Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
HepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4–POR–UGT1A1–CYP1A2–CYP2C19–CYP2C9–CYP2D6 (CYPs–UGT1...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-05-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/11/10/1677 |
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| author | Ryosuke Negoro Mitsuki Tasaka Sayaka Deguchi Kazuo Takayama Takuya Fujita |
| author_facet | Ryosuke Negoro Mitsuki Tasaka Sayaka Deguchi Kazuo Takayama Takuya Fujita |
| author_sort | Ryosuke Negoro |
| collection | DOAJ |
| description | HepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4–POR–UGT1A1–CYP1A2–CYP2C19–CYP2C9–CYP2D6 (CYPs–UGT1A1) knock-in (KI)-HepG2 cells using a PITCh system to evaluate whether they could be a new hepatocyte model for pharmaceutical studies. To evaluate whether CYPs–UGT1A1 KI-HepG2 cells express and function with CYPs and UGT1A1, gene expression levels of <i>CYPs</i> and <i>UGT1A1</i> were analyzed by using real-time PCR, and metabolites of CYPs or UGT1A1 substrates were quantified by HPLC. The expression levels of <i>CYPs</i> and <i>UGT1A1</i> in the CYPs–UGT1A1 KI-HepG2 cells were comparable to those in primary human hepatocytes (PHHs) cultured for 48 h. The CYPs and UGT1A1 activity levels in the CYPs–UGT1A1 KI-HepG2 cells were much higher than those in the wild-type (WT)-HepG2 cells. These results suggest that the CYPs–UGT1A1 KI-HepG2 cells expressed functional CYPs and UGT1A1. We also confirmed that the CYPs–UGT1A1 KI-HepG2 cells were more sensitive to drug-induced liver toxicity than the WT-HepG2 cells. CYPs–UGT1A1 KI-HepG2 cells could be used to predict drug metabolism and drug-induced liver toxicity, and they promise to be a helpful new hepatocyte model for drug discovery research. |
| first_indexed | 2024-03-10T03:09:29Z |
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| id | doaj.art-35f328aa1a3741669e773a1d4d2fbe7d |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T03:09:29Z |
| publishDate | 2022-05-01 |
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| record_format | Article |
| series | Cells |
| spelling | doaj.art-35f328aa1a3741669e773a1d4d2fbe7d2023-11-23T10:28:04ZengMDPI AGCells2073-44092022-05-011110167710.3390/cells11101677Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh SystemRyosuke Negoro0Mitsuki Tasaka1Sayaka Deguchi2Kazuo Takayama3Takuya Fujita4Laboratory of Molecular Pharmacokinetics, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, JapanLaboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, JapanCenter for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, JapanCenter for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, JapanLaboratory of Molecular Pharmacokinetics, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, JapanHepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4–POR–UGT1A1–CYP1A2–CYP2C19–CYP2C9–CYP2D6 (CYPs–UGT1A1) knock-in (KI)-HepG2 cells using a PITCh system to evaluate whether they could be a new hepatocyte model for pharmaceutical studies. To evaluate whether CYPs–UGT1A1 KI-HepG2 cells express and function with CYPs and UGT1A1, gene expression levels of <i>CYPs</i> and <i>UGT1A1</i> were analyzed by using real-time PCR, and metabolites of CYPs or UGT1A1 substrates were quantified by HPLC. The expression levels of <i>CYPs</i> and <i>UGT1A1</i> in the CYPs–UGT1A1 KI-HepG2 cells were comparable to those in primary human hepatocytes (PHHs) cultured for 48 h. The CYPs and UGT1A1 activity levels in the CYPs–UGT1A1 KI-HepG2 cells were much higher than those in the wild-type (WT)-HepG2 cells. These results suggest that the CYPs–UGT1A1 KI-HepG2 cells expressed functional CYPs and UGT1A1. We also confirmed that the CYPs–UGT1A1 KI-HepG2 cells were more sensitive to drug-induced liver toxicity than the WT-HepG2 cells. CYPs–UGT1A1 KI-HepG2 cells could be used to predict drug metabolism and drug-induced liver toxicity, and they promise to be a helpful new hepatocyte model for drug discovery research.https://www.mdpi.com/2073-4409/11/10/1677genome editingCRISPR-Cas9PITCh systemHepG2 cellprimary human hepatocytesCYP1A2 |
| spellingShingle | Ryosuke Negoro Mitsuki Tasaka Sayaka Deguchi Kazuo Takayama Takuya Fujita Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System Cells genome editing CRISPR-Cas9 PITCh system HepG2 cell primary human hepatocytes CYP1A2 |
| title | Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System |
| title_full | Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System |
| title_fullStr | Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System |
| title_full_unstemmed | Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System |
| title_short | Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System |
| title_sort | generation of hepg2 cells with high expression of multiple drug metabolizing enzymes for drug discovery research using a pitch system |
| topic | genome editing CRISPR-Cas9 PITCh system HepG2 cell primary human hepatocytes CYP1A2 |
| url | https://www.mdpi.com/2073-4409/11/10/1677 |
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