Characterization of tamoxifen and 4-hydroxytamoxifen glucuronidation by human UGT1A4 variants

INTRODUCTION: Tamoxifen (TAM) is an antiestrogen widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism of TAM and one of its major active metabolites, 4-hydroxytamoxifen (4-OH-TAM), is via glucuronidation. In the present study, the glucuronidating activities of three common variant isoforms encoded by the human UDP-glucuronosyltransferase (UGT) 1A4 gene were examined against TAM, trans-4-OH-TAM and cis-4-OH-TAM. METHODS: HPLC was used to detect glucuronide conjugates in microsomes from UGT1A4-overexpressing HK293 cells. The UGT1A4 wild-type cDNA was synthesized by RT-PCR using normal human liver total RNA. The UGT1A4(24Thr/48Leu )and UGT1A4(24Pro/48Val )variants were generated by site-directed mutagenesis of the pcDNA3.1/V5-His-TOPO plasmid expressing wild-type UGT1A4(24Pro/48Leu). Levels of UGT1A4 expression in UGT-overexpressing cell lines were measured by western blot analysis. RESULTS: Microsomes from wild-type UGT1A4(24Pro/48Leu)-overexpressing HK293 cells exhibited significant levels of activity against TAM, trans-4-OH-TAM and cis-4-OH-TAM, forming exclusively the tamoxifen quaternary ammonium glucuronide (TAM-N(+)-glucuronide) and the 4-hydroxytamoxifen quaternary ammonium glucuronides (trans-4-OH-TAM-N(+)-glucuronide and cis-4-OH-TAM-N(+)-glucuronide) with apparent K(m )values of 2.0 μM, 2.2 μM, and 2.1 μM, respectively. Higher glucuronidation activities were found by kinetic analysis for microsomes from the variant UGT1A4(24Pro/48Val)-overexpressing cell line as compared with microsomes from wild-type UGT1A4(24Pro/48Leu)-overexpressing cells against TAM and against both the trans and cis isomers of 4-OH-TAM. A significantly (P < 0.02) lower K(m )value (~1.6-fold to 1.8-fold) was observed for both 4-OH-TAM isomers, while a near-significant (P = 0.053) decrease in K(m )was observed for TAM for the UGT1A4(24Pro/48Val )variant as compared with wild-type UGT1A4. The V(max)/K(m )ratio for the UGT1A4(24Pro/48Val )variant was significantly (P ≤ 0.005) higher than that observed for the wild-type UGT1A4 isoform for both the trans and cis isomers of 4-OH-TAM after normalization for UGT1A4 expression by western blotting. No significant effect on enzyme kinetics was observed for the UGT1A4(24Thr/48Leu )variant against either isomer of 4-OH-TAM or with TAM. CONCLUSION: These data suggest that the UGT1A4 codon 48 Leu>Val polymorphism significantly alters glucuronidation rates against TAM and its active hydroxylated metabolites, and that this polymorphism may play an important role in individual pharmacological response to TAM therapy.