Facile screening of potential xenoestrogens by an estrogen receptor-based reusable optical biosensor.

PubMed

Liu, Lanhua; Zhou, Xiaohong; Lu, Yun; Shan, Didi; Xu, Bi; He, Miao; Shi, Hanchang; Qian, Yi

2017-11-15

The apparent increase in hormone-induced cancers and disorders of the reproductive tract has led to a growing demand for new technologies capable of screening xenoestrogens. We reported an estrogen receptor (ER)-based reusable fiber biosensor for facile screening estrogenic compounds in environment. The bioassay is based on the competition of xenoestrogens with 17β-estradiol (E 2 ) for binding to the recombinant receptor of human estrogen receptor α (hERα) protein, leaving E 2 free to bind to fluorophore-labeled anti-E 2 monoclonal antibody. Unbound anti-E 2 antibody then binds to the immobilized E 2 -protein conjugate on the fiber surface, and is detected by fluorescence emission induced by evanescent field. As expected, the stronger estrogenic activity of xenoestrogen would result in the weaker fluorescent signal. Three estrogen-agonist compounds, diethylstilbestrol (DES), 4-n-nonylphenol (NP) and 4-n-octylphenol (OP), were chosen as a paradigm for validation of this assay. The rank order of estrogenic potency determined by this biosensor was DES>OP>NP, which were consistent with the published results in numerous studies. Moreover, the E 2 -protein conjugate modified optical fiber was robust enough for over 300 sensing cycles with the signal recoveries ranging from 90% to 100%. In conclusion, the biosensor is reusable, reliable, portable and amenable to on-line operation, providing a facile, efficient and economical alternative to screen potential xenoestrogens in environment. Copyright © 2017 Elsevier B.V. All rights reserved.

COMPARISON OF THE ESTROGENIC POTENCIES OF ESTRADIOL, ETHYNYLESTRADIOL, DIETHYLSTILBESTROL, NONYLPHENOL AND METHOXYCHLOR IN VIVO AND IN VITRO

EPA Science Inventory

Five natural, pharmaceutical, or xenobiotic chemicals (17b-estradiol, ethynylestradiol, diethystilbestrol, nonylphenol, methoxychlor) were tested in two in vitro (MCF-7 breast tumor cell proliferation [E-screen], yeast estrogen system [YES]), and one in vivo (male sheepshead min...

Development of a New Decision Tree to Rapidly Screen Chemical Estrogenic Activities of Xenopus laevis.

PubMed

Wang, Ting; Li, Weiying; Zheng, Xiaofeng; Lin, Zhifen; Kong, Deyang

2014-02-01

During the last past decades, there is an increasing number of studies about estrogenic activities of the environmental pollutants on amphibians and many determination methods have been proposed. However, these determination methods are time-consuming and expensive, and a rapid and simple method to screen and test the chemicals for estrogenic activities to amphibians is therefore imperative. Herein is proposed a new decision tree formulated not only with physicochemical parameters but also a biological parameter that was successfully used to screen estrogenic activities of the chemicals on amphibians. The biological parameter, CDOCKER interaction energy (Ebinding ) between chemicals and the target proteins was calculated based on the method of molecular docking, and it was used to revise the decision tree formulated by Hong only with physicochemical parameters for screening estrogenic activity of chemicals in rat. According to the correlation between Ebinding of rat and Xenopus laevis, a new decision tree for estrogenic activities in Xenopus laevis is finally proposed. Then it was validated by using the randomly 8 chemicals which can be frequently exposed to Xenopus laevis, and the agreement between the results from the new decision tree and the ones from experiments is generally satisfactory. Consequently, the new decision tree can be used to screen the estrogenic activities of the chemicals, and combinational use of the Ebinding and classical physicochemical parameters can greatly improves Hong's decision tree. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A simple heterogeneous one-step assay for screening estrogenic compounds.

PubMed

Huovinen, Tuomas; Rytkönen, Kalle; Lamminmäki, Urpo; Pellinen, Teijo

2013-01-01

Estrogen receptor (ER) modulators are a serious health issue but estrogenic compounds, especially antagonists of ER function, are widely screened for in search of novel therapeutics against hormonal diseases such as the breast cancer. Here we report a novel and a simple bioassay for estrogenic and anti-estrogenic compounds based on ligand-dependent recruitment of ER co-activator steroid receptor co-activator 1 (SRC-1) to purified Renilla luciferase-tagged ERα. In this assay, in vivo-biotinylated (E. coli) SRC-1, purified Renilla luciferase-ERα, and the analyte sample are mixed and incubated for 2 h in a streptavidin-coated microtiter wells, and after one washing step, luminescence is measured with a simple instrument. The assay does not require chemical labeling of the components and shows good sensitivity (25 pM E(2)) and wide dynamic range of more than four orders of magnitude.

A PROTEOMIC (SELDI-TOF-MS) APPROACH TO ESTROGEN AGONIST SCREENING

EPA Science Inventory

A small fish model and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were place...

Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay.

PubMed Central

Miller, D; Wheals, B B; Beresford, N; Sumpter, J P

2001-01-01

We used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery. Thirty-two of these compounds displayed activity: 22 with potencies relative to 17beta-estradiol, ranging from 1/3,000 to < 1/3,000,000, and 10 compounds with an impaired response that could not be directly compared with 17beta-estradiol. Forty-one compounds were inactive. The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da. PMID:11266322

In vitro and in vivo estrogenicity of UV screens.

PubMed Central

Schlumpf, M; Cotton, B; Conscience, M; Haller, V; Steinmann, B; Lichtensteiger, W

2001-01-01

Ultraviolet (UV) screens are increasingly used as a result of growing concern about UV radiation and skin cancer; they are also added to cosmetics and other products for light stability. Recent data on bioaccumulation in wildlife and humans point to a need for in-depth analyses of systemic toxicology, in particular with respect to reproduction and ontogeny. We examined six frequently used UVA and UVB screens for estrogenicity in vitro and in vivo. In MCF-7 breast cancer cells, five out of six chemicals, that is, benzophenone-3 (Bp-3), homosalate (HMS), 4-methyl-benzylidene camphor (4-MBC), octyl-methoxycinnamate (OMC), and octyl-dimethyl-PABA (OD-PABA), increased cell proliferation with median effective concentrations (EC(50)) values between 1.56 and 3.73 microM, whereas butyl-methoxydibenzoylmethane (B-MDM) was inactive. Further evidence for estrogenic activity was the induction of pS2 protein in MCF-7 cells and the blockade of the proliferative effect of 4-MBC by the estrogen antagonist ICI 182,780. In the uterotrophic assay using immature Long-Evans rats that received the chemicals for 4 days in powdered feed, uterine weight was dose-dependently increased by 4-MBC (ED(50 )309mg/kg/day), OMC (ED(50) 935 mg/kg/day), and weakly by Bp-3 (active at 1,525 mg/kg/day). Three compounds were inactive by the oral route in the doses tested. Dermal application of 4-MBC to immature hairless (hr/hr) rats also increased uterine weight at concentrations of 5 and 7.5% in olive oil. Our findings indicate that UV screens should be tested for endocrine activity, in view of possible long-term effects in humans and wildlife. PMID:11333184

Lack of activity of cadmium in in vitro estrogenicity assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silva, Elisabete; Lopez-Espinosa, Maria Jose; Molina-Molina, Jose-Manuel

2006-10-01

Prompted by reports about strong estrogenic effects of cadmium, attempts were made to reproduce these observations using the yeast estrogen screen (YES) and the E-Screen assays. For the first time, possible activation of the Src/MAPK pathway was also investigated. In the YES, only a slight activation (10% of a maximal effect) of the estrogen receptor alpha (ER{alpha}) was observed at cadmium concentrations between 5 x 10{sup -7} M and 5 x 10{sup -6} M. In the E-Screen assay, carried out by two laboratories, the heavy metal was without observable cell proliferative effects when tested in the range between 6 xmore » 10{sup -11} M and 1 x 10{sup -5} M. However, in both assays, cadmium led to a reduction of the effects of 17{beta}-estradiol (E2). Treatment of MCF-7 human breast cancer cells with 1 x 10{sup -7} M cadmium failed to induce phosphorylation of Src and the MAP kinases Erk1 and Erk2-effects shown to occur with E2 and epidermal growth factor (EGF). In summary, we were unable to confirm the strong estrogenicity of cadmium reported recently by a number of laboratories. This apparent absence of effects in our hands is not due to a lack of uptake of the metal or to effective protection against cadmium by high levels of glutathione or metallothionein, since toxicity and an antagonism of E2 responses were observed both in the YES and the E-Screen.« less

Lupinalbin A as the most potent estrogen receptor α- and aryl hydrocarbon receptor agonist in Eriosema laurentii de Wild. (Leguminosae).

PubMed

Ateba, Sylvin Benjamin; Njamen, Dieudonné; Medjakovic, Svjetlana; Zehl, Martin; Kaehlig, Hanspeter; Jungbauer, Alois; Krenn, Liselotte

2014-08-09

Eriosema laurentii De Wild. (Leguminosae) is a plant used in Cameroon against infertility and gynecological or menopausal complaints. In our previous report, a methanol extract of its aerial parts was shown to exhibit estrogenic and aryl hydrocarbon receptor agonistic activities in vitro and to prevent menopausal symptoms in ovariectomized Wistar rats. In order to determine the major estrogen receptor α (ERα) agonists in the extract, an activity-guided fractionation was performed using the ERα yeast screen. To check whether the ERα active fractions/compounds also accounted for the aryl hydrocarbon receptor (AhR) agonistic activity of the crude methanol extract, they were further tested on the AhR yeast screen. This study led to the identification of 2'-hydroxygenistein, lupinalbin A and genistein as major estrogenic principles of the extract. 2'-hydroxygenistein and lupinalbin A were, for the first time, also shown to possess an AhR agonistic activity, whereas genistein was not active in this assay. In addition, it was possible to deduce structure-activity relationships. These results suggest that the identified compounds are the major active principles responsible for the estrogenic and AhR agonistic activities of the crude methanol extract of the aerial parts of Eriosema laurentii.

Occurrence and change of estrogenic activity in the process of drinking water treatment and distribution.

PubMed

Xiao, Sanhua; Lv, Xuemin; Lu, Yi; Yang, Xiaoming; Dong, Xiaorong; Ma, Kunpeng; Zeng, Yifan; Jin, Tao; Tang, Fei

2016-09-01

From 2010 to 2012, the Yangtze River and Hanjiang River (Wuhan section) were monitored for estrogenic activities during various water level periods. Using a recombinant yeast estrogen screen (YES) assay, 54 water samples were evaluated over the course of nine sampling campaigns. The mean 17β-estradiol equivalent (EEQ) value of raw water from the Yangtze River was 0-5.20 ng/L; and the EEQ level from the Hanjiang River was 0-3.22 ng/L. In Wuhan, drinking water treatment plants (DWTPs) using conventional treatments reduced estrogenic activities by more than 89 %. In general, water samples collected during the level period showed weaker estrogenic activities compared to those collected during the dry period. The samples collected in 2010 showed the strongest estrogenic activities of the 3-year period. The lack of correlations between estrogenic activities and selected common water quality parameters showed that estrogenic activity cannot be tied to common water quality parameters.

Nonylphenol and estrogenic activity in aquatic environmental samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanghe, T.; Devriese, G.; Verstraete, W.

1999-03-01

The authors surveyed a series of surface waters and sewage treatment plants in Flanders (north of Belgium) for the presence of estrogenic activity and a xeno-estrogenic compound para-nonylphenol (NP), respectively. The surface waters of rural origin, used for drinking water production were free of significant levels of estrogenic activity and NP. Domestic sewage, after proper treatment, appeared to be no major source of this chemical. Yet, in some industrial effluents and surface waters of highly industrialized regions, NP and/or estrogenic activity was prominent, that is, <1 to 122 {micro}g NP/L and 11 to 42 {micro}g NP/L, respectively. This is becausemore » of the ongoing use of NP polyethoxylates in industry. The response of the recombinant yeast estrogen assay to the environmental samples tested was not consistent with the detected concentrations of NP. Standard addition of a natural estrogen, 17{beta}-estradiol, generated no or a reduced response compared to the standard curve concentration. Application of humic acids to standard series of NP and 17{beta}-estradiol resulted in a dose-dependent decrease of the estrogenic response. It appears that this bioassay is subject to considerable interferences due to the complexity of environmental samples. Parallel implementation of extensive chemical screening for xenobiotics and use of the bioassay are needed for adequate assessment of the potential estrogenic hazard to avoid false negative evaluations.« less

Mixture Effects of Estrogenic Pesticides at the Human Estrogen Receptor α and β

PubMed Central

Seeger, Bettina; Klawonn, Frank; Nguema Bekale, Boris; Steinberg, Pablo

2016-01-01

Consumers of fruits and vegetables are frequently exposed to small amounts of hormonally active pesticides, some of them sharing a common mode of action such as the activation of the human estrogen receptor α (hERα) or β (hERβ). Therefore, it is of particular importance to evaluate risks emanating from chemical mixtures, in which the individual pesticides are present at human-relevant concentrations, below their corresponding maximum residue levels. Binary and ternary iso-effective mixtures of estrogenic pesticides at effect concentrations eliciting a 1 or 10% effect in the presence or absence of 17β-estradiol were tested experimentally at the hERα in the yeast-based estrogen screen (YES) assay as well as in the human U2-OS cell-based ERα chemical-activated luciferase gene expression (ERα CALUX) assay and at the hERβ in the ERβ CALUX assay. The outcome was then compared to predictions calculated by means of concentration addition. In most cases, additive effects were observed with the tested combinations in all three test systems, an observation that supports the need to expand the risk assessment of pesticides and consider cumulative risk assessment. An additional testing of mixture effects at the hERβ showed that most test substances being active at the hERα could also elicit additive effects at the hERβ, but the hERβ was less sensitive. In conclusion, effects of the same ligands at the hERα and the hERβ could influence the estrogenic outcome under physiological conditions. PMID:26812056

Estrogenic activities of chemicals related to food contact plastics and rubbers tested by the yeast two-hybrid assay.

PubMed

Ogawa, Yuko; Kawamura, Yoko; Wakui, Chiseko; Mutsuga, Motoh; Nishimura, Tetsuji; Tanamoto, Kenichi

2006-04-01

Food contact plastics and rubbers possibly contain many kinds of chemicals such as monomers, oligomers, additives, degradation products of polymers and additives, and impurities. Among them, bisphenol A, nonylphenol, benzylbutyl phthalate, styrene oligomers and hydroxylated benzophenones have been reported to possess estrogenic activities. In this study, other chemicals related to food contact plastics and rubbers, and their metabolites induced by the S9-mixture were tested for their estrogenic activities using the yeast two-hybrid assay. Among the 150 chemicals, 10 chemicals such as bis(4-hydroxyphenyl) methane, 4-cyclohexylphenol, 4-phenylphenol, 4,4'-isopropylidenediphenol alkylphosphite, two type of styrenated phenol (including mono type), tris(nonylphenyl) phosphite, 2,2'-dihydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone and 2,4-diphenyl-4-methyl-1-pentene, their metabolites and the metabolites of 6 other chemicals, such as 2-(phenylmethyl) phenol, styrenated phenol (di and tri type), 1-(N-phenylamino)naphthalene, 4-tert-butylphenylsalicylate, nonylphenol ethoxylates and 2-methyl-6-tert-butylphenol, displayed estrogenic activities. All of them contained a phenol group in their chemical structures or formed one easily by hydrolysis or metabolism. However, most of the chemicals related to food contact plastics and rubbers, and their metabolites did not show any estrogenicity.

Changes in estrogenicity and micropollutant concentrations across unit processes in a biological wastewater treatment system.

PubMed

Chen, Jian Lin; Ravindran, Shanthinie; Swift, Simon; Singhal, Naresh

2018-03-01

The behavior of 10 micropollutants, i.e. four estrogens (estrone, 17β-estradiol, estriol, 17α-ethynylestradiol), carbamazepine (CBZ), sulfamethoxazole (SMX), triclosan, oxybenzone, 4-nonylphenol, and bisphenol A, was investigated in a typical domestic wastewater treatment plant. LC-MS and yeast estrogen screen bioassay were used to study the changes in micropollutants and estrogenicity across unit processes in the treatment system. Primary treatment via sedimentation showed that only 4-nonylphenol was removed, but led to no significant change in estrogenicity. Secondary treatment by the biological nitrification-dentrification process showed complete removal of oxybenzone and partial removal of the estrogens, which led to a decrease in estrogenic activity from 80 to 48 ng/L as estradiol equivalent (EEq). Ultraviolet treatment completely degraded the estrogens and triclosan, but failed to lower the concentrations of bisphenol A, SMX, and CBZ; a decrease in estrogenic activity from 48 to 5 ng/L EEq across the unit, a value that was only slightly larger than the observed EEq of 1 ng/L for the deionized control. Similarly, the anaerobic digestion of sludge completely degraded estrogens, oxybenzone, and SMX, but had no impact on bisphenol A, triclosan, and CBZ. The study emphasises the need to complement chemical analyses with estrogenic bioassays to evaluate the efficacy of waste water treatment plants.

Overview of a workshop on screening methods for detecting potential (anti-) estrogenic/androgenic chemicals in wildlife

USGS Publications Warehouse

Ankley, Gerald T.; Mihaich, Ellen; Stahl, Ralph G.; Tillitt, Donald E.; Colborn, Theo; McMaster, Suzzanne; Miller, Ron; Bantle, John; Campbell, Pamela; Denslow, Nancy; Dickerson, Richard L.; Folmar, Leroy C.; Fry, Michael; Giesy, John P.; Gray, L. Earl; Guiney, Patrick; Hutchinson, Thomas; Kennedy, Sean W.; Kramer, Vincent; LeBlanc, Gerald A.; Mayes, Monte; Nimrod, Alison; Patino, Reynaldo; Peterson, Richard; Purdy, Richard; Ringer, Robert; Thomas, Peter C.; Touart, Les; Van Der Kraak, Glen; Zacharewski, Tim

1998-01-01

The U.S. Congress has passed legislation requiring the U.S. Environmental Protection Agency (U.S. EPA) to develop, validate, and implement screening tests for identifying potential endocrine-disrupting chemicals within 3 years. To aid in the identification of methods suitable for this purpose, the U.S. EPA, the Chemical Manufacturers Association, and the World Wildlife Fund sponsored several workshops, including the present one, which dealt with wildlife species. This workshop was convened with 30 international scientists representing multiple disciplines in March 1997 in Kansas City, Missouri, USA. Participants at the meeting identified methods in terms of their ability to indicate (anti-) estrogenic/androgenic effects, particularly in the context of developmental and reproductive processes. Data derived from structure-activity relationship models and in vitro test systems, although useful in certain contexts, cannot at present replace in vivo tests as the sole basis for screening. A consensus was reached that existing mammalian test methods (e.g., with rats or mice) generally are suitable as screens for assessing potential (anti-) estrogenic/ androgenic effects in mammalian wildlife. However, due to factors such as among-class variation in receptor structure and endocrine function, it is uncertain if these mammalian assays would be of broad utility as screens for other classes of vertebrate wildlife. Existing full and partial life-cycle tests with some avian and fish species could successfully identify chemicals causing endocrine disruption; however, these long-term tests are not suitable for routine screening. However, a number of short-term tests with species from these two classes exist that could serve as effective screening tools for chemicals inducing (anti-) estrogenic/androgenic effects. Existing methods suitable for identifying chemicals with these mechanisms of action in reptiles and amphibians are limited, but in the future, tests with species from

Validation of the REA bioassay to detect estrogenic activity in the water cycle.

PubMed

Nguyen, Mai Thao; van der Oost, Ron; Bovee, Toine F H

2011-12-01

Endocrine disrupting compounds (EDCs) with estrogenic potency contaminate water and might eventually cause adverse effects to the aquatic environment. Many estrogenic compounds are not completely removed by wastewater treatment systems and, together with the run-off from agricultural areas, they enter surface waters. Chemical analytical methods to determine these compounds are usually expensive and laborious. Therefore, screening bioassays which are able to detect compounds based on their effects offer a solution for prior selection of samples that need to be chemically analyzed. In this study, the REA (RIKILT yeast Estrogen bioAssay), which has been developed to detect estrogenic compounds in calf urine and animal feed at RIKILT, is validated at the Water Board Laboratory of Waterproef for water samples. According to EC Decision 2002/657, detection capability CCβ, specificity and stability have to be determined for the internal validation of a qualitative screening test. In addition, surface water and effluent samples were analyzed to further demonstrate the applicability of the validated test procedure. Results demonstrate that the REA assay is reproducible and specific for estrogenic compounds in water and meets the criteria as prescribed in EC Decision 2002/657. The assay was sensitive enough to detect estrogenic activity of pollutants in water with a limit of quantification (LOQ) below 1 ng EEQ/L. This means that samples can be compared with preliminary threshold levels for drinking water and surface waters (7 and 1 ng EEQ/L, respectively). The stability of estrogenic activity in water samples is at least 4 weeks, when stored at 4 °C. Copyright © 2011 Elsevier Ltd. All rights reserved.

Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction.

PubMed

Zhou, Jingwen; Zhou, Haiyan; Du, Guocheng; Liu, Liming; Chen, Jian

2010-09-01

To obtain a thiamine-auxotrophic yeast strain that overproduces alpha-ketoglutaric acid (alpha-KG) from glycerol and to investigate nutrient effects on alpha-KG production. Yeast strain WSH-Z06, a thiamine auxotroph that gave high yields of alpha-KG from glycerol, was obtained by screening for ampicillin/kanamycin resistance and thiamine auxotrophy. The strain was identified as Yarrowia lipolytica based on physiological, chemical, and phylogenetic analysis. The ability of the strain to convert glycerol to alpha-KG was analysed by investigating the effects of nutritional factors, including thiamine, riboflavin, nitrogen sources, and calcium ion. Thiamine and calcium ion concentration had the greatest effect on alpha-KG accumulation. Under optimal conditions, a yield of 39.2 g l(-1)alpha-KG was obtained from 100 g l(-1) glycerol, with 16.84 g l(-1) pyruvate as a by-product. The current work provides a method for screening for an alpha-KG overproducer. Nutrients have a significant impact on alpha-KG production in the yeast strain presented here. The alpha-KG-overproducing yeast strain Y. lipolytica WSH-Z06 is a promising parent strain for further metabolic engineering to lower by-product accumulation and accelerate glycerol utilization.

Estrogenicity and androgenicity screening of PCB sulfate monoesters in human breast cancer MCF-7 cells.

PubMed

Flor, Susanne; He, Xianran; Lehmler, Hans-Joachim; Ludewig, Gabriele

2016-02-01

Recent studies identified polychlorinated biphenyl (PCB) sulfate esters as a major product of PCB metabolism. Since hydroxy-PCBs (HO-PCBs), the immediate precursors of PCB sulfates and important contributors to PCB toxicity, were shown to have estrogenic activity, we investigated the estrogenicity/androgenicty of a series of PCB sulfate metabolites. We synthesized the five possible structural sulfate monoester metabolites of PCB 3, a congener shown to be biotransformed to sulfates, a sulfate ester of the paint-specific congener PCB 11, and sulfate monoesters of two HO-PCBs reported to interact with sulfotransferases (PCB 39, no ortho chlorines, and PCB 53, 3 ortho chlorines). We tested these PCB sulfates and 4'-HO-PCB 3 as positive control for estrogenic, androgenic, anti-estrogenic, and anti-androgenic activity in the E- and A-screen with human breast cancer MCF7-derived cells at 100 μM-1 pM concentrations. Only 4'-HO-PCB 3 was highly cytotoxic at 100 μM. We observed structure-activity relationships: compounds with a sulfate group in the chlorine-containing ring of PCB 3 (2PCB 3 and 3PCB 3 sulfate) showed no interaction with the estrogen (ER) and androgen (AR) receptor. The 4'-HO-PCB 3 and its sulfate ester had the highest estrogenic effect, but at 100-fold different concentrations, i.e., 1 and 100 μM, respectively. Four of the PCB sulfates were estrogenic (2'PCB 3, 4'PCB 3, 4'PCB 39, and 4'PCB 53 sulfates; at 100 μM). These sulfates and 3'PCB 3 sulfate also exhibited anti-estrogenic activity, but at nM and pM concentrations. The 4'PCB 3 sulfate (para-para' substituted) had the strongest androgenic activity, followed by 3'PCB 3, 4'PCB 53, 4PCB11, and 4PCB 39 sulfates and the 4'HO-PCB 3. In contrast, anti-androgenicity was only observed with the two compounds that have the sulfate group in ortho- or meta- position in the second ring (2'PCB 3 and 3'PCB 3 sulfate). No dose-response was observed in any screen, but, with exception of estrogenic activity (only seen

Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians--screening for estrogen, androgen and thyroid hormone disruption.

PubMed

Scholz, S; Renner, P; Belanger, S E; Busquet, F; Davi, R; Demeneix, B A; Denny, J S; Léonard, M; McMaster, M E; Villeneuve, D L; Embry, M R

2013-01-01

Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability.

Comprehensive assessment of hormones, phytoestrogens, and estrogenic activity in an anaerobic swine waste lagoon

USGS Publications Warehouse

Yost, Erin E.; Meyer, Michael T.; Dietze, Julie E.; Meissner, Benjamin M.; Williams, Mike; Worley-Davis, Lynn; Lee, Boknam; Kullman, Seth W.

2013-01-01

In this study, the distribution of steroid hormones, phytoestrogens, and estrogenic activity was thoroughly characterized within the anaerobic waste lagoon of a typical commercial swine sow operation. Three independent rounds of sampling were conducted in June 2009, April 2010, and February 2011. Thirty-seven analytes in lagoon slurry and sludge were assessed using LC/MS-MS, and yeast estrogen screen was used to determine estrogenic activity. Of the hormone analytes, steroidal estrogens were more abundant than androgens or progesterone, with estrone being the predominant estrogen species. Conjugated hormones were detected only at low levels. The isoflavone metabolite equol was by far the predominant phytoestrogen species, with daidzein, genistein, formononetin, and coumestrol present at lower levels. Phytoestrogens were often more abundant than steroidal estrogens, but contributed minimally towards total estrogenic activity. Analytes were significantly elevated in the solid phases of the lagoon; although low observed log KOC values suggest enhanced solubility in the aqueous phase, perhaps due to dissolved or colloidal organic carbon. The association with the solid phase, as well as recalcitrance of analytes to anaerobic degradation, results in a markedly elevated load of analytes and estrogenic activity within lagoon sludge. Overall, findings emphasize the importance of adsorption and transformation processes in governing the fate of these compounds in lagoon waste, which is ultimately used for broadcast application as a fertilizer.

Comprehensive Assessment of Hormones, Phytoestrogens, and Estrogenic Activity in an Anaerobic Swine Waste Lagoon

PubMed Central

2013-01-01

In this study, the distribution of steroid hormones, phytoestrogens, and estrogenic activity was thoroughly characterized within the anaerobic waste lagoon of a typical commercial swine sow operation. Three independent rounds of sampling were conducted in June 2009, April 2010, and February 2011. Thirty-seven analytes in lagoon slurry and sludge were assessed using LC/MS-MS, and yeast estrogen screen was used to determine estrogenic activity. Of the hormone analytes, steroidal estrogens were more abundant than androgens or progesterone, with estrone being the predominant estrogen species. Conjugated hormones were detected only at low levels. The isoflavone metabolite equol was by far the predominant phytoestrogen species, with daidzein, genistein, formononetin, and coumestrol present at lower levels. Phytoestrogens were often more abundant than steroidal estrogens, but contributed minimally toward total estrogenic activity. Analytes were significantly elevated in the solid phases of the lagoon; although low observed log KOC values suggest enhanced solubility in the aqueous phase, perhaps due to dissolved or colloidal organic carbon. The association with the solid phase, as well as recalcitrance of analytes to anaerobic degradation, results in a markedly elevated load of analytes and estrogenic activity within lagoon sludge. Overall, findings emphasize the importance of adsorption and transformation processes in governing the fate of these compounds in lagoon waste, which is ultimately used for broadcast application as a fertilizer. PMID:24144340

Comparison of five in vitro bioassays to measure estrogenic activity in environmental waters.

PubMed

Leusch, Frederic D L; de Jager, Christiaan; Levi, Yves; Lim, Richard; Puijker, Leo; Sacher, Frank; Tremblay, Louis A; Wilson, Vickie S; Chapman, Heather F

2010-05-15

Bioassays are well established in the pharmaceutical industry and single compound analysis, but there is still uncertainty about their usefulness in environmental monitoring. We compared the responses of five bioassays designed to measure estrogenic activity (the yeast estrogen screen, ER-CALUX, MELN, T47D-KBluc, and E-SCREEN assays) and chemical analysis on extracts from four different water sources (groundwater, raw sewage, treated sewage, and river water). All five bioassays displayed similar trends and there was good agreement with analytical chemistry results. The data from the ER-CALUX and E-SCREEN bioassays were robust and predictable, and well-correlated with predictions from chemical analysis. The T47D-KBluc appeared likewise promising, but with a more limited sample size it was less compelling. The YES assay was less sensitive than the other assays by an order of magnitude, which resulted in a larger number of nondetects. The MELN assay was less predictable, although the possibility that this was due to laboratory-specific difficulties cannot be discounted. With standardized bioassay data analysis and consistency of operating protocols, bioanalytical tools are a promising advance in the development of a tiered approach to environmental water quality monitoring.

Development of an image analysis screen for estrogen receptor alpha (ERα) ligands through measurement of nuclear translocation dynamics.

PubMed

Dull, Angie; Goncharova, Ekaterina; Hager, Gordon; McMahon, James B

2010-11-01

We have developed a robust high-content assay to screen for novel estrogen receptor alpha (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen utilizes a green fluorescent protein tagged-glucocorticoid/estrogen receptor (GFP-GRER) chimera which consisted of the N-terminus of the glucocorticoid receptor fused to the human ER ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands, and translocated to the nucleus in response to stimulation with ERα agonists or antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. The assay was validated with known ERα agonists and antagonists, and the Library of Pharmacologically Active Compounds (LOPAC 1280). Additionally, screening of crude natural product extracts demonstrated the robustness of the assay, and the ability to quantitate the effects of toxicity on nuclear translocation dynamics. The GFP-GRER nuclear translocation assay was very robust, with z' values >0.7, CVs <5%, and has been validated with known ER ligands, and inclusion of cytotoxicity filters will facilitate screening of natural product extracts. This assay has been developed for future primary screening of synthetic, pure natural products, and natural product extracts libraries available at the National Cancer Institute at Frederick. Copyright © 2010 Elsevier Ltd. All rights reserved.

Estrogenicity and androgenicity screening of PCB sulfate monoesters in human breast cancer MCF-7 cells

PubMed Central

Flor, Susanne; He, Xianran; Lehmler, Hans-Joachim; Ludewig, Gabriele

2015-01-01

Recent studies identified PCB sulfate esters as a major product of PCB metabolism. Since hydroxy-PCBs (HO-PCBs), the immediate precursors of PCB sulfates and important contributors to PCB toxicity, were shown to have estrogenic activity, we investigated the estrogenicity/androgenicty of a series of PCB sulfate metabolites. We synthesized the five possible structural sulfate monoester metabolites of PCB 3, a congener shown to be biotransformed to sulfates, a sulfate ester of the paint-specific congener PCB 11, and sulfate monoesters of two HO-PCBs reported to interact with sulfotransferases (PCB 39, no ortho chlorines, and PCB 53, 3 ortho chlorines). We tested these PCB sulfates and 4’-HO-PCB 3 as positive control for estrogenic, androgenic, anti-estrogenic and anti-androgenic activity in the E- and A-screen with human breast cancer MCF7 derived cells at 100 μM – 1 pM concentrations. Only 4’-HO-PCB 3 was highly cytotoxic at 100 μM. We observed structure-activity relationships: compounds with a sulfate group in the chlorine-containing ring of PCB 3 (2PCB 3 and 3PCB 3 sulfate) showed no interaction with the estrogen (ER) and androgen (AR) receptor. The 4’-HO-PCB 3 and its sulfate ester had the highest estrogenic effect, but at 100 fold different concentrations, i.e. 1 μM and 100 μM, respectively. Four of the PCB sulfates were estrogenic (2’PCB 3, 4’PCB 3, 4PCB 39, 4PCB 53 sulfates; at 100 μM). These sulfates and 3’PCB 3 sulfate also exhibited anti-estrogenic activity, but at nM and pM concentrations. The 4’PCB 3 sulfate (para-para’ substituted) had the strongest androgenic activity, followed by 3’PCB 3, 4PCB 53, 4PCB11, and 4PCB 39 sulfates and the 4’HO-PCB 3. In contrast, anti-androgenicity was only observed with the two compounds that have the sulfate group in ortho- or meta- position in the second ring (2’PCB 3 and 3’PCB 3 sulfate). No dose-response was observed in any screen, but, with exception of estrogenic activity (only seen at

Combined zebrafish-yeast chemical-genetic screens reveal gene-copper-nutrition interactions that modulate melanocyte pigmentation.

PubMed

Ishizaki, Hironori; Spitzer, Michaela; Wildenhain, Jan; Anastasaki, Corina; Zeng, Zhiqiang; Dolma, Sonam; Shaw, Michael; Madsen, Erik; Gitlin, Jonathan; Marais, Richard; Tyers, Mike; Patton, E Elizabeth

2010-01-01

Hypopigmentation is a feature of copper deficiency in humans, as caused by mutation of the copper (Cu(2+)) transporter ATP7A in Menkes disease, or an inability to absorb copper after gastric surgery. However, many causes of copper deficiency are unknown, and genetic polymorphisms might underlie sensitivity to suboptimal environmental copper conditions. Here, we combined phenotypic screens in zebrafish for compounds that affect copper metabolism with yeast chemical-genetic profiles to identify pathways that are sensitive to copper depletion. Yeast chemical-genetic interactions revealed that defects in intracellular trafficking pathways cause sensitivity to low-copper conditions; partial knockdown of the analogous Ap3s1 and Ap1s1 trafficking components in zebrafish sensitized developing melanocytes to hypopigmentation in low-copper environmental conditions. Because trafficking pathways are essential for copper loading into cuproproteins, our results suggest that hypomorphic alleles of trafficking components might underlie sensitivity to reduced-copper nutrient conditions. In addition, we used zebrafish-yeast screening to identify a novel target pathway in copper metabolism for the small-molecule MEK kinase inhibitor U0126. The zebrafish-yeast screening method combines the power of zebrafish as a disease model with facile genome-scale identification of chemical-genetic interactions in yeast to enable the discovery and dissection of complex multigenic interactions in disease-gene networks.

Transport of steroid hormones, phytoestrogens, and estrogenic activity across a swine lagoon/sprayfield system.

PubMed

Yost, Erin E; Meyer, Michael T; Dietze, Julie E; Williams, C Michael; Worley-Davis, Lynn; Lee, Boknam; Kullman, Seth W

2014-10-07

The inflow, transformation, and attenuation of natural steroid hormones and phytoestrogens and estrogenic activity were assessed across the lagoon/sprayfield system of a prototypical commercial swine sow operation. Free and conjugated steroid hormones (estrogens, androgens, and progesterone) were detected in urine and feces of sows across reproductive stages, with progesterone being the most abundant steroid hormone. Excreta also contained phytoestrogens indicative of a soy-based diet, particularly, daidzein, genistein, and equol. During storage in barn pits and the anaerobic lagoon, conjugated hormones dissipated, and androgens and progesterone were attenuated. Estrone and equol persisted along the waste disposal route. Following application of lagoon slurry to agricultural soils, all analytes exhibited attenuation within 2 days. However, analytes including estrone, androstenedione, progesterone, and equol remained detectable in soil at 2 months postapplication. Estrogenic activity in the yeast estrogen screen and T47D-KBluc in vitro bioassays generally tracked well with analyte concentrations. Estrone was found to be the greatest contributor to estrogenic activity across all sample types. This investigation encompasses the most comprehensive suite of natural hormone and phytoestrogen analytes examined to date across a livestock lagoon/sprayfield and provides global insight into the fate of these analytes in this widely used waste management system.

Transport of Steroid Hormones, Phytoestrogens, and Estrogenic Activity across a Swine Lagoon/Sprayfield System

PubMed Central

Yost, Erin E.; Meyer, Michael T.; Dietze, Julie E.; Williams, C. Michael; Worley-Davis, Lynn; Lee, Boknam; Kullman, Seth W.

2017-01-01

The inflow, transformation, and attenuation of natural steroid hormones, phytoestrogens, and estrogenic activity was assessed across the lagoon/sprayfield system of a prototypical commercial swine sow operation. Free and conjugated steroid hormones (estrogens, androgens, and progesterone) were detected in urine and feces of sows across reproductive stages, with progesterone being the most abundant steroid hormone. Excreta also contained phytoestrogens indicative of a soy-based diet; particularly daidzein, genistein, and equol. During storage in barn pits and the anaerobic lagoon, conjugated hormones dissipated, and androgens and progesterone were attenuated. Estrone and equol persisted along the waste disposal route. Following application of lagoon slurry to agricultural soils, all analytes exhibited attenuation within 2 days. However, analytes including estrone, androstenedione, progesterone, and equol remained detectable in soil at two months post-application. Estrogenic activity in the yeast estrogen screen and T47D-KBluc in vitro bioassays generally tracked well with analyte concentrations. Estrone found to be the greatest contributor to estrogenic activity across all sample types. This investigation encompasses the most comprehensive suite of natural hormone and phytoestrogen analytes examined to date across a lagoon/sprayfield system, and provides global insight into the fate of these analytes in this widely used waste management system. PMID:25148584

Database for High Throughput Screening Hits (dHITS): a simple tool to retrieve gene specific phenotypes from systematic screens done in yeast.

PubMed

Chuartzman, Silvia G; Schuldiner, Maya

2018-03-25

In the last decade several collections of Saccharomyces cerevisiae yeast strains have been created. In these collections every gene is modified in a similar manner such as by a deletion or the addition of a protein tag. Such libraries have enabled a diversity of systematic screens, giving rise to large amounts of information regarding gene functions. However, often papers describing such screens focus on a single gene or a small set of genes and all other loci affecting the phenotype of choice ('hits') are only mentioned in tables that are provided as supplementary material and are often hard to retrieve or search. To help unify and make such data accessible, we have created a Database of High Throughput Screening Hits (dHITS). The dHITS database enables information to be obtained about screens in which genes of interest were found as well as the other genes that came up in that screen - all in a readily accessible and downloadable format. The ability to query large lists of genes at the same time provides a platform to easily analyse hits obtained from transcriptional analyses or other screens. We hope that this platform will serve as a tool to facilitate investigation of protein functions to the yeast community. © 2018 The Authors Yeast Published by John Wiley & Sons Ltd.

Heterocyclic aromatic hydrocarbons show estrogenic activity upon metabolization in a recombinant transactivation assay.

PubMed

Brinkmann, Markus; Maletz, Sibylle; Krauss, Martin; Bluhm, Kerstin; Schiwy, Sabrina; Kuckelkorn, Jochen; Tiehm, Andreas; Brack, Werner; Hollert, Henner

2014-05-20

Heterocyclic aromatic hydrocarbons (hetero-PAHs) are increasingly studied at contaminated sites; especially at former industrial facilities where coal tar-oil was handled, e.g., wood treatment plants, high concentrations of hetero-PAHs are frequently detected in groundwater plumes. In previous studies, fractions of groundwater with high estrogenic activity contained hetero-PAHs and their hydroxylated metabolites. To evaluate this preliminary evidence, selected hetero-PAHs were screened for their estrogenic activity in lyticase yeast estrogen screen (LYES) and ER CALUX. All tested substances were inactive in the LYES. Hetero-PAHs such as acridine, xanthene, indole, 2-methylbenzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophene, quinoline, and 6-methylquinoline were positive in the ER CALUX, with estradiol equivalence factors (EEFs) from 2.85 × 10(-7) to 3.18 × 10(-5). The EEF values of these substances were comparable to those of other xenoestrogens (e.g., alkylphenols or bisphenol A) that are sometimes found in surface water. Chemical analyses revealed that T47Dluc cells could metabolize most of the substances. Among the metabolites (tentatively) identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were hydroxides and their keto tautomers, sulfates, sulfoxides, and N-oxides. Because of their high concentrations measured in groundwater, we conclude that hetero-PAHs and metabolites may be a potential risk and should be the subject of further research.

An exposure:activity profiling method for interpreting high-throughput screening data for estrogenic activity--proof of concept.

PubMed

Becker, Richard A; Friedman, Katie Paul; Simon, Ted W; Marty, M Sue; Patlewicz, Grace; Rowlands, J Craig

2015-04-01

Rapid high throughput in vitro screening (HTS) assays are now available for characterizing dose-responses in assays that have been selected for their sensitivity in detecting estrogen-related endpoints. For example, EPA's ToxCast™ program recently released endocrine assay results for more than 1800 substances and the interagency Tox21 consortium is in the process of releasing data for approximately 10,000 chemicals. But such activity measurements alone fall short for the purposes of priority setting or screening because the relevant exposure context is not considered. Here, we extend the method of exposure:activity profiling by calculating the exposure:activity ratios (EARs) using human exposure estimates and AC50 values for a range of chemicals tested in a suite of seven estrogenic assays in ToxCast™ and Tox21. To provide additional context, relative estrogenic exposure:activity quotients (REEAQ) were derived by comparing chemical-specific EARs to the EAR of the ubiquitous dietary phytoestrogen, genistein (GEN). Although the activity of a substance in HTS-endocrine assays is not a measure of health hazard or risk, understanding how such a dose compares to human exposures provides a valuable additional metric that can be used in decision-making; substances with small EARs and REEAQs would indicate low priority for further endocrine screening or testing. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

PubMed

Petit, F G; Métivier, R; Valotaire, Y; Pakdel, F

1999-01-01

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

Screening for new brewing yeasts in the non-Saccharomyces sector with Torulaspora delbrueckii as model.

PubMed

Michel, Maximilian; Kopecká, Jana; Meier-Dörnberg, Tim; Zarnkow, Martin; Jacob, Fritz; Hutzler, Mathias

2016-04-01

This study describes a screening system for future brewing yeasts focusing on non-Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off-flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by-products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre-fermentation as a bio-flavouring agent for beers that have been post-fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour-forming properties. Copyright © 2015 John Wiley & Sons, Ltd.

Estrogenic and androgenic activities of TBBA and TBMEPH, metabolites of novel brominated flame retardants, and selected bisphenols, using the XenoScreen XL YES/YAS assay.

PubMed

Fic, Anja; Žegura, Bojana; Gramec, Darja; Mašič, Lucija Peterlin

2014-10-01

The present study investigated and compared the estrogenic and androgenic activities of the three different classes of environmental pollutants and their metabolites using the XenoScreen XL YES/YAS assay, which has advantages compared with the original YES/YAS protocol. Contrary to the parent brominated flame retardants TBB and TBPH, which demonstrated no or very weak (anti)estrogenic or (anti)androgenic activities, their metabolites, TBBA and TBMEPH, exhibited anti-estrogenic (IC50 for TBBA=31.75 μM and IC50 for TBMEPH=0.265 μM) and anti-androgenic (IC50 for TBBA=73.95 μM and IC50 for TBMEPH=2.92 μM) activities. These results reveal that metabolism can enhance the anti-estrogenic and anti-androgenic effects of these two novel brominated flame retardants. Based on the activities of BPAF, BPF, BPA and MBP, we can conclude that the XenoScreen XL YES/YAS assay gives comparable results to the (anti)estrogenic or (anti)androgenic assays that are reported in the literature. For BPA, it was confirmed previously that the metabolite formed after an ipso-reaction (hydroxycumyl alcohol) exhibited higher estrogenic activity compared with the parent BPA, but this was not confirmed for BPAF and BPF ipso-metabolites, which were not active in the XenoScreen YES/YAS assay. Among the substituted BPA analogues, bis-GMA exhibited weak anti-estrogenic activity, BADGE demonstrated weak anti-estrogenic and anti-androgenic activities (IC50=13.73 μM), and the hydrolysed product BADGE·2H2O demonstrated no (anti)estrogenic or (anti)androgenic activities. Copyright © 2014. Published by Elsevier Ltd.

Conversion and Estrogenicity of 17β-estradiol During Photolytic/Photocatalytic Oxidation and Catalytic Wet-air Oxidation.

PubMed

Bistan, Mirjana; Tišler, Tatjana; Pintar, Albin

2012-06-01

Estrogen 17β-estradiol (E2), produced by human body and excreted into municipal wastewaters, belongs to the group of endocrine disrupting compounds that are resistant to biological degradation. The aim of this study was to assess the efficiency of E2 removal from aqueous solutions by means of catalytic wet-air oxidation (CWAO) and photolytic/photocatalytic oxidation. CWAO experiments were conducted in a trickle-bed reactor at temperatures up to 230 °C and oxygen partial pressure of 10 bar over TiO2 and Ru/TiO2 solids. Photolytic/photocatalytic oxidation was carried out in a batch slurry reactor employing a TiO2 P-25 (Degussa) catalyst under visible or UV light. HPLC analysis and yeast estrogen screen assay were used to evaluate the removal of E2 and estrogenicity of treated samples. The latter was completely removed during photolytic/photocatalytic oxidation under UV (365 nm) light and photocatalytic oxidation under visible light. In CWAO experiments, complete removal of both E2 and estrogenicity from the feed solution were noticed in the presence of TiO2 and Ru/TiO2 catalysts.

Mutagenicity and estrogenicity of raw water and drinking water in an industrialized city in the Yangtze River Delta.

PubMed

Xiao, Sanhua; Lv, Xuemin; Zeng, Yifan; Jin, Tao; Luo, Lan; Zhang, Binbin; Zhang, Gang; Wang, Yanhui; Feng, Lin; Zhu, Yuan; Tang, Fei

2017-10-01

Public concern was aroused by frequently reported water pollution incidents in Taihu Lake and the Yangtze River. The pollution also caught and sustained the attention of the scientific community. From 2010 to 2016, raw water and drinking water samples were continually collected at Waterworks A and B (Taihu Lake) and Waterworks C (Yangtze River). The non-volatile organic pollutants in the water samples were extracted by solid phase extraction. Ames tests and yeast estrogen screen (YES) assays were conducted to evaluate the respective mutagenic and estrogenic effects. Water samples from the Yangtze River-based Waterworks C possessed higher mutagenicity than those from Taihu Lake-based Waterworks A (P＜0.001) and Waterworks B (P = 0.026). Water treatment enhanced the direct mutagenicity (P = 0.022), and weakened the estrogenicity of the raw water (P＜0.001) with a median removal rate of 100%. In fact, very few of the finished samples showed estrogenic activity. Raw water samples from Waterworks A showed weaker estrogenicity than those from Waterworks B (P = 0.034) and Waterworks C (P = 0.006). In summary, mutagenic effects in drinking water and estrogenic effects in raw water merited sustained attention. The Yangtze River was more seriously polluted by mutagenic and estrogenic chemicals than Taihu Lake was. Copyright © 2017 Elsevier Ltd. All rights reserved.

Determination of water quality, toxicity and estrogenic activity in a nearshore marine environment in Rio de Janeiro, Southeastern Brazil.

PubMed

do Nascimento, Marilia Teresa Lima; Santos, Ana Dalva de Oliveira; Felix, Louise Cruz; Gomes, Giselle; de Oliveira E Sá, Mariana; da Cunha, Danieli Lima; Vieira, Natividade; Hauser-Davis, Rachel Ann; Baptista Neto, José Antonio; Bila, Daniele Maia

2018-03-01

Endocrine disrupting compounds (EDCs) can be found in domestic sewage, wastewater treatment plant effluents, natural water, rivers, lakes and in the marine environment. Jurujuba Sound, located in the state of Rio de Janeiro, Southeastern Brazil, receives untreated sewage into its waters, one the main sources of aquatic contamination in this area. In this context, the aim of the present study was to evaluate the estrogenic potential of water sampled from different depths and from areas with differential contamination levels throughout Jurujuba Sound. Water quality was evaluated and acute toxicity assays using Allviibrio fischeri were conducted, while estrogenic activity of the water samples was determined by a Yeast Estrogen Screening assay (YES). Water quality was mostly within the limits established for marine waters by the Brazilian legislation, with only DOC and ammoniacal nitrogen levels above the maximum permissible limits. No acute toxicity effects were observed in the Allivibrio fisheri assay. The YES assay detected moderate estrogenic activity in bottom water samples from 3 sampling stations, ranging from 0.5 to 3.2ngL -1 , as well as in one surface water sample. Estrogenic activity was most frequently observed in samples from the bottom of the water column, indicating adsorption of estrogenic compounds to the sediment. Copyright © 2017 Elsevier Inc. All rights reserved.

Screening Chemicals for Estrogen Receptor Bioactivity Using a Computational Model.

PubMed

Browne, Patience; Judson, Richard S; Casey, Warren M; Kleinstreuer, Nicole C; Thomas, Russell S

2015-07-21

The U.S. Environmental Protection Agency (EPA) is considering high-throughput and computational methods to evaluate the endocrine bioactivity of environmental chemicals. Here we describe a multistep, performance-based validation of new methods and demonstrate that these new tools are sufficiently robust to be used in the Endocrine Disruptor Screening Program (EDSP). Results from 18 estrogen receptor (ER) ToxCast high-throughput screening assays were integrated into a computational model that can discriminate bioactivity from assay-specific interference and cytotoxicity. Model scores range from 0 (no activity) to 1 (bioactivity of 17β-estradiol). ToxCast ER model performance was evaluated for reference chemicals, as well as results of EDSP Tier 1 screening assays in current practice. The ToxCast ER model accuracy was 86% to 93% when compared to reference chemicals and predicted results of EDSP Tier 1 guideline and other uterotrophic studies with 84% to 100% accuracy. The performance of high-throughput assays and ToxCast ER model predictions demonstrates that these methods correctly identify active and inactive reference chemicals, provide a measure of relative ER bioactivity, and rapidly identify chemicals with potential endocrine bioactivities for additional screening and testing. EPA is accepting ToxCast ER model data for 1812 chemicals as alternatives for EDSP Tier 1 ER binding, ER transactivation, and uterotrophic assays.

In vitro OECD test methods applied to screen the estrogenic effect of chemicals, used in Korea.

