Benzene and phenol metabolism by mouse and rat liver microsomes

Benzene, an important industrial solvent and constituent of unleaded gasoline, causes leukemia and aplastic anemia in humans. Mice are more sensitive than rats to benzene toxicity, though neither species has been shown to respond consistently with benzene-induced leukemia. Benzene biotransformation in liver to phenol, hydroquinone, catechol and/or muconaldehyde is thought to be necessary for its hematotoxicity and/or genotoxicity. Our goal is to develop a mathematical simulation model capable of describing the pathways and kinetics of benzene metabolism by rat and mouse liver microsomes and to assess the role of species metabolic differences in species sensitivity. Microsomes were incubated with 4 microM [U-14C]-benzene or 4 microM [U-14C]phenol. Metabolite production was quantified by extraction into ethyl acetate, HPLC separation and liquid scintillation spectroscopy. After 45 min, mouse liver microsomes converted 20% of the benzene to phenol, 31% to hydroquinone and 2% to catechol. Rat liver microsomes converted 23% of benzene to phenol, 8% to hydroquinone and 0.5% to catechol. Production of hydroquinone and catechol continued for 90 min for mouse liver microsomes, while production by rat liver microsomes had virtually ceased by 90 min. Muconic acid production by mouse liver microsomes was < 0.2% and < 0.04% from benzene and phenol respectively after 90 min. A quantitative simulation model was constructed to describe the in vitro metabolism of benzene, incorporating the reaction sequences: benzene-->phenol-->catechol-->trihydroxybenzene and phenol-->hydroquinone-->trihydroxybenzene. In the model, all of the reaction steps are assumed to be catalyzed by the same enzyme(s), cytochrome(s) P450, and benzene, phenol, hydroquinone and catechol in solution are all assumed to compete, through reversible binding, for the same reaction site(s) on cytochrome(s) P450. The simulation model accurately described both the benzene and phenol kinetic data, supporting this proposed mechanism. In particular, this model suggests that the observed inhibition of benzene on phenol metabolism, and of phenol on benzene metabolism, occurs through competition for a common reaction site, which can also bind catechol and hydroquinone.