S-Phenyl
mercapturic acid is a
minor
metabolite of benzene used as a bio
marker for hu
manbenzene exposures. The reaction of intracellular glutathione with benzene oxide-oxepin, theinitial
metabolite of benzene, is presu
med to give 1-(
S-glutathionyl)-cyclohexa-3,5-dien-2-ol,which undergoes dehydration to
S-phenylglutathione, the precursor of
S-phenyl
mercapturicacid. To validate the proposed route to
S-phenylglutathione, reactions of benzene oxide-oxepinwith glutathione and other sulfur nucleophiles have been studied. The reaction of benzeneoxide with an excess of aqueous sodiu
m 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 D
2O with(CD
3)
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
mages/entities/deg.gif">C and pD 7.0. The half-life was not affectedin the range of 2-15
mM glutathione in the presence and absence of a co
mmercial sa
mple ofhu
man 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 spectro
metry and by its acid-catalyzed deco
mposition to
S-phenylglutathione. Incubationof benzene oxide with
N-acetyl-
L-cysteine at 37
mages/entities/deg.gif">C and pH 10.0 and subsequent
massspectro
metric analysis of the
mixture showed for
mation of pre
-S-phenyl
mercapturic acid andthe dehydration product,
S-phenyl
mercapturic acid. The data validate the pre
mise that benzeneoxide-oxepin can be captured by glutathione to give (1
R,2
R)- and/or (1
S,2
S)-1-(
S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which dehydrate to
S-phenylglutathione. The capture is a relativelyinefficient process at pH 7 that is accelerated at higher pH. These studies account for theobservation that the
metabolis
m of benzene is do
minated by the for
mation of phenol. Thepathway leading to
S-phenyl
mercapturic acid is necessarily
minor on account of the lowefficiency of benzene oxide capture by glutathione at pH 7 vs spontaneous rearrange
ment tophenol.