamine. X-ray Powder Diffraction. Black precipitate from the above tubes was collected by filtration through a 0.45 µm hydrophilic polypropylene membrane filter (PALL Life Sciences), washed with copious amounts of milli-Q water, and dried at room temperature for 3 days. Powder XRD patterns of the solid were obtained using a Philips PW3830/40 X-ray generator with PW1710 XRD system and a Rigaku diffractometer with Cu/K R1 radiation source (k ) 1.54059 Å). MDI Datascan/Jade (v. 7.5) data collection and processing software were used. PDF 4.0 database was cross checked to compare and identify the XRD pattern and compound, respectively. Synthesis of Bis(methylmercuric)selenide. (CH 3 Hg) 2 Se was synthesized following the method reported by Naganuma and Imura * Corresponding author. Tel: 1-204-474-6250. Fax: 1-204-474-7608. E-mail: wangf@ms.umanitoba.ca. † Department of Chemistry. ‡ Department of Environment and Geography. Chem. Res. Toxicol. 2010, 23, 1202–1206 1202 10.1021/tx100080s  2010 American Chemical Society Published on Web 05/25/2010