Dongxiao Sun

Glucuronidation of Active Tamoxifen Metabolites by the Human UDP Glucuronosyltransferases

Tamoxifen (TAM) is an antiestrogen that has been widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism and elimination of TAM and its major active metabolites 4-hydroxytamoxifen (4-OH-TAM) and 4-OH-N-desmethyl-TAM (endoxifen; 4-hydroxy-N-desmethyl-tamoxifen) is via glucuronidation. Although limited studies have been performed characterizing the glucuronidation of 4-OH-TAM, no studies have been performed on endoxifen. In the present study, characterization of the glucuronidating activities of human UDP glucuronosyltransferases (UGTs) against isomers of 4-OH-TAM and endoxifen was performed. Using homogenates of individual UGT-overexpressing cell lines, UGTs 2B7 ∼ 1A8 > UGT1A10 exhibited the highest overall O-glucuronidating activity against trans-4-OH-TAM as determined by Vmax/KM, with the hepatic enzyme UGT2B7 exhibiting the highest binding affinity and lowest KM (3.7 μM). As determined by Vmax/KM, UGT1A10 exhibited the highest overall O-glucuronidating activity against cis-4-OH-TAM, 10-fold higher than the next-most active UGTs 1A1 and 2B7, but with UGT1A7 exhibiting the lowest KM. Although both N- and O-glucuronidation occurred for 4-OH-TAM in human liver microsomes, only O-glucuronidating activity was observed for endoxifen; no endoxifen-N-glucuronidation was observed for any UGT tested. UGTs 1A10 ∼ 1A8 > UGT2B7 exhibited the highest overall glucuronidating activities as determined by Vmax/KM for trans-endoxifen, with the extrahepatic enzyme UGT1A10 exhibiting the highest binding affinity and lowest KM (39.9 μM). Similar to that observed for cis-4-OH-TAM, UGT1A10 also exhibited the highest activity for cis-endoxifen. These data suggest that several UGTs, including UGTs 1A10, 2B7, and 1A8 play an important role in the metabolism of 4-OH-TAM and endoxifen.

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

IntroductionTamoxifen (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.MethodsHPLC 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/48Leu and UGT1A424Pro/48Val variants 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.ResultsMicrosomes from wild-type UGT1A424Pro/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 Km 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 UGT1A424Pro/48Val-overexpressing cell line as compared with microsomes from wild-type UGT1A424Pro/48Leu-overexpressing cells against TAM and against both the trans and cis isomers of 4-OH-TAM. A significantly (P < 0.02) lower Km 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 Km was observed for TAM for the UGT1A424Pro/48Val variant as compared with wild-type UGT1A4. The Vmax/Km ratio for the UGT1A424Pro/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 UGT1A424Thr/48Leu variant against either isomer of 4-OH-TAM or with TAM.ConclusionThese 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.

Functional Significance of UDP-Glucuronosyltransferase Variants in the Metabolism of Active Tamoxifen Metabolites

Tamoxifen (TAM) is a selective estrogen receptor modulator widely used in the prevention and treatment of breast cancer. A major mode of metabolism of the major active metabolites of TAM, 4-OH-TAM and endoxifen, is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To examine whether polymorphisms in the UGT enzymes responsible for the glucuronidation of active TAM metabolites play an important role in interindividual differences in TAM metabolism, cell lines overexpressing wild-type or variant UGTs were examined for their activities against TAM metabolites in vitro. For variants of active extrahepatic UGTs, the UGT1A8(173Ala/277Tyr) variant exhibited no detectable glucuronidation activity against the trans isomers of either 4-OH-TAM or endoxifen. Little or no difference in TAM glucuronidating activity was observed for the UGT1A8(173Gly/277Cys) or UGT1A10(139Lys) variants compared with their wild-type counterparts. For active hepatic UGTs, the UGT2B7(268Tyr) variant exhibited significant (P < 0.01) 2- and 5-fold decreases in activity against the trans isomers of 4-OH-TAM and endoxifen, respectively, compared with wild-type UGT2B7(268His). In studies of 111 human liver microsomal specimens, the rate of O-glucuronidation against trans-4-OH-TAM and trans-endoxifen was 28% (P < 0.001) and 27% (P = 0.002) lower, respectively, in individuals homozygous for the UGT2B7 Tyr(268)Tyr genotype compared with subjects with the UGT2B7 His(268)His genotype, with a significant (P < 0.01) trend of decreasing activity against both substrates with increasing numbers of the UGT2B7(268His) allele. These results suggest that functional polymorphisms in TAM-metabolizing UGTs, including UGT2B7 and potentially UGT1A8, may be important in interindividual variability in TAM metabolism and response to TAM therapy.

