Exploring chemically-induced oxidative stress: A comparative study of zebrafish embryos and maturated HepG2 cells

To advance next-generation risk assessment of non-genotoxic carcinogens, robust mechanism-based assays are essential. A recognized mode of action for non-genotoxic carcinogens is induction of oxidative stress leading to regenerative proliferation. Most of the currently available New Approach Methodologies (NAMs) rely on simple high-throughput cell models with limited biological complexity and often lack metabolic capacity. In this study, we quantify chemically-induced oxidative stress in zebrafish embryos (ZFE), Danio rerio, to evaluate the added value of a whole-organism model with functional metabolism over a simple high-throughput hepatocyte cell line. Four-day-old ZFE were exposed to a set of 22 chemicals, including fifteen chemicals inducing oxidative stress and seven with another primary mode of action. Following 24 h of exposure, reactive oxygen species (ROS) were quantified in the ZFE using the dichloro-dihydro-fluorescein (DCFH) assay. Using analytically determined internal concentrations, chemicals were ranked based on their ROS-inducing potential. Results were compared with ROS induction in maturated HepG2 cells, where DCFH fluorescence was quantified during the first hour of chemical exposure. Both models identified nine chemicals with ROS-inducing potency, although the identified chemicals did not completely overlap. Of the fifteen chemicals reported to primarily induce oxidative stress, only four were not detected by either model. In ZFE, three of the seven chemicals reported to have another primary mode of action than oxidative stress were flagged for ROS production, whereas none of these seven were flagged in maturated HepG2 cells. We explore potential explanations for discrepancies between the models and discuss their applicability in a regulatory context.