Mise au point de techniques analytiques pour la spéciation du sélénium dans les boues de stations d'épuration d'eaux résiduaires urbaines

Les stations d'épuration d'eaux résiduaires sont une des étapes du cycle du sélénium dans l'environnement et contribuent à sa redistribution dans le milieu naturel. Très peu étudié jusqu'à présent dans ces milieux, le sélénium n'en est pas moins un élément très important du point de vue écotoxicologique, sa teneur dans les boues de stations d'épuration destinées à l'épandage agricole faisant par ailleurs l'objet d'une norme.Nous avons mis au point des techniques permettant la détermination spécifique de l'élément total dans ce type d'échantillon, par minéralisation classique ou assistée par micro-ondes et dosage par Voltamétrie de Redissolution Cathodique Différentielle Pulsée (DPCSV) et Spectrométrie d'Absorption Atomique ElectroThermique (ETAAS). Le contrôle qualité a été effectué sur deux échantillons certifiés fournis par le Bureau Communautaire de Référence (BCR) : la boue CRM 145 R et la boue CRM 007.Cependant, lorsqu'on parle de risque toxicologique, il est important de s'intéresser à la détermination des différentes formes sous lesquelles cet élément peut être présent. Nous avons pour cela réalisé des extractions parallèles (spéciation de phases) du sélénium contenu dans les boues afin de déterminer quel pourcentage du sélénium total est réellement et potentiellement disponible pour les végétaux lors d'un épandage sur sol agricole. La spéciation d'espèces a été brièvement abordée dans le but de déterminer les teneurs en Se(IV) et Se(VI), espèces les plus toxiques.The great effort undertaken for about twenty years to improve the quality of surface waters has led to the construction of numerous waste water treatment plants, generating an increasing amount of sludge. Waste water and sludge treatment processes represent an important point in the hydrological cycle at which the disposal of substantial quantities of trace elements to the environment may be regulated. From the law on waste recovery and disposal in 1975 to the European guideline about wastes in 1991, the priority has been given to waste recovery and recycling. With increasing pressure to ban all sludge dumping at sea, and considering the prohibitive costs of land-filling and incineration, there is a great tendency to dispose of sludge on land (40% in 1988 to 60% in 1992).Although numerous studies have demonstrated the intrinsic value of sludge for soil amendment, given its nitrogen, phosphorus and homogeneous organic matter content, evidence has accumulated in recent years that numerous environmental problems can arise because of the presence in sludges of high amounts of certain trace elements (potentially toxic to plants and to human beings and liable to be concentrated along the food chain), among which selenium is particularly interesting.Selenium presents a complex case, as it is also an essential element for living organisms (including humans). The amendment with sewage sludge is sometimes used to increase the selenium content in crops, and afterwards in cattle, when there is a proven lack of this element in a given place. Nevertheless the boundary between essentiality and toxicity is relatively narrow and is expressed at trace levels. It is thus particularly important to survey the selenium concentrations encountered in sewage sludge, especially as guidelines and regulations concerning these data will probably be strengthened. Presently, in France, sludge must not contain more than 200 mg Se·kg-1 dry weight and must not be used on soils containing more than 10 mg Se·kg-1 dry weight (AFNOR U 44-041 norm). This norm concerns only the total amount of selenium contained in sludge and does not take into account the different species (organic and inorganic Se(-II), Se(0), Se(IV) and Se(VI)) that could be present.First of all we had to develop methods for the classical and microwave-assisted wet digestion of sewage sludge, and the determination of their total selenium concentration by Differential Pulse Cathodic Stripping Voltammetry (DPCSV) and ElectroThermal Atomic Absorption Spectrometry (ETAAS). Quality assurance involved the analysis of two BCR (Community Bureau of Reference) certified sewage sludge reference materials (CRM 145 R and CRM 007) and the different techniques were then applied to natural samples from a representative French sewage treatment plant located in the city of Tarbes (South-West of France). The mixture HNO3-H2O2-H2O led to the best results for the digestion and analysis of certified samples, caused few problems for the analysis by DPCSV and ETAAS, and was therefore retained. The decrease of the digestion duration obtained by the use of microwaves was particularly interesting (from one to three days on a hot plate to less than one hour by the Microdigest 301 (PROLABO, France)), and reproducibility was also acceptable (between 3 and 10%). Concentrations obtained for the sewage sludge from the Tarbes treatment plant were very much lower than those for NF U 44-041: 1.08±0.11 mg Se·kg-1 dry weight.However knowledge of speciation, that is to say the determination of the different physicochemical forms of selenium present in a given medium, is necessary when speaking of the toxicological risk represented by an element. The mobility of selenium and its toxicity to the biosphere are related to its association with various sludge or soil constituents as well as to its total concentration. "Soft" or partial extraction techniques are necessary when the aim of the study to determine trace element speciation. The extractants used must separate selenium from the matrix without inducing any loss or change in the partitioning of individual chemical species. In parallel extractions the mechanisms involved for each extractant must correspond to processes occurring in nature and are then associated with special fractions of selenium: soluble, exchangeable, "oxidizable", and "mineral" fractions .Parallel extractions with three types of extractants were chosen for this study and applied first to CRM 007: warm water (soluble fraction), ammonium phosphate-citric acid (soluble + exchangeable fraction) and sodium hydroxide (soluble + exchangeable + "oxidizable" fraction). The soluble, exchangeable, "oxidizable" and "mineral" fractions represent respectively : 11%, 14%, 39% and 36%. The same procedure was then applied to natural samples from Tarbes giving the following results: 36% soluble, 22% exchangeable, 42% "oxidizable". The sodium hydroxide extraction procedure allowed us to extract the entire Se content of this sludge (1.07±0.03 mg Se·kg-1 dry weight), showing that all the selenium present is potentially available after agricultural land application. It was then possible in this fraction to deal with the species speciation of selenium by the mean of a separation of inorganic and organic species on an Amberlite CG-400 resin and a specific analysis by DPCSV. Se(IV) and Se(VI) represent respectively between 30 and 40% and between 2 and 20% of total selenium in the sludges from Tarbes

