Abstract
The cytochrome P450 enzyme CYP2B6 plays an important role in the metabolism of structurally diverse drugs, including the anticancer drug cyclophosphamide, and may be an important determinant of clinical responses to these agents. A spectrofluorometric method is described for the determination of CYP2B6-catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity in human liver microsomes. The specificity of this method for CYP2B6 is increased by the use of inhibitory antibodies to CYP1A2, CYP2C, and CYP2E1, which block the contributions of these higher-K m enzymes to human liver microsomal metabolism of 7-ethoxy-4-trifluoromethylcoumarin. The enzymatic product, 7-hydroxy-4-trifluoromethylcoumarin, is monitored by fluorescence using an excitation wavelength of 410 nm and an emission wavelength of 510 nm. This approach can be modified to assay the catalytic activity of cDNA-expressed CYP2B6.
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References
Mimura, M., Baba, T., Yamazaki, H., et al. (1993) Characterization of cytochrome P-450 2B6 in human liver microsomes. Drug Metab. Dispos. 21, 1048–1056.
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.
Yamano, S., Nhamburo, P. T., Aoyama, T., et al. (1989) cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver. Biochemistry 28, 7340–7348.
Code, E. L., Crespi, C. L., Penman, B. W., Gonzalez, F. J., Chang, T. K. H., and Waxman, D. J. (1997) Human cytochrome P450 2B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation. Drug Metab. Dispos. 25, 985–993.
Chang, T. K. H., Yu, L., Maurel, P., and Waxman, D. J. (1997) Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines. Cancer Res. 57, 1946–1954.
Faucette, S. R., Wang, H., Hamilton, G. A., et al. (2004) Regulation of CYP2B6 in primary human hepatocytes by prototypical inducers. Drug Metab. Dispos. 32, 348–358.
Strom, S. C., Pisarov, L. A., Dorko, K., Thompson, M. T., Schuetz, J. D., and Schuetz, E. G. (1996) Use of human hepatocytes to study P450 gene induction. Methods Enzymol. 272, 388–401.
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.
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.
Shou, M., Korzekwa, K. R., Crespi, C. L., Gonzalez, F. J., and Gelboin, H. V. (1994) The role of 12 cDNA-expressed human rodent and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7,8-diol. Carcinogenesis 10, 159–168.
Shou, M., Korzekwa, K. R., Krausz, K. W., Crespi, C. L., Gonzalez, F. J., and Gelboin, H. V. (1994) Regio-and stereo-selective metabolism of phenathrene by twelve cDNA-expressed human, rodent and rabbit cytochromes P-450. Cancer Lett. 83, 305–313.
Dehal, S. S. and Kupfer, D. (1994) Metabolism of the proestrogenic pesticide methoxychlor by hepatic P450 monooxygenases in rat and humans: dual pathways involving novel ortho ring-hydroxylation by CYP2B. Drug Metab. Dispos. 22, 937–946.
Court, M. H., Duan, S. X., Hesse, L. M., Venkatakrishnan, K., and Greenblatt, D. J. (2001) Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes. Anesthesiology 94, 110–119.
Oda, Y., Hamaoka, N., Hiroi, T., et al. (2001) Involvement of human liver cytochrome P450 2B6 in the metabolism of propofol. Br. J. Clin. Pharmacol. 51, 281–285.
Hesse, L. M., Venkatakrishnan, K., Court, M. H., et al. (2000) CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metab. Dispos. 28, 1176–1183.
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.
Yuan, R., Madani, S., Wei, X. X., Reynolds, K., and Huang, S. M. (2002) Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab. Dispos. 30, 1311–1319.
Ko, J. W., Desta, Z., and Flockhart, D. A. (1998) Human N-demethylation of (S)-mephenytoin by cytochrome P450s 2C9 and 2B6. Drug Metab. Dispos. 26, 775–778.
Faucette, S. R., Hawke, R. L., LeCluyse, E. L., et al. (2000) Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab. Dispos. 28, 1222–1230.
Faucette, S. R., Hawke, R. L., Shord, S. S., LeCluyse, E. L., and Lindley, C. M. (2001) Evaluation of the contribution of cytochrome P450 3A4 to human liver microsomal bupropion hydroxylation. Drug Metab. Dispos. 29, 1123–1129.
Buters, J. T. M., Schiller, C. D., and Chou, R. C. (1993) A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man: direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin. Biochem. Pharmacol. 46, 1577–1584.
Ekins, S., VandenBranden, M., Ring, B. J., and Wrighton, S. A. (1997) Examination of purported probes of human CYP2B6. Pharmacogenetics 7, 165–179.
Sridar, C., Kent, U. M., Notley, L. M., Gillam, E. M. J., and Hollenberg, P. F. (2002) Effect of tamoxifen on the enzymatic activity of human cytochrome P450 2B6. J. Pharmacol. Exp. Ther. 301, 945–952.
Jushchyshyn, M. I., Kent, U. M., and Hollenberg, P. F. (2003) The mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine. Drug Metab. Dispos. 31, 46–52.
DeLuca, J. G., Dysart, G. R., Rasnick, D., and Bradley, M. O. (1988) A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog. Biochem. Pharmacol. 37, 1731–1739.
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.
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.
Acknowledgments
This work was supported in part by the Canadian Institutes of Health Research (grant MOP-42385) and the National Institutes of Health (grant CA49248).
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Chang, T.K.H., Crespi, C.L., Waxman, D.J. (2006). Determination of CYP2B6 Component of 7-Ethoxy-4-Trifluoromethylcoumarin O-Deethylation Activity in Human Liver Microsomes. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-998-2:97
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DOI: https://doi.org/10.1385/1-59259-998-2:97
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