- Kouichi Yoshinari (Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka / Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University)
1) Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka , 2) Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University
Peroxisome proliferator-activated receptor α (PPARα) belongs to the nuclear receptor superfamily and exerts hypolipidemic and anti-inflammatory functions when activated by ligand-binding. To screen its ligands, cell-based reporter assays have been widely used, but it is difficult to investigate the effects of the metabolites of test compounds on PPARα due to very low drug-metabolizing capability of cell lines generally used in those assays. The aim of this study was to construct a convenient PPARα reporter assay system with drug-metabolizing capability by using 9,000 x g supernatant (S9) of rat liver homogenate, which abundantly includes various drug-metabolizing enzymes. We used clofibrate as a model compound since it requires hydrolysis to clofibric acid to activate PPARα. In cell-based reporter assays using a PPARα-responsive luciferase reporter plasmid and a rat PPARα expression plasmid, reporter activity was increased by treatment with bezafibrate and clofibric acid, which directly activate PPARα as ligands, but not with clofibrate. The addition of S9 to culture media increased reporter activity of the cells treated with clofibrate, as expected. When heat-denatured S9 was used or a carboxylesterase inhibitor was included in the system, clofibrate-induced PPARα activation was not observed, suggesting that carboxylesterases are responsible for the hydrolysis of clofibrate to clofibric acid. Taken together, we have established a convenient PPARα reporter assay system with drug-metabolizing capability to assess PPARα-activating potency of both test compounds and their metabolites.