Diaryl naphthyl methanes a novel class of anti-implantation agents

Diaryl naphthyl methanes and the corresponding 1, 2, 3, 4- and 5, 6, 7, 8-tetrahydro naphthyl methane derivatives have been synthesized as novel estrogen receptor binding ligands. The secondary and tertiary amino alkoxy derivatives of diaryl naphthyl and tetrahydro naphthyl methane interact with the estrogen receptor to elicit promising estrogenic, antiestrogenic and implantation inhibition activities in rats. The most active compounds in this series are 7, 9 and 20, cent percent active in preventing implantation in rats at 2.5 mgkg(-1) dose.     

Quantitative appreciation of steroidogenic gene expression in mouse tissues: new roles for type 2 5alpha-reductase, 20alpha-hydroxysteroid dehydrogenase and estrogen sulfotransferase

We have recently developed an improved method for the RealTime PCR quantification of reversed transcribed mRNA (Q_RTPCR) that allows to obtain absolute mRNA expression levels with high sensitivity and accuracy. Using this Q_RTPCR method to assess the mRNA expression levels of genes encoding steroidogenic enzymes in male and female mouse tissues allows us to gain quantitative appreciation of the function of these genes. We could thus identify the existence of two types of steroidogenic tissues: those of classical endocrine glands such as the testis, ovary and adrenals which deliver steroids into the circulation, and in which millions of copies/mug total RNA are detected, and those of peripheral intracrine tissues where steroids are synthesized locally and exert their action at the site where they are produced (prostate, uterus, etc.), and in which the expression level of steroidogenic enzymes is much lower. We also observed an abnormally high expression levels of type 2 5alpha-reductase and 20alpha-HSD in the male and female adrenals, respectively, thus indirectly suggesting new roles for these sex-specific enzymes. On the other hand estrogen sulfotransferase, the enzyme that inactivates estrogen, has been found selectively expressed in male tissues, thus suggesting a role for this enzyme to protect male-specific tissues against estrogenic activity.     

Effects of a normal, human-concentration, phytoestrogen diet on rat uterine growth.

The estrogenic action of the prototype natural phytoestrogen coumestrol was examined in rats in in vitro and in vivo tests. To establish the binding specificity of coumestrol and its relation to biological activities, saturation analyses and uterine weight assays were performed. These assays indicated that coumestrol competitively inhibited binding to the estrogen receptor and induced increases in uterine weight in keeping with its estrogen receptor affinity constant. Most importantly, coumestrol was uterotrophic when incorporated in a semipurified diet at natural dietary concentrations. Significant increases occurred in both uterine wet weight and dry weight, indicating that coumestrol produces true uterine growth. Effects appeared to be cumulative, raising questions of time-related interactions with other estrogen-sensitive mechanisms and clearance of isoflavonoids. Coumestrol induced uterine growth over a 90-hour period at dietary concentrations of 0.01 to 0.1%. Lower doses not active over this period were active when provided over a longer period of time: a 0.005% concentration was not active over a 90-hour period, but was active when provided over a 180-hour period. Coumestrol-induced uterine growth was accompanied by the induction of cytosolic progestin receptors and increases in nuclear estrogen binding. Scatchard analyses verified that these changes were due to changes in receptor number. These studies show that the naturally occurring phytoestrogens have dramatic estrogenic effects at natural dietary levels. These actions may be expressed via traditional receptor-mediated actions and therefore may have the same implications for development, health, and disease as do the steroidal estrogens produced by the body. Because rats have no sex hormone-binding globulin, further studies must be conducted in humans. However, these findings suggest that the natural dietary phytoestrogen coumestrol is a potent estrogen that must be considered in calculating the total estrogenic load to which humans are exposed during normal life.     

