The VATO project: Development and validation of a dynamic transfer model of tritium in grassland ecosystem.

In this paper, a dynamic compartment model with a high temporal resolution has been investigated to describe tritium transfer in grassland ecosystems exposed to atmospheric 3H releases from nuclear facilities under normal operating or accidental conditions. TOCATTA-χ model belongs to the larger framework of the SYMBIOSE modelling and simulation platform that aims to assess the fate and transport of a wide range of radionuclides in various environmental systems. In this context, the conceptual and mathematical models of TOCATTA-χ have been designed to be relatively simple, minimizing the number of compartments and input parameters required. In the same time, the model achieves a good compromise between easy-to-use (as it is to be used in an operational mode), explicative power and predictive accuracy in various experimental conditions. In the framework of the VATO project, the model has been tested against two-year-long in situ measurements of 3H activity concentration monitored by IRSN in air, groundwater and grass, together with meteorological parameters, on a grass field plot located 2 km downwind of the AREVA NC La Hague nuclear reprocessing plant, as was done in the past for the evaluation of transfer of 14C in grass. By considering fast exchanges at the vegetation-air canopy interface, the model correctly reproduces the observed variability in TFWT activity concentration in grass, which evolves in accordance with spikes in atmospheric HTO activity concentration over the previous 24 h. The average OBT activity concentration in grass is also correctly reproduced. However, the model has to be improved in order to reproduce punctual high concentration of OBT activity, as observed in December 2013. The introduction of another compartment with a fast kinetic (like TFWT) - although outside the model scope - improves the predictions by increasing the correlation coefficient from 0.29 up to 0.56 when it includes this particular point. Further experimental investigation will be undertaken by IRSN and EDF next year to better evaluate (and properly model) other aspects of tritium transfer where knowledge gaps have been identified in both experimental and modelling areas.

The VATO project: An original methodology to study the transfer of tritium as HT and HTO in grassland ecosystem.

Tritium (3H) is mainly released into the environment by nuclear power plants, military nuclear facilities and nuclear reprocessing plants. The construction of new nuclear facilities in the world as well as the evolution of nuclear fuel management might lead to an increase of 3H discharges from the nuclear industry. The VATO project was set up by IRSN (Institut de Radioprotection et de Sûreté Nucléaire) and EDF (Electricité de France) to reduce the uncertainties in the knowledge about transfers of 3H from an atmospheric source (currently releasing HT and HTO) to a grassland ecosystem. A fully instrumented technical platform with specifically designed materials was set up downwind of the AREVA NC La Hague reprocessing plant (Northwest of the France). This study, started in 2013, was conducted in four main steps to provide an hourly data set of 3H concentrations in the environment, adequate to develop and/or validate transfer models. It consisted first in characterizing the physico-chemical forms of 3H present in the air around the plant. Then, 3H transfer kinetics to grass were quantified regarding contributions from various compartments of the environment. For this purpose, an original experimental procedure was provided to take account for biases due to rehydration of freeze-dried samples for the determination of OBT activity concentrations in biological samples. In a third step, the 3H concentrations measured in the air and in rainwater were reconstructed at hourly intervals. Finally, a data processing technique was used to determine the biological half-lives of OBT in grass.

Inter-comparison of dynamic models for radionuclide transfer to marine biota in a Fukushima accident scenario.

We report an inter-comparison of eight models designed to predict the radiological exposure of radionuclides in marine biota. The models were required to simulate dynamically the uptake and turnover of radionuclides by marine organisms. Model predictions of radionuclide uptake and turnover using kinetic calculations based on biological half-life (TB1/2) and/or more complex metabolic modelling approaches were used to predict activity concentrations and, consequently, dose rates of (90)Sr, (131)I and (137)Cs to fish, crustaceans, macroalgae and molluscs under circumstances where the water concentrations are changing with time. For comparison, the ERICA Tool, a model commonly used in environmental assessment, and which uses equilibrium concentration ratios, was also used. As input to the models we used hydrodynamic forecasts of water and sediment activity concentrations using a simulated scenario reflecting the Fukushima accident releases. Although model variability is important, the intercomparison gives logical results, in that the dynamic models predict consistently a pattern of delayed rise of activity concentration in biota and slow decline instead of the instantaneous equilibrium with the activity concentration in seawater predicted by the ERICA Tool. The differences between ERICA and the dynamic models increase the shorter the TB1/2 becomes; however, there is significant variability between models, underpinned by parameter and methodological differences between them. The need to validate the dynamic models used in this intercomparison has been highlighted, particularly in regards to optimisation of the model biokinetic parameters.

Estimation of marine source-term following Fukushima Dai-ichi accident.

Contamination of the marine environment following the accident in the Fukushima Dai-ichi nuclear power plant represented the most important artificial radioactive release flux into the sea ever known. The radioactive marine pollution came from atmospheric fallout onto the ocean, direct release of contaminated water from the plant and transport of radioactive pollution from leaching through contaminated soil. In the immediate vicinity of the plant (less than 500 m), the seawater concentrations reached 68,000 Bq.L(-1) for (134)Cs and (137)Cs, and exceeded 100,000 Bq.L(-1) for (131)I in early April. Due to the accidental context of the releases, it is difficult to estimate the total amount of radionuclides introduced into seawater from data obtained in the plant. An evaluation is proposed here, based on measurements performed in seawater for monitoring purposes. Quantities of (137)Cs in seawater in a 50-km area around the plant were calculated from interpolation of seawater measurements. The environmental halftime of seawater in this area is deduced from the time-evolution of these quantities. This halftime appeared constant at about 7 days for (137)Cs. These data allowed estimation of the amount of principal marine inputs and their evolution in time: a total of 27 PBq (12 PBq-41 PBq) of (137)Cs was estimated up to July 18. Even though this main release may be followed by residual inputs from the plant, river runoff and leakage from deposited sediments, it represents the principal source-term that must be accounted for future studies of the consequences of the accident on marine systems. The (137)Cs from Fukushima will remain detectable for several years throughout the North Pacific, and (137)Cs/(134)Cs ratio will be a tracer for future studies.

Molecular cloning of a new member of the p53 family from the Pacific oyster Crassostrea gigas and seasonal pattern of its transcriptional expression level.

Like other sessile filter-feeding molluscs, oysters may be exposed in the natural environment to a variety of contaminants. Long-term exposure to pollutants may be one factor affecting prevalence of cancerous-like disorders, such as neoplasia. Environmentally induced alterations in p53 protein expression, in relation to leukemia, have been reported in various mollusc species inhabiting polluted water, suggesting that p53 proteins can also be used as a marker for environmental research. This work reports the cloning and sequencing of a p53-like cDNA in the mollusc bivalve Crassostreagigas. The deduced amino acid sequences of p53 shared a high degree of homology with the homologues from other mollusc species, including typical eukaryotic p53 signature sequences. We examined the p53 transcription expression pattern during the annual cycle in oyster gills and whole soft tissues in four locations along the French coasts. Real-time PCR analysis suggested that strong variations at p53 mRNA level are probably synchronized with the seasonal cycle at the four locations investigated.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s).

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

[Examples of the keeping of drugs during the XVIth, XVIIth and XVIIIth centuries: silenes and drawers]. / Deux exemples de conditionnement pour conservation des drouges aux XVI, XV et XVIII siècles: silènes et tiroirs.

Silenes are wooden boxes with colourful paintings for the keeping of drugs and can be found nowadays in three collections in France: Hotel-Dieu in Troyes (Aube), a pharmacy in Bauge (Maine-et-Loire) and St-Roch museum in Issoudun (Indre). Drawers can be related to silenes but they are more ordinary and less attractive.

Regulation of Na+/H+ antiporter in trout red blood cells.

The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself. Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP. In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that CIA may be able to revive "sequestered' antiporters. We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.

Association of the band 3 protein with a volume-activated, anion and amino acid channel: a molecular approach.

