More precisely, the book presents the results obtained during the development and application of biological markers as indicators of exposure/effect to toxic chemicals in marine environments, using diverse sentinel species such as fish, bivalves and crustaceans. An important aspect is also the publication of technical annexes that describe in detail the experimental procedures developed for both chemical and biochemical measurement.
Many previous studies and books have been dedicated to fundamental and developmental aspects of biomarkers. The purpose of this book is to provide, through various case studies, an overview of the practical use of biological markers in marine animals to evaluate the health effects of environmental contamination in marine ecosystems. More precisely, the book presents the results obtained during the development and application of biological markers as indicators of exposure/effect to toxic chemicals in marine environments, using diverse sentinel species such as fish, bivalves and crustaceans. An important aspect is also the publication of technical annexes that describe in detail the experimental procedures developed for both chemical and biochemical measurement.
Cover 1
Biomarkers in Marine Organisms: A Practical Approach 4
Copyright Page 5
Contents 8
Preface 6
Contents 8
List of Participants of European Projects 18
Abbreviations 22
Chapter 1. Induction of Molluscan Cytochrome P450 Monooxygenase System as a Biomarker of Organic Pollution in Environmental Monitoring 24
1. The uptake of xenobiotics and bioaccumulation in aquatic molluscs 25
2. Mixed function oxidase (MFO)-dependent metabolism 25
3. CYP1A as a biomonitoric tool 26
4. The molluscan MFO system: physical and catalytic properties 27
5. Multiple CYP forms in Mytilus sp 29
6. Regulation of CYP forms in Mytilus sp 30
7. Xenobiotic-dependent changes in Mytilus sp. MFO properties: laboratory studies 32
8. Xenobiotic-dependent changes in Mytilus sp. MFO properties: field studies 37
9. Final comments 45
References 45
Chapter 2. Sensitivity and Specificity of Metallothionein as a Biomarker for Aquatic Environment Biomonitoring 52
1. Introduction 53
2. Materials and methods 54
3. Results and discussion 55
References 62
Chapter 3. Genotoxicity Biomarkers in Aquatic Organisms as Indictors of Carcinogenic Marine Pollutants 68
1. Introduction 69
2. Materials and methods 71
3. Results 74
4. Discussion 81
References 83
Chapter 4. DNA Adduct Detection in Mussels Exposed to Bulky Aromatic Compounds in Laboratory and Field Conditions 88
1. Introduction 89
2. Formation and detection of DNA adducts in mussels 91
3. Conclusion 100
References 103
Chapter 5. Developmental, Cytogenetic and Biochemical Effects of Spiked or Environmentally Polluted Sediments in Sea Urchin Bioassays 108
1. Introduction 110
2. Materials and methods 111
3. Results 114
Conclusions 143
Appendix 145
References 151
Chapter 6. Comparative Study of Sediment and Mussel Aromatic Compound Content in European Coastal Environments. Relationship with Specific Biomarkers 154
1. Introduction 156
2. Sediment PAH contamination 159
3. Mussel PAH contamination 173
4. Impact of sediment contamination on organisms 189
Conclusion 195
References 196
Chapter 7. Monitoring of Biological Effects of Pollutants: Field Application 202
1. Monitoring biological effects along French coasts 204
2. Crassostrea Gigas Embryo and Larval Bioassays in Waters and Sediments 212
3. Testing biomarkers at sea: an in situ approach in the Northwestern Mediterranean Sea 219
4. Molecular epidemiology studies using ras oncogene as a genetic marker of malignancy for dragonet callionymus lyra along the French coast. The Seine estuary 226
5. Prospects 229
References 232
Chapter 8. Biochemical Markers in Mussel, Mytilus sp., and Pollution Monitoring in European Coasts: Data Analysis 238
1. Introduction 239
2. Materials and methods 240
References 257
Chapter 9. Investigation of Genotoxicity and Immunotoxicity for Monitoring Marine Pollution in the Baltic Sea and Mediterranean Sea 260
1. Introduction 262
2. Materials and methods 263
3. Results 267
4. Discussion 276
References 278
Chapter 10. Biochemical Tools for the Assessment of Pesticide Exposure in a Deltaic Environment: The Use of Cholinesterases and Carboxylesterases 282
1. The Ebro delta: environmental threats 284
