Molecular Toxicology Protocols M E T H O D S I N M O L E C U L A R B I O L O G Y™ John M. Walker, SERIES EDITOR 330077. Phosphodiesterase Mehtods and Protocols, 228844. Signal Transduction Protocols, Second Edition, edited by Claire Lugnier, 2005 edited by Robert C. Dickson and Michael D. Mendenhall, 2004 330066. Receptor Binding Techniques: Second Edition, edited by Anthony P. Davenport, 2005 228833. Bioconjugation Protocols, edited by Christof M. Niemeyer, 2004 330055. Protein–Ligand Interactions: Methods and 228822. Apoptosis Methods and Protocols, edited by Protocols,edited by G. Ulrich Nienhaus, 2005 Hugh J. M. Brady, 2004 330044. Human Retrovirus Protocols:Virology and 228811. Checkpoint Controls and Cancer, Volume 2: Molecular Biology,edited by Tuofu Zhu, 2005 Activation and Regulation Protocols,edited by 330033. NanoBiotechnology Protocols,edited by Sandra Axel H. Schönthal, 2004 J. Rosenthal and David W. Wright, 2005 228800. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 ISSN: 1064-3745 eISBN:1-59259-840-4 Library of Congress Cataloging-in-Publication Data Molecular toxicology protocols / edited by Phouthone Keohavong, Stephen G. Grant. p. ; cm. -- (Methods in molecular biology ; 291) Includes bibliographical references and index. ISBN 1-58829-084-0 (alk. paper) 1. Molecular toxicology--Laboratory manuals. [DNLM: 1. DNA Adducts--analysis. 2. DNA Mutational Analysis. 3. Apoptosis. 4. DNA Repair. QU 58.5 M7185 2005] I. Keohavong, Phouthone. II. Grant, Stephen G. III. Series: Methods in molecular biology (Clifton, N.J.) ; v. 291. RA1220.3.M66 2005 615.9--dc22 2004005911 Preface It seems fashionable today to simply place the word “molecular” in front of a traditional field and consider it reinvented. This, without a clear consensus on what the “molecular” actually means. Certainly chemists working in the field of toxicology have always considered that they worked at the “molecular” level. It has not been so clear on the biological side, however, where there has been a history of ongoing discovery and characterization of toxic mechanisms. In other biological fields, “molecular” really implies using the tools of “molecular” biology, i.e., recombinant DNA. Just as the adoption of molecular biological techniques first invaded, then transformed such biological fields as genetics, physiology, and developmental biology, so too have these new methods begun to transform toxicology. Molecular Toxicology Protocols is a book about science on the interface, and a science that is about to explode upon the clinical and popular horizon. Toxicology, a subdiscipline of pharmacology, is actually the interface of chemistry and biology. As most practice it, this field also extends into nonchemical “agents” of deleterious biological effects, especially radiation, the purview of the radiobiologist and health physicist. With the huge increase in computational power made available over the last ten years it has become possible to model and predict the potential toxicity of as yet unmade chemicals. Perhaps the greatest change in the practice of toxicology has been application of the the tools of the trade directly to the human population, in what are known clinically as “translational” studies, opening the new frontier of epidemiology through the more conventional portal of biostatistics. These studies expand the traditional public health aspect of toxicology, screening of synthetic agents for toxicological potential prior to their introduction into the environment, attempting to define “normal” or “background,” perhaps unavoidable, exposures as mechanisms of human disease, and to design methods of preclinical intervention (“chemo- prevention”). Thus, for our purposes, we will define “molecular” toxicology as either any study of toxicological mechanism, or any translation of toxicological practice into the human population. Today, such “molecular” toxicology is mostly genetic toxicology, where the genetic material itself, the DNA, is the target molecule. Of course DNA is found throughout the human body, such that all of the traditional modulators of toxicological effect, uptake, distribution, metabolism, and so on, must be taken into account. Although genetic damage can have many outcomes, the one outcome most clearly linking exposure and disease is cancer. During the past several years, important progress has been made in the understanding of the molecular biology of the cell, the responses of cells to genotoxic agents, and the molecular biology of human cancer. This progress has been achieved thanks to the ongoing development of new state-of-the-art techniques, as well as v vi Preface improvements made upon existing methods to study changes not only in cellular morphology, but also in the cellular genetic material, the DNA, the cellular transcript, the mRNA, and the translated product, proteins. These molecular methods are now opening up many areas to potential clinical applications. Several books are currently available on the applications of molecular methods to