Covalent binding of benzene and its metabolites to DNA in rabbit bone marrow mitochondria in vitro

Abstract

Rabbit bone marrow mitochondria, stripped of their outer membrane (mitoplasts), have been shown to carry out the NADPH-dependent bioactivation of radiolabelled benzene in vitro to metabolites capable of covalently binding to mtDNA, thereby inhibiting transcription. The metabolites of benzene produced in bone marrow cells by the microsomal cytochrome P-450 are thought to be phenol, catechol, hydroquinone and p-benzoquinone (Andrews et al., Life Sci., 25 (1979) 567; Irons et al., Chem.-Biol. Interact., 30 (1980) 241). Incubation of mitoplasts from rabbit bone marrow cells in vitro with varying concentrations of the putative microsomal metabolites showed a concentration-dependent inhibition of RNA synthesis. The 50% inhibitory molar concentration (IC50) for each metabolite was determined to be: 1,2,4- benzenetriol , 6.3 X 10(-7); p-benzo-quinone, 2 X 10(-6); phenol, 2.5 X 10(-5); hydroquinone, 5 X 10(-5); catechol, 2 X 10(-3); benzene, 1.6 X 10(-2). DNA, isolated from rabbit bone marrow cell or rat liver mitoplasts prelabelled in DNA with [3H]dGTP and exposed to [14C]benzene in vitro, was enzymatically hydrolyzed to nucleosides which were chromatographed on a Sephadex LH-20 column to separate free nucleosides from nucleoside-adducts. The elution profiles indicated that rat liver mtDNA contained six guanine nucleoside-adducts and rabbit bone marrow cell mtDNA contained seven guanine nucleoside-adducts. Incubation of bone marrow mitoplasts in vitro in the presence of benzene and the hydroxyl radical scavenger, mannitol, resulted in the inhibition of formation of four of the guanosine-adducts. When [3H]dATP was substituted as the prelabelled precursor nucleotide, the LH-20 column profile indicated that two adenine nucleoside-adducts were also formed from benzene in vitro. Furthermore, a comparison of the Sephadex LH-20 column profiles of purine adducts derived from [14C]benzene- and [3H]dGMP-labelled mtDNA with profiles generated by individually incubating each of the putative unlabelled metabolites with bone marrow mitoplasts in vitro has indicated that p-benzoquinone, phenol, hydroquinone and 1,2,4- benzenetriol form adducts with guanine. One of the two adenosine-adducts may arise from hydroquinone; the compound forming the other adduct is unknown at the present time. Exposure of mitoplasts to catechol in vitro resulted in the formation of a guanine nucleoside-adduct that was present in rat liver mtDNA but absent from the DNA isolated from rabbit bone marrow cell mitoplasts exposed to [14C]benzene in vitro. This suggests that catechol is probably not a major metabolite of benzene formed in bone marrow cell mitochondria.