Cytochrome P450 Protocols

Preface ..............................................................................................................v Contributors .....................................................................................................xi 1 Cytochrome P450 Nomenclature, 2004 David R. Nelson .................................................................................... 1 2 Spectral Analyses of Cytochromes P450 John B. Schenkman and Ingela Jansson .............................................. 11 3 Establishment of Functional Human Cytochrome P450 Monooxygenase Systems in Escherichia coli Michael P. Pritchard, Lesley McLaughlin, and Thomas Friedberg .................................................................... 19 4 Purification of Cytochromes P450: Products of Bacterial Recombinant Expression Systems F. Peter Guengerich and Martha V. Martin ........................................ 31 5 Expression of Recombinant Flavin-Containing Monooxygenases in a Baculovirus/Insect Cell System Azara Janmohamed, Prapimpuk Thaunsukon, Elizabeth A. Shephard, and Ian R. Phillips ..................................... 39 6 Cytochrome P450 Reconstitution Systems Hiroshi Yamazaki and Tsutomu Shimada ........................................... 61 7 Catalytic Assays for Human Cytochrome P450: An Introduction Thomas K. H. Chang and David J. Waxman ....................................... 73 8 Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 With 7-Ethoxyresorufin as Substrate Thomas K. H. Chang and David J. Waxman ....................................... 85 9 Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin 7-Hydroxylation David J. Waxman and Thomas K. H. Chang ....................................... 91 10 Determination of CYP2B6 Component of 7-Ethoxy-4-Trifluoromethylcoumarin O-Deethylation Activity in Human Liver Microsomes Thomas K. H. Chang, Charles L. Crespi, and David J. Waxman ..................................................................... 97 viii Contents 11 High-Performance Liquid Chromatography Analysis of CYP2C8-Catalyzed Paclitaxel 6α-Hydroxylation Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman ................................................................... 103 12 Determination of CYP2C9-Catalyzed Diclofenac 4'-Hydroxylation by High-Performance Liquid Chromatography Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman ................................................................... 109 13 CYP2C19-Mediated (S)-Mephenytoin 4'-Hydroxylation Assayed by High-Performance Liquid Chromatography With Radiometric Detection Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman ................................................................... 115 14 CYP2D6-Dependent Bufuralol 1'-Hydroxylation Assayed by Reverse-Phase Ion-Pair High-Performance Liquid Chromatography With Fluorescence Detection Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman ................................................................... 121 15 Spectrophotometric Analysis of Human CYP2E1-Catalyzed p-Nitrophenol Hydroxylation Thomas K. H. Chang, Charles L. Crespi, and David J. Waxman ................................................................... 127 16 Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6⇓-Hydroxylation David J. Waxman and Thomas K. H. Chang ..................................... 133 17 Determination of CYP4A11-Catalyzed Lauric Acid 12-Hydroxylation by High-Performance Liquid Chromatography With Radiometric Detection Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman ................................................................... 143 18 An Isocratic High-Performance Liquid Chromatography Assay for CYP7A1-Catalyzed Cholesterol 7α-Hydroxylation David J. Waxman and Thomas K. H. Chang ..................................... 149 19 Use of 7-Ethoxycoumarin to Monitor Multiple Enzymes in the Human CYP1, CYP2, and CYP3 Families David J. Waxman and Thomas K. H. Chang ..................................... 153 20 Benzydamine N-Oxygenation as a Measure of Flavin-Containing Monooxygenase Activity Catherine K. Yeung and Allan E. Rettie ............................................ 157 21 Prochiral Sulfoxidation as a Probe for Flavin-Containing Monooxygenases Catherine K. Yeung and Allan E. Rettie ............................................ 163 22 Targeting Antipeptide Antibodies Toward Cytochrome P450 Enzymes Robert J. Edwards.............................................................................. 173 23 The Human Cytochrome P450 Allele Nomenclature Committee Web Site: Submission Criteria, Procedures, and Objectives Sarah C. Sim and Magnus Ingelman-Sundberg ................................. 183 24 Genotyping for Cytochrome P450 Polymorphisms Ann K. Daly, Barry P. King, and Julian B. S. Leathart ...................... 193 25 Hepatocyte Cultures in Drug Metabolism and Toxicological Research and Testing Tamara Vanhaecke and Vera Rogiers ............................................... 209 26 Isolation of Rat Hepatocytes Peggy Papeleu, Tamara Vanhaecke, Tom Henkens, Greetje Elaut, Mathieu Vinken, Sarah Snykers, and Vera Rogiers .......................................................................... 229 27 Rat Hepatocyte Cultures: Conventional Monolayer and Cocultures With Rat Liver Epithelial Cells Tom Henkens, Tamara Vanhaecke, Peggy Papeleu, Greetje Elaut, Mathieu Vinken, Sarah Snykers, and Vera Rogiers .......................................................................... 239 28 Rat Hepatocyte Cultures: Collagen Gel Sandwich and Immobilization Cultures Mathieu Vinken, Greetje Elaut, Tom Henkens, Peggy Papeleu, Sarah Snykers, Tamara Vanhaecke, and Vera Rogiers .......................................................................... 247 29 Hepatocytes in Suspension Greetje Elaut, Peggy Papeleu, Mathieu Vinken, Tom Henkens, Sarah Snykers, Tamara Vanhaecke, and Vera Rogiers ................. 255 30 Isolation of Rat Bone Marrow Stem Cells Sarah Snykers, Tamara Vanhaecke, and Vera Rogiers ..................... 265 31 Transfection of Primary Cultures of Rat Hepatocytes Mina Edwards, Siew Cheng Wong, Roongsiri Chotpadiwetkul, Despina Smirlis, Ian R. Phillips, and Elizabeth A. Shephard ........................................................... 273 32 Human Hepatocyte Culture Lydiane Pichard, Edith Raulet, Gérard Fabre, Jean Bernard Ferrini, Jean-Claude Ourlin, and Patrick Maurel ......................... 283 Contents ix x Contents 33 Determination of Cellular Localization of the Expression of Flavin-Containing Monooxygenase Genes in Mouse Tissues by In Situ Hybridization Azara Janmohamed, Ian R. Phillips, and Elizabeth A. Shephard ........................................................... 295 34 Deletion of Genes From the Mouse Genome Using Cre/loxP Technology Diana Hernandez, Pritpal Chandan, Azara Janmohamed, Ian R. Phillips, and Elizabeth A. Shephard ................................... 307 35 Characterization of Targeted Mouse Embryonic Stem Cell Chromosomes: Karyotyping and Fluorescence In Situ Hybridization of Metaphase Spreads Diana Hernandez, Ian R. Phillips, and Elizabeth A. Shephard ........................................................... 321 36 Microinjection of Targeted Embryonic Stem Cells and Establishment of Knockout Mouse Lines for Fmo Genes Diana Hernandez, Anna N. Melidoni, Ian R. Phillips, and Elizabeth A. Shephard ........................................................... 329 37 Use of Reporter Genes to Measure Xenobiotic-Mediated Activation of CYP Gene Transcription Nick Plant ......................................................................................... 343 Index ............................................................................................................ 355