Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro.

Alternaria molds can produce a variety of different mycotoxins, often resulting in food contamination with chemical mixtures, posing a challenge for risk assessment. Some of these metabolites possess estrogenic properties, an effect whose toxicological relevance is questioned in the light of the strong genotoxic and cytotoxic properties of co-occurring toxins. Thus, we tested a complex extract from A. alternata for estrogenic properties in Ishikawa cells. By assessing alkaline phosphatase activity, we did not observe estrogen receptor (ER) activation at non-cytotoxic concentrations (≤ 10 µg/ml). Furthermore, an extract stripped of highly genotoxic perylene quinones also did not mediate estrogenic effects, despite diminished genotoxic properties in the comet assay (≥ 10 µg/ml). Interestingly, both extracts impaired the estrogenicity of 17ß-estradiol (E2) at non-cytotoxic concentrations (5-10 µg/ml), indicating anti-estrogenic effects which could not be explained by the presence of known mycoestrogens. A mechanism for this unexpected result might be the activation of the aryl hydrocarbon receptor (AhR) by Alternaria metabolites, as indicated by the induction of CYP1A1 transcription. While a direct influence on the metabolism of E2 could not be confirmed by LC-MS/MS, literature describing a direct interplay of the AhR with estrogenic pathways points to a corresponding mode of action. Taken together, the present study indicates AhR-mediated anti-estrogenic effects as a novel mechanism of naturally co-occurring Alternaria toxin mixtures. Furthermore, our results confirm their genotoxic activity and raise questions about the contribution of still undiscovered metabolites to toxicological properties.

Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation.

Five new polyalkoxylated furofuranone lignan glucosides, terminalosides L-P (1-5), were isolated from EtOAc extracts of the leaves of Terminalia citrina, a Bangladeshi medicinal plant. The structures of the isolates were deduced primarily by NMR spectroscopy, and four of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. The absolute configurations and conformations of the furofuranone ring were confirmed by ECD spectroscopy. All of the isolates were evaluated for their estrogenic and/or antiestrogenic properties using two estrogen responsive breast cancer cell lines, T47D and MCF-7. At a concentration of 10nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common. This is the first report of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity.

Phenolic constituents of the Bangladeshi medicinal plant Pothos scandens and their anti-estrogenic, hyaluronidase inhibition, and histamine release inhibitory activities.

Extracts from the stem and roots of the Bangladeshi medicinal plant Pothos scandens L. (Araceae) were isolated, and three hemiterpene glucoside aromatic esters, pothobanosides A (1), B (2), and C (3), and a phenylisobutanoid, pothobanol (4), along with 14 known compounds, were characterized. The isolates were tested for their estrogenic/anti-estrogenic activity using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D, and syringoyl derivatives (2, 3, and canthoside B) showed strong inhibitory activity against both cell lines. Their less oxygenated analogs (1, and markhamioside F) were almost inactive. The isolates were also evaluated for hyaluronidase and histamine release inhibitory activities, and pothobanoside A (1) showed significant hyaluronidase inhibitory activity among the isolated compounds, which was similar to that of the positive control rosmarinic acid. Because hyaluronidase produces an angiogenic response that has been implicated in tumor invasiveness and metastasis, 1 could be valuable as an anti-tumor compound with a different mechanism of action from related compounds (2, 3). Pothobanoside C (3) and pothobanol (4) were also found to inhibit histamine release to a similar degree to the positive control epigallocatechin 3-O-(3"-O-methyl)-gallate. The histamine release inhibitory potency of these isolates may support the traditional uses of this plant in folk medicine.

Benzocoumarins: isolation, synthesis, and biological activities.

Benzocoumarins are coumarins with a phenyl group bonded to 3,4-, 5,6-, 6,7-, or 7,8-positions. Over the past years, significant efforts have been made not only to isolate the novel structural analogs of benzocoumarins with prominent bioactivities but also to design new synthetic methods to synthesize benzocoumarins with better or novel biological properties. The aim of this review is to provide the readers with an overview of the research progress of benzocoumarins from 1953 to May 2014, covering its isolation, synthesis, and biological activities.

Estrogenic and anti-estrogenic activities of hispolon from Phellinus lonicerinus (Bond.) Bond. et sing.

