Comparison of metabolism-mediated effects of pyrrolizidine alkaloids in a HepG2/C3A cell-S9 co-incubation system and quantification of their glutathione conjugates

Toxicity of pyrrolizidine alkaloids (PAs) largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrrolic derivatives. These then spontaneously release the esterifying acids to generate carbonium ions that form covalent adducts with cellular nucleophiles to exhibit toxicity.In our investigation, metabolism-mediated toxicity of monocrotaline, retrorsine, lycopsamine, echimidine (retronecine-type PAs), heliotrine (a heliotridine-type PA) and senkirkine (an otonecine-type PA) was studied using an in vitro co-incubation assay.Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver S9 fraction and the toxicity was assessed as lowered mitochondrial activity.Bioactivation potential was measured by incubating PAs with rat liver S9 fraction, NADPH and GSH in a cell free system. Pyrrolic metabolites generated were entrapped as glutathione conjugates (7-GSH-DHP and 7,9-di-GSH-DHP) which were quantifi...