Table Of ContentMUTAGENICITY:
ASSAYS AND
APPLICATIONS
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MUTAGENICITY:
ASSAYS AND
APPLICATIONS
Edited by
ASHUTOSH KUMAR
VASILY N. DOBROVOLSKY
ALOK DHAWAN
RISHI SHANKER
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CONTENTS
List of Contributors xi
Foreword xv
Preface xvii
Acknowledgments xix
1. Mutagenesis, Genetic Disorders and Diseases 1
Manisha Dixit and Amit Kumar
1. Introduction 1
2. Mutagens 2
3. Mutations 3
4. DNA Damage Response and Repair 9
5. DNA Repair Pathways 10
6. Genetic Disorders and Diseases 20
7. Summary 30
Acknowledgment 31
References 31
2. Detection of Mutation in Prokaryotic Cells 35
Ashutosh Kumar, Rishi Shanker, and Alok Dhawan
1. Introduction 35
2. Materials and Methodologies 38
3. Result Evaluation 44
4. Result Interpretation 44
5. Experimental Design 45
6. Precaution 46
References 47
Further Reading 48
3. Detection of Gene Mutation in Cultured Mammalian Cells 49
Jayant Dewangan, Prabhash Kumar Pandey, Aman Divakar, Sakshi Mishra,
Sonal Srivastava, and Srikanta Kumar Rath
1. Introduction 49
2. PCR-Based Mutation Detection 50
3. Detection of Mutations by Denaturing Gradient Gel Electrophoresis 52
4. Single-Strand Conformation Polymorphism 53
5. Heteroduplex Analysis 54
v
vi Contents
6. Microarray 55
7. Arrayed Primer Extension Technology 56
8. Single-Base Extension-TAGS Technology 56
9. Atomic Force Microscopy 57
10. Fluorescence In Situ Hybridization 57
11. DNA Sequencing 59
12. Karyotyping 60
13. Hypoxanthine Phosphoribosyl Transferase, Thymidine Kinase, and Xanthine–
Guanine Phosphoribosyl Transferase Gene Mutation Detection Methods 60
14. Conclusion 61
Acknowledgment 62
References 62
4. Chromosomal Aberrations 69
Abhishek K. Jain, Divya Singh, Kavita Dubey, Renuka Maurya,
and Alok K. Pandey
1. Introduction 69
2. Characteristics of Human Chromosomes 70
3. Sources of Specimen for Cytogenetic Analysis 71
4. Cytogenetic Analysis 71
5. Types of Chromosome Aberration 72
6. Mechanism of the Formation of Chromosome Aberration 76
7. Causes of Chromosome Aberration 78
8. Frequency of Chromosome Aberration 80
9. Methods for Detection of Chromosomal Aberration 81
10. Clinical Manifestation of Chromosomal Abnormalities 85
11. Applications of Chromosomal Aberration Analyses 88
12. Future Perspective 89
13. Conclusion 90
References 90
5. In Vivo Cytogenetic Assays 93
Pasquale Mosesso and Serena Cinelli
1. Introduction 93
2. Cytogenetic End points 95
3. Treatment Protocols 96
4. Analysis 100
References 108
Contents vii
6. Mutagenicity and Genotoxicity Testing in Environmental Pollution
Control 113
Amit Bafana, Kannan Krishnamurthi, Saravanadevi Sivanesan,
and Pravin K. Naoghare
1. Introduction 113
2. Types and Sources of Pollution 114
3. Impact of Environmental Pollution 117
4. Need of Mutagenicity Assays to Assess Environmental Pollution 123
5. Application of Mutagenicity Assays to Control
Environmental Pollution 125
References 128
7. Mutagens in Food 133
Payal Mandal, Ankita Rai, Sakshi Mishra, Anurag Tripathi, and Mukul Das
1. Introduction 133
2. Additives 134
3. Contaminants 138
4. Adulterants in Food 145
5. Mutagens Produced as an Outcome of Food Processing 148
6. Summary 151
Acknowledgments 151
References 151
Further Reading 160
