Planarian behavioral screening of ToxCast Phase I chemicals to determine context of use - Objectives. The developing brain is highly vulnerable to chemical toxicants, but <2% of environmental chemicals have been tested for developmental neurotoxicity (DNT). This data deficit results from the extreme cost and time requirement of mammalian testing. There is an urgent need to develop high-throughput screening (HTS) models to replace, refine, and/or reduce (“3Rs”) vertebrate testing. The development of HTS models is especially challenging for neurotoxicity (NT) and DNT where the functional relevancy of adverse outcomes needs to be assessed on the whole organism level. The overarching goal of this research is to develop a cost-effective invertebrate organismal HTS test method to identify NT and DNT to complement existing in vitro tests. The specific objective is to determine the applicability domain and utility of HTS using the asexual freshwater planarian Dugesia japonica as a first-tier behavioral screening platform for NT/DNT. This will be achieved by screening the ToxCast Phase I library, consisting of 293 unique, data-rich compounds, and comparing the results to existing data from mammalian, in vitro, and alternative organismal models. Planarians are invertebrates of intermediate neural and anatomical complexity compared to nematodes and zebrafish and have tractable, evolutionarily conserved neuronal circuits. Planarians uniquely allow for direct comparison of xenobiotic effects on the adult and developing nervous systems. We hypothesize that planarian behavioral HTS adds complementary value to existing testing methods because of its unique strengths (full metabolism at all developmental stages, screening into adulthood, parallel screening of adult and developing organisms). Experimental approach. In Aim 1, we will use the ToxCast Phase I library to profile behavioral phenotypes in intact and regenerating planarians to identify the neurotoxic effects of different chemicals’ domains. We will first test each chemical at 100 µM. Chemicals that elicit effects at this concentration will then be tested across 7 concentrations. Effects will be quantified as benchmark concentrations for 37 readouts over two time points. Development-specific effects will be extracted by comparison of adverse outcomes in intact vs. regenerating planarians. In Aim 2, we will compare concordance with mammalian guideline data and publicly accessible ToxCast Phase I data from in vitro, nematode, and developing zebrafish assays. We will also determine planarian HTS performance for specific chemical domains to assess context of use. These comparisons will inform on whether planarian HTS recapitulates animal NT/DNT, adds complementary information to NT/DNT in vitro tests, and how it performs compared to more established non-animal organismal models. Expected results: This project will evaluate how the planarian system captures the chemical subspace of this chemically diverse library and how our results compare to data in existing models. This will determine the context of use of this promising first-tier NT/DNT screening system and bolster the development of a comprehensive non-animal DNT testing battery, to meet the urgent need to fill the DNT data gap and reduce animal testing.
