Anne-Yvonne Jeantet

Bioaccumulation, toxicity and physico-chemical speciation of silver in bivalve molluscs: ecotoxicological and health consequences

Abstract The toxicity and bioaccumulation of silver have been studied comparatively in the freshwater bivalve Dreissena polymorpha and in four marine species: the oyster Crassostrea gigas, the mussel Mytilus galloprovincialis, the scallop Chlamys varia and the clam Scrobicularia plana. In the freshwater and marine mussels, the silver absorption was very limited but M. galloprovincialis was more susceptible than D. polymorpha. The oysters and scallops were silver accumulators but the effect of the metal was much more noxious in the second species. These data have demonstrated that no direct relationships exist between the global concentration in soft tissues and the susceptibility of the different bivalves. Thus the physico-chemical forms of storage of silver (silver sulphide, metal-binding proteinic compounds) were determined in each species and related to the high variability of biological responses. The modes of exposure (through food or water) influenced the binding of silver with cytosolic compounds (in oysters) and its biological effects (in scallops). Changes in salinity had only a moderate effect on silver bioaccumulation in oysters, a fact which is interesting since pollutions due to this metal often affect estuarine and coastal areas. In the light of these data, the potential injuries due to silver pollution may be attributable: (1) to noxious effects on susceptible species with a resulting imbalance of biocoenosis structure; and (2) to the existence of silver accumulation with a risk of transfer to the food web. However, the knowledge of the chemical speciation of silver in organisms permit to predict the importance of this risk.

A suitable model for the biomonitoring of trace metal bioavailabilities in estuarine sediments: the annelid polychaete Nereis diversicolor

The need to use biomonitors representative of the sedimentary compartment has been recognized, particularly in estuaries. Thus, trace metal contamination has been monitored in an infaunal polychaete worm Nereis diversicolor and sediments in the Seine estuary and comparatively in the relatively clean Authie estuary (French coast of the English Channel) over two years taking samples every three months at both sites. No correlations were shown between total metal (Ag, Cd, Cu, Pb, Zn) concentrations in raw sediments and ragworms. Because these worms are known to be good biomonitors of the bioavailabilities of sedimentary trace metals, it follows that total sediment metal concentrations have a poor predictive ecotoxicological value. Using a correction factor to minimize the influence of weight, it is possible to make a good estimation of the average metal concentrations in a population of worms at a given site, sampling only a limited number of specimens. Metallothioneins are often considered to be good biomarkers of the presence of significant availabilities of trace metals. Metallothionein-like proteins (MTLPs) are present in N. diversicolor, but there is no significant relationship between MTLPs and metal concentrations. This situation might result from the importance of metal-containing granules, both extra- and intracellular, in ragworms.

Testicular wild-type p53 expression in transgenic mice induces spermiogenesis alterations ranging from differentiation defects to apoptosis

While p53 is dispensable for development, an excess of p53 has dramatic consequences on the embryogenesis and on the cell differentiation. In an attempt to analyse in vivo the effects of p53 activity, we have generated transgenic mice expressing the wild-type p53 under the control of the metallothionein I promoter. In the three transgenic lines established, exogenous p53 is expressed constitutively in the postmeiotic cells of transgenic males and two lines are subfertile. Transgenic males expressing the upper level of p53 produce few spermatozoa since the majority of developing spermatids undergo apoptosis. In the subfertile males exhibiting an intermediate amount of p53, teratozoospermia is obvious suggesting an altered terminal differentiation of postmeiotic cells. In contrast lower level of p53 does not lead the third line to sterility. These results suggest that the activity of p53 is dependent in vivo on the amount of p53 present within cells, as it has been already demonstrated in vitro.

Bioavailability and toxicity of sediment-bound lead to a filter-feeder bivalve Crassostrea gigas (Thunberg)

Two different approaches were used to study the bioavailability of sediment-bound lead. In vitro techniques simulating the potential metal desorption under conditions prevailing in the digestive tract were assayed on a contaminated sediment. An experimental model of a food chain was designed to determine the retention of lead in the soft tissues of oysters according to the environmental source of the metal (water or sediment). Neither enzymatic action nor leaching at low pH (both aspects of digestion) induce the release of important lead amounts from particles. Therefore, after 3 weeks of exposure, the retention of lead from the trophic source is lower (1%) compared with direct contamination (5%). Lysosomes are the major intracellular structures responsible for lead storage in the gills, digestive tract and digestive gland. The abundance of lysosomes and their lead amount vary according to the gross concentrations in the soft tissues. The cytopathological data are in agreement with the results about lead accumulation: in oysters exposed to sediment-bound lead, impairments are not so marked as in individuals contaminated directly from water but the same organelles are concerned. Mitochondrial impairments may be related to the effect of lead on cellular respiration processes and changes involving the granular endoplasmic reticulum may have an effect on the level of protein synthesis. Cellular extrusions carrying away numerous lysosomes loaded with lead could account for the balancing of lead incorporation between 2 and 3 weeks of exposure.

Fate and effects of soluble or sediment-bound arsenic in oysters (Crassostrea gigas Thun.).

