A methoxyflavonoid, chrysoeriol, selectively inhibits the formation of a carcinogenic estrogen metabolite in MCF-7 breast cancer cells

Article abstract of DOI:10.1016/j.jsbmb.2009.10.002

A 17β-estradiol (E₂) is hydrolyzed to 2-hydroxy-E₂ (2-OHE₂) and 4-hydroxy-E₂ (4-OHE₂) via cytochrome P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries, and uterus, CYP1B1 is highly expressed, and 4-OHE₂ is predominantly formed in cancerous tissues. In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3′-methoxy ether), which is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive manner. Additionally, chrysoeriol inhibited E₂ hydroxylation was catalyzed by CYP1B1, but not by CYP1A1. Methylation of 4-OHE₂, which is thought to be a detoxification process, was not affected by the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E₂ without any effects on the formation of 2-methoxy E₂. In conclusion, we present the first report to show that chrysoeriol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the formation of carcinogenic 4-OHE₂ from E_2..

Full text of DOI:10.1016/j.jsbmb.2009.10.002

Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 70–76
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
A methoxyflavonoid, chrysoeriol, selectively inhibits the formation of a
carcinogenic estrogen metabolite in MCF-7 breast cancer cells
Hitomi Takemuraa,d, Harue Uchiyamab, Takeshi Ohuraa,b, Hiroyuki Sakakibaraa,b
,
Ryoko Kurutoe, Takashi Amagaia,b, Kayoko Shimoia,b,c,∗
a Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422-8526, Japan
b Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422-8526, Japan
c Global COE Program, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422-8526, Japan
d Department of Health and Nutritional Science, Faculty of Human Health Sciences, Matsumoto University, 2095-1 Niimura, Matsumoto 390-1295, Japan
e Faculty of Education, Tokoha Gakuen University, 1-22-1 Sena, Aoi-ku, Shizuoka 420-0911, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2009
Received in revised form 1 October 2009
Accepted 6 October 2009
A 17␤-estradiol (E2) is hydrolyzed to 2-hydroxy-E2 (2-OHE2) and 4-hydroxy-E2 (4-OHE2) via cytochrome
P450 (CYP) 1A1 and 1B1, respectively. In estrogen target tissues including the mammary gland, ovaries,
and uterus, CYP1B1 is highly expressed, and 4-OHE2 is predominantly formed in cancerous tissues.
In this study, we investigated the inhibitory effects of chrysoeriol (luteorin-3ꢀ-methoxy ether), which
is a natural methoxyflavonoid, against activity of CYP1A1 and 1B1 using in vitro and cultured cell
techniques. Chrysoeriol selectively inhibited human recombinant CYP1B1-mediated 7-ethoxyresorufin-
O-deethylation (EROD) activity 5-fold more than that of CYP1A1-mediated activity in a competitive
manner. Additionally, chrysoeriol inhibited E2 hydroxylation was catalyzed by CYP1B1, but not by
CYP1A1. Methylation of 4-OHE2, which is thought to be a detoxification process, was not affected by
the presence of chrysoeriol. In human breast cancer MCF-7 cells, chrysoeriol did not affect the gene
expression of CYP1A1 and 1B1, but significantly inhibited the formation of 4-methoxy E2 without any
effects on the formation of 2-methoxy E2. In conclusion, we present the first report to show that chrysoe-
riol is a chemopreventive natural ingredient that can selectively inhibit CYP1B1 activity and prevent the
formation of carcinogenic 4-OHE2 from E2.
Keywords:
Estrogen
CYP1B1
4-Hydroxyestradiol
Chrysoeriol
Chemoprevention
© 2009 Elsevier Ltd. All rights reserved.
