Targeted RNA-seq (TempO-Seq) of temporal concentration responses to drug exposures in PHH and HepaRG cells

For the purpose of mechanism-based risk assessment, we investigated temporal concentration-dependent responses in cultures of PHH and HepaRG cells exposed to three drugs, acetaminophen (APAP), cyclosporine A (CSA) and valproic acid (VPA), that have a high liability for drug-induced liver injury. To facilitate the identification of early KEs and visualisation of their development over time, samples were collected at 4 time points, namely 8 and 24 hours (single exposure), 48 and 72 hours (daily repeated exposure), after exposure to a broad concentration range. Concentrations were selected based on the reported total Cmax of each drug. As these are approved drugs currently on the market, they are not expected to induce overt adverse effects around Cmax. Therefore, the selected maximum concentration was set at approximately 30x Cmax, whilst the minimum tested concentration was approximately 0.1x Cmax. The large concentration range allowed to apply benchmark concentration (BMC) modelling on individual genes as well as gene co-expression networks to derive in vitro transcriptomics benchmark concentrations (BMCs) and assess their suitability to be used as PoD in chemical risk assessment.