CYP4A1 gene transfection studies and the peroxisome proliferator-activated receptor: Development of a high-throughput assay to detect peroxisome proliferators

An in vitro reporter gene assay has been established to examine cytochrome P4504A1 (CYP4A1) induction. A response element from the upstream region of the rat CYP4A1 gene containing a peroxisome proliferator response element (PPRE) has been linked to the chloramphenicol acetyl-transferase (CAT) gene in a reporter vector (1). This CYP4A1 reporter construct has been co-transfected into human HepG2 cells in the presence and absence of expression vectors encoding the transcription factors PPARα and RXRα. The assay employs calcium phosphate-DNA co-precipitate mediated transfection. Reporter gene products have been quantitated using chemiluminescent based assays. We have shown that, in the presence of PPARα, the above CYP4A1 construct is transcriptionally activated by a range of structurally different peroxisome proliferators including Wy-14643, ciprofibrate, clofibric acid and nafenopin. Our future efforts will focus on the establishment of a high-throughput assay for the detection of peroxisome proliferators. Such an assay would provide an invaluable in vitro test for the screening of developmental drug candidates prior to in vivo studies.