Potential beneficial metabolic interactions between tamoxifen and isoflavones via cytochrome P450-mediated pathways in female rat liver microsomes

Article abstract of DOI:10.1023/B:PHAM.0000048202.92930.61

Purpose. This study aims to evaluate a cytochrome P450-based tamoxifen-isoflavone interaction and to determine the mechanisms responsible for inhibitory effects of isoflavones (e.g., genistein) on the formation of α-hydroxytamoxifen. Methods. Metabolism studies were performed in vitro using female rat liver microsomes. The effects of genistein and an isoflavone mixture on tamoxifen metabolism and the inhibition mechanism were determined using standard kinetic analysis, preincubation, and selective chemical inhibitors of P450. Results. Metabolism of tamoxifen was saturable with K _m values of 4.9 ± 0.6, 14.6 ± 2.2, 25 ± 5.9 μM and V_max values of 34.7 ± 1.4, 297.5 ± 19.2, 1867 ± 231 pmol min^-1 mg^-1 for α-hydroxylation, N-desmethylation, and N-oxidation, respectively. Genistein (25 μM) inhibited α-hydroxylation at 2.5 μM tamoxifen by 64% (p < 0.001) but did not affect the 4-hydroxylation, N-desmethylation, and N-oxidation. A combination of three (genistein, daidzein, and glycitein) to five isoflavones (plus biochanin A and formononetin) inhibited tamoxifen α-hydroxylation to a greater extent but did not decrease the formation of identified metabolites. The inhibition on α-hydroxylation by genistein was mixed-typed with a K_{i ,} value of 10.6 μM. Studies using selective chemical inhibitors showed that tamoxifen α-hydroxylation was mainly mediated by rat CYP1A2 and CYP3A1/2 and that genistein 3'-hydroxylation was mainly mediated by rat CYP1A2, CYP2C6 and CYP2D1. Conclusions. Genistein and its isoflavone analogs have the potential to decrease side effects of tamoxifen through metabolic interactions that inhibit the formation of α-hydroxytamoxifen via inhibition of CYP1A2.

Full text of DOI:10.1023/B:PHAM.0000048202.92930.61

Pharmaceutical Research, Vol. 21, No. 11, November 2004 (© 2004)
Research Paper
be one of the likely reasons for endometrial cancer (6), but
tissue specific proliferative effects of tamoxifen and its me-
tabolites may also be the causes (1,7). A minor metabolite of
tamoxifen (Scheme 1), ␣-hydroxytamoxifen, and its sulfate
are thought to be responsible for DNA adducts formation
(8,9). In contrast, major metabolites of tamoxifen such as
4-hydroxytamoxifen and N-desmethyltamoxifen (Scheme 1)
are at least as active as tamoxifen but are not known to cause
cancer (6,10).
Potential Beneficial Metabolic
Interactions Between Tamoxifen and
Isoflavones via Cytochrome
P450–Mediated Pathways in Female
Rat Liver Microsomes
Tamoxifen undergoes extensive phase I metabolism to
form numerous metabolites (see Scheme 1 for major ones) in
vivo (11–13) and in vitro (14–16). Tamoxifen metabolism is
mainly mediated via P450-catalyzed biotransformation pro-
cesses. Tamoxifen ␣-hydroxylation is primarily catalyzed by
CYP3A in human hepatic microsomes (16), whereas 4-hy-
droxy, N-desmethyl, and N-oxide metabolites of tamoxifen
are mainly formed by the action of CYP2D6, CYP3A4, and
flavin-containing monooxygenase (FMO), respectively
(14,17).
Jun Chen,1 Steven C. Halls,2 Joshua F. Alfaro,2
Zhaohui Zhou,2 and Ming Hu1,3
Received January 17, 2004; accepted July 14, 2004
Purpose. This study aims to evaluate a cytochrome P450–based
tamoxifen-isoflavone interaction and to determine the mechanisms
responsible for inhibitory effects of isoflavones (e.g., genistein) on the
formation of ␣-hydroxytamoxifen.
Genistein, an isoflavone, is the main component of soy
isoflavones that may exert a variety of health benefits. The
claimed benefits include reduction of cancer risk, relief of
postmenopausal symptoms, and maintenance of cardiovascu-
lar health (18–21). Genistein undergoes extensive phase I
(CYP mediated) and phase II (UDP-glucuronosyltransferases
and sulfotransferases mediated) metabolisms in intestine and
liver, resulting in low bioavailability (22–27). Several oxidized
metabolites of genistein have been isolated from human
plasma (24,26,28). Total plasma concentrations of isoflavones
can reach low micromolar range (1–5 ␮M), after a consump-
tion of soy rich food or soy isoflavone supplement (22–24).
Postmenopausal women are increasing their consump-
tion of isoflavone-containing products, particularly as the re-
sult of failed hormone replacement therapy (HRT) trial. For
breast cancer survivors, the need for an alternative is more
urgent. As a consequence, more than 50% of them use alter-
native medicine for HRT (29), and isoflavones is one of their
favorable choices (30–32). Breast cancer survivors may have a
higher chance of using isoflavones as HRT because tamoxifen
increases the incidence of hot flashes (1).
Methods. Metabolism studies were performed in vitro using female
rat liver microsomes. The effects of genistein and an isoflavone mix-
ture on tamoxifen metabolism and the inhibition mechanism were
determined using standard kinetic analysis, preincubation, and selec-
tive chemical inhibitors of P450.
Results. Metabolism of tamoxifen was saturable with Km values of 4.9
0.6, 14.6 2.2, 25 5.9 ␮M and Vmax values of 34.7 1.4, 297.5
19.2, 1867
231 pmol min−1 mg−1 for ␣-hydroxylation, N-
desmethylation, and N-oxidation, respectively. Genistein (25 ␮M)
inhibited ␣-hydroxylation at 2.5 ␮M tamoxifen by 64% (p < 0.001)
but did not affect the 4-hydroxylation, N-desmethylation, and N-
oxidation. A combination of three (genistein, daidzein, and glycitein)
to five isoflavones (plus biochanin A and formononetin) inhibited
tamoxifen ␣-hydroxylation to a greater extent but did not decrease
the formation of identified metabolites. The inhibition on ␣-hydrox-
ylation by genistein was mixed-typed with a Ki, value of 10.6 ␮M.
