Enrichment of soy isoflavone extracts through macroporous resin for characterization of toxicity and estrogenic activities.

Soy isoflavone extracts are widely researched for their distinctive potential in contributing to various functional foods. The research work focuses on testing the toxicity of purified soy isoflavone extracts in mice models. With an agreement of the animal ethics, acute toxicity is firstly used to screen the effects of test compounds in mice for therapeutic purposes. Moreover, tests were conducted on BALB/c for estrogen in vivo and MCF7 for in vitro, screening active protection of liver cells, lipid peroxidation and scavenging free radicals 2,2-diphenyl-1-picrylhydrazyl (DPPH). Genistin and daidzin were found to be the two major compounds accounting for 47% and 35% of total purified soy isoflavones. The acute toxicity test results exhibited no effect against physiological accretion of BALB/c after 7-day administration with the given dose of 10 g/kgBW. Moreover, modified E-screen assay on MCF7 cells proved that the estrogen of isoflavone extracts induces cell proliferation by 15% compared with other non-steroid culture techniques. Therefore, this research contributes to helping researchers apply soy isoflavones in functional food, to alleviate the difficulties in menopausal symptoms for women in the future. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05491-4.

In vitro and in vivo estrogenic activity of triclosan.

Triclosan (TCS) is an antibacterial and antifungal agent used in many consumer products and exhibits a chemical structure similar to non-steroidal estrogen, which is known to induce endocrine disruption. Triclosan has been found in human plasma, urine, and breast milk, and the safety of TCS-containing products has been disputed. Although studies attempted to determine the estrogenic activity of TCS, no clear results have emerged. The aim of the present study was to examine estrogenic activity of TCS using an in vitro E-screen assay and an in vivo uterotrophic assay. The in vitro E-screen assay demonstrated that TCS significantly enhanced proliferation of MCF-7 breast cancer cells, although not in a concentration-dependent manner. The in vivo uterotrophic results showed no significant change in the weight of uteri obtained from TCS-administered Sprague-Dawley rats. Further, to understand the estrogenic activity attributed to TCS at the molecular level, gene-expression profiling of uterus samples was performed from both TCS- or estrogen-treated rats and the genes and cellular processes affected by TCS or estrogen were compared. Data demonstrated that both the genes and cellular processes affected by TCS or estrogen were significantly similar, indicating the possibility that TCS-mediated estrogenic activity occurred at the global transcriptome level. In conclusion, in vitro and gene-profiling results suggested that TCS exhibited estrogenic activity.

Efficacy and reusability of mixed-phase TiO2-ZnO nanocomposites for the removal of estrogenic effects of 17ß-Estradiol and 17α-Ethinylestradiol from water.

Photocatalytic removal of estrogenic compounds (ECs), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2) were assessed using a TiO2-ZnO nanocomposite (NC) over a range of initial EC concentration (Co; 10 mg/L - 0.05 mg/L). Photocatalytic removal was evaluated under UV and visible irradiation using 10 mg/L NC over 240 min duration. After 240 min, analysis using GCxGC TOF MS revealed 100% transformation at Co ≤ 1 mg/L and ≥25% transformation at Co ≤ 10 mg/L under visible irradiation. Degradation was accompanied by breakdown of the fused ring structure of E2, generating smaller molecular weight by-products which were subsequently mineralized as revealed through TOC removal. With UV photocatalysis, ~30% and ~20% mineralization was attained for E2 and EE2, respectively, for Co of 10 mg/L. Under visible irradiation, ~25% and ~10% mineralization was achieved for E2 and EE2, respectively. Estrogenicity variation was estimated using the E-screen assay conducted with estrogen receptor-positive MCF-7 breast cancer cells. Complete removal of estrogenicity of ECs was confirmed after 240 min of photocatalysis under UV and visible irradiation. FTIR spectroscopy-based analysis of the NC after E2 photocatalysis revealed the presence of sorbed organics. Desorption, followed by GC × GC TOF-MS analysis revealed these organics as by-products of photocatalysis. Desorption of sorbed organics followed by recalcination at 600 °C for 1 h regenerated the active sites on the NC, enabling its efficient reuse for 3 cycles under visible irradiation without loss in activity.

Contaminants of emerging concern in drinking water: Quality assessment by combining chemical and biological analysis.

Drinking water quality is a priority issue of the environmental policy agenda, however regulation on Contaminants of Emerging Concern (CECs) is limited. A proposal to revise the Drinking Water Directive has recently been approved (EU Council 2020), which updates the quality standards and introduces the watch list mechanism, including for the first time endocrine disruptors and pharmaceuticals. The purpose of this study was to evaluate the occurrence of selected CECs in surface water at the entrance of drinking water treatment plants (DWTPs) and in treated water, ready for distribution in the network. Samples were collected at three different DWTPs (Italy) and CECs assessed by LC-MS/MS were the following: bisphenol A (BPA), nonylphenol (NP), octylphenol, perfluorooctanesulfonic and perfluorooctanoic acids (PFOS and PFOA), atenolol, caffeine (CFF), carbamazepine (CBZ), estrone, 17-ß-estradiol, 17-α-ethinyl estradiol, diclofenac, and ibuprofen. In addition, biological analyses were performed to ascertain cumulative estrogenic and/or genotoxic potential of the samples. CFF, NP, PFOA, BPA, and CBZ were the most frequently detected contaminants, found in treated water in the following ranges: CFF 12.47-66.33 ng/L, NP 7.90-53.62 ng/L, PFOA

In vitro estrogen-disrupting effects of organophosphate flame retardants.

Organophosphate flame retardants (OPFRs), as substitutes for polybrominated diphenyl ethers (PBDEs), are frequently detected in the environment and biota due to their widespread use. Thus, there is a need to investigate their potential estrogen-disrupting effects and possible mechanisms of action in an effort to obtain a better risk assessment. In this study, we characterized the activities on estrogen receptor α (ERα) and the estrogen-disrupting potential of fourteen OPFRs, TMP, TEP, TPP, TnBP, TiBP, THP, TPhP, TCP, DPK, MDPP, IDPP, CDP, IPPDP and MPhP, using three in vitro assays representing different specific modes of action (MoAs). In the yeast two-hybrid assay, no OPFRs induced agonistic activity, but TiBP, DPK, TPhP, MDPP, CDP and IPPDP were shown to be hydrophobicity-dependent antagonists and to compete with E2 for binding to ERα. In the MVLN cell assay, TPhP was the only OPFR among the 14 tested that was able to activate ERα-estrogen responsive element (ERE) pathways. The results from the E-SCREEN assay showed that all tested OPFRs except TMP had estrogenic properties, and G protein-coupled receptor 30 (GPR30) was involved in the estrogenicity of eight OPFRs, TiBP, THP, TPhP, TCP, MDPP, IPPDP, CDP and MPhP. It was also found that in the E-SCREEN assay, the estrogenicity of alkyl-OPFRs but not aryl-OPFRs was closely correlated to hydrophobicity. Our research suggested that most OPFRs were estrogen disruptors, but their related mechanisms were complex and might involve ERα-mediated and/or ERα-independent pathways. Further in vitro studies concerning the estrogenic effects and involved mechanisms of OPFRs, as well as comprehensive evaluations of OPFRs including health and ecological assessments are needed to determine whether they are safe substitutes for PBDEs.

Interactions between plant-derived oestrogenic substances and the mycoestrogen zearalenone in a bioassay with MCF-7 cells.

Human and animal diets may contain several non-steroidal oestrogenic compounds which originate either from plants (phytoestrogens) or from fungi that infect plants (mycoestrogens such as zearalenone (ZEN)). Phytoestrogens may compete with ZEN in binding to the oestrogen receptor ß and thereby may counteract the oestrogenic activity of ZEN. Using a modified version of the E-screen assay, plant-derived oestrogenic substances were tested for their proliferative or anti-proliferative effect on oestrogen-dependent MCF-7 cells. The samples were additionally tested for their ability to influence the oestrogenic activity of ZEN (1 µM). Among the individual substances tested, 8-prenylnaringenin had the strongest effect, as cell proliferation was increased by 78% at the lowest concentration (0.23 µM), and by 167% at the highest concentration (29.4 µM). Coumestrol (5.83 µM) increased cell proliferation by 39%, and genistein (370 µM) by 61%, respectively. Xanthohumol and enterolactone did not stimulate cell proliferation significantly. In the co-incubation experiments with ZEN, none of the single substances was able to decrease the oestrogenic activity of ZEN. Only for 8-prenylnaringenin (14.7 and 29.4 µM) was a trend towards an increase in the ZEN-induced cell proliferation up to 72% observed. In conclusion, with the exception of 8-prenylnaringenin, no substantial interaction between phytoestrogens and the mycotoxin ZEN could be detected using a bioassays with MCF-7 cells.

Estrogen-like and tissue-selective effects of 7-methoxycoumarin from Ficus umbellata (Moraceae): an in vitro and in vivo study.

BACKGROUND: Ficus umbellata is a medicinal plant previously shown to endow estrogenic properties. Its major component was isolated and characterized as 7-methoxycoumarin (MC). Noteworthy, coumarins and the respective active metabolite 7-hydroxycoumarin analogs have shown aromatase inhibitory activity, which is of particular interest in the treatment of estrogen-dependent cancers. The present work aimed at evaluating the estrogenic/antiestrogenic effects of MC in vitro and in vivo. METHODS: To do so, in vitro assays using E-screen and reporter gene were done. In vivo, a 3-day uterotrophic assay followed by a postmenopausal-like rat model to characterize MC as well as F. umbellata aqueous extract in ovariectomized Wistar rats was performed. The investigations focused on histological (vaginal and uterine epithelial height) and morphological (uterine wet weight, vagina stratification and cornification) endpoints, bone mass, biochemical parameters and lipid profile. RESULTS: MC induced a significant (p < 0.05) MCF-7 cell proliferation at a concentration of 0.1 µM, but did not inhibit the effect induced by estradiol in both E-screen and reporter gene assays. In vivo, MC treatment did not show an uterotrophic effect in both rat models used. However, MC (1 mg/kg) induced a significant increase (p < 0.01) of vaginal epithelial height. No significant change was observed with MC in abdominal fat weight, serum lipid levels and bone weight. CONCLUSION: These results suggest that MC has a weak estrogenic activity in vitro and in vivo that accounts only in part to the estrogenicity of the whole plant extract. MC could be beneficial with regard to vagina dryness as it showed a tissue specific effect without exposing the uterus to a potential tumorigenic growth.

Estrogenic activity of zinc pyrithione: an in vivo and in vitro study.

Zinc pyrithione (ZP) is commonly used to prevent dandruff and seborrheic dermatitis. Many consumers are exposed daily to high doses of ZP, causing serious concerns about its toxicity. The reproductive and developmental toxicities were previously reported in pregnant rats. However, the estrogenic activity of ZP at varying degrees of exposure has been rarely studied. Thus, we performed an uterotrophic assay, E-screen assay, and gene expression profiling to assess the estrogenic activity of ZP. For the uterotrophic assay, ZP (2, 10, or 50 mg/kg/d) was subcutaneously administered to ovariectomized rats every day for three days. Uteri were extracted 24 hours after the last dose. Then, wet and blotted uterine weights were measured. For the E-screen essay, MCF-7 cells (a breast cancer cell line) were exposed to 10-9 to 10-6 M of ZP, and cell proliferation was then measured. For the gene expression analysis, changes of gene expression levels in uterine samples taken for the uterotrophic assay were analyzed. In the uterotrophic assay, the concentration of ZP had no significant effect on uterine weight. In the E-screen assay, ZP at any concentration showed no significant increase in MCF-7 cell proliferation, compared to the control group. However, 10-6 M of ZP significantly reduced cell viability. The changes in gene expression slightly differed between the ZP and control groups. The in vivo and in vitro assays, together with gene expression analysis, demonstrated that ZP showed no significant estrogenic activity.

Ethanol-extracted Cameroonian propolis exerts estrogenic effects and alleviates hot flushes in ovariectomized Wistar rats.

BACKGROUND: Since the biological properties of propolis depend to the plants that can be found in a specific region, propolis from unexplored regions attracts the attention of scientists. Ethanolic extract of Cameroonian propolis (EEP) is used to treat various ailments including gynecological problems and amenorrhea. Since there were no scientific data to support the above claims, the present study was therefore undertaken to assess estrogenic properties of Cameroonian propolis. METHODS: To achieve our goal, the ability of EEP to induce MCF-7 cells proliferation in E-screen assay as well as to activate estrogen receptors α (ERα) and ß (ERß) in cell-based reporter gene assays using human embryonic kidney cells (HEK293T) transfected with ERs was tested. Further, a 3-day uterotrophic assay was performed and the ability of EEP to alleviate hot flushes in ovariectomized adult rats was evaluated. RESULTS: In vitro, EEP showed an antiestrogenic activity in both HEK293T ER-α and ER-ß cells. In vivo, EEP induced a significant increase in a bell shape dose response manner of the uterine wet weight, the total protein levels in the uterus, the uterine and vaginal epithelium height and acini border cells of mammary gland with the presence of abundant eosinophil secretions. Moreover, EEP induced a significant decrease in the total number, average duration as well as frequency of hot flushes after 3 days of treatment in rat (equivalent to a month in woman). The dose of 150 mg/kg exhibited the most potent estrogenic effects among all the tested doses. The UPLC-HRMS analysis showed the presence of caffeic acid derivatives and trirtepernoids in EEP, which are well known endowed with estrogenic properties. CONCLUSION: These results suggest that Ethanolic extract of Cameroonian propolis has estrogen-like effects in vivo and may alleviate some menopausal problems such as vaginal dryness and hot flushes. Ethanol-extracted Cameroobian propolis exhibited in vitro and in vivo estrogen-like effects. This extract may contain promising phytoestrogens.

Estrogenic activity of zinc pyrithione: an in vivo and in vitro study

Zinc pyrithione (ZP) is commonly used to prevent dandruff and seborrheic dermatitis. Many consumers are exposed daily to high doses of ZP, causing serious concerns about its toxicity. The reproductive and developmental toxicities were previously reported in pregnant rats. However, the estrogenic activity of ZP at varying degrees of exposure has been rarely studied. Thus, we performed an uterotrophic assay, E-screen assay, and gene expression profiling to assess the estrogenic activity of ZP. For the uterotrophic assay, ZP (2, 10, or 50 mg/kg/d) was subcutaneously administered to ovariectomized rats every day for three days. Uteri were extracted 24 hours after the last dose. Then, wet and blotted uterine weights were measured. For the E-screen essay, MCF-7 cells (a breast cancer cell line) were exposed to 10⁻⁹ to 10⁻⁶ M of ZP, and cell proliferation was then measured. For the gene expression analysis, changes of gene expression levels in uterine samples taken for the uterotrophic assay were analyzed. In the uterotrophic assay, the concentration of ZP had no significant effect on uterine weight. In the E-screen assay, ZP at any concentration showed no significant increase in MCF-7 cell proliferation, compared to the control group. However, 10⁻⁶ M of ZP significantly reduced cell viability. The changes in gene expression slightly differed between the ZP and control groups. The in vivo and in vitro assays, together with gene expression analysis, demonstrated that ZP showed no significant estrogenic activity.

Estrogenic activity of zinc pyrithione: an in vivo and in vitro study

Zinc pyrithione (ZP) is commonly used to prevent dandruff and seborrheic dermatitis. Many consumers are exposed daily to high doses of ZP, causing serious concerns about its toxicity. The reproductive and developmental toxicities were previously reported in pregnant rats. However, the estrogenic activity of ZP at varying degrees of exposure has been rarely studied. Thus, we performed an uterotrophic assay, E-screen assay, and gene expression profiling to assess the estrogenic activity of ZP. For the uterotrophic assay, ZP (2, 10, or 50 mg/kg/d) was subcutaneously administered to ovariectomized rats every day for three days. Uteri were extracted 24 hours after the last dose. Then, wet and blotted uterine weights were measured. For the E-screen essay, MCF-7 cells (a breast cancer cell line) were exposed to 10⁻⁹ to 10⁻⁶ M of ZP, and cell proliferation was then measured. For the gene expression analysis, changes of gene expression levels in uterine samples taken for the uterotrophic assay were analyzed. In the uterotrophic assay, the concentration of ZP had no significant effect on uterine weight. In the E-screen assay, ZP at any concentration showed no significant increase in MCF-7 cell proliferation, compared to the control group. However, 10⁻⁶ M of ZP significantly reduced cell viability. The changes in gene expression slightly differed between the ZP and control groups. The in vivo and in vitro assays, together with gene expression analysis, demonstrated that ZP showed no significant estrogenic activity.

Estrogenic and cytotoxic potentials of compounds isolated from Millettia macrophylla Benth (Fabaceae): towards a better understanding of its underlying mechanisms.

BACKGROUND: Millettia macrophylla was previously reported to have estrogenic effects and to prevent postmenopausal osteoporosis in Wistar rats. So, the study deals with the identification of its secondary metabolites and the evaluation of their estrogenicity and cytotoxicity toward tumoural cells. Thus, 13 known compounds were obtained from successive chromatographic columns and identified by NMR data compared to those previously reported. METHODS: In vitro estrogenicity of the isolates and the phenolic fraction (PF) of M. macrophylla were performed by E-screen and reporter gene assays, while their cytotoxicity was evaluated by Alamar Blue (resazurin) assay. A 3-days uterotrophic assay and the ability of PF to alleviate hot flushes in ovariectomized adult rats were tested in vivo. RESULTS: Seven of the 13 secondary metabolites turned to be estrogenic. Only two exhibited cytotoxic effects on MCF-7 and MDA-MB-231 with CC50 values of 110 µM and 160 µM, respectively. PF induced a significant (p < 0.01) MCF-7 cells proliferation and transactivated both ERα and ERß in the reported gene assay at 10-2 µg/mL. In vivo, PF acted more efficiently than the methanol crude extract, resulting to a significant (p < 0.01) increase in the uterine wet weight, uterine protein level, uterine and vaginal epithelial height at the dose of 10 mg/kg BW. In addition, PF reduced the average duration and frequency of hot flushes induced in rat. CONCLUSION: These aforementioned results indicate that PF is a good candidate for the preparation of an improved traditional medicine able to alleviate some menopausal complaints such as vaginal dryness and hot flushes. Estrogenic and cytotoxic potentials of compounds isolated from Millettia macrophylla Benth. (Fabaceae): towards a better understanding of its underlying mechanism.

Estrogenicity of novel phase I and phase II metabolites of zearalenone and cis-zearalenone.

Zearalenone and its cis-isomer, cis-zearalenone, are nonsteroidal mycotoxins that elicit an estrogenic response upon binding to the estrogen receptor. This study compares the estrogenicity of eleven congeners including novel metabolites as 15-OH-zearalenone, zearalenone-14-sulfate, α-cis-zearalenol and ß-cis-zearalenol using the E-Screen assay. Overall, a change in the configuration from trans to cis retains significant estrogenic activity. In contrast, alterations of the aromatic moiety including hydroxylation and sulfation showed a markedly decreased estrogenicity when compared to zearalenone.

Estrogenic activities of four components from pregnant mare’s urine extract / 中国药理学通报

Aim To investigate the estrogenic activities of four components from pregnant mare’s urine extract. Methods The estrogenic activities of four components were assessed using two in vitro tests:the MCF-7 cell proliferation assay (E-screen test)and the luciferase transfected CHO cell gene reporter assay.In the lucifer-ase reporter gene assay,the reporter gene plasmids PGM-ERE-Luc and ERαor ERβand a control plasmid (pRL-cmv)were transiently co-transfected into CHO cells to establish an ERα-or ERβ-cell screening system which was used to measure estrogenic activity of four compounds.Results MCF-7 cells treated with HP, DHP,PT and HA significantly proliferated,thereby of-fering in vitro evidence for the estrogenic activities of HP,DHP,PT and HA,and they showed dose-depend-ent activities.Compared EC50 of PE and RPE with that of E2 ,HP,DHP,PT and HA exerted relatively weak estrogenic activities.The in vitro ER-mediated reporter gene assay revealed that HP,DHP,PT and HA dis-played estrogenic activities mediated by ERβor ERα. Compared with the EC50 of E2 ,HP,DHP,PT and HA exhibited lower estrogenic potencies.Conclusion HP, DHP,PT and HA possess weaker estrogenic activities than E2 .

Estrogenic activity of constituents of underarm deodorants determined by E-Screen assay.

The purpose of this study was to ascertain whether different kinds of underarm deodorants commercially available in Germany might contain substances with estrogenic potential which after use enter the aquatic environment via wastewater. Twenty five deodorants produced by ten different manufacturers in the form of sprays, roll-ons and sticks were investigated using an in vitro-test system (E-Screen assay) for the determination of estrogenic activity based on the human breast cancer cell line MCF-7. Seven out of ten spray deodorant samples showed a quantifiable estrogenic activity. In the case of the sticks and roll-ons it was only one out of six and one out of nine, respectively. The 17ß-estradiol equivalent concentrations (EEQs) of the samples ranged from 0.1 ng g(-1) to 9 ng g(-1) deodorant. Spray deodorant samples showed the highest activities in the E-Screen assay compared to the stick and roll-on deodorants. In order to identify substances possibly contributing to the observed biological activity the samples were additionally analyzed by GC/MS. The obtained results of this non-target screening led to the selection of 62 single substances present in the deodorants which for their part were analyzed by E-Screen assay. Eight of these single substances, all of them fragrances, showed estrogenic effects with estradiol equivalence factors (EEFs) similar to parabens, a group of 4-hydroxybenzoic acid esters commonly used as preservatives in personal care products, which are known to have a slight estrogenic effect. Thus, these fragrances are obviously responsible to a substantial degree for the observed estrogenic activity of the deodorants.