THE EFFECTIVENESS OF THE COMBINATIONS OF CALCITRIOL AND ETHYNIL ETHYL ESTRADIOL TO DECREASE OSTEOPOROSIS AND ENDOMETRIAL CANCER RISKS IN OVARIECTOMIZED RATS

The objective of this research was to study the effectiveness of calcitriol and ethynil ethyl estradiol combinations to decrease osteoporosis and endometrial cancer risks in ovariectomized Wistar rats. Twenty five 8-week old female Wistar rats were randomly divided into five groups (normal controlled rats/NK, ovariectomized rats/OVK, ovariectomized rats + calcitriol supplementation/OVD, ovariectomized rats + ethynil ethyl estradiol supplementation/OVE, and ovariectomized rats + calcitriol + ethynil ethyl estradiol supplementation/OVDE). At the end of the study, all rats were weighed and euthanized with 10% ketamine and 2% xylazine. Uterus and left femur were taken and fixed in 10% formaldehyde solution for histopathological examination using hematoxylin and eosin stain. Uterus was weighed before the fixation. The results showed that the percentage of uterus weight in OVK was significantly decreased compared to NK. Meanwhile, the percentage of uterus weight in OVDE was significantly increased compared to OVK. Histopathological features of the uterus in OVK were atrophy, reduction of myometrial and endometrial layers thickness, and formation of cuboidal epithelium in the endometrial lumen. However, the thickness of myometrial and endometrial layers in OVDE were increased, and its endometrial lumen was lined with metaplastic and hyperplastic squamous cell. Histopathological features of distal femur epiphysis in OVK ras showed fewer trabecular spiculums and more adipocyte in the bone marrow compared to NK. However, OVDE had more trabecular bone spiculum and less adipocyte in the bone marrow compared to OVK. In conclusion, the combination of calcitriol and ethynil ethyl estradiol supplementation could reduce osteoporosis, but increased the endometrial cancer risk in ovariectomized Wistar rats. ____________________________________________________________________________________________________________________


INTRODUCTION
Low level of estrogen causes osteoporosis either in animals or humans.There is a relationship between low circulating estrogen concentrations in menopausal period with osteoporotic fracture in post-menopausal women (Doherty et al., 2001).Estrogen plays a role in the suppression of bone remodeling (Manolagas, 2002) by suppressing bone resorption, decreasing osteoclast production (Hughes et al., 1996;Teitelbaum, 2000), and inducing bone formation by increasing osteoblast production, despite the variation depends on its animal model (Qu et al., 1998).Estrogen also activates the kidney to convert vitamin D into its active form (Notelovitz, 1997).The decreased estrogen level causes the reduction of 1,25-dihydroxyvitamin D 3 (Notelovitz, 1997).Hormone replacement therapy using estradiol can inhibit bone resorption, increase bone density, and reduce the risk of bone fracture (Rossouw et al., 2002;Anderson et al., 2004).However, it may cause stroke, pulmonary embolism, breast cancer, and endometrial cancer (Rodan and Martin, 2000;Rossouw et al., 2002).Estradiol supplementation causes hypertrophy and hyperplasia of all uterus structure (Gallo et al., 2008;Pan et al., 2010).According to Amant et al. (2005) and Kong et al. (1997), endometrial hyperplasia was the precursor of endometrial cancer.Several study showed that 1,25dihydroxyvitamin D 3 increased bone formation (Hendy et al., 2006) and decreased the risk of endometrial cancer (Salazar-Martinez et al., 2005;Mohr et al., 2007).Supplementation of 8 ng calcitriol for six weeks in ovariectomized rats reduced the estrogen concentration and cause osteoporosis, which was marked by reduced trabecular bone spiculum, increased bone marrow cavity, and domination of adipocyte tissue of the distal femur (Hartiningsih et al., 2012).The effect of combined calcitriol and ethynil ethyl estradiol in reducing osteoporosis and its safety for uterus in ovariectomized rats had never been reported.The effectiveness of combined calcitriol and ethynil ethyl estradiol was analyzed using histopathological examination of the distal femur and the uterus.Combined estrogen and ethynil ethyl estradiol are expected to reduce osteoporosis, which is marked by increased trabecular bone spiculum and reduced adipocyte tissue in bone marrow cavity.They are also expected to be safe for the uterus, with no effect of inducing hypertrophy and hyperplasia on the uterus structure.

MATERIALS AND METHOD
In this research, twenty five female Wistar rats with 8 weeks of age were used and placed in individual cages with 25 C room temperature.The rats were given standard feeding which contained 20% protein, 0.6% calcium, and 0.4% phosphate.The standard feeding, for each g/100 g, consist of 78% corn, 20% anchovy, 0.3% CaCO 3 , 0.7% molase, 1.0% mineralvitamin, and sufficient distilled water.
The rats were divided into five groups (normal control/NK, ovariectomized control/OVK, ovariectomized + 20 ng/day/rat oral calcitriol/OVD, ovariectomized + 25 µg/day/rat oral ethynil ethyl estradiol/OVE, and ovariectomized + combined 20 ng/day/rat oral calcitriol and 25 µg/day/rat oral ethynil ethyl estradiol/OVDE.One week after environmental adaptation, ovariectomy was performed using incision in the caudal midline.Combination of 10% ketamine (50 mg/kg) and 2% xylazine (5 mg/kg) was injected intramuscularly for anesthesia.The same procedure was performed in the controlled Wistar rats without ovariectomy.One week after surgery, all Wistar rats were started to be given the treatment for eight weeks.
At the end of treatment period, rats were weighed and euthanized using combination of ketamine 10% and xylazine 2% intramuscularly.Left femoral bone was taken and fixated in formalin 10% for histopathological examination.Before the fixation in formalin 10%, the uterus was weighed and the result was analyzed using one way Anova and Duncan's test, while the result of histopathological examination was analyzed descriptively.

RESULTS AND DISCUSSION
Ovariectomy caused a significant decrease in uterus weight percentage of OVK rats (0.068±0.02%) compared to that of the NK rats (0.22±0.02%).Supplementation of ethynil ethyl estradiol caused uterus weight percentage of OVE rats (0.24±0.04%) increased 3.5 folds more than that of OVK rats (0.068±0.02%).Supplementation of combined calcitriol and ethynil ethyl estradiol caused uterus weight percentage of OVDE rats (0.17±0.03%) to increased 2.5 folds greater than OVK rats.Supplementation of calcitriol caused uterus weight percentage of OVD rats (0.058%±0.01%) decreased 0.85 fold than OVK rats (Figure 1).Saruhan et al. (2006) reported that the ovariectomy reduced the uterus weight.Reduction of uterus weight percentage in OVK rats was reciprocal to the study by Li et al. (2003), Pan et al. (2010), andGallo et al. (2008), which reported that ovariectomy in rats reduced serum estradiol concentration and uterus weight.The higher percentage of uterus weight of OVE rats compared to OVK rats, and OVDE rats compared to OVD rats were also consistent to the study done by Li et al. (2003), Pan et al. (2010), andGallo et al. (2008), which reported that ethynil ethyl estradiol in ovariectomized rats increased serum estradiol concentration and increased uterus weight.Uterus weight percentage of OVD rats, which was less than OVK rats, in spite of its non-significant result, was caused by the lower estrogen concentration in OVD rats compared to OVK rats.Hartiningsih et al. (2006) reported that supplementation of calcitriol for 3 months after ovariohysterectomy reduced estrogen.OVDE rats had a lower uterus weight percentage compared to OVE rats, despite its non-significant result, and was caused by its lower estrogen concentration compared to OVE rats.
