High-Throughput Screening Methods in Toxicity Testing

HIGH-THROUGHPUT SCREENING METHODS IN TOXICITY TESTING......Page 3
CONTENTS......Page 7
PREFACE......Page 11
CONTRIBUTORS......Page 13
PART I GENERAL ASPECTS......Page 19
1.1 INTRODUCTION......Page 21
1.2.2 Pesticidal Inerts......Page 24
1.2.5 Food Additives/Ingredients......Page 25
1.2.6 Water Contaminants......Page 26
1.3 THE CHEMICAL LIBRARIES......Page 27
1.4 THE BIOLOGICAL ASSAYS......Page 29
1.5 IN VIVO TOXICITY DATABASE......Page 34
1.6 PREDICTIVE MODELS......Page 38
1.7 CHEMICAL PRIORITIZATION......Page 42
1.8 TARGETED TESTING......Page 43
1.9 CONCLUSION......Page 44
REFERENCES......Page 45
2.1 INTRODUCTION......Page 51
2.2 DRUG TOXICITY FAILURE IN (PRE)CLINICAL DEVELOPMENT......Page 52
2.3 STRATEGY FOR IMPLEMENTATION OF IN VITRO TOXICITY TESTING......Page 53
2.4 ASSAYS FOR IN VITRO GENOTOXICITY SCREENING......Page 56
2.5 ASSAYS FOR IN VITRO CYTOTOXICITY SCREENING......Page 59
2.6 ASSAYS FOR NUCLEAR RECEPTOR-INDUCED ACTIVATION OF PHASE I AND II ENZYMES......Page 64
2.7 ASSAYS FOR NUCLEAR STEROID RECEPTOR ACTIVATION AND ENDOCRINE DISRUPTION......Page 68
2.8 ASSAYS FOR NUCLEAR RECEPTOR ACTIVATION AND NON-GENOTOXIC CARCINOGENICITY......Page 70
2.9 ASSAYS FOR NUCLEAR RECEPTOR ACTIVATION AND EMBRYOTOXICITY......Page 71
2.10 TOXICOGENOMICS: A DIFFERENT MULTIPLE SIZE HTS FORMAT TO STUDY MECHANISMS OF ACTION......Page 75
2.11 CONCLUSIONS......Page 76
REFERENCES......Page 77
3.1 INTRODUCTION......Page 95
3.2.1 Approach and Design of the ToxCast Research Project......Page 97
3.2.2 Application of Pharmacokinetic Data in the In Vitro-to-In Vivo Extrapolation of Oral Equivalent Doses......Page 98
3.2.3 Estimation of Human Oral Exposures......Page 100
3.3.1 Comparison of the IVIVE Determinations with Existing In Vivo Data......Page 101
3.3.2 Comparison of Dosimetry-Adjusted Oral Equivalent Doses and Exposure Estimates......Page 102
3.4 DISCUSSION......Page 107
FUNDING......Page 108
REFERENCES......Page 109
4.1 INTRODUCTION......Page 115
4.2 POTENTIAL ROLE OF hESCs IN HIGH-THROUGHPUT SCREENING STRATEGIES FOR TOXICITY......Page 116
4.3.1 hESC Applications for Cardiotoxicity HTS......Page 117
4.3.2 hESC Applications for Neurotoxicity HTS......Page 118
4.3.3 hESC Applications for Hepatoxicity HTS......Page 119
ACKNOWLEDGMENTS......Page 120
REFERENCES......Page 121
PART II HIGH-THROUGHPUT ASSAYS TO ASSESS DIFFERENT CYTOTOXICITY ENDPOINTS......Page 125
5.1 INTRODUCTION......Page 127
5.2 BASIC CONSIDERATIONS FOR MEASUREMENTS OF CELLULAR HEALTH......Page 128
5.3.1 Metabolism-Based Assays......Page 131
5.3.2 Nonmetabolism-Based Assays......Page 133
5.4.1 Examination of Primary Cytotoxicity (for MOA)......Page 136
5.4.2 Secondary Examination of Cytotoxicity (Normalization)......Page 139
5.5 SUMMARY......Page 140
REFERENCES......Page 142
6 HIGH-THROUGHPUT FLOW CYTOMETRY ANALYSIS OF APOPTOSIS......Page 147
REFERENCES......Page 158
7.1 INTRODUCTION......Page 161
7.2 AUTOMATED IMAGING: PREDEFINED UNBIASED MICROSCOPY FIT FOR HCS......Page 162
7.3 END-POINT HCS ASSAYS......Page 165
7.4 TIME-LAPSE MICROSCOPY OF APOPTOSIS......Page 167
7.5 APPLICATION OF LIVE APOPTOSIS IMAGING TO FUNCTIONAL GENOMICS SCREENING......Page 168
7.6 TIME-LAPSE MICROSCOPY OF CELL STRESS DYNAMICS......Page 169
7.7 HIGH CONTENT ANALYSIS......Page 171
REFERENCES......Page 172
8.1 SKIN SENSITIZATION AND THE NEED TO SCREEN NOVEL CHEMICALS......Page 177
8.2 THE MECHANISTIC BACKGROUND OF THE KERATINOSENS ASSAY......Page 179
8.3 THE CREATION OF THE KERATINOSENS REPORTER CELL LINE......Page 180
8.4 THE STANDARD OPERATING PROCEDURE OF THE KERATINOSENS ASSAY......Page 181
8.5 DATA ANALYSIS AND PREDICTION MODEL......Page 182
8.7 THE PREDICTIVITY FOR STANDARD LISTS OF REFERENCE CHEMICALS......Page 184
8.8 THE APPLICABILITY DOMAIN AND LIMITATIONS OF THE ASSAYS AS DETERMINED THROUGH THE SCREENING......Page 185
8.9 CASE STUDIES ON SPECIFIC CHEMICAL CLASSES......Page 186
8.10 THE USE OF THE ASSAY IN AN INTEGRATED TESTING STRATEGY (ITS) FOR A COMPLETE REPLACEMENT OF ANIMAL TESTING......Page 188
REFERENCES......Page 189
9.1 INTRODUCTION......Page 195
9.2 PHOTOTOXIC PATHWAYS......Page 196
9.3 SCREENING SYSTEMS FOR PHOTOSAFETY ASSESSMENT......Page 197
9.3.2 Photochemical Evaluation......Page 198
9.3.3 In Vitro Phototoxic Assessment......Page 199
9.4 3T3 NRU PHOTOXICITY TEST......Page 200
9.5.1 Theoretical Basis of the ROS Assay and Assay Reliability......Page 201
9.5.2 Prediction Capacity......Page 202
REFERENCES......Page 205
PART III HIGH-THROUGHPUT ASSAYS TO ASSESS DNA DAMAGE AND CARCINOGENESIS......Page 209
10.1 INTRODUCTION......Page 211
10.2.1 Principle of the AmesTM II Assay......Page 213
10.2.3 Strain Genotypes......Page 214
10.2.5 Advantages of the Ames IITM System......Page 215
10.2.7 Detailed Method......Page 216
10.3 AMES LIQUID FORMAT ASSAY......Page 221
10.3.2 Advantages of the Ames Liquid Format Assay......Page 223
10.3.4 Detailed Method......Page 224
REFERENCES......Page 228
11.1 INTRODUCTION......Page 231
11.3 PRINCIPLE OF THE VITOTOX TEST......Page 232
11.4 CONSTRUCTION OF recN-luxCDABE FUSIONS......Page 233
11.5 CONSTRUCTION OF pr1-LUXCDABE FUSION......Page 234
11.6 VITOTOXR  TEST PROCEDURE......Page 235
11.7 EXAMPLES OF VITOTOX TEST RESULTS......Page 236
11.8.1 Testing Chemicals with Known Genotoxic Properties and New Drug Candidates......Page 239
11.8.2 Testing Secondary Metabolites and Crude Extracts of Biological Control Agents......Page 240
11.8.3 Further Comparative Studies: Technotox Workshop......Page 241
11.8.4 Further Comparative Studies: EILATox-Oregon Workshop......Page 243
11.8.5 Environmental Samples......Page 244
11.8.6 Smoke-Water and Isolated Smoke-Water Compounds......Page 245
11.8.8 Investigating Genotoxicity of Nonionizing Radiation......Page 246
REFERENCES......Page 247
12.1 INTRODUCTION......Page 251
12.2 GENOTOXICITY......Page 253
12.4 REGULATORY TESTS TO DETECT PHARMACEUTICALS WITH GENOTOXIC AND CARCINOGENIC POTENTIAL......Page 254
12.4.1 The Ames Assay......Page 256
12.4.3 The Mouse Lymphoma TK Assay (MLA)......Page 257
12.4.5 The Alkaline Comet Assay (Single-Cell Gel Electrophoresis Assay)......Page 258
12.4.6 The Impact of Positive Findings for Genotoxic Potential and Follow-up Testing Strategies......Page 259
12.5 SCREENING FOR COMPOUNDS WITH GENOTOXIC AND CARCINOGENIC POTENTIAL WITHIN THE LEAD OPTIMIZATION PHASE OF DRUG DEVELOPMENT......Page 262
12.5.1 The Detection of Genotoxicity with Bacterial Screens......Page 264
12.5.3 The Detection of Genotoxicity with Rodent and Human Cell Line-Based Screens......Page 265
12.5.4 Human HepG2 and TK6 Cells......Page 266
12.5.5 HCS Micronucleus Assays in the Rodent Cell Line CHO-k1 and the Human Cell Line HepG2......Page 269
12.5.7 Luminescence-Based Reporter Assays in the HepG2 Cell Line......Page 270
12.6.1 Sensitivity and Specificity of Combinations of Regulatory In Vitro Genotoxicity Assays......Page 273
12.6.2 Sensitivity and Specificity of Combinations of the Novel Higher Throughput Genotoxicity Assays and Comparison with the Regulatory In Vitro Screening Battery......Page 277
REFERENCES......Page 281
13 HIGH-THROUGHPUT GENOTOXICITY TESTING: THE GREENSCREEN ASSAY......Page 289
REFERENCES......Page 299
14.1.1 Procedure......Page 303
14.2 FLUORIMETRIC DETECTION OF ALKALINE DNA UNWINDING......Page 304
14.2.3 Automation and High Throughput......Page 306
14.3.3 Automation and High Throughput......Page 307
REFERENCES......Page 308
15.1 INTRODUCTION......Page 313
15.2 DESCRIPTION OF THE DIFFERENT VERSIONS OF THE HIGH-THROUGHPUT COMET ASSAY......Page 314
15.3 VALIDATION OF THE HIGH-THROUGHPUT ASSAYS......Page 317
15.4 ADVANTAGES OF THE HIGH-THROUGHPUT COMET ASSAYS COMPARED TO THE CONVENTIONAL VERSION......Page 318
15.5 APPLICATION AREAS......Page 319
15.6 COMPARISON OF THE HIGH-THROUGHPUT COMET ASSAY AND THE HIGH-THROUGHPUT MICRONUCLEUS TEST......Page 322
REFERENCES......Page 323
16.1 INTRODUCTION......Page 327
16.2 96-WELL SOFT AGAR COLONY FORMATION ASSAY......Page 328
16.3 384-WELL SOFT AGAR COLONY FORMATION ASSAY......Page 329
16.4 AUTOMATED 96-WELL SOFT AGAR COLONY FORMATION ASSAY......Page 330
REFERENCES......Page 333
17.1 INTRODUCTION......Page 335
17.2.2 Reagents......Page 337
17.3 REAGENT SETUP......Page 338
17.4.1 Overview of the Bhas 42 Cell Transformation Assay by the Hydrogen Peroxide Method......Page 339
17.4.2 Solubility Test......Page 341
17.4.4 Cell Culture and Passage......Page 342
17.4.6 Medium Change......Page 343
17.4.7 Preliminary Cell Growth Assay......Page 344
17.4.8 Initiation Assay......Page 345
17.4.9 Promotion Assay......Page 347
17.4.10 Measuring Fluorescence Using AlamarBlue......Page 349
17.5.1 Procedures......Page 350
17.5.2 Examples of Results Obtained......Page 351
17.6 DISCUSSION......Page 352
REFERENCES......Page 354
PART IV HIGH-THROUGHPUT ASSAYS TO ASSESS REPRODUCTIVE TOXICITY, CARDIOTOXICITY, AND HAEMATOTOXICITY......Page 359
18.1 INTRODUCTION......Page 361
18.2 ESTABLISHING THE ReProGlo ASSAY PROTOCOL......Page 362
18.3 PARTICIPATION IN THE ReProTect FEASIBILITY STUDY......Page 366
18.5 INTEGRATION OF A METABOLIC ACTIVATION SYSTEM......Page 368
18.6 CONCLUSIONS......Page 370
REFERENCES......Page 371
19.1 INTRODUCTION......Page 375
19.2 THE IMPLEMENTATION AND ASSESSMENT OF FOCUSED MOLECULAR-BASED ENDPOINTS IN THE EST......Page 377
19.3 IMPLEMENTING GLOBAL MOLECULAR-BASED ENDPOINTS IN THE EST......Page 379
19.4 COMPARISON OF EST TO TRADITIONAL DEVELOPMENTAL TOXICITY IN VIVO DATA......Page 381
19.5 COMPARISONS ACROSS IN VITRO AND IN VIVO MODELS USING OMIC APPROACHES......Page 382
19.6 CONCLUSION......Page 383
REFERENCES......Page 385
20.1 INTRODUCTION......Page 389
20.3 DEVELOPMENTAL CONCORDANCE......Page 390
20.4 EXAMPLES OF SCREENS AND VALIDATION APPROACHES......Page 391
20.5 THE QUESTION OF BIOAVAILABILITY......Page 394
REFERENCES......Page 397
21.1 INTRODUCTION......Page 403
21.2.1 Introduction......Page 404
21.2.2 Construction of a Single Cell Imaging Cytometry System......Page 407
21.2.3 Data Acquisition of Single Cell Imaging Cytometry......Page 408
21.3.1 Potassium Ion Channel......Page 409
21.3.2 Concentration of Intracellular Sodium Ion......Page 413
21.3.3 Cell Death: Apoptosis and Necrosis......Page 414
21.4 APPLICATION OF HCS TO ANALYZE DRUG-INDUCED CARDIOTOXICITY BY USING A SINGLE CELL IMAGING CYTOMETRY SYSTEM AND FLUORESCENT INDICATORS......Page 417
REFERENCES......Page 419
22.1 INTRODUCTION......Page 421
22.2 THE MICROELECTRODE ARRAY SYSTEMS......Page 427
22.3 THE USE OF CELLULAR OXYGEN UPTAKE RATES FOR MONITORING THE STATE OF CARDIOMYOCYTES......Page 432
22.5 CONCLUSIONS......Page 434
REFERENCES......Page 435
23.1 INTRODUCTION......Page 439
23.2 HEMATOPOIESIS......Page 440
23.5 LIQUID CULTURE ASSAYS......Page 441
23.8 THE HALOR  SYSTEM......Page 442
23.10 APPLICATIONS OF HIGH-THROUGHPUT ASSAYS TO ESTIMATE THE HEMATOTOXIC POTENTIAL OF DRUGS......Page 443
23.11 LIMITATIONS OF IN VITRO ASSAYS TO ESTIMATE THE HEMATOTOXIC POTENTIAL OF DRUGS......Page 444
REFERENCES......Page 445
PART V HIGH-THROUGHPUT ASSAYS TO ASSESS DRUG METABOLISM AND RECEPTOR-RELATED TOXICITY......Page 449
24.1 INTRODUCTION TO METABOLITE-BASED TOXICITY TESTING......Page 451
24.2 BIOCOLLOID REACTOR PARTICLES......Page 455
24.3 HIGH-THROUGHPUT ANALYSIS WITH BIOCOLLOID REACTORS......Page 461
24.4 SUMMARY AND FUTURE PROSPECTS......Page 466
REFERENCES......Page 467
25.2 CYTOCHROME P450 INHIBITION METHODS......Page 471
25.2.1 Cytochrome P450 Inhibition Testing with Fluorimetric and Luminometric Substrates......Page 472
25.2.2 Cytochrome P450 Inhibition Testing Using Drug Probe Substrates and Human Liver Microsomes......Page 474
