HPTLC Fingerprint Authentication of Selected Sideritis spp. Using a Pharmacognostic Approach.

The genus Sideritis (Lamiaceae) comprises around 150 species, of which many are popular herbal remedies in Mediterranean folk medicine. Already mentioned by Dioscorides and Theophrastus, the "ironwort" or "Greek mountain tea" has been receiving increased attention in recent years. A European Union herbal monograph and assessment report (HMPC) has been issued, covering the species Sideritis scardica, S. clandestina, S. raeseri, and S. syriaca. This study presents results of a first pharmacognostic examination of the botanical and phytochemical differences among and between these emerging commercial species, and other, less studied species. An HPTLC method is proposed for normal phase separation of the species; this means applying two mobile phases on silica plates and subsequent derivatization with natural product reagent (NP/PEG) for visualization of phenolic compounds and anisaldehyde for a broader detection. With the help of selected reference compounds, a system suitability test was established for proper chromatographic separation. The method was applied to specimens from botanical gardens and commercial raw material in order to test its suitability for differentiation and authentication. The HPTLC analysis also includes, for the first time, S. hyssopifolia and other less used Sideritis species. The results might enable the development of a validated phytochemical fingerprint authentication procedure for quality assurance of Sideritis herba.

Manipulating Estrogenic/Anti-Estrogenic Activity of Triphenylethylenes towards Development of Novel Anti-Neoplastic SERMs.

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ERα) agonists or antagonists depending on the target issue. Tamoxifen (TAM) (a non-steroidal triphenylethylene derivative) was the first SERM approved as anti-estrogen for the treatment of metastatic breast cancer. On the hunt for novel SERMs with potential growth inhibitory activity on breast cancer cell lines yet no potential to induce endometrial carcinoma, we designed and synthesized 28 novel TAM analogs. The novel analogs bear a triphenylethylene scaffold. Modifications on rings A, B, and C aim to attenuate estrogenic/anti-estrogenic activities of the novel compounds so they can potentially inhibit breast cancer and provide positive, beneficial estrogenic effects on other tissues with no risk of developing endometrial hyperplasia. Compound 12 (E/Z-1-(2-{4-[1-(4-Chloro-phenyl)-2-(4-methoxy-phenyl)-propenyl]-phenoxy}-ethyl)-piperidine) showed an appreciable relative ERα agonistic activity in a yeast estrogen screen (YES) assay. It successfully inhibited the growth of the MCF-7 cell line with GI50 = 0.6 µM, and it was approximately three times more potent than TAM. It showed no potential estrogenicity on Ishikawa endometrial adenocarcinoma cell line via assaying alkaline phosphatase (AlkP) activity. Compound 12 was tested in vivo to assess its estrogenic properties in an uterotrophic assay in an ovariectomized rat model. Compared to TAM, it induced less increase in wet uterine wet weight and showed no uterotrophic effect. Compound 12 is a promising candidate for further development due to its inhibition activity on MCF-7 proliferation with moderate AlkP activity and no potential uterotrophic effects. The in vitro estrogenic activity encourages further investigations toward potential beneficial properties in cardiovascular, bone, and brain tissues.

6-Prenylnaringenin from Hops Disrupts ERα-Mediated Downregulation of CYP1A1 to Facilitate Estrogen Detoxification.

