Development of an in vitro reporter gene assay to assess xenobiotic induction of the human CYP3A4 gene.
The current work concerns the development and validation of an in vitro reporter gene assay system for the assessment of induction of human CYP3A4. A plasmid containing approximately 1 kb of the CYP3A4 regulatory region (which contains several recognised regulatory elements including glucocorticoid responsive elements) coupled to the reporter gene for human secreted placental alkaline phosphatase (SPAP) was transfected into the human hepatoblastoma cell line HepG2. Calcium phosphate precipitation was the method of choice for transfection. The transfected cells were dosed with known inducers of CYP3A4 and the levels of SPAP in the medium were subsequently measured using a chemiluminescent assay, as an indirect measure of CYP3A4 induction. The inducers used in this study included dexamethasone, phenytoin, triacetyloleandomycin (TAO), rifampicin, carbamazepine, phenylbutazone and sulfinpyrazone. These compounds activated CYP3A4 by between 1.5-4.5-fold thus representing a major advance in assessing the induction of human CYP genes in vitro.
Ogg
M S
MS
Molecular Toxicology Group, School of Biological Sciences, University of Surrey, Guildford, UK.
Gray
T J
TJ
Gibson
G G
GG
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D015262:Xenobiotics / Q000494:pharmacology*
D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D000469:Alkaline Phosphatase