Glucuronidation of Tobacco-Specific Nitrosamines by UGT2B10

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important tobacco-specific nitrosamine (TSNA) in the etiology of tobacco-related cancers, and N-glucuronidation is an important mechanism of NNAL detoxification. In the present study, an analysis of the UDP-glucuronosyltransferases (UGTs) responsible for the N-glucuronidation of the TSNAs N′-nitrosonornicotine, N′-nitrosoanabasine, and N′-nitrosoanatabine was performed. Using human embryonic kidney 293 cells overexpressing UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, only UGT1A4 and UGT2B10 exhibited N-glucuronidating activity against these TSNAs. The KMs for UGT2B10 were 15 to 22-fold lower than those of UGT1A4 against the three TSNAs and were similar to those observed for microsomes prepared from human liver specimens. The overall activity of UGT2B10 was 3.6 to 27-fold higher than UGT1A4 against the three TSNAs as determined by Vmax/KM after normalization by levels of UGT2B10 versus UGT1A4 mRNA. Similarly high levels of activity were also observed for UGT2B10 against a fourth TSNA, NNAL, exhibiting a 6.3-fold lower KM and 3-fold higher normalized Vmax/KM than that observed for UGT1A4. Real-time polymerase chain reaction analysis showed that UGT2B10 was expressed at a level that, on average, was 26% higher than that observed for UGT1A4 in a screening of normal liver tissue specimens from 20 individual subjects. These data suggest that UGT2B10 is likely the most active UGT isoform in human liver for the N-glucuronidation of TSNAs.

Characterization of UGTs Active against SAHA and Association between SAHA Glucuronidation Activity Phenotype with UGT Genotype

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor used in the treatment of cutaneous T-cell lymphoma and in clinical trials for treatment of multiple other cancers. A major mode of SAHA metabolism is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To characterize the UGTs active against SAHA, homogenates from HEK293 cell lines overexpressing UGT wild-type or variant UGT were used. The hepatic UGTs 2B17 and 1A9 and the extrahepatic UGTs 1A8 and 1A10 exhibited the highest overall activity against SAHA as determined by V(max)/K(M) (16+/-6.5, 7.1+/-2.2, 33+/-6.3, and 24+/-2.4 nL x min(-1) x microg UGT protein(-1), respectively), with UGT2B17 exhibiting the lowest K(M) (300 micromol/L) against SAHA of any UGT in vitro. Whereas the UGT1A8p.Ala173Gly variant exhibited a 3-fold (P<0.005) decrease in glucuronidation activity against SAHA compared with wild-type UGT1A8, the UGT1A8p.Cys277Tyr variant exhibited no detectable glucuronidation activity; a similar lack of detectable glucuronidation activity was observed for the UGT1A10p.Gly139Lys variant. To analyze the effects of the UGT2B17 gene deletion variant (UGT2B17*2) on SAHA glucuronidation phenotype, human liver microsomes (HLM) were analyzed for glucuronidation activity against SAHA and compared with UGT2B17 genotype. HLM from subjects homozygous for UGT2B17*2 exhibited a 45% (P<0.01) decrease in glucuronidation activity and a 75% (P<0.002) increase in K(M) compared with HLMs from subjects homozygous for the wild-type UGT2B17*1 allele. Overall, these results suggest that several UGTs play an important role in the metabolism of SAHA and that UGT2B17-null individuals could potentially exhibit altered SAHA clearance rates with differences in overall response.

