Name: high content screening analysis to evaluate the toxicological effects of harmful and potentially harmful constituents (hphc)
Uploaded: 2016-05-10T15:00:00.000+00:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC at JoVE.com

笔者想感谢Karsta Luettich和Gregory Vuillaume他们的手稿的审查。

<ol>
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所有作者都是菲莫国际的员工。菲利普莫里斯国际公司是该项目的资金和赞助商的唯一来源。

用于替代动物实验和新的高通量测试方法的需求在过去几年里得到了广泛的讨论。这已使科学家和监管当局调查标准毒性试验的替代方法，利用细胞试验紧密模仿靶组织的生理学。在这项研究中，我们已经证明结合具有高含量筛选（HCS）平台实时细胞分析仪（RTCA）评估暴露于单个CS成分对人肺上皮细胞的影响的适用性。这种设置可以类似用于评估其他各种空气污染物，空气中的颗粒，和纳米粒子诱导的细胞毒性。此外，所获得的结果可以与来自全基因组转录和计算方法基于因果生物网络相匹配。据此前报道，这种方法使我们能够证实的分子通路数据在CS曝光5与HCS终点的扰动，解决这些途径扰动也表型。 为流程图测定中，实时细胞分析提供以剂量和时间依赖性的分辨率，这允许它的剂量和暴露时间点可以是用于下游分析14有利更好的决策细胞活力相关的信息。分析仪的原理依赖于在由细胞产生的，因为它们附着和扩散上覆盖有金微电极的培养以及表面电阻抗的变化。阻抗被转换成称为小区索引一个无量纲参数，其可用于监测细胞粘附，扩散，形态和最终细胞活力。虽然该技术不提供关于细胞毒性机制的信息，其灵敏度能的，即使在非常低的剂量在所述HCS并不翔实形态学细胞变化的检测（数据未显示）。基于前雇主OU中的实验中，我们已经注意到，RTCA方法能够检测在较低剂量相比，HCS端点形态学变化。 在与实时细胞分析初步筛选中，HCS平台被用来获取关于每个HPHC引起的那种细胞毒性的更深入的信息。允许个人资料HPHCs对他们在细胞车厢潜在影响HCS检测板/细胞器以及识别那些引发遗传毒性和氧化应激。该分析揭示了不同的配置文件，从而选定HPHCs诱导NHBE细胞毒性。在一般情况下，所有的化合物中，除苯酚，发现在最高测试剂量以诱导坏死。在H2AX的癌症发展1 aminonaphtalene诱导的磷酸化的潜在作用，作为遗传毒性标志物，但是HCS面板中的线粒体毒性读出这个HPHC中还发现了活性（质量增加，细胞色素C relea一致本身）和氧化应激（GSH耗竭）。同样，如先前所描述，苯酚被确定以诱导线粒体功能障碍，并导致DNA损伤以及谷胱甘肽耗竭。铬（VI），列为I组致癌的化合物之一，和巴豆醛也都鉴定为基因毒性，特别是铬（VI）也诱导细胞凋亡（半胱天冬级联活化）和巴豆醛引起增加ROS产生。最后砷（V）中，被发现会引起cJun磷酸化这是应力激酶途径激活的标志。 在这项研究中，我们利用NHBE细胞在体外肺上皮细胞的模型。在HCS设置使用这些细胞是前所未有的，使更广泛的端点，包括遗传毒性和氧化应激标志物的调查。两个活细胞和固定细胞染色方法被我们的协议内描述的，示范的整体技术的灵活性。 f中行为，非常相同的协议可以被应用到更广泛的目标，这可以通过使用任何荧光染料或抗体的加以处理。对的活染色方案的成功执行，则尊重培养时间，因为一些染料具有有限的半衰期和荧光信号完成图像采集之前可能降低是重要的。同样重要的是要考虑到，如果使用不同的细胞类型中，所有的染色条件，应重新评估，作为最佳的染料浓度和温育时间可以是不同的。 在当前的论文中，我们描述了只有五个化合物，其中的HCS方法筛选的场景。考虑到先前描述的板布局，他们被给予超过2种不同的板集总共24板（6测定和2的时间点）板.The数量也可以增加，从而允许更多的化合物或同时筛选在不变拟多个端点的tigation。在这样做之前，但是，应该考虑到，某些端点（GSH和ROS）需要立即采集，并作为结果，板的给药应以交错的方式来执行，以允许采集先前板。另一方面，使用固定的细胞染色协议表示有利的，因为在板可堆叠，在定影后的任何步骤中断协议，用于染色过程完成在稍后阶段。这种方法，例如，将提供操作员的时间来完成所有的活细胞染色板而不损害数据质量。 通过减少板的数量进一步优化工作流程，它也将是可能的复用多个端点在一起。例如，在这种情况下DNA损伤和应激激酶可以一起研究简单地使用两个二级抗体与荧光发射的不同的Channels。所述HCS平台的不断发展，其中包括全自动细胞接种，化合物稀释，剂量和染色，以及加入的新的端点将进一步扩大HCS平台的能力，作为对上皮HPHCs和其他细胞类型的有力的分析工具。

