Background: The hepatotoxic pyrrolizidine alkaloids (PAs) were reported to increase bile acid (BA)
levels in the rat. However, it is still unclear whether the production of highly reactive dehydropyrrolizidine through
CYP450s is directly relevant to BA changes.
Objective: To further explore the mechanism by which metabolic activation of PAs induced BA changes, the effect
of impaired or enhanced metabolic activation on the BA profiling and BA-related synthesis and to investigate transport
genes, and explore the involvement of the Nrf2 pathway.
Methods: Blood and liver samples were collected after intragastrical administration of 35 mg/kg retrorsine or saline
for seven days in wild-type (WT) and Nrf2 KO mice. CYP450 inhibitor, 1-aminobenzotriazole (ABT), or gammaglutamylcysteine
synthetase inhibitor, L-buthionine-sulfoximine (BSO) were employed in WT mice. Retrorsineinduced
hepatotoxicity was evaluated by a biochemical method and H&E staining method. Serum BAs were quantified
by high-performance liquid chromatography/triple quadrupole mass spectrometry. Blood pyrrole-protein adducts
were semi quantified by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The
gene and protein expression of BA-related transporters and enzymes in the liver were measured by a quantitative
real-time PCR method and western blotting method.
Results: The BA concentrations in serum were increased in the retrorsine-treated WT mice, along with the upregulation
of BA transporters, Ostβ, Mrp3, Mrp4, and Mrp2. When ABT was co-administered, the altered BA levels
and Mrp4 mRNA and protein levels were reversed, accompanied by a 50% reduction of 6,7-dihydro-7-hydroxy-1-
hydroxymethyl-5H-pyrrolizine (DHP) formation. When BSO was co-administered, serum BAs were not further
increased, but Ostβ, Mrp3, Mrp4 mRNA, and Mrp4 protein levels continuously increased. The induction of Mrp4 by
retrorsine among the tested BA transporters was the only one that was abolished or enhanced in the presence of ABT
or BSO. The Nrf2 protein levels in the nucleus increased in the retrorsine-treated WT mice, which were remarkably
repressed by co-administration of ABT and enhanced by co-administration of BSO. In Nrf2 KO mice receiving retrorsine,
the bile acids and the mRNA and protein levels of Mrp2, Mrp3, Mrp4, and Ostβ were hardly changed, indicating
the direct role of Nrf2 in retrorsine-induced BA changes in WT mice.
Conclusion: The activation of Nrf2 translocation by forming the reactive metabolite of PAs induced the expressions
of BA transporters and changed serum BA levels. Mrp4 was a sensitive biomarker for the perturbation of redox
status caused by the formation of dehydropyrrolizidine.