Chem.-Biol. Interactions, 33 (1980) 1--17 1 © Elsevier/North-Holland Scientific Publishers Ltd. MULTI-STEP METABOLIC ACTIVATION OF BENZENE. EFFECT OF SUPEROXIDE DISMUTASE ON COVALENT BINDING TO MICROSOMAL MACROMOLECULES, AND IDENTIFICATION OF GLUTATHIONE CONJUGATES USING HIGH PRESSURE LIQUID CHROMATOGRAPHY AND FIELD DESORPTION MASS SPECTROMETRY A. TUNEKa, K.L. PLATTc, M. PRZYBYLSKI b and F. OESCHc.* alnstitute of Environmental Health, University of Lund, SSlvegatan 21, S-223 62 Lund (Sweden) blnstitut fiir Organische Chemie der Universit~t, Johann-Joachim-Becher- Weg 18-20, D-6500 Mainz and cSection on Biochemical Pharmacology, Institute of Pharmacology, Obere-Zahlbacher-Strasse 67, D-6500 Mainz (F.R.G.) (Received July 17th, 1980) (Accepted August 15th, 1980) SUMMARY Incubation of [14C]benzene or [14C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accu~nulation of [~4C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectro- metry (FDMS) as 2-(S-glutathionyl)hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydro- quinone or p-benzoquinone in the presence of [3H]glutathione or, alterna- tively, with [14C]benzene or [~4C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of *To whom correspondence should be sent. Abbreviations: GSH, reduced glutathione; HPLC, high performance liquid chromato- graphy; FDMS, field desorption mass spectrometry; TLC, thin-layer chromatography; BAT, best anode temperature (temperature of the field ion emitter at which the highest intensity of the molecular ion is observed).