1. Introduction
compared with 2-OHE2 in mice [5]. Additionally, ratios of 4-
OHE2/2-OHE2 formation in neoplastic tissue were higher than that
The prolonged exposure of estrogens, especially in post-
menopausal women, is well known to play a role in breast cancer
etiology [1]. The conceivable mechanisms that could partially play
a part in the mutagenic effects of the endogenous metabolites of
estrogens within the catechol estrogens (CEs) pathway are shown
in Fig. 1. This pathway is initially promoted via cytochrome P450
(CYP) families, especially CYP1A1 and CYP1B1. 17␤-Estradiol (E2),
one of the major estrogens, is hydrolyzed for CEs by both CYP1A1
and CYP1B1, and mainly converted to 2-hydroxy-E2 (2-OHE2) and
4-hydroxy-E2 (4-OHE2), respectively [2,3]. 4-OHE2 was carcino-
genic in the hamster kidney, whereas 2-OHE2 did not induce any
tumors [4]. However, in mouse uterine, both hydroxyl metabo-
lites were carcinogenic, and 4-OHE2 had a higher tumor incidence
in normal breast tissue [6]. Quinone intermediates derived by oxi-
dation of 4-OHE2 have been reported to react with purine bases
of DNA to form depurinating adducts that generate highly muta-
genic apurinic sites, although quinones from 2-OHE2 produce less
harmful and stable DNA adducts [7]. Furthermore, the metabolites
of CEs may also generate potentially mutagenic oxygen radicals
by metabolic redox cycling or other mechanisms [7]. Several types
of indirect DNA damage are caused by estrogen-induced oxidants,
such as oxidized DNA bases, DNA strand breakage, and adduct for-
mation by reactive aldehydes derived from lipid hydroperoxides
[8,9]. It was found that 4-OHE2 was capable of causing a loss of
heterozygosity at doses as low as 0.007 nM [10,11]. On the other
hand, catechol-O-methyltransferase (COMT) is involved in methy-
lating CEs such as 2-OHE2 and 4-OHE2 (Fig. 1), and decrease their
detrimental effects [12]. This background information indicates
that inhibition of hydroxylation of E2 by the CYP family, especially
CYP1B1, without affecting the methylation pathway has been pos-
tulated to be important for the estrogen related carcinogenesis such
as breast cancer.
∗
Corresponding author at: Institute for Environmental Sciences, 52-1 Yada,
Suruga, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5787; fax: +81 54 264 5787.
E-mail address: shimoi@u-shizuoka-ken.ac.jp (K. Shimoi).
0960-0760/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2009.10.002

H. Takemura et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 70–76
71
Recently, many types of natural compounds have been reported
to be chemopreventive ingredients that have selective inhibitory
effects against the activity of CYPs. For example, some kinds of
flavonoids, which are one of the polyphenol groups that exist
abundantly in vegetables, fruits and teas [13], are able to modu-
late the effects of the CYPs system [14]. The flavonoid acacetin, a
methoxyflavonoid, showed an extremely strong inhibitory effect
for activation of the CYP enzymes [15]. Interestingly, methoxy
flavonoids including acacetin, diosmetin, hesperetin, homoeriod-
ictyol, and 2,3ꢀ,4,5ꢀ-tetramethoxystilbene have been reported to
selectively inhibit the activation of CYP1B1 [15–17], indicating
that the methoxy substituent may be important for inhibition of
CYP activation, especially for CYP1B1. However, there has been no
report about the inhibitory effects on estrogen metabolism acti-
vated by CYP1A1 or CYP1B1 in human breast cells or in vivo.
In the present study, we investigated the effects of chrysoe-
riol (luteolin 3ꢀ-methoxy ether) on metabolism of E2 via CYP1A1
and CYP1B1, because there has been no report concerning the
inhibitory effects of CYPs by chrysoeriol. Additionally, this methoxy
flavonoid has been reported to be abundantly available in our daily
foods. First, we analyzed effects of chrysoeriol on 7-ethoxyresorufin
O-deethylation (EROD) activity induced by human recombinant
CYP1A1 and CYP1B1. Next, we evaluated the protective effects
of chrysoeriol on the formation of individual CEs (2-OHE2 and
4-OHE2) from E2, and also methylation of 4-OHE2. Finally, these
effects were evaluated using the human breast cancer cell line MCF-
7. To our knowledge, this is the first report to find that physiological
levels of chrysoeriol act as a selective inhibitor for CYP1B1 in human
breast cancer cells.
Fig. 1. Scheme of estrogen metabolism in breast cells.
2. Materials and methods
2.3. Enzyme inhibition kinetics
2.1. Chemicals
The enzyme kinetics for CYP1B1-catalyzed EROD was mea-
sured at increasing concentrations of ethoxyresorufin (0, 0.19, 0.37,
0.75, 1.5, 3.0, and 6.0 ␮M), or chrysoeriol (0, 25, 50 and 75 nM).
