A. G. H. Ederveen

Intestinal calcium transporter genes are upregulated by estrogens and the reproductive cycle through vitamin D receptor-independent mechanisms.

1α,25(OH)2‐vitamin D strongly regulates the expression of the epithelial calcium channel CaT1. CaT1 expression is reduced in ERKOα mice and induced by estrogen treatment, pregnancy, or lactation in VDR WT and KO mice. Estrogens and vitamin D are thus independent potent regulators of the expression of this calcium influx mechanism, which is involved in active intestinal calcium absorption.

Tibolone: a compound with tissue specific inhibitory effects on sulfatase.

The aim was to test whether sulfatase activity is differently regulated by tibolone in human bone, endometrium and breast cells since selective inhibition of sulfatases in various tissues may contribute to the tissue-specificity of tibolone. Tibolone, its 3 alpha- and 3 beta-hydroxy metabolites and their 3-sulfated forms, and its Delta(4)-isomer strongly (70-90%) inhibited the sulfatase activity in human breast cell lines (two T-47D clones) and intermediately (8-43%) in human endometrial cells (HEC-1A). In contrast, they did not inhibit sulfatase in two human osteoblast-like cell lines (MG 63, HOS TE-85). The specific sulfatase inhibitor, EMATE, showed inhibition in all cell lines. Just as estrone sulfate, 3 alpha-sulfated tibolone was also converted by sulfatase to the unconjugated 3 alpha-hydroxy-tibolone intracellularly in all cell lines. The tissue specific inhibition pattern of sulfatase activity by tibolone and its metabolites suggest that tibolone could be protective against development of mammary carcinomas, whereas it retains favorable estrogenic effects on bone.

Skeletal effects of estrogen deficiency as induced by an aromatase inhibitor in an aged male rat model.

Aromatization of androgens into estrogens may be important for maintenance of the male skeleton. To address this hypothesis, we evaluated the skeletal effects of selective estrogen deficiency as induced by the aromatase inhibitor vorozole (Vor), with or without 17beta-estradiol (E(2)) administration (1.35 microg/day), in aged (12-month-old) male rats. A baseline group was killed at the start of the experiment (Base). The control group (Control), the group treated with vorozole alone (Vor), the group treated with E(2) alone (E(2)), or the group with a combination of both (Vor + E(2)) were killed 15 weeks later. Vorozole significantly increased serum testosterone (T) and reduced serum E(2) compared with Control. Body weight gain and serum insulin-like growth factor-I (IGF-I) were also lower in Vor, whereas significant weight loss and decrease of serum IGF-I occurred as a result of E(2) administration. Bone formation as assessed by serum osteocalcin was unaffected but osteoid surface in the proximal metaphysis of the tibia was increased in Vor-treated rats. Bone resorption as evaluated by urinary deoxypyridinoline excretion was increased in Vor. Biochemical parameters of bone turnover were reduced significantly in all E(2) treated rats. Premature closure of the growth plates and decreased osteoid and mineralizing surfaces were also observed in E(2) and Vor + E(2). Apparent bone density of lumbar vertebrae and femur, as measured by dual-energy X-ray absorptiometry (DXA), was significantly reduced in Vor. Vorozole decreased femoral bone density mainly in the distal femur (trabecular and cortical region). This decrease of bone density was not present in E(2) and Vor + E(2). Similar findings were observed when bone density was assessed by peripheral quantitative computed tomography (pQCT); that is, trabecular density of the distal femur, the proximal tibia, and the distal lumbar vertebra were all lower in Vor. This decrease in density was not observed in all E(2)-treated animals. In conclusion, administration of the aromatase inhibitor, vorozole, to aged male rats induces net trabecular bone loss in both the appendicular and axial skeleton, despite a concomitant increase in serum testosterone. E(2) administration is able to prevent this trabecular bone loss in vorozole-treated animals.

