UGTs For Tamoxifen

NIH Public Access
Author Manuscript
Cancer Res. Author manuscript; available in PMC 2010 March 1.
Published in final edited form as:
NIH-PA Author Manuscript
Cancer Res. 2009 March 1; 69(5): 1892–1900. doi:10.1158/0008-5472.CAN-08-3708.
Functional significance of UDP-glucuronosyltransferase (UGT)

variants in the metabolism of active tamoxifen metabolites
Andrea S. Blevins-Primeau*, Dongxiao Sun*, Gang Chen, Arun K. Sharma, Carla J.

Gallagher, Shantu Amin, and Philip Lazarus
Cancer Control and Population Sciences (A.S.B-P., D.S., G.C., C.J.G., P.L.) and Chemical
Carcinogenesis and Chemoprevention (A.K.S., S.A.) Programs, Penn State Cancer Institute;
Departments of Pharmacology (A.S.B-P., D.S., A.K.S., S.A., P.L.) and Public Health Sciences (G.C.,
C.J.G., P.L.), Penn State University College of Medicine, Hershey, PA 17033
Abstract
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 inter-individual differences in
TAM metabolism, cell lines over-expressing wild-type or variant UGTs were examined for their
activities against TAM metabolites in vitro. For variants of active extra-hepatic UGTs, the
UGT1A8173Ala/277Tyr variant exhibited no detectable glucuronidation activity against the trans
isomers of either 4-OH-TAM or endoxifen. No difference in TAM glucuronidating activity was
observed for the UGT1A8173Gly/277Cys or UGT1A10139Lys variants as compared to their wild-type
counterparts. For active hepatic UGTs, the UGT2B7268Tyr variant exhibited significant (p<0.01) 2-
and 5-fold decreases in activity against the trans isomers of 4-OH-TAM and endoxifen, respectively,
as compared to wild-type UGT2B7268His. 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 Tyr268Tyr genotype
as compared to subjects with the UGT2B7 His268His genotype, with a significant (p<0.01) trend of
decreasing activity against both substrates with increasing numbers of the UGT2B7268His allele.
These results suggest that functional polymorphisms in TAM-metabolizing UGTs, including
UGT2B7 and potentially UGT1A8, may be important in inter-individual variability in TAM
metabolism and response to TAM therapy.
INTRODUCTION
TAM (1-[4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1(Z)-ene)a is a non-steroidal
anti-estrogen that has been commonly used for the treatment and prevention of estrogen-
dependent breast cancer (1–4). Adjuvant TAM treatment increases recurrence-free survival
Corresponding author: Philip Lazarus, Ph.D., Penn State University College of Medicine, Rm. C3739D, MC-H069, 500 University Drive,
Hershey, PA 17033. Fax: (717) 531-0480; Email: E-mail: [email protected].
*Denotes equal contributions to this work.
aAbbreviations: TAM, tamoxifen; 4-OH-TAM, 4-hydroxytamoxifen; endoxifen, 4-hydroxy-N-desmethylTAM; UGT, UDP-
glucuronosyltransferase; SNP, single nucleotide polymorphism; UDPGA, UDP-glucuronic acid; 4-OH-TAM-N+-Gluc, 4-
hydroxytamoxifen quaternary ammonium glucuronide; HLM, human liver microsomes; HPLC, high pressure liquid chromatography;
LC-MS, liquid chromatography-mass spectrometry; UPLC/MS/MS, ultra-performance liquid chromatography attached sequentially to
tandem mass spectrometry; 4-MU, 4-methylumbelliferone; SNP, single nucleotide polymorphism; KM, Michaelis-Menten equilibrium
constant; Vmax, maximal velocity.
Blevins-Primeau et al. Page 2
and overall survival in breast cancer patients with hormone receptor-positive tumors
irrespective of their nodal status, menopausal status or age (5). In addition to its anti-estrogenic
properties, which have been related to symptoms such as hot flashes, vaginal bleeding and
pruritus vulvae (2,6), TAM also has partial estrogen-agonistic effects that may be linked to
reduced risk for ischemic heart disease and osteoporosis (7,8), but may also increase the risk
for endometrial cancer (9,10) and venous thromboembolism (11). Although TAM is generally
well-tolerated, there is significant inter-individual variability in the clinical efficacy as well as
toxicities of TAM. For instance, about 30% of patients acquire TAM resistance and relapse
(12). In addition, the relative risk of endometrial cancer in patients treated with TAM is
estimated to be two- to three-fold that of controls (10,13–15). The mechanisms underlying
variability in response to TAM and to TAM-related toxicities remains obscure. Since there is
compelling evidence that TAM is converted to anti-estrogenic metabolites that are more potent
than TAM itself, altered patterns of metabolism of TAM and/or its active metabolites might
contribute to this inter-individual variability.
TAM is metabolized via cytochrome P450s, primarily CYP2D6 and CYPs 3A4/3A5, into
several metabolites after oral administration including the hydroxylated TAM metabolites, 4-
OH-TAM and endoxifen. Both 4-OH-TAM and endoxifen are abundant in the serum of TAM-
treated patients, with the levels of serum endoxifen approaching 6- to 12-fold that observed
for 4-OH-TAM. Since the trans isomers of both 4-OH-TAM and endoxifen exhibit up to 100-
fold the levels of anti-estrogenic activity as compared to TAM (16–21), it is thought that they
may be the major contributors to TAM’s anti-estrogenic properties. While cis-4-OH-TAM

exhibits anti-estrogenic activity in vitro in the presence of estradiol, it has also been suggested
to possess some estrogen agonist activity (22–24). The trans isomers of 4-OH-TAM and
endoxifen are more abundant than the cis isomers, possibly at a ratio of 70:30, at physiological
pH (25,26).
An important route of elimination and detoxification of TAM and its metabolites is via
glucuronidation. TAM is excreted predominantly through the bile primarily by conjugation to
glucuronic acid (27), with most of the 4-OH-TAM found in the bile of TAM-treated patients
as a glucuronide conjugate (27,28). TAM glucuronides have also been identified in the urine
and serum of TAM-treated patients (27,28), and it has been suggested that glucuronidation
within target tissues like the adipose tissue of the breast may also be important in terms of
TAM metabolism and overall TAM activity (29).
N-glucuronidation occurs for both TAM and 4-OH-TAM while O-glucuronidation occurs for
4-OH-TAM and endoxifen (30,31). In vitro studies have demonstrated that the hepatic
UGT1A4 is the only active enzyme responsible for the N-glucuronidation of TAM and 4-OH-
TAM while UGT2B7 and, to a lesser extent UGT1A1, are the major hepatic enzymes involved
in the O-glucuronidation of the trans isomers of 4-OH-TAM and endoxifen (31). UGT2B7
exhibited higher levels of activity against the trans isomers of 4-OH-TAM and endoxifen; other
hepatic UGTs (including UGTs 1A3, 1A9, 2B15, and 2B17) were significantly more active
against cis TAM metabolites (31). The extra-hepatic UGTs 1A10 and 1A8 are expressed in
target tissues including breast and were also demonstrated to be highly active against isomers
of 4-OH-TAM and endoxifen in vitro (31). While previous studies have demonstrated that the
UGT1A448Val variant exhibits increased N-glucuronidation activity in vitro against 4-OH-
TAM as compared with the wild-type UGT1A448Leu isoform (30), no studies have been
performed examining UGT variants and O-glucuronidation activity against 4-OH-TAM or
endoxifen. The goal of the present study was to examine whether prevalent missense SNPs in
the most active TAM metabolite O-glucuronidating enzymes alters their activity against the
trans isomers of 4-OH-TAM and endoxifen and could therefore potentially play an important
role in patient response to TAM.

