title: Development of a highly sensitive cytotoxicity assay system for CYP3A4-mediated metabolic activation.
abstract: Drug-induced hepatotoxicity, which is a rare but serious adverse reaction to a large number of pharmaceutical drugs, is sometimes associated with reactive metabolites produced by drug-metabolizing enzymes. In the present study, we constructed a cell-based system to evaluate the cytotoxicity of reactive metabolites produced by CYP3A4 using human hepatoma cells infected with an adenovirus vector expressing human CYP3A4 (AdCYP3A4). When seven hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7, and Fa2N4 cells) were infected with AdCYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and testosterone 6β-hydroxylase activity (670 pmol · min(-1) · mg(-1)). With the use of AdCYP3A4-infected HepG2 cells, the cytotoxicities of 23 drugs were evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, and the cell viability when treated with 11 drugs (amiodarone, desipramine, felbamate, isoniazid, labetalol, leflunomide, nefazodone, nitrofurantoin, tacrine, terbinafine, and tolcapone) was significantly decreased. Moreover, the transfection of siRNA for nuclear factor erythroid 2-related factor 2 (Nrf2) to decrease the cellular expression level of Nrf2 exacerbated the cytotoxicity of some drugs (troglitazone, flutamide, acetaminophen, clozapine, terbinafine, and desipramine), suggesting that the genes regulated by Nrf2 are associated with the detoxification of the cytotoxicities mediated by CYP3A4. We constructed a highly sensitive cell-based system to detect the drug-induced cytotoxicity mediated by CYP3A4. This system would be beneficial in preclinical screening in drug development and increase our understanding of the drug-induced cytotoxicity associated with CYP3A4.
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hosomi
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Fukami
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iwamura
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D064420:Drug-Related Side Effects and Adverse Reactions; D004789:Enzyme Activation; D006801:Humans; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human