Name: comprehensive assessment of germline chemical toxicity using the nematode caenorhabditis elegans
Uploaded: 2015-02-22T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans at JoVE.com

The authors would like to thank the following funding sources NIH ES020353 and the Colgate-Palmolive Alternative Research Grant award for making this work possible.

1.准备喂食细菌注意:本节介绍喂食细菌（ 大肠杆菌菌株OP50）的准备。 <ol><li>隔离大肠杆菌的单个菌落从LB培养基（LB）琼脂平板上，并在无菌条件下大肠杆菌菌株OP50接种入300ml高压灭菌的LB肉汤。 </li><li>允许接种培养过夜在摇床在200rpm和37℃下生长，直到达到饱和。 </li><li>转移OP50成6无菌，预先称重的50ml锥形管中。通过在6000转（5000 XG）离心5分钟沉淀细菌。 </li><li>弃去上清液，并用50ml无菌的M9洗细菌。重复两次。 </li><li>第二次洗涤后除去残留的M9，确保没有M9被留在管中。 </li><li>通过称量含有细菌沉淀管确定粒料的重量，并减去在50ml锥形管中的重量。 </li><li>悬浮颗粒我Ñ​​M9以100mg / ml的浓度。 </li><li>储存在4℃的OP50溶液，直到它被用于蠕虫培养。 </li></ol> 2.制备线虫生长培养基（NGM）培养皿注意:这部分描述NGM培养皿的制备，这是所述板，其中所述C。线虫蠕虫例行维持在实验室。 <ol><li>高压灭菌的NGM琼脂培养基。 </li><li>使用无菌的程序，免除17毫升的NGM琼脂溶液倒入用蠕动泵6厘米培养皿。 注:琼脂中平板恒定量减少了需要从一个板切换到另一个时再聚焦显微镜。 </li><li>离开所述板在室温下放置2-3天使用前，让琼脂固化，允许多余的水分蒸发。 </li><li>种子用无菌技术的NGM板。应用1-2滴OP50液体培养物并扩散用玻璃棒。确保不一路蔓延细菌草坪到板的边缘。 </li><li>允许OP50草坪在室温下用板成长蠕虫之前生长1-2天。 </li></ol> 3.准备一个同步蠕虫人口注:C。的生命周期线虫是由萌芽阶段，四幼虫阶段（L1-L4）和成年。本节描述了蠕虫的时代同步人口的制备。所有的材料来在漂白处理后与线虫接触必须是无菌的。 <ol><li> 第1天-妊娠蠕虫人口的制备 <ol><li>使用NGM板中的大多数蠕虫人口由饿死L1幼虫。 </li><li>收集L1幼虫用消毒虫挑，并将它们转移到新鲜6厘米NGM板接种OP50。孵育约65小时，在20℃，直到大部分的蠕虫是妊娠成人。到避免板拥挤并允许蠕虫未耗尽的细菌生长，不L1幼虫多于一个或两个簇转移到每个新鲜NGM板。 </li></ol></li><li> 4日-建立同步L1幼虫人口 <ol><li>通过与M9 1-2毫升冲洗它们收集来自NGM板的妊娠蠕虫和它们转移到一个锥形15ml试管。 </li><li>让妊娠蠕虫沉淀到15毫升锥形管的底部为约5-10分钟。去除上清，而不会干扰病毒颗粒。 </li><li>该蠕虫病毒颗粒转移到2-4离心管，并添加了漂白剂/氢氧化钠溶液1ml。孵育在室温2-3分钟。监视立体显微镜下的反应的进行，以确认所有的蠕虫都死了。不要漂白超过5分钟，因为胚胎会死亡。 </li><li>通过在3000转（900 XG）离心1分钟收集蠕虫。除去上清液，并添加无菌的M9 1ml至中和反应。 </li><li>通过在3000转（900 XG）离心1分钟洗两次的蠕虫。 </li><li>除去上清液，并添加M9与高达100-200微升。 </li><li>使用玻璃巴斯德吸管加一滴蠕虫/ M9混合以清除NGM板孵育它们至少24小时，在20℃，以允许胚胎孵化。 注:没有食物的幼虫的生长将在L1阶段停止。 </li></ol></li><li> 5日-建立同步L4幼虫人口 <ol><li>通过洗涤该板用1-2的M9毫升收集来自明确NGM板在L1幼虫，并将它们传送到一圆锥形15ml试管。 </li><li>让死成虫沉淀到15毫升锥形管的底部为约5分钟。 </li><li>收集上清液，其中所述L1蠕虫，在一个新的锥形15ml试管，并通过在2600转（1000 XG）离心2分钟沉淀出蠕虫。 </li><li>除去上清液乐AVING约500微升。 </li><li>通过滴用立体显微镜计数在10微升的蠕虫数目确定在M9溶液蠕虫的浓度。计数至少5滴。 </li><li>调整蠕虫的浓度为20-25蠕虫/微升，添加50微升的蠕虫/ M9混合物来接种OP50 NGM板。 </li><li>孵育65小时，在15℃（用于通过周末孵育），以允许在L1同步的人口增长，直到它们到达L4阶段。 </li></ol></li></ol> 4.暴露蠕虫在化学物质在96孔板注:本节介绍了使用Pxol1的含:: C.绿色荧光蛋白的转录记者线虫菌株筛选非整倍体的诱导。 <ol><li>通过洗涤该板用1-2的M9毫升收集来自明确NGM板的L4幼虫，并将它们传送到一圆锥形15ml试管。 </li><li>让L4蠕虫沉积物的底部的15毫​​升锥形管中大约5-10分钟。去除上清，而不会干扰病毒颗粒。 </li><li>加3-5的M9毫升，并确定由滴用立体显微镜计数在10微升的蠕虫数目，在M9溶液蠕虫的浓度。计数至少5滴每个样品。 </li><li>重悬在蠕虫中的M9 1000蠕虫/ ml的浓度。 </li><li>从稀释第1 OP50细菌10倍与M9。确保悬浮的细菌达到室温，因为较低的温度会影响蠕虫的发展。 </li><li>使用多道移液器，先添加100微升蜗杆/ M9混合物（来自步骤4.4）中，然后添加400微升的稀释OP50菌（来自步骤4.5），以对一个2毫升深圆底96孔板各孔中。在结束时，每个孔将具有在500μl100蠕虫。 </li><li>加入0.5微升的任一测试的化学品或适当的控制，以所期望的孔，以实现一个建议的最终精矿100μMATION，浓度一般在C.化学屏幕中使用线虫 12。暴露乙醇或DMSO溶剂对照为0.1％的最终浓度。 注意:我们建议使用诺考达唑（100μM）作为阳性对照。 </li><li>封用粘接膜的板。一定要密封板以及防止井之间的交叉污染。 </li><li>包装板用铝箔。 </li><li>板转移到适当的温度的摇床（170-180转）为适当长度的时间（24或65小时）。室内的温度影响的蠕虫的生长;维持20℃左右的温度下进行。 </li></ol>在384孔板5.图像采集妊娠蠕虫注意:这部分描述了使用具有高含量的显微镜图像的露出Pxol1 :: GFP蠕虫，可视化在成人雌雄同体的子宫内的胚胎的GFP的表达。为每384孔板进行筛选，用从4×96孔板蠕虫。 <ol><li>在摇床化学暴露后，让96孔板休息10-15分钟，以允许成虫沉降到板的底部。 </li><li>使用多道移液器，去除350微升的M9的，是非常小心，不要打扰蠕虫在板的底部。 </li><li> 1毫升M9和重复步骤5.1洗蠕虫。 </li><li>使用多道移液器，去除1毫升了M9的，是合租小心，不要打扰蠕虫在板的底部。 </li><li>使用多道移液器，悬浮蠕虫在剩余M9和收集100微升的蠕虫/ M9混合物从96孔板中，并将其加载到一个黑色壁/透明底384孔板的孔中。重复使用蠕虫从4×96孔板的过程中，直到已加载该384孔板的所有孔中。 注意:这是重要的，所有的孔中，以具有相同的体积，以便增加效率和自动聚焦的速度在图像采集过程中。每口井应该包含在80到100蠕虫。 </li><li>加入1μl左旋咪唑（100μM）的每个孔中，让蠕虫孵育大约30分钟。 注:左旋咪唑充当乙酰胆碱受体激动剂和固定不变的蠕虫。 </li><li>板块转移到能够提供自动成像的广角含量高的显微镜。 </li><li>进行图像采集，使用根据制造商的指导方针的高含量的图像采集和分析软件。选择4X目标收购每孔1的图像。 </li><li>启动图像采集前，调整GFP成像参数到45毫秒的曝光和图像分辨率为2,160 X 2,160的。 </li><li>收集数据作为GFP阳性蠕虫蠕虫的总数在井分割数，后者被测量井之间相对一致。 </li></ol>

