Identification of benzo[a]pyrene-7,8-dione as an authentic metabolite of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in isolated rat hepatocytes

Dihydrodiol dehydrogenase (DD) has been shown to catalyze theoxidation of (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-diol) to yield benzo[a]pyrene-7,8-dione (BPQ) in uninducedfortified rat liver S100 fractions but the formation of BPQhas not been observed in whole cells. In these studies [³H]BP-diolwas incubated with isolated hepatocytes from uninduced ratsfor 0–20 min at 37°C. Organic-extractable radioactivityin the cell media accounted for 20% of the total [³H]BP-dioladded. Reverse phase (RP)-HPLC analysis of this fraction revealedthe formation of an unknown metabolite that co-chromatographedwith an authentic synthetic standard of BPQ. The identity ofthe unknown metabolite was further established by: (i) co-chromatographywith synthetic BPQ under both RP- and normal phase-HPLC conditionsusing diode array detection, which indicated that the metaboliteshared UV/ vis spectral identity with standard BPQ; and by (ii)electron impact mass spectrometry of the unknown metabolitewhich gave the same parent and fragment ions as the syntheticstandard. The formation of BPQ by isolated hepatocytes was foundto be 0.50 nmol/3x10⁶ cells/10 min, and represented 7% of thetotal organic-soluble metabolites in the extracellular media.Its formation was abolished by the addition of indomethacin,a competitive inhibitor of DD, indicating that this enzyme wasresponsible for BPQ formation. Other organic-soluble metabolitesformed corresponded to BP-tetraols (hydrolysis products of theanti-and syn-diol epoxides). Examination of the aqueous phaseof the extracellular media indicated that a large portion ofBP-diol was converted to glucuronide and sulfate conjugates.Under the conditions employed BP-tetraols and BPQ were formedto an equal extent implying that in hepatocytes isolated fromuninduced rats, DD and CYP1A1 contributed equally to the metabolismof BP-diol.