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Endocrinology Vol. 138, No. 5 1794-1800
Copyright © 1997 by The Endocrine Society

Articles

Clomiphene Prevents Cancellous Bone Loss from Tibia of Ovariectomized Rats

M. A. Jimenez, D. E. Magee, H. U. Bryant and R. T. Turner

Department of Orthopedics and Biochemistry and Molecular Biology (M.A.J., R.T.T.), Mayo Clinic, Rochester, Minnesota 55905; and the Department of Skeletal Diseases (D.E.M., H.U.B.), Lilly Corporate Center, Indianapolis, Indiana 46285

Address all correspondence and requests for reprints to: Russell T. Turner, 3-69 Medical Science Building, Department of Orthopedic Research, Mayo Clinic, Rochester, Minnesota 55905.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen inhibits postmenopausal bone loss and decreases fracture risk. Unfortunately, estrogen replacement therapy has many undesirable side effects, the majority of which are due to stimulation of reproductive tissues. Tissue specific estrogen agonists provide a promising new alternative to natural estrogens for hormone replacement. Clomiphene (CLO) is a substituted triphenylethylene antiestrogen based on its ability to antagonize estrogen-mediated uterine growth in rodents. CLO is used clinically for the treatment of disorders of ovulation in patients wishing to become pregnant. In order to determine whether CLO has tissue selective actions, we performed a dose-response study in adult (6-month-old) ovariectomized (OVX’d) rats. The rats received daily (gavage) doses of either 17 {alpha}-ethynyl estradiol (E) (0.1 mg/kg) or CLO (0.01–10 mg/kg) daily for 5 weeks. Long-term loss of ovarian function had no effect on serum cholesterol, greatly decreased uterine weight, cancellous bone area and trabecular number, and increased bone formation rate (BFR) and osteoblast and osteoclast perimeters. E treatment of OVX’d rats prevented uterine atrophy, greatly lowered cholesterol, and prevented many of the bone changes. CLO was a very weak estrogen agonist in supporting uterine weight, a partial agonist in reducing serum cholesterol, and an excellent agonist in maintaining normal bone mass and indices of bone turnover. We conclude from these studies that CLO exhibits pronounced tissue selective estrogen agonism in the rat. Specifically, CLO is effective in preventing cancellous bone loss in the OVX’d rats and has minimal uterotrophic activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOPOROSIS is one of the most important disorders associated with aging. More than 1.5 million Americans have fractures related to this abnormality. During the course of a lifetime, women typically lose 50 percent of their cancellous and 30 percent of their cortical bone (1). Much of this bone loss is related to gonadal insufficiency. Surgical or natural menopause results in elevated bone turnover, an imbalance between bone formation and bone resorption and net bone loss (1, 2). Similar skeletal changes occur in rats after ovariectomy (OVX) (3, 4). Estrogen replacement is effective in preventing bone loss in menopausal women (5, 6, 7) and rats after OVX (4, 8, 9). However, estrogen therapy in women is associated with an increased risk of both breast and uterine cancers (10). Moreover, other nonthreatening side effects may lead to poor compliance and contraindications in some patients (11, 12).

The limitations of estrogen replacement therapy have led to a search for safe, effective alternatives. One group of promising agents that has been investigated is collectively known as the antiestrogens (13). Antiestrogens can be structurally dissimilar and include steroid, substituted triphenylethylene, and benzothiophene-derived compounds. Antiestrogens are functionally similar in that they antagonize uterine growth and vaginal cornification induced by estrogen in rodents (14). In contrast, antiestrogens have highly variable effects on nonreproductive estrogen target tissues. The antiestrogens studied to date include: Tamoxifen (TAM) (15), Raloxifene (RAL) (16), Droloxifene (DRO) (17), Clomiphene (CLO), ICI 182, 780 and ZM 189, 154 (18, 19). Treatment of growing and skeletally mature OVX rats with TAM reduces the changes in bone architecture and bone mineral content that are expected after OVX but may also result in partial estrogen antagonism to the skeleton in ovary intact animals (20, 21, 22, 23). DRO is 3-hydroxy tamoxifen and has actions similar to the parent compound (17). RAL was similar to TAM in that it prevented OVX-induced cancellous osteopenia and increases in radial bone growth, bone resorption, and blood cholesterol. However, RAL was less effective than TAM in suppressing cancellous bone turnover and more effective in antagonizing uterine growth and differentiation (16, 24). Limited studies suggest that CLO has effects on bone mineral content and uterine tissue weight, similar to tamoxifen (25, 26). The effects of CLO on bone growth, cell numbers and activities, cancellous bone mass and architecture, and turnover are, however, unknown. ICI 182, 780 and ZM 189, 154 differ from the above mentioned antiestrogens in that they do not prevent bone loss in OVX rats and may induce cancellous osteopenia in ovary intact rats (18, 19, 27).

Clinically, CLO is an established agent for the induction of ovulation in subfertile women (28). The most common side effects of CLO are hot flashes (mild), headaches, constipation, breast soreness, and weight gain. The use of estrogen has similar but more extreme side effects and also include more potentially serious side effects such as cancer and coagulability disorders. The effects of CLO on bone are of interest because of its current clinical use and because of the noted limitations of conventional hormone replacement therapy. The purpose of this study in adult OVX’d rats was to compare the actions of estrogen and CLO on bone architecture and turnover.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Forty-two 6-month-old virgin Sprague-Dawley rats, weighing between 247–353 g, were purchased from Charles River Laboratories (Portage, MI). The rats were housed in hanging cages, maintained at a temperature of 22.2C and on 12-h light cycle, and had continuous access to food [TekVad Diet, TD89222 (0.5% calcium and 0.4% phosphorous), TekVad, Madison, WI].

The animals were ovariectomized under ketamine hydrochloride (120 mg/kg) and Xylazine 5 hydrochloride (24 mg/kg) anesthesia via a dorsal midline incision. The animals were permitted 4 days to recover from the surgery. Then they were divided into seven weight-matched groups, each containing six animals. At the beginning of the study, all animals received 20 mg/kg of Calcein via tail vein injection. In addition, all rats also received 20 mg/kg of Tetracycline HCl and 20 mg/kg of Alizarin via tail injection at 27 days and 33 days after start of experiment, respectively. The OVX control rats and age-matched sham-operated rats were given equivalent volumes of vehicles (20% ß-cyclodextrin) by gavage. All other groups received either 17 {alpha}-ethyl estradiol (E) [0.1 mg/kg · day], or clomiphene citrate by gavage for 35 days. Animals that were treated with CLO received either 0.01, 0.1, 1.0, or 10.0 mg/kg daily. The dose of E was determined in earlier studies that showed that, at this dose, E prevents the fall in bone mineral density from proximal tibia in OVX rats (23).

The animals were fasted the night before death (day 35) and anesthetized with a mixture of ketamine and xylazine (67 and 6.7 mg/kg, respectively). Blood was collected via cardiac puncture, and the rats were killed by carbon dioxide gas. The uterus was quickly excised for wet weight, and tibiae were fixed in 70% ethanol for cortical and cancellous bone histomorphometry.

Calcein, alizarin complexone, tetracycline HCL, clomiphene citrate, and 17 {alpha}-ethynyl estradiol were all obtained from Sigma (St. Louis, MO).

Cholesterol assay
Blood samples were allowed to cool at room temperature for 2 h, and serum was obtained after centrifugation for 10 min at 3000 rpm. Serum cholesterol was determined using a Boehringer Mannheim Diagnostics high performance cholesterol assay. Briefly, cholesterol esters were hydrolyzed into free cholesterol and fatty acids by a microbial cholesterol esterase. The cholesterol was then oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide was then reacted with phenol and 4-aminophenazone in the presence of a peroxidase to produce a p-quinone imine dye, which was read spectrophotometrically at 500 nm.

Bone histomorphometry
Histomorphometric measurements were performed with the OsteoMeasure Analysis System (OsteoMetrics, Atlanta, GA), which consisted of a computer CRT101 + (OsteoMetrics) coupled to a photomicroscope and image analysis system. This image system consisted of a high resolution color video camera (JVC TK-10704, Japan) that records the image specimen through the microscope (Olympus BH-2, New Hyde Park, NY) and displays the image on a video monitor (Electrohome 38-D051MA-YU, Ontario, Canada) that registers the movement of a digitizing pen on a graphics tablet (Osteotablet, OsteoMetrics, Atlanta, GA). The region of interest is traced, and the line lengths and area bounded by lines are calculated automatically.

