In Vitro Toxicity Indicators (eBook)

Front Cover 1
In Vitro Toxicity Indicators, Part B 4
Copyright Page 5
Table of Contents 8
Dedication 6
Contributors 14
Preface 20
Section I: Perspectives 22
Chapter 1. Cellular and Molecular Pathobiology of Reversible and Irreversible Injury 22
INTRODUCTION 22
DEFINITIONS 22
METHODS FOR DETERMINING REVERSIBLE AND IRREVERSIBLE RESPONSES TO INJURY 27
SUBCELLULAR CHARACTERISTICS OF REVERSIBLE INJURY 36
SUBCELLULAR CHARACTERISTICS OF IRREVERSIBLE INJURY 40
CONCLUSIONS 41
SUMMARY 43
ACKNOWLEDGMENTS 44
REFERENCES 44
Chapter 2. Molecular Mechanisms of Toxic Cell Death: An Overview 46
INTRODUCTION 46
DISRUPTION OF CYTOSKELETAL ARCHITECTURE IN TOXIC CELL KILLING 47
MITOCHONDRIAL DAMAGE AND ENERGY DEPLETION 48
GLUTATHIONE AND PROTEIN THIOL MODIFICATIONS 49
Ca2+ OVERLOAD AND ACTIVATION OF Ca2+-DEPENDENT CATABOLIC PROCESSES 50
APOPTOSIS 51
ALTERATIONS IN CELL SIGNALING: Ca2+ SIGNALS 52
PROTEIN KINASE C AND GROWTH STIMULATION 53
CONCLUSIONS 54
REFERENCES 54
Chapter 3. Membrane Lipid Peroxidation: Cellular Mechanisms and Toxicological Implications 56
INTRODUCTION 56
BACKGROUND 56
GENERAL MECHANISMS 59
ROLE OF IRON 60
ROLE OF IRON CHELATORS 61
ROLE OF ENZYMES 62
OCCURRENCE OF MEMBRANE LIPID PEROXIDATION IN VIVO 64
CONSEQUENCES OF MEMBRANE LIPID PEROXIDATION 65
CONCLUSIONS 66
ACKNOWLEDGMENTS 66
REFERENCES 67
Section II: Membrane Integrity and Function 69
Chapter 4. Enzyme Leakage as an Indicator of Cytotoxicity in Cultured Cells 69
INTRODUCTION 69
MATERIALS/REAGENTS 69
PROCEDURES 70
COMMENTS 71
ACKNOWLEDGMENTS 72
REFERENCES 72
Chapter 5. Cellular K+ 73
INTRODUCTION 73
MATERIALS, EQUIPMENT, AND REAGENTS 73
PROCEDURE 74
COMMENTS 77
REFERENCES 79
Chapter 6. Morphological and Biochemical Investigations on Plasma Membrane Blebbing during Cell Injury 81
INTRODUCTION 81
METHODOLOGIES FOR INVESTIGATING CELL BLEBBING 83
MECHANISMS OF OXIDATIVE STRESS-INDUCED MEMBRANE BLEBBING 92
ACNOWLEDGMENTS 93
REFERENCES 94
Chapter 7. Morphological Evaluation of Gap Junctional Intercellular Communication 95
INTRODUCTION 95
MATERIALS 97
PROCEDURE 99
COMMENTS 101
REFERENCES 102
Chapter 8. Fluorescence Microscopic Examination of Cytoskeletal Elements 104
INTRODUCTION 104
MATERIALS AND REAGENTS 105
PROCEDURES 105
COMMENTS 115
REFERENCES 118
Chapter 9. Energy Coupled Na+Cotransport 120
INTRODUCTION 120
MATERIALS AND REAGENTS 121
PROCEDURES 122
COMMENTS 123
REFERENCES 129
Chapter 10. Renal Organic Ion Transport 131
INTRODUCTION 131
MATERIALS AND REAGENTS 132
PROCEDURES 134
COMMENTS 138
ACKNOWLEDGMENTS 143
REFERENCES 143
Chapter 11. Endoplasmic Reticulum Calcium Pump 144
INTRODUCTION 144