Studies using selective chemical inhibitors showed that tamoxifen
␣-hydroxylation was mainly mediated by rat CYP1A2 and CYP3A1/2
and that genistein 3Ј-hydroxylation was mainly mediated by rat
CYP1A2, CYP2C6 and CYP2D1.
Conclusions. Genistein and its isoflavone analogs have the potential
to decrease side effects of tamoxifen through metabolic interactions
that inhibit the formation of ␣-hydroxytamoxifen via inhibition of
CYP1A2.
KEY WORDS: cytochrome P450; drug interaction; genistein; rat
liver microsomes; tamoxifen.
INTRODUCTION
Tamoxifen, the most prescribed drug for cancer treat-
ment, is also used as a chemopreventive agent for women with
a high risk of developing breast cancer (1). Additionally,
tamoxifen can treat other hormone-related cancers such as
ovarian (2) and refractory prostate cancers (3). However,
tamoxifen increases the incidence of endometrial cancer in
women (1,4,5) and causes liver cancer in rats (6). DNA ad-
duct formation with tamoxifen metabolites is hypothesized to
1 Department of Pharmaceutical Sciences, College of Pharmacy,
Washington State University, Pullman, Washington 99164, USA.
2 Department of Chemistry, Washington State University, Pullman,
Washington 99164, USA.
3 To whom correspondence should be addressed. (e-mail: minghu@
wsu.edu)
Scheme 1. Tamoxifen and some of its metabolites in vivo.
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0724-8741/04/1100-2095/0 © 2004 Springer Science+Business Media, Inc.

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Chen et al.
A combination of tamoxifen and isoflavones may be de- the Bio-Rad protein assay (Bio-Rad laboratories, Hercules,
sirable because they have synergistic antiproliferative and cy- CA, USA) using bovine serum albumin as the standard.
totoxic actions against breast cancer (33,34). However, stud-
ies concerning their metabolic interactions have not been re-
ported, and whether genistein would favorably or adversely
affect the metabolism of tamoxifen is unknown. Hence, the
main objectives of this study were to 1) evaluate the phase I
metabolism of tamoxifen in female rat liver microsomes, 2)
investigate the influence of genistein and its analogs on the
formation of active or toxic metabolites of tamoxifen, 3) ex-
plore the mechanisms of their possible interactions, and 4)
provide useful mechanistic information describing which P450
isoforms may be involved in potential metabolic interactions
between tamoxifen and isoflavones.
Microsomal Metabolism Experiments
Microsomes were incubated at 37°C with a NADPH re-
generating system and different concentrations of a substrate
with or without a potential inhibitor at a selected concentra-
tion for 20 min in a 100 mM pH 7.4 potassium phosphate
buffer as described previously (25). The reaction was termi-
nated at the end of an incubation period, and 6-hydroxytest-
esterone (2.5 nmol, used as the internal standard) was added.
Tamoxifen and its metabolites were extracted with 8 ml meth-
ylene chloride, where was then evaporated, and the dried
samples were reconstituted in 50% (v/v) methanol for HPLC
analysis. When tamoxifen samples were processed in the pres-
ence of isoflavones, 0.5 ml of 1 M NaOH was added to each
sample thereby ensuring that isoflavones and their metabo-
lites would not be extracted into methylene chloride, since
preliminary studies showed that some of the isoflavones and/
or their metabolites interfered with the quantification of
tamoxifen metabolites (not shown). In genistein metabolism
experiments, the experimental conditions were the same as
described previously (25).
MATERIALS AND METHODS
Materials
Tamoxifen, 4-hydroxytamoxifen, NADP, glucose-6-
phosphate sodium, glucose-6-phosphate dehydrogenase, and
P450 chemical inhibitors or substrates (␣-naphthoflavone, fu-
rafylline, sulfaphenazole, quinidine, tranylcypromine, lovas-
tatin, indomethacin, ketoconazole, troleandomycin, and quer-
cetin) were bought from Sigma Chemical Co. (St. Louis, MO,
USA). SK&F525 was obtained from SmithKline Laboratories
(Philadelphia, PA, USA). Tamoxifen metabolite standards:
␣-hydroxy and N-desmethyl tamoxifen were kindly provided
by Dr. Fredric Beland of FDA (Little Rock, AK, USA) and
Dr. Judy Bolton of University of Illinois (Chicago, IL, USA).
Tamoxifen-N-oxide was synthesized (see Appendix). Genis-
tein, daidzein, glycitein, biochanin A, and formononetin were
purchased from Indofine Chemicals (Wayne, NJ, USA). 3Ј-
Hydroxygenistein was synthesized as described (25). HPLC
grade acetonitrile and methylene chloride were purchased
from Fisher Scientific (Pittsburgh, PA, USA). Other materi-
als were used as received.
HPLC Analysis
The metabolic samples were examined by HPLC using
an Agilent 1090 system, running ChemStation software,
which consisted of a quaternary pump, a photodiode array
detector and an autosampler (Wilmington, DE, USA). Ex-
tracted metabolites were separated using a C18 column
(Aqua 5 µm, 150 × 4.6 mm; Phenomenex, Torrance, CA,
USA) at a flow rate of 1 ml/min. Tamoxifen and its metabo-
lites were monitored at 244 and 210 nm and eluted with a
gradient method (0–18 min, mobile phase A 25–43%, linear
gradient; 18–24 min, 43% mobile phase A; 24–28 min, mobile
phase A 43–49%, linear gradient; 28–38 min, mobile phase A
49–69%, linear gradient; 38–41 min, 25% mobile phase A)
using mobile phase A (100% acetonitrile) and mobile phase B
(pH 3.0 potassium phosphate buffer 50 mM). A re-
equilibrium time of 5 min was allowed between injections.
