Using a novel "Integrated Biomarker Proteomic" index to assess the effects of freshwater pollutants in European eel peripheral blood mononuclear cells.

Using proteomic data as biomarkers of environmental pollution has the potential to be of a great interest in ecological risk assessment as they constitute early warning indicators of ecologically relevant effects on biological systems. To develop such specific and sensitive biomarkers, the use of a set of proteins is required and the identification of protein expression signatures (PES) may reflect the exposure to specific classes of pollutants. Using 2D-DIGE (Differential in Gel Electrophoresis) methodology, this study aimed at identifying specific PES on European eel (Anguilla anguilla) peripheral blood mononuclear cells (PBMC) after 48 h in vitro exposure to two sublethal concentrations of dichlorodiphenyltrichloroethane (p,p'-DDT) (10 µg/L and 1mg/L) or cadmium (Cd) (1 µg/L and 100 µg/L). The present results have been supplemented with data of a first in vitro study on cells exposed to perfluorooctane sulfonate (PFOS) (10 µg/L and 1mg/L). A total of thirty-four protein spots, belonging to 18 different identified proteins found in all conditions, have been selected as possible biomarkers to develop a synthetic Integrated Biomarker Proteomic (IBP) index. IBP follows a dose-response relationship with higher values at the highest tested concentration for each pollutant (Cd: 9.96; DDT: 7.44; PFOS: 7.94) compared to the lowest tested concentration (Cd: 3.81; DDT: 2.91; PFOS: 2.06). In a second step, star plot graphs have been applied to proteomic data in order to allow visual integration of a set of early warning responses measured with protein biomarkers. Such star plots permit to discriminate the type of pollutant inducing a proteomic response. We conclude that using IBP is relevant in environmental risk assessment, giving to this index the potential to be applied as a global index of proteome alteration in endangered species such as the European eel. BIOLOGICAL SIGNIFICANCE: In this study, 34 protein spots have been selected as possible biomarkers to develop a synthetic Integrated Biomarker Proteomic index (IBP). Results show that IBP follows a dose-response relationship with higher values at the highest tested concentration for each pollutant compared to the lowest tested concentration. Star plot graphs have also been applied to proteomic data in order to allow visual integration of a set of early warning responses measured with protein biomarkers. Such star plots permit to discriminate the type of pollutant inducing a proteomic response. IBP is relevant in environmental risk assessment, giving to this index the potential to be applied as a global index of proteome alteration in endangered species such as the European eel.

Postglacial climate changes and rise of three ecotypes of harbour porpoises, Phocoena phocoena, in western Palearctic waters.

Despite no obvious barriers to gene flow in the marine realm, environmental variation and ecological specializations can lead to genetic differentiation in highly mobile predators. Here, we investigated the genetic structure of the harbour porpoise over the entire species distribution range in western Palearctic waters. Combined analyses of 10 microsatellite loci and a 5085 base-pair portion of the mitochondrial genome revealed the existence of three ecotypes, equally divergent at the mitochondrial genome, distributed in the Black Sea (BS), the European continental shelf waters, and a previously overlooked ecotype in the upwelling zones of Iberia and Mauritania. Historical demographic inferences using approximate Bayesian computation (ABC) suggest that these ecotypes diverged during the last glacial maximum (c. 23-19 kilo-years ago, kyrbp). ABC supports the hypothesis that the BS and upwelling ecotypes share a more recent common ancestor (c. 14 kyrbp) than either does with the European continental shelf ecotype (c. 28 kyrbp), suggesting they probably descended from the extinct populations that once inhabited the Mediterranean during the glacial and post-glacial period. We showed that the two Atlantic ecotypes established a narrow admixture zone in the Bay of Biscay during the last millennium, with highly asymmetric gene flow. This study highlights the impacts that climate change may have on the distribution and speciation process in pelagic predators and shows that allopatric divergence can occur in these highly mobile species and be a source of genetic diversity.