MATERIALS AND REAGENTS 146
PROCEDURES 147
COMMENTS 149
ACKNOWLEDGMENT 150
REFERENCES 150
Section III: Energy Metabolism 151
Chapter 12. Measurement of Oxygen Consumption 151
EQUIPMENT 153
CHEMICALS 155
BUFFERS 155
SOLUTIONS 155
MEASUREMENT OF OXYGEN CONSUMPTION 156
DIRECT MEASUREMENT OF MITOCHONDRIAL FUNCTION IN CELLS 159
ACKNOWLEDGMENTS 161
REFERENCES 161
Chapter 13. ATP and Other Energetic Parameters as Indicators of Cell Injury 163
INTRODUCTION 163
MATERIALS AND REAGENTS 164
PROCEDURES 165
RESULTS AND INTERPRETATION 169
COMMENTS 172
ACKNOWLEDGMENTS 173
REFERENCES 173
Chapter 14. Evaluation of Glucose Metabolism in Vitro: Altered Competence of Intermediary Metabolism as an Index of Hepatotoxicity 175
INTRODUCTION 175
MATERIALS AND REAGENTS 176
PROCEDURES 178
COMMENTS 182
ACKNOWLEDGMENT 185
REFERENCES 185
Section IV: DNA-Related Indices 187
Chapter 15. Quantitation of DNA by Fluorometry 187
INTRODUCTION 187
STANDARD ASSAY 188
mRNA EXTRACTION AND DNA QUANTITATION 190
CAPILLARY ASSAY 192
MICROWELL ASSAY 195
LINEAR REGRESSION 197
COMMENTS 198
CONCLUSION 199
ACKNOWLEDGMENTS 200
REFERENCES 200
Chapter 16. Techniques for Measuring Cell Proliferation 201
INTRODUCTION 201
APPLICATIONS 205
MATERIALS 207
INSTRUMENTS 211
PROCEDURES 212
CONTROLS AND STANDARDS 218
CRITICAL ASPECTS OF THE PROCEDURES 219
SUMMARY 222
ACKNOWLEDGMENT 222
REFERENCES 222
Chapter 17. Nuclease P1-Enhanced 32P-Postlabeling Assay for Aromatic Carcinogen-DNA Adducts 224
INTRODUCTION 224
MATERIALS AND REAGENTS 225
PROCEDURES 230
TLC (12,15) 232
COMMENTS 235
ACKNOWLEDGMENTS 237
REFERENCES 237
Chapter 18. Genomic Hypomethylation 239
INTRODUCTION 239
MATERIALS AND REAGENTS 240
PROCEDURES 242
TLC 244
COMMENTS 249
REFERENCES 250
Chapter 19. DNA Degradation: Double-Strand Breaks 251
INTRODUCTION 251
MATERIALS AND REAGENTS 253
EQUIPMENT 254
PROCEDURES 255
REFERENCES 259
Chapter 20. Genotypic Mutation Analysis by RFLP/PCR 260
INTRODUCTION 260
PROCEDURES 261
COMMENTS: LIMITATIONS OF MUTATION ANALYSIS BY RFLP/PCR 268
ACKNOWLEDGMENTS 269
REFERENCES 270
Section V: 
271 
Chapter 21. Gluconeogenesis in Liver Cells 271
REGULATION OF GLUCONEOGENESIS 274
CHOICE OF ASSAYS FOR GLUCONEOGENESIS IN LIVER CELLS 274
PROTOCOL FOR DETERMINATION OF LIVER CELL GLUCONEOGENESIS 275
COMMENTARY 277
REFERENCES 279
Chapter 22. Measurement of Protein Synthesis 281
MEASUREMENT OF PROTEIN SYNTHESIS BY RADIOACTIVE AMINO ACID INCORPORATION 281
EVALUATION OF PROTEIN SYNTHESIS BY ELECTROPHORESIS AND FLUOROGRAPHY 285
SUMMARY 291
ACKNOWLEDGMENT 291
REFERENCES 292
Chapter 23. Measuring Collagen Synthesis 293
INTRODUCTION 293
MATERIALS AND METHODS 294