The reaction mixtures were preincubated at 37 ◦C for 5 min, and
the reactions were initiated by addition of recombinant micro-
somes. Incubations were performed in a shaking water bath at
37 ◦C for 10 min. The formation of resorufin was determined as
described above. For the inhibition kinetics studies, Vmax and Km
values were determined by the nonlinear regression curve fit using
the Michaelis–Menten equation by GraphPad Prism 4 (GraphPad
Software, Inc., San Diego, CA).
HPLC grade chrysoeriol was purchased from Extrasynthese
(Genay, Cedex, France). Ethoxyresorufin, resorufin, 2-OHE2 and
4-OHE2 were obtained from Sigma Chemical Co. (St. Louis, MO). 2-
Methoxyestradiol (2-MeOE2) and 4-methoxyestradiol (4-MeOE2)
were purchased from Steraids Inc. (Newport, RI). ␤-NADPH, ␤-
NADH and glucose-6-phosphate (Oriental Yeast Co., Ltd., Osaka,
Japan), recombinant human CYP1A1 and CYP1B1 supersomes
(Gentest Corporation via BD Biosciences, San Jose, CA), human
liver cytosol (XenoTech, LLC, Lenexa, KS), S-adenosyl-l-methionine
(SAM) (New England Biolabs, Inc., Ipswich, MA), and [methyl-
3H] SAM (PerkinElmer Life and Analytical Sciences, Boston, MA)
were used in this study. All the other chemicals and reagents
were obtained from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan).
2.4. Hydroxylation of E2 by recombinant CYP1A1 and 1B1
The CYP1A1 and 1B1-mediated hydroxylation of E2 was carried
out by a modified EROD enzyme assay. Briefly, the reaction mixture,
described above but with ethoxyresorufin substituted for 20 ␮M E2,
was incubated at 37 ◦C for 40 min, and then immediately extracted
with 5 mL of dichloromethane. After centrifugation at 3000 rpm for
10 min, the sodium sulfate was added to the lower fraction, then fil-
tered through an Ekicrodisc (0.2 ␮m, PTFE, Gelman Sciences, Tokyo,
Japan), and finally concentrated to ca. 1 mL by nitrogen gas. After
50 ␮L of bistrimethylsilyltrifluoroacetamide (BSTFA) was added to
the solution, the mixture was kept at room temperature for 60 min,
and the solvent was used for the quantitative analysis.
The reactant mixtures were analyzed on a Hewlett-Packard (HP)
model 6890 gas chromatograph -HP 5972A mass spectrometry
(GC/MS) system (Agilent Technologies, Palo Alto, CA). The GC/MS
instrument was equipped with an automated sample introduc-
tion system and a splitless injector. A DB-5 capillary column was
used (5% phenylmethylpolysiloxane, 60 m × 0.320 mm × 0.25 ␮m,
2.2. 7-Ethoxyresorufin O-deethylation (EROD) enzyme assay
The EROD enzyme assay was employed as a method for eval-
uation of CYP1A1 and 1B1 activity with slight modifications [18].
The reaction mixture (200 ␮L), which was composed 4 mM NADPH,
4 mM NADH, 20 mM MgCl2, 0.1 M potassium phosphate buffer
(pH 7.4), individual CYP supersomes and various concentrations
of chrysoeriol was incubated at 37 ◦C for 5 min, and then the reac-
tion was initiated by the addition of 40 ␮L of ethoxyresorufin. After
incubation at 37 ◦C for 10 min, the formation of resorufin was deter-
mined fluorometrically (530 nm excitation and 590 nm emission)
with a spectrofluorometer (Thermo Fisher Scientific Inc., Worces-
ter, MA). IC50 values were determined graphically by plotting
percent of control enzyme activity versus inhibitor concentra-
tion.

