Cytochrome P450 3A-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 T 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 alanine transaminase, aspartate transaminase, alkaline phosphatase, blood ammonia, and total bile acid levels in mice, accompanied by hepatocellular degeneration, necrosis, and inflammatory infiltration. Pretreatment with the cytochrome P450 3A inhibitor ketoconazole (100 mg/kg) markedly alleviated the liver injury induced by FBL. Liquid chromatography-tandem mass spectrometry 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 glutathione depletion and protein adduction. SIGNIFICANCE STATEMENT: Tinospora sagittata is widely used, however, its hepatotoxic principles remain undefined. This study identifies fibleucin as a previously unrecognized hepatotoxic constituent and demonstrates that it undergoes cytochrome P450 3A-mediated metabolic activation to generate a reactive intermediate consistent with cis-2-butene-1,4-dial, leading to glutathione depletion and hepatic protein adduction. These findings establish a mechanistic link between bioactivation of a herbal constituent and liver injury, highlighting metabolic risk in furanoditerpenoid-containing traditional medicines.