Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Chemical toxicity testing in vitro using cytochrome P450–expressing cell lines, such as human CYP1B1

Nature Protocols volume 6, pages 677–688 (2011)Cite this article

Subjects

Abstract

This protocol describes how to use cytochrome P450–dependent monooxygenase (CYP)-expressing cell lines in toxicity testing of chemicals in vitro. Selected cells amenable to permanently grow in culture are genetically manipulated to stably express single CYP enzymes originating from any species of interest. This expression can be characterized by, for example, determining CYP mRNA content, CYP protein level (western blotting or in situ immunofluorescence) and CYP-mediated enzyme activity (substrate conversion assays). These cells can be used to determine substrate specificities and species differences, e.g., in the bioactivation of drugs. Once constructed, CYP-expressing cells can serve as a straightforward and reliable tool in toxicity testing and the corresponding assays could be adapted for high-throughput analysis. Using these cells, enzyme assays can be performed in a matter of hours. This protocol is exemplified with V79 fibroblasts from Chinese hamster (Cricetulus griseus), modified to express human cytochrome P450 1B1 (CYP1B1). These cells are characterized for their CYP1B1-linked properties by in situ immunofluorescence and their activity in the 7-ethoxyresorufin-O-deethylase enzyme assay. This is followed by an assay showing metabolic activation of the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene by CYP1B1, along with the toxicological endpoints of cytotoxicity and micronucleus formation.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Flowchart of the methods and the assays applied.
Figure 2: Toxic effects of DBP and its 11,12-diol enantiomers on CYP1B1-expressing V79 cells.
Figure 3: Postlabeling assay and DNA adduct formation in DBP metabolizing V79 cells.
Figure 4: Example of the analysis of DBP metabolites resulting from the catalytic activity of human CYP1B1 expressed in V79 cells (adapted from ref.

Similar content being viewed by others

References

  1. Contopoulos-Ioannidis, D.G., Alexiou, G.A., Gouvias, T.C. & Ioannidis, J.P. Medicine. Life cycle of translational research for medical interventions. Science 321, 1298–1299 (2008).

    Article  CAS  Google Scholar 

  2. Schoonen, W.G., Westerink, W.M. & Horbach, G.J. High-throughput screening for analysis of in vitro toxicity. In Molecular, Clinical and Environmental Toxicology Vol. 1 (ed. Luch, A.) 401–452 (Birkhäuser Publishing, Switzerland, 2009).

  3. Smith, D.A. & Schmid, E.F. Drug withdrawals and the lessons within. Curr. Opin. Drug Discov. Devel. 9, 38–46 (2006).

    CAS  PubMed  Google Scholar 

  4. Guengerich, F.P. Cytochrome P450s and other enzymes in drug metabolism and toxicity. AAPS J. 8, E101–111 (2006).

    Article  CAS  Google Scholar 

  5. Dekant, W. The role of biotransformation and bioactivation in toxicity. In Molecular, Clinical and Environmental Toxicology Vol. 1 (ed. Luch, A.) 57–86 (Birkhäuser Publishing, Switzerland, 2009).

  6. Hart, S.N. & Zhong, X.B. P450 oxidoreductase: genetic polymorphisms and implications for drug metabolism and toxicity. Expert Opin. Drug Metab. Toxicol. 4, 439–452 (2008).

    Article  CAS  Google Scholar 

  7. Nebert, D.W. & Dalton, T.P. The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis. Nat. Rev. Cancer 6, 947–960 (2006).

    Article  CAS  Google Scholar 

  8. Guengerich, F.P. Cytochrome P450 and chemical toxicology. Chem. Res. Toxicol. 21, 70–83 (2008).

    Article  CAS  Google Scholar 

  9. Nelson, D.R. et al. Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 14, 1–18 (2004).

    Article  CAS  Google Scholar 

  10. Ingelman-Sundberg, M. Pharmacogenetics of cytochrome P450 and its applications in drug therapy: the past, present and future. Trends Pharmacol. Sci. 25, 193–200 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Doehmer, J. et al. Cytochrome P450 mediated reactions studied in genetically engineered V79 Chinese hamster cells. Pharmacogenetics 5, S91–96 (1995).

