gestodene and alpha-naphthoflavone

Kinetics of the 2- and 4-hydroxylations of estradiol (E2) by human liver microsomal samples were studied to determine the major P450 isoform involved in these endogenous reactions. Thirty human liver microsomal samples were analysed. Metabolism of 25 microM [14C]E2 produced 2-hydroxy and 4-hydroxy derivatives with a ratio of 3.2 +/- 1.5 and a great inter-individual variation. Kinetic analysis of the 2- and 4-hydroxylations of E2 exhibited a curvilinear double reciprocal plot with an apparent Km of 15 microM. Further experiments demonstrated that alpha-naphthoflavone, testosterone and progesterone increased the 2-hydroxylation activity, suggesting the involvement of a substrate activation mechanism. These two hydroxylations of E2 were shown to be catalysed by cytochrome P450 with an apparent dissociation constant Ks of 0.8 microM. These 2- and 4-hydroxylations inter-correlated significantly (r = 0.93; N = 30). The 2-hydroxylation of E2 correlated with four monooxygenase activities known to be supported by P450 3A4/3A5, namely nifedipine oxidation (r = 0.78; N = 29); erythromycin N-demethylation (r = 0.69; N = 27), testosterone 6 beta-hydroxylation (r = 0.66; N = 25) and tamoxifen N-demethylation (r = 0.64; N = 29). On the other hand, E2-hydroxylations did not correlate with activities supported by P450 1A2 and P450 2E1. Furthermore, drugs as cyclosporin, diltiazem, triacetyl-oleandomycin and 17 alpha-ethynylestradiol inhibited more than 90% of the E2-hydroxylations at concentrations < 250 microM, while weak inhibition was shown with 500 microM cimetidine and no significant inhibition with caffeine, phenacetin and omeprazole. Finally, 2- and 4-hydroxylations of E2 correlated significantly with the content of P450 3A4/3A5 immunodetected by a monoclonal antibody anti-human P450-nifedipine (r = 0.84; N = 28). E2-hydroxylation activities were inhibited by more than 80% with polyclonal anti-human anti-P450-nifedipine. Preincubation of human liver microsomes with 100 microM gestodene (a suicide substrate of P450 3A4) inactivated this P450 isoform and accordingly allowed evaluation of the contribution of other P450 isoforms to the E2 metabolism to about 21% (+/- 17%, N = 29). All these results taken together suggest that P450 3A4/3A5 are the major forms involved in the formation of catecholestrogens in the human liver microsomes.

Topics: Adolescent; Adult; Benzoflavones; Caffeine; Child, Preschool; Cytochrome P-450 CYP1A2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Activation; Estradiol; Female; Humans; Hydroxylation; Infant; Infant, Newborn; Isoenzymes; Kinetics; Male; Microsomes, Liver; Middle Aged; Norpregnenes; Oxidoreductases; Phenacetin; Progesterone Congeners; Tamoxifen; Testosterone

The major oxidation product of the classic polycyclic hydrocarbon carcinogen benzo(a)pyrene [B(a)P] is 3-hydroxy B(a)P. Numerous studies have been concerned with the measurement of B(a)P 3-hydroxylation activity in experimental animals and human tissues. Although human liver is the main site of this reaction, systematic studies had not been carried out to define the roles of individual cytochrome P-450 (P-450) enzymes involved. Purified human P4502C8 and P4503A4 showed appreciable catalytic activity; purified human P4501A2 and yeast recombinant (human) P4502C9 and P4502C10 had less activity. No B(a)P 3-hydroxylation activity was observed with purified human P4502A6, P4502D6, P45602E1, or P4502CMP. When microsomes prepared from different human liver samples were compared, B(a)P 3-hydroxylation activity was well correlated with nifedipine 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 (P4502CMP), and coumarin 7-hydroxylation (P4502A6). In three of the liver microsomal samples with relatively high B(a)P 3-hydroxylation activity, immunoinhibition was observed with anti-P4503A greater than anti-P4502C (and no inhibition with several other antibodies). The selective chemical inhibitors gestodene and troleandomycin (P4503A enzymes) and sulfaphenazole (P4502C enzymes) reduced the B(a)P 3-hydroxylation activity of the more active microsomal preparations to rates seen in the preparations with low activity. This residual activity (and most of the activity in the low activity samples) was refractory to all of the chemical inhibitors and antibodies. The addition of 7,8-benzoflavone dramatically stimulated B(a)P 3-hydroxylation in all of the microsomal samples (and also stimulated purified P4503A4), arguing against an important role for P4501A1 or P4501A2. We conclude that roles of human P-450 enzymes for B(a)P 3-hydroxylation follow the order P4503A4 greater than or equal to P4502C8 greater than P4502C9/10 in human liver and that the other P-450s examined here do not have major roles. P4502C8 and P4502CMP (but not P4503A4) were found to activate B(a)P to products genotoxic in Salmonella typhimurium; this pathway would appear to involve products other than 3-hydroxy B(a)P and B(a)P 7,8-dihydrodiols.

Topics: Antibodies; Benzo(a)pyrene; Benzoflavones; Cytochrome P-450 Enzyme System; Gene Expression; Humans; Hydroxylation; Isoenzymes; Kinetics; Liver; Microsomes, Liver; Norpregnenes; Recombinant Proteins; Saccharomyces cerevisiae; Sulfaphenazole

One of the major routes of elimination of dapsone (4,4'-diaminodiphenylsulfone) is by N-oxidation, to produce a hydroxylamine metabolite. The specific form of cytochrome P-450 (P-450) involved in this oxidation reaction was examined in human liver microsomal preparations previously characterized with respect to their content of several known P-450 enzymes. Among five preparations, the rank order of activity for dapsone hydroxylamine formation was most well correlated with the immunochemically determined level of P-4503A4 (r = 0.94, p less than 0.03). Moreover, inhibition of microsomal oxidation was observed with antibodies specific to P-4503A, with a maximum reduction of greater than 90%, but was not produced by antibodies specific to P-4501A2, P-4502CMP, or P-4502E1. Prior incubation of microsomes with gestodene (100 microM) or troleandomycin (20 microM), known selective mechanism-based inhibitors of P-4503A enzymes (in the presence of NADPH), led to 75% and 40% reductions in catalytic activity, respectively. In contrast, preincubation with increasing concentrations of alpha-naphthoflavone, a known activator of P-4503A4, increased dapsone N-hydroxylation in a concentration-dependent manner, with 5-fold activation being observed at 50 microM alpha-naphthoflavone. Finally, P-4503A4 isolated from human liver microsomes and cDNA-expressed P-4503A4 (in yeast) were both able to catalyze dapsone N-hydroxylation, with the latter preparation exhibiting a 3-fold activation in the presence of 100 microM alpha-naphthoflavone. Collectively, these findings demonstrate that N-oxidation of dapsone in human liver is predominantly mediated by P-4503A4, and they suggest that quantitative measurement of this metabolic pathway in vivo might serve as an index of the activity of this enzyme.

Topics: Antibodies; Benzoflavones; Cytochrome P-450 Enzyme System; Humans; Hydroxylation; Isoenzymes; Kinetics; Microsomes, Liver; Norpregnenes; Troleandomycin

Using a new sensitive reverse-phase HPLC assay relying on UV detection at 344 nm, the capacity of 18 human liver microsomal samples to support NADPH-dependent, cytochrome P450-mediated oxidation and arachidonic acid-dependent oxidation of the enantiomers of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-DHD) was determined. The (-)-7R,8R-enantiomer, the preferred substrate of cytochrome P450, formed 94% diolepoxide 2 (anti-isomer; 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene) measured as derived alcohols, and the (+)-7S,8S-enantiomer formed 67% diolepoxide 1 (syn-isomer; 7S,8R-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). Arachidonic acid-supported oxidations gave approximately 70% diolepoxide 2 from each enantiomer. The involvement of different sets of cytochrome P450 isozymes was supported by incubations in the presence of alpha-naphthoflavone (alpha-NF) (50 microM) and correlation studies. In the absence of alpha-NF, a positive correlation was found between the metabolism of the (-)-enantiomer but not the (+)-isomer of B[a]P-7,8,-DHD and the relative content of P450IA2. In the presence of alpha-NF, the P450IIIA3/4 content correlated positively with the metabolism of both the (+)-enantiomer and the (-)-enantiomer. Gestodene (100 microM) inhibited the alpha-NF-stimulated metabolism, confirming the involvement of cytochrome P450IIIA3/4. No difference was found between the extent of arachidonic acid-supported, peroxyl radical-mediated metabolism of the (+)- and (-)-enantiomers of B[a]P-7,8-DHD. The metabolism was almost completely abolished by 2 microM butylatedhydroxyanisole and 100 microM nordihydroguaiaretic acid, confirming the free radical nature of the reaction.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Adolescent; Adult; Aged; Arachidonic Acid; Benzoflavones; Biotransformation; Butylated Hydroxyanisole; Child; Cytochrome P-450 Enzyme System; Humans; Male; Masoprocol; Microsomes, Liver; Middle Aged; NADP; Norpregnenes; Oxidation-Reduction; Progesterone Congeners; Stereoisomerism