CYP2D6-mediated catalysis of tamoxifen aromatic hydroxylation with an NIH shift: similar hydroxylation mechanism in chicken, rat and human liver microsomes

Abstract

1. 4-Tritiated-tamoxifen (4- [3 H]-tamoxifen) and 4-deuterated-tamoxifen (4- [2 H]-tamoxifen) were synthesized to examine tamoxifen metabolism by human P450 (CYP) forms and also for the possibility of determining tamoxifen-4-hydroxylation in humans in vivo. 2. Liver microsomes from several species and cDNA-expressed human P450s were incubated with 4- [3 H]-tamoxifen and the reaction monitored by assaying 4-hydroxytamoxifen (4-OH-tam) and 3 H 2 O formed. However, tamoxifen-4-hydroxylation did not generate stoichiometric amounts of 3 H 2 O and the expected unlabelled 4-OH-tam but instead yielded radiolabelled 4-OH-tam, apparently from [3 H]-migration to the ortho -position, referred to as the NIH shift. 3. CYP2D6 was the prime catalyst of tam-4-hydroxylation, whereas CYP2B6, 2C9 and 2C19 yielded only low levels of 4-OH-tam; nevertheless, in all cases the 4-OH-tam was radioactive, apparently resulting from reactions involving an NIH shift. 4. Chicken liver microsomal preparation, being catalytically the most active in tamoxifen-4-hydroxylation, was incubated with deuterated tamoxifen (4- [2 H]-tamoxifen) in order to determine whether an NIH shift occurs. Ion-trap mass-spectrometry of the HPLC-purified 4-OH-tam, from that incubation, indicated about 60% of [2 H]-retention in 4-OH-tam, signifying an NIH shift. These findings indicate that the aromatic hydroxylation of tamoxifen does not entail hydroxyl insertion with an Sn2-displacement of hydrogen or a hydrogen isotope (2 H or 3 H), but apparently involves epoxidation followed by migration of the 3 H, 2 H or 1 H to the ortho -position, and dissociation of the 1 H in preference to 3 H or 2 H, i.e. retention of the hydrogen isotope appears to be related to the bond strengths: C- 3 H>C- 2 H>C- 1 H.