| [Objective] The term "Food non-nutrients" is named compared with the traditional nutrients, such as lipids, proteins, carbohydrates, vitamins and minerals. Non-nutrients are divided into different categories, including phytoestrogens, chlorophyll, carotenoids, protease inhibitors, polyphenols, saponins, and so on. Certain non-nutrients possess anticarcinogenic activity, antioxidative potential, and prevention on chronic diseases. The food non-nutrients have been used widely in fields of dietary supplements, medicine and commodity. The trend of increase in the consumption of fruits and vegetables, and dietary supplements and herbal products has substantially increased human exposure to non-nutrients. Cytochrome P450 family plays a critical role in the metabolism of xenobiotics, and the CYP3A4 subfamily is involved in the metabolism of steroid hormones, food, drugs, environment pollutants and procarcinogens. The alteration of CYP3A4 gene expression and activity may influence the metabolism of substrates. Some compounds have been shown to induce CYP3A4 and food-drug interactions occurred. The aim of present study is to establish a cell-based PXRdependent reporter gene assay used for screening CYP3A4 inducers. The broad application of non-nutrients increase the human exposure and may modulate drug-metabolising enzyme, such as CYP3A4, leading to clinically relevant drug-food interactions. Pregnane X receptor (PXR), also called steroid and xenobiotic receptor (SXR), a key transcriptional regulator of CYP3A4, can be bind to the XRE in the promoter in CYP3A4 gene. Many compounds induce the CYP3A4 via PXR. Because of the potential for induction-based interactions, especially for food-drug interactions and it is important to examine the non-nutrients' induction potential for CYP3A4. The study can help us to illustrate the possible mechanisms interactions between the food non-nutrients and other compounds (drug), and to predict possible adverse reactions and contribution to safety evaluation of food, dietary supplement and drug. Six non-nutrients genistein (GST), coumestrol (COM), quercetin (QU), zearalenone (ZEA), sodium copper chlorophyllin (CHL) and |3-carotene (P-C) were choosed to examine for their induction potential for CYP3A4 because of their broad applications and vital concernments to human.[Methods] PXR-dependent reporter gene assay was established for examining the potential induction of six non-nutrients, genistein (GST), coumestrol (COM), quercetin (QU), zearalenone (ZEA), sodium copper chlorophyllin (CHL) and p-carotene (P-C). Transient cotransfection reporter gene assays in HepG2 cells were performed with the hPXR expression plasmid and the reporter plasmid containing the XRE in the promoter of CYP3A4 linked to luciferase. Plasmids used in transfections were prepared by QIAprep Spin Miniprep Kit. HepG2 cells were seeded in 24-well dishes at 1 X 105 cells per well in Dulbecco's modified Eagle's medium without phenol red supplemented with 10% dextran-charcoal treated fetal bovine serum (FBS). After 16-24 h, the cells were transfected with lipofectamin?000 according to manufactures and then the cells were 90%-95% confluent. For each well, 450 ng Reporter plasmid, 150 ng hPXR expression plasmid and 160 ng CMV gal control plasmid were used. The culture median wasreplaced with fresh media with varied concentrations of non-nutrients after 4-6 h of transfection. The compounds were dissolved with DMSO or distilled water and diluted to end concentrations with completed DMEM. The concentration of DMSO is controlled below 0.1%. The negative and positive cells received with 0.1% DMSO and rifampicin respectively. 12 h, 24 h and 48 h incubation with the compounds, the cells were rinsed in PBS and the lysates were analysed for both luciferase and p-galactosidase in order to correct for transfection efficiency. Luciferase activities of cell lysates were determined using LS6500 Multi-Purpose Scintillation Counter (Beckman) and the p-galactosidase were determined with spectrophotometer at 420nm. Results were...
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