MATERIALS AND METHODS

Chemicals and materials
trans-4-OH-TAM (98% pure), UDPGA, alamethicin, β-glucuronidase and bovine serum

albumin were purchased from Sigma-Aldrich (St. Louis, MO). Endoxifen was synthesized in
the Organic Synthesis Facility at the Penn State College of Medicine, with the trans-endoxifen
isomer purified as described previously (31). HPLC-grade ammonium acetate, acetonitrile and
peptide synthesis grade triethylamine were purchased from Fisher Scientific (Pittsburgh, PA)
and were used after filtration. Dulbecco’s modified Eagles medium, Dulbecco’s phosphate-
buffered saline (minus calcium-chloride and magnesium-chloride), fetal bovine serum,
penicillin-streptomycin and geneticin (G418) were purchased from Gibco (Grand Island, NY).
The Platinum® Pfx DNA polymerase and the pcDNA3.1/V5-His-TOPO mammalian
expression vector were obtained from Invitrogen (Carlsbad, CA) while the restriction enzymes
DpnI and StuI were purchased from New England Biolabs (Beverly, MA). The BCA protein
assay kit was purchased from Pierce (Rockford, IL) while the QIAEX® II gel extraction kit
was purchased from Qiagen (Valencia, CA). The human UGT1A western blotting kit and anti-
UGT1A antibody were purchased from Gentest (Woburn, MA). All other chemicals used were
purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise specified.
UGT-over-expressing cell lines

The HEK293 cell lines over-expressing the wild-type UGT1A10139Glu, UGT2B7268His and
UGT1A8173Ala/277Cys isoforms and the UGT1A10139Gly and UGT2B7268Tyr variants used in

this study have been described previously (32–34). The UGT1A8173Gly/277Cys and
UGT1A8173Ala/277Tyr variants were generated by site-directed mutagenesis of the pcDNA3.1/
V5-His-TOPO plasmid expressing wild-type the UGT1A8 gene as previously described (31,
33) using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The primers used to
change UGT1A8 codon 173 from Ala to Gly were: sense, 5′-
TTTAACTTATTTTTTTCGCATTGCAGGAG-3′, and antisense, 5′-
CTCCTGCAATGCGAAAAAAATAAGTTAAA-3′, corresponding to nucleotides +349 to
+377 relative to the translation start site. The primers used to change UGT1A8 codon 277 from
Cys to Tyr were sense, 5′-GTGGTATCAACTACCATCAGGGAAAGCC-3′, and antisense,
5′-GGCTTTCCCTGATGGTAGTTGATACCAC-3′, corresponding to nucleotides +815 to
+843 relative to the translation start site. The underlined base for each primer indicates the
base-pair change. Similar to that described previously for site-directed mutagenesis-generated
UGT variants (31,33), the UGT1A8173Gly/277Cys and UGT1A8173Ala/277Tyr cDNA sequences
were confirmed by dideoxy sequencing prior to transfection by electroporation into the
HEK293 (human embryonic kidney fibroblast) cell line as previously described (31,33). Cells
were grown in Dulbecco’s Modified Eagle’s medium to 80% confluence prior to the
preparation of cell homogenates as previously described (34). Total homogenate protein

concentrations were measured using the BCA protein assay.
UGT protein levels were determined by Western blot analysis for all UGT-over-expressing
cell lines examined in this study as previously described (33). For UGT1A-over-expressing
cells, the UGT1A antibody from Gentest was utilized; for UGT2B-over-expressing cells, a
previously described UGT2B-specific antibody was used (31). Relative UGT protein levels
were expressed as the mean of three independent experiments, and all activity assays were
normalized relative to UGT expression in the respective UGT-over-expressing cell line.
HLM
Normal human liver tissue specimens (n=111) were obtained from the Tissue Procurement
Facility at the H. Lee Moffitt Cancer Center (Tampa, FL) and include 78 liver specimens that
were examined in previous studies (34,35). Microsomes (HLM) were prepared as previously

described (34) and stored at 10–20 mg protein/mL at −80°C. Matching genomic DNA was
prepared from nuclei isolated during the microsomal differential centrifugation preparation
procedure for each specimen using standard phenol chloroform techniques. Microsomal
protein concentrations were measured using the BCA protein assay. Matching total RNA was
obtained for each specimen directly from Moffitt’s Tissue Procurement Facility and was stored
at −80°C.
Glucuronidation assays
The glucuronidation activities of homogenates from HEK293 cell lines over-expressing UGTs
1A8, 1A10 and 2B7 against trans-4-OH-TAM and trans-endoxifen were performed as
previously described (30,31). Briefly, after an initial incubation of cell homogenate protein
(100–1000 μg) or HLM (2.5 μg total protein) with alamethicin (50 μg/mg protein) for 15 min
in an ice bath, glucuronidation reactions were performed in a final reaction volume of 100 μL
(cell homogenates) or 25 μL (HLM) at 37°C in 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 4
mM UDPGA, and 1 to 250 μM trans-4-OH-TAM or 8 to 725 μM trans-endoxifen for cell
homogenate glucuronidation assays, and 4 μM trans-4-OH-TAM or 30 μM trans-endoxifen
for HLM glucuronidation assays. Kinetic analysis of HLM from subjects with varying
UGT2B7 genotypes was performed using 0.5–15 μM trans-4-OH-TAM and 2–60 μM trans-
endoxifen in five HLM specimens from individual subjects from each UGT2B7 genotype
group (15 HLM specimens total). Reactions were terminated by the addition of 100 μl cold
methanol on ice. Mixtures were centrifuged for 10 min at 4°C at 16,100 g prior to the collection
of the supernatant. All glucuronidation assays were performed in triplicate for cell homogenate
assays and duplicate for HLM assays in independent experiments.
TAM metabolites were assessed for cell homogenate glucuronidation assays by HPLC identical
to that described previously (30,31). Assays of TAM metabolite formation in HLM were
analyzed by UPLC/MS/MS using a Waters (Milford, MA) Aquity UPLC consisting of a binary
gradient pump, an autosampler (maintained at 4°C), and a UV detector operated at 254 nm,
attached to a Waters TQD triple quadrupole mass spectrometer that was purchased after the
analysis of UGT-over-expressing cell lines (described above). Similar to that described
previously (26), each sample was injected onto an Aquity UPLC BEH C18 1.7 μM, 2.1X100
mm column (Waters) with the following gradient elution conditions for trans-4-OH-TAM:
starting with 69% buffer A (0.01mol/L NH4Ac, pH 5.0)/31% acetonitrile for 2 min with a
subsequent linear gradient to 75% acetonitrile over 2 min. The gradient elution conditions for
trans-endoxifen, using the same buffers, were as follows: starting with 30% acetonitrile for 4
min and a subsequent linear gradient to 75% acetonitrile for 2 min. The elution flow rate was
0.5 mL/min and 5 μL of the reaction was injected for all assays. Electrospray ionization mass
spectrometry (ESI-MS) daughter scans of 564 and 550 (m+1/z) verified the glucuronides of
trans-4-OH-TAM and trans-endoxifen. The formation of both O- and N+-glucuronides of 4-
OH-TAM and endoxifen were quantified by UPLC based on the ratio of the glucuronide versus
free trans-4-OH-TAM or trans-endoxifen. HLM assays without TAM metabolite were
regularly analyzed as negative controls for glucuronidation activity as previously described
(34).
For 4-MU, glucuronidation assays were performed as described above with an incubation time
of 120 min. HPLC analysis utilized the following gradient program: starting with 98% buffer
A (100 mM NH4Ac, pH 5.0)/2% acetonitrile for 5 min, a linear gradient to 70% acetonitrile
over 17.5 min was performed and then maintained at 70% for 10 min.
UGT Genotyping
Genomic DNA from the 111 liver specimens examined for glucuronidation activity in this
study were used to genotype the UGT2B7 codon 268 (His>Tyr) polymorphism, the

