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17β-amino-1,3,5(10)estratrien-3-ol (17βAE2), is the 17β-aminoestrogens prototype possessing anticoagulant activity, contrasting with the procoagulant effects of 17β-estradiol (17βE2). Its estrogenicity profile has not been reported, and it was evaluated by uterotrophic assay, estrogen receptor binding affinity and its ability to induce gene transcription of the human estrogen receptor (hER)α mediated in a Saccharomyces cerevisiae yeast expression system. Additionally, 17βAE2 and 17αAE2 were compared with 17βE2 in HeLa cells co-transfected with expression vectors for hERα or hERβ subtypes and for an estrogen-responsive reporter gene. Immature female CD1 mice and Wistar rats (21 days old) were treated for three days with 17βAE2 (10-5000 μg/kg), 17βE2 (0.001-1000 μg/kg) or vehicle (propylenglycol 10 ml/kg) and uterine weights were estimated. 17βAE2 increased uterine weight in a dose-dependent manner. The effective dose (ED)50 uterine weight values: 17βAE2=552 and 764 μg/kg (17βE2=4.8 and 16 μg/kg) and their relative uterotrophic potency were 0.86 and 2.1 (17βE2=100) in mice and rats, respectively. 17βAE2 competed with [(3)H]E2 for the estrogen receptor. The 17βAE2 relative binding affinities (RBAs) were: 0.074; Ki=2.2×10(-6)M (17βE2=100; Ki=1.6×10(-9)M); 0.029 and Ki=3.8×10(-6)M (17βE2=100; Ki=1.1×10(-9)M) for mice and rats uteri respectively. 17βAE2 activated hERα-mediated β-galactosidase transcription activity in the yeast system co-transfected with hERα gene. 17βAE2 effective concentration (EC)50=1.82 μM (17βE2=2.14 nM) with a relative potency of 0.12 (17βE2=100). These transactivation effects were abolished by the antagonist fulvestrant (ICI 182,780), similarly to 17βE2. 17βAE2 and 17αAE2 bind with low relative affinity to hERα and hERβ. Both induced hER-mediated reporter gene transactivation in a dose-response manner. The overall results provide evidence that 17βAE2 has a weak agonist estrogenic action greatly mediated through the hERβ and to a lesser extent the hERα at genomic level., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Testosterone (T) restores bone mass loss in postmenopausal women and osteoporotic men mainly through its bioconversion to estradiol (E2). In target tissues, T is also biotransformed to the A-ring-reduced metabolites 3α,5α-androstanediol (3α,5α-diol) and 3β,5α-androstanediol (3β,5α-diol), which are potent estrogen receptor (ER) agonists; however, their biological role in bone has not been completely elucidated. To assess if osteoblasts bioconvert T to 3α,5α-diol and to 3β,5α-diol, we studied in cultured neonatal rat osteoblasts the metabolism of [14C]-labeled T. In addition, the intrinsic estrogenic potency of diols on cell proliferation and differentiation in neonatal calvarial rat osteoblasts was also investigated. Osteoblast function was assessed by determining cell DNA, cell-associated osteocalcin, and calcium content, as well as alkaline phosphatase activity and Alp1 gene expression. The results demonstrated that diols were the major bioconversion products of T, with dihydrotestosterone being an obligatory intermediary, thus demonstrating in the rat osteoblasts the activities of 5α-steroid reductase and 3α- and 3β-hydroxysteroid dehydrogenases. The most important finding was that 3β,5α- and 3α,5α-diols induced osteoblast proliferation and differentiation, mimicking the effect of E2. The observation that osteoblast differentiation induced by diols was abolished by the presence of the antiestrogen ICI 182,780, but not by the antiandrogen 2-hydroxyflutamide, suggests that diols effects are mediated through an ER mechanism. The osteoblast capability to bioconvert T into diols with intrinsic estrogen-like potency offers new insights to understand the mechanism of action of T on bone cells and provides new avenues for hormone replacement therapy to maintain bone mass density.

Background: Buame [17β-(butylamino)-1,3,5(10)-estratrien-3-ol] possesses anticoagulant and antiplatelet activities that are potentially antithrombotic. Since its estrogenicity is unknown, it was evaluated by established methods., Methods: Buame (10, 100, 500, and 1,000 μg/kg), 17β-estradiol (E(2)) (100 μg/kg), or propylene glycol (10 ml/kg) were subcutaneously (sc) administered for three days to immature Wistar female rats (21 days old). The relative uterotrophic effect to E(2) was 78 (E(2) = 100) with a relative uterotrophic potency of 1.48 (E(2) = 100). Adult ovariectomized Wistar rats received an sc injection at 8:00 h (reversed cycle) of: 7.5 μg of E(2) (≈ 30 μg/kg), buame (≈ 750, 1,500, 3,000 μg/kg), or corn oil (≈ 1.2 ml/kg). After 24 h, progesterone (4-5 mg/kg) was administered. Sexual receptivity was assessed 5 to 7 h later, and the lordosis quotient (LQ; number lordosis/number mounts x 100) was evaluated., Results: Buame induced lordosis (LQmax 85 ± 9; ED50 952 ± 19 μg/kg) and E(2) LQmax 56 ± 8; ED50 10 ± 2 μg/kg; the relative LQ-potency was 0.51 (E(2) = 100). Buame competed with [(3)H]E(2) for the estrogen receptor (Buame RBA= 0.15 and Ki = 5.9 x 10(-7) M; E(2) RBA= 100;Ki = 6.6 x 10(-9) M). Buame increased MCF-7 cells proliferation, from 10(-11) to 10(-)9 M, its proliferative effect was 1.73-1.79 (E(2) = 3.0-3.9); its relative proliferative effect to E(2) was 33-40% (E(2) = 100%) and relative potency 10.4-10.7 (E(2) = 100). Tamoxifen and fulvestrant (ICI 182,780) inhibited buame's proliferation indicating mediation through estrogen receptors in this response., Conclusion: Buame is therefore an estrogen partial agonist with a weak estrogenic activity.

Breast cancer is a sex steroid hormone-dependent malignant neoplasia. The role of oestradiol in this malignancy has been well documented; however, the involvement of androgens has remained controversial. To determine the role of non-phenolic androgen metabolites in human breast cancer, we studied the metabolism of [(14)C] testosterone and [(14)C] androstenedione in oestrogen-dependent MCF-7 cells and non-oestrogen-dependent MDA-MB 231 cells, at different substrate concentrations (1-10 muM) and time periods (30 min-48 h). Cultured non-oestrogen-dependent HeLa and yeast cells served as controls. Metabolites were identified and quantified by reverse isotope dilution. A distinctive pattern of androgen metabolism was identified in MCF-7 cells, being the 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-diol) and its 3beta epimer (3beta,5alpha-diol), the major conversion products of testosterone (48.3%), with 5alpha-dihydrotestosterone as intermediary. The formation of 3alpha,5alpha-diol and 3beta,5alpha-diol (diols) was substrate concentration- and time-dependent, and abolished by finasteride. In contrast, very little of any diol formation was observed in MDA-MB 231, HeLa and yeast cell incubations. Additional enzyme gene expression studies revealed an overexpression of 5alpha-steroid reductase type-1 in MCF-7 cells, as compared with MDA-MB 231 cells. The oestrogen-like activities of diols were assessed in HeLa cells co-transfected with expression vectors for alpha or beta subtypes of the human oestrogen receptor (hER) genes and for an oestrogen-responsive reporter gene. The results show that 3beta, 5alpha-diol and to a lesser extent 3alpha,5alpha-diol bind with high relative affinity to hERalpha and hERbeta. Both diols induced hER-mediated reporter gene transactivation in a dose-response manner, similar to that induced by oestradiol, though with lower potency, an effect that was abolished by ICI-182 780. Furthermore, 3beta,5alpha-diol and to lesser extent 3alpha,5alpha-diol induced MCF-7 cell proliferation. The overall results demonstrated that MCF-7 cells exhibit enhanced expression and activity of androgen-metabolising enzymes, leading to rapid and large diol formation, and provide evidence that these androgen metabolites exert a potent oestrogen-agonistic effect, at genomic level, in oestrogen-dependent breast cancer cells. The data suggest that diols may act as in situ intracrine factors in breast cancer and that its formation can be pharmacologically inhibited.

The binding of estradiol (E(2)) to estrogen receptors (ER) is followed by conformational changes resulting in coactivator or corepressor recruitment that influences gene transcription. A series of synthetic A-ring reduced 19-nortestosterone-derived progestins has the capacity to selectively bind and activate transcription through the ERalpha. Herein, the molecular mechanisms involved in ER subtype-selective interactions of these compounds as assessed by their effects upon both ERalpha and ERbeta structural conformation and their ability to induce recruitment of steroid receptor coactivator-1 (SRC-1) to ERalpha were investigated. The results demonstrated that all synthetic A-ring 3beta,5alpha-tetrahydro-reduced derivatives of 19-nortestosterone induced an ERalpha trypsin digestion pattern similar to that seen with E(2), without effects upon ERbeta. In addition, these compounds had the ability to recruit SRC-1 to the ligand-binding domain of ERalpha similar to E(2). Our data indicate that A-ring 3beta,5alpha-tetrahydro-reduced 19-nortestosterone-derived progestins behave as selective ERalpha agonists with ligand-receptor structural and functional responses similar to those induced with natural E(2).

Levonorgestrel (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-gonen-3-one), a potent contraceptive progestin stimulates growth and proliferation of cultured breast cancer cells through a receptor-mediated mechanism, even though levonorgestrel does not bind to the oestrogen receptor (ER). To assess whether the oestrogen-like effects induced by this synthetic progestin are exerted via its metabolic conversion products, we studied the binding affinity of three A-ring levonorgestrel derivatives to the ER and their capability to transactivate an oestrogen-dependent yeast system co-transfected with the human ER gene and oestrogen responsive elements fused to a beta-galactosidase reporter vector. The results demonstrated that the 3beta,5alpha reduced levonorgestrel derivative and to a lesser extent its 3alpha isomer interact with the oestrogen receptor, with a significantly lower relative binding affinity (2.4% and 0.4%, respectively) than that of oestradiol (100%), while levonorgestrel does not. Both levonorgestrel metabolites were able to activate, in a dose-dependent manner, the beta-galactosidase reporter gene in the yeast expression system, an effect that was precluded by a steroidal antioestrogen. The oestrogenic potency of levonorgestrel metabolites was significantly lower (750-fold) than that of oestradiol. Furthermore, high doses of 3beta,5alpha levonorgestrel (2.5 mg/day/6 days) induced an increase of oestrogen-dependent progestin receptor in the anterior pituitary of castrated rats. The overall data offer a plausible explanation for the weak oestrogenic effects induced by high, non-pharmacological doses of levonorgestrel.

The estrogen-like effects of norethisterone (NET) seem to be mediated by the interaction of 3 beta 5 alpha- and 3 alpha 5 alpha-tetrahydronorethisterone (3 beta 5 alpha- and 3 alpha 5 alpha-NET, respectively) with the estrogen receptor. Considering that the in vitro uterine contractile response to serotonin (5-HT) is specifically dependent on estrogen, the aim of the present study was to investigate whether NET and its A-ring reduced metabolites administered in vivo to ovariectomized rats induce uterine sensitivity to 5-HT in vitro. The administration of 3 beta NET in vivo, which is the NET metabolite with the highest affinity for the estrogen receptor, induced a maximal contractile response to serotonin similar to that of 17 beta-estradiol treatment. The other metabolites induced less uterine activity. According to the effective dose 50, the order of estrogenic potency was 17 beta-estradiol > 3 beta 5 alpha NET > 3 alpha 5 alpha NET > NET > 5 alpha NET. The estrogenic effect of 3 beta 5 alpha- and 3 alpha 5 alpha NET may be exerted through their interaction with the estrogen receptor, whereas NET and 5 alpha NET, which do not bind to the estrogen receptor and display a minor estrogenic activity, require prior bioconversion to 3 beta 5 alpha NET and perhaps to 3 alpha 5 alpha NET. The A-ring reduced metabolites of NET, mainly the 3 beta 5 alpha NET, may be exerting estrogenic responses and modulating uterine activity when administered in vivo.