Cortical bone measurements
Ground transverse sections were used for histomorphometric analysis of cortical bone. Cross-sections 150 µm thick were cut just proximal to the tibia-fibula synostosis with a low speed saw (Isomet, Buehler, Lake Bluff, IL) equipped with a diamond wafer blade. The sections were ground to a thickness of 15–20 µm on a roughened glass plate and mounted in glycerin. The samples were microscopically examined under UV light to visualize fluorochrome labels. The following measurements were performed: 1) cross-sectional area, defined as the area of bone and marrow cavity bounded by the periosteal surface of the specimen; 2) medullary area, defined as the area delineated by the endocortical surface of the specimen; 3) cortical bone area, calculated as the difference between cross-sectional and medullary area; 4) periosteal perimeter, defined as the total perimeter enclosing the cross section (includes fluorochrome-labeled and nonlabeled perimeters); 5) endocortical perimeter, defined as the total perimeter enclosing the medullary cavity, including fluorochrome-labeled and nonlabeled perimeters; 6) periosteal bone formation rate, calculated as the area bounded by the calcein label and divided by the labeling period of 35 days; 7) periosteal mineral apposition rate, defined as the periosteal bone formation rate divided by the label perimeter; 8) periosteal label perimeter, defined as the periosteal perimeter labeled with alizarin.

Cancellous bone measurements
The metaphysis was dehydrated in a series of increasing concentrations of ethanol, embedded without demineralization in a mixture of methylmethacrylate-2-hydroxyethyl-methacrylate (12.5:1) to retain the fluorochrome labels, and sectioned at a thickness of 5 µm.

A standard sampling site was established in the secondary spongiosa of the metaphyseal region of the proximal tibia, 1 mm distal to the calcein label that was deposited in the mineralizing cartilage at the epiphyseal growth plate at the beginning of the study, at a point where its center was perpendicular to and on the long axis of the bone. The sampling site is situated in the secondary spongiosa and extends bilaterally in each section but excludes the cortical edges. A total metaphyseal area of 2.8 mm2 was sampled for each section.

Measurements related to bone growth
Longitudinal bone growth was determined by measuring the distance from trabeculae containing calcein label to the mineralizing growth plate cartilage. The mean longitudinal growth rate (LGR) was calculated by dividing this distance by the 35-day duration of the experiment.

Measurements related to bone volume
Cancellous bone area was determined as the area of total cancellous bone per mm2 metaphyseal area within the sampling site and expressed as a percentage. Cancellous bone perimeter was determined as the perimeter of cancellous bone per mm2 metaphyseal area.

Calculations related to bone architecture
Trabecular thickness (Tb.Th.) was calculated as Tb.Th = 2/B.Pm/B.Ar, where B.Ar and B.Pm are cancellous bone area and perimeter, respectively.

Trabecular separation (Tb.Sp) was calculated as Tb.Sp = Tb.Th/(B.Ar/T.Ar) - Tb.Th, where T.Ar is tissue area.

Trabecular number (Tb.N) is Tb.N = (B.Ar/T.Ar)/Tb.Th.

Measurements and calculated values related to bone formation
The bone formation rate (tissue referent) was calculated as the double (labels 2 and 3) labeled perimeter plus 1/2 single labeled perimeter per mm2 metaphyseal area times the mineral apposition rate.

The bone formation rate (perimeter referent) was calculated as the double (labels 2 and 3) labeled perimeter plus 1/2 single-labeled perimeter per mm cancellous bone perimeter times mineral apposition rate.

The mineral apposition rate was the mean distance between the tetracycline label and the alizarin label, measured every 50 µm, divided by the labeling interval of 6 days. This method gives a weighted average for the mineral apposition rate by taking into account label length. Methods of calculating mineral apposition rate that give the intralabel distance for all double-labeled regions equal weight will be unduly influenced by the shorter labels.

Osteoblast perimeter was determined in 5-µm thick toluidine blue-stained sections. Osteoblasts were identified as a palisade of large basophilic cuboidal cells directly lining a bone perimeter.

Measurement related bone resorption
Calcein label (label 1) perimeter (tissue area referent) was measured in the growth-adjusted metaphyseal sampling site, as described (16). The calcein-labeled perimeter (perimeter referent) was determined as the cancellous bone perimeter with calcein label and expressed as a percentage of the total cancellous bone perimeter.

Osteoclast perimeter was determined in 5-µm thick toluidine blue-stained sections. Osteoclasts were identified as large, multinucleated cells with a foamy cytoplasm and usually were located within a Howship’s lacunae.

Statistical analysis
One-way ANOVA was performed with Fisher protected least significant difference post-hoc multiple comparison tests to establish significance. The treated groups were compared with the OVX and sham groups to establish the effects of treatment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of OVX, E, and CLO on body weight and serum cholesterol are shown in Table 1Go. OVX had no significant effect on serum cholesterol and increased body weight. E treatment after OVX significantly reduced serum cholesterol and prevented the increase in body weight. Serum cholesterol levels were partially reduced by CLO, compared with estrogen-treated animals, but with higher doses the reduction was more pronounced.