2. Toxicity o f organophosphorus pesticides: inhibition of cholinesterases 285
3. The bivalve farms: Mytilus galloprovincialis 286
References 298
Chapter 11. Environmental Monitoring in the North Sea by Combining Biomarker Studies in the Sea Star Asterias Rubens with Sediment Quality Assessment Based on Sea Urchin Bioassays 302
1. Introduction 304
2. Materials and methods 308
3. Results 316
4. Discussion 333
Appendix 342
References 349
Chapter 12. Cholinesterase Activity as a Bioindicator for Monitoring Marine Pollution in the Baltic Sea and the Mediterranean Sea 354
1. Introduction 356
2. Materials and methods 356
3. Results 358
4. Discussion 362
References 363
Chapter 13. Evaluation of Various Biomarkers in the Wild Fish Serranus Cabrilla Collected in the NW Mediterranean Sea 366
1. Introduction 368
2. Materials and methods 369
3. Results and discussion 371
References 378
Chapter 14. Microbiological Indicators for Monitoring Marine Pollution in the Baltic Sea and the Mediterranean Sea 380
1. Introduction 381
2. Materials and methods 381
3. Results 383
4. Discussion 386
References 388
Chapter 15. Isolation of Cytochrome P450 CDNAS (CYP1A1 and CYP4T2) from the Sea Bass (Dicentrarchus Labrax): Tools for Biomonitoring Sea Pollution 390
1. Introduction 392
2. Results and discussion 393
References 413
Chapter 16. Inhibitory Effects of Heavy Metals on CYP1A1 Induction in Black Seabream (Spondyliosoma Cantharus) Hepatocyte Cultures 416
1. Introduction 418
2. Materials and methods 419
3. Results 421
References 429
Chapter 17. Biochemical Responses of Crabs (Carcinus spp ) to Polycyclic Aromatic Hydrocarbons (PAHs) as the Basis for New Biomarker Assays 432
1. Introduction 433
2. Induction of proteins 435
3. Genotoxicity 439
4. Linked biomarker responses 445
5. Conclusions 446
References 449
Chapter 18. Cloning of Metallothionein cDNAS in Carcinus Maenas 454
1. Introduction 455
2. Experimental work 456
3. Results and discussion 456
4. Concluding remarks 459
References 460
Chapter 19. Development of Cytochrome P450 Biomarkers from Posidinia Oceanica 462
1. Introduction 463
2. Materals and methods 464
3. Results 466
4. Discussion 467
5. Future studies 468
References 468
Technical Annexes 470
Measurement of Total Cytochrome P450 Content in Digestive Gland Microsomes of Mussel (Mytilus sp.) 472
Measurement of Cytochrome P450 Immunopositive Proteins in Digestive Gland Microsomes of Mussel (Mytilus sp.) by Western Analysis 474
Metallothionein Assay 476
Evaluation of Lysosomal Membrane Stability 478
Alkaline Filter Elution Method 484
Mussel Micronucleus Test 490
Detection of Bulky Aromatic DNA Adducts by 32 P-postlabelling 494
Embryotoxicity on Sea Urchins 498
Sediment Spiking Technique 500
Measurement of Oxidative Activity: Luminol-dependent Chemiluminescence and 8-Oxodesoxyguanine 501
Chemical Analyses of PAH in Sediment and Mussels 504
Procedures for Cholinesterase Determination in Fish and Mussel 510
Microplate Method for Measurement of EthoxyResorufin-O-Deethylase (EROD) in Fish 514
Benzo(a)pyrene Hydroxylase Activity Measurement in Microsomes from Mytilus sp. 518
Determination of Cholinesterase Activity in Mussel Gills 522
Chemical Analysis: PAHs, PCBs and Pesticides 524
Biochemical Markers Acetylcholinesterase Activity and Cytochrome P450 526
Measurement of DNA-Strandbreaks in Tissue of the Sea Star Asterias Rubens 528
Measurement of Sediment-Toxicity Using a Fertilization and Embryonic Development Assay with the Sea Urchins (Psammechinus Miliaris, Paracentrotus Lividus) 532
Determination of EROD, GST and AchE Activities in Painted Comber (Serranus Cabrilla) 536
Miniaturized DNA Unwinding Assay Employing Hydroxyapatite "Batch" Elution 540
Molecular Cloning of CYP Genes in the Sea Bass (Dicentrarchus Labrax): Experimental Procedures 542
Fish Hepatocyte Cultures 550
Determination of Cytochromes P450 Expression and Induction in Black Seabream (Spondyliosama cantharus): Enzyme Activities, Northern and Esters Blots 552
Biochemical Responses of Crabs (Carcinus spp) to Polycyclic Aromatic Hydrocarbons (PAHs) as the Basis for New Biomarkers Assays 556
Cloning of Metallothionein cDNAs in Carcinus Maenas 560
Development of Biomarkers from Seagrass Posidonia Oceanica 562
List of Contributors 566
Index 572
SENSITIVITY AND SPECIFICITY OF METALLOTHIONEIN AS A BIOMARKER FOR AQUATIC ENVIRONMENT BIOMONITORING.
A. Viarengo, B. Burlando, V. Evangelisti, S. Mozzone and F. Dondero, Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale “Amedeo Avogadro”, Sede di Alessandria, Corso Borsalino 54, 15100 Alessandria, Italy
ABSTRACT
This chapter contains a general description of the biochemical and physiological features of metallothioneins. In addition, original data are presented showing the sensitivity of metallothionein response in mussels exposed to different heavy metals such as copper, zinc and cadmium in comparison with metal effects on lysosomal membrane stability, a well known biomarker of stress. Finally, concerning the specificity of metallothionein response, data are presented indicating that metallothionein should be considered a biomarker of heavy metals exposure in molluscs and a biomarker of stress particularly sensitive to metals in fish.
Keywords
Heavy metals
metallothionein
lysosomal activity
mussel
fish
1. INTRODUCTION
Metallothioneins are low molecular weight, soluble, heat stable metalloproteins, able to bind extremely high concentrations (6-12 g at/mol) of essential heavy metal cations, such as Zn and Cu, or heavy metals without a known biological function, such as Hg, Cd, Ag, etc. Metallothioneins have a peculiar amino acid composition, characterized by the absence of aromatic amino acids (involving a virtual lack of absorbance at 280 nm), of histidine and in molluscs also of methionine. On the contrary, cysteinyl residues are present in large amount (about 30% in mammal metallothioneins) showing repeated motives along the sequence such as Cys-X-Cys, Cys-Cys, Cys-X-Y-Cys, where X, Y are amino acid different from cysteine (Kägi & Kojima, 1987; Kägi & Shaffer, 1988). The sulfhydryl residues of cysteine occur in the reduced form and are co-ordinated to heavy metals through mercaptide bonds, giving rise to spectroscopic features typical of metal tetrathiolate clusters (Kägi & Kojima, 1987). Metallothioneins have been subdivided into three different classes (I, II and III), depending on the structural and biochemical similarities with mammalian metallothioneins (class I) (Kägi & Kojima, 1987; Kägi, 1993). A remarkable feature of metallothioneins is their inducibility. It is in fact well known that different factors, and in particular heavy metal cations, can stimulate the synthesis of mRNA encoding for metallothioneins. Owing to this property and due to the high affinity of metallothioneins for heavy metals, these proteins play an important role in regulating the physiological concentration of essential metals, such as Zn and Cu, and in detoxifying noxious metal cations penetrating into the cells (George, 1983; Viarengo et al., 1985; Hamer, 1986; Bremner, 1987; Engel & Brouwer, 1989; Roesijadi, 1992; Viarengo & Nott, 1993).
However, other roles have also been proposed for metallothioneins. Firstly, they have been considered a component of the antioxidant defence system involved in the protection of cells from cytotoxic effects of reactive oxygen species (ROS) (Thornalley & Vasak, 1985; Thomas et al., 1986; Chubatsu & Meneghini, 1993; Woo & Lazo, 1997; Brouwer & Brouwer, 1998). Secondly, metallothioneins could be implicated in the regulation of basal transcription processes by modulating the binding to DNA of zinc finger peptides (Roesijadi et al., 1998).
The inducibility by heavy metals renders metallothioneins a possible “biomarker” able to evidentiate the exposure of aquatic organisms to heavy metal pollution (Engel & Roesijadi, 1987; Viarengo et al., 1988a; Hogstrand & Haux, 1990; Viarengo & Canesi, 1991; Viarengo et al., 1999). In this paper, data concerning the response of mussels Mytilus galloprovincialis to heavy metal pollutants are presented. As known, mussels are intertidal filter-feeding lamellibranch molluscs able to concentrate in their tissues chemical pollutants present in the surrounding seawater (Phillips, 1976; Goldberg et al., 1978; Viarengo & Canesi, 1991). Mussels are, therefore, frequently used in biomonitoring programs as sentinel organisms. The aim of this study is to make a comparison between the sensitivity of metallothioneins and that of another largely used biomarker, i.e. lysosomal membrane stability, in evaluating the effects of heavy metal exposure on marine organisms. Lysosomal membrane stability is a biological parameter which is often used to assess at the cellular level the stress syndrome induced by common marine pollutants (Moore et al., 1978; Moore et al., 1980; Lowe et al., 1981; Moore et al. 1986; Viarengo et al., 1987; Moore, 1990; Khöler, 1991). Finally, some indications about the specificity of metallothioneins in the evaluation of the biological effects of heavy metal pollution in different sentinel organisms are also reported.
2. MATERIALS AND METHODS
Chemicals: Leupeptin, PMSF (phenylmethylsulfonyl fluoride), EDTA (Ethylendiaminotetracetic acid), GSH (reduced glutathione), DTNB (5,5′-dithiobis-2-nitrobenzoic acid), Naphtol As-BI N-acetyl-D-glucosaminide, Polypep and Fast Violet B salt were purchased from SIGMA-ALDRICH s.r.l. (Milan, Italy); β-mercaptoethanol and BSA (bovine serum albumine) were obtained from Merck (Milan, Italy). Thiolyte® monobromobimane was purchased from Calbiochem. All the other reagents were of analytical grade.
Animals and Treatments.: Specimens of mussels Mytilus galloprovincialis Lam. (4-6 cm shell length) were collected from La Spezia, Italy and acclimated to laboratory conditions at 15°C for three days in EDTA-free synthetic seawater (1 litre/animal), pH 7.9-8.0, 35%o salinity (La Roche et al., 1970). Mussels were exposed for five days to different copper (as CuCl2) or cadmium (as CdCl2) concentrations ranging respectively from 0.078 μM to 1.258 μM Cu2+ and from 0.016 μM to 1.636 μM Cd2+. Alternatively, mussels were treated for five days with a mixture of copper, cadmium and mercury at a concentration of 0.16 μM each. In all experiments seawater changes and metal additions were done daily.
Metallothionein evaluation.: Mussel gills and digestive gland were rapidly dissected out and metallothionein samples prepared as previously published (Viarengo et al., 1997a).
Lysosomal membrane stability.: Pieces of digestive gland were placed on cryostat chucks, cooled in n-hexane pre-cooled to −70°C with liquid nitrogen and then stored at −80°C as previously described by Moore (1976). Sections of 10 μm were cut using a cryostat, collected on slides and analysed as previously described (Moore, 1976; Viarengo et al., 1987).
Statistical analysis.: Data, representing the means ± SD of at least four measurements, were compared by the Bonferroni t-test.
3. RESULTS AND DISCUSSION
The evaluation of metallothionein concentration in animal tissues was often considered a sophisticated biochemical approach achievable only by specialized research groups (Piotrowski et al., 1973; Olafson and Sim, 1979; Suzuki, 1980; Eaton and Toal, 1982; Thompson and Cosson, 1984; Lehman and Klaassen, 1986; Nolan and Shaikh, 1986; Scheuhammer and Cherian 1986; Lobel and Payne, 1987; Sunaga et al., 1987; Mason et al., 1990; Mazzucotelli et al., 1991; Martinez et al., 1993). Recently, a simple, sensible, accurate and low-cost spectrophotometric metallothionein assay has been developed in our lab. By this procedure, tissue metallothionein amounts are estimated by using a partially purified metalloprotein fraction obtained through acidic ethanol/chloroform fractionation of the homogenate. The method involves precautions to obtain complete metallothionein precipitation and to avoid sulfhydryl oxidation, contamination by soluble low-molecular weight thiols, and enzymatic protein degradation. In the final extract, the concentration of metallothioneins, denatured by low pH and high ionic strength, is evaluated spectrophotometrically using the Ellman’s SH reagent (Viarengo et al., 1997a).
The above mentioned method was used to evaluate the metallothionein content in the digestive gland of mussels exposed for five days to different copper concentrations (Fig. 1). As shown in figure 1, when mussels...
| Erscheint lt. Verlag | 7.11.2001 |
|---|---|
| Sprache | englisch |
| Themenwelt | Medizin / Pharmazie ► Gesundheitsfachberufe |
| Studium ► 2. Studienabschnitt (Klinik) ► Pharmakologie / Toxikologie | |
| Naturwissenschaften ► Biologie ► Biochemie | |
| Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie | |
| Naturwissenschaften ► Biologie ► Limnologie / Meeresbiologie | |
| Naturwissenschaften ► Chemie ► Analytische Chemie | |
| Naturwissenschaften ► Chemie ► Technische Chemie | |
| Technik ► Umwelttechnik / Biotechnologie | |
| ISBN-10 | 0-08-052804-X / 008052804X |
| ISBN-13 | 978-0-08-052804-5 / 9780080528045 |
| Haben Sie eine Frage zum Produkt? |
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