Glyphosate induces epithelial mesenchymal transition-related changes in human endometrial Ishikawa cells via estrogen receptor pathway.

Glyphosate based herbicides are the most commonly used herbicide in the world. We aimed to determine whether glyphosate (Gly) induces epithelial mesenchymal transition (EMT) - related changes in a human endometrial carcinoma cell line (Ishikawa cells), and whether the estrogen receptor (ER) pathway is involved in these changes. Ishikawa cells were exposed to Gly (0.2 µM and 2 µM) or 17ß-estradiol (E2: 10-9 M). We detected that Gly increased cell migration and invasion ability compared to vehicle, as did E2. Moreover, a down regulation of E-cadherin mRNA expression was determined in response to Gly, similar to E2-effects. These results show that Gly promotes EMT-related changes in Ishikawa cells. When an ER antagonist (Fulvestrant: 10-7 M) was co-administrated with Gly, all changes were reversed, suggesting that Gly might promote EMT-related changes via ER-dependent pathway. Our results are interesting evidences of Gly effects on endometrial cancer progression via the ER-dependent pathway.

Effects of isoflavones on breast tissue and the thyroid hormone system in humans: a comprehensive safety evaluation.

Isoflavones are secondary plant constituents of certain foods and feeds such as soy, linseeds, and red clover. Furthermore, isoflavone-containing preparations are marketed as food supplements and so-called dietary food for special medical purposes to alleviate health complaints of peri- and postmenopausal women. Based on the bioactivity of isoflavones, especially their hormonal properties, there is an ongoing discussion regarding their potential adverse effects on human health. This review evaluates and summarises the evidence from interventional and observational studies addressing potential unintended effects of isoflavones on the female breast in healthy women as well as in breast cancer patients and on the thyroid hormone system. In addition, evidence from animal and in vitro studies considered relevant in this context was taken into account along with their strengths and limitations. Key factors influencing the biological effects of isoflavones, e.g., bioavailability, plasma and tissue concentrations, metabolism, temporality (pre- vs. postmenopausal women), and duration of isoflavone exposure, were also addressed. Final conclusions on the safety of isoflavones are guided by the aim of precautionary consumer protection.

The effect of estrogen on tendon and ligament metabolism and function.

Tendons and ligaments are crucial structures inside the musculoskeletal system. Still many issues in the treatment of tendon diseases and injuries have yet not been resolved sufficiently. In particular, the role of estrogen-like compound (ELC) in tendon biology has received until now little attention in modern research, despite ELC being a well-studied and important factor in the physiology of other parts of the musculoskeletal system. In this review we attempt to summarize the available information on this topic and to determine many open questions in this field.

Oestradiol-induced synapse formation in the female hippocampus: roles of oestrogen receptor subtypes.

During the oestrus cycle, varying spine synapse density correlates positively with varying local synthesis of oestradiol in the hippocampus. In this context, the roles of the oestrogen receptor (ER) subtypes ERα and ß are not fully understood. In the present study, we used neonatal hippocampal slice cultures from female rats because these cultures synthesise oestradiol and express both receptor subtypes, and inhibition of oestradiol synthesis in these cultures results in spine synapse loss. Using electron microscopy, we tested the effects on spine synapse density in response to agonists of both ERα and ERß. Application of agonists to the cultures had no effect. After inhibition of oestradiol synthesis, however, agonists of ERα induced spine synapse formation, whereas ERß agonists led to a reduction in spine synapse density in the CA1 region of these cultures. Consistently, up-regulation of ERß in the hippocampus of adult female aromatase-deficient mice is paralleled by hippocampus-specific spine synapse loss in this mutant. Finally, we found an increase in spine synapses in the adult female ERß knockout mouse, but no effect in the adult female ERα knockout mouse. Our data suggest antagonistic roles of ERß and ERα in spine synapse formation in the female hippocampus, which may contribute to oestrus cyclicity of spine synapse density in the hippocampus.

Responses of estrogen sensitive tissues in female Wistar rats to pre- and postnatal isoflavone exposure.

Effects of isoflavones on estrogen sensitive tissues are discussed controversially. This study was designed to investigate tissue specific effects of an isoflavone exposure through different periods of life in female Wistar rats and to compare the effects of genistein (GEN) to those of mixed dietary isoflavones, GEN and daidzein (DAI). One group received an isoflavone-free diet (IDD), another was fed an isoflavone-rich diet (IRD) and the third group an IDD supplemented with GEN (GEN(d)) prior to mating, throughout pregnancy and up to weaning. The offspring were kept on the respective diets during growth, puberty and adulthood. The weight of the uterus, the height of the uterine and vaginal epithelium, the bone mineral density of the tibia, and the expression of the estrogen sensitive gene CaBP9K in the liver were determined. At d21, the uterine weight, the uterine epithelium and the expression of CaBP9K in the liver were significantly stimulated in GEN(d) animals compared to IDD and IRD. Interestingly, bone mineral density was increased in GEN(d) and in IRD animals. Around puberty (d50) neither uterine wet weights nor trabecular bone density differed significantly among the isoflavone groups and the IDD control. At d80 no significant differences in uterine weight were observed among IDD, GEN(d) and IRD animals. However, bone mineral density was increased in GEN(d) and IRD animals. In summary, our results demonstrate that lifelong dietary exposure to isoflavones can affect estrogen sensitive tissues, apparently in a tissue selective manner. With respect to health risk and benefit our data indicate that an increased bone mineral density can be achieved by lifelong exposure to an IRD, which, in contrast to GEN supplementation, does not seem to stimulate the proliferation of the uterine epithelium.

Effects of the extracts of some tropical medicinal plants on estrogen inducible yeast and Ishikawa screens, and on ovariectomized Wistar rats.

A total of 33 extracts from 18 Cameroonians plants were studied in two in vitro test systems to determine potential estrogenic activities. The estrogenic activities of the extracts, which have shown promising activity on both in vitro screens were further investigated in vivo on ovariectomized Wistar rats. All 33 extracts were screened in the yeast test-system. Five of these extracts, namely the ethyl acetate extract of the stem bark of Millettia conraui, the ethyl acetate extract of the stem bark of Millettia drastica, the methanol extract of the leaves of Bridelia ferruginea, the methanol extract of the roots of Pseudarthria hookeri and the methanol extract of the roots of Nauclea latifolia showed interesting estrogenic properties, and were therefore further investigated on alkaline Phosphase induction in Ishikawa cells. The extracts of Millettia conraui, Millettia drastica, Pseudarthria hoockeri and Nauclea latifolia showed significant stimulatory effects at 10 and 100 mg/ml doses. The extract of Bridelia ferruginea was not further evaluated because of its toxicity on Ishikawa cells. This stimulatory effect was completely inhibited by a combined treatment with the pure antiestrogen ICI (Faslodex, 5 x 10(-7) M). In vivo experiments showed that per os administration of 200 mg/kg bw of the extracts of Millettia conraui and Bridelia ferruginea significantly increased uterine epithelial height by 17.93% and 28.08% respectively compared with uteri of ovariectomized controls after 7 days of treatment. Uterine epithelial height of animals treated with 100 rg/kg bw/d of ethinylestradiol increased by 242.3% in the same experiment. Extracts of Nauclea latifolia and Millettia drastica had no effect on the uterine epithelial height of ovariectomised rats. 200 mg/kg bw/d of the extracts of Nauclea latifolia, Millettia drastica, Bridelia ferruginea and Millettia conraui given orally significantly increased vaginal epithelial height by 15.64%, 24.06%, 51.02% and 58.12% following the same treatment regiment compared to untreated controls. In line with these data was the finding that vaginal epithelial height and vaginal cornification in the presence of each of these extracts was more advanced than in ovariectomized controls although not as prominent as in response to ethinylestradiol treatment. These results suggest that some constituents of the extracts of Millettia conraui, Millettia drastica, Pseudarthria hookeri, Nauclea latifolia and Bridelia ferruginea may have estrogenic activity.

