Intermediary metabolism of estriol in pregnancy

Estriol (E₃), the most abundant estrogen in pregnancy is produced predominantly in the placenta from androgen precursors of fetal origin. The estriol so formed is secreted efficiently into the maternal circulation where it is converted to 4 conjugates—estriol-3-sulfate (E₃-3S), estriol-16-glucosiduronate (E₃-16G), estriol-3-glucosiduronate (E₃-3G) and estriol-3-sulfate-16-glucosiduronate (E₃-SG). The order of renal clearances is E₃-16G > E₃-3G > E₃-3S ~ E₃-SG. Unconjugated E₃ and E₃-3G differ from the other forms of estriol in that their removal from the blood compartment is essentially irreversible. E₃-3S, E₃-16G and E₃-SG undergo interconversions during enterohepatic circulation and eventual partial conversion to E₃-3G. Following delivery of the fetus and placenta, unconjugated E₃ is no longer detectable in the maternal serum within l–2h, whereas the concentrations of the conjugates decline more slowly, the rates being determined by the rates of renal clearance and enterohepatic interconversions. E₃-3G levels were dramatically elevated in a case of Group C polycystic kidney disease, providing evidence that this conjugate is indeed an end-product of estriol metabolism.     

Biological responses and histological studies of estriol and estriol sulfate in fetal and newborn uteri of guinea pig

The biological responses of estriol (E₃) and of estriol-3-sulfate (Ea₃-S) in the fetal and newborn uteri of guinea pig were studied. After a treatment of E₃ (1 mg/kg/day) or E₃-S (1.4 mg/kg/day) to pregnant guinea pigs (49–60 days of gestation) for 6 days, both estrogens provoke a significant uterotrophic effect in the fetal uterus which increases in weight 1.8–2.5 times in relation to the non-treated animals. The stimulation of progesterone receptor (PR) is also very intense, 7–12 times in relation to the control animals.In another series of experiments newborn guinea pigs (2-day old) were treated with 100μg/animal of E₃ or 140 μg/animal of E₃-S for a short (2 days) or a long (12 days) period. Concerning the uterotrophic effect, the weight of the uterus increases 1.8–2.5 times (in relation to the non-treated animals) after a 2-day treatment and the effect continues to increase up to 4–5 times in the 12-day treated animals. In opposition to the fetal uterus, the effect on PR provoked by E₃ or E₃-S is very limited (only an increase of 1 time in relation to the non-treated animals); the effect is even less intense after the 12-day treatment. Histological studies show an intense hypertrophic effect particularly in the epithelium of the endometrium in the fetal and newborn uteri for both E₃ and E₃-S. In the newborns the effect is also intense on the epithelium of the uterine gland.It is concluded that: (1) Estriol and estriol sulfate are very active and with similar intensity on the uterine weight before and after birth; (2) The stimulatory effect on PR decreases very significantly after birth and after a long treatment; (3) E₃S can act as a potent hormonal precursor.     

Estriol and estradiol interactions with the estrogen receptor in vivo and in vitro

