TOXICOKINETICS AND METABOLISM Vessela Nedelcheva á Ivan Gut á Pavel Sou cek Bronislava Tichavska á Lucie TyÂnkova á Jaroslav MraÂz F. Peter Guengerich á Magnus Ingelman-Sundberg Metabolism of benzene in human liver microsomes: individual variations in relation to CYP2E1 expression Received: 8 September 1998 / Accepted: 24 November 1998 Abstract In human liver microsomes the oxidations of benzene, chlorzoxazone, aniline, dimethylformamide, and 4-nitrophenol were signi®cantly 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 spe- ci®c inhibitor of CYP2E1 at this level. None of these metabolic rates correlated with immunochemically de- termined levels of CYP1A2, 2C9, and 3A4 nor oxidation of 7-ethoxyresoru®n, tolbutamide, and nifedipine. Ben- zene oxidation to water-soluble metabolites was char- acterized by typical Michaelis-Menten kinetics. The dierent 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 ben- zene 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. Key words Benzene á Chlorzoxazone á Covalent binding á Cytochrome P450 á Enzyme kinetics Introduction Benzene is a human carcinogen (IARC 1987): continu- ing occupational exposures to benzene in some countries still cause leukaemia (Yin 1995). Benzene toxicity is postulated to be due to its oxidation to benzene oxide, 1,4-benzoquinone, 1,2,4-trihydroxybenzene and trans,- trans-muconaldehyde, which can alkylate proteins and DNA (Irons 1985; Sou cek et al. 1994). The putative rearrangement of benzene oxide to phenol and cleavage to trans,trans-muconaldehyde are considered to be very rapid, while benzene and phenol are oxidized by cyto- chrome P450 (CYP; EC 1.14.14.1) enzymes (for no- menclature see Nelson et al. 1996). Hydroquinone oxidation to benzoquinone is either non-enzymatic in the presence of dioxygen or catalysed by peroxidases in bone marrow (Greenlee et al. 1981; Smith et al. 1989). Rat and rabbit CYP2E1, major catalysts in the oxi- dation of c. 75 carcinogenic and toxic chemicals, most eciently oxidize benzene to soluble products, mainly phenol and hydroquinone (Johansson and Ingelman- Sundberg 1983; Guengerich et al. 1991; Gut et al. 1993) and to covalently binding metabolites (Gut et al. 1996). In contrast, other rat and rabbit CYP enzymes except 2B1 apparently do not eectively oxidize benzene (Koop et al. 1989, Gut et al. 1993). CYP2E1 was also the most ecient CYP enzyme in benzene oxidation in human liver microsomes (Guengerich et al. 1991; Schlosser et al. 1993; Seaton et al. 1994), but was measured only at one low benzene level. The evidence that human CYP2E1 is the principal enzyme producing reactive products co- valently bound to biomacromolecules is limited. CYP1A1 which oxidizes phenol to hydroquinone in rabbits, is absent in human liver. Rat but not human Arch Toxicol (1999) 73: 33±40 Ó Springer-Verlag 1999 V. Nedelcheva á I. Gut (&) á P. Sou cek á B. Tichavska á L. TyÂnkova á J. MraÂz National Institute of Public Health, Department of Occupational Medicine, S Ï robaÂrova 48, Praha 10, 10042, Czech Republic e-mail: ivan.gut@ecn.cz Tel.: (4202) 6708 2765; Fax: (4202) 673 11 236 F.P. Guengerich Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37232-0146, USA M. Ingelman-Sundberg Karolinska Institutet, Department of Biological Chemistry, Berzelius Laboratory, Stockholm, S-171 77, Sweden