Cristina Lemini

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.

Anticoagulant and estrogenic effects of two new 17β-aminoestrogens, butolame [17β-(4-hydroxy-1-butylamino)-1, 3,5(10)-estratrien-3-ol] and pentolame [17β-(5-hydroxy-1-pentylamino)-1, 3,5(10)-estratrien-3-ol]

Abstract The syntheses and characterizations of two new 17β-aminoestrogens, butolame [17β-(4-hydroxy-1-butylamino)-1, 3,5(10)-estratrien-3-ol] and pentolame [17β-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol], are presented. Both compounds, when administered in single subcutaneous injections to male mice and rats, produce dose-dependent increases in blood clotting times that may last several days. The estrogenic effects assessed by the vaginal cornification test are of relatively short duration.

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.

Synthesis and molecular structure of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17β-yl)-3-hydroxypropylamine; an amino-estrogen with prolonged anticoagulant and brief estrogenic effects

The synthesis and molecular structure of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-3-hydroxypropylamine, is described. It was characterized by ir, nmr, mass spectrometry and chemical analysis. The crystal structure of this compound was determined by single-crystal x-ray diffraction. Prolame belongs to space group P212121. Cell dimensions are: a = 8.356(2), b = 13.343(4) and c = 16.119(4) A. Z = 4; R = 4.1%.

In vivo estrogen bioactivities and in vitro estrogen receptor binding and transcriptional activities of anticoagulant synthetic 17β-aminoestrogens

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.

Estrogenic effects of the synthetic aminoestrogen 17β-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol (pentolame)

In this study, we investigated the effects of pentolame, a 17 beta-aminoestrogen derivative, upon coagulation, serum LH, pituitary progestin receptors, uterine weight, and endometrium morphological changes in the castrated female rat. Groups of animals were subcutaneously (s.c.) injected with either estradiol (E2) (0.1 up to 1000 micrograms/animal), pentolame (1 up to 1000 micrograms/animal), or the vehicle alone daily for 5 consecutive days starting 2 weeks following ovariectomy. Administration of pentolame (10 to 1000 micrograms/animal) increased significantly (p < 0.05) the blood clotting time when compared with that obtained in the group of control animals (EC50 582 micrograms). Pentolame (500 and 1000 micrograms/rat for 5 days) caused a significant inhibition (p < 0.01) of serum LH levels (IC50 860 micrograms), which remained suppressed until Day 5 post last injection. In addition, treatment with pentolame was able to restore in the castrated female rat the presence of specific estrogen-dependent progestin binding sites at the anterior pituitary level. The affinity constants and the number of binding sites of pentolame-induced progestin receptors were similar to those obtained with estradiol at equipotent doses (860 micrograms vs. 1 microgram/animal, respectively). Administration of the 17 beta-aminoestrogen derivative resulted in a significant increase in uterine weight (EC50 420 micrograms) and endometrial characteristics were indistinguishable from those observed in the group of rats treated with E2.

Synthesis of 2H-1-Benzopyrans from Aryllithium and a,b-Unsaturated Aldehydes

The synthesis of 2,2-dialkyl-2H-1-benzopyrans (23), (25), (27), (30), (31) and (29) was performed employing the 1,2-addition reaction of aryllithium salts to cyclocitral, 3-methyl-2-butenal, citral and farne- sal respectively. Subsequent deprotection with a Mexican bentonitic earth (Tonsil) in wet acetone was followed by ring closure reaction as the key step. Interesting intermediate products were isolated in the formation of benzopyrans (30) and (31)

The anticoagulant effect of prolame, N-(3-hydroxy-1,3,5(10) estratrien-17β-yl)-3-hydroxypropylamine, a novel amino-estrogen

The anticoagulant and estrogenic effects of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-3-hydroxypropylamine, are described. A single subcutaneous injection of prolame in male mice, ovariectomized mice, adult and infant male rats, produced dose-dependent increases of blood clotting time, which could be observed with the larger doses even after 4 days. In ovariectomized mice, prolame produced vaginal cornifications of shorter duration than those produced by estradiol-17 beta. The evidence suggests that, in contrast with currently used estrogens, prolame would not generate cardiovascular accidents if used for the treatment of prostatic carcinoma; it could also be exceptionally effective for the prevention of thrombosis.

A comparative structural study of the steroid epimers: 17β-amino-1,3,5(10)- estratrien-3-ol, 17α-amino-1,3,5(10)-estratrien-3-ol, and some derivatives by 1H NMR, and x-ray diffraction analysis

The epimers 17 beta-amino-1,3,5(10)-estratrien-3-ol and 17 alpha-amino-1,3,5(10)-estratrien-3-ol were synthesized. 17 beta-Amino-1,3,5(10)-estratrien-3-ol was prepared by catalytic hydrogenation of the estrone-oxime. The 17 alpha-amino epimer was obtained from estradiol, via tosylate, followed by nucleophilic displacement by sodium azide and subsequent reduction with LiAlH4. They were characterized by spectroscopic methods. Determination of the crystal structures of 3-(toluene-4-sulfonyloxy)-17 alpha-1,3,5(10)-estratriene, 3-(toluene-4-sulfonyloxy)-17-alpha-amino,1,3,5(10)-estratriene hydrochloride, 17 beta-acetylamino-1,3,5(10)-estratriene-3-ol, and 3-acetoxy-17 beta-acetylamino-1,3,5(10)-estratriene enabled us to characterize the structure of the 17 alpha and 17 beta amino epimers for the first time.