Abstract
We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.
Highlights
The human liver is the main site for drug metabolism and toxicities caused by the generation of reactive metabolites [1]
Vector was introduced by the measles virus envelope protein-mediated microcell-mediated chromosome transfer (MV-Microcell-Mediated Chromosome Transfer (MMCT)) method into CYPs-HepG2 cells established in a previous study [8], in which four major drug-metabolizing CYP enzymes (CYP2C9, CYP2C19, CY2D6, and CYP3A4) and CYP oxidoreductase (POR) were constitutively expressed under the control of CAG promoter, generating CYP-expressing luminescent HepG2 cells
No remarkable changes were observed in the bioluminescence measurement and the water-soluble tetrazolium-1 (WST-1) assay when non-toxicant D-mannitol was used to treat the two cell lines (Figure 2C). These results demonstrate that the sensitivities of Emerald Luc (ELuc)-HepG2 and CYPs-ELuc-HepG2 cells to a toxicant whose toxicity does not depend on CYP metabolism are very similar, and that the decrease of bioluminescence intensity of cells well correlates with cytotoxicity assessed by the conventional cell viability assay, as reported previously by us [19,20] and others [16,31]
Summary
The human liver is the main site for drug metabolism and toxicities caused by the generation of reactive metabolites [1]. The utility of HepG2 cells for assessing drug metabolism and toxicity is limited by the low expression of drugmetabolizing enzymes [6,7]. To solve this problem, we previously generated HepG2 cells using the mouse artificial chromosome (MAC) vector, in which major drug-metabolizing. We considered that the dynamics of cytotoxicity expression can be and precisely analyzed by applying the real-time bioluminescence measurement method. Brazilian click beetle luciferase (Emerald Luc; ELuc) [27] into previously generated CYPsHepG2 cells, and succeeded in monitoring time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity by real-time bioluminescence measurement
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