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Abstract: Metallothionein (MTs) is a family of low molecular weight peptides with high cysteine content. In aquatic invertebrates, MTs play an important role in the detoxification of metals and they are often cited as useful biomarkers for toxic metal stress. The change in concentrations of metallothionein in response to aluminum and cadmium exposure was investigated in the soft tissues of two freshwater molluscs: Lymnaea stagnalis and Dreissena polymorpha that have contrasting feeding behavior and therefore metal accumulation profiles. The majority of added Al had precipitated in the bottom of tanks within 24h, with an average of only 15.4% and 26.2% remaining in the water column of tanks containing snails and mussels, respectively. In contrast, most of the Cd did not precipitate but remained in solution (81.1% and 82.7%, respectively) between water changes. Both metals accumulated in the soft tissues of both animals, but only cadmium exposure led to an increase in metallothionein concentrations in the soft tissues. However, no significant difference was observed in the level of total protein in both tested molluscs upon exposure to Al and Cd compared to the corresponding control. Therefore, care should be taken in the use of metallothioneins as biomarkers for metal stress, since not all metallic stressors induce their expression. [Copyright &y& Elsevier]

Abstract: The present study was undertaken to elucidate the cellular mechanisms, which govern metal sequestration and detoxification in gastropods. For this purpose the pond snail, Lymnaea stagnalis was exposed to environmentally relevant concentrations of three species of metals (Al, Zn and Cd) for 30 days and the localization and fate of these metals were followed in different tissues of the snails. The measurement of relative distribution of metals between tissues revealed that the digestive gland and kidney account for most of the accumulated metals. Al and Cd (non-essential metals) were redistributed to the digestive gland, possibly because of the presence of specific binding entities in the digestive glands of the herein species. This study focuses on the role of intracellular metal-containing granules on metal sequestration. Three main types of granules were identified in the digestive gland cells namely small, green and yellow granules. The morphological examination and the progressive accumulation of elements within these granules revealed that they are developmental stages with the yellow granule being the mature one. The total number of these granules was found to be significantly increased upon exposure of the snails to Al only. This increase may be a response to the large amount of Al that is accumulated through feeding route of this grazing snail. X-ray microanalysis (XRMA) revealed that metals were localized in all three types of digestive gland granules. The increased amount of ligands (P and S) in the granules may give evidence for their role in metal sequestration. Levels of Al and P were positively correlated in the digestive gland granules. It is possible that aluminium is bound to phosphorus to render it insoluble and so to both immobilize it within the lysosome and to be excreted in a highly insoluble form. On the other hand, both Zn and Cd induced marked upregulation of S in mature (yellow) granules by 26- and 11-folds, respectively. The lysosomal codeposition of S and either Cd or Zn in the lysosomal granules in addition to the increase in RER cisternae may indicate that the exposure to these metals could induce metallothionein synthesis in the cells. The microscopical examinations in the present study revealed that metal detoxification from the digestive gland cells may occur via faeces or via basal exocytosis towards hemocytes dispersed by the connective tissue in the visceral mass. In the kidney, one type of granules, the excretory concretions, was identified in the nephrocytes. The significant increase in the number of these concretions in the snail L. stagnalis upon exposure to metals may give further evidence for their role in metal excretion. [Copyright &y& Elsevier]

Silicon (Si) has a marked affinity for aluminium (Al(III)), but not other trace metals such as cadmium (Cd(II)) and zinc (Zn(II)). Exogenous orthosilicic acid (Si(OH)4) ameliorates the toxicity of Al(III) to the pond snail Lymnaea stagnalis, but its mechanism of action is unclear. Here, studies were conducted to ascertain whether interaction between orthosilicic acid and Al(III) occurs in the water column to prevent Al(III) uptake, or in the tissues to reduce the toxicity of accumulated metal. Silicon did not reduce the accumulation of Al(III) by the digestive gland (the main ‘sink’ for trace metals in L. stagnalis) following exposure of the snail for 30 days to 500 μg l−1 added Al(III) and 13-fold molar excess of orthosilicic acid. However, Si concentrations correlated well with Al(III) levels in the digestive gland (R2=0.77), giving a ratio of 2.5:1 (Al(III):Si). Exposure to Zn(II) or Cd(II) and 13-fold molar excess of orthosilicic acid did not prevent uptake of these metals, or result in a correlation between metal and Si concentrations of the snail digestive gland. These data show that aquated orthosilicic acid does not prevent Al(III) accumulation by L. stagnalis. However, following exposure, the ratio of Al(III) to Si in the digestive gland is suggestive of the early formation of hydroxyaluminosilicates, probably proto-imogolites (2–3:1 Al(III):Si). Whether hydroxyaluminates are formed ex vivo in the water column and taken up by snails into the digestive gland, or formed in situ within the digestive gland remains to be established. Either way, orthosilicic acid clearly prevents the in vivo toxicity of Al(III) rather than reducing its uptake. Silicon appears to have an important role in the handling Al(III) by the pond snail which may also have wider relevance in understanding the role of Si in ameliorating Al(III) toxicity. [Copyright &y& Elsevier]

Examines the mechanism of aluminum-dependent regulation of intracellular silicon in freshwater snails (Lymnaea stagnalis). Use of X-ray microanalysis; Effect of aluminum exposure on the accumulation of lysosomal granules; Liability of aluminum in a ligand competition assay.