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Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

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Abstract

A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1 % dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC 50 within 2.5-fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.

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References

  • Atkinson A, Kenny JR, Grime K. Automated assessment of time-dependent inhibition of human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometry analysis. Drug Metab Dispos. 2005;33:1637–47.

    Article  CAS  PubMed  Google Scholar 

  • Chan WK, Delucchi AB. Resveratrol, a red wine constituent, is a mechanism-based inactivator of cytochrome P450 3A4. Life Sci. 2000;67:3103–12.

    Article  CAS  PubMed  Google Scholar 

  • Choi S, Sainz Jr B, Corcoran P, Uprichard S, Jeong H. Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells. Xenobiotica. 2009;39:205–17.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Donato MT, Jover R, Gómez-Lechón MJ. Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering. Curr Drug Metab. 2013;14:946–68.

    Article  CAS  PubMed  Google Scholar 

  • Gerets HH, Tilmant K, Gerin B, Chanteux H, Depelchin BO, Dhalluin S, et al. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol Toxicol. 2012;28:69–87.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gordon DW, Rosenthal G, Hart J, Sirota R, Baker AL. Chaparral ingestion. The broadening spectrum of liver injury caused by herbal medications. JAMA. 1995;273:489–90.

    Article  CAS  PubMed  Google Scholar 

  • Guguen-Guillouzo C, Guillouzo A. General review on in vitro hepatocyte models and their applications. Methods Mol Biol. 2010;640:1–40.

    Article  CAS  PubMed  Google Scholar 

  • Gustafsson F, Foster AJ, Sarda S, Bridgland-Taylor MH, Kenna JG. A correlation between the in vitro drug toxicity of drugs to cell lines that express human P450s and their propensity to cause liver injury in humans. Toxicol Sci. 2014;137:189–211.

    Article  CAS  PubMed  Google Scholar 

  • Hisaka A, Ohno Y, Yamamoto T, Suzuki H. Prediction of pharmacokinetic drug-drug interaction caused by changes in cytochrome P450 activity using in vivo information. Pharmacol Ther. 2010;125:230–48.

    Article  CAS  PubMed  Google Scholar 

  • Hosomi H, Fukami T, Iwamura A, Nakajima M, Yokoi T. Development of a highly sensitive cytotoxicity assay system for CYP3A4-mediated metabolic activation. Drug Metab Dispos. 2011;39:1388–95.

    Article  CAS  PubMed  Google Scholar 

  • Isom HC, Secott T, Georgoff I, Woodworth C, Mummaw J. Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci U S A. 1985;82:3252–6.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kaplowitz N. Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov. 2005;4:489–99.

    Article  CAS  PubMed  Google Scholar 

  • Lambert JD, Zhao D, Meyers RO, Kuester RK, Timmermann BN, Dorr RT. Nordihydroguaiaretic acid: hepatotoxicity and detoxification in the mouse. Toxicon. 2002;40:1701–8.

    Article  CAS  PubMed  Google Scholar 

  • Lin J, Schyschka L, Mühl-Benninghaus R, Neumann J, Hao L, Nussler N, et al. Comparative analysis of phase I and II enzyme activities in 5 hepatic cell lines identifies Huh-7 and HCC-T cells with the highest potential to study drug metabolism. Arch Toxicol. 2012;86:87–95.

    Article  CAS  PubMed  Google Scholar 

  • Liu YT, Hao HP, Liu CX, Wang GJ, Xie HG. Drugs as CYP3A probes, inducers, and inhibitors. Drug Metab Rev. 2007;39:699–721.

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Flynn TJ, Ferguson MS, Hoagland EM, Yu LL. Effects of dietary phenolics and botanical extracts on hepatotoxicity-related endpoints in human and rat hepatoma cells and statistical models for prediction of hepatotoxicity. Food Chem Toxicol. 2011;49:1820–7.

    Article  CAS  PubMed  Google Scholar 

  • Lübberstedt M, Müller-Vieira U, Mayer M, Biemel KM, Knöspel F, Knobeloch D, et al. HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro. J Pharmacol Toxicol Methods. 2011;63:59–68.

    Article  PubMed  Google Scholar 

  • Malinen MM, Kanninen LK, Corlu A, Isoniemi HM, Lou YR, Yliperttula ML, et al. Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels. Biomaterials. 2014;35:5110–21.

    Article  CAS  PubMed  Google Scholar 

  • McGinnity DF, Berry AJ, Kenny JR, Grime K, Riley RJ. Evaluation of time-dependent cytochrome P450 inhibition using cultured human hepatocytes. Drug Metab Dispos. 2006;34:1291–300.

    Article  CAS  PubMed  Google Scholar 

  • Meisenheimer PL, Uyeda HT, Ma D, Sobol M, McDougall MG, Corona C, et al. Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition. Drug Metab Dispos. 2011;39:2403–10.

    Article  CAS  PubMed  Google Scholar 

  • Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982;42:3858–63.

    CAS  PubMed  Google Scholar 

  • Promega technical bulletin TB325. P450-GloTM assays. Madison: Promega Corporation. 2015; 1-TM 53.

  • Sainz Jr B, Chisari FV. Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells. J Virol. 2006;80:10253–7.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Santos NC, Figueira-Coelho J, Martins-Silva J, Saldanha C. Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects. Biochem Pharmacol. 2003;65:1035–41.

    Article  CAS  PubMed  Google Scholar 

  • Sivertsson L, Ek M, Darnell M, Edebert I, Ingelman-Sundberg M, Neve EP. CYP3A4 catalytic activity is induced in confluent Huh7 hepatoma cells. Drug Metab Dispos. 2010;38:995–1002.

    Article  CAS  PubMed  Google Scholar 

  • Sjogren AK, Liljevald M, Glinghammar B, Sagemark J, Li XQ, Jonebring A, et al. Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes. Arch Toxicol. 2014;88:1427–37.

    Article  CAS  PubMed  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76–85.

    Article  CAS  PubMed  Google Scholar 

  • Templeton IE, Houston JB, Galetin A. Predictive utility of in vitro rifampin induction data generated in fresh and cryopreserved human hepatocytes, Fa2N-4, and HepaRG cells. Drug Metab Dispos. 2011;39:1921–9.

    Article  CAS  PubMed  Google Scholar 

  • Voican CS, Corruble E, Naveau S, Perlemuter G. Antidepressant-induced liver injury: a review for clinicians. Am J Psychiatry. 2014;171:404–15.

    Article  PubMed  Google Scholar 

  • Wang SF, Chou YC, Mazumder N, Kao FJ, Nagy LD, Guengerich FP, et al. 7-Ketocholesterol induces P-glycoprotein through PI3K/mTOR signaling in hepatoma cells. Biochem Pharmacol. 2013;86:548–60.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zhang X, Guo S, Fan R, Yu M, Li F, Zhu C, et al. Dual-functional liposome for tumor targeting and overcoming multidrug resistance in hepatocellular carcinoma cells. Biomaterials. 2012;33:7103–14.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Michael F. Santillo for image processing and critical review of the paper. The authors thank Ms. Shelia PughBishop for technical assistance. This research was supported by the US Food and Drug Administration. This research report is not an official US Food and Drug Administration guidance or policy statement. No official support or endorsement by the US Food and Drug Administration is intended, nor should it be inferred.

Conflict of interest

The authors declare no conflicts of interest.

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Authors and Affiliations

  1. Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Laurel, MD, USA

    Yitong Liu, Thomas J. Flynn & Paddy L. Wiesenfeld

  2. Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA

    Menghang Xia

  3. Division of Public Health Informatics & Analytics, Office of Analytics and Outreach, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD, USA

    Martine S. Ferguson

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  1. Yitong Liu
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  2. Thomas J. Flynn
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  4. Paddy L. Wiesenfeld
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  5. Martine S. Ferguson
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Correspondence to Yitong Liu.

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Liu, Y., Flynn, T.J., Xia, M. et al. Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells. Cell Biol Toxicol 31, 221–230 (2015). https://doi.org/10.1007/s10565-015-9306-9

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  • DOI: https://doi.org/10.1007/s10565-015-9306-9

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