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The authors declare that they have no competing financial interests.

此处所描述的方法构成第一大规模策略种系毒物的鉴定。它需要使用一个GFP转基因Pxol-1含有应变:: GFP的忠实报告非整倍体的诱导，其中用作种系功能障碍的代理早期胚胎。该方法涉及一个C的仔细同步线虫蠕虫人口和蠕虫暴露于化学品中的96孔格式接着GFP阳性蠕虫通过自动化高含量显微镜成像。 几个步骤这个协议是至关重要的结果的一致性。首先，?...

有在美国注册的商业约87000化学品，但只有一小部分，这些已经过测试，潜在的健康影响1。那些已被测试过的，仅一部分被评定为生殖健康的影响，部分原因是在确定改变哺乳动物早期生殖活动的困难，特别是在雌性生殖细胞发育和分化。事实上，第一减数分裂事件发生期间，在雌性哺乳动物胚胎发育的早期阶段，因此难以进入并收集在适于筛选目的号码。 生殖细胞提供了两代人之间的关键环节，其相应的功能是依赖于细胞和染色体分裂的复杂程序的精确执行称为减数分裂。减数分裂过程的失调可导致减少的生育力和生产配子和胚胎染色体数目异常，这种情况被称为非整倍体。减数分裂中染色体分离错误是高度相关的人体健康。染色体异常是常见的，具有一个频率的1在150个活产，21三体，18和13以及X和Y染色体的误差是最普遍的类型2,3。此外，先天性畸形，包括那些染色体来源的，是婴儿死亡的首要原因，在美国4中的环境影响可以影响染色体分离和行为的概念并不是新的5，但仍知之甚少。因此，关键是要考察其引入到我们的环境中的化学物质会干扰人的生育能力，早期的发展和整体生殖健康。 在光的哺乳动物模型的这些限制，我们开发了一种高通量筛选测定法中的蛔虫，以测试生殖毒性&lt;EM&gt;温度。线虫。我们已动员本常用的遗传模型系统提供了几个重要的功能，例如它的尺寸小，成本低，短的再现周期，生殖细胞的比例高，并易于操作6。蠕虫可以生长在96孔板或在高容量液体培养物并由于其透明性，可直接上成像板检测荧光报道的。下面所描述的实验利用了这些特点优势，以含荧光记者Pxol-1 :: GFP检测生殖干扰和诱导胚胎非整倍体的虫株的优势。 使用该报告菌株的是基于通常罕见的男性在一个主要雌雄同体蠕虫人口。这些男性（&lt;0.2％），自然地从错误中X染色体7的隔离起源。然而，由于种系破坏经常导致错误的常染色体的偏析heterochromosomes的和，它与雄性表型（Ⅹmissegregation）以及胚胎致死（常染色体missegregation）升高的发病率相关两者。容易地检测男性的感应而绕过胚胎致死的问题，一个雄性特异性启动子（XOL-1）用于驱动的GFP表达在早期胚胎仍含有蠕虫的子宫内。因此，表达GFP的胚胎的外观被用作用于非整倍体胚胎的存在的代理。此方法已被以前用于确定基因牵连种系维护和减数分裂8,9。适用于化学筛选，这株被采用在中高通量筛选。重要的是，应变忠实报告的化学品的aneugenicity，因此相关的哺乳动物的生殖端点10。这里描述的检测将是特别有用的毒理学家医药和化工等行业的设置看迅速评估朝生殖端点的化学品的毒性。此外，此法完全对准了毒性，在21 世纪11报告强调了政府的优先事项。

