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Inter- and intraspecies differences in biomarker responses and contaminant levels in two mysticete species (Balaenoptera physalus and Balaenoptera edeni) of Gulf of California (Mexico) and Mediterranean Sea
Comparative Biochemistry and Physiology, Part A 157 (2010) S22–S28
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
27th Congress of the NewEuropean Society of Comparative Biochemistry and Physiology Alessandria (Italy) — Sept. 5–9, 2010 Biological effects of climatic changes and pollution: From biomarkers to system biology
Session 5: Biomarker in aquatic organisms from protists to top predators INVITED LECTURE 1. Assessment of toxic effects of metal nanoparticles using biomarkers and in vitro toxicity tests M.P. Cajaraville (Dept. Zoology & Animal Cell Biology, School of Science & Technology, University of the Basque Country, Sarriena z/g, E-48940, Leioa, Basque Country, Spain) Due to the great increase in the use of nanomaterials for a variety of biomedical, domestic and industrial applications, the expected input of nanoparticles (NP) and other nanomaterials in the aquatic environment is expected to rise in the following years. In spite of this, there is only limited information on the fate, distribution and toxicity of NPs to aquatic organisms. In the framework of the EU-funded project Nanoretox, the aims of our research group were to investigate the bioavailability of metal NPs in mussels Mytilus galloprovincialis and zebrafish Danio rerio and to determine their possible adverse effects on the same target organisms. For this, a two-tiered strategy was developed combining both in vitro and in vivo studies. In vitro techniques provide a quick and reproducible tool for the screening of nanoparticle toxicity. In step 1, cytotoxicity of a variety of NPs at a wide range of concentrations was tested in isolated mussel hemocytes and gill cells using neutral red uptake and MTT assays and LC50s were calculated. In step 2, sublethal concentrations below the LC25 were selected to investigate in vitro uptake and reactivity of NPs and to discover putative mechanisms of toxicity in both cell types. In vivo studies with mussels comprised short-term 1–3 days experiments to determine bioavailability and lysosomal membrane stability as a general indicator of health (step 1) and 21 days experiments to assess adverse effects using a battery of molecular and cellular biomarkers (step 2). Similarly, studies with zebrafish started with short-term embryo toxicity tests (step 1) followed by 21 days experiments (step 2) in case significant toxicity was found in step 1. The developed strategy will be illustrated with results 1095-6433/$ – see front matter
obtained with CuO NPs, in comparison with bulk CuO and ionic Cu, and results obtained with different TiO2 NPs compared to the corresponding bulk forms. Funded by EU 7th FP (project Nanoretox), Spanish Ministry of Science and Innovation (project Nanocancer) and Basque Government through a grant to consolidated research groups (GIC07/26-IT393-07). doi:10.1016/j.cbpa.2010.06.061
ORAL PRESENTATIONS 2. Inter- and intraspecies differences in biomarker responses and contaminant levels in two mysticete species (Balaenoptera physalus and Balaenoptera edeni) of Gulf of California (Mexico) and Mediterranean Sea M.C. Fossi, S. Maltese, L. Mazzi, D. Coppola, S. Casini, C. Panti, L. Marsili (University of Siena, Italy); J. Urban, C. Torres (Universidad Autónoma de Baja California Sur, Mexico); L. Rojas-Bracho (Instituto Nacional de Ecologia, Mexico); B. Jimenez, J. Muñoz (CSIC, Spain) Inter- and intra-species differences in biomarker responses and contaminant levels were investigated in two mysticete species, fin whale (Balaenoptera physalus) and the unexplored species Bryde whale (Balaenoptera edeni) of Gulf of California (Mexico) and fin whale of Mediterranean Sea, using skin biopsy as diagnostic tool. We developed a non lethal “multi-trial-biomarker-tool”, combining protein (western blot of CYP1A1, CYP2B) and gene expression biomarkers (qRT-PCR of CYP1A1, ER, AhR, and E2F-1) with analysis of OCs, PAHs and PBDEs. In the first phase of the project we explored the level and effects of contaminants in skin biopsies of the two species of Gulf of California in comparison to fin whale of Mediterranean Sea. In the second, in-vitro, phase we applied this
Abstracts / Comparative Biochemistry and Physiology, Part A 157 (2010) S22–S28
S23
approach to fin whale biopsy slices treated with mixtures of OCs and PBDEs in order to explore the different toxicological effects of contaminants. This “multi-trial-diagnostic-tool”, applied to skin biopsies, underlined differences in contaminant levels and biomarker responses between the two species of Gulf of California and also between the two fin whale populations. Higher levels of OCs and PAHs were detected in the zooplankton-eating species (fin whale) in comparison to the fish-eating species (Bryde whale); on the opposite, higher levels of CYP1A1 and CYP2B were detected in the fish-eating species, similar to odontocete species. The interspecies investigation showed the presence of a higher “toxicological stress” in the Mediterranean fin whale population, highlighted by warning signals such as CYP1A1 induction and up-regulation of ERα and E2F-1 genes, combined with a lack of CYP2B induction in field and in vitro experiments.
