Identification of the reactive glutathione conjugate S-(2-chloroethyl)glutathione in the bile of 1-bromo-2-chloroethane-treated rats by high-pressure liquid chromatography and precolumn derivatization with o-phthalaldehyde.
The conjugation of glutathione with 1,2-dihaloethanes leads to the formation of S-(2-haloethyl)glutathione which, following intramolecular cyclization, produces an electrophilic thiiranium ion. The extent to which the formation of the thiiranium ion is responsible for the toxicity associated with 1,2-dihaloethanes has been difficult to determine because of the inherent instability of the compound under physiological conditions. The goal of this study was to attempt to identify a putative precursor of the thiiranium ion, S-(2-chloroethyl)glutathione (CEG), in the bile of rats treated with 1,2-dihaloethanes such as 1-bromo-2-chloroethane (BCE). In order to detect the presence of CEG, a precolumn procedure for derivatizing the amine of CEG with o-phthalaldehyde/2-mercaptoethanol (OPA/MCE) was developed. Studies with a model compound, S-ethylglutathione, indicated that the derivatization reaction between S-ethylglutathione and OPA/MCE proceeded rapidly and under mild conditions. The resulting fluorescent adduct of S-ethylglutathione was detected at low concentrations following separation by reverse-phase HPLC. Derivatization of CEG with OPA/MCE followed by preparative HPLC and mass spectral analysis revealed that the major fluorescent adduct in the reaction mixture was the expected 1-[(2-hydroxyethyl)thio]-2-substituted-isoindole derivative of CEG. Also present in the derivatization reaction mixture were small quantities of S-(2-hydroxyethyl)glutathione, the product of CEG hydrolysis, and a product involving the addition of MCE to CEG. Analysis of the bile samples obtained from bile-cannulated rats treated with BCE showed the presence of a peak corresponding to CEG. Over a 3-h interval, 2% of the BCE administered was excreted into the bile as CEG.
Marchand
D H
DH
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-6504.
Reed
D J
DJ
D000818:Animals; D001646:Bile / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D006842:Hydrocarbons, Brominated / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008623:Mercaptoethanol / Q000737:chemistry; D051379:Mice; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D009764:o-Phthalaldehyde
D006842:Hydrocarbons, Brominated; D008623:Mercaptoethanol; D009764:o-Phthalaldehyde; D005978:Glutathione; C037311:bromoethane; C046570:S-(2-chloroethyl)glutathione