Lack of Cell Proliferative and Tumorigenic Effects of 4-Hydroxyestradiol in the Anterior Pituitary of Rats: Role of Ultrarapid O-Methylation Catalyzed by Pituitary Membrane-Bound Catechol-O-Methyltransferase

Article abstract of DOI:10.1021/acs.chemrestox.7b00096

In animal models, estrogens are complete carcinogens in certain target sites. 4-Hydroxyestradiol (4-OH-E₂), an endogenous metabolite of 17β-estradiol (E₂), is known to have prominent estrogenic activity plus potential genotoxicity and mutagenicity. We report here our finding that 4-OH-E₂ does not induce pituitary tumors in ACI female rats, whereas E₂ produces 100% pituitary tumor incidence. To probe the mechanism, we conducted a short-term animal experiment to compare the proliferative effect of 4-OH-E₂ in several organs. We found that, whereas 4-OH-E₂ had little ability to stimulate pituitary cell proliferation in ovariectomized female rats, it strongly stimulates cell proliferation in certain brain regions of these animals. Further, when we used in vitro cultured rat pituitary tumor cells as models, we found that 4-OH-E₂ has similar efficacy as E₂ in stimulating cell proliferation, but its potency is approximately 3 orders of magnitude lower than that of E₂. Moreover, we found that the pituitary tumor cells have the ability to selectively metabolize 4-OH-E₂ (but not E₂) with ultrahigh efficiency. Additional analysis revealed that the rat pituitary expresses a membrane-bound catechol-O-methyltransferase that has an ultralow K_m value (in nM range) for catechol estrogens. On the basis of these observations, it is concluded that rapid metabolic disposition of 4-OH-E₂ through enzymatic O-methylation in rat anterior pituitary cells largely contributes to its apparent lack of cell proliferative and tumorigenic effects in this target site.

Full text of DOI:10.1021/acs.chemrestox.7b00096

Article
pubs.acs.org/crt
Lack of Cell Proliferative and Tumorigenic Effects of
4‑Hydroxyestradiol in the Anterior Pituitary of Rats: Role of
Ultrarapid O‑Methylation Catalyzed by Pituitary Membrane-Bound
Catechol‑O‑Methyltransferase
Pan Wang,†,§ Laura H. Mills,† Ji-Hoon Song,† Jina Yu,† and Bao-Ting Zhu†,‡,*
†Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160,
United States
§State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences,
Beijing 100101, China
‡Kobilka Institute of Innovative Drug Discovery, The Chinese University of Hong Kong (Shenzhen), Shenzhen, Guangdong 518172,
China
S
* Supporting Information
ABSTRACT: In animal models, estrogens are complete carcinogens in
certain target sites. 4-Hydroxyestradiol (4-OH-E2), an endogenous
metabolite of 17β-estradiol (E2), is known to have prominent estrogenic
activity plus potential genotoxicity and mutagenicity. We report here our
finding that 4-OH-E2 does not induce pituitary tumors in ACI female
rats, whereas E2 produces 100% pituitary tumor incidence. To probe the
mechanism, we conducted a short-term animal experiment to compare
the proliferative effect of 4-OH-E2 in several organs. We found that,
whereas 4-OH-E2 had little ability to stimulate pituitary cell proliferation
in ovariectomized female rats, it strongly stimulates cell proliferation in
certain brain regions of these animals. Further, when we used in vitro
cultured rat pituitary tumor cells as models, we found that 4-OH-E2 has
similar efficacy as E2 in stimulating cell proliferation, but its potency is
approximately 3 orders of magnitude lower than that of E2. Moreover, we found that the pituitary tumor cells have the ability to
selectively metabolize 4-OH-E2 (but not E2) with ultrahigh efficiency. Additional analysis revealed that the rat pituitary expresses
a membrane-bound catechol-O-methyltransferase that has an ultralow Km value (in nM range) for catechol estrogens. On the
basis of these observations, it is concluded that rapid metabolic disposition of 4-OH-E2 through enzymatic O-methylation in rat
anterior pituitary cells largely contributes to its apparent lack of cell proliferative and tumorigenic effects in this target site.
rats and mice.11−14 An earlier study showed that the pituitary
ER has a dissociation constant of 0.15 0.06 nM for 4-OH-E2,
INTRODUCTION
■
Estrogens have diverse physiological and pathophysiological
which is essentially the same as the value 0.13
0.03 nM
effects in humans and animals. The endogenous estrogen 17β-
estradiol (E2) is among the most potent nonpeptidal mitogen
in many target organs.1 Excessive stimulation of cell
proliferation mediated by the estrogen receptors (ERs) has
been recognized as an important mechanism for estrogen-
induced tumorigenesis in certain target organs (e.g., uterus,
breast, and pituitary) in experimental animal models.2
However, estrogen may also exert antiproliferative or apoptotic
actions depending on the types of estrogen and its doses used
as well as the cell/tissue types involved.3−5
In humans, catechol estrogens, such as 2- and 4-
hydroxyestradiol (2-OH-E2 and 4-OH-E2), are major oxidative
metabolites of E2 formed by cytochrome P450 enzymes.6 On
the basis of earlier studies, 4-OH-E2 has a relative binding
affinities for ERα and ERβ ranging from 42−150% of E2 in
vitro,7−10 and it also has strong uterotropic activity in female
determined for the uterine ER.15 Another study showed that
the in vivo translocating capacities of the various catechol
estrogens (including 4-OH-E2) correlated well with their
binding affinities for pituitary cytosol receptors determined in
vitro, indicating that 4-OH-E2 not only binds ER but also
activates ER in the pituitary in the same way as E2.16 In addition
to the significant estrogenic activity, catechol estrogens are also
capable of undergoing redox cycling, which generates reactive
oxygen species that may cause DNA damage, mutagenesis, and
ultimately tumorigenesis.17,18
Earlier studies showed that 4-OH-E2 is complete carcino-
genic in inducing tumorigenesis in the kidneys of male Syrian
Received: April 13, 2017
© XXXX American Chemical Society
A
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hamsters.19,20 In recent years, the facile induction of pituitary
and breast tumors in intact female ACI rats by estrogens such
as E2 has been widely used as an animal model for studying the
mechanism of estrogen tumorigenesis.21−23 Surprisingly, an
earlier study reported that 4-OH-E2 failed to induce mammary
tumorigenesis in this animal model, whereas E2 under the same
experimental conditions was a strong inducer of mammary
tumorigenesis.24 The question as to whether 4-OH-E2 can
induce pituitary tumor formation, however, is not known,
which is the focus of our present study.
Metabolic O-methylation of catechol estrogens catalyzed by
catechol-O-methyltransferase (COMT) has received consid-
erable attention in the past 30 years because it abolishes the
potential genotoxicity and mutagenicity of these chemically
reactive estrogen intermediates and thus is considered to be an
important protective mechanism against estrogen tumori-
genesis.25 COMT catalyzes the metabolic O-methylation of
catechol substrates using S-adenosyl-L-methionine (AdoMet) as
methyl donor.26−28 COMT has low substrate specificity and
can catalyze the O-methylation of many substrates, including
endogenous catecholamines, endogenous catechol estrogens,
and various catechol-containing xenobiotics.29,30
COMT is present in most mammals and exists in two
different forms, namely, the soluble form (S-COMT) and the
membrane-bound form (MB-COMT).31,32 These two forms
are encoded by a single gene using two separate promoters P1
and P2.33 The rat S-COMT protein is composed of 221 amino
acids with a molecular weight of 24 kDa.34 The rat MB-COMT
protein contains 43 additional amino acid residues at the N-
terminus, which contains a hydrophobic signal-anchor peptide
anchoring the MB-COMT polypeptide to the endoplasmic
reticulum membrane.35 Earlier studies showed that S-COMT
and MB-COMT prepared from several tissues showed similar
affinity for the O-methylation of catechol estrogens.36 Similarly,
the recombinant human S-COMT and MB-COMT have
similar Km values for the O-methylation of catechol estrogens,30
although MB-COMT displays a 10-fold higher affinity than S-
COMT for the O-methylation of catecholamines.30,37
In the present study, we found that 4-OH-E2 did not induce
pituitary cell proliferation and tumorigenesis in female rats.
Data are presented to suggest that the apparent lack of cell
proliferative and tumorigenic activities of 4-OH-E2 in the rat
pituitary may be partly due to its ultrarapid metabolic
disposition in this target site, likely catalyzed by the pituitary
MB-COMT, which has an ultralow Km value for the O-
methylation of catechol estrogens.
solvents used in this study were of HPLC grade or better and obtained
from Fisher Scientific (Springfield, NJ).
Animal Experiments. All experimental protocols involving the use
of live animals were approved by the Institutional Animal Care and
Use Committee of the University of Kansas Medical Center. The
animal experiments were carried out in accordance with the National
Institutes of Health guidelines for humane treatment of animals. After
arrival, the animals were allowed to acclimatize for a week before being
used in the experiments. The animals were housed under controlled
conditions of temperature and photoperiod (12 h light/12 h dark
cycle) and had free access to standard laboratory rodent chow and
water throughout the experimental period.
For the estrogen-induced tumorigenesis experiment, 62-day-old
intact female ACI rats were randomly divided into 3 groups (with 25−
26 animals per group), and they were implanted s.c. with a small pellet
containing a known amount of E2, 4-OH-E2, or vehicle alone (as
control). Each of the estrogen pellets, in 20 mg total weight, contained
18 μmol of E2 (4.9 mg) or 4-OH-E2 (5.2 mg) plus cholesterol or
contained only cholesterol for the control group. The dose of
estrogens used in this study was comparable to the dose used in an
earlier study, where a pellet containing 4 mg of E2 was used.24 The
pelleting components were thoroughly mixed and manually com-
pressed into a small cylindrical pellet using a Parr Pellet Press (Parr
Instrument Company Moline, IL). These pellets were surgically
implanted under the back skin of the animals under halothane
anesthesia.
The animals were weighed weekly and also examined for the
presence of palpable mammary tumors. The decision to terminate an
animal during the course of the long-term tumorigenesis experiment
was made when apparent moribund signs were observed (usually due
to the presence of large pituitary tumors). At the end of the
experiment, the animals were sacrificed with CO2 overdose followed
by decapitation. The trunk blood was collected in the Vacutainer test
tubes (Fisher Scientific, Springfield, NJ) containing sodium heparin,
and the plasma samples were prepared for storage at −80 °C for future
analysis of hormone levels. At the time of death, the size and location
of the tumors in each animal were recorded, and the weights of the
tumor(s) and of the pituitary were also determined. The pituitary was
removed for histopathological analysis. The removed tissues were
stored in 10% buffered neutral formalin overnight followed by
dehydration through a sequential transfer through 80−100% ethanol
and then 100% xylene. The tissues were embedded in paraffin blocks,
cut into 7 μm sections, and placed on Superfrost microscope slides
(Fisher Scientific, Springfield, NJ). The sections were stained with
hematoxylin and eosin and evaluated under a light microscope by a
board-certified pathologist with expertise in cancer pathology.
For the short-term biological activity study, 6-week-old ovariec-
tomized female Sprague−Dawley (SD) rats (from Harlan Sprague−
Dawley Laboratory, Houston, TX) were used. Ovariectomized SD rats
have been widely used as the animal model for accessing the estrogenic
activity of chemicals.38,39 The animals were randomly divided into
three groups: control, E2 (20 μg/rat), and 4-OH-E2 (20 μg/rat). The
dose of the estrogens was selected according to earlier reports.40,41 All
steroids were injected i.m. into the animals once every 2 days for 7
days. To quantify the rate of cell proliferation in the pituitary and also
other target organs, we performed immunohistochemical detection of
BrdU-stained cells as described in our earlier study.42 In brief, 2 h
before sacrifice, the animals were injected i.p. with BrdU at 50 μg/g
body weight (dissolved in sterile saline). The pituitary and brain were
removed, fixed, dehydrated, embedded in paraffin blocks, and cut into
5 μm sections. The sections were rehydrated and blocked using 1%
normal horse serum. The sections were incubated with anti-BrdU
primary antibody (Novocastra, Newcastle, UK) for 60 min followed by
a biotinylated horse antimouse secondary antibody (Vector Labo-
ratories, Burlingame, CA) for 45 min at room temperature. Detection
was performed using ABC Elite reagent (Vector Laboratories), and
color was developed using diaminobenzidine (Sigma Chemical Co.)
for 5 min. Hematoxylin was used as a counterstain. The number of
cells that incorporated BrdU were determined using a light
microscope.
