title: 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.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-2324, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.forename: S S
authors.initials: SS
authors.lastname: Dehal
authors.forename: G
authors.initials: G
authors.lastname: Hynd
authors.forename: G B
authors.initials: GB
authors.lastname: Jones
authors.forename: D
authors.initials: D
authors.lastname: Kupfer
mesh_terms: D000818:Animals; D002384:Catalysis; D002645:Chickens / Q000378:metabolism*; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D013629:Tamoxifen / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D018076:DNA, Complementary; D013629:Tamoxifen; D019389:Cytochrome P-450 CYP2D6