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

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Abstract
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 andcis-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 UGT1A424Thr/48Leuand UGT1A424Pro/48Valvariants were generated by site-directed mutagenesis of the pcDNA3.1/V5-His-TOPO plasmid expressing wild-type UGT1A424Pro/48Leu. Levels of UGT1A4 expression in UGT-overexpressing cell lines were measured by western blot analysis.Results: Microsomes from wild-type UGT1A424Pro/48Leu-overexpressing HK293 cells exhibited significant levels of activity against TAM,trans-4-OH-TAM andcis-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 andcis-4-OH-TAM-N+-glucuronide) with apparentKmvalues 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 UGT1A424Pro/48Val-overexpressing cell line as compared with microsomes from wild-type UGT1A424Pro/48Leu-overexpressing cells against TAM and against both thetransandcisisomers of 4-OH-TAM. A significantly (P< 0.02) lowerKmvalue (~1.6-fold to 1.8-fold) was observed for both 4-OH-TAM isomers, while a near-significant (P= 0.053) decrease inKmwas observed for TAM for the UGT1A424Pro/48Valvariant as compared with wild-type UGT1A4. TheVmax/Kmratio for the UGT1A424Pro/48Valvariant was significantly (P≤ 0.005) higher than that observed for the wild-type UGT1A4 isoform for both thetransandcisisomers of 4-OH-TAM after normalization for UGT1A4 expression by western blotting. No significant effect on enzyme kinetics was observed for the UGT1A424Thr/48Leuvariant 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.