Cytochrome P450 Protocols / Edition 2

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Cytochromes P450 (CYPs) comprise a large superfamily of proteins that are of central importance in the detoxification or activation of a tremendous number of natural and synthetic hydrophobic xenobiotics, including many therapeutic drugs, chemical carcinogens and environmental pollutants. CYPs are important in mediating interactions between an organism and its chemical environment and in the regulation of physiological processes. Cytochrome P450 Protocols, Third Edition focuses on high-throughput methods for the simultaneous analysis of multiple CYPs, substrates or ligands. Although the emphasis is on CYPs of mammalian origin, it reflects an increasing interest in CYPs of bacterial species. Also included are chapters on cytochrome P450 reductase (the redox partner of CYPs) and the flavin-containing monooxygenases (FMOs), and metabolomic and lipidomic approaches for identification of endogenous substrates of CYPs (‘de-orphanizing’ CYP substrates). Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and easily accessible, Cytochrome P450 Protocols, Third Edition provides a wide range of techniques accessible to researchers in fields as diverse as biochemistry, molecular biology, pharmacology, toxicology, environmental biology and genetics.

The book contains black-and-white illustrations.

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Editorial Reviews

A collection of techniques for investigating the large superfamily of proteins that are of central importance in detoxifying or activating many foreign hydrophobic compounds, among them therapeutic drugs, chemical carcinogens, and environmental pollutants. The focus is on P450 species from mammals, but the methods are also suitable for those from other sources. The techniques described are used for purifying, spectral analysis, expression in heterologous cells, determining functional capabilities, detecting and quantifying, gene expression and its regulation, and cell culture. Addressed to biologists and chemists with no previous knowledge of the techniques. Annotation c. by Book News, Inc., Portland, Or.
Doody's Review Service
Reviewer: Jed N. Lampe, Ph.D.(University of Kansas Medical Center)
Description: With this third edition, the authors and contributors continue to provide what has become a tour de force of bench top manuals for the cytochrome P450 field. Building on the success of the first two editions, this book expands and diversifies a set of well-established, validated protocols for the production and analysis of cytochrome P450 enzymes (CYPs) and their diverse metabolic products.
Purpose: The authors focus on high-throughput techniques for the examination of multiple CYPs in both in vitro and in vivo models, which is a notable expansion and extension of the fundamental techniques established in the earlier two editions.
Audience: Although predominantly useful for an industrial audience due to the high-throughput nature of most of the techniques, this book should also prove of interest to graduate students and postdoctoral researchers who are new to the field of P450 science, or who would like to expand their research directions.
Features: The highlight of this edition, as well as the previous editions, is the step-by-step protocols with extensive troubleshooting notes at the end of each chapter. These are augmented with selected and appropriate color illustrations that enhance readers' understanding of each technique.
Assessment: This stands alone as the premier book for this field, which is sorely lacking in publications of this type. Researchers in the P450 field will find this to be a useful bench top guide that will provide them with the tools to explore and understand CYPs, both in vitro and in vivo.
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Product Details

  • ISBN-13: 9781617375729
  • Publisher: Springer-Verlag New York, LLC
  • Publication date: 11/19/2010
  • Series: Methods in Molecular Biology Series, #320
  • Edition description: 2nd ed. 2006
  • Edition number: 2
  • Pages: 364
  • Product dimensions: 6.00 (w) x 9.00 (h) x 0.85 (d)

Table of Contents

Cytochrome P450 Nomenclature, 2004

David R. Nelson

Spectral Analyses of Cytochromes P450

John B. Schenkman and Ingela Jansson

Establishment of Functional Human Cytochrome P450 Monooxygenase Systems in Escherichia coli

Michael P. Pritchard, Lesley McLaughlin, and Thomas Friedberg

Purification of Cytochromes P450: Products of Bacterial Recombinant Expression Systems

F. Peter Guengerich and Martha V. Martin

Expression of Recombinant Flavin-Containing Monooxygenases in a Baculovirus/Insect Cell System