Botanical dietary supplements (BDS) containing hops are sold as women's health supplements due to the potent hop phytoestrogen, 8-prenylnaringenin (8-PN), and the cytoprotective chalcone, xanthohumol. Previous studies have shown a standardized hop extract to beneficially influence chemical estrogen carcinogenesis in vitro by fostering detoxified 2-hydroxylation over genotoxic 4-hydroxylation estrogen metabolism. In this study, hop extract and its bioactive compounds were investigated for its mechanism of action within the chemical estrogen carcinogenesis pathway, which is mainly mediated through the 4-hydroxylation pathway catalyzed by CYP1B1 that can form gentoxic quinones. Aryl hydrocarbon receptor (AhR) agonists induce CYP1A1 and CYP1B1, while estrogen receptor alpha (ERα) inhibits transcription of CYP1A1, the enzyme responsible for 2-hydroxylated estrogens and the estrogen detoxification pathway. An In-Cell Western MCF-7 cell assay revealed hop extract and 6-prenylnaringenin (6-PN) degraded ERα via an AhR-dependent mechanism. Reverse transcription PCR and xenobiotic response element luciferase assays showed hop extract and 6-PN-mediated activation of AhR and induction of CYP1A1. A reduction in estrogen-mediated DNA (cytosine-5)-methyltransferase 1 (DNMT1) downregulation of CYP1A1 accompanied this activity in a chromatin immunoprecipitation assay. Ultimately, hop extract and 6-PN induced preferential metabolism of estrogens to their detoxified form in vitro. These results suggest that the standardized hop extract and 6-PN activate AhR to attenuate epigenetic inhibition of CYP1A1 through degradation of ERα, ultimately increasing 2-hydroxylated estrogens. A new mechanism of action rationalizes the positive influence of hop BDS and 6-PN on oxidative estrogen metabolism in vitro and, thus, potentially on chemical estrogen carcinogenesis. The findings underscore the importance of elucidating various biological mechanisms of action and standardizing BDS to multiple phytoconstituents for optimal resilience promoting properties.

Is There a Role for Environmental and Metabolic Factors Predisposing to Severe COVID-19?

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic affects people around the world. However, there have been striking differences in the number of infected individuals and deaths in different countries. Particularly, within Central Europe in countries that are similar in ethnicity, age, and medical standards and have performed similar steps of containment, such differences in mortality rates remain inexplicable. We suggest to consider and explore environmental factors to explain these intriguing variations. Countries like Northern Italy, France, Spain, and UK have suffered from 5 times more deaths from the corona virus infection than neighboring countries like Germany, Switzerland, Austria, and Denmark related to the size of their respective populations. There is a striking correlation between the level of environmental pollutants including pesticides, dioxins, and air pollution such as NO2 known to affect immune function and healthy metabolism with the rate of mortality in COVID-19 pandemic in these European countries. There is also a correlation with the use of chlorination of drinking water in these regions. In addition to the improvement of environmental protective programs, there are possibilities to lower the blood levels of these pollutants by therapeutic apheresis. Furthermore, therapeutic apheresis might be an effective method to improve metabolic inflammation, altered vascular perfusion, and neurodegeneration observed as long-term complications of COVID-19 disease.

Novel insight in estrogen homeostasis and bioactivity in the ACI rat model of estrogen-induced mammary gland carcinogenesis.

Despite being widely used to investigate 17ß-estradiol (E2)-induced mammary gland (MG) carcinogenesis and prevention thereof, estrogen homeostasis and its significance in the female August Copenhagen Irish (ACI) rat model is unknown. Thus, levels of 12 estrogens including metabolites and conjugates were determined mass spectrometrically in 38 plasmas and 52 tissues exhibiting phenotypes ranging from normal to palpable tumor derived from a representative ACI study using two different diets. In tissues, 40 transcripts encoding proteins involved in estrogen (biotrans)formation, ESR1-mediated signaling, proliferation and oxidative stress were analyzed (TaqMan PCR). Influence of histo(patho)logic phenotypes and diet on estrogen and transcript levels was analyzed by 2-way ANOVA and explanatory variables influencing levels and bioactivity of estrogens in tissues were identified by multiple linear regression models. Estrogen profiles in tissue and plasma and the influence of Hsd17b1 levels on intra-tissue levels of E2 and E1 conclusively indicated intra-mammary formation of E2 in ACI tumors by HSD17B1-mediated conversion of E1. Proliferation in ACI tumors was influenced by Egfr, Igf1r, Hgf and Met levels. 2-MeO-E1, the only oxidative estrogen metabolite detected above 28-42 fmol/g, was predominately observed in hyperplastic tissues and intra-tissue conversion of E1 seemed to contribute to its levels. The association of the occurrence of 2-MeO-E1 with higher levels of oxidative stress observed in hyperplastic and tumor tissues remained equivocal. Thus, the present study provides mechanistic explanation for previous and future results observed in the ACI model.

