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.收集和处理水样（从埃舍尔等修改。9） <ol><li>填充含有1g叠氮化钠和50mg抗坏血酸与感兴趣水样品的顶部干净的1L的琥珀色玻璃瓶中。 72小时内，在4℃储存样品和过程。 注:叠氮化钠是剧毒，必须谨慎处理。使用护具（眼睛/面部，手套，服装）和正常工作的通风橱权衡。不要使用金属铲进行称量。 </li><li>通过一1.6微米玻璃纤维过滤器，然后通过在5-10毫升/分钟的流速预处理固相萃取（SPE）柱通过样品。调整真空泵的压力来控制流速。 </li><li>真空干燥该盒15分钟。 </li><li>用10毫升甲醇，随后用10毫升丙酮中洗脱盒:己烷（1:1，体积/体积）。 </li><li>下的高纯度氮气的轻柔气流集中洗脱液至〜1毫升溶剂交换通过加入500微升二甲基亚砜（DMSO），并蒸发该萃取液下降到500微升。 </li><li>转移提取物到琥珀色玻璃自动进样瓶中。储存于-20℃。 </li></ol> 2.准备分析具体参考化学和水提取物的稀释液<ol><li> 制备9稀释的校准曲线。 <ol><li>使在100％DMSO测定具体的参考，化学品的储液。最终浓度必须为2微米17β雌二醇对雌激素受体分析，和100μM的地塞米松为GR测定。存储储备溶液在-20℃。 </li><li>添加15微升适当的参考化学库存到285微升测定培养基的在无菌管（管＃1）。通过上下吹打混匀。 </li><li>加入200μl5％DMSO在测定培养基中的溶液在8个附加管（管＃2-9）。 </li><li>通过浴缸传输距离地铁1号100微升等分至管＃2È＃9以执行3倍稀释系列。每个稀释样品必须彻底用吸管取等分试样，并将其加入到下一个管之前进行混合。 </li></ol></li><li>由190微升检测培养基混合10微升DMSO的准备溶剂对照样本。 </li><li>制备四种稀释液每个水提取物。 <ol><li>在第一管中，加入5μL水提取物的95微升分析培养基调匀。加入50μl5％DMSO在检测培养基中的其他三个管。 </li><li>通过从一个管转移50微升溶液到下执行2倍稀释。 </li></ol></li></ol> 3.准备细胞悬液进行FRET生物测定<ol><li>根据制造商的说明准备在测定特定媒体。测定培养基必须储存在4℃下，并在使用前水浴加热到37℃。 </li><li>以ER或GR分裂被捕细胞的小瓶出低温无ZER并通过将小瓶在37℃水浴中2分钟，轻轻摇动快速解冻细胞。净化用70％乙醇小瓶，并将其放置在II级生物安全柜。 </li><li>打开使用无菌技术的小瓶中，并将细胞转移到10ml测定培养基中。 </li><li>离心5分钟200×g的。 </li><li>使用无菌玻璃吸管吸出上清液和重悬细胞沉淀于6ml测定培养基中。 </li><li>混合5微升细胞悬浮液与5μl的一个至关重要的染色液并加入等分试样到计数室。 </li><li>算使用光学显微镜或自动细胞计数器的活细胞的数目，并估计活细胞的密度在细胞悬浮液中。稀释如果必要，得到每ml测定培养基550000活细胞的最终密度。 </li></ol> 4.在96孔黑色壁透明底板板细胞及加稀水提取物<ol><li>创建板布局包括了9点检测特定的校准曲线，QA / QC样品和稀释倍数为每个水提取物。一个96孔板布局的一个例子示于图1。 </li><li>添加90微升测定培养基的到复制无细胞对照孔。 </li><li>倒入细胞悬浮在无菌移液贮存器，并添加90微升的细胞悬浮液，使用多道移液器其它井。 </li><li>加入10微升第2步到适当的孔中制备的稀释的样品。每孔DMSO的最终浓度必须为最大0.5％。 </li><li>覆盖有盖板，并放置在一个5％CO 2培养箱在37℃下16小时。 </li></ol><img alt="图1" src="/files/ftp_upload/54725/54725fig1.jpg" /> 图1:96孔板布局的实施例的多穴板被设计为包括一个测定特定的校准曲线，3种类型QA / QC控制（仅介质，细胞在清洁中，细胞在DMSO飙升介质），每个水提取物稀释4。每个控制和水提取物的稀释一式三份井进行了分析。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54725/54725fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5.准备装载解决方案，并添加到每口井<ol><li>温育之后，使板平衡至室温。对于此FRET生物测定，步骤5.2通过5.6应在不存在直接的光的情况下进行。 </li><li>根据制造商的说明制备6×加载溶液。 </li><li>加入20μl的6倍加载溶液的各孔中。 </li><li>添加10微升的细胞活力试剂至各孔以评价稀释的水提取物的细胞毒性。 </li><li>密封采用铝胶膜板。 </li><li>孵育在室温在黑暗中板2小时。 </li></ol> 6.测量细胞毒性和内分泌活动响应<ol><li>设置了以下制造商的说明底部阅读功能酶标仪。 </li><li>对于FRET转录生物测定，测量蓝色（（前）/发射器（EM）波长四百六十零分之四百○九纳米激发）和绿色（409例/ 530 EM NM）渠道的荧光。 </li><li>对于细胞毒性试验，测量在560例/ 590 EM纳米荧光。 </li></ol> 7.评估QA / QC检查以确定数据质量<ol><li>比较无细胞（媒体只）和细胞仅（在清洁测定培养基的细胞）对照的平均生荧光。唯一的媒体背景荧光应该比仅细胞控制的响应至少低25％。 </li><li>处理原始数据FRET。对于这两种"蓝"和"绿"的数据集，从含有以及所有细胞中减去无细胞对照孔的平均荧光。计算蓝/绿比每个实验井。 </li><li>唯一比较细胞和细胞DMSO对照的蓝/绿比值。这些控件的荧光值应为15％的相对标准偏差（RSD）内。 </li><li>积测定特定的校准曲线作为对表示为日志分子量（日志M）的样品浓度蓝/绿比值。计算斜率，R 2和登录EC 50（50％有效浓度）。校准参数应于表1中所列的预期范围之内。 </li><li>计算RSD为所有一式三份井。重复中的变异性应低于20％。 </li><li>对于细胞毒性数据（560实施例/ 590 EM纳米），从所有含细胞的孔中减去无细胞控制平均（即媒体背景）。 </li><li>计算各样品的平均扣除背景荧光。积所得的荧光数据如DMSO对照的百分比。样品稀释不应该表现出相比于细胞仅与DMSO对照的20％以上的细胞死亡。 </li></ol> 8.数据分析<ol><li>计算检测限（LOD）的测定极限的最低校准响应加上响应的平均值的两个标准偏差。 </li><li>为了呈现出剂量反应的样品，获得了欧共体50 &#160;使用10和50中的参考化学的效应％浓度之间的校准曲线的线性回归的斜率的水萃取物。 </li><li>用计算公式生物分析当量浓度（BEQ）:参考化学BEQ = EC 50 / EC水样50。 </li></ol>

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The authors have nothing to disclose.

环境雌激素，如17β雌二醇（E2），这些化学品在纳克/升浓度23,24权证筛查的有案可稽效力。在这项研究中，废水排放对ER响应（BEQ范围:2.3至17毫微克的E2 / L）的比报道的从澳大利亚污水处理厂20二级出水稍高，而BEQs地表水（&lt;0.5至4纳克E2 / L的）为报道的表面和雨水别处（&lt;1至11毫微克的E2 / L），16的范围内。尽管地表水样本中测得ER活动水平低，此法代表了水质评价是有?...

当前水质监测的前提是要准确和精确地测量化学污染物的产生，作为暴露于野生动物和人类的代理的能力。然而，这种化学物质通过化学监测和评估模式不能跟上，我们所面临的不断变化的化学世界。当我们了解的命运和合成和天然化学物质的影响，我们继续寻找测量工具，解决预期生物的影响，而在同一时间的免疫化学品生产，使用和环保投入的变化。这样的工具是特别了解相关未知或新的化学品，并转化产物是否值得我们注意。此外，存在于水中的化学物质的复杂混合物由个体化学监测不善处理。因此，我们面临的现有监控工具箱地表水THA这些问题，现代化，更好地解决的挑战收不到处理后的废水污水和城市/雨水径流排放。 近年来，生物分析技术已经表明希望作为筛选工具水质评估。如果对通过称为起作用的化学反应尤其in&#39;vitro生物测定，动作1,2的具体模式是极大的兴趣，环境监测社区3。大量调查在体外生物测定用来量化饮用水，地表废水4-6的内分泌活动。此外，一些生物测定目标分子发起事件（ 例如，受体活化），其可以潜在地通过不利后果通路连接于有害影响分析7,8。 生物筛选水质评价的发展一直相对较快，与数百种不同的体外生物分析端点已被评估他们的实用9,10。目前，仅生物测定的少数已显示以达到良好的测量精度（实验室内）同时展现水质5,6-之间区分的能力。特别是对于处理后的废水污水，雌激素和糖皮质类固醇的发生已经成功占据了利用体外转录实验11,12。然而，大多数研究迄今为止已采用的生物测定，其细胞系是专有的（并且因此没有被广泛可得），需要连续护理和操纵，或两者兼而有之。这样一来，能够标准化的协议，进行实验室间的校准练习，并最终该筛选技术转移到水资源的社会仍然受阻。 通过美国ToxCast程序审核体外生物检测中的至少一个供应商是容易商购的13用"冻融4;格式。这些除法被捕细胞"试剂盒"已被证明是一种用于测定从水中提取的化学品代表不同程度的处理14的活性健壮。虽然商协议可用来筛选个别化学品或混合物的生物活性，其中一些需要修改，才可以被应用到的水样。处理后的废水出水15，雨水径流16，受纳水体17,18和最近的循环水19,20是感兴趣的水质社区水介质的最好的例子。 这项研究提供了一个单一的，标准化的协议使用市售测量水样中的内分泌活动，除被捕体外转录生物测定。我们通过后台，剂量响应和可重复性响应为TW的全面评估证明该协议的健壮性特别感兴趣雌激素与糖皮质激素受体转录（ER和GR，分别）邻端点。该协议在加州应用于处理后的废水污水和地表水幕样本淡水系统。

<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

体外转录生物测定表明希望作为水质监测工具，但他们采用和广泛应用已部分阻碍由于缺乏规范的方法和强大的，用户友好的技术可用性。在这项研究中，使用可商购的，分裂停滞的细胞系，以定量筛选本感兴趣的环境质量的专业人员水样中的化学物质的内分泌活性。其中包括全面的质量保证/质量控制一个单一的，标准化的协议（QA / QC）检查是为雌激素和糖皮质激素受体的活性（ER和GR分别）使用基于细胞的荧光共振能量转移（FRET）分析开发。在加利福尼亚州（美国）处理城市废水和地表水从淡水系统的样品，采用固相萃取提取和使用标准化原为内分泌活动分析山坳。背景和特定端点引用的化学物质的剂量 - 反应满足必要的可靠的测量QA / QC准则。地表水样品的生物测定筛选反应，主要是检测不到的。与此相反，从二级处理厂流出物样品具有最高的可测量的活性，与估计的生物测定当量浓度（BEQs）到392纳克地塞米松/升的GR和17纳克17β雌二醇/升的ER。为三级污水样品的生物测定的反应比对二级污水测量低，说明经过深度处理一个较低的残余内分泌活性化学物质。这个协议表明 ，利用市售的体外转录生物测定，除法被捕细胞"试剂盒"，可适于筛选在水中内分泌活动。

在本研究中，选择了处理城市废水的4倍24小时的复合材料样品，从淡水系统在南加州地表水6抓取样品，包括超纯水的字段为空，说明该协议。 4流出物样品3是从以往的活性污泥的废水处理厂（"二次流出物"），并从与砂/碳过滤后加入生物处理（"叔流出物"）的先进废水处理厂的第四个。地表水样品从代表不同的土地用途（开，农业和城市）流域收集。 <p class="jove_content"...

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.

在用水筛查内分泌活动市售的<em&gt;体外</em&gt;式激活生物测定

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

screening-for-endocrine-activity-water-using-commercially-available

Universidad San Sebastian

Research

JoVE Journal

Environment

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

7.5K 次观看.  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.

视频: 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.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

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

科研

环境科学

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.

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

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).

使用Tg（Vtg1:mcherry）斑马鱼胚胎测试内分泌干扰化合物的雌激素效应

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.

使用基于细胞的雌激素受体对植物雌激素进行筛查β记者分析

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 ...

生物化学

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.

在内分泌干扰物计算水生毒理学的Silico建模方法:使用QSAR工具箱的基于软件的方法

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

生物工程

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.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

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

生物学

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.

高通量筛选转录后调控基因的化学调节剂

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.

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</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 ...

甲状腺激素作用指示小鼠的成像以评估内分泌干扰

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.

作者聚焦：使用 THAI 小鼠模型对甲状腺激素干扰进行体内评估

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.

通过甲状腺激素作用指示剂小鼠体内表征内分泌干扰化学效应

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

在用水筛查内分泌活动市售的

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