Hispolon was the main antitumor active ingredient in Phellinus sensu lato species. In order to confirm the dual regulating estrogenic ingredient and obtain more effective natural estrogen replacement drugs, hispolon was separated from Phellinus lonicerinus (Bond.) Bond. et sing. Hispolon exhibited significant anti-proliferative effect against estrogen-sensitive ER (+) MCF-7 cells in the absence of estrogen, and exhibits antagonistic effects on 17ß-estradiol (E2)-induced MCF-7 cell proliferation when E2 and the different concentrations of hispolon were treated simultaneously. Hispolon also inhibited the proliferation of estrogen-negative ER (-) MDA-MB-231 cells at the concentration of 5.00×10(-5) M. The yeast two-hybrid experiments showed that hispolon had strong and non-selective effects on the estrogen receptor (ER) α and ERß at a concentration of 1.00×10(-6) M. The ERß-binding ability of hispolon was larger than ERα in the concentration range of 1.00×10(-9) M and 1.00×10(-7) M. Hispolon could increase the body weight coefficient, serum E2 and progesterone contents in immature female mice at dose of 9.10×10(-6) mol/kg, and increase coefficient of thymus and spleen in mice. The Gscores of hispolon-ERα and hispolon-ERß docked complexes were -7.93 kcal/mol and -7.79 kcal/mol in docking simulations. Hispolon presented dual regulating estrogenic activities, which showed estrogenic agonist activity at low concentration or lack of endogenous estrogen, and the estrogenic antagonistic effect was stimulated at high concentrations or too much endogenous estrogen. Hispolon could be used for treating the estrogen deficiency-related disease with the benefit of non-toxic to normal cells, good antitumor effects and estrogenic activity.

Anti-estrogenic activity of mansonone G and mansorin A derivatives.

CONTEXT: Mansonone G and mansorin A are major bioactive constituents from Mansonia gagei Drumm (Sterculiaceae) wood, and their mild anti-estrogenic activity was reported previously by the authors. OBJECTIVE: In order to increase the potency of their anti-estrogenic effect and to clarify their binding way to estrogen receptor on a molecular level, several derivatives of both compounds will be prepared and a docking study of the original compounds and their derivatives on estrogen receptor alpha (ERα) was carried out. MATERIALS AND METHODS: The original compounds were isolated from the heartwood of M. gagei. Nine alkyl derivatives were prepared by acetylation, methylation, or adding a basic side chain to the free hydroxyl group of both compounds. The estrogenic/anti-estrogenic activities of the derivatives compared to the original compounds were carried out using ERα competitive binding screen and yeast two-hybrid assay expressing ERα and ERß using concentrations ranging from 10 to 100 µM. RESULTS: Acetyl mansonone G showed a 10-fold increase in its binding ability to ERα compared to mansonone G with an IC50 630 µM. Similarly, methyl mansonone G and acetyl mansonone G showed 50% and 35% inhibition of 17ß-estradiol-induced ß-galactosidase activity at 10 µM in the yeast expressing ERα, and 42% and 30%, respectively, at 10 µM in the yeast expressing ERß. Virtual docking of acetyl mansonone G to ERα showed that it binds, with its acetyl oxygen, in a similar way to the 17ß-OH of estradiol. DISCUSSION AND CONCLUSION: The phenolic hydroxyl group in mansonones and mansorins was not essential for binding to estrogen receptors. In addition, acetyl mansonone G could represent a promising starting material for the synthesis of anti-estrogenic agents.

Advancement in optimization tactic achieved by newly developed chromatographic response function: application to LC separation of raloxifene and its impurities.

In this paper a new chromatographic response function (CRF) is designed and proposed for utilization in the optimization strategies. The function capability to represent the overall quality of a experimentally obtained chromatograms was compared to the other two objective functions and proved to give more accurate and reliable results. The new CRF has improved concept of separation and time term estimation. It reflects all important defects of the chromatogram such as the appearance of asymmetrical or overlapping peaks and prolonged elution time and allows the appropriate weighting of each of them. The LC separation of raloxifene and its four impurities was evaluated through the central composite design experimental plan choosing the new CRF to be the only output of the system. The function demonstrated the ability to judge the impact of the complex interactions of the selected chromatographic parameters (acetonitrile content in the mobile phase, sodium dodecyl sulfate concentration in the water phase, pH of the mobile phase and column temperature) on the mixture behavior and led to the determination of the optimal separation conditions. The newly developed CRF proved to have the advanced performances and it presents the important step forward in the optimization of the chromatographic separation.

