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.

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.

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.

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.

Receptor activated C kinase is down-regulated in the male gonad of the marine bivalve mollusc Mya arenaria exposed to tributyltin (TBT).

This study was aimed at investigating the molecular mechanisms by which tributyltin (TBT) impairs the reproductive processes in the marine bivalve Mya arenaria. The suppression polymerase chain reaction subtractive hybridization (SSH) method was used to identify differentially expressed transcripts in the gonads of adult M. arenaria 72 h after a single injection of 160 ng TBT in the adductor muscle.

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.

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).

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.

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.

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.

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.

Functional analysis of chemically-induced mutations at the flounder TP53 locus, the FACIM assay.

A functional assay was developed in yeast to identify mutations induced by DNA-damaging agents at the flounder TP53 locus. This assay named FACIM for functional analysis of chemically-induced p53 mutations, is based on the assumption that most genotoxin-induced mutations inactivate transcriptional activity of the TP53 protein. The functional status of the protein expressed in yeast was measured using a p53-responsive reporter gene.

Molecular cloning of superoxide dismutase (Cu/Zn-SOD) from aquatic molluscs.

The potential of the first line of the active oxygen-scavenging system, partial cDNA encoding Cu/Zn superoxide dismutase (SOD) was isolated in three aquatic mollusc species: Ruditapes decussatus (marine clam), Dreissena polymorpha (continental water mussel) and Bathymodiolus azoricus (hydrothermal vent mussel). These SOD cDNA fragments were amplified by PCR with degenerate oligonucleotide primers derived from the amino acid sequence conserved in the Cu/Zn-SOD from several other organisms. A partial cDNA of CuZn-SOD was obtained for R.

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.

The European flounder (Platichthys flesus) TP53 functions as a temperature-sensitive transcription factor which inhibits cell growth in yeast.

Numerous studies focus on biological roles of the TP53 tumor suppressor gene in mammals but little is known about the actual function of TP53 in lower vertebrates. In this study, we used an in vivo functional assay in yeast to address the transactivation capacity of the flounder TP53 protein. We showed that the flounder TP53 acts as a sequence-specific transcription factor which is able to transactivate various human promoters containing a p53-responsive element (RE).

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.

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.

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.

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.

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.

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.

Genetic and immunological characterisation of a multixenobiotic resistance system in the turbot (Scophthalmus maximus).

Pleiotropic resistance driven by transport proteins constitutes a very ubiquitous protection mechanism against natural or synthetic toxic compounds. The multidrug (MDR) or multixenobiotic (MXR) system has been identified in many different species, and may be used as a biomarker for pollution assessment. Here we report the existence of a gene encoding a MXR-related protein in a benthic fish species, the turbot Scophthalmus maximus, and its constitutive expression in several tissues.

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.

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.