Investigation of Metabolism-Mediated Toxicity of Pyrrolizidine Alkaloids from Comfrey

Comfrey (Symphytum officinale), has long been used internally and externally as an herbal remedy. Comfrey roots and leaves contain varying levels of the hepatotoxic pyrrolizidine alkaloids (PAs) that have been reported to induce veno-occlusive disease in humans [1]. The hepatotoxicity of the PAs largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrroles that form covalent adducts with cellular nucleophiles including DNA [2, 3]. In our investigation, metabolism-mediated toxicity of PAs from comfrey was studied using an in vitro assay. Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver microsomes (S9 fraction) for 6 hrs and the cytotoxicity was assessed as lowered mitochondrial activity (reduction of MTT). In the presence of glutathione, the pyrrolic metabolites form conjugates (7-GSH-DHR and 7,9-di-GSH-DHR) which can be quantified using LC/MS. Our results indicate that PAs are metabolized by rat liver microsomes into reactive pyrroles and show cytotoxicity in HepG2/C3A cells. The cytotoxic effects can be correlated with the amount of GSH adducts detected by LC/MS. By following this method, the amounts of the GSH-adducts quantified in LC-MS can be used to determine toxicity of the various pyrrolizidine alkaloids.