METHODS
■
Chemicals and Reagents. E2, 2-OH-E2, and 4-OH-E2 were
purchased from Steraloids (Newport, RI). 4-OH-E2 was purified with
high-performance liquid chromatography (HPLC) to remove residual
E2 contamination prior to use in the experiments. The purities of 4-
OH-E2, E2, and 2-OH-E2 used in this study were higher than 99%
based on HPLC analysis. Additional analysis of 4-OH-E2 by gas
chromatography−mass spectrometry (GC/MS) found no detectable
E2 as a contaminant. Dulbecco’s modified Eagle’s medium (DMEM),
horse serum, fetal bovine serum, AdoMet, and 1,4-dithiothreitol were
obtained from Sigma-Aldrich (St. Louis, MO). [Methyl-3H]AdoMet
(specific activity 11.2−13.5 Ci/mmol) was obtained from PerkinElmer
(Waltham, MA). Monoclonal mouse anti-BrdU primary antibody,
biotinylated antimouse IgG, Vectastain ABC kit, and DAB kit were
obtained from Vector Laboratories (Burlingame, CA). Anti-COMT
antibody was obtained from Millipore (Billerica, MA). Sulfatase from
Helix pomatia was purchased from Sigma-Aldrich (St. Louis, MO). All
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Radioimmunoassay (RIA) of the Plasma Levels of Prolactin
(PRL), Luteinizing Hormone (LH), and Follicle Stimulating
Hormone (FSH). To measure the plasma levels of E2, PRL, LH, and
FSH, whole blood samples were collected from each animal after
decapitation and placed in Vacutainer test tubes containing heparin
sodium (Fisher Scientific, Springfield, NJ). The collected whole blood
samples were centrifuged for 20 min at 1000g. The plasma samples
were transferred precisely to another set of small vials with sealed caps
and stored at −80 °C. The plasma levels of rat LH, FSH, and PRL
(ng/mL) were determined by highly sensitive, specific, and
quantitative RIAs using the RIA reagent sets prepared and distributed
by the National Hormone and Peptide Program of the National
Institute of Diabetes and Digestive and Kidney Diseases.
Tumor Cell Culture. Rat pituitary tumor cell lines GH3 and RC-
4B/C and human breast cancer cell line MCF-7 were obtained from
the American Type Culture Collection (Manassas, VA). The culture
medium for GH3 is DMEM supplemented with 12.5% horse serum
and 2.5% fetal bovine serum (FBS). The culture medium for RC-4B/C
cells is DMEM supplemented with 10% FBS, 0.2 mg/mL of bovine
serum albumin, and 2.5 ng/mL of epidermal growth factor. MCF-7
cells are maintained in EMEM containing 10% FBS supplemented
with 100 units/ml of penicillin, 100 μg/mL of streptomycin, 2 μg/mL
of insulin, 0.5 mM sodium pyruvate, 10 mM nonessential amino acids,
and 2 mM L-glutamine. To study the proliferative effect of E2 and 4-
OH-E2 in GH3, RC-4B/C, and MCF-7 cells, we used phenol red-free
medium supplemented with dextran-coated charcoal-stripped FBS and
horse serum. The charcoal-stripping procedure was carried out as
described earlier,43 which was employed to remove the endogenous
estrogens present in the serum.
pher with a 7683 autosampler and the HP-5MS capillary column (30
m × 0.25 mm × 0.25 μm) coupled with an Agilent-5973 mass
spectrometer. Helium was used as the carrier gas. The injector and
detector temperatures were 260 and 280 °C, respectively. During
analysis, the column temperature was increased from 180−260 °C at a
rate of 4 °C/min and then maintained isothermally at 260 °C for 5
min. Then, the temperature was increased to 300 °C at a rate of 5 °C/
min and then maintained isothermally at 300 °C for 5 min. The mass
spectrometer was operated in the electron impact mode (70 eV). Mass
abundance was determined by the SIM mode. The E2 and 4-OH-E2
were quantified according to the area of the peak and normalized with
internal standard E2-d5.
Preparation of GH3 Cell Lysates and Rat Pituitary and Liver
Homogenates. GH3 cells were propagated in 75 cm2 flasks to
approximately 80% confluence. They were then detached from the
flasks by treatment with 3 mL of a trypsin-EDTA solution for 5 min.
The cell pellets were washed in PBS once and then kept at −80 °C
until they were used for preparation of cellular lysates. The pituitary
glands from female Sprague−Dawley rats were purchased from Sierra
for Medical Science (Whittier, CA).
Cell pellets, rat pituitaries, and livers were defrosted on ice and
immersed in 50 mM Tris-HCl buffer (pH 7.5) containing a protease
inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell pellets were
lysed with ultrasound sonication on ice for 5 min. Rat pituitary and
liver tissues were homogenized on ice using a Wheaton homogenizer
(Millville, NJ) for 10 min. The whole homogenates were then
centrifuged at 4 °C at 10,000g for 15 min to remove intact cells and
nuclei. The supernatant was used as the cell lysates or crude tissue
homogenates in the COMT-mediated methylation assays.
The cells were first propagated in the 75 cm2 flasks to approximately
80% confluence under 37 °C air with 5% CO2 and 95% humidity.
They were then detached from the flask by treatment with 3 mL of the
trypsin-EDTA solution for 5 min. Cell suspensions were centrifuged.
and the cell sediments were resuspended in the culture medium at the
desired 105 cells/mL density. A 100 μL aliquot of the cell suspension
was then added to each well of the 96-well microplate usually at a final
density of 104 cells per well. After the cells were allowed to attach and
grow for 24 h, the cell culture medium was changed, and different drug
treatments were introduced at that time. In most experiments, the
drug treatment lasted for 6 days with one medium change on the
fourth day following the initial drug treatment.
The cell viability in the 96-well microplates was determined using
the MTT assay. Briefly, 22 μL of 5 mg/mL MTT was added to each
well of the 96-well microplate, and the plate was incubated at 37 °C for
1 h. The medium was removed at the end of the incubation, and 100
μL of DMSO was added to dissolve the formazan crystals. The
absorbance value of each well was measured at 560 nm with a UVmax
microplate reader (Molecular Device, Palo Alto, CA).
In some of the experiments, the microsomal and cytosolic fractions
were further isolated from the cell lysates or crude tissue homogenates
as described earlier.46 The GH3 cell lysates or crude tissue
homogenates were subjected to centrifugation at 4 °C at 100,000g
for 2 h, and the supernatant fraction (cytosolic fraction) was used as
the source of S-COMT. The pellet (microsomal fraction) was
suspended in Tris-HCl buffer (pH 7.5) containing a protease inhibitor
cocktail and it was used as the source of MB-COMT.
Fragmentation of GH3 Cell Lysates with Gel Filtration. The
GH3 whole cell lysates containing 100 mg of total protein were
applied on a column (20 cm × 3 cm) packed with Bio-Rad P60 gel.
Elution was performed on an FPLC with 50 mM Tris-HCl as the
mobile phase and a UV detector for protein monitoring. The fraction
was collected at a flow rate of 0.1 mL/min and a volume of 1 mL/tube.
The whole procedure was carried out at 4 °C.