Characterization of 17-dihydroexemestane glucuronidation: potential role of the UGT2B17 deletion in exemestane pharmacogenetics

Objective Exemestane is a third-generation aromatase inhibitor used in the treatment of breast cancer in postmenopausal women. Reduction to form 17-dihydroexemestane and subsequent glucuronidation to exemestane-17-O-glucuronide is a major pathway for exemestane metabolism. The goal of this study was to analyze 17-dihydroexemestane anti-aromatase activity, characterize the 17-dihydroexemestane glucuronidation pathway, and determine whether the functional polymorphisms in active UGTs could play a role in altered 17-dihydroexemestane glucuronidation. Methods Homogenates from a HEK293 aromatase-overexpressing cell line (HEK293-aro) were used to examine exemestane versus 17-dihydroexemestane anti-aromatase activities. UGT-overexpressing cell lines and a panel (n=110) of human liver microsome (HLM) were screened for glucuronidation activity against 17-dihydroexemestane. UGT2B17 genotyping and liver mRNA expression were performed by real-time PCR. Results The inhibition of estrone formation from androst-4-ene-3,17-dione in HEK293-aro cell homogenates was similar for 17-dihydroexemestane (IC50=2.3±0.83 μmol/l) and exemestane (IC50=1.4±0.42 μmol/l). UGTs 2B17 and 1A4 were high-expression hepatic UGTs that exhibited activity against 17-dihydroexemestane, with UGT2B17 exhibiting a 17-fold higher Vmax/KM than UGT1A4. The rate of exemestane-17-O-glucuronide formation was shown to be significantly (P<0.001) decreased (14-fold) in HLMs exhibiting the UGT2B17(*2/*2) deletion genotype versus wild-type UGT2B17(*1/*1) HLMs; a 36-fold lower Vmax/KM (P=0.023) was observed in UGT2B17(*2/*2) versus UGT2B17(*1/*1) HLMs. A significant (P<0.0001, R2=0.72) correlation was observed between HLM exemestane-17-O-glucuronide formation and liver UGT2B17 expression. Conclusion These data suggest that 17-dihydroexemestane is an active metabolite of exemestane and that the UGT2B17 deletion polymorphism could play an important role in determining levels of excretion of 17-dihydroexemestane and overall exemestane metabolism.

Elimination of antiestrogenic effects of active tamoxifen metabolites by glucuronidation.

1-[4-(2-Dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1-(Z)-ene (tamoxifen, TAM) is a nonsteroidal antiestrogen that has been commonly used for the prevention and treatment of estrogen receptor-positive breast cancer. TAM is extensively metabolized into several primary active metabolites including 4-hydroxy-TAM (4-OH-TAM) and endoxifen. Glucuronidation is the major phase II metabolic pathway important in their excretion. Whereas high antiestrogenic activity has been reported for both 4-OH-TAM and endoxifen, studies examining the effect of glucuronide conjugation of these metabolites have not previously been performed. In the present study, the antiestrogenic activities of glucuronidated TAM metabolites were determined by examining their effect on the induction of the estrogen-responsive progesterone receptor (PGR) gene. 17β-Estradiol (E2)-mediated PGR gene expression in MCF-7 cells was determined by real-time reverse transcriptase-polymerase chain reaction for each TAM metabolite isomer. E2 (1 × 10-10 M) induction of PGR mRNA was 6-fold after a 12-h incubation; only unconjugated TAM metabolites inhibited this effect. A virtually identical dose-dependent inhibition of E2-induced PGR gene expression was found for both the trans- and cis-isomers of 4-OH-TAM and endoxifen, with maximal inhibition attained at 1 × 10-6 M of TAM metabolite. The glucuronide conjugates of all 4-OH-TAM and endoxifen isomers exhibited no effect on E2-mediated induction of PGR expression at all concentrations of TAM metabolite examined in this study. These data indicate that isomers of both 4-OH-TAM and endoxifen exhibit roughly equipotent antiestrogenic effects on E2-induced gene expression and that glucuronide conjugates of the same metabolites effectively negate this activity. This result may have important implications in terms of both whole-body and target tissue-specific glucuronidation pathways and individual responses to TAM therapy and cancer prevention.

