title: Reactions of benzene oxide with thiols including glutathione.
abstract: S-Phenylmercapturic acid is a minor metabolite of benzene used as a biomarker for human benzene exposures. The reaction of intracellular glutathione with benzene oxide-oxepin, the initial metabolite of benzene, is presumed to give 1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which undergoes dehydration to S-phenylglutathione, the precursor of S-phenylmercapturic acid. To validate the proposed route to S-phenylglutathione, reactions of benzene oxide-oxepin with glutathione and other sulfur nucleophiles have been studied. The reaction of benzene oxide with an excess of aqueous sodium sulfide, followed by acetylation, gave bis-(6-trans-5-acetoxycyclohexa-1,3-dienyl)sulfide, the structure of which was proved by X-ray crystallography. Reactions of benzene oxide-oxepin in a 95:5 (v/v) mixture of phosphate buffer in D2O with (CD3)2SO were monitored by 1H NMR spectroscopy. In the absence of glutathione, the half-life of benzene oxide-oxepin was ca. 34 min at 25 degrees C and pD 7.0. The half-life was not affected in the range of 2-15 mM glutathione in the presence and absence of a commercial sample of human glutathione S-transferase (at pH 7.0, 8.0, 8.5, or 10.0). The adduct 1-(S-glutathionyl)-cyclohexa-3,5-diene-2-ol was identified in these reaction mixtures, especially at higher pH, by mass spectrometry and by its acid-catalyzed decomposition to S-phenylglutathione. Incubation of benzene oxide with N-acetyl-L-cysteine at 37 degrees C and pH 10.0 and subsequent mass spectrometric analysis of the mixture showed formation of pre-S-phenylmercapturic acid and the dehydration product, S-phenylmercapturic acid. The data validate the premise that benzene oxide-oxepin can be captured by glutathione to give (1R,2R)- and/or (1S,2S)-1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which dehydrate to S-phenylglutathione. The capture is a relatively inefficient process at pH 7 that is accelerated at higher pH. These studies account for the observation that the metabolism of benzene is dominated by the formation of phenol. The pathway leading to S-phenylmercapturic acid is necessarily minor on account of the low efficiency of benzene oxide capture by glutathione at pH 7 vs spontaneous rearrangement to phenol.
authors.affiliation: School of Natural Sciences--Chemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NEI 7RU, United Kingdom. b.t.golding@ncl.ac.uk
authors.forename: Alistair P
authors.initials: AP
authors.lastname: Henderson
authors.forename: Martine L
authors.initials: ML
authors.lastname: Barnes
authors.forename: Christine
authors.initials: C
authors.lastname: Bleasdale
authors.forename: Richard
authors.initials: R
authors.lastname: Cameron
authors.forename: William
authors.initials: W
authors.lastname: Clegg
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Heath
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Lindstrom
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Rappaport
authors.forename: Suramya
authors.initials: S
authors.lastname: Waidyanatha
authors.forename: William P
authors.initials: WP
authors.lastname: Watson
authors.forename: Bernard T
authors.initials: BT
authors.lastname: Golding
mesh_terms: D003510:Cyclohexanes / Q000138:chemical synthesis* / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D013438:Sulfhydryl Compounds / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D003510:Cyclohexanes; D013438:Sulfhydryl Compounds; D005978:Glutathione; C023372:benzene oxide