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Endocrinology Vol. 144, No. 3 785-792
Copyright © 2003 by The Endocrine Society

ARTICLE

Dose-Response Effects of 2-Methoxyestradiol on Estrogen Target Tissues in the Ovariectomized Rat

J. D. Sibonga, S. Lotinun, G. L. Evans, V. S. Pribluda, S. J. Green and R. T. Turner

Departments of Orthopedics (J.D.S., S.L., G.L.E., R.T.T.) and Biochemistry and Molecular Biology, (R.T.T.), Mayo Clinic, Rochester, Minnesota 55905; and EntreMed Inc. (V.S.P., S.J.G.), Rockville, Maryland 20850

Address all correspondence and requests for reprints to: Jean D. Sibonga, Ph.D., Division of Orthopedic Research, Mayo Clinic, 200 First Street SW, Medical Sciences Building 3-69, Rochester, Minnesota 55905. E-mail: sibonga.jean{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
In three experiments, we evaluated the pharmacological effects of 2-methoxyestradiol (2ME2) on several estrogen target tissues. Experiment 1: we gavaged recently ovariectomized (OVX) 9.5-wk-old rats with 2ME2 at doses of 0, 0.1, 1, 4, 20, and 75 mg/kg in a 21-d dose-response study. 2ME2 reduced body weight and serum cholesterol, increased uterine weight and epithelial cell height, and inhibited longitudinal and radial bone growth compared with values in the untreated OVX rat. All doses of 2ME2 maintained cancellous bone mass at the baseline level, the lowest effective dose being 20-fold less than a uterotrophic dose. Experiment 2: in an 8-wk experiment in adult OVX rats, a nonuterotrophic dose of 2ME2 (4 mg/kg·d) suppressed body weight gain, inhibited bone formation in cancellous bone and partially prevented bone loss in the tibial metaphysis. Experiment 3: in weanling rats, ICI 182,780 did not antagonize the effect of 2ME2. We conclude that 2ME2 antagonizes the skeletal changes that follow OVX at doses that have minimal or no effects in the uterus in both young and adult rats; 2ME2 does not appear to act via estrogen receptors and is active on bone at doses well below those required for tumor suppression in mice. 2ME2, through a novel pathway, may be a useful alternative to conventional hormone replacement therapy for prevention of postmenopausal bone loss.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
2-METHOXYESTRADIOL (2ME2) IS a metabolite of 17ß-estradiol (E2) that reportedly makes a minimal contribution to E2’s hormonal actions (1). This is based upon 2ME2’s low circulating levels and its 2000-fold lower binding affinity (than the parent compound) for the estrogen receptor (2). 2ME2 is derived from the O-methylation at the 2-position of 2-hydroxyestradiol, which is the major catechol estrogen produced by the cytochrome P450 system in the liver. Some of the unique physiological actions of estrogens may be the result of further conjugation of catechol estrogens by local enzymes in target tissues and cells.

2ME2 is recognized for its unique and profound actions on tumor cells, particularly for its inhibitory effects on angiogenesis and cell proliferation (3, 4, 5, 6, 7). Because of its antiproliferative activity in established cancer cell lines, its tumorstatic actions in animal models for both primary and metastatic tumors, and its minimal toxicity, 2ME2 has been introduced into clinical phase I trials for the suppression of solid tumor growth. These actions of 2ME2 are not associated with E2 signaling pathways and appear selective for rapidly replicating tumor cells, although there are reports of its effects in normal cells (8, 9, 10). Although 2ME2’s mechanism of action remains uncertain, several reports have shown that it may induce apoptosis through p53 activation (11), up-regulate death receptor 5 (12), affect microtubule dynamics in vitro (13), and inhibit the activity of superoxide dismutase (14). More recently, 2ME2 has been shown to be toxic to MG63, TE85, and ROS cells but has no effect on the survival of primary osteoblasts (15). These findings suggest that 2ME2 may be useful for treatment of osteosarcoma.

The effect of pharmacological levels of 2ME2 on normal physiological systems is not well characterized. This laboratory has recently reported that 2ME2, at a dose that inhibits tumor growth in several models (100 mg/kg) dramatically suppressed longitudinal bone growth in ovary-intact young, growing rats (16). 2ME2 resulted in a dramatic reduction in the height of the growth plate, an action that is superficially similar to E2. No effects of 2ME2 were detected in either the turnover of cancellous bone in the tibial metaphysis or in the radial growth of cortical bone at the tibial diaphysis (16). It may be possible that ovary-intact rats are insensitive to the additional effects of weak estrogens. Thus, it is important to characterize the possible effects of 2ME2 therapy on bone modeling and remodeling in animal models for juvenile and adult cancer patients.

