Antioxidant effect of the active metabolites of tibolone.

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10(-7), 10(-8), 10(-9 )M) of either estradiol, tibolone, 3α-hydroxytibolone, 3ß-hydroxytibolone, Δ(4)-tibolone, 3α-sulfated-tibolone, 3α-17ß-disulfated-tibolone, 3ß-sulfated-tibolone or 3ß-17ß-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine-xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10(-9 )M of steroids. Using 10(-8 )M concentration: estradiol (80.9 ± 2.5%); 3ß-sulfated-tibolone (83.3 ± 4.7%); 3ß-17ß-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10(-7 )M, 3ß-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine-xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17ß-disulfated-tibolone (71.9 ± 2.5%), 3ß-sulfated-tibolone (73.9 ± 5.0%), and 3ß-17ß-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

Difference in signalling between various hormone therapies in endometrium, myometrium and upper part of the vagina.

BACKGROUND: Combined hormone treatments in post-menopausal women have different clinical responses on uterus and vagina; therefore, we investigated differences in steroid signalling between various hormone therapies in these tissues. METHODS: A total of 30 post-menopausal women scheduled for hysterectomy were distributed into four subgroups: control-group (n = 9), Tibolone-group (n = 8); estradiol (E(2))-group (n = 7); E(2) + medroxyprogesterone acetate (MPA)-group (n = 6). Medication was administered orally every day for 21 days prior to removal of uterus and upper part of the vagina. Tissue RNA was isolated, and gene expression profiles were generated using GeneChip technology and analysed by cluster analysis and significance analysis of microarrays. Apoptosis and cell proliferation assays, as well as immunohistochemistry for hormone receptors were performed. RESULTS: 21-days of treatment with E(2), E(2) + MPA or tibolone imposes clear differential gene expression profiles on endometrium and myometrium. Treatment with E(2) only results in the most pronounced effect on gene expression (up to 1493 genes differentially expressed), proliferation and apoptosis. Tibolone, potentially metabolized to both estrogenic and progestagenic metabolites, shows some resemblance to E(2) signalling in the endometrium and, in contrast, shows significant resemblance to E(2) + MPA signalling in the myometrium. In the vagina the situation is entirely different; all three hormonal treatments result in regulation of a small number (4-73) of genes, in comparison to signalling in endometrium and myometrium. CONCLUSION: Endometrium and myometrium differentially respond to the hormone therapies and use completely different sets of genes to regulate similar biological processes, while in this experiment the upper part of the vagina is hardly hormone responsive.

3beta-OH-tibolone acutely dilates rat muscle arterioles similar to 3alpha-OH-tibolone.

In an earlier study, we focused on the vasoactive effect of 3alpha-OH-tibolone on spontaneously constricted isolatedfemale rat gracilis muscle arterioles. Vasodilator effects (from 10 to 10 M) of 3alpha-OH-tibolone were similar to those of 17beta-estradiol. It was reported that 3beta-OH-tibolone like estradiol altered GABAB activation in neurons through a membrane estrogen receptor, whereas the 3alpha-OH metabolite did not. We therefore hypothesized that the 3beta-OH metabolite may also have a vasodilating effect in our isolated arteriole model. The results indicate that 3beta-OH-tibolone induces a vasodilator effect in small arterioles that is comparable with that of 3alpha-OH-tibolone at the same concentration. This is intriguing because the binding affinity of 3alpha-OH-tibolone to the estrogen receptor is almost twice that of 3beta-OH-tibolone. Other mechanisms may play a role.

7alpha-Methyl-ethinyl estradiol is not a metabolite of tibolone but a chemical stress artifact.

OBJECTIVE: This study was conducted to establish whether 7alpha-methyl-ethinyl estradiol (7alpha-MEE) in plasma from postmenopausal women treated with tibolone is a metabolite or an artifact. DESIGN: Clinical samples with known levels of tibolone metabolites, plus plasma samples spiked with tibolone and metabolites, were analyzed for levels of 7alpha-MEE using liquid chromatography-mass spectometry (LC-MS/MS) with and without derivatization. RESULTS: Approximately 20 to 40 pg/mL 7alpha-MEE was detected using LC-MS/MS with derivatization in plasma samples from postmenopausal women treated with tibolone. In plasma samples spiked with 200 ng/mL tibolone or Delta-tibolone, LC-MS/MS with derivatization revealed the generation of around 200 and 36 pg/mL 7alpha-MEE, respectively, whereas LC-MS/MS without derivatization showed no detectable chemical conversion of tibolone to 7alpha-MEE. Generation of 7alpha-MEE is increased by the "stress conditions" used in the derivatization procedure; simply drying the sample also shows this artifactual conversion. The major active and sulfated 3-hydroxy metabolites of tibolone are not converted to 7alpha-MEE. Without derivatization, and avoiding stress conditions, no detectable levels (<20 pg/mL) of 7alpha-MEE were found in plasma samples from postmenopausal women treated with single (eight participants at 13 time points) or multiple (seven participants at 18 time points) doses of tibolone. CONCLUSIONS: 7alpha-MEE is not a metabolite of tibolone but is a chemical artifact generated during analytical procedures with derivatization. Using LC-MS/MS without derivatization, 7alpha-MEE cannot be demonstrated in plasma from postmenopausal women after single or multiple doses of tibolone.

Conversion of tibolone to 7alpha-methyl-ethinyl estradiol using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry: interpretation and clinical implications.

OBJECTIVE: Tibolone, a hormone therapy drug, is used to treat climacteric symptoms. This drug is rapidly metabolized into three major metabolites (3alpha-hydroxytibolone, 3beta-hydroxytibolone, and Delta4-tibolone). One clinical study provided evidence of conversion of tibolone to another estrogenic metabolite, 7alpha-methyl-ethinyl estradiol (MEE). However, no evidence of MEE formation was found in another study using the human aromatase enzyme. Because MEE was analyzed by gas chromatography-mass spectrometry (GC-MS), which requires derivatization, together with the fact that derivatization of some steroids may lead to aromatization, it is feasible that the MEE detected resulted from an artifact generated during the derivatization process. Hence, our objective was to assess whether tibolone is converted to MEE. DESIGN: We assayed MEE formation in a nonbiological system using GC-MS after derivatization and by analyzing MEE formation using liquid chromatography-mass spectrometry (LC-MS) in nonderivatized samples. RESULTS: MEE formation was evident in tibolone samples derivatized with either pentafluoropropionic anhydride or trimethylsilyl and analyzed by GC-MS. The amount of MEE formed increased with increasing amounts of tibolone (0.5, 1, 2.5, and 5 microg) derivatized; however, relative to tibolone, the percentage of MEE formed remained constant and ranged between 0.22% and 0.29% of tibolone. In contrast to GC-MS, no MEE formation was seen when tibolone was analyzed by liquid chromatography-mass spectrometry without derivatization. CONCLUSIONS: Our findings prove that conversion of tibolone to MEE is an artifact that is generated in a GC-MS system and is largely due to the intense heating step involved in GC-MS. Caution should be exercised to extrapolate clinical implications from existing data on MEE formation using a GC-MS system.

Effect of tibolone administration on central and peripheral levels of allopregnanolone and beta-endorphin in female rats.