PubMed

Lee, Hee-Seok; Park, Eun-Jung; Han, Songyi; Oh, Gyeong-Yong; Kim, Min-Hee; Kang, Hui-Seung; Suh, Jin-Hyang; Oh, Jae-Ho; Lee, Kwang-Soo; Hwang, Myung-Sil; Moon, Guiim; Hong, Jin-Hwan; Hwang, In-Gyun

2016-09-01

In this study, 27 chemicals found in household products, which became an issue in Korea were screened for the agonistoc and antagonistic effects against human estrogen receptor using official Organization for Economic Cooperation and Development (OECD) in vitro assays, STTA assay using ERα-HeLa-9903 cell line and BG1Luc ER TA assay. In the case of human ER agonist screening by two assays, all tested chemicals did not show agonist effect against ER. In ER antagonist test by BG1Luc ER TA assay, five surfactants α-dodecyl-ω-hydroxypoly(oxyethylene), alcohols C16-18 ethoxylated, nonylphenol, ethoxylated, 3,6,9,12,15,18,21-heptaoxatritriacontan-1-ol, and α-dodecyl-ω-hydroxypoly(oxy-1,2-ethanediyl)) were found to exhibit weak antagonistic activities. The agonist/antagonist effects against human estrogen receptor of various chemicals, used in Korea by OECD test guideline are reported in this study. These results indicated that two OECD in vitro assays will can be applied in Korea by screening of agonistic/antagonistic effects against human ER of various chemicals. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry.

PubMed

Chen, Jun; Young, Susan M; Allen, Chris; Seeber, Andrew; Péli-Gulli, Marie-Pierre; Panchaud, Nicolas; Waller, Anna; Ursu, Oleg; Yao, Tuanli; Golden, Jennifer E; Strouse, J Jacob; Carter, Mark B; Kang, Huining; Bologa, Cristian G; Foutz, Terry D; Edwards, Bruce S; Peterson, Blake R; Aubé, Jeffrey; Werner-Washburne, Margaret; Loewith, Robbie J; De Virgilio, Claudio; Sklar, Larry A

2012-04-20

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded, and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high-throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in a manner analogous to that of rapamycin. We have shown that CID 3528206 inhibited yeast cell growth and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC(50)'s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.

Metabolomics-based prediction models of yeast strains for screening of metabolites contributing to ethanol stress tolerance

NASA Astrophysics Data System (ADS)

Hashim, Z.; Fukusaki, E.

2016-06-01

The increased demand for clean, sustainable and renewable energy resources has driven the development of various microbial systems to produce biofuels. One of such systems is the ethanol-producing yeast. Although yeast produces ethanol naturally using its native pathways, production yield is low and requires improvement for commercial biofuel production. Moreover, ethanol is toxic to yeast and thus ethanol tolerance should be improved to further enhance ethanol production. In this study, we employed metabolomics-based strategy using 30 single-gene deleted yeast strains to construct multivariate models for ethanol tolerance and screen metabolites that relate to ethanol sensitivity/tolerance. The information obtained from this study can be used as an input for strain improvement via metabolic engineering.

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

PubMed

Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

2015-08-25

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

Characterization of Estrogen and Androgen Activity of Food Contact Materials by Different In Vitro Bioassays (YES, YAS, ERα and AR CALUX) and Chromatographic Analysis (GC-MS, HPLC-MS)

PubMed Central

Osorio, Veronica; Grininger, Angelika; Richter, Alexander; Bergmair, Johannes; Pyerin, Michael; Washüttl, Michael; Tacker, Manfred

2014-01-01

Endocrine active substances (EAS) show structural similarities to natural hormones and are suspected to affect the human endocrine system by inducing hormone dependent effects. Recent studies with in vitro tests suggest that EAS can leach from packaging into food and may therefore pose a risk to human health. Sample migrates from food contact materials were tested for estrogen and androgen agonists and antagonists with different commonly used in vitro tests. Additionally, chemical trace analysis by GC-MS and HPLC-MS was used to identify potential hormone active substances in sample migrates. A GC-MS method to screen migrates for 29 known or potential endocrine active substances was established and validated. Samples were migrated according to EC 10/2011, concentrated by solid phase extraction and tested with estrogen and androgen responsive reporter gene assays based on yeast cells (YES and YAS) or human osteoblast cells (ERα and AR CALUX). A high level of agreement between the different bioassays could be observed by screening for estrogen agonists. Four out of 18 samples tested showed an estrogen activity in a similar range in both, YES and ERα CALUX. Two more samples tested positive in ERα CALUX due to the lower limits of detection in this assay. Androgen agonists could not be detected in any of the tested samples, neither with YAS nor with AR CALUX. When testing for antagonists, significant differences between yeast and human cell-based bioassays were noticed. Using YES and YAS many samples showed a strong antagonistic activity which was not observed using human cell-based CALUX assays. By GC-MS, some known or supposed EAS were identified in sample migrates that showed a biological activity in the in vitro tests. However, no firm conclusions about the sources of the observed hormone activity could be obtained from the chemical results. PMID:25000404

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances.

PubMed

Navas, José M; Segner, Helmut

2006-10-25

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to

Joint effects of heterogeneous estrogenic chemicals in the E-screen--exploring the applicability of concentration addition.

PubMed

Silva, Elisabete; Rajapakse, Nissanka; Scholze, Martin; Backhaus, Thomas; Ermler, Sibylle; Kortenkamp, Andreas

2011-08-01

In the last few years, significant advances have been made toward understanding the joint action of endocrine disrupting chemicals (EDCs). A number of studies have demonstrated that the combined effects of different types of EDCs (e.g., estrogenic, antiandrogenic, or thyroid-disrupting agents) can be predicted by the model of concentration addition (CA). However, there is still limited information on the effects of mixtures of large numbers of chemicals with varied structural features, which are more representative of realistic human exposure scenarios. The work presented here aims at filling this gap. Using a breast cancer cell proliferation assay (E-Screen), we assessed the joint effects of five mixtures, containing between 3 and 16 estrogenic agents, including compounds as diverse as steroidal hormones (endogenous and synthetic), pesticides, cosmetic additives, and phytoestrogens. CA was employed to predict mixture effects, which were then compared with experimental outcomes. The effects of two of the mixtures tested were additive, being accurately predicted by CA. However, for the three other mixtures, CA slightly overestimated the experimental observations. In view of these results, we hypothesized that the deviations were due to increased metabolism of steroidal estrogens in the mixture setting. We investigated this by testing the impact of two such mixtures on the activation and expression of steroidal estrogen metabolizing enzymes, such as cytochrome P450 (CYP) 1A1, CYP 1B1, and CYP 3A4. Activation of CYP 1B1 and, consequently, a reduction in the levels of steroidal estrogens in the mixture could contribute to the shortfall from the additivity prediction that we observed.

The anti-estrogenic activity of sediments from agriculturally-intense watersheds: Assessment using in vivo and in vitro assays

PubMed Central

Sellin Jeffries, Marlo K.; Conoan, Nicholas H.; Cox, Marc B.; Sangster, Jodi L.; Balsiger, Heather A.; Bridges, Andrew A.; Cowman, Tim; Knight, Lindsey A.; Bartelt-Hunt, Shannon L.; Kolok, Alan S.

2015-01-01

The goal of the current study was to determine whether sediments from agriculturally-intense watersheds can act as a potential source of anti-estrogenic endocrine-disrupting compounds. The specific objectives of the current study were to determine 1) whether female fathead minnows (Pimephales promelas) experience alterations in endocrine function when exposed to sediments collected from agriculturally-intense watersheds and 2) if these sediments display anti-estrogenic activity in an in vitro assay. In addition, sediment samples were analyzed for the presence of steroid hormones and pesticides associated with local agricultural practices. To accomplish this, sediments and water were collected from three sites within two agriculturally-intense Nebraska watersheds (Bow Creek and the Elkhorn River). In 2009, minnows were exposed to sediment and/or water collected from the two Bow Creek sites (East Bow Creek and the Confluence) in the laboratory, while in 2010, minnows were exposed to sediment and/or water from East Bow Creek, the Confluence and the Elkhorn River. Following the 7-d exposure period, the hepatic mRNA expression of two-estrogen responsive genes, estrogen receptor α (ERα) and vitellogenin (Vtg) was determined. In 2009, females exposed to Confluence sediments, in the presence of laboratory water or Confluence water, experienced significant reductions in ERα expression relative to unexposed and Confluence water-exposed females. The defeminization of these females suggests the presence of a biologically-available anti-estrogenic compound in sediments collected from this site. In 2010, sediments were assessed for anti-estrogenic activity on days 0 and 7 of the exposure period using a four-hour yeast estrogen screen. Lipophilic extracts (LEs) of day 0 sediments collected from the Confluence and the Elkhorn River induced significant reductions in the estrogenic reporter activity of treated yeast cultures suggesting the presence of a lipophilic anti-estrogenic

Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

PubMed

Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G

2008-02-01

The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

Integrated assessment of wastewater treatment plant effluent estrogenicity in the Upper Murray River, Australia, using the native Murray rainbowfish (Melanotaenia fluviatilis)

USGS Publications Warehouse

Vajda, Alan M.; Kumar, Anupama; Woods, Marianne; Williams, Mike; Doan, Hai; Tolsher, Peter; Kookana, Rai S.; Barber, Larry B.

2016-01-01

The contamination of major continental river systems by endocrine-active chemicals (EACs) derived from the discharge of wastewater treatment plant (WWTP) effluents can affect human and ecosystem health. As part of a long-term effort to develop a native fish model organism for assessment of endocrine disruption in Australia's largest watershed, the Murray-Darling River Basin, the present study evaluated endocrine disruption in adult males of the native Australian Murray rainbowfish (Melanotaenia fluviatilis) exposed to effluent from an activated sludge WWTP and water from the Murray River during a 28-d, continuous-flow, on-site experiment. Analysis of the WWTP effluent and river water detected estrone and 17β-estradiol at concentrations up to approximately 25 ng L−1. Anti-estrogenicity of effluent samples was detected in vitro using yeast-based bioassays (yeast estrogen screen) throughout the experiment, but estrogenicity was limited to the first week of the experiment. Histological evaluation of the testes indicated significant suppression of spermatogenesis by WWTP effluent after 28 d of exposure. Plasma vitellogenin concentrations and expression of vitellogenin messenger RNA in liver were not significantly affected by exposure to WWTP effluent. The combination of low contaminant concentrations in the WWTP effluent, limited endocrine disrupting effects in the Murray rainbowfish, and high in-stream dilution factors (>99%) suggest minimal endocrine disruption impacts on native Australian fish in the Murray River downstream from the WWTP outfall. 

Occurrence and in vitro bioactivity of estrogen, androgen, and glucocorticoid compounds in a nationwide screen of United States stream waters

EPA Science Inventory

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a suite of chemical a...

Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

PubMed

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma; Lafontaine, Denis L J; Urbonavičius, Jaunius

2012-01-01

Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

PubMed Central

Helsdingen, Richard J. R.; Hamers, Astrid R. M.; van Duursen, Majorie B. M.; Nielen, Michel W. F.; Hoogenboom, Ron L. A. P.

2007-01-01

Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action. PMID:17849102

Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

EPA Science Inventory

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

High-throughput screening and mechanism-based evaluation of estrogenic botanical extracts

PubMed Central

Overk, Cassia R.; Yao, Ping; Chen, Shaonong; Deng, Shixing; Imai, Ayano; Main, Matthew; Schinkovitz, Andreas; Farnsworth, Norman R.; Pauli, Guido F.; Bolton, Judy L.

2009-01-01

Symptoms associated with menopause can greatly affect the quality of life for women. Botanical dietary supplements have been viewed by the public as safe and effective despite a lack of evidence indicating a urgent necessity to standardize these supplements chemically and biologically. Seventeen plants were evaluated for estrogenic biological activity using standard assays: competitive estrogen receptor (ER) binding assay for both alpha and beta subtypes, transient transfection of the estrogen response element luciferase plasmid into MCF-7 cells expressing either ER alpha or ER beta, and the Ishikawa alkaline phosphatase induction assay for both estrogenic and antiestrogenic activities. Based on the combination of data pooled from these assays, the following was determined: a) a high rate of false positive activity for the competitive binding assays, b) some extracts had estrogenic activity despite a lack of ability to bind the ER, c) one extract exhibited selective estrogen receptor modulator (SERM) activity, and d) several extracts show additive/synergistic activity. Taken together, these data indicate a need to reprioritize the order in which the bioassays are performed for maximal efficiency of programs involving bioassay-guided fractionation. In addition, possible explanations for the conflicts in the literature over the estrogenicity of Cimicifuga racemosa (black cohosh) are suggested. PMID:18473738

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals

PubMed Central

Brennan, Jennifer C.; Bassal, Arzoo; He, Guochun; Denison, Michael S.

2016-01-01

Estrogenic endocrine disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, there is a critical need for rapidly detecting these chemicals. We developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the USEPA and OECD as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only one of the two known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells and qRT-PCR confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα and ERβ-selective chemicals. PMID:26139245

Evaluation of estrogenic, antiestrogenic and genotoxic activity of nemorosone, the major compound found in brown Cuban propolis

PubMed Central

2013-01-01

Background Brown propolis is the major type of propolis found in Cuba; its principal component is nemorosone, the major constituent of Clusia rosea floral resins. Nemorosone has received increasing attention due to its strong in vitro anti-cancer action. The citotoxicity of nemorosone in several human cancer cell lines has been reported and correlated to the direct action it has on the estrogen receptor (ER). Breast cancer can be treated with agents that target estrogen-mediated signaling, such as antiestrogens. Phytoestrogen can mimic or modulate the actions of endogenous estrogens and the treatment of breast cancer with phytoestrogens may be a valid strategy, since they have shown anti-cancer activity. Methods The aim of the present investigation was to assess the capacity of nemorosone to interact with ERs, by Recombinant Yeast Assay (RYA) and E-screen assays, and to determine by comet assay, if the compound causes DNA-damaging in tumoral and non-tumoral breast cells. Results Nemorosone did not present estrogenic activity, however, it inhibited the 17-β-estradiol (E2) action when either of both methods was used, showing their antiestrogenicity. The DNA damage induced by the benzophenone in cancer and normal breast cells presented negative results. Conclusion These findings suggest that nemorosone may have therapeutic application in the treatment of breast cancer. PMID:23902919

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen

PubMed Central

2013-01-01

Background HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. Results Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. Conclusions Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I. PMID:24229420

Screening Estrogenic Activities of Chemicals or Mixtures In Vivo Using Transgenic (cyp19a1b-GFP) Zebrafish Embryos

PubMed Central

Brion, François; Le Page, Yann; Piccini, Benjamin; Cardoso, Olivier; Tong, Sok-Keng; Chung, Bon-chu; Kah, Olivier

2012-01-01

The tg(cyp19a1b-GFP) transgenic zebrafish expresses GFP (green fluorescent protein) under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i) it is only expressed in radial glial progenitors in the brain of fish and (ii) it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture), including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective. PMID:22586461

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

PubMed

Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

2018-02-08

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

PubMed Central

Kalay, Gizem; Lusk, Richard; Dome, Mackenzie; Hens, Korneel; Deplancke, Bart; Wittkopp, Patricia J.

2016-01-01

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming. PMID:27527791

Estrogenic effects of nonylphenol and octylphenol isomers in vitro by recombinant yeast assay (RYA) and in vivo with early life stages of zebrafish.

PubMed

Puy-Azurmendi, E; Olivares, A; Vallejo, A; Ortiz-Zarragoitia, M; Piña, B; Zuloaga, O; Cajaraville, M P

2014-01-01

Commercial OP and NP are complex isomer mixtures that can be individually present in the environment, showing different estrogenic potencies. The aims of this study were to establish the estrogenic potency of some AP isomers in comparison to the commercial NP (cNP) mixture in vitro and to investigate in vivo their possible effects during the embryo and larval development of zebrafish. An in vitro estrogen receptor-based recombinant yeast assay was used to test the estrogenicity of specific AP isomers (22-OP, 33-OP, 22-NP, 33-NP and 363-NP) and cNP. The EC₅₀ was in the range of 0.6-7.7 mg/L. Both OP isomers and 363-NP exhibited higher estrogenic activity than cNP. For in vivo experiments, one-day postfertilisation (dpf) embryos were exposed to cNP (50, 250 and 500 μg/L), 363-NP and 33-OP (50 μg/L), 17β-estradiol (100 ng/L) and DMSO (0.01% v/v) for 4weeks. After exposure fish were maintained for 2 weeks in clean water in order to evaluate a possible recovery. Fish of groups exposed to cNP and 363-NP were the last to hatch. Histological alterations were not observed after 7, 28 or 42 dpf. Exposure to 33-OP increased transcriptional levels of erα, vtg and cyp19a1b genes. However, transcriptional response in E2 exposure was observed at later stages and with higher fold induction levels. Exposure to cNP decreased levels of erα whereas increased levels of rxrγ and cyp19a1b. Exposure to 363-NP did not cause changes in transcriptional levels of studied genes. The differences in response of the OP isomer compared to the NP isomer in zebrafish could be related to the rapid decay in concentration of the latter. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of watershed densities of animal feeding operations on nutrient concentrations and estrogenic activity in agricultural streams.

PubMed

Ciparis, Serena; Iwanowicz, Luke R; Voshell, J Reese

2012-01-01

Application of manures from animal feeding operations (AFOs) as fertilizer on agricultural land can introduce nutrients and hormones (e.g. estrogens) to streams. A landscape-scale study was conducted in the Shenandoah River watershed (Virginia, USA) in order to assess the relationship between densities of AFOs in watersheds of agricultural streams and in-stream nutrient concentrations and estrogenic activity. The effect of wastewater treatment plants (WWTPs) on nutrients and estrogenic activity was also evaluated. During periods of high and low flow, dissolved inorganic nitrogen (DIN) and orthophosphate (PO(4)-P) concentrations were analyzed and estrogens/estrogenic compounds were extracted and quantified as17β-estradiol equivalents (E2Eq) using a bioluminescent yeast estrogen screen. Estrogenic activity was measurable in the majority of collected samples, and 20% had E2Eq concentrations >1 ng/L. Relatively high concentrations of DIN (>1000 μg/L) were also frequently detected. During all sampling periods, there were strong relationships between watershed densities of AFOs and in-stream concentrations of DIN (R(2) = 0.56-0.81) and E2Eq (R(2) = 0.39-0.75). Relationships between watershed densities of AFOs and PO(4)-P were weaker, but were also significant (R(2) = 0.27-0.57). When combined with the effect of watershed AFO density, streams receiving WWTP effluent had higher concentrations of PO(4)-P than streams without WWTP discharges, and PO(4)-P was the only analyte with a consistent relationship to WWTPs. The results of this study suggest that as the watershed density of AFOs increases, there is a proportional increase in the potential for nonpoint source pollution of agricultural streams and their receiving waters by nutrients, particularly DIN, and compounds that can cause endocrine disruption in aquatic organisms. Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of watershed densities of animal feeding operations on nutrient concentrations and estrogenic activity in agricultural streams

USGS Publications Warehouse

Ciparis, S.; Iwanowicz, L.R.; Voshell, J.R.

2012-01-01

Application of manures from animal feeding operations (AFOs) as fertilizer on agricultural land can introduce nutrients and hormones (e.g. estrogens) to streams. A landscape-scale study was conducted in the Shenandoah River watershed (Virginia, USA) in order to assess the relationship between densities of AFOs in watersheds of agricultural streams and in-stream nutrient concentrations and estrogenic activity. The effect of wastewater treatment plants (WWTPs) on nutrients and estrogenic activity was also evaluated. During periods of high and low flow, dissolved inorganic nitrogen (DIN) and orthophosphate (PO 4-P) concentrations were analyzed and estrogens/estrogenic compounds were extracted and quantified as17??-estradiol equivalents (E2Eq) using a bioluminescent yeast estrogen screen. Estrogenic activity was measurable in the majority of collected samples, and 20% had E2Eq concentrations >1ng/L. Relatively high concentrations of DIN (>1000??g/L) were also frequently detected. During all sampling periods, there were strong relationships between watershed densities of AFOs and in-stream concentrations of DIN (R 2=0.56-0.81) and E2Eq (R 2=0.39-0.75). Relationships between watershed densities of AFOs and PO 4-P were weaker, but were also significant (R 2=0.27-0.57). When combined with the effect of watershed AFO density, streams receiving WWTP effluent had higher concentrations of PO 4-P than streams without WWTP discharges, and PO 4-P was the only analyte with a consistent relationship to WWTPs. The results of this study suggest that as the watershed density of AFOs increases, there is a proportional increase in the potential for nonpoint source pollution of agricultural streams and their receiving waters by nutrients, particularly DIN, and compounds that can cause endocrine disruption in aquatic organisms. ?? 2011 Elsevier B.V.

Effects of watershed densities of animal feeding operations on nutrient concentrations and estrogenic activity in agricultural streams

USGS Publications Warehouse

Ciparis, Serena; Iwanowicz, Luke R.; Voshell, J. Reese

2012-01-01

Application of manures from animal feeding operations (AFOs) as fertilizer on agricultural land can introduce nutrients and hormones (e.g. estrogens) to streams. A landscape-scale study was conducted in the Shenandoah River watershed (Virginia, USA) in order to assess the relationship between densities of AFOs in watersheds of agricultural streams and in-stream nutrient concentrations and estrogenic activity. The effect of wastewater treatment plants (WWTPs) on nutrients and estrogenic activity was also evaluated. During periods of high and low flow, dissolved inorganic nitrogen (DIN) and orthophosphate (PO4-P) concentrations were analyzed and estrogens/estrogenic compounds were extracted and quantified as17β-estradiol equivalents (E2Eq) using a bioluminescent yeast estrogen screen. Estrogenic activity was measurable in the majority of collected samples, and 20% had E2Eq concentrations > 1 ng/L. Relatively high concentrations of DIN (> 1000 μg/L) were also frequently detected. During all sampling periods, there were strong relationships between watershed densities of AFOs and in-stream concentrations of DIN (R2 = 0.56–0.81) and E2Eq (R2 = 0.39–0.75). Relationships between watershed densities of AFOs and PO4-P were weaker, but were also significant (R2 = 0.27–0.57). When combined with the effect of watershed AFO density, streams receiving WWTP effluent had higher concentrations of PO4-P than streams without WWTP discharges, and PO4-P was the only analyte with a consistent relationship to WWTPs. The results of this study suggest that as the watershed density of AFOs increases, there is a proportional increase in the potential for nonpoint source pollution of agricultural streams and their receiving waters by nutrients, particularly DIN, and compounds that can cause endocrine disruption in aquatic organisms.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals.

PubMed

Brennan, Jennifer C; Bassal, Arzoo; He, Guochun; Denison, Michael S

2016-01-01

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals. © 2015 SETAC.

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

PubMed

Fujimoto, Nariaki; Honda, Hiroaki; Kitamura, Shigeyuki

2004-01-01

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

Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

PubMed Central

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma

2012-01-01

Background Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal Findings We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action. PMID:23227207

Dietary Exposure of Nigerians to Mutagens and Estrogen-Like Chemicals

PubMed Central

Omoruyi, Iyekhoetin Matthew; Ahamioje, Derek; Pohjanvirta, Raimo

2014-01-01

Food and drinking water are poorly delineated sources of human exposure to chemical food mutagens and endocrine-disrupting chemicals. In this study, we investigated the presence of mutagens and chemicals exhibiting estrogenic activity in the daily diet of Nigerians, using in vitro assays. Commercially processed foods or snacks and various brands of pure water sachets were extracted by solid-phase extraction and liquid-liquid extraction, respectively. Mutagenicity was determined by the conventional Ames test and two complementary assays on two strains of Salmonella (TA 100 and TA 98), while the estrogenic activity was assessed by a yeast bioluminescent assay, using two recombinant yeast strains (Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc). A third of the food varieties investigated (chin-chin, hamburger, suya and bean cake) were mutagenic in all three assays, either in the presence or absence of S9 mix. Of the packed water samples, five out of the sixteen investigated (31%), were found to be estrogenic, with estradiol and bisphenol A equivalents ranging from 0.79 to 44.0 ng/L and 124.2 to 1,000.8 ng/L, respectively. Hence, although the current situation in Nigeria does not appear to be substantially worse than, e.g., in Europe, regular monitoring is warranted in the future. PMID:25153465

Comparison of in vitro estrogenic activity and estrogen concentrations in source and treated waters from 25 U.S. drinking water treatment plants

EPA Science Inventory

In vitro bioassays have been successfully used to screen for estrogenic activity in wastewater and surface water, however, few have been applied to treated drinking water. Here, extracts of source and treated drinking water samples were assayed for estrogenic activity using T47D...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

PubMed Central

Beresford, Nicola; Baynes, Alice; Kanda, Rakesh; Mills, Matthew R.; Arias-Salazar, Karla; Collins, Terrence J.; Jobling, Susan

2016-01-01

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies. PMID:27684328

Fluorescent characteristics of estrogenic compounds in landfill leachate.

PubMed

Zhanga, Hua; Changb, Cheng-Hsuan; Lü, Fan; Su, Ay; Lee, Duu-Jong; He, Pin-Jing; Shao, Li-Ming

2009-08-01

Estrogens in landfill leachate could probably contaminate receiving water sources if not properly polished before discharge. This work measured, using an estrogen receptor-alpha competitor screening assay, the estrogenic potentials of leachate samples collected at a local sanitary landfill in Shanghai, China and their compounds fractionated by molecular weights. The chemical structures of the constituent compounds were characterized using fluorescence excitation and emission matrix (EEM). The organic matters of molecular weight <600 Da and of 3000-14,000 Da contributed most of the estrogenic potentials of the raw leachates. The former were considered as the typical endocrine disrupting compounds in dissolved state; while the latter the fulvic acids with high aromaticity that were readily adsorbed with estrogens (bound state). Statistical analysis on EEM peaks revealed that the chemical structures of noted estrogens in dissolved state and in bound state were not identical. Aerobic treatment effectively removed dissolved estrogens, but rarely removed those bound estrogens.

Effect-related monitoring: estrogen-like substances in groundwater.

PubMed

Kuch, Bertram; Kern, Frieder; Metzger, Jörg W; von der Trenck, Karl Theo

2010-02-01

Concentration monitoring as a basis for risk assessment is a valid approach only if there is an unambiguous relation between concentration and effect. In many cases, no such unambiguous relation exists, since various substances can exert the same effect with differing potencies. If some or all of these substances contributing to a biological effect are unknown, effect-related monitoring becomes indispensable. Endocrine-disrupting substances in water bodies, including the groundwater, are a prominent example of such a case. The aim of the investigations described here was to detect hormonally active substances in the groundwater downstream of obsolete landfills by using the E-screen assay and to possibly assign the biological effect to individual chemical compounds by means of instrumental analyses carried out in parallel. Grab samples of the groundwater were collected downstream from abandoned landfills and prepared by liquid/liquid extraction. The total estrogenic activity in these samples was determined in vitro by applying the E-screen assay. The human breast cancer cells (MCF-7) used in the E-screen proliferate in response to the presence of estrogenically active compounds. Expressed in concentration units of the reference substance 17beta-estradiol (E2), the test system allows the quantification of estrogenicity with a limit of detection (LOD) in the range of 0.1 ng/L. Aliquots of the samples were screened using gas chromatography/mass spectrometry (GC/MS) in order to quantify known estrogenically active substances and to identify unknown compounds. Estrogen-positive samples were extracted at different pH values, split into acidic, neutral, and basic fractions and analyzed by GC/MS, searching for individual components that display estrogenic activity. Estrogenic activity exceeding the LOD and the provisional benchmark of 0.5 ng E2/L was found at three out of seven abandoned waste disposal sites tested. The low concentrations of known xenoestrogens such as

Discovery of estrogen receptor α modulators from natural compounds in Si-Wu-Tang series decoctions using estrogen-responsive MCF-7 breast cancer cells.

PubMed

Liu, Li; Ma, Hongyue; Tang, Yuping; Chen, Wenxing; Lu, Yin; Guo, Jianming; Duan, Jin-Ao

2012-01-01

The binding between the estrogen receptor α (ER-α) and a variety of compounds in traditional Chinese formulae, Si-Wu-Tang (SWT) series decoctions, was studied using a stably-transfected human breast cancer cell line (MVLN). In 38 compounds tested from SWT series decoctions, the estrogen-like activity of 22 compounds was above 60% in 20 μg mL(-1). Furthermore, theoretical affinity of these compounds was certificated using the functional virtual screen of ER-α modulators by FlexX-Pharm. The accuracy of functional virtual screening of ER-α modulators could reach to 77.27%. The results showed that some compounds, such as organic acids and flavones in SWT series decoctions could be used as selective estrogen receptor modulators (SERMs) and could be selected for further development as potential agents for estrogen related diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

PubMed Central

Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

2015-01-01

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

PubMed Central

Vizeacoumar, Franco J.; van Dyk, Nydia; S.Vizeacoumar, Frederick; Cheung, Vincent; Li, Jingjing; Sydorskyy, Yaroslav; Case, Nicolle; Li, Zhijian; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan; Bloom, Kerry

2010-01-01

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. PMID:20065090

A strategy for rapid production and screening of yeast artificial chromosome libraries.

PubMed

Strauss, W M; Jaenisch, E; Jaenisch, R

1992-01-01

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture "pools," and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4-5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the alpha 1(I) collagen (Col1a1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.

CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

EPA Pesticide Factsheets

Data from a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) demonstrating using predictive computational models on high-throughput screening data to screen thousands of chemicals against the estrogen receptor.This dataset is associated with the following publication:Mansouri , K., A. Abdelaziz, A. Rybacka, A. Roncaglioni, A. Tropsha, A. Varnek, A. Zakharov, A. Worth, A. Richard , C. Grulke , D. Trisciuzzi, D. Fourches, D. Horvath, E. Benfenati , E. Muratov, E.B. Wedebye, F. Grisoni, G.F. Mangiatordi, G.M. Incisivo, H. Hong, H.W. Ng, I.V. Tetko, I. Balabin, J. Kancherla , J. Shen, J. Burton, M. Nicklaus, M. Cassotti, N.G. Nikolov, O. Nicolotti, P.L. Andersson, Q. Zang, R. Politi, R.D. Beger , R. Todeschini, R. Huang, S. Farag, S.A. Rosenberg, S. Slavov, X. Hu, and R. Judson. (Environmental Health Perspectives) CERAPP: Collaborative Estrogen Receptor Activity Prediction Project. ENVIRONMENTAL HEALTH PERSPECTIVES. National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA, 1-49, (2016).

Erythrina excelsa exhibits estrogenic effects in vivo and in vitro and is cytotoxic on breast and colon cancer cell lines.

PubMed

Tchoukouegno Ngueu, Sadrine; Tchoumtchoua, Job; Njamen, Dieudonné; Halabalaki, Maria; Laudenbach-Leschowski, Ute; Diel, Patrick

2016-01-01

Eythrina excelsa Baker (Fabaceae) is a medicinal plant used to treat various ailments including those of the female genital tract. The objective of this study is to investigate the estrogenic and cytotoxic effects of the ethanol extract of the stem bark of E. excelsa. Erythrina excelsa was evaluated in vitro using the yeast estrogen screen (YES). The extract was then tested in a 3-day uterotrophic assay on ovariectomised Wistar rats at doses of 50 and 100 mg/kg BW/d. Cytotoxic effects were assessed on breast (MCF-7) and colon (HT-29) cancer cell lines using the MTT cell viability assay. Additionally, a LC-PDA-ESI (+)-HRMS and HRMS/MS method was developed and applied for the identification of representative secondary metabolites scaffolds in the extract. In the YES, the extract stimulated the transactivation of the estrogen receptor in a dose-dependent manner with an EC50 value of 1.8 μg/mL. In rats, E. excelsa increased uterine wet weight, uterine epithelial height, and the mRNA expression of estrogen-responsive genes in the uterus and liver at 50 whereas at 100 mg/kg BW/d anti-estrogenic effects were observed. In the MTT-assay, a dose-dependent decrease of the viability of both cell lines was observed with EC50 values of 13.6 μg/mL (MCF-7) and 27.7 μg/mL (HT-29). The phytochemical analysis revealed that the extract is rich in isoflavonoids, mainly prenylated and pyran-derivatives thereof. Erythrina excelsa is rich in prenylated and pyran-substituted isoflavonoids, exhibits estrogenic/anti-estrogenic and cytotoxic effects and warrant sufficient interest for deeper investigations.

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

PubMed Central

Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

2016-01-01

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae.

PubMed

Zhang, Chenlu; Acosta-Sampson, Ligia; Yu, Vivian Yaci; Cate, Jamie H D

2017-01-01

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

PubMed

Shohdy, Nadim; Efe, Jem A; Emr, Scott D; Shuman, Howard A

2005-03-29

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

A SIMPLE AND RAPID MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME OF FLIGHT MASS SPECTROMETRY METHOD TO SCREEN FISH PLASMA SAMPLES FOR ESTROGEN-RESPONSIVE BIOMARKERS

EPA Science Inventory

In this study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix assisted laster desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) couopled with a short...

Pesticide residues and estrogenic activity in fruit and vegetables sampled from major fresh produce markets in South Africa.

PubMed

Mutengwe, Mbulaheni Thomas; Aneck-Hahn, Natalie Hildegard; Korsten, Lise; Van Zijl, Magdalena Catherina; De Jager, Christiaan

2016-01-01

Food is likely to be one of the major pathways through which people are exposed to endocrine-disrupting chemicals. With the exception of residual effects, there are concerns that a number of naturally occurring and synthetic chemicals exert adverse effects upon endocrine systems in wildlife and humans. The current study reports selected pesticide concentrations and the total estrogenic activity of fruit and vegetables using the recombinant yeast oestrogen screen (YES) and T47D-KBluc reporter gene assays. A total of 53 food samples (27 fruit and 26 vegetables) from Johannesburg and Tshwane fresh produce markets (in South Africa) were analysed. Of these, 17 contained one to three different pesticide residues with concentrations ranging between 0.01 and 0.68 mg kg(-1), whereas in the rest of the samples no residues were detected. All pesticides detected except in one sample were below the maximum residue level (MRL), but others were unauthorised for use in specified fruit and vegetables. Estrogenic activity was detected in 26.4% (14 samples) of the samples tested, and the estradiol equivalents ranged from 0.007 to 2 pg g(-1). Although the estrogenic activity was low, it may contribute to adverse health effects. Continuous monitoring for pesticides in fruit and vegetables is important in view of the unauthorised pesticides detected in produce from South Africa and the endocrine-disrupting chemical activity found.

Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease.

PubMed

Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Alvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A

2012-11-01

MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. According to our results, supplementation with riboflavin or coenzyme Q(10) effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease

PubMed Central

Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Álvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A

2012-01-01

BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. PMID:22747838

Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay.

PubMed

Heintz-Buschart, Anna; Eickhoff, Holger; Hohn, Erwin; Bilitewski, Ursula

2013-03-10

Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. As it is not primarily a pathogen, but can exist in a commensal state, we aimed at the identification of new anti-infective compounds which do not eradicate the fungus, but primarily disable a virulence determinant. The yeast–hyphae-dimorphism of C. albicans is considered a major contributor to fungal disease, as mutants locked into either yeast or hyphal state have been shown to be less virulent in the mouse-model. We devised a high-throughput screening procedure which allows us to find inhibitors of the induction of hyphae. Hyphae-formation was induced by nitrogen starvation at 37 °C and neutral pH in a reporter strain, which couples promoter activity of the hyphae-specific HWP1 to β-galactosidase expression. In a pilot screening of 720 novel synthetic compounds, we identified substances which inhibited the outgrowth of germ tubes. They belonged to chemical classes not yet known for antimycotic properties, namely methyl aryl-oxazoline carboxylates, dihydrobenzo[d]isoxazolones and thiazolo[4,5-e]benzoisoxazoles. In conclusion we developed a novel screening assay, which addresses the morphological switch from the yeast form of C. albicans to its hyphal form and identified novel chemical structures with activity against C. albicans. Copyright © 2012 Elsevier B.V. All rights reserved.

Yeast as a tool to identify anti-aging compounds

PubMed Central

Zimmermann, Andreas; Hofer, Sebastian; Pendl, Tobias; Kainz, Katharina; Madeo, Frank; Carmona-Gutierrez, Didac

2018-01-01

Abstract In the search for interventions against aging and age-related diseases, biological screening platforms are indispensable tools to identify anti-aging compounds among large substance libraries. The budding yeast, Saccharomyces cerevisiae, has emerged as a powerful chemical and genetic screening platform, as it combines a rapid workflow with experimental amenability and the availability of a wide range of genetic mutant libraries. Given the amount of conserved genes and aging mechanisms between yeast and human, testing candidate anti-aging substances in yeast gene-deletion or overexpression collections, or de novo derived mutants, has proven highly successful in finding potential molecular targets. Yeast-based studies, for example, have led to the discovery of the polyphenol resveratrol and the natural polyamine spermidine as potential anti-aging agents. Here, we present strategies for pharmacological anti-aging screens in yeast, discuss common pitfalls and summarize studies that have used yeast for drug discovery and target identification. PMID:29905792

Isolation of lignans and biological activity studies of Ephedra viridis.

PubMed

Pullela, Srinivas V; Takamatsu, Satoshi; Khan, Shabana I; Khan, Ikhlas A

2005-08-01

Phytochemical investigation of Ephedra viridis (whole plant) led to the isolation of four lignans including (+)-9-acetoxyisolariciresinol, which is a new lignan, lariciresinol, 9-acetoxylariciresinol and isolariciresinol. All the isolates were tested for their antioxidant activity and cytotoxicity against a panel of solid tumor and human leukemia cells. They were also screened for estrogenic activity using a recombinant yeast estrogen screening (YES) assay. Most of the lignans exhibited moderate antioxidant activity without any cytotoxicity. None of them were estrogenic in the YES assay.

Circulating microparticles and endogenous estrogen in newly menopausal women

PubMed Central

Jayachandran, M.; Litwiller, R. D.; Owen, W. G.; Miller, V. M.

2011-01-01

Background Estrogen modulates antithrombotic characteristics of the vascular endothelium and the interaction of blood elements with the vascular surface. A marker of these modulatory activities is formation of cell-specific microparticles. This study examined the relationship between blood-borne microparticles and endogenous estrogen at menopause. Methods Platelet activation and plasma microparticles were characterized from women being screened (n = 146) for the Kronos Early Estrogen Prevention Study. Women were grouped according to serum estrogen (< 20 pg/ml; low estrogen, n = 21 or > 40 pg/ml; high estrogen, n = 11). Results Age, body mass index, blood pressure and blood chemistries were the same in both groups. No woman was hypertensive, diabetic or a current smoker. Platelet counts, basal and activated expression of P-selectin on platelet membranes were the same, but activated expression of glycoprotein IIb/IIIa was greater in the high-estrogen group. Numbers of endothelium-, platelet-, monocyte- and granulocyte-derived microparticles were greater in the low-estrogen group. Of the total numbers of microparticles, those positive for phosphatidylserine and tissue factor were also greater in the low-estrogen group. Conclusion These results suggest that, with declines in endogenous estrogen at menopause, numbers of procoagulant microparticles increase and thus may provide a means to explore mechanisms for cardiovascular risk development in newly menopausal women. PMID:19051075

Estrogen-associated severe hypertriglyceridemia with pancreatitis.

PubMed

Aljenedil, Sumayah; Hegele, Robert A; Genest, Jacques; Awan, Zuhier

Estrogen, whether therapeutic or physiologic, can cause hypertriglyceridemia. Hypertriglyceridemia-induced pancreatitis is a rare complication. We report 2 women who developed estrogen-associated severe hypertriglyceridemia with pancreatitis. The first patient developed pancreatitis secondary to hypertriglyceridemia associated with in vitro fertilization cycles. Marked reduction in her triglyceride was achieved with dietary restrictions and fibrate. The second patient developed pancreatitis secondary to hypertriglyceridemia during her pregnancies. She was noncompliant with the treatment; therefore, her triglyceride remained high after delivery. In both patients, no hypertriglyceridemia-associated genes mutations were identified, although the second patient had strong polygenic susceptibility to hypertriglyceridemia. Estrogen-induced severe hypertriglyceridemia with pancreatitis can be a life-threatening condition. Screening in high-risk patients is crucial to prevent subsequent complications. Copyright © 2016 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement

Yeast diversity and native vigor for flavor phenotypes.

PubMed

Carrau, Francisco; Gaggero, Carina; Aguilar, Pablo S

2015-03-01

Saccharomyces cerevisiae, the yeast used widely for beer, bread, cider, and wine production, is the most resourceful eukaryotic model used for genetic engineering. A typical concern about using engineered yeasts for food production might be negative consumer perception of genetically modified organisms. However, we believe the true pitfall of using genetically modified yeasts is their limited capacity to either refine or improve the sensory properties of fermented foods under real production conditions. Alternatively, yeast diversity screening to improve the aroma and flavors could offer groundbreaking opportunities in food biotechnology. We propose a 'Yeast Flavor Diversity Screening' strategy which integrates knowledge from sensory analysis and natural whole-genome evolution with information about flavor metabolic networks and their regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Integral assessment of estrogenic potentials in sediment-associated samples: Part 2: Study of estrogen and anti-estrogen receptor-binding potentials of sediment-associated chemicals under different salinity conditions using the salinity-adapted enzyme-linked receptor assay.

PubMed

Kase, Robert; Hansen, Peter D; Fischer, Birgit; Manz, Werner; Heininger, Peter; Reifferscheid, Georg

2009-01-01

receptor-binding effects. According to the results of sediment testing, and given the integrative power to detect different directions of effects, the ELRA shows sufficient sensitivity and salinity tolerance to discriminate receptor-binding potentials in environmental samples. The optimised ELRA assay is a fast, cost-effective, reliable and highly reproducible tool that can be used for high-throughput screening in a microplate format in detecting both estrogenic and anti-estrogenic effects. Additionally, the ELRA is robust against microbial contaminations, and is not susceptible towards cytotoxic interferences like the common cell-culture methods. The general applicability and sufficient sensitivity of the ELRA was shown in freshwater environments. Marine and brackish samples can be measured up to salinity levels of 20.5 per thousand. In view of the proven sensitivity, functionality and the fastness of the ELRA, it is recommendable to standardise the test method. At the moment, no adequate in vitro test procedure exists which is standardised to DIN or ISO levels. The E-screen and the yeast estrogen/androgen screens (YES/YAS) sometimes underlie strong cytotoxic effects, as reported in the first part of this study. Further development of an ELRA assay using human androgen receptors appears to be very promising to gain information about androgenic and anti-androgenic effects, too. This would offer a possibility to use the ELRA as a fast and reliable pre-screening tool for the detection of endocrine potentials, thus minimising time and cost-expensive animal experiments.

T-screen and yeast assay for the detection of the thyroid-disrupting activities of cadmium, mercury, and zinc.

PubMed

Li, Jian; Liu, Yun; Kong, Dongdong; Ren, Shujuan; Li, Na

2016-05-01

In the present study, a two-hybrid yeast bioassay and a T-screen were used to screen for the thyroid receptor (TR)-disrupting activity of select metallic compounds (CdCl2, ZnCl2, HgCl2, CuSO4, MnSO4, and MgSO4). The results reveal that none of the tested metallic compounds showed TR-agonistic activity, whereas ZnCl2, HgCl2, and CdCl2 demonstrated TR antagonism. For the yeast assay, the dose-response relationship of these metallic compounds was established, and the concentrations producing 20 % of the maximum effect of ZnCl2, HgCl2, and CdCl2 were 9.1 × 10(-5), 3.2 × 10(-6), and 1.2 × 10(-6) mol/L, respectively. The T-screen also supported the finding that ZnCl2, HgCl2, and CdCl2 decreased the cell proliferation at concentrations ranging from 10(-6) to 10(-4) mol/L. Furthermore, the thyroid-disrupting activity of metallic compounds in environmental water samples collected from the Guanting Reservoir, Beijing, China was evaluated. Solid-phase extraction was used to separate the organic extracts, and a modified two-hybrid yeast bioassay revealed that the metallic compounds in the water samples could affect thyroid hormone-induced signaling by decreasing the binding of the thyroid hormone. The addition of ethylenediaminetetraacetic acid (30 mg/L) could eliminate the effects. Thus, the cause(s) of the thyroid toxicity in the water samples appeared to be partly related to the metallic compounds.

Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh

2013-10-01

Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα bindingmore » affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and

Comparison of in vitro estrogenic activity and estrogen concentrations in source and treated waters from 25 U.S. drinking water treatment plants.

PubMed

Conley, Justin M; Evans, Nicola; Mash, Heath; Rosenblum, Laura; Schenck, Kathleen; Glassmeyer, Susan; Furlong, Ed T; Kolpin, Dana W; Wilson, Vickie S

2017-02-01

In vitro bioassays have been successfully used to screen for estrogenic activity in wastewater and surface water, however, few have been applied to treated drinking water. Here, extracts of source and treated water samples were assayed for estrogenic activity using T47D-KBluc cells and analyzed by liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) for natural and synthetic estrogens (including estrone, 17β-estradiol, estriol, and ethinyl estradiol). None of the estrogens were detected above the LC-FTMS quantification limits in treated samples and only 5 source waters had quantifiable concentrations of estrone, whereas 3 treated samples and 16 source samples displayed in vitro estrogenicity. Estrone accounted for the majority of estrogenic activity in respective samples, however the remaining samples that displayed estrogenic activity had no quantitative detections of known estrogenic compounds by chemical analyses. Source water estrogenicity (max, 0.47ng 17β-estradiol equivalents (E2Eq) L -1 ) was below levels that have been linked to adverse effects in fish and other aquatic organisms. Treated water estrogenicity (max, 0.078ngE2EqL -1 ) was considerably below levels that are expected to be biologically relevant to human consumers. Overall, the advantage of using in vitro techniques in addition to analytical chemical determinations was displayed by the sensitivity of the T47D-KBluc bioassay, coupled with the ability to measure cumulative effects of mixtures, specifically when unknown chemicals may be present. Published by Elsevier B.V.

An Assessment of the Model of Concentration Addition for Predicting the Estrogenic Activity of Chemical Mixtures in Wastewater Treatment Works Effluents

PubMed Central

Thorpe, Karen L.; Gross-Sorokin, Melanie; Johnson, Ian; Brighty, Geoff; Tyler, Charles R.

2006-01-01

The effects of simple mixtures of chemicals, with similar mechanisms of action, can be predicted using the concentration addition model (CA). The ability of this model to predict the estrogenic effects of more complex mixtures such as effluent discharges, however, has yet to be established. Effluents from 43 U.K. wastewater treatment works were analyzed for the presence of the principal estrogenic chemical contaminants, estradiol, estrone, ethinylestradiol, and nonylphenol. The measured concentrations were used to predict the estrogenic activity of each effluent, employing the model of CA, based on the relative potencies of the individual chemicals in an in vitro recombinant yeast estrogen screen (rYES) and a short-term (14-day) in vivo rainbow trout vitellogenin induction assay. Based on the measured concentrations of the four chemicals in the effluents and their relative potencies in each assay, the calculated in vitro and in vivo responses compared well and ranged between 3.5 and 87 ng/L of estradiol equivalents (E2 EQ) for the different effluents. In the rYES, however, the measured E2 EQ concentrations in the effluents ranged between 0.65 and 43 ng E2 EQ/L, and they varied against those predicted by the CA model. Deviations in the estimation of the estrogenic potency of the effluents by the CA model, compared with the measured responses in the rYES, are likely to have resulted from inaccuracies associated with the measurement of the chemicals in the extracts derived from the complex effluents. Such deviations could also result as a consequence of interactions between chemicals present in the extracts that disrupted the activation of the estrogen response elements in the rYES. E2 EQ concentrations derived from the vitellogenic response in fathead minnows exposed to a series of effluent dilutions were highly comparable with the E2 EQ concentrations derived from assessments of the estrogenic potency of these dilutions in the rYES. Together these data support the

Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance.

PubMed

Weckesser, S; Engel, K; Simon-Haarhaus, B; Wittmer, A; Pelz, K; Schempp, C M

2007-08-01

There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide [CO2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea CO2-extract effectively inhibited the growth of several Gram-positive bacteria like Staphylococcus aureus (including methicillin-resistant strains - MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.

Screening and characterizing of xylanolytic and xylose-fermenting yeasts isolated from the wood-feeding termite, Reticulitermes chinensis

PubMed Central

Xie, Rongrong; Zhou, Feng; Huang, Miao

2017-01-01

The effective fermentation of xylose remains an intractable challenge in bioethanol industry. The relevant xylanase enzyme is also in a high demand from industry for several biotechnological applications that inevitably in recent times led to many efforts for screening some novel microorganisms for better xylanase production and fermentation performance. Recently, it seems that wood-feeding termites can truly be considered as highly efficient natural bioreactors. The highly specialized gut systems of such insects are not yet fully realized, particularly, in xylose fermentation and xylanase production to advance industrial bioethanol technology as well as industrial applications of xylanases. A total of 92 strains from 18 yeast species were successfully isolated and identified from the gut of wood-feeding termite, Reticulitermes chinensis. Of these yeasts and strains, seven were identified for new species: Candida gotoi, Candida pseudorhagii, Hamamotoa lignophila, Meyerozyma guilliermondii, Sugiyamaella sp.1, Sugiyamaella sp. 2, and Sugiyamaella sp.3. Based on the phylogenetic and phenotypic characterization, the type strain of C. pseudorhagii sp. nov., which was originally designated strain SSA-1542T, was the most frequently occurred yeast from termite gut samples, showed the highly xylanolytic activity as well as D-xylose fermentation. The highest xylanase activity was recorded as 1.73 and 0.98 U/mL with xylan or D-xylose substrate, respectively, from SSA-1542T. Among xylanase-producing yeasts, four novel species were identified as D-xylose-fermenting yeasts, where the yeast, C. pseudorhagii SSA-1542T, showed the highest ethanol yield (0.31 g/g), ethanol productivity (0.31 g/L·h), and its fermentation efficiency (60.7%) in 48 h. Clearly, the symbiotic yeasts isolated from termite guts have demonstrated a competitive capability to produce xylanase and ferment xylose, suggesting that the wood-feeding termite gut is a promising reservoir for novel xylanases

Light-mediated control of DNA transcription in yeast

PubMed Central

Hughes, Robert M.; Bolger, Steven; Tapadia, Hersh; Tucker, Chandra L.