We describe the effects of the metabolite 2ME2 on tibial histomorphometry of weanling, adolescent and adult female rats. In a 3-wk experiment, ovariectomized (OVX) adolescent rats were employed to evaluate the dose response for estrogen targets (bone, uterus, and serum cholesterol) to 2ME2 while in the estrogen-deficient state. A nonuterotrophic dose of 2ME2 was further evaluated in adult OVX rats following longer-term treatment (8 wk). The effect of 1-wk treatment with 2ME2 on the growth of skeletal and reproductive tissue, in the presence and absence of the estrogen receptor ligand, ICI 182,780 (ICI), was analyzed in the weanling rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
The institutional animal care and use committee approved all animal protocols in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. In the experiments, all rats were fed standard rat chow ad libitum except that the OVX pair-fed control group was fed the mean value of food consumed by the group treated with the highest dose of 2ME2 (75 mg/kg) in experiment 1.

Drugs
Stock preparations of 2ME2 (Tetrionics, Madison, WI) and of 17{alpha}-ethynyl estradiol (17{alpha}-EE2) (Sigma, St. Louis, MO) were prepared as liposomal suspensions with dimyristoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) at 15 mg/ml and 20 µg/ml stock solutions, respectively. At the highest concentration assessed, the molar ratio of 2ME2:dimyristoylphosphatidylcholine was 1:1.75. Dilutions of stock solution were performed with control liposomes. All liposomes were stored at 4 C until used. Liposomes were administered to rats by oral gavage. ICI was supplied and custom prepared into controlled-release pellets by Innovative Research of America (Sarasota, FL) to deliver a dose of 1.5 mg/kg·d for 7 d.

Experiment 1 (adolescent rats)
All rats were virgin females that were OVX or sham-operated by the supplier (Harlan Sprague Dawley, Inc., Indianapolis, IN) 1 wk before treatment. A mean body weight of 199 ± 1 was recorded at the commencement of the experiment (9.5 wk of age). Rats were divided into weight-matched groups with 8–10 rats/group, except for the OVX control group, which had 20 rats.

The following control groups used: OVX controls (n = 20), OVX pair-fed (n = 10), OVX baseline (n = 8), sham control (n = 9). OVX treatment groups (n = 9/group) were: 1) 17{alpha}-EE2, 10 µg/kg; 2) 17{alpha}-EE2, 100 µg/kg; 3) 2ME2, 0.1 mg/kg; 4) 2ME2, 1 mg/kg; 5) 2ME2, 4 mg/kg; 6) 2ME2, 20 mg/kg; and 7) 2ME2, 75 mg/kg. The OVX baseline group was killed at the beginning of the experiment, 1 wk post OVX. Drug-free liposomes were administered to OVX control (i.e. 0 mg/kg 2ME2), OVX pair-fed, and the sham control groups. All rats received standard rat chow (LabDiet, Brentwood, MO), which includes 0.95% calcium and 0.67% phosphorus.

For the first week, rats were acclimated to gavaging of liposomes by gradually increasing the delivery volume (0.25-ml increments) until final treatment doses were delivered in a 1-ml volume by d 7. Rats were gavaged this volume (i.e. treatment dose) for the next 15 d until euthanized on d 22. Body weights were recorded every day for the first week and then periodically, every 3–4 d.

For bone histomorphometry, rats were administered fluorochromes (20 mg/kg) perivascularly at the base of the tail. Tetracycline-hydrochloride (Sigma) was delivered on the first day of treatment, calcein (Sigma) 9 d before, and demeclocycline (Sigma) 2 d before the rats were killed.

Experiment 2 (adult rats)
Virgin female rats (Harlan Sprague Dawley, Inc.) were ovariectomized (OVX) by the supplier 1 wk before treatment commenced. OVX rats, at 6 months of age, had a mean body weight of 248 ± 4 at the beginning of the experiment. The OVX rats were divided into weight-matched groups (n = 9–10) of control and 2ME2-treated rats. In contrast to experiment 1, the 2ME2-treated rats in experiment 2 were gavaged with liposomes (0.1 ml/0.1 kg body weight)—for a final dose of 4 mg/kg—each day for the first 10 d and then 5 d/wk until the end of the experiment. Control rats were untreated. Control and 2ME2-treated rats were euthanized at 8 months of age following an experiment duration of 60 d. Separate groups of sham-operated rats were euthanized at 6 months of age (290 ± 6, n = 5) and at 8 months of age (281 ± 4 g, n = 10) to provide age-matched reference values for ovary-intact rats.

For bone histomorphometry, rats were administered fluorochromes (20 mg/kg) perivascularly at the base of the tail: tetracycline-HCl (Sigma) was delivered on the day before treatment commenced, calcein (Sigma) 12 d before the rats were killed, and demeclocycline (Sigma) 2 d before they were killed.

Experiment 3 (weanling rats)
Female rats (Harlan Sprague Dawley, Inc.) were obtained at 21 d of age (45.2 ± 0.5 g body weight) and divided into four groups (n {approx} 9/group): control, 2ME2, ICI, and 2ME2 + ICI. 2ME2 (4 mg/kg) was given daily by gavage for 1 wk. Rats were weighed every day and the volume of liposomes to be gavaged was adjusted per body weight (0.1 ml/0.1 kg body weight) as previously mentioned in experiment 2. Non-2ME2-treated rats were given drug-free liposomes. ICI and placebo pellets (3 mm diameter size) were implanted at an sc site on the first day of treatment. An additional group of weanling rats (n = 17) was implanted with either ICI pellets (n = 8) or with placebo (n = 9), as previously described; these rats were gavaged with 17{alpha}-EE2 containing liposomes at 100 µg/kg.

Rats were injected perivascularly at the base of the tail with fluorochromes: tetracycline-HCl (Sigma, 20 mg/kg) on the first day of treatment and calcein (Sigma, 20 mg/kg) 1 d before they were killed. Rats were euthanized on morning of d 8. At necropsy, rats were killed by decapitation following anesthesia with a mixture of ketamine:xylazine (90:9 mg/kg, ip injection), uteri were removed and wet weights recorded.

Tissues at necropsy
Blood was drained from the carcass following decapitation and allowed to clot at room temperature. Serum was stored at -70 C before assay.

Uteri were removed, wet weights recorded, and fixed in 10% neutral buffered formalin. Fixed uteri were transferred to the Histology Laboratory (Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN), where they were processed and embedded in paraffin blocks.

Tibiae were defleshed at the time of necropsy and preserved by immersion in 70% ethanol for histomorphometry.

Bone histomorphometry
Tibiae were dehydrated by daily changes of ethanol (1 d in 95% and 6 d in 100%) before infiltration with a methylmethacrylate mixture (methylmethacrylate-2-hydroxyethyl and methylmethacrylate 12.5:1). Undecalcified bones were embedded in the methylmethacrylate and longitudinal sections (5 µm) were cut from the center of the proximal tibiae with a Reichert Jung microtome (model 2065, Heidelberg, Germany) as previously described (16, 17).

All tissue measurements were performed with the OsteoMeasure Analysis System (OsteoMetrics, Atlanta, GA), that consists of a Pentium 133 computer coupled to a photomicroscope and image analysis system. This image system includes a high-resolution color video camera (Sony DXC-970 MD) that transmits the image of the specimen through the microscope (Olympus Corp. BH-2, New Hyde Park, NY) to a video monitor that registers the movement of a digitizing mouse on a graphics tablet (OsteoTablet, OsteoMetrics). The regions of interest, e.g. bone perimeters and fluorochrome labels, are traced by the operator; lengths of tracings, areas enclosed by traced lines and average distances between traced lines are calculated automatically by the software. All histomorphometry data are generated and derived in accordance with standardized formulae, methods, and nomenclature (18).

Cortical bone
Cross-sections of the tibial diaphysis ({approx}150 µm) were cut at the tibia-fibula synostosis with a diamond-edge saw (Isomet, Buehler, Lake Bluff, IL) and then ground on a roughened glass surface to an approximate 25-µm thickness for visualization of fluorochrome label under UV light, as previously described (16, 17). Medullary areas, cross-sectional areas and cortical bone areas were quantified under light microscopy. Double-labeled periosteal surfaces were measured for calculations of periosteal mineral apposition rate and periosteal bone formation rate from the tetracycline label (20-d interval for experiment 1, 58-d interval for experiment 2, and 7-d interval for experiment 3).

Cancellous bone
All measurements of cancellous bone were conducted on unstained sections in a standardized sampling site as previously described (16, 17). In brief, the site is located between 1.9 mm and 1.0 mm from the most distal point of the growth plate for experiments 1 and 2, respectively. This site encompassed an approximately 2.5-mm2 area and excluded the endocortical surface. Cancellous bone area was expressed as a percent of total sampling area. Cancellous bone formation rate (surface based) was estimated from data derived from the measurements of double-labeled (calcein and demeclocycline) bone perimeters as per standard formulae (18). Mineralizing perimeter was defined as perimeter with double label. Interlabeling periods were 7 d and 10 d for experiments 1 and 2, respectively. Longitudinal sections of selected groups in experiment 1 were stained with toluidine blue (1%) for the measurements of eroded and osteoblast-covered perimeters (perimeter-based).

Longitudinal bone growth
The longitudinal growth rate in experiment 1 was derived from the averaged perpendicular length measured between the calcein and demeclocycline labels, appearing immediately distal to the growth plate, divided by the inter-labeling period of 7 d. This value is reported as µm/d as per standard nomenclature (18).

Measurement of epithelial cell height
Paraffin blocks of uteri were sectioned longitudinally with a Leica Corp. (Wetzlar, Germany) microtome and a section (5–7 µm thick) obtained from the middle of the tissue was stained with hematoxylin-eosin. The average height of an epithelial cell layer was determined for approximately 1.5 mm in length of luminal epithelia.

Serum chemistry
The Immunochemical Core Laboratory (Mayo Clinic) performed serum cholesterol measurements with an automated procedure (Hitachi 912; Roche Diagnostics Corp., Indianapolis, IN).

Statistical analysis
Group values were expressed as mean ± SE. Statistical differences between groups were determined by Fisher’s protected least significant difference post hoc test following determination of significance by one-way ANOVA. The dose rates for 17{alpha}-EE2 and 2ME2 that were considered effective at preventing OVX-induced changes were obtained from the dose response curves and defined as the dose which resulted in a value equivalent to the response of the age-matched, sham-operated control. Two-factor ANOVA (SuperAnova, Abacus Concepts, Berkeley, CA) was used to evaluate the significant effects of 2ME2, of ICI and of interaction between the two factors in weanling rats. Significance was considered at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Experiment 1
The body weights and body weight gain are shown in Table 1Go. The initial body weight was comparable for all groups. Ovariectomy increased growth and final body weight compared with sham animals. 17{alpha}-EE2 and 2ME2 reduced final body weight and growth in a dose-dependent manner at doses of 10 µg/kg and 1 mg/kg, respectively. 17{alpha}-EE2 and 2ME2 maintained body weight and growth at the sham value. OVX, compared with sham operation, significantly increased the longitudinal growth rate of rats (see Fig. 5Go), but no statistical difference could be detected in tibial bone length (data not shown).


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  		Table 1. Effect of 2ME2 on body weight gain (experiment 1)

 


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  		Figure 5. Effect of 2ME2 on longitudinal growth rate (experiment 1). All values are expressed as mean ± SEM (n = 7–10 except for OVX control, which was n = 18 measured sections). The values for reference controls are: SHAM rats (65 ± 4 µm/d) and OVX rats (100 ± 3 µm/d) (P <= 0.0001 SHAM vs. OVX). Significant differences (P < 0.05) are denoted as (a) compared with OVX rats and (b) compared with SHAM rats. The absence of error bars denotes that error is less than the thickness of the data point.

 
As shown in Fig. 1Go, 17{alpha}-EE2 and 2ME2 reduced serum cholesterol in a dose-dependent manner with 17{alpha}-EE2 demonstrating greater potency. 17{alpha}-EE2 and 2ME2 maintained serum cholesterol at the sham value at less than 0.01 mg/kg and approximately 2 mg/kg, respectively.



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  		Figure 1. Effect of 2ME2 on serum total cholesterol (experiment 1). All values are expressed as mean ± SEM (n = 7–11, except for OVX control, which was n = 19 serum samples). The measured levels for reference controls are: SHAM rats (103.2 ± 3.6 mg/dl) and OVX rats (109.8 ± 2.6 mg/dl) (NS). Significant differences are denoted as (a) compared with OVX rats and (b) compared with SHAM rats. The absence of error bars denotes that error is less than the thickness of the data point.

 
The effects of 17{alpha}-EE2 and 2ME2 on uterine wet weight and epithelial cell height are shown in Fig. 2Go, A and B, respectively. OVX resulted in uterine atrophy. 17{alpha}-EE2 and 2ME2 increased wet weight in a dose-dependent manner with restoration to the sham value occurring at approximately 40 mg/kg for 2ME2. The restoration dose for 17{alpha}-EE2 could not be determined from the curve. Epithelial cell height was decreased following OVX. 17{alpha}-EE2 and 2ME2 increased epithelial cell height in a dose-responsive manner with restoration to the sham values occurring at approximately 0.08 mg/kg (17{alpha}-EE2) and approximately 20 mg/kg (2ME2).



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  		Figure 2. Effect of 2ME2 on (A) uterine wet weight and (B) epithelial cell height (experiment 1). All values are expressed as mean ± SEM (n = 7–11, except for OVX control, which was n = 20 uterine tissue). The uterine wet weight recorded for reference controls are: SHAM rats (0.442 ± 0.033 g) and OVX rats (0.118 ± 0.005 g) (P <= 0.0001 SHAM vs. OVX). The epithelial cell heights for reference controls are: SHAM rats (26.12 ± 2.13 µm) and OVX rats (13.63 ± 0.28 µm) (P <= 0.0001 SHAM vs. OVX). Significant differences (P < 0.05) are denoted as (a) compared with OVX rats and (b) compared with SHAM rats. The absence of error bars denotes that error is less than the thickness of the data point.

 
Table 2Go outlines the effects of treatment on cancellous bone area and architecture. OVX resulted in cancellous osteopenia. Pair-feeding had no effect in OVX rats. All doses of 17{alpha}-EE2 and 2ME2 prevented the decrease in BA/TA compared with baseline value and only the highest dose of 2ME2 increased BA/TA to the level in aged-matched sham-operated rats (20.7 ± 2.1%, n = 9). When compared with OVX, all doses of 2ME2 >=1 mg/kg reduced trabecular separation and increased trabecular number; and with the exception of the 1- and 20-mg/kg doses, 2ME2 increased trabecular thickness. When compared with baseline values, trabecular thickness was greater for all doses of 2ME2; trabecular separation was unaffected and only the highest dose of 2ME2 prevented a reduction in trabecular number.


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  		Table 2. Effect of 2ME2 on tibial cancellous bone volume and architecture (experiment 1)

 
The effects of 17{alpha}-EE2 and 2ME2 on cancellous bone formation are shown in Fig. 3Go. Although OVX did not significantly increase cancellous bone formation rates over sham, dose-dependent decreases were observed. Restoration of sham values occurred at approximately 0.04 mg/kg (17{alpha}-EE2) and approximately 10 mg/kg (2ME2), respectively. Similarly, the 20- and 75-mg/kg doses of 2ME2 and the 100-µg/kg dose of 17{alpha}-EE2 reduced the mineralizing perimeter (M.Pm/B.Pm%) compared with OVX control (data not shown). Mineral apposition rate, though, was reduced by nearly all doses of 2ME2 (not 1 mg/kg) and by both doses of 17{alpha}-EE2 (data not shown).



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  		Figure 3. Effect of 2ME2 on cancellous bone formation rate (surface referent) (experiment 1). All values are expressed as mean ± SEM (n = 7–10, except for OVX control, which was n = 18 measured sections). The values measured for reference controls are: SHAM rats (0.23 ± 0.02 µm3/µm2·d) and OVX rats (0.28 ± 0.02 µm3/µm2·d) (NS, SHAM vs. OVX). Significant differences (P < 0.05) are denoted as (a) compared with OVX rats and (b) compared with SHAM rats.

 
There was no effect of treatment on the static cortical bone histomorphometry of OVX rats (data not shown).

17{alpha}-EE2 and 2ME2 resulted in dose-dependent decreases in the periosteal mineral apposition rate (data not shown). 17{alpha}-EE2 reduced periosteal bone formation rates (Fig. 4Go) and 2ME2 lowered rates dose dependently. Restoration of sham values occurred at less than 0.01 mg/kg (17{alpha}-EE2) and approximately 1.5 mg/kg (2ME2).



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  		Figure 4. Effect of 2ME2 on periosteal bone formation rate (experiment 1). All values are expressed as mean ± SEM (n = 7–11, except for OVX control, which was n = 20). The bone formation rate measured for reference controls was: SHAM rats (18.58 ± 1.02 mm2·d) and OVX rats (23.15 ± 1.81 mm2·d) (P < 0.01 SHAM vs. OVX). Significant differences (P < 0.05) are denoted as (a) compared with OVX rats and (b) compared with SHAM rats. The absence of error bars denotes that error is less than the thickness of the data point.

 
17{alpha}-EE2 and 2ME2 resulted in dose-dependent decreases in the longitudinal bone growth (Fig. 5Go). Restoration of sham values occurred at 0.01 mg/kg (17{alpha}-EE2) and approximately 2 mg/kg (2ME2).

Pair-feeding of OVX rats significantly depressed the following parameters compared with OVX rats fed ad libitum: body weight gain, rates of mineral apposition (data not shown) and bone formation (Fig. 4Go) at the periosteum and longitudinal growth rate (Fig. 5Go). Pair-feeding had no effect on uterine tissue. Restrictions in body weight gain may account for the changes in bone toward values measured in sham rats.

Measurement of eroded and osteoblast-covered perimeters in OVX, sham, and 17{alpha}-EE2-treated rats (n = 9–11/group) revealed that OVX increased the percent eroded perimeter compared with sham (9.2 ± 1.5% OVX control vs. 5.6 ± 0.7% sham, P < 0.01) and that 2ME2 (75 mg/kg) and 17{alpha}-EE2 (100 µg/kg) prevented this increase (4.4 ± 0.6% 2ME2 and 4.1 ± 0.7% 17{alpha}-EE2 vs. sham, NS). Similarly, OVX increased the percent of osteoblast-covered perimeter vs. sham (7.9 ± 2.2% OVX control vs. 2.0 ± 0.9% sham, P <= 0.002) and 2ME2 and 17{alpha}-EE2 suppressed this increase in osteoblasts (2.1 ± 0.8% 2ME2 and 1.3 ± 0.8% 17{alpha}-EE2 vs. sham, NS).

Experiment 2
The initial body weights were comparable for all groups of adult rats. OVX increased final body weight compared with baseline OVX rats (323 ± 7 g OVX control vs. 276 ± 6 g OVX baseline, P <= 0.05), but body weight remained near baseline values in 2ME2-treated (277 ± 7 g) and in sham (281 ± 4 g) rats. Uterine wet weight was reduced by OVX (127 ± 7 mg OVX control P <= 0.05 vs. sham 584 ± 87 mg). Uterine wet weights in 2ME2-treated rats were not statistically different from OVX control (194 ± 12 mg, NS).

Cancellous bone histomorphometry is summarized in Figs. 6Go and 7Go. Static measurements of 8-month-old rats (Fig. 6Go, A–D) indicated that OVX reduced cancellous bone area and trabecular number, and increased trabecular separation. 2ME2 treatment suppressed these changes in OVX rats but not to the level measured in sham rats. 2ME2 had no effect on trabecular thickness in OVX rats. Dynamic measurement of cancellous bone (Fig. 7Go, A and B) indicated that 2ME2 reduced the percent of mineralizing perimeter, the mineral apposition rate (data not shown) and the bone formation rate. Mineralizing perimeter and bone formation rate were also reduced in sham rats compared with OVX control.



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  		Figure 6. Effects of 2ME2 on cancellous bone area and architecture of the OVX adult rat (experiment 2). Adult rats were treated with 2ME2 (4 mg/kg) 5 d/wk for 8 wk. Values expressed as mean ± SEM (n = 9–10, except for sham rats at 6 months, where n = 5). Baseline values are measurements in rats of 6 months in age (solid bars). Statistical effect of ovariectomy in rats at 6 months of age is denoted by *. Significant differences (P < 0.05) for groups at 8 months of age (open bars) are denoted as (a) vs. OVX group at 6 months, (b) vs. OVX control at 8 months, and (c) vs. SHAM group at 8 months.

 


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  		Figure 7. Effects of 2ME2 on OVX bone formation rate (surface referent) and mineralizing perimeter of the OVX adult rat (experiment 2). Adult rats were treated with 2ME2 (4 mg/kg) 5 d/wk for 8 wk. Measurements were double labeled perimeter based and all values are expressed as mean ± SEM (n = 9–10). Significant differences (P < 0.05) are denoted as (b) vs. OVX control at 8 months and (c) vs. SHAM control at 8 months.

 
Dynamic histomorphometry for cortical bone are depicted in Fig. 8Go. 2ME2 suppressed rates of periosteal mineral apposition (Fig. 8AGo) and bone formation (Fig. 8BGo) stimulated by OVX but not to the levels in sham-operated rats.



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  		Figure 8. Effects of 2ME2 on cortical bone histomorphometry of the OVX adult rat (experiment 2). Adult rats were treated with 2ME2 (4 mg/kg) 5 d/wk for 8 wk. Cross-sections were obtained at the tibial diaphysis at the tibia-fibula synostosis. Dynamic measurements and estimations of rat were conducted for growth between the baseline fluorochrome label and killing. All values are expressed as mean ± SEM (n = 9–10). Significant differences (P < 0.05) are denoted as (b) vs. OVX control at 8 months and (c) vs. SHAM control at 8 months.

 
Experiment 3
Table 3Go summarizes the effects of 2ME2, ICI, and the combination of 2ME2 and ICI on weanling rats. By two-way ANOVA, 2ME2 had no effect on uterine wet weight but suppressed periosteal mineral apposition rate and periosteal bone formation rate. ICI decreased uterine wet weight, increased periosteal mineral apposition, and increased periosteal bone formation rate. ICI had no effect on longitudinal growth rate (data not shown). Effects of 2ME2 were not influenced by the presence of ICI, and vice versa, for any of these parameters. One-way ANOVA and post hoc analysis indicated that 2ME2 significantly reduced MAR and BFR in the –ICI weanling rats but did not significantly increase uterine wet weight. Similarly, in the + ICI weanling rats, treatment with 2ME2 had no effect on uterine wet weight but reduced MAR and BFR.


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  		Table 3. Effects of 2ME2 and ICI in weanling rats (experiment 3)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
We conducted a histomorphometric evaluation of the effects of 2ME2 in the tibia of the OVX growing and adult rats. We report that this estrogen metabolite, which is recognized for its antiproliferative and toxic actions on tumor cells, can partially prevent cortical and cancellous bone changes induced by OVX at the doses required for optimal tumorstatic activity in mice (5). The results of this study suggest that bone metabolism in estrogen-deficient women may be affected during the course of tumor treatment with 2ME2 but that these effects are likely to be beneficial.

Consistent with the well-known effects of OVX (19, 20, 21), the rats in this study exhibited uterine atrophy, cancellous osteopenia, and increased turnover of cancellous bone. Increased longitudinal and radial bone growth was evident with OVX. These changes were largely prevented by 17{alpha}-EE2, implying that the altered metabolism associated with OVX is due to estrogen deficiency. Additionally, and confirming previous reports, 2ME2 was effective in reducing serum cholesterol, another well-recognized action of E2 (16, 22). Unexpected, however, were the dramatic effects of 2ME2 on bone metabolism. Furthermore, a nonuterotrophic dose suppressed bone remodeling in the adult rat by reducing the extent of mineralizing perimeter and activity of osteoblasts. 2ME2 appears to preserve bone mass in the juvenile rats by suppressing bone remodeling.

The different sensitivity of the uterus and bone to 2ME2—as reflected by different potency of 2ME2 to restore measurements to sham values—may indicate different underlying mechanisms of action. There are several potential mechanisms by which 2ME2 could mimic the actions of E2 on bone and uterus. First, 2ME2 could act as an estrogen receptor agonist. A review of 2ME2’s estrogenicity (23) and an assessment of its interaction with estrogen receptors (24) suggest that this is not the case. The reported affinity of 2ME2 for both estrogen receptor subtypes is exceptionally weak (12), which could account for 2ME2’s lower potency in vivo. The affinity of 2ME2 for estrogen receptor ß was even lower than for estrogen receptor {alpha} (24). In addition, the in vitro antiproliferative activity of 2ME2 is independent of estrogen receptor expression, and not affected by agonists (E2) or competitive inhibitors (ICI) of estrogen receptors (15). Our data from the weanling rats suggested the presence of circulating estrogen, as indicated by the antagonism of uterine wet weights by ICI. However, the presence of estrogen is unlikely to have an impact on the failure of ICI, the high affinity estrogen receptor ligand (25), to antagonize 2ME2’s actions on cortical bone and on cartilage. In addition, 2ME2 neither antagonizes nor potentiates the action of circulating E2 in growing ovary-intact rats (16). Thus, the in vivo and in vitro observations suggest that at least some of the observed actions of 2ME2 are not mediated by conventional estrogen receptors.

Second, 2ME2 could undergo demethylation to 2-hydroxyestradiol, which in turn could act as an estrogen receptor agonist. The estrogenic effects induced by high doses of 2ME2 in several species correlated with its conversion into 2-hydroxyestradiol and 2-hydroxyestrone by liver microsomes of those same species (EntreMed, data on file). In addition, the ability of 2ME2 to sustain the growth of an estrogen-dependent cell line was abolished by inhibitors of its demethylation to 2-hydroxyestradiol, suggesting that the estrogenic activity was not intrinsic to 2ME2 (25). 2-Hydroxylation of the metabolite estrone essentially inactivates its estrogenic activity (26, 27). However, until the in vivo actions of 2-hydroxyestradiol on bone cells have been characterized, we cannot evaluate whether the observed effects of 2ME2 could occur through this mechanism. Although demethylation might occur in different tissues, circulating 2-hydroxyestradiol is maintained at a very low level due to the high content of catechol-O-methyl transferase in erythrocytes (23). Finally, 2ME2, through a novel, estrogen receptor-independent mechanism, could regulate either the same signaling pathways activated by E2 or alternative pathways that result in similar phenotypic changes.