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements.

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.

Estrogenic and anti-estrogenic compounds from the Thai medicinal plant, Smilax corbularia (Smilacaceae).

From the rhizomes of Smilax corbularia Kunth. (Smilacaceae), 11 compounds, (2R,3R)-2″-acetyl astilbin, (2R,3R)-3″-acetyl astilbin, (2R,3R)-4″-acetyl astilbin, (2R,3R)-3″-acetyl engeletin, (2R,3S)-4″-acetyl isoastilbin, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10R)-2H,8H-benzo [1,2-b:3,4-b'] dipyran-8-one, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10S)-2H, 8H-benzo [1,2-b:3,4-b'] dipyran-8-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxy-phenyl)-5-[(1E)-2-(3,4-dihydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, and 5,7,3',4'-tetrahydroxy-3-phenylcoumarin along with 34 known compounds were isolated and characterized as 19 flavonoids, 14 catechin derivatives, 6 stilbene derivatives, and 6 miscellaneous substances. All isolates had their estrogenic and anti-estrogenic activities determined using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D. The major constituents were recognized as flavanonol rhamnosides by the suppressive effect on estradiol induced cell proliferation at a concentration of 1µM. Meanwhile, flavanonol rhamnoside acetates demonstrated estrogenic activity in both MCF-7 and T47D cells at a concentration of 100µM, and they enhanced the effects of co-treated E2 on T47D cell proliferation at concentrations of more than 0.1µM.

Estrogenic and antiestrogenic activities of phytoalexins from red kidney bean (Phaseolus vulgaris L.).

Legumes are the predominant source of isoflavones considered to be phytoestrogens that mimic the hormone 17ß-estradiol (E2). Due to the risks associated with hormone replacement therapy, there is a growing need for alternative sources of estrogenic formulations for the treatment of menopausal symptoms. Legume phytoalexins (induced isoflavones) are produced under conditions of stress that include insect damage, wounding, or application of elicitors. The estrogenic and antiestrogenic activities of methanolic extracts obtained from red kidney bean treated with the fungus Aspergillus sojae were compared with those of untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay. A. sojae-treated red kidney bean extracts displayed both estrogenic and antiestrogenic activities. Analysis of elicitor-treated red kidney bean extracts showed that A. sojae treatments achieved maximal levels of kievitone at 1199 ± 101 µg/g and phaseollin at 227.8 ± 44 µg/g. The phytoalexins kievitone and phaseollin were isolated from A. sojae-treated red kidney bean extracts and analyzed for estrogenic activity using ERα and ERß binding, ERE luciferase assays in MCF-7 and HEK 293 cells, and MCF-7 cell proliferation. Kievitone showed the highest relative binding affinity to ERα with kievitone (0.48%) > phaseollin (0.21%), and phaseollin showed the highest relative binding affinity to ERß with phaseollin (0.53%) > kievitone (0.42%). In an ERE luciferase assay in MCF-7 cells, kievitone displayed high ER transactivation at 10 µM; phaseollin displayed low ER transactivation. Both kievitone and phaseollin stimulated MCF-7 cell proliferation, with kievitone displaying agonist activity between 0.1 and 10 µM. Cotransfection reporter assays performed in HEK 293 demonstrated that phaseollin selectively increased ERE transcriptional activity of ERß and kievitone selectively increased ERE transcriptional activity of ERα. Although phaseollin displayed attenuation of ER transactivation in the ERE luciferase assay in MCF-7 cells, both phytoalexins attenuated the effects of E2 in an MCF-7 cell colonial survival assay. This work provides evidence that the red kidney bean phytoalexins kievitone and phaseollin possess both estrogenic and antiestrogenic activities.

Comparative X-ray and conformational analysis of a new crystal of 13α,21-dihydroeurycomanone with eurycomanone from Eurycoma longifolia and their anti-estrogenic activity using the uterotrophic assay.

13 α,21-Dihydroeurycomanone (1), a known quassinoid of Eurycoma longifolia Jack was recrystallized from chloroform into a novel crystal structure in space group P2 (1). Its X-ray data were compared with those of eurycomanone ( 2). Following intraperioneal injections at similar doses of 2.44 µmol/kg/day for 3 consecutive days, 2 displayed comparable potency with tamoxifen but was more potent than 1 in the anti-estrogenic effect against 17 α-ethynylestradiol (EE)-induced uterotrophy of immature rats.

