Richard P. Cosson

Influence of metal exposure on metallothionein synthesis and lipid peroxidation in two bivalve mollusks: the oyster (Crassostrea gigas) and the mussel (Mytilus edulis)

The impact of metals (silver, cadmium, copper, mercury and zinc) on metallothionein (MT) and malondialdehyde (MDA) levels of the oyster (Crassostrea gigas) and the mussel (Mytilus edulis) was studied after 4 or 21 days of metal exposure. Moreover, total protein levels were determined. After 4 days of metal exposure, although C. gigas and M. edulis accumulated cadmium and mercury concentrations in the gills and digestive gland, no significant variation of total protein level was occurred. After 21 days of exposure, metals were bioaccumulated in the gills and the digestive gland of both mollusks. A decrease of total protein concentrations in the gills of oysters and the digestive gland of mussels and an increase on metallothionein concentrations in the gills of both mollusks were observed. An increase of MDA levels was noticed for the gills and the digestive gland of mussels exposed for 21 days to either cadmium, silver or mercury whereas a decrease of MDA levels was observed in the gills of the oysters exposed for the same time to the same metals. The levels of proteins, MDA and MT were metal, species or organ dependent.

Cadmium detoxification processes in the digestive gland of cephalopods in relation to accumulated cadmium concentrations.

The high concentrations of cadmium recorded in the digestive gland of cephalopods from various temperate and subpolar waters suggest that these molluscs have developed efficient cadmium detoxification mechanisms. The subcellular distribution of cadmium in the digestive gland cells was investigated in seven cephalopod species from the Bay of Biscay (France) and the Faroe Islands. In most species, cadmium was mainly found in the cytosolic fraction of the digestive gland cells, reaching up to 86% of the total cadmium for the squid Loligo vulgaris from the Bay of Biscay. But species with the highest total level of cadmium showed a higher percentage of cadmium associated to insoluble compounds. The quantification of metallothioneins (MTs) by the polarographic method was performed in order to evaluate the involvement of these proteins in the detoxification of the high amounts of bioaccumulated cadmium. Metallothionein levels in cephalopods ranged form 742 +/- 270 to 3478 +/- 1572 microg/g wet weight. No relationship could be established between total cadmium, cytosolic cadmium and MT levels suggesting the occurrence of other Cd-binding ligands. Although these proteins have not been characterised, as cadmium in the digestive gland of cephalopods is mainly associated with soluble ligands, a high potential transfer to predators can be predicted.

METALLOTHIONEINS AND DETOXIFICATION. IS THE USE OF DETOXICATION PROTEIN FOR MTs A LANGUAGE ABUSE

Our own results and a literature review led us to reconsider the detoxifying function of MTs in living organisms. Despite the fact that many authors have observed a synthesis or a level increase of MTs as a response to toxic metal uptake, arguments exist which tend us to give to MTs a strict zinc homeostasic function. Many experiments have been conducted using non-natural routes of exposure and/or concentrations far from those observed even in heavily polluted environments. Zinc is the only metal for which a primary induction has been established. Correlations between zinc and MTs levels are frequently observed, specially at the early stages of life. Our knowledge about metallic cluster structure and our experimental results about inter-metallic competition for binding-sites on the apoprotein, support the idea of substitution processes instead of de novo synthesis in most cases of contamination, leading frequently to acclimation.

Spatial variation of metal bioaccumulation in the hydrothermal vent mussel Bathymodiolus azoricus

The variability of the bioaccumulation of metals (Ag, Cd, Cu, Fe, Mn and Zn) was extensively studied in the mussel Bathymodiolus azoricus from five hydrothermal vent sites inside three main vent fields of increasing depth along the Mid-Atlantic Ridge: Menez Gwen, Lucky Strike and Rainbow. Metal bioaccumulation varied greatly between vent fields and even between sites inside a vent field with B. azoricus showing a great capacity to accumulate metals. The bioaccumulation of these metals also varied significantly among tissues. The main target was the gills where metals were mainly associated with soluble compounds whereas in the digestive gland they were mainly associated with insoluble compounds. Storage of metals under insoluble forms in B. azoricus seems to be a major pathway for the detoxification of both essential and non-essential metals. Mussels from the studied fields can be discriminated following their metallic load but the segregation relies partially on the composition of the metal-enriched fluids.

Responses of wild small mammals to a pollution gradient: host factors influence metal and metallothionein levels.

We investigated how host factors (species, age, gender) modulated Cd, Pb, Zn, and Cu concentrations, metallothionein levels (MTs) and their relationships in 7 sympatric small mammal species along a pollution gradient. Cd concentrations in liver and kidneys increased with age in all species. Age effect on other metals and MTs differs among species. Gender did not influence metal and MT levels except in the bank vole. Three patterns linking internal metal concentrations and MTs were observed along the gradient: a low metal accumulation with a (i) high (wood mouse) or (ii) low (bank vole) level of MTs accompanied by a slight or no increase of MTs with Cd accumulation; (iii) an elevated metal accumulation with a sharp increase of MTs (common and pygmy shrews). In risk assessment and biomonitoring perspectives, we conclude that measurements of MTs and metals might be associated because they cannot be interpreted properly when considered separately.

Effects of Lipid Extraction on δ13C and δ15N Values in Seabird Muscle, Liver and Feathers

Abstract We determined whether lipid extraction of seabird muscle, liver and feathers is required for δ13C and δ15N analysis, based on a study of Barau’s Petrels (Pterodroma baraui) and White-tailed Tropicbirds (Phaethon lepturus). Samples were analyzed for stable isotopes before and after lipid removal. Lipid-free muscle and liver were significantly enriched in 13C compared to those containing lipids (0.52‰ and 0.61‰ mean differences in δ13C respectively) implying that lipids should be extracted from these tissues to avoid effects of uncontrolled differential lipid content. However lipid extraction also slightly increased muscle δ15N values in tropicbirds. Researchers should thus run samples separately for δ13C and δ15N analysis, extracting lipids only in aliquots in which δ13C is measured, or apply arithmetic δ13C normalization methods on non lipid-extracted samples. Predictive models developed from our data, inferring δ13C values of lipid-extracted material from δ13C values and C:N ratios of non lipid-extracted material are presented and compared to five normalization methods taken from literature. Our models showed the best prediction efficiencies followed by that developed by McConnaughey and McRoy (1979). In feathers, no difference in stable isotopic measurements were noted between aliquots treated with sodium hydroxide or 2:1 chloroform:methanol. Both protocols seem suitable for preparing feather samples for isotopic analysis.

Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: field and laboratory investigations

Hg bioaccumulation was investigated in the king scallop Pecten maximus in the laboratory and in the field. In controlled conditions, scallops were exposed to (203)Hg through seawater, sediment and food in order to determine its uptake and depuration kinetics. In the field, Hg and metallothionein (MT) concentrations and the metal subcellular distribution were determined in scallops from two sites of the Bay of Seine (France) differently subjected to the Seine river inputs. While Hg concentrations in the whole soft parts and kidneys (viz. the highest accumulator organ) did not differ between scallops from both sites (74-156 ng g(-1)dry wt), they did for the digestive gland and the gills. According to the experimental results, a higher exposure to dissolved Hg might occur in the site close to the estuary whereas Hg would be mainly incorporated via the dietary pathway in the site away from the estuary. Within the cells of wild scallops, Hg was mainly associated to the cytosolic fraction in the digestive gland and gills (60-100%). However, the lack of relationship between Hg and MT levels suggests that Hg detoxification in P. maximus involves other, non-MT, soluble compounds. In kidneys, insoluble compounds played an important role in Hg sequestration. No effect of scallop age was observed neither on Hg and MT concentrations nor on the subcellular distribution of the metal. Finally, according to FAO/WHO recommendations (maximum weekly Hg intake), our results clearly indicate that the low Hg contents in the edible part of the king scallops from the Bay of Seine prevent any risk for human consumers.

Insight on trace element detoxification in the Black-tailed Godwit (Limosa limosa) through genetic, enzymatic and metallothionein analyses

Trace element concentrations (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn) were investigated in the liver, kidneys, muscle and feathers of 31 black-tailed godwits (Limosa limosa) accidentally killed during catches by mist net in the Pertuis Charentais, Atlantic coast of France. Analyses of carbon and nitrogen stable isotope ratios were carried out in liver, muscle and feathers in order to elucidate dietary patterns and to determine whether differences in diet explained the variation in elemental uptake. This study also aimed to have a preliminary assessment of sub-lethal effects triggered by trace elements through the investigation of gene expressions by quantitative real-time PCR, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione peroxidase), and metallothionein (MT) levels. The results showed that Cr and Ni concentrations in tissues of adults were lower than in juveniles in part because adults may have eliminated these trace elements through moulting. Except for Cd and Ni, trace element concentrations were negatively correlated to the body mass of godwits. Ag, As, Hg and Se concentrations were positively linked with the trophic position of birds. The diet could be considered as a fundamental route of exposure for these elements demonstrating therefore the qualitative linkage between dietary habits of godwits and their contaminant concentrations. Our results strongly suggest that even though trace element concentrations were mostly below toxicity threshold level, the elevated concentrations of As, Ag, Cd, Cu, Fe and Se may however trigger sub-lethal effects. Trace elements appear to enhance expression of genes involved in oxidative stress defence, which indicates the production of reactive oxygen species. Moreover, birds with the highest concentrations appeared to have an increased mitochondrial metabolism suggesting that the fight against trace element toxicity requires additional energetic needs notably to produce detoxification mechanisms such as metallothioneins.

Diatom artificial medium (DAM): a new artificial medium for the diatom Haslea ostrearia and other marine microalgae

Artificial media are used in physiological studies of microalgae to maintain consistent conditions from one experiment to another and these media must be adapted to the needs of the organism studied. The artificial medium, in this case named diatom artificial medium (DAM), was designed to maintain long-term cultures of Haslea ostrearia and 19 other planktonic microalgae, and to allow physiological studies related to metal metabolism. The biomass and biochemical composition of H. ostrearia grown in the DAM and in a modified Provasoli medium were compared to assess the suitability of this new artificial medium for the culture of this diatom. The DAM provided sufficient nutrients to allow H. ostrearia to grow as efficiently as in the enriched seawater medium, without negative impact on metabolism. The DAM was tested with 19 other microalgae in order to widen its potential use, and 18 of the 19 showed a good adaptation to this medium. The chemical speciation of metals (Cd, Cu, Pb, Zn) was assessed using a speciation mathematical model. The presence of EDTA resulted in the total complexation of the trace metals implying that they were present in a sole chemical species in the DAM.

Evidence of species-specific detoxification processes for trace elements in shorebirds.

This study investigated sub-lethal effects and detoxification processes activated in free-ranging Red Knots (RKs) (Calidris canutus) from the Pertuis Charentais on the Atlantic coast of France, and compared the results with previous data obtained on another shorebird species, the Black-tailed Godwit (Limosa limosa). The concentrations of 13 trace elements (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn) were assessed in the liver, kidneys, muscle and feathers. Stable isotope analyses of carbon and nitrogen were carried out to determine whether differences in diet explained variations in elemental uptake. The mRNA expression of relevant genes (cytochrome c oxidase 1, acetyl-CoA carboxylase, Cu/Zn and Mn superoxide dismutase, catalase, metallothionein, malic enzyme), antioxidant enzyme activities (catalase, glutathione peroxidase (GPx), superoxide dismutase), and metallothionein (MT) levels were investigated to shed light on trace element detoxification and toxic effects. Although Red Knots were characterized by elevated As and Se concentrations which were potentially toxic, most elements were usually below toxicity threshold levels. The results strongly suggested a dietary specialization of Red Knots, with individuals feeding on higher trophic status prey experiencing higher As, Hg and Se burdens. Red Knots and Godwits also showed discrepancies in elemental accumulation and detoxification processes. Higher As and Se concentrations in Red Knots enhanced catalase gene expression and enzyme activity, while Godwits had higher Ag, Cu, Fe and Zn levels and showed higher MT production and GPx activity. The results strongly suggest that detoxification pathways are essentially trace element- and species-specific.