Name: screening for endocrine activity in water using commercially-available in vitro transactivation bioassays
Uploaded: 2016-12-04T13:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays at JoVE.com

Funding was provided by State Water Resources Control Board (Agreements No. 10-096-250 and 14-090-270). We thank S. Abbasi, M. Connor, S. Engelage, K. North, J. Armstrong, S. Asato, M. Dojiri, D. Schlenk, S. Snyder, S. Westerheide, B. Escher, F. Leusch, G. Pelanek, K. Bi, and J. Printen. The authors declare no conflict of interest, and reference to trade names does not imply endorsement.

1. 수집 및 처리 물 샘플 (에셔 등의 수정. 9) <ol><li> 관심의 물 샘플 정상에 1g 나트륨 아 지드 50 mg의 아스코르브 산을 포함하는 깨끗한 1 L 황색 유리 병을 채 웁니다. 72 시간 내 매장 4 ° C에서 샘플 및 프로세스. 주 : 아 지드 화 나트륨은 매우 독성이주의를 처리해야합니다. 보호 장비 (눈 / 얼굴, 장갑, 의류)을 사용하고, 제대로 작동 흄 후드에 무게. 계량 금속 주걱을 사용하지 마십시오. </li><li> 1.6 μM 유리 섬유 필터를 통해 다음 50-10 ml / 분의 유속에서 컨디셔닝 고체상 추출 (SPE) 카트리지를 통해 샘플을 통과한다. 유량을 제어하기위한 진공 펌프의 압력을 조정한다. </li><li> 진공을 15 분 동안 카트리지를 건조. </li><li> 아세톤 10 ㎖ 다음 메탄올 10 ㎖와 카트리지 용리 : 헥산 (1 : 1, V / V). </li><li> 고순도 질소의 부드러운 흐름하에 ~ 1 mL의 용출액을 농축시킨다. 용제디메틸 설폭 사이드 (DMSO) 500 μl를 첨가하고, 다운 500 μL에 추출물을 증발시켜 교환. </li><li> 황색 유리 오토 샘플러 바이알에 추출물을 전송합니다. -20 ° C에서 보관하십시오. </li></ol> 2. 분석 특정 참조 화학 물 추출물의 희석을 준비합니다 <ol><li> 교정 곡선 (9) 희석을 준비합니다. <ol><li> 100 % DMSO의 분석 특정 기준 화학 물질의 원액을 확인합니다. 최종 농도는 ER 분석 2 μM 17β-에스트라 디올 및 GR 분석 100 μM 덱사메타손해야합니다. -20 ° C에서 재고 솔루션을 저장합니다. </li><li> 멸균 튜브에 분석 매체의 285 μL에 대한 적절한 기준 화학 주식의 15 μl를 추가 (튜브 # 1). 상하로 피펫 팅하여 샘플을 섞는다. </li><li> 8 추가 튜브 (튜브 # 2-9)에 분석 배지에서 5 % DMSO 용액 200 μl를 추가합니다. </li><li> 욕조 통해 튜브 # 2 관 # 1에서 100 μL 나누어지는 이동예 # 9는 3 배의 일련의 희석을 수행한다. 각각의 희석 시료를 분취 량을 복용하고 다음 튜브에 추가하기 전에 피펫 충분히 혼합해야합니다. </li></ol></li><li> 분석 배지 190 μL의 DMSO 10 μL를 혼합 한 용매 대조군 샘플을 준비한다. </li><li> 각각의 물 추출물에 대한 네 희석을 준비합니다. <ol><li> 제 1 튜브에 분석 배지 95 μL의 물 추출물 5 μL를 첨가하고 잘 혼합한다. 다른 세 튜브에 분석 매체에 5 % DMSO의 50 μl를 추가합니다. </li><li> 다음에 하나의 튜브에서 용액 50 μl를 전송하여 2 배 희석을 수행합니다. </li></ol></li></ol> 3. FRET 생물 검정을 실시하기 위해 세포 현탁액을 준비합니다 <ol><li> 제조업체의 지침에 따라 분석 특정 매체를 준비합니다. 분석 매체는 4 ° C에서 보관 및 사용 전에 물을 욕조에 37 ° C로 예열해야합니다. </li><li> 극저온 무료에서 ER 또는 GR 분할 체포 세포의 유리 병을 가지고ZER 부드러운 교반 2 분 동안 37 ° C의 물을 욕조에 유리 병을 배치하여 신속하게 세포를 해동합니다. 70 % 에탄올로 유리 병의 오염을 제거하고 클래스 II 생물 안전 캐비닛에 넣습니다. </li><li> 무균 기술을 사용하여 유리 병을 열고 분석 배지 10 ㎖에 세포를 옮긴다. </li><li> 5 분 동안 200 X g에서 원심 분리기. </li><li> 멸균 유리 피펫을 사용하여 상등액을 흡인하고 분석 배지 6 ㎖의 세포 펠렛을 재현 탁. </li><li> 중요한 얼룩 솔루션의 5 μL와 세포 현탁액의 5 μl를 혼합하고 계산 챔버로 나누어지는을 추가합니다. </li><li> 광학 현미경 또는 자동 세포 계수기를 사용하여 살아있는 세포의 개수를 계산하고, 세포 현탁액의 생균의 농도를 추정한다. 