Metabolism of benzene in human liver microsomes: individual variations in relation to CYP2E1 expression.

In human liver microsomes the oxidations of benzene, chlorzoxazone, aniline, dimethylformamide, and 4-nitrophenol were significantly correlated with each other and with the level of cytochrome P450 (CYP) 2E1 estimated by immunoblotting. Moreover, benzene oxidation to water-soluble metabolites was suppressed by 0.1 mM diethyldithiocarbamate, supposedly a specific inhibitor of CYP2E1 at this level. None of these metabolic rates correlated with immunochemically determined levels of CYP1A2, 2C9, and 3A4 nor oxidation of 7-ethoxyresorufin, tolbutamide, and nifedipine. Benzene oxidation to water-soluble metabolites was characterized by typical Michaelis-Menten kinetics. The different benzene K(m) values seen in individual human microsomal samples were not correlated with the level or activity of CYP1A2, 2C9, 2E1, and 3A4 but could be due to CYP2E1 microheterogeneity. The lowest K(m) for benzene oxidation could be related to C/D and/or c1/c2 polymorphism of CYP2E1 gene. Covalent binding of benzene reactive metabolites to microsomal proteins was also correlated with the CYP2E1 metabolic rates and immunochemical levels. At high concentrations of benzene covalent binding was inversely related to benzene concentrations (as well as to formation of water-soluble metabolites) in agreement with the view that secondary metabolites, mainly benzoquinone, are responsible for the covalent binding.

Cytochromes P450 in benzene metabolism and involvement of their metabolites and reactive oxygen species in toxicity.

Cytochrome P450 (CYP) 2E1 was the most efficient CYP enzyme that oxidized benzene to soluble and covalently bound metabolites in rat and human liver microsomes. The covalent binding was due mostly to the formation of benzoquinone (BQ), the oxidation product of hydroquinone (HQ), and was inversely related to the formation of soluble metabolites. In rats, inhalation of benzene (4 mg/liter of air) caused a rapid destruction of CYP2B1 previously induced by phenobarbital. The ability of benzene metabolites to destroy liver microsomal CYP in vitro decreased in the order BQ > HQ > catechol > phenol. The destruction was reversed by ascorbate and diminished by alpha-tocopherol, suggesting that HQ was not toxic, whereas BQ and semiquinone radical (SQ) caused the effect. In the presence of nicotinamide adenine dinucleotide phosphate, reduced (NADPH) the microsomes did not oxidize HQ to BQ, while the formation of superoxide anion radical from both HQ and BQ was markedly quenched. Destruction of CYP in vitro caused by HQ or BQ was not mediated by hydroxyl radical formation or by lipid peroxidation. On the contrary, HQ and BQ inhibited NADPH-mediated lipid peroxidation. Ascorbate induced high levels of hydroxyl radical formation and lipid peroxidation, which were differentially affected by quinones, indicating different mechanisms. Despite reducing the toxicity of HQ and BQ, ascorbate appeared to induce its own toxicity, reflected in high levels of lipid peroxidation. Iron redox cycling played a significant role in the NADPH-induced hydroxyl radical formation but not in that caused by ascorbate; however, lipid peroxidation induced by NADPH or ascorbate was suppressed by ethylenediaminetraacetate, indicating a crucial role of iron. Thus, the data indicate that the quinones destroyed CYP directly and not via oxygen activation or lipid peroxidation.