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H. Takemura et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 70–76
J&W Scientific). Helium was used as a carrier gas at a flow rate
of 1 mL/min. The oven temperature was kept at 100 ◦C for 5 min,
increased from 100 to 300 ◦C at a rate of 15 ◦C/min, and then kept
at 300 ◦C for 20 min. The temperature of the injector and the GC/MS
transfer line was kept at 300 ◦C. The MSD was run in electron impact
ionization mode, and the electron energy was 70 eV. Quantifica-
tions of the metabolites were performed in selected-ion monitoring
(SIM) mode. The following ions were used by the SIM program for
qualification and identification of the metabolites (m/z): 432 and
504 for trimethylsilylated 2-OHE2 (retention time, 23.95 min); 432
and 504 for trimethylsilylated 4-OHE2 (retention time, 24.59 min);
and 340 for Equilin (retention time, 22.42 min) as internal standard.
by a CoulArray HPLC system (ESA, Inc., Chelmsford, MA) equipped
with 5600A CoulArray detector set at 440 mV. The column, Capcell
Pak C18-UG120 column (250 mm × Ø 4.6 mm, S-5, 5 ␮m, Shiseido
Co. Ltd., Tokyo, Japan) was used at 40 ◦C. Linear gradient elution was
performed with solution A (0.1 M ammonium acetate, 15% acetoni-
trile, 5% methanol) and solution B (0.1 M ammonium acetate, 50%
acetonitrile, 20% methanol) delivered at a flow rate of 1.0 mL/min
as follows: initially 100% of solution A; for the next 50 min, 10% A;
and finally, 10% A for 10 min. The injected volume of the extract
was 50 ␮L.
2.8. Gene expression of CYP1A1, 1B1, and COMT
Total RNA was extracted from MCF-7 cells treated with chryso-
eriol using the RNA Protect Cell Reagent (Qiagen Inc., Valencia,
CA) and the RNeasy Plus Mini Kit (Qiagen Inc.) according to the
protocol included. Following isolation, RNA quantity, purity, and
concentration were determined using a Gene Quant pro spec-
trophotometer (Amersham Biosciences, Foster City, CA). Gene
expression of CYP1A1 and 1B1 were measured using quantitative
RT-PCR method as described previously [21]. Briefly, the RNA sam-
ple (300 ng) was added to 20 ␮L of reaction mixture containing
random hexamers, MuLv Reverse Transcriptase, RNase inhibitors,
25 mM MgCl2, 10 × PCR Buffer II (Applied Biosystems), and 10 mM
dNTP mix (Promega Co., Madison, WI). Synthesis of cDNA was per-
formed at 42 ◦C for 60 min, and the reverse transcription reaction
was stopped by heating to 95 ◦C for 7 min followed by chilling on ice.
The cDNA was stored at −20 ◦C until further use. A total of 2 ␮L of
cDNA was added to the 18 ␮L of PCR mixture containing 10 ␮L Taq
Man Gene Expression Master Mix (Applied Biosystems), 6 ␮L dis-
tilled water DNase RNase Free (Invitrogen Corp., Carlsbad, CA), 1 ␮L
house-keeping gene solution (glyceraldehyde-3-phosphate dehy-
drogenase; GAPDH), and 1 ␮L individual target gene expression
reagents: CYP1A1, Assay ID, Hs00153120 m1; CYP1B1, Assay ID,
Hs00164383 m1; COMT, Assay ID, Hs00984971 m1. Quantitative
RT-PCR was performed on a 7500 Real-Time PCR System (Applied
Biosystems). The samples were amplified by incubation for 2 min at
50 ◦C, then 10 min at 95 ◦C, followed by 40 cycles of 95 ◦C for 15 s and
60 ◦C for 1 min. The relative expression level of the target gene prod-
uct was calculated by the comparative automatic threshold cycles
method, using the house-keeping gene, GAPDH, as a calibrator. The
relative differences in expression between groups were expressed
using cycle time values and the relative differences between groups
were expressed as relative increases, setting the control as 100%.
2.5. Methylation of 4-OHE2 by human hepatic cytosolic COMT
The catechol-O-methyltransferase (COMT)-mediated O-
methylation of estrogens was carried out as described previously
[19]. The reaction was initiated by the addition of human liver
cytosolic protein (0.5 mg) in to the reaction mixture consisted with
1.2 mM MgCl2, 250 ␮M SAM (containing 0.5 ␮Ci [methyl-3H]SAM),
1 mM dithiothreitol, and 10 ␮M 4-OHE2 in 125 ␮L of Tris–HCl
buffer (10 mM, pH 7.4) and incubated at 37 ◦C for 20 min. After the
incubation, the reaction mixture was immediately cooled on ice,
500 ␮L of ice-cold distilled water was added, and extraction with
3 mL of ice-cold n-heptane was performed. After centrifugation
at 1000 × g for 10 min, the organic fractions were dissolved in
5 mL of Ultima Gold scintillation cocktail (PerkinElmer, Japan) and
the radioactivity was measured by a liquid scintillation analyzer
(LSC-5100; Aloka, Co., Ltd., Tokyo, Japan).