<table><tbody><tr><td>60 mm Vented, sharp edge Petri dishes</td><td>Tritech</td><td>T3315</td><td></td></tr><tr><td>Agar</td><td>Apex</td><td>20-274</td><td></td></tr><tr><td>Axygen 96-well clear round bottom 2 ml polypropylene deep well plate</td><td>Corning</td><td>P-DW-20-C-S</td><td></td></tr><tr><td>Bactopeptone</td><td>Apex</td><td>20-261</td><td></td></tr><tr><td>Bacto-tryptone</td><td>Fisher Scientific</td><td>BP1421-2</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td></td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>VWR</td><td>AA12316-A1</td><td></td></tr><tr><td>Cholesterol</td><td>Fisher Scientific</td><td>ICN10138225</td><td></td></tr><tr><td>DMSO</td><td>VWR</td><td>IC19018680</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; (OP50)</td><td>Caenorhabditis Genetics Center</td><td></td><td>&lt;a href=&quot;http://www.cbs.umn.edu/cgc&quot; target=&quot;_blank&quot;&gt;http://www.cbs.umn.edu/cgc&lt;/a&gt;</td></tr><tr><td>Ethanol 200 proof</td><td>VWR</td><td>EM-4455S</td><td></td></tr><tr><td>Greiner CELLSTAR 384 well plates</td><td>Sigma-Aldrich</td><td>M1937-32EA</td><td></td></tr><tr><td>ImageXpress Micro XLS System</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Levamisole hydrochloride</td><td>Fluka</td><td>31742</td><td></td></tr><tr><td>Magnesium sulfate anhydrous (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>VWR</td><td>97061-438</td><td></td></tr><tr><td>MetaXpress High Content Image Acquisition &amp;amp; Analysis Software</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Rayon films for biological cultures</td><td>VWR</td><td>60941-086</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>VWR</td><td>BDH0316</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ 745</td><td>This microscope has a total magnification form 3.35X to 300X. Any microscope with similar characteristics will work.</td></tr><tr><td>TY2441 &lt;em&gt;C. elegans&lt;/em&gt;, &lt;em&gt;Pxol-1::GFP&lt;/em&gt; reporter strain</td><td>Caenorhabditis Genetics Center</td><td></td><td>&lt;a href=&quot;http://www.cbs.umn.edu/cgc&quot; target=&quot;_blank&quot;&gt;http://www.cbs.umn.edu/cgc&lt;/a&gt;</td></tr><tr><td>Yeast extract</td><td>Becton Dickinson</td><td>212750</td><td></td></tr></tbody></table>

comprehensive assessment of germline chemical toxicity using the nematode caenorhabditis elegans

识别数以千计的出现在我们的环境中的化学物质的生殖毒性已经在环境健康领域中最诱人的挑战之一。这部分对模型系统，可以（1）准确地概括生殖过程的键的功能和（2）这样做的一个中高通量方式的缺乏是由于，而不需要大量的脊椎动物的。 我们在这里描述的线虫C.化验线虫 ，它允许快速鉴定生殖系毒物通过监测非整倍体胚胎的诱导。通过利用GFP报告线，在染色体分离从种系的破坏而导致的错误很容易可视化并用自动荧光显微镜定量。由此一组特定于它的毒性化合物的筛选可以在96至384孔板格式在几天之内进行。积极H第二分析其可以被执行以确定是否染色体异常起源于减数分裂中断或从早期胚胎染色体分离错误。总之，此法代表了一个快速第一遍战略的生殖功能障碍的化学品接触的快速评估。