stressor on the biomarker VTG in rainbow trout. To this end, juvenile fish were exposed to two doses of the prototypic estrogen-active compound, 17-beta-estradiol (E2) in combination with the fishpathogenic parasite Tetracapsuloides bryosalmonae, the etiological agent of the proliferative kidney disease (PKD). The VTG response was assessed at the mRNA level using qRT-PCR; in parallel, the liver transcriptomic response to the single stressors and their combinations was examined. Combined chemical/biological exposure resulted in a significant inhibition of both the rate and the magnitude of the VTG response, while neither the parasite nor the fish response to the parasite was affected by the presence of E2. The observed attenuation of the VTG biomarker response in diseases may have implications for the utility of this marker in environmental scenarios with low estrogenic exposure.
doi:10.1016/j.cbpa.2010.06.062
doi:10.1016/j.cbpa.2010.06.064
3. Non-vertebrate animals have only one gene of the p53 family which is most similar to vertebrate p63
5. Assessing the effects of the selective serotonin reuptake inhibitor fluoxetine on Carcinus maenas using locomotor behaviour and biomarkers as effect criteria
M. Štifanić, A. Baričević, R. Batel (Ruđer Bošković Institute, Croatia) Genes of the p53 family are known to be critical regulators of the cell cycle. They have already been established as possible biomarkers. Elaborate regulation mechanisms result in numerous mRNA and protein isoforms being expressed from each gene of the family. A relatively recent discovery of p53 paralogs (p63 and p73) and high similarity of isoforms expressed from all three p53 family genes caused confusion in their nomenclature in non-vertebrate species. We analyzed all molluscan p53-similar sequences found in the GenBank database and compared their sequences and genomic structures to p53 family genes from other animals. Our results lead to the conclusion that invertebrates have only one gene of the p53 family which radiated into three genes only in vertebrates. This ancestral (molluscan) p53 family gene shows to be most similar to p63 in vertebrates. Furthermore, our analyses indicate p53 family genes to be single-copy genes thus increasing their value as potential biomarkers. doi:10.1016/j.cbpa.2010.06.063
4. Pathogenic infection attenuates the response of the estrogenic biomarker, vitellogenin, in rainbow trout R. Burki, T. Wahli, H. Segner (University of Bern, Switzerland); P. Burkhardt-Holm (University of Basel, Switzerland); C.E. Rexroad, S. Afanasyev, M. Antikainen, A. Krasnov (AKVAFORSK, Norway) In the environment, aquatic organisms are exposed multiple instead of single stressors. During recent years, considerable progress has been achieved in assessing combination effects of chemical mixtures. For instance, it has been demonstrated that the induction of vitellogenin (VTG) in (male) fish – an established biomarker of exposure to estrogen-active compounds – responds additively to combinations of estrogen-active compounds, while simultaneous exposure to ligands of the estrogen receptor and of the arylhydrocarbon receptor has an antagonistic effect on VTG induction. In contrast to the progress in understanding combination effects of chemical mixtures, approaches to assess the combined effects of chemical and physical and/or biological stressors are less developed. Here, we study the combined impact of a chemical and a biological