Myosin binding protein C: implications for signal-transduction

Myosin binding protein C (MYBPC) is a crucial component of the sarcomere and an important regulator of muscle function. While mutations in different myosin binding protein C (MYBPC) genes are well known causes of various human diseases, such as hypertrophic (HCM) and dilated (DCM) forms of cardiomyopathy as well as skeletal muscular disorders, the underlying molecular mechanisms remain not well understood. A variety of MYBPC3 (cardiac isoform) mutations have been studied in great detail and several corresponding genetically altered mouse models have been generated. Most MYBPC3 mutations may cause haploinsufficiency and with it they may cause a primary increase in calcium sensitivity which is potentially able to explain major features observed in HCM patients such as the hypercontractile phenotype and the well known secondary effects such as myofibrillar disarray, fibrosis, myocardial hypertrophy and remodelling including arrhythmogenesis. However the presence of poison peptides in some cases cannot be fully excluded and most probably other mechanisms are also at play. Here we shall discuss MYBPC interacting proteins and possible pathways linked to cardiomyopathy and heart failure

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Impacts of exposure to the toxic dinoflagellate Karenia brevis on reproduction of the northern quahog, Mercenaria mercenaria

International audienceThe Gulf of Mexico, including the southwest Florida coast, USA, experience recurrent blooms of the brevetoxin (PbTx)-producing dinoflagellate, Karenia brevis. Northern quahogs (hard clams) Mercenaria mercenaria, are an important commercial species in this region. This study examined the effects of field and laboratory exposure of adult clams to K. brevis during their reproductive period, and effects on their subsequently produced offspring. Ripe adult clams were collected from a site which had been exposed to an eight-month natural bloom of K. brevis and an unaffected reference site. Ripe adult clams were also exposed to bloom concentrations of K. brevis for 10 days in the laboratory. Clams exposed to K. brevis accumulated PbTx at concentrations of 1508 (field exposure), 1444 (1000 cells mL−1 laboratory treatment) and 5229 ng g−1 PbTx-3 eq (5000 cells mL−1 laboratory treatment). Field-exposed clams showed histopathological effects: a significantly higher prevalence of mucus in the stomach/ intestine (23.3%), edema in gill tissues (30%) and presence of the cestode parasite, Tylocephalum spp. in whole tissue (40%), compared to non-exposed clams (0, 3.3 and 6.7% respectively). These clams also showed reduced gonadal allocation (23% gonadal area) and a higher prevalence of clams of undetermined sex (20%) compared to those sampled from the non-exposed site (43% and 0%, respectively). It is hypothesized that less energy may be channeled into reproduction as more is allocated for homeostasis or tissue repair. The fertilization success of gametes obtained from both field and laboratory-exposed adults was significantly lower in clams that had been exposed to K. brevis and development of these offspring was negatively affected at Days 1 and 4 post-fertilization (in field- and laboratory-exposed clams at the higher K. brevis concentration and in laboratory-exposed clams at the higher K. brevis concentration, respectively). Negative effects may be due to toxin accumulation in the gametes of field-exposed clams (244 ± 50 ng PbTx g−1 and 470 ± 82 ng g−1 wet weight in oocytes and sperm, respectively). Adverse effects in M. mercenaria are compared to those previously reported in oysters, Crassostrea virginica, under similar conditions of exposure. This study provides further evidence of the impacts of K. brevis and its associated toxins on the adults and offspring of exposed shellfish. Site-selection for the collection of broodstock and aquaculture grow-out efforts should therefore consider the local occurrence of K. brevis blooms