UGT1A1*28 TATAA box polymorphism (see GenBank wild-type accession number

NM_000463 and SNP rs8175347), and the UGT1A4 codon 24 (Pro>Thr) and codon 48
(Leu>Val) polymorphisms. UGT2B7 and UGT1A4 genotypes were determined by direct
sequencing of PCR-amplified PCR products spanning the codon 268 polymorphism for
UGT2B7, and codons 24 and 48 for UGT1A4. The same primers were used for both PCR
amplification and sequencing; UGT2B7: sense, 5′-
CTATAGTGCTTTACTTTGACTTTTGGTTCG-3′ and antisense, 5′-
GCTAGAAAAGCAAAGAAGGGAAAAAATGATTAGTTATATCTGA-3′, corresponding
to nucleotides +642–+670 and +1555–+1597, respectively, relative to the UGT2B7 translation
start site (Genbank accession #NM_001074); UGT1A4: sense, 5′-
GGCTTCTGCTGAGATGGCCAG-3′, and antisense, 5′-
CCTTGAGTGTAGCCCAGCGT-3′, corresponding to nucleotides located −13 to +8 and +277
to +306, respectively, relative to the UGT1A4 translation start site (Genbank accession
#NM_007120). Sequencing was performed using an ABI 3130 Capillary Sequencer at the
Functional Genomics Core Facility at the Penn State College of Medicine.
The UGT1A1*28 polymorphism was genotyped utilizing DNA fragment analysis by capillary
electrophoresis on the ABI 3130 Capillary Sequencer at the Penn State Molecular Genetics
Core Facility using primers and PCR conditions similar to those described previously (36)
using 0.5 μL of a size standard (GeneScan 500 LIZ Size Standard, Applied Biosystems, Foster
City, CA) as a DNA size marker. Informative results were obtained for 105 of the 111 liver
specimens examined in this study.
UGT2B7 codon 268 genotypes were determined primarily by real-time PCR assays using the
TaqMan Drug Metabolism Genotyping Assay C_32449742_20 (Applied Biosystems, Foster
City, CA) in the ABI 7900HT sequence detection system equipped with an autoloader in the
Functional Genomics Core Facility at the Penn State College of Medicine. Forty percent of all
samples within each of the three potential UGT2B7 genotype groups (His268His, His268Tyr
and Tyr268Tyr; as identified by real-time PCR) were further confirmed by standard PCR and
direct sequencing. The same primers were used for both PCR and sequencing: sense, 5′-
CTATAGTGCTTTACTTTGACTTTTGGTTCG-3′, located +642–+670 from the UGT2B7
translation start site; and antisense, 5′-
GCTAGAAAAGCAAAGAAGGGAAAAAATGATTAGTTATATCTGA-3′, located
+1555–+1597 from the UGT2B7 translation start site. For those samples for which real-time
assays of UGT2B7 genotypes were inconclusive (n=13), PCR/direct sequencing analysis was
performed as described above. For eight of these samples, both real-time and direct sequencing
failed to provide informative results, and direct sequencing of UGT2B7 cDNA was then
performed for these samples using matching total RNA as template. Briefly, after reverse
transcription-PCR was performed using standard conditions with 5 μg total liver RNA as
template and oligo(dT) as primer, PCR was performed using 2 μL of cDNA as template and
sense (5′-TGCAGATGCTATTTTTCCCTGTA-3′) and antisense (5′-
GAACCTTTTGTGGGATCTGGGCC-3′) primers located +456 to +478 and +984 to +1006,
respectively, from the UGT2B7 translation start site. Direct sequencing of these RT-PCR-
amplified fragments was then performed using the same primers used for PCR.
UGT2B7 expression analysis

Matching total RNA was available for expression analysis for 99 of the 111 liver specimens
analyzed in this study. Five ug of RNA was used for cDNA synthesis using standard reverse
transcription methods as described above, with 20 ng of cDNA used for expression analysis
using the ABI gene expression kit assay for UGT2B7 (Hs02556232_s1; Applied Biosystems,
Foster City, CA) with the ABI 7900HT detection system equipped with an autoloader in the
Functional Genomics Core Facility at the Penn State College of Medicine. Expression assays

were performed in triplicate with expression normalized relative to the expression levels of the
housekeeping gene PPIA within the same samples.
Statistical analysis
The Student’s t-test (2-sided) was used for comparing kinetic values of glucuronidation
formation for UGT wild-type versus variant over-expressing cell lines, and for HLM
glucuronidation rates stratified by UGT genotypes. The one-way ANOVA trend test was used
to compare HLM glucuronidation rates across multiple UGT genotypes. Kinetic constants were
determined using Graphpad Prism4 software.
RESULTS
Kinetic studies of TAM metabolite glucuronidation by UGT variants
Results from previous studies demonstrated that the hepatic UGT2B7 and the extra-hepatic
UGTs 1A8 and 1A10 exhibited the highest overall glucuronidating activities against trans-4-
OH-TAM and trans-endoxifen (31). Known missense polymorphisms have been identified for
each of these UGTs, including a highly-prevalent SNP at codon 268 of the UGT2B7 gene
(32), a codon 139 SNP in the UGT1A10 gene that is most prevalent in African Americans
(37), and two coding region SNPs that result in amino acid changes of Ala to Gly at codon 173
and Cys to Tyr at codon 277 of the UGT1A8 gene (38). To determine whether any of these
SNPs result in differential activities against the trans isomers of 4-OH-TAM or endoxifen, in
vitro kinetic analysis of HEK293 cells over-expressing the wild-type or variant isoforms of
each of these three UGT enzymes was performed. While the levels of expression of wild-type
versus variant UGT1A10 protein was determined for UGT1A10 over-expressing cell lines in
previous studies (33), semi-quantitative Western blot analysis was performed for the UGT1A8-
and UGT2B7-over-expressing cell lines. As shown in Figure 1, each of the UGT1A8-over-
expressing cell lines developed for the present analysis exhibited similar levels of expression
(8.0, 6.3 and 7.0 ng UGT1A8/μg total protein for the UGT1A8173Ala/277Cys-,
UGT1A8173Gly/277Tyr- and UGT1A8173Ala/277Tyr-over-expressing lines, respectively). As
described previously in other laboratories for the UGT2B7-over-expressing cell lines (32),
UGT2B7268His was consistently expressed at a level that was 3.3-fold that of the
UGT2B7268Tyr variant in the present analysis (results not shown). These values were used for
normalization of UGT1A8 and UGT2B7 levels in their respective UGT-over-expressing cell
lines in in vitro kinetic analysis.
Representative HPLC traces of glucuronidation formation by UGT-over-expressing cell

homogenates are shown in Figure 2. As described previously for cell lines over-expressing
wild-type UGTs 1A8, 1A10 or 2B7 (31), significant levels of O-glucuronidation of the trans
isomers of 4-OH-TAM or endoxifen were observed in the present studies; no N-

glucuronidation of these TAM metabolites was observed for these UGTs. The
UGT1A8173Gly/277Cys variant exhibited no difference in overall glucuronidation activity
(Vmax/KM) against trans-4-OH-TAM and exhibited a small (1.25-fold) but significant (p<
0.05) decrease in overall activity (manifested primarily by a higher KM) against trans-
endoxifen as compared to wild-type UGT1A8173Ala/277Cys (Table 1). In contrast, the
UGT1A8173Ala/277Tyr variant exhibited no detectable glucuronidation against the trans isomers
of either 4-OH-TAM or endoxifen (Table 1). However, the UGT1A8173Ala/277Tyr variant did
exhibit detectable levels of activity against 4-MU (Figure 2, panel D). No difference in overall
glucuronidation activity was observed for the UGT1A10139Lys variant versus wild-type
UGT1A10 against the trans isomers of 4-OH-TAM and endoxifen.
Kinetic analysis demonstrated that significantly higher glucuronidation activities were

observed for the wild-type UGT2B7268His as compared to the UGT2B7268Tyr variant against

the trans isomers of both 4-OH-TAM (p<0.05) and endoxifen (p<0.01; Table 1). This was
manifested by a higher KM (2.4-fold) and a lower Vmax/KM (2.4-fold) for 4-OH-TAM, as well
as a lower Vmax (5.5-fold) and lower Vmax/KM (5.0-fold) for endoxifen.
Glucuronidation activities of HLM against the trans isomers of 4-OH-TAM and endoxifen
stratified by UGT genotypes
To determine the rate of glucuronidation of trans-4-OH-TAM and trans-endoxifen,
glucuronidation assays were performed for 111 HLMs and analyzed by UPLC/MS/MS. The
concentrations of 4-OH-TAM or endoxifen used in the HLM glucuronidation activity assays
was determined after kinetic analysis of three randomly-chosen HLM specimens - the resulting
KM’s were 4 μM and 30 μM for trans-4-OH-TAM and trans-endoxifen, respectively (data not
shown). Using 4 μM trans-4-OH-TAM as substrate in HLM glucuronidation activity assays,
two major putative glucuronide peaks were observed, the TAM-4-O-glucuronide and the 4-
OH-TAM-N+-glucuronide, which exhibited retention times of 1.76 and 3.35 min, respectively,
distinct from free trans-4-OH-TAM which eluted at 3.95 min (Figure 3, panel A). Using 30
μM trans-endoxifen as substrate in HLM glucuronidation activity assays, a single major
putative glucuronide peak was observed at a retention time of 1.95 min which was distinct
from the endoxifen peak eluting at 3.90 min (Figure 3, panel B). All putative glucuronide peaks
were sensitive to treatment with β-glucuronidase (results not shown), eluted at retention times
identical to previously characterized TAM-glucuronide standards (26); results not shown), and
were confirmed by tandem MS (Figure 3, panels C–D; the MS/MS pattern observed for
trans-TAM-4-O-glucuronide and trans-4-OH-TAM-N+-glucuronide are identical). For 4-OH-

TAM glucuronidation assays, a third, smaller peak eluting at a retention time of 2.02 min was
confirmed to be cis-TAM-4-O-glucuronide, likely formed due to spontaneous interconversion
between the trans and cis 4-OH-TAM isomers (34). Similar to that observed in previous studies
for three HLM specimens (31), no endoxifen-N-glucuronide was observed with any of the 111
HLM examined in the present analysis. The mean rate of formation of TAM-4-O-glucuronide,
4-OH-TAM-N+-glucuronide and endoxifen-O-glucuronide in HLM was 141 ± 45, 175 ± 52
and 168 ± 66 pmol•min−1•mg−1, respectively. A 4.5-, 10-, and 17-fold range in glucuronide
formation was observed for TAM-4-O-glucuronide, 4-OH-TAM-N+-glucuronide, and
endoxifen-O- glucuronide, respectively. The range of the ratio of TAM-4-O-glucuronide : 4-
OH-TAM-N+-glucuronide in the HLM samples was 8.0-fold.
As described above, previous studies have indicated that UGT2B7 is the major hepatic enzyme
that performs O-glucuronidation of the trans isomers of both 4-OH-TAM and endoxifen
(31). When stratifying HLM O-glucuronidation activities by UGT2B7 codon 268 genotype,
there was a near-significant (p=0.059) 13% decrease in TAM-4-O-glucuronide formation in
HLM with the UGT2B7 (His268Tyr) genotype and a significant (p<0.001) 28% decrease in
TAM-4-O-glucuronide formation in HLM with the UGT2B7 (Tyr268Tyr) genotype as

compared to HLM with the UGT2B7 (His268His) genotype (Figure 4, panel A). A significant
(p=0.01) 17% decrease in TAM-4-O-glucuronide formation was observed in HLM with the
UGT2B7 His268Tyr genotype versus HLM with the UGT2B7 (Tyr268Tyr) genotype. A
significant trend of decreasing O-glucuronidation of trans-4-OH-TAM was observed in HLM
with increasing numbers of the UGT2B7268Tyr allele (p<0.001).
Similar to that observed for trans-4-OH-TAM, a significant (p=0.002) 27% decrease in O-

glucuronidation of trans-endoxifen was observed in HLM with the UGT2B7 (Tyr268Tyr)
genotype as compared to HLM with the UGT2B7 (His268His) genotype (Figure 4, panel B).
In addition, a significant trend of decreasing O-glucuronidation of trans-endoxifen was
observed in HLM with increasing numbers of the UGT2B7268Tyr allele (p=0.009). The mean
level of UGT2B7 expression was similar in liver specimens within each of the UGT2B7
genotype groups, with specimens with the UGT2B7 (His268His) genotype exhibiting a 4%

higher level of expression than liver specimens with the UGT2B7 (Tyr>Tyr) genotype (data
not shown). In an analysis of five randomly-chosen HLM specimens from subjects from each
UGT2B7 genotype group (ie, 5 HLM per genotype = 15 total HLM specimens), the resulting
KM’s for trans-4-OH-TAM followed a non-significant decreasing trend of 9, 7, and 5 μM,