S.R. Mesquita, L. Guilhermino, L. Guimarães (University of Porto, CIIMAR/ICBAS, Portugal) The serotonin reuptake inhibitor drug fluoxetine is currently found in effluent and surface waters. Considering its antidepressant and other effects in humans, biochemical, physiological and behaviour alterations are also expected to occur in wild organisms exposed to the substance. In the present study, intermoult Carcinus maenas were exposed for seven days to five fluoxetine concentrations ranging from 0.49 to 750 μg/L in a laboratorial bioassay. The following endpoints were assessed at the end of the test: spontaneous locomotor behaviour (SLB) using the classical openfield test; epidermis chitobiase activity (CTB), muscle cholinesterase (ChE), lactate dehydrogenase (LDH) and NADP+-dependent isocitrate dehydrogenase (IDH) activities; the activities of the enzymes glutathione S-transferases (GST), glutathione peroxidase (Gpx), glutathione reductase (GR), and the levels of total glutathiones (TG) and lipid peroxidation (LPO) in the hepatopancreas. Animals exposed to 120 and 750 μg/L spent significantly more time moving (TM) in the open-field and crossed a higher number of squares (NS) than controls. ChE, GST and GR activities, and TG levels, were also significantly increased in crabs exposed to 120 and 750 μg/L. Interestingly, significant correlations were found between TM and ChE (38.6%), TM and TG (31.7%), NS and ChE (47.1%), and NS and TG (34.4%). These results show that SLB was a sensitive endpoint regarding fluoxetine exposure, suggesting that bioassays based on it may be suitable tools to assess the toxicity of environmental contaminants to C. maenas. This work was supported by national-FCT and EU-FEDER funds through the project CRABTHEMES (FCOMP-01-0124-FEDER-007383). doi:10.1016/j.cbpa.2010.06.065
POSTER PRESENTATIONS 6. A non-lethal multi-biomarker approach to investigate the ecotoxicological status of Mediterranean loggerhead sea turtle (Caretta caretta, Linneo, 1758) S. Casini, I. Caliani, L. Marsili, M. Giannetti, S. Maltese, S. Ancora, N. Bianchi, C. Panti, T. Campani, L. Carletti, D. Coppola, M.C. Fossi (University of Siena, Italy); A. Cañadas (Alnitak, Spain); M. Parga (Submon, Spain)
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
27th Congress of the NewEuropean Society of Comparative Biochemistry and Physiology Alessandria (Italy) — Sept. 5–9, 2010 Biological effects of climatic changes and pollution: From biomarkers to system biology
Session 5: Biomarker in aquatic organisms from protists to top predators INVITED LECTURE 1. Assessment of toxic effects of metal nanoparticles using biomarkers and in vitro toxicity tests M.P. Cajaraville (Dept. Zoology & Animal Cell Biology, School of Science & Technology, University of the Basque Country, Sarriena z/g, E-48940, Leioa, Basque Country, Spain) Due to the great increase in the use of nanomaterials for a variety of biomedical, domestic and industrial applications, the expected input of nanoparticles (NP) and other nanomaterials in the aquatic environment is expected to rise in the following years. In spite of this, there is only limited information on the fate, distribution and toxicity of NPs to aquatic organisms. In the framework of the EU-funded project Nanoretox, the aims of our research group were to investigate the bioavailability of metal NPs in mussels Mytilus galloprovincialis and zebrafish Danio rerio and to determine their possible adverse effects on the same target organisms. For this, a two-tiered strategy was developed combining both in vitro and in vivo studies. In vitro techniques provide a quick and reproducible tool for the screening of nanoparticle toxicity. In step 1, cytotoxicity of a variety of NPs at a wide range of concentrations was tested in isolated mussel hemocytes and gill cells using neutral red uptake and MTT assays and LC50s were calculated. In step 2, sublethal concentrations below the LC25 were selected to investigate in vitro uptake and reactivity of NPs and to discover putative mechanisms of toxicity in both cell types. In vivo studies with mussels comprised short-term 1–3 days experiments to determine bioavailability and lysosomal membrane stability as a general indicator of health (step 1) and 21 days experiments to assess adverse effects using a battery of molecular and cellular biomarkers (step 2). Similarly, studies with zebrafish started with short-term embryo toxicity tests (step 1) followed by 21 days experiments (step 2) in case significant toxicity was found in step 1. The developed strategy will be illustrated with results 1095-6433/$ – see front matter