15. - Etude qualitative et quantitative de l’effet de berge : application à la nappe alluviale de Flins-Aubergenville

Since 1989 a research program on bank filtration has been set up by : the Lyonnaise des Eaux-Dumez group (LED), "Ecole des Mines de Paris" (CIG), "Agence de l'Eau Seine-Normandie" (AESN) and the Institut de Protection et de Sûreté Nucléaire (IPSN). The object of this program is to study transfer of pollutants between a river and it's connected aquifer. This program is a major concern for water resources managers in the Seine-Normandie area. In fact, these aquifers provide more than 50% of drinking water for the urban communities. This communication is an overview of the research program. Three sorts of pollutants are analyzed : • Nitrogen, due to the increase of nitrate concentration in the river Seine, especially downstream the Achères sewage treatment plant ; • Radio elements coming from normal and accidental operating of nuclear power plant (Co, Cs, Ag, Sb, I, Sr, Cs, Ru, Te) ; • Pesticides (Atrazine and Simazine). High concentrations were detected in aquifers and rivers within agricultural areas, at spring time. First results show an important biochemical activity in the first centimeters of river Seine deposits as well as a considerable water purification in the first meters of the river bank.Depuis 1989, à l’initiative de la Lyonnaise des Eaux-Dumez (LED), un programme de recherche consacré à l’effet de berges a été entrepris avec l’École des Mines de Paris (CIG), l’Agence de l’Eau Seine-Normandie (AESN) et l’Institut de Protection et de Sûreté Nucléaire (IPSN). Il a pour objectif d’étudier dans le détail le transfert de polluants entre une rivière et la nappe alluviale connectée répondant ainsi, à une préoccupation majeure des gestionnaires de l’eau. En effet, ces aquifères fournissent, en Seine-Normandie, plus de 50 % de l’eau potable destinée aux collectivités. Cette communication présente brièvement le programme de recherche et donne un aperçu des résultats acquis. L’étude entreprise intéresse trois familles de polluants : • l’azote, du fait de l’augmentation sensible des teneurs en nitrates dans les cours d’eau, en particulier en Seine en aval de la station d’épuration d’Achères ; • des radioéléments, issus des rejets normaux ou accidentels d’une installation nucléaire (Co, Cs, Ag, Sb, I, Sr, Cs, Ru, Te) ; • des pesticides (Atrazine et Simazine) dont il a été détecté des concentrations importantes au printemps dans les aquifères et les cours d’eau des zones cultivées. Les premiers résultats montrent, notamment, une activité biochimique importante dans les premiers centimètres de boues de Seine ainsi qu’une épuration notable dans les premiers mètres de berge.Detay Michel, Doussan C., Grenet B., Ledoux Emmanuel, Poitevin G., Picat P., Vignier V. 15. - Etude qualitative et quantitative de l’effet de berge : application à la nappe alluviale de Flins-Aubergenville. In: L'avenir de l'eau. Quelques réponses des sciences hydrotechniques à une inquiétude mondiale. Vingt deuxièmes journées de l'hydraulique. Paris, 15-17 septembre 1992. Tome 3, 1992

Effects of the red tide dinoflagellate, Karenia brevis, on early development of the eastern oyster Crassostrea virginica and northern quahog Mercenaria mercenaria.

