Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

Abstract

The utility of HepG2 cells to assess drug metabolism and toxicity induced by chemical compounds is hampered by their low cytochrome P450 (CYP) activities. To overcome this limitation, we established HepG2 cell lines expressing major CYP enzymes involved in drug metabolism (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and CYP oxidoreductase (POR) using the mammalian-derived artificial chromosome vector. Transchromosomic HepG2 (TC-HepG2) cells expressing four CYPs and POR were used to determine time- and concentration-dependent inhibition and toxicity of several compounds by luminescence detection of CYP-specific substrates and cell viability assays. Gene expression levels of all four CYPs and POR, as well as the CYP activities, were higher in TC-HepG2 clones than in parental HepG2 cells. Additionally, the activity levels of all CYPs were reduced in a concentration-dependent manner by specific CYP inhibitors. Furthermore, preincubation of TC-HepG2 cells with CYP inhibitors known as time-dependent inhibitors (TDI) prior to the addition of CYP-specific substrates determined that CYP inhibition was enhanced in the TDI group than in the non-TDI group. Finally, the IC50 of bioactivable compound aflatoxin B1 was lower in TC-HepG2 cells than in HepG2 cells. In conclusion, the TC-HepG2 cells characterized in the current study are a highly versatile model to evaluate drug-drug interactions and hepatotoxicity in initial screening of candidate drug compounds, which require a high degree of processing capacity and reliability.

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells

“…Non-destructive bioluminescence measurement revealed strong bioluminescence of both cell lines with almost the same intensity ( Figure 1C). Finally, we also confirmed the remarkable activities of the four CYPs in CYPs-ELuc-HepG2 cells (Supplementary Figure S1B), as reported in a previous study [8].…”

Development of Caco-2 cells expressing four CYPs via a mammalian artificial chromosome

“…The gene expression and activity of CYPs in TC-HepG2 carrying the 4CYPs-MAC are either comparable to or higher than those in primary human hepatocytes [ 15 ]. In this study, in contrast to the other CYPs, CYP3A4 mRNA expression was still low in Caco-2 carrying the 4CYPs-MAC compared with the level in human adult intestine, despite the significant enhancement of mRNA expression.…”

“…Recently, a mouse artificial chromosome (MAC) vector constructed from native mouse chromosome 11 was used to increase the activity of multiple CYPs in HepG2 cells, which are a liver cancer cell line typically exhibiting low CYP activity [ 14 ]. In this study, four CYP genes (CYP3A4, CYP2C9, CYP2C19, CYP2D6) and a POR gene were loaded on the MAC (4CYPs-MAC) and transferred to HepG2 cells (TC-HepG2) to make the cells more suitable as a model to evaluate drug–drug interactions (DDIs) and hepatotoxicity in the initial screening of candidate drugs [ 15 ]. The expression and activity of CYPs in TC-HepG2 were comparable to those in human hepatocytes and this expression was sustained after a long culture period because of the stability of the MAC in human cells [ 16 ].…”

Humanized UGT2 and CYP3A transchromosomic rats for improved prediction of human drug metabolism

“…A MAC vector stably maintained in mice has been developed and various small- to intermediate-size genes of interest from circular vectors such as plasmids, PACs, and BACs have been transferred to the MAC vector by a site-specific recombination system for several applications (28–30). However, the loading of a large Mb-sized gene cluster to the MAC vectors has not been demonstrated yet.…”