Metabolism of benzo[a]pyrene in primary cultures of human hepatocytes: dose–response over a four-log range

Cultures of isolated human hepatocytes from three different human liver specimens were exposed for 24 h to media containing [3H]benzo[a]pyrene (BP) (0.1, 1.0, 10, 100 μM). The cells and media were harvested and extracted. Subsequent incubations of the aqueous phase with β-glucuronidase and aryl sulfatase, followed by acetone/ethyl acetate extraction, were utilized to determine specific conjugation. Separation of the BP and its metabolites in the residues of the extracts was achieved by h.p.l.c. The capacity of human hepatocytes to metabolize BP was not saturated at up to 100 μM of BP, and the predominant metabolites produced were eluted in the void volume and were a mixture of highly polar BP forms. The next four most prevalent forms of BP metabolites were the 3-hydroxy BP, BP-4,5-dihydrodiol, BP-9,10-dihydrodiol, and BP-7,8-dihydrodiol. These metabolites all increased nearly linearly with dose. Conjugation varied for each different case, ranging from 31 to 91%, but a general trend clearly appeared; if β-glucuronidation decreased, then sulfation increased and vice versa. BP metabolite binding to DNA was associated wth the amount of unconjugated BP-7,8-dihydrodiol metabolite. BP metabolite binding to DNA was nearly linear from 0.1 to 10 μM BP; however, binding to DNA at 100 μM increased 64- to 844-fold over the binding occurring at 10 μM. Thus, human hepatocytes have a strong tendency to form highly polar BP metabolites, and total binding of BP to DNA over a four-log dose range is much less at 0.1–10 μM than one would predict from extrapolation from the high concentration (100 μM).