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

摘要： 发育和生殖毒理学(DART)研究通常使用大量的 用基因近亲繁殖的啮齿动物和兔子品系来预测化学物质对人类健康的不利影响。当在 体外或体外方法被用于早期药物发现领域，它们面临着DART研究的几个挑战 由于缺乏系统水平的生物学、多器官生理学和男性/女性生殖器官。线虫是 一种在遗传和分子上具有吸引力的模式生物，为高通量提供了许多优势 DART研究，包括高度保守的基因和生化途径，明确和 具有生殖系统快、生命和生殖周期快、维护成本低等特点。而C. 基于优雅的化验显示与高等哺乳动物(大鼠和兔)有显著的一致性，有一个紧急的 需要更灵敏的分析来提高灵敏度和特异度，以广泛接受线虫作为 用于DART测试的替代动物模型。这项提案的目标是展示第一批线虫 基于高含量DART分析将减少假阴性和捕获的多种表型 来自一组遗传不同菌株的个体间毒理学反应。NeWormics的活体芯片技术 是唯一能够以高通量提供线虫菌株高分辨率成像的筛选平台。 它将使我们能够准确地评估多个端点，如进食行为、身体大小、外阴 未麻醉成人的发育、动物应激和胚胎健康。使用多参数DART分析 使用遗传背景，我们可以减少似乎不同的实验结果的出现 单一菌株研究，增强重复性，捕捉潜在的个体间变异，并减少虚假 底片。在目标1中，我们将对建议的高含量DART检测的检测质量进行表征(7 表型，包括胚胎特征)，用8个剂量测试6种已知毒物，并使用 N_2野生型菌株。然后我们将筛选33种化学物质，其中包括10种假阴性物质，以证明 我们的多重表型空间的可预测性和使用线虫的DART预测的改进的敏感性。在……里面 目标2，我们将测试一组化学物质与12个不同菌株的组合，以捕获个体间的 毒理学反应的变化，并提高了物种间的可预测性。我们将计算广义的 通过测量毒物表型的变异并确定其遗传贡献来确定遗传力 方差值。在第二阶段，我们计划扩大我们的检测的应用范围，以测试更大的化学药物库 并加入一组更大的遗传差异菌株进行QTL分析，以准确定位 似乎与飞镖效果有关。随着这一提议的成功，NeWormics将处于独特的地位 提供一套全面的体内毒理学服务，帮助降低整体成本和动物数量 用于识别新化学品的毒性。

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.