A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides

Biochem Pharmacol. 2004 Dec 15;68(12):2347-58. doi: 10.1016/j.bcp.2004.07.041.


A stable hepatoma cell line expressing the human pregnane X receptor (hPXR) and the cytochrome P4503A4 (CYP3A4) distal and proximal promoters plus the luciferase reporter gene was developed to assess the ability of several xenobiotic agents to induce CYP3A4 and CYP2B6. After selection for neomycin resistance, one clone, displaying high luciferase activity in response to rifampicin (RIF), was isolated and the stable expression of hPXR was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Dose-response curves were generated by treating these cells with increasing concentrations of RIF, phenobarbital (PB), clotrimazole (CLOT) or 5beta-pregnane-3,20-dione (5beta-PREGN). The effective concentrations for half maximal response (EC50) were determined for each of these compounds. RIF was the most effective compound, with maximal luciferase activity induced at 10 microM. The agonist activities of PXR-specific inducers measured using our stable model were consistent with those measured in transient transfectants. The abilities of organochlorine (OC), organophosphate (OP) and pyrethroid pesticides (PY) to activate hPXR were also assessed and found to be consistent with the abilities of these compounds to induce CYP3A4 and CYP2B6 in primary culture of human hepatocytes. These results suggest that CYP3A4 and CYP2B6 regulation through PXR activation by persistent pesticides may have an impact on the metabolism of xenobiotic agents and endogenous steroid hormones. Our model provides a useful tool for studying hPXR activation and for identifying agents capable of inducing CYP3A4 and CYP2B6.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aryl Hydrocarbon Hydroxylases / metabolism*
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / metabolism*
  • Enzyme Induction / drug effects
  • Genes, Reporter / drug effects
  • Hepatocytes / drug effects*
  • Hepatocytes / enzymology
  • Humans
  • Oxidoreductases, N-Demethylating / metabolism*
  • Pesticides / pharmacology*
  • Pregnane X Receptor
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Steroid / genetics
  • Transfection
  • Tumor Cells, Cultured


  • Pesticides
  • Pregnane X Receptor
  • Receptors, Cytoplasmic and Nuclear
  • Receptors, Steroid
  • Cytochrome P-450 Enzyme System
  • Aryl Hydrocarbon Hydroxylases
  • CYP2B6 protein, human
  • CYP3A protein, human
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Oxidoreductases, N-Demethylating