Oestrogen-mediated cardioprotection following ischaemia and reperfusion is mimicked by an oestrogen receptor (ER)alpha agonist and unaffected by an ER beta antagonist

Oestrogen protects the heart from ischaemic injury. The current study aims to characterise two novel oestrogen receptor (ER) ligands, an ERα agonist ERA-45 and an ERβ antagonist ERB-88, and then use them to investigate the roles of ERα and ERβ in mediating the cardioprotection by E from ischaemia–reperfusion injury in the rat. The ER ligands were characterised by gene transactivation assay using ER-transfected Chinese hamster ovary (CHO) cells and in bioavailability studies in vivo. Female rats (n=48) were ovariectomised and implanted with 17β-oestradiol (17βE2) releasing or placebo pellets. ERA-45, ERB-88 or vehicle was administered for 5 days prior to ischaemia–reperfusion studies. Necrosis, neutrophil infiltration (myeloperoxidase activity) and oxidant stress production (electron paramagnetic resonance) from the area-at-risk were measured to assess reperfusion injury. The ERα agonist ERA-45 showed more than 35-fold selectivity for ERα compared with ERβ gene transactivation. In vitro, the ERβ antagonist ERB-88 inhibited transactivation by 17βE2 via ERβ with 46-fold selectivity relative to inhibition via ERα. In vivo, 17βE2 significantly reduced neutrophil infiltration, oxidant stress and necrosis following ischaemia and reperfusion. Cardioprotection by 17βE2 was not inhibited by ERB-88 but was completely reproduced by ERA-45. In conclusion, protection of the rat heart after ischaemia–reperfusion by 17βE2 is achieved through the reduction of cardiomyocyte death, neutrophil infiltration and oxygen-free radical availability.The results of this study indicate that these effects are primarily mediated via activation of ERα.

Tibolone Exerts Its Protective Effect on Trabecular Bone Loss Through the Estrogen Receptor

Tibolone (Org OD14) has estrogenic, progestogenic, and/or androgenic activity depending on the tissue. In postmenopausal women, tibolone prevents bone loss without stimulating the endometrium. Tibolone is effective in preventing trabecular bone loss from the peripheral and axial skeleton of young and old ovariectomized (OVX) rats by reducing bone turnover, that is, bone resorption, like estrogens. We evaluated the contribution of the various hormonal activities to tibolones bone‐conserving effect. Three‐month‐old OVX rats received tibolone (125 μg/rat or 500 μg/rat, twice daily), alone or combined with an antiestrogen, antiandrogen, or antiprogestogen, and the effects on trabecular bone mass and bone turnover were evaluated. Sham‐operated and control OVX groups were treated with vehicle. The remaining OVX groups received oral doses of tibolone twice daily, alone or with twice daily (a) antiestrogen ICI 164.384, (b) antiandrogen flutamide, or (c) antiprogestogen Org 31710. For comparison, the effects of 17β‐estradiol and testosterone were examined also. After 4 weeks, trabecular bone mineral density (BMD) in the distal femur, plasma osteocalcin, and urinary deoxypyridinoline/creatinine ratio (Dpyr/Cr) were measured. Tibolone or 17β‐estradiol significantly blocked ovariectomy‐induced loss of trabecular BMD and inhibited bone resorption and bone turnover as judged by reduced Dpyr/Cr ratio and osteocalcin, respectively. These effects of both compounds were counteracted by the antiestrogen. This suggests a major involvement of the estrogen receptor in the action of tibolone on bone metabolism. However, the antiandrogen and the antiprogestogen did not counteract the effects of tibolone, excluding a major role of the androgenic and progestogenic activities of tibolone in its action against trabecular bone loss. The results indicate that tibolone acts on bone almost entirely through activation of the estrogen receptor.

Ovariectomy and orchidectomy induce a transient increase in the osteoclastogenic potential of bone marrow cells in the mouse.

