Skip to main content
SpringerLink
Log in

Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin 7-Hydroxylation

  • Protocol
Cytochrome P450 Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 320))

Abstract

A spectrofluorometric method is described for the determination of CYP2A6-catalyzed coumarin 7-hydroxylation. Following acidification of the reaction mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and assayed using an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This assay is applicable to enzymatic studies of cDNA-expressed CYP2A6 and can be used to monitor coumarin 7-hydroxylation activity in microsomes prepared from liver and other tissues and in isolated hepatocytes and cultured cells that express this cytochrome P450 activity.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Maurice, M., Emiliani, S., Dalet-Beluche, I., Derancourt, J., and Lange, R. (1991) Isolation and characterization of a cytochrome P450 of the IIA subfamily from human liver microsomes. Eur. J. Biochem. 200, 511–517.

    Article  CAS  PubMed  Google Scholar 

  2. Yun, C. H., Shimada, T., and Guengerich, F. P. (1991) Purification and characterization of human liver microsomal cytochrome P-450 2A6. Mol. Pharmacol. 40, 679–685.

    CAS  PubMed  Google Scholar 

  3. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther. 270, 414–423.

    CAS  PubMed  Google Scholar 

  4. Pearce, R., Greenway, D., and Parkinson, A. (1992) Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation. Arch. Biochem. Biophys. 298, 211–225.

    Article  CAS  PubMed  Google Scholar 

  5. Imaoka, S., Yamada, T., Hiroi, T., et al. (1996) Multiple forms of human P450 expressed in Saccharomyces cerevisiae: systematic characterization and comparison with those of the rat. Biochem. Pharmacol. 51, 1041–1050.

    Article  CAS  PubMed  Google Scholar 

  6. Pelkonen, O., Rautio, A., Raunio, H., and Pasanen, M. (2000) CYP2A6: a human coumarin 7-hydroxylase. Toxicology 144, 139–147.

    Article  CAS  PubMed  Google Scholar 

  7. Le Gal, A., Dreano, Y., Lucas, D., and Berthou, F. (2003) Diversity of selective environmental substrates for human cytochrome P450 2A6: alkoxyethers, nicotine, coumarin, N-nitrosodiethylamine, and N-nitrosobenzylmethylamine. Toxicol. Lett. 144, 77–91.

    Article  PubMed  Google Scholar 

  8. Dalet-Beluche, I., Boulenc, X., Fabre, G., Maurel, P., and Bonfils, C. (1992) Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes: cross reactivity with human forms. Eur. J. Biochem. 204, 641–648.

    Article  CAS  PubMed  Google Scholar 

  9. Messina, E. S., Tyndale, R. F., and Sellers, E. M. (1997) A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes. J. Pharmacol. Exp. Ther. 282, 1608–1614.

    CAS  PubMed  Google Scholar 

  10. Gemma, S., Vittozzi, L., and Testai, E. (2003) Metabolism of chloroform in the human liver and identification of the competent P450s. Drug Metab. Dispos. 31, 266–274.

    Article  CAS  PubMed  Google Scholar 

  11. Waxman, D. J., Lapenson, D. P., Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Korzekwa, K. (1991) Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s. Arch. Biochem. Biophys. 290, 160–166.

    Article  CAS  PubMed  Google Scholar 

  12. Sai, Y., Yang, T. J., Krausz, K. W., Gonzalez, F. J., and Gelboin, H. V. (1999) An inhibitory monoclonal antibody to human cytochrome P450 2A6 defines its role in the metabolism of coumarin, 7-ethoxycoumarin and 4-nitroanisole in human liver. Pharmacogenetics 9, 229–237.

    CAS  PubMed  Google Scholar 

  13. Draper, A. J., Madan, A., and Parkinson, A. (1997) Inhibition of coumarin 7-hydroxylase activity in human liver microsomes. Arch. Biochem. Biophys. 341, 47–61.

    Article  CAS  PubMed  Google Scholar 

  14. Pelkonen, O., Sotaniemi, E. A., and Ahokas, J. T. (1985) Coumarin 7-hydroxylase activity in human liver microsomes: properties of the enzyme and interspecies comparisons. Br. J. Clin. Pharmacol. 19, 59–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yamazaki, H., Inui, Y., Yun, C. H., Mimura, M., Guengerich, F. P., and Shimada, T. (1992) Cytochrome P450 2E1 and 2A6 enzymes as major catalyts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis 13, 1789–1794.

