چكيده لاتين :
CYP3A4 probably has the broadest catalytic activity of any cytochrome P4S0 , It is a crucial
task to test new drug candidates in a reliable system for their ability to induce expression
of 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 ligated
into 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 plasmid
DNA (between XREM and alkaline phosphatase gene), creating pXP-SEAP2 plasmid. Reporter
constructs were then co-transfected with an hPXR expression vector into human liver and
intestinal cells in culture. Xenobiotic modulation ofCYP3A4 promoter activity was determined
by chemiluminescent secretory alkaline phosphatase assay. Significant CYP3A4 induction at
the transcriptional level using three different cell lines and four classical CYP3A4 inducers was
observed. 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 vitro
approach for chara cterization of possible CYP3A4 inducers.