various types of technology. However, to our knowledge, there is no book emphasizing the application of molecular methods to genetic toxicology. Therefore, the aim of Molecular Toxicology Protocols is to bring together a series of articles, each describing commonly used methods to elucidate specific molecular aspects of toxicology. With such content, this book addresses not only molecular biologists and molecular toxicologists, but also all individuals interested in applying molecular methods to clinical populations, including geneticists, pathologists, biochemists, and epidemiologists. The volume is divided into seven parts, roughly corresponding to the spectrum of biomarkers intermediate between exposure and disease outcome as proposed in molecular epidemiological models. Thus, Part I includes chapters describing methods of detecting premutagenic lesions in the genetic material, while Part II contains chapters describing the applications of methods to assess gross or macroscopic genetic damage. Parts III and IV focus on detection and characterization of viable mutations, in surrogate markers and cancer-related genes, respectively. The chapters of Part V describe methods for the analysis of the various pathways of DNA repair, an important modulator of genotoxicity. Part VI addresses the application of the new array technologies to genetic toxicology, including methods for the analysis of individual variation in biotransformation and the effects of genotoxic exposure on gene expression. Finally, Part VII describes methods for analysis of cytotoxicity caused by the induction of apoptosis, because cell death can either protect the organism from a transforming cell or cause distinct health effects itself. We have no doubt that as time goes by “molecular” approaches will play an expanding role in all types of toxicology, not just genetic toxicology. Moreover, genetic toxicology will undoubtedly be found to play a role in many more diseases of aging than cancer alone; it is probably a fundamental mechanism of aging itself. Therefore, while the current focus of Molecular Toxicology Protocols is genetic toxicology, and more specifically the genetic toxicology of cancer, we believe this represents just the tip of the iceberg with respect to how the field of molecular toxicology will eventually be understood. Phouthone Keohavong Stephen G. Grant Contents Preface ..............................................................................................................v Contributors .....................................................................................................xi PART I: ANALYSIS OF DNA ADDUCTS 1 32P-Postlabeling Analysis of DNA Adducts David H. Phillips, Alan Hewer, and Volker M. Arlt.............................3 2 Modification of the 32P-Postlabeling Method to Detect a Single Adduct Species as a Single Spot Masako Ochiai, Takashi Sugimura, and Minako Nagao.....................13 3 DNA Isolation and Sample Preparation for Quantification of Adduct Levels by Accelerator Mass Spectrometry Karen H. Dingley, Esther A. Ubick, John S. Vogel, and Kurt W. Haack.........................................................................21 4 Fluoroimaging-Based Immunoassay of DNA Photoproducts in Ultraviolet-B-Irradiated Tadpoles Iovanna Pandelova, Stephen R. Hewitt, and John B. Hays................29 5 Analysis of DNA Strand Cleavage at Abasic Sites Walter A. Deutsch and Vijay Hegde...................................................39 PART II: DETECTION OF CHROMOSOMAL AND GENOME-WIDE DAMAGE 6 Premature Chromosome Condensation in Human Resting Peripheral Blood Lymphocytes for Chromosome Aberration Analysis Using Specific Whole-Chromosome DNA Hybridization Probes Pataje G. S. Prasanna and William F. Blakely....................................49 7 Mutagen-Induced Chromatid Breakage as a Marker of Cancer Risk Xifeng Wu, Yun-Ling Zheng, and T. C. Hsu........................................59 8 Flow Cytometric Analysis of Micronuclei in Erythrocytes Jan Grawé...........................................................................................69 9 The Comet Assay: A Sensitive Genotoxicity Test for the Detection of DNA Damage Günter Speit and Andreas Hartmann.................................................85 10 Computerized Image Analysis Software for the Comet Assay R. C. Chaubey.....................................................................................97 vii viii Contents 11 The Comet–FISH Technique: A Tool for Detection of Specific DNA Damage and Repair Alexander Rapp, Michael Hausmann, and Karl Otto Greulich........107 12 DNA Double-Strand Break Damage and Repair Assessed by Pulsed-Field Gel Electrophoresis Nina Joshi and Stephen G. Grant.....................................................121 PART III: DETECTION AND CHARACTERIZATION OF SURROGATE GENE MUTATION 13 Analysis of In Vivo Mutation in the Hprt and Tk Genes of Mouse Lymphocytes Vasily N. Dobrovolsky, Joseph G. Shaddock, and Robert H. Heflich.................................................................133 