François Leboulenger

A pollution-monitoring pilot study involving contaminant and biomarker measurements in the Seine Estuary, France, using zebra mussels (Dreissena polymorpha).

Zebra mussel (Dreissena polymorpha) is an invasive species that has proliferated in European and North American rivers and lakes during the last century. In this study, D. polymorpha has been used to provide information on contamination levels and biological effects in the Seine Estuary (France). The bivalves accumulated polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) to a high degree with values reaching 800 ng/g dry weight for PCBs (sum of 20 congeners), and 1,000 ng/g dry weight of PAHs (sum of 14 compounds) in the whole body. These values are among the highest reported of PCBs and, to a lesser extent, of PAHs in other contaminated areas in the world. Toxic equivalent quantities of PCBs and PAHs detected in zebra mussels varied from 20 to 40 pg dioxin equivalents/g dry weight for PCBs and up to 120 ng benzo[a]pyrene equivalents/ g dry weight for PAHs, indicating a high potential risk for animals feeding on them. Biological impacts, such as altered condition index, decreased lysosomal stability, and high levels of multixenobiotic resistance (MXR) proteins also were detected in mussels living downstream of Rouen, the main city of the Seine Estuary. Taken together, these results indicate that the Seine Estuary is a heavily polluted area with the potential to cause deleterious health effects in some endogenous living organisms. This study also shows that chemical and biological measurements bring different but complementary results that can help diagnose environmental health.

Characterization of an inducible isoform of the Cu/Zn superoxide dismutase in the blue mussel Mytilus edulis

Aerobic organisms are protected against oxidative stress by antioxidant systems which mobilise enzymes such as the Cu/Zn superoxide dismutase (Cu/Zn-SOD) which transfers O2(.-) to H2O2. In this paper, we report the characterization of three isoforms of Cu/Zn-SOD in the blue mussel Mytilus edulis and we show that one of these isoforms is strongly inducible. Cytosolic extracts of digestive gland and gills from adult blue mussels were analysed by polyacrylamide gel electrophoresis or isoelectric focusing followed by in situ staining for SOD activity. Two main bands of Cu/Zn-SOD were obtained at pI 4.7 and 4.6 corresponding to native apparent molecular weight values of 205 and 155 kDa. Blue mussels from chemically contaminated area in Le Havre harbour exhibited a third Cu/Zn-SOD isoform characterized by a more acidic isoelectric point (pI 4.55) and a native apparent molecular weight of 130 kDa. When maintained in clean marine water, mussels from this area showed a transitory decrease in total SOD activity accompanied by the disappearance of the SOD-3 band. Conversely, the exposure (4 and 8 h, and 3 and 7 days) of control blue mussels to copper (25 microg l(-1)) markedly increased SOD-3 band while the total SOD activity did not systematically change. Taken together our results suggest that the variations of SOD expression pattern in Mytihus edulis could be used as a tool for the marine environment monitoring.

Validation of an analytical procedure for polychlorinated biphenyls, coplanar polychlorinated biphenyls and polycyclic aromatic hydrocarbons in environmental samples

This work describes an efficient analytical procedure for the analysis of PCBs (polychlorinated biphenyls), coplanar PCBs and PAHs (polycyclic aromatic hydrocarbons) from the same sample. The method includes a solvent extraction followed by a combined purification-separation step on an alumina-silica column. Coplanar PCBs are isolated from the first fraction (PCBs) by a further high-performance liquid chromatography (HPLC) fractionation on a PYE [2-(1-pyrenyl)ethyldimethylsilylated silica gel] column. PCBs are identified and quantified by gas chromatography (GC) with electron-capture detection whereas GC with flame ionization detection or mass spectrometry are used for PAH determinations. This method allows the measurement of these contaminants in biota and sediment at trace levels as low as 1 pg g(-1) for coplanar PCBs with a precision better than 20%.

Flounder health status in the Seine Bay. A multibiomarker study

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.

VARIATIONS OF FATTY ACID CONTENTS IN SELECTED TISSUES OF THE FEMALE DOG COCKLE (GLYCYMERIS GLYCYMERIS L., MOLLUSCA, BIVALVIA) DURING THE ANNUAL CYCLE

Abstract The seasonal variations of fatty acid concentrations were studied during two consecutive years in five tissues of female dog cockles ( Glycymeris glycymeris ) originating from Douarnenez Bay (South Brittany, France). In all tissues, saturated fatty acids (SFA) were the major forms of fatty acids, followed by polyunsaturated fatty acids (PUFA). The concentrations of monounsaturated fatty acids (MUFA) were low. The C20:0 was most often the predominant SFA in all tissues. The total amount of fatty acids in each of the SFA, MUFA and PUFA series underwent important fluctuations in the foot and in the visceral mass, whereas only slight variations were recorded in the adductor muscles, the tunic coat and the mantle. A main peak of SFA was measured in the foot at the beginning of winter and peak values of SFA were recorded in the visceral mass in April, June and in autumn. The highest concentrations of fatty acids were measured in the visceral mass, in April just before the first and main annual spawning period which occurs in late April or in May, depending of the year. Our results suggest that G. glycymeris feeds both on bacteria-enriched detritic matter and on phytoplankton. Important fluctuations of fatty acids recorded in the visceral mass can, at least in part, be related to certain phases of the reproductive cycle. The foot may constitute a temporary reserve tissue for fatty acids, especially for SFA, at the beginning of winter.

Characterisation of Mytilus edulis hemocyte subpopulations by single cell time-lapse motility imaging.

In bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 mum min(-1). Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte-hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed.

Identification of differentially expressed genes in Dreissena polymorpha exposed to contaminants.

Development of transcriptome analysis methods such as differential display PCR and construction of subtractive libraries now makes it possible to profile gene expression in response to xenobiotic exposure. As an example of application of these methods, zebra mussels (Dreissena polymorpha) were treated with various contaminants such as Aroclor 1254, 3-methylcholanthrene, chrysene and atrazine. A total of 242 mRNAs were identified as differentially expressed. Analysis of these mRNAs should provide valuable information regarding detoxification mechanisms in this bivalve species. In addition, the use of cDNA array technology applied to these gene products may constitute a multi-marker approach to monitor the effect of contamination on this aquatic species.

Data processing and classification analysis of proteomic changes: a case study of oil pollution in the mussel, Mytilus edulis

BackgroundProteomics may help to detect subtle pollution-related changes, such as responses to mixture pollution at low concentrations, where clear signs of toxicity are absent. The challenges associated with the analysis of large-scale multivariate proteomic datasets have been widely discussed in medical research and biomarker discovery. This concept has been introduced to ecotoxicology only recently, so data processing and classification analysis need to be refined before they can be readily applied in biomarker discovery and monitoring studies.ResultsData sets obtained from a case study of oil pollution in the Blue mussel were investigated for differential protein expression by retentate chromatography-mass spectrometry and decision tree classification. Different tissues and different settings were used to evaluate classifiers towards their discriminatory power. It was found that, due the intrinsic variability of the data sets, reliable classification of unknown samples could only be achieved on a broad statistical basis (n > 60) with the observed expression changes comprising high statistical significance and sufficient amplitude. The application of stringent criteria to guard against overfitting of the models eventually allowed satisfactory classification for only one of the investigated data sets and settings.ConclusionMachine learning techniques provide a promising approach to process and extract informative expression signatures from high-dimensional mass-spectrometry data. Even though characterisation of the proteins forming the expression signatures would be ideal, knowledge of the specific proteins is not mandatory for effective class discrimination. This may constitute a new biomarker approach in ecotoxicology, where working with organisms, which do not have sequenced genomes render protein identification by database searching problematic. However, data processing has to be critically evaluated and statistical constraints have to be considered before supervised classification algorithms are employed.

Tidal height influences the levels of enzymatic antioxidant defences in Mytilus edulis

We investigated the potential variability of enzymatic antioxidant activities in blue mussels Mytilus edulis from a single intertidal population but living at different tidal heights. Activity levels of antioxidant enzymes (Cu/Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase) were measured in the gills and digestive gland of mussels sampled at high shore (HS, air-exposure>6h/12h) and low shore (LS, air-exposure<2h/12h) of an intertidal zone (Yport, Normandie, France) for two consecutive autumns. In both tissues, levels of each enzymatic activity (except GST) were clearly higher in HS mussels than in LS for the two years. These results suggest an ability to acclimate the enzymatic antioxidant defences to the degree of undergone stress, confirming the importance of environmental conditions in the antioxidant responses. Therefore, the location of organisms on the shore should be taken into account in sampling for ecotoxicological studies.

Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas.

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.