Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 With 7-Ethoxyresorufin as Substrate

  • Thomas K. H. Chang
  • David J. Waxman
Part of the Methods in Molecular Biology book series (MIMB, volume 320)

Abstract

Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYP1B1. The formation of the enzymatic product, resorufin, is monitored continuously by fluorescence using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. This method can be applied to assay P450-catalyzed formation of resorufin from other alkoxyresorufins, such as 7-methoxyresorufin, 7-benzyloxyresorufin, and 7-pentoxyresorufin. It can also be used to assay 7-ethoxyresorufin O-dealkylation activity in isolated hepatocytes and cultured cells that express this P450 activity.

Key Words

Cytochrome P450 CYP1A1 CYP1A2 CYP1B1 7-ethoxyresorufin 7-ethoxyresorufin O-dealkylation 7-methoxyresorufin, 7-benzyloxyresorufin 7-pentoxyresorufin 

Notes

Acknowledgments

This work was supported in part by the Canadian Institutes of Health Research (grant MOP-42385) and by the National Institutes of Health (grant 5 P42 ES07381).

References

  1. 1.
    Nelson, D. R., Zeldin, D. C., Hoffman, S. M. G., Maltais, L. J., Wain, H. M., and Nebert, D. W. (2004) 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.CrossRefPubMedGoogle Scholar
  2. 2.
    Murray, B. P., Edwards, R. J., Murray, S., Singleton, A. M., Davies, D. S., and Boobis, A. R. (1993) Human hepatic CYP1A1 and CYP1A2 content, determined with specific antipeptide antibodies, correlates with the mutagenic activation of PhIP. Carcinogenesis 14, 585–592.CrossRefPubMedGoogle Scholar
  3. 3.
    Schweikl, H., Taylor, J. A., Kitareewan, S., Linko, P., Nagorney, D., and Goldstein, J. A. (1993) Expression of CYP1A1 and CYP1A2 genes in human liver. Pharmacogenetics 3, 239–249.CrossRefPubMedGoogle Scholar
  4. 4.
    Wheeler, C. W., Park, S. S., and Guenthner, T. M. (1990) Immunochemical analysis of a cytochrome P-450IA1 homologue in human lung microsomes. Mol. Pharmacol. 38, 634–643.PubMedGoogle Scholar
  5. 5.
    Shimada, T., Yun, C. H., Yamazaki, H., Gautier, J. C., Beaune, P. H., and Guengerich, F. P. (1992) Characterization of human lung microsomal cytochrome P-450 1A1 and its role in the oxidation of chemical carcinogens. Mol. Pharmacol. 41, 856–864.PubMedGoogle Scholar
  6. 6.
    Chang, T. K. H., Chen, J., Pillay, V., Ho, J. Y., and Bandiera, S. M. (2003) Real-time polymerase chain reaction analysis of CYP1B1 gene expression in human liver. Toxicol. Sci. 71, 11–19.CrossRefPubMedGoogle Scholar
  7. 7.
    Shimada, T., Hayes, C. L., Yamazaki, H., et al. (1996) Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res. 56, 2979–2984.PubMedGoogle Scholar
  8. 8.
    Sesardic, D., Boobis, A. R., Edwards, R. J., and Davies, D. S. (1988) A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O-deethylation of phenacetin and is inducible by cigarette smoking. Br. J. Clin. Pharmacol. 26, 363–372.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dohr, O., Vogel, C., and Abel, J. (1995) Different response of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-sensitive genes in human breast cancer MCF-7 and MDA-MB 231 cells. Arch. Biochem. Biophys. 321, 405–412.CrossRefPubMedGoogle Scholar
  10. 10.
    Kress, S. and Greenlee, W. F. (1997) Cell-specific regulation of human CYP1A1 and CYP1B1 genes. Cancer Res. 57, 1264–1269.PubMedGoogle Scholar
  11. 11.
    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.CrossRefPubMedGoogle Scholar
  12. 12.
    Shimada, T., Gillam, E. M. J., Sutter, T. R., Strickland, P. T., Guengerich, F. P., and Yamazaki, H. (1997) Oxidation of xenobiotics by recombinant human cytochrome P450 1B1. Drug Metab. Dispos. 29, 617–622.Google Scholar
  13. 13.
    Eugster, H. P., Probst, M., Wurgler, F. E., and Sengstag, C. (1993) Caffeine, estradiol, and progesterone interact with human CYP1A1 and CYP1A2. Evidence from cDNA-directed expression in Saccharomyces cerevisiae. Drug Metab. Dispos. 21, 43–49.PubMedGoogle Scholar
  14. 14.
    Guo, Z., Gillam, E. M. J., Ohmori, S., Tukey, R. H., and Guengerich, F. P. (1994) Expression of modified human cytochrome P450 1A1 in Escherichia coli: effects of 5′ substitution, stabilization, purification, spectral characterization, and catalytic properties. Arch. Biochem. Biophys. 312, 436–446.CrossRefPubMedGoogle Scholar
  15. 15.
    Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R. (1996) 17β-Estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc. Natl. Acad. Sci. USA 93, 9776–9781.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chang, T. K. H., Chen, J., and Lee, W. B. K. (2001) Differential inhibition and inactivation of human CYP1 enzymes by trans-resveratrol: evidence for mechanism-based inactivation of CYP1A2. J. Pharmacol. Exp. Ther. 299, 874–882.PubMedGoogle Scholar
  17. 17.
    Bourdi, M., Larrey, D., Nataf, J., et al. (1990) Anti-liver endoplasmic reticulum autoantibodies are directed against human cytochrome P-4501A2: a specific marker of dihydralazineinduced hepatitis. J. Clin. Invest. 85, 1967–1973.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Burke, M. D., Thompson, S., Weaver, R. J., Wolf, C. R., and Mayer, R. T. (1994) Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem. Pharmacol. 48, 923–936.CrossRefPubMedGoogle Scholar
  19. 19.
    Burke, M. D. and Mayer, R. T. (1983) Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins). Chem. Biol. Interact. 45, 243–258.CrossRefPubMedGoogle Scholar
  20. 20.
    McManus, M. E., Burgess, W. M., Veronese, M. E., Huggett, A., Quattrochi, L. C., and Tukey, R. H. (1990) Metabolism of 2-acetylaminofluorene and benzo[a]pyrene and activation of food-derived heterocyclic amine mutagens by human cytochromes P-450. Cancer Res. 50, 3367–3376.PubMedGoogle Scholar
  21. 21.
    Lee, Q. P., Fantel, A. G., and Juchau, M. R. (1991) Human embryonic cytochrome P450s: phenoxazone ethers as probes for expression of functional isoforms during organogenesis. Biochem. Pharmacol. 42, 2377–2385.CrossRefPubMedGoogle Scholar
  22. 22.
    Waxman, D. J., Lapenson, D. P., Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Korzekwa, K. (1991) Steroid hormone hydroxylase specificities of eleven cDNAexpressed human cytochrome P450s. Arch. Biochem. Biophys. 290, 160–166.CrossRefPubMedGoogle Scholar
  23. 23.
    Stevens, J. C., Shipley, L. A., Cashman, J. R., VandenBranden, M., and Wrighton, S. A. (1993) Comparison of human and rhesus monkey in vitro phase I and phase II hepatic drug metabolism activities. Drug Metab. Dispos. 21, 753–760.PubMedGoogle Scholar
  24. 24.
    Pohl, R. J. and Fouts, J. R. (1980) A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Anal. Biochem. 107, 150–155.CrossRefPubMedGoogle Scholar
  25. 25.
    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.CrossRefPubMedGoogle Scholar
  26. 26.
    Donato, M. T., Gomez-Lechon, M. J., and Castell, J. V. (1993) A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal. Biochem. 213, 29–33.CrossRefPubMedGoogle Scholar
  27. 27.
    Kennedy, S. W., Lorenzen, A., James, C. A., and Collins, B. T. (1993) Ethoxyresorufin O-deethylase and porphyrin analysis in chicken embryo hepatocyte cultures with a fluorescence multiwell plate reader. Anal. Biochem. 211, 102–112.CrossRefPubMedGoogle Scholar
  28. 28.
    Buters, J. T., Shou, M., Hardwick, J. P., Korzekwa, K. R., and Gonzalez, F. J. (1995) cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus: purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification. Drug Metab. Dispos. 23, 696–701.PubMedGoogle Scholar
  29. 29.
    Sandhu, P., Guo, Z., Baba, T., Martin, M. V., Tukey, R. H., and Guengerich, F. P. (1994) Expression of modified human cytochrome P450 1A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme. Arch. Biochem. Biophys. 309, 168–177.CrossRefPubMedGoogle Scholar
  30. 30.
    Burke, M. D. and Mayer, R. T. (1974) Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Dispos. 2, 583–588.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Thomas K. H. Chang
    • 1
  • David J. Waxman
    • 2
  1. 1.Faculty of Pharmaceutical SciencesUniversity of British ColumbiaVancouverCanada
  2. 2.Division of Cell and Molecular Biology, Department of BiologyBoston UniversityBostonUSA

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