ROLES OF HUMAN LIVER CYTOCHROME-P4502C AND CYTOCHROME-P4503A ENZYMES IN THE 3-HYDROXYLATION OF BENZO(A)PYRENE

The major oxidation product of the classic polycyclic hydrocarbon carc inogen benzo(a)pyrene [B(a)P] is 3-hydroxy B(a)P. Numerous studies hav e been concerned with the measurement of B(a)P 3-hydroxylation activit y in experimental animals and human tissues. Although human liver is t he main site of this reaction, systematic studies had not been carried out to define the roles of individual cytochrome P450 (P450) enzymes involved. Purified human P4502C8 and P4503A4 showed appreciable cataly tic activity; purified human P4501A2 and yeast recombinant (human) P45 02C9 and P4502C10 had less activity. No B(a)P 3-hydroxylation activity was observed with purified human P4502A6, P4502D6, P45602E1, or P4502 C(MP). When microsomes prepared from different human liver samples wer e compared, B(a)P 3-hydroxylation activity was well correlated with ni fedipine oxidation (a P4503A4 marker) but not markers of other P-450s, including tolbutamide hydroxylation (P4502C9 and 2C10), chlorzoxazone 6-hydroxylation (P4502E1), (S)-mephenytoin 4'-hydroxylation (P4502C(M P)), and coumarin 7-hydroxylation (P4502A6). In three of the liver mic rosomal samples with relatively high B(a)P 3-hydroxylation activity, i mmunoinhibition was observed with anti-P4503A > anti-P4502C (and no in hibition with several other antibodies). The selective chemical inhibi tors gestodene and troleandomycin (P4503A enzymes) and sulfaphenazole (P4502C enzymes) reduced the B(a)P 3-hydroxylation activity of the mor e active microsomal preparations to rates seen in the preparations wit h low activity. This residual activity (and most of the activity in th e low activity samples) was refractory to all of the chemical inhibito rs and antibodies. The addition of 7,8-benzoflavone dramatically stimu lated B(a)P 3-hydroxylation in all of the microsomal samples (and also stimulated purified P4503A4), arguing against an important role for P 4501A1 or P4501A2. We conclude that roles of human P450 enzymes for B( a)P 3-hydroxylation follow the order P4503A4 greater-than-or-equal-to P4502C8 > P4502C9/10 in human liver and that the other P450s examined here do not have major roles. P4502C8 and P4502C(MP) (but not P4503A4) were found to activate B(a)P to products genotoxic in Salmonella typh imurium; this pathway would appear to involve products other than 3-hy droxy B(a)P and B(a)P 7,8-dihydrodiols.