Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies
Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies
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DOI：
10.1371/journal.pone.0187072
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发表时间：
2017-10-24
期刊：
影响因子：
3.7
通讯作者：
Kazuki, Yasuhiro
中科院分区：
文献类型：
--
作者：
Satoh, Daisuke;Iwado, Satoru;Kazuki, Yasuhiro
HepG 2细胞的低细胞色素P450（CYP 450）活性阻碍了HepG 2细胞评估化合物诱导的药物代谢和毒性的效用。为了克服这一局限性，我们建立了HepG 2细胞系表达主要的酶参与药物代谢（CYP 2C 9，CYP 2C 19，CYP 2D 6，和CYP 3A 4）和β-氧化还原酶（POR）使用拟南芥衍生的人工染色体载体。使用表达四种CYP和POR的Transchromosomic HepG 2（TC-HepG 2）细胞，通过CYP特异性底物的发光检测和细胞活力测定来确定几种化合物的时间和浓度依赖性抑制和毒性。TC-HepG 2克隆中所有四种CYP和POR的基因表达水平以及抑制活性均高于亲本HepG 2细胞。此外，所有CYP的活性水平均被特异性CYP抑制剂以浓度依赖性方式降低。此外，在加入CYP特异性底物之前，将TCHepG 2细胞与时间依赖性抑制剂（TDI）一起预孵育，确定TDI组的TCHepG 2抑制作用比非TDI组增强。最后，生物活性化合物黄曲霉毒素B1在TC-HepG 2细胞中的IC 50低于在HepG 2细胞中。总之，本研究中表征的TC-HepG 2细胞是在候选药物化合物的初始筛选中评估药物-药物相互作用和肝毒性的高度通用模型，这需要高度的处理能力和可靠性。
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 TCHepG2 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.