Induction of tamoxifen-4-hydroxylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) in avian liver: identification of P450 TCDDAA as catalyst of 4-hydroxylation induced by TCDD and beta NF.

Abstract

Tamoxifen has been found to be metabolized by liver primarily into three metabolites, tamoxifen-N-oxide, formed by the flavin-containing monooxygenase, and N-desmethyl- and 4-hydroxytamoxifen, formed by cytochrome P450. The N-demethylation was demonstrated to be catalyzed by P4503A in rat and human liver; however, the P450s catalyzing the 4-hydroxylation have not been identified. Although 4-hydroxytamoxifen exhibits more potent estrogen agonist/antagonist activity than tamoxifen, the relative contributions of the parent drug and its 4-hydroxy metabolite(s) to the activity of tamoxifen in vivo have not been established. We report here that the rate of tamoxifen 4-hydroxylation is higher in livers of adult chicken and chick embryos than in livers of mammalian species. Tamoxifen 4-hydroxylation was increased by treatment of chick embryos with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and to a lesser extent by phenobarbital (PB). The major effect of PB treatment was an increase in tamoxifen N-demethylation. Tamoxifen 4-hydroxylase activity of reconstituted purified chicken P450s was highest for TCDDAA, a P450 active in arachidonate epoxygenation and estradiol 2-hydroxylation, and one of the two major P450s induced by TCDD and beta NF in chick embryo liver. The second P450, TCDDAHH, which is active in aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase was inactive in tamoxifen 4-hydroxylation. Anti-TCDDAA IgG immunoinhibited tamoxifen 4-hydroxylation in microsomes from beta NF-treated embryos by over 80%, but was ineffective against this reaction in the controls. The immunochemical findings together with the reconstitution data identify TCDDAA as the P450 responsible for TCDD/beta NF-induced tamoxifen 4-hydroxylation in chick liver. In PB-treated livers, a P450 fraction containing CYP2H1/H2, the major PB-induced P450s, had the highest tamoxifen 4-hydroxylase and N-demethylase activities, a finding compatible with one or both of those P450s being responsible for the PB-induced tamoxifen 4-hydroxylation and N-demethylation. The findings reported here raise the possibility that exposure of women undergoing tamoxifen therapy to agents that induce human CYP1A2 or CYPB1/2 analogues may produce increased levels of 4-hydroxytamoxifen and that this may affect the therapeutic potency of tamoxifen.