Assay of the COMT-Mediated O-Methylation of Catechol
Substrates. The COMT-mediated O-methylation of catechol estro-
gens (4-OH-E2 or 2-OH-E2) was carried out as described earlier.30
The reaction mixture consisted of 0.5 mg/mL of total protein, different
concentrations of 4-OH-E2 or 2-OH-E2 as substrate, 250 μM AdoMet
(containing 0.5 mCi [methyl-3H]AdoMet), 1.2 mM MgCl2, and 1 mM
dithiothreitol in a final volume of 200 μL of 50 mM Tris-HCl buffer
(pH 7.5). The reaction was initiated by adding the enzyme preparation
and was carried out at 37 °C for 30 min. After incubation, the reaction
was arrested by immediately placing the tubes on ice and followed by
addition of 500 μL of ice-cold saline and then extracted with 2 mL of
n-heptane. After a brief centrifugation at 1000g, 1 mL of the organic
extracts was measured for radioactivity content in a liquid scintillation
counter. The enzyme kinetic parameter Km values for the O-
methylation of catechol estrogens were calculated using the
commercial enzyme kinetics curve regression program of the
SigmaPlot software (version 9; Systat Software, Inc., San Jose, CA),
which is based on the Michaelis−Menten equation and its curve
pattern.
Analysis of Estrogen Levels in Cell Culture Medium. GH3
cells were seeded in 6-well plates in regular culture medium, and 100
nM of 4-OH-E2 or E2 in DMEM (without serum) was introduced. The
medium was collected after 60 min of incubation. Aliquots (1 mL) of
the culture medium were transferred to a 1.5 mL microcentrifuge tube
containing 200 μL of Na2CO3 buffer (2 M, pH 5.0). The mixture was
centrifuged at 10, 000g for 5 min, and 1 mL of supernatant was
transferred to a small glass tube. Ten microliters of ascorbic acid (0.15
mg/μL) was added as antioxidant; 20 μL of 0.5 ng/μL deuterated 17β-
estradiol-d5 (E2-d5) (in 200 proof ethanol) was added as internal
standard, and 10 μL of H-2 sulfatase (2,000 units/mL) was added as
the enzyme for hydrolysis of sulfated estrogens. The reaction mixture
was incubated at 37 °C for 4 h. After incubation, the tubes were
centrifuged at 1500g for 10 min, and the supernatant was transferred
to another set of test tubes and extracted with 5 mL of ethyl acetate.
The organic extracts (4.5 mL) were precisely transferred and dried
under a stream of nitrogen gas. For derivatization, bis(trimethylsilyl)-
trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)
(50 μL) was added to the dried sample in a small glass vial, and the
mixture was incubated at 65 °C for 0.5 h.
Western Blotting Analysis for COMT. Cytosolic and microsomal
fractions that were prepared from the whole homogenates of GH3
cells and rat pituitary tissues were boiled in the SDS sample buffer
(containing 2% SDS, 10% glycerol, 1.2% 2-mercaptoethanol, and
0.02% bromophenol blue in 50 mM Tris-HCl, pH 6.8) for 5 min at
100 °C. Equal amounts of total proteins (approximately 30 μg/lane)
were electrophoresed in 12% polyacrylamide gel and then transferred
The GC/MS was carried out as described earlier.44,45 Briefly, the
GC/MS apparatus consisted of an Agilent 6890N gas chromatogra-
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Figure 1. Effect of chronic administration of E2 or 4-OH-E2 on pituitary tumor induction in female ACI rats. (A) Weights of rat pituitary glands
following chronic treatment with E2 or 4-OH-E2. The y-axis values represent the mean SEM of the pituitary weights of the animals, and the x-axis
values represent the mean SD of the treatment days for all animals. The filled circle, filled triangle, and empty circle indicate the treatment groups
that received vehicle only (control), E2, or 4-OH-E2, respectively. (B) Plasma levels of PRL, FSH, and LH at the end of the long-term experiment,
which were analyzed at the National Hormone and Pituitary Program using the 125I-labeled RIAs. Each value is the mean SEM (N = 25). *P <
#
0.05 compared to the control, and P < 0.05 compared to the E2 group.
to PVDF membranes. The membranes were treated with 5% nonfat
milk for 1 h to block nonspecific binding and then rinsed and
incubated with the COMT antibody (at 1:5000 dilution). The
membranes were then treated with horseradish peroxidase-conjugated
antirabbit IgG (at 1:5000 dilution) for 1 h. The protein bands were
detected on X-ray films with the aid of a chemiluminescence substrate.
Statistical Analysis. The quantitative data are expressed as means
SEM. The Student’s t-tests were used to compare paired data and
the one-way analysis of variance followed by Dunnett’s tests was used
for multiple comparisons. For the BrdU staining data, the Chi-square
test was used. P < 0.05 was considered statistically significant.
scopic examination of the pituitary revealed that not a single
animal in these two groups developed a pituitary tumor. The
average pituitary weights in 4-OH-E2-treated animals were 17.3
1.3 mg, which were slightly increased over the control group
(11.7 0.8 mg) (P < 0.05) (Figure 1A).
The plasma levels of prolactin (PRL) in control animals and
in animals chronically treated with 4-OH-E2 were 70.0 20.6
and 71.1 11.0 ng/mL, respectively, whereas the plasma levels
of prolactin in E2-treated animals were 6050
467 ng/mL
(Figure 1B). Histopathological examination showed the
presence of adenomas (prolactinoma) with focal hemorrhage,
cystic change, and apoplectic necrosis in E2-treated animals, but
no appreciable histological abnormalities were observed in the
pituitary for 4-OH-E2-treated animals (data not shown). The
drastic change in plasma prolactin levels is consistent with the
histological observation of the presence of prolactinoma in the
anterior pituitary. Moreover, chronic E2 administration
markedly reduced the plasma levels of FSH and LH (Figure
1B), and this effect is known to be an estrogen receptor-
mediated action (commonly referred to as estrogen’s feedback
regulation) on the anterior pituitary cells that secrete FSH and
LH.47 In comparison, 4-OH-E2 had a detectable but weaker
effect than E2 in regulating FSH and LH release (Figure 1B),
RESULTS
■
4-OH-E2 Fails to Induce Pituitary Tumorigenesis in
Female ACI Rats. First, we compared the tumorigenic activity
of 4-OH-E2 and E2 in the pituitary of intact female ACI rats. All
animals implanted with an E2 pellet developed a large pituitary
tumor with an average weight of 254 19 mg (Figure 1A). All
animals in this group became severely moribund at 5−6.5
months after E2 implantation, most likely due to the presence
of a large pituitary tumor, and they had to be euthanized. In
comparison, all the control animals (implanted with a vehicle
pellet) and those implanted with a 4-OH-E2 pellet remained
healthy during the entire course of the experiment. Macro-
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which is consistent with the lower estrogenic activity of 4-OH-
E2 in vivo.
to stimulate rat pituitary cell proliferation is due to an intrinsic
lack of sensitivity of pituitary cells to this estrogen metabolite or
due to the low bioavailability of 4-OH-E2 inside these cells, we
conducted in vitro experiments to compare the proliferative
effect of 4-OH-E2 and E2 using two ER-positive pituitary tumor
cell lines (GH3 and RC-4B/C) as models. Our basic
assumption is that, if the pituitary cells indeed cannot respond
to 4-OH-E2’s proliferative action, then we should not be able to
see significant stimulation of cell proliferation in these cells
even when very high concentrations of 4-OH-E2 are present in
the culture medium. However, if it is simply due to the low
bioavailability of 4-OH-E2 inside the pituitary cells, then we
should still be able to see significant cell proliferation when
sufficiently high concentrations of 4-OH-E2 are present in the
culture medium.
