Yazarlar, primat bitki gıdalarının östrojenik aktivitesini belirlemek için geçici transfeksiyon testlerinin kullanımındaki ilk eğitim için Dale Leitman'a minnettardır. Bradford Westrich ve C. Eric Johnson'a laboratuvar ekipmanlarının kurulmasına ve öğrencilerin ekstraksiyon yöntemlerine eğitmelerine yardımcı oldular. Son olarak, bu araştırmayı finanse ettiğiniz için Indiana Üniversitesi'ne teşekkür ederiz.

1. Bitki malzemelerinin hazırlanması
<ol><li>Bir liyofilzer kullanarak taze toplanan kuru bitki öğelerini dondurun.<ol>
<li>Numuneleri ışıktan korumak için, kurutma işlemi sırasında odaları alüminyum folyo ile örtün.</li>
		<li>Numunelerin tamamen kuru olduğundan emin olmak için, odalar dokunmak için artık soğuk hissetmeyene ve bitki malzemeleri tartıldığında kütle kaybetmeyene kadar liyofilize edin.</li>
		<li>Kurutulmuş bitkileri öğütülene kadar ışık yokluğunda steril düşük kalıntı torbalarında saklayın.</li>
	</ol>
</li>
	<li>0,85 mm örgü ekranlı bir taşlama değirmeni kullanarak numuneleri ince öğütün.<ol>
<li>Zemin örneklerini ekstraksiyona kadar ışık yokluğunda torbalarda saklayın.</li>
	</ol>
</li>
</ol>2. Bitki ikincil metabolitlerinin ekstraksiyonu
<ol><li>İkincil bitki metabolitlerini çıkarmak için, 1 g kurutulmuş numune ile 10 mL HPLC sınıfı metanol oranı kullanın.<ol>
<li>Örneği analitik bir teraziye tartın ve uygun boyutta bir Erlenmeyer şişesine (125 – 250 mL) ekleyin. Daha sonra uygun miktarda metanol ekleyin. Ayıklanan numune kütlesini kaydedin.</li>
		<li>Bitki-metanol çözeltisini alüminyum folyo ile örtün ve daha sonra yörüngesel bir çalkalayıcıda 3 gün boyunca oda sıcaklığında (RT) 100 rpm hızında dönmeye ayarlayın ve potansiyel olarak östrojenik bileşiklerin metanol içinde çözünmesini sağlar.</li>
		<li>Süpernatantı filtre kağıdı (125 mm) kullanarak damlama filtrasyon sistemine dekantasyon.</li>
		<li>Bir döner evaporatör kullanarak, numune kalınlaşana kadar bitki özünü kurutun, ancak 300 mL yuvarlak tabanlı bir şişede dökülebilir. Numuneyi 50 mL yuvarlak tabanlı bir şişeye dökün, büyük şişeyi az miktarda metanol ile durulayın. Metanol tamamen buharlaşana kadar numuneyi küçük şişede kurutmaya devam edin.</li>
		<li>Analitik bir terazi kullanarak numune kalıntısını tartın. Kayıt kalıntısı kütlesi.</li>
		<li>Bitki ekstresini 0,1 g'lık bir ekstrakt ile 2 mL DMSO konsantrasyonunda dimetil sülfit (DMSO) içinde çözün. Homojenleşene kadar girdap.</li>
		<li>Bitki özü-DMSO çözeltisini test edilene kadar kehribar cam şişelerde 4 °C'de saklayın.</li>
	</ol>
</li>
</ol>DİkKAT: Bitkiler bilinmeyen biyolojik olarak aktif kimyasallar üretebilir ve DMSO onları hücre zarları arasında taşıyabilen bir araçtır. Bu numuneleri kullanırken uygun kişisel koruyucu ekipman ve özeni kullanın.
3. İnsan östrojen reseptörü β transfeksiyon tahlil12
NOT: Test protokolünün 1.
<ol><li>Standart eğri için 17β-Estradiol seyreltmeleri hazırlayın.<ol>
<li>Hücre Geri Kazanımı Orta ve Bileşik Tarama Ortamını (CSM) dondurucu deposundan aktarın ve 37 °C'lik bir su banyosunda çözün.</li>