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  		Table 1. Effects of OVX, estrogen, and clomiphene on serum cholesterol and body weight

 
The effects OVX, E, and CLO on uterine weight are shown graphically in Fig. 1Go. OVX significantly reduced uterine weight. E treatment following OVX maintained uterine weight. CLO at low doses (0.01 and 0.1 mg/kg) had no effect on uterine weight. At higher doses (1.0 and 10.0 mg/kg), CLO-treated rats had uterine weights that exceeded OVX but were significantly lighter than the sham-operated group and the estrogen-treated group.



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  		Figure 1. The effects of OVX, estrogen, and clomiphene on uterine weight. Values are mean ± SE. a P < 0.05 compared with OVX; b P < 0.05 compared with sham.

 
Cortical bone histomorphometry data is shown in Table 2Go. OVX, E and CLO had no significant effect on cross-sectional area, medullary area, or cortical bone area, but OVX increased P.s.BFR. This increase in bone formation was due to nonsignificant increases in Ps.L.Pm. and Ps.MAR. E inhibited Ps.BFR, and there was a dose dependent inhibition of P.s.BFR. by CLO.


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  		Table 2. Effects of OVX, estrogen, and clomiphene on cortical bone histomorphometry

 
Indices of cancellous bone architecture are shown in Table 3Go and Fig. 2Go. OVX significantly reduced cancellous bone area, trabecular number and perimeter, and increased trabecular separation. E prevented the changes in trabecular number and separation after OVX but only partially blocked the reduction in cancellous bone area and trabecular perimeter. CLO, at the lowest concentration (0.01 mg/kg), did not significantly alter cancellous architecture. However, higher doses of CLO prevented the decrease in trabecular number, perimeter, and cancellous bone area, and increase in trabecular separation. At a dose of 1 mg/kg, CLO significantly increased trabecular thickness compared with the sham operated group.


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  		Table 3. Effects of OVX, estrogen and clomiphene on cancellous bone architecture

 


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  		Figure 2. The effects of OVX, estrogen, and clomiphene on cancellous bone area (%) in the proximal tibial metaphysis. Values are mean ± SE. a P < 0.05 compared with OVX; b P < 0.05 compared with sham.

 
Measurements related to bone growth and cancellous bone formation and resorption are summarized in Table 4Go. OVX resulted in an increase in the LGR that was prevented by treatment with E. CLO resulted in a dose-related decrease in LGR to values that did not differ significantly from the sham rats. OVX resulted in an increase in MAR (perimeter referent), BFR (perimeter referent), and labeled perimeter. Both E and CLO (at doses >=0.1 mg/kg) prevented these changes. OVX resulted in increases in osteoblast and osteoclast perimeters, changes that were prevented by E and CLO (all doses). In addition, OVX resulted in the tendency to reduce calcein label (Pre-OVX label) perimeter and treatment with E and CLO tended to maintain calcein label perimeter. However, these tendencies did not achieve statistical significance.


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  		Table 4. Effects of OVX, estrogen, and clomiphene on cancellous bone dynamic histomorphometry

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CLO was shown to reduce changes in bone mineral density in young OVX’d rats (25, 26), but these earlier studies did not investigate the cellular mechanism of action. Furthermore, bone mineral density can be difficult to interpret in growing OVX rats because gonadal hormone insufficiency results in changes in bone geometry as well as bone mineral content. It was therefore deemed important to examine the effects of CLO on skeletally mature adult rats. Bone histomorphometry was analyzed in the present study to provide a more detailed analysis of cellular and architectural changes in bone.

OVX resulted in changes in cortical and cancellous bone consistent with previous studies (3, 4, 8, 9). The cortical bone changes consisted of increases in medullary area and radial bone growth due to enhanced periosteal bone formation (4). The OVX-induced increase in the medullary area observed in this study, although similar in magnitude to published studies, failed to reach significance by ANOVA. As a result, the effects of CLO on OVX enhanced endocortical bone resorption were not determined. The changes in cancellous bone following OVX included the expected increases in BFR (perimeter referent), MAR, osteoblast perimeter, and osteoclast perimeter and decreases in bone area, total perimeter, and trabecular number. As expected, BFR (tissue referent) was not increased because of the severe cancellous osteopenia.

E treatment prevented most of the skeletal changes observed following OVX as well as the uterine atrophy and increased weight gain. However, E replacement did not normalize all measurements; E was only partially effective in preventing cancellous osteopenia following OVX, whereas the hormone reduced serum cholesterol to values below the sham-operated controls. These findings suggest that administration of E by gavage does not mimic normal ovarian function. In this regard, gavage may be less effective than sc implantation of controlled release pellets in preventing cancellous osteopenia following OVX (23).

