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derivatives of tamoxifen and clomiphene in ovariectomized rats
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Specific Bone-Protective Effects of Metabolites/Derivatives of Tamoxifen and Clomiphene in Ovariectomized Rats P. C. RUENITZ,1 Y. SHEN,2 M. LI,2 H. LIANG,2 R. D. WHITEHEAD, JR.,1 S. PUN,2 and T. J. WRONSKI2 1 2
College of Pharmacy, University of Georgia, Athens, GA, USA Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
abnormalities in bone remodeling, the most common of which is loss of gonadal steroid action, which can occur in menopause or in male or female hypogonadism. Thus, postmenopausal bone loss is not itself a disease but rather a consequence of endocrine imbalance. When estrogen replacement therapy is indicated for prevention of postmenopausal bone loss, there are several types of therapeutic agents available or in development. The most important of these suppress bone resorption and formation in a manner that maintains net bone balance, probably as a consequence of interaction with estrogen receptors (ER) in osteoblasts and osteoclasts, cell types responsible for bone maintenance.3,8,30 Thus, 17b-estradiol and its orally active analogs, including conjugated equine estrogens, are widely used in estrogen replacement therapy.29 A major drawback to long-term administration of estrogens for prevention of osteoporosis is undesirable effects on the reproductive tract. Thus, an increased risk of developing uterine cancer has been associated with estrogen replacement therapy.4 In addition, a perceived link between estrogen use and breast cancer is of concern.13 An approach to this problem is identification of substances whose estrogenicity is restricted to nonreproductive tissues. Thus, tamoxifen (TAM) and clomiphene (CLO) (Figure 1), nonsteroidal ER ligands previously thought of in terms of their estrogen antagonist effects on the reproductive axis, and on estrogen-dependent cancers,29 have been found to act like estrogen mimetics in prevention of bone loss in postmenopausal women.9,20,32,42 Consequently, analogs of these triphenylethylenes with decreased residual uterotrophic effects are of continuing interest.26 The ovariectomized (ovx) rat has become an established animal model of osteopenia associated with estrogen deficiency. Histomorphometric analysis of bone specimens from ovx rats receiving 17b-estradiol, TAM, or CLO has shown a decreased rate of bone turnover and maintenance of normal bone mass compared with that observed in untreated ovx rats.2,15,21,33,40 Similarly, estrogen or TAM administration to ovx rats resulted in decreased serum levels of osteocalcin.37 Osteocalcin is a bone matrix protein31 released into the serum during bone formation, thus serving as a specific indicator of bone turnover.6 Clarification of the ways that TAM, CLO, and their structural analogs exert their bone-protective effects is complicated by the fact that these drugs can undergo extensive oxidative biotransformation to metabolites having divergent profiles of activity relative to the parent drugs. Thus, in the rat, CLO was converted to 4-hydroxy CLO (Figure 1), which had greatly increased antiuterotrophic potency/efficacy compared with CLO itself.23 In
In the ovariectomized (ovx) rat, the nonsteroidal antiestrogens, clomiphene (CLO) and tamoxifen (TAM), at dose levels that prevent development of osteopenia to a degree approaching that of 17b-estradiol are, in contrast to 17bestradiol, only weakly uterotrophic. Metabolites of CLO and TAM might contribute differentially to these effects. Thus, we have evaluated bone protective and uterine effects in ovx rats of two such metabolites: 4-hydroxy CLO, produced by p-hydroxylation of CLO; and 4HTA, produced from TAM by stepwise replacement of its dimethylaminoethyl side chain with an acetic acid moiety, accompanied by p-hydroxylation. Also reported are effects of D4HTA, the dihydrodesethyl derivative of 4HTA previously characterized as a full estrogen mimetic in vitro. Administration of 4-hydroxy CLO (2.5 mg/kg subcutaneously) 5 days/week for 5 weeks to 3-monthold ovx rats resulted in complete prevention of bone loss and suppression of bone turnover to levels comparable to those of intact controls and to those of ovx animals similarly receiving 17b-estradiol (10 mg/kg). However, uterine weight in animals receiving 4-hydroxy CLO was 64% less than that in 17bestradiol-treated animals. Although 4HTA (3.7 mg/kg s.c.) had a modest uterotrophic effect, it did not prevent bone loss associated with ovariectomy. In contrast, D4HTA (3.6 mg/kg s.c.) partially reduced bone turnover indicators and cancellous bone loss in a manner similar in many ways to that observed in TAM-treated ovx animals, but it had no uterotrophic effect. These results suggest that, although 4HTA does not contribute to the bone-protective effect of TAM, 4-hydroxy CLO might augment that of CLO. (Bone 23: 537–542; 1998) © 1998 by Elsevier Science Inc. All rights reserved. Key Words: Antiestrogen; Clomiphene; Estrogen; Osteoporosis; Osteopenia; Tamoxifen. Introduction Osteoporosis is characterized by a progressive decrease in bone density, which can lead to an increased incidence of bone fractures.36 This condition results when the rate of bone resorption exceeds that of bone formation. Several disorders induce Address for correspondence and reprints: Dr. Peter C. Ruenitz, College of Pharmacy, University of Georgia, Athens, GA 30602. E-mail: [email protected] © 1998 by Elsevier Science Inc. All rights reserved.
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Materials and Methods Chemicals 4-Hydroxy CLO {4-[1-(p-hydroxyphenyl)-2-phenyl-2-chloroethenyl]phenoxyethyl-N,N-diethylamine}, 4HTA {4-[1-(p-hydroxyphenyl)-2-phenyl-1-butenyl]phenoxyacetic acid}, and D4HTA {4[1-(p-hydroxyphenyl)-2-phenylethyl]phenoxyacetic acid} were used as prepared and characterized previously.23,28 All other chemicals were purchased from Sigma Co. (St. Louis, MO). Animals and Dosing
Figure 1. Structures of TAM and CLO and specified metabolites, and dihydrodesethyl-4HTA (D4HTA). 4HTA and CLO are composed of the respective trans isomers (shown), plus nearly equal amounts of the corresponding cis isomers. In MCF-7 cells, TAM, CLO, and 4-hydroxy CLO were estrogen antagonists; 4HTA and D4HTA were estrogen mimetics.
