Authors are thankful to Dale Leitman for initial training in use of transient transfection assays to determine estrogenic activity of primate plant foods. Thanks to Bradford Westrich and C. Eric Johnson for helping to set up laboratory equipment and training students in extraction methods. Finally, thank you to Indiana University for funding this research.

1. Preparation of plant materials
<ol>
	<li>Freeze dry plant items that were collected fresh using a lyophilizer.
	<ol>
		<li>To protect samples from light, cover chambers with aluminum foil during drying process.</li>
		<li>To ensure that samples are completely dry, lyophilize until chambers no longer feel cold to touch and plant materials no longer lose mass when weighed.</li>
		<li>Store dried plants in sterile low residue bags in absence of light until grinding.</li>
	</ol>
	</li>
	<li>Finely grind samples using a grinding mill with 0.85 mm mesh screen.
	<ol>
		<li>Store the ground samples in the bags in the absence of light until extraction.</li>
	</ol>
	</li>
</ol>
2. Extraction of plant secondary metabolites
<ol>
	<li>To extract the secondary plant metabolites, use a ratio of 1 g of dried sample to 10 mL of HPLC grade methanol.
	<ol>
		<li>Weigh sample on an analytical balance and add it to an appropriately sized Erlenmeyer flask (125 &#8211; 250 mL). Then add appropriate volume of methanol. Record the mass of sample extracted.</li>
		<li>Cover the plant-methanol solution with aluminum foil, and then set to rotate at 100 rpm speed at room temperature (RT) for 3 days on an orbital shaker, allowing the potentially estrogenic compounds to dissolve into the methanol.</li>
		<li>Decant the supernatant into a drip filtration system using filter paper (125 mm).</li>
		<li>Using a rotary evaporator, dry the plant extract until the sample is thickened, but pourable, in a 300 mL round-bottom flask. Pour sample into a 50 mL round-bottom flask, rinsing the large flask with a small amount of methanol. Continue to dry the sample in the small flask until the methanol is completely evaporated.</li>
		<li>Weigh the sample residue using an analytical balance. Record residue mass.</li>
		<li>Dissolve the plant extract in dimethyl sulfoxide (DMSO) at a concentration of 0.1 g of extract to 2 mL of DMSO. Vortex until homogenized.</li>
		<li>Store the plant extract-DMSO solution at 4 &#176;C in amber glass vials until the assay.</li>
	</ol>
	</li>
</ol>
CAUTION: Plants can produce unknown biologically active chemicals, and DMSO is a vehicle that can transport them across cell membranes. Use appropriate personal protective equipment and care when handling these samples.
3. Human estrogen receptor &#946; transfection assay12
NOTE: Aseptic technique and a laminar flow hood is required for Day 1 of the assay protocol.
<ol>
	<li>Prepare dilutions of 17&#946;-Estradiol for the standard curve.
	<ol>
		<li>Transfer the Cell Recovery Medium and Compound Screening Medium (CSM) from the freezer storage and thaw in a 37 &#176;C water bath.</li>
		<li>Label microcentrifuge tubes Intermediate 1 and 2 (INT1, INT2) and 1-8.</li>
		<li>Fill INT1 with 995 &#956;L of CSM, INT2 with 615 &#956;L of CSM, tube 1 with 900 &#956;L of CSM, and tubes 2-8 with 600 &#956;L of CSM. Set tube 8 aside.</li>
		<li>Transfer 5 &#956;L of 100 &#956;M 17&#946;-Estradiol Stock into INT1. Discard the tip. Vortex.</li>
		<li>Before each transfer, rinse pipette 3 times, and then transfer 10 &#956;L from INT1 into INT2. Discard the tip.</li>
		<li>Rinse pipette 3 times, and then transfer 100 &#956;L from INT2 into tube 1. Discard tip. Transfer 300 &#956;L from tube 1 into tube 2. Repeat for tubes 3 through 7. Discard 300 &#956;L from tube 7 into waste container. Tube 8 is a Zero and does not receive estradiol. Final concentrations of plated standards are: 400, 133.3, 44.44, 14.815, 4.938, 1.646, 0.5487, and 0 pM estradiol.</li>
	</ol>
	</li>
	<li>Prepare sample compounds.
	<ol>
		<li>Vortex samples.</li>
		<li>Take 4 &#956;L of each plant sample in DMSO and add to 496 &#956;L of CSM to yield a 0.8% DMSO solution.</li>
	</ol>
	</li>
	<li>Rapidly thaw Reporter Cells.
	<ol>
		<li>Retrieve the tube of Cell Recovery Medium from the 37 &#176;C water bath. Disinfect the outside surface using 70% ethanol.</li>
		<li>Retrieve Reporter Cells from -80 &#176;C storage and thaw by transferring 10 mL of the pre-warmed CRM into the tube of frozen cells.</li>
		<li>Close the tube of Reporter Cells and transfer to a 37&#176;C water bath for 5-10 min.</li>
		<li>Retrieve the tube of Reporter Cell Suspension from the water bath. Invert the tube of cells several times gently to break up aggregates of cells and produce a homogenous suspension. Clean the surface of the tube with 70% ethanol.</li>
	</ol>
	</li>
	<li>Assay plating
	<ol>
		<li>Dispense 100 &#956;L of the Reporter Cell Suspension into each well using a multichannel pipette.</li>
		<li>Dispense 100 &#956;L of samples in triplicate into appropriate assay wells.</li>
		<li>Transfer the plate into a 37 &#176;C, humidified 5% CO2 incubator for 22-24 h.</li>
	</ol>
	</li>
	<li>Thaw Detection Substrate and Detection Buffer in a dark refrigerator overnight to prepare for Day 2.</li>
	<li>Just prior to the end of the plate incubation, remove Detection Substrate and Detection Buffer from refrigerator and place in low light area until equilibrated to RT. Once at RT, invert each tube gently several times to thoroughly mix solutions.
	<ol>
		<li>Immediately before the incubation is complete, pour the entire contents of the Detection Buffer into the tube of Detection Substrate to create Luciferase Detection Reagent. Mix gently so as not to produce foam.</li>
		<li>Once the incubation is complete, invert the plate to discard content into an appropriate waste container. Gently tap the plate on a clean absorbent paper towel to remove the last droplets from the wells.</li>
		<li>Add 100 &#956;L of the Luciferase Detection Reagent to each well. Allow the assay plate to rest at RT for 15 min. Do not shake the plate.</li>
	</ol>
	</li>
	<li>Quantify luminescence using a 96-well plate-reading luminometer.</li>
</ol>

<ol>
	<li>Wasserman, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+plant+consumption+predicts+red+colobus+monkey+(Procolobus+rufomitratus)+hormonal+state+and+behavior.">Estrogenic plant consumption predicts red colobus monkey (Procolobus rufomitratus) hormonal state and behavior.</a> Hormones and Behavior. 62 (5), 553-562 (2012).</li><li>Wasserman, M. D., Milton, K., Chapman, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+phytoestrogens+in+primate+ecology+and+evolution.">The roles of phytoestrogens in primate ecology and evolution.</a> International Journal of Primatology. 34 (5), 861-878 (2013).</li><li>DeGabriel, J. L., Moore, B. D., Foley, W. J., Johnson, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+plant+defensive+chemistry+on+nutrient+availability+predict+reproductive+success+in+a+mammal.">The effects of plant defensive chemistry on nutrient availability predict reproductive success in a mammal.</a> Ecology. 90 (3), 711-719 (2009).</li><li>Wasserman, M. D., Steiniche, T., Despr&#233;s-Einspenner, M. -. L., Lambert, J. E., Rothman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primate Diet &amp; Nutrition. , (2020).</li><li>Benavidez, K. M., Chapman, C. A., Leitman, D. C., Harris, T. R., Wasserman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intergroup+variation+in+oestrogenic+plant+consumption+by+black-and-white+colobus+monkeys.">Intergroup variation in oestrogenic plant consumption by black-and-white colobus monkeys.</a> African Journal of Ecology. , (2019).</li><li>Bennetts, H. W., Underwood, E. J., Shier, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+breeding+problem+of+sheep+on+subterranean+clover+pastures+in+Western+Australia.">A specific breeding problem of sheep on subterranean clover pastures in Western Australia.</a> Australian Veterinary Journal. 22 (1), 2-12 (1946).</li><li>Tubbs, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenicity+of+captive+southern+white+rhinoceros+diets+and+their+association+with+fertility.">Estrogenicity of captive southern white rhinoceros diets and their association with fertility.</a> General and Comparative Endocrinology. 238, 32-38 (2016).</li><li>Shen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+the+influences+of+diosgenin+on+aging+ovarian+reserve+and+function+in+a+mouse+model.">Observation of the influences of diosgenin on aging ovarian reserve and function in a mouse model.</a> European Journal of Medical Research. 22 (1), 42 (2017).</li><li>Bou&#233;, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+estrogenic+effects+of+legume+extracts+containing+phytoestrogens.">Evaluation of the estrogenic effects of legume extracts containing phytoestrogens.</a> Journal of Agricultural and Food Chemistry. 51 (8), 2193-2199 (2003).</li><li>Klinge, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+interaction+with+estrogen+response+elements.">Estrogen receptor interaction with estrogen response elements.</a> Nucleic Acids Research. 29 (14), 2905-2919 (2001).</li><li>Nishikawa, J. -. i., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+screening+methods+for+chemicals+with+hormonal+activities+using+interaction+of+nuclear+hormone+receptor+with+coactivator.">New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator.</a> Toxicology and Applied Pharmacology. 154 (1), 76-83 (1999).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Estrogen Receptor Beta (ERb; ESR2; NR3A2) Reporter Assay System. , (2020).</li><li>Wasserman, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+plant+foods+of+red+colobus+monkeys+and+mountain+gorillas+in+uganda.">Estrogenic plant foods of red colobus monkeys and mountain gorillas in uganda.</a> American Journal of Physical Anthropology. 148 (1), 88-97 (2012).</li><li>Vivar, O. I., Saunier, E. F., Leitman, D. C., Firestone, G. L., Bjeldanes, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+activation+of+estrogen+receptor-&#946;+target+genes+by+3,+3'-diindolylmethane.">Selective activation of estrogen receptor-&#946; target genes by 3, 3'-diindolylmethane.</a> Endocrinology. 151 (4), 1662-1667 (2010).</li><li>Whitten, P. L., Patisaul, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+and+interassay+comparisons+of+phytoestrogen+action.">Cross-species and interassay comparisons of phytoestrogen action.</a> Environmental Health Perspectives. 109, 5-20 (2001).</li><li>Di Gioia, F., Petropoulos, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Food and Nutrition Research. , (2019).</li><li>Lutz, I., Kloas, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibians+as+a+model+to+study+endocrine+disruptors:+I.+Environmental+pollution+and+estrogen+receptor+binding.">Amphibians as a model to study endocrine disruptors: I. Environmental pollution and estrogen receptor binding.</a> Science of The Total Environment. 225 (1), 49-57 (1999).</li><li>Felcyn, J. R., Davis, J. C. C., Tran, L. H., Berude, J. C., Latch, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+Photochemistry+of+Isoflavone+Phytoestrogens:+Degradation+Kinetics+and+Pathways.">Aquatic Photochemistry of Isoflavone Phytoestrogens: Degradation Kinetics and Pathways.</a> Environmental Science &amp; Technology. 46 (12), 6698-6704 (2012).</li><li>Jeng, Y. -. J., Kochukov, M. Y., Watson, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+estrogen+receptor-alpha-mediated+nongenomic+actions+of+phytoestrogens+in+GH3/B6/F10+pituitary+tumor+cells.">Membrane estrogen receptor-alpha-mediated nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells.</a> Journal of Molecular Signaling. 4, 2-2 (2009).</li><li>Dixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytoestrogens.">Phytoestrogens.</a> Annual Review of Plant Biology. 55, (2004).</li><li>Kuiper, G. G. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+Estrogenic+Chemicals+and+Phytoestrogens+with+Estrogen+Receptor+&#946;.">Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor &#946;.</a> Endocrinology. 139 (10), 4252-4263 (1998).</li><li>Wasserman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Feeding on Phytoestrogens: Implications of Estrogenic Plants for Primate Ecology. , (2011).</li><li>Jefferson, W. N., Patisaul, H. B., Williams, C. 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The authors have nothing to disclose.

