Journal of Physiology and Biochemistry

, Volume 66, Issue 1, pp 23–28

Effects of raloxifene and estradiol on bone turnover parameters in intact and ovariectomized rats

Authors

  • S. Canpolat
    • Faculty of Medicine, Department of PhysiologyYeditepe University
  • N. Tug
    • Faculty of Medicine, Department of Obstetrics and GynaecologyFırat University
  • A. D. Seyran
    • Faculty of Medicine, Department of PhysiologyFırat University
  • S. Kumru
    • Faculty of Medicine, Department of Obstetrics and GynaecologyFırat University
    • Faculty of Medicine, Department of PhysiologyYeditepe University
Original Paper

DOI: 10.1007/s13105-010-0008-8

Cite this article as:
Canpolat, S., Tug, N., Seyran, A.D. et al. J Physiol Biochem (2010) 66: 23. doi:10.1007/s13105-010-0008-8

Abstract

This study was designed to investigate effects of raloxifene (RLX) and estradiol on bone formation and resorption in intact and ovariectomized (ovx) rat models. In the intact model, a total of 24 adult female rats were divided into three groups: Controls subcutaneously received saline alone. RLX (2 mg/kg) and estradiol (30 μg/kg) were injected to two groups of animals for a period of 6 weeks at two daily intervals. In the second model, rats (n = 24) were ovx and allowed to recover for a period of at least 3 weeks. Control group received vehicle alone. Remaining rats were divided into two groups and injected with RLX (2 mg/kg) and estradiol (30 µg/kg) for 6 weeks. Urine samples were collected from all animals 24 h after the last drug administration. Urinary deoxypyridinoline (DPD) was measured by ELISA. Serum parathyroid hormone (PTH), calcitonin, and osteocalcin levels were measured by immunoradiometric method. Serum concentrations of alkaline phosphatase (ALP), Ca, and inorganic phosphate were determined by enzymatic–colorimetric method. Lumbar vertebrae (L2) of all animals were dissected out and processed for histopathological evaluation. Removal of ovaries significantly elevated urinary DPD levels (p < 0.01) compared with intact controls. Treatment of both intact and ovx rats with estradiol resulted in significant decreases (p < 0.01) in DPD values. RLX administration had no significant effect in the intact rats, but it remarkably reduced bone turnover in the ovx animals (p < 0.001). Both estradiol and RLX produced conflicting effects on serum ALP, osteocalcin, and PTH levels in both animal models. These findings suggest that RLX exerts its protective effects by reducing bone resorption, similar to that of estradiol, in ovx rats.

Keywords

RaloxifeneDeoxypyridinolineBone turnoverEstradiolRat

Introduction

Raloxifene (RLX) is a nonsteroidal selective estrogen modulator (SERM) developed as a therapeutic agent for postmenopausal osteoporosis [3]. RLX is a benzothiophene-derived compound originally classified as antiestrogens, but later termed as SERM on the basis of their tissue-selective estrogen agonist or antagonist actions [3]. It has estrogenic effects on the skeleton and serum lipids, but has anti-estrogenic properties on endometrial and breast tissue [13]. RLX has been reported to prevent bone loss and to reduce vertebral fracture risk in postmenopausal women [25, 29, 30]. In addition to protective actions on osteoporosis, beneficial effects of RLX on the cardiovascular system have also been reported [31]. However, there are also contradictory findings to suggest that RLX is not a good SERM since it induces hypertrophy of uterine epithelium [9, 24].

Bone remodeling cycle consists of a balance in the bone-resorbing functions of osteoclasts, coupled to that of osteoblasts, which subsequently form new bone in absorbed sites [33]. Osteoporosis results from a net imbalance between bone formation and resorption [22, 35]. Biochemical markers of bone turnover used in clinical and experimental studies include osteocalcin, alkaline phosphatase (ALP), and urinary deoxypyridinoline (DPD) [10, 33]. There are several clinical and experimental studies to indicate that estrogen prevents bone resorption [17, 21]. It has been suggested that administration of RLX to estrogen-deficient rats is associated with reduced bone turnover, increased bone mineral density, and maintenance of normal bone architecture [16]. A recent randomized trial conducted in osteopenic postmenopausal women has shown that effects of 60 mg/day RLX was less effective on the lumbar spine bone mineral density than 1.25 mg/day tibolone [12].