Under this condition, the retention time for ␣-hydroxy-
tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen,
tamoxifen-N-oxide, and tamoxifen were 12.7, 19.4, 27.9, 34.0,
and 29.2 min, respectively. The internal standard 6-hydroxy-
testosterone had a retention time of 7.1 min (Fig. 1A).
Rat Liver Microsomes Preparation
Female rat liver microsomes (FRLM) were prepared
from adult female Sprague-Dawley rats (200∼250 g) using a
procedure adopted from the literature with minor modifica-
tion (35). Briefly, eight fresh rat livers were harvested from
euthanized rats using a procedure approved by Washington
State University’s Institutional Animal Care and Uses Com-
mittee. The livers were perfused with and washed with ice-
cold saline, weighed and minced. Minced livers were homog-
enized using a motorized homogenizer (4 strokes) in ice-cold
homogenization buffer (50 mM potassium phosphate, 250
mM sucrose, 1 mM EDTA, pH 7.4) and centrifuged at 7700 ×
Identification of Tamoxifen Metabolites by Tandem
Mass Spectrometry
Dried samples were dissolved in 50% methanol and ap-
g for 15 min at 4°C. The supernatant collected was then cen- plied to the same chromatographic conditions described
trifuged again at 18,500 × g for 15 min at 4°C. After the pellet above for HPLC analysis of metabolites, with the following
was discarded, the supernatant was centrifuged again at modifications: the mobile phase contained 1% formic acid
85,600 × g for 1 h at 4°C to yield microsomal pellets. The rather than a phosphate buffer and an Agilent 1100 HPLC
microsomes were resuspended in washing buffer (10 mM po- system (Waldbronn, Germany) with binary pump, autosam-
tassium phosphate, 0.1 mM EDTA, 150 mM KCl, pH 7.4), pler, and UV detector. The effluent was introduced into an
and re-pelleted by centrifugation at 85,600 × g for 1 h at 4°C API 4000 mass spectrometer (Applied Biosystems, Foster
to yield microsomes. The microsomal pellets were then resus- City, CA, USA) using turbo ion spray. Ionization and frag-
pended in 250 mM sucrose, aliquoted into vials (0.5 ml per mentation parameters (collision energy 30 V) were optimized
vial) and stored at −80°C until use. The concentration of mi- using a tamoxifen standard infused into a constant flow (1
crosomal protein (normally 25–35 mg/ml) was determined by ml/min) of 1% (v/v) formic acid in H2O: acetonitrile (3:1 v/v).

Metabolic Interaction Between Tamoxifen and Isoflavones
2097
Fig. 2 HPLC chromatogram of genistein metabolism in female rat
liver microsomes (FRLM). Method was described under “Micro-
somal Metabolism Experiment.” Microsomes were incubated with 25
␮M genistein for 45 min at 37°C in the presence of a NADPH re-
generating system. Genistein metabolites were extracted with meth-
ylene chloride, and solvent was evaporated. Dried samples were re-
constituted by 50% (v/v) methanol and analyzed by HPLC. 3Ј-
Hydroxygenistein was the major new peak identified by its retention
time in comparison with a synthesized chemical standard. The com-
pound 7-hydroxycoumarin (10 nmol) was used as the internal stan-
dard.
oxide formation were obtained at different tamoxifen concen-
trations (C) of 1, 2, 4, 8, 12, 20, and 40 µM. The apparent Km
(Michaelis constant) and Vmax (maximum reaction rate) val-
ues were estimated using a packaged nonlinear regression
analysis method (SigmaPlot, SPSS Science, Chicago, IL,
USA) and the Michaelis-Menten model:
Fig. 1. HPLC chromatogram of tamoxifen metabolism in female rat
liver microsomes (FRLM) (panel A) and tandem mass spectra of
tamoxifen metabolites (panel B). Method used to generate these
chromatograms was described under “Microsomal Metabolism Ex-
periment.” Briefly, microsomes were incubated with 10 ␮M tamoxi-
fen for 20 min at 37°C in the presence of a NADPH regenerating
system. Tamoxifen metabolites were extracted with methylene chlo-
ride, and then dried. Dried samples were reconstituted by 50% (v/v)
methanol/water and analyzed by HPLC. There were 10 new peaks
Vmax × C
Km + C
V =
(1)
Analysis of Inhibition of Tamoxifen Metabolism
by Genistein
Four concentrations (C) of tamoxifen (1.25, 2.5, 5.0, 10
␮M) and four concentrations (I) of genistein (0, 12.5, 18.75, 25
from the HPLC chromatogram and they were labeled as ␣-OH-tam ␮M) were used to determine the mode of inhibition. A slope
(␣-hydroxytamoxifen), 4-OH-tam (4-hydroxytamoxifen), N-dm-tam
(N-desmethyltamoxifen), tam-N-oxide (tamoxifen-N-Oxide), and un-
known metabolites M1-M6. We used 6-hydroxytestesterone (2.5
nmol) as the internal standard, which was added prior to extraction.
Panel B presents the tandem mass spectra of standard ␣-hydroxyl,
4-hydroxyl, N-desmethyl, and N-oxide of tamoxifen. Insets show the
mass spectra of the parent ion used to generate the product ion
spectra.
was obtained from the Lineweaver-Burk plot (36) for each of
the genistein concentrations. The apparent Ki value (the equi-
librium dissociation constant for enzyme–inhibitor complex)
was obtained from the x-intercept of a plot of slope vs. in-
hibitor concentrations using the following equation (36):
Km
1
Km
Slope =
×
+
× I
(2)
ͩ ͪ
Vmax Ki
Vmax
The Ki values were calculated by linear regression using Mi-
The effluent was monitored for a total MS and product (MS/
MS) of the most intense parent ions in both positive and
negative mode.
crosoft Excel program.
Statistical Analysis
One-way ANOVA or unpaired Student’s t test was used
to analyze the data. The prior level of significance was set at
5% or p < 0.05.
Identification of Genistein and 3؅-Hydroxygenistein
These two compounds were monitored at 254 and 220
nm, and eluted with gradient method as described (25), gen-
erating the HPLC chromatogram shown in Fig. 2.