Estrogenic and anti-estrogenic activities of the Thai traditional herb, Butea superba Roxb.

This study evaluated the estrogenic and antiestrogenic activities of native and in vitro hepatic metabolized tuberous extracts of wild Butea superba collected from 23 out of the 76 provinces in Thailand by yeast estrogen screening (YES). The YES screen used consisted of the human estrogen receptors hERα and hERß and the human transcriptional intermediary factor 2 or human steroid receptor coactivator 1, respectively, together with the ß-galactosidase expression cassette as the reporter. The relative potency, effectiveness and relative inductive efficiency were evaluated by determining the ß-galactosidase activity (EC(50)) of each tuberous extract in relation to that induced by 17ß-estradiol. Six pure compounds isolated from B. superba were tested in parallel and exhibited a maximum relative potency compared to 17ß-estradiol of 15.5% and 5.27% in the respective hERα and hERß assays. Eighteen and seventeen plant extracts were respectively found to interact with the hERα and hERß receptors in the YES assays with higher relative potency and relative inductive efficiency with hERß than with hERα. The selected plant extracts tested exhibited antiestrogenic activity. Coincubation with the rat liver S9 mixture also elevated the estrogenic potency of these plant extracts.

Estrogenic activity of a naringinase-treated extract of Sophora japonica cultivated in Egypt.

The naringinase-treated methanol extract of Sophora japonica L. (Fabaceae) seeds showed potent estrogen agonist activity. Through bioassay-guided isolation of the main active constituents from the naringinase-treated methanol extract of S. japonica, the aglycones genistein and kaempferol were found to be the main phytoestrogens in the naringinase-treated extract. In addition, kaempferol was nearly equipotent to genistein as an estrogen agonist. Concerning the compounds isolated from the untreated methanol extract, sophoricoside showed weak estrogenic activity on ERbeta only.

Schistosoma haematobium: identification of new estrogenic molecules with estradiol antagonistic activity and ability to inactivate estrogen receptor in mammalian cells.

We have previously identified the expression of an estradiol (E2)-related molecule by Schistosoma haematobium total antigen (Sh). We now show that this molecule has an antagonistic effect of estradiol in vitro. Our results are consistent with the existence of an estrogenic molecule that antagonizes the activity of estradiol. We found evidence for this molecule as we identified and characterized by mass spectrometry new estrogenic molecules previously unknown, present in schistosome worm extracts and sera of Schistosoma-infected individuals. We also show that Sh is able to interact in vitro with estrogen receptor (ER), explaining how host endocrine system can favor the establishment of schistosomes. These findings highlight the exploitation of the host endocrine system by schistosomes and represent an additional regulatory component of schistosome development that defines a novel paradigm enabling host-parasite interactions. The identification of these molecules opens new ways for the development of alternative drugs to treat schistosomiasis.

A set of time-resolved fluorescence resonance energy transfer assays for the discovery of inhibitors of estrogen receptor-coactivator binding.

Therapeutic block of estrogen action is typically achieved with conventional antagonists (CAs), compounds that displace estradiol from the estrogen receptor (ER) and induce formation of an ER conformation that cannot bind to coactivator proteins, such as the steroid receptor coactivators (SRCs). As an alternative mode for blocking estrogen action, the authors seek small molecules that act as coactivator binding inhibitors (CBIs)-that is, they compete directly with SRC3 for interaction with estradiol-bound ER. CBIs would be interesting mechanistic probes of estrogen action and might also provide an alternative, more durable endocrine therapy for hormone-responsive breast cancer, where cellular adaptations lead to resistance to CAs. The authors have designed and optimized a set of time-resolved fluorescence resonance energy transfer (TR-FRET) assays to monitor the interaction of ER with SRC3 and ligands, and they have used them in high-throughput screens to discover small-molecule CBIs that are able to disrupt this interaction. These assays also distinguish CBIs from CAs. These robust and sensitive "mix-and-measure" assays use low concentrations of ER labeled with a europium chelate as FRET donor and a Cy5-labeled SRC as acceptor. This multiplexed protocol produces excellent signal-to-noise ratios (>100) and Z' values (>0.8).

Anti-estrogenic activity of mansorins and mansonones from the heartwood of Mansonia gagei DRUMM.