Looking for protein expression signatures in European eel peripheral blood mononuclear cells after in vivo exposure to perfluorooctane sulfonate and a real world field study.

The decline of European eel population can be attributed to many factors such as pollution by xenobiotics present in domestic and industrial effluents. Perfluorooctane sulfonate (PFOS) is a ubiquitous compound of a particular concern in Europe. PFOS can reach high concentrations in tissues of organisms and many toxic effects have been reported in fish. This study aimed at evaluating the toxicological effects of PFOS in European eel peripheral blood mononuclear cells (PBMCs) at the protein expression level. To identify proteins whose expression was modified by PFOS, we performed a proteomic analysis on the post-nuclear fraction of PBMCs after a chronic exposure (28 days) of yellow eels to zero, 1 or 10 µg/L PFOS. This in vivo study was completed by a proteomic field study on eels sampled in Belgian rivers presenting different PFOS pollution degrees. Proteins were separated by two-dimensional in-gel electrophoresis (2D-DIGE) to compare the post-nuclear fraction of PBMCs from the reference group with cells from fish exposed to the pollutant of interest. On the 28 spots that were significantly (p < 0.05; ANOVA followed by a Dunnett post-hoc test) affected by PFOS in the in vivo experiment, a total of 17 different proteins were identified using nano-LC ESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. In the field experiment, 18 significantly (p < 0.05; ANOVA followed by Dunnett's test) affected spots conducted to the identification of 16 different proteins. Interestingly, only three proteins were found in common between in vivo and in situ experiments: plastin-2, alpha-enolase and glyceraldehyde 3-phosphate dehydrogenase. Comparing the results with a previous study, plastin-2 and alpha-enolase were also been found to be affected after in vitro exposure of PBMCs during 48 h to either 10 µg or 1 mg PFOS/L. Potential use of these proteins as biomarkers of PFOS exposure in European eel could indicate early warning signals.

Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure.

Since the 1980s, the stocks of European eel have been declining in most of their geographical distribution area. Many factors can be attributed to this decline such as pollution by xenobiotics like perfluorooctane sulfonate (PFOS). This study aimed at evaluating the in vitro toxicity of eel peripheral blood mononuclear cells (PBMC) exposed to PFOS. Exposure time and two concentrations were chosen to avoid cell mortality (48 h exposure at 10 µg PFOS/L and 1mg PFOS/L). After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed to compare PBMC from the control group with cells exposed to the pollutant. On the 158 spots that were significantly affected by PFOS exposure, a total of 48 different proteins were identified using nano-LCESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. These proteins can be categorized into diverse functional classes, related to cytoskeleton, protein folding, cell signaling, proteolytic pathway and carbohydrate and energy metabolism, which provide clues on the cellular pathways mainly affected by PFOS. Some of the identified proteins are rarely found in other ecotoxicological proteomic studies and could constitute potential biomarkers of exposure to PFOS in fish.

Fish peripheral blood mononuclear cells preparation for future monitoring applications.

Fish species possess many specific characteristics that support their use in ecotoxicology. Widely used in clinical research, peripheral blood mononuclear cells (PBMCs) can reasonably be exploited as relevant target cells in the assessment of environmental chemical toxicity. The current article focuses on the methods necessary to isolate, characterize, and culture fish PBMCs. These procedures were successfully applied on an endangered species, the European eel (Anguilla anguilla L.), and on an economically important and worldwide exported species, the Asian catfish (Pangasianodon hypophthalmus S.). Proteomic approaches can be useful to screen xenobiotic exposure at the protein expression level, giving the opportunity to develop early warning signals thanks to molecular signatures of toxicity. To date, a major limitation of proteomic analyses is that most protein expression profiles often reveal the same predominant and frequently differentially expressed families of proteins regardless of the experimental stressing conditions. The current study describes a methodology to get a postnuclear fraction of high quality isolated from fish PBMCs in order to perform subsequent subproteomic analyses. Applied on samples from eel, the subproteomic analysis (two-dimensional differential in-gel electrophoresis) allowed the identification by liquid chromatography-tandem mass spectrometry and searches in the full NCBInr (National Center for Biotechnology Information nonredundant) database of 66 proteins representing 36 different proteins validated through Peptide and Protein Prophet of Scaffold software.

Phylogeny and androgenesis in the invasive Corbicula clams (Bivalvia, Corbiculidae) in Western Europe.

BACKGROUND: The genus Corbicula is one of the most invasive groups of molluscs. It includes both sexual and androgenetic lineages. The present study re-assessed the different morphotypes and haplotypes of West European Corbicula in order to clarify their taxonomic identification and phylogenetic relationships with American and Asian Corbicula clams. We studied several populations from West European river basins (Meuse, Seine, Rhine and Rhône) through an "integrative taxonomy" approach. We combined morphology, partial mitochondrial COI and cyt b sequences and eleven microsatellite loci. Furthermore, we looked for discrepancies between mtDNA and nrDNA/morphology, indicative of androgenesis between lineages. RESULTS: There are three Corbicula morphotypes in Western Europe associated to three mitochondrial lineages and three genotypes. Form R shares the same COI haplotype as the American form A and the Japanese C. leana. Form S and the American form C have the same haplotype, although their morphologies seem divergent. The European form Rlc belongs to the same mitochondrial lineage as both the American form B and the Asian C. fluminea.Interestingly, within each haplotype/genotype or lineage, no genetic diversity was found although their invasive success is high. Moreover, we detected rare mismatches between mtDNA and nrDNA/morphology, indicative of androgenesis and mitochondrial capture between form R and form S and therefore challenging the phylogenetic relatedness and the species status within this genus. The global phylogenetic analysis revealed that the sexual Corbicula lineages seem restricted to the native areas while their androgenetic relatives are widespread and highly invasive. CONCLUSIONS: We clarified the discrepancies and incongruent results found in the literature about the European morphotypes of Corbicula and associated mitochondrial lineages. The three West European morphotypes belong to three distinct nuclear and mitochondrial lineages. However mitochondrial capture occurs in sympatric populations of forms R and S. The species status of the morphotypes therefore remains doubtful. Moreover the androgenetic lineages seem widely distributed compared to their sexual relatives, suggesting that androgenesis and invasive success may be linked in the genus Corbicula.

Eco-, geno- and human toxicology of bio-active nanoparticles for biomedical applications.

Gene delivery has become an increasingly important strategy for treating a variety of human diseases, including infections, genetic disorders and tumours. To avoid the difficulties of using viral carriers, more and more non-viral gene delivery nanoparticles are developed. Among these new approaches polyethylene imine (PEI) is currently considered as one of the most effective polymer based method solution and considered as the gold standard. The toxicity of nanoparticles is a major concern when used for medical application. In this study we chose two nanoparticles for an in depth toxicological and ecotoxicological evaluation, one well characterized, PEI, and another novel polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). In the present study we have assessed the toxicity of these cation nanoparticles as such and of the polyplexes - nanoparticles covered with DNA. As these nanoparticles are also frequently used in high volumes in various industries and as such may enter in the environment, we also made an initial assessment of ecotoxicological effects assessment. The following nanoparticles related aspects have been studied during the project: development and characterization, ecotoxicity, general toxicity and specific toxicity. To this end a battery of different tests was used. The conclusion of these tests is that toxicity is varying between different nanoparticles and between different DNA covering ratios. In general, in the different systems tested, the PEI polymer is more toxic than the PDMAEMA polymer. The same difference is seen for the polyplexes and the higher the charge ratio, the more toxic are the polyplexes. Our study also clearly shows the need for a broad spectrum of toxicity assays for a comprehensive risk assessment. Our study has performed such a comprehensive analysis of two biomedical nanoparticles.