Effects of the chemically synthesized flavanone 7-(O-prenyl)naringenin-4'-acetate on the estrogen signaling pathway in vivo and in vitro

The flavanone naringenin is known to possess only weak estrogenic properties, but some of its derivatives such as 8-prenylnaringenin are potent phytoestrogens. The aim of this study was to further clarify structure-function relationships of flavanones regarding their estrogenic or antiestrogenic properties by characterizing the new chemically synthesized naringenin derivative 7-(O-prenyl)naringenin-4'-acetate (7-O-PN). A yeast based reporter gene assay and MVLN cells, a MCF-7-derived cell line that possesses a luciferase reporter gene under the control of a vitellogenin estrogen responsive element, were used to investigate estrogenic actions of 7-O-PN in vitro. Estradiol (E2) has been used as a positive control. Subsequently a 3-day rat uterotrophic assay was performed to test for estrogenic effects. In addition, mRNA expression of estrogen sensitive genes in the uteri of these rats was measured using real time rtPCR. While E2 leads to a strong dose dependent signal in the yeast based reporter gene assay and in MVLN cells, 7-O-PN shows mild E2 antagonistic properties at concentrations 10(-8) and 10(-7)M, E2 agonistic properties at 10(-6) and 10(-5)M in MVLN cells and no effects on the yeast based system. In contrast to E2 treatment, 7-O-PN treatment did not increase uterus wet weight compared to the negative control. These findings are supported by mRNA expression studies of proliferation markers. Additionally, mRNA expression studies of estrogen regulated genes revealed very strong antiestrogenic properties of 7-O-PN regarding regulation of complement C3 expression while some estrogenic effects could be observed on the expression of estrogen receptor beta, clusterin and possibly on progesterone receptor and vascular endothelial growth factor.     

Effect of estrogens on glucose phosphate dehydrogenase activity of liver and oviduct in the amphibian Bufo arenarum.

Glucose- 6phosphate dehydrogenase (G-6PDH) stimulation by estradiol- 17beta has been studied in oviduct and liver of Bufa arenarum. OviducalG-6PDH has been found to be stimulated by a single dose of estradiol- 17beta (100 mug/100 g body weight), the stimulation being dependent on season. Hepatic G-6PDH of females is susceptible to hormonal stimulation, without seasonal variation, while in males the enzymatic activity is not modified under the same conditions. The stimulating effect of estrogen on oviducal and hepatic G- 6PDH was inhibited by Actynomicin D. The susceptibility of G- 6PDH to estrogenic action would assure NADPH production, indispensable for the biosynthesis of lipids which are required for cell growth and for hepatic vitellogenesis.     

Ovarian and adrenal influence on the ontogenesis of uterine cytosol and nuclear receptors of estrogens

Adrenal and ovarian relative influence over rat uterus estrogenic receptor, both cytosolic (RcE) and nuclear (RnE) ontogenesis, has been studied. Ovarian influence has been studied by practising bilateral ovariectomy the first day of life and examining estrogenic receptor content evolution from, birth to puberty in these ovariectomized animals (OVX1) by comparison with the normal ones. This influence seems to be of scarce importance until the 20th day of life, since estrogenic receptors content is practically coincident in the OVX1 animals and in the control ones. From the 20th day, ovarian secretion influence increases and estrogenic receptor evolution starts evolving in a different way in the two types of animals. Adrenal influence has been studied by practising bilateral adrenalectomy on the 10th or 30th day of life to OVX1 animals or else OVX and ADX on the 10th or 30th day. Adrenal influence in the upkeep of high estrogen receptor levels on the 10th day, seems to be important, since in the absence of these glands it decreases in a considerable way. The situation is different on the 30th day. At this age ovarian secretion seems to be the most important in maintaining estrogen receptor levels, while adrenal secretion effects tend to inhibit them, especially RnE.     

