Changes in metallothionein concentrations in response to variation in natural factors (salinity, sex, weight) and metal contamination in crabs from a metal-rich estuary

Intermoult male and female crabs Pachygrapsus marmoratus and Carcinus maenas were sampled from three sites between the mouth and 25 km upstream in the Gironde, the most Cd-contaminated estuary in France, in order to study the relative importance of natural factors (salinity, sex, weight) and accumulated metal concentrations on metallothionein (MT) concentrations. In the two species studied, higher metal, total protein and MT concentrations were observed in the hepatopancreas than in the gills. In P. marmoratus, MT concentrations were mainly related to changes in the natural factors even if MT and Zn concentrations were positively correlated in the hepatopancreas whereas in C. maenas, the main relationships were with accumulated metal levels. In the case of the natural factors, the most important ones were weight in gills of both crab species, and salinity changes in both hepatopancreas and gills of P. marmoratus. Cd and Cu concentrations in both organs of the two species were inversely related to salinity. The same observation was found for Zn concentrations in C. maenas but not in P. marmoratus. In the hepatopancreas of both species, the highest total protein concentrations were found in crabs from the site with the highest salinity, whereas there were no such differences in the gills. It seems that changes in MT concentrations are linked more to changes in general protein metabolism than to changes in metal accumulation. Thus it was important to examine the storage of metals in other tissue compartments, particularly the insoluble fraction which includes mineral granules which is known to also contribute to trace metal detoxification in invertebrates. In the gills of the crabs, Zn was present mainly in the insoluble fraction, whereas Cd was nearly equally distributed between soluble and insoluble fractions. In contrast, Cu in the gills and all three metals in the hepatopancreas of both species were mainly cytosolic, but this does not necessarily imply a predominant role for MT since the cytosolic fraction also includes other macromolecules which may be the target binding site for accumulated trace metals.     

The potential use of metallothionein in the clam Ruditapes decussatus as a biomarker of in situ metal exposure

This work aimed to validate the relationship between metallothioneins (MTs) and metals (Cd, Cu and Zn) in field conditions. Specimens of the marine bivalve Ruditapes decussatus (Linné, 1758) from Gargour were transferred in two sites: Gargour and Sidi Mansour, both situated along the south-eastern coast of Tunisia. The bivalves were removed from pairs of cages at day 0 (date of transplantation), day 62 and day 132. Metals (Cd, Cu and Zn) and MTs were determined in the subcellular fractions of the digestive gland. In Gargour, metal and MT levels increased significantly after 62 days of transplantation. However, they showed modest and non-significant variations in Sidi Mansour. Zn was mainly associated with the insoluble fraction, whereas Cd and Cu percentages in the soluble and the insoluble fractions were equivalent. Simple correlation analysis showed a positive and significant relationship between MTs and each metal. If all metals were taken together, multiple correlations showed that MTs were significantly correlated with Cd and Zn, with an important coefficient for Cd, but no significant relationship was observed for Cu. Gel filtration chromatography showed that in the heat stable fraction, the only cytosolic SH rich compounds have an apparent low molecular mass (about 15 kDa), which could correspond to metallothioneins. In the digestive gland of R. decussatus MTs responded to moderate increases of metal contamination, without interference with other factors, and could be a promising biochemical indicator of metal exposure.     

Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica

Exposure to a sublethal concentration of cadmium (Cd; 50 microg L(-1)) resulted in significantly increased Cd concentrations in the gill and digestive gland of the Antarctic bivalve Laternula elliptica. Continuous accumulation of Cd in the two organs during the 14-day exposure period was associated with sequestration of Cd to both the soluble cytosolic and insoluble particulate cell fractions. However, the contribution of each cell fraction to Cd sequestration differed between the two organs; in the gill, a larger portion of Cd was associated with the insoluble fraction, while in the digestive gland, both the soluble and insoluble fractions sequestered similar amounts of Cd. Metal-binding components in the insoluble cell fraction were not identified in this study. On the other hand, a metallothionein-like protein (MTLP) was the major Cd-detoxifying component in the soluble cell fraction of the gill and digestive gland. The amount of MTLP increased linearly with exposure time and the amount of Cd accumulated in the tissue, which suggests a potential utility of MTLP as a biomarker for exposure to Cd and possibly other metals.     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

