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Catalytic Assays for Human Cytochrome P450

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Cytochrome P450 Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 107))

Abstract

Cytochrome P450 (P450) enzymes catalyze the blotransformatlon of a broad range of structurally diverse foreign chemicals and endogenous substances. Many P450 enzymes have been isolated and identified in liver and other tlssues, including kidney, lung, intestines, and brain. Although these enzymes have a high degree of similarity in their ammo-acid sequences, many of them are subject to differential regulation and have distinct catalytic functions With the development of P450 form-specific (or P450 subfamlly-specific) inhlbltory antibodies, the discovery of enzyme-selective chemical inhibitors, and the general availability of catalytically active, mdivldual P450 cDNA-expressed proteins, several substrates, including drugs and endogenous substances, have been identified as useful catalytic monitors for specific P450 enzymes or subfamilies (see Table 1). These catalytic monitors can serve as useful expenmental tools in a variety of studies. These include studies designed: 1 To identify individual P45Os that catalyze the actlvatlon or detoxification of a particular compound, 2 To quantify the expression and catalytic activity of an mdlvrdual P450 in tissue samples and In cultured cells, 3. To analyze the enzyme kinetics of a particular P450; and 4 To eluctdate the mechanism of interaction between xenoblotlcs and mdlvldual P450 enzymes

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References

  1. Carrrere, V., Goasduff, T., Ratanasavanh, D., Morel, F., Gautter, J. C, Guillouzo, A., Beaune, P., and Bet-thou, F. (1993) Both cytochromes P4502El and IA1 are mvolved in the metabolism of chlorzoxazone Chem. Res Toxzcol 6, 852–857.

    Article  Google Scholar 

  2. Yamazaki, H., Guo, Z., and Guengerich, F. P. (1995) Selectivtty of cytochrome P4502E1 in chlorzoxazone 6-hydroxylation. Drug Metab Dispos 23, 438–440.

    PubMed  CAS  Google Scholar 

  3. Chang, T K. H., Yu, L, Goldstein, J. A, and Waxman, D J. (1997) Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-Ile359 allele as low-Km catalysts of cyclophosphamtde and ifosfamide acttvatron. Pharmacogenetzcs 7, 211–221.

    Article  CAS  Google Scholar 

  4. Chang, T. K. H., Gonzalez, F. J., and Waxman, D J. (1994) Evaluatron of tnacetyloleandomycm, a-naphthoflavone and diethyldithiocarbamate as selective chemical probes for mhtbitton of human cytochromes P450 Arch Bzochem Bzophys. 311, 437–442

    Article  CAS  Google Scholar 

  5. 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 Odeethylation of phenacetin and IS inducible by cigarette smoking Br J Clzn Pharmacol 26, 363–372.

    CAS  Google Scholar 

  6. Boobis, A. R., Kahn, G. C., Whyte, C., Brodie, M J, and Davies, D. S (1981) Biphastc 0-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver mtcrosomal fractions. Bzochem. Pharmacol. 30, 2451–2456

    Article  CAS  Google Scholar 

  7. Butler, M. A., Iwasaki, M., Guengerich, F. P., and Kadlubar, F. F (1989) Human cytochrome P-450PA (P-450JA2), the phenacetin 0-deethylase, is prtmarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carctnogenrc arylamines Proc Nat1 Acad Scz USA 86, 7696–7700

    Article  CAS  Google Scholar 

  8. Sesardic, D., Pasanen, M., Pelkonen, 0, and Boobrs, A R. (1990) Differential expression and regulation of members of the cytochrome P450 IA gene subfamily in human tissues Carcznogeneszs 11, 1183–1188.

    CAS  Google Scholar 

  9. Waxman, D. J, Lapenson, D P., Aoyama, T, Gelbom, H V., Gonzalez, F. J., and Korzekwa, K. (1991) Steroid hormone hydroxylase specificities of eleven cDNAexpressed human cytochrome P45Os Arch Biochem Bzophys 290, 160–166.

    Article  CAS  Google Scholar 

  10. Chang, T. K H., Weber, G. F., Crespi, C L., and Waxman, D. J. (1993) Dtfferenteal activation of cyclophosphamtde and ifosphamide by cytochromes P-4502B and 3A in human liver microsomes. Cancer Res. 53, 5629–5637.

    PubMed  CAS  Google Scholar 

  11. Butler, A. M and Murray, M (1997) Biotransformation of parathion in human liver: participation of CYP3A4 and its inactivation during mtcrosomal parathion oxtdation J Pharmacol Exp Ther. 280, 966–973

    PubMed  CAS  Google Scholar 

  12. Burke, M D, Thompson, S, Weaver, R. J, Wolf, C. R., and Mayer, R T. (1994) Cytochrome P450 spectticities of alkoxyresorufin O-dealkylatron in human and rat liver. Bzochem. Pharmacol 48, 923–936

    Article  CAS  Google Scholar 

  13. Distlerath, L. M., Reilly, P. E. B., Martin, M. V., Davis, G. G, Wilkmson, G R., and Guengertch, F P. (1985) Purification and characterizatton of the human ltver cytochrome P-450 involved n-r debrisoquine 4-hydroxylation and phenacetin O-deethylatton, two prototypes for genetic polymorphtsm in oxrdatlve drug metabolism. J Brol. Chem. 260, 9057–9067

    CAS  Google Scholar 

  14. Tang, B. K. and Kalow, W. (1996) Assays for CYPlA2 by testmg zn vzvo metabolism of caffeme in humans Methods Enzymol 272, 124–131.

    Article  PubMed  CAS  Google Scholar 

  15. Shimada, T., Grllam, E. M. J., Sutter, T R., Strickland, P T., Guengench, F. P., and Yamazaki, H (1997) Oxidation of xenobiottcs by recombinant human cytochrome P4501B1. DrugMetab Dzspos 29, 617–622.

    Google Scholar 

  16. Spink, D. C., Hayes, C L, Young, N. R., Christou, M., Sutter, T. R, Jefcoate, C. R., and Grerthy, J. F. (1994) The effects of 2,3,7,8-tetrachlorodtbenzo-p-dtoxm on estrogen metabolism in MCF-7 breast cancer cells. evidence for mductton of a novel 17 beta-estradiol4-hydroxylase. J Steroid Blochem Mol. Btol 51, 251–258

    Article  CAS  Google Scholar 

  17. Aoyama, T., Korzekwa, K., Nagata, K., Grllette, J, Gelbom, H. V., and Gonzalez, F. J. (1990) Estradtol metabolism by complementary deoxyrtbonuclerc acidexpressing human cytochrome P45Os Endocrznology 126, 3101–3106.

    Article  CAS  Google Scholar 

  18. Suchar, L. A., Chang, R L., Rosen, R. T, Lech, J, and Conney, A. H (1995) High-performance hqutd chromatography separation of hydroxylated estradtol metabolites: Formatton of estradtol metabohtes by liver mrcrosomes from male and female rats. J Pharmacol Exp Ther 272, 197–206

    PubMed  CAS  Google Scholar 

  19. Yun, C H, Shtmada, T., and Guengertch, F. P. (1991) Purrficatron and characterization of human liver microsomal cytochrome P-4502A6 Mol. Pharmacol 40, 679–685

    PubMed  CAS  Google Scholar 

  20. Code, E L., Crespt, C L, Penman, B W., Gonzalez, F J., Chang, T K. H, and Waxman, D. J. (1997) Human cytochrome P4502B6: Intermdtvldual hepattc expression, substrate specificity and role in procarcmogen activation Drug Metab Dtspos. 25, 985–993

    CAS  Google Scholar 

  21. Rahman, A., Korzekwa, K. R., Grogan, J., Gonzalez, F. J, and Harris, J W. (1994) Selective btotransformatton of taxol to 6cL-hydroxytaxol by human cytochrome P4502C8. Cancer Res. 54, 5543–5546

    PubMed  CAS  Google Scholar 

  22. Leemann, T., Transon, C, and Dayer, P (1992) Cytochrome P450TB (CYP2C) a maJor monooxygenase catalyzing dtclofenac 4’-hydroxylatron in human liver. Life Sct 52, 29–34

    Article  Google Scholar 

  23. Brian, W R., Srivastava, P K, Umbenhauer, D. R, Lloyd, R S., and Guengertch, F P. (1989) Expression of a human liver cytochrome P-450 protein with tolbutamtde hydroxylase activity in Saccaromyces cerevwae. Bzochemlstry 28, 4993–4999.

    CAS  Google Scholar 

  24. Doecke, C. J, Veronese, M. E., Pond, S. M, Miners, J O., Btrkett, D J., Sansom, L. N., and McManus, M E. (1991) Relationship between phenytom and tolbutamtde hydroxylattons in human liver mtcrosomes. Br J Clin. Pharmacol. 31, 125–130

    PubMed  CAS  Google Scholar 

  25. Miners, J. O and Bukett, D J. (1996) Use of tolbutamrde as a substrate probe for human hepattc cytochrome P4502C9. Methods Enzymol 272, 139–145

    Article  PubMed  CAS  Google Scholar 

  26. Rettie, A. E., Korzekwa, K R, Kunze, K. L, Lawrence, R F, Eddy, A C, Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Trager, W. F. (1992) Hydroxylatton of warfarin by human cDNA-expressed cytochrome P-450: a role for P-4502C9 in the etiology of (S)-warfarm drug tnteractlons. Chem. Res. Toxzcol 5, 54–59.

    Article  CAS  Google Scholar 

  27. Kaminsky, L S., Fasco, M J, and Guengerich, F P (1981) Productton and apphcatton ofanttbodtes to rat liver cytochrome P-450 Methods Enzymol 74, 262–273

    Article  PubMed  CAS  Google Scholar 

  28. Lang, D and Backer, R. (1995) Highly sensitive and specific high-performance liquid chromatographtc analysts of 7-hydroxywarfarm, a marker for human cytochrome P-4502C9 activity. J. Chromatogr 672, 305–309.

    Article  CAS  Google Scholar 

  29. Goldstein, J A., Faletto, M. B., Romkes-Sparks, M., Sullivan, T, Kttareewan, S, Raucy, J. L., Lasker, J M, and Ghanayem, B I(1994) Evidence that CYP2Cl9 is the major (S)-mephenytom 4’-hydroxylase in humans. Bzochemzstry 33, 1743–1752

    Article  CAS  Google Scholar 

  30. Gut, J, Catin, T, Dayer, P., Kronbach, T., Zanger, U., and Meyer, U. A (1986) Debrisoqume/sparteme-type polymorphtsm of drug oxldatton purtficatton and characterizatton of two functtonally different human liver cytochrome P-450 isozymes mvolved in impaired hydroxylatton of the prototype substrate bufuralol J Blol Chem 261, 11,734–11,743.

    CAS  Google Scholar 

  31. Kronbach, T. (1991) Bufuralol, dextromethorphan and debrisoqume as prototype substrates for human P450IID6. Methods Enzymol 206, 509–517.

    Article  PubMed  CAS  Google Scholar 

  32. Dayer, P, Leemann, T., and Striberni, R. (1989) Dextromethorphan O-demethylatton in liver microsomes as a prototype reaction to monitor cytochrome P-450 db, activity Cin. Pharmacol Ther 45, 34–40.

    Article  CAS  Google Scholar 

  33. Rodrtgues, A. D. (1996) Measurement of human liver mtcrosomal cytochrome P4502D6 actrvrty usmg [O-methyl-14C]dextromethorphan as substrate Methods Enzymol 272, 186–195

    Article  Google Scholar 

  34. Tassaneeyakul, W, Veronese, M E., Btrkett, D J., Gonzalez, F. J., and Mmers, J 0 (1993) Validation of 4-mtrophenol as an zn vitro substrate probe for human liver CYP2El using cDNA expresston and microsomal kinetic techniques Bzochem Pharmacol 46, 1975–1981.

    Article  CAS  Google Scholar 

  35. Peter, R, Backer, R., Beaune, P. H., Iwasaki, M, Guengertch, F P., and Yang, C S (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-45011E1 Chem Res Toxicol 3, 566–573

    Article  PubMed  CAS  Google Scholar 

  36. Lucas, D., Menez, J F., and Bet-thou, F. (1996) Chlorzoxazone an zn vitro and zn vlvo substrate probe for liver CYP2E1. Methods Enzymol 272, 115–123

    Article  PubMed  CAS  Google Scholar 

  37. Amet, Y., Berthou, F., Band, S., Dreano, Y., Bad, J P, and Menez, J. F (1995) Validation of the (o-I)-hydroxylation of lauric acid as an zn vitro substrate probe for human liver CYP2El Bzochem. Pharmacol 50, 1775–1782.

    CAS  Google Scholar 

  38. Wrtghton, S. A., Thomas, P. E., Molowa, D. T., Hanm, M., Shively, J. E., Mames, S. L., Watkins, P B., Parker, G., Mendezptcon, G., Levm, W., and Guzehan, P. S. (1986) Characterization of ethanol-inducible human liver N-nitrosodtmethylamine demethylase. Bzochemzstry 25, 6731–6735.

    Article  Google Scholar 

  39. Guengerich, F. P, Ktm, D. H., and Iwasakt, M (1991) Role of human cytochrome P-450 IIEl in the oxtdatton of many low molecular weight cancer suspects. Chem Res. Toxicol 4, 168–179

    Article  PubMed  CAS  Google Scholar 

  40. Yoo, J. S. H., Guengertch, F. P., and Yang, C S. (1988) Metabolism of N-mtrosodtalkylammes by human ltver mtcrosomes Cancer Res 88, 1499–1504.

    Google Scholar 

  41. Waxman, D. J, Attisano, C., Guengerich, F. P, and Lapenson, D P. (1988) Human hver microsomal steroid metabolism: Identificatton of the maJor microsoma1 steroid hormone Go-hydroxylase cytochrome P-450 enzyme Arch Blochem Bzophys. 263, 424–436.