Although beneficial effects of estrogen on bone are generally accepted, its overall value is frequently questioned due to hormone-associated side effects such as uterine stimulation and increased risk of breast cancer [19, 36]. In recent years, use of SERMs for alternative treatment of osteoporosis has been suggested. Therefore, the aim of this study was to compare effects of RLX and estradiol-17-β (E2) on bone formation and resorption markers in intact and ovariectomized (ovx) rat models.

Material and methods

Adult female Wistar rats weighing 210–230 g were used in this study. In the intact model, a total of 24 adult female rats were divided into three groups (n = 8/group). Controls subcutaneously (sc) received saline alone. RLX (2 mg/kg) and E2 (30 μg/kg; Sigma Chemical Co. St. Louis, MO, USA) were injected to two groups of animals every other day for a period of 6 weeks.

In the second experimental model, rats (n = 24) were bilaterally ovx and allowed to recover for a period of 3 weeks. Sham ovx rats in the first group received vehicle alone. Remaining rats were divided into two groups and sc injected with RLX (2 mg/kg) and E2 (30 µg/kg) for 6 weeks. All experiments were performed in compliance with institutional and international guidelines for laboratory animal care.

Urine samples were collected prior to end of the experiments from all animals. Then, the rats were decapitated and trunk blood collected. Serum samples were obtained after centrifugation of blood samples at 3,000 rpm (4°C) for 10 min. Urinary DPD concentrations were measured by ELISA (Metra Biosystems; Mountain View, CA, USA). Serum parathyroid hormone (PTH), calcitonin, and osteocalcin levels were determined by immunoradiometric method (Immunotopics International, San Clemente, CA, USA). Serum concentrations of ALP, Ca, and inorganic phosphate were determined by enzymatic–colorimetric method in an Olympus AU600 auto-analyzer using commercially available kits from Olympus Corp. (Hamburg, Germany). Samples were studied in duplicate in the tests, and a mean was taken.

Statistical analysis

One-way analysis of variance (ANOVA) was performed on the data. The results were expressed as mean±SEM. P < 0.05 was considered to be statistically significant.

Results

Levels of urinary DPD, serum osteocalcin, ALP, calcitonin, and PTH are shown in Figs. 1, 2, 3, and 4. Removal of ovaries significantly elevated urinary DPD levels compared with intact controls (Fig. 1). Treatment of both intact and ovx rats with E2 resulted in significant decreases in DPD values. RLX administration had no significant effect in the intact rats, but it remarkably reduced concentrations of this bone absorption marker in the ovx animals.
https://static-content.springer.com/image/art%3A10.1007%2Fs13105-010-0008-8/MediaObjects/13105_2010_8_Fig1_HTML.gif
Fig. 1

Urinary DPD levels in intact and ovx rats following administration of raloxifene (RLX) or estradiol (E2). Values are mean±SEM; ***p < 0.001 vs. control group; +++p < 0.05; ++p < 0.01 vs. ovx group, using one-way ANOVA

https://static-content.springer.com/image/art%3A10.1007%2Fs13105-010-0008-8/MediaObjects/13105_2010_8_Fig2_HTML.gif
Fig. 2

Effect of administration of raloxifene (RLX), estradiol (E2), and serum osteocalcin levels in intact and ovx rats. Values are mean±SEM. *p < 0.05; **p < 0.01 vs. intact control group; and +++p < 0.001 vs. ovx group, using one-way ANOVA

https://static-content.springer.com/image/art%3A10.1007%2Fs13105-010-0008-8/MediaObjects/13105_2010_8_Fig3_HTML.gif
Fig. 3