Contaminated sediments are a possible source of stress to the benthic biota. In order to examine this way of transfer concurrently with direct exposure, oysters were exposed to As dissolved in natural seawater or loaded to particles. The sediment used as a vector of transfer was a mud from a coastal area devoted to oyster culture, the finest particles of which have been selected. It was submitted to experimental contamination then to in vitro desorption tests, in which enzymes and pH changes were used to mimic the digestive processes in Molluscs. Although different enzymes or pH induced the desorption of 3 to 24% of sediment-bound arsenic, the accumulation of this element in the soft tissues of oysters remained low after exposure to contaminated particles. The uptake of soluble arsenic was also limited although checking the level of arsenic in seawater every day revealed no significant decrease of the contaminant in the experimental medium. However, cytological effects were noted in oysters exposed to sediment-bound arsenic and moreover to soluble arsenic. They consisted of structural alterations of mitochondria and nuclei, suggesting a disturbance of both the cellular respiratory metabolism and nucleotid incorporation.

Trophosomal sulfur in vestimentifera: A methodologic approach

Si la structure de la fibre nucltosomique, structure de base de la chromatine eucaryote, est bien connue, ses niveaux d’organisation supCrieurs sent encore ma1 compris. Nous avons entrepris une ttude de l’organisation supramoltculaire des phases condenskes de nucltosomes. l Phases condensees de nuclCosomes en conditions de bon solvant : AprPs extraction et purification biochimique, les mononucltosomes sont concent& soit par ultrafiltration su; membrane de cellulose, soit par melange avec un polymkre neutre (Poly Ethylkne Glycol), jusqtt’a I’obtention de solutions dont la gamme de concentration, 200 & 500 mglml est proche de celle rencontree localement dans un noyau cellulaire. Les solutions observtes au microscope polarisant montrent la formation de structures fluides et ordonnees, en particulier des germes en forme d’ttoile ou de fleur, dont la symCtrie d’ordre six rCvtle la symCtrie hexagonale d’une phase colonnaire discotique, dont chaque colonne correspondrait j une pile de nucliosomes. En microscopic Clectronique, l’observation de rCpliques de cryofracturc permet de visualiser les colonnes de nuclCosomes et le rtseau hexagonal qu’elles forment. Les &arts au mod&le d’empilement hexagonal dense r&lent l’existence de twist entre colonnes. Ces formes cristallines liquides sont compartes aux vrais cristaux de nuclCosomes [J.T. Finch et al (1977) Nutlcre 269, 29-31 ; J. Dubochet & M. Nell (1978) Scierzce 202, 280-2861. l Phases condensCes de nuclCosomes en conditions de mauvais solvant : Nous montrons que la condensation des nuclCosomes peut Cgalement Ctre obtenue par neutralisation des charges en surface du nucltosome, & I’aide d’un polycation organique, la spermidine. Les phases condendes, sCgr6gCes h partir de la solution dilute, sont analystes par deux techniques complCmentaires : la microscopic optique et la cryomicroscopie electronique de films minces hydrates [Adrian et al (1984), Nature 308, 32-341, qui permettent d’avoir ac&s aussi bien 2 l’ordre B grande distance qu’8 l’ordre local entre nuclCosomes dans ces phases. Les rCsultats obtenus suggkrent la formation d’une phase cristalline liquide discotique hexagonale dont la structure serait similaire 2 celle observCe en conditions de bon solvant. L’analyse de ces phases de nucle’osomes devrait permettre, par une comparaison de leurs gComCtries avec les formes condensCes de la chromatine itt vivo, de mieux en comprendre les niveaux d’organisation supCrieurs de la chromatine des Eucaryotes. TROPHOSOMAL SULFUR IN VESTIMENTIFERA: A METHODOLOGIC APPROACII.

Heteroplasmy in the European rabbit: Mitochondria and mitochondrial DNA evolution through the germ line

In the Eurooean rabbit. Orvctolams cuniculrs. a svstematic heteroplasmy has been described wh&e every animal carl1es.a population of mitochondrial DNA (mtDNA) molecules differing in iheir length. Such an observation urged for the examination of&e evolution of these molecules and the organites which contain them, through generations. A genetic study, conducted on controled animal lineages (NeoCaledoman breed, INRA) has revealed that the transmission of mtDNA depends on three forces: drift, high level of mutations and selection which may vary according to the tissue and the age. A cytological analysis has been initiated, in parallel, to monitor the dynamics of mitochondria along the different steps of development. This work has been devoted, first, to the establishment of the germ line. Three days after birth, ovaries exhibit a vast majority of oogonia (SO%), the ram oocytes being at the beginning of meiotic prophase. At 40 days and further, all the germ cells are oocytes the nucleus of which is at diplotene and the cytoplasm in the growing phase. The ovaries from females (Neo-Caledonian breed) of different ages were taken out. The right ovary was fixed; the left one, incubated one hour in an enzymatic solution (D. Sz6lll)si pers. comm.), was dissected in order to isolate oogonia and oocytes for a genetic study. Ultrastructural examination of serial sections allowed the observation of whole germ cells. Skape, number and distribution of mitochondria were recorded according to the various stages (oogonia and oocytes). The results will be discussed in connection with the genetti analysis conclusions.