Metallothionein-like Proteins Research Articles

Are Accumulated Sulfide-Bound Metals Metabolically Available in the Benthic OligochaeteTubifex tubifex?

The present study evaluates the relationship between metal-binding sediment characteristics like acid volatile sulfides (AVS), metal accumulation, and internal metal distribution in the benthic oligochaete Tubifex tubifex and relates this accumulation to the induction of metallothionein-like proteins (MTLPs). In total, 15 Flemish lowland rivers were sampled. Cd, Cu, Zn, Pb, Ni, As, Cr, Co, and Ag concentrations were measured in environmental fractions (water and sediment) and worm tissue (both total and subcellular fractions). Furthermore, total cytosolic MTLP concentrations were measured in the worm tissue. Our results showed that Cd, Pb, Ni, and Cr were mainly stored as biological detoxified metal (BDM) while Cu, Zn, As, and Ag were mostly available in the metal sensitive fraction (MSF). A remarkable difference in the subcellular distribution of accumulated Cd, Ni, and Co between anoxic (SEMMe-AVS<0; mostly stored as BDM) and oxic (SEMMe-AVS>0; mostly stored in the MSF) sediments was noticed. Moreover, a rapid increase in MTLP induction was found when SEMTot-AVS>0. Our results indicate that the accumulated sulfide-bound metals were detoxified and little available to the metabolism of T. tubifex under anoxic conditions.

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate “sink” for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10μgL−1) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO3. Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.

Identification of differentially expressed genes in a resistant versus a susceptible blueberry cultivar after infection by Colletotrichum acutatum.

Anthracnose fruit rot, caused by the fungus Colletotrichum acutatum, is an important disease of blueberry worldwide. The cultivar Elliott is resistant, severely restricting fungal growth and sporulation relative to the susceptible cultivar Jersey. The objectives of this research were as follows: (i) to analyse pre-penetration events in 'Elliott' and 'Jersey' at different stages of fruit development; (ii) to identify putative defence genes in 'Elliott' fruit; and (iii) to monitor the timing of the oxidative burst in both cultivars. Light microscopy revealed no differences in the pre-penetration activities of C. acutatum on the immature fruit of both cultivars. However, at fruit ripening, conidia germinated and formed appressoria more rapidly on 'Jersey' than on 'Elliott' fruit. Using suppression subtractive hybridization, 37 differentially expressed sequence tags (ESTs) were detected in 'Elliott' versus 'Jersey' upon infection. Several of the ESTs had homology to known plant defence genes, such as a class II chitinase, pathogenesis-related protein 10 (PR10) and β-1,3-glucanase. Two putative genes involved in oxidative stress were identified: a metallothionein-like protein and monodehydroascorbate reductase. ESTs of fungal origin were also detected. Many ESTs had no homology to known genes. Using semi-quantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR), the expression of most of the candidate genes was detected earlier in 'Elliott' than in 'Jersey', some within 24 h post inoculation (hpi). Monitoring of the oxidative burst showed that the overall H(2) O(2) concentration was two to three times higher in 'Elliott' than in 'Jersey' at 24 hpi. The elucidation of the basis of resistance to C. acutatum in blueberry will facilitate the development of new anthracnose fruit rot-resistant cultivars.

Differential roles of metallothionein‐like proteins in cadmium uptake and elimination by the scallop Chlamys nobilis

Metallothionein-like proteins (MTLPs) are important inducible ligands for Cd detoxification, but whether MTLPs are involved in Cd bioaccumulation remains unclear. The present study investigated the potential interrelationships between MTLP turnover and Cd biokinetics in the scallop Chlamys nobilis with or without dissolved Cd pre-exposure. After Cd pre-exposure, increased synthesis rate and standing stock concentration of MTLPs as well as increased dissolved Cd uptake rate, were found in C. nobilis. However, the level of newly sequestrated Cd by MTLPs in the pre-exposed scallops was similar to that of the controls, indicating that MTLPs had no effect on Cd uptake from the dissolved phase. Instead, Cd uptake rate was positively related to the Cd binding rate of cellular debris and organelles. In addition, no significant difference was observed in the Cd dietary assimilation efficiency of the scallops, showing that MTLPs played a minimal role in Cd uptake from the dietary phase. Conversely, the Cd efflux rate constant decreased by 68 to 75% in the pre-exposed scallops in comparison with that in the control. Correspondingly, progressive elevations of MTLP-bound Cd and standing stock MTLP concentration were observed in the pre-exposed individuals during the Cd elimination period. In the pre-exposed scallops, the faster de novo MTLP synthesis could remobilize and recycle more Cd from other intracellular pools, effectively reducing the Cd elimination rate. The present study thus showed the differential roles of MTLPs in Cd uptake and elimination in the bivalves.