vitro evaluation of 4-hydroxy CLO in MCF-7 cells, whose proliferation is dependent on the presence of estrogens, showed it to be a more potent growth suppressor than CLO.24 TAM, in addition to undergoing 4-hydroxylation and/or N-demethylation,29 is in part further metabolized by formal replacement of its aminomethyl group with a carboxyl group, affording 4HTA (Figure 1).25,27 In MCF-7 cells, 4HTA, in contrast to TAM and its N-desmethyl and 4-hydroxy metabolites, was an estrogen mimetic.28,39 However, unlike most other substances characterized as estrogens in MCF-7 cells, 4HTA was not uterotrophic in the ovx rat (unpublished findings). The effects of 4HTA and 4-hydroxy CLO in estrogen-responsive cells and tissues indicated that these metabolites could participate in the observed bone-protective effects of their respective parent drugs. Thus, we decided to investigate the effects of these compounds on bone mass, bone turnover, and uterine weight in the ovx rat. We also chose to examine the dihydrodesethyl analog of 4HTA (D4HTA) (Figure 1) in this study because its in vitro estrogenic potency and efficacy were comparable to those of 4HTA.28
In the first experiment, 50 female Sprague-Dawley rats that were approximately 90 days of age and weighed an average of 240 g at the beginning of the study were used. Animal care and handling were carried out solely at the AAALAC-accredited animal facility at the University of Georgia College of Pharmacy, in accordance with a protocol approved by the University of Georgia Institutional Animal Care and Use Committee. On the day of surgery (day 0), all rats were anesthetized with an intraperitoneal (i.p.) injection of ketamine hydrochloride and xylazine at doses of 50 and 10 mg/kg body weight, respectively. Ten rats were sham-operated, during which the ovaries were exteriorized but replaced intact. Bilateral ovariectomies were performed in the remaining 40 rats from a dorsal approach. Each rat was housed individually at 25°C with a light/dark cycle of 13 h/11 h. Food (Teklad 22/5 Rodent Diet, Madison, WI), with calcium and phosphate contents of 0.95% and 0.67%, respectively, was available ad libitum to all animals. Sham-operated rats (n 5 10) and one of the four groups of ovx rats (n 5 10) were injected subcutaneously (s.c.) with vehicle (5% benzyl alcohol in corn oil) 5 days per week for 5 weeks. The remaining 30 ovx rats (n 5 10 per group) were subjected to the same treatment regimen with 17b-estradiol, 4-hydroxy CLO, or D4HTA at respective doses of 10 mg/kg per day, 2.5 mg/kg per day, or 3.6 mg/kg per day, administered s.c. in 5% benzyl alcohol/corn oil vehicle. The aforementioned treatments were initiated on the first day after surgery. The dose level of 17b-estradiol was believed to be optimal based on earlier studies.40 Those of the hydroxytriphenylethylenes approximated maximally effective bone protective dose levels of TAM and CLO.2,15,21,33 Each rat was injected s.c. with demeclocycline and calcein (Sigma Co.) at a dose of 15 mg/kg body weight on days 10 and 3 before killing, respectively, to label sites of bone formation. A second experiment, using 40 animals, was performed in exactly the same manner as just described, in which effects of the s.c. administration of 3.7 mg/kg per day of 4HTA to one group of ovx animals was compared with the effects of vehicle or 17b-estradiol in three other groups (vehicle-treated sham ovx, vehicle-treated ovx, and 17b-estradiol-treated ovx rats). Necropsy Procedures All rats were killed by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. Failure to detect ovarian tissue and observation of marked atrophy of the uterine horns confirmed the success of ovariectomy. The body weight of each animal was recorded, as was uterine wet weight. Blood samples were allowed to coagulate at room temperature (2 h) in a Vacutainer tube. Serum was obtained by centrifugation for 10 min at 3000 rpm, and samples were stored at 280°C until analyzed. The right tibia was removed at necropsy, dissected free of muscle, and cut in half cross sectionally with a hand-held saw (Dremel Moto Tool, Racine, WI). The bone samples were then
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placed in 10% phosphate-buffered formalin for 24 h for tissue fixation.
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Table 1. Effect of various treatment regimens on whole body weight, uterine weight, and serum osteocalcin (OC) level (results from experiment 2 are in italics)a
Cancellous Bone Histomorphometry The proximal halves of the tibiae were dehydrated by immersion in ethanol, and embedded undecalcified in methylmethacrylate.1 Longitudinal sections (4 and 8 mm thick) were cut with AO Autocut/Jung 1150 or 2050 microtomes. The 4-mm-thick sections were stained according to the von Kossa method with a tetrachrome counterstain (Polysciences, Warrington, PA). Bone measurements were performed in cancellous bone tissue of the proximal tibial metaphysis beginning at distances 1 mm distal to the growth plate-metaphyseal junction to exclude the primary spongiosa. In general, two sections of the proximal tibia, with a total of 40 –50 mm of cancellous bone perimeter, were sampled in each animal with an appreciable amount of cancellous bone. Additional sections were sampled in osteopenic animals to approximate the cancellous bone perimeter sampled in animals with greater cancellous bone mass. Bone measurements were performed with the Bioquant bone morphometry system (R&M Biometrics, Nashville, TN) as previously described.41 Cancellous bone volume as a percentage of bone tissue area and osteoblast and osteoclast surfaces as percentages of total cancellous perimeter were measured in 4-mmthick, stained sections. Fluorochrome-based indices of bone formation were measured in unstained, 8-mm-thick sections of the proximal tibial metaphysis. The percentage of cancellous bone surface with a double fluorochrome label (mineralizing surface) and mineral apposition rate were measured with the Bioquant system. In addition, bone formation rate (tissue level, total surface referent) was calculated by multiplying mineralizing surface by mineral apposition rate.11 Cortical Bone Histomorphometry The distal halves of the tibiae were dehydrated in ten changes of 100% ethanol, defatted in ten changes of acetone (at least 2 h per change), and embedded undecalcified in a styrene monomer that polymerizes into a polyester resin (Tap Plastics, San Jose, CA). The tibial diaphysis 1–2 mm proximal to the tibiofibular junction was sawed into 100-mm-thick cross sections with an Isomet low-speed saw (Buehler, Lake Bluff, IL). Bone measurements were performed with the Bioquant system. Cortical bone tissue area and bone marrow area were measured in one section per animal at a magnification of 320. Cortical bone area was calculated by subtracting marrow area from cortical bone tissue area. Cortical width was measured from the periosteal to the endocortical surfaces at four equally spaced sites at the anterior (cranial), posterior (caudal), medial, and lateral aspects of each cross section. The four measurements were averaged to obtain a mean cortical width for each animal. Fluorochrome-based indices of bone formation were measured under UV illumination at magnifications of 3100 and 3200 for mineralizing surface and mineral apposition rate, respectively. The percentage of periosteal surface with a double fluorochrome label (mineralizing surface) and interlabel distances along the double-labeled surfaces were measured with the Bioquant system. The mineral apposition rate (MAR) and bone formation rate, surface referent (BFR/BS), were calculated according to the following formulas: MAR 5 interlabel distance/ time interval between labels (7 days) and BFR/BS 5 mineralizing surface 3 MAR.
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Treatment group Sham vehicle Ovx vehicle 17b-estradiol 4-Hydroxy CLO D4HTA 4HTA
Body weight (g)
Uterine weight (mg)
Relative serum OC levelb
323 (16) 304 (14) 379 (23)d 364 (25)d 310 (24)c 303 (12)c 271 (8)d 342 (23) 305 (12)
776 (101) 613 (38) 99 (19)d 86 (13)d 269 (48)c 225 (30)c 161 (21)c,d 112 (7)d 124 (12)c,d
108 (21) 127 (33) 175 (21)d 176 (29)d 100 (33)c 100 (16)c 113 (28)c 145 (27)c,d 188 (42)d
a
Data are expressed as mean (SD). The mean amount of OC in serum from 17b-estradiol-treated animals was 4.20 6 1.39 ng/mL (experiment 1), and 2.08 6 0.33 ng/mL (experiment 2). c p , 0.05 compared with ovx vehicle. d p , 0.05 compared with sham vehicle. b
Serum Rat Osteocalcin Radioimmunoassay Materials, as well as standard protocols for their use, were obtained from Biomedical Technologies (Stoughton MA). Serum samples from each member of the different treatment groups were diluted 1:20. Duplicate aliquots (100 mL) of each diluted sample were incubated in an orbital shaker at 80 rpm (4°C) for 16 h with the first antibody and nonimmune serum in RIA buffer (final volume 0.5 mL). An aliquot of [125I]-osteocalcin (10 nCi, 0.034 ng) in 100 mL of radioimmunoassay (RIA) buffer was then added to each tube and incubation was continued for 24 h. Then the second (precipitating) antibody was added in 1 mL of modified buffer, and incubation was continued for 4 h. Tubes were then centrifuged at 1500g for 15 min. Pellets were washed with cold distilled water and recentrifuged as before. Supernatant was decanted and radioactivity in pellets was determined using a gamma counter. Serum osteocalcin levels were determined by comparing the sample values (cpm) to the linear region of a standard curve of cpm/pellet vs. the amount of osteocalcin present, obtained from incubations to which known amounts (0 – 0.35 ng) of osteocalcin had been added. All values were corrected for nonspecific binding in standard incubations. Mean values (and SD) for treatment groups in each experiment are expressed relative to those of respective ovx 17b-estradioltreated groups. Statistical Analysis Data are expressed as the mean 6 SD for each group. Statistical differences among groups were evaluated by one-way ANOVA followed by Fisher’s protected least significant difference (PLSD) test for multiple comparisons.22 Differences were considered significant at p , 0.05. Results With respect to sham-operated controls, ovariectomy resulted in an average 18.5% increase in overall body weight over the 5 week course of the two studies (Table 1). This increase was not observed in ovx animals receiving subcutaneous 17b-estradiol, 4HTA, or D4HTA 5 days/week. Ovx animals similarly receiving 4-hydroxy CLO experienced a 16% loss of body weight, in comparison with sham-operated controls.
