Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism.

F P Guengerich,M V Martin,P H Beaune,P Kremers,T Wolff,D J Waxman

doi:10.1016/s0021-9258(19)89213-x

Abstract

The metabolism of the dihydropyridine calcium antagonist and vasodilator nifedipine has been reported to exhibit polymorphism among individual humans (Kleinbloesem, C. H., van Brummelen, P., Faber, H., Danhof, M., Vermeulen, N. P. E., and Breimer, D.D. (1984) Biochem. Pharmacol. 33, 3721-3724). Nifedipine oxidation has been shown to be catalyzed by cytochrome P-450 (P-450) enzymes. Reconstitution, immunoinhibition, and induction studies with rat liver indicated that the forms designated P-450UT-A and P-450PCN-E are the major contributors to microsomal nifedipine oxidation. The P-450 which oxidizes nifedipine (P-450NF) was purified to electrophoretic homogeneity from several human liver samples. Antibodies raised to P-450NF were highly specific as judged by immunoblotting analysis and inhibited greater than 90% of the nifedipine oxidase activity in human liver microsomes. A monoclonal antibody raised to the human P-450 preparation reacted with both human P-450NF and rat P-450PCN-E. Immunoblotting analysis of 39 human liver microsomal samples using anti-P-450NF antibodies revealed the same 52,000-dalton polypeptide, corresponding to P-450NF, with only one of the microsomal samples showing an additional immunoreactive protein. The level of nifedipine oxidase activity was highly correlated with the amount of P-450NF thus detected using either polyclonal (r = 0.78) or monoclonal (r = 0.65) antibodies, suggesting that the amount of the P-450NF polypeptide may be a major factor in influencing the level of catalytic activity in humans as well as rats. Cytochrome b5 enhanced the catalytic activity of reconstituted P-450NF, and anti-cytochrome b5 inhibited nifedipine oxidase activity in human liver microsomes. P-450NF also appears to be a major contributor to human liver microsomal aldrin epoxidation, d-benzphetamine N-demethylation, 17 beta-estradiol 2- and 4-hydroxylation, and testosterone 6 beta-hydroxylation, the major pathway for oxidation of this androgen in human liver microsomes.

Highlights

0.65) antibodies, suggesting that theamount of the P450NFpolypeptide may be a major factor ininfluencing the levelof catalytic activity in humaansswell as rats
Drugs have been recognized and are probably due at least in part to polymorphisms in the cytochrome P-450’ enzymes (Kupfer and Preisig, 1984; Guengerich et al, 1986). These polymorphisms usually contain a strong genetic component and, in addition to predisposing individuals to potential drug toxicities, may contribute to theinfluence of host factors in carcinogenesis, as the P-450s are involved in thebiotransformation of a variety of environmental pollutantsp, esticides, and cancer-causing agents as well as drugs (Wislocki et al, 1980).Such polymorphisms have been characterized in animal models (Simmons and Kasper, 1983; Larrey et al, 1984; Johnson and Schwab, 1984) but only recently have the molecular details been considered in humans
In some cases where experiments in this report were done with preparations of these P-450s made in Dr Waxman’s laboratory, a combined designation is used (e.g. P-450PCN-E(PB.2a)).The human P-450s are designated P-450NF, P - 4 5 0 ~(D~istlerath et al, 1985),P - 4 5 0 ~(D~istlerath et al, 1985), and P - 4 5 0 ~(G~uengerich et al, 1986) to signify their respective involvements in the genetic polymorphisms of nifedipine oxidation (NF), debrisoquine 4-hydroxylation (DB), phenacetin 0deethylation (PA), andS-mephenytoin-4-hydroxylation(MP) activities

Summary

3.79 Isosafrole

’Results are expressed as means of duplicate assays. * Results are expressed as means (+ S.D.) of triplicate assays. Immunochemical Inhibition of Catalytic Activity in Liver Microsomes-The studies with microsomes and reconstituted P-450 systems described above suggested that P-~~OIJT-AP, 0 p ~ ~a.n~d, P-450pB.B might be the major forms of P-450 contributing to nifedipine oxidation in rat liver. In order to examine the roles of these enzymes more closely, inhibitory antibodies specific for each of these P-450s were incubated with microsomes prepared from untreated male rats (Fig. 10). Both anti-P-450uT.Aand anti-P-450pcN.Eshowed extensive inhibition,but anti-P-450pB.n did not. Roleof Cytochrome b5 in Human Microsomal Nifedipine Oxidase Activity-Preliminary studies indicated that the inclusion of either purified rat or human liver cytochrome b5 in the reconstituted P-45oNF resulted in avariable enhancement of nifedipine oxidase activity.

DISCUSSION

Findings

RESULTS