Comparison of metallothionein concentrations and tissue distribution of trace metals in crabs (Pachygrapsus marmoratus) from a metal-rich estuary,in and out of the reproductive season

Crabs, Pachygrapsus marmoratus, were sampled in June 1997 and February 1998 from two sites (at the mouth and 25 km upstream) in the metal-rich Gironde estuary, France. Gills and hepatopancreas were analysed for metal (Cd, Cu, Zn) and metallothionein (MT) contents, in order to examine the influence of both biological and environmental factors on the physico-chemical forms of detoxified metal storage in the crabs. The concentrations of MT and both cytosolic and insoluble metals were not greatly different between males and females, and the influence of organ weights was also minimal. Intersite differences were observed, probably resulting from the gradient of salinity in the estuary, which interacts with both the chemical speciation and bioavailability of metals, and the general protein metabolism of the crabs. Seasonal changes were also important, probably in interaction with the moult and reproductive cycles. In February, concentrations of insoluble metals were generally higher than in June, in both organs, suggesting that essential metals, particularly Zn, are stored during winter then remobilised during the breeding season. The natural variability in the concentrations of MT often concealed any relationship with accumulated metal concentrations. Thus MT in crabs cannot be considered as a useful biomarker of metal pollution.     

Metals in squid,Loligo forbesi,adults, eggs and hatchlings. No evidence for a role for Cu- or Zn-metallothionein

An adult squid Loligo forbesi had the following metals in its liver/digestive gland: Mn, Fe, Ni, Zn, Cu, As, Cd, Ba and Pb in the range of 1-110 ppm wet wt. Adult mantle muscle, adult eyes, eggs and hatchlings contained a lesser number of these metals at concentrations above 1 ppm. Chromatographic analysis of non-heat-treated cytosols (in the presence of 5 mM 2-mercaptoethanol) gave no evidence for the presence of copper- or zinc-containing fractions with the molecular weights of mollusc metallothioneins in any of the above tissues. Copper and Zn were bound to either the particulate fraction or to very low molecular weight species.     

Metallothioneins and trace elements in digestive gland,gills, kidney and gonads of Octopus vulgaris

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (µg g^− 1, dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation.     

Inhibition of carbonic anhydrase in the gills of two euryhaline crabs,Callinectes sapidus and Carcinus maenas,by heavy metals

Two subcellular fractions of gill tissue, cytoplasm and basolateral membranes, from two species of euryhaline decapod crustaceans, Callinectes sapidus and Carcinus maenas, acclimated to low salinity, were isolated via differential centrifugation. Carbonic anhydrase activity from both fractions was titrated against a variety of heavy metals in vitro. The metals Ag(+), Cd(2+), Cu(2+) and Zn(+) showed inhibitory action against the enzyme. Ki values for these metals against cytoplasmic CA from C. sapidus were in the range of 0.05-0.5 microM (for Ag(+), Cd(2+) and Cu(2+)) and 2-6 microM for Zn(+), some of the highest sensitivities reported for CA from an aquatic organism. The Ki values for these same metals were approximately 2-3 orders of magnitude higher for cytoplasmic CA from C. maenas, indicating that there are significant differences in heavy metal sensitivity in branchial CA from the two species, and that C. maenas possesses a metal-resistant CA isoform. It required concentrations of metals in the millimolar range, however, to inhibit CA activity from the membrane fraction of the gill of both species. There were no effects on either mortality or on hemolymph osmotic and ionic concentrations in C. maenas that were exposed to 10 microM Cd or Zn(+) at 32 per thousand salinity and subsequently transferred to 10 per thousand. The presence of a metal-resistant CA isoform in the gills of C. maenas suggests that this species would not be restricted from its normal estuarine environment by heavy metal pollution.     

Comparative study of the bioaccumulation and elimination of trace metals (Cd,Pb, Zn,Mn and Fe) in the digestive gland,gills and muscle of bivalve Pinna nobilis during a field transplant experiment

