Preface ........................................................ xv
List of contributors .......................................... xix
List of abbreviations ....................................... xxiii
List of figures .............................................. xxxi
List of tables ............................................... xxxv
List of colour plates ...................................... xxxvii
1 Introduction: basic requirements for in vitro applications
in aquatic eco-toxicology
(C. Mothersill and B. Austin) ................................ 1
1.1 References .............................................. 5
PART I CULTURE TECHNIQUES ..................................... 7
2 Basic methods and media for eukaryotic cells in vitro
(B. Austin and C. Mothersill) ................................ 9
2.1 Animals ................................................ 10
2.2 Equipment .............................................. 10
2.3 Procedures ............................................. 11
2.4 Culture media .......................................... 15
2.5 Conclusions ............................................ 16
2.6 References ............................................. 16
3 Development of primary tissue culture techniques for use
in ecotoxicology: some considerations necessary prior to
model selection
(C Mothersill and C. Seymour) ............................... 17
3.1 Overview ............................................... 17
3.2 Introduction ........................................... 18
3.2.1 Problems with in vitro approaches and
background philosophies ......................... 18
3.2.2 Historical perspectives ......................... 20
3.3 Methodology ............................................ 21
3.4 Cell population-based in vitro approaches to normal
tissue toxicology ...................................... 23
3.5 Validation of data from primary epithelial culture
systems ................................................ 28
3.6 Conclusions ............................................ 29
3.7 References ............................................. 29
4 Cultured epithelia from fish gills
(P. Part and C.M. Wood) ..................................... 35
4.1 Introduction ........................................... 35
4.2 Gill cells in monolayer cultures ....................... 36
4.3 Cultured gill epithelia ................................ 37
4.3.1 The experimental system ......................... 37
4.3.2 Preparation of epithelia ........................ 38
4.3.3 Development of epithelial properties ............ 41
4.3.4 Exposure to freshwater .......................... 42
4.3.5 Hormonal effects on cultured gill epithelia ..... 45
4.3.6 The cultured epithelium in aquatic toxicology ... 46
4.3.7 In vitro screening and testing of chemicals ..... 48
4.4 Concluding remarks ..................................... 48
4.5 References ............................................. 49
PART II END POINT AND TOOLS FOR IN VITRO TOXICOLOGY ........... 53
5 Applications of in vitro techniques in studies of
biomarkers and eco toxicology
(D. Sheehan) ................................................ 55
5.1 Introduction ........................................... 55
5.1.1 Chemical threat posed by xenobiotics ............ 55
5.1.2 Biological xenobiotic defence mechanisms ........ 56
5.1.3 Enzymatic detoxification ........................ 57
5.2 Detoxification systems of invertebrates and aquatic
vertebrates as bioindicators of environmental
pollution .............................................. 60
5.2.1 Sentinel species ................................ 60
5.2.2 Potential of invertebrate species for in
vitro toxicology ................................ 61
5.3 Primary cell cultures .................................. 61
5.4 Uses of cell lines in toxicology ....................... 65
5.4.1 Cytotoxicity .................................... 65
5.4.2 Ultrastructural effects ......................... 66
5.4.3 Formation of protein adducts .................... 66
5.4.4 Genotoxicity .................................... 67
5.5 Non-mammalian tissue culture ........................... 68
5.6 Potential of lower vertebrate and invertebrate
tissue culture in toxicology ........................... 69
5.7 Future perspectives .................................... 69
5.8 References ............................................. 70
6 End points for in vitro toxicity testing with fish cells
(H. Segner and T. Braunbeck) ................................ 77
6.1 Introduction ........................................... 77
6.2 Cell systems from teleost fish ......................... 78
6.2.1 Primary cells ................................... 78
6.2.2 Permanent cell lines ............................ 80
6.2.3 Recombinant fish cell lines ..................... 81
6.3 End points in in vitro assays .......................... 82
6.3.1 Molecular end points ............................ 83
6.3.2 Morphological end points ........................ 97
6.3.3 Cytotoxicity assays ............................ 114
6.4 Perspectives .......................................... 124
6.5 References ............................................ 125
7 Toxicogenomic technologies for in vitro aquatic
toxicology
(L.E.J. Lee, M.M. Vijayan and B. Dixon) .................... 143
7.1 Introduction .......................................... 143
7.2 Toxicogenomics ........................................ 144
7.3 Genomic and transgenic technologies ................... 144
7.3.1 Transgenic technologies ........................ 145
7.3.2 Genomic studies ................................ 148
7.4 Microarrays ........................................... 149
7.4.1 Toxicant fingerprinting ........................ 151
7.4.2 Current limitations of microarrays ............. 151
7.5 Proteomics ............................................ 152
7.5.1 Methodology .................................... 153
7.5.2 Fish proteomes ................................. 154
7.6 Conclusions ........................................... 155
7.7 References ............................................ 155
8 Delayed and indirect genotoxicity: a paradigm shift in
toxicology?