obtained with CuO NPs, in comparison with bulk CuO and ionic Cu, and results obtained with different TiO2 NPs compared to the corresponding bulk forms. Funded by EU 7th FP (project Nanoretox), Spanish Ministry of Science and Innovation (project Nanocancer) and Basque Government through a grant to consolidated research groups (GIC07/26-IT393-07). doi:10.1016/j.cbpa.2010.06.061
ORAL PRESENTATIONS 2. Inter- and intraspecies differences in biomarker responses and contaminant levels in two mysticete species (Balaenoptera physalus and Balaenoptera edeni) of Gulf of California (Mexico) and Mediterranean Sea M.C. Fossi, S. Maltese, L. Mazzi, D. Coppola, S. Casini, C. Panti, L. Marsili (University of Siena, Italy); J. Urban, C. Torres (Universidad Autónoma de Baja California Sur, Mexico); L. Rojas-Bracho (Instituto Nacional de Ecologia, Mexico); B. Jimenez, J. Muñoz (CSIC, Spain) Inter- and intra-species differences in biomarker responses and contaminant levels were investigated in two mysticete species, fin whale (Balaenoptera physalus) and the unexplored species Bryde whale (Balaenoptera edeni) of Gulf of California (Mexico) and fin whale of Mediterranean Sea, using skin biopsy as diagnostic tool. We developed a non lethal “multi-trial-biomarker-tool”, combining protein (western blot of CYP1A1, CYP2B) and gene expression biomarkers (qRT-PCR of CYP1A1, ER, AhR, and E2F-1) with analysis of OCs, PAHs and PBDEs. In the first phase of the project we explored the level and effects of contaminants in skin biopsies of the two species of Gulf of California in comparison to fin whale of Mediterranean Sea. In the second, in-vitro, phase we applied this
Abstracts / Comparative Biochemistry and Physiology, Part A 157 (2010) S22–S28
S23
approach to fin whale biopsy slices treated with mixtures of OCs and PBDEs in order to explore the different toxicological effects of contaminants. This “multi-trial-diagnostic-tool”, applied to skin biopsies, underlined differences in contaminant levels and biomarker responses between the two species of Gulf of California and also between the two fin whale populations. Higher levels of OCs and PAHs were detected in the zooplankton-eating species (fin whale) in comparison to the fish-eating species (Bryde whale); on the opposite, higher levels of CYP1A1 and CYP2B were detected in the fish-eating species, similar to odontocete species. The interspecies investigation showed the presence of a higher “toxicological stress” in the Mediterranean fin whale population, highlighted by warning signals such as CYP1A1 induction and up-regulation of ERα and E2F-1 genes, combined with a lack of CYP2B induction in field and in vitro experiments.
stressor on the biomarker VTG in rainbow trout. To this end, juvenile fish were exposed to two doses of the prototypic estrogen-active compound, 17-beta-estradiol (E2) in combination with the fishpathogenic parasite Tetracapsuloides bryosalmonae, the etiological agent of the proliferative kidney disease (PKD). The VTG response was assessed at the mRNA level using qRT-PCR; in parallel, the liver transcriptomic response to the single stressors and their combinations was examined. Combined chemical/biological exposure resulted in a significant inhibition of both the rate and the magnitude of the VTG response, while neither the parasite nor the fish response to the parasite was affected by the presence of E2. The observed attenuation of the VTG biomarker response in diseases may have implications for the utility of this marker in environmental scenarios with low estrogenic exposure.