The cytosolic estrogen receptor (calf uterus) bound to estradiol (E₂) at 0°C changes from a state with fast into a state with slow E₂ dissociation rates when placed at 28°C. This temperature accelerated transition in receptor affinity for its ligand takes place within 10 min at 28°C. Similarly, receptor bound to estriol (E₃) at 0°C changes, when heated, from a state with fast into a state with slow E₃ dissociation. The main difference between RE₂ and RE₃ was that E₃ dissociates from unheated 8S RE₃ and heat-transformed 5S RE₃ at a much faster rate than E₂ from RE₂;In the mature ovariectomized rat a slow dissociating 5S receptor estrogen complex is found in nuclei 1 h after injection of [³H]E₂ or [³H]E₃. In vitro dissociation of these 2 estrogens from this nuclear bound receptor formed in vivo takes place at rates similar to those from heat-transformed cytosolic RE₂ or RE₃ complexes.Addition of pyridoxal 5'-phosphate (PLP) to the slow-dissociating heat-transformed 5S estrogen receptor complexes causes rapid dissociation of E₂ or E₃; this effect is dose-dependent and is not due to disruption of 5S dimers, since after PLP addition RE₂; and RE₃ sediment unchanged as 5S dimers.The presence of a large excess of non-radioactive 4S RE₃ does not interfere with the temperature induced rapid transition of 4S R[³H]E₂ complexes from the state with fast into a state with slow E₂ dissociation kinetics.A model is presented to explain the temperature induced biphasic estrogen dissociation from the receptor. It is proposed that the low affinity 4S RE₂ monomer undergoes a temperature and estrogen dependent conformation change, such that the ligand is “locked” into the receptor's binding site. This conformational change results in the formation of a high affinity 4S monomer from which estrogen dissociates at a slower rate. This reaction is independent from subsequent 4S to 5S dimerization (transformation). The different rates of ligand dissociation from the low and high affinity 4S receptors reflect the different interactions (hydrophobic and hydrogen bonding) of E₂ and E₃ with the estrogen binding domain.     

Relative distribution of estrone,estradiol and estriol between fetal and maternal perfusates during perfusions of human term placentas with labeled C19 precursors

Published results from in vivo experiments carried out in Rhesus monkeys and from in vitro perfusions of human term placentas have indicated that placental estradiol (E₂) is preferentially released towards the mother whereas estrone (E₁) is about evenly distributed between fetal and maternal circulation.In order to examine the distribution of estriol, relative to that of E₁ and E₂, we have now prepared [³H]16-hydroxyandrostenedione by incubation of [6,7-³H]androstenedione with Streptomyces roseochromogenus and perfused placental cotyledons with mixtures of these two labeled precursors. Measurement of the concentrations of tritiated E₁, E₂ and E₃ in the maternal and fetal perfusates, flowing at approx 10 and 5 ml/min, respectively, indicated that the distribution of E₃ is different from that of E₂ and resembles the distribution of E₁.The simple perfusion system being used shows differences in the distribution of various estrogens between fetal and maternal perfusates which may reflect the in vivo situation and offers the opportunity for experimental examination of various explanations for these differences, e.g. existence of specific carrier systems in the syncytial membranes, specific binding of the estrogens to secreted placental proteins, and actions of placental and decidual 17β-hydroxysteroid dehydrogenases.     

Estriol in pregnancy. III. Development, comparison and use of specific antisera for rapid radioimmunoassay of unconjugated estriol in pregnancy plasma

Estriol-6-(0-carboxymethyl) oxime (E₃-CMO) and estriol-4-azobenzoic acid (E₃-4-ABA) were linked to bovine serum albumin (BSA). Twelve rabbits were immunized, six with each E₃-BSA conjugate. All six E₃-6-CMO-BSA rabbits, but only one E₃-4-ABA-BSA animal, responded with useful antibody titers. All antisera exhibited good Ring D specificity. E₃-6-CMO-BSA (type 1) antisera cross-reacted up to 220 percent with 6-oxoestriol while the E₃-4-ABA-BSA (type 2) antiserum cross-reacted only 3.8 percent with this steroid. Neither type of antiserum cross-reacted with neutral steroids nor with estriol-16-glucosiduronate and estriol-3-sulfate-16-glucosiduronate, but both cross-reacted with estriol-3-sulfate and estriol-3-glucosiduronate.Both types of antisera could be utilized for a rapid and specific radioimmunoassay (RIA) of unconjugated E₃ in third trimester pregnancy plasma without need for further purification of the plasma extract. Blanks were negligible, sensitivity was sufficient, recovery was virtually complete by using ³H-E₃ as an internal standard, and precision was satisfactory. The measurements of unconjugated plasma E₃ concentrations in ninety apparently normal women between 29 and 40 weeks or gestation obtained by this RIA averaged 7.6, 10.2 and 16.7 ng/ml at 29 to 32, 33 to 36 and 37 to 40 weeks of gestation, respectively.The results obtained in this study indicate that antisera against E₃-6-CMO-BSA, despite their appreciable cross-reaction with 6-oxoestriol, are as useful for a rapid RIA of plasma unconjugated E₃ as antisera against E₃-4-ABA-BSA because very little, if any, 6-oxoestriol is present in late pregnancy plasma. As anti-E₃ titers were much higher and much more readily obtained in response to immunization with E₃-6-CMO-BSA than with E₃-4-ABA-BSA, E₃-6-CMO-BSA appears to be the preferable antigen to develop antisera for a rapid, yet specific, E₃ RIA.     