(E.G. Wright) .............................................. 161
8.1 Introduction .......................................... 161
8.2 The biological effects of ionizing radiation .......... 162
8.2.1 The concept of radiation-induced genomic
instability .................................... 162
8.2.2 Radiation-induced chromosomal instability ...... 164
8.2.3 Radiation-induced delayed gene mutations ....... 165
8.2.4 Radiation-induced genomic instability in
vivo ........................................... 166
8.2.5 Radiation-induced instability in the germ
line ........................................... 167
8.2.6 Radiation-induced genomic instability in
a non-mammalian system ......................... 168
8.2.7 Mechanisms underlying inducible instability .... 168
8.2.8 Instability associated with exposures other
than ionizing radiation ........................ 168
8.3 Radiation-induced bystander effects ................... 169
8.3.1 Bystander effects associated with a-particle
irradiations ................................... 170
8.3.2 Mechanisms underlying bystander effects ........ 171
8.3.3 Clastogenic factors: long-range acting
mechanisms ..................................... 173
8.4 A link between radiation-induced genomic
instability and bystander effects ..................... 174
8.5 Conclusions ........................................... 175
8.6 References ............................................ 175
PART III SPECIFIC TOXINS AND TOXIC GROUPS ..................... 183
9 Toxicogenomic applications in environmental toxicology
(L.N. Moens, K. Van der Ven, M. Caturla and W.M.
De Coen) ................................................... 185
9.1 Introduction .......................................... 185
9.2 Methodological approaches for differential gene
expression ............................................ 187
9.2.1 Differential display (DD) ...................... 187
9.3 Restriction endonuclease-facilitated analysis of
gene expression ....................................... 188
9.3.1 Serial analysis of gene expression (SAGE) ...... 188
9.4 Subtractive hybridization (SH) methods ................ 189
9.4.1 Traditional SH (physical separation) ........... 189
9.4.2 Chemical crosslinking subtraction (CCLS) ....... 189
9.4.3 Representational difference analysis (RDA) ..... 190
9.4.4 Suppression subtractive hybridization (SSH) .... 190
9.4.5 DNA arrays and differential cDNA library
screening ...................................... 191
9.4.6 Real-time PCR: quantitative analysis of
changes in molecular targets ................... 193
9.5 A focus on SSH, DNA arrays and real-time PCR:
methods and toxicological applications ................ 194
9.5.1 Suppression subtractive hybridization SSH ...... 194
9.5.2 DNA arrays ..................................... 201
9.5.3 Real-time PCR .................................. 207
9.6 Evaluation ............................................ 213
9.7 References ............................................ 215
10 Biomarkers to assess endocrine disruption of
reproduction in bivalves
(F. Gagne and C. Blaise) ................................... 221
10.1 Introduction .......................................... 221
10.2 Hormonal regulation of reproduction ................... 223
10.2.1 Role of estradiol-17β in vitellogenesis ....... 225
10.3 Biomarkers to assess endocrine effects on
reproduction .......................................... 225
10.3.1 Steroidogenesis and hormone metabolism ......... 225
10.3.2 Hormone receptor studies ....................... 226
10.3.3 Assays for vitellogenin ........................ 228
10.3.4 Spawning activity .............................. 231
10.3.5 General indicators of gamete activity .......... 232
10.3.6 Biomarkers to monitor fertilization ............ 233
10.4 Case studies .......................................... 233
10.4.1 Case study 1: masculinization of the bivalve
Mya arenaria ................................... 233
10.4.2 Case study 2: induced feminization of
freshwater mussels by a municipal effluent ..... 234
10.5 General conclusions ................................... 235
10.6 References ............................................ 236
11 Fish cells used to detect aquatic carcinogens and
genotoxic agents
{A. Castano, C. Becerril and M.T. Llorente) ................ 241
11.1 Introduction .......................................... 241
11.2 In vitro assays for the evaluation of mutagenicity/
carcinogenicity of chemicals .......................... 243
11.2.1 Advantages of in vitro testing using fish
cells ......................................... 252
11.3 Fish cells in the genetic assessment of
contaminants to wild populations of aquatic
organisms ............................................. 257
11.3.1 In vitro assays in the understanding of
contamination on populations ................... 257
11.3.2 Fish cells from sentinel species used as
biomarkers of exposure to contaminants ......... 259
11.4 Concluding remarks .................................... 264
11.5 References ............................................ 266
12 In vitro biomarkers of radiation exposure in the
environment
{B.A. Ulsh and D.R. Boreham) ............................... 279
12.1 Introduction .......................................... 279
12.2 Chromosome aberrations ................................ 280
