In vitro test battery for testing molecular initiating events in chemical-induced cholestasis

Cholestatic liver injury is a complex adversity leading to the toxic accumulation of noxious bile salts in the liver and systemic circulation. Cholestasis can be instigated by a plethora of chemicals originating from several applicability domains. Current efforts fail to predict the cholestatic potential of chemicals due to, at least in part, gaps in the mechanistic understanding of this type of adversity. A recently introduced adverse outcome pathway (AOP) network on cholestatic liver injury generated using artificial intelligence pulls up transporter changes, bile canalicular changes and hepatocellular changes as molecular initiating events (MIEs). The present study used this AOP network as the mechanistic basis for the development of an in vitro test battery to predict MIEs of cholestatic hepatotoxicity, including assays to monitor transporter changes at the sinusoidal uptake, canalicular efflux and basolateral efflux pole as well as bile canalicular changes. For this purpose, human HepaRG cells were exposed to known cholestatic chemicals covering various MIEs, non-cholestatic hepatotoxic chemicals and non-hepatotoxic chemicals. Subsequent application of the MIE test battery shows great potential for identifying cholestatic chemicals, while correctly predicting all negative control chemicals. In conclusion, the established in vitro test battery shows potential for early prediction of cholestatic chemicals.