Synthesis and biological properties of 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl- and 16alpha-[125I]iodo-estradiols

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl-(CIVE) and 16alpha-[125I]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17alpha,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17alpha-ethynyl group of the 7alpha-cyano-17alpha-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [125I]-CIVE isomers were prepared via the same stereospecific reaction. [125I]-CIE was prepared from 7alpha-cyano-16beta-bromoestradiol via halogen exchange with Na125I. Addition of the 7alpha-cyano group to 16alpha-iodoestradiol did not affect estrogen receptor binding affinity (RBA of CIE is 115). However the estrogenic potential of CIE, as measured by the capacity to stimulate the expression of the pS2 gene, was reduced to 1% as compared to that of estradiol. Addition of a 7alpha-cyano group to the (17alpha,20E/Z)-IVE isomers reduced the RBA to 21 and 36, respectively, while the estrogenic potential was reduced to 2-3% of that of estradiol. Uterus uptake in immature rats of the 125I-labeled CIVE 20E-isomer and the 16alpha-iodo CIE peaked at 0.5h post injection while the (17alpha,20Z)-CIVE isomer showed a maximum only past 5h post injection. Uptake of all three 125I-labeled 7alpha-cyanoestrogens was suppressed by the co-injection of non-radioactive estradiol confirming the role of estrogen receptors in the localization process. Uterus retention pattern differ substantially from those of the analogues 7alpha-methylestrogens, which were previously shown to give high maximum 125I-uptake values at 2h post injection. Overall our data indicate that addition of a 7alpha-cyano group to 123I-labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.     

Aromatic acids derived from phenylalanine in the tissues of rats with experimentally induced phenylketonuria-like characteristics

1. Aromatic acids were extracted from brain and liver of rats with phenylketonuria-like characteristics produced by administration of phenylalanine, either alone or in combination with p-chlorophenylalanine. The metabolism of the aromatic acids in these tissues was measured by gas chromatography. 2. At 1h after an intraperitoneal injection of l-phenylalanine (1g/kg) in 23-day-old rats, the phenyl-lactate concentration was 2.2mug/g in the liver and 0.43mug/g in the brain, and the concentration of o-hydroxyphenylacetate was 0.26mug/g in the liver. 3. Phenylacetate concentrations in brain and liver were 0.26 and 0.14mug/g respectively. 4. Suckling rats produced phenyl-lactate less rapidly than weanling rats, but accumulated higher concentrations in longer-term experiments. 5. Intraperitoneal injections of phenyl-lactic acid showed that this compound could directly penetrate the blood-brain barrier, and could produce similar brain/liver ratios of phenyllactate to those found after phenylalanine injection. 6. Qualitative and quantitative similarities in urinary excretion of aromatic acids between the rats used in this study and human patients with uncontrolled phenylketonuria indicate that a patient with a circulating phenylalanine concentration of the order of those achieved in the experimental animal may have aromatic acid concentrations in brain and liver comparable with those found in the rats used in the present study.     

Estrogenic activity of an environmental pollutant, 2-nitrofluorene,after metabolic activation by rat liver microsomes

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.     

Effect of 17alpha-ethinylestradiol on biliary excretion of bile acids.

The effect of 17alpha-ethinylestradiol on biliary bile acids has been investigated. The ratio of cholate to chenodeoxycholate was diminished by the estrogen in cholestyramine-treated rats. With low doses, this effect was due to increased excretion of chenodeoxycholate. With the highest dose, the decreased ratio was due to a reduction in the levels of cholic acid. In the intermediate dosage range, both factors contributed to the decreased ratio. Prolonged treatment with 500 mug daily of 17alpha-ethinylestradiol produced a reduction in the excretory rate of both bile acids in animals treated or not treated with cholestyramine.     

Dietary soy isoflavone-aglycone lowers food intake in female rats with and without ovariectomy

Objective: Estrogens downregulate eating behavior, and soy isoflavones are known to be estrogenic agents. We aimed to examine whether the estrogenic property of soy isoflavones can affect food intake and body weight. Methods and Procedures: Seven‐week‐old male, female, and ovariectomized (OVX) Sprague‐Dawley rats were given free access to a diet containing 100–300 mg total isoflavone/kg diet, or to a control diet, either with or without concurrent administration of estradiol by subcutaneous implantation. Results: Dietary soy isoflavone was shown to lower food intake in female rats, whether or not the animals had undergone ovariectomy. Administration of estradiol lowered the food intake in male rats and in OVX female rats. The decrease in weekly food intake in female rats led to a reduction in their weekly gain in body weight. Dietary soy isoflavone significantly increased the concentration of serum isoflavones, especially equol (a metabolite of daidzein), regardless of gender or ovariectomy. Dietary soy isoflavone did not affect either serum estradiol concentration or uterine and didymus weights, but estradiol administration improved the uterine atrophy in OVX rats, and decreased the didymus weight in male rats. Discussion: Soy isoflavone lowers the food intake in female rats, but not in the male animals. Contrary to the hypothesis currently in vogue, the reduction in food intake caused by soy isoflavone may not be a purely estrogenic effect. This follows from the finding that the effects of soy isoflavones on food intake and on the reproductive organs differ from the corresponding effects produced by estrogen.     

Estrogenic effects of the ethyl-acetate extract of the stem bark of Erythrina lysistemon Hutch (Fabaceae)

The stem bark of Erythrina lysistemon, one of the traditionally used "women remedies", has been assessed for its estrogenic activity. The ethyl-acetate extract of the stem bark of E. lysistemon showed estrogenic activities in vitro either in a yeast-based estrogen receptor assay or on the estrogen-dependent stimulation of alkaline phosphatase activity in the human endometrial carcinoma cell line Ishikawa. The estrogenic activity was investigated in vivo in young ovariectomized Wistar female rats after a 7-day treatment. The estrogenicity was evaluated through the proliferative status of target sex organs such as uterus and vagina. The results obtained showed that oral administration of 200 mg/kg BW/d of E. lysistemon extract in comparison to untreated ovariectomized rats significantly increased the vaginal epithelial height by 47.23% (from 8.71+/-0.47 to 12.34+/-1.31 microm); and induced a weak increase of uterine epithelial height by 6.76% (from 5.42+/-0.52 to 5.84+/-0.91 microm). Both were not as pronounced as those elicited in the positive control of 100 microg/kg BW/d of ethinylestradiol given orally. Overall our results suggest that the extract of E. lysistemon contains secondary metabolites endowed with estrogenic activity.     

Effects of environmental estrogenic chemicals on AP1 mediated transcription with estrogen receptors alpha and beta

There has been much discussion concerning endocrine disrupting chemicals suspected of exerting adverse effects in both wildlife and humans. Since the majority of these compounds are estrogenic, a large number of in vitro tests for estrogenic characteristics have been developed for screening purpose. One reliable and widely used method is the reporter gene assay employing estrogen receptors (ERs) and a reporter gene with a cis-acting estrogen responsive element (ERE). Other elements such as AP1 also mediate estrogenic signals and the manner of response could be quite different from that of ERE. Since this has yet to be explored, the ER mediated AP1 activity in response to a series of environmental estrogens was investigated in comparison with ERE findings. All the compounds exhibited estrogenic properties with ERE-luc and their AP1 responses were quite similar. These was one exception, however, p,p'-DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane) did not exert any AP1-luc activity, while it appeared to be estrogenic at 10(-7) to 10(-5)M with the ERE action. None of the compounds demonstrated ER beta:AP1 activity. These data suggest that significant differences can occur in responses through the two estrogen pathways depending on environmental chemicals.     

