Skip to main content
Log in

Comparative genotoxicity investigation using comet and gammaH2AX assays for screening of genotoxicants in HepG2 human hepatoma cells

  • Published:
Toxicology and Environmental Health Sciences Aims and scope Submit manuscript

Abstract

As public interest in safety has increased the toxicity evaluation of chemicals become more important. In this study, the DNA-damaging effect of genotoxicants was examined in HepG2 cell line originated from human hepatocellular carcinoma by widely used genotoxicity assays: the comet assay and gammaH2AX immunostaining. Four different direct/indirect genotoxicants were tested in dose-/time-dependent manner. The comet assay and the gammaH2AX immunostaining enables detection of DNA damages in the form of DNA strand breaks with different sensitivity. Therefore, the combination of comet assay and gammaH2AX immunostaining will be complementary tool for evaluation of various forms and degree of DNA damage. Our result also suggested that HepG2 cells could be a suitable model for assessing the genotoxicity of various mutagens and for determining the lowest genotoxic concentration. Further analysis using a larger number of chemicals is warranted to determine the sensitivity and the specificity of HepG2 with in vitro genotoxicity test.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Khoury, L., Zalko, D. & Audebert, M. Complementarity of phosphorylated histones H2AX and H3 quantification in different cell lines for genotoxicity screening. Arch. Toxicol., doi:10.1007/s00204-015-1599-1 (2015).

    Google Scholar 

  2. Kirkland, D. Improvements in the reliability of in vitro genotoxicity testing. Expert Opin Drug Metab. Toxicol. 7, 1513–1520, doi:10.1517/17425255.2011.627855 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. Collins, A. R. et al. The comet assay: topical issues. Mutagenesis 23, 143–151, doi:10.1093/mutage/gem051 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Audebert, M., Dolo, L., Perdu, E., Cravedi, J. P. & Zalko, D. Use of the gammaH2AX assay for assessing the genotoxicity of bisphenol A and bisphenol F in human cell lines. Arch. Toxicol. 85, 1463–1473 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Ostling, O. & Johanson, K. J. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123, 291–298 (1984).

    Article  CAS  PubMed  Google Scholar 

  6. Brendler-Schwaab, S., Hartmann, A., Pfuhler, S. & Speit, G. The in vivo comet assay: use and status in genotoxicity testing. Mutagenesis 20, 245–254 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Collins, A. R. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol. Biotechnol. 26, 249–261 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Singh, N. P., McCoy, M. T., Tice, R. R. & Schneider, E. L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191 (1988).

    Article  CAS  PubMed  Google Scholar 

  9. Zhou, C. et al. DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors. Mutat. Res. 604, 8–18 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pilch, D. R. et al. Characteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem. Cell Biol. 81, 123–129 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Paull, T. T. et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Wang, H. et al. Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors. J. Cell Physiol. 202, 492–502 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Furuta, T. et al. Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J. Biol. Chem. 278, 20303–20312 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Ward, I. M., Minn, K., Jorda, K. G. & Chen, J. Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J. Biol. Chem. 278, 19579–19582 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Rothkamm, K. & Lobrich, M. Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc. Natl. Acad. Sci. U.S.A. 100, 5057–5062 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bosco, E. E. et al. RB signaling prevents replicationdependent DNA double-strand breaks following genotoxic insult. Nucleic Acids Res. 32, 25–34 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ha, L., Ceryak, S. & Patierno, S. R. Generation of S phase-dependent DNA double-strand breaks by Cr(VI) exposure: involvement of ATM in Cr(VI) induction of gamma-H2AX. Carcinogenesis 25, 2265–2274 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Huang, X. et al. Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin. Cytometry A 58, 99–110 (2004).

    Article  PubMed  Google Scholar 

  19. Banath, J. P. & Olive, P. L. Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks. Cancer Res. 63, 4347–4350 (2003).

    CAS  PubMed  Google Scholar 

  20. Taneja, N. et al. Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy. J. Biol. Chem. 279, 2273–2280 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Guillouzo, A. & Guguen-Guillouzo, C. Evolving concepts in liver tissue modeling and implications for in vitro toxicology. Expert. Opin. Drug Metab. Toxicol. 4, 1279–1294 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Knasmuller, S. et al. Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens. Mutat. Res. 402, 185–202 (1998).

    Article  CAS  PubMed  Google Scholar 

  23. Uhl, M., Helma, C. & Knasmuller, S. Single-cell gel electrophoresis assays with human-derived hepatoma (Hep G2) cells. Mutat. Res. 441, 215–224 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Valentin-Severin, I. et al. Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays. Mutat. Res. 536, 79–90 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Tice, R. R. et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen 35, 206–221 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Alexandrov, K. et al. CYP1A1 and GSTM1 genotypes affect benzo[a]pyrene DNA adducts in smokers’ lung: comparison with aromatic/hydrophobic adduct formation. Carcinogenesis 23, 1969–1977 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Kim, J. H. et al. Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1. Carcinogenesis 19, 1847–1853 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. Uno, S. et al. Oral exposure to benzo[a]pyrene in the mouse: Detoxication by inducible cytochrome P450 is more important than metabolic activation. Mol. Pharmacol. 65, 1225–1237 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  30. Koedrith, P. & Seo, Y. R. Enhancement of the efficacy of mitomycin C-mediated apoptosis in human colon cancer cells with RNAi-based thioredoxin reductase 1 deficiency. Exp. Ther. Med. 2, 873–878 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Young Rok Seo.

Additional information

These authors contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, Y.J., Koedrith, P., Kim, H.S. et al. Comparative genotoxicity investigation using comet and gammaH2AX assays for screening of genotoxicants in HepG2 human hepatoma cells. Toxicol. Environ. Health Sci. 8, 68–78 (2016). https://doi.org/10.1007/s13530-016-0263-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13530-016-0263-3

Keywords

Navigation