32P-postlabelling analysis of DNA adducts of benzo[a]pyrene formed in complex metabolic activation systems in vitro

The influence of cytochrome P-450-linked monooxygenase, epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases on the metabolic activation of benzo[a]pyrene (BP) was studied by incubating BP with preparations of rat-liver microsomal and cytosolic fractions in the presence of exogenous DNA. 32P-Postlabelling analysis of the DNA revealed the presence of covalently bound adducts formed by BP, which were visualised as radioactive spots on autoradiographs of thin-layer chromatograms. The effects on the adduct profile of adding different combinations of 1,2-epoxy-3,3,3-trichloropropane (TCPO), UDP-glucoronic acid (UDPGA) and glutathione (GSH) to the incubation mixture were determined. As many as 14 different DNA adducts were resolved and quantitated on the chromatograms, the numbers and quantities of which varied within a large range depending on the incubation conditions. The influence of the enzyme inhibitor and cofactors on the adduct patterns reflects the complex effects of simultaneous enzyme interactions on the metabolic activation of BP.