Sex Differences in UDP-Glucuronosyltransferase 2B17 Expression and Activity

UDP-glucuronosyltransferases (UGTs) are enzymes involved in the metabolism of steroid hormones, carcinogens, cancer chemotherapy agents, and addictive agents from cigarettes. Because the UGT2B family of genes has been linked to hormonal regulation in human cell lines in vitro, we hypothesized that there may be sex-related differences in the expression and activity of these genes in human tissues. To evaluate whether there are sex differences in UGT2B expression and activity, we examined 103 normal human liver specimens for UGT2B expression by real-time polymerase chain reaction and in vitro glucuronidation activities in human liver microsomes (HLM). Men exhibited an approximately 4-fold higher level of expression of UGT2B17 than women (p = 0.007). Consistent with the increased expression of UGT2B17 in men, HLM from men also had a higher level of glucuronidation activity than HLM from women against three UGT2B17 substrates: 3-fold higher for 17-dihydroexemestane (p = 0.002); 3-fold higher for 3-hydroxycotinine (p < 0.001); and 1.5-fold higher for suberoylanilide hydroxamic acid (p = 0.014). When we stratified by UGT2B17 gene deletion genotype, similar patterns were observed for all three substrates, with HLM from men with the UGT2B17 (+/+) or (+/0) genotypes exhibiting significantly higher levels of glucuronidation activity against all three substrates compared with HLM from women. These data suggest that men have a higher amount of UGT2B17 glucuronidation activity then women. This sex difference in UGT2B17 gene expression and corresponding protein activity could potentially result in different levels of carcinogen detoxification or drug elimination in men versus women.

Potential Role of UGT Pharmacogenetics in Cancer Treatment and Prevention: Focus on Tamoxifen

Tamoxifen (TAM) is a selective estrogen receptor modulator that is widely used in the prevention and treatment of estrogen receptor‐positive (ER+) breast cancer. Its use has significantly contributed to a decline in breast cancer mortality, since breast cancer patients treated with TAM for 5 years exhibit a 30–50% reduction in both the rate of disease recurrence after 10 years of patient follow‐up and occurrence of contralateral breast cancer. However, in patients treated with TAM there is substantial interindividual variability in the development of resistance to TAM therapy, and in the incidence of TAM‐induced adverse events, including deep vein thrombosis, hot flashes, and the development of endometrial cancer. This article will focus on the UDP glucuronosyltransferases, a family of metabolizing enzymes that are responsible for the deactivation and clearance of TAM and TAM metabolites, and how interindividual differences in these enzymes may play a role in patient response to TAM.

Identification of a prevalent functional missense polymorphism in the UGT2B10 gene and its association with UGT2B10 inactivation against tobacco-specific nitrosamines.

Objective To study the potential association between UDP-glucuronosyltransferase (UGT)2B10 genotypes and [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol] NNAL-N-glucuronidation activity in human liver microsomes (HLM) and to identify potential functional polymorphisms. Methods A total of 77 subjects were genotyped for three UGT2B10 tagging single nucleotide polymorphisms NNAL-N-glucuronidation activity in HLM was determined by high-pressure liquid chromatography and analyzed by UGT2B10 haplotypes. Results Four common UGT2B10 haplotypes (termed A through D) were identified. Haplotype C was found to be significantly (P<0.001) associated with lower NNAL-N-glucuronidation in HLM. A 1.8-fold and 12-fold reduction in NNAL-N-glucuronidation levels and a 1.7-fold and 11-fold reduction in the ratio of NNAL-N-Gluc: NNAL-O-Gluc, were observed in HLM from subjects with one and two copies of UGT2B10 haplotype C, respectively. A novel polymorphism resulting in an aspartic acid to tyrosine amino acid change at codon 67 of the UGT2B10 complementary DNA was identified exclusively in subjects with a haplotype C. Unlike the high activity observed in microsomes from HEK293 cells over expressing the wild-type UGT2B1067Asp variant, microsomes from HEK293 cells over expressing the UGT2B1067Tyr variant exhibited minimal glucuronide formation activity against NNAL or other tobacco-specific nitrosamines tested in vitro. Conclusions The UGT2B1067Tyr variant corresponding to the UGT2B10 haplotype C is a functional single nucleotide polymorphism that may be responsible for inter individual variation in NNAL-N-glucuronidation activity and may increase susceptibility to smoking-related cancers.