CYP3A-mediated metabolic activation of fibleucin from Tinospora sagittata initiates liver injury in mice

Tinospora sagittata is a widely used medicinal herb whose safety has raised increasing concerns in recent years. Fibleucin (FBL), a furanoditerpenoid isolated from Tinospora sagittata , has not been systematically evaluated for its safety and toxicological mechanisms despite its presence as a major bioactive constituent in this medicinal plant. To address this toxicological knowledge gap, we comprehensively investigated the hepatotoxicity and/or metabolic activation of FBL in vivo (mice) and in vitro (mouse and human liver microsomal incubations as well as primary mouse hepatocytes). Oral administration of FBL (single intragastric dose) dose-dependently elevated serum ALT, AST, ALP, blood ammonia, and total bile acid levels in mice, accompanied by hepatocellular degeneration, necrosis, and inflammatory infiltration. Pretreatment with the CYP3A inhibitor ketoconazole (100 mg/kg) markedly alleviated the liver injury induced by FBL. LC-MS/MS analysis of mouse liver microsomal incubations and primary hepatocyte exposure (1.0–50 μM FBL) revealed that FBL was bioactivated to an electrophilic intermediate reactive to thiols and amines. Similar observation was obtained in human liver microsomal incubations. The reactive intermediate was found to react with nucleophilic sites of hepatic glutathione and proteins to form the corresponding conjugates and adducts. The observed adduction correlated with the liver injury and cytotoxicity induced by FBL. Chemical synthesis was carried out to probe the mechanism of toxic action. The mechanistic investigation suggests that FBL was metabolized to the corresponding cis -2-butene-1,4-dial intermediate responsible for the GSH depletion and protein adduction.