    Article  CAS  Google Scholar 

  13. Schober, W. et al. On the species-specific biotransformation of dibenzo[a,l]pyrene. Chem. Biol. Interact. 161, 37–48 (2006).

    Article  CAS  Google Scholar 

  14. Doehmer, J. et al. Building a biotechnology platform for metabolism studies in pharmacology and toxicology. In Progress in the Reduction, Refinement and Replacement of Animal Experimentation (eds. Balls, M., van Zeller, A.M. & Halders, M.E.) 139–146 (Elsevier Science, 2000).

  15. Parikh, A., Gillam, E.M. & Guengerich, F.P. Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15, 784–788 (1997).

    Article  CAS  Google Scholar 

  16. Pritchard, M.P., McLaughlin, L. & Friedberg, T. Establishment of functional human cytochrome P450 monooxygenase systems in Escherichia coli. Methods Mol. Biol. 320, 19–29 (2006).

    CAS  PubMed  Google Scholar 

  17. Luch, A. et al. Stable expression of human cytochrome P450 1B1 in V79 Chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo[a,l]pyrene. Chem. Res. Toxicol. 11, 686–695 (1998).

    Article  CAS  Google Scholar 

  18. Luch, A. et al. Metabolic activation of dibenzo[a,l]pyrene by human cytochrome P450 1A1 and P450 1B1 expressed in V79 Chinese hamster cells. Chem. Res. Toxicol. 12, 353–364 (1999).

    Article  CAS  Google Scholar 

  19. Krahn, D.F. Chinese hamster cell mutagenesis: a comparison of the CHO and V79 systems. Ann. NY Acad. Sci. 407, 231–238 (1983).

    Article  CAS  Google Scholar 

  20. Liu, Y. & Glatt, H. Human cytochrome P450 2E1 and sulfotransferase 1A1 coexpressed in Chinese hamster V79 cells enhance spontaneous mutagenesis. Environ. Mol. Mutagen. 51, 23–30 (2010).

    CAS  PubMed  Google Scholar 

  21. Teubner, W., Meinl, W. & Glatt, H. Stable expression of rat sulfotransferase 1B1 in V79 cells: activation of benzylic alcohols to mutagens. Carcinogenesis 23, 1877–1884 (2002).

    Article  CAS  Google Scholar 

  22. Luch, A. et al. The K-region trans-8,9-diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1. Cancer Res. 59, 4603–4609 (1999).

    CAS  PubMed  Google Scholar 

  23. Doehmer, J. Predicting drug metabolism-dependent toxicity for humans with a genetically engineered cell battery. Altern. Lab. Anim. 34, 561–575 (2006).

    CAS  PubMed  Google Scholar 

  24. Coecke, S. et al. Metabolism: a bottleneck in in vitro toxicological test development. The report and recommendations of ECVAM workshop 54. Altern. Lab. Anim. 34, 49–84 (2006).

    CAS  PubMed  Google Scholar 

  25. Borenfreund, E. & Puerner, J.A. A simple quantitative procedure using monolayer culture for toxicity assays. J. Tissue Cult. Meth. 9, 7–9 (1984).

    Article  Google Scholar 

  26. Ralston, S.L., Seidel, A., Luch, A., Platt, K.L. & Baird, W.M. Stereoselective activation of dibenzo[a,l]pyrene to (−)-anti(11R,12S,13S,14R)- and (+)-syn(11S,12R,13S,14R)-11,12-diol-13,14-epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary carcinoma cell line MCF-7. Carcinogenesis 16, 2899–2907 (1995).

