17ß-estradiol induces hyperresponsiveness in guinea pig airway smooth muscle by inhibiting the plasma membrane Ca2+-ATPase.

High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17ß-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17ß-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.

Cardiovascular disrupting effects of bisphenols, phthalates, and parabens related to endothelial dysfunction: Review of toxicological and pharmacological mechanisms.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. CVDs are promoted by the accumulation of lipids and immune cells in the endothelial space resulting in endothelial dysfunction. Endothelial cells are important components of the vascular endothelium, that regulate the vascular flow. The imbalance in the production of vasoactive substances results in the loss of vascular homeostasis, leading the endothelial dysfunction. Thus, endothelial dysfunction plays an essential role in the development of atherosclerosis and can be triggered by different cardiovascular risk factors. On the other hand, the 17ß-estradiol (E2) hormone has been related to the regulation of vascular tone through different mechanisms. Several compounds can elicit estrogenic actions similar to those of E2. For these reasons, they have been called endocrine-disrupting compounds (EDCs). This review aims to provide up-to-date information about how different EDCs affect endothelial function and their mechanistic roles in the context of CVDs.

The Interplay of GPER1 with 17ß-Aminoestrogens in the Regulation of the Proliferation of Cervical and Breast Cancer Cells: A Pharmacological Approach.

The G-protein-coupled receptor for estrogen (GPER1) is a transmembrane receptor involved in the progression and development of various neoplasms whose ligand is estradiol (E2). 17ß-aminoestrogens (17ß-AEs) compounds, analogs to E2, are possible candidates for use in hormone replacement therapy (HRT), but our knowledge of their pharmacological profile is limited. Thus, we explored the molecular recognition of GPER1 with different synthetic 17ß-AEs: prolame, butolame, and pentolame. We compared the structure and ligand recognition sites previously reported for a specific agonist (G1), antagonists (G15 and G36), and the natural ligand (E2). Then, the biological effects of 17ß-AEs were analyzed through cell viability and cell-cycle assays in two types of female cancer. In addition, the effect of 17ß-AEs on the phosphorylation of the oncoprotein c-fos was evaluated, because this molecule is modulated by GPER1. Molecular docking analysis showed that 17ß-AEs interacted with GPER1, suggesting that prolame joins GPER1 in a hydrophobic cavity, similarly to G1, G15, and E2. Prolame induced cell proliferation in breast (MCF-7) and cervical cancer (SIHA) cells; meanwhile, butolame and pentolame did not affect cell proliferation. Neither 17ß-AEs nor E2 changed the activation of c-fos in MCF-7 cells. Meanwhile, in SIHA cells, E2 and 17ß-AEs reduced c-fos phosphorylation. Thus, our data suggest that butolame and pentolame, but not prolame, could be used for HRT without presenting a potential risk of inducing breast- or cervical-cancer-cell proliferation. The novelty of this work lies in its study of compound analogs to E2 that may represent important therapeutic strategies for women in menopause, with non-significant effects on the cell viability of cancer cells. The research focused on the interactions of GPER1, a molecule recently associated with promoting and maintaining various neoplasms.

Effects of post-ovariectomy time frame and age on the antidepressant-like actions of estradiol and prolame in female rats.

Estrogen replacement therapy (ERT) is an effective treatment for symptoms associated with climacteric and depression some women experience during perimenopause and menopause. The antidepressant-like effects of ERT may depend on the type of estrogen, age, and time when restitution is initiated after hormonal decline. Prolame is a synthetic steroid with estrogenic and antidepressant-like effects that may produce fewer adverse effects. We hypothesize that such actions of prolame on females depend on age and the duration of hormone deprivation period. We assessed the antidepressant-like effects of 17ß-estradiol (E2) and prolame in young and middle-aged rats across different post-ovariectomy (Ovx) time frames. Independent groups of young adults and middle-aged female rats were tested in the forced swimming test (FST) at 3, 8, 16, and 24 weeks post-Ovx. Prolame and E2 were administered in a sub-chronic schedule consisting of three injections before the FST. Likewise, the utero-trophic effects of these hormones were analyzed. We found that E2 and prolame reduced immobility in young rats 3 and 8 weeks after Ovx; in contrast, only prolame produced this effect in middle-aged rats three weeks post-Ovx. E2 and prolame increased the animals' utero-somatic index at all post-Ovx times, but the action of E2 and prolame produced a greater response in young adult rats. Our findings showed that the antidepressant-like effects of E2 and prolame depend on the post-Ovx time frame, age, and estrogen type. Interestingly, our results indicate that, in contrast to E2, prolame maintained its antidepressant effect in middle-aged rats.