14 Quantifying In Vivo Somatic Mutations Using Transgenic Mouse Model Systems Roy R. Swiger....................................................................................145 15 Methods for Detecting Somatic Mutations In Vitro: The Human T-Cell Cloning Assay Selecting for HPRTMutants Sai-Mei Hou..................................................................................... 155 16 Molecular Analysis of Mutations in the Human HPRT Gene Phouthone Keohavong, Liqiang Xi, and Stephen G. Grant..............161 17 Simultaneous Quantification of t(14;18) and HPRT Exon 2/3 Deletions in Human Lymphocytes James C. Fuscoe................................................................................171 18 The GPA In Vivo Somatic Mutation Assay Stephen G. Grant..............................................................................179 19 Flow Cytometric Measurement of Mutant T Cells With Altered Expression of TCR:Detecting Somatic Mutations in Humans and Mice Seishi Kyoizumi, Yoichiro Kusunoki, and Tomonori Hayashi..........197 PART IV: DETECTION AND CHARACTERIZATION OF CANCER GENE MUTATION 20 Mutation Screening of the TP53Gene by Temporal Temperature Gradient Gel Electrophoresis Therese Sørlie, Hilde Johnsen, Phuong Vu, Guro Elisabeth Lind, Ragnhild Lothe, and Anne-Lise Børresen-Dale.............................207 21 Analysis of K-RAS and P53 Mutations in Sputum Samples Weimin Gao and Phouthone Keohavong.........................................217 22 Allele-Specific Competitive Blocker–PCR Detection of Rare Base Substitution Barbara L. Parsons, Page B. McKinzie, and Robert H. Heflich........235 Contents ix 23 Gel-Based Nonradioactive Single-Strand Conformational Polymorphism and Mutation Detection: Limitations and Solutions Vibhuti Gupta, Reetakshi Arora, Anand Ranjan, Narendra K. Bairwa, Dheeraj K. Malhotra, P. T. Udhayasuriyan, Anjana Saha, and Ramesh Bamezai..............247 24 Detection and Characterization of Oncogene Mutations in Preneoplastic and Early Neoplastic Lesions Toshinari Minamoto..........................................................................263 25 Detection of DNA Double-Strand Breaks and Chromosome Translocations Using Ligation-Mediated PCR and Inverse PCR Michael J. Villalobos, Christopher J. Betti, and Andrew T. M. Vaughan..........................................................279 PART V: ANALYSIS OF DNA REPAIR MECHANISMS 26 Microsatellite Instability: An Indirect Assay to Detect Defects in the Cellular Mismatch Repair Machinery Anjana Saha, Narendra K. Bairwa, and Ramesh Bamezai................293 27 Unscheduled DNA Synthesis: A Functional Assay for Global Genomic Nucleotide Excision Repair Crystal M. Kelly and Jean J. Latimer.................................................303 28 Analysis of DNA Repair Using Transfection-Based Host Cell Reactivation Jennifer M. Johnson and Jean J. Latimer...........................................321 29 An Immunoassay for Measuring Repair of Ultraviolet Photoproducts Shirley McCready..............................................................................337 30 Analysis of DNA Double-Strand Break Repair by Nonhomologous End Joining in Cell-Free Extracts From Mammalian Cells Petra Pfeiffer, Elke Feldmann, Andrea Odersky, Steffi Kuhfittig-Kulle, and Wolfgang Goedecke...........................351 31 Measuring Recombination Proficiency in Mouse Embryonic Stem Cells Andrew J. Pierce and Maria Jasin.....................................................373 PART VI: ARRAY TECHNOLOGIES 32 Strategies for Measurement of Biotransformation Enzyme Gene Expression Marjorie Romkes and Shama C. Buch..............................................387 33 Genotyping Technologies: Application to Biotransformation Enzyme Genetic Polymorphism Screening Marjorie Romkes and Shama C. Buch..............................................399 x Contents 34 TaqManTM Fluorogenic Detection System to Analyze Gene Transcription in Autopsy Material Kaori Shintani-Ishida, Bao-Li Zhu, and Hitoshi Maeda....................415 35 Development of Quantitative Reverse Transcriptase PCR Assays for Measuring Gene Expression Tony E. Godfrey and Lori A. Kelly....................................................423 PART VII: APOPTOSIS 36 Quantification of Selective Phosphatidylserine Oxidation During Apoptosis James P. Fabisiak, Yulia Y. Tyurina, Vladimir A. Tyurin, and Valerian E. Kagan...................................................................449 37 Quantitative Method of Measuring Phosphatidylserine Externalization During Apoptosis Using Electron Paramagnetic Resonance Spectroscopy and Annexin-Conjugated Iron James P. Fabisiak, Grigory G. Borisenko, and Valerian E. Kagan.....457 38 Detection of Programmed Cell Death in Cells Exposed to Genotoxic Agents Using a Caspase Activation Assay Michael E. Gehring and Patrick P. Koty...........................................465 Index............................................................................................................473