NTP MediumThroughput C. elegans Screening Facility

目前，在使用的化学品超过8万种，每年约有2000种新化学品投入使用。监管机构已经认识到替代毒理学方法和模型的必要性，以减少当前毒性测试方案的时间和费用。与国家毒理学计划(NTP)合作，我们小组正在开发秀丽线虫作为体内毒理学测试的替代生物。短的生命周期、简单而廉价的维护和培养以及详细的生物学知识使快速、低成本的毒性测试得以开发，这些测试很容易对有毒行为进行机械性研究。由于应激反应和其他相关途径在进化上是保守的，线虫引起的反应很可能适用于理解包括人类在内的高等生物体中的类似过程。 这个小组是国家毒理学计划的一部分，也是生物分子筛选分部的一部分。该筛选设施参与了线虫作为毒理学测试替代生物的开发。由于信号转导和胁迫反应途径在进化上的保守性，线虫引起的反应很可能适用于理解高等生物体中的类似过程。该小组有两项主要活动：研究和开发线虫的MTS和HTS技术，以及参与NTP、EPA和NCGC之间的谅解备忘录，命名为Tox21(Science 15 2月2008319：906-907DOI：10.1126/Science.1154619)。 已开发出线虫中通量筛选试验--用于监测生长、大小、繁殖、取食和移动的方案，并已用于测试100种毒物。在制定监测协议的过程中，专门为线虫数据制定了统计分析程序。我们已经提交了一份关于摄食分析的手稿，并正在完成其他研究，这些研究将包括在目前正在准备的关于生殖和生长分析的手稿中。我们与Grace Kissling(EDBP)、Marjolein Smith(SRA)合作开发了一个数学模型，描述了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第二阶段文库(700)中的化学物质，此外还有镉、汞、二甲基亚砜、百草枯、毒死蜱、MNNG和衣霉素。 我们创造了一种自动化、高含量、高通量的体内毒理学检测方法。在胁迫诱导的线虫基因启动子的驱动下，检测了含有荧光蛋白的几个转基因线虫株的转录反应。这个系统既捕捉到了通路激活的变化，也捕捉到了基因表达的组织特异性。 目前，我们有14个基因结构可供测试--ce-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和UGT-13。每个转基因菌株都已被证实对至少一种刺激有反应，通常是热休克；然而，这种反应往往被高基线荧光水平所掩盖。 为了选择敏感和特异的应激反应基因，我们使用了一系列众所周知的化合物来比较应激反应基因的转录--氧化应激源百草枯；DNA损伤剂N-甲基-N‘-硝基-N-亚硝基；重金属毒素镉；有机磷神经毒素毒死蜱；内质网应激源衣霉素；以及常见的线虫控制应激源热休克。通过肉眼观察荧光的变化，我们发现在被测试的前6个基因(CYP-35A2、GST-4、HSP-16.2、HSP-16.4、HSP-4和HSP-60)中，至少有一个基因在每个毒物的响应下上调。这种反应一般是机械相关的，例如，HSP-4(已知对内质网胁迫做出反应)转基因线虫在衣霉素处理后信号增加。 用6种浓度的毒死蜱处理pCyp-35A2：：mCherry、Punc-47：：GFP线虫，对方法进行了优化。这种组合代表了荧光强度和位置的变化。用高含量成像仪拍摄的转基因线虫图像测量了花期数据，并使用CellProfilers WormToolbox进行了分析。该软件是专门为线虫设计的高通量成像分析程序。 初步研究表明，新的检测结果在趋势上与定量实时聚合酶链式反应(qRT-PCR)相似，尽管灵敏度较低。未来的研究将通过qRT-PCR和新的高含量、高通量测试来比较目前可用于七种应激源的所有转基因线虫。 我们还在开发一种通过测量体内ATP水平来测量线粒体功能的新方法。利用一种高度表达萤火虫荧光素酶的线虫菌株，我们正在开发一种跟踪ATP池水平的AN分析方法，作为生理状态的标志。毒物暴露后发光的变化将反映三磷酸腺苷水平的变化。这项测试将被用来确定各种化学物质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. Because of the evolutionarily conserved nature of signal transduction and stress-response pathways, it is likely that responses elicited in C. elegans will be applicable to understanding similar processes in higher organisms. 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 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. 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 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 eye, 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 my measuring ATP in vivo levels. Using a highly strain of C. elegans which constitutively expresses firefly luciferase, we are developing a 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.