Splanchnic and intestinal uptake and formation of estriol and estriol conjugates in the dog in vivo.

A loading dose of 3H-estriol was given to male dogs followed by a constant infusion. The concentrations of total radioactivity, conjugated estriol metabolites, estriol, estriol-o-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-3-sulfate and estriol-3-sulfate, 16alpha-glucosiduronate were determined in plasma from the femoral artery(A), hepatic vein(HV) and superior mesenteric vein (SMV). From these values the splanchnic (100[1-HV/A]) and intestinal (100[1-SMV/A]) extractions were calculated. The mean splanchnic extraction of total radioactivity was positive (23, SE 3, P less than .01), indicating net uptake by the splanchnic area, possibly due to biliary excretion. The mean splanchnic extraction of estriol was 77, SE 1, P less than .01, also indicating net uptake. The splachnic extractions of estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate and estriol-3-sulfate were negative (-15, SE 3, P less than .01; -23, SE 6, P less than .01; -31, SE 8, P less than .01 respectively) indicating net formation of these conjugates for release into the systemic circulation. The mean intestinal extraction of estriol was 12, SE 4, P less than .01, indicating net uptake by the intestine. This net uptake was associated with mean negative intestinal extractions of estriol-3-glucosiduronate (-15, SE 7, P approximately .05), estriol-3-sulfate (-33, SE 10, P less than .01) and estriol-3-sulfate, 16alpha-glucosiduronate (-53, SE 13, P less than .01), indicating net formation of these conjugates by the intestine.     

Effect of estriol treatment on the menstrual cycle and prolactin secretion

In order to study the biological effects of estriol in women 20 mg estriol was administered daily to 7 young women. Plasma luteinizing hormone (LH), estradiol (E₂), progesterone (Pg) and prolactin (Prl) were measured during a treatment and a control cycle every second or third day. Further 3, 6 or 20 mg estriol was administered in a single dose to 5 women and plasma Prl, unconjugated and conjugated estriol (E₃) measured over 24 h at 2–3 h intervals. In 2 experiments with 20 mg E₃, blood samples were taken more frequently, over 6h.When 20 mg E₂ was administered daily, 2 of the 7 young women had anovulatory cycles. The mean plasma E₂ was lower during the follicular and ovulatory phases (P < 0.025) and mean plasma LH was higher (P < 0.005) during the luteal phase, when E₃ was given. Because of the 2 anovulatory cycles the mean Pg value during the luteal phase was lower (P < 0.05) during treatment. There was a slight decrease in mean Prl in 5 out of 6 women (P < 0.0005), but in only 1 woman was this decrease substantial (from a mean value of 27.6–18.9 ng/ml; P < 0.01). When 6 or 20 mg E₃ was administered orally in the morning a significant negative correlation (P < 0.01) between plasma Prl and unconjugated E₃ was found. The correlation coefficient was highest (r = −0.74) with 6mg E₃. When 3mg was administered no obvious effect on Prl section was seen. However, when results from all experiments with identical time schedules were pooled (two with 3 mg, two with 6 mg and one with 20 mg E₃) and the mean values for plasma Prl calculated and compared with the mean values obtained in 7 control experiments, it was found that E₃ administration in the morning almost abolishes the Prl rise during the following night. There was a statistically significant (P < 0.0125) decrease in the difference between the maximum value during the night and the minimum value during the day. The minimum value was significantly higher (P < 0.01) and the maximum value significantly lower (P < 0.025) after e₃) treatment, compared to the control values.It is concluded that long-term administration of 20 mg e₃ usually has only a slight but significant decreasing effect on mean plasma Prl concentration measured in the morning, before the next dose is taken. However, E₃ has an inhibiting effect on Prl secretion during the night and a stimulating effect during the day, if administered in the morning, resulting in a considerable reduction in the amplitude of the circadian rhythm. Continuous administration of high doses (20 mg/day) of E₃ may result in anovulatory cycles, but has little effect on plasma hormone values in the cycles which remain ovulatory.     