The prohormone 19-norandrostenedione displays selective androgen receptor modulator (SARM) like properties after subcutaneous administration.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (4-estrene-3,17-dione, NOR), which is, after oral administration, readily metabolised to nortestosterone, also known as nandrolone (durabolin). In this study we have characterised molecular mechanisms of its action determined its tissue specific androgenic and anabolic potency after subcutaneous (s.c.) administration and investigated potential adverse effects. Receptor binding tests demonstrate that NOR binds with high selectivity to the AR. The potency of NOR to transactivate androgen receptor (AR) dependent reporter gene expression was 10 times lower as compared to dihydrotestosterone (DHT). In vivo experiments in orchiectomised rats demonstrated that s.c. treatment with NOR resulted only in a stimulation of the weight of the levator ani muscle; the prostate and seminal vesicle weights remained completely unaffected. Like testosterone, administration of NOR resulted in a stimulation of AR and myostatin mRNA expression in the gastrocnemius muscle. NOR does not affect prostate proliferation, the liver weight and the expression of the tyrosine aminotransferase gene (TAT) in the liver. Summarizing these data it is obvious that NOR, if administrated s.c. and in contrast to its metabolite nandrolone, highly selectively stimulates the growth of the skeletal muscle but has only weak androgenic properties. This observation may have relevance with respect to therapeutic aspects but also doping prevention.

Characterisation of the pharmacological profile of desoxymethyltestosterone (Madol), a steroid misused for doping.

Desoxymethyltestosterone (DMT), also known as Madol, is a steroid recently identified to be misused as a doping agent. Since, the knowledge of functions of this substance is rather limited, it was our aim to characterise the pharmacological profile of DMT and to identify potential adverse side effects. DMT was synthesised, its purity was confirmed and its biological activity was tested. The potency of Madol (DMT) to transactivate androgen receptor (AR) dependent reporter gene expression was two times lower as compared to dihydrotestosterone (DHT). Receptor binding tests demonstrate that DMT binds with high selectivity to the AR, binding to the progesterone receptor (PR) was low. In vivo experiments in orchiectomised rats demonstrated that treatment with DMT resulted only in a stimulation of the weight of the levator ani muscle; the prostate and seminal vesicle weights remained unaffected. Like testosterone, administration of DMT resulted in a stimulation of IGF-1 and myostatin mRNA expression in the gastrocnemius muscle. In the prostate proliferation was stimulated by TP (testosteronepropionate), but remained unaffected by DMT. Remarkably, treatment with DMT, in contrast to TP, resulted in a significant increase of the heart weight. In the liver, DMT slightly stimulates the expression of the tyrosine aminotransferase gene (TAT). Our results demonstrate that DMT is a potent AR agonist with an anabolic activity. Besides the levator ani weight, DMT also modulates the gene expression in the musculus gastrocnemius. The observed stimulation of TAT expression in the liver and the significant increase of the heart weight after DMT treatment can be taken as an indication for side effects. Summarizing these data it is obvious that DMT is a powerful anabolic steroid with selective androgen receptor modulators (SARM) like properties and some indications for toxic side effects. Therefore, there is a need for a strict control of a possible misuse.

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.

17beta-hydroxy-5alpha-androst-1-en-3-one (1-testosterone) is a potent androgen with anabolic properties.

Since the begining of the year 2005, the use of steroid precursors (prohormones) is illegal in the United States; nevertheless, there is still an enormous abuse of such substances. One of the most frequently misused steroids, often declared to be a prohormone, is 1-testosterone (17beta-hydroxy-5alpha-androst-1-en-3-one, 1-Testo). In this study, we have characterised molecular mechanisms of its action, determined its tissue specific androgenic and anabolic potency and investigated potential adverse effects. 1-Testo binds highly selective to the androgen receptor (AR) and has a high potency to stimulate AR dependent transactivation. In vivo an equimolar dose of 1-Testo has the same potency to stimulate the growth of the prostate, the seminal vesicles and the androgen sensitive levator ani muscle as the reference compound testosterone propionate (TP). Administration of 1-Testo, in contrast to TP, results in a significant increase of liver weight. Our results demonstrate that 1-Testo, even without being metabolised, is a very potent androgen. It binds selectively to the AR and transactivates AR dependent reporter genes. In vivo it has a high androgenic and anabolic potency and increases liver weight. In summary 1-Testo can be characterised as a typical anabolic steroid. It has to be assumed that consumption of this substance is associated with adverse side effects typical for this class of compounds. Therefore, a strict control of its ban is essential.