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Figure 2. Cancellous bone volume (A), osteoclast surface (B), osteoblast surface (C), and bone formation rate (D) in the proximal tibial metaphysis for the five groups of rats. Each bar is the mean 6 SD for ten animals. aSignificantly different from vehicle-treated sham group (p , 0.05); bsignificantly different from vehicle-treated ovx group (p , 0.05); csignificantly different from 17b-estradiol-treated ovx group (p , 0.05); dsignificantly different from 4-hydroxy CLO-treated ovx group (p , 0.05).
17b-estradiol treatment resulted in 167% greater uterine weight (average of both experiments), compared with vehicletreated ovx animals. This increase was not as great as has been observed in closely related studies, in which higher doses and/or more potent derivatives of 17b-estradiol were administered. Similar comparative uterine weights in animals receiving 4HTA and 4-hydroxy CLO were, in turn, 25% and 61% higher than respective ovx vehicle controls. The uterine weight of ovx animals receiving D4HTA did not differ from that of ovx animals receiving vehicle. Cancellous bone volume was decreased by 68% in vehicletreated ovx rats compared with vehicle-treated control rats (Figure 2A). In contrast, cancellous bone volume remained at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO, and treatment with D4HTA partially prevented the ovx-induced cancellous bone loss. In the second experiment, cancellous bone volume in ovx rats treated with 4HTA (8.3 6 3.7%) did not differ from that of vehicle-treated ovx animals (7.5 6 3.6%), but was lower than that of 17b-estradiol-treated ovx animals (24.0 6 5.5%). This lack of effect on cancellous bone volume, and on serum OC levels (see subsequent text), precluded further detailed histomorphometric evaluation of 4HTA effects. Osteoclast surface (Figure 2B) and osteoblast surface (Figure 2C) of vehicle-treated ovx rats increased significantly compared with that of vehicle-treated control rats at 35 days after surgery. In contrast, these cellular indices of bone resorption and formation, respectively, were suppressed at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO. Treatment
with D4HTA partially suppressed the OVX-induced increase in osteoclast and osteoblast surfaces. The effects of ovariectomy and the various treatments on mineralizing surface (data not shown) were similar to those described earlier for osteoblast surface with the exception that ovx rats treated with 17b-estradiol had a significantly lower mineralizing surface than the vehicle-treated control rats. Mineral apposition rate (MAR) was significantly increased in vehicle-treated ovx rats compared with vehicle-treated controls rats at 35 days postsurgery (1.7 vs. 1.1 mm/day, p , 0.05). In contrast, MAR was maintained at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO. Treatment with D4HTA had no effect on MAR in ovx rats. Cancellous bone formation rate (BFR/BS; Figure 2D) was significantly increased in vehicle-treated ovx rats when compared with vehicle-treated control rats. However, BFR/BS was suppressed at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO, whereas treatment with D4HTA partially suppressed the ovx-induced increase in BFR/BS. Cortical bone area and marrow area did not differ significantly among groups as the mean values ranged from 5.2 to 5.4 mm2 for the former variable and from 1.2 to 1.3 mm2 for the latter variable. Mean values for cortical width were also similar at 0.8 – 0.9 mm for all five groups. However, periosteal bone formation rate (Figure 3) was significantly increased in vehicletreated ovx rats when compared with vehicle-treated control rats. 17b-estradiol treatment of ovx rats inhibited periosteal bone formation rate to a level below that of vehicle-treated control
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Figure 3. Periosteal bone formation rate in the tibial diaphysis for the five groups of rats. Each bar is the mean 6 SD for ten animals. a Significantly different from vehicle-treated sham group (p , 0.05); b significantly different from vehicle-treated ovx group (p , 0.05); c significantly different from 17b-estradiol-treated ovx group (p , 0.05); d significantly different from 4-hydroxy CLO-treated ovx group (p , 0.05).
rats. Ovx rats treated with 4-hydroxy CLO exhibited a similar inhibition of periosteal bone formation, but treatment of ovx rats with D4HTA did not inhibit this skeletal process. Ovariectomy resulted in an average 51% elevation of serum OC levels with respect to sham-operated controls in the two experiments (Table 1). Administration of 17b-estradiol prevented this increase, as did administration of 4-hydroxy CLO. Ovx animals receiving D4HTA exhibited a less pronounced (34%) increase in serum OC with respect to ovx vehicle controls, but serum OC levels in 4HTA-treated ovx animals did not differ from those in vehicle-treated ovx animals. Discussion As indicated in Figure 2A, 4-hydroxy CLO was as effective as 17b-estradiol in maintenance of cancellous bone volume, suppression of bone turnover indicators including serum OC (Figure 2B,C, Table 1), and suppression of periosteal bone formation (Figure 3). But, its effect on uterine weight was less than that of 17b-estradiol. Care must be taken in interpreting these data because the dose level of 4-hydroxy CLO at which these effects were seen was considerably greater than that of estradiol. Nevertheless, at the single dose level tested, 4-hydroxy CLO expressed bone specific estrogenicity comparable to that observed with approximately equimolar dose levels of structural analogs droloxifene and raloxifene.10,17 In the ovx rat, these two ER ligands, like 4-hydroxy CLO, exhibited full bone-protective effects similar to 17b-estradiol, but had less effect than 17bestradiol on uterine weight. Also, like droloxifene and raloxifene and TAM and CLO, 4-hydroxy CLO treatment resulted in overall weight loss compared with sham ovx controls.10,15,17 Neither the mechanistic basis nor the pharmacologic/toxicologic significance of this effect has been reported. The observation that 4-hydroxy CLO prevents bone loss in ovx rats has implications concerning the bone-protective effect of CLO. In the ovx rat, CLO was a weak estrogen agonist in supporting uterine weight, and an excellent agonist in maintaining bone mass and indices of bone turnover.15 Based on our studies, these effects might be in part due to its biotransformation to 4-hydroxy CLO. Although 4-hydroxy CLO is a rat liver microsomal CLO metabolite,23 the degree to which it accompanies CLO in blood and tissues of the ovx rat has not been determined.
