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.

摘要： 发育和生殖毒理学（DART）研究通常使用大量的 基因近交啮齿动物和兔子品系，以预测化学品对人类健康的不利影响。而在 体外或离体方法用于早期药物发现领域，它们面临DART研究的几个挑战 由于缺乏系统级生物学、多器官生理学和雄性/雌性生殖器官。C.埃莱甘斯群岛 一种遗传和分子上有吸引力的模式生物，为高通量生物学提供了许多优势， DART研究，包括高度保守的基因和生化途径，一个明确的， 其特点是生殖系统，生命和生殖周期快，维护成本低。而C. 基于线虫的测定显示与高等哺乳动物（大鼠和兔）的显著一致性，因此迫切需要 需要更灵敏的测定来提高灵敏度和特异性以广泛接受C.雅丽甘斯 DART测试的替代动物模型。本提案的目的是证明第一个C。elegans 基于高含量DART分析来分析多种表型，这将减少假阴性并捕获 来自一组遗传多样性菌株的个体间毒理学反应。Newormics的vivoChip技术 是目前唯一能提供C. elegans菌株at high高throughput通量. 它将使我们能够准确地评估多个终点，如进食行为，身体大小，外阴 发育、动物应激和未麻醉成年人的胚胎健康。多参数DART分析 利用遗传背景，我们可以减少看似不同的实验结果的发生， 单菌株研究，提高重现性，捕捉潜在个体间变异，并减少假 底片。在目标1中，我们将表征所提出的高含量DART测定的测定质量（具有7 表型，包括胚胎性状），使用 N2野生型菌株。然后，我们将筛选33种化学品，包括10种假阴性，以证明 我们的多表型空间的可预测性和使用C.优美的在 目标2，我们将测试一个子集的化学品对一组12个不同的菌株，以捕捉个体间的 毒理学反应的变化，并提高种间的可预测性。我们将计算广义 通过测量毒理学表型的方差来确定遗传性，并确定遗传贡献， 差异。在第二阶段，我们计划扩大我们的检测方法的应用，以测试更大的化学品库 并结合一个更大的基因分歧菌株面板进行QTL分析，以查明基因， 似乎与DART效应有关。随着这一提议的成功，Newormics将处于一个独特的地位。 提供一套全面的体内毒理学服务，以帮助降低总体成本和动物数量 用于识别新化学品的毒性。