respectively, for the UGT2B7 His268His, His268Tyr, and Tyr268Tyr genotypes (data not
shown). The resulting KM’s (16 μM) for trans-endoxifen were similar across UGT2B7
genotypes (data not shown).
To determine whether genotypes in other hepatic UGTs shown previously to exhibit activity
against the trans isomers of 4-OH-TAM or endoxifen were similarly linked to altered HLM
glucuronidation phenotype, HLM glucuronidation activities were stratified by UGT1A1 and
UGT1A4 genotypes (30,31). Non-significant decreases in O-glucuronidation activity of 14 and
11% were observed against the trans isomers of 4-OH-TAM and endoxifen, respectively, in
HLM with the UGT1A1 (*28/*28) genotype as compared to HLM with the UGT1A1 (*1/*1)
genotype; this decrease remained non-significant when combining HLM with either the
UGT1A1 (*28/*28) or (*1/*28) genotypes (results not shown). No significant associations
were observed for either the UGT1A4 codons 24 (Pro>Thr) or 48 (Leu>Val) polymorphisms
and HLM N-glucuronidation activity against 4-OH-TAM (results not shown).
Discussion
The present study examines the potential role of UGT polymorphisms on the metabolism of
the trans isomers of 4-OH-TAM and endoxifen, the major active metabolites of tamoxifen.
Several UGTs were previously shown to be active against these metabolites, with UGT2B7
the most active hepatic UGT. As UGT2B7 expression has been detected in a variety of tissues
including liver, the gastrointestinal tract and breast (39–43), variations in UGT2B7 function
or expression could potentially significantly impact individual response to drugs or
chemotherapeutic agents. The data presented in this study demonstrate that O-glucuronidation
of both trans-4-OH-TAM and trans-endoxifen in HLM was significantly associated with
UGT2B7 genotype, with lower activities correlated with increasing numbers of the
UGT2B7268Tyr allele. These data were consistent with the observation that HEK293 cells that
over-expressed the UGT2B7268Tyr variant exhibited lower activity in vitro against both TAM
metabolites as compared to cells over-expressing wild-type UGT2B7268His. These results are
also consistent with a functional role for this polymorphism against other substrates including
tobacco carcinogen metabolites like 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL;
(34)].
Previous studies have observed a SNP located at −161(T>C) relative to the ATG transcriptional
start site in the promoter of UGT2B7 that is in complete linkage disequilibrium with the SNP
at UGT2B7 codon 268 (42). In the present study, UGT2B7 genotype was also not associated
with a change in UGT2B7 expression, as there was less than a 4% difference in expression
levels between HLM with the UGT2B7 (His268His) versus (Tyr268Tyr) genotypes, a pattern
similar to that observed in previous studies (44). While recent studies have indicated that the
intronic SNP IVS1 + 985A>G is associated with altered UGT2B7 expression and the formation
of morphine glucuronide metabolites (44), in an analysis of a subset of the liver specimens
examined in the present study (n=45), specimens containing the IVS1 + 985G variant did not
result in a change in UGT2B7 expression or a change in activity against TAM metabolites as
compared to HLM containing the wild-type IVS1 + 985A (results not shown). These data
suggest that the decrease in O-glucuronidation activity against TAM metabolites in HLM
associated with the UGT2B7 codon 268 polymorphism is indeed due to functional changes
within the UGT2B7 enzyme.

In addition to UGT2B7, polymorphisms in UGTs 1A1 and 1A4, which were previously shown
to be active against TAM, trans--4-OH-TAM and/or trans-endoxifen (30,31), were examined
for their effect on HLM glucuronidation activity. A microsatellite (TA)-repeat polymorphism
present in the TATAA-box of the UGT1A1 promoter has been linked to lower UGT1A1
expression (45,46) and lower activity against a variety of endogenous and exogenous
substrates, including bilirubin (45,47,48), carcinogens such as metabolites of benzo(a)pyrene
(45), and chemotherapeutic agents such as SN-38, the major metabolite of irinotecan (49,50).
The non-significant trend of decreased glucuronidation activity against TAM metabolites that
was observed in HLMs from subjects with one or more UGT1A1*28 alleles is consistent with
previous studies indicating that UGT1A1 exhibits only weak relative activity against these
substrates as compared to UGT2B7 (31) and may therefore play a more minor role in TAM
metabolism.
The N-glucuronidation of TAM and 4-OH-TAM was previously shown to be performed

exclusively by UGT1A4 (30,51). While previous studies also demonstrated that the UGT1A4
codon 48 polymorphism was linked to a small but significant alteration in N-glucuronidation
activity against TAM and 4-OH-TAM in vitro (30), no significant difference was observed in
HLM against trans-4-OH-TAM in the present study. This may be due to the relatively low
number of HLM specimens that were heterozygous (n=13) or homozygous (n=1) for the
UGT1A448Val variant in this study.
The extra-hepatic UGTs 1A10 and 1A8 exhibited the highest levels of activity in vitro against
the trans isomers of 4-OH-TAM and endoxifen in previous studies (31). In the present study,
no effect on TAM metabolite glucuronidation activity was observed in vitro for the
UGT1A10139Lys variant as compared to wild-type UGT1A10139Glu. Similarly, the
UGT1A8173Gly variant exhibited a marginally significant lower overall in vitro
glucuronidating activity against trans-endoxifen as determined by Vmax/KM as compared to
wild-type UGT1A8173Ala; no significant difference was observed for this variant against
trans-4-OH-TAM. This relatively minor effect on TAM metabolite glucuronidating activities
is consistent with the fact that the Ala>Gly amino acid substitution at UGT1A8 codon 173 is
a conservative non-polar amino acid change and with data from previous in vitro metabolic
studies that revealed that UGT1A8173Ala and UGT1A8173Gly exhibit similar catalytic
properties (38,52). Interestingly, the UGT1A8277Tyr variant exhibited no detectable
glucuronidating activity against both trans-4-OH-TAM and trans-endoxifen. This is consistent
with previous data indicating that this variant exhibited dramatically reduced activity towards
other substrates (38,52). While the prevalence of this polymorphism is low in the population
[~2% in Caucasians; (38)], the observation that UGT1A8 is highly active against TAM
metabolites and is well-expressed in the breast (53,54) suggests that, like the UGT2B7 codon
268 polymorphism, the UGT1A8 codon 277 polymorphism could potentially be important in
individual response to TAM.
The results described above are consistent with that observed previously for functional
polymorphisms in the CYP2D6 gene. Decreased levels of endoxifen were observed in the
serum of TAM-treated women after stratifying patients by the CYP2D6 deletion genotype
(55), a decrease also observed when CYP2D6 inhibitors were co-administered with TAM
(19). These data suggest an important role for endoxifen in TAM therapeutic efficacy. The
CYP2D6*4 deletion allele has been associated with time until breast cancer recurrence,
relapse-free survival, disease-free survival, and overall survival in patients treated with TAM
(19,55,56). In addition, patients with the CYP2D6*4 genotype report few, if any, occurrences
of hot flashes (55). Despite a strong correlation between CYP2D6 genotype and serum levels
of endoxifen in patients treated with TAM, large variability in circulating endoxifen levels are
still observed after controlling for CYP2D6 genotype (19,55). The evidence presented in the

present study suggests that inter-individual differences in TAM glucuronidation pathways may
help explain this variability.
In summary, results from this study suggest that genetic variants in UGTs that are highly active
against TAM metabolites significantly alter TAM metabolism in vitro and, potentially, its
elimination in TAM-treated individuals. Similar to that described above for CYP2D6, this
could potentially affect overall patient response to TAM. Additional studies examining the
effect of UGT1A8 and UGT2B7 genotypes on breast microsomal glucuronidation activity
against TAM metabolites, plasma TAM metabolite levels, and overall patient response to TAM
will be required to further examine the role of UGT polymorphisms on the therapeutic efficacy
of TAM.
Acknowledgements
We thank the Functional Genomics and Molecular Biology Core Facilities at the Penn State University College of
Medicine for DNA genotyping, DNA sequencing, and usage of densitometric equipment. These studies were supported
by Public Health Service (PHS) grants R01-DE13158 (National Institute for Dental and Craniofacial Research) from
the National Institutes of Health, Department of Health and Human Services (to P. Lazarus).
Grant support: USPHS grants R01-DE13158 (National Institute for Dental and Craniofacial Research) from the NIH,
Department of Health and Human Services (P. Lazarus), and a formula grant under the Pennsylvania Department of
Health’s Health Research Formula Funding Program, State of PA, Act 2001-77–part of the PA Tabacco Settlement
Legislation (P. Lazarus).
References
1. Fisher B, Costantino JP, Wickerham DL, et al. Tamoxifen for prevention of breast cancer: report of
the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 1998;90:1371–
88. [PubMed: 9747868]
2. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998;339:1609–18. [PubMed:
9828250]
3. Cuzick J, Powles T, Veronesi U, et al. Overview of the main outcomes in breast-cancer prevention
trials. Lancet 2003;361:296–300. [PubMed: 12559863]
4. Howell A, Howell SJ, Evans DG. New approaches to the endocrine prevention and treatment of breast
cancer. Cancer Chemother Pharmacol 2003;52 Suppl 1:S39–44. [PubMed: 12819938]
5. Ghosh SS, Sappal BS, Kalpana GV, Lee SW, Chowdhury JR, Chowdhury NR. Homodimerization of
human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its
functional implications. J Biol Chem 2001;276:42108–15. [PubMed: 11546782]
6. Nechushtan H, Peretz T. Tamoxifen and breast cancer. Harefuah 2002;141:718–20. 61, 60. [PubMed:
12222137]
7. McDonald CC, Stewart HJ. Fatal myocardial infarction in the Scottish adjuvant tamoxifen trial. The
Scottish Breast Cancer Committee. Bmj 1991;303:435–7. [PubMed: 1912833]