2012-01-01

A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light / cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems. PMID:22922268

Exploration for the Salinity Tolerance-Related Genes from Xero-Halophyte Atriplex canescens Exploiting Yeast Functional Screening System

PubMed Central

Li, Jingtao; Sun, Xinhua; Liu, Yanzhi; Wang, Xueliang; Zhang, Hao; Pan, Hongyu

2017-01-01

Plant productivity is limited by salinity stress, both in natural and agricultural systems. Identification of salt stress-related genes from halophyte can provide insights into mechanisms of salt stress tolerance in plants. Atriplex canescens is a xero-halophyte that exhibits optimum growth in the presence of 400 mM NaCl. A cDNA library derived from highly salt-treated A. canescens plants was constructed based on a yeast expression system. A total of 53 transgenic yeast clones expressing enhanced salt tolerance were selected from 105 transformants. Their plasmids were sequenced and the gene characteristics were annotated using a BLASTX search. Retransformation of yeast cells with the selected plasmids conferred salt tolerance to the resulting transformants. The expression patterns of 28 of these stress-related genes were further investigated in A. canescens leaves by quantitative reverse transcription-PCR. In this study, we provided a rapid and robust assay system for large-scale screening of genes for varied abiotic stress tolerance with high efficiency in A. canescens. PMID:29149055

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays.

PubMed

Martins, Flaviano S; Miranda, Ieso C; Rosa, Carlos A; Nicoli, Jacques R; Neves, Maria J

2008-01-01

Probiotics are viable defined microorganisms (bacteria or yeasts) that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.

Removal of estrogens and estrogenicity through drinking water treatment.

PubMed

Schenck, Kathleen; Rosenblum, Laura; Wiese, Thomas E; Wymer, Larry; Dugan, Nicholas; Williams, Daniel; Mash, Heath; Merriman, Betty; Speth, Thomas

2012-03-01

Estrogenic compounds have been shown to be present in surface waters, leading to concerns over their possible presence in finished drinking waters. In this work, two in vitro human cell line bioassays for estrogenicity were used to evaluate the removal of estrogens through conventional drinking water treatment using a natural water. Bench-scale studies utilizing chlorine, alum coagulation, ferric chloride coagulation, and powdered activated carbon (PAC) were conducted using Ohio River water spiked with three estrogens, 17β-estradiol, 17α-ethynylestradiol, and estriol. Treatment of the estrogens with chlorine, either alone or with coagulant, resulted in approximately 98% reductions in the concentrations of the parent estrogens, accompanied by formation of by-products. The MVLN reporter gene and MCF-7 cell proliferation assays were used to characterize the estrogenic activity of the water before and after treatment. The observed estrogenic activities of the chlorinated samples showed that estrogenicity of the water was reduced commensurate with removal of the parent estrogen. Therefore, the estrogen chlorination by-products did not contribute appreciably to the estrogenic activity of the water. Coagulation alone did not result in significant removals of the estrogens. However, addition of PAC, at a typical drinking water plant dose, resulted in removals ranging from approximately 20 to 80%.

Screening wild yeast strains for alcohol fermentation from various fruits.

PubMed

Lee, Yeon-Ju; Choi, Yu-Ri; Lee, So-Young; Park, Jong-Tae; Shim, Jae-Hoon; Park, Kwan-Hwa; Kim, Jung-Wan

2011-03-01

Wild yeasts on the surface of various fruits including grapes were surveyed to obtain yeast strains suitable for fermenting a novel wine with higher alcohol content and supplemented with rice starch. We considered selected characteristics, such as tolerance to alcohol and osmotic pressure, capability of utilizing maltose, and starch hydrolysis. Among 637 putative yeast isolates, 115 strains exhibiting better growth in yeast-peptone-dextrose broth containing 30% dextrose, 7% alcohol, or 2% maltose were selected, as well as five α-amylase producers. Nucleotide sequence analysis of the 26S rDNA gene classified the strains into 13 species belonging to five genera; Pichia anomala was the most prevalent (41.7%), followed by Wickerhamomyces anomalus (19.2%), P. guilliermondii (15%), Candida spp. (5.8%), Kodamaea ohmeri (2.5%), and Metschnikowia spp. (2.5%). All of the α-amylase producers were Aureobasidium pullulans. Only one isolate (NK28) was identified as Saccharomyces cerevisiae. NK28 had all of the desired properties for the purpose of this study, except α-amylase production, and fermented alcohol better than commercial wine yeasts.

A demonstration of the uncertainty in predicting the estrogenic activity of individual chemicals and mixtures from an in vitro estrogen receptor transcriptional activation assay (T47D-KBluc) to the in vivo uterotrophic assay using oral exposure

EPA Science Inventory

In vitro estrogen receptor assays are valuable screening tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently...

Screening of binding proteins that interact with Chinese sacbrood virus VP3 capsid protein in Apis cerana larvae cDNA library by the yeast two-hybrid method.

PubMed

Fei, Dongliang; Wei, Dong; Yu, Xiaolei; Yue, Jinjin; Li, Ming; Sun, Li; Jiang, Lili; Li, Yijing; Diao, Qingyun; Ma, Mingxiao

2018-03-15

Chinese sacbrood virus (CSBV) causes larval death and apiary collapse of Apis cerana. VP3 is a capsid protein of CSBV but its function is poorly understood. To determine the function of VP3 and screen for novel binding proteins that interact with VP3, we conducted yeast two-hybrid screening, glutathione S-transferase pull-down, and co-immunoprecipitation assays. Galectin (GAL) is a protein involved in immune regulation and host-pathogen interactions. The yeast two-hybrid screen implicated GAL as a major VP3-binding candidate. The assays showed that the VP3 interacted with GAL. Identification of these cellular targets and clarifying their contributions to the host-pathogen interaction may be useful for the development of novel therapeutic and prevention strategies against CSBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Estrogen and colorectal cancer incidence and mortality.

PubMed

Lavasani, Sayeh; Chlebowski, Rowan T; Prentice, Ross L; Kato, Ikuko; Wactawski-Wende, Jean; Johnson, Karen C; Young, Alicia; Rodabough, Rebecca; Hubbell, F Allan; Mahinbakht, Ali; Simon, Michael S

2015-09-15

The preponderance of observational studies describe an association between the use of estrogen alone and a lower incidence of colorectal cancer. In contrast, no difference in the incidence of colorectal cancer was seen in the Women's Health Initiative (WHI) randomized, placebo-controlled trial with estrogen alone after a mean intervention of 7.1 years and cumulative follow-up of 13.2 years. This study extends these findings by providing detailed analyses of the effects of estrogen alone on the histology, grade, and stage of colorectal cancer, relevant subgroups, and deaths from and after colorectal cancer. The WHI study was a randomized, double-blind, placebo-controlled trial involving 10,739 postmenopausal women with prior hysterectomy. Participants were assigned to conjugated equine estrogen at 0.625 mg/d (n = 5279) or a matching placebo (n = 5409). Rates of colorectal cancer diagnoses and deaths from and after colorectal cancer were assessed throughout the study. Colorectal cancer rates in the estrogen-alone and placebo groups were comparable: 0.14% and 0.12% per year, respectively (hazard ratio [HR], 1.13; 95% confidence interval [CI], 0.83-1.58; P = .43). Bowel screening examinations were comparable between the 2 groups throughout the study. The grade, stage, and location of colorectal cancer did not differ between the randomization groups. There were more colorectal cancer deaths in the estrogen-alone group (34 [0.05%] vs 24 [0.03%]; HR, 1.46, 95% CI, 0.86-2.46; P = .16), but the difference was not statistically significant. The colorectal cancer incidence was higher for participants with a history of colon polyp removal in the estrogen-alone group (0.23% vs 0.02%; HR, 13.47; nominal 95% CI, 1.76-103.0; P < .001). The use of estrogen alone in postmenopausal women with prior hysterectomy does not influence the incidence of colorectal cancer or deaths from or after colorectal cancer. A possibly higher risk of colorectal cancer in women with

Yeast for virus research

PubMed Central

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

CERAPP: Collaborative Estrogen Receptor Activity Prediction ...

EPA Pesticide Factsheets

Humans potentially are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Many of these chemicals never have been tested for their ability to interact with the estrogen receptor (ER). Risk assessors need tools to prioritize chemicals for assessment in costly in vivo tests, for instance, within the EPA Endocrine Disruptor Screening Program. Here, we describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) demonstrating the efficacy of using predictive computational models on high-throughput screening data to screen thousands of chemicals against the ER. CERAPP combined multiple models developed in collaboration among 17 groups in the United States and Europe to predict ER activity of a common set of 32,464 chemical structures. Quantitative structure-activity relationship models and docking approaches were employed, mostly using a common training set of 1677 compounds provided by EPA, to build a total of 40 categorical and 8 continuous models for binding, agonist, and antagonist ER activity. All predictions were tested using an evaluation set of 7522 chemicals collected from the literature. To overcome the limitations of single models, a consensus was built weighting models using a scoring function (0 to 1) based on their accuracies. Individual model scores ranged from 0.69 to 0.85, showing

Comparison of estrogen mixtures in vitro vs. in vivo

EPA Science Inventory

Numerous sources contribute to widespread contamination of drinking water sources with both natural and synthetic estrogens, which isa concern for potential ecological and human health effects. In vitro screening assays are valuable tools for identifying mechanisms of toxicity bu...

A yeast-based genetic screening to identify human proteins that increase homologous recombination.

PubMed

Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

2008-05-01

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

A robust high-throughput fungal biosensor assay for the detection of estrogen activity.

PubMed

Zutz, Christoph; Wagener, Karen; Yankova, Desislava; Eder, Stefanie; Möstl, Erich; Drillich, Marc; Rychli, Kathrin; Wagner, Martin; Strauss, Joseph

2017-10-01

Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which β-galactosidase gene expression is controlled by an estrogen-responsive-element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre-treatment by chemical extraction was needed. For 17β-estradiol, the biosensor showed a limit of detection of 1ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high-throughput formats without the necessity for organic solvents. Copyright © 2017 Elsevier Inc. All rights reserved.

Chemical Probes of Rapid Estrogen Signaling in Breast Cancer Treatment and Chemoprevention

DTIC Science & Technology

2006-04-01

conjugated to cell-impermeable polyacrylate polymers that should allow for selective targeting of membrane-initiated responses of estrogen receptor. It...dilutions of the different compounds were prepared in ES2 screening buffer (100 mM potassium phosphate, pH7.4, 100 µg/ml bovine gamma globulin) and 50 µl...W81XWH-04-1-0447 TITLE: Chemical Probes of Rapid Estrogen Signaling in Breast Cancer Treatment and Chemoprevention PRINCIPAL

Nutrient supplements boost yeast transformation efficiency

PubMed Central

Yu, Sheng-Chun; Dawson, Alexander; Henderson, Alyssa C.; Lockyer, Eloise J.; Read, Emily; Sritharan, Gayathri; Ryan, Marjah; Sgroi, Mara; Ngou, Pok M.; Woodruff, Rosie; Zhang, Ruifeng; Ren Teen Chia, Travis; Liu, Yu; Xiang, Yiyu; Spanu, Pietro D.

2016-01-01

Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per μg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening. PMID:27760994

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

PubMed

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Occurrence and In Vitro Bioactivity of Estrogen, Androgen, and Glucocorticoid Compounds in a Nationwide Screen of United States Stream Waters.

PubMed

Conley, Justin M; Evans, Nicola; Cardon, Mary C; Rosenblum, Laura; Iwanowicz, Luke R; Hartig, Phillip C; Schenck, Kathleen M; Bradley, Paul M; Wilson, Vickie S

2017-05-02

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a broad suite of chemical analytes, in streamwater from 35 well-characterized sites (3 reference and 32 impacted) across 24 states and Puerto Rico. ER agonism was the most frequently detected with nearly all sites (34/35) displaying activity (range, 0.054-116 ng E2Eq L -1 ). There was a strong linear relationship (r 2 = 0.917) between in vitro ER activity and concentrations of steroidal estrogens after correcting for the in vitro potency of each compound. AR agonism was detected in 5/35 samples (range, 1.6-4.8 ng DHTEq L -1 ) but concentrations of androgenic compounds were largely unable to account for the in vitro activity. Similarly, GR agonism was detected in 9/35 samples (range, 6.0-43 ng DexEq L -1 ); however, none of the recognized GR-active compounds on the target-chemical analyte list were detected. The utility of in vitro assays in water quality monitoring was evident from both the quantitative agreement between ER activity and estrogen concentrations, as well as the detection of AR and GR activity for which there were limited or no corresponding target-chemical detections to explain the bioactivity. Incorporation of in vitro bioassays as complements to chemical analyses in standard water quality monitoring efforts would allow for more complete assessment of the chemical mixtures present in many surface waters.

Occurrence and in vitro bioactivity of estrogen, androgen, and glucocorticoid compounds in a nationwide screen of United States stream waters

USGS Publications Warehouse

Conley, Justin M.; Evans, Nicola; Cardon, Mary C.; Rosenblum, Laura; Iwanowicz, Luke R.; Hartig, Phillip C.; Schenck, Kathleen M.; Bradley, Paul M.; Wilson, Vickie S.

2017-01-01

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a broad suite of chemical analytes, in streamwater from 35 well-characterized sites (3 reference and 32 impacted) across 24 states and Puerto Rico. ER agonism was the most frequently detected with nearly all sites (34/35) displaying activity (range, 0.054–116 ng E2Eq L–1). There was a strong linear relationship (r2 = 0.917) between in vitro ER activity and concentrations of steroidal estrogens after correcting for the in vitro potency of each compound. AR agonism was detected in 5/35 samples (range, 1.6–4.8 ng DHTEq L–1) but concentrations of androgenic compounds were largely unable to account for the in vitro activity. Similarly, GR agonism was detected in 9/35 samples (range, 6.0–43 ng DexEq L–1); however, none of the recognized GR-active compounds on the target-chemical analyte list were detected. The utility of in vitro assays in water quality monitoring was evident from both the quantitative agreement between ER activity and estrogen concentrations, as well as the detection of AR and GR activity for which there were limited or no corresponding target-chemical detections to explain the bioactivity. Incorporation of in vitro bioassays as complements to chemical analyses in standard water quality monitoring efforts would allow for more complete assessment of the chemical mixtures present in many surface waters.

Characterizing the Estrogenic Potential of 1060 Environmental ...

EPA Pesticide Factsheets

In order to detect environmental chemicals that pose a risk of endocrine disruption, high-throughput screening (HTS) tests capable of testing thousands of environmental chemicals are needed. Alteration of estrogen signaling has been implicated in a variety of adverse health effects including cancer promotion, reproductive deficits, and vascular effects. Here we investigate the estrogenic potential of 1060 chemicals of environmental relevance using a real-time measure of growth kinetics by electrode impedance in the estrogen-responsive human ductal carcinoma, T47D cell line. Cells were treated in concentration response and measurements of cellular impedance were recorded every hour for six days. Progestens, androgens, and mineralocortocoids (progesterone, dihydrotestosterone, aldosterone) invoked a biphasic impedance signature that contrasted with the anticipated exponential impedance observed in response to known estrogen receptor agonists (17β-estradiol, genestein, bisphenol-A, nonylphenol, 4-tert-octylphenol). Several compounds, including bisphenol-A, and genestein caused impedance comparable to that of 17β-estradiol, although at much higher concentrations. Additionally, trenbolone and cyproterone acetate invoked the characteristic biphasic signature observed with other endogenous steroid hormones. The continuous real-time nature of this assay allows for the rapid detection of differential growth characteristics not easily detected by traditional cell prol

FDA-Approved Selective Estrogen Receptor Modulators Inhibit Ebola Virus Infection

PubMed Central

Johansen, Lisa M.; Brannan, Jennifer M.; Delos, Sue E.; Shoemaker, Charles J.; Stossel, Andrea; Lear, Calli; Hoffstrom, Benjamin G.; DeWald, Lisa Evans; Schornberg, Kathryn L.; Scully, Corinne; Lehár, Joseph; Hensley, Lisa E.; White, Judith M.; Olinger, Gene G.

2014-01-01

Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)– and ex–US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections. PMID:23785035

Is Estrogen a Therapeutic Target for Glaucoma?

PubMed Central

Dewundara, Samantha; Wiggs, Janey; Sullivan, David A.; Pasquale, Louis R.

2016-01-01

Objective To provide an overview of the association between estrogen and glaucoma. Methods A literature synthesis of articles published in peer review journals screened through May 05, 2015 using the PubMed database. Key words used were “estrogen and glaucoma,” “reproductive factors and glaucoma,” “estrogen, nitric oxide and eye.” Forty three journal articles were included. Results Markers for lifetime estrogen exposure have been measured by several studies and show that the age of menarche onset, oral contraceptive (OC) use, bilateral oophorectomy, age of menopause onset and duration between menarche to menopause are associated with primary open angle (POAG) risk. The Blue Mountain Eye Study found a significantly increased POAG risk with later (>13 years) compared with earlier (≤12 years) age of menarche. Nurses’ Health Study (NHS) investigators found that OC use of greater than 5 years was associated with a 25% increased risk of POAG. The Mayo Clinic Cohort Study of Oophorectomy and Aging found that women who underwent bilateral oophorectomy before age 43 had an increased risk of glaucoma. The Rotterdam Study found that women who went through menopause before reaching the age of 45 years had a higher risk of open-angle glaucoma (2.6-fold increased risk) while the NHS showed a reduced risk of POAG among women older than 65 who entered menopause after age ≥ 54 years. Increased estrogen states may confer a reduced risk of glaucoma or glaucoma related traits such as reduced intraocular pressure (IOP). Pregnancy, a hyperestrogenemic state, is associated with decreased IOP during the third trimester. Though the role of post-menopausal hormone (PMH) use in the reduction of IOP is not fully conclusive, PMH use may reduce the risk of POAG. From a genetic epidemiologic perspective, estrogen metabolic pathway single nucleotide polymorphisms (SNPs) were associated with POAG in women and polymorphisms in endothelial nitric oxide synthase, a gene receptive to

Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts.

PubMed

Carrasco, Mario; Villarreal, Pablo; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

2016-02-19

Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases. All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi. Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting

Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells

PubMed Central

2010-01-01

Background Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens. Methods We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone) at 10 pM (representing pre-development levels), and 1 nM (representing higher cycle-dependent and pregnancy levels) in combinations with the same levels of xenoestrogens in GH3/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2) phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERβ, and GPER) were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively). Results All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation) of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses. Conclusions Responses mediated by endogenous estrogens representing different life stages are vulnerable to very

DEVELOPMENT AND CHARACTERIZATION OF A CELL LINE THAT STABLY EXPRESSES AN ESTROGEN-RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ESTROGEN RECEPTOR AGONIST AND ANTAGONISTS

EPA Science Inventory

Screening for endocrine disrupting chemicals (EDCs) that act as estrogens or antiestrogens relies on the use of in vitro binding and gene expression assays coupled with short-term diagnostic in vivo assays. Although binding assays are useful to identify chemicals that are competi...

Occurrence and estrogenic activity of steroid hormones in Chinese streams: A nationwide study based on a combination of chemical and biological tools.

PubMed

Yao, Bo; Li, Rui; Yan, Shuwen; Chan, Shen-An; Song, Weihua

2018-05-18

Steroid hormones (SHs) are continuously released into the aquatic environment through various pathways after being excreted by humans and animals, interfere with the normal function of the endocrine system and may affect the physiology and reproduction of exposed aquatic life. To conduct a nationwide investigation of the occurrence and biological effects of SHs in surface river/steam water in China, we quantitated 27 selected SHs in 217 surface water samples by solid-phase extraction (SPE) tandem LC-MS/MS and used a recombinant yeast estrogen assay to screen extracts of the water samples for estrogenic activities. SHs were commonly found in the surface water samples, and their levels were typically in the ng L -1 range. Estrone (E1) and estriol (E3) were normally present in several to dozens of times higher concentrations than estradiol (E2) and 17-a-Ethinylestradiol (EE2). The high concentrations (mean > 1 μg L -1 ) of Sum SHs were primarily obtained in areas under extreme water stress, specifically the eastern coastal areas. Source apportionment based on the profiles of the target compounds indicated that 54.5% of the SHs in target samples came from freshly discharged untreated sewage. The estrogen equivalent (EEQ (bio) ) values ranged from 0.01 to 40.27 ng L -1 , and the calculated EEQ (EEQ (cal) ) values were generally lower than the corresponding EEQ (bio) values for all samples. E2 was the main contributor to the estrogenicity among the three estrogens, with a contribution ratio of 82.8%. The risk quotient values of E2 were highest and ranged from 1.55 to 782.95, and 76.0% of the target surface samples displayed the greatest environmental risk. We concluded that the impacts of SHs on humans in Chinese surface waters should not be ignored and that certain actions should be taken to decrease the levels of SHs in source waters, especially measures targeting SHs in untreated wastewater from the vast rural areas. Copyright © 2018. Published by

Screening protocol for Torulopsis (Candida) glabrata.

PubMed Central

Land, G; Burke, J; Shelby, C; Rhodes, J; Collett, J; Bennett, I; Johnson, J

1996-01-01

A screening test has been developed for the presumptive identification of Torulopsis (Candida) glabrata from other common clinical isolates of yeast-like fungi. An interlaboratory comparison of a protocol consisting of morphology on cornmeal Tween 80 agar and trehalose fermentation at 42 degrees C was successful in differentiating T. glabrata from other taxa that are frequent or possible clinical isolates. The screening results for 517 clinical yeast isolates, 241 of which were T. glabrata, were compared with their final identification via commercial systems (API20C Yeast Identification System [bioMERIEUX, Hazelwood, Mo.] and Rapid Yeast Identification Panel [Dade Microscan, Sacramento, Calif.]). The trehalose screening test has a sensitivity and a specificity of 97.8 and 95.8%, respectively, and a positive predictive value of 97.4% and a negative predictive value of 96.5%. Overall, the trehalose screen had an efficiency rating of 93.9% for ruling in or out T. glabrata. Since T. glabrata represents a substantial part of the workload in a clinical laboratory, a significant reduction in direct and indirect costs should be realized. PMID:8862605

The yeast two hybrid system in a screen for proteins interacting with axolotl (Ambystoma mexicanum) Msx1 during early limb regeneration.

PubMed

Abuqarn, Mehtap; Allmeling, Christina; Amshoff, Inga; Menger, Bjoern; Nasser, Inas; Vogt, Peter M; Reimers, Kerstin

2011-07-01

Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration. 2011 Elsevier B.V. All rights reserved.

Estrogen regulation of gene expression in the teleost fish immune system.

PubMed

Burgos-Aceves, Mario Alberto; Cohen, Amit; Smith, Yoav; Faggio, Caterina

2016-11-01

Elucidating the mechanisms of estrogens-induced immunomodulation in teleost fish is of great importance due to the observed worldwide continuing decrease in pristine environments. However, little is know about the immunotoxicological consequences of exposure to these chemicals in fish, or of the mechanisms through which these effects are mediated. In this review, we summarize the results showing estrogens (natural or synthetic) acting through estrogen receptors and regulating specific target genes, also through microRNAs (miRNAs), leading to modulation of the immune functioning. The identification and characterization of miRNAs will provide new opportunities for functional genome research on teleost immune system and can also be useful when screening for novel molecule biomarkers for environmental pollution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes.

PubMed

Goldberg, Alexander A; Richard, Vincent R; Kyryakov, Pavlo; Bourque, Simon D; Beach, Adam; Burstein, Michelle T; Glebov, Anastasia; Koupaki, Olivia; Boukh-Viner, Tatiana; Gregg, Christopher; Juneau, Mylène; English, Ann M; Thomas, David Y; Titorenko, Vladimir I

2010-07-01

In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA.

Estrogen

MedlinePlus

... life', the end of monthly menstrual periods). Some brands of estrogen are also used to treat vaginal ... prevent osteoporosis should consider a different treatment. Some brands of estrogen are also to relieve symptoms of ...

Removal of Estrogens and Estrogenicity through Drinking Water Treatment

EPA Science Inventory

Estrogenic compounds have been shown to be present in surface waters, leading to concerns over their possible presence in finished drining waters. In this work, two in vitro human cell line bioassays for estrogenicity were used to evaluate the removal of estrogens through conven...

Identification of superior lipid producing Lipomyces and Myxozyma yeasts

USDA-ARS?s Scientific Manuscript database

Oleaginous yeasts are of interest for production of single cell oils from sugars. Here 17 members of the Lipomyces and Myxozyma clade were screened for lipid production when cultured on glucose. The highest ranking yeasts included L. tetrasporus (21 g/l), L. kononenkoae (19.6 g/l), and L. lipofer (1...

Alcoholic Fermentation of d-Xylose by Yeasts

PubMed Central

Toivola, Ansa; Yarrow, David; van den Bosch, Eduard; van Dijken, Johannes P.; Scheffers, W. Alexander

1984-01-01

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used. PMID:16346558

Accelerating Yeast Prion Biology using Droplet Microfluidics

NASA Astrophysics Data System (ADS)

Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

2012-02-01

Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

Estrogenicity of sugar beet by-products used as animal feeds

USDA-ARS?s Scientific Manuscript database

A veterinarian observed a reduction in embryo transfer success rates on beef and dairy farms in Minnesota, which were both feeding sugar beet by-products. Beet tailings and pelleted post-extraction beet pulp, associated with the affected farms were analyzed for estrogenicity by E-Screen (proliferati...

On Selecting a Minimal Set of In Vitro Assays to Reliably Determine Estrogen Agonist Activity

EPA Science Inventory

The US EPA is charged with screening chemicals for their ability to be endocrine disruptors through interaction with the estrogen, androgen and thyroid axes. The agency is starting to explore the use of high-throughput in vitro assays to use in the Endocrine Disruptor Screening P...

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

PubMed

Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

2004-06-01

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

Characterizing the Growth Kinetics in Estrogen Responsive ...

EPA Pesticide Factsheets

There is a need to develop high-throughput screening (HTS) tests capable of testing thousands of environmental chemicals for endocrine disrupting potential. The estrogen signaling pathway is a known xenobiotic target that has been implicated in a variety of adverse health effects including reproductive deficits and cancer promotion. Using real-time measurements of growth kinetics by electrode impedance, the estrogen-responsive human ductal carcinoma cell line, T47D, was treated with 2000 chemicals of environmental relevance. Cells were treated in concentration response and measurements of cellular impedance were recorded every hour for six days. Exponential impedance, signifying increased proliferation, was observed by prototypical estrogen receptor agonists (17β-estradiol, genestein, bisphenol-A, nonylphenol, 4-tert-octylphenol). Several compounds, including bisphenol-A and genestein, induced cell proliferation at comparable levels to 17β-estradiol, although at much higher concentrations. Progestins, and mineralocortocoids (progesterone, dihydrotestosterone, aldosterone) invoked a biphasic impedance signature. In conclusion, the real-time nature of this assay allows for rapid detection of differential growth characteristics shows potential, in combination with other ToxCast HTS assays, to detect environmental chemicals with potential endocrine activity. [This abstract does not necessarily reflect Agency policy]. Several compounds, including bisphenol-A and

Evaluating Endocrine Disruption Activity of Deposits on Firefighting Gear Using a Sensitive and High Throughput Screening Method.

PubMed

Stevenson, Max; Alexander, Barbara; Baxter, C Stuart; Leung, Yuet-Kin

2015-12-01

Adverse health outcomes related to exposure to endocrine disrupting chemicals, including increased incidences of coronary heart disease, prostate and testicular cancers, and congenital disabilities, have been reported in firefighters or their offspring. We, therefore, measured the estrogenic and antiestrogenic activity of extracts of used firefighter gear to assess exposure to these agents. Extracts and known chemical contaminants were examined for estrogenicity and antiestrogenicity in yeast cells expressing the estrogen receptor. Most extracts of used gear and phthalate diesters detectable on this gear displayed strong antiestrogenic effects. Notably, new glove and hood extracts showed significant estrogenic activity. Overall, our data suggest that firefighters are exposed to both estrogenic and antiestrogenic agents, possibly phthalates that may lead to health risks observed in this occupation as a result of perturbation of hormone homeostasis.

Estrogen Degraders and Estrogen Degradation Pathway Identified in an Activated Sludge.

PubMed

Chen, Yi-Lung; Fu, Han-Yi; Lee, Tzong-Huei; Shih, Chao-Jen; Huang, Lina; Wang, Yu-Sheng; Ismail, Wael; Chiang, Yin-Ru

2018-05-15

The environmental release and fate of estrogens are becoming an increasing public concern. Bacterial degradation has been considered the main process for eliminating estrogens from wastewater treatment plants. Various bacterial isolates are reportedly capable of aerobic estrogen degradation, and several estrogen degradation pathways have been proposed in proteobacteria and actinobacteria. However, the ecophysiological relevance of estrogen-degrading bacteria in the environment is unclear. In this study, we investigated the estrogen degradation pathway and corresponding degraders in activated sludge collected from the Dihua Sewage Treatment Plant, Taipei, Taiwan. Cultivation-dependent and cultivation-independent methods were used to assess estrogen biodegradation in the collected activated sludge. Estrogen metabolite profile analysis revealed the production of pyridinestrone acid and two A/B-ring cleavage products in activated sludge incubated with estrone (1 mM), which are characteristic of the 4,5- seco pathway. PCR-based functional assays detected sequences closely related to alphaproteobacterial oecC , a key gene of the 4,5- seco pathway. Metagenomic analysis suggested that Novosphingobium spp. are major estrogen degraders in estrone-amended activated sludge. Novosphingobium sp. strain SLCC, an estrone-degrading alphaproteobacterium, was isolated from the examined activated sludge. The general physiology and metabolism of this strain were characterized. Pyridinestrone acid and the A/B-ring cleavage products were detected in estrone-grown strain SLCC cultures. The production of pyridinestrone acid was also observed during the aerobic incubation of strain SLCC with 3.7 nM (1 μg/liter) estrone. This concentration is close to that detected in many natural and engineered aquatic ecosystems. The presented data suggest the ecophysiological relevance of Novosphingobium spp. in activated sludge. IMPORTANCE Estrogens, which persistently contaminate surface water

Isolation and characterization of ethanol tolerant yeast strains

PubMed Central

Tikka, Chiranjeevi; Osuru, Hari Prasad; Atluri, Navya; Raghavulu, Praveen Chakravarthi Veera; yellapu, Nanda Kumar; Mannur, Ismail Shaik; Prasad, Uppu Venkateswara; Aluru, Sudheer; K, Narasimha Varma; Bhaskar, Matcha

2013-01-01

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains. PMID:23750092

RELATIVE BINDING AFFINITY OF ALKYLPHENOLS TO RAINBOW TROUT ESTROGEN RECEPTOR

EPA Science Inventory

RELATIVE BINDING AFFINITY OF ALKYLPHENOLS TO RAINBOW TROUT ESTROGEN RECEPTOR. T R Henry1, J S Denny2 and P K Schmieder2. USEPA, ORD, NHEERL, 1Experimental Toxicology Division and 2Mid-Continent Ecology Division, Duluth, MN, USA.
The USEPA has been mandated to screen industria...

In Vitro Assessment of Estrogenic Activity in Source and Treated Drinking Water Extracts from 25 U.S. Drinking Water Plants

EPA Science Inventory

The presence of estrogenic compounds in environmental water samples and their potential impact on fish, wildlife and human reproductive health has been of concern for some time. In vitro assays have been successfully used to screen for estrogenic activity in many types of water s...

From drug to protein: using yeast genetics for high-throughput target discovery.

PubMed

Armour, Christopher D; Lum, Pek Yee

2005-02-01

The budding yeast Saccharomyces cerevisiae has long been an effective eukaryotic model system for understanding basic cellular processes. The genetic tractability and ease of manipulation in the laboratory make yeast well suited for large-scale chemical and genetic screens. Several recent studies describing the use of yeast genetics for high-throughput drug target identification are discussed in this review.

Estrogens and aging skin.

PubMed

Thornton, M Julie

2013-04-01

Estrogen deficiency following menopause results in atrophic skin changes and acceleration of skin aging. Estrogens significantly modulate skin physiology, targeting keratinocytes, fibroblasts, melanocytes, hair follicles and sebaceous glands, and improve angiogenesis, wound healing and immune responses. Estrogen insufficiency decreases defense against oxidative stress; skin becomes thinner with less collagen, decreased elasticity, increased wrinkling, increased dryness and reduced vascularity. Its protective function becomes compromised and aging is associated with impaired wound healing, hair loss, pigmentary changes and skin cancer.   Skin aging can be significantly delayed by the administration of estrogen. This paper reviews estrogen effects on human skin and the mechanisms by which estrogens can alleviate the changes due to aging. The relevance of estrogen replacement, selective estrogen receptor modulators (SERMs) and phytoestrogens as therapies for diminishing skin aging is highlighted. Understanding estrogen signaling in skin will provide a basis for interventions in aging pathologies.

NATURE OF BINDING INTERACTION OF SELECTED CHEMICALS WITH RAT ESTROGEN RECEPTORS

EPA Science Inventory

The US EPA is currently validating a rat uterine estrogen receptor (ER) binding assay as part of the Tier 1 Screening Battery for the Endocrine Disruptor Program. An eventual goal is to use interactive data to create computerized structure-activity models. However, more informati...

The E-screen test and the MELN gene-reporter assay used for determination of estrogenic activity in fruits and vegetables in relation to pesticide residues.

PubMed

Schilirò, Tiziana; Porfido, Arianna; Longo, Annalisa; Coluccia, Sara; Gilli, Giorgio

2013-12-01

Endocrine-disrupting chemicals (EDCs) may lead to adverse systemic effects by interfering with normal hormone homeostasis, and diet is considered to be among the main routes of EDC exposure. The present study investigated the total estrogenic activity of fruits and vegetables by calculating the 17-β-estradiol equivalent quantity (EEQ) using two in vitro tests: the human breast cancer cell line (MCF-7 BUS) proliferation assay (E-screen test) and the luciferase-transfected human breast cancer cell line (MELN) gene-reporter assay. Of the 24 analyzed fruits and vegetables, 14 contained from 1 to 4 pesticide residues in concentrations ranging from 0.02 to 1.19 ppm, whereas the other 10 did not contain any pesticide residues. The EEQ values for all positive samples ranged from 0.010 to 0.616 μg/100g for the above in vitro tests. Our study demonstrates that estrogenic activity was present in fruits and vegetables and that the concentration of allowable pesticide residues and EEQ values were positively correlated; however, no correlation was found by comparing the estrogenic activity and the intrinsic content of phytoestrogens obtained from the available literature. A theoretical adult dietary intake of 0.7-0.9 ng EEQ/L/day from fruits and vegetables was calculated. Copyright © 2013 Elsevier Ltd. All rights reserved.

Estrogenic activity, chemical levels and health risk assessment of municipal distribution point water from Pretoria and Cape Town, South Africa.

PubMed

Van Zijl, Magdalena Catherina; Aneck-Hahn, Natalie Hildegard; Swart, Pieter; Hayward, Stefan; Genthe, Bettina; De Jager, Christiaan

2017-11-01

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and have been detected in drinking water from various countries. Although various water treatment processes can remove EDCs, chemicals can also migrate from pipes that transport water and contaminate drinking water. This study investigated the estrogenic activity in drinking water from various distribution points in Pretoria (City of Tshwane) (n = 40) and Cape Town (n = 40), South Africa, using the recombinant yeast estrogen screen (YES) and the T47D-KBluc reporter gene assay. The samples were collected seasonally over four sampling periods. The samples were also analysed for bisphenol A (BPA), nonylphenol (NP), di(2-ethylhexyl) adipate (DEHA), dibutyl phthalate (DBP), di(2-ethylhexyl) phthalate (DEHP), diisononylphthalate (DINP), 17β-estradiol (E 2 ), estrone (E 1 ) and ethynylestradiol (EE 2 ) using ultra-performance liquid chromatography-tandem mass spectrophotometry (UPLC-MS/MS). This was followed by a scenario based health risk assessment to assess the carcinogenic and toxic human health risks associated with the consumption of distribution point water. None of the water extracts from the distribution points were above the detection limit in the YES bioassay, but the EEq values ranged from 0.002 to 0.114 ng/L using the T47D-KBluc bioassay. BPA, DEHA, DBP, DEHP, DINP E 1 , E 2, and EE 2 were detected in distribution point water samples. NP was below the detection limit for all the samples. The estrogenic activity and levels of target chemicals were comparable to the levels found in other countries. Overall the health risk assessment revealed acceptable health and carcinogenic risks associated with the consumption of distribution point water. Copyright © 2017 Elsevier Ltd. All rights reserved.

Editor’s Highlight: A Genome-wide Screening of Target Genes Against Silver Nanoparticles in Fission Yeast

PubMed Central

Lee, Sook-Jeong; Lee, Minho; Nam, Miyoung; Lee, Sol; Choi, Jian; Lee, Hye-Jin; Kim, Dong-Uk; Hoe, Kwang-Lae

2018-01-01

Abstract To identify target genes against silver nanoparticles (AgNPs), we screened a genome-wide gene deletion library of 4843 fission yeast heterozygous mutants covering 96% of all protein encoding genes. A total of 33 targets were identified by a microarray and subsequent individual confirmation. The target pattern of AgNPs was more similar to those of AgNO3 and H2O2, followed by Cd and As. The toxic effect of AgNPs on fission yeast was attributed to the intracellular uptake of AgNPs, followed by the subsequent release of Ag+, leading to the generation of reactive oxygen species (ROS). Next, we focused on the top 10 sensitive targets for further studies. As described previously, 7 nonessential targets were associated with detoxification of ROS, because their heterozygous mutants showed elevated ROS levels. Three novel essential targets were related to folate metabolism or cellular component organization, resulting in cell cycle arrest and no induction in the transcriptional level of antioxidant enzymes such as Sod1 and Gpx1 when 1 of the 2 copies was deleted. Intriguingly, met9 played a key role in combating AgNP-induced ROS generation via NADPH production and was also conserved in a human cell line. PMID:29294138

Elimination of estrogenic activity of thermal paper using laccase from Trichoderma sp NFCCI-2745.

PubMed

Divya, L M; Prasanth, G K; Sadasivan, C

2013-02-01

In thermal printing, bisphenol A (BPA) functions chemically as a developer and reacts with white or colorless dyes in the presence of heat, converting them to a dark color. BPA can transfer readily to skin in small amounts from these papers. Its damage to environment and organisms has caused an extensive concern. In the present study, thermal paper used at the local automated teller machine counters of India were analyzed for the presence of BPA, and the capability of the paper to produce estrogenicity were assessed using a yeast two-hybrid assay experimental system. The study also focused on eliminating the endocrine-disrupting properties with partially purified laccase from newly isolated ascomycete fungi. The results indicate that these papers can produce estrogen hormone-like effect on experimental systems. It should be noted that on a daily basis, tons of such receipts are being dumped in the environment. Estrogenic properties of thermal paper were effectively removed from the reaction mixture within 3 h of incubation with the partially purified enzyme. We propose the utilization of waste thermal paper as a cheap substrate for laccase production for a safer and cleaner environment.

An original method for producing acetaldehyde and diacetyl by yeast fermentation.

PubMed

Rosca, Irina; Petrovici, Anca Roxana; Brebu, Mihai; Stoica, Irina; Minea, Bogdan; Marangoci, Narcisa

In this study a natural culture medium that mimics the synthetic yeast peptone glucose medium used for yeast fermentations was designed to screen and select yeasts capable of producing high levels of diacetyl and acetaldehyde. The presence of whey powder and sodium citrate in the medium along with manganese and magnesium sulfate enhanced both biomass and aroma development. A total of 52 yeasts strains were cultivated in two different culture media, namely, yeast peptone glucose medium and yeast acetaldehyde-diacetyl medium. The initial screening of the strains was based on the qualitative reaction of the acetaldehyde with Schiff's reagent (violet color) and diacetyl with Brady's reagent (yellow precipitate). The fermented culture media of 10 yeast strains were subsequently analyzed by gas chromatography to quantify the concentration of acetaldehyde and diacetyl synthesized. Total titratable acidity values indicated that a total titratable acidity of 5.5°SH, implying culture medium at basic pH, was more favorable for the acetaldehyde biosynthesis using strain D15 (Candida lipolytica; 96.05mgL -1 acetaldehyde) while a total titratable acidity value of 7°SH facilitated diacetyl flavor synthesis by strain D38 (Candida globosa; 3.58mgL -1 diacetyl). Importantly, the results presented here suggest that this can be potentially used in the baking industry. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Sources, mechanisms, and fate of steroid estrogens in wastewater treatment plants: a mini review.

PubMed

Ting, Yien Fang; Praveena, Sarva Mangala

2017-04-01

Steroid estrogens, such as estrone (E 1 ), 17β-estradiol (E 2 ), estriol (E 3 ), and 17α-ethinylestradiol (EE 2 ), are natural and synthetic hormones released into the environment through incomplete sewage discharge. This review focuses on the sources of steroid estrogens in wastewater treatment plants (WWTPs). The mechanisms and fate of steroid estrogens throughout the entire wastewater treatment system are also discussed, and relevant information on regulatory aspects is given. Municipal, pharmaceutical industry, and hospitals are the main sources of steroid estrogens that enter WWTPs. A typical WWTP comprises primary, secondary, and tertiary treatment units. Sorption and biodegradation are the main mechanisms for removal of steroid estrogens from WWTPs. The fate of steroid estrogens in WWTPs depends on the types of wastewater treatment systems. Steroid estrogens in the primary treatment unit are removed by sorption onto primary sludge, followed by sorption onto micro-flocs and biodegradation by microbes in the secondary treatment unit. Tertiary treatment employs nitrification, chlorination, or UV disinfection to improve the quality of the secondary effluent. Activated sludge treatment systems for steroid estrogens exhibit a removal efficiency of up to 100%, which is higher than that of the trickling filter treatment system (up to 75%). Moreover, the removal efficiency of advance treatment systems exceeds 90%. Regulatory aspects related to steroid estrogens are established, especially in the European Union. Japan is the only Asian country that implements a screening program and is actively involved in endocrine disruptor testing and assessment. This review improves our understanding of steroid estrogens in WWTPs, proposes main areas to be improved, and provides current knowledge on steroid estrogens in WWTPs for sustainable development.

Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer.

PubMed

Giamas, Georgios; Filipović, Aleksandra; Jacob, Jimmy; Messier, Walter; Zhang, Hua; Yang, Dongyun; Zhang, Wu; Shifa, Belul Assefa; Photiou, Andrew; Tralau-Stewart, Cathy; Castellano, Leandro; Green, Andrew R; Coombes, R Charles; Ellis, Ian O; Ali, Simak; Lenz, Heinz-Josef; Stebbing, Justin

2011-06-01

Therapies targeting estrogen receptor α (ERα, encoded by ESR1) have transformed the treatment of breast cancer. However, large numbers of women relapse, highlighting the need for the discovery of new regulatory targets modulating ERα pathways. An siRNA screen identified kinases whose silencing alters the estrogen response including those previously implicated in regulating ERα activity (such as mitogen-activated protein kinase and AKT). Among the most potent regulators was lemur tyrosine kinase-3 (LMTK3), for which a role has not previously been assigned. In contrast to other modulators of ERα activity, LMTK3 seems to have been subject to Darwinian positive selection, a noteworthy result given the unique susceptibility of humans to ERα+ breast cancer. LMTK3 acts by decreasing the activity of protein kinase C (PKC) and the phosphorylation of AKT (Ser473), thereby increasing binding of forkhead box O3 (FOXO3) to the ESR1 promoter. LMTK3 phosphorylated ERα, protecting it from proteasomal degradation in vitro. Silencing of LMTK3 reduced tumor volume in an orthotopic mouse model and abrogated proliferation of ERα+ but not ERα- cells, indicative of its role in ERα activity. In human cancers, LMTK3 abundance and intronic polymorphisms were significantly associated with disease-free and overall survival and predicted response to endocrine therapies. These findings yield insights into the natural history of breast cancer in humans and reveal LMTK3 as a new therapeutic target.

Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

PubMed

Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

2018-01-16

"A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of

Membrane estrogen receptors - is it an alternative way of estrogen action?

PubMed

Soltysik, K; Czekaj, P

2013-04-01

The functions of estrogens are relatively well known, however the molecular mechanism of their action is not clear. The classical pathway of estrogen action is dependent on ERα and ERβ which act as transcription factors. The effects of this pathway occur within hours or days. In addition, so-called, non-classical mechanism of steroid action dependent on membrane estrogen receptors (mER) was described. In this mechanism the effects of estrogen action are observed in a much shorter time. Here we review the structure and cellular localization of mER, molecular basis of non-classical mER action, physiological role of mER as well as implications of mER action for cancer biology. Finally, some concerns about the new estrogen receptor - GPER and candidates for estrogen receptors - ER-X and ERx, are briefly discussed. It seems that mER is a complex containing signal proteins (signalosome), as IGF receptor, EGF receptor, Ras protein, adaptor protein Shc, non-receptor kinase c-Src and PI-3K, what rationalizes production of second messengers. Some features of membrane receptors are almost identical if compared to nuclear receptors. Probably, membrane and nuclear estrogen receptors are not separate units, but rather the components of a complex mechanism in which they both cooperate with each other. We conclude that the image of the estrogen receptor as a simple transcription factor is a far-reaching simplification. A better understanding of the mechanisms of estrogen action will help us to design more effective drugs affecting signal pathways depending on both membrane and nuclear receptors.

Egg wash wastewater: estrogenic risk or environmental asset?

PubMed

Shappell, Nancy W

2013-07-01

Commercial production of eggs and egg products requires the washing of eggs to remove urinary-fecal material and broken egg residue. In the case of one Ohio farming facility, 1.6 million birds produce 1.4 million eggs per day, using approximately 50 mL of wash water/egg or approximately 70,000 L per day. The aqueous waste stream was evaluated for estrogenicity to determine if potential for endocrine disruption would result from agricultural application of such wastewater. Samples collected the Fall (October) of 2010 included: water from 2 egg washers operating in series, inlet pipe to the treatment lagoon, a lagoon composite, and products used within the facility in the cleaning of equipment and treatment of the waste. In February 2011, the treatment lagoon was fitted with an extensive aeration system and subsequent sample sets were collected on 3 consecutive days in May and November. Samples were extracted by solid phase extraction and assayed for estrogenic activity using the in vitro E-Screen assay. Raw untreated wastewater from the egg washers contained 17β-estradiol equivalents (E2 Eqs) ranging from 9 to 18 ng/L, pipe grab samples entering into the treatment lagoon ranged from <0.14 to 4.4 ng/L (variability related to time of emptying of egg wash tanks), whereas treatment lagoon water contained 0.3 to 4.0 ng/L E2 Eq. Addition of an aeration system to the treatment lagoon eliminated surface "frothing," reduced noxious odor emission, and E2 Eqs were lower than the pre-aeration concentrations (4 ng/L [n = 1, no statistical comparison possible] vs 0.3 to 1.4 ng/L in 2011). Because of matrix effects, estrogens were not quantifiable by LC-MS2 in even egg washwater extracts, at concentrations in which internal deuterated estrogen standards were quantifiable. Estrone and E2 parent ions were detected in egg washwater samples only, and confirmatory ion fragments were detected in only one of these samples. Estrogenicity of the wastewater from the treatment lagoon was

Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

PubMed Central

Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

2010-01-01

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

A Novel Yeast Surface Display Method for Large-Scale Screen Inhibitors of Sortase A.

PubMed

Wu, Lin; Li, Huijun; Tang, Tianle

2017-01-24

Fluorescence resonance energy transfer substrates of sortase A are too expensive to be used to roughly screen high-throughput sortase A inhibitors. This makes therapeutic strategies difficult to realize in a clinical therapeutic use. Instead, we design here an LPETG-EGFP (leucine, proline, glutamic, threonine and glycine-enhanced green fluorescence) protein displayed on a yeast surface as a substrate by adaptively reducing the cost. We do this by optimizing the induction conditions of sortase A expression in Escherichia coli DE3(BL21) and catalyzing LPETG proteins, which are displayed on surface of Pichia pastoris . Different expression conditions of sortase A include: induction temperature (22 °C, 28 °C, 37 °C and 40 °C), induction time (4 h, 5 h, 6 h and 7 h) and induction concentration of isopropyl β-d-thiogalactoside IPTG (0.25 mmol/L, 0.5 mmol/L, 1 mmol/L, and 2 mmol/L). The fluorescence change of the LPETG-EGFP protein on the surface of P. pastoris over time was detected by flow cytometry and fluorescence spectrophotometry, and then the sensitivities of the two methods were compared. Using berberine chloride as an inhibitor, the activity of sortase A was investigated with the substrates of LPETG-EGFP protein, and compared to Dabcyl-QALPETGEE-Edans. A high yield of sortase A was achieved by inducing 1.0 mmol/L IPTG at 28 °C for 6 h. The intensity of green fluorescence of substrates displayed on the yeast surface was increased over time, while the stability was decreased slightly. Both fluorescence spectrophotometery and flow cytometry were fit for detection because of their high sensitivity. We utilized two different substrates of sortase A to investigate sortase A activity, which resulted in the increase of fluorescence intensity with respect to the increased time of growth. However, the method with Dabcyl-QALPETGEE-Edans as its substrate was more robust. Thus, the method described in this paper is a simple and cheap method which is very suitable for

Effect-directed identification of endocrine disruptors in plastic baby teethers.

PubMed

Berger, Elisabeth; Potouridis, Theodoros; Haeger, Astrid; Püttmann, Wilhelm; Wagner, Martin

2015-11-01

Concerns have been raised regarding the human health effects of endocrine disrupting chemicals (EDCs), many of which are associated with and leaching from plastics. As infants are particularly vulnerable to EDCs, we have investigated whether plastic teethers for babies represent a relevant source of exposure. Applying effect-directed analysis, we use bioassays to screen teethers, toys used to soothe a baby's teething ache, for endocrine activity and chemical analysis to identify the causative compounds. We detected significant endocrine activity in two of 10 plastic teethers. Those samples leached estrogenic and/or antiandrogenic activity as detected in the Yeast Estrogen Screen and Yeast Antiandrogen Screen. After sample fractionation, gas chromatography-mass spectrometry non-target screening revealed that methyl-, ethyl- and propylparaben were responsible for the observed estrogenic and antiandrogenic activity in one product. The second product is likely to contain at least six different antiandrogenic compounds that remain so far unidentified. This study demonstrates that plastic teethers can be a source of infant exposure to well-established and unknown EDCs. Because of their limited value to the product, but potential toxicity, manufacturers should critically revisit the use of parabens in plastic teethers and further toys. Moreover, plastic teethers might leach EDCs that escape routine analysis and, thus, toxicological evaluation. The resulting uncertainty in product safety poses a problem to consumers, producers and regulators that remain to be resolved. Copyright © 2015 John Wiley & Sons, Ltd.