25.2.3 Time-Dependent Inhibition of Cytochrome P450 and Toxicity......Page 476
25.2.4 Methods to Detect Time-Dependent Inhibition of Cytochrome P450 In Vitro......Page 477
25.3 CYTOCHROME P450 INDUCTION METHODS......Page 478
25.3.2 Cytochrome P450 Induction as a Cause of Drug Interactions......Page 479
25.3.3 Mechanisms of Cytochrome P450 Induction......Page 480
25.3.4 Assays to Detect Human Cytochrome P450 Induction......Page 481
25.3.5 The Measurement of Enzyme Activity as a Cytochrome P450 Induction End Point......Page 482
25.3.6 The Measurement of mRNA as a Cytochrome P450 End Point......Page 484
25.3.7 Parameters Affecting Outcomes in Human Hepatocyte Induction Assays......Page 485
25.4 CYTOCHROME P450 INHIBITION AND INDUCTION BY THERAPEUTIC PROTEINS......Page 486
REFERENCES......Page 487
26.1 INTRODUCTION......Page 497
26.2 YEAST AS A NUCLEAR RECEPTOR STUDY ORGANISM......Page 498
26.3 PRINCIPLE OF NUCLEAR RECEPTOR YEAST ASSAYS......Page 500
26.4.1 Estrogen Receptor Assays......Page 502
26.4.2 Androgen Receptor Assays......Page 504
26.4.4 Aryl Hydrocarbon Receptor Assays......Page 506
26.4.5 Retinoid Receptor Assays......Page 509
26.5 YEAST NUCLEAR RECEPTOR ASSAYS AND HIGH-THROUGHPUT SCREENING......Page 510
REFERENCES......Page 511
27.1 INTRODUCTION......Page 519
27.2.1 Other Beneficial Peroxisomal Functions and Human Disease......Page 520
27.2.3 Alternative Cell Models......Page 521
27.3.1 Cell Plating......Page 522
27.3.2 Compound Addition......Page 523
27.4.1 Hoechst-33342 for Nuclear and Cytoplasmic Delineation......Page 524
27.5 ASSAY VALIDATION......Page 526
27.6 IMPLEMENTATION OF NOVEL PHOTOSWITCHABLE/ PHOTOBLEACHABLE FLUORESCENT REPORTERS FOR HIGH CONTENT INVESTIGATION OF PROTEIN DYNAMICS......Page 527
27.7 COMPOUND LIBRARIES FOR ASSAY DEVELOPMENT......Page 529
27.8 EXPLORATORY MULTIVARIATE DATA ANALYSIS......Page 530
27.9 DISCUSSION AND SUMMARY......Page 532
REFERENCES......Page 533
28.1 BACKGROUND......Page 537
28.2 GENERAL APPROACH......Page 538
28.3 PRINCIPLE OF CALUXR  REPORTER GENE ASSAYS TO MEASURE MAJOR TOXICITY PATHWAYS......Page 539
28.4 SELECTIVE REPORTER GENE ASSAYS IN THE CALUXR  PANEL......Page 540
28.6 METABOLISM......Page 541
28.7 APPLICATIONS OF PANELS OF CALUX  REPORTER GENE ASSAYS......Page 544
28.8 HIGH-THROUGHPUT (HTP) SCREENING......Page 546
28.9 QUALITY CONTROL AND VALIDATION OF HIGH-THROUGHPUT METHODS......Page 547
REFERENCES......Page 548
29.1.1 Dioxin Contaminations......Page 551
29.1.3 Toxic Equivalency Factors and European Regulation of Food and Feed Dioxin Contamination......Page 552
29.2.2 The DR-CALUX  Reporter Gene Assay......Page 554
29.2.3 Perspectives of the DR-CALUX......Page 556
29.2.4 Improvements and Further Development......Page 558
29.3 CONCLUSIONS AND OUTLOOK......Page 559
REFERENCES......Page 561
INDEX......Page 565
Supplemental Images......Page 571