Effect of cadmium on cytosine hydroxymethylation in gastropod hepatopancreas

5-Hydroxymethylcytosine (5hmC) is an important, yet poorly understood epigenetic DNA modification, especially in invertebrates. Aberrant genome-wide 5hmC levels have been associated with cadmium (Cd) exposure in humans, but such information is lacking for invertebrate bioindicators. Here, we aimed to determine whether this epigenetic mark is present in DNA of the hepatopancreas of the land snail Cantareus aspersus and is responsive to Cd exposure. Adult snails were reared under laboratory conditions and exposed to graded amounts of dietary cadmium for 14 days. Weight gain was used as a sublethal endpoint, whereas survival as a lethal endpoint. Our results are the first to provide evidence for the presence of 5hmC in DNA of terrestrial mollusks; 5hmC levels are generally low with the measured values falling below 0.03%. This is also the first study to investigate the interplay of Cd with DNA hydroxymethylation levels in a non-human animal study system. Cadmium retention in the hepatopancreas of C. aspersus increased from a dietary Cd dose of 1 milligram per kilogram dry weight (mg/kg d. wt). For the same treatment, we identified the only significant elevation in percentage of samples with detectable 5hmC levels despite the lack of significant mortalities and changes in weight gain among treatment groups. These findings indicate that 5hmC is an epigenetic mark that may be responsive to Cd exposure, thereby opening a new aspect to invertebrate environmental epigenetics

Spatially Explicit Analysis of Metal Transfer to Biota: Influence of Soil Contamination and Landscape

Concepts and developments for a new field in ecotoxicology, referred to as “landscape ecotoxicology,” were proposed in the 1990s; however, to date, few studies have been developed in this emergent field. In fact, there is a strong interest in developing this area, both for renewing the concepts and tools used in ecotoxicology as well as for responding to practical issues, such as risk assessment. The aim of this study was to investigate the spatial heterogeneity of metal bioaccumulation in animals in order to identify the role of spatially explicit factors, such as landscape as well as total and extractable metal concentrations in soils. Over a smelter-impacted area, we studied the accumulation of trace metals (TMs: Cd, Pb and Zn) in invertebrates (the grove snail Cepaea sp and the glass snail Oxychilus draparnaudi) and vertebrates (the bank vole Myodes glareolus and the greater white-toothed shrew Crocidura russula). Total and CaCl2-extractable concentrations of TMs were measured in soils from woody patches where the animals were captured. TM concentrations in animals exhibited a high spatial heterogeneity. They increased with soil pollution and were better explained by total rather than CaCl2-extractable TM concentrations, except in Cepaea sp. TM levels in animals and their variations along the pollution gradient were modulated by the landscape, and this influence was species and metal specific. Median soil metal concentrations (predicted by universal kriging) were calculated in buffers of increasing size and were related to bioaccumulation. The spatial scale at which TM concentrations in animals and soils showed the strongest correlations varied between metals, species and landscapes. The potential underlying mechanisms of landscape influence (community functioning, behaviour, etc.) are discussed. Present results highlight the need for the further development of landscape ecotoxicology and multi-scale approaches, which would enhance our understanding of pollutant transfer and effects in ecosystems

Biochemical characterization of metallothionein isoforms in terrestrial snails: Relationship between amino acid sequence and metal binding specificity

International audienceTerrestrial helicid snails (Helicidae) accumulate and store huge amounts of metallic trace elements (Cu, Cd) directly from their environment by binding the metals to Metallothioneins (MTs). Previous studies with different helicid species (Helix pomatia, Arianta arbustorum) have revealed that these animals possess two strictly metal-specific MT isoforms. While one of them is directly involved in the maintenance of intracellular Cu+ homeostasis, the second isoform is strictly associated with Cd2+ and plays an important role in detoxification of this harmful metal. The purpose of the present study was to identify and characterizeMT isoforms from another helicid species, Cornu aspersum. Three full-length MTcDNAswere characterized from this species.Two of them exhibited a high degree of identity with the formerly known Cu+- and Cd2+-specific MT isoforms from Helix pomatia. The third isoform isolated from C. aspersum seems to be associated with both metals (Cd and Cu). Analysis of the translated amino acid sequence of the intermediate Cd/Cu isoform exhibited major differences in the primary sequence compared to the other two isoforms. The three amino acids Arg, Lys and Asn (prevailing in the other two isoforms) were replaced by the neutral amino acid Ser. We suggest therefore, that the molar content and position of the amino acids mentioned within the sequence of the three isoforms may contribute significantly to metal specificity. Computational analysis of several biochemical parameters indicates, moreover, that the novel Cd/Cu isoform may represent an intermediate evolutionary stage between the two other, metal-specific isoforms

Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata).

International audienceThe aim of the present work was to study the effect of Cd2+ exposure on metallothionein (MT) induction and on the distribution of metals (Cd, Cu, and Zn) in the terrestrial pulmonate Helix aspersa. In particular, the soluble and nonsoluble pools of the accumulated metals and their tissue distribution in uncontaminated and contaminated edible snails were investigated after a two-week exposure to Cd2+. In the soluble cytosolic pool of the midgut gland of H. aspersa, three metal-specific putative MT isoforms were separated following a fractionation protocol with diethylaminoethyl cellulose, size-exclusion chromatography, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Interestingly, one of the above isoforms seems to bind both Cd and Cu, which may in addition mobilize, after induction by Cd2+, some of the intracellular Cu and, thus, perhaps increase the Cu pool in the cytosolic fraction. The cDNA and its translated amino acid sequence of a Cd2+-binding MT isoform from the snail midgut gland was characterized and attributed to one of the putative MT isoforms obtained by RP-HPLC. The amino acid sequence of this Cd-MT isoform of H. aspersa differed from similar sequences described in other terrestrial pulmonates, such as Helix pomatia or Arianta arbustorum, by only a few amino acids (n = 4 and 8, respectively). That the identified Cd-MT from H. aspersa is inducible by Cd2+ also was shown, chromatographic evidence aside, by a specific polymerase chain reaction protocol on a cDNA basis, which included a noninducible housekeeping gene as a control

Biodisponibilité et transfert d'éléments traces métalliques dans les réseaux trophiques : Apports d'une approche simplifiée via une chaîne alimentaire expérimentale

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Differential liver proteome mapping of control and cadmium-fed rats.

International audienceA comparative study of proteome maps from control and Cd-exposed rat liver was performed using a new technology of two-dimensional liquid chromatography separation method (PF-2D system, Beckman Coulter). Rats were fed for one month 0 or 100 μg Cd g(-1). The between-replicate and between-sample variations showed good repeatability and suitable reproducibility for the two dimensions of separation of proteins. In this complex mixture, PF-2D led to the separation of two major peaks which differed between control and Cd-exposed rat livers, one being identified by mass spectrometry as Cu/Zn superoxide dismutase (SOD), a well-known biomarker of Cd exposure, the other as phosphatidylethanolamine binding protein (PEBP). SOD content was decreased in Cd-exposed rat liver, compared to the control group which was corroborated by a significant decrease of SOD activity. PEBP content also tended to be decreased after Cd exposure. Present results demonstrate interest but also limitations of proteomic approach using PF-2D system to analyze effects of chemicals on organisms

Comparison of transfer and effects of Cd on rats exposed in a short experimental snail-rat food chain or to CdCl2 dosed food.

International audienceTransfer and toxic effects of two cadmium (Cd) forms, inorganic (CdCl2 dosed rat food) or organic (contaminated snail-based rat food) were studied in Wistar rat. Cd concentrations in rat food were 0 and 2.5 microg Cd g(-1) for both inorganic and organic forms and a high concentration of 100 microg Cd g(-1) was also tested for the inorganic form. Rats were exposed for four weeks to contaminated food. Both forms of Cd were bioavailable to rats, with a percentage of transfer from food to rats of around 1% for all contaminated groups. Cd concentrations in rat tissues increased with increasing Cd concentrations in the food. Rats fed with organic form of Cd accumulated significantly more Cd in the main organ for Cd toxicity, the kidney, than those eating the inorganic form. Survival was not affected for any rat group but a decrease in growth and food consumption was observed for the inorganic form. As a defence system against Cd toxicity, rats increased their metallothionein (MT) synthesis at the highest Cd concentration in the target organs (kidney, liver and small intestine) and even did the same at low Cd concentrations (2.5 microg Cd g(-1)) in the kidney. At this low Cd concentration, MT induction was lower in the small intestine of rats ingesting organic Cd than those ingesting inorganic Cd. Bioavailability of organic and inorganic forms of Cd was similar, but subsequent Cd distribution within organs was different. This quantification of the trophic transfer of both inorganic and organic forms of a toxicant is a basis for a better assessment of the fate and effects of chemicals in food webs

Effects of subchronic digestive exposure to organic or inorganic cadmium on biomarkers in rat tissues.

International audienceIn an experimental food chain, Wistar rats were fed cadmium (Cd) in an inorganic (CdCl(2)) or organic (mainly associated with metallothionein from Helix aspersa snail viscera) form. After 1 month of exposure to 100 microg inorganic Cd g(-1) in food, an induction of metallothionein was observed in all target tissues. In liver, glutathione peroxidase (GSH-Px) activity decreased and alanine aminotransferase (ALAT) activity increased, suggesting that Cd causes hepatotoxicity. However, lipid peroxidation as well as catalase and caspase 3 (a marker of apoptosis) activities were not modified. At a rather low exposure (2.5 microg Cd g(-1)), metallothionein level in the kidney was found to be the most sensitive biomarker of exposure for both Cd forms. In the small intestine of rats ingesting inorganic Cd, metallothionein expression was significantly higher than that observed for rats fed organic Cd. Present results allowed proposing a simple design to assess the effect of a chemical in a trophic transfer approach