Expression pattern and signalling pathways in neutrophil like HL-60 cells after treatment with estrogen receptor selective ligands.

PubMed

Blesson, Chellakkan Selvanesan; Sahlin, Lena

2012-09-25

Estrogens play a role in the regulation of genes associated with inflammation and immunity in neutrophils. Estrogen signalling is mediated by estrogen receptor (ER)α, ERβ, and G-protein-coupled estrogen receptor-1 (GPER). The mechanisms by which estrogen regulate genes in neutrophils are poorly understood. Our aim was to identify the presence of ERs and to characterize estrogen responsive genes in terminally differentiated neutrophil like HL-60 (nHL-60) cells using estradiol and selective ER agonists. ERs were identified by Western blotting and immunocytochemistry. Microarray technique was used to screen for differentially expressed genes and the selected genes were verified by quantitative PCR. We show the presence of functional ERα, ERβ and GPER. Microarray analysis showed the presence of genes that are uniquely regulated by a single ligand and also genes that are regulated by multiple ligands. We conclude that ERs are functionally active in nHL-60 cells regulating genes involved in key physiological functions. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Yeast Genomics for Bread, Beer, Biology, Bucks and Breath

NASA Astrophysics Data System (ADS)

Sakharkar, Kishore R.; Sakharkar, Meena K.

The rapid advances and scale up of projects in DNA sequencing dur ing the past two decades have produced complete genome sequences of several eukaryotic species. The versatile genetic malleability of the yeast, and the high degree of conservation between its cellular processes and those of human cells have made it a model of choice for pioneering research in molecular and cell biology. The complete sequence of yeast genome has proven to be extremely useful as a reference towards the sequences of human and for providing systems to explore key gene functions. Yeast has been a ‘legendary model’ for new technologies and gaining new biological insights into basic biological sciences and biotechnology. This chapter describes the awesome power of yeast genetics, genomics and proteomics in understanding of biological function. The applications of yeast as a screening tool to the field of drug discovery and development are highlighted and the traditional importance of yeast for bakers and brewers is discussed.

The Yeast Deletion Collection: A Decade of Functional Genomics

PubMed Central

Giaever, Guri; Nislow, Corey

2014-01-01

The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general. PMID:24939991

Species comparisons in molecular and functional attributes of the androgen and estrogen receptor

EPA Science Inventory

While endocrine disrupting compounds (EDCs) have the potential to act via several mechanisms of action, one of the most widely studied is the ability of environmental chemicals to interact directly with either the estrogen (ER) or androgen receptor (AR). In vitro screening assay...

Discovery of synthesis and secretion of polyol esters of fatty acids by four basidiomycetous yeast species in the order Sporidiobolales.

PubMed

Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Fiehn, Oliver; Cathcart, Erin; Fry, Russell W; Kanti, Atit; Joko Nugroho, Agustinus; Faulina, Sarah Asih; Stephanandra, Sira; German, J Bruce; Boundy-Mills, Kyria L

2017-06-01

Polyol esters of fatty acids (PEFA) are amphiphilic glycolipids produced by yeast that could play a role as natural, environmentally friendly biosurfactants. We recently reported discovery of a new PEFA-secreting yeast species, Rhodotorula babjevae, a basidiomycetous yeast to display this behavior, in addition to a few other Rhodotorula yeasts reported on the 1960s. Additional yeast species within the taxonomic order Sporidiobolales were screened for secreted glycolipid production. PEFA production equal or above 1 g L -1 were detected in 19 out of 65 strains of yeast screened, belonging to 6 out of 30 yeast species tested. Four of these species were not previously known to secrete glycolipids. These results significantly increase the number of yeast species known to secrete PEFA, holding promise for expanding knowledge of PEFA synthesis and secretion mechanisms, as well as setting the groundwork towards commercialization.

Yeast as a potential vehicle for neglected tropical disease drug discovery.

PubMed

Denny, P W; Steel, P G

2015-01-01

High-throughput screening (HTS) efforts for neglected tropical disease (NTD) drug discovery have recently received increased attention because several initiatives have begun to attempt to reduce the deficit in new and clinically acceptable therapies for this spectrum of infectious diseases. HTS primarily uses two basic approaches, cell-based and in vitro target-directed screening. Both of these approaches have problems; for example, cell-based screening does not reveal the target or targets that are hit, whereas in vitro methodologies lack a cellular context. Furthermore, both can be technically challenging, expensive, and difficult to miniaturize for ultra-HTS [(u)HTS]. The application of yeast-based systems may overcome some of these problems and offer a cost-effective platform for target-directed screening within a eukaryotic cell context. Here, we review the advantages and limitations of the technologies that may be used in yeast cell-based, target-directed screening protocols, and we discuss how these are beginning to be used in NTD drug discovery. © 2014 Society for Laboratory Automation and Screening.

Estrogenicity and Nutrient Concentration of Surface Waters Surrounding a Large Confinement Dairy Operation Using Best Management Practices for Land Application of Animal Wastes

USDA-ARS?s Scientific Manuscript database

The impact of a confinement dairy operation (> 2,000 head) using best management practices for land application of animal wastes, on estrogenic activity (E-Screen), estrogens, and nutrients of associated surface waters and tile drain runoff were evaluated. Farm tile drain and creek samples were col...

Estrogens and development

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLachlan, J.A.; Newbold, R.R.

1987-11-01

The normal development of the genital organs of mammals, including humans, is under hormonal control. A role for the female sex hormone estrogen in this process is still unclear. However, exposure of experimental animals or humans to the potent exogenous estrogen, diethylstilbestrol (DES), results in persistent differentiation effects. Since many chemicals in the environment are weakly estrogenic, the possibility of hormonally altered differentiation must be considered.

Characterization of Hyaluronan-Degrading Enzymes from Yeasts.

PubMed

Smirnou, Dzianis; Krčmář, Martin; Kulhánek, Jaromír; Hermannová, Martina; Bobková, Lenka; Franke, Lukáš; Pepeliaev, Stanislav; Velebný, Vladimír

2015-10-01

Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.

20180312 - Retrofitting an Estrogen Receptor Transactivation Assay with Metabolic Competence Using Alginate Immobilization of Metabolic Enzymes (AIME) (SOT)

EPA Science Inventory

The VM7Luc4E2 estrogen receptor (ER) transactivation assay is an OECD approved method (TG 457) for the detection of ER agonists and antagonists, and is also part of the Tox21 high-throughput screening (HTS) portfolio. Despite its international acceptance as a screening assay, imm...

A yeast functional screen predicts new candidate ALS disease genes

PubMed Central

Couthouis, Julien; Hart, Michael P.; Shorter, James; DeJesus-Hernandez, Mariely; Erion, Renske; Oristano, Rachel; Liu, Annie X.; Ramos, Daniel; Jethava, Niti; Hosangadi, Divya; Epstein, James; Chiang, Ashley; Diaz, Zamia; Nakaya, Tadashi; Ibrahim, Fadia; Kim, Hyung-Jun; Solski, Jennifer A.; Williams, Kelly L.; Mojsilovic-Petrovic, Jelena; Ingre, Caroline; Boylan, Kevin; Graff-Radford, Neill R.; Dickson, Dennis W.; Clay-Falcone, Dana; Elman, Lauren; McCluskey, Leo; Greene, Robert; Kalb, Robert G.; Lee, Virginia M.-Y.; Trojanowski, John Q.; Ludolph, Albert; Robberecht, Wim; Andersen, Peter M.; Nicholson, Garth A.; Blair, Ian P.; King, Oliver D.; Bonini, Nancy M.; Van Deerlin, Vivianna; Rademakers, Rosa; Mourelatos, Zissimos; Gitler, Aaron D.

2011-01-01

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery. PMID:22065782

Estrogenic activity of natural and synthetic estrogens in human breast cancer cells in culture.

PubMed Central

Zava, D T; Blen, M; Duwe, G

1997-01-01

We investigated the estrogenic activity of various environmental pollutants (xenobiotics), in particular the xenoestrogen o,p-DDT, and compared their effects with those of endogenous estrogens, phytoestrogens, and mycoestrogens on estrogen receptor binding capacity, induction of estrogen end products, and activation of cell proliferation in estrogen-sensitive human breast cancer cells in monolayer culture. We also quantified the levels of phytoestrogens in extracts of some common foods, herbs, and spices and in human saliva following consumption of a high phytoestrogen food source (soy milk) to compare phytoestrogen abundance and bioavailability relative to the reported xenoestrogen burden in humans. Results show that natural endogenous estrogens, phytoestrogens, mycoestrogens, and xenoestrogens bind estrogen receptor (ER) in intact cells, but demonstrate marked differences in their ability to induce end products of estrogen action and to regulate cell proliferation. All of the different classes of estrogens stimulated cell proliferation at concentrations that half-saturated ER, but only some classes were able to induce estrogen-regulated end products. Genistein, a common phytoestrogen found in soy foods, differed from the xenoestrogen DDT in its effects on cell proliferation and ability to induce estrogen-regulated end products. Moreover, we found that many of the foods, herbs, and spices commonly consumed by humans contain significant amounts of phytoestrogens, and consumption of soy milk, a phytoestrogen-rich food, markedly increases the levels of phytoestrogens in saliva. In conclusion, our in vitro results predict that a diet high in phytoestrogens would significantly reduce the binding of weak xenoestrogens to ER in target tissues in vivo. PMID:9168008

Estrogen-withdrawal migraine. I. Duration of exposure required and attempted prophylaxis by premenstrual estrogen administration.

PubMed

Somerville, B W

1975-03-01

The minimum exposure to estrogen required to cause estrogen-withdrawal migraine has been studied by giving long-acting estradiol valerate to four women and short-acting estradiol benzoate to two women. It was found that several days of exposure to high estrogen levels were needed to cause migraine on estrogen withdrawal. Oral administration of estrogen supplements in the form of estradiol valerate or as conjugated equine estrogens during the premenstrual phase in four women did not significantly affect plasma levels of estradiol, nor was it effective in preventing menstrual migraine.

Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae.

PubMed

Bellon, Jennifer R; Schmid, Frank; Capone, Dimitra L; Dunn, Barbara L; Chambers, Paul J

2013-01-01

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.

Repellent activity of estrogenic compounds toward zoospores of the phytopathogenic fungus Aphanomyces cochlioides.

PubMed

Islam, M T; Tahara, S

2001-01-01

Screening chemical compounds, we found that a xenoestrogen, bisphenol A, showed potent repellent activity against the zoospores of Aphanomyces cochlioides. Based on this finding, we tested a number of androgenic and estrogenic compounds (e.g. testosterone, progesterone, estradiols, diethylstilbestrol, estrone, estriol, pregnenolone, dienestrol etc.) on the motility behavior of A. cochlioides zoospores. Interestingly, most of the estrogenic compounds exhibited potent repellent activity (1 microg/ml or less by the "particle method") toward the motile zoospores of A. cochlioides. We derivatized some of the estrogens and discussed the relationship between the structure of active molecules and their repellent activity. Apparently, aromatization of the A ring with a free hydroxyl group at C-3 position of a steroidal structure is necessary for higher repellent activity. Interestingly, methylation of diethylstilbestrol (DES) yielded completely different activity i.e. both mono- and di-methyl ethers of DES showed attractant activity. Moreover, the attracted zoospores were encysted and then germinated in the presence of di-methyl ether of DES. The potential usefulness of this repellent test is discussed for the detection of estrogenic activity of naturally occurring compounds, and the possible role of phytoestrogens in host/parasite interactions. So far, this will be the first report of repellent activity of estrogenic compounds toward trivial fungal zoospores.

Impact of Estrogens and Estrogen Receptor Alpha (ESR1) in Brain Lipid Metabolism.

PubMed

Morselli, Eugenia; de Souza Santos, Roberta; Gao, Su; Ávalos, Yenniffer; Criollo, Alfredo; Palmer, Biff F; Clegg, Deborah J

2018-03-06

Estrogens and their receptors play key roles in regulating body weight, energy expenditure, and metabolic homeostasis. It is known that lack of estrogens promotes increased food intake and induces the expansion of adipose tissues, for which much is known. An area of estrogenic research that has received less attention is the role of estrogens and their receptors in influencing intermediary lipid metabolism in organs such as the brain. In this review, we highlight the actions of estrogens and their receptors in regulating their impact on modulating fatty acid content, utilization, and oxidation through their direct impact on intracellular signaling cascades within the central nervous system.

Screening of multiple hormonal activities in surface water and sediment from the Pearl River system, South China, using effect-directed in vitro bioassays.

PubMed

Zhao, Jian-Liang; Ying, Guang-Guo; Yang, Bin; Liu, Shan; Zhou, Li-Jun; Chen, Zhi-Feng; Lai, Hua-Jie

2011-10-01

This paper reports screening of multiple hormonal activities (estrogenic and androgenic activities, antiestrogenic and antiandrogenic activities) for surface water and sediment from the Pearl River system (Liuxi, Zhujiang, and Shijing rivers) in South China, using in vitro recombinant yeast bioassays. The detection frequencies for estrogenic and antiandrogenic activities were both 100% in surface water and 81 and 93% in sediment, respectively. The levels of estrogenic activity were 0.23 to 324 ng 17β-estradiol equivalent concentration (EEQ)/L in surface water and 0 to 101 ng EEQ/g in sediment. Antiandrogenic activities were in the range of 20.4 to 935 × 10(3) ng flutamide equivalent concentration (FEQ)/L in surface water and 0 to 154 × 10(3) ng FEQ/g in sediment. Moreover, estrogenic activity and antiandrogenic activity in sediment showed good correlation (R(2) = 0.7187), suggesting that the agonists of estrogen receptor and the antagonists of androgen receptor co-occurred in sediment. The detection frequencies for androgenic and antiestrogenic activities were 41 and 29% in surface water and 61 and 4% in sediment, respectively. The levels of androgenic activities were 0 to 45.4 ng dihydrotestosterone equivalent concentration (DEQ)/L in surface water, and the potency was very weak in the only detected sediment site. The levels of antiestrogenic activity were 0 to 1,296 × 10(3) ng tamoxifen equivalent concentration (TEQ)/L in surface water and 0 to 89.5 × 10(3) ng TEQ/g in sediment. The Shijing River displayed higher levels of hormonal activities than the Zhujiang and Liuxi rivers, indicating that the Shijing River had been suffering from heavy contamination with endocrine-disrupting chemicals. The equivalent concentrations of hormonal activities in some sites were greater than the lowest-observed-effect concentrations reported in the literature, suggesting potential adverse effects on aquatic organisms. Copyright © 2011 SETAC.

SPECIES DIFFERENCES IN ANDROGEN AND ESTROGEN RECEPTOR STRUCTURE AND FUNCTION AMONG VERTEBRATES AND INVERTEBRATES FOR INTERSPECIES EXTRAPOLATION OF ENDOCRINE DISRUPTING CHEMICALS

EPA Science Inventory

In vitro screening assays designed to identify hormone minics or antagonists, including the EDSTAC Tier 1 Screening (TIS) Battery, typically use only mammalian estrogen (ER) and androgen receptors (AR). However, there is uncertainty concerning species differences in binding affin...

Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes

PubMed Central

Sliva, Anna; Kuang, Zheng; Meluh, Pamela B.; Boeke, Jef D.

2016-01-01

The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating. PMID:26837954

Estrogen, vascular estrogen receptor and hormone therapy in postmenopausal vascular disease.

PubMed

Khalil, Raouf A

2013-12-15

Cardiovascular disease (CVD) is less common in premenopausal women than men of the same age or postmenopausal women, suggesting vascular benefits of estrogen. Estrogen activates estrogen receptors ERα, ERβ and GPR30 in endothelium and vascular smooth muscle (VSM), which trigger downstream signaling pathways and lead to genomic and non-genomic vascular effects such as vasodilation, decreased VSM contraction and growth and reduced vascular remodeling. However, randomized clinical trials (RCTs), such as the Women's Health Initiative (WHI) and Heart and Estrogen/progestin Replacement Study (HERS), have shown little vascular benefits and even adverse events with menopausal hormone therapy (MHT), likely due to factors related to the MHT used, ER profile, and RCT design. Some MHT forms, dose, combinations or route of administration may have inadequate vascular effects. Age-related changes in ER amount, distribution, integrity and post-ER signaling could alter the vascular response to MHT. The subject's age, preexisting CVD, and hormone environment could also reduce the effects of MHT. Further evaluation of natural and synthetic estrogens, phytoestrogens, and selective estrogen-receptor modulators (SERMs), and the design of appropriate MHT combinations, dose, route and 'timing' could improve the effectiveness of conventional MHT and provide alternative therapies in the peri-menopausal period. Targeting ER using specific ER agonists, localized MHT delivery, and activation of specific post-ER signaling pathways could counter age-related changes in ER. Examination of the hormone environment and conditions associated with hormone imbalance such as polycystic ovary syndrome may reveal the causes of abnormal hormone-receptor interactions. Consideration of these factors in new RCTs such as the Kronos Early Estrogen Prevention Study (KEEPS) could enhance the vascular benefits of estrogen in postmenopausal CVD. Copyright © 2013 Elsevier Inc. All rights reserved.

Estrogen, Vascular Estrogen Receptor and Hormone Therapy in Postmenopausal Vascular Disease

PubMed Central

Khalil, Raouf A.

2013-01-01

Cardiovascular disease (CVD) is less common in premenopausal women than men of the same age or postmenopausal women, suggesting vascular benefits of estrogen. Estrogen activates estrogen receptors ERα, ERβ and GPR30 in endothelium and vascular smooth muscle (VSM), which trigger downstream signaling pathways and lead to genomic and non-genomic vascular effects such as vasodilation, decreased VSM contraction and growth and reduced vascular remodeling. However, randomized clinical trials (RCTs), such as the Women’s Health Initiative (WHI) and Heart and Estrogen/progestin Replacement Study (HERS), have shown little vascular benefits and even adverse events with menopausal hormone therapy (MHT), likely due to factors related to the MHT used, ER profile, and RCT design. Some MHT forms, dose, combinations or route of administration may have inadequate vascular effects. Age-related changes in ER amount, distribution, integrity and post-ER signaling could alter the vascular response to MHT. The subject’s age, preexisting CVD, and hormone environment could also reduce the effects of MHT. Further evaluation of natural and synthetic estrogens, phytoestrogens, and selective estrogen-receptor modulators (SERMs), and the design of appropriate MHT combinations, dose, route and 'timing' could improve the effectiveness of conventional MHT and provide alternative therapies in the peri-menopausal period. Targeting ER using specific ER agonists, localized MHT delivery, and activation of specific post-ER signaling pathways could counter age-related changes in ER. Examination of the hormone environment and conditions associated with hormone imbalance such as polycystic ovary syndrome may reveal the causes of abnormal hormone-receptor interactions. Consideration of these factors in new RCTs such as the Kronos Early Estrogen Prevention Study (KEEPS) could enhance the vascular benefits of estrogen in postmenopausal CVD. PMID:24099797

Estrogenic Compounds, Estrogen Receptors and Vascular Cell Signaling in the Aging Blood Vessels

PubMed Central

Smiley, Dia A.; Khalil, Raouf A.

2010-01-01

The cardiovascular benefits of menopausal hormone therapy (MHT) remain controversial. The earlier clinical observations that cardiovascular disease (CVD) was less common in MHT users compared to non-users suggested cardiovascular benefits of MHT. Also, experimental studies have identified estrogen receptors ERα, ERβ and GPR30, which mediate genomic or non-genomic effects in vascular endothelium, smooth muscle, and extracellular matrix (ECM). However, data from randomized clinical trials (RCTs), most notably the Women's Health Initiative (WHI) study, have challenged the cardiovascular benefits and highlighted adverse cardiovascular events with MHT. The discrepancies have been attributed to the design of RCTs, the subjects' advanced age and preexisting CVD, and the form of estrogen used. The discrepancies may also stem from age-related changes in vascular ER amount, distribution, integrity, and post-receptor signaling pathways as well as structural changes in the vasculature. Age-related changes in other sex hormones such as testosterone may also alter the hormonal environment and influence the cardiovascular effects of estrogen. Investigating the chemical properties, structure-activity relationship and pharmacology of natural and synthetic estrogens should improve the effectiveness of conventional MHT. Further characterization of phytoestrogens, selective estrogen-receptor modulators (SERMs), and specific ER agonists may provide substitutes to conventional MHT. Conditions with excess or low estrogen levels such as polycystic ovary syndrome (PCOS) and Turner syndrome may provide insight into the development and regulation of ER and the mechanisms of aberrant estrogen-ER interactions. The lessons learned from previous RCTs have led to more directed studies such as the Kronos Early Estrogen Prevention Study (KEEPS). Careful design of experimental models and RCTs, coupled with the development of specific ER modulators, hold the promise of improving the actions of

Evaluation and characterization of anti-estrogenic and anti-androgenic activities in soil samples along the Second Songhua River, China.

PubMed

Li, Jian; Wang, Yafei; Kong, Dongdong; Wang, Jinsheng; Teng, Yanguo; Li, Na

2015-11-01

In the present study, re-combined estrogen receptor (ER) and androgen receptor (AR) gene yeast assays combined with a novel approach based on Monte Carlo simulation were used for evaluation and characterization of soil samples collected from Jilin along the Second Songhua River to assess their antagonist/agonist properties for ER and AR. The results showed that estrogenic activity only occurred in the soil samples collected in the agriculture area, but most soil samples showed anti-estrogenic activities, and the bioassay-derived 4-hydroxytamoxifen equivalents ranged from N.D. to 23.51 μg/g. Hydrophilic substance fractions were determined as potential contributors associated with anti-estrogenic activity in these soil samples. Moreover, none of the soil samples exhibited AR agonistic potency, whereas 54% of the soil samples exhibited AR antagonistic potency. The flutamide equivalents varied between N.D. and 178.05 μg/g. Based on Monte Carlo simulation-related mass balance analysis, the AR antagonistic activities were significantly correlated with the media polar and polar fractions. All of these results support that this novel calculation method can be adopted effectively to quantify and characterize the ER/AR agonists and antagonists of the soil samples, and these data could help provide useful information for future management and remediation efforts.

Distinct Effects of Estrogen on Mouse Maternal Behavior: The Contribution of Estrogen Synthesis in the Brain

PubMed Central

Murakami, Gen

2016-01-01

Estrogen surge following progesterone withdrawal at parturition plays an important role in initiating maternal behavior in various rodent species. Systemic estrogen treatment shortens the latency to onset of maternal behavior in nulliparous female rats that have not experienced parturition. In contrast, nulliparous laboratory mice show rapid onset of maternal behavior without estrogen treatment, and the role of estrogen still remains unclear. Here the effect of systemic estrogen treatment (for 2 h, 1 day, 3 days, and 7 days) after progesterone withdrawal was examined on maternal behavior of C57BL/6 mice. This estrogen regimen led to different effects on nursing, pup retrieval, and nest building behaviors. Latency to nursing was shortened by estrogen treatment within 2 h. Moreover, pup retrieval and nest building were decreased. mRNA expression was also investigated for estrogen receptor α (ERα) and for genes involved in regulating maternal behavior, specifically, the oxytocin receptor (OTR) and vasopressin receptor in the medial amygdala (MeA) and medial preoptic area (MPOA). Estrogen treatment led to decreased ERα mRNA in both regions. Although OTR mRNA was increased in the MeA, OTR and vasopressin receptor mRNA were reduced in the MPOA, showing region-dependent transcription regulation. To determine the mechanisms for the actions of estrogen treatment, the contribution of estrogen synthesis in the brain was examined. Blockade of estrogen synthesis in the brain by systemic letrozole treatment in ovariectomized mice interfered with pup retrieval and nest building but not nursing behavior, indicating different contributions of estrogen synthesis to maternal behavior. Furthermore, letrozole treatment led to an increase in ERα mRNA in the MeA but not in the MPOA, suggesting that involvement of estrogen synthesis is brain region dependent. Altogether, these results suggest that region-dependent estrogen synthesis leads to differential transcriptional activation due

CLONING, EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR ALPHA FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

EPA Science Inventory

In vitro screening assays designed to identify hormone mimics or antagonists, including those recommended for use in the EPA's Tier 1 screening battery, typically use mammalian estrogen (ER) and androgen receptors (AR) such as rat or human. Although we know that the amino acid s...

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches’ Broom disease

PubMed Central

de Souza Cabral, Anderson; de Carvalho, Patricia Maria Barroso; Pinotti, Tatiana; Hagler, Allen Norton; Mendonça-Hagler, Leda Cristina Santana; Macrae, Andrew

2009-01-01

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth. PMID:24031327

Beneficial effects of Lepidium meyenii (Maca) on psychological symptoms and measures of sexual dysfunction in postmenopausal women are not related to estrogen or androgen content.

PubMed

Brooks, Nicole A; Wilcox, Gisela; Walker, Karen Z; Ashton, John F; Cox, Marc B; Stojanovska, Lily

2008-01-01

To examine the estrogenic and androgenic activity of Lepidium meyenii (Maca) and its effect on the hormonal profile and symptoms in postmenopausal women. Fourteen postmenopausal women completed a randomized, double-blind, placebo-controlled, crossover trial. They received 3.5 g/day of powered Maca for 6 weeks and matching placebo for 6 weeks, in either order, over a total of 12 weeks. At baseline and weeks 6 and 12 blood samples were collected for the measurement of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin, and the women completed the Greene Climacteric Scale to assess the severity of menopausal symptoms. In addition, aqueous and methanolic Maca extracts were tested for androgenic and estrogenic activity using a yeast-based hormone-dependent reporter assay. No differences were seen in serum concentrations of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin between baseline, Maca treatment, and placebo (P > 0.05). The Greene Climacteric Scale revealed a significant reduction in scores in the areas of psychological symptoms, including the subscales for anxiety and depression and sexual dysfunction after Maca consumption compared with both baseline and placebo (P < 0.05). These findings did not correlate with androgenic or alpha-estrogenic activity present in the Maca as no physiologically significant activity was observed in yeast-based assays employing up to 4 mg/mL Maca extract (equivalent to 200 mg/mL Maca). Preliminary findings show that Lepidium meyenii (Maca) (3.5 g/d) reduces psychological symptoms, including anxiety and depression, and lowers measures of sexual dysfunction in postmenopausal women independent of estrogenic and androgenic activity.

Estrogens in Male Physiology.

PubMed

Cooke, Paul S; Nanjappa, Manjunatha K; Ko, CheMyong; Prins, Gail S; Hess, Rex A

2017-07-01

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17β-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues. Copyright © 2017 the American Physiological Society.

QSAR Classification of ToxCast and Tox21 Chemicals on the Basis of Estrogen Receptor Assays (FutureToxII)

EPA Science Inventory

The ToxCast and Tox21 programs have tested ~8,200 chemicals in a broad screening panel of in vitro high-throughput screening (HTS) assays for estrogen receptor (ER) agonist and antagonist activity. The present work uses this large in vitro data set to develop in silico QSAR model...

20180416 - Retrofitting an Estrogen Receptor Transactivation Assay with Metabolic Competence Using Alginate Immobilization of Metabolic Enzymes (AIME) (SETAC HTS)

EPA Science Inventory

The VM7Luc4E2 estrogen receptor (ER) transactivation assay is an OECD approved method (TG 457) for the detection of ER agonists and antagonists, and is also part of the Tox21 high-throughput screening (HTS) portfolio. Despite international acceptance as a screening assay, immorta...

Identification of estrogenic and antiestrogenic activities of respirable diesel exhaust particles by bioassay-directed fractionation.

PubMed

Oh, Seung Min; Ryu, Byung Taek; Chung, Kyu Hyuck

2008-01-01

Bioassay-directed fractionation was performed to identify causative chemical groups of DEPs with estrogenic and antiestrogenic activities. Bioassay-directed fractionation consists of a cell bioassay (E-SCREEN) in conjunction with acid-base partitioning (F1 and F2) and silica gel column fractionation of neutral fractions (F3-F7). Crude extract (CE) of DEPs in dichloromethane (DCM) exhibited both estrogenic and antiestrogenic activity. Estrogenic activity of CE and some fractions (F1, F2, F3, F5 and F6) was induced through estrogen receptor (ER)-mediated pathways. In particular, the acid polar fraction (F2) of DEPs, which contains phenols, induced high levels of estrogenic activity compared to other fractions. The estrogenic activity of F2 (610.80 pg-bio-EEQ/g-DEPs) was higher than that of the total estrogenic activity of CE (222.22 pg-bio-EEQ/g-DEPs). This result indicates that the estrogenic activity induced by causative estrogenic fraction (F2) may be antagonized by unidentified chemicals in DEPs. On the other hand, non-polar fractions (F3 and F4) of DEPs include aliphatic and chlorinated hydrocarbon, polyaromatic hydrocarbons, and their alkyl derivatives, which play an important role in the antiestrogenic activity of DEPs. In particular, F4, which contains PAH and its derivatives, showed the highest antiestrogenic activity. Since in our previous study, dibenzo(a, h)anthracene and chrysene were identified in F4, and these chemicals have antiestrogenic activity, we assume that these chemicals are the major causative chemicals with antiestrogenic activity in DEPs. In contrast to the estrogenic activity of DEPs, antiestrogenic activity of CE was stronger than that of antiestrogenic fractions (F3 and F4) at non-cytotoxic concentrations, indicating that additive or synergistic effects by unidentified chemicals contained in DEPs occurred.

Estrogen and Osteoporosis.

ERIC Educational Resources Information Center

Lindsay, Robert

1987-01-01

This article reviews the use of estrogen in the prevention and treatment of osteoporosis. Dosage levels, interactions with other factors, side effects, and the mechanism of estrogen action are discussed. (Author/MT)

Gene expression profiling in Ishikawa cells: A fingerprint for estrogen active compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boehme, Kathleen; Simon, Stephanie; Mueller, Stefan O.

2009-04-01

Several anthropogenous and naturally occurring substances, referred to as estrogen active compounds (EACs), are able to interfere with hormone and in particular estrogen receptor signaling. EACs can either cause adverse health effects in humans and wildlife populations or have beneficial effects on estrogen-dependent diseases. The aim of this study was to examine global gene expression profiles in estrogen receptor (ER)-proficient Ishikawa plus and ER-deficient Ishikawa minus endometrial cancer cells treated with selected well-known EACs (Diethylstilbestrol, Genistein, Zearalenone, Resveratrol, Bisphenol A and o,p'-DDT). We also investigated the effect of the pure antiestrogen ICI 182,780 (ICI) on the expression patterns caused bymore » these compounds. Transcript levels were quantified 24 h after compound treatment using Illumina BeadChip Arrays. We identified 87 genes with similar expression changes in response to all EAC treatments in Ishikawa plus. ICI lowered the magnitude or reversed the expression of these genes, indicating ER dependent regulation. Apart from estrogenic gene regulation, Bisphenol A, o,p'-DDT, Zearalenone, Genistein and Resveratrol displayed similarities to ICI in their expression patterns, suggesting mixed estrogenic/antiestrogenic properties. In particular, the predominant antiestrogenic expression response of Resveratrol could be clearly distinguished from the other test compounds, indicating a distinct mechanism of action. Divergent gene expression patterns of the phytoestrogens, as well as weaker estrogenic gene expression regulation determined for the anthropogenous chemicals Bisphenol A and o,p'-DDT, warrants a careful assessment of potential detrimental and/or beneficial effects of EACs. The characteristic expression fingerprints and the identified subset of putative marker genes can be used for screening chemicals with an unknown mode of action and for predicting their potential to exert endocrine disrupting effects.« less

The Molecular, Cellular and Clinical Consequences of Targeting the Estrogen Receptor Following Estrogen Deprivation Therapy

PubMed Central

Fan, Ping; Maximov, Philipp Y.; Curpan, Ramona F.; Abderrahman, Balkees; Jordan, V. Craig

2015-01-01

During the past twenty years our understanding of the control of breast tumor development, growth and survival has changed dramatically. The once long forgotten application of high dose synthetic estrogen therapy as the first chemical therapy to treat any cancer has been resurrected, refined and reinvented as the new biology of estrogen-induced apoptosis. High dose estrogen therapy was cast aside once tamoxifen, from its origins as a failed “morning after pill”, was reinvented as the first targeted therapy to treat any cancer. The current understanding of the mechanism of estrogen-induced apoptosis is described as a consequence of acquired resistance to long term antihormone therapy in estrogen receptor (ER) positive breast cancer. The ER signal transduction pathway remains a target for therapy in breast cancer despite “antiestrogen” resistance, but becomes a regulator of resistance. Multiple mechanisms of resistance come into play: Selective ER Modulator (SERM) stimulated growth, growth factor/ER crosstalk, estrogen-induced apoptosis and mutations of ER. But it is with the science of estrogen-induced apoptosis that the next innovation in women’s health will be developed. Recent evidence suggests that the glucocorticoid properties of medroxyprogesterone acetate blunt estrogen-induced apoptosis in estrogen deprived breast cancer cell populations. As a result breast cancer develops during long-term Hormone Replacement Therapy (HRT). A new synthetic progestin with estrogen-like properties, such as the 19 nortestosterone derivatives used in oral contraceptives, will continue to protect the uterus from unopposed estrogen stimulation but at the same time, reinforce apoptosis in vulnerable populations of nascent breast cancer cells. PMID:26052034

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

PubMed Central

Bellon, Jennifer R.; Schmid, Frank; Capone, Dimitra L.; Dunn, Barbara L.; Chambers, Paul J.

2013-01-01

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment. PMID:23614011

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

PubMed

Yang, Fang; Lei, Yingying; Zhou, Meiling; Yao, Qili; Han, Yichao; Wu, Xiang; Zhong, Wanshun; Zhu, Chenghang; Xu, Weize; Tao, Ran; Chen, Xi; Lin, Da; Rahman, Khaista; Tyagi, Rohit; Habib, Zeshan; Xiao, Shaobo; Wang, Dang; Yu, Yang; Chen, Huanchun; Fu, Zhenfang; Cao, Gang

2018-02-16

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

VASCULAR ACTIONS OF ESTROGENS: FUNCTIONAL IMPLICATIONS

PubMed Central

Miller, Virginia M.; Duckles, Sue P.

2009-01-01

The impact of estrogen exposure in preventing or treating cardiovascular disease is controversial. But it is clear that estrogen has important effects on vascular physiology and pathophysiology, with potential therapeutic implications. Therefore, it is the goal of this review to summarize, using an integrated approach, current knowledge of the vascular effects of estrogen, both in humans and in experimental animals. Aspects of estrogen synthesis and receptors, as well as general mechanisms of estrogenic action are reviewed with an emphasis on issues particularly relevant to the vascular system. Recent understanding of the impact of estrogen on mitochondrial function suggests that the longer lifespan of women compared to men may depend in part on the ability of estrogen to decrease production of reactive oxygen species in mitochondria. Mechanisms by which estrogen increases endothelial vasodilator function, promotes angiogenesis and modulates autonomic function are summarized. Key aspects of the relevant pathophysiology of inflammation, atherosclerosis, stroke, migraine and thrombosis are reviewed concerning current knowledge of estrogenic effects. A number of emerging concepts are addressed throughout. These include the importance of estrogenic formulation and route of administration and the impact of genetic polymorphisms, either in estrogen receptors or in enzymes responsible for estrogen metabolism, on responsiveness to hormone treatment. The importance of local metabolism of estrogenic precursors and the impact of timing for initiation of treatment and its duration are also considered. While consensus opinions are emphasized, controversial views are presented in order to stimulate future research. PMID:18579753

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis

PubMed Central

Pillon, Arnaud; Boussioux, Anne-Marie; Escande, Aurélie; Aït-Aïssa, Sélim; Gomez, Elena; Fenet, Hélène; Ruff, Marc; Moras, Dino; Vignon, Françoise; Duchesne, Marie-Josèphe; Casellas, Claude; Nicolas, Jean-Claude; Balaguer, Patrick

2005-01-01

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis. PMID:15743715

A-C Estrogens as Potent and Selective Estrogen Receptor-Beta Agonists (SERBAs) to Enhance Memory Consolidation under Low-Estrogen Conditions.

PubMed

Hanson, Alicia M; Perera, K L Iresha Sampathi; Kim, Jaekyoon; Pandey, Rajesh K; Sweeney, Noreena; Lu, Xingyun; Imhoff, Andrea; Mackinnon, Alexander Craig; Wargolet, Adam J; Van Hart, Rochelle M; Frick, Karyn M; Donaldson, William A; Sem, Daniel S

2018-06-14

Estrogen receptor-beta (ERβ) is a drug target for memory consolidation in postmenopausal women. Herein is reported a series of potent and selective ERβ agonists (SERBAs) with in vivo efficacy that are A-C estrogens, lacking the B and D estrogen rings. The most potent and selective A-C estrogen is selective for activating ER relative to seven other nuclear hormone receptors, with a surprising 750-fold selectivity for the β over α isoform and with EC 50 s of 20-30 nM in cell-based and direct binding assays. Comparison of potency in different assays suggests that the ER isoform selectivity is related to the compound's ability to drive the productive conformational change needed to activate transcription. The compound also shows in vivo efficacy after microinfusion into the dorsal hippocampus and after intraperitoneal injection (0.5 mg/kg) or oral gavage (0.5 mg/kg). This simple yet novel A-C estrogen is selective, brain penetrant, and facilitates memory consolidation.

Effect of halogenated substituents on the metabolism and estrogenic effects of the equine estrogen, equilenin.

PubMed

Liu, Xuemei; Zhang, Fagen; Liu, Hong; Burdette, Joanna E; Li, Yan; Overk, Cassia R; Pisha, Emily; Yao, Jiaqin; van Breemen, Richard B; Swanson, Steven M; Bolton, Judy L

2003-06-01

Estrogen replacement therapy has been correlated with an increased risk for developing breast and endometrial cancers. One potential mechanism of estrogen carcinogenesis involves metabolism of estrogens to 2- and 4-hydroxylated catechols, which are further oxidized to electrophilic/redox active o-quinones that have the potential to both initiate and promote the carcinogenic process. Previously, we showed that the equine estrogens, equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are primarily metabolized to the catechol, 4-hydroxyequilenin. This catechol was found to autoxidize to an o-quinone causing oxidation and alkylation of DNA in vitro and in vivo. To block catechol formation from equilenin, 4-halogenated equilenin derivatives were synthesized. These derivatives were tested for their ability to bind to the estrogen receptor, induce estrogen sensitive genes, and their potential to form catechol metabolites. We found that the 4-fluoro derivatives were more estrogenic than the 4-chloro and 4-bromo derivatives as demonstrated by a higher binding affinity for estrogen receptors alpha and beta, an enhanced induction of alkaline phosphatase activity in Ishikawa cells, pS2 expression in S30 cells, and PR expression in Ishikawa cells. Incubation of these compounds with tyrosinase in the presence of GSH showed that the halogenated equilenin compounds formed less catechol GSH conjugates than the parent compounds, equilenin and 17beta-hydroxyequilenin. In addition, these halogenated compounds showed less cytotoxicity in the presence of tyrosinase than the parent compounds in S30 cells. Also, as stated above, the 4-fluoro derivatives showed similar estrogenic effects as compared with parent compounds; however, they were less toxic in S30 cells as compared to equilenin and 17beta-equilenin. Because 17beta-hydroxy-4-halogenated equilenin derivatives showed higher estrogenic effects than the halogenated

Dissolved organic matter and estrogen interactions regulate estrogen removal in the aqueous environment: A review.

PubMed

Ma, Li; Yates, Scott R

2018-06-03

This review summarizes the characterization and quantification of interactions between dissolved organic matter (DOM) and estrogens as well as the effects of DOM on aquatic estrogen removal. DOM interacts with estrogens via binding or sorption mechanisms like π-π interaction and hydrogen bonding. The binding affinity is evaluated in terms of organic-carbon-normalized sorption coefficient (Log K OC ) which varies with types and composition of DOM. DOM has been suggested to be a more efficient sorbent compared with other matrices, such as suspended particulate matter, sediment and soil; likely associated with its large surface area and concentrated carbon content. As a photosensitizer, DOM enhanced estrogen photodegradation when the concentration of DOM was below a threshold value, and when above, the acceleration effect was not observed. DOM played a dual role in affecting biodegradation of estrogens depending on the recalcitrance of the DOM and the nutrition status of the degraders. DOM also acted as an electron shuttle (redox mediator) mediating the degradation of estrogens. DOM hindered enzyme-catalyzed removal of estrogens while enhanced their transformation during the simultaneous photo-enzymatic process. Membrane rejection of estrogens was pronounced for hydrophobic DOM with high aromaticity and phenolic moiety content. Elimination of estrogens via photolysis, biodegradation, enzymolysis and membrane rejection in the presence of DOM is initiated by sorption, accentuating the role of DOM as a mediator in regulating aquatic estrogen removal. Published by Elsevier B.V.

Selection of oleaginous yeasts for fatty acid production.

PubMed

Lamers, Dennis; van Biezen, Nick; Martens, Dirk; Peters, Linda; van de Zilver, Eric; Jacobs-van Dreumel, Nicole; Wijffels, René H; Lokman, Christien

2016-05-27

Oleaginous yeast species are an alternative for the production of lipids or triacylglycerides (TAGs). These yeasts are usually non-pathogenic and able to store TAGs ranging from 20 % to 70 % of their cell mass depending on culture conditions. TAGs originating from oleaginous yeasts can be used as the so-called second generation biofuels, which are based on non-food competing "waste carbon sources". In this study the selection of potentially new interesting oleaginous yeast strains is described. Important selection criteria were: a broad maximum temperature and pH range for growth (robustness of the strain), a broad spectrum of carbon sources that can be metabolized (preferably including C-5 sugars), a high total fatty acid content in combination with a low glycogen content and genetic accessibility. Based on these selection criteria, among 24 screened species, Schwanniomyces occidentalis (Debaromyces occidentalis) CBS2864 was selected as a promising strain for the production of high amounts of lipids.

Endoxifen, 4-Hydroxytamoxifen and an Estrogenic Derivative Modulate Estrogen Receptor Complex Mediated Apoptosis in Breast Cancer.

PubMed

Maximov, Philipp Y; Abderrahman, Balkees; Fanning, Sean W; Sengupta, Surojeet; Fan, Ping; Curpan, Ramona F; Quintana Rincon, Daniela Maria; Greenland, Jeffery A; Rajan, Shyamala S; Greene, Geoffrey L; Jordan, V Craig

2018-05-08

Estrogen therapy was used to treat advanced breast cancer in postmenopausal women for decades until the introduction of tamoxifen. Resistance to long-term estrogen deprivation (LTED) with tamoxifen and aromatase inhibitors used as a treatment for breast cancer inevitably occurs, but unexpectedly low dose estrogen can cause regression of breast cancer and increase disease free survival in some patients. This therapeutic effect is attributed to estrogen-induced apoptosis in LTED breast cancer. Here we describe modulation of the estrogen receptor liganded with antiestrogens (endoxifen, 4-hydroxytamoxifen) and an estrogenic triphenylethylene (TPE) EthoxyTPE (EtOXTPE) on estrogen-induced apoptosis in LTED breast cancer cells. Our results show that the angular TPE estrogen (EtOXTPE) is able to induce the ER-mediated apoptosis only at a later time compared to planar estradiol in these cells. Using RT-PCR, ChIP, Western blotting, molecular modelling and X-ray crystallography techniques we report novel conformations of the ER complex with an angular estrogen EtOXTPE and endoxifen. We propose that alteration of the conformation of the ER complexes, with changes in coactivator binding, governs estrogen-induced apoptosis through the PERK sensor system to trigger an Unfolded Protein Response (UPR). The American Society for Pharmacology and Experimental Therapeutics.

Protein–protein interactions and selection: yeast-based approaches that exploit guanine nucleotide-binding protein signaling.

PubMed

Ishii, Jun; Fukuda, Nobuo; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

2010-05-01

For elucidating protein–protein interactions, many methodologies have been developed during the past two decades. For investigation of interactions inside cells under physiological conditions, yeast is an attractive organism with which to quickly screen for hopeful candidates using versatile genetic technologies, and various types of approaches are now available.Among them, a variety of unique systems using the guanine nucleotide-binding protein (G-protein) signaling pathway in yeast have been established to investigate the interactions of proteins for biological study and pharmaceutical research. G-proteins involved in various cellular processes are mainly divided into two groups: small monomeric G-proteins,and heterotrimeric G-proteins. In this minireview, we summarize the basic principles and applications of yeast-based screening systems, using these two types of G-protein, which are typically used for elucidating biological protein interactions but are differentiated from traditional yeast two-hybrid systems.

Estrogen signaling through the G protein-coupled estrogen receptor regulates granulocyte activation in fish.

PubMed

Cabas, Isabel; Rodenas, M Carmen; Abellán, Emilia; Meseguer, José; Mulero, Victoriano; García-Ayala, Alfonsa

2013-11-01

Neutrophils are major participants in innate host responses. It is well known that estrogens have an immune-modulatory role, and some evidence exists that neutrophil physiology can be altered by these molecules. Traditionally, estrogens act via classical nuclear estrogen receptors, but the identification of a G protein-coupled estrogen receptor (GPER), a membrane estrogen receptor that binds estradiol and other estrogens, has opened up the possibility of exploring additional estrogen-mediated effects. However, information on the importance of GPER for immunity, especially, in neutrophils is scant. In this study, we report that gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils, express GPER at both mRNA and protein levels. By using a GPER selective agonist, G1, it was found that GPER activation in vitro slightly reduced the respiratory burst of acidophilic granulocytes and drastically altered the expression profile of several genes encoding major pro- and anti-inflammatory mediators. In addition, GPER signaling in vivo modulated adaptive immunity. Finally, a cAMP analog mimicked the effects of G1 in the induction of the gene coding for PG-endoperoxide synthase 2 and in the induction of CREB phosphorylation, whereas pharmacological inhibition of protein kinase A superinduced PG-endoperoxide synthase 2. Taken together, our results demonstrate for the first time, to our knowledge, that estrogens are able to modulate vertebrate granulocyte functions through a GPER/cAMP/protein kinase A/CREB signaling pathway and could establish therapeutic targets for several immune disorders in which estrogens play a prominent role.

Estrogenic Activity of Hyperforin in MCF-7 Human Breast Cancer Cells Transfected with Estrogen Receptor.

PubMed

Kwon, Joseph; Oh, Kyung Seo; Cho, Se-Young; Bang, Mi Ae; Kim, Hwan Seon; Vaidya, Bipin; Kim, Duwoon

2016-11-01

Hyperforin, a major active compound of St. John's wort extract, affects estrogenic activity. In this study, the compound evoked estrogen response element-dependent luciferase activity and cell proliferation in MCF-7 cells. Hyperforin-induced cell proliferation was significantly inhibited by the estrogen receptor antagonist ICI 182,780. These results suggested that hyperforin had estrogenic and cell proliferation activities, which were stimulated via the estrogen receptor. Compared to 17 β -estradiol, hyperforin showed significantly lower estrogenic activity and cell proliferation. The mechanism underlying the estrogenic activity of hyperforin was unknown, therefore, in this study, for the first time, the expression and post-translational modification of proteins were determined and compared among control, 17 β -estradiol-treated, and hyperforin-treated cells using proteomic techniques. A total of 453 proteins were identified, of which 282 proteins were significantly modulated in hyperforin-treated cells compared to 17 β -estradiol-treated cells. Ingenuity pathway analysis also demonstrated that hyperforin treatment induced less cell proliferation than 17 β -estradiol by downregulating estrogen receptor 1. Protein network analysis showed that cell proliferation was regulated mainly by cyclin D1 and extracellular signal-regulated kinases. In conclusion, although, hyperforin exhibited lower estrogenic activity than 17 β -estradiol, the compound induced lower levels of cancer cell proliferation in vitro . Georg Thieme Verlag KG Stuttgart · New York.

Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast.

PubMed

Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A

2016-06-01

Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3' end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. Copyright © 2016 Larson et al.

Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

PubMed Central

Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

2016-01-01

Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

Fecal microbial determinants of fecal and systemic estrogens and estrogen metabolites: a cross-sectional study.

PubMed

Flores, Roberto; Shi, Jianxin; Fuhrman, Barbara; Xu, Xia; Veenstra, Timothy D; Gail, Mitchell H; Gajer, Pawel; Ravel, Jacques; Goedert, James J

2012-12-21

High systemic estrogen levels contribute to breast cancer risk for postmenopausal women, whereas low levels contribute to osteoporosis risk. Except for obesity, determinants of non-ovarian systemic estrogen levels are undefined. We sought to identify members and functions of the intestinal microbial community associated with estrogen levels via enterohepatic recirculation. Fifty-one epidemiologists at the National Institutes of Health, including 25 men, 7 postmenopausal women, and 19 premenopausal women, provided urine and aliquots of feces, using methods proven to yield accurate and reproducible results. Estradiol, estrone, 13 estrogen metabolites (EM), and their sum (total estrogens) were quantified in urine and feces by liquid chromatography/tandem mass spectrometry. In feces, β-glucuronidase and β-glucosidase activities were determined by realtime kinetics, and microbiome diversity and taxonomy were estimated by pyrosequencing 16S rRNA amplicons. Pearson correlations were computed for each loge estrogen level, loge enzymatic activity level, and microbiome alpha diversity estimate. For the 55 taxa with mean relative abundance of at least 0.1%, ordinal levels were created [zero, low (below median of detected sequences), high] and compared to loge estrogens, β-glucuronidase and β-glucosidase enzymatic activity levels by linear regression. Significance was based on two-sided tests with α=0.05. In men and postmenopausal women, levels of total urinary estrogens (as well as most individual EM) were very strongly and directly associated with all measures of fecal microbiome richness and alpha diversity (R≥0.50, P≤0.003). These non-ovarian systemic estrogens also were strongly and significantly associated with fecal Clostridia taxa, including non-Clostridiales and three genera in the Ruminococcaceae family (R=0.57-0.70, P=0.03-0.002). Estrone, but not other EM, in urine correlated significantly with functional activity of fecal β-glucuronidase (R=0.36, P=0

Estrogen-mediated inactivation of FOXO3a by the G protein-coupled estrogen receptor GPER.

PubMed

Zekas, Erin; Prossnitz, Eric R

2015-10-15

Estrogen (17β-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERβ as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear. MCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation. In the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions. Our results suggest that non-genomic signaling by GPER contributes

The Measurement of Estrogens

NASA Astrophysics Data System (ADS)

Holder, Geoff; Makin, Hugh L. J.; Bradlow, H. Leon

Biologists use the word ‘estrogen' when referring to molecules which have the ability to induce uterine growth or vaginal cornification in the immature or ovariectomized rodent. The word estrogen was derived from two Greek words - oistros meaning frenzy and gennein - to beget. Chemists and biochemists, however, often restrict their use of this term to molecules that contain a characteristic 18-carbon steroid nucleus with an aromatic (phenolic) A-ring, both those that are biologically active estrogens and those without biologic activity but which are of intrinsic interest, such as the estrogen conjugates. This chapter is concerned only with these steroid compounds. The structure and inter-relationship of some common estrogens are given in Fig. 8.1. In addition to the biological estrogens, there are a wide variety of both natural and synthetic compounds which have estrogenic activity when measured by one or another parameter. While many of the assay procedures described in this review are applicable to these compounds, their application to non C18-steroids will not be discussed here. Methodology for these non-steroidal compounds can be found in reviews by Wang et al. (2002), Wu et al. (2004), Muir (2006), and Delmonte and Rader (2006). While not wishing to downgrade the importance of previous work in the estrogen field, the authors have taken a deliberate decision to exclude most publications prior to 1975, not because these do not have value but simply because space is not unlimited and readers of the present chapter might be expected to be seeking information about methodology which is less than 30 years old. Readers seeking pre-1975 information in this area can find it in Oakey and Holder (1995).

Estrogen and the female heart.

PubMed

Knowlton, A A; Korzick, D H

2014-05-25

Estrogen has a plethora of effects in the cardiovascular system. Studies of estrogen and the heart span human clinical trials and basic cell and molecular investigations. Greater understanding of cell and molecular responses to estrogens can provide further insights into the findings of clinical studies. Differences in expression and cellular/intracellular distribution of the two main receptors, estrogen receptor (ER) α and β, are thought to account for the specificity and differences in responses to estrogen. Much remains to be learned in this area, but cellular distribution within the cardiovascular system is becoming clearer. Identification of GPER as a third ER has introduced further complexity to the system. 17β-estradiol (E2), the most potent human estrogen, clearly has protective properties activating a signaling cascade leading to cellular protection and also influencing expression of the protective heat shock proteins (HSP). E2 protects the heart from ischemic injury in basic studies, but the picture is more involved in the whole organism and clinical studies. Here the complexity of E2's widespread effects comes into play and makes interpretation of findings more challenging. Estrogen loss occurs primarily with aging, but few studies have used aged models despite clear evidence of differences between the response to estrogen deficiency in adult and aged animals. Thus more work is needed focusing on the effects of aging vs. estrogen loss on the cardiovascular system. Published by Elsevier Ireland Ltd.

Estrogen supports urothelial defense mechanisms.

PubMed

Lüthje, Petra; Brauner, Hanna; Ramos, Nubia L; Ovregaard, Amanda; Gläser, Regine; Hirschberg, Angelica Lindén; Aspenström, Pontus; Brauner, Annelie

2013-06-19

Epidemiological data imply a role of estrogen in the pathogenesis of urinary tract infections (UTIs), although the underlying mechanisms are not well understood. However, it is thought that estrogen supplementation after menopause decreases the risk of recurrent infections. We sought to investigate the influence of estrogen on host-pathogen interactions and the consequences for UTI pathogenesis. We analyzed urothelial cells from menstruating and postmenopausal women before and after a 2-week period of estrogen supplementation, and also studied the influence of estradiol during Escherichia coli UTI in a mouse infection model. Important findings were confirmed in two human urothelial cell lines. We identified two epithelial defense mechanisms modulated by estrogen. Estrogen induced the expression of antimicrobial peptides, thereby enhancing the antimicrobial capacity of the urothelium and restricting bacterial multiplication. In addition, estrogen promoted the expression and redistribution of cell-cell contact-associated proteins, thereby strengthening the epithelial integrity and preventing excessive loss of superficial cells during infection. These two effects together may prevent bacteria from reaching deeper layers of the urinary tract epithelium and developing reservoirs that can serve as a source for recurrent infections. Thus, this study presents some underlying mechanisms for the beneficial effect of estradiol after menopause and supports the application of estrogen in postmenopausal women suffering from recurrent UTI.

Estrogen Effects on Wound Healing

PubMed Central

Horng, Huann-Cheng; Chang, Wen-Hsun; Yeh, Chang-Ching; Huang, Ben-Shian; Chang, Chia-Pei; Chen, Yi-Jen; Tsui, Kuan-Hao

2017-01-01

Wound healing is a physiological process, involving three successive and overlapping phases—hemostasis/inflammation, proliferation, and remodeling—to maintain the integrity of skin after trauma, either by accident or by procedure. Any disruption or unbalanced distribution of these processes might result in abnormal wound healing. Many molecular and clinical data support the effects of estrogen on normal skin homeostasis and wound healing. Estrogen deficiency, for example in postmenopausal women, is detrimental to wound healing processes, notably inflammation and re-granulation, while exogenous estrogen treatment may reverse these effects. Understanding the role of estrogen on skin might provide further opportunities to develop estrogen-related therapy for assistance in wound healing. PMID:29099810

Virtual and biomolecular screening converge on a selective agonist for GPR30.

PubMed

Bologa, Cristian G; Revankar, Chetana M; Young, Susan M; Edwards, Bruce S; Arterburn, Jeffrey B; Kiselyov, Alexander S; Parker, Matthew A; Tkachenko, Sergey E; Savchuck, Nikolay P; Sklar, Larry A; Oprea, Tudor I; Prossnitz, Eric R

2006-04-01

Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the seven-transmembrane G protein-coupled receptor GPR30 binds estrogen with high affinity and resides in the endoplasmic reticulum, where it activates multiple intracellular signaling pathways. To differentiate between the functions of ERalpha or ERbeta and GPR30, we used a combination of virtual and biomolecular screening to isolate compounds that selectively bind to GPR30. Here we describe the identification of the first GPR30-specific agonist, G-1 (1), capable of activating GPR30 in a complex environment of classical and new estrogen receptors. The development of compounds specific to estrogen receptor family members provides the opportunity to increase our understanding of these receptors and their contribution to estrogen biology.

Occurrence of killer yeasts in leaf-cutting ant nests.

PubMed

Carreiro, S C; Pagnocca, F C; Bacci, M; Bueno, O C; Hebling, M J A; Middelhoven, W J

2002-01-01

Killer activity was screened in 99 yeast strains isolated from the nests of the leaf-cutting ant Atta sexdens against 6 standard sensitive strains, as well as against each other. Among this yeast community killer activity was widespread since 77 strains (78%) were able to kill or inhibit the growth of at least one standard strain or nest strain. Toxin production was observed in representatives of all the studied genera including Aureobasidium, Rhodotorula, Tremella and Trichosporon, whose killer activity has not yet been described.

Estrogen-related receptor gamma disruption of source water and drinking water treatment processes extracts.

PubMed

Li, Na; Jiang, Weiwei; Rao, Kaifeng; Ma, Mei; Wang, Zijian; Kumaran, Satyanarayanan Senthik

2011-01-01

Environmental chemicals in drinking water can impact human health through nuclear receptors. Additionally, estrogen-related receptors (ERRs) are vulnerable to endocrine-disrupting effects. To date, however, ERR disruption of drinking water potency has not been reported. We used ERRgamma two-hybrid yeast assay to screen ERRgamma disrupting activities in a drinking water treatment plant (DWTP) located in north China and in source water from a reservoir, focusing on agonistic, antagonistic, and inverse agonistic activity to 4-hydroxytamoxifen (4-OHT). Water treatment processes in the DWTP consisted of pre-chlorination, coagulation, coal and sand filtration, activated carbon filtration, and secondary chlorination processes. Samples were extracted by solid phase extraction. Results showed that ERRgamma antagonistic activities were found in all sample extracts, but agonistic and inverse agonistic activity to 4-OHT was not found. When calibrated with the toxic equivalent of 4-OHT, antagonistic effluent effects ranged from 3.4 to 33.1 microg/L. In the treatment processes, secondary chlorination was effective in removing ERRgamma antagonists, but the coagulation process led to significantly increased ERRgamma antagonistic activity. The drinking water treatment processes removed 73.5% of ERRgamma antagonists. To our knowledge, the occurrence of ERRgamma disruption activities on source and drinking water in vitro had not been reported previously. It is vital, therefore, to increase our understanding of ERRy disrupting activities in drinking water.

Endocrine disrupting activities in sewage effluent and river water determined by chemical analysis and in vitro assay in the context of granular activated carbon upgrade.

PubMed

Grover, D P; Balaam, J; Pacitto, S; Readman, J W; White, S; Zhou, J L

2011-09-01

As part of endocrine disruption in catchments (EDCAT) programme, this work aims to assess the temporal and spatial variations of endocrine disrupting chemicals (EDCs) in River Ray, before and after the commissioning of a full-scale granular activated carbon (GAC) plant at a sewage treatment works (STW). Through spot and passive sampling from effluent and river sites, estrogenic and anti-androgenic activities were determined by chemical analysis and in vitro bio-assay. A correlation was found between chemical analyses of the most potent estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2)) and yeast estrogen screen (YES) measurement, both showing clearly a reduction in estrogenic activity after the commissioning of the GAC plant at the STW. During the study period, the annual average concentrations of E1, E2 and EE2 had decreased from 3.5 ng L(-1), 3.1 ng L(-1) and 0.5 ng L(-1) to below their limit of detection (LOD), respectively, with a concentration reduction of at least 91%, 81% and 60%. Annual mean estrogenic activity measured by YES of spot samples varied from 1.9 ng L(-1) to 0.4 ng L(-1) E2 equivalent between 2006 and 2008 representing a 79% reduction. Similarly, anti-androgenic activity measured by yeast anti-androgen screen (anti-YAS) of spot samples was reduced from 148.8 to 22.4 μg flutamide L(-1), or by 85%. YES and anti-YAS values were related to each other, suggesting co-existence of both types of activities from chemical mixtures in environmental samples. The findings confirm the effectiveness of a full-scale GAC in removing both estrogenic and anti-androgenic activities from sewage effluent. Copyright © 2011 Elsevier Ltd. All rights reserved.

Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-β

PubMed Central

Lu, Jun-Yu; Jin, Peng; Gao, Wei; Wang, De-Zhi; Sheng, Jian-Qiu

2017-01-01

Epidemiological data demonstrated that hormone replace treatment has protective effect against colorectal cancer (CRC). Our previous studies showed that this effect may be associated with DNA mismatch repair. This study aims to investigate the mechanism of estrogen induction of MLH1, and whether colorectal tumor proliferation can be inhibited through induction of MLH1 by estrogen signal pathway. Human CRC cell lines were used to examine the regulation of MLH1 expression by over-expression and depletion of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), under the treatment with 17β-estradiol or β-Estradiol 6-(O-carboxy-methyl)oxime:BSA, followed by a real-time Q-PCR and Western blotting analysis. Luciferase reporter and chromatin immunoprecipitation assays were used to identify the estrogen response elements in the proximal promoter of MLH1 gene. Then, the influence of estrogen-induced MLH1 on CRC tumor growth were determined in vitro and in vivo. We found that mismatch repair ability and microsatellite stability of cells were enhanced by estrogen via induction of MLH1 expression, which was mediated by ERβ, through a transcriptional activation process. Furthermore, we identified that ERβ exerted an inhibitory effect on CRC tumor proliferation in vitro and in vivo, combined with 5-FU, through up-regulation of MLH1 expression. Finally, we concluded that estrogen enhances mismatch repair ability and tumor inhibition effect in vitro and in vivo, via induction of MLH1 expression mediated by ERβ. PMID:28404976

Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-β.

PubMed

Lu, Jun-Yu; Jin, Peng; Gao, Wei; Wang, De-Zhi; Sheng, Jian-Qiu

2017-06-13

Epidemiological data demonstrated that hormone replace treatment has protective effect against colorectal cancer (CRC). Our previous studies showed that this effect may be associated with DNA mismatch repair. This study aims to investigate the mechanism of estrogen induction of MLH1, and whether colorectal tumor proliferation can be inhibited through induction of MLH1 by estrogen signal pathway. Human CRC cell lines were used to examine the regulation of MLH1 expression by over-expression and depletion of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), under the treatment with 17β-estradiol or β-Estradiol 6-(O-carboxy-methyl)oxime:BSA, followed by a real-time Q-PCR and Western blotting analysis. Luciferase reporter and chromatin immunoprecipitation assays were used to identify the estrogen response elements in the proximal promoter of MLH1 gene. Then, the influence of estrogen-induced MLH1 on CRC tumor growth were determined in vitro and in vivo. We found that mismatch repair ability and microsatellite stability of cells were enhanced by estrogen via induction of MLH1 expression, which was mediated by ERβ, through a transcriptional activation process. Furthermore, we identified that ERβ exerted an inhibitory effect on CRC tumor proliferation in vitro and in vivo, combined with 5-FU, through up-regulation of MLH1 expression. Finally, we concluded that estrogen enhances mismatch repair ability and tumor inhibition effect in vitro and in vivo, via induction of MLH1 expression mediated by ERβ.

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pinto, Caroline; Grimaldi, Marina; Boulahtouf, Abdelhay

2014-10-01

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28 °C as compared tomore » 37 °C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology. - Highlights: • Zebrafish is increasingly used to study the effects of estrogens. • We assessed the activity of pharmaceutical and environmental estrogens on zfERs. • Environmental estrogens displayed greater potency for zfERα compared to zfERβs. • hERβ selective agonists displayed greater

Quantitative maps of genetic interactions in yeast - comparative evaluation and integrative analysis.

PubMed

Lindén, Rolf O; Eronen, Ville-Pekka; Aittokallio, Tero

2011-03-24

High-throughput genetic screening approaches have enabled systematic means to study how interactions among gene mutations contribute to quantitative fitness phenotypes, with the aim of providing insights into the functional wiring diagrams of genetic interaction networks on a global scale. However, it is poorly known how well these quantitative interaction measurements agree across the screening approaches, which hinders their integrated use toward improving the coverage and quality of the genetic interaction maps in yeast and other organisms. Using large-scale data matrices from epistatic miniarray profiling (E-MAP), genetic interaction mapping (GIM), and synthetic genetic array (SGA) approaches, we carried out here a systematic comparative evaluation among these quantitative maps of genetic interactions in yeast. The relatively low association between the original interaction measurements or their customized scores could be improved using a matrix-based modelling framework, which enables the use of single- and double-mutant fitness estimates and measurements, respectively, when scoring genetic interactions. Toward an integrative analysis, we show how the detections from the different screening approaches can be combined to suggest novel positive and negative interactions which are complementary to those obtained using any single screening approach alone. The matrix approximation procedure has been made available to support the design and analysis of the future screening studies. We have shown here that even if the correlation between the currently available quantitative genetic interaction maps in yeast is relatively low, their comparability can be improved by means of our computational matrix approximation procedure, which will enable integrative analysis and detection of a wider spectrum of genetic interactions using data from the complementary screening approaches.

The role of lager beer yeast in oxidative stability of model beer.

PubMed

Berner, T S; Arneborg, N

2012-03-01

In this study, we investigated the relationship between the ability of lager brewing yeast strains to tolerate oxidative stress and their ability to produce oxidative stable model beer. Screening of 21 lager brewing yeast strains against diamide and paraquat showed that the oxidative stress resistance was strain dependent. Fermentation of model wort in European Brewing Convention tubes using three yeast strains with varying oxidative stress resistances resulted in three model beers with different rates of radical formation as measured by electron spin resonance in forced ageing experiments. Interestingly, the strain with the lowest oxidative stress resistance and lowest secretion of thioredoxin, as measured by Western blotting, resulted in the highest uptake of iron, as measured by inductively coupled plasma-mass spectrometry, and the slowest formation of radicals in the model beers. A more oxidative stable beer is not obtained by a more-oxidative-stress-tolerant lager brewing yeast strain, exhibiting a higher secretion of thioredoxin, but rather by a less-oxidative-stress-tolerant strain, exhibiting a higher iron uptake. To obtain lager beers with enhanced oxidative stability, yeast strains should be screened for their low oxidative stress tolerance and/or high ability to take up iron rather than for their high oxidative stress tolerance and/or high ability to secrete thioredoxin. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Environmental estrogenic effects of alkylphenol ethoxylates.

PubMed

Nimrod, A C; Benson, W H

1996-05-01

Alkylphenol ethoxylates (APEs) and related compounds recently have been reported to be estrogenic because it has been demonstrated in laboratory studies that they mimic the effects of estradiol both in vitro and in vivo. Chemicals referred to as "environmental estrogens" are suspected of causing health effects in both humans and wildlife through disruption of the endocrine system. In this review, the occurrence, environmental fate, and biological effects of APEs are presented. To provide understanding of the potential for endocrine disruption due to environmental estrogens, the physiology of estrogens in mammals and fish is also reviewed. The estrogenic potency of other environmental estrogens is compared to the potency of APE degradation products. The reproductive effects of estrogenic compounds are considered when evaluating the potential health effects of APEs. Given the reported environmental concentrations and bioconcentration factors of APE products, the potential for these compounds to produce estrogenic effects in the environment appears low. Although questions concerning the physiological effects of APEs and other environmental estrogens remain unanswered, there are indications that research is in progress that will lead to better understanding of the risks to humans and wildlife.

CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

PubMed Central

Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra; Roncaglioni, Alessandra; Tropsha, Alexander; Varnek, Alexandre; Zakharov, Alexey; Worth, Andrew; Richard, Ann M.; Grulke, Christopher M.; Trisciuzzi, Daniela; Fourches, Denis; Horvath, Dragos; Benfenati, Emilio; Muratov, Eugene; Wedebye, Eva Bay; Grisoni, Francesca; Mangiatordi, Giuseppe F.; Incisivo, Giuseppina M.; Hong, Huixiao; Ng, Hui W.; Tetko, Igor V.; Balabin, Ilya; Kancherla, Jayaram; Shen, Jie; Burton, Julien; Nicklaus, Marc; Cassotti, Matteo; Nikolov, Nikolai G.; Nicolotti, Orazio; Andersson, Patrik L.; Zang, Qingda; Politi, Regina; Beger, Richard D.; Todeschini, Roberto; Huang, Ruili; Farag, Sherif; Rosenberg, Sine A.; Slavov, Svetoslav; Hu, Xin; Judson, Richard S.

2016-01-01

Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. Objectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) and demonstrate the efficacy of using predictive computational models trained on high-throughput screening data to evaluate thousands of chemicals for ER-related activity and prioritize them for further testing. Methods: CERAPP combined multiple models developed in collaboration with 17 groups in the United States and Europe to predict ER activity of a common set of 32,464 chemical structures. Quantitative structure–activity relationship models and docking approaches were employed, mostly using a common training set of 1,677 chemical structures provided by the U.S. EPA, to build a total of 40 categorical and 8 continuous models for binding, agonist, and antagonist ER activity. All predictions were evaluated on a set of 7,522 chemicals curated from the literature. To overcome the limitations of single models, a consensus was built by weighting models on scores based on their evaluated accuracies. Results: Individual model scores ranged from 0.69 to 0.85, showing high prediction reliabilities. Out of the 32,464 chemicals, the consensus model predicted 4,001 chemicals (12.3%) as high priority actives and 6,742 potential actives (20.8%) to be considered for further testing. Conclusion: This project demonstrated the possibility to screen large libraries of chemicals using a consensus of different in silico approaches. This concept will be applied in future projects related to other

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

PubMed Central

2011-01-01

Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy. PMID:22014111

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.

PubMed

Le Breton, Marc; Meyniel-Schicklin, Laurène; Deloire, Alexandre; Coutard, Bruno; Canard, Bruno; de Lamballerie, Xavier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent; Davoust, Nathalie

2011-10-20

The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

Estrogenic activity, estrogens, and calcium in runoff post-layer litter application from rainfall simulated events

USDA-ARS?s Scientific Manuscript database

Estrogens in runoff from fields fertilized with animal wastes have been implicated as endocrine disruptors of fish in recipient surface waters. The goal of this study was to measure estrogenic activity in runoff post-application of animal waste with the greatest potential for estrogenic activity - ...

Estrogen, aging and the cardiovascular system

PubMed Central

Stice, James P.; Lee, Jennifer S.; Pechenino, Angela S.; Knowlton, Anne A.

2014-01-01

Estrogen is a powerful hormone with pleiotropic effects. Estrogens have potent antioxidant effects and are able to reduce inflammation, induce vasorelaxation and alter gene expression in both the vasculature and the heart. Estrogen treatment of cultured cardiac myocytes and endothelial cells rapidly activates NFκB, induces heat-shock protein (HSP)-72, a potent intracellular protective protein, and protects cells from simulated ischemia. In in vivo models, estrogens protect against ischemia and trauma/hemorrhage. Estrogens may decrease the expression of soluble epoxide hydrolase, which has deleterious effects on the cardiovascular system through metabolism of epoxyeicosatrienoic acids. Natural (endogenous) estrogens in premenopausal women appear to protect against cardiovascular disease and yet controlled clinical trials have not indicated a benefit from estrogen replacement postmenopause. Much remains to be understood in regards to the many properties of this powerful hormone and how changes in this hormone interact with aging-associated changes. The unexpected negative results of trials of estrogen replacement postmenopause probably arise from our lack of understanding of the many effects of this hormone. PMID:19371207

Visualization and Image Analysis of Yeast Cells.

PubMed

Bagley, Steve

2016-01-01

When converting real-life data via visualization to numbers and then onto statistics the whole system needs to be considered so that conversion from the analogue to the digital is accurate and repeatable. Here we describe the points to consider when approaching yeast cell analysis visualization, processing, and analysis of a population by screening techniques.

Estrogen promotes megakaryocyte polyploidization via estrogen receptor beta-mediated transcription of GATA1.

PubMed

Du, C; Xu, Y; Yang, K; Chen, S; Wang, X; Wang, S; Wang, C; Shen, M; Chen, F; Chen, M; Zeng, D; Li, F; Wang, T; Wang, F; Zhao, J; Ai, G; Cheng, T; Su, Y; Wang, J

2017-04-01

Estrogen is reported to be involved in thrombopoiesis and the disruption of its signaling may cause myeloproliferative disease, yet the underlying mechanisms remain largely unknown. GATA-binding factor 1 (GATA1) is a key regulator of megakaryocyte (MK) differentiation and its deficiency will lead to megakaryoblastic leukemia. Here we show that estrogen can dose-dependently promote MK polyploidization and maturation via activation of estrogen receptor beta (ERβ), accompanied by a significant upregulation of GATA1. Chromatin immunoprecipitation and a dual luciferase assay demonstrate that ERβ can directly bind the promoter region of GATA1 and activate its transcription. Steroid receptor coactivator 3 (SRC3) is involved in ERβ-mediated GATA1 transcription. The deficiency of ERβ or SRC3, similar to the inhibition of GATA1, leads to the impediment of estrogen-induced MK polyploidization and platelet production. Further investigations reveal that signal transducer and activator of transcription 1 signaling pathway downstream of GATA1 has a crucial role in estrogen-induced MK polyploidization, and ERβ-mediated GATA1 upregulation subsequently enhances nuclear factor erythroid-derived 2 expression, thereby promoting proplatelet formation and platelet release. Our study provides a deep insight into the molecular mechanisms of estrogen signaling in regulating thrombopoiesis and the pathogenesis of ER deficiency-related leukemia.

Analysis of emerging contaminants in water and solid samples using high resolution mass spectrometry with a Q Exactive orbital ion trap and estrogenic activity with YES-assay.

PubMed

Comtois-Marotte, Simon; Chappuis, Thomas; Vo Duy, Sung; Gilbert, Nicolas; Lajeunesse, André; Taktek, Salma; Desrosiers, Mélanie; Veilleux, Éloïse; Sauvé, Sébastien

2017-01-01

Trace emerging contaminants (ECs) occur in both waste and surface waters that are rich in particulates that have been found to sorb several organic contaminants. An analytical method based on off-line solid-phase extraction (SPE) followed by liquid chromatography-mass spectrometry (LC-MS) analysis was developed for the detection and quantification of 31 ECs from surface water, wastewater, suspended particulate matter (SPM) as well as sediments. Lyophilized sediments and air-dried SPM were subjected to ultrasonic extraction. Water samples and extracts were then concentrated and cleaned-up by off-line SPE. Quantification was realized using a Q Exactive mass spectrometer in both full scan (FS) and MS 2 modes. These two modes were optimized and compared to determine which one was the most suitable for each matrix studied. Yeast estrogen screen assay (YES-assay) adapted from the direct measurement of estrogenic activity without sample extraction was tested on filtered wastewater samples. An endocrine disrupting effect was detected in all effluent samples analyzed with estradiol equivalent concentrations ranging from 4.4 to 720 ng eq E2 L -1 for the WWTP-1 and 6.5-42 ng eq E2 L -1 for the WWTP-2. The analytical methods were also applied on six samples of surface water, the corresponding SPM, the sediments and thirty-nine effluent samples from two wastewater treatment plants (WWTPs) sampled over a period of five months (February to June 2014). Copyright © 2016 Elsevier Ltd. All rights reserved.

Estrogens and Cognition: Friends or Foes?

PubMed Central

Korol, Donna L.; Pisani, Samantha L.

2015-01-01

Estrogens are becoming well known for their robust enhancement on cognition particularly for learning and memory that relies upon functioning of the hippocampus and related neural systems. What is also emerging is that estrogen modulation of cognition is not uniform, at times enhancing yet at other times impairing learning. This review explores the bidirectional effects of estrogens on learning from a multiple memory systems view, focusing on the hippocampus and striatum, whereby modulation by estrogens sorts according to task attributes and neural systems engaged during cognition. We highlight our findings that show the ability to solve hippocampus-sensitive tasks typically improves under relatively high estrogen status while the ability to solve striatum-sensitive tasks degrades with estrogen exposures. Though constrained by dose and timing of exposure, these opposing enhancements and impairments of cognition can be observed following treatments with different estrogenic compounds including the hormone estradiol, the isoflavone genistein found in soybeans, and agonists that are selective for specific estrogen receptors, suggesting that activation of a single receptor type is sufficient to produce the observed shifts in learning strategies. Using this multi-dimensional framework will allow us to extend our thinking of the relationship between estrogens and cognition to other brain regions and cognitive functions. PMID:26149525

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP.

PubMed

Järvinen, Erkka; Deng, Feng; Kidron, Heidi; Finel, Moshe

2018-04-01

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E 1 -G), estradiol-3- and 17-glucuronides (E 2 -3G and E 2 -17G), as well as estriol-3- and 16-glucuronides (E 3 -3G and E 3 -16G) are found in human plasma and urine. Unlike E 2 -17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E 1 -G, E 2 -3G, E 3 -3G, E 3 -16G and estrone-3-sulfate (E 1 -S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E 1 -S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E 1 -G and E 2 -3G, were still transported by BCRP at 10-fold higher rates than E 1 -S. BCRP also transported E 3 -16G at higher rates than the studied MRPs, while it transported E 3 -3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E 1 -G, E 2 -3G, E 3 -3G and E 3 -16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high K m values, between 180 and 790 μM. MRP3 transported all the tested glucuronides at rather similar rates, at K m values below 20 μM, but lower V max values than other transporters. In the case of E 3 -3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E 3 -16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans. Copyright © 2017 Elsevier Ltd. All

Genome-wide bisulfite sensitivity profiling of yeast suggests bisulfite inhibits transcription.

PubMed

Segovia, Romulo; Mathew, Veena; Tam, Annie S; Stirling, Peter C

2017-09-01

Bisulfite, in the form of sodium bisulfite or metabisulfite, is used commercially as a food preservative. Bisulfite is used in the laboratory as a single-stranded DNA mutagen in epigenomic analyses of DNA methylation. Recently it has also been used on whole yeast cells to induce mutations in exposed single-stranded regions in vivo. To understand the effects of bisulfite on live cells we conducted a genome-wide screen for bisulfite sensitive mutants in yeast. Screening the deletion mutant array, and collections of essential gene mutants we define a genetic network of bisulfite sensitive mutants. Validation of screen hits revealed hyper-sensitivity of transcription and RNA processing mutants, rather than DNA repair pathways and follow-up analyses support a role in perturbation of RNA transactions. We propose a model in which bisulfite-modified nucleotides may interfere with transcription or RNA metabolism when used in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterizing the Estrogenic Potential of 1060 Environmental Chemicals by Assessing Growth Kinetics in T47D Cells

EPA Science Inventory

In order to detect environmental chemicals that pose a risk of endocrine disruption, high-throughput screening (HTS) tests capable of testing thousands of environmental chemicals are needed. Alteration of estrogen signaling has been implicated in a variety of adverse health effec...

Estrogen via estrogen receptor beta partially inhibits mandibular condylar cartilage growth.

PubMed

Chen, J; Kamiya, Y; Polur, I; Xu, M; Choi, T; Kalajzic, Z; Drissi, H; Wadhwa, S

2014-11-01

Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in wild type (WT) and ER beta Knockout (KO) mice. 21-day-old female WT (n = 37) and ER beta KO mice (n = 36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double ethynyl-2'-deoxyuridine/bromodeoxyuridine (EdU/BrdU) labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed. In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression. Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression. Copyright © 2014 China University of Geosciences (Beijing) and Peking University. Published by Elsevier Ltd. All rights reserved.

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

PubMed

Benko, Zsigmond; Elder, Robert T; Li, Ge; Liang, Dong; Zhao, Richard Y

2016-01-01

HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

Synthetic Genetic Arrays: Automation of Yeast Genetics.

PubMed

Kuzmin, Elena; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2016-04-01

Genome-sequencing efforts have led to great strides in the annotation of protein-coding genes and other genomic elements. The current challenge is to understand the functional role of each gene and how genes work together to modulate cellular processes. Genetic interactions define phenotypic relationships between genes and reveal the functional organization of a cell. Synthetic genetic array (SGA) methodology automates yeast genetics and enables large-scale and systematic mapping of genetic interaction networks in the budding yeast,Saccharomyces cerevisiae SGA facilitates construction of an output array of double mutants from an input array of single mutants through a series of replica pinning steps. Subsequent analysis of genetic interactions from SGA-derived mutants relies on accurate quantification of colony size, which serves as a proxy for fitness. Since its development, SGA has given rise to a variety of other experimental approaches for functional profiling of the yeast genome and has been applied in a multitude of other contexts, such as genome-wide screens for synthetic dosage lethality and integration with high-content screening for systematic assessment of morphology defects. SGA-like strategies can also be implemented similarly in a number of other cell types and organisms, includingSchizosaccharomyces pombe,Escherichia coli, Caenorhabditis elegans, and human cancer cell lines. The genetic networks emerging from these studies not only generate functional wiring diagrams but may also play a key role in our understanding of the complex relationship between genotype and phenotype. © 2016 Cold Spring Harbor Laboratory Press.

Chemical Probes of Rapid Estrogen Signaling in Breast Cancer Treatment and Chemoprevention

DTIC Science & Technology

2007-04-01

The analogs will also be conju- gated to cell-impermeable polyacrylate polymers that should allow for selective targeting of membrane-initiated...the GW7604 analogs. Briefly, serial dilutions of the different compounds were prepared in ES2 screening buffer (100 mM potassium phosphate, pH7.4, 100...AD_________________ Award Number: W81XWH-04-1-0447 TITLE: CHEMICAL PROBES OF RAPID ESTROGEN

Yeast Two-Hybrid and One-Hybrid Screenings Identify Regulators of hsp70 Gene Expression.

PubMed

Saito, Youhei; Nakagawa, Takanobu; Kakihana, Ayana; Nakamura, Yoshia; Nabika, Tomomi; Kasai, Michihiro; Takamori, Mai; Yamagishi, Nobuyuki; Kuga, Takahisa; Hatayama, Takumi; Nakayama, Yuji

2016-09-01

The mammalian stress protein Hsp105β, which is specifically expressed during mild heat shock and localizes to the nucleus, induces the major stress protein Hsp70. In the present study, we performed yeast two-hybrid and one-hybrid screenings to identify the regulators of Hsp105β-mediated hsp70 gene expression. Six and two proteins were detected as Hsp105β- and hsp70 promoter-binding proteins, respectively. A luciferase reporter gene assay revealed that hsp70 promoter activation is enhanced by the transcriptional co-activator AF9 and splicing mediator SNRPE, but suppressed by the coiled-coil domain-containing protein CCDC127. Of these proteins, the knockdown of SNRPE suppressed the expression of Hsp70 irrespective of the presence of Hsp105β, indicating that SNRPE essentially functions as a transcriptional activator of hsp70 gene expression. The overexpression of HSP70 in tumor cells has been associated with cell survival and drug resistance. We here identified novel regulators of Hsp70 expression in stress signaling and also provided important insights into Hsp70-targeted anti-cancer therapy. J. Cell. Biochem. 117: 2109-2117, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Characterizing the Growth Kinetics in Estrogen Responsive T47D Cells After Exposure to 2000 Environmental Chemicals

EPA Science Inventory

There is a need to develop high-throughput screening (HTS) tests capable of testing thousands of environmental chemicals for endocrine disrupting potential. The estrogen signaling pathway is a known xenobiotic target that has been implicated in a variety of adverse health effects...

Novel drugs that target the estrogen-related receptor alpha: their therapeutic potential in breast cancer

PubMed Central

May, Felicity EB

2014-01-01

The incidence of breast cancer continues to rise: 1.7 million women were diagnosed with and 521,000 women died from breast cancer in 2012. This review considers first current treatment options: surgery; radiotherapy; and systemic endocrine, anti-biological, and cytotoxic therapies. Clinical management includes prevention, early detection by screening, treatment with curative intent, management of chronic disease, and palliative control of advanced breast cancer. Next, the potential of novel drugs that target DNA repair, growth factor dependence, intracellular and intercellular signal transduction, and cell cycle are considered. Estrogen-related receptor alpha has attracted attention as a therapeutic target in triple-negative breast cancers with de novo resistance to, and in breast cancers with acquired resistance to, endocrine therapies such as antiestrogens and aromatase inhibitors. Estrogen-related receptor alpha is an orphan receptor and transcription factor. Its activity is regulated by coregulator proteins and posttranslational modification. It is an energy sensor that controls adaptation to energy demand and may facilitate glycolytic metabolism and mitochondrial oxidative respiration in breast cancer cells. Estrogen-related receptor alpha increases breast cancer cell migration, proliferation, and tumor development. It is expressed at high levels in estrogen receptor-negative tumors, and is proposed to activate estrogen-responsive genes in endocrine-resistant tumors. The structures and functions of the ligand-binding domains of estrogen receptor alpha and estrogen-related receptor alpha, their ability to bind estrogens, phytoestrogens, and synthetic ligands, and the effects of ligand agonists, antagonists, and inverse agonists on biological activity, are evaluated. Synthetic ligands of estrogen-related receptor alpha have activity in preclinical models of metabolic disorders, diabetes, osteoporosis, and oncology. The clinical settings in which these novel

Estrogen Metabolism and Breast Cancer

PubMed Central

Samavat, Hamed; Kurzer, Mindy S

2015-01-01

There is currently accumulating evidence that endogenous estrogens play a critical role in the development of breast cancer. Estrogens and their metabolites have been studied in both pre- and postmenopausal women with more consistent results shown in the latter population, in part because of large hormonal variations during the menstrual cycle and far fewer studies having been performed in premenopausal women. In this review we describe in detail estrogen metabolism and associated genetic variations, and provide a critical review of the current literature regarding the role of estrogens and their metabolites in breast cancer risk. PMID:24784887

Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.

2011-02-01

Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17{beta} to the four rainbow trout ER isoformsmore » with that of three known environmental estrogens 17{alpha}-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ER{alpha} subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17{beta}, bisphenol A binds less strongly to all four receptors, 17{alpha}-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the {alpha} subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.« less

[Equine estrogens vs. esterified estrogens in the climacteric and menopause. The controversy arrives in Mexico].

PubMed

Velasco-Murillo, V

2001-01-01

It exists controversies about if the effects and benefits of the esterified estrogens could be similar to those informed for equines, because its chemical composition and bioavailability are different. Esterified estrogens has not delta 8,9 dehydroestrone, and its absorption and level of maximum plasmatic concentrations are reached very fast. In United States of America and another countries, esterified estrogens has been marketed and using for treatment of climacteric syndrome and prevention of postmenopausal osteoporosis, based on the pharmacopoiea of that country, but the Food and Drug administration (FDA) has not yet authorized up today, a generic version of conjugated estrogens. In Instituto Mexicano del Seguro Social (IMSS) and another institutions of health sector in Mexico, starting in year 2000, it has been used esterified estrogens for medical treatment of climacteric and menopausal conditions. For this reason, in this paper we revised the most recent information about pharmacology, chemical composition, clinical use and costs of the conjugated estrogens with the purpose to guide the decisions to purchase this kind of drugs in Mexican heath institutions.

Gene-nutrient interaction markedly influences yeast chronological lifespan.

PubMed

Smith, Daniel L; Maharrey, Crystal H; Carey, Christopher R; White, Richard A; Hartman, John L

2016-12-15

Research into the genetic mechanisms of aging has expanded rapidly over the past two decades. This has in part been the result of the use of model organisms (particularly yeast, worms and flies) and high-throughput technologies, combined with a growing interest in aging research. Despite this progress, widespread consensus regarding the pathways that are fundamental to the modulation of cellular aging and lifespan for all organisms has been limited due to discrepancies between different studies. We have compared results from published genome-wide, chronological lifespan (CLS) screens of individual gene deletion strains in Saccharomyces cerevisiae in order to identify gene deletion strains with consistent influences on longevity as possible indicators of fundamental aging processes from this single-celled, eukaryotic model organism. Three previous reports have described genetic modifiers of chronological aging in the budding yeast (S. cerevisiae) using the yeast gene deletion strain collection. We performed a comparison among the data sets using correlation and decile distribution analysis to describe concordance between screens and identify strains that consistently increased or decreased CLS. We used gene enrichment analysis in an effort to understand the biology underlying genes identified in multiple studies. We attempted to replicate the different experimental conditions employed by the screens to identify potential sources of variability in CLS worth further investigating. Among 3209 strains present in all three screens, nine deletions strains were in common in the longest-lived decile (2.80%) and thirteen were in common in the shortest-lived decile (4.05%) of all three screens. Similarly, pairwise overlap between screens was low. When the same comparison was extended to three deciles to include more mutants studied in common between the three screens, enrichment of cellular processes based on gene ontology analysis in the long-lived strains remained very

Gene-Nutrient Interaction Markedly Influences Yeast Chronological Lifespan

PubMed Central

Smith, Daniel L.; Maharrey, Crystal H.; Carey, Christopher R.; White, Richard A.; Hartman, John L.

2016-01-01

Purpose Research into the genetic mechanisms of aging has expanded rapidly over the past two decades. This has in part been the result of the use of model organisms (particularly yeast, worms and flies) and high-throughput technologies, combined with a growing interest in aging research. Despite this progress, widespread consensus regarding the pathways that are fundamental to the modulation of cellular aging and lifespan for all organisms has been limited due to discrepancies between different studies. We have compared results from published genome-wide, chronological lifespan (CLS) screens of individual gene deletion strains in S. cerevisiae in order to identify gene deletion strains with consistent influences on longevity as possible indicators of fundamental aging processes from this single-celled, eukaryotic model organism. Methods Three previous reports have described genetic modifiers of chronological aging in the budding yeast (S. cerevisiae) using the yeast gene deletion strain collection. We performed a comparison among the data sets using correlation and decile distribution analysis to describe concordance between screens and identify strains that consistently increased or decreased CLS. We used gene enrichment analysis in an effort to understand the biology underlying genes identified in multiple studies. We attempted to replicate the different experimental conditions employed by the screens to identify potential sources of variability in CLS worth further investigating. Results Among 3209 strains present in all three screens, nine (2.80%) deletions strains were in common in the longest-lived decile and thirteen (4.05%) were in common in the shortest-lived decile for all three screens. Similarly, pairwise overlap between screens was low. When the same comparison was extended to three deciles to include more mutants studied in common between the three screens, enrichment of cellular processes based on gene ontology analysis in the long-lived strains

Sex Hormones and Cardiometabolic Health: Role of Estrogen and Estrogen Receptors.

PubMed

Clegg, Deborah; Hevener, Andrea L; Moreau, Kerrie L; Morselli, Eugenia; Criollo, Alfredo; Van Pelt, Rachael E; Vieira-Potter, Victoria J

2017-05-01

With increased life expectancy, women will spend over three decades of life postmenopause. The menopausal transition increases susceptibility to metabolic diseases such as obesity, diabetes, cardiovascular disease, and cancer. Thus, it is more important than ever to develop effective hormonal treatment strategies to protect aging women. Understanding the role of estrogens, and their biological actions mediated by estrogen receptors (ERs), in the regulation of cardiometabolic health is of paramount importance to discover novel targeted therapeutics. In this brief review, we provide a detailed overview of the literature, from basic science findings to human clinical trial evidence, supporting a protective role of estrogens and their receptors, specifically ERα, in maintenance of cardiometabolic health. In so doing, we provide a concise mechanistic discussion of some of the major tissue-specific roles of estrogens signaling through ERα. Taken together, evidence suggests that targeted, perhaps receptor-specific, hormonal therapies can and should be used to optimize the health of women as they transition through menopause, while reducing the undesired complications that have limited the efficacy and use of traditional hormone replacement interventions. Copyright © 2017 Endocrine Society.

Importance of Extranuclear Estrogen Receptor-α and Membrane G Protein–Coupled Estrogen Receptor in Pancreatic Islet Survival

PubMed Central

Liu, Suhuan; Le May, Cedric; Wong, Winifred P.S.; Ward, Robert D.; Clegg, Deborah J.; Marcelli, Marco; Korach, Kenneth S.; Mauvais-Jarvis, Franck

2009-01-01

OBJECTIVE We showed that 17β-estradiol (E2) favors pancreatic β-cell survival via the estrogen receptor-α (ERα) in mice. E2 activates nuclear estrogen receptors via an estrogen response element (ERE). E2 also activates nongenomic signals via an extranuclear form of ERα and the G protein–coupled estrogen receptor (GPER). We studied the contribution of estrogen receptors to islet survival. RESEARCH DESIGN AND METHODS We used mice and islets deficient in estrogen receptor-α (αERKO−/−), estrogen receptor-β (βERKO−/−), estrogen receptor-α and estrogen receptor-β (αβERKO−/−), and GPER (GPERKO−/−); a mouse lacking ERα binding to the ERE; and human islets. These mice and islets were studied in combination with receptor-specific pharmacological probes. RESULTS We show that ERα protection of islet survival is ERE independent and that E2 favors islet survival through extranuclear and membrane estrogen receptor signaling. We show that ERβ plays a minor cytoprotective role compared to ERα. Accordingly, βERKO−/− mice are mildly predisposed to streptozotocin-induced islet apoptosis. However, combined elimination of ERα and ERβ in mice does not synergize to provoke islet apoptosis. In αβERKO−/− mice and their islets, E2 partially prevents apoptosis suggesting that an alternative pathway compensates for ERα/ERβ deficiency. We find that E2 protection of islet survival is reproduced by a membrane-impermeant E2 formulation and a selective GPER agonist. Accordingly, GPERKO−/− mice are susceptible to streptozotocin-induced insulin deficiency. CONCLUSIONS E2 protects β-cell survival through ERα and ERβ via ERE-independent, extra-nuclear mechanisms, as well as GPER-dependent mechanisms. The present study adds a novel dimension to estrogen biology in β-cells and identifies GPER as a target to protect islet survival. PMID:19587358

Importance of extranuclear estrogen receptor-alpha and membrane G protein-coupled estrogen receptor in pancreatic islet survival.

PubMed

Liu, Suhuan; Le May, Cedric; Wong, Winifred P S; Ward, Robert D; Clegg, Deborah J; Marcelli, Marco; Korach, Kenneth S; Mauvais-Jarvis, Franck

2009-10-01

We showed that 17beta-estradiol (E(2)) favors pancreatic beta-cell survival via the estrogen receptor-alpha (ERalpha) in mice. E(2) activates nuclear estrogen receptors via an estrogen response element (ERE). E(2) also activates nongenomic signals via an extranuclear form of ERalpha and the G protein-coupled estrogen receptor (GPER). We studied the contribution of estrogen receptors to islet survival. We used mice and islets deficient in estrogen receptor-alpha (alphaERKO(-/-)), estrogen receptor-beta (betaERKO(-/-)), estrogen receptor-alpha and estrogen receptor-beta (alphabetaERKO(-/-)), and GPER (GPERKO(-/-)); a mouse lacking ERalpha binding to the ERE; and human islets. These mice and islets were studied in combination with receptor-specific pharmacological probes. We show that ERalpha protection of islet survival is ERE independent and that E(2) favors islet survival through extranuclear and membrane estrogen receptor signaling. We show that ERbeta plays a minor cytoprotective role compared to ERalpha. Accordingly, betaERKO(-/-) mice are mildly predisposed to streptozotocin-induced islet apoptosis. However, combined elimination of ERalpha and ERbeta in mice does not synergize to provoke islet apoptosis. In alphabetaERKO(-/-) mice and their islets, E(2) partially prevents apoptosis suggesting that an alternative pathway compensates for ERalpha/ERbeta deficiency. We find that E(2) protection of islet survival is reproduced by a membrane-impermeant E(2) formulation and a selective GPER agonist. Accordingly, GPERKO(-/-) mice are susceptible to streptozotocin-induced insulin deficiency. E(2) protects beta-cell survival through ERalpha and ERbeta via ERE-independent, extra-nuclear mechanisms, as well as GPER-dependent mechanisms. The present study adds a novel dimension to estrogen biology in beta-cells and identifies GPER as a target to protect islet survival.

Estrogenic effect of leachates and soil extracts from lysimeters spiked with sewage sludge and reference endocrine disrupters.

PubMed

Dizer, Halim; Fischer, Birgit; Sepulveda, Isabel; Loffredo, Elisabetta; Senesi, Nicola; Santana, Fernando; Hansen, Peter-D

2002-01-01

Several experiments were conducted to evaluate the behavior and performance of some potential endocrine disrupters (ECDs). Two in vitro screening assays, one based on MCF7-cell proliferation (E-screen test) and the other on estrogenic receptor activity [enzyme-linked receptor assay (ELRA)], were used for the tests, which were done in lysimeters 80 cm in diameter with depth of 30 cm (shallow) or 90 cm (deep). A sandy soil was used to fill in all lysimeters, which were spiked on the surface with either: (a) a sewage sludge (SS) at a dose equivalent to 20 tons ha-1; (b) a mixture of reference ECDs, comprising 17 alpha- and 17 beta-estradiol (E2), nonylphenol, octylphenol, and bisphenol A at doses 100 times higher than the maximum concentrations respectively found in the applied SS; or (c) a mixture of ECDs and SS. After percolation of the lysimeters with rain and/or artificial water, five leachates were sampled from each lysimeter during a period of 210 days. Immediately after the lysimeter percolation experiments, four and six soil fractions were dissected from, respectively, the 30-cm and 90-cm lysimeters and extracted by water. Both the leachate and soil extract samples were analyzed for their estrogenicity using the assays indicated above. The E-screen assay was highly sensitive only for some leachate and extract samples but gave no response for most leachates and soil extracts. The results of the ELRA assay suggests a significantly higher estrogenicity of leachate samples from shallow lysimeters compared with that of leachates from deep lysimeters. In contrast, the estrogenic effect measured for soil extracts of shallow lysimeters was lower than that measured for soil extracts of deep lysimeters. The results of the E-screen assay suggests the occurrence of a fast mobilization of applied ECDs and a moderate retardation effect of native ECDs contained in applied SS in the sandy soil used in the lysimeters. In lysimeters spiked with a mixture of SS and ECDs, the

Identification, contribution, and estrogenic activity of potential EDCs in a river receiving concentrated livestock effluent in Southern Taiwan.

PubMed

Liu, Yung-Yu; Lin, Yi-Siou; Yen, Chia-Hung; Miaw, Chang-Ling; Chen, Ting-Chien; Wu, Meng-Chun; Hsieh, Chi-Ying

2018-04-27

We assessed 22 selected endocrine-disrupting compounds (EDCs) and other emerging, potentially endocrine-active compounds with estrogenic activity from the waters of the Wuluo River, southern Taiwan. This watershed receives high amounts of livestock and untreated household wastewaters. The river is surrounded by concentrated animal feedlot operations (CAFOs). River water samples were analyzed for selected compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS), T47D-KBluc reporter gene assay, and E-screen cell proliferation in vitro bioassay. Total concentrations of ∑alkylphenolic compounds (bisphenol A, 4-nonylphenol, t-nonylphenol, octylphenol, nonylphenol mono-ethoxylate, nonylphenol di-ethoxylate) were much higher than ∑estrogens (estrone, 17 β-estradiol, estriol, 17ß-ethynylestradiol, diethylstilbestrol), ∑preservatives (methyl paraben, ethyl paraben, propyl paraben, butyl paraben), ∑UV-filters (benzophenone, methyl benzylidene camphor, benzophenone-3), ∑antimicrobials (triclocarben, triclosan, chloroxylenol), and an insect repellent (diethyltoluamide) over four seasonal sampling periods. The highest concentration was found for bisphenol A with a mean of 302 ng/L. However, its contribution to estrogenic activity was not significant due to its relatively low estrogenic potency. Lower detection rates were found for BP, EE2, TCS, and PCMX, while DES and EP were not detected. E1 and E2 levels in raw water samples were 50% higher than the predicted no-effect concentrations (PNEC) for aquatic organisms of 6 and 2 ng/L, respectively. The potency of estrogenic activity ranged from 11.7 to 190.1 ng/L E2 T47D-Kbluc and 6.63 to 84.5 ng/L E2 E-Screen for extracted samples. Importantly, estrone contributed 50% of the overall activity in 60% and 44% of the samples based on T47D-KBluc and MCF-7 bioassays, followed by 17 ß-estradiol, highlighting the importance of total steroid estrogen loading. This study demonstrates that the estrogenic

(GTG)5 MSP-PCR fingerprinting as a technique for discrimination of wine associated yeasts?

PubMed

Ramírez-Castrillón, Mauricio; Mendes, Sandra Denise Camargo; Inostroza-Ponta, Mario; Valente, Patricia

2014-01-01

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.

(GTG)5 MSP-PCR Fingerprinting as a Technique for Discrimination of Wine Associated Yeasts?

PubMed Central

Inostroza-Ponta, Mario; Valente, Patricia

2014-01-01

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure. PMID:25171185

Chemical signaling and insect attraction is a conserved trait in yeasts.

PubMed

Becher, Paul G; Hagman, Arne; Verschut, Vasiliki; Chakraborty, Amrita; Rozpędowska, Elżbieta; Lebreton, Sébastien; Bengtsson, Marie; Flick, Gerhard; Witzgall, Peter; Piškur, Jure

2018-03-01

Yeast volatiles attract insects, which apparently is of mutual benefit, for both yeasts and insects. However, it is unknown whether biosynthesis of metabolites that attract insects is a basic and general trait, or if it is specific for yeasts that live in close association with insects. Our goal was to study chemical insect attractants produced by yeasts that span more than 250 million years of evolutionary history and vastly differ in their metabolism and lifestyle. We bioassayed attraction of the vinegar fly Drosophila melanogaster to odors of phylogenetically and ecologically distinct yeasts grown under controlled conditions. Baker's yeast Saccharomyces cerevisiae , the insect-associated species Candida californica , Pichia kluyveri and Metschnikowia andauensis , wine yeast Dekkera bruxellensis , milk yeast Kluyveromyces lactis , the vertebrate pathogens Candida albicans and Candida glabrata , and oleophilic Yarrowia lipolytica were screened for fly attraction in a wind tunnel. Yeast headspace was chemically analyzed, and co-occurrence of insect attractants in yeasts and flowering plants was investigated through a database search. In yeasts with known genomes, we investigated the occurrence of genes involved in the synthesis of key aroma compounds. Flies were attracted to all nine yeasts studied. The behavioral response to baker's yeast was independent of its growth stage. In addition to Drosophila , we tested the basal hexapod Folsomia candida (Collembola) in a Y-tube assay to the most ancient yeast, Y. lipolytica, which proved that early yeast signals also function on clades older than neopteran insects. Behavioral and chemical data and a search for selected genes of volatile metabolites underline that biosynthesis of chemical signals is found throughout the yeast clade and has been conserved during the evolution of yeast lifestyles. Literature and database reviews corroborate that yeast signals mediate mutualistic interactions between insects and yeasts

Targeted Radiotherapy of Estrogen Receptor Positive Tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raghavan Rajagopalan

The overall objectives of the proposal were to develop estrogen receptor (ER) binding small molecule radiopharmaceuticals for targeted radiotherapy of ER positive (ER+) tumors. In particular, this proposal focused on embedding a {sup 186,188}Re or a {sup 32}P radionuclide into an estrogen steroidal framework by isosteric substitution such that the resulting structure is topologically similar to the estrogen (estrogen mimic). The estrogen mimic molecules expected to bind to the ER and exhibit biodistribution akin to that of native estrogen due to structural mimicry. It is anticipated that the {sup 186,188}Re- or a {sup 32}P-containing estrogen mimics will be useful formore » targeted molecular radiotherapy of ER+ tumors. It is well established that the in vivo target tissue uptake of estrogen like steroidal molecules is related to the binding of the steroids to sex hormone binding globulin (SHBG). SHBG is important in the uptake of estrogens and testosterone in target tissues by SHBG receptors on the cell surface. However, hitherto the design of estrogen like small molecule radiopharmaceuticals was focused on optimizing ER binding characteristics without emphasis on SHBG binding properties. Consequently, even the molecules with good ER affinity in vitro, performed poorly in biodistribution studies. Based on molecular modeling studies the proposal focused on developing estrogen mimics 1-3 which were topologically similar to native estrogens, and form hydrogen bonds in ER and SHBG in the same manner as those of native estrogens. To this end the technical objectives of the proposal focused on synthesizing the rhenium-estrone and estradiol mimics 1 and 2 respectively, and phosphorous estradiol mimic 3 and to assess their stability and in vitro binding characteristics to ER and SHBG.« less

Estrogen Signaling in Metabolic Inflammation

PubMed Central

Monteiro, Rosário; Teixeira, Diana; Calhau, Conceição

2014-01-01

There is extensive evidence supporting the interference of inflammatory activation with metabolism. Obesity, mainly visceral obesity, is associated with a low-grade inflammatory state, triggered by metabolic surplus where specialized metabolic cells such as adipocytes activate cellular stress initiating and sustaining the inflammatory program. The increasing prevalence of obesity, resulting in increased cardiometabolic risk and precipitating illness such as cardiovascular disease, type 2 diabetes, fatty liver, cirrhosis, and certain types of cancer, constitutes a good example of this association. The metabolic actions of estrogens have been studied extensively and there is also accumulating evidence that estrogens influence immune processes. However, the connection between these two fields of estrogen actions has been underacknowledged since little attention has been drawn towards the possible action of estrogens on the modulation of metabolism through their anti-inflammatory properties. In the present paper, we summarize knowledge on the modification inflammatory processes by estrogens with impact on metabolism and highlight major research questions on the field. Understanding the regulation of metabolic inflammation by estrogens may provide the basis for the development of therapeutic strategies to the management of metabolic dysfunctions. PMID:25400333

Modulation of pain by estrogens.

PubMed

Craft, Rebecca M

2007-11-01

It has become increasingly apparent that women suffer a disproportionate amount of pain during their lifetime compared to men. Over the past 15 years, a growing number of studies have suggested a variety of causes for this sex difference, from cellular to psychosocial levels of analysis. From a biological perspective, sexual differentiation of pain appears to occur similarly to sexual differentiation of other phenomena: it results in large part from organizational and activational effects of gonadal steroid hormones. The focus of this review is the activational effects of a single group of ovarian hormones, the estrogens, on pain in humans and animals. The effects of estrogens (estradiol being the most commonly examined) on experimentally induced acute pain vs. clinical pain are summarized. For clinical pain, the review is limited to a few syndromes for which there is considerable evidence for estrogenic involvement: migraine, temporomandibular disorder (TMD) and arthritis. Because estrogens can modulate the function of the nervous, immune, skeletal, and cardiovascular systems, estrogenic modulation of pain is an exceedingly complex, multi-faceted phenomenon, with estrogens producing both pro- and antinociceptive effects that depend on the extent to which each of these systems of the body is involved in a particular type of pain. Forging a more complete understanding of the myriad ways that estrogens can ameliorate vs. facilitate pain will enable us to better prevent and treat pain in both women and men.

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1.

PubMed

Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Altelaar, A F Maarten; van Leeuwen, Fred

2016-12-06

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features.

Characterization of butter spoiling yeasts and their inhibition by some spices.

PubMed

Sagdic, Osman; Ozturk, Ismet; Bayram, Okan; Kesmen, Zulal; Yilmaz, Mustafa Tahsin

2010-01-01

This study was designed to identify the yeasts in packaged and unpackaged butters and screen antiyeast activity of spices, including marjoram (Origanum majorana L.), summer savory (Satureja hortensis L.), and black cumin (Nigella sativa L.) against the most dominant yeast species in the packaged and unpackaged butters. Mean total yeast populations were 5.40 log CFU/g in unpackaged butter samples and 2.22 log CFU/g in packaged butter samples, indicating better hygienic quality of packaged samples. Forty-nine yeast species were isolated and identified from butter samples with the most prevalent isolates belonging to genera Candida-C. kefyr, C. zeylanoides, and C. lambica-and with moderate number of isolates belonging to genera Cryptococcus, Rhodotorula, Saccharomyces, and Zygosaccharomyces. Black cumin exhibited the highest antiyeast activity against C. zeylanoides and C. lambica species, even inhibited these species, while summer savory inhibited C. kefyr. The results of this study revealed clear antimicrobial potential of black cumin against the yeast species isolated from butters. Marjoram, summer savory, and black cumin could be used as natural antimicrobial agents against spoilage yeasts in food preservation, especially in butter. © 2010 Institute of Food Technologists®

Impact of smoking on estrogenic efficacy.

PubMed

Ruan, X; Mueck, A O

2015-02-01

Depending on the type, duration and intensity of cigarette smoking, the efficacy of endogenous and exogenous estrogen can be reduced or completely cancelled. Not only does smoking diminish the beneficial effects of estrogen on hot flushes and urogenital symptoms and its positive effects on lipid metabolism, but smoking also can reduce estrogen's ability to prevent osteoporosis and perhaps also cardiovascular diseases. This is mainly caused by dose-dependent elevated hepatic clearance, partially in conjunction with lower estrogen levels, and has been demonstrated so far only with oral estrogen applications. Compensation for the failure of therapeutic action should not be made by increasing the dose in smokers since this might result in the production of potentially mutagenic estrogen metabolites associated with a higher risk of breast cancer. Since the favorable effects of estrogens seem to be not lost in smokers when estrogens are applied transdermally, this route should be preferred in smokers. The most important conclusion from the data presented is that the effects of smoking are very complex and dependent on a multiplicity of factors, so that different types of clinically relevant negative effects must be expected. Women who continue to smoke despite all warnings should be informed that smoking, in addition to all its other negative effects, can also jeopardize the success of hormone replacement therapy.

Critical review and evaluation of the uterotrophic bioassay for the identification of possible estrogen agonists and antagonists: in support of the validation of the OECD uterotrophic protocols for the laboratory rodent. Organisation for Economic Co-operation and Development.

PubMed

Owens, J William; Ashby, John

2002-01-01

A current issue for regulatory agencies is endocrine-related modes of action such as those mediated by the estrogen, androgen, and thyroid nuclear receptors. At the national and international levels, the consensus recommendation for the assessment of such modes of action is a tiered series of in vitro and in vivo protocols. The tiered framework begins with screens for structural alerts and then moves to rapid, mechanistic in vitro screening assays, and then to in vivo screening bioassays. The objective of these screens is to identify substances that may warrant testing for endocrine-mediated adverse effects. The final framework tier as needed is to test these substances in long-term bioassays for adverse endocrine-mediated reproductive and/or developmental effects. The subject of this review, the rodent uterotrophic bioassay, is intended to be a rapid in vivo screening bioassay for possible estrogen agonists and based on the response of the estrogen-sensitive uterus. The central metric of bioassay is a statistically significant increase in the weight of the uterus after 3 consecutive days of test substance administration. The extensive background literature is summarized in this review on the mode of action underlying the bioassay and the uterine response to estrogens. The review includes the bioassay's history of development and how its employment has changed and evolved over time. The review describes two major uterotrophic bioassay versions, the intact, immature female and the mature, ovariectomized female, and the protocol factors likely to influence relevance, reproducibility, and reliability of bioassay. The emphasis of the review is the ability of the uterotrophic bioassay to identify the substances of current interest: weak estrogen agonists with binding affinities relative to the natural 17beta-estradiol in the log 0 to log -3 range. Using selected model substances having RBAs in this target range, the bioassay's performance in a hierarchical, tiered

Estrogen promotes cutaneous wound healing via estrogen receptor β independent of its antiinflammatory activities

PubMed Central

Campbell, Laura; Emmerson, Elaine; Davies, Faith; Gilliver, Stephen C.; Krust, Andre; Chambon, Pierre; Ashcroft, Gillian S.

2010-01-01

Post-menopausal women have an increased risk of developing a number of degenerative pathological conditions, linked by the common theme of excessive inflammation. Systemic estrogen replacement (in the form of hormone replacement therapy) is able to accelerate healing of acute cutaneous wounds in elderly females, linked to its potent antiinflammatory activity. However, in contrast to many other age-associated pathologies, the detailed mechanisms through which estrogen modulates skin repair, particularly the cell type–specific role of the two estrogen receptors, ERα and ERβ, has yet to be determined. Here, we use pharmacological activation and genetic deletion to investigate the role of both ERα and ERβ in cutaneous tissue repair. Unexpectedly, we report that exogenous estrogen replacement to ovariectomised mice in the absence of ERβ actually delayed wound healing. Moreover, healing in epidermal-specific ERβ null mice (K14-cre/ERβL2/L2) largely resembled that in global ERβ null mice. Thus, the beneficial effects of estrogen on skin wound healing are mediated by epidermal ERβ, in marked contrast to most other tissues in the body where ERα is predominant. Surprisingly, agonists to both ERα and ERβ are potently antiinflammatory during skin repair, indicating clear uncoupling of inflammation and overall efficiency of repair. Thus, estrogen-mediated antiinflammatory activity is not the principal factor in accelerated wound healing. PMID:20733032

Yeast as a model system for mammalian seven-transmembrane segment receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeansonne, N.E.

1994-05-01

Investigators have used the budding yeast Saccharomyces cerevisiae as a model system in which to study the {beta}-adrenergic receptor, the T-cell receptor pathway, initiation of mammalian DNA replication, initiation of mammalian transcription, secretion, the CDC2 kinase system, cell cycle control, and aging, as well as the function of oncogenes. This list continues to growth with the discovery of an immunoglobulin heavy-chain binding homologue in yeast, an Rb binding protein homologue, and a possible yeast arrestin. Yeast is relatively easy to maintain, to grow, and to genetically manipulate. A single gene can be overexpressed, selectively mutated or deleted from its chromosomalmore » location. In this way, the in vivo function of a gene can be studied. It has become reasonable to consider yeast as a model system for studying the seven transmembrane segments (7-TMS) receptor family. Currently, subtypes of the {beta}-adrenergic receptor are being studied in yeast. The receptor and its G{sub {alpha}}-G-protein, trigger the mating pheromone receptor pathway. This provides a powerful assay for determining receptor function. Studies expressing the muscarinic cholinergic receptor in yeast are underway. The yeast pheromone receptor belongs to this receptor family, sharing sequences and secondary structure homology. An effective strategy has been to identify a yeast pathway or process which is homologous to a mammalian system. The pathway is delineated in yeast, identifying other genetic components. Then yeast genes are used to screen for human homologues of these components. The putative human homologues are then expressed in yeast and in mammalian cells to determine function. When this type of {open_quotes}mixing and matching{close_quotes} works, yeast genetics can be a powerful tool. 115 refs.« less

Estrogens and cognition: Friends or foes?: An evaluation of the opposing effects of estrogens on learning and memory.

PubMed

Korol, Donna L; Pisani, Samantha L

2015-08-01

This article is part of a Special Issue "Estradiol and cognition". Estrogens are becoming well known for their robust enhancement on cognition particularly for learning and memory that relies upon functioning of the hippocampus and related neural systems. What is also emerging is that estrogen modulation of cognition is not uniform, at times enhancing yet at other times impairing learning. This review explores the bidirectional effects of estrogens on learning from a multiple memory systems view, focusing on the hippocampus and striatum, whereby modulation by estrogens sorts according to task attributes and neural systems engaged during cognition. We highlight our findings showing that the ability to solve hippocampus-sensitive tasks typically improves under relatively high estrogen status while the ability to solve striatum-sensitive tasks degrades with estrogen exposures. Though constrained by dose and timing of exposure, these opposing enhancements and impairments of cognition can be observed following treatments with different estrogenic compounds including the hormone estradiol, the isoflavone genistein found in soybeans, and agonists that are selective for specific estrogen receptors, suggesting that activation of a single receptor type is sufficient to produce the observed shifts in learning strategies. Using this multi-dimensional framework will allow us to extend our thinking of the relationship between estrogens and cognition to other brain regions and cognitive functions. Copyright © 2015 Elsevier Inc. All rights reserved.

A demonstration of the uncertainty in predicting the estrogenic ...

EPA Pesticide Factsheets

In vitro estrogen receptor assays are valuable screening tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently unable to fully account for adsorption, distribution, metabolism, and excretion. To explore this issue, we calculated relative potency factors (RPF) for several chemicals and mixtures in the T47D-KBluc estrogen receptor transactivation assay. The in vitro RPF values were then used to predict rat uterotrophic assay responses following oral administration of individual chemicals and mixtures. 17β-estradiol (E2), 17α-ethinyl estradiol (EE2), benzyl-butyl phthalate (BBP), bisphenol-A (BPA), bisphenol-AF (BPAF), bisphenol-C (BPC), bisphenol-S (BPS), and methoxychlor (MET) were tested individually, while BPS+MET, BPAF+MET, and BPAF+BPC+BPS+EE2+MET were tested as equipotent mixtures. In vivo ED50 values for BPA, BPAF, and BPC were accurately predicted using in vitro data; however, E2 was less potent than predicted, BBP was a false positive, and BPS and MET were 76.6 and 368.3-fold more active in vivo than predicted from the in vitro potency assessment, respectively. Further, mixture ED50 values were more accurately predicted by the dose addition model using individual chemical in vivo uterotrophic data (0.7-1.5-fold difference from observed) than in vitro data (1.4-86.8-fold). Overall,

Vascular Effects of Estrogenic Menopausal Hormone Therapy

PubMed Central

Reslan, Ossama M.; Khalil, Raouf A.

2011-01-01

Cardiovascular disease (CVD) is more common in men and postmenopausal women (Post-MW) than premenopausal women (Pre-MW). Despite recent advances in preventive measures, the incidence of CVD in women has shown a rise that matched the increase in the Post-MW population. The increased incidence of CVD in Post-MW has been related to the decline in estrogen levels, and hence suggested vascular benefits of endogenous estrogen. Experimental studies have identified estrogen receptor ERα, ERβ and a novel estrogen binding membrane protein GPR30 (GPER) in blood vessels of humans and experimental animals. The interaction of estrogen with vascular ERs mediates both genomic and non-genomic effects. Estrogen promotes endothelium-dependent relaxation by increasing nitric oxide, prostacyclin, and hyperpolarizing factor. Estrogen also inhibits the mechanisms of vascular smooth muscle (VSM) contraction including [Ca2+]i, protein kinase C and Rho-kinase. Additional effects of estrogen on the vascular cytoskeleton, extracellular matrix, lipid profile and the vascular inflammatory response have been reported. In addition to the experimental evidence in animal models and vascular cells, initial observational studies in women using menopausal hormonal therapy (MHT) have suggested that estrogen may protect against CVD. However, randomized clinical trials (RCTs) such as the Heart and Estrogen/progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI), which examined the effects of conjugated equine estrogens (CEE) in older women with established CVD (HERS) or without overt CVD (WHI), failed to demonstrate protective vascular effects of estrogen treatment. Despite the initial set-back from the results of MHT RCTs, growing evidence now supports the ‘timing hypothesis’, which suggests that MHT could increase the risk of CVD if started late after menopause, but may produce beneficial cardiovascular effects in younger women during the perimenopausal period. The choice of

Estrogens, Neuroinflammation, and Neurodegeneration

PubMed Central

Villa, Alessandro; Vegeto, Elisabetta; Poletti, Angelo

2016-01-01

Inflammatory activation of microglia is a hallmark of several disorders of the central nervous system. In addition to protecting the brain against inflammatory insults, microglia are neuroprotective and play a significant role in maintaining neuronal connectivity, but the prolongation of an inflammatory status may limit the beneficial functions of these immune cells. The finding that estrogen receptors are present in monocyte-derived cells and that estrogens prevent and control the inflammatory response raise the question of the role that this sex steroid plays in the manifestation and progression of pathologies that have a clear sex difference in prevalence, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. The present review aims to provide a critical review of the current literature on the actions of estrogen in microglia and on the involvement of estrogen receptors in the manifestation of selected neurological disorders. This current understanding highlights a research area that should be expanded to identify appropriate replacement therapies to slow the progression of such diseases. PMID:27196727

Strategies for identifying new prions in yeast

PubMed Central

MacLea, Kyle S

2011-01-01

The unexpected discovery of two prions, [URE3] and [PSI+], in Saccharomyces cerevisiae led to questions about how many other proteins could undergo similar prion-based structural conversions. However, [URE3] and [PSI+] were discovered by serendipity in genetic screens. Cataloging the full range of prions in yeast or in other organisms will therefore require more systematic search methods. Taking advantage of some of the unique features of prions, various researchers have developed bioinformatic and experimental methods for identifying novel prion proteins. These methods have generated long lists of prion candidates. The systematic testing of some of these prion candidates has led to notable successes; however, even in yeast, where rapid growth rate and ease of genetic manipulation aid in testing for prion activity, such candidate testing is laborious. Development of better methods to winnow the field of prion candidates will greatly aid in the discovery of new prions, both in yeast and in other organisms, and help us to better understand the role of prions in biology. PMID:22052351

Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

PubMed

Mishra, Sweta; Tai, Qin; Gu, Xiang; Schmitz, James; Poullard, Ashley; Fajardo, Roberto J; Mahalingam, Devalingam; Chen, Xiaodong; Zhu, Xueqiong; Sun, Lu-Zhe

2015-12-29

The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERß) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

Rapid Parallel Screening for Strain Optimization

DTIC Science & Technology

2013-08-16

fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can...reporter, and, 2) a yeast TAR cloning shuttle vector for transferring catabolic clusters to E. coli. 15. SUBJECT TERMS NA 16. SECURITY CLASSIFICATION OF... fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can utilize

Rapid Parallel Screening for Strain Optimization

DTIC Science & Technology

2013-05-16

fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can...reporter, and, 2) a yeast TAR cloning shuttle vector for transferring catabolic clusters to E. coli. 15. SUBJECT TERMS NA 16. SECURITY CLASSIFICATION OF... fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can utilize

SCIENTIFIC AND TECHNOLOGICAL SUPPORT ON IN VITRO ASSAYS FOR THE AGENCY'S ENDOCRINE DISRUPTOR SCREENING PROGRAM

EPA Science Inventory

In response to the 1996 legislative mandate for an endocrine screening and testing program, we are helping develop, standardize and validate relatively sensitive, robust and relatively simple methods for in vitro screening of chemicals that affect estrogen, and androgen function ...

The role of estrogens and estrogen receptor signaling pathways in cancer and infertility: the case of schistosomes.

PubMed

Botelho, Mónica C; Alves, Helena; Barros, Alberto; Rinaldi, Gabriel; Brindley, Paul J; Sousa, Mário

2015-06-01

Schistosoma haematobium, a parasitic flatworm that infects more than 100 million people, mostly in the developing world, is the causative agent of urogenital schistosomiasis, and is associated with a high incidence of squamous cell carcinoma (SCC) of the bladder. Schistosomiasis haematobia also appears to negatively influence fertility, and is particularly associated with female infertility. Given that estrogens and estrogen receptors are key players in human reproduction, we speculate that schistosome estrogen-like molecules may contribute to infertility through hormonal imbalances. Here, we review recent findings on the role of estrogens and estrogen receptors on both carcinogenesis and infertility associated with urogenital schistosomiasis and discuss the basic hormonal mechanisms that might be common in cancer and infertility. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Estrogen Receptors: An Overview from Different Perspectives.

PubMed

Eyster, Kathleen M

2016-01-01

The estrogen receptors, ERα, ERβ, and GPER, mediate the effects of estrogenic compounds on their target tissues. Estrogen receptors are located in the tissues of the female reproductive tract and breast as one would expect, but also in tissues as diverse as bone, brain, liver, colon, skin, and salivary gland. The purpose of this discussion of the estrogen receptors is to provide a brief overview of the estrogen receptors and estrogen action from perspectives such as the historical, physiological, pharmacological, pathological, structural, and ligand perspectives.

A Yeast Model of FUS/TLS-Dependent Cytotoxicity

PubMed Central

Ju, Shulin; Tardiff, Daniel F.; Han, Haesun; Divya, Kanneganti; Zhong, Quan; Maquat, Lynne E.; Bosco, Daryl A.; Hayward, Lawrence J.; Brown, Robert H.; Lindquist, Susan; Ringe, Dagmar; Petsko, Gregory A.

2011-01-01

FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression. PMID:21541368

The applications of machine learning algorithms in the modeling of estrogen-like chemicals.

PubMed

Liu, Huanxiang; Yao, Xiaojun; Gramatica, Paola

2009-06-01

Increasing concern is being shown by the scientific community, government regulators, and the public about endocrine-disrupting chemicals that, in the environment, are adversely affecting human and wildlife health through a variety of mechanisms, mainly estrogen receptor-mediated mechanisms of toxicity. Because of the large number of such chemicals in the environment, there is a great need for an effective means of rapidly assessing endocrine-disrupting activity in the toxicology assessment process. When faced with the challenging task of screening large libraries of molecules for biological activity, the benefits of computational predictive models based on quantitative structure-activity relationships to identify possible estrogens become immediately obvious. Recently, in order to improve the accuracy of prediction, some machine learning techniques were introduced to build more effective predictive models. In this review we will focus our attention on some recent advances in the use of these methods in modeling estrogen-like chemicals. The advantages and disadvantages of the machine learning algorithms used in solving this problem, the importance of the validation and performance assessment of the built models as well as their applicability domains will be discussed.

Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells.

PubMed

Mesnage, Robin; Phedonos, Alexia; Arno, Matthew; Balu, Sucharitha; Corton, J Christopher; Antoniou, Michael N

2017-08-01

Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in "BPA-free" products. We have compared estrogenic activity of BPA with 6 bisphenol analogues [bisphenol S (BPS); bisphenol F (BPF); bisphenol AP (BPAP); bisphenol AF (BPAF); bisphenol Z (BPZ); bisphenol B (BPB)] in 3 human breast cancer cell lines. Estrogenicity was assessed (10-11-10-4 M) by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element-mediated transcription in a luciferase assay. BPAF was the most potent bisphenol, followed by BPB > BPZ ∼ BPA > BPF ∼ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs. Data mining of ToxCast high-throughput screening assays confirm our results but also show divergence in the sensitivities of the assays. Gene expression profiles were determined in MCF-7 cells by microarray analysis. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα gene expression biomarker further indicates that all BPA alternatives act as ERα agonists in MCF-7 cells. These results were confirmed by Illumina-based RNA sequencing. In conclusion, BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. These findings point to the importance of better understanding the risk of adverse effects from exposure to BPA alternatives, including hormone-dependent breast cancer. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.

COMPARATIVE ESTROGENICITY OF ESTRADIOL, ETHYNYL ESTRADIOL AND DIETHYLSTILBESTROL IN AN IN VIVO, MALE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS), VITELLOGENIN BIOASSAY

EPA Science Inventory

An in vivo bioassay for vitellogenin (VTG) synthesis was developed to screen individual chemicals or mixtures of chemicals for potentially estrogenic effects in a marine teleost model. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate VTG synthesis in male sheep...

Evaluation of toxicity and estrogenicity of the landfill-concentrated leachate during advanced oxidation treatment: chemical analyses and bioanalytical tools.

PubMed

Wang, Guifang; Lu, Gang; Zhao, Jiandi; Yin, Pinghe; Zhao, Ling

2016-08-01

Landfill-concentrated leachate from membrane separation processes is a potential pollution source for the surroundings. In this study, the toxicity and estrogenicity potentials of concentrated leachate prior to and during UV-Fenton and Fenton treatments were assessed by a combination of chemical (di (2-ethylhexyl) phthalate and dibutyl phthalate were chosen as targets) and biological (Daphnia magna, Chlorella vulgaris, and E-screen assay) analyses. Removal efficiencies of measured di (2-ethylhexyl) phthalate and dibutyl phthalate were more than 97 % after treatment with the two methods. Biological tests showed acute toxicity effects on D. magna tests in untreated concentrated leachate samples, whereas acute toxicity on C. vulgaris tests was not observed. Both treatment methods were found to be efficient in reducing acute toxicity effects on D. magna tests. The E-screen test showed concentrated leachate had significant estrogenicity, UV-Fenton and Fenton treatment, especially the former, were effective methods for reducing estrogenicity of concentrated leachate. The EEQchem (estradiol equivalent concentration) of all samples could only explain 0.218-5.31 % range of the EEQbio. These results showed that UV-Fenton reagent could be considered as a suitable method for treatment of concentrated leachate, and the importance of the application of an integrated (biological + chemical) analytical approach for a comprehensive evaluation of treatment suitability.

Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants.

PubMed

Rodrigues, Andre; Cable, Rachel N; Mueller, Ulrich G; Bacci, Maurício; Pagnocca, Fernando C

2009-10-01

We investigate the diversity of yeasts isolated in gardens of the leafcutter ant Atta texana. Repeated sampling of gardens from four nests over a 1-year time period showed that gardens contain a diverse assemblage of yeasts. The yeast community in gardens consisted mostly of yeasts associated with plants or soil, but community composition changed between sampling periods. In order to understand the potential disease-suppressing roles of the garden yeasts, we screened isolates for antagonistic effects against known microfungal garden contaminants. In vitro assays revealed that yeasts inhibited the mycelial growth of two strains of Escovopsis (a specialized attine garden parasite), Syncephalastrum racemosum (a fungus often growing in gardens of leafcutter lab nests), and the insect pathogen Beauveria bassiana. These garden yeasts add to the growing list of disease-suppressing microbes in attine nests that may contribute synergistically, together with actinomycetes and Burkholderia bacteria, to protect the gardens and the ants against diseases. Additionally, we suggest that garden immunity against problem fungi may therefore derive not only from the presence of disease-suppressing Pseudonocardia actinomycetes, but from an enrichment of multiple disease-suppressing microorganisms in the garden matrix.

Comparison of Estrogen-Responsive Plasma Protein Biomarkers and Reproductive Endpoints in Sheepshead Minnows Exposed to 17B-Trenbolone

EPA Science Inventory

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies, mass spectral analysis revealed four peptides (2950.5, 2972.5, 3003.4, 3025.5 m/z) in the plasma of estrogen ag...

A new series of estrogen receptor modulators that display selectivity for estrogen receptor beta.

PubMed

Henke, Brad R; Consler, Thomas G; Go, Ning; Hale, Ron L; Hohman, Dana R; Jones, Stacey A; Lu, Amy T; Moore, Linda B; Moore, John T; Orband-Miller, Lisa A; Robinett, R Graham; Shearin, Jean; Spearing, Paul K; Stewart, Eugene L; Turnbull, Philip S; Weaver, Susan L; Williams, Shawn P; Wisely, G Bruce; Lambert, Millard H

2002-12-05

A series of 1,3,5-triazine-based estrogen receptor (ER) modulators that are modestly selective for the ERbeta subtype are reported. Compound 1, which displayed modest potency and selectivity for ERbeta vs ERalpha, was identified via high-throughput screening utilizing an ERbeta SPA-based binding assay. Subsequent analogue preparation resulted in the identification of compounds such as 21 and 43 that display 25- to 30-fold selectivity for ERbeta with potencies in the 10-30 nM range. These compounds profile as full antagonists at ERbeta and weak partial agonists at ERalpha in a cell-based reporter gene assay. In addition, the X-ray crystal structure of compound 15 complexed with the ligand binding domain of ERbeta has been solved and was utilized in the design of more conformationally restrained analogues such as 31 in an attempt to increase selectivity for the ERbeta subtype.

Interactions between the cytomegalovirus promoter and the estrogen response element: implications for design of estrogen-responsive reporter plasmids.

PubMed

Derecka, K; Wang, C K; Flint, A P F

2006-07-01

We aimed to produce an estrogen-responsive reporter plasmid that would permit monitoring of estrogen receptor function in the uterus in vivo. The plasmid pBL-tk-CAT(+)ERE was induced by estrogen in bovine endometrial stromal cells. When the CAT gene was replaced by the secreted alkaline phosphatase SeAP, the resulting construct pBL-tk-SeAP(+)ERE remained estrogen responsive. However when the tk promoter was replaced by the cytomegalovirus (cmv) promoter, the resulting plasmid (pBL-cmv-SeAP(+)ERE) was not estrogen responsive. Inhibition of ERE function was not due to an effect in trans or due to lack of estrogen receptor. It was not due to an interaction between the cmv promoter and the SeAP gene. cmv promoter function was dependent on NF-kappaB, and mutagenesis in the NF-kappaB sites reduced basal reporter expression without imparting responsiveness to estrogen. A mutation in the TATA box also failed to impart estrogen responsiveness. Modeling of DNA accessibility indicated the ERE was inserted at a site accessible to transcription factors. We conclude that the cmv promoter inhibits ERE function in cis when the two sequences are located in the same construct, and that this effect does not involve an interaction between cmv and reporter gene, NF-kappaB sites or the TATA box, or DNA inaccessibility.

Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains.

PubMed

Kong, In Iok; Turner, Timothy Lee; Kim, Heejin; Kim, Soo Rin; Jin, Yong-Su

2018-02-01

Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeasts, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites, and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotolerance, increased resistances towards fermentation inhibitors and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared 21 industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency and ability to ferment lignocellulosic sugars. These data may be useful for the selection of a parental strain for specific biotechnological applications of engineered yeast. © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.

PubMed

Wang, Dan; Xie, Jiangbi; Zhu, Xiaocui; Li, Jinqiu; Zhao, Dongqin; Zhao, Meiping

2014-05-15

In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERα36 and GPR30/GPER1.

PubMed

Pelekanou, Vasiliki; Kampa, Marilena; Kiagiadaki, Foteini; Deli, Alexandra; Theodoropoulos, Panayiotis; Agrogiannis, George; Patsouris, Efstratios; Tsapis, Andreas; Castanas, Elias; Notas, George

2016-02-01

Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti-inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30/G-protein estrogen receptor 1, in a sex-independent manner. 17-β-Estradiol inhibits the LPS-induced IL-6 inflammatory response, resulting in inhibition of NF-κB transcriptional activity. This is achieved via a direct physical interaction of ligand-activated estrogen receptor-α 36-kDa splice variant with the p65 component of NF-κB in the nucleus. G-protein coupled receptor 30/G-protein estrogen receptor 1, which also physically interacts with estrogen receptor-α 36-kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL-6 expression. However, its activation does not mimic the effect of estrogens, on neither IL-6 nor NF-κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage-dependent inflammatory processes. © Society for Leukocyte Biology.

Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene.

PubMed

Hyder, S M; Stancel, G M; Nawaz, Z; McDonnell, D P; Loose-Mitchell, D S

1992-09-05

We have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyltransferase, linked to regions of mouse c-fos, to identify a specific estrogen response element (ERE) in this protooncogene. This element is located in the untranslated 3'-flanking region of the c-fos gene, 5 kilobases (kb) downstream from the c-fos promoter and 1.5 kb downstream of the poly(A) signal. This element confers estrogen responsiveness to chloramphenicol acetyltransferase reporters linked to both the herpes simplex virus thymidine kinase promoter and the homologous c-fos promoter. Deletion analysis localized the response element to a 200-base pair fragment which contains the element GGTCACCACAGCC that resembles the consensus ERE sequence GGTCACAGTGACC originally identified in Xenopus vitellogenin A2 gene. A synthetic 36-base pair oligodeoxynucleotide containing this c-fos sequence conferred estrogen inducibility to the thymidine kinase promoter. The corresponding sequence also induced reporter activity when present in the c-fos gene fragment 3 kb from the thymidine kinase promoter. Gel-shift experiments demonstrated that synthetic oligonucleotides containing either the consensus ERE or the c-fos element bind human estrogen receptor obtained from a yeast expression system. However, the mobility of the shifted band is faster for the fos-ERE-complex than the consensus ERE complex suggesting that the three-dimensional structure of the protein-DNA complexes is different or that other factors are differentially involved in the two reactions. When the 5'-GGTCA sequence present in the c-fos ERE is mutated to 5'-TTTCA, transcriptional activation and receptor binding activities are both lost. Mutation of the CAGCC-3' element corresponding to the second half-site of the c-fos sequence also led to the loss of receptor binding activity, suggesting that both half-sites of this element are involved in this function. The estrogen induction mediated by either the c-fos or

Navigating yeast genome maintenance with functional genomics.

PubMed

Measday, Vivien; Stirling, Peter C

2016-03-01

Maintenance of genome integrity is a fundamental requirement of all organisms. To address this, organisms have evolved extremely faithful modes of replication, DNA repair and chromosome segregation to combat the deleterious effects of an unstable genome. Nonetheless, a small amount of genome instability is the driver of evolutionary change and adaptation, and thus a low level of instability is permitted in populations. While defects in genome maintenance almost invariably reduce fitness in the short term, they can create an environment where beneficial mutations are more likely to occur. The importance of this fact is clearest in the development of human cancer, where genome instability is a well-established enabling characteristic of carcinogenesis. This raises the crucial question: what are the cellular pathways that promote genome maintenance and what are their mechanisms? Work in model organisms, in particular the yeast Saccharomyces cerevisiae, has provided the global foundations of genome maintenance mechanisms in eukaryotes. The development of pioneering genomic tools inS. cerevisiae, such as the systematic creation of mutants in all nonessential and essential genes, has enabled whole-genome approaches to identifying genes with roles in genome maintenance. Here, we review the extensive whole-genome approaches taken in yeast, with an emphasis on functional genomic screens, to understand the genetic basis of genome instability, highlighting a range of genetic and cytological screening modalities. By revealing the biological pathways and processes regulating genome integrity, these analyses contribute to the systems-level map of the yeast cell and inform studies of human disease, especially cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen.

PubMed

Santiago, Ednalise; Akamine, Pearl; Snider, Jamie; Wong, Victoria; Jessulat, Matthew; Deineko, Viktor; Gagarinova, Alla; Aoki, Hiroyuki; Minic, Zoran; Phanse, Sadhna; San Antonio, Andrea; Cubano, Luis A; Rymond, Brian C; Babu, Mohan; Stagljar, Igor; Rodriguez-Medina, Jose R

2016-05-03

Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis. Copyright © 2016 Santiago et al.

Evaluation of OASIS QSAR Models Using ToxCast™ in Vitro Estrogen and Androgen Receptor Binding Data and Application in an Integrated Endocrine Screening Approach

PubMed Central

Bhhatarai, Barun; Wilson, Daniel M.; Price, Paul S.; Marty, Sue; Parks, Amanda K.; Carney, Edward

2016-01-01

Background: Integrative testing strategies (ITSs) for potential endocrine activity can use tiered in silico and in vitro models. Each component of an ITS should be thoroughly assessed. Objectives: We used the data from three in vitro ToxCast™ binding assays to assess OASIS, a quantitative structure-activity relationship (QSAR) platform covering both estrogen receptor (ER) and androgen receptor (AR) binding. For stronger binders (described here as AC50 < 1 μM), we also examined the relationship of QSAR predictions of ER or AR binding to the results from 18 ER and 10 AR transactivation assays, 72 ER-binding reference compounds, and the in vivo uterotrophic assay. Methods: NovaScreen binding assay data for ER (human, bovine, and mouse) and AR (human, chimpanzee, and rat) were used to assess the sensitivity, specificity, concordance, and applicability domain of two OASIS QSAR models. The binding strength relative to the QSAR-predicted binding strength was examined for the ER data. The relationship of QSAR predictions of binding to transactivation- and pathway-based assays, as well as to in vivo uterotrophic responses, was examined. Results: The QSAR models had both high sensitivity (> 75%) and specificity (> 86%) for ER as well as both high sensitivity (92–100%) and specificity (70–81%) for AR. For compounds within the domains of the ER and AR QSAR models that bound with AC50 < 1 μM, the QSAR models accurately predicted the binding for the parent compounds. The parent compounds were active in all transactivation assays where metabolism was incorporated and, except for those compounds known to require metabolism to manifest activity, all assay platforms where metabolism was not incorporated. Compounds in-domain and predicted to bind by the ER QSAR model that were positive in ToxCast™ ER binding at AC50 < 1 μM were active in the uterotrophic assay. Conclusions: We used the extensive ToxCast™ HTS binding data set to show that OASIS ER and AR QSAR models had

Estrogen action and prostate cancer

PubMed Central

Nelles, Jason L; Hu, Wen-Yang; Prins, Gail S

2011-01-01

Early work on the hormonal basis of prostate cancer focused on the role of androgens, but more recently estrogens have been implicated as potential agents in the development and progression of prostate cancer. In this article, we review the epidemiological, laboratory and clinical evidence that estrogen may play a causative role in human prostate cancer, as well as rodent and grafted in vivo models. We then review recent literature highlighting potential mechanisms by which estrogen may contribute to prostate cancer, including estrogenic imprinting and epigenetic modifications, direct genotoxicity, hyperprolactinemia, inflammation and immunologic changes, and receptor-mediated actions. We discuss the work performed so far separating the actions of the different known estrogen receptors (ERs), ERα and ERβ, as well as G-protein-coupled receptor 30 and their specific roles in prostate disease. Finally, we predict that future work in this field will involve more investigations into epigenetic changes, experiments using new models of hormonal dysregulation in developing human prostate tissue, and continued delineation of the roles of the different ER subtypes, as well as their downstream signaling pathways that may serve as therapeutic targets. PMID:21765856

Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

PubMed

Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E

2016-10-01

Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

XX ovarian dysgenesis is caused by a PSMC3IP/HOP2 mutation that abolishes coactivation of estrogen-driven transcription.

PubMed

Zangen, David; Kaufman, Yotam; Zeligson, Sharon; Perlberg, Shira; Fridman, Hila; Kanaan, Moein; Abdulhadi-Atwan, Maha; Abu Libdeh, Abdulsalam; Gussow, Ayal; Kisslov, Irit; Carmel, Liran; Renbaum, Paul; Levy-Lahad, Ephrat

2011-10-07

XX female gonadal dysgenesis (XX-GD) is a rare, genetically heterogeneous disorder characterized by lack of spontaneous pubertal development, primary amenorrhea, uterine hypoplasia, and hypergonadotropic hypogonadism as a result of streak gonads. Most cases are unexplained but thought to be autosomal recessive. We elucidated the genetic basis of XX-GD in a highly consanguineous Palestinian family by using homozygosity mapping and candidate-gene and whole-exome sequencing. Affected females were homozygous for a 3 bp deletion (NM_016556.2, c.600_602del) in the PSMC3IP gene, leading to deletion of a glutamic acid residue (p.Glu201del) in the highly conserved C-terminal acidic domain. Proteasome 26S subunit, ATPase, 3-Interacting Protein (PSMC3IP)/Tat Binding Protein Interacting Protein (TBPIP) is a nuclear, tissue-specific protein with multiple functions. It is critical for meiotic recombination as indicated by the known role of its yeast ortholog, Hop2. Through the C terminus (not present in yeast), PSMC3IP also coactivates ligand-driven transcription mediated by estrogen, androgen, glucocorticoid, progesterone, and thyroid nuclear receptors. In cell lines, the p.Glu201del mutation abolished PSMC3IP activation of estrogen-driven transcription. Impaired estrogenic signaling can lead to ovarian dysgenesis both by affecting the size of the follicular pool created during fetal development and by failing to counteract follicular atresia during puberty. PSMC3IP joins previous genes known to be mutated in XX-GD, the FSH receptor, and BMP15, highlighting the importance of hormonal signaling in ovarian development and maintenance and suggesting a common pathway perturbed in isolated XX-GD. By analogy to other XX-GD genes, PSMC3IP is also a candidate gene for premature ovarian failure, and its role in folliculogenesis should be further investigated. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The Dual Estrogen Receptor α Inhibitory Effects of the Tissue-Selective Estrogen Complex for Endometrial and Breast Safety

PubMed Central

Han, Sang Jun; Begum, Khurshida; Foulds, Charles E.; Hamilton, Ross A.; Bailey, Suzanna; Malovannaya, Anna; Chan, Doug; Qin, Jun

2016-01-01

The conjugated estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) is designed to minimize the undesirable effects of estrogen in the uterus and breast tissues and to allow the beneficial effects of estrogen in other estrogen-target tissues, such as the bone and brain. However, the molecular mechanism underlying endometrial and breast safety during TSEC use is not fully understood. Estrogen receptor α (ERα)–estrogen response element (ERE)–DNA pull-down assays using HeLa nuclear extracts followed by mass spectrometry–immunoblotting analyses revealed that, upon TSEC treatment, ERα interacted with transcriptional repressors rather than coactivators. Therefore, the TSEC-mediated recruitment of transcriptional repressors suppresses ERα-mediated transcription in the breast and uterus. In addition, TSEC treatment also degraded ERα protein in uterine tissue and breast cancer cells, but not in bone cells. Interestingly, ERα-ERE-DNA pull-down assays also revealed that, upon TSEC treatment, ERα interacted with the F-box protein 45 (FBXO45) E3 ubiquitin ligase. The loss-of- and gain-of-FBXO45 function analyses indicated that FBXO45 is involved in TSEC-mediated degradation of the ERα protein in endometrial and breast cells. In preclinical studies, these synergistic effects of TSEC on ERα inhibition also suppressed the estrogen-dependent progression of endometriosis. Therefore, the endometrial and breast safety effects of TSEC are associated with synergy between the selective recruitment of transcriptional repressors to ERα and FBXO45-mediated degradation of the ERα protein. PMID:26487511

In Vitro Estrogenic and Breast Cancer Inhibitory Activities of Chemical Constituents Isolated from Rheum undulatum L.

PubMed

Lee, Dahae; Park, SeonJu; Choi, Sungyoul; Kim, Seung Hyun; Kang, Ki Sung

2018-05-18

We investigated the estrogenic and breast cancer inhibitory activities of chemical constituents isolated from Rhei undulati Rhizoma (roots of Rheum undulatum L.), which is used as a laxative, an anti-inflammatory, and an anti-blood stagnation agent. Estrogen-like activity was studied using the well characterized E-screen assay in estrogen receptor (ER)-positive MCF-7 cells. The mechanism underlying the breast cancer inhibitory activity of the compounds was studied using human ER-negative MDA-MB-231 and ER-positive MCF-7 cells. The activation of apoptosis pathway-related proteins was investigated by western blotting, using extracts of R. undulatum prepared in three solvent conditions (EX1, EX2, and EX3). The R. undulatum chemical constituents (compounds 1 ⁻ 3 ) showed estrogen-like activity in the concentration range of 10 to 50 μM, by increasing the proliferation of human ER-positive MCF-7 cells. These effects were attenuated by co-treatment with 100 nM fulvestrant, an ER antagonist. Compounds 1 ⁻ 3 decreased the viability of MCF-7 cells in a concentration-dependent manner. Compounds 1 (aloe emodin) and 2 (rhapontigenin) induced mitochondria-independent apoptosis by activating the caspase-8 pathway, whereas the cytotoxic effect of compound 3 (chrysophanol 1- O -β-d-glucopyranoside) was mediated through the mitochondria-dependent apoptotic pathway.

Aging, Estrogens, and Episodic Memory in Women

PubMed Central

Henderson, Victor W.

2009-01-01

Objective To review the relation in midlife and beyond between estrogen exposures and episodic memory in women. Background Episodic memory performance declines with usual aging, and impairments in episodic memory often portend the development of Alzheimer's disease. In the laboratory, estradiol influences hippocampal function and animal learning. However, it is controversial whether estrogens affect memory after a woman's reproductive years. Method Focused literature review, including a summary of a systematic search of clinical trials of estrogens in which outcomes included an objective measure of episodic memory. Results The natural menopause transition is not associated with objective changes in episodic memory. Strong clinical trial evidence indicates that initiating estrogen-containing hormone therapy after about age 60 years does not benefit episodic memory. Clinical trial findings in middle-age women before age 60 are limited by smaller sample sizes and shorter treatment durations, but these also do not indicate substantial memory effects. Limited short-term evidence, however, suggests that estrogens may improve verbal memory after surgical menopause. Although hormone therapy initiation in old age increases dementia risk, observational studies raise the question of an early critical window during which midlife estrogen therapy reduces late-life Alzheimer's disease. However, almost no data address whether midlife estrogen therapy affects episodic memory in old age. Conclusions Episodic memory is not substantially impacted by the natural menopause transition or improved by use of estrogen-containing hormone therapy after age 60. Further research is needed to determine whether outcomes differ after surgical menopause or whether episodic memory later in life is modified by midlife estrogenic exposures. PMID:19996872

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1

PubMed Central

Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Maarten Altelaar, AF; van Leeuwen, Fred

2016-01-01

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features. DOI: http://dx.doi.org/10.7554/eLife.18919.001 PMID:27922451

ANALYSIS OF LAGOON SAMPLES FROM DIFFERENT CONCENTRATED ANIMAL FEEDING OPERATIONS FOR ESTROGENS AND ESTROGEN CONJUGATES

EPA Science Inventory

Although Concentrated Animal Feeding Operations CAFOs) have been identified as potentially important sources for the release of estrogens into the environment, information is lacking on the concentrations of estrogens in whole lagoon effluents (including suspended solids)which ar...

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women I: Serum levels, variability and the basis for urinary biomonitoring of serum estrogenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fleck, Stefanie C.; Twaddle, Nathan C.; Churchwell, Mona I.

Biomonitoring of human exposure to estrogens most frequently focuses on environmental and dietary estrogens, and infrequently includes measures of exposure to potent endogenous estrogens present in serum. Pregnancy is a developmentally sensitive period during which “added” serum estrogenicity exceeding normal intra-individual daily variability may be of particular relevance. Here, we made repeated measurements of serum concentrations of estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), daidzein (DDZ), genistein (GEN) and bisphenol A (BPA) in thirty pregnant women using ultra-performance liquid chromatography coupled with tandem mass spectrometry detection (UPLC-MS/MS) and electrospray ionization (ESI). Serum E1, E2, and E3 concentrations varied significantlymore » (coefficients of variation 9–10%) with broad ranges across the cohort: 1.61–85.1 nM, 9.09–69.7 nM, and 1.5–36.3 nM respectively. BPA (undetected, estimated from total exposure), DDZ and GEN concentrations were 1-5 orders of magnitude lower. The 24-h urinary elimination profiles of endogenous estrogens were each strongly correlated with their corresponding serum concentrations (Pearson's Correlation Coefficients of 0.83 (E1), 0.84 (E2) and 0.94 (E3)). Lastly, a multivariate regression analysis produced equations for estimating serum concentrations of E1, E2, E3, E4, GEN and DDZ from urinary elimination rates and gestation period, an important step towards non-invasive biomonitoring for assessment of “added” estrogenicity during pregnancy.« less

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women I: Serum levels, variability and the basis for urinary biomonitoring of serum estrogenicity

DOE PAGES

Fleck, Stefanie C.; Twaddle, Nathan C.; Churchwell, Mona I.; ...

2018-03-13

Biomonitoring of human exposure to estrogens most frequently focuses on environmental and dietary estrogens, and infrequently includes measures of exposure to potent endogenous estrogens present in serum. Pregnancy is a developmentally sensitive period during which “added” serum estrogenicity exceeding normal intra-individual daily variability may be of particular relevance. Here, we made repeated measurements of serum concentrations of estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), daidzein (DDZ), genistein (GEN) and bisphenol A (BPA) in thirty pregnant women using ultra-performance liquid chromatography coupled with tandem mass spectrometry detection (UPLC-MS/MS) and electrospray ionization (ESI). Serum E1, E2, and E3 concentrations varied significantlymore » (coefficients of variation 9–10%) with broad ranges across the cohort: 1.61–85.1 nM, 9.09–69.7 nM, and 1.5–36.3 nM respectively. BPA (undetected, estimated from total exposure), DDZ and GEN concentrations were 1-5 orders of magnitude lower. The 24-h urinary elimination profiles of endogenous estrogens were each strongly correlated with their corresponding serum concentrations (Pearson's Correlation Coefficients of 0.83 (E1), 0.84 (E2) and 0.94 (E3)). Lastly, a multivariate regression analysis produced equations for estimating serum concentrations of E1, E2, E3, E4, GEN and DDZ from urinary elimination rates and gestation period, an important step towards non-invasive biomonitoring for assessment of “added” estrogenicity during pregnancy.« less

EADB: An Estrogenic Activity Database for Assessing ...

