CYP2C19-Mediated Metabolic Activation and OAT3/P-gp-Associated Transport Contribute to Dioscorea bulbifera L.-Induced Hepatotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea bulbifera L. (DBL) is a traditional medicinal herb, but its clinical use is often limited by its potential to cause herb-induced liver injury. 8-Epidiosbulbin E acetate (EEA) is recognized as the major hepatotoxic constituent in DBL; however, the mechanisms underlying the drug transport and metabolic activation of EEA leading to hepatotoxicity are unclear, which severely impedes the understanding of its toxicity risk and safety control measures. AIM OF THE STUDY: This study aimed to identify the key EEA transporters and P450 enzymes mediating EEA metabolic activation in vivo to systematically clarify the contribution of transport and metabolism in DBL/EEA-induced hepatotoxicity. MATERIALS AND METHODS: Transporter and P450-overexpressing cell models were used, combined with CCK-8, apoptosis, and cell cycle assays and LC-MS/MS technology, to evaluate EEA cytotoxicity, intracellular content, and EEA-GSH conjugate formation. In animal experiments, mice were pretreated with ticlopidine, probenecid, novobiocin, or verapamil to examine the roles of CYP2C19-mediated metabolic activation, OAT-sensitive uptake, and P-gp-mediated efflux in EEA- and DBL extract-induced hepatotoxicity. EEA or DBL extract was then administered orally, and the extent of liver injury was evaluated by histopathological examination and serum aminotransferase measurements. RESULTS: CYP2C19 metabolically activated EEA, leading to decreased cell viability, increased apoptosis, cell cycle arrest, and increased EEA-GSH conjugate levels. OAT3 overexpression increased EEA cellular uptake, whereas P-gp overexpression promoted EEA efflux. In mice, CYP2C19 or OAT3 inhibition attenuated DBL/EEA-induced liver injury, whereas P-gp inhibition exacerbated hepatotoxicity. CONCLUSION: These findings suggest that OAT3-sensitive cellular uptake, P-gp-mediated efflux, and CYP2C19-mediated bioactivation are important determinants of EEA hepatotoxicity, which is characterized by GSH depletion, cell-cycle perturbations, and apoptosis. This study provides a mechanistic explanation for DBL/EEA-induced hepatotoxicity and highlights that the interplay between metabolism and transport is a key determinant of its toxicity.