6533b7d3fe1ef96bd12614a5

RESEARCH PRODUCT

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

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Abstract Incubation of [ 14 C]benzene or [ 14 C]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 accumulation of [ 14 C]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 spectrometry (FDMS) as 2-( S -glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p -benzoquinone in the presence of [ 3 H]glutathione or, alternatively, with [ 14 C]benzene or [ 14 C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-( S -glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [ 3 H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p -benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-( S -glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH + ) ( m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities. The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p -benzosemiquinone and/or p -benzoquinone as quantitatively important reactive metabolites.