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Benzene is a ubiquitous environmental pollutant that is known to cause hematotoxicity and leukemia in humans. The initial oxidative metabolite of benzene has long been suspected to be benzene oxide (3,5-cyclohexadiene-1,2-oxide). During in vitro experiments designed to characterize the oxidative metabolism of [14C]benzene, a metabolite was detected by HPLC-radioactivity analysis that did not elute with other known oxidative metabolites. The purpose of our investigation was to prove the hypothesis that this metabolite was benzene oxide. Benzene (1 mM) was incubated with liver microsomes from human donors, male B6C3F1 mice, or male Fischer-344 rats, NADH (1 mM), and NADPH (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) and then extracted with methylene chloride. Gas chromatography-mass spectrometry analysis of incubation extracts for mice, rats, and humans detected a metabolite whose elution time and mass spectrum matched that of synthetic benzene oxide. The elution time of the benzene oxide peak was approximately 4.1 min, while phenol eluted at approximately 8 min. Benzene oxide also coeluted with the HPLC peak of the previously unidentified metabolite. Based on the 14C activity of this peak, the concentration of benzene oxide was determined to be approximately 18 microM, or 7% of total benzene metabolites, after 18 min of incubation of mouse microsomes with 1 mM benzene. The metabolite was not observed in incubations using heat-inactivated microsomes. This is the first demonstration that benzene oxide is a product of hepatic benzene metabolism in vitro. The level of benzene oxide detected suggests that benzene oxide is sufficiently stable to reach significant levels in the blood of mice, rats, and humans and may be translocated to the bone marrow. Therefore benzene oxide should not be excluded as a possible metabolite involved in benzene-induced leukemogenesis. [ABSTRACT FROM PUBLISHER]

1,3-Butadiene (BD) is a more potent tumor inducer in mice than in rats. BD also shows striking differences in metabolic activation, with substantially higher blood concentrations of 1,2:3,4-diepoxybutane (butadiene diepoxide; BDE) in BD-exposed mice than in similarly exposed rats. The objective of this study was to develop a single mechanistic model structure capable of describing BD disposition in both species. To achieve this objective, known pathways of 1,2-epoxy-3-butene (butadiene monoepoxide; BMO) and BDE metabolism were incorporated into a physiologically based pharmacokinetic model by scaling rates determined in vitro. With this model structure, epoxide clearance was underestimated for both rats and mice. Improved simulation of blood epoxide concentrations was achieved by addition of first-order metabolism in the slowly perfused tissues, verified by simulation of data on the time course for BMO elimination after i.v. injection of BMO. Blood concentrations of BD were accurately predicted for mice and rats exposed by inhalation to constant concentrations of BD. However, if all BD was assumed to be metabolized to BMO, blood concentrations of BMO were overpredicted. By assuming that only a fraction of BD metabolism produces BMO, blood concentrations of BMO could be predicted over a range of BD exposure concentrations for both species. In vitro and in vivo studies suggest an alternative cytochrome P-450-mediated pathway for BD metabolism that does not yield BMO. Including an alternative pathway for BD metabolism in the model also gave accurate predictions of blood BDE concentrations after inhalation of BD. Blood concentrations of BMO and BDE observed in both mice and rats are best explained by the existence of an alternative pathway for BD metabolism which does not produce BMO. [ABSTRACT FROM PUBLISHER]

Low levels of benzene from sources including cigarette smoke and automobile emissions are ubiquitous in the environment. Since the toxicity of benzene probably results from oxidative metabolites, an understanding of the profile of biotransformation of low levels of benzene is critical in making a valid risk assessment. To that end, we have investigated metabolism of a low concentration of [14C]benzene (3.4 microM) by microsomes from human, mouse and rat liver. The extent of phase I benzene metabolism by microsomal preparations from 10 human liver samples and single microsomal preparations from both mice and rats was then related to measured activities of cytochrome P450 (CYP) 2E1. Measured CYP 2E1 activities, as determined by hydroxylation of p-nitrophenol, varied 13-fold (0.253-3.266 nmol/min/mg) for human samples. The fraction of benzene metabolized in 16 min ranged from 10% to 59%. Also at 16 min, significant amounts of oxidative metabolites were formed. Phenol was the main metabolite formed by all but two human microsomal preparations. In those samples, both of which had high CYP 2E1 activity, hydroquinone was the major metabolite formed. Both hydroquinone and catechol formation showed a direct correlation with CYP 2E1 activity over the range of activities present. A simulation model was developed based on a mechanism of competitive inhibition between benzene and its oxidized metabolites, and was fit to time-course data for three human liver preparations. Model calculations for initial rates of benzene metabolism ranging from 0.344 to 4.442 nmol/mg/min are directly proportional to measured CYP 2E1 activities. The model predicted the dependence of benzene metabolism on the measured CYP 2E1 activity in human liver samples, as well as in mouse and rat liver samples. These results suggest that differences in measured hepatic CYP 2E1 activity may be a major factor contributing to both interindividual and interspecies variations in hepatic metabolism of benzene. Validation of this system in vivo should lead to more accurate assessment of the risk of benzene's toxicity following low-level exposure.

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.