OBJECTIVE: We evaluated the effects of tibolone oral administration on neuroendocrine function by investigating the modulation exerted by tibolone administration on allopregnanolone and central and peripheral beta-endorphin (beta-EP) levels in ovariectomized rats. DESIGN: Female Wistar rats (N = 64) were included: 48 rats were ovariectomized, 8 cycling rats were included as controls, and 8 cycling rats were treated with placebo. The ovariectomized animals were divided into six groups: untreated rats and those that received 14-day oral treatment with either placebo, estradiol valerate (E2V) 0.05 mg/kg/d, or tibolone (0.1, 0.5, or 2 mg/kg/d. beta-EP levels were assessed in the frontal lobe, parietal lobe, hippocampus, hypothalamus, anterior pituitary, neurointermediate pituitary, and plasma, whereas allopregnanolone levels were measured in the frontal lobe, parietal lobe, hippocampus, hypothalamus, anterior pituitary, adrenal glands, and serum. RESULTS: The administration of tibolone (0.5 and 2 mg/kg/d) in ovariectomized rats induces a significant increase of allopregnanolone in the frontal lobe, parietal lobe, hippocampus, hypothalamus, whereas in serum a significant increase of allopregnanolone occurs only with the dose of 2 mg/kg/d, a significant decrease in allopregnanolone levels occurs in the adrenal glands. No changes occurred in the anterior pituitary. Tibolone doses of 0.5 and 2 mg/kg/d induced a significant increase in beta-EP content in the frontal lobe, hypothalamus, and neurointermediate lobe; and, at doses of 2 mg/kg/d, in the parietal lobe, anterior pituitary, and plasma, without changes in the hippocampus. Compared with E2V, 0.5 mg/kg/d tibolone showed a similar effect on allopregnanolone and beta-EP in most brain regions. CONCLUSIONS: Tibolone administration affects beta-EP and allopregnanolone levels, playing a role as a neuroendocrine modulator.

Effects of tibolone on nuclear receptors in human endometrial cells.

OBJECTIVE: Tibolone regulates estrogenic activity in a tissue-selective manner. The purpose of this study was to evaluate the effects of tibolone on the mRNA content of nuclear receptors, estrogen receptor-alpha and beta (ERalpha and ERbeta), progesterone receptor (PR), and androgen receptor (AR) in human endometrial stromal and glandular cells. STUDY DESIGN: Human endometrial stromal and glandular cells were isolated from endometrial tissue fragments and separately incubated with tibolone and its metabolites. Nuclear receptor mRNA was determined using real-time polymerase chain reaction (PCR). RESULTS: In endometrial stromal cells, tibolone, Delta4-tibolone, and 3betaOH-tibolone, but not 3alphaOH-tibolone, significantly reduced ERalpha mRNA by approximately 60% and ERalpha protein by approximately 80%. No reduction of ERalpha was observed in endometrial glandular cells. Tibolone induced PR mRNAs to various extents and reached up to 6-fold in glandular cells, but only a moderate increase (approximately 1.5-fold) in stromal cells. Tibolone increased ERbeta and had little effect on AR mRNA in endometrial cells. CONCLUSION: The results showed the majority of the nuclear receptors were not significantly altered. However, tibolone significantly reduced ERalpha in stromal cells and increased PR in glandular cells. These biological effects may play essential roles in averting stimulation of the endometrium in tibolone users.

Endothelium-dependent dilatory effects of 3alpha-OH-tibolone in gracilis muscle arterioles of the female Wistar rat.

OBJECTIVE: Tibolone is a synthetic steroid used for the treatment of the symptoms of menopause that, once metabolized, has estrogenic, progestogenic, and androgenic properties. We investigated the direct vasodilatory effects of the major active tibolone metabolite 3alpha-OH-tibolone and its sulfated form on female rat skeletal muscle arterioles, which play an important role in the control of blood pressure. DESIGN: In isolated, pressurized spontaneously constricted arterioles (mean passive diameter 83 +/- 3 microm), we investigated the vasodilatory effect of 3alpha-OH-tibolone and its sulfated form. To study the role of the endothelium and in particular that of nitric oxide, we repeated the experiments with 3alpha-OH-tibolone after removal of the endothelium and on vessels pretreated with the nitric oxide synthesis inhibitor, Nomega-nitro-L-arginine (L-Na). Finally we compared the vasodilatory effect of 3alpha-OH-tibolone with 17beta-estradiol. RESULTS: A dose-dependent dilatation to 3alpha-OH-tibolone was observed starting at a concentration of 10 M. With the sulfated form of 3alpha-OH-tibolone, dilatation was only present at the highest concentration (10 M). In the denuded vessels, the vasodilatory effect was absent at concentrations from 10 to 10 M. The dilatation induced by 3alpha-OH-tibolone was not significantly reduced by L-Na. The vasodilatory effect of 3alpha-OH-tibolone did not differ from that of 17beta-estradiol. CONCLUSIONS: 3alpha-OH-tibolone has a dose-dependent vasodilatory effect on isolated skeletal muscle arterioles from the rat. The sulfated form has no vasodilatory effect in this setup. This finding suggests that during this short incubation time there was no conversion of the sulfated metabolite into its active form by the vascular endothelium. The vasodilatory effect of 3alpha-OH-tibolone is endothelium dependent at physiologic concentrations and comparable to that of 17beta-estradiol.