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On the other hand, it is unlikely that 4HTA, a significant metabolite of TAM in the ovx rat,27 contributes to the boneprotective effect of its parent drug. Although 4HTA exhibited effects on body and uterine weight suggestive of estrogenic activity, it was ineffective in preventing loss of cancellous bone volume or elevation of serum osteocalcin after ovariectomy (Table 1). The basis for the modest uterotrophic effect of 4HTA observed in the current study has not been studied systematically. D4HTA, a saturated analog of 4HTA (Figure 1), did not prevent uterine weight loss associated with ovariectomy (Table 1), but partially prevented cancellous bone loss (Figure 2A) and suppressed bone turnover indicators (Figure 2B–D, Table 1). Skeletal effects were less pronounced than those produced by a substantially lower dose level of 17b-estradiol, but were in general similar to those produced by equivalent dose levels of TAM.10,17,21 Further studies carried out using a range of increased D4HTA dose levels will be needed to determine: (a) whether skeletal effects approaching those of 17b-estradiol can be achieved; and (b) the degree to which skeletal and uterine effects are separated. Substances that stimulate growth of estrogen-responsive cells in vitro usually exhibit reproductive tract estrogenicity.29 Thus, antiestrogens such as TAM, which exhibit partial agonist effects in MCF-7 cells, also have partial uterotrophic effects in the rat. But D4HTA, a full estrogen agonist in MCF-7 cells,28 did not appear to be uterotrophic in this animal model. These findings suggest a divergence in the mechanistic basis for differential systemic effects of D4HTA compared with that of TAM and related antiestrogens. These last compounds probably owe their specific extra-reproductive tract estrogenicity to variations in coactivators and/or corepressors, in bone as opposed to uterine tissue, that modulate effectiveness (estrogenicity) of their ERliganded complexes.16 Receptor heterogeneity probably does not account for the lack of a uterine effect of D4HTA either. The ratio of the two ER isoforms in the rat uterus does not seem to vary greatly from that in MCF-7 cells,18,35 and differential affinity of close structural analogs of D4HTA for the ER isoforms was not observed.18 Two ER-independent factors might account for D4HTA’s differential systemic effects. First, carboxylic acid metabolites of TAM structurally similar to D4HTA were found to be excluded from reproductive tissue in immature female rats, in contrast to TAM and its nonacidic metabolites, yet significant levels of carboxylic acid metabolites were found in other tissues.27 The molecular basis for reproductive tract exclusion of these substances is not known. Second, D4HTA, being structurally related to substances capable of interacting with calcium cations, might likewise be accumulated in bone tissues and may stabilize bone matrix.5,19 Despite the fact that 4-hydroxy CLO was shown to exert its effects exclusively via ER in vitro,23,24 the present study has not eliminated other receptors/mechanisms besides interaction with ER through which it could, in part, express its bone protective effects. The regulatory protein, calmodulin, has been suggested as an alternate “receptor” through which the experimental and clinical bone-protective effects of TAM and structurally similar substances like 4-hydroxy CLO could be mediated.14,34,38 Confirmation of the degree to which at least some of the skeletal effects of 4-hydroxy CLO, and those of D4HTA as well, are independent of ER awaits assessment of (a) dose-response and clearance studies and (b) the extent to which these effects are attenuated by a pure estrogen antagonist.7,12
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Acknowledgment: This research was supported by Grant AR 42069 from the National Institutes of Health.
References 1. Baron, R., Vignery, A., Neff, L., Silvergate, A., and Santa Maria, A. Processing of undecalcified bone specimens for bone histomorphometry. In: Recker, R. R., Ed. Bone Histomorphometry: Techniques and Interpretation. Boca Raton, FL: CRC; 1983; 13–35. 2. Beall, P. T., Misra, L. K., Young, R. L., Spjut, H. J., Evans, H. J., and LeBlanc, A. Clomiphene protects against osteoporosis in the mature ovariectomized rat. Calcif Tissue Int 36:123–125; 1984. 3. Brubaker, K. D. and Gay, C. V. Specific binding of estrogen to osteoclast surfaces. Biochem Biophys Res Commun 200:899 –907; 1994. 4. Buring, J. E., Bain, C. J., and Ehrmann, R. L. Conjugated estrogen use and risk of endometrial cancer. Am J Epidemiol 124:434 – 441; 1986. 5. Caselli, G., Mantovanini, M., Gandolfi, C. A., Allegretti, M., Fiorentino, S., Pellegrini, L., Melillo, G., Bertini, R., Sabbatini, W., Anacardio, R., Clavenna, G., Sciortino, G., and Teti, A. Tartronates—a new generation of drugs affecting bone metabolism. J Bone Miner Res 12:972–981; 1997. 6. Delmas, P. D., Malaval, L., Arlot, M. E., and Meunier, P. J. Serum bone gla-protein compared to bone histomorphometry in endocrine diseases. Bone 6:339 –341; 1985. 7. Dukes, M., Chester, R., Yarwood, L., and Wakeling, A. E. Effects of a non-steroidal pure antiestrogen, ZM 189,154 on estrogen target organs of the rat including bones. J Endocrinol 141:335–341; 1994. 8. Eriksen, E. F., Colvard, D. S., Berg, N. J., Graham, M. L., Mann, K. G., Spelsberg, T. C., and Riggs, B. L. Evidence of estrogen receptors in normal human osteoblast-like cells. Science 241:84 – 86; 1988. 9. Fornander, T., Rutqvist, L. E., Sjoberg, H. E., Blomqvist, L., Mattsson, A., and Glas, U. Long term tamoxifen in early breast cancer: Effect on bone mineral density in postmenopausal women. J Clin Oncol 8:1019 –1024; 1990. 10. Frolik, C. A., Bryant, H. U., Black, E. C., Magee, D. E., and Chandrasekhar, S. Time-dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: Effects of raloxifene HCl, tamoxifen, estrogen, and alendronate. Bone 18:621– 627; 1996. 11. Frost, H. M. Bone histomorphometry: Analysis of trabecular bone dynamics. In: Recker, R. R., Ed. Bone Histomorphometry: Techniques and Interpretation. Boca Raton, FL: CRC; 1983; 109 –132. 12. Gallagher, A., Chambers, T. J., and Tobias, J. H. The estrogen antagonist ICI 182,780 reduces cancellous bone volume in female rats. Endocrinology 133: 2787–2791; 1993. 13. Grady, D. and Rubin, S. M. The postmenopausal estrogen/breast cancer controversy. JAMA 269:990 –991; 1993. 14. Hall, T. J. and Schaueblin, M. Promethazine inhibits osteoclastic bone resorption in vitro. Calcif Tissue Int 55:68 –70; 1994. 15. Jimenez, M. A., Magee, D. E., Bryant, H. U., and Turner, R. T. Clomiphene prevents cancellous bone loss from tibia of ovariectomized rats. Endocrinology 138:1794 –1800; 1997. 16. Katzenellenbogen, J. A., O’Malley, B. W., and Katzenellenbogen, B. S. Tripartite steroid hormone receptor pharmacology: Interaction with multiple effector sites as a basis for the cell- and promoter-specific action of these hormones. Mol Endocrinol 10:119 –131; 1996. 17. Ke, H. Z., Chen, H. K., Simmons, H. A., Qi, H., Crawford, D. T., Pirie, C. M., Chidsey-Frink, K. L., Ma, Y. F., Jee, W. S. S., and Thompson, D. D. Comparative effects of droloxifene, tamoxifen, and estrogen on bone, serum cholesterol, and uterine histology in the ovariectomized rat model. Bone 20:31–39; 1997. 18. Kuiper, G. G., Carlsson, B., Grandien, K., Enmark, E., Ha¨ggblad, J., Nilsson, S., and Gustafsson, J. Å. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors a and b. Endocrinology 138:863– 870; 1997. 19. Lin, J. H., Duggan, D. E., Chen, I.-W., and Ellsworth, R. L. Physiological disposition of alendronate, a potent anti-osteolytic bisphosphonate, in laboratory animals. Drug Metab Dispos 19:926 –932; 1991. 20. Love, R. R., Mazess, R. B., Tormey, D. C., Barden, H. S., Newcomb, P. A., and Jordan, V. C. Bone mineral density in women with breast cancer treated with adjuvant tamoxifen for at least two years. Breast Cancer Res Treat 12:297–302; 1988.
Bone Vol. 23, No. 6 December 1998:537–542 21. Moon, L. Y., Wakley, G. K., and Turner, R. T. Dose-dependent effects of tamoxifen on long bones in growing rats: Influence of ovarian status. Endocrinology 129:1568 –1574; 1991. 22. Neter, J., Wasserman, W., and Whitmore, G. A. Applied Statistics. Boston: Allyn and Bacon; 1982. 23. Ruenitz, P. C., Bagley, J. R., and Mokler, C. M. Metabolism of clomiphene in the rat: Estrogen receptor affinity and antiestrogenic activity of clomiphene metabolites. Biochem Pharmacol 32:2941–2947; 1983. 24. Ruenitz, P. C., Bagley, J. R., Watts, C. K. W., Hall, R. E., and Sutherland, R. L. Substituted-vinyl hydroxytriarylethylenes: Synthesis and effects on MCF 7 breast cancer cell proliferation. J Med Chem 29:2511–2519; 1986. 25. Ruenitz, P. C. and Nanavati, N. T. Identification and distribution in the rat of acidic metabolites of tamoxifen. Drug Metab Dispos 18:645– 648; 1990. 26. Ruenitz, P. C. Drugs for osteoporosis prevention. Mechanisms of bone maintenance. Curr Med Chem 2:791– 802; 1995. 27. Ruenitz, P. C. and Bai, X. Acidic metabolites of tamoxifen: Aspects of formation and fate in the female rat. Drug Metab Dispos 23:993–998; 1995. 28. Ruenitz, P. C., Bourne, C. S., Sullivan, K. J., and Moore, S. A. Estrogenic triarylethylene acetic acids: Effect of structural variation on estrogen receptor affinity and estrogenic potency and efficacy in MCF-7 cells. J Med Chem 39:4853– 4859; 1996. 29. Ruenitz, P. C. Female sex hormones and analogs. In: Wolff, M. E., Ed. Burger’s Medicinal Chemistry and Drug Discovery, 5th Ed. Vol. 4. New York: Wiley; 1997; 553–587. 30. Schot, L. P. C. and Schuurs, A. H. W. Sex steroids and osteoporosis: Effects of deficiencies and substitutive treatments. J Ster Biochem Mol Biol 37:167–182; 1990. 31. Tarallo, P., Henny, J., Fournier, B., and Siest, G. Plasma osteocalcin: Biological variations and reference limits. Scand J Clin Lab Invest 50:649 – 655; 1990. 32. Turken, S., Siris, E., Seldin, D., Flaster, E., Hyman, G., and Lindsay, R. Effects of tamoxifen on spinal bone density in women with breast cancer. J Natl Cancer Inst 81:1086 –1088; 1989. 33. Turner, R. T., Wakley, G. K., Hannon, K. S., and Bell, N. H. Tamoxifen inhibits osteoclast-mediated resorption of trabecular bone in ovarian hormonedeficient rats. Endocrinology 122:1146 –1150; 1988. 34. Tyan, M. L. Effect of promethazine on lumbar vertebral bone mass in postmenopausal women. J Intern Med 234:143–148; 1993. 35. Watanabe, T., Inoue, S., Ogawa, S., Ishii, Y., Hiroi, H., Ikeda, K., Orimo, A., and Muramatsu, M. Agonistic effect of tamoxifen is dependent on cell-type, ere-promoter context, and estrogen-receptor subtype—functional difference between estrogen-receptor-alpha and estrogen-receptor-beta. Biochem Biophys Res Commun 236:140 –145; 1997. 36. Wiita, B. Osteoporosis— causes and consequences. Curr Sci 68:446 – 450; 1995. 37. Williams, D. C., Paul, D. C., and Black, L. J. Effects of estrogen and tamoxifen on serum osteocalcin levels in ovariectomized rats. Bone Miner 14:205–220; 1991. 38. Williams, J. P., Blair, H. C., McKenna, M. A., Jordan, S. E., and McDonald, J. M. Regulation of avian osteoclastic H1-ATPase and bone resorption by tamoxifen and calmodulin antagonists: Effects independent of steroid receptors. J Biol Chem 271:12488 –12495; 1996. 39. Wilson, S., Ruenitz, P. C., and Ruzicka, J. A. Estrogen receptor affinity and effects on MCF-7 cell growth of triarylethylene carboxylic acids related to tamoxifen. J Ster Biochem Mol Biol 42:613– 616; 1992. 40. Wronski, T. J., Cintro´n, M., Doherty, A. L., and Dann, L. M. Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats. Endocrinology 123:681– 686; 1988. 41. Wronski, T. J., Dann, L. M., Qi, H., and Yen, C. F. Skeletal effects of withdrawal of estrogen and diphosphonate treatment in ovariectomized rats. Calcif Tissue Int 53:210 –216; 1993. 42. Young, R. L., Goldzieher, J. W., Elkind-Hirsch, K., Hickox, P. G., and Chakraborty, P. K. A short-term comparison of the effects of clomiphene citrate and conjugated equine estrogen in menopausal/castrate women. Int J Fertil 36:167–171; 1991.
Date Received: May 19, 1998 Date Revised: July 13, 1998 Date Accepted: August 20, 1998
Specific Bone-Protective Effects of Metabolites/Derivatives of Tamoxifen and Clomiphene in Ovariectomized Rats P. C. RUENITZ,1 Y. SHEN,2 M. LI,2 H. LIANG,2 R. D. WHITEHEAD, JR.,1 S. PUN,2 and T. J. WRONSKI2 1 2
College of Pharmacy, University of Georgia, Athens, GA, USA Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
abnormalities in bone remodeling, the most common of which is loss of gonadal steroid action, which can occur in menopause or in male or female hypogonadism. Thus, postmenopausal bone loss is not itself a disease but rather a consequence of endocrine imbalance. When estrogen replacement therapy is indicated for prevention of postmenopausal bone loss, there are several types of therapeutic agents available or in development. The most important of these suppress bone resorption and formation in a manner that maintains net bone balance, probably as a consequence of interaction with estrogen receptors (ER) in osteoblasts and osteoclasts, cell types responsible for bone maintenance.3,8,30 Thus, 17b-estradiol and its orally active analogs, including conjugated equine estrogens, are widely used in estrogen replacement therapy.29 A major drawback to long-term administration of estrogens for prevention of osteoporosis is undesirable effects on the reproductive tract. Thus, an increased risk of developing uterine cancer has been associated with estrogen replacement therapy.4 In addition, a perceived link between estrogen use and breast cancer is of concern.13 An approach to this problem is identification of substances whose estrogenicity is restricted to nonreproductive tissues. Thus, tamoxifen (TAM) and clomiphene (CLO) (Figure 1), nonsteroidal ER ligands previously thought of in terms of their estrogen antagonist effects on the reproductive axis, and on estrogen-dependent cancers,29 have been found to act like estrogen mimetics in prevention of bone loss in postmenopausal women.9,20,32,42 Consequently, analogs of these triphenylethylenes with decreased residual uterotrophic effects are of continuing interest.26 The ovariectomized (ovx) rat has become an established animal model of osteopenia associated with estrogen deficiency. Histomorphometric analysis of bone specimens from ovx rats receiving 17b-estradiol, TAM, or CLO has shown a decreased rate of bone turnover and maintenance of normal bone mass compared with that observed in untreated ovx rats.2,15,21,33,40 Similarly, estrogen or TAM administration to ovx rats resulted in decreased serum levels of osteocalcin.37 Osteocalcin is a bone matrix protein31 released into the serum during bone formation, thus serving as a specific indicator of bone turnover.6 Clarification of the ways that TAM, CLO, and their structural analogs exert their bone-protective effects is complicated by the fact that these drugs can undergo extensive oxidative biotransformation to metabolites having divergent profiles of activity relative to the parent drugs. Thus, in the rat, CLO was converted to 4-hydroxy CLO (Figure 1), which had greatly increased antiuterotrophic potency/efficacy compared with CLO itself.23 In
In the ovariectomized (ovx) rat, the nonsteroidal antiestrogens, clomiphene (CLO) and tamoxifen (TAM), at dose levels that prevent development of osteopenia to a degree approaching that of 17b-estradiol are, in contrast to 17bestradiol, only weakly uterotrophic. Metabolites of CLO and TAM might contribute differentially to these effects. Thus, we have evaluated bone protective and uterine effects in ovx rats of two such metabolites: 4-hydroxy CLO, produced by p-hydroxylation of CLO; and 4HTA, produced from TAM by stepwise replacement of its dimethylaminoethyl side chain with an acetic acid moiety, accompanied by p-hydroxylation. Also reported are effects of D4HTA, the dihydrodesethyl derivative of 4HTA previously characterized as a full estrogen mimetic in vitro. Administration of 4-hydroxy CLO (2.5 mg/kg subcutaneously) 5 days/week for 5 weeks to 3-monthold ovx rats resulted in complete prevention of bone loss and suppression of bone turnover to levels comparable to those of intact controls and to those of ovx animals similarly receiving 17b-estradiol (10 mg/kg). However, uterine weight in animals receiving 4-hydroxy CLO was 64% less than that in 17bestradiol-treated animals. Although 4HTA (3.7 mg/kg s.c.) had a modest uterotrophic effect, it did not prevent bone loss associated with ovariectomy. In contrast, D4HTA (3.6 mg/kg s.c.) partially reduced bone turnover indicators and cancellous bone loss in a manner similar in many ways to that observed in TAM-treated ovx animals, but it had no uterotrophic effect. These results suggest that, although 4HTA does not contribute to the bone-protective effect of TAM, 4-hydroxy CLO might augment that of CLO. (Bone 23: 537–542; 1998) © 1998 by Elsevier Science Inc. All rights reserved. Key Words: Antiestrogen; Clomiphene; Estrogen; Osteoporosis; Osteopenia; Tamoxifen. Introduction Osteoporosis is characterized by a progressive decrease in bone density, which can lead to an increased incidence of bone fractures.36 This condition results when the rate of bone resorption exceeds that of bone formation. Several disorders induce Address for correspondence and reprints: Dr. Peter C. Ruenitz, College of Pharmacy, University of Georgia, Athens, GA 30602. E-mail: [email protected] © 1998 by Elsevier Science Inc. All rights reserved.
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Materials and Methods Chemicals 4-Hydroxy CLO {4-[1-(p-hydroxyphenyl)-2-phenyl-2-chloroethenyl]phenoxyethyl-N,N-diethylamine}, 4HTA {4-[1-(p-hydroxyphenyl)-2-phenyl-1-butenyl]phenoxyacetic acid}, and D4HTA {4[1-(p-hydroxyphenyl)-2-phenylethyl]phenoxyacetic acid} were used as prepared and characterized previously.23,28 All other chemicals were purchased from Sigma Co. (St. Louis, MO). Animals and Dosing
Figure 1. Structures of TAM and CLO and specified metabolites, and dihydrodesethyl-4HTA (D4HTA). 4HTA and CLO are composed of the respective trans isomers (shown), plus nearly equal amounts of the corresponding cis isomers. In MCF-7 cells, TAM, CLO, and 4-hydroxy CLO were estrogen antagonists; 4HTA and D4HTA were estrogen mimetics.
vitro evaluation of 4-hydroxy CLO in MCF-7 cells, whose proliferation is dependent on the presence of estrogens, showed it to be a more potent growth suppressor than CLO.24 TAM, in addition to undergoing 4-hydroxylation and/or N-demethylation,29 is in part further metabolized by formal replacement of its aminomethyl group with a carboxyl group, affording 4HTA (Figure 1).25,27 In MCF-7 cells, 4HTA, in contrast to TAM and its N-desmethyl and 4-hydroxy metabolites, was an estrogen mimetic.28,39 However, unlike most other substances characterized as estrogens in MCF-7 cells, 4HTA was not uterotrophic in the ovx rat (unpublished findings). The effects of 4HTA and 4-hydroxy CLO in estrogen-responsive cells and tissues indicated that these metabolites could participate in the observed bone-protective effects of their respective parent drugs. Thus, we decided to investigate the effects of these compounds on bone mass, bone turnover, and uterine weight in the ovx rat. We also chose to examine the dihydrodesethyl analog of 4HTA (D4HTA) (Figure 1) in this study because its in vitro estrogenic potency and efficacy were comparable to those of 4HTA.28
In the first experiment, 50 female Sprague-Dawley rats that were approximately 90 days of age and weighed an average of 240 g at the beginning of the study were used. Animal care and handling were carried out solely at the AAALAC-accredited animal facility at the University of Georgia College of Pharmacy, in accordance with a protocol approved by the University of Georgia Institutional Animal Care and Use Committee. On the day of surgery (day 0), all rats were anesthetized with an intraperitoneal (i.p.) injection of ketamine hydrochloride and xylazine at doses of 50 and 10 mg/kg body weight, respectively. Ten rats were sham-operated, during which the ovaries were exteriorized but replaced intact. Bilateral ovariectomies were performed in the remaining 40 rats from a dorsal approach. Each rat was housed individually at 25°C with a light/dark cycle of 13 h/11 h. Food (Teklad 22/5 Rodent Diet, Madison, WI), with calcium and phosphate contents of 0.95% and 0.67%, respectively, was available ad libitum to all animals. Sham-operated rats (n 5 10) and one of the four groups of ovx rats (n 5 10) were injected subcutaneously (s.c.) with vehicle (5% benzyl alcohol in corn oil) 5 days per week for 5 weeks. The remaining 30 ovx rats (n 5 10 per group) were subjected to the same treatment regimen with 17b-estradiol, 4-hydroxy CLO, or D4HTA at respective doses of 10 mg/kg per day, 2.5 mg/kg per day, or 3.6 mg/kg per day, administered s.c. in 5% benzyl alcohol/corn oil vehicle. The aforementioned treatments were initiated on the first day after surgery. The dose level of 17b-estradiol was believed to be optimal based on earlier studies.40 Those of the hydroxytriphenylethylenes approximated maximally effective bone protective dose levels of TAM and CLO.2,15,21,33 Each rat was injected s.c. with demeclocycline and calcein (Sigma Co.) at a dose of 15 mg/kg body weight on days 10 and 3 before killing, respectively, to label sites of bone formation. A second experiment, using 40 animals, was performed in exactly the same manner as just described, in which effects of the s.c. administration of 3.7 mg/kg per day of 4HTA to one group of ovx animals was compared with the effects of vehicle or 17b-estradiol in three other groups (vehicle-treated sham ovx, vehicle-treated ovx, and 17b-estradiol-treated ovx rats). Necropsy Procedures All rats were killed by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. Failure to detect ovarian tissue and observation of marked atrophy of the uterine horns confirmed the success of ovariectomy. The body weight of each animal was recorded, as was uterine wet weight. Blood samples were allowed to coagulate at room temperature (2 h) in a Vacutainer tube. Serum was obtained by centrifugation for 10 min at 3000 rpm, and samples were stored at 280°C until analyzed. The right tibia was removed at necropsy, dissected free of muscle, and cut in half cross sectionally with a hand-held saw (Dremel Moto Tool, Racine, WI). The bone samples were then
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placed in 10% phosphate-buffered formalin for 24 h for tissue fixation.
