Human CYP3A4-introduced HepG2 cells:In vitroscreening system of new chemicals for the evaluation of CYP3A4-inhibiting activity

Abstract

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.

Evaluation of EROD and CYP3A4 activities in earthworm Eisenia fetida as biomarkers for soil heavy metal contamination

“…While CYP3A4 activity, like EROD activity, could be a suitable biomarkers of heavy metal exposure, studies on CYP3A4 activity have focused on the mechanisms of drug metabolism and their interactions in humans or rats [56][57][58][59][60][61][62][63][64], but not on the effects of environmental pollutants. In our study, the inverted U-shaped dose-response of CYP3A4 activity to Cd exposure seemed to be consistent with the hormetic hypothesis [65].The phenomenon of hormesis is probably related to the activation of adaptive pathways that are responsible for cellular and physiological homeostasis [66].…”

Inhibition of CYP3A4 by 6′,7′-dihydroxybergamottin in human CYP3A4 over-expressed hepG2 cells

“…One‐hundred‐thousand cells were seeded onto 24‐well culture plates and grown to confluence. We previously reported that enzymatic reactions by CYP3A4, such as diazepam N‐desmethylation, diazepam 3‐hydroxylation, lidocaine monodeethylation, atorvastatin acid 2‐hydroxylation and nifedipine oxidation, were increased about 100 fold in HepG2‐GS‐3A4 cells compared with those in HepG2‐GS cells and that these metabolites were easily detected in the culture medium of the HepG2‐GS‐3A4 cells [10] . We also reported that the inhibitory effects of ketoconazole and cimetidine on CYP3A4 activity were adequately evaluated by nifedipine as a substrate of CYP3A4.…”

“…HepG2 cells, from a cell line that originated from human hepatoblastoma, were obtained from RIKEN Cell Bank (Tsukuba, Japan). HepG2‐GS‐3A4 cells, transduced with both hamster glutamine synthetase (GS) gene and human CYP3A4 gene, were established previously [8–10] . In short, pBudCE4 plasmid (Invitrogen, Carlsbad, CA, USA), which had two multi‐cloning sites with human cytomegalovirus gene and human elongation 1‐alpha subunit gene as promoters, was used for the vector after ligation with hamster Gs and human CYP3A4 genes; it was introduced by lipofection and positive clones were selected using the index of resistance against zeocin.…”

“…We also reported that the inhibitory effects of ketoconazole and cimetidine on CYP3A4 activity were adequately evaluated by nifedipine as a substrate of CYP3A4. Furthermore, oxidised nifedipine in the culture medium was easier to detect by HPLC than metabolites of other substrates [10] . Therefore, in this study, we used nifedipine as a probe of CYP3A4 activity.…”

“…By using these novel cells and new technology for three‐dimensional cell culture, we developed a new bioreactor for successful treatment of acute liver failure and concomitant diazepam intoxication in an animal model [9] . Because the capacity for drug metabolism via CYP3A4 in these cells is extremely high and we could detect the metabolites of CYP3A4 substrates in culture medium, we further showed that our cells can be applied for screening inhibitors of CYP3A4 in vitro [10] . Because these cells contain a large amount of CYP3A4 protein, they can also be used as a tool to investigate the interaction of CYP3A4 with other chemicals and might be useful to evaluate the mechanism of furanocoumarin‐induced inhibition of CYP3A4.…”