{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T02:48:17Z","timestamp":1790822897350,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2021,3,11]],"date-time":"2021-03-11T00:00:00Z","timestamp":1615420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Regional Innovation Strategy Support Program","award":["Ministry of Education, Culture, Sports, Science and Technology of Japan"],"award-info":[{"award-number":["Ministry of Education, Culture, Sports, Science and Technology of Japan"]}]},{"name":"Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)","award":["Japan Agency for Medical Research and Development (AMED)"],"award-info":[{"award-number":["Japan Agency for Medical Research and Development (AMED)"]}]},{"name":"AI-SHIPS project","award":["Ministry of Economy, Trade and Industry (METI), Japan"],"award-info":[{"award-number":["Ministry of Economy, Trade and Industry (METI), Japan"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ijms22062843","type":"journal-article","created":{"date-parts":[[2021,3,11]],"date-time":"2021-03-11T05:38:22Z","timestamp":1615441102000},"page":"2843","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells"],"prefix":"10.3390","volume":"22","author":[{"given":"Satoru","family":"Iwado","sequence":"first","affiliation":[{"name":"Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1153-0720","authenticated-orcid":false,"given":"Satoshi","family":"Abe","sequence":"additional","affiliation":[{"name":"Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitsuo","family":"Oshimura","sequence":"additional","affiliation":[{"name":"Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4818-4710","authenticated-orcid":false,"given":"Yasuhiro","family":"Kazuki","sequence":"additional","affiliation":[{"name":"Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan"},{"name":"Division of Genome and Cellular Functions, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7422-730X","authenticated-orcid":false,"given":"Yoshihiro","family":"Nakajima","sequence":"additional","affiliation":[{"name":"Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan"},{"name":"Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu 761-0395, Kagawa, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1315","DOI":"10.1007\/s00204-013-1078-5","article-title":"Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME","volume":"87","author":"Godoy","year":"2013","journal-title":"Arch. Toxicol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1039\/C2TX20051A","article-title":"In vitro models for liver toxicity testing","volume":"2","author":"Soldatow","year":"2013","journal-title":"Toxicol. Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/s13287-020-1555-8","article-title":"Different approaches for transformation of mesenchymal stem cells into hepatocyte-like cells","volume":"11","author":"Afshari","year":"2020","journal-title":"Stem Cell Res. Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1517\/17425255.2012.685159","article-title":"The HepaRG cell line: A unique in vitro tool for understanding drug metabolism and toxicology in human","volume":"8","author":"Andersson","year":"2012","journal-title":"Expert Opin. Drug Metab. Toxicol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1186\/s41232-019-0102-4","article-title":"Generation of functional human hepatocytes in vitro: Current status and future prospects","volume":"39","author":"Yamaguchi","year":"2019","journal-title":"Inflamm. Regen."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1035","DOI":"10.1124\/dmd.31.8.1035","article-title":"Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties","volume":"31","author":"Wilkening","year":"2003","journal-title":"Drug Metab. Dispos."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.tiv.2016.02.019","article-title":"High content analysis assay for prediction of human hepatotoxicity in HepaRG and HepG2 cells","volume":"33","author":"Saito","year":"2016","journal-title":"Toxicol. Vitro"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Satoh, D., Iwado, S., Abe, S., Kazuki, K., Wakuri, S., Oshimura, M., and Kazuki, Y. (2017). Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0187072"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"880","DOI":"10.1124\/dmd.105.008672","article-title":"The human intestinal cytochrome P450 \u201cpie\u201d","volume":"34","author":"Paine","year":"2006","journal-title":"Drug Metab. Dispos."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1016\/j.molcel.2015.03.002","article-title":"Molecular spies for bioimaging--fluorescent protein-based probes","volume":"58","author":"Miyawaki","year":"2015","journal-title":"Mol. Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1146\/annurev-physiol-022516-034055","article-title":"A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging","volume":"79","author":"Specht","year":"2017","journal-title":"Annu. Rev. Physiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/s00216-011-5455-8","article-title":"Analytical chemiluminescence and bioluminescence: Latest achievements and new horizons","volume":"402","author":"Roda","year":"2012","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1517\/17460441.2010.506213","article-title":"Bioluminescence assays: Multicolor luciferase assay, secreted luciferase assay and imaging luciferase assay","volume":"5","author":"Nakajima","year":"2010","journal-title":"Expert Opin. Drug Discov."},{"key":"ref_14","first-page":"5","article-title":"Spying on cancer: Molecular imaging in vivo with genetically encoded reporters","volume":"7","author":"Gross","year":"2005","journal-title":"Cancer Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1177\/2211068214560608","article-title":"High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter","volume":"20","author":"Class","year":"2015","journal-title":"J. Lab. Autom."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1007\/s00204-020-02934-9","article-title":"The modified IL-8 Luc assay, an in vitro skin sensitisation test, can significantly improve the false-negative judgment of lipophilic sensitizers with logKow values > 3.5","volume":"95","author":"Kimura","year":"2021","journal-title":"Arch. Toxicol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1038\/s41598-017-18606-1","article-title":"Development and characterization of a novel luciferase based cytotoxicity assay","volume":"8","author":"Matta","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Tsuji, S., Ohbayashi, T., Yamakage, K., Oshimura, M., and Tada, M. (2016). A Cytoplasmic Form of Gaussia luciferase Provides a Highly Sensitive Test for Cytotoxicity. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0156202"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.ab.2017.01.015","article-title":"Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase","volume":"522","author":"Wakuri","year":"2017","journal-title":"Anal. Biochem."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Yasunaga, M., Fujita, Y., Saito, R., Oshimura, M., and Nakajima, Y. (2017). Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement. BMC Biotechnol., 17.","DOI":"10.1186\/s12896-017-0374-1"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1002\/bio.3454","article-title":"Bioluminescence-based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells","volume":"33","author":"Uno","year":"2018","journal-title":"Luminescence"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1002\/bio.1170090603","article-title":"Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells","volume":"9","author":"Gandelman","year":"1994","journal-title":"J. Biolumin. Chemilumin."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1002\/bit.20059","article-title":"Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells","volume":"86","author":"Ignowski","year":"2004","journal-title":"Biotechnol. Bioeng."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.antiviral.2008.01.158","article-title":"Applications of bioluminescence imaging to antiviral research and therapy: Multiple luciferase enzymes and quantitation","volume":"78","author":"Luker","year":"2008","journal-title":"Antivir. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1376","DOI":"10.1039\/c0mb00328j","article-title":"Illuminating intracellular signaling and molecules for single cell analysis","volume":"7","author":"Awais","year":"2011","journal-title":"Mol. Biosyst."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"top070607","DOI":"10.1101\/pdb.top070607","article-title":"Cellular bioluminescence imaging","volume":"2012","author":"Welsh","year":"2012","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Nakajima, Y., Yamazaki, T., Nishii, S., Noguchi, T., Hoshino, H., Niwa, K., Viviani, V.R., and Ohmiya, Y. (2010). Enhanced beetle luciferase for high-resolution bioluminescence imaging. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0010011"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.bbrc.2013.10.171","article-title":"Highly stable maintenance of a mouse artificial chromosome in human cells and mice","volume":"442","author":"Kazuki","year":"2013","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1007\/s10577-014-9459-z","article-title":"A pathway from chromosome transfer to engineering resulting in human and mouse artificial chromosomes for a variety of applications to bio-medical challenges","volume":"23","author":"Oshimura","year":"2015","journal-title":"Chromosome Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e00540","DOI":"10.1002\/prp2.540","article-title":"No evidence for interactions of dimethylfumarate (DMF) and its main metabolite monomethylfumarate (MMF) with human cytochrome P450 (CYP) enzymes and the P-glycoprotein (P-gp) drug transporter","volume":"7","author":"Aubets","year":"2019","journal-title":"Pharmacol. Res. Perspect."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1093\/toxsci\/kfr237","article-title":"An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8","volume":"124","author":"Takahashi","year":"2011","journal-title":"Toxicol. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1635","DOI":"10.1007\/s00204-014-1312-9","article-title":"Metabolism of aflatoxins: Key enzymes and interindividual as well as interspecies differences","volume":"88","author":"Dohnal","year":"2014","journal-title":"Arch. Toxicol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1021\/tx050358e","article-title":"Dominant contribution of P450 3A4 to the hepatic carcinogenic activation of aflatoxin B1","volume":"19","author":"Kamdem","year":"2006","journal-title":"Chem. Res. Toxicol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1046\/j.1365-2125.2003.01718.x","article-title":"Effect of the CYP3A4 inhibitor erythromycin on the pharmacokinetics of lignocaine and its pharmacologically active metabolites in subjects with normal and impaired liver function","volume":"55","author":"Orlando","year":"2003","journal-title":"Br. J. Clin. Pharmacol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1111\/bcpt.13343","article-title":"Interaction potential between clarithromycin and individual statins-A systematic review","volume":"126","author":"Christensen","year":"2020","journal-title":"Basic Clin. Pharmacol. Toxicol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.pharmthera.2016.03.011","article-title":"Primaquine pharmacology in the context of CYP 2D6 pharmacogenomics: Current state of the art","volume":"161","author":"Marcsisin","year":"2016","journal-title":"Pharmacol. Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2380","DOI":"10.1128\/AAC.00015-15","article-title":"Differential CYP 2D6 metabolism alters primaquine pharmacokinetics","volume":"59","author":"Potter","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1016\/S0009-9236(99)70065-2","article-title":"Investigation of terbinafine as a CYP2D6 inhibitor in vivo","volume":"65","author":"Gotschall","year":"1999","journal-title":"Clin. Pharmacol. Ther."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/j.dmpk.2015.04.003","article-title":"Variation in the inhibitory potency of terbinafine among genetic variants of CYP2D6","volume":"30","author":"Akiyoshi","year":"2015","journal-title":"Drug Metab. Pharmacokinet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"398","DOI":"10.2144\/96213bm12","article-title":"Improved dicistronic mRNA expression vectors for efficient selection of transfectants highly expressing foreign genes","volume":"21","author":"Kobayashi","year":"1996","journal-title":"BioTechniques"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1111\/j.1751-1097.2010.00777.x","article-title":"Quantum yields and kinetics of the firefly bioluminescence reaction of beetle luciferases","volume":"86","author":"Niwa","year":"2010","journal-title":"Photochem. Photobiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"043104","DOI":"10.1063\/1.4979812","article-title":"Absolute optical responsivity down to the photon counting level with a photomultiplier tube","volume":"88","author":"Tanabe","year":"2017","journal-title":"Rev. Sci. Instrum."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Katoh, M., Kazuki, Y., Kazuki, K., Kajitani, N., Takiguchi, M., Nakayama, Y., Nakamura, T., and Oshimura, M. (2010). Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer. BMC Biotechnol., 10.","DOI":"10.1186\/1472-6750-10-37"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1016\/j.bbrc.2004.06.145","article-title":"Construction of a novel human artificial chromosome vector for gene delivery","volume":"321","author":"Katoh","year":"2004","journal-title":"Biochem. Biophys. Res. Commun."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/6\/2843\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:33:57Z","timestamp":1760160837000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/6\/2843"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,11]]},"references-count":44,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2021,3]]}},"alternative-id":["ijms22062843"],"URL":"https:\/\/doi.org\/10.3390\/ijms22062843","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,11]]}}}