Skip to main content

Quantitative comparison of in vitro genotoxicity between metabolically competent HepaRG cells and HepG2 cells using the high-throughput high-content CometChip assay

Archives of Toxicology volume 93, pages 1433–1448 (2019)Cite this article

Abstract

In vitro genotoxicity testing that employs metabolically active human cells may be better suited for evaluating human in vivo genotoxicity than current bacterial or non-metabolically active mammalian cell systems. In the current study, 28 compounds, known to have different genotoxicity and carcinogenicity modes of action (MoAs), were evaluated over a wide range of concentrations for the ability to induce DNA damage in human HepG2 and HepaRG cells. DNA damage dose–responses in both cell lines were quantified using a combination of high-throughput high-content (HTHC) CometChip technology and benchmark dose (BMD) quantitative approaches. Assays of metabolic activity indicated that differentiated HepaRG cells had much higher levels of cytochromes P450 activity than did HepG2 cells. DNA damage was observed for four and two out of five indirect-acting genotoxic carcinogens in HepaRG and HepG2 cells, respectively. Four out of seven direct-acting carcinogens were positive in both cell lines, with two of the three negatives being genotoxic mainly through aneugenicity. The four chemicals positive in both cell lines generated HTHC Comet data in HepaRG and HepG2 cells with comparable BMD values. All the non-genotoxic compounds, including six non-genotoxic carcinogens, were negative in HepaRG cells; five genotoxic non-carcinogens also were negative. Our results indicate that the HTHC CometChip assay detects a greater proportion of genotoxic carcinogens requiring metabolic activation (i.e., indirect carcinogens) when conducted with HepaRG cells than with HepG2 cells. In addition, BMD genotoxicity potency estimate is useful for quantitatively evaluating CometChip assay data in a scientifically rigorous manner.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Antherieu S, Chesne C, Li R et al (2010) Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab Dispos 38(3):516–525

    Article  CAS  PubMed  Google Scholar 

  • Ates G, Mertens B, Heymans A et al (2018) A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG cell line as a rapid and reliable tool for improved in vitro hazard assessment. Arch Toxicol 92(4):1593–1608

    Article  CAS  PubMed  Google Scholar 

  • Badisa VL, Latinwo LM, Odewumi CO et al (2007) Mechanism of DNA damage by cadmium and interplay of antioxidant enzymes and agents. Environ Toxicol 22(2):144–151

    Article  CAS  PubMed  Google Scholar 

  • Bryce SM, Bernacki DT, Bemis JC, Dertinger SD (2016) Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach. Environ Mol Mutagen 57(3):171–189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cerec V, Glaise D, Garnier D et al (2007) Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor. Hepatology 45(4):957–967

    Article  CAS  PubMed  Google Scholar 

  • Cheung YL, Snelling J, Mohammed NN, Gray TJ, Ioannides C (1996) Interaction with the aromatic hydrocarbon receptor, CYP1A induction, and mutagenicity of a series of diaminotoluenes: implications for their carcinogenicity. Toxicol Appl Pharmacol 139(1):203–211

    Article  CAS  PubMed  Google Scholar 

  • Coppinger WJ, Brennan SA, Carver JH, Thompson ED (1984) Locus specificity of mutagenicity of 2,4-diaminotoluene in both L5178Y mouse lymphoma and AT3-2 Chinese hamster ovary cells. Mutat Res 135(2):115–123

    Article  CAS  PubMed  Google Scholar 

  • Dierks EA, Stams KR, Lim HK, Cornelius G, Zhang H, Ball SE (2001) A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry. Drug Metab Dispos 29(1):23–29

    CAS  PubMed  Google Scholar 

  • Ge J, Chow DN, Fessler JL, Weingeist DM, Wood DK, Engelward BP (2015) Micropatterned comet assay enables high throughput and sensitive DNA damage quantification. Mutagenesis 30(1):11–19

    Article  CAS  PubMed  Google Scholar 

  • Gollapudi BB, Johnson GE, Hernandez LG et al (2013) Quantitative approaches for assessing dose–response relationships in genetic toxicology studies. Environ Mol Mutagen 54(1):8–18

    Article  CAS  PubMed  Google Scholar 

  • Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C (2007) The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact 168(1):66–73

    Article  CAS  PubMed  Google Scholar 

  • Guo X, Heflich RH, Dial SL, Richter PA, Moore MM, Mei N (2015) Quantitative analysis of the relative mutagenicity of five chemical constituents of tobacco smoke in the mouse lymphoma assay. Mutagenesis 31(3):287–296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guo X, Heflich RH, Dial SL, De M, Richter PA, Mei N (2018a) Quantitative differentiation of whole smoke solution-induced mutagenicity in the mouse lymphoma assay. Environ Mol Mutagen 59(2):103–113

    Article  CAS  PubMed  Google Scholar 

  • Guo X, Seo JE, Bryce SM et al (2018b) Comparative genotoxicity of TEMPO and three of its derivatives in mouse lymphoma cells. Toxicol Sci 163(1):214–225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hardy A, Benford D, Halldorsson T et al (2017) Update: use of the benchmark dose approach in risk assessment. EFSA Journal 15(1):4658

    Google Scholar 

  • Hartwig A (2010) Mechanisms in cadmium-induced carcinogenicity: recent insights. Biometals 23(5):951–960

    Article  CAS  PubMed  Google Scholar 

  • Hong YH, Jeon HL, Ko KY et al (2018) Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells. Mutat Res 827:59–67

    Article  CAS  Google Scholar 

  • ICH (2011) Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1). ICH Expert Working Group. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf. Accessed 12 Nov 2018

  • Jennen DG, Magkoufopoulou C, Ketelslegers HB, van Herwijnen MH, Kleinjans JC, van Delft JH (2010) Comparison of HepG2 and HepaRG by whole-genome gene expression analysis for the purpose of chemical hazard identification. Toxicol Sci 115(1):66–79

    Article  CAS  PubMed  Google Scholar 

  • Josse R, Rogue A, Lorge E, Guillouzo A (2012) An adaptation of the human HepaRG cells to the in vitro micronucleus assay. Mutagenesis 27(3):295–304

    Article  CAS  PubMed  Google Scholar 

  • Kawaguchi S, Nakamura T, Yamamoto A, Honda G, Sasaki YF (2010) Is the comet assay a sensitive procedure for detecting genotoxicity? J Nucleic Acids 2010:541050

  • Khoury L, Zalko D, Audebert M (2013) Validation of high-throughput genotoxicity assay screening using gammaH2AX in-cell western assay on HepG2 cells. Environ Mol Mutagen 54(9):737–746

    Article  CAS  PubMed  Google Scholar 

  • Khoury L, Zalko D, Audebert M (2016) Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening. Mutagenesis 31(1):83–96

    CAS  PubMed  Google Scholar 

  • Kirkland D, Aardema M, Henderson L, Muller L (2005) Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity. Mutat Res 584(1–2):1–256

    CAS  PubMed  Google Scholar 

  • Kirkland D, Kasper P, Muller L, Corvi R, Speit G (2008) Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop. Mutat Res 653(1–2):99–108

    Article  CAS  PubMed  Google Scholar 

  • Kirkland D, Kasper P, Martus HJ et al (2016) Updated recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests. M utat Res Genet Toxicol Environ Mutagen 795:7–30

    Article  CAS  Google Scholar 

  • Krewski D, Acosta D Jr, Andersen M et al (2010) Toxicity testing in the 21st century: a vision and a strategy. J Toxicol Environ Health B Crit Rev 13(2–4):51–138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Le Hegarat L, Dumont J, Josse R et al (2010) Assessment of the genotoxic potential of indirect chemical mutagens in HepaRG cells by the comet and the cytokinesis-block micronucleus assays. Mutagenesis 25(6):555–560

    Article  CAS  PubMed  Google Scholar 

  • Le Hegarat L, Mourot A, Huet S et al (2014) Performance of comet and micronucleus assays in metabolic competent HepaRG cells to predict in vivo genotoxicity. Toxicol Sci 138(2):300–309

    Article  CAS  PubMed  Google Scholar 

  • Luch A (2005) Nature and nurture—lessons from chemical carcinogenesis. Nat Rev Cancer 5(2):113–125

    Article  CAS  PubMed  Google Scholar 

  • MacGregor JT, Frotschl R, White PA et al (2015) IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure–response relationships and points of departure (PoDs). Mutat Res Genet Toxicol Environ Mutagen 783:55–65

    Article  CAS  PubMed  Google Scholar 

  • Marrone AK, Tryndyak V, Beland FA, Pogribny IP (2016) MicroRNA responses to the genotoxic carcinogens aflatoxin B1 and benzo[a]pyrene in human HepaRG cells. Toxicol Sci 149(2):496–502

    Article  CAS  PubMed  Google Scholar 

  • OECD (2015) Guidance document on revisions to OECD genetic toxicology test guidelines. OECD Workgroup of National Coordinators for Test 42 Guidelines (WNT) https://www.oecd.org/chemicalsafety/testing/Genetic%20Toxicology%20Guidance%20Document%20Aug%2031%202015.pdf. Accessed 12 Nov 2018

  • Quesnot N, Rondel K, Audebert M et al (2016) Evaluation of genotoxicity using automated detection of gammaH2AX in metabolically competent HepaRG cells. Mutagenesis 31(1):43–50

    CAS  PubMed  Google Scholar 

  • Robison TW, Jacobs A (2009) Metabolites in safety testing. Bioanalysis 1(7):1193–1200

    Article  CAS  PubMed  Google Scholar 

  • Sand S, Parham F, Portier CJ, Tice RR, Krewski D (2017) Comparison of points of departure for health risk assessment based on high-throughput screening data. Environ Health Perspect 125(4):623–633

    Article  CAS  PubMed  Google Scholar 

  • Sasaki YF, Nakamura T, Kawaguchi S (2007) What is better experimental design for in vitro comet assay to detect chemical genotoxicity. AATEX 14:499–504

    Google Scholar 

  • Severin I, Jondeau A, Dahbi L, Chagnon MC (2005) 2,4-Diaminotoluene (2,4-DAT)-induced DNA damage, DNA repair and micronucleus formation in the human hepatoma cell line HepG2. Toxicology 213(1–2):138–146

    Article  CAS  PubMed  Google Scholar 

  • Sison-Young RL, Mitsa D, Jenkins RE et al (2015) Comparative proteomic characterization of 4 human liver-derived single cell culture models reveals significant variation in the capacity for drug disposition, bioactivation, and detoxication. Toxicol Sci 147(2):412–424

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Skipper A, Sims JN, Yedjou CG, Tchounwou PB (2016) Cadmium chloride induces DNA damage and apoptosis of human liver carcinoma cells via oxidative stress. Int J Environ Res Public Health 13(1):88

    Article  CAS  PubMed Central  Google Scholar 

  • Sykora P, Witt KL, Revanna P et al (2018) Next generation high throughput DNA damage detection platform for genotoxic compound screening. Sci Rep 8(1):2771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tryndyak V, Kindrat I, Dreval K, Churchwell MI, Beland FA, Pogribny IP (2018) Effect of aflatoxin B1, benzo[a]pyrene, and methapyrilene on transcriptomic and epigenetic alterations in human liver HepaRG cells. Food Chem Toxicol 121:214–223

    Article  CAS  PubMed  Google Scholar 

  • Uhl M, Helma C, Knasmuller S (1999) Single-cell gel electrophoresis assays with human-derived hepatoma (HepG2) cells. Mutat Res 441(2):215–224

    Article  CAS  PubMed  Google Scholar 

  • Valentin-Severin I, Le Hegarat L, Lhuguenot JC, Le Bon AM, Chagnon MC (2003) Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays. Mutat Res 536(1–2):79–90

    Article  CAS  PubMed  Google Scholar 

  • VICH (2013) VICH topic GL23(R): Studies to evaluate the safety of residues of veterinary drugs in human food: Genotoxicity testing. https://www.ema.europa.eu/documents/scientific-guideline/international-cooperation-harmonisation-technical-requirements-registration-veterinary-medicinal_en-2.pdf. Accessed 12 Nov 2018

  • Westerink WM, Schoonen WG (2007) Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol In Vitro 21(8):1581–1591

    Article  CAS  PubMed  Google Scholar 

  • Wills JW, Long AS, Johnson GE et al (2016) Empirical analysis of BMD metrics in genetic toxicology part II: in vivo potency comparisons to promote reductions in the use of experimental animals for genetic toxicity assessment. Mutagenesis 31(3):265–275

    Article  CAS  PubMed  Google Scholar 

  • Wood DK, Weingeist DM, Bhatia SN, Engelward BP (2010) Single cell trapping and DNA damage analysis using microwell arrays. Proc Natl Acad Sci USA 107(22):10008–10013

    Article  PubMed  Google Scholar 

  • Xu J, Oda S, Yokoi T (2018) Cell-based assay using glutathione-depleted HepaRG and HepG2 human liver cells for predicting drug-induced liver injury. Toxicol In Vitro 48:286–301

    Article  CAS  PubMed  Google Scholar 

  • Zeilinger K, Freyer N, Damm G, Seehofer D, Knospel F (2016) Cell sources for in vitro human liver cell culture models. Exp Biol Med (Maywood) 241(15):1684–1698

    Article  CAS  Google Scholar 

  • Zeller A, Duran-Pacheco G, Guerard M (2017) An appraisal of critical effect sizes for the benchmark dose approach to assess dose–response relationships in genetic toxicology. Arch Toxicol 91(12):3799–3807

    Article  CAS  PubMed  Google Scholar 

  • Zhang R, Niu Y, Zhou Y (2010) Increase the cisplatin cytotoxicity and cisplatin-induced DNA damage in HepG2 cells by XRCC1 abrogation related mechanisms. Toxicol Lett 192(2):108–114

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

J.E.S. and K.D. were supported by appointments to the Postgraduate Research Program at the National Center for Toxicological Research (NCTR) administered by the Oak Ridge Institute for Science Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration (FDA). We greatly appreciate Dr. Lei Guo (DBT/NCTR) for generously providing HepG2 cells and thank Drs. Robert H. Heflich, Dayton Petibone, and Elvis-Yane Cuevas-Martinez for their critical review of this article.

Author information

Author notes

  1. Kostiantyn Dreval

    Present address: Department of Internal Medicine, Division of Molecular Medicine, Program in Cancer Genetics, Epigenetics and Genomics, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, 87131, USA

Affiliations

  1. Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR, 72079, USA

    Ji-Eun Seo, Nan Mei & Xiaoqing Guo

  2. Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR, 72079, USA

    Volodymyr Tryndyak, Qiangen Wu, Kostiantyn Dreval, Igor Pogribny & Matthew Bryant

  3. Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD, 20855, USA

    Tong Zhou

  4. Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, 20993, USA

    Timothy W. Robison

Authors
  1. Ji-Eun Seo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Volodymyr Tryndyak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qiangen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kostiantyn Dreval
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Igor Pogribny
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Matthew Bryant
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tong Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Timothy W. Robison
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nan Mei
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xiaoqing Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaoqing Guo.

Ethics declarations

Conflict of interest

There was no conflict of interest declared.

Disclaimer

The information in this paper is not a formal dissemination of information by the U.S. FDA and does not represent the agency position or policy.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 1244 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Seo, JE., Tryndyak, V., Wu, Q. et al. Quantitative comparison of in vitro genotoxicity between metabolically competent HepaRG cells and HepG2 cells using the high-throughput high-content CometChip assay. Arch Toxicol 93, 1433–1448 (2019). https://doi.org/10.1007/s00204-019-02406-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-019-02406-9

Keywords

  • High-throughput high-content
  • CometChip assay
  • HepaRG cells
  • HepG2 cells
  • Benchmark dose