An Alkaline Phosphatase Reporter Gene Assay for Induction of CYP3A4 in Vitro

Cyp3a4 probably has the broadest catalytic activity of any cytochrome p4s0 , it is a crucialtask to test new drug candidates in a reliable system for their ability to induce expressionof this enzyme, firstly, a total of 300 bp core dist al enhancer of cyp3a4 xrem region(-7972/-7673) were amplified from human genomic dna. the pcr product was then ligatedinto a human secretory alkaline phosphatase edna-containing reporter vector (pseap2-1)creating px-seap2 plasmid. secondly, 1143 bp of the cyp3a4 proximal promoter region(-1203/-61) was amplified from the genomic dna and then ligated into px-seap2 plasmiddna (between xrem and alkaline phosphatase gene), creating pxp-seap2 plasmid. reporterconstructs were then co-transfected with an hpxr expression vector into human liver andintestinal cells in culture. xenobiotic modulation ofcyp3a4 promoter activity was determinedby chemiluminescent secretory alkaline phosphatase assay. significant cyp3a4 induction atthe transcriptional level using three different cell lines and four classical cyp3a4 inducers wasobserved. transfection of reporter constructs in hepg2, huh7 and caco-2 cells , in general,produced similar pattern of induction by the same drugs with the exception ofphenobarbital.the results suggest that, carefully designed reporter gene systems can provide a useful in vitroapproach for chara cterization of possible cyp3a4 inducers.