In vitro hepatotoxicity of Petasites hybridus extract (Ze 339) depends on the concentration, intrinsic cytochrome activity and the species used

Ze 339, a CO₂ extract prepared from the leaves of Petasites hybridus, possesses antispasmodic and anti-inflammatory effects and is proven to be effective in the treatment of allergic rhinitis. To study possible hepatotoxic effects of Ze 339, its main constituents and metabolites, a series of in vitro investigations were performed. Furthermore, different reconstituted fractions of the extract were examined in three in vitro test systems using hepatocytes: Two human cell lines, with lower and higher intrinsic activity of cytochrome P450 enzymes (HepG2, HepaRG) as well as a rodent cell line with high intrinsic activity (H-4-II-E) were used. Metabolic activity, assessed by the WST-1 assay, was chosen as indicator of cytotoxicity. To assess potential bioactivation of Ze 339 compounds, metabolic experiments using S9 fractions from rats, dogs and humans, and isolated cytochromes (human/rat) were performed and the formation of reactive metabolites was assessed by measuring cellular concentrations of glutathione and glutathione disulphide. Apoptotic behavior was examined by determining caspase activity of the extrinsic and the intrinsic pathway. Modification of mRNA expression of genes involved in adaptive, cellular defense mechanisms was examined by quantitative real-time polymerase chain reaction (RT-PCR) in HepaRG cells. Our data revealed that the cytotoxicity of Ze 339, its single constituents and main metabolites depends on the concentration, the intrinsic cytochrome activity of the cell system and the species used (rat > dog > human).