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Linkage of Exposure and Effects using Genomics, Proteomics and Metabolomics in Small Fish Models

Project Purpose:
The overall goal of this research is to utilize two small fish species, the zebrafish and fathead minnow, as a basis for the development of (a) mechanistic biomarkers, and (b) techniques for extrapolation of toxicological effects across endpoints, species and chemicals. To achieve this, a systems-based approach is being used to define toxicity pathways for model chemicals, including a number of human and veterinary pharmaceuticals, with well-defined modes/mechanisms of action (MOA) within the vertebrate hypothalamic-pituitary-gonadal (HPG) axis.

Project Description(s):
This project is using a combination of whole organism endpoints, transcriptomic, proteomic, and metabolomic approaches, and computational modeling to (a) identify new molecular biomarkers of exposure to endocrine disrupting compounds (EDCs) representing several modes/mechanisms of action (MOA) that encompass different classes of human and veterinary pharmaceuticals, and (b) link those biomarkers to effects that are relevant for both diagnostic and predictive risk assessments using small fish models. These goals are being achieved through a three-phase approach that incorporates expertise across EPA/ORD and capitalizes on partnerships with other federal and non-federal laboratories. During Phase 1, effects of a candidate list of 11 compounds having different MOA within the HPG axis are being characterized using the fathead minnow (Pimephales promelas). These chemicals include estrogens, androgens, anti-androgens and various inhibitors of steroidogenesis. The fathead minnow represents the small fish model most commonly used as the Agency's aquatic toxicological standard for both lab testing and field monitoring. Phase 1 results provide input for population modeling and provide crucial data for anchoring for the markers identified in other phases. Phase 2 takes advantage of the well characterized zebrafish (Danio rerio) genome, to identify transcriptome and proteome level changes in addition to metabolite changes, associated with zebrafish exposure to the same suite of EDCs. Phase 2 data are being used to identify relevant molecular changes that could (a) serve as diagnostic markers for various types of EDC exposure and (b) begin to inform a systems-level characterization of the responses to those exposures. In Phase 3, candidate genes/diagnostic markers identified in zebrafish (Phase 2) are being validated in fathead minnows through focused toxicological testing aimed at examination of changes in specific gene expression and protein levels. Metabolite profiles in fathead minnows are compared to those detected in zebrafish exposed to the same EDCs. In this way, changes at the genomic, proteomic, and metabonomic level can be linked to one another, linked across multiple teleost species, and ultimately linked to adverse effects at the individual- and, through modeling, the population-level.

Project Outcomes:
This study is focused on the identification of mechanistic biomarkers of exposure and effects for chemicals that impact the HPG axis. Test chemicals used include pharmaceuticals representative of different MOA, including estrogens, androgens, anti-androgens and inhibitors of steroidogenesis. The outputs of this project also serve to characterize source-to-outcome linkages critical for effective risk assessments for HPG-active chemicals. Furthermore, comparison to complementary mammalian studies will facilitate improved cross-species extrapolation.

Garald Ankley at ankley.garald@epa.gov

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