EPA Pesticide Factsheets

Endocrine-active chemicals can potentially have adverse effects on both humans and wildlife. They can interfere with the body’s endocrine system through direct or indirect interactions with many protein targets. Estrogen receptors (ERs) are one of the major targets, and many endocrine disruptors are estrogenic and affect the normal estrogen signaling pathways. However, ERs can also serve as therapeutic targets for various medical conditions, such as menopausal symptoms, osteoporosis, and ER-positive breast cancer. Because of the decades-long interest in the safety and therapeutic utility of estrogenic chemicals, a large number of chemicals have been assayed for estrogenic activity, but these data exist in various sources and different formats that restrict the ability of regulatory and industry scientists to utilize them fully for assessing risk-benefit. To address this issue, we have developed an Estrogenic Activity Database (EADB; http://www.fda.gov/ScienceResearch/ BioinformaticsTools/EstrogenicActivityDatabaseEADB/default. htm) and made it freely available to the public. EADB contains 18,114 estrogenic activity data points collected for 8212 chemicals tested in 1284 binding, reporter gene, cell proliferation, and in vivo assays in 11 different species. The chemicals cover a broad chemical structure space and the data span a wide range of activities. A set of tools allow users to access EADB and evaluate potential endocrine activity of

Lipase production in lipolytic yeast from Wonorejo mangrove area

NASA Astrophysics Data System (ADS)

Alami, Nur Hidayatul; Nasihah, Liziyatin; Umar, Rurin Luswidya Artaty; Kuswytasari, Nengah Dwianita; Zulaika, Enny; Shovitri, Maya

2017-06-01

Lipase is an enzyme that is often used in industry and become a commercial enzyme. One group of microorganisms capable of producing lipase is a yeast. This study aims to screen yeast from Wonorejo mangrove that potential to produce lipase and to optimize the production of these enzymes. Screening test include the measurement of lipolytic index and value of fatty acid. Yeast with the best value of fatty acid will be continued to the measurement of lipase activity. It is affected by several environmental factors, such as pH, temperature, and incubation time. This research was conducted to observe the optimization variation on environmental factors combination to produce lipase. Lipase activity was tested by using p-Nitrophenyl Palmitate (pNPP). Absorbency was measured by spectrofotometer on wavelength of 410 nm. Measurement of the enzyme activity was done by interpolating the absorbance values on the p-nitrophenol standard curve then calculated by the formula. All data were analyzed by using descriptive quantitative method. The results show that the highest lypolityc index was 2.08. The highest value of fatty acid was 0.49 that was reached on 168 hours of incubation. Candida W3.8 expressed the highest lypolylitic potential. The optimum environment to produce lipase by Candida W 3.8 was on 120 hours of incubation time, in temperature range of 27°C - 45°C and pH range of 4,5 - 7.

Functional mapping of yeast genomes by saturated transposition

PubMed Central

Michel, Agnès H; Hatakeyama, Riko; Kimmig, Philipp; Arter, Meret; Peter, Matthias; Matos, Joao; De Virgilio, Claudio; Kornmann, Benoît

2017-01-01

Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput. DOI: http://dx.doi.org/10.7554/eLife.23570.001 PMID:28481201

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor.

PubMed

Bahadur, Urvashi; Ganjam, Goutham K; Vasudevan, Nandini; Kondaiah, Paturu

2005-02-28

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-alpha) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ERalpha antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the

MODELING THE EFFECTS OF FLEXIBILITY ON THE BINDING OF ENVIRONMENTAL ESTROGENS TO THE ESTROGEN RECEPTOR

EPA Science Inventory

Modeling the effects of flexibility on the binding of environmental estrogens to the estrogen receptor
There are many reports of environmental endocrine disruption in the literature, yet it has been difficult to identify the specific chemicals responsible for these effects. ...

Yeast Based Sensors

NASA Astrophysics Data System (ADS)

Shimomura-Shimizu, Mifumi; Karube, Isao

Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.

[The biological and clinical relevance of estrogen metabolome].

PubMed

Kovács, Krisztián; Vásárhelyi, Barna; Mészáros, Katalin; Patócs, Attila; Karvaly, Gellért

2017-06-01

Considerable knowledge has been gathered on the physiological role of estrogens. However, fairly little information is available on the role of compounds produced in the breakdown process of estrone and estradiol wich may play a role in various diseases associated with estrogen impact. To date, approximately 15 extragonadal estrogen-related compounds have been identified. These metabolites may exert protective, or, instead, pro-inflammatory and/or pro-oncogenic activity in a tissue-specific manner. Systemic and local estrogen metabolite levels are not necesserily correlated, which may promote the diagnostic significance of the locally produced estrogen metabolites in the future. The aim of the present study is a bibliographic review of the extragonadal metabolome in peripheral tissues, and to highlight the role of the peripheral tissue homeostasis of estrogens as well as the non-hormonal biological activity and clinical significance of the estrogen metabolome. Orv Hetil. 2017; 158(24): 929-937.

Isolation of linoleic acid as an estrogenic compound from the fruits of Vitex agnus-castus L. (chaste-berry).

PubMed

Liu, J; Burdette, J E; Sun, Y; Deng, S; Schlecht, S M; Zheng, W; Nikolic, D; Mahady, G; van Breemen, R B; Fong, H H S; Pezzuto, J M; Bolton, J L; Farnsworth, N R

2004-01-01

A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERalpha) and beta (ERbeta). The extract at 46 +/- 3 microg/ml displaced 50% of estradiol from ERalpha and 64 +/- 4 microg/ml from ERbeta. Treatment of the ER+ hormone-dependent T47D:A18 breast cancer cell line with the extract induced up-regulation of ERbeta mRNA. Progesterone receptor (PR) mRNA was upregulated in the Ishikawa endometrial cancer cell line. However, chaste-tree berry extract did not induce estrogen-dependent alkaline phosphatase (AP) activity in Ishikawa cells. Bioassay-guided isolation, utilizing ER binding as a monitor, resulted in the isolation of linoleic acid as one possible estrogenic component of the extract. The use of pulsed ultrafiltration liquid chromatography-mass spectrometry, which is an affinity-based screening technique, also identified linoleic acid as an ER ligand based on its selective affinity, molecular weight, and retention time. Linoleic acid also stimulated mRNA ERbeta expression in T47D:A18 cells, PR expression in Ishikawa cells, but not AP activity in Ishikawa cells. These data suggest that linoleic acid from the fruits of Vitex agnus-castus can bind to estrogen receptors and induce certain estrogen inducible genes.

Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells.

PubMed Central

Kojima, Hiroyuki; Katsura, Eiji; Takeuchi, Shinji; Niiyama, Kazuhito; Kobayashi, Kunihiko

2004-01-01

We tested 200 pesticides, including some of their isomers and metabolites, for agonism and antagonism to two human estrogen receptor (hER) subtypes, hERalpha and hERbeta, and a human androgen receptor (hAR) by highly sensitive transactivation assays using Chinese hamster ovary cells. The test compounds were classified into nine groups: organochlorines, diphenyl ethers, organophosphorus pesticides, pyrethroids, carbamates, acid amides, triazines, ureas, and others. These pesticides were tested at concentrations < 10-5 M. Of the 200 pesticides tested, 47 and 33 showed hER- and hERbeta-mediated estrogenic activities, respectively. Among them, 29 pesticides had both hERalpha and hERbeta agonistic activities, and the effects of the organochlorine insecticides beta-benzene hexachloride (BHC) and delta-BHC and the carbamate insecticide methiocarb were predominantly hERbeta rather than hERalpha agonistic. Weak antagonistic effects toward hERalpha and hERbeta were shown in five and two pesticides, respectively. On the other hand, none of tested pesticides showed hAR-mediated androgenic activity, but 66 of 200 pesticides exhibited inhibitory activity against the transcriptional activity induced by 5alpha-dihydrotestosterone. In particular, the antiandrogenic activities of two diphenyl ether herbicides, chlornitrofen and chlomethoxyfen, were higher than those of vinclozolin and p,p -dichlorodiphenyl dichloroethylene, known AR antagonists. The results of our ER and AR assays show that 34 pesticides possessed both estrogenic and antiandrogenic activities, indicating pleiotropic effects on hER and hAR. We also discussed chemical structures related to these activities. Taken together, our findings suggest that a variety of pesticides have estrogenic and/or antiandrogenic potential via ER and/or AR, and that numerous other manmade chemicals may also possess such estrogenic and antiandrogenic activities. PMID:15064155

Melatonin affects the dynamic steady-state equilibrium of estrogen sulfates in human umbilical vein endothelial cells by regulating the balance between estrogen sulfatase and sulfotransferase.

PubMed

González, Alicia; Martínez-Campa, Carlos; Alonso-González, Carolina; Cos, Samuel

2015-12-01

Melatonin is known to reduce the growth of endocrine-responsive breast cancers by interacting with estrogen signaling pathways. Estrogens play an important role in breast cancer, but also in various types of tissues, including vascular tissue. Estrogen sulfatase (STS) converts inactive estrogen sulfates into active estrogens, whereas estrogen sulfotransferase (EST) sulfonates estrogens to estrogen sulfates. Therefore, STS and EST are considered to be involved in the regulation of local estrogen levels in hormone‑dependent tumors and in non-pathologic tissues, such as those of the vascular system. Estrogens have a major impact on the vasculature, influencing vascular function, the expression of adhesion proteins, angiogenesis and the inflammatory state. In this study, we investigated the status of STS and EST in human umbilical vein endothelial cells (HUVECs) and the modulatory effects of melatonin. Both STS and EST were highly expressed in the HUVECs. The enzymatic activity correlated with the expression levels in these cells. Our findings also demonstrated that melatonin, at physiological concentrations, modulated the synthesis and transformation of biologically active estrogens in HUVECs through the inhibition of STS activity and expression, and the stimulation of EST activity and expression. Since melatonin decreased the STS levels and increased the EST levels, it modified the dynamic steady‑state equilibrium of estrogen sulfates by increasing the inactive estrogen levels and decreasing the active estrogen levels. Therefore, melatonin may modulate the known different biological actions of estrogens in endothelial cells, as well as in estrogen-dependent tumors and non-pathologic tissues.

75 FR 67963 - Endocrine Disruptor Screening Program (EDSP); Announcing the Availability of a Draft for Weight...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-04

... ENVIRONMENTAL PROTECTION AGENCY [EPA-HQ-OPPT-2010-0877; FRL-8849-8] Endocrine Disruptor Screening...-tiered paradigm for screening and testing chemicals for endocrine activity (i.e., estrogen, androgen, and... 5417), e.g., persons who conduct testing of chemical substances for endocrine effects. This listing is...

Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

PubMed

Kobayashi, Takehiko; Sasaki, Mariko

2017-01-01

The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors.

PubMed

Madureira, Tânia Vieira; Malhão, Fernanda; Pinheiro, Ivone; Lopes, Célia; Ferreira, Nádia; Urbatzka, Ralph; Castro, L Filipe C; Rocha, Eduardo

2015-12-01

Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50μM; (2) an ER antagonist (ICI 182,780) at 10 and 50μM; and (3) mixtures of both (Mix I-10μM EE2 and 50μM ICI; Mix II-1μM EE2 and 10μM ICI and Mix III-1μM EE2 and 50μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17β-hydroxysteroid dehydrogenase 4-17β-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERβ-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic

Antioxidant properties and global metabolite screening of the probiotic yeast Saccharomyces cerevisiae var. boulardii.

PubMed

Datta, Suprama; Timson, David J; Annapure, Uday S

2017-07-01

Saccharomyces cerevisiae var. boulardii is the only yeast species with probiotic properties. It is considered to have therapeutic significance in gastrointestinal disorders. In the present study, a comparative physiological study between this yeast and Saccharomyces cerevisiae (BY4742) was performed by evaluating two prominent traits of probiotic species, responses to different stress conditions and antioxidant capacity. A global metabolite profile was also developed aiming to identify which therapeutically important secondary metabolites are produced. Saccharomyces cerevisiae var. boulardii showed no significant difference in growth patterns but greater stress tolerance compared to S. cerevisiae. It also demonstrated a six- to 10-fold greater antioxidant potential (judged by the 1,1-diphenyl-2-picrylhydrazyl assay), with a 70-fold higher total phenolic content and a 20-fold higher total flavonoid content in the extracellular fraction. These features were clearly differentiated by principal component analysis and further indicated by metabolite profiling. The extracellular fraction of the S. cerevisiae var. boulardii cultures was found to be rich in polyphenolic metabolites: vanillic acid, cinnamic acid, phenyl ethyl alcohol (rose oil), erythromycin, amphetamine and vitamin B 6 , which results in the antioxidant capacity of this strain. The present study presents a new perspective for differentiating the two genetically related strains of yeast, S. cerevisiae and S. cerevisiae var. boulardii by assessing their metabolome fingerprints. In addition to the correlation of the phenotypic properties with the secretory metabolites of these two yeasts, the present study also emphasizes the potential to exploit S. cerevisiae var. boulardii in the industrial production of these metabolites. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

GPER Mediates Non-Genomic Effects of Estrogen.

PubMed

Pupo, Marco; Maggiolini, Marcello; Musti, Anna Maria

2016-01-01

Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a number of lines of evidence correlate the sustained exposure to exogenous estrogen with increased risk of the onset of various cancers. Mainly these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors, and upon activation they regulate the expression of different target genes by binding directly to specific DNA sequences. On the other hand, in recent years it has become clear that the G protein-coupled estrogen receptor 30 (GPR30/GPER) is able to mediate non-genomic action of estrogens in different cell contexts. In particular, GPER has been shown to specifically bind estrogens, and in turn to functionally cross-react with diverse cell signaling systems such as the epidermal growth factor receptor (EGFR) pathway, the Notch signaling pathway and the mitogen-activated protein kinases (MAPK) pathway. In this chapter we will present some of the different experimental techniques currently used to demonstrate the functional role of GPER in mediating non-genomic actions of estrogens, such as the dual luciferase assay, assessment of the involvement of GPER in the stimulation of cell migration in breast cancer cell lines and in cancer-associated fibroblasts, and chromatin immunoprecipitation assay. Overall, the experimental procedures described herein represent key instruments for assessing the biological role of GPER in mediating non-genomic signals of estrogen.

Interference of Paraben Compounds with Estrogen Metabolism by Inhibition of 17β-Hydroxysteroid Dehydrogenases

PubMed Central

Engeli, Roger T.; Rohrer, Simona R.; Vuorinen, Anna; Herdlinger, Sonja; Kaserer, Teresa; Leugger, Susanne; Schuster, Daniela

2017-01-01

Parabens are effective preservatives widely used in cosmetic products and processed food, with high human exposure. Recent evidence suggests that parabens exert estrogenic effects. This work investigated the potential interference of parabens with the estrogen-activating enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) 1 and the estrogen-inactivating 17β-HSD2. A ligand-based 17β-HSD2 pharmacophore model was applied to screen a cosmetic chemicals database, followed by in vitro testing of selected paraben compounds for inhibition of 17β-HSD1 and 17β-HSD2 activities. All tested parabens and paraben-like compounds, except their common metabolite p-hydroxybenzoic acid, inhibited 17β-HSD2. Ethylparaben and ethyl vanillate inhibited 17β-HSD2 with IC50 values of 4.6 ± 0.8 and 1.3 ± 0.3 µM, respectively. Additionally, parabens size-dependently inhibited 17β-HSD1, whereby hexyl- and heptylparaben were most active with IC50 values of 2.6 ± 0.6 and 1.8 ± 0.3 µM. Low micromolar concentrations of hexyl- and heptylparaben decreased 17β-HSD1 activity, and ethylparaben and ethyl vanillate decreased 17β-HSD2 activity. However, regarding the very rapid metabolism of these compounds to the inactive p-hydroxybenzoic acid by esterases, it needs to be determined under which conditions low micromolar concentrations of these parabens or their mixtures can occur in target cells to effectively disturb estrogen effects in vivo. PMID:28925944

Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians – interactions with estrogens, androgens, and thyroid hormones

EPA Science Inventory

Endocrine disruption is considered a highly relevant endpoint for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening for endocrine disruption – with focus on vertebrates (fish and amphibians) and estrogen, and...

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

PubMed

Nuñez, S B; Medin, J A; Braissant, O; Kemp, L; Wahli, W; Ozato, K; Segars, J H

1997-03-14

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

The Estrogen Receptor-α in Osteoclasts Mediates the Protective Effects of Estrogens on Cancellous But Not Cortical Bone

PubMed Central

Martin-Millan, Marta; Almeida, Maria; Ambrogini, Elena; Han, Li; Zhao, Haibo; Weinstein, Robert S.; Jilka, Robert L.; O'Brien, Charles A.; Manolagas, Stavros C.

2010-01-01

Estrogens attenuate osteoclastogenesis and stimulate osteoclast apoptosis, but the molecular mechanism and contribution of these effects to the overall antiosteoporotic efficacy of estrogens remain controversial. We selectively deleted the estrogen receptor (ER)α from the monocyte/macrophage cell lineage in mice (ERαLysM−/−) and found a 2-fold increase in osteoclast progenitors in the marrow and the number of osteoclasts in cancellous bone, along with a decrease in cancellous bone mass. After loss of estrogens these mice failed to exhibit the expected increase in osteoclast progenitors, the number of osteoclasts in bone, and further loss of cancellous bone. However, they lost cortical bone indistinguishably from their littermate controls. Mature osteoclasts from ERαLysM−/− were resistant to the proapoptotic effect of 17β-estradiol. Nonetheless, the effects of estrogens on osteoclasts were unhindered in mice bearing an ERα knock-in mutation that prevented binding to DNA. Moreover, a polymeric form of estrogen that is not capable of stimulating the nuclear-initiated actions of ERα was as effective as 17β-estradiol in inducing osteoclast apoptosis in cells with the wild-type ERα. We conclude that estrogens attenuate osteoclast generation and life span via cell autonomous effects mediated by DNA-binding-independent actions of ERα. Elimination of these effects is sufficient for loss of bone in the cancellous compartment in which complete perforation of trabeculae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming osteoblasts. However, additional effects of estrogens on osteoblasts, osteocytes, and perhaps other cell types are required for their protective effects on the cortical compartment, which constitutes 80% of the skeleton. PMID:20053716

Targeting estrogen/estrogen receptor alpha enhances Bacillus Calmette-Guérin efficacy in bladder cancer

PubMed Central

Shang, Zhiqun; Li, Yanjun; Hsu, Iawen; Zhang, Minghao; Tian, Jing; Wen, Simeng; Han, Ruifa; Messing, Edward M.; Chang, Chawnshang; Niu, Yuanjie; Yeh, Shuyuan

2016-01-01

Recent studies showed the potential linkage of estrogen/estrogen receptor signaling with bladder tumorigenesis, yet detailed mechanisms remain elusive. Here we found a new potential therapy with the combination of Bacillus Calmette Guerin (BCG) and the anti-estrogen ICI 182,780 led to better suppression of bladder cancer (BCa) than BCG alone. Mechanism dissection found ICI 182,780 could promote BCG attachment/internalization to the BCa cells through increased integrin-α5β1 expression and IL-6 release, which may enhance BCG-induced suppression of BCa cell growth via recruiting more monocytes/macrophages to BCa cells and increased TNF-α release. Consistently, in vivo studies found ICI 182,780 could potentiate the anti-BCa effects of BCG in the carcinogen-induced mouse BCa models. Together, these in vitro and in vivo results suggest that combining BCG with anti-estrogen may become a new therapeutic approach with better efficacy to suppress BCa progression and recurrence. PMID:27092883

Targeting estrogen/estrogen receptor alpha enhances Bacillus Calmette-Guérin efficacy in bladder cancer.

PubMed

Shang, Zhiqun; Li, Yanjun; Hsu, Iawen; Zhang, Minghao; Tian, Jing; Wen, Simeng; Han, Ruifa; Messing, Edward M; Chang, Chawnshang; Niu, Yuanjie; Yeh, Shuyuan

2016-05-10

Recent studies showed the potential linkage of estrogen/estrogen receptor signaling with bladder tumorigenesis, yet detailed mechanisms remain elusive. Here we found a new potential therapy with the combination of Bacillus Calmette-Guerin (BCG) and the anti-estrogen ICI 182,780 led to better suppression of bladder cancer (BCa) than BCG alone. Mechanism dissection found ICI 182,780 could promote BCG attachment/internalization to the BCa cells through increased integrin-α5β1 expression and IL-6 release, which may enhance BCG-induced suppression of BCa cell growth via recruiting more monocytes/macrophages to BCa cells and increased TNF-α release. Consistently, in vivo studies found ICI 182,780 could potentiate the anti-BCa effects of BCG in the carcinogen-induced mouse BCa models. Together, these in vitro and in vivo results suggest that combining BCG with anti-estrogen may become a new therapeutic approach with better efficacy to suppress BCa progression and recurrence.

Contaminants assessment in the coral reefs of Virgin Islands National Park and Virgin Islands Coral Reef National Monument

USGS Publications Warehouse

Bargar, Timothy A.; Garrison, Virginia H.; Alvarez, David A.; Echols, Kathy

2013-01-01

Coral, fish, plankton, and detritus samples were collected from coral reefs in Virgin Islands National Park (VIIS) and Virgin Islands Coral Reef National Monument (VICR) to assess existing contamination levels. Passive water sampling using polar organic chemical integrative samplers (POCIS) and semi-permeable membrane devices found a few emerging pollutants of concern (DEET and galaxolide) and polynuclear aromatic hydrocarbons. Very little persistent organic chemical contamination was detected in the tissue or detritus samples. Detected contaminants were at concentrations below those reported to be harmful to aquatic organisms. Extracts from the POCIS were subjected to the yeast estrogen screen (YES) to assess potential estrogenicity of the contaminant mixture. Results of the YES (estrogen equivalency of 0.17–0.31 ng/L 17-β-estradiol) indicated a low estrogenicity likelihood for contaminants extracted from water. Findings point to low levels of polar and non-polar organic contaminants in the bays sampled within VICR and VIIS.

A systematic screen for morphological abnormalities during fission yeast sexual reproduction identifies a mechanism of actin aster formation for cell fusion

PubMed Central

Groux, Raphaël; Vincenzetti, Vincent

2017-01-01

In non-motile fungi, sexual reproduction relies on strong morphogenetic changes in response to pheromone signaling. We report here on a systematic screen for morphological abnormalities of the mating process in fission yeast Schizosaccharomyces pombe. We derived a homothallic (self-fertile) collection of viable deletions, which, upon visual screening, revealed a plethora of phenotypes affecting all stages of the mating process, including cell polarization, cell fusion and sporulation. Cell fusion relies on the formation of the fusion focus, an aster-like F-actin structure that is marked by strong local accumulation of the myosin V Myo52, which concentrates secretion at the fusion site. A secondary screen for fusion-defective mutants identified the myosin V Myo51-associated coiled-coil proteins Rng8 and Rng9 as critical for the coalescence of the fusion focus. Indeed, rng8Δ and rng9Δ mutant cells exhibit multiple stable dots at the cell-cell contact site, instead of the single focus observed in wildtype. Rng8 and Rng9 accumulate on the fusion focus, dependent on Myo51 and tropomyosin Cdc8. A tropomyosin mutant allele, which compromises Rng8/9 localization but not actin binding, similarly leads to multiple stable dots instead of a single focus. By contrast, myo51 deletion does not strongly affect fusion focus coalescence. We propose that focusing of the actin filaments in the fusion aster primarily relies on Rng8/9-dependent cross-linking of tropomyosin-actin filaments. PMID:28410370

Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

NASA Astrophysics Data System (ADS)

van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

Characterization of diverse internal binding specificities of PDZ domains by yeast two-hybrid screening of a special peptide library.

PubMed

Mu, Yi; Cai, Pengfei; Hu, Siqi; Ma, Sucan; Gao, Youhe

2014-01-01

Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.

Yeast Infection (Vaginal)

MedlinePlus

Yeast infection (vaginal) Overview A vaginal yeast infection is a fungal infection that causes irritation, discharge and intense itchiness ... symptoms Causes The fungus candida causes a vaginal yeast infection. Your vagina naturally contains a balanced mix of yeast, including ...

Nuclear size as estrogen-responsive chromatin quality parameter of mouse spermatozoa.

PubMed

Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Viggiano, Andrea; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

2013-11-01

Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1(-/-)) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1(+/+), Cnr(+/-) and Cnr1(-/-) epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1(-/-) sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality. Copyright © 2013 Elsevier Inc. All rights reserved.

In vitro assessment of estrogen, androgen, and glucocorticoid bioactivity in a nationwide screen of United States stream waters

EPA Science Inventory

In vitro bioassays can identify environmental samples contaminated with bioactive chemicals that interact with steroid receptors and provide a cumulative, effect-based measurement of contamination. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR...

In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay##

EPA Science Inventory

Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms of...

In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay

EPA Science Inventory

Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms o...

Estrogen synthesis and signaling pathways during ageing: from periphery to brain

PubMed Central

Cui, Jie; Shen, Yong; Li, Rena

2012-01-01

Estrogens are the primary female sex hormones and play important roles in both reproductive and non-reproductive systems. Estrogens can be synthesized in non-reproductive tissue as liver, heart, muscle, bone and brain. The tissue-specific estrogen synthesis is consistent with a diversity of estrogen actions. Here, we will focus on tissue and cell-specific estrogen synthesis and estrogen receptor signaling. This review will include three parts: (I) tissue and cell-specific estrogen synthesis and metabolism, (II) tissue and cell-specific distribution of estrogen receptors and signaling and (III) tissue-specific estrogen function and related disorders, including cardiovascular diseases, osteoporosis, Alzheimer's disease and Parkinson disease. This comprehensive review provides new insights into estrogens by giving a better understanding of the tissue-specific estrogen effects and their roles in various diseases. PMID:23348042

Estrogen and Progestin (Hormone Replacement Therapy)

MedlinePlus

... progestin are two female sex hormones. Hormone replacement therapy works by replacing estrogen hormone that is no ... Progestin is added to estrogen in hormone replacement therapy to reduce the risk of uterine cancer in ...

High-throughput analysis of yeast replicative aging using a microfluidic system

PubMed Central

Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

2015-01-01

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

Protocols and programs for high-throughput growth and aging phenotyping in yeast.

PubMed

Jung, Paul P; Christian, Nils; Kay, Daniel P; Skupin, Alexander; Linster, Carole L

2015-01-01

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

Identification of Breast Cancer Inhibitors Specific for G Protein-Coupled Estrogen Receptor (GPER)-Expressing Cells.

PubMed

Aiello, Francesca; Carullo, Gabriele; Giordano, Francesca; Spina, Elena; Nigro, Alessandra; Garofalo, Antonio; Tassini, Sabrina; Costantino, Gabriele; Vincetti, Paolo; Bruno, Agostino; Radi, Marco

2017-08-22

Together with estrogen receptors ERα and ERβ, the G protein-coupled estrogen receptor (GPER) mediates important pathophysiological signaling pathways induced by estrogens and is currently regarded as a promising target for ER-negative (ER-) and triple-negative (TN) breast cancer. Only a few selective GPER modulators have been reported to date, and their use in cancer cell lines has often led to contradictory results. Herein we report the application of virtual screening and cell-based studies for the identification of new chemical scaffolds with a specific antiproliferative effect against GPER-expressing breast cancer cell lines. Out of the four different scaffolds identified, 8-chloro-4-(4-chlorophenyl)pyrrolo[1,2-a]quinoxaline 14 c was found to be the most promising compound able to induce: 1) antiproliferative activity in GPER-expressing cell lines (MCF7 and SKBR3), similarly to G15; 2) no effect on cells that do not express GPER (HEK293); 3) a decrease in cyclin D1 expression; and 4) a sustained induction of cell-cycle negative regulators p53 and p21. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aerobic Exercise, Estrogens, and Breast Cancer Risk

DTIC Science & Technology

2011-11-01

on endogenous sex hormone levels, menstrual cycle characteristics, and estrogen metabolism in sedentary, eumenorrheic, healthy premenopausal women...changes in menstrual cycle length, and 4) limited changes in estrogen metabolism. The resulting increases in urinary 2-hydroxyestrone levels and 2...effects of a 16-week, aerobic exercise intervention on endogenous sex hormone levels, menstrual cycle characteristics, and estrogen metabolism of young

Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts.

PubMed

Gonçalves, Margarida; Pontes, Ana; Almeida, Pedro; Barbosa, Raquel; Serra, Marta; Libkind, Diego; Hutzler, Mathias; Gonçalves, Paula; Sampaio, José Paulo

2016-10-24

Beer is one of the oldest alcoholic beverages and is produced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S. cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single or multiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked to brewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequence of fundamental differences in the modes of beer and wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Environmental estrogen(s) induced swimming behavioural alterations in adult zebrafish (Danio rerio).

PubMed

Goundadkar, Basavaraj B; Katti, Pancharatna

2017-09-01

The present study is an attempt to investigate the effects of long-term (75days) exposure to environmental estrogens (EE) on the swimming behaviour of zebrafish (Danio rerio). Adult zebrafish were exposed semi-statically to media containing commonly detected estrogenic water contaminants (EE2, DES and BPA) at a concentration (5ng/L) much lower than environmentally recorded levels. Time spent in swimming, surface preference, patterns and path of swimming were recorded (6mins) for each fish using two video cameras on day 15, 30 60 and 75. Video clips were analysed using a software program. Results indicate that chronic exposure to EE leads to increased body weight and size of females, reduced (P<0.05) swimming time, delay in latency, increased (P<0.05) immobility, erratic movements and freezing episodes. We conclude that estrogenic contamination of natural aquatic systems induces alterations in locomotor behaviour and associated physiological disturbances in inhabitant fish fauna. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of transdermal estrogen replacement therapy on cardiovascular risk factors.

PubMed

Menon, Dileep V; Vongpatanasin, Wanpen

2006-01-01

The prevalence of hypertension and cardiovascular disease increases dramatically after menopause in women, implicating estrogen as having a protective role in the cardiovascular system. However, recent large clinical trials have failed to show cardiovascular benefit, and have even demonstrated possible harmful effects, of opposed and unopposed estrogen in postmenopausal women. While these findings have led to a revision of guidelines such that they discourage the use of estrogen for primary or secondary prevention of heart disease in postmenopausal women, many investigators have attributed the negative results in clinical trials to several flaws in study design, including the older age of study participants and the initiation of estrogen late after menopause.Because almost all clinical trials use oral estrogen as the primary form of hormone supplementation, another question that has arisen is the importance of the route of estrogen administration with regards to the cardiovascular outcomes. During oral estrogen administration, the concentration of estradiol in the liver sinusoids is four to five times higher than that in the systemic circulation. This supraphysiologic concentration of estrogen in the liver can modulate the expression of many hepatic-derived proteins, which are not observed in premenopausal women. In contrast, transdermal estrogen delivers the hormone directly into the systemic circulation and, thus, avoids the first-pass hepatic effect.Although oral estrogen exerts a more favorable influence than transdermal estrogen on traditional cardiovascular risk factors such as high- and low-density lipoprotein-cholesterol levels, recent studies have indicated that oral estrogen adversely influences many emerging risk factors in ways that are not seen with transdermal estrogen. Oral estrogen significantly increases levels of acute-phase proteins such as C-reactive protein and serum amyloid A; procoagulant factors such as prothrombin fragments 1+2; and several

Estrogen in cardiovascular disease during systemic lupus erythematosus.

PubMed

Gilbert, Emily L; Ryan, Michael J

2014-12-01

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease that disproportionately affects women during their childbearing years. Cardiovascular disease is the leading cause of mortality in this patient population at an age when women often have low cardiovascular risk. Hypertension is a major cardiovascular disease risk factor, and its prevalence is markedly increased in women with SLE. Estrogen has traditionally been implicated in SLE disease progression because of the prevalence of the disease in women; however, its role in cardiovascular risk factors such as hypertension is unclear. The objective of this review is to discuss evidence for the role of estrogen in both human and murine SLE with emphasis on the effect of estrogen on cardiovascular risk factors, including hypertension. PubMed was used to search for articles with terms related to estradiol and SLE. The references of retrieved publications were also reviewed. The potential permissive role of estrogen in SLE development is supported by studies from experimental animal models of lupus in which early removal of estrogen or its effects leads to attenuation of SLE disease parameters, including autoantibody production and renal injury. However, data about the role of estrogens in human SLE are much less clear, with most studies not reaching firm conclusions about positive or negative outcomes after hormonal manipulations involving estrogen during SLE (ie, oral contraceptives, hormone therapy). Significant gaps in knowledge remain about the effect of estrogen on cardiovascular risk factors during SLE. Studies in women with SLE were not designed to determine the effect of estrogen or hormone therapy on blood pressure even though hypertension is highly prevalent, and risk of premature ovarian failure could necessitate use of hormone therapy in women with SLE. Recent evidence from an experimental animal model of lupus found that estrogen may protect against cardiovascular risk factors in

Estrogen in Cardiovascular Disease during Systemic Lupus Erythematosus

PubMed Central

Gilbert, Emily L.; Ryan, Michael J.

2015-01-01

Purpose Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease that disproportionately affects women during their childbearing years. Cardiovascular disease is the leading cause of mortality in this patient population at an age when women often have low cardiovascular risk. Hypertension is a major cardiovascular disease risk factor, and its prevalence is markedly increased in women with SLE. Estrogen has traditionally been implicated in SLE disease progression because of the prevalence of the disease in women; however, its role in cardiovascular risk factors such as hypertension is unclear. The objective of this review is to discuss evidence for the role of estrogen in both human and murine SLE with emphasis on the effect of estrogen on cardiovascular risk factors, including hypertension. Methods PubMed was used to search for articles with terms related to estradiol and SLE. The references of retrieved publications were also reviewed. Findings The potential permissive role of estrogen in SLE development is supported by studies from experimental animal models of lupus in which early removal of estrogen or its effects leads to attenuation of SLE disease parameters, including autoantibody production and renal injury. However, data about the role of estrogens in human SLE are much less clear, with most studies not reaching firm conclusions about positive or negative outcomes after hormonal manipulations involving estrogen during SLE (ie, oral contraceptives, hormone therapy). Significant gaps in knowledge remain about the effect of estrogen on cardiovascular risk factors during SLE. Studies in women with SLE were not designed to determine the effect of estrogen or hormone therapy on blood pressure even though hypertension is highly prevalent, and risk of premature ovarian failure could necessitate use of hormone therapy in women with SLE. Recent evidence from an experimental animal model of lupus found that estrogen may protect against

GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

PubMed Central

Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

2010-01-01

Summary Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition. PMID:17623774

Estrogen receptor status of breast cancer in Ontario

PubMed Central

McKeown-Eyssen, Gail E.; Rogers-Melamed, Iris; Clarke, E. Aileen

1985-01-01

Data from a number of studies of breast cancer have suggested that after the ages associated with the menopause the rates of estrogen-receptor-positive tumours increase with age, whereas the rates of estrogen-receptor-negative tumours do not. Previous investigators studied cases in specific treatment centres, so there was a possibility that the findings were influenced by differences in patterns of case referral by age. A review of all the cases of breast cancer diagnosed in Ontario women in 1981 and assayed for estrogen receptors, however, confirmed the earlier findings. The results showed that the incidence of estrogen-receptor-positive and estrogen-receptor-negative tumours increased at about the same rate before age 45, but thereafter an increase in incidence was seen only for estrogen-receptor-positive tumours. These differences in patterns of incidence suggest the possibility that the two types of tumour may have different etiologic factors. PMID:4063915

Mapping protein-protein interactions using yeast two-hybrid assays.

PubMed

Mehla, Jitender; Caufield, J Harry; Uetz, Peter

2015-05-01

Yeast two-hybrid (Y2H) screens are an efficient system for mapping protein-protein interactions and whole interactomes. The screens can be performed using random libraries or collections of defined open reading frames (ORFs) called ORFeomes. This protocol describes both library and array-based Y2H screening, with an emphasis on array-based assays. Array-based Y2H is commonly used to test a number of "prey" proteins for interactions with a single "bait" (target) protein or pool of proteins. The advantage of this approach is the direct identification of interacting protein pairs without further downstream experiments: The identity of the preys is known and does not require further confirmation. In contrast, constructing and screening a random prey library requires identification of individual prey clones and systematic retesting. Retesting is typically performed in an array format. © 2015 Cold Spring Harbor Laboratory Press.

Estrogen Injection

MedlinePlus

... used to treat hot flushes (hot flashes; sudden strong feelings of heat and sweating) and/or vaginal ... the symptoms of certain types of prostate (a male reproductive organ) cancer. The conjugated estrogens form of ...

Insights into Rapid Modulation of Neuroplasticity by Brain Estrogens

PubMed Central

Woolfrey, Kevin M.; Penzes, Peter

2013-01-01

Converging evidence from cellular, electrophysiological, anatomic, and behavioral studies suggests that the remodeling of synapse structure and function is a critical component of cognition. This modulation of neuroplasticity can be achieved through the actions of numerous extracellular signals. Moreover, it is thought that it is the integration of different extracellular signals regulation of neuroplasticity that greatly influences cognitive function. One group of signals that exerts powerful effects on multiple neurologic processes is estrogens. Classically, estrogens have been described to exert their effects over a period of hours to days. However, there is now increasing evidence that estrogens can rapidly influence multiple behaviors, including those that require forebrain neural circuitry. Moreover, these effects are found in both sexes. Critically, it is now emerging that the modulation of cognition by rapid estrogenic signaling is achieved by activation of specific signaling cascades and regulation of synapse structure and function, cumulating in the rewiring of neural circuits. The importance of understanding the rapid effects of estrogens on forebrain function and circuitry is further emphasized as investigations continue to consider the potential of estrogenic-based therapies for neuropathologies. This review focuses on how estrogens can rapidly influence cognition and the emerging mechanisms that underlie these effects. We discuss the potential sources and the biosynthesis of estrogens within the brain and the consequences of rapid estrogenic-signaling on the remodeling of neural circuits. Furthermore, we argue that estrogens act via distinct signaling pathways to modulate synapse structure and function in a manner that may vary with cell type, developmental stage, and sex. Finally, we present a model in which the coordination of rapid estrogenic-signaling and activity-dependent stimuli can result in long-lasting changes in neural circuits

The use of a whole animal biophotonic model as a screen for the angiogenic potential of estrogenic compounds

USDA-ARS?s Scientific Manuscript database

Vascular endothelial growth factor (VEGF) is essential for normal vascular growth and development during wound repair. VEGF is estrogen responsive and capable of regulating its own receptor, vascular endothelial growth factor receptor-2 (VEGFR-2). Several agricultural pesticides (e.g., methoxychlor)...

Estrogens and Androgens in Skeletal Physiology and Pathophysiology

PubMed Central

Almeida, Maria; Laurent, Michaël R.; Dubois, Vanessa; Claessens, Frank; O'Brien, Charles A.; Bouillon, Roger; Vanderschueren, Dirk

2016-01-01

Estrogens and androgens influence the growth and maintenance of the mammalian skeleton and are responsible for its sexual dimorphism. Estrogen deficiency at menopause or loss of both estrogens and androgens in elderly men contribute to the development of osteoporosis, one of the most common and impactful metabolic diseases of old age. In the last 20 years, basic and clinical research advances, genetic insights from humans and rodents, and newer imaging technologies have changed considerably the landscape of our understanding of bone biology as well as the relationship between sex steroids and the physiology and pathophysiology of bone metabolism. Together with the appreciation of the side effects of estrogen-related therapies on breast cancer and cardiovascular diseases, these advances have also drastically altered the treatment of osteoporosis. In this article, we provide a comprehensive review of the molecular and cellular mechanisms of action of estrogens and androgens on bone, their influences on skeletal homeostasis during growth and adulthood, the pathogenetic mechanisms of the adverse effects of their deficiency on the female and male skeleton, as well as the role of natural and synthetic estrogenic or androgenic compounds in the pharmacotherapy of osteoporosis. We highlight latest advances on the crosstalk between hormonal and mechanical signals, the relevance of the antioxidant properties of estrogens and androgens, the difference of their cellular targets in different bone envelopes, the role of estrogen deficiency in male osteoporosis, and the contribution of estrogen or androgen deficiency to the monomorphic effects of aging on skeletal involution. PMID:27807202

Estrogens and Androgens in Skeletal Physiology and Pathophysiology.

PubMed

Almeida, Maria; Laurent, Michaël R; Dubois, Vanessa; Claessens, Frank; O'Brien, Charles A; Bouillon, Roger; Vanderschueren, Dirk; Manolagas, Stavros C

2017-01-01

Estrogens and androgens influence the growth and maintenance of the mammalian skeleton and are responsible for its sexual dimorphism. Estrogen deficiency at menopause or loss of both estrogens and androgens in elderly men contribute to the development of osteoporosis, one of the most common and impactful metabolic diseases of old age. In the last 20 years, basic and clinical research advances, genetic insights from humans and rodents, and newer imaging technologies have changed considerably the landscape of our understanding of bone biology as well as the relationship between sex steroids and the physiology and pathophysiology of bone metabolism. Together with the appreciation of the side effects of estrogen-related therapies on breast cancer and cardiovascular diseases, these advances have also drastically altered the treatment of osteoporosis. In this article, we provide a comprehensive review of the molecular and cellular mechanisms of action of estrogens and androgens on bone, their influences on skeletal homeostasis during growth and adulthood, the pathogenetic mechanisms of the adverse effects of their deficiency on the female and male skeleton, as well as the role of natural and synthetic estrogenic or androgenic compounds in the pharmacotherapy of osteoporosis. We highlight latest advances on the crosstalk between hormonal and mechanical signals, the relevance of the antioxidant properties of estrogens and androgens, the difference of their cellular targets in different bone envelopes, the role of estrogen deficiency in male osteoporosis, and the contribution of estrogen or androgen deficiency to the monomorphic effects of aging on skeletal involution. Copyright © 2017 the American Physiological Society.

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

PubMed

Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

2016-01-01

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

Engineered yeast for enhanced CO2 mineralization†

PubMed Central

Barbero, Roberto; Carnelli, Lino; Simon, Anna; Kao, Albert; Monforte, Alessandra d’Arminio; Riccò, Moreno; Bianchi, Daniele; Belcher, Angela

2014-01-01

In this work, a biologically catalyzed CO2 mineralization process for the capture of CO2 from point sources was designed, constructed at a laboratory scale, and, using standard chemical process scale-up protocols, was modeled and evaluated at an industrial scale. A yeast display system in Saccharomyces cerevisae was used to screen several carbonic anhydrase isoforms and mineralization peptides for their impact on CO2 hydration, CaCO3 mineralization, and particle settling rate. Enhanced rates for each of these steps in the CaCO3 mineralization process were confirmed using quantitative techniques in lab-scale measurements. The effect of these enhanced rates on the CO2 capture cost in an industrial scale CO2 mineralization process using coal fly ash as the CaO source was evaluated. The model predicts a process using bCA2- yeast and fly ash is ~10% more cost effective per ton of CO2 captured than a process with no biological molecules, a savings not realized by wild-type yeast and high-temperature stable recombinant CA2 alone or in combination. The levelized cost of electricity for a power plant using this process was calculated and scenarios in which this process compares favorably to CO2 capture by MEA absorption process are presented. PMID:25289021

ANALYSIS OF LAGOON SAMPLES FROM DIFFERENT CONCENTRATED ANIMAL FEEDING OPERATIONS (CAFOS) FOR ESTROGENS AND ESTROGEN CONJUGATES (PRESENTATION)

EPA Science Inventory

Although Concentrated Animal Feeding Operations (CAFOs) have been identified as potentially important sources for the release of estrogens into the environment, information is lacking on the concentrations of estrogens in whole lagoon effluents (including suspended solids) which ...

Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

PubMed

Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

2016-09-01

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).

Non-interferometric quantitative phase imaging of yeast cells

NASA Astrophysics Data System (ADS)

Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

Estrogenic modulation of auditory processing: a vertebrate comparison

PubMed Central

Caras, Melissa L.

2013-01-01

Sex-steroid hormones are well-known regulators of vocal motor behavior in several organisms. A large body of evidence now indicates that these same hormones modulate processing at multiple levels of the ascending auditory pathway. The goal of this review is to provide a comparative analysis of the role of estrogens in vertebrate auditory function. Four major conclusions can be drawn from the literature: First, estrogens may influence the development of the mammalian auditory system. Second, estrogenic signaling protects the mammalian auditory system from noise- and age-related damage. Third, estrogens optimize auditory processing during periods of reproductive readiness in multiple vertebrate lineages. Finally, brain-derived estrogens can act locally to enhance auditory response properties in at least one avian species. This comparative examination may lead to a better appreciation of the role of estrogens in the processing of natural vocalizations and may provide useful insights toward alleviating auditory dysfunctions emanating from hormonal imbalances. PMID:23911849

Estrogenic activity of lambda-cyhalothrin in the MCF-7 human breast carcinoma cell line.

PubMed

Zhao, Meirong; Zhang, Ying; Liu, Weiping; Xu, Chao; Wang, Lumei; Gan, Jianying

2008-05-01

Synthetic pyrethroids are widely used in both agricultural and urban environments for insect control. Lambda-cyhalothrin (LCT) is one of the most common pyrethroids and is used mainly for controlling mosquitoes, fleas, cockroaches, flies, and ants around households. Previous studies have addressed the environmental behaviors and acute toxicities of LCT, but little is known about its chronic toxicity, such as estrogen-like activity. In the present study, the estrogenic potential of LCT was evaluated using the MCF-7 human breast carcinoma cell line. The in vitro E-screen assay showed that 10(-7) M LCT could significantly promote MCF-7 cell proliferation, with a relative proliferative effect ratio of 45%. The cell proliferation induced by LCT could be blocked completely, however, by the addition of 10(-9) M of the estrogen receptor (ER)-antagonist ICI 182,780. The semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) results showed that the Trefoil factor 1 (pS2) and progesterone receptor gene expression were up-regulated by 10(-7) M LCT for 2- and 1.5-fold, respectively. On the other hand, RT-PCR, Western blot analysis, and immunofluorescent assay demonstrated that LCT significantly repressed the mRNA and protein expression levels of ERalpha and ERbeta. These observations indicate that LCT possesses estrogenic properties and may function as a xenoestrogen, likely via a mechanism similar to that of 17beta-estradiol. The endocrine-disruption potential of LCT should be considered when assessing the safety of this compound in sensitive environmental compartments.

Extracellular matrix integrity: a possible mechanism for differential clinical effects among selective estrogen receptor modulators and estrogens?

PubMed

Cox, David A; Helvering, Leah M

2006-03-09

Recent gene microarray studies have illustrated heterogeneity in gene expression changes not only between estrogens and selective estrogen receptor modulators (SERMs), but also across different SERM molecules. In ovariectomized rats, this phenomenon was observed with respect to a number of genes involved in collagen turnover and extracellular matrix (ECM) integrity in the uterus and vaginal tissues. Preliminary mechanistic data suggest that these effects on ECM integrity may have relevance in the context of the effect of estrogens and some SERMs to increase the risk of pelvic organ prolapse and the incidence of urinary incontinence in postmenopausal women. Given the pivotal role of ECM integrity and collagen turnover in other tissues and disease states, these processes may provide a fruitful target for future research into the mechanisms for the heterogeneous pharmacology of estrogens and SERMs across different cell types and target tissues.

Serum factors and clinical characteristics associated with serum E-Screen activity

PubMed Central

Wang, Jue; Trentham-Dietz, Amy; Hemming, Jocelyn D. C.; Hedman, Curtis J.; Sprague, Brian L.

2013-01-01

Background The E-Screen bioassay can measure the mitogenicity of human serum and thus may be useful as a biomarker in epidemiologic studies of breast cancer. While the assay’s MCF-7 cells are known to proliferate in response to estrogen, the specific determinants of variation in E-Screen activity in human serum samples are poorly understood. We sought to identify serum molecules and patient characteristics associated with serum E-Screen activity among postmenopausal women. Methods Postmenopausal women (N=219) aged 55–70 with no history of postmenopausal hormone use or breast cancer completed a questionnaire and provided a blood sample. Serum was analyzed for E-Screen activity and a variety of molecules including sex hormones, growth factors, and environmental chemicals. Stepwise selection procedures were used to identify correlates of E-Screen activity. Results Serum samples from all women had detectable E-Screen activity, with a median estradiol equivalents value of 0.027 ng/mL and interquartile range of 0.018–0.036 ng/mL. In the final multivariable-adjusted model, serum E-Screen activity was positively associated with serum estradiol, estrone, IGFBP-3, and testosterone levels (p<0.05), as well as body mass index (p=0.03). Serum E-Screen activity was lower among women with higher SHBG (p<0.0001) and progesterone levels (p=0.03). Conclusion Serum E-Screen activity varies according to levels of endogenous estrogens and other serum molecules. Obesity appears to confer additional serum mitogenicity beyond its impact on the measured hormones and growth factors. Impact By capturing mitogenicity due to a variety of patient and serum factors, the E-Screen may provide advantages for use as a biomarker in breast cancer studies. PMID:23588007

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

PubMed

Zingue, Stéphane; Nde, Chantal Beatrice Magne; Michel, Thomas; Ndinteh, Derek Tantoh; Tchatchou, Jules; Adamou, Moïse; Fernandez, Xavier; Fohouo, Fernand-Nestor Tchuenguem; Clyne, Colin; Njamen, Dieudonné

2017-01-21

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. 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 β (ERβ) 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. In vitro, EEP showed an antiestrogenic activity in both HEK293T ER-α and ER-β 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. 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

Persistent aryl hydrocarbon receptor inducers increase with altitude, and estrogen-like disrupters are low in soils of the Alps.