Synaptopodin is regulated by aromatase activity.

Locally synthesized estradiol plays an important role in synaptic plasticity in the hippocampus. We have previously shown that in hippocampal neurons, activity of the enzyme aromatase, which converts testosterone into estradiol, is reduced via Ca2+ -dependent phosphorylation. Synaptopodin is a highly estrogen responsive protein, and it has been shown that it is an important regulator of synaptic plasticity, mediated by its close association with internal calcium stores. In this study, we show that the expression of synaptopodin is stronger in the hippocampus of female animals than in that of male animals. Phosphorylation of aromatase, using letrozole, however, down-regulates synaptopodin immunohistochemistry in the hippocampus of both male and females. Similarly, in aromatase knock-out mice synaptopodin expression in the hippocampus is reduced sex independently. Using primary-dissociated hippocampal neurons, we found that evoked release of Ca2+ from internal stores down-regulates aromatase activity, which is paralleled by reduced expression of synaptopodin. Opposite effects were achieved after inhibition of the release. Calcium-dependent regulation of synaptopodin expression was abolished when the control of aromatase activity by the Ca2+ transients was disrupted. Our data suggest that the regulation of aromatase activity by Ca2+ transients in neurons contributes to synaptic plasticity in the hippocampus of male and female animals as an on-site regulatory mechanism.

[Isoflavone-containing dietary supplements]. / Isoflavonhaltige Nahrungsergänzungsmittel.

Isoflavones (IFs) from soy and other legumes have weak estrogenic properties. Isolated IFs are available as dietary supplements and advertised to alleviate symptoms of menopause. The present chapter provides an overview of the occurrence, the chemical structure of IFs and their metabolites, the market situation and reviews the current evidence on the efficacy and safety of IF-containing dietary supplements.The biological effectiveness of IFs is attributable to the activation of the estrogen receptor (ER). Studies on the influence of IFs on endogenous estrogen levels in women show inconsistent results. So far, the European Food Safety Authority (EFSA) has rejected all submitted health claims for IFs due to insufficient scientific evidence for any of the postulated health effects. Based on the results of their recent risk assessment, the EFSA concluded that the available human studies did not support the hypothesis of adverse effects of isolated IFs on the human mammary gland, uterus or thyroid in healthy postmenopausal women. However, the assessment does not allow a general statement on the safety of IF-containing dietary supplements. Studies in animal models are often not comparable with the complex interactions in humans due to differences in the metabolism of IFs, in the developmental stage at time of consumption and in the temporarily restricted uptake of IFs during certain stages of life. CONCLUSION: So far, for none of the advertised functions is unequivocal scientific evidence available. On the basis of available data, potential unwanted side effects cannot be fully excluded. This holds particularly true for women with undiagnosed diseases, especially for those with undetected precancerous lesions in the mammary gland.

Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross-species and sex comparison.

Soy isoflavones (IF) are in the focus of biomedical research since more than two decades. To assess their bioactivity, IF are investigated in rats and mice as a model. As the biological activity of IF is affected by their biotransformation, our aim was to comprehensively compare the conjugative and microbial metabolism of daidzein and genistein in adult humans, rats and mice of both sexes. One identical soy extract and a validated LC-MS method were used for all studies. We detected considerable differences between the three species. In rats and mice, sex-specific differences were observed in addition. The major plasma phase II metabolites in humans were the 7-sulfo-4'-glucuronides (39-49 %) and, in case of genistein, also the diglucuronide (34 %), whereas in mice monosulfates (33-41 %) and monoglucuronides (30-40 %) predominated. In male rats the disulfates (23-62 %) and 7-sulfo-4'-glucuronides (19-54 %) were predominant, while in female rats the 7-glucuronides (81-93 %) exhibited highest concentrations. The portion of aglycones was low in humans (0.5-1.3 %) and rats (0.5-3.1 %) but comparatively high in mice (3.1-26.0 %), especially in the case of daidzein. Furthermore, substantial differences were observed between daidzein and genistein metabolism. In contrast to humans, all rats and mice were equol producer, independent of their sex. In conclusion, there are marked differences between humans, rats and mice in the profile of major metabolites following IF phase II metabolism. These differences may contribute to resolve inconsistencies in results concerning the bioactivity of IF and should be considered when applying findings of animal studies to humans, e.g., for risk assessment.