COMMENTS 298
REFERENCES 301
Chapter 24. Measurement of Macrophage Phagocytosis Using a Sheep Red Blood Cell Assay 302
INTRODUCTION 302
MATERIALS AND REAGENTS 303
PROCEDURES 303
COMMENTS 307
ACKNOWLEDGMENTS 308
REFERENCES 309
Section VI: Biochemical Indices 310
Chapter 25. Cell Function Studies Using Fluorescent Ca2+ Indicators 310
INTRODUCTION 310
PRINCIPLES OF FLUORESCENT Ca2+ INDICATOR USE 310
RELATIVE ADVANTAGES OF FLUORESCENT Ca2+ INDICATORS COMPARED TO OTHER TECHNIQUES 313
PROPERTIES OF AVAILABLE Ca2+ INDICATORS 314
FLUORESCENCE MEASUREMENT SYSTEMS 319
METHODS FOR LOADING DYES INTO CELLS 323
CALIBRATION OF Ca2+ SIGNALS 323
POTENTIAL DIFFICULTIES IN THE USE AND INTERPRETATION OF Ca2+ INDICATOR DATA 329
MEASUREMENTS OF Ca2+ CHANGES DURING CELL INJURY 334
ACKNOWLEDGEMENT 336
REFERENCES 336
Chapter 26. Ca2+ -Bioluminescent Indicators 338
INTRODUCTION 338
METHOD 339
INTRACELLULAR INCORPORATION OF AEQUORIN 339
MEASUREMENT AEQUORIN LUMINESCENCE 341
APPLICATIONS 345
COMPARISON OF TWO CLASSES OF Ca2+ INDICATORS 347
REFERENCES 348
Chapter 27. Measurement of Receptor-Activated Accumulation of Inositol Phosphates as an Index of Phospholipase C Activity 351
INTRODUCTION 351
MATERIALS 352
USE OF [3H]INOSITOL TO MEASURE LEVELS OF [3H]INOSITOL PHOSPHATES IN ISOLATED CELLS AND TISSUE SLICES 353
METHODOLOGY FOR QUENCHING INCUBATIONS AND EXTRACTING THE SOLUBLE INOSITOL PHOSPHATES 356
BATCH ELUTION OF INOSITOL PHOSPHATES FROM GRAVITY-FED ION-EXCHANGE COLUMNS 357
HPLC ANALYSIS OF INOSITOL PHOSPHATES 359
REFERENCES 361
Chapter 28. Covalent Binding to Proteins 363
INTRODUCTION 363
MATERIALS 364
PROCEDURES 365
COMMENTS 368
REFERENCES 370
Chapter 29. Glutathione, Oxidized Glutathione, and Mixed Disulfides in Biological Samples 372
INTRODUCTION 372
SAMPLE PREPARATION 373
METHOD I: DETERMINATION OF GSH AND GSSG BY ENZYMATIC RECYCLING 374
METHOD II: MONOBROMOBIMANE LABELING OF GSH FOLLOWED BY HPLC/FLUORESCENCE DETECTION 378
METHOD III: HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DUAL ELECTROCHEMICAL DETECTION 382
METHOD IV: FLOW-CYTOMETRIC EVALUATION OF GLUTATHIONE 385
OTHER METHODS 387
REFERENCES 388
Section VII: Lipid Peroxidation 390
Chapter 30. Thiobarbituric Acid Assay Reactants 390
INTRODUCTION 390
ASSAYS FOR LIPID PEROXIDATION 391
MECHANISM OF LIPID PEROXIDATION 392
APPLICATION OF THE TBA ASSAY TO IN VITRO LIPID PEROXIDATION STUDIES 395
METHOD: TBA REAGENT 396
PROCEDURE 396
COMMENTS 396
REFERENCES 397
Chapter 31. Volatile Hydrocarbons 400
INTRODUCTION 400
PROCEDURES 400
COMMENTS 404
REFERENCES 405
Chapter 32. Electronically Excited State Formation 407
INTRODUCTION 407