분석 배지 1 ㎖ 당 550,000 생균의 최종 농도를 얻기 위해 필요한 경우 희석. </li></ol> 4. 플레이트 96 웰 블랙 벽 클리어 바닥 플레이트의 세포 및 희석 물 추출물 추가 <ol><li> 접시 만들기각각의 물 추출물에 대한 9 점 분석 특정 교정 곡선, QA / QC 샘플과 다수의 희석을 포함 레이아웃. 96- 웰 플레이트 레이아웃의 예는도 1에 도시되어있다. </li><li> 복제 무 세포 제어 우물에 분석 매체의 90 μl를 추가합니다. </li><li> 멸균 피펫 저수지에 세포 현탁액을 붓고 멀티 채널 피펫을 사용하여 다른 우물에 세포 현탁액의 90 μl를 추가합니다. </li><li> 적절한 우물 2 단계 동안 준비 희석 된 샘플의 10 μl를 추가합니다. 웰 당 DMSO의 최종 농도는 0.5 %의 최대해야합니다. </li><li> 뚜껑 접시를 덮고 16 시간 동안 37 ° C에서 5 % CO 2 배양기에 넣습니다. </li></ol><img alt="그림 1" src="/files/ftp_upload/54725/54725fig1.jpg" /> 그림 1. 96- 웰 플레이트 레이아웃 예제 멀티 웰 플레이트 분석법 특정 검량선 3 유형을 포함하도록 설계QA / QC 컨트롤의 물 추출물 당 4 희석 (미디어 만, DMSO 깨끗한 배지에서 세포와 세포 매체 아군). 각각의 제어 및 물 추출물의 희석이 세중의 우물에서 분석된다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54725/54725fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 5.로드 솔루션을 준비하고 각 음에 추가 <ol><li> 인큐베이션 후, 플레이트를 실온으로 평형을 허용한다. 이 FRET의 생물 검정의 경우, 5.6을 통해 5.2 직접 빛의 부재에서 수행되어야 단계를 반복합니다. </li><li> 제조자의 지시에 따라 6X 로딩 용액을 제조 하였다. </li><li> 각 웰에 6 배 로딩 용액 20 μl를 추가합니다. </li><li> 희석 물 추출물의 세포 독성을 평가하기 위해 각 웰에 세포 생존 시약 10 μL를 추가한다. </li><li> 알루미늄 접착 필름 접시를 밀봉. </li><li> 2 시간 동안 실온에서 어둠 속에서 접시를 품어. </li></ol> 6. 측정 세포 독성 및 내분비 활동 응답 <ol><li> 제조업체의 지침에 따라 하단 판독 기능을 갖춘 마이크로 플레이트 리더를 설정합니다. </li><li> 프렛 전이 활성화 생물 검정법를 들어, 파란색 형광 (460분의 409 nm의 여기 (예) / 방출 (엠) 파장)과 녹색 (409 예 / 530 엠 ㎚) 채널을 측정한다. </li><li> 세포 독성 분석의 경우, 560 예 / 590 엠 nm에서 형광을 측정한다. </li></ol> 7. 데이터의 품질을 결정하는 QA / QC 검사 평가 <ol><li> 셀 전용 (클린 분석 배지에서 세포)를 제어하여 무 세포 (전용 미디어)의 평균 원시 형광을 비교합니다. 미디어 전용 배경 형광은 세포 전용 제어의 응답보다 25 % 이상 낮아야한다. </li><li> 원시 FRET 데이터를 처리합니다. 모두 &#39;파란색&#39;과 &#39;녹색&#39;데이터 세트의 경우, 잘가 포함 된 모든 세포에서 세포가없는 대조군 웰의 평균 형광을 뺍니다. 을 계산각 실험 아니라 블루 / 그린 비율. </li><li> DMSO 컨트롤 만 세포 및 세포의 파란색 / 녹색 비율을 비교. 이러한 컨트롤의 형광 값은 15 %의 상대 표준 편차 (RSD)이어야한다. </li><li> 로그 분자량 (M 로그)로 표현 샘플 농도에 대해서 청색 / 녹색 비율로 특정 분석 검량선을 그린다. 기울기, R 2을 계산하고 EC 50 (50 % 영향 농도)를 기록합니다. 교정 파라미터는 표 1에 열거 된 예상 범위 내이어야한다. </li><li> 모든 세중 우물의 RSD를 계산합니다. 복제 중 변동성은 20 % 이하이어야한다. </li><li> 세포 독성 데이터 (560 예 / 590 엠 ㎚)의 경우, 모든 세포 함유 우물에서 무 세포 제어 평균 (즉, 미디어 배경)를 뺍니다. </li><li> 각 샘플의 평균 배경-감산 형광을 계산합니다. DMSO를 제어의 비율로 얻어진 형광 데이터를 플롯. 샘플 희석하지 말아야셀 전용 및 DMSO 대조군에 비해 20 % 이상의 세포 치사율을 나타낸다. </li></ol> 8. 데이터 분석 <ol><li> 최소 교정 응답을 더한 그 응답의 평균의 두 표준 편차로 검출 (LOD)의 분석 한계를 계산합니다. </li><li> 투여 량 반응을 나타낸 샘플의 경우, EC (50) 도출 &#160; 기준 화학적 10 50 % 효과 농도의 보정 곡선의 선형 회귀의 기울기를 이용하여 물 추출물. </li><li> / EC 물 샘플의 50 참조 화학 물질의 BEQ = EC 50 : 수식을 사용하여 생체 시료 분석 등가 농도 (BEQ)를 계산합니다. </li></ol>