The purpose of this study was to investigate the long-term bioaccumulation and elimination of Cd, Pb, Mn, Zn and Fe by Pinna nobilis tissues after their 90 day-transplantation period at Téboulba fishing harbor. During the transplantation period, the Cd, Pb, Mn, Zn and Fe concentrations in the different tissues of the mussels were measured before and after exposure period. Metal (Cd, Pb, Mn, Zn and Fe) accumulation in P. nobilis mussels varied depending on the analyzed tissue and the caging times. Notable differences in Cd, Pb, Mn, Zn and Fe accumulation patterns within the digestive gland, gills and muscle were found and may be due to the ability of each tissue to accumulate metals. During the depuration phase, the elimination of Cd, Pb, Mn, Zn and Fe depended on the target tissue and the metal speciation. Cd, Pb, Mn and Fe were eliminated rapidly from one organ and increased in other when compared to those of 90 day transplanted mussels. The increase of metal loads during the elimination phase is not clear and particularly what kind of processes is responsible for such response. However, it is reasonable to assume that metals increase is related to the existence of an accumulation/detoxification mechanism, which involves the transport of metals from an organ to another. The data obtained indicate that because of the significantly high quantities of Cd, Pb, Mn, Zn and Fe accumulated during the exposure phase, the transplanted mussels are suitable bioindicators for monitoring trace metals in marine ecosystem.     

Distribution,bioavailability, and potential risk assessment of the metals in tributary sediments of Three Gorges Reservoir: The impact of water impoundment

To investigate the impact of water impoundment on the metal contamination in sediments cores from the three tributaries of Three Gorges Reservoir (TGR), the concentrations, distribution, bioavailability, and potential risk of eight trace metals between summer and winter were analyzed using sequential analysis. The mean contents of all studied metals were higher than the geochemical background value, and were higher in summer than in winter. The results of the partitioning study indicated that Cr and Ni prevailed in the residual fraction, while a small proportion was found in the easily soluble fractions. Cu and Zn were distributed mainly in the residual and reducible fraction, while Cd and Pb were predominantly associated with non-residual fractions. These observations suggested that the most easily mobilized metals in the study area were Cd and Pb. The mean enrichment factors (EF) of Cu, Zn, Cd and Hg were higher than 1.5, revealing the potential anthropogenic inputs, whilst the EF of other metals remained within the range of natural variability. The positive correlation between non-residual Cu, Zn and Cd and their EF values further indicated that anthropogenic inputs were the potentially major contributor for the enrichment of Cu, Zn and Cd in TGR sediments. The results evaluated by both potential ecological risk index and modified risk assessment code (mRAC) of all sampling sites demonstrated the relatively high potential risk of sediment contamination effect in TGR.     

Leaching Behavior of Heavy Metals and Transformation of Their Speciation in Polluted Soil Receiving Simulated Acid Rain

Heavy metals that leach from contaminated soils under acid rain are of increasing concern. In this study, simulated acid rain (SAR) was pumped through columns of artificially contaminated purple soil. Column leaching tests and sequential extraction were conducted for the heavy metals Cu, Pb, Cd, and Zn to determine the extent of their leaching as well as to examine the transformation of their speciation in the artificially contaminated soil columns. Results showed that the maximum leachate concentrations of Cu, Pb, Cd, and Zn were less than those specified in the Chinese Quality Standards for Groundwater (Grade IV), thereby suggesting that the heavy metals that leached from the polluted purple soil receiving acid rain may not pose as risks to water quality. Most of the Pb and Cd leachate concentrations were below their detection limits. By contrast, higher Cu and Zn leachate concentrations were found because they were released by the soil in larger amounts as compared with those of Pb and Cd. The differences in the Cu and Zn leachate concentrations between the controls (SAR at pH 5.6) and the treatments (SAR at pH 3.0 and 4.5) were significant. Similar trends were observed in the total leached amounts of Cu and Zn. The proportions of Cu, Pb, Cd, and Zn in the EXC and OX fractions were generally increased after the leaching experiment at three pH levels, whereas those of the RES, OM, and CAR fractions were slightly decreased. Acid rain favors the leaching of heavy metals from the contaminated purple soil and makes the heavy metal fractions become more labile. Moreover, a pH decrease from 5.6 to 3.0 significantly enhanced such effects.     

Assessment of Heavy Metal Pollution in Water and Sediments from the Ghalechay River,Baychebagh Copper Mine Area,Iran

The concentrations of selected heavy metals in sediments and waters in Baychebagh copper mine were determined using ICP-OES. Except for Co, the average concentrations of Cd, Cu, Pb, and Zn in sediments from the Ghalechay River in the district exceed the world-average shale and continental upper crust value. Enrichment factors for Pb, Cu, and Cd were significantly enriched in sediments, indicating environmental contamination. Geoaccumulation index calculated for different sampling stations indicates that the sediments are unpolluted with respect to Co and Zn while unpolluted to moderately polluted with Cu and highly polluted with Pb and Cd. The Sediment Quality Guidelines (SQGs) suggest that Cd and Pb may pose the highest risk for the environment. Sequential extraction analyses of sediments revealed that Cu, Co, Pb, and Zn bound to extractable, carbonate, reducible and oxidizable fractions are lower than residual fraction. About 10% of the total Pb was associated with the exchangeable fraction, indicating remobilization, while Cd (89%), Pb (73%) Co (58%), Cu (76%), and Zn (68%) closely associated with the residual and oxidizable fractions, resulting in their environmental immobility. The residual forms are not expected to be released under normal conditions in the river and could be considered an inert phase.     

Partitioning of Zn,Cd, Pb,and Cu in organic-rich soil profiles in the vicinity of a zinc smelter

Abstract

A five-step sequential extraction procedure was applied to organic-rich soil samples from five soil profiles situated 1–8 km from a zinc smelter. The partitioning of Zn, Cd, Pb, and Cu into five operationally defined fractions (exchangeable, “carbonate’’-bound, reducible, oxidizable, and residual) was studied at different soil depths down to 35cm. In the surface soil (0–1 cm) a major part of Pb and Cu was extracted in the oxidizable fraction, whereas for Zn and Cd slightly more was extracted in the ‘‘carbonate”-fraction than in the other four fractions. Extracted metal proportions in the oxidizable fraction were respectively of the order of 30%, 20%, 50%, and 80% for Zn, Cd, Pb, and Cu in the surface soil for all sites, but these proportions decreased with soil depth. In the surface soil less than 20% of all the elements were extracted in the residual fraction, but the proportions associated with this fraction generally increased with soil depth. In the C-horizon, differences in extracted proportions of Pb and Cu in the residual fraction were probably due to geochemical factors, whereas for Zn the low extracted proportion at a highly contaminated site (20%) may be due to Zn migration to the C-horizon at this site. For Cd the extracted proportions in the C-horizon were lower than for the other elements, generally below 20%, presumably because Cd is weaker in terms of its adsorption to the soil than the other elements studied. Total concentrations of the metals decreased strongly with increasing distance from the smelter, but less systematic differences were observed for their distributions among fractions. Potentially bioavailable metal proportions (exchangeable + “carbonate”-bound fraction) in the surface soil were about 50%, 60%, 20%, and 10% for Zn, Cd, Pb, and Cu, respectively. In C-horizon soil the mobility sequence Cd>Zn>Pb = Cu was generally observed. The present results indicate that the concentrations and chemical fractionation of Zn, Pb, and Cd in these soils represent a considerable risk to natural terrestrial food chains.     

Influence of some metal concentrations on the activity of antioxidant enzymes and concentrations of vitamin E and SH-groups in the digestive gland and gills of the freshwater bivalve Unio tumidus from the Serbian part of Sava River

We examined whether the freshwater bivalve Unio tumidus from the Sava River can serve as a bioindicator organism for long-term biomonitoring of river ecosystems for the presence of metal pollutants. To this end, we assessed in the digestive glands and gills of mussels, changes in activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST)), and changes in the concentrations of the non-enzymatic components of the antioxidant system (vitamin E and sulfhydryl groups (-SH), after exposure to metals in the environment. Mussels were collected at four sites where the concentrations of dissolved metals (Cu, Cd, Zn, Fe, Mn, Hg, Ni, As, Pb) were quantified. Cu, Ni and As exerted concentration-dependent inhibitory effects on CAT and GST activities. Increasing concentrations of Cd promoted increases in GSH-Px activity and -SH concentration. In response to increased Zn concentration GR activity increased whereas Fe promoted decreased enzymatic activity. Negative correlations between the concentrations of Cu and Cd and vitamin E, and a positive correlation between Mn and vitamin E concentrations were detected. The described correlations between components of the antioxidant system and metal levels in the environment reveal a high physiological sensitivity of freshwater mussels to pollution, supporting their use in biomonitoring of metal contamination in river ecosystems.     

Metal exposures to native populations of the caddisfly Hydropsyche (Trichoptera: Hydropsychidae) determined from cytosolic and whole body metal concentrations

Metal concentrations of the soluble fraction of the cytoplasm (cytosol) and the whole body were determined in the caddisfly Hydropsyche spp. (Trichoptera). Metal accumulation in the cytosol and the whole body were compared in samples collected along 380 kms of a contamination gradient in the Clark Fork river in four consecutive years (1992–1995), and from a contaminated tributary (Flint Creek). Samples from the contaminated sites were compared to an uncontaminated tributary (Blackfoot River). Relations between cytosolic metal concentration and cytosolic protein (used as a general biomarker of protein metabolism) also were examined in 1994 and 1995. Relative to whole body concentrations, cytosolic metal concentrations varied among metals and years. Spatial patterns in whole body and cytosolic Cd, Cu and Pb concentrations were qualitatively similar each year, and these concentrations generally corresponded to contamination levels measured in bed sediments. The proportions of metals recovered in the cytosol of ranged from 12 to 64% for Cd and Cu and from 2 to 38% for Pb. Zinc in the whole body also was consistent with contamination levels, but cytosolic Zn concentrations increased only at the highest whole body Zn concentrations. As a result, the proportion of Zn recovered in the cytosol ranged from 16 to 63% and tended to be inversely related to whole body Zn concentrations. The proportions of cytosolic metals varied significantly among years and, as a result, interannual differences in metal concentrations were greater in the cytosol than in the whole body. The results demonstrated that Hydropsyche in the river were chronically exposed to biologically available metals. Some features of this exposure were not evident from whole body concentrations. In general, protein levels did not correspond to cytosolic metal concentrations. A variety of environmental factors could interact with metal exposures to produce complex responses in protein metabolism. Systematic study will be necessary to differentiate the effects of multiple environmental stressors on organisms living in contaminated ecosystems. This revised version was published online in July 2006 with corrections to the Cover Date.     

Risk Element (As,Cd, Cu,Pb, and Zn) Contamination of Soils and Edible Vegetables in the Vicinity of Guixi Smelter,South China

Risk element (As, Cd, Cu, Pb, and Zn) contamination in soils and in two edible vegetables (Solanum melongena L. and Capsicum annum L.) was investigated in the vicinity of Guixi Smelter, South China. Soil As concentrations averaged 23.9 mg/kg. Sites near the smelter tailings recorded the highest levels of As and heavy metals in soils. The concentration order of heavy metals in soils was Cd < Pb < Zn < Cu. Cu and Cd in soils were abundant in the exchangeable and bound to carbonate fraction, while Pb and Zn were in the residual fraction, limiting their potential toxicity as pollutants. The proportions of the metals in the mobile fraction followed the order Pb < Zn < Cu < Cd. In Solanum melongena L. and Capsicum annum L., Zn concentration was the highest, followed by Cu, Cd, and Pb, different from that in soils and in the mobile fraction. Concentrations of heavy metals in the labile fractions in soils and in vegetables presented significant correlation (p < 0.05). Both of the two vegetables are not the Cu and Zn accumulators. As for Cd and As, Capsicum annum L. poses a higher risk to animal and human health than Solanum melongena L., with soil-plant transfer coefficients more than three. Root-stem is the main barrier for most of the heavy metals and As in the two vegetables, resulting in higher metal concentrations in roots relative to other plant tissues. The low stem-fruit transfer coefficients for Zn in Solanum melongena L. and for Pb in Capsicum annum L. suggested that very few of them could reach the fruits.     

Characterization of Heavy Metal Fractions in Agricultural Soils by Sequential Extraction Procedure: The Relationship Between Soil Properties and Heavy Metal Fractions

The present research was conducted to determine heavy metals in agricultural soils from Çanakkale, Turkey, using a sequential extraction procedure (acid soluble, reducible, oxidizable, and residual) as proposed by the Community Bureau of Reference (BCR) of the European Commission. Soil samples were taken from 12 different cultivated sites and analyzed for Cd, Co, Cr, Cu, Mn, Ni, Pb, and Zn concentrations. The results revealed an order of Mn > Cd > Pb > Co > Ni > Cu > Zn > Cr for the heavy metals based on the sum of the first three fractions (acid soluble + reducible + oxidizable). The relationships between soil properties and each metal fraction were identified through Pearsons's correlation analysis. Hierarchical cluster analysis was performed to determine the behaviors and similarities of metals in each fraction. While Mn, Pb, and Zn exhibited subjective behaviors in the acid-soluble fraction, Cd, Co, Cu, Cr, and Ni exhibited similar behaviors with each other.     

Heavy Metal Accumulation and Distribution in Organs of the Mussel Crenomytilus grayanus from Upwelling Areas of the Sea of Okhotsk and the Sea of Japan

The subcellular and cytosolic distribution of Zn, Fe, Ni, Cu, Mn, Cd, and Pb in the digestive gland and kidney of the mussel Crenomytilus grayanus from upwelling areas of the Sea of Okhotsk and the Sea of Japan was studied. Cd, Zn, Pb, and Ni have accumulated in the kidneys of mussels from these areas. When the concentrations of both essential and toxic metals in the mussel organs had increased significantly, their redistribution into the cytosol took place. Gel chromatography of cytosolic proteins in the kidneys of mussels sampled in the area with a strong stationary upwelling revealed metallothioneins. This is uncommon for bivalve mollusks from unpolluted areas. High molecular weight proteins act as preliminary ligands for Cd.     

Autometallographical localisation of Cu and Zn within target cell compartments of winkles following exposure to Cu&Zn mixtures

This investigation attempts to determine the usefulness of autometallography to localise particular metals in certain key tissues of molluscs exposed to metal mixtures. For this purpose, winkles (Littorina littorea) removed from shell were exposed to very high concentrations of either copper (Cu), zinc (Zn) or a mixture of both metals (Cu&Zn) dissolved in sea-water for short periods of time. Protein-bound metals were detected by autometallography as black silver deposits (BSD) on histological sections of gills, foot, mantle, digestive gland/gonad complex, stomach and kidney. Copper was localised within cytoplasmic granules of gill ciliated cells, nephrocytes and stomach epithelial cells as well as within digestive cell lysosomes. Zinc was essentially found in the basal lamina (histological sense) of gill, stomach, kidney and digestive gland epithelia. BSD were also evidenced in cytoplasmic granules of pore cells present in parenchymal connective tissue of mantle, foot, gill, digestive gland and stomach. Copper and zinc concentrations were additionally calculated for the whole soft body as well as for certain organs by atomic absorption spectrophotometry (AAS). According to AAS, a synergistic phenomenon would contribute to increase the rate of Cu and Zn accumulation in presence of each other. However, after exposure to Cu&Zn autometallography did not evidence any synergistic phenomenon, and Cu and Zn were localised in their respective accumulation sites. In conclusion, autometallography might indicate the presence of certain metals in the environment irrespective of factors, such as "metal-metal interaction-like" phenomena, affecting metal concentrations in soft tissues.     

First report of phytochelatins in a mushroom: induction of phytochelatins by metal exposure in Boletus edulis

Some species of macromycetes (mushrooms) consistently are found to contain high concentrations of toxic metals such as cadmium (Cd) and mercury (Hg), and consumption of wild-growing mushrooms is acknowledged as a significant source for Cd and Hg in humans. Yet little is known about the speciation of Cd and Hg in mushroom tissues. Here we present the first evidence of peptides of the phytochelatin family being responsible for binding a large fraction of Cd in caps of the macromycete Boletus edulis exposed to excess metals. Concentrations of Cd, Zn, Cu and Hg, as well as cytosolic Cd-binding capacity (CCBC), glutathione (GSH) and free proline (Pro) were quantified in fruiting bodies of B. edulis differentially exposed to a wide range of metals. Metal distribution among cytosolic compounds were investigated by size exclusion chromatography (SEC), followed by metal determinations with atomic absorption chromatography (AAS) and HR-ICP-MS. Cd-binding compounds in SEC elutates were investigated further by high performance liquid chromatography-mass spectrometry (HPLC-MS). CCBC was >90 times higher in the exposed group relative to the reference group (Mann-Whitney's P < 0.001), whereas concentrations of free Pro were almost identical for the two groups. For the whole study selection, CCBC correlated positively with metal exposure (Spearman's P < 0.001 for all four metals), suggesting dose-dependent induction of Cd-binding compounds by exposure to these metals, possibly as a defense mechanism. The presence of phytochelatins (PCs), a family of cystein-rich oligopeptides, was confirmed in Cd-containing SEC fractions by HPLC-MS. The appearance of more complex PCs was coupled to declining concentrations of GSH. To our knowledge this is the first report demonstrating the presence of PCs in a macromycete.