2.6. Cell culture and treatment
Human breast cancer MCF-7 cells kindly provided by Dr. H.
Hagenmaier (University of Tuebingen, Germany) were cultured in
Dulbecco’s modified Eagle medium (DMEM) supplemented with
10% fetal bovine serum, 0.1 mg/mL kanamycin and 0.1 mg/mL
ampicillin at 37 ◦C under 5% CO2. When cells were 90% confluent
in Ø 100 mm dishes, the medium was changed to estrogen free
DMEM including 50 or 100 nM chrysoeriol. After 15 min of treat-
ment, 1 ␮M E2 was added into the dishes and then incubated again
for 24 h. Dimethyl sulfoxide (DMSO; 0.1% of final volume) was used
as a vehicle control in the experiment. The media were isolated
by centrifugation at 3000 rpm for 10 min and stored at −20 ◦C. To
evaluate gene expression of CYP1A1 and 1B1, sub-confluent cells
were incubated with estrogen free DMEM including 100 or 500 nM
chrysoeriolfor 15 min, and then10 nME2 was addedintothe dishes.
Two micromolar of benzo[a]pyrene (BaP) was used as positive con-
trol to induce CYP1A1 and 1B1. The cells were collected, and then
RNA extraction was immediately carried out using the method
described below.
2.9. Statistical analysis
Results were expressed as mean standard deviation (SD) for
experiments performed at least in triplicate. Statistical significance
of differences was evaluated using two-tailed unpaired ANOVA fol-
lowed by Dunnett’s multiple comparison test.
2.7. Deconjugation, extraction and HPLC analysis
3. Results
To determine total estrogen (unconjugated and conjugated
metabolites), 2 mL aliquots from the media were incubated with
1000 U of ␤-glucuronidase/sulfatase at 37 ◦C for 17 h after addi-
tion of 0.5 mL of 2 M ammonium acetate buffer. The mixtures were
then extracted 2 times with 2.5 mL of dichloromethane. The com-
bined dichloromethane extracts were evaporated to dryness and
then assayed by a CoulArray HPLC system as described below. Rates
of metabolite formation were normalized to cellular protein con-
tent, which was determined by the BCA Protein Assay Kit (Pierce,
Rockford, IL).
3.1. Inhibitory effects of chrysoeriol on EROD activity induced by
CYP1A1 or 1B1
The inhibitory effects of chrysoeriol were determined from
EROD activities induced by human recombinant CYP1A1 and
CYP1B1. As shown in Table 1, chrysoeriol dose-dependently
decreased EROD activity induced by each CYP. The individual
inhibitory activities (IC50 values) of CYP1A1 and 1B1 were 94.7 and
19.7 nM, respectively.
HPLC analysis was carried out according the method reported
by Lavigne et al. [20] with some modifications. Briefly, the HPLC
system employed in this study was analyzed chromatographically
A kinetic study of formation of resorufin from 7-ethoxyresorufin
catalyzed by human recombinant CYP1B1 was analyzed in the pres-
ence of chrysoeriol (0, 25, 50, and 75 nM) using a Lineweaver–Burk

H. Takemura et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 70–76
73
Table 1
Effects of chrysoeriol on human recombinant CYP1A1- and CYP1B1-mediated EROD
activity.
Chrysoeriol (nM)
CYP1A1
CYP1B1
0
100.0 7.9
95.0 1.9
72.2 3.8**
41.7 2.2**
100.0 3.7
66.3 2.8**
36.2 5.1**
17.9 2.9**
10
40
100
IC50 valuesa
94.7 5.7
19.7 1.5
Microsomes containing individual CYP1A1 and 1B1 were incubated with increasing
concentrations of chrysoeriol for 5 min, and then the reaction was started with the
ethoxyresorufin substrate. The appearance of resorufin was measured over 10 min
by a fluorescence plate reader (530 nm excitation and 590 nm emission) at 37 ◦C.
