Characterizing toxicity pathways of fluoxetine to predict adverse outcomes in adult fathead minnows (Pimephales promelas)

Abstract

Current ecotoxicity testing programs mandated by regulatory agencies are impeded as they predominantly rely on slow and expensive animal tests measuring traditional adverse outcomes such as mortality, growth, disease, reproductive failure, or developmental dysfunction. To address these concerns and support environmental risk assessment, the development of new approach methodologies (NAMs) is increasingly involving short-term mechanistic assays that employ molecular endpoints, such as transcriptomics and proteomics, to predict adverse outcomes of regulatory relevance. The research in this thesis aimed to use fluoxetine (FLX) as the model compound for the development of a novel mechanism-based toxicity assay through elucidation of its molecular toxicity pathways in adult fathead minnows. Specifically, the objectives of this study were to characterize the relationships between molecular response patterns using whole proteomics and transcriptomics and apical level effects of regulatory relevance (fecundity and histopathology). In two parallel studies, fish were exposed to three FLX concentrations (measured: 2.42, 10.7, and 56.7 µgL-1) and a control. After the 96-hour exposure, molecular response signatures were characterized using whole proteomics and transcriptomics analyses in livers and brains of exposed male fish. Following the 21-day exposure, fish were sampled and assessed for liver histopathology and morphometric measurements. Fecundity was monitored throughout the study and revealed a significant reduction at all FLX-treatment levels. Hepatic histopathological assessment found presence of lipid-type vacuolation in two of five specimens of fish exposed to 56.7 µgL-1 FLX. Whole transcriptomic analysis in the liver revealed dysregulation of pathways associated with biosynthesis and metabolism of fatty acids, which may be an upstream molecular response that led to lipid-type vacuolation of hepatocytes, as observed in the histology analysis. Whole proteome analysis of the same fish revealed dysregulation of several processes including PPAR signalling. These molecular signatures may be upstream responses that led to lipid-type vacuolation of hepatocytes. Upregulated genes in the brain suggested alterations in serotonin-related signalling processes and reproductive behaviour, which may explain the observed significant decrease in fecundity. While the relationships between molecular responses and adverse outcomes remain complex, this research provided important insights into the mechanistic toxicity of FLX. This work achieved the research objectives in demonstrating the potential of large-scale omics data to elucidate the complex physiological response of adult fathead minnows to FLX as well as added to the growing body of literature on the utility of these methods in support of chemical hazard assessment.