International audienceThe brevetoxin-producing dinoflagellate, Karenia brevis, adversely affects many shellfish species including the commercially and ecologically important bivalve molluscs, the northern quahog (=hard clam) Mercenaria mercenaria and eastern oyster Crassostrea virginica, in the Gulf of Mexico, USA. This study assessed the effects of exposure of these bivalves to K. brevis during their early development. In separate experiments, embryos of 2-4 cell stage of M. mercenaria and C. virginica were exposed to both whole and lysed K. brevis cells isolated from Manasota Key, Florida. Low bloom concentrations of 500 to 3000 cellsmL(-1) were simulated for 96h. Shell length, percent abnormality (and normality), and percent mortality of resulting larvae were measured. Percentages were recorded after 6, 24, and 96h of exposure; larval shell length was measured at 24 and 96h. For both quahogs and oysters, the effects of exposing embryos to K. brevis on all larval responses were generally dose- and time-dependent. Percent mortalities and abnormalities of both clam and oyster embryos increased significantly after only 6h of exposure to whole cells of K. brevis. For clams, these parameters were significantly higher in whole and lysed treatments (at 3000 cellsmL(-1)) than in controls. Percent mortalities of oysters were significantly higher in the whole-cell treatment (3000 cellsmL(-1)) than under control conditions. After 24h of exposure, mean larval shell length of both bivalve species was significantly reduced relative to controls. This was evident for clam larvae in both the lysed treatment at 1500 cellsmL(-1) and in whole and lysed treatments at 3000 cellsmL(-1), and for oyster larvae in the lysed treatment at 3000 cellsmL(-1). After 96h, both species exposed to the lysed cell treatment at 3000 cellsmL(-1) had significantly smaller larvae compared to those in the control. Overall, lysed cells of K. brevis had a more pronounced effect on shell length, percent abnormality, and mortality in both clams and oysters than did whole cells. Given the fact that blooms of K. brevis overlap with the spawning periods of these two bivalves, and that cells of this naked dinoflagellate are readily lysed by wave action, these results suggest that exposure to K. brevis during the early life history stages of clams and oysters could adversely affect their population recruitment. Further, the presence of whole or lysed cells of K. brevis in hatcheries could have a major negative impact on production

Susceptibility of gametes and embryos of the eastern oyster, Crassostrea virginica, to Karenia brevis and its toxins

The bivalve mollusc, Crassostrea virginica, is frequently exposed to blooms of Karenia brevis along the west coast of Florida during periods of spawning and early larval development. A continuous 4-day exposure of gametes and 2-4 cell stage embryos of C virginica to whole-cell and culture filtrate of K. brevis at 500 and 5000 cells mL(-1), was followed by a 4-day 'recovery' period. Larval growth, percent of normal, abnormal and dead larvae, and the presence of food in the larval gut were measured throughout the exposure period. Results suggest that negative effects mainly occur during embryogenesis and early development. Damage to feeding apparatus/gut may occur during embryonic development or exposure to toxins may act as a feeding deterrent on non-toxic algae. Following 2-h in vitro exposure of gametes, differences in oocyte and sperm cell parameters were investigated using flow cytometry. The reduced sperm viability in the whole-cell 5000 cells mL(-1) treatment suggests the involvement of extracellular brevetoxins (PbTx) and perhaps other harmful, uncharacterized compounds associated with the K. brevis cell membrane. The cumulative effects of reduced sperm viability, fertilization success, embryonic and larval survival, and the near-annual exposure to blooms of K brevis could cause significant bottlenecks on oyster recruitment

Effects of field and laboratory exposure to the toxic dinoflagellate Karenia brevis on the reproduction of the eastern oyster, Crassostrea virginica, and subsequent development of offspring

00000International audienceBlooms of the brevetoxin-producing dinoflagellate, Karenia brevis, are a recurrent and sometimes devastating phenomenon in the Gulf of Mexico. The eastern oyster, Crassostrea virginica, is exposed regularly to these blooms, yet little is known about the impacts of K. brevis upon this important species. The present study considered the effects of exposure to both a natural bloom and cultured K. brevis on the reproductive development of C. virginica. Oysters had been exposed to a bloom of K. brevis that occurred in Lee County, Florida, from September 2012 through May 2013, during a period of gametogenesis and gamete ripening. Ripe adult oysters were collected from this bloom-exposed site and from a site 200 miles north which was not exposed to any bloom. In addition, responses to two 10-day laboratory exposures of either unripe or ripe adult oysters to whole cells of K. brevis at high bloom concentrations (1000 and 5000 cells mL−1) were determined. Both field- and laboratory-exposed adult oysters accumulated PbTx (attaining ∼22 × 103 ng g−1 and 922 ng g−1 PbTx-3 equivalents in the laboratory and the field, respectively), and significant mucal, edematous, and inflammatory features, indicative of a defense response, were recorded in adult tissues in direct contact with K. brevis cells. Laboratory-exposed oysters also showed an increase in the total number of circulating hemocytes suggesting that: (1) new hemocytes may be moving to sites of tissue inflammation, or, (2) hemocytes are released into the circulatory system from inflamed tissues where they may be produced. The area of oyster tissue occupied by gonad (representative of reproductive effort) and reactive oxygen species production in the spermatozoa of oysters exposed to the natural bloom of K. brevis were significantly lower compared to oysters that were not exposed to K. brevis. Additionally, following 10-day exposure of ripe oysters, a significant, 46% reduction in the prevalence of individuals with ripe gametes was obtained in the 5000 cells mL−1 K. brevis treatment.ăăBrevetoxin (PbTx) was recorded within the spermatozoa and oocytes of naturally exposed oysters and was estimated to be 18 and 26% of the adult PbTx load, respectively. Larvae derived from gametes containing PbTx showed significantly higher mortalities and attained a smaller larval size for the first 6 days post-fertilization. These negative effects on larval development may be due to the presence of PbTx in the lipid droplets of the oocytes, which is mobilized by the larvae during embryonic and lecithotrophic larval development. Provision of a non-contaminated food source to larvae however, appeared to mitigate the early negative effects of this neonatal PbTx exposure.ăăResults herein show that adult eastern oysters and their offspring are susceptible to exposure to K. brevis. Caution should therefore be exercised when identifying oyster reef restoration areas and in efforts to establish aquaculture in areas prone to red tides

Influencia del medio de cultivo sobre la cinética de crecimiento y expresión de la proteína recombinante Rv2626c de Mycobacterium tuberculosis H37Rv expresada en Streptomyces lividans TK24

Streptomyces lividans, ha ganado gran atención en los últimos tiempos, como vector de expresión y producción de proteínas de Mycobacterium tuberculosis de interés biomédico, como alternativa a su obtención tradicional en E. coli. La proteína Rv2626c, Proteína de Respuesta Hipóxica 1, es codificada por el gen Rv2626c (hrp1) de M. tuberculosis, perteneciente al regulón de la fase de latencia DosR. Esta proteína se sobre-expresa en la fase de latencia de la tuberculosis bajo condiciones de estrés como hipoxia y bajos niveles, de óxido nítrico. Se ha demostrado la inmunogenicidad y capacidad de esta proteína, de inducir citocinas características del patrón Th-1, tales como el interferón gamma. Por ello, nuestro grupo logró la obtención de Rv2626c por la tecnología del ADN recombinante usando como cepa hospedera Streptomyces lividans TK24. Con el objetivo de aumentar el nivel de expresión de la proteína recombinante, rRv2626c en este trabajo se ensayaron diferentes medios de cultivo, evaluando el crecimiento de la cepa transformada en condiciones de zaranda. Se determinó la cinética de crecimiento en el medio definido, formulado industrialmente en el Centro Nacional de Biopreparados: caldo Triptona Soya (TSB-BioCen) y en medio de cultivo equivalente preparado en el laboratorio. Para establecer la cinética de crecimiento, se utilizó el cálculo del peso seco y la determinación de la concentración de proteínas totales por la técnica de ácido bicinconinico (BCA) a diferentes tiempos del cultivo. Posteriormente, se identificó la proteína clonada mediante SDS-PAGE y Western Blotting, así como los niveles de expresión mediante análisis densitométrico. Los resultados indican que se alcanzaron los máximos niveles de densidad celular a las 36 h de cultivo y los más altos niveles de expresión proteica total y específica entre las 42 y 54 h. Con el medio químicamente definido TSB-Biocen preformulado en lugar del preparado en el laboratorio partiendo de los componentes, se logró reducir el tiempo óptimo para la expresión-secreción de la proteína rv2626c de 96 a 54 h. Los mejores resultados en la promoción de la expresión de la proteína recombinante se lograron con el medio definido TSB-BioCen, a las 48 h con 8,5% de rendimiento específico, superando en más de 10 veces los niveles de crecimiento celular obtenidos con el medio elaborado en el laboratorio y más de dos veces los niveles de secreción de la proteína recombinante