In response to swelling, cells recover their initial volume by releasing intracellular solutes via volume-sensitive pathways. There is increasing evidence that structurally dissimilar organic osmolytes (amino acids, polyols, methyl amines), which are lost from cells in response to swelling, share a single pathway having the characteristics of an anion channel. However, the molecular identity of this pathway remains to be established. It has been suggested that the erythrocyte anion exchanger (AE1) or some AE1-related proteins could be involved. A direct evaluation of this possibility has been made by comparing the functional properties of two AE1s when expressed in Xenopus laevis oocytes: tAE1 is from a fish erythrocyte which releases taurine when swollen, and mAE1 is from a mammalian erythrocyte which does not regulate its volume when swollen. While mAE1 performs exclusively Cl-/Cl- exchange, tAE1 behaves as a bifunctional protein with both anion exchange and Cl-/taurine channel functions. Construction of diverse tAE1/mAE1 chimaeras allows the identification of protein domains associated with this channel activity. Thus, some AE1 isoforms could act as a swelling-activated osmolyte channel, a result having a potentially important implication in malaria. This review also discusses the possibility that several different proteins might function as swelling-activated osmolyte channels.

A role for the anion exchanger AE1 (band 3 protein) in cell volume regulation.

In response to swelling cells recover their volume by releasing ions (mainly K+, Cl-) and different organic solutes (e.g. taurine) via volume-sensitive pathways. Depending on the cause of swelling (net uptake of electrolytes or decrease in external osmolality) cells use specifically some of these pathways. Previous data indicate that the anion exchanger (AE1) is involved in the choice of the regulatory pattern the cells adopt. Molecular cloning and functional expression of AE1 from the trout erythrocyte shows that this anion exchanger can function as a channel mediating taurine fluxes. In the erythrocyte, the channel activation depends on the conditions as the cell is swollen: when swelling is caused by an accumulation of electrolytes (resulting in an increase of the intracellular ionic strength) the channel is not activated and the regulatory volume decrease occurs exclusively by a release of K and Cl via a KCl cotransporter. When swelling is caused by hypotonic shock (resulting in a decrease in intracellular ionic strength) the KCl cotransporter is then mainly inactivated or even silent; conversely the channel is activated and allows volume recovery by mediating the release of both taurine and probably K and Cl. The possibility that AEs function as volume-activated taurine channels in other cell types and as a malaria-induced channel in malaria-infected human red cells is considered.

Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr.

Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.

Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis.

Most, but not all, cell types release intracellular organic solutes (e.g. taurine) in response to cell swelling to achieve cell volume regulation. Although this efflux is blocked by classical inhibitors of the electroneutral anion exchanger band 3 (AE1), it is thought to involve an anion channel. The role of band 3 in volume-dependent taurine transport was determined by expressing, in Xenopus oocytes, band 3 from erythrocytes which do (trout) or do not (mouse) release taurine when swollen. AE1 of both species elicited anion exchange activity, but only trout band 3 showed chloride channel activity and taurine transport. Chimeras constructed from trout and mouse band 3 allowed the identification of some protein domains critically associated with channel activity and taurine transport. The data provide evidence that swelling-induced taurine movements occur via an anion channel which is dependent on, or controlled by, band 3. They suggest the involvement of proteins of the band 3 (AE) family in cell volume regulation.

The cytoplasmic domain of the Na+/H+ exchangers (NHEs) dictates the nature of the hormonal response: behavior of a chimeric human NHE1/trout beta NHE antiporter.

Studies of the effect of cAMP on the cloned Na+/H+ exchangers (NHEs) are difficult to interpret as variable results have been reported for the different isoforms when expressed in various cell types. We took advantage of the fact that the human NHE1 and the trout erythrocyte beta NHE, when expressed in the same cell line, PS120, respond differently to cAMP (NHE1 is insensitive, beta NHE is activated) to analyze the molecular mechanisms of cAMP activation. We constructed both a chimera between NHE1 and beta NHE and a set of beta NHE mutants to delineate the critical parts of the molecule involved in the activation process. NHE1 becomes cAMP stimulated when its cytoplasmic domain is replaced by the cytoplasmic domain of beta NHE; thus, the cytoplasmic C terminus of beta NHE, which contains two cAMP-dependent consensus sequences, is essential to confer cAMP dependence. Serine to glycine substitution of only one of the two protein kinase A (PKA) consensus sites decreased by 60% the ability of cAMP to activate Na+/H+ exchange. Simultaneous Ser to Gly substitution of the two PKA consensus sites decreased the cAMP-mediated activation by 72%. The residual activation required a cytoplasmic fragment (aa 559-661) that contains four sequences considered likely as putative PKA consensus sites. The results obtained with the chimeric NHE also demonstrated that if the cytoplasmic C terminus is crucially involved in the hormonal activation, the rate of Na+/H+ exchange so induced can be modulated by the nature of the interaction between the N- and C-terminal domains.

Evidence for a K(+)-H+ exchange in trout red blood cells.

1. Exposure of trout red blood cells to beta-adrenergic agonist isoprenaline activates a cAMP-dependent Na(+)-H+ antiport, the movements of protons being compensated by a Cl(-)-OH- (or HCO3-) exchange mediated by band 3 protein. The absorption of water osmotically linked to sodium and chloride induces cell swelling. 2. In the presence of acetazolamide, anionic exchange is inhibited and activation of cationic exchange resulted in the first 2 min in a strong external acidification and a large internal alkalinization leading to a reversal of the transmembrane pH gradient. Then, for at least 1 h and despite the inhibition of Cl- entry, a net Na+ uptake occurred which was balanced by an equivalent K+ loss, with the result that cell volume and pH gradient remained unchanged. 3. In such conditions, the inactivation of the Na(+)-H+ exchanger by a beta-antagonist, propranolol, blocked Na+ entry while K+ continued to be lost. This volume-independent K+ efflux, which is thus independent of the Na(+)-H+ exchanger, was not accompanied by a Cl- efflux but was associated with large internal and external pH changes consistent with K(+)-H+ exchange. 4. The K+ loss and the related pH changes are inhibited by compounds which are known to inhibit the K(+)-anion co-transporter in trout red cells, i.e. 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (Dids) and niflumic acid.

Regulation of Na+/H+ exchange and pH in erythrocytes of fish.

1. The function of trout RBC Na+/H+ antiport is unrelated to cell volume or cell pH regulation. Its role is to improve oxygen transport capacity when the supply of oxygen becomes limited. 2. Antiport activation, mediated by cAMP, promotes complex changes in blood pH which have been analyzed in vivo and in vitro. 3. The regulation of antiport (activation, desensitization, control by molecular oxygen and by a newly discovered cytosolic protein, arrestin) is presented. 4. Molecular cloning of the antiport shows that two typical site motifs of phosphorylation by cAMP-dependent protein kinase are localized on the cytoplasmic region.

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner.

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.

Catecholamine release controlled by blood oxygen tension during deep hypoxia in trout: effect on red blood cell Na/H exchanger activity.

Changes in plasma catecholamine levels were measured in trout exposed to acute hypoxia, in order to correlate with acid-base disturbances due to activation of the cAMP-dependent Na+/H+ antiporters of red blood cells, as previously described (Fiévet B., Respir. Physiol. 74, 99-114, 1988). The extracellular acidosis corresponding with the stimulation of the exchangers, occurred when arterial oxygen partial pressure (PaO2) reached around 15 Torr (Thomas S., Respir. Physiol. 74, 77-90, 1988). This blood pH drop coincided with a marked increase in plasma catecholamine levels. The catecholamine secretion was transient and the hormones were cleared provided PaO2 remained above 10 Torr. On the other hand, when PaO2 remained below 10 Torr, there was a persistent secretion of catecholamines. This is in agreement with the fact that the exchangers are 'turned off' or sustained when PaO2 remains above or below 10 Torr respectively, as previously described. Following the transient hormone peak when PaO2 stabilized above 10 Torr, it was possible to trigger the second pattern of continuous catecholamine secretion by controlling water PO2 so that PaO2 declined below 10 Torr. We conclude that the blood oxygen level controls catecholamine secretion during deep hypoxia.

Na+-H+ exchange and pH regulation in red blood cells: role of uncatalyzed H2CO3 dehydration.

Erythrocytes of rainbow trout respond to adrenergic stimulation by activation of a Na+-H+ exchange. When red blood cells are suspended in their own plasma and equilibrated with a convenient gas mixture in a tonometer, the extrusion of H+ induces a fast, very strong acidification of the blood (by 0.5-0.7 pH units), explained as follows. Excretion of H+ into a medium containing HCO3- causes the formation of H2CO3. The uncatalyzed dehydration of H2CO3 is slow so that H+ accumulates above the level that would prevail at equilibrium, promoting a strong acid disequilibrium pH. Then the blood pH progressively returns to a value close to its initial value because of the slow uncatalyzed dehydration of H2CO3 and washout of the CO2 so produced. The period of acid disequilibrium pH, however, is lengthened because part of the CO2 generated by the spontaneous dehydration is not washed out by tonometry but diffuses into the red cells where it is rapidly converted into HCO3- and H+ by carbonic anhydrase and then excreted by Na+-H+ and Cl-HCO3- exchangers. This recycling process "refuels" the ionic reaction, increasing the time needed to reach equilibrium. The anion exchanger does not sense this strong acid disequilibrium pH, since the external HCO3- concentration is practically unchanged at that time. During the extracellular pH (pHe) recovery period, simultaneously extracellular HCO3- content decreases and intracellular Cl- content increases. Thus intracellular pH and pHe appear to be uncoupled. This overall interpretation is confirmed by experiments using carbonic anhydrase and drugs such as propranolol and amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptive respiratory responses of trout to acute hypoxia. I. Effects of water ionic composition on blood acid-base status response and gill morphology.

The effects of various levels of hypoxia (PWO2 ranging from 10 to 60 Torr) on arterial blood gases (PaO2 and PaCO2) and acid-base status were investigated in trout at 15 degrees C. The hypoxic responses of two stocks of trout living in natural waters having very different levels of NaCl (1.0 mmol.L-1 and 0.1 mmol.L-1) and carbonate alkalinity (0.4 mmol.L-1 and 2.4 mmol.L-1) were compared. The use of an extracorporeal circulation method made it possible to continuously monitor the pH changes. The different patterns of the acid-base status observed in response to hypoxia depend on the evolution of PaO2. Two critical PaO2 thresholds were defined. Crossing the upper (about 15 Torr) induces metabolic acidosis which is normally followed by pH recovery, while crossing the lower (about 10 Torr) promotes loss of capacity to compensate acidosis. The NaCl concentration of the water drastically modifies the fish sensitivity to hypoxia: fish living in water with a low NaCl concentration have less resistance to hypoxic exposure. This may be explained by the fact that in fish living in low NaCl concentrations, the secondary gill lamellae are surrounded by chloride cells, which considerably reduce the surface area available for gas exchange. Consequently a modest fall in PWO2 induces a drastic reduction of the arterial oxygen tension which crosses the lower critical PaO2 threshold.

Adaptive respiratory responses of trout to acute hypoxia. II. Blood oxygen carrying properties during hypoxia.

The effects of deep and acute hypoxia (PwO2 = 25 Torr) on oxygen transport characteristics (Hill number (n) and P50) were investigated in trout at 15 degrees C. When a fish is submitted to such an acute and deep hypoxia, a metabolic acidosis develops as soon as the arterial oxygen tension drops to about 15 Torr. We first showed that the hemoglobin of blood sampled at the end of the acidification period has an increased oxygen affinity. This improved affinity could be explained by the internal alkalisation of erythrocytes due to the extrusion of protons via a beta-adrenergic stimulation of Na+/H+ exchanges occurring at the onset of hypoxia and responsible for extracellular acidosis. Secondly we observed a significant increase (about 20%) of the number of blood cells per volume of blood during the acidosis. This cell number stays constant afterwards. The dual effects of a higher hemoglobin oxygen affinity and a greater amount of available hemoglobin improving blood oxygen loading at the fish gills appear to be a fast adaptive response to acute hypoxia. Surprisingly, we found that the elevated affinity occurring during acidosis remained constant as long as the fish were maintained in hypoxia, in spite of possible large variations of extracellular pH (pHe). This result is difficult to reconcile with the idea that the increase in affinity is imposed by intracellular pH (pHi), since in red blood cells pHi depends on pHe, thus any modification of pHe would in this case modify oxygen affinity.