Epicutaneous Administration of 17ß-Estradiol Induces Langerhans Cells Depletion.

BACKGROUND: Langerhans cells (LC) number and function in mouse vaginal mucosa are affected by 17ß-estradiol (E2) application; nonetheless, its effect on epidermal LC has not been studied. The purpose of this study was to evaluate the effect of topical administration of E2 on the number, phenotype, and migratory ability of LC in mouse skin. METHODS: Ears of adult CD1 male mice were topically treated once with several doses. Immunohistochemical staining for CD207 and TUNEL staining were performed. LC migration to lymph nodes and the effect on the expression of costimulatory molecules on cultured dendritic cells (DC) were also evaluated. RESULTS: E2 decreased the number of CD207+ LC in a dose-dependent manner. One hour after treatment, 1 and 10 µg/mL E2 significantly reduced the LC number by 21% and 26%, respectively, after two hours, the reduction was 23% and 41%, respectively. After 48 hours, LC recovered, and after 96 hours of treatment, the CD207+/MHCII+ DC numbers were increased in regional lymph nodes. However, CD86 and CD40 molecules were expressed at lower levels than in positive control. The TUNEL assay did not show apoptotic cells. Furthermore, in cultured DC, E2 promoted a decrease in CD40 and CD86 expression and an increase in CD273, CD274, MHCII, and CCR7. CONCLUSIONS: The topical administration of E2 induced a transitory local diminution of LC population and a tolerogenic phenotype. This decrease in epidermal LC suggests that E2 may affect skin immune responses, inducing an inhibitory response, which should be considered when prescribing topical E2 medications.

Determining Factors in the Therapeutic Success of Checkpoint Immunotherapies against PD-L1 in Breast Cancer: A Focus on Epithelial-Mesenchymal Transition Activation.

Breast cancer is the most common neoplasm diagnosed in women around the world. Checkpoint inhibitors, targeting the programmed death receptor-1 or ligand-1 (PD-1/PD-L1) axis, have dramatically changed the outcome of cancer treatment. These therapies have been recently considered as alternatives for treatment of breast cancers, in particular those with the triple-negative phenotype (TNBC). A further understanding of the regulatory mechanisms of PD-L1 expression is required to increase the benefit of PD-L1/PD-1 checkpoint immunotherapy in breast cancer patients. In this review, we will compile the most recent studies evaluating PD-1/PD-L1 checkpoint inhibitors in breast cancer. We review factors that determine the therapeutic success of PD-1/PD-L1 immunotherapies in this pathology. In particular, we focus on pathways that interconnect the epithelial-mesenchymal transition (EMT) with regulation of PD-L1 expression. We also discuss the relationship between cellular metabolic pathways and PD-L1 expression that are involved in the promotion of resistance in TNBC.

Potential Estrogenic Properties of 17ß-Hydroxy-ethylimine Estradiol Derivative Targeted to Menopause Stage.

BACKGROUND/AIM: Hormone replacement therapy during menopause increases the risk of thromboembolic diseases and cancer, so safety alternative therapeutic strategies are needed. 17ß-Aminoestrogens are a synthetic estrogens group that possess mild anticoagulant activity that contrasts with the pro-coagulant effects showed by estradiol's (E2) in rodents. Being considered an alternative to conventional hormone replacement therapy during menopause without thrombogenic risks producing. The present study aimed to determine the estrogenic profile and anxiolytic activity of 17ß-[hydroxy-ethylimine]-1,3,5(10)-estratrien-3-ol (IE2), a related compound unknown until now. METHODS: IE2 was assessed in immature rats by uterotrophic assay administering IE2, E2, or vehicle. In ovariectomized adult Wistar rats (Ovx) to facilitating the lordotic behavior compared with E2, estradiol benzoate, or vehicle. The effect of IE2 on anxiety was estimated in Ovx animals treated with IE2, E2, or vehicle group and evaluated in the elevated plus-maze model. RESULTS AND CONCLUSION: IE2 produced an uterotrophic effect, lordotic behavior, and anxiolytic effect in a dose-dependent manner, similar to E2. IE2 depicted estrogenicity, indicating potential clinical use as hormone replacement therapy during menopause.