PubMed

Levy, Walkiria; Henkelmann, Bernhard; Bernhöft, Silke; Bovee, Toine; Buegger, Franz; Jakobi, Gert; Kirchner, Manfred; Bassan, Rodolfo; Kräuchi, Norbert; Moche, Wolfgang; Offenthaler, Ivo; Simončič, Primoz; Weiss, Peter; Schramm, Karl-Werner

2011-01-01

Soil samples from remote Alpine areas were analyzed for polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans and polychlorinated biphenyls by high-resolution gas chromatography/high-resolution gas spectrometry. Additionally, the EROD micro-assay and a genetically modified yeast estrogen bioassay were carried out to determine persistent aryl hydrocarbon receptors (AhR) and estrogen receptors (ER) agonists, respectively. Regarding the AhR agonists, the toxicity equivalents of analytical and EROD determined values were compared, targeting both altitude of samples and their soil organic content. The ratio between bioassay derived equivalents and analytical determinations suggested no significant contribution of unknown AhR inducers in these sampling sites and some antagonism in soils with relatively high PCB loading. More CYP1A1 expression was induced at the highest sites or about 1400-1500 m a.s.l. along the altitude profiles. Surprisingly, no clear tendencies with the soil organic content were found for dioxin-like compounds. Mean values obtained in the present study were for ER agonists, 2: 0.37±0.12ng 17ß-estradiol EQ g-1 dry soil [corrected] and 6.1 ± 4.2 pg TCDD-EQ g⁻¹ dry soil for AhR agonists. Low bioassay responses with a higher relative amount of ER disrupters than AhR inducers were detected,indicating the higher abundance of estrogen-like than persistent dioxin-like compounds in these forested areas [corrected].

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

USGS Publications Warehouse

Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki

2014-01-01

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

High-Throughput Screening to Identify Regulators of Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

PubMed

Kassir, Yona

2017-01-01

Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.

Estrogen-induced myelotoxicity in dogs: A review

PubMed Central

Sontas, Hasan B.; Dokuzeylu, Banu; Turna, Ozge; Ekici, Hayri

2009-01-01

Exogenous estrogens used for therapeutic purposes or endogenous estrogen sources such as functional Sertoli cell or ovarian granulosa cell tumors may cause bone marrow toxicity in dogs. The condition is characterized by hematologic abnormalities including thrombocytopenia, anemia, and leukocytosis or leukopenia. Despite intensive therapy with blood or platelet-rich transfusions, broad-spectrum antibiotics, steroids, and bone marrow stimulants, prognosis is unfavorable. Due to the the risk of stimulating the development of uterine diseases and the potential for inducing aplastic anemia, estrogen use in dogs is best avoided where possible. This paper describes the causes of estrogen-induced myelotoxicity, the clinical presentation of the patients, the diagnosis, and the treatment options in the dog. PMID:20046604

Quantum chemical studies of estrogenic compounds

USDA-ARS?s Scientific Manuscript database

Quantum chemical methods are potent tools to provide information on the chemical structure and electronic properties of organic molecules. Modern computational chemistry methods have provided a great deal of insight into the binding of estrogenic compounds to estrogenic receptors (ER), an important ...

CLONING AND IN VITRO EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR α FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

EPA Science Inventory

In vitro screening assays designed to identify hormone mimics or antagonists typically use mammalian (rat, human) estrogen (ER) and androgen receptors (AR). Although we know that the amino acid sequences of steroid receptors in nonmammalian vertebrates are not identical to the ma...

Estrogen modulates sexually dimorphic contextual fear extinction in rats through estrogen receptor beta.

PubMed

Chang, Yao-Ju; Yang, Chih-Hao; Liang, Ying-Ching; Yeh, Che-Ming; Huang, Chiung-Chun; Hsu, Kuei-Sen

2009-11-01

Females and males are different in brain and behavior. These sex differences occur early during development due to a combination of genetic and hormonal factors and continue throughout the lifespan. Previous studies revealed that male rats exhibited significantly higher levels of contextual fear memory than female rats. However, it remains unknown whether a sex difference exists in the contextual fear extinction. To address this issue, male, normally cycling female, and ovariectomized (OVX) female Sprague-Dawley rats were subjected to contextual fear conditioning and extinction trials. Here we report that although male rats exhibited higher levels of freezing than cycling female rats after contextual fear conditioning, female rats subjected to conditioning in the proestrus and estrus stage exhibited an enhancement of fear extinction than male rats. An estrogen receptor (ER) beta agonist diarylpropionitrile but not an ERalpha agonist propyl-pyrazole-triol administration also enhanced extinction of contextual fear in OVX female rats, suggesting that estrogen-mediated facilitation of extinction involves the activation of ERbeta. Intrahippocampal injection of estradiol or diarylpropionitrile before extinction training in OVX female rats remarkably reduced the levels of freezing response during extinction trials. In addition, the locomotion or anxiety state of female rats does not vary across the ovarian cycle. These results reveal a crucial role for estrogen in mediating sexually dimorphic contextual fear extinction, and that estrogen-mediated enhancement of fear extinction involves the activation of ERbeta.

Estrogens and progression of diabetic kidney damage.

PubMed

Doublier, Sophie; Lupia, Enrico; Catanuto, Paola; Elliot, Sharon J

2011-01-01

It is generally accepted that estrogens affect and modulate the development and progression of chronic kidney diseases (CKD) not related to diabetes. Clinical studies have indeed demonstrated that the severity and rate of progression of renal damage tends to be greater among men, compared with women. Experimental studies also support the notion that female sex is protective and male sex permissive, for the development of CKD in non-diabetics, through the opposing actions of estrogens and testosterone. However, when we consider diabetes-induced kidney damage, in the setting of either type 1 or type 2 diabetes, the contribution of gender to the progression of renal disease is somewhat uncertain. Previous studies on the effects of estrogens in the pathogenesis of progressive kidney damage have primarily focused on mesangial cells. More recently, data on the effects of estrogens on podocytes, the cell type whose role may include initiation of progressive diabetic renal disease, became available. The aim of this review will be to summarize the main clinical and experimental data on the effects of estrogens on the progression of diabetes-induced kidney injury. In particular, we will highlight the possible biological effects of estrogens on podocytes, especially considering those critical for the pathogenesis of diabetic kidney damage.

Isoliquiritigenin exhibits anti-proliferative properties in the pituitary independent of estrogen receptor function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weis, Karen E.; Raetzman, Lori T., E-mail: raetzma

The plant flavonoid isoliquiritigenin (ISL) is a botanical estrogen widely taken as an herbal supplement to ease the symptoms of menopause. ISL has been also shown to have anti-tumor properties in a number of cancer cell backgrounds. However, the effects of ISL on normal cells are less well known and virtually unstudied in the context of the pituitary gland. We have established a pituitary explant culture model to screen chemical agents for gene expression changes within the pituitary gland during a period of active proliferation and differentiation. Using this whole-organ culture system we found ISL to be weakly estrogenic basedmore » on its ability to induce Cckar mRNA expression, an estrogen receptor (ER) mediated gene. Using a range of ISL from 200 nM to 200 μM, we discovered that ISL promoted cell proliferation at a low concentration, yet potently inhibited proliferation at the highest concentration. ICI 182,780 failed to antagonize ISL's repression of pituitary cell proliferation, indicating the effect is independent of ER signaling. Coincident with a decrease in proliferating cells, we observed down-regulation of transcript for cyclin D2 and E2 and a strong induction of mRNA and protein for the cyclin dependent kinase inhibitor Cdkn1a (p21). Importantly, high dose ISL did not alter the balance of progenitor vs. differentiated cell types within the pituitary explants and they seemed otherwise healthy; however, TUNEL staining revealed an increase in apoptotic cell death in ISL treated cultures. Our results merit further examination of ISL as an anti-tumor agent in the pituitary gland. - Highlights: • Isoliquiritigenin possesses weak estrogenic activity based on induction of Cckar. • ISL can be anti-proliferative in pituitary explants without altering cell lineages. • Anti-proliferative behavior of ISL is not estrogen receptor mediated. • ISL induces p21 expression leading to cell cycle arrest and apoptosis.« less

Exploring the Yeast Acetylome Using Functional Genomics

PubMed Central

Duffy, Supipi Kaluarachchi; Friesen, Helena; Baryshnikova, Anastasia; Lambert, Jean-Philippe; Chong, Yolanda T.; Figeys, Daniel; Andrews, Brenda

2014-01-01

SUMMARY Lysine acetylation is a dynamic posttranslational modification with a well-defined role in regulating histones. The impact of acetylation on other cellular functions remains relatively uncharacterized. We explored the budding yeast acetylome with a functional genomics approach, assessing the effects of gene overexpression in the absence of lysine deacetylases (KDACs). We generated a network of 463 synthetic dosage lethal (SDL) interactions involving class I and II KDACs, revealing many cellular pathways regulated by different KDACs. A biochemical survey of genes interacting with the KDAC RPD3 identified 72 proteins acetylated in vivo. In-depth analysis of one of these proteins, Swi4, revealed a role for acetylation in G1-specific gene expression. Acetylation of Swi4 regulates interaction with its partner Swi6, both components of the SBF transcription factor. This study expands our view of the yeast acetylome, demonstrates the utility of functional genomic screens for exploring enzymatic pathways, and provides functional information that can be mined for future studies. PMID:22579291

A gene expression biomarker accurately predicts estrogen ...

EPA Pesticide Factsheets

The EPA’s vision for the Endocrine Disruptor Screening Program (EDSP) in the 21st Century (EDSP21) includes utilization of high-throughput screening (HTS) assays coupled with computational modeling to prioritize chemicals with the goal of eventually replacing current Tier 1 screening tests. The ToxCast program currently includes 18 HTS in vitro assays that evaluate the ability of chemicals to modulate estrogen receptor α (ERα), an important endocrine target. We propose microarray-based gene expression profiling as a complementary approach to predict ERα modulation and have developed computational methods to identify ERα modulators in an existing database of whole-genome microarray data. The ERα biomarker consisted of 46 ERα-regulated genes with consistent expression patterns across 7 known ER agonists and 3 known ER antagonists. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression data sets from experiments in MCF-7 cells. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% or 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) OECD ER reference chemicals including “very weak” agonists and replicated predictions based on 18 in vitro ER-associated HTS assays. For 114 chemicals present in both the HTS data and the MCF-7 c

Estrogen Abolishes Latent Inhibition in Ovariectomized Female Rats

ERIC Educational Resources Information Center

Nofrey, Barbara S.; Ben-Shahar, Osnat M.; Brake, Wayne G.

2008-01-01

Estrogen is frequently prescribed as a method of birth control and as hormone replacement therapy for post-menopausal women with varied effects on cognition. Here the effects of estrogen on attention were examined using the latent inhibition (LI) behavioral paradigm. Ovariectomized (OVX) female rats were given either estrogen benzoate (EB, 10 or…

The yeast genome may harbor hypoxia response elements (HRE).

PubMed

Ferreira, Túlio César; Hertzberg, Libi; Gassmann, Max; Campos, Elida Geralda

2007-01-01

The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated when cells are submitted to hypoxia. The heterodimer is composed of two subunits, HIF-1alpha and the constitutively expressed HIF-1beta. During normoxia, HIF-1alpha is degraded by the 26S proteasome, but hypoxia causes HIF-1alpha to be stabilized, enter the nucleus and bind to HIF-1beta, thus forming the active complex. The complex then binds to the regulatory sequences of various genes involved in physiological and pathological processes. The specific regulatory sequence recognized by HIF-1 is the hypoxia response element (HRE) that has the consensus sequence 5'BRCGTGVBBB3'. Although the basic transcriptional regulation machinery is conserved between yeast and mammals, Saccharomyces cerevisiae does not express HIF-1 subunits. However, we hypothesized that baker's yeast has a protein analogous to HIF-1 which participates in the response to changes in oxygen levels by binding to HRE sequences. In this study we screened the yeast genome for HREs using probabilistic motif search tools. We described 24 yeast genes containing motifs with high probability of being HREs (p-value<0.1) and classified them according to biological function. Our results show that S. cerevisiae may harbor HREs and indicate that a transcription factor analogous to HIF-1 may exist in this organism.

Bioflavoring by non-conventional yeasts in sequential beer fermentations.

PubMed

Holt, Sylvester; Mukherjee, Vaskar; Lievens, Bart; Verstrepen, Kevin J; Thevelein, Johan M

2018-06-01

Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compounds and their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomyces species and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ultrasensitive environmental assessment of xeno-estrogens in water samples using label-free graphene immunosensors.

PubMed

Barton, Huw; Berbel-Filho, Waldir M; Consuegra, Sofia; Francis, Lewis; Tizaoui, Chedly; Conlan, R Steven; Teixeira, Sofia Rodrigues

2018-05-01

There is a growing interest in the possible environmental health impact posed by endocrine-disrupting chemicals (EDCs). A challenge to the field of endocrine disruption is that these substances are diverse and may not appear to share any structural similarity other than usually being low molecular mass (<1000 Da) compounds. Here we demonstrate the effectiveness of sensor device for the detection of low molecular weight, poorly water soluble, estrogenic compounds E1, E2 and EE2, fabricated by electropolymerization over graphene screen printed electrode (SPE). The PANI/Gr-SPE-devices displayed linear responses to estrogenic substances, in EIS assays, from 0.0975 ng/L to 200 ng/L in water samples, with a detection limit of 0.043 pg/L for E1, 0.19 ng/L for E2 and 0.070 pg/L for EE2 which is lower than other current biosensing techniques. This portable, disposable immunosensor offers a solution for immediate measurement at sample collection sites, due to its excellent sensitivity and selectivity when testing water samples obtained directly from rivers and waste water treatment facilities. The simple screen printing production method will enable the low cost, high volume production required for this type of environmental analysis. Copyright © 2018 Elsevier Inc. All rights reserved.

Estrogen-dependent changes in serum iron levels as a translator of the adverse effects of estrogen during infection: a conceptual framework.

PubMed

Hamad, Mawieh; Awadallah, Samir

2013-12-01

Elevated levels of estrogen often associate with increased susceptibility to infection. This has been attributed to the ability of estrogen to concomitantly enhance the growth and virulence of pathogens and suppress host immunity. But the exact mechanism of how estrogen mediates such effects, especially in cases where the pathogen and/or the immune components in question do not express estrogen receptors, has yet to be elucidated. Here we propose that translating the adverse effects of estrogen during infection is dependent to a significant degree upon its ability to manipulate iron homeostasis. For elevated levels of estrogen alter the synthesis and/or activity of several factors involved in iron metabolism including hypoxia inducible factor 1α (HIF-1α) and hepcidin among others. This leads to the inhibition of hepcidin synthesis in hepatocytes and the maintenance of ferroportin (FPN) integrity on the surface of iron-releasing duodenal enterocytes, hepatocytes, and macrophages. Intact FPN permits the continuous efflux of dietary and stored iron into the circulation, which further enhances pathogen growth and virulence on the one hand and suppresses host immunity on the other. This new conceptual framework may help explain a multitude of disparate clinical and experimental observations pertinent to the relationship between estrogen and infection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Metabolite profiling of microfluidic cell culture conditions for droplet based screening.

PubMed

Bjork, Sara M; Sjostrom, Staffan L; Andersson-Svahn, Helene; Joensson, Haakan N

2015-07-01

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

PubMed

Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

2014-09-01

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines. Published by Elsevier Ltd.

Activation of the G protein-coupled estrogen receptor, but not estrogen receptor α or β, rapidly enhances social learning.

PubMed

Ervin, Kelsy Sharice Jean; Mulvale, Erin; Gallagher, Nicola; Roussel, Véronique; Choleris, Elena

2015-08-01

Social learning is a highly adaptive process by which an animal acquires information from a conspecific. While estrogens are known to modulate learning and memory, much of this research focuses on individual learning. Estrogens have been shown to enhance social learning on a long-term time scale, likely via genomic mechanisms. Estrogens have also been shown to affect individual learning on a rapid time scale through cell-signaling cascades, rather than via genomic effects, suggesting they may also rapidly influence social learning. We therefore investigated the effects of 17β-estradiol and involvement of the estrogen receptors (ERs) using the ERα agonist propyl pyrazole triol, the ERβ agonist diarylpropionitrile, and the G protein-coupled ER 1 (GPER1) agonist G1 on the social transmission of food preferences (STFP) task, within a time scale that focused on the rapid effects of estrogens. General ER activation with 17β-estradiol resulted in a modest facilitation of social learning, with mice showing a preference up to 30min of testing. Specific activation of the GPER1 also rapidly enhanced social learning, with mice showing a socially learned preference up to 2h of testing. ERα activation instead shortened the expression of a socially learned food preference, while ERβ activation had little to no effects. Thus, rapid estrogenic modulation of social learning in the STFP may be the outcome of competing action at the three main receptors. Hence, estrogens' rapid effects on social learning likely depend on the specific ERs present in brain regions recruited during social learning. Copyright © 2015 Elsevier Ltd. All rights reserved.

Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

PubMed Central

Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

2012-01-01

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

Brain Sex Matters: estrogen in cognition and Alzheimer’s disease

PubMed Central

Li, Rena; Cui, Jie; Shen, Yong

2014-01-01

Estrogens are the primary female sex hormones and play important roles in both reproductive and non-reproductive systems. Estrogens can be synthesized in non-reproductive tissues such as liver, heart, muscle, bone and the brain. During the past decade, increasing evidence suggests that brain estrogen can not only be synthesized by neurons, but also by astrocytes. Brain estrogen also works locally at the site of synthesis in paracrine and/or intracrine fashion to maintain important tissue-specific functions. Here, we will focus on the biology of brain estrogen and its impact on cognitive function and Alzheimer’s disease. This comprehensive review provides new insights into brain estrogens by presenting a better understanding of the tissue-specific estrogen effects and their roles in healthy ageing and cognitive function. PMID:24418360

Estrogens and Coronary Artery Disease: New Clinical Perspectives.

PubMed

Meyer, M R; Barton, M

2016-01-01

In premenopausal women, endogenous estrogens are associated with reduced prevalence of arterial hypertension, coronary artery disease, myocardial infarction, and stroke. Clinical trials conducted in the 1990s such as HERS, WHI, and WISDOM have shown that postmenopausal treatment with horse hormone mixtures (so-called conjugated equine estrogens) and synthetic progestins adversely affects female cardiovascular health. Our understanding of rapid (nongenomic) and chronic (genomic) estrogen signaling has since advanced considerably, including identification of a new G protein-coupled estrogen receptor (GPER), which like the "classical" receptors ERα and ERβ is highly abundant in the cardiovascular system. Here, we discuss the role of estrogen receptors in the pathogenesis of coronary artery disease and review natural and synthetic ligands of estrogen receptors as well as their effects in physiology, on cardiovascular risk factors, and atherosclerotic vascular disease. Data from preclinical and clinical studies using nonselective compounds activating GPER, which include selective estrogen receptor modulators such as tamoxifen or raloxifene, selective estrogen receptor downregulators such as Faslodex™ (fulvestrant/ICI 182,780), vitamin B3 (niacin), green tea catechins, and soy flavonoids such as genistein or resveratrol, strongly suggest that activation of GPER may afford therapeutic benefit for primary and secondary prevention in patients with or at risk for coronary artery disease. Evidence from preclinical studies suggest similar efficacy profiles for selective small molecule GPER agonists such as G-1 which are devoid of uterotrophic activity. Further clinical research in this area is warranted to provide opportunities for future cardiovascular drug development. © 2016 Elsevier Inc. All rights reserved.

Mechanism of estrogen activation of c-myc oncogene expression.

PubMed

Dubik, D; Shiu, R P

1992-08-01

The estrogen receptor complex is a known trans-acting factor that regulates transcription of specific genes through an interaction with a specific estrogen-responsive cis-acting element (ERE). In previous studies we have shown that in estrogen-responsive human breast cancer cells estrogen rapidly activates c-myc expression. This activated expression occurs through enhanced transcription and does not require the synthesis of new protein intermediates; therefore, an ERE is present in the human c-myc gene regulatory region. To localize the ERE, constructs containing varying lengths of the c-myc 5'-flanking region ranging from -2327 to +25 (relative to the P1 promoter) placed adjacent to the chloramphenicol acetyl transferase reporter gene (CAT) were prepared. They were used in transient transfection studies in MCF-7 and HeLa cells co-transfected with an estrogen receptor expression vector. These studies reveal that all constructs containing the P2 promoter region exhibited estrogen-regulated CAT expression and that a 116-bp region upstream and encompassing the P2 TATA box is necessary for this activity. Analysis of this 116-bp region failed to identify a cis-acting element with sequences resembling the consensus ERE; however, co-transfection studies with mutant estrogen receptor expression vectors showed that the DNA-binding domain of the receptor is essential for estrogen-regulated CAT gene expression. We have also observed that anti-estrogen receptor complexes can weakly trans-activate from this 116-bp region but fail to do so from the ERE-containing ApoVLDLII-CAT construct. To explain these results we propose a new mechanism of estrogen trans-activation in the c-myc gene promoter.

Postmenopausal estrogen and progestin effects on the serum proteome

PubMed Central

2009-01-01

Background Women's Health Initiative randomized trials of postmenopausal hormone therapy reported intervention effects on several clinical outcomes, with some important differences between estrogen alone and estrogen plus progestin. The biologic mechanisms underlying these effects, and these differences, have yet to be fully elucidated. Methods Baseline serum samples were compared with samples drawn 1 year later for 50 women assigned to active hormone therapy in both the estrogen-plus-progestin and estrogen-alone randomized trials, by applying an in-depth proteomic discovery platform to serum pools from 10 women per pool. Results In total, 378 proteins were quantified in two or more of the 10 pooled serum comparisons, by using strict identification criteria. Of these, 169 (44.7%) showed evidence (nominal P < 0.05) of change in concentration between baseline and 1 year for one or both of estrogen-plus-progestin and estrogen-alone groups. Quantitative changes were highly correlated between the two hormone-therapy preparations. A total of 98 proteins had false discovery rates < 0.05 for change with estrogen plus progestin, compared with 94 for estrogen alone. Of these, 84 had false discovery rates <0.05 for both preparations. The observed changes included multiple proteins relevant to coagulation, inflammation, immune response, metabolism, cell adhesion, growth factors, and osteogenesis. Evidence of differential changes also was noted between the hormone preparations, with the strongest evidence in growth factor and inflammation pathways. Conclusions Serum proteomic analyses yielded a large number of proteins similarly affected by estrogen plus progestin and by estrogen alone and identified some proteins and pathways that appear to be differentially affected between the two hormone preparations; this may explain their distinct clinical effects. PMID:20034393

Cumulative estrogen exposure and prospective memory in older women.

PubMed

Hesson, Jacqueline

2012-10-01

This study looked at cumulative lifetime estrogen exposure, as estimated with a mathematical index (Index of Cumulative Estrogen Exposure (ICEE)) that included variables (length of time on estrogen therapy, age at menarche and menopause, postmenopausal body mass index, time since menopause, nulliparity and duration of breastfeeding) known to influence estrogen levels across the life span, and performance on prospective and retrospective memory measures in a group of 50 postmenopausal women (mean age=69.3years) who, if they were current or former users of estrogen therapy, had started therapy within 5years of menopause. The ICEE was found to be a significant predictor of performance on the Prospective Memory task (F(1)=4.21, p=.046, η(p)(2)=.084). No significant relationship was noted between the ICEE and performance on measures of retrospective memory. The results suggest that the level of cumulative lifetime exposure to estrogen a woman has influences her prospective memory performance later in life and that the influence of reproductive and biological markers of endogenous estrogen exposure are relevant factors to consider when studying the effect of estrogen therapy on cognitive functioning in postmenopausal women. In addition, the finding that performance on a measure of prospective memory, but not performance on measures of retrospective memory, was associated with the ICEE adds further support to the theory that the frontal cortex may be especially sensitive to estrogen. Copyright © 2012 Elsevier Inc. All rights reserved.

The Endocrine Role of Estrogens on Human Male Skeleton

PubMed Central

Rochira, Vincenzo; Kara, Elda; Carani, Cesare

2015-01-01

Before the characterization of human and animal models of estrogen deficiency, estrogen action was confined in the context of the female bone. These interesting models uncovered a wide spectrum of unexpected estrogen actions on bone in males, allowing the formulation of an estrogen-centric theory useful to explain how sex steroids act on bone in men. Most of the principal physiological events that take place in the developing and mature male bone are now considered to be under the control of estrogen. Estrogen determines the acceleration of bone elongation at puberty, epiphyseal closure, harmonic skeletal proportions, the achievement of peak bone mass, and the maintenance of bone mass. Furthermore, it seems to crosstalk with androgen even in the determination of bone size, a more androgen-dependent phenomenon. At puberty, epiphyseal closure and growth arrest occur when a critical number of estrogens is reached. The same mechanism based on a critical threshold of serum estradiol seems to operate in men during adulthood for bone mass maintenance via the modulation of bone formation and resorption in men. This threshold should be better identified in-between the ranges of 15 and 25 pg/mL. Future basic and clinical research will optimize strategies for the management of bone diseases related to estrogen deficiency in men. PMID:25873947

GPR30: A G protein-coupled receptor for estrogen.

PubMed

Prossnitz, Eric R; Arterburn, Jeffrey B; Sklar, Larry A

2007-02-01

Estrogen is a critical steroid in human physiology exerting its effect both at the transcriptional level as well as at the level of rapid intracellular signaling through second messengers. Many of estrogen's transcriptional effects have long been known to be mediated through classical nuclear steroid receptors but recent studies also demonstrate the existence of a 7-transmembrane G protein-coupled receptor, GPR30 that responds to estrogen with rapid cellular signaling. There is currently controversy over the ability of classical estrogen receptors to recapitulate GPR30-mediated signaling mechanisms and vice versa. This article will summarize recent literature and address the relationship between GPR30 and conventional estrogen receptor signaling.

SCREENING CALIFORNIA SURFACE WATERS FOR ESTROGENIC ENDOCRINE DISRUPTING CHEMICALS (EEDC) WITH A JUVENILE RAINBOW TROUT LIVER VITELLOGENIN MRNA PROCEDURE

EPA Science Inventory

Concern regarding the occurrence of chemicals that disrupt endocrine system functions in aquatic species has heightened over the last 15 years. However, little attention has been given to monitoring for estrogenic endocrine disrupting chemicals (EEDCs) in California's freshwater ...

21 CFR 862.1270 - Estrogens (total, in pregnancy) test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Estrogens (total, in pregnancy) test system. 862... Test Systems § 862.1270 Estrogens (total, in pregnancy) test system. (a) Identification. As estrogens (total, in pregnancy) test system is a device intended to measure total estrogens in plasma, serum, and...

Differential neonatal imprinting and regulation by estrogen of estrogen receptor subtypes alpha and beta and of the truncated estrogen receptor product (TERP-1) mRNA expression in the male rat pituitary.

PubMed

Tena-Sempere, M; Barreiro, M L; González, L C; Pinilla, L; Aguilar, E

2001-11-01

Two distinct nuclear estrogen receptors (ERs) have been identified, the classical one, renamed ERalpha, and the more recently cloned ERbeta. In a variety of tissues, gene expression of both receptor subtypes results in the generation of multiple transcripts encoding the full-length as well as several alternately spliced isoforms. In the rat pituitary, a truncated, tissue-specific variant of ERalpha, called TERP-1, has been identified and found able to modulate ERalpha and ERbeta activity. So far, its pattern of expression and hormonal regulation have been mostly studied in females. The present study was designed to analyze the pattern of expression of TERP-1 mRNA in the male rat pituitary at different stages of postnatal development, and to evaluate the impact of neonatal imprinting and estrogen treatment upon TERP-1 expression in the male pituitary. Assessment of TERP-1 mRNA levels by semi-quantitative RT-PCR, using a variant-specific primer pair, revealed that TERP-1 is also expressed in the male rat pituitary. Relative mRNA expression levels changed markedly during postnatal development, with moderate expression of the TERP-1 transcript at birth, barely detectable levels during the infantile-prepubertal period, and maximal values in adulthood. Expression of TERP-1 was sensitive to neonatal estrogen exposure, which resulted in a significant, persistent increase in mRNA levels from the infantile period until puberty. This phenomenon was not mimicked by neonatal blockade of endogenous GnRH. In addition, estrogen was able to acutely up-regulate pituitary TERP-1 mRNA expression levels in prepubertal (30-day-old) and adult (75-day-old) males. Interestingly, neonatal imprinting as well as acute estrogen treatment resulted in opposite effects on TERP-1 and full-length ERalpha and ERbeta transcripts, the latter being decreased under both conditions. In conclusion, our data indicate that TERP-1 mRNA is expressed in a developmentally regulated manner in the male rat

Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.

PubMed Central

McCormick, M K; Campbell, E; Deaven, L; Moyzis, R

1993-01-01

Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach. Images PMID:8430075

Effect of vaginal estrogen on pessary use

PubMed Central

Dessie, Sybil G.; Armstrong, Katherine; Modest, Anna M.; Hacker, Michele R.

2016-01-01

Introduction and hypothesis Many providers recommend concurrent estrogen therapy with pessary use to limit complications; however, limited data exist to support this practice. We hypothesized that vaginal estrogen supplementation decreases incidence of pessary-related complications and discontinuation. Methods We performed a retrospective cohort study of women who underwent a pessary fitting from 1 January 2007 through 1 September 2013 at one institution; participants were identified by billing code and were eligible if they were post-menopausal and had at least 3 months of pessary use and 6 months of follow-up. All tests were two sided, and P values < 0.05 were considered statistically significant. Results Data from 199 women were included; 134 used vaginal estrogen and 65 did not. Women who used vaginal estrogen had a longer median follow-up time (29.5 months) compared with women who did not (15.4 months) and were more likely to have at least one pessary check (98.5 % vs 86.2 %, P < 0.001). Those in the estrogen group were less likely to discontinue using their pessary (30.6 % vs 58.5 %, P < 0.001) and less likely to develop increased vaginal discharge than women who did not [hazard ratio (HR) 0.31, 95 % confidence interval (CI) 0.17–0.58]. Vaginal estrogen was not protective against erosions (HR 0.93, 95 % CI 0.54–1.6) or vaginal bleeding (HR 0.78, 95 % CI 0.36–1.7). Conclusions Women who used vaginal estrogen exhibited a higher incidence of continued pessary use and lower incidence of increased vaginal discharge than women who did not. PMID:26992727

Oleaginous yeasts from Antarctica: Screening and preliminary approach on lipid accumulation.

PubMed

Viñarta, Silvana C; Angelicola, M Virginia; Barros, J Maximiliano; Fernández, Pablo M; Mac Cormak, Walter; Aybar, Manuel J; de Figueroa, Lucía I C

2016-12-01

The capability of 17 Rhodotorula spp. isolated from Antarctica to accumulate intracellular lipids in nitrogen-limited medium was investigated. As results, 10 isolates were selected by Nile red staining, while 12 isolates were selected as oleaginous by analysis of total lipid content (20.4-73%, w/w of dry biomass). The higher lipid production and accumulation was exhibited for six strains belonging to three species of Rhodotorula (Rhodotorula glutinis, Rhodotorula glacialis, and Rhodotorula laryngis). This is the first report where R. laryngis have been identified within oleaginous specie. Lipid accumulation was evaluated comparatively in two nitrogen-limited glucose-based media (MI and MII). MI (low C/N ratio) was more suitable for biomass and lipid production while in MII (high C/N ratio) total lipid content was improved. R. glutinis R4, R. glacialis R15, and R. glutinis R48 showed high lipid concentrations (4.65-6.93 g L -1 ) and they were able to accumulate large amounts of lipids per gram of biomass (47-77%, w/w). A similar profile in fatty acids composition and content of neutral lipids to vegetable oils was observed, indicating that lipids produced by oleaginous Antarctic yeasts can be considered an alternative feedstock for biodiesel production. Antarctica represents an important source of oleaginous yeasts with adaptive capabilities to accumulate considerable amounts of lipids with biotechnological interest at 15 °C and 25 °C. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A YEAST ESTROGEN SCREEN FOR EXAMINING THE RELATIVE EXPOSURE OF CELLS TO NATURAL AND XENO ESTROGENS. (R824760)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Treatment of BG-1 Ovarian Cancer Cells Expressing Estrogen Receptors with Lambda-cyhalothrin and Cypermethrin Caused a Partial Estrogenicity Via an Estrogen Receptor-dependent Pathway

PubMed Central

Kim, Cho-Won; Go, Ryeo-Eun

2015-01-01

Synthetic pyrethroids (SPs) are the most common pesticides which are recently used for indoor pest control. The widespread use of SPs has resulted in the increased exposure to wild animals and humans. Recently, some SPs are suspected as endocrine disrupting chemicals (EDCs) and have been assessed for their potential estrogenicity by adopting various analyzing assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LC) and cypermethrin (CP), the most commonly used pesticides in Korea, using BG-1 ovarian cancer cells expressing estrogen receptors (ERs). To evaluate the estrogenic activities of two SPs, LC and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT-PCR) in LC or CP treated BG-1 ovarian cancer cells. In MTT assay, LC (10−6 M) and CP (10−5 M) significantly induced the growth of BG-1 cancer cells. LC or CP-induced cell growth was antagonized by addition of ICI 182,720 (10−8 M), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT-PCR results showed that transcriptional level of cyclin D1, a cell cycle-regulating gene, was significantly up-regulated by LC and CP, while these effects were reversed by co-treatment of ICI 182,780. However, p21, a cyclin D-ckd-4 inhibitor gene, was not altered by LC or CP. Moreover, ERα expression was not significantly changed by LC and CP, while downregulated by E2. Finally, in xenografted mouse model transplanted with human BG-1 ovarian cancer cells, E2 significantly increased the tumor volume compare to a negative control, but LC did not. Taken together, these results suggest that LC and CP may possess estrogenic potentials by stimulating the growth of BG-1 ovarian cancer cells via partially ER signaling pathway associated with cell cycle as did E2, but this estrogenic effect was not found in in vivo mouse model. PMID:26877835

USE OF THE LABORATORY RAT AS A MODEL IN ENDOCRINE DISRUPTOR SCREENING AND TESTING

EPA Science Inventory

The screening and testing program the US Environmental Protection Agency is currently developing to detect endocrine-disrupting chemicals (EDCs) is described. EDCs have been shown to alter the following activities: hypothalamic-pituitary-gonadal [HPG] function; estrogen, androge...

Caffeine, coffee and tea intake and urinary estrogens and estrogen metabolites in premenopausal women

PubMed Central

Sisti, Julia S.; Hankinson, Susan E.; Caporaso, Neil E.; Gu, Fangyi; Tamimi, Rulla M.; Rosner, Bernard; Xu, Xia; Ziegler, Regina; Eliassen, A. Heather

2015-01-01

Background Prior studies have found weak inverse associations between breast cancer and caffeine and coffee intake, possibly mediated through their effects on sex hormones. Methods High-performance liquid chromatography/tandem mass spectrometry was used to quantify levels of 15 individual estrogens and estrogen metabolites (EM) among 587 premenopausal women in the Nurses’ Health Study II with mid-luteal phase urine samples and caffeine, coffee and/or tea intakes from self-reported food frequency questionnaires. Multivariate linear mixed models were used to estimate geometric means of individual EM, pathways and ratios by intake categories, and P-values for tests of linear trend. Results Compared to women in the lowest quartile of caffeine consumption, those in the top quartile had higher urinary concentrations of 16α-hydroxyestrone (28% difference; P-trend=0.01) and 16-epiestriol (13% difference; P-trend=0.04), and a decreased parent estrogens/2-, 4-, 16-pathway ratio (P-trend=0.03). Coffee intake was associated with higher 2-catechols, including 2-hydroxyestradiol (57% difference, ≥4 cups/day vs. ≤6 cups/week; P-trend=0.001) and 2-hydroxyestrone (52% difference; P-trend=0.001), and several ratio measures. Decaffeinated coffee was not associated with 2-pathway metabolism, but women in the highest (vs. lowest) category of intake (≥2 cups/day vs. ≤1–3 cups/month) had significantly lower levels of two 16-pathway metabolites, estriol (25% difference; P-trend=0.01) and 17-epiestriol (48% difference; Ptrend=0.0004). Tea intake was positively associated with 17-epiestriol (52% difference; Ptrend=0.01). Conclusion Caffeine and coffee intake were both associated with profiles of estrogen metabolism in premenopausal women. Impact Consumption of caffeine and coffee may alter patterns of premenopausal estrogen metabolism. PMID:26063478

Caffeine, coffee, and tea intake and urinary estrogens and estrogen metabolites in premenopausal women.

PubMed

Sisti, Julia S; Hankinson, Susan E; Caporaso, Neil E; Gu, Fangyi; Tamimi, Rulla M; Rosner, Bernard; Xu, Xia; Ziegler, Regina; Eliassen, A Heather

2015-08-01

Prior studies have found weak inverse associations between breast cancer and caffeine and coffee intake, possibly mediated through their effects on sex hormones. High-performance liquid chromatography/tandem mass spectrometry was used to quantify levels of 15 individual estrogens and estrogen metabolites (EM) among 587 premenopausal women in the Nurses' Health Study II with mid-luteal phase urine samples and caffeine, coffee, and/or tea intakes from self-reported food frequency questionnaires. Multivariate linear mixed models were used to estimate geometric means of individual EM, pathways, and ratios by intake categories, and P values for tests of linear trend. Compared with women in the lowest quartile of caffeine consumption, those in the top quartile had higher urinary concentrations of 16α-hydroxyestrone (28% difference; Ptrend = 0.01) and 16-epiestriol (13% difference; Ptrend = 0.04), and a decreased parent estrogens/2-, 4-, 16-pathway ratio (Ptrend = 0.03). Coffee intake was associated with higher 2-catechols, including 2-hydroxyestradiol (57% difference, ≥4 cups/day vs. ≤6 cups/week; Ptrend = 0.001) and 2-hydroxyestrone (52% difference; Ptrend = 0.001), and several ratio measures. Decaffeinated coffee was not associated with 2-pathway metabolism, but women in the highest (vs. lowest) category of intake (≥2 cups/day vs. ≤1-3 cups/month) had significantly lower levels of two 16-pathway metabolites, estriol (25% difference; Ptrend = 0.01) and 17-epiestriol (48% difference; Ptrend = 0.0004). Tea intake was positively associated with 17-epiestriol (52% difference; Ptrend = 0.01). Caffeine and coffee intake were both associated with profiles of estrogen metabolism in premenopausal women. Consumption of caffeine and coffee may alter patterns of premenopausal estrogen metabolism. ©2015 American Association for Cancer Research.

Estrogen-DNA Adducts as Novel Biomarkers for Ovarian Cancer Risk and for Use in Prevention

DTIC Science & Technology

2013-03-01

genes for four selected estrogen-metabolizing enzymes : cytochrome P450 (CYP)1A1 (I462V), CYP1B1 (V432L),catechol-O-methyltransferase (COMT) (V158M...homozygous for the catechol-O-methyltransferase allele and the cytochrome P450 1B1 high activity allele had significantly increased DNA adduct ratios and... enzyme polymorphisms to serve as biomarkers to screen for ovarian cancer . Task 1. Obtain approval of the protocol from the OCRP Human Research

Tissue-Specific Effects of Loss of Estrogen during Menopause and Aging.

PubMed

Wend, Korinna; Wend, Peter; Krum, Susan A

2012-01-01

The roles of estrogens have been best studied in the breast, breast cancers, and in the female reproductive tract. However, estrogens have important functions in almost every tissue in the body. Recent clinical trials such as the Women's Health Initiative have highlighted both the importance of estrogens and how little we know about the molecular mechanism of estrogens in these other tissues. In this review, we illustrate the diverse functions of estrogens in the bone, adipose tissue, skin, hair, brain, skeletal muscle and cardiovascular system, and how the loss of estrogens during aging affects these tissues. Early transcriptional targets of estrogen are reviewed in each tissue. We also describe the tissue-specific effects of selective estrogen receptor modulators (SERMs) used for the treatment of breast cancers and postmenopausal symptoms.

The human oxytocin gene promoter is regulated by estrogens.

PubMed

Richard, S; Zingg, H H

1990-04-15

Gonadal steroids affect brain function primarily by altering the expression of specific genes, yet the specific mechanisms by which neuronal target genes undergo such regulation are unknown. Recent evidence suggests that the expression of the neuropeptide gene for oxytocin (OT) is modulated by estrogens. We therefore examined the possibility that this regulation occurred via a direct interaction of the estrogen-receptor complex with cis-acting elements flanking the OT gene. DNA-mediated gene transfer experiments were performed using Neuro-2a neuroblastoma cells and chimeric plasmids containing portions of the human OT gene 5'-glanking region linked to the chloramphenicol acetyltransferase gene. We identified a 19-base pair region located at -164 to -146 upstream of the transcription start site which is capable of conferring estrogen responsiveness to the homologous as well as to a heterologous promoter. The hormonal response is strictly dependent on the presence of intracellular estrogen receptors, since estrogen induced stimulation occurred only in Neuro-2a cells co-transfected with an expression vector for the human estrogen receptor. The identified region contains a novel imperfect palindrome (GGTGACCTTGACC) with sequence similarity to other estrogen response elements (EREs). To define cis-acting elements that function in synergism with the ERE, sequences 3' to the ERE were deleted, including the CCAAT box, two additional motifs corresponding to the right half of the ERE palindrome (TGACC), as well as a CTGCTAA heptamer similar to the "elegans box" found in Caenorhabditis elegans. Interestingly, optimal function of the identified ERE was fully independent of these elements and only required a short promoter region (-49 to +36). Our studies define a molecular mechanism by which estrogens can directly modulate OT gene expression. However, only a subset of OT neurons are capable of binding estrogens, therefore, direct action of estrogens on the OT gene may be

Photocatalytic ozonation of urban wastewater and surface water using immobilized TiO2 with LEDs: Micropollutants, antibiotic resistance genes and estrogenic activity.

PubMed

Moreira, Nuno F F; Sousa, José M; Macedo, Gonçalo; Ribeiro, Ana R; Barreiros, Luisa; Pedrosa, Marta; Faria, Joaquim L; Pereira, M Fernando R; Castro-Silva, Sérgio; Segundo, Marcela A; Manaia, Célia M; Nunes, Olga C; Silva, Adrián M T

2016-05-01

Photocatalytic ozonation was employed for the first time in continuous mode with TiO2-coated glass Raschig rings and light emitting diodes (LEDs) to treat urban wastewater as well as surface water collected from the supply area of a drinking water treatment plant (DWTP). Different levels of contamination and types of contaminants were considered in this work, including chemical priority substances (PSs) and contaminants of emerging concern (CECs), as well as potential human opportunistic antibiotic resistant bacteria and their genes (ARB&ARG). Photocatalytic ozonation was more effective than single ozonation (or even than TiO2 catalytic ozonation) in the degradation of typical reaction by-products (such as oxalic acid), and more effective than photocatalysis to remove the parent micropollutants determined in urban wastewater. In fact, only fluoxetine, clarithromycin, erythromycin and 17-alpha-ethinylestradiol (EE2) were detected after photocatalytic ozonation, by using solid-phase extraction (SPE) pre-concentration and LC-MS/MS analysis. In surface water, this treatment allowed the removal of all determined micropollutants to levels below the limit of detection (0.01-0.20 ng L(-1)). The efficiency of this process was then assessed based on the capacity to remove different groups of cultivable microorganisms and housekeeping (16S rRNA) and antibiotic resistance or related genes (intI1, blaTEM, qnrS, sul1). Photocatalytic ozonation was observed to efficiently remove microorganisms and ARGs. Although after storage total heterotrophic and ARB (to ciprofloxacin, gentamicin, meropenem), fungi, and the genes 16S rRNA and intI1, increased to values close to the pre-treatment levels, the ARGs (blaTEM, qnrS and sul1) were reduced to levels below/close to the quantification limit even after 3-days storage of treated surface water or wastewater. Yeast estrogen screen (YES), thiazolyl blue tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) assays were also performed

Behaviour of estrogenic endocrine-disrupting chemicals in permeable carbonate sands.

PubMed

Shepherd, Benjamin O; Erler, Dirk V; Tait, Douglas R; van Zwieten, Lukas; Kimber, Stephen; Eyre, Bradley D

2015-08-01

The remediation of four estrogenic endocrine-disrupting compounds (EDCs), estrone (E1), estradiol (E2), ethinylestradiol (EE2) and estriol (E3), was measured in saturated and unsaturated carbonate sand-filled columns dosed with wastewater from a sewage treatment plant. The estrogen equivalency (EEQ) of inlet wastewater was 1.2 ng L(-1) and was remediated to an EEQ of 0.5 ng L(-1) through the unsaturated carbonate sand-filled columns. The high surface area of carbonate sand and associated high microbial activity may have assisted the degradation of these estrogens. The fully saturated sand columns showed an increase in total estrogenic potency with an EEQ of 2.4 ng L(-1), which was double that of the inlet wastewater. There was a significant difference (P < 0.05) in total estrogenic potency between aerobic and anaerobic columns. The breakdown of conjugated estrogens to estrogenic EDCs formed under long residence time and reducing conditions may have been responsible for the increase in the fully saturated columns. This may also be explained by the desorption of previously sorbed estrogenic EDCs. The effect of additional filter materials, such as basalt sediment and coconut fibre, on estrogenic EDC reduction was also tested. None of these amendments provided improvements in estrogen remediation relative to the unamended unsaturated carbonate sand columns. Aerobic carbonate sand filters have good potential to be used as on-site wastewater treatment systems for the reduction of estrogenic EDCs. However, the use of fully saturated sand filters, which are used to promote denitrification, and the loss of nitrogen as N2 were shown to cause an increase in EEQ. The potential for the accumulation of estrogenic EDCs under anaerobic conditions needs to be considered when designing on-site sand filtration systems required to reduce nitrogen. Furthermore, the accumulation of estrogens under anaerobic conditions such as under soil absorption systems or leachate fields has the

Estrogen Deficiency Promotes Cerebral Aneurysm Rupture by Upregulation of Th17 Cells and Interleukin-17A Which Downregulates E-Cadherin.

PubMed

Hoh, Brian L; Rojas, Kelley; Lin, Li; Fazal, Hanain Z; Hourani, Siham; Nowicki, Kamil W; Schneider, Matheus B; Hosaka, Koji

2018-04-13

Estrogen deficiency is associated with the development of cerebral aneurysms; however, the mechanism remains unknown. We explored the pathway of cerebral aneurysm development by investigating the potential link between estrogen deficiency and inflammatory factors. First, we established the role of interleukin-17 (IL-17)A. We performed a cytokine screen demonstrating that IL-17A is significantly expressed in mouse and human aneurysms ( P =0.03). Likewise, IL-17A inhibition was shown to prevent aneurysm formation by 42% ( P =0.02) and rupture by 34% ( P <0.05). Second, we found that estrogen deficiency upregulates T helper 17 cells and IL-17A and promotes aneurysm rupture. Estrogen-deficient mice had more ruptures than control mice (47% versus 7%; P =0.04). Estradiol supplementation or IL-17A inhibition decreased the number of ruptures in estrogen-deficient mice (estradiol 6% versus 37%; P =0.04; IL-17A inhibition 18% versus 47%; P =0.018). Third, we found that IL-17A-blockade protects against aneurysm formation and rupture by increased E-cadherin expression. IL-17-inhibited mice had increased E-cadherin expression ( P =0.003). E-cadherin inhibition reversed the protective effect of IL-17A inhibition and increased the rate of aneurysm formation (65% versus 28%; P =0.04) and rupture (12% versus 0%; P =0.22). However, E-cadherin inhibition alone does not significantly increase aneurysm formation in normal mice or in estrogen-deficient mice. In cell migration assays, E-cadherin inhibition promoted macrophage infiltration across endothelial cells ( P <0.05), which may be the mechanism for the estrogen deficiency/IL-17/E-cadherin aneurysm pathway. Our data suggest that estrogen deficiency promotes cerebral aneurysm rupture by upregulating IL-17A, which downregulates E-cadherin, encouraging macrophage infiltration in the aneurysm vessel wall. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Evaluation of yeasts from Tibetan fermented products as agents for biocontrol of blue mold of Nashi pear fruits*

PubMed Central

Hu, Hao; Xu, Yang; Lu, Huang-ping; Xiao, Rui; Zheng, Xiao-dong; Yu, Ting

2015-01-01

A total of 20 strains of yeast isolated from Tibetan fermented products were screened for antagonism against blue mold of pear caused by Penicillium expansum. Six isolates that inhibited incidence of postharvest decay by 35% or more were selected for further screening. Among them, the most effective was Rhodotorula mucilaginosa. The results showed that washed cell suspensions of R. mucilaginosa yielded better antagonistic efficacy than unwashed cell-culture mixtures, cell-free culture filtrates, and autoclaved cell cultures. Biocontrol activity improved with increasing concentrations of incubated cells. The best concentration was 1×108 cells/ml, at which the incidence of decay was only 16.7% after 6 d of incubation. The germination of conidia of P. expansum in vitro was significantly inhibited by both washed cell-suspensions and unwashed cell-culture mixtures. Rapid colonization by yeast at different concentrations showed a relationship between yeast-cell concentration and biocontrol activity. Although the titratable acidity of pear fruits increased after treatment, R. mucilaginosa did not affect the total soluble solids or ascorbic acid content. This is the first study to report that the yeast R. mucilaginosa from Tibet Autonomous Region of China may have potential as an antagonist to control the postharvest decay of pear fruits. PMID:25845361

Evaluation of yeasts from Tibetan fermented products as agents for biocontrol of blue mold of Nashi pear fruits.

PubMed

Hu, Hao; Xu, Yang; Lu, Huang-ping; Xiao, Rui; Zheng, Xiao-dong; Yu, Ting

2015-04-01

A total of 20 strains of yeast isolated from Tibetan fermented products were screened for antagonism against blue mold of pear caused by Penicillium expansum. Six isolates that inhibited incidence of postharvest decay by 35% or more were selected for further screening. Among them, the most effective was Rhodotorula mucilaginosa. The results showed that washed cell suspensions of R. mucilaginosa yielded better antagonistic efficacy than unwashed cell-culture mixtures, cell-free culture filtrates, and autoclaved cell cultures. Biocontrol activity improved with increasing concentrations of incubated cells. The best concentration was 1×10(8) cells/ml, at which the incidence of decay was only 16.7% after 6 d of incubation. The germination of conidia of P. expansum in vitro was significantly inhibited by both washed cell-suspensions and unwashed cell-culture mixtures. Rapid colonization by yeast at different concentrations showed a relationship between yeast-cell concentration and biocontrol activity. Although the titratable acidity of pear fruits increased after treatment, R. mucilaginosa did not affect the total soluble solids or ascorbic acid content. This is the first study to report that the yeast R. mucilaginosa from Tibet Autonomous Region of China may have potential as an antagonist to control the postharvest decay of pear fruits.