毒理学风险评估历来依赖于使用动物模型当中，虽然在生命科学基础，也有不足之处联系，如不一致译​​人类和成本高。此外，出现了越来越多的努力，找到"3R原则"2（替代，减少和优化）的精神动物试验替代方案。这种努力已经加速，在过去的几年中，这不仅是因为最近的进展，如高通量的技术和系统生物学方法，但由于立法限制使用动物试验也，特别是在欧洲联盟。 的细胞信号途径调节到毒性损伤的响应的复杂性使得它显然使用单一毒理学终点将不足以描述的某些化合物的毒理学基础。对于这一点，数百相互作用的p的相互有助于生物网络roteins也将需要被考虑在内。研究毒物对这些网络的影响，一个系统毒理学方法与表型中，高通量筛选测定组合是有用的推断效能并同时提供对个别毒物作用机制的更多信息。 在这项研究中，我们采用HCS作为强大的筛选工具，它是由一个自动显微镜和生物软件应用的，可以采集，处理和分析来自特定的基于荧光的细胞分析而得的图像数据。这允许在小区内的视觉变化进行量化，在单细胞或亚细胞水平，并可以同时分析许多参数。3例如，DNA双链断裂是使用组蛋白H2AX的磷酸化的基于抗体的鉴定评估，并使用细胞烫发活性氧（ROS）的定量eable超敏感的染料。 因为肺上皮细胞代表针对吸入毒物，包括香烟烟雾中的第一生物屏障，我们利用主支气管上皮细胞作为体外模型来分析由美国食品和药品管理局公布HPHCs的效果。4本手稿是一个后续-最多上以前的研究5中，我们评估HPHCs的不同子集的生物学影响。 作为我们工作流以评估在体外的细胞毒性的一部分，我们最初评估选择的15 HPHC的的效力，使用基于阻抗的实时蜂窝分析使我们能够建立剂量范围，适合于随后的HCS（RTCA）系统分析（ 图1）。毒理学评估HCS然后用细胞毒性九个多参数进行端点，每次在两个时间点（4和24小时）监测。所使用的标记物指示线粒体毒性，DNA损伤，应激激酶，活性氧（ROS），谷胱甘肽（GSH）的含量，蛋白酶3 - 7活性，细胞色素C释放和细胞膜的通透性，如表1所述。 我们的方法使识别和香烟烟雾成分通过剂量和时间依赖性的采样的效果的表征。最终，这产生每个HPHC 的体外毒理学特性。多组学方法也可以被用来进一步补充HCS分析。这最后还提供了在细胞信号和/或转录水平的影响有了更深的了解。

<table><tbody><tr><td>Cellomics ArrayScan VTI HCS Reader</td><td>Thermo</td><td>N01-0002B</td><td></td></tr><tr><td>xCelligence RTCA MP</td><td>ACEA</td><td>05331625001</td><td></td></tr><tr><td>Screener (HCS)</td><td>Genedata</td><td>NA</td><td></td></tr><tr><td>CASY counter TTC</td><td>Roche</td><td>05 651 719 001</td><td></td></tr><tr><td>e-Plates VIEW 96</td><td>ACEA</td><td>06 472 451 001</td><td></td></tr><tr><td>RTCA Frame 96</td><td>ACEA</td><td>05232392001</td><td></td></tr><tr><td>RTCA Cardio Temperature Tool</td><td>ACEA</td><td>2801171</td><td></td></tr><tr><td>Plate sealer breathseal</td><td>Greiner bio-one</td><td>676051</td><td></td></tr><tr><td>Normal Human Bronchial Epithelial cells (NHBE)</td><td>Lonza</td><td>CC-2540</td><td>non-smoking 60-year-old Caucasian male donor&amp;nbsp;</td></tr><tr><td>BEGM BulletKit</td><td>Lonza</td><td>CC-3170</td><td>Warm at 37 &amp;deg;C before use</td></tr><tr><td>ReagentPack Subculture Reagents kit</td><td>Lonza</td><td>CC-5034</td><td>Warm at 37 &amp;deg;C before use</td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Easy Flask filter cap 75&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Thermo Scientific</td><td>12-565-349</td><td></td></tr><tr><td>96 well assay plate&amp;nbsp; black&amp;nbsp;</td><td>Corning</td><td>3603</td><td></td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Fisher Scientific</td><td>PI-62249</td><td></td></tr><tr><td>Draq5 (For Far Red Nuclear Staining)</td><td>Biostatus</td><td>DR50200</td><td></td></tr><tr><td>Mitochondrial Dye: MitoTracker Red CMXRos</td><td>Life technologies</td><td>M-7512</td><td></td></tr><tr><td>Mitochondrial Dye: MitoTracker Red CM-H2XRos</td><td>Life technologies</td><td>M-7513</td><td></td></tr><tr><td>ROS Dye: Dihydroethidium</td><td>Sigma</td><td>D7008</td><td></td></tr><tr><td>ROS Dye: CellROX</td><td>Life technologies</td><td>C10422</td><td></td></tr><tr><td>ROS Dye: MitoSOX</td><td>Life technologies</td><td>M36008</td><td></td></tr><tr><td>GSH Dye: Monochlorobimane</td><td>Sigma</td><td>69899</td><td>Toxic</td></tr><tr><td>GSH Dye: Monobromobimane</td><td>Life technologies</td><td>M-1378</td><td>Toxic</td></tr><tr><td>Membrane permeability Dye: YO-PRO-1&amp;nbsp;</td><td>Life technologies</td><td>Y3603</td><td>Irritating&amp;nbsp;</td></tr><tr><td>Membrane permeability Dye: TO-PRO-1&amp;nbsp;</td><td>Life technologies</td><td>T3602</td><td>Irritating&amp;nbsp;</td></tr><tr><td>Membrane permeability Dye: TOTO-1&amp;nbsp;</td><td>Life technologies</td><td>T3600</td><td>Irritating&amp;nbsp;</td></tr><tr><td>Caspase Dye: Cellevent Caspase 3/7 green</td><td>Life technologies</td><td>C10423</td><td>Irritating&amp;nbsp;</td></tr><tr><td>Anti-Cytochrome C antibody (Mouse)</td><td>Thermo</td><td>MA5-11823</td><td></td></tr><tr><td>Anti-phospho-c-Jun antibody (Mouse)</td><td>Thermo</td><td>MA5-15889</td><td></td></tr><tr><td>Anti-phospho-H2AX antibody (Mouse)</td><td>Thermo</td><td>MA1-2022</td><td></td></tr><tr><td>Goat anti-Mouse IgG DyLight 650&amp;nbsp;</td><td>Abcam</td><td>ab96878</td><td></td></tr><tr><td>10x permeabilization buffer</td><td>Fisher</td><td>8408400</td><td></td></tr><tr><td>4% Formaldehyde solution</td><td>Sigma</td><td>F1635</td><td>Toxic</td></tr><tr><td>10x blocking buffer</td><td>Fisher</td><td>8408500</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline&amp;nbsp;</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution</td><td>Sigma</td><td>H8264</td><td></td></tr><tr><td>Staurosporine</td><td>Sigma</td><td>S4400</td><td>Toxic</td></tr><tr><td>Valinomycin</td><td>Sigma</td><td>V0627</td><td>Toxic</td></tr><tr><td>Paraquat</td><td>Sigma</td><td>36541</td><td>Toxic</td></tr><tr><td>Anisomycin</td><td>Sigma</td><td>A9789</td><td>Toxic</td></tr><tr><td>Ethacrynic acid</td><td>Sigma</td><td>E4754</td><td>Toxic</td></tr><tr><td>1-Aminonaphthalene</td><td>Sigma</td><td>34390</td><td>Toxic</td></tr><tr><td>2-Nitropropane</td><td>Sigma</td><td>130265</td><td>Toxic</td></tr><tr><td>Acetamide</td><td>Sigma</td><td>695122</td><td>Toxic</td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td>Toxic</td></tr><tr><td>Acrylamide</td><td>Sigma</td><td>A9099</td><td>Toxic</td></tr><tr><td>Arsenic (V)</td><td>Sigma</td><td>A6756</td><td>Toxic</td></tr><tr><td>Benzene</td><td>Sigma</td><td>12540</td><td>Toxic</td></tr><tr><td>Chromium (VI)</td><td>Sigma</td><td>216623</td><td>Toxic</td></tr><tr><td>Crotonaldehyde</td><td>Sigma</td><td>262668</td><td>Toxic</td></tr><tr><td>Methyl ethyl ketone</td><td>Sigma</td><td>34861</td><td>Toxic</td></tr><tr><td>Nickel&amp;nbsp;</td><td>Sigma</td><td>203866</td><td>Toxic</td></tr><tr><td>Nitrobenzene</td><td>Sigma</td><td>48547</td><td>Toxic</td></tr><tr><td>Phenol</td><td>Sigma</td><td>P5566</td><td>Toxic</td></tr><tr><td>Quinoline</td><td>Sigma</td><td>241571</td><td>Toxic</td></tr><tr><td>Toluene</td><td>Sigma</td><td>34866</td><td>Toxic</td></tr></tbody></table>

high content screening analysis to evaluate the toxicological effects of harmful and potentially harmful constituents (hphc)

香烟烟雾（CS）是心血管和肺疾病的主要危险因素。因为CS是一个包含7000多化学品1这是具有挑战性的评估个别成分其总体毒性的贡献复杂的气溶胶。个别成分以及混合物的毒理学配置文件可以然而，建立了体外 ，由通量筛选工具，这使烟草烟雾的危害和潜在的有害成分（HPHCs）的分析施加高，受美国食品和药品的定义管理局（FDA）。2 对于初步评估，使用该化合物的毒性的实时，无标记评估的基于阻抗的工具。该仪器读数依赖于细胞黏附，活力和形态，所有共同提供的电池状态的概述。无量纲参数，命名的单元格指数，用来量化。一组的DIF同的染色方法是为基于荧光成像开发的调查和HCS平台被用来获取对每个HPHC引起的一种细胞毒性更深入的信息。 测试的15个成分中，只有五人入选基于HCS-分析，他们注册了一个可计算的LD 50（&lt;20毫米）。这些包括1- aminonaphtalene，砷（V），铬（VI），巴豆醛和苯酚。基于它们在HCS效果，1- aminonaphtalene和酚可确定以诱导线粒体功能障碍，并连同铬（VI），为基于增加组蛋白H2AX的磷酸化基因毒性。巴豆醛被确定为氧化应激诱导剂和砷作为应力激酶途径的活化剂。 这项研究表明，阻抗和基于HCS技术的组合提供了用于在体外的CS成分的评估一个强大的工具。

