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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.)

A number of clinical studies have demonstrated that norethisterone (NET), a potent synthetic progestin, restores postmenopausal bone loss, although its mode of action on bone cells is not fully understood, while the effect of naturally occurring progesterone in bone has remained controversial. A recent report claims that the potent effects of NET on osteoblastic cell proliferation and differentiation, mimicking the action of estrogens, are mediated by non-phenolic NET derivatives. To determine whether osteoblasts possess the enzymes required to bioconvert a progesterone receptor (PR) agonist into A-ring reduced metabolites with affinity to bind estrogen receptor (ER), we studied the in vitro metabolism of [(3)H]-labeled NET in cultured neonatal rat osteoblasts and the interaction of its metabolic conversion products with cytosolic -osteoblast ER, employing a competition analysis. Results indicated that NET was extensively bioconverted (36.4%) to 5 alpha-reduced metabolites, including 5 alpha-dihydro NET, 3 alpha,5 alpha-tetrahydro NET (3 alpha,5 alpha-NET) and 3beta,5 alpha-tetrahydro NET (3beta,5 alpha-NET), demonstrating the activities of 5 alpha-steroid reductase and two enzymes of the aldo-keto reductases family. Expression of Srd5a1 in neonatal osteoblast was well demonstrated, whereas Srd5a2 expression was not detected. The most striking finding was that 3beta,5 alpha-NET and 3 alpha,5 alpha-NET were efficient competitors of [(3)H]-estradiol for osteoblast ER binding sites, exhibiting affinities similar to that of estradiol. The results support the concept that the interplay of 5 alpha-steroid reductase and aldo-keto reductases in osteoblastic cells, acting as an intracrine modulator system is capable to bioconvert a PR agonist into ER agonists, offering an explanation of the molecular mechanisms NET uses to enhance osteoblastic cell activities.

The alkyl esters of p-hydroxybenzoic acid known as parabens (Pbens) are used as preservatives in food, pharmaceutical and cosmetic formulations. They have been reported as estrogenic. Here, we present evidence for the in vivo and in vitro bioactivities and receptor binding affinities of methylparaben (MePben), ethylparaben (EtPben), propylparaben (PrPben), and butylparaben (BuPben) compared with those of estradiol (E2). Estrogenicity was studied using the uterotrophic assay in immature (Im) and adult ovariectomized (Ovx) CD1 mice, and in immature female Wistar rats (IW). Animals were subcutaneously (sc) treated for three consecutive days with different molar equivalent doses ranging from 3.62 to 1086 micromol/kg body weight of Pbens, E2 (0.036 micromol/kg), or vehicle. Pbens increased uterine weight in Im and Ovx animals and their relative uterotrophic effect to E2 (100) (RUEE2) were from 34 to 91. The relative uterotrophic potencies related to E2 (100) (RUPE2) of these compounds were from 0.003 to 0.007. The E2 ED50 for CD1 animals able to increase the uterine weight was 7 microg/kg (0.9-55 confidence limits); and that of Pbens ranged from 18 to 74 mg/kg. In IW rats, the ED50 were from 33 to 338 mg/kg. All Pbens, except MePb, competed with [3H]E2 for the estrogen receptor binding sites. The uterotrophic effects of Pbens in Im mice have a positive correlation with the side-chain length of the ester group of these compounds. The E2 and Pbens relative binding affinities (RBA) and Ki values correlated to their estrogenic activity. The NOELs values for Pbens uterotrophic activity in Im were from 0.6 to 6.5 mg/kg per day; and Ovx from 6 to 55 mg/kg. The NOELs IW ranged from 16.5 to 70 mg/kg indicating that Im were more susceptible than Ovx and IW to these effects. The data shown here confirm the estrogenicity of Pbens.

Levonorgestrel (LNG), a 19-nor-testosterone derivative, is widely used in contraceptive formulations. This compound does not bind to the estrogen receptor (ER), but it shows estrogen-like effects under in vivo and in vitro conditions. The estrogenicity of LNG may be attributed to its bio-transformation into non-phenolic metabolites. In this study, the ability of A-ring reduced LNG metabolites to activate transcription via an estrogenic mechanism of action, including differences between ER alpha and ER beta subtypes, were investigated. Transactivation assays were performed in HeLa cells transfected with expression vectors for ER alpha and ER beta and an estrogen-responsive reporter gene. Cells were also transfected with expression vectors for both progesterone receptor (PR) isoforms (A or B). As expected, the tetrahydro derivatives of LNG (3 alpha,5 alpha- and 3 beta,5 alpha-LNG) showed significantly lower PR-mediated transcriptional activities through both isoforms when compared with progesterone (P(4)) and LNG. In contrast, the 3 beta,5 alpha-tetrahydro derivative resulted in a significant activation of estrogen-dependent gene transcription. This effect was selectively confined to the ER alpha, since little if any activity could be observed with the ER beta and no antagonistic activities were demonstrated. This study provides structural and molecular clues for the well documented in vitro and in vivo intrinsic estrogenicity of 19-nor-testosterone-derived progestins and ligand requirements for ER alpha recognition.

It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ER alpha and ER beta. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5 alpha-dihydro (5 alpha-norethisterone and 5 alpha-Gestodene), or 3 alpha,5 alpha-tetrahydro or 3 beta,5 alpha-tetrahydro derivatives (3 alpha,5 alpha-norethisterone/3 alpha,5 alpha-Gestodene and 3 beta,5 alpha-norethisterone/3beta,5 alpha-Gestodene, respectively) on estrogen-mediated transcriptional regulation, the 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene showed the highest induction when HeLa cells were transiently transfected with an expression vector for ER alpha. This activity could be inhibited with tamoxifen. These compounds did not activate gene transcription via ER beta, and none of them showed antagonistic activities through either ER subtype. The 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene were active in other cells in addition to HeLa cells and activated reporter expression through the oxytocin promoter. In summary, two ER alpha selective agonists have been identified. These compounds, with ER alpha vs. ER beta selective agonist activity, may be useful in evaluating the distinct role of these receptors as well as in providing useful insights into ER action.

Estrogenic activities of the two 17beta-aminoestrogen (AE) derivatives, prolame and butolame, were studied upon coagulation, serum luteinizing hormone (LH) and uterine weight, including endometrial morphology in castrated female rats. We have also investigated the ability of these two compounds, as well as another AE pentolame, to activate transcription through the estrogen receptor alpha (ERalpha) and the estrogen receptor beta (ERbeta). Administration of prolame and butolame to castrated animals increased significantly (P < 0.01) the mean clotting time when compared with that obtained in the group of control animals. Butolame was a more potent anticoagulant than prolame (P < 0.01), as judged by their corresponding IC(50) (5.4 +/- 0.65 and 66.6 +/- 2.57 micro;g/animal, respectively). In contrast, estradiol significantly shortened blood clotting times (P < 0.005). Both prolame and butolame caused a significant inhibition of serum LH levels (EC(50) 8.10 +/- 0.79 and 17 +/- 64 microg/animal, respectively), and restored castration-induced reduction in uterine weight of ovariectomized rats (EC(50) 4.14 +/- 1.57 and 17.0 +/- 1.78 microg/animal, respectively). In terms of the effects of prolame, butolame and pentolame in transient transfection assays, all the three AE activated ER dependent reporter gene expression, however, only at high concentrations. Prolame had the highest activity followed by butolame and pentolame. Induction of transcription by these compounds was preferentially mediated through the ERalpha, especially in the case of pentolame where little, if any, activation occurred through the ERbeta. None of the compounds showed antagonistic activities through either ER subtype. The overall data suggest that modifications in the structure and length of the amino-alcohol side-chain at C-17 might have an impact on the affinity and estrogenic intrinsic properties of AE at the level of diverse target tissues.

Levonorgestrel (LNG) is a synthetic steroid that displays potent progestational and androgenic effects but it lacks estrogen-like activity. To examine the mode of action of this progestin, we studied its metabolism in vitro in target organs and the specific interactions of LNG and its metabolites with putative steroid receptors. The results demonstrated that [3H]LNG was efficiently converted to A-ring reduced derivatives when incubated with rat hypothalamus and pituitary. Under optimal incubation conditions, [3H]5 alpha-dihydro LNG (5 alpha-LNG) and [3H]3 alpha,5 alpha-tetrahydro LNG (3 alpha,5 alpha-LNG) were identified as the major metabolic conversion products, while [3H]3 beta,5 alpha-LNG formation occurred to a lesser extent. A-ring reduction of LNG was NADPH-dependent. Assessment of the relative binding affinities of LNG and its derivatives to progesterone (PR), androgen (AR) and estrogen (ER) receptors by displacement analysis revealed that unchanged LNG binds with high affinity to PR and AR but not to ER. 5 alpha-LNG exhibited a diminished though significant interaction with PR and an enhanced binding affinity for AR as compared with LNG, indicating that 5 alpha-reduction of LNG increases its affinity for AR. The most striking finding was that further reduction of the 5 alpha-LNG molecule at C-3 abolished its binding activity to PR, AR, and even to ER. The overall data provides a plausible explanation for the lack of estrogen agonistic action of LNG and for its potent progestational and androgenic effects.