Correlation between <b>
                     <i>UDP-Glucuronosyltransferase</i>
                  </b> Genotypes and 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Glucuronidation Phenotype in Human Liver Microsomes

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117

ABSTRACT: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 ϳ 1A8 > UGT1A10 exhibited the highest overall O-glucuronidating activity against trans-4-OH-TAM as determined by V max /K M , with the hepatic enzyme UGT2B7 exhibiting the highest binding affinity and lowest K M (3.7 M). As determined by V max /K M , 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 K M . 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 ϳ 1A8 > UGT2B7 exhibited the highest overall glucuronidating activities as determined by V max /K M for trans-endoxifen, with the extrahepatic enzyme UGT1A10 exhibiting the highest binding affinity and lowest K M (39.9 M). 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.

Section: Methodsmentioning

confidence: 99%

Glucuronidation of Active Tamoxifen Metabolites by the Human UDP Glucuronosyltransferases

Sun¹,

Sharma²,

Dellinger³

et al. 2007

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117

“…4-OH-TAM isomers can be glucuronidated at the N-amino position as well as O-glucuronidated at the 4-hydroxyl position, whereas endoxifen isomers are glucuronidated solely at the 4-hydroxyl position (Sun et al, 2007). The O-Gluc products of trans-4-OH-TAM, cis-4-OH-TAM, trans-endoxifen, and cis-endoxifen were prepared by incubation with pig liver microsomes, which were prepared essentially as described previously for human liver microsomes (Fang and Lazarus, 2004;Wiener et al, 2004b). After preincubation with alamethicin (on ice for 15 min), pig liver microsomes (100 g) were incubated in 50 mM Tris-HCI (pH 7.4), 10 mM MgCI 2 , 4 mM UDP-glucuronic acid with either the trans-4-OH-TAM/cis-4-OH-TAM (70:30) or trans-endoxifen/cis-endoxifen mixtures (concentration of ϳ100 M each) at 37°C for 4 h in a total reaction volume of 200 l. Reactions were terminated by addition of 200 l of cold methanol, mixtures were centrifuged at 16,100g at 4°C for 10 min, and 200 l of supernatant was injected onto the HPLC column (Gemini C 18 250 mm ϫ 4.6 mm, 5 m; Phenomenex).…”

Section: Methodsmentioning

confidence: 99%

Elimination of Antiestrogenic Effects of Active Tamoxifen Metabolites by Glucuronidation

Yan

Sun²,

Sharma

et al. 2007

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TAM and endoxifen isomers exhibited no effect on E 2 -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 E 2 -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.

“…The rate of glucuronidation by UGT1A9-overexpressing cell microsomes was determined essentially as described previously (Fang et al, 2002;Wiener et al, 2004;Al-Zoughool and Talaska, 2005;Dellinger et al, 2006Dellinger et al, , 2007. For glucuronidation rate determinations, substrate concentrations, microsomal protein levels, and incubation times for individual assays were chosen to maximize levels of detection within a linear range of uptake and were similar to established protocols (Fang et al, 2002;Wiener et al, 2004;Dellinger et al, 2007;Sun et al, 2007). For O-glucuronidated substrates, kinetic analysis against 3-OH-B[a]P was performed using UGT1A9-overexpressing cell microsomes (1 g of protein) preincubated with alamethicin (50 g/mg protein) for 10 min on ice.…”

Section: Figmentioning

confidence: 99%

Functional Characterization of Low-Prevalence Missense Polymorphisms in the UDP-Glucuronosyltransferase 1A9 Gene

Olson¹,

Dellinger²,

Zhong³

et al. 2009

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ABSTRACT:The UDP-glucuronosyltransferase (UGT) 1A9 has been shown to play an important role in the detoxification of several carcinogens and clearance of anticancer and pain medications. The goal of the present study was to identify novel polymorphisms in UGT1A9 and characterize their effect on glucuronidation activity. The UGT1A9 gene was analyzed by direct sequencing of buccal cell genomic DNA from 90 healthy subjects. In addition to a previously identified single nucleotide polymorphism (SNP) at codon 33 resulting in an amino acid substitution (Met>Thr), two low-prevalence (<0.02) novel missense SNPs at codons 167 (Val>Ala) and 183 (Cys>Gly) were identified and are present in both white and African-American subjects. Glucuronidation activity assays using HEK293