Proliferative Properties of 17ß-aminoestrogens in MCF-7 Human Breast Cancer Cells.

The 17ß-aminoestrogens (AEs) prolame, butolame and pentolame are weakly oestrogenic in rodents and display anticoagulant properties in contrast to oestradiol (E2 ) which presents pro-coagulant effects, potentially thrombogenic. They possess anti-anxiety and antidepressive properties, being good candidates for menopausal hormone therapy (MHT). Their capability to induce proliferation of MCF-7 human breast cancer cells, in which proliferative rate depends on oestrogens, has not been explored; thus, the aim of this work was to characterize it. AEs' proliferation properties were evaluated compared with E2 in MCF-7 carcinoma cell line cultures using established methods. Receptor mediation on cell proliferation was studied by co-incubating them with the oestrogen receptor antagonists tamoxifen, ICI 182,780 and the selective antagonists MPP (ERα) and PHTPP (ERß). E2 and AEs increased MCF-7 cell proliferation; their proliferative effect was between 1.5-2 and E2 = 3.6 compared with controls (0); their relative proliferative effect was 18-38% (E2 = 100%) with a relative proliferative potency of 4.5-8.9 (E2 = 100). The ERα antagonist MPP inhibited the MCF-7 cell proliferation induced by E2 and AEs; on the contrary, the ERß antagonist PHTPP exacerbated the proliferative response, showing that the AEs' proliferative activity was mainly ERα-mediated and apparently controlled by ERß. Preliminary cytometric DNA flow analysis showed that AEs' cell cycle S phase inducer property was lower than E2 following the proliferative order: E2 > butolame > prolame > pentolame, indicating pentolame with the weakest proliferative properties and being the safest of this series as a candidate for MHT.

17ß-Aminoestrogens induce guinea pig airway smooth muscle hyperresponsiveness through L-type Ca(2+) channels activation.

Therapy with estrogens is frequently used in menopausal women and as hormonal contraception. Because of its thrombotic effects, long term estrogen administration used in hormonal replacement therapy (HRT) and contraception could represent a health hazard. In this regard, 17ß-aminoestrogens such as aminoestrol, butolame and pentolame have shown promising HRT potential, because they have a weak agonist estrogenic action and antithrombotic activity. Additionally, estrogens play a protective role in airway smooth muscle, but the effect of 17ß-aminoestrogens on the airway smooth muscle has not been tested yet. In guinea pig tracheal smooth muscle pentolame and butolame induced hyperresponsiveness to histamine (His), carbachol (Cch) and KCl. Interestingly, aminoestrol did not show this effect at the highest concentration studied, it even lowered the contraction induced by Cch. The hyperresponsiveness induced by pentolame to His was abolished by nifedipine. In single tracheal myocytes, KCl induced an increment in the intracellular Ca(2+) concentration [Ca(2+)]i, pentolame also showed an increase in [Ca(2+)]i and the addition of KCl in the plateau of this rise further significantly augmented the [Ca(2+)]i response. Additionally, in patch clamp experiments pentolame increased the L-type Ca(2+) currents. Thus, 17ß-aminoestrogens such as pentolame and butolame, but not aminoestrol, activate L-type Ca(2+) channel to induced hyperresponsiveness to Cch, His and KCl in guinea pig tracheal smooth muscle. Due to its lack of effect on airways and to its anticoagulant characteristics, aminoestrol seems to be the best alternative in the HRT among the 17ß-aminoestrogens studied.

In vivo and in vitro estrogenic profile of 17ß-amino-1,3,5(10)estratrien-3-ol.