Estrogenic properties of isoflavones derived from Millettia griffoniana.

In most developing countries, 70-80% of the population still resort to traditional medicine for their primary health care. This medicine utilises medicinal plants which are traditionally taken as concoction and infusion. The root and stem bark of Millettia griffoniana (Leguminosae), has been reported to contain isoflavonoids, alkaloids, and diterpenoids. The possible benefit of some bioactive isoflavones derived from M. griffoniana prompted us to screen them for estrogenic activity. Six isoflavones and coumarin derived from M. griffoniana (bail) namely, compound nos. 1-6 (Fig. 1) were tested for their potential estrogenic activities in three different estrogen receptor alpha (ERalpha)-dependent assays. In a yeast-based ERalpha assay, all test substances and 17beta-estradiol as endogenous agonist, showed a significant induction of beta-galactosidase activity. The test compounds at the concentration of 5 x 10(-6) M could achieve 59-121% of the beta-galactosidase induction obtained with 10(-8) M 17beta-estradiol (100%). In the reporter gene assay based on stably transfected MCF-7 cells (MVLN cells), the estrogen responsive induction of luciferase was also stimulated by the M. griffoniana isoflavones. In Ishikawa cells, all substances exhibited estrogenic activity revealed by the induction of alkaline phosphatase (AlkP) activity. The estrogenic activities of isoflavones from M. griffoniana could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ERalpha. Although all substances showed estrogenic effects, 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone (7-O-DHF), Griffonianone C (GRIF-C), and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO) were found to be the most potent of tested substances. In summary, estrogenic activities of the isoflavones derived from M. griffoniana were described for the first time using reporter gene assays and the estrogen-inducible AlkP Ishikawa model.

Tetrahydrogestrinone is a potent but unselective binding steroid and affects glucocorticoid signalling in the liver.

Tetrahydrogestrinone (THG) is a steroid recently identified to be misused as doping agent. However, the knowledge on functions of this substance in humans or animal models is rather limited. Therefore, it was our aim to further characterize the pharmacological profile of THG and identify potential adverse side effects. THG was synthesized, the purity was confirmed and its biological activity was tested. The potency of THG to transactivate AR dependent reporter gene expression was two orders of magnitude lower compared to dihydrotestosterone. THG binds with high affinity but unselective to the androgen (AR), progesterone (PR), glucocorticoid (GR) and mineralocorticoid (MR) receptor. Treatment of orchiectomised rats with THG resulted in a stimulation of prostate, seminal vesicle and levator ani muscle, indicating androgenic and anabolic properties. In the liver THG, in contrast to testosteronepropionate (TP), down regulates the expression of the GR dependent tyrosine aminotransferase gene (TAT). In summary, our results demonstrate that THG is not a specific AR agonist. THG exhibits a high binding affinity to all tested steroid hormone receptors and binds with highest affinity to the GR. Our in vivo data are indicative of an anabolic and androgenic potency of THG, but the repression of TAT demonstrates that THG also interferes with the glucocorticoid hormone system. Therefore, it is conceivable that an intake will result in adverse side effects.

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453).

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.

Is there an estrogenic component in the metabolic syndrome?

One of the major upcoming concerns leading to health related problems in the industrialized societies is the metabolic syndrome which is characterized by central obesity, hypertension, raised fasting glucose and triglyceride levels. The focus of this review is on a potential estrogenic linkage between the metabolic mechanisms involved into the development of this disease cluster and specific estrogen related regulatory pattern. The candidate molecules for this link are insulin and insulin-like growthfactor, C-reactive protein, peroxisome-proliferation-activatingreceptorgamma, and leptin which all seem to interact with each other and show a responsiveness to changing estrogen levels. From this perspective they might also represent target molecules for a phytochemical intervention with phytoestrogens.

Comparative responses of three rat strains (DA/Han, Sprague-Dawley and Wistar) to treatment with environmental estrogens.

The rat uterotrophic assay is a widely used screening test for the detection of estrogenic, endocrine-disrupting chemicals. Although much attention has been paid to identifying protocol variables and reproducibility between laboratories the question whether toxicodynamic and toxicokinetic variations of different strains may affect their sensitivity to estrogenic stimuli has been rarely addressed. We have compared the estrogenic activity of the environmental chemicals genistein (GEN), bisphenol A (BPA) and p- tert-octylphenol (OCT) in DA/Han (DA), Sprague-Dawley (SD) and Wistar (WIS) rats after repeated oral application. Rats were treated per os for 3 days with different doses of these weakly estrogenic compounds and the potent reference estrogen ethinylestradiol (EE). Then uterine wet weight, thickness of the uterine epithelium, uterine gene expression of clusterin (CLU), and thickness of the vaginal epithelium were examined as parameters for estrogenic potency of the test compounds in the three strains of rats. The uterotrophic response to treatment with BPA, OCT and GEN was similar in the three strains, and allowed us to rank them as GEN being more potent than OCT, and BPA being the weakest estrogen. This was confirmed by analysis of other biological endpoints, despite some differences in the magnitude of their response among strains and to distinct compounds. For instance, the uterus wet weight response to EE treatment indicated lower sensitivity of SD rats than that of DA and WIS rats, but this was not observed for responses of the uterine or vaginal epithelium. Moreover, blood concentrations were assessed at the time of killing and related to biological responses: plasma levels of total and unconjugated BPA and GEN depended upon the dose administered and varied to some extent within treatment groups and among the three rat strains. However, there was no good correlation in the three strains between individual compound concentrations analysed 24 h after the last dose and the uterotrophic wet weights. Summarising our results, we conclude that the sensitivity of various biological endpoints can differ slightly between strains of rats. On the other hand, our data demonstrate that the choice of the rat strain does not lead to pronounced differences in the evaluation of estrogenic activities of chemicals, especially when different biological endpoints are included in the analysis.

Endometrial cancer: experimental models useful for studies on molecular aspects of endometrial cancer and carcinogenesis.

There is definitely a need for the development of new drugs for the treatment and cure of endometrial cancer. In addition there are various new drugs or phyto-remedies under development which are intended for use in the treatment and prevention of breast cancer, for the treatment of menopausal symptoms and for hormone replacement therapy. The efficacy of novel drugs targeting steroid receptors in endometrial cancers has to be evaluated and the safety of other endocrine measures on endometrial cancers or on endometrial carcinogenesis has to be assessed. For these experimental purposes five main classes of experimental models are available: spontaneous endometrial tumorigenesis models in inbred animals (Donryu rats, DA/Han rats, BDII/Han rats), inoculation tumors from chunks of tumors (rat EnDA-tumor, human EnCa 101 tumor) or from inoculated tumor cell lines (rat RUCA-I cells, human Ishikawa and ECC-1 cells), developmental estrogenic exposure or chemical carcinogen exposure of CD-1 and ICR mice, transgenic approaches such as mice heterozygous regarding the tumor suppressor gene PTEN (pten(+/-)-mice) and endometrial tumor cell lines cultured under conditions promoting in vivo-like morphology and functions e.g. cell culture on reconstituted basement membrane. Although the number of models is comparatively small, most aspects related to functions of estrogenic or gestagenic substances are assessable, particularly if various experimental models are combined. Whereas models based on human endometrial adenocarcinoma cells are widely used, the properties and advantages of animal-derived models have mainly been ignored so far.

