Xiaoyan Pang, Chongzhuang Tang, Peng Cao , Lei Zhou and Xiaoyan Chen*
Background: The hepatotoxic pyrrolizidine alkaloids (PAs) were reported to increase bile acid (BA) levels in 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 transport genes were investigated and the involvement of Nrf2 pathway were explored.
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 gamma-glutamylcysteine synthetase inhibitor, L-buthionine-sulfoximine (BSO) were employed in WT mice. Retrorsine-induced 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 semiquantified 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 up-regulation 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 level 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 the formation of 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.
retrorsine, bile acid, oxidative stress, reactive metabolite, transporter, Nrf2
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, 201203, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, 201203, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210028, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, 201203, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai, 201203