Involvement of astrocytic CYP1A1 isoform in the metabolism and toxicity of the alkaloid pyrrolizidine monocrotaline

摘要

Monocrotaline (MCT) and its pyrrole derivative, dehydromonocrotaline (DHMC), interact with molecular targets in cells of the central nervous system. DHMC presents higher toxicity than MCT indicating that its metabolism of MCT is a critical step of this alkaloid toxicity. This study sought to elucidate the metabolism and the toxicity of MCT in C6 astrocyte cell line and primary cultures of rat astrocytes by investigating metabolic enzymatic mechanisms of the cytochrome P450 (CYP) system and conjugation with glutathione. Treatment with omeprazole (OMP) (20 mu M), a non-specific inducer of CYP450 induced approximately 10-fold increase in CYP1A1 activity after 2 h of treatment. Similarly, the 7-Ethoxyresorufin-O-deethylase (EROD) activity was induced by treatment with MCT (100-500 mu M), indicating that the P450 CYP1A1 isoform was active and involved in the metabolism of MCT. Analysis of conjugation with glutathione showed a significant depletion of GSH after MCT (500 mu M) treatment, and this was partially reversed by pretreatment with a P450 inhibitor (cimetidine 100 mu M). These results suggest that not only the alkaloid MCT but, also its metabolite may deplete GSH. Rosenfeld staining showed intense vacuolization after MCT treatment, which was partially inhibited in the presence of a P450 activator. MTT test showed that association of MCT with OMP induced a reduction in cell viability in C6 and primary astrocytic cells. These results demonstrate that MCT is metabolized by astrocytic CYP1A1 to generate metabolites that can deplete GSH. Moreover, changes in the activity of the P450 enzymes interfere with the cytotoxic effects induced by the alkaloid. (C) 2017 Elsevier Ltd. All rights reserved.