17ß-amino-1,3,5(10)estratrien-3-ol (17ßAE2), is the 17ß-aminoestrogens prototype possessing anticoagulant activity, contrasting with the procoagulant effects of 17ß-estradiol (17ßE2). 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, 17ßAE2 and 17αAE2 were compared with 17ßE2 in HeLa cells co-transfected with expression vectors for hERα or hERß subtypes and for an estrogen-responsive reporter gene. Immature female CD1 mice and Wistar rats (21 days old) were treated for three days with 17ßAE2 (10-5000 µg/kg), 17ßE2 (0.001-1000 µg/kg) or vehicle (propylenglycol 10 ml/kg) and uterine weights were estimated. 17ßAE2 increased uterine weight in a dose-dependent manner. The effective dose (ED)50 uterine weight values: 17ßAE2=552 and 764 µg/kg (17ßE2=4.8 and 16 µg/kg) and their relative uterotrophic potency were 0.86 and 2.1 (17ßE2=100) in mice and rats, respectively. 17ßAE2 competed with [(3)H]E2 for the estrogen receptor. The 17ßAE2 relative binding affinities (RBAs) were: 0.074; Ki=2.2×10(-6)M (17ßE2=100; Ki=1.6×10(-9)M); 0.029 and Ki=3.8×10(-6)M (17ßE2=100; Ki=1.1×10(-9)M) for mice and rats uteri respectively. 17ßAE2 activated hERα-mediated ß-galactosidase transcription activity in the yeast system co-transfected with hERα gene. 17ßAE2 effective concentration (EC)50=1.82 µM (17ßE2=2.14 nM) with a relative potency of 0.12 (17ßE2=100). These transactivation effects were abolished by the antagonist fulvestrant (ICI 182,780), similarly to 17ßE2. 17ßAE2 and 17αAE2 bind with low relative affinity to hERα and hERß. Both induced hER-mediated reporter gene transactivation in a dose-response manner. The overall results provide evidence that 17ßAE2 has a weak agonist estrogenic action greatly mediated through the hERß and to a lesser extent the hERα at genomic level.

Ovariectomy differential influence on some hemostatic markers of mice and rats.

Rodent ovariectomy is an experimental method to eliminate the main source of sexual steroids. This work explored for the first time the ovariectomy temporal changes induced in the hemostatic coagulation markers: prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration (FIB) along with uterine weight on adult female CD1 mice and Wistar rats. Uterine weight (Uw) was assessed before ovariectomy (control), and 1, 3, 5, 7, 9, 16, and 21 days after surgery. PT, aPTT, TT and FIB were estimated the same days, using reported standard techniques. Ovariectomy decreased Uw, since day 1; and from day 10 to 21 reached the lowest values for both species. After day 1, mice hemostatic parameters changed (PT +10%, P<0.05; aPTT +53%, P<0.05; TT -24%, P<0.05; FIB +67%, P<0.05). Rats showed significant changes only in TT and FIB (TT -13%, P<0.001; FIB +65%, P<0.001). Neither mice PT, aPTT and TT, recovered control values after 21 days. In the rats from day 5 to 16 aPTT diminished (18-23%, P<0.05) recovering to control values on day 21, TT after 9 days and PT on day 16. In both species, FIB returned to its control values after 9 days. Ovariectomy differentially altered the PT hemostatic parameter of mice and rats indicating a non-equivalence among both species behaviour for experimental studies of blood coagulation.

In vivo profile of the anticoagulant effect of 17ß-amino-1,3,5(10)estratrien-3-ol.

The anticoagulant activity of 17ß-amino-1,3,5(10)estratrien-3-ol (AE(2)) was established for the first time. Experiment 1: mice groups were treated with a single subcutaneous (s.c.) AE(2) injection (0.5, 1, 2, 4, and 8 mg/100 g BW) or vehicle (propylenglycol; 0.5 ml/100 g). After 24 h, AE(2) produced dose-dependent blood clotting time increases related to control, Emax=+121% (P<0.01) finishing the sixth day. Experiment 2: four groups received a single s.c. administration of AE(2) (4 or 8 mg/100g BW) or 17ß-estradiol (E(2); 3mg/100g BW) or vehicle. After 24 and 48 h post-administration, the times of blood clotting, prothrombin, thrombin, and activated partial thromboplastin and fibrinogen concentrations were assessed. Both AE(2) doses increased blood clotting and fibrinogen similarly, blood clotting time: 64, 94%; fibrinogen: 71, 107% (P<0.01). Prothrombin, activated partial thromboplastin and thrombin times, increased 13-15%, 27-55%, and 15-29%, respectively (P<0.01). Meanwhile E(2) decreased blood clotting 20% (P<0.01) and thrombin 23% (P<0.01) after 48 h. Experiment 3: for five consecutive days, mice received AE(2) or E(2) (0.1, 1, 10, 100, and 1000 µg/kg/day), or vehicle. Blood clotting time was assessed at 1, 2, 3, 4, 5, 8, and 11 days after treatment. AE(2) at all doses were anticoagulant for 2-3 days after administration whereas E(2) was procoagulant for 8-11 days. These opposite effects were: AE(2) Emax=+29%; E(2) Emax=-30%; (P<0.01). AE(2) is the parent compound of the 17ß-aminoestrogens, with the largest and longest anticoagulant effect until now reported.

Gender differences in response to chronic treatment with 17ß-oestradiol and 17ß-aminoestrogen pentolame on hemostasis in rats.

OBJECTIVES: This work evaluated chronic treatment with 17ß-oestradiol (E2) and 17ß-aminoestrogen pentolame (AEP) on prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration (FIB). Male (M) and ovariectomized (Ovx) Wistar rats were used to explore gender differences in the pharmacological response. MATERIALS AND METHODS: Rats (n=12-18) were treated every third day during three months with E2 (1, 10, 100 µg/kg), AEP (1, 10, 100, 500 µg/kg) or vehicle (propylenglycol 1 ml/ kg). PT, aPTT, TT, and FIB were measured using standardized techniques. RESULTS: Chronic treatment with E2 in male rats increased PT (4-7%; P<0.05), decreased aPTT (9%; 100 µg/kg; P<0.05) and decreased TT (5% at 100 µg/Kg; P<0.05). Chronic treatment with E2 in ovariectomized female rats decreased PT (3-4%; P<0.05), did not induce significant changes on aPTT and decreased TT in a dose dependent manner (12-27%; P<0.05). Chronic treatment with AEP in male rats did not alter PT, increased aPTT in a dose dependent manner (5-16%; P<0.05), and decreased TT (5%; 500 µg/Kg; P<0.05) while in female ovariectomized rats it decreased PT (5-9%; P<0.05), increased aPTT (8-13%; P<0.05) and decreased TT (6-13%; P<0.05). E2 and AEP decreased FIB in M and Ovx animals. Decreases in FIB by E2 were more pronounced in male (15-18% P<0.05) than in ovariectomized rats (10-14% P<0.05). E2 showed more potency than AEP, lowering FIB at 1 and 10 µg/kg doses. Both estrogens decreased FIB in ovariectomized animals (E2, 10-14%, P<0.05; AEP, 9% P<0.05) and were reverted by increasing dosage. CONCLUSIONS: Gender influenced response to chronic treatment with E2 and AEP on hemostatic parameters. PT and aPTT were the most affected parameters, demonstrating non-equivalence in the pharmacological response of M and Ovx rats.

In vivo and in vitro evaluation of the estrogenic properties of the 17ß-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17ß-estradiol.

BACKGROUND: Buame [17ß-(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), 17ß-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.

Differential effects of esculetin and daphnetin on in vitro cell proliferation and in vivo estrogenicity.

Esculetin (6,7-dihydroxycoumarin) and daphnetin (7,8-dihydroxycoumarin) are secondary metabolites of plants used in folk medicine. These compounds have showed great antiproliferative activity in several tumor cell lines and have been proposed as potential anticancer agents. However, the estrogenic potential of these two compounds has to date not been reported. The present study compared esculetin and daphnetin on the inhibition of cell proliferation and cell cycle progression of the MCF-7 estrogen-responsive human carcinoma cell line. In vivo and in vitro estrogenic activity for both compounds was also evaluated. Esculetin inhibited cell proliferation after 72 h exposure (IC50=193 ± 6.6 µM), while daphnetin evidenced inhibiting effects starting at 24-h exposure (72 h, IC50=73 ± 4.1 µM). Both effects showed changes in cyclin D1 gene expression. In non-estrogenic conditions (E-screening assay), esculetin produced biphasic response on proliferation of the MCF-7 cells; at 10(-8)-10(-6)M, concentrations induced proliferative effects as EC50=4.07 × 10(-9)M (E(2)=2.91 × 10(-12)M); at higher concentrations (10(-5)-10(-4)M), cell proliferation was inhibited. Relative proliferative effect at E(2) was 52% (E(2)=100), relative proliferative potency was 0.072 (E(2)=100). Additionally, esculetin tested in vivo showed estrogenic effects at 50-100mg/kg doses; relative uterotrophic effect at E(2) was 37%, with relative uterotrophic potency registered at 0.003. In contrast, daphnetin did not induce estrogenic effects in vitro or with in vivo models. The low estrogenic activity of esculetin could prove useful in postmenopausal therapy but not as a safe antitumor agent in estrogen-dependent tumors. Daphnetin-based antiproliferative selectivity with MCF-7 cells showed that daphnetin is a promising antitumoral agent also acting on estrogen dependent tumors.

Induction of sexual behavior in female rats by the 17beta-aminoestrogens prolame, butolame and pentolame.

17beta-aminoestrogens (AEs) decrease luteinizing hormone levels, increase uterine weight, activate transcription through ERalpha and ERbeta receptors and induce progesterone receptor expression in the anterior pituitary. This work evaluates the influence of single and multiple administration of a homologous series of the AEs: prolame, butolame and pentolame on rat female sexual behavior to explore their capability of inducing lordosis by themselves. In a reversed cycle the animals received one or three subcutaneous (s.c.) injections at 8:00 hr of: 7.5 microg E2/day (approximately 30 microg/kg), or 10 microg BE/day (approximately 40 microg/kg), or 1 mg/day prolame, butolame, pentolame (approximately 4000 microg/kg), or 300 microL/day of corn oil (approximately 1.2 ml/kg). Twenty-four hr following treatment, progesterone (P; 1 mg/0.1 ml of corn oil/rat) was administered; and 5 to 7 hr later, rats were tested for sexual receptivity and the lordosis quotient (LQ) was estimated (number of lordosis displays/number of mounts x 100). Single administration by themselves did not facilitate lordosis, 24 hr after the second injection, AEs-LQs values were: 24, 30, 21, E2 = 13 and EB = 11. administrations of three AEs increased LQs to: 48, 50, 45 E2 = 33 and EB = 57. The sequential P administration facilitated lordosis; after one injection: LQs 50-90 (p<0.01), meanwhile E2 and BE inducing LQs of 43 and 81 respectively. After the second and third injections and P administration the AEs LQs were 92-100 (p<0.01) similarly to E2 (95; p<0.01) and BE (96; p<0.01). The facilitation of sexual behavior by AEs was time and dose-dependent.

Effect of the 17beta-aminoestrogen pentolame on bone mineral levels in ovariectomized rats.

Estrogens are fundamental to maintaining bone mineral balance. 17beta-aminoestrogens produce low estrogenic effects through ERalpha and ERbeta receptors, however their effects on bone tissue are unknown. This work evaluates the effects of the 17beta-aminoestrogen pentolame (AEP) and estradiol (E2) on the mineral profile of rat femur. Six months after ovariectomy (Ovx) adult Wistar rats (200-250g) were treated every third day for 30 days with subcutaneous (s.c.) injections of E2 (1, 10, 100 microg/kg), AEP (1, 10, 100, 500 microg/kg) or vehicle (propylenglycol; 1 ml/kg). After treatment, femur samples were prepared and Ca, P, Mg, Si, Fe, S, Na, K, and Cl concentration profiles were estimated using an X-ray analysis system coupled to an scanning electron microscope. Ovariectomy significantly decreased Ca, P, Mg and Si and increased Fe and S. Treatment with E2 restored Ca, P, Mg, and Si to the control values and decreased Fe and S in a dose dependent manner. AEP restored the levels of Ca, P, Mg and Si at all doses administered. AEP increased the levels of Fe and restored S to the basal level. The other minerals showed great variability and no significant differences were detected. Our results indicate differential action of AEP related to E2 in the restitution of bone mineral content.

