In vitro screening of acute hepatic cytotoxicity of pyrrolizidine alkaloids in human and rodent hepatic cell lines

Pyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential. The aim of this work was to develop a predictive screening tool of their relative hepatotoxicity. Methods: Different human and rodent hepatocyte cell lines (H-4-II-E, HepG2, HepaRG) were used to assess cytotoxicity of lasiocarpine, seneciphylline, and monocrotaline with WST-1 assay. Results: Incubation over 72h at concentrations from 25µM up to even 2400µM, resulted in no toxic effects in neither cell line. In a galactose-based culture medium (11.1mM) which increases cell susceptibility to mitochondrial toxicants, showed a significant toxicity at 900µM in H 4-II-E and HepG2 cells. Inhibition of carboxylesterase-mediated PA detoxification (specific carboxylesterase 2 inhibitor loperamide (2.5µM) and unspecific carboxylesterase inhibitor bis-p-nitrophenyl-phosphate (BNPP, 100µM)) revealed that loperamide enhanced toxic effects of lasiocarpine in both cell lines, whereas BNPP had a weaker effect. Inhibition of glutathione-mediated detoxification by etacrynic acid (100µM) did not enhance toxicity. Comparison of lasiocarpine, seneciphylline and monocrotaline in galactose-based medium with loperamide showed the following rank order of toxicity in HepG2 cells: lasiocarpine > seneciphylline ≥ monocrotaline. Conclusion: If no toxicity is observed under standard conditions, sensitisation with galactose is useful to assess relative acute cytotoxicity of PAs in different cell lines. The results also suggest that carboxylesterases are involved in the detoxification of PAs.