Differential tolerance of two Gammarus pulex populations transplanted from different metallogenic regions to a polymetal gradient

The River Hayle, Cornwall, UK exhibits pronounced Cu and Zn concentration gradients which were used to compare the metal handling abilities of two populations of Gammarus pulex (Crustacea: Amphipoda). One population was native to the Hayle region (Drym) and presumably has been historically impacted by elevated Cu and Zn levels, whilst naïve gammarids were collected from the River Cray, Kent, UK. Both populations were subject to a 32 day in situ exposure at four R. Hayle sites (Drym, Godolphin, Relubbus and St. Erth). Mortality (LT50), Cu and Zn accumulation and sub-cellular distribution, and oxidative stress (malondialdehyde production) increased with the expected Cu and Zn bioavailabilities at the four sites (i.e. Godolphin > Relubbus > St. Erth > Drym). The naïve population experienced greater metal induced effects in terms of Cu and Zn accumulation, oxidative stress responses and lower LT50s. Analysis of Cu and Zn sub-cellular distribution, however, revealed no significant differences in metal handling. In both populations each metal was localised predominantly to the sub-cellular fraction containing metal bound to metallothionein-like proteins (MTLP) or that holding both metal-rich granules (MRG) and exoskeleton, MTLP and MRG binding being indicative of metal detoxification. However, a greater capacity for detoxified metal storage is not a mechanism implicated in the perceived tolerance of the historically impacted gammarids. Instead our results suggest that the historically impacted population was adapted for lower uptake of Cu and Zn leading to lower bioaccumulation, stress response and ultimately mortality. These results demonstrate not only the usefulness of the in situ methodology, but also that differences in population exposure history can cause significant differences in metal responses during exposure at higher concentrations.

Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses

Cicer microphyllum, a wild relative of cultivated chickpea, is a high altitude cold desert-adapted species distributed in western and trans-Himalayas. A complementary DNA (cDNA) encoding metallothionein-like protein has been identified from a cold-induced subtraction cDNA library from C. microphyllum. The sequence of the cloned metallothionein gene from C. microphyllum (GQ900702) contains 240-bp-long open reading frame and encodes predicted 79-amino acid protein of 7.9kDa. Sequence analysis identified the motifs characteristic of type II metallothionein and designated as CmMet-2. Southern hybridization confirms a single copy of the CmMet-2 gene in C. microphyllum genome. In situ hybridization indicated spatial transcript regulation of CmMet-2 in root and aerial parts and also confirmed through real-time PCR-based quantitative transcript analysis. The data revealed a significantly low level of transcript in the aerial parts than the roots. Quantitative analysis using real-time PCR assay revealed induction of transcript in all parts of plants in response to cold stress at 4°C. The transcript abundance was found to increase exponentially with time course from 6 to 24h after exposure. Further, regulation of transcript accumulation in response to abscisic acid application, polyethylene glycol (100μM)-induced osmotic stress, or ZnSO(4) (1μM) foliar spray indicated by Northern hybridization suggests the involvement of CmMet-2 in multiple stress response.

