Developing adverse outcome pathways underlying CAR activation-induced liver injuries using HepG2 spheroid model

The constitutive androstane receptor (CAR) plays a key role in hepatic xenobiotic responses, yet its toxicity mechanisms remain unclear due to limitations in current models. This study employed a HepG2 spheroid model to systematically delineate chemical-induced liver injuries that are induced by CAR activation. By assessing the expression levels of metabolizing enzymes and functional biomarkers, the advantages of the 3D culture were shown as superior to 2D culture. Immunofluorescence confirmed CAR cytoplasmic localization in untreated status, and activation-induced transnucleic-activation upon exposure. Transcriptomics demonstrated the 3D-HepG2's superiority for hepatic studies involving metabolizing pathways. Knockdown experiments demonstrated CAR's critical role in hepatotoxicity that were induced by acetaminophen (5, 10, and 20 mM) and 2,2',4,4'-tetrabromodiphenyl ether (100, 150, and 200 μM). The detection of early DNA damage markers and liver injury indicators revealed that CAR mediates aristolochic acids (25, 50, and 100 μM)-induced DNA damage. Lipidomics was firstly applied to the 3D-HepG2, proving its superiority for researching hepatic lipid metabolism. Further, the detection of lipid droplet formation and key enzyme alterations proved that CAR mediates lipid accumulation that was induced by perfluorooctanoic acid (100, 150, and 300 μM) and nonylphenol (15, 30, and 45 μM). Eventually, adverse outcome pathways were constructed linking CAR activations to chemical-induced hepatotoxicity, dyslipidemia, and DNA damage, respectively. The present study provides scientific insights for studies concerning exogenous chemicals-induced liver injuries.