METHODOLOGY 408
FORMATION OF ELECTRONICALLY EXCITED STATES DURING LIPID PEROXIDATION 411
LIPID PEROXIDATION AND LOW-LEVEL CHEMILUMINESCENCE 414
COMMENTS 418
REFERENCES 419
Chapter 33. Spectrophotometric Detection of Lipid-Conjugated Dienes 423
DIFFERENCE SPECTRUM METHOD 424
ANALYSIS FOR CONCENTRATION OF LIPID 427
REFERENCES 429
Chapter 34. Quantitation of Conjugated Dienes by a Diels-Alder Reaction 430
INTRODUCTION 430
MATERIALS AND REAGENTS 431
EQUIPMENT 431
EXTRACTION OF LIPID FROM TISSUE 432
SEPARATION OF PHOSPHOLIPID FROM TOTAL LIPID 432
PHOSPHATE ASSAY FOR PHOSPHOLIPID CONTENT 433
HYDROLYSIS OF PHOSPHOLIPID WITH LITHIUM ALUMINUM HYDRIDE 433
REACTION OF [14C]-TCNE WITH LIPID-CONJUGATED DIENES 434
SYNTHESIS OF [l,2-14C]TETRACYANOETHYLENE (OR TETRA-[14C]CYANOETHYLENE) 435
ACKNOWLEDGMENTS 437
REFERENCES 437
Chapter 35. Quantitation of Conjugated Dienes by Second-Derivative UV Spectroscopy 438
INTRODUCTION 438
CONSTRUCTION OF A STANDARD CURVE AND OPTIMIZATION OF INSTRUMENT SETTINGS 439
ANALYSIS OF BIOLOGICAL LIPID SAMPLES 440
REFERENCES 442
Section VIII: Specialized Techniques 444
Chapter 36. A CENTRIFUGE FILTRATION SEPARATION OF CELLS, AND THE MEASUREMENT OF CONSTITUENTS 444
INTRODUCTION 444
SAMPLE PREPARATION 447
APPLICATION OF THE CENTRIFUGATION TECHNIQUE TO THE MEASUREMENT OF INTRACELLULAR CONSTITUENTS 448
COMMENTS 451
ACKNOWLEDGMENTS 452
REFERENCES 452
Chapter 37. Flow Cytometry 454
INTRODUCTION 454
MEASUREMENT OF ANTIBODY BINDING BY DIRECT OR INDIRECT IMMUNOFLUORESCENCE 454
DNA CELL CYCLE ANALYSIS AND PLOIDY DETERMINATION USING PROPIDIUM IODIDE 457
MEASUREMENT OF INTRACELLULAR OXIDATIVE METABOLISM USING DICHLOROFLUORESCIN DIACETATE (DCFH-DA) 458
REFERENCES 460
Chapter 38. Single-Cell Microscopic Techniques for Studying Toxic Injury 461
INTRODUCTION 461
EQUIPMENT 461
BRIGHT-FIELD MICROSCOPY 463
FLUORESCENT PROBES 463
CELL VIABILITY 464
CELL SIZE AND SHAPE 466
ORGANELLE MARKERS 467
PROBES FOR FREE THIOLS AND REACTIVE OXYGEN SPECIES 467
OXYGEN RADICAL PROBES 468
ION-INDICATING PROBES 468
SUBCELLULAR LOCALIZATION 472
CONFOCAL MICROSCOPY 473
COMMENTS 476
ACKNOWLEDGMENTS 477
REFERENCES 477
Chapter 39. Measurement of Macrophage and Neutrophil Chemotaxis 479
INTRODUCTION 479
METHOD I: MEASUREMENT OF CHEMOTAXIS IN MICROWELL CHAMBERS 480
PROCEDURE 481
DISTINGUISHING BETWEEN CHEMOTAXIS AND CHEMOKINESIS 482
METHOD II: MEASUREMENT OF CHEMOTAXIS UNDER AGAROSE 484
COMMENTS 485
REFERENCES 485
Chapter 40. Two-Dimensional Electrophoresis as Applied to Problems of in Vitro and in Vivo Toxicology 486
INTRODUCTION 486
MATERIALS AND REAGENTS 487
PROCEDURE 488
COMMENTS 495
REFERENCES 496
Index 498