The ER&#946;&#160;reporter assay developed to individually screen pharmaceutical agents is also suitable for screening plant foods for phytoestrogens biologically active through the ER&#946;. Important considerations in the protocol include treating the plant samples with care: fresh plant material needs to be dried swiftly to prevent molding or other biological degradation, and it needs to be kept away from light to prevent photolysis of the compounds18. The assay protocol12</su...

Plants are a necessary source of food for many animals, providing calories and nutrients critical to survival, reproduction, growth, development, and behavior1. Plants produce thousands of chemicals, many as adaptations for their own growth, stomatic maintenance, and reproduction. Other compounds, deemed plant secondary metabolites (PSMs), have functions that are less clear, though some are toxic and likely used as a defense against herbivory and parasitism (e.g., alkaloids, tannins)2,3. Some of these chemicals have the ability to affect long term physiological processes in animals, such as endocrine functioning, although why these endocrine-active phytochemicals interact with the vertebrate endocrine system is still unclear2,4.
Phytoestrogens, the most well studied endocrine-active phytochemicals, are polyphenolic PSMs that structurally and functionally mimic estrogens, directly interacting with the hypothalomo-pituitary gonadal axis of the vertebrate endocrine system5. Ingestion of phytoestrogens in the human diet is associated with protection against some cancers, heart disease, and menopausal symptoms, though other effects include fertility problems. In fact, the physiological effects of these compounds were discovered in the 1940s when infertility in sheep was attributed to their grazing on phytoestrogen-rich clover (Trifolium subterrareum)6. When ingested, phytoestrogens can pass into cells and mimic the effects of estrogen. While phytoestrogens had negative effects on sheep fertility, the relationship between phytoestrogens and physiology is not simple. Like sheep, southern white rhinoceros display sensitivity to estrogenic compounds in feed derived from high quantities of soy and alfalfa. Daughters of females fed this diet during pregnancy are less likely to reproduce7. However, other studies have shown that phytoestrogens may have positive effects as well, including maturation of ovarian follicles in older mice8, prevention of certain cancers, antioxidant activity, and antiproliferative effects9.
The breadth of effects of phytoestrogens are not surprising given that estrogens affect a wide array of biological functions, including growth,&#160;development, and regulation of the reproductive and central nervous systems10. Although there are many mechanisms of action, phytoestrogens often have the ability to modify, enhance, or disrupt estrogen signaling through their ability to act as ligands for the intranuclear estrogen receptors alpha and beta (ER&#945; and ER&#946;). Many phytoestrogens have a phenolic ring structure similar to estrogens that allows them to bind estrogen receptors. Those with agonistic estrogenic activity function like estrogen, forming an activated ER-ligand complex that can dimerize and bind to an estrogen response element (ERE) and trigger gene transcription11. Thus, estrogens and phytoestrogens regulate cell activity and system functions through their actions as transcription factors.
Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses Chinese hamster ovary CHO cells engineered to highly express ER&#946;, which have been transfected with the firefly (Photinus pyralis) luciferase gene linked to an ERE promoter12. When estrogenic compounds are present, they bind to the ER, dimerize, and bind to the ERE, leading to transcription of the luciferase gene. Upon addition of a substrate solution, the luciferase catalyzes a reaction leading to photon emission. Therefore, positive samples produce light and negative samples do not.
This commercially available assay eliminates the need for laboratories to transfect the mammalian cells with the reporter gene and estrogen receptor13,14, which was unstable and variable in efficacy. The assay provides a stable transfection platform that allows for quickly and simply determining whether a plant has estrogenic activity via receptor binding.
We test the hypothesis that soybeans have higher estrogenic activity than all other foods given their known concentrations of estrogenic isoflavones15 using human foods from local grocers.