doi:10.1016/j.cbpa.2010.06.062
doi:10.1016/j.cbpa.2010.06.064
3. Non-vertebrate animals have only one gene of the p53 family which is most similar to vertebrate p63
5. Assessing the effects of the selective serotonin reuptake inhibitor fluoxetine on Carcinus maenas using locomotor behaviour and biomarkers as effect criteria
M. Štifanić, A. Baričević, R. Batel (Ruđer Bošković Institute, Croatia) Genes of the p53 family are known to be critical regulators of the cell cycle. They have already been established as possible biomarkers. Elaborate regulation mechanisms result in numerous mRNA and protein isoforms being expressed from each gene of the family. A relatively recent discovery of p53 paralogs (p63 and p73) and high similarity of isoforms expressed from all three p53 family genes caused confusion in their nomenclature in non-vertebrate species. We analyzed all molluscan p53-similar sequences found in the GenBank database and compared their sequences and genomic structures to p53 family genes from other animals. Our results lead to the conclusion that invertebrates have only one gene of the p53 family which radiated into three genes only in vertebrates. This ancestral (molluscan) p53 family gene shows to be most similar to p63 in vertebrates. Furthermore, our analyses indicate p53 family genes to be single-copy genes thus increasing their value as potential biomarkers. doi:10.1016/j.cbpa.2010.06.063
4. Pathogenic infection attenuates the response of the estrogenic biomarker, vitellogenin, in rainbow trout R. Burki, T. Wahli, H. Segner (University of Bern, Switzerland); P. Burkhardt-Holm (University of Basel, Switzerland); C.E. Rexroad, S. Afanasyev, M. Antikainen, A. Krasnov (AKVAFORSK, Norway) In the environment, aquatic organisms are exposed multiple instead of single stressors. During recent years, considerable progress has been achieved in assessing combination effects of chemical mixtures. For instance, it has been demonstrated that the induction of vitellogenin (VTG) in (male) fish – an established biomarker of exposure to estrogen-active compounds – responds additively to combinations of estrogen-active compounds, while simultaneous exposure to ligands of the estrogen receptor and of the arylhydrocarbon receptor has an antagonistic effect on VTG induction. In contrast to the progress in understanding combination effects of chemical mixtures, approaches to assess the combined effects of chemical and physical and/or biological stressors are less developed. Here, we study the combined impact of a chemical and a biological
S.R. Mesquita, L. Guilhermino, L. Guimarães (University of Porto, CIIMAR/ICBAS, Portugal) The serotonin reuptake inhibitor drug fluoxetine is currently found in effluent and surface waters. Considering its antidepressant and other effects in humans, biochemical, physiological and behaviour alterations are also expected to occur in wild organisms exposed to the substance. In the present study, intermoult Carcinus maenas were exposed for seven days to five fluoxetine concentrations ranging from 0.49 to 750 μg/L in a laboratorial bioassay. The following endpoints were assessed at the end of the test: spontaneous locomotor behaviour (SLB) using the classical openfield test; epidermis chitobiase activity (CTB), muscle cholinesterase (ChE), lactate dehydrogenase (LDH) and NADP+-dependent isocitrate dehydrogenase (IDH) activities; the activities of the enzymes glutathione S-transferases (GST), glutathione peroxidase (Gpx), glutathione reductase (GR), and the levels of total glutathiones (TG) and lipid peroxidation (LPO) in the hepatopancreas. Animals exposed to 120 and 750 μg/L spent significantly more time moving (TM) in the open-field and crossed a higher number of squares (NS) than controls. ChE, GST and GR activities, and TG levels, were also significantly increased in crabs exposed to 120 and 750 μg/L. Interestingly, significant correlations were found between TM and ChE (38.6%), TM and TG (31.7%), NS and ChE (47.1%), and NS and TG (34.4%). These results show that SLB was a sensitive endpoint regarding fluoxetine exposure, suggesting that bioassays based on it may be suitable tools to assess the toxicity of environmental contaminants to C. maenas. This work was supported by national-FCT and EU-FEDER funds through the project CRABTHEMES (FCOMP-01-0124-FEDER-007383). doi:10.1016/j.cbpa.2010.06.065
POSTER PRESENTATIONS 6. A non-lethal multi-biomarker approach to investigate the ecotoxicological status of Mediterranean loggerhead sea turtle (Caretta caretta, Linneo, 1758) S. Casini, I. Caliani, L. Marsili, M. Giannetti, S. Maltese, S. Ancora, N. Bianchi, C. Panti, T. Campani, L. Carletti, D. Coppola, M.C. Fossi (University of Siena, Italy); A. Cañadas (Alnitak, Spain); M. Parga (Submon, Spain)