A reporter gene assay to assess the molecular mechanisms of xenobiotic-dependent induction of the human CYP3A4 gene in vitro.
1. A plasmid containing 1 kb of the CYP3A4 regulatory (promoter) region coupled to a reporter gene for secretary placental alkaline phosphatase (SPAP) was transfected into HepG2 cells. Transfected cells were dosed with several known inducers of CYP3A4 and the levels of SPAP were measured. The effect of co-transfecting a plasmid encoding the human glucocorticoid receptor on reporter gene activity was also examined. 2. Dexamethasone induced CYP3A4-dependent reporter gene expression in a concentration-dependent manner and induction was approximately doubled in the presence of the glucocorticoid receptor. Dexamethasone-dependent induction was blocked by RU-486 (a glucocorticoid receptor antagonist), in the presence of the co-transfected glucocorticoid receptor. 3. Induction of CYP3A4-dependent reporter gene expression and enhancement of the induction by the glucocorticoid receptor was also observed with pregnenolone-16alpha-carbonitrile (PCN), rifampicin, phenytoin, carbamazepine, phenylbutazone and phenobarbitone, all known in vivo inducers of CYP3A4 in man. 4. Metyrapone and sulfinpyrazone induced CYP3A4-dependent reporter gene expression, but induction was not enhanced by the glucocorticoid receptor. 5. Clotrimazole, erythromycin and triacetyloleandomycin (TAO) did not induce CYP3A4-dependent reporter gene expression, consistent with the observation that these inducers act through post-transcriptional mechanisms. 6. These results highlight differences in the molecular mechanisms of induction of CYP3A4 by the xenobiotics studied and indicate that the glucocorticoid receptor is involved in the induction of the CYP3A4 gene by some, but not all, CYP3A4 inducers. 7. We propose that the approach described here provides a useful in vitro approach for the identification of transcriptional regulators of the CYP3A4 gene.
Ogg
M S
MS
Molecular Toxicology Group, School of Biological Sciences, University of Surrey, Guildford, UK.
Williams
J M
JM
Tarbit
M
M
Goldfarb
P S
PS
Gray
T J
TJ
Gibson
G G
GG
D000469:Alkaline Phosphatase / Q000235:genetics; D002110:Caffeine / Q000494:pharmacology; D002220:Carbamazepine / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics; D002994:Clofibrate / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects* / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D015735:Mifepristone / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000187:drug effects* / Q000235:genetics*; D010634:Phenobarbital / Q000494:pharmacology; D010653:Phenylbutazone / Q000494:pharmacology; D010672:Phenytoin / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects; D015262:Xenobiotics / Q000494:pharmacology*; D019324:beta-Naphthoflavone / Q000494:pharmacology
D005938:Glucocorticoids; D006727:Hormone Antagonists; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D015262:Xenobiotics; D015735:Mifepristone; D002220:Carbamazepine; D002110:Caffeine; D019324:beta-Naphthoflavone; D010672:Phenytoin; D003907:Dexamethasone; 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; D010653:Phenylbutazone; D002994:Clofibrate; D007538:Isoniazid; D012293:Rifampin; D010634:Phenobarbital