The data was indicated as a percentage compared with controls that were treated
with vehicle solvent but without chrysoeriol (mean SD, n = 3).
Fig. 4. Effects of chrysoeriol on the reaction of human hepatic COMT-mediated O-
methylation of 4-OHE2. The incubation mixture consisted of 10 ␮M 4-OHE2, 250 ␮M
SAM (containing 0.5 ␮Ci [methyl-3H]), 0.5 mg/mL human cytosolic protein, individ-
ual amounts of chrysoeriol, 1 mM dithiothreitol, and 1.2 mM MgCl2 in a final volume
of 1.25 mL adjusted using Tris–HCl buffer (10 mM, pH 7.4). Incubations were carried
out at 37 ◦C for 10 min. Data are presented as the mean SD (n = 3). The result-
ing methylated estrogens were measured as radioactivity content using a liquid
scintillation analyzer.
a
Each IC50 value was calculated by plotting the suppression of EROD activity
against the dose, indicating that the amount (nM) required for 50% suppression of
EROD activity.
**
Significant differences vs. control (p < 0.01) by ANOVA and Dunnett’s multiple
comparison test.
3.2. In vitro effects of chrysoeriol on hydroxylation of E2 by
CYP1A1 and 1B1
There was no inhibitory effect of chrysoeriol to the formation of
2-OHE2 by CYP1A1 up to 100 nM (Fig. 3A), whereas chrysoeriol sig-
nificantly inhibited the formation of 4-OHE2 catalyzed by CYP1B1
at 50 and 100 nM (Fig. 3B).
3.3. Effects of chrysoeriol on methylation of 4-OHE2 by human
hepatic cytosolic COMT
The effects of chrysoeriol on O-methylation of 4-OHE2 were
evaluated using human hepatic cytosolic COMT, which catalyzes
an inactivation pathway for CE as shown in Fig. 1. The incubation of
4-OHE2 with COMT increased methylation activity, while addition
of 1, 10, and 100 ␮M chrysoeriol indicated no significant inhibitory
effect against this methylation activity (Fig. 4).
Fig. 2. Lineweaver–Burk plots showing inhibition kinetics of CYP1B1-catalyzed
EROD activity by the presence of chrysoeriol. Human recombinant CYP1B1 was
incubated with 7-ethoxyresorufin in the absence (᭹), or presence or various concen-
trations of chrysoeriol 25 nM (ꢀ), 50 nM (ꢁ), 75 nM (ꢂ) at increasing concentration
of 7-ethoxyresorufin from 0 to 6 ␮M. Resorufin formation was determined fluoro-
metrically.
3.4. Effects of chrysoeriol on E2 hydroxylation and gene
expression of CYP1A1, 1B1, and COMT in MCF-7 cells
E2 introduced in human breast cancer MCF-7 cells could be
hydrolyzed to 2-OHE2 and 4-OHE2 by endogenous CYP1A1 and
CYP1B1, respectively, as shown in Fig. 1. Following such metabolic
processes, O-methylation, glucuronidation and sulfation for these
2-OHE2 and 4-OHE2 occurred in the cells. Indeed, when human
breast cancer MCF-7 cells were exposed to 1 ␮M E2 for 24 h, both
2-MeOE2 and 4-MeOE2 were observed in the medium after treat-
plot (Fig. 2). The Vmax and Ki values were estimated at 2.08
pmol/␮g protein/min and 8.3 nM, respectively. The plot also
showed that chrysoeriol acts as a competitive inhibitor against
CYP1B1-catalyzed conversion from 7-ethoxyresorufin to resorufin.
Fig. 3. Effects of chrysoeriol on catechol estrogen formation catalyzed by recombinant human CYP1A1 or CYP1B1. E2 (20 ␮M) was incubated with recombinant human
CYP1A1 or CYP1B1 microsomes in the presence or absence of chrysoeriol at 37 ◦C for 30 min, and the resulting catechol estrogens were detected by GC/MS analysis. Data are
presented as the mean SD (n = 3). Static analysis for single comparison was performed using Dunnett’s test (**P < 0.01 vs. control).