    Article  CAS  PubMed  Google Scholar 

  16. Chang, T. K. H., Gonzalez, F. J., and Waxman, D. J. (1994) Evaluation of triacetyloleandomycin, α-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450. Arch. Biochem. Biophys. 311, 437–442.

    Article  CAS  PubMed  Google Scholar 

  17. Koenigs, L. L., Peter, R. M., Thompson, S. J., Rettie, A. E., and Trager, W. F. (1997) Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen. Drug Metab. Dispos. 25, 1407–1415.

    CAS  PubMed  Google Scholar 

  18. Waxman, D. J. and Walsh, C. (1982) Phenobarbital-induced rat liver cytochrome P450: purification and characterization of two closely related isozymic forms. J. Biol. Chem. 257, 10,446–10,457.

    CAS  PubMed  Google Scholar 

  19. Greenlee, W. F. and Poland, A. (1978) An improved assay of 7-ethoxycoumarin O-deethylase activity: induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8,-tetrachlorodibenzo-p-dioxin. J. Pharmacol. Exp. Ther. 205, 596–605.

    CAS  PubMed  Google Scholar 

  20. Yamazaki, H., Tanaka, M., and Shimada, T. (1999) High sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes. J. Chromatogr. B 721, 13–19.

    Article  CAS  Google Scholar 

  21. Walsky, R. L. and Obach, R. S. (2004) Validated assays for human cytochrome P450 activities. Drug Metab. Dispos. 32, 647–660.

    Article  CAS  PubMed  Google Scholar 

  22. Tan, Y., Patten, C. J., Smith, T., and Yang, C. S. (1997) Competitive interactions between cytochromes P450 2A6 and 2E1 for NADPH-cytochrome P450 oxidoreductase in the microsomal membranes produced by a baculovirus expression system. Arch. Biochem. Biophys. 342, 82–91

    Article  CAS  PubMed  Google Scholar 

  23. Crespi, C. L., Penman, B. W., Leakey, J. A. E., et al. (1990) Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1. Carcinogenesis 11, 1293–1300.

    Article  CAS  PubMed  Google Scholar 

  24. Pearce, R. E., McIntyre, C. J., Madan, A., et al. (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch. Biochem. Biophys. 331, 145–169.

    Article  CAS  PubMed  Google Scholar 

  25. Chen, L., Buters, J. T. M., Hardwick, J. P., et al. (1997) Coexpression of cytochrome P450 2A6 and human NADPH-P450 oxidoreductase in the baculovirus system. Drug Metab. Dispos. 25, 399–405.

    CAS  PubMed  Google Scholar 

  26. Donato, M. T., Jimenez, N., Castell, J. V., and Gomez-Lechon, M. J. (2004) Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab. Dispos. 32, 699–706.

    Article  CAS  PubMed  Google Scholar 

  27. Chang, T. K. H., Weber, G. F., Crespi, C. L., and Waxman, D. J. (1993) Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Res. 53, 5629–5637.

    CAS  PubMed  Google Scholar 

  28. Prough, R. A., Burke, M. D., and Mayer, R. T. (1978) Direct fluorometric methods for measuring mixed-function oxidase activity. Methods Enzymol. 52, 372–377.

    Article  CAS  PubMed  Google Scholar 

  29. Chang, T. K. H. and Waxman, D. J. (1996) The CYP2A subfamily, in Cytochromes P450: Metabolic and Toxicological Aspects. (Ioannides, C., ed.), CRC Press, Boca Raton, FL, pp. 99–134.

    Google Scholar 

  30. Chauret, N., Gauthier, A., and Nicoll-Griffith, D. A. (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab. Dispos. 26, 1–4.

    CAS  PubMed  Google Scholar 

  31. Busby, W. F. Jr., Ackermann, J. M., and Crespi, C. L. (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab. Dispos. 27, 246–249.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the National Institutes of Health (grant CA49248) and the Canadian Institutes of Health Research (grant MOP-42385).

Author information

Authors and Affiliations

  1. Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA, USA

    David J. Waxman

  2. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada

    Thomas K. H. Chang

Authors
  1. David J. Waxman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas K. H. Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. School of Biological Sciences, Queen Mary University of London, London, UK

    Ian R. Phillips

  2. Department of Biochemistry and Molecular Biology, University College London, London, UK

    Elizabeth A. Shephard

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Waxman, D.J., Chang, T.K.H. (2006). Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin 7-Hydroxylation. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-998-2:91

Download citation

  • DOI: https://doi.org/10.1385/1-59259-998-2:91

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-441-8

  • Online ISBN: 978-1-59259-998-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation