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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.

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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, which is essentially the same as the value 0.13 ± 0.03 nM 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 carcinogenic in inducing tumorigenesis in the kidneys of male Syrian

INTRODUCTION

Estrogens have diverse physiological and pathophysiological effects in humans and animals. The endogenous estrogen 17βestradiol (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 estrogeninduced 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 4hydroxyestradiol (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 © 2017 American Chemical Society

Received: April 13, 2017 Published: June 15, 2017 1448

DOI: 10.1021/acs.chemrestox.7b00096 Chem. Res. Toxicol. 2017, 30, 1448−1462

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Chemical Research in Toxicology 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 considerable 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 tumorigenesis.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 Nterminus, 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 SCOMT 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 Omethylation of catechol estrogens.

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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 compressed 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 ovariectomized 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 Laboratories, 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 4OH-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 1449


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Chemical Research in Toxicology 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 RC4B/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 4OH-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. 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). 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 17βestradiol-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. The GC/MS was carried out as described earlier.44,45 Briefly, the GC/MS apparatus consisted of an Agilent 6890N gas chromatogra-

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. 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 estrogens (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 Omethylation 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. 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 1450


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Chemical Research in Toxicology

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 receptormediated 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),

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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. Macro1451


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Chemical Research in Toxicology which is consistent with the lower estrogenic activity of 4-OHE2 in vivo. 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

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. 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-OHE2’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 Kinetics 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 4OH-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 twocomponent 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 4OH-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 E2treated 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. 4OH-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 1452


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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).

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 SCOMT, 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 singlecomponent 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 SCOMT, we separated the microsomal fraction (containing mostly MB-COMT) from the cytosolic fraction (containing mostly S-COMT) using the standard high-speed centrifugation

For comparison, we also determined the COMT in rat liver homogenates and found that the liver COMT had singlecomponent 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, SCOMT 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 1454


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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).

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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 MBCOMT 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 MBCOMT 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 1455


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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 4OH-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 ERpositive pituitary prolactinoma cell lines (GH3 and RC-4B/C). 1456


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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.

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 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 4OH-E2 in pituitary cells in vivo, then we should still be able to see a full-range of growth stimulation when higher concentrations of 4-OH-E2 are used under controlled in vitro conditions. 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 1457


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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 Omethylation 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 Omethylation 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 Omethylation of catechol estrogens in selected tissues (summarized in Table 2).30,36,56,57 They all reported quite 1458


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Chemical Research in Toxicology 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

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 It is worth noting that an earlier report also showed that 4OH-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 Omethylation 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 approximately 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 COMTmediated 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 tumorigenesis.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 superoxide, 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-

Km value substrates

enzyme source

4-OH-E2

GH3 pituitary liver GH3 pituitary liver

2-OH-E2

MB-COMT

S-COMT

72 nM 3.2 μM 123.3 nM 3.1 μM 10.3 μM (crude homogenates) 94 nM 1.8 μM 75 nM 1.2 μM 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 substrate

enzyme source

2-OH-E2 2-OH-E2

rabbit aorta recombinant human COMT recombinant human COMT pig endometrium pig endometrium hamster kidney rat kidney hamster liver rat liver hamster kidney rat kidney hamster liver rat liver

4-OH-E2 4-OH-E2 2-OH-E2 4-OH-E2 4-OH-E2 4-OH-E2 4-OH-E2 2-OH-E2 2-OH-E2 2-OH-E2 2-OH-E2 a

Km for MBCOMT (μM)a

Km for SCOMT (μM)

ref

0.15 3.2

0.27 3.6

36 30

6.4

4.5

30

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

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 MBCOMT 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 modifications of MB-COMT in the pituitary gland. However, in breast cancer cells, the single-component kinetics indicates that MBand S-COMT had the same K m 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). 1459


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Chemical Research in Toxicology Funding

OH-E2 fails to induce tumor formation due to its rapid Omethylation, 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-induced 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), from Shenzhen City Peacock Team Project (No. 201619854), and from a Shenzhen city municipality grant (No. JCYJ20140714151402768). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AdoMet, S-adenosyl-L-methionine; BSTFA, bis(trimethylsilyl)trifluoroacetamide; COMT, catechol-O-methyltransferase; DMEM, Dulbecco’s modified Eagle’s medium; E2, 17βestradiol; ER, estrogen receptor; E2-d5, deuterated 17βestradiol-d5; FBS, fetal bovine serum; FSH, follicle-stimulating hormone; HPLC, high -performance liquid chromatography; 4OH-E2, 4-hydroxyestradiol; 2-OH-E2, 2-hydroxyestradiol; LH, luteinizing hormone; MB-COMT, membrane-bound catecholO-methyltransferase; S-COMT, soluble catechol-O-methyltransferase; SD, Sprague−Dawley; RIA, radioimmunoassay; PRL, prolactin; SVZ, subventricular zone; TMCS, trimethylchlorosilane

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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-OHE2-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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(1) Spady, T. J., McComb, R. D., and Shull, J. D. (1999) Estrogen action in the regulation of cell proliferation, cell survival, and tumorigenesis in the rat anterior pituitary gland. Endocr. J. 11, 217− 233. (2) Yager, J. D., and Leihr, J. G. (1996) Molecular mechanisms of estrogen carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 36, 203−232. (3) Figueira, M. I., Correia, S., Vaz, C. V., Cardoso, H. J., Gomes, I. M., Marques, R., Maia, C. J., and Socorro, S. (2016) Estrogens downregulate the stem cell factor (SCF)/c-KIT system in prostate cells: Evidence of antiproliferative and proapoptotic effects. Biochem. Pharmacol. 99, 73−87. (4) Seilicovich, A. (2010) Cell life and death in the anterior pituitary gland: role of oestrogens. J. Neuroendocrinol. 22, 758−764. (5) Fukui, M., and Zhu, B. T. (2009) Mechanism of 2methoxyestradiol-induced apoptosis and growth arrest in human breast cancer cells. Mol. Carcinog. 48, 66−78. (6) Zhu, B. T., and Conney, A. H. (1998) Functional role of estrogen in target cells: review and perspectives. Carcinogenesis 19, 1−27. (7) Zhu, B. T., Han, G. Z., Shim, J. Y., Wen, Y., and Jiang, X. R. (2006) Quantitative structure-activity relationship of various endogenous estrogen metabolites for human estrogen receptor alpha and beta subtypes: Insights into the structural determinants favoring a differential subtype binding. Endocrinology 147, 4132−4150. (8) Schütze, N., Vollmer, G., Wünsche, W., Grote, A., Feit, B., and Knuppen, R. (1994) Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to the estrogen receptor of MCF-7 cells in cytosolic extracts and in nuclei of intact cells. Exp. Clin. Endocrinol. Diabetes 102, 399− 408. (9) Van Aswegen, C. H., Purdy, R. H., and Wittliff, J. L. (1989) Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers. J. Steroid Biochem. 32, 485−492. (10) Notides, A. C. (1970) The binding affinity and specificity of the estrogen receptor of the rat uterus and anterior pituitary. Endocrinology 87, 987−992. (11) Newbold, R. R., and Liehr, J. G. (2000) Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens. Cancer Res. 60, 235−237. (12) Liu, D., and Bachmann, K. A. (1998) An investigation of the relationship between estrogen, estrogen metabolites and blood cholesterol levels in ovariectomized rats. J. Pharmacol. Exp. Ther. 286, 561−568.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00096. Figure S1, BrdU staining of anterior pituitary cells in ovariectomized female Sprague−Dawley rats after treatment with E2 or 4-OH-E2 for 7 days; Figure S2, enzyme kinetics for the O-methylation of 4-OH-E2 catalyzed by whole cell lysates (containing both cytosol and microsomes) prepared from MCF-7 breast cancer cells; and Figure S3, effect of E2 or 4-OH-E2 on MCF-7 breast cancer cell proliferation in culture (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 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. 1460


Article

Chemical Research in Toxicology (13) Franks, S., MacLusky, N. J., and Naftolin, F. (1982) Comparative pharmacology of oestrogens and catechol oestrogens: actions on the immature rat uterus in vivo and in vitro. J. Endocrinol. 94, 91−98. (14) Emons, G., Knuppen, R., and Ball, P. (1981) 4-Hydroxyestradiol-17 beta and 4-hydroxyestradiol-17 alpha: comparative studies on central and peripheral effects of two epimeric catecholestrogens. Endocrinology 109, 1799−1801. (15) Merriam, G. R., MacLusky, N. J., Picard, M. K., and Naftolin, F. (1980) Comparative properties of the catechol estrogens, I: methylation by catechol-O-methyltransferase and binding to cytosol estrogen receptors. Steroids 36, 1−11. (16) Kirchhoff, J., Hornung, E., Ghraf, R., Ball, P., and Knuppen, R. (1981) Interactions of catecholestrogens with cytoplasmic and nuclear estrogen receptors in rat pituitary gland and hypothalamus. J. Neurochem. 37, 1540−1547. (17) Cavalieri, E., Frenkel, K., Liehr, J. G., Rogan, E., and Roy, D. (2000) Estrogens as endogenous genotoxic agents–DNA adducts and mutations. J. Natl. Cancer Inst. Monogr. 27, 75−93. (18) Liehr, J. G. (2000) Is estradiol a genotoxic mutagenic carcinogen? Endocr. Rev. 21, 40−54. (19) Li, J. J., Li, S. A., Oberley, T. D., and Parsons, J. A. (1995) Carcinogenic activities of various steroidal and nonsteroidal estrogens in the hamster kidney: relation to hormonal activity and cell proliferation. Cancer Res. 55, 4347−4351. (20) Liehr, J. G., Fang, W. F., Sirbasku, D. A., and Ari-Ulubelen, A. (1986) Carcinogenicity of catechol estrogens in Syrian hamsters. J. Steroid Biochem. 24, 353−356. (21) Harvell, D. M., Strecker, T. E., Tochacek, M., Xie, B., Pennington, K. L., McComb, R. D., Roy, S. K., and Shull, J. D. (2000) Rat strain-specific actions of 17b-estradiol in the mammary gland: Correlation between estrogen-induced lobuloalveloar hyperplasia and susceptibility to estrogen-induced mammary cancers. Proc. Natl. Acad. Sci. U. S. A. 97, 2779−2784. (22) Harvell, D. M., Strecker, T. E., Xie, B., Pennington, K. L., McComb, R. D., and Shull, J. D. (2002) Dietary energy restriction inhibits estrogen-induced mammary, but not pituitary, tumorigenesis in the ACI rat. Carcinogenesis 23, 161−169. (23) Shull, J. D., Spady, T. J., Snyder, M. C., Johansson, S. L., and Pennington, K. L. (1997) Ovary-intact, but not ovariectomized female ACI rats treated with 17b-estradiol rapidly develop mammary carcinoma. Carcinogenesis 18, 1595−1601. (24) Turan, V. K., Sanchez, R. I., Li, J. J., Li, S. A., Reuhl, K. R., Thomas, P. E., Conney, A. H., Gallo, M. A., Kauffman, F. C., and Mesia-Vela, S. (2004) The effects of steroidal estrogens in ACI rat mammary carcinogenesis: 17beta-estradiol, 2-hydroxyestradiol, 4hydroxyestradiol, 16alpha-hydroxyestradiol, and 4-hydroxyestrone. J. Endocrinol. 183, 91−99. (25) Yager, J. D. (2015) Mechanisms of estrogen carcinogenesis: The role of E2/E1-quinone metabolites suggests new approaches to preventive intervention–A review. Steroids 99, 56−60. (26) Axelrod, J., and Tomchick, R. (1958) Enzymatic O-methylation of epinephrine and other catechols. J. Biol. Chem. 233, 702−705. (27) Thakker, D. R., and Creveling, C. R. (1990) in Reactions in Drug Metaolism, pp 193−232, Taylor & Francis. (28) Zhu, B. T. (2002) Catechol-O-methyltransferase (COMT)mediated methylation metabolism of endogenous bioactive catechols and modulation by endobiotics and xenobiotics: Importance in pathophysiology and pathogenesis. Curr. Drug Metab. 3, 321−349. (29) Zhu, B. T., and Liehr, J. G. (1996) Inhibition of catechol O methyltransferase-catalyzed O-methylation of 2 and 4-hydroxyestradiol by quercetin. Possible role in estradiol-induced tumorigenesis. J. Biol. Chem. 271, 1357−1363. (30) Bai, H. W., Shim, J. Y., Yu, J., and Zhu, B. T. (2007) Biochemical and molecular modeling studies of the O-methylation of various endogenous and exogenous catechol substrates catalyzed by recombinant human soluble and membrane-bound catechol-Omethyltransferases. Chem. Res. Toxicol. 20, 1409−1425.

(31) Salminen, M., Lundström, K., Tilgmann, C., Savolainen, R., Kalkkinen, N., and Ulmanen, I. (1990) Molecular cloning and characterization of rat liver catechol-O-methyltransferase. Gene 93, 241−247. (32) Tenhunen, J., and Ulmanen, I. (1993) Production of rat soluble and membrane-bound catechol O-methyltransferase forms from bifunctional mRNAs. Biochem. J. 296, 595−600. (33) Tenhunen, J., Salminen, M., Jalanko, A., Ukkonen, S., and Ulmanen, I. (1993) Structure of the rat catechol-O-methyltransferase gene: separate promoters are used to produce mRNAs for soluble and membrane-bound forms of the enzyme. DNA Cell Biol. 12, 253−263. (34) Tilgmann, C., and Kalkkinen, N. (1991) Purification and partial sequence analysis of the soluble catechol-O-methyltransferase from human placenta: comparison to the rat liver enzyme. Biochem. Biophys. Res. Commun. 174, 995−1002. (35) Tilgmann, C., Melen, K., Lundström, K., Jalanko, A., Julkunen, I., Kalkkinen, N., and Ulmanen, I. (1992) Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain. Eur. J. Biochem. 207, 813−821. (36) Reid, J. J., Stitzel, R. E., and Head, R. J. (1986) Characterization of the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof. Naunyn-Schmiedeberg's Arch. Pharmacol. 334, 17−28. (37) Lotta, T., Vidgren, J., Tilgmann, C., Ulmanen, I., Melén, K., Julkunen, I., and Taskinen, J. (1995) Kinetics of human soluble and membrane-bound catechol O-methyltransferase: a revised mechanism and description of the thermolabile variant of the enzyme. Biochemistry 34, 4202−4210. (38) Schweikart, K. M., Eldridge, S. R., Safgren, S. L., Parman, T., Reid, J. M., Ames, M. M., Goetz, M. P., and Davis, M. A. (2014) Comparative uterotrophic effects of endoxifen and tamoxifen in ovariectomized Sprague-Dawley rats. Toxicol. Pathol. 42, 1188−1196. (39) Zhou, H., Qin, L. Q., Ma, D. F., Wang, Y., and Wang, P. Y. (2010) Uterotrophic effects of cow milk in immature ovariectomized Sprague-Dawley rats. Environ. Health Prev. Med. 15, 162−168. (40) Lawson, D. M., Sensui, N., and Gala, R. R. (1984) Ovariectomized Sprague-Dawley and Long-Evans rats release prolactin differently in response to estrogen. Life Sci. 34, 455−459. (41) Obradovic, M., Sudar, E., Zafirovic, S., Stanimirovic, J., Labudovic-Borovic, M., and Isenovic, E. R. (2015) Estradiol in vivo induces changes in cardiomyocytes size in obese rats. Angiology 66, 25−35. (42) Mills, L. H., Yu, J., Xu, X. M., Lee, A. J., and Zhu, B. T. (2008) Naturally-occurring estradiol-17β-fatty acid esters, but not estradiol17β, preferentially induce mammary tumorigenesis in female rats: implications for an important role in human breast cancer. Toxicol. Appl. Pharmacol. 15, 332−341. (43) Liu, Z. J., and Zhu, B. T. (2004) Concentration-dependent mitogenic and antiproliferative actions of 2-methoxyestradiol in estrogen receptor-positive human breast cancer cells. J. Steroid Biochem. Mol. Biol. 88, 265−275. (44) Lee, A. J., Cai, M. X., Thomas, P. E., Conney, A. H., and Zhu, B. T. (2003) Characterization of the oxidative metabolites of 17betaestradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms. Endocrinology 144, 3382−3398. (45) Lee, A. J., Kosh, J. W., Conney, A. H., and Zhu, B. T. (2001) Characterization of the NADPH-dependent metabolism of 17βestradiol to multiple metabolites by human liver microsomes and selectively expressed human cytochrome P450 3A4 and 3A5. J. Pharmacol. Exp. Ther. 298, 420−432. (46) Zhu, B. T., Gallo, M. A., Burger, C. W., Jr, Meeker, R. J., Cai, M. X., Xu, S., and Conney, A. H. (2008) Effect of 2,3,7,8tetrachlorodibenzo-p-dioxin administration and high-fat diet on the body weight and hepatic estrogen metabolism in female C3H/HeN mice. Toxicol. Appl. Pharmacol. 226, 107−118. (47) Christian, C. A., Glidewell-Kenney, C., Jameson, J. L., and Moenter, S. M. (2008) Classical estrogen receptor alpha signaling 1461


Article

Chemical Research in Toxicology mediates negative and positive feedback on gonadotropin-releasing hormone neuron firing. Endocrinology 149, 5328−5334. (48) Geary, N., Asarian, L., Korach, K. S., Pfaff, D. W., and Ogawa, S. (2001) Deficits in E2-dependent control of feeding, weight gain, and cholecystokinin satiation in ER-alpha null mice. Endocrinology 142, 4751−4757. (49) Farinetti, A., Tomasi, S., Foglio, B., Ferraris, A., Ponti, G., Gotti, S., Peretto, P., and Panzica, G. C. (2015) Testosterone and estradiol differentially affect cell proliferation in the subventricular zone of young adult gonadectomized male and female rats. Neuroscience 286, 162−170. (50) Khan, M. M., Wakade, C., de Sevilla, L., and Brann, D. W. (2015) Selective estrogen receptor modulators (SERMs) enhance neurogenesis and spine density following focal cerebral ischemia. J. Steroid Biochem. Mol. Biol. 146, 38−47. (51) Li, J., Siegel, M., Yuan, M., Zeng, Z., Finnucan, L., Persky, R., Hurn, P. D., and McCullough, L. D. (2011) Estrogen enhances neurogenesis and behavioral recovery after stroke. J. Cereb. Blood Flow Metab. 31, 413−425. (52) De Nicola, A. F., von Lawzewitsch, I., Kaplan, S. E., and Libertun, C. (1978) Biochemical and ultrastructural studies on estrogen-induced pituitary tumors in F344 rats. J. Natl. Cancer Inst. 61, 753−763. (53) Wiklund, J., Wertz, N., and Gorski, J. (1981) A comparison of estrogen effects on uterine and pituitary growth and prolactin synthesis in F344 and Holtzman rats. Endocrinology 109, 1700−1707. (54) Sarkar, D. K. (2006) Genesis of prolactinomas: studies using estrogen-treated animals. Front. Horm. Res. 35, 32−49. (55) Chorich, L., Rosser, M., Howard, E., Zamorano, P., and Maheshi, V. B. (1992) Rat anterior pituitary estrogen receptor mRNA levels after estrogen and progesterone treatment. Mol. Cell. Neurosci. 3, 487−496. (56) Chakraborty, C., Davis, D. L., and Dey, S. K. (1990) The Omethylation of catechol oestrogens by pig conceptuses and endometrium during the peri-implantation period. J. Endocrinol. 127, 77−84. (57) Li, S. A., Purdy, R. H., and Li, J. J. (1989) Variations in catechol O-methyltransferase activity in rodent tissues: possible role in estrogen carcinogenicity. Carcinogenesis 10, 63−67. (58) Raftogianis, R., Creveling, C., Weinshilboum, R., and Weisz, J. (2000) Estrogen metabolism by conjugation. J. Natl. Cancer Inst. Monogr. 27, 113−124. (59) Ben-Jonathan, N., and Hnasko, R. (2001) Dopamine as a prolactin (PRL) inhibitor. Endocr. Rev. 22, 724−763. (60) Bui, Q. D., Neisz, J., and Hershey, M. S. (1989) Monooxygenase mediating catecholestrogen formation by rat anterior pituitary is an estrogen-4-hydroxylase. Endocrinology 124, 1085−1087. (61) Parada-Bustamante, A., Orihuela, P. A., Ríos, M., NavarreteGómez, P. A., Cuevas, C. A., Velasquez, L. A., Villalón, M. J., and Croxatto, H. B. (2007) Catechol-O-methyltransferase and methoxyestradiols participate in the intraoviductal nongenomic pathway through which estradiol accelerates egg transport in cycling rats. Biol. Reprod. 77, 934−941. (62) Vijayanathan, V., Venkiteswaran, S., Nair, S. K., Verma, A., Thomas, T. J., Zhu, B. T., and Thomas, T. (2006) Physiologic levels of 2-methoxyestradiol interfere with nongenomic signaling of 17βestradiol in human breast cancer cells. Clin. Cancer Res. 12, 2038− 2048. (63) Tworoger, S. S., and Hankinson, S. E. (2008) Prolactin and breast cancer etiology: an epidemiologic perspective. J. Mammary Gland Biol. Neoplasia 13, 41−53. (64) Clevenger, C. V., Furth, P. A., Hankinson, S. E., and Schuler, L. A. (2003) The role of prolactin in mammary carcinoma. Endocr. Rev. 24, 1−27. (65) Rose-Hellekant, T. A., Arendt, L. M., Schroeder, M. D., Gilchrist, K., Sandgren, E. P., and Schuler, L. A. (2003) Prolactin induces ERalpha-positive and ERalpha-negative mammary cancer in transgenic mice. Oncogene 22, 4664−4674.

(66) Henderson, B. E., and Feigelson, H. S. (2000) Hormonal carcinogenesis. Carcinogenesis 21, 427−433. (67) Bolton, J. L. (2002) Quinoids, quinoid radicals, and phenoxyl radicals formed from estrogens and antiestrogens. Toxicology 177, 55− 65. (68) Bolton, J. L., and Dunlap, T. (2017) Formation and biological targets of quinones: cytotoxic versus cytoprotective effects. Chem. Res. Toxicol. 30, 13−37. (69) Bolton, J. L. (2014) Quinone methide bioactivation pathway: Contribution to Toxicity and/or Cytoprotection? Curr. Org. Chem. 18, 61−69. (70) Kolbanovskiy, A., Kuzmin, V., Shastry, A., Kolbanovskaya, M., Chen, D., Chang, M., Bolton, J. L., and Geacintov, N. E. (2005) Base selectivity and effects of sequence and DNA secondary structure on the formation of covalent adducts derived from the equine estrogen metabolite 4-hydroxyequilenin. Chem. Res. Toxicol. 18, 1737−1747. (71) Bolton, J. L., and Thatcher, G. R. (2008) Potential mechanisms of estrogen quinone carcinogenesis. Chem. Res. Toxicol. 21, 93−101. (72) Wang, Z., Wijewickrama, G. T., Peng, K. W., Dietz, B. M., Yuan, L., van Breemen, R. B., Bolton, J. L., and Thatcher, G. R. (2009) Estrogen receptor alpha enhances the rate of oxidative DNA damage by targeting an equine estrogen catechol metabolite to the uucleus. J. Biol. Chem. 284, 8633−8642. (73) Wang, Z., Chandrasena, E. R., Yuan, Y., Peng, K. W., van Breemen, R. B., Thatcher, G. R., and Bolton, J. L. (2010) Redox cycling of catechol estrogens generating apurinic/apyrimidinic sites and 8-oxo-deoxyguanosine via reactive oxygen species differentiates equine and human estrogens. Chem. Res. Toxicol. 23, 1365−1373. (74) Cavalieri, E. L., and Rogan, E. G. (2012) The etiology and prevention of breast cancer. Drug Discovery Today: Dis. Mech. 9, e55− e69. (75) Cavalieri, E. L., and Rogan, E. G. (2016) Depurinating estrogenDNA adducts, generators of cancer initiation: their minimization leads to cancer prevention. Clin. Transl. Med. 5, 12.

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