Abstract

When [³H]benzo[a]pyrene is incubated in vitro together with deproteinized salmon sperm DNA, NADPH, and mouse liver microsomes, the covalent binding of benzo[a]pyrene metabolites to DNA occurs. The metabolite-nucleoside complexes can be resolved into at least nine distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. These peaks are arbitrarily designated A (most polar) through I (least polar). With the use of synthetic and biologically produced metabolites, seven of nine peaks are tentatively assigned to one or more metabolites of benzo[a]pyrene. Peaks A and C are unidentified. Peaks B, D, F, and I include products of benzo[a]pyrene quinones that are further metabolized. Peak E reflects almost exclusively both the cis- and trans-7,8-diol 9,10-epoxides of benzo[a]pyrene. Peak G represents predominantly the K-region metabolite (the 4,5-oxide), interacting with one or more nucleosides. Peak H comprises reactive intermediates resulting from the further metabolism of benzo[a]pyrene phenols. The 7,8-oxide and the 9,10-oxide contribute to peaks E, F, G, and H. Benzo[a] pyrene thus may be metabolized to four different "types" of reactive intermediates capable of binding to DNA: (a) primary arene oxides, (b) diol epoxides, (c) phenols oxygenated further, and (d) quinones oxygenated further (or quinone-derived free radicals). These last three types of microsomally activated intermediates are therefore the result of two- or three-step enzymatic processes in which cytochrome P-450-mediated monooxygenations occur at least twice.