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H. Takemura et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 70–76
Fig. 5. Effects of chrysoeriol on estrogen metabolites in MCF-7 cells. Cells were treated with chrysoeriol for 24 h. Media was incubated with ␤-glucuronidase/sulfatase for
12 h, and then extracted according to the method described in Materials and methods. Concentrations of 2-MeOE2 and 4-MeOE2 were determined using a CoulArray HPLC
system. Each preparation of these metabolites was normalized to the cellular protein concentration. Data presented as the mean SD (n = 3). Statistical analysis for single
comparisons was performed using Dunnett’s test (***P < 0.001 vs. vehicle control).
ment with ␤-glucuronidase/sulfatase (Fig. 5). Furthermore, the
cells were co-treated with chrysoeriol and E2, which resulted in
the formation of 4-MeOE2 at concentrations of 50 and 100 nM, the
effects of chrysoeriol treatment alone was significantly inhibited
(Fig. 5B), but not for 2-MeOE2 (Fig. 5A). The effects of chrysoe-
riol on constitutive CYP1A1, CYP1B1 and COMT mRNA expression
were investigated in MCF-7 cells using quantitative RT-PCR analy-
sis. BaP significantly increased the gene expression of CYP1A1 and
CYP1B1 (Fig. 6A and B) in a similar manner to our previous data
[21]. On the other hand, chrysoeriol (100 and 500 nM) in the pres-
ence of E2 did not affect gene expression of either CYP. Moreover,
chrysoeriol showed no effect on the genetic expression of COMT
(Fig. 6C).
followed by transformation into an inactive form, 2-MeOE2, at a
faster rate than 4-OHE2 [27]. Also, 2-MeOE2 has an inhibitory effect
on cell proliferation [28], indicating that CYP1A1 is important for
detoxification of estrogen. Therefore, the compounds, which selec-
tively inhibit CYP1B1 but not CYP1A1, would be considered to be
useful for the chemoprevention of estrogen related carcinogenesis,
as typified by breast cancer.
Several kinds of flavonoids, which exist widely in our foods,
vegetables, fruits and teas [13], have been reported to affect
CYP enzymes [14]. Among the flavonoids, myricetin, apigenin,
kaempferol, quercetin, amentoflavone, quercitrin and rutin were
slightly more selective for a CYP1B1 EROD inhibitor compared
with CYP1A1 [18]. Another study has indicated that isorham-
netin was the most potent CYP1B1 inhibitor compared with major
flavonoids, quercetin and kaempferol [29]. In contrast, flavonol
glucoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside,
and isorhamnetin-3-O-rutinoside did not inhibit CYP1 enzymes
[29]. Interestingly, methoxy flavonoids, acacetin, diosmetin, hes-
peretin, homoeriodictyol, and 2,3ꢀ,4,5ꢀ-tetramethoxystilbene are
more selective inhibitors for CYP1B1 than CYP1A1 [15–17], indicat-
ing that the methoxy substituents may be important for selective
inhibition of CYP1B1.
4. Discussion
Endogenous and/or exogenous estrogens are metabolized by
CYP oxidation, glucuronidation by UDP-glucuronosyl transferase,
sulfation by sulfotransferase, and O-methylation by COMT under
physiological conditions (Fig. 1). Finally, estrogens are eliminated
from the body as urine and/or feces after these metabolic trans-
formations to estrogenically inactive metabolites. Hydroxylation,
which is the first step in the metabolism of estrogens, is initiated
by CYP enzymes. The hydroxides of estrogen were mainly found in
liver. In liver, approximately 80% of E2 is biotransformed to 2-OHE2
and 20% to 4-OHE2 [2,3,22]. On the other hand, in human cancerous
breast tissues and cells, CYP1B1 is highly expressed, and therefore,
formation of 4-OHE2 was more dominant compared with 2-OHE2
[6,23,24]. As we stated in the Introduction, 2-OHE2 and 4-OHE2 are
carcinogenic, and especially, 4-OHE2 has a higher tumor incidence
compared with 2-OHE2 [4,5]. Additionally, 4-OHE2 generates free
radicals from the reductive-oxidative cycling with the correspond-
ing semiquinone and quinone forms, which cause cellular damage
[7–9,25,26]. On the other hand, 2-OHE2 is methylated by COMT,
Chrysoeriol (luteolin-3ꢀ-methoxy ether), found in Rooibos tea,
celery, Chinese celery, celery seed, and leaves of Digitalis pur-
purea [30–33], is a bioactive flavonoid known for antioxidant,
anti-inflammatory [30], bronchodilatory [31], antimutagenic [33],
free radical scavenging activities [34], and the inhibition of 17␤-
hydroxysteroid dehydrogenase type 1 [35]. As described in the
Introduction, inhibitory effects of methoxy flavonoids on estrogen
metabolism are activated by CYP1A1 and CYP1B1 in breast cells
in spite of many reports on their inhibitory effect against CYP 1
enzyme activity. Therefore, in the present study, we focused on
chrysoeriol and investigated its effects on the metabolism of E2 via
CYP1A1 and CYP1B1 in in vitro enzyme and cell systems.
Fig. 6. Effects of chrysoeriol on CYP1A, 1B1, and COMT mRNA expression in MCF-7 cells. Cells were treated with 100 or 500 nM chrysoeriol with 10 nM E2 for 12 h.
Benzo[a]pyrene (BaP) was used as a positive control for the induction of CYP1A1 and 1B1. Levels were measured by quantitative RT-PCR and normalized to those of
controls (10 nM E2). Each column represents the mean of at least three independent experiments SD. Statistical analysis for single comparisons was performed using
Dunnett’s test (***P < 0.001 vs. vehicle control).

H. Takemura et al. / Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 70–76
75
Chrysoeriol inhibited human recombinant CYP1A1- and
5. Conflict of interest
CYP1B1-mediated EROD activity with an IC50 value of 94.7 and
19.7 nM, respectively (Table 1), indicating a 5-fold selectivity in its
inhibition of CYP1B1 rather than CYP1A1, in a competitive man-
ner (Fig. 2). Chrysoeriol inhibited E2 hydroxylation catalyzed by
CYP1B1 at a lower concentration (Fig. 3B), but not hydroxylation
via CYP1A1 (Fig. 3A). Our results strongly suggest that chrysoeriol
is a selective inhibitor against CYP1B1 activity and prevents the
formation of carcinogenic 4-OHE2 from E2.
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors would like to thank Ms. Yumiko Usui for her helpful
HPLC analysis. This work was supported partly by a Grant-in-aid for
Young Scientists (B) (No. 17700563) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
On the other hand, 2-OHE2 and 4-OHE2 are rapidly O-
methylated to form monomethyl ethers catalyzed by COMT as
shown in Fig. 1, which is thought to be a detoxification pro-
cess. COMT is an important enzyme that protects cells from the
genotoxicity and cytotoxicity of catechol estrogens, by prevent-
ing their conversion to quinones by CYP1. The low activity allele
of COMT confers an increased risk for developing breast can-
cer in certain women [36]. Human COMT has been shown to
have a higher catalytic activity toward 2-OHE2 compared with
4-OHE2, which may contribute to the comparatively stronger car-
cinogenicity of 4-OHE2 [19]. Inhibition of the COMT-mediated
O-methylation of endogenous 2- and 4-hydroxylated estrogens
by these catechol-containing dietary polyphenols is expected
to result in an increase in the tissue levels of the procar-
cinogenic 4-OHE2 plus a decrease in the tissue levels of the
anticarcinogenic 2-MeOE2. Zhu et al. have previously shown that
quercetin, fisetin, catechin, epicatechin and (−)-epigallocatechin-
3-O-gallate (EGCG) are substrates and also strong inhibitors for
the O-methylation of catechol estrogens [19,37]. However, our
results indicated that chrysoeriol, which does not have catechol
structure due to a methoxy substitution at the 3ꢀ position in
the flavone structure, showed no effect on the COMT-mediated
O-methylation of 4-OHE2 until the amounts exceeded 100 ␮M
(Fig. 4).
Furthermore, we found that chrysoeriol significantly inhibited
the formation of 4-MeOE2 but not 2-MeOE2 at concentrations under
50 nM in human breast MCF-7 cells (Fig. 5). On the other hand, the
same amount of chrysoeriol had no effects on gene expression of
CYP1A1, 1B1 or COMT (Fig. 6). Chrysoeriol could not disrupt the
AhR/ARNT pathway at such a concentration, and furthermore, it
could not inhibit the methylation of 4-OHE2 as described above.
Hence, these results indicate that chrysoeriol can inhibit the enzy-
matic activity of CYP1B1, and consequently decrease the formation
of 4-MeOE2 in the cells.
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