RESULTS
Kinetic Analysis of Tamoxifen Metabolism
Preliminary Studies
The initial rates (V) of ␣-hydroxytamoxifen, 4-hy-
droxytamoxifen, N-desmethyltamoxifen, and tamoxifen-N-
Effects of time of incubation and amounts of microsomes
used in incubation were determined to optimize tamoxifen

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Chen et al.
microsomal metabolism experiments. The results indicated
complex, which prevented determination of its kinetic param-
that an incubation time of 20 min and a microsomal concen- eters using a simple Michaelis-Menten model (Fig. 3B).
tration of 0.15 mg/ml are the best for the present study and Eadie-Hoffstee plots generated suggested the dominance of
were used subsequently unless otherwise specified. A micro- more than one isoform in the formation of all the metabolites
somal concentration of 0.4 mg/ml was occasionally used to except N-desmethyltamoxifen, which appeared to have one
maintain analytical sensitivity in inhibition studies, but more dominant form.
microsomes were not used since higher concentration of pro-
tein did not always lead to higher metabolism (37).
Effect of Genistein and Its Analogs on Tamoxifen
Metabolite Formation
Identification of Tamoxifen Metabolites
To determine possible metabolic interactions between
genistein and tamoxifen, we measured the formation rates of
four known metabolites of tamoxifen (2.5 µM) in the presence
of increasing concentrations of genistein (Fig. 4A). The re-
sults indicated that formation rate of ␣-hydroxytamoxifen de-
creased (maximum ס 64%) when the genistein concentration
increased (maximum ס 50 ␮M). The inhibition was concen-
tration-dependent, and reached the maximum at 25 ␮M. In
contrast, increasing genistein concentration did not affect (p >
0.1) the formation rates of 4-hydroxy (p > 0.05), N-desmethyl
(p > 0.1), and N-oxide (p > 0.1) metabolites of tamoxifen.
We also determined the effect of several isoflavone com-
binations since popular soy and herbal supplements always
contain a mixture of these isoflavones. The results indicated
that a mixture of three soy isoflavones (genistein, daidzein,
and glycitein at 5 and 10 ␮M each) inhibited ␣-hy-
droxytamoxifen formation by 36% (p < 0.001) and 46% (p <
0.001), respectively (Fig. 4B). A mix of five isoflavones (genis-
tein, daidzein, glycitein, biochanin A, and formononetin at 5
and 10 ␮M each), which are contained in popular red clover
extracts, inhibited ␣-hydroxytamoxifen formation by more
than 60% (Fig. 4B). Similar to the effects of genistein, for-
mation rates of active tamoxifen metabolites such as 4-hy-
droxytamoxifen and tamoxifen-N-oxide were not altered.
We also determined the effect of these isoflavones on the
formation of 6-hydroxy-testosterone, a typical CYP3A-
catalyzed reaction. The results indicated that 200 ␮M of genis-
tein, formononetin, daidzein, and prunetin did not inhibit the
metabolism of testosterone (20 ␮M) to 6-hydroxy-
testosterone (not shown).
We identified known tamoxifen metabolites for the pur-
pose of showing similarities and differences between metabo-
lism in rat and human microsomes. Tamoxifen metabolites
found in FRLM incubation were determined to be similar to
those identified previously in humans and rats (14,38,39). At
an initial tamoxifen concentration of 10 ␮M, more than 10
new metabolites peaks were found in the HPLC chromato-
gram (Fig. 1A). The known metabolites of tamoxifen were
labeled as ␣-hydroxytamoxifen (m/z ס 388.3, [M+H]+), 4-hy-
d r o x y t a m o x i f e n ( m / z
ס
3 8 8 . 3 , [ M + H ] + ) , N -
desmethyltamoxifen (m/z ס 358.3, [M+H]+) and tamoxifen-
N-oxide (m/z ס 388.3, [M+H]+), which were identified by
their retention times in comparison with authentic standards
and confirmed by LC/MS/MS (Fig. 1B). In addition to the
above four metabolites, several other metabolites were de-
tected by LC/MS/MS. However, specific assignments for
these metabolites were not made, as metabolite identification
was not the focus of the current study. Formation rates of
␣-hydroxy, 4-hydroxy, N-desmethyl, and N-oxide metabolites
of tamoxifen were 22.9 0.8, 80.8 3, 126 10, and 559 33.4
pmol min−1 mg−1 of microsomal protein, respectively.
Tamoxifen-N-oxide formation rate was the most rapid,
whereas ␣-hydroxytamoxifen formation was the slowest
among the identified metabolites.
Identification of Genistein Metabolites
The compound 3Ј-hydroxygenistein was identified as the
major phase I metabolite in FRLM incubation (Fig. 2), using
an authentic synthetic standard. The formation rate of 3Ј-
hydroxygenistein (@25 ␮M genistein) was 84.5
3.4 pmol
min−1 mg protein−1. The formation of 3Ј-hydroxygenistein
increased linearly with time (p < 0.001) for up to an hour, and
the formation rate also increased linearly with microsome
amounts for up to 0.9 mg/ml (data not shown). Therefore,
subsequent studies of genistein metabolism used 0.4 mg/ml
microsomal protein and a 45 min incubation time.
Mode of Inhibition of ␣-Hydroxytamoxifen Formation
by Genistein
To reveal the type of metabolism involved, the data were
graphed using a classic Lineweaver-Burk plot (36). This was
determined by using four concentrations of tamoxifen and
four concentrations of genistein. The results indicated that
formation rate of ␣-hydroxytamoxifen decreased as genistein
concentration increased (Fig. 5A). The Lineweaver-Burk
plots revealed that the lines did not intercept at the x- or
Determination of Kinetic Parameters of
Tamoxifen Metabolism
We examined formation rates of four known metabolites y-axis, suggesting that the inhibition was mix-typed or par-
of tamoxifen as a function of tamoxifen concentration (1–40 tially competitive (36). The apparent Ki was 10.6 ␮M accord-
µM) (Fig. 3), in order to determine kinetic descriptors of ing to linear regression.
CYP-catalyzed metabolism. The results indicated that the
rates were dependent upon tamoxifen concentration with ap-
parent Km values of 4.9 0.6, 14.6 2.2, 25 5.9 ␮M, and
apparent Vmax values of 34.7 1.4, 298 19, 1867 231 pmol
min−1 mg protein−1 for ␣-hydroxy, N-desmethyl, and N-oxide
Absence of Mechanism-based Inhibition of
␣-Hydroxytamoxifen Formation by Genistein
Mechanism-based inhibition often displayed time-
metabolites of tamoxifen (Figs. 3A, 3C, and 3D), respectively dependence as the result of enzyme inactivation by an inhibi-
(Table I). The relationship between the formation rate of tor. Therefore, formation rates of ␣-hydroxytamoxifen (10
4-hydroxytamoxifen and tamoxifen concentration was more and 20 µM) were determined in microsomes preincubated

Metabolic Interaction Between Tamoxifen and Isoflavones
2099
Fig. 3 Kinetic characterization of tamoxifen metabolism catalyzed by female rat liver microsomes using
conventional (i.e., rate vs. concentration) plots and eadie-hoffstee plots. Formation rates of ␣-hydroxy
(A), 4-hydroxy (B), N-desmethyl (C), and N-oxide (D) metabolites of tamoxifen were determined as
described under “Materials and Methods.” Each point is the average of three determinations (labeled
as “Investigated”) and error bars are standard deviations of the mean. The solid curve (labeled as
“Estimated”) in Fig.3 were fitted using Km and Vmax values listed in Table I.
with genistein (25 ␮M) for different length of time. The re- Effects of Various Prototypical Inhibitors of Cytochrome
sults indicated that preincubation with genistein for an in- P450 Isoforms on Tamoxifen Metabolism
creasing length of time (zero to 30 min) did not significantly
(p > 0.2) increase the extent of its inhibitory effect on the
formation of ␣-hydroxytamoxifen.
Prototypical selective inhibitors were used to determine
which CYP isoform(s) contributes to the metabolism of

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Chen et al.
Table I. Enzyme Kinetic Parameters of Tamoxifen Metabolism in
Female Rat Liver Microsomes
Apparent
parameter
␣-hydroxy- N-desmethyl- Tamoxifen-
tamoxifen
tamoxifen
N-oxide
Km (␮M)
Vmax (pmol min−1 mg−1
Vmax/Km (␮l min−1 mg−1 a
4.9 0.6
34.7 1.4
6.6
14.6 2.2
298 19
20
25 5.9
1867 231
75
)
)
a This ratio is commonly known as “intrinsic clearance.” It is the rate
constant at a substrate concentration that is substantially less than
the Km value.
tamoxifen. In order to get the maximum possible inhibition
for each of the isoforms, the final inhibitor concentrations in
the reaction mixtures were usually much higher than reported
Ki values. The concentrations were 50 ␮M for SKF-525A
(general P450 inhibitor) (40,41), diethyldithiocarbamic acid
(CYP2E1 inhibitor) (42,43), troleandomycine (CYP3A2 in-
hibitor) (42), and indomethacin (CYP3A2 inhibitor) (44); 25
␮M for ␣-naphthoflavone (CYP1A2 inhibitor) (41), querce-
tin, sulfaphenazole (CYP2C6 inhibitor) (43,45), lovastatin
(CYP3A substrate in rat) (46), and tranylcypromine
(CYP2C11 inhibitor) (41,47); and 5 ␮M for quinidine
(CYP2D1 inhibitor) (41,45) and 2 ␮M for ketoconazole
(CYP3A2 inhibitor) (43,45).
SKF-525A is a commonly used classic noncompetitive
inhibitor for all cytochrome P450s (40,41). Its use resulted in
89%, 100%, and 53% inhibition of the formation of ␣-hy-
droxy, 4-hydroxy, and N-desmethyl metabolites of tamoxifen,
respectively. However, SKF-525A did not inhibit the forma-
tion of tamoxifen-N-oxide, which was the reason we did not
follow the formation of tamoxifen-N-oxide further.
The formation of ␣-hydroxytamoxifen was inhibited sig-
nificantly (p < 0.05) by ␣-naphthoflavone (67%), lovastatin
(58%), tranylcypromine (55%), quinidine (55%), ketocona-
zole (68%), troleandomycin (38%), and indomethacin (42%),
but was not inhibited by sulfaphenazole and diethyldithiocar-
bamic acid (Table II). The extent of inhibition by quercetin
was not obtained because of an unexpected interference to
the measurement of ␣-hydroxytamoxifen by HPLC.
The formation of 4-hydroxytamoxifen was also inhibited
significantly (p < 0.05) by ␣-naphthoflavone (62%), quercetin
(61%), tranylcypromine (75%), quinidine (90%), ketocona-
zole (83%), sulfaphenazole (37%), troleandomycin (55%),
and indomethacin (57%), respectively (Fig. 7B). Similar to
inhibition of 4-hydroxytamoxifen formation, N-desmethyl-
tamoxifen formation was significantly inhibited by ␣-naph-
thoflavone (67%), quercetin (49%), tranylcypromine (28%),
indomethacin (49%), quinidine (27%), ketoconazole (59%),
and troleandomycin (31%), respectively (Table II).
Fig. 4 Effect of genistein (A) and isoflavone mixture (B) on the
formation of tamoxifen metabolites. Tamoxifen (2.5 ␮M) was incu-
bated with microsomes and the NADPH regenerating system in the
presence of 12.5, 25, 50 ␮M genistein or an isoflavones mixture at 5
or 10 ␮M each. A mixture of genistein (ge), daidzein (da), and gly-
citein (gl) are present in soy products. A mixture of three soy isofla-
vones plus biochanin A (bi) and formononetin (fo) are normally
present in popular isoflavone extract products such as red clover.
Controlled experiments used same volume and concentration of or-
ganic solvent in the place of genistein. Formation rates of ␣-hydroxy,
4-hydroxl, N-desmethyl, and N-oxide of tamoxifen were determined
as described under “Materials and Methods.” Each point or column
represents mean percent control of three formation rates of these
four metabolites and the error bars are standard deviations of the
mean. The control values ( SD) for the formation rates of ␣-hydroxy,
4-hydroxy, N-desmethyl, and N-oxide metabolites of tamoxifen (at
1.25, 2.5, 5 and 10 ␮M of tamoxifen) were 25.7 0.01, 72.7 7.2, 146
27, and 274 11 pmol min−1 mg protein−1, respectively.
together decreased the formation of ␣-hydroxytamoxifen by
To characterize further the contributions of various CYP 80% (p < 0.05) (Table II).
isoforms, combinations of chemical inhibitors (␣-naphtho-
For tamoxifen 4-hydroxylation, the use of combinatorial
flavone 25 ␮M, quinidine 5 ␮M, tranylcypromine 25 ␮M and chemicals inhibitors (␣-naphthoflavone, quinidine, tranylcy-
ketoconazole 2 ␮M) were used to determine their effect on promine and ketoconazole) resulted in total of 90% (p < 0.05)
tamoxifen metabolism. The results indicated that ␣-naphtho- inhibition (Table II). All these four inhibitors contributed to
flavone inhibited ␣-hydroxylation by 67%. The addition of the maximal inhibitory effects. Last, the same combinatorial
quinidine and/or tranylcypromine did not further significantly approach generated a 73% (p < 0.05) decrease in N-
decrease the formation of ␣-hydroxytamoxifen. However, the demethylation, and the inhibition appeared to be primarily
addition of ketoconazole further inhibited ␣-hydroxylation by contributed by ␣-naphthoflavone (Table II) because it alone
50% (p < 0.05). Therefore, the use of these four inhibitors inhibited the metabolism by more than 62%.

Metabolic Interaction Between Tamoxifen and Isoflavones
2101
DISCUSSION
The goals of this study were to determine the effects of
genistein and its analogs on tamoxifen metabolism and to
delineate possible mechanisms of tamoxifen-isoflavone inter-
actions using prototypical chemical inhibitors.
Our study is designed to take into account the physi-
ologic concentrations of tamoxifen and genistein (12,24,48).
In vivo plasma concentrations of total genistein (mostly con-
jugated) in humans may reach as high as 10 ␮M after a single
dose of soy isoflavone product (24,49). Despite this, hepatic
concentrations of intact isoflavones or aglycones could reach
quite a high level in liver, since these isoflavones are rapidly
absorbed. On the other hand, the physiologic concentrations
of tamoxifen are in a low to sub-micromolar range (12). Thus,
we have chosen in vitro concentrations of tamoxifen and
genistein/isoflavones that are close to the observed in vivo
concentrations.
The results indicated that major metabolites (e.g., 4-hy-
droxytamoxifen) found in human plasma were also found in
our rat microsomal studies, as was the minor metabolite of
interest, ␣-hydroxytamoxifen (Fig. 1). On the other hand,
tamoxifen-N-oxide was found as a major metabolite in FRLM
even though it was identified as a minor metabolite in other in
vitro studies (13), perhaps due to species difference.
We also found that the formation of these metabolites
was concentration-dependent (Fig. 3) with Km values (5–25
␮M, Table I) significantly higher than in vivo concentrations
of tamoxifen (1 ␮M or less), suggesting the intrinsic clearance
value is a better predictor of in vivo clearance.
Our studies also indicated that phase I metabolism of
genistein in FRLM and human liver microsomes are similar
with cytochrome P450-mediated formation of 3Ј-hydroxy-
genistein as the primary metabolic pathway (24–26). Addi-
tionally, O-demethylation of methylated isoflavones (e.g.,
biochanin A) was also similar. A major isoform responsible
for the observed phase I metabolism appears to be CYP1A2
(25). Taken together, these studies using FRLM could pro-
vide clinically relevant information about metabolic interac-
tions between tamoxifen and isoflavones, although verifica-
tion in humans would be ultimately necessary.
To determine the potential interactions, we focused our
studies on the effects of isoflavones on tamoxifen metabolism.
The results indicated that genistein and a mixture of its ana-
logs significantly inhibit the formation of the toxic metabolite
␣-hydroxytamoxifen, but did not decrease the formation of
other active metabolites (e.g., 4-hydroxytamoxifen) (Fig. 4).
There was also a small but significant activation of tamoxifen-
N-oxide formation (Fig. 4A). We are excited by this discovery
since this is a rare instance of a potentially beneficial drug-
supplement/herbal interaction. In contrast, there are numer-
ous literature reports that have shown adverse interactions.
We next determined the mechanisms responsible for the
inhibition of ␣-hydroxylation of tamoxifen by genistein. The
results indicated that the inhibition is concentration-
dependent, and mixed-typed (both competitive and non-
competitive) (Fig. 5B) (36). The results also showed that pre-
incubation of FRLM with genistein (up to 30 min) did not
decrease the extent of inhibition, suggesting that genistein
was not a mechanism-based inhibitor (i.e., inactivator) of
␣-hydroxylation of tamoxifen (Fig. 6).
Fig. 5 Inhibition of ␣-hydroxytamoxifen formation by increasing
concentrations of genistein (A). The Lineweaver-Burk plot is used
here to determine the inhibitory mechanism by which genistein in-
hibits the formation of ␣-hydroxytamoxifen or mode of inhibition
(B). Four concentrations of tamoxifen (1.25, 2.5, 5, and 10 ␮M) were
incubated with microsomes and the NADPH regenerating system in
the presence of genistein (12.5, 18.75, 25, and 50 ␮M) respectively.
Control used the same incubation with %0.25 stock solution solvent
instead of genistein. Formation rate of ␣-hydroxytamoxifen were de-
termined as described under “Materials and Methods.” Each point
represents the mean of three determinations and the error bars are
the standard deviation of the mean. The mean control values ( SD)
for ␣-hydroxylation (at 1.25, 2.5, 5, and 10 ␮M of tamoxifen) were
18.5 1.3, 25.7 0.01, 36.7 1.34, 39 2.1 pmol min−1 mg protein−1
respectively (A). Lines in (B) were generated by linear regression.
,
Effects of Prototypical Chemical Inhibitors on
3؅-Hydroxygenistein Formation
Tranylcypromine, an inhibitor of CYP2C11, was the
most potent among all the inhibitors tested, inhibiting the
formation of 3Ј-hydroxygenistein by 89% (p < 0.05). Other
inhibitors also decreased the formation of 3Ј-hydroxy-
genistein and the extent of the inhibition was 46% (p < 0.05)
for furafylline (CYP1A2 inhibitor) (43,45), 39% (p < 0.05) for
quinidine, and 33% (p < 0.05) for both sulfaphenazole and
ketoconazole. The extent of inhibition was similar at two
tested concentrations (25 or 50 ␮M for inhibitors other than
quinidine, which were used at 5 or 10 ␮M) (Fig. 6).
We used various selective inhibitors of CYP isoforms to

2102
Chen et al.
Table II. Effects of Various Prototypical Chemical Inhibitors on Tamoxifen Metabolism
Inhibitors
Tamoxifen (% control)a
4-hydroxyl
␣-hydroxyl
N-desmethyl
CYP
Conc.
Chemicals
isoform
(␮M)
%
SD
%
SD
%
SD
Control
SKF-525
␣-Naphthoflavone (N)
Quercetin
Sulfaphenazole
Lovastatin
Tranylcypromine (T)
Indomethacin
Quinidine (Q)
Diethyldithiocarbamic acid
Ketoconazole (K)
Troleandomycin
N + Q
100
7.09
1.72
2.26
6.92
6.63
4.05
3.32
6.48
6.69
5.07
5.10
2.26
0.26
3.49
0.43
100
0.00
37.8
7.09
0.00
0.44
1.88
0.63
15.2
2.44
1.39
0.67
0.29
0.88
1.54
8.38
1.87
0.70
100
47.2
2.45
2.77
2.18
4.26
5.82
9.58
4.06
15.06
11.87
3.32
4.41
0.62
2.55
4.75
1.49
All CYP
CYP1A2
CYP2C6
CYP2C6
CYP3A
CYP2C11
CYP3A2
CYP2D1
CYP2E1
CYP3A2
CYP3A2
50
25
25
25
25
25
50
5
12.3
32.8
33.2
50.8
202.5
126.1
62.4
51.1
72.8
90.1
41.4
69.1
78.0
61.1
26.6
CNDa
107.8
42.2
38.7
62.8
87.1
25.3
82.4
10.1
217.6
17.5
45.7
28.2
16.8
9.7
45.1
57.82
44.7
82.4
41.9
62.4
35.5
40.3
19.7
50
2
50
N + Q + T
N + Q + T + K
CND stands for “cannot be determined” because of technical difficulty. In this case, a contamination peak from quercetin.
a The results are expressed as the remaining rate of metabolism after normalizing for control.
␣-hydroxylation of tamoxifen. The CYP3A1/2 inhibitor keto-
conazole (2 ␮M), which is a potent CYP3A1/2 inhibitor in rat
with an IC50 value of 0.29 ␮M (44), inhibited ␣-hydroxylation
by 58%. However, at concentrations at or above 1 ␮M, ke-
toconazole also inhibits CYP2C6 (45). In this case, however,
CYP2C6 was not involved since sulfaphenazole (45), the most
potent and highly specific CYP2C6 inhibitor, did not cause
any inhibition. Therefore, CYP3A1/2 and CYP1A2 are likely
the main CYP isoforms responsible for ␣-hydroxylation of
tamoxifen. This is supported by the facts that addition of
ketoconazole to the inhibitor mixture further inhibited the
formation of ␣-hydroxytamoxifen by 50% (Table II). Fur-
thermore, the relationship shown in the Eadie-Hoffstee plot
suggested the involvement of at least two isoforms for the
formation of ␣-hydroxytamoxifen (Fig. 3A). Additionally, the
involvement of CYP3A1/2 is supported by inhibitory action
of a more specific CYP3A1/2 inhibitor, troleandomycine (41),
and another CYP3A1/2 inhibitor indomethacin (Table II).
Although tranylcypromine and quinidine were effective
inhibitors of ␣-hydroxylation of tamoxifen, addition of these
two compounds to the inhibitor mixture did not result in
greater extent of inhibition, suggesting that the contribution
of CYP2D1 and CYP2C11 is minimal (Table II). The ob-
served inhibitory effects may be due to the cross inhibition of
other CYP isoforms by these compounds. For example, quin-
idine, a potent CYP2D1 inhibitor in rat liver microsomes (41),
also inhibited rat CYP3A1/2 and CYP2C6 at 1 µM (45).
We then determined the contribution of various CYP
isoforms on the formation of 3Ј-hydroxygenistein. The inhibi-
tor results suggest that CYP1A2, CYP2C6, CYP2D1 and
CYP3A1/2 subfamilies could contribute to the formation of
3Ј-hydroxygenistein. However, the involvement of CYP3A1/2
may be small since genistein did not inhibit the formation of
6-hydroxytestosterone or 4-hydroxytamoxifen, both involving
CYP3A1/2. Previously, we have shown that CYP1A and
CYP2C subfamilies are involved in the 3Ј-hydroxylation of
genistein in human liver microsomes (25).
further confirm which CYP isoforms were responsible for the
observed drug-isoflavone interactions. To accomplish this ob-
jective, we needed to determine CYP isoforms involved in
tamoxifen and genistein metabolism separately and jointly.
We first determined the maximal contribution of differ-
ent CYP isoforms to the formation of ␣-hydroxytamoxifen.