12.3 Cytogenetic assays .................................... 282
12.3.1 Challenges ..................................... 282
12.3.2 Micronucleus assay ............................. 283
12.3.3 Dicentric analysis ............................. 284
12.3.4 Fluorescence in situ hybridization (FISH) ...... 285
12.4 Using cytogenetic biomarkers for environmental
biodosimetry .......................................... 288
12.5 References ............................................ 289
13 Ecotoxicological characterization of complex mixtures
(G. Repetto, A. del Peso and A. Jos) ....................... 295
13.1 Introduction .......................................... 295
13.2 Alternatives in ecotoxicology ......................... 296
13.3 Principal environmental samples used for
ecotoxicological evaluation ........................... 300
13.4 Global ecotoxicity evaluation ......................... 302
13.5 Non-fish cell cultures ................................ 304
13.6 Fish cell cultures .................................... 309
13.6.1 Cell and tissue morphology ..................... 309
13.6.2 Cellular viability ............................. 310
13.6.3 Genotoxicity ................................... 312
13.6.4 Biotransformation and defence systems .......... 313
13.6.5 Endocrine disruption ........................... 314
13.7 Toxicity identification and reduction evaluation
procedures ............................................ 315
13.8 Conclusions and likely future trends .................. 319
13.9 References ............................................ 320
14 In vitro ecotoxicological assessment of aquatic
sediments
(S. Ni Shuilleabhain, M. Davoren, M.G.J. Hartl and
J. O'Hallorari) ............................................ 327
14.1 Introduction .......................................... 327
14.1.1 What are sediments? ........................... 328
14.2 Assessing the characteristics of aquatic sediments .... 329
14.2.1 Sediment types ................................. 329
14.2.2 The significance of sediments in
ecotoxicology .................................. 330
14.3 Sediment collection, storage and preparation—general
consider ations ....................................... 331
14.3.1 Control or reference sediment .................. 332
14.3.2 Sediment collection ............................ 332
14.3.3 Whole sediment storage and preservation ........ 334
14.3.4 Health and safety .............................. 334
14.4 Sediment preparation methods .......................... 334
14.4.1 Spiking sediments .............................. 334
14.4.2 Aqueous extraction procedures .................. 335
14.4.3 Pore water extraction procedures ............... 335
14.4.4 Elutriate extraction procedures ................ 343
14.4.5 Solvent extraction ............................. 344
14.4.6 Novel approaches to sediment extraction ........ 346
14.5 In vitro tests for the toxicological assessment of
sediments ............................................. 347
14.5.1 Bacterial bioassays for assessing sediment
toxicity ....................................... 347
14.5.2 Bioassays with primary producers for
assessing sediment toxicity .................... 349
14.6 Use of microscale tests for assessing sediment
toxicity .............................................. 349
14.6.1 Submitochondrial particle (SMP) technology ..... 352
14.7 Use of in vitro cell cultures in determining
sediment toxicity ..................................... 353
14.7.1 Cytotoxicity studies ........................... 356
14.7.2 Genotoxicity studies ........................... 356
14.7.3 Mechanistic and biomarker studies .............. 357
14.8 Conclusions ........................................... 358
14.9 Perspectives and future research ...................... 359
14.10 References ........................................... 360
PART IV PRACTICAL ISSUES ..................................... 375
15 In vitro I in vivo bridging approaches—validating the
relevance of in vitro techniques with references to the
whole organism in the natural environment
(M. Kilemade and B. Quinn) ................................ 377
15.1 Preface ............................................... 377
15.2 Guidelines for in vitro/in vivo comparisons ........... 378
15.3 Inherent advantages of in vitro aquatic toxicology
research models ....................................... 379
15.4 Inherent problems with in vitro responses in
aquatic toxicological studies ......................... 380
15.5 Aquatic toxicology—in vitro-in vivo responses ......... 381
15.6 Cell lines versus primary culture ..................... 382
15.7 Reduction and replacement of in vivo tests ............ 383
15.7.1 Relative toxicity .............................. 383
15.7.2 Absolute sensitivity ........................... 384
15.8 Bridging the in vivo/in vitro divide .................. 388
15.8.1 Quantitative structure-activity relationship
(QSAR) ......................................... 389
15.8.2 Toxicokinetic models ........................... 389
15.9 Why primary cell cultures? ............................ 390
15.10 In vitro/in vivo end points and responses ............ 391
15.11 Future research ...................................... 391
15.12 Conclusions .......................................... 392
