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 PPAR alpha and RXR alpha. 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 PPAR alpha, the above CYP4A1 construct is transcriptionally activated by a range of structurally different peroxisome proliferators including Wy-14,643, 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.
Giddings
S J
SJ
Molecular Toxicology Group, School of Biological Sciences, University of Surrey, Guildford, UK.
Clarke
S E
SE
Gibson
G G
GG
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000235:genetics; D008830:Microbodies / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics
D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D015500:Chloramphenicol O-Acetyltransferase