Identification and transcriptional analysis of genes involved in Bacillus cereus-induced systemic resistance in Lilium

Bacillus cereus C1L was demonstrated to induce systemic resistance in Lilium formosanum against leaf blight caused by Botrytis elliptica. Suppression subtractive hybridization library of L. formosanum triggered by B. cereus C1L were screened and 3 differentially expressed genes were identified. Based on sequence analysis, these genes encoding putative glycine-rich protein, metallothionein-like protein, and PsbR protein of photosystem 2, were designated LfGRP1, LfMT1, and LfPsbR, respectively. The results of Northern blot analysis showed that expressions of LfGRP1, LfMT1 and LfPsbR increased in response to B. elliptica infection. On the other hand, expression of LfMT1 increased but expressions of LfGRP1 and LfPsbR decreased when the rhizosphere of L. formosanum was drenched with suspension of B. cereus C1L with or without subsequent challenge with B. elliptica on lily leaves. Similar expression profiles of homologues of LfGRP1, LfMT1, and LfPsbR (named LsGRP1, LsMT1, and LsPsbR, respectively) were presented in Lilium oriental hybrid Star Gazer. In addition, application of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, on the leaves reduced disease severity and expressions of LsGRP1 and LsPsbR just as that in response to B. cereus C1L treatment.

Differential Gene Expression Analysis of Yunnan Red Pear, Pyrus Pyrifolia, During Fruit Skin Coloration

The color of fruit skin is an important quality parameter, and in many plants, it is the result of coordinative regulation of the anthocyanin pathway. To characterize the mechanism involved in fruit peel coloration of Yunnan red pear (Pyrus pyrifolia), we constructed a subtractive cDNA library using the suppression subtractive hybridization (SSH) technology. cDNA of red peel exposed to sunlight (for 2, 4, 6, and 8 days) was subtracted from that of white skin unexposed to sunlight. Over 100 differentially expressed ESTs were obtained, putatively involved in primary and secondary metabolism, stress, and defense response. Expression analysis using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) for 13 genes was performed with two pear cultivars, light-skinned ‘Zaobaimi’ and red-skinned ‘Yunhong-1’, which had been bagged and then exposed to sunlight for 0, 1, 2, 3, 5, and 7 days before harvest. This analysis showed that genes encoding for a metallothionein-like protein and a NADP-malic acid enzyme were constitutively expressed, whereas other selected genes were either down- or up-regulated. Semiquantitative RT-PCR analysis for 7 anthocyanin biosynthetic pathway genes and 3 putative regulatory genes was also performed. Results showed that an R2R3 MYB transcription factor PyMYB10 was up-regulated in both the less-colored pear ‘Zaobaimi’ and well-colored red pear Yunhong-1 after the bag was removed, but that kinetics differed between cultivars. Other anthocyanin-related genes appeared to be coordinately regulated by the MYB–bHLH–WD40 complex. DFR and ANS genes seemed to be limiting factors for the peel coloration of less-colored pear ‘Zaobaimi’, while all biosynthetic steps are up-regulated by 7 days after bag removal in red fruit. This study suggests the regulation of red pear coloring is via differential effects of the MYB–bHLH–WD40 complex on the pear anthocyanin pathway genes.

Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve ( Unio pictorum) transplanted in a metal polluted river basin

The freshwater painter's mussel ( Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H 2O 2) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that some oxidative biomarkers, as well as those evaluating chromosomal and cell damages, are highly sensitive and could be profitably applied to caged painter's mussels for environmental quality assessment in freshwater.

Short-term metal accumulation and MTLP induction in the digestive glands of Perna virdis exposed to Zn and Cd

Time-dependent Zn and Cd accumulation and metallothionein like protein (MTLP) induction in the digestive glands of mussels, Perna virdis, were measured under different exposure conditions. The initial uptake rate at start of chase (ρ 0) and mean residence time (τ) were calculated to determine the physiological response of organisms and their potential detoxification mechanisms. It was found that in digestive glands, Zn had obviously higher ρ 0 and shorter mean residence time than Cd, indicating that these two metals had different accumulation dynamics even though they were very close in the periodic element table. MTLP levels in digestive glands varied from 0.51 to 1.05 μg/g ww (wet weight). The MTLP level increased continuously when mussels were exposed to low and middle levels of Zn and Cd media, and reached maximal levels at day 4, then decreased when they were exposed to high level Zn and Cd solutions. With regard to the fraction of Zn and Cd accumulated in the digestive glands, the ratios of soluble metal to total metal decreased continuously after exposure in low and middle levels of Zn and Cd media, and decreased continuously in the first 4 days and then to level off when mussels were exposed to media with high concentration of Zn and Cd. Results suggested that both MTLP induction and metal insolubilization were detoxification processes in digestive glands of mussels.

Differential uptake and oxidative stress response in zebrafish fed a single dose of the principal copper and zinc enriched sub-cellular fractions of Gammarus pulex

The sub-cellular compartmentalisation of trace metals and its effect on trophic transfer and toxicity in the aquatic food chain has been a subject of growing interest. In the present study, the crustacean Gammarus pulex was exposed to either 11 μg Cu l −1, added solely as the enriched stable isotope 65Cu, or 660 μg Zn l −1, radiolabeled with 2MBq 65Zn, for 16 days. Post-exposure the heat stable cytosol containing metallothionein-like proteins (MTLP) and a combined granular and exoskeletal (MRG + exo) fractions were isolated by differential centrifugation, incorporated into gelatin and fed to zebrafish as a single meal. Assimilation efficiency (AE) and intestinal lipid peroxidation, as malondialdehyde (MDA) were measured. There was a significant difference ( p < 0.05) between the retention of the MTLP-Zn (39.0 ± 6.4%) and MRG + exo-Zn (17.2 ± 3.7%) and of this zinc retained by the zebrafish a significantly greater proportion of the MTLP-Zn feed had been transported away from the site of uptake. For 65Cu, although the results pointed towards greater bioavailability of the MTLP fraction compared to MRG + exo during the slow elimination phase (24–72 h) these results were not significant ( p = 0.155). Neither zinc feed provoked a lipid peroxidation response in the intestinal tissue of zebrafish compared to control fish (gelatin fed), but both 65Cu labeled feeds did. The greater effect was exerted by the MRG + exo (2.96 ± 0.29 nmol MDA mg protein −1) feed which three-fold greater than control ( p < 0.01) and almost twice the MDA concentration of the MTLP feed (1.76 ± 0.21 nmol MDA mg protein −1, p < 0.05). The oxidative stress response produced by Zn and Cu is in keeping with their respective redox potentials; Zn being oxidatively inert and Cu being redox active. These results are similar, in terms of bioavailability and stress response of each feed, to those in our previous study in which 109Cd labeled G. pulex fractions were fed to zebrafish. Thus it appears that when a metal (Cu or Cd) has the potential to cause cytotoxicity via lipid peroxidation, a feed consisting of a largely unavailable fraction (MRG + exo) causes a greater intestinal stress response than the more bioavailable (MTLP) feed.

Responses of abalone Haliotis diversicolor to sublethal exposure of waterborne and dietary silver and cadmium

In this study, we examined the chronic waterborne and dietary exposure of silver (Ag) and cadmium (Cd) to the abalone Haliotis diversicolor using various endpoints such as growth and feeding rates, metal body burden, subcellular distribution, and metallothionein (MT) concentration over a period of 7 weeks of exposure. The growth and feeding rates of abalones were inhibited during the early stage of exposure to different extents, but then recovered to nearly the control levels. A large portion of Ag was redistributed to organelles and metal-rich granules from the cellular debris fraction, whereas cellular debris and metallothionein-like protein were the dominant pools for the storage of Cd, which remained comparable during the exposure period. The MT concentrations were significantly elevated (in a dose-dependent manner) within the first 2 weeks of exposure, after which the MT concentrations started to decrease. All these results implied that abalones respond rapidly to metal exposure, but apparently developed subsequent acclimation.

Metal concentrations and metallothionein-like protein levels in deep-sea fishes captured near hydrothermal vents in the Mid-Atlantic Ridge off Azores

