NTP MediumThroughput C. elegans Screening Facility

目前，有超过80，000种化学品在使用，每年大约有2000种新化学品投入使用。 监管机构已经认识到需要替代毒理学方法和模型，以减少当前毒性测试方案的时间和费用。 在国家毒理学计划（NTP）的合作下，我们的小组正在开发C。线虫作为体内毒理学试验的替代生物。短的生命周期，容易和廉价的维护和培养，以及详细的生物学知识，允许开发快速，低成本的毒性测试，很容易借给自己的机制研究毒物的行动。由于应激反应和其他相关途径在进化上的保守性，C. elegans将适用于理解包括人类在内的高等生物的类似过程。 该小组是国家毒理学计划的一部分，属于生物分子筛选分支。 筛选设施参与了C。线虫作为毒理学测试的替代生物。 该小组的主要活动有两个：研究和开发C。elegans MTS和HTS技术以及参与NTP、EPA和NCGC之间的谅解备忘录，指定为Tox 21（Science 15 February 2008 319：906-907 DOI：10.1126/science.1154619）。 C.线虫中等通量筛选试验-已经开发了用于监测生长、大小、繁殖、进食和运动的方案，并且已经用于测试超过100种毒物。 在监测方案的制定中，专门为C。elegans数据。 我们已提交了一份关于摄食试验的手稿，并正在完成其他研究，这些研究将纳入目前正在编写的关于繁殖和生长试验的手稿中，已发表的关于摄食、繁殖和生长试验开发和应用的手稿以及关于其联合使用的综述。 在与Grace Kissling（EDBP），Marjolein Smith（SRA）的合作中，我们开发了一个描述C。线虫的生长和毒物对生长参数的影响。今年，WormTox小组测试了ToxCAST第二阶段库（700）中的化学品，以及镉、汞、DMSO、百草枯、毒死蜱、MNNG和衣霉素。 我们还创建了一种自动化、高含量、高通量的体内毒理学试验。在几株转基因C. elegans含有由胁迫诱导型C. elegans基因 该系统捕获了通路激活的变化以及基因表达的组织特异性。目前，我们有14个基因构建体可用于测试- ced-3，cep-35 A2，gcs-1，gst-38，gst-4，hsp-16.2，hsp-16.4，hsp-17，hsp-4，hsp-6，hsp-60，mtl-2，ugt-1和ugt-13。 每个转基因菌株已被证实响应至少一个刺激，通常是热休克;然而，反应往往是模糊的高基线荧光水平。 为了选择具有敏感和特异性应激反应的基因，我们使用一系列众所周知的化合物-百草枯，一种氧化应激因子; N-甲基-N '-硝基-N-亚硝基胍，一种DNA损伤剂;镉，一种重金属毒素;毒死蜱，一种有机磷酸盐神经毒素;衣霉素，一种内质网应激因子;和热休克，一种常见的C。elegans控制应激源。 通过显微镜观察荧光的变化，我们发现，至少有一个前6个基因测试（cep-35 A2，GST-4，HSP-16.2，HSP-16.4，HSP-4，HSP-60）上调响应每种毒物。 这种反应通常是机械相关的;例如，HSP-4（已知对内质网应激有反应）转基因线虫在衣霉素处理后增加了信号。 使用用6种浓度的毒死蜱处理的pcyp-35 A2：：mCherry、punc-47：：GFP线虫优化测定。 这种组合代表了荧光强度和位置的变化。 从用高含量成像仪拍摄的转基因线虫的图像测量开花数据，并使用CellProfilers Wormplant进行分析。 该软件是一个高通量的成像分析程序，专门设计用于线虫。 初步研究表明，新检测方法的结果与定量实时聚合酶链反应（qRT-PCR）的结果趋势相似，但灵敏度较低。 未来的研究将通过qRT-PCR和新的高含量、高通量测定来比较目前可获得的所有转基因线虫对七种应激物的反应。 我们还在开发一种新的检测方法，通过测量体内ATP水平来测量线粒体功能。利用C.为了研究组成型表达萤火虫荧光素酶的线虫，我们正在开发一种检测方法，该方法可以跟踪ATP池的水平，作为生理状态的标志物。毒物暴露后发光的变化将反映ATP水平的变化。该试验将用于确定各种化学品对生理健康的相对影响，而不是更常见的形态特征变化。

Currently, there are over 80,000 chemicals in use and approximately 2000 new chemicals are introduced into use every year. Regulatory agencies have recognized the need for alternative toxicological methods and models to decrease the time and expense of current toxicity testing protocols. In association with the National Toxicology Program (NTP), our group is developing C. elegans as an alternative organism for in vivo toxicological testing. Short life cycles, easy and inexpensive maintenance and culturing, and detailed biological knowledge has allowed for the development of rapid, low-cost toxicity tests that readily lend themselves to mechanistic studies of toxicant actions. Because of the evolutionarily conserved nature of the stress-response and other relevant pathways, it is likely that responses elicited in C. elegans will be applicable to understanding similar processes in higher organisms, including humans. This group is part of the National Toxicology Program and within the Biomolecular Screening Branch. The screening facility is involved in the development of C. elegans as an alternative organism for toxicological testing. There are two major activities of this group: research and development of C. elegans MTS and HTS technologies and participation in the MOU among the NTP, EPA, and NCGC, designated Tox21 (Science 15 February 2008 319: 906-907 DOI: 10.1126/science.1154619). Development of C. elegans medium-throughput screening assays - Protocols for the monitoring of growth, size, reproduction, feeding, and movement have been developed and have been used to test over 100 toxicants. Included in the creation of monitoring protocols, statistical analysis routines have been developed specifically for the C. elegans data. We have submitted one