Among these inhibitors, the CYP1A2 inhibitor ␣-naphthofla-
vone appeared to significantly decrease its formation, sug-
gesting that CYP1A2 makes a significant contribution to
Fig. 6 Effects of selective cytochrome P450 inhibitors on genistein
metabolism. Genistein (25 ␮M) was incubated with microsomes and
the NADPH regenerating system in the presence of P450 inhibitor
furafylline or sulfaphenazole or tranylcypromine or ketoconazole at
concentration of 25 and 50 ␮M, or quinidine at 5 and 10 ␮M, as
described. Equivalent amounts of organic solvent (0.15% in this case)
were added instead of inhibitors in the control experiment. Forma-
tion rate of 3Ј-hydroxylgenistein (3Ј-OH-Gen) were determined as
described under “Materials and Methods.” Each column represents
the mean of three determinations and the error bars are the standard
deviation of the mean. The mean control value ( SD) for formation
rates of 3Ј-hydroxylgenistein was 84.5 3.4 pmol min−1 mg protein−1
.
Together, these results indicated ␣-hydroxylation of

Metabolic Interaction Between Tamoxifen and Isoflavones
2103
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cytochrome P450 pathways. The involvement of CYP1A2 is
likely since genistein could compete for the same metabolic
pathways.
Finally, we would like to briefly comment on the signifi-
cance of our research from a clinical point of view, since there
is no information about these compounds’ potential interac-
tions in humans. We also want to point out that our studies
were conducted in rat liver microsomes, which are very dif-
ferent from humans in vivo. Nevertheless, we believe that we
have for the first time observed potential beneficial metabolic
interactions between drugs and herbal supplements, which is
rarely reported. Therefore, our discovery suggests that addi-
tional studies in humans may be warranted to determine if the
combination is beneficial for patients. Another important
point is that different inhibitors of CYP isoforms could po-
tential change the nature of interactions (i.e., toxic or benefi-
cial). That change could influence which combination of
tamoxifen and prescription drugs (e.g., ketoconazole, lovas-
tatin) or over-the-counter medications (e.g., quercetin is sold
as a herbal product) is beneficial vs. harmful to the patients.
In conclusion, this study demonstrates that isoflavones
decrease the formation of toxic ␣-hydroxytamoxifen through
metabolic interactions using rat liver microsomes. The
mechanism responsible for tamoxifen-genistein metabolic in-
teractions appears to be mediated via competitive and non-
competitive inhibition of CYP1A2. Even though the potential
benefit of co-administering isoflavones and tamoxifen for hu-
man remained to be demonstrated, as there is evidence that
␣-hydroxytamoxifen is mainly formed via the action of
CYP3A4 in human microsomes, this study shows the poten-
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ACKNOWLEDGMENT
This work was supported by NIH AT00182 and NIH CA
87779. We thank Dr. Fredrick Beland of FDA (Little Rock,
AK) and Dr. Judy Bolton (University of Illinois) for provid-
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In a round-bottom flask wrapped with aluminum foil,
tamoxifen (27 mg, 0.072 mmol) was dissolved in 0.5 ml of
CH2Cl2. After the solution was cooled down to 0°C, 3-chlo-
roperoxybenzoic acid (mCPBA) (26 mg, maximal 77%, ∼0.12
mmol, dissolved in 0.25 ml of CH2Cl2) was added dropwise to
the solution. After ∼10 min, tamoxifen was completely con-
verted to a more polar compound, as judged by thin-layer
chromatography (TLC). After an additional 50 min, the re-
action mixture was diluted with 40 ml of CH2Cl2, and was
washed with a mixture of 10 ml of saturated aqueous
42. J. Schmider, D. J. Greenblatt, S. M. Fogelman, L. L. von Moltke, NaHCO3 and 10 ml of 10% aqueous Na2CO3 (final pH was 9)
and R. I. Shader. Metabolism of dextromethorphan in vitro: in-
to remove mCPBA and 3-chlorobenzoic acid, then with brine.
volvement of cytochromes P450 2D6 and 3A3/4, with a possible
The organic extract was dried with MgSO4 overnight (the
role of 2E1. Biopharm. Drug Dispos. 18:227–240 (1997).
flask was covered with aluminum foil and placed in the re-
43. V. A. Eagling, J. F. Tjia, and D. J. Back. Differential selectivity of
frigerator). The MgSO4 was filtered off and the solvent re-
moved in vacuum. An off white solid was obtained as the
desired product (22 mg, 79% yield). TLC: Rf 0.35 (Methanol).
1H NMR (CDCl3, 300 MHz): 0.93 (t, 3H), 2.45 (q, 2H), 3.28
(s, 6H), 3.65 (dd, 2H), 4.4 (dd, 2H), 6.55 (d, 2H), 6.78 (d, 2H),
7.08-7.26 (m, 8H), 7.27- 7.38 (m, 2H). 13C NMR (CDCl3, 75
MHz): 12.55, 28.01, 58.89, 60.79, 69.07, 75.59, 112.31, 125.10,
125.58, 126.90, 127.12, 128.41, 128.64, 131.02, 135.41, 136.92,
140.71, 141.22, 142.60, 154.49. DEPT: (CH): 112.31, 125.10,
125.58, 126.90, 127.12, 128.41, 128.64, 131.02; (CH2): 28.01,
cytochrome P450 inhibitors against probe substrates in human
and rat liver microsomes. Br. J. Clin. Pharmacol. 45:107–114
(1998).
44. Y. Masubuchi, E. Masuda, and T. Horie. Multiple mechanisms in
indomethacin-induced impairment of hepatic cytochrome P450
enzymes in rats. Gastroenterology 122:774–783 (2002).
45. K. Kobayashi, K. Urashima, N. Shimada, and K. Chiba. Selec-
tivities of human cytochrome P450 inhibitors toward rat P450
isoforms: study with cDNA-expressed systems of the rat. Drug
Metab. Dispos. 31:833–836 (2003).
46. R. W. Wang, P. H. Kari, A. Y. Lu, P. E. Thomas, F. P.
Guengerich, and K. P. Vyas. Biotransformation of lovastatin. IV.
Identification of cytochrome P450 3A proteins as the major en- 60.79, 69.07; and (CH3): 12.56, 58.89.