Time course of anterior pituitary estrogen receptor after low 17 beta-estradiol doses in ovariectomized rats

The present paper describes the effect of three 17-beta-estradiol (E2) doses (1, 10 and 500 ng E2/kg) on the cytosolic and nuclear estrogen receptor content of anterior pituitary (Ap) of ovariectomized rats. The estrogen receptors were measured by [3H]E2 exchange in cytosol and crude nuclear fractions. Two hours after the administration of 10 or 500 ng E2/kg the Rc showed a depletion to 20-30% of preinjection level. The 1 ng E2/kg dose did not provoke any Rc depletion. The Rc replenishment was completed 5 h after injection of 10 ng E2/kg, but it was delayed to 10 h after injection of 500 ng E2/kg. An increased amount of Rc over the control levels was produced by 1 and 10 ng E2/kg doses, but not by the 500 ng E2/kg. The Rn level in Ap increased significantly after all E2 doses, and their highest levels were similar for 1, 10 and 500 ng E2/kg. These results suggest that some estrogenic responses like synthesis of the estrogen receptor proteins, can be elicited without previous significant Rc depletion. The relationships between Rc and Rn in Ap suggest an autoregulatory mechanism for the control of the cellular level of unbound estrogen receptors, that can be altered by the exogenous E2.     

Cardiac norepinephrine release: modulation by ovariectomy and estrogen

Previously, we have demonstrated that in contrast to male rats, female rats do not show an age-related reduction of depolarization-elicited norepinephrine (NE) release from cardiac synaptosomes (resealed nerve terminals). These results suggest that sex hormones such as estrogen may modulate NE release from cardiac synaptosomes prepared from female rats. The present study was designed to test the hypotheses that long-term estrogen depletion, resulting from ovariectomy, and estrogen replacement alters depolarization-elicited NE release from cardiac synaptosomes. Female F344 rats were divided into two groups, one of which underwent bilateral ovariectomy, whereas the other underwent a sham operation. Three ovariectomized subgroups received daily injections of conjugated equine estrogens, delta8,9-dehydroestrone or 17 alpha-dihydroequilenin. Another ovariectomized control subgroup and the sham-operated animals received daily injections of vehicle. After 90 or 270 days of treatment, the animals were sacrificed. Cardiac synaptosomes were prepared from each heart, incubated with [(3)H]-NE, and used to evaluate NE release capacity by exposure to 50 mM K(+). The effectiveness of the ovariectomy and the estrogenic actions of the test compounds was confirmed by evaluating vaginal smears, determining uterine weights, and measuring serum luteinizing hormone (LH) concentrations. Ovariectomy (after both 90 and 270 days) significantly increased depolarization-induced NE release compared with sham-operated rats. Treatment with all three estrogenic preparations reduced NE release in ovariectomized rats to values similar to those observed in sham-operated animals. Interestingly, NE release rates from rats treated with conjugated estrogens for 270 but not 90 days were significantly below that observed in age-matched sham animals. These results demonstrate that estrogen modulates depolarization-elicited NE release from cardiac nerve terminals. Such modulation may represent a protective action by estrogen at the cardiac synapse.     

Estrogen-controlled gene expression in tissue culture cells by zearalenone

In two estrogen-sensitive cell lines, Le42 and MCF-7, the estrogenic potential of the nonsteroidal mycotoxin zearalenone has been investigated. The chloramphenicol acetyltransferase (CAT) gene expression in Le42 cells is induced by zearalenone after transfection with a CAT-gene construct controlled by an estrogen responsive element [(1986) Cell 46, 1053-1061]. In MCF-7 cells zearalenone induces at least 2 exoproteins (52 and 160 kDa) which are estrogen-specific [(1980) Cell 20, 353-362). These data suggest that zearalenone acts by activating the estrogen receptor. Due to the high sensitivity of these cell lines for zearalenone both test systems are proposed as assays for a quantitative estimation of the biological (estrogenic) activity of this widespread mycotoxin.     

Oxysterol regulation of estrogen receptor alpha-mediated gene expression in a transcriptional activation assay system using HeLa cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor alpha-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17beta-estradiol > genistein > beta-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = beta-epoxysitosterol = 7beta-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.