Effect of raloxifene (RLX) and estradiol (E2) administration on serum ALP levels in intact and ovx rats. Values are mean±SEM, ***p < 0.001 vs. intact control group, ++p < 0.01 vs. ovx group, using one-way ANOVA

https://static-content.springer.com/image/art%3A10.1007%2Fs13105-010-0008-8/MediaObjects/13105_2010_8_Fig4_HTML.gif
Fig. 4

Serum calcitonin (nanograms per milliliter) and PTH levels (units per liter) in intact and ovx rats following administration of raloxifene (RLX) or estradiol (E2). Results are mean±SEM *p < 0.05; ***p < 0.001 vs. corresponding control group, ++p < 0.01, +++p < 0.001 vs. corresponding ovx group, using one-way ANOVA

Administration of E2 significantly decreased serum osteocalcin levels in both intact and ovx rats (Fig. 2). RLX treatment significantly increased osteocalcin levels in the intact model and decreased in the ovx rats. Serum ALP levels were elevated by RLX in the ovx animals. Administration of E2 decreased serum ALP values, but these reductions were statistically significant only in the intact group. Serum ALP levels were not significantly affected by removal of ovaries (Fig. 3).

E2 treatment significantly increased and decreased serum calcitonin levels in the intact and ovx rats, respectively (Fig. 4). Levels of this hormone were significantly reduced by RLX only in the ovx model. Injection of E2 caused a small but significant increase of PTH only in the intact rat model (Fig. 4), whereas RLX administration only provoked a significant enhancement of PTH levels.

Serum calcium and inorganic phosphate concentrations are summarized in Table 1. Serum values of Ca (milligrams per deciliter) were significantly elevated by exogenous E2 administration in both intact and ovx rats compared with the corresponding control groups. Administration of RLX had no significant effect on serum Ca in any of the experimental model.
Table 1

Serum calcium and inorganic phosphate concentrations (milligrams per deciliter) in intact and ovariectomized (ovx) rats treated with oestradiol (E2) and raloxifene (RLX) for a period of 6 weeks

Groups

Calcium (mg/dL)

Inorganic phosphate (mg/dL)

Control

10.1 ± 0.1

5.7 ± 0.4

E2

11.4 ± 0.1*

6.3 ± 0.1

RLX

10.3 ± 0.2

7.4 ± 0.2*

Ovx

9.7 ± 0.4

4.6 ± 0.4

Ovx + E2

11.0 ± 0.1**

6.6 ± 0.2***

Ovx + RLX

9.8 ± 0.1

6.1 ± 0.2****

*p<0.005 compared with control group; **p<0.005 compared with ovx group; ***p<0.01; and ****p<0.05, using one-way ANOVA

RLX treatment brought about significant increases in serum inorganic phosphate levels (milligrams per deciliter) in both intact and ovx animals. The elevation of phosphate concentrations by E2-treatment was significant only in the ovx group (Table 1).

Discussion

DPD is a product of collagen breakdown and is primarily excreted in urine [37]. It is known that estrogen decreases serum and urinary levels of this resorption marker. In contrast, cessation of estrogen production is associated with loss of the restraining effect of this gonadal hormone on osteoclastic bone resorption, which is reflected by increased levels of urinary DPD [26]. In the present study, urinary DPD levels were reduced by exogenous administration of E2 in both models. RLX had no significant effect on DPD concentrations in the intact rats, but significantly reduced its elevated levels in the ovx model. These findings thus indicate that RLX prevents degradation of collagen content of bone in ovx animals and is ineffective in the presence of endogenous estrogen. Previously, administration of RLX to ovx rats for 6 weeks also reduced urinary levels [20, 23]. It has recently been shown that RLX treatment for 12 months (60 mg/day) brought about decreased urinary DPD levels in osteoporotic postmenopausal women [28]. Taken together, we suggest that RLX exerts anti-resorptive effects on bone when estrogen levels are low. Indeed, a very recent study has shown that 1 year treatment with two different doses of RLX to intact female dogs did not alter any collagen parameters [1].