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Table 1. Effect of various treatment regimens on whole body weight, uterine weight, and serum osteocalcin (OC) level (results from experiment 2 are in italics)a
Cancellous Bone Histomorphometry The proximal halves of the tibiae were dehydrated by immersion in ethanol, and embedded undecalcified in methylmethacrylate.1 Longitudinal sections (4 and 8 mm thick) were cut with AO Autocut/Jung 1150 or 2050 microtomes. The 4-mm-thick sections were stained according to the von Kossa method with a tetrachrome counterstain (Polysciences, Warrington, PA). Bone measurements were performed in cancellous bone tissue of the proximal tibial metaphysis beginning at distances 1 mm distal to the growth plate-metaphyseal junction to exclude the primary spongiosa. In general, two sections of the proximal tibia, with a total of 40 –50 mm of cancellous bone perimeter, were sampled in each animal with an appreciable amount of cancellous bone. Additional sections were sampled in osteopenic animals to approximate the cancellous bone perimeter sampled in animals with greater cancellous bone mass. Bone measurements were performed with the Bioquant bone morphometry system (R&M Biometrics, Nashville, TN) as previously described.41 Cancellous bone volume as a percentage of bone tissue area and osteoblast and osteoclast surfaces as percentages of total cancellous perimeter were measured in 4-mmthick, stained sections. Fluorochrome-based indices of bone formation were measured in unstained, 8-mm-thick sections of the proximal tibial metaphysis. The percentage of cancellous bone surface with a double fluorochrome label (mineralizing surface) and mineral apposition rate were measured with the Bioquant system. In addition, bone formation rate (tissue level, total surface referent) was calculated by multiplying mineralizing surface by mineral apposition rate.11 Cortical Bone Histomorphometry The distal halves of the tibiae were dehydrated in ten changes of 100% ethanol, defatted in ten changes of acetone (at least 2 h per change), and embedded undecalcified in a styrene monomer that polymerizes into a polyester resin (Tap Plastics, San Jose, CA). The tibial diaphysis 1–2 mm proximal to the tibiofibular junction was sawed into 100-mm-thick cross sections with an Isomet low-speed saw (Buehler, Lake Bluff, IL). Bone measurements were performed with the Bioquant system. Cortical bone tissue area and bone marrow area were measured in one section per animal at a magnification of 320. Cortical bone area was calculated by subtracting marrow area from cortical bone tissue area. Cortical width was measured from the periosteal to the endocortical surfaces at four equally spaced sites at the anterior (cranial), posterior (caudal), medial, and lateral aspects of each cross section. The four measurements were averaged to obtain a mean cortical width for each animal. Fluorochrome-based indices of bone formation were measured under UV illumination at magnifications of 3100 and 3200 for mineralizing surface and mineral apposition rate, respectively. The percentage of periosteal surface with a double fluorochrome label (mineralizing surface) and interlabel distances along the double-labeled surfaces were measured with the Bioquant system. The mineral apposition rate (MAR) and bone formation rate, surface referent (BFR/BS), were calculated according to the following formulas: MAR 5 interlabel distance/ time interval between labels (7 days) and BFR/BS 5 mineralizing surface 3 MAR.
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Treatment group Sham vehicle Ovx vehicle 17b-estradiol 4-Hydroxy CLO D4HTA 4HTA
Body weight (g)
Uterine weight (mg)
Relative serum OC levelb
323 (16) 304 (14) 379 (23)d 364 (25)d 310 (24)c 303 (12)c 271 (8)d 342 (23) 305 (12)
776 (101) 613 (38) 99 (19)d 86 (13)d 269 (48)c 225 (30)c 161 (21)c,d 112 (7)d 124 (12)c,d
108 (21) 127 (33) 175 (21)d 176 (29)d 100 (33)c 100 (16)c 113 (28)c 145 (27)c,d 188 (42)d
a
Data are expressed as mean (SD). The mean amount of OC in serum from 17b-estradiol-treated animals was 4.20 6 1.39 ng/mL (experiment 1), and 2.08 6 0.33 ng/mL (experiment 2). c p , 0.05 compared with ovx vehicle. d p , 0.05 compared with sham vehicle. b
Serum Rat Osteocalcin Radioimmunoassay Materials, as well as standard protocols for their use, were obtained from Biomedical Technologies (Stoughton MA). Serum samples from each member of the different treatment groups were diluted 1:20. Duplicate aliquots (100 mL) of each diluted sample were incubated in an orbital shaker at 80 rpm (4°C) for 16 h with the first antibody and nonimmune serum in RIA buffer (final volume 0.5 mL). An aliquot of [125I]-osteocalcin (10 nCi, 0.034 ng) in 100 mL of radioimmunoassay (RIA) buffer was then added to each tube and incubation was continued for 24 h. Then the second (precipitating) antibody was added in 1 mL of modified buffer, and incubation was continued for 4 h. Tubes were then centrifuged at 1500g for 15 min. Pellets were washed with cold distilled water and recentrifuged as before. Supernatant was decanted and radioactivity in pellets was determined using a gamma counter. Serum osteocalcin levels were determined by comparing the sample values (cpm) to the linear region of a standard curve of cpm/pellet vs. the amount of osteocalcin present, obtained from incubations to which known amounts (0 – 0.35 ng) of osteocalcin had been added. All values were corrected for nonspecific binding in standard incubations. Mean values (and SD) for treatment groups in each experiment are expressed relative to those of respective ovx 17b-estradioltreated groups. Statistical Analysis Data are expressed as the mean 6 SD for each group. Statistical differences among groups were evaluated by one-way ANOVA followed by Fisher’s protected least significant difference (PLSD) test for multiple comparisons.22 Differences were considered significant at p , 0.05. Results With respect to sham-operated controls, ovariectomy resulted in an average 18.5% increase in overall body weight over the 5 week course of the two studies (Table 1). This increase was not observed in ovx animals receiving subcutaneous 17b-estradiol, 4HTA, or D4HTA 5 days/week. Ovx animals similarly receiving 4-hydroxy CLO experienced a 16% loss of body weight, in comparison with sham-operated controls.
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Figure 2. Cancellous bone volume (A), osteoclast surface (B), osteoblast surface (C), and bone formation rate (D) in the proximal tibial metaphysis for the five groups of rats. Each bar is the mean 6 SD for ten animals. aSignificantly different from vehicle-treated sham group (p , 0.05); bsignificantly different from vehicle-treated ovx group (p , 0.05); csignificantly different from 17b-estradiol-treated ovx group (p , 0.05); dsignificantly different from 4-hydroxy CLO-treated ovx group (p , 0.05).