In vivo test systems for the quantitative and qualitative analysis of the biological activity of phytoestrogens.

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. One of the most extensively used in vivo assays to characterise the estrogenic potency of these phytoestrogens and mechanisms of their action is the rodent uterotrophic assay. Various protocols exist for this test system, using immature, hypophysectomized, or ovariectomized rats and mice and oral or subcutaneous administration of the test compound. However, just monitoring the ability of a compound to stimulate uterine growth is not sufficient to characterize its estrogenicity. Over the last decades, an increasing number of estrogen sensitive tissues has been identified. Moreover, a variety of different molecular mechanisms have been discovered for the action of estrogens, including non-genomic actions. Therefore, an in vivo test design for estrogenicity should include an analysis of several estrogen sensitive parameters in different estrogen sensitive tissues. To distinguish between agonistic and antagonistic properties of a substance, combinations of the test compound with estrogens and antiestrogens should be analyzed. A reasonable supplement to this enhanced uterotrophic assay are selected estrogen sensitive tumor models, which can be used to test for potential chemopreventive properties of phytoestrogens.

Phytoestrogens and carcinogenesis-differential effects of genistein in experimental models of normal and malignant rat endometrium.

The phytoestrogen genistein was studied in normal and malignant experimental uterine models in vivo. The action of genistein on the uterus and vagina of ovariectomized DA/Han rats after 3 day oral administration (25, 50 or 100 mg/kg/BW/d) was compared to ethinyl oestradiol (0.1 mg/kg/BW/d). Effects on uterine and vaginal morphology, uterine growth and uterine gene expression were studied. A dose dependent increase of the uterine wet weight and the uterine and vaginal epithelial height, a dose dependent up-regulation of complement C3, down-regulation of clusterin mRNA expression and a stimulation of the vaginal cornification was observed after administration of genistein. Uterine gene expression and vaginal epithelium respond to genistein at doses where no significant effects on uterine wet weight were detectable. In general the vagina was more sensitive to genistein than the uterus. To analyse the action of genistein in malignant uterine tissue, the impact of a 28 d treatment with 50 mg/kg/d of genistein on the in-vivo tumour growth of RUCA I endometrial adenocarcinoma cells, following subcutaneous inoculation into syngeneic DA/Han rats, was assessed. In contrast to ethinyl oestradiol (0.1 mg/kg/BW/d), a dose of 50 mg/kg/BW/d of genistein did not affect tumour growth. Nevertheless C3 and TRPM2 mRNA expression in the tumour were both significantly stimulated by ethinyl oestradiol and genistein. In comparison to ovariectomized animals genistein up-regulated uterine wet weight and uterine dependent gene expression in tumour bearing animals. In conclusion, four independent uterine and vaginal parameters indicate genistein is a weak oestrogen receptor agonist in the uterus and vagina of female DA/Han rats, and evidence is provided for a selective oestrogen receptor modulator (SERM)-like action of genistein in normal and malignant uterine tissue.

Expression of l-3-phosphoserine phosphatase is regulated by reconstituted basement membrane.

Reconstituted basement membrane (Matrigel) promotes differentiation of endometrial adenocarcinoma cells in vitro. However, little is known about the molecular basis of these in vitro differentiation processes. Using differential display RT-PCR to search for potential molecular markers we screened for genes which respond to contact to basement membrane by alteration of expression levels. Here we report that the cDNA MT32 represents an mRNA with a time dependent biphasic response pattern to contact to basement membrane. Characterizing MT32 revealed that the sequence of MT32 is identical to l-3-phosphoserine phosphatase. PCR analysis of l-3-phosphoserine phosphatase expression surprisingly revealed at least three variants of this enzyme. In summary, and in view of the literature, l-3-phosphoserine phosphatase and potential variants or family members represent molecular markers to study regulation of gene expression by components of the extracellular matrix. In conclusion, l-3-phosphoserine phosphatase(s) may be important in endometrial carcinogenesis since this enzyme synthesizes important metabolic intermediates which serve both as building blocks for peptide synthesis and for signal transducing molecules.