2026/06/15 by Masaki Kumondai, Ayaka Yagi, Masahiro Watanabe +10
Pharmacology, Toxicology and Pharmaceutics · Medicine · #Drug-Induced Hepatotoxicity and Protection #Liver physiology and pathology #Liver Disease Diagnosis and Treatment
paper · doi:10.1016/j.dmd.2026.100343
Drug-induced liver injury (DILI) is one of the most serious adverse drug reactions in clinical practice and a major concern in toxicity assessment during drug development. In particular, DILI caused by antifungals can become life-threatening in severe cases; therefore, early detection and appropriate intervention are essential. Here, potential early biomarkers of DILI induced by azole antifungals were explored by performing metabolomic analysis of the supernatants from spheroid cultures of primary human hepatocytes. Primary human hepatocytes obtained from 2 different donors were independently cultured under 3-dimensional spheroid conditions and exposed to 9 antifungal agents. Culture supernatants were collected and analyzed using untargeted metabolomics. The sampling time point was determined based on longitudinal evaluation of cell viability, and the latest time point at which concentration-dependent cytotoxicity was observed was selected for analysis. In parallel, the levels of conventional liver injury markers, aspartate aminotransferase and alanine aminotransferase, were also evaluated in the supernatants. Cell viability was assessed at the time of supernatant collection. Several candidate biomarkers associated with hepatotoxicity were identified, whereas conventional liver injury markers, including aspartate aminotransferase and alanine aminotransferase, showed no clear concentration-dependent changes. Among these, one compound was identified as glycocholic acid through comparison with an authentic standard. Another metabolite detected in positive ion mode could not be conclusively identified and requires further characterization. These findings support the development of novel biomarkers for the early detection of DILI and highlight their potential applications in toxicity screening during drug development and in clinical biomarker research. SIGNIFICANCE STATEMENT: This study identifies novel metabolite-based biomarkers for azole antifungal-induced liver injury using 3-dimensional-cultured primary human hepatocytes and untargeted metabolomics. Glycocholic acid and an additional unidentified metabolite showed stronger associations with hepatotoxicity than conventional markers (aspartate aminotransferase and alanine aminotransferase). These findings highlight the potential of advanced in vitro models and metabolomics to improve early detection of drug-induced liver injury in drug development and clinical settings.