17b-estradiol treatment resulted in 167% greater uterine weight (average of both experiments), compared with vehicletreated ovx animals. This increase was not as great as has been observed in closely related studies, in which higher doses and/or more potent derivatives of 17b-estradiol were administered. Similar comparative uterine weights in animals receiving 4HTA and 4-hydroxy CLO were, in turn, 25% and 61% higher than respective ovx vehicle controls. The uterine weight of ovx animals receiving D4HTA did not differ from that of ovx animals receiving vehicle. Cancellous bone volume was decreased by 68% in vehicletreated ovx rats compared with vehicle-treated control rats (Figure 2A). In contrast, cancellous bone volume remained at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO, and treatment with D4HTA partially prevented the ovx-induced cancellous bone loss. In the second experiment, cancellous bone volume in ovx rats treated with 4HTA (8.3 6 3.7%) did not differ from that of vehicle-treated ovx animals (7.5 6 3.6%), but was lower than that of 17b-estradiol-treated ovx animals (24.0 6 5.5%). This lack of effect on cancellous bone volume, and on serum OC levels (see subsequent text), precluded further detailed histomorphometric evaluation of 4HTA effects. Osteoclast surface (Figure 2B) and osteoblast surface (Figure 2C) of vehicle-treated ovx rats increased significantly compared with that of vehicle-treated control rats at 35 days after surgery. In contrast, these cellular indices of bone resorption and formation, respectively, were suppressed at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO. Treatment
with D4HTA partially suppressed the OVX-induced increase in osteoclast and osteoblast surfaces. The effects of ovariectomy and the various treatments on mineralizing surface (data not shown) were similar to those described earlier for osteoblast surface with the exception that ovx rats treated with 17b-estradiol had a significantly lower mineralizing surface than the vehicle-treated control rats. Mineral apposition rate (MAR) was significantly increased in vehicle-treated ovx rats compared with vehicle-treated controls rats at 35 days postsurgery (1.7 vs. 1.1 mm/day, p , 0.05). In contrast, MAR was maintained at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO. Treatment with D4HTA had no effect on MAR in ovx rats. Cancellous bone formation rate (BFR/BS; Figure 2D) was significantly increased in vehicle-treated ovx rats when compared with vehicle-treated control rats. However, BFR/BS was suppressed at the control level in ovx rats treated with either 17b-estradiol or 4-hydroxy CLO, whereas treatment with D4HTA partially suppressed the ovx-induced increase in BFR/BS. Cortical bone area and marrow area did not differ significantly among groups as the mean values ranged from 5.2 to 5.4 mm2 for the former variable and from 1.2 to 1.3 mm2 for the latter variable. Mean values for cortical width were also similar at 0.8 – 0.9 mm for all five groups. However, periosteal bone formation rate (Figure 3) was significantly increased in vehicletreated ovx rats when compared with vehicle-treated control rats. 17b-estradiol treatment of ovx rats inhibited periosteal bone formation rate to a level below that of vehicle-treated control
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Figure 3. Periosteal bone formation rate in the tibial diaphysis for the five groups of rats. Each bar is the mean 6 SD for ten animals. a Significantly different from vehicle-treated sham group (p , 0.05); b significantly different from vehicle-treated ovx group (p , 0.05); c significantly different from 17b-estradiol-treated ovx group (p , 0.05); d significantly different from 4-hydroxy CLO-treated ovx group (p , 0.05).
rats. Ovx rats treated with 4-hydroxy CLO exhibited a similar inhibition of periosteal bone formation, but treatment of ovx rats with D4HTA did not inhibit this skeletal process. Ovariectomy resulted in an average 51% elevation of serum OC levels with respect to sham-operated controls in the two experiments (Table 1). Administration of 17b-estradiol prevented this increase, as did administration of 4-hydroxy CLO. Ovx animals receiving D4HTA exhibited a less pronounced (34%) increase in serum OC with respect to ovx vehicle controls, but serum OC levels in 4HTA-treated ovx animals did not differ from those in vehicle-treated ovx animals. Discussion As indicated in Figure 2A, 4-hydroxy CLO was as effective as 17b-estradiol in maintenance of cancellous bone volume, suppression of bone turnover indicators including serum OC (Figure 2B,C, Table 1), and suppression of periosteal bone formation (Figure 3). But, its effect on uterine weight was less than that of 17b-estradiol. Care must be taken in interpreting these data because the dose level of 4-hydroxy CLO at which these effects were seen was considerably greater than that of estradiol. Nevertheless, at the single dose level tested, 4-hydroxy CLO expressed bone specific estrogenicity comparable to that observed with approximately equimolar dose levels of structural analogs droloxifene and raloxifene.10,17 In the ovx rat, these two ER ligands, like 4-hydroxy CLO, exhibited full bone-protective effects similar to 17b-estradiol, but had less effect than 17bestradiol on uterine weight. Also, like droloxifene and raloxifene and TAM and CLO, 4-hydroxy CLO treatment resulted in overall weight loss compared with sham ovx controls.10,15,17 Neither the mechanistic basis nor the pharmacologic/toxicologic significance of this effect has been reported. The observation that 4-hydroxy CLO prevents bone loss in ovx rats has implications concerning the bone-protective effect of CLO. In the ovx rat, CLO was a weak estrogen agonist in supporting uterine weight, and an excellent agonist in maintaining bone mass and indices of bone turnover.15 Based on our studies, these effects might be in part due to its biotransformation to 4-hydroxy CLO. Although 4-hydroxy CLO is a rat liver microsomal CLO metabolite,23 the degree to which it accompanies CLO in blood and tissues of the ovx rat has not been determined.
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On the other hand, it is unlikely that 4HTA, a significant metabolite of TAM in the ovx rat,27 contributes to the boneprotective effect of its parent drug. Although 4HTA exhibited effects on body and uterine weight suggestive of estrogenic activity, it was ineffective in preventing loss of cancellous bone volume or elevation of serum osteocalcin after ovariectomy (Table 1). The basis for the modest uterotrophic effect of 4HTA observed in the current study has not been studied systematically. D4HTA, a saturated analog of 4HTA (Figure 1), did not prevent uterine weight loss associated with ovariectomy (Table 1), but partially prevented cancellous bone loss (Figure 2A) and suppressed bone turnover indicators (Figure 2B–D, Table 1). Skeletal effects were less pronounced than those produced by a substantially lower dose level of 17b-estradiol, but were in general similar to those produced by equivalent dose levels of TAM.10,17,21 Further studies carried out using a range of increased D4HTA dose levels will be needed to determine: (a) whether skeletal effects approaching those of 17b-estradiol can be achieved; and (b) the degree to which skeletal and uterine effects are separated. Substances that stimulate growth of estrogen-responsive cells in vitro usually exhibit reproductive tract estrogenicity.29 Thus, antiestrogens such as TAM, which exhibit partial agonist effects in MCF-7 cells, also have partial uterotrophic effects in the rat. But D4HTA, a full estrogen agonist in MCF-7 cells,28 did not appear to be uterotrophic in this animal model. These findings suggest a divergence in the mechanistic basis for differential systemic effects of D4HTA compared with that of TAM and related antiestrogens. These last compounds probably owe their specific extra-reproductive tract estrogenicity to variations in coactivators and/or corepressors, in bone as opposed to uterine tissue, that modulate effectiveness (estrogenicity) of their ERliganded complexes.16 Receptor heterogeneity probably does not account for the lack of a uterine effect of D4HTA either. The ratio of the two ER isoforms in the rat uterus does not seem to vary greatly from that in MCF-7 cells,18,35 and differential affinity of close structural analogs of D4HTA for the ER isoforms was not observed.18 Two ER-independent factors might account for D4HTA’s differential systemic effects. First, carboxylic acid metabolites of TAM structurally similar to D4HTA were found to be excluded from reproductive tissue in immature female rats, in contrast to TAM and its nonacidic metabolites, yet significant levels of carboxylic acid metabolites were found in other tissues.27 The molecular basis for reproductive tract exclusion of these substances is not known. Second, D4HTA, being structurally related to substances capable of interacting with calcium cations, might likewise be accumulated in bone tissues and may stabilize bone matrix.5,19 Despite the fact that 4-hydroxy CLO was shown to exert its effects exclusively via ER in vitro,23,24 the present study has not eliminated other receptors/mechanisms besides interaction with ER through which it could, in part, express its bone protective effects. The regulatory protein, calmodulin, has been suggested as an alternate “receptor” through which the experimental and clinical bone-protective effects of TAM and structurally similar substances like 4-hydroxy CLO could be mediated.14,34,38 Confirmation of the degree to which at least some of the skeletal effects of 4-hydroxy CLO, and those of D4HTA as well, are independent of ER awaits assessment of (a) dose-response and clearance studies and (b) the extent to which these effects are attenuated by a pure estrogen antagonist.7,12
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Acknowledgment: This research was supported by Grant AR 42069 from the National Institutes of Health.
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Date Received: May 19, 1998 Date Revised: July 13, 1998 Date Accepted: August 20, 1998