Abstract
It has been shown that when [14C]benzene is incubated with rat liver microsomes in the presence of a NADPH-generating system, metabolites which irreversibly bind to biomacromolecules are formed. This binding occurs mainly to microsomal protein rather than ribonucleic acids. The addition of 2 mM reduced glutathione or cysteine to the incubation mixture prevented 90-95% of the binding but had only a small effect on the formation of phenol, the main metabolite of benzene and rearrangement product of the putative reactive intermediate benzene oxide. The rate of phenol formation was approximately the same when fully deuterated benzene and normal benzene were used as substrates. This is compatible with phenol formation via benzene oxide and not possible if the C-H cleaving of a direct insertion reaction were the rate-limiting step. The decrease in binding in presence of reduced glutathione was accompanied by a corresponding increase in the amount of water soluble metabolites. In contrast to the situation with metabolically activated benzene, the putative reactive metabolite benzene oxide led to negligible binding and reduced glutathione had no effect on the formation of water soluble metabolites, i.e., the spontaneous conjugation of benzene oxide and reduced glutathione was practically zero at the physiological pH of the experiments. However, when [3H]-phenol was incubated with rat liver microsomes and a NADPH-generating system, effects similar to those with benzene were observed in that marked irreversible binding occurred and this binding was prevented by reduced glutathione. The extent of binding was much greater when using phenol. These results are compatible with the assumption that an immediate metabolite of phenol rather than of benzene is responsible for the binding. This assumption is supported by the fact that addition of uridine-diphosphate-glucuronic acid to incubations with [14C]benzene decreased the amount of both phenol and the irreversibly bound metabolites. Metabolism of benzene to 1,2-dihydro-1,2-dihydroxybenzene was observable, but only when pure epoxide hydratase was added in amounts greatly exceeding those already present in the microsomes. Increased formation of this diol upon addition of homogeneous epoxide hydratase is unequivocal proof for the metabolic formation of benzene oxide, which has so far eluded isolation. The observations (i) prove that reactive metabolite(s) which irreversibly bind to microsomal protein are formed during microsomal metabolism of benzene and that this binding is prevented by low molecular weight nucleophiles such as glutathione or cysteine; (ii) exclude benzene oxide as the metabolite predominantly responsible for this binding; and (iii) strongly indicate that a secondary metabolite of benzene, namely a metabolite of phenol, is mainly responsible for this binding.
- Copyright © 1978 by Academic Press, Inc.
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