Exploring modulation of action potential firing by artificial graft of fast GABAergic autaptic afferences in hypophyseal neuroendocrine melanotrope cells.

Excitable cells are connected via electrical and chemical synapses to form a complex plastic network that transmit and modify action potential trains. In vivo and in vitro studies suggest that powerful type A GABAergic self-innervations, known as autapses, play a central role in the shaping of spike discharges. Herein, we have investigated the effects of artificial autaptic activity on action potential firing in cultured hypophyseal neuroendocrine melanotrope cells removed from any synaptic input. Type A autaptic conductances were introduced by using a dedicated dynamic-clamp device, based on a digital signal processor. The results indicate that cells grafted with autaptic dynamic-clamp are subjected to a modulation of both evoked firing and artificial GABA(A)-currents. The autaptic feedback reduced the action potentials amplitude, decreasing both the overshoot and the after-hyperpolarization potential (AHP). In return, the reduced voltage changes diminished the autaptic current amplitudes. The overall effect was a decrease of the cell firing rate. The introduction of a variable autaptic delay strongly altered the cell responses. Under certain conditions, the artificial autaptic current formed an additional component of the AHP which was markedly augmented. This action resulted in a stabilization of the action potential train leading to a more regular firing. In conclusion, it appeared that the autaptic feedback was very dependent on functional parameters such as the autaptic distance. Further investigations are in progress to complete our model, taking the autaptic plasticity into account.

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.

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.

Differential pattern of Cu/Zn superoxide dismutase isoforms in relation to tidal spatio-temporal changes in the blue mussel Mytilus edulis.

Inducible antioxidant defences in marine organisms such as mussel bivalves are commonly used as biomarkers of pollutant-induced oxidative stress and their variations proposed as one of the biological effect measurements for assessment of contamination impact in aquatic environments. Among them, the copper/zinc superoxide dismutases (Cu/Zn-SODs) are metalloenzymes which play a key role in the protection of cells in case of oxidative stress. In order to observe possible variations of an antioxidant response in relation to tidal oscillations, the copper/zinc superoxide dismutase activity (Cu/Zn-SOD) was characterized in the digestive gland and gills of blue mussels sampled at high and low shore throughout the tidal cycle. Determination of SOD activity was performed on gels after isoelectro-focusing, allowing the revelation of three isoforms. In both tissues, high-shore mussels exhibited a higher level of total SOD activity than low-shore mussels. During emersion, a decrease of total SOD activity appeared in digestive gland for both groups. In high-shore mussels, the less acidic form contributed to 75% of the total activity, the second one to 20% and the more acidic one to 5% in both tissues before air exposure. During emersion, the relative contribution of the three isoforms to the total activity was markedly changed with a significant decrease in intensity of the first isoform and parallel increases in the two other ones. After re-immersion a progressive recovery of proportions of SOD isoforms was observed. In low-shore mussels, the relative contribution of the three isoforms to the total SOD activity showed similar changes. The observed variations could correspond to changes in the redox status of the mussels during tidal oscillations.

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

BACKGROUND: Proteomics 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. RESULTS: Data 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. CONCLUSION: Machine 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.

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.

Proteome modifications of blue mussel (Mytilus edulis L.) gills as an effect of water pollution.

The discharge of chemicals such as oil associated or not with derived products constitutes a real threat for the environment. We report here the differential expression of the blue mussel (Mytilus edulis) gill proteins corresponding to two contaminated environmental conditions: crude oil and offshore produced water. In order to evaluate and understand contaminants, effects and adaptive response of these organisms, we identified proteins using MS. The latter can be grouped into three main classes: proteins involved in the cellular structure, in metabolism, and in defence proteins.

Regulation of volume-sensitive Cl- channels in multi-drug resistant MCF7 cells.

The P-glycoprotein (P-gp) is thought to be involved in the regulation of volume-sensitive chloride channels. In this study, the possible coupling between P-gp and swelling-activated chloride channels has been examined in MCF7 cells with sensitive (MDR-), resistant (MDR+), and reversed resistant (MDR(REV)) phenotypes. Western blot analysis showed that incubation of cells with doxorubicin induced P-gp expression in a reversible manner. Exposure of MDR+ cells to hypotonicity resulted in an inhibition of P-gp activity while hypotonic challenges induced swelling-activated chloride currents (I(Cl-swell)) in MDR-, MDR+, and MDR(REV) MCF7 cells. While verapamil inhibited I(Cl-swell) in all cell types, doxorubicin and vincristine rapidly and reversibly inhibited I(Cl-swell) uniquely in MDR+. Intracellular dialysis of MDR+ cells with C219 anti-P-gp antibody abolished the sensitivity of I(Cl-swell) to doxorubicin and led to a response pattern very close to that of MDR- cells. Taken together, these results strongly suggest that the P-glycoprotein regulates I(Cl-swell) in resistant MCF7.

Impact of endocrine toxicants on survival, development, and reproduction of the estuarine copepod Eurytemora affinis (Poppe).

Given recent reports suggesting that certain contaminants may be present in sewage effluents at levels, which may exert a deleterious impact on fish, it seems pertinent to extend ecological hazard evaluation for such substances to aquatic invertebrates. For this reason, we sought to determine whether 17beta-estradiol (E2), benzo(a)pyrene (BaP), 4-nonylphenol (NP), di(ethyl-hexyl)-phthalate (DEHP), and atrazine (A), individually or in binary mixture, can inhibit the survival, development, or reproductive output of the estuarine copepod Eurytemora affinis. In the first experiment nauplii were exposed to graded concentrations of individual contaminants to determine the 96-h LC50, 10-day no observed effect concentration (NOEC) and 10-day lowest observed effect concentration of each compound. In the second experiment newly released (<24-h-old) nauplii were exposed either to an individual contaminant at the NOEC or to binary mixtures, where each compound was used at half NOEC. The effects were monitored daily for development and sex ratio. After 10 days of exposure, adult males and females were paired and exposures continued to investigate effects on reproductive output (maximum 28 day total exposure). Based on these life cycle parameters the lowest 10-day NOECs were 6+/-4 microg L(-1) for E2, 7+/-3 microg L(-1) for NP, 12+/-3 microg L(-1) for BaP, 25+/-3 microg L(-1) for A, and 109+/-29 microg L(-1) for DEHP. BaP, NP, and DEHP inhibited naupliar development, but in binary mixture with E2 these compounds did not inhibit larval development. The results suggest that endocrine disruption could occur in copepods following exposure to estrogenic compounds, especially if they are exposed starting from embryonic development.

Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase.

In the present work, we investigated in the blue mussel (Mytilus edulis) the seasonal variations in the activity of several enzymes, which participate in the cellular defence system that is involved in the adaptive response of organisms to pollution. The activity levels of glutathione S-transferase, glutathione peroxidase, glutathione reductase and three isoforms of Cu/Zn-superoxide dismutase in gills and digestive glands of this bivalve species were used as biomarkers. Adult wild mussels were collected in Le Havre harbour (north-west coast of France) from four sites with different environmental conditions. Measurements of enzymatic activities were performed on tissue homogenates except for Cu/Zn-superoxide dismutase for which the activity of each isoform was detected on gel after isoelectric focusing. Seasonal variations in antioxidant enzyme activities were observed, characterized by low activity levels during winter, a period where oxidative stress is known to be high in bivalves. A clear-cut discrepancy between tissues was noted concerning inter-individual variability of data, which was low in gills but high in digestive gland, leading to the conclusion that gills could preferentially be used in biomonitoring studies dealing with oxidative stress in the blue mussel. As compared to animals from the reference site, mussels from the most polluted sites exhibited changes in the Cu/Zn-superoxide dismutase pattern characterized by an increase in the activity of the more acidic isoform without significant variation of the total activity of the enzyme. The most striking data were recorded in mussels collected at the outlet of a thermoelectric power plant. When compared to animals from the reference site, not only their gills showed a highly significant induction of the most acidic isoform of the Cu/Zn-superoxide dismutase (+340%, P < 0.001) but also high levels of glutathione S-transferase activity (+269%, P < 0.001). This study points out the usefulness of Cu/Zn-superoxide dismutase expression pattern as a biomarker of exposure to environmental stress rather than measurement of total activity of the enzyme, in field studies using Mytilus edulis. It also indicates the informative potential for glutathione S-transferase measurements in gills and underlines the advantages of selecting a battery of biomarkers for evaluating the impact of contamination on marine organisms.

Cell responses to xenobiotics: comparison of MCF7 multi-drug- and mussel blood cell multi-xenobiotic-defense mechanisms.

Multi-drug resistance (MDR) in MCF7 breast cancer cells and multi-xenobiotic resistance (MXR) in mussel (Mytilus edulis) blood cells (MBC) are well known mechanisms that contribute to the decrease in intracellular concentrations of many unrelated but cytotoxic compounds. In the present work, we have carried out comparative investigations of the MDR/MXR protective mechanisms using a rapid colorimetric assay for cell viability and calcein accumulation for MDR/MXR activities. These studies were performed using cultured MCF7 and MBC before and after in vitro exposure to xenobiotics. Our results indicate that a 5-day exposure to doxorubicin or vincristine decreased calcein accumulation in MBC which is consistent with an induction of multi-xenobiotic resistance. The increase in calcein accumulation provoked by 1-h treatment with 50 microM verapamil was much lower in MBC when compared to the P-glycoprotein overexpressing MCF7 cell line. We conclude that such microplate assays could be used in primary cultures of MBC to estimate the effects of various chemicals on MXR activity.

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.

Molecular cloning of flounder Xp18, a newly identified highly conserved protein mainly expressed in the ovary.

Screening of a flounder ovary cDNA library with a rainbow trout p53 probe led to the isolation of a p53-unrelated cDNA encoding an unknown 161 amino acid protein. In view of its apparent molecular weight and yet unknown function, the deduced protein was named Xp18. Corresponding orthologous cDNAs or expressed sequence tags have been identified in several species including human, rodents, bovine, chicken and zebrafish and a related cDNA has also been isolated in the fruit fly. Deduced amino acid sequences appeared to be extremely well conserved throughout vertebrate evolution. Structure predictions suggested that Xp18 may correspond to an integral protein comprising four transmembrane domains. The charged C-termini of all known vertebrate Xp18-like proteins displayed a characteristic KXKXX motif which is considered as an endoplasmic reticulum targeting sequence. Gene expression, as shown by Northern blot and quantitative reverse transcription-polymerase chain reaction analysis, was significantly higher in the ovary and to a lesser extent in the brain. Xp18 transcripts were also detected by in situ hybridization in most of the circumventricular regions of the brain of adult flounders. The gene encoding the human protein is located on chromosome Xq22.1, a genome region involved in numerous genetic diseases including premature ovarian failure.

Rhodamine exclusion activity in primary cultured turbot (Scophthalmus maximus) hepatocytes.

Cellular detoxification by direct processes has been investigated in fish by studying the ability of hepatocytes prepared from juvenile aquarium-reared turbot (Scophthalmus maximus) to actively exclude the fluorescent dye rhodamine B (RB). Cell viability was studied by measurements of non-specific esterase activity using fluorescein diacetate. This revealed that turbot hepatocytes can be cultured for a few days with a viability decreasing to 38% after 24 h. The 24-h cultured cells have been used to study the rhodamine B exclusion activity using confocal laser microscopy. Hepatocytes accumulated the dye in a competitive manner with verapamil, thus suggesting that they express a transport system similar to the P-glycoprotein-mediated multixenobiotic resistance process. Incubation of cells with 1 microM RB and 20 microM verapamil led to a 26% increase of cellular fluorescence as compared to the accumulation in absence of competitor. Rhodamine B accumulated in the whole cytoplasm, with more concentrated areas that might correspond to the lysosomal compartment and the cell membrane.

Partial purification and characterization of acetylcholinesterase (AChE) from the estuarine copepod Eurytemora affinis (Poppe).

Oligohaline copepods such as Eurytemora affinis are widespread in estuaries of northwestern Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a sublethal biomarker of exposure to neurotoxic compounds supposes that ChE has been defined in copepods. This study reports the partial purification and characterization of ChE extracted from E. affinis. Analysis by non-denaturing PAGE and by isoelectric focusing indicated that the enzyme is probably a single dimeric form of 140 KDa, with a pI of 6.2. This enzyme is likely an acetylcholinesterase (AChE) since it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates, such as butyrylthiocholine and propionylthiocholine, at pH 7.0 and 25 degrees C, and is inhibited by eserine but not by iso-OMPA. The enzyme exhibited high sensitivity to some of the various pollutants tested. The kinetic properties of this ChE were compared with those of other invertebrate ChEs.