Tibolone and its metabolites enhance tissue factor and PAI-1 expression in human endometrial stromal cells: Evidence of progestogenic effects.

Tibolone is a highly effective postmenopausal hormone treatment that has its biological activity dependent on metabolism to 3alpha- and 3beta-OH tibolone, which bind solely to the estrogen receptor. Despite the high levels of estrogen receptor-binding metabolites in the circulation, the endometrium becomes atrophic, suggesting inactivation of the estrogen response in this tissue which may be due to the progestogenic activity of tibolone and the Delta-4 tibolone metabolite. We evaluated the effects of tibolone and its metabolites on tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) expression in human endometrial stromal cells (HESCs). Since TF and PAI-1 exhibit long-term in vivo and in vitro up-regulation by progestin they serve as endpoints for assessing chronic effects of progestin exposure. Confluent HESCs were primed in serum-containing medium with vehicle control, 10(-8)mol/L estradiol, 10(-7)mol/L medroxyprogesterone acetate, or 10(-8) to 10(-6)mol/L tibolone or its metabolites, then switched to a defined medium with corresponding vehicle or steroids. After 24h, ELISAs indicated that the progestin elevated TF (6.2-fold +/-3.0; p<0.05) and PAI-1 (eight-fold +/-2.1; p<0.05) levels, whereas the cells were refractory to estradiol exposure. Tibolone and Delta-4 tibolone (10(-8) to 10(-6)mol/L) were as effective as 10(-7)mol/L medroxyprogesterone acetate in enhancing TF and PAI-1 output (p<0.05). Unexpectedly, at the higher concentrations 3alpha- and 3beta-OH tibolone also elevated TF and PAI-1 expression (p<0.05). Western blotting confirmed the ELISA results. Our findings suggest that HESCs metabolize 3alpha- and 3beta-OH tibolone to tibolone and subsequently to Delta-4 tibolone, which can both stimulate the progesterone receptor. Since TF and PAI-1 promote hemostasis by complementary mechanisms, our findings account for the reduced occurrence of abnormal uterine bleeding associated with tibolone therapy.

Tibolone and its metabolites induce antimitogenesis in human coronary artery smooth muscle cells: role of estrogen, progesterone, and androgen receptors.

Tibolone, a hormone replacement drug, protects postmenopausal women against osteoporosis and climacteric symptoms without inducing adverse effects on the endometrium and breast. Compared with other estrogens, little is known about the cardiovascular effects of tibolone. Because abnormal growth of smooth muscle cells (SMCs) is a prerequisite for coronary artery disease, here we investigated the effects of tibolone on SMC growth. We examined the effects of tibolone and its metabolites on human arterial SMC growth (DNA synthesis, cellular proliferation, cell migration, collagen synthesis) and MAPK expression. Fetal calf serum-induced SMC growth, phosphorylated MAPK expression, and platelet-derived growth factor-induced SMC-migration were concentration-dependently inhibited by tibolone and its endogenous estrogenic and progestogenic/androgenic metabolites in the following order of potency: Delta 4-tibolone>3 beta-OH-tibolone congruent with 3 alpha-OH-tibolone. The antimitogenic effects of tibolone were partially blocked by ER antagonist (ICI182780), progesterone receptor antagonist (RU486) but not by the androgen receptor antagonist (flutamide); moreover, RU486 was more potent than ICI182780. The antimitogenic effects of tibolone were completely blocked by RU486 plus ICI182780. In addition, the inhibitory effects of equimolar concentrations of the three tibolone metabolites summed up to the inhibitory effects of tibolone. In conclusion, tibolone inhibits SMC growth and MAPK phosphorylation via both its estrogenic and progestogenic metabolites, and these inhibitory effects involve both progesterone and ERs. Hence, tibolone may induce antivasoocclusive actions and protect women against coronary artery disease.

Oestradiol and mirtazapine restore the disturbed tail-temperature of oestrogen-deficient rats.

The purpose of the present study was to further evaluate the tail-temperature test as a tool to test potential steroidal and non-steroidal compounds for the treatment of hot flushes. Ovariectomized rats were implanted with a temperature sensitive probe. After a recovery period of 5 weeks, the effect of oestradiol (given via a silastic tube) and the 5-HT(2) receptor antagonist mirtazapine (10 mg/kg i.p.) on the tail-temperature in the active phase of the animals was measured. Oestradiol completely restored the disturbed tail temperature after 3 days. Treatment with mirtazapine also restored the oestrogen withdrawal-induced disturbed tail-temperature. The effect of mirtazapine was already seen on the first day of treatment. These experiments confirm and extend the idea that measuring the oestradiol withdrawal-induced disturbance of tail-temperature may be a useful tool to select compounds that might have beneficial effects in the treatment of hot flushes. Blockade of the 5-HT(2A) receptors prevented or reduced the ovariectomy-induced disturbance of the rat tail-temperature, which may validate this model to evaluate the effect of compounds on hot flushes.

In vitro studies of tibolone in breast cells.

OBJECTIVE: To investigate the effects of tibolone and its main metabolites on breast homeostasis. DESIGN: In vitro studies in primary cultures of normal breast cells and in breast cancer cell lines. SETTING: Hospital-based academic research center. PATIENT(S): Human breast cells were obtained from women undergoing surgery for hypermastia. Breast cancer cell lines (MCF-7, T47-D, and ZR75-1) were routinely obtained from subcultures. INTERVENTION(S): Cells were incubated with tibolone, its various metabolites, the pure pregnane Org 2058, and the androgen dihydrotestosterone. MAIN OUTCOME MEASURE(S): Proliferation was determined by using a morphometric growth index, apoptosis by using morphologic analysis and flow cytometry, and antiapoptotic proteins bcl-2 and bclx(L) by using Western blot assay. Activity of 17beta-hydroxysteroid dehydrogenase was measured as an epithelial differentiation marker. RESULT(S): Tibolone and its delta(4) isomer were antiproliferative in normal breast cells. Tibolone and its delta(4) isomer increased apoptosis in breast cells. These proapoptotic effects were at least partially mediated through decreased expression of the antiapoptotic proteins bcl-2 and bclx(L). An increase in HSD activity was observed after tibolone administration. CONCLUSION(S): Tibolone is antiproliferative and proapoptotic and induces differentiation in normal breast cells. It is also proapoptotic in breast cancer cell lines.

Tibolone is not converted by human aromatase to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE): analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS.

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.

Receptor profiling and endocrine interactions of tibolone.

The receptor profiles and in vivo activity of tibolone, and its primary metabolites, Delta(4)-isomer, and 3alpha- and 3beta-hydroxytibolone, were studied and compared to those of structurally related compounds. The Delta(4)-isomer was the strongest binder and activator of the progesterone receptor (PR); tibolone was 10 times weaker in binding and half as potent in transactivation of PR; 3alpha- and 3beta-hydroxytibolone did not bind or activate PR. In rabbits oral tibolone produced a minor progestagenic effect in the endometrium, whereas co-administration of tibolone and the anti-estrogen ICI 164,384 unmasked tibolone's progestagenic effect. 3-Hydroxytibolones were the strongest binders and activators of the estrogen receptors (ERs), with greater affinity for ERalpha than for ERbeta. Tibolone showed weaker binding and activation of both ERs and the Delta(4)-isomer has a binding and activation activity of less than 0.1% of E2 for ERalpha or ERbeta. Tamoxifen and 4-hydroxytamoxifen showed partial ERalpha agonistic effects with a maximal response of 12% and raloxifene of 3-5%. Oral administration of 1mg tibolone to ovariectomized rats induced an estrogenic effect on vaginal epithelium. The Delta(4)-isomer was a stronger binder and activator of the androgen receptor (AR) than tibolone; both 3-hydroxytibolones did not bind or activate AR. Introducing a 7alpha-methyl group decreased progestagenic and increased androgenic activity. We conclude that the progestagenic and androgenic activities of tibolone are mediated by the Delta(4)-isomer, and the estrogenic activity, by the 3-hydroxytibolones. The estrogenic activity of the 3-hydroxytibolones masked the progestagenic activity of tibolone in rabbit endometrium. Full estrogenic response was observed in rat vaginal tissue after oral administration of tibolone.

Tissue-selectivity: the mechanism of action of tibolone.

Tibolone is effective in preventing bone loss and treating climacteric symptoms, without stimulating the endometrium. The effects on bone, brain and vagina can be accurately explained by the oestrogenic activity of tibolone, but oestrogenic activity is not expressed in the endometrium. Tibolone behaves differently from oestrogen plus progestogen combinations on the breast. Therefore, tibolone can be characterised as a selective oestrogen activity regulator. The objective of this review is to characterise the typical properties of tibolone in order to explain its tissue-selective action. Tibolone is rapidly converted into three major metabolites: 3 alpha- and 3 beta-hydroxy-tibolone, which have oestrogenic effects, and the Delta(4)-isomer, which has progestogenic and androgenic effects. The 3-hydroxy metabolites are present in the circulation, predominantly in their inactive sulphated form. The tissue-selective effects of tibolone are the result of metabolism, enzyme regulation and receptor activation that vary in different tissues. The bone preserving effects are the result of oestradiol receptor activation, whilst other steroid receptors, notably the progesterone and androgen receptor, are not involved. Breast tissue of monkeys is not stimulated, as occurs with oestrogen plus progestogen, because tibolone and its metabolites inhibit sulphatase and 17 beta-hydroxysteroid dehydrogenase (HSD) type I and stimulate sulphotransferase and 17 beta-HSD type II, the combined effects of which prevent conversion to active oestrogens. In addition, tibolone affects cellular homeostasis in the breast by inhibiting proliferation and stimulating apoptosis. Tibolone does not stimulate the endometrium because of the action of the highly stable progestogenic metabolite (Delta(4)-isomer) in combination with an effect on the sulphatase (inhibition)-sulphotransferase (stimulation) system. The oestrogenic metabolites of tibolone have direct favourable effects on the cardiovascular system and, in in vivo models, tibolone has shown no adverse consequences. In conclusion, tibolone shows oestrogenic effects in brain, vagina and bone and has direct oestrogenic effects on the cardiovascular system. In the endometrium, the progestogenic activity of the Delta(4)-metabolite and the effect on oestrogen-inactivating enzymes prevent oestrogenic stimulation. The mammary gland is not stimulated in currently used animal models. Tibolone appears to regulate estrogenic activity in the various tissues by influencing the availability of estrogenic compounds for the estradiol receptor in a tissue-selective manner.

Estetrol prevents and suppresses mammary tumors induced by DMBA in a rat model.

BACKGROUND: Estetrol (E4) is a pregnancy-specific estrogenic steroid hormone produced by the human fetal liver in both male and female fetuses. During pregnancy, E4 plasma values increase exponentially until parturition and decrease thereafter. The synthesis of E4 in the liver of a newborn ceases during the first weeks after birth. MATERIALS AND METHODS: Here we report the effect of E4 on the initiation and growth of mammary tumors chemically induced by 7,12 dimethylbenz(a)anthracene (DMBA) in female Sprague-Dawley rats in two different protocols. Two prevention studies to test the effect on initiation and growth of induced tumors and one intervention study to test the effect on tumor growth were performed. In the prevention studies, the effect of oral doses of E4 over a dose range of 0.5-3.0 mg/kg was investigated. In the intervention study, oral doses of 1, 3 and 10 mg/kg E4 were used. The anti-estrogen tamoxifen (TAM) and ethinylestradiol (EE) were used as reference compounds. In all studies, a group with ovariectomized animals (OVX) was included. RESULTS: In the prevention studies, 2.5 mg and 3 mg/kg E4 showed a significant effect on the number and growth of induced tumors by DMBA, and the effects were comparable to those of TAM, whereas EE had no effect. In the intervention study, the effect of a high dose of E4 (10 mg/kg) on tumor number was similar to that of OVX and better than TAM and high-dose EE. The 3 mg/kg E4 had an effect comparable to high-dose EE. The treatment effects were largely due to complete regression of existing tumors. CONCLUSIONS: The natural fetal estrogen E4 prevents tumor initiation by DMBA and inhibits the growth of existing DMBA-induced tumors.

Metabolism of the synthetic progestogen norethynodrel by human ketosteroid reductases of the aldo-keto reductase superfamily.

Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17ß-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3ß-hydroxymetabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3ß-hydroxy NOR, while AKR1C3 gave 3ß-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ(4)-isomer of NOR and the most commonly used synthetic progestogen, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3ß-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB.

Hormone replacement therapy dependent changes in breast cancer-related gene expression in breast tissue of healthy postmenopausal women.

Risk assessment of future breast cancer risk through exposure to sex steroids currently relies on clinical scorings such as mammographic density. Knowledge about the gene expression patterns in existing breast cancer tumors may be used to identify risk factors in the breast tissue of women still free of cancer. The differential effects of estradiol, estradiol together with gestagens, or tibolone on breast cancer-related gene expression in normal breast tissue samples taken from postmenopausal women may be used to identify gene expression profiles associated with a higher breast cancer risk. Breast tissue samples were taken from 33 healthy postmenopausal women both before and after a six month treatment with either 2mg micronized estradiol [E2], 2mg micronized estradiol and 1mg norethisterone acetate [E2+NETA], 2.5mg tibolone [T] or [no HRT]. Except for [E2], which was only given to women after hysterectomy, the allocation to each of the three groups was randomized. The expression of 102 mRNAs and 46 microRNAs putatively involved in breast cancer was prospectively determined in the biopsies of 6 women receiving [no HRT], 5 women receiving [E2], 5 women receiving [E2+NETA], and 6 receiving [T]. Using epithelial and endothelial markers genes, non-representative biopsies from 11 women were eliminated. Treatment of postmenopausal women with [E2+NETA] resulted in the highest number of differentially (p<0.05) regulated genes (16.2%) compared to baseline, followed by [E2] (10.1%) and [T] (4.7%). Among genes that were significantly down-regulated by [E2+NETA] ranked estrogen-receptor-1 (ESR1, p=0.019) and androgen receptor (AR, p=0.019), whereas CYP1B1, a gene encoding an estrogen-metabolizing enzyme, was significantly up-regulated (p=0.016). Mammary cells triggered by [E2+NETA] and [E2] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. In this prospective study, prolonged administration of [E2+NETA] and to a lesser extent of [E2] but not [T] were associated in otherwise healthy breast tissue with a change in the expression of genes putatively involved in breast cancer. Our data suggest that normal mammary cells triggered by [E2+NETA] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. This feasibility study provides the basis for whole genome analyses to identify novel markers involved in increased breast cancer risk.

Progestins induce catalase activities in breast cancer cells through PRB isoform: correlation with cell growth inhibition.

Reactive oxygen species (ROS) have been suggested to participate in tumor emergence due to their mitogenic and apoptotic signaling, and as contributors to DNA structural damage. Here we report that progesterone and various synthetic steroids with progestin potencies (norethisterone acetate, MPA, and Tibolone) counteract cell growth induced by hydrogen peroxide (H(2)O(2)), through a potent induction of catalase activities, in breast cancer cells and normal human epithelial breast cells. At physiological concentrations, progesterone and the pure progestin, Org2058, displayed the most potent H(2)O(2) detoxification ability suggesting its effect was characteristic of its progestin potency. We also report on the enhancement of catalase activities by progesterone receptor isoform B (PRB), as determined from experiments using antiprogestins and MDA-MB-231, cells engineered for the selective expression of progesterone receptor isoform A or B. The potent action of progesterone on catalase activities indicates its contribution to a beneficial role in breast cell homeostasis.