Osteocalcin levels are generally increased in menopausal women, and they drastically decrease following hormone replacement therapy [5, 10]. In our study, osteocalcin levels were significantly elevated in ovx animals in comparison to the intact control group. Furthermore, osteocalcin values were significantly reduced by administration of E2 in both experimental models. Similar findings have recently been reported that ovx resulted in elevated levels of ALP and osteocalcin, and E2 supplementation reversed these effects [15, 34]. RLX significantly increased osteocalcin levels in the presence of endogenous estrogen, but had decreasing effects in the ovx model. Similarly, Li et al. [23] previously reported that removal of ovaries increased, and RLX treatment reduced serum osteocalcin levels in rats. Clinical findings have shown that chronic treatment of postmenopausal women with RLX resulted in decreased osteocalcin profile [11, 28] while others found no effect [4]. RLX treatment elevated serum ALP in only the ovx rats in the present study. These findings appear to be consistent with a previous report that this SERM increases bone remodeling markers following ovx [8]. However, a previous clinical survey has indicated that treatment with RLX (150 mg/day) for 24 months reduced serum ALP levels in the postmenopausal women [11]. The high dose and long period of treatment may account for the discrepancy in ALP concentrations between these studies. Indeed, serum ALP levels were increased at week 4 and started to decline at week 12 following RLX treatment (60 mg/day) of postmenopausal women [29].

It is interesting to note that RLX had dual effects on some of the bone turnover markers in the presence or absence of endogenous E2. For instance, reduction of urinary DPD by RLX treatment in intact rats was slight, and RLX was more effective in decreasing collagen loss in the ovx group. These dual effects of RLX have not been elucidated previously, and the underlying mechanisms are currently unknown. Several cytokines such as interleukin-1, interleukin-6, and transforming growth factor-beta are involved in high turnover bone resorption following estrogen deficiency in rats [7] which can be suppressed by both RLX and E2 [6]. RLX exerts its effects by binding with high affinity to both ER-alpha and ER-beta [2]. Perhaps, a competition between RLX and estrogen in binding to ERs may account for the dual effects of RLX in the two different models employed in the present study.

It has been reported that serum PTH levels were unaffected following ovx for 55 days post-operatively [32]. Our findings confirm this study since serum PTH concentrations in ovx group did not significantly differ from those seen in the intact rats. Exogenous E2 supplementation increased PTH mRNA levels in ovx rats [14]. A clinical study has also indicated that hormone replacement therapy resulted in increased levels of serum PTH in postmenopausal women [27]. In the present study, increases in serum PTH levels were observed following E2 administration, but these changes were significant in only the intact group. RLX treatment for 12 weeks (60 mg/day) had no significant effect on serum PTH levels in postmenopausal women [18]. To date, effects of RLX on PTH function have not been elucidated in laboratory animals. In our study, although this SERM had no significant effect in the intact rats, it caused a significant increase in serum PTH levels in the ovx animals. In the same groups, serum calcium levels were unchanged, but significant increases were observed in inorganic phosphate levels. Similar observations have been reported in RLX-treated postmenopausal women [4]. Our findings suggest that RLX has no effect on calcium absorption in intact or ovx rat models.

In conclusion, results of the present study indicate that RLX exerts its protective effects by reducing bone resorption, similar to that of E2, in ovx rats. Our findings also suggest that RLX treatment does not have a considerable impact on bone mineralization and the hormones involved in its regulation.

Acknowledgements

We would like to thank Novartis Turkey for providing RLX used in the present study and to Dr. Yusuf Turkoz of Inonu University Medical School for his courtesy in biochemical assays.

Copyright information

© University of Navarra 2010