SETAC Europe 35th Annual Meeting

Comprehensive Characterization of Rainbow Trout Hepatic 3D Spheroids: Morphological and Transcriptional Insights

Abstract: The demand for alternative ecotoxicology methods is increasing, especially with a focus on New Approach Methodologies (NAMs) to provide reliable in vitro models for chemical hazard assessments. Many existing cell-based assays rely on primary or continuous cell cultures effective for acute toxicity but often lack the necessary complexity for long-term exposure studies. However, few alternative models support comprehensive, long-term toxicity evaluations, essential for understanding chronic impacts.

A novel 3D spheroid model derived from rainbow trout (Oncorhynchus mykiss) primary hepatocyte (RT-HEP-SP) has emerged as a promising NAM, preserving key morphological, physiological, and biochemical properties for weeks, making it suitable for chronic toxicity studies. However, broader application of the RT-HEP-SP system requires thorough characterization and validation. In this study, high-resolution confocal microscopy was employed to assess various markers, such as spheroid morphology (cytoskeleton, DNA and nuclear integrity, bile canaliculi formation), viability (metabolic activity, hypoxia within the spheroid core), and physiological response (reactive oxygen species [ROS] induction, mitochondrial membrane potential, ethoxyresorufin-O-deethylase [EROD] activity, and Phase II metabolism components like uridine 5′-diphospho-glucuronosyltransferase [UGT]) during chemical exposure.

RNA sequencing (RNA-seq) and targeted bioassays were used to evaluate RT-HEP-SPs’ responses to various toxicants. A Biomarker Toolbox was developed with sensitive and reproducible markers for oxidative stress, biotransformation (e.g., aryl hydrocarbon receptor, EROD,), and viability (e.g., lactate dehydrogenase). These biomarkers were validated through short-term (24–96 hours) exposures to model chemicals with distinct toxic modes of action (MOAs), including copper, 17 β-ethinyl estradiol (EE2), benzo[a]pyrene, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), confirming the model’s responsiveness and reproducibility.

Initial findings indicate that RT-HEP-SP are a reliable model for assessing cytotoxicity, membrane integrity, and oxidative stress, with no core hypoxia detected. RNA-seq analyses reveal their molecular complexity and suitability for the assessment of chemicals with different MOAs. This model shows strong potential as an ecotoxicity screening tool for various chemicals.

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Author(s):

Maria Hultman Norwegian institute for water research Norway This Author Is the Presenter

Prem Chand Norwegian institute for water research, University of Oslo Norway

You Song Norwegian institute for water research Norway

Awadhesh Jha University of Plymouth United Kingdom

Coen Campsteijn University of Oslo Norway