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

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.Project Narrative: Presently, toxicology studies rely on the use of genetically inbred laboratory animals to characterize hazard information for chemicals and cannot fully predict response to genetic variabilities in humans. To enhance the reliability of toxicology results to accurately reflect interindividual variations in humans while reducing the number of animals used in such studies, this project proposes an inexpensive and rapid screening assay utilizing an invertebrate small organism model, C. elegans, with a survey of hundreds of diverse genetic strains. This approach will facilitate an advanced toxicology testing of developmental and reproductive toxicology (DART) risks and thus reduce false-negatives and save millions of animals.