    Article  CAS  Google Scholar 

  27. Phillips, D.H. & Arlt, V.M. The 32P-postlabeling assay for DNA adducts. Nat. Protoc. 2, 2772–2781 (2007).

    Article  CAS  Google Scholar 

  28. Seidel, A. et al. Regio- and stereoselectivity in the metabolism of benzo[c]phenanthrene mediated by genetically engineered V79 Chinese hamster cells expressing rat and human cytochromes P450. Environ. Toxicol. Pharmacol. 5, 179–196 (1998).

    Article  CAS  Google Scholar 

  29. Luch, A., Glatt, H., Platt, K.L., Oesch, F. & Seidel, A. Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo[a,l]pyrene. Carcinogenesis 15, 2507–2516 (1994).

    Article  CAS  Google Scholar 

  30. Luch, A., Seidel, A., Glatt, H. & Platt, K.L. Metabolic activation of the (+)-S,S- and (−)-R,R-enantiomers of trans-11,12-dihydroxy-11,12-dihydro-dibenzo[a,l]pyrene: stereoselectivity, DNA adduct formation, and mutagenicity in Chinese hamster V79 cells. Chem. Res. Toxicol. 10, 1161–1170 (1997).

    Article  CAS  Google Scholar 

  31. Sutter, T.R. et al. Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2. J. Biol. Chem. 269, 13092–13099 (1994).

    CAS  PubMed  Google Scholar 

  32. Schneider, A. et al. Stable expression of human cytochrome P450 3A4 in conjunction with human NADPH-cytochrome P450 oxidoreductase in V79 Chinese hamster cells. Arch. Biochem. Biophys. 332, 295–304 (1996).

    Article  CAS  Google Scholar 

  33. Ford, D.K. & Yerganian, G. Observations on the chromosomes of Chinese hamster cells in tissue culture. J. Natl. Cancer Inst. 21, 393–425 (1958).

    CAS  PubMed  Google Scholar 

  34. Graham, F.L. & van der Eb, A.J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467 (1973).

    Article  CAS  Google Scholar 

  35. Doehmer, J. et al. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1. Proc. Natl. Acad. Sci. USA 85, 5769–5773 (1988).

    Article  CAS  Google Scholar 

  36. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951).

    CAS  Google Scholar 

  37. Organisation for Economic Cooperation and Development (OECD). In vitro 3T3 NRU phototoxicity test. OECD guideline 432 for testing of chemicals 15 http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECDtg432.pdf (13 April 2004).

  38. Schmalix, W.A. et al. Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells. Eur. J. Pharmacol. 293, 123–131 (1995).

    Article  CAS  Google Scholar 

  39. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, 1989).

  40. Luch, A. et al. Metabolic activation of dibenzo[a,l]pyrene by cytochrome P450 enzymes to stable DNA adducts occurs exclusively through the formation of the (−)-trans-(11R,12R)-diol. Polycyl. Aromat. Compds. 21, 87–98 (2000).

    Article  CAS  Google Scholar 

  41. Luch, A. On the impact of the molecule structure in chemical carcinogenesis. In Molecular, Clinical and Environmental Toxicology, Vol. 1 (ed. Luch, A.) 151–179 (Birkhäuser Publishing, Switzerland,, 2009).

  42. Engst, W., Landsiedel, R., Hermersdorfer, H., Doehmer, J. & Glatt, H. Benzylic hydroxylation of 1-methylpyrene and 1-ethylpyrene by human and rat cytochromes P450 individually expressed in V79 Chinese hamster cells. Carcinogenesis 20, 1777–1785 (1999).

    Article  CAS  Google Scholar 

  43. Philip, P.A. et al. Use of V79 cells with stably transfected cytochrome P450 cDNAs in studying the metabolism and effects of cytotoxic drugs. Cancer Chemother. Pharmacol. 43, 59–67 (1999).

    Article  CAS  Google Scholar 

  44. Frei, E., Bieler, C.A., Arlt, V.M., Wiessler, M. & Stiborova, M. Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes. Biochem. Pharmacol. 64, 289–295 (2002).

    Article  CAS  Google Scholar 

  45. Gabelova, A., Farkasova, T., Bacova, G. & Robichova, S. Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450. Mutat. Res. 517, 135–145 (2002).

    Article  CAS  Google Scholar 

  46. Arlt, V.M. et al. Activation of 3-nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells. Int. J. Cancer 105, 583–592 (2003).

    Article  CAS  Google Scholar 

  47. Bieler, C.A., Arlt, V.M., Wiessler, M. & Schmeiser, H.H. DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes. Cancer Lett. 200, 9–18 (2003).

    Article  CAS  Google Scholar 

  48. Krebsfaenger, N., Murdter, T.E., Zanger, U.M., Eichelbaum, M.F. & Doehmer, J. V79 Chinese hamster cells genetically engineered for polymorphic cytochrome P450 2D6 and their predictive value for humans. ALTEX 20, 143–154 (2003).

    PubMed  Google Scholar 

  49. Glatt, H. et al. Bioactivation of the heterocyclic aromatic amine 2-amino-3-methyl-9H-pyrido [2,3-b]indole (MeAαC) in recombinant test systems expressing human xenobiotic-metabolizing enzymes. Carcinogenesis 25, 801–807 (2004).

    Article  CAS  Google Scholar 

  50. Glatt, H., Schneider, H. & Liu, Y. V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals. Mutat. Res. 580, 41–52 (2005).

    Article  CAS  Google Scholar 

  51. Carmo, H. et al. CYP2D6 increases toxicity of the designer drug 4-methylthioamphetamine (4-MTA). Toxicology 229, 236–244 (2007).

    Article  CAS  Google Scholar 

  52. Liu, Y. & Glatt, H. Mutagenicity of N-nitrosodiethanolamine in a V79-derived cell line expressing two human biotransformation enzymes. Mutat. Res. 643, 64–69 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

A.L. gratefully acknowledges the help and advice of former colleagues at the Universities of Mainz, Germany (K.-L. Platt and A. Seidel), and Munich, Germany (H. Greim, J. Doehmer, A. Schneider, V. Soballa, W. Schober and N. Krebsfänger), as well as colleagues at the Cancer Center of Purdue University, Indiana, USA (W.M. Baird, S.L. Ralston, S.L. Coffing, B. Mahadevan and V. Melendez-Colon). Most of the work upon which this protocol is based was performed in the laboratories of J. Doehmer (Munich) and W.M. Baird (Purdue).

Author information

Author notes

  1. Robert Landsiedel, Tewes Tralau & Andreas Luch

    Present address: Present addresses: Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany (R.L.); German Federal Institute for Risk Assessment, Department of Product Safety, Berlin, Germany (T.T. and A.L.).,

Authors and Affiliations

  1. Department of Nutritional Toxicology, German Institute of Human Nutrition (DIfE), Nuthetal, Germany

    Robert Landsiedel

  2. Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany

    Eric Fabian

  3. University of Manchester, Manchester Interdisciplinary Biocentre, Manchester, UK

    Tewes Tralau

  4. Massachusetts Institute of Technology, Center for Cancer Research, Cambridge, Massachusetts, USA

    Andreas Luch

Authors
  1. Robert Landsiedel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric Fabian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tewes Tralau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andreas Luch
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.L. produced the compounds and its enantiomerically pure derivatives, contributed to the characterization of the transgenic V79 cell line and the design and performance of the assays described and conducted the DNA binding and adduct analyses; R.L. and E.F. provided additional experimental data, and T.T. produced the manuscript.

Corresponding author

Correspondence to Andreas Luch.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Landsiedel, R., Fabian, E., Tralau, T. et al. Chemical toxicity testing in vitro using cytochrome P450–expressing cell lines, such as human CYP1B1. Nat Protoc 6, 677–688 (2011). https://doi.org/10.1038/nprot.2011.316

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.316

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research