Induction of apoptosis in mussel Mytilus galloprovincialis gills by model cytotoxic agents.

Apoptosis signaling pathway was investigated in the marine mussel Mytilus galloprovincialis exposed to various stressors. Analyses were performed in mussels exposed to two major pollutants of the aquatic environment: tributyltin and the water soluble fraction of diesel oil, for 1 h and animals were then maintained in sea water for a recovery period of 6 and 24 h. Apoptosis was evaluated at several levels of the cell signaling cascade by measuring Bcl-xS expression, caspase-3 activity and DNA damage (Fast micromethod(®) and TUNEL techniques). H(2)O(2) was used as a control of apoptosis induction for validation of the assays. Results showed an induction of Bcl-xS expression, a protein implicated in apoptosis, after 1 h exposure to all concentrations of chemicals. Moreover, in the same manner, apoptotic DNA damage was induced with all chemicals tested. Besides, caspase 3 activity was detected after 1 h exposure to low doses of TBT and diesel oil while the high concentrations induced this protein after 6 h. The achieved data were also correlated with our previous study, demonstrating an induction of the mitogen-activated protein kinase (MAPK) activity in the mussel M. galloprovincialis exposed to the same conditions. In conclusion, this study was one of the first characterizing the MAP kinase cell signaling pathway leading to apoptosis in the mussel M. galloprovincialis exposed to chemicals. It showed for the first time that the Bcl-xS protein was present in these mussels as in other species and played a role in apoptosis mediation. Moreover, the main originality of this work was that it showed that two apoptotic pathways might be present in the mussel: a caspase 3-dependent and a caspase 3-independent pathways.

MAP kinase cell signaling pathway as biomarker of environmental pollution in the sponge Suberites domuncula.

In the present study, we analyzed the effects of two major pollutants of the environment, tributyltin (TBT) and water-accommodated fraction (WAF) of diesel oil, on MAP kinase activation, apoptosis induction and DNA damage, in the marine sponge Suberites domuncula. Our results clearly demonstrated a differential activation of the MAPKs depending on the chemicals tested. TBT induced the activation of p38 and JNK while diesel oil enhanced activation of both ERK and p38. The activation of MAPKs was observed after 1 h exposure and 6 and 24 h of recovery in seawater. In addition, DNA fragmentation, assessed by two techniques, the Fast micromethod(®) and the TUNEL assay, was detected after sponges were treated with both chemicals. Moreover, the study of caspase 3/7 activity showed that apoptosis was induced and triggered with all concentrations of TBT but only at high diesel oil concentrations. After TBT exposure, a correlation was observed between JNK activation, caspase 3 activity and DNA damage while p38 activation followed the two latter parameters at high concentrations of diesel oil, suggesting that sponges enhanced a specific apoptotic pathway depending on the xenobiotic tested. This study demonstrated a high signal response by the sponge Suberites domuncula to the tested chemicals. Cell signaling pathway studies may thus be of use in water quality biomonitoring programs.

Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus.

Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I (K slow)) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I (K fast)) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I (KCa)) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I (Ca)) inhibited by verapamil at 5 × 10(-4) M, T-type Ca(2+) current, rapidly activated and inactivated, and sodium current (I (Na)) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 µM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2'-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.

Sodium channel Na(V)1.5 expression is enhanced in cultured adult rat skeletal muscle fibers.