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	<li>Dix, D. J., Houck, K. A., Martin, M. T., Richard, M. A., Setzer, R. W., Kavlock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ToxCast+program+for+prioritizing+toxicity+testing+of+environmental+chemicals.">The ToxCast program for prioritizing toxicity testing of environmental chemicals.</a> Toxicol. Sci. 95 (1), 5-12 (2007).</li><li>Reif, D. 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=Endocrine+profiling+and+prioritization+of+environmental+chemicals+using+ToxCast+data.">Endocrine profiling and prioritization of environmental chemicals using ToxCast data.</a> Environ. Health Perspect. 118 (12), 1714-1720 (2010).</li><li>Maruya, K. A., 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=A+tiered,+integrated+biological+and+chemical+monitoring+framework+for+contaminants+of+emerging+concern+(CECs)+in+aquatic+ecosystems.">A tiered, integrated biological and chemical monitoring framework for contaminants of emerging concern (CECs) in aquatic ecosystems.</a> Integr. Environ. Assess. Manag. , (2015).</li><li>Van der Linden, S. C., 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=Detection+of+multiple+hormonal+activities+in+wastewater+effluents,+surface+water,+using+a+panel+of+steroid+receptor+CALUX+bioassays.">Detection of multiple hormonal activities in wastewater effluents, surface water, using a panel of steroid receptor CALUX bioassays.</a> Environ. Sci. Technol. 42 (15), 5814-5820 (2008).</li><li>Leusch, F. D. L., 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=Comparison+of+five+in+vitro+bioassays+to+measure+estrogenic+activity+in+environmental+waters.">Comparison of five in vitro bioassays to measure estrogenic activity in environmental waters.</a> Environ. Sci. Technol. 44 (10), 3853-3860 (2010).</li><li>Jarosova, B., 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=Europe-wide+survey+of+estrogenicity+in+wastewater+treatment+plant+effluents:+the+need+for+effect-based+monitoring.">Europe-wide survey of estrogenicity in wastewater treatment plant effluents: the need for effect-based monitoring.</a> Environ. Sci. Pollut. Res. 21 (18), 10970-10982 (2014).</li><li>Sonneveld, E., 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=Comparison+of+in+vitro+and+in+vivo+screening+models+for+androgenic+and+estrogenic+activities.">Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities.</a> Toxicol. Sci. 89 (1), 173-187 (2006).</li><li>Piersma, A. H., 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+an+alternative+in+vitro+test+battery+for+detecting+reproductive+toxicants.">Evaluation of an alternative in vitro test battery for detecting reproductive toxicants.</a> Reprod. Toxicol. 38, 53-64 (2013).</li><li>Escher, B. 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=Benchmarking+organic+micropollutants+in+wastewater,+recycled+water+and+drinking+water+with+in+vitro+bioassays.">Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.</a> Environ. Sci. Technol. 48 (3), 1940-1956 (2014).</li><li>U.S. Environmental Protection Agency (USEPA) Endocrine Disruptor Screening Program. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prioritization of the endocrine disruptor screening program universe of chemicals for an estrogen receptor adverse outcome pathway using computational toxicology tools. , (2012).</li><li>Leusch, F. D. L., 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=Assessment+of+wastewater+and+recycled+water+quality:+a+comparison+of+lines+of+evidence+from+in+vitro,+in+vivo+and+chemical+analyses.">Assessment of wastewater and recycled water quality: a comparison of lines of evidence from in vitro, in vivo and chemical analyses.</a> Water Res. 50, 420-431 (2014).</li><li>Jia, A., Wu, S., Daniels, K. D., Snyder, 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=Balancing+the+budget:+accounting+for+glucocorticoid+bioactivity+and+fate+during+water+treatment.">Balancing the budget: accounting for glucocorticoid bioactivity and fate during water treatment.</a> Environ. Sci. Technol. 50 (6), 2870-2880 (2016).</li><li>Huang, R., 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=Chemical+genomics+profiling+of+environmental+chemical+modulation+of+human+nuclear+receptors.">Chemical genomics profiling of environmental chemical modulation of human nuclear receptors.</a> Environ. Health Perspect. 119 (8), 1142-1148 (2011).</li><li>Mehinto, A. C., 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=Interlaboratory+comparison+of+in+vitro+bioassays+for+screening+of+endocrine+active+chemicals+in+recycled+water.">Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water.</a> Water Res. 83, 303-309 (2015).</li><li>Ternes, T. A., Joss, A., Siegrist, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrutinizing+pharmaceuticals+and+personal+care+products+in+wastewater+treatment.">Scrutinizing pharmaceuticals and personal care products in wastewater treatment.</a> Environ. Sci. Technol. 38 (20), 392A-399A (2004).</li><li>Tang, J. Y. 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=Toxicity+characterization+of+urban+stormwater+with+bioanalytical+tools.">Toxicity characterization of urban stormwater with bioanalytical tools.</a> Water Res. 47, 5594-5606 (2013).</li><li>Scott, P. 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=An+assessment+of+endocrine+activity+in+Australian+rivers+using+chemical+and+in+vitro+analyses.">An assessment of endocrine activity in Australian rivers using chemical and in vitro analyses.</a> Environ. Sci. Pollut. Res. 21 (22), 12951-12967 (2014).</li><li>Vidal-Dorsch, D. E., Bay, S. M., Maruya, K., Snyder, S. A., Trenholm, R. A., Vanderford, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contaminants+of+emerging+concern+in+municipal+wastewater+effluents+and+marine+receiving+water.">Contaminants of emerging concern in municipal wastewater effluents and marine receiving water.</a> Environ. Toxicol. Chem. 31 (12), 2674-2682 (2012).</li><li>WateReuse Research Foundation (WRRF). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Direct potable reuse: a path forward. , (2011).</li><li>Leusch, F. D. L., 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=Assessment+of+the+application+of+bioanalytical+tools+as+surrogate+measure+of+chemical+contaminants+in+recycled+water.">Assessment of the application of bioanalytical tools as surrogate measure of chemical contaminants in recycled water.</a> Water Res. 49, 300-315 (2014).</li><li>Schriks, 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=Occurrence+of+glucocorticoid+activity+in+various+surface+waters+in+the+Netherlands.">Occurrence of glucocorticoid activity in various surface waters in the Netherlands.</a> Chemosphere. 93 (2), 450-454 (2013).</li><li>Suzuki, G., Sato, K., Isobe, T., Takigami, H., Brouwer, A., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+glucocorticoid+receptor+agonist+in+effluents+from+sewage+treatment+plants+in+Japan.">Detection of glucocorticoid receptor agonist in effluents from sewage treatment plants in Japan.</a> Sci. Tot. Environ. 527-528, 328-334 (2015).</li><li>Purdom, C. E., Hardiman, P. A., Byea, V. V. J., Enoa, N. C., Tyler, C. R., Sumpter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+effects+of+effluents+from+sewage+treatment+works.">Estrogenic effects of effluents from sewage treatment works.</a> Chemistry and Ecology. 8 (4), 275-285 (1994).</li><li>Kidd, K. A., 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=Collapse+of+a+fish+population+after+exposure+to+a+synthetic+estrogen.">Collapse of a fish population after exposure to a synthetic estrogen.</a> Proc. Natl. Acad. Sci. 104 (21), 8897-8901 (2007).</li><li>Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., Kobayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+estrogen+and+androgen+receptor+activities+in+200+pesticides+by+in+vitro+reporter+gene+assays+using+Chinese+hamster+ovary+cells.">Screening of estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells.</a> Environ. Health Perspect. 112 (5), 524-531 (2004).</li><li>Kugathas, S., Sumpter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+glucocorticoids+in+the+environment:+First+results+on+their+potential+impacts+on+fish.">Synthetic glucocorticoids in the environment: First results on their potential impacts on fish.</a> Environ. Sci. Technol. 45, 2377-2383 (2011).</li><li>Van der Linden, S. C., 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=Development+of+a+panel+of+high-throughput+reporter-gene+assays+to+detect+genotoxicity+and+oxidative+stress.">Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 760, 23-32 (2014).</li><li>Cwiertny, D. M., Snyder, S. A., Schlenk, D., Kolodziej, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+designer+drugs:+when+transformation+may+not+eliminate+risk.">Environmental designer drugs: when transformation may not eliminate risk.</a> Environ. Sci. Technol. 48, 11737-11745 (2014).</li></ol>

The authors have nothing to disclose.

이러한 17β-에스트라 디올 (E2), NG /의 L 농도 23, 24에서 이러한 화학 물질에 대한 보증 심사 등의 환경 에스트로겐의 잘 문서화 힘. 본 연구에서는 폐수 유출 물에 대한 ER 응답 (BEQ 범위 : 2.3-17 ng의 E2 / L)이 E2 / L NG 4 0.5 &lt;지표수 (대한 BEQs 반면, 호주 WWTPs 20 차 유출에 대해보고보다 다소 높았다 ) E2 NG 11 / L) (16)에 다른 표면과 우수 (&lt;1에 대해보고 된 범위 내에 있었다. 표면 ?...

현재 수질 모니터링은 정확하고 정밀 야생 동물 및 인체 노출 프록시로 화학적 오염 물질의 발생을 측정하는 능력을 전제한다. 그러나,이 화학 물질 별 화학 물질 모니터링 및 평가 패러다임은 우리가 직면 끊임없이 변화하는 화학 우주와 보조를 맞출 수 없습니다. 우리가 운명 합성 및 천연 화학 물질의 효과에 대한 자세한 내용은, 우리는 생물학적 영향을 예상 다루도록 측정 도구를 검색을 계속하고, 동시에 그 화학 물질의 생산, 사용 및 환경 입력의 변화에 ​​면역이다. 이러한 도구는 알 수 없거나 새로운 화학 물질 및 변환 제품은 우리의 관심을받을 자격이 있는지 이해에 특히 적합하다. 또한, 물에 존재하는 화학 물질의 복잡한 혼합물이 잘못 개별 화학 물질 모니터링에 의해 해결된다. 따라서, 우리는 더 나은 주소로 지표수 그쪽에서 이러한 문제를 기존의 모니터링 도구 상자를 현대화의 도전에 직면t이 처리 된 폐수 유출 및 도시 / 강우 유출수의 방전을받을 수 있습니다. 최근, 생물 분석 기법 수질 평가 스크리닝 도구로서 가능성을 보여 주었다. 알려진 통해 작용하는 화학 물질에 반응 특히, in&#39;vitro 생물 검정에서, 행동 1,2의 특정 모드는 환경 모니터링 지역 3 큰 관심이다. 다수의 연구는 음주, 표면 및 폐수 4 -6의 내분비 활동을 정량화하는 체외 생물 검정에 사용하고있다. 또한, 생물 검정의 수가 분자 개시 이벤트 (예, 수용체 활성화) 잠재적으로 불리한 결과 경로를 통해 악영향 연결될 수 -7,8- 분석 대상. 수질 평가를위한 bioscreening의 진화에 대해 평가 된 시험 관내 생물 검정 엔드 포인트에서 다른 수백, 상대적으로 신속하고있다 그들의유틸리티 9,10. 현재 생물 검정의 소수는 수질 5,6-를 구별하는 능력을 보여주는 반면 (실험실 내에서) 양호한 측정 정밀도를 달성하는 것으로 나타났다. 특히 처리 된 폐수 유출의 경우, 에스트로겐과 글루코 코르티코이드 스테로이드의 발생은 성공적이었다 체외 전이 활성화 분석 (11, 12)에서 사용하기를 차지했다. 누구의 세포 라인 독점 (때문에 널리 사용되지 않음)입니다 그러나 지금까지 대부분의 연구가 고용 한 생물 검정은, 지속적인 관리 및 조작, 또는 두 가지 모두를 필요로한다. 그 결과, 능력은 사회가 장애 된 남아있는 물 자원이 선별 기술을 전송하는 궁극적 프로토콜을 표준화 간 실험실 교정 연습을 수행하고 있습니다. 미국 ToxCast 프로그램을 통해 심사 체외 생물 검정 중 적어도 하나의 공급 업체는 &quot;동결과 해동을 사용하기 쉬운 13 상업적으로 사용할 수 있습니다4; 형식. 이러한 분할 셀 구속 &quot;키트&quot;는 처리 (14)의 서로 다른 레벨을 나타내는 물로부터 추출 된 화학 물질의 활성을 측정하는 강력한 것으로 나타났다. 공급 프로토콜은 개별 화학 물질 또는 이들의 혼합물의 생활 성을 스크리닝 할 수 있지만 이들이 물 샘플에 적용하기 전에, 그들 중 일부는 변경이 필요하다. 처리 된 폐수 유출 (15), 우수 유출 (16), 수신 물은 17, 18 그리고 최근 재활용 물 (19, 20) 수질 커뮤니티에 관심있는 수성 매질의 주요 예입니다. 이 연구는 시판 사용하여 물 샘플에서 내분비 활동을 측정하는 하나의 표준화 된 프로토콜을 제시 분할 체포 체외 전이 활성화 생물 검정에. 우리는 TW에 대한 응답의 배경, 복용량 응답 성 및 반복성의 포괄적 인 평가를 통해 프로토콜의 견고 함을 보여 주었다특히 관심의 에스트로겐과 글루코 코르티코이드 수용체의 전이 활성화 (ER 및 GR, 각각)의 오 엔드 포인트. 이 프로토콜은 캘리포니아에서 담수 시스템에서 처리 된 폐수 유출 및 지표수의 화면 샘플에 적용 하였다.

<table><tbody><tr><td>GeneBLAzer ER alpha DA assay kit</td><td>ThermoFisher</td><td>K1393</td><td>Kit includes ER division arrested (DA) cells and LiveBLAzer FRET loading kit.</td></tr><tr><td>GeneBLAzer GR DA assay kit</td><td>ThermoFisher</td><td>K1391</td><td>Kit includes GR division arrested (DA) cells and LiveBLAzer FRET loading kit.</td></tr><tr><td>PrestoBlue cell viability reagent&amp;nbsp;</td><td>ThermoFisher</td><td>A-13261</td><td></td></tr><tr><td>Trypan blue, 0.4% in PBS</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T8154</td><td>Also available at ThermoFisher</td></tr><tr><td>Corning 96-well black wall, clear-bottom plate</td><td>Corning</td><td>3603</td><td>Individually wrapped, sterile with lid</td></tr><tr><td>Whatman glass fiber filters, GF/A, 1.6 &amp;micro;M</td><td>Sigma-Aldrich&amp;nbsp;</td><td>WHA1820025</td><td></td></tr><tr><td>Microplate aluminum sealing film</td><td>E&amp;amp;K Scientific</td><td>T592100</td><td></td></tr><tr><td>Oasis HLB 6 cc cartridge, 200 mg sorbent</td><td>Waters</td><td>WAT106202</td><td></td></tr><tr><td>17&amp;beta; Estradiol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>E2758</td><td>CAS #50-28-2</td></tr><tr><td>Ascorbic acid</td><td>Fisher Scientific</td><td>A61-100</td><td>Also available at Sigma-Aldrich</td></tr><tr><td>Dexamethasone&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D4902</td><td>CAS #50-02-2</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D8418</td><td>Molecular grade</td></tr><tr><td>Solvents (acetone, hexane, methanol)</td><td>Fisher Scientific</td><td></td><td>HPLC grade</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S2002</td><td>Chemical is highly toxic and must be handled with caution. Use protective clothing and weigh under a fume-hood. Also available at EMD Millipore.</td></tr><tr><td>Automated cell counter or hemocytometer</td><td>Various*</td><td></td><td>Suppliers include Bio-Rad, Fisher Scientific, Sigma-Aldrich and ThermoFisher.</td></tr><tr><td>Class II biological safety cabinet</td><td>Various*</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Various*</td><td></td><td></td></tr><tr><td>Cryogenic freezer&amp;nbsp;</td><td>Various*</td><td></td><td>Liquid nitrogen storage dewar is recommended.&amp;nbsp;</td></tr><tr><td>Fluorescence microplate-reader</td><td>Various*</td><td></td><td>&amp;nbsp;The reader must have bottom read capabilities.</td></tr><tr><td>* No recommended source, the choice of this equipment depends on budget, frequency of use, and lab space.</td><td></td><td></td><td></td></tr></tbody></table>

screening for endocrine activity in water using commercially-available in vitro transactivation bioassays

체외 전이 활성화 생물 검정에서 그러나 자신의 채택 및 광범위한 응용 프로그램으로 인해 표준화 된 방법과 강력하고 사용자 친화적 인 기술의 가용성의 부족으로 부분적으로 방해하고있다, 수질 모니터링 도구로 약속을 보여 주었다. 본 연구에서는 시판 부문 체포 세포주 정량적으로 환경 품질 전문가에게 관심의 물 샘플에 존재하는 화학 물질의 내분비 활동을 선별하기 위해 사용되었다. 포괄적 인 품질 보증 / 품질 관리를 포함 하나, 표준화 된 프로토콜 (QA / QC) 검사 셀 기반 형광 공명 에너지 전달 (FRET) 분석을 사용하여 (각각, ER 및 GR) 에스트로겐과 글루코 코르티코이드 수용체의 활동을 위해 개발되었다. 캘리포니아 (미국) 담수 시스템에서 처리 된 하수 폐수 및 지표수의 샘플은, 고상 추출을 사용하여 추출하고, 표준화 된 프로토를 사용하여 내분비 활동을 분석 하였다안부. 배경 및 엔드 포인트 별 기준 화학 물질에 대한 용량 - 반응은 신뢰할 수있는 측정을 위해 필요하다고 인정 QA / QC 가이드 라인을 만났다. 표면 물 샘플에 대한 생물 검정 심사의 반응은 크게 감지되지 않았습니다. 대조적으로, 2 차 처리 공장에서 폐수 샘플 ER 392 ng를 덱사메타손 GR에 대한 / L 및 17 ng를 17β-에스트라 디올 / L까지 추정 된 생물 검정 등가 농도 (BEQs)로, 가장 높은 측정 활동을했다. 급 유출 샘플에 대한 생물 검정 응답은 고도 처리 후 내분비 활성 화학 물질의 낮은 잔류를 나타내는 보조 폐수를 측정보다 낮았다. 이 프로토콜은 시판 활용 체외 전이 활성화 생물 검정에, 분할 체포 셀 &quot;키트&quot;물에 내분비 활동에 대한 선별 적용 할 수 있음을 보여 주었다.

본 연구에서는, 처리시 폐수 유출의 4 배 24 시간 복합 샘플, 남부 캘리포니아에서 담수 시스템의 지표수 6 잡아 샘플 및 초순수로 구성된 필드를 비워는이 프로토콜을 설명하기 위해 선정되었다. 4 유출 물 샘플 3은 기존의 활성 슬러지 폐수 처리 시설 ( &quot;보조 유출&quot;), 모래 / 탄소 여과 추가 후 생물학적 처리 ( &quot;차 유출&quot;)와 고급 폐수 처리 공장에서 네 번째로 ?...

Watch this Scientific Journal Video about Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays at JoVE.com

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.

물 사용에 내분비 활동에 대한 선별 시판<em&gt; 체외</em&gt; 전이 활성화 생물학적 정량

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

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

7.5K 조회수.  Southern California Coastal Water Research Project Authority. 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.

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

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

작은 오염 물질의 면역 학적 분석을위한 물 샘플의 자동화 된 고체상 추출을위한 간단한 방법

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

연구

환경과학

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.

폐수 처리 프로세스의 효능의 평가를위한 화학 및 생체 독성 방법의 배터리를 사용하면 에스트로겐 효능을 제거하는 방법

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

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, &#945;- and &#946;-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

Tg를 사용 하 여 (Vtg1:mcherry) 제브라피시 배아 내 분 비 방해 화합물의 에스트로겐 효과 테스트

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

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.

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.

세포 기반 에스트로겐 수용체를 사용하여 식물에스트로겐에 대한 검사 β 리포터 분석

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 ...

생화학

In Silico Modeling Method for Computational Aquatic Toxicology of Endocrine Disruptors: A Software-Based Approach Using QSAR Toolbox

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological systems. In particular, quantitative structure-activity relationship (QSAR) models have been increasingly applied to assess the environmental effects of a plethora of toxic materials. In recent years, some more highlighted types of toxicants are endocrine disruptors (EDs, which are chemicals that can interfere with any hormone-related metabolism). Because EDs may significantly affect animal development and reproduction, rapidly predicting the adverse effects of EDs using in silico techniques is required. This study presents an in silico method to generate prediction data on the effects of representative EDs in aquatic vertebrates, particularly fish species. The protocol describes an example utilizing the automated workflow of the QSAR Toolbox software developed by the Organization for Economic Co-operation and Development (OECD) to enable acute ecotoxicity predictions of EDs. As a result, the following are determined: (1) calculation of the numerical correlations between the concentration for 50% of lethality (LC50) and octanol-water partition coefficient (Kow), (2) output performances in which the LC50 values determined in experiments are compared to those generated by computations, and (3) the dependence of estrogen receptor binding affinity on the relationship between Kow and LC50.

Quantitative structure-activity relationship (QSAR) modeling is a representative bioinformatics-assisted method in toxicological screening. This protocol demonstrates how to computationally assess the risks of endocrine disruptors (EDs) in aquatic environments. Utilizing the OECD QSAR Toolbox, the protocol implements an in silico assay for analyzing toxicity of EDs in fish.

내분비 분열기의 전산 수생 독성학을 위한 실리코 모델링 방법: QSAR 툴박스를 사용하는 소프트웨어 기반 접근 방식

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological ...

생체공학

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Zebrafish의 유충 루시 페라 제 리포터 시스템과 글루코 코르티코이드 신호에 대한 화학적 스크리닝 절차

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

생물학

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

Screenable<em&gt; 생체</em&gt; 형질 전환 발광 생물을 사용 미토콘드리아 변조기에 대한 분석<em&gt; 예쁜 꼬마 선충</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq). TF ChIP-seq generates a large data set (TF-regulatome) that includes PRSs and background interactions. These approaches require secondary low throughput validation in model systems that replicate the temporal and spatial specificity of the PRS. 
In this report, a protocol is described to rapidly validate activity of PRSs in their developmental context. Following selection of candidate PRSs, the associated sequences are isolated, cloned into a GFP reporter construct, and then transfected into chick embryos. For PRSs that are anticipated to be functional during early development, from gastrulation through early organogenesis, whole embryo electroporation (EP) is recommended. With this technique transfection, incubation and monitoring occurs ex ovo during early gastrulation. The entire embryo is transfected and as structures form, they can be monitored live for PRS activity. This ex ovo approach supports development up to early limb bud outgrowth. 
If the functional activity of a PRS is expected to be later in development or if the investigation is to ascertain its activity during the development of a specific organ, targeted regional electroporation (TREP) is used. This approach requires injection of construct DNA directly into the developing organ, precursor or adjacent cavity prior to electroporation. An approach to transfect the presumptive limb bud using TREP is also outlined and illustrated. TREP supports a rapid organ-specific validation of a PRS' activity. Both techniques can also be used to investigate regulatory domains by site-directed mutagenesis of the PRS-reporter construct. 
The chick as a bioassay offers a rapid effective tool for PRS validation, functional domain determination, and further characterization of the regulatory events responsible for development.

With the increased use of transcription factor-specific ChIP-seq technology, techniques to validate potential regulatory regions are critical. The authors describe a rapid chick bioassay to validate the activity of regulatory sequences functioning during development.

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin ...

내분비 교란을 평가하기 위한 갑상선 호르몬 작용 지표 마우스의 이미징

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

저자 스포트라이트: THAI 마우스 모델을 사용한 갑상선 호르몬 파괴의 생체 내 평가

The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

갑상선 호르몬 작용 지표 마우스를 통한 내분비 교란 화학 효과의 생체 내 특성 분석

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that ...

물 사용에 내분비 활동에 대한 선별 시판

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