		<li>Etiket mikrosantrifüj tüpleri Orta 1 ve 2 (INT1, INT2) ve 1-8.</li>
		<li>INT1'i 995 μL CSM ile, INT2'yi 615 μL CSM ile, tüp 1'i 900 μL CSM ile ve 2-8 tüpleri 600 μL CSM ile doldurun. Tüp 8'i kenara koyun.</li>
		<li>100 μM 17β-Estradiol Stoğunun 5 μL'lik kısmını INT1'e aktarın. Ucu atın. Girdap.</li>
		<li>Her aktarımdan önce pipetleri 3 kez durulayın ve ardından INT1'den INT2'ye 10 μL aktarın. Ucu atın.</li>
		<li>Pipetleri 3 kez durulayın ve ardından INT2'den 100 μL'yi tüp 1'e aktarın. Ucu atın. 300 μL'i tüp 1'den tüp 2'ye aktarın. 3 ile 7 arasında tüpler için tekrarlayın. 300 μL'yi tüp 7'den atık kabına atın. Tüp 8 bir Sıfır'dır ve estradiol almaz. Kaplamalı standartların son konsantrasyonları şunlardır: 400, 133.3, 44.44, 14.815, 4.938, 1.646, 0.5487 ve 0 pM estradiol.</li>
	</ol>
</li>
	<li>Numune bileşikleri hazırlayın.<ol>
<li>Girdap örnekleri.</li>
		<li>DMSO'daki her tesis örneğinden 4 μL alın ve %0,8 DMSO çözeltisi elde etmek için 496 μL CSM ekleyin.</li>
	</ol>
</li>
	<li>Muhabir Hücrelerini hızla çözün.<ol>
<li>Hücre Kurtarma Ortamı tüpünü 37 °C su banyosundan alın. % 70 etanol kullanarak dış yüzeyi dezenfekte edin.</li>
		<li>Önceden ısıtılmış CRM'nin 10 mL'lik kısmını donmuş hücrelerin tüpüne aktararak -80 °C depolamadan Reporter Hücrelerini alın ve çözün.</li>
		<li>Reporter Cells tüpünü kapatın ve 5-10 dakika boyunca 37°C su banyosuna aktarın.</li>
		<li>Muhabir Hücre Süspansiyonu tüpünü su banyosundan alın. Hücre agregalarını parçalamak ve homojen bir süspansiyon üretmek için hücre tüpünü birkaç kez nazikçe ters çevirin. Tüpün yüzeyini% 70 etanol ile temizleyin.</li>
	</ol>
</li>
	<li>Tahlil kaplaması<ol>
<li>Çok kanallı pipet kullanarak her kuyuya 100 μL Reporter Hücre Süspansiyonu dağıtın.</li>
		<li>100 μL numuneleri uygun tahlil kuyularına üç taraflı olarak dağıtın.</li>
		<li>Plakayı22-24 saat boyunca nemlendirilmiş% 5 CO 2 inkübatörü olan 37 °C'ye aktarın.</li>
	</ol>
</li>
	<li>2. Güne hazırlanmak için bir gecede koyu renkli bir buzdolabında Çözülme Algılama Substratı ve Algılama Tamponu.</li>
	<li>Plaka inkübasyonunun bitiminden hemen önce, Algılama Substratı ve Algılama Tamponunu buzdolabından çıkarın ve RT'ye dengeleninceye kadar düşük ışık alanına yerleştirin. RT'ye girdikten sonra, çözeltileri iyice karıştırmak için her tüpü birkaç kez hafifçe ters çevirin.<ol>
<li>İnkübasyon tamamlanmadan hemen önce, Luciferaz Algılama Reaktifi oluşturmak için Algılama Arabelleği'nin tüm içeriğini Algılama Substratı tüpüne dökün. Köpük üretmemek için hafifçe karıştırın.</li>
		<li>Kuluçka tamamlandıktan sonra, içeriği uygun bir atık kabına atmak için plakayı ters çevirin. Kuyulardan son damlacıkları çıkarmak için plakaya temiz emici bir kağıt havluya hafifçe dokunun.</li>
		<li>Her kuyuya 100 μL Lucifease Algılama Reaktifi ekleyin. Test plakasının RT'de 15 dakika dinlenmesine izin verin. Tabağı sallamayın.</li>
	</ol>
</li>
	<li>96 kuyulu plaka okuma luminometresi kullanarak lüminesansı ölçün.</li>
</ol>

<ol>
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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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Yazarların açıklayacak bir şeyi yok.

Farmasötik ajanları tek tek taramak için geliştirilen ERβ muhabir testi, ERβ aracılığıyla biyolojik olarak aktif olan fitoöstrojenler için bitkisel gıdaların taranmasını da uygundur. Protokoldeki önemli hususlar arasında bitki örneklerinin özenle ele alınması yer almaktadır: küflenmeyi veya diğer biyolojik bozulmaları önlemek için taze bitki materyalinin hızlı bir şekilde kurutulması ve bileşiklerin fotolizini önlemek için ışıktan uzak tutulması gerekir18. Ü...

Bitkiler birçok hayvan için gerekli bir besin kaynağıdır, hayatta kalma, üreme, büyüme, gelişme ve davranış için kritik öneme sahip kalori ve besin maddeleri sağlar1. Bitkiler, birçoğu kendi büyümeleri, stomatik bakımları ve üremeleri için adaptasyonlar olarak binlerce kimyasal üretir. Bitki ikincil metabolitleri (PSM' ler) olarak kabul edilen diğer bileşikler, daha az net işlevlere sahiptir, ancak bazıları toksiktir ve muhtemelen otçul ve parazite karşı bir savunma olarak kullanılır (örneğin, alkaloidler, tanenler)2,3. Bu kimyasalların bazıları, hayvanlarda endokrin işleyişi gibi uzun süreli fizyolojik süreçleri etkileme yeteneğine sahiptir, ancak bu endokrin aktif fitokimyasalların omurgalı endokrin sistemle neden etkileşime girdiği hala belirsizdir2,4.
En iyi çalışılan endokrin-aktif fitokimyasallar olan fitoöstrojenler, yapısal ve işlevsel olarak östrojenleri taklit eden, omurgalı endokrin sistemin hipotaomo-hipofiz gonadal ekseni ile doğrudan etkileşime giren polifenolikPSM'lerdir 5. fitoöstrojenlerin insan diyetinde yutulması, bazı kanserlere, kalp hastalıklarına ve menopoz semptomlarına karşı koruma ile ilişkilidir, ancak diğer etkileri doğurganlık problemlerini içerir. Aslında, bu bileşiklerin fizyolojik etkileri 1940'larda koyunlardaki kısırlığın fitoöstrojen bakımından zengin yonca (Trifolium subterrareum)6üzerinde otlatmalarına atfedildiği zaman keşfedilmiştir. Yutildiğinde, fitoöstrojenler hücrelere geçebilir ve östrojenin etkilerini taklit edebilir. Fitoöstrojenlerin koyun doğurganlığı üzerinde olumsuz etkileri olsa da, fitoöstrojenler ve fizyoloji arasındaki ilişki basit değildir. Koyunlar gibi, güney beyaz gergedanları da yüksek miktarlarda soya ve yoncadan elde edilen yemlerdeki östrojenik bileşiklere duyarlılık gösterir. Hamilelik sırasında bu diyeti besleyen kadınların kızlarının üreme olasılığı daha düşüktür7. Bununla birlikte, diğer çalışmalar fitoöstrojenlerin, yaşlı farelerde yumurtalık köklerinin olgunlaşması da dahil olmak üzere olumlu etkileri olabileceğini göstermiştir8, bazı kanserlerin önlenmesi, antioksidan aktivite ve antiproliferatif etkiler9.
Östrojenlerin üreme, gelişme ve üreme ve merkezi sinir sistemlerinin düzenlenmesi de dahil olmak üzere çok çeşitli biyolojik işlevleri etkilediği göz önüne alındığında, fitoöstrojenlerin etkilerinin genişliği şaşırtıcı değildir10. Birçok etki mekanizması olmasına rağmen, fitoöstrojenler genellikle intranükleer östrojen reseptörleri alfa ve beta (ERα ve ERβ) için ligand görevi görebilme yetenekleri sayesinde östrojen sinyalini değiştirme, geliştirme veya bozma yeteneğine sahiptir. Birçok fitoöstrojen, östrojen reseptörlerini bağlamalarını sağlayan östrojenlere benzer fenolik bir halka yapısına sahiptir. Östrojen gibi agonistik östrojenik aktivite işlevine sahip olanlar, bir östrojen yanıt elemanına (ERE) küçültebilen ve bağlanabilen ve gen transkripsiyonunu tetikleyebilen aktif bir ER-ligand kompleksi oluşturur11. Böylece östrojenler ve fitoöstrojenler transkripsiyon faktörleri olarak yaptıkları eylemlerle hücre aktivitesini ve sistem fonksiyonlarını düzenlerler.
Burada, östrojenik biyolojik aktiviteye sahip bileşiklerin varlığı için bitki özlerini taramak için hücre tabanlı bir testin yeni kullanımını sunuyoruz. Bu tahlil, ere promotörü12ile bağlantılı ateş böceği (Photinus pyralis) luciferaz geni ile transfekte edilmiş ERβ'yi yüksek oranda ifade etmek için tasarlanmış Çin hamster yumurtalık CHO hücrelerini kullanır. Östrojenik bileşikler mevcut olduğunda, acil servise bağlanırlar, küçülürler ve ERE'ye bağlanırlar, bu da luciferaz geninin transkripsiyonuna yol açtı. Bir substrat çözeltisi eklenmeden sonra, luciferaz foton emisyonuna yol açan bir reaksiyonu katalize eder. Bu nedenle, pozitif numuneler ışık üretir ve negatif numuneler üretmez.
Piyasada bulunan bu tahlil, laboratuvarların memeli hücrelerini muhabir geni ve östrojen reseptörü13,14ile dönüştürme ihtiyacını ortadan kaldırır. Test, bir bitkinin reseptör bağlama yoluyla östrojenik aktiviteye sahip olup olmadığını hızlı ve basit bir şekilde belirlemeye izin veren kararlı bir transfeksiyon platformu sağlar.
Soya fasulyesinin bilinen östrojenik izoflavon15 konsantrasyonları göz önüne alındığında, yerel bakkallardan gelen insan gıdalarını kullanarak diğer tüm gıdalardan daha yüksek östrojenik aktiviteye sahip olduğu hipotezini test ediyoruz.

<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

Bitkiler birçok hayvan için bir besin kaynağıdır ve binlerce kimyasal üretebilirler. Bu bileşiklerden bazıları, endokrin fonksiyon gibi onları tüketen omurgalılardaki fizyolojik süreçleri etkiler. En iyi çalışılan endokrin-aktif fitokimyasallar olan fitoöstrojenler, omurgalı endokrin sistemin hipotalamo-hipofiz gonadal ekseni ile doğrudan etkileşime girer. Burada, östrojenik biyolojik aktiviteye sahip bileşiklerin varlığı için bitki özlerini taramak için hücre tabanlı bir testin yeni kullanımını sunuyoruz. Bu tahlil, östrojen reseptör betasını (ERβ) yüksek oranda ifade etmek için tasarlanmış ve luciferaz geni ile trans enfekte olmuş memeli hücrelerini kullanır. Östrojenik aktiviteye sahip bileşiklere maruz kalmak, hücrelerin ışık üretmesine neden olur. Bu tahlil, biyolojik östrojenik aktiviteyi test etmenin güvenilir ve basit bir yoludur. Geçici transfeksiyon tahlilleri, özellikle kullanım kolaylığı, hücrelerin stabilitesi ve tahlil hassasiyeti üzerinde çeşitli iyileştirmelere sahiptir.

İnsan diyetlerinde yaygın olarak bulunan yirmi iki meyve ve sebze özü östrojenik bileşiklerin varlığı için tarandı. Bezelye ailesi bilinen bir fitoöstrojen kaynağı olduğu için soya fasulyesi, kar bezelyesi ve snap bezelye gibi baklagiller de dahil olmak üzere çeşitli yiyecekler test edildi16, ayrıca incir, hurma, mısır, havuç, elma, muz, çilek, domates, lahana ve lahana. Endokrin bozucu bileşikler ortak maddelerde (örneğin plastikler ve pestisitler) bulunur ve bazıları...

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

Ticari olarak mevcut bir östrojen reseptörü β muhabir testlerini, insan ve insan dışı primat gıdaları östrojenik aktivite için taramak için optimize ettik. Bilinen östrojenik insan gıda soyasının yüksek olduğunu, diğer gıdaların ise hiçbir aktivite göstermediğini göstererek bu tahlilini doğruladık.

Hücre Tabanlı Östrojen Reseptörü Kullanarak Fitoöstrojen taraması β 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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

4.8K Görüntüleme.  Indiana University. Ticari olarak mevcut bir östrojen reseptörü β muhabir testlerini, insan ve insan dışı primat gıdaları östrojenik aktivite için taramak için optimize ettik. Bilinen östrojenik insan gıda soyasının yüksek olduğunu, diğer gıdaların ise hiçbir aktivite göstermediğini göstererek bu tahlilini doğruladık.

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.

Atıksu Arıtma Süreçlerinin Etkinliğinin Değerlendirilmesi için Kimya ve Ekotoksikolojik Yöntemlerinin bir pil kullanılması östrojenik Potency çıkarmak

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

Çevre

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.

Su Kullanımı Endokrin Aktivitesi Tarama Piyasada satılan<em&gt; İn Vitro</em&gt; Transaktivasyon biyolojik tayinler

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.

Ligand bağlayıcı etki Dimerization etkinliği östrojen reseptör Alfa memeli iki-hibrid tahlil kullanarak algılama

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

Kanser Araştırmaları

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.

Menopoz Sonrası Kardiyovasküler Disfonksiyon Exogenous Östrojen Tedaviler Tarama için Bir In Vivo Östrojen Eksikliği Fare Modeli

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

Biyokimya

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.

Transkripsiyonel Aktif Alellerin Tek Molekül FISH ile Tek Hücre Analizi

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.

Meme Kanseri Hücrelerinde östrojen düzenlenmiş microRNA profil

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

Tıp

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.

Dişi Yumurtalıklı Farelerde İnsan Meme Kanseri Ksenogreftlerinde Östrojen Reseptörü α Ekspresyonunu Görselleştirmek için 16α-[18F]-Floro-17β-Estradiol PET Kullanımı

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.

Meme Kanserinde Östrojen Reseptör Sinyali tarafından Metabolik Yönetmelik Sistem Biyolojisi

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.

Kişisel bakım ürünleri için lisans öğretim laboratuvar bir maya östrojen ekran optimize kullanarak östrojenik ligandlar saptamak

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

Biyoloji

Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity

Source: Fender, E. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

Fitoöstrojen Östrojenik Aktiviteyi Taramak için Östrojen Reseptör-Raportör Aktivite Testi

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