The knowledge of metal contamination in deep-sea fishes living in the surroundings of hydrothermal vents is very scarce, along with the detoxification mechanisms that allow them to live near one of the most metal contaminated marine environments. Six deep-sea fish species, although not vent endemic were collected near three Mid-Atlantic Ridge (MAR) hydrothermal vents (Menez Gwen, Lucky Strike and Rainbow) and the gills, muscle and liver were selected for this study due to their importance in metal metabolism and storage. The concentrations of seven metals (Ag, Cd, Cr, Cu, Fe, Mn, and Ni) and a metal-related biomarker (metallothionein-like proteins–MTL) were assessed. Major differences in metal accumulation among fish species are related to their feeding habits and vent site of their capture. The liver and gills are in general the most important tissues for metal accumulation compared to the muscle, but tissue partitioning is very dependent on the fish species considered. Compared to other deep-sea fishes, fish capture in the vicinity of hydrothermal vents accumulates higher amounts of metals in general. However, MTL levels are not considerably different from what is found in commercial coastal fishes, and is poorly correlated with metal concentrations in the tissues. Therefore, MTL may not constitute one major detoxification system for deep-sea species living in the vicinity of three important MAR vent sites.

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 (µgg−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.

Experimental exposure to cadmium affects metallothionein-like protein levels but not survival and growth in wolf spiders from polluted and reference populations

Both local adaptation and acclimation in tolerance mechanisms may allow populations to persist under metal pollution. However, both mechanisms are presumed to incur (energetic) costs and to trade-off with other life-history traits. To test this hypothesis, we exposed Pardosa saltans (Lycosidae) spiderlings originating from metal-polluted and unpolluted sites to a controlled cadmium (Cd) treatment, and compared contents of metal-binding metallothionein-like proteins (MTLPs), internal metal concentrations, and individual survival and growth rates with a reference treatment. While increased MTLP concentrations in offspring originating from both polluted and unpolluted populations upon exposure indicates a plastic tolerance mechanism, survival and growth rates remain largely unaffected, independent of the population of origin. However, MTLP and Cd concentrations were not significantly correlated. We suggest that MTLP production may be an important mechanism enabling P. saltans populations to persist in ecosystems polluted with heavy metals above a certain level.

Cadmium accumulation and loss in the Pacific oyster Crassostrea gigas along the west coast of the USA

Oysters can accumulate cadmium (Cd) in very high concentrations in their tissues and there is now increasing concern for the seafood safety of farmed oysters worldwide. Bioaccumulation and biokinetics of Cd (dietary assimilation, uptake from the dissolved phase, and efflux) were mea- sured in different populations of Pacific oyster Crassostrea gigas collected along the west coast of the USA in 2 different seasons. Triploid juvenile oysters were first transplanted to 5 Washington sites and 1 Oregon site for 3 mo, then transported back to the laboratory for tissue Cd measurements and bio- kinetic experiments. A second group of seed oysters was collected from California and Washington for additional analysis. Oysters from all sites had Cd concentrations lower than the food standard limit and there was spatial difference in tissue Cd concentrations. A significant fraction of Cd was associ- ated with metallothionein-like proteins, implying that the oysters can detoxify Cd by induction of metallothionein. The difference in Cd dietary assimilation (using diatoms as a food source) and efflux in oysters transplanted to different locations was not significant. The uptake rate constants for Cd from the dissolved phase, however, differed markedly among the transplanted populations, and appeared to be affected by the oysters' clearance rate, depending on the population. This uptake rate was also negatively affected by the size of oysters. Dietary assimilation of Cd was comparable at diatom food concentrations below 2 to 5 mg l -1 , and was lower with ingested sediments than with diatoms. Measurements of Cd kinetics in different populations of Pacific oysters under a variety of environmental conditions provide an insight into the Cd accumulation in this commercially important species.

Metallothionein-Like Proteins and Energy Reserve Levels after Ni and Pb Exposure in the Pacific White PrawnPenaeus vannamei

This study analyzed the changes in metallothionein-like proteins (MTLPs) and Energy Reserves (ERs) in hepatopancreas and abdominal muscle of the white prawn Penaeus vannamei. Realistic metal concentration exposure for 10 days to Ni and Pb in solution revealed that juvenile prawns partially induce MTLP in hepatopancreas after Pb exposure. Ni was distributed equally between soluble and insoluble fractions, while Pb was present only in the insoluble fraction, suggesting different detoxification strategy. No changes in lipids and glycogen concentration were detected under these experimental conditions in both tissues analyzed. MTLP could not be considered as a suitable indicator for lead exposure in hepatopancreas.

Subcellular compartmentalization of cadmium, nickel, and lead in Gammarus fossarum: Comparison of methods

Knowledge of the subcellular behavior of toxicants, mainly metals, in living organisms is fundamental for understanding their toxicological consequences at individual and trans-generation levels and throughout the food web. However, different partitioning methodologies are available in the literature, particularly concerning the cytosolic fraction. Different cytosolic compounds, metallothionein-like proteins (MTLPs), and non-metallothionein-like proteins (non-MTLPs) could be separated by size exclusion chromatography or by heat treatment and centrifugation. Our results show changes of metal partitioning in the cytosolic fraction according to the methodologies used. After heat treatment, the majority of cytosolic cadmium (Cd) was found with MTLPs compounds and the opposite results were observed with size exclusion chromatography. Metal partitioning could also play an important role in the metal trophic transfer. Therefore, metal partitioning in Gammarus fossarum, an ecologically relevant species and good metal bioaccumulator, defining the trophically available metal fraction, is also discussed.

Comparative toxicity of cadmium in the commercial fish species Sparus aurata and Solea senegalensis

The induction of metallothionein-like proteins (MTLPs) as well as cadmium levels (Cd) was studied in tissues of gilthead sea bream Sparus aurata and flatfish Solea senegalensis after an intraperitoneal (i.p.) injection of cadmium (2.5 mg kg −1 body weight). The liver, gills, intestine and blood of S. aurata as well as the liver and intestine of S. senegalensis were collected for analysis at 0, 3 and 6 days after the injection. Cd levels significantly increased in all tissues of the treated animals, and the highest accumulation was found in the liver in both species ( p<0.05). Cadmium administration provoked significant inductions of MTLP synthesis in the liver and intestine of S. aurata ( p<0.05), while no increase in these protein levels was recorded in tissues of S. senegalensis. Our findings imply that MTLP synthesis in S. aurata probably led to an enhanced ability to cope with the toxicant, whereas the MTLP induction in S. senegalensis was inhibited by the accumulated cadmium, as reflected by the mortality rate of this species. Under the experimental conditions, hepatic MTLPs appear to be a good indicator of the Cd level in S. aurata; however, no link was found between the cadmium concentration and MTLP induction in S. senegalensis, which suggests that MTLPs did not play a main role in detoxification by metal sequestration.

Cadmium bound to metal rich granules and exoskeleton from Gammarus pulex causes increased gut lipid peroxidation in zebrafish following single dietary exposure

There has been a growing interest in establishing how the sub-cellular distribution of metals in macro-invertebrate prey affects metal trophic bioavailability and toxicity. In this study, the crustacean Gammarus pulex was exposed to 300 μg Cd l −1 spiked with 109Cd for 13 days, from which the two principal metal containing sub-cellular fractions, the metallothionein-like protein (MTLP) and the metal rich granule and exoskeleton (MRG + exo) were isolated. These fractions were produced at equal metal content, incorporated into gelatin and fed to zebrafish as a single meal; assimilation efficiency (AE), carcass and gut tissue metal concentrations and gut lipid peroxidative damage measured as malondialdehyde (MDA) were assessed. The AE of cadmium bound to the MTLP fraction was 32.1 ± 5.6% which was significantly greater than the AE of MRG + exo bound Cd, 13.0 ± 2.1% ( p < 0.05). Of the metal retained by the fish at 72 h post-feeding, 94% of MTLP-Cd had been incorporated into the carcass, whereas a significant proportion (46%) of MRG + exo-Cd, although assimilated, appeared to remain associated with intestinal tissue. However, this did not translate into a gut tissue concentration difference with 6.8 ± 1.2 ng Cd g −1 in fish fed MTLP-Cd compared to 9.5 ± 1.4 ng Cd g −1 in fish fed MRG + exo fraction. Both feeds led to significantly increased MDA levels compared to the control group (gelatin only feed), but MRG + exo feed caused significantly more oxidative damage than the MTLP feed ( p < 0.01). Thus, MTLP-Cd is more bioavailable than the cadmium bound to granules and exoskeleton, but it was the latter fraction, largely considered as having limited bioavailability, that appeared to exert a greater localised oxidative injury to the digestive tract of zebrafish.