Chronic administration of E2 is known to decrease an
animal’s body weight gain in a dose-dependent manner, and
this effect was reported to be associated with the activity of
ERα.48 Although 4-OH-E2 had no tumorigenic activity in the
pituitary, it surprisingly produced a slightly stronger reduction
in body weight during the first 150 days of estrogen treatment
(Figure 2). We observed in this study that 4-OH-E2 has a
As shown in Figure 3B, E2 strongly stimulated the
proliferation of GH3 and RC-4B/C cells in vitro with EC50
values of 0.02 and 0.11 nM, respectively. The EC50 values of E2
in these two cell lines are consistent with the known
mechanism of ERα-associated stimulation of cell proliferation.
In comparison, 4-OH-E2 did not have an appreciable
stimulatory effect on cell proliferation in these cells until its
concentration reached >10 nM. As such, the potency of 4-OH-
E2’s proliferative effect in GH3 and RC-4B/C cells is
approximately 3 orders of magnitude lower than that of E2.
Notably, despite the very low potency of 4-OH-E2, its dose−
response curve patterns in stimulating the proliferation of GH3
and RC-4B/C cells closely resemble those of E2, which suggests
that the proliferative effects of both estrogens are mediated by
the same ERs and that 4-OH-E2 has a similar efficacy to that of
E2. Together, these results suggest that the very low
bioavailability of 4-OH-E2 in rat pituitary cells is the major
underlying cause for its apparent lack of a cell proliferative
effect in vivo.
Rat Pituitary Cells Metabolize 4-OH-E2 Much Faster
than E2. To provide experimental evidence for the suggestion
that the apparent lack of a cell proliferative effect by 4-OH-E2 in
the rat pituitary is due to its rapid local metabolic disposition,
we used cultured GH3 tumor cells as an in vitro model and
examined the rate of metabolic disposition of E2 and 4-OH-E2.
We found that the disappearance of 4-OH-E2 in the GH3 cell
culture medium was far faster than the disappearance of E2
under the same cell culture conditions (Figure 4). These data
suggest that the rapid metabolism of 4-OH-E2 may be
responsible for the abrogation of the proliferative and
tumorigenic activities of this highly estrogenic E2 metabolite
in rat pituitary cells.
Rat Pituitary COMT Displays Two-Component Ki-
netics for the O-Methylation of Catechol Estrogens. It
is known that the COMT-mediated O-methylation is a selective
pathway for the metabolic inactivation of catechol estrogens but
not for the inactivation of E2.28 Therefore, we hypothesized that
the pituitary may be able to very rapidly methylate catechol
estrogens. To test this hypothesis, we first sought to
characterize the enzyme kinetics for the O-methylation of 4-
OH-E2 when the crude lysates prepared from GH3 cells were
used as the enzyme source (Figure 5A). We found that the
enzyme kinetics had two components as revealed by the
Eadie−Hofstee plot: one with a very low Km at 51 nM and the
other with a high Km at 7.7 μM. Similarly, the COMT present
in rat pituitary homogenates also showed similar two-
component kinetics with Km values of 40 nM and 6.7 μM,
respectively, for the O-methylation of 4-OH-E2 (Figure 5B).
Figure 2. Body weight changes after chronic administration of E2 or 4-
OH-E2 in female ACI rats. Each value is the mean SEM (N = 7).
stronger effect than E2 for reducing the daily food intake by the
animals (data not shown). The body weight reduction in E2-
treated animals accelerated after the first 150 days following E2
treatment, likely due to the rapid development of pituitary
tumors.
4-OH-E2 Fails to Stimulate Pituitary Cell Proliferation.
To determine whether 4-OH-E2 can stimulate cell proliferation
in the pituitary or other estrogen target organs or tissues, we
used the BrdU-labeling method and compared the proliferative
effect of E2 and 4-OH-E2 in several target tissues of female
ovariectomized rats. Previous studies have reported that E2 can
strongly stimulate cell proliferation in both the pituitary and
subventricular zone (SVZ) of the brain.1,49−51 Administration
of 4-OH-E2 for 7 days did not show a significant stimulation of
anterior pituitary cell proliferation (Figure 3A, Figure S1), but it
strongly stimulated cell proliferation in the SVZ of the brain. 4-
OH-E2 also increased uterine wet weight gain (Figure 3A). The
effect of 4-OH-E2 in SVZ cell proliferation and uterus wet
weight gain was slightly weaker than those of E2, which is in line
with their known ERα-binding affinities.7 On the basis of these
in vivo observations, it is apparent that the lack of proliferative
effect of 4-OH-E2 in the rat pituitary is an organ-selective
phenomenon.
4-OH-E2 has High Efficacy but Low Potency in
Stimulating Rat Pituitary Tumor Cell Proliferation in
Culture. To probe whether the selective inability of 4-OH-E2
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Figure 3. Effect of E2 or 4-OH-E2 on pituitary cell proliferation in vitro and in vivo. (A) Effect of E2 and 4-OH-E2 on the uterine wet weight (left)
and cell proliferation in the anterior pituitary (middle) and subventricular zone (SVZ) (right) in ovariectomized female rats. Each value is the mean
SEM (N = 7). (B) Effect of E2 and 4-OH-E2 on the proliferation of GH3 and RC-4B/C cells in culture. Cells were treated with different
concentrations of E2#or 4-OH-E2 for 6 days with one medium change on the third day. Each value is the mean SEM (N = 6). *P < 0.05 compared
to the control, and P < 0.05 compared to the E2 group (Chi-square test).
Figure 4. Metabolism of 4-OH-E2 and E2 in GH3 cell culture medium. Cells were treated with 100 nM 4-OH-E2 or E2 for 60 min and, then 1 mL of
cell culture medium was retrieved and processed for GC/MS analysis. The quantity (in ng) of each estrogen was normalized to the internal standard
E2-d5. The control value was derived from the respective cell culture medium containing 100 nM of 4-OH-E2 or E2 right before it was added to the
cell culture. Each value is the mean SEM (N = 4).
