Molecular Toxicology Protocols

477 Pages·2005·3.326 MB·English

by  David H. Phillips, Alan Hewer, Volker M. Arlt (auth.), Phouthone Keohavong, Stephen G. Grant (eds.)

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™
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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™
Molecular Toxicology
Protocols
Edited by
Phouthone Keohavong
Stephen G. Grant
Department of Environmental and Occupational Health
University of Pittsburgh, Pittsburgh, PA
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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