Withdrawal of gender steroids in both women and men is associated with an increase in bone turnover with bone resorption exceeding bone formation leading to bone loss. To further investigate this process, the osteoclastogenic potential of mouse bone marrow cells was assessed at different timepoints after ovariectomy (ovx) or orchidectomy (orx). Cocultures of osteoclast-free fetal mouse long bones together with bone marrow from ovariectomized or orchidectomized mice indicated that the withdrawal of gender steroids in female and male mice induces a transient increase in osteoclastogenesis. The osteoclastogenic potential of the bone marrow cells was increased 7 days after ovx or orx. However, osteoclastic resorption was not increased at 3 days after surgery and had normalized 30 days after either ovx or orx. These results suggest that the withdrawal of gender steroids induces a transient increase in osteoclastogenesis in mice of both genders, which is associated with the early phase of rapid bone loss.

Effect of 16 Months of Treatment with Tibolone on Bone Mass, Turnover, and Biomechanical Quality in Mature Ovariectomized Rats

Tibolone (Org OD14) is a tissue‐specific steroid with estrogenic effects on the bone and vagina but not endometrium or breast and has been shown to prevent ovariectomy‐induced bone loss in young and old rats. We evaluated the effect of long‐term tibolone treatment on bone parameters in mature ovariectomized (OVX) rats. Six‐month‐old rats were allotted to one of six groups (n = 8). Sham‐operated and control OVX groups received vehicle, whereas other groups (all OVX) received tibolone (125, 250, or 500 μg/day orally) or 17α‐ethinylestradiol (EE; 24 μg/day orally) for 16 months. Treatment with tibolone prevented ovariectomy‐induced bone loss in peripheral (femur and tibia) and axial (L1‐L2 and L4) skeleton. In peripheral skeleton, tibolone and EE prevented loss of bone mass and quality to a similar extent. Tibolone dose‐dependently inhibited trabecular bone volume loss in L1‐L2 and tibia, and at 500 μg/day it inhibited 88% of L1‐L2 and 55% of tibial volume loss (p ≤ 0.05 in each case). Tibolone, 500 μg, resulted in 10% greater cortical strength of femur (p ≤ 0.05) and 60% greater compressive strength of L4 (p ≤ 0.05) compared with vehicle‐treated OVX rats. Tibolone and EE inhibited bone resorption and turnover, assessed by urinary deoxypyridinoline/creatinine and plasma osteocalcin, respectively. We conclude that 16 months of tibolone treatment prevents ovariectomy‐induced deterioration of axial and peripheral skeleton and preserves cortical and trabecular bone strength by reducing bone resorption.

Tibolone, a steroid with a tissue-specific hormonal profile, completely prevents ovariectomy-induced bone loss in sexually mature rats

Tibolone (Org OD 14) is a synthetic steroid with combined estrogenic, progestagenic, and androgenic properties and behaves as a tissue‐specific steroid. In the current study, we determined the effects of a 4‐week treatment with different doses of tibolone on estrogen deficiency–induced bone loss in mature 3‐month‐old rats. As a reference, 17α‐ethinyl estradiol (EE2) was used. The frequency of administration, once or twice a day, was also studied. Bone parameters were determined in sham operated controls, ovariectomized (OVX) controls and OVX‐treated rats. Bone loss was assessed by peripheral quantitative computed tomography directly and by quantitative Roentgen densitometry after defatting to exclude influence of fat changes. Femoral bone geometric parameters, plasma osteocalcin level, and urinary deoxypyridinoline/creatinine ratio were also determined. Ovariectomy caused a significant decrease in trabecular bone mineral density in the distal metaphyseal part of the femur using both methods, whereas no change in cortical bone density was found. Trabecular bone loss was prevented in a dose‐dependent manner by tibolone (250, 1000, and 4000 μg/rat/day) when given once or twice daily. EE2 also prevented trabecular bone loss but its efficacy was dependent upon the frequency of dosing. Both tibolone and EE2 induced a significant reduction in the urinary deoxypyridinoline/creatinine ratio and plasma osteocalcin level. Tibolone and EE2 had no effect on other femoral bone parameters except a reduction in femoral length. In conclusion, treatment with tibolone for 4 weeks prevented OVX‐induced bone loss by suppressing both bone resorption and bone turnover in a similar way as EE2. However, the frequency of dosing is more important for EE2 than for tibolone. Tibolone acts in this animal model for postmenopausal bone loss as an estrogen agonist on bone.