RTCA 因为没有检测到毒性作用，当HCS端点不会信息，没有显示的那些化合物细胞活力下降到在RCTA不是由HCS（ 图3b，C，D，G，K，L，M所测试的最高浓度页 ）。化合物显示仅在最高浓度细胞活力下降（ 图3e，O）也被取消了HCS。最后，只用一个可计算的LD 50（&lt;20毫摩尔）的成分被选择用于进一步的HCS分析（ 图 3a，F，H，J，N）。符合上述标准HPHCs是:1-aminonaphtalene，砷（V），铬（VI），巴豆醛和苯酚。 HCS 作为质量检查（QC），阳性对照是科幻首先进行分析，以保证正确地进行该染色步骤。阳性对照处理的细胞的代表性图片示于图6中 ，数据值是如前所述标准化为车辆。没有剂量反应曲线被绘制为只有三个剂量测试，不是所有的三个剂量都在每一个时间点考虑。阳性对照的剂量被选择（基于先前的实验中，未示出的数据），使适当的反应是为在两个4小时和24小时的每个端点观察。在特定的剂量1和2中使用而剂量2和3用于评估在24小时的效果，在4小时来评估的效果。如果为阳性对照的剂量没有观察到反应板被丢弃。注意，对于所有的端点，除非线粒体膜电位和GSH含量时，信号强度的增加，预计。 所有的化合物，除苯酚，引起坏死性苯氧<html>型的基础上，增加细胞膜的通透性（ 图7a，F，H，1）。 1- aminonaphtalene，铬（VI），巴豆醛和苯酚被确定为基础上，增加组蛋白H2AX的磷酸化（ 图7E，J，N，P）为基因毒性。苯酚和1- aminonaphtalene被发现诱发严重线粒体功能障碍（ 图7b，邻 ），其与1-aminonaphtalene，导致了增加的细胞色素C的释放（ 图7c）。增加的胱天蛋白酶3/7活性的检测在铬曝光提供凋亡事件的证据。氧化应激诱导（ROS或GSH）也处理后与1- aminonaphtalene，巴豆醛和苯酚（ 图7d，M，Q）进行检测。最后，砷诱导细胞应激的转录因子cJun（ 图7克 ）增加的磷酸化证明。 负载/ 53987 / 53987fig1.jpg"/&gt; 图1.化合物TOX-探查工作流。 一个）的工作流程的示意图，随后在这项研究。首先，使用RTCA平台来选择适当的剂量为后续HCS来表征特定化合物的毒性特征。b）该研究的实验设计进行剂量范围发现。播种后24小时，细胞剂量和阻抗值在接下来的24小时连续监控，而HCS终点进行了调查4和给药后24小时。 <a href="https://www.jove.com/files/ftp_upload/53987/53987fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53987/53987fig2.jpg" /> 图2. RTCA曝光板。首先被执行五步1点10连续稀释生成的复合主盘。每种化合物，包括车辆控制（剂量0），然后在一式三份与介质和星形孢菌素作为对照加入到曝光板在一起。需要注意的是序列传输时保持剂量，最高剂量是行号1，而车辆控制是行数7. <a href="https://www.jove.com/files/ftp_upload/53987/53987fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53987/53987fig3.jpg" /> 图3代表RTCA细胞活性的结果。a） 第 1-aminonaphtalene， 二）2-硝基丙烷， 三）乙酰胺， 四）丙酮， 电子）丙烯酰胺，f）的砷（V）中，G）苯，h）的铬（VI） ，j）的巴豆醛，k）的甲基乙基酮， 升）NickeL（II）中，m）硝基苯，n）的苯酚， 邻）喹啉， 对）甲苯。在24小时的血样，曲线（AUC）下面积计算各剂量（包括阳性对照和载体）从0到100％的活性（y轴），其中0反映的活性和标准化的范围内车辆与阳性对照-100。然后值描绘，并用四参数Hill方程，并在可能，LD 50计算拟合。浓度对数标度（X轴）表示。 <a href="https://www.jove.com/files/ftp_upload/53987/53987fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53987/53987fig4.jpg" /> 图4.稀释方案为HCS试验得到阳性对照化合物A）阳性对照的加入和已经hicle到串行稀释板。b）该阳性对照的系列稀释。 三）200X阳性对照的剂量。 德）的200倍阳性对照的剂量在介质（1:40稀释），产生含有5倍剂量阳性对照板。请注意，每个剂量是一式三份稀释，以反映在曝光板的最后布局）。 <a href="https://www.jove.com/files/ftp_upload/53987/53987fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53987/53987fig5.jpg" /> 图5. HCS曝光板。首先被执行五步稀释生成复合主盘。每个化合物，包括车辆控制（剂量0），然后在一式三份与阳性对照加入到曝光板在一起。请注意剂量订单转移后保持，最高剂量是行号1，而车辆控制是行数7. <a href="https://www.jove.com/files/ftp_upload/53987/53987fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53987/53987fig6.jpg" /> 图6.代表性的荧光抗体照片或染料染色的细胞的 ） 核参数 - 核染料 :它与DNA结合的活的或固定细胞中的渗透染料。此染色剂用于识别各个细胞标记的核区域b） 坏死 - 细胞膜渗透性染料 :的细胞膜完整性的基于染料的检测。试剂在本质上是不可渗透的细胞膜。期间坏死，膜变得可渗透和染料进入细胞并与DNA结合产生强光S。ignal 三） 凋亡 - 细胞色素C:细胞色素C释放，早期凋亡的一个著名标志性的基于抗体的检测。在细胞凋亡的诱导，细胞色素C从线粒体释放并扩散到细胞核四）DNA 损伤 - pH2AX:组蛋白H2AX的磷酸化的基于抗体的检测，双链DNA断裂的一个公知的标志E） 胁迫激酶 - cJun:在丝氨酸73 cJun的，细胞应激的一个公知的标志的磷酸化的基于抗体的检测六） 氧化应激 - DHE:超氧自由基的基于染料的检测。本身二氢乙荧光在细胞质蓝色，而氧化型荧光乙锭在红DNA嵌入G）GSH - MBCL:免费GSH分子染料检测。 MBCL与谷胱甘肽反应来产生高荧光产品高） 细胞凋亡 - 胱天蛋白酶激活3/7:3/7蛋白酶的活性基染料检测。试剂是非荧光与抑制DNA结合一个4个氨基酸的肽。在胱天蛋白酶3/7的活化，该肽被裂解使染料与DNA结合，并产生一个明亮，荧光响应。面板BH显示阳性对照处理的细胞。 <a href="https://www.jove.com/files/ftp_upload/53987/53987fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53987/53987fig7.jpg" /> 图7.代表性的HCS结果:1 aminonaphtalene（AE），砷（V）（f和g），铬（VI）（ 香港 ），巴豆醛（LN） ​​和苯酚（OQ）。 4小时（蓝线）和24小时（橙色线）信号计算各剂量和归一化到车辆活性（0％）。这不包括在曲线拟合计算值显示为灰色。浓度对数标度（X轴）表示。 <a href="https://www.jove.com/files/ftp_upload/53987/53987fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>化验</td><td>端点＃ </td><td>生物终点</td><td>细胞室</td><td>输出功能</td></tr><tr><td rowspan="4">细胞毒性</td><td> 1 </td><td>线粒体质量6 </td><td>细胞质</td><td>现货平均面积</td></tr><tr><td> 2 </td><td>线粒体膜电位6 </td><td>细胞质</td><td>现货平均强度</td></tr><tr><td> 3 </td><td>细胞色素c释放<sup&gt; 7 </td><td>核</td><td>平均强度</td></tr><tr><td> 4 </td><td>细胞膜的通透性8 </td><td>核</td><td>平均强度</td></tr><tr><td> DNA损伤</td><td>五</td><td>磷酸H2AX 9 </td><td>核</td><td>平均强度</td></tr><tr><td>应激激酶</td><td> 6 </td><td>磷酸cJun 10 </td><td>核</td><td>平均强度</td></tr><tr><td> ROS </td><td> 7 </td><td> ROS 11 </td><td>核</td><td>平均强度</td></tr><tr><td> GSH含量</td><td> 8 </td><td> GSH 12 </td><td>细胞质</td><td>现货平均强度</td></tr><tr><td>细胞凋亡</td><td> 9 </td><td>胱天蛋白酶3 13 </td><td>细胞质</td><td>现货平均强度</td></tr></tbody></table> 表1. HCS一个列表ssays和端点。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"></td><td rowspan="2">车辆</td><td colspan="6" rowspan="2"> RTCA剂量（μM） </td><td rowspan="2"> LD 50 </td><td colspan="5"> HCS剂量</td></tr><tr><td colspan="2"></td><td colspan="4">细胞活力选定（​​μM） </td><td> 3R4F（NM） </td></tr><tr><td colspan="2"> 1 Aminonaphtalene </td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 280μM </td><td> 2000 </td><td> 500 </td><td> 200 </td><td> 150 </td><td> 0.27 </td></tr><tr><td colspan="2"> 2-硝基丙烷</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">乙酰胺</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">丙酮</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">丙烯酰胺</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">砷（V） </td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 160μM </td><td> 200 </td><td> 100 </td><td> 50 </td><td> 25 </td> &lt;TD&gt; 0.17 </td></tr><tr><td colspan="2">苯</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">铬（VI） </td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 20μM </td><td> 100 </td><td> 50 </td><td> 20 </td><td> 10 </td><td> 0.06 </td></tr><tr><td colspan="2">巴豆醛</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 200μM </td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2000 </td></tr><tr><td colspan="2">甲乙酮</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt;20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">镍</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">硝基苯</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">苯酚</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 2300μM </td><td> 5000 </td><td> 2000 </td><td> 1000 </td><td> 500 </td><td> 240 </td></tr><tr><td colspan="2">喹啉</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td&gt; <td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">甲苯</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表2. 在治疗24小时 相对LD 50 测试HPHC化合物列表 。选择HCS分析化合物是橙色高亮显示和测试的剂量也给予。该3R4F剂量相当于构成本，在一个棒的从基准香烟3R4F烟雾的量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>化验</td><td>复合</td><td>原液</td><td>溶剂</td&gt; <td colspan="3">剂量（多个）（μM） </td></tr><tr><td>细胞活力</td><td>星形孢菌素</td><td> 10毫</td><td> DMSO </td><td colspan="3"> 50 </td></tr><tr><td>细胞毒性</td><td>霉素</td><td> 10毫</td><td> DMSO </td><td> 50 </td><td> 20 </td><td>五</td></tr><tr><td> DNA损伤</td><td>百草枯</td><td> 100毫米</td><td> DMSO </td><td> 500 </td><td> 200 </td><td> 50 </td></tr><tr><td>应激激酶</td><td>茴香</td><td> 2毫米</td><td> DMSO </td><td> 10 </td><td> 4 </td><td> 1 </td></tr><tr><td> ROS </td><td>鱼藤酮</td><td> 200毫米</td><td> DMSO </td><td> 1000 </td><td> 400 </td><td> 100 </td></tr><tr><td> GSH含量</td><td>利尿酸</td><td> 200毫米</td><td> DMSO </td><td> 1000 </td><td> 400 </td><td> 100 </td></tr><tr><td>细胞凋亡</td><td>星形孢菌素</td><td> 40毫米</td><td> DMSO </td><td> 200 &lt;/ TD&gt; <td> 50 </td><td> 20 </td></tr></tbody></table> 表3列出用于每个检测阳性对照和浓度。

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High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

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High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

12.1K 次观看.  Philip Morris International R&D. The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds. 

视频: High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

基于发育毒性测定化学品安全筛选和系统生物学数据生成人类多能干细胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

科研

发育生物学

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

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

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

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

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 ...

环境科学

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

利用目标筛选和非靶向筛选高分辨率质谱相结合的工作流程识别跨氟和多氟化学物种

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

化学

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite their high environmental occurrence, there is very limited information regarding their toxicological effects. It is urgent to develop high-throughput screening (HTS) methods to guide toxicological studies. In this study, a receptor-ligand binding assay using an HTS system was developed to determine the binding potency of environmental pollutants on nuclear receptors. The test is conducted using a microplate reader (i.e., a 96-well plate containing various chemicals) by measuring the fluorescence polarization (FP) of a specific fluorescent probe. This assay consists of four parts: the construction and transformation of recombinant vectors, the expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor. The binding potency of two environmental pollutants, perfluorooctanesulfonic acid (PFOS) and triphenyl phosphate (TPHP), with peroxisome proliferator-activated receptor gamma (PPAR&#947;) was determined to illustrate the assay procedure. Finally, the advantages and disadvantages of this method and its potential applications were also discussed.

This protocol describes a high-throughput screening system that uses fluorescence polarization of a specific fluorescent probe binding to a nuclear receptor as a readout for screening environmental pollutants.

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

癌症研究

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

免疫与感染

通过电电池-衬底传感评估 MOF 诱导的电池行为变化

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

作者聚焦:评估人肺上皮细胞对金属有机框架反应的进展 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

用于实时评估金属有机框架毒理学特征的电池-基底传感

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

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