<table><tbody><tr><td>1000 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>20 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176; water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176;, humidified 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>analytical balance</td><td></td><td></td><td></td></tr><tr><td>cell culture-rated laminar flow hood</td><td></td><td></td><td></td></tr><tr><td>dimethyl sulfoxide</td><td></td><td></td><td></td></tr><tr><td>disposable media basin, sterile</td><td></td><td></td><td></td></tr><tr><td>drip filtration system</td><td></td><td></td><td></td></tr><tr><td>Erlenmeyer flasks</td><td></td><td></td><td>125 mL and 250 mL</td></tr><tr><td>HPLC grade methanol</td><td></td><td></td><td></td></tr><tr><td>Human ER&amp;beta; Reporter Assay System, 1 x 96-well format assays</td><td>Indigo Biosciences</td><td>IB00411</td><td>Assay kit - analyzes 24 samples plus standard curve</td></tr><tr><td>lyophilizer</td><td></td><td></td><td></td></tr><tr><td>multi-channel pipette</td><td></td><td></td><td></td></tr><tr><td>orbital shaker</td><td></td><td></td><td></td></tr><tr><td>plate-reading luminometer</td><td></td><td></td><td>ex. Bioteck Synergy HTX</td></tr><tr><td>rotory evaporator</td><td></td><td></td><td></td></tr><tr><td>round bottom flasks</td><td></td><td></td><td>50 mL and 300 mL</td></tr><tr><td>sterile microcentrifuge tubes or sterile multi-channel media basins</td><td></td><td></td><td></td></tr><tr><td>sterile tips</td><td></td><td></td><td>200 &amp;micro;L and 1000 &amp;micro;L</td></tr><tr><td>Whatman grade 1 paper</td><td></td><td></td><td></td></tr><tr><td>whirl-pak bags</td><td></td><td></td><td>sterile polyethylene bags</td></tr></tbody></table>

screening for phytoestrogens using a cell-based estrogen receptor  &#946; reporter assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

Twenty-two extracts of fruits and vegetables commonly found in human diets were screened for the presence of estrogenic compounds. A variety of foods were assayed, including legumes, such as soybeans, snow peas, and snap peas, as the pea family is a known source of phytoestrogens16, as well as figs, dates, corn, carrots, apples, bananas, strawberries, tomato, kale, and cabbage. Endocrine disrupting compounds are found in common substances (e.g., plastics and pesticides) and some are biologically a...

Watch this Scientific Journal Video about Screening for Phytoestrogens using a Cell-based Estrogen Receptor  Î² Reporter Assay at JoVE.com

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

screening-for-phytoestrogens-using-cell-based-estrogen-receptor

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

4.8K Views.  Indiana University. We have optimized a commercially available estrogen receptor β reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Video: Screening for Phytoestrogens using a Cell-based Estrogen Receptor  β Reporter Assay

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

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.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Environment

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

Cancer Research

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Medicine

Using 16&#945;-[18F]-Fluoro-17&#946;-Estradiol PET to Visualize Estrogen Receptor &#945; Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ER&#945;-positive breast cancer cells (MCF-7, 3 &#215; 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ER&#945;-negative breast cancer cells (MDA-MB-231, 1 &#215; 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 &#181;g of 17&#946;-estradiol (20 &#181;g/20 &#181;L; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17&#946;-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ER&#945;. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ER&#945;-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.

Here, we demonstrate a protocol to use 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts.

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

Biology

Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity

Source: Fender, E. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61005">Screening for Phytoestrogens using a Cell-based Estrogen Receptor &#946; Reporter Assay.</a> J. Vis. Exp. (2021).
This video describes a cell-based reporter assay to determine the estrogenic activity of plant-derived compounds. A compound with estrogen-like activity mimics its binding to estrogen receptors and drives downstream reporter enzyme gene transcription. The reporter enzyme generates bioluminescence in a reaction, indicating the estrogenic activity of the compound.

Source: Fender, E. et al., Screening for Phytoestrogens using a Cell-based Estrogen Receptor &#946; Reporter Assay. J. Vis. Exp. (2021).
This video ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay

Summary

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Chapters in this video

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

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Using 16α-[18F]-Fluoro-17β-Estradiol PET to Visualize Estrogen Receptor α Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity