High-content screening imaging and real-time cellular impedance monitoring for the assessment of chemical’s bio-activation with regards hepatotoxicity

•Neutral red uptake was the most robust assay used for the drug cytotoxicity evaluation on murine liver models.•Real-time cellular impedance could detect bio-activation through revealing specific curve profiles and response kinetics.•Real-time cellular impedance seems unsuitable for use in monitoring viability in primary cultures of rat hepatocytes.•The contribution of HCS appears necessary to highlight disruptions to cellular function in response to drug exposure.•Coupling neutral red assay, real-time cellular impedance and HCS represents a complementary strategy for drug assessment. Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HCS) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals.