Human Pregnane X Receptor-mediated Transcriptional Regulation Of CYP3A4 By Some Phytochemicals

[Objective] Phytochemicals are small molecular compounds which widely existed in corns, vegetables, fruits and herbs. Their physiological or biomedical effects have been used in prevention and treatment of diseases. The interaction of phytochemials and other xenobiotics is an important issue in safety evaluation. Most phytochemials are metabolized in liver and intestinal by cytochrome P450 among which CYP3A4 is the most important enzyme in metabolism and is responsible for approximately 60% P450-mediated metabolism of drugs. CYP3A4 can be induced by various xenobiotics. The induction of CYP3A4 is essential for interaction between two xenobiotics. Pregnane X receptor ( PXR) is a key regulator of CYP3A4 transcription, it can bind to the XRE in promoter of in CYP3A4 gene and activates its transcription. Many xenobiotics induce CYP3A4 via PXR. To understand whether a xenobiotic can induce CYP3A4 enzyme or not could forecast potential interactions so as to reduce or avoid kickbacks resulting from interactions. The cell-based PXR reporter gene assay is an effective means for assessing xenobiotics potential to induce CYP3A4 activity and related mechanisms and for predicting the potential interactions between xenobiotics. The aim of the present study is to evaluate whether phytochemicals selected would affect the transcription of CYP3A4 and its possible mechanisms and provide foundations for evaluation and safe use of phytochemicals.[Methods] Transient cotransfection reporter gene assays in HepG₂ cells were performed with the hPXR expression plasmid and the reporter plasmid which contains XRE in the promoter of CYP3A4 linked to luciferase . The culture medium was replaced with fresh medium with varied concentrations of six phytochemicals (kaempferol, isohamnetin, soybeam isoflavone, luteolin, curcumin and rutin) after 6 h of transfection. The negative and positive cells received with 0.1% DMSO and rifampicin respectively. After incubation with the compounds for 12 h, 24 h, and 48 h, the cells were rinsed in PBS and the lysates were analysed for both luciferase and β -galactosidase which was used to correct for transfection efficiency. The phytochemicals, which could transactivate CYP3A4 expression after 24 h continue to be tested by PXR reporter-gene assays by 12 h and 48 h, respectively. Luciferase activities of cell lysates were measured using a Multilabel Counter and the β -galactosidase activities were determined with spectrophotometer at 420 nm. Results were expressed as fold compared to the luciferase activities of 0.1% DMSO treated cells. All transfection experiments were repeated at least twice in triplicate. Data were shown as means±S.D. Statistical evaluation of the data was analyzed by Student's t-test . [Results] In the study of dose-effect relationship, kaempferol, soybean isoflavone, luteolin and curcumin can induce the CYP3A4 transcription via PXR with evident dose-dependent manners, but isorhamnetin and rutin have no such results. The ability of induction by kaempferol increases with concentrations between 10 u mol/L and 50 u mol/L. 0.01 u mol/L kaempferol and 10 μmol/L kaempferol exhibit 1.45-fold and 7.93-fold luciferase activity of 0.1%DMSO treated cells, respectively. The inducibilities of soybeam isoflavone, luteolin and curcumin also increase with concentrations between 1 μmol/L and 50 μmol/L. 24h after induction, 50 μmol/L soybeam isoflavone, luteolin and curcumin exhibit 5.46-fold, 2.87-fold, and 2.07-fold compared to 0.1% DMSO treated cells, respectively. In the study of time-effect relationship, kaempferol (1μ mol/L,10μ mol/L), soybean isoflavone (10μ mol/L, 50μ mol/L) and curcumin (10μ mol/L ,50 μmol/L) all can induce CYP3A4 transcription between 12h and 48h, and their strongest abilities of induction appear in 48h. 48h after induction, 10 μmol/Lkaempferol, 50 μmol/L soybean isoflavone, 50μmol/L luteolin and 50 μmol/L curcumin exhibit 8.42-fold, 6.72-fold, 3.24-fold, and 2.13-fold compared to 0.1% DMSO treated cells, respectively.[Conclussion] Four phytochemicals, i.e. kaemp...