Histopathological figure of uterus in NK rats showed columnar endometrial epithelium, which was reciprocal to Saruhan et al. (2006).Histopathological figure of uterus in OVK and OVD rats showed atrophy, reduction in diameter, reduction in endometrialmyometrial layer, and cuboidal epithelium of the uterus (Figure 2).Several researches showed atrophic and cuboidal epithelium of uterus, two months after ovariectomy (Sonmez et al., 2000;Saruhan et al., 2006;Pan et al., 2010).Histopathological figure of uterus in OVE and OVDE rats uterus showed an increase of luminal diameter and endometrial-myometrial layer.However, in OVE rats, endometrial lumen was lined by complex and hyperplastic squamous epithelium, while in OVDE rats, the uterus was lined by metaplastic, complex, and hyperplastic squamous epithelium.According to Pan et al. (2010) and Gallo et al. (2008), the supplementation of estradiol caused epithelial hyperplasia of the uterus, and hypertrophy in all of its structure.Amant et al. (2005) and Kong et al. (1997) reported the hyperplasia of endometrium was a precursor of type I endometrial cancer.Eighty percent of all endometrial carcinoma or type I endometrial can- cer was reported to be caused by hormone replacement therapy or estrogen.Histopathological figure of uterus in OVE and OVDE rats showed an increased risk of endometrial cancer.

Histopathological Analysis of Distal Femoral Epiphysis
Histopathological analysis of distal femoral epiphysis of normal control rats NK showed a normal shape (Figure 3A), in agreement to what Jee (1983) and Bullock and Rosendahl (1984) have explained, that proximal epiphysis and distal part of long bones consisted of the trabeculae and the cleft that connects trabeculae are filled with bone marrow.The use of trabeculae spicules to mark the presence of osteoblast is in accordance with a report by Shiraishi et al. (2000) which stated that recruitment, osteoblast differentiation, and bone formation by osteoblast were marked by the thickening of trabeculae spicules.The use of adipose tissue to mark the presence of osteoclast in bone marrow cavity is in accordance with Rosen et al. (2009) whose report stated that bone marrow cavity was filled with hematopoietic tissue, bone tissue, and adipose tissue.It has been reported by Dazzi et al. (2006) and Minguell et al. (2001) that the balance of microenvironment in bone marrow is an important factor for hematopoietic process, osteogenesis process, and other processes.
Histopathologic analysis of distal femoral epiphysis of OVK rats (Figure 3B) and OVD rats (Figure 3C) showed a little amount of trabeculae spicules and the bone marrow cavity was dominated by adipose tissue.On OVDE rats (Figure 3D) and OVE rats (Figure 3E) showed more trabeculae spicules, and bone marrow cavity of OVE rats was dominated by hematopoietic tissue, while bone marrow cavity of OVDE rats was dominated by adipose tissue.
Domination of adipose tissue in bone marrow cavity of OVK rats femoral epiphysis marked osteoporosis.It had been reported by Syed et al. (2008) and Benayahu et al. (2000) that estrogen deficiency caused an increase in the accumulation, number, and size of adipose cells in bone marrow of post-menopausal women that suffer from osteoporosis and ovariectomized mice.Domination of adipose tissue in bone marrow cavity of OVK rats femoral epiphysis marked the increasing osteoclast activity in bone resorption which was thought to be related to the and hematopoietic cells predominance in bone marrow cavity (h); OVK rats (B) and OVD rats (C) were showed few trabecular spicules (s), and adipocytes predominance in bone marrow (a); OVDE rats (D) and OVE rats (E) were showed more trabecular spicules (s), adipocytes predominance in bone marrow of OVDE rats (a), and hematopoietic cells predominance in bone marrow cavity of OVE rats (h) (HE 100x) decreasing level of estrogen.According to Cooke and Naaz (2004) and Riggs et al. (2002), estrogen had a fundamental role in control of osteoblast and adipose cells.It had also been reported by Verma et al. (2002), Justesen et al. (2001) and Rodriguez et al. (1999) that the decrease of estrogen concentration affects proliferation and estrogenic activities in bone marrow and will cause a higher conversion of stromal cells into adipose cells rather than into osteoblasts.According to Weisberg et al. (2003) not only did the presence of adipose cells in bone marrow cavity suppress bone formation and inhibit osteoblastogenesis, but it also increase bone resorption.Some researchers reported that the decrease of estrogen concentration increase bone resorption until it exceeds bone formation (Manolagas et al., 2000;Khosla et al., 2002;Riggs et al., 2002) which causes wider resorption of trabeculae bone, loss of trabeculae connectivity (Manolagas et al., 2000;Khosla et al., 2002;Riggs et al., 2002), and the absence of trabeculae structure (Rosen and Bouxsein, 2006;Syed et al., 2008).The decrease of distal femoral bone trabecular spicules in OVK rats also marked bone formation.Cigiela et al. (2012) reported that ovariectomy decreases trabeculae spicules and causes bone marrow cavity to become wider.According to Nakamura et al. (2003) and Notelovitz (1997), in normal condition, bone will maintain normal blood calcium level through the balance between bone resorption by osteoclast and formation of new bone by osteoblast.According to some researchers, bone resorption and formation imbalance (Goltzman, 2002;Teitelbaum and Ross, 2003;Martin and Sims, 2005;Seeman et al., 2006;Karsdal et al., 2007), higher rate of bone resorption rate than that of bone formation (Doige, 1988;Palmer, 1993), increasing bone resorption and decreasing bone formation or both (Banks, 1981;Bullock and Rosendahl, 1984;Jubb et al., 1985) might cause osteoporosis.Therefore, it could be said that ovariectomy on OVK rats cause osteoporosis.
Calcitriol suplementation on OVD rats caused more adipose tissue to form in bone marrow cavity and trabeculae spicules of the distal epiphyseal plate of the femur were decreased compared to that of the NK rats, but appeared to have fewer amount of adipose tissue and more trabeculae spicules than OVK rats (Figure 3).The amount of adipose tissue in the bone marrow of femur distal epiphyseal plate of OVD rats, which was lesser than that of OVK rats but greater than NK rats, revealed that calcitriol suplementation decrease adipose tissue even in OVD rats that have lower concentration of estrogen.It was reported by Duque et al. (2004) that calcitriol inhibits adipogenesis in bone marrow cavity by lowering the expression of peroxisome proliferatoractivated receptor gamma (PPARγ) (Duque et al., 2004).Zhou et al. (2006), Kong and Li (2006), and Sun and Zemel (2004), also reported that 1,25dihydroxyvitamin D 3 induced apoptosis adipogenesis and inhibited adipogenesis in cells.Adipose tissue in the bone marrow cavity of femur the distal epiphyseal plate in OVD rats, which was lesser than that of the OVK rats but greater than the NK rats revealed that calcitriol supplementation caused OVD rats's bone resorption activities become lower than that of OVK rats, but higher than that of NK rats.
Trabecular spicules of femur distal epiphyseal plate in OVD rats, which was greater in OVK rats but lesser in NK rats, thought to be related with a higher level of calcitriol.Chang et al. (2013) and Anderson et al. (2006) reported that supplementation of 1,25dihydroxyvitamin D 3 increased preosteoblast proliferation to be osteoblast.Chang et al. (2013) also reported that supplementation of 1,25dihydroxyvitamin D 3 caused higher marker of osteoclasin formation and alkaline phosphatase as well as caused higher mineral density of femoral and trabeculae femoral volume although still lower than rats that had been treated with estradiol.According to Zhou et al. (2006) 1,25-dihydroxyvitamin D 3 works directly on osteoblast to increase bone formation.Therefore, in this study, calcitriol supplementation causes bone formation activity on OVD rats to become higher than that of OVK rats, even though it's still lower than that of the NK rats.
Ethynil ethyl estradiol supplementation on OVE rats and OVDE rats were thought to cause estrogen concentration of OVE rats raised above that of OVK rats, and estrogen concentration of OVDE rats to become higher than that of OVD rats.Histopathological analysis of OVE rats showed fewer adipose tissues in bone marrow cavity and more spiculum trabeculae in the distal epiphyseal plate of the femur than that of OVK rats.The same thing was occurred in OVDE rats when compared to OVD rats (Figure 3).The fewer number of adipose tissue in bone marrow cavity of OVE rats and OVDE rats marked a lowered activity of bone resorption that was thought to have a relationship with the higher level of estrogen.Parikka et al. (2001) and Kameda et al. (1997) reported that estradiol lowers osteoclast activity for bone resorption.The more number of trabecular spicules that found in femur distal epiphyseal plate of OVE rats and OVDE rats were considered related with the higher level of estrogen in OVE rats and OVDE rats.Some researchers reported that estrogen could increase bone formation by increasing osteoblast formation, differentiation, proliferation, and osteoblast function, although it depends on the animal used (Chow et al., 1992;Majeska et al., 1994;Qu et al., 1998).Chang et al. (2013) reported that estrogen has direct impact on osteoblast by increasing the number of osteoblast and hasten osteoblast in forming new bones.In conclusion, estradiol supplementation lowers bone resorption activity and increase bone formation activity.
The combination of calcitriol ethynil ethyl estradiol suplementation on OVDE rats caused rats OVDE to have higher amount of adipose tissue within their bone marrow cavity and higher number of trabeculae spicules than OVK rats (Figure 3).The lesser amount of adipose tissue in the bone marrow cavity of OVDE rats compared to that of OVK rats but greater than NK rats revealed that the combination of calcitriol and ethynil ethyl estradiol suplementation caused the bone resorption activity of OVDE rats to become lower than OVK rats, but higher than NK rats.The higher number of trabecular spicules on femur distal epiphyseal plate of OVDE rats than in OVK rats and NK rats were showed an increment of bone formation process of OVDE rats than in OVK rats and NK rats.Adipose tissue in bone marrow cavity of OVDE rats was looked abundant than in NK rats, meanwhile, adipose tissue in OVDE rats looked fewer than OVK rats.These differences might be due to the higher number of estrogen found in OVDE rats than OVK rats.The more number of trabecular spicules in distal femoral epiphysis of OVDE rats than OVK rats was assumed related with the higher number of 1,25dihydroxyvitamin D 3 and estrogen of OVDE rats than OVK rats.Furthermore, the more number of trabecular spicules in distalis femoral epiphysis of OVDE rats than NK rats is assumed related with higher number of 1,25-dihydroxyvitamin D 3 and estrogen of OVDE rats than NK rats.According to the result, it showed that the combination of calcitriol and ethynil ethyl estradiol in OVDE rats causes bone resorption activity of OVDE rats lower than OVK rats, but higher than NK rats, and causes bone formation activities of OVDE rats higher than OVK rats and NK rats.

CONCLUSION
It can be concluded that the combination of calcitriol 20 g/day/rat and ethynil ethyl estradiol 25 µg/day/rat administered orally for 8 weeks in ovariectomized rats lower osteoporosis risk but increase endometrium cancer risk.

Figure 1 .
Figure 1.Percentage of uterus weight (g/body weight) after 8 weeks of treatment

Figure 3 .
Figure 3. Histopathological figure of distal epiphyseal femoral bone of NK rats (A) showed no change in trabecular spicules (s)and hematopoietic cells predominance in bone marrow cavity (h); OVK rats (B) and OVD rats (C) were showed few trabecular spicules (s), and adipocytes predominance in bone marrow (a); OVDE rats (D) and OVE rats (E) were showed more trabecular spicules (s), adipocytes predominance in bone marrow of OVDE rats (a), and hematopoietic cells predominance in bone marrow cavity of OVE rats (h)(HE 100x)