8. Rutqvist LE, Mattsson A. Cardiac and thromboembolic morbidity among postmenopausal women with
early-stage breast cancer in a randomized trial of adjuvant tamoxifen. The Stockholm Breast Cancer
Study Group. J Natl Cancer Inst 1993;85:1398–406. [PubMed: 8350363]
9. van Leeuwen FE, Benraadt J, Coebergh JW, et al. Risk of endometrial cancer after tamoxifen treatment
of breast cancer. Lancet 1994;343:448–52. [PubMed: 7905955]
10. Rutqvist LE, Johansson H, Signomklao T, Johansson U, Fornander T, Wilking N. Adjuvant tamoxifen
therapy for early stage breast cancer and second primary malignancies. Stockholm Breast Cancer
Study Group. J Natl Cancer Inst 1995;87:645–51. [PubMed: 7752269]
11. Meier CR, Jick H. Tamoxifen and risk of idiopathic venous thromboembolism. Br J Clin Pharmacol
1998;45:608–12. [PubMed: 9663819]
12. Early Breast Cancer Trialists’ Collaborative Group. Tamoxifen for early breast cancer: an overview
of the randomised trials. Lancet 1998;351:1451–67. [PubMed: 9605801]

13. Fisher B, Costantino JP, Redmond CK, Fisher ER, Wickerham DL, Cronin WM. Endometrial cancer
in tamoxifen-treated breast cancer patients: findings from the National Surgical Adjuvant Breast and
Bowel Project (NSABP) B-14. J Natl Cancer Inst 1994;86:527–37. [PubMed: 8133536]
14. Bernstein L, Deapen D, Cerhan JR, et al. Tamoxifen therapy for breast cancer and endometrial cancer
risk. J Natl Cancer Inst 1999;91:1654–62. [PubMed: 10511593]
15. Bergman L, Beelen ML, Gallee MP, Hollema H, Benraadt J, van Leeuwen FE. Risk and prognosis
of endometrial cancer after tamoxifen for breast cancer. Comprehensive Cancer Centres’ ALERT
Group. Assessment of Liver and Endometrial cancer Risk following Tamoxifen. Lancet
2000;356:881–7. [PubMed: 11036892]
16. Jordan VC, Collins MM, Rowsby L, Prestwich G. A monohydroxylated metabolite of tamoxifen with
potent antioestrogenic activity. J Endocrinol 1977;75:305–16. [PubMed: 591813]
17. Furr BJ, Jordan VC. The pharmacology and clinical uses of tamoxifen. Pharmacol Ther 1984;25:127–
205. [PubMed: 6438654]
18. Katzenellenbogen BS, Norman MJ, Eckert RL, Peltz SW, Mangel WF. Bioactivities, estrogen
receptor interactions, and plasminogen activator-inducing activities of tamoxifen and hydroxy-
tamoxifen isomers in MCF-7 human breast cancer cells. Cancer Res 1984;44:112–9. [PubMed:
6537799]
19. Borges S, Desta Z, Li L, et al. Quantitative effect of CYP2D6 genotype and inhibitors on tamoxifen
metabolism: implication for optimization of breast cancer treatment. Clin Pharmacol Ther
2006;80:61–74. [PubMed: 16815318]
20. Johnson MD, Zuo H, Lee KH, et al. Pharmacological characterization of 4-hydroxy-N-desmethyl
tamoxifen, a novel active metabolite of tamoxifen. Breast Cancer Res Treat 2004;85:151–9.
[PubMed: 15111773]
21. Lim YC, Desta Z, Flockhart DA, Skaar TC. Endoxifen (4-hydroxy-N-desmethyltamoxifen) has anti-
estrogenic effects in breast cancer cells with potency similar to 4-hydroxy-tamoxifen. Cancer
Chemother Pharmacol 2005;55:471–8. [PubMed: 15685451]
22. Buckley MM, Goa KL. Tamoxifen. A reappraisal of its pharmacodynamic and pharmacokinetic
properties, and therapeutic use. Drugs 1989;37:451–90. [PubMed: 2661195]
23. Chu W, Fyles A, Sellers EM, et al. Association between CYP3A4 genotype and risk of endometrial
cancer following tamoxifen use. Carcinogenesis 2007;28:2139–42. [PubMed: 17434921]
24. Robertson DW, Katzenellenbogen JA, Long DJ, Rorke EA, Katzenellenbogen BS. Tamoxifen
antiestrogens. A comparison of the activity, pharmacokinetics, and metabolic activation of the cis
and trans isomers of tamoxifen. J Steroid Biochem 1982;16:1–13. [PubMed: 7062732]
25. Malet C, Spritzer P, Cumins C, Guillaumin D, Mauvais-Jarvis P, Kuttenn F. Effect of 4-
hydroxytamoxifen isomers on growth and ultrastructural aspects of normal human breast epithelial
(HBE) cells in culture. J Steroid Biochem Mol Biol 2002;82:289–96. [PubMed: 12589935]
26. Zheng Y, Sun D, Sharma AK, Chen G, Amin S, Lazarus P. Elimination of antiestrogenic effects of
active tamoxifen metabolites by glucuronidation. Drug Metab Dispos 2007;35:1942–8. [PubMed:
17620345]
27. Lien EA, Solheim E, Lea OA, Lundgren S, Kvinnsland S, Ueland PM. Distribution of 4-hydroxy-N-
desmethyltamoxifen and other tamoxifen metabolites in human biological fluids during tamoxifen
treatment. Cancer Res 1989;49:2175–83. [PubMed: 2702659]
28. Lien EA, Solheim E, Kvinnsland S, Ueland PM. Identification of 4-hydroxy-N-desmethyltamoxifen
as a metabolite of tamoxifen in human bile. Cancer Res 1988;48:2304–8. [PubMed: 3349495]
29. Nowell SA, Ahn J, Rae JM, et al. Association of genetic variation in tamoxifen-metabolizing enzymes
with overall survival and recurrence of disease in breast cancer patients. Breast Cancer Res Treat
2005;91:249–58. [PubMed: 15952058]
30. Sun D, Chen G, Dellinger RW, Duncan K, Fang JL, Lazarus P. Characterization of tamoxifen and 4-
hydroxytamoxifen glucuronidation by human UGT1A4 variants. Breast Cancer Res 2006;8:R50.
[PubMed: 16884532]
31. Sun D, Sharma AK, Dellinger RW, et al. Glucuronidation of active tamoxifen metabolites by the
human UDP-glucuronosyltransferases (UGTs). Drug Metab Dispos. 2007