Inability of [3H]estriol to induce maximal cooperativity of the estrogen receptor

The interaction of [³H]estriol with the partially purified estrogen receptor from calf uterus shows positive cooperativity that is dependent upon receptor concentration and temperature. At a receptor concentration of 1 nM and 25°C the [³H]estriol-receptor cooperativity was low, the Hill coefficient (n_H) was 1.03 ± 0.02; however, with increasing receptor concentrations the receptor's cooperativity increased until at approximately intracellular receptor concentration (20 nM) the n_H = 1.20 ± 0.04. At 0°C and a receptor concentration of 10 nM the [³H]estriol-receptor interaction was highly cooperative, the Scatchard plot was convex and n_H = 1.58 ±0.04 while at 30°C the Scatchard plot approached linearity and n_H = 1.03 ± 0.02. In comparison, [³H]estradiol was capable of inducing, at 0 or 30°C and at a receptor concentration of 1 nM or greater, maximal receptor cooperativity, n_{H = 1.63}.These data demonstrate: (a) the receptor's conformation and binding mechanism change in a specific manner with temperature, so that receptor analysis at 0°C does not necessarily reflect the receptor's properties at biologically relevant temperatures; (b) the dependence of the receptor's cooperativity on receptor concentration, which suggests interaction between dissociable subunits; and (c) the lower cooperativity induced by estriol, in comparison with estradiol, which indicates estriol is less efficient in shifting the receptor toward a higher affinity or the activated state of the receptor.     

Estrogenic action of estriol fatty acid esters

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.     

An enzyme-immunoassay for estriol.

A practical method was developed for enzyme-immunoassay of serum estriol, with alkaline-phosphatase as a marker enzyme. Alkaline-phosphatase was conjugated with estriol-6-(O-carboxymethyl) oxime using water soluble carbodiimide. The estriol-alkaline-phosphatase complex, which has both enzyme activity and capacity to bind anti-estriol serum, was obtained by Sephadex G-200 gel filtration. This complex, which was stable for at least 3 months at 4 degrees C, was used as enzyme-labelled estriol. Anti-estriol serum raised against estriol-6-(O-carboxymethyl) oxime bovine serum albumin was employed. "Bound and free" estriol were separated by the double antibody method. A linear relation was obtained between estriol concentration and antibody-bound alkaline-phosphatase activity in the range of 0.2-100 ng estriol/ml. In this assay system, cross-reactivity with other steroids was negligible under physiological conditions, and endogenous alkaline-phosphatase, which increases during the late pregnancy, caused no interference. The coefficients of variation were 3.3-14.2% (within assays), and less than 22% (between assays), and the mean recovery rate was 77.5%. Serum estriol values determined by the present method correlated well with those determined by radioimmunoassay (r=0.90 for total estriol; r=0.98 for free estriol). The present method of enzyme-immunoassay is suitable for measurement of serum estriol during pregnancy.     

Predicting in vivo anti-hepatofibrotic drug efficacy based on in vitro high-content analysis

Background/Aims

Many anti-fibrotic drugs with high in vitro efficacies fail to produce significant effects in vivo. The aim of this work is to use a statistical approach to design a numerical predictor that correlates better with in vivo outcomes.