This study analyzes changes in the distribution, electrophysiological properties, and proteic composition of voltage-gated sodium channels (Na(V)) in cultured adult rat skeletal muscle fibers. Patch clamp and molecular biology techniques were carried out in flexor digitorum brevis (FDB) adult rat skeletal muscle fibers maintained in vitro after cell dissociation with collagenase. After 4 days of culture, an increase of the Na(V)1.5 channel type was observed. This was confirmed by an increase in TTX-resistant channels and by Western blot test. These channels exhibited increased activation time constant (tau(m)) and reduced conductance, similar to what has been observed in denervated muscles in vivo, where the density of Na(V)1.5 was increasing progressively after denervation. By real-time polymerase chain reaction, we found that the expression of beta subunits was also modified, but only after 7 days of culture: increase in beta(1) without beta(4) modifications. beta(1) subunit is known to induce a negative shift of the inactivation curve, thus reducing current amplitude and duration. At day 7, tau(h) was back to normal and tau(m) still increased, in agreement with a decrease in sodium current and conductance at day 4 and normalization at day 7. Our model is a useful tool to study the effects of denervation in adult muscle fibers in vitro and the expression of sodium channels. Our data evidenced an increase in Na(V)1.5 channels and the involvement of beta subunits in the regulation of sodium current and fiber excitability.

Mechanisms of the sensory effects of tacrolimus on the skin.

BACKGROUND: Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported. OBJECTIVES: To understand the mechanisms underlying these burning sensations. METHODS: We examined the impact of tacrolimus on substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation. Because phosphorylation of TRPV1 (transient receptor potential subtype vanilloid 1) plays a role in the induction of pain, we investigated whether tacrolimus regulates the phosphorylation state of TRPV1. Finally, we used a macropatch to evaluate the impact of tacrolimus on voltage-gated calcium currents of sensory neurons. RESULTS: Tacrolimus was able to induce initial SP release by extracellular calcium influx and inhibited SP release induced by capsaicin after 1, 24 and 72 h of pretreatment. Analysis of TRPV1 phosphorylation by Western blot confirmed the capacity of tacrolimus to favour phosphorylation. An electrophysiological study showed inhibitory effects on calcium currents. CONCLUSIONS: The efficacy of tacrolimus in pruritus, as well as the sensory side-effects, could be explained by a direct effect on neurons through an effect on calcineurin, possibly by a desensitization of TRPV1 and calcium currents through the PIP(2) regulation pathway.

Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution.

Comet assay and early apoptosis.

The comet assay is a single cell gel electrophoresis test currently used as a qualitative and quantitative genotoxicity test. However, some of the results from this comet assay and current knowledge on apoptosis lead us to suspect the presence of some false positive results. The aim of this study was to ascertain if apoptotic cells can yield comet images that might distort the interpretation of the results. Using Jurkat cells, that hardly express Fas antigen, and apoptosis induction with anti-Fas antibody, it was possible to show that apoptosis can generate typical comet pictures as soon as the cells enter the apoptosis process. Therefore, comet images cannot be interpreted as a genotoxicity indicator when an apoptosis risk is present. Yopro-1 staining, that is also nearly immediate after apoptosis induction, can be used to balance comet assay results.

A comparative study between classical stirred and ultrasonically-assisted dead-end ultrafiltration.

The aim of this work was to determine mass transfer coefficients in the cases of ultrasonically-assisted and classical stirred dead-end ultrafiltration. A comparative study was then performed, and mass transfer coefficients obtained under ultrasonic conditions are described by an empirical model. This correlation results from an analogy with what is observed using a stirred cell and involves the ultrasonic power as the main parameter. The hydrodynamics are assumed to depend on the intensity of the ultrasound effects illustrated by the agitation arising within the cell. This agitation is due to convective currents as well as physical effects due to cavitation. The concentration polarization phenomenon is therefore affected by this action of ultrasonic waves.

Effects of osmolality, morphology perturbations and intracellular nucleotide content during the movement of sea bass (Dicentrarchus labrax) spermatozoa.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20 degrees C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg-1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 +/- 1.8% of spermatozoa were motile with a swimming velocity of 141.8 +/- 1.2 microns s-1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+ modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.

Primary cultures of heart cells from the scallop Pecten maximus (mollusca-bivalvia).

Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.

Nucleotide content, oxidative phosphorylation, morphology, and fertilizing capacity of turbot (Psetta maxima) spermatozoa during the motility period.