Metabolism of tibolone and its metabolites in uterine and vaginal tissue of rat and human origin

In postmenopausal women, tibolone shows clear tissue differences in its stimulatory effects on the vagina and uterus. In rats, however, it has stimulatory effects on both tissues, with a different, more estrogenic, effect on the uterus than in humans. This may be due to differences in local metabolism. Therefore, in the present study, the metabolism of tibolone was analyzed in incubations of uterine and vaginal tissue from postmenopausal women and ovariectomized rats using radiolabeled tibolone in order to understand the tissue- and species-specific metabolism. In the rat, tibolone (50 nM) was mainly 3alpha-reduced to the estrogenic 3alpha-OH-tibolone in the uterus and vagina. The 3beta-OH tibolone can be isomerized to 3alpha-OH-tibolone with tibolone as intermediate. In contrast, in the same tissues from postmenopausal women, the progestagenic Delta4-isomer and estrogenic 3beta-OH-tibolone were the major metabolites of tibolone. The formation of the Delta4-isomer was higher in uterine tissue. The 3beta-hydroxysteroid dehydrogenase (HSD) inhibitor epostane had no effect on tibolone metabolism in human uterine and vaginal tissue microsomes and HEK293 cells expressing the human 3beta-HSD types 1 and 2 isoforms did not metabolize tibolone. Moreover, the 3beta-reduction of tibolone to 3beta-OH-tibolone was NADPH dependent, while the isomerization of tibolone to the Delta4-isomer did not require a cofactor. It was therefore concluded that human 3beta-HSD isoforms are not involved in the metabolism of tibolone, and that the 3beta-reduction and the Delta5-10 to Delta4 isomerization may be catalyzed by different enzymes. In conclusion, we showed that, in hormone therapy target tissues of the rat as compared with the human, different metabolic pathways for tibolone exist and therefore result in metabolites with different pharmacological properties. The rat is therefore a poor model to predict the effects of tibolone on the uterus in postmenopausal women.

The estrogenic component of tibolone reduces adiposity in female aromatase knockout mice

Objective: To explore the effects of tibolone on adiposity in the absence of aromatase and determine which of the hormonal properties of tibolone are exerting these effects. Methods: In this study, vehicle; tibolone; estrogenic (ethinyl estradiol [EE]), progestogenic (ORG2058), or androgenic (dihydrotestosterone) compounds; or a combination of ORG2058 + EE was administered to 6-month-old ovariectomized aromatase knockout (ArKO) mice for a period of 6 weeks. Results: In response to tibolone or EE-alone treatments, omental adipose tissue and infrarenal adipose tissue weights were significantly reduced (P = 0.004 and P = 0.01; P = 0.009 and P = 0.014, respectively) compared with those in ovariectomized and vehicle-treated ArKO mice. In contrast, adipose tissue weight tended to increase after ORG2058-alone treatment. Furthermore, EE in the presence of ORG2058 (ORG2058 + EE group) results in little effect on adiposity when compared with that in ovariectomized and vehicle-treated ArKO mice, showing that ORG2058 can negate the effect of EE. Dihydrotestosterone treatment did not have an impact on adipose tissue mass. Adipocyte volume and numbers followed the same treatment trends. Conclusions: In summary, our study in the ArKO mouse has confirmed the efficacy of tibolone as a hormone therapy to reduce adipose tissue accumulation after menopause and also shows that aromatization of tibolone is not required to elicit these estrogenic effects.