CLO treatment prevented all of the bone changes that follow OVX. Additionally, the higher doses of CLO increased trabecular thickness, an action not typically observed in E-treated OVX rats. The precise mechanism for the difference is not clear; E and CLO each decreased indices of bone turnover. However, a small but persistent positive difference in the balance between bone formation and bone resorption would be sufficient to account for the observed small increase in trabecular thickness.

CLO had biological activities similar to but not identical to several other tissue selective estrogen agonists. Tamoxifen slowed but, unlike CLO, was unable to prevent cancellous osteopenia in adult OVX rats (29). CLO and RAL also appeared to differ in their respective actions on cancellous bone. The CLO-induced increase in trabecular thickness observed in this study was not described for RAL (16). On the other hand, CLO appears to be a more potent inhibitor of bone formation than RAL (16).

CLO had estrogen agonistic effects on body weight and serum cholesterol. OVX results in increased body weight (3, 4), and this change was prevented by treatment with either E or CLO. OVX has had variable effects on serum cholesterol in rats. For unexplained reasons, cholesterol is increased in some studies (16) but not others (17). In either situation, estrogen treatment reduced serum cholesterol in OVX rats. CLO initiated a dose-dependent decrease in serum cholesterol, with the maximum response resulting in serum values that were depressed compared with ovary intact rats. Moreover, studies in postmenopausal women have noted an increase in HDL cholesterol following treatment with CLO (30). Thus, CLO may share the cardiovascular protective qualities attributed to estrogen.

We did not investigate uterine histology, and no studies to date in CLO-treated rats have examined the epithelial height and stromal eosinophilia of uteri. Studies in humans treated with CLO have identified specific sites of uterine action. In a study of infertile women, CLO significantly altered cervical mucous quality and late luteal phase endometrial morphology despite physiological levels of plasma estrogen (31). In another study of postmenopausal women, CLO was found to increase the incidence of endometrial atrophy compared with control and estrogen-treated groups and also decreased the concentration of cytosol estrogen receptor in the endometrium (32).

Most of the effects of estrogen on reproductive tissues are believed to be mediated by the binding of the hormone to a specific receptor (33). The estrogen-estrogen receptor complex then functions as a gene specific transcription factor (34, 35). In contrast to the well characterized and abundant receptor in reproductive tissues, localization of estrogen receptor in the skeletal tissues has proven difficult (36). The messenger RNA for the estrogen receptor was recently detected in rat periosteum (37), offering support that the described skeletal effects of E and CLO are estrogen receptor mediated. Additionally, some of the skeletal effects of estrogen are rapid (38, 39, 40) in rats and birds and are antagonized by TAM in Japanese quail, a species in which the antiestrogen functions as a pure estrogen receptor antagonist (38).

The mechanisms for tissue-selective actions of antiestrogens have not been elucidated. Antiestrogens compete with estrogen for binding to estrogen receptors and fail to initiate gene transcription (41, 42). In the absence of the hormone, the receptor resides in the target cell nuclei as part of a large macromolecular complex comprising heat-shock protein 90 and other proteins (41). Some studies suggest that estrogen receptor agonists and antagonists promote dissociation of the heat shock protein and permit the interaction of receptor with specific DNA sequences in the regulatory regions of target gene promoters (42, 43). Consequently, it appears that events that follow DNA binding are likely responsible for distinguishing between agonist and antagonist activated receptors (44). Moreover, a recent discovery of two distinct transcriptional regulatory sites on the estrogen receptor gene may shed important light on the mechanism for tissue selective actions of antiestrogens. These two sites appear to be differentially regulated in a target cell context-dependent manner by estrogen and antiestrogens (42, 43, 44, 45, 46).

The survival advantage of adjavent TAM therapy due to reduction in the incidence of tumor recurrence in women with breast cancer is well established (47). Numerous studies in postmenopausal women with breast cancer have also demonstrated the beneficial effects of TAM in reducing bone loss (48, 49, 50, 51). However, TAM has detrimental side effects. A recent study demonstrated that 37% of women taking TAM showed histological changes in the endometrium, similar to unopposed estrogen replacement (47). Additionally, studies in ovary intact adult rats and premenopausal women suggest that TAM may have detrimental effects on the skeleton of women with normal ovarian function (29, 51).

The beneficial effects and limitations of TAM has stimulated research on related compounds. The results of this study with CLO and previous studies with RAL (16, 24), DRO (17), and TAM (23) have demonstrated similarities as well as differences between the skeletal actions of individual antiestrogens with each other and with estrogen. These differences may be very important in optimizing the efficacy and minimizing the incidence of detrimental side effects of tissue selective estrogen agonists when applied clinically for preventing and treating postmenopausal bone loss.

Received September 30, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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