The interdependence between motility, respiration, ATP production, and utilization was investigated in intact spermatozoa of turbot (Psetta maxima), a marine teleost. When spermatozoa were diluted in a hyperosmotic medium (>300 mOsmol/kg), they immediately became motile, and the intracellular concentration of ATP as well as the adenylate energy charge ratio dropped concomitant with the straight-line velocity. The ADP and AMP levels increased from 1.4 to 8.0 nmole/10(8) cells and from 0.6 to 6.0 nmole/10(8) cells, respectively. Moreover, 31P-NMR spectra recorded prior to the swimming phase revealed the presence of phosphomonoesters (PMEs) and phosphodiesters (PDEs), intracellular inorganic phosphate (Pi), and phosphocreatine (PCr). At the end of the motility period, PCr, PDE, and PME decreased, while the Pi level increased markedly. Following initiation of motility, O2 consumption of spermatozoa increased from 34.9 to 124.8 O2 nmole/10(9) spermatozoa/min. FCCP, an uncoupler of oxidative phosphorylation, did not significantly affect the respiratory rate of motile spermatozoa. Ouabain, a specific inhibitor of (Na+/K+)/ATPase, slightly decreased the respiration rate of motile spermatozoa, indicating that the major part of ATP catabolism was linked to dynein ATPase. Inhibitors of the respiratory chain (KCN, NaN3, NaHCO3-, oligomycin) reduced sperm respiration, percentage of motile cells, velocity, and adenylate contents. Following the reactivation of motility of demembranated spermatozoa, KCN, NaN3, NaHCO3- altered the flagellar beat frequency, demonstrating that these respiratory inhibitors possess action sites other than mitochondria. Mitochondrial oxidative phosphorylation is highly requested to produce energy required during motion. Nevertheless it is insufficient to maintain endogenous ATP stores. A second phase of motility was induced by a transfer of exhausted spermatozoa into an ionic medium of low osmolality (200 mOsmol/kg) for 30 min. Spermatozoa, once reactivated in AM, recovered 55% of initial motility and 31% of initial fertilization rate. In hypo-osmotic medium, mitochondrial oxidative phosphorylation also induced ATP regeneration. Following activation of movement, several morphological changes were observed in the mitochondria and the midpiece.

Cryopreservation of pecten maximus heart cells

A dissociation protocol for Pecten maximus heart has been established that makes it possible to obtain functional primary cultures routinely (9, 10); freezing assays of isolated cells were performed with the aim of making it possible to cultivate these cells in vitro after thawing to provide a constant standardized source of cells for applied research. Various parameters such as the nature and the concentration of the cryoprotectant, the cooling rate, and the incubation time of cells with the cryoprotective agent were evaluated. Best results were obtained by freezing cells in 12% dimethyl sulfoxide (Me2SO) in Leibovitz L15 medium at a cooling rate of approximately 2-3 degreesC/min. Thawed cells in culture attached to the substrate and survived for at least 3 weeks. They exhibited similar morphology and synthesised proteins, DNA, and lipids in vitro at levels close to those observed in fresh cells. To our knowledge, it is the first time that cultures have been obtained from cryopreserved marine invertebrate cells. Copyright 1998 Academic Press.

Transient expression of luciferase reporter gene after lipofection in oyster (Crassostrea gigas) primary cell cultures.

Transient expression of the luciferase gene, under transcriptional control of several heterologous promoters, was obtained in heart primary cell cultures of the Pacific oyster, Crassostrea gigas. Drosophila heat shock protein 70 promoter (hsp70), cytomegalovirus, and simian virus early promoters, controlling the luciferase gene, were transfected into the cell cultures using liposomes. Two culture media were used to establish primary cell cultures and tested as transfection media. Parameters such as the quantity of DNA and the ratio of DNA to liposome were analyzed to define the best transfection conditions. In oysters, the Drosophila inducible hsp70 promoter behaved in a way similar to that observed in other animal species. Moreover, for this study, hsp70 was more efficient than the cytomegalovirus and simian virus promoters.