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

How to Cite:Hossein HamzeiyMohammad Ali EghbalAn Alkaline Phosphatase Reporter Gene Assay for Induction of CYP3A4 In Vitro.Iran J Pharm Res.5(4):e128298.https://doi.org/10.22037/ijpr.2010.688.

Abstract

CYP3A4 probably has the broadest catalytic activity of any cytochrome P450. 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 distal 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 cDNA-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 of CYP3A4 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 characterization of possible CYP3A4 inducers.

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Author(s):

Hossein Hamzeiy:[PubMed][Scholar]
Mohammad Ali Eghbal:[PubMed][Scholar]