Multidimensional in Vivo Hazard Assessment Using Zebrafish

The U.S. National Research Council issued Toxicity Testing in the 21st Century: A Vision and a Strategy in 2007 to challenge traditional approaches for toxicity testing. The report described the need to refocus toxicity testing on relevant human doses and on identifying the early molecular response pathways that are perturbed to produce toxicity. Application of this approach would replace reliance on primarily high-dose, gross phenotypic responses in high-cost, low-throughput mammalian models. The goal in focusing on molecular and cellular pathways that are targets for chemicals is to gain insights into toxic mechanisms underlying apical endpoints. To directly address this need and to help prioritize chemicals for testing, in 2008, the U.S. EPA National Center for Computational Toxicology (NCCT), National Toxicology Program, and National Human Genome Research Institute’s NIH Chemical Genomics Center developed a partnership, “Tox21” (http://epa.gov/ncct/Tox21/), to test a larger set of compounds (10 000) that were broadly characterized and may have toxicological concerns. In 2010, the U.S. Food and Drug Administration joined the partnership to bring together expertise in experimental toxicology, computational toxicology, high-throughput technologies, and animal models of human diseases. As an additional effort, the EPA-NCCT developed the ToxCast program in 2007 to assess a large number of chemicals in a diverse set of in vitro assays. The long-term goal was to predict the potential toxicity of chemicals and to develop cost-effective approaches to prioritize the thousands of chemicals that have little to no hazard safety information. As a “Proof of Concept,” phase 1 of the ToxCast program was completed in 2009 and consisted of approximately 300 well-studied chemicals with existing toxicity information run across approximately 600 high-throughput in vitro assays (Judson et al., 2010). Phase 1 consisted primarily of pesticides, many having over 30 years of data from traditional toxicology methods and definitive toxicity endpoint(s) (ie, target organ, reproductive, or developmental). The ToxCast program established multidimensional, multiassay signatures to predict animal toxicity using the traditional toxicity data to gauge accuracy. Phase 2 is currently ongoing and consists of approximately 700 chemicals from a broad range of sources such as industrial and consumer products, food additives, “green” products, cosmetic-related chemicals, and failed pharmaceutical drugs. The traditional toxicity data are lacking in phase 2, but human clinical data and other toxicology studies are available to assess and test the performance of predictive models developed in phase 1. Although there is a growing effort to utilize molecular and pathway-focused assays in toxicology, it has proven difficult to translate in vitro data to predict whole animal toxicity. The high-throughput screening (HTS) assays used in the ToxCast program included both biochemical and cell-based systems that investigated protein function or binding, transcriptional activity, fundamental cellular processes, and systemic readouts. Cultured cells lack biological complexity; they express limited gene products and intrinsically represent an artificial biological environment for testing. These inherent limitations have tempered some of the enthusiasm for the Tox21 initiatives. The zebrafish is a small complex organism that is amenable to large-scale in vivo genetic and chemical studies (Pardo-Martin et al., 2010). Zebrafish have a short generation time and rapid development and short life cycle (Kimmel et al., 1995). The embryos develop externally and are transparent for the first few days of life, and due to their small size, only a small quantity of chemical is needed for a full evaluation of biological response. There is significant physiological and genetic homology between humans and zebrafish (70% gene homology), and approximately 84% of human genes known to be associated with human diseases are also present in zebrafish (Howe et al., 2013). The versatility of zebrafish makes it an ideal model to address Toxicity Testing for the 21st century, providing an essential bridge between in vitro and mammalian data. By combining the utility of the embryonic zebrafish as the first tier to identify potential toxicity and the application of HTS in vitro assays to gain insight into toxicity mechanisms, we can begin to address the paradigm shift in toxicity testing. Here, we describe a rapid in vivo approach to discover chemical hazard potential using embryonic zebrafish. We examined all 1078 ToxCast phase 1 and 2 chemicals (1060 unique chemicals) for developmental and neurotoxicity in the embryonic zebrafish. Each chemical was tested in a broad concentration range spanning 4 orders of magnitude (6.4nM to 64μM) with multiple replicates at each concentration (n = 32). Simultaneous evaluation of 22 endpoints identified distinct patterns of chemical response that can help identify mechanistic pathways. By utilizing the zebrafish as a biological sensor, and these data as a reference set, we are better positioned to build predictive toxicity frameworks and accelerate chemical testing.