    Article  CAS  Google Scholar 

  42. Guengertch, F P, Martin, M V, Beaune, P H, Kremers, P, Wolff, T, and Waxman, D. J. (1986) Characterization of rat and human liver mtcrosomal cytochrome P-450 forms involved in mfedipme oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J Bzol Chem 261, 5051–5060

    Google Scholar 

  43. Gonzalez, F. J., Schmid, B., Umeno, M., McBride, 0. W., Hardwtck, J. P, Meyer, U A, Gelbom, H V, and Idle, J R (1988) Human P450PCNl sequence, chromosome localization and direct evidence through cDNA expression that P450PCNl IS mfedtpme oxidase DNA 7, 79–86.

    Article  PubMed  CAS  Google Scholar 

  44. Powell, P. K., Wolf, I., and Lasker, J. M. (1996) Identrficatton of CYP4All as the maJor lauric acid o-hydroxylase in human liver microsomes Arch Bzochem Blophys. 335, 219–226

    Article  CAS  Google Scholar 

  45. Maeda, Y., Eggertsen, G., Nyberg, B., Setogucht, T, Okuda, K, Emarsson, K, and Bjorkhem, I. (1995) Immunochemical determination of human cholesterol 7a-hydroxylase. Eur J Biochem 228, 144–148

    Article  PubMed  CAS  Google Scholar 

  46. Bourdt, M, Larrey, D, Nataf, J, Bernuau, J., Pessayre, D, Iwasaki, M, Guengerich, F P, and Beaune, P H. (1990) Anti-liver endoplasmic reticulum autoantibodtes are directed against human cytochrome P-4501A2: a specific marker of dihydralazme-induced hepatms J. Clan Invest 85, 1967–1973.

    Article  Google Scholar 

  47. Sonnichsen, D S, Lm, Q., Schuetz, E. G., Schuetz, J. D, Pappo, A., and Relling, M V (1995) Variabihty in human cytochrome P450 pachtaxel metabolism J Pharmacol. Exp Ther 275, 566–575.

    PubMed  CAS  Google Scholar 

  48. Richardson, T. H, Jung, F, Griffin, K J, Wester, M, Raucy, J L, Kemper, B, Bornheim, L. M., Hassett, C., Omiecmski, C J, and Johnson, E. F.(1995) A universal approach to the expresston of human and rabbit cytochrome P45Os of the 2C subfamily in Escherlchla COIL Arch. Blochem Biophys 323, 87–96.

    Article  CAS  Google Scholar 

  49. Yamazakt, H., Guo, Z., Persmark, M, Mimura, M., Inoue, K., Guengerich, F. P, and Shimada, T. (1994) Bufuralol hydroxylation by cytochrome P4502D6 and IA2 enzymes in human liver microsomes. Mol Pharmacol 46, 568–577

    Google Scholar 

  50. Draper, A. J., Madan, A., and Parkinson, A. (1997) Inhibttton of coumarm 7-hydroxylase activity in human liver microsomes Arch Blochem Bzophys 341, 47–61

    Article  CAS  Google Scholar 

  51. Gorski, J C, Jones, D. R., Wrighton, S A, and Hall, S D (1997) Contributton of human CYP3A subfamtly members to the 6-hydroxylation of chlorzoxazone Xenoblotica 27, 243–256

    Article  CAS  Google Scholar 

  52. Wang, R W and Lu, A Y H (1997) Inhibitory anti-peptide antibody against human CYP3A4. Drug Metab Dispos 25, 762–767.

    PubMed  CAS  Google Scholar 

  53. Clarke, S E., Baldwin, S. J., Bloomer, J C., Ayrton, A. D., Sozto, R S., and Chenery, R. J (1994) Laurie acid as a model substrate for the simultaneous determmation of cytochrome P4502El and 4A in hepattc microosomes. Chem Res. Toxlcol. 7, 836–842.

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

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

    Thomas K. H. Chang

  2. Department of Biology, Boston University, Boston, MA

    David J. Waxman

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  1. Thomas K. H. Chang
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  2. David J. Waxman
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Editor information

Editors and Affiliations

  1. Department of Biochemistry, Queen Mary and Westfie1d College, University of London, UK

    Ian R. Phillips

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

    Elizabeth A. Shephard

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© 1998 Humana Press Inc.

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K. H. Chang, T., J. Waxman, D. (1998). Catalytic Assays for Human Cytochrome P450. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology™, vol 107. Humana Press. https://doi.org/10.1385/0-89603-519-0:95

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  • DOI: https://doi.org/10.1385/0-89603-519-0:95

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-519-5

  • Online ISBN: 978-1-59259-580-8

  • eBook Packages: Springer Protocols

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