manuscript on the feeding assay and are completing additional studies that will be included in manuscripts currently being prepared on the reproduction and growth assays published manuscripts on the development and application of the feeding, reproduction, and growth assays as well as reviews on their combined use. In collaboration with Grace Kissling (EDBP), Marjolein Smith (SRA) we have developed a mathematical model that described C. elegans growth and the effects of toxicants on growth parameters. This year the WormTox group tested the chemicals in the ToxCAST phase 2 library (700), in addition to cadmium, mercurical, DMSO, paraquat, chlorpyrifos, MNNG and tunicamycin. We have also created an automated, high-content, high-throughput in vivo toxicological assay. Transcriptional response was measured in several strains of transgenic C. elegans containing fluorescent proteins driven by the promoters of stress-inducible C. elegans genes. This system captured both changes in pathway activation, as well as tissue-specificity of gene expression. Currently, we have 14 gene constructs available for testing -- ced-3, cyp-35A2, gcs-1, gst-38, gst-4, hsp-16.2, hsp-16.4, hsp-17, hsp-4, hsp-6, hsp-60, mtl-2, ugt-1 and ugt-13. Each transgenic strain has been confirmed to respond to at least one stimulus, usually heat shock; however, the reaction was often obscured by high baseline fluorescence levels. To select genes with a sensitive and specific stress response, we compared transcription of stress response genes using an array of well understood compounds -- paraquat, an oxidative stressor; N-methyl-N'-nitro-N-nitrosoguanidine, a DNA damaging agent; cadmium, a heavy metal toxin; chlorpyrifos, an organophosphate neurotoxin; tunicamycin, an endoplasmic reticulum stressor; and heat shock, a common C. elegans control stressor. By observing changes in fluorescence by microscope, we showed that at least one of the first 6 genes tested (cyp-35A2, gst-4, hsp-16.2, hsp-16.4, hsp-4, and hsp-60) upregulated in response to each toxicant. The response was generally mechanistically relevant; for example, hsp-4 (known to respond to endoplasmic reticulum stress) transgenic nematodes increased signal after tunicamycin treatment. The assay was optimized using pcyp-35A2::mCherry, punc-47::GFP nematodes treated with 6 concentrations of chlorpyrifos. This combination typified both changes in intensity and location of fluorescence. Florescence data were measured from images of transgenic nematodes taken with a high content imager and analyzed using CellProfilers WormToolbox. The software is a high-throughput imaging analysis program specifically designed for use with nematodes. Initial studies indicate that the results of the new assay are similar in trend, although less sensitive, than quantitative real-time polymerase chain reaction (qRT-PCR). Future studies will compare all transgenic nematodes currently available to the seven stressors by both qRT-PCR and the novel high-content, high-throughput assay. We are also developing a new assay to measure mitochondrial function by measuring ATP in vivo levels. Using a strain of C. elegans which constitutively expresses firefly luciferase, we are developing an assay that tracks levels of ATP pools as a marker for physiological status. Changes in luminescence following toxicant exposure will reflect changes in ATP levels. This assay will be used to determine the relative effects of a variety of chemicals on physiological health instead of the more common changes in morphological traits.