Human CYP3A4-introduced HepG2 cells: in vitro screening system of new chemicals for the evaluation of CYP3A4-inhibiting activity.

1. The aims were to attest whether HepG2-GS-3A4, a cell line into which the human CYP3A4 gene was introduced, can be used for a screening of chemicals that will inhibit CYP3A4 activity. 2. The capacity of the cells for metabolizing CYP3A4 substrates in vitro was evaluated. Also determined was the effect of CYP3A4 inhibitors and non-inhibitors on nifedipine hydroxylation. Western blot, immunohistochemostry and determination of beta-nicotinamide adenine dinucleotide phosphate (NADPH)-reductase activity were performed. 3. HepG2-GS-3A4 selectively metabolized substrates of CYP3A4 (diazepam, nordiazepam, lidocaine, atorvastatin, and nifedipine) to a greater degree than control. The metabolites were easily detected in the culture medium. Values of V(max) of HepG2-GS-3A4 were about 30- to 100-fold higher than those of the control, while values of K(m) were comparable. Pre-incubation of cimetidine and ketoconazole significantly inhibited nifedipine hydroxylation, while addition of inhibitors specific to other isoforms of CYPs had no substantial effect. The HepG2-GS-3A4 expressed a higher amount of CYP3A4 protein and mRNA than control. Most NADPH reductase activity was detected in microsomal fractions. 4 In conclusion, HepG2-GS-3A4 sufficiently and selectively metabolize substrates of CYP3A4, and inhibitors of CYP3A4 reduced the metabolism. Because the metabolites were easily detected in the culture medium, this cell might be useful for the new and easy screening of new drugs for the evaluation of CYP3A4-inhibiting activity in vitro.