32. Coffman BL, King CD, Rios GR, Tephly TR. The glucuronidation of opioids, other xenobiotics, and
androgens by human UGT2B7Y(268) and UGT2B7H(268). Drug Metab Dispos 1998;26:73–7.
[PubMed: 9443856]
33. Dellinger RW, Fang JL, Chen G, Weinberg R, Lazarus P. Importance of UDP-glucuronosyltransferase
1A10 (UGT1A10) in the detoxification of polycyclic aromatic hydrocarbons: decreased
glucuronidative activity of the UGT1A10139Lys isoform. Drug Metab Dispos 2006;34:943–9.
[PubMed: 16510539]
34. Wiener D, Fang JL, Dossett N, Lazarus P. Correlation between UDP-glucuronosyltransferase
genotypes and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronidation phenotype in
human liver microsomes. Cancer Res 2004;64:1190–6. [PubMed: 14871856]
35. Lazarus P, Zheng Y, Aaron Runkle E, Muscat JE, Wiener D. Genotype-phenotype correlation between
the polymorphic UGT2B17 gene deletion and NNAL glucuronidation activities in human liver
microsomes. Pharmacogenet Genomics 2005;15:769–78. [PubMed: 16220109]
36. Huang CK, Dulau A, Su-Rick CJ, Pan Q. Validation of rapid polymerase chain reaction-based
detection of all length polymorphisms in the UGT 1A1 gene promoter. Diagn Mol Pathol 2007;16:50–
3. [PubMed: 17471158]
37. Elahi A, Bendaly J, Zheng Z, et al. Detection of UGT1A10 polymorphisms and their association with
orolaryngeal carcinoma risk. Cancer 2003;98:872–80. [PubMed: 12910533]
38. Huang YH, Galijatovic A, Nguyen N, et al. Identification and functional characterization of UDP-
glucuronosyltransferases UGT1A8*1, UGT1A8*2 and UGT1A8*3. Pharmacogenetics
2002;12:287–97. [PubMed: 12042666]
39. Nakamura A, Nakajima M, Yamanaka H, Fujiwara R, Yokoi T. Expression of UGT1A and UGT2B
mRNA in human normal tissues and various cell lines. Drug Metab Dispos. 2008
40. Ren Q, Murphy SE, Zheng Z, Lazarus P. O-Glucuronidation of the lung carcinogen 4-
(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL) by human UDP-glucuronosyltransferases
2B7 and 1A9. Drug Metab Dispos 2000;28:1352–60. [PubMed: 11038164]
41. Strassburg CP, Strassburg A, Nguyen N, Li Q, Manns MP, Tukey RH. Regulation and function of
family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus.
Biochem J 1999;338( Pt 2):489–98. [PubMed: 10024527]
42. Turgeon D, Carrier JS, Levesque E, Hum DW, Belanger A. Relative enzymatic activity, protein
stability, and tissue distribution of human steroid-metabolizing UGT2B subfamily members.
Endocrinology 2001;142:778–87. [PubMed: 11159850]
43. Zheng Z, Fang JL, Lazarus P. Glucuronidation: an important mechanism for detoxification of benzo
[a]pyrene metabolites in aerodigestive tract tissues. Drug Metab Dispos 2002;30:397–403. [PubMed:
11901093]
44. Innocenti F, Liu W, Fackenthal D, et al. Single nucleotide polymorphism discovery and functional
assessment of variation in the UDP-glucuronosyltransferase 2B7 gene. Pharmacogenet Genomics
2008;18:683–97. [PubMed: 18622261]
45. Fang JL, Lazarus P. Correlation between the UDP-glucuronosyltransferase (UGT1A1) TATAA box
polymorphism and carcinogen detoxification phenotype: significantly decreased glucuronidating
activity against benzo(a)pyrene-7,8-dihydrodiol(-) in liver microsomes from subjects with the

UGT1A1*28 variant. Cancer Epidemiol Biomarkers Prev 2004;13:102–9. [PubMed: 14744740]
46. Hsieh TY, Shiu TY, Huang SM, et al. Molecular pathogenesis of Gilbert’s syndrome: decreased
TATA-binding protein binding affinity of UGT1A1 gene promoter. Pharmacogenet Genomics
2007;17:229–36. [PubMed: 17496722]
47. Bosma PJ, Chowdhury JR, Bakker C, et al. The genetic basis of the reduced expression of bilirubin
UDP-glucuronosyltransferase 1 in Gilbert’s syndrome. N Engl J Med 1995;333:1171–5. [PubMed:
7565971]
48. Monaghan G, Ryan M, Seddon R, Hume R, Burchell B. Genetic variation in bilirubin UPD-
glucuronosyltransferase gene promoter and Gilbert’s syndrome. Lancet 1996;347:578–81. [PubMed:
8596320]
49. Iyer L, King CD, Whitington PF, et al. Genetic predisposition to the metabolism of irinotecan
(CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation

of its active metabolite (SN-38) in human liver microsomes. J Clin Invest 1998;101:847–54.
[PubMed: 9466980]
50. Iyer L, Hall D, Das S, et al. Phenotype-genotype correlation of in vitro SN-38 (active metabolite of
irinotecan) and bilirubin glucuronidation in human liver tissue with UGT1A1 promoter
polymorphism. Clin Pharmacol Ther 1999;65:576–82. [PubMed: 10340924]
51. Ogura K, Ishikawa Y, Kaku T, et al. Quaternary ammonium-linked glucuronidation of trans-4-
hydroxytamoxifen, an active metabolite of tamoxifen, by human liver microsomes and UDP-
glucuronosyltransferase 1A4. Biochem Pharmacol 2006;71:1358–69. [PubMed: 16480962]
52. Bernard O, Tojcic J, Journault K, Perusse L, Guillemette C. Influence of nonsynonymous
polymorphisms of UGT1A8 and UGT2B7 metabolizing enzymes on the formation of phenolic and
acyl glucuronides of mycophenolic acid. Drug Metab Dispos 2006;34:1539–45. [PubMed:
16790554]
53. Lehmann L, Wagner J. Gene expression of 17beta-estradiol-metabolizing isozymes: comparison of
normal human mammary gland to normal human liver and to cultured human breast adenocarcinoma
cells. Adv Exp Med Biol 2008;617:617–24. [PubMed: 18497089]
54. Thibaudeau J, Lepine J, Tojcic J, et al. Characterization of common UGT1A8, UGT1A9, and UGT2B7
variants with different capacities to inactivate mutagenic 4-hydroxylated metabolites of estradiol and
estrone. Cancer Res 2006;66:125–33. [PubMed: 16397224]
55. Goetz MP, Rae JM, Suman VJ, et al. Pharmacogenetics of tamoxifen biotransformation is associated
with clinical outcomes of efficacy and hot flashes. J Clin Oncol 2005;23:9312–8. [PubMed:
16361630]
56. Goetz MP, Knox SK, Suman VJ, et al. The impact of cytochrome P450 2D6 metabolism in women
receiving adjuvant tamoxifen. Breast Cancer Res Treat 2007;101:113–21. [PubMed: 17115111]

Figure 1. Western blot analysis of UGT 1A8 variants from UGT-overexpressing cell lines
Equal amounts of protein (30 μg) from untransfected HEK293 cells, and from
UGT1A8173Ala/277Cys-, UGT1A8173Gly/277Cys-, and UGT1A8173Ala/277Tyr-over-expressing
HEK293 cells, were loaded into each lane for Western blot analysis. β-Actin was examined as
an internal control for protein loading for each lane, and 200 or 300 ng of UGT1A protein
standards (Gentest) were loaded as a gel-loading reference for UGT protein quantification. The
relative ratios of UGT1A8 : β-actin protein expression are from three independent experiments
and their expression relative to the wild-type UGT1A8173Ala/277Cys are indicated below each
lane.

Figure 2. HPLC analyses of over-expressing cell line glucuronidating activities

Shown are HPLC traces of trans-TAM-4-O-glucuronide and trans-endoxifen-O-glucuronide
formation by, (A) UGT1A8173Ala/277Cys-, (B) UGT1A10139Glu-, and (C) UGT2B7268His-over-
expressing HEK293 cells using 500 μg homogenate protein in glucuronidation activity assays
as described in the Materials and Methods. Panel D shows 4-MU-O-glucuronide formation
using 1 mg UGT1A8173Ala/277Tyr-over-expressing cell homogenate. Over-expressing cell
homogenates were incubated at 37°C for 60 or 120 min for assays with TAM metabolites or
4-MU, respectively, prior to analysis by HPLC as described in the Materials and Methods.
Peak 1, trans-4-TAM-O-glucuronide; peak 2, trans-4-OH-TAM; peak 3, trans-endoxifen-O-
glucuronide; peak 4, trans-endoxifen; peak 5, 4-MU-O-glucuronide; peak 6, 4-MU.

Figure 3. UPLC analyses of HLM glucuronidating activities against TAM metabolites

Representative UPLC traces of glucuronidation assays incubated with HLM (40 μg) and either
(A) trans-4-OH-TAM (4 μM) or (B) trans-endoxifen (30 μM). Tandem MS/MS traces of
putative glucuronide peaks from HLM glucuronidation activity assays are shown in panels
(C) for trans-TAM-4-O-glucuronide, and (D) for trans-endoxifen-O-glucuronide. Peak 1,
trans-TAM-4-O-glucuronide; peak 2, cis-TAM-4-O-glucuronide; peak 3, trans-4-OH-TAM-
N+-glucuronide; peak 5, trans-endoxifen-O-glucuronide; peak 6, trans-endoxifen.

Figure 4. Analysis of glucuronidation activities against trans-4-OH-TAM and trans-endoxifen in

HLM stratified by UGT2B7 genotypes
Glucuronidation activity assays were performed and 4-OH-TAM- and endoxifen-glucuronides
were separated by UPLC as described in the Materials and Methods. Shown are the rate of O-
glucuronide formation from, (A) trans-4-OH-TAM, stratified by UGT2B7 codon 268
genotypes; and (B) trans-endoxifen, stratified by UGT2B7 codon 268 genotypes. Comparative
analysis was performed using the wild-type UGT2B7268His as the referent; * denotes p< 0.001;
** denotes p < 0.002, and error bars represent standard error.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Table 1
Kinetic analyses of O-glucuronidation of the trans isomers of 4-OH-TAM and endoxifen by UGT variants.a
trans-4-OH-TAM trans-endoxifen
Vmax KM Vmax/KM Vmax Km Vmax/KM

UGT Variant (pmol•min−1•μg−1)b (μM) (μl•min−1•μg−1)b (pmol•min−1•μg−1)b (μM) (μl•min−1•μg−1)b
UGT2B7268His 0.55 ± 0.18 3.7 ± 0.6 0.15 ± 0.03 3.0 ± 0.44 101 ± 17 0.030 ± 0.004
Blevins-Primeau et al.
UGT2B7268Tyr 0.54 ± 0.09* 8.7 ± 0.8** 0.062 ± 0.01** 0.55 ± 0.01** 101 ± 15 0.006 ± 0.001**
UGT1A10139Glu 4.7 ± 0.3 96 ± 8 0.049 ± 0.006 5.7 ± 0.7 40 ± 3 0.14 ± 0.005
UGT1A10139Lys 2.1 ± 0.2** 52 ± 6** 0.040 ± 0.006 1.9 ± 0.2** 13 ± 2** 0.14 ± 0.004
173Ala/277Cys
UGT1A8 2.3 ± 0.1 23 ± 2 0.10 ± 0.02 5.4 ± 0.2 98 ± 9 0.060 ± 0.004
173Gly/277Cys
UGT1A8 5.4 ± 0.2** 43 ± 7** 0.13 ± 0.03 5.9 ± 0.4 135 ± 26 0.040 ± 0.005*
UGT1A8173Ala/277Tyr no detectable activity no detectable activity
a
All data are the mean ± S.D. based on three independent experiments. Homogenates from cells over-expressing UGT1A8173Ala/277Tyr exhibited no detectable activity against trans-4-OH-TAM and
trans-endoxifen.
b
Data are expressed per μg UGT protein as determined by Western blot analysis.
*
p ≤ 0.05;
**
p<0.01.

Page 18

UGTs For Tamoxifen

Uploaded by

Copyright:

Available Formats

UGTs For Tamoxifen

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

UGTs For Tamoxifen

Uploaded by

Copyright:

Available Formats

NIH Public Access

Cancer Res. 2009 March 1; 69(5): 1892–1900. doi:10.1158/0008-5472.CAN-08-3708.

Functional significance of UDP-glucuronosyltransferase (UGT)

Andrea S. Blevins-Primeau*, Dongxiao Sun*, Gang Chen, Arun K. Sharma, Carla J.

metabolism and response to TAM therapy.

may be the major contributors to TAM’s anti-estrogenic properties. While cis-4-OH-TAM

Cancer Res. Author manuscript; available in PMC 2010 March 1.

MATERIALS AND METHODS

trans-4-OH-TAM (98% pure), UDPGA, alamethicin, β-glucuronidase and bovine serum

UGT-over-expressing cell lines

UGT1A8173Ala/277Cys isoforms and the UGT1A10139Gly and UGT2B7268Tyr variants used in

preparation of cell homogenates as previously described (34). Total homogenate protein

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Cancer Res. Author manuscript; available in PMC 2010 March 1.

UGT1A1*28 TATAA box polymorphism (see GenBank wild-type accession number

specimens examined in this study.

UGT2B7 expression analysis

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Representative HPLC traces of glucuronidation formation by UGT-over-expressing cell

isomers of 4-OH-TAM or endoxifen were observed in the present studies; no N-

Kinetic analysis demonstrated that significantly higher glucuronidation activities were

Cancer Res. Author manuscript; available in PMC 2010 March 1.

trans-TAM-4-O-glucuronide and trans-4-OH-TAM-N+-glucuronide are identical). For 4-OH-

TAM-4-O-glucuronide formation in HLM with the UGT2B7 (Tyr268Tyr) genotype as

Similar to that observed for trans-4-OH-TAM, a significant (p=0.002) 27% decrease in O-

Cancer Res. Author manuscript; available in PMC 2010 March 1.

KM’s for trans-4-OH-TAM followed a non-significant decreasing trend of 9, 7, and 5 μM,

Cancer Res. Author manuscript; available in PMC 2010 March 1.

The N-glucuronidation of TAM and 4-OH-TAM was previously shown to be performed

individual response to TAM.

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Scottish Breast Cancer Committee. Bmj 1991;303:435–7. [PubMed: 1912833]

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Cancer Res. Author manuscript; available in PMC 2010 March 1.

activity against benzo(a)pyrene-7,8-dihydrodiol(-) in liver microsomes from subjects with the

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Figure 2. HPLC analyses of over-expressing cell line glucuronidating activities

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Figure 3. UPLC analyses of HLM glucuronidating activities against TAM metabolites

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Figure 4. Analysis of glucuronidation activities against trans-4-OH-TAM and trans-endoxifen in

Cancer Res. Author manuscript; available in PMC 2010 March 1.

Vmax KM Vmax/KM Vmax Km Vmax/KM

Cancer Res. Author manuscript; available in PMC 2010 March 1.

You might also like

Andrea S. Blevins-Primeau, Dongxiao Sun, Gang Chen, Arun K. Sharma, Carla J.