The Next-Generation Developmental and Reproductive Toxicology (DART) Assay using High-Content Analysis of Genetically Diverse C. elegans Populations

使用遗传多样性线虫种群高内涵分析进行下一代发育和生殖毒理学 (DART) 测定

• 批准号: 10326002
• 负责人: Evan Hegarty
• 金额: $ 25.37万
• 依托单位: VIVOVERSE, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326002
• 关键词: Adult; Adverse effects; Agreement; Animal Model; Animal Testing; Animal Testing Alternatives; Animals; Biochemical Pathway; Biological; Biological Assay; Biology; Body Size; Caenorhabditis elegans; Caliber; Chemicals; Confidence Intervals; Development; Dimensions; Dose; Embryo; Evaluation; Face; Feeding behaviors; Female; Genes; Genetic; Genomic Segment; Goals; Health; Heavy Metals; Heritability; Human; Image; In Vitro; Inbreeding; Industry; Invertebrates; Laboratory Animals; Length; Life; Life Cycle Stages; Link; Maintenance; Mammals; Measures; Methods; Modeling; Molecular; Nature; Nematoda; Optics; Oryctolagus cuniculus; Pathway interactions; Performance; Pesticides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenotype; Physiology; Population; Positioning Attribute; Principal Component Analysis; Quantitative Trait Loci; Rapid screening; Rattus; Reproducibility; Reproductive system; Resolution; Risk; Rodent; Safety; Sensitivity and Specificity; Services; Signal Transduction; Stress; Surveys; System; Technology; Testing; Time; Toxic effect; Toxicology; Toxin; Variant; Vulva; Whole Organism; base; cost; cost effective; density; developmental toxicity; drug discovery; feeding; genetic strain; genome wide association study; genome-wide analysis; graphical user interface; hazard; high resolution imaging; high throughput screening; improved; in vivo; individual response; insight; inter-individual variation; interest; male; neurotoxicity; next generation; novel; predicting response; reproductive; reproductive organ; response; screening; small molecule libraries; success; toxicant; trait

Abstract: Developmental and reproductive toxicology (DART) studies are commonly conducted using a large number of genetically inbred rodent and rabbit strains to predict adverse effects of chemicals on human health. While in vitro or ex vivo methods are used in early drug discovery fields, they face several challenges for DART studies due to lack of system-level biology, multi-organ physiology, and male/female reproductive organs. C. elegans is a genetically and molecularly attractive model organism that provides many advantages for high-throughput DART studies, including a high degree of conserved genes and biochemical pathways, a well-defined and characterized reproductive system, a rapid life and reproductive cycle, and low maintenance costs. While C. elegans based assays show a significant agreement with higher mammals (rat and rabbit), there is an urgent need for more sensitive assays to improve both sensitivity and specificity for wide acceptance of C. elegans as an alternative animal model for DART testing. The goal of this proposal is to demonstrate the first C. elegans based high-content DART assay to analyze multiple phenotypes that will reduce false negatives and capture interindividual toxicology responses from a panel of genetically diverse strains. Newormics’ vivoChip technology is the only screening platform that can provide high-resolution imaging of C. elegans strains at high throughputs. It will allow us to assess accurately multiple endpoints, such as the feeding behavior, body size, vulva development, animal stress, and embryo health in non-anesthetized adults. With multi-parametric DART analysis using genetic backgrounds, we can reduce the occurrence of seemingly disparate experimental results from single-strain studies, enhance reproducibility, capture potential interindividual variabilities, and reduce false- negatives. In Aim 1, we will characterize the assay-quality of the proposed high-content DART assay (with 7 phenotypes, including embryonic traits) by testing 6 known toxicants with 8 doses and in 10 replicates using the N2 wild-type strain. We will then screen 33 chemicals including 10 false negatives to demonstrate the predictability of our multiple phenotype space and improved sensitivity of DART predictions using C. elegans. In Aim 2, we will test a subset of chemicals against a panel of 12 divergent strains to capture the interindividual variations in toxicological responses and improve interspecies predictability. We will calculate broad-sense heritability by measuring the variance in toxicology phenotypes and identify genetic contributions for such variances. In the Phase II, we plan to expand the application of our assay to test a larger library of chemicals and incorporate a larger panel of genetically divergent strains to perform QTL analysis to pinpoint genes that appear to be linked to DART effects. With the success of this proposal, Newormics will be in a unique position to offer a comprehensive set of in vivo toxicology services to help reduce overall cost and number of animals required for identifying toxicity of new chemicals.

抽象的： 发育和生殖毒理学（DART）研究通常使用大量 遗传上的近交啮齿动物和兔子菌株可以预测化学物质对人类健康的不利影响。同时在 体外或体内方法用于早期药物发现领域，它们面临着飞镖研究的几个挑战 由于缺乏系统水平的生物学，多器官生理学和雄性/女性生殖器官。秀丽隐杆线虫是 一种一般且分子吸引人的模型生物，为高通量提供了许多优势 DART研究，包括高度保守基因和生化途径，一个明确的和 表征生殖系统，快速寿命和生殖周期以及维护成本较低。而C. 基于秀丽隐杆线的阿萨斯与较高的哺乳动物（大鼠和兔子）一致，紧急 需要更敏感的测定法以提高敏感性和特异性，以广泛接受秀丽隐杆线虫作为 DART测试的替代动物模型。该提议的目的是演示第一个C.秀丽隐杆线虫 基于高含量的DART测定法以分析多种表型，这些表型将减少假阴性并捕获 个体间毒理学的反应来自一组遗传多样的菌株。 Newormics的Vivochip技术 是唯一可以在高吞吐量下提供秀丽隐杆线虫菌株的高分辨率成像的筛选平台。 它将允许我们准确评估多个终点，例如喂养行为，身体大小，外阴 非麻醉成年人的发育，动物压力和胚​​胎健康。通过多参数DART分析 使用遗传背景，我们可以减少从看似不同的实验结果的发生 单元研究，提高可重复性，捕获潜在的个体差异，并减少假 负面。在AIM 1中，我们将表征拟议的高含量DART测定法的评估质量（有7个 表型，包括胚胎特征），通过测试6种具有8剂的已知毒物，并使用10个重复的毒物 N2野生型菌株。然后，我们将筛选33种化学物质，包括10种假阴性，以证明 使用秀丽隐杆线虫的多种表型空间的可预测性和提高DART预测的灵敏度。在 AIM 2，我们将针对12个发散菌株的面板测试一部分化学物质，以捕获个体 毒理学反应的变化并改善种间的可预测性。我们将计算广泛的 通过衡量毒理学表型的差异并确定这种遗传贡献来遗传性 差异。在第二阶段，我们计划扩大评估的应用以测试较大的化学物质库 并结合了较大的遗传菌株，以执行QTL分析，以查明基因 似乎与飞镖效应有关。随着该提议的成功，新象征将处于独特的位置 提供一套全面的体内毒理学服务，以帮助减少整体成本和动物数量 确定新化学物质的毒性所需。