Soy isoflavone exposure through all life stages accelerates 17ß-estradiol-induced mammary tumor onset and growth, yet reduces tumor burden, in ACI rats.

There is an ongoing debate whether the intake of soy-derived isoflavones (sISO) mediates beneficial or adverse effects with regard to breast cancer risk. Therefore, we investigated whether nutritional exposure to a sISO-enriched diet from conception until adulthood impacts on 17ß-estradiol (E2)-induced carcinogenesis in the rat mammary gland (MG). August-Copenhagen-Irish (ACI) rats were exposed to dietary sISO from conception until postnatal day 285. Silastic tubes containing E2 were used to induce MG tumorigenesis. Body weight, food intake, and tumor growth were recorded weekly. At necropsy, the number, position, size, and weight of each tumor were determined. Plasma samples underwent sISO analysis, and the morphology of MG was analyzed. Tumor incidence and multiplicity were reduced by 20 and 56 %, respectively, in the sISO-exposed rats compared to the control rats. Time-to-tumor onset was shortened from 25 to 20 weeks, and larger tumors developed in the sISO-exposed rats. The histological phenotype of the MG tumors was independent of the sISO diet received, and it included both comedo and cribriform phenotypes. Morphological analyses of the whole-mounted MGs also showed no diet-dependent differences. Lifelong exposure to sISO reduced the overall incidence of MG carcinomas in ACI rats, although the time-to-tumor was significantly shortened.

Progesterone, as well as 17ß-estradiol, is important for regulating AHR battery homoeostasis in the rat uterus.

Several studies indicate that the aryl hydrocarbon receptor (AHR), which plays an important role in mediating the toxicity of many industrial chemicals, plays an important role in the physiology of female reproductive tract organs. This makes it likely that the AHR and additional components of the AHR signalling pathway are under the control of female sex steroids. In a previous study, we could already demonstrate the regulation of many members of the AHR battery by 17ß-estradiol (E2) in the uterus of rats. In this study, we addressed the potential role of progesterone (P4) in this context. In a comparative approach using ovariectomized rats which were treated for 3 days with either vehicle control, E2, progesterone (P4) or the combination of both hormones in addition to sham-operated animals, we could demonstrate that in addition to E2, P4 is also an important factor in regulating AHR signalling in the rat uterus. P4 has effects similar to E2 on uterine Ahr, Arnt and Arnt2 mRNA levels, resulting in a downregulation of these genes, while the E2-mediated downregulation of key AHR response genes Cyp1a1, Gsta2 and Ugt1 is completely antagonized by P4. As with E2, P4 leads to an increase in uterine AHR levels, especially in the endometrial epithelium despite the decrease in corresponding mRNA levels. This indicates a complex gene-specific regulatory network involving E2, P4 and possibly AHR itself to maintain all components of the AHR signalling cascade at the required levels during all stages of the oestrous cycle and pregnancy.

Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or ß, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or ß, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor ß) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/ß cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes.

Impact of a functionalized olive oil extract on the uterus and the bone in a model of postmenopausal osteoporosis.

PURPOSE: The Mediterranean diet rich in fruits, vegetables and olive oil has been related to a lower osteoporosis incidence and accordingly to a reduced fracture risk. These observations might be mediated by the active constituents of extra virgin olive oil, and especially polyphenols. In the context of exploring the features of olive oil active constituents on postmenopausal osteoporosis, an extra virgin olive oil total polyphenolic fraction (TPF) was isolated and its effect on the bone loss attenuation was investigated. METHODS: Female Lewis rats were ovariectomized and fed a diet enriched with a total phenolic extract of extra virgin olive oil in a concentration of 800 mg/kg diet. RESULTS: Oleocanthal, one compound of the polyphenolic fraction, showed a higher relative estrogen receptor binding affinity to the ERα compared to the ERß. While the TPF only slightly induced the uterine wet weight (490.7 ± 53.7 vs. 432.7 ± 23, p = 0.058), TPF regulated estrogen response genes in the uterus (progesterone receptor, antigen identified by monoclonal antibody Ki67, complement C3). Comparing the quantified bone parameters, the oral TPF substitution did not attenuate the ovariectomy-induced bone loss. CONCLUSIONS: The administration of extra virgin olive oil polyphenols regulated uterine estrogen response marker genes in an E2-agonistic manner. The bone loss induced by estrogen ablation was not mitigated by treatment with the polyphenolic extract.

Erythroidine alkaloids: a novel class of phytoestrogens.

Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, ß-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-ß-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and ß-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010% and 0.005 ± 0.010%, respectively, whereas only ß-erythroidine bound to ERß (0.006 ± 0.010%). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and ß-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of phytoestrogens.

Phytotherapy and women's reproductive health: the Cameroonian perspective.

Approximately 80 % of the population in Africa use traditional medicinal plants to improve their state of health. The reason of such a wide use of medicinal plants has been mainly attributed to their accessibility and affordability. Expectation of little if any side effects, of a "natural" and therefore safe treatment regimen, as well as traditional beliefs additionally contribute to their popularity. Several of these plants are used by women to relieve problems related to their reproductive health, during or after their reproductive life, during pregnancy, or following parturition. The African pharmacopoeia thus provides plants used for preventing and/or treating gynecological infections, dysmenorrhea, irregular menstruations, oligomenorrhea or protracted menstruation, and infertility. Such plants may then be used as antimicrobians, emmenagogues, or as suppressors of uterine flow. African medicinal plants are also used during pregnancy for prenatal care, against fetal malposition or malpresentation, retained dead fetus, and against threatened abortion. Some others are used as anti-fertilizing drugs for birth control. Such plants may exert various activities, namely, anti-implantation or early abortifacient, anti-zygotic, blastocytotoxic, and anti-ovulatory effects. Some herbs could also act as sexual drive suppressors or as a post-coital contraceptive by reducing the fertility index. A number of these plants have already been subject to scientific investigations and many of their properties have been assessed as estrogenic, oxytocic, or anti-implantation. Taking into account the diversity of the African pharmacopoeia, we are still at an early stage in the phytochemical and pharmacological characterization of these medicinal plants that affect the female reproductive system, in order to determine, through in vitro and in vivo studies, their pharmacological properties and their active principles.

Effect of 17ß-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line.

Due to the health risks attributed to perimenopausal hormone therapy, phytoestrogens such as flavonoids are receiving widespread attention to help alleviate menopausal symptoms, including hormone-driven mood disorders. Based on our previous reporter gene study regarding their transactivational activity in raphe nuclei cells from a brain region involved in regulation of mood disturbances, we herein study their effects on the regulation of expression of 17ß-estradiol (E2)-regulated genes. DNA microarray was used to globally assess E2-induced gene expression in RNDA cells, a rat raphe nuclei-derived cellular model expressing oestrogen receptor ß. Out of 212 regulated genes, six were selected for verification and as endpoints for the effect of flavonoids on the regulation of mRNA expression in proliferating as well as differentiating RNDA cells. Under proliferative conditions, E2 up-regulated mRNA expression of Cml-5, Sox-18 and Krt-19. Similar effects were observed in response to 8-prenylnaringenin (8-PN), genistein (GEN), daidzein (DAI) and equol (EQ). In line with E2, mRNA expression of Nefm and Zdhhc-2 was down-regulated following 8-PN, GEN, DAI, EQ and naringenin treatment. No regulation was observed on Slc6a4 mRNA expression in response to E2 or the flavonoids in proliferating RNDA cells. When cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards E2 and the tested flavonoids were noticed. These expression studies additionally highlighted some of the genes as markers for RNDA cellular differentiation. RNDA cells should prove useful to elucidate molecular and cellular mechanisms of exogenous oestrogen receptor ligands with neural cell populations.

Regulation of uterine AHR battery gene expression by 17ß-Estradiol is predominantly mediated by estrogen receptor α.

The aryl hydrocarbon receptor (AHR) is known to mediate the cellular response to numerous xenobiotics including dioxin. Surprisingly AHR knockout mice provide evidence for the involvement of the AHR signalling cascade in estrogen regulated physiological functions of the female reproductive system. Several studies already aimed to investigate the impact of the AHR mediated xenobiotic response pathway on estrogen receptor (ER) signalling, whereas on contrary availability of data describing the effect of 17ß-Estradiol (E2) on the AHR signalling cascade is rather limited. In this study we observed an inhibitory effect of E2 treatment on uterine Ahr, Arnt, Arnt2, Ahrr, Cyp1a1, Ugt1 and Nfe2l2 gene expression in ovariectomized Wistar rats, whereas Cyp1b1, Nqo1 and Gsta2 displayed an increased transcription. The usage of the ER selective agonists, 16α-LE(2) (ERα selective) and 8ß-VE(2) (ERß selective), enabled us to distinguish between ER subtype specific responses. On mRNA level the observed changes in gene expression were mainly mediated by ERα except for the expression of Nqo1. In most cases the activation of ERß caused effects opposite to the ones observed following activation of ERα. Despite the significant changes in AHR mRNA levels immunohistochemical staining uterine tissue section did not reveal changes of the AHR protein level. Taken together our results validate, support and extend the hypothesis of uterine crosstalk between AHR and ER signalling pathways. Furthermore they give an insight into how the AHR and its related genes may participate in E2 dependent uterine physiological processes and provide another potential mechanism of action for xenoestrogens.

The rat prepubertal uterine myometrium and not the luminal epithelium is predominantly affected by a chronic dietary genistein exposure.

Current knowledge about dietary soy isoflavone-induced hormonal effects and potential priming effects for the responsiveness of the organism to other estrogens is insufficient. The present study examined the effects of pre- and postnatal soy isoflavone exposure on estrogen responsiveness by estrogen receptor agonists in the uteri of prepubertal Wistar rats. To this end, offspring were generated from dams already maintained on three dietary groups, (1) a phytoestrogen-free diet, (2) a soy isoflavone-rich diet with 232 ppm daidzein and 240 ppm genistein or (3) a custom-made diet supplemented with 700 ppm genistein (GEN). Then, F1 females continuously exposed to isoflavones from GD1 to PND21 and non-exposed controls were subjected to an immature uterotrophic assay to compare physiological parameters and the response to subcutaneous treatment with 17ß-estradiol, GEN or an estrogen receptor subtype (ERα and ERß)-specific agonist. Uterine wet weight (UWW), luminal epithelial height (LEH) and myometrial thickness (MMT) were determined. In addition, isoflavone plasma levels and mRNA expression profiles of relevant steroid receptors and of molecular markers for proliferation and estrogenicity were assessed for all groups. The influence of dietary isoflavones on the sensitivity to various estrogenic stimuli in these prepubertal animals was minor. Yet, the uterus of immature rats with high chronic GEN exposure alone showed already an increase in UWW, LEH and MMT. The myometrial response to GEN was more pronounced than that of the luminal epithelium, which may be due to a non-uniform distribution of steroid receptors, in particular the progesterone receptor. In conclusion, although the impact of a continuous, prenatally initiated exposure to dietary isoflavones on the uterine physiology of juvenile rats is modest, the possible priming effects of this exposure for beneficial or adverse late-onset consequences in adults should not be neglected.

Characterization of identity, metabolism and androgenic activity of 17-hydroxyandrosta-3,5-diene by GC-MS and a yeast transactivation system.

Anabolic-androgenic steroids are frequently misused compounds in sports, and they belong to the controlled substances according to the requirements of the World Anti-Doping Agency. The classical techniques of steroid detection are mass spectrometry coupled to gas or liquid chromatography. Biological methods that base on the ability of substances to bind the steroid receptor are not applied in routine doping control procedures so far, but they appear to be useful for characterization of steroid androgenic potential. In this study we used the yeast androgen receptor reporter system (YAS), which in the past has already successfully been applied to both various androgenic substances and also urine samples. Giving attention to the androgenic potential of steroidal dietary supplements, we exemplified the analysis using both mass spectrometry techniques and the YAS-based assay on the product "Syntrax Tetrabol" which was a confiscated dietary supplement and marketed as a steroid precursor. Identification, structure and the kinetic behavior of its excreted metabolites were analyzed by NMR, GC-MS and LC-MS/MS. The androgenic potential of the parent compound as well as its metabolites in urine was evaluated with the help of the YAS. The application of urine samples with a previous deconjugation and the inclusion of urine density values were carried out and led to increased responses on the YAS. Further, the possibility of a complementary application of structure-based instrumental analysis and biological detection of androgenicity with the help of the YAS seems to be desirable and is discussed.

Flexible Etherified and Esterified Triphenylethylene Derivatives and Their Evaluation on ER-positive and Triple-Negative Breast Cancer Cell Lines.

Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) with potential clinical benefits for all stages of breast cancer. TAM is primarily metabolized to more potent metabolites via polymorphic CYP2D6. This affects the clinical outcome of TAM treatment. Herein we report novel TAM analogues that can avoid metabolism via CYP2D6. The novel analogues bear a flexible skeleton. Compounds have either an ester group on ring C or homodiaminoalkoxy groups on rings B and C. Compound 6 (E/Z-4-[1-[4-(2-diethylaminoethoxy)phenyl]-3-(4-methoxyphenyl)-2-methyl[propenyl]phenol) was found to be ten-fold more potent than TAM on MCF-7 cells (GI50 =0.15 µM). It showed fivefold greater inhibitory activity on MDA-MB-231 cells than TAM (GI50 =1.71 µM). Compound 13 (4-{3,3-bis-[4-(3-dimethylaminopropoxy)phenyl]-2-methylallyl}methoxybenzene) was the most potent among the homodiaminoalkoxy derivatives (GI50 =0.44) on both MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, the COMPARE algorithm suggested that it has different molecular targets from those of some other reported anticancer drugs.

AHR agonistic effects of 6-PN contribute to potential beneficial effects of Hops extract.

Hops (Humulus lupulus) is used as an alternative to hormone replacement therapy due to the phytoestrogen, 8-prenylnaringenin (8-PN). To examine the potential risks/benefits of hops extract and its compounds (8-PN and 6-prenylnaringenin, 6-PN), we aimed to evaluate the estrogen receptor α (ERα) and aryl hydrocarbon receptor (AHR) signaling pathways in human endometrial cancer cells. Hops extract, 8-PN and 6-PN showed estrogenic activity. Hops extract and 6-PN activated both ERα and AHR pathways. 6-PN increased the expression of the tumor suppressor gene (AHRR), and that of genes involved in the estrogen metabolism (CYP1A1, CYP1B1). Although 6-PN might activate the detoxification and genotoxic pathways of estrogen metabolism, hops extract as a whole only modulated the genotoxic pathway by an up-regulation of CYP1B1 mRNA expression. These data demonstrate the relevant role of 6-PN contained in the hops extract as potential modulator of estrogen metabolism due to its ERα and AHR agonist activity.