曝光Pxol-1 :: GFP报告菌株的化学试剂，如微管毒物诺考达唑（ ​​图1）导致了表达GFP的胚胎中相比DMSO对照露出成年雌雄同体的子宫高比例的诱导。 GFP阳性胚胎是比在其他胚胎以及自发荧光，在动物的肠道观察中观察到的弱的背景荧光显著亮。暴露蠕虫被直接成像在384孔板和蠕虫的含有至少一个GFP阳性胚胎的数量以及计算每个井和归由该蠕虫的总数。从化学屏幕正击中意味着诱...

Watch this Scientific Journal Video about Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans at JoVE.com

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

comprehensive-assessment-germline-chemical-toxicity-using-nematode

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

8.0K 次观看.  University of California, Los Angeles. We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

视频: Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

科研

生物化学

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

一种用自动表型分析预测线虫化学毒性的高通量测定

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

环境科学

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

癌症研究

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

胚胎暴露于低剂量毒物的长期行为和生殖后果

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

发育生物学

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

生物学

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

测定化学物质对线虫线虫生长和繁殖的影响

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

医学

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

神经科学

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

Caenorhabditis elegans is a powerful model organism used in many research laboratories to understand the consequences of exposure to chemical pollutants, pesticides, and a wide variety of toxic substances. These nematodes are easy to work with and can be used to generate novel research findings, even in the undergraduate biology laboratory. A multi-week laboratory series of authentic, student-driven research projects trains students in a toolkit of techniques and approaches in behavioral measurements, cell biology, and microscopy that they then apply to their projects. One technique in that toolkit is quantifying the percentage of neurons exhibiting neurodegenerative damage following exposure to a chemical toxicant like a pesticide. Young adult C. elegans nematodes can be exposed to different concentrations of commercially available pesticides or other types of toxicants for 2-24 h. Then, undergraduate students can visualize different neuron subtypes using fluorescent-expressing strains of C. elegans. These techniques do not require sophisticated image processing software and are effective at even low magnifications, making the need for expensive confocal microscopy unnecessary. This paper demonstrates how to treat the nematodes with pesticides and how to image and score the neurons. It also provides a straightforward protocol for the microscopy and analysis of neuron morphology. The materials used for this technique are inexpensive and readily available in most undergraduate biology departments. This technique can be combined with behavioral measures like locomotion, basal slowing, or egg-laying to conduct a potentially publishable series of experiments and give undergraduate students an authentic research experience at a very low cost.

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

Young adult Caenorhabditis elegans nematodes are exposed to different concentrations of commercial pesticides or other toxicants for 2-24 h. Then, different neurons can be visualized using fluorescent-expressing strains. This paper demonstrates how to expose nematodes to pesticides and assess neuron damage.

研究杀虫剂对 秀丽隐杆线虫 神经元的影响

Caenorhabditis elegans is a powerful model organism used in many research laboratories to understand the consequences of exposure to chemical pollutants, ...

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.

生殖系化学毒性的利用线虫综合评估

摘要

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此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

一种用自动表型分析预测线虫化学毒性的高通量测定

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

胚胎暴露于低剂量毒物的长期行为和生殖后果

高通量筛选和生物传感与荧光 C。线虫菌株

测定化学物质对线虫线虫生长和繁殖的影响

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

研究杀虫剂对秀丽隐杆线虫神经元的影响

可筛选的

生殖系化学毒性的利用线虫综合评估

摘要

探索更多视频

此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

一种用自动表型分析预测线虫化学毒性的高通量测定

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

胚胎暴露于低剂量毒物的长期行为和生殖后果

高通量筛选和生物传感与荧光 C。线虫菌株

测定化学物质对线虫线虫生长和繁殖的影响

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

研究杀虫剂对 秀丽隐杆线虫 神经元的影响

可筛选的

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

研究杀虫剂对秀丽隐杆线虫神经元的影响