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 ...

<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 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 border="0" cellpadding="0" cellspacing="0" style="width:721px;" width="722">
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1000 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">200 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">37 &#8451; water bath</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">37 &#8451;, humidified 5% CO2 incubator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70% ethanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">analytical balance</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">cell culture-rated laminar flow hood</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">dimethyl sulfoxide</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">disposable media basin, sterile</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">drip filtration system</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Erlenmeyer flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>125 mL and 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HPLC grade methanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Human ER&#946; Reporter Assay System, 1 x 96-well format assays</td>
			<td style="width:80px;">Indigo Biosciences</td>
			<td style="width:119px;">IB00411</td>
			<td>Assay kit - analyzes 24 samples plus standard curve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">lyophilizer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">multi-channel pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">orbital shaker</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">plate-reading luminometer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>ex. Bioteck Synergy HTX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">rotory evaporator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">round bottom flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>50 mL and 300 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">sterile microcentrifuge tubes or sterile multi-channel media basins</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">sterile tips</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>200 &#181;L and 1000 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Whatman grade 1 paper</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">whirl-pak bags</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></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  ÃŽÂ² Reporter Assay at JoVE.com

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  ÃƒÅ½Ã‚Â² 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.

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 ...

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