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1. The uptake of heavy metals via the alimentary tract can be an important factor for the metal budget of fish. 2. Concepts such as biomagnification, bioaccumulation, biotransference, or concentration factors, convey little information about the real threat originating from heavy metals in an aquatic food chain. 3. In polluted aquatic ecosystems the transfer of metals through food chains can be high enough to bring about harmful concentrations in the tissues of fish. This relationship is called the food chain effect. 4. Two kinds of ecological factors influence the food chain effect: firstly, high levels of contamination of the food, and, secondly, the reduction of species diversity. When susceptible species are eliminated, metal-tolerant food organisms may become dominant. Their tolerance may be based either on their ability to accumulate excessive amounts of metals or to exclude heavy metals from the tissues. These two strategies represent feedback mechanisms which may enhance or weaken the food chain effect. 5. It is concluded that future investigations on transference of heavy metals to fish must take into more careful consideration the specific ecological situation of a given environment.

BackgroundThe degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion.ResultsHpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions.ConclusionThe Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Species of the family Tubificidae represent a major faunal element in benthic freshwater communities throughout the world. Some of them are considered particularly tolerant of the influence of toxicants such as cadmium. One of the most abundant species, "Tubifex tubifex," is frequently used as an indicator of environmental pollution, despite considerable taxonomic problems caused by phenotypic plasticity and genetic heterogeneity. Our study provides a phylogeny of "T. tubifex" based on a segment of the mitochondrial 16S rDNA and presents a rapid PCR-based method of genotype screening which was then applied in cadmium toxicity studies on natural populations. Phylogenetic analysis identified five major mitochondrial lineages, some of them separated by large genetic distances (up to 13%) but morphologically indistinguishable, thus highly suggestive of the existence of cryptic species. All lineages were present at different frequencies in the European river populations studied, with a tendency of the more resistant lineages to occur at higher frequencies in the more tolerant populations. In fact, lineage-specific toxicity experiments showed that individuals of different mitochondrial lineages consistently varied in cadmium resistance, suggesting that in benthic oligochaetes, evolution seems to proceed predominantly through natural selection acting on physiological, rather than morphological, characters. In consequence, toxicological studies involving "T. tubifex" as a monitoring or test organism should allow for the possibility of genetic inhomogeneity of this mudworm group by combining both toxicological and genetic methods.

1. The concentrations of Zn, Cu, Cd, Pb, Cr, Ni, and Mn have been measured in water, sediments, and organisms from two contaminated rivers situated in an industrial area and near a motorway on the southern city boundary of Bolzano, Italy. 2. The low metal contents of water are in contrast to the elevated concentrations of sediments indicating a chronic rather than an acute contamination of the rivers. 3. The metal concentrations of the dominant moss Fontinalis antipyretrica are higher than those of sediments, the lower branches of the plant being more concentrated than its upper parts. 4. Among benthic invertebrates the dominant animals are the isopod Asellus aquaticus and the snail Lymnaea truncatula. Both species feed on contaminated moss or detritus and accumulate the heavy metals to an even greater extent than the water plants. 5. The dominant predator is the rainbow trout (Salmo gairdneri) which feeds mainly on metal enriched isopods and snails. 6. As a consequence, there is an absorption of heavy metals from lumen of fish into the gut tissue and a flux to other organs such as liver, kidney, and muscle. 7. Since the metal concentrations of water are low it is suggested that the absorption through the gills of fish may be of secondary importance compared with the excessive supply through the food.

A cadmium-binding metallothionein has been purified from metal-exposed Roman snails (Helix pomatia) using gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography. The S-methylated protein was digested with trypsin and the endoproteinases Asp-N, Glu-C and Arg-C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N-terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N-terminal amino acid was an acetylated serine residue. Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys-Xaa-Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N-terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N-terminal sequences of histones H2A and H4 which may be important for regulatory functions.

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Concentrations of cadmium, lead, copper and zinc were measured in individuals of Arianta arbustorum from different urban sampling sites. In comparison to snails from a reference site, the animals collected in the city showed higher concentrations of cadmium, lead, and copper, indicating elevated levels of metal pollution. The most pronounced difference in tissue concentrations between control animals and contaminated snails was observed for lead. Within the city, metal levels in snails differed significantly, even between adjacent populations. Arianta arbustorum is a suitable species for biomonitoring, because it is widespread, resident and easy to collect; it has a high capacity for metal accumulation and shows different concentrations depending on metal contamination of the sampling area. An interspecific comparison of metal concentrations in terrestrial gastropods was conducted to define background levels and classes of burden. Three pollution levels are distinguished on the basis of the snails' metal burden: no pollution (class 1: reference sites), moderate (class 2: traffic and other human activities in urban areas), and high pollution (class 3: mining and heavy industry).