Methods

High-content analysis (HCA) was performed with 49 drugs on hepatic stellate cells (HSCs) LX-2 stained with 10 fibrotic markers. ∼0.3 billion feature values from all cells in >150,000 images were quantified to reflect the drug effects. A systematic literature search on the in vivo effects of all 49 drugs on hepatofibrotic rats yields 28 papers with histological scores. The in vivo and in vitro datasets were used to compute a single efficacy predictor (E_predict).

Results

We used in vivo data from one context (CCl₄ rats with drug treatments) to optimize the computation of E_predict. This optimized relationship was independently validated using in vivo data from two different contexts (treatment of DMN rats and prevention of CCl₄ induction). A linear in vitro-in vivo correlation was consistently observed in all the three contexts. We used E_predict values to cluster drugs according to efficacy; and found that high-efficacy drugs tended to target proliferation, apoptosis and contractility of HSCs.

Conclusions

The E_predict statistic, based on a prioritized combination of in vitro features, provides a better correlation between in vitro and in vivo drug response than any of the traditional in vitro markers considered.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Effects of oestriol: Preliminary results on receptor kinetics in target tissues of postmenopausal women

This in vivo investigation was done to study the effects of intravaginal oestriol (E₃) administration on endometrial, myometrial and vaginal tissue of normal postmenopausal women.All women received intravaginal E₃-suppositoria (containing 0.5 mg E₃) once a day for 3 weeks prior to hysterectomy. The medication was continued until the day of operation. At the time of operation both uterine and vaginal tisue was obtained.The receptor content in the cytosol was measured by a multiple point-dextran-coated-charcoal assay using [³h]e₂ and [³H]ORG-2058 as ligands. The receptor content in the nucleus was measured by incubating purified whole nuclei in 10 nM [³h]e₂ for 18 h at 0°C. We have shown that under these conditions there is a total exchange of all occupied receptors.Preliminary data on 4 patients are available. Vaginal cytology clearly showed an increase of the maturation value. Oestrogen receptor concentrations in the cytosol of all three tissues studied were lower than those obtained in untreated women, suggesting nuclear transformation of the receptor as a consequence of treatment. The nuclear E₂ receptor levels cannot be compared with normal women yet.Progesterone receptors in endometrial and myometrial cytosol seemed to be higher than those in untreated women, indicating effects of the treatment. In the human, vaginal progesterone receptor cannot be used as a marker for oestrogenic stimulation because only exceptionally could their presence be detected in either treated or untreated women.     

16-sulfates of estriol in body fluids of human pregnancy at term.

A method was developed for the assay of estriol-16-sulfate (E3-16S) and estriol-3, 16-disulfate (E3-3,16-diS) in maternal serum, cord serum and amniotic fluid at delivery in human pregnancy. Tritiated E3-16S and E3-3,16-diS are added to the fluid being analyzed. The conjugates are separated and purified by sequential chromatography on alumina, Celite and Sephadex LH-20. Each conjugate is hydrolyzed with Glusulase and the released estriol is quantified by radioimmmunoassay. E3-3,16-diS was found in each fluid, most concentrated in the cord serum. Small amounts of E3-16S were found in some amniotic fluids, and this conjugate was virtually absent from the sera. These new estriol conjugates comprise less than 1 percent of total, estriol, apparently too low to be of diagnostic value in human pregnancy.     

Effect of estriol, estriol-3-sulfate and estriol-17-sulfate on progesterone and estrogen receptors of MCF-7 human breast cancer cells

The levels of progesterone receptors (PR [cytosol (Cy) and nuclear (N)] and estrogen receptors (ER) [cytosol and nuclear; occupied and unoccupied specific binding sites] were evaluated in the MCF-7 cancer cell line incubated with estriol (E3), estriol-3-sulfate (E3-3-S) or estriol-17-sulfate (E3-17-S) for 7 days in culture. Cells were grown in MEM medium containing 2 mM glutamine, 10% v/v dialysed calf serum and penicillin-streptomycin (100 U/ml) in the absence (control) or in the presence of 5 X 10(-8) M E3, E3-3-S or E3-17-S. The total PR (Cy + N) concentration which was 0.47 +/- 0.10 (SE) pmol/mg DNA in the non-treated cells, increased to 1.95 +/- 0.48 in the E3 and to 1.55 +/- 0.26 in the E3-3-S treated cells. No effect (PR: 0.47 +/- 0.15 pmol/mg DNA) was observed with the E3-17-S treatment. Total ER (Cy + N, occupied + unoccupied binding sites) in pmol/mg DNA +/- SE, were as follows: control 0.79 +/- 0.17; + E3: 0.33 +/- 0.09; +E3-3-S: 0.90 +/- 0.18 and +E3-17-S: 1.82 +/- 0.58. The measurement by radioimmunoassay of unconjugated estriol in the culture medium indicated that after incubation with E3-3-S, a fraction (0.5-1%) of the sulfate was hydrolyzed but no hydrolysis was observed in the incubations with E3-17-S. It is concluded that in the MCF-7 human mammary cancer cell line E3 and E3-3-S stimulate PR very significantly, but it is suggested that E3-3-S acts through the hydrolyzed E3. On the other hand, E3-17-S is inactive because it is not hydrolyzed. Consequently, E3-3-S can play an important role in the biological responses of this mammary cancer cell line.     

Synthesis and evaluation of (17α,20E)21-[125I]Iodo-11-substituted-19-norpregna-1,3,5(10),20-tetraene-3,17β-diols: the influence of 11-stereochemistry on tissue distribution of radioiodinated estrogens

The 21-tri-n-butylstannyl derivatives of (17α,20E)-11α and β-methoxy-19-norpregna-1,3,5(10),20-tetraene-3,17β-diol were synthesized and characterized. These compounds, as well as the 11-unsubstituted compound were converted via electrophilic ipso radioiododestannylation to the corresponding 21[¹²⁵I]iodo analogs at the no-carrier-added level in 73–90% isolated radiochemical yields. The radiochemical 4c [IVαME₂, (17,20E)-21[¹²⁵I]iodo-11α-methoxy-19-norpregna-1,3,5(10) ,20-tetraene-3,17β-diol] was evaluated in immature female rats and the results compared to those previously reported for 4a (IVE₂) and 4b (IVβ ME₂) to determine the influence of 11-substitution on the ability of the compounds to function as estrogen receptor-seeking agents in vivo. The results indicated that the uptake of 11α-methoxy derivative in the target organ was substantially lower, of shorter duration, with a much smaller specific receptor binding component than the other two radioligands. The distribution profile of the three 17α-iodovinyl estrogens paralleled that previously reported for the corresponding 17α-ethynyl estrogens and this study suggests that the in vivo pharmacological results reported for the 17α-ethynyl estrogens may be used to predict the in vivo behavior of the corresponding 17α-iodovinyl analogs.     

Determination of estriol, estriol sulfate, progesterone and neutral steroid mono- and disulfates in umbilical cord blood plasma

Fractions of unconjugated steroids, and steroid mono- and disulfates were isolated from cord plasma, and the concentrations of estriol, estriol sulfate, progesterone, 13 neutral steroid monosulfates (MoS) and 10 neutral steroid disulfates (DiS) were determined by gas-liquid chromatography. The mean concentrations in 30 cord plasma samples at term after normal pregnancy and delivery were as follows (μg/100 ml of free steroid ±standard deviation): estriol 16±5; estriol monosulfate 135±43; progesterone 59±19; dehydroepiandrosterone MoS 76±23; 5-androstene-3_β,17_α-diol DiS 279±77; 5-androstene-3_β,17_β-diol DiS 211±109; 16_α-hydroxydehydroepiandrosterone MoS 305±97; 16_β-hydroxy-dehydroepiandrosterone DiS 8±25; 33,17_β-dihydroxy-5-androsten-16-one MoS 37±16, DiS 29±15 5-androstene-3_β,16_α,17_β-triol MoS 25±9; 5-androstene-3_β,16_β,17_α-triol DiS 31±14; pregnenolone MoS 4±33; 5-pregnene-3_β,20_α-diol MoS 41±14, DiS 68±43; 16_α-hydroxypregnenolone MoS 101±42; 17-hydroxypregnenolone MoS 56±30; 21-hydroxypregnenolone DiS 26±15; 5-pregnene-3_β,20_α,21-triol MoS 37±18; 5_α-pregnane-3_α,20_α-diol MoS 21±10, DiS 54±21; 5_α-pregnane-3_β,20_α-diol MoS 18±9, DiS 7±39; 5_β-pregnane-3_α,20_α-diol MoS 17±7; 5_α-pregnane-3_α,20_α,21-triol MoS 110±56, DiS 22±19.The total amount of steroid monosulfates in the cord plasma pool was 1 mg/100 ml and that of steroid disulfates 0.5 mg/100 ml. 3_β-Hydroxy-Δ⁵-steroids predominated. Considerable amounts of saturated c₂₁ steroids were also detected. No statistically significant differences were found in the concentrations of any of the steroids studied, when a group of male and female fetuses were compared.     

The estriol-induced inhibition of the estrogen receptor's positive cooperativity

The effect of estriol on the positive cooperativity of [³H]estradiol binding to the partially purified calf uterine estrogen receptor was investigated using the kinetic analysis of Sasson and Notides (J. biol. Chem. 257 1982, 11540). The receptor was titrated with variable concentrations of [³H]estradiol with or without estriol; the estriol was maintained in a constant molar ratio to the [³H]estradiol concentration. A 4-fold molar excess of estriol above the [³H]estradiol concentrations inhibited the receptor's cooperative [³H]estradiol binding. In the absence of estriol, the [³H]estradiol receptor interaction was highly cooperative, the Scatchard plot was convex and the Hill coefficient was 1.61 ± 0.02a. In the presence of sufficient estriol to reduce the maximally bound [³H]estradiol to 77%, the Scatchard plot was linear and the Hill coefficient was 1.04 ± 0.04. The inhibition of the cooperative [³H]estradiol binding by estriol was not due to isotope dilution of the specifically bound [³H]estradiol by the unlabeled estriol.These data demonstrate that the cooperative binding of ³H]estradiol by the receptor that is characteristic of the equilibrium between the two states of the receptor (active and nonactive) is eliminated by the presence of estriol. This finding is consistent with the agonist/antagonist activity of estriol observed in vivo.     

Sulphamoylated estradiol analogue induces antiproliferative activity and apoptosis in breast cell lines

Research into potential anticancer agents has shown that 2-methoxyestradiol exerts antiproliferative activity in vitro and in vivo in an estrogen receptor-independent manner. Due to its limited biological accessibility and rapid metabolic degradation, several new analogues have been developed in recent years. This study investigated the in vitro effects of a novel in silicodesigned compound (C16) in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7), an estrogen receptor-negative breast adenocarcinoma epithelial cell line (MDA-MB-231) and a nontumorigenic breast cell line (MCF-12A). Light microscopy revealed decreased cell density, cells blocked in metaphase and the presence of apoptotic characteristics in all three cell lines after exposure to C16 for 24 h. Polarizationoptical transmitted light differential interference contrast revealed the presence of several rounded cells and decreased cell density. The xCELLigence real-time label-independent approach revealed that C16 exerted antiproliferative activity. Significant inhibition of cell growth was demonstrated after 24 h of exposure to 0.2 ??M C16 in all three cell lines. However, the non-tumorigenic MCF-12A cell line recovered extremely well after 48 h when compared to the tumorigenic cell lines. This indicates that C16 acts as an antiproliferative agent, possesses antimitotic activity and induces apoptosis in vitro. These features warrant further investigation.