Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: Effects on coumarin, dicumarol, and testosterone oxidation

Abstract

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7α- and 15α-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7α- and 15α-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7α- and/or 15α-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (>70%) in human and monkey, but only a minor metabolite (<1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme ( K m 0.2 – 0.6 μM), which was markedly inhibited (>95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied ~17-fold among liver microsomes from 22 human subjects. This variation was highly correlated ( r 2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with β-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied ~5-fold among the human liver microsomal samples, but this variation correlated poorly ( r 2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity. Under conditions where coumarin 7-hydroxylation was markedly inhibited (>95%), antibody against rat CYP2A1 failed to inhibit the metabolism of dicumarol by human liver microsomes, although dicumarol metabolism was inhibited up to 60% by antibodies against CYP2C enzymes. A 100-fold molar excess of dicumarol, R-warfarin, or S-warfarin caused little or no inhibition of coumarin 7-hydroxylation by human liver microsomes. These results suggest that, despite being the principal catalyst of coumarin 7-hydroxylation, CYP2A6 contributes negligibly, if at all, to the metabolism of dicumarol and warfarin by human liver microsomes and that, in contrast to rat CYP2A1 and CYP2A2, CYP2A6 does not catalyze the 7α- or 15α-hydroxylation of testosterone.