Toxicoproteomics of APAP-exposed HepG2 cells identifies candidate translational biomarkers of liver injury and recovery

Introduction Drug‐induced liver injury (DILI) is a leading cause of acute liver failure (ALF) and a major obstacle in drug development, reflecting the lack of robust, mechanism‐based biomarkers. Human in vitro systems, coupled with unbiased omics, provide controlled, human‐relevant and mechanistically informative platforms that can accelerate biomarker discovery. Here, we explore quantitative toxicoproteomics of paracetamol (acetaminophen; APAP)‐exposed HepG2 cells as a platform for the identification of candidate translational biomarkers of APAP‐liver injury and recovery. Methods HepG2 cells were exposed to the APAP IC 50 concentration for 8 h (injury phase), followed by drug withdrawal and a 24 h recovery phase. Injury‐induced cell responses were evaluated through RT‐qPCR analysis and intracellular ROS quantification. Proteomic changes associated with injury and recovery were characterized by quantitative LC‐MS/MS and selected proteins were validated by immunoblotting. Results LC‐MS/MS quantified 1,478 proteins and revealed increased protein secretion upon APAP exposure. Notably, comparison of the proteomic dataset against published proteomic profiles of a large cohort of ALF patients supported the potential clinical relevance of the obtained signature. Injury‐phase upregulated proteins, including KRT18 and HMGB1, recapitulated known clinical DILI biomarkers. Similarly, recovery‐phase was marked by clinical markers of liver regeneration, e.g ., AFP, APOA1 and HP, enabling discrimination between injury and regeneration. HPD, S100A6 and TXNRD1 emerged as candidate diagnostic biomarkers, whilst SERPINA1 and complement components (C3, C5) showed prognostic potential. Conclusion Collectively, this study demonstrated how toxicoproteomics applied to human in vitro hepatic models, validated against clinical evidence, provide a relevant platform for the discovery of novel translational biomarkers of liver injury and recovery.