Sub-acute effects of the 17beta-aminoestrogen pentolame and estradiol on hemostasis in the rat.

This work describes the sub-acute treatment of male Wistar rats with estradiol (E2) and the 17beta-aminoestrogen pentolame (AEP) on the hemostatic screening tests: blood clotting time (BCT), prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and fibrinogen concentrations (FIB). Animals received five consecutive subcutaneous (s.c.) doses of E2 (1 and 10 mg/kg) and AEP (5, 10 and 20 mg/kg) and the course of blood clotting times (BTC) was followed 24, 48, 72 and 96 hr after finishing treatment. Five injections of equivalent doses (0.1, 1, 10, 100 or 1000 microg/kg) of both steroids were performed. In all experiments a control group (propylenglycol 1 ml/kg) was included. Individual blood samples were obtained (under anesthesia) from the iliac artery and PT, aPTT, TT, and FIB were determined according to previous reported techniques. Forty-eight hr after treatment, E2 decreased BCT (10%) while AEP increased BCT 87% in a dose dependent manner. Only E2 significantly decreased FIB concentration (p<0.5; 25%) while AEP induced a dose dependent increase in FIB from 18 to 88%, (p < 0.05). AEP produced biphasic responses on aPTT (+24 % and -15%; p<0.05) and decreased TT from 11 to 14%, (p<0.01). AEP aPTT vs. FIB showed a r2=-0.99, (p < 0.05). Pentolame showed disparate effects compared with E2 on the blood clotting parameters and exerts its most important effects on the common coagulation pathway.

Gender and inter-species influence on coagulation tests of rats and mice.

INTRODUCTION: Rats and mice have been used to evaluate effects of natural and synthetic oestrogens. However, data about oestrogen's effects on haemostasis in rodents is very limited. The aim of this work was to standardize blood coagulation screening tests in adult male, female, and ovariectomized (Ovx) Wistar rats and CD1 mice in an effort to evaluate the influence of gender and species differences on haemostasis. MATERIALS AND METHODS: Values were obtained for the following haemostatic parameters: prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin clotting time (TT), and fibrinogen (FIB), through modifications of the conventional techniques used for human blood coagulation analysis. RESULTS: Both rats and mice showed gender intra-species and inter-species differences of high significance in PT, aPTT, TT, and FIB values. Intra-species differences were found in TT (+10% p<0.01) and FIB concentration (-21% p<0.001) between male and Ovx rats. Male vs. Ovx mice showed a TT difference of -20% (p<0.001). The main inter-species differences found were PT values of male rats vs. male mice (-39%) and female rats vs. female mice (-35%, both p<0.001). Female rats and mice aPTT values vs. those corresponding to Ovx animals showed differences of +15% and +32% (p<0.001), respectively. CONCLUSIONS: These data reveal the great importance of gender intra- and inter-species differences on the values of haemostatic screening tests, which should be taken into consideration when evaluating the effects of oestrogens and other drugs on the coagulation system.

Estrogen counteracts ozone-induced oxidative stress and nigral neuronal death.

Oxidative stress is implicated in the premature death of dopamine neurons in substantia nigra in Parkinson's disease. The incidence of Parkinson's disease is higher in men than in women, and estrogen may provide neuroprotection against oxidative damage. We examined the protective effects of estrogen on rat nigral death after chronic ozone inhalation. Ozone inhalation produced impaired nigral cell morphology and loss of dopamine neurons in ovariectomized rats. This was counteracted after 60 days of 17beta-estradiol treatment, when blood levels were highest. These results indicate that ozone exposure may be a useful Parkinson's disease model and neuroprotection afforded by 17beta-estradiol is dependent on the high levels achieved after its prolonged administration.