María Estela Ávila

In vivo and in vitro estrogen bioactivities of alkyl parabens.

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 mmol/kg body weight of Pbens, E2 (0.036 mmol/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 mg/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.

Morphometric analysis of mice uteri treated with the preservatives methyl, ethyl, propyl, and butylparaben

The alkyl esters of p-hydroxybenzoic acid (PHBA) known as parabens (Pbens) are widely used as preservatives in food, pharmaceuticals, and cosmetics. Several in vivo and in vitro studies have shown these compounds to be estrogenic. Here, for the first time, we present evidence of their estrogenicity using a morphometric analysis of uteri from mice treated with the preservatives methylparaben (MePben), ethylparaben (EtPben), propylparaben (PrPben), and butylparaben (BuPben) compared with estradiol (E2). Different groups of adult ovariectomized (Ovx) CD1 mice were subcutaneously (sc) treated daily for three days with two different equimolar doses (362 and 1086 mmol/kg) of the Pbens: MePben (55 and 165 mg/kg), EtPben (60 and 180 mg/kg), PrPben (65 and 195 mg/kg), BuPben (70 and 210 mg/kg), E2 (10 mg/kg; 0.036 mmol/kg), and vehicle (propyleneglycol; V, 10 mL/kg). On the fourth day, uteri were dissected, blotted, weighed, and placed in a fixative solution for 24 h. The paraffin embeded uteri were cut to obtain 7 mm thick transversal sections. Luminal epithelium heights (LEH), glandular epithelium heights (GEH), and myometrium widths (MW) were measured. The highest Pbens dose was able to produce uterotrophic effects (38 to 76%) compared to E2 efects (100%). The relative uterotrophic potency to E2 (100) was from 0.02 to 0.009. Significant increases (P <0.05) in LEH, GEH, and MW as compared with V were obtained: LEH from 87 to 113% (E2 153%), GEH from 10 to 40% (E2 60%), and MW from 35 to 43% (E2 88%). These results confirm that Pbens at the doses assayed here induce estrogenic histological changes in the uteri of Ovx mice.

Proliferative Properties of 17β‐aminoestrogens in MCF‐7 Human Breast Cancer Cells

The 17β‐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 (ERβ). 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 ERβ antagonist PHTPP exacerbated the proliferative response, showing that the AEs’ proliferative activity was mainly ERα‐mediated and apparently controlled by ERβ. 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.

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 17β-oestradiol and 17β-aminoestrogen pentolame on hemostasis in rats

Objectives: This work evaluated chronic treatment with 17β-oestradiol (E2) and 17β-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 17β-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17β-estradiol

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 buames proliferation indicating mediation through estrogen receptors in this response. CONCLUSION Buame is therefore an estrogen partial agonist with a weak estrogenic activity.

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

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.