Through an anti-estrogenic bioassay-guided fractionation of the methanol extract of Mansonia gagei, three new coumarins, called mansorins I (1), II (2) and III (3) and a new naphthoquinone, mansonone I (4), were isolated. Their structures were determined based on their NMR data and CD spectroscopy. The anti-estrogenic activity of the fractions and the isolated compounds were investigated using a yeast two-hybrid assay method expressing estrogen receptors alpha (ERalpha) and beta (ERbeta). In addition, an ERalpha competitor screening system (ligand binding screen) was used to verify the binding affinities of the isolated compounds to the estrogen receptor. 1,2-Naphthoquinones (mansonones) showed more binding affinities to ER in both assay systems. All the tested compounds showed higher binding affinities to ERbeta than to ERalpha in the yeast two-hybrid assay. Mansonones F and S showed the most potent estrogen binding and estrogen antagonistic effects.

Improved antitumoral properties of pure antiestrogen RU 58668-loaded liposomes in multiple myeloma.

In most of multiple myeloma (MM) cells, the "pure" antiestrogen (AE) RU 58668 (RU) induced either a G1-arrest (LP-1, OPM-2, NCI-H929, U266 cells) or apoptosis (RPMI 8226 cells). In RPMI 8226 cells, RU activates a caspase-dependent cell death pathway leading to the release of cytochrome c, the decrease of the essential MM survival factor Mcl-1, the cleavage of Bid and the activation of caspases-3 and -8. Incorporation of RU in pegylated cholesterol-containing liposomes allowed a controlled RU release, improving its anti-proliferative and apoptotic effects in cells. In RPMI 8226 xenografts, i.v. injected RU-liposomes but not free RU, exhibited antitumor activity. In vivo, RU-liposomes triggered the mitochondrial death pathway, concomitantly with a down-regulation of Mcl-1 and Bid cleavage. The decrease of CD34 immunoreactivity indicated a reduction of angiogenesis. The decrease of VEGF secretion in vitro supported a direct effect of RU on angiogenesis. These pro-apoptotic and antiangiogenic effects were explained by a prolonged exposure to the drug and to the endocytosis capacity of liposomes which might increase RU uptake and bypass a membrane export of free RU. Thus, these combined enhanced activities of RU-liposomes support that such a delivery of an AE may constitute a strategy of benefit for MM treatment.

The transgenic Arabidopsis plant system, pER8-GFP, as a powerful tool in searching for natural product estrogen-agonists/antagonists.

The transgenic Arabidopsis plant system, pER8-GFP, may be used as a powerful tool in searching for natural estrogen-agonists/antagonists. Among selected plant extracts and natural products, the method was able to distinguish active extracts (e.g., Glycine max and Pueraria lobata) and pure compounds (e.g., 17beta-estradiol (1), genistein (10), and daidzein (11)) and also to distinguish effects of structural changes on activity. Thus, this rapid sensitive system was proven to be suitable for the discovery of natural products with estrogenic activity.

Anti-estrogenic activity of lignans from Acanthopanax chiisanensis root.

Anti-estrogenic activity of (-)-sesamin (1), helioxanthin (2), savinin (3), taiwanin C (4), and 3-(3,4-dimethoxybenzyl)-2-(3,4-methylenedioxybenzyl)butyrolactone (5) isolated from the root of Acanthopanax chiisanensis was tested using Ishikawa cells. Among them, compound 3 exhibited anti-estrogenic activity (IC50 = 4.86 microM).

Estrogenic and antiestrogenic activities of the roots of Moghania philippinensis and their constituents.

In the course of our search for natural estrogenic compounds from medicinal plants, we found that the methanolic extract from the roots of Moghania philippinensis (Fabaceae) showed significant effects on the proliferation of MCF-7 cells (human breast cancer) and induction of beta-galactosidase activity in a yeast two-hybrid assay. Through estrogenic activity-guided fractionation, we isolated several active flavonoids including prenylated ones. The CHCl(3) fraction and its new constituent, 8-(1,1-dimethylallyl)genistein (9), appreciably increased the uterine weight in ovariectomized rats when administered orally for 14 consecutive days, in which compound 9 showed stronger estrogenic activity than genistein. Antiestrogenic activities were also examined based on the inhibition of MCF-7 cell proliferation and beta-galactosidase activity in the yeast two-hybrid assay, mediated by 17beta-estradiol. 5,7,3',4'-Tetrahydroxy-6,8-diprenylisoflavone (6) showed the strongest antiestrogenic activity.