Azara Janmohamed, Prapimpuk Thaunsukon, Elizabeth A. Shephard, and Ian R. Phillips

Cytochrome P450 Reconstitution Systems

Hiroshi Yamazaki and Tsutomu Shimada

Catalytic Assays for Human Cytochrome P450: An Introduction

Thomas K. H. Chang and David J. Waxman

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

Thomas K. H. Chang and David J. Waxman

Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin 7-Hydroxylation

David J. Waxman and Thomas K. H. Chang

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

High-Performance Liquid Chromatography Analysis of CYP2C8-Catalyzed Paclitaxel 6a-Hydroxylation

Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman

Determination of CYP2C9-Catalyzed Diclofenac 4'-Hydroxylation by High-Performance Liquid Chromatography

Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman

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

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

Spectrophotometric Analysis of Human CYP2E1-Catalyzed p-Nitrophenol Hydroxylation

Thomas K. H. Chang, Charles L. Crespi, and David J. Waxman

Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6b-Hydroxylation

David J. Waxman and Thomas K. H. Chang

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

An Isocratic High-Performance Liquid Chromatography Assay for CYP7A1-Catalyzed Cholesterol 7a-Hydroxylation

David J. Waxman and Thomas K. H. Chang

Use of 7-Ethoxycoumarin to Monitor Multiple Enzymes in the Human CYP1, CYP2, and CYP3 Families

David J. Waxman and Thomas K. H. Chang

Benzydamine N-Oxygenation as a Measure of Flavin-Containing Monooxygenase Activity

Catherine K. Yeung and Allan E. Rettie

Prochiral Sulfoxidation as a Probe for Flavin-Containing Monooxygenases

Catherine K. Yeung and Allan E. Rettie

Targeting Antipeptide Antibodies Toward Cytochrome P450 Enzymes

Robert J. Edwards

The Human Cytochrome P450 Allele Nomenclature Committee Web Site: Submission Criteria, Procedures, and Objectives

Sarah C. Sim and Magnus Ingelman-Sundberg

Genotyping for Cytochrome P450 Polymorphisms

Ann K. Daly, Barry P. King, and Julian B. S. Leathart

Hepatocyte Cultures in Drug Metabolism and Toxicological Research and Testing

Tamara Vanhaecke and Vera Rogiers

Isolation of Rat Hepatocytes

Peggy Papeleu, Tamara Vanhaecke, Tom Henkens, Greetje Elaut, Mathieu Vinken, Sarah Snykers, and Vera Rogiers

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

Rat Hepatocyte Cultures: Collagen Gel Sandwich and Immobilization Cultures

Mathieu Vinken, Greetje Elaut, Tom Henkens, Peggy Papeleu, Sarah Snykers, Tamara Vanhaecke, and Vera Rogiers

Hepatocytes in Suspension

Greetje Elaut, Peggy Papeleu, Mathieu Vinken, Tom Henkens, Sarah Snykers, Tamara Vanhaecke, and Vera Rogiers

Isolation of Rat Bone Marrow Stem Cells

Sarah Snykers, Tamara Vanhaecke, and Vera Rogiers

Transfection of Primary Cultures of Rat Hepatocytes

Mina Edwards, Siew Cheng Wong, Roongsiri Chotpadiwetkul, Despina Smirlis, Ian R. Phillips, and Elizabeth A. Shephard

Human Hepatocyte Culture

Lydiane Pichard, Edith Raulet, Gérard Fabre, Jean Bernard Ferrini, Jean-Claude Ourlin, and Patrick Maurel

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

Deletion of Genes From the Mouse Genome Using Cre/loxP Technology

Diana Hernandez, Pritpal Chandan, Azara Janmohamed, Ian R. Phillips, and Elizabeth A. Shephard

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

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

Use of Reporter Genes to Measure Xenobiotic-Mediated Activation of CYP Gene Transcription

Nick Plant


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