15.13 References ........................................... 393
16 Cell culture approaches in aquatic immunotoxicology
(N.C. Bols, S.J. DeWitte-Orr, J.L. Brubacher, B. Dixon
and R.C. Ganassin) ......................................... 399
16.1 Introduction .......................................... 399
16.1.1 Defining aquatic immunotoxicology ............. 399
16.2 Cell cultures and aquatic immunotoxicology ............ 400
16.2.1 Primary cell cultures versus cell lines ........ 401
16.2.2 Cell types ..................................... 401
16.2.3 In vivo versus in vitro exposure ............... 402
16.2.4 Value of in vitro approaches ................... 403
16.3 Haemopoietic cell cultures ............................ 403
16.3.1 Primary long-term haemopoietic cultures
(LTHCs) ........................................ 403
16.3.2 Colony formation in semi-solid matrix .......... 404
16.4 Lymphocyte cultures ................................... 404
16.4.1 Lymphocyte blastogenesis in primary cultures ... 404
16.4.2 Non-specific cytotoxic cells (NCCs) and
natural killer (NK) cells ...................... 406
16.4.3 Lymphocyte cell lines .......................... 407
16.5 Phagocyte cultures .................................... 407
16.5.1 Primary phagocyte cultures ..................... 409
16.5.2 Monocyte/Macrophage cell lines ................. 412
16.6 Conclusions ........................................... 413
16.7 References ............................................ 413
17 Validation of alternative methods to animal testing
(M. Haider and A. Worth) ................................... 421
17.1 Introduction .......................................... 421
17.1.1 What does validation mean? ..................... 422
17.1.2 Is formal validation necessary? ................ 422
17.1.3 Validation guidelines .......................... 422
17.2 The ECVAM validation process .......................... 423
17.2.1 Test development ............................... 424
17.2.2 Prevalidation .................................. 425
17.2.3 Validation ..................................... 425
17.2.4 Independent assessment and regulatory
acceptance ..................................... 426
17.3 The ICCVAM test method evaluation process ............. 427
17.3.1 Test method submission ......................... 427
17.3.2 ICCVAM interagency working groups .............. 427
17.3.3 Independent scientific peer review panel ....... 429
17.3.4 ICCVAM test recommendations .................... 429
17.3.5 Regulatory agency considerations of ICCVAM
recommendations ................................ 429
17.4 Regulatory tests ...................................... 429
17.4.1 Chemicals ...................................... 429
17.4.2 Effluents ...................................... 431
17.5 Conclusions ........................................... 431
17.6 References ............................................ 431
18 Legal and ethical aspects of environmental toxicology
with specific references to in vitro approaches
(C. Seymour) ............................................... 435
18.1 The background philosophy ............................. 435
18.2 Why do we control toxicological experimentation? ...... 436
18.3 Is there an automatic right to life for any species,
even man? ............................................. 436
18.4 The Nuremberg Code and its impact on animal
toxicology ............................................ 437
18.5 Present day consequences of the Nuremberg Code;
the anthropocentric view is made legal ................ 438
18.6 The anthropocentric view is evolving .................. 438
18.7 The problem of assessment of values by one group or
species on behalf of another .......................... 439
18.8 Deep ecology and the biocentric viewpoint applied
to toxicology ......................................... 440
18.9 Altruism toward animals in research ................... 441
18.10 The way forward? ..................................... 441
18.11 References ........................................... 442
19 The mathematics of cell growth in culture
(R. Ocone, A. Kummer and X. Yang) .......................... 443
19.1 Introduction .......................................... 443
19.2 What is growth in cell culture? ....................... 443
19.3 How is growth expressed? .............................. 444
19.3.1 Kinetics ...................................... 445
19.4 Significance of cell growth modelling ................. 446
19.4.1 Formulating a mathematical model ............... 447
19.4.2 Single-cell modelling .......................... 448
19.4.3 Modelling cell population in culture ........... 449
19.4.4 Determination of model's parameters ............ 451
19.5 Linking organizational levels ......................... 452
19.5.1 Development of a regulatory thermodynamic
theory ......................................... 452
19.5.2 Constitutive relations ......................... 456
19.6 Conclusions ........................................... 457
19.7 References ............................................ 457
20 Conclusions
(B. Austin, C. Seymour and C. Mother sill) ................. 459
Index ......................................................... 465
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