8. Emerging Computational Methods for Predicting Chemically
Induced Mutagenicity 161
Shraddha Pandit, Alok Dhawan, and Ramakrishnan Parthasarathi
1. Introduction 161
2. Relationship Between Mutagens and Their Ability to Cause Cancer 162
3. Crucial Aspects of Computational Predictive Modeling 163
4. In Silico Tools and Techniques for Chemical Mutagenic
Prediction 164
5. Framework of Computational Approach for Understanding
Mutagenicity 166
6. Summary 173
Acknowledgments 174
References 174
viii Contents
9. Overview of Nonclinical Aspects for Investigational New Drugs
Submission: Regulatory Perspectives 177
Ranjeet Prasad Dash and Manish Nivsarkar
1. Introduction 177
2. Target Identification 179
3. Target Validation 179
4. Hit Series Identification 179
5. Assay Development 180
6. Hit to Lead Identification 180
7. Lead Optimization 180
8. Investigational New Drug Submission 181
9. Conclusions 187
References 188
10. Mutagenicity Testing: Regulatory Guidelines and
Current Needs 191
Rajesh Sundar, Mukul R. Jain, and Darshan Valani
1. B ackground 191
2. Mutagenicity Endpoints 192
3. Regulatory Framework 194
4. Regulatory Studies for Mutagenicity Testing 197
5. Regulatory Requirements for Registration 198
6. Predictive Mutagenicity Testing 210
7. Limitations and Challenges of Mutagenicity Assessment 210
8. Current Needs in Mutagenicity Testing 217
References 223
Further Reading 228
11. Detecting Mutations In Vivo 229
Vasily N. Dobrovolsky and Robert H. Heflich
1. I ntroduction 229
2. Regulatory Applications of Mutation Data 230
3. Detection of Mutation in the Hprt Gene 233
4. Detection of Mutation in the Tk1 and Aprt Genes 236
5. T ransgenic Models for Detecting In Vivo Mutation 237
6. D etection of Mutation in the Endogenous Pig-a Gene 239
7. T he Future of In Vivo Mutation Detection 245
Disclaimer 247
References 247
Contents ix
12. An In Vitro Male Germ Cell Assay and Its Application for
Detecting Phase Specificity of Genotoxins/Mutagens 251
Khaled Habas, Martin H. Brinkworth, and Diana Anderson
1. I ntroduction 251
2. S permatogenesis 252
3. A ssessment of Mutagenic Agents Causing DNA Damage in Male Germ
Cells in the STA-PUT System 254
4. M ethods Existing for Detection Signals From Potential Germ Cell
Mutagens 256
5. M utagenicity and Genotoxicity Testing in Germ Cells Using STA-PUT
Velocity Sedimentation 257
6. P rotocol of the STA-PUT System 258
7. S etting Up the STA-PUT Apparatus 259
8. I solating Spermatogenic Cells From
Whole Testes 259
9. C ell Loading and Sedimentation 260
10. A nalysis of Fractions 260
11. C oncluding Remarks 260
References 262
13. Fluorescence In Situ Hybridization in Genotoxicity Testing 265
Dayton M. Petibone and Wei Ding
1. I ntroduction 265
2. Integration of FISH Into the Comet Assay 268
3. Integration of FISH With the Micronucleus Assay 272
4. Application of FISH to Detect Structural Chromosome
Aberrations 276
5. Conclusions 282
Disclaimer 282
References 282
14. DNA Damage, Repair, and Maintenance of Telomere Length:
Role of Nutritional Supplements 287
Krupa Kansara and Souvik Sen Gupta
1. I ntroduction: DNA Repair and Telomere 287
2. V itamins: DNA Repair and Telomere Length 289
3. M inerals: DNA Repair and Telomere Length 295
4. O ther Bioactive Dietary Components and Telomere Length 297