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Figure 5. GH3 pituitary tumor cells and rat pituitary tissue display two-component kinetics for the O-methylation of 4-OH-E2. Enzyme kinetics for
the O-methylation of 4-OH-E2 when (A) GH3 cell, (B) rat pituitary, or (C) rat liver crude homogenate was used as the enzyme source. The right
panels are the Eadie−Hofstee plots that are converted from the data in the corresponding left panels. Each value is the mean SEM (N = 4).
For comparison, we also determined the COMT in rat liver
homogenates and found that the liver COMT had single-
component kinetics with an apparent Km value of 10.3 μM
(Figure 5C).
Similarly, when 2-OH-E2 was used as substrate, the GH3 cell
lysates and the rat pituitary tissue homogenates also displayed
two-component kinetics with low Km values of 135 and 68 nM,
respectively, and high Km values of 7.1 and 4.2 μM, respectively
(Figure 6A and B). In comparison, the rat liver crude
homogenates showed similar single-component kinetics with
an apparent Km value of 13.3 μM (Figure 6C).
MB-COMT Shows Ultrahigh Activity for Catechol
Estrogens. COMT exists in two different forms, namely, S-
COMT and MB-COMT.31,32 To determine which protein was
responsible for the low and high Km COMT activity, we used a
gel filtration column (Bio-Rad P60 gel) to separate the whole
cellular protein preparation from GH3 cells according to the
protein size. Then, we assayed the COMT activity of the
separated fractions (data shown in Figure 7A). It is apparent
that there are two different proteins (labeled COMT I and
COMT II) that contain the catalytic activity for O-methylation.
As revealed by Western blotting analysis (Figure 7B), the
COMT I fractions contained the MB-COMT, whereas the
COMT II fractions contained S-COMT. The MB-COMT was
eluted much earlier through the gel filtration column, indicating
that its gross molecular weight is much larger than that of S-
COMT, probably due to its association with the membrane
lipids.
Next, we separately analyzed the enzyme kinetics of the
COMT I and COMT II fractions using 4-OH-E2 as substrate.
As shown in Figure 7C, COMT I displayed the single-
component kinetics with a low Km value of 76 nM for 4-OH-E2,
whereas COMT II displayed the single-component kinetics
with a high Km value of 15 μM for 4-OH-E2.
To further characterize the kinetics of MB-COMT and S-
COMT, we separated the microsomal fraction (containing
mostly MB-COMT) from the cytosolic fraction (containing
mostly S-COMT) using the standard high-speed centrifugation
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Figure 6. GH3 pituitary tumor cells and rat pituitary tissue display two-component kinetics for the O-methylation of 2-OH-E2. Enzyme kinetics for
the O-methylation of 2-OH-E2 when (A) GH3 cell, (B) rat pituitary, or (C) rat liver crude homogenates were used as the enzyme source. The right
panels are the Eadie−Hofstee plots that are converted from the data in the corresponding left panels. Each value is the mean SEM (N = 4).
for both GH3 cells and rat pituitary tissues. As shown by
Western blotting analysis (Figure 8A), MB-COMT was
concentrated in the microsomal fraction, whereas S-COMT
was concentrated in the cytosolic fraction prepared from both
GH3 cells and the rat pituitary tissue. In addition, GH3 cells
and rat pituitary tissues contained roughly equal levels of MB-
COMT and S-COMT (Figure 8A). Next, we separately
determined the kinetics of MB-COMT and S-COMT using
4-OH-E2 and 2-OH-E2 as substrates (Figures 8B and C and 9A
and B). The Km values are summarized in Table 1. Both MB-
COMT and S-COMT displayed single-component kinetics
when 4-OH-E2 or 2-OH-E2 was used as substrate. MB-COMT
had a much lower Km value compared to that of S-COMT for
both 4-OH-E2 and 2-OH-E2. MB-COMT had no preference for
4-OH-E2 over 2-OH-E2, but S-COMT displayed a slight
difference (approximately 1-fold higher preference for 2-OH-E2
over 4-OH-E2).
DISCUSSION
■
It has been known for many years that estrogen can induce
pituitary cell proliferation and tumorigenesis in some of the rat
strains in both male and female rats.1,2,52,53 Pathological
analysis revealed that the pituitary tumors induced by chronic
treatment with an estrogen are usually of the prolactinoma
type,54 which is also confirmed in the present study. 4-OH-E2 is
an endogenous estrogen metabolite with strong estrogenic
activity and high binding affinity for the ERs.6,7 Moreover, this
estrogen metabolite is chemically reactive, capable of causing
genotoxicity and mutagenicity.17,18,20 Earlier studies have
shown that 4-OH-E2, like E2, could induce tumorigenesis in
male Syrian hamsters with a high tumor incidence.19 Therefore,
it is rather unexpected that this highly tumorigenic estrogen
derivative fails to induce tumorigenesis in the pituitary of
female ACI rats, whereas E2 serves as a strong tumor inducer.
In line with the observation of a lack of tumorigenic activity, we
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Figure 7. Separation of COMT proteins in GH3 cell lysates for analysis of the catalytic activity. (A) COMT activity for each fraction eluted from the
gel filtration column. The estimated protein size of each fraction was labeled under the elution volume. (B) Western blotting analysis of the COMT
proteins contained in each fraction eluted from the gel filtration column. “T” represents the cell lysate. (C) Enzyme kinetics for O-methylation with
4-OH-E2 catalyzed by the COMT I and COMT II fractions.
also found that 4-OH-E2 fails to stimulate cell proliferation in
the pituitary of female ACI rats. It is apparent that this lack of
proliferative effect in rat pituitary is organ-selective, because 4-
OH-E2 is able to induce cell proliferation in the SVZ brain
region in female rats.
Because it is known that rat pituitary expresses ERα and that
both E2 and 4-OH-E2 have high binding affinities for ERα,7,55 it
is puzzling that even though E2 is a strong mitogen and a strong
tumor inducer in rat pituitary, 4-OH-E2 fails to induce cell
proliferation and tumorigenesis in this organ. A more plausible
explanation for this discrepancy is because 4-OH-E2 may have
very low bioavailability in pituitary cells. Another less likely
possibility may be that there is a unique modification in
pituitary ERs that renders the cells selectively insensitive to the
proliferative action of 4-OH-E2. To experimentally probe these
two possibilities, we designed an in vitro experiment to
compare the proliferative effect of 4-OH-E2 and E2 in two ER-
positive pituitary prolactinoma cell lines (GH3 and RC-4B/C).
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Figure 8. Enzyme kinetics for the O-methylation of 4-OH-E2 catalyzed by microsomal and cytosolic fractions prepared from GH3 cells and rat
pituitary tissues. (A) Western blotting analysis of the COMT proteins in the microsomal (M) and cytosolic (C) fractions prepared from GH3 cells
and rat pituitary tissues. (B and C) Enzyme kinetics for the O-methylation of 4-OH-E2 catalyzed by the microsomal and cytosolic fractions prepared
from GH3 cells (B) and rat pituitary tissues (C). The fractionation of cell lysates and crude tissue homogenates was carried out using the differential
centrifugation method as described in the Methods section.
Our basic premise is that, if the pituitary cells indeed cannot
respond to 4-OH-E2’s proliferative action due to the presence
of a unique modification in the ERs, then we should not be able
to see significant stimulation of proliferation in these cells even
when very high concentrations of 4-OH-E2 are present.
However, if it is simply due to the low bioavailability of 4-
OH-E2 in pituitary cells in vivo, then we should still be able to
see a full-range of growth stimulation when higher concen-
trations of 4-OH-E2 are used under controlled in vitro
conditions.
with the known mechanism of ERα-mediated stimulation of
cell proliferation.43 In comparison, 4-OH-E2 does not have an
appreciable growth-stimulatory effect in these cells until its
concentrations reaches >10 nM. Notably, the overall dose−
response curve patterns of 4-OH-E2’s proliferative effect in both
GH3 and RC-4B/C cells are very similar to those of E2,
although the estimated potency of 4-OH-E2 is approximately 3
orders of magnitude lower than that of E2. Considering the fact
that the difference in the ER-binding affinity of E2 and 4-OH-E2
are very small in vitro,7 these results suggest that the very low
bioavailability of 4-OH-E2 in rat pituitary cells likely is the main
underlying cause for its apparent lack of proliferative effect in
vivo. Providing experimental support for this suggestion, we
found that approximately 99% of 4-OH-E2 disappeared from
Our results showed that E2 can strongly stimulate the
proliferation of GH3 and RC-4B/C cells in vitro with EC50
values at 0.02 and 0.11 nM, respectively. The effects of E2 in
these cells are as expected, and the EC50 values are consistent
J
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Figure 9. Enzyme kinetics for the O-methylation of 2-OH-E2 catalyzed by microsomal (M) and cytosolic (C) fractions prepared from (A) GH3 cells
and (B) rat pituitary tissues. The fractionation of the cell lysates and crude tissue homogenates was carried out using the differential centrifugation
method as described in the Methods section.
the culture medium of rat pituitary tumor cells within the first
hour of addition, whereas ∼90% of E2 remained in the culture
medium. However, it is also possible that an estrogen may exert
some of its pituitary effects in vivo via its initial action in the
hypothalamus, and this possibility cannot be ruled out.
Considering the fact that the metabolic machinery present in
pituitary cells can only rapidly dispose of 4-OH-E2 but not E2, it
naturally prompted us to probe the COMT-mediated O-
methylation in pituitary cells because COMT can only catalyze
the metabolic disposition of catechol estrogens (including 4-
OH-E2), but not E2. We found, for the first time, that rat
pituitary MB-COMT displays an ultrahigh affinity for the O-
methylation of catechol estrogens with Km values around 40−
51 nM, whereas the pituitary S-COMT has an affinity
approximately 150-times lower with a Km of approximately 7
μM.
Notably, there were several earlier studies that had also
determined the Km values for the COMT-mediated O-
methylation of catechol estrogens in selected tissues
(summarized in Table 2).30,36,56,57 They all reported quite
K
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One of the biological functions of the ultralow Km pituitary
COMT may serve as a built-in mechanism to effectively protect
this organ against highly tumorigenic catechol estrogens. It is
also possible that the ultralow Km pituitary COMT may rapidly
metabolize dopamine, which is an important neurotransmitter
in the anterior pituitary and can inhibit prolactin release and
lactotroph proliferation.59 On the other hand, it should be
noted that the rat pituitary expresses high levels of catechol
estrogen-forming enzymes,60 which can convert endogenous
estrogens to catechol estrogens. Moreover, the pituitary also
has the unique ability to rapidly convert catechol estrogens to
methoxyestrogens as shown in this study. It is possible that
methoxyestrogens may be endogenously formed estrogen
derivatives with some unique biological functions in certain
target sites (such as the pituitary). In line with this speculation,
it is of note that methoxyestrogens were reported to have a
unique ability to activate the nongenomic pathways in certain
systems61,62 despite the fact that they are devoid of significant
binding affinity for the ERs.7
Table 1. Km Values for the MB- and S-COMTs Present in the
GH3 Cells, Rat Pituitary Tissue, and Rat Liver Tissue When
2-OH-E2 and 4-OH-E2 Were Used as Substrates
Km value
substrates
4-OH-E2
enzyme source
MB-COMT
S-COMT
GH3
72 nM
3.2 μM
3.1 μM
pituitary
liver
123.3 nM
10.3 μM (crude homogenates)
2-OH-E2
GH3
94 nM
75 nM
1.8 μM
1.2 μM
pituitary
liver
13.3 μM (crude homogenates)
Table 2. Km Values for the MB- and S-COMTs Present in
Different Tissues When 2-OH-E2 and 4-OH-E2 Were Used
as Substrates
Km for MB-
COMT (μM)
Km for S-
COMT (μM)
a
substrate
enzyme source
ref
2-OH-E2 rabbit aorta
2-OH-E2 recombinant
0.15
3.2
0.27
3.6
36
30
It is worth noting that an earlier report also showed that 4-
OH-E2 failed to induce mammary tumor formation in female
ACI rats, whereas E2 under the same experimental conditions
strongly induced mammary tumorigenesis.24 The lack of a
mammary tumorigenic effect of 4-OH-E2 is likely due to a
combination of two contributing factors. One is the lack of
elevated prolactin release in animals treated with 4-OH-E2
(Figure 1); epidemiological studies revealed that circulating
PRL is an important risk factor for breast cancer.63,64 In rodent
animal models, overexpression of prolactin induces breast
cancer development.65 Another important factor (data shown
in Figure S2) is that 4-OH-E2 is also very rapidly O-methylated
by COMT in mammary glandular cells, which abrogates its
estrogenicity and cell proliferative activity. We found that the
COMT proteins contained in the crude homogenates from
MCF-7 human breast cancer cells have high affinity for the O-
methylation of 4-OH-E2 with an apparent Km of 310 nM with
single-component kinetics (Figure S2). Moreover, the EC50 of
4-OH-E2 for stimulating MCF-7 cell proliferation is approx-
imately 1 nM, which is 50-fold higher than the EC50 of E2 in
these cells (approximately 0.02 nM) (Figure S3), whereas the
ERα-binding affinity of 4-OH-E2 is approximately 70% of E2.7
Together, these data likely suggest that the rapid COMT-
mediated O-methylation of 4-OH-E2 may also be a contributing
factor for its apparent lack of tumorigenicity in the breast.
The carcinogenesis of estrogens mainly involves two
mechanisms. First, excessive stimulation of cell proliferation
mediated by the estrogen receptors (ERs) has been recognized
as an important mechanism for estrogen-induced tumori-
genesis.66 Second, numerous studies have shown that the
cytochrome P450-catalyzed formation of chemically reactive
metabolites of endogenous estrogens, i.e., catechol estrogens, is
capable of undergoing redox cycling between o-quinones and
semiquinone radicals.2,17,18,67 This redox cycling leads to the
formation of reactive oxygen species (ROS) including super-
oxide, hydrogen peroxide, and ultimately the hydroxyl
radical.2,68,69 These free radicals cause oxidation of the
nucleotide residues of DNA and oxidative cleavage of the
DNA backbone, which results in the formation of DNA
adducts, DNA strand breaks, and generated DNA mutations
that can lead to cancer initiation.67,70−75 Thus, the damage of
DNA by estrogens is mostly dependent on the conversion of
estrogens to catechol estrogens, whereas the cell proliferative
effect is not. Our results suggest that, in the pituitary where 4-
human COMT
4-OH-E2 recombinant
human COMT
6.4
4.5
30
4-OH-E2 pig endometrium
2-OH-E2 pig endometrium
4-OH-E2 hamster kidney
4-OH-E2 rat kidney
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.44
0.77
2.99
5.81
4.55
7.16
1.46
3.84
4.93
10.0
56
56
57
57
57
57
57
57
57
57
4-OH-E2 hamster liver
4-OH-E2 rat liver
2-OH-E2 hamster kidney
2-OH-E2 rat kidney
2-OH-E2 hamster liver
2-OH-E2 rat liver
a
ND: not determined.
high Km values, which range from 0.77 to 10 μM. Although an
earlier study reported relatively low Km values for both MB-
COMT and S-COMT in rabbit aorta for 2-OH-E2 (0.15 and
0.27 μM, respectively), no drastic differences were noted for the
Km values of these two COMTs.36 Therefore, the results of our
present study are the very first report of an ultralow Km value
for pituitary MB-COMT with catechol estrogens as substrates.
In fact, to our knowledge, this is the lowest Km value ever
reported for COMT-mediated O-methylation of all known
substrates.
Most of the known metabolizing enzymes have rather low
specificity with relatively high Km values.27,58 It is very rare to
have a general-purpose metabolizing enzyme with a substrate’s
Km in the nM range. Usually, only highly specialized enzymes
such as those involved in the biosynthesis of endogenous
hormones (such as steroid-synthesizing cytochrome P450
enzymes) have very low Km values, i.e., very high affinity for
the substrates.6 The possible mechanisms underlying the
ultrahigh activity of pituitary MB-COMT may include unique
protein structures and/or post-translational protein modifica-
tions of MB-COMT in the pituitary gland. However, in breast
cancer cells, the single-component kinetics indicates that MB-
and S-COMT had the same Km values (Figure S2).
Nevertheless, the low Km value of COMT in breast cancer
cells may also lead to rapid metabolic inactivation of 4-OH-E2,
resulting in drastically reduced cell proliferative activity in
breast tissues compared to that of E2 (Figure S3).
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Funding
OH-E2 fails to induce tumor formation due to its rapid O-
methylation, E2’s carcinogenicity is probably not dependent on
its conversion to 4-OH-E2. This indicates that the cell
proliferative effect of estrogen may play a relatively more
dominant role in estrogen-induced tumorigenesis in the rat
pituitary. Somewhat in line with this suggestion, it is of note
here that estrogen-induced tumorigenesis in the rat pituitary is
known to be mostly benign in nature (partly due to the lack of
high estrogen-induced mutagenicity), whereas estrogen-in-
duced mammary tumors in rats are mostly malignant in nature,
which is consistent with the presence of mutagenic catechol
estrogens and the accumulation of oncogenic DNA mutations.
This research was supported by grants RO1-CA97109 and
RO1-CA92391 from the National Cancer Institute of the
National Institutes of Health, grants from the Natural Science
Foundation of China (No. 81473224) and from Shenzhen City
Peacock Team Project (No. 201619854).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
AdoMet, S-adenosyl-L-methionine; BSTFA, bis(trimethylsilyl)-
trifluoroacetamide; COMT, catechol-O-methyltransferase;
DMEM, Dulbecco’s modified Eagle’s medium; E2, 17β-
estradiol; ER, estrogen receptor; E2-d5, deuterated 17β-
estradiol-d5; FBS, fetal bovine serum; FSH, follicle-stimulating
hormone; HPLC, high -performance liquid chromatography; 4-
OH-E2, 4-hydroxyestradiol; 2-OH-E2, 2-hydroxyestradiol; LH,
luteinizing hormone; MB-COMT, membrane-bound catechol-
O-methyltransferase; S-COMT, soluble catechol-O-methyl-
transferase; SD, Sprague−Dawley; RIA, radioimmunoassay;
PRL, prolactin; SVZ, subventricular zone; TMCS, trimethyl-
chlorosilane
CONCLUSIONS
■
The results of this study show that, unlike E2, 4-OH-E2 does
not stimulate cell proliferation and tumorigenesis in the
pituitary of ACI female rats. However, using cultured rat
pituitary tumor cells as in vitro models, we found that 4-OH-E2
can still stimulate cell proliferation just like E2, but its potency is
approximately 3 orders of magnitude lower than that of E2.
Moreover, we find that the rat pituitary tumor cells in culture
have a unique ability to selectively metabolize 4-OH-E2 (but
not E2) with exceptionally high efficiency. Additional studies
show that the rat pituitary MB-COMT has an ultralow Km value
for the O-methylation of catechol estrogens with Km values of
40−51 nM. This finding may suggest that the rat pituitary has
an important defense mechanism for protection against 4-OH-
E2-induced tumorigenesis. On the basis of these findings, it is
suggested that the apparent lack of proliferative and
tumorigenic activity of 4-OH-E2 in the pituitary is due to the
ultrarapid metabolic inactivation of this endogenous estrogen
metabolite. The results of this study also raise the possibility
that some of the estrogen derivatives, in particular the catechol
estrogens and methoxyestrogens, may serve unique biological
functions in the pituitary. This possibility is intriguing and
merits further investigation.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.chemres-
tox.7b00096.
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Figure S1, BrdU staining of anterior pituitary cells in
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Figure S3, effect of E2 or 4-OH-E2 on MCF-7 breast
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AUTHOR INFORMATION
Corresponding Author
*E-mail: BTZhu@CUHK.edu.cn. Phone: +86-755-84273851.
ORCID
Bao-Ting Zhu: 0000-0001-8791-8460
Author Contributions
B.T.Z. conceived the initial ideas. P.W. and B.T.Z. designed the
experiments. P.W., L.H.M., J.-H.S., and J.Y. performed the
experiments, and P.W. and B.T.Z. analyzed the data and wrote
the paper.
■
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