The possibility of a novel, estrogen receptor-independent mechanism is supported by several observations. There is a high degree of structural specificity required to confer the unique actions of 2ME2; the estrogen metabolite has actions that are not mimicked by E2, 2-hydroxyestradiol, or 2-methoxyestrone (15). These unique actions include antiproliferative activity in a wide variety of cell types, antiangiogenic activity in vivo and in vitro (6), and the killing of osteosarcoma and of immortalized osteoblastic cells (15). The killing of immortalized osteoblasts is independent of the number and subtype of estrogen receptor (15). Finally, the respective effects of E2 and 2ME2 on gene expression differ dramatically in skeletal tissue (28). Although a limited dose range was used in this study to establish tissue-specific responses to E2, our observations were consistent with other investigations using extended dose ranges for both orally administered 17{alpha}-EE2 (29) and sc administered E2 (30).

The findings of the present study demonstrate that 2ME2 has tissue selective actions superficially similar to selective estrogen receptor modulators (SERMs). Most SERMs exhibit largely estrogen agonistic activity on bone and largely antagonistic activity on reproductive tissues. 2ME2, however, differs from tamoxifen, raloxifene, ICI, and other SERMs in that the administration of a high dose to intact rats (16) does not antagonize endogenous estrogen’s stimulation of growth and differentiation in reproductive tissues (16). This observation demonstrates that 2ME2 acts by a mechanism different from SERMs. The results of recent studies suggest that local metabolism of estrogens can lead to tissue selectivity (27). It is possible that the observed tissue selectivity of 2ME2 is due to its local metabolism to a more potent estrogen.

In agreement with Liu and Bachmann (22), 2ME2 decreased serum cholesterol. Oral administration of 17{alpha}-EE2 also reduced serum cholesterol. We have previously shown that E2 by oral administration lowers cholesterol (31) but that administration by sc injection or continuous infusion does not (26, 27). E2 is extensively metabolized before its absorption (32), suggesting that a metabolite is responsible for the hypocholesteremia, rather than the parent compound. A precedent for this mechanism exists in the uterus of the estrogen receptor {alpha} knockout mouse where lactoferrin, an estrogen-response gene, is up-regulated by a catecholestrogen metabolite (33). Thus, estrogen metabolites may actually mediate some of estrogen’s actions in target tissues, including bone. But, the significance of this would be difficult to ascertain until methods to measure levels of metabolites in target tissue cells become available.

2ME2 is currently being evaluated in phase I clinical trials for breast cancer (34). As potential therapy for the postmenopausal women with mammary tumors, 2ME2 may offer additional protection against menopause-induced bone loss due to accelerated skeletal remodeling. Animal experiments have suggested that long-term breast cancer treatment with the SERM tamoxifen may promote the selective growth of hormone-independent tumors (35). The tumor suppressive activity of 2ME2, in contrast, appears to occur through a nonestrogen receptor mechanism and thereby is an alternative to SERMs such as tamoxifen and raloxifene for breast cancer treatment.

In summary, 2ME2 treatment—a potential new chemotherapy with low toxicity, partial tissue selectivity, and nonestrogen receptor-dependent antitumor activity—has desirable actions on bone metabolism in OVX rats with reduced effects on uterine growth and differentiation. 2ME2 would particularly benefit postmenopausal women at risk, or undergoing treatment, for breast cancer. We conclude that 2ME2 is a potential substitute for estrogen and SERMs in the prevention of postmenopausal osteoporosis. The excellent potential for lower risk-to-benefit characteristics of the weak estrogens warrant further evaluation of estrogen metabolites as treatments for osteoporosis.


   Note Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
2ME2 is also being evaluated in phase I/II clinical trials for both breast and prostate cancer (36, 37, 38).


   Acknowledgments
 
The authors extend their appreciation to Ms. Minzhi Zhang, Ms. Julie A. Burgess, Ms. Angela M. Kennedy, and Dr. Theresa E. Hefferan for their technical assistance in conducting the animal experiments; to Mr. Glenn M. Swartz (EntreMed, Rockville, MD) for the preparation of liposomes containing 2ME2 and 17{alpha}-EE2; and to Ms. Lori Rolbiecki and Ms. Peggy Backup for editorial assistance.


   Footnotes
 
This work was funded by NIH Grant AR-45233 and the Mayo Foundation.

Abbreviations: E2, 17ß-Estradiol; 17{alpha}-EE2, 17{alpha}-ethynyl estradiol; ICI, ICI 182,780; 2ME2, 2